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Sample records for stromal metalloproteases expression

  1. COMPARATIVE ANALYSIS AND CLINICAL VALUE OF THE EXPRESSION OF METALLOPROTEASES AND THEIR INHIBITORS BY INTRATUMOR STROMAL MONONUCLEAR INFLAMMATORY CELLS AND THOSE AT THE INVASIVE FRONT OF BREAST CARCINOMAS

    OpenAIRE

    Gonzalez , Luis O; Gonzalez-Reyes , Salome; Marin , Laura; Gonzalez , Lucia; Gonzales , José-Manuel; Lamelas , M Luz; Merino , Antonio; Pidal , Ivan; Alvarez , Elena; Andicoechea , Alejandro; Del Casar , José M; Vizoso , Francisco J

    2010-01-01

    Abstract Aims: Matrix metalloproteases (MMPs) and their inhibitors (TIMPs) play an essential role in the degradation of stromal connective tissue and basement membrane components. The dynamic analysis of these components might help predict tumor agressiveness. Methods and Results: An immunohistochemical study was performed using tissue arrays and specific antibodies against MMPs -1, 2, 7, 9, 11, 13, 14, and TIMPs -1, 2 and 3. More than 5,000 determinations on cancer specimens ...

  2. Efficient Extracellular Expression of Metalloprotease for Z-Aspartame Synthesis.

    Science.gov (United States)

    Zhu, Fucheng; Liu, Feng; Wu, Bin; He, Bingfang

    2016-12-28

    Metalloprotease PT121 and its mutant Y114S (Tyr114 was substituted to Ser) are effective catalysts for the synthesis of Z-aspartame (Z-APM). This study presents the selection of a suitable signal peptide for improving expression and extracellular secretion of proteases PT121 and Y114S by Escherichia coli. Co-inducers containing IPTG and arabinose were used to promote protease production and cell growth. Under optimal conditions, the expression levels of PT121 and Y114S reached >500 mg/L, and the extracellular activity of PT121/Y114S accounted for 87/82% of the total activity of proteases. Surprisingly, purer protein was obtained in the supernatant, because arabinose reduced cell membrane permeability, avoiding cell lysis. Comparison of Z-APM synthesis and caseinolysis between proteases PT121 and Y114S showed that mutant Y114S presented remarkably higher activity of Z-APM synthesis and considerably lower activity of caseinolysis. The significant difference in substrate specificity renders these enzymes promising biocatalysts.

  3. Regulation of Metalloprotease Gene Expression in Vibrio vulnificus by a Vibrio harveyi LuxR Homologue

    Science.gov (United States)

    Shao, Chung-Ping; Hor, Lien-I

    2001-01-01

    Expression of the Vibrio vulnificus metalloprotease gene, vvp, was turned up rapidly when bacterial growth reached the late log phase. A similar pattern of expression has been found in the metalloprotease gene of Vibrio cholerae, and this has been shown to be regulated by a Vibrio harveyi LuxR-like transcriptional activator. To find out whether a LuxR homologue exists in V. vulnificus, a gene library of this organism was screened by colony hybridization using a probe derived from a sequence that is conserved in various luxR-like genes of vibrios. A gene containing a 618-bp open reading frame was identified and found to be identical to the smcR gene of V. vulnificus reported previously. An isogenic SmcR-deficient (RD) mutant was further constructed by an in vivo allelic exchange technique. This mutant exhibited an extremely low level of vvp transcription compared with that of the parent strain. On the other hand, the cytolysin gene, vvhA, was expressed at a higher level in the RD mutant than in the parent strain during the log phase of growth. These data suggested that SmcR might not only be a positive regulator of the protease gene but might also be involved in negative regulation of the cytolysin gene. Virulence of the RD mutant in either normal or iron-overloaded mice challenged by intraperitoneal injection was comparable to that of the parent strain, indicating that SmcR is not required for V. vulnificus virulence in mice. PMID:11157950

  4. Active matrix metalloproteases are expressed early on and are high during the Barrett's esophagus malignancy sequence

    NARCIS (Netherlands)

    Davelaar, Akueni L.; Straub, Daniëlle; Buttar, Navtej S.; Fockens, Paul; Krishnadath, Kausilia K.

    2015-01-01

    Objective. Molecular processes underlying Barrett's malignant development are poorly understood. Matrix metalloproteases (MMPs) are enzymes involved in inflammation, tissue remodeling, and malignant development. Therefore, active MMPs may have a role in early metaplasia development and Barrett's

  5. Higher Expression of Epidermal Growth Factor Receptor Is Associated with Extracellular Matrix Metalloprotease Inducer in Colorectal Adenocarcinoma: Tissue Microarray Analysis of Immunostaining Score with Clinicopathological Parameters

    Directory of Open Access Journals (Sweden)

    Jong-Shiaw Jin

    2006-01-01

    Full Text Available Aim: Extracellular matrix metalloprotease inducer (EMMPRIN expression was demonstrated in several cancers, but its expression profile in colorectal cancers remains unclear. Epidermal growth factor receptor (EGFR was reported to regulate EMMPRIN expression in human epithelial cancers. Our purpose was to determine EMMPRIN expression and its relationship with EGFR in colorectal cancers.

  6. Stromal matrix metalloprotease-13 knockout alters Collagen I structure at the tumor-host interface and increases lung metastasis of C57BL/6 syngeneic E0771 mammary tumor cells

    International Nuclear Information System (INIS)

    Perry, Seth W; Schueckler, Jill M; Burke, Kathleen; Arcuri, Giuseppe L; Brown, Edward B

    2013-01-01

    Matrix metalloproteases and collagen are key participants in breast cancer, but their precise roles in cancer etiology and progression remain unclear. MMP13 helps regulate collagen structure and has been ascribed largely harmful roles in cancer, but some studies demonstrate that MMP13 may also protect against tumor pathology. Other studies indicate that collagen’s organizational patterns at the breast tumor-host interface influence metastatic potential. Therefore we investigated how MMP13 modulates collagen I, a principal collagen subtype in breast tissue, and affects tumor pathology and metastasis in a mouse model of breast cancer. Tumors were implanted into murine mammary tissues, and their growth analyzed in Wildtype and MMP13 KO mice. Following extraction, tumors were analyzed for collagen I levels and collagen I macro- and micro-structural properties at the tumor-host boundary using immunocytochemistry and two-photon and second harmonic generation microscopy. Lungs were analyzed for metastases counts, to correlate collagen I changes with a clinically significant functional parameter. Statistical analyses were performed by t-test, analysis of variance, or Wilcoxon-Mann–Whitney tests as appropriate. We found that genetic ablation of host stromal MMP13 led to: 1. Increased mammary tumor collagen I content, 2. Marked changes in collagen I spatial organization, and 3. Altered collagen I microstructure at the tumor-host boundary, as well as 4. Increased metastasis from the primary mammary tumor to lungs. These results implicate host MMP13 as a key regulator of collagen I structure and metastasis in mammary tumors, thus making it an attractive potential therapeutic target by which we might alter metastatic potential, one of the chief determinants of clinical outcome in breast cancer. In addition to identifying stromal MMP13 is an important regulator of the tumor microenvironment and metastasis, these results also suggest that stromal MMP13 may protect against

  7. Stromal matrix metalloprotease-13 knockout alters Collagen I structure at the tumor-host interface and increases lung metastasis of C57BL/6 syngeneic E0771 mammary tumor cells.

    Science.gov (United States)

    Perry, Seth W; Schueckler, Jill M; Burke, Kathleen; Arcuri, Giuseppe L; Brown, Edward B

    2013-09-05

    Matrix metalloproteases and collagen are key participants in breast cancer, but their precise roles in cancer etiology and progression remain unclear. MMP13 helps regulate collagen structure and has been ascribed largely harmful roles in cancer, but some studies demonstrate that MMP13 may also protect against tumor pathology. Other studies indicate that collagen's organizational patterns at the breast tumor-host interface influence metastatic potential. Therefore we investigated how MMP13 modulates collagen I, a principal collagen subtype in breast tissue, and affects tumor pathology and metastasis in a mouse model of breast cancer. Tumors were implanted into murine mammary tissues, and their growth analyzed in Wildtype and MMP13 KO mice. Following extraction, tumors were analyzed for collagen I levels and collagen I macro- and micro-structural properties at the tumor-host boundary using immunocytochemistry and two-photon and second harmonic generation microscopy. Lungs were analyzed for metastases counts, to correlate collagen I changes with a clinically significant functional parameter. Statistical analyses were performed by t-test, analysis of variance, or Wilcoxon-Mann-Whitney tests as appropriate. We found that genetic ablation of host stromal MMP13 led to: 1. Increased mammary tumor collagen I content, 2. Marked changes in collagen I spatial organization, and 3. Altered collagen I microstructure at the tumor-host boundary, as well as 4. Increased metastasis from the primary mammary tumor to lungs. These results implicate host MMP13 as a key regulator of collagen I structure and metastasis in mammary tumors, thus making it an attractive potential therapeutic target by which we might alter metastatic potential, one of the chief determinants of clinical outcome in breast cancer. In addition to identifying stromal MMP13 is an important regulator of the tumor microenvironment and metastasis, these results also suggest that stromal MMP13 may protect against breast

  8. Expression and immunological cross-reactivity of LALP3, a novel astacin-like metalloprotease from brown spider (Loxosceles intermedia) venom.

    Science.gov (United States)

    Morgon, Adriano M; Belisario-Ferrari, Matheus R; Trevisan-Silva, Dilza; Meissner, Gabriel O; Vuitika, Larissa; Marin, Brenda; Tashima, Alexandre K; Gremski, Luiza H; Gremski, Waldemiro; Senff-Ribeiro, Andrea; Veiga, Silvio S; Chaim, Olga M

    2016-01-01

    Loxosceles spiders' venom comprises a complex mixture of biologically active toxins, mostly consisting of low molecular mass components (2-40 kDa). Amongst, isoforms of astacin-like metalloproteases were identified through transcriptome and proteome analyses. Only LALP1 (Loxosceles Astacin-Like protease 1) has been characterized. Herein, we characterized LALP3 as a novel recombinant astacin-like metalloprotease isoform from Loxosceles intermedia venom. LALP3 cDNA was cloned in pET-SUMO vector, and its soluble heterologous expression was performed using a SUMO tag added to LALP3 to achieve solubility in Escherichia coli SHuffle T7 Express LysY cells, which express the disulfide bond isomerase DsbC. Protein purification was conducted by Ni-NTA Agarose resin and assayed for purity by SDS-PAGE under reducing conditions. Immunoblotting analyses were performed with specific antibodies recognizing LALP1 and whole venom. Western blotting showed linear epitopes from recombinant LALP3 that cross-reacted with LALP1, and dot blotting revealed conformational epitopes with native venom astacins. Mass spectrometry analysis revealed that the recombinant expressed protein is an astacin-like metalloprotease from L. intermedia venom. Furthermore, molecular modeling of LALP3 revealed that this isoform contains the zinc binding and Met-turn motifs, forming the active site, as has been observed in astacins. These data confirmed that LALP3, which was successfully obtained by heterologous expression using a prokaryote system, is a new astacin-like metalloprotease isoform present in L. intermedia venom. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  9. CpxR-Dependent Thermoregulation of Serratia marcescens PrtA Metalloprotease Expression and Its Contribution to Bacterial Biofilm Formation.

    Science.gov (United States)

    Bruna, Roberto E; Molino, María Victoria; Lazzaro, Martina; Mariscotti, Javier F; García Véscovi, Eleonora

    2018-04-15

    PrtA is the major secreted metalloprotease of Serratia marcescens Previous reports implicate PrtA in the pathogenic capacity of this bacterium. PrtA is also clinically used as a potent analgesic and anti-inflammatory drug, and its catalytic properties attract industrial interest. Comparatively, there is scarce knowledge about the mechanisms that physiologically govern PrtA expression in Serratia In this work, we demonstrate that PrtA production is derepressed when the bacterial growth temperature decreases from 37°C to 30°C. We show that this thermoregulation occurs at the transcriptional level. We determined that upstream of prtA , there is a conserved motif that is directly recognized by the CpxR transcriptional regulator. This feature is found along Serratia strains irrespective of their isolation source, suggesting an evolutionary conservation of CpxR-dependent regulation of PrtA expression. We found that in S. marcescen s, the CpxAR system is more active at 37°C than at 30°C. In good agreement with these results, in a cpxR mutant background, prtA is derepressed at 37°C, while overexpression of the NlpE lipoprotein, a well-known CpxAR-inducing condition, inhibits PrtA expression, suggesting that the levels of the activated form of CpxR are increased at 37°C over those at 30°C. In addition, we establish that PrtA is involved in the ability of S. marcescens to develop biofilm. In accordance, CpxR influences the biofilm phenotype only when bacteria are grown at 37°C. In sum, our findings shed light on regulatory mechanisms that fine-tune PrtA expression and reveal a novel role for PrtA in the lifestyle of S. marcescens IMPORTANCE We demonstrate that S. marcescens metalloprotease PrtA expression is transcriptionally thermoregulated. While strongly activated below 30°C, its expression is downregulated at 37°C. We found that in S. marcescens , the CpxAR signal transduction system, which responds to envelope stress and bacterial surface adhesion, is

  10. Androgen Receptor Expression in Epithelial and Stromal Cells of Prostatic Carcinoma and Benign Prostatic Hyperplasia.

    Science.gov (United States)

    Filipovski, Vanja; Kubelka-Sabit, Katerina; Jasar, Dzengis; Janevska, Vesna

    2017-08-15

    Prostatic carcinoma (PCa) derives from prostatic epithelial cells. However stromal microenvironment, associated with malignant epithelium, also plays a role in prostatic carcinogenesis. Alterations in prostatic stromal cells contribute to the loss of growth control in epithelial cells that lead to progression of PCa. To analyse the differences between Androgen Receptor (AR) expression in both epithelial and stromal cells in PCa and the surrounding benign prostatic hyperplasia (BPH) and to compare the results with tumour grade. Samples from 70 cases of radical prostatectomy specimens were used. The expression and intensity of the signal for AR was analysed in the epithelial and stromal cells of PCa and BPH, and the data was quantified using histological score (H-score). AR showed significantly lower expression in both epithelial and stromal cells of PCa compared to BPH. In PCa a significant positive correlation of AR expression was found between stromal and epithelial cells of PCa. AR expression showed a correlation between the stromal cells of PCa and tumour grade. AR expression is reduced in epithelial and stromal cells of PCa. Expression of AR in stromal cells of PCa significantly correlates with tumour grade.

  11. Differential gene expression by 1,25(OH)2D3 in an endometriosis stromal cell line.

    Science.gov (United States)

    Ingles, Sue Ann; Wu, Liang; Liu, Benjamin T; Chen, Yibu; Wang, Chun-Yeh; Templeman, Claire; Brueggmann, Doerthe

    2017-10-01

    Endometriosis is a common female reproductive disease characterized by invasion of endometrial cells into other organs, frequently causing pelvic pain and infertility. Alterations of the vitamin D system have been linked to endometriosis incidence and severity. To shed light on the potential mechanism for these associations, we examined the effects of 1,25(OH) 2 D 3 on gene expression in endometriosis cells. Stromal cell lines derived from endometriosis tissue were treated with 1,25(OH) 2 D 3 , and RNA-seq was used to identify genes differentially expressed between treated and untreated cells. Gene ontology and pathway analyses were carried out using Partek Flow and Ingenuity software suites, respectively. We identified 1627 genes that were differentially expressed (886 down-regulated and 741 up-regulated) by 1,25(OH) 2 D 3 . Only one gene, CYP24A1, was strongly up-regulated (369-fold). Many genes were strongly down-regulated. 1,25(OH) 2 D 3 treatment down-regulated several genetic pathways related to neuroangiogenesis, cellular motility, and invasion, including pathways for axonal guidance, Rho GDP signaling, and matrix metalloprotease inhibition. These findings support a role for vitamin D in the pathophysiology of endometriosis, and provide new targets for investigation into possible causes and treatments. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Stromal cell markers are differentially expressed in the synovial tissue of patients with early arthritis

    NARCIS (Netherlands)

    Choi, Ivy Y.; Karpus, Olga N.; Turner, Jason D.; Hardie, Debbie; Marshall, Jennifer L.; de Hair, Maria J. H.; Maijer, Karen I.; Tak, Paul P.; Raza, Karim; Hamann, Jörg; Buckley, Christopher D.; Gerlag, Danielle M.; Filer, Andrew

    2017-01-01

    Previous studies have shown increased expression of stromal markers in synovial tissue (ST) of patients with established rheumatoid arthritis (RA). Here, ST expression of stromal markers in early arthritis in relationship to diagnosis and prognostic outcome was studied. ST from 56 patients included

  13. Stromal cells expressing hedgehog-interacting protein regulate the proliferation of myeloid neoplasms

    International Nuclear Information System (INIS)

    Kobune, M; Iyama, S; Kikuchi, S; Horiguchi, H; Sato, T; Murase, K; Kawano, Y; Takada, K; Ono, K; Kamihara, Y; Hayashi, T; Miyanishi, K; Sato, Y; Takimoto, R; Kato, J

    2012-01-01

    Aberrant reactivation of hedgehog (Hh) signaling has been described in a wide variety of human cancers including cancer stem cells. However, involvement of the Hh-signaling system in the bone marrow (BM) microenvironment during the development of myeloid neoplasms is unknown. In this study, we assessed the expression of Hh-related genes in primary human CD34 + cells, CD34 + blastic cells and BM stromal cells. Both Indian Hh (Ihh) and its signal transducer, smoothened (SMO), were expressed in CD34 + acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS)-derived cells. However, Ihh expression was relatively low in BM stromal cells. Remarkably, expression of the intrinsic Hh-signaling inhibitor, human Hh-interacting protein (HHIP) in AML/MDS-derived stromal cells was markedly lower than in healthy donor-derived stromal cells. Moreover, HHIP expression levels in BM stromal cells highly correlated with their supporting activity for SMO + leukemic cells. Knockdown of HHIP gene in stromal cells increased their supporting activity although control cells marginally supported SMO + leukemic cell proliferation. The demethylating agent, 5-aza-2′-deoxycytidine rescued HHIP expression via demethylation of HHIP gene and reduced the leukemic cell-supporting activity of AML/MDS-derived stromal cells. This indicates that suppression of stromal HHIP could be associated with the proliferation of AML/MDS cells

  14. Prostate stromal cells express the progesterone receptor to control cancer cell mobility.

    Science.gov (United States)

    Yu, Yue; Lee, Jennifer Suehyun; Xie, Ning; Li, Estelle; Hurtado-Coll, Antonio; Fazli, Ladan; Cox, Michael; Plymate, Stephen; Gleave, Martin; Dong, Xuesen

    2014-01-01

    Reciprocal interactions between epithelium and stroma play vital roles for prostate cancer development and progression. Enhanced secretions of cytokines and growth factors by cancer associated fibroblasts in prostate tumors create a favorable microenvironment for cancer cells to grow and metastasize. Our previous work showed that the progesterone receptor (PR) was expressed specifically in prostate stromal fibroblasts and smooth muscle cells. However, the expression levels of PR and its impact to tumor microenvironment in prostate tumors are poorly understood. Immunohistochemistry assays are applied to human prostate tissue biopsies. Cell migration, invasion and proliferation assays are performed using human prostate cells. Real-time PCR and ELISA are applied to measure gene expression at molecular levels. Immunohistochemistry assays showed that PR protein levels were decreased in cancer associated stroma when compared with paired normal prostate stroma. Using in vitro prostate stromal cell models, we showed that conditioned media collected from PR positive stromal cells inhibited prostate cancer cell migration and invasion, but had minor suppressive impacts on cancer cell proliferation. PR suppressed the secretion of stromal derived factor-1 (SDF-1) and interlukin-6 (IL-6) by stromal cells independent to PR ligands. Blocking PR expression by siRNA or supplementation of exogenous SDF-1 or IL-6 to conditioned media from PR positive stromal cells counteracted the inhibitory effects of PR to cancer cell migration and invasion. Decreased expression of the PR in cancer associated stroma may contribute to the elevated SDF-1 and IL-6 levels in prostate tumors and enhance prostate tumor progression.

  15. Matrix metalloprotease-3 expression in the medial plica and pannus-like tissue in knees from patients with medial compartment osteoarthritis.

    Science.gov (United States)

    Wang, Hwai-Shi; Kuo, Pei-Yin; Yang, Chih-Chang; Lyu, Shaw-Ruey

    2011-03-01

    The severity of cartilage degeneration is positively correlated with the severity of the pathologic change of medial plica. However, knowledge of the pathogenic mechanisms and the impact of plica on cartilage destruction is limited. The aim of the present study was therefore to investigate matrix metalloprotease-3 (MMP-3) expression in the plica isolated from patients with medial compartment osteoarthritis of the knee. Immunohistochemistry showed that MMP-3 was highly expressed in pannus-like tissue and the plica. Western blotting of culture supernatants showed that interleukin-1β (IL-1β) treatment induced MMP-3 release by cells isolated from pannus tissue or the plica. Furthermore, reverse transcriptase polymerase chain reaction and real-time polymerase chain reaction analysis showed that MMP-3 mRNA levels were increased after IL-1β treatment of the cultured cells. MMP-3 and IL-1β mRNAs were expressed in the plica and pannus-like tissue, with MMP-3 mRNA being expressed at significantly higher levels in the plica than in normal synovial membrane and highly expressed in the plica at different stages in osteoarthritis (OA) patients. Pannus-like tissue and the plica express IL-1β and MMP-3. Moreover, MMP-3 mRNA and protein expression in the plica may contribute to the pathogenesis of OA. © 2011 Blackwell Publishing Limited.

  16. Expression of tyrosine kinase gene in mouse thymic stromal cells

    NARCIS (Netherlands)

    Rinke de Wit, T. F.; Izon, D. J.; Revilla, C.; Oosterwegel, M.; Bakker, A. Q.; van Ewijk, W.; Kruisbeek, A. M.

    1996-01-01

    Amongst the most important signal transduction molecules involved in regulating growth and differentiation are the protein tyrosine kinases (PTK). Since T cell development is a consequence of interactions between thymic stromal cells (TSC) and thymocytes, identification of the PTK in both

  17. Bone marrow stromal elements in murine leukemia; Decreased CSF-producing fibroblasts and normal IL-1 expression by macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Ben-Ishay, Z [Laboratory of Experimental Hematology, Department of Anatomy and Embryology, Hebrew University-Hadassah Medical School (Israel); Barak, V [Laboratory of Immunology, Department of Oncology, Hadassah University Hospital (Israel); Shoshan, S [Faculty of Dental Medicine, Connective Tissue Research Laboratory, Hebrew University, Jerusalem (Israel); Prindull, G [Department of Pediatrics, University of Gottingen, Gottingen (Germany, F.R.)

    1990-01-01

    A study of bone marrow stromal elements in murine acute myeloid leukemia (AML) was carried out. Our previous studies had indicated marrow stromal deficiency in murine AML. In the current investigation, separate stromal cells were cultured and the results obtained have shown that, while marrow stromal macrophages are normal in leukemia and express adequate amounts of IL-1, the fibroblasts are markedly reduced. However, if sufficient fibroblasts are pooled in vitro, they produce adequate amounts of CSF. Test of TNF{alpha} in leukemic cells CM, as possible cause of marrow stromal inhibition in leukemia, had not disclosed this cytokine. Further, it was observed that total body lethal irradiation of leukemic mice aggravates the stromal deficiency, confirming results of our previous investigations. It is concluded that bone marrow stromal deficiency in murine AML is due to decreased fibroblasts and, implicity, reduced CSF production. (author).

  18. Prostate stromal cells express the progesterone receptor to control cancer cell mobility.

    Directory of Open Access Journals (Sweden)

    Yue Yu

    Full Text Available Reciprocal interactions between epithelium and stroma play vital roles for prostate cancer development and progression. Enhanced secretions of cytokines and growth factors by cancer associated fibroblasts in prostate tumors create a favorable microenvironment for cancer cells to grow and metastasize. Our previous work showed that the progesterone receptor (PR was expressed specifically in prostate stromal fibroblasts and smooth muscle cells. However, the expression levels of PR and its impact to tumor microenvironment in prostate tumors are poorly understood.Immunohistochemistry assays are applied to human prostate tissue biopsies. Cell migration, invasion and proliferation assays are performed using human prostate cells. Real-time PCR and ELISA are applied to measure gene expression at molecular levels.Immunohistochemistry assays showed that PR protein levels were decreased in cancer associated stroma when compared with paired normal prostate stroma. Using in vitro prostate stromal cell models, we showed that conditioned media collected from PR positive stromal cells inhibited prostate cancer cell migration and invasion, but had minor suppressive impacts on cancer cell proliferation. PR suppressed the secretion of stromal derived factor-1 (SDF-1 and interlukin-6 (IL-6 by stromal cells independent to PR ligands. Blocking PR expression by siRNA or supplementation of exogenous SDF-1 or IL-6 to conditioned media from PR positive stromal cells counteracted the inhibitory effects of PR to cancer cell migration and invasion.Decreased expression of the PR in cancer associated stroma may contribute to the elevated SDF-1 and IL-6 levels in prostate tumors and enhance prostate tumor progression.

  19. Functional features of gene expression profiles differentiating gastrointestinal stromal tumours according to KIT mutations and expression

    International Nuclear Information System (INIS)

    Ostrowski, Jerzy; Dobosz, Anna Jerzak Vel; Jarosz, Dorota; Ruka, Wlodzimierz; Wyrwicz, Lucjan S; Polkowski, Marcin; Paziewska, Agnieszka; Skrzypczak, Magdalena; Goryca, Krzysztof; Rubel, Tymon; Kokoszyñska, Katarzyna; Rutkowski, Piotr; Nowecki, Zbigniew I

    2009-01-01

    Gastrointestinal stromal tumours (GISTs) represent a heterogeneous group of tumours of mesenchymal origin characterized by gain-of-function mutations in KIT or PDGFRA of the type III receptor tyrosine kinase family. Although mutations in either receptor are thought to drive an early oncogenic event through similar pathways, two previous studies reported the mutation-specific gene expression profiles. However, their further conclusions were rather discordant. To clarify the molecular characteristics of differentially expressed genes according to GIST receptor mutations, we combined microarray-based analysis with detailed functional annotations. Total RNA was isolated from 29 frozen gastric GISTs and processed for hybridization on GENECHIP ® HG-U133 Plus 2.0 microarrays (Affymetrix). KIT and PDGFRA were analyzed by sequencing, while related mRNA levels were analyzed by quantitative RT-PCR. Fifteen and eleven tumours possessed mutations in KIT and PDGFRA, respectively; no mutation was found in three tumours. Gene expression analysis identified no discriminative profiles associated with clinical or pathological parameters, even though expression of hundreds of genes differentiated tumour receptor mutation and expression status. Functional features of genes differentially expressed between the two groups of GISTs suggested alterations in angiogenesis and G-protein-related and calcium signalling. Our study has identified novel molecular elements likely to be involved in receptor-dependent GIST development and allowed confirmation of previously published results. These elements may be potential therapeutic targets and novel markers of KIT mutation status

  20. Pilot Comparison of Stromal Gene Expression among Normal Prostate Tissues and Primary Prostate Cancer Tissues in White and Black Men

    National Research Council Canada - National Science Library

    Bova, G. S

    2006-01-01

    ..., and expression analysis of prostate-stroma specific cells in normal and cancerous prostates, and aims to develop preliminary data sufficient to identify potential differences in stromal RNA expression in normal and cancerous...

  1. Stromal Expression of Hypoxia Regulated Proteins Is an Adverse Prognostic Factor in Colorectal Carcinomas

    Directory of Open Access Journals (Sweden)

    Arjen H. G. Cleven

    2007-01-01

    Full Text Available Background: Hypoxia modifies the phenotype of tumors in a way that promotes tumor aggressiveness and resistance towards chemotherapy and radiotherapy. However, the expression and influence of hypoxia-regulated proteins on tumor biology are not well characterized in colorectal tumors. We studied the role of protein expression of hypoxia-inducible factor (HIF-1α, HIF-2α, carbonic anhydrase 9 (CA9 and glucose transporter 1 (GLUT1 in patients with colorectal adenocarcinomas. Methods: Expression of HIF-1α, HIF-2α, CA9 and GLUT1 was quantified by immunohistochemistry in 133 colorectal adenocarcinomas. The expression of hypoxia markers was correlated with clinicopathological variables and overall patient survival. Results: Expression of these hypoxia markers was detected in the epithelial compartment of the tumor cells as well as in tumor-associated stromal cells. Although tumor cells frequently showed expression of one or more of the investigated hypoxia markers, no correlation among these markers or with clinical response was found. However, within the tumor stroma, positive correlations between the hypoxia markers HIF-2α, CA9 and GLUT1 were observed. Furthermore expression of HIF-2α and CA9 in tumor-associated stroma were both associated with a significantly reduced overall survival. In the Cox proportional hazard model, stromal HIF-2α expression was an independent prognostic factor for survival. Conclusion: These observations show, that expression of hypoxia regulated proteins in tumor-associated stromal cells, as opposed to their expression in epithelial tumor cells, is associated with poor outcome in colorectal cancer. This study suggests that tumor hypoxia may influence tumor-associated stromal cells in a way that ultimately contributes to patient prognosis.

  2. IL-8 and MCP Gene Expression and Production by LPS-Stimulated Human Corneal Stromal Cells

    Directory of Open Access Journals (Sweden)

    Roni M. Shtein

    2012-01-01

    Full Text Available Purpose. To determine time course of effect of lipopolysaccharide (LPS on production of interleukin-8 (IL-8 and monocyte chemotactic protein (MCP by cultured human corneal stromal cells. Methods. Human corneal stromal cells were harvested from donor corneal specimens, and fourth to sixth passaged cells were used. Cell cultures were stimulated with LPS for 2, 4, 8, and 24 hours. Northern blot analysis of IL-8 and MCP gene expression and ELISA for IL-8 and MCP secretion were performed. ELISA results were analyzed for statistical significance using two-tailed Student's t-test. Results. Northern blot analysis demonstrated significantly increased IL-8 and MCP gene expression after 4 and 8 hours of exposure to LPS. ELISA for secreted IL-8 and MCP demonstrated statistically significant increases (P<0.05 after corneal stromal cell stimulation with LPS. Conclusions. This paper suggests that human corneal stromal cells may participate in corneal inflammation by secreting potent leukocyte chemotactic and activating proteins in a time-dependent manner when exposed to LPS.

  3. Ursolic acid reduces the metalloprotease/anti-metalloprotease imbalance in cerebral ischemia and reperfusion injury.

    Science.gov (United States)

    Wang, Yanzhe; He, Zhiyi; Deng, Shumin

    2016-01-01

    Activators of PPARs, particularly PPARγ, may be effective neuroprotective drugs against inflammatory responses in cerebral ischemia and reperfusion injury. Ursolic acid (UA) may act as a PPARγ agonist and serve as an anti-inflammatory agent. In this study, we used a rat middle cerebral artery occlusion and reperfusion model to examine how UA acts as a neuroprotective agent to modulate the metalloprotease/anti-metalloprotease balance. The middle cerebral artery occlusion and reperfusion model (occlusion for 2 hours followed by reperfusion for 48 hours) was induced in male Sprague Dawley rats. UA was administered intragastrically 0.5, 24, and 47 hours after reperfusion. Bisphenol A diglycidyl ether (a PPARγ antagonist) was intraperitoneally administered 1, 24.5, and 47.5 hours after reperfusion. Forty-eight hours after reperfusion, neurological deficits and infarct volume were estimated. The PPARγ level and the metalloprotease/anti-metalloprotease balance were examined by Western blotting and immunohistochemistry. The activation of MAPK signaling pathways was also assessed. UA-treated (5, 10, or 20 mg/kg) rats showed significant improvement in neurological deficit score, infarct volume, and the number of intact neurons compared with control rats (Pprotective effects in a dose-dependent manner. Co-treatment with UA and bisphenol A diglycidyl ether completely abolished the UA-induced changes in PPARγ expression; however UA continued to exert a significant but partial neuroprotective effect. UA can act as a PPARγ agonist to improve the metalloprotease/anti-metalloprotease balance, possibly by inhibiting the activation of the MAPK signaling pathway, thereby attenuating cerebral ischemia and reperfusion injury. Therefore, UA may serve as a novel neuroprotective therapeutic agent.

  4. Identification of metalloprotease gene families in sugarcane

    Directory of Open Access Journals (Sweden)

    O.H.P. Ramos

    2001-12-01

    Full Text Available Metalloproteases play a key role in many physiological processes in mammals such as cell migration, tissue remodeling and processing of growth factors. They have also been identified as important factors in the patho-physiology of a number of human diseases, including cancer and hypertension. Many bacterial pathogens rely on proteases in order to infect the host. Several classes of metalloproteases have been described in humans, bacteria, snake venoms and insects. However, the presence and characterization of plant metalloproteases have rarely been described in the literature. In our research, we searched the sugarcane expressed sequence tag (SUCEST DNA library in order to identify, by homology with sequences deposited in other databases, metalloprotease gene families expressed under different conditions. Protein sequences from Arabidopsis thaliana and Glycine max were used to search the SUCEST data bank. Conserved regions corresponding to different metalloprotease domains and sequence motifs were identified in the reads to characterize each group of enzymes. At least four classes of sugarcane metalloproteases have been identified, i.e. matrix metalloproteases, zincins, inverzincins, and ATP-dependent metalloproteases. Each enzyme class was analyzed for its expression in different conditions and tissues.Metaloproteases exercem papéis importantes em muitos processos fisiológicos em mamíferos tais como migração celular, remodelamento tecidual e processamento de fatores de crescimento. Estas enzimas estão envolvidas também na pato-fisiologia de um grande número de doenças humanas como hipertensão e câncer. Muitas bactérias patogênicas dependem de proteases para infectar o hospedeiro. Diversas classes de metaloproteases foram descritas em seres humanos, bactérias, venenos de serpentes e insetos. No entanto, a presença e a caracterização de metaloproteases em plantas estão pouco descritas na literatura. Neste trabalho, foi

  5. Prognostic value of stromal decorin expression in patients with breast cancer: a meta-analysis.

    Science.gov (United States)

    Li, Shuang-Jiang; Chen, Da-Li; Zhang, Wen-Biao; Shen, Cheng; Che, Guo-Wei

    2015-11-01

    Numbers of studies have investigated the biological functions of decorin (DCN) in oncogenesis, tumor progression, angiogenesis and metastasis. Although many of them aim to highlight the prognostic value of stromal DCN expression in breast cancer, some controversial results still exist and a consensus has not been reached until now. Therefore, our meta-analysis aims to determine the prognostic significance of stromal DCN expression in breast cancer patients. PubMed, EMBASE, the Web of Science and China National Knowledge Infrastructure (CNKI) databases were searched for full-text literatures met out inclusion criteria. We applied the hazard ratio (HR) with 95% confidence interval (CI) as the appropriate summarized statistics. Q-test and I(2) statistic were employed to estimate the level of heterogeneity across the included studies. Sensitivity analysis was conducted to further identify the possible origins of heterogeneity. The publication bias was detected by Begg's test and Egger's test. There were three English literatures (involving 6 studies) included into our meta-analysis. On the one hand, both the summarized outcomes based on univariate analysis (HR: 0.513; 95% CI: 0.406-0.648; Panalysis (HR: 0.544; 95% CI: 0.388-0.763; Panalysis (HR: 0.504; 95% CI: 0.389-0.651; Panalysis (HR: 0.568; 95% CI: 0.400-0.806; P=0.002) also indicated that stromal DCN expression was positively associated with high disease-free survival (DFS) of breast cancer patients. No significant heterogeneity or publication bias was observed within this meta-analysis. The present evidences indicate that high stromal DCN expression can significantly predict the good prognosis in patients with breast cancer. The discoveries from our meta-analysis have better be confirmed in the updated review pooling more relevant investigations in the future.

  6. Designed Surface Topographies Control ICAM-1 Expression in Tonsil-Derived Human Stromal Cells

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    Aliaksei S. Vasilevich

    2018-06-01

    Full Text Available Fibroblastic reticular cells (FRCs, the T-cell zone stromal cell subtype in the lymph nodes, create a scaffold for adhesion and migration of immune cells, thus allowing them to communicate. Although known to be important for the initiation of immune responses, studies about FRCs and their interactions have been impeded because FRCs are limited in availability and lose their function upon culture expansion. To circumvent these limitations, stromal cell precursors can be mechanotranduced to form mature FRCs. Here, we used a library of designed surface topographies to trigger FRC differentiation from tonsil-derived stromal cells (TSCs. Undifferentiated TSCs were seeded on a TopoChip containing 2176 different topographies in culture medium without differentiation factors, then monitored cell morphology and the levels of ICAM-1, a marker of FRC differentiation. We identified 112 and 72 surfaces that upregulated and downregulated, respectively, ICAM-1 expression. By monitoring cell morphology, and expression of the FRC differentiation marker ICAM-1 via image analysis and machine learning, we discovered correlations between ICAM-1 expression, cell shape and design of surface topographies and confirmed our findings by using flow cytometry. Our findings confirmed that TSCs are mechano-responsive cells and identified particular topographies that can be used to improve FRC differentiation protocols.

  7. Analysis of gene expression in prostate cancer epithelial and interstitial stromal cells using laser capture microdissection

    International Nuclear Information System (INIS)

    Gregg, Jennifer L; Brown, Kathleen E; Mintz, Eric M; Piontkivska, Helen; Fraizer, Gail C

    2010-01-01

    The prostate gland represents a multifaceted system in which prostate epithelia and stroma have distinct physiological roles. To understand the interaction between stroma and glandular epithelia, it is essential to delineate the gene expression profiles of these two tissue types in prostate cancer. Most studies have compared tumor and normal samples by performing global expression analysis using a mixture of cell populations. This report presents the first study of prostate tumor tissue that examines patterns of differential expression between specific cell types using laser capture microdissection (LCM). LCM was used to isolate distinct cell-type populations and identify their gene expression differences using oligonucleotide microarrays. Ten differentially expressed genes were then analyzed in paired tumor and non-neoplastic prostate tissues by quantitative real-time PCR. Expression patterns of the transcription factors, WT1 and EGR1, were further compared in established prostate cell lines. WT1 protein expression was also examined in prostate tissue microarrays using immunohistochemistry. The two-step method of laser capture and microarray analysis identified nearly 500 genes whose expression levels were significantly different in prostate epithelial versus stromal tissues. Several genes expressed in epithelial cells (WT1, GATA2, and FGFR-3) were more highly expressed in neoplastic than in non-neoplastic tissues; conversely several genes expressed in stromal cells (CCL5, CXCL13, IGF-1, FGF-2, and IGFBP3) were more highly expressed in non-neoplastic than in neoplastic tissues. Notably, EGR1 was also differentially expressed between epithelial and stromal tissues. Expression of WT1 and EGR1 in cell lines was consistent with these patterns of differential expression. Importantly, WT1 protein expression was demonstrated in tumor tissues and was absent in normal and benign tissues. The prostate represents a complex mix of cell types and there is a need to analyze

  8. Progesterone receptor expression during prostate cancer progression suggests a role of this receptor in stromal cell differentiation.

    Science.gov (United States)

    Yu, Yue; Yang, Ou; Fazli, Ladan; Rennie, Paul S; Gleave, Martin E; Dong, Xuesen

    2015-07-01

    The progesterone receptor, like the androgen receptor, belongs to the steroid receptor superfamily. Our previous studies have reported that the PR is expressed specifically in prostate stroma. PR inhibits proliferation of, and regulates cytokine secretion by stromal cells. However, PR protein expression in cancer-associated stroma during prostate cancer progression has not been profiled. Since the phenotypes of prostate stromal cells change dynamically as tumors progress, whether the PR plays a role in regulating stromal cell differentiation needs to be investigated. Immunohistochemistry assays measured PR protein levels on human prostate tissue microarrays containing 367 tissue cores from benign prostate, prostate tumors with different Gleason scores, tumors under various durations of castration therapy, and tumors at the castration-resistant stage. Immunoblotting assays determined whether PR regulated the expression of alpha smooth muscle actin (α-SMA), vimentin, and fibroblast specific protein (FSP) in human prostate stromal cells. PR protein levels decreased in cancer-associated stroma when compared with that in benign prostate stroma. This reduction in PR expression was not correlated with Gleason scores. PR protein levels were elevated by castration therapy, but reduced to pre-castration levels when tumors progressed to the castration-resistant stage. Enhanced PR expression in human prostate stromal cells increased α-SMA, but decreased vimentin and FSP protein levels ligand-independently. These results suggest that PR plays an active role in regulating stromal cell phenotypes during prostate cancer progression. © 2015 Wiley Periodicals, Inc.

  9. Ursolic acid reduces the metalloprotease/anti-metalloprotease imbalance in cerebral ischemia and reperfusion injury

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    Wang Y

    2016-05-01

    Full Text Available Yanzhe Wang, Zhiyi He, Shumin Deng Department of Neurology, The First Affiliated Hospital of China Medical University, Shenyang, People’s Republic of China Background: Activators of PPARs, particularly PPARγ, may be effective neuroprotective drugs against inflammatory responses in cerebral ischemia and reperfusion injury. Ursolic acid (UA may act as a PPARγ agonist and serve as an anti-inflammatory agent. In this study, we used a rat middle cerebral artery occlusion and reperfusion model to examine how UA acts as a neuroprotective agent to modulate the metalloprotease/anti-metalloprotease balance. Methods: The middle cerebral artery occlusion and reperfusion model (occlusion for 2 hours followed by reperfusion for 48 hours was induced in male Sprague Dawley rats. UA was administered intragastrically 0.5, 24, and 47 hours after reperfusion. Bisphenol A diglycidyl ether (a PPARγ antagonist was intraperitoneally administered 1, 24.5, and 47.5 hours after reperfusion. Forty-eight hours after reperfusion, neurological deficits and infarct volume were estimated. The PPARγ level and the metalloprotease/anti-metalloprotease balance were examined by Western blotting and immunohistochemistry. The activation of MAPK signaling pathways was also assessed. Results: UA-treated (5, 10, or 20 mg/kg rats showed significant improvement in neurological deficit score, infarct volume, and the number of intact neurons compared with control rats (P<0.01. Both the PPARγ protein level and the percentage of PPARγ-positive cells were increased in the UA-treated groups (P<0.01. Compared with the control group, the UA-treated groups exhibited reduced protein levels of MMP2, MMP9, and activated MAPKs (P<0.01 but an increased level of TIMP1 (P<0.01. UA exerted its protective effects in a dose-dependent manner. Co-treatment with UA and bisphenol A diglycidyl ether completely abolished the UA-induced changes in PPARγ expression; however UA continued to exert a

  10. Radiation effect on the generation and expression of bone marrow stromal cells

    International Nuclear Information System (INIS)

    Xu Xiaoya; Jin Weilao; Gao Jianjun; Zhou Yi; Sheng Hui

    2009-01-01

    In order to investigate focal flushing dose radiation effect on the generation, differentiation and gene expression of bone marrow stromal cells the femoral head of rats was irradiated at 30 Gy by 137 Cs γ-rays (dose rate: 0.83 Gy/min). Then the bone marrow stromal cells (BMSCs) was cultured. The ability of proliferation and colony formation was observed and the expression level of Cbf-α1, PPAR-γ, VEGF-a and KDR was detected by RT-PCR technology in two weeks later. It has been found that after partly irradiated by 137 Cs γ-rays the proliferation and the number of colony of BMSCs in irradiated group decreases obviously meanwhile the expression level of Cbf-α1, PPAR-γ and VEGF-a in irradiated BMSCs obviously decreases by 18.98 %, 9.46 %, 57.34 % and 5.56 % respectively compared to the normal BMSCs (p<0.05). It shows that the focal great radiation could damage the BMSCs and depress the generation, differentiation and the expression of the related genes obviously. (authors)

  11. CD117 expression in fibroblasts-like stromal cells indicates unfavorable clinical outcomes in ovarian carcinoma patients.

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    Ruixia Huang

    Full Text Available The stem cell factor (SCF receptor CD117 (c-kit, is widely used for identification of hematopoietic stem cells and cancer stem cells. Moreover, CD117 expression in carcinoma cells indicates a poor prognosis in a variety of cancers. However the potential expression in tumor microenvironment and the biological and clinical impact are currently not reported. The expression of CD117 was immunohistochemically evaluated in a serial of 242 epithelial ovarian cancer (EOC cases. Thirty-eight out of 242 cases were CD117 positive in fibroblast-like stromal cells and 22 cases were positive in EOC cells. Four cases were both positive in fibroblast-like stromal cells and EOC cells for CD117. CD117 expression in fibroblast-like stromal cells in ovarian carcinoma was closely linked to advanced FIGO stage, poor differentiation grade and histological subtype (p<0.05, and it was significantly associated with poor overall survival (OS and progression free survival (PFS (Kaplan-Meier analysis; p<0.05, log-rank test. CD117 expression in ovarian carcinoma cells was not associated with these clinicopathological variables. The CD117 positive fibroblast-like stromal cells were all positive for mesenchymal stem/stromal cell (MSC marker CD73 but negative for fibroblast markers fibroblast activation protein (FAP and α smooth muscle actin (α-SMA, indicating that the CD117+/CD73+ fibroblast-like stromal cells are a subtype of mesenchymal stem cells in tumor stroma, although further characterization of these cells are needed. It is concluded herewith that the presence of CD117+/CD73+ fibroblast-like stromal cells in ovarian carcinoma is an unfavorable clinical outcome indication.

  12. N-cadherin Expression in Testicular Germ Cell and Gonadal Stromal Tumors

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    Daniel J. Heidenberg, Joel H. Barton, Denise Young, Michael Grinkemeyer, Isabell A. Sesterhenn

    2012-01-01

    Full Text Available Neural-cadherin is a member of the cadherin gene family encoding the N-cadherin protein that mediates cell adhesion. N-cadherin is a marker of Sertoli cells and is also expressed in germ cells of varying stages of maturation. The purpose of this study was to determine the presence and distribution of this protein by immunohistochemistry in 105 germ cell tumors of both single and mixed histological types and 12 gonadal stromal tumors. Twenty-four germ cell tumors consisted of one cell type and the remaining were mixed. Of the 23 seminomas in either pure or mixed tumors, 74% were positive. Two spermatocytic seminomas were positive. Of the 83 cases with yolk sac tumor, 99% were positive for N-cadherin. The teratomas were positive in 73% in neuroectodermal and / or glandular components. In contrast, 87% of embryonal carcinomas did not express N-cadherin. Only 17% of the syncytiotrophoblastic cells were positive for N-cadherin. In conclusion, N-cadherin expression is very helpful in the identification of yolk sac tumors. In addition to glypican-3 and Sal-like protein 4, N-cadherin can be beneficial for the diagnosis and classification of this subtype of testicular germ cell tumor. Nine of the 12 gonadal stromal tumors were positive to a variable extent.

  13. Early passage bone marrow stromal cells express genes involved in nervous system development supporting their relevance for neural repair

    NARCIS (Netherlands)

    Nandoe Tewarie, R.D.S.; Bossers, K.; Ritfeld, G.J.; Blits, B.; Grotenhuis, J.A.; Verhaagen, J.; Oudega, M.

    2011-01-01

    PURPOSE: The assessment of the capacity of bone marrow stromal cells (BMSC) to repair the nervous system using gene expression profiling. The evaluation of effects of long-term culturing on the gene expression profile of BMSC. METHODS: Fourty four k whole genome rat microarrays were used to study

  14. Regulation of cholesterol 25-hydroxylase expression by vitamin D3 metabolites in human prostate stromal cells

    International Nuclear Information System (INIS)

    Wang, J.-H.; Tuohimaa, Pentti

    2006-01-01

    Vitamin D 3 plays an important role in the control of cell proliferation and differentiation. Cholesterol 25-hydroxylase (CH25H) is an enzyme converting cholesterol into 25-hydroxycholesterol. Vitamin D 3 as well as 25-hydroxycholesterol has been shown to inhibit cell growth and induce cell apoptosis. Here we show that 10 nM 1α,25(OH) 2 D 3 and 500 nM 25OHD 3 upregulate CH25H mRNA expression in human primary prostate stromal cells (P29SN). Protein synthesis inhibitor cycloheximide does not block 1α,25(OH) 2 D 3 mediated upregulation of CH25H mRNA. Transcription inhibitor actinomycin D blocks basal level as well as 1α,25(OH) 2 D 3 induced CH25H mRNA expression. 1α,25(OH) 2 D 3 has no effect on CH25H mRNA stability. 25-Hydroxycholesterol significantly decreased the P29SN cell number. A CH25H enzyme inhibitor, desmosterol, increases basal cell number but has no significant effect on vitamin D 3 treated cells. Our data suggest that ch25h could be a vitamin D 3 target gene and may partly mediate anti-proliferative action of vitamin D 3 in human primary prostate stromal cells

  15. Despite differential gene expression profiles pediatric MDS derived mesenchymal stromal cells display functionality in vitro.

    Science.gov (United States)

    Calkoen, F G J; Vervat, C; van Pel, M; de Haas, V; Vijfhuizen, L S; Eising, E; Kroes, W G M; 't Hoen, P A C; van den Heuvel-Eibrink, M M; Egeler, R M; van Tol, M J D; Ball, L M

    2015-03-01

    Pediatric myelodysplastic syndrome (MDS) is a heterogeneous disease covering a spectrum ranging from aplasia (RCC) to myeloproliferation (RAEB(t)). In adult-type MDS there is increasing evidence for abnormal function of the bone-marrow microenvironment. Here, we extensively studied the mesenchymal stromal cells (MSCs) derived from children with MDS. MSCs were expanded from the bone-marrow of 17 MDS patients (RCC: n=10 and advanced MDS: n=7) and pediatric controls (n=10). No differences were observed with respect to phenotype, differentiation capacity, immunomodulatory capacity or hematopoietic support. mRNA expression analysis by Deep-SAGE revealed increased IL-6 expression in RCC- and RAEB(t)-MDS. RCC-MDS MSC expressed increased levels of DKK3, a protein associated with decreased apoptosis. RAEB(t)-MDS revealed increased CRLF1 and decreased DAPK1 expressions. This pattern has been associated with transformation in hematopoietic malignancies. Genes reported to be differentially expressed in adult MDS-MSC did not differ between MSC of pediatric MDS and controls. An altered mRNA expression profile, associated with cell survival and malignant transformation, of MSC derived from children with MDS strengthens the hypothesis that the micro-environment is of importance in this disease. Our data support the understanding that pediatric and adult MDS are two different diseases. Further evaluation of the pathways involved might reveal additional therapy targets. Copyright © 2015. Published by Elsevier B.V.

  16. Extracellular protease mRNAs are predominantly expressed in the stromal areas of microdissected mouse breast carcinomas

    DEFF Research Database (Denmark)

    Pedersen, Tanja Xenia; Pennington, Caroline J; Almholt, Kasper

    2005-01-01

    Solid tumors synthesize a number of extracellular matrix-degrading proteases that are important for tumor progression. Based on qualitative in situ hybridization studies in human cancer tissue, a range of components involved in proteolysis appear to be expressed by stromal cells rather than cancer...

  17. Fetal Mesenchymal Stromal Cells Differentiating towards Chondrocytes Acquire a Gene Expression Profile Resembling Human Growth Plate Cartilage

    NARCIS (Netherlands)

    van Gool, S.A.; Emons, J.A.M.; Leijten, Jeroen Christianus Hermanus; Decker, E.; Sticht, C.; van Houwelingen, J.C.; Goeman, J.J.; Kleijburg, C.; Scherjon, S.; Gretz, N.; Wit, J.M.; Rappold, G.; Post, Janine Nicole; Karperien, Hermanus Bernardus Johannes

    2012-01-01

    Abstract We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs) differentiating towards chondrocytes as an alternative model for the human growth plate (GP). Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether

  18. Gene expression in gastrointestinal stromal tumors is distinguished by KIT genotype and anatomic site.

    Science.gov (United States)

    Antonescu, Cristina R; Viale, Agnes; Sarran, Lisa; Tschernyavsky, Sylvia J; Gonen, Mithat; Segal, Neil H; Maki, Robert G; Socci, Nicholas D; DeMatteo, Ronald P; Besmer, Peter

    2004-05-15

    Gastrointestinal stromal tumors (GISTs) are specific KIT expressing and KIT-signaling driven mesenchymal tumors of the human digestive tract, many of which have KIT-activating mutations. Previous studies have found a relatively homogeneous gene expression profile in GIST, as compared with other histological types of sarcomas. Transcriptional heterogeneity within clinically or molecularly defined subsets of GISTs has not been previously reported. We tested the hypothesis that the gene expression profile in GISTs might be related to KIT genotype and possibly to other clinicopathological factors. An HG-U133A Affymetrix chip (22,000 genes) platform was used to determine the variability of gene expression in 28 KIT-expressing GIST samples from 24 patients. A control group of six intra-abdominal leiomyosarcomas was also included for comparison. Statistical analyses (t tests) were performed to identify discriminatory gene lists among various GIST subgroups. The levels of expression of various GIST subsets were also linked to a modified version of the growth factor/KIT signaling pathway to analyze differences at various steps in signal transduction. Genes involved in KIT signaling were differentially expressed among wild-type and mutant GISTs. High gene expression of potential drug targets, such as VEGF, MCSF, and BCL2 in the wild-type group, and Mesothelin in exon 9 GISTs were found. There was a striking difference in gene expression between stomach and small bowel GISTs. This finding was validated in four separate tumors, two gastric and two intestinal, from a patient with familial GIST with a germ-line KIT W557R substitution. GISTs have heterogeneous gene expression depending on KIT genotype and tumor location, which is seen at both the genomic level and the KIT signaling pathway in particular. These findings may explain their variable clinical behavior and response to therapy.

  19. Bone regeneration by implantation of adipose-derived stromal cells expressing BMP-2

    International Nuclear Information System (INIS)

    Li Huiwu; Dai Kerong; Tang Tingting; Zhang Xiaoling; Yan Mengning; Lou Jueren

    2007-01-01

    In this study, we reported that the adipose-derived stromal cells (ADSCs) genetically modified by bone morphogenetic protein 2 (BMP-2) healed critical-sized canine ulnar bone defects. First, the osteogenic and adipogenic differentiation potential of the ADSCs derived from canine adipose tissue were demonstrated. And then the cells were modified by the BMP-2 gene and the expression and bone-induction ability of BMP-2 were identified. Finally, the cells modified by BMP-2 gene were applied to a β-tricalcium phosphate (TCP) carrier and implanted into ulnar bone defects in the canine model. After 16 weeks, radiographic, histological, and histomorphometry analysis showed that ADSCs modified by BMP-2 gene produced a significant increase of newly formed bone area and healed or partly healed all of the bone defects. We conclude that ADSCs modified by the BMP-2 gene can enhance the repair of critical-sized bone defects in large animals

  20. Defining the expression of marker genes in equine mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Deborah J Guest

    2008-11-01

    Full Text Available Deborah J Guest1, Jennifer C Ousey1, Matthew RW Smith21Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk, CB8 7UU; 2Reynolds House Referrals, Greenwood Ellis and Partners, 166 High Street, Newmarket, Suffolk, CB8 9WS, UKAbstract: Mesenchymal stromal (MS cells have been derived from multiple sources in the horse including bone marrow, adipose tissue and umbilical cord blood. To date these cells have been investigated for their differentiation potential and are currently being used to treat damage to horse musculoskeletal tissues. However, no work has been done in horse MS cells to examine the expression profile of proteins and cell surface antigens that are expressed in human MS cells. The identification of such profiles in the horse will allow the comparison of putative MS cells isolated from different laboratories and different tissues. At present it is difficult to ascertain whether equivalent cells are being used in different reports. Here, we report on the expression of a range of markers used to define human MS cells. Using immunocytochemistry we show that horse MS cells homogenously express collagens, alkaline phosphatase activity, CD44, CD90 and CD29. In contrast, CD14, CD79α and the embryonic stem cell markers Oct-4, SSEA (stage specific embryonic antigen -1, -3, -4, TRA (tumor rejection antigen -1–60 and -1–81 are not expressed. The MS cells also express MHC class I antigens but do not express class II antigens, although they are inducible by treatment with interferon gamma (IFN-γ.Keywords: mesenchymal stem cells, equine, gene expression

  1. Adult rat bone marrow stromal cells express genes associated with dopamine neurons

    International Nuclear Information System (INIS)

    Kramer, Brian C.; Woodbury, Dale; Black, Ira B.

    2006-01-01

    An intensive search is underway to identify candidates to replace the cells that degenerate in Parkinson's disease (PD). To date, no suitable substitute has been found. We have recently found that adult rat bone marrow stromal cells (MSCs) can be induced to assume a neuronal phenotype in vitro. These findings may have particular relevance to the treatment of PD. We now report that adult MSCs express multiple dopaminergic genes, suggesting that they are potential candidates for cell therapy. Using RT-PCR, we have examined families of genes that are associated with the development and/or survival of dopaminergic neurons. MSCs transcribe a variety of dopaminergic genes including patched and smoothened (components of the Shh receptor), Gli-1 (downstream mediator of Shh), and Otx-1, a gene associated with formation of the mesencephalon during development. Furthermore, Shh treatment elicits a 1.5-fold increase in DNA synthesis in cultured MSCs, suggesting the presence of a functional Shh receptor complex. We have also found that MSCs transcribe and translate Nurr-1, a nuclear receptor essential for the development of dopamine neurons. In addition, MSCs express a variety of growth factor receptors including the glycosyl-phosphatidylinositol-anchored ligand-binding subunit of the GDNF receptor, GFRα1, as well as fibroblast growth factor receptors one and four. The expression of genes that are associated with the development and survival of dopamine neurons suggests a potential role for these cells in the treatment of Parkinson's disease

  2. Bone marrow stromal and vascular smooth muscle cells have chemosensory capacity via bitter taste receptor expression.

    Directory of Open Access Journals (Sweden)

    Troy C Lund

    Full Text Available The ability of cells to detect changes in the microenvironment is important in cell signaling and responsiveness to environmental fluctuations. Our interest is in understanding how human bone marrow stromal-derived cells (MSC and their relatives, vascular smooth muscle cells (VSMC, interact with their environment through novel receptors. We found, through a proteomics screen, that MSC express the bitter taste receptor, TAS2R46, a protein more typically localized to the taste bud. Expression was also confirmed in VSMCs. A prototypical bitter compound that binds to the bitter taste receptor class, denatonium, increased intracellular calcium release and decreased cAMP levels as well as increased the extracellular release of ATP in human MSC. Denatonium also bound and activated rodent VSMC with a change in morphology upon compound exposure. Finally, rodents given denatonium in vivo had a significant drop in blood pressure indicating a vasodilator response. This is the first description of chemosensory detection by MSC and VSMCs via a taste receptor. These data open a new avenue of research into discovering novel compounds that operate through taste receptors expressed by cells in the marrow and vascular microenvironments.

  3. EGF-R is Expressed and AP-1 and NF-κ:B Are Activated in Stromal Myofibroblasts Surrounding Colon Adenocarcinomas Paralleling Expression of COX-2 and VEGF

    Directory of Open Access Journals (Sweden)

    Panagiotis A. Konstantinopoulos

    2007-01-01

    Full Text Available Background: COX-2 and VEGF are important triggers of colon cancer growth, metastasis and angiogenesis. Cox-2 promoter contains transcriptional regulatory elements for AP-1 and NF-κ:B transcription factors whilst vegf is a known AP-1 downstream target gene. We investigated whether stromal myofibroblasts surrounding colon adenocarcinomas express COX-2 and VEGF and whether activation of AP-1 and NF-κ:B, as well as expression of EGF-R parallel expression of COX-2 and VEGF in these cells. Methods: Immunohistochemical methodology was performed on archival sections from 40 patients with colon adenocarcinomas. We evaluated c-FOS, p-c-JUN (phosphorylated c-JUN, p-Iκ:B-α (phosphorylated Iκ:B-α, EGF-R, COX-2, NF-κ:B and VEGF expression in stromal myofibroblasts surrounding colon adenocarcinomas. Double immunostaining with a-smooth muscle actin and each antibody was done to verify the expression of these molecules in stromal myofibroblasts. Results: VEGF, p-Iκ:B-α, NF-κ:B, c-FOS, p-c-JUN, EGF-R and COX-2 were expressed in stromal myofibroblasts surrounding colon adenocarcinomas in the majority of cases. EGF-R, p-Iκ:B-α, NF-κ:B, c-FOS and p-c-JUN correlated positively with COX-2 and VEGF expression. Conclusion: Stromal myofibroblasts surrounding colon adenocarcinomas are an important source of VEGF and COX-2 production, while AP-1 and NF-κ:B transcription factors are activated and EGF-R is expressed in these cells and associated with COX-2 and VEGF production.

  4. ERα and ERK1/2 MAP kinase expression in microdissected stromal and epithelial endometrial cells

    Directory of Open Access Journals (Sweden)

    Said Abu Alkhair Mohamed

    2014-03-01

    Total and phosphorylated levels for ERK1/2 and ERα were measured by quantitation of signals from Western blots using specific antibodies against the active and total forms of ERK1/2 and against ERα. When the level of the proteins was quantitated and normalized to β actin from microdissected stroma and epithelium, no significant difference was detected in the levels of these proteins between the two tissue compartments. There was a trend toward higher expression in the stroma vs. epithelium, respectively (active ERK1/2 0.45 ± 0.17 vs. 0.2 ± 0.65; total ERK1/2 0.54 ± 0.35 vs. 0.28 ± 0.23; ERα 0.82 ± 0.28 vs. 0.54 ± 0.18; n = 6. These data demonstrate that there are comparable levels of ERα (P = 0.41, total ERK1/2 (P = 0.18 and active ERK1/2 (P = 0.13 in the stroma and epithelium of proliferative phase endometrium with a trend toward higher expression of these proteins in the stromal compartment.

  5. In vitro expression of erythropoietin by transfected human mesenchymal stromal cells.

    Science.gov (United States)

    Mok, P-L; Cheong, S-K; Leong, C-F; Othman, A

    2008-01-01

    Mesenchymal stromal cells (MSC) are pluripotent progenitor cells that can be found in human bone marrow (BM). These cells have low immunogenicity and could suppress alloreactive T-cell responses. In the current study, MSC were tested for their capacity to carry and deliver the erythropoietin (EPO) gene in vitro. Expanded BM MSC was transfected with EPO-encoded plasmid pMCV1.2 and EPO-encoded MIDGE (minimalistic immunologically defined gene expression) vector by electroporation. The expressed EPO was used to induce hematopoietic stem cells (HSC) into erythroid colonies. The results showed that the MIDGE vector was more effective and stable than the plasmid (pMCV1.2) in delivering EPO gene into MSC. The supernatants containing EPO obtained from the transfected cell culture were able to induce the differentiation of HSC into erythroid colonies. MSC hold promise as a cell factory for the production of biologic molecules, and MIDGE vector is more effective and stable than the plasmid in nucleofection involving the EPO gene.

  6. Distinct Patterns of Stromal and Tumor Expression of ROR1 and ROR2 in Histological Subtypes of Epithelial Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    C.E. Henry

    2017-06-01

    Full Text Available OBJECTIVE: The ROR1 and ROR2 receptor tyrosine kinases have both been implicated in ovarian cancer progression and have been shown to drive migration and invasion. There is an increasing importance of the role of stroma in ovarian cancer metastasis; however, neither ROR1 nor ROR2 expression in tumor or stromal cells has been analyzed in the same clinical cohort. AIM: To determine ROR1 and ROR2 expression in ovarian cancer and surrounding microenvironment and examine associations with clinicopathological characteristics. METHODS: Immunohistochemistry for ROR1 and ROR2 was used to assess receptor expression in a cohort of epithelial ovarian cancer patients (n = 178. Results were analyzed in relation to clinical and histopathological characteristics and survival. Matched patient sample case studies of normal, primary, and metastatic lesions were used to examine ROR expression in relation to ovarian cancer progression. RESULTS: ROR1 and ROR2 are abnormally expressed in malignant ovarian epithelium and stroma. Higher ROR2 tumor expression was found in early-stage, low-grade endometrioid carcinomas. ROR2 stromal expression was highest in the serous subtype. In matched patient case studies, metastatic samples had higher expression of ROR2 in the stroma, and a recurrent sample had the highest expression of ROR2 in both tumor and stroma. CONCLUSION: ROR1 and ROR2 are expressed in tumor-associated stroma in all histological subtypes of ovarian cancer and hold potential as therapeutic targets which may disrupt tumor and stroma interactions.

  7. Altered AIB1 or AIB1Δ3 Expression Impacts ERα Effects on Mammary Gland Stromal and Epithelial Content

    Science.gov (United States)

    Nakles, Rebecca E.; Shiffert, Maddalena Tilli; Díaz-Cruz, Edgar S.; Cabrera, M. Carla; Alotaiby, Maram; Miermont, Anne M.; Riegel, Anna T.

    2011-01-01

    Amplified in breast cancer 1 (AIB1) (also known as steroid receptor coactivator-3) is a nuclear receptor coactivator enhancing estrogen receptor (ER)α and progesterone receptor (PR)-dependent transcription in breast cancer. The splice variant AIB1Δ3 demonstrates increased ability to promote ERα and PR-dependent transcription. Both are implicated in breast cancer risk and antihormone resistance. Conditional transgenic mice tested the in vivo impact of AIB1Δ3 overexpression compared with AIB1 on histological features of increased breast cancer risk and growth response to estrogen and progesterone in the mammary gland. Combining expression of either AIB1 or AIB1Δ3 with ERα overexpression, we investigated in vivo cooperativity. AIB1 and AIB1Δ3 overexpression equivalently increased the prevalence of hyperplastic alveolar nodules but not ductal hyperplasia or collagen content. When AIB1 or AIB1Δ3 overexpression was combined with ERα, both stromal collagen content and ductal hyperplasia prevalence were significantly increased and adenocarcinomas appeared. Overexpression of AIB1Δ3, especially combined with overexpressed ERα, led to an abnormal response to estrogen and progesterone with significant increases in stromal collagen content and development of a multilayered mammary epithelium. AIB1Δ3 overexpression was associated with a significant increase in PR expression and PR downstream signaling genes. AIB1 overexpression produced less marked growth abnormalities and no significant change in PR expression. In summary, AIB1Δ3 overexpression was more potent than AIB1 overexpression in increasing stromal collagen content, inducing abnormal mammary epithelial growth, altering PR expression levels, and mediating the response to estrogen and progesterone. Combining ERα overexpression with either AIB1 or AIB1Δ3 overexpression augmented abnormal growth responses in both epithelial and stromal compartments. PMID:21292825

  8. Endothelial marker-expressing stromal cells are critical for kidney formation.

    Science.gov (United States)

    Mukherjee, Elina; Maringer, Katherine; Papke, Emily; Bushnell, Daniel; Schaefer, Caitlin; Kramann, Rafael; Ho, Jacqueline; Humphreys, Benjamin D; Bates, Carlton; Sims-Lucas, Sunder

    2017-09-01

    Kidneys are highly vascularized and contain many distinct vascular beds. However, the origins of renal endothelial cells and roles of the developing endothelia in the formation of the kidney are unclear. We have shown that the Foxd1-positive renal stroma gives rise to endothelial marker-expressing progenitors that are incorporated within a subset of peritubular capillaries; however, the significance of these cells is unclear. The purpose of this study was to determine whether deletion of Flk1 in the Foxd1 stroma was important for renal development. To that end, we conditionally deleted Flk1 (critical for endothelial cell development) in the renal stroma by breeding-floxed Flk1 mice ( Flk1 fl/fl ) with Foxd1cre mice to generate Foxd1cre; Flk1 fl/fl ( Flk1 ST-/- ) mice. We then performed FACsorting, histological, morphometric, and metabolic analyses of Flk1 ST-/- vs. control mice. We confirmed decreased expression of endothelial markers in the renal stroma of Flk1 ST-/- kidneys via flow sorting and immunostaining, and upon interrogation of embryonic and postnatal Flk1 ST-/- mice, we found they had dilated peritubular capillaries. Three-dimensional reconstructions showed reduced ureteric branching and fewer nephrons in developing Flk1 ST-/- kidneys vs. Juvenile Flk1 ST-/- kidneys displayed renal papillary hypoplasia and a paucity of collecting ducts. Twenty-four-hour urine collections revealed that postnatal Flk1 ST-/- mice had urinary-concentrating defects. Thus, while lineage-tracing revealed that the renal cortical stroma gave rise to a small subset of endothelial progenitors, these Flk1-expressing stromal cells are critical for patterning the peritubular capillaries. Also, loss of Flk1 in the renal stroma leads to nonautonomous-patterning defects in ureteric lineages. Copyright © 2017 the American Physiological Society.

  9. Neural differentiation potential of human bone marrow-derived mesenchymal stromal cells: misleading marker gene expression

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    Montzka Katrin

    2009-03-01

    Full Text Available Abstract Background In contrast to pluripotent embryonic stem cells, adult stem cells have been considered to be multipotent, being somewhat more restricted in their differentiation capacity and only giving rise to cell types related to their tissue of origin. Several studies, however, have reported that bone marrow-derived mesenchymal stromal cells (MSCs are capable of transdifferentiating to neural cell types, effectively crossing normal lineage restriction boundaries. Such reports have been based on the detection of neural-related proteins by the differentiated MSCs. In order to assess the potential of human adult MSCs to undergo true differentiation to a neural lineage and to determine the degree of homogeneity between donor samples, we have used RT-PCR and immunocytochemistry to investigate the basal expression of a range of neural related mRNAs and proteins in populations of non-differentiated MSCs obtained from 4 donors. Results The expression analysis revealed that several of the commonly used marker genes from other studies like nestin, Enolase2 and microtubule associated protein 1b (MAP1b are already expressed by undifferentiated human MSCs. Furthermore, mRNA for some of the neural-related transcription factors, e.g. Engrailed-1 and Nurr1 were also strongly expressed. However, several other neural-related mRNAs (e.g. DRD2, enolase2, NFL and MBP could be identified, but not in all donor samples. Similarly, synaptic vesicle-related mRNA, STX1A could only be detected in 2 of the 4 undifferentiated donor hMSC samples. More significantly, each donor sample revealed a unique expression pattern, demonstrating a significant variation of marker expression. Conclusion The present study highlights the existence of an inter-donor variability of expression of neural-related markers in human MSC samples that has not previously been described. This donor-related heterogeneity might influence the reproducibility of transdifferentiation protocols as

  10. Transforming growth factor-β inhibits CCAAT/enhancer-binding protein expression and PPARγ activity in unloaded bone marrow stromal cells

    International Nuclear Information System (INIS)

    Ahdjoudj, S.; Kaabeche, K.; Holy, X.; Fromigue, O.; Modrowski, D.; Zerath, E.; Marie, P.J.

    2005-01-01

    The molecular mechanisms regulating the adipogenic differentiation of bone marrow stromal cells in vivo remain largely unknown. In this study, we investigated the regulatory effects of transforming growth factor beta-2 (TGF-β2) on transcription factors involved in adipogenic differentiation induced by hind limb suspension in rat bone marrow stromal cells in vivo. Time course real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis of gene expression showed that skeletal unloading progressively increases the expression of CCAAT/enhancer-binding protein (C/EBP)α and C/EBPβ α at 5 days in bone marrow stromal cells resulting in increased peroxisome proliferator-activated receptor γ (PPARγ2) transcripts at 7 days. TGF-β2 administration in unloaded rats corrected the rise in C/EBPα and C/EBPβ transcripts induced by unloading in bone marrow stromal cells. This resulted in inhibition of PPARγ2 expression that was associated with increased Runx2 expression. Additionally, the inhibition of C/EBPα and C/EBPβ expression by TGF-β2 was associated with increased PPARγ serine phosphorylation in bone marrow stromal cells, a mechanism that inhibits PPARγ transactivating activity. The sequential inhibitory effect of TGF-β2 on C/EBPα, C/EBPβ, and PPARγ2 resulted in reduced LPL expression and abolition of bone marrow stromal cell adipogenic differentiation, which contributed to prevent bone loss induced by skeletal unloading. We conclude that TGF-β2 inhibits the excessive adipogenic differentiation of bone marrow stromal cells induced by skeletal unloading by inhibiting C/EBPα, C/EBPβ, and PPARγ expression and activity, which provides a sequential mechanism by which TGF-β2 regulates adipogenic differentiation of bone marrow stromal cells in vivo

  11. Impaired Expression of Focal Adhesion Kinase in Mesenchymal Stromal Cells from Low-Risk Myelodysplastic Syndrome Patients

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    Yuenv Wu

    2017-08-01

    Full Text Available The pathogenic role of mesenchymal stromal cells (MSCs in myelodysplastic syndromes (MDS development and progression has been investigated by numerous studies, yet, it remains controversial in some aspects (1, 2. In the present study, we found distinct features of MSCs from low-risk (LR-MDS stromal microenvironment as compared to those from healthy subjects. At the molecular level, focal adhesion kinase, a key tyrosine kinase in control of cell proliferation, survival, and adhesion process, was found profoundly suppressed in expression and activation in LR-MDS MSC. At a functional level, LR-MDS MSCs showed impaired growth and clonogenic capacity, which were independent of cellular senescence and apoptosis. The pro-adipogenic differentiation and attenuated osteogenic capacity along with reduced SDF-1 expression could be involved in creating an unfavorable microenvironment for hematopoiesis. In conclusion, our experiments support the theory that the stromal microenvironment is fundamentally altered in LR-MDS, and these preliminary data offer a new perspective on LR-MDS pathophysiology.

  12. Leukemia inhibitory factor increases the proliferation of human endometrial stromal cells and expression of genes related to pluripotency

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    Mojdeh Salehnia

    2017-08-01

    Full Text Available Background: Concerning the low population of human endometrial mesenchymal cells within the tissue and their potential application in the clinic and tissue engineering, some researches have been focused on their in vitro expansion. Objective: The aim of this study was to evaluate the effect of leukemia inhibitory factor (LIF as a proliferative factor on the expansion and proliferation of human endometrial stromal cells. Materials and Methods: In this experimental study, the isolated and cultured human endometrial stromal cells from women at ovulatory phase aged 20-35 years, after fourth passage were divided into control and LIF-treated groups. In the experimental group, the endometrial cells were treated by 10 ng/ml LIF in culture media and the cultured cells without adding LIF considered as control group. Both groups were evaluated and compared for proliferation rate using MTT assay, for CD90 marker by flow cytometric analysis and for the expression of Oct4, Nanog, PCNA and LIFr genes using real-time RT-PCR. Results: The proliferation rate of control and LIF-treated groups were 1.17±0.17 and 1.61±0.06 respectively and there was a significant increase in endometrial stromal cell proliferation following in vitro treatment by LIF compared to control group (p=0.049. The rate of CD90 positive cells was significantly increased in LIFtreated group (98.96±0.37% compared to control group (94.26±0.08% (p=0.0498. Also, the expression ratio of all studied genes was significantly increased in the LIFtreated group compared to control group (p=0.0479. Conclusion: The present study showed that LIF has a great impact on proliferation, survival, and maintenance of pluripotency of human endometrial stromal cells and it could be applicable in cell therapies.

  13. Differential expression of metabolic genes in tumor and stromal components of primary and metastatic loci in pancreatic adenocarcinoma.

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    Nina V Chaika

    Full Text Available Pancreatic cancer is the fourth leading cause of cancer related deaths in the United States with a five-year survival rate of 6%. It is characterized by extremely aggressive tumor growth rate and high incidence of metastasis. One of the most common and profound biochemical phenotypes of animal and human cancer cells is their ability to metabolize glucose at high rates, even under aerobic conditions. However, the contribution of metabolic interrelationships between tumor cells and cells of the surrounding microenvironment to the progression of cancer is not well understood. We evaluated differential expression of metabolic genes and, hence, metabolic pathways in primary tumor and metastases of patients with pancreatic adenocarcinoma.We analyzed the metabolic gene (those involved in glycolysis, tri-carboxylic acid pathway, pentose-phosphate pathway and fatty acid metabolism expression profiles of primary and metastatic lesions from pancreatic cancer patients by gene expression arrays. We observed two principal results: genes that were upregulated in primary and most of the metastatic lesions; and genes that were upregulated only in specific metastatic lesions in a site-specific manner. Immunohistochemical (IHC analyses of several metabolic gene products confirmed the gene expression patterns at the protein level. The IHC analyses also revealed differential tumor and stromal expression patterns of metabolic enzymes that were correlated with the metastasis sites.Here, we present the first comprehensive studies that establish differential metabolic status of tumor and stromal components and elevation of aerobic glycolysis gene expression in pancreatic cancer.

  14. Increased COX-2 expression in epithelial and stromal cells of high mammographic density tissues and in a xenograft model of mammographic density.

    Science.gov (United States)

    Chew, G L; Huo, C W; Huang, D; Hill, P; Cawson, J; Frazer, H; Hopper, J L; Haviv, I; Henderson, M A; Britt, K; Thompson, E W

    2015-08-01

    Mammographic density (MD) adjusted for age and body mass index is one of the strongest known risk factors for breast cancer. Given the high attributable risk of MD for breast cancer, chemoprevention with a safe and available agent that reduces MD and breast cancer risk would be beneficial. Cox-2 has been implicated in MD-related breast cancer risk, and was increased in stromal cells in high MD tissues in one study. Our study assessed differential Cox-2 expression in epithelial and stromal cells in paired samples of high and low MD human breast tissue, and in a validated xenograft biochamber model of MD. We also examined the effects of endocrine treatment upon Cox-2 expression in high and low MD tissues in the MD xenograft model. Paired high and low MD human breast tissue samples were immunostained for Cox-2, then assessed for differential expression and staining intensity in epithelial and stromal cells. High and low MD human breast tissues were separately maintained in biochambers in mice treated with Tamoxifen, oestrogen or placebo implants, then assessed for percentage Cox-2 staining in epithelial and stromal cells. Percentage Cox-2 staining was greater for both epithelial (p = 0.01) and stromal cells (p tissues. In high MD biochamber tissues, percentage Cox-2 staining was greater in stromal cells of oestrogen-treated versus placebo-treated tissues (p = 0.05).

  15. A standardized extract of Butea monosperma (Lam.) flowers suppresses the IL-1β-induced expression of IL-6 and matrix-metalloproteases by activating autophagy in human osteoarthritis chondrocytes.

    Science.gov (United States)

    Ansari, Mohammad Y; Khan, Nazir M; Haqqi, Tariq M

    2017-12-01

    Osteoarthritis (OA) is a leading cause of joint dysfunction, disability and poor quality of life in the affected population. The underlying mechanism of joint dysfunction involves increased oxidative stress, inflammation, high levels of cartilage extracellular matrix degrading proteases and decline in autophagy-a mechanism of cellular defense. There is no disease modifying therapies currently available for OA. Different parts of the Butea monosperma (Lam.) plant have widely been used in the traditional Indian Ayurvedic medicine system for the treatment of various human diseases including inflammatory conditions. Here we studied the chondroprotective effect of hydromethanolic extract of Butea monosperma (Lam.) flowers (BME) standardized to the concentration of Butein on human OA chondrocytes stimulated with IL-1β. The hydromethanolic extract of Butea monosperma (Lam.) (BME) was prepared with 70% methanol-water mixer using Soxhlet. Chondrocytes viability after BME treatment was measured by MTT assay. Gene expression levels were determined by quantitative polymerase chain reaction (qPCR) using TaqMan assays and immunoblotting with specific antibodies. Autophagy activation was determined by measuring the levels of microtubule associated protein 1 light chain 3-II (LC3-II) by immunoblotting and visualization of autophagosomes by transmission electron and confocal microscopy. BME was non-toxic to the OA chondrocytes at the doses employed and suppressed the IL-1β induced expression of inerleukin-6 (IL-6) and matrix metalloprotease-3 (MMP-3), MMP-9 and MMP-13. BME enhanced autophagy in chondrocytes as determined by measuring the levels of LC3-II by immunoblotting and increased number of autophagosomes in BME treated chondrocytes by transmission electron microscopy and confocal microscopy. BME upregulated the expression of several autophagy related genes and increased the autophagy flux in human OA chondrocytes under pathological conditions. Further analysis revealed that

  16. Prognostic significance of epithelial/stromal caveolin‐1 expression in prostatic hyperplasia, high grade prostatic intraepithelial hyperplasia and prostatic carcinoma and its correlation with microvessel density

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    Dareen A. Mohammed

    2017-03-01

    Full Text Available Caveolin-1 may play a role in cancer development and progression. The aim was to record the expression and localization of caveolin-1 in benign prostatic hyperplasia (BPH, high grade prostatic intraepithelial neoplasia (HGPIN and prostatic carcinoma (PCa. Microvessel density was evaluated with CD34 immunostain. Correlations with known prognostic factors of PCa were recorded. Immunohistochemical expression of caveolin-1 and the MVD was evaluated in 65 cases; BPH (25, HGPIN (20 and PCa (20. Stromal caveolin-1expression was significantly higher in BPH than HGPIN and PCca. There was significant inverse relation between stromal caveolin-1 expression and extension to lymph node and seminal vesicle in carcinoma cases. Epithelial caveolin-1 was significantly higher in carcinomas than in BPH and HGPIN. Epithelial expression in carcinoma was significantly associated with preoperative PSA, Gleason score and lymph node extension. MVD was significantly higher in PCa than in BPH and HGPIN. There were significant relations between MVD and preoperative PSA, Gleason score, lymph node and seminal vesicle extension. Stromal caveolin-1 was associated with low MVD while epithelial caveolin-1 with high MVD. Conclusions: Caveolin-1 plays an important role in prostatic carcinogenesis and metastasis. Stromal expression of caveolin-1 in PCa is lowered in relation to BPH and HGPIN. In PCa; stromal caveolin-1 was associated with good prognostic parameters. Epithelial caveolin-1 is significantly increased in PCa than BPH and HGPIN. It is associated with clinically aggressive disease. Caveolin-1 may play a role in angiogenesis.

  17. Multilineage differentiation of porcine bone marrow stromal cells associated with specific gene expression pattern

    DEFF Research Database (Denmark)

    Zou, Lijin; Zou, Xuenong; Chen, Li

    2007-01-01

    There are increasing reports regarding differentiation of bone marrow stromal cells (BMSC) from human and various species of animals including pigs. The phenotype and function of BMSC along a mesenchymal lineage differentiation are well characterized by specific transcription factors and marker g...

  18. T-Cadherin Expression in Melanoma Cells Stimulates Stromal Cell Recruitment and Invasion by Regulating the Expression of Chemokines, Integrins and Adhesion Molecules

    International Nuclear Information System (INIS)

    Rubina, Kseniya A.; Surkova, Ekaterina I.; Semina, Ekaterina V.; Sysoeva, Veronika Y.; Kalinina, Natalia I.; Poliakov, Alexei A.; Treshalina, Helena M.; Tkachuk, Vsevolod A.

    2015-01-01

    T-cadherin is a glycosyl-phosphatidylinositol (GPI) anchored member of the cadherin superfamily involved in the guidance of migrating cells. We have previously shown that in vivo T-cadherin overexpression leads to increased melanoma primary tumor growth due to the recruitment of mesenchymal stromal cells as well as the enhanced metastasis. Since tumor progression is highly dependent upon cell migration and invasion, the aim of the present study was to elucidate the mechanisms of T-cadherin participation in these processes. Herein we show that T-cadherin expression results in the increased invasive potential due to the upregulated expression of pro-oncogenic integrins, chemokines, adhesion molecules and extracellular matrix components. The detected increase in chemokine expression could be responsible for the stromal cell recruitment. At the same time our previous data demonstrated that T-cadherin expression inhibited neoangiogenesis in the primary tumors. We demonstrate that T-cadherin overexpression leads to the increase in the expression of anti-angiogenic molecules and reduction in pro-angiogenic factors. Thus, T-cadherin plays a dual role in melanoma growth and progression: T-cadherin expression results in anti-angiogenic effects in melanoma, however, this also stimulates transcription of genes responsible for migration and invasion of melanoma cells

  19. T-Cadherin Expression in Melanoma Cells Stimulates Stromal Cell Recruitment and Invasion by Regulating the Expression of Chemokines, Integrins and Adhesion Molecules

    Energy Technology Data Exchange (ETDEWEB)

    Rubina, Kseniya A., E-mail: rkseniya@mail.ru; Surkova, Ekaterina I.; Semina, Ekaterina V.; Sysoeva, Veronika Y.; Kalinina, Natalia I. [Department of Biochemistry and Molecular Medicine, Faculty of Medicine, M.V. Lomonosov Moscow State University, Lomonosovsky av., 31/5, Moscow 119192 (Russian Federation); Poliakov, Alexei A. [Division of Developmental Neurobiology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA (United Kingdom); Treshalina, Helena M. [Federal State Budgetary Scietific Institution «N.N. Blokhin Russian Cancer Research Center» (FSBSI “N.N.Blokhin RCRC”), Kashirskoe Shosse 24, Moscow 115478 (Russian Federation); Tkachuk, Vsevolod A. [Department of Biochemistry and Molecular Medicine, Faculty of Medicine, M.V. Lomonosov Moscow State University, Lomonosovsky av., 31/5, Moscow 119192 (Russian Federation)

    2015-07-21

    T-cadherin is a glycosyl-phosphatidylinositol (GPI) anchored member of the cadherin superfamily involved in the guidance of migrating cells. We have previously shown that in vivo T-cadherin overexpression leads to increased melanoma primary tumor growth due to the recruitment of mesenchymal stromal cells as well as the enhanced metastasis. Since tumor progression is highly dependent upon cell migration and invasion, the aim of the present study was to elucidate the mechanisms of T-cadherin participation in these processes. Herein we show that T-cadherin expression results in the increased invasive potential due to the upregulated expression of pro-oncogenic integrins, chemokines, adhesion molecules and extracellular matrix components. The detected increase in chemokine expression could be responsible for the stromal cell recruitment. At the same time our previous data demonstrated that T-cadherin expression inhibited neoangiogenesis in the primary tumors. We demonstrate that T-cadherin overexpression leads to the increase in the expression of anti-angiogenic molecules and reduction in pro-angiogenic factors. Thus, T-cadherin plays a dual role in melanoma growth and progression: T-cadherin expression results in anti-angiogenic effects in melanoma, however, this also stimulates transcription of genes responsible for migration and invasion of melanoma cells.

  20. Increased expression of chemerin in squamous esophageal cancer myofibroblasts and role in recruitment of mesenchymal stromal cells.

    Directory of Open Access Journals (Sweden)

    J Dinesh Kumar

    Full Text Available Stromal cells such as myofibroblasts influence tumor progression. The mechanisms are unclear but may involve effects on both tumor cells and recruitment of bone marrow-derived mesenchymal stromal cells (MSCs which then colonize tumors. Using iTRAQ and LC-MS/MS we identified the adipokine, chemerin, as overexpressed in esophageal squamous cancer associated myofibroblasts (CAMs compared with adjacent tissue myofibroblasts (ATMs. The chemerin receptor, ChemR23, is expressed by MSCs. Conditioned media (CM from CAMs significantly increased MSC cell migration compared to ATM-CM; the action of CAM-CM was significantly reduced by chemerin-neutralising antibody, pretreatment of CAMs with chemerin siRNA, pretreatment of MSCs with ChemR23 siRNA, and by a ChemR23 receptor antagonist, CCX832. Stimulation of MSCs by chemerin increased phosphorylation of p42/44, p38 and JNK-II kinases and inhibitors of these kinases and PKC reversed chemerin-stimulated MSC migration. Chemerin stimulation of MSCs also induced expression and secretion of macrophage inhibitory factor (MIF that tended to restrict migratory responses to low concentrations of chemerin but not higher concentrations. In a xenograft model consisting of OE21 esophageal cancer cells and CAMs, homing of MSCs administered i.v. was inhibited by CCX832. Thus, chemerin secreted from esophageal cancer myofibroblasts is a potential chemoattractant for MSCs and its inhibition may delay tumor progression.

  1. T-Cadherin Expression in Melanoma Cells Stimulates Stromal Cell Recruitment and Invasion by Regulating the Expression of Chemokines, Integrins and Adhesion Molecules

    Directory of Open Access Journals (Sweden)

    Kseniya A. Rubina

    2015-07-01

    Full Text Available T-cadherin is a glycosyl-phosphatidylinositol (GPI anchored member of the cadherin superfamily involved in the guidance of migrating cells. We have previously shown that in vivo T-cadherin overexpression leads to increased melanoma primary tumor growth due to the recruitment of mesenchymal stromal cells as well as the enhanced metastasis. Since tumor progression is highly dependent upon cell migration and invasion, the aim of the present study was to elucidate the mechanisms of T-cadherin participation in these processes. Herein we show that T-cadherin expression results in the increased invasive potential due to the upregulated expression of pro-oncogenic integrins, chemokines, adhesion molecules and extracellular matrix components. The detected increase in chemokine expression could be responsible for the stromal cell recruitment. At the same time our previous data demonstrated that T-cadherin expression inhibited neoangiogenesis in the primary tumors. We demonstrate molecules and reduction in pro-angiogenic factors. Thus, T-cadherin plays a dual role in melanoma growth and progression: T-cadherin expression results in anti-angiogenic effects in melanoma, however, this also stimulates transcription of genes responsible for migration and invasion of melanoma cells.

  2. Expression of Siglec-11 by human and chimpanzee ovarian stromal cells, with uniquely human ligands: implications for human ovarian physiology and pathology

    Science.gov (United States)

    Wang, Xiaoxia; Chow, Renee; Deng, Liwen; Anderson, Dan; Weidner, Noel; Godwin, Andrew K; Bewtra, Chanda; Zlotnik, Albert; Bui, Jack; Varki, Ajit; Varki, Nissi

    2011-01-01

    Siglecs (Sialic acid-binding Immunoglobulin Superfamily Lectins) are cell surface signaling receptors of the I-type lectin group that recognize sialic acid-bearing glycans. CD33-related-Siglecs are a subset with expression primarily in cells of hematopoietic origin and functional relevance to immune reactions. Earlier we reported a human-specific gene conversion event that markedly changed the coding region for the extracellular domain of Siglec-11, associated with human-specific expression in microglia (Hayakawa T, Angata T, Lewis AL, Mikkelsen TS, Varki NM, Varki A. 2005. A human-specific gene in microglia. Science. 309:1693). Analyzing human gene microarrays to define new patterns of expression, we observed high levels of SIGLEC11 transcript in the ovary and adrenal cortex. Thus, we examined human and chimpanzee tissues using a well-characterized anti-Siglec-11 mouse monoclonal antibody. Although adrenal expression was variable and confined to infiltrating macrophages in capillaries, ovarian expression of Siglec-11 in both humans and chimpanzees was on fibroblasts, the first example of Siglec expression on mesenchyme-derived stromal cells. Cytokines from such ovarian stromal fibroblasts play important roles in follicle development and ovulation. Stable transfection of SIGLEC11 into a primary human ovarian stromal fibroblast cell line altered the secretion of growth-regulated oncogene α, interleukin (IL)-10, IL-7, transforming growth factor β1 and tumor necrosis factor-α, cytokines involved in ovarian physiology. Probing for Siglec-11 ligands revealed distinct and strong mast cell expression in human ovaries, contrasting to diffuse stromal ligands in chimpanzee ovaries. Interestingly, there was a trend of increased Siglec-11 expression in post-menopausal ovaries compared with pre-menopausal ones. Siglec-11 expression was also found on human ovarian stromal tumors and in polycystic ovarian syndrome, a human-specific disease. These results indicate potential

  3. High Stromal Carbonic Anhydrase IX Expression Is Associated With Decreased Survival in p16-Negative Head-and-Neck Tumors

    International Nuclear Information System (INIS)

    Brockton, Nigel; Dort, Joseph; Lau, Harold; Hao, Desiree; Brar, Sony; Klimowicz, Alexander; Petrillo, Stephanie; Diaz, Roman; Doll, Corinne; Magliocco, Anthony

    2011-01-01

    Purpose: Head-and-neck squamous cell carcinoma (HNSCC) is the fifth most common malignancy worldwide. Alcohol use and tobacco use are the most established risk factors; however, human papilloma virus (HPV) infection is a major risk factor for a subset of HNSCCs. Although HPV-positive tumors typically present at a more advanced stage at diagnosis, they are associated with a better prognosis. Tumor hypoxia confers poor prognosis and treatment failure, but direct tumor oxygen measurement is challenging. Endogenous markers of hypoxia (EMHs) have been proposed but have not replicated the prognostic utility of direct oxygen measurement. The expression of endogenous markers of hypoxia may be influenced by oxygen-independent factors, such as the HPV status of the tumor. Methods and Materials: Consecutive cases of locally advanced HNSCC, treated with a uniform regimen of combined radiotherapy and chemotherapy, were identified. Tissue microarrays were assembled from triplicate 0.6-mm cores of archived tumor tissue. HPV status was inferred from semiquantitative p16 immunostaining and directly measured by use of HPV-specific chromogenic in situ hybridization and polymerase chain reaction. Automated quantitative fluorescent immunohistochemistry was conducted to measure epithelial and stromal expression of carbonic anhydrase IX (CAIX) and glucose transporter 1 (GLUT1). Results: High stromal CAIX expression was associated with significantly reduced overall survival (p = 0.03) in patients with p16-negative tumors. Conclusions: This is the first study to use quantitative immunohistochemistry to examine endogenous markers of hypoxia stratified by tumor p16/HPV status. Assessment of CAIX expression in p16-negative HNSCC could identify patients with the least favorable prognosis and inform therapeutic strategies.

  4. Mesenchymal stromal cells reverse hypoxia-mediated suppression of α-smooth muscle actin expression in human dermal fibroblasts

    International Nuclear Information System (INIS)

    Faulknor, Renea A.; Olekson, Melissa A.; Nativ, Nir I.; Ghodbane, Mehdi; Gray, Andrea J.; Berthiaume, François

    2015-01-01

    During wound healing, fibroblasts deposit extracellular matrix that guides angiogenesis and supports the migration and proliferation of cells that eventually form the scar. They also promote wound closure via differentiation into α-smooth muscle actin (SMA)-expressing myofibroblasts, which cause wound contraction. Low oxygen tension typical of chronic nonhealing wounds inhibits fibroblast collagen production and differentiation. It has been suggested that hypoxic mesenchymal stromal cells (MSCs) secrete factors that promote wound healing in animal models; however, it is unclear whether these factors are equally effective on the target cells in a hypoxic wound environment. Here we investigated the impact of MSC-derived soluble factors on the function of fibroblasts cultured in hypoxic fibroblast-populated collagen lattices (FPCLs). Hypoxia alone significantly decreased FPCL contraction and α-SMA expression. MSC-conditioned medium restored hypoxic FPCL contraction and α-SMA expression to levels similar to normoxic FPCLs. (SB431542), an inhibitor of transforming growth factor-β 1 (TGF-β 1 )-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-β 1 at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-β 1 is a major paracrine signal secreted by MSCs that can restore fibroblast functions relevant to the wound healing process and that are impaired in hypoxia. - Highlights: • Fibroblasts were cultured in collagen lattices (FPCLs) as model contracting wounds. • Hypoxia decreased FPCL contraction and fibroblast α-smooth muscle actin expression. • Mesenchymal stromal cells (MSCs) restored function of hypoxic fibroblasts. • MSCs regulate fibroblast function mainly via secreted transforming growth factor-β 1

  5. Mesenchymal stromal cells reverse hypoxia-mediated suppression of α-smooth muscle actin expression in human dermal fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Faulknor, Renea A.; Olekson, Melissa A.; Nativ, Nir I.; Ghodbane, Mehdi; Gray, Andrea J.; Berthiaume, François, E-mail: fberthia@rci.rutgers.edu

    2015-02-27

    During wound healing, fibroblasts deposit extracellular matrix that guides angiogenesis and supports the migration and proliferation of cells that eventually form the scar. They also promote wound closure via differentiation into α-smooth muscle actin (SMA)-expressing myofibroblasts, which cause wound contraction. Low oxygen tension typical of chronic nonhealing wounds inhibits fibroblast collagen production and differentiation. It has been suggested that hypoxic mesenchymal stromal cells (MSCs) secrete factors that promote wound healing in animal models; however, it is unclear whether these factors are equally effective on the target cells in a hypoxic wound environment. Here we investigated the impact of MSC-derived soluble factors on the function of fibroblasts cultured in hypoxic fibroblast-populated collagen lattices (FPCLs). Hypoxia alone significantly decreased FPCL contraction and α-SMA expression. MSC-conditioned medium restored hypoxic FPCL contraction and α-SMA expression to levels similar to normoxic FPCLs. (SB431542), an inhibitor of transforming growth factor-β{sub 1} (TGF-β{sub 1})-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-β{sub 1} at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-β{sub 1} is a major paracrine signal secreted by MSCs that can restore fibroblast functions relevant to the wound healing process and that are impaired in hypoxia. - Highlights: • Fibroblasts were cultured in collagen lattices (FPCLs) as model contracting wounds. • Hypoxia decreased FPCL contraction and fibroblast α-smooth muscle actin expression. • Mesenchymal stromal cells (MSCs) restored function of hypoxic fibroblasts. • MSCs regulate fibroblast function mainly via secreted transforming growth factor-β{sub 1}.

  6. Human ADAM 12 (meltrin alpha) is an active metalloprotease

    DEFF Research Database (Denmark)

    Loechel, F; Gilpin, B J; Engvall, E

    1998-01-01

    The ADAMs (a disintegrin and metalloprotease) are a family of multidomain proteins with structural homology to snake venom metalloproteases. We recently described the cloning and sequencing of human ADAM 12 (meltrin alpha). In this report we provide evidence that the metalloprotease domain of ADAM...

  7. Fetal mesenchymal stromal cells differentiating towards chondrocytes acquire a gene expression profile resembling human growth plate cartilage.

    Directory of Open Access Journals (Sweden)

    Sandy A van Gool

    Full Text Available We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs differentiating towards chondrocytes as an alternative model for the human growth plate (GP. Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether chondrocytes derived from hfMSCs are a suitable model for studying the development and maturation of the GP. hfMSCs efficiently formed hyaline cartilage in a pellet culture in the presence of TGFβ3 and BMP6. Microarray and principal component analysis were applied to study gene expression profiles during chondrogenic differentiation. A set of 232 genes was found to correlate with in vitro cartilage formation. Several identified genes are known to be involved in cartilage formation and validate the robustness of the differentiating hfMSC model. KEGG pathway analysis using the 232 genes revealed 9 significant signaling pathways correlated with cartilage formation. To determine the progression of growth plate cartilage formation, we compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development.

  8. Transitions from mono- to co- to tri-culture uniquely affect gene expression in breast cancer, stromal, and immune compartments.

    Science.gov (United States)

    Regier, Mary C; Maccoux, Lindsey J; Weinberger, Emma M; Regehr, Keil J; Berry, Scott M; Beebe, David J; Alarid, Elaine T

    2016-08-01

    Heterotypic interactions in cancer microenvironments play important roles in disease initiation, progression, and spread. Co-culture is the predominant approach used in dissecting paracrine interactions between tumor and stromal cells, but functional results from simple co-cultures frequently fail to correlate to in vivo conditions. Though complex heterotypic in vitro models have improved functional relevance, there is little systematic knowledge of how multi-culture parameters influence this recapitulation. We therefore have employed a more iterative approach to investigate the influence of increasing model complexity; increased heterotypic complexity specifically. Here we describe how the compartmentalized and microscale elements of our multi-culture device allowed us to obtain gene expression data from one cell type at a time in a heterotypic culture where cells communicated through paracrine interactions. With our device we generated a large dataset comprised of cell type specific gene-expression patterns for cultures of increasing complexity (three cell types in mono-, co-, or tri-culture) not readily accessible in other systems. Principal component analysis indicated that gene expression was changed in co-culture but was often more strongly altered in tri-culture as compared to mono-culture. Our analysis revealed that cell type identity and the complexity around it (mono-, co-, or tri-culture) influence gene regulation. We also observed evidence of complementary regulation between cell types in the same heterotypic culture. Here we demonstrate the utility of our platform in providing insight into how tumor and stromal cells respond to microenvironments of varying complexities highlighting the expanding importance of heterotypic cultures that go beyond conventional co-culture.

  9. Characterization and role of a metalloprotease induced by chitin in Serratia sp. KCK.

    Science.gov (United States)

    Kim, Hyun-Soo; Golyshin, Peter N; Timmis, Kenneth N

    2007-11-01

    A metalloprotease induced by chitin in a new chitinolytic bacterium Serratia sp. Strain KCK was purified and characterized. Compared with other Serratia enzymes, it exhibited a rather broad pH activity range (pH 5.0-8.0), and thermostability. The cognate ORF, mpr, was cloned and expressed. Its deduced amino acid sequence showed high similarity to those of bacterial zinc-binding metalloproteases and a well-conserved serralysin family motif. Pretreatment of chitin with the Mpr protein promoted chitin degradation by chitinase A, which suggests that Mpr participates in, and facilitates, chitin degradation by this microorganism.

  10. Prognostic significance of epithelial/stromal caveolin-1 expression in prostatic hyperplasia, high grade prostatic intraepithelial hyperplasia and prostatic carcinoma and its correlation with microvessel density.

    Science.gov (United States)

    Mohammed, Dareen A; Helal, Duaa S

    2017-03-01

    Caveolin-1 may play a role in cancer development and progression. The aim was to record the expression and localization of caveolin-1 in benign prostatic hyperplasia (BPH), high grade prostatic intraepithelial neoplasia (HGPIN) and prostatic carcinoma (PCa). Microvessel density was evaluated with CD34 immunostain. Correlations with known prognostic factors of PCa were recorded. Immunohistochemical expression of caveolin-1 and the MVD was evaluated in 65 cases; BPH (25), HGPIN (20) and PCa (20). Stromal caveolin-1expression was significantly higher in BPH than HGPIN and PCca. There was significant inverse relation between stromal caveolin-1 expression and extension to lymph node and seminal vesicle in carcinoma cases. Epithelial caveolin-1 was significantly higher in carcinomas than in BPH and HGPIN. Epithelial expression in carcinoma was significantly associated with preoperative PSA, Gleason score and lymph node extension. MVD was significantly higher in PCa than in BPH and HGPIN. There were significant relations between MVD and preoperative PSA, Gleason score, lymph node and seminal vesicle extension. Stromal caveolin-1 was associated with low MVD while epithelial caveolin-1 with high MVD. Caveolin-1 plays an important role in prostatic carcinogenesis and metastasis. Stromal expression of caveolin-1 in PCa is lowered in relation to BPH and HGPIN. In PCa; stromal caveolin-1 was associated with good prognostic parameters. Epithelial caveolin-1 is significantly increased in PCa than BPH and HGPIN. It is associated with clinically aggressive disease. Caveolin-1 may play a role in angiogenesis. Copyright © 2017 National Cancer Institute, Cairo University. Production and hosting by Elsevier B.V. All rights reserved.

  11. Physiology and pathophysiology of matrix metalloproteases

    NARCIS (Netherlands)

    Klein, T.; Bischoff, R.

    Matrix metalloproteases (MMPs) comprise a family of enzymes that cleave protein substrates based on a conserved mechanism involving activation of an active site-bound water molecule by a Zn(2+) ion. Although the catalytic domain of MMPs is structurally highly similar, there are many differences with

  12. Physiology and pathophysiology of matrix metalloproteases

    NARCIS (Netherlands)

    Klein, T; Bischoff, Rainer

    2010-01-01

    Matrix metalloproteases (MMPs) comprise a family of enzymes that cleave protein substrates based on a conserved mechanism involving activation of an active site-bound water molecule by a Zn(2+) ion. Although the catalytic domain of MMPs is structurally highly similar, there are many differences with

  13. The temporal expression of estrogen receptor alpha-36 and runx2 in human bone marrow derived stromal cells during osteogenesis

    International Nuclear Information System (INIS)

    Francis, W.R.; Owens, S.E.; Wilde, C.; Pallister, I.; Kanamarlapudi, V.; Zou, W.; Xia, Z.

    2014-01-01

    Highlights: • ERα36 is the predominant ERα isoform involved in bone regulation in human BMSC. • ERα36 mRNA is significantly upregulated during the process of osteogenesis. • The pattern of ERα36 and runx2 mRNA expression is similar during osteogenesis. • ERα36 appears to be co-localised with runx2 during osteogenesis. - Abstract: During bone maintenance in vivo, estrogen signals through estrogen receptor (ER)-α. The objectives of this study were to investigate the temporal expression of ERα36 and ascertain its functional relevance during osteogenesis in human bone marrow derived stromal cells (BMSC). This was assessed in relation to runt-related transcription factor-2 (runx2), a main modulatory protein involved in bone formation. ERα36 and runx2 subcellular localisation was assessed using immunocytochemistry, and their mRNA expression levels by real time PCR throughout the process of osteogenesis. The osteogenically induced BMSCs demonstrated a rise in ERα36 mRNA during proliferation followed by a decline in expression at day 10, which represents a change in dynamics within the culture between the proliferative stage and the differentiative stage. The mRNA expression profile of runx2 mirrored that of ERα36 and showed a degree subcellular co-localisation with ERα36. This study suggests that ERα36 is involved in the process of osteogenesis in BMSCs, which has implications in estrogen deficient environments

  14. Expression of podoplanin in stromal fibroblasts plays a pivotal role in the prognosis of patients with pancreatic cancer.

    Science.gov (United States)

    Hirayama, Kazuyoshi; Kono, Hiroshi; Nakata, Yuuki; Akazawa, Yoshihiro; Wakana, Hiroyuki; Fukushima, Hisataka; Fujii, Hideki

    2018-01-01

    To investigate the role of podoplanin (PDPN) expression in invasive ductal carcinoma of the pancreas (IDCP) in humans. Tumor samples were obtained from 95 patients with IDCP. Immunohistochemical staining was done to evaluate the expression of PDPN in cancer tissues. PDPN was detected predominantly in stromal fibroblasts, stained with α-smooth muscle actin. The cutoff value of PDPN-positive areas was calculated according to a histogram. There was no significant difference in clinicopathologic factors between patients with high vs. those with low PDPN expression. The high PDPN group showed significantly poorer disease-free and disease-specific survival rates than the low PDPN group. Among patients from the high PDPN group, those with lymph node metastases and those with a tumor larger than 20 cm in diameter had significantly poorer prognoses than similar patients from the low PDPN group. Multivariate Cox proportional hazards analysis indicated that a high expression of PDPN was an independent risk factor for disease-specific survival. PDPN expression in cancer-related fibrotic tissues is associated with a poor prognosis, especially in patients with large tumors or lymph node metastases.

  15. Do Matrix Metalloproteases and Tissue Inhibitors of Metalloproteases in Tenocytes of the Rotator Cuff Differ with Varying Donor Characteristics?

    Directory of Open Access Journals (Sweden)

    Franka Klatte-Schulz

    2015-06-01

    Full Text Available An imbalance between matrix metalloproteases (MMPs and the tissue inhibitors of metalloproteases (TIMPs may have a negative impact on the healing of rotator cuff tears. The aim of the project was to assess a possible relationship between clinical and radiographic characteristics of patients such as the age, sex, as well as the degenerative status of the tendon and the MMPs and TIMPs in their tenocyte-like cells (TLCs. TLCs were isolated from ruptured supraspinatus tendons and quantitative Real-Time PCR and ELISA was performed to analyze the expression and secretion of MMPs and TIMPs. In the present study, MMPs, mostly gelatinases and collagenases such as MMP-2, -9 and -13 showed an increased expression and protein secretion in TLCs of donors with higher age or degenerative status of the tendon. Furthermore, the expression and secretion of TIMP-1, -2 and -3 was enhanced with age, muscle fatty infiltration and tear size. The interaction between MMPs and TIMPs is a complex process, since TIMPs are not only inhibitors, but also activators of MMPs. This study shows that MMPs and TIMPs might play an important role in degenerative tendon pathologies.

  16. Low/Negative Expression of PDGFR-α Identifies the Candidate Primary Mesenchymal Stromal Cells in Adult Human Bone Marrow

    DEFF Research Database (Denmark)

    Li, Hongzhe; Ghazanfari, Roshanak; Zacharaki, Dimitra

    2014-01-01

    Human bone marrow (BM) contains a rare population of nonhematopoietic mesenchymal stromal cells (MSCs), which are of central importance for the hematopoietic microenvironment. However, the precise phenotypic definition of these cells in adult BM has not yet been reported. In this study, we show...... exhibited high levels of genes associated with mesenchymal lineages and HSC supportive function. Moreover, lin(-)/CD45(-)/CD271(+)/CD140a(low/-) cells effectively mediated the ex vivo expansion of transplantable CD34(+) hematopoietic stem cells. Taken together, these data indicate that CD140a is a key...... that low/negative expression of CD140a (PDGFR-α) on lin(-)/CD45(-)/CD271(+) BM cells identified a cell population with very high MSC activity, measured as fibroblastic colony-forming unit frequency and typical in vitro and in vivo stroma formation and differentiation capacities. Furthermore, these cells...

  17. Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells

    DEFF Research Database (Denmark)

    Simonsen, Janne Lytoft; Rosada, Cecilia; Serakinci, Nedime

    2002-01-01

    Human bone marrow stromal cells (hMSCs) were stably transduced by a retroviral vector containing the gene for the catalytic subunit of human telomerase (hTERT). Transduced cells (hMSC-TERTs) had telomerase activity, and the mean telomere length was increased as compared with that of control cells....... The transduced cells have now undergone more than 260 population doublings (PD) and continue to proliferate, whereas control cells underwent senescence-associated proliferation arrest after 26 PD. The cells maintained production of osteoblastic markers and differentiation potential during continuous subculturing......, did not form tumors, and had a normal karyotype. When implanted subcutaneously in immunodeficient mice, the transduced cells formed more bone than did normal cells. These results suggest that ectopic expression of telomerase in hMSCs prevents senescence-associated impairment of osteoblast functions....

  18. Persistence of human parvovirus B19 in multipotent mesenchymal stromal cells expressing the erythrocyte P antigen: implications for transplantation

    Science.gov (United States)

    Sundin, Mikael; Lindblom, Anna; Örvell, Claes; Barrett, A.John; Sundberg, Berit; Watz, Emma; Wikman, Agneta; Broliden, Kristina; Le Blanc, Katarina

    2014-01-01

    Multipotent mesenchymal stromal cells (MSC) are used to improve the outcome of hematopoietic stem cell transplantation and in regenerative medicine. However, MSC may harbor persistent viruses that may compromise their clinical benefit. Retrospectively screened, 1 of 20 MSC from healthy donors contained parvovirus B19 (B19) DNA. We found that MSC express the B19 receptor (the globoside P antigen) and a co-receptor (Ku 80), and can transmit B19 to bone marrow cells in vitro, suggesting that the virus can persist in the marrow stroma of healthy individuals. Two stem cell transplant patients received the B19 positive MSC as treatment for graft-versus-host disease. Neither developed viremia nor symptomatic B19 infection. These results demonstrate for the first time that persistent B19 in MSC can infect hematopoietic cells and underscore the importance of monitoring B19 transmission by MSC products. PMID:18804048

  19. Effect of Different Preconditioning Regimens on the Expression Profile of Murine Adipose-Derived Stromal/Stem Cells

    Directory of Open Access Journals (Sweden)

    Patrick C. Baer

    2018-06-01

    Full Text Available Stem cell-based therapies require cells with a maximum regenerative capacity in order to support regeneration after tissue injury and organ failure. Optimization of this regenerative potential of mesenchymal stromal/stem cells (MSC or their conditioned medium by in vitro preconditioning regimens are considered to be a promising strategy to improve the release of regenerative factors. In the present study, MSC were isolated from inguinal adipose tissue (mASC from C57BL/6 mice, cultured, and characterized. Then, mASC were either preconditioned by incubation in a hypoxic environment (0.5% O2, or in normoxia in the presence of murine epidermal growth factor (EGF or tumor necrosis factor α (TNFα for 48 h. Protein expression was measured by a commercially available array. Selected factors were verified by PCR analysis. The expression of 83 out of 308 proteins (26.9% assayed was found to be increased after preconditioning with TNFα, whereas the expression of 61 (19.8% and 70 (22.7% proteins was increased after incubation with EGF or in hypoxia, respectively. Furthermore, we showed the proliferation-promoting effects of the preconditioned culture supernatants on injured epithelial cells in vitro. Our findings indicate that each preconditioning regimen tested induced an individual expression profile with a wide variety of factors, including several growth factors and cytokines, and therefore may enhance the regenerative potential of mASC for cell-based therapies.

  20. Mesenchymal stromal cells express GARP/LRRC32 on their surface: effects on their biology and immunomodulatory capacity.

    Science.gov (United States)

    Carrillo-Galvez, Ana Belén; Cobo, Marién; Cuevas-Ocaña, Sara; Gutiérrez-Guerrero, Alejandra; Sánchez-Gilabert, Almudena; Bongarzone, Pierpaolo; García-Pérez, Angélica; Muñoz, Pilar; Benabdellah, Karim; Toscano, Miguel G; Martín, Francisco; Anderson, Per

    2015-01-01

    Mesenchymal stromal cells (MSCs) represent a promising tool for therapy in regenerative medicine, transplantation, and autoimmune disease due to their trophic and immunomodulatory activities. However, we are still far from understanding the mechanisms of action of MSCs in these processes. Transforming growth factor (TGF)-β1 is a pleiotropic cytokine involved in MSC migration, differentiation, and immunomodulation. Recently, glycoprotein A repetitions predominant (GARP) was shown to bind latency-associated peptide (LAP)/TGF-β1 to the cell surface of activated Foxp3(+) regulatory T cells (Tregs) and megakaryocytes/platelets. In this manuscript, we show that human and mouse MSCs express GARP which presents LAP/TGF-β1 on their cell surface. Silencing GARP expression in MSCs increased their secretion and activation of TGF-β1 and reduced their proliferative capacity in a TGF-β1-independent manner. Importantly, we showed that GARP expression on MSCs contributed to their ability to inhibit T-cell responses in vitro. In summary, we have found that GARP is an essential molecule for MSC biology, regulating their immunomodulatory and proliferative activities. We envision GARP as a new target for improving the therapeutic efficacy of MSCs and also as a novel MSC marker. © 2014 AlphaMed Press.

  1. Expression profile analysis of aorta-gonad-mesonephros region-derived stromal cells reveals genes that regulate hematopoiesis

    International Nuclear Information System (INIS)

    Nagao, Kenji; Ohta, Takayuki; Hinohara, Atsushi; Tahara, Tomoyuki; Hagiwara, Tetsuya; Maeda, Yoshitake; Yoneya, Takashi; Sohma, Yoshiaki; Heike, Toshio; Nakahata, Tatsutoshi; Inagaki, Yoshimasa; Nishikawa, Mitsuo

    2008-01-01

    The aorta-gonad-mesonephros (AGM) region is involved in the generation and maintenance of the first definitive hematopoietic stem cells (HSCs). A mouse AGM-derived cell line, AGM-S3, was shown to support the development of HSCs. To elucidate the molecular mechanisms regulating early hematopoiesis, we obtained subclones from AGM-S3, one of which was hematopoiesis supportive (S3-A9) and the other one of which was non-supportive (S3-A7), and we analyzed their gene expression profiles by gene chip analysis. In the present study, we found that Glypican-1 (GPC1) was highly expressed in the supportive subclone AGM-S3-A9. Over-expression of GPC1 in non-supportive cells led to the proliferation of progenitor cells in human cord blood when cocultured with the transfected-stromal cells. Thus, GPC1 may have an important role in the establishment of a microenvironment that supports early events in hematopoiesis

  2. Bone marrow stromal cells with a combined expression of BMP-2 and VEGF-165 enhanced bone regeneration

    International Nuclear Information System (INIS)

    Xiao Caiwen; Zhou Huifang; Fu Yao; Gu Ping; Fan Xianqun; Liu Guangpeng; Zhang Peng; Hou Hongliang; Tang Tingting

    2011-01-01

    Bone graft substitutes with osteogenic factors alone often exhibit poor bone regeneration due to inadequate vascularization. Combined delivery of osteogenic and angiogenic factors from biodegradable scaffolds may enhance bone regeneration. We evaluated the effects of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF), combined with natural coral scaffolds, on the repair of critical-sized bone defects in rabbit orbits. In vitro expanded rabbit bone marrow stromal cells (BMSCs) were transfected with human BMP2 and VEGF165 genes. Target protein expression and osteogenic differentiation were confirmed after gene transduction. Rabbit orbital defects were treated with a coral scaffold loaded with BMP2-transduced and VEGF-transduced BMSCs, BMP2-expressing BMSCs, VEGF-expressing BMSCs, or BMSCs without gene transduction. Volume and density of regenerated bone were determined by micro-computed tomography at 4, 8, and 16 weeks after implantation. Neovascularity, new bone deposition rate, and new bone formation were measured by immunostaining, tetracycline and calcein labelling, and histomorphometric analysis at different time points. The results showed that VEGF increased blood vessel formation relative to groups without VEGF. Combined delivery of BMP2 and VEGF increased new bone deposition and formation, compared with any single factor. These findings indicate that mimicking the natural bone development process by combined BMP2 and VEGF delivery improves healing of critical-sized orbital defects in rabbits.

  3. Stromal expression of heat-shock protein 27 is associated with worse clinical outcome in patients with colorectal cancer lung metastases.

    Directory of Open Access Journals (Sweden)

    Thomas Schweiger

    Full Text Available Pulmonary metastases are common in patients with primary colorectal cancer (CRC. Heat-shock protein 27 (Hsp27 is upregulated in activated fibroblasts during wound healing and systemically elevated in various diseases. Cancer-associated fibroblasts (CAFs are also thought to play a role as prognostic and predictive markers in various malignancies including CRC. Surprisingly, the expression of Hsp27 has never been assessed in CAFs. Therefore we aimed to investigate the expression level of Hsp27 in CAFs and its clinical implications in patients with CRC lung metastases.FFPE tissue samples from 51 pulmonary metastases (PMs and 33 paired primary tumors were evaluated for alpha-SMA, CD31, Hsp27 and vimentin expression by immunohistochemistry and correlated with clinicopathological variables. 25 liver metastases served as control group. Moreover, serum samples (n=10 before and after pulmonary metastasectomy were assessed for circulating phospho-Hsp27 and total Hsp27 by ELISA.Stromal expression of Hsp27 was observed in all PM and showed strong correlation with alpha-SMA (P<0.001 and vimentin (P<0.001. Strong stromal Hsp27 was associated with higher microvessel density in primary CRC and PM. Moreover, high stromal Hsp27 and αSMA expression were associated with decreased recurrence-free survival after pulmonary metastasectomy (P=0.018 and P=0.008, respectively and overall survival (P=0.031 and P=0.017, respectively. Serum levels of phospho- and total Hsp27 dropped after metastasectomy to levels comparable to healthy controls.Herein we describe for the first time that Hsp27 is highly expressed in tumor stroma of CRC. Stromal α-SMA and Hsp27 expressions correlate with the clinical outcome after pulmonary metastasectomy. Moreover, serum Hsp27 might pose a future marker for metastatic disease in CRC.

  4. Neoplastic and stromal cells contribute to an extracellular matrix gene expression profile defining a breast cancer subtype likely to progress.

    Directory of Open Access Journals (Sweden)

    Tiziana Triulzi

    Full Text Available We recently showed that differential expression of extracellular matrix (ECM genes delineates four subgroups of breast carcinomas (ECM1, -2, -3- and -4 with different clinical outcome. To further investigate the characteristics of ECM signature and its impact on tumor progression, we conducted unsupervised clustering analyses in 6 additional independent datasets of invasive breast tumors from different platforms for a total of 643 samples. Use of four different clustering algorithms identified ECM3 tumors as an independent group in all datasets tested. ECM3 showed a homogeneous gene pattern, consisting of 58 genes encoding 43 structural ECM proteins. From 26 to 41% of the cases were ECM3-enriched, and analysis of datasets relevant to gene expression in neoplastic or corresponding stromal cells showed that both stromal and breast carcinoma cells can coordinately express ECM3 genes. In in vitro experiments, β-estradiol induced ECM3 gene production in ER-positive breast carcinoma cell lines, whereas TGFβ induced upregulation of the genes leading to ECM3 gene classification, especially in ER-negative breast carcinoma cells and in fibroblasts. Multivariate analysis of distant metastasis-free survival in untreated breast tumor patients revealed a significant interaction between ECM3 and histological grade (p = 0.001. Cox models, estimated separately in grade I-II and grade III tumors, indicated a highly significant association between ECM3 and worse survival probability only in grade III tumors (HR = 3.0, 95% CI = 1.3-7.0, p = 0.0098. Gene Set Enrichment analysis of ECM3 compared to non-ECM3 tumors revealed significant enrichment of epithelial-mesenchymal transition (EMT genes in both grade I-II and grade III subsets of ECM3 tumors. Thus, ECM3 is a robust cluster that identifies breast carcinomas with EMT features but with accelerated metastatic potential only in the undifferentiated (grade III phenotype. These findings support the

  5. Stromal Clues in Endometrial Carcinoma: Loss of Expression of β-Catenin, Epithelial-Mesenchymal Transition Regulators, and Estrogen-Progesterone Receptor.

    Science.gov (United States)

    Senol, Serkan; Sayar, Ilyas; Ceyran, Ayse B; Ibiloglu, Ibrahim; Akalin, Ibrahim; Firat, Ugur; Kosemetin, Duygu; Engin Zerk, Pinar; Aydin, Abdullah

    2016-05-01

    Epithelial-stroma interactions in the endometrium are known to be responsible for physiological functions and emergence of several pathologic lesions. Periglandular stromal cells act on endometrial cells in a paracrine manner through sex hormones. In this study, we immunohistochemically evaluated the expression of epithelial-mesenchymal transition regulators (SNAIL/SLUG, TWIST, ZEB1), adhesion molecules (β-catenin and E-cadhenin), estrogen (ER)-progesterone (PR) receptor and their correlation with each other in 30 benign, 148 hyperplastic (EH), and 101 endometrioid-type endometrial carcinoma (EC) endometria. In the epithelial component, loss of expression in E-cadherin, ER and PR, and overexpression of TWIST and ZEB1 were significantly higher in EC than in EH (P<0.01). In the periglandular stromal component, β-catenin and SNAIL/SLUG expression were significantly higher in normal endometrium and simple without atypical EH compared with complex atypical EH and EC (P<0.01). In addition, periglandular stromal TWIST expression was significantly higher in EH group compared with EC (P<0.05). There was significantly negative correlation between β-catenin and ER, TWIST and ER, and TWIST and PR in hyperplastic and carcinomatous glandular epithelium, whereas there was a significantly positive correlation between β-catenin and SNAIL-SLUG, β-catenin and TWIST, β-catenin and ER, β-catenin and PR, SNAIL-SLUG and ER, SNAIL-SLUG and PR, TWIST and ER, TWIST and PR, in periglandular/cancer-associated stromal cells (P<0.01). In conclusion, the pattern of positive and negative correlations in the expression of epithelial-mesenchymal transition regulators (SNAIL-SLUG and TWIST), sex hormone receptors (ER and PR), and β-catenin between ECs and hyperplasia, as well as between epithelium and stroma herein, is suggestive of a significant role for these proteins and their underlying molecular processes in the development of endometrial carcinomas.

  6. Molecular signature and in vivo behavior of bone marrow endosteal and subendosteal stromal cell populations and their relevance to hematopoiesis

    Energy Technology Data Exchange (ETDEWEB)

    Balduino, Alex, E-mail: balduino@uva.edu.br [School of Dentistry, Veiga de Almeida University, Rio de Janeiro, RJ (Brazil); Mello-Coelho, Valeria [Biomedical Science Institute, Federal University of Rio de Janeiro, Rio de Janeiro, RJ (Brazil); National Institute on Aging, National Institute of Health, Baltimore, MD (United States); Wang, Zhou; Taichman, Russell S.; Krebsbach, Paul H. [Department of Periodontics, Prevention and Geriatrics, University of Michigan School of Dentistry, Ann Arbor, MI (United States); Weeraratna, Ashani T.; Becker, Kevin G. [National Institute on Aging, National Institute of Health, Baltimore, MD (United States); Mello, Wallace de [Instituto Oswaldo Cruz, Rio de Janeiro, RJ (Brazil); Taub, Dennis D. [National Institute on Aging, National Institute of Health, Baltimore, MD (United States); Borojevic, Radovan [Biomedical Science Institute, Federal University of Rio de Janeiro, Rio de Janeiro, RJ (Brazil)

    2012-11-15

    In the bone marrow cavity, hematopoietic stem cells (HSC) have been shown to reside in the endosteal and subendosteal perivascular niches, which play specific roles on HSC maintenance. Although cells with long-term ability to reconstitute full hematopoietic system can be isolated from both niches, several data support a heterogenous distribution regarding the cycling behavior of HSC. Whether this distinct behavior depends upon the role played by the stromal populations which distinctly create these two niches is a question that remains open. In the present report, we used our previously described in vivo assay to demonstrate that endosteal and subendosteal stromal populations are very distinct regarding skeletal lineage differentiation potential. This was further supported by a microarray-based analysis, which also demonstrated that these two stromal populations play distinct, albeit complementary, roles in HSC niche. Both stromal populations were preferentially isolated from the trabecular region and behave distinctly in vitro, as previously reported. Even though these two niches are organized in a very close range, in vivo assays and molecular analyses allowed us to identify endosteal stroma (F-OST) cells as fully committed osteoblasts and subendosteal stroma (F-RET) cells as uncommitted mesenchymal cells mainly represented by perivascular reticular cells expressing high levels of chemokine ligand, CXCL12. Interestingly, a number of cytokines and growth factors including interleukin-6 (IL-6), IL-7, IL-15, Hepatocyte growth factor (HGF) and stem cell factor (SCF) matrix metalloproteases (MMPs) were also found to be differentially expressed by F-OST and F-RET cells. Further microarray analyses indicated important mechanisms used by the two stromal compartments in order to create and coordinate the 'quiescent' and 'proliferative' niches in which hematopoietic stem cells and progenitors reside.

  7. Molecular signature and in vivo behavior of bone marrow endosteal and subendosteal stromal cell populations and their relevance to hematopoiesis

    International Nuclear Information System (INIS)

    Balduino, Alex; Mello-Coelho, Valeria; Wang, Zhou; Taichman, Russell S.; Krebsbach, Paul H.; Weeraratna, Ashani T.; Becker, Kevin G.; Mello, Wallace de; Taub, Dennis D.; Borojevic, Radovan

    2012-01-01

    In the bone marrow cavity, hematopoietic stem cells (HSC) have been shown to reside in the endosteal and subendosteal perivascular niches, which play specific roles on HSC maintenance. Although cells with long-term ability to reconstitute full hematopoietic system can be isolated from both niches, several data support a heterogenous distribution regarding the cycling behavior of HSC. Whether this distinct behavior depends upon the role played by the stromal populations which distinctly create these two niches is a question that remains open. In the present report, we used our previously described in vivo assay to demonstrate that endosteal and subendosteal stromal populations are very distinct regarding skeletal lineage differentiation potential. This was further supported by a microarray-based analysis, which also demonstrated that these two stromal populations play distinct, albeit complementary, roles in HSC niche. Both stromal populations were preferentially isolated from the trabecular region and behave distinctly in vitro, as previously reported. Even though these two niches are organized in a very close range, in vivo assays and molecular analyses allowed us to identify endosteal stroma (F-OST) cells as fully committed osteoblasts and subendosteal stroma (F-RET) cells as uncommitted mesenchymal cells mainly represented by perivascular reticular cells expressing high levels of chemokine ligand, CXCL12. Interestingly, a number of cytokines and growth factors including interleukin-6 (IL-6), IL-7, IL-15, Hepatocyte growth factor (HGF) and stem cell factor (SCF) matrix metalloproteases (MMPs) were also found to be differentially expressed by F-OST and F-RET cells. Further microarray analyses indicated important mechanisms used by the two stromal compartments in order to create and coordinate the “quiescent” and “proliferative” niches in which hematopoietic stem cells and progenitors reside.

  8. Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation.

    Science.gov (United States)

    Sharma, Sunita; Sapkota, Dipak; Xue, Ying; Sun, Yang; Finne-Wistrand, Anna; Bruland, Ove; Mustafa, Kamal

    2016-01-01

    Selection of appropriate osteoinductive growth factors, suitable delivery method and proper supportive scaffold are critical for a successful outcome in bone tissue engineering using bone marrow stromal cells (BMSC). This study examined the molecular and functional effect of a combination of adenoviral mediated expression of bone morphogenetic protein-2 (BMP2) in BMSC and recently developed and characterized, biodegradable Poly(L-lactide-co-є-caprolactone){poly(LLA-co-CL)}scaffolds in osteogenic molecular changes and ectopic bone formation by using in vitro and in vivo approaches. Pathway-focused custom PCR array, validation using TaqMan based quantitative RT-PCR (qRT-PCR) and ALP staining showed significant up-regulation of several osteogenic and angiogenic molecules, including ALPL and RUNX2 in ad-BMP2 BMSC group grown in poly(LLA-co-CL) scaffolds both at 3 and 14 days. Micro CT and histological analyses of the subcutaneously implanted scaffolds in NOD/SCID mice revealed significantly increased radiopaque areas, percentage bone volume and formation of vital bone in ad-BMP2 scaffolds as compared to the control groups both at 2 and 8 weeks. The increased bone formation in the ad-BMP2 group in vivo was paralleled at the molecular level with concomitant over-expression of a number of osteogenic and angiogenic genes including ALPL, RUNX2, SPP1, ANGPT1. The increased bone formation in ad-BMP2 explants was not found to be associated with enhanced endochondral activity as evidenced by qRT-PCR (SOX9 and FGF2) and Safranin O staining. Taken together, combination of adenoviral mediated BMP-2 expression in BMSC grown in the newly developed poly(LLA-co-CL) scaffolds induced expression of osteogenic markers and enhanced bone formation in vivo.

  9. A stable explant culture of HER2/neu invasive carcinoma supported by alpha-Smooth Muscle Actin expressing stromal cells to evaluate therapeutic agents

    Directory of Open Access Journals (Sweden)

    Piechocki Marie P

    2008-04-01

    Full Text Available Abstract Background To gain a better understanding of the effects of therapeutic agents on the tumor microenvironment in invasive cancers, we developed a co-culture model from an invasive lobular carcinoma. Tumor cells expressing HER2/neu organize in nests surrounded by alpha-Smooth Muscle Actin (α-SMA expressing tumor stroma to resemble the morphology of an invading tumor. This co-culture, Mammary Adenocarcinoma Model (MAM-1 maintains a 1:1 ratio of HER2/neu positive tumor cells to α-SMA-reactive stromal cells and renews this configuration for over 20 passages in vitro. Methods We characterized the cellular elements of the MAM-1 model by microarray analysis, and immunocytochemistry. We developed flow cytometric assays to evaluate the relative responses of the tumor and stroma to the tyrosine kinase inhibitor, Iressa. Results The MAM-1 gene expression profile contains clusters that represent the ErbB-2 breast cancer signature and stroma-specific clusters associated with invasive breast cancers. The stability of this model and the ability to antigenically label the tumor and stromal fractions allowed us to determine the specificity of Iressa, a receptor tyrosine kinase inhibitor, for targeting the tumor cell population. Treatment resulted in a selective dose-dependent reduction in phospho-pMEK1/2 and pp44/42MAPK in tumor cells. Within 24 h the tumor cell fraction was reduced 1.9-fold while the stromal cell fraction increased >3-fold, consistent with specific reductions in phospho-pp44/42 MAPK, MEK1/2 and PCNA in tumor cells and reciprocal increases in the stromal cells. Erosion of the tumor cell nests and augmented growth of the stromal cells resembled a fibrotic response. Conclusion This model demonstrates the specificity of Iressa for HER2/neu expressing tumor cells versus the tumor associated myofibroblasts and is appropriate for delineating effects of therapy on signal transduction in the breast tumor microenvironment and improving

  10. A stable explant culture of HER2/neu invasive carcinoma supported by alpha-Smooth Muscle Actin expressing stromal cells to evaluate therapeutic agents

    International Nuclear Information System (INIS)

    Piechocki, Marie P

    2008-01-01

    To gain a better understanding of the effects of therapeutic agents on the tumor microenvironment in invasive cancers, we developed a co-culture model from an invasive lobular carcinoma. Tumor cells expressing HER2/neu organize in nests surrounded by alpha-Smooth Muscle Actin (α-SMA) expressing tumor stroma to resemble the morphology of an invading tumor. This co-culture, Mammary Adenocarcinoma Model (MAM-1) maintains a 1:1 ratio of HER2/neu positive tumor cells to α-SMA-reactive stromal cells and renews this configuration for over 20 passages in vitro. We characterized the cellular elements of the MAM-1 model by microarray analysis, and immunocytochemistry. We developed flow cytometric assays to evaluate the relative responses of the tumor and stroma to the tyrosine kinase inhibitor, Iressa. The MAM-1 gene expression profile contains clusters that represent the ErbB-2 breast cancer signature and stroma-specific clusters associated with invasive breast cancers. The stability of this model and the ability to antigenically label the tumor and stromal fractions allowed us to determine the specificity of Iressa, a receptor tyrosine kinase inhibitor, for targeting the tumor cell population. Treatment resulted in a selective dose-dependent reduction in phospho-pMEK1/2 and pp44/42MAPK in tumor cells. Within 24 h the tumor cell fraction was reduced 1.9-fold while the stromal cell fraction increased >3-fold, consistent with specific reductions in phospho-pp44/42 MAPK, MEK1/2 and PCNA in tumor cells and reciprocal increases in the stromal cells. Erosion of the tumor cell nests and augmented growth of the stromal cells resembled a fibrotic response. This model demonstrates the specificity of Iressa for HER2/neu expressing tumor cells versus the tumor associated myofibroblasts and is appropriate for delineating effects of therapy on signal transduction in the breast tumor microenvironment and improving strategies that can dually or differentially target the tumor and stromal

  11. Low/Negative Expression of PDGFR-α Identifies the Candidate Primary Mesenchymal Stromal Cells in Adult Human Bone Marrow

    Directory of Open Access Journals (Sweden)

    Hongzhe Li

    2014-12-01

    Full Text Available Human bone marrow (BM contains a rare population of nonhematopoietic mesenchymal stromal cells (MSCs, which are of central importance for the hematopoietic microenvironment. However, the precise phenotypic definition of these cells in adult BM has not yet been reported. In this study, we show that low/negative expression of CD140a (PDGFR-α on lin−/CD45−/CD271+ BM cells identified a cell population with very high MSC activity, measured as fibroblastic colony-forming unit frequency and typical in vitro and in vivo stroma formation and differentiation capacities. Furthermore, these cells exhibited high levels of genes associated with mesenchymal lineages and HSC supportive function. Moreover, lin−/CD45−/CD271+/CD140alow/− cells effectively mediated the ex vivo expansion of transplantable CD34+ hematopoietic stem cells. Taken together, these data indicate that CD140a is a key negative selection marker for adult human BM-MSCs, which enables to prospectively isolate a close to pure population of candidate human adult stroma stem/progenitor cells with potent hematopoiesis-supporting capacity.

  12. Pilot Comparison of Stromal Gene Expression among Normal Prostate Tissues and Primary Prostate Cancer Tissues in White and Black Men

    National Research Council Canada - National Science Library

    Bova, G. S

    2006-01-01

    Recent advances in prostate biology suggest that stromal cells surrounding prostate epithelia may play a key role in permitting or stimulating epithelial cells to lose control and form precancerous and cancerous lesions...

  13. Despite differential gene expression profiles pediatric MDS derived mesenchymal stromal cells display functionality in vitro

    Directory of Open Access Journals (Sweden)

    F.G.J. Calkoen

    2015-03-01

    An altered mRNA expression profile, associated with cell survival and malignant transformation, of MSC derived from children with MDS strengthens the hypothesis that the micro-environment is of importance in this disease. Our data support the understanding that pediatric and adult MDS are two different diseases. Further evaluation of the pathways involved might reveal additional therapy targets.

  14. Succinate Dehydrogenase Subunit B (SDHB Is Expressed in Neurofibromatosis 1-Associated Gastrointestinal Stromal Tumors (Gists: Implications for the SDHB Expression Based Classification of Gists

    Directory of Open Access Journals (Sweden)

    Jeanny H. Wang, Jerzy Lasota, Markku Miettinen

    2011-01-01

    Full Text Available Gastrointestinal Stromal Tumor (GIST is the most common mesenchymal tumor of the digestive tract. GISTs develop with relatively high incidence in patients with Neurofibromatosis-1 syndrome (NF1. Mutational activation of KIT or PDGFRA is believed to be a driving force in the pathogenesis of familial and sporadic GISTs. Unlike those tumors, NF1-associated GISTs do not have KIT or PGDFRA mutations. Similarly, no mutational activation of KIT or PDGFRA has been identified in pediatric GISTs and in GISTs associated with Carney Triad and Carney-Stratakis Syndrome. KIT and PDGFRA-wild type tumors are expected to have lesser response to imatinib treatment. Recently, Carney Triad and Carney-Stratakis Syndrome -associated GISTs and pediatric GISTs have been shown to have a loss of expression of succinate dehydrogenase subunit B (SDHB, a Krebs cycle/electron transport chain interface protein. It was proposed that GISTs can be divided into SDHB- positive (type 1, and SDHB-negative (type 2 tumors because of similarities in clinical features and response to imatinib treatment. In this study, SDHB expression was examined immunohistochemically in 22 well-characterized NF1-associated GISTs. All analyzed tumors expressed SDHB. Based on SDHB-expression status, NF1-associated GISTs belong to type 1 category; however, similarly to SDHB type 2 tumors, they do not respond well to imatinib treatment. Therefore, a simple categorization of GISTs into SDHB-positive and-negative seems to be incomplete. A classification based on both SDHB expression status and KIT and PDGFRA mutation status characterize GISTs more accurately and allow subdivision of SDHB-positive tumors into different clinico-genetic categories.

  15. Gene expression pattern of functional neuronal cells derived from human bone marrow mesenchymal stromal cells

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    Bron Dominique

    2008-04-01

    Full Text Available Abstract Background Neuronal tissue has limited potential to self-renew or repair after neurological diseases. Cellular therapies using stem cells are promising approaches for the treatment of neurological diseases. However, the clinical use of embryonic stem cells or foetal tissues is limited by ethical considerations and other scientific problems. Thus, bone marrow mesenchymal stomal cells (BM-MSC could represent an alternative source of stem cells for cell replacement therapies. Indeed, many studies have demonstrated that MSC can give rise to neuronal cells as well as many tissue-specific cell phenotypes. Methods BM-MSC were differentiated in neuron-like cells under specific induction (NPBM + cAMP + IBMX + NGF + Insulin. By day ten, differentiated cells presented an expression profile of real neurons. Functionality of these differentiated cells was evaluated by calcium influx through glutamate receptor AMPA3. Results Using microarray analysis, we compared gene expression profile of these different samples, before and after neurogenic differentiation. Among the 1943 genes differentially expressed, genes down-regulated are involved in osteogenesis, chondrogenesis, adipogenesis, myogenesis and extracellular matrix component (tuftelin, AGC1, FADS3, tropomyosin, fibronectin, ECM2, HAPLN1, vimentin. Interestingly, genes implicated in neurogenesis are increased. Most of them are involved in the synaptic transmission and long term potentialisation as cortactin, CASK, SYNCRIP, SYNTL4 and STX1. Other genes are involved in neurite outgrowth, early neuronal cell development, neuropeptide signaling/synthesis and neuronal receptor (FK506, ARHGAP6, CDKRAP2, PMCH, GFPT2, GRIA3, MCT6, BDNF, PENK, amphiregulin, neurofilament 3, Epha4, synaptotagmin. Using real time RT-PCR, we confirmed the expression of selected neuronal genes: NEGR1, GRIA3 (AMPA3, NEF3, PENK and Epha4. Functionality of these neuron-like cells was demonstrated by Ca2+ influx through glutamate

  16. In vitro differentiation of bone marrow stromal cells into neurons and glial cells and differential protein expression in a two-compartment bone marrow stromal cell/neuron co-culture system.

    Science.gov (United States)

    Qi, Xu; Shao, Ming; Peng, Haisheng; Bi, Zhenggang; Su, Zhiqiang; Li, Hulun

    2010-07-01

    This study was performed to establish a bone marrow stromal cell (BMSC)/neuron two-compartment co-culture model in which differentiation of BMSCs into neurons could occur without direct contact between the two cell types, and to investigate protein expression changes during differentiation of this entirely BMSC-derived population. Cultured BMSCs isolated from Wistar rats were divided into three groups: BMSC culture, BMSC/neuron co-culture and BMSC/neuron two-compartment co-culture. Cells were examined for neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) expression. The electrophysiological behavior of the BMSCs was examined using patch clamping. Proteins that had significantly different expression levels in BMSCs cultured alone and co-cultured with neurons were studied using a protein chip-mass spectroscopy technique. Expression of NSE and GFAP were significantly higher in co-culture cells than in two-compartment co-culture cells, and significantly higher in both co-culture groups than in BMSCs cultured alone. Five proteins showed significant changes in expression during differentiation: TIP39_RAT and CALC_RAT underwent increases, and INSL6_RAT, PNOC_RAT and PCSK1_RAT underwent decreases in expression. We conclude that BMSCs can differentiate into neurons during both contact co-culture with neurons and two-compartment co-culture with neurons. The rate at which BMSCs differentiated into neurons was higher in contact co-culture than in non-contact co-culture.

  17. Genome-wide analysis of gene expression during adipogenesis in human adipose-derived stromal cells reveals novel patterns of gene expression during adipocyte differentiation

    Directory of Open Access Journals (Sweden)

    Melvin Anyasi Ambele

    2016-05-01

    Full Text Available We have undertaken an in-depth transcriptome analysis of adipogenesis in human adipose-derived stromal cells (ASCs induced to differentiate into adipocytes in vitro. Gene expression was assessed on days 1, 7, 14 and 21 post-induction and genes differentially expressed numbered 128, 218, 253 and 240 respectively. Up-regulated genes were associated with blood vessel development, leukocyte migration, as well as tumor growth, invasion and metastasis. They also shared common pathways with certain obesity-related pathophysiological conditions. Down-regulated genes were enriched for immune response processes. KLF15, LMO3, FOXO1 and ZBTB16 transcription factors were up-regulated throughout the differentiation process. CEBPA, PPARG, ZNF117, MLXIPL, MMP3 and RORB were up-regulated only on days 14 and 21, which coincide with the maturation of adipocytes and could possibly serve as candidates for controlling fat accumulation and the size of mature adipocytes. In summary, we have identified genes that were up-regulated only on days 1 and 7 or days 14 and 21 that could serve as potential early and late-stage differentiation markers.

  18. Differential evolution and neofunctionalization of snake venom metalloprotease domains.

    Science.gov (United States)

    Brust, Andreas; Sunagar, Kartik; Undheim, Eivind A B; Vetter, Irina; Yang, Daryl C; Yang, Dary C; Casewell, Nicholas R; Jackson, Timothy N W; Koludarov, Ivan; Alewood, Paul F; Hodgson, Wayne C; Lewis, Richard J; King, Glenn F; Antunes, Agostinho; Hendrikx, Iwan; Fry, Bryan G

    2013-03-01

    Snake venom metalloproteases (SVMP) are composed of five domains: signal peptide, propeptide, metalloprotease, disintegrin, and cysteine-rich. Secreted toxins are typically combinatorial variations of the latter three domains. The SVMP-encoding genes of Psammophis mossambicus venom are unique in containing only the signal and propeptide domains. We show that the Psammophis SVMP propeptide evolves rapidly and is subject to a high degree of positive selection. Unlike Psammophis, some species of Echis express both the typical multidomain and the unusual monodomain (propeptide only) SVMP, with the result that a lower level of variation is exerted upon the latter. We showed that most mutations in the multidomain Echis SVMP occurred in the protease domain responsible for proteolytic and hemorrhagic activities. The cysteine-rich and disintegrin-like domains, which are putatively responsible for making the P-III SVMPs more potent than the P-I and P-II forms, accumulate the remaining variation. Thus, the binding sites on the molecule's surface are evolving rapidly whereas the core remains relatively conserved. Bioassays conducted on two post-translationally cleaved novel proline-rich peptides from the P. mossambicus propeptide domain showed them to have been neofunctionalized for specific inhibition of mammalian a7 neuronal nicotinic acetylcholine receptors. We show that the proline rich postsynaptic specific neurotoxic peptides from Azemiops feae are the result of convergent evolution within the precursor region of the C-type natriuretic peptide instead of the SVMP. The results of this study reinforce the value of studying obscure venoms for biodiscovery of novel investigational ligands.

  19. TGFβ1 attenuates expression of prolactin and IGFBP-1 in decidualized endometrial stromal cells by both SMAD-dependent and SMAD-independent pathways.

    Directory of Open Access Journals (Sweden)

    Nicole M Kane

    Full Text Available BACKGROUND: Decidualization (differentiation of the endometrial stromal cells during the secretory phase of the menstrual cycle is essential for successful implantation. Transforming Growth Factor β1 (TGFβ1 canonically propagates its actions via SMAD signalling. A role for TGFβ1 in decidualization remains to be established and published data concerning effects of TGFβ1 on markers of endometrial decidualization are inconsistent. METHODOLOGY/PRINCIPAL FINDINGS: Non-pregnant endometrial stromal cells (ESC and first trimester decidual stromal cells (DSC were cultured in the presence or absence of a decidualizing stimulus. Incubation of ESCs with TGFβ1 (10 ng/ml down-regulated the expression of transcripts encoding the decidual marker proteins prolactin (PRL, insulin-like growth factor binding protein-1 (IGFBP-1 and tissue factor (TF. TGFβ1 also inhibited secretion of PRL and IGFBP-1 proteins by ESCs and surprisingly this response preceded down-regulation of their mRNAs. In contrast, DSCs were more refractory to the actions of TGFβ1, characterized by blunted and delayed down-regulation of PRL, IGFBP-1, and TF transcripts, which was not associated with a significant reduction in secretion of PRL or IGFBP-1 proteins. Addition of an antibody directed against TGFβ1 increased expression of IGFBP-1 mRNA in decidualised cells. Knockdown of SMAD 4 using siRNAs abrogated the effect of TGFβ1 on expression of PRL in ESCs but did not fully restore expression of IGFBP-1 mRNA and protein. CONCLUSIONS/SIGNIFICANCE: TGFβ1 inhibits the expression and secretion of decidual marker proteins. The impact of TGFβ1 on PRL is SMAD-dependent but the impact on IGFBP1 is via an alternative mechanism. In early pregnancy, resistance of DSC to the impact of TGFβ1 may be important to ensure tissue homeostasis.

  20. A Disintegrin and Metalloprotease 17 in the Cardiovascular and Central Nervous Systems.

    Science.gov (United States)

    Xu, Jiaxi; Mukerjee, Snigdha; Silva-Alves, Cristiane R A; Carvalho-Galvão, Alynne; Cruz, Josiane C; Balarini, Camille M; Braga, Valdir A; Lazartigues, Eric; França-Silva, Maria S

    2016-01-01

    ADAM17 is a metalloprotease and disintegrin that lodges in the plasmