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Sample records for vegf independent mechanism

  1. A gastrin precursor, gastrin-gly, upregulates VEGF expression in colonic epithelial cells through an HIF-1-independent mechanism

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    Bertrand, Claudine; Kowalski-Chauvel, Aline; Do, Catherine; Résa, Cécile; Najib, Souad; Daulhac, Laurence; Wang, Timothy C.; Ferrand, Audrey; Seva, Catherine

    2013-01-01

    One of the major angiogenic factor released by tumor cells is VEGF. Its high expression is correlated with poor prognosis in colorectal tumors. In colon cancer, gastrin gene expression is also upregulated. In these tumors, gastrin precursors are mainly produced and act as growth factors. Recently, a study has also shown that the gastrin precursor, G-gly induced in vitro tubules formation by vascular endothelial cells suggesting a potential proangiogenic role. Here, we demonstrate that stimulation of human colorectal cancer cell lines with G-gly increases the expression of the proangiogenic factor VEGF at the mRNA and protein levels. In addition, blocking the progastrin autocrine loop leads to a downregulation of VEGF. Although HIF-1 is a major transcriptional activator for VEGF our results suggest an alternative mechanism for VEGF regulation in normoxic conditions, independent of HIF-1 that involves the PI3K/AKT pathway. Indeed we show that G-gly does not lead to HIF-1 accumulation in colon cancer cells. Moreover, we found that G-gly activates the PI3K/AKT pathway and inhibition of this pathway reverses the effects of G-gly observed on VEGF mRNA and protein levels. In correlation with these results, we observed in vivo, on colon tissue sections from transgenic mice overexpressing G-gly, an increase in VEGF expression in absence of HIF-1 accumulation. In conclusion, our study demonstrates that gastrin precursors, known to promote colon epithelial cells proliferation and survival can also contribute to the angiogenesis process by stimulating the expression of the proangiogenic factor VEGF via the PI3K pathway and independently of hypoxia conditions. PMID:19876923

  2. p38 MAP kinase inhibition promotes primary tumour growth via VEGF independent mechanism.

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    O'Sullivan, Adrian W; Wang, Jiang H; Redmond, Henry P

    2009-11-15

    The surgical insult induces an inflammatory response that activates P38 MAP kinases and solid tumours can also release cytokines. Therfore inhibition of these pathways may reduce tumour growth We set out to examine the effects of P38-MAPK inhibition on apoptosis, proliferation, VEGF release and cell cycle effects in-vitro and on primary tumour growth in-vivo. 4T-1 cells (2 x 105 cells/well) were incubated, in 24 well plates with control, 25, 50 or 100 ng/ml of SB-202190 for 24 hours. Cells were subsequently asessed for apoptosis, proliferation, VEGF release and cell cycle analysis. Balb-c mice each received 1 x 106 4T 1 cells subcutaneously in the flank and were then randomised to receive control or SB202190 (2.5 microM/kg) by intraperitoneal injection daily. Tumour size was measured alternate days and at day 24 animals were sacrificed and serum VEGF assessed. P38-MAPK inhibition in-vitro resulted in a significant reduction in proliferation (75.2 +/- 8.4% vs. 100 +/- 4.3%, p etag/ml compared to 158.6 +/- 27.1 etag/ml) These findings demonstrate that P38-MAPK inhibition in-vitro reduces proliferation and G1 cell cycle phase as well as promoting primary tumour growth in-vivo. These effects would appear to be independent of VEGF.

  3. Aberrant, ectopic expression of VEGF and VEGF receptors 1 and 2 in malignant colonic epithelial cells. Implications for these cells growth via an autocrine mechanism

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    Ahluwalia, Amrita [Veterans Affairs Long Beach Healthcare System, Long Beach, CA (United States); Jones, Michael K. [Veterans Affairs Long Beach Healthcare System, Long Beach, CA (United States); Department of Medicine, University of California, Irvine, CA (United States); Szabo, Sandor [Veterans Affairs Long Beach Healthcare System, Long Beach, CA (United States); Department of Pathology, University of California, Irvine, CA (United States); Tarnawski, Andrzej S., E-mail: amrita.ahluwalia@va.gov [Veterans Affairs Long Beach Healthcare System, Long Beach, CA (United States); Department of Medicine, University of California, Irvine, CA (United States)

    2013-08-09

    Highlights: •Malignant colonic epithelial cells express VEGF and its receptors. •Cultured colon cancer cells secrete VEGF into the medium. •Inhibition of VEGF receptor significantly decreases colon cancer cell proliferation. •VEGF is critical for colon cancer cell growth. -- Abstract: Vascular endothelial growth factor A (referred to as VEGF) is implicated in colon cancer growth. Currently, the main accepted mechanism by which VEGF promotes colon cancer growth is via the stimulation of angiogenesis, which was originally postulated by late Judah Folkman. However, the cellular source of VEGF in colon cancer tissue; and, the expression of VEGF and its receptors VEGF-R1 and VEGF-R2 in colon cancer cells are not fully known and are subjects of controversy. Material and methods: We examined and quantified expression of VEGF, VEGF-R1 and VEGF-R2 in three different human colonic tissue arrays containing sections of adenocarcinoma (n = 43) and normal mucosa (n = 41). In human colon cancer cell lines HCT116 and HT29 and normal colon cell lines NCM356 and NCM460, we examined expression of VEGF, VEGF-R1 and VEGF-R2 mRNA and protein, VEGF production and secretion into the culture medium; and, the effect of a potent, selective inhibitor of VEGF receptors, AL-993, on cell proliferation. Results: Human colorectal cancer specimens had strong expression of VEGF in cancer cells and also expressed VEGF-R1 and VEGF-R2.In vitro studies showed that human colon cancer cell lines, HCT116 and HT29, but not normal colonic cell lines, express VEGF, VEGF-R1 and VEGF-R2 and secrete VEGF into the medium up to a concentration 2000 pg/ml within 48 h. Furthermore, we showed that inhibition of VEGF receptors using a specific VEGF-R inhibitor significantly reduced proliferation (by >50%) of cultured colon cancer cell lines. Conclusions: Our findings support the contention that VEGF generated by colon cancer cells stimulates their growth directly through an autocrine mechanism that is

  4. Lymphatic vessel density and VEGF-C expression as independent predictors of melanoma metastases.

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    Špirić, Zorica; Eri, Živka; Erić, Mirela

    2017-11-01

    In many patients, the clinical behaviour of cutaneous melanoma is very difficult to predict by traditional histologic and clinical parameters. This study aimed to examine the role of quantitative parameters of tumour lymphangiogenesis and vascular endothelial growth factor (VEGF)-C in predicting metastatic risk in patients with cutaneous melanoma. One hundred melanoma specimens were stained with lymphatic-specific antibody D2-40 and with anti-VEGF-C antibody. Quantitative parameters of lymphangiogenesis-lymphatic vessel density (LVD) and lymphatic vessel area (LVA)-were determined by computer-assisted morphometric analysis. Moderate or strong staining was assessed as a positive expression of VEGF-C in tumour cells. Univariate analysis revealed that intratumoural LVD, peritumoural LVD, VEGF-C expression in tumour cells, melanoma thickness, Clark level, ulceration, gender and histologic type were significant predictors of lymph node metastasis (p = 0.000, p = 0.000, p = 0.000, p = 0.000, p = 0.005, p = 0.005, p = 0.011 and p = 0.027, respectively). No significant association of intratumoural and peritumoural LVA with metastases was found. In multivariate analysis, independent predictors of metastatic risks were melanoma thickness [odds ratio OR = 1.655, 95% confidence interval (CI) 1.102-2.484, p = 0.015], intratumoural LVD (OR = 1.086, 95% CI 1.027-1.148, p = 0.004), peritumoural LVD (OR = 1.050, 95% CI 1.008-1.094, p = 0.020) and a positive VEGF-C expression in tumour cells (OR = 20.337, 95% CI 2.579-160.350, p = 0.004). This study identified intratumoural and peritumoural LVD and the VEGF-C expression in tumour cells as more significant predictors of metastatic risk than melanoma thickness, ulceration and other clinical-pathological parameters. Copyright © 2017 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.

  5. Cellular and molecular mechanisms involved in the neuroprotective effects of VEGF on motoneurons

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    Jerònia eLladó

    2013-10-01

    Full Text Available Vascular endothelial growth factor (VEGF, originally described as a factor with a regulatory role in vascular growth and development, it is also known for its direct effects on neuronal cells. The discovery in the past decade that transgenic mice expressing reduced levels of VEGF developed late-onset motoneuron pathology, reminiscent of amyotrophic lateral sclerosis (ALS, opened a new field of research on this disease. VEGF has been shown to protect motoneurons from excitotoxic death, which is a relevant mechanism involved in motoneuron degeneration in ALS. Thus, VEGF delays motoneuron degeneration and increases survival in animal models of ALS. VEGF exerts its anti-excitotoxic effects on motoneurons through molecular mechanisms involving the VEGF receptor-2 resulting in the activation of the PI3-K/Akt signaling pathway, upregulation of GluR2 subunit of AMPA receptors, inhibition of p38MAPK and induction of the anti-apoptotic molecule Bcl-2. In addition, VEGF acts on astrocytes to reduce astroglial activation and to induce the release of growth factors. The potential use of VEGF as a therapeutic tool in ALS is counteracted by its vascular effects and by its short effective time frame. More studies are needed to assess the optimal isoform, route of administration and time frame for using VEGF in the treatment of ALS.

  6. Interaction between VEGF and Calcium-Independent Phospholipase A(2) in Proliferation and Migration of Retinal Pigment Epithelium

    DEFF Research Database (Denmark)

    Toft-Kehler, Anne Katrine; Andersen, Emelie Cammilla; Andreasen, Jens Rovelt

    2012-01-01

    Purpose: Inhibition of VEGF in the eye is an important treatment modality for reducing proliferation and migration of retinal pigment epithelium (RPE) in age-related macular degeneration (AMD). Additionally, previous studies suggest calcium-independent phospholipase A2 group VIA (iPLA2-VIA...

  7. PPARγ-Independent Mechanism

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    Christopher M. Hogan

    2011-01-01

    Full Text Available Acute and chronic lung inflammation is associated with numerous important disease pathologies including asthma, chronic obstructive pulmonary disease and silicosis. Lung fibroblasts are a novel and important target of anti-inflammatory therapy, as they orchestrate, respond to, and amplify inflammatory cascades and are the key cell in the pathogenesis of lung fibrosis. Peroxisome proliferator-activated receptor gamma (PPARγ ligands are small molecules that induce anti-inflammatory responses in a variety of tissues. Here, we report for the first time that PPARγ ligands have potent anti-inflammatory effects on human lung fibroblasts. 2-cyano-3, 12-dioxoolean-1, 9-dien-28-oic acid (CDDO and 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2 inhibit production of the inflammatory mediators interleukin-6 (IL-6, monocyte chemoattractant protein-1 (MCP-1, COX-2, and prostaglandin (PGE2 in primary human lung fibroblasts stimulated with either IL-1β or silica. The anti-inflammatory properties of these molecules are not blocked by the PPARγ antagonist GW9662 and thus are largely PPARγ independent. However, they are dependent on the presence of an electrophilic carbon. CDDO and 15d-PGJ2, but not rosiglitazone, inhibited NF-κB activity. These results demonstrate that CDDO and 15d-PGJ2 are potent attenuators of proinflammatory responses in lung fibroblasts and suggest that these molecules should be explored as the basis for novel, targeted anti-inflammatory therapies in the lung and other organs.

  8. Activation of protease-activated receptor 2 induces VEGF independently of HIF-1.

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    Jeppe Grøndahl Rasmussen

    Full Text Available BACKGROUND: Human adipose stem cells (hASCs can promote angiogenesis through secretion of proangiogenic factors such as vascular endothelial growth factor (VEGF. In other cell types, it has been shown that induction of VEGF is mediated by both protease activated receptor 2 (PAR2 and hypoxia inducible factor 1(HIF-1. The present study hypothesized that PAR2 stimulation through activation of kinase signaling cascades lead to induction of HIF-1 and secretion of VEGF. METHODOLOGY/PRINCIPAL FINDINGS: Immunohistochemistry revealed the expression of PAR2 receptors on the surface of hASCs. Blocking the PAR2 receptors with a specific antibody prior to trypsin treatment showed these receptors are involved in trypsin-evoked increase in VEGF secretion from hASCs. Blocking with specific kinase inhibitors suggested that that activation of MEK/ERK and PI3-kinase/Akt pathways are involved in trypsin-eveoked induction of VEGF. The effect of the trypsin treatment on the transcription of VEGF peaked at 6 hours after the treatment and was comparable to the activation observed after keeping hASCs for 24 hours at 1% oxygen. In contrast to hypoxia, trypsin alone failed to induce HIF-1 measured with ELISA, while the combination of trypsin and hypoxia had an additive effect on both VEGF transcription and secretion, results which were confirmed by Western blot. CONCLUSION: In hASCs trypsin and hypoxia induce VEGF expression through separate pathways.

  9. TNF-α upregulates HIF-1α expression in pterygium fibroblasts and enhances their susceptibility to VEGF independent of hypoxia.

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    Kim, Kyoung Woo; Lee, Soo Jin; Kim, Jae Chan

    2017-11-01

    The clinical manifestations of pterygium are characterized by rapid growth and postoperative recurrences. We had previously proposed that hypoxia-inducible factor (HIF)-1α recruits progenitor cells during the development and progression of pterygia. Recently, it was reported that various stimuli, including inflammation, could activate HIF-1α even under normoxic conditions. The ocular surface directly faces external environments, and is thus frequently exposed to inflammatory insults. First, we examined the gene expression of HIF-1α, its downstream molecule, vascular endothelial growth factor (VEGF)-A, and VEGF receptor (VEGFR)-2 in corneal and conjunctival cells compared with cultured human umbilical vein endothelial cells. Corneal fibroblasts had high expression of VEGFR-2 in the presence of TNF-α, and HIF-1α was activated by TNF-α in diverse ocular surface cells. The HIF-1α/VEGF/VEGFR signaling pathway in response to TNF-α was evaluated in cultured human pterygium fibroblasts (HPFs) at the gene and protein levels and was compared to treatment with cobalt chloride (CoCl 2 ), a hypoxic mimetic, to exclude the effect of hypoxia. Although VEGF-A expression was not changed by TNF-α, expression of HIF-1α and VEGFR-2 was enhanced in HPFs treated with TNF-α, independent of hypoxia conditioning. In addition, VEGF-C gene expression was activated solely by TNF-α in HPF, but VEGF-B levels were not significantly affected. These results may provide mechanistic explanations for the uniquely vigorous proliferation of pterygium fibrovascular tissue during TNF-α-induced ocular surface inflammation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. VEGFR2-mediated vascular dilation as a mechanism of VEGF-induced anemia and bone marrow cell mobilization.

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    Lim, Sharon; Zhang, Yin; Zhang, Danfang; Chen, Fang; Hosaka, Kayoko; Feng, Ninghan; Seki, Takahiro; Andersson, Patrik; Li, Jingrong; Zang, Jingwu; Sun, Baocun; Cao, Yihai

    2014-10-23

    Molecular mechanisms underlying tumor VEGF-induced host anemia and bone marrow cell (BMC) mobilization remain unknown. Here, we report that tumor VEGF markedly induced sinusoidal vasculature dilation in bone marrow (BM) and BMC mobilization to tumors and peripheral tissues in mouse and human tumor models. Unexpectedly, anti-VEGFR2, but not anti-VEGFR1, treatment completely blocked VEGF-induced anemia and BMC mobilization. Genetic deletion of Vegfr2 in endothelial cells markedly ablated VEGF-stimulated BMC mobilization. Conversely, deletion of the tyrosine kinase domain from Vegfr1 gene (Vegfr1(TK-/-)) did not affect VEGF-induced BMC mobilization. Analysis of VEGFR1(+)/VEGFR2(+) populations in peripheral blood and BM showed no significant ratio difference between VEGF- and control tumor-bearing animals. These findings demonstrate that vascular dilation through the VEGFR2 signaling is the mechanism underlying VEGF-induced BM mobilization and anemia. Thus, our data provide mechanistic insights on VEGF-induced BMC mobilization in tumors and have therapeutic implications by targeting VEGFR2 for cancer therapy. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Mechanism of selective VEGF-A binding by neuropilin-1 reveals a basis for specific ligand inhibition.

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    Matthew W Parker

    Full Text Available Neuropilin (Nrp receptors function as essential cell surface receptors for the Vascular Endothelial Growth Factor (VEGF family of proangiogenic cytokines and the semaphorin 3 (Sema3 family of axon guidance molecules. There are two Nrp homologues, Nrp1 and Nrp2, which bind to both overlapping and distinct members of the VEGF and Sema3 family of molecules. Nrp1 specifically binds the VEGF-A(164/5 isoform, which is essential for developmental angiogenesis. We demonstrate that VEGF-A specific binding is governed by Nrp1 residues in the b1 coagulation factor domain surrounding the invariant Nrp C-terminal arginine binding pocket. Further, we show that Sema3F does not display the Nrp-specific binding to the b1 domain seen with VEGF-A. Engineered soluble Nrp receptor fragments that selectively sequester ligands from the active signaling complex are an attractive modality for selectively blocking the angiogenic and chemorepulsive functions of Nrp ligands. Utilizing the information on Nrp ligand binding specificity, we demonstrate Nrp constructs that specifically sequester Sema3 in the presence of VEGF-A. This establishes that unique mechanisms are used by Nrp receptors to mediate specific ligand binding and that these differences can be exploited to engineer soluble Nrp receptors with specificity for Sema3.

  12. Cancer-Associated Systemic Syndrome (CASS:The Mechanism of VEGF in Tumor-Bearing Mice

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    Fang CHEN

    2009-04-01

    Full Text Available Background and objective VEGF plays an important role in the development of cancer. The aim of this study is to observe the structural alterations in multiple organs of high VEGF expression mice, and insight into the role of tumor-derived VEGF in the development of CASS. Methods Murine fibrosarcoma of T241-VEGF and T241- Vector tumor cells were transplanted subcutaneously in mice to construct the xenograft tumor model. The mice gross examinations were observed and the percentage of survival animals in each group is presented. The level of hemoglobin, the numbers of erythrocytes and serum concentration of VEGF in peripheral blood were analyzed. Histological analysis of liver, spleen, adrenal gland and bone-marrow were applied. Vascular networks in tumors were analyzed under a confocal microscope. Results The VEGF-expressing tumor bearing mice manifested CASS by severe anemia, hepatosplenomegaly and loss of body weight. The survival rate of mice was decreased. The level of hemoglobin and erythrocytes in circulating blood were significantly reduced (P<0.01, with the increased serum concentration of VEGF. The blood vessels of tumorappeared as primitive and dilated sinusoidal vascular structures. Conclusion The tumor-produced VEGF affect multiple tissues, organs and resulted in CASS in mice model. It suggest that VEGF might be involved in the occurrence and development of CASS. It might be helpful for anti-VEGF therapy in clinical CASS and combing anti-VEGF therapy in advanced cancer patients.

  13. Discontinuation of anti-VEGF cancer therapy promotes metastasis through a liver revascularization mechanism

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    Yang, Yunlong; Zhang, Yin; Iwamoto, Hideki

    2016-01-01

    The impact of discontinuation of anti-VEGF cancer therapy in promoting cancer metastasis is unknown. Here we show discontinuation of anti-VEGF treatment creates a time-window of profound structural changes of liver sinusoidal vasculatures, exhibiting hyper-permeability and enlarged open-pore size...

  14. Effect and possible mechanism of monocyte-derived VEGF on monocyte-endothelial cellular adhesion after electrical burns.

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    Ruan, Qiongfang; Zhao, Chaoli; Ye, Ziqing; Ruan, Jingjing; Xie, Qionghui; Xie, Weiguo

    2015-06-01

    One of the major obstacles in the treatment of severe electrical burns is properly handling the resulting uncontrolled inflammation. Such inflammation often causes secondary injury and necrosis, thus complicating patient outcomes. Vascular endothelial grow factor (VEGF) has emerged as an important mediator for the recruitment of monocytes to the site inflammation. This study was designed to explore the effects and possible mechanism of VEGF on monocyte-endothelial cellular adhesion. To do so, we used a cultured human monocytic cell line (THP-1) that was stimulated with serum derived from rats that had received electrical burns. Serum was obtained from rats that had received electrical burns. Both the VEGF and soluble flt-1 (sflt-1) concentrations of the serum were determined by double-antibody sandwich ELISA. The concentrations of VEGF, sflt-1, and TNF-α obtained from the cell-free cultured supernatant of THP-1 cells that had been exposed to the serum were then determined by double-antibody sandwich ELISA. Serum-stimulated THP-1 cells were added to wells with a monolayer of endothelial cells to detect the level of monocyte-endothelial cells adhesion. Finally, the state of phosphorylation of AKT was determined by Western blotting. Both in vivo and in vitro studies showed that compared to controls, the levels of VEGF were significantly increased after electrical burns. This increased was accompanied by a reduction of sflt-1 levels. Furthermore, the serum of rats that had received electrical burns was able to both activate monocytes to secrete TNF-α and enhance monocyte-endothelial cell adhesion. Treatment with the serum also resulted in an up-regulation of the phosphorylation of AKT, but had no effect on the total levels of AKT. Phosphatidylinositide 3-kinases (PI3K) inhibition decreased the number of THP-1 cells that were adhered to endothelial cells. Finally, sequestering VEGF with sflt-1 was able to reduce the effect on monocyte-endothelial cells adhesion by

  15. Inhibition of VEGF: a novel mechanism to control angiogenesis by Withania somnifera's key metabolite Withaferin A.

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    Saha, Sanjib; Islam, Md Khirul; Shilpi, Jamil A; Hasan, Shihab

    2013-01-01

    Angiogenesis, or new blood vessel formation from existing one, plays both beneficial and detrimental roles in living organisms in different aspects. Vascular endothelial growth factor (VEGF), a signal protein, well established as key regulator of vasculogenesis and angiogenesis. VEGF ensures oxygen supply to the tissues when blood supply is not adequate, or tissue environment is in hypoxic condition. Limited expression of VEGF is necessary, but if it is over expressed, then it can lead to serious disease like cancer. Cancers that have ability to express VEGF are more efficient to grow and metastasize because solid cancers cannot grow larger than a limited size without adequate blood and oxygen supply. Anti-VEGF drugs are already available in the market to control angiogenesis, but they are often associated with severe side-effects like fetal bleeding and proteinuria in the large number of patients. To avoid such side-effects, new insight is required to find potential compounds as anti-VEGF from natural sources. In the present investigation, molecular docking studies were carried out to find the potentiality of Withaferin A, a key metabolite of Withania somnifera, as an inhibitor of VEGF. Molecular Docking studies were performed in DockingServer and SwissDock. Bevacizumab, a commercial anti-VEGF drug, was used as reference to compare the activity of Withaferin A. X-ray crystallographic structure of VEGF, was retrieved from Protein Data Bank (PDB), and used as drug target protein. Structure of Withaferin A and Bevacizumab was obtained from PubChem and ZINC databases. Molecular visualization was performed using UCSF Chimera. Withaferin A showed favorable binding with VEGF with low binding energy in comparison to Bevacizumab. Molecular Docking studies also revealed potential protein-ligand interactions for both Withaferin A and Bevacizumab. Conclusively our results strongly suggest that Withaferin A is a potent anti-VEGF agent as ascertained by its potential

  16. High early uterine vascular resistance values increase the risk of adverse pregnancy outcome independently from placental VEGF and VEGFR1 reactivities.

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    Valent, Sándor; Németh, Júlia; Sára, Levente; Gidai, János; Tóth, Péter; Schaff, Zsuzsanna; Paulin, Ferenc; Pajor, Attila

    2011-06-01

    From data in the literature, we hypothesized that high vascular resistance values in the uterine arteries at the end of the first trimester would increase adverse pregnancy outcomes and therefore might be accompanied by changes in VEGF/VEGFR1 immunoreactivities. In our university hospital 82 women (Study I n=62 and Study II n=20) were divided into two groups according to their uterine vascular resistance values. Uterine vascular resistance values were measured in the 10-13th weeks of gestation by color-Doppler ultrasonography. Women were divided into low and high vascular resistance groups. In the prospective follow-up study (Study I) the data of the pregnancy outcome were recorded. In cross-sectional study (Study II), VEGF and VEGFR1 immunoreactivities were measured on the tissue samples from women who underwent termination of pregnancy. In the high vascular resistance group (PI>2.3), the probability of adverse pregnancy outcome was significantly higher (40.0% vs. 12.8%). No differences in VEGF and VEGFR1 immunoreactivities were observed between groups. In both groups, intense VEGF immunoreactivity was observed in the maternal glandular epithelium and in the decidual cells. Weak reactivity was observed in the villous trophoblast. VEGFR1 immunoreactivity was intense in all regions. Our data suggest that high vascular resistance values in the first trimester are independent from VEGF/VEGFR1 immunoreactivities and markedly increase the probability of adverse pregnancy outcomes. This may be used for early screening of pregnant women in the first trimester. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  17. [VEGF as an angiogenic, neurotrophic, and neuroprotective factor].

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    Namiecińska, Magdalena; Marciniak, Katarzyna; Nowak, Jerzy Z

    2005-01-01

    Vascular endothelial growth factor (VEGF, occurring in several isoforms: VEGF-A, -B, -C, -D) is a well-known endothelial cell mitogen and vascular growth and permeability factor. Recent work done over the last few years has elucidated the important role of VEGF, which participates in the regulation of normal (physiological or therapeutic) and pathological angiogenesis (VEGF-A, VEGF-B) and lymphangiogenesis (VEGF-C, VEGF-D). VEGF has also been implicated in practically every stage of angiogenesis, yet its role in the initiation of new blood vessel creation appears to be the most important. In addition to its role as a key angiogenic factor, VEGF also possesses neurotrophic and neuroprotective activity both in the peripheral and in the central nervous system, exerting a direct action on neurons, Schwann cells, astrocytes, neural stem cells, and microglia. VEGF interacts with three subtypes of VEGF receptors occurring on the cellular membrane known as VEGFR-1 (Flt-1), VEGFR-2 (Flk-1/KDR), and VEGFR-3 (Flt-4). All these receptor types possess an internal tyrosin kinase domain. Interaction of VEGF with particular subtypes of receptors activates a circuit of signaling pathways, e.g. PI3K/Akt, Ras/Raf-MEK/Erk, eNOS/NO, and IP3/Ca2+. These participate in the generation of specific biological responses connected with proliferation, migration, increasing vascular permeability, or promoting endothelial cell survival. Recent findings from experiments performed on animals with experimentally evoked focal cerebral ischemia suggest that the neuroprotective activity of VEGF runs in parallel with its ability to promote neurogenesis and angiogenesis and that these effects may operate independently through multiple mechanisms. The above-mentioned three major features characterizing the neurobiological activity of VEGF, i.e. neuroprotection, neurogenesis, and angiogenesis, together with their possible functional link(s), provide the rationale for considering VEGF-based therapy as a

  18. Clathrin-independent endocytosis: mechanisms and function

    DEFF Research Database (Denmark)

    Sandvig, Kirsten; Pust, Sascha; Skotland, Tore

    2011-01-01

    It is now about 20 years since we first wrote reviews about clathrin-independent endocytosis. The challenge at the time was to convince the reader about its existence. Then the suggestion came up that caveolae might be responsible for the uptake. However, clearly this could not be the case since ...... having several functions of their own. This article aims at providing a brief update on the importance of clathrin-independent endocytic mechanisms, how the processes are regulated differentially, for instance on the poles of polarized cells, and the challenges in studying them....

  19. Voriconazole Enhances the Osteogenic Activity of Human Osteoblasts In Vitro through a Fluoride-Independent Mechanism

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    Allen, Kahtonna C.; Sanchez, Carlos J.; Niece, Krista L.; Wenke, Joseph C.

    2015-01-01

    Periostitis, which is characterized by bony pain and diffuse periosteal ossification, has been increasingly reported with prolonged clinical use of voriconazole. While resolution of clinical symptoms following discontinuation of therapy suggests a causative role for voriconazole, the biological mechanisms contributing to voriconazole-induced periostitis are unknown. To elucidate potential mechanisms, we exposed human osteoblasts in vitro to voriconazole or fluconazole at 15 or 200 μg/ml (reflecting systemic or local administration, respectively), under nonosteogenic or osteogenic conditions, for 1, 3, or 7 days and evaluated the effects on cell proliferation (reflected by total cellular DNA) and osteogenic differentiation (reflected by alkaline phosphatase activity, calcium accumulation, and expression of genes involved in osteogenic differentiation). Release of free fluoride, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) was also measured in cell supernatants of osteoblasts exposed to triazoles, with an ion-selective electrode (for free fluoride) and enzyme-linked immunosorbent assays (ELISAs) (for VEGF and PDGF). Voriconazole but not fluconazole significantly enhanced the proliferation and differentiation of osteoblasts. In contrast to clinical observations, no increases in free fluoride levels were detected following exposure to either voriconazole or fluconazole; however, significant increases in the expression of VEGF and PDGF by osteoblasts were observed following exposure to voriconazole. Our results demonstrate that voriconazole can induce osteoblast proliferation and enhance osteogenic activity in vitro. Importantly, and in contrast to the previously proposed mechanism of fluoride-stimulated osteogenesis, our findings suggest that voriconazole-induced periostitis may also occur through fluoride-independent mechanisms that enhance the expression of cytokines that can augment osteoblastic activity. PMID:26324277

  20. Voriconazole Enhances the Osteogenic Activity of Human Osteoblasts In Vitro through a Fluoride-Independent Mechanism.

    Science.gov (United States)

    Allen, Kahtonna C; Sanchez, Carlos J; Niece, Krista L; Wenke, Joseph C; Akers, Kevin S

    2015-12-01

    Periostitis, which is characterized by bony pain and diffuse periosteal ossification, has been increasingly reported with prolonged clinical use of voriconazole. While resolution of clinical symptoms following discontinuation of therapy suggests a causative role for voriconazole, the biological mechanisms contributing to voriconazole-induced periostitis are unknown. To elucidate potential mechanisms, we exposed human osteoblasts in vitro to voriconazole or fluconazole at 15 or 200 μg/ml (reflecting systemic or local administration, respectively), under nonosteogenic or osteogenic conditions, for 1, 3, or 7 days and evaluated the effects on cell proliferation (reflected by total cellular DNA) and osteogenic differentiation (reflected by alkaline phosphatase activity, calcium accumulation, and expression of genes involved in osteogenic differentiation). Release of free fluoride, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) was also measured in cell supernatants of osteoblasts exposed to triazoles, with an ion-selective electrode (for free fluoride) and enzyme-linked immunosorbent assays (ELISAs) (for VEGF and PDGF). Voriconazole but not fluconazole significantly enhanced the proliferation and differentiation of osteoblasts. In contrast to clinical observations, no increases in free fluoride levels were detected following exposure to either voriconazole or fluconazole; however, significant increases in the expression of VEGF and PDGF by osteoblasts were observed following exposure to voriconazole. Our results demonstrate that voriconazole can induce osteoblast proliferation and enhance osteogenic activity in vitro. Importantly, and in contrast to the previously proposed mechanism of fluoride-stimulated osteogenesis, our findings suggest that voriconazole-induced periostitis may also occur through fluoride-independent mechanisms that enhance the expression of cytokines that can augment osteoblastic activity. Copyright © 2015

  1. VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

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    Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico); Gonzalez Espinosa, Claudia, E-mail: cgonzal@cinvestav.mx [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico)

    2010-10-15

    Research highlights: {yields} Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. {yields} CoCl{sub 2}-induced VEGF secretion in mast cells occurs by a Ca{sup 2+}-insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. {yields} Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits Fc{epsilon}RI-dependent anaphylactic degranulation in mast cells. {yields} Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl{sub 2}) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl{sub 2} promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl{sub 2}-induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl{sub 2}-induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl{sub 2} in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals

  2. The growth and aggressive behavior of human osteosarcoma is regulated by a CaMKII-controlled autocrine VEGF signaling mechanism.

    Directory of Open Access Journals (Sweden)

    Paul G Daft

    Full Text Available Osteosarcoma (OS is a hyperproliferative malignant tumor that requires a high vascular density to maintain its large volume. Vascular Endothelial Growth Factor (VEGF plays a crucial role in angiogenesis and acts as a paracrine and autocrine agent affecting both endothelial and tumor cells. The alpha-Ca2+/Calmodulin kinase two (α-CaMKII protein is an important regulator of OS growth. Here, we investigate the role of α-CaMKII-induced VEGF in the growth and tumorigenicity of OS. We show that the pharmacologic and genetic inhibition of α-CaMKII results in decreases in VEGF gene expression (50% and protein secretion (55%, while α- CaMKII overexpression increases VEGF gene expression (250% and protein secretion (1,200%. We show that aggressive OS cells (143B express high levels of VEGF receptor 2 (VEGFR-2 and respond to exogenous VEGF (100nm by increasing intracellular calcium (30%. This response is ameliorated by the VEGFR inhibitor CBO-P11, suggesting that secreted VEGF results in autocrine stimulated α-CaMKII activation. Furthermore, we show that VEGF and α-CaMKII inhibition decreases the transactivation of the HIF-1α and AP-1 reporter constructs. Additionally, chromatin immunoprecipitation assay shows significantly decreased binding of HIF-1α and AP-1 to their responsive elements in the VEGF promoter. These data suggest that α-CaMKII regulates VEGF transcription by controlling HIF-1α and AP-1 transcriptional activities. Finally, CBO-P11, KN-93 (CaMKII inhibitor and combination therapy significantly reduced tumor burden in vivo. Our results suggest that VEGF-induced OS tumor growth is controlled by CaMKII and dual therapy by CaMKII and VEGF inhibitors could be a promising therapy against this devastating adolescent disease.

  3. The growth and aggressive behavior of human osteosarcoma is regulated by a CaMKII-controlled autocrine VEGF signaling mechanism.

    Science.gov (United States)

    Daft, Paul G; Yang, Yang; Napierala, Dobrawa; Zayzafoon, Majd

    2015-01-01

    Osteosarcoma (OS) is a hyperproliferative malignant tumor that requires a high vascular density to maintain its large volume. Vascular Endothelial Growth Factor (VEGF) plays a crucial role in angiogenesis and acts as a paracrine and autocrine agent affecting both endothelial and tumor cells. The alpha-Ca2+/Calmodulin kinase two (α-CaMKII) protein is an important regulator of OS growth. Here, we investigate the role of α-CaMKII-induced VEGF in the growth and tumorigenicity of OS. We show that the pharmacologic and genetic inhibition of α-CaMKII results in decreases in VEGF gene expression (50%) and protein secretion (55%), while α- CaMKII overexpression increases VEGF gene expression (250%) and protein secretion (1,200%). We show that aggressive OS cells (143B) express high levels of VEGF receptor 2 (VEGFR-2) and respond to exogenous VEGF (100nm) by increasing intracellular calcium (30%). This response is ameliorated by the VEGFR inhibitor CBO-P11, suggesting that secreted VEGF results in autocrine stimulated α-CaMKII activation. Furthermore, we show that VEGF and α-CaMKII inhibition decreases the transactivation of the HIF-1α and AP-1 reporter constructs. Additionally, chromatin immunoprecipitation assay shows significantly decreased binding of HIF-1α and AP-1 to their responsive elements in the VEGF promoter. These data suggest that α-CaMKII regulates VEGF transcription by controlling HIF-1α and AP-1 transcriptional activities. Finally, CBO-P11, KN-93 (CaMKII inhibitor) and combination therapy significantly reduced tumor burden in vivo. Our results suggest that VEGF-induced OS tumor growth is controlled by CaMKII and dual therapy by CaMKII and VEGF inhibitors could be a promising therapy against this devastating adolescent disease.

  4. Myeloid cells expressing VEGF and arginase-1 following uptake of damaged retinal pigment epithelium suggests potential mechanism that drives the onset of choroidal angiogenesis in mice.

    Directory of Open Access Journals (Sweden)

    Jian Liu

    Full Text Available Whilst data recognise both myeloid cell accumulation during choroidal neovascularisation (CNV as well as complement activation, none of the data has presented a clear explanation for the angiogenic drive that promotes pathological angiogenesis. One possibility that is a pre-eminent drive is a specific and early conditioning and activation of the myeloid cell infiltrate. Using a laser-induced CNV murine model, we have identified that disruption of retinal pigment epithelium (RPE and Bruch's membrane resulted in an early recruitment of macrophages derived from monocytes and microglia, prior to angiogenesis and contemporaneous with lesional complement activation. Early recruited CD11b(+ cells expressed a definitive gene signature of selective inflammatory mediators particularly a pronounced Arg-1 expression. Accumulating macrophages from retina and peripheral blood were activated at the site of injury, displaying enhanced VEGF expression, and notably prior to exaggerated VEGF expression from RPE, or earliest stages of angiogenesis. All of these initial events, including distinct VEGF (+ Arg-1(+ myeloid cells, subsided when CNV was established and at the time RPE-VEGF expression was maximal. Depletion of inflammatory CCR2-positive monocytes confirmed origin of infiltrating monocyte Arg-1 expression, as following depletion Arg-1 signal was lost and CNV suppressed. Furthermore, our in vitro data supported a myeloid cell uptake of damaged RPE or its derivatives as a mechanism generating VEGF (+ Arg-1(+ phenotype in vivo. Our results reveal a potential early driver initiating angiogenesis via myeloid-derived VEGF drive following uptake of damaged RPE and deliver an explanation of why CNV develops during any of the stages of macular degeneration and can be explored further for therapeutic gain.

  5. Autocrine VEGF signaling promotes cell proliferation through a PLC-dependent pathway and modulates Apatinib treatment efficacy in gastric cancer

    OpenAIRE

    Lin, Yi; Zhai, Ertao; Liao, Bing; Xu, Lixia; Zhang, Xinhua; PENG, Sui; He, Yulong; Cai, Shirong; Zeng, Zhirong; Chen, Minhu

    2017-01-01

    Background Tumor cells produce vascular endothelial growth factor (VEGF) which interact with the membrane or cytoplasmic VEGF receptors (VEGFRs) to promote cell growth in an angiogenesis-independent fashion. Apatinib, a highly selective VEGFR2 inhibitor, is the only effective drug for patients with terminal gastric cancer (GC) who have no other chemotherapeutic options. However, its treatment efficacy is still controversy and the mechanism behind remains undetermined. In this study, we aimed ...

  6. Tissue inhibitor of metalloproteinase-3 (TIMP3) promotes endothelial apoptosis via a caspase-independent mechanism.

    Science.gov (United States)

    Qi, Jian Hua; Anand-Apte, Bela

    2015-04-01

    Tissue inhibitor of metalloproteinases-3 (TIMP3) is a tumor suppressor and a potent inhibitor of angiogenesis. TIMP3 exerts its anti-angiogenic effect via a direct interaction with vascular endothelial growth factor (VEGF) receptor-2 (KDR) and inhibition of proliferation, migration and tube formation of endothelial cells (ECs). TIMP3 has also been shown to induce apoptosis in some cancer cells and vascular smooth muscle cells via MMP inhibition and caspase-dependent mechanisms. In this study, we examined the molecular mechanisms of TIMP3-mediated apoptosis in endothelial cells. We have previously demonstrated that mice developed smaller tumors with decreased vascularity when injected with breast carcinoma cells overexpressing TIMP3, than with control breast carcinoma cells. TIMP3 overexpression resulted in increased apoptosis in human breast carcinoma (MDA-MB435) in vivo but not in vitro. However, TIMP3 could induce apoptosis in ECs in vitro. The apoptotic activity of TIMP3 in ECs appears to be independent of MMP inhibitory activity. Furthermore, the equivalent expression of functional TIMP3 promoted apoptosis and caspase activation in ECs expressing KDR (PAE/KDR), but not in ECs expressing PDGF beta-receptor (PAE/β-R). Surprisingly, the apoptotic activity of TIMP3 appears to be independent of caspases. TIMP3 inhibited matrix-induced focal adhesion kinase (FAK) tyrosine phosphorylation and association with paxillin and disrupted the incorporation of β3 integrin, FAK and paxillin into focal adhesion contacts on the matrix, which were not affected by caspase inhibitors. Thus, TIMP3 may induce apoptosis in ECs by triggering a caspase-independent cell death pathway and targeting a FAK-dependent survival pathway.

  7. Candesartan, an angiotensin II type 1 receptor antagonist, inhibits pathological retinal neovascularization by downregulating VEGF receptor-2 expression.

    Science.gov (United States)

    Nakamura, Shinsuke; Tsuruma, Kazuhiro; Shimazawa, Masamitsu; Hara, Hideaki

    2012-06-15

    Several studies have examined the anti-angiogenic effects of angiotensin II type 1 (AT(1)) receptor antagonists; however, the mechanisms underlying these effects are currently unclear. In the present study, we examined the efficacy and the mechanism of candesartan, an AT(1) receptor antagonist, in suppressing pathological retinal neovascularization. We used an in vivo murine oxygen-induced retinopathy (OIR) model and also studied the in vitro proliferation and migration of human retinal microvascular endothelial cells (HRMECs) induced by vascular endothelial growth factor (VEGF)-A. The regulation of angiogenesis-associated genes such as hypoxia-inducible factor (HIF-1α), VEGF-A, VEGF receptor-1, and VEGF receptor-2 was evaluated with real-time RT-PCR in the OIR model. In the OIR model, candesartan suppressed the pathological neovascularization in a dose-dependent manner, but did not prevent the physiological angiogenesis. However, candesartan did not inhibit VEGF-A-induced proliferation or migration in HRMECs in the in vitro study. When administered interperitoneally in the OIR model, candesartan reduced the upregulation of VEGF receptor-2 in the retina, but had no effects in the other angiogenesis-related genes, such as HIF-1α, VEGF-A, and VEGF receptor-1. These findings indicate that candesartan inhibited the retinal pathological neovascularization, at least in part, by suppressing the expression of VEGF receptor-2, independent of VEGF signaling cascade. Therefore, candesartan may be a useful therapeutic target for the inhibition of retinal neovascularization that has a low risk of serious side effects. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Suppression of alpha-tocopherol ether-linked acetic acid in VEGF-induced angiogenesis and the possible mechanisms in human umbilical vein endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Chuang, Cheng-Hung, E-mail: chchuang@hk.edu.tw [Department of Nutrition, Master Program of Biomedical Nutrition, Hungkuang University, 1018 Sec. 6 Taiwan Boulevard, Taichung 43302, Taiwan, ROC (China); Liu, Chia-Hua [Department of Food Science and Biotechnology, National Chung-Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan, ROC (China); Lu, Ta-Jung [Department of Chemistry, Institute of Technology and Innovation Management, National Chung-Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan, ROC (China); Hu, Miao-Lin, E-mail: mlhuhu@dragon.nchu.edu.tw [Department of Food Science and Biotechnology, National Chung-Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan, ROC (China)

    2014-12-15

    Alpha-tocopherol ether-linked acetic acid (α-TEA) has been reported to exhibit both anti-tumor and anti-metastatic activities in cell culture and animal studies. However, it is unclear whether α-TEA possesses anti-angiogenic effects. In this study, we investigated the effect of α-TEA on vascular endothelial growth factor (VEGF)-induced angiogenesis and matrix metalloproteinase (MMP) expression both in vitro and ex vivo. We found that the α-TEA inhibited tube formation, invasion, and migration in human umbilical vein endothelial cells (HUVECs) and that such actions were accompanied by reduced expression of MMP-2. α-TEA also inhibited ex vivo angiogenesis, as indicated by chicken egg chorioallantoic membrane assay. We further showed that α-TEA attenuated protein expression of VEGF receptor-2 (VEGFR-2)-mediated p38 mitogen-activated protein kinase (p38), phosphorylated p38, and focal adhesion kinase (FAK). Moreover, α-TEA (30 μM) significantly up-regulated protein expression of tissue inhibitors of MMP (TIMP)-2 (by 138%) and the metastasis suppressor gene nm23-H1 (by 54%). These results demonstrate that the anti-angiogenic effect of α-TEA both in vitro and ex vivo and its possible mechanistic action appears to involve the inhibition of MMP-2 level through VEGFR-2-mediated FAK and p38 signaling pathways and through up-regulation of TIMP-2 and nm23-H1 expression. - Graphical abstract: Possible mechanisms of α-TEA on inhibited angiogenesis of human umbilical vein endothelial cells. Brief summary In the present study, we have demonstrated that VEGF-mediated angiogenesis is significantly inhibited by α-TEA, and that this effect involves inhibition of MMP-2 level through VEGFR-2-mediated FAK and p38 signaling pathways related to invasion and migration. - Highlights: • The anti-angiogenic effect and the mechanistic action of α-TEA were investigated. • α-TEA significantly inhibited VEGF-mediated angiogenesis both in vitro and ex vivo. • α-TEA down

  9. A possible mechanism for exonuclease 1-independent eukaryotic mismatch repair

    Science.gov (United States)

    Kadyrov, Farid A.; Genschel, Jochen; Fang, Yanan; Penland, Elisabeth; Edelmann, Winfried; Modrich, Paul

    2009-01-01

    Mismatch repair contributes to genetic stability, and inactivation of the mammalian pathway leads to tumor development. Mismatch correction occurs by an excision-repair mechanism and has been shown to depend on the 5′ to 3′ hydrolytic activity exonuclease 1 (Exo1) in eukaryotic cells. However, genetic and biochemical studies have indicated that one or more Exo1-independent modes of mismatch repair also exist. We have analyzed repair of nicked circular heteroduplex DNA in extracts of Exo1-deficient mouse embryo fibroblast cells. Exo1-independent repair under these conditions is MutLα-dependent and requires functional integrity of the MutLα endonuclease metal-binding motif. In contrast to the Exo1-dependent reaction, we have been unable to detect a gapped excision intermediate in Exo1-deficient extracts when repair DNA synthesis is blocked. A possible explanation for this finding has been provided by analysis of a purified system comprised of MutSα, MutLα, replication factor C, proliferating cell nuclear antigen, replication protein A, and DNA polymerase δ that supports Exo1-independent repair in vitro. Repair in this system depends on MutLα incision of the nicked heteroduplex strand and dNTP-dependent synthesis-driven displacement of a DNA segment spanning the mismatch. Such a mechanism may account, at least in part, for the Exo1-independent repair that occurs in eukaryotic cells, and hence the modest cancer predisposition of Exo1-deficient mammalian cells. PMID:19420220

  10. Serum vascular endothelial growth factor (VEGF) is elevated in GH deficient adults.

    Science.gov (United States)

    Murray, Robert D; Randeva, Harpal S; Lewandowski, Krzysztof C; Komorowski, Jan; Lawrance, Jeremy A; Adams, Judith E; Shalet, Stephen M

    2011-04-01

    GHD adults exhibit a number of adverse surrogate markers of vascular risk culminating in excess vascular morbidity and mortality. Vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of a number of vascular morbidities. Furthermore, serum levels decrease following GH replacement in GHD adults, though it remains unclear if levels are significantly elevated in untreated individuals. A cross-sectional case-control study. We measured fasting serum VEGF, MMP2, and MMP9 in 27 patients with GHD, 24 with partial GHD (GHI), and 25 sex- and age-matched controls. GHD (483±334 vs 326±180ng/l, P=0.04), but not GHI (354±192 vs 326±180ng/l, P=n/s) adults had significantly elevated VEGF levels compared with controls. Neither MMP2, nor MMP9 levels were elevated in the patient groups. Serum VEGF levels correlated positively with LDL-cholesterol (R=0.34, P=0.004) and serum MMP9 values (R=0.36, P=0.002), and negatively with IGF-I values, however, no correlation was observed with MMP2. Multiple regression analysis with VEGF levels as the dependent variable, and age, gender, % fat mass, LDL-C, insulin and IGF-I as independent variables revealed VEGF levels to be dependent on LDL-C alone (P=0.003, R=0.36). GHD adults have elevated VEGF levels, which correlate with MMP9 levels. Both VEGF and MMP9 are associated with vascular pathologies and may provide insight in to the pathophysiological mechanisms underlying the increased vascular morbidity and mortality observed in GHD adults. Copyright © 2011 Growth Hormone Research Society. Published by Elsevier Ltd. All rights reserved.

  11. Red wine polyphenol compounds favor neovascularisation through estrogen receptor α-independent mechanism in mice.

    Directory of Open Access Journals (Sweden)

    Matthieu Chalopin

    Full Text Available Red wine polyphenol compounds (RWPC exert paradoxical effects depending on the dose on post-ischemic neovascularisation. Low dose RWPC (0.2 mg/kg/day is pro-angiogenic, whereas high dose (20 mg/kg/day is anti-angiogenic. We recently reported that the endothelial effect of RWPC is mediated through the activation of a redox-sensitive pathway, mitochondrial biogenesis and the activation of α isoform of the estrogen receptor (ERα. Here, we investigated the implication of ERα on angiogenic properties of RWPC. Using ovariectomized mice lacking ERα treated with high dose of RWPC after hindlimb ischemia, we examined blood flow reperfusion, vascular density, nitric oxide (NO production, expression and activation of proteins involved in angiogenic process and muscle energy sensing network. As expected, high dose of RWPC treatment reduced both blood flow and vascular density in muscles of mice expressing ERα. These effects were associated with reduced NO production resulting from diminished activity of eNOS. In the absence of RWPC, ERα deficient mice showed a reduced neo-vascularisation associated with a decreased NO production. Surprisingly in mice lacking ERα, high dose of RWPC increased blood flow and capillary density in conjunction with increased NO pathway and production as well as VEGF expression. Of particular interest is the activation of Sirt-1, AMPKα and PGC-1α/β axis in ischemic hindlimb from both strains. Altogether, the results highlight a pro-angiogenic property of RWPC via an ERα-independent mechanism that is associated with an up-regulation of energy sensing network. This study brings a corner stone of a novel pathway for RWPC to correct cardiovascular diseases associated with failed neovascularisation.

  12. Current biology of VEGF-B and VEGF-C.

    Science.gov (United States)

    Olofsson, B; Jeltsch, M; Eriksson, U; Alitalo, K

    1999-12-01

    Endothelial growth factors and their receptors may provide important therapeutic tools for the treatment of pathological conditions characterised by defective or aberrant angiogenesis. Vascular endothelial growth factor (VEGF) is pivotal for vasculogenesis and for angiogenesis in normal and pathological conditions. VEGF-B and VEGF-C provide this gene family with additional functions, for example, VEGF-C also regulates lymphangiogenesis.

  13. Sulforaphane inhibits multiple inflammasomes through an Nrf2-independent mechanism.

    Science.gov (United States)

    Greaney, Allison J; Maier, Nolan K; Leppla, Stephen H; Moayeri, Mahtab

    2016-01-01

    The inflammasomes are intracellular complexes that have an important role in cytosolic innate immune sensing and pathogen defense. Inflammasome sensors detect a diversity of intracellular microbial ligands and endogenous danger signals and activate caspase-1, thus initiating maturation and release of the proinflammatory cytokines interleukin-1β and interleukin-18. These events, although crucial to the innate immune response, have also been linked to the pathology of several inflammatory and autoimmune disorders. The natural isothiocyanate sulforaphane, present in broccoli sprouts and available as a dietary supplement, has gained attention for its antioxidant, anti-inflammatory, and chemopreventive properties. We discovered that sulforaphane inhibits caspase-1 autoproteolytic activation and interleukin-1β maturation and secretion downstream of the nucleotide-binding oligomerization domain-like receptor leucine-rich repeat proteins NLRP1 and NLRP3, NLR family apoptosis inhibitory protein 5/NLR family caspase-1 recruitment domain-containing protein 4 (NAIP5/NLRC4), and absent in melanoma 2 (AIM2) inflammasome receptors. Sulforaphane does not inhibit the inflammasome by direct modification of active caspase-1 and its mechanism is not dependent on protein degradation by the proteasome or de novo protein synthesis. Furthermore, sulforaphane-mediated inhibition of the inflammasomes is independent of the transcription factor nuclear factor erythroid-derived 2-like factor 2 (Nrf2) and the antioxidant response-element pathway, to which many of the antioxidant and anti-inflammatory effects of sulforaphane have been attributed. Sulforaphane was also found to inhibit cell recruitment to the peritoneum and interleukin-1β secretion in an in vivo peritonitis model of acute gout and to reverse NLRP1-mediated murine resistance to Bacillus anthracis spore infection. These findings demonstrate that sulforaphane inhibits the inflammasomes through a novel mechanism and contributes to

  14. Targeting VEGF-A with a vaccine decreases inflammation and joint destruction in experimental arthritis.

    Science.gov (United States)

    Semerano, Luca; Duvallet, Emilie; Belmellat, Nadia; Marival, Nicolas; Schall, Nicolas; Monteil, Maëlle; Grouard-Vogel, Géraldine; Bernier, Emilie; Lecouvey, Marc; Hlawaty, Hanna; Muller, Sylviane; Boissier, Marie-Christophe; Assier, Eric

    2016-01-01

    Inflammation and angiogenesis are two tightly linked processes in arthritis, and therapeutic targeting of pro-angiogenic factors may contribute to control joint inflammation and synovitis progression. In this work, we explored whether vaccination against vascular endothelial growth factor (VEGF) ameliorates collagen-induced arthritis (CIA). Anti-VEGF vaccines were heterocomplexes consisting of the entire VEGF cytokine (or a VEGF-derived peptide) linked to the carrier protein keyhole limpet hemocyanin (KLH). Two kinds of vaccines were separately tested in two independent experiments of CIA. In the first, we tested a kinoid of the murine cytokine VEGF (VEGF-K), obtained by conjugating VEGF-A to KLH. For the second, we selected two VEGF-A-derived peptide sequences to produce heterocomplexes (Vpep1-K and Vpep2-K). DBA/1 mice were immunized with either VEGF-K, Vpep1-K, or Vpep2-K, before CIA induction. Clinical and histological scores of arthritis, anti-VEGF, anti-Vpep Ab titers, and anti-VEGF Abs neutralizing capacity were determined. Both VEGF-K and Vpep1-K significantly ameliorated clinical arthritis scores and reduced synovial inflammation and joint destruction at histology. VEGF-K significantly reduced synovial vascularization. None of the vaccines reduced anti-collagen Ab response in mice. Both VEGF-K and Vpep1-K induced persistently high titers of anti-VEGF Abs capable of inhibiting VEGF-A bioactivity. Vaccination against the pro-angiogenic factor VEGF-A leads to the production of anti-VEGF polyclonal Abs and has a significant anti-inflammatory effect in CIA. Restraining Ab response to a single peptide sequence (Vpep1) with a peptide vaccine effectively protects immunized mice from joint inflammation and destruction.

  15. Pancreatic duct cells as a source of VEGF in mice.

    Science.gov (United States)

    Xiao, Xiangwei; Prasadan, Krishna; Guo, Ping; El-Gohary, Yousef; Fischbach, Shane; Wiersch, John; Gaffar, Iljana; Shiota, Chiyo; Gittes, George K

    2014-05-01

    Vascular endothelial growth factor (VEGF) is essential for proper pancreatic development, islet vascularisation and insulin secretion. In the adult pancreas, VEGF is thought to be predominantly secreted by beta cells. Although human duct cells have previously been shown to secrete VEGF at angiogenic levels in culture, an analysis of the kinetics of VEGF synthesis and secretion, as well as elucidation of an in vivo role for this ductal VEGF in affecting islet function and physiology, has been lacking. We analysed purified duct cells independently prepared by flow cytometry, surgical isolation or laser-capture microdissection. We infected duct cells in vivo with Vegf (also known as Vegfa) short hairpin RNA (shRNA) in an intrapancreatic ductal infusion system and examined the effect of VEGF knockdown in duct cells in vitro and in vivo. Pancreatic duct cells express high levels of Vegf mRNA. Compared with beta cells, duct cells had a much higher ratio of secreted to intracellular VEGF. As a bioassay, formation of tubular structures by human umbilical vein endothelial cells was essentially undetectable when cultured alone and was substantially increased when co-cultured with pancreatic duct cells but significantly reduced when co-cultured with duct cells pretreated with Vegf shRNA. Compared with islets transplanted alone, improved vascularisation and function was detected in the islets co-transplanted with duct cells but not in islets co-transplanted with duct cells pretreated with Vegf shRNA. Human islet preparations for transplantation typically contain some contaminating duct cells and our findings suggest that the presence of duct cells in the islet preparation may improve transplantation outcomes.

  16. On strain-rate independent damping in continuum mechanics

    Science.gov (United States)

    Mulder, Gerben

    2017-10-01

    Strain-rate independent damping is a theory of energy dissipation in solids. It is based on the assumption that an increase or decrease in the strain-energy density correlates with a multiplication of 1+η or 1-η respectively, of the material stiffness matrix, with 0≤ η derived for strain-rate independent damping can be solved for 1, 2 or 3 dimensions via direct integration, provided that the software supports PDE coefficients that are functions of the solution and its space and time derivatives. A 3D problem with 22,000 DOF's and 10,000 time steps was solved successfully and convincingly.

  17. Targeting Activin Receptor-Like Kinase 1 (ALK1) Inhibits Angiogenesis and Tumorigenesis Through a Mechanism of Action Complementary to Anti-VEGF Therapies

    Science.gov (United States)

    Hu-Lowe, Dana D.; Chen, Enhong; Zhang, Lianglin; Watson, Katherine D.; Mancuso, Patrizia; Lappin, Patrick; Wickman, Grant; Chen, Jeffrey H.; Wang, Jianying; Jiang, Xin; Amundson, Karin; Simon, Ronald; Erbersdobler, Andreas; Bergqvist, Simon; Feng, Zheng; Swanson, Terri A.; Simmons, Brett H.; Lippincott, John; Casperson, Gerald F.; Levin, Wendy J.; Stampino, Corrado Gallo; Shalinsky, David R.; Ferrara, Katherine W.; Fiedler, Walter; Bertolini, Francesco

    2011-01-01

    Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1) plays an important role in vascular development, remodeling, and pathologic angiogenesis. Here we investigated the role of ALK1 in angiogenesis in the context of common pro-angiogenic factors (PAFs; vascular endothelial growth factor [VEGF] A and basic fibroblast growth factor [bFGF]). We observed that PAFs stimulated ALK1-mediated signaling, including Smad1/5/8 phosphorylation, nuclear translocation and Id-1 expression, cell spreading, and tubulogenesis of endothelial cells (ECs). An antibody specifically targeting ALK1 (anti-ALK1) markedly inhibited these events. In mice, anti-ALK1 suppressed MatrigelTM angiogenesis stimulated by PAFs, and inhibited xenograft tumor growth by attenuating both blood and lymphatic vessel angiogenesis. In a human melanoma model with acquired resistance to a VEGF receptor kinase inhibitor, anti-ALK1 also delayed tumor growth and disturbed vascular normalization associated with VEGF receptor inhibition. In a human/mouse chimera tumor model, targeting human ALK1 decreased human vessel density, and improved antitumor efficacy when combined with bevacizumab (anti-VEGF). Anti-angiogenesis and antitumor efficacy were associated with disrupted colocalization of ECs with desmin+ perivascular cells, and reduction of blood flow primarily in large/mature vessels as assessed by contrast-enhanced ultrasonography. Thus, ALK1 may play a role in stabilizing angiogenic vessels and contribute to resistance to anti-VEGF therapies. Given our observation of its expression in the vasculature of many human tumor types and in circulating ECs from patients with advanced cancers, ALK1 blockade may represent an effective therapeutic opportunity complementary to the current anti-angiogenic modalities in the clinic. PMID:21212415

  18. Mammalian Target of Rapamycin Inhibitors Induce Tumor Cell Apoptosis In Vivo Primarily by Inhibiting VEGF Expression and Angiogenesis

    Directory of Open Access Journals (Sweden)

    Patrick Frost

    2013-01-01

    Full Text Available We found that rapalog mTOR inhibitors induce G1 arrest in the PTEN-null HS Sultan B-cell lymphoma line in vitro, but that administration of rapalogs in a HS Sultan xenograft model resulted in significant apoptosis, and that this correlated with induction of hypoxia and inhibition of neoangiogenesis and VEGF expression. Mechanistically, rapalogs prevent cap-dependent translation, but studies have shown that cap-independent, internal ribosome entry site (IRES-mediated translation of genes, such as c-myc and cyclin D, can provide a fail-safe mechanism that regulates tumor survival. Therefore, we tested if IRES-dependent expression of VEGF could likewise regulate sensitivity of tumor cells in vivo. To achieve this, we developed isogenic HS Sultan cell lines that ectopically express the VEGF ORF fused to the p27 IRES, an IRES sequence that is insensitive to AKT-mediated inhibition of IRES activity and effective in PTEN-null tumors. Mice challenged with p27-VEGF transfected tumor cells were more resistant to the antiangiogenic and apoptotic effects of the rapalog, temsirolimus, and active site mTOR inhibitor, pp242. Our results confirm the critical role of VEGF expression in tumors during treatment with mTOR inhibitors and underscore the importance of IRES activity as a resistance mechanism to such targeted therapy.

  19. [Role of VEGF in diseases of the retina].

    Science.gov (United States)

    Barquet, Luis Arias

    2015-03-01

    Angiogenesis is the process through which new blood vessels are formed, based on preexisting vessels, and is the paradigm of diseases such as cancer and exudative ageassociated macular degeneration (ARMD). Several proangiogenic factors have been identified, such as vascular endothelial growth factor (VEGF), especially VEGF-A, which activates endothelial cells and promotes cell proliferation, migration, and an increase in vascular permeability. VEGF is also involved in the etiopathogenesis of other retinal diseases, such as diabetic macular edema and macular edema secondary to retinal vein occlusion. Likewise, there is increasing evidence that placental growth factor (PIGF) acts recepsynergetically with VEGF in promoting these diseases. Currently, the main treatment for these diseases are the anti-VEGF drugs, aflibercept, ranibizumab and bevacizumab. These agents differ in their molecular structure and mechanism of action. Copyright © 2015 Sociedad Española de Oftalmología. Published by Elsevier España, S.L.U. All rights reserved.

  20. Density-dependence as a size-independent regulatory mechanism

    NARCIS (Netherlands)

    De Vladar, H.P.

    2006-01-01

    The growth function of populations is central in biomathematics. The main dogma is the existence of density-dependence mechanisms, which can be modelled with distinct functional forms that depend on the size of the Population. One important class of regulatory functions is the theta-logistic, which

  1. Pain without Nociceptors? Nav1.7-Independent Pain Mechanisms

    Science.gov (United States)

    Minett, Michael S.; Falk, Sarah; Santana-Varela, Sonia; Bogdanov, Yury D.; Nassar, Mohammed A.; Heegaard, Anne-Marie; Wood, John N.

    2014-01-01

    Summary Nav1.7, a peripheral neuron voltage-gated sodium channel, is essential for pain and olfaction in mice and humans. We examined the role of Nav1.7 as well as Nav1.3, Nav1.8, and Nav1.9 in different mouse models of chronic pain. Constriction-injury-dependent neuropathic pain is abolished when Nav1.7 is deleted in sensory neurons, unlike nerve-transection-related pain, which requires the deletion of Nav1.7 in sensory and sympathetic neurons for pain relief. Sympathetic sprouting that develops in parallel with nerve-transection pain depends on the presence of Nav1.7 in sympathetic neurons. Mechanical and cold allodynia required distinct sets of neurons and different repertoires of sodium channels depending on the nerve injury model. Surprisingly, pain induced by the chemotherapeutic agent oxaliplatin and cancer-induced bone pain do not require the presence of Nav1.7 sodium channels or Nav1.8-positive nociceptors. Thus, similar pain phenotypes arise through distinct cellular and molecular mechanisms. Therefore, rational analgesic drug therapy requires patient stratification in terms of mechanisms and not just phenotype. PMID:24440715

  2. Pain without Nociceptors? Nav1.7-Independent Pain Mechanisms

    Directory of Open Access Journals (Sweden)

    Michael S. Minett

    2014-01-01

    Full Text Available Nav1.7, a peripheral neuron voltage-gated sodium channel, is essential for pain and olfaction in mice and humans. We examined the role of Nav1.7 as well as Nav1.3, Nav1.8, and Nav1.9 in different mouse models of chronic pain. Constriction-injury-dependent neuropathic pain is abolished when Nav1.7 is deleted in sensory neurons, unlike nerve-transection-related pain, which requires the deletion of Nav1.7 in sensory and sympathetic neurons for pain relief. Sympathetic sprouting that develops in parallel with nerve-transection pain depends on the presence of Nav1.7 in sympathetic neurons. Mechanical and cold allodynia required distinct sets of neurons and different repertoires of sodium channels depending on the nerve injury model. Surprisingly, pain induced by the chemotherapeutic agent oxaliplatin and cancer-induced bone pain do not require the presence of Nav1.7 sodium channels or Nav1.8-positive nociceptors. Thus, similar pain phenotypes arise through distinct cellular and molecular mechanisms. Therefore, rational analgesic drug therapy requires patient stratification in terms of mechanisms and not just phenotype.

  3. Differential expression of VEGF ligands and receptors in prostate cancer.

    Science.gov (United States)

    Woollard, David J; Opeskin, Kenneth; Coso, Sanja; Wu, Di; Baldwin, Megan E; Williams, Elizabeth D

    2013-05-01

    Prostate cancer disseminates to regional lymph nodes, however the molecular mechanisms responsible for lymph node metastasis are poorly understood. The vascular endothelial growth factor (VEGF) ligand and receptor family have been implicated in the growth and spread of prostate cancer via activation of the blood vasculature and lymphatic systems. The purpose of this study was to comprehensively examine the expression pattern of VEGF ligands and receptors in the glandular epithelium, stroma, lymphatic vasculature and blood vessels in prostate cancer. The localization of VEGF-A, VEGF-C, VEGF-D, VEGF receptor (VEGFR)-1, VEGFR-2, and VEGFR-3 was examined in cancerous and adjacent benign prostate tissue from 52 subjects representing various grades of prostate cancer. Except for VEGFR-2, extensive staining was observed for all ligands and receptors in the prostate specimens. In epithelial cells, VEGF-A and VEGFR-1 expression was higher in tumor tissue compared to benign tissue. VEGF-D and VEGFR-3 expression was significantly higher in benign tissue compared to tumor in the stroma and the endothelium of lymphatic and blood vessels. In addition, the frequency of lymphatic vessels, but not blood vessels, was lower in tumor tissue compared with benign tissue. These results suggest that activation of VEGFR-1 by VEGF-A within the carcinoma, and activation of lymphatic endothelial cell VEGFR-3 by VEGF-D within the adjacent benign stroma may be important signaling mechanisms involved in the progression and subsequent metastatic spread of prostate cancer. Thus inhibition of these pathways may contribute to therapeutic strategies for the management of prostate cancer. Copyright © 2012 Wiley Periodicals, Inc.

  4. VEGF121b and VEGF165b are weakly angiogenic isoforms of VEGF-A

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    Pio Ruben

    2010-12-01

    Full Text Available Abstract Background Different isoforms of VEGF-A (mainly VEGF121, VEGF165 and VEGF189 have been shown to display particular angiogenic properties in the generation of a functional tumor vasculature. Recently, a novel class of VEGF-A isoforms, designated as VEGFxxxb, generated through alternative splicing, have been described. Previous studies have suggested that these isoforms may inhibit angiogenesis. In the present work we have produced recombinant VEGF121/165b proteins in the yeast Pichia pastoris and constructed vectors to overexpress these isoforms and assess their angiogenic potential. Results Recombinant VEGF121/165b proteins generated either in yeasts or mammalian cells activated VEGFR2 and its downstream effector ERK1/2, although to a lesser extent than VEGF165. Furthermore, treatment of endothelial cells with VEGF121/165b increased cell proliferation compared to untreated cells, although such stimulation was lower than that induced by VEGF165. Moreover, in vivo angiogenesis assays confirmed angiogenesis stimulation by VEGF121/165b isoforms. A549 and PC-3 cells overexpressing VEGF121b or VEGF165b (or carrying the PCDNA3.1 empty vector, as control and xenotransplanted into nude mice showed increased tumor volume and angiogenesis compared to controls. To assess whether the VEGFxxxb isoforms are differentially expressed in tumors compared to healthy tissues, immunohistochemical analysis was conducted on a breast cancer tissue microarray. A significant increase (p xxxb and total VEGF-A protein expression in infiltrating ductal carcinomas compared to normal breasts was observed. A positive significant correlation (r = 0.404, p = 0.033 between VEGFxxxb and total VEGF-A was found. Conclusions Our results demonstrate that VEGF121/165b are not anti-angiogenic, but weakly angiogenic isoforms of VEGF-A. In addition, VEGFxxxb isoforms are up-regulated in breast cancer in comparison with non malignant breast tissues. These results are to be taken

  5. The prognosis was poorer in colorectal cancers that expressed both VEGF and PROK1 (No correlation coefficient between VEGF and PROK1).

    Science.gov (United States)

    Goi, Takanori; Nakazawa, Toshiyuki; Hirono, Yasuo; Yamaguchi, Akio

    2015-10-06

    The angiogenic proteins vascular endothelial growth factor (VEGF) and prokineticin1 (PROK1) proteins are considered important in colorectal cancer, the relationship between their simultaneous expression and prognosis was investigated in the present study. VEGF and PROK1 expression in 620 primary human colorectal cancer lesions was confirmed via immunohistochemical staining with anti-VEGF and anti-PROK1 antibodies, and the correlation between the expression of these 2 proteins and recurrence/prognosis were investigated. VEGF protein was expressed in 329 (53.1%) and PROK1 protein was expressed in 223 (36.0%). PROK1 and VEGF were simultaneously expressed in 116 (18.7%) of the 620 cases. The correlation coefficient between VEGF expression and PROK1 expression was r = 0.11, and therefore correlation was not observed. Clinical pathology revealed that substantially lymphnode matastasis, hematogenous metastasis, or TMN advanced-stage IV was significantly more prevalent in cases that expressed both VEGF and PROK1 than in the cases negative for both proteins or those positive for only 1 of the proteins. Also the cases positive for both proteins exhibited the worst recurrence and prognosis. In the Cox proportional hazards model, VEGF and PROK1 expression was an independent prognostic factor. The prognosis was poorer in colorectal cancers that expressed both PROK1 and VEGF relative to the cases that expressed only 1 protein, and the expression of both proteins was found to be an independent prognostic factor.

  6. COX-2, VEGF and tumour angiogenesis.

    LENUS (Irish Health Repository)

    Toomey, D P

    2009-06-01

    Epidemiological evidence suggests a protective effective of regular NSAID use against developing cancer. Cyclooxygenase-2, a target of NSAIDs, is upregulated in many cancers and has been associated with increased VEGF production and angiogenesis. Angiogenesis is the formation of new vessels from existing vasculature and as an essential process for tumour development represents an important therapeutic target. Following an extensive review of the literature this article details the current knowledge on the role of COX-2 in tumorigenesis focusing on its relationship to angiogenesis and VEGF production by tumour cells. While COX-2 is clearly detrimental to prognosis and NSAIDs have a beneficial effect, the possibility of COX-2 independent effects being partly or wholly responsible for this benefit cannot be excluded.

  7. Mathematical Modeling of Cellular Cross-Talk Between Endothelial and Tumor Cells Highlights Counterintuitive Effects of VEGF-Targeted Therapies.

    Science.gov (United States)

    Jain, Harsh; Jackson, Trachette

    2017-04-24

    Tumor growth and progression are critically dependent on the establishment of a vascular support system. This is often accomplished via the expression of pro-angiogenic growth factors, including members of the vascular endothelial growth factor (VEGF) family of ligands. VEGF ligands are overexpressed in a wide variety of solid tumors and therefore have inspired optimism that inhibition of the different axes of the VEGF pathway-alone or in combination-would represent powerful anti-angiogenic therapies for most cancer types. When considering treatments that target VEGF and its receptors, it is difficult to tease out the differential anti-angiogenic and anti-tumor effects of all combinations experimentally because tumor cells and vascular endothelial cells are engaged in a dynamic cross-talk that impacts key aspects of tumorigenesis, independent of angiogenesis. Here we develop a mathematical model that connects intracellular signaling responsible for both endothelial and tumor cell proliferation and death to population-level cancer growth and angiogenesis. We use this model to investigate the effect of bidirectional communication between endothelial cells and tumor cells on treatments targeting VEGF and its receptors both in vitro and in vivo. Our results underscore the fact that in vitro therapeutic outcomes do not always translate to the in vivo situation. For example, our model predicts that certain therapeutic combinations result in antagonism in vivo that is not observed in vitro. Mathematical modeling in this direction can shed light on the mechanisms behind experimental observations that manipulating VEGF and its receptors is successful in some cases but disappointing in others.

  8. Brain alterations and clinical symptoms of dementia in diabetes: Abeta/tau-dependent and independent mechanisms

    Directory of Open Access Journals (Sweden)

    Naoyuki eSato

    2014-09-01

    Full Text Available Emerging evidence suggests that diabetes affects cognitive function and increases the incidence of dementia. However, the mechanisms by which diabetes modifies cognitive function still remains unclear. Morphologically, diabetes is associated with neuronal loss in the frontal and temporal lobes including the hippocampus, and aberrant functional connectivity of the posterior cingulate cortex and medial frontal/temporal gyrus. Clinically, diabetic patients show decreased executive function, information processing, planning, visuospatial construction, and visual memory. Therefore, in comparison with the characteristics of AD brain structure and cognition, diabetes seems to affect cognitive function through not only simple AD pathological feature-dependent mechanisms, but also independent mechanisms. As an Abeta/tau-independent mechanism, diabetes compromises cerebrovascular function, increases subcortical infarction and might alter the blood brain barrier (BBB. Diabetes also affects glucose metabolism, insulin signaling and mitochondrial function in the brain. Diabetes also modifies metabolism of Abeta and tau and causes Abeta/tau-dependent pathological changes. Moreover, there is evidence that suggests an interaction between Abeta/tau-dependent and independent mechanisms. Therefore, diabetes modifies cognitive function through Abeta/tau-dependent and independent mechanisms. Interaction between these two mechanisms forms a vicious cycle.

  9. Rational design of soft mechanical metamaterials: Independent tailoring of elastic properties with randomness

    Science.gov (United States)

    Mirzaali, M. J.; Hedayati, R.; Vena, P.; Vergani, L.; Strano, M.; Zadpoor, A. A.

    2017-07-01

    The elastic properties of mechanical metamaterials are direct functions of their topological designs. Rational design approaches based on computational models could, therefore, be used to devise topological designs that result in the desired properties. It is of particular importance to independently tailor the elastic modulus and Poisson's ratio of metamaterials. Here, we present patterned randomness as a strategy for independent tailoring of both properties. Soft mechanical metamaterials incorporating various types of patterned randomness were fabricated using an indirect additive manufacturing technique and mechanically tested. Computational models were also developed to predict the topology-property relationship in a wide range of proposed topologies. The results of this study show that patterned randomness allows for independent tailoring of the elastic properties and covering a broad area of the elastic modulus-Poisson's ratio plane. The uniform and homogenous topologies constitute the boundaries of the covered area, while topological designs with patterned randomness fill the enclosed area.

  10. Time influence of mechanical ventilation on functional independence in patients submitted to cardiac surgery: literature review

    Directory of Open Access Journals (Sweden)

    André Luiz Cordeiro

    Full Text Available Abstract Introduction: Patients submitted to cardiovascular surgery present motor and respiratory complications mainly due to high surgery manipulation and the use of mechanical ventilation. Reducing the weaning start time and disconnecting patient's ventilation system prematurely can decrease the pulmonary complications and hospitalization time. Motor complications are the most relevant as they have a direct effect on functional independence provoked by immobility time. Objectives : Identify if mechanical ventilation time has an impact on functional capacity on patients submitted to cardiac surgery in order to contribute to the establishment of reliable evidence to practice through this patient's profile. Materials and methods : Original articles were analyzed, published between 2000 and 2014, which focused on the influence of mechanical ventilation time concerning the functional independence on patients submitted to cardiac surgery, contained in the following electronic database: Scielo, BIREME (LILACS, PubMed e CAPES. Results : It was observed that the length of stay in the intensive care unit in cardiac surgery was influenced directly by CPB, VM and pulmonary dysfunction. Functional independence was compromised in patients with longer duration of mechanical ventilation, postoperative pain and prolonged bed rest. It was also found that there is no consensus on the protocol for improved functional capacity. Conclusion : There is a functional decline in patients undergoing cardiac surgery, especially those at increased length of stay in mechanical ventilation, reflecting a direct and negative impact on their functional independence and quality of life.

  11. Sanguinarine is a novel VEGF inhibitor involved in the suppression of angiogenesis and cell migration

    Science.gov (United States)

    XU, JIA-YING; MENG, QING-HUI; CHONG, YU; JIAO, YANG; ZHAO, LIN; ROSEN, ELIOT M.; FAN, SAIJUN

    2013-01-01

    Vascular endothelial growth factor (VEGF) is a main angiogenic factor which is known to be upregulated in lung cancer. In the present study, it was demonstrated that sanguinarine, an alkaloid obtained from the bloodroot plant, markedly repressed the VEGF-induced tube formation of human microvascular endothelial cells (HMVECs) and the migration of human A549 lung cancer cells. Furthermore, sanguinarine decreased VEGF secretion and expression in HMVECs and A549 lung cancer cells in a dose- and time-dependent manner. Additionally, sanguinarine inhibited the activation of serum starvation- and hypoxia-induced VEGF promoter activity. Sanguinarine also inhibited the VEGF-mediated Akt and p38 activation, as well as VE-cadherin protein phosphorylation. To the best of our knowledge, this is the first study demonstrating that VEGF inhibition appears to be an important mechanism involved in the antiangiogenic and anti-invasive activities of sanguinarine in lung cancer treatment. PMID:24649171

  12. Pigment patterns in adult fish result from superimposition of two largely independent pigmentation mechanisms.

    Science.gov (United States)

    Ceinos, Rosa M; Guillot, Raúl; Kelsh, Robert N; Cerdá-Reverter, José M; Rotllant, Josep

    2015-03-01

    Dorso-ventral pigment pattern differences are the most widespread pigmentary adaptations in vertebrates. In mammals, this pattern is controlled by regulating melanin chemistry in melanocytes using a protein, agouti-signalling peptide (ASIP). In fish, studies of pigment patterning have focused on stripe formation, identifying a core striping mechanism dependent upon interactions between different pigment cell types. In contrast, mechanisms driving the dorso-ventral countershading pattern have been overlooked. Here, we demonstrate that, in fact, zebrafish utilize two distinct adult pigment patterning mechanisms - an ancient dorso-ventral patterning mechanism, and a more recent striping mechanism based on cell-cell interactions; remarkably, the dorso-ventral patterning mechanism also utilizes ASIP. These two mechanisms function largely independently, with resultant patterns superimposed to give the full pattern. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. An increase of VEGF plasma levels is associated with restenosis of drug-eluting stents.

    Science.gov (United States)

    Katsaros, Katharina M; Kastl, Stefan P; Krychtiuk, Konstantin A; Hutter, Randolph; Zorn, Gerlinde; Maurer, Gerald; Huber, Kurt; Wojta, Johann; Christ, Günter; Speidl, Walter S

    2014-06-01

    Drug-eluting stents (DES) reduce late lumen loss compared to bare metal stents but were not able to eradicate in-stent restenosis (ISR) fully. Vascular endothelial growth factor (VEGF) may inhibit late lumen loss through accelerated reendothelialisation, but may also promote neointima formation by proinflammatory effects. The aim of this study was to evaluate whether endogenous plasma levels of VEGF are associated with development of ISR after implantation of DES. We studied 85 patients who were treated with 159 DES. VEGF plasma levels were determined before and 24 hours after PCI. During the eight-month follow-up period, two patients (2.4%) died of cardiovascular causes and 12 patients (14.5% of patients, 7.6% of stents) developed angiographic ISR. Basal VEGF plasma levels were not different in patients with and without ISR at follow-up. In contrast to patients without ISR, VEGF increased significantly upon PCI in patients with ISR (p<0.005). Patients with a decrease of VEGF after PCI had a restenosis rate of 2.4% compared to a restenosis rate of 26.2% in patients with an increase of VEGF after the procedure (p<0.05). This was independent from clinical and angiographic risk factors. Basal plasma levels of VEGF are not associated with the development of ISR. However, an increase of VEGF after PCI is associated with a dramatically increased ISR rate after implantation of DES.

  14. Exercise-induced capillary growth in human skeletal muscle and the dynamics of VEGF.

    Science.gov (United States)

    Hoier, Birgitte; Hellsten, Ylva

    2014-05-01

    In skeletal muscle, growth of capillaries is an important adaptation to exercise training that secures adequate diffusion capacity for oxygen and nutrients even at high-intensity exercise when increases in muscle blood flow are profound. Mechanical forces present during muscle activity, such as shear stress and passive stretch, lead to cellular signaling, enhanced expression of angiogenic factors, and initiation of capillary growth. The most central angiogenic factor in skeletal muscle capillary growth is VEGF. During muscle contraction, VEGF increases in the muscle interstitium, acts on VEGF receptors on the capillary endothelium, and thereby stimulates angiogenic processes. A primary source of muscle interstitial VEGF during exercise is the skeletal muscle fibers which contain large stores of VEGF within vesicles. We propose that, during muscle activity, these VEGF-containing vesicles are redistributed toward the sarcolemma where the contents are secreted into the extracellular fluid. VEGF mRNA expression is increased primarily after exercise, which allows for a more rapid replenishment of VEGF stores lost through secretion during exercise. Future studies should focus on elucidating mechanisms and regulation of VEGF secretion. © 2014 John Wiley & Sons Ltd.

  15. Mechanisms of Glioma Formation: Iterative Perivascular Glioma Growth and Invasion Leads to Tumor Progression, VEGF-Independent Vascularization, and Resistance to Antiangiogenic Therapy

    Directory of Open Access Journals (Sweden)

    Gregory J. Baker

    2014-07-01

    Full Text Available As glioma cells infiltrate the brain they become associated with various microanatomic brain structures such as blood vessels, white matter tracts, and brain parenchyma. How these distinct invasion patterns coordinate tumor growth and influence clinical outcomes remain poorly understood. We have investigated how perivascular growth affects glioma growth patterning and response to antiangiogenic therapy within the highly vascularized brain. Orthotopically implanted rodent and human glioma cells are shown to commonly invade and proliferate within brain perivascular space. This form of brain tumor growth and invasion is also shown to characterize de novo generated endogenous mouse brain tumors, biopsies of primary human glioblastoma (GBM, and peripheral cancer metastasis to the human brain. Perivascularly invading brain tumors become vascularized by normal brain microvessels as individual glioma cells use perivascular space as a conduit for tumor invasion. Agent-based computational modeling recapitulated biological perivascular glioma growth without the need for neoangiogenesis. We tested the requirement for neoangiogenesis in perivascular glioma by treating animals with angiogenesis inhibitors bevacizumab and DC101. These inhibitors induced the expected vessel normalization, yet failed to reduce tumor growth or improve survival of mice bearing orthotopic or endogenous gliomas while exacerbating brain tumor invasion. Our results provide compelling experimental evidence in support of the recently described failure of clinically used antiangiogenics to extend the overall survival of human GBM patients.

  16. A Therapeutically Targetable Mechanism of BCR-ABL-Independent Imatinib Resistance in Chronic Myeloid Leukemia

    Science.gov (United States)

    Ma, Leyuan; Shan, Yi; Bai, Robert; Xue, Liting; Eide, Christopher A.; Ou, Jianhong; Zhu, Lihua J.; Hutchinson, Lloyd; Cerny, Jan; Khoury, Hanna Jean; Sheng, Zhi; Druker, Brian J.; Li, Shaoguang; Green, Michael R.

    2014-01-01

    Resistance to the BCR-ABL inhibitor imatinib mesylate (IM) poses a major problem for the treatment of chronic myeloid leukemia (CML). IM resistance often results from a secondary mutation in BCR-ABL that interferes with drug binding. However, in many instances there is no mutation in BCR-ABL, and the basis of such BCR-ABL-independent IM resistance remains to be elucidated. To gain insight into BCR-ABL-independent IM resistance mechanisms, we performed a large-scale RNA interference (RNAi) screen and identified IM-sensitizing genes (IMSGs) whose knockdown renders BCR-ABL+ cells IM-resistant. In these IMSG knockdown cells, RAF/MEK/ERK signaling is sustained after IM treatment due to upregulation of PRKCH, which encodes the protein kinase C (PKC) family member PKCη, an activator of CRAF. PRKCH is also upregulated in samples from CML patients with BCR-ABL-independent IM resistance. Combined treatment with IM and trametinib, an FDA-approved MEK inhibitor, synergistically kills BCR-ABL+ IMSG knockdown cells and prolongs survival in mouse models of BCR-ABL-independent IM-resistant CML. Finally, we showed that CML stem cells contain high levels of PRKCH and this contributes to their intrinsic IM resistance. Combined treatment with IM and trametinib synergistically kills CML stem cells with negligible effect on normal hematopoietic stem cells. Collectively, our results identify a therapeutically targetable mechanism of BCR-ABL-independent IM resistance in CML and CML stem cells. PMID:25186176

  17. PGC-1alpha mediates exercise-induced skeletal muscle VEGF expression in mice

    DEFF Research Database (Denmark)

    Leick, Lotte; Hellsten, Ylva; Fentz, Joachim

    2009-01-01

    The aim of the present study was to test the hypothesis that PGC-1alpha is required for exercise-induced VEGF expression in both young and old mice and that AMPK activation leads to increased VEGF expression through a PGC-1alpha-dependent mechanism. Whole body PGC-1alpha knockout (KO) and litterm...

  18. Delineating multiple functions of VEGF-A in the adult brain.

    Science.gov (United States)

    Licht, Tamar; Keshet, Eli

    2013-05-01

    Vascular endothelial growth factor-A (abbreviated throughout this review as VEGF) is mostly known for its angiogenic activity, for its activity as a vascular permeability factor, and for its vascular survival activity [1]. There is a growing body of evidence, however, that VEGF fulfills additional less 'traditional' functions in multiple organs, both during development, as well as homeostatic functions in fully developed organs. This review focuses on the multiple roles of VEGF in the adult brain and is less concerned with the roles played by VEGF during brain development, functions described elsewhere in this review series. Most functions of VEGF that are essential for proper brain development are, in fact, dispensable in the adult brain as was clearly demonstrated using a conditional brain-specific VEGF loss-of-function (LOF) approach. Thus, in contrast to VEGF LOF in the developing brain, a process which is detrimental for the growth and survival of blood vessels and leads to massive neuronal apoptosis [2-4], continued signaling by VEGF in the mature brain is no longer required for maintaining already established cerebral vasculature and its inhibition does not cause appreciable vessel regression, hypoxia or apoptosis [4-7]. Yet, VEGF continues to be expressed in the adult brain in a constitutive manner. Moreover, VEGF is expressed in the adult brain in a region-specific manner and in distinctive spatial patterns incompatible with an angiogenic role (see below), strongly suggesting angiogenesis-independent and possibly also perfusion-independent functions. Here we review current knowledge on some of these 'non-traditional', often unexpected homeostatic VEGF functions, including those unrelated to its effects on the brain vasculature. These effects could be mediated directly (on non-vascular cells expressing cognate VEGF receptors) or indirectly (via the endothelium). Experimental approaches aimed at distinguishing between these possibilities for each particular

  19. Clostridium difficile modulates host innate immunity via toxin-independent and dependent mechanism(s.

    Directory of Open Access Journals (Sweden)

    Nazila V Jafari

    Full Text Available Clostridium difficile infection (CDI is the leading cause of hospital and community-acquired antibiotic-associated diarrhoea and currently represents a significant health burden. Although the role and contribution of C. difficile toxins to disease pathogenesis is being increasingly understood, at present other facets of C. difficile-host interactions, in particular, bacterial-driven effects on host immunity remain less studied. Using an ex-vivo model of infection, we report that the human gastrointestinal mucosa elicits a rapid and significant cytokine response to C. difficile. Marked increase in IFN-γ with modest increase in IL-22 and IL-17A was noted. Significant increase in IL-8 suggested potential for neutrophil influx while presence of IL-12, IL-23, IL-1β and IL-6 was indicative of a cytokine milieu that may modulate subsequent T cell immunity. Majority of C. difficile-driven effects on murine bone-marrow-derived dendritic cell (BMDC activation were toxin-independent; the toxins were however responsible for BMDC inflammasome activation. In contrast, human monocyte-derived DCs (mDCs released IL-1β even in the absence of toxins suggesting host-specific mediation. Infected DC-T cell crosstalk revealed the ability of R20291 and 630 WT strains to elicit a differential DC IL-12 family cytokine milieu which culminated in significantly greater Th1 immunity in response to R20291. Interestingly, both strains induced a similar Th17 response. Elicitation of mucosal IFN-γ/IL-17A and Th1/Th17 immunity to C. difficile indicates a central role for this dual cytokine axis in establishing antimicrobial immunity to CDI.

  20. The Effect of Depression on Serum VEGF Level in Alzheimer’s Disease

    Directory of Open Access Journals (Sweden)

    JaeHoon Jung

    2015-01-01

    Full Text Available Objective. Growing evidence suggests that angiogenesis might represent a new pathogenic mechanism involved in the progression of Alzheimer’s disease (AD. Among angiogenic cytokines, vascular endothelial growth factor (VEGF levels in AD patients have been evaluated, but the results are controversial among studies. We investigated serum levels of VEGF in AD patients with depression, AD patients without depression, and the controls, respectively. The aim of this study is to elucidate the relationship between VEGF, depression, and cognitive impairment in AD. Methods. The CDR (Clinical Dementia Rating, MMSE-KC (the Mini-Mental Status Examination-Korean version, and SGDS-K (the Korean version of the Geriatric Depression Scale-Short Form were measured in the subjects. Serum VEGF levels were measured in 24 AD patients with depression, 25 AD patients without depression, and 26 controls, using an enzyme-linked immunosorbent assay kit. Results. Serum VEGF levels in AD patients with depression were significantly higher than AD patients without depression or the control. A correlation was observed between VEGF and scores on SGDS-K, but no correlation was detected between VEGF and MMSE-KC scores. Conclusion. Serum VEGF levels in AD patients with depression were higher than those without depression. Depression might be associated with changes in serum levels of VEGF in AD patients.

  1. Specific imaging of VEGF-A expression with radiolabeled anti-VEGF monoclonal antibody.

    NARCIS (Netherlands)

    Stollman, T.H.; Scheer, M.G.W.; Leenders, W.P.J.; Verrijp, K.C.; Soede, A.C.; Oyen, W.J.G.; Ruers, T.J.M.; Boerman, O.C.

    2008-01-01

    Vascular endothelial growth factor-A (VEGF-A) is one of the most important angiogenic factors. Here, we studied in a nude mouse model whether the expression of VEGF-A in a tumor could be imaged with a radiolabeled anti-VEGF antibody. The humanized anti-VEGF-A antibody A.4.6.1. (bevacizumab), which

  2. Interferon decreases VEGF levels in patients with chronic myeloid leukemia treated with imatinib.

    Science.gov (United States)

    Legros, L; Guilhot, J; Huault, S; Mahon, F X; Preudhomme, C; Guilhot, F; Hueber, A O

    2014-06-01

    In chronic myeloid leukemia (CML), evidence is supporting the role of VEGF in growth, and survival of leukemia cells. The evaluation of plasma VEGF levels in 403 CML patients randomized within SPIRIT study to received imatinib-400mg versus imatinib+cytarabine versus imatinib+interferon (IFN) versus imatinib-600mg demonstrated that VEGF is an independent factor of BCR-ABL burden. VEGF low levels at diagnosis were associated with a progression-free survival of 100% at 48 months. Under treatment, significant lowest levels were observed in imatinib+IFN arm. These results support the use of VEGF as a parameter to predict CML evolution and let us to speculate about antiangiogenic properties of IFN. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Anti-VEGF Agents for Ocular Angiogenesis and Vascular Permeability

    Directory of Open Access Journals (Sweden)

    Kenichi Kimoto

    2012-01-01

    Full Text Available We review articles describing intravitreal injection of anti-VEGF drug trials, while discussing the mechanisms of the action of anti-VEGF antibodies, and also evaluating their outcomes. Intraocular injections of anti-VEGF drug are considered to be an effective treatment for macular edema after retinal vein occlusion, however, recurrent/persistent edema is common. The recent reports may lead to a shift in treatment paradigm for DME, from laser photocoagulation, to newer approaches using anti-VEGF drugs. There have been several well-publicized prospective, randomized studies that demonstrated the efficacy of intravitreal injection of anti-VEGF drugs for patients with AMD. Adjuvant bevacizumab for neovascular glaucoma may prevent further PAS formation, and it is likely to open up a therapeutic window for a panretinal photocoagulation and trabeculectomy. Intravitreal injection of bevacizumab (IVB results in a substantial decrease in bleeding from the retinal vessels or new vessels during a standard vitrectomy. IVB has also been reported to be effective for inducing the regression of new vessels in proliferative diabetic retinopathy. The use of bevacizumab in stage 4 or 5 retinopahty of permaturity (ROP is to reduce the plus sign to help reduce hemorrhage during the subsequent vitrectomy. Some authors reported cases of resolution of stage 4 A ROP after bevacizumab injection.

  4. A checkpoint-independent mechanism delays entry into mitosis after UV irradiation.

    Science.gov (United States)

    Rothe, Christiane; Rødland, Gro Elise; Anda, Silje; Stonyte, Vilte; Boye, Erik; Lopez-Aviles, Sandra; Grallert, Beáta

    2017-12-01

    When cells are exposed to stress they delay entry into mitosis. The most extensively studied mechanism behind this delay is the DNA-damage-induced G2/M checkpoint. Here, we show the existence of an additional stress-response pathway in Schizosaccharomyces pombe that is independent of the classic ATR/Rad3-dependent checkpoint. This novel mechanism delays entry mitosis independently of the spindle assembly checkpoint and the mitotic kinases Fin1, Ark1 and Plo1. The pathway delays activation of the mitotic cyclin-dependent kinase (CDK) Cdc2 after UV irradiation. Furthermore, we demonstrate that translation of the mitotic cyclin Cdc13 is selectively downregulated after UV irradiation, and we propose that this downregulation of Cdc13 contributes to the delayed activation of Cdc2 and the delayed mitosis. © 2017. Published by The Company of Biologists Ltd.

  5. Anticoagulant independent mechanical heart valves: viable now or still a distant holy grail.

    Science.gov (United States)

    Chaux, Aurelio; Gray, Richard J; Stupka, Jonathan C; Emken, Michael R; Scotten, Lawrence N; Siegel, Rolland

    2016-12-01

    Valvular heart disease remains a large public health problem for all societies; it attracts the attention of public health organizations, researchers and governments. Valve substitution is an integral part of the treatment for this condition. At present, the choice of valve prosthesis is either tissue or mechanical. Tissue valves have become increasingly popular in spite of unresolved problems with durability, hemodynamics, cost and need for anticoagulation therapy. As a consequence, mechanical valve innovation has virtually ceased; the last successful mechanical design is 25 years old. We postulate that with improved technology, knowledge and experience gained over the last quarter century, the best possible solution to the problem of valve substitution can be achieved with a mechanical valve that is anticoagulant independent, durable, hemodynamically and cost efficient. At present, it is possible to design, test and produce a valve that can accomplish these goals.

  6. Celecoxib Down-Regulates the Hypoxia-Induced Expression of HIF-1α and VEGF Through the PI3K/AKT Pathway in Retinal Pigment Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Yi-zhou Sun

    2017-12-01

    Full Text Available Background/Aims: The goal of this study was to detect the expression of hypoxia-inducible factor 1α (HIF-1α and vascular endothelial growth factor (VEGF in human retinal pigmented epithelial (RPE cells treated with celecoxib, a selective cyclooxygenase-2 (COX-2 inhibitor, under hypoxic and normoxic conditions and to explore the signaling mechanism involved in regulating the hypoxia-induced expression of HIF-1α and VEGF in RPE cells. Methods: D407 cells were cultured in normoxic or hypoxic conditions, with or without celecoxib or a PI3K inhibitor (LY294002. The anti-proliferative effect of celecoxib was assessed using the MTT assay. RT-PCR, Western blotting and ELISA were performed to detect the levels of PI3K, phosphorylated AKT (p-AKT, HIF-1α, VEGF and COX-2. Results: Celecoxib inhibited the proliferation of RPE cells in a dose-dependent manner. Celecoxib suppressed the expression of VEGF at both the mRNA and protein levels and decreased HIF-1α protein expression. HIF-1α activation was regulated by the PI3K/AKT pathway. The celecoxib-induced down-regulation of HIF-1α and VEGF required the suppression of the hypoxia-induced PI3K/AKT pathway. However, the down-regulation of COX-2 did not occur in cells treated with celecoxib. Conclusions: The antiangiogenic effects of celecoxib in RPE cells under hypoxic conditions resulted from the inhibition of HIF-1α and VEGF expression, which may be partly mediated by a COX-2-independent, PI3K/AKT-dependent pathway.

  7. A proteomic study of potential VEGF-C-associated proteins in bladder cancer T24 cells.

    Science.gov (United States)

    Zhang, Hui-hui; Qi, Fan; Zu, Xiong-bing; Cao, You-han; Miao, Jian-guang; Xu, Liang; Qi, Lin

    2012-11-01

    Overexpression of vascular endothelial growth factor-C (VEGF-C) has been found to play an important role in malignant progression of various cancer cells, in addition to lymphangiogenesis. However, the mechanisms involved are still largely unknown. Our early research has confirmed that the expression of VEGF-C in bladder cancer was markedly higher than that in normal bladder tissues. VEGF-C can also obviously promote proliferation and invasion of bladder cancer T24 cells. In the present work, we attempted to use proteomic analysis to screen out potential VEGF-C-associated proteins involved in malignant progression of the bladder cancer T24 cells. Lentivirus vector-based RNA interference (RNAi) was employed to diminish VEGF-C expression of bladder cancer T24 cells. Then we performed comparative proteome analysis to explore differentially expressed proteins in T24 cells with and without VEGF-C siRNA, by two-dimensional difference gel electrophoresis (2D-DIGE). Twenty-three proteins were identified. Some proteins (matrix metalloproteinase-9, Keratin 8, Serpin B5, Annexin A8) with significant differences were further confirmed by Western blotting. The 23 potential VEGF-C-associated proteins identified in our study provide us with further insights into the mechanism of VEGF-C promoting malignant progression of bladder cancer cells.

  8. New Insights on the Mechanism of the K+-Independent Activity of Crenarchaeota Pyruvate Kinases

    Science.gov (United States)

    De la Vega-Ruíz, Gustavo; Domínguez-Ramírez, Lenin; Riveros-Rosas, Héctor; Guerrero-Mendiola, Carlos; Torres-Larios, Alfredo; Hernández-Alcántara, Gloria; García-Trejo, José J.; Ramírez-Silva, Leticia

    2015-01-01

    Eukarya pyruvate kinases have glutamate at position 117 (numbered according to the rabbit muscle enzyme), whereas in Bacteria have either glutamate or lysine and in Archaea have other residues. Glutamate at this position makes pyruvate kinases K+-dependent, whereas lysine confers K+-independence because the positively charged residue substitutes for the monovalent cation charge. Interestingly, pyruvate kinases from two characterized Crenarchaeota exhibit K+-independent activity, despite having serine at the equivalent position. To better understand pyruvate kinase catalytic activity in the absence of K+ or an internal positive charge, the Thermofilum pendens pyruvate kinase (valine at the equivalent position) was characterized. The enzyme activity was K+-independent. The kinetic mechanism was random order with a rapid equilibrium, which is equal to the mechanism of the rabbit muscle enzyme in the presence of K+ or the mutant E117K in the absence of K+. Thus, the substrate binding order of the T. pendens enzyme was independent despite lacking an internal positive charge. Thermal stability studies of this enzyme showed two calorimetric transitions, one attributable to the A and C domains (Tm of 99.2°C), and the other (Tm of 105.2°C) associated with the B domain. In contrast, the rabbit muscle enzyme exhibits a single calorimetric transition (Tm of 65.2°C). The calorimetric and kinetic data indicate that the B domain of this hyperthermophilic enzyme is more stable than the rest of the protein with a conformation that induces the catalytic readiness of the enzyme. B domain interactions of pyruvate kinases that have been determined in Pyrobaculum aerophilum and modeled in T. pendens were compared with those of the rabbit muscle enzyme. The results show that intra- and interdomain interactions of the Crenarchaeota enzymes may account for their higher B domain stability. Thus the structural arrangement of the T. pendens pyruvate kinase could allow charge-independent

  9. New insights on the mechanism of the K(+- independent activity of crenarchaeota pyruvate kinases.

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    Gustavo De la Vega-Ruíz

    Full Text Available Eukarya pyruvate kinases have glutamate at position 117 (numbered according to the rabbit muscle enzyme, whereas in Bacteria have either glutamate or lysine and in Archaea have other residues. Glutamate at this position makes pyruvate kinases K+-dependent, whereas lysine confers K+-independence because the positively charged residue substitutes for the monovalent cation charge. Interestingly, pyruvate kinases from two characterized Crenarchaeota exhibit K+-independent activity, despite having serine at the equivalent position. To better understand pyruvate kinase catalytic activity in the absence of K+ or an internal positive charge, the Thermofilum pendens pyruvate kinase (valine at the equivalent position was characterized. The enzyme activity was K+-independent. The kinetic mechanism was random order with a rapid equilibrium, which is equal to the mechanism of the rabbit muscle enzyme in the presence of K+ or the mutant E117K in the absence of K+. Thus, the substrate binding order of the T. pendens enzyme was independent despite lacking an internal positive charge. Thermal stability studies of this enzyme showed two calorimetric transitions, one attributable to the A and C domains (Tm of 99.2°C, and the other (Tm of 105.2°C associated with the B domain. In contrast, the rabbit muscle enzyme exhibits a single calorimetric transition (Tm of 65.2°C. The calorimetric and kinetic data indicate that the B domain of this hyperthermophilic enzyme is more stable than the rest of the protein with a conformation that induces the catalytic readiness of the enzyme. B domain interactions of pyruvate kinases that have been determined in Pyrobaculum aerophilum and modeled in T. pendens were compared with those of the rabbit muscle enzyme. The results show that intra- and interdomain interactions of the Crenarchaeota enzymes may account for their higher B domain stability. Thus the structural arrangement of the T. pendens pyruvate kinase could allow charge-independent

  10. New insights on the mechanism of the K(+-) independent activity of crenarchaeota pyruvate kinases.

    Science.gov (United States)

    De la Vega-Ruíz, Gustavo; Domínguez-Ramírez, Lenin; Riveros-Rosas, Héctor; Guerrero-Mendiola, Carlos; Torres-Larios, Alfredo; Hernández-Alcántara, Gloria; García-Trejo, José J; Ramírez-Silva, Leticia

    2015-01-01

    Eukarya pyruvate kinases have glutamate at position 117 (numbered according to the rabbit muscle enzyme), whereas in Bacteria have either glutamate or lysine and in Archaea have other residues. Glutamate at this position makes pyruvate kinases K+-dependent, whereas lysine confers K+-independence because the positively charged residue substitutes for the monovalent cation charge. Interestingly, pyruvate kinases from two characterized Crenarchaeota exhibit K+-independent activity, despite having serine at the equivalent position. To better understand pyruvate kinase catalytic activity in the absence of K+ or an internal positive charge, the Thermofilum pendens pyruvate kinase (valine at the equivalent position) was characterized. The enzyme activity was K+-independent. The kinetic mechanism was random order with a rapid equilibrium, which is equal to the mechanism of the rabbit muscle enzyme in the presence of K+ or the mutant E117K in the absence of K+. Thus, the substrate binding order of the T. pendens enzyme was independent despite lacking an internal positive charge. Thermal stability studies of this enzyme showed two calorimetric transitions, one attributable to the A and C domains (Tm of 99.2°C), and the other (Tm of 105.2°C) associated with the B domain. In contrast, the rabbit muscle enzyme exhibits a single calorimetric transition (Tm of 65.2°C). The calorimetric and kinetic data indicate that the B domain of this hyperthermophilic enzyme is more stable than the rest of the protein with a conformation that induces the catalytic readiness of the enzyme. B domain interactions of pyruvate kinases that have been determined in Pyrobaculum aerophilum and modeled in T. pendens were compared with those of the rabbit muscle enzyme. The results show that intra- and interdomain interactions of the Crenarchaeota enzymes may account for their higher B domain stability. Thus the structural arrangement of the T. pendens pyruvate kinase could allow charge-independent

  11. Kronecker compressive sensing-based mechanism with fully independent sampling dimensions for hyperspectral imaging

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    Zhao, Rongqiang; Wang, Qiang; Shen, Yi

    2015-11-01

    We propose a new approach for Kronecker compressive sensing of hyperspectral (HS) images, including the imaging mechanism and the corresponding reconstruction method. The proposed mechanism is able to compress the data of all dimensions when sampling, which can be achieved by three fully independent sampling devices. As a result, the mechanism greatly reduces the control points and memory requirement. In addition, we can also select the suitable sparsifying bases and generate the corresponding optimized sensing matrices or change the distribution of sampling ratio for each dimension independently according to different HS images. As the cooperation of the mechanism, we combine the sparsity model and low multilinear-rank model to develop a reconstruction method. Analysis shows that our reconstruction method has a lower computational complexity than the traditional methods based on sparsity model. Simulations verify that the HS images can be reconstructed successfully with very few measurements. In summary, the proposed approach can reduce the complexity and improve the practicability for HS image compressive sensing.

  12. Prickle isoforms control the direction of tissue polarity by microtubule independent and dependent mechanisms

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    Katherine A. Sharp

    2016-03-01

    Full Text Available Planar cell polarity signaling directs the polarization of cells within the plane of many epithelia. While these tissues exhibit asymmetric localization of a set of core module proteins, in Drosophila, more than one mechanism links the direction of core module polarization to the tissue axes. One signaling system establishes a polarity bias in the parallel, apical microtubules upon which vesicles containing core proteins traffic. Swapping expression of the differentially expressed Prickle isoforms, Prickle and Spiny-legs, reverses the direction of core module polarization. Studies in the proximal wing and the anterior abdomen indicated that this results from their differential control of microtubule polarity. Prickle and Spiny-legs also control the direction of polarization in the distal wing (D-wing and the posterior abdomen (P-abd. We report here that this occurs without affecting microtubule polarity in these tissues. The direction of polarity in the D-wing is therefore likely determined by a novel mechanism independent of microtubule polarity. In the P-abd, Prickle and Spiny-legs interpret at least two directional cues through a microtubule-polarity-independent mechanism.

  13. Engineering interpenetrating network hydrogels as biomimetic cell niche with independently tunable biochemical and mechanical properties.

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    Tong, Xinming; Yang, Fan

    2014-02-01

    Hydrogels have been widely used as artificial cell niche to mimic extracellular matrix with tunable properties. However, changing biochemical cues in hydrogels developed-to-date would often induce simultaneous changes in mechanical properties, which do not support mechanistic studies on stem cell-niche interactions. Here we report the development of a PEG-based interpenetrating network (IPN), which is composed of two polymer networks that can independently and simultaneously crosslink to form hydrogels in a cell-friendly manner. The resulting IPN hydrogel allows independently tunable biochemical and mechanical properties, as well as stable and more homogeneous presentation of biochemical ligands in 3D than currently available methods. We demonstrate the potential of our IPN platform for elucidating stem cell-niche interactions by modulating osteogenic differentiation of human adipose-derived stem cells. The versatility of such IPN hydrogels is further demonstrated using three distinct and widely used polymers to form the mechanical network while keeping the biochemical network constant. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Trolox inhibits osteolytic bone metastasis of breast cancer through both PGE2-dependent and independent mechanisms.

    Science.gov (United States)

    Lee, Jong-Ho; Kim, Bongjun; Jin, Won Jong; Kim, Jung-Wook; Kim, Hong-Hee; Ha, Hyunil; Lee, Zang Hee

    2014-09-01

    Bone is a preferred site of metastasis from breast cancer, and increased osteoclast activity is implicated in breast cancer outgrowth in the bone microenvironment. Our previous observation of an anti-osteoclastic activity of Trolox, a vitamin E analog, led us to investigate whether Trolox inhibits bone metastasis and osteolysis caused by breast cancer. Administration of Trolox markedly inhibited osteolytic bone metastasis in an experimental metastasis model by intracardiac injection of 4T1 breast cancer cells. Trolox inhibited proliferation of 4T1 cells in the bone marrow but not in the mammary fat pad. In addition, Trolox could reduce tumor burden, osteolysis, and prostaglandin E2 (PGE2) production induced by direct inoculation of 4T1 cells into the marrow cavity of the tibia. Trolox decreased the migratory and invasive activities of 4T1 cells via PGE2-dependent and independent mechanisms. It also inhibited the ability of 4T1 cells to stimulate the expression of receptor activator of nuclear factor-κB ligand (RANKL), a key cytokine for osteoclast differentiation factor, in osteoblasts. In addition, Trolox suppressed RANKL expression in osteoblasts induced by soluble factors from 4T1 cells. Furthermore, Trolox suppressed 4T1 cell-induced osteoclast differentiation in the co-culture of bone marrow cells and osteoblasts via both PGE2-dependent and independent mechanisms. Taken together, these results suggest that Trolox inhibits breast cancer cell-induced osteoclast differentiation and the invasive behavior of cancer cells through PGE2-dependent and independent mechanisms, thereby suppressing osteolytic bone metastasis of breast cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Tumour cells expressing single VEGF isoforms display distinct growth, survival and migration characteristics.

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    Chryso Kanthou

    Full Text Available Vascular endothelial growth factor-A (VEGF is produced by most cancer cells as multiple isoforms, which display distinct biological activities. VEGF plays an undisputed role in tumour growth, vascularisation and metastasis; nevertheless the functions of individual isoforms in these processes remain poorly understood. We investigated the effects of three main murine isoforms (VEGF188, 164 and 120 on tumour cell behaviour, using a panel of fibrosarcoma cells we developed that express them individually under endogenous promoter control. Fibrosarcomas expressing only VEGF188 (fs188 or wild type controls (fswt were typically mesenchymal, formed ruffles and displayed strong matrix-binding activity. VEGF164- and VEGF120-producing cells (fs164 and fs120 respectively were less typically mesenchymal, lacked ruffles but formed abundant cell-cell contacts. On 3D collagen, fs188 cells remained mesenchymal while fs164 and fs120 cells adopted rounded/amoeboid and a mix of rounded and elongated morphologies respectively. Consistent with their mesenchymal characteristics, fs188 cells migrated significantly faster than fs164 or fs120 cells on 2D surfaces while contractility inhibitors accelerated fs164 and fs120 cell migration. VEGF164/VEGF120 expression correlated with faster proliferation rates and lower levels of spontaneous apoptosis than VEGF188 expression. Nevertheless, VEGF188 was associated with constitutively active/phosphorylated AKT, ERK1/2 and Stat3 proteins. Differences in proliferation rates and apoptosis could be explained by defective signalling downstream of pAKT to FOXO and GSK3 in fs188 and fswt cells, which also correlated with p27/p21 cyclin-dependent kinase inhibitor over-expression. All cells expressed tyrosine kinase VEGF receptors, but these were not active/activatable suggesting that inherent differences between the cell lines are governed by endogenous VEGF isoform expression through complex interactions that are independent of tyrosine

  16. Simvastatin may induce insulin resistance through a novel fatty acid mediated cholesterol independent mechanism

    Science.gov (United States)

    Kain, Vasundhara; Kapadia, Bandish; Misra, Parimal; Saxena, Uday

    2015-01-01

    Statins are a class of oral drugs that are widely used for treatment of hypercholesterolemia. Recent clinical data suggest that chronic use of these drugs increases the frequency of new onset diabetes. Studies to define the risks of statin-induced diabetes and its underlying mechanisms are clearly necessary. We explored the possible mechanism of statin induced insulin resistance using a well-established cell based model and simvastatin as a prototype statin. Our data show that simvastatin induces insulin resistance in a cholesterol biosynthesis inhibition independent fashion but does so by a fatty acid mediated effect on insulin signaling pathway. These data may help design strategies for prevention of statin induced insulin resistance and diabetes in patients with hypercholesterolemia. PMID:26345110

  17. Culture-Independent Molecular Methods for Detection of Antifungal Resistance Mechanisms and Fungal Identification.

    Science.gov (United States)

    Perlin, David S; Wiederhold, Nathan P

    2017-08-15

    Resistance to azoles and echinocandins has emerged as a significant factor affecting the clinical management of patients with invasive fungal infections. Immunosuppressed patients at high risk for invasive fungal infections often have prolonged or repeated exposure to antifungals resulting in either the well-documented selection of naturally occurring, less susceptible fungal species, or the in situ development of specific resistance mechanisms. Nucleic acid-based molecular diagnostics are particularly well suited for the rapid detection of low-abundance fungal pathogens and identification of the infecting pathogen to the genus and species levels, as well as assessment of resistance mechanisms. A wide range of molecular probing technologies involving real-time polymerase chain reaction (PCR) assays that facilitate direct analysis of a single infecting genome in a sterile blood specimen are available and have recently been commercialized (eg, Roche LightCycler SeptiFast and T2 Biosystems T2Candida). One of the exciting applications of molecular technology is the direct detection of specific resistance mechanisms that evolve during therapy. In principle, the detection of resistance mechanisms that have been independently validated to cause resistance provides a culture-independent biomarker for potential therapeutic failure. The emergence of real-time PCR assays utilizing allele-specific molecular detection technology that is highly sensitive, robust, and high-throughput has the potential to improve patient care by providing faster detection of drug-resistant infecting strains and to help inform therapeutic management. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  18. The Nigeria Independent Accountability Mechanism for maternal, newborn, and child health.

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    Garba, Aminu Magashi; Bandali, Sarah

    2014-10-01

    Since the 2010 launch of the UN Secretary-General's Global Strategy for Women's and Children's Health, worldwide political energy coalesced around improving the health of women and children. Nigeria acted on a key recommendation emerging from the Global Strategy and became one of the first countries to establish an independent group known as the Nigeria Independent Accountability Mechanism (NIAM). NIAM aims to track efforts on progress related to Nigeria's roadmap for the health of women and children. It includes eminent people from outside government to ensure independence, and is recognized within government to analyze and report on progress. The concept of NIAM received approval at various national and international forums, as well as from the Nigeria Federal Ministry of Health. This experience provides an example of connecting expertise and groups with the government to influence and accelerate progress in maternal, newborn, and child health. Engagement between government and civil society should become the norm rather than the exception to achieve national goals. Copyright © 2014. Published by Elsevier Ireland Ltd.

  19. Chromatin contributes to structural integrity of promyelocytic leukemia bodies through a SUMO-1-independent mechanism.

    Science.gov (United States)

    Eskiw, Christopher H; Dellaire, Graham; Bazett-Jones, David P

    2004-03-05

    Promyelocytic leukemia (PML) protein is implicated in transcriptional regulation, apoptosis, DNA repair, and tumor suppression. It is not known, however, whether PML and other components of PML bodies function within the vicinity of the bodies or elsewhere in the nucleoplasm. In this study, we demonstrate that chromatin organization around PML bodies influences their morphology, dynamics, and structural integrity by a SUMO-1-independent mechanism. Following transcriptional inhibition and during early apoptosis, chromatin retracts from the periphery of PML bodies, coinciding with the formation of new PML-containing structures through fission of supramolecular PML-containing microbodies. Both fission and fusion of microbodies with parental PML bodies indicate a loss of structural integrity of the bodies, dependent on the state of the surrounding chromatin. This is supported by the observation that treatment of live cells with DNase I could reproduce the structural instability of PML bodies. In addition, PML bodies, which are normally surrounded by chromatin and are positionally stable, become more dynamic following these treatments, presumably due to the loss of chromatin contacts. Overexpression of SUMO-1, a modification required for PML body formation, did not prevent PML body fission, indicating that chromatin-based integrity of PML body structure occurs through a SUMO-1-independent mechanism.

  20. Thermal fluctuations affect the transcriptome through mechanisms independent of average temperature.

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    Sørensen, Jesper Givskov; Schou, Mads Fristrup; Kristensen, Torsten Nygaard; Loeschcke, Volker

    2016-08-04

    Terrestrial ectotherms are challenged by variation in both mean and variance of temperature. Phenotypic plasticity (thermal acclimation) might mitigate adverse effects, however, we lack a fundamental understanding of the molecular mechanisms of thermal acclimation and how they are affected by fluctuating temperature. Here we investigated the effect of thermal acclimation in Drosophila melanogaster on critical thermal maxima (CTmax) and associated global gene expression profiles as induced by two constant and two ecologically relevant (non-stressful) diurnally fluctuating temperature regimes. Both mean and fluctuation of temperature contributed to thermal acclimation and affected the transcriptome. The transcriptomic response to mean temperatures comprised modification of a major part of the transcriptome, while the response to fluctuations affected a much smaller set of genes, which was highly independent of both the response to a change in mean temperature and to the classic heat shock response. Although the independent transcriptional effects caused by fluctuations were relatively small, they are likely to contribute to our understanding of thermal adaptation. We provide evidence that environmental sensing, particularly phototransduction, is a central mechanism underlying the regulation of thermal acclimation to fluctuating temperatures. Thus, genes and pathways involved in phototransduction are likely of importance in fluctuating climates.

  1. COX-independent mechanisms of cancer chemoprevention by anti-inflammatory drugs

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    Evrim eGurpinar

    2013-07-01

    Full Text Available Epidemiological and clinical studies suggest that non-steroidal anti-inflammatory drugs (NSAIDs, including cyclooxygenase (COX-2 selective inhibitors, reduce the risk of developing cancer. Experimental studies in human cancer cell lines and rodent models of carcinogenesis support these observations by providing strong evidence for the antineoplastic properties of NSAIDs. The involvement of COX-2 in tumorigenesis and its overexpression in various cancer tissues suggest that inhibition of COX-2 is responsible for the chemopreventive efficacy of these agents. However, the precise mechanisms by which NSAIDs exert their antiproliferative effects are still a matter of debate. Numerous other studies have shown that NSAIDs can act through COX-independent mechanisms. This review provides a detailed description of the major COX-independent molecular targets of NSAIDs and discusses how these targets may be involved in their anticancer effects. Toxicities resulting from COX inhibition and the suppression of prostaglandin synthesis preclude the long-term use of NSAIDs for cancer chemoprevention. Furthermore, chemopreventive efficacy is incomplete and treatment often leads to the development of resistance. Identification of alternative NSAID targets and elucidation of the biochemical processes by which they inhibit tumor growth could lead to the development of safer and more efficacious drugs for cancer chemoprevention.

  2. Independent mechanisms recruit the cohesin loader protein NIPBL to sites of DNA damage

    Science.gov (United States)

    Bot, Christopher; Pfeiffer, Annika; Giordano, Fosco; Dantuma, Nico P.; Ström, Lena

    2017-01-01

    ABSTRACT NIPBL is required to load the cohesin complex on to DNA. While the canonical role of cohesin is to couple replicated sister chromatids together until the onset of mitosis, it also promotes tolerance to DNA damage. Here, we show that NIPBL is recruited to DNA damage throughout the cell cycle via independent mechanisms, influenced by type of damage. First, the heterochromatin protein HP1γ (also known as CBX3) recruits NIPBL to DNA double-strand breaks (DSBs) through the corresponding HP1-binding motif within the N-terminus. By contrast, the C-terminal HEAT repeat domain is unable to recruit NIPBL to DSBs but independently targets NIPBL to laser microirradiation-induced DNA damage. Each mechanism is dependent on the RNF8 and RNF168 ubiquitylation pathway, while the recruitment of the HEAT repeat domain requires further ATM or ATR activity. Thus, NIPBL has evolved a sophisticated response to damaged DNA that is influenced by the form of damage, suggesting a highly dynamic role for NIPBL in maintaining genomic stability. PMID:28167679

  3. A transcription-independent epigenetic mechanism is associated with antigenic switching in Trypanosoma brucei.

    Science.gov (United States)

    Aresta-Branco, Francisco; Pimenta, Silvia; Figueiredo, Luisa M

    2016-04-20

    Antigenic variation in Trypanosoma brucei relies on periodic switching of variant surface glycoproteins (VSGs), which are transcribed monoallelically by RNA polymerase I from one of about 15 bloodstream expression sites (BES). Chromatin of the actively transcribed BES is depleted of nucleosomes, but it is unclear if this open conformation is a mere consequence of a high rate of transcription, or whether it is maintained by a transcription-independent mechanism. Using an inducible BES-silencing reporter strain, we observed that chromatin of the active BES remains open for at least 24 hours after blocking transcription. This conformation is independent of the cell-cycle stage, but dependent upon TDP1, a high mobility group box protein. For two days after BES silencing, we detected a transient and reversible derepression of several silent BESs within the population, suggesting that cells probe other BESs before commitment to one, which is complete by 48 hours. FACS sorting and subsequent subcloning confirmed that probing cells are switching intermediates capable of returning to the original BES, switch to the probed BES or to a different BES. We propose that regulation of BES chromatin structure is an epigenetic mechanism important for successful antigenic switching. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. Delineation of VEGF-regulated genes and functions in the cervix of pregnant rodents by DNA microarray analysis

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    Papka Raymond E

    2008-12-01

    Full Text Available Abstract Background VEGF-regulated genes in the cervices of pregnant and non-pregnant rodents (rats and mice were delineated by DNA microarray and Real Time PCR, after locally altering levels of or action of VEGF using VEGF agents, namely siRNA, VEGF receptor antagonist and mouse VEGF recombinant protein. Methods Tissues were analyzed by genome-wide DNA microarray analysis, Real-time and gel-based PCR, and SEM, to decipher VEGF function during cervical remodeling. Data were analyzed by EASE score (microarray and ANOVA (Real Time PCR followed by Scheffe's F-test for multiple comparisons. Results Of the 30,000 genes analyzed, about 4,200 genes were altered in expression by VEGF, i.e., expression of about 2,400 and 1,700 genes were down- and up-regulated, respectively. Based on EASE score, i.e., grouping of genes according to their biological process, cell component and molecular functions, a number of vascular- and non-vascular-related processes were found to be regulated by VEGF in the cervix, including immune response (including inflammatory, cell proliferation, protein kinase activity, and cell adhesion molecule activity. Of interest, mRNA levels of a select group of genes, known to or with potential to influence cervical remodeling were altered. For example, real time PCR analysis showed that levels of VCAM-1, a key molecule in leukocyte recruitment, endothelial adhesion, and subsequent trans-endothelial migration, were elevated about 10 folds by VEGF. Further, VEGF agents also altered mRNA levels of decorin, which is involved in cervical collagen fibrillogenesis, and expression of eNO, PLC and PKC mRNA, critical downstream mediators of VEGF. Of note, we show that VEGF may regulate cervical epithelial proliferation, as revealed by SEM. Conclusion These data are important in that they shed new insights in VEGF's possible roles and mechanisms in cervical events near-term, including cervical remodeling.

  5. Serum VEGF levels are related to the presence of pulmonary arterial hypertension in systemic sclerosis

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    Sakkas Lazaros

    2009-05-01

    Full Text Available Abstract Background The association between systemic sclerosis and pulmonary arterial hypertension (PAH is well recognized. Vascular endothelial growth factor (VEGF has been reported to play an important role in pulmonary hypertension. The aim of the present study was to examine the relationship between systolic pulmonary artery pressure, clinical and functional manifestations of the disease and serum VEGF levels in systemic sclerosis. Methods Serum VEGF levels were measured in 40 patients with systemic sclerosis and 13 control subjects. All patients underwent clinical examination, pulmonary function tests and echocardiography. Results Serum VEGF levels were higher in systemic sclerosis patients with sPAP ≥ 35 mmHg than in those with sPAP LCO were independent predictors of systolic pulmonary artery pressure. Conclusion Serum VEGF levels are increased in systemic sclerosis patients with sPAP ≥ 35 mmHg. The correlation between VEGF levels and systolic pulmonary artery pressure may suggest a possible role of VEGF in the pathogenesis of PAH in systemic sclerosis.

  6. VEGF and IL-18 in induced sputum of lung cancer patients.

    Science.gov (United States)

    Rovina, Nikoletta; Hillas, Georgios; Dima, Efrossini; Vlastos, Fotis; Loukides, Stylianos; Veldekis, Dimitrios; Roussos, Charis; Alhanatis, Manos; Bakakos, Petros

    2011-06-01

    Cytokines are key players in the biological processes of malignant tumors and special interest has been focused on cytokines exerting tumor and anti-tumor properties, such as vascular endothelial growth factor (VEGF) and Interleukin-18 (IL-18). Aim of this study was to assess IL-18 and VEGF levels in induced sputum of lung cancer patients at diagnosis, and assess their possible association with the histological type of cancer, the stage and the overall patient survival. Seventy six patients with a diagnosis of lung cancer were recruited and were followed up for 48months. Thirteen healthy smokers and 16 healthy non-smokers were used as control groups. VEGF and IL-18 were measured by ELISA in sputum supernatants at the time of diagnosis. Lung cancer patients had significantly higher baseline IL-18 and VEGF levels compared to healthy controls (p<0.001). No difference was found in IL-18 and VEGF levels between the various stages in non-small cell lung cancer (NSCLC) and between limited and extended small cell lung cancer (SCLC). However, the ratio of VEGF/IL-18 was significantly higher in NSCLC compared to SCLC patients (p=0.018). In extended SCLC overall survival was inversely associated with baseline sputum VEGF levels (p=0.034) and estimated mortality risk was 1.14 (95% CI 1.006-1.283) for an increase of 100pg/ml in VEGF levels. Such association was not found regarding baseline IL-18 levels. VEGF levels in induced sputum may have a prognostic role in the survival of SCLC. The ratio VEGF/IL-18 in induced sputum differs between NSCLC and SCLC, indicating differences in angiogenesis mechanisms and/or immunological response in these two major histological types of lung cancer. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. Post-radiation increase in VEGF enhances glioma cell motility in vitro

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    Kil Whoon

    2012-02-01

    Full Text Available Abstract Background Glioblastoma multiforme (GBM is among the most lethal of all human tumors, with frequent local recurrences after radiation therapy (RT. The mechanism accounting for such a recurrence pattern is unclear. It has classically been attributed to local recurrence of treatment-resistant cells. However, accumulating evidence suggests that additional mechanisms exist that involve the migration of tumor or tumor stem cells from other brain regions to tumor bed. VEGFs are well-known mitogens and can be up-regulated after RT. Here, we examine the effect of irradiation-induced VEGF on glioma cell motility. Materials and methods U251 and LN18 cell lines were used to generate irradiated-conditioned medium (IR-CM. At 72 h after irradiation, the supernatants were harvested. VEGF level in IR-CM was quantified by ELISA, and expression levels for VEGF mRNA were detected by RT-PCR. In vitro cancer cell motility was measured in chambers coated with/without Matrigel and IR-CM as a cell motility enhancer and a VEGF antibody as a neutralizer of VEGF bioactivity. Immunoblots were performed to evaluate the activity of cell motility-related kinases. Proliferation of GBM cells after treatment was measured by flow cytometry. Results Irradiation increased the level of VEGF mRNA that was mitigated by pre-RT exposure to Actinomycin D. U251 glioma cell motility (migration and invasion was enhanced by adding IR-CM to un-irradiated cells (174.9 ± 11.4% and 334.2 ± 46% of control, respectively. When we added VEGF antibody to IR-CM, this enhanced cell motility was negated (110.3 ± 12.0% and 105.7 ± 14.0% of control, respectively. Immunoblot analysis revealed that IR-CM increased phosphorylation of VEGF receptor-2 (VEGFR2 secondary to an increase in VEGF, with a concomitant increase of phosphorylation of the downstream targets (Src and FAK. Increased phosphorylation was mitigated by adding VEGF antibody to IR-CM. There was no difference in the mitotic index of

  8. Kinase-Independent Mechanisms of Resistance of Leukemia Stem Cells to Tyrosine Kinase Inhibitors

    Science.gov (United States)

    2014-01-01

    Summary Tyrosine kinase inhibitors such as imatinib mesylate have changed the clinical course of chronic myeloid leukemia; however, the observation that these inhibitors do not target the leukemia stem cell implies that patients need to maintain lifelong therapy. The mechanism of this phenomenon is unclear: the question of whether tyrosine kinase inhibitors are inactive inside leukemia stem cells or whether leukemia stem cells do not require breakpoint cluster region (Bcr)-Abl signaling is currently under debate. Herein, I propose an alternative model: perhaps the leukemia stem cell requires Bcr-Abl, but is dependent on its kinase-independent functions. Kinases such as epidermal growth factor receptor and Janus kinase 2 possess kinase-independent roles in regulation of gene expression; it is worth investigating whether Bcr-Abl has similar functions. Mechanistically, Bcr-Abl is able to activate the Ras, phosphatidylinositol 3-kinase/Akt, and/or the Src-kinase Hck/Stat5 pathways in a scaffolding-dependent manner. Whereas the scaffolding activity of Bcr-Abl with Grb2 is dependent on autophosphorylation, kinases such as Hck can use Bcr-Abl as substrate, inducing phosphorylation of Y177 to enable scaffolding ability in the absence of Bcr-Abl catalytic activity. It is worth investigating whether leukemia stem cells exclusively express kinases that are able to use Bcr-Abl as substrate. A kinase-independent role for Bcr-Abl in leukemia stem cells would imply that drugs that target Bcr-Abl’s scaffolding ability or its DNA-binding ability should be used in conjunction with current therapeutic regimens to increase their efficacy and eradicate the stem cells of chronic myeloid leukemia PMID:24598782

  9. Validation of a Grid Independent Spray Model and Fuel Chemistry Mechanism for Low Temperature Diesel Combustion

    Directory of Open Access Journals (Sweden)

    Takeshi Yoshikawa

    2009-09-01

    Full Text Available Spray and combustion submodels used in a Computational Fluid Dynamics (CFD code, KIVACHEMKIN, were validated for Low Temperature Combustion (LTC in a diesel engine by comparing measured and model predicted fuel spray penetrations, and in-cylinder distributions of OH and soot. The conditions considered were long ignition delay, early and late fuel injection cases. It was found that use of a grid independent spray model, called the GASJET model, with an improved n-heptane chemistry mechanism can well predict the heat release rate, not only of the main combustion stage, but also of the cool flame stage. Additionally, the GASJET model appropriately predicts the distributions of OH and soot in the cylinder even when the resolution of the computational mesh is decreased by half, which significantly reduces the required computational time.

  10. Research on Shifting Control Method of Positive Independent Mechanical Split Path Transmission for the Starting Gear

    Directory of Open Access Journals (Sweden)

    JunQiang Xi

    2013-01-01

    Full Text Available To realize a smooth and quick shift of the positive independent mechanical split path transmission (PIMSPT equipped with automatic shifting control system (ASCS, the research on the feasibility of improving shift quality by dynamic and cooperative controlling engine, steering clutches, and brakes has been conducted. The shifting control method suited to starting gear of PIMSPT has been proposed. The control method is based on control parameters, such as the driving shaft speed and its derivative. The control laws of steering clutches and brakes are presented during each gear and stage of shifting. Bench and road test results show that the proposed shifting control method can not only shorten the shift time, but also decrease the jerk of shifting effectively.

  11. Dot1 binding induces chromatin rearrangements by histone methylation-dependent and -independent mechanisms

    Directory of Open Access Journals (Sweden)

    Stulemeijer Iris JE

    2011-02-01

    Full Text Available Abstract Background Methylation of histone H3 lysine 79 (H3K79 by Dot1 is highly conserved among species and has been associated with both gene repression and activation. To eliminate indirect effects and examine the direct consequences of Dot1 binding and H3K79 methylation, we investigated the effects of targeting Dot1 to different positions in the yeast genome. Results Targeting Dot1 did not activate transcription at a euchromatic locus. However, chromatin-bound Dot1 derepressed heterochromatin-mediated gene silencing over a considerable distance. Unexpectedly, Dot1-mediated derepression was established by both a H3K79 methylation-dependent and a methylation-independent mechanism; the latter required the histone acetyltransferase Gcn5. By monitoring the localization of a fluorescently tagged telomere in living cells, we found that the targeting of Dot1, but not its methylation activity, led to the release of a telomere from the repressive environment at the nuclear periphery. This probably contributes to the activity-independent derepression effect of Dot1. Conclusions Targeting of Dot1 promoted gene expression by antagonizing gene repression through both histone methylation and chromatin relocalization. Our findings show that binding of Dot1 to chromatin can positively affect local gene expression by chromatin rearrangements over a considerable distance.

  12. Molecular mechanism of metal-independent decomposition of lipid hydroperoxide 13-HPODE by halogenated quinoid carcinogens.

    Science.gov (United States)

    Qin, Hao; Huang, Chun-Hua; Mao, Li; Xia, Hai-Ying; Kalyanaraman, Balaraman; Shao, Jie; Shan, Guo-Qiang; Zhu, Ben-Zhan

    2013-10-01

    Halogenated quinones are a class of carcinogenic intermediates and newly identified chlorination disinfection by-products in drinking water. 13-Hydroperoxy-9,11-octadecadienoic acid (13-HPODE) is the most extensively studied endogenous lipid hydroperoxide. Although it is well known that the decomposition of 13-HPODE can be catalyzed by transition metal ions, it is not clear whether halogenated quinones could enhance its decomposition independent of metal ions and, if so, what the unique characteristics and similarities are. Here we show that 2,5-dichloro-1,4-benzoquinone (DCBQ) could markedly enhance the decomposition of 13-HPODE and formation of reactive lipid alkyl radicals such as pentyl and 7-carboxyheptyl radicals, and the genotoxic 4-hydroxy-2-nonenal (HNE), through the complementary application of ESR spin trapping, HPLC-MS, and GC-MS methods. Interestingly, two chloroquinone-lipid alkoxyl conjugates were also detected and identified from the reaction between DCBQ and 13-HPODE. Analogous results were observed with other halogenated quinones. This represents the first report that halogenated quinoid carcinogens can enhance the decomposition of the endogenous lipid hydroperoxide 13-HPODE and formation of reactive lipid alkyl radicals and genotoxic HNE via a novel metal-independent nucleophilic substitution coupled with homolytic decomposition mechanism, which may partly explain their potential genotoxicity and carcinogenicity. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Contraction induced secretion of VEGF from skeletal muscle cells is mediated by adenosine

    DEFF Research Database (Denmark)

    Høier, Birgitte; Olsen, Karina; Nyberg, Michael Permin

    2010-01-01

    The role of adenosine and contraction for secretion of VEGF in skeletal muscle was investigated in human subjects and rat primary skeletal muscle cells. Microdialysis probes were inserted into the thigh muscle of seven male subjects and dialysate was collected at rest, during infusion of adenosine...... and during knee extensor exercise. The dialysate was analyzed for content of VEGF protein and adenosine. The mechanism of VEGF secretion from muscle cells in culture was examined in resting and electro stimulated cells, and in response to the adenosine analogue NECA, and the adenosine A(2A) receptor specific...... infusion enhanced (Pmuscle cells, NECA...

  14. Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A

    Science.gov (United States)

    Boo, Yong Chool; Sorescu, George; Boyd, Nolan; Shiojima, Ichiro; Walsh, Kenneth; Du, Jie; Jo, Hanjoong

    2002-01-01

    Recently, we have shown that shear stress stimulates NO(*) production by the protein kinase B/Akt (Akt)-dependent mechanisms in bovine aortic endothelial cells (BAEC) (Go, Y. M., Boo, Y. C., Park, H., Maland, M. C., Patel, R., Pritchard, K. A., Jr., Fujio, Y., Walsh, K., Darley-Usmar, V., and Jo, H. (2001) J. Appl. Physiol. 91, 1574-1581). Akt has been believed to regulate shear-dependent production of NO(*) by directly phosphorylating endothelial nitric-oxide synthase (eNOS) at the Ser(1179) residue (eNOS-S(1179)), but a critical evaluation using specific inhibitors or dominant negative mutants (Akt(AA) or Akt(AAA)) has not been reported. In addition, other kinases, including protein kinase A (PKA) and AMP kinase have also shown to phosphorylate eNOS-S(1179). Here, we show that shear-dependent phosphorylation of eNOS-S(1179) is mediated by an Akt-independent, but a PKA-dependent, mechanism. Expression of Akt(AA) or Akt(AAA) in BAEC by using recombinant adenoviral constructs inhibited phosphorylation of eNOS-S(1179) if cells were stimulated by vascular endothelial growth factor (VEGF), but not by shear stress. As shown before, expression of Akt(AA) inhibited shear-dependent NO(*) production, suggesting that Akt is still an important regulator in NO production. Further studies showed that a selective inhibitor of PKA, H89, inhibited shear-dependent phosphorylation of eNOS-S(1179) and NO(*) production. In contrast, H89 did not inhibit phosphorylation of eNOS-S(1179) induced by expressing a constitutively active Akt mutant (Akt(Myr)) in BAEC, showing that the inhibitor did not affect the Akt pathway. 8-Bromo-cAMP alone phosphorylated eNOS-S(1179) within 5 min without activating Akt, in an H89-sensitive manner. Collectively, these results demonstrate that shear stimulates phosphorylation of eNOS-S(1179) in a PKA-dependent, but Aktindependent manner, whereas the NO(*) production is regulated by the mechanisms dependent on both PKA and Akt. A coordinated interaction

  15. A new experiment-independent mechanism to persistify and serve the detector geometry of ATLAS

    Science.gov (United States)

    Bianchi, Riccardo Maria; Boudreau, Joseph; Vukotic, Ilija

    2017-10-01

    The complex geometry of the whole detector of the ATLAS experiment at LHC is currently stored only in custom online databases, from which it is built on-the-fly on request. Accessing the online geometry guarantees accessing the latest version of the detector description, but requires the setup of the full ATLAS software framework “Athena”, which provides the online services and the tools to retrieve the data from the database. This operation is cumbersome and slows down the applications that need to access the geometry. Moreover, all applications that need to access the detector geometry need to be built and run on the same platform as the ATLAS framework, preventing the usage of the actual detector geometry in stand-alone applications. Here we propose a new mechanism to persistify (in software development in general, and in HEP computing in particular, persistifying means taking an object which lives in memory only - for example because it was built on-the-fly while processing the experimental data, - serializing it and storing it on disk as a persistent object) and serve the geometry of HEP experiments. The new mechanism is composed by a new file format and the modules to make use of it. The new file format allows to store the whole detector description locally in a file, and it is especially optimized to describe large complex detectors with the minimum file size, making use of shared instances and storing compressed representations of geometry transformations. Then, the detector description can be read back in, to fully restore the in-memory geometry tree. Moreover, a dedicated REST API is being designed and developed to serve the geometry in standard exchange formats like JSON, to let users and applications download specific partial geometry information. With this new geometry persistification a new generation of applications could be developed, which can use the actual detector geometry while being platform-independent and experiment-independent.

  16. Posture-independent piston valve: a novel valve mechanism that actuates based on intracranial pressure alone.

    Science.gov (United States)

    Medow, Joshua E; Luzzio, Christopher C

    2012-01-01

    Shunt valves are intended to maintain physiological intracranial pressure (ICP). A variety of mechanisms have been designed to accomplish this goal but have had limited success. Siphoning, in particular, has been a problem not effectively solved by proposed or manufactured valves. Poor control of ICP results in headache, neurological disturbances, decreased cognition, shunt malfunction, slit ventricles, subdural hematomas, decreased cranial volume, and maldevelopment. The authors of this study describe a prototype valve that was machined and tested ex vivo and that actuates based on ICP alone regardless of the presence of a siphon. Their object was to determine if a novel shunt mechanism that actuates perpendicular to the flow of fluid would eliminate the effect of siphoning in a valve for the treatment of hydrocephalus. A posture-independent piston valve (PIPV) was anchored to a graduated reservoir. Opening pressure was measured by noting the fluid level in the reservoir when the piston moved. Measurements were made using a 90-cm and a 120-cm water-filled siphon tube (1.3-mm standard distal catheter) to simulate an upright posture. A recumbent posture was simulated by the absence of a siphon. Opening pressure of the valve did not change regardless of the presence or absence of a water-filled siphon. The PIPV was triggered only by the pressure head at the inlet and did not actuate in the presence of a siphon, demonstrating proof of principle of the perpendicular actuating mechanism. The PIPV is a purely mechanical device that has practical application in the treatment of hydrocephalus.

  17. VEGF(121)b, a new member of the VEGF(xxx)b family of VEGF-A splice isoforms, inhibits neovascularisation and tumour growth in vivo.

    Science.gov (United States)

    Rennel, E S; Varey, A H R; Churchill, A J; Wheatley, E R; Stewart, L; Mather, S; Bates, D O; Harper, S J

    2009-10-06

    The key mediator of new vessel formation in cancer and other diseases is VEGF-A. VEGF-A exists as alternatively spliced isoforms - the pro-angiogenic VEGF(xxx) family generated by exon 8 proximal splicing, and a sister family, termed VEGF(xxx)b, exemplified by VEGF(165)b, generated by distal splicing of exon 8. However, it is unknown whether this anti-angiogenic property of VEGF(165)b is a general property of the VEGF(xxx)b family of isoforms. The mRNA and protein expression of VEGF(121)b was studied in human tissue. The effect of VEGF(121)b was analysed by saturation binding to VEGF receptors, endothelial migration, apoptosis, xenograft tumour growth, pre-retinal neovascularisation and imaging of biodistribution in tumour-bearing mice with radioactive VEGF(121)b. The existence of VEGF(121)b was confirmed in normal human tissues. VEGF(121)b binds both VEGF receptors with similar affinity as other VEGF isoforms, but inhibits endothelial cell migration and is cytoprotective to endothelial cells through VEGFR-2 activation. Administration of VEGF(121)b normalised retinal vasculature by reducing both angiogenesis and ischaemia. VEGF(121)b reduced the growth of xenografted human colon tumours in association with reduced microvascular density, and an intravenous bolus of VEGF(121)b is taken up into colon tumour xenografts. Here we identify a second member of the family, VEGF(121)b, with similar properties to those of VEGF(165)b, and underline the importance of the six amino acids of exon 8b in the anti-angiogenic activity of the VEGF(xxx)b isoforms.

  18. Using Anti-VEGF in Diabetic Retinopathy

    OpenAIRE

    Marashi, Ameen

    2016-01-01

    Vascular endothelium growth factor is the main pathological factor in diabetic retinopathy and diabetic macular edema (DME), Anti-VEGF agents are safe and effective in DME treatment, there are multiple Anti-VEGF agents, choosing between them is essential to individualize treatment for each patient to achieve the optimum results.

  19. Akt-dependent and independent mechanisms of mTOR regulation in cancer

    Science.gov (United States)

    Memmott, Regan M.; Dennis, Phillip A.

    2009-01-01

    The protein kinase mTOR (mammalian target of rapamycin) is a critical regulator of cellular metabolism, growth, and proliferation. These processes contribute to tumor formation, and many cancers are characterized by aberrant activation of mTOR. Although activating mutations in mTOR itself have not been identified, deregulation of upstream components that regulate mTOR are prevalent in cancer. The prototypic mechanism of mTOR regulation in cells is through activation of the PI3K/Akt pathway, but mTOR receives input from multiple signaling pathways. This review will discuss Akt-dependent and independent mechanisms of mTOR regulation in response to mitogenic signals, as well as its regulation in response to energy and nutrient-sensing pathways. Preclinical and clinical studies have demonstrated that tumors bearing genetic alterations that activate mTOR are sensitive to pharmacologic inhibition of mTOR. Elucidation of novel pathways that regulate mTOR may help identify predictive factors for sensitivity to mTOR inhibitors and could provide new therapeutic targets for inhibiting the mTOR pathway in cancer. This review will also highlight pharmacologic approaches that inhibit mTOR via activation of the AMP-activated protein kinase (AMPK), an important inhibitor of the mTOR pathway and an emerging target in cancer. PMID:19166931

  20. Right occipital cortex suppresses male rat testosterone secretion by a pituitary-independent mechanism.

    Science.gov (United States)

    Lepore, Gianluca; Zedda, Marco; Gerendai, Ida; Csernus, Valér; Gadau, Sergio; Manca, Paolo; Chisu, Valentina; Farina, Vittorio

    2006-08-01

    In addition to being regulated by the hypothalamo-hypophyseal system, testosterone (T) secretion is influenced by a number of less understood mechanisms. The aim of the present study was to examine whether defined areas of the right cerebral cortex could modulate T production. In adult male Wistar rats right frontal or occipital decortication, anterior or posterior callosotomy and corresponding sham-operations were performed. After 7-day survival time, T secretion in vitro, serum T and LH concentrations were measured by RIA. Right occipital decortication and posterior callosotomy resulted in an increase in T secretion in vitro when compared to the corresponding sham-operated controls. In contrast, right frontal decortication or anterior callosotomy did not interfere with steroidogenesis. Serum LH concentration was not altered by any interventions. The right occipital but not the right frontal cortex is involved in the control of T secretion. The caudal part of the corpus callosum accommodating the fibers originating from the occipital cortex might have a similar function. The fact that LH remained unchanged in all experimental groups suggests that the right occipital cortex and the caudal part of the corpus callosum influence testicular steroidogenesis by a pituitary-independent mechanism.

  1. Polyphenols modulate calcium-independent mechanisms in human arterial tissue-engineered vascular media.

    Science.gov (United States)

    Diebolt, Myriam; Laflamme, Karina; Labbé, Raymond; Auger, François A; Germain, Lucie; Andriantsitohaina, Ramaroson

    2007-10-01

    In the present study, an arterial tissue-engineered vascular media (TEVM) was produced from cultured human smooth muscle cells of the umbilical artery and we took advantage of this model to evaluate the regulation of contraction and the signalling pathways of polyphenols in arteries. Cultured human smooth muscle cells of the umbilical artery were used to produce arterial TEVMs. Contraction experiments were performed to determine intracellular targets involved in the modulation of contraction by polyphenols extract from red wine, Provinols (SEPPIC Groupe Air Liquide, Paris, France). Smooth muscle cells in arterial TEVM displayed a differentiated phenotype as demonstrated by the expression of alpha-smooth muscle actin, a vascular smooth muscle-specific marker, and tissue contraction in response to vasoconstrictor and vasodilator agents. Contractions caused by histamine were associated with an increase in [Ca(2+)](i) and a Ca(2+)-independent signalling pathway. The latter pathway involved mechanisms sensitive to protein kinase C, myosin light chain kinase, and Rho-associated protein kinase inhibitors. The regulation of contraction induced by Provinols shows that treatment of arterial TEVM with this compound significantly decreased histamine-induced contraction. This effect was associated with the inhibition of the Rho-associated protein kinase pathway and the decrease in alpha-smooth muscle actin expression. The use of arterial TEVM, brings new insights into the mechanisms by which polyphenols regulate vascular contraction in the human artery.

  2. Potential mechanism for pentachlorophenol-induced carcinogenicity: a novel mechanism for metal-independent production of hydroxyl radicals.

    Science.gov (United States)

    Zhu, Ben-Zhan; Shan, Guo-Qiang

    2009-06-01

    The hydroxyl radical ((*)OH) has been considered to be one of the most reactive oxygen species produced in biological systems. It has been shown that (*)OH can cause DNA, protein, and lipid oxidation. One of the most widely accepted mechanisms for (*)OH production is through the transition metal-catalyzed Fenton reaction. Pentachlorophenol (PCP) was one of the most widely used biocides, primarily for wood preservation. PCP is now ubiquitously present in our environment and even found in people who are not occupationally exposed to it. PCP has been listed as a priority pollutant by the U.S. Environmental Protection Agency (EPA) and classified as a group 2B environmental carcinogen by the International Association for Research on Cancer (IARC). The genotoxicity of PCP has been attributed to its two major quinoid metabolites: tetrachlorohydroquinone and tetrachloro-1,4-benzoquinone (TCBQ). Although the redox cycling of PCP quinoid metabolites to generate reactive oxygen species is believed to play an important role, the exact molecular mechanism underlying PCP genotoxicity is not clear. Using the salicylate hydroxylation assay and electron spin resonance (ESR) secondary spin-trapping methods, we found that (*)OH can be produced by TCBQ and H(2)O(2) independent of transition metal ions. Further studies showed that TCBQ, but not its corresponding semiquinone radical, the tetrachlorosemiquinone radical (TCSQ(*)), is essential for (*)OH production. The major reaction product between TCBQ and H(2)O(2) was identified to be trichloro-hydroxy-1,4-benzoquinone (TrCBQ-OH), and H(2)O(2) was found to be the source and origin of the oxygen atom inserted into this reaction product. On the basis of these data, we propose that (*)OH production by TCBQ and H(2)O(2) is not through a semiquinone-dependent organic Fenton reaction but rather through the following novel mechanism: a nucleophilic attack of H(2)O(2) to TCBQ, leading to the formation of an unstable trichloro-hydroperoxyl-1

  3. CCL5 promotes VEGF-C production and induces lymphangiogenesis by suppressing miR-507 in human chondrosarcoma cells.

    Science.gov (United States)

    Wang, Li-Hong; Lin, Chih-Yang; Liu, Shih-Chia; Liu, Guan-Ting; Chen, Yen-Ling; Chen, Jih-Jung; Chan, Chia-Han; Lin, Ting-Yi; Chen, Chi-Kuan; Xu, Guo-Hong; Chen, Shiou-Sheng; Tang, Chih-Hsin; Wang, Shih-Wei

    2016-06-14

    Chondrosarcoma is the second most frequently occurring type of bone malignancy that is characterized by the distant metastasis propensity. Vascular endothelial growth factor-C (VEGF-C) is the major lymphangiogenic factor, and makes crucial contributions to tumor lymphangiogenesis and lymphatic metastasis. Chemokine CCL5 has been reported to facilitate angiogenesis and metastasis in chondrosarcoma. However, the effect of chemokine CCL5 on VEGF-C regulation and lymphangiogenesis in chondrosarcoma has largely remained a mystery. In this study, we showed a clinical correlation between CCL5 and VEGF-C as well as tumor stage in human chondrosarcoma tissues. We further demonstrated that CCL5 promoted VEGF-C expression and secretion in human chondrosarcoma cells. The conditioned medium (CM) from CCL5-overexpressed cells significantly induced tube formation of human lymphatic endothelial cells (LECs). Mechanistic investigations showed that CCL5 activated VEGF-C-dependent lymphangiogenesis by down-regulating miR-507. Moreover, inhibiting CCL5 dramatically reduced VEGF-C and lymphangiogenesis in the chondrosarcoma xenograft animal model. Collectively, we document for the first time that CCL5 induces tumor lymphangiogenesis by the induction of VEGF-C in human cancer cells. Our present study reveals miR-507/VEGF-C signaling as a novel mechanism in CCL5-mediated tumor lymphangiogenesis. Targeting both CCL5 and VEGF-C pathways might serve as the potential therapeutic strategy to block cancer progression and metastasis in chondrosarcoma.

  4. VEGF-A isoform-specific regulation of calcium ion flux, transcriptional activation and endothelial cell migration

    Directory of Open Access Journals (Sweden)

    Gareth W. Fearnley

    2015-07-01

    Full Text Available Vascular endothelial growth factor A (VEGF-A regulates many aspects of vascular physiology such as cell migration, proliferation, tubulogenesis and cell-cell interactions. Numerous isoforms of VEGF-A exist but their physiological significance is unclear. Here we evaluated two different VEGF-A isoforms and discovered differential regulation of cytosolic calcium ion flux, transcription factor localisation and endothelial cell response. Analysis of VEGF-A isoform-specific stimulation of VEGFR2-dependent signal transduction revealed differential capabilities for isoform activation of multiple signal transduction pathways. VEGF-A165 treatment promoted increased phospholipase Cγ1 phosphorylation, which was proportional to the subsequent rise in cytosolic calcium ions, in comparison to cells treated with VEGF-A121. A major consequence of this VEGF-A isoform-specific calcium ion flux in endothelial cells is differential dephosphorylation and subsequent nuclear translocation of the transcription factor NFATc2. Using reverse genetics, we discovered that NFATc2 is functionally required for VEGF-A-stimulated endothelial cell migration but not tubulogenesis. This work presents a new mechanism for understanding how VEGF-A isoforms program complex cellular outputs by converting signal transduction pathways into transcription factor redistribution to the nucleus, as well as defining a novel role for NFATc2 in regulating the endothelial cell response.

  5. Telomerase inhibition targets clonogenic multiple myeloma cells through telomere length-dependent and independent mechanisms.

    Directory of Open Access Journals (Sweden)

    Sarah K Brennan

    2010-09-01

    Full Text Available Plasma cells constitute the majority of tumor cells in multiple myeloma (MM but lack the potential for sustained clonogenic growth. In contrast, clonotypic B cells can engraft and recapitulate disease in immunodeficient mice suggesting they serve as the MM cancer stem cell (CSC. These tumor initiating B cells also share functional features with normal stem cells such as drug resistance and self-renewal potential. Therefore, the cellular processes that regulate normal stem cells may serve as therapeutic targets in MM. Telomerase activity is required for the maintenance of normal adult stem cells, and we examined the activity of the telomerase inhibitor imetelstat against MM CSC. Moreover, we carried out both long and short-term inhibition studies to examine telomere length-dependent and independent activities.Human MM CSC were isolated from cell lines and primary clinical specimens and treated with imetelstat, a specific inhibitor of the reverse transcriptase activity of telomerase. Two weeks of exposure to imetelstat resulted in a significant reduction in telomere length and the inhibition of clonogenic MM growth both in vitro and in vivo. In addition to these relatively long-term effects, 72 hours of imetelstat treatment inhibited clonogenic growth that was associated with MM CSC differentiation based on expression of the plasma cell antigen CD138 and the stem cell marker aldehyde dehydrogenase. Short-term treatment of MM CSC also decreased the expression of genes typically expressed by stem cells (OCT3/4, SOX2, NANOG, and BMI1 as revealed by quantitative real-time PCR.Telomerase activity regulates the clonogenic growth of MM CSC. Moreover, reductions in MM growth following both long and short-term telomerase inhibition suggest that it impacts CSC through telomere length-dependent and independent mechanisms.

  6. A compartment model of VEGF distribution in humans in the presence of soluble VEGF receptor-1 acting as a ligand trap.

    Directory of Open Access Journals (Sweden)

    Florence T H Wu

    Full Text Available Vascular endothelial growth factor (VEGF, through its activation of cell surface receptor tyrosine kinases including VEGFR1 and VEGFR2, is a vital regulator of stimulatory and inhibitory processes that keep angiogenesis--new capillary growth from existing microvasculature--at a dynamic balance in normal physiology. Soluble VEGF receptor-1 (sVEGFR1--a naturally-occurring truncated version of VEGFR1 lacking the transmembrane and intracellular signaling domains--has been postulated to exert inhibitory effects on angiogenic signaling via two mechanisms: direct sequestration of angiogenic ligands such as VEGF; or dominant-negative heterodimerization with surface VEGFRs. In pre-clinical studies, sVEGFR1 gene and protein therapy have demonstrated efficacy in inhibiting tumor angiogenesis; while in clinical studies, sVEGFR1 has shown utility as a diagnostic or prognostic marker in a widening array of angiogenesis-dependent diseases. Here we developed a novel computational multi-tissue model for recapitulating the dynamic systemic distributions of VEGF and sVEGFR1. Model features included: physiologically-based multi-scale compartmentalization of the human body; inter-compartmental macromolecular biotransport processes (vascular permeability, lymphatic drainage; and molecularly-detailed binding interactions between the ligand isoforms VEGF(121 and VEGF(165, signaling receptors VEGFR1 and VEGFR2, non-signaling co-receptor neuropilin-1 (NRP1, as well as sVEGFR1. The model was parameterized to represent a healthy human subject, whereupon we investigated the effects of sVEGFR1 on the distribution and activation of VEGF ligands and receptors. We assessed the healthy baseline stability of circulating VEGF and sVEGFR1 levels in plasma, as well as their reliability in indicating tissue-level angiogenic signaling potential. Unexpectedly, simulated results showed that sVEGFR1 - acting as a diffusible VEGF sink alone, i.e., without sVEGFR1-VEGFR heterodimerization

  7. M-CSF signals through the MAPK/ERK pathway via Sp1 to induce VEGF production and induces angiogenesis in vivo.

    Directory of Open Access Journals (Sweden)

    Jennifer M Curry

    Full Text Available BACKGROUND: M-CSF recruits mononuclear phagocytes which regulate processes such as angiogenesis and metastases in tumors. VEGF is a potent activator of angiogenesis as it promotes endothelial cell proliferation and new blood vessel formation. Previously, we reported that in vitro M-CSF induces the expression of biologically-active VEGF from human monocytes. METHODOLOGY AND RESULTS: In this study, we demonstrate the molecular mechanism of M-CSF-induced VEGF production. Using a construct containing the VEGF promoter linked to a luciferase reporter, we found that a mutation reducing HIF binding to the VEGF promoter had no significant effect on luciferase production induced by M-CSF stimulation. Further analysis revealed that M-CSF induced VEGF through the MAPK/ERK signaling pathway via the transcription factor, Sp1. Thus, inhibition of either ERK or Sp1 suppressed M-CSF-induced VEGF at the mRNA and protein level. M-CSF also induced the nuclear localization of Sp1, which was blocked by ERK inhibition. Finally, mutating the Sp1 binding sites within the VEGF promoter or inhibiting ERK decreased VEGF promoter activity in M-CSF-treated human monocytes. To evaluate the biological significance of M-CSF induced VEGF production, we used an in vivo angiogenesis model to illustrate the ability of M-CSF to recruit mononuclear phagocytes, increase VEGF levels, and enhance angiogenesis. Importantly, the addition of a neutralizing VEGF antibody abolished M-CSF-induced blood vessel formation. CONCLUSION: These data delineate an ERK- and Sp1-dependent mechanism of M-CSF induced VEGF production and demonstrate for the first time the ability of M-CSF to induce angiogenesis via VEGF in vivo.

  8. Leukemia inhibitory factor protects axons in experimental autoimmune encephalomyelitis via an oligodendrocyte-independent mechanism.

    Directory of Open Access Journals (Sweden)

    Melissa M Gresle

    Full Text Available Leukemia inhibitory factor (LIF and Ciliary Neurotrophic factor (CNTF are members of the interleukin-6 family of cytokines, defined by use of the gp130 molecule as an obligate receptor. In the murine experimental autoimmune encephalomyelitis (EAE model, antagonism of LIF and genetic deletion of CNTF worsen disease. The potential mechanism of action of these cytokines in EAE is complex, as gp130 is expressed by all neural cells, and could involve immuno-modulation, reduction of oligodendrocyte injury, neuronal protection, or a combination of these actions. In this study we aim to investigate whether the beneficial effects of CNTF/LIF signalling in EAE are associated with axonal protection; and whether this requires signalling through oligodendrocytes. We induced MOG₃₅₋₅₅ EAE in CNTF, LIF and double knockout mice. On a CNTF null background, LIF knockout was associated with increased EAE severity (EAE grade 2.1±0.14 vs 2.6±0.19; P<0.05. These mice also showed increased axonal damage relative to LIF heterozygous mice, as indicated by decreased optic nerve parallel diffusivity on MRI (1540±207 µm²-/s vs 1310±175 µm²-/s; P<0.05, and optic nerve (-12.5% and spinal cord (-16% axon densities; and increased serum neurofilament-H levels (2.5 fold increase. No differences in inflammatory cell numbers or peripheral auto-immune T-cell priming were evident. Oligodendrocyte-targeted gp130 knockout mice showed that disruption of CNTF/LIF signalling in these cells has no effect on acute EAE severity. These studies demonstrate that endogenous CNTF and LIF act centrally to protect axons from acute inflammatory destruction via an oligodendrocyte-independent mechanism.

  9. The Stapled AKAP Disruptor Peptide STAD-2 Displays Antimalarial Activity through a PKA-Independent Mechanism.

    Directory of Open Access Journals (Sweden)

    Briana R Flaherty

    Full Text Available Drug resistance poses a significant threat to ongoing malaria control efforts. Coupled with lack of a malaria vaccine, there is an urgent need for the development of new antimalarials with novel mechanisms of action and low susceptibility to parasite drug resistance. Protein Kinase A (PKA has been implicated as a critical regulator of pathogenesis in malaria. Therefore, we sought to investigate the effects of disrupted PKA signaling as a possible strategy for inhibition of parasite replication. Host PKA activity is partly regulated by a class of proteins called A Kinase Anchoring Proteins (AKAPs, and interaction between HsPKA and AKAP can be inhibited by the stapled peptide Stapled AKAP Disruptor 2 (STAD-2. STAD-2 was tested for permeability to and activity against Plasmodium falciparum blood stage parasites in vitro. The compound was selectively permeable only to infected red blood cells (iRBC and demonstrated rapid antiplasmodial activity, possibly via iRBC lysis (IC50 ≈ 1 μM. STAD-2 localized within the parasite almost immediately post-treatment but showed no evidence of direct association with PKA, indicating that STAD-2 acts via a PKA-independent mechanism. Furosemide-insensitive parasite permeability pathways in the iRBC were largely responsible for uptake of STAD-2. Further, peptide import was highly specific to STAD-2 as evidenced by low permeability of control stapled peptides. Selective uptake and antiplasmodial activity of STAD-2 provides important groundwork for the development of stapled peptides as potential antimalarials. Such peptides may also offer an alternative strategy for studying protein-protein interactions critical to parasite development and pathogenesis.

  10. Extension of Drosophila lifespan by Rhodiola rosea through a mechanism independent from dietary restriction.

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    Schriner, Samuel E; Lee, Kevin; Truong, Stephanie; Salvadora, Kathyrn T; Maler, Steven; Nam, Alexander; Lee, Thomas; Jafari, Mahtab

    2013-01-01

    Rhodiola rosea has been extensively used to improve physical and mental performance and to protect against stress. We, and others, have reported that R. rosea can extend lifespan in flies, worms, and yeast. However, its molecular mechanism is currently unknown. Here, we tested whether R. rosea might act through a pathway related to dietary restriction (DR) that can extend lifespan in a range of model organisms. While the mechanism of DR itself is also unknown, three molecular pathways have been associated with it: the silent information regulator 2 (SIR2) proteins, insulin and insulin-like growth factor signaling (IIS), and the target of rapamycin (TOR). In flies, DR is implemented through a reduction in dietary yeast content. We found that R. rosea extract extended lifespan in both sexes independent of the yeast content in the diet. We also found that the extract extended lifespan when the SIR2, IIS, or TOR pathways were genetically perturbed. Upon examination of water and fat content, we found that R. rosea decreased water content and elevated fat content in both sexes, but did not sensitize flies to desiccation or protect them against starvation. There were some sex-specific differences in response to R. rosea. In female flies, the expression levels of glycolytic genes and dSir2 were down-regulated, and NADH levels were decreased. In males however, R. rosea provided no protection against heat stress and had no effect on the major heat shock protein HSP70 and actually down-regulated the mitochondrial HSP22. Our findings largely rule out an elevated general resistance to stress and DR-related pathways as mechanistic candidates. The latter conclusion is especially relevant given the limited potential for DR to improve human health and lifespan, and presents R. rosea as a potential viable candidate to treat aging and age-related diseases in humans.

  11. Extension of Drosophila lifespan by Rhodiola rosea through a mechanism independent from dietary restriction.

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    Samuel E Schriner

    Full Text Available Rhodiola rosea has been extensively used to improve physical and mental performance and to protect against stress. We, and others, have reported that R. rosea can extend lifespan in flies, worms, and yeast. However, its molecular mechanism is currently unknown. Here, we tested whether R. rosea might act through a pathway related to dietary restriction (DR that can extend lifespan in a range of model organisms. While the mechanism of DR itself is also unknown, three molecular pathways have been associated with it: the silent information regulator 2 (SIR2 proteins, insulin and insulin-like growth factor signaling (IIS, and the target of rapamycin (TOR. In flies, DR is implemented through a reduction in dietary yeast content. We found that R. rosea extract extended lifespan in both sexes independent of the yeast content in the diet. We also found that the extract extended lifespan when the SIR2, IIS, or TOR pathways were genetically perturbed. Upon examination of water and fat content, we found that R. rosea decreased water content and elevated fat content in both sexes, but did not sensitize flies to desiccation or protect them against starvation. There were some sex-specific differences in response to R. rosea. In female flies, the expression levels of glycolytic genes and dSir2 were down-regulated, and NADH levels were decreased. In males however, R. rosea provided no protection against heat stress and had no effect on the major heat shock protein HSP70 and actually down-regulated the mitochondrial HSP22. Our findings largely rule out an elevated general resistance to stress and DR-related pathways as mechanistic candidates. The latter conclusion is especially relevant given the limited potential for DR to improve human health and lifespan, and presents R. rosea as a potential viable candidate to treat aging and age-related diseases in humans.

  12. Genetics of VEGF serum variation in human isolated populations of cilento: importance of VEGF polymorphisms.

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    Daniela Ruggiero

    Full Text Available Vascular Endothelial Growth Factor (VEGF is the main player in angiogenesis. Because of its crucial role in this process, the study of the genetic factors controlling VEGF variability may be of particular interest for many angiogenesis-associated diseases. Although some polymorphisms in the VEGF gene have been associated with a susceptibility to several disorders, no genome-wide search on VEGF serum levels has been reported so far. We carried out a genome-wide linkage analysis in three isolated populations and we detected a strong linkage between VEGF serum levels and the 6p21.1 VEGF region in all samples. A new locus on chromosome 3p26.3 significantly linked to VEGF serum levels was also detected in a combined population sample. A sequencing of the gene followed by an association study identified three common single nucleotide polymorphisms (SNPs influencing VEGF serum levels in one population (Campora, two already reported in the literature (rs3025039, rs25648 and one new signal (rs3025020. A fourth SNP (rs41282644 was found to affect VEGF serum levels in another population (Cardile. All the identified SNPs contribute to the related population linkages (35% of the linkage explained in Campora and 15% in Cardile. Interestingly, none of the SNPs influencing VEGF serum levels in one population was found to be associated in the two other populations. These results allow us to exclude the hypothesis that the common variants located in the exons, intron-exon junctions, promoter and regulative regions of the VEGF gene may have a causal effect on the VEGF variation. The data support the alternative hypothesis of a multiple rare variant model, possibly consisting in distinct variants in different populations, influencing VEGF serum levels.

  13. Chronic central administration of Ghrelin increases bone mass through a mechanism independent of appetite regulation.

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    Hyung Jin Choi

    Full Text Available Leptin plays a critical role in the central regulation of bone mass. Ghrelin counteracts leptin. In this study, we investigated the effect of chronic intracerebroventricular administration of ghrelin on bone mass in Sprague-Dawley rats (1.5 μg/day for 21 days. Rats were divided into control, ghrelin ad libitum-fed (ghrelin ad lib-fed, and ghrelin pair-fed groups. Ghrelin intracerebroventricular infusion significantly increased body weight in ghrelin ad lib-fed rats but not in ghrelin pair-fed rats, as compared with control rats. Chronic intracerebroventricular ghrelin infusion significantly increased bone mass in the ghrelin pair-fed group compared with control as indicated by increased bone volume percentage, trabecular thickness, trabecular number and volumetric bone mineral density in tibia trabecular bone. There was no significant difference in trabecular bone mass between the control group and the ghrelin ad-lib fed group. Chronic intracerebroventricular ghrelin infusion significantly increased the mineral apposition rate in the ghrelin pair-fed group as compared with control. In conclusion, chronic central administration of ghrelin increases bone mass through a mechanism that is independent of body weight, suggesting that ghrelin may have a bone anabolic effect through the central nervous system.

  14. Ddc2 mediates Mec1 activation through a Ddc1- or Dpb11-independent mechanism.

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    Amitava Bandhu

    2014-02-01

    Full Text Available The protein kinase Mec1 (ATR ortholog and its partner Ddc2 (ATRIP ortholog play a key role in DNA damage checkpoint responses in budding yeast. Previous studies have established the model in which Ddc1, a subunit of the checkpoint clamp, and Dpb11, related to TopBP1, activate Mec1 directly and control DNA damage checkpoint responses at G1 and G2/M. In this study, we show that Ddc2 contributes to Mec1 activation through a Ddc1- or Dpb11-independent mechanism. The catalytic activity of Mec1 increases after DNA damage in a Ddc2-dependent manner. In contrast, Mec1 activation occurs even in the absence of Ddc1 and Dpb11 function at G2/M. Ddc2 recruits Mec1 to sites of DNA damage. To dissect the role of Ddc2 in Mec1 activation, we isolated and characterized a separation-of-function mutation in DDC2, called ddc2-S4. The ddc2-S4 mutation does not affect Mec1 recruitment but diminishes Mec1 activation. Mec1 phosphorylates histone H2A in response to DNA damage. The ddc2-S4 mutation decreases phosphorylation of histone H2A more significantly than the absence of Ddc1 and Dpb11 function does. Our results suggest that Ddc2 plays a critical role in Mec1 activation as well as Mec1 localization at sites of DNA damage.

  15. Antibody-independent mechanisms regulate the establishment of chronic Plasmodium infection

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    Lin, Jingwen; Cunningham, Deirdre; Tumwine, Irene; Kushinga, Garikai; McLaughlin, Sarah; Spence, Philip; Böhme, Ulrike; Sanders, Mandy; Conteh, Solomon; Bushell, Ellen; Metcalf, Tom; Billker, Oliver; Duffy, Patrick E.; Newbold, Chris; Berriman, Matthew; Langhorne, Jean

    2017-01-01

    Malaria is caused by parasites of the genus Plasmodium. All human-infecting Plasmodium species can establish long-lasting chronic infections1–5, creating an infectious reservoir to sustain transmission1,6. It is widely accepted that maintenance of chronic infection involves evasion of adaptive immunity by antigenic variation7. However, genes involved in this process have been identified in only two of five human-infecting species: P. falciparum and P. knowlesi. Furthermore, little is understood about the early events in establishment of chronic infection in these species. Using a rodent model we demonstrate that only a minority of parasites from among the infecting population, expressing one of several clusters of virulence-associated pir genes, establishes a chronic infection. This process occurs in different species of parasite and in different hosts. Establishment of chronicity is independent of adaptive immunity and therefore different from the mechanism proposed for maintainance of chronic P. falciparum infections7–9. Furthermore, we show that the proportions of parasites expressing different types of pir genes regulate the time taken to establish a chronic infection. Since pir genes are common to most, if not all, species of Plasmodium10, this process may be a common way of regulating the establishment of chronic infections. PMID:28165471

  16. Sulforaphane induces oxidative stress and death by p53-independent mechanism: implication of impaired glutathione recycling.

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    José Miguel P Ferreira de Oliveira

    Full Text Available Sulforaphane (SFN is a naturally-occurring isothiocyanate best known for its role as an indirect antioxidant. Notwithstanding, in different cancer cell lines, SFN may promote the accumulation of reactive oxygen species (ROS and cause cell death e.g. by apoptosis. Osteosarcoma often becomes chemoresistant, and new molecular targets to prevent drug resistance are needed. Here, we aimed to determine the effect of SFN on ROS levels and to identify key biomarkers leading to ROS unbalance and apoptosis in the p53-null MG-63 osteosarcoma cell line. MG-63 cells were exposed to SFN for up to 48 h. At 10 μM concentration or higher, SFN decreased cell viability, increased the%early apoptotic cells and increased caspase 3 activity. At these higher doses, SFN increased ROS levels, which correlated with apoptotic endpoints and cell viability decline. In exposed cells, gene expression analysis revealed only partial induction of phase-2 detoxification genes. More importantly, SFN inhibited ROS-scavenging enzymes and impaired glutathione recycling, as evidenced by inhibition of glutathione reductase (GR activity and combined inhibition of glutathione peroxidase (GPx gene expression and enzyme activity. In conclusion, SFN induced oxidative stress and apoptosis via a p53-independent mechanism. GPx expression and activity were found associated with ROS accumulation in MG-63 cells and are potential biomarkers for the efficacy of ROS-inducing agents e.g. as co-adjuvant drugs in osteosarcoma.

  17. The vasorelaxant effect of gallic acid involves endothelium-dependent and -independent mechanisms.

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    de Oliveira, Lais Moraes; de Oliveira, Thiago Sardinha; da Costa, Rafael Menezes; de Souza Gil, Eric; Costa, Elson Alves; Passaglia, Rita de Cassia Aleixo Tostes; Filgueira, Fernando Paranaíba; Ghedini, Paulo César

    2016-06-01

    The mechanisms of action involved in the vasorelaxant effect of gallic acid (GA) were examined in the isolated rat thoracic aorta. GA exerted a relaxant effect in the highest concentrations (0.4-10mM) in both endothelium-intact and endothelium-denuded aortic rings. Pre-incubation with L-NAME, ODQ, calmidazolium, TEA, 4-aminopyridine, and barium chloride significantly reduced the pEC50 values. Moreover, this effect was not modified by indomethacin, wortmannin, PP2, glibenclamide, or paxillin. Pre-incubation of GA (1, 3, and 10mM) in a Ca(2+)-free Krebs solution attenuated CaCl2-induced contractions and blocked BAY K8644-induced vascular contractions, but it did not inhibit a contraction induced by the release of Ca(2+) from the sarcoplasmatic reticulum stores. In addition, a Western blot analysis showed that GA induces phosphorylation of eNOS in rat thoracic aorta. These results suggest that GA induces relaxation in rat aortic rings through an endothelium-dependent pathway, resulting in eNOS phosphorylation and opening potassium channels. Additionally, the relaxant effect by an endothelium-independent pathway involves the blockade of the Ca(2+) influx via L-type Ca(2+) channels. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Estrogen modulates xanthine dehydrogenase/xanthine oxidase activity by a receptor-independent mechanism.

    Science.gov (United States)

    Budhiraja, Rohit; Kayyali, Usamah S; Karamsetty, Mallik; Fogel, Michael; Hill, Nicholas S; Chalkley, Roger; Finlay, Geraldine A; Hassoun, Paul M

    2003-12-01

    Hypoxia causes up-regulation and activation of xanthine dehydrogenase/xanthine oxidase (XDH/XO) in vitro and in the lungs in vivo. This up-regulation, and the likely corresponding production of reactive oxygen species, may underlie the pathogenesis of an array of disorders. Thus, compounds that prevent hypoxia-induced increase in XDH/XO activity may provide a therapeutic strategy in such disorders. The antioxidant properties of estrogens have been demonstrated in several studies. However, the effect of these compounds on XDH/XO has not been explored previously. The aim of this study was to investigate the effects of estrogen on hypoxia-induced increase in XDH/XO activity. Rat pulmonary artery microvascular endothelial cells were exposed to normoxia or hypoxia in the presence or absence of 17beta- or 17alpha-estradiol. The XDH/XO enzyme and gene promoter activities were measured in different groups of cells. Hypoxia caused a twofold increase in XDH/XO enzymatic and promoter activity. Either of the estradiol stereoisomers prevented the hypoxia-induced increase in XDH/XO enzymatic activity, but not the promoter activity. ICI 182,780, an antagonist of the estrogen receptor, failed to block the inhibitory effect of estradiol on XDH/XO. In conclusion, 17alpha- and 17beta-estradiol modulate the hypoxia-induced regulation of XDH/XO activity at a posttranscriptional level by a receptor-independent mechanism.

  19. Nuclear factor kappa-B signaling is integral to ocular neovascularization in ischemia-independent microenvironment.

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    Michael DeNiro

    Full Text Available Retinal ischemia promotes the upregulation of VEGF expression and accounts for most pathological features of retinal neovascularization (NV. Paradoxically, VEGF remains the pivotal stimulator of ocular NV, despite the absence of ischemia. Therefore, the central question arises as to how the various molecular mechanisms interplay in ischemia-independent NV. It's been suggested that NFκB plays a crucial role in the pathogenesis of diabetic vasculopathies. Here, we dissected the molecular mechanism of ocular NV in the rho/VEGF transgenic mouse model, which develops subretinal NV in ischemia-independent microenvironment. Furthermore, we examined whether intravitreal administration of YC-1, a HIF-1 inhibitor, can modulate the activation of NFκB and its downstream angiogenic signaling in the mouse retina. We demonstrated that YC-1 inhibited retinal NFκB/p65 DNA binding activity and downregulated NFκB/p65, FAK, α5β1, EPO, ET-1, and MMP-9 expression at the message and the protein levels. In addition, YC-1 significantly inhibited subretinal NV by reducing the number of neovascular lesions, the area of each lesion and the total area of NV per retina. We further investigated the influence of VEGF signaling pathway on HIF-1α transcriptional activity to substantiate that this mouse model develops subretinal NV in an ischemia-independent microenvironment. Our data demonstrated that VEGF overexpression didn't have any impact on HIF-1α transcriptional activity, whereas treatment with YC-1 significantly inhibited endogenous HIF-1 activity. Our study suggests that retinal NFκB transcriptional activity is pivotal to ischemia-independent mechanisms, which lead to the local activation of angiogenic cascades. Our data also indicate that the nexus between VEGF and NFκB is implicated in triggering the angiogenic cascade that promotes retinal NV. Hence, targeting the VEGF/NFκB axis may act in a negative feedback loop to suppress ocular NV. This study suggests

  20. Mechanical stimulation induces mTOR signaling via an ERK-independent mechanism: implications for a direct activation of mTOR by phosphatidic acid.

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    Jae Sung You

    Full Text Available Signaling by mTOR is a well-recognized component of the pathway through which mechanical signals regulate protein synthesis and muscle mass. However, the mechanisms involved in the mechanical regulation of mTOR signaling have not been defined. Nevertheless, recent studies suggest that a mechanically-induced increase in phosphatidic acid (PA may be involved. There is also evidence which suggests that mechanical stimuli, and PA, utilize ERK to induce mTOR signaling. Hence, we reasoned that a mechanically-induced increase in PA might promote mTOR signaling via an ERK-dependent mechanism. To test this, we subjected mouse skeletal muscles to mechanical stimulation in the presence or absence of a MEK/ERK inhibitor, and then measured several commonly used markers of mTOR signaling. Transgenic mice expressing a rapamycin-resistant mutant of mTOR were also used to confirm the validity of these markers. The results demonstrated that mechanically-induced increases in p70(s6k T389 and 4E-BP1 S64 phosphorylation, and unexpectedly, a loss in total 4E-BP1, were fully mTOR-dependent signaling events. Furthermore, we determined that mechanical stimulation induced these mTOR-dependent events, and protein synthesis, through an ERK-independent mechanism. Similar to mechanical stimulation, exogenous PA also induced mTOR-dependent signaling via an ERK-independent mechanism. Moreover, PA was able to directly activate mTOR signaling in vitro. Combined, these results demonstrate that mechanical stimulation induces mTOR signaling, and protein synthesis, via an ERK-independent mechanism that potentially involves a direct interaction of PA with mTOR. Furthermore, it appears that a decrease in total 4E-BP1 may be part of the mTOR-dependent mechanism through which mechanical stimuli activate protein synthesis.

  1. Mechanical stimulation induces mTOR signaling via an ERK-independent mechanism: implications for a direct activation of mTOR by phosphatidic acid.

    Science.gov (United States)

    You, Jae Sung; Frey, John W; Hornberger, Troy A

    2012-01-01

    Signaling by mTOR is a well-recognized component of the pathway through which mechanical signals regulate protein synthesis and muscle mass. However, the mechanisms involved in the mechanical regulation of mTOR signaling have not been defined. Nevertheless, recent studies suggest that a mechanically-induced increase in phosphatidic acid (PA) may be involved. There is also evidence which suggests that mechanical stimuli, and PA, utilize ERK to induce mTOR signaling. Hence, we reasoned that a mechanically-induced increase in PA might promote mTOR signaling via an ERK-dependent mechanism. To test this, we subjected mouse skeletal muscles to mechanical stimulation in the presence or absence of a MEK/ERK inhibitor, and then measured several commonly used markers of mTOR signaling. Transgenic mice expressing a rapamycin-resistant mutant of mTOR were also used to confirm the validity of these markers. The results demonstrated that mechanically-induced increases in p70(s6k) T389 and 4E-BP1 S64 phosphorylation, and unexpectedly, a loss in total 4E-BP1, were fully mTOR-dependent signaling events. Furthermore, we determined that mechanical stimulation induced these mTOR-dependent events, and protein synthesis, through an ERK-independent mechanism. Similar to mechanical stimulation, exogenous PA also induced mTOR-dependent signaling via an ERK-independent mechanism. Moreover, PA was able to directly activate mTOR signaling in vitro. Combined, these results demonstrate that mechanical stimulation induces mTOR signaling, and protein synthesis, via an ERK-independent mechanism that potentially involves a direct interaction of PA with mTOR. Furthermore, it appears that a decrease in total 4E-BP1 may be part of the mTOR-dependent mechanism through which mechanical stimuli activate protein synthesis.

  2. Six Novel Loci Associated with Circulating VEGF Levels Identified by a Meta-analysis of Genome-Wide Association Studies.

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    Seung Hoan Choi

    2016-02-01

    Full Text Available Vascular endothelial growth factor (VEGF is an angiogenic and neurotrophic factor, secreted by endothelial cells, known to impact various physiological and disease processes from cancer to cardiovascular disease and to be pharmacologically modifiable. We sought to identify novel loci associated with circulating VEGF levels through a genome-wide association meta-analysis combining data from European-ancestry individuals and using a dense variant map from 1000 genomes imputation panel. Six discovery cohorts including 13,312 samples were analyzed, followed by in-silico and de-novo replication studies including an additional 2,800 individuals. A total of 10 genome-wide significant variants were identified at 7 loci. Four were novel loci (5q14.3, 10q21.3, 16q24.2 and 18q22.3 and the leading variants at these loci were rs114694170 (MEF2C, P = 6.79 x 10(-13, rs74506613 (JMJD1C, P = 1.17 x 10(-19, rs4782371 (ZFPM1, P = 1.59 x 10(-9 and rs2639990 (ZADH2, P = 1.72 x 10(-8, respectively. We also identified two new independent variants (rs34528081, VEGFA, P = 1.52 x 10(-18; rs7043199, VLDLR-AS1, P = 5.12 x 10(-14 at the 3 previously identified loci and strengthened the evidence for the four previously identified SNPs (rs6921438, LOC100132354, P = 7.39 x 10(-1467; rs1740073, C6orf223, P = 2.34 x 10(-17; rs6993770, ZFPM2, P = 2.44 x 10(-60; rs2375981, KCNV2, P = 1.48 x 10(-100. These variants collectively explained up to 52% of the VEGF phenotypic variance. We explored biological links between genes in the associated loci using Ingenuity Pathway Analysis that emphasized their roles in embryonic development and function. Gene set enrichment analysis identified the ERK5 pathway as enriched in genes containing VEGF associated variants. eQTL analysis showed, in three of the identified regions, variants acting as both cis and trans eQTLs for multiple genes. Most of these genes, as well as some of those in the associated loci, were involved in platelet biogenesis

  3. WRN loss induces switching of telomerase-independent mechanisms of telomere elongation.

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    April Renee Sandy Gocha

    Full Text Available Telomere maintenance can occur in the presence of telomerase or in its absence, termed alternative lengthening of telomeres (ALT. ALT adds telomere repeats using recombination-based processes and DNA repair proteins that function in homologous recombination. Our previous work reported that the RecQ-like BLM helicase is required for ALT and that it unwinds telomeric substrates in vitro. WRN is also a RecQ-like helicase that shares many biochemical functions with BLM. WRN interacts with BLM, unwinds telomeric substrates, and co-localizes to ALT-associated PML bodies (APBs, suggesting that it may also be required for ALT processes. Using long-term siRNA knockdown of WRN in three ALT cell lines, we show that some, but not all, cell lines require WRN for telomere maintenance. VA-13 cells require WRN to prevent telomere loss and for the formation of APBs; Saos-2 cells do not. A third ALT cell line, U-2 OS, requires WRN for APB formation, however WRN loss results in p53-mediated apoptosis. In the absence of WRN and p53, U-2 OS cells undergo telomere loss for an intermediate number of population doublings (50-70, at which point they maintain telomere length even with the continued loss of WRN. WRN and the tumor suppressor BRCA1 co-localize to APBs in VA-13 and U-2 OS, but not in Saos-2 cells. WRN loss in U-2 OS is associated with a loss of BRCA1 from APBs. While the loss of WRN significantly increases telomere sister chromatid exchanges (T-SCE in these three ALT cell lines, loss of both BRCA1 and WRN does not significantly alter T-SCE. This work demonstrates that ALT cell lines use different telomerase-independent maintenance mechanisms that variably require the WRN helicase and that some cells can switch from one mechanism to another that permits telomere elongation in the absence of WRN. Our data suggest that BRCA1 localization may define these mechanisms.

  4. Neural crest cell-derived VEGF promotes embryonic jaw extension

    Science.gov (United States)

    Wiszniak, Sophie; Mackenzie, Francesca E.; Anderson, Peter; Kabbara, Samuela; Ruhrberg, Christiana; Schwarz, Quenten

    2015-01-01

    Jaw morphogenesis depends on the growth of Meckel’s cartilage during embryogenesis. However, the cell types and signals that promote chondrocyte proliferation for Meckel’s cartilage growth are poorly defined. Here we show that neural crest cells (NCCs) and their derivatives provide an essential source of the vascular endothelial growth factor (VEGF) to enhance jaw vascularization and stabilize the major mandibular artery. We further show in two independent mouse models that blood vessels promote Meckel’s cartilage extension. Coculture experiments of arterial tissue with NCCs or chondrocytes demonstrated that NCC-derived VEGF promotes blood vessel growth and that blood vessels secrete factors to instruct chondrocyte proliferation. Computed tomography and X-ray scans of patients with hemifacial microsomia also showed that jaw hypoplasia correlates with mandibular artery dysgenesis. We conclude that cranial NCCs and their derivatives provide an essential source of VEGF to support blood vessel growth in the developing jaw, which in turn is essential for normal chondrocyte proliferation, and therefore jaw extension. PMID:25922531

  5. [Suppression of VEGF protein expression by arctigenin in oral squamous cell carcinoma].

    Science.gov (United States)

    Pu, Guang-rui; Liu, Fa-yu; Wang, Bo

    2015-08-01

    To observe arctigenin's inhibitory effect on oral squamous cell carcinoma, and explore the possible mechanism. The expression of VEGF in 32 cases of oral squamous cell cancer and 20 adjacent tissue specimen were detected with immunohistochemistry. Human nude mouse transplantation tumor model of oral squamous cell cancer was prepared with HSC-3 cells line. Transplanted tumor growth and VEGF expression in transplanted tumor tissues were assayed after treatment with arctigenin. One-way ANOVA was used for comparison between groups with SPSS 16.0 software package. Compared with the adjacent tissue, immunohistochemical staining score of VEGF was significantly higher (Parctigenin, the growth of oral squamous cell transplanted tumors in nude mouse was inhibited (Parctigenin group (PArctigenin can dose-dependently inhibit the growth of oral squamous cell carcinomas, and this effect may be related to down regulation of VEGF expression.

  6. VEGF and endothelial guidance in angiogenic sprouting.

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    Gerhardt, Holger

    2008-10-01

    The cellular actions of VEGF need to be coordinated to guide vascular patterning during sprouting angiogenesis. Individual endothelial tip cells lead and guide the blood vessel sprout, while neighbouring stalk cells proliferate and form the vascular lumen. Recent studies illustrate how endothelial DLL4/NOTCH signalling, stimulated by VEGF, regulates the sprouting response by limiting tip cell formation in the stalk. The spatial distribution of VEGF, in turn, regulates the shape of the ensuing sprout by directing tip cell migration and determining stalk cell proliferation.

  7. Incomplete Large Vessel Occlusions in Mechanical Thrombectomy: An Independent Predictor of Favorable Outcome in Ischemic Stroke.

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    Maus, Volker; You, Seunghwa; Kalkan, Alev; Borggrefe, Jan; Kabbasch, Christoph; Barnikol, Utako Birgit; Stetefeld, Henning; Dohmen, Christian; Liebig, Thomas; Fink, Gereon Rudolf; Mpotsaris, Anastasios

    2017-01-01

    Cerebral large vessel occlusion (LVO) in acute ischemic stroke (AIS) may be complete (CLVO) or incomplete (ILVO). The influence of ILVO on clinical outcome after mechanical thrombectomy (MT) remains unclear. We investigated primarily the clinical outcome in patients with AIS due to ILVO or CLVO. Five hundred three consecutive AIS patients with LVO treated with stent-retriever or direct aspiration-based MT between 2010 and 2016 were analyzed. The primary endpoint was favorable clinical outcome (modified Rankin Scale ≤2) at 90 days; secondary endpoints were periprocedural parameters. Forty-nine patients (11.3%) with a median National Institutes of Health Stroke Scale (NIHSS) of 11 presented with ILVO and the remainder presented with CLVO and median NIHSS of 15 (p < 0.001). The median groin puncture-to-reperfusion time was 30 vs. 67 min, respectively (p < 0.001). Successful reperfusion was reached in 47 out of 49 ILVO (95.9%) vs. 298 out of 381 CLVO (78.2%; p < 0.005) with less retrieval maneuvers (1.7 ± 2.2 vs. 3.0 ± 2.5; p < 0.001). The favorable outcome at 90 days was 81% in patients with ILVO vs. 29.1% in CLVO (p < 0.001); respective all-cause mortality rates were 6.4 vs. 28.5% (p < 0.001). Periprocedural complications (6.9%) occurred exclusively in CLVO patients (p < 0.05). ILVO was associated with favorable clinical outcome independent of age and NIHSS in multivariate logistic regression both in the anterior (OR 3.6; 95% CI 1.8-6.9; p < 0.001) and posterior circulation (OR 3.5; 95% CI 1.8-6.9; p < 0.001). AIS due to ILVO is frequent and is associated with a nearly threefold higher chance of favorable clinical outcome at 90 days, independent of age and initial NIHSS compared to CLVO. © 2017 S. Karger AG, Basel.

  8. S 47445 Produces Antidepressant- and Anxiolytic-Like Effects through Neurogenesis Dependent and Independent Mechanisms

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    Indira Mendez-David

    2017-07-01

    rat model, S 47445 (from 1 mg/kg demonstrated a rapid onset of effect on anhedonia compared to venlafaxine and imipramine. In the CORT model, S 47445 demonstrated significant neurogenic effects on proliferation, survival and maturation of hippocampal newborn neurons at doses inducing an antidepressant-like effect. It also corrected CORT-induced deficits of growth and arborization of dendrites. Finally, the antidepressant/anxiolytic-like activities of S 47445 required adult hippocampal neurogenesis in the novelty suppressed feeding test contrary to OF, EPM and ST. The observed increase in hippocampal BDNF levels could be one of the mechanisms of S 47445 responsible for the adult hippocampal neurogenesis increase. Altogether, S 47445 displays robust antidepressant-anxiolytic-like properties after chronic administration through neurogenesis dependent/independent mechanisms and neuroplastic activities. The AMPA-PAM S 47445 could have promising therapeutic potential for the treatment of major depressive disorders or generalized anxiety disorders.

  9. NLRP3 mediates osteolysis through inflammation-dependent and -independent mechanisms

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    Qu, Chao; Bonar, Sheri L.; Hickman-Brecks, Cynthia L.; Abu-Amer, Samer; McGeough, Matthew D.; Peña, Carla A.; Broderick, Lori; Yang, Chang; Grimston, Susan K.; Kading, Jacqueline; Abu-Amer, Yousef; Novack, Deborah V.; Hoffman, Hal M.; Civitelli, Roberto; Mbalaviele, Gabriel

    2015-01-01

    Activating-mutations in NOD-like receptor (NLR) family, pyrin domain-containing 3 (NLRP3) cause neonatal-onset multisystem inflammatory disease. However, the ontogeny of skeletal anomalies in this disorder is poorly understood. Mice globally expressing the D301N mutation in Nlrp3 (D303N in human) model the human phenotype, including systemic inflammation and skeletal deformities. To gain insights into the skeletal manifestations, we generated mice in which the expression of D301N Nlrp3 (Nlrp3 D301N) is restricted to myeloid cells. These mice exhibit systemic inflammation and severe osteopenia (∼60% lower bone mass) similar to mice globally expressing the knock-in mutation, consistent with the paradigm of innate immune-driven cryopyrinopathies. Because systemic inflammation may indirectly affect bone homeostasis, we engineered mice in which Nlrp3 D301N is expressed specifically in osteoclasts, the cells that resorb bone. These mice also develop ∼50% lower bone mass due to increased osteolysis, but there is no systemic inflammation and no change in osteoclast number. Mechanistically, aside from its role in IL-1β maturation, Nlrp3 D301N expression enhances osteoclast bone resorbing ability through reorganization of actin cytoskeleton while promoting the degradation of poly(ADP-ribose) polymerase 1, an inhibitor of osteoclastogenesis. Thus, NLRP3 inflammasome activation is not restricted to the production of proinflammatory mediators but also leads to cytokine-autonomous responses.—Qu, C., Bonar, S. L., Hickman-Brecks, C. L., Abu-Amer, S., McGeough, M. D., Peña, C. A., Broderick, L., Yang, C., Grimston, S., K., Kading, J., Abu-Amer, Y., Novack, D. V., Hoffman, H. M., Civitelli, R., Mbalaviele, G. NLRP3 mediates osteolysis through inflammation-dependent and -independent mechanisms. PMID:25477279

  10. Mouse Oocytes Acquire Mechanisms That Permit Independent Cell Volume Regulation at the End of Oogenesis.

    Science.gov (United States)

    Richard, Samantha; Tartia, Alina P; Boison, Detlev; Baltz, Jay M

    2017-09-01

    Mouse embryos employ a unique mechanism of cell volume regulation in which glycine is imported via the GLYT1 transporter to regulate intracellular osmotic pressure. Independent cell volume regulation normally becomes active in the oocyte after ovulation is triggered. This involves two steps: the first is the release of the strong adhesion between the oocyte and zona pellucida (ZP) while the second is the activation of GLYT1. In fully-grown oocytes, release of adhesion and GLYT1 activation also occur spontaneously in oocytes removed from the follicle. It is unknown, however, whether the capacity to release oocyte-ZP adhesion or activate GLYT1 first arises in the oocyte after ovulation is triggered or instead growing oocytes already possess these capabilities but they are suppressed in the follicle. Here, we assessed when during oogenesis oocyte-ZP adhesion can be released and when GLYT1 can be activated, with adhesion assessed by an osmotic assay and GLYT1 activity determined by [ 3 H]-glycine uptake. Oocyte-ZP adhesion could not be released by growing oocytes until they were nearly fully grown. Similarly, the amount of GLYT1 activity that can be elicited in oocytes increased sharply at the end of oogenesis. The SLC6A9 protein that is responsible for GLYT1 activity and Slc6a9 transcripts are present in growing oocytes and increased over the course of oogenesis. Furthermore, SLC6A9 becomes localized to the oocyte plasma membrane as the oocyte grows. Thus, oocytes acquire the ability to regulate their cell volume by releasing adhesion to the ZP and activating GLYT1 as they approach the end of oogenesis. J. Cell. Physiol. 232: 2436-2446, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  11. Mechanistic insights into Mg2+-independent prenylation by CloQ from classical molecular mechanics and hybrid quantum mechanics/molecular mechanics molecular dynamics simulations.

    Science.gov (United States)

    Bayse, Craig A; Merz, Kenneth M

    2014-08-05

    Understanding the mechanism of prenyltransferases is important to the design of engineered proteins capable of synthesizing derivatives of naturally occurring therapeutic agents. CloQ is a Mg(2+)-independent aromatic prenyltransferase (APTase) that transfers a dimethylallyl group to 4-hydroxyphenylpyruvate in the biosynthetic pathway for clorobiocin. APTases consist of a common ABBA fold that defines a β-barrel containing the reaction cavity. Positively charged basic residues line the inside of the β-barrel of CloQ to activate the pyrophosphate leaving group to replace the function of the Mg(2+) cofactor in other APTases. Classical molecular dynamics simulations of CloQ, its E281G and F68S mutants, and the related NovQ were used to explore the binding of the 4-hydroxyphenylpyruvate (4HPP) and dimethylallyl diphosphate substrates in the reactive cavity and the role of various conserved residues. Hybrid quantum mechanics/molecular mechanics potential of mean force (PMF) calculations show that the effect of the replacement of the Mg(2+) cofactor with basic residues yields a similar activation barrier for prenylation to Mg(2+)-dependent APTases like NphB. The topology of the binding pocket for 4HPP is important for selective prenylation at the ortho position of the ring. Methylation at this position alters the conformation of the substrate for O-prenylation at the phenol group. Further, a two-dimensional PMF scan shows that a "reverse" prenylation product may be a possible target for protein engineering.

  12. Loss of autocrine endothelial-derived VEGF significantly reduces hemangiosarcoma development in conditional p53-deficient mice.

    Science.gov (United States)

    Farhang Ghahremani, Morvarid; Radaelli, Enrico; Haigh, Katharina; Bartunkova, Sonia; Haenebalcke, Lieven; Marine, Jean-Christophe; Goossens, Steven; Haigh, Jody J

    2014-01-01

    Malignant transformation of the endothelium is rare, and hemangiosarcomas comprise only 1% of all sarcomas. For this reason and due to the lack of appropriate mouse models, the genetic mechanisms of malignant endothelial transformation are poorly understood. Here, we describe a hemangiosarcoma mouse model generated by deleting p53 specifically in the endothelial and hematopoietic lineages. This strategy led to a high incidence of hemangiosarcoma, with an average latency of 25 weeks. To study the in vivo roles of autocrine or endothelial cell autonomous VEGF signaling in the initiation and/or progression of hemangiosarcomas, we genetically deleted autocrine endothelial sources of VEGF in this mouse model. We found that loss of even a single conditional VEGF allele results in substantial rescue from endothelial cell transformation. These findings highlight the important role of threshold levels of autocrine VEGF signaling in endothelial malignancies and suggest a new approach for hemangiosarcoma treatment using targeted autocrine VEGF inhibition.

  13. Penduliflaworosin, a Diterpenoid from Croton crassifolius, Exerts Anti-Angiogenic Effect via VEGF Receptor-2 Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Yeyin Liang

    2017-01-01

    Full Text Available Anti-angiogenesis targeting vascular endothelial growth factor receptor-2 (VEGFR-2 has been considered as an important strategy for cancer therapy. Penduliflaworosin is a diterpenoid isolated from the plant Croton crassifolius. Our previous study showed that this diterpenoid possesses strong anti-angiogenic activity by inhibiting vessel formation in zebrafish. This study was conducted to further investigate the anti-angiogenic activity and mechanism of penduliflaworosin. Results revealed that penduliflaworosin significantly inhibited VEGF-induced angiogenesis processes including proliferation, invasion, migration, and tube formation of human umbilical vein endothelial cells (HUVECs. Moreover, it notably inhibited VEGF-induced sprout formation of aortic rings and blocked VEGF-induced vessel formation in mice. Western blotting studies showed that penduliflaworosin inhibited phosphorylation of the VEGF receptor-2 and its downstream signaling mediators in HUVECs, suggesting that the anti-angiogenic activity was due to an interference with the VEGF/VEGF receptor-2 pathway. In addition, molecular docking simulation indicated that penduliflaworosin could form hydrogen bonds within the ATP-binding region of the VEGF receptor-2 kinase unit. Finally, cytotoxicity assay showed that penduliflaworosin possessed little toxicity toward both cancer and normal cells. Taken together, our findings demonstrate that penduliflaworosin exerts its anti-angiogenic effect via the VEGF receptor-2 signaling pathway. The anti-angiogenic property and low cytotoxicity of penduliflaworosin suggest that it may be useful in cancer treatments.

  14. Deleterious effects of endogenous and exogenous testosterone on mesenchymal stem cell VEGF production.

    Science.gov (United States)

    Ray, Rinki; Herring, Christine M; Markel, Troy A; Crisostomo, Paul R; Wang, Meijing; Weil, Brent; Lahm, Tim; Meldrum, Daniel R

    2008-05-01

    Modulating the paracrine effects of bone marrow mesenchymal stem cells (BMSCs) may be important for the treatment of ischemic myocardial tissue. In this regard, endogenous estrogen may enhance BMSC vascular endothelial growth factor (VEGF) production. However, little information exists regarding the effect of testosterone on stem cell function. We hypothesized that 1) endogenous or exogenous estrogen will enhance stem cell production of VEGF and 2) endogenous or exogenous testosterone will inhibit BMSC VEGF production. BMSCs were collected from adult male, female, castrated male, and ovariectomized female rats. One hundred thousand cells were incubated with testosterone (1, 10, or 100 nM) or estrogen (0.15, 1.5, or 15 nM) for 48 h. Cell supernatants were collected, and VEGF was measured by ELISA. BMSCs harvested from castrated males, normal females, and ovariectomized females produced more VEGF compared with normal males. Castration was associated with the highest level (1,018 +/- 98.26 pg/ml) of VEGF production by BMSCs, which was significantly more than that produced by BMSCs harvested from normal male and normal female animals. Exogenous testosterone significantly reduced VEGF production in BMSCs harvested from ovariectomized females in a dose-dependent manner. Exogenous estrogen did not alter BMSC VEGF production. These findings suggest that testosterone may work on BMSCs to decrease protective growth factor production and that effective removal of testosterone's deleterious effects via castration may prove to be beneficial in terms of protective factor production. By manipulating the mechanisms that BMSCs use to produce growth factors, we may be able to engineer stem cells to produce maximum growth factors during therapeutic use.

  15. Excess HB-EGF, which promotes VEGF signaling, leads to hydrocephalus

    Science.gov (United States)

    Shim, Joon W.; Sandlund, Johanna; Hameed, Mustafa Q.; Blazer-Yost, Bonnie; Zhou, Feng C.; Klagsbrun, Michael; Madsen, Joseph R.

    2016-01-01

    Heparin binding epidermal growth factor-like growth factor (HB-EGF) is an angiogenic factor mediating radial migration of the developing forebrain, while vascular endothelial growth factor (VEGF) is known to influence rostral migratory stream in rodents. Cell migratory defects have been identified in animal models of hydrocephalus; however, the relationship between HB-EGF and hydrocephalus is unclear. We show that mice overexpressing human HB-EGF with β-galactosidase reporter exhibit an elevated VEGF, localization of β-galactosidase outside the subventricular zone (SVZ), subarachnoid hemorrhage, and ventriculomegaly. In Wistar polycystic kidney rats with hydrocephalus, alteration of migratory trajectory is detected. Furthermore, VEGF infusions into the rats result in ventriculomegaly with an increase of SVZ neuroblast in rostral migratory stream, whereas VEGF ligand inhibition prevents it. Our results support the idea that excess HB-EGF leads to a significant elevation of VEGF and ventricular dilatation. These data suggest a potential pathophysiological mechanism that elevated HB-EGF can elicit VEGF induction and hydrocephalus. PMID:27243144

  16. Tumor VEGF:VEGFR2 autocrine feed-forward loop triggers angiogenesis in lung cancer.

    Science.gov (United States)

    Chatterjee, Sampurna; Heukamp, Lukas C; Siobal, Maike; Schöttle, Jakob; Wieczorek, Caroline; Peifer, Martin; Frasca, Davide; Koker, Mirjam; König, Katharina; Meder, Lydia; Rauh, Daniel; Buettner, Reinhard; Wolf, Jürgen; Brekken, Rolf A; Neumaier, Bernd; Christofori, Gerhard; Thomas, Roman K; Ullrich, Roland T

    2013-04-01

    The molecular mechanisms that control the balance between antiangiogenic and proangiogenic factors and initiate the angiogenic switch in tumors remain poorly defined. By combining chemical genetics with multimodal imaging, we have identified an autocrine feed-forward loop in tumor cells in which tumor-derived VEGF stimulates VEGF production via VEGFR2-dependent activation of mTOR, substantially amplifying the initial proangiogenic signal. Disruption of this feed-forward loop by chemical perturbation or knockdown of VEGFR2 in tumor cells dramatically inhibited production of VEGF in vitro and in vivo. This disruption was sufficient to prevent tumor growth in vivo. In patients with lung cancer, we found that this VEGF:VEGFR2 feed-forward loop was active, as the level of VEGF/VEGFR2 binding in tumor cells was highly correlated to tumor angiogenesis. We further demonstrated that inhibition of tumor cell VEGFR2 induces feedback activation of the IRS/MAPK signaling cascade. Most strikingly, combined pharmacological inhibition of VEGFR2 (ZD6474) and MEK (PD0325901) in tumor cells resulted in dramatic tumor shrinkage, whereas monotherapy only modestly slowed tumor growth. Thus, a tumor cell-autonomous VEGF:VEGFR2 feed-forward loop provides signal amplification required for the establishment of fully angiogenic tumors in lung cancer. Interrupting this feed-forward loop switches tumor cells from an angiogenic to a proliferative phenotype that sensitizes tumor cells to MAPK inhibition.

  17. CD47 signaling regulates the immunosuppressive activity of VEGF in T cells*

    Science.gov (United States)

    Kaur, Sukhbir; Chang, Tiffany; Singh, Satya P.; Lim, Langston; Mannan, Poonam; Garfield, Susan H.; Pendrak, Michael L.; Pantoja, David R. Soto; Rosenberg, Avi Z.; Jin, Shelly; Roberts, David D.

    2014-01-01

    Thromobospondin-1 inhibits angiogenesis in part by interacting with the ubiquitous cell surface receptor CD47. In endothelial cells, CD47 interacts directly with vascular endothelial growth factor receptor-2 (VEGFR2), and thrombospondin-1 inhibits VEGFR2 phosphorylation and signaling by disrupting this association. We now show that CD47 similarly associates with and regulates VEGFR2 in T cells. Thrombospondin-1 inhibits phosphorylation of VEGFR2 and its downstream target Src in wild type but not in CD47-deficient human Jurkat and primary murine T cells. VEGFR2 signaling inhibits proliferation and TCR signaling in wild type T cells. However, ligation of CD47 by thrombospondin-1 or loss of CD47 expression reverses some inhibitory effects of VEGF on proliferation and T cell activation. We further found that VEGF and VEGFR2 expression are up-regulated in CD47-deficient murine CD4+ and human Jurkat T cells, and the resulting autocrine VEGFR2 signaling enhances proliferation and some TCR responses in the absence of CD47. Thus, CD47 signaling modulates the ability of VEGF to regulate proliferation and TCR signaling, and autocrine production of VEGF by T cells contributes to this regulation. This provides a mechanism to understand the context-dependent effects of thrombospondin-1 and VEGF on T cell activation and reveals an important role for CD47 signaling in regulating T cell production of the major angiogenic factor VEGF. PMID:25200950

  18. CD47 signaling regulates the immunosuppressive activity of VEGF in T cells.

    Science.gov (United States)

    Kaur, Sukhbir; Chang, Tiffany; Singh, Satya P; Lim, Langston; Mannan, Poonam; Garfield, Susan H; Pendrak, Michael L; Soto-Pantoja, David R; Rosenberg, Avi Z; Jin, Shelly; Roberts, David D

    2014-10-15

    Thrombospondin-1 (TSP1) inhibits angiogenesis, in part, by interacting with the ubiquitous cell-surface receptor CD47. In endothelial cells, CD47 interacts directly with vascular endothelial growth factor receptor (VEGFR)-2, and TSP1 inhibits VEGFR2 phosphorylation and signaling by disrupting this association. We show that CD47 similarly associates with and regulates VEGFR2 in T cells. TSP1 inhibits phosphorylation of VEGFR2 and its downstream target Src in wild type but not in CD47-deficient human Jurkat and primary murine T cells. VEGFR2 signaling inhibits proliferation and TCR signaling in wild type T cells. However, ligation of CD47 by TSP1 or loss of CD47 expression reverses some inhibitory effects of VEGF on proliferation and T cell activation. We further found that VEGF and VEGFR2 expression are upregulated in CD47-deficient murine CD4(+) and human Jurkat T cells, and the resulting autocrine VEGFR2 signaling enhances proliferation and some TCR responses in the absence of CD47. Thus, CD47 signaling modulates the ability of VEGF to regulate proliferation and TCR signaling, and autocrine production of VEGF by T cells contributes to this regulation. This provides a mechanism to understand the context-dependent effects of TSP1 and VEGF on T cell activation, and reveals an important role for CD47 signaling in regulating T cell production of the major angiogenic factor VEGF.

  19. Bee products prevent VEGF-induced angiogenesis in human umbilical vein endothelial cells

    Directory of Open Access Journals (Sweden)

    Mishima Satoshi

    2009-11-01

    Full Text Available Abstract Background Vascular endothelial growth factor (VEGF is a key regulator of pathogenic angiogenesis in diseases such as cancer and diabetic retinopathy. Bee products [royal jelly (RJ, bee pollen, and Chinese red propolis] from the honeybee, Apis mellifera, have been used as traditional health foods for centuries. The aim of this study was to investigate the anti-angiogenic effects of bee products using human umbilical vein endothelial cells (HUVECs. Methods In an in vitro tube formation assay, HUVECs and fibroblast cells were incubated for 14 days with VEGF and various concentrations of bee products [RJ, ethanol extract of bee pollen, ethanol extract of Chinese red propolis and its constituent, caffeic acid phenethyl ester (CAPE]. To clarify the mechanism of in vitro angiogenesis, HUVEC proliferation and migration were induced by VEGF with or without various concentrations of RJ, bee pollen, Chinese red propolis, and CAPE. Results RJ, bee pollen, Chinese red propolis, and CAPE significantly suppressed VEGF-induced in vitro tube formation in the descending order: CAPE > Chinese red propolis >> bee pollen > RJ. RJ and Chinese red propolis suppressed both VEGF-induced HUVEC proliferation and migration. In contrast, bee pollen and CAPE suppressed only the proliferation. Conclusion Among the bee products, Chinese red propolis and CAPE in particular showed strong suppressive effects against VEGF-induced angiogenesis. These findings indicate that Chinese red propolis and CAPE may have potential as preventive and therapeutic agents against angiogenesis-related human diseases.

  20. The mechanism of kaolin clay flocculation by a cation-independent bioflocculant produced by Chryseobacterium daeguense W6

    Directory of Open Access Journals (Sweden)

    Weijie Liu

    2015-03-01

    Full Text Available In recent years, several novel cation-independent bioflocculants have been reported, which can avoid the secondary contamination caused by addition of cations. However, compared with cation-dependent bioflocculants, the flocculating mechanism of cation-independent bioflocculants is largely unknown. In this study, a cation-independent bioflocculant MBF-W6 produced by Chryseobacterium daeguense W6 was used as a model to investigate the flocculating mechanism. The results showed that the major flocculating component of MBF-W6 is a complex of proteins and polysaccharides. The zeta potential results indicated that kaolin clay particles were not precipitated due to charge neutralization and the bridging mediated by cations did not play a major role in the flocculating process. These results are consistent with the fact that MBF-W6 is a cation-independent bioflocculant. Further scanning electron microscopic observation showed that MBF-W6 induced flocs formed tight packed structure, suggesting that the kaolin clay particles maybe directly attached and bridged by bioflocculant MBF-W6. In addition, we also found out that Fe3+ ions inhibit the flocculating activity of MBF-W6 by affecting –COO− and –NH groups. Therefore this study can improve our understanding on flocculating mechanism of cation-independent bioflocculants.

  1. Reduced size-independent mechanical properties of cortical bone in high-fat diet-induced obesity.

    Science.gov (United States)

    Ionova-Martin, S S; Do, S H; Barth, H D; Szadkowska, M; Porter, A E; Ager, J W; Ager, J W; Alliston, T; Vaisse, C; Ritchie, R O

    2010-01-01

    Overweight and obesity are rapidly expanding health problems in children and adolescents. Obesity is associated with greater bone mineral content that might be expected to protect against fracture, which has been observed in adults. Paradoxically, however, the incidence of bone fractures has been found to increase in overweight and obese children and adolescents. Prior studies have shown some reduced mechanical properties as a result of high-fat diet (HFD) but do not fully address size-independent measures of mechanical properties, which are important to understand material behavior. To clarify the effects of HFD on the mechanical properties and microstructure of bone, femora from C57BL/6 mice fed either a HFD or standard laboratory chow (Chow) were evaluated for structural changes and tested for bending strength, bending stiffness and fracture toughness. Here, we find that in young, obese, high-fat fed mice, all geometric parameters of the femoral bone, except length, are increased, but strength, bending stiffness, and fracture toughness are all reduced. This increased bone size and reduced size-independent mechanical properties suggests that obesity leads to a general reduction in bone quality despite an increase in bone quantity; yield and maximum loads, however, remained unchanged, suggesting compensatory mechanisms. We conclude that diet-induced obesity increases bone size and reduces size-independent mechanical properties of cortical bone in mice. This study indicates that bone quantity and bone quality play important compensatory roles in determining fracture risk. Copyright (c) 2009 Elsevier Inc. All rights reserved.

  2. Independent risk of mechanical ventilation for AIDS-related Pneumocystis carinii pneumonia associated with bronchoalveolar lavage neutrophilia

    DEFF Research Database (Denmark)

    Bang, D; Emborg, J; Elkjaer, J

    2001-01-01

    %). In a logistic regression analysis, higher age, increased bronchoalveolar lavage (BAL) neutrophilia and a positive BAL cytomegalovirus CMV culture were associated with the need of MV. In multivariate analyses, only BAL neutrophilia remained independently predictive of mechanical ventilation. In conclusion, short...

  3. Importance of bacterial replication and alveolar macrophage-independent clearance mechanisms during early lung infection with Streptococcus pneumoniae.

    Science.gov (United States)

    Camberlein, Emilie; Cohen, Jonathan M; José, Ricardo; Hyams, Catherine J; Callard, Robin; Chimalapati, Suneeta; Yuste, Jose; Edwards, Lindsey A; Marshall, Helina; van Rooijen, Nico; Noursadeghi, Mahdad; Brown, Jeremy S

    2015-03-01

    Although the importance of alveolar macrophages for host immunity during early Streptococcus pneumoniae lung infection is well established, the contribution and relative importance of other innate immunity mechanisms and of bacterial factors are less clear. We have used a murine model of S. pneumoniae early lung infection with wild-type, unencapsulated, and para-amino benzoic acid auxotroph mutant TIGR4 strains to assess the effects of inoculum size, bacterial replication, capsule, and alveolar macrophage-dependent and -independent clearance mechanisms on bacterial persistence within the lungs. Alveolar macrophage-dependent and -independent (calculated indirectly) clearance half-lives and bacterial replication doubling times were estimated using a mathematical model. In this model, after infection with a high-dose inoculum of encapsulated S. pneumoniae, alveolar macrophage-independent clearance mechanisms were dominant, with a clearance half-life of 24 min compared to 135 min for alveolar macrophage-dependent clearance. In addition, after a high-dose inoculum, successful lung infection required rapid bacterial replication, with an estimated S. pneumoniae doubling time of 16 min. The capsule had wide effects on early lung clearance mechanisms, with reduced half-lives of 14 min for alveolar macrophage-independent and 31 min for alveolar macrophage-dependent clearance of unencapsulated bacteria. In contrast, with a lower-dose inoculum, the bacterial doubling time increased to 56 min and the S. pneumoniae alveolar macrophage-dependent clearance half-life improved to 42 min and was largely unaffected by the capsule. These data demonstrate the large effects of bacterial factors (inoculum size, the capsule, and rapid replication) and alveolar macrophage-independent clearance mechanisms during early lung infection with S. pneumoniae. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  4. Two independent mechanisms for motion-in-depth perception: evidence from individual differences

    Directory of Open Access Journals (Sweden)

    Harold T Nefs

    2010-10-01

    Full Text Available Our forward-facing eyes allow us the advantage of binocular visual information: using the tiny differences between right and left eye views to learn about depth and location in three dimensions. Our visual systems also contain specialized mechanisms to detect motion-in-depth from binocular vision, but the nature of these mechanisms remains controversial. Binocular motion-in-depth perception could theoretically be based on first detecting binocular disparity and then monitoring how it changes over time. The alternative is to monitor the motion in the right and left eye separately and then compare these motion signals. Here we used an individual differences approach to test whether the two sources of information are processed via dissociated mechanisms, and to measure the relative importance of those mechanisms. Our results suggest the existence of two distinct mechanisms, each contributing to the perception of motion in depth in most observers. Additionally, for the first time, we demonstrate the relative prevalence of the two mechanisms within a normal population. In general, visual systems appear to rely mostly on the mechanism sensitive to changing binocular disparity, but perception of motion in depth is augmented by the presence of a less sensitive mechanism that uses interocular velocity differences. Occasionally, we find observers with the opposite pattern of sensitivity. More generally this work showcases the power of the individual differences approach in studying the functional organisation of cognitive systems.

  5. Expression of CXCR4 and VEGF-C is correlated with lymph node metastasis in non-small cell lung cancer.

    Science.gov (United States)

    Bi, Ming Ming; Shang, Bin; Wang, Zhou; Chen, Gang

    2017-11-01

    This study investigated the correlations between CXCR4 and VEGF-C expression and lymph node metastasis in non-small cell lung cancer (NSCLC). Tumor specimens, lymph nodes, and normal lung tissues were obtained from 110 NSCLC patients who underwent complete resection. Quantitative reverse transcription-PCR and immunohistochemistry assays were conducted to evaluate messenger RNA (mRNA) and protein expression of CXCR4 and VEGF-C. Logistic regression analysis was performed to determine the independent risk factors for lymph node metastasis in NSCLC. CXCR4 and VEGF-C mRNA expression were observed in 78 (70.9%) and 64 (58.2%) lung cancer tissues, while CXCR4 and VEGF-C protein expression were observed in 76 (69.9%) and 58 (52.7%) lung cancer tissues, respectively. The expression rates of CXCR4 and VEGF-C mRNA in metastatic lymph nodes were 84.8% and 66.7%, which were higher than that in non-metastatic lymph nodes (27.3% and 18.2%), respectively. Logistic regression analysis revealed that positive expressions of CXCR4 and VEGF-C mRNA were independent risk factors for lymph node metastasis in NSCLC. Furthermore, combined expression of CXCR4 and VEGF-C showed a much higher odds ratio than CXCR4 or VEGF-C expression alone. CXCR4 and VEGF-C were highly expressed in lung cancer tissues and metastatic lymph nodes. CXCR4 and VEGF-C expression levels were significantly correlated with lymph node metastasis in NSCLC. CXCR4 and VEGF-C might synergically promote lymphatic metastasis in lung cancer and might be a clinical predictor of lymph node metastasis in NSCLC patients. © 2017 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

  6. Mycobacterial PIMs inhibit host inflammatory responses through CD14-dependent and CD14-independent mechanisms.

    Directory of Open Access Journals (Sweden)

    Nathalie Court

    Full Text Available Mycobacteria develop strategies to evade the host immune system. Among them, mycobacterial LAM or PIMs inhibit the expression of pro-inflammatory cytokines by activated macrophages. Here, using synthetic PIM analogues, we analyzed the mode of action of PIM anti-inflammatory effects. Synthetic PIM(1 isomer and PIM(2 mimetic potently inhibit TNF and IL-12 p40 expression induced by TLR2 or TLR4 pathways, but not by TLR9, in murine macrophages. We show inhibition of LPS binding to TLR4/MD2/CD14 expressing HEK cells by PIM(1 and PIM(2 analogues. More specifically, the binding of LPS to CD14 was inhibited by PIM(1 and PIM(2 analogues. CD14 was dispensable for PIM(1 and PIM(2 analogues functional inhibition of TLR2 agonists induced TNF, as shown in CD14-deficient macrophages. The use of rough-LPS, that stimulates TLR4 pathway independently of CD14, allowed to discriminate between CD14-dependent and CD14-independent anti-inflammatory effects of PIMs on LPS-induced macrophage responses. PIM(1 and PIM(2 analogues inhibited LPS-induced TNF release by a CD14-dependent pathway, while IL-12 p40 inhibition was CD14-independent, suggesting that PIMs have multifold inhibitory effects on the TLR4 signalling pathway.

  7. Mycobacterial PIMs inhibit host inflammatory responses through CD14-dependent and CD14-independent mechanisms.

    Science.gov (United States)

    Court, Nathalie; Rose, Stéphanie; Bourigault, Marie-Laure; Front, Sophie; Martin, Olivier R; Dowling, Jennifer K; Kenny, Elaine F; O'Neill, Luke; Erard, François; Quesniaux, Valerie F J

    2011-01-01

    Mycobacteria develop strategies to evade the host immune system. Among them, mycobacterial LAM or PIMs inhibit the expression of pro-inflammatory cytokines by activated macrophages. Here, using synthetic PIM analogues, we analyzed the mode of action of PIM anti-inflammatory effects. Synthetic PIM(1) isomer and PIM(2) mimetic potently inhibit TNF and IL-12 p40 expression induced by TLR2 or TLR4 pathways, but not by TLR9, in murine macrophages. We show inhibition of LPS binding to TLR4/MD2/CD14 expressing HEK cells by PIM(1) and PIM(2) analogues. More specifically, the binding of LPS to CD14 was inhibited by PIM(1) and PIM(2) analogues. CD14 was dispensable for PIM(1) and PIM(2) analogues functional inhibition of TLR2 agonists induced TNF, as shown in CD14-deficient macrophages. The use of rough-LPS, that stimulates TLR4 pathway independently of CD14, allowed to discriminate between CD14-dependent and CD14-independent anti-inflammatory effects of PIMs on LPS-induced macrophage responses. PIM(1) and PIM(2) analogues inhibited LPS-induced TNF release by a CD14-dependent pathway, while IL-12 p40 inhibition was CD14-independent, suggesting that PIMs have multifold inhibitory effects on the TLR4 signalling pathway.

  8. Reducing VEGF-B Signaling Ameliorates Renal Lipotoxicity and Protects against Diabetic Kidney Disease.

    Science.gov (United States)

    Falkevall, Annelie; Mehlem, Annika; Palombo, Isolde; Heller Sahlgren, Benjamin; Ebarasi, Lwaki; He, Liqun; Ytterberg, A Jimmy; Olauson, Hannes; Axelsson, Jonas; Sundelin, Birgitta; Patrakka, Jaakko; Scotney, Pierre; Nash, Andrew; Eriksson, Ulf

    2017-03-07

    Diabetic kidney disease (DKD) is the most common cause of severe renal disease, and few treatment options are available today that prevent the progressive loss of renal function. DKD is characterized by altered glomerular filtration and proteinuria. A common observation in DKD is the presence of renal steatosis, but the mechanism(s) underlying this observation and to what extent they contribute to disease progression are unknown. Vascular endothelial growth factor B (VEGF-B) controls muscle lipid accumulation through regulation of endothelial fatty acid transport. Here, we demonstrate in experimental mouse models of DKD that renal VEGF-B expression correlates with the severity of disease. Inhibiting VEGF-B signaling in DKD mouse models reduces renal lipotoxicity, re-sensitizes podocytes to insulin signaling, inhibits the development of DKD-associated pathologies, and prevents renal dysfunction. Further, we show that elevated VEGF-B levels are found in patients with DKD, suggesting that VEGF-B antagonism represents a novel approach to treat DKD. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. VEGF, sVEGFR-1 and Endostatin Serum Levels and VEGF Polymorphisms in Recurrent Aphthous Stomatitis

    Directory of Open Access Journals (Sweden)

    Salim Yuce

    2016-09-01

    Full Text Available Aim: Recurrent aphthous stomatitis (RAS is one of the most frequent diseases of the oral mucosa, characterized by chronic, painful, recurrent, and necrotizing ulcerations. The precise etiology and pathogenesis of RAS have not been clarified. Therefore, we aimed to investigate serum levels of VEGF, sVEGFR-1, and endostatin as well as the frequencies of VEGF 936 C/T and -1154 G/A single nucleotide polymorphisms (SNPs in Turkish patients with recurrent aphthous stomatitis. Material and Method: Forty-two patients with RAS (24 minor RAS and 18 major RAS and 37 healthy subjects were included in the study. Serum levels of VEGF, sVEGFR-1, and endostatin were measured using the ELISA method. VEGF 936 C/T and -1154 G/A SNPs were determined by the PCR-RFLP method. Results: The mean serum level of VEGF was found higher in bearing CC genotype of 936 C/T SNP compared with CT genotype (639.5 ± 309.1 vs 442.1 ± 197.8; p = 0.032. VEGF -1154 GA genotype was found to be more frequent in patients with minor RAS and GG genotype was more frequent in patients with major RAS (p = 0.022. There was a significant difference between minor RAS and major RAS with regard to mean VEGF serum levels (677.1 ± 316.7 vs 492.9 ± 242.7; p = 0.032.Discussion: The T allele of 936 C/T SNP is associated with decreased serum VEGF level, and this decrease may have a contributory role in impaired neovascularization and re-epithelialization in the etiology and pathogenesis of RAS. Further studies are needed to determine the role of VEGF in RAS susceptibility and its clinical manifestations.

  10. Independent risk of mechanical ventilation for AIDS-related Pneumocystis carinii pneumonia associated with bronchoalveolar lavage neutrophilia

    DEFF Research Database (Denmark)

    Bang, D.; Emborg, J.; Elkjaer, J.

    2001-01-01

    The use of mechanical ventilation (MV) for AIDS-related Pneumocystis carinii pneumonia (PCP) has varied over time. The introduction of adjunctive corticosteroid therapy has changed the pathophysiology of PCP. In the present study, we attempted to identify factors predictive of severe respiratory ......-term mortality remained high after the introduction of adjunctive corticosteroid therapy. BAL neutrophilia may be a useful prognostic marker to identify patients at high risk of requiring mechanical ventilation Udgivelsesdato: 2001/8......The use of mechanical ventilation (MV) for AIDS-related Pneumocystis carinii pneumonia (PCP) has varied over time. The introduction of adjunctive corticosteroid therapy has changed the pathophysiology of PCP. In the present study, we attempted to identify factors predictive of severe respiratory......%). In a logistic regression analysis, higher age, increased bronchoalveolar lavage (BAL) neutrophilia and a positive BAL cytomegalovirus CMV culture were associated with the need of MV. In multivariate analyses, only BAL neutrophilia remained independently predictive of mechanical ventilation. In conclusion, short...

  11. Resonant transmission in one-dimensional quantum mechanics with two independent point interactions: Full parameter analysis

    Science.gov (United States)

    Konno, Kohkichi; Nagasawa, Tomoaki; Takahashi, Rohta

    2017-10-01

    We discuss the scattering of a quantum particle by two independent successive point interactions in one dimension. The parameter space for two point interactions is given by U(2) × U(2) , which is described by eight real parameters. We perform an analysis of perfect resonant transmission on the whole parameter space. By investigating the effects of the two point interactions on the scattering matrix of plane wave, we find the condition under which perfect resonant transmission occurs. We also provide the physical interpretation of the resonance condition.

  12. The bps signal: embryonic arrest from an auxin-independent mechanism in bypass triple mutants.

    Science.gov (United States)

    Lee, Dong-Keun; Sieburth, Leslie E

    2012-06-01

    Long-distance signaling is essential for coordination of plant development and environmental responses. We originally isolated a tiny mutant named bypass1 (bps1), which has defects in shoot and root development. The bps1 roots overproduce a mobile signal (bps signal) that arrests both root and shoot development. Our recent study demonstrated that all three BPS gene family members prevent ectopic synthesis of the same bps signal.bps multiple mutants show progressively more severe developmental defects. An embryogenesis analysis revealed abnormal cell divisions in all meristem lineages of bps triple mutants. These defects appear to be auxin independent, and arise prior to changes in PLT1 and PLT2 expression.

  13. Serotonin regulates C. elegans fat and feeding through independent molecular mechanisms

    DEFF Research Database (Denmark)

    Srinivasan, Supriya; Sadegh, Leila; Elle, Ida C

    2008-01-01

    We investigated serotonin signaling in C. elegans as a paradigm for neural regulation of energy balance and found that serotonergic regulation of fat is molecularly distinct from feeding regulation. Serotonergic feeding regulation is mediated by receptors whose functions are not required for fat...... feeding behavior. These findings suggest that, as in mammals, C. elegans feeding behavior is regulated by extrinsic and intrinsic cues. Moreover, obesity and thinness are not solely determined by feeding behavior. Rather, feeding behavior and fat metabolism are coordinated but independent responses...

  14. Quercetin enhances adiponectin secretion by a PPAR-gamma independent mechanism

    DEFF Research Database (Denmark)

    Wein, Silvia; Behm, Norma; Petersen, Rasmus Koefoed

    2010-01-01

    To study possible insulin sensitizing, anti-inflammatory and anti-oxidative effects of the flavonol quercetin, rats were fed a high-fat diet (19%, w/w) with (HFQ) or without (HF) 0.03% quercetin or a flavonoid-poor low-fat (5%, w/w) maintenance diet (LF) over 4 weeks. Body weight was measured...... composition and plasma leptin levels were not affected. Neither quercetin nor its metabolites induced PPAR-gamma-mediated transactivation in vitro. Adiponectin stimulating effects of quercetin are PPAR-gamma-independent and prevent impairment of insulin sensitivity without affecting body weight...... and composition....

  15. Roxithromycin inhibits VEGF-induced human airway smooth muscle cell proliferation: Opportunities for the treatment of asthma

    Energy Technology Data Exchange (ETDEWEB)

    Pei, Qing-Mei, E-mail: 34713316@qq.com [Department of Radiology, Tianjin Hospital of Integrated Traditional Chinese and Western Medicine, Tianjin (China); Jiang, Ping, E-mail: jiangping@163.com [Department of Respiration, Tianjin First Central Hospital, Tianjin (China); Yang, Min, E-mail: YangMin@163.com [Department of Respiration, Tianjin First Central Hospital, Tianjin (China); Qian, Xue-Jiao, E-mail: qianxuejiao@163.com [Department of Respiration, Tianjin First Central Hospital, Tianjin (China); Liu, Jiang-Bo, E-mail: LJB1984@163.com [Department of Respiration, Tianjin First Central Hospital, Tianjin (China); Kim, Sung-Ho, E-mail: chenghao0726@hotmail.com [Department of Respiration, Tianjin First Central Hospital, Tianjin (China)

    2016-10-01

    Asthma is a chronic respiratory disease characterized by reversible airway obstruction with persistent airway inflammation and airway remodelling, which is associated with increased airway smooth muscle (ASM) mass. Roxithromycin (RXM) has been widely used in asthma treatment; however, its mechanism of action is poorly understood. Vascular endothelial growth factor (VEGF) has been implicated in inflammatory and airway blood vessel remodelling in patients with asthma, and shown to promote ASM cell proliferation. Here, we investigated the effect of RXM on VEGF-induced ASM cell proliferation and attempted to elucidate the underlying mechanisms of action. We tested the effect of RXM on proliferation and cell cycle progression, as well as on the expression of phospho-VEGF receptor 2 (VEGFR2), phospho-extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-Akt, and caveolin-1 in VEGF-stimulated ASM cells. RXM inhibited VEGF-induced ASM cell proliferation and induced cell cycle arrest. Additionally, VEGF-induced ASM cell proliferation was suppressed by inhibiting the activity of ERK1/2, but not that of Akt. Furthermore, RXM treatment inhibits VEGF-induced activation of VEGFR2 and ERK and downregulation of caveolin-1 in a dose-dependent manner. RXM also inhibited TGF-β-induced VEGF secretion by ASM cells and BEAS-2B cells. Collectively, our findings suggest that RXM inhibits VEGF-induced ASM cell proliferation by suppression of VEGFR2 and ERK1/2 activation and caveolin-1 down-regulation, which may be involved in airway remodelling. Further elucidation of the mechanisms underlying these observations should enable the development of treatments for smooth muscle hyperplasia-associated diseases of the airway such as asthma. - Highlights: • RXM inhibited VEGF-induced ASM cell proliferation and induced cell cycle arrest. • VEGF-induced cell proliferation was suppressed by inhibiting the activity of ERK1/2. • RXM inhibits activation of VEGFR2 and ERK and downregulation

  16. Bone cell-independent benefits of raloxifene on the skeleton: a novel mechanism for improving bone material properties.

    Science.gov (United States)

    Gallant, Maxime A; Brown, Drew M; Hammond, Max; Wallace, Joseph M; Du, Jiang; Deymier-Black, Alix C; Almer, Jonathan D; Stock, Stuart R; Allen, Matthew R; Burr, David B

    2014-04-01

    Raloxifene is an FDA approved agent used to treat bone loss and decrease fracture risk. In clinical trials and animal studies, raloxifene reduces fracture risk and improves bone mechanical properties, but the mechanisms of action remain unclear because these benefits occur largely independent of changes to bone mass. Using a novel experimental approach, machined bone beams, both from mature male canine and human male donors, were depleted of living cells and then exposed to raloxifene ex vivo. Our data show that ex vivo exposure of non-viable bone to raloxifene improves intrinsic toughness, both in canine and human cortical bone beams tested by 4-point bending. These effects are cell-independent and appear to be mediated by an increase in matrix bound water, assessed using basic gravimetric weighing and sophisticated ultrashort echo time magnetic resonance imaging. The hydroxyl groups (OH) on raloxifene were shown to be important in both the water and toughness increases. Wide and small angle X-ray scattering patterns during 4-pt bending show that raloxifene alters the transfer of load between the collagen matrix and the mineral crystals, placing lower strains on the mineral, and allowing greater overall deformation prior to failure. Collectively, these findings provide a possible mechanistic explanation for the therapeutic effect of raloxifene and more importantly identify a cell-independent mechanism that can be utilized for novel pharmacological approaches for enhancing bone strength. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Expression of HIF-1A/VEGF/ING-4 Axis in Pulmonary Sarcoidosis.

    Science.gov (United States)

    Piotrowski, W J; Kiszałkiewicz, J; Pastuszak-Lewandoska, D; Górski, P; Antczak, A; Migdalska-Sęk, M; Górski, W; Czarnecka, K H; Domańska, D; Nawrot, E; Brzeziańska-Lasota, E

    2015-01-01

    Angiogenesis/angiostasis regulated by hypoxia inducible factor-1A (HIF-1A)/vascular endothelial growth factor (VEGF)/inhibitor of growth protein 4 (ING-4) axis may be crucial for the course and outcome of sarcoidosis. Overexpression of angiogenic factors (activation of VEGF through HIF-1A) may predispose to chronic course and lung fibrosis, whereas immunoangiostasis (related to an overexpression of inhibitory ING-4) may be involved in granuloma formation in early sarcoid inflammation, or sustained or recurrent formation of granulomas. In this work we investigated gene expression of HIF-1A, VEGF and ING-4 in bronchoalveolar fluid (BALF) cells and in peripheral blood (PB) lymphocytes of sarcoidosis patients (n=94), to better understand mechanisms of the disease and to search for its biomarkers. The relative gene expression level (RQ value) was analyzed by qPCR. The results were evaluated according to the presence of lung parenchymal involvement (radiological stage I vs. II-IV), acute vs. insidious onset, lung function tests, calcium metabolism parameters, percentage of lymphocytes (BALL%) and BAL CD4+/CD8+ in BALF, age, and gender. In BALF cells, the ING-4 and VEGF RQ values were increased, while HIF-1A expression was decreased. In PB lymphocytes all studied genes were overexpressed. Higher expression of HIF-1A in PB lymphocytes of patients with abnormal spirometry, and in BALF cells of patients with lung volume restriction was found. VEGF gene expression in BALF cells was also higher in patients with abnormal spirometry. These findings were in line with previous data on the role of HIF-1A/VEGF/ING-4 axis in the pathogenesis of sarcoidosis. Up-regulated HIF-1A and VEGF genes are linked to acknowledged negative prognostics.

  18. Corticosteroid suppression of VEGF-A in infantile hemangioma-derived stem cells.

    Science.gov (United States)

    Greenberger, Shoshana; Boscolo, Elisa; Adini, Irit; Mulliken, John B; Bischoff, Joyce

    2010-03-18

    Corticosteroids are commonly used to treat infantile hemangioma, but the mechanism of action of this therapy is unknown. We investigated the effect of corticosteroids in a previously described in vivo model of infantile hemangioma and in cultured hemangioma-derived cells. We tested hemangioma-derived stem cells for vasculogenic activity in vivo after implantation into immune-deficient (nude) mice. We studied dexamethasone treatment of both the cells before implantation and the mice after implantation. We also tested hemangioma-derived stem cells for expression of vascular endothelial growth factor A (VEGF-A) in vitro and studied the inhibition of VEGF-A expression, using short hairpin RNA (shRNA) in vivo and in vitro. Systemic treatment with dexamethasone led to dose-dependent inhibition of tumor vasculogenesis in the murine model. Pretreatment of hemangioma-derived stem cells in vitro before implantation also inhibited vasculogenesis. Dexamethasone suppressed VEGF-A production by hemangioma-derived stem cells in vitro but not by hemangioma-derived endothelial cells or human umbilical-vein endothelial cells. Silencing VEGF-A in hemangioma-derived stem cells reduced vasculogenesis in vivo. VEGF-A was detected in hemangioma specimens in the proliferating phase but not in the involuting phase and was shown by immunostaining to reside outside of vessels. Corticosteroid treatment suppressed other proangiogenic factors in hemangioma-derived stem cells, including urokinase plasminogen activator receptor, interleukin-6, monocyte chemoattractant protein 1, and matrix metalloproteinase 1. In a murine model, dexamethasone inhibited the vasculogenic potential of stem cells derived from human infantile hemangioma. The corticosteroid also inhibited the expression of VEGF-A by hemangioma-derived stem cells, and silencing of VEGF-A expression in these cells inhibited vasculogenesis in vivo. 2010 Massachusetts Medical Society

  19. Topoisomerase 1 Regulates Gene Expression in Neurons through Cleavage Complex-Dependent and -Independent Mechanisms.

    Directory of Open Access Journals (Sweden)

    Angela M Mabb

    Full Text Available Topoisomerase 1 (TOP1 inhibitors, including camptothecin and topotecan, covalently trap TOP1 on DNA, creating cleavage complexes (cc's that must be resolved before gene transcription and DNA replication can proceed. We previously found that topotecan reduces the expression of long (>100 kb genes and unsilences the paternal allele of Ube3a in neurons. Here, we sought to evaluate overlap between TOP1cc-dependent and -independent gene regulation in neurons. To do this, we utilized Top1 conditional knockout mice, Top1 knockdown, the CRISPR-Cas9 system to delete Top1, TOP1 catalytic inhibitors that do not generate TOP1cc's, and a TOP1 mutation (T718A that stabilizes TOP1cc's. We found that topotecan treatment significantly alters the expression of many more genes, including long neuronal genes, immediate early genes, and paternal Ube3a, when compared to Top1 deletion. Our data show that topotecan has a stronger effect on neuronal transcription than Top1 deletion, and identifies TOP1cc-dependent and -independent contributions to gene expression.

  20. Mechanisms of Rad52-independent spontaneous and UV-induced mitotic recombination in Saccharomyces cerevisiae.

    Science.gov (United States)

    Coïc, Eric; Feldman, Taya; Landman, Allison S; Haber, James E

    2008-05-01

    In wild-type diploid cells, heteroallelic recombination between his4A and his4C alleles leads mostly to His+ gene conversions that have a parental configuration of flanking markers, but approximately 22% of recombinants have associated reciprocal crossovers. In rad52 strains, gene conversion is reduced 75-fold and the majority of His+ recombinants are crossover associated, with the largest class being half-crossovers in which the other participating chromatid is lost. We report that UV irradiating rad52 cells results in an increase in overall recombination frequency, comparable to increases induced in wild-type (WT) cells, and surprisingly results in a pattern of recombination products quite similar to RAD52 cells: gene conversion without exchange is favored, and the number of 2n - 1 events is markedly reduced. Both spontaneous and UV-induced RAD52-independent recombination depends strongly on Rad50, whereas rad50 has no effect in cells restored to RAD52. The high level of noncrossover gene conversion outcomes in UV-induced rad52 cells depends on Rad51, but not on Rad59. Those outcomes also rely on the UV-inducible kinase Dun1 and Dun1's target, the repressor Crt1, whereas gene conversion events arising spontaneously depend on Rad59 and Crt1. Thus, there are at least two Rad52-independent recombination pathways in budding yeast.

  1. Mechanism of action of bombesin on amylase secretion. Evidence for a Ca2+-independent pathway.

    Science.gov (United States)

    Bruzzone, R

    1989-02-01

    The mode of action of bombesin on amylase secretion was investigated in rat pancreatic acini. Bombesin induced a dose-dependent increase in inositol 1,4,5-trisphosphate and cytosolic free Ca2+. The threshold concentration capable of inducing both effects was 0.1 nM and the half-maximal dose of the peptide for Ca2+ mobilization was approximately 0.6 nM. By contrast, amylase release was approximately 30 times more sensitive than inositol 1,4,5-trisphosphate production and Ca2+ mobilization to bombesin action, with 1 pM being the first stimulatory concentration and a half-maximal effect at approximately 20 pM. The ability of low bombesin doses to trigger enzyme secretion was unaffected by chelation of extracellular Ca2+ with EGTA. In order to test whether the stimulation of amylase release was truly a Ca2+-independent response, the intracellular Ca2+ stores were depleted by pretreating acini with EGTA plus ionomycin, the Ca2+ ionophore. Under these conditions bombesin was still capable of eliciting a significant twofold enhancement of the secretory activity. These results indicate that bombesin, an agonist thought to activate secretion mainly through mobilization of Ca2+ from intracellular stores, elicits amylase release at low concentrations, independently of a concomitant rise in cytosolic free Ca2+. The relevance of these findings to the physiological regulation of pancreatic exocrine secretion is discussed.

  2. Gauge-independent Higgs mechanism and the implications for quark confinement

    Directory of Open Access Journals (Sweden)

    Kondo Kei-Ichi

    2017-01-01

    Full Text Available We propose a gauge-invariant description for the Higgs mechanism by which a gauge boson acquires the mass. We do not need to assume spontaneous breakdown of gauge symmetry signaled by a non-vanishing vacuum expectation value of the scalar field. In fact, we give a manifestly gauge-invariant description of the Higgs mechanism in the operator level, which does not rely on spontaneous symmetry breaking. For concreteness, we discuss the gauge-Higgs models with U(1 and SU(2 gauge groups explicitly. This enables us to discuss the confinement-Higgs complementarity from a new perspective.

  3. VEGF and eNOS Expression in Umbilical Cord from Pregnancy Complicated by Hypertensive Disorder with Different Severity

    Directory of Open Access Journals (Sweden)

    K. Bhavina

    2014-01-01

    Full Text Available Background. Reduced blood flow in hypertensive pregnancy may influence the production vasoconstrictors; subsequently the vessel remains in highly contracted state. NO is a vasodilator; VEGF influences its synthesis by regulating eNOS production. Aim of our study was to evaluate the expression of VEGF and eNOS in different severity of hypertensive pregnancy. Methods. Study was conducted in 4 groups with 40 members: group 1—control, group 2—gestational hypertension, group 3—mild preeclampsia, and group 4—severe preeclampsia. Fetal end of umbilical cord was taken and follows IHC staining protocol for VEGF and eNOS antibody. Staining intensity were measured by semiquantitative scoring method. Mann Whitney U test was used to compare each group. Results. Decreased expression of both VEGF and eNOS was found in hypertensive condition than in normal condition. Among hypertensive group, severe preeclamptic group showed more intensity in staining than gestational hypertension and mild preeclampsia. Conclusion. Reduction of VEGF and eNOS in gestational hypertension may lead to hypoperfusion and subsequent hypoxia of fetus in hypertensive pregnancy. The developed hypoxic state may upregulate the synthesis of VEGF and thereby eNOS. Increased expression of VEGF and eNOS in severe group may be a compensatory mechanism to dilate the blood vessels and to improve blood flow of fetus.

  4. Albendazole inhibits HIF-1α-dependent glycolysis and VEGF expression in non-small cell lung cancer cells.

    Science.gov (United States)

    Zhou, Fang; Du, Jin; Wang, Jianjun

    2017-04-01

    Albendazole (ABZ) has an anti-tumor ability and inhibits HIF-1α activity. HIF-1α is associated with glycolysis and vascular endothelial cell growth factor (VEGF) expression, which plays an important role in cancer progression. These clues indicate that ABZ exerts an anti-cancer effect by regulating glycolysis and VEGF expression. The aim of this study is to clarify the effects of ABZ on non-small cell lung cancer (NSCLC) cells and explore the underlying molecular mechanisms. The expression levels of HIF-1α and VEGF were detected using western blot analysis, and the effect of ABZ on glycolysis was evaluated by measuring the relative activities of hexokinase (HK), pyruvate kinase (PK), and lactate dehydrogenase (LDH) and detecting the production of lactate in A549 and H1299 cells. The results showed that ABZ decreased the expression levels of HIF-1α and VEGF and suppressed glycolysis in under hypoxia, but not normoxic condition. Inhibiting HIF-1α also suppressed glycolysis and VEGF expression. Additionally, ABZ inhibited the volume and weight, decreased the relative activities of HK, PK, and LDH, and reduced the levels of HIF-1α and VEGF of A549 xenografts in mouse models. In conclusion, ABZ inhibited growth of NSCLC cells by suppressing HIF-1α-dependent glycolysis and VEGF expression.

  5. Polymer Mechanics as a Model for Short-Term and Flow-Independent Cartilage Viscoelasticity

    Science.gov (United States)

    June, R. K.; Neu, C. P.; Barone, J. R.; Fyhrie, D. P.

    2011-01-01

    Articular cartilage is the load bearing soft tissue that covers the contacting surfaces of long bones in articulating joints. Healthy cartilage allows for smooth joint motion, while damaged cartilage prohibits normal function in debilitating joint diseases such as osteoarthritis. Knowledge of cartilage mechanical function through the progression of osteoarthritis, and in response to innovative regeneration treatments, requires a comprehensive understanding of the molecular nature of interacting extracellular matrix constituents and interstitial fluid. The objectives of this study were therefore to (1) examine the timescale of cartilage stress-relaxation using different mechanistic models and (2) develop and apply a novel (termed “sticky”) polymer mechanics model to cartilage stress-relaxation based on temporary binding of constituent macromolecules. Using data from calf cartilage samples, we found that different models captured distinct timescales of cartilage stress-relaxation: monodisperse polymer reptation best described the first second of relaxation, sticky polymer mechanics best described data from ∼1-100 seconds of relaxation, and a model of inviscid fluid flow through a porous elastic matrix best described data from 100 seconds to equilibrium. Further support for the sticky polymer model was observed using experimental data where cartilage stress-relaxation was measured in either low or high salt concentration. These data suggest that a complete understanding of cartilage mechanics, especially in the short time scales immediately following loading, requires appreciation of both fluid flow and the polymeric behavior of the extracellular matrix. PMID:21552375

  6. Utilizing a reference material for assessing absolute tumor mechanical properties in modality independent elastography

    Science.gov (United States)

    Kim, Dong Kyu; Weis, Jared A.; Yankeelov, Thomas E.; Miga, Michael I.

    2014-03-01

    There is currently no reliable method for early characterization of breast cancer response to neoadjuvant chemotherapy (NAC) [1,2]. Given that disruption of normal structural architecture occurs in cancer-bearing tissue, we hypothesize that further structural changes occur in response to NAC. Consequently, we are investigating the use of modalityindependent elastography (MIE) [3-8] as a method for monitoring mechanical integrity to predict long term outcomes in NAC. Recently, we have utilized a Demons non-rigid image registration method that allows 3D elasticity reconstruction in abnormal tissue geometries, making it particularly amenable to the evaluation of breast cancer mechanical properties. While past work has reflected relative elasticity contrast ratios [3], this study improves upon that work by utilizing a known stiffness reference material within the reconstruction framework such that a stiffness map becomes an absolute measure. To test, a polyvinyl alcohol (PVA) cryogel phantom and a silicone rubber mock mouse tumor phantom were constructed with varying mechanical stiffness. Results showed that an absolute measure of stiffness could be obtained based on a reference value. This reference technique demonstrates the ability to generate accurate measurements of absolute stiffness to characterize response to NAC. These results support that `referenced MIE' has the potential to reliably differentiate absolute tumor stiffness with significant contrast from that of surrounding tissue. The use of referenced MIE to obtain absolute quantification of biomarkers is also translatable across length scales such that the characterization method is mechanics-consistent at the small animal and human application.

  7. B cells contribute to MS pathogenesis through antibody-dependent and antibody-independent mechanisms

    Directory of Open Access Journals (Sweden)

    Wilson HL

    2012-05-01

    Full Text Available Heather L Wilson1,21Vaccine and Infectious Disease Organization-International Vaccine Center, 2Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, CanadaAbstract: For many years, central dogma defined multiple sclerosis (MS as a T cell-driven autoimmune disorder; however, over the past decade there has been a burgeoning recognition that B cells contribute to the pathogenesis of certain MS disease subtypes. B cells may contribute to MS pathogenesis through production of autoantibodies (or antibodies directed at foreign bodies, which unfortunately cross-react with self-antigens, through promotion of T cell activation via antigen presentation, or through production of cytokines. This review highlights evidence for antibody-dependent and antibody-independent B cell involvement in MS pathogenesis.Keywords: autoantibodies, antibody targets, clinically isolated MS, primary progressive MS, secondary progressive MS, relapsing and remitting MS, T cells, T regulatory cells

  8. Coinfection. Virus-helminth coinfection reveals a microbiota-independent mechanism of immunomodulation.

    Science.gov (United States)

    Osborne, Lisa C; Monticelli, Laurel A; Nice, Timothy J; Sutherland, Tara E; Siracusa, Mark C; Hepworth, Matthew R; Tomov, Vesselin T; Kobuley, Dmytro; Tran, Sara V; Bittinger, Kyle; Bailey, Aubrey G; Laughlin, Alice L; Boucher, Jean-Luc; Wherry, E John; Bushman, Frederic D; Allen, Judith E; Virgin, Herbert W; Artis, David

    2014-08-01

    The mammalian intestine is colonized by beneficial commensal bacteria and is a site of infection by pathogens, including helminth parasites. Helminths induce potent immunomodulatory effects, but whether these effects are mediated by direct regulation of host immunity or indirectly through eliciting changes in the microbiota is unknown. We tested this in the context of virus-helminth coinfection. Helminth coinfection resulted in impaired antiviral immunity and was associated with changes in the microbiota and STAT6-dependent helminth-induced alternative activation of macrophages. Notably, helminth-induced impairment of antiviral immunity was evident in germ-free mice, but neutralization of Ym1, a chitinase-like molecule that is associated with alternatively activated macrophages, could partially restore antiviral immunity. These data indicate that helminth-induced immunomodulation occurs independently of changes in the microbiota but is dependent on Ym1. Copyright © 2014, American Association for the Advancement of Science.

  9. Virus-helminth co-infection reveals a microbiota-independent mechanism of immuno-modulation

    Science.gov (United States)

    Osborne, Lisa C.; Monticelli, Laurel A.; Nice, Timothy J.; Sutherland, Tara E.; Siracusa, Mark C.; Hepworth, Matthew R.; Tomov, Vesselin T.; Kobuley, Dmytro; Tran, Sara V.; Bittinger, Kyle; Bailey, Aubrey G.; Laughlin, Alice L.; Boucher, Jean-Luc; Wherry, E. John; Bushman, Frederic D.; Allen, Judith E.; Virgin, Herbert W.; Artis, David

    2015-01-01

    The mammalian intestine is colonized by beneficial commensal bacteria and is a site of infection by pathogens, including helminth parasites. Helminths induce potent immuno-modulatory effects, but whether these effects are mediated by direct regulation of host immunity or indirectly through eliciting changes in the microbiota is unknown. We tested this in the context of virus-helminth co-infection. Helminth co-infection resulted in impaired antiviral immunity and was associated with changes in the microbiota and STAT6-dependent helminth-induced alternative activation of macrophages. Notably, helminth-induced impairment of antiviral immunity was evident in germ-free mice but neutralization of Ym1, a chitinase-like molecule that is associated with alternatively-activated macrophages, could partially restore antiviral immunity. These data indicate that helminth-induced immuno-modulation occurs independently of changes in the microbiota but is dependent on Ym1. PMID:25082704

  10. Evidence that episodic memory impairment during tobacco abstinence is independent of attentional mechanisms.

    Science.gov (United States)

    Merritt, Paul S; Cobb, Adam R; Moissinac, Luke; Hirshman, Elliot

    2010-01-01

    Previous studies have demonstrated reductions in episodic memory during nicotine withdrawal. However, these studies have been unable to dissociate memory reductions from losses in attention associated with tobacco abstinence. In the present study, the authors sought to determine whether episodic memory reduction is a primary effect of nicotine withdrawal during tobacco abstinence. Heavy smokers were tested when smoking normally and following 24 hrs of abstinence. Participants were tested with a recognition memory task in which items were studied under full and divided attention conditions. Forward digit span and backward digit span were also included as control measures. Withdrawal was associated with a reduction in memory performance that was independent of attention at encoding. The authors conclude that impairment of episodic memory is a primary effect of nicotine withdrawal during tobacco abstinence. Further research is required to determine if this is associated with continued use of tobacco and cessation failures.

  11. Acetylcholinesterase Regulates Skeletal In Ovo Development of Chicken Limbs by ACh-Dependent and -Independent Mechanisms.

    Directory of Open Access Journals (Sweden)

    Janine Spieker

    Full Text Available Formation of the vertebrate limb presents an excellent model to analyze a non-neuronal cholinergic system (NNCS. Here, we first analyzed the expression of acetylcholinesterase (AChE by IHC and of choline acetyltransferase (ChAT by ISH in developing embryonic chicken limbs (stages HH17-37. AChE outlined formation of bones, being strongest at their distal tips, and later also marked areas of cell death. At onset, AChE and ChAT were elevated in two organizing centers of the limb anlage, the apical ectodermal ridge (AER and zone of polarizing activity (ZPA, respectively. Thereby ChAT was expressed shortly after AChE, thus strongly supporting a leading role of AChE in limb formation. Then, we conducted loss-of-function studies via unilateral implantation of beads into chicken limb anlagen, which were soaked in cholinergic components. After varying periods, the formation of cartilage matrix and of mineralizing bones was followed by Alcian blue (AB and Alizarin red (AR stainings, respectively. Both acetylcholine (ACh- and ChAT-soaked beads accelerated bone formation in ovo. Notably, inhibition of AChE by BW284c51, or by the monoclonal antibody MAB304 delayed cartilage formation. Since bead inhibition of BChE was mostly ineffective, an ACh-independent action during BW284c51 and MAB304 inhibition was indicated, which possibly could be due to an enzymatic side activity of AChE. In conclusion, skeletogenesis in chick is regulated by an ACh-dependent cholinergic system, but to some extent also by an ACh-independent aspect of the AChE protein.

  12. Comparing protein VEGF inhibitors: In vitro biological studies

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Lanlan; Liang, Xiao Huan [Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080 (United States); Ferrara, Napoleone, E-mail: nf@gene.com [Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080 (United States)

    2011-05-06

    Highlights: {yields} VEGF is a mediator of angiogenesis. {yields} VEGF inhibitors have clinical applications in cancer and eye disorders. {yields} Five protein VEGF inhibitors were compared for their ability to inhibit. {yields} VEGF-induced activities in cultured endothelial cells. -- Abstract: VEGF inhibitors are widely used as a therapy for tumors and intravascular neovascular disorders, but limited and conflicting data regarding their relative biological potencies are available. The purpose of the study is to compare different protein VEGF inhibitors for their ability to inhibit VEGF-stimulated activities. We tested ranibizumab, the full-length variant of ranibizumab (Mab Y0317), bevacizumab, the VEGF-TrapR1R2 and Flt(1-3)-IgG in bioassays measuring VEGF-stimulated proliferation of bovine retinal microvascular endothelial cells or chemotaxis of human umbilical vein endothelial cells (HUVEC). The inhibitors were also compared for their ability to inhibit MAP kinase activation in HUVECs following VEGF addition. Ranibizumab, VEGF-TrapR1R2 and Flt(1-3)-IgG had very similar potencies in the bioassays tested. Bevacizumab was over 10-fold less potent than these molecules. Mab Y0317 was over 30-fold more potent than bevacizumab. The findings reported in this manuscript describe important intrinsic characteristics of several VEGF inhibitors that may be useful to design and interpret preclinical or clinical studies.

  13. Naringenin inhibits seed germination and seedling root growth through a salicylic acid-independent mechanism in Arabidopsis thaliana.

    Science.gov (United States)

    Hernández, Iker; Munné-Bosch, Sergi

    2012-12-01

    Flavonoids fulfill an enormous range of biological functions in plants. In seeds, these compounds play several roles; for instance proanthocyanidins protect them from moisture, pathogen attacks, mechanical stress, UV radiation, etc., and flavonols have been suggested to protect the embryo from oxidative stress. The present study aimed at determining the role of flavonoids in Arabidopsis thaliana (L.) seed germination, and the involvement of salicylic acid (SA) and auxin (indole-3-acetic acid), two phytohormones with the same biosynthetic origin as flavonoids, the shikimate pathway, in such a putative role. We show that naringenin, a flavanone, strongly inhibits the germination of A. thaliana seeds in a dose-dependent and SA-independent manner. Altered auxin levels do not affect seed germination in Arabidopsis, but impaired auxin transport does, although to a minor extent. Naringenin and N-1-naphthylphthalamic acid (NPA) impair auxin transport through the same mechanisms, so the inhibition of germination by naringenin might involve impaired auxin transport among other mechanisms. From the present study it is concluded that naringenin inhibits the germination of Arabidopsis seeds in a dose-dependent and SA-independent manner, and the results also suggest that such effects are exerted, at least to some extent, through impaired auxin transport, although additional mechanisms seem to operate as well. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  14. Thermal fluctuations affect the transcriptome through mechanisms independent of average temperature

    OpenAIRE

    Jesper Givskov Sørensen; Mads Fristrup Schou; Torsten Nygaard Kristensen; Volker Loeschcke

    2016-01-01

    Terrestrial ectotherms are challenged by variation in both mean and variance of temperature. Phenotypic plasticity (thermal acclimation) might mitigate adverse effects, however, we lack a fundamental understanding of the molecular mechanisms of thermal acclimation and how they are affected by fluctuating temperature. Here we investigated the effect of thermal acclimation in Drosophila melanogaster on critical thermal maxima (CTmax) and associated global gene expression profiles as induced by ...

  15. Iron-Induced Damage in Cardiomyopathy: Oxidative-Dependent and Independent Mechanisms

    Directory of Open Access Journals (Sweden)

    Elena Gammella

    2015-01-01

    Full Text Available The high incidence of cardiomyopathy in patients with hemosiderosis, particularly in transfusional iron overload, strongly indicates that iron accumulation in the heart plays a major role in the process leading to heart failure. In this context, iron-mediated generation of noxious reactive oxygen species is believed to be the most important pathogenetic mechanism determining cardiomyocyte damage, the initiating event of a pathologic progression involving apoptosis, fibrosis, and ultimately cardiac dysfunction. However, recent findings suggest that additional mechanisms involving subcellular organelles and inflammatory mediators are important factors in the development of this disease. Moreover, excess iron can amplify the cardiotoxic effect of other agents or events. Finally, subcellular misdistribution of iron within cardiomyocytes may represent an additional pathway leading to cardiac injury. Recent advances in imaging techniques and chelators development remarkably improved cardiac iron overload detection and treatment, respectively. However, increased understanding of the pathogenic mechanisms of iron overload cardiomyopathy is needed to pave the way for the development of improved therapeutic strategies.

  16. Psychostimulants affect dopamine transmission through both dopamine transporter-dependent and independent mechanisms

    Science.gov (United States)

    dela Peña, Ike; Gevorkiana, Ruzanna; Shi, Wei-Xing

    2015-01-01

    The precise mechanisms by which cocaine and amphetamine-like psychostimulants exert their reinforcing effects are not yet fully defined. It is widely believed, however, that these drugs produce their effects by enhancing dopamine neurotransmission in the brain, especially in limbic areas such as the nucleus accumbens, by inducing dopamine transporter-mediated reverse transport and/or blocking dopamine reuptake though the dopamine transporter. Here, we present the evidence that aside from dopamine transporter, non-dopamine transporter-mediated mechanisms also participate in psychostimulant-induced dopamine release and contribute to the behavioral effects of these drugs, such as locomotor activation and reward. Accordingly, psychostimulants could increase norepinephrine release in the prefrontal cortex, the latter then alters the firing pattern of dopamine neurons resulting in changes in action potential-dependent dopamine release. These alterations would further affect the temporal pattern of dopamine release in the nucleus accumbens, thereby modifying information processing in that area. Hence, a synaptic input to a nucleus accumbens neuron may be enhanced or inhibited by dopamine depending on its temporal relationship to dopamine release. Specific temporal patterns of dopamine release may also be required for certain forms of synaptic plasticity in the nucleus accumbens. Together, these effects induced by psychostimulants, mediated through a non-dopamine transporter-mediated mechanism involving norepinephrine and the prefrontal cortex, may also contribute importantly to the reinforcing properties of these drugs. PMID:26209364

  17. Alginate hydrogels allow for bioactive and sustained release of VEGF-C and VEGF-D for lymphangiogenic therapeutic applications.

    Science.gov (United States)

    Campbell, Kevin T; Hadley, Dustin J; Kukis, David L; Silva, Eduardo A

    2017-01-01

    Lymphatic dysfunction is associated with the progression of many cardiovascular disorders due to their role in maintaining tissue fluid homeostasis. Promoting new lymphatic vessels (lymphangiogenesis) is a promising strategy to reverse these cardiovascular disorders via restoring lymphatic function. Vascular endothelial growth factor (VEGF) members VEGF-C and VEGF-D are both potent candidates for stimulating lymphangiogenesis, though maintaining spatial and temporal control of these factors represents a challenge to developing efficient therapeutic lymphangiogenic applications. Injectable alginate hydrogels have been useful for the controlled delivery of many angiogenic factors, including VEGF-A, to stimulate new blood vasculature. However, the utility of these tunable hydrogels for delivering lymphangiogenic factors has never been closely examined. Thus, the objective of this study was to utilize ionically cross-linked alginate hydrogels to deliver VEGF-C and VEGF-D for potential lymphangiogenic applications. We demonstrated that lymphatic endothelial cells (LECs) are sensitive to temporal presentation of VEGF-C and VEGF-D but with different responses between the factors. The greatest LEC mitogenic and sprouting response was observed for constant concentrations of VEGF-C and a high initial concentration that gradually decreased over time for VEGF-D. Additionally, alginate hydrogels provided sustained release of radiolabeled VEGF-C and VEGF-D. Finally, VEGF-C and VEGF-D released from these hydrogels promoted a similar number of LEC sprouts as exogenously added growth factors and new vasculature in vivo via a chick chorioallantoic membrane (CAM) assay. Overall, these findings demonstrate that alginate hydrogels can provide sustained and bioactive release of VEGF-C and VEGF-D which could have applications for therapeutic lymphangiogenesis.

  18. TNF-α-Induced VEGF and MMP-9 Expression Promotes Hemorrhagic Transformation in Pituitary Adenomas

    Directory of Open Access Journals (Sweden)

    Qin Liu

    2011-06-01

    Full Text Available Pituitary apoplexy is a clinical syndrome with unknown pathogenesis. Therefore, identifying the underlying mechanisms is of high clinical relevance. Tumor necrosis factor alpha (TNF-α is a critical cytokine mediating various hemorrhagic events, but little is known about its involvement in pituitary apoplexy. Here we show that TNF-α may be an important regulator of hemorrhagic transformation in pituitary adenomas. In this study, sixty surgical specimens of hemorrhagic and non-hemorrhagic human pituitary adenomas were examined. Hemorrhagic pituitary adenomas displayed higher protein and mRNA levels of TNF-α, vascular endothelial growth factor (VEGF and matrix metalloproteinase-9 (MMP-9 compared with those of non-hemorrhagic tumors. Exposure of MMQ pituitary adenoma cells to TNF-α induced VEGF and MMP-9 expression in vitro. Additionally, TNF-α administration caused hemorrhagic transformation and enhanced VEGF and MMP-9 expression in MMQ pituitary adenoma cell xenografts in mice. Blockers of VEGF or MMP-9, either alone or in combination, attenuated but not abrogated TNF-α mediated hemorrhagic transformation in xenografts. This study suggests that TNF-α may play a role in the development of intratumoral hemorrhage in pituitary adenomas via up-regulation of VEGF and MMP-9.

  19. Low Molecular Weight Fucoidan Inhibits Tumor Angiogenesis through Downregulation of HIF-1/VEGF Signaling under Hypoxia

    Directory of Open Access Journals (Sweden)

    Meng-Chuan Chen

    2015-07-01

    Full Text Available Activation of hypoxia-induced hypoxia-inducible factors-1 (HIF-1 plays a critical role in promoting tumor angiogenesis, growth and metastasis. Low molecular weight fucoidan (LMWF is prepared from brown algae, and exhibits anticancer activity. However, whether LMWF attenuates hypoxia-induced angiogenesis in bladder cancer cells and the molecular mechanisms involved remain unclear. This is the first study to demonstrate that LMWF can inhibit hypoxia-stimulated H2O2 formation, HIF-1 accumulation and transcriptional activity vascular endothelial growth factor (VEGF secretion, and the migration and invasion in hypoxic human bladder cancer cells (T24 cells. LMWF also downregulated hypoxia-activated phosphorylation of PI3K/AKT/mTOR/p70S6K/4EBP-1 signaling in T24 cells. Blocking PI3K/AKT or mTOR activity strongly diminished hypoxia-induced HIF-1α expression and VEGF secretion in T24 cells, supporting the involvement of PI3K/AKT/mTOR in the induction of HIF-1α and VEGF. Additionally, LMWF significantly attenuated angiogenesis in vitro and in vivo evidenced by reduction of tube formation of hypoxic human umbilical vascular endothelial cells and blood capillary generation in the tumor. Similarly, administration of LMWF also inhibited the HIF-1α and VEGF expression in vivo, accompanied by a reduction of tumor growth. In summary, under hypoxia conditions, the antiangiogenic activity of LMWF in bladder cancer may be associated with suppressing HIF-1/VEGF-regulated signaling pathway.

  20. Autocrine VEGF and IL-8 Promote Migration via Src/Vav2/Rac1/PAK1 Signaling in Human Umbilical Vein Endothelial Cells.

    Science.gov (United States)

    Ju, Li; Zhou, Zhiwen; Jiang, Bo; Lou, Yue; Guo, Xirong

    2017-01-01

    Pro-angiogenic factors VEGF and IL-8 play a major role in modulating the migratory potential of endothelial cells. The goal of this study was to investigate the effect of autocrine VEGF and IL-8 in the form of self-conditioned medium (CM) on human umbilical vein endothelial cells (HUVECs). Enzyme-linked immunosorbent assay (ELISA) examined the automatic secretion of VEGF and IL-8 protein by HUVECs. Western blot, small interfering RNA (siRNA), pulldown and Transwell assays were used to explore the role and the mechanism of autocrine VEGF and IL-8 in migration of HUVECs. Neutralizing VEGF and IL-8 in CM significantly abrogated CM-induced migration of HUVECs. Autocrine VEGF and IL-8 increased Src phosphorylation, Rac1 activity and PAK1 phosphorylation in a time dependent manner. Additionally, blocking Rac1 activity with Rac1 siRNA largely abolished autocrine VEGF and IL-8-induced cell migration. Vav2 siRNA suppressed autocrine VEGF and IL-8-induced Rac1 activation and cell migration. Furthermore, blocking Src signaling with PP2, a specific inhibitor for Src, markedly prevented autocrine VEGF and IL-8-induced Vav2 and Rac1 activation as well as consequently cell migration. PAK1 siRNA also significantly abolished autocrine VEGF and IL-8-induced cell migration. We demonstrated for the first time that autocrine VEGF and IL-8 promoted endothelial cell migration via the Src/Vav2/Rac1/PAK1 signaling pathway. This finding reveals the molecular mechanism in the increase of endothelial cell migration induced by autocrine growth factors and cytokines, which is expected to provide a novel therapeutic target in vascular diseases. © 2017 The Author(s)Published by S. Karger AG, Basel.

  1. Akt-dependent and independent mechanisms of mTOR regulation in cancer

    OpenAIRE

    Memmott, Regan M.; Dennis, Phillip A.

    2009-01-01

    The protein kinase mTOR (mammalian target of rapamycin) is a critical regulator of cellular metabolism, growth, and proliferation. These processes contribute to tumor formation, and many cancers are characterized by aberrant activation of mTOR. Although activating mutations in mTOR itself have not been identified, deregulation of upstream components that regulate mTOR are prevalent in cancer. The prototypic mechanism of mTOR regulation in cells is through activation of the PI3K/Akt pathway, b...

  2. [The clinical picture of rheumatoid arthritis--the complex of three independent mechanisms].

    Science.gov (United States)

    Fassbender, Hans Georg; Meyer-Scholten, Carola; Zorn, Kati

    2009-01-01

    The assumption of inflamation as the only cause of the complex clinical picture of rheumatoid atrhritis does not correspond to facts. We have found and proven the existence of three seemingly unconnected mechanisms, and only their combination can account for the general clinical picture of rheumatoid arthritis. They are: 1. immunologic synovitis, responsible for pain, swelling and stiffnes; 2. oncological process ("tumorlike proliferation"), responsible for the destruction of joints; 3. Primary necrotizing process, responsible for the (sometimes lethal) destructions in the heart and blood vessels.

  3. Glucosensing in liver and Brockmann bodies of rainbow trout through glucokinase-independent mechanisms.

    Science.gov (United States)

    Otero-Rodiño, Cristina; Librán-Pérez, Marta; Velasco, Cristina; Álvarez-Otero, Rosa; López-Patiño, Marcos A; Míguez, Jesús M; Soengas, José L

    2016-09-01

    We hypothesize that glucosensor mechanisms other than that mediated by glucokinase (GK) are present in the liver and Brockmann bodies (BB) of rainbow trout, and are affected by stress. We evaluated in these tissues changes in parameters related to putative glucosensor mechanisms based on liver X receptor (LXR), mitochondrial activity, sweet taste receptor, and SGLT-1 6h after intraperitoneal injection of saline solution alone (normoglycaemic treatment) or containing insulin (hypoglycaemic treatment), or d-glucose (hyperglycaemic treatment). Half of tanks were kept at normal stocking density (NSD; 10kgfishmass·m(-3)) whereas the remaining tanks were kept at high stocking density (HSD; 70kgfishmass·m(-3)). The results provide for the first time in fish evidence for the presence of putative glucosensor systems based on mitochondrial activity and sweet taste receptor in liver whereas in BB systems based on LXR, mitochondrial activity, sweet taste receptor, and SGLT-1 could be operative. We also obtained for the first time in fish evidence for the functioning of integrative metabolic sensors in response to changes in nutrient levels since changes in the mRNA abundance of sirtuin 1 (SIRT-1) were observed in response to increased glucose levels. The stress conditions elicited by HSD altered the response of the glucosensor systems based on mitochondrial activity, sweet taste receptor, and SGLT-1 in the liver, and LXR and SGLT-1 in the BB. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Cannabinoids Modulate Neuronal Activity and Cancer by CB1 and CB2 Receptor-Independent Mechanisms.

    Science.gov (United States)

    Soderstrom, Ken; Soliman, Eman; Van Dross, Rukiyah

    2017-01-01

    Cannabinoids include the active constituents of Cannabis or are molecules that mimic the structure and/or function of these Cannabis-derived molecules. Cannabinoids produce many of their cellular and organ system effects by interacting with the well-characterized CB1 and CB2 receptors. However, it has become clear that not all effects of cannabinoid drugs are attributable to their interaction with CB1 and CB2 receptors. Evidence now demonstrates that cannabinoid agents produce effects by modulating activity of the entire array of cellular macromolecules targeted by other drug classes, including: other receptor types; ion channels; transporters; enzymes, and protein- and non-protein cellular structures. This review summarizes evidence for these interactions in the CNS and in cancer, and is organized according to the cellular targets involved. The CNS represents a well-studied area and cancer is emerging in terms of understanding mechanisms by which cannabinoids modulate their activity. Considering the CNS and cancer together allow identification of non-cannabinoid receptor targets that are shared and divergent in both systems. This comparative approach allows the identified targets to be compared and contrasted, suggesting potential new areas of investigation. It also provides insight into the diverse sources of efficacy employed by this interesting class of drugs. Obtaining a comprehensive understanding of the diverse mechanisms of cannabinoid action may lead to the design and development of therapeutic agents with greater efficacy and specificity for their cellular targets.

  5. Cannabinoids Modulate Neuronal Activity and Cancer by CB1 and CB2 Receptor-Independent Mechanisms

    Directory of Open Access Journals (Sweden)

    Ken Soderstrom

    2017-10-01

    Full Text Available Cannabinoids include the active constituents of Cannabis or are molecules that mimic the structure and/or function of these Cannabis-derived molecules. Cannabinoids produce many of their cellular and organ system effects by interacting with the well-characterized CB1 and CB2 receptors. However, it has become clear that not all effects of cannabinoid drugs are attributable to their interaction with CB1 and CB2 receptors. Evidence now demonstrates that cannabinoid agents produce effects by modulating activity of the entire array of cellular macromolecules targeted by other drug classes, including: other receptor types; ion channels; transporters; enzymes, and protein- and non-protein cellular structures. This review summarizes evidence for these interactions in the CNS and in cancer, and is organized according to the cellular targets involved. The CNS represents a well-studied area and cancer is emerging in terms of understanding mechanisms by which cannabinoids modulate their activity. Considering the CNS and cancer together allow identification of non-cannabinoid receptor targets that are shared and divergent in both systems. This comparative approach allows the identified targets to be compared and contrasted, suggesting potential new areas of investigation. It also provides insight into the diverse sources of efficacy employed by this interesting class of drugs. Obtaining a comprehensive understanding of the diverse mechanisms of cannabinoid action may lead to the design and development of therapeutic agents with greater efficacy and specificity for their cellular targets.

  6. A gamma interferon independent mechanism of CD4 T cell mediated control of M. tuberculosis infection in vivo.

    Science.gov (United States)

    Gallegos, Alena M; van Heijst, Jeroen W J; Samstein, Miriam; Su, Xiaodi; Pamer, Eric G; Glickman, Michael S

    2011-05-01

    CD4 T cell deficiency or defective IFNγ signaling render humans and mice highly susceptible to Mycobacterium tuberculosis (Mtb) infection. The prevailing model is that Th1 CD4 T cells produce IFNγ to activate bactericidal effector mechanisms of infected macrophages. Here we test this model by directly interrogating the effector functions of Th1 CD4 T cells required to control Mtb in vivo. While Th1 CD4 T cells specific for the Mtb antigen ESAT-6 restrict in vivo Mtb growth, this inhibition is independent of IFNγ or TNF and does not require the perforin or FAS effector pathways. Adoptive transfer of Th17 CD4 T cells specific for ESAT-6 partially inhibited Mtb growth while Th2 CD4 T cells were largely ineffective. These results imply a previously unrecognized IFNγ/TNF independent pathway that efficiently controls Mtb and suggest that optimization of this alternative effector function may provide new therapeutic avenues to combat Mtb through vaccination.

  7. DCYTB is a predictor of outcome in breast cancer that functions via iron-independent mechanisms.

    Science.gov (United States)

    Lemler, David J; Lynch, Miranda L; Tesfay, Lia; Deng, Zhiyong; Paul, Bibbin T; Wang, Xiaohong; Hegde, Poornima; Manz, David H; Torti, Suzy V; Torti, Frank M

    2017-03-07

    Duodenal cytochrome b (DCYTB) is a ferrireductase that functions together with divalent metal transporter 1 (DMT1) to mediate dietary iron reduction and uptake in the duodenum. DCYTB is also a member of a 16-gene iron regulatory gene signature (IRGS) that predicts metastasis-free survival in breast cancer patients. To better understand the relationship between DCYTB and breast cancer, we explored in detail the prognostic significance and molecular function of DCYTB in breast cancer. The prognostic significance of DCYTB expression was evaluated using publicly available microarray data. Signaling Pathway Impact Analysis (SPIA) of microarray data was used to identify potential novel functions of DCYTB. The role of DCYTB was assessed using immunohistochemistry and measurements of iron uptake, iron metabolism, and FAK signaling. High DCYTB expression was associated with prolonged survival in two large independent cohorts, together totaling 1610 patients (cohort #1, p = 1.6e-11, n = 741; cohort #2, p = 1.2e-05, n = 869; log-rank test) as well as in the Gene expression-based Outcome for Breast cancer Online (GOBO) cohort (p iron or total cellular iron in breast cancer cells. Because SPIA pathway analysis of patient microarray data revealed an association between DCYTB and the focal adhesion pathway, we examined the influence of DCYTB on FAK activation in breast cancer cells. These experiments reveal that DCYTB reduces adhesion and activation of focal adhesion kinase (FAK) and its adapter protein paxillin. DCYTB is an important predictor of outcome and is associated with response to therapy in breast cancer patients. DCYTB does not affect intracellular iron in breast cancer cells. Instead, DCYTB may retard cancer progression by reducing activation of FAK, a kinase that plays a central role in tumor cell adhesion and metastasis.

  8. Compound C inhibits macrophage chemotaxis through an AMPK-independent mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Youngyi [College of Pharmacy, Woosuk University, Wanju, Jeonbuk 55338 (Korea, Republic of); Department of Biochemistry, Chonbuk National University Medical School, Jeonju, Jeonbuk 54896 (Korea, Republic of); Park, Byung-Hyun, E-mail: bhpark@jbnu.ac.kr [Department of Biochemistry, Chonbuk National University Medical School, Jeonju, Jeonbuk 54896 (Korea, Republic of); Bae, Eun Ju, E-mail: ejbae@woosuk.ac.kr [College of Pharmacy, Woosuk University, Wanju, Jeonbuk 55338 (Korea, Republic of)

    2016-01-15

    Macrophage infiltration in adipose tissue is a well-established cause of obesity-linked insulin resistance. AMP-activated protein kinase (AMPK) activation in peripheral tissues such as adipose tissue has beneficial effects on the protection against obesity-induced insulin resistance, which is mainly mediated by prevention of adipose tissue macrophage infiltration and inflammation. In examining the role of AMPK on adipose tissue inflammation, we unexpectedly found that compound C (CC), despite its inhibition of AMPK, robustly inhibited macrophage chemotaxis in RAW 264.7 cells when adipocyte conditioned medium (CM) was used as a chemoattractant. Here, we report that CC inhibition of macrophage migration occurred independently of AMPK. Mechanistically, this inhibitory effect of cell migration by CC was mediated by inhibition of the focal adhesion kinase, AKT, nuclear factor κB pathways. Moreover, the expression of chemokine monocyte chemoattractant protein-1 and pro-inflammatory genes such as tumor necrosis factor α and inducible nitric oxide synthase were prevented by CC treatment in RAW 264.7 cells stimulated with either adipocyte CM or lipopolysaccharide. Lastly, in accord with the findings of the anti-inflammatory effect of CC, we demonstrated that CC functioned as a repressor of macrophage CM-mediated insulin resistance in adipocytes. Taken together, our results suggest that CC serves as a useful inhibitory molecule against macrophage chemotaxis into adipose tissue and thus might have therapeutic potential for the treatment of obesity-linked adipose inflammation. - Highlights: • Compound C (CC) inhibits macrophage chemotaxis regardless of AMPK suppression. • CC enhances insulin sensitivity in adipocytes. • CC inhibits focal adhesion kinase, AKT, and NF-κB signaling in RAW 264.7 cells.

  9. Performance pressure and caffeine both affect cognitive performance, but likely through independent mechanisms.

    Science.gov (United States)

    Boere, Julia J; Fellinger, Lizz; Huizinga, Duncan J H; Wong, Sebastiaan F; Bijleveld, Erik

    2016-02-01

    A prevalent combination in daily life, performance pressure and caffeine intake have both been shown to impact people's cognitive performance. Here, we examined the possibility that pressure and caffeine affect cognitive performance via a shared pathway. In an experiment, participants performed a modular arithmetic task. Performance pressure and caffeine intake were orthogonally manipulated. Findings indicated that pressure and caffeine both negatively impacted performance. However, (a) pressure vs. caffeine affected performance on different trial types, and (b) there was no hint of an interactive effect. So, though the evidence is indirect, findings suggest that pressure and caffeine shape performance via distinct mechanisms, rather than a shared one. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. P70S6K 1 regulation of angiogenesis through VEGF and HIF-1{alpha} expression

    Energy Technology Data Exchange (ETDEWEB)

    Bian, Chuan-Xiu; Shi, Zhumei [Department of Pathology, Cancer Center, Nanjing Medical University, Nanjing 210029 (China); Meng, Qiao; Jiang, Yue; Liu, Ling-Zhi [Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107 (United States); Jiang, Bing-Hua, E-mail: binghjiang@yahoo.com [Department of Pathology, Cancer Center, Nanjing Medical University, Nanjing 210029 (China); Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107 (United States)

    2010-07-30

    Research highlights: {yields} P70S6K1 regulates VEGF expression; {yields} P70S6K1 induces transcriptional activation through HIF-1{alpha} binding site; {yields} P70S6K1 regulates HIF-1{alpha}, but not HIF-1{beta} protein expression; {yields} P70S6K1 mediates tumor growth and angiogenesis through HIF-1{alpha} and VEGF expression. -- Abstract: The 70 kDa ribosomal S6 kinase 1 (p70S6K1), a downstream target of phosphoinositide 3-kinase (PI3K) and ERK mitogen-activated protein kinase (MAPK), is an important regulator of cell cycle progression, and cell proliferation. Recent studies indicated an important role of p70S6K1 in PTEN-negative and AKT-overexpressing tumors. However, the mechanism of p70S6K1 in tumor angiogenesis remains to be elucidated. In this study, we specifically inhibited p70S6K1 activity in ovarian cancer cells using vector-based small interfering RNA (siRNA) against p70S6K1. We found that knockdown of p70S6K1 significantly decreased VEGF protein expression and VEGF transcriptional activation through the HIF-1{alpha} binding site at its enhancer region. The expression of p70S6K1 siRNA specifically inhibited HIF-1{alpha}, but not HIF-1{beta} protein expression. We also found that p70S6K1 down-regulation inhibited ovarian tumor growth and angiogenesis, and decreased cell proliferation and levels of VEGF and HIF-1{alpha} expression in tumor tissues. Our results suggest that p70S6K1 is required for tumor growth and angiogenesis through HIF-1{alpha} and VEGF expression, providing a molecular mechanism of human ovarian cancer mediated by p70S6K1 signaling.

  11. Mechanism of metal-independent decomposition of organic hydroperoxides and formation of alkoxyl radicals by halogenated quinones

    Science.gov (United States)

    Zhu, Ben-Zhan; Zhao, Hong-Tao; Kalyanaraman, Balaraman; Liu, Jun; Shan, Guo-Qiang; Du, Yu-Guo; Frei, Balz

    2007-01-01

    The metal-independent decomposition of organic hydroperoxides and the formation of organic alkoxyl radicals in the absence or presence of halogenated quinones were studied with electron spin resonance (ESR) and the spin-trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO). We found that 2,5-dichloro-1,4-benzoquinone (DCBQ) markedly enhanced the decomposition of tert-butylhydroperoxide (t-BuOOH), leading to the formation of the DMPO adducts with t-butoxyl radicals (t-BuO•) and methyl radicals (•CH3). The formation of DMPO/t-BuO• and DMPO/•CH3 was dose-dependent with respect to both DCBQ and t-BuOOH and was not affected by iron- or copper-specific metal chelators. Comparison of the data obtained with DCBQ and t-BuOOH with those obtained in a parallel study with ferrous iron and t-BuOOH strongly suggested that t-BuO• was produced by DCBQ and t-BuOOH through a metal-independent mechanism. Other halogenated quinones were also found to enhance the decomposition of t-BuOOH and other organic hydroperoxides such as cumene hydroperoxide, leading to the formation of the respective organic alkoxyl radicals in a metal-independent manner. Based on these data, we propose a mechanism for DCBQ-mediated t-BuOOH decomposition and formation of t-BuO•: a nucleophilic attack of t-BuOOH on DCBQ, forming a chloro-t-butylperoxyl-1,4-benzoquinone intermediate, which decomposes homolytically to produce t-BuO•. This represents a mechanism of organic alkoxyl radical formation not requiring the involvement of redox-active transition metal ions. PMID:17360415

  12. uPA dependent and independent mechanisms of wound healing by C-phycocyanin

    Science.gov (United States)

    Madhyastha, H K; Radha, K S; Nakajima, Y; Omura, S; Maruyama, M

    2008-01-01

    Wound repair requires both recruitment and well co-ordinated actions of many cell types including inflammatory cells, endothelial cells, epithelial cells and importantly fibroblast cells. Urokinase-type plasminogen activator (uPA) system plays a vital role in wound healing phenomenon. We have previously demonstrated that C-phycocyanin (C-pc), a biliprotein from blue-green algae, transcriptionally regulates uPA through cAMP-dependent protein kinase A (PKA) pathway. To date, a role for C-pc in wound-healing scenario is not elucidated. This study was designed to examine the wound-healing property of C-pc in relation to fibroblast proliferation and migration. C-pc increased fibroblast proliferation in a dose-dependent manner. It also enhanced G1 phase of cell cycle and increased the expressions of cyclin-dependent kinases 1 and 2, which facilitate cell cycle progression, in a uPA-independent manner. In vitro wound healing and migration assays revealed the pro-migratory properties of C-pc. Short-interference RNA studies demonstrated that uPA was necessary for C-pc-induced fibroblast migration. C-pc also significantly elevated the expressions of chemokines (MDC, RANTES, Eotaxin, GRO α, ENA78 and TARC) and Rho-GTPases (Cdc 42 and Rac 1) in a uPA-dependent manner. Pre-treatment of C-pc-stimulated cells with pharmacological inhibitor of PI-3K (LY294002) annulled the expression of GTPases implying that Rac 1 and Cdc 42 were induced through PI-3K pathway. C-pc-induced cellular migration towards wounded area was also negatively affected by PI-3K inhibition. In vivo wound-healing experiments in mice validated our finding that C-pc accelerates wound healing. Our data provides conclusive evidence of a novel therapeutic usage for C-pc as a wound-healing agent. C-pc is a food and drug administration (FDA)-approved health supplement. We believe this compound can also be beneficial in healing of internal wounds, such as ulcers. PMID:18266963

  13. Geranylated 4-Phenylcoumarins Exhibit Anticancer Effects against Human Prostate Cancer Cells through Caspase-Independent Mechanism

    Science.gov (United States)

    Suparji, Noor Shahirah; Chan, Gomathi; Sapili, Hani; Arshad, Norhafiza M.; In, Lionel L. A.; Awang, Khalijah; Hasima Nagoor, Noor

    2016-01-01

    Geranylated 4-phenylcoumarins, DMDP-1 & -2 isolated from Mesua elegans were investigated for anticancer potential against human prostate cancer cells. Treatment with DMDP-1 & -2 resulted in cell death in a time and dose dependent manner in an MTT assay on all cancer cell lines tested with the exception of lung adenocarcinoma cells. DMDP-1 showed highest cytotoxic efficacy in PC-3 cells while DMDP-2 was most potent in DU 145 cells. Flow cytometry indicated that both coumarins were successful to induce programmed cell death after 24 h treatment. Elucidation on the mode-of-action via protein arrays and western blotting demonstrated death induced without any significant expressions of caspases, Bcl-2 family proteins and cleaved PARP, thus suggesting the involvement of caspase-independent pathways. In identifying autophagy, analysis of GFP-LC3 showed increased punctate in PC-3 cells pre-treated with CQ and treated with DMDP-1. In these cells decreased expression of autophagosome protein, p62 and cathepsin B further confirmed autophagy. In contrary, the DU 145 cells pre-treated with CQ and treated with DMDP-2 has reduced GFP-LC3 punctate although the number of cells with obvious GFP-LC3 puncta was significantly increased in the inhibitor-treated cells. The increase level of p62 suggested leakage of cathepsin B into the cytosol to trigger potential downstream death mediators. This correlated with increased expression of cathepsin B and reduced expression after treatment with its inhibitor, CA074. Also auto-degradation of calpain-2 upon treatment with DMDP-1 &-2 and its inhibitor alone, calpeptin compared with the combination treatment, further confirmed involvement of calpain-2 in PC-3 and DU 145 cells. Treatment with DMDP-1 & -2 also showed up-regulation of total and phosphorylated p53 levels in a time dependent manner. Hence, DMDP-1 & -2 showed ability to activate multiple death pathways involving autophagy, lysosomal and endoplasmic reticulum death proteins which could

  14. VEGF receptor antagonist Cyclo-VEGI reduces inflammatory reactivity and vascular leakiness and is neuroprotective against acute excitotoxic striatal insult

    Directory of Open Access Journals (Sweden)

    McLarnon James G

    2008-05-01

    conferred by Cyclo-VEGI treatment (33% increase in NeuN and 38% decrease in Fluoro-Jade. Conclusion An antagonist for VEGF receptor-mediated signaling, Cyclo-VEGI, has shown efficacy in a broad spectrum of activity against striatal excitotoxic insult including inhibition of microgliosis, reduction in leakiness of BBB and parenchymal infiltration of plasma fibrinogen and in conferring significant protection for striatal neurons. Antagonism of VEGF-mediated activity, possibly targeting VEGF receptors on reactive microglia, is suggested as a neuroprotective mechanism against inflammatory reactivity and a novel strategy to attenuate acute excitotoxic damage.

  15. Deletions linked to TP53 loss drive cancer through p53-independent mechanisms.

    Science.gov (United States)

    Liu, Yu; Chen, Chong; Xu, Zhengmin; Scuoppo, Claudio; Rillahan, Cory D; Gao, Jianjiong; Spitzer, Barbara; Bosbach, Benedikt; Kastenhuber, Edward R; Baslan, Timour; Ackermann, Sarah; Cheng, Lihua; Wang, Qingguo; Niu, Ting; Schultz, Nikolaus; Levine, Ross L; Mills, Alea A; Lowe, Scott W

    2016-03-24

    Mutations disabling the TP53 tumour suppressor gene represent the most frequent events in human cancer and typically occur through a two-hit mechanism involving a missense mutation in one allele and a 'loss of heterozygosity' deletion encompassing the other. While TP53 missense mutations can also contribute gain-of-function activities that impact tumour progression, it remains unclear whether the deletion event, which frequently includes many genes, impacts tumorigenesis beyond TP53 loss alone. Here we show that somatic heterozygous deletion of mouse chromosome 11B3, a 4-megabase region syntenic to human 17p13.1, produces a greater effect on lymphoma and leukaemia development than Trp53 deletion. Mechanistically, the effect of 11B3 loss on tumorigenesis involves co-deleted genes such as Eif5a and Alox15b (also known as Alox8), the suppression of which cooperates with Trp53 loss to produce more aggressive disease. Our results imply that the selective advantage produced by human chromosome 17p deletion reflects the combined impact of TP53 loss and the reduced dosage of linked tumour suppressor genes.

  16. Deletions linked to TP53 loss drive cancer through p53–independent mechanisms

    Science.gov (United States)

    Xu, Zhengmin; Scuoppo, Claudio; Rillahan, Cory D.; Gao, Jianjiong; Spitzer, Barbara; Bosbach, Benedikt; Kastenhuber, Edward R.; Baslan, Timour; Ackermann, Sarah; Cheng, Lihua; Wang, Qingguo; Niu, Ting; Schultz, Nikolaus; Levine, Ross L.; Mills, Alea A.; Lowe, Scott W.

    2016-01-01

    Mutations disabling the TP53 tumour suppressor gene represent the most frequent events in human cancer and typically occur through a two-hit mechanism involving a missense mutation in one allele and a ‘loss of heterozygosity’ deletion encompassing the other. While TP53 missense mutations can also contribute gain-of-function activities that impact tumour progression, it remains unclear whether the deletion event, which frequently includes many genes, impacts tumorigenesis beyond TP53 loss alone. Here we show that somatic heterozygous deletion of mouse chromosome 11B3, a 4-megabase region syntenic to human 17p13.1, produces a greater effect on lymphoma and leukaemia development than Trp53 deletion. Mechanistically, the effect of 11B3 loss on tumorigenesis involves co-deleted genes such as Eif5a and Alox15b (also known as Alox8), the suppression of which cooperates with Trp53 loss to produce more aggressive disease. Our results imply that the selective advantage produced by human chromosome 17p deletion reflects the combined impact of TP53 loss and the reduced dosage of linked tumour suppressor genes. PMID:26982726

  17. An Independent Review and Accountability Mechanism for the Sustainable Development Goals

    Science.gov (United States)

    2016-01-01

    Abstract The Framework Convention on Global Health (FCGH), a proposed global treaty to be rooted in the right to health and aimed at health equity, could establish a nuanced, layered, and multi-faceted regime of compliance with, and accountability to, the right to health. In so doing, it would significantly strengthen accountability for the health-related Sustainable Development Goals (SDGs), which it would encompass. Legally binding, the FCGH could facilitate accountability through the courts and catalyze comprehensive domestic accountability regimes, requiring national strategies that include transparency, community and national mechanisms for accountability and participation and an enabling environment for social empowerment. A “Right to Health Capacity Fund” could ensure resources to implement these strategies. Inclusive national processes could establish targets, benchmarks, and indicators consistent with FCGH guidance, with regular reporting to a treaty body, which could also hear individual cases. State reports could be required to include plans to overcome implementation gaps, subjecting poorly complying states to penalties and targeted capacity building measures. Regional special rapporteurs could facilitate compliance through regular country visits, while also responding to serious violations. And reaching beyond government compliance, from capacity building to the courts and contractual obligations, the FCGH could establish nationally enforceable right to health obligations on the private sector. PMID:27781005

  18. Lipin 2 binds phosphatidic acid by the electrostatic hydrogen bond switch mechanism independent of phosphorylation.

    Science.gov (United States)

    Eaton, James M; Takkellapati, Sankeerth; Lawrence, Robert T; McQueeney, Kelley E; Boroda, Salome; Mullins, Garrett R; Sherwood, Samantha G; Finck, Brian N; Villén, Judit; Harris, Thurl E

    2014-06-27

    Lipin 2 is a phosphatidic acid phosphatase (PAP) responsible for the penultimate step of triglyceride synthesis and dephosphorylation of phosphatidic acid (PA) to generate diacylglycerol. The lipin family of PA phosphatases is composed of lipins 1-3, which are members of the conserved haloacid dehalogenase superfamily. Although genetic alteration of LPIN2 in humans is known to cause Majeed syndrome, little is known about the biochemical regulation of its PAP activity. Here, in an attempt to gain a better general understanding of the biochemical nature of lipin 2, we have performed kinetic and phosphorylation analyses. We provide evidence that lipin 2, like lipin 1, binds PA via the electrostatic hydrogen bond switch mechanism but has a lower rate of catalysis. Like lipin 1, lipin 2 is highly phosphorylated, and we identified 15 phosphosites. However, unlike lipin 1, the phosphorylation of lipin 2 is not induced by insulin signaling nor is it sensitive to inhibition of the mammalian target of rapamycin. Importantly, phosphorylation of lipin 2 does not negatively regulate either membrane binding or PAP activity. This suggests that lipin 2 functions as a constitutively active PA phosphatase in stark contrast to the high degree of phosphorylation-mediated regulation of lipin 1. This knowledge of lipin 2 regulation is important for a deeper understanding of how the lipin family functions with respect to lipid synthesis and, more generally, as an example of how the membrane environment around PA can influence its effector proteins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Abnormal Histone Methylation is Responsible for Increased VEGF165a Secretion from Airway Smooth Muscle Cells in Asthma

    Science.gov (United States)

    Clifford, Rachel L.; John, Alison E.; Brightling, Christopher E.; Knox, Alan J.

    2012-01-01

    Vascular Endothelial Growth Factor (VEGF), a key angiogenic molecule, is aberrantly expressed in several diseases including asthma where it contributes to bronchial vascular remodelling and chronic inflammation. Asthmatic human airway smooth muscle (HASM) cells hypersecrete VEGF but the mechanism is unclear. Here we defined the mechanism in HASM cells from non-asthmatic (NA) and asthmatic (A) patients. We found that asthmatic cells lacked a repression complex at the VEGF promoter which was present in non-asthmatic cells. Recruitment of G9A, trimethylation of histone H3 at lysine 9 (H3K9me3) and a resultant decrease in RNA polymerase II (RNA pol II) at the VEGF promoter was critical to repression of VEGF secretion in non-asthmatic cells. At the asthmatic promoter H3K9me3 was absent due to failed recruitment of G9a; RNA pol II binding, in association with TAF1, was increased, H3K4me3 was present and Sp1 binding was exaggerated and sustained. In contrast DNA methylation and histone acetylation were similar in A and NA cells. This is the first study to show that airway cells in asthma have altered epigenetic regulation of remodelling gene(s). Histone methylation at genes such as VEGF may be an important new therapeutic target. PMID:22689881

  20. Dopamine induces neutrophil apoptosis through a dopamine D-1 receptor-independent mechanism.

    LENUS (Irish Health Repository)

    Sookhai, S

    2012-02-03

    BACKGROUND: For the normal resolution of an acute inflammatory response, neutrophil (PMN) apoptosis is essential to maintain immune homeostasis and to limit inappropriate host tissue damage. A delay in PMN apoptosis has been implicated in the pathogenesis of the systemic inflammatory response syndrome (SIRS). Dopamine, a biogenic amine with known cardiovascular and neurotransmitter properties, is used in patients with SIRS to maintain hemodynamic stability. We sought to determine whether dopamine may also have immunoregulatory properties capable of influencing PMN apoptosis, function, and activation state in patients with SIRS. METHODS: PMNs were isolated from healthy volunteers and patients with SIRS and treated with varying doses of dopamine and a dopamine D-1 receptor agonist, fenoldopam. PMN apoptosis was assessed every 6 hours with use of propidium iodide DNA staining and PMN function was assessed with use of respiratory burst activity, phagocytosis ability, and CD11a, CD11b, and CD18 receptor expression as functional markers. RESULTS: There was a significant delay in PMN apotosis in patients with SIRS compared with controls. Treatment of isolated PMNs from both healthy controls and patients with SIRS with 10 and 100 mumol\\/L dopamine induced apoptosis. PMN ingestive and cytocidal capacity were both decreased in patients with SIRS compared with controls. Treatment with dopamine significantly increased phagocytic function. Fenoldopam did not induce PMN apoptosis. CONCLUSION: Our data demonstrate for the first time that dopamine induces PMN apoptosis and modulates PMN function both in healthy controls and in patients with SIRS. These results indicate that dopamine may be beneficial during SIRS through a nonhemodynamic PMN-dependent proapoptotic mechanism.

  1. Autocrine VEGF isoforms differentially regulate endothelial cell behavior

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    Hideki Yamamoto

    2016-09-01

    Full Text Available Vascular endothelial growth factor A (VEGF is involved in all the essential biology of endothelial cells, from proliferation to vessel function, by mediating intercellular interactions and monolayer integrity. It is expressed as three major alternative spliced variants. In mice, these are VEGF120, VEGF164, and VEGF188, each with different affinities for extracellular matrices and cell surfaces, depending on the inclusion of heparin-binding sites, encoded by exons 6 and 7. To determine the role of each VEGF isoform in endothelial homeostasis, we compared phenotypes of primary endothelial cells isolated from lungs of mice expressing single VEGF isoforms in normoxic and hypoxic conditions. The differential expression and distribution of VEGF isoforms affect endothelial cell functions, such as proliferation, adhesion, migration and integrity, which are dependent on the stability of and affinity to VEGF receptor 2 (VEGFR2. We found a correlation between autocrine VEGF164 and VEGFR2 stability, which is also associated with increased expression of proteins involved in cell adhesion. Endothelial cells expressing only VEGF188, which localizes to extracellular matrices or cell surfaces, presented a mesenchymal morphology and weakened monolayer integrity. Cells expressing only VEGF120 lacked stable VEGFR2 and dysfunctional downstream processes, rendering the cells unviable. Endothelial cells expressing these different isoforms in isolation also had differing rates of apoptosis, proliferation, and signaling via nitric oxide (NO synthesis. These data indicate that autocrine signaling of each VEGF isoform has unique functions on endothelial homeostasis and response to hypoxia, due to both distinct VEGF distribution and VEGFR2 stability, which appears to be, at least partly, affected by differential NO production. This study demonstrates that each autocrine VEGF isoform has a distinct effect on downstream functions, namely VEGFR2-regulated endothelial cell

  2. Collisionless Hall MHD Reconnection Dynamics: Is the Nonlinear Reconnection Rate Independent of the Mechanism that Breaks Field Lines?

    Science.gov (United States)

    Bhattacharjee, A.; Germaschewski, K.; Wang, X.; Linde, T.; Rosner, R.; Siegel, A.

    2002-12-01

    There has been considerable interest in recent years in collisionless reconnection dynamics governed by the generalized Ohm's law in which electron inertia provides the mechanism for breaking field lines. It has been suggested in several theoretical studies that the nonlinear reconnection dynamics, to leading order, is independent of the mechanism that breaks field lines (that is, electron inertia). We test this suggestion carefully using the new Magnetic Reconnection Code (MRC) developed at the Center for Magnetic Reconnection Studies. The MRC is a new massively parallel code with Adaptive Mesh Refinement (AMR) that integrates the equations of Hall MHD. The use of AMR enables unprecedented levels of resolution of the current and vorticity layers and uncovers interesting secondary dynamics not seen in previous studies. We apply the MRC to the study of two problems, one involving free reconnection caused by a spontaneous and fast collisionless instability, the other involving forced reconnection induced by boundary perturbations on a stable plasma. In the case of free reconnection, over the range of parameters covered by our simulations, it is shown that the nonlinear reconnection rate is near-explosive, and furthermore, that the nonlinear magnetic island width is an invariant function of a dimensionless variable which is the product of the linear growth rate and time. Now, since the linear growth rate is a function of the ion sound radius as well as the electron skin depth, we conclude that the nonlinear reconnection rate is not independent of electron inertia. In the case of forced reconnection, after a slow growth phase, the dynamics exhibits an impulsive growth in the amplitude of the thin current sheet, and a subsequent current disruption mediated by secondary instabilities. These results, in which electron inertia provides the mechanism for breaking field lines, are contrasted with resistive Hall MHD simulations in which resistivity provides the mechanism for

  3. Molecular mechanism of metal-independent decomposition of organic hydroperoxides by halogenated quinoid carcinogens and the potential biological implications.

    Science.gov (United States)

    Huang, Chun-Hua; Ren, Fu-Rong; Shan, Guo-Qiang; Qin, Hao; Mao, Li; Zhu, Ben-Zhan

    2015-05-18

    Halogenated quinones (XQ) are a class of carcinogenic intermediates and newly identified chlorination disinfection byproducts in drinking water. Organic hydroperoxides (ROOH) can be produced both by free radical reactions and enzymatic oxidation of polyunsaturated fatty acids. ROOH have been shown to decompose to alkoxyl radicals via catalysis by transition metal ions, which may initiate lipid peroxidation or transform further to the reactive aldehydes. However, it is not clear whether XQ react with ROOH in a similar manner to generate alkoxyl radicals metal-independently. By complementary applications of ESR spin-trapping, HPLC/high resolution mass spectrometric and other analytical methods, we found that 2,5-dichloro-1,4-benzoquinone (DCBQ) could significantly enhance the decomposition of a model ROOH tert-butylhydroperoxide, resulting in the formation of t-butoxyl radicals independent of transition metals. On the basis of the above findings, we detected and identified, for the first time, an unprecedented C-centered quinone ketoxy radical. Then, we extended our study to the more physiologically relevant endogenous ROOH 13-hydroperoxy-9,11-octadecadienoic acid and found that DCBQ could also markedly enhance its decomposition to generate the reactive lipid alkyl radicals and the genotoxic 4-hydroxy-2-nonenal (HNE). Similar results were observed with other XQ. In summary, these findings demonstrated that XQ can facilitate ROOH decomposition to produce reactive alkoxyl, quinone ketoxy, lipid alkyl radicals, and genotoxic HNE via a novel metal-independent mechanism, which may explain partly their potential genotoxicity and carcinogenicity.

  4. Dual delivery of VEGF and ANG-1 in ischemic hearts using an injectable hydrogel.

    Science.gov (United States)

    Rufaihah, Abdul Jalil; Johari, Nurul Azizah; Vaibavi, Srirangam Ramanujam; Plotkin, Marian; Di Thien, Do Thi; Kofidis, Theodoros; Seliktar, Dror

    2017-01-15

    Acute myocardial infarction (MI) caused by ischemia is the most common cause of cardiac dysfunction. While growth factor therapy is promising, the retention in the highly vascularized myocardium is limited and prevents sustained activation needed for adequate cellular responses. Here, we demonstrated the use of polyethylene glycol-fibrinogen (PF) hydrogels for sustained dual delivery of vascular endothelial growth factor (VEGF) and angiopoietin-1 (ANG-1) to enhance myocardial repair and function. VEGF and ANG-1 were incorporated in PF hydrogels and their in vitro characteristics were studied. Acute MI was generated in a rodent model with rats randomly assigned to 4 groups; sham, saline, PF and PF-VEGF-ANG1 (n=10 each group). Saline or hydrogel was injected in infarct and peri-infarct areas of the myocardium. After 4weeks, myocardial function was assessed using echocardiography. Tissue samples were harvested for Hematoxylin and Eosin, Masson Trichrome and capillary staining to assess the extent of fibrotic scar and arteriogenesis. Both VEGF and ANG-1 were released in a sustained and controlled manner over 30days. PF-VEGF-ANG1 treated animals showed the best improvement in cardiac function, highest degree of cardiac muscle preservation, and arteriogenesis. This study demonstrates that PF hydrogels can simultaneously provide mechanical support to attenuate adverse myocardial remodelling, and a pro-angiogenic benefit from the sustained VEGF and ANG1 delivery that culminates in a restorative effect following MI. The utility of this synergistic, biomaterial-based growth factor delivery may have clinical implications in the prevention of post-MI cardiac dysfunction. Acute myocardial infarction (MI) caused by ischemia is the most common cause of cardiac dysfunction. Here, we demonstrated the use of polyethylene glycol-fibrinogen (PF) hydrogels for sustained dual delivery of vascular endothelial growth factor (VEGF) and angiopoietin-1 (ANG-1) to enhance myocardial repair

  5. Pazopanib and anti-VEGF therapy

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    Harry A Drabkin

    2010-03-01

    Full Text Available Harry A DrabkinMedical University of South Carolina and Hollings Cancer Center, Charleston, SC, USAAbstract: Pazopanib (VotrientTM, GlaxoSmithKline, a multi-kinase inhibitor with activity against VEGFR and other receptors, was recently approved by the FDA for the treatment of advanced renal cell carcinoma (RCC. Here, we review the history of its development, together with an overview of VEGF and its receptors and co-receptors. Results from selected clinical trial data in RCC and other malignant diseases are presented. Based on available evidence, pazopanib is an effective VEGFR inhibitor with demonstrable clinical activity in metastatic RCC and promising activity in other diseases. Like most kinase inhibitors, its activity is not restricted to VEGF receptors, which is reflected in its side-effect profile.Keywords: pazopanib, VEGFR, renal cell carcinoma

  6. The response of VEGF-stimulated endothelial cells to angiostatic molecules is substrate-dependent

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    Polverini Peter J

    2005-10-01

    Full Text Available Abstract Background The microenvironment surrounding cells can exert multiple effects on their biological responses. In particular the extracellular matrix surrounding cells can profoundly influence their behavior. It has been shown that the extracellular matrix composition in tumors is vastly different than that found in normal tissue with increased amounts of certain matrices such as collagen I. It has been previously demonstrated that VEGF stimulation of endothelial cells growing on type I collagen results in the induction of bcl-2 expression and enhanced endothelial cell survival. We sought to investigate whether this increased endothelial cell survival resulted in the failure of angiostatic molecules to inhibit angiogenesis. Results We now demonstrate that VEGF-induced survival on collagen I impairs the ability of three known angiostatic molecules, TSP-1, IP-10 and endostatin to inhibit endothelial cell proliferation. Apoptosis of endothelial cells, growing on collagen I, induced by TSP-1 and IP-10 was also inhibited following VEGF stimulation. In contrast, endostatin induced apoptosis in these same cells. Further analysis determined that endostatin did not decrease the expression of bcl-2 nor did it increase activation of caspase-3 in the presence of VEGF. Alternatively, it appeared that in the presence of VEGF, endostatin induced the activation of caspase-8 in endothelial cells grown on collagen I. Furthermore, only endostatin had the ability to inhibit VEGF-induced sprout formation in collagen I gels. Conclusion These data suggest that TSP-1, IP-10 and endostatin inhibit endothelial cells via different mechanisms and that only endostatin is effective in inhibiting angiogenic activities in the presence of collagen I. Our results suggest that the efficacy of angiostatic treatments may be impaired depending on the context of the extracellular matrix within the tumor environment and thus could impede the efficacy of angiostatic therapies.

  7. Expression of VEGF(xxx)b, the inhibitory isoforms of VEGF, in malignant melanoma.

    Science.gov (United States)

    Pritchard-Jones, R O; Dunn, D B A; Qiu, Y; Varey, A H R; Orlando, A; Rigby, H; Harper, S J; Bates, D O

    2007-07-16

    Malignant melanoma is the most lethal of the skin cancers and the UK incidence is rising faster than that of any other cancer. Angiogenesis - the growth of new vessels from preexisting vasculature - is an absolute requirement for tumour survival and progression beyond a few hundred microns in diameter. We previously described a class of anti-angiogenic isoforms of VEGF, VEGF(xxx)b, that inhibit tumour growth in animal models, and are downregulated in some cancers, but have not been investigated in melanoma. To determine whether VEGF(xxx)b expression was altered in melanoma, PCR and immunohistochemistry of archived human tumour samples were used. In normal epidermis and in a proportion of melanoma samples, VEGF(xxx)b staining was seen. Some melanomas had much weaker staining. Subsequent examination revealed that expression was significantly reduced in primary melanoma samples (both horizontal and vertical growth phases) from patients who subsequently developed tumour metastasis compared with those who did not (analysis of variance (ANOVA) Pxxx)b expression appears to predict metastatic spread in patients with primary melanoma. These results suggest that there is a switch in splicing as part of the metastatic process, from anti-angiogenic to pro-angiogenic VEGF isoforms. This may form part of a wider metastatic splicing phenotype.

  8. An increase in vascular endothelial growth factor (VEGF and VEGF soluble receptor-1 (sFlt-1 are associated with early recurrent spontaneous abortion.

    Directory of Open Access Journals (Sweden)

    Lihong Pang

    Full Text Available Recurrent spontaneous abortion (RSA is a health problem that affects approximately 1% to 5% reproductive age woman. Yet, in around half of these patients, the mechanism for RSA is unexplained. Recent studies have indicated that placental ischemia/hypoxia and endothelial dysfunction are important factors in miscarriage. Other studies have indicated that the level and expression of soluble FMS-like tyrosine kinase-1 (sFlt1 is increased under a hypoxic environment. However, decreased sFlt-1 in the maternal circulation during the first trimester has recently been proposed as a potential marker for identifying risk of pregnancy loss. In this prospective study clinical samples were obtained within a short time after the fetal death, protein expression and maternal serum levels of sFlt1 were assessed and compared to samples taken from those with normal pregnancies. Our results indicate that levels of VEGF and sFlt-1 are both increased in women during early pregnancy compared women that are not pregnant (p<0.05 indicating that VEGF and sFlt-1 are both associated with pregnancy. More importantly, we detected a significant (p<0.05 increase in sFlt1 and VEGF levels and expression in the RSA patients who suffered subsequent miscarriages compare to controls. These results demonstrate that there is likely a relationship between VEGF, sFlt-1 and RSA suggesting that the high levels and over expression of sFlt-1 and VEGF might be associated with the pathogenesis of RSA.

  9. VEGF Expression in Patellar Tendinopathy: A Preliminary Study

    Science.gov (United States)

    Lian, Øystein; Bahr, Roald; Hart, David A.; Duronio, Vincent

    2008-01-01

    Vascular function and angiogenesis are regulated by vascular endothelial growth factor-A (VEGF). The purpose of this preliminary study was to address the following questions: Is VEGF expression in the patellar tendon more prevalent in patients with patellar tendinopathy than in individuals with normal, pain-free patellar tendons? Which cell populations express VEGF in normal and tendinopathic tendon? Is there a difference in symptom duration between VEGF+ and VEGF− tendons? We collected patellar tendon tissue from 22 patients undergoing open débridement of the patellar tendon and from 10 patients undergoing intramedullary nailing of the tibia. VEGF expression was assessed immunohistochemically. Relevant inflammatory and repair cell types were immunolabeled. VEGF expression was absent from control tendons, but was present in a subset of patients with histopathological evidence of angiofibroblastic tendinosis. VEGF was expressed in the intimal layer of tendon vessels, but was absent in other cell types. Patients demonstrating VEGF expression in the patellar tendon had a shorter symptom duration (12 ± 7.8 months) than patients with no detectable VEGF (32.8 ± 23.5 months). VEGF may contribute to the vascular hyperplasia that is a cardinal feature of symptomatic tendinosis, particularly in cases with more recent onset. PMID:18459027

  10. Prognostic Relevance of the Expression of CA IX, GLUT-1, and VEGF in Ovarian Epithelial Cancers.

    Science.gov (United States)

    Kim, Kyungbin; Park, Won Young; Kim, Jee Yeon; Sol, Mee Young; Shin, Dong Hun; Park, Do Youn; Lee, Chang Hun; Lee, Jeong Hee; Choi, Kyung Un

    2012-12-01

    Tumor hypoxia is associated with malignant progression and treatment resistance. Hypoxia-related factors, such as carbonic anhydrase IX (CA IX), glucose transporter-1 (GLUT-1), and vascular endothelial growth factor (VEGF) permit tumor cell adaptation to hypoxia. We attempted to elucidate the correlation of these markers with variable clinicopathological factors and overall prognosis. Immunohistochemistry for CA IX, GLUT-1, and VEGF was performed on formalin-fixed, paraffin-embedded tissues from 125 cases of ovarian epithelial cancer (OEC). CA IX expression was significantly associated with an endometrioid and mucinous histology, nuclear grade, tumor necrosis, and mitosis. GLUT-1 expression was associated with tumor necrosis and mitosis. VEGF expression was correlated only with disease recurrence. Expression of each marker was not significant in terms of overall survival in OECs; however, there was a significant correlation between poor overall survival rate and high coexpression of these markers. The present study suggests that it is questionable whether CA IX, GLUT-1, or VEGF can be used alone as independent prognostic factors in OECs. Using at least two markers helps to predict patient outcomes in total OECs. Moreover, the inhibition of two target gene combinations might prove to be a novel anticancer therapy.

  11. Heat Shock Protein 70 Negatively Regulates TGF-β-Stimulated VEGF Synthesis via p38 MAP Kinase in Osteoblasts

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    Go Sakai

    2017-11-01

    Full Text Available Background/Aims: We previously demonstrated that transforming growth factor-β (TGF-β stimulates the synthesis of vascular endothelial growth factor (VEGF through the activation of p38 mitogen-activated protein (MAP kinase in osteoblast-like MC3T3-E1 cells. Heat shock protein70 (HSP70 is a ubiquitously expressed molecular chaperone. In the present study, we investigated the involvement of HSP70 in the TGF-β-stimulated VEGF synthesis and the underlying mechanism in these cells. Methods: Culture MC3T3-E1 cells were stimulated by TGF-β. Released VEGF was measured using an ELISA assay. VEGF mRNA level was quantified by RT-PCR. Phosphorylation of each protein kinase was analyzed by Western blotting. Results: VER-155008 and YM-08, both of HSP70 inhibitors, significantly amplified the TGF-β-stimulated VEGF release. In addition, the expression level of VEGF mRNA induced by TGF-β was enhanced by VER-155008. These inhibitors markedly strengthened the TGF-β-induced phosphorylation of p38 MAP kinase. The TGF-β-induced phosphorylation of p38 MAP kinase was amplified in HSP70-knockdown cells. SB203580, an inhibitor of p38 MAP kinase, significantly suppressed the amplification by these inhibitors of the TGF-β-induced VEGF release. Conclusion: These results strongly suggest that HSP70 acts as a negative regulator in the TGF-β-stimulated VEGF synthesis in osteoblasts, and that the inhibitory effect of HSP70 is exerted at a point upstream of p38 MAP kinase.

  12. Cinnamon extract reduces VEGF expression via suppressing HIF-1α gene expression and inhibits tumor growth in mice.

    Science.gov (United States)

    Zhang, Keqiang; Han, Ernest S; Dellinger, Thanh H; Lu, Jianming; Nam, Sangkil; Anderson, Richard A; Yim, John H; Wen, Wei

    2017-02-01

    Although many anti-VEGF agents are available for cancer treatment, side effects of these agents limit their application for cancer treatment and prevention. Here we studied the potential use of a diet-based agent as an inhibitor for VEGF production. Using a VEGF reporter assay, our data showed that an extract from cinnamon (CE) was a potent inhibitor of VEGF production in human cancer cells and suggested inhibition might be mediated through the suppression of HIF-1α gene expression and protein synthesis. Furthermore, CE treatment was found to inhibit expression and phosphorylation of STAT3 and AKT, which are key factors in the regulation of HIF-1α expression, and significantly reduce angiogenesis potential of cancer cells by migration assay. Consistent with these results, we observed significant suppression of VEGF expression, blood vessel formation, and tumor growth in a human ovarian tumor model in mice treated with CE. Cinnamaldehyde, a major component in cinnamon, was identified as one active component in CE that inhibits VEGF expression. Taken together, our findings provide a novel mechanism underlying anti-angiogenic and anti-tumor actions of CE and support the potential use of CE in cancer prevention and treatment. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  13. Src Kinase becomes preferentially associated with the VEGFR, KDR/Flk-1, following VEGF stimulation of vascular endothelial cells

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    Wang Jing

    2002-12-01

    Full Text Available Abstract Background The cytoplasmic tyrosine kinase, Src, has been found to play a crucial role in VEGF (vascular endothelial growth factor – dependent vascular permeability involved in angiogenesis. The two main VEGFRs present on vascular endothelial cells are KDR/Flk-1 (kinase insert domain-containing receptor/fetal liver kinase-1 and Flt-1 (Fms-like tyrosine kinase-1. However, to date, it has not been determined which VEGF receptor (VEGFR is involved in binding to and activating Src kinase following VEGF stimulation of the receptors. Results In this report, we demonstrate that Src preferentially associates with KDR/Flk-1 rather than Flt-1 in human umbilical vein endothelial cells (HUVECs, and that VEGF stimulation resulted in an increase of Src activity associated with activated KDR/Flk-1. These findings were determined through immunoprecipitation-kinase experiments and coimmunoprecipitation studies, and were further confirmed by GST-pull-down assays and Far Western studies. However, Fyn and Yes, unlike Src, were found to associate preferentially with Flt-1. Conclusions Thus, Src preferentially associates with KDR/Flk-1, rather than with Flt-1, upon VEGF stimulation in endothelial cells. Our findings further highlight the potential significance of upregulated KDR/Flk-1-associated Src activity in the process of angiogenesis, and help to elucidate more clearly the specific roles and mechanisms involving Src family tyrosine kinase in VEGF-stimulated signal transduction events.

  14. Decreased VEGF-A and sustained PEDF expression in a human retinal pigment epithelium cell line cultured under hypothermia

    Directory of Open Access Journals (Sweden)

    Masayuki Takeyama

    2015-01-01

    Full Text Available BACKGROUND: Previous reports have described a decrease in retinal temperature and clinical improvement of wet age-related macular degeneration (AMD after vitrectomy. We hypothesized that the retinal temperature decrease after vitrectomy plays a part in the suppression of wet AMD development. To test this hypothesis, we evaluated the temperature dependence of the expression of vascular endothelial growth factor-A (VEGF-A and in vitro angiogen-esis in retinal pigment epithelium (RPE. RESULTS: We cultured ARPE-19 cells at 37, 35, 33 and 31°C and measured the expression of VEGF-A, VEGF-A splicing variants, and pigment epithelium-derived factor (PEDF. We performed an in vitro tube formation assay. The dehydrogenase activity was also evaluated at each temperature. Expression of VEGF-A significantly decreased with decreased temperature while PEDF expression did not. VEGF165 expression and in vitro angiogenesis also were temperature dependent. The dehydrogenase activity significantly decreased as the culture temperature decreased. CONCLUSIONS: RPE cultured under hypothermia that decreased cellular metabolism also had decreased VEGF-A and sustained PEDF expression, creating an anti-angiogenic environment. This mechanism may be associated with a beneficial effect after vitrectomy in patients with wet AMD.

  15. Comparative integromics on VEGF family members.

    Science.gov (United States)

    Katoh, Yuriko; Katoh, Masaru

    2006-06-01

    VEGF, Hedgehog, FGF, Notch, and WNT signaling pathways network together for vascular remodeling during embryogenesis, tissue regeneration, and carcinogenesis. VEGFA (VEGF), VEGFB, VEGFC, VEGFD (FIGF) and PGF (PlGF) are VEGF family ligands for receptor tyrosine kinases, including VEGFR1 (FLT1), VEGFR2 (KDR) and VEGFR3 (FLT4). Bevacizumab (Avastin), Sunitinib (Sutent) and Sorafenib (Nexavar) are anti-cancer drugs targeted to VEGF signaling pathway. TCF/LEF binding sites within the promoter region of human VEGF family members were searched for by using bioinformatics and human intelligence (Humint). Because four TCF/LEF-binding sites were identified within the 5'-promoter region of human VEGFD gene within AC095351.5 genome sequence, comparative genomics analyses on VEGFD orthologs were further performed. ASB9-ASB11-VEGFD locus at human chromosome Xp22.2 and ASB5-VEGFC locus at human chromosome 4q34 were paralogous regions within the human genome. Human VEGFD mRNA was expressed in lung, small intestine, uterus, breast, neural tissues, and neuroblastoma. Mouse Vegfd mRNA was expressed in kidney, pregnant oviduct, and neural tissues. Chimpanzee VEGFD promoter, cow Vegfd promoter, mouse Vegfd promoter and rat Vegfd promoter were identified within NW_121675.1, AC161065.2, AL732475.6 and AC130036.3 genome sequences, respectively. Three out of four TCF/LEF-binding sites within human VEGFD promoter were conserved in chimpanzee VEGFD promoter, and one in cow Vegfd promoter. TCF/LEF-binding site, not conserved in human VEGFD promoter, occurred in cow, mouse and rat Vegfd promoters. At least five out of six bHLH-binding sites within human VEGFD proximal promoter region were conserved in chimpanzee VEGFD proximal promoter region, while only one in cow Vegfd proximal promoter region. Together these facts indicate that relatively significant promoter evolution occurred among mammalian VEGFD orthologs. Human VEGFD was characterized as a potent target gene of WNT

  16. Intermittent fasting promotes adipose thermogenesis and metabolic homeostasis via VEGF-mediated alternative activation of macrophage.

    Science.gov (United States)

    Kim, Kyoung-Han; Kim, Yun Hye; Son, Joe Eun; Lee, Ju Hee; Kim, Sarah; Choe, Min Seon; Moon, Joon Ho; Zhong, Jian; Fu, Kiya; Lenglin, Florine; Yoo, Jeong-Ah; Bilan, Philip J; Klip, Amira; Nagy, Andras; Kim, Jae-Ryong; Park, Jin Gyoon; Hussein, Samer Mi; Doh, Kyung-Oh; Hui, Chi-Chung; Sung, Hoon-Ki

    2017-11-01

    Intermittent fasting (IF), a periodic energy restriction, has been shown to provide health benefits equivalent to prolonged fasting or caloric restriction. However, our understanding of the underlying mechanisms of IF-mediated metabolic benefits is limited. Here we show that isocaloric IF improves metabolic homeostasis against diet-induced obesity and metabolic dysfunction primarily through adipose thermogenesis in mice. IF-induced metabolic benefits require fasting-mediated increases of vascular endothelial growth factor (VEGF) expression in white adipose tissue (WAT). Furthermore, periodic adipose-VEGF overexpression could recapitulate the metabolic improvement of IF in non-fasted animals. Importantly, fasting and adipose-VEGF induce alternative activation of adipose macrophage, which is critical for thermogenesis. Human adipose gene analysis further revealed a positive correlation of adipose VEGF-M2 macrophage-WAT browning axis. The present study uncovers the molecular mechanism of IF-mediated metabolic benefit and suggests that isocaloric IF can be a preventive and therapeutic approach against obesity and metabolic disorders.

  17. Independent Reporting Mechanism (IRM)

    DEFF Research Database (Denmark)

    Eberholst, Mads Kæmsgaard

    2017-01-01

    To raise the impact of OGP in Denmark, the government can broaden the inclusivity of the OGP process and deepen in-person engagement with a more diverse group of stakeholders. Future action plans may include commitments regarding political party fnancing, lobbying regulations and access to inform...

  18. Impaired angiogenesis and endochondral bone formation in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188.

    Science.gov (United States)

    Maes, Christa; Carmeliet, Peter; Moermans, Karen; Stockmans, Ingrid; Smets, Nico; Collen, Désiré; Bouillon, Roger; Carmeliet, Geert

    2002-02-01

    Vascular endothelial growth factor (VEGF)-mediated angiogenesis is an important part of bone formation. To clarify the role of VEGF isoforms in endochondral bone formation, we examined long bone development in mice expressing exclusively the VEGF120 isoform (VEGF120/120 mice). Neonatal VEGF120/120 long bones showed a completely disturbed vascular pattern, concomitant with a 35% decrease in trabecular bone volume, reduced bone growth and a 34% enlargement of the hypertrophic chondrocyte zone of the growth plate. Surprisingly, embryonic hindlimbs at a stage preceding capillary invasion exhibited a delay in bone collar formation and hypertrophic cartilage calcification. Expression levels of marker genes of osteoblast and hypertrophic chondrocyte differentiation were significantly decreased in VEGF120/120 bones. Furthermore, inhibition of all VEGF isoforms in cultures of embryonic cartilaginous metatarsals, through the administration of a soluble receptor chimeric protein (mFlt-1/Fc), retarded the onset and progression of ossification, suggesting that osteoblast and/or hypertrophic chondrocyte development were impaired. The initial invasion by osteoclasts and endothelial cells into VEGF120/120 bones was retarded, associated with decreased expression of matrix metalloproteinase-9. Our findings indicate that expression of VEGF164 and/or VEGF188 is important for normal endochondral bone development, not only to mediate bone vascularization but also to allow normal differentiation of hypertrophic chondrocytes, osteoblasts, endothelial cells and osteoclasts.

  19. Anti-VEGF Therapy for Diabetic Eye Diseases.

    Science.gov (United States)

    Bahrami, Bobak; Hong, Thomas; Gilles, Mark C; Chang, Andrew

    2017-01-01

    Diabetic retinopathy (DR) is a leading cause of vision impairment and blindness in the working-age population. The identification of vascular endothelial growth factor (VEGF) as a key mediator in the pathogenesis of DR has revolutionized the management of this vision-threatening disease. There is now strong evidence supporting intravitreal anti-VEGF therapy as first line in the management of sight-threatening diabetic macular edema (DME), along with a growing body of evidence to support the use of anti-VEGF drugs for proliferative DR. This review summarizes the role of VEGF in DR, the evidence for anti-VEGF therapy, safety considerations, and the future of anti-VEGF therapy for the management of DR. Copyright 2017 Asia-Pacific Academy of Ophthalmology.

  20. Inhibition of different histone acetyltransferases (HATs) uncovers transcription-dependent and -independent acetylation-mediated mechanisms in memory formation.

    Science.gov (United States)

    Merschbaecher, Katja; Hatko, Lucyna; Folz, Jennifer; Mueller, Uli

    2016-02-01

    Acetylation of histones changes the efficiency of the transcription processes and thus contributes to the formation of long-term memory (LTM). In our comparative study, we used two inhibitors to characterize the contribution of different histone acetyl transferases (HATs) to appetitive associative learning in the honeybee. For one we applied garcinol, an inhibitor of the HATs of the p300 (EP300 binding protein)/CBP (CREB-binding protein) family, and the HATs of the PCAF (p300/CBP-associated factor) family. As comparative agent we applied C646, a specific inhibitor that selectively blocks HATS of the p300/CBP family. Immunochemical analysis reveals differences in histone H3 acetylation in the honeybee brain, in response to the injection of either C646 or garcinol. Behavioral assessment reveals that the two drugs cause memory impairment of different nature when injected after associative conditioning: processes disturbed by garcinol are annihilated by the established transcription blocker actinomycin D and thus seem to require transcription processes. Actions of C646 are unaltered by actinomycin D, and thus seem to be independent of transcription. The outcome of our different approaches as summarized suggests that distinct HATs contribute to different acetylation-mediated processes in memory formation. We further deduce that the acetylation-mediated processes in memory formation comprise transcription-dependent and transcription-independent mechanisms. © 2016 Merschbaecher et al.; Published by Cold Spring Harbor Laboratory Press.

  1. Membrane-mediated action of the endocannabinoid anandamide on membrane proteins: implications for understanding the receptor-independent mechanism

    Science.gov (United States)

    Medeiros, Djalma; Silva-Gonçalves, Laíz Da Costa; da Silva, Annielle Mendes Brito; Dos Santos Cabrera, Marcia Perez; Arcisio-Miranda, Manoel

    2017-01-01

    Endocannabinoids are amphiphilic molecules that play crucial neurophysiological functions acting as lipid messengers. Antagonists and knockdown of the classical CB1 and CB2 cannabinoid receptors do not completely abolish many endocannabinoid activities, supporting the idea of a mechanism independent of receptors whose mode of action remains unclear. Here we combine gramicidin A (gA) single channel recordings and membrane capacitance measurements to investigate the lipid bilayer-modifying activity of endocannabinoids. Single channel recordings show that the incorporation of endocannabinoids into lipid bilayers reduces the free energy necessary for gramicidin channels to transit from the monomeric to the dimeric conformation. Membrane capacitance demonstrates that the endocannabinoid anandamide has limited effects on the overall structure of the lipid bilayers. Our results associated with the theory of membrane elastic deformation reveal that the action of endocannabinoids on membrane proteins can involve local adjustments of the lipid/protein hydrophobic interface. The current findings shed new light on the receptor-independent mode of action of endocannabinoids on membrane proteins, with important implications towards their neurobiological function.

  2. More than one way to evolve a weed: parallel evolution of US weedy rice through independent genetic mechanisms.

    Science.gov (United States)

    Qi, Xinshuai; Liu, Yan; Vigueira, Cynthia C; Young, Nelson D; Caicedo, Ana L; Jia, Yulin; Gealy, David R; Olsen, Kenneth M

    2015-07-01

    Many different crop species were selected for a common suite of 'domestication traits', which facilitates their use for studies of parallel evolution. Within domesticated rice (Oryza sativa), there has also been independent evolution of weedy strains from different cultivated varieties. This makes it possible to examine the genetic basis of parallel weed evolution and the extent to which this process occurs through shared genetic mechanisms. We performed comparative QTL mapping of weediness traits using two recombinant inbred line populations derived from crosses between an indica crop variety and representatives of each of the two independently evolved weed strains found in US rice fields, strawhull (S) and blackhull awned (B). Genotyping-by-sequencing provided dense marker coverage for linkage map construction (average marker interval <0.25 cM), with 6016 and 13 730 SNPs mapped in F5 lines of the S and B populations, respectively. For some weediness traits (awn length, hull pigmentation and pericarp pigmentation), QTL mapping and sequencing of underlying candidate genes confirmed that trait variation was largely attributable to individual loci. However, for more complex quantitative traits (including heading date, panicle length and seed shattering), we found multiple QTL, with little evidence of shared genetic bases between the S and B populations or across previous studies of weedy rice. Candidate gene sequencing revealed causal genetic bases for 8 of 27 total mapped QTL. Together these findings suggest that despite the genetic bottleneck that occurred during rice domestication, there is ample genetic variation in this crop to allow agricultural weed evolution through multiple genetic mechanisms. © 2015 John Wiley & Sons Ltd.

  3. Novel naproxen/esomeprazole magnesium compound pellets based on acid-independent mechanism: in vitro and in vivo evaluation.

    Science.gov (United States)

    Lu, Jing; Kan, Shuling; Zhao, Yi; Zhang, Wenli; Liu, Jianping

    2016-09-01

    The purpose of this study was to develop the novel naproxen/esomeprazole magnesium compound pellets (novel-NAP/EMZ) depending on EMZ acid-independent mechanism which has been proved to be predominate in the mechanism of co-therapy with nonsteroidal anti-inflammatory drug. The novel-NAP/EMZ compound pellets, composed of NAP colon-specific pellets (NAP-CSPs) and EMZ modified-release pellets (EMZ-MRPs), were prepared by fluid-bed coating technology with desired in vitro release profiles. The resulting pellets were filled into hard gelatin capsules for in vivo evaluation in rats and compared with the reference compound pellets, consisted of NAP enteric-coated pellets (NAP-ECPs) and EMZ immediate-release pellets (EMZ-IRPs). The reference compound pellets were prepared simulating the drug delivery system of VIMOVO(®). In vivo pharmacokinetics, EMZ-MRPs had significantly larger AUC0-t (p pellets, the novel-NAP/EMZ compound pellets did not show distinct differences in histological mucosal morphology. However, biochemical tests exhibited enhanced total antioxidant capacity, increased nitric oxide content and reduced malondialdehyde level for novel-NAP/EMZ compound pellets, indicating that the acid-independent action took effect. The gastric pH values of novel-NAP/EMZ compound pellets were at a low and stable level, which could ensure normal physiological range of human gastric pH. As a result, the novel-NAP/EMZ compound pellets may be a more suitable formulation with potential advantages by improving bioavailability of drug and further reducing undesirable gastrointestinal damages.

  4. Engineered microenvironments for synergistic VEGF - Integrin signalling during vascularization.

    Science.gov (United States)

    Moulisová, Vladimíra; Gonzalez-García, Cristina; Cantini, Marco; Rodrigo-Navarro, Aleixandre; Weaver, Jessica; Costell, Mercedes; Sabater I Serra, Roser; Dalby, Matthew J; García, Andrés J; Salmerón-Sánchez, Manuel

    2017-05-01

    We have engineered polymer-based microenvironments that promote vasculogenesis both in vitro and in vivo through synergistic integrin-growth factor receptor signalling. Poly(ethyl acrylate) (PEA) triggers spontaneous organization of fibronectin (FN) into nanonetworks which provide availability of critical binding domains. Importantly, the growth factor binding (FNIII12-14) and integrin binding (FNIII9-10) regions are simultaneously available on FN fibrils assembled on PEA. This material platform promotes synergistic integrin/VEGF signalling which is highly effective for vascularization events in vitro with low concentrations of VEGF. VEGF specifically binds to FN fibrils on PEA compared to control polymers (poly(methyl acrylate), PMA) where FN remains in a globular conformation and integrin/GF binding domains are not simultaneously available. The vasculogenic response of human endothelial cells seeded on these synergistic interfaces (VEGF bound to FN assembled on PEA) was significantly improved compared to soluble administration of VEGF at higher doses. Early onset of VEGF signalling (PLCγ1 phosphorylation) and both integrin and VEGF signalling (ERK1/2 phosphorylation) were increased only when VEGF was bound to FN nanonetworks on PEA, while soluble VEGF did not influence early signalling. Experiments with mutant FN molecules with impaired integrin binding site (FN-RGE) confirmed the role of the integrin binding site of FN on the vasculogenic response via combined integrin/VEGF signalling. In vivo experiments using 3D scaffolds coated with FN and VEGF implanted in the murine fat pad demonstrated pro-vascularization signalling by enhanced formation of new tissue inside scaffold pores. PEA-driven organization of FN promotes efficient presentation of VEGF to promote vascularization in regenerative medicine applications. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  5. Hypoxia promotes adipose-derived stem cell proliferation via VEGF

    Directory of Open Access Journals (Sweden)

    Phuc Van Pham

    2016-01-01

    Full Text Available Adipose-derived stem cells (ADSCs are a promising mesenchymal stem cell source with therapeutic applications. Recent studies have shown that ADSCs could be expanded in vitro without phenotype changes. This study aimed to evaluate the effect of hypoxia on ADSC proliferation in vitro and to determine the role of vascular endothelial growth factor (VEGF in ADSC proliferation. ADSCs were selectively cultured from the stromal vascular fraction obtained from adipose tissue in DMEM/F12 medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic. ADSCs were cultured under two conditions: hypoxia (5% O2 and normal oxygen (21% O2. The effects of the oxygen concentration on cell proliferation were examined by cell cycle and doubling time. The expression of VEGF was evaluated by the ELISA assay. The role of VEGF in ADSC proliferation was studied by neutralizing VEGF with anti-VEGF monoclonal antibodies. We found that the ADSC proliferation rate was significantly higher under hypoxia compared with normoxia. In hypoxia, ADSCs also triggered VEGF expression. However, neutralizing VEGF with anti-VEGF monoclonal antibodies significantly reduced the proliferation rate. These results suggest that hypoxia stimulated ADSC proliferation in association with VEGF production. [Biomed Res Ther 2016; 3(1.000: 476-482

  6. Molecular Imaging of VEGF Receptors in Graft Arteriosclerosis

    Science.gov (United States)

    Zhang, Jiasheng; Razavian, Mahmoud; Tavakoli, Sina; Nie, Lei; Tellides, George; Backer, Joseph M.; Backer, Marina V.; Bender, Jeffrey R.; Sadeghi, Mehran M.

    2012-01-01

    Objectives Vascular endothelial growth factor (VEGF) signaling plays a key role in the pathogenesis of vascular remodeling, including graft arteriosclerosis (GA). GA is the major cause of late organ failure in cardiac transplantation. We used molecular near-infrared fluorescent (NIRF) imaging with an engineered Cy5.5-labeled single-chain VEGF tracer (scVEGF/Cy) to detect VEGF receptors (VEGFRs) and vascular remodeling in human coronary artery grafts by molecular imaging. Methods and Results VEGFR-specificity of probe uptake was shown by flow cytometry in endothelial cells. In severe combined immunodeficiency mice, transplantation of human coronary artery segments into the aorta followed by adoptive transfer of allogeneic human peripheral blood mononuclear cells (PBMCs) led to significant neointima formation in the grafts over a period of 4 weeks. NIRF imaging of transplant recipients at 4 weeks demonstrated focal uptake of scVEGF/Cy in remodeling artery grafts. Uptake specificity was demonstrated using an inactive homologue of scVEGF/Cy. scVEGF/Cy uptake predominantly localized in the neointima of remodeling coronary arteries and correlated with VEGFR-1, but not VEGFR-2 expression. There was a significant correlation between scVEGF/Cy uptake and transplanted artery neointima area. Conclusions Molecular imaging of VEGF receptors may provide a non-invasive tool for detection of GA in solid organ transplantation. PMID:22723442

  7. Endostatin inhibits VEGF-A induced osteoclastic bone resorption in vitro

    Directory of Open Access Journals (Sweden)

    Ilvesaro Joanna

    2006-07-01

    Full Text Available Abstract Background Endostatin is a C-terminal fragment of collagen XVIII which is a component of basement membranes with the structural properties of both collagens and proteoglycans. Endostatin has a major role in angiogenesis which is intimately associated with bone development and remodeling. Signaling between the endothelial cells and the bone cells, for example, may have a role in recruitment of osteoclastic precursor cells. Our study aims at exploring a possibility that endostatin, either as a part of basement membrane or as a soluble molecule, may control osteoclastogenesis and osteoclastic bone resorption in vitro. Methods Rat pit formation assay was employed in order to examine the effect of endostatin alone or in combination with vascular endothelial growth factor-A (VEGF-A on bone resorption in vitro. Effect of these agents on osteoclast differentiation in vitro was also tested. Osteoclastogenesis and the number of osteoclasts were followed by tartrate resistant acid phosphatase (TRACP staining and resorption was evaluated by measuring the area of excavated pits. Results Endostatin inhibited the VEGF-A stimulated osteoclastic bone resorption, whereas endostatin alone had no effect on the basal resorption level in the absence of VEGF-A. In addition, endostatin could inhibit osteoclast differentiation in vitro independent of VEGF-A. Conclusion Our in vitro data indicate that collagen XVIII/endostatin can suppress VEGF-A induced osteoclastic bone resorption to the basal level. Osteoclastogenesis is also inhibited by endostatin. The regulatory effect of endostatin, however, is not critical since endostatin alone does not modify the basal bone resorption.

  8. Clinical risk factors for the development of hypertension in patients treated with inhibitors of the VEGF signaling pathway.

    Science.gov (United States)

    Hamnvik, Ole-Petter R; Choueiri, Toni K; Turchin, Alexander; McKay, Rana R; Goyal, Lipika; Davis, Michael; Kaymakcalan, Marina D; Williams, Jonathan S

    2015-01-15

    VEGF signaling pathway inhibitor (anti-VEGF) therapy is associated with hypertension, but little is known about predisposing clinical characteristics. This study describes the real-world association between baseline clinical characteristics, blood pressure (BP) response, and survival in patients prescribed anti-VEGF therapies. Clinical data from Partners HealthCare in Massachusetts was obtained from adults treated with anti-VEGF therapies (2002-2013). Treatment-induced hypertensive response was defined as worsening of preexisting hypertension or new diagnosis of hypertension (if no prior hypertension history). Data from 1120 patients with renal cell carcinoma (32.2%), hepatocellular carcinoma (11.6%), gastrointestinal stromal tumors (12.5%), and other sarcomas (15.3%) were analyzed. Most patients received sunitinib (52%), sorafenib (25.9%), or pazopanib (18%). A treatment-induced hypertensive response was identified in 49.7% of treated patients. Preexisting hypertension, present in 65.4%, was an independent risk factor for BP elevation (odds ratio [OR], 1.56; 95% confidence interval [CI], 1.27-1.92); other risk factors included age ≥60 years (OR, 1.26; 95% CI, 1.06-1.52), and body mass index (BMI) ≥25 kg/m(2) (OR, 1.26; 95% CI, 1.04-1.53). Race, sex, anti-VEGF therapy prescribed, and baseline antihypertensive class were not significant risk factors. The absolute observed mean increase in BP was 21 mm Hg (systolic)/15 mm Hg (diastolic), both in patients with and without preexisting hypertension. The development of hypertension predicted improved survival (hazard ratio, 0.76; 95% CI, 0.65-0.89). Preexisting hypertension, age, and BMI identify patients at risk for significant anti-VEGF therapy-induced BP elevation. Hypertension appears to be a clinical biomarker of efficacy of anti-VEGF therapies in a broad range of malignancies. © 2014 American Cancer Society.

  9. 5-azacytidine induces transcriptome changes in Escherichia coli via DNA methylation-dependent and DNA methylation-independent mechanisms.

    Science.gov (United States)

    Militello, Kevin T; Simon, Robert D; Mandarano, Alexandra H; DiNatale, Anthony; Hennick, Stacy M; Lazatin, Justine C; Cantatore, Sarah

    2016-06-27

    Escherichia coli K-12 strains contain DNA cytosine methyltransferase (Dcm), which generates 5-methylcytosine at 5'CCWGG3' sites. Although the role of 5-methylcytosine in eukaryotic gene expression is relatively well described, the role of 5-methylcytosine in bacterial gene expression is largely unknown. To identify genes that are controlled by 5-methylcytosine in E. coli, we compared the transcriptomes of cells grown in the absence and presence of the DNA methylation inhibitor 5-azacytidine. We observed expression changes for 63 genes. The majority of the gene expression changes occurred at early stationary phase and were up-regulations. To identify gene expression changes due to a loss of DNA methylation, we compared the expression of selected genes in a wild-type and dcm knockout strain via reverse transcription quantitative PCR. Our data indicate that 5-azacytidine can influence gene expression by at least two distinct mechanisms: DNA methylation loss and a mechanism that is independent of DNA methylation loss. In addition, we have identified new targets of 5-methylcytosine-mediated regulation of gene expression. In summary, our data indicate that 5-azacytidine impacts the composition of the bacterial transcriptome, and the primary effect is increased gene expression at early stationary phase.

  10. Sucralose, an activator of the glucose-sensing receptor, increases ATP by calcium-dependent and -independent mechanisms.

    Science.gov (United States)

    Li, Longfei; Ohtsu, Yoshiaki; Nakagawa, Yuko; Masuda, Katsuyoshi; Kojima, Itaru

    2016-08-31

    Sucralose is an artificial sweetener and activates the glucose-sensing receptor expressed in pancreatic β-cells. Although sucralose does not enter β-cells nor acts as a substrate for glucokinase, it induces a marked elevation of intracellular ATP ([ATP]c). The present study was conducted to identify the signaling pathway responsible for the elevation of [ATP]c induced by sucralose. Previous studies have shown that sucralose elevates cyclic AMP (cAMP), activates phospholipase C (PLC) and stimulates Ca(2+) entry by a Na(+)-dependent mechanism in MIN6 cells. The addition of forskolin induced a marked elevation of cAMP, whereas it did not affect [ATP]c. Carbachol, an activator of PLC, did not increase [ATP]c. In addition, activation of protein kinase C by dioctanoylglycerol did not affect [ATP]c. In contrast, nifedipine, an inhibitor of the voltage-dependent Ca(2+) channel, significantly reduced [ATP]c response to sucralose. Removal of extracellular Na(+) nearly completely blocked sucralose-induced elevation of [ATP]c. Stimulation of Na(+) entry by adding a Na(+) ionophore monensin elevated [ATP]c. The monensin-induced elevation of [ATP]c was only partially inhibited by nifedipine and loading of BAPTA, both of which completely abolished elevation of [Ca(2+)]c. These results suggest that Na(+) entry is critical for the sucralose-induced elevation of [ATP]c. Both calcium-dependent and -independent mechanisms are involved in the action of sucralose.

  11. MiRNA-directed regulation of VEGF and other angiogenic factors under hypoxia.

    Directory of Open Access Journals (Sweden)

    Zhong Hua

    Full Text Available MicroRNAs (miRNAs are a class of 20-24 nt non-coding RNAs that regulate gene expression primarily through post-transcriptional repression or mRNA degradation in a sequence-specific manner. The roles of miRNAs are just beginning to be understood, but the study of miRNA function has been limited by poor understanding of the general principles of gene regulation by miRNAs. Here we used CNE cells from a human nasopharyngeal carcinoma cell line as a cellular system to investigate miRNA-directed regulation of VEGF and other angiogenic factors under hypoxia, and to explore the principles of gene regulation by miRNAs. Through computational analysis, 96 miRNAs were predicted as putative regulators of VEGF. But when we analyzed the miRNA expression profile of CNE and four other VEGF-expressing cell lines, we found that only some of these miRNAs could be involved in VEGF regulation, and that VEGF may be regulated by different miRNAs that were differentially chosen from 96 putative regulatory miRNAs of VEGF in different cells. Some of these miRNAs also co-regulate other angiogenic factors (differential regulation and co-regulation principle. We also found that VEGF was regulated by multiple miRNAs using different combinations, including both coordinate and competitive interactions. The coordinate principle states that miRNAs with independent binding sites in a gene can produce coordinate action to increase the repressive effect of miRNAs on this gene. By contrast, the competitive principle states when multiple miRNAs compete with each other for a common binding site, or when a functional miRNA competes with a false positive miRNA for the same binding site, the repressive effects of miRNAs may be decreased. Through the competitive principle, false positive miRNAs, which cannot directly repress gene expression, can sometimes play a role in miRNA-mediated gene regulation. The competitive principle, differential regulation, multi-miRNA binding sites, and false

  12. Dietary compound isoliquiritigenin inhibits breast cancer neoangiogenesis via VEGF/VEGFR-2 signaling pathway.

    Directory of Open Access Journals (Sweden)

    Zhiyu Wang

    Full Text Available Angiogenesis is crucial for cancer initiation, development and metastasis. Identifying natural botanicals targeting angiogenesis has been paid much attention for drug discovery in recent years, with the advantage of increased safety. Isoliquiritigenin (ISL is a dietary chalcone-type flavonoid with various anti-cancer activities. However, little is known about the anti-angiogenic activity of isoliquiritigenin and its underlying mechanisms. Herein, we found that ISL significantly inhibited the VEGF-induced proliferation of human umbilical vein endothelial cells (HUVECs at non-toxic concentration. A series of angiogenesis processes including tube formation, invasion and migration abilities of HUVECs were also interrupted by ISL in vitro. Furthermore, ISL suppressed sprout formation from VEGF-treated aortic rings in an ex-vivo model. Molecular mechanisms study demonstrated that ISL could significantly inhibit VEGF expression in breast cancer cells via promoting HIF-1α (Hypoxia inducible factor-1α proteasome degradation and directly interacted with VEGFR-2 to block its kinase activity. In vivo studies further showed that ISL administration could inhibit breast cancer growth and neoangiogenesis accompanying with suppressed VEGF/VEGFR-2 signaling, elevated apoptosis ratio and little toxicity effects. Molecular docking simulation indicated that ISL could stably form hydrogen bonds and aromatic interactions within the ATP-binding region of VEGFR-2. Taken together, our study shed light on the potential application of ISL as a novel natural inhibitor for cancer angiogenesis via the VEGF/VEGFR-2 pathway. Future studies of ISL for chemoprevention or chemosensitization against breast cancer are thus warranted.

  13. Anti-VEGF treatment for myopic choroid neovascularization: from molecular characterization to update on clinical application

    Directory of Open Access Journals (Sweden)

    Zhang Y

    2015-07-01

    Full Text Available Yan Zhang,1 Qian Han,2 Yusha Ru,1 Qiyu Bo,1 Rui Hua Wei1 1Tianjin Medical University Eye Hospital, Tianjin Medical University Eye Institute, College of Optometry and Ophthalmology, Tianjin Medical University, Tianjin, 2Tangshan Eye Hospital, Tangshan, Hebei Province, People’s Republic of China Abstract: Choroidal neovascularization (CNV secondary to pathologic myopia has a very high incidence in global, especially in Asian, populations. It is a common cause of irreversible central vision loss, and severely affects the quality of life in the patients with pathologic myopia. The traditional therapeutic modalities for CNV secondary to pathologic myopia include thermal laser photocoagulation, surgical management, transpupillary thermotherapy, and photodynamic therapy with verteporfin. However, the long-term outcomes of these modalities are disappointing. Recently, intravitreal administration of anti-VEGF biological agents, including bevacizumab, ranibizumab, pegaptanib, aflibercept, and conbercept, has demonstrated promising outcomes for this ocular disease. The anti-VEGF regimens are more effective on improving visual acuity, reducing central fundus thickness and central retina thickness than the traditional modalities. These anti-VEGF agents thus hold the potential to become the first-line medicine for treatment of CNV secondary to pathologic myopia. This review follows the trend of “from bench to bedside”, initially discussing the pathogenesis of myopic CNV, delineating the molecular structures and mechanisms of action of the currently available anti-VEGF drugs, and then systematically comparing the up to date clinical applications as well as the efficacy and safety of the anti-VEGF drugs to the CNV secondary to pathologic myopia. Keywords: formation of new vessels, choroid membrane, pathologic myopia, vascular endothelial growth factor, molecular mechanisms, clinical trials

  14. [Human umbilic mesenchymal stromal cells repairs diabetic foot in rats associated with VEGF expressional change].

    Science.gov (United States)

    Wang, Ying; Dan, Qi-Qin; Wang, Qing-Ping; Zhou, Ning; Jin, Xing-Fang; Hou, Zong-Liua; Peng, Bao-Kun; Wang, Ting-Hua

    2014-01-01

    OBJECTITVE: To explore the mechanism of human umbilic mesenchymal cells (HUMSCs) implantation for the treatment of diabetic foot in rats associate with vascular endothelia growth factor (VEGF) expression changes. After diabetic foot model in rats were established by administration of streptozotozin (STZ) in intraperitoneal injection (2 weeks), ulceration in foot was induced by incision injury combined with swearing staphylococcus aureas. Then, HUMSCs were smeared on the ulceration of foot in diabetic rats. Ten days later, the densities of blood vessel and the level of VEGF expression were determined by using immunohistochemistry, RT-PCR and Western blot. HUMSC grafts reduced significantly the volume of ulceration in diabetic foot rats (P < 0.05). RT-PCR and Western blot showed that VEGF and its mRNA were significantly upregulated (P < 0.05). VEGF immunstaining was found in blood vessels and the densities of blood vessels in HUMSC group were increased significantly (P < 0.05). HUMSC implantation showed a positive role in promoting the recovery of the ulceration in foot with diabetic rats.

  15. Rapamycin reversal of VEGF-C-driven lymphatic anomalies in the respiratory tract.

    Science.gov (United States)

    Baluk, Peter; Yao, Li-Chin; Flores, Julio C; Choi, Dongwon; Hong, Young-Kwon; McDonald, Donald M

    2017-08-17

    Lymphatic malformations are serious but poorly understood conditions that present therapeutic challenges. The goal of this study was to compare strategies for inducing regression of abnormal lymphatics and explore underlying mechanisms. CCSP-rtTA/tetO-VEGF-C mice, in which doxycycline regulates VEGF-C expression in the airway epithelium, were used as a model of pulmonary lymphangiectasia. After doxycycline was stopped, VEGF-C expression returned to normal, but lymphangiectasia persisted for at least 9 months. Inhibition of VEGFR-2/VEGFR-3 signaling, Notch, β-adrenergic receptors, or autophagy and antiinflammatory steroids had no noticeable effect on the amount or severity of lymphangiectasia. However, rapamycin inhibition of mTOR reduced lymphangiectasia by 76% within 7 days without affecting normal lymphatics. Efficacy of rapamycin was not increased by coadministration with the other agents. In prevention trials, rapamycin suppressed VEGF-C-driven mTOR phosphorylation and lymphatic endothelial cell sprouting and proliferation. However, in reversal trials, no lymphatic endothelial cell proliferation was present to block in established lymphangiectasia, and rapamycin did not increase caspase-dependent apoptosis. However, rapamycin potently suppressed Prox1 and VEGFR-3. These experiments revealed that lymphangiectasia is remarkably resistant to regression but is responsive to rapamycin, which rapidly reduces and normalizes the abnormal lymphatics without affecting normal lymphatics.

  16. Targeting VEGF in canine oxygen-induced retinopathy - a model for human retinopathy of prematurity

    Directory of Open Access Journals (Sweden)

    McLeod DS

    2016-05-01

    Full Text Available D Scott McLeod, Gerard A Lutty Department of Ophthalmology, Wilmer Ophthalmological Institute, Johns Hopkins Hospital, Baltimore, MD, USA Abstract: Development of the dog superficial retinal vasculature is similar to the mechanism of human retinal vasculature development; they both develop by vasculogenesis, differentiation, and assembly of vascular precursors called angioblasts. Canine oxygen-induced retinopathy (OIR was first developed by Arnall Patz in an effort to experimentally determine the effects of hyperoxia on the development of the retinal vasculature. The canine OIR model has many characteristics in common with human retinopathy of prematurity. Exposure of 1-day-old dogs to hyperoxia for 4 days causes a vaso-obliteration throughout the retina. Vasoproliferation, after the animals have returned to room air, is robust. The initial small preretinal neovascular formations anastomose to form large preretinal membranes that eventually cause tractional retinal folds. The end-stage pathology of the canine model is similar to stage IV human retinopathy of prematurity. Therefore, canine OIR is an excellent forum to evaluate the response to drugs targeting VEGF and its receptors. Evaluation of an antibody to VEGF-R2 and the VEGF-Trap demonstrated that doses should be titered down so that preretinal neovascularization is inhibited but retinal revascularization is able to proceed, vascularizing peripheral retina and preventing it from being a source of VEGF. Keywords: angioblasts, blood vessels, endothelial cells, oxygen, retinopathy, retina, vascular endothelial cell growth factor

  17. Mechanical prophylaxis is a heparin-independent risk for anti–platelet factor 4/heparin antibody formation after orthopedic surgery

    Science.gov (United States)

    Bito, Seiji; Migita, Kiyoshi; Nakamura, Mashio; Shinohara, Kazuhito; Sato, Tomotaro; Tonai, Takeharu; Shimizu, Motoyuki; Shibata, Yasuhiro; Kishi, Kazuhiko; Kubota, Chikara; Nakahara, Shinnosuke; Mori, Toshihito; Ikeda, Kazuo; Ota, Shusuke; Minamizaki, Takeshi; Yamada, Shigeru; Shiota, Naofumi; Kamei, Masataka; Motokawa, Satoru

    2016-01-01

    Platelet-activating antibodies, which recognize platelet factor 4 (PF4)/heparin complexes, induce spontaneous heparin-induced thrombocytopenia (HIT) syndrome or fondaparinux-associated HIT without exposure to unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH). This condition mostly occurs after major orthopedic surgery, implying that surgery itself could trigger this immune response, although the mechanism is unclear. To investigate how surgery may do so, we performed a multicenter, prospective study of 2069 patients who underwent total knee arthroplasty (TKA) or hip arthroplasty. Approximately half of the patients received postoperative thromboprophylaxis with UFH, LMWH, or fondaparinux. The other half received only mechanical thromboprophylaxis, including dynamic (intermittent plantar or pneumatic compression device), static (graduated compression stockings [GCSs]), or both. We measured anti-PF4/heparin immunoglobulins G, A, and M before and 10 days after surgery using an immunoassay. Multivariate analysis revealed that dynamic mechanical thromboprophylaxis (DMT) was an independent risk factor for seroconversion (odds ratio [OR], 2.01; 95% confidence interval [CI], 1.34-3.02; P = .001), which was confirmed with propensity-score matching (OR, 1.99; 95% CI, 1.17-3.37; P = .018). For TKA, the seroconversion rates in patients treated with DMT but no anticoagulation and in patients treated with UFH or LMWH without DMT were similar, but significantly higher than in patients treated with only GCSs. The proportion of patients with ≥1.4 optical density units appeared to be higher among those treated with any anticoagulant plus DMT than among those not treated with DMT. Our study suggests that DMT increases risk of an anti-PF4/heparin immune response, even without heparin exposure. This trial was registered to www.umin.ac.jp/ctr as #UMIN000001366. PMID:26659923

  18. Novel VEGF decoy receptor fusion protein conbercept targeting multiple VEGF isoforms provide remarkable anti-angiogenesis effect in vivo.

    Directory of Open Access Journals (Sweden)

    Qin Wang

    Full Text Available VEGF family factors are known to be the principal stimulators of abnormal angiogenesis, which play a fundamental role in tumor and various ocular diseases. Inhibition of VEGF is widely applied in antiangiogenic therapy. Conbercept is a novel decoy receptor protein constructed by fusing VEGF receptor 1 and VEGF receptor 2 extracellular domains with the Fc region of human immunoglobulin. In this study, we systematically evaluated the binding affinity of conbercept with VEGF isoforms and PlGF by using anti-VEGF antibody (Avastin as reference. BIACORE and ELISA assay results indicated that conbercept could bind different VEGF-A isoforms with higher affinity than reference. Furthermore, conbercept could also bind VEGF-B and PlGF, whereas Avastin showed no binding. Oxygen-induced retinopathy model showed that conbercept could inhibit the formation of neovasularizations. In tumor-bearing nude mice, conbercept could also suppress tumor growth very effectively in vivo. Overall, our study have demonstrated that conbercept could bind with high affinity to multiple VEGF isoforms and consequently provide remarkable anti-angiogenic effect, suggesting the possibility to treat angiogenesis-related diseases such as cancer and wet AMD etc.

  19. Metformin suppresses adipogenesis through both AMP-activated protein kinase (AMPK)-dependent and AMPK-independent mechanisms.

    Science.gov (United States)

    Chen, Suet Ching; Brooks, Rebecca; Houskeeper, Jessica; Bremner, Shaun K; Dunlop, Julia; Viollet, Benoit; Logan, Pamela J; Salt, Ian P; Ahmed, S Faisal; Yarwood, Stephen J

    2017-01-15

    People with Type 2 diabetes mellitus (T2DM) have reduced bone mineral density and an increased risk of fractures due to altered mesenchymal stem cell (MSC) differentiation in the bone marrow. This leads to a shift in the balance of differentiation away from bone formation (osteogenesis) in favour of fat cell development (adipogenesis). The commonly used anti-diabetic drug, metformin, activates the osteogenic transcription factor Runt-related transcription factor 2 (Runx2), which may suppress adipogenesis, leading to improved bone health. Here we investigate the involvement of the metabolic enzyme, AMP-activated protein kinase (AMPK), in these protective actions of metformin. The anti-adipogenic actions of metformin were observed in multipotent C3H10T1/2 MSCs, in which metformin exerted reciprocal control over the activities of Runx2 and the adipogenic transcription factor, PPARγ, leading to suppression of adipogenesis. These effects appeared to be independent of AMPK activation but rather through the suppression of the mTOR/p70S6K signalling pathway. Basal AMPK and mTOR/p70S6K activity did appear to be required for adipogenesis, as demonstrated by the use of the AMPK inhibitor, compound C. This observation was further supported by using AMPK knockout mouse embryo fibroblasts (MEFs) where adipogenesis, as assessed by reduced lipid accumulation and expression of the adipogeneic transcription factor, C/EBPβ, was found to display an absolute requirement for AMPK. Further activation of AMPK in wild type MEFS, with either metformin or the AMPK-specific activator, A769662, was also associated with suppression of adipogenesis. It appears, therefore, that basal AMPK activity is required for adipogenesis and that metformin can inhibit adipogenesis through AMPK-dependent or -independent mechanisms, depending on the cellular context. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  20. NF-κB dependent and independent mechanisms of quartz-induced proinflammatory activation of lung epithelial cells

    Science.gov (United States)

    2010-01-01

    In the initiation and progression of pulmonary inflammation, macrophages have classically been considered as a crucial cell type. However, evidence for the role of epithelial type II cells in pulmonary inflammation has been accumulating. In the current study, a combined in vivo and in vitro approach has been employed to investigate the mechanisms of quartz-induced proinflammatory activation of lung epithelial cells. In vivo, enhanced expression of the inflammation- and oxidative stress-related genes HO-1 and iNOS was found on the mRNA level in rat lungs after instillation with DQ12 respirable quartz. Activation of the classical NF-κB pathway in macrophages and type II pneumocytes was indicated by enhanced immunostaining of phospho-IκBα in these specific lung cell types. In vitro, the direct, particle-mediated effect on proinflammatory signalling in a rat lung epithelial (RLE) cell line was compared to the indirect, macrophage product-mediated effect. Treatment with quartz particles induced HO-1 and COX-2 mRNA expression in RLE cells in an NF-κB independent manner. Supernatant from quartz-treated macrophages rapidly activated the NF-κB signalling pathway in RLE cells and markedly induced iNOS mRNA expression up to 2000-fold compared to non-treated control cells. Neutralisation of TNFα and IL-1β in macrophage supernatant did not reduce its ability to elicit NF-κB activation of RLE cells. In addition the effect was not modified by depletion or supplementation of intracellular glutathione. The results from the current work suggest that although both oxidative stress and NF-κB are likely involved in the inflammatory effects of toxic respirable particles, these phenomena can operate independently on the cellular level. This might have consequences for in vitro particle hazard testing, since by focusing on NF-κB signalling one might neglect alternative inflammatory pathways. PMID:20492675

  1. Identification and characterization of VEGF and FGF from Hydra.

    Science.gov (United States)

    Krishnapati, Lakshmi-Surekha; Ghaskadbi, Surendra

    2013-01-01

    Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) play important roles in the formation of the blood vascular system and in axon guidance, nervous system development and function. Here, we report isolation and characterization of VEGF and FGF homologues from Hydra vulgaris Ind-Pune, a Cnidarian which exhibits an organized nervous system and primitive epithelio-muscular cells. VEGF expression was prominent in the endoderm of the peduncle region and tentacles, as evident from in situ hybridization of whole polyps and its transverse sections. High levels of FGF were detected in the ectoderm of the budding region. The expression of VEGF in endodermal and FGF in interstitial cells was confirmed using sf-1 hydra, a temperature-sensitive mutant strain of Hydra magnipapillata. Tissue-specific expression of VEGF and FGF was confirmed by semi quantitative RT-PCR for ectodermal and endodermal tissues in H. vulgaris Ind-Pune. Treatment with SU5416, a specific inhibitor of the VEGF receptor, did not affect the whole polyp, but did delay both budding and head regeneration, suggesting a possible role of VEGF in nerve cell development, tube formation and/or in branching. FGF expression in the ectoderm of budding region, where the majority of interstitial stem cells reside suggests its role in interstitial stem cell maintenance. Further, activation of canonical Wnt signalling with the glycogen synthase kinase-3β (GSK-3β) inhibitor alsterpaullone caused down-regulation of VEGF and FGF, suggesting an antagonistic relationship between the Wnt and VEGF/FGF pathways. Our results indicate that VEGF and FGF evolved early in evolution, before the development of the blood vascular system, and open up the possibility of elucidating the evolutionarily ancient functions of VEGF and FGF.

  2. The mechanism of epipodophyllotoxin-induced thymocyte apoptosis: possible role of a novel Ca(2+)-independent protein kinase.

    Science.gov (United States)

    Ye, X; Georgoff, I; Fleisher, S; Coffman, F D; Cohen, S; Fresa, K L

    1993-10-15

    The epipodophyllotoxins, etoposide (VP-16) and teniposide (VM-26), inhibit topoisomerase II activity by stabilization of the cleavable complex between the enzyme and DNA and formation of protein-bound double-stranded DNA breaks. While it is thought that these agents are cytotoxic by preventing cells from completing the S phase or undergoing mitosis, recent evidence suggests that these agents are also potent inducers of programmed cell death or apoptosis in both normal and malignant cells. We have examined the intracellular pathway leading to epipodophyllotoxin-induced apoptosis in normal mouse thymocytes. Epipodophyllotoxin-induced apoptosis may proceed via a mechanism that is independent of inhibition of topoisomerase activity per se because novobiocin and coumermycin, which inhibit the ATPase subunit of topoisomerase II, were relatively inefficient inducers of apoptosis in these cells, under conditions where strong apoptosis by the epipodophyllotoxins and dexamethasone could be observed. In addition, camptothecin, which inhibits topoisomerase I by stabilization of the cleavable complex between that enzyme and DNA, was also a poor inducer of apoptosis in these cells. Our data suggest that epipodophyllotoxin-induced mouse thymocyte apoptosis, like that induced by dexamethasone, proceeds via a mechanism that involves protein kinase C (PKC) or a similar enzyme. Apoptosis induced by VM-26 or by dexamethasone was inhibited by 1-(5-isoquinolinylsulfonyl)-2- methylpiperazine dihydrochloride (H7), an inhibitor of both PKC and cAMP-dependent protein kinases, but was relatively unaffected by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a more specific inhibitor of cAMP-dependent protein kinases. A more specific inhibitor of PKC, sangivamycin, also inhibited both VM-26-induced and dexamethasone-induced apoptosis. Both VM-26- and dexamethasone-induced apoptosis were unaffected by EGTA, a calcium (Ca2+) chelator, under conditions that inhibited apoptosis induced by

  3. β-adrenergic receptor activation in immortalized human urothelial cells stimulates inflammatory responses by PKA-independent mechanisms

    Directory of Open Access Journals (Sweden)

    Porter James E

    2005-08-01

    -AR agonist isoproterenol. However, the increased production of iNOS and COX-2 by isoproterenol is not blocked when UROtsa cells are preincubated with inhibitors of PKA. Therefore, UROtsa cell β-AR activation significantly increases the amount of iNOS and COX-2 produced by a PKA-independent mechanism. Consequently, this immortalized human urothelial cell line can be useful in characterizing potential AR signaling mechanisms associated with chronic inflammatory diseases of the bladder.

  4. Circulating VEGF as a biological marker in patients with rheumatoid arthritis? Preanalytical and biological variability in healthy persons and in patients

    DEFF Research Database (Denmark)

    Hetland, Merete Lund; Christensen, Ib Jarle; Lottenburger, Tine

    2008-01-01

    BACKGROUND: Soluble vascular endothelial growth factor (VEGF) is a promising biomarker in monitoring rheumatoid arthritis (RA), but studies of pre-analytical and biologic variability are few. METHODS: VEGF was measured by ELISA methods in serum and plasma from healthy persons and RA patients. Pre....../ml (range: non-detectable to 352); serum: 328 pg/ml (53-1791)) were independent of gender and age. Short- and long-term biologic variability included diurnal variation (sampling should take place after 7 AM) and impact of exercise (increased VEGF immediately after bicycling normalised within 1 hour......). CONCLUSIONS: Pre-analytical factors and biologic variability including diurnal variation and impact of exercise should be accounted for in future studies that include circulating VEGF as a biological marker....

  5. Involvement of Potassium Channels and Calcium-Independent Mechanisms in Hydrogen Sulfide-Induced Relaxation of Rat Mesenteric Small Arteries.

    Science.gov (United States)

    Hedegaard, Elise R; Gouliaev, Anja; Winther, Anna K; Arcanjo, Daniel D R; Aalling, Mathilde; Renaltan, Nirthika S; Wood, Mark E; Whiteman, Matthew; Skovgaard, Nini; Simonsen, Ulf

    2016-01-01

    Endogenous hydrogen sulfide (H2S) is involved in the regulation of vascular tone. We hypothesized that the lowering of calcium and opening of potassium (K) channels as well as calcium-independent mechanisms are involved in H2S-induced relaxation in rat mesenteric small arteries. Amperometric recordings revealed that free [H2S] after addition to closed tubes of sodium hydrosulfide (NaHS), Na2S, and GYY4137 [P-(4-methoxyphenyl)-P-4-morpholinyl-phosphinodithioic acid] were, respectively, 14%, 17%, and 1% of added amount. The compounds caused equipotent relaxations in isometric myographs, but based on the measured free [H2S], GYY4137 caused more relaxation in relation to released free H2S than NaHS and Na2S in rat mesenteric small arteries. Simultaneous measurements of [H2S] and tension showed that 15 µM of free H2S caused 61% relaxation in superior mesenteric arteries. Simultaneous measurements of smooth muscle calcium and tension revealed that NaHS lowered calcium and caused relaxation of NE-contracted arteries, while high extracellular potassium reduced NaHS relaxation without corresponding calcium changes. In NE-contracted arteries, NaHS (1 mM) lowered the phosphorylation of myosin light chain, while phosphorylation of myosin phosphatase target subunit 1 remained unchanged. Protein kinase A and G, inhibitors of guanylate cyclase, failed to reduce NaHS relaxation, whereas blockers of voltage-gated KV7 channels inhibited NaHS relaxation, and blockers of mitochondrial complex I and III abolished NaHS relaxation. Our findings suggest that low micromolar concentrations of free H2S open K channels followed by lowering of smooth muscle calcium, and by another mechanism involving mitochondrial complex I and III leads to uncoupling of force, and hence vasodilation. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  6. Carbachol-Mediated Endocytosis of NHE3 Involves a Clathrin-Independent Mechanism Requiring Lipid Rafts and Cdc42

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    Nicholas C. Zachos

    2014-03-01

    Full Text Available Background: In intestinal epithelial cells, acute regulation of the brush border Na+/H+ exchanger, NHE3, usually occurs by changes in endocytosis and/or exocytosis. Constitutive NHE3 endocytosis involves clathrin. Carbachol (CCH, which elevates intracellular Ca2+ ([Ca2+]i, decreases NHE3 activity and stimulates endocytosis; however, the mechanism involved in calcium-mediated endocytosis of NHE3 is unclear. A pool of NHE3 resides in lipid rafts, which contributes to basal, but not cAMP-mediated, NHE3 trafficking, suggesting that an alternative mechanism exists for NHE3 endocytosis. Cdc42 was demonstrated to play an integral role in some cases of cholesterol-sensitive, clathrin-independent endocytosis. Therefore, the current study was designed to test the hypotheses that (1 clathrin-mediated endocytosis (CME is involved in constitutive, but not CCH-mediated, endocytosis of NHE3, and (2 CCH-mediated endocytosis of NHE3 occurs through a lipid raft, activated Cdc42-dependent pathway that does not involve clathrin. Methods: The role of Cdc42 and lipid rafts on NHE3 activity and endocytosis were investigated in polarized Caco-2/BBe cells using pharmacological and shRNA knockdown approaches. Results: Basal NHE3 activity was increased in the presence of CME blockers (chlorpromazine; K+ depletion supporting previous reports that constitutive NHE3 endocytosis is clathrin dependent. In contrast, CCH-inhibition of NHE3 activity was abolished in Caco-2/BBe cells treated with MβCD (to disrupt lipid rafts as well as in Cdc42 knockdown cells but was unaffected by CME blockers. Conclusion: CCH-mediated inhibition of NHE3 activity is not dependent on clathrin and involves lipid rafts and requires Cdc42.

  7. Elevated serum and tissue VEGF associated with poor outcome in ...

    African Journals Online (AJOL)

    Enas Mohamed Ali

    Abstract Vascular endothelial growth factor (VEGF) has a potent angiogenesis functions in experimental models, although their role in the progression of human breast cancer is unclear. The aim of the current study was to examine the expression pattern of VEGF in serum and tissues of breast cancer patients, examine the ...

  8. Increased vascular endothelial growth factor (VEGF) expression in ...

    African Journals Online (AJOL)

    Vascular endothelial growth factor (VEGF), a well known angiogenic factor, has been shown to have direct and/or indirect influence on spinal cord injury (SCI). The purpose of this study is to observe VEGF expression changes in rats with SCI by bone marrow stromal cells (BMSCs) treatment. The mRNA expression of VEGF ...

  9. VEGF, the underlying factor for metabolic syndrome; fact or fiction?

    Science.gov (United States)

    Mazidi, Mohsen; Rezaie, Peyman; Kengne, A P; Stathopoulou, Maria G; Azimi-Nezhad, Mohsen; Siest, Sophie

    2017-11-01

    Metabolic syndrome (MetS) is currently diagnosed by the co-presence of at least three of the five following abnormalities: abdominal obesity, dysglycaemia, elevated serum triglycerides, low high-density cholesterol (HDL) and finally elevated blood pressure. Metabolic syndrome increases the risk of developing cardiovascular disease and diabetes. This review is on the associations between MetS and vascular endothelial growth factor (VEGF). VEGF induces migration and proliferation of endothelial cells (ECs), increases vascular permeability and has a role in tumor growth, adipose tissue expansion, age-related macular degeneration and diabetic retinopathy. Circulating levels of VEGFs are elevated in obese individuals and it has also been suggested that VEGF is secreted from adipose tissues, especially from intra-abdominal adipose tissue. There is abundant evidence to support that poor glycemic control in diabetic patients is associated with increased plasma VEGF, which in turn may cause hypertension and several vascular complications in diabetic patients. Circulating VEGF levels are increased in children and young adults with type 1 diabetes mellitus and middle-aged diabetic patients with proliferative retinopathy. It has been revealed that plasma VEGF increases in patients with hyperlipidemia and may trigger the development of atherosclerosis. It can be concluded that there is a positive association between VEGF and components of MetS. Because of the importance of this relationship, more investigations are needed in this field. Copyright © 2016. Published by Elsevier Ltd.

  10. Anti-VEGF antibodies mitigate the development of radiation necrosis in mouse brain

    Science.gov (United States)

    Jiang, Xiaoyu; Engelbach, John A; Yuan, Liya; Cates, Jeremy; Gao, Feng; Drzymala, Robert E; Hallahan, Dennis E; Rich, Keith M; Schmidt, Robert E; Ackerman, Joseph JH; Garbow, Joel R

    2014-01-01

    Purpose To quantify the effectiveness of anti-VEGF antibodies (bevacizumab and B20-4.1.1) as mitigators of radiation-induced, CNS (brain) necrosis in a mouse model. Experimental Design Cohorts of mice were irradiated with single-fraction 50- or 60-Gy doses of radiation targeted to the left hemisphere (brain) using the Leksell Perfexion Gamma Knife. The onset and progression of radiation necrosis were monitored longitudinally by in vivo, small-animal MRI, beginning four weeks post-irradiation. MRI-derived necrotic volumes for antibody (Ab)-treated and untreated mice were compared. MRI results were supported by correlative histology. Results Hematoxylin and eosin stained sections of brains from irradiated, non-Ab-treated mice confirmed profound tissue damage, including regions of fibrinoid vascular necrosis, vascular telangiectasia, hemorrhage, loss of neurons, and edema. Treatment with the murine anti-VEGF antibody B20-4.1.1 mitigated radiation-induced changes in an extraordinary, highly statistically-significant manner. The development of radiation necrosis in mice under treatment with bevacizumab (a humanized anti-VEGF antibody) was intermediate between that for B20-4.1.1-treated and non-Ab-treated animals. MRI findings were validated by histologic assessment, which confirmed that anti-VEGF-antibody treatment dramatically reduced late-onset necrosis in irradiated brain. Conclusions The single-hemispheric-irradiation mouse model, with longitudinal MRI monitoring, provides a powerful platform for studying the onset and progression of radiation necrosis and for developing and testing new therapies. The observation that anti-VEGF antibodies are effective mitigants of necrosis in our mouse model will enable a wide variety of studies aimed at dose optimization and timing and mechanism of action with direct relevance to ongoing clinical trials of bevacizumab as a treatment for radiation necrosis. PMID:24647570

  11. TAK1 (MAP3K7 signaling regulates hematopoietic stem cells through TNF-dependent and -independent mechanisms.

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    Giichi Takaesu

    Full Text Available A cytokine/stress signaling kinase Tak1 (Map3k7 deficiency is known to impair hematopoietic progenitor cells. However, the role of TAK1 signaling in the stem cell function of the hematopoietic system is not yet well defined. Here we characterized hematopoietic stem cells (HSCs harboring deletion of Tak1 and its activators, Tak1 binding proteins 1 and 2 (Tab1 and Tab2 using a competitive transplantation assay in a mouse model. Tak1 single or Tab1/Tab2 double deletions completely eliminated the reconstitution activity of HSCs, whereas Tab1 or Tab2 single deletion did not cause any abnormality. Tak1 single or Tab1/Tab2 double deficient lineage-negative, Sca-1(+, c-Kit(+ (LSK cells did not proliferate and underwent cell death. We found that Tnfr1 deficiency restored the reconstitution activity of Tak1 deficient bone marrow cells for 6-18 weeks. However, the reconstitution activity of Tak1- and Tnfr1-double deficient bone marrow cells declined over the long term, and the number of phenotypically identified long-term hematopoietic stem cells were diminished. Our results indicate that TAB1- or TAB2-dependent activation of TAK1 is required for maintenance of the hematopoietic system through two mechanisms: one is prevention of TNF-dependent cell death and the other is TNF-independent maintenance of long-term HSC.

  12. Mycobacterium tuberculosis inhibits macrophage responses to IFN-gamma through myeloid differentiation factor 88-dependent and -independent mechanisms.

    Science.gov (United States)

    Fortune, Sarah M; Solache, Alejandra; Jaeger, Alejandra; Hill, Preston J; Belisle, John T; Bloom, Barry R; Rubin, Eric J; Ernst, Joel D

    2004-05-15

    Mycobacterium tuberculosis overcomes macrophage bactericidal activities and persists intracellularly. One mechanism by which M. tuberculosis avoids macrophage killing might be through inhibition of IFN-gamma-mediated signaling. In this study we provide evidence that at least two distinct components of M. tuberculosis, the 19-kDa lipoprotein and cell wall peptidoglycan (contained in the mycolylarabinogalactan peptidoglycan (mAGP) complex), inhibit macrophage responses to IFN-gamma at a transcriptional level. Moreover, these components engage distinct proximal signaling pathways to inhibit responses to IFN-gamma: the 19-kDa lipoprotein inhibits IFN-gamma signaling in a Toll-like receptor (TLR)2-dependent and myeloid differentiation factor 88-dependent fashion whereas mAGP inhibits independently of TLR2, TLR4, and myeloid differentiation factor 88. In addition to inhibiting the induction of specific IFN-gamma responsive genes, the 19-kDa lipoprotein and mAGP inhibit the ability of IFN-gamma to activate murine macrophages to kill virulent M. tuberculosis without inhibiting production of NO. These results imply that inhibition of macrophage responses to IFN-gamma may contribute to the inability of an apparently effective immune response to eradicate M. tuberculosis.

  13. The thiazolidinedione pioglitazone increases cholesterol biosynthetic gene expression in primary cortical neurons by a PPARgamma-independent mechanism.

    Science.gov (United States)

    Cocks, Graham; Wilde, Jonathan I; Graham, Simon J; Bousgouni, Vicky; Virley, David; Lovestone, Simon; Richardson, Jill

    2010-01-01

    In a recent clinical study, the thiazolidinedione (TZD) pioglitazone (Actos was reported to preserve cognitive function in patients with mild to moderate Alzheimer's disease and type II diabetes mellitus. TZDs are agonists of the nuclear hormone receptor peroxisome proliferator-activated receptor-gamma (PPARgamma), are peripheral insulin sensitizers, and have recently been reported to increase mitochondrial biogenesis in the central nervous system and dendritic spine density. We report a transcriptional profile of the TZD pioglitazone and the non-TZD PPARgamma agonist GW347845 in primary cortical culture. We observed that pioglitazone, but not GW347845, increased cholesterol biosynthetic and lipogenic gene expression after 6 h, and the expression of the cholesterol efflux transporters Abca1 and Abcg1 after 24 h. Co-treatment of pioglitazone with the PPARgamma antagonist GW9662 did not significantly reduce these effects, suggesting a PPARgamma-independent mechanism. These findings suggest a novel effect of TZDs in neurons that may be of relevance as a novel approach against Alzheimer's disease.

  14. Coral-Derived Compound WA-25 Inhibits Angiogenesis by Attenuating the VEGF/VEGFR2 Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Shih-Wei Lin

    2015-02-01

    Full Text Available Background: WA-25 (dihydroaustrasulfone alcohol, a synthetic derivative of marine compound WE-2 suppresses atherosclerosis in rats by reducing neointima formation. Because angiogenesis plays a critical role in the pathogenesis of atherosclerosis, the present study investigated the angiogenic function and mechanism of WA-25. Methods: The angiogenic effect of WA-25 was evaluated using a rat aortic ring assay and transgenic zebrafish models were established using transgenic Tg(fli-1:EGFPy1 and Tg(kdrl:mCherryci5-fli1a:negfpy7 zebrafish embryos. In addition, the effect of WA-25 on distinct angiogenic processes, including matrix metalloproteinase (MMP expression, endothelial cell proliferation and migration, as well as tube formation, was studied using human umbilical vein endothelial cells (HUVECs. The effect of WA-25 on the endothelial vascular endothelial growth factor (VEGF signaling pathway was elucidated using qRT-PCR, immunoblot analysis, immunofluorescence and flow cytometric analyses. Results: The application of WA-25 perturbed the development of intersegmental vessels in transgenic zebrafish. Moreover, WA-25 potently suppressed microvessel sprouting in organotypic rat aortic rings. Among cultured endothelial cells, WA-25 significantly and dose-dependently inhibited MMP-2/MMP-9 expression, proliferation, migration and tube formation in HUVECs. Mechanistic studies revealed that WA-25 significantly reduced the VEGF release by reducing VEGF expression at the mRNA and protein levels. In addition, WA-25 reduced surface VEGF receptor 2 (VEGFR2/Flk-1 expression by repressing the VEGFR2 mRNA level. Finally, an exogenous VEGF supply partially rescued the WA-25-induced angiogenesis blockage in vitro and in vivo. Conclusions: WA-25 is a potent angiogenesis inhibitor that acts through the down-regulation of VEGF and VEGFR2 in endothelial cells. General Significance: WA-25 may constitute a novel anti-angiogenic drug that acts by targeting endothelial

  15. The expression of COX-2 in VEGF-treated endothelial cells is mediated through protein tyrosine kinase

    Directory of Open Access Journals (Sweden)

    Pravit Akarasereenont

    2002-01-01

    Full Text Available Cyclooxygenase (COX, existing as the COX-1 and COX-2 isoforms, converts arachidonic acid to prostaglandin H2, which is then further metabolized to various prostaglandins. Vascular endothelial growth factor (VEGF has been shown to play important roles in inflammation and is upregulated by the prostaglandin E series through COX-2 in several cell types. Here, we have investigated the effects of VEGF on the COX isoform expressed in human umbilical vein endothelial cells (HUVEC. The signalling mechanism of the COX isoform expressed in endothelial cells activated with VEGF will be also investigated using the tyrosine kinase inhibitor, genistein, and protein kinase C inhibitor, staurosporine. The activity of COX2 was assessed by measuring the production of 6-keto-prostaglandin F1α in the presence of exogenous arachidonic acids (10 μM, 10 min by enzyme immunoassay. The expression of COX isoform protein was detected by immunoblot using specific antibodies. Untreated HUVEC contained no COX-2 protein. In HUVEC treated with VEGF (0.01-50 ng/ml, COX-2 protein, but not COX-1, and COX activity were increased in a dose-dependent manner. Interestingly, the increased COX-2 protein and activity in response to VEGF (10 ng/ml was inhibited by the tyrosine kinase inhibitor, genistein (0.05-5 μg/ml, but not by the protein kinase C inhibitor, staurosporine (0.1-10 ng/ml. Thus, the induction of COX-2 by VEGF in endothelial cells was mediated through protein tyrosine kinase, and the uses of specific COX-2 inhibitors in these conditions, in which VEGF was involved, might have a role.

  16. Changing paradigms of anti-VEGF in the Indian scenario

    Directory of Open Access Journals (Sweden)

    P Mahesh Shanmugam

    2014-01-01

    Full Text Available Anti-vascular endothelial growth factors (VEGF agents have revolutionized the treatment of retinal diseases. Use of anti-VEGF agents in the Indian Scenario present some unique challenges considering the absence of compounding pharmacies, poor penetrance of health insurance and limited affordability of the citizens of a developing economy. To study the changing paradigms of anti-VEGF use in the Indian scenario, all articles published by Indian authors, data from web-based surveys amongst Indian vitreo-retinal specialists were reviewed. In the paucity of compounding pharmacies in India, fractionation and injection techniques differ from those of developed countries. Frequent anti-VEGF monotherapy offers the best anatomical and visual results, but economics of scale do not allow the same in the Indian scenario, resulting in PRN dosing and combination of anti-VEGF with laser photocoagulation, being the commonly employed treatment protocols.

  17. Additional file 5: Figure S5. of Anti-tumor effects of ONC201 in combination with VEGF-inhibitors significantly impacts colorectal cancer growth and survival in vivo through complementary non-overlapping mechanisms

    OpenAIRE

    Wagner, Jessica; Leah Kline, C.; Zhou, Lanlan; Khazak, Vladimir; El-Deiry, Wafik

    2018-01-01

    Analysis of regorafenib and ONC201 mechanism in combination. A) HUVEC representative images of sprouting from HUVECs grown on Matrigel. B) Quantitation of HUVEC sprouting and branching after 12 hours. C) HCT116 cells from live cell imaging using CHOP-800 and Actin-700 on LiCor Odyssey. Cells treated for 48 hours. ONC201: 5 μM. HUVECS N=4, ONC201 treatment 5 μM, Regorafenib 1 mg/ml. (PPTX 623 kb)

  18. An Increase in Vascular Endothelial Growth Factor (VEGF) and VEGF Soluble Receptor-1 (sFlt-1) Are Associated with Early Recurrent Spontaneous Abortion

    Science.gov (United States)

    Pang, Lihong; Wei, Zhouling; Li, Ouyang; Huang, Rudian; Qin, Junzhen; Chen, Hongyan; Fan, Xiaojing; Chen, Zi-Jiang

    2013-01-01

    Recurrent spontaneous abortion (RSA) is a health problem that affects approximately 1% to 5% reproductive age woman. Yet, in around half of these patients, the mechanism for RSA is unexplained. Recent studies have indicated that placental ischemia/hypoxia and endothelial dysfunction are important factors in miscarriage. Other studies have indicated that the level and expression of soluble FMS-like tyrosine kinase-1 (sFlt1) is increased under a hypoxic environment. However, decreased sFlt-1 in the maternal circulation during the first trimester has recently been proposed as a potential marker for identifying risk of pregnancy loss. In this prospective study clinical samples were obtained within a short time after the fetal death, protein expression and maternal serum levels of sFlt1 were assessed and compared to samples taken from those with normal pregnancies. Our results indicate that levels of VEGF and sFlt-1 are both increased in women during early pregnancy compared women that are not pregnant (pmiscarriages compare to controls. These results demonstrate that there is likely a relationship between VEGF, sFlt-1 and RSA suggesting that the high levels and over expression of sFlt-1 and VEGF might be associated with the pathogenesis of RSA. PMID:24098721

  19. Long-Term Continuous Corticosterone Treatment Decreases VEGF Receptor-2 Expression in Frontal Cortex

    OpenAIRE

    Howell, Kristy R.; Kutiyanawalla, Ammar; Pillai, Anilkumar

    2011-01-01

    Objective Stress and increased glucocorticoid levels are associated with many neuropsychiatric disorders including schizophrenia and depression. Recently, the role of vascular endothelial factor receptor-2 (VEGFR2/Flk1) signaling has been implicated in stress-mediated neuroplasticity. However, the mechanism of regulation of VEGF/Flk1 signaling under long-term continuous glucocorticoid exposure has not been elucidated. Material and Methods We examined the possible effects of long-term continuo...

  20. The PARP inhibitor PJ-34 sensitizes cells to UVA-induced phototoxicity by a PARP independent mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Lakatos, Petra; Hegedűs, Csaba [Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, Debrecen (Hungary); Salazar Ayestarán, Nerea; Juarranz, Ángeles [Department of Biology, Faculty of Sciences, Universidad Autónoma of Madrid, 28049-Madrid (Spain); Kövér, Katalin E. [Department of Inorganic and Analytical Chemistry, Faculty of Sciences, University of Debrecen, Debrecen (Hungary); Szabó, Éva [Department of Dermatology, Faculty of Medicine, University of Debrecen, Debrecen (Hungary); Virág, László, E-mail: lvirag@med.unideb.hu [Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, Debrecen (Hungary); MTA-DE Cell Biology and Signaling Research Group, Debrecen (Hungary)

    2016-08-15

    Highlights: • PARP-1 is not a key regulator of photochemotherapy. • The PARP inhibitor PJ-34 sensitizes cells to UVA-induced phototoxicity by a PARP independent mechanism. • Photosensitization by PJ-34 is associated with increased ROS production and DNA damage. • Cells sensitized by PJ-34 undergo caspase-mediated apoptosis. - Abstract: A combination of a photosensitizer with light of matching wavelength is a common treatment modality in various diseases including psoriasis, atopic dermatitis and tumors. DNA damage and production of reactive oxygen intermediates may impact pathological cellular functions and viability. Here we set out to investigate the role of the nuclear DNA nick sensor enzyme poly(ADP-ribose) polymerase 1 in photochemical treatment (PCT)-induced tumor cell killing. We found that silencing PARP-1 or inhibition of its enzymatic activity with Veliparib had no significant effect on the viability of A431 cells exposed to 8-methoxypsoralen (8-MOP) and UVA (2.5 J/cm{sup 2}) indicating that PARP-1 is not likely to be a key player in either cell survival or cell death of PCT-exposed cells. Interestingly, however, another commonly used PARP inhibitor PJ-34 proved to be a photosensitizer with potency equal to 8-MOP. Irradiation of PJ-34 with UVA caused changes both in the UV absorption and in the 1H NMR spectra of the compound with the latter suggesting UVA-induced formation of tautomeric forms of the compound. Characterization of the photosensitizing effect revealed that PJ–34 + UVA triggers overproduction of reactive oxygen species, induces DNA damage, activation of caspase 3 and caspase 8 and internucleosomal DNA fragmentation. Cell death in this model could not be prevented by antioxidants (ascorbic acid, trolox, glutathione, gallotannin or cell permeable superoxide dismutase or catalase) but could be suppressed by inhibitors of caspase-3 and −8. In conclusion, PJ-34 is a photosensitizer and PJ–34 + UVA causes DNA damage and caspase

  1. A local anesthetic, ropivacaine, suppresses activated microglia via a nerve growth factor-dependent mechanism and astrocytes via a nerve growth factor-independent mechanism in neuropathic pain

    Directory of Open Access Journals (Sweden)

    Sakamoto Atsuhiro

    2011-01-01

    Full Text Available Abstract Background Local anesthetics alleviate neuropathic pain in some cases in clinical practice, and exhibit longer durations of action than those predicted on the basis of the pharmacokinetics of their blocking effects on voltage-dependent sodium channels. Therefore, local anesthetics may contribute to additional mechanisms for reversal of the sensitization of nociceptive pathways that occurs in the neuropathic pain state. In recent years, spinal glial cells, microglia and astrocytes, have been shown to play critical roles in neuropathic pain, but their participation in the analgesic effects of local anesthetics remains largely unknown. Results Repetitive epidural administration of ropivacaine reduced the hyperalgesia induced by chronic constrictive injury of the sciatic nerve. Concomitantly with this analgesia, ropivacaine suppressed the increases in the immunoreactivities of CD11b and glial fibrillary acidic protein in the dorsal spinal cord, as markers of activated microglia and astrocytes, respectively. In addition, epidural administration of a TrkA-IgG fusion protein that blocks the action of nerve growth factor (NGF, which was upregulated by ropivacaine in the dorsal root ganglion, prevented the inhibitory effect of ropivacaine on microglia, but not astrocytes. The blockade of NGF action also abolished the analgesic effect of ropivacaine on neuropathic pain. Conclusions Ropivacaine provides prolonged analgesia possibly by suppressing microglial activation in an NGF-dependent manner and astrocyte activation in an NGF-independent manner in the dorsal spinal cord. Local anesthetics, including ropivacaine, may represent a new approach for glial cell inhibition and, therefore, therapeutic strategies for neuropathic pain.

  2. The production of VEGF involving MAP kinase activation by low level laser therapy in human granulosa cells

    Science.gov (United States)

    Utsunomiya-Kai, Yufuko; Kai, Kentaro; Miyakawa, Isao; Ohshiro, Toshio; Narahara, Hisashi

    2012-01-01

    Objective: The function of granulosa cells is regulated by various hormones and growth factors. Our aim is to clarify the regulation of vascular endothelial growth factor (VEGF) production via mitogen-activated protein kinase (MAPK) induced by low level laser therapy (LLLT) in human granulosa cells. Methods: A human granulosa cell line, KGN cells, were cultured and incubated after LLLT (60mW, GaAlAs 830nm). The levels of VEGF in the culture media were determined by an enzyme-linked immunosorbent assay. The activation of MAP kinase in KGN cells was detected by western blot analysis. Results: VEGF production was significantly increased by LLLT in a time-dependent manner. MAP kinase activity was increased by LLLT. In addition it was enhanced by LLLT and follicle-stimulating hormone (FSH) stimulation. Conclusions: The results suggested that VEGF is induced by LLLT through mechanisms involving MAPK. The increase in VEGF may contribute to neovascularization, which in turn would promote various ovulation phenomena as well as follicular growth. PMID:24511196

  3. Plumbagin Alleviates Capillarization of Hepatic Sinusoids In Vitro by Downregulating ET-1, VEGF, LN, and Type IV Collagen

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    Guiyu Li

    2017-01-01

    Full Text Available Critical roles for liver sinusoidal endothelial cells (LSECs in liver fibrosis have been demonstrated, while little is known regarding the underlying molecular mechanisms of drugs delivered to the LSECs. Our previous study revealed that plumbagin plays an antifibrotic role in liver fibrosis. In this study, we investigated whether plumbagin alleviates capillarization of hepatic sinusoids by downregulating endothelin-1 (ET-1, vascular endothelial growth factor (VEGF, laminin (LN, and type IV collagen on leptin-stimulated LSECs. We found that normal LSECs had mostly open fenestrae and no organized basement membrane. Leptin-stimulated LSECs showed the formation of a continuous basement membrane with few open fenestrae, which were the features of capillarization. Expression of ET-1, VEGF, LN, and type IV collagen was enhanced in leptin-stimulated LSECs. Plumbagin was used to treat leptin-stimulated LSECs. The sizes and numbers of open fenestrae were markedly decreased, and no basement membrane production was found after plumbagin administration. Plumbagin decreased the levels of ET-1, VEGF, LN, and type IV collagen in leptin-stimulated LSECs. Plumbagin promoted downregulation of ET-1, VEGF, LN, and type IV collagen mRNA. Altogether, our data reveal that plumbagin reverses capillarization of hepatic sinusoids by downregulation of ET-1, VEGF, LN, and type IV collagen.

  4. Corelease of bioactive VEGF and HGF from viscous liquid poly(5-ethylene ketal ε-caprolactone-co-D,L-lactide).

    Science.gov (United States)

    Babasola, Iyabo Oladunni; Rooney, Meghan; Amsden, Brian G

    2013-12-02

    The potential of a viscous liquid injectable delivery system composed of poly(5-ethylene ketal ε-caprolactone-co-D,L-lactide) (PEKCDLLA) to release bioactive vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) using an osmotic pressure release mechanism for the purpose of treating critical limb ischemia was investigated. VEGF and HGF were lyophilized separately with trehalose and bovine serum albumin (BSA) and incorporated into the polymer by simple mixing. VEGF and HGF were released by convective flow through superhydrated regions formed within the polymer as a result of the osmotic activity generated upon dissolution of the particles, along with the contributions of polymer degradation at later time points. A sustained release of highly bioactive VEGF and HGF for over 40 days with minimal burst was achieved under conditions of multidirectional delivery. The solubility of the growth factors in the concentrated trehalose solution formed upon dissolution of the particle within the polymer was determined to be a key parameter governing the rate and extent of growth factor release. This formulation approach, of using a low viscosity polymer delivery vehicle, is potentially useful for localized delivery of acid and temperature sensitive proteins, such as VEGF and HGF. This system may also serve as a platform for controlled and predictable delivery patterns for other therapeutic proteins in other clinical settings.

  5. Impact of Alendronate and VEGF- antisense combined treatment on highly VEGF-expressing A431 cells

    OpenAIRE

    Mongerard-Coulanges, Medge; Migianu-Griffoni, Evelyne; Lecouvey, Marc; Jolles, Béatrice

    2009-01-01

    Abstract Bisphosphonates, and more specially nitrogen- containing bisphosphonates, which are in current use for the treatment of bone diseases, demonstrate proapoptotic, antiproliferative, antiangiogenic and anti-invasive properties on tumor cells. The amino- bisphosphonate alendronate is considered as a potential anticancer drug. In the case of A431 cells, which express high levels of VEGF, it had a two-step effect. At 24h, the antitumor properties of alendronate were counterbalan...

  6. Antioxidant administration attenuates mechanical ventilation-induced rat diaphragm muscle atrophy independent of protein kinase B (PKB–Akt) signalling

    Science.gov (United States)

    McClung, J M; Kavazis, A N; Whidden, M A; DeRuisseau, K C; Falk, D J; Criswell, D S; Powers, S K

    2007-01-01

    Oxidative stress promotes controlled mechanical ventilation (MV)-induced diaphragmatic atrophy. Nonetheless, the signalling pathways responsible for oxidative stress-induced muscle atrophy remain unknown. We tested the hypothesis that oxidative stress down-regulates insulin-like growth factor-1–phosphotidylinositol 3-kinase–protein kinase B serine threonine kinase (IGF-1–PI3K–Akt) signalling and activates the forkhead box O (FoxO) class of transcription factors in diaphragm fibres during MV-induced diaphragm inactivity. Sprague–Dawley rats were randomly assigned to one of five experimental groups: (1) control (Con), (2) 6 h of MV, (3) 6 h of MV with infusion of the antioxidant Trolox, (4) 18 h of MV, (5) 18 h of MV with Trolox. Following 6 h and 18 h of MV, diaphragmatic Akt activation decreased in parallel with increased nuclear localization and transcriptional activation of FoxO1 and decreased nuclear localization of FoxO3 and FoxO4, culminating in increased expression of the muscle-specific ubiquitin ligases, muscle atrophy factor (MAFbx) and muscle ring finger-1 (MuRF-1). Interestingly, following 18 h of MV, antioxidant administration was associated with attenuation of MV-induced atrophy in type I, type IIa and type IIb/IIx myofibres. Collectively, these data reveal that the antioxidant Trolox attenuates MV-induced diaphragmatic atrophy independent of alterations in Akt regulation of FoxO transcription factors and expression of MAFbx or MuRF-1. Further, these results also indicate that differential regulation of diaphragmatic IGF-1–PI3K–Akt signalling exists during the early and late stages of MV. PMID:17916612

  7. PD123319 augments angiotensin II-induced abdominal aortic aneurysms through an AT2 receptor-independent mechanism.

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    Alan Daugherty

    Full Text Available AT2 receptors have an unclear function on development of abdominal aortic aneurysms (AAAs, although a pharmacological approach using the AT2 receptor antagonist PD123319 has implicated a role. The purpose of the present study was to determine the role of AT2 receptors in AngII-induced AAAs using a combination of genetic and pharmacological approaches. We also defined effects of AT2 receptors in AngII-induced atherosclerosis and thoracic aortic aneurysms.Male AT2 receptor wild type (AT2 +/y and deficient (AT2 -/y mice in an LDL receptor -/- background were fed a saturated-fat enriched diet, and infused with either saline or AngII (500 ng/kg/min. AT2 receptor deficiency had no significant effect on systolic blood pressure during AngII-infusion. While AngII infusion induced AAAs, AT2 receptor deficiency did not significantly affect either maximal width of the suprarenal aorta or incidence of AAAs. The AT2 receptor antagonist PD123319 (3 mg/kg/day and AngII were co-infused into male LDL receptor -/- mice that were either AT2 +/y or -/y. PD123319 had no significant effect on systolic blood pressure in either wild type or AT2 receptor deficient mice. Consistent with our previous findings, PD123319 increased AngII-induced AAAs. However, this effect of PD123319 occurred irrespective of AT2 receptor genotype. Neither AT2 receptor deficiency nor PD123319 had any significant effect on AngII-induced thoracic aortic aneurysms or atherosclerosis.AT2 receptor deficiency does not affect AngII-induced AAAs, thoracic aortic aneurysms and atherosclerosis. PD123319 augments AngII-induced AAAs through an AT2 receptor-independent mechanism.

  8. Leptin Acts via Lateral Hypothalamic Area Neurotensin Neurons to Inhibit Orexin Neurons by Multiple GABA-Independent Mechanisms

    Science.gov (United States)

    Goforth, Paulette B.; Leinninger, Gina M.; Patterson, Christa M.

    2014-01-01

    The adipocyte-derived hormone leptin modulates neural systems appropriately for the status of body energy stores. Leptin inhibits lateral hypothalamic area (LHA) orexin (OX; also known as hypocretin)-producing neurons, which control feeding, activity, and energy expenditure, among other parameters. Our previous results suggest that GABAergic LHA leptin receptor (LepRb)-containing and neurotensin (Nts)-containing (LepRbNts) neurons lie in close apposition with OX neurons and control Ox mRNA expression. Here, we show that, similar to leptin, activation of LHA Nts neurons by the excitatory hM3Dq DREADD (designer receptor exclusively activated by designer drugs) hyperpolarizes membrane potential and suppresses action potential firing in OX neurons in mouse hypothalamic slices. Furthermore, ablation of LepRb from Nts neurons abrogated the leptin-mediated inhibition, demonstrating that LepRbNts neurons mediate the inhibition of OX neurons by leptin. Leptin did not significantly enhance GABAA-mediated inhibitory synaptic transmission, and GABA receptor antagonists did not block leptin-mediated inhibition of OX neuron activity. Rather, leptin diminished the frequency of spontaneous EPSCs onto OX neurons. Furthermore, leptin indirectly activated an ATP-sensitive potassium (KATP) channel in OX neurons, which was required for the hyperpolarization of OX neurons by leptin. Although Nts did not alter OX activity, galanin, which is coexpressed in LepRbNts neurons, inhibited OX neurons, whereas the galanin receptor antagonist M40 (galanin-(1–12)-Pro3-(Ala-Leu)2-Ala amide) prevented the leptin-induced hyperpolarization of OX cells. These findings demonstrate that leptin indirectly inhibits OX neurons by acting on LHA LepRbNts neurons to mediate two distinct GABA-independent mechanisms of inhibition: the presynaptic inhibition of excitatory neurotransmission and the opening of KATP channels. PMID:25143620

  9. A clathrin independent macropinocytosis-like entry mechanism used by bluetongue virus-1 during infection of BHK cells.

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    Sarah Gold

    2010-06-01

    Full Text Available Acid dependent infection of Hela and Vero cells by BTV-10 occurs from within early-endosomes following virus uptake by clathrin-mediated endocytosis (Forzan et al., 2007: J Virol 81: 4819-4827. Here we report that BTV-1 infection of BHK cells is also dependent on a low endosomal pH; however, virus entry and infection were not inhibited by dominant-negative mutants of Eps15, AP180 or the 'aa' splice variant of dynamin-2, which were shown to inhibit clathrin-mediated endocytosis. In addition, infection was not inhibited by depletion of cellular cholesterol, which suggests that virus entry is not mediated by a lipid-raft dependent process such as caveolae-mediated endocytosis. Although virus entry and infection were not inhibited by the dominant-negative dynamin-2 mutant, entry was inhibited by the general dynamin inhibitor, dynasore, indicating that virus entry is dynamin dependent. During entry, BTV-1 co-localised with LAMP-1 but not with transferrin, suggesting that virus is delivered to late-endosomal compartments without first passing through early-endosomes. BTV-1 entry and infection were inhibited by EIPA and cytochalasin-D, known macropinocytosis inhibitors, and during entry virus co-localised with dextran, a known marker for macropinocytosis/fluid-phase uptake. Our results extend earlier observations with BTV-10, and show that BTV-1 can infect BHK cells via an entry mechanism that is clathrin and cholesterol-independent, but requires dynamin, and shares certain characteristics in common with macropinocytosis.

  10. Cancer cachexia causes skeletal muscle damage via transient receptor potential vanilloid 2-independent mechanisms, unlike muscular dystrophy.

    Science.gov (United States)

    Iwata, Yuko; Suzuki, Nobuyuki; Ohtake, Hitomi; Kamauchi, Shinya; Hashimoto, Naohiro; Kiyono, Tohru; Wakabayashi, Shigeo

    2016-06-01

    Muscle wasting during cancer cachexia contributes to patient morbidity. Cachexia-induced muscle damage may be understood by comparing its symptoms with those of other skeletal muscle diseases, but currently available data are limited. We modelled cancer cachexia in mice bearing Lewis lung carcinoma/colon adenocarcinoma and compared the associated muscle damage with that in a murine muscular dystrophy model (mdx mice). We measured biochemical and immunochemical parameters: amounts/localization of cytoskeletal proteins and/or Ca(2+) signalling proteins related to muscle function and abnormality. We analysed intracellular Ca(2+) mobilization and compared results between the two models. Involvement of Ca(2+)-permeable channel transient receptor potential vanilloid 2 (TRPV2) was examined by inoculating Lewis lung carcinoma cells into transgenic mice expressing dominant-negative TRPV2. Tumourigenesis caused loss of body and skeletal muscle weight and reduced muscle force and locomotor activity. Similar to mdx mice, cachexia muscles exhibited myolysis, reduced sarcolemmal sialic acid content, and enhanced lysosomal exocytosis and sarcolemmal localization of phosphorylated Ca(2+)/CaMKII. Abnormal autophagy and degradation of dystrophin also occurred. Unlike mdx muscles, cachexia muscles did not exhibit regeneration markers (centrally nucleated fibres), and levels of autophagic proteolytic pathway markers increased. While a slight accumulation of TRPV2 was observed in cachexia muscles, Ca(2+) influx via TRPV2 was not elevated in cachexia-associated myotubes, and the course of cachexia pathology was not ameliorated by dominant-negative inhibition of TRPV2. Thus, cancer cachexia may induce muscle damage through TRPV2-independent mechanisms distinct from those in muscular dystrophy; this may help treat patients with tumour-induced muscle wasting.

  11. Fructose sensitizes Jurkat cells oxidative stress-induced apoptosis via caspase-dependent and caspase-independent mechanisms.

    Science.gov (United States)

    Diaz-Aguirre, Viviana; Velez-Pardo, Carlos; Jimenez-Del-Rio, Marlene

    2016-11-01

    Whether fructose (FRU), as the sole energy source, confers a metabolic advantage on cancer cells against noxious stimuli is unknown. The aim of this study was to evaluate the effects of low (11 mM), moderate (25 mM), and high (55 mM) FRU concentrations alone or in combination with rotenone (ROT) or doxorubicin (DOX) in Jurkat cells, an acute lymphoblastic leukemia cell model. Glucose (GLU) was used as a control. Using different cell analysis techniques, we demonstrated that FRU was predominantly metabolized via oxidative phosphorylation (∼95%) (i.e., lactate production was reduced >120-fold), resulting in endogenous oxidative stress-induced conditions. The cells were characterized by generation of O2(•)(-) (43%)/ H2 O2 (40%) and activation of NF-κB (∼95-fold increase, fi), c-Jun-N terminal kinase (JNK), p53 (40-fi), and c-Jun (9-fi). In addition, we observed a loss of ΔΨm (10%), activation of caspase-3 (50-fi) and apoptosis-inducing factor (AIF, 2-fi), and condensation and fragmentation of the nuclei [20% by acridine orange/ethidium bromide/Hoechst (AO/EB/H) staining, 15% by flow cytometry] compared to those of GLU 11 at 24 h. Although DOX killed Jurkat cells independent of sugar content in the culture medium, leukemic cells in low, but not high, FRU were extremely sensitive to ROT. Taken together, our findings suggest that Jurkat cells are more susceptible to cell death if forced to shift from GLU metabolism (i.e., aerobic glycolysis) to FRU metabolism (i.e., oxidative phosphorylation) after treatment with mitochondria-targeting molecules. These observations may help elucidate the cell death mechanism of leukemic cells cultured in FRU. © 2016 International Federation for Cell Biology.

  12. Two different avian cold-sensitive sensory neurons: Transient receptor potential melastatin 8 (TRPM8)-dependent and -independent activation mechanisms.

    Science.gov (United States)

    Yamamoto, A; Takahashi, K; Saito, S; Tominaga, M; Ohta, T

    2016-12-01

    Sensing the ambient temperature is an important function for survival in animals. Some TRP channels play important roles as detectors of temperature and irritating chemicals. There are functional differences of TRP channels among species. TRPM8 in mammals is activated by cooling compounds and cold temperature, but less information is available on the functional role of TRPM8 in avian species. Here we investigated the pharmacological properties and thermal sensitivities of chicken TRPM8 (cTRPM8) and cold-sensitive mechanisms in avian sensory neurons. In heterologously expressed cTRPM8, menthol and its derivative, WS-12 elicited [Ca 2+ ] i increases, but icilin did not. In chicken sensory neurons, icilin increased [Ca 2+ ] i, in a TRPA1-dependent manner. Icilin selectively stimulated heterologously expressed chicken TRPA1 (cTRPA1). Similar to mammalian orthologue, cTRPM8 was activated by cold. Both heterologous and endogenous expressed cTRPM8 were sensitive to mammalian TRPM8 antagonists. There are two types of cold-sensitive cells regarding menthol sensitivity in chicken sensory neurons. The temperature threshold of menthol-insensitive neurons was significantly lower than that of menthol-sensitive ones. The population of menthol-insensitive neurons was large in chicken but almost little in mammals. The cold-induced [Ca 2+ ] i increases were not abolished by the external Ca 2+ removal or by blockades of PLC-IP 3 pathways and ryanodine channels. The cold stimulation failed to evoke [Ca 2+ ] i increases after intracellular Ca 2+ store-depletion. These results indicate that cTRPM8 acts as a cold-sensor similar to mammals. It is noteworthy that TRPM8-independent cold-sensitive neurons are abundant in chicken sensory neurons. Our results suggest that most of the cold-induced [Ca 2+ ] i increases are mediated via Ca 2+ release from intracellular stores and that these mechanisms may be specific to avian species. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Co-expression of α9β1 integrin and VEGF-D confers lymphatic metastatic ability to a human breast cancer cell line MDA-MB-468LN.

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    Mousumi Majumder

    Full Text Available INTRODUCTION AND OBJECTIVES: Lymphatic metastasis is a common occurrence in human breast cancer, mechanisms remaining poorly understood. MDA-MB-468LN (468LN, a variant of the MDA-MB-468GFP (468GFP human breast cancer cell line, produces extensive lymphatic metastasis in nude mice. 468LN cells differentially express α9β1 integrin, a receptor for lymphangiogenic factors VEGF-C/-D. We explored whether (1 differential production of VEGF-C/-D by 468LN cells provides an autocrine stimulus for cellular motility by interacting with α9β1 and a paracrine stimulus for lymphangiogenesis in vitro as measured with capillary-like tube formation by human lymphatic endothelial cells (HMVEC-dLy; (2 differential expression of α9 also promotes cellular motility/invasiveness by interacting with macrophage derived factors; (3 stable knock-down of VEGF-D or α9 in 468LN cells abrogates lymphangiogenesis and lymphatic metastasis in vivo in nude mice. RESULTS: A comparison of expression of cyclo-oxygenase (COX-2 (a VEGF-C/-D inducer, VEGF-C/-D and their receptors revealed little COX-2 expression by either cells. However, 468LN cells showed differential VEGF-D and α9β1 expression, VEGF-D secretion, proliferative, migratory/invasive capacities, latter functions being stimulated further with VEGF-D. The requirement of α9β1 for native and VEGF-D-stimulated proliferation, migration and Erk activation was demonstrated by treating with α9β1 blocking antibody or knock-down of α9. An autocrine role of VEGF-D in migration was shown by its impairment by silencing VEGF-D and restoration with VEGF-D. 468LN cells and their soluble products stimulated tube formation, migration/invasiveness of HMVEC-dLy cell in a VEGF-D dependent manner as indicated by the loss of stimulation by silencing VEGF-D in 468LN cells. Furthermore, 468LN cells showed α9-dependent stimulation of migration/invasiveness by macrophage products. Finally, capacity for intra-tumoral lymphangiogenesis and

  14. More than one way to evolve a weed: parallel evolution of U.S. weedy rice through independent genetic mechanisms

    Science.gov (United States)

    Many crop species were independently selected for a shared suite of “domestication traits,” which facilitates their use for studies of parallel evolution. Within domesticated rice (Oryza sativa), there has also been independent evolution of weedy strains from different cultivated varieties. This mak...

  15. Correlation between the expression of vegf and survival in osteosarcoma.

    Science.gov (United States)

    Baptista, André Mathias; Camargo, André Ferrari De França; Filippi, Renée Zon; Oliveira, Cláudia Regina Gomes Cardim Mendes De; Azevedo Neto, Raymundo Soares De; Camargo, Olavo Pires De

    2014-01-01

    To present a series of 50 consecutive patients with non-metastatic extremity osteosarcoma, and attempt to correlate expression of the vascular endothelial growth factor (VEGF) protein in biopsy tissue to their prognosis regarding overall survival, disease-free survival and local recurrence. Fifty cases of non-metastatic osteosarcoma of the extremities treated between 1986 and 2006 at Instituto de Ortopedia e Traumatologia da Universidade de São Paulo, São Paulo, Brasil, were evaluated regarding expression of the VEGF protein. There were 19 females and 31 males. The mean age was 16 years old (range 5-28 years old) and the mean follow-up was 60.6 months (range 25-167 months). The variables studied were age, gender, anatomic location, type of surgery, surgical margins, tumor size, post chemotherapy necrosis, local recurrence, pulmonary metastasis and death. Thirty-six patients showed VEGF expression on 30% or less cells (low), and the remaining 14 cases had VEGF expression above 30% (high). Among the 36 patients with low VEGF expression, nine developed pulmonary metastasis and four died (11.1%). Among the 14 patients with high VEGF expression, six developed pulmonary metastasis and three died (21.4%). There was no statistically significant correlation between the expression of VEGF and any of the variables studied. Level of Evidence IV, Therapeutic Study.

  16. Vacuum-assisted closure increases ICAM-1, MIF, VEGF and collagen I expression in wound therapy.

    Science.gov (United States)

    Wang, Weiyang; Pan, Zhenyu; Hu, Xiang; Li, Zonghuan; Zhao, Yong; Yu, Ai-Xi

    2014-05-01

    Severe traumatic wounds are challenging to manage during surgery. The introduction of vacuum-assisted closure (VAC) is a breakthrough in wound management. The aim of the present study was to investigate the effect of VAC on cytokines in wounds during the management of severe traumatic wounds following initial debridement. VAC and conventional wound care (CWC) were independently applied to severe traumatic wounds on pigs. The expression levels of intercellular adhesion molecule-1 (ICAM-1), migration inhibitory factor (MIF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor, collagen I and human fibroblast collagenase 1 were detected by quantitative polymerase chain reaction and western blotting. VAC significantly increased the expression of ICAM-1, MIF, VEGF and collagen I compared with that induced by CWC at the protein and mRNA levels. Therefore, the results of the present study indicate that VAC therapy is an effective method for treating severe traumatic wounds, as it increases the expression of cytokines in wounds. VAC significantly increases the expression of ICAM-1, MIF, VEGF and collagen I to manage severe traumatic wounds.

  17. Synergism of MSC-secreted HGF and VEGF in stabilising endothelial barrier function upon lipopolysaccharide stimulation via the Rac1 pathway.

    Science.gov (United States)

    Yang, Yi; Chen, Qi-Hong; Liu, Ai-Ran; Xu, Xiu-Ping; Han, Ji-Bin; Qiu, Hai-Bo

    2015-12-16

    Mesenchymal stem cells (MSCs) stabilise endothelial barrier function in acute lung injury via paracrine hepatocyte growth factor (HGF). Vascular endothelial growth factor (VEGF), which is secreted by MSCs, is another key regulator of endothelial permeability; however, its role in adjusting permeability remains controversial. In addition, whether an interaction occurs between HGF and VEGF, which are secreted by MSCs, is not completely understood. We introduced a co-cultured model of human pulmonary microvascular endothelial cells (HPMECs) and MSC conditioned medium (CM) collected from MSCs after 24 h of hypoxic culture. The presence of VEGF and HGF in the MSC-CM was neutralised by anti-VEGF and anti-HGF antibodies, respectively. To determine the roles and mechanisms of MSC-secreted HGF and VEGF, we employed recombinant humanised HGF and recombinant humanised VEGF to co-culture with HPMECs. Additionally, we employed the RhoA inhibitor C3 transferase and the Rac1 inhibitor NSC23766 to inhibit the activities of RhoA and Rac1 in HPMECs treated with MSC-CM or VEGF/HGF with the same dosage as in the MSC-CM. Then, endothelial paracellular and transcellular permeability was detected. VE-cadherin, occludin and caveolin-1 protein expression in HPMECs was measured by western blot. Adherens junction proteins, including F-actin and VE-cadherin, were detected by immunofluorescence. MSC-CM treatment significantly decreased lipopolysaccharide-induced endothelial paracellular and transcellular permeability, which was significantly inhibited by pretreatment with HGF antibody or with both VEGF and HGF antibodies. Furthermore, MSC-CM treatment increased the expression of the endothelial intercellular adherence junction proteins VE-cadherin and occludin and decreased the expression of caveolin-1 protein. MSC-CM treatment also decreased endothelial apoptosis and induced endothelial cell proliferation; however, the effects of MSC-CM treatment were inhibited by pretreatment with HGF

  18. Genome-wide pathway analysis identifies VEGF pathway association with oral ulceration in systemic lupus erythematosus.

    Science.gov (United States)

    Aterido, Adrià; Julià, Antonio; Carreira, Patricia; Blanco, Ricardo; López-Longo, José Javier; Venegas, José Javier Pérez; Olivé, Àlex; Andreu, José Luís; Aguirre-Zamorano, Maria Ángeles; Vela, Paloma; Nolla, Joan M; Marenco-de la Fuente, José Luís; Zea, Antonio; Pego, José María; Freire, Mercedes; Díez, Elvira; López-Lasanta, María; López-Corbeto, Mireia; Palau, Núria; Tortosa, Raül; Gelpí, Josep Lluís; Absher, Devin; Myers, Richard M; Fernández-Nebro, Antonio; Marsal, Sara

    2017-06-15

    Systemic lupus erythematosus (SLE) is a genetically complex rheumatic disease characterized by heterogeneous clinical manifestations of unknown etiology. Recent studies have suggested the existence of a genetic basis for SLE heterogeneity. The objective of the present study was to identify new genetic variation associated with the clinically relevant phenotypes in SLE. A two-stage pathway-based approach was used to identify the genetic variation associated with the main clinical phenotypes in SLE. In the discovery stage, 482 SLE patients were genotyped using Illumina Human Quad610 microarrays. Association between 798 reference genetic pathways from the Molecular Signatures Database and 11 SLE phenotypes was tested using the set-based method implemented in PLINK software. Pathways significantly associated after multiple test correction were subsequently tested for replication in an independent cohort of 425 SLE patients. Using an in silico approach, we analyzed the functional effects of common SLE therapies on the replicated genetic pathways. The association of known SLE risk variants with the development of the clinical phenotypes was also analyzed. In the discovery stage, we found a significant association between the vascular endothelial growth factor (VEGF) pathway and oral ulceration (P value for false discovery rate (P FDR ) oral ulceration. Therapies commonly used to treat mucocutaneous phenotypes in SLE were found to strongly influence VEGF pathway gene expression (P = 4.60e-4 to 5.38e-14). Analysis of known SLE risk loci identified a strong association between PTPN22 and the risk of hematologic disorder and with the development of antinuclear antibodies. The present study has identified VEGF genetic pathway association with the risk of oral ulceration in SLE. New therapies targeting the VEGF pathway could be more effective in reducing the severity of this phenotype. These findings represent a first step towards the understanding of the genetic basis

  19. Mechanism of Metal-Independent Decomposition of Organic Hydroperoxides and Formation of Alkoxyl Radicals by Halogenated Quinones

    National Research Council Canada - National Science Library

    Ben-Zhan Zhu; Hong-Tao Zhao; Balaraman Kalyanaraman; Jun Liu; Guo-Qiang Shan; Yu-Guo Du; Balz Frei

    2007-01-01

    The metal-independent decomposition of organic hydroperoxides and the formation of organic alkoxyl radicals in the absence or presence of halogenated quinones were studied with electron spin resonance (ESR...

  20. Resveratrol Increases Serum BDNF Concentrations and Reduces Vascular Smooth Muscle Cells Contractility via a NOS-3-Independent Mechanism

    Directory of Open Access Journals (Sweden)

    Michał Wiciński

    2017-01-01

    Full Text Available Resveratrol is a polyphenol that presents both antineuroinflammatory properties and the ability to interact with NOS-3, what contributes to vasorelaxation. Brain-derived neurotrophic factor (BNDF, a molecule associated with neuroprotection in many neurodegenerative disorders, is considered as an important element of maintaining stable cerebral blood flow. Vascular smooth muscle cells (VSMCs are considered to be an important element in the pathogenesis of neurodegeneration and a potential preventative target by agents which reduce the contractility of the vessels. Our main objectives were to define the relationship between serum and long-term oral resveratrol administration in the rat model, as well as to assess the effect of resveratrol on phenylephrine- (PHE- induced contraction of vascular smooth muscle cells (VSMCs. Moreover, we attempt to define the dependence of contraction mechanisms on endothelial NO synthase. Experiments were performed on Wistar rats (n=17 pretreated with resveratrol (4 weeks; 10 mg/kg p.o. or placebo. Serum BDNF levels were quantified after 2 and 4 weeks of treatment with ELISA. Contraction force was measured on isolated and perfused tail arteries as the increase of perfusion pressure with a constant flow. Values of serum BNDF in week 0 were 1.18±0.12 ng/mL (treated and 1.17±0.13 ng/mL (control (p = ns. After 2 weeks of treatment, BDNF in the treatment group was higher than in controls, 1.52±0.23 ng/mL and 1.24±0.13 ng/mL, respectively. (p=0.02 Following 4 weeks of treatment, BDNF values were higher in the resveratrol group compared to control 1.64±0.31 ng/mL and 1.32±0.26 ng/mL, respectively (p=0.031. EC50 values obtained for PHE in resveratrol pretreated arteries were significantly higher than controls (5.33±1.7 × 10−7 M/L versus 4.53±1.2 × 10−8 M/L, p<0.05. These results show a significant increase in BDNF concentration in the resveratrol pretreated group. The reactivity of resistant

  1. K20E, an oxidative-coupling compound of methyl caffeate, exhibits anti-angiogenic activities through down-regulations of VEGF and VEGF receptor-2

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Chun-Hsu [Department of Pharmacy, Taipei Medical University, Taipei 11031, Taiwan (China); Lin, Wen-Hsin; Chien, Yi-Chung; Liu, Fon-Chang; Sheu, Ming-Jyh [School of Pharmacy, China Medical University, Taichung 40402, Taiwan (China); Kuo, Yueh-Hsiung, E-mail: kuoyh@mail.cmu.edu.tw [Tsuzuki Institute for Traditional Medicine, China Medical University, Taichung 40402, Taiwan (China); Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung 40402, Taiwan (China); Department of Biotechnology, Asia University, Taichung 41354, Taiwan (China); Wu, Chieh-Hsi, E-mail: chhswu@tmu.edu.tw [Department of Pharmacy, Taipei Medical University, Taipei 11031, Taiwan (China); School of Pharmacy, China Medical University, Taichung 40402, Taiwan (China); Department of Biological Science and Technology, China Medical University, Taichung 40402, Taiwan (China)

    2015-01-15

    Anti-angiogenesis is one of the most popular clinical interventions for cancer chemotherapy. A series of synthesized derivative of methyl caffeate were used to evaluate the anti-angiogenic activity and to investigate possible pharmacological mechanisms in the present study. The most potent anti-angiogenic compound was evaluated in the experiments of murine allograft tumor model and Matrigel plug assay as well as cell models in the human umbilical vascular endothelial cells (HUVECs) and the LLC1 lung cancer cells. Our results suggested that K20E suppressed the tumor growth in the allograft tumor model and exhibited anti-angiogenic activity in Matrigel plug assay. Besides, HUVEC viability was found to be significantly reduced by arresting cell cycle at G{sub 2}/M phase and apoptosis. Cell migration, invasion, and tube formation of the HUVECs were also markedly suppressed by K20E treatment. K20E largely down-regulated the intracellular and secreted vascular endothelial growth factor (VEGF) in the LLC1 cancer cells. Besides, VEGF receptor-2 (VEGFR-2) and its downstream signaling cascades (AKT-mTOR and MEK1/2-ERK1/2) as well as gelatinases were all evidently reduced in the HUVECs treated with K20E. Inversely, K20E can up-regulate the expression levels of p53 and p21 proteins in the HUVECs. Based on these results, our study suggested that K20E possessed inhibiting angiogenesis through regulation of VEGF/VEGFR-2 and its downstream signaling cascades in the vascular endothelial cells (VECs). - Highlights: • K20E is an oxidative-coupling compound of methyl caffeate. • K20E exhibits anti-tumor and anti-angiogenesis effects. • K20E suppresses the expressions of VEGF and VEGF receptor-2 (VEGFR-2) proteins. • K20E deactivates VEGFR-2-mediated downstream signaling pathways to inhibit angiogenesis. • K20E up-regulates p53-p21 pathway to induce apoptosis and cell arrest at G2/M phase.

  2. Synthesis of the human VEGF165 gene based on overlap PCR and ...

    African Journals Online (AJOL)

    Yomi

    2012-01-16

    Jan 16, 2012 ... human VEGF165 (hVEGF165) gene based on overlap PCR method and recombinant expressed in Chinese's hamster ovary (CHO) ... recombinant human VEGF165 (rhVEGF165) protein in CHO cells. Key words: Overlap .... Stable transfected positive clones appeared after 14 days when the. CHO cells not ...

  3. Cell type-specific dependency on the PI3K/Akt signaling pathway for the endogenous Epo and VEGF induction by baicalein in neurons versus astrocytes.

    Directory of Open Access Journals (Sweden)

    Yu-Yo Sun

    Full Text Available The neuroprotective effect of baicalein is generally attributed to inhibition of 12/15-lipoxygenase (12/15-LOX and suppression of oxidative stress, but recent studies showed that baicalein also activates hypoxia-inducible factor-α (HIF1α through inhibition of prolyl hydrolase 2 (PHD2 and activation of the phosphatidylinositide-3 kinase (PI3K/Akt signaling pathway. Yet, the significance and regulation of prosurvival cytokines erythropoietin (Epo and vascular endothelial growth factor (VEGF, two transcriptional targets of HIF1α, in baicalein-mediated neuroprotection in neurons and astrocytes remains unknown. Here we investigated the causal relationship between the PI3K/Akt signaling pathway and Epo/VEGF expression in baicalein-mediated neuroprotection in primary rat cortical neurons and astrocytes. Our results show that baicalein induced Epo and VEGF expression in a HIF1α- and PI3K/Akt-dependent manner in neurons. Baicalein also protected neurons against excitotoxicity in a PI3K- and Epo/VEGF-dependent manner without affecting neuronal excitability. In contrast, at least a 10-fold higher concentration of baicalein was needed to induce Epo/VEGF production and PI3K/Akt activity in astrocytes for protection of neurons. Moreover, only baicalein-induced astrocytic VEGF, but not Epo expression requires HIF1α, while PI3K/Akt signaling had little role in baicalein-induced astrocytic Epo/VEGF expression. These results suggest distinct mechanisms of baicalein-mediated Epo/VEGF production in neurons and astrocytes for neuroprotection, and provide new insights into the mechanisms and potential of baicalein in treating brain injury in vivo.

  4. miR-184 exhibits angiostatic properties via regulation of Akt and VEGF signaling pathways.

    Science.gov (United States)

    Park, Jong Kook; Peng, Han; Yang, Wending; Katsnelson, Julia; Volpert, Olga; Lavker, Robert M

    2017-01-01

    Corneal avascularity is critical for achieving transparency necessary for proper transmission of light to the lens and visual acuity. Although much is known about angiogenesis and angiostasis, the precise regulation of these processes in the cornea is unclear. MicroRNA (miR)-184, the most abundant corneal epithelial miRNA, has been suggested to function in corneal angiostasis by altering VEGF signaling; however, the mechanism(s) underlying this regulation have not been addressed. Using a combination of in vitro and in vivo assays to evaluate angiogenesis, we demonstrated that human limbal epithelial keratinocytes (HLEKs) engineered to overexpress miR-184 secreted lower amounts of angiogenic mitogens. Human dermal microvascular cells exposed to conditioned medium from miR-184-overexpressing HLEKs were less proliferative and failed to seal linear scratch wounds. The in vivo Matrigel plug assay showed that conditioned medium from miR-184-expressing HLEKs elicited a lesser degree of neovascularization compared with controls. We found that miR-184 directly targets and represses the proangiogenic factors, friend of Gata 2 (FOG2), platelet-derived growth factor (PDGF)-β, and phosphatidic acid phosphatase 2b (PPAP2B). FOG2 regulates VEGF expression, whereas PDGF-β and PPAP2B regulate Akt activity. By attenuating both VEGF and Akt signaling, miR-184 acts as a broad-spectrum negative regulator of corneal angiogenesis.-Park, J. K., Peng, H., Yang, W., Katsnelson, J., Volpert, O., Lavker, R. M. miR-184 exhibits angiostatic properties via regulation of Akt and VEGF signaling pathways. © FASEB.

  5. Clinical significance of pretreatment serum levels of VEGF and its receptors, IL- 8, and their prognostic value in type I and II endometrial cancer patients.

    Directory of Open Access Journals (Sweden)

    Beata Kotowicz

    Full Text Available The study aimed to assess the usefulness of the determination of cytokines: IL-8, VEGF and its soluble receptors: VEGF-R1, VEGF-R2 in patients with endometrial cancer (EC.The study group consisted of 118 patients with EC subjected to surgical treatment. Before the treatment we determined the serum levels of cytokines IL-8, and VEGF as well as VEGFR1 and VEGFR2 receptors. For comparison, the concentration of CA 125 was also measured. VEGFR1 and CA 125 were determined in the COBAS e601 system using Roche Diagnostics kits, while IL-8, VEGF and VEGFR2 were measured by ELISA assay using R&D Systems kits.The concentrations of IL-8, VEGF, VEGFR1 and CA 125 allowed to distinguish patients for the control group. The highest diagnostic sensitivity has been shown for the concentrations of VEGF (AUC = 0.904 and IL-8 (AUC = 0.818. Among all studied parameters only CA125 concentrations increased with the clinical stage; being significantly higher in patients in FIGO III-IV, than FIGO I-IB. In patients at the FIGO stage I-IB, complementary determinations of CA 125 and VEGF resulted in the largest increase of diagnostic sensitivity. Patients with metastases to the para-aortic lymph nodes had significantly higher levels of VEGF compared to subjects without such lesions. The concentrations of IL-8 were an independent prognostic factor in the assessment of overall survival in patients with type I endometrial cancer, while the concentrations of VEGFR2 in those with type II.In patients with endometrial cancer, the clinical usefulness of IL-8 and VEGFR2 measurements as the potential prognostic factors has been demonstrated. In type I, the concentrations of IL-8 determined before treatment can be helpful in predicting overall survival. In patients qualified to type II EC, the concentrations of VEGFR2 have the value of an independent prognostic factor for overall survival, this requires research on larger groups of patients. The increased levels of VEGF may be useful

  6. Therapeutic lymphangiogenesis using stem cell and VEGF-C hydrogel.

    Science.gov (United States)

    Hwang, Ji Hye; Kim, In Gul; Lee, Ji Young; Piao, Shuyu; Lee, David S; Lee, Tae Seung; Ra, Jeong Chan; Lee, Ji Youl

    2011-07-01

    Lymphedema is a manifestation of lymphatic system insufficiency. It arises from primary lymphatic dysplasia or secondary obliteration after lymph node dissection or irradiation. Although improvement of swelling can be achieved by comprehensive non-operative therapy, treatment of this condition requires lifelong care and good compliance. Recently molecular-based treatments using VEGF-C have been investigated by several researchers. We designed the present study to determine whether the therapeutic efficacy of implanted human adipose-derived stem cells (hADSCs) could be improved by applying a gelatin hydrogel containing VEGF-C (VEGF-C hydrogel) to the site of tissue injury in a lymphedema mouse model. Four weeks after the operation, we evaluated edema and determined lymphatic vessel density at various post-operative time points. Mice treated with hADSCs and VEGF-C hydrogel showed a significantly decreased dermal edema depth compared to the groups of mice that received hADSCs only or VEGF-C hydrogel only. Immunohistochemical analysis also revealed that the hADSC/VEGF-C hydrogel group showed significantly greater lymphatic vessel regeneration than all the other groups. hADSCs were detected in the implantation sites of all mice in the hADSC/VEGF-C group, and exhibited a lymphatic endothelial differentiation phenotype as determined by co-staining PKH-labeled hADSCs for the lymphatic marker LYVE-1. Our results suggest that co-administration of hADSCs and VEGF-C hydrogel has a substantial positive effect on lymphangiogenesis. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. Uteroplacental insufficiency affects kidney VEGF expression in a model of IUGR with compensatory glomerular hypertrophy and hypertension.

    Science.gov (United States)

    Baserga, Mariana; Bares, Allyson L; Hale, Merica A; Callaway, Christopher W; McKnight, Robert A; Lane, Pascale H; Lane, Robert H

    2009-06-01

    Low nephron endowment secondary to intrauterine growth restriction (IUGR) results in compensatory hypertrophy of the remaining glomeruli, which in turn is associated with hypertension. However, gender differences exist in the response of the kidney to injury, and IUGR female offspring seems protected from an unfavorable outcome. We previously reported differences in gender-specific gene expression in the IUGR kidney as well as increased circulating corticosterone levels following uteroplacental insufficiency (UPI). Vascular endothelial growth factor (VEGF), which is critical for renal development, is an important candidate in the IUGR kidney since its expression can be regulated by sex-steroids and glucocorticoids. We hypothesize that IUGR leads to altered kidney VEGF expression in a gender-specific manner. Following uterine ligation in the pregnant rat, UPI decreases renal VEGF levels in male and female IUGR animals at birth and through postnatal day 21. However, by day 120 of life, IUGR females have increased kidney VEGF expression, not present in the IUGR males. In addition, IUGR males exhibit increased serum testosterone levels as well as proteinuria. These findings are intriguing in light of the difference in glomerular hypertrophy observed: IUGR males show increased glomerular area when compared to IUGR females. In this model characterized by decreased nephron number and adult onset hypertension, UPI decreases renal VEGF expression during nephrogenesis. Our most intriguing finding is the increased renal VEGF levels in adult IUGR females, associated with a more benign phenotype. We suggest that the mechanisms underlying renal disease in response to IUGR are most likely regulated in a gender specific manner.

  8. 1-o-acetylbritannilactone (ABL) inhibits angiogenesis and lung cancer cell growth through regulating VEGF-Src-FAK signaling

    Energy Technology Data Exchange (ETDEWEB)

    Zhengfu, He; Hu, Zhang; Huiwen, Miao; Zhijun, Li [Department of Thoracic Surgery, Sir Run Run Shaw Hospital of Zhejiang University School of Medicine, Hangzhou (China); Jiaojie, Zhou [Zhejiang University School of Medicine, Hangzhou (China); Xiaoyi, Yan, E-mail: xiaoyiyan163@163.com [Zhejiang University School of Medicine, Hangzhou (China); Xiujun, Cai, E-mail: xiujuncaomaj@163.com [Sir Run Run Shaw Hospital of Zhejiang University School of Medicine, Hangzhou (China)

    2015-08-21

    The search for safe, effective and affordable therapeutics against non-small cell lung cancer (NSCLC) and other lung cancers is important. Here we explored the potential effect of 1-o-acetylbritannilactone (ABL), a novel extract from Inula britannica-F, on angiogenesis and lung cancer cell growth. We demonstrated that ABL dose-dependently inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration, and capillary structure formation of cultured human umbilical vascular endothelial cells (HUVECs). In vivo, ABL administration suppressed VEGF-induced new vasculature formation in Matrigel plugs. For the mechanism investigations, we found that ABL largely inhibited VEGF-mediated activation of Src kinase and focal adhesion kinase (FAK) in HUVECs. Furthermore, treatment of A549 NSCLC cells with ABL resulted in cell growth inhibition and Src-FAK in-activation. Significantly, administration of a single dose of ABL (12 mg/kg/day) remarkably suppressed growth of A549 xenografts in nude mice. In vivo microvessels formation and Src activation were also significantly inhibited in ABL-treated xenograft tumors. Taken together, our findings suggest that ABL suppresses angiogenesis and lung cancer cell growth possibly via regulating the VEGFR-Src-FAK signaling. - Highlights: • 1-o-acetylbritannilactone (ABL) inhibits VEGF-induced angiogenesis in vivo. • ABL inhibits VEGF-induced HUVEC migration, proliferation, capillary tube formation. • ABL inhibits VEGF-mediated activation of Src and FAK in HUVECs. • ABL inhibits growth and Src-FAK activation in A549 cells. • ABL administration inhibits A549 tumor angiogenesis and growth in nude mice.

  9. Valproic acid inhibits the angiogenic potential of cervical cancer cells via HIF-1α/VEGF signals.

    Science.gov (United States)

    Zhao, Y; You, W; Zheng, J; Chi, Y; Tang, W; Du, R

    2016-11-01

    Cervical cancer is one of the most prevalent malignancies in women worldwide. Therefore, the investigation about the molecular pathogenesis and related therapy targets of cervical cancer is an emergency. The objective of the present study is to investigate the effects of valproic acid (VPA), a histone deacetylase inhibitor, on the angiogenesis of cervical cancer. The effects and mechanisms of VPA on in vitro angiogenesis and vascular endothelial growth factor (VEGF) expression of human cervical cancer HeLa and SiHa cells were investigated. Our present study reveals that 1 mM VPA can significantly inhibit the in vitro angiogenic potential and VEGF expression of human cervical cancer HeLa and SiHa cells. Further, the transcription and protein levels of hypoxia inducible factor-1α (HIF-1α), and not HIF-1β, are significantly inhibited in VPA-treated cervical cancer cells. Over expression of HIF-1α can obviously reverse VPA-induced VEGF down regulation. VPA-treatment decreases the activation of Akt and ERK1/2 in both HeLa and SiHa cells in a time-dependent manner. The inhibitor of Akt (LY 294002) or ERK1/2 (PD98059) can inhibit VEGF alone and cooperatively reinforce the suppression effects of VPA on HIF-1α and VEGF expression. Collectively, our data reveal that the inhibition of PI3K/Akt and ERK1/2 signals are involved in VPA-induced HIF-1α and VEGF suppression of cervical cancer cells.

  10. Thrombospondin-1 and VEGF in inflammatory bowel disease

    Directory of Open Access Journals (Sweden)

    Canan Alkim

    2012-01-01

    Full Text Available Angiogenesis is an important process in the pathogenesis of chronic inflammation. We aimed to study the angiogeneic balance in inflammatory bowel disease (IBD by evaluating the expression of vascular endothelial growth factor (VEGF and thrombospondin-1 (TSP-1 on colonic epithelial cells, together with the expression of inducible nitric oxide synthase (iNOS.Twenty-one ulcerative colitis (UC, 14 Crohn's disease (CD, 11 colorectal cancer patients, and 11 healthy controls colonic biopsy samples were evaluated immunohistochemically.The expressions of TSP-1, VEGF, and iNOS in UC and CD groups were higher than expression in healthy control group, all with statistical significance. However, in colorectal cancer group, VEGF and iNOS expressions were increased importantly, but TSP-1 expression was not statistically different from healthy control group's expression. Both TSP-1 and VEGF expressions were correlated with iNOS expression distinctly but did not correlate with each other.Both pro-angiogeneic VEGF and antiangiogeneic TSP-1 expressions were found increased in our IBD groups, but in colorectal cancer group, only VEGF expression was increased. TSP-1 increases in IBD patients as a response to inflammatory condition, but this increase was not enough to suppress pathologic angiogenesis and inflammation in IBD.

  11. Human CAFs promote lymphangiogenesis in ovarian cancer via the Hh-VEGF-C signaling axis.

    Science.gov (United States)

    Wei, Rui; Lv, Mengqin; Li, Fei; Cheng, Teng; Zhang, Zhengzhong; Jiang, Guiying; Zhou, Ying; Gao, Ruiqiu; Wei, Xiao; Lou, Jicheng; Wu, Xizi; Luo, Danfeng; Ma, Xiangyi; Jiang, Jin; Ma, Ding; Xi, Ling

    2017-09-15

    Cancer-associated fibroblasts (CAFs) play a pivotal role in the development and progression of many human cancers. Recent studies have shown that Hedgehog (Hh) signalling modulates the stromal microenvironment and prepares a suitable niche for tumour metastasis. However, the detailed molecular mechanisms underlying CAF-mediated lymphangiogenesis have not been fully elucidated. Therefore, our goal is to illustrate whether Hh ligands can activate Hh signalling in CAFs in a paracrine fashion and elucidate the effect of CAFs on lymphangiogenesis. We determined here that Sonic Hedgehog (SHH) secreted by ovarian cancer (OC) cells activated Hh signalling in CAFs and promoted the proliferation of CAFs. Moreover, we co-injected SHH-overexpressing OC cells and CAFs in a xenograft model and found that the CAFs accelerated tumourigenesis and lymphangiogenesis in OC. Mechanistically, we found that SHH secreted by the OC cells induced VEGF-C expression in CAFs. Inhibition of Hh signalling in CAFs decreased VEGF-C expression and diminished the positive role of CAFs in supporting tumourigenesis and lymphangiogenesis in a murine xenograft model. Our results demonstrate that CAFs constitute a supportive niche for cancer lymphangiogenesis via the Hh/VEGF-C signalling axis and provide evidence for the clinical application of Hh inhibitors in the treatment of OC.

  12. A Conserved Role for VEGF Signaling in Specification of Homologous Mesenchymal Cell Types Positioned at Spatially Distinct Developmental Addresses in Early Development of Sea Urchins.

    Science.gov (United States)

    Erkenbrack, Eric M; Petsios, Elizabeth

    2017-07-01

    Comparative studies of early development in echinoderms are revealing the tempo and mode of alterations to developmental gene regulatory networks and to the cell types they specify. In euechinoid sea urchins, skeletogenic mesenchyme (SM) ingresses prior to gastrulation at the vegetal pole and aligns into a ring-like array with two bilateral pockets of cells, the sites where spiculogenesis will later occur. In cidaroid sea urchins, the anciently diverged sister clade to euechinoid sea urchins, a homologous SM cell type ingresses later in development, after gastrulation has commenced, and consequently at a distinct developmental address. Thus, a heterochronic shift of ingression of the SM cell type occurred in one of the echinoid lineages. In euechinoids, specification and migration of SM are facilitated by vascular endothelial growth factor (VEGF) signaling. We describe spatiotemporal expression of vegf and vegfr and experimental manipulations targeting VEGF signaling in the cidaroid Eucidaris tribuloides. Spatially, vegf and vegfr mRNA localizes similarly as in euechinoids, suggesting conserved deployment in echinoids despite their spatially distinct development addresses of ingression. Inhibition of VEGF signaling in E. tribuloides suggests its role in SM specification is conserved in echinoids. Temporal discrepancies between the onset of vegf expression and SM ingression likely result in previous observations of SM "random wandering" behavior. Our results indicate that, although the SM cell type in echinoids ingresses into distinct developmental landscapes, it retains a signaling mechanism that restricts their spatial localization to a conserved developmental address where spiculogenesis later occurs. © 2017 Wiley Periodicals, Inc.

  13. Pien Tze Huang Inhibits Hypoxia-Induced Angiogenesis via HIF-1α/VEGF-A Pathway in Colorectal Cancer

    Directory of Open Access Journals (Sweden)

    Hongwei Chen

    2015-01-01

    Full Text Available Hypoxia-induced angiogenesis plays an important role in the development and metastasis of solid tumors and is highly regulated by HIF-1α/VEGF-A pathway. Therefore, inhibiting tumor angiogenesis via suppression of HIF-1α/VEGF-A signaling represents a promising strategy for anticancer treatment. As a traditional Chinese medicine formula, Pien Tze Huang (PZH has long been used as a folk remedy for cancer in China and Southeast Asia. Previously, we reported that PZH inhibits colorectal cancer (CRC growth both in vivo and in vitro. To elucidate the antitumor mechanisms of PZH, in the present study we used human umbilical vein endothelial cells (HUVEC and colorectal carcinoma HCT-8 cells to evaluate the effects of PZH on hypoxia-induced angiogenesis and investigated the underlying molecular mechanisms. We found that PZH could inhibit hypoxia-induced migration and tube formation of HUVEC cells in a dose-dependent manner, although the low concentrations of PZH had no effect on HUVEC viability. Moreover, PZH inhibited hypoxia-induced activation of HIF-1α signaling and the expression of VEGF-A and/or VEGFR2 in both HCT-8 and HUVEC cells. Collectively, our findings suggest that PZH can inhibit hypoxia-induced tumor angiogenesis via suppression of HIF-1α/VEGF-A pathway.

  14. CXCL7-Mediated Stimulation of Lymphangiogenic Factors VEGF-C, VEGF-D in Human Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Minghuan Yu

    2010-01-01

    Full Text Available Increased expression of lymphangiogenesis factors VEGF-C/D and heparanase has been correlated with the invasion of cancer. Furthermore, chemokines may modify matrix to facilitate metastasis, and they are associated with VEGF-C and heparanase. The chemokine CXCL7 binds heparin and the G-protein-linked receptor CXCR2. We investigated the effect of CXCR2 blockade on the expression of VEGF-C/D, heparanase, and on invasion. CXCL7 siRNA and a specific antagonist of CXCR2 (SB225002 were used to treat CXCL7 stably transfected MCF10AT cells. Matrigel invasion assays were performed. VEGF-C/D expression and secretion were determined by real-time PCR and ELISA assay, and heparanase activity was quantified by ELISA. SB225002 blocked VEGF-C/D expression and secretion (P<.01. CXCL7 siRNA knockdown decreased heparanase (P<.01. Both SB225002 and CXCL7 siRNA reduced the Matrigel invasion (P<.01. The MAP kinase signaling pathway was not involved. The CXCL7/CXCR2 axis is important for cell invasion and the expression of VEGF-C/D and heparanase, all linked to invasion.

  15. Quercetin and epigallocatechin gallate inhibit glucose uptake and metabolism by breast cancer cells by an estrogen receptor-independent mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Moreira, Liliana, E-mail: lilianam87@gmail.com [Department of Biochemistry (U38-FCT), Faculty of Medicine of University of Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto (Portugal); Araújo, Isabel, E-mail: isa.araujo013@gmail.com [Department of Biochemistry (U38-FCT), Faculty of Medicine of University of Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto (Portugal); Costa, Tito, E-mail: tito.fmup16@gmail.com [Department of Biochemistry (U38-FCT), Faculty of Medicine of University of Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto (Portugal); Correia-Branco, Ana, E-mail: ana.clmc.branco@gmail.com [Department of Biochemistry (U38-FCT), Faculty of Medicine of University of Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto (Portugal); Faria, Ana, E-mail: anafaria@med.up.pt [Department of Biochemistry (U38-FCT), Faculty of Medicine of University of Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto (Portugal); Chemistry Investigation Centre (CIQ), Faculty of Sciences of University of Porto, Rua Campo Alegre, 4169-007 Porto (Portugal); Faculty of Nutrition and Food Sciences of University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto (Portugal); Martel, Fátima, E-mail: fmartel@med.up.pt [Department of Biochemistry (U38-FCT), Faculty of Medicine of University of Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto (Portugal); Keating, Elisa, E-mail: keating@med.up.pt [Department of Biochemistry (U38-FCT), Faculty of Medicine of University of Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto (Portugal)

    2013-07-15

    In this study we characterized {sup 3}H-2-deoxy-D-glucose ({sup 3}H -DG) uptake by the estrogen receptor (ER)-positive MCF7 and the ER-negative MDA-MB-231 human breast cancer cell lines and investigated the effect of quercetin (QUE) and epigallocatechin gallate (EGCG) upon {sup 3}H-DG uptake, glucose metabolism and cell viability and proliferation. In both MCF7 and MDA-MB-231 cells {sup 3}H-DG uptake was (a) time-dependent, (b) saturable with similar capacity (V{sub max}) and affinity (K{sub m}), (c) potently inhibited by cytochalasin B, an inhibitor of the facilitative glucose transporters (GLUT), (d) sodium-independent and (e) slightly insulin-stimulated. This suggests that {sup 3}H-DG uptake by both cell types is mediated by members of the GLUT family, including the insulin-responsive GLUT4 or GLUT12, while being independent of the sodium-dependent glucose transporter (SGLT1). QUE and EGCG markedly and concentration-dependently inhibited {sup 3}H-DG uptake by MCF7 and by MDA-MB-231 cells, and both compounds blocked lactate production by MCF7 cells. Additionally, a 4 h-treatment with QUE or EGCG decreased MCF7 cell viability and proliferation, an effect that was more potent when glucose was available in the extracellular medium. Our results implicate QUE and EGCG as metabolic antagonists in breast cancer cells, independently of estrogen signalling, and suggest that these flavonoids could serve as therapeutic agents/adjuvants even for ER-negative breast tumors. -- Highlights: • Glucose uptake by MCF7 and MDA-MB-231 cells is mainly mediated by GLUT1. • QUE and EGCG inhibit cellular glucose uptake thus abolishing the Warburg effect. • This process induces cytotoxicity and proliferation arrest in MCF7 cells. • The flavonoids’ effects are independent of estrogen receptor signalling.

  16. Irradiation-induced regulation of plasminogen activator inhibitor type-1 and vascular endothelial growth factor in six human squamous cell carcinoma lines of the head and neck; Bestrahlungsinduzierte Regulation des Plasminogenaktivator-Inhibitor Typ 1 (PAI-1) und des vaskulaeren endothelialen Wachstumsfaktors (VEGF) in sechs Plattenepithelkarzinomzelllinien der Kopf-Hals-Region

    Energy Technology Data Exchange (ETDEWEB)

    Artman, Meri Tuuli

    2014-01-29

    Radiation therapy is frequently used to treat squamous cell carcinoma of the head and neck (SCCHN), although, it can be unsuccessful due to radiation resistance of the tumor. Currently, there are no established predictive markers for radiation resistance in SCCHN. The aim of this work was to investigate PAI-1 and VEGF secretion as markers for radiation resistance in six human SCCHN cell lines. The cell lines differed in their basal secretion levels and in their in vitro radiation sensitivity. PAI-1 and VEGF levels increased after irradiation in a dose-dependent manner. A significant correlation was detected between radiation-induced PAI-1 and VEGF secretion, which suggests that irradiation-induced secretion of PAI-1 and VEGF are partially regulated by related mechanisms. However, neither basal levels nor radiation-induced PAI-1 and VEGF secretion correlated with radiation resistance. Therefore, PAI-1 and VEGF are most likely not predictive markers for radiation resistance in SCCHN.

  17. Breast-cancer extracellular vesicles induce platelet activation and aggregation by tissue factor-independent and -dependent mechanisms.

    Science.gov (United States)

    Gomes, Fausto G; Sandim, Vanessa; Almeida, Vitor H; Rondon, Araci M R; Succar, Barbara B; Hottz, Eugenio D; Leal, Ana Carolina; Verçoza, Brunno Renato F; Rodrigues, Juliany Cola F; Bozza, Patrícia T; Zingali, Russolina B; Monteiro, Robson Q

    2017-11-01

    Cancer-associated thrombosis is one of the major causes of worse prognosis among tumor-bearing patients. Extracellular vesicles derived from cancer cells, which can be divided mainly into microvesicles and exosomes, can participate in several tumor progression phenomena. Tumor-derived microvesicles positive for tissue factor (TF) have been associated with thrombotic risk in certain cancer types. Cancer cell-derived exosomes, however, have not. In this study we evaluated the capacity of extracellular vesicles (EVs, containing both microvesicles and exosomes) derived from breast-cancer cell lines in promoting platelet activation, aggregation and plasma coagulation, in experiments that access both TF-dependent and -independent activities. EVs were isolated from the conditioned media of two human mammary carcinoma cell lines: MDA-MB-231 (highly invasive) and MCF-7 (less invasive). TF-independent EV/platelet interaction, platelet P-selectin exposure and aggregation were evaluated. Western blotting, plasma clotting and platelet aggregation in the presence of plasma were performed for the measurement of TF-dependent activity in EVs. Interaction between MDA-MB-231 EVs and washed platelets led to increased platelet P-selectin exposure and platelet aggregation compared to MCF-7 EVs. MDA-MB-231 EVs had higher TF protein levels and TF-dependent procoagulant activity than MCF-7 EVs. Consequently, TF-dependent platelet aggregation was also induced by MDA-MB-231 EVs, but not by MCF-7 EVs. Our results suggest that MDA-MB-231 EVs induce TF-independent platelet activation and aggregation, as well as TF-dependent plasma clotting and platelet aggregation by means of thrombin generation. In this context, aggressive breast cancer-derived EVs may contribute to cancer-associated thrombosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. NCoR1-independent mechanism plays a role in the action of the unliganded thyroid hormone receptor.

    Science.gov (United States)

    Mendoza, Arturo; Astapova, Inna; Shimizu, Hiroaki; Gallop, Molly R; Al-Sowaimel, Lujain; MacGowan, S M Dileas; Bergmann, Tim; Berg, Anders H; Tenen, Danielle E; Jacobs, Christopher; Lyubetskaya, Anna; Tsai, Linus; Hollenberg, Anthony N

    2017-10-03

    Nuclear receptor corepressor 1 (NCoR1) is considered to be the major corepressor that mediates ligand-independent actions of the thyroid hormone receptor (TR) during development and in hypothyroidism. We tested this by expressing a hypomorphic NCoR1 allele (NCoR1ΔID), which cannot interact with the TR, in Pax8-KO mice, which make no thyroid hormone. Surprisingly, abrogation of NCoR1 function did not reverse the ligand-independent action of the TR on many gene targets and did not fully rescue the high mortality rate due to congenital hypothyroidism in these mice. To further examine NCoR1's role in repression by the unliganded TR, we deleted NCoR1 in the livers of euthyroid and hypothyroid mice and examined the effects on gene expression and enhancer activity measured by histone 3 lysine 27 (H3K27) acetylation. Even in the absence of NCoR1 function, we observed strong repression of more than 43% of positive T3 (3,3',5-triiodothyronine) targets in hypothyroid mice. Regulation of approximately half of those genes correlated with decreased H3K27 acetylation, and nearly 80% of these regions with affected H3K27 acetylation contained a bona fide TRβ1-binding site. Moreover, using liver-specific TRβ1-KO mice, we demonstrate that hypothyroidism-associated changes in gene expression and histone acetylation require TRβ1. Thus, many of the genomic changes mediated by the TR in hypothyroidism are independent of NCoR1, suggesting a role for additional signaling modulators in hypothyroidism.

  19. SREBP inhibits VEGF expression in human smooth muscle cells.

    Science.gov (United States)

    Motoyama, Koka; Fukumoto, Shinya; Koyama, Hidenori; Emoto, Masanori; Shimano, Hitoshi; Maemura, Koji; Nishizawa, Yoshiki

    2006-03-31

    Sterol regulatory element-binding proteins (SREBPs) are transcription factors that regulate expression of genes encoding enzymes for lipid biosynthesis. SREBPs are activated by HMG-CoA reductase inhibitors (statins). Statins have been also reported to suppress vascular endothelial growth factor (VEGF) expression in vascular smooth muscle cells (VSMCs). Therefore, we hypothesized that SREBPs are involved in statin-mediated regulation of VEGF production in VSMCs. SREBP1 was robustly expressed, and was activated by atorvastatin in VSMCs, as demonstrated by increased levels of the mature nuclear form of SREBP1, and increased promoter activities of a reporter containing sterol regulatory elements by atorvastatin. Moreover, overexpression of SREBP1a dose-dependently suppressed VEGF promoter activity. Site-specific mutation or deletion of the proximal Sp1 sites reduced the inhibitory effects of SREBP1a on VEGF promoter activity. These data demonstrated that SREBP1, activated by atorvastatin, suppressed VEGF expression through the indirect interaction with the proximal tandem Sp1 sites in VSMCs.

  20. Osteopontin and MMP9: Associations with VEGF Expression/Secretion and Angiogenesis in PC3 Prostate Cancer Cells

    Energy Technology Data Exchange (ETDEWEB)

    Gupta, Aditi; Zhou, Cindy Q.; Chellaiah, Meenakshi A., E-mail: mchellaiah@umaryland.edu [Department of Oncology and Diagnostic Sciences, Dental School, University of Maryland, Baltimore, MD 21201 (United States)

    2013-05-27

    Osteopontin and MMP9 are implicated in angiogenesis and cancer progression. The objective of this study is to gain insight into the molecular mechanisms underlying angiogenesis, and to elucidate the role of osteopontin in this process. We report here that osteopontin/αvβ3 signaling pathway which involves ERK1/2 phosphorylation regulates the expression of VEGF. An inhibitor to MEK or curcumin significantly suppressed the phosphorylation of ERK1/2 and expression of VEGF. MMP9 knockdown reduces the secretion but not the expression of VEGF. Moreover, MMP9 knockdown increases the release of angiostatin, a key protein that suppresses angiogenesis. Conditioned media from PC3 cells treated with curcumin or MEK inhibitor inhibited tube formation in vitro in human microvascular endothelial cells. Similar inhibitory effect on tube formation was found with conditioned media collected from PC3 cells expressing mutant-osteopontin at integrin-binding site and knockdown of osteopontin or MMP9. We conclude that MMP9 activation is associated with angiogenesis via regulation of secretion of VEGF and angiostatin in PC3 cells. Curcumin is thus a potential drug for cancer treatment because it demonstrated anti-angiogenic and anti-invasive properties.

  1. Low energy shock wave therapy induces angiogenesis in acute hind-limb ischemia via VEGF receptor 2 phosphorylation.

    Science.gov (United States)

    Holfeld, Johannes; Tepeköylü, Can; Blunder, Stefan; Lobenwein, Daniela; Kirchmair, Elke; Dietl, Marion; Kozaryn, Radoslaw; Lener, Daniela; Theurl, Markus; Paulus, Patrick; Kirchmair, Rudolf; Grimm, Michael

    2014-01-01

    Low energy shock waves have been shown to induce angiogenesis, improve left ventricular ejection fraction and decrease angina symptoms in patients suffering from chronic ischemic heart disease. Whether there is as well an effect in acute ischemia was not yet investigated. Hind-limb ischemia was induced in 10-12 weeks old male C57/Bl6 wild-type mice by excision of the left femoral artery. Animals were randomly divided in a treatment group (SWT, 300 shock waves at 0.1 mJ/mm2, 5 Hz) and untreated controls (CTR), n = 10 per group. The treatment group received shock wave therapy immediately after surgery. Higher gene expression and protein levels of angiogenic factors VEGF-A and PlGF, as well as their receptors Flt-1 and KDR have been found. This resulted in significantly more vessels per high-power field in SWT compared to controls. Improvement of blood perfusion in treatment animals was confirmed by laser Doppler perfusion imaging. Receptor tyrosine kinase profiler revealed significant phosphorylation of VEGF receptor 2 as an underlying mechanism of action. The effect of VEGF signaling was abolished upon incubation with a VEGFR2 inhibitor indicating that the effect is indeed VEGFR 2 dependent. Low energy shock wave treatment induces angiogenesis in acute ischemia via VEGF receptor 2 stimulation and shows the same promising effects as known from chronic myocardial ischemia. It may therefore develop as an adjunct to the treatment armentarium of acute muscle ischemia in limbs and myocardium.

  2. Exercise induced capillary growth in human skeletal muscle and the dynamics of VEGF

    DEFF Research Database (Denmark)

    Høier, Birgitte; Hellsten, Ylva

    2014-01-01

    , these VEGF containing vesicles are redistributed towards the sarcolemma where the contents are secreted into the extracellular fluid. VEGF mRNA expression is increased primarily after exercise, which allows for a more rapid replenishment of VEGF stores lost through secretion during exercise. Future studies...... in the muscle interstitium, acts on VEGF receptors on the capillary endothelium and thereby stimulates angiogenic processes. A primary source of muscle interstitial VEGF during exercise is the skeletal muscle fibers which contain large stores of VEGF within vesicles. We propose that, during muscle activity...

  3. Investigation of Antiangiogenic Tumor Therapy Potential of Microencapsulated HEK293 VEGF165b Producing Cells

    Directory of Open Access Journals (Sweden)

    Fatemeh Afkhami

    2010-01-01

    encapsulated and non-encapsulated cells was similar. The effect of VEGF165b harvested from encapsulated cells on Human Umbilical Vein Endothelial cells (HUVECs proliferation were also examined.The same inhibitory effects on HUVECs proliferation was seen when the cells were incubated with a mixture of VEGF165b and a 2-fold VEGF165b or with VEGF165b and 2-fold excess VEGF165b released from encapsulated cells. Subcutaneous injection of microencapsulated VEGF165b producing cells in tumor site of nude mice resulted in the reduction of the number of vessels around the tumors.

  4. DNA methylation regulates expression of VEGF-C, and S-adenosylmethionine is effective for VEGF-C methylation and for inhibiting cancer growth

    Energy Technology Data Exchange (ETDEWEB)

    Da, M.X. [Department of Surgical Oncology, Gansu Provincial Hospital, Lanzhou (China); Zhang, Y.B. [Department of Surgery, Ningxia Medical University, Yinchuan (China); Yao, J.B. [Department of Surgical Oncology, Gansu Provincial Hospital, Lanzhou (China); Duan, Y.X. [Department of Surgery, Ningxia Medical University, Yinchuan (China)

    2014-09-30

    DNA hypomethylation may activate oncogene transcription, thus promoting carcinogenesis and tumor development. S-adenosylmethionine (SAM) is a methyl donor in numerous methylation reactions and acts as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA. The main objectives of this study were to determine whether DNA hypomethylation correlated with vascular endothelial growth factor-C (VEGF-C) expression, and the effect of SAM on VEGF-C methylation and gastric cancer growth inhibition. VEGF-C expression was assayed by Western blotting and RT-qPCR in gastric cancer cells, and by immunohistochemistry in tumor xenografts. VEGF-C methylation was assayed by bisulfite DNA sequencing. The effect of SAM on cell apoptosis was assayed by flow cytometry analyses and its effect on cancer growth was assessed in nude mice. The VEGF-C promoters of MGC-803, BGC-823, and SGC-7901 gastric cancer cells, which normally express VEGF-C, were nearly unmethylated. After SAM treatment, the VEGF-C promoters in these cells were highly methylated and VEGF-C expression was downregulated. SAM also significantly inhibited tumor growth in vitro and in vivo. DNA methylation regulates expression of VEGF-C. SAM can effectively induce VEGF-C methylation, reduce the expression of VEGF-C, and inhibit tumor growth. SAM has potential as a drug therapy to silence oncogenes and block the progression of gastric cancer.

  5. How the inverse seesaw mechanism can reveal itself natural, canonical, and independent of the right-handed neutrino mass

    Science.gov (United States)

    Dias, A. G.; de S. Pires, C. A.; Rodrigues da Silva, P. S.

    2011-09-01

    The common lore in the literature of neutrino mass generation is that the canonical seesaw mechanism beautifully offers an explanation for the tiny neutrino mass but at the cost of introducing right-handed neutrinos at a scale that is out of range for the current experiments. The inverse seesaw mechanism is an interesting alternative to the canonical one once it leads to tiny neutrino masses with the advantage of being testable at the TeV scale. However, this last mechanism suffers from an issue of naturalness concerning the scale responsible for such small masses, namely, the parameter μ that is related to lepton number violation and is supposed to be at the keV scale, much lower than the electroweak one. However, no theoretical framework was built that offers an explanation for obtaining this specific scale. In this work, we propose a variation of the inverse seesaw mechanism by assuming a minimal scalar and fermionic set of singlet fields, along with a Z5⊗Z2 symmetry, that allows a dynamical explanation for the smallness of μ, recovering the neat canonical seesaw formula and with right-handed (RH) neutrinos free to be at the electroweak scale, thus testable at LHC and current neutrino experiments.

  6. Mechanisms of biopower and neoliberal governmentality in precarious work: Mobilizing the dependent self-employed as independent business owners

    NARCIS (Netherlands)

    Moisander, J.; Groß, C.; Eräranta, K.

    2018-01-01

    In the contemporary conditions of neoliberal governmentality, and the emerging ‘gig economy,’ standard employment relationships appear to be giving way to precarious work. This article examines the mechanisms of biopower and techniques of managerial control that underpin—and produce consent

  7. Mechanical stress triggers cardiomyocyte autophagy through angiotensin II type 1 receptor-mediated p38MAP kinase independently of angiotensin II.

    Directory of Open Access Journals (Sweden)

    Li Lin

    Full Text Available Angiotensin II (Ang II type 1 (AT1 receptor is known to mediate a variety of physiological actions of Ang II including autophagy. However, the role of AT1 receptor in cardiomyocyte autophagy triggered by mechanical stress still remains elusive. The aim of this study was therefore to examine whether and how AT1 receptor participates in cardiomyocyte autophagy induced by mechanical stresses. A 48-hour mechanical stretch and a 4-week transverse aorta constriction (TAC were imposed to cultured cardiomyocytes of neonatal rats and adult male C57B/L6 mice, respectively, to induce cardiomyocyte hypertrophy prior to the assessment of cardiomyocyte autophagy using LC3b-II. Losartan, an AT1 receptor blocker, but not PD123319, the AT2 inhibitor, was found to significantly reduce mechanical stretch-induced LC3b-II upregulation. Moreover, inhibition of p38MAP kinase attenuated not only mechanical stretch-induced cardiomyocyte hypertrophy but also autophagy. To the contrary, inhibition of ERK and JNK suppressed cardiac hypertrophy but not autophagy. Intriguingly, mechanical stretch-induced autophagy was significantly inhibited by Losartan in the absence of Ang II. Taken together, our results indicate that mechanical stress triggers cardiomyocyte autophagy through AT1 receptor-mediated activation of p38MAP kinase independently of Ang II.

  8. Model-based unsupervised learning informs metformin-induced cell-migration inhibition through an AMPK-independent mechanism in breast cancer.

    Science.gov (United States)

    Athreya, Arjun P; Kalari, Krishna R; Cairns, Junmei; Gaglio, Alan J; Wills, Quin F; Niu, Nifang; Weinshilboum, Richard; Iyer, Ravishankar K; Wang, Liewei

    2017-04-18

    We demonstrate that model-based unsupervised learning can uniquely discriminate single-cell subpopulations by their gene expression distributions, which in turn allow us to identify specific genes for focused functional studies. This method was applied to MDA-MB-231 breast cancer cells treated with the antidiabetic drug metformin, which is being repurposed for treatment of triple-negative breast cancer. Unsupervised learning identified a cluster of metformin-treated cells characterized by a significant suppression of 230 genes (p-value metformin action: a) pathway analysis indicated known mechanisms related to metformin action, including the citric acid (TCA) cycle, oxidative phosphorylation, and mitochondrial dysfunction (p-value metformin response; c) among remaining lesser functionally-studied genes for metformin-response was CDC42, down-regulated in breast cancer treated with metformin. However, CDC42's mechanisms in metformin response remained unclear. Our functional studies showed that CDC42 was involved in metformin-induced inhibition of cell proliferation and cell migration mediated through an AMPK-independent mechanism. Our results points to 230 genes that might serve as metformin response signatures, which needs to be tested in patients treated with metformin and, further investigation of CDC42 and AMPK-independence's role in metformin's anticancer mechanisms.

  9. VEGF gene polymorphisms and outcome of epithelial ovarian cancer patients.

    Science.gov (United States)

    Camerin, Gabriela Ribeiro; Brito, Angelo Borsarelli Carvalho; Vassallo, José; Derchain, Sophie Françoise Mauricette; Lima, Carmen Silvia Passos

    2017-02-01

    Since VEGF polymorphisms were associated with variable protein production, we analyzed herein their roles in outcome of epithelial ovarian cancer (EOC) patients. Genotypes of 85 patients with primary EOC were identified in DNA by real-time PCR. Progression-free survival and overall survival were analyzed using Kaplan-Meier method, univariate Cox model and bootstrap resampling study. At 60 months of follow-up, progression-free survival was shorter in patients with VEGF c.-2578 CC genotype compared with others (52.7 vs 82.2%; p = 0.04). Those patients had 2.15 more chance of presenting disease progression than others (p = 0.04); bootstrap study validated the result (p = 0.03). Our data suggest that VEGF c.-2578C>A polymorphism acts as a prognostic factor in EOC.

  10. On the definition of the time evolution operator for time-independent Hamiltonians in non-relativistic quantum mechanics

    Science.gov (United States)

    Amaku, Marcos; Coutinho, Francisco A. B.; Masafumi Toyama, F.

    2017-09-01

    The usual definition of the time evolution operator e-i H t /ℏ=∑n=0∞1/n ! (-i/ℏHt ) n , where H is the Hamiltonian of the system, as given in almost every book on quantum mechanics, causes problems in some situations. The operators that appear in quantum mechanics are either bounded or unbounded. Unbounded operators are not defined for all the vectors (wave functions) of the Hilbert space of the system; when applied to some states, they give a non-normalizable state. Therefore, if H is an unbounded operator, the definition in terms of the power series expansion does not make sense because it may diverge or result in a non-normalizable wave function. In this article, we explain why this is so and suggest, as an alternative, another definition used by mathematicians.

  11. Inhibition of histone deacetylase 1 or 2 reduces induced cytokine expression in microglia through a protein synthesis independent mechanism.

    Science.gov (United States)

    Durham, Benjamin S; Grigg, Ronald; Wood, Ian C

    2017-10-01

    Histone deacetylase (HDAC) inhibitors prevent neural cell death in in vivo models of cerebral ischaemia, brain injury and neurodegenerative disease. One mechanism by which HDAC inhibitors may do this is by suppressing the excessive inflammatory response of chronically activated microglia. However, the molecular mechanisms underlying this anti-inflammatory effect and the specific HDAC responsible are not fully understood. Recent data from in vivo rodent studies have shown that inhibition of class I HDACs suppresses neuroinflammation and is neuroprotective. In our study, we have identified that selective HDAC inhibition with inhibitors apicidin, MS-275 or MI-192, or specific knockdown of HDAC1 or 2 using siRNA, suppresses the expression of cytokines interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α) in BV-2 murine microglia activated with lipopolysaccharide (LPS). Furthermore, we found that in the absence of HDAC1, HDAC2 is up-regulated and these increased levels are compensatory, suggesting that these two HDACs have redundancy in regulating the inflammatory response of microglia. Investigating the possible underlying anti-inflammatory mechanisms suggests an increase in protein expression is not important. Taken together, this study supports the idea that inhibitors selective towards HDAC1 or HDAC2, may be therapeutically useful for targeting neuroinflammation in brain injuries and neurodegenerative disease. © 2017 International Society for Neurochemistry.

  12. The Regulatory T Cell Lineage Factor Foxp3 Regulates Gene Expression through Several Distinct Mechanisms Mostly Independent of Direct DNA Binding.

    Directory of Open Access Journals (Sweden)

    Xin Xie

    2015-06-01

    Full Text Available The lineage factor Foxp3 is essential for the development and maintenance of regulatory T cells, but little is known about the mechanisms involved. Here, we demonstrate that an N-terminal proline-rich interaction region is crucial for Foxp3's function. Subdomains within this key region link Foxp3 to several independent mechanisms of transcriptional regulation. Our study suggests that Foxp3, even in the absence of its DNA-binding forkhead domain, acts as a bridge between DNA-binding interaction partners and proteins with effector function permitting it to regulate a large number of genes. We show that, in one such mechanism, Foxp3 recruits class I histone deacetylases to the promoters of target genes, counteracting activation-induced histone acetylation and thereby suppressing their expression.

  13. The retrohoming of linear group II intron RNAs in Drosophila melanogaster occurs by both DNA ligase 4-dependent and -independent mechanisms.

    Directory of Open Access Journals (Sweden)

    Travis B White

    Full Text Available Mobile group II introns are bacterial retrotransposons that are thought to have invaded early eukaryotes and evolved into introns and retroelements in higher organisms. In bacteria, group II introns typically retrohome via full reverse splicing of an excised intron lariat RNA into a DNA site, where it is reverse transcribed by the intron-encoded protein. Recently, we showed that linear group II intron RNAs, which can result from hydrolytic splicing or debranching of lariat RNAs, can retrohome in eukaryotes by performing only the first step of reverse splicing, ligating their 3' end to the downstream DNA exon. Reverse transcription then yields an intron cDNA, whose free end is linked to the upstream DNA exon by an error-prone process that yields junctions similar to those formed by non-homologous end joining (NHEJ. Here, by using Drosophila melanogaster NHEJ mutants, we show that linear intron RNA retrohoming occurs by major Lig4-dependent and minor Lig4-independent mechanisms, which appear to be related to classical and alternate NHEJ, respectively. The DNA repair polymerase θ plays a crucial role in both pathways. Surprisingly, however, mutations in Ku70, which functions in capping chromosome ends during NHEJ, have only moderate, possibly indirect effects, suggesting that both Lig4 and the alternate end-joining ligase act in some retrohoming events independently of Ku. Another potential Lig4-independent mechanism, reverse transcriptase template switching from the intron RNA to the upstream exon DNA, occurs in vitro, but gives junctions differing from the majority in vivo. Our results show that group II introns can utilize cellular NHEJ enzymes for retromobility in higher organisms, possibly exploiting mechanisms that contribute to retrotransposition and mitigate DNA damage by resident retrotransposons. Additionally, our results reveal novel activities of group II intron reverse transcriptases, with implications for retrohoming mechanisms and

  14. In vitro evidence supports the presence of glucokinase-independent glucosensing mechanisms in hypothalamus and hindbrain of rainbow trout.

    Science.gov (United States)

    Otero-Rodiño, Cristina; Velasco, Cristina; Álvarez-Otero, Rosa; López-Patiño, Marcos A; Míguez, Jesús M; Soengas, José L

    2016-06-01

    We previously obtained evidence in rainbow trout for the presence and response to changes in circulating levels of glucose (induced by intraperitoneal hypoglycaemic and hyperglycaemic treatments) of glucosensing mechanisms based on liver X receptor (LXR), mitochondrial production of reactive oxygen species (ROS) leading to increased expression of uncoupling protein 2 (UCP2), and sweet taste receptor in the hypothalamus, and on sodium/glucose co-transporter 1 (SGLT-1) in hindbrain. However, these effects of glucose might be indirect. Therefore, we evaluated the response of parameters related to these glucosensing mechanisms in a first experiment using pooled sections of hypothalamus and hindbrain incubated for 6 h at 15°C in modified Hanks' medium containing 2, 4 or 8 mmol l(-1) d-glucose. The responses observed in some cases were consistent with glucosensing capacity. In a second experiment, pooled sections of hypothalamus and hindbrain were incubated for 6 h at 15°C in modified Hanks' medium with 8 mmol l(-1) d-glucose alone (control) or containing 1 mmol l(-1) phloridzin (SGLT-1 antagonist), 20 µmol l(-1) genipin (UCP2 inhibitor), 1 µmol l(-1) trolox (ROS scavenger), 100 µmol l(-1) bezafibrate (T1R3 inhibitor) and 50 µmol l(-1) geranyl-geranyl pyrophosphate (LXR inhibitor). The response observed in the presence of these specific inhibitors/antagonists further supports the proposal that critical components of the different glucosensing mechanisms are functioning in rainbow trout hypothalamus and hindbrain. © 2016. Published by The Company of Biologists Ltd.

  15. Left ventricular mechanics in humans with high aerobic fitness: adaptation independent of structural remodelling, arterial haemodynamics and heart rate

    Science.gov (United States)

    Stöhr, Eric J; McDonnell, Barry; Thompson, Jane; Stone, Keeron; Bull, Tom; Houston, Rory; Cockcroft, John; Shave, Rob

    2012-01-01

    Individuals with high aerobic fitness have lower systolic left ventricular strain, rotation and twist (‘left ventricular (LV) mechanics’) at rest, suggesting a beneficial reduction in LV myofibre stress and more efficient systolic function. However, the mechanisms responsible for this functional adaptation are not known and the influence of aerobic fitness on LV mechanics during dynamic exercise has never been studied. We assessed LV mechanics, LV wall thickness and dimensions, central augmentation index (AIx), aortic pulse wave velocity (aPWV), blood pressure and heart rate in 28 males (age: 21 ± 2 years SD) with a consistent physical activity level (no change >6 months). Individuals were examined at rest and during exercise (40% peak exercise capacity) and separated post hoc into a moderate and high aerobic fitness group (: 49 ± 5 and 63 ± 7 ml kg−1 min−1, respectively, P 0.05). However, for the same AIx, the high group had significantly lower LV apical rotation (P = 0.002) and LV twist (P = 0.003) while basal rotation and strain indices did not differ between groups (P > 0.05). We conclude that young males with high aerobic fitness have lower LV apical rotation at rest and during submaximal exercise that can occur without changes in gross LV structure, arterial haemodynamics or heart rate. The findings suggest a previously unknown type of physiological adaptation of the left ventricle that may have important implications for exercise training in older individuals and patient populations in which exercise training has previously failed to show clear benefits for LV function. PMID:22431336

  16. In the absence of BYPASS1-related gene function, the bps signal disrupts embryogenesis by an auxin-independent mechanism.

    Science.gov (United States)

    Lee, Dong-Keun; Van Norman, Jaimie M; Murphy, Caroline; Adhikari, Emma; Reed, Jason W; Sieburth, Leslie E

    2012-02-01

    Development is often coordinated by biologically active mobile compounds that move between cells or organs. Arabidopsis mutants with defects in the BYPASS1 (BPS1) gene overproduce an active mobile compound that moves from the root to the shoot and inhibits growth. Here, we describe two related Arabidopsis genes, BPS2 and BPS3. Analyses of single, double and triple mutants revealed that all three genes regulate production of the same mobile compound, the bps signal, with BPS1 having the largest role. The triple mutant had a severe embryo defect, including the failure to properly establish provascular tissue, the shoot meristem and the root meristem. Aberrant expression of PINFORMED1, DR5, PLETHORA1, PLETHORA2 and WUSCHEL-LIKE HOMEOBOX5 were found in heart-stage bps triple-mutant embryos. However, auxin-induced gene expression, and localization of the PIN1 auxin efflux transporter, were intact in bps1 mutants, suggesting that the primary target of the bps signal is independent of auxin response. Thus, the bps signal identifies a novel signaling pathway that regulates patterning and growth in parallel with auxin signaling, in multiple tissues and at multiple developmental stages.

  17. Both overlapping and independent mechanisms determine how diet and insulin-ligand knockouts extend lifespan of Drosophila melanogaster.

    Science.gov (United States)

    Zandveld, Jelle; van den Heuvel, Joost; Zwaan, Bastiaan J; Piper, Matthew D W

    2017-01-01

    Lifespan in many organisms, including Drosophila melanogaster, can be increased by reduced insulin-IGF-like signaling (IIS) or by changes in diet. Most studies testing whether IIS is involved in diet-mediated lifespan extension employ only a few diets, but recent data shows that a broad range of nutritional environments is required. Here, we present lifespan data of long-lived Drosophila, lacking three of the eight insulin-like peptides [Drosophila insulin-like peptides 2,3,5 (dilp2-3,5)] on nine different diets that surround the optimum for lifespan. Their nutritional content was varied by manipulating sugar and yeast concentrations independently, and thus incorporated changes in both diet restriction and nutrient balance. The mutants were substantially longer-lived than controls on every diet, but the effects on the lifespan response to sugar and yeast differed. Our data illustrates how a greater coverage of diet balance (DB) and restriction can unify differing interpretations of how IIS might be involved in the response of lifespan to diet.

  18. Curcumol Promotes Vascular Endothelial Growth Factor (VEGF)-Mediated Diabetic Wound Healing in Streptozotocin-Induced Hyperglycemic Rats.

    Science.gov (United States)

    Zhou, Jie; Ni, Maowei; Liu, Xia; Ren, Zeming; Zheng, Zhiguo

    2017-01-31

    BACKGROUND Wound healing in chronic diabetic mellitus is mainly associated with the management of angiogenesis. The angiogenic mechanism of vascular endothelial growth factor (VEGF) has been widely studied in the context of diabetic ulcers. The aim of this study was to investigate the wound-healing potential of curcumol in streptozotocin-induced diabetic rats. MATERIAL AND METHODS Sixty male SD (Sprague Dawley) rats were purchased and randomly assigned into four groups: a control group and a model group treated with blank ointment, a high-dose curcumol group, and a low-dose curcumol group. The number of animals in each group was 15. Diabetes was induced by an intraperitoneal injection of streptozotocin. Two cutaneous wounds were incised at the dorsal region of all the experimental animals. Wound healing was assessed for all animal groups by observing the rate of wound closure. The expression of VEGF at the wound sites was studied by immunohistochemical staining to evaluate the vascular endothelial cell reaction. VEGF protein and related mRNA levels were analyzed by Western blotting and RT-PCR (reverse transcription-polymerase chain reaction). RESULTS Curcumol treatment significantly increased the rates of wound closure in treated animals, and hence wound healing was drastically enhanced for treatment groups compared to control groups. Histological observations and related mRNA and protein levels showed a higher VEGF expression in the treatment groups. CONCLUSIONS Our analyses clearly suggested that the observed enhancement in wound healing as a result of curcumol administration was attributable to VEGF-mediated angiogenesis.

  19. Prolonged presence of VEGF promotes vascularization in 3D bioprinted scaffolds with defined architecture

    NARCIS (Netherlands)

    Poldervaart, Michelle T; Gremmels, Hendrik; van Deventer, Kelly; Fledderus, Joost O; Oner, F Cumhur; Verhaar, Marianne C; Dhert, Wouter J A; Alblas, Jacqueline

    2014-01-01

    Timely vascularization is essential for optimal performance of bone regenerative constructs. Vascularization is efficiently stimulated by vascular endothelial growth factor (VEGF), a substance with a short half-life time. This study investigates the controlled release of VEGF from gelatin

  20. Recombinant Goat VEGF164 Increases Hair Growth by Painting Process on the Skin of Shaved Mouse

    Directory of Open Access Journals (Sweden)

    Wenlei Bao

    2014-09-01

    Full Text Available To detect goat vascular endothelial growth factor (VEGF-mediated regrowth of hair, full-length VEGF164 cDNA was cloned from Inner Mongolia cashmere goat (Capra hircus into the pET-his prokaryotic expression vector, and the recombinant plasmid was transferred into E. coli BL21 cells. The expression of recombinant 6×his-gVEGF164 protein was induced by 0.5 mM isopropyl thio-β-D-galactoside at 32°C. Recombinant goat VEGF164 (rgVEGF164 was purified and identi ed by western blot using monoclonal anti-his and anti-VEGF antibodies. The rgVEGF164 was smeared onto the dorsal area of a shaved mouse, and we noted that hair regrowth in this area was faster than in the control group. Thus, rgVEGF164 increases hair growth in mice.

  1. Delphinidin Inhibits Tumor Growth by Acting on VEGF Signalling in Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Thérèse Keravis

    Full Text Available The vasculoprotective properties of delphinidin are driven mainly by its action on endothelial cells. Moreover, delphinidin displays anti-angiogenic properties in both in vitro and in vivo angiogenesis models and thereby might prevent the development of tumors associated with excessive vascularization. This study was aimed to test the effect of delphinidin on melanoma-induced tumor growth with emphasis on its molecular mechanism on endothelial cells. Delphinidin treatment significantly decreased in vivo tumor growth induced by B16-F10 melanoma cell xenograft in mice. In vitro, delphinidin was not able to inhibit VEGFR2-mediated B16-F10 melanoma cell proliferation but it specifically reduced basal and VEGFR2-mediated endothelial cell proliferation. The anti-proliferative effect of delphinidin was reversed either by the MEK1/2 MAP kinase inhibitor, U-0126, or the PI3K inhibitor, LY-294002. VEGF-induced proliferation was reduced either by U-0126 or LY-294002. Under these conditions, delphinidin failed to decrease further endothelial cell proliferation. Delphinidin prevented VEGF-induced phosphorylation of ERK1/2 and p38 MAPK and decreased the expression of the transcription factors, CREB and ATF1. Finally, delphinidin was more potent in inhibiting in vitro cyclic nucleotide phosphodiesterases (PDEs, PDE1 and PDE2, compared to PDE3-PDE5. Altogether delphinidin reduced tumor growth of melanoma cell in vivo by acting specifically on endothelial cell proliferation. The mechanism implies an association between inhibition of VEGF-induced proliferation via VEGFR2 signalling, MAPK, PI3K and at transcription level on CREB/ATF1 factors, and the inhibition of PDE2. In conjunction with our previous studies, we demonstrate that delphinidin is a promising compound to prevent pathologies associated with generation of vascular network in tumorigenesis.

  2. An RNA editing/dsRNA binding-independent gene regulatory mechanism of ADARs and its clinical implication in cancer.

    Science.gov (United States)

    Qi, Lihua; Song, Yangyang; Chan, Tim Hon Man; Yang, Henry; Lin, Chi Ho; Tay, Daryl Jin Tai; Hong, HuiQi; Tang, Sze Jing; Tan, Kar Tong; Huang, Xi Xiao; Lin, Jaymie Siqi; Ng, Vanessa Hui En; Maury, Julien Jean Pierre; Tenen, Daniel G; Chen, Leilei

    2017-10-13

    Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by Adenosine DeAminases acting on double-stranded RNA(dsRNA) (ADAR), occurs predominantly in the 3' untranslated regions (3'UTRs) of spliced mRNA. Here we uncover an unanticipated link between ADARs (ADAR1 and ADAR2) and the expression of target genes undergoing extensive 3'UTR editing. Using METTL7A (Methyltransferase Like 7A), a novel tumor suppressor gene with multiple editing sites at its 3'UTR, we demonstrate that its expression could be repressed by ADARs beyond their RNA editing and double-stranded RNA (dsRNA) binding functions. ADARs interact with Dicer to augment the processing of pre-miR-27a to mature miR-27a. Consequently, mature miR-27a targets the METTL7A 3'UTR to repress its expression level. In sum, our study unveils that the extensive 3'UTR editing of METTL7A is merely a footprint of ADAR binding, and there are a subset of target genes that are equivalently regulated by ADAR1 and ADAR2 through their non-canonical RNA editing and dsRNA binding-independent functions, albeit maybe less common. The functional significance of ADARs is much more diverse than previously appreciated and this gene regulatory function of ADARs is most likely to be of high biological importance beyond the best-studied editing function. This non-editing side of ADARs opens another door to target cancer. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Cadmium-induced glutathionylation of actin occurs through a ROS-independent mechanism: Implications for cytoskeletal integrity

    Energy Technology Data Exchange (ETDEWEB)

    Choong, Grace; Liu, Ying; Xiao, Weiqun; Templeton, Douglas M., E-mail: doug.templeton@utoronto.ca

    2013-10-15

    Glutathionylation requires glutathione synthesis but is independent of ROS. • Glutathionylation is protective against cytoskeletal disruption at low cadmium.

  4. Auxin promotes susceptibility to Pseudomonas syringae via a mechanism independent of suppression of salicylic acid-mediated defenses.

    Science.gov (United States)

    Mutka, Andrew M; Fawley, Stephen; Tsao, Tiffany; Kunkel, Barbara N

    2013-06-01

    Auxin is a key plant growth regulator that also impacts plant-pathogen interactions. Several lines of evidence suggest that the bacterial plant pathogen Pseudomonas syringae manipulates auxin physiology in Arabidopsis thaliana to promote pathogenesis. Pseudomonas syringae strategies to alter host auxin biology include synthesis of the auxin indole-3-acetic acid (IAA) and production of virulence factors that alter auxin responses in host cells. The application of exogenous auxin enhances disease caused by P. syringae strain DC3000. This is hypothesized to result from antagonism between auxin and salicylic acid (SA), a major regulator of plant defenses, but this hypothesis has not been tested in the context of infected plants. We further investigated the role of auxin during pathogenesis by examining the interaction of auxin and SA in the context of infection in plants with elevated endogenous levels of auxin. We demonstrated that elevated IAA biosynthesis in transgenic plants overexpressing the YUCCA 1 (YUC1) auxin biosynthesis gene led to enhanced susceptibility to DC3000. Elevated IAA levels did not interfere significantly with host defenses, as effector-triggered immunity was active in YUC1-overexpressing plants, and we observed only minor effects on SA levels and SA-mediated responses. Furthermore, a plant line carrying both the YUC1-overexpression transgene and the salicylic acid induction deficient 2 (sid2) mutation, which impairs SA synthesis, exhibited additive effects of enhanced susceptibility from both elevated auxin levels and impaired SA-mediated defenses. Thus, in IAA overproducing plants, the promotion of pathogen growth occurs independently of suppression of SA-mediated defenses. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  5. Goal-independent mechanisms for free response generation: creative and pseudo-random performance share neural substrates.

    Science.gov (United States)

    de Manzano, Örjan; Ullén, Fredrik

    2012-01-02

    To what extent free response generation in different tasks uses common and task-specific neurocognitive processes has remained unclear. Here, we investigated overlap and differences in neural activity during musical improvisation and pseudo-random response generation. Brain activity was measured using fMRI in a group of professional classical pianists, who performed musical improvisation of melodies, pseudo-random key-presses and a baseline condition (sight-reading), on either two, six or twelve keys on a piano keyboard. The results revealed an extensive overlap in neural activity between the two generative conditions. Active regions included the dorsolateral and dorsomedial prefrontal cortices, inferior frontal gyrus, anterior cingulate cortex and pre-SMA. No regions showed higher activity in improvisation than in pseudo-random generation. These findings suggest that the activated regions fulfill generic functions that are utilized in different types of free generation tasks, independent of overall goal. In contrast, pseudo-random generation was accompanied by higher activity than improvisation in several regions. This presumably reflects the participants' musical expertise as well as the pseudo-random generation task's high load on attention, working memory, and executive control. The results highlight the significance of using naturalistic tasks to study human behavior and cognition. No brain activity was related to the size of the response set. We discuss that this may reflect that the musicians were able to use specific strategies for improvisation, by which there was no simple relationship between response set size and neural activity. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. Low Molecular Weight Heparin Inhibits Plasma Thrombin Generation via Direct Targeting of Factor IXa: Contribution of the Serpin-independent Mechanism

    Science.gov (United States)

    Buyue, Yang; Misenheimer, Tina M.; Sheehan, John P.

    2012-01-01

    Background While heparin possesses multiple mechanisms of action, enhanced factor Xa inhibition by antithrombin is accepted as the predominant therapeutic mechanism. The contribution of factor IXa inhibition to heparin activity in human plasma remains incompletely defined. Objectives To determine the relevance of factor IXa as a therapeutic target for heparins, particularly serpin-independent inhibition of intrinsic tenase (factor IXa-factor VIIIa) activity. Patient/Methods Thrombin generation was detected by fluorogenic substrate cleavage. Inhibitory potency (EC50) of low molecular weight heparin (LMWH), super-sulfated LMWH (ssLMWH), Fondaparinux, and unfractionated heparin (UFH) was determined by plotting concentration versus relative velocity index (ratio +/− heparin). Inhibition was compared under factor IX-dependent and independent conditions (0.2 or 4 pM TF, respectively) in normal plasma, and in mock- or antithrombin/factor IX-depleted plasma supplemented with recombinant factor IX. Results UFH and Fondaparinux demonstrated similar potency under factor IX-dependent and independent conditions, whereas LMWH (2.9-fold) and ssLMWH (5.1-fold) demonstrated increased potency with limiting TF. UFH (62-fold) and Fondaparinux (42-fold) demonstrated markedly increased EC50 values in antithrombin-depleted plasma, whereas LMWH (9.4-fold) and ssLMWH (2-fold) were less affected, with an EC50 within the therapeutic range for LMWH. The molecular target for LMWH/ssLMWH was confirmed by supplementing factor IX/antithrombin-depleted plasma with 90 nM recombinant factor IX possessing mutations in the heparin-binding exosite. Mutated factor IX demonstrated resistance to inhibition of thrombin generation by LMWH and ssLMWH that paralleled the effect of these mutations on intrinsic tenase inhibition. Conclusions Therapeutic LMWH concentrations inhibit plasma thrombin generation via antithrombin-independent interaction with the factor IXa heparin-binding exosite. PMID:22905983

  7. Tumor cell–associated tissue factor and circulating hemostatic factors cooperate to increase metastatic potential through natural killer cell–dependent and–independent mechanisms

    Science.gov (United States)

    Palumbo, Joseph S.; Talmage, Kathryn E.; Massari, Jessica V.; La Jeunesse, Christine M.; Flick, Matthew J.; Kombrinck, Keith W.; Hu, Zhiwei; Barney, Kelley A.

    2007-01-01

    Tumor cell–associated tissue factor (TF) is a powerful determinant of metastatic potential. TF may increase metastasis by supporting thrombin-mediated proteolysis, through intracellular signaling events mediated by the TF cytoplasmic domain, through TF/fVIIa/fXa–mediated activation of protease-activated receptors, or through a combination of these processes. To better define the relationship between tumor cell-associated TF and circulating hemostatic factors in malignancy, we generated a set of C57Bl/6-derived tumor lines genetically lacking TF, expressing wild-type murine TF, or expressing a mutant TF lacking the cytoplasmic domain. Comparison of the metastatic potential of these cells in immunocompetent mice with genetic deficits in prothrombin, platelet function, or fibrinogen revealed that TF supports metastasis through mechanisms independent of the cytoplasmic domain, but dependent on each of these distal hemostatic factors. TF was neither required for primary tumor growth nor necessary for initial localization of embolized tumor cells within the lungs. Rather, tumor cell fate studies indicated TF supports metastasis by increasing the survival of micrometastases. One mechanism linking TF to metastasis is through a fibrin(ogen)-dependent and platelet-dependent restriction in natural killer cell–mediated clearance of micrometastases. However, TF also supported the early success of micrometastases through an additional mechanism independent of natural killer cells, but coupled to circulating prothrombin. PMID:17371949

  8. Tumor cell-associated tissue factor and circulating hemostatic factors cooperate to increase metastatic potential through natural killer cell-dependent and-independent mechanisms.

    Science.gov (United States)

    Palumbo, Joseph S; Talmage, Kathryn E; Massari, Jessica V; La Jeunesse, Christine M; Flick, Matthew J; Kombrinck, Keith W; Hu, Zhiwei; Barney, Kelley A; Degen, Jay L

    2007-07-01

    Tumor cell-associated tissue factor (TF) is a powerful determinant of metastatic potential. TF may increase metastasis by supporting thrombin-mediated proteolysis, through intracellular signaling events mediated by the TF cytoplasmic domain, through TF/fVIIa/fXa-mediated activation of protease-activated receptors, or through a combination of these processes. To better define the relationship between tumor cell-associated TF and circulating hemostatic factors in malignancy, we generated a set of C57Bl/6-derived tumor lines genetically lacking TF, expressing wild-type murine TF, or expressing a mutant TF lacking the cytoplasmic domain. Comparison of the metastatic potential of these cells in immunocompetent mice with genetic deficits in prothrombin, platelet function, or fibrinogen revealed that TF supports metastasis through mechanisms independent of the cytoplasmic domain, but dependent on each of these distal hemostatic factors. TF was neither required for primary tumor growth nor necessary for initial localization of embolized tumor cells within the lungs. Rather, tumor cell fate studies indicated TF supports metastasis by increasing the survival of micrometastases. One mechanism linking TF to metastasis is through a fibrin(ogen)-dependent and platelet-dependent restriction in natural killer cell-mediated clearance of micrometastases. However, TF also supported the early success of micrometastases through an additional mechanism independent of natural killer cells, but coupled to circulating prothrombin.

  9. A new human NHERF1 mutation decreases renal phosphate transporter NPT2a expression by a PTH-independent mechanism.

    Directory of Open Access Journals (Sweden)

    Marie Courbebaisse

    Full Text Available BACKGROUND: The sodium-hydrogen exchanger regulatory factor 1 (NHERF1 binds to the main renal phosphate transporter NPT2a and to the parathyroid hormone (PTH receptor. We have recently identified mutations in NHERF1 that decrease renal phosphate reabsorption by increasing PTH-induced cAMP production in the renal proximal tubule. METHODS: We compared relevant parameters of phosphate homeostasis in a patient with a previously undescribed mutation in NHERF1 and in control subjects. We expressed the mutant NHERF1 protein in Xenopus Oocytes and in cultured cells to study its effects on phosphate transport and PTH-induced cAMP production. RESULTS: We identified in a patient with inappropriate renal phosphate reabsorption a previously unidentified mutation (E68A located in the PDZ1 domain of NHERF1.We report the consequences of this mutation on NHERF1 function. E68A mutation did not modify cAMP production in the patient. PTH-induced cAMP synthesis and PKC activity were not altered by E68A mutation in renal cells in culture. In contrast to wild-type NHERF1, expression of the E68A mutant in Xenopus oocytes and in human cells failed to increase phosphate transport. Pull down experiments showed that E68A mutant did not interact with NPT2a, which robustly interacted with wild type NHERF1 and previously identified mutants. Biotinylation studies revealed that E68A mutant was unable to increase cell surface expression of NPT2a. CONCLUSIONS: Our results indicate that the PDZ1 domain is critical for NHERF1-NPT2a interaction in humans and for the control of NPT2a expression at the plasma membrane. Thus we have identified a new mechanism of renal phosphate loss and shown that different mutations in NHERF1 can alter renal phosphate reabsorption via distinct mechanisms.

  10. Structural evidence for a fatty acid-independent myotoxic mechanism for a phospholipase A2-like toxin.

    Science.gov (United States)

    Salvador, Guilherme H M; Dos Santos, Juliana I; Borges, Rafael J; Fontes, Marcos R M

    2018-03-01

    The myotoxic mechanism for PLA2-like toxins has been proposed recently to be initiated by an allosteric change induced by a fatty acid binding to the protein, leading to the alignment of the membrane docking site (MDoS) and membrane disrupting site (MDiS). Previous structural studies performed by us demonstrated that MjTX-II, a PLA2-like toxin isolated from Bothrops moojeni, presents a different mode of ligand-interaction caused by natural amino acid substitutions and an insertion. Herein, we present four crystal structures of MjTX-II, in its apo state and complexed with fatty acids of different lengths. Analyses of these structures revealed slightly different oligomeric conformations but with both MDoSs in an arrangement that resembles an active-state PLA2-like structure. To explore the structural transitions between apo protein and fatty-acid complexes, we performed Normal Mode Molecular Dynamics simulations, revealing that oligomeric conformations of MjTX-II/fatty acid complexes may be reached in solution by the apo structure. Similar simulations with typical PLA2-like structures demonstrated that this transition is not possible without the presence of fatty acids. Thus, we hypothesize that MjTX-II does not require fatty acids to be active, although these ligands may eventually help in its stabilization by the formation of hydrogen bonds. Therefore, these results complement previous findings for MjTX-II and help us understand its particular ligand-binding properties and, more importantly, its particular mechanism of action, with a possible impact on the design of structure-based inhibitors for PLA2-like toxins in general. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Pharmacokinetics and tolerability of cediranib, a potent VEGF signalling inhibitor, in cancer patients with hepatic impairment

    DEFF Research Database (Denmark)

    van Herpen, Carla M L; Lassen, Ulrik; Desar, Ingrid M E

    2013-01-01

    Vascular endothelial growth factor (VEGF) signalling plays a key role in tumour angiogenesis. Cediranib (AZD2171) is a small-molecule VEGF signalling inhibitor with potent activity against all three VEGF receptors. In this phase I, open-label, parallel-group study, adults with advanced solid...

  12. Biological variations in plasma VEGF and VEGFR-1 may compromise their biomarker value in colorectal cancer

    DEFF Research Database (Denmark)

    Svendsen, Mads N.; Brunner, Nils; Christensen, Ib Jarle

    2010-01-01

    Vascular Endothelial Growth Factor (VEGF) plays a prominent role in tumor angiogenesis and plasma VEGF concentration may carry prognostic information in colorectal cancer. The VEGF receptor 1 (VEGFR-1) is a regulatory receptor which is shredded into plasma of patients with colorectal cancer. For ...

  13. In vivo VEGF imaging with radiolabeled bevacizumab in a human ovarian tumor xenograft

    NARCIS (Netherlands)

    Nagengast, Wouter B.; Hospers, Geke A.; Mulder, Nanno H.; de Jong, Johan R.; Hollema, Harry; Brouwers, Adrienne H.; van Dongen, Guns A.; Perk, Lars R.; Lub-de Hooge, Marjolijn N.

    Vascular endothelial growth factor (VEGF), released by tumor cells, is an important growth factor in tumor angiogenesis. The humanized monoclonal antibody bevacizumab blocks VEGF-induced tumor angiogenesis by binding, thereby neutralizing VEGF. Our aim was to develop radiolabeled bevacizumab for

  14. VEGF Polymorphisms Are Associated With Endocardial Cushion Defects : A Family-Based Case-Control Study

    NARCIS (Netherlands)

    Smedts, Huberdina P. M.; Isaacs, Aaron; de Costa, Dominique; Uitterlinden, Andre G.; van Duijn, Cornelia M.; Gittenberger-de Groot, Adriana C.; Helbing, Willem A.; Steegers, Eric A. P.; Steegers-Theunissen, Regina P. M.

    Endocardial cushion defects (ECDs) of the cardiac outflow tract are among the most common congenital heart disease phenotypes. VEGF is essential for endocardial cushion formation and derangements in VEGF synthesis lead to ECD. Three functional single nucleotide polymorphisms (SNPs) in the VEGF gene

  15. VEGF expression and microvascular density in relation to high-risk ...

    African Journals Online (AJOL)

    VEGF expression and microvascular density in relation to high-risk-HPV infection in cervical carcinoma – An immunohistochemical study. ... Eleven cases were low grade and 19 were high-grade cases. VEGF expression was detected in 100% of cases. The relation between carcinoma grade and VEGF expression and ...

  16. IFN-Gamma-Dependent and Independent Mechanisms of CD4+ Memory T Cell-Mediated Protection from Listeria Infection

    Directory of Open Access Journals (Sweden)

    Stephanie M. Meek

    2018-02-01

    Full Text Available While CD8+ memory T cells can promote long-lived protection from secondary exposure to intracellular pathogens, less is known regarding the direct protective mechanisms of CD4+ T cells. We utilized a prime/boost model in which mice are initially exposed to an acutely infecting strain of lymphocytic choriomeningitis virus (LCMV, followed by a heterologous rechallenge with Listeria monocytogenes recombinantly expressing the MHC Class II-restricted LCMV epitope, GP61–80 (Lm-gp61. We found that heterologous Lm-gp61 rechallenge resulted in robust activation of CD4+ memory T cells and that they were required for rapid bacterial clearance. We further assessed the relative roles of TNF and IFNγ in the direct anti-bacterial function of CD4+ memory T cells. We found that disruption of TNF resulted in a complete loss of protection mediated by CD4+ memory T cells, whereas disruption of IFNγ signaling to macrophages results in only a partial loss of protection. The protective effect mediated by CD4+ T cells corresponded to the rapid accumulation of pro-inflammatory macrophages in the spleen and an altered inflammatory environment in vivo. Overall, we conclude that protection mediated by CD4+ memory T cells from heterologous Listeria challenge is most directly dependent on TNF, whereas IFNγ only plays a minor role.

  17. An acetylcholinesterase-independent mechanism for neurobehavioral impairments after chronic low level exposure to dichlorvos in rats.

    Science.gov (United States)

    Verma, Suresh Kumar; Kumar, Vijay; Gill, Kiran Dip

    2009-03-01

    The present study was designed to explore an alternate mechanism of action other than acetylcholinesterase inhibition for the chronic, low-level exposure to dichlorvos, an organophosphate, in vivo. Dichlorvos, at dose of 1.0 as well as 6.0 mg/kg b. wt. for 12 weeks to rats showed impairment in neurobehavioral indices viz. rota rod, passive avoidance and water maze tests. Though higher dose of dichlorvos had a detrimental effect on acetylcholinesterase activity, no significant inhibition was seen with lower dose of dichlorvos for the same period of exposure i.e. 12 weeks. Muscarinic acetylcholine receptor binding studies revealed a decrease in the number of binding sites (B(max)) in low as well as high dose groups but the dissociation constant (K(d)) value was unaffected with both doses of dichlorvos. Use of selective ligands against M(1), M(2) and M(3) receptor subtypes indicated that M(2) is the major receptor subtype being affected by chronic low-level exposure to dichlorvos. Western blot analysis and immunofluorescence studies also confirmed these biochemical findings. Thus, the present study suggests that M(2) receptors may play a major role in the development of neurobehavioral impairments after chronic exposure to dichlorvos.

  18. DEHP exposure impairs mouse oocyte cyst breakdown and primordial follicle assembly through estrogen receptor-dependent and independent mechanisms

    Energy Technology Data Exchange (ETDEWEB)

    Mu, Xinyi [Laboratory of Reproductive Biology, Chongqing Medical University, Chongqing 400016 (China); Department of Histology and Embryology, College of Basic Medicine, Chongqing Medical University, Chongqing 400016 (China); Liao, Xinggui; Chen, Xuemei; Li, Yanli; Wang, Meirong; Shen, Cha; Zhang, Xue; Wang, Yingxiong; Liu, Xueqing [Laboratory of Reproductive Biology, Chongqing Medical University, Chongqing 400016 (China); He, Junlin, E-mail: hejunlin_11@aliyun.com [Laboratory of Reproductive Biology, Chongqing Medical University, Chongqing 400016 (China)

    2015-11-15

    Highlights: • DEHP inhibits primordial folliculogenesis in vivo and in vitro. • Estrogen receptors participate in the effect of DEHP on early ovarian development. • DEHP exposure impairs the expression of Notch2 signaling components. • DEHP exposure disrupts the proliferation of pregranulosa precursor cells. - Abstract: Estrogen plays an essential role in the development of mammalian oocytes, and recent studies suggest that it also regulates primordial follicle assembly in the neonatal ovaries. During the last decade, potential exposure of humans and animals to estrogen-like endocrine disrupting chemicals has become a growing concern. In the present study, we focused on the effect of diethylhexyl phthalate (DEHP), a widespread plasticizer with estrogen-like activity, on germ-cell cyst breakdown and primordial follicle assembly in the early ovarian development of mouse. Neonatal mice injected with DEHP displayed impaired cyst breakdown. Using ovary organ cultures, we revealed that impairment was mediated through estrogen receptors (ERs), as ICI 182,780, an efficient antagonist of ER, reversed this DEHP-mediated effect. DEHP exposure reduced the expression of ERβ, progesterone receptor (PR), and Notch2 signaling components. Finally, DEHP reduced proliferation of pregranulosa precursor cells during the process of primordial folliculogenesis. Together, our results indicate that DEHP influences oocyte cyst breakdown and primordial follicle formation through several mechanisms. Therefore, exposure to estrogen-like chemicals during fetal or neonatal development may adversely influence early ovarian development.

  19. Probenecid blocks human P2X7 receptor-induced dye uptake via a pannexin-1 independent mechanism.

    Science.gov (United States)

    Bhaskaracharya, Archana; Dao-Ung, Phuong; Jalilian, Iman; Spildrejorde, Mari; Skarratt, Kristen K; Fuller, Stephen J; Sluyter, Ronald; Stokes, Leanne

    2014-01-01

    P2X7 is a ligand-gated ion channel which is activated by ATP and displays secondary permeability characteristics. The mechanism of development of the secondary permeability pathway is currently unclear, although a role for the hemichannel protein pannexin-1 has been suggested. In this study we investigated the role of pannexin-1 in P2X7-induced dye uptake and ATP-induced IL-1β secretion from human monocytes. We found no pharmacological evidence for involvement of pannexin-1 in P2X7-mediated dye uptake in transfected HEK-293 cells with no inhibition seen for carbenoxolone and the pannexin-1 mimetic inhibitory peptide, 10Panx1. However, we found that probenecid inhibited P2X7-induced cationic and anionic dye uptake in stably transfected human P2X7 HEK-293 cells. An IC50 value of 203 μM was calculated for blockade of ATP-induced responses at human P2X7. Probenecid also reduced dye uptake and IL-1β secretion from human CD14+ monocytes whereas carbenoxolone and 10Panx1 showed no inhibitory effect. Patch clamp and calcium indicator experiments revealed that probenecid directly blocks the human P2X7 receptor.

  20. Probenecid blocks human P2X7 receptor-induced dye uptake via a pannexin-1 independent mechanism.

    Directory of Open Access Journals (Sweden)

    Archana Bhaskaracharya

    Full Text Available P2X7 is a ligand-gated ion channel which is activated by ATP and displays secondary permeability characteristics. The mechanism of development of the secondary permeability pathway is currently unclear, although a role for the hemichannel protein pannexin-1 has been suggested. In this study we investigated the role of pannexin-1 in P2X7-induced dye uptake and ATP-induced IL-1β secretion from human monocytes. We found no pharmacological evidence for involvement of pannexin-1 in P2X7-mediated dye uptake in transfected HEK-293 cells with no inhibition seen for carbenoxolone and the pannexin-1 mimetic inhibitory peptide, 10Panx1. However, we found that probenecid inhibited P2X7-induced cationic and anionic dye uptake in stably transfected human P2X7 HEK-293 cells. An IC50 value of 203 μM was calculated for blockade of ATP-induced responses at human P2X7. Probenecid also reduced dye uptake and IL-1β secretion from human CD14+ monocytes whereas carbenoxolone and 10Panx1 showed no inhibitory effect. Patch clamp and calcium indicator experiments revealed that probenecid directly blocks the human P2X7 receptor.

  1. An Independent Review and Accountability Mechanism for the Sustainable Development Goals: The Possibilities of a Framework Convention on Global Health.

    Science.gov (United States)

    Friedman, Eric A

    2016-06-01

    The Framework Convention on Global Health (FCGH), a proposed global treaty to be rooted in the right to health and aimed at health equity, could establish a nuanced, layered, and multi-faceted regime of compliance with, and accountability to, the right to health. In so doing, it would significantly strengthen accountability for the health-related Sustainable Development Goals (SDGs), which it would encompass. Legally binding, the FCGH could facilitate accountability through the courts and catalyze comprehensive domestic accountability regimes, requiring national strategies that include transparency, community and national mechanisms for accountability and participation and an enabling environment for social empowerment. A "Right to Health Capacity Fund" could ensure resources to implement these strategies. Inclusive national processes could establish targets, benchmarks, and indicators consistent with FCGH guidance, with regular reporting to a treaty body, which could also hear individual cases. State reports could be required to include plans to overcome implementation gaps, subjecting poorly complying states to penalties and targeted capacity building measures. Regional special rapporteurs could facilitate compliance through regular country visits, while also responding to serious violations. And reaching beyond government compliance, from capacity building to the courts and contractual obligations, the FCGH could establish nationally enforceable right to health obligations on the private sector.

  2. Genetic variations in VEGF and VEGFR2 and glioblastoma outcome

    DEFF Research Database (Denmark)

    Sjöström, S; Wibom, C; Andersson, U

    2011-01-01

    significantly associated to survival, namely rs2071559 and rs12502008. However, these results are likely to be false positives due to multiple testing and could not be confirmed in a separate dataset. Overall, this study provides little evidence that VEGF and VEGFR2 polymorphisms are important for glioblastoma...

  3. Genetic variations in VEGF and VEGFR2 and glioblastoma outcome

    DEFF Research Database (Denmark)

    Sjöström, S; Wibom, C; Andersson, U

    2010-01-01

    significantly associated to survival, namely rs2071559 and rs12502008. However, these results are likely to be false positives due to multiple testing and could not be confirmed in a separate dataset. Overall, this study provides little evidence that VEGF and VEGFR2 polymorphisms are important for glioblastoma...

  4. Elevated serum and tissue VEGF associated with poor outcome in ...

    African Journals Online (AJOL)

    Serum and tissue vascular endothelial growth factor was strongly associated with grade III tumor, large tumor size, positive lymph node, negative hormone receptor status, +ve HER2 neu and poor survival, the data of the present study showed significant increase in mean serum level of VEGF in patients with positive ...

  5. (VEGF) expression in rats with spinal cord injury by transplantation ...

    African Journals Online (AJOL)

    User

    2011-05-16

    May 16, 2011 ... was quantified by means of western blot and immunohistochemistry technology. It was found that .... 0.6× volume of isopropyl alcohol and a 0.1× volume of 3 M sodium ... To detect expression and localization of VEGF in spinal cord tissue, .... ischemic brain (Chen et al., 2003) have provided their beneficial ...

  6. Thrombospondin-1 and VEGF in inflammatory bowel disease | Alkim ...

    African Journals Online (AJOL)

    Background and aim: Angiogenesis is an important process in the pathogenesis of chronic inflammation. We aimed to study the angiogeneic balance in inflammatory bowel disease (IBD) by evaluating the expression of vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1) on colonic epithelial cells, ...

  7. The complex I subunit NDUFA10 selectively rescues Drosophila pink1 mutants through a mechanism independent of mitophagy.

    Science.gov (United States)

    Pogson, Joe H; Ivatt, Rachael M; Sanchez-Martinez, Alvaro; Tufi, Roberta; Wilson, Emma; Mortiboys, Heather; Whitworth, Alexander J

    2014-11-01

    Mutations in PINK1, a mitochondrially targeted serine/threonine kinase, cause autosomal recessive Parkinson's disease (PD). Substantial evidence indicates that PINK1 acts with another PD gene, parkin, to regulate mitochondrial morphology and mitophagy. However, loss of PINK1 also causes complex I (CI) deficiency, and has recently been suggested to regulate CI through phosphorylation of NDUFA10/ND42 subunit. To further explore the mechanisms by which PINK1 and Parkin influence mitochondrial integrity, we conducted a screen in Drosophila cells for genes that either phenocopy or suppress mitochondrial hyperfusion caused by pink1 RNAi. Among the genes recovered from this screen was ND42. In Drosophila pink1 mutants, transgenic overexpression of ND42 or its co-chaperone sicily was sufficient to restore CI activity and partially rescue several phenotypes including flight and climbing deficits and mitochondrial disruption in flight muscles. Here, the restoration of CI activity and partial rescue of locomotion does not appear to have a specific requirement for phosphorylation of ND42 at Ser-250. In contrast to pink1 mutants, overexpression of ND42 or sicily failed to rescue any Drosophila parkin mutant phenotypes. We also find that knockdown of the human homologue, NDUFA10, only minimally affecting CCCP-induced mitophagy, and overexpression of NDUFA10 fails to restore Parkin mitochondrial-translocation upon PINK1 loss. These results indicate that the in vivo rescue is due to restoring CI activity rather than promoting mitophagy. Our findings support the emerging view that PINK1 plays a role in regulating CI activity separate from its role with Parkin in mitophagy.

  8. Telmisartan, a possible PPAR-δ agonist, reduces TNF-α-stimulated VEGF-C production by inhibiting the p38MAPK/HSP27 pathway in human proximal renal tubular cells

    Energy Technology Data Exchange (ETDEWEB)

    Kimura, Hideki, E-mail: hkimura@u-fukui.ac.jp [Division of Nephrology, Department of General Medicine, School of Medicine, Faculty of Medical Sciences, University of Fukui, Fukui (Japan); Department of Clinical Laboratories and Nephrology, University of Fukui Hospital, Fukui (Japan); Mikami, Daisuke; Kamiyama, Kazuko [Division of Nephrology, Department of General Medicine, School of Medicine, Faculty of Medical Sciences, University of Fukui, Fukui (Japan); Sugimoto, Hidehiro [Department of Clinical Laboratories and Nephrology, University of Fukui Hospital, Fukui (Japan); Kasuno, Kenji; Takahashi, Naoki [Division of Nephrology, Department of General Medicine, School of Medicine, Faculty of Medical Sciences, University of Fukui, Fukui (Japan); Yoshida, Haruyoshi [Division of Nephrology, Department of General Medicine, School of Medicine, Faculty of Medical Sciences, University of Fukui, Fukui (Japan); Division of Nephrology, Obama Municipal Hospital, Obama, Fukui (Japan); Iwano, Masayuki [Division of Nephrology, Department of General Medicine, School of Medicine, Faculty of Medical Sciences, University of Fukui, Fukui (Japan)

    2014-11-14

    Highlights: • TNF-α increased VEGF-C expression by enhancing phosphorylation of p38MAPK and HSP27. • Telmisartan decreased TNF-α-stimulated expression of VEGF-C. • Telmisartan suppressed TNF-α-induced phosphorylation of p38MAPK and HSP27. • Telmisartan activated endogenous PPAR-δ protein. • Telmisartan suppressed p38MAPK phosphorylation in a PPAR-δ-dependent manner. - Abstract: Vascular endothelial growth factor-C (VEGF-C) is a main inducer of inflammation-associated lymphangiogenesis in various inflammatory disorders including chronic progressive kidney diseases, for which angiotensin II receptor type 1 blockers (ARBs) are widely used as the main treatment. Although proximal renal tubular cells may affect the formation of lymphatic vessels in the interstitial area by producing VEGF-C, the molecular mechanisms of VEGF-C production and its manipulation by ARB have not yet been examined in human proximal renal tubular epithelial cells (HPTECs). In the present study, TNF-α dose-dependently induced the production of VEGF-C in HPTECs. The TNF-α-induced production of VEGF-C was mediated by the phosphorylation of p38MAPK and HSP27, but not by that of ERK or NFkB. Telmisartan, an ARB that can activate the peroxisome proliferator-activated receptor (PPAR), served as a PPAR-δ activator and reduced the TNF-α-stimulated production of VEGF-C. This reduction was partially attributed to a PPAR-δ-dependent decrease in p38MAPK phosphorylation. Our results indicate that TNF-α induced the production of VEGF-C in HPTECs by activating p38MAPK/HSP27, and this was partially inhibited by telmisartan in a PPAR-δ dependent manner. These results provide a novel insight into inflammation-associated lymphangiogenesis.

  9. BMP-non-responsive Sca1+ CD73+ CD44+ mouse bone marrow derived osteoprogenitor cells respond to combination of VEGF and BMP-6 to display enhanced osteoblastic differentiation and ectopic bone formation.

    Directory of Open Access Journals (Sweden)

    Vedavathi Madhu

    Full Text Available Clinical trials on fracture repair have challenged the effectiveness of bone morphogenetic proteins (BMPs but suggest that delivery of mesenchymal stem cells (MSCs might be beneficial. It has also been reported that BMPs could not increase mineralization in several MSCs populations, which adds ambiguity to the use of BMPs. However, an exogenous supply of MSCs combined with vascular endothelial growth factor (VEGF and BMPs is reported to synergistically enhance fracture repair in animal models. To elucidate the mechanism of this synergy, we investigated the osteoblastic differentiation of cloned mouse bone marrow derived MSCs (D1 cells in vitro in response to human recombinant proteins of VEGF, BMPs (-2, -4, -6, -9 and the combination of VEGF with BMP-6 (most potent BMP. We further investigated ectopic bone formation induced by MSCs pre-conditioned with VEGF, BMP-6 or both. No significant increase in mineralization, phosphorylation of Smads 1/5/8 and expression of the ALP, COL1A1 and osterix genes was observed upon addition of VEGF or BMPs alone to the cells in culture. The lack of CD105, Alk1 and Alk6 expression in D1 cells correlated with poor response to BMPs indicating that a greater care in the selection of MSCs is necessary. Interestingly, the combination of VEGF and BMP-6 significantly increased the expression of ALP, COL1A1 and osterix genes and D1 cells pre-conditioned with VEGF and BMP-6 induced greater bone formation in vivo than the non-conditioned control cells or the cells pre-conditioned with either VEGF or BMP-6 alone. This enhanced bone formation by MSCs correlated with higher CADM1 expression and OPG/RANKL ratio in the implants. Thus, combined action of VEGF and BMP on MSCs enhances osteoblastic differentiation of MSCs and increases their bone forming ability, which cannot be achieved through use of BMPs alone. This strategy can be effectively used for bone repair.

  10. The N1 effect of temporal attention is independent of sound location and intensity: implications for possible mechanisms of temporal attention.

    Science.gov (United States)

    Lange, Kathrin

    2012-11-01

    It has been repeatedly shown that the auditory N1 is enhanced for sounds presented at an attended time point. The present study investigated the underlying mechanisms using a temporal cuing paradigm. In each trial, an auditory cue indicated at which time point a second sound could be relevant for response selection. Crucially, in addition to temporal attention, two physical sound features with known effects on the sensory N1 were manipulated: location and intensity. Positive evidence for conjoint effects of attention and location or attention and intensity would corroborate the notion that the sensory N1 was modulated by temporal attention, thus supporting a gain mechanism. However, the N1 effect of temporal attention was not similarly lateralized as the sensory N1, and, moreover, it was independent of sound intensity. Thus, the present results do not provide compelling evidence that temporal attention involves an increase in sensory gain. Copyright © 2012 Society for Psychophysiological Research.

  11. Potent DNA damage by polyhalogenated quinones and H2O2 via a metal-independent and Intercalation-enhanced oxidation mechanism

    Science.gov (United States)

    Yin, Ruichuan; Zhang, Dapeng; Song, Yuling; Zhu, Ben-Zhan; Wang, Hailin

    2013-01-01

    Polyhalogenated quinones are a class of carcinogenic intermediates. We found recently that the highly reactive and biologically/environmentally important ·OH can be produced by polyhalogenated quinones and H2O2 independent of transition metal ions. However, it is not clear whether this unusual metal-independent ·OH producing system can induce potent oxidative DNA damage. Here we show that TCBQ and H2O2 can induce oxidative damage to both dG and dsDNA; but surprisingly, it was more efficient to induce oxidative damage in dsDNA than in dG. We found that this is probably due to its strong intercalating ability to dsDNA through competitive intercalation assays. The intercalation of TCBQ in dsDNA may lead to ·OH generation more adjacent to DNA. This is the first report that polyhalogenated quinoid carcinogens and H2O2 can induce potent DNA damage via a metal-independent and intercalation-enhanced oxidation mechanism, which may partly explain their potential genotoxicity, mutagenesis, and carcinogenicity. PMID:23429247

  12. Potent DNA damage by polyhalogenated quinones and H₂O₂ via a metal-independent and Intercalation-enhanced oxidation mechanism.

    Science.gov (United States)

    Yin, Ruichuan; Zhang, Dapeng; Song, Yuling; Zhu, Ben-Zhan; Wang, Hailin

    2013-01-01

    Polyhalogenated quinones are a class of carcinogenic intermediates. We found recently that the highly reactive and biologically/environmentally important ·OH can be produced by polyhalogenated quinones and H₂O₂ independent of transition metal ions. However, it is not clear whether this unusual metal-independent ·OH producing system can induce potent oxidative DNA damage. Here we show that TCBQ and H₂O₂ can induce oxidative damage to both dG and dsDNA; but surprisingly, it was more efficient to induce oxidative damage in dsDNA than in dG. We found that this is probably due to its strong intercalating ability to dsDNA through competitive intercalation assays. The intercalation of TCBQ in dsDNA may lead to ·OH generation more adjacent to DNA. This is the first report that polyhalogenated quinoid carcinogens and H₂O₂ can induce potent DNA damage via a metal-independent and intercalation-enhanced oxidation mechanism, which may partly explain their potential genotoxicity, mutagenesis, and carcinogenicity.

  13. Isoliquiritigenin induces apoptosis of human bladder cancer T24 cells via a cyclin-dependent kinase-independent mechanism.

    Science.gov (United States)

    Si, Lingling; Yang, Xinhui; Yan, Xinyan; Wang, Yanming; Zheng, Qiusheng

    2017-07-01

    that, with increasing ISL concentration, the Bcl-2 expression level was significantly decreased (PT24 cells. The mechanism by which ISL induces T24 cell apoptosis in vitro may be associated with an increase in CDK2 activity, downregulation of the ΔΨm and activation of caspase-3/caspase-9-mediated mitochondrial apoptotic signaling pathways.

  14. PAC-1 and its derivative WF-210 Inhibit Angiogenesis by inhibiting VEGF/VEGFR pathway.

    Science.gov (United States)

    Wang, Fangyang; Wang, Lihui; Li, Yi; Wang, Nannan; Wang, Yating; Cao, Qi; Gong, Ping; Yang, Jingyu; Wu, Chunfu

    2017-12-19

    Procaspase Activating Compound-1 (PAC-1) and its derivative WF-210 induce apoptosis in cancer cells by activating procaspase-3 to caspase-3. The aim of this study was to extend current knowledge about the mechanisms of PAC-1 and WF-210, particularly about their effects on tumor angiogenesis. PAC-1 and WF-210 restrained VEGF-induced human umbilical vascular endothelial cells (HUVECs) proliferation, invasion, and tube formation. PAC-1 and WF-210 abrogated VEGF-induced vessel sprouting from rat aortic rings and inhibited vascular formation in the Matrigel plug assay. PAC-1 and WF-210 suppressed phosphorylation of VEGFR2 and its downstream protein kinases c-Src, FAK, and AKT in both HUVECs and U-87 cells. When given to mice bearing subcutaneous or orthotopic xenograft, PAC-1 and WF-210 inhibited the tumor growth and tumor angiogenesis. Further tests showed that PAC-1 and WF-210 inhibited stemness and induced autophagy flux of U-87 cells. This study revealed mechanisms of PAC-1 and WF-210 other than inducing apoptosis, which provides additional support for their using in the clinic. Copyright © 2017. Published by Elsevier B.V.

  15. ScVEGF-PEG-HBED-CC and scVEGF-PEG-NOTA conjugates: comparison of easy-to-label recombinant proteins for [{sup 68}Ga]PET imaging of VEGF receptors in angiogenic vasculature

    Energy Technology Data Exchange (ETDEWEB)

    Eder, Matthias [German Cancer Research Center (DKFZ), 69120 Heidelberg (Germany)], E-mail: m.eder@dkfz.de; Krivoshein, Arcadius V.; Backer, Marina; Backer, Joseph M. [SibTech, Inc., Brookfield, CT 06804 (United States); Haberkorn, Uwe [Department of Nuclear Medicine, University of Heidelberg, 69120 Heidelberg (Germany); Eisenhut, Michael [German Cancer Research Center (DKFZ), 69120 Heidelberg (Germany)

    2010-05-15

    Introduction: VEGF receptors play a key role in angiogenesis and are important targets for several approved and many experimental drugs. Imaging of VEGF receptor expression in malignant tumors would provide important information, which can influence patient management. The aim of this study was the development of an easy-to-label positron-emitting tracer for imaging VEGF receptors. The tracer is based on engineered single-chain VEGF (scVEGF), expressed with cysteine-containing fusion tag (Cys-tag) for site-specific conjugation of PEGylated bifunctional chelating agents, HBED-CC or NOTA, suitable for labeling with {sup 68}Ga at ambient temperature. Methods: scVEGF-PEG-HBED-CC was synthesized by activating a single carboxyl group of the [Fe(HBED-CC)]{sup -} complex with N-hydroxysuccinimide. Reaction of the activated complex with NH{sub 2}-PEG-maleimide was followed by site-specific conjugation of PEGylated chelator to a thiol group in Cys-tag of scVEGF. The scVEGF-PEG-NOTA conjugate was synthesized using NHS-PEG-maleimide and p-NH{sub 2}-Bn-NOTA. {sup 68}Ga complexation was performed in HEPES buffer (pH 4.2) at room temperature. The functional activity after labeling was tested by radioligand cell binding assays. Biodistribution and PET studies in tumor-bearing mice were performed after 1, 2, 3 and 4 h postinjection. Results: The radiolabeling of scVEGF-PEG-HBED-CC proved more efficient than scVEGF-PEG-NOTA allowing to stop the reaction after 4 min (>97% radiochemical yield). Radioligand cell binding assays performed on HEK-293 cells overexpressing VEGFR-2 revealed no change in the binding properties of {sup 68}Ga-radiolabeled scVEGF relative to other scVEGF-based tracers. Both tracers showed comparable results in biodistribution, such as tumor accumulation and low liver uptake. The tracers were stable in 50% human serum for at least 72 h. Conclusions: The conjugates scVEGF-PEG-HBED-CC and scVEGF-PEG-NOTA revealed comparable in vivo characteristics and allowed easy

  16. House dust mite major allergens Der p 1 and Der p 5 activate human airway-derived epithelial cells by protease-dependent and protease-independent mechanisms

    Directory of Open Access Journals (Sweden)

    Timmerman J André B

    2006-03-01

    Full Text Available Abstract House dust mite allergens (HDM cause bronchoconstriction in asthma patients and induce an inflammatory response in the lungs due to the release of cytokines, chemokines and additional mediators. The mechanism how HDM components achieve this is largely unknown. The objective of this study was to assess whether HDM components of Dermatophagoides pteronissinus with protease activity (Der p 1 and unknown enzymatic activity (Der p 2, Der p 5 induce biological responses in a human airway-derived epithelial cell line (A549, and if so, to elucidate the underlying mechanism(s of action. A549 cells were incubated with HDM extract, Der p 1, recombinant Der p 2 and recombinant Der p 5. Cell desquamation was assessed by microscopy. The proinflammatory cytokines, IL-6 and IL-8, were measured by ELISA. Intracellular Ca2+ levels were assessed in A549 cells and in mouse fibroblasts expressing the human protease activated receptor (PAR1, PAR2 or PAR4. HDM extract, Der p 1 and Der p 5 dose-dependently increased the production of IL-6 and IL-8. Added simultaneously, Der p 1 and Der p 5 further increased the production of IL-6 and IL-8. The action of Der p 1 was blocked by cysteine-protease inhibitors, while that of Der p 5 couldn't be blocked by either serine- or cysteine protease inhibitors. Der p 5 only induced cell shrinking, whereas HDM extract and Der p1 also induced cell desquamation. Der p 2 had no effect on A549 cells. Der p 1's protease activity causes desquamation and induced the release of IL6 and IL-8 by a mechanism independent of Ca2+ mobilisation and PAR activation. Der p 5 exerts a protease-independent activation of A549 that involves Ca2+ mobilisation and also leads to the production of these cytokines. Together, our data indicate that allergens present in HDM extracts can trigger protease-dependent and protease-independent signalling pathways in A549 cells.

  17. Energy Independence

    Science.gov (United States)

    Abelson, Philip H.

    1973-01-01

    Discusses President Nixon's proposed national endeavor for energy self-sufficiency in the United States by 1980, to be known as Project Independence. Examines some of the factors that will be involved in attempting to attain energy independence. (JR)

  18. Mesenchymal stem cells protect cigarette smoke-damaged lung and pulmonary function partly via VEGF-VEGF receptors.

    Science.gov (United States)

    Guan, Xiao-Jun; Song, Lin; Han, Feng-Feng; Cui, Zhi-Lei; Chen, Xi; Guo, Xue-Jun; Xu, Wei-Guo

    2013-02-01

    Progressive pulmonary inflammation and emphysema have been implicated in the progression of chronic obstructive pulmonary disease (COPD), while current pharmacological treatments are not effective. Transplantation of bone marrow mesenchymal stem cells (MSCs) has been identified as one such possible strategy for treatment of lung diseases including acute lung injury (ALI) and pulmonary fibrosis. However, their role in COPD still requires further investigation. The aim of this study is to test the effect of administration of rat MSCs (rMSCs) on emphysema and pulmonary function. To accomplish this study, the rats were exposed to cigarette smoke (CS) for 11 weeks, followed by administration of rMSCs into the lungs. Here we show that rMSCs infusion mediates a down-regulation of pro-inflammatory mediators (TNF-α, IL-1β, MCP-1, and IL-6) and proteases (MMP9 and MMP12) in lung, an up-regulation of vascular endothelial growth factor (VEGF), VEGF receptor 2, and transforming growth factor (TGFβ-1), while reducing pulmonary cell apoptosis. More importantly, rMSCs administration improves emphysema and destructive pulmonary function induced by CS exposure. In vitro co-culture system study of human umbilical endothelial vein cells (EA.hy926) and human MSCs (hMSCs) provides the evidence that hMSCs mediates an anti-apoptosis effect, which partly depends on an up-regulation of VEGF. These findings suggest that MSCs have a therapeutic potential in emphysematous rats by suppressing the inflammatory response, excessive protease expression, and cell apoptosis, as well as up-regulating VEGF, VEGF receptor 2, and TGFβ-1. Copyright © 2012 Wiley Periodicals, Inc.

  19. Independent suspension

    National Research Council Canada - National Science Library

    Chaikin, Don

    1992-01-01

    ... independent suspension. INDEPENDENCE! An independent system is simply one in which each of the vehicle's wheels is free to react totally separate from any of the other wheels. If the right rear wheel hits a bump, the left rear wheel is undisturbed. Since the whole car does not bounce and shake every time one of the wheels hits a potho...

  20. VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis

    Directory of Open Access Journals (Sweden)

    Gareth W. Fearnley

    2016-05-01

    Full Text Available Vascular endothelial growth factor A (VEGF-A binding to the receptor tyrosine kinase VEGFR2 triggers multiple signal transduction pathways, which regulate endothelial cell responses that control vascular development. Multiple isoforms of VEGF-A can elicit differential signal transduction and endothelial responses. However, it is unclear how such cellular responses are controlled by isoform-specific VEGF-A–VEGFR2 complexes. Increasingly, there is the realization that the membrane trafficking of receptor–ligand complexes influences signal transduction and protein turnover. By building on these concepts, our study shows for the first time that three different VEGF-A isoforms (VEGF-A165, VEGF-A121 and VEGF-A145 promote distinct patterns of VEGFR2 endocytosis for delivery into early endosomes. This differential VEGFR2 endocytosis and trafficking is linked to VEGF-A isoform-specific signal transduction events. Disruption of clathrin-dependent endocytosis blocked VEGF-A isoform-specific VEGFR2 activation, signal transduction and caused substantial depletion in membrane-bound VEGFR1 and VEGFR2 levels. Furthermore, such VEGF-A isoforms promoted differential patterns of VEGFR2 ubiquitylation, proteolysis and terminal degradation. Our study now provides novel insights into how different VEGF-A isoforms can bind the same receptor tyrosine kinase and elicit diverse cellular outcomes.

  1. Activation of parallel fiber feedback by spatially diffuse stimuli reduces signal and noise correlations via independent mechanisms in a cerebellum-like structure.

    Directory of Open Access Journals (Sweden)

    Benjamin Simmonds

    2015-01-01

    Full Text Available Correlations between the activities of neighboring neurons are observed ubiquitously across systems and species and are dynamically regulated by several factors such as the stimulus' spatiotemporal extent as well as by the brain's internal state. Using the electrosensory system of gymnotiform weakly electric fish, we recorded the activities of pyramidal cell pairs within the electrosensory lateral line lobe (ELL under spatially localized and diffuse stimulation. We found that both signal and noise correlations were markedly reduced (>40% under the latter stimulation. Through a network model incorporating key anatomical features of the ELL, we reveal how activation of diffuse parallel fiber feedback from granule cells by spatially diffuse stimulation can explain both the reduction in signal as well as the reduction in noise correlations seen experimentally through independent mechanisms. First, we show that burst-timing dependent plasticity, which leads to a negative image of the stimulus and thereby reduces single neuron responses, decreases signal but not noise correlations. Second, we show trial-to-trial variability in the responses of single granule cells to sensory input reduces noise but not signal correlations. Thus, our model predicts that the same feedback pathway can simultaneously reduce both signal and noise correlations through independent mechanisms. To test this prediction experimentally, we pharmacologically inactivated parallel fiber feedback onto ELL pyramidal cells. In agreement with modeling predictions, we found that inactivation increased both signal and noise correlations but that there was no significant relationship between magnitude of the increase in signal correlations and the magnitude of the increase in noise correlations. The mechanisms reported in this study are expected to be generally applicable to the cerebellum as well as other cerebellum-like structures. We further discuss the implications of such

  2. Activation of parallel fiber feedback by spatially diffuse stimuli reduces signal and noise correlations via independent mechanisms in a cerebellum-like structure.

    Science.gov (United States)

    Simmonds, Benjamin; Chacron, Maurice J

    2015-01-01

    Correlations between the activities of neighboring neurons are observed ubiquitously across systems and species and are dynamically regulated by several factors such as the stimulus' spatiotemporal extent as well as by the brain's internal state. Using the electrosensory system of gymnotiform weakly electric fish, we recorded the activities of pyramidal cell pairs within the electrosensory lateral line lobe (ELL) under spatially localized and diffuse stimulation. We found that both signal and noise correlations were markedly reduced (>40%) under the latter stimulation. Through a network model incorporating key anatomical features of the ELL, we reveal how activation of diffuse parallel fiber feedback from granule cells by spatially diffuse stimulation can explain both the reduction in signal as well as the reduction in noise correlations seen experimentally through independent mechanisms. First, we show that burst-timing dependent plasticity, which leads to a negative image of the stimulus and thereby reduces single neuron responses, decreases signal but not noise correlations. Second, we show trial-to-trial variability in the responses of single granule cells to sensory input reduces noise but not signal correlations. Thus, our model predicts that the same feedback pathway can simultaneously reduce both signal and noise correlations through independent mechanisms. To test this prediction experimentally, we pharmacologically inactivated parallel fiber feedback onto ELL pyramidal cells. In agreement with modeling predictions, we found that inactivation increased both signal and noise correlations but that there was no significant relationship between magnitude of the increase in signal correlations and the magnitude of the increase in noise correlations. The mechanisms reported in this study are expected to be generally applicable to the cerebellum as well as other cerebellum-like structures. We further discuss the implications of such decorrelation on the neural

  3. VEGF increases the permeability of sheep pleura ex vivo through VEGFR2 stimulation.

    Science.gov (United States)

    Peppa, Vasiliki I; Arsenopoulou, Zoi V; Zarogiannis, Sotirios G; Deligiorgi, Triantafyllia; Jagirdar, Rajesh; Makantasis, Ioannis; Stefanidis, Ioannis; Liakopoulos, Vassilios; Molyvdas, Paschalis-Adam; Gourgoulianis, Konstantinos I; Hatzoglou, Chrissi

    2014-10-01

    Vascular endothelial growth factor (VEGF), a cytokine that increases vascular permeability to water and proteins and induces angiogenesis, has been implicated in the development of pleural effusions. Inflammatory and malignant pleural effusions are rich in VEGF content while mesothelial cells produce and excrete VEGF. In this report we aimed at investigating by means of electrophysiology the direct effects of VEGF on the parietal and visceral sheep pleura as well as the type of receptors that mediate this effect. Our findings show that VEGF has a direct effect on the pleural mesothelium rendering it more permeable and this effect is mediated through the stimulation of VEGF receptor 2. Our findings shed more light to the role of VEGF in the pathogenesis of pleural effusions and provide functional evidence for a role of VEGFR2 on the pleural mesothelium that has never been studied before. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. [Study on RhBMP-2 induced osteoporosis rat BMSCs in vitro osteogenesis and VEGF expression].

    Science.gov (United States)

    Li, Jun; Wang, Yun; Bao, Xiao-ming; Wei, Peng-bin; Zhang, Min

    2015-05-01

    To observe the impact of bone morphogenetic protein-2 (rhBMP-2) on bone marrow stromal cells (BMSCs) osteogenesis in vitro and vascular endothelial growth factors (VEGF) expression in bone osteoporotic to prevent and treat the osteoporosis. Twenty 6-month-old female SD rats weighted (300±20) g underwent bilateral ovariectomized. At 3 months after operation, dual-energy X-ray absorptiometry was used to measure bone mineral density of rats,the values were compared with preoperative to ensure the model successfully, and the osteoporosis rats' BMSCs were cultured by bone marrow adherent cultured and the BMSCs morphology was observed under a phase contrast microscope upside down. The osteoporosis rats' BMSCs at the 2nd generation (p2) were randomly divided into experimental and control groups and were added complete medium (containing rhBMP-2) and osteogenic induced liquid, respectively. Two weeks later, the formation of cell calcium nodules were detected by Alizarin red staining method,alkaline phosphatase activity was measured by enzyme standard instrument and the expression of VEGF was detected by RT-PCT method. (1)Whole body bone mineral density of rats before and after operation were (0.179±0.007), (0.158±0.006) g/cm2,there was statistically significant (t=4.180,Pinduced by BMSCs (P2) in the experimental group had more strong dyeing effect than the control group obviously. (3)Alkaline phosphatase activity at 2 weeks after osteogenesis induced by BMSCs (P2) of the experimental group (15.62±1.27) ug/gprot was significantly higher than that of the control group (8.62±0.93) ug/gprot,there was statistically significant (t=7.709, Pinduced by BMSCs (P2) of the experimental group 3.723±0.143 was significantly higher than that of the control group 0.950±0.072, there was statistically significant (t=29.462, Posteoporosis rat BMSCs, promote the VEGF expression of osteogenesis factor. Regulating the VEGF expression may be one of the mechanisms of BMP-2 to participate in

  5. The class-I HDAC inhibitor MGCD0103 induces apoptosis in Hodgkin lymphoma cell lines and synergizes with proteasome inhibitors by an HDAC6-independent mechanism.

    Science.gov (United States)

    Buglio, Daniela; Mamidipudi, Vidya; Khaskhely, Noor M; Brady, Helen; Heise, Carla; Besterman, Jeffrey; Martell, Robert E; MacBeth, Kyle; Younes, Anas

    2010-11-01

    Inhibition of histone deacetylase 6 (HDAC6)-dependent aggresome function by pan HDAC inhibitors was recently reported to be a key mechanism underlying the synergistic activity between proteasome inhibitors and HDAC inhibitors in a variety of tumour types. Because these combinations induce significant thrombocytopenia in vivo, we examined whether less toxic, isotype-selective HDAC inhibitors may still synergize with proteasome inhibitors, and if so, by what mechanisms. Here, we showed that the class I HDAC inhibitor, MGCD0103, has a potent antiproliferative activity in Hodgkin lymphoma (HL) cell lines. Furthermore, MGCD0103 induced tumour necrosis factor α (TNF-α) expression and secretion, which was associated with nuclear factor (NF)-κB activation. Selective inhibition of TNF-α expression by short interfering mRNA, or inhibition of MGCD0103-induced NF-kB activation by proteasome inhibitors enhanced MGCD0103-induced cell death. Thus, our results demonstrate that MGCD0103 may synergize with proteasome inhibitors by HDAC6-independent mechanisms, providing mechanistic rationale for exploring this potentially less toxic combination for the treatment of lymphoma. © 2010 Blackwell Publishing Ltd.

  6. Manipulation of the HIF–Vegf pathway rescues methyl tert-butyl ether (MTBE)-induced vascular lesions

    Energy Technology Data Exchange (ETDEWEB)

    Bonventre, Josephine A., E-mail: josephine.bonventre@oregonstate.edu [Rutgers, The State University of New Jersey, Joint Graduate Program in Toxicology, 170 Frelinghuysen Road, Piscataway, NJ 08854 (United States); Rutgers, The State University of New Jersey, Department of Biochemistry and Microbiology, 76 Lipman Dr., New Brunswick, NJ 08901 (United States); Oregon State University, Department of Environmental and Molecular Toxicology, 1011 Agricultural and Life Sciences Bldg, Corvallis, OR 97331 (United States); Kung, Tiffany S., E-mail: tiffany.kung@rutgers.edu [Rutgers, The State University of New Jersey, Joint Graduate Program in Toxicology, 170 Frelinghuysen Road, Piscataway, NJ 08854 (United States); Rutgers, The State University of New Jersey, Department of Biochemistry and Microbiology, 76 Lipman Dr., New Brunswick, NJ 08901 (United States); White, Lori A., E-mail: lawhite@aesop.rutgers.edu [Rutgers, The State University of New Jersey, Joint Graduate Program in Toxicology, 170 Frelinghuysen Road, Piscataway, NJ 08854 (United States); Rutgers, The State University of New Jersey, Department of Biochemistry and Microbiology, 76 Lipman Dr., New Brunswick, NJ 08901 (United States); Cooper, Keith R., E-mail: cooper@aesop.rutgers.edu [Rutgers, The State University of New Jersey, Joint Graduate Program in Toxicology, 170 Frelinghuysen Road, Piscataway, NJ 08854 (United States); Rutgers, The State University of New Jersey, Department of Biochemistry and Microbiology, 76 Lipman Dr., New Brunswick, NJ 08901 (United States)

    2013-12-15

    Methyl tert-butyl ether (MTBE) has been shown to be specifically anti-angiogenic in piscine and mammalian model systems at concentrations that appear non-toxic in other organ systems. The mechanism by which MTBE targets developing vascular structures is unknown. A global transcriptome analysis of zebrafish embryos developmentally exposed to 0.00625–5 mM MTBE suggested that hypoxia inducible factor (HIF)-regulated pathways were affected. HIF-driven angiogenesis via vascular endothelial growth factor (vegf) is essential to the developing vasculature of an embryo. Three rescue studies were designed to rescue MTBE-induced vascular lesions: pooled blood in the common cardinal vein (CCV), cranial hemorrhages (CH), and abnormal intersegmental vessels (ISV), and test the hypothesis that MTBE toxicity was HIF–Vegf dependent. First, zebrafish vegf-a over-expression via plasmid injection, resulted in significantly fewer CH and ISV lesions, 46 and 35% respectively, in embryos exposed to 10 mM MTBE. Then HIF degradation was inhibited in two ways. Chemical rescue by N-oxaloylglycine significantly reduced CCV and CH lesions by 30 and 32% in 10 mM exposed embryos, and ISV lesions were reduced 24% in 5 mM exposed zebrafish. Finally, a morpholino designed to knock-down ubiquitin associated von Hippel–Lindau protein, significantly reduced CCV lesions by 35% in 10 mM exposed embryos. In addition, expression of some angiogenesis related genes altered by MTBE exposure were rescued. These studies demonstrated that MTBE vascular toxicity is mediated by a down regulation of HIF–Vegf driven angiogenesis. The selective toxicity of MTBE toward developing vasculature makes it a potentially useful chemical in the designing of new drugs or in elucidating roles for specific angiogenic proteins in future studies of vascular development. - Highlights: • Global gene expression of MTBE exposed zebrafish suggested altered HIF1 signaling. • Over expression of zebrafish vegf-a rescues MTBE

  7. Are Independent Probes Truly Independent?

    Science.gov (United States)

    Camp, Gino; Pecher, Diane; Schmidt, Henk G.; Zeelenberg, Rene

    2009-01-01

    The independent cue technique has been developed to test traditional interference theories against inhibition theories of forgetting. In the present study, the authors tested the critical criterion for the independence of independent cues: Studied cues not presented during test (and unrelated to test cues) should not contribute to the retrieval…

  8. MnTBAP Inhibits Bleomycin-Induced Pulmonary Fibrosis by Regulating VEGF and Wnt Signaling

    Science.gov (United States)

    Venkatadri, Rajkumar; Krishnan V. Iyer, Anand; Ramesh, Vani; Wright, Clayton; Castro, Carlos A.; Yakisich, Juan S.; Azad, Neelam

    2017-01-01

    Cellular oxidative stress is implicated not only in lung injury but also in contributing to the development of pulmonary fibrosis. We demonstrate that a cell-permeable superoxide dismutase (SOD) mimetic and peroxynitrite scavenger, manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) significantly inhibited bleomycin-induced fibrogenic effects both in vitro and in vivo. Further investigation into the underlying mechanisms revealed that MnTBAP targets canonical Wnt and non-canonical Wnt/Ca2+ signaling pathways, both of which were upregulated by bleomycin treatment. The effect of MnTBAP on canonical Wnt signaling was significant in vivo but inconclusive in vitro and the non-canonical Wnt/Ca2+ signaling pathway was observed to be the predominant pathway regulated by MnTBAP in bleomycin-induced pulmonary fibrosis. Furthermore, we show that the inhibitory effects of MnTBAP involve regulation of VEGF which is upstream of the Wnt signaling pathway. Overall, the data show that the superoxide scavenger MnTBAP attenuates bleomycin-induced pulmonary fibrosis by targeting VEGF and Wnt signaling pathways. PMID:27649046

  9. Erythropoietin protects the in vitro blood-brain barrier against VEGF-induced permeability.

    Science.gov (United States)

    Martínez-Estrada, Ofelia María; Rodríguez-Millán, Elisabeth; González-De Vicente, Esther; Reina, Manuel; Vilaró, Senén; Fabre, Myriam

    2003-11-01

    The blood-brain barrier (BBB) ensures the homeostasis of the brain microenvironment, mostly through complex tight junctions between brain endothelial cells that prevent the passage of hydrophilic molecules from blood to brain and vice versa. A recent study has shown in vivo that systemic administration of erythropoietin (Epo) protects against brain injury. Using an in vitro model of the bovine BBB, we observed that the expression of the Epo receptor is modulated by its ligand and hypoxic stimuli such as vascular endothelial growth factor (VEGF) treatment. In addition, Epo protects against the VEGF-induced permeability of the BBB, decreases the levels of endothelial nitric oxide synthase and restores junction proteins. The kinetic transport experiments revealed the capacity of Epo to cross the in vitro BBB in a saturable and specific way. Our results suggest a new mechanism for Epo-induced neuroprotection, in which circulating Epo controls and maintains the BBB through an Epo receptor signalling pathway and the re-establishment of cell junctions.

  10. Rho-kinase inhibition and electromechanical coupling in rat and guinea-pig ureter smooth muscle: Ca2+-dependent and -independent mechanisms

    Science.gov (United States)

    Shabir, S; Borisova, L; Wray, Susan; Burdyga, T

    2004-01-01

    Recent data have shown Ca2+-dependent activation of Rho-kinase by sustained depolarization of arterial smooth muscle. Visceral smooth muscles, however, contract phasically in response to action potentials and it is unclear whether Ca2+-dependent or -independent Rho-kinase activation occurs. We have therefore investigated this, under physiologically relevant conditions, in intact ureter. Action potentials, ionic currents, Ca2+ transients, myosin light chain (MLC) phosphorylation and phasic contraction evoked by action potentials in guinea-pig and rat ureter were investigated. In rat, but not guinea-pig ureter, three Rho-kinase inhibitors, Y-27632, HA-1077 and H-1152, significantly decreased phasic contractions and Ca2+ transients. Voltage- and current-clamp data showed that Rho-kinase inhibition reduced the plateau component of the action potential, inhibited Ca2+-channels and, indirectly, Ca2+-activated Cl− channels. The Ca2+ channel agonist Bay K8644 could reverse these effects. The K+ channel blocker TEA could also reverse the inhibitory effect of Y-27632 on the action potential and Ca2+ transient. Ca2+ transients and inward current, activated by carbachol-induced sarcoplasmic reticulum Ca2+release, were not affected by Rho-kinase inhibition. Rho-kinase inhibition produced a Ca2+-independent increase in the relaxation rate of contraction, associated with acceleration of MLC dephosphorylation, which was sensitive to calyculin A. These data show for the first time that: (1) Rho-kinase has major effects on Ca2+ signalling associated with the action potential, (2) this effect is species dependent and (3) Rho-kinase controls relaxation of phasic contraction of myogenic origin. Thus Rho-kinase can modulate phasic smooth muscle in the absence of agonist, and the mechanisms are both Ca2+-dependent, involving ion channels, and Ca2+-independent, involving MLC phosphorylation activity. PMID:15331677

  11. Deriving time dependent Schrödinger equation from Wave-Mechanics, Schrödinger time independent equation, Classical and Hamilton-Jacobi equations

    Directory of Open Access Journals (Sweden)

    Nilesh P. BARDE

    2015-05-01

    Full Text Available The concept of time dependent Schrödinger equation (TDSE illustrated in literature and even during class room teaching is mostly either complex or meant for advanced learners. This article is intended to enlighten the concept to the beginners in the field and further to improve knowledge about detailed steps for abstract mathematical formulation used which helps in understanding to derive TDSE using various tools and in more comprehensible manner. It is shown that TDSE may be derived using wave mechanics, time independent equation, classical & Hamilton-Jacobi’s equations. Similar attempts have been done earlier by some researchers. However, this article provides a comprehensive, lucid and well derived derivation, derived using various approaches, which would make this article unique.

  12. Loss of p19(Arf facilitates the angiogenic switch and tumor initiation in a multi-stage cancer model via p53-dependent and independent mechanisms.

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    Danielle B Ulanet

    2010-08-01

    Full Text Available The Arf tumor suppressor acts as a sensor of oncogenic signals, countering aberrant proliferation in large part via activation of the p53 transcriptional program, though a number of p53-independent functions have been described. Mounting evidence suggests that, in addition to promoting tumorigenesis via disruptions in the homeostatic balance between cell proliferation and apoptosis of overt cancer cells, genetic alterations leading to tumor suppressor loss of function or oncogene gain of function can also incite tumor development via effects on the tumor microenvironment. In a transgenic mouse model of multi-stage pancreatic neuroendocrine carcinogenesis (PNET driven by inhibition of the canonical p53 and Rb tumor suppressors with SV40 large T-antigen (Tag, stochastic progression to tumors is limited in part by a requirement for initiation of an angiogenic switch. Despite inhibition of p53 by Tag in this mouse PNET model, concomitant disruption of Arf via genetic knockout resulted in a significantly accelerated pathway to tumor formation that was surprisingly not driven by alterations in tumor cell proliferation or apoptosis, but rather via earlier activation of the angiogenic switch. In the setting of a constitutional p53 gene knockout, loss of Arf also accelerated tumor development, albeit to a lesser degree. These findings demonstrate that Arf loss of function can promote tumorigenesis via facilitating angiogenesis, at least in part, through p53-independent mechanisms.

  13. Ras oncogene-independent activation of RALB signaling is a targetable mechanism of escape from NRAS(V12) oncogene addiction in acute myeloid leukemia.

    Science.gov (United States)

    Pomeroy, E J; Lee, L A; Lee, R D W; Schirm, D K; Temiz, N A; Ma, J; Gruber, T A; Diaz-Flores, E; Moriarity, B S; Downing, J R; Shannon, K M; Largaespada, D A; Eckfeldt, C E

    2017-06-08

    Somatic mutations that lead to constitutive activation of NRAS and KRAS proto-oncogenes are among the most common in human cancer and frequently occur in acute myeloid leukemia (AML). An inducible NRAS(V12)-driven AML mouse model has established a critical role for continued NRAS(V12) expression in leukemia maintenance. In this model genetic suppression of NRAS(V12) expression results in rapid leukemia remission, but some mice undergo spontaneous relapse with NRAS(V12)-independent (NRI) AMLs providing an opportunity to identify mechanisms that bypass the requirement for Ras oncogene activity and drive leukemia relapse. We found that relapsed NRI AMLs are devoid of NRAS(V12) expression and signaling through the major oncogenic Ras effector pathways, phosphatidylinositol-3-kinase and mitogen-activated protein kinase, but express higher levels of an alternate Ras effector, Ralb, and exhibit NRI phosphorylation of the RALB effector TBK1, implicating RALB signaling in AML relapse. Functional studies confirmed that inhibiting CDK5-mediated RALB activation with a clinically relevant experimental drug, dinaciclib, led to potent RALB-dependent antileukemic effects in human AML cell lines, induced apoptosis in patient-derived AML samples in vitro and led to a 2-log reduction in the leukemic burden in patient-derived xenograft mice. Furthermore, dinaciclib potently suppressed the clonogenic potential of relapsed NRI AMLs in vitro and prevented the development of relapsed AML in vivo. Our findings demonstrate that Ras oncogene-independent activation of RALB signaling is a therapeutically targetable mechanism of escape from NRAS oncogene addiction in AML.

  14. Inhibition of protein kinase C delta attenuates allergic airway inflammation through suppression of PI3K/Akt/mTOR/HIF-1 alpha/VEGF pathway.

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    Yun Ho Choi

    Full Text Available Vascular endothelial growth factor (VEGF is supposed to contribute to the pathogenesis of allergic airway disease. VEGF expression is regulated by a variety of stimuli such as nitric oxide, growth factors, and hypoxia-inducible factor-1 alpha (HIF-1α. Recently, inhibition of the mammalian target of rapamycin (mTOR has been shown to alleviate cardinal asthmatic features, including airway hyperresponsiveness, eosinophilic inflammation, and increased vascular permeability in asthma models. Based on these observations, we have investigated whether mTOR is associated with HIF-1α-mediated VEGF expression in allergic asthma. In studies with the mTOR inhibitor rapamycin, we have elucidated the stimulatory role of a mTOR-HIF-1α-VEGF axis in allergic response. Next, the mechanisms by which mTOR is activated to modulate this response have been evaluated. mTOR is known to be regulated by phosphoinositide 3-kinase (PI3K/Akt or protein kinase C-delta (PKC δ in various cell types. Consistent with these, our results have revealed that suppression of PKC δ by rottlerin leads to the inhibition of PI3K/Akt activity and the subsequent blockade of a mTOR-HIF-1α-VEGF module, thereby attenuating typical asthmatic attack in a murine model. Thus, the present data indicate that PKC δ is necessary for the modulation of the PI3K/Akt/mTOR signaling cascade, resulting in a tight regulation of HIF-1α activity and VEGF expression. In conclusion, PKC δ may represent a valuable target for innovative therapeutic treatment of allergic airway disease.

  15. Adiponectin promotes VEGF-C-dependent lymphangiogenesis by inhibiting miR-27b through a CaMKII/AMPK/p38 signaling pathway in human chondrosarcoma cells.

    Science.gov (United States)

    Huang, Chun-Yin; Chang, An-Chen; Chen, Hsien-Te; Wang, Shih-Wei; Lo, Yuan-Shun; Tang, Chih-Hsin

    2016-09-01

    Chondrosarcoma is the second most frequently occurring type of bone malignancy characterized by distant metastatic propensity. Vascular endothelial growth factor-C (VEGF-C) is the major lymphangiogenic factor, and makes crucial contributions to tumour lymphangiogenesis and lymphatic metastasis. Adiponectin is a protein hormone secreted predominantly by differentiated adipocytes. In recent years, adiponectin has also been indicated as facilitating tumorigenesis, angiogenesis and metastasis. However, the effect of adiponectin on VEGF-C regulation and lymphangiogenesis in chondrosarcoma has remained largely a mystery. In the present study, we have shown a clinical correlation between adiponectin and VEGF-C, as well as tumour stage, in human chondrosarcoma tissues. We further demonstrated that adiponectin promoted VEGF-C expression and secretion in human chondrosarcoma cells. The conditioned medium from adiponectin-treated cells significantly induced tube formation and migration of human lymphatic endothelial cells. In addition, adiponectin knock down inhibited lymphangiogenesis in vitro and in vivo We also found that adiponectin-induced VEGF-C is mediated by the calmodulin-dependent protein kinase II (CaMKII), AMP-activated protein kinase (AMPK) and p38 signaling pathway. Furthermore, the expression of miR-27b was negatively regulated by adiponectin via the CaMKII, AMPK and p38 cascade. The present study is the first to describe the mechanism of adiponectin-promoted lymphangiogenesis by up-regulating VEGF-C expression in chondrosarcomas. Thus, adiponectin could serve as a therapeutic target in chondrosarcoma metastasis and lymphangiogenesis. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  16. VEGF Promotes Malaria-Associated Acute Lung Injury in Mice

    Science.gov (United States)

    Carapau, Daniel; Pena, Ana C.; Ataíde, Ricardo; Monteiro, Carla A. A.; Félix, Nuno; Costa-Silva, Artur; Marinho, Claudio R. F.; Dias, Sérgio; Mota, Maria M.

    2010-01-01

    The spectrum of the clinical presentation and severity of malaria infections is broad, ranging from uncomplicated febrile illness to severe forms of disease such as cerebral malaria (CM), acute lung injury (ALI), acute respiratory distress syndrome (ARDS), pregnancy-associated malaria (PAM) or severe anemia (SA). Rodent models that mimic human CM, PAM and SA syndromes have been established. Here, we show that DBA/2 mice infected with P. berghei ANKA constitute a new model for malaria-associated ALI. Up to 60% of the mice showed dyspnea, airway obstruction and hypoxemia and died between days 7 and 12 post-infection. The most common pathological findings were pleural effusion, pulmonary hemorrhage and edema, consistent with increased lung vessel permeability, while the blood-brain barrier was intact. Malaria-associated ALI correlated with high levels of circulating VEGF, produced de novo in the spleen, and its blockage led to protection of mice from this syndrome. In addition, either splenectomization or administration of the anti-inflammatory molecule carbon monoxide led to a significant reduction in the levels of sera VEGF and to protection from ALI. The similarities between the physiopathological lesions described here and the ones occurring in humans, as well as the demonstration that VEGF is a critical host factor in the onset of malaria-associated ALI in mice, not only offers important mechanistic insights into the processes underlying the pathology related with malaria but may also pave the way for interventional studies. PMID:20502682

  17. TNF-α mediates choroidal neovascularization by upregulating VEGF expression in RPE through ROS-dependent β-catenin activation.

    Science.gov (United States)

    Wang, Haibo; Han, Xiaokun; Wittchen, Erika S; Hartnett, M Elizabeth

    2016-01-01

    Inflammation, oxidative stress, and angiogenesis have been proposed to interact in age-related macular degeneration. It has been postulated that external stimuli that cause oxidative stress can increase production of vascular endothelial growth factor (VEGF) in retinal pigment epithelial (RPE) cells. In this study, we tested the hypothesis that the inflammatory cytokine, tumor necrosis factor alpha (TNF-α), contributed to choroidal neovascularization (CNV) by upregulating VEGF in RPE through intracellular reactive oxygen species (ROS)-dependent signaling and sought to understand the mechanisms involved. In a murine laser-induced CNV model, 7 days after laser treatment and intravitreal neutralizing mouse TNF-α antibody or isotype immunoglobulin G (IgG) control, the following measurements were made: 1) TNF-α protein and VEGF protein in RPE/choroids with western blot, 2) CNV volume in RPE/choroidal flatmounts, and 3) semiquantification of oxidized phospholipids stained with E06 antibody within CNV with immunohistochemistry (IHC). In cultured human RPE cells treated with TNF-α or PBS control, 1) ROS generation was measured using the 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence assay, and 2) NOX4 protein and VEGF protein or mRNA were measured with western blot or quantitative real-time PCR in cells pretreated with apocynin or nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) inhibitor, VAS 2870, or transfected with p22phox siRNA, and each was compared to its appropriate control. Western blots of phosphorylated p65 (p-p65), total p65 and β-actin, and quantitative real-time PCR of VEGF mRNA were measured in human RPE cells treated with TNF-α and pretreatment with the nuclear factor kappa B inhibitor, Bay 11-7082 or control. Western blots of β-catenin, VEGF, and p22phox and coimmunoprecipitation of β-catenin and T-cell transcriptional factor were performed in human RPE cells treated with TNF-α following pretreatment with

  18. Role of VEGF family members and receptors in coronary vessel formation.

    Science.gov (United States)

    Tomanek, Robert J; Holifield, Jennifer S; Reiter, Rebecca S; Sandra, Alexander; Lin, Jim J-C

    2002-11-01

    The specific roles of vascular endothelial growth factor (VEGF) family members and their receptors (VEGFRs) in coronary vessel formation were studied. By using the quail heart explant model, we found that neutralizing antibodies to VEGF-B or VEGF-C inhibited tube formation on the collagen gel more than anti-VEGF-A. Soluble VEGFR-1, a receptor for VEGF-A and -B, inhibited tube formation by 87%, a finding consistent with that of VEGF-B inhibition. In contrast, addition of soluble VEGFR-2, a receptor for VEGF family members A, C, D, and E, inhibited tube formation by only 43%. Acidic FGF-induced tube formation dependency on VEGF was demonstrated by the attenuating effect of a soluble VEGFR-1 and -2 chimera. The localization of VEGF R-2 and R-3 was demonstrated by in situ hybridization of serial sections, which documented marked accumulations of transcripts for both receptors at the base of the truncus arteriosus coinciding with the temporal and spatial formation of the coronary arteries by means of ingrowth of capillary plexuses. This finding suggests that both VEGFR-2 and R-3 may play a role in the formation of the coronary artery roots. In summary, these experiments document a role for multiple members of the VEGF family and their receptors in formation of the coronary vascular bed. Copyright 2002 Wiley-Liss, Inc.

  19. Clinicopathological and prognostic significance of HER-2/neu and VEGF expression in colon carcinomas

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    Li Jing

    2011-06-01

    Full Text Available Abstract Background HER-2/neu and VEGF expression is correlated with disease behaviors in various cancers. However, evidence for their expression in colon cancer is rather contradictory both for the protein expression status and prognostic value. HER-2/neu is found to participate in VEGF regulation, and has known correlation with VEGF expression in some tumors. In this study, we investigated HER-2/neu and VEGF expression in Chinese colon patients and explored whether there was any correlation between their expression patterns. Methods HER-2/neu and VEGF were investigated immunohistochemically using tumor samples obtained from 317 colon cancer patients with all tumor stages. Correlation of the degree of staining with clinicopathological parameters and survival was investigated. Results Positive expression rates of HER-2/neu and VEGF in colon cancer were 15.5% and 55.5% respectively. HER-2/neu expression was significantly correlated with tumor size and distant metastases (P (P > 0.05. Expression of VEGF was significantly correlated with tumor size, tumor stage, lymph node metastases, and distant metastases (P (P = 0.146. No correlation between HER-2/neu and VEGF expression was detected (P = 0.151. Conclusions HER-2/neu and VEGF are not important prognostic markers of colon cancer. The present results do not support any association between HER2/neu and VEGF expression in this setting.

  20. Chronic hypoxia attenuates VEGF signaling and angiogenic responses by downregulation of KDR in human endothelial cells.

    Science.gov (United States)

    Olszewska-Pazdrak, Barbara; Hein, Travis W; Olszewska, Paulina; Carney, Darrell H

    2009-05-01

    Coronary artery disease results in progressive vascular stenosis associated with chronic myocardial ischemia. Vascular endothelial growth factor (VEGF) stimulates endothelial cell angiogenic responses to revascularize ischemic tissues; however, the effect of chronic hypoxia on the responsiveness of endothelial cells to VEGF remains unclear. We, therefore, investigated whether hypoxia alters VEGF-stimulated signaling and angiogenic responses in primary human coronary artery endothelial (HCAE) cells. Exposure of HCAE cells to hypoxia (1% O(2)) for 24 h decreased VEGF-stimulated endothelial cell migration ( approximately 82%), proliferation ( approximately 30%), and tube formation. Hypoxia attenuated VEGF-stimulated activation of endothelial nitric oxide (NO) synthase (eNOS) ( approximately 72%) and reduced NO production in VEGF-stimulated cells from 237 +/- 38.8 to 61.3 +/- 28.4 nmol/l. Moreover, hypoxia also decreased the ratio of phosphorylated eNOS to total eNOS in VEGF-stimulated cells by approximately 50%. This effect was not observed in thrombin-stimulated cells, suggesting that hypoxia specifically inhibited VEGF signaling upstream of eNOS phosphorylation. VEGF-induced activation of Akt, ERK1/2, p38, p70S6 kinases, and S6 ribosomal protein was also attenuated in hypoxic cells. Moreover, VEGF-stimulated phosphorylation of VEGF receptor-2 (KDR) at Y996 and Y1175 was decreased by hypoxia. This decrease correlated with a 70 +/- 12% decrease in KDR protein expression. Analysis of mRNA from these cells showed that hypoxia reduced steady-state levels of KDR mRNA by 52 +/- 16% and decreased mRNA stability relative to normoxic cells. Our findings demonstrate that chronic hypoxia attenuates VEGF-stimulated signaling in HCAE cells by specific downregulation of KDR expression. These data provide a novel explanation for the impaired angiogenic responses to VEGF in endothelial cells exposed to chronic hypoxia.

  1. Peptide vaccines and peptidomimetics targeting HER and VEGF proteins may offer a potentially new paradigm in cancer immunotherapy

    Science.gov (United States)

    Kaumaya, Pravin TP; Foy, Kevin Chu

    2013-01-01

    The ErbB family (HER-1, HER-2, HER-3 and HER-4) of receptor tyrosine kinases has been the focus of cancer immunotherapeutic strategies while antiangiogenic therapies have focused on VEGF and its receptors VEGFR-1 and VEGFR-2. Agents targeting receptor tyrosine kinases in oncology include therapeutic antibodies to receptor tyrosine kinase ligands or the receptors themselves, and small-molecule inhibitors. Many of the US FDA-approved therapies targeting HER-2 and VEGF exhibit unacceptable toxicities, and show problems of efficacy, development of resistance and unacceptable safety profiles that continue to hamper their clinical progress. The combination of dif ferent peptide vaccines and peptidomimetics targeting specific molecular pathways that are dysregulated in tumors may potentiate anticancer immune responses, bypass immune tolerance and circumvent resistance mechanisms. The focus of this review is to discuss efforts in our laboratory spanning two decades of rationally developing peptide vaccines and therapeutics for breast cancer. This review highlights the prospective benefit of a new, untapped category of therapies biologically targeted to EGF receptor (HER-1), HER-2 and VEGF with potential peptide ‘blockbusters‘ that could lay the foundation of a new paradigm in cancer immunotherapy by creating clinical breakthroughs for safe and efficacious cancer cures. PMID:22894670

  2. Peptide vaccines and targeting HER and VEGF proteins may offer a potentially new paradigm in cancer immunotherapy.

    Science.gov (United States)

    Kaumaya, Pravin T P; Foy, Kevin Chu

    2012-08-01

    The ErbB family (HER-1, HER-2, HER-3 and HER-4) of receptor tyrosine kinases has been the focus of cancer immunotherapeutic strategies while antiangiogenic therapies have focused on VEGF and its receptors VEGFR-1 and VEGFR-2. Agents targeting receptor tyrosine kinases in oncology include therapeutic antibodies to receptor tyrosine kinase ligands or the receptors themselves, and small-molecule inhibitors. Many of the US FDA-approved therapies targeting HER-2 and VEGF exhibit unacceptable toxicities, and show problems of efficacy, development of resistance and unacceptable safety profiles that continue to hamper their clinical progress. The combination of different peptide vaccines and peptidomimetics targeting specific molecular pathways that are dysregulated in tumors may potentiate anticancer immune responses, bypass immune tolerance and circumvent resistance mechanisms. The focus of this review is to discuss efforts in our laboratory spanning two decades of rationally developing peptide vaccines and therapeutics for breast cancer. This review highlights the prospective benefit of a new, untapped category of therapies biologically targeted to EGF receptor (HER-1), HER-2 and VEGF with potential peptide 'blockbusters' that could lay the foundation of a new paradigm in cancer immunotherapy by creating clinical breakthroughs for safe and efficacious cancer cures.

  3. Fluid shear promotes chondrosarcoma cell invasion by activating matrix metalloproteinase 12 via IGF-2 and VEGF signaling pathways

    Science.gov (United States)

    Wang, P; Chen, S-H; Hung, W-C; Paul, C; Zhu, F; Guan, P-P; Huso, DL; Kontrogianni-Konstantopoulos, A; Konstantopoulos, K

    2015-01-01

    Interstitial fluid flow in and around the tumor tissue is a physiologically relevant mechanical signal that regulates intracellular signaling pathways throughout the tumor. Yet, the effects of interstitial flow and associated fluid shear stress on the tumor cell function have been largely overlooked. Using in vitro bioengineering models in conjunction with molecular cell biology tools, we found that fluid shear (2 dyn/cm2) markedly upregulates matrix metalloproteinase 12 (MMP-12) expression and its activity in human chondrosarcoma cells. MMP-12 expression is induced in human chondrocytes during malignant transformation. However, the signaling pathway regulating MMP-12 expression and its potential role in human chondrosarcoma cell invasion and metastasis have yet to be delineated. We discovered that fluid shear stress induces the synthesis of insulin growth factor-2 (IGF-2) and vascular endothelial growth factor (VEGF) B and D, which in turn transactivate MMP-12 via PI3-K, p38 and JNK signaling pathways. IGF-2-, VEGF-B- or VEGF-D-stimulated chondrosarcoma cells display markedly higher migratory and invasive potentials in vitro, which are blocked by inhibiting MMP-12, PI3-K, p38 or JNK activity. Moreover, recombinant human MMP-12 or MMP-12 overexpression can potentiate chondrosarcoma cell invasion in vitro and the lung colonization in vivo. By reconstructing and delineating the signaling pathway regulating MMP-12 activation, potential therapeutic strategies that interfere with chondrosarcoma cell invasion may be identified. PMID:25435370

  4. 1-o-acetylbritannilactone (ABL) inhibits angiogenesis and lung cancer cell growth through regulating VEGF-Src-FAK signaling.

    Science.gov (United States)

    Zhengfu, He; Hu, Zhang; Huiwen, Miao; Zhijun, Li; Jiaojie, Zhou; Xiaoyi, Yan; Xiujun, Cai

    2015-08-21

    The search for safe, effective and affordable therapeutics against non-small cell lung cancer (NSCLC) and other lung cancers is important. Here we explored the potential effect of 1-o-acetylbritannilactone (ABL), a novel extract from Inula britannica-F, on angiogenesis and lung cancer cell growth. We demonstrated that ABL dose-dependently inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration, and capillary structure formation of cultured human umbilical vascular endothelial cells (HUVECs). In vivo, ABL administration suppressed VEGF-induced new vasculature formation in Matrigel plugs. For the mechanism investigations, we found that ABL largely inhibited VEGF-mediated activation of Src kinase and focal adhesion kinase (FAK) in HUVECs. Furthermore, treatment of A549 NSCLC cells with ABL resulted in cell growth inhibition and Src-FAK in-activation. Significantly, administration of a single dose of ABL (12 mg/kg/day) remarkably suppressed growth of A549 xenografts in nude mice. In vivo microvessels formation and Src activation were also significantly inhibited in ABL-treated xenograft tumors. Taken together, our findings suggest that ABL suppresses angiogenesis and lung cancer cell growth possibly via regulating the VEGFR-Src-FAK signaling. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Anti-rheumatic agent auranofin induced apoptosis in chronic myeloid leukemia cells resistant to imatinib through both Bcr/Abl-dependent and -independent mechanisms

    Science.gov (United States)

    Li, Xiaofen; Lan, Xiaoying; Liu, Shouting; Huang, Hongbiao; Liu, Ningning; Liao, Siyan; Zang, Dan; Song, Wenbin; Liu, Quentin; Carter, Bing Z.; Dou, Q. Ping; Wang, Xuejun; Liu, Jinbao

    2014-01-01

    Resistance to Imatinib mesylate (IM) is an emerging problem for patients with chronic myelogenous leukemia (CML). T315I mutation in the Bcr-Abl is the predominant mechanism of the acquired resistance to IM and second generation tyrosine kinase inhibitors (TKI). Therefore it is urgent to search for new measures to overcome TKI-resistance. Auranofin (AF), clinically used to treat rheumatic arthritis, was recently approved by US Food and Drug Administration for Phase II clinical trial to treat cancer. In contrast to the reports that AF induces apoptosis by increasing intracellular reactive oxygen species (ROS) levels via inhibiting thioredoxin reductase, our recent study revealed that AF-induced apoptosis depends on inhibition of proteasomal deubiquitinases (UCHL5 and USP14). Here we report that (i) AF induces apoptosis in both Bcr-Abl wild-type cells and Bcr-Abl-T315I mutation cells and inhibits the growth of IM-resistant Bcr-Abl-T315I xenografts in vivo; (ii) AF inhibits Bcr-Abl through both downregulation of Bcr-Abl gene expression and Bcr-Abl cleavage mediated by proteasome inhibition-induced caspase activation; (iii) proteasome inhibition but not ROS is required for AF-induced caspase activation and apoptosis. These findings support that AF overcomes IM resistance through both Bcr/Abl-dependent and -independent mechanisms, providing great clinical significance for cancer treatment. PMID:25193854

  6. Production, characterization, and flocculation mechanism of cation independent, pH tolerant, and thermally stable bioflocculant from Enterobacter sp. ETH-2.

    Directory of Open Access Journals (Sweden)

    Wei Tang

    Full Text Available Synthetic high polymer flocculants, frequently utilized for flocculating efficiency and low cost, recently have been discovered as producing increased risk to human health and the environment. Development of a more efficient and environmentally sound alternative flocculant agent is investigated in this paper. Bioflocculants are produced by microorganisms and may exhibit a high rate of flocculation activity. The bioflocculant ETH-2, with high flocculating activity (2849 mg Kaolin particle/mg ETH-2, produced by strain Enterobacter sp. isolated from activated sludge, was systematically investigated with regard to its production, characterization, and flocculation mechanism. Analyses of microscopic observation, zeta potential and ETH-2 structure demonstrates the bridging mechanism, as opposed to charge neutralization, was responsible for flocculation of the ETH-2. ETH-2 retains high molecular weight (603 to 1820 kDa and multi-functional groups (hydroxyl, amide and carboxyl that contributed to flocculation. Polysaccharides mainly composed of mannose, glucose, and galactose, with a molar ratio of 1:2.9:9.8 were identified as the active constituents in bioflocculant. The structure of the long backbone with active sites of polysaccharides was determined as a primary basis for the high flocculation activity. Bioflocculant ETH-2 is cation independent, pH tolerant, and thermally stable, suggesting a potential fit for industrial application.

  7. The independent roles of mechanical, structural and adhesion characteristics of 3D hydrogels on the regulation of cancer invasion and dissemination.

    Science.gov (United States)

    Beck, Jennifer N; Singh, Anirudha; Rothenberg, Ashley R; Elisseeff, Jennifer H; Ewald, Andrew J

    2013-12-01

    Metastasis begins with the escape, or dissemination, of cancer cells from the primary tumor. We recently demonstrated that tumors preferentially disseminate into collagen I and not into basement membrane protein gels (Matrigel). In this study, we used synthetic polymer systems to define material properties that could induce dissemination into Matrigel. We first specifically varied rigidity by varying the crosslinking density of poly(ethylene glycol) (PEG) networks within Matrigel scaffolds. Increased microenvironmental rigidity limited epithelial growth but did not promote dissemination. We next incorporated adhesive signals into the PEG network using peptide-conjugated cyclodextrin (α-CDYRGDS) rings. The α-CDYRGDS rings threaded along the PEG polymers, enabling independent control of matrix mechanics, adhesive peptide composition, and adhesive density. Adhesive PEG networks induced dissemination of normal and malignant mammary epithelial cells at intermediate values of adhesion and rigidity. Our data reveal that microenvironmental signals can induce dissemination of normal and malignant epithelial cells without requiring the fibrillar structure of collagen I or containing collagen I-specific adhesion sequences. Finally, the nanobiomaterials and assays developed in this study are generally useful both in 3D culture of primary mammalian tissues and in the systematic evaluation of the specific role of mechanical and adhesive inputs on 3D tumor growth, invasion, and dissemination. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Docosahexaenoic acid inhibits vascular endothelial growth factor (VEGF)-induced cell migration via the GPR120/PP2A/ERK1/2/eNOS signaling pathway in human umbilical vein endothelial cells.

    Science.gov (United States)

    Chao, Che-Yi; Lii, Chong-Kuei; Ye, Siou-Yu; Li, Chien-Chun; Lu, Chia-Yang; Lin, Ai-Hsuan; Liu, Kai-Li; Chen, Haw-Wen

    2014-05-07

    Cell migration plays an important role in angiogenesis and wound repair. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that is essential for endothelial cell survival, proliferation, and migration. Docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, shows both anti-inflammatory and antioxidant activities in vitro and in vivo. This study investigated the molecular mechanism by which DHA down-regulates VEGF-induced cell migration. HUVECs were used as the study model, and the MTT assay, Western blot, wound-healing assay, and phosphatase activity assay were used to explore the effects of DHA on cell migration. GPR120 is the putative receptor for DHA action. The results showed that DHA, PD98059 (an ERK1/2 inhibitor), and GW9508 (a GPR120 agonist) inhibited VEGF-induced cell migration. In contrast, pretreatment with okadaic acid (OA, a PP2A inhibitor) and S-nitroso-N-acetyl-DL-penicillamine (an NO donor) reversed the inhibition of cell migration by DHA. VEGF-induced cell migration was accompanied by phosphorylation of ERK1/2 and eNOS. Treatment of HUVECs with DHA increased PP2A enzyme activity and decreased VEGF-induced phosphorylation of ERK1/2 and eNOS. However, pretreatment with OA significantly decreased DHA-induced PP2A enzyme activity and reversed the DHA inhibition of VEGF-induced ERK1/2 and eNOS phosphorylation. These results suggest that stimulation of PP2A activity and inhibition of the VEGF-induced ERK1/2/eNOS signaling pathway may be involved in the DHA suppression of VEGF-induced cell migration. Thus, the effect of DHA on angiogenesis and wound repair is at least partly by virtue of its attenuation of cell migration.

  9. Manipulation of the HIF-Vegf pathway rescues methyl tert-butyl ether (MTBE)-induced vascular lesions.

    Science.gov (United States)

    Bonventre, Josephine A; Kung, Tiffany S; White, Lori A; Cooper, Keith R

    2013-12-15

    Methyl tert-butyl ether (MTBE) has been shown to be specifically anti-angiogenic in piscine and mammalian model systems at concentrations that appear non-toxic in other organ systems. The mechanism by which MTBE targets developing vascular structures is unknown. A global transcriptome analysis of zebrafish embryos developmentally exposed to 0.00625-5mM MTBE suggested that hypoxia inducible factor (HIF)-regulated pathways were affected. HIF-driven angiogenesis via vascular endothelial growth factor (vegf) is essential to the developing vasculature of an embryo. Three rescue studies were designed to rescue MTBE-induced vascular lesions: pooled blood in the common cardinal vein (CCV), cranial hemorrhages (CH), and abnormal intersegmental vessels (ISV), and test the hypothesis that MTBE toxicity was HIF-Vegf dependent. First, zebrafish vegf-a over-expression via plasmid injection, resulted in significantly fewer CH and ISV lesions, 46 and 35% respectively, in embryos exposed to 10mM MTBE. Then HIF degradation was inhibited in two ways. Chemical rescue by N-oxaloylglycine significantly reduced CCV and CH lesions by 30 and 32% in 10mM exposed embryos, and ISV lesions were reduced 24% in 5mM exposed zebrafish. Finally, a morpholino designed to knock-down ubiquitin associated von Hippel-Lindau protein, significantly reduced CCV lesions by 35% in 10mM exposed embryos. In addition, expression of some angiogenesis related genes altered by MTBE exposure were rescued. These studies demonstrated that MTBE vascular toxicity is mediated by a down regulation of HIF-Vegf driven angiogenesis. The selective toxicity of MTBE toward developing vasculature makes it a potentially useful chemical in the designing of new drugs or in elucidating roles for specific angiogenic proteins in future studies of vascular development. © 2013.

  10. Extra virgin olive oil rich in polyphenols modulates VEGF-induced angiogenic responses by preventing NADPH oxidase activity and expression.

    Science.gov (United States)

    Calabriso, Nadia; Massaro, Marika; Scoditti, Egeria; D'Amore, Simona; Gnoni, Antonio; Pellegrino, Mariangela; Storelli, Carlo; De Caterina, Raffaele; Palasciano, Giuseppe; Carluccio, Maria Annunziata

    2016-02-01

    Previous studies have shown the antiinflammatory, antioxidant and antiangiogenic properties by pure olive oil polyphenols; however, the effects of olive oil phenolic fraction on the inflammatory angiogenesis are unknown. In this study, we investigated the effects of the phenolic fraction (olive oil polyphenolic extract, OOPE) from extra virgin olive oil and related circulating metabolites on the VEGF-induced angiogenic responses and NADPH oxidase activity and expression in human cultured endothelial cells. We found that OOPE (1-10 μg/ml), at concentrations achievable nutritionally, significantly reduced, in a concentration-dependent manner, the VEGF-induced cell migration, invasiveness and tube-like structure formation through the inhibition of MMP-2 and MMP-9. OOPE significantly (Poxidase activity, p47phox membrane translocation and the expression of Nox2 and Nox4. Moreover, the treatment of endothelial cells with serum obtained 4 h after acute intake of extra virgin olive oil, with high polyphenol content, decreased VEGF-induced NADPH oxidase activity and Nox4 expression, as well as, MMP-9 expression, as compared with fasting control serum. Overall, native polyphenols and serum metabolites of extra virgin olive oil rich in polyphenols are able to lower the VEGF-induced angiogenic responses by preventing endothelial NADPH oxidase activity and decreasing the expression of selective NADPH oxidase subunits. Our results provide an alternative mechanism by which the consumption of olive oil rich in polyphenols may account for a reduction of oxidative stress inflammatory-related sequelae associated with chronic degenerative diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. VEGF-conjugated alginate hydrogel prompt angiogenesis and improve pancreatic islet engraftment and function in type 1 diabetes

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Nina; Han, Yongming [Department of Anatomy, Basic Medical College, Hubei University of Chinese Medicine, Wuhan, Hubei (China); Xu, Hanlin [Pharmacy Faculty, Hubei University of Chinese Medicine, Wuhan, Hubei (China); Gao, Yisen; Yi, Tao [Acupuncture and Moxibustion College, Hubei University of Chinese Medicine, Wuhan, Hubei (China); Yao, Jiale; Dong, Li; Cheng, Dejun [Basic Medical College, Hubei University of Chinese Medicine, Wuhan, Hubei (China); Chen, Zebin, E-mail: chenzebin-hbtcm@outlook.com [Acupuncture and Moxibustion College, Hubei University of Chinese Medicine/Hubei Provincial Collaborative Innovation Center of Preventive Treatment by Acupuncture and Moxibustion, Wuhan, Hubei (China)

    2016-02-01

    Type 1 diabetes was a life-long disease that affected numerous people around the world. Insulin therapy has its limitations that may involve hyperglycemia and heavy burden of patient by repeated dose. Islet transplantation emerged as a promising approach to reach periodical reverse of diabetes, however, transplanted islets suffer from foreign body reaction and lack of nutrition and oxygen supply, especially in the blood-vessel-shortage subcutaneous site which was preferred by patient and surgeon. In this study, we designed and synthesized a vascular endothelial growth factor (VEGF) conjugated alginate material to encapsulate the transplanted islets via 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) reaction, and successful conjugation was confirmed by Nuclear Magnetic Resonance H1 spectrum. The best VEGF concentration (100 ng/ml) was determined by the combined studies of the mechanical property and endothelial cell growth assay. In vivo study, conjugated VEGF on alginate exhibited sustained promoting angiogenesis property after subcutaneous transplantation by histology study and islets encapsulated in this material achieved long term therapeutic effect (up to 50 days) in the diabetic mice model. In conclusion, this study establishes a simple biomaterial strategy for islet transplantation to enhance islet survival and function, which could be a feasible therapeutic alternative for type 1 diabetes. - Highlights: • We synthesized VEGF-conjugated alginate material to encapsulate the transplanted islets. • The biomaterials improve islet engraftment and function due to angiogenesis. • The biomaterials could be a strong support for cell therapy with islet transplantation in type 1 diabetes.

  12. Low energy shock wave therapy induces angiogenesis in acute hind-limb ischemia via VEGF receptor 2 phosphorylation.

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    Johannes Holfeld

    Full Text Available OBJECTIVES: Low energy shock waves have been shown to induce angiogenesis, improve left ventricular ejection fraction and decrease angina symptoms in patients suffering from chronic ischemic heart disease. Whether there is as well an effect in acute ischemia was not yet investigated. METHODS: Hind-limb ischemia was induced in 10-12 weeks old male C57/Bl6 wild-type mice by excision of the left femoral artery. Animals were randomly divided in a treatment group (SWT, 300 shock waves at 0.1 mJ/mm2, 5 Hz and untreated controls (CTR, n = 10 per group. The treatment group received shock wave therapy immediately after surgery. RESULTS: Higher gene expression and protein levels of angiogenic factors VEGF-A and PlGF, as well as their receptors Flt-1 and KDR have been found. This resulted in significantly more vessels per high-power field in SWT compared to controls. Improvement of blood perfusion in treatment animals was confirmed by laser Doppler perfusion imaging. Receptor tyrosine kinase profiler revealed significant phosphorylation of VEGF receptor 2 as an underlying mechanism of action. The effect of VEGF signaling was abolished upon incubation with a VEGFR2 inhibitor indicating that the effect is indeed VEGFR 2 dependent. CONCLUSIONS: Low energy shock wave treatment induces angiogenesis in acute ischemia via VEGF receptor 2 stimulation and shows the same promising effects as known from chronic myocardial ischemia. It may therefore develop as an adjunct to the treatment armentarium of acute muscle ischemia in limbs and myocardium.

  13. The relative importance of nitric oxide and nitric oxide-independent mechanisms in acetylcholine-evoked dilatation of the rat mesenteric bed.

    Science.gov (United States)

    Parsons, S J; Hill, A; Waldron, G J; Plane, F; Garland, C J

    1994-12-01

    1. The relative contribution of nitric oxide (NO) to acetylcholine-induced smooth muscle relaxation was investigated in the rat perfused mesenteric vasculature and in isolated segments of second, third and fourth order arterial branches. 2. The EC50 values and maximal relaxation to acetylcholine were not significantly different in the sequential arterial branches, being approximately 0.05 microM and 85%, respectively. 3. The NO synthase inhibitor L-NG-nitro-L-arginine methyl ester (L-NAME; 100 microM) reduced acetylcholine-evoked endothelium-dependent dilatation and relaxation in the perfused mesenteric bed and in isolated arterial segments. The maximum response to acetylcholine in both preparations was reduced by between 35% to 40% while the EC50 values were increased by 5-6 fold. L-NAME had no effect on basal smooth muscle tone in either case. 4. In contrast, endothelium-dependent dilatation of the perfused mesenteric bed evoked by A23187 (0.002-20 nmol), was unaffected by exposure to L-NAME. The EC50 values and maximal responses elicited by A23187 (20 nmol) before and after exposure to L-NAME were 0.96 +/- 0.5 nmol and 67.0 +/- 7.0% (n = 4), and 0.7 +/- 0.4 nmol and 70.0 +/- 5.0% (n = 4; P > 0.01), respectively. 5. Perfusion of the isolated mesenteric bed with raised K(+)-Krebs buffer (25 mM) had no effect on basal tone, but reduced the amplitude of both acetylcholine- and A23187-evoked dilatation. The maximum responses to acetylcholine (2 micromol) and A23187 (20 nmol) were reduced from 67.5 +/- 7.3% and 65.4+/-8.2% to 18.9 +/-11.0% (n=5; P<0.01) and 13.5 +/-12.0% (n=4; P<0.01), respectively.6. Exposure of the mesenteric bed to L-NAME in the presence of raised K+-Krebs further reduced the maximal response elicited by acetylcholine to only 8.9 +/- 2.8% (n =4; P< 0.01).7. These results indicate that acetylcholine-evoked vasodilatation of the rat mesenteric vasculature is mediated by both NO-dependent and -independent mechanisms. The relative contribution made by

  14. 10-Hydroxy-2-decenoic Acid, a Major Fatty Acid from Royal Jelly, Inhibits VEGF-Induced Angiogenesis in Human Umbilical Vein Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Hiroshi Izuta

    2009-01-01

    Full Text Available Vascular endothelial growth factor (VEGF is reported to be a potent pro-angiogenic factor that plays a pivotal role in both physiological and pathological angiogenesis. Royal jelly (RJ is a honeybee product containing various proteins, sugars, lipids, vitamins and free amino acids. 10-Hydroxy-2-decenoic acid (10HDA, a major fatty acid component of RJ, is known to have various pharmacological effects; its antitumor activity being especially noteworthy. However, the mechanism underlying this effect is unclear. We examined the effect of 10HDA on VEGF-induced proliferation, migration and tube formation in human umbilical vein endothelial cells (HUVECs. Our findings showed that, 10HDA at 20 µM or more significantly inhibited such proliferation, migration and tube formation. Similarly, 10 µM GM6001, a matrix metalloprotease inhibitor, prevented VEGF-induced migration and tube formation. These findings indicate that 10HDA exerts an inhibitory effect on VEGF-induced angiogenesis, partly by inhibiting both cell proliferation and migration. Further experiments will be needed to clarify the detailed mechanism.

  15. Time-course of sFlt-1 and VEGF-A release in neutropenic patients with sepsis and septic shock: a prospective study

    Directory of Open Access Journals (Sweden)

    De Souza Carmino A

    2011-03-01

    Full Text Available Abstract Background Septic shock is the most feared complication of chemotherapy-induced febrile neutropenia. So far, there are no robust biomarkers that can stratify patients to the risk of sepsis complications. The VEGF-A axis is involved in the control of microvascular permeability and has been involved in the pathogenesis of conditions associated with endothelial barrier disruption such as sepsis. sFlt-1 is a soluble variant of the VEGF-A receptor VEGFR-1 that acts as a decoy receptor down-regulating the effects of VEGF-A. In animal models of sepsis, sFlt-1 was capable to block the barrier-breaking negative effects of VEGF-A and to significantly decrease mortality. In non-neutropenic patients, sFlt-1 has been shown to be a promising biomarker for sepsis severity. Methods We prospectively evaluated concentrations of sFlt-1 and VEGF-A at different time-points during febrile neutropenia, and evaluated the association of these levels with sepsis severity and septic shock development. Results Neutropenic patients that evolved with septic shock (n = 10 presented higher levels of sFlt-1 and VEGF-A measured 48 hours after fever onset than patients with non-complicated sepsis (n = 31 and levels of these biomarkers correlated with sepsis severity scores. Estimation of the diagnostic accuracy of sFlt-1 levels for the discrimination of patients that evolved to septic shock yielded promising results in our study population. Discussion Our data suggest that sFlt-1 and VEGF-A could be useful biomarkers for sepsis severity in patients with febrile neutropenia. In addition, the kinetics of sFlt-1 release in patients that evolve to septic shock suggest that the sFlt-1 could be a salvage compensatory mechanism in patients with septic shock, but that the magnitude of the sFlt-1 release observed in human sepsis is not sufficient to reproduce the beneficial anti-VEGF-A effects observed in animal models of sepsis.

  16. Caveolin-1 plays a crucial role in inhibiting neuronal differentiation of neural stem/progenitor cells via VEGF signaling-dependent pathway.

    Directory of Open Access Journals (Sweden)

    Yue Li

    Full Text Available In the present study, we aim to elucidate the roles of caveolin-1(Cav-1, a 22 kDa protein in plasma membrane invaginations, in modulating neuronal differentiation of neural progenitor cells (NPCs. In the hippocampal dentate gyrus, we found that Cav-1 knockout mice revealed remarkably higher levels of vascular endothelial growth factor (VEGF and the more abundant formation of newborn neurons than wild type mice. We then studied the potential mechanisms of Cav-1 in modulating VEGF signaling and neuronal differentiation in isolated cultured NPCs under normoxic and hypoxic conditions. Hypoxic embryonic rat NPCs were exposed to 1% O₂ for 24 h and then switched to 21% O₂ for 1, 3, 7 and 14 days whereas normoxic NPCs were continuously cultured with 21% O₂. Compared with normoxic NPCs, hypoxic NPCs had down-regulated expression of Cav-1 and up-regulated VEGF expression and p44/42MAPK phosphorylation, and enhanced neuronal differentiation. We further studied the roles of Cav-1 in inhibiting neuronal differentiation by using Cav-1 scaffolding domain peptide and Cav-1-specific small interfering RNA. In both normoxic and hypoxic NPCs, Cav-1 peptide markedly down-regulated the expressions of VEGF and flk1, decreased the phosphorylations of p44/42MAPK, Akt and Stat3, and inhibited neuronal differentiation, whereas the knockdown of Cav-1 promoted the expression of VEGF, phosphorylations of p44/42MAPK, Akt and Stat3, and stimulated neuronal differentiation. Moreover, the enhanced phosphorylations of p44/42MAPK, Akt and Stat3, and neuronal differentiation were abolished by co-treatment of VEGF inhibitor V1. These results provide strong evidence to prove that Cav-1 can inhibit neuronal differentiation via down-regulations of VEGF, p44/42MAPK, Akt and Stat3 signaling pathways, and that VEGF signaling is a crucial target of Cav-1. The hypoxia-induced down-regulation of Cav-1 contributes to enhanced neuronal differentiation in NPCs.

  17. HIF-VEGF pathways are critical for chronic otitis media in Junbo and Jeff mouse mutants.

    Directory of Open Access Journals (Sweden)

    Michael T Cheeseman

    2011-10-01

    Full Text Available Otitis media with effusion (OME is the commonest cause of hearing loss in children, yet the underlying genetic pathways and mechanisms involved are incompletely understood. Ventilation of the middle ear with tympanostomy tubes is the commonest surgical procedure in children and the best treatment for chronic OME, but the mechanism by which they work remains uncertain. As hypoxia is a common feature of inflamed microenvironments, moderation of hypoxia may be a significant contributory mechanism. We have investigated the occurrence of hypoxia and hypoxia-inducible factor (HIF mediated responses in Junbo and Jeff mouse mutant models, which develop spontaneous chronic otitis media. We found that Jeff and Junbo mice labeled in vivo with pimonidazole showed cellular hypoxia in inflammatory cells in the bulla lumen, and in Junbo the middle ear mucosa was also hypoxic. The bulla fluid inflammatory cell numbers were greater and the upregulation of inflammatory gene networks were more pronounced in Junbo than Jeff. Hif-1α gene expression was elevated in bulla fluid inflammatory cells, and there was upregulation of its target genes including Vegfa in Junbo and Jeff. We therefore investigated the effects in Junbo of small-molecule inhibitors of VEGFR signaling (PTK787, SU-11248, and BAY 43-9006 and destabilizing HIF by inhibiting its chaperone HSP90 with 17-DMAG. We found that both classes of inhibitor significantly reduced hearing loss and the occurrence of bulla fluid and that VEGFR inhibitors moderated angiogenesis and lymphangiogenesis in the inflamed middle ear mucosa. The effectiveness of HSP90 and VEGFR signaling inhibitors in suppressing OM in the Junbo model implicates HIF-mediated VEGF as playing a pivotal role in OM pathogenesis. Our analysis of the Junbo and Jeff mutants highlights the role of hypoxia and HIF-mediated pathways, and we conclude that targeting molecules in HIF-VEGF signaling pathways has therapeutic potential in the treatment of

  18. Nicotine promotes proliferation of human nasopharyngeal carcinoma cells by regulating α7AChR, ERK, HIF-1α and VEGF/PEDF signaling.

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    Dingbo Shi

    Full Text Available Nicotine, the major component in cigarette smoke, can promote tumor growth and angiogenesis, but the precise mechanisms involved remain largely unknown. Here, we investigated the mechanism of action of nicotine in human nasopharyngeal carcinoma (NPC cells. Nicotine significantly promoted cell proliferation in a dose and time-dependent manner in human NPC cells. The mechanism studies showed that the observed stimulation of proliferation was accompanied by the nicotine-mediated simultaneous modulation of α7AChR, HIF-1α, ERK and VEGF/PEDF signaling. Treatment of NPC cells with nicotine markedly upregulated the expression of α7AChR and HIF-1α proteins. Transfection with a α7AChR or HIF-1α-specific siRNA or a α7AChR-selective inhibitor significantly attenuated the nicotine-mediated promotion of NPC cell proliferation. Nicotine also promoted the phosphorylation of ERK1/2 but not JNK and p38 proteins, thereby induced the activation of ERK/MAPK signaling pathway. Pretreatment with an ERK-selective inhibitor effectively reduced the nicotine-induced proliferation of NPC cells. Moreover, nicotine upregulated the expression of VEGF but suppressed the expression of PEDF at mRNA and protein levels, leading to a significant increase of the ratio of VEGF/PEDF in NPC cells. Pretreatment with a α7AChR or ERK-selective inhibitor or transfection with a HIF-1α-specific siRNA in NPC cells significantly inhibited the nicotine-induced HIF-1α expression and VEGF/PEDF ratio. These results therefore indicate that nicotine promotes proliferation of human NPC cells in vitro through simultaneous modulation of α7AChR, HIF-1α, ERK and VEGF/PEDF signaling and suggest that the related molecules such as HIF-1α might be the potential therapeutic targets for tobacco-associated diseases such as nasopharyngeal carcinomas.

  19. Guided bone regeneration (GBR) utilizing injectable Vascular Endothelial Growth Factor (VEGF) delivery gel

    Science.gov (United States)

    Kaigler, Darnell; Silva, Eduardo A.; Mooney, David J.

    2013-01-01

    Background Vascularization underlies the success of guided bone regeneration (GBR) procedures. This study evaluated the regenerative potential of GBR in combination with Vascular Endothelial Growth Factor (VEGF) delivery, via an injectable hydrogel system. Methods Critical-sized defects were created in rat calvariae and GBR procedures were performed with a collagen membrane either alone (control), plus bolus delivery of VEGF, or plus application of VEGF releasing hydrogels (VEGF - Alg). Four and eight weeks following treatment, defect sites were evaluated with microcomputed tomographic and histomorphometric analyses for blood vessel and bone formation. Results At four weeks, relative to the control condition, the bolus addition of VEGF did not affect blood vessel density within the defect site; yet, the application of the VEGF+ Alg significantly (pGBR may be a promising strategy for enhancing outcomes of GBR. PMID:22668339

  20. VEGF family members regulate myocardial tubulogenesis and coronary artery formation in the embryo.

    Science.gov (United States)

    Tomanek, Robert J; Ishii, Yasuo; Holifield, Jennifer S; Sjogren, Christina L; Hansen, Heidi K; Mikawa, Takashi

    2006-04-14

    This study tested the hypothesis that coronary tubulogenesis and coronary artery formation require VEGF family members. Quail embryos were injected with soluble vascular endothelial growth factor (VEGF) receptors R1 (Flt-1), R2 (Flk-1), R3 (Flt-4), VEGF-Trap (a chimera of R1 and R2), or neutralizing antibodies to VEGF-A, VEGF-B, or fibroblast growth factor (FGF)-2. Our data document that tubulogenesis is temporally dependent on multiple VEGF family members, because the early stage of tubulogenesis was markedly inhibited by VEGF-Trap and to a lesser extent by soluble VEGFR-1. Some inhibition of tubulogenesis was documented when anti-FGF-2, but not anti-VEGF-A, antibodies were injected at embryonic day 6 (E6). Most importantly, we found that VEGF-Trap injected at either E6 or E7 prevented the formation of coronary arteries. Soluble VEGFR-1 and soluble VEGFR-2 modified the formation of coronary arteries, whereas soluble VEGFR-3 was without effect. Antibodies to VEGF-B, but not VEGF-A, had a strong inhibitory effect on coronary artery development. The absence of coronary artery stems, and thus a functional coronary circulation, in the embryos injected with VEGF-Trap caused an accumulation of erythrocytes in the subepicardium and muscular interventricular septum. Using retroviral cell tagging, we showed that some of the erythrocytes in blood islands and small vascular tubes were progeny of the proepicardium. Thus, another salient finding of this study is the first definitive documentation of proepicardially derived hemangioblasts, which can differentiate into erythrocytes.

  1. VEGF Polymorphisms Related to Higher Serum Levels of Protein Identify Patients with Hepatocellular Carcinoma

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    Maria Eduarda Lopes Baitello

    2016-01-01

    Full Text Available Hepatocellular carcinoma (HCC is the most common primary neoplasia of the liver. Major risk factors for hepatocellular carcinoma include chronic liver diseases, carcinogenic agents, and genetic alterations as well as vascular endothelial growth factor (VEGF involved in angiogenesis process. The aim of this study was to evaluate the association of VEGF-A (C936T and A1154G with HCC and cirrhosis, in addition to serum levels of VEGF, clinical profile, lifestyle habits, and comorbidities. A total of 346 individuals were studied: 102 with HCC (G1, 117 with cirrhosis (G2, and 127 controls (G3. Polymorphisms were analysed by PCR/RFLP and serum levels of VEGF by ELISA. Alpha error was set at 5%. The wild-type genotype of both polymorphisms prevailed (P>0.05. In G1, 23% of the patients died, with no relation to genetic profile (P>0.05. Increased VEGF level was observed in G1 and G3, related to the mutant allele of VEGF-C936T and VEGF-A1154G, respectively, and compared with the wild-type genotype (P=0.0285; P=0.0284, resp. as well as G1 versus G2 and G3 for VEGF-C936T and G1 versus G2 for VEGF-A1154G (P<0.05 for both. In conclusion, there is a relationship between mutant alleles of VEGF-C936T and VEGF-A1154G polymorphisms and higher VEGF level, making them potential markers for HCC.

  2. Circulating VEGF as a biological marker in patients with rheumatoid arthritis? Preanalytical and biological variability in healthy persons and in patients

    DEFF Research Database (Denmark)

    Hetland, Merete Lund; Christensen, Ib Jarle; Lottenburger, Tine

    2008-01-01

    and contamination of plasma with cellular elements lead to significant increases in VEGF levels, whereas storage for up to 2 years at -80 degrees C or up to 10 freeze/thaw cycles did not affect VEGF levels. Serum VEGF levels were 7-10 fold higher than plasma VEGF levels. Reference intervals for VEGF (plasma: 45 pg...

  3. Estimation of Immunohistochemical Expression of VEGF in Ductal Carcinomas of the Breast

    DEFF Research Database (Denmark)

    Maae, Else; Nielsen, Martin; Dahl Steffensen, Karina

    2011-01-01

    Vascular endothelial growth factor A (VEGF-A) is a very important growth factor in angiogenesis and holds the potential as both a predictive marker for anti-angiogenic cancer treatment and as a prognostic variable. Consequently, reliable estimation of VEGF expression is crucial. We immunostained...... whole tumor sections for VEGF-A, -B, and VEGFR-1 of invasive ductal carcinomas of the breast and scored the tumors manually with staining intensity as the only parameter and by a combination of qualitative and quantitative information. We also introduce an automated method for analyzing VEGF expression...

  4. Modified VEGF targets the ischemic myocardium and promotes functional recovery after myocardial infarction.

    Science.gov (United States)

    Yang, Yun; Shi, Chunying; Hou, Xianglin; Zhao, Yannan; Chen, Bing; Tan, Bo; Deng, Zongwu; Li, Qingguo; Liu, Jianzhou; Xiao, Zhifeng; Miao, Qi; Dai, Jianwu

    2015-09-10

    Vascular endothelial growth factor (VEGF) promotes angiogenesis and improves cardiac function after myocardial infarction (MI). However, the non-targeted delivery of VEGF decreases its therapeutic efficacy due to an insufficient local concentration in the ischemic myocardium. In this study, we used a specific peptide to modify VEGF and determined that this modified VEGF (IMT-VEGF) localized to the ischemic myocardium through intravenous injection by interacting with cardiac troponin I (cTnI). When IMT-VEGF was used to mediate cardiac repair in a rat model of ischemia-reperfusion (I-R) injury, we observed a decreased scar size, enhanced angiogenesis and improved cardiac function. Moreover, an alternative treatment using the repeated administration of a low-dose IMT-VEGF also promoted angiogenesis and functional recovery. The therapeutic effects of IMT-VEGF were further confirmed in a pig model of MI as the result of the conserved properties of its interacting protein, cTnI. These results suggest a promising therapeutic strategy for MI based on the targeted delivery of IMT-VEGF. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. A Biomimic Reconstituted High Density Lipoprotein Nanosystem for Enhanced VEGF Gene Therapy of Myocardial Ischemia

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    Xiaotian Sun

    2015-01-01

    Full Text Available A biomimic reconstituted high density lipoprotein (rHDL based system, rHDL/Stearic-PEI/VEGF complexes, was fabricated as an advanced nanovector for delivering VEGF plasmid. Here, Stearic-PEI was utilized to effectively condense VEGF plasmid and to incorporate the plasmid into rHDL. The rHDL/Stearic-PEI/VEGF complexes with diameter under 100 nm and neutral surface charge demonstrated enhanced stability under the presence of bovine serum albumin. Moreover, in vitro cytotoxicity and transfection assays on H9C2 cells further revealed their superiority, as they displayed lower cytotoxicity with much higher transfection efficiency when compared to PEI 10K/VEGF and Lipos/Stearic-PEI/VEGF complexes. In addition, in vivo investigation on ischemia/reperfusion rat model implied that rHDL/Stearic-PEI/VEGF complexes possessed high transgene capacity and strong therapeutic activity. These findings indicated that rHDL/Stearic-PEI/VEGF complexes could be an ideal gene delivery system for enhanced VEGF gene therapy of myocardial ischemia, which might be a new promising strategy for effective myocardial ischemia treatment.

  6. Protein kinase D1 signaling in angiogenic gene expression and VEGF-mediated angiogenesis

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    Bin eRen MD, Phd, FAHA

    2016-05-01

    Full Text Available Protein kinase D 1 (PKD-1 is a signaling kinase important in fundamental cell functions including migration, proliferation and differentiation. PKD-1 is also a key regulator of gene expression and angiogenesis that is essential for cardiovascular development and tumor progression. Further understanding molecular aspects of PKD-1 signaling in the regulation of angiogenesis may have translational implications in obesity, cardiovascular disease and cancer. The author will summarize and provide the insights into molecular mechanisms by which PKD-1 regulates transcriptional expression of angiogenic genes, focusing on the transcriptional regulation of CD36 by PKD-1-FoxO1 signaling axis along with the potential implications of this axis in arterial differentiation and morphogenesis. He will also discuss a new concept of dynamic balance between proangiogenic and antiangiogenic signaling in determining angiogenic switch, and stress how PKD-1 signaling regulates VEGF signaling-mediated angiogenesis.

  7. Low concentrations of o,p'-DDT inhibit gene expression and prostaglandin synthesis by estrogen receptor-independent mechanism in rat ovarian cells.

    Directory of Open Access Journals (Sweden)

    Jing Liu

    Full Text Available o,p'-DDT is an infamous xenoestrogen as well as a ubiquitous and persistent pollutant. Biomonitoring studies show that women have been internally exposed to o,p'-DDT at range of 0.3-500 ng/g (8.46×10(-10 M-1.41×10(-6 M in blood and other tissues. However, very limited studies have investigated the biological effects and mechanism(s of o,p'-DDT at levels equal to or lower than current exposure levels in human. In this study, using primary cultures of rat ovarian granulosa cells, we determined that very low doses of o,p'-DDT (10(-12-10(-8 M suppressed the expression of ovarian genes and production of prostaglandin E2 (PGE2. In vivo experiments consistently demonstrated that o,p'-DDT at 0.5-1 mg/kg inhibited the gene expression and PGE2 levels in rat ovary. The surprising results from the receptor inhibitors studies showed that these inhibitory effects were exerted independently of either classical estrogen receptors (ERs or G protein-coupled receptor 30 (GPR30. Instead, o,p'-DDT altered gene expression or hormone action via inhibiting the activation of protein kinase A (PKA, rather than protein kinase C (PKC. We further revealed that o,p'-DDT directly interfered with the PKA catalytic subunit. Our novel findings support the hypothesis that exposure to low concentrations of o,p'-DDT alters gene expression and hormone synthesis through signaling mediators beyond receptor binding, and imply that the current exposure levels of o,p'-DDT observed in the population likely poses a health risk to female reproduction.

  8. Low concentrations of o,p'-DDT inhibit gene expression and prostaglandin synthesis by estrogen receptor-independent mechanism in rat ovarian cells.

    Science.gov (United States)

    Liu, Jing; Zhao, Meirong; Zhuang, Shulin; Yang, Yan; Yang, Ye; Liu, Weiping

    2012-01-01

    o,p'-DDT is an infamous xenoestrogen as well as a ubiquitous and persistent pollutant. Biomonitoring studies show that women have been internally exposed to o,p'-DDT at range of 0.3-500 ng/g (8.46×10(-10) M-1.41×10(-6) M) in blood and other tissues. However, very limited studies have investigated the biological effects and mechanism(s) of o,p'-DDT at levels equal to or lower than current exposure levels in human. In this study, using primary cultures of rat ovarian granulosa cells, we determined that very low doses of o,p'-DDT (10(-12)-10(-8) M) suppressed the expression of ovarian genes and production of prostaglandin E2 (PGE2). In vivo experiments consistently demonstrated that o,p'-DDT at 0.5-1 mg/kg inhibited the gene expression and PGE2 levels in rat ovary. The surprising results from the receptor inhibitors studies showed that these inhibitory effects were exerted independently of either classical estrogen receptors (ERs) or G protein-coupled receptor 30 (GPR30). Instead, o,p'-DDT altered gene expression or hormone action via inhibiting the activation of protein kinase A (PKA), rather than protein kinase C (PKC). We further revealed that o,p'-DDT directly interfered with the PKA catalytic subunit. Our novel findings support the hypothesis that exposure to low concentrations of o,p'-DDT alters gene expression and hormone synthesis through signaling mediators beyond receptor binding, and imply that the current exposure levels of o,p'-DDT observed in the population likely poses a health risk to female reproduction.

  9. Low Concentrations of o,p’-DDT Inhibit Gene Expression and Prostaglandin Synthesis by Estrogen Receptor-Independent Mechanism in Rat Ovarian Cells

    Science.gov (United States)

    Liu, Jing; Zhao, Meirong; Zhuang, Shulin; Yang, Yan; Yang, Ye; Liu, Weiping

    2012-01-01

    o,p’-DDT is an infamous xenoestrogen as well as a ubiquitous and persistent pollutant. Biomonitoring studies show that women have been internally exposed to o,p’-DDT at range of 0.3–500 ng/g (8.46×10−10 M−1.41×10−6 M) in blood and other tissues. However, very limited studies have investigated the biological effects and mechanism(s) of o,p’-DDT at levels equal to or lower than current exposure levels in human. In this study, using primary cultures of rat ovarian granulosa cells, we determined that very low doses of o,p’-DDT (10−12−10−8 M) suppressed the expression of ovarian genes and production of prostaglandin E2 (PGE2). In vivo experiments consistently demonstrated that o,p’-DDT at 0.5–1 mg/kg inhibited the gene expression and PGE2 levels in rat ovary. The surprising results from the receptor inhibitors studies showed that these inhibitory effects were exerted independently of either classical estrogen receptors (ERs) or G protein-coupled receptor 30 (GPR30). Instead, o,p’-DDT altered gene expression or hormone action via inhibiting the activation of protein kinase A (PKA), rather than protein kinase C (PKC). We further revealed that o,p’-DDT directly interfered with the PKA catalytic subunit. Our novel findings support the hypothesis that exposure to low concentrations of o,p’-DDT alters gene expression and hormone synthesis through signaling mediators beyond receptor binding, and imply that the current exposure levels of o,p’-DDT observed in the population likely poses a health risk to female reproduction. PMID:23209616

  10. CAT-1 as a novel CAM stabilizes endothelial integrity and mediates the protective actions of L-Arg via a NO-independent mechanism.

    Science.gov (United States)

    Guo, Lu; Tian, Shuang; Chen, Yuguo; Mao, Yun; Cui, Sumei; Hu, Aihua; Zhang, Jianliang; Xia, Shen-Ling; Su, Yunchao; Du, Jie; Block, Edward R; Wang, Xing Li; Cui, Zhaoqiang

    2015-10-01

    Interendothelial junctions play an important role in the maintenance of endothelial integrity and the regulation of vascular functions. We report here that cationic amino acid transporter-1 (CAT-1) is a novel interendothelial cell adhesion molecule (CAM). We identified that CAT-1 protein localized at cell-cell adhesive junctions, similar to the classic CAM of VE-cadherin, and knockdown of CAT-1 with siRNA led to an increase in endothelial permeability. In addition, CAT-1 formed a cis-homo-dimer and showed Ca(2+)-dependent trans-homo-interaction to cause homophilic cell-cell adhesion. Co-immunoprecipitation assays showed that CAT-1 can associate with β-catenin. Furthermore, we found that the sub-cellular localization and function of CAT-1 are associated with cell confluency, in sub-confluent ECs CAT-1 proteins distribute on the entire surface and function as L-Arg transporters, but most of the CAT-1 in the confluent ECs are localized at interendothelial junctions and serve as CAMs. Further functional characterization has disclosed that extracellular L-Arg exposure stabilizes endothelial integrity via abating the cell junction disassembly of CAT-1 and blocking the cellular membrane CAT-1 internalization, which provides the new mechanisms for L-Arg paradox and trans-stimulation of cationic amino acid transport system (CAAT). These results suggest that CAT-1 is a novel CAM that directly regulates endothelial integrity and mediates the protective actions of L-Arg to endothelium via a NO-independent mechanism. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Resveratrol Ameliorates Palmitate-Induced Inflammation in Skeletal Muscle Cells by Attenuating Oxidative Stress and JNK/NF-κB Pathway in a SIRT1-Independent Mechanism.

    Science.gov (United States)

    Sadeghi, Asie; Seyyed Ebrahimi, Shadi Sadat; Golestani, Abolfazl; Meshkani, Reza

    2017-09-01

    Resveratrol has been shown to exert anti-inflammatory and anti-oxidant effects in a variety of cell types, however, its role in prevention of inflammatory responses mediated by palmitate in skeletal muscle cells remains unexplored. In the present study, we investigated the effects of resveratrol on palmitate-induced inflammation and elucidated the underlying mechanisms in skeletal muscle cells. The results showed that palmitate significantly enhanced TNF-α and IL-6 mRNA expression and protein secretion from C2C12 cells at 12, 24, and 36 h treatments. Increased expression of cytokines was accompanied by an enhanced phosphorylation of JNK, P38, ERK1/2, and IKKα/IKKβ. In addition, JNK and P38 inhibitors could significantly attenuate palmitate-induced mRNA expression of TNF-α and IL-6, respectively, whereas NF-κB inhibitor reduced the expression of both cytokines in palmitate-treated cells. Resveratrol pretreatment significantly prevented palmitate-induced TNF-α and IL-6 mRNA expression and protein secretion in C2C12 cells. Importantly, pre-treatment of the cells with resveratrol completely abrogated the phosphorylation of ERK1/2, JNK, and IKKα/IKKβ in palmitate treated cells. The protection from palmitate-induced inflammation by resveratrol was accompanied by a decrease in the generation of reactive oxygen species (ROS). N-acetyl cysteine (NAC), a known scavenger of ROS, could protect palmitate-induced expression of TNF-α and IL-6. Furthermore, inhibition of SIRT1 by shRNA or sirtinol demonstrated that the anti-inflammatory effect of resveratrol in muscle cells is mediated through a SIRT1-independent mechanism. Taken together, these findings suggest that resveratrol may represent a promising therapy for prevention of inflammation in skeletal muscle cells. J. Cell. Biochem. 118: 2654-2663, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  12. Cbx2 stably associates with mitotic chromosomes via a PRC2- or PRC1-independent mechanism and is needed for recruiting PRC1 complex to mitotic chromosomes.

    Science.gov (United States)

    Zhen, Chao Yu; Duc, Huy Nguyen; Kokotovic, Marko; Phiel, Christopher J; Ren, Xiaojun

    2014-11-15

    Polycomb group (PcG) proteins are epigenetic transcriptional factors that repress key developmental regulators and maintain cellular identity through mitosis via a poorly understood mechanism. Using quantitative live-cell imaging in mouse ES cells and tumor cells, we demonstrate that, although Polycomb repressive complex (PRC) 1 proteins (Cbx-family proteins, Ring1b, Mel18, and Phc1) exhibit variable capacities of association with mitotic chromosomes, Cbx2 overwhelmingly binds to mitotic chromosomes. The recruitment of Cbx2 to mitotic chromosomes is independent of PRC1 or PRC2, and Cbx2 is needed to recruit PRC1 complex to mitotic chromosomes. Quantitative fluorescence recovery after photobleaching analysis indicates that PRC1 proteins rapidly exchange at interphasic chromatin. On entry into mitosis, Cbx2, Ring1b, Mel18, and Phc1 proteins become immobilized at mitotic chromosomes, whereas other Cbx-family proteins dynamically bind to mitotic chromosomes. Depletion of PRC1 or PRC2 protein has no effect on the immobilization of Cbx2 on mitotic chromosomes. We find that the N-terminus of Cbx2 is needed for its recruitment to mitotic chromosomes, whereas the C-terminus is required for its immobilization. Thus these results provide fundamental insights into the molecular mechanisms of epigenetic inheritance. © 2014 Zhen et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  13. CD40 ligand exhibits a direct antiviral effect on Herpes Simplex Virus type-1 infection via a PI3K-dependent, autophagy-independent mechanism.

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    Vlahava, Virginia-Maria; Eliopoulos, Aristides G; Sourvinos, George

    2015-06-01

    The interaction between CD40 and its ligand, CD40L/CD154, is crucial for the efficient initiation and regulation of immune responses against viruses. Herpes Simplex Virus type-1 (HSV-1) is a neurotropic virus capable of manipulating host responses and exploiting host proteins to establish productive infection. Herein we have examined the impact of CD40L-mediated CD40 activation on HSV-1 replication in U2OS cells stably expressing the CD40 receptor. Treatment of these cells with CD40L significantly reduced the HSV-1 progeny virus compared to non-treated cells. The activation of CD40 signaling did not affect the binding of HSV-1 virions on the cell surface but rather delayed the translocation of VP16 to the nucleus, affecting all stages of viral life cycle. Using pharmacological inhibitors and RNAi we show that inhibition of PI3 kinase but not autophagy reverses the effects of CD40L on HSV-1 replication. Collectively, these data demonstrate that CD40 activation exerts a direct inhibitory effect on HSV-1, initiating from the very early stages of the infection by exploiting PI3 kinase-dependent but autophagy-independent mechanisms. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Insulin-Like Growth Factor-I Induces Arginase Activity in Leishmania amazonensis Amastigote-Infected Macrophages through a Cytokine-Independent Mechanism

    Directory of Open Access Journals (Sweden)

    Celia Maria Vieira Vendrame

    2014-01-01

    Full Text Available Leishmania (Leishmania amazonensis exhibits peculiarities in its interactions with hosts. Because amastigotes are the primary form associated with the progression of infection, we studied the effect of insulin-like growth factor (IGF-I on interactions between L. (L. amazonensis amastigotes and macrophages. Upon stimulation of infected macrophages with IGF-I, we observed decreased nitric oxide production but increased arginase expression and activity, which lead to increased parasitism. However, stimulation of amastigote-infected macrophages with IGF-I did not result in altered cytokine levels compared to unstimulated controls. Because IGF-I is present in tissue fluids and also within macrophages, we examined the possible effect of this factor on phosphatidylserine (PS exposure on amastigotes, seen previously in tissue-derived amastigotes leading to increased parasitism. Stimulation with IGF-I induced PS exposure on amastigotes but not on promastigotes. Using a PS-liposome instead of amastigotes, we observed that the PS-liposome but not the control phosphatidylcholine-liposome led to increased arginase activity in macrophages, and this process was not blocked by anti-TGF-β antibodies. Our results suggest that in L. (L. amazonensis amastigote-infected macrophages, IGF-I induces arginase activity directly in amastigotes and in macrophages through the induction of PS exposure on amastigotes in the latter, which could lead to the alternative activation of macrophages through cytokine-independent mechanisms.

  15. Heterotrimeric G proteins-mediated resistance to necrotrophic pathogens includes mechanisms independent of salicylic acid-, jasmonic acid/ethylene- and abscisic acid-mediated defense signaling.

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    Trusov, Yuri; Sewelam, Nasser; Rookes, James Edward; Kunkel, Matt; Nowak, Ekaterina; Schenk, Peer Martin; Botella, José Ramón

    2009-04-01

    Heterotrimeric G proteins are involved in the defense response against necrotrophic fungi in Arabidopsis. In order to elucidate the resistance mechanisms involving heterotrimeric G proteins, we analyzed the effects of the Gβ (subunit deficiency in the mutant agb1-2 on pathogenesis-related gene expression, as well as the genetic interaction between agb1-2 and a number of mutants of established defense pathways. Gβ-mediated signaling suppresses the induction of salicylic acid (SA)-, jasmonic acid (JA)-, ethylene (ET)- and abscisic acid (ABA)-dependent genes during the initial phase of the infection with Fusarium oxysporum (up to 48 h after inoculation). However, at a later phase it enhances JA/ET-dependent genes such as PDF1.2 and PR4. Quantification of the Fusarium wilt symptoms revealed that Gβ- and SA-deficient mutants were more susceptible than wild-type plants, whereas JA- and ET-insensitive and ABA-deficient mutants demonstrated various levels of resistance. Analysis of the double mutants showed that the Gβ-mediated resistance to F. oxysporum and Alternaria brassicicola was mostly independent of all of the previously mentioned pathways. However, the progressive decay of agb1-2 mutants was compensated by coi1-21 and jin1-9 mutations, suggesting that at this stage of F. oxysporum infection Gβ acts upstream of COI1 and ATMYC2 in JA signaling. © 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd.

  16. Blockade of TRPA1 with HC-030031 attenuates visceral nociception by a mechanism independent of inflammatory resident cells, nitric oxide and the opioid system.

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    Pereira, L M S; Lima-Júnior, R C P; Bem, A X C; Teixeira, C G; Grassi, L S; Medeiros, R P; Marques-Neto, R D; Callado, R B; Aragão, K S; Wong, D V T; Vale, M L; Brito, G A C; Ribeiro, R A

    2013-02-01

    Some studies have shown a somatic nociceptive response due to the activation of transient receptor potential A1 channels (TRPA1), which is modulated by the TRPA1 antagonist HC-030031. However, a few studies report the role of TRPA1 in visceral pain. Therefore, we investigated the participation of TRPA1 in visceral nociception and the involvement of nitric oxide, the opioid system and resident cells in the modulation of these channels. Mice were treated with vehicle or HC-030031 (18.75-300 mg/kg) before ifosfamide (400 mg/kg), 0.75% mustard oil (50 μL/colon), acetic acid 0.6% (10 mL/kg), zymosan (1 mg/cavity) or misoprostol (1 μg/cavity) injection. Visceral nociception was assessed through the electronic von Frey test or the writhing response. Ifosfamide-administered mice were euthanized for bladder analysis. The involvement of nitric oxide and the opioid system were investigated in mice injected with ifosfamide and mustard oil, respectively. The participation of resident peritoneal cells in acetic acid-, zymosan- or misoprostol-induced nociception was also evaluated. HC-030031 failed to protect animals against ifosfamide-induced bladder injury (p > 0.05). However, a marked antinociceptive effect against ifosfamide, mustard oil, acetic acid, zymosan and misoprostol was observed (p nociception by a mechanism independent of the modulation of resident cells, nitric oxide and opioid pathways. © 2012 European Federation of International Association for the Study of Pain Chapters.

  17. Carotid Artery Stenosis Contralateral to Acute Tandem Occlusion: An Independent Predictor of Poor Clinical Outcome after Mechanical Thrombectomy with Concomitant Carotid Artery Stenting.

    Science.gov (United States)

    Maus, Volker; Behme, Daniel; Borggrefe, Jan; Kabbasch, Christoph; Seker, Fatih; Hüseyin, Cicek; Barnikol, Utako Birgit; Yeo, Leonard Leong Litt; Brouwer, Patrick; Söderman, Michael; Möhlenbruch, Markus; Psychogios, Marios Nikos; Liebig, Thomas; Dohmen, Christian; Fink, Gereon Rudolf; Mpotsaris, Anastasios

    2017-11-24

    Cerebral ischemic strokes due to extra-/intracranial tandem occlusions (TO) of the anterior circulation are responsible for causing mechanical thrombectomy (MT). The impact of concomitant contralateral carotid stenosis (CCS) upon outcome remains unclear in this stroke subtype. Retrospective analysis of prospectively collected data of 4 international stroke centers between 2011 and 2017. One hundred ninety-seven consecutive patients with anterior TO were treated with MT and acute carotid artery stenting (CAS). Clinical (including demographics and National Institutes of Health Stroke Scale [NIHSS]), imaging (including angiographic evaluation of CCS) and procedural data were evaluated. Favorable clinical outcome was defined as modified Rankin Scale (mRS) ≤2 at 90 days. In 186 out of 197 TO patients preinterventional CT angiography was available for analysis, thereof 49 patients (26%) presented with CCS. Median admission NIHSS and procedural timings did not differ between groups. Reperfusion was successful in 38 out of 49 patients (78%) vs. 113 out of 148 patients (76%) without CCS. In stark contrast, rate of favorable outcome at 90 days differed significantly between groups (22 vs. 44%; p 50% is an independent predictor of poor clinical outcome. This most likely cause is due to poorer collateral flow to the affected tissue. © 2017 S. Karger AG, Basel.

  18. SFRP2 Is Associated with Increased Adiposity and VEGF Expression.

    Science.gov (United States)

    Crowley, Rachel K; O'Reilly, Michael W; Bujalska, Iwona J; Hassan-Smith, Zaki K; Hazlehurst, Jonathan M; Foucault, Danielle R; Stewart, Paul M; Tomlinson, Jeremy W

    The aim of this study was to assess depot-specific expression and secretion of secreted frizzled-related protein 2 (sFRP2) by adipose tissue and its effect on adipocyte biology. We measured serum sFRP2 concentrations in 106 patients in vivo to explore its relationship to fat mass, glycaemia and insulin resistance. Expression of sFRP2 in mouse and human tissues was assessed using polymerase chain reaction and Western blot. Western blot confirmed secretion of sFRP2 by adipose tissue into cell culture medium. Effects of recombinant sFRP2 on lipogenesis and preadipocyte proliferation were measured. Preadipocyte expression of the angiogenic genes vascular endothelial growth factor (VEGF) and nuclear factor of activated T-cells 3 (NFATC3) was measured after recombinant sFRP2 exposure. Complementary clinical studies correlating human serum sFRP2 with age, gender, adiposity and insulin secretion were also performed. sFRP2 messenger RNA (mRNA) was expressed in mouse and human adipose tissue. In humans, sFRP2 mRNA expression was 4.2-fold higher in omental than subcutaneous adipose. Omental adipose tissue secreted 63% more sFRP2 protein than subcutaneous. Treatment with recombinant sFRP2 did not impact on lipogenesis or preadipocyte proliferation but was associated with increased VEGF mRNA expression. In human subjects, circulating insulin levels positively correlated with serum sFRP2, and levels were higher in patients with abnormal glucose tolerance (34.2ng/ml) compared to controls (29.5ng/ml). A positive correlation between sFRP2 and BMI was also observed. Circulating sFRP2 is associated with adipose tissue mass and has a potential role to drive adipose angiogenesis through enhanced VEGF expression.

  19. Corneal avascularity is due to soluble VEGF receptor-1.

    Science.gov (United States)

    Ambati, Balamurali K; Nozaki, Miho; Singh, Nirbhai; Takeda, Atsunobu; Jani, Pooja D; Suthar, Tushar; Albuquerque, Romulo J C; Richter, Elizabeth; Sakurai, Eiji; Newcomb, Michael T; Kleinman, Mark E; Caldwell, Ruth B; Lin, Qing; Ogura, Yuichiro; Orecchia, Angela; Samuelson, Don A; Agnew, Dalen W; St Leger, Judy; Green, W Richard; Mahasreshti, Parameshwar J; Curiel, David T; Kwan, Donna; Marsh, Helene; Ikeda, Sakae; Leiper, Lucy J; Collinson, J Martin; Bogdanovich, Sasha; Khurana, Tejvir S; Shibuya, Masabumi; Baldwin, Megan E; Ferrara, Napoleone; Gerber, Hans-Peter; De Falco, Sandro; Witta, Jassir; Baffi, Judit Z; Raisler, Brian J; Ambati, Jayakrishna

    2006-10-26

    Corneal avascularity-the absence of blood vessels in the cornea-is required for optical clarity and optimal vision, and has led to the cornea being widely used for validating pro- and anti-angiogenic therapeutic strategies for many disorders. But the molecular underpinnings of the avascular phenotype have until now remained obscure and are all the more remarkable given the presence in the cornea of vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, and the proximity of the cornea to vascularized tissues. Here we show that the cornea expresses soluble VEGF receptor-1 (sVEGFR-1; also known as sflt-1) and that suppression of this endogenous VEGF-A trap by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/- mice and Pax6+/- patients with aniridia are deficient in sflt-1, and recombinant sflt-1 administration restores corneal avascularity in corn1 and Pax6+/- mice. Manatees, the only known creatures uniformly to have vascularized corneas, do not express sflt-1, whereas the avascular corneas of dugongs, also members of the order Sirenia, elephants, the closest extant terrestrial phylogenetic relatives of manatees, and other marine mammals (dolphins and whales) contain sflt-1, indicating that it has a crucial, evolutionarily conserved role. The recognition that sflt-1 is essential for preserving the avascular ambit of the cornea can rationally guide its use as a platform for angiogenic modulators, supports its use in treating neovascular diseases, and might provide insight into the immunological privilege of the cornea.

  20. Vitamin D Decreases Serum VEGF Correlating with Clinical Improvement in Vitamin D-Deficient Women with PCOS: A Randomized Placebo-Controlled Trial.

    Science.gov (United States)

    Irani, Mohamad; Seifer, David B; Grazi, Richard V; Irani, Sara; Rosenwaks, Zev; Tal, Reshef

    2017-03-28

    Vascular endothelial growth factor (VEGF) has been suggested to play a role in the pathophysiology of polycystic ovary syndrome (PCOS) and may contribute to increased risk of ovarian hyperstimulation syndrome (OHSS) in affected individuals. Vitamin D (VitD) supplementation improves multiple clinical parameters in VitD-deficient women with PCOS and decreases VEGF levels in several other pathologic conditions. Unveiling the basic mechanisms underlying the beneficial effects of vitamin D on PCOS may enhance our understanding of the pathophysiology of this syndrome. It may also suggest a new treatment for PCOS that can improve it through the same mechanism as vitamin D and can be given regardless of vitamin D levels. Therefore, we aimed to explore the effect of VitD supplementation on serum VEGF levels and assess whether changes in VEGF correlate with an improvement in characteristic clinical abnormalities of PCOS. This is a randomized placebo-controlled trial conducted between October 2013 and March 2015. Sixty-eight VitD-deficient women with PCOS were recruited. Women received either 50,000 IU of oral VitD3 or placebo once weekly for 8 weeks. There was a significant decrease in serum VEGF levels (1106.4 ± 36.5 to 965.3 ± 42.7 pg·mL -1 ; p PCOS. This is a novel molecular explanation for the beneficial effects of VitD treatment. It also suggests the need to investigate a potential role of VitD treatment in reducing the incidence or severity of OHSS in VitD-deficient women with PCOS.

  1. The antihypertension drug doxazosin inhibits tumor growth and angiogenesis by decreasing VEGFR-2/Akt/mTOR signaling and VEGF and HIF-1α expression.

    Science.gov (United States)

    Park, Mi Sun; Kim, Boh-Ram; Dong, Seung Myung; Lee, Seung-Hoon; Kim, Dae-Yong; Rho, Seung Bae

    2014-07-15

    Doxazosin is an α1 adrenergic receptor blocker that also exerts antitumor effects. However, the underlying mechanisms by which it modulates PI3K/Akt intracellular signaling are poorly understood. In this study, we reveal that doxazosin functions as a novel antiangiogenic agent by inhibiting vascular endothelial growth factor (VEGF)-induced cell migration and proliferation. It also inhibited VEGF-induced capillary-like structure tube formation in vitro. Doxazosin inhibited the phosphorylation of VEGF receptor-2 (VEGFR-2) and downstream signaling, including PI3K, Akt, 3-phosphoinositide-dependent protein kinase 1 (PDK1), mammalian target of rapamycin (mTOR), and hypoxia-inducible factor 1 (HIF-1α). However, it had no effect on VEGF-induced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Furthermore, doxazosin reduced tumor growth and suppressed tumor vascularization in a xenograft human ovarian cancer model. These results provide evidence that doxazosin functions in the endothelial cell system to modulate angiogenesis by inhibiting Akt and mTOR phosphorylation and interacting with VEGFR-2.

  2. VEGF production and signaling in Müller glia are critical to modulating vascular function and neuronal integrity in diabetic retinopathy and hypoxic retinal vascular diseases.

    Science.gov (United States)

    Le, Yun-Zheng

    2017-10-01

    Müller glia (MG) are major retinal supporting cells that participate in retinal metabolism, function, maintenance, and protection. During the pathogenesis of diabetic retinopathy (DR), a neurovascular disease and a leading cause of blindness, MG modulate vascular function and neuronal integrity by regulating the production of angiogenic and trophic factors. In this article, I will (1) briefly summarize our work on delineating the role and mechanism of MG-modulated vascular function through the production of vascular endothelial growth factor (VEGF) and on investigating VEGF signaling-mediated MG viability and neural protection in diabetic animal models, (2) explore the relationship among VEGF and neurotrophins in protecting Müller cells in in vitro models of diabetes and hypoxia and its potential implication to neuroprotection in DR and hypoxic retinal diseases, and (3) discuss the relevance of our work to the effectiveness and safety of long-term anti-VEGF therapies, a widely used strategy to combat DR, diabetic macular edema, neovascular age-related macular degeneration, retinopathy of prematurity, and other hypoxic retinal vascular disorders. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Dilated thin-walled blood and lymphatic vessels in human endometrium: a potential role for VEGF-D in progestin-induced break-through bleeding.

    Directory of Open Access Journals (Sweden)

    Jacqueline F Donoghue

    Full Text Available Progestins provide safe, effective and cheap options for contraception as well as the treatment of a variety of gynaecological disorders. Episodes of irregular endometrial bleeding or breakthrough bleeding (BTB are a major unwanted side effect of progestin treatment, such that BTB is the leading cause for discontinued use of an otherwise effective and popular medication. The cellular mechanisms leading to BTB are poorly understood. In this study, we make the novel finding that the large, dilated, thin walled vessels characteristic of human progestin-treated endometrium include both blood and lymphatic vessels. Increased blood and lymphatic vessel diameter are features of VEGF-D action in other tissues and we show by immunolocalisation and Western blotting that stromal cell decidualisation results in a significant increase in VEGF-D protein production, particularly of the proteolytically processed 21 kD form. Using a NOD/scid mouse model with xenografted human endometrium we were able to show that progestin treatment causes decidualisation, VEGF-D production and endometrial vessel dilation. Our results lead to a novel hypothesis to explain BTB, with stromal cell decidualisation rather than progestin treatment per se being the proposed causative event, and VEGF-D being the proposed effector agent.

  4. Avian leukosis virus subgroup J induces VEGF expression via NF-κB/PI3K-dependent IL-6 production.

    Science.gov (United States)

    Gao, Yanni; Zhang, Yao; Yao, Yongxiu; Guan, Xiaolu; Liu, Yongzhen; Qi, Xiaole; Wang, Yongqiang; Liu, Changjun; Zhang, Yanping; Gao, Honglei; Nair, Venugopal; Wang, Xiaomei; Gao, Yulong

    2016-12-06

    Avian leukosis virus subgroup J (ALV-J) is an oncogenic virus causing hemangiomas and myeloid tumors in chickens. Interleukin-6 (IL-6) is a multifunctional pro-inflammatory interleukin involved in many types of cancer. We previously demonstrated that IL-6 expression was induced following ALV-J infection in chickens. The aim of this study is to characterize the mechanism by which ALV-J induces IL-6 expression, and the role of IL-6 in tumor development. Our results demonstrate that ALV-J infection increases IL-6 expression in chicken splenocytes, peripheral blood lymphocytes, and vascular endothelial cells. IL-6 production is induced by the ALV-J envelope protein gp85 and capsid protein p27 via PI3K- and NF-κB-mediated signaling. IL-6 in turn induced expression of vascular endothelial growth factor (VEGF)-A and its receptor, VEGFR-2, in vascular endothelial cells and embryonic vascular tissues. Suppression of IL-6 using siRNA inhibited the ALV-J induced VEGF-A and VEGFR-2 expression in vascular endothelial cells, indicating that the ALV-J-induced VEGF-A/VEGFR-2 expression is mediated by IL-6. As VEGF-A and VEGFR-2 are important factors in oncogenesis, our findings suggest that ALV-J hijacks IL-6 to promote tumorigenesis, and indicate that IL-6 could potentially serve as a therapeutic target in ALV-J infections.

  5. Effects of xuesetong soft capsules on angiogenesis and VEGF mRNA expression in ischemic myocardium in rats with myocardial infarction.

    Science.gov (United States)

    Wang, Zhen-Tao; Zhang, Shu-Juan; Han, Li-Hua; Chai, Song-Bo

    2012-03-01

    To observe the effects of Xuesetong Soft Capsules, Notoginseng total saponin) on angiogenesis and vascular endothelial growth factor (VEGF) mRNA expression in ischemic myocardium of rats with myocardial infarction. The left coronary artery of rats was ligated to establish the animal model of acute myocardial infarction. Rats were randomly divided into Xuesetong Soft Capsule, Shexiangbaoxin Pill (positive control), model (negative control) and sham operation groups. After 6 weeks, microvessel count (MVC), microvessel density (MVD) and VEGF mRNA expression in ischemic myocardium were evaluated. MVC and MVD in the myocardial infarct border area in model, Shexiangbaoxin Pill and Xuesetong Soft Capsule groups significantly increased compared with those of the sham operation group (P myocardial infarct border area in Xuesetong Soft Capsule and Shexiangbaoxin Pill groups significantly increased compared with those of the model group (P 0.05). The model group showed significantly higher VEGF mRNA expression than that in the sham operation group (P 0.05). Xuesetong Soft Capsules promote angiogenesis in ischemic myocardium after myocardial infarction and the mechanism may be associated with VEGF mRNA expression.

  6. Potent small molecule Hedgehog agonists induce VEGF expression in vitro.

    Science.gov (United States)

    Seifert, Katrin; Büttner, Anita; Rigol, Stephan; Eilert, Nicole; Wandel, Elke; Giannis, Athanassios

    2012-11-01

    Here, we describe the synthesis, SAR studies as well as biological investigations of the known Hedgehog signaling agonist SAG and a small library of its analogues. The SAG and its derivatives were analyzed for their potency to activate the expression of the Hh target gene Gli1 in a reporter gene assay. By analyzing SAR important molecular descriptors for Gli1 activation have been identified. SAG as well as compound 10c proven to be potent activators of VEGF expression in cultivated dermal fibroblasts. Importantly and in contrast to SAG, derivative 10c displayed no toxicity in concentrations up to 250 μm. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Independent Directors

    DEFF Research Database (Denmark)

    Ringe, Wolf-Georg

    2013-01-01

    that they did not prevent firms' excessive risk taking; further, these directors sometimes showed serious deficits in understanding the business they were supposed to control, and remained passive in addressing structural problems. A closer look reveals that under the surface of seemingly unanimous consensus......This paper re-evaluates the corporate governance concept of ‘board independence’ against the disappointing experiences during the 2007-08 financial crisis. Independent or outside directors had long been seen as an essential tool to improve the monitoring role of the board. Yet the crisis revealed...... about board independence in Western jurisdictions, a surprising disharmony prevails about the justification, extent and purpose of independence requirements. These considerations lead me to question the benefits of the current system. Instead, this paper proposes a new, ‘functional’ concept of board...

  8. Detection of VEGF-A(xxx)b isoforms in human tissues.

    Science.gov (United States)

    Bates, David O; Mavrou, Athina; Qiu, Yan; Carter, James G; Hamdollah-Zadeh, Maryam; Barratt, Shaney; Gammons, Melissa V; Millar, Ann B; Salmon, Andrew H J; Oltean, Sebastian; Harper, Steven J

    2013-01-01

    Vascular Endothelial Growth Factor-A (VEGF-A) can be generated as multiple isoforms by alternative splicing. Two families of isoforms have been described in humans, pro-angiogenic isoforms typified by VEGF-A165a, and anti-angiogenic isoforms typified by VEGF-A165b. The practical determination of expression levels of alternative isoforms of the same gene may be complicated by experimental protocols that favour one isoform over another, and the use of specific positive and negative controls is essential for the interpretation of findings on expression of the isoforms. Here we address some of the difficulties in experimental design when investigating alternative splicing of VEGF isoforms, and discuss the use of appropriate control paradigms. We demonstrate why use of specific control experiments can prevent assumptions that VEGF-A165b is not present, when in fact it is. We reiterate, and confirm previously published experimental design protocols that demonstrate the importance of using positive controls. These include using known target sequences to show that the experimental conditions are suitable for PCR amplification of VEGF-A165b mRNA for both q-PCR and RT-PCR and to ensure that mispriming does not occur. We also provide evidence that demonstrates that detection of VEGF-A165b protein in mice needs to be tightly controlled to prevent detection of mouse IgG by a secondary antibody. We also show that human VEGF165b protein can be immunoprecipitated from cultured human cells and that immunoprecipitating VEGF-A results in protein that is detected by VEGF-A165b antibody. These findings support the conclusion that more information on the biology of VEGF-A165b isoforms is required, and confirm the importance of the experimental design in such investigations, including the use of specific positive and negative controls.

  9. Evaluation of anticancer peptide VEGF111b secretion in HEK293 human cells

    Directory of Open Access Journals (Sweden)

    Morteza Sadeghi

    2017-04-01

    Full Text Available Background: VEGF111b is a new isoform of vascular endothelial growth factor (VEGF recently considered as a new anticancer drug. The aim of this study was to evaluate the VEGF111b secretion from HEK293 cell wall in order to commercial production of this recombinant factor. Materials and Methods: After the design of VEGF111b sequence using OLIGO software and NCBI gene bank data, it was cloned into the pBUD.cE4.1 vector. The pBUD.VEGF111b recombinant vector was transfected into HEK293 cells using lipofectamine kit. Forty-eight hours after the transfection the production of VEGF111b was estimated by Western blotting and Human anti VEGF antibody. The VEGF111b secretion into cell culture and cell lysate extract was measured using ELISA. Results: The correct cloning of VEGF111b into pBUD.cE4.1vector was confirmed using enzymatic digestion and gel electrophoresis. The observed production of recombinant peptide in HEK293 was confirmed with 12KDa band in cell lysate extract of Western blotting. The ELISA results at 450 nanometer absorbance for cell culture media and cell lysate extract were 19.20±2.81 pg/ml and 32.87±7.42 pg/ml, respectively. However, no VEGF111b expression was observed in negative controls. Conclusion: The findings of this study indicate the powerful ability of transformation and secretion of VEGF111b from HEK293 cell wall to cell culture media with no breaking and proteolytic digestion. It seems that the commercial production and purification of this therapeutic peptide from HEK293 cell culture would be possible with high efficiency.

  10. Epithelial membrane protein 2 controls VEGF expression in ARPE-19 cells.

    Science.gov (United States)

    Morales, Shawn A; Telander, David G; Leon, Deanna; Forward, Krisztina; Braun, Jonathan; Wadehra, Madhuri; Gordon, Lynn K

    2013-03-28

    VEGF production by RPE cells has been shown to be important in regulating aberrant angiogenesis in the retina, which is responsible for multiple types of ocular pathology. EMP2 is highly expressed in the RPE and has been shown to regulate FAK activation, which is implicated in VEGF expression in other cell lines. The purpose of this study was to determine whether EMP2 regulates VEGF expression in the RPE cell line, ARPE-19. ARPE-19 cells were engineered to overexpress EMP2. EMP2 siRNA was used to decrease EMP2 expression. The small molecule inhibitor PP2 was used to inhibit FAK activation. VEGF levels were measured by Western blot and ELISA. Functional differences in secreted VEGF were assayed using HUVEC migration. VEGF expression levels correlated with levels of EMP2. An increase of VEGF by 150% was observed in EMP2 overexpressing cells as compared with ARPE-19 cells. Concordantly, EMP2 knockdown resulted in a 57% decrease in VEGF expression. HUVEC migration (P = 0.01) and vessel tube formation (P < 0.01) were significantly increased when exposed to cell culture supernatants from EMP2 overexpressing cells. This study establishes a novel connection between EMP2 and VEGF and may reflect either a direct effect through the tetraspan web or an indirect change through FAK activation. This connection is functionally significant. In addition to the direct use of anti-VEGF antibodies, modulation of EMP2 with impact on VEGF is potentially a distinct therapeutic target for the treatment of neovascularization associated with retinal diseases that involve pathologic angiogenesis.

  11. Differential responsiveness in VEGF receptor subtypes to hypoxic stress in various tissues of plateau animals.

    Science.gov (United States)

    Xie, Hui-Chun; Li, Jin-Gang; He, Jian-Ping

    2017-05-04

    With hypoxic stress, hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) are elevated and their responses are altered in skeletal muscles of plateau animals [China Qinghai-Tibetan plateau pikas (Ochotona curzoniae)] as compared with control animals [normal lowland Sprague-Dawley (SD) rats]. The results indicate that HIF-1alpha and VEGF are engaged in physiological functions under hypoxic environment. The purpose of the current study was to examine the protein levels of VEGF receptor subtypes (VEGFRs: VEGFR-1, VEGFR-2 and VEGFR-3) in the end organs, namely skeletal muscle, heart and lung in response to hypoxic stress. ELISA and Western blot analysis were employed to determine HIF-1alpha and the protein expression of VEGFRs in control animals and plateau pikas. We further blocked HIF-1alpha signal to determine if HIF-1alpha regulates alternations in VEGFRs in those tissues. We hypothesized that responsiveness of VEGFRs in the major end organs of plateau animals is differential with insult of hypoxic stress and is modulated by low oxygen sensitive HIF-1alpha. Our results show that hypoxic stress induced by exposure of lower O(2) for 6 h significantly increased the levels of VEGFR-2 in skeletal muscle, heart and lung and the increases were amplified in plateau pikas. Our results also demonstrate that hypoxic stress enhanced VEGFR-3 in lungs of plateau animals. Nonetheless, no significant alternations in VEGFR-1 were observed in those tissues with hypoxic stress. Moreover, we observed decreases of VEGFR-2 in skeletal muscle, heart and lung; and decreases of VEGFR-3 in lung following HIF-1alpha inhibition. Overall, our findings suggest that in plateau animals 1) responsiveness of VEGFRs is different under hypoxic environment; 2) amplified VEGFR-2 response appears in skeletal muscle, heart and lung, and enhanced VEGFR-3 response is mainly observed in lung; 3) HIF-1alpha plays a regulatory role in the levels of VEGFRs. Our results

  12. Molecular Analysis of Vascular Endothelial Growth Factor (VEGF) Receptors in EUS-guided Samples Obtained from Patients with Pancreatic Adenocarcinoma.

    Science.gov (United States)

    Costache, Madalin Ionut; Iordache, Sevastita; Costache, Cornelia Alexandra; Dragos, Ene; Dragos, Alexandru; Saftoiu, Adrian

    2017-03-01

    Vascular endothelial growth factor (VEGF) and its receptors (VEGF-R1 and VEGF-R2) are the most important angiogenesis stimulating factors in pancreatic cancer. This study aims to assess VEGF-R1 and VEGF-R2 gene expression in EUS-FNA samples and identify prognostic markers in pancreatic adenocarcinoma. This was a retrospective study of prospectively collected data of 88 consecutive patients, with clinical and imaging suspicion of pancreatic neoplasms, based on samples obtained through endoscopic ultrasound-guided fine needle aspiration (EUS-FNA). EUS had an accuracy of 93.2% for the diagnosis of pancreatic cancer. Based on real-time qPCR analysis, VEGF-R1 and VEGF-R2 expressions were present in 90% and 65% of the analysed malignant samples, respectively; 89% of the patients died during the study, with a median survival rate of only 9 months. The survival was correlated with the initial stage and with the presence of VEGF-R1 and VEGF-R2 gene expression. We found that there are significant correlations between death/survival and T stage, N stage, resectability status, VEGF-R1, VEGF-R2 and VEGF-R1/VEGF-R2 coexpression. Using a Cox model regression our study demonstrates that VEGF-R1/VEGF-R2 coexpression might be considered as a poor prognostic factor in pancreatic cancer. EUS is a very effective technique for the diagnosis and staging of pancreatic adenocarcinoma in patients with clinical and imaging suspicion of pancreatic neoplasm, with an accuracy of 93.2%. Furthermore, the role of molecular analysis of EUS-guided FNA samples was established by the assessment of VEGF-R1, VEGF-R2 gene expression, which might be considered prognostic markers in pancreatic cancer.

  13. Independent preferences

    DEFF Research Database (Denmark)

    Vind, Karl

    1991-01-01

    A simple mathematical result characterizing a subset of a product set is proved and used to obtain additive representations of preferences. The additivity consequences of independence assumptions are obtained for preferences which are not total or transitive. This means that most of the economic...

  14. [Macular Edema in Uveitis - Steroids or VEGF Inhibitors?

    Science.gov (United States)

    Heinz, Carsten; Heiligenhaus, Arnd

    2017-06-09

    Macular edema in uveitis patients is certainly the most frequent complication leading to a permanent and irreversible reduction in vision during the course of the disease. Thanks to optical coherence tomography (OCT) technology and fluorescein angiography (FAG), significantly more macular edemas are detected. Macular edema can be found in various uveitis varieties and can show different clinical patterns. All macular edema should be treated. Macular edema with active inflammation usually reacts very well to general uveitis treatment. In the case of eyes without visible inflammation, however, the response to such therapy is usually less effective. According to the latest treatment recommendations, dexamethasone implants should be used as the first intravitreal therapy. Vascular endothelial growth factor inhibitors (VEGF inhibitors) are second-line treatment regimens. The choice of therapy is, therefore, primarily based on the degree of inflammation and the individual complications, such as glaucoma, lens situation or previous increase in IOP after steroid administration. These individual complications may allow using VEGF inhibitors as first line treatment. An improvement in the macular edema can be achieved with both groups of active substances. Georg Thieme Verlag KG Stuttgart · New York.

  15. Galectin-9 controls CD40 signaling through a Tim-3 independent mechanism and redirects the cytokine profile of pathogenic T cells in autoimmunity.

    Directory of Open Access Journals (Sweden)

    Gisela M Vaitaitis

    Full Text Available While it has long been understood that CD40 plays a critical role in the etiology of autoimmunity, glycobiology is emerging as an important contributor. CD40 signaling is also gaining further interest in transplantation and cancer therapies. Work on CD40 signaling has focused on signaling outcomes and blocking of its ligand, CD154, while little is known about the actual receptor itself and its control. We demonstrated that CD40 is in fact several receptors occurring as constellations of differentially glycosylated forms of the protein that can sometimes form hybrid receptors with other proteins. An enticing area of autoimmunity is differential glycosylation of immune molecules leading to altered signaling. Galectins interact with carbohydrates on proteins to effect such signaling alterations. Studying autoimmune prone NOD and non-autoimmune BALB/c mice, here we reveal that in-vivo CD40 signals alter the glycosylation status of non-autoimmune derived CD4 T cells to resemble that of autoimmune derived CD4 T cells. Galectin-9 interacts with CD40 and, at higher concentrations, prevents CD40 induced proliferative responses of CD4(loCD40(+ effector T cells and induces cell death through a Tim-3 independent mechanism. Interestingly, galectin-9, at lower concentrations, alters the surface expression of CD3, CD4, and TCR, regulating access to those molecules and thereby redirects the inflammatory cytokine phenotype and CD3 induced proliferation of autoimmune CD4(loCD40(+ T cells. Understanding the dynamics of the CD40 receptor(s and the impact of glycosylation status in immunity will gain insight into how to maintain useful CD40 signals while shutting down detrimental ones.

  16. Nitric oxide increases the spontaneous firing rate of rat medial vestibular nucleus neurons in vitro via a cyclic GMP-mediated PKG-independent mechanism.

    Science.gov (United States)

    Podda, Maria Vittoria; Marcocci, Maria Elena; Oggiano, Leonardo; D'Ascenzo, Marcello; Tolu, Eusebio; Palamara, Anna Teresa; Azzena, Gian Battista; Grassi, Claudio

    2004-10-01

    The effects of nitric oxide (NO) on the discharge rate of medial vestibular nucleus neurons (MVNn) were investigated in rat brainstem slices. The NO-donor sodium nitroprusside (SNP, 200 microM) caused a marked enhancement (+36.7%) of MVNn spontaneous firing rate, which was prevented by the NO-scavenger, carboxy-PTIO (300 microM). The SNP effects were not modified (+37.4%) by synaptic uncoupling, suggesting that NO influences intrinsic membrane properties of MVNn rather than the synaptic input they receive. The excitatory action of SNP was virtually abolished by slice pretreatment with the soluble guanylyl cyclase inhibitor, ODQ (10 microM), and it was mimicked (+33.1%) by the cGMP analogue 8-Br-cGMP (400 microM). Protein kinase G (PKG) and cAMP/protein kinase A (PKA) were both excluded as downstream effectors of the NO/cGMP-induced excitation. However, the cyclic nucleotide-gated (CNG) channel blockers, L-cis-diltiazem (LCD, 100 microM) and Sp-8-Br-PET-cGMPS (100 microM), significantly reduced the firing rate increase produced by 8-Br-cGMP. Moreover, LCD alone decreased spontaneous MVNn firing (-19.7%), suggesting that putative CNG channels may contribute to the tonic control of resting MVNn discharge. 8-Br-cAMP (1 mM) also elicited excitatory effects in MVNn (+40.8%), which occluded those induced by 8-Br-cGMP, indicating that the two nucleotides share a common target. Finally, nested-polymerase chain reaction assay revealed the expression of CNG channel alpha subunit transcript in MVNn. Our data provide the first demonstration that NO/cGMP signalling modulates MVNn spontaneous firing through a mechanism that is independent of PKG or PKA and probably involves activation of CNG channels.

  17. beta3-Adrenergic-dependent and -independent mechanisms participate in cold-induced modulation of insulin signal transduction in brown adipose tissue of rats.

    Science.gov (United States)

    Gasparetti, Alessandra L; Alvarez-Rojas, Fernanda; de Araujo, Eliana P; Hirata, Aparecida E; Saad, Mário J A; Velloso, Lício A

    2005-03-01

    During cold exposure, homeothermic animals mobilize glucose with higher efficiency than at thermoneutrality. An interaction between the insulin signal transduction machinery and high sympathetic tonus is thought to play an important role in this phenomenon. In the present study, rats were exposed to cold during 8 days and treated, or not, with a beta3-adrenergic agonist, BRL37344 sodium 4-2-2-(3-chlorophenyl)-2-hydroxyethyl amino propyl phenoxy-acetic acid sodium (BRL37344), or antagonist, SR59230A 3-(2-ethylphenoxy)-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-(2S)-2-propanol oxalate (SR59230A), to evaluate the cross-talk between insulin and beta3-adrenergic intracellular signaling in brown adipose tissue. The drugs did not modify food ingestion, body temperature, and body weight in control and cold-exposed rats. Treatment of control rats with BRL37344 led to higher insulin-induced tyrosine phosphorylation of the insulin receptors, insulin receptor substrate (IRS)-1 and ERK, higher insulin-induced IRS-1/PI3-kinase association, and higher [Ser(473)] phosphorylation of Akt. Cold exposure alone promoted higher insulin-induced tyrosine phosphorylation of the insulin receptors, IRS-1, IRS-2, and ERK, and higher insulin-induced IRS-1 and IRS-2/PI3-kinase association. Except for the regulation of ERK, SR59230A abolished all the cold-induced effects upon the insulin signal transduction pathway. However, this antagonist only partially inhibited the cold-induced increase of glucose uptake. Thus, the sympathetic tonus generated during cold-exposure acts, in brown adipose tissue, through the beta3-adrenergic receptor and modulates insulin signal transduction, with the exception of ERK. However, insulin-independent mechanisms other than beta3-adrenergic activation participate in cold-induced glucose uptake in brown adipose tissue of rats.

  18. Drosophila TG-A transglutaminase is secreted via an unconventional Golgi-independent mechanism involving exosomes and two types of fatty acylations.

    Science.gov (United States)

    Shibata, Toshio; Hadano, Jinki; Kawasaki, Daichi; Dong, Xiaoqing; Kawabata, Shun-Ichiro

    2017-06-23

    Transglutaminases (TGs) play essential intracellular and extracellular roles by covalently cross-linking many proteins. Drosophila TG is encoded by one gene and has two alternative splicing-derived isoforms, TG-A and TG-B, which contain distinct N-terminal 46- and 38-amino acid sequences, respectively. The TGs identified to date do not have a typical endoplasmic reticulum (ER)-signal peptide, and the molecular mechanisms of their secretion under physiologic conditions are unclear. Immunocytochemistry revealed that TG-A localizes to multivesicular-like structures, whereas TG-B localizes to the cytosol. We also found that TG-A, but not TG-B, was modified concomitantly by N-myristoylation and S-palmitoylation, and N-myristoylation was a pre-requisite for S-palmitoylation. Moreover, TG-A, but not TG-B, was secreted in response to calcium signaling induced by Ca(2+) ionophores and uracil, a pathogenic bacteria-derived substance. Brefeldin A and monensin, inhibitors of the ER/Golgi-mediated conventional pathway, did not suppress TG-A secretion, whereas inhibition of S-palmitoylation by 2-bromopalmitate blocked TG-A secretion. Ultracentrifugation, electron microscopy analyses, and treatments with inhibitors of multivesicular body formation revealed that TG-A was secreted via exosomes together with co-transfected mammalian CD63, an exosomal marker, and the secreted TG-A was taken up by other cells. The 8-residue N-terminal fragment of TG-A containing the fatty acylation sites was both necessary and sufficient for the exosome-dependent secretion of TG-A. In conclusion, TG-A is secreted through an unconventional ER/Golgi-independent pathway involving two types of fatty acylations and exosomes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Transcriptional activation of the Axl and PDGFR-α by c-Met through a ras- and Src-independent mechanism in human bladder cancer

    Directory of Open Access Journals (Sweden)

    Tseng Vincent S

    2011-04-01

    Full Text Available Abstract Background A cross-talk between different receptor t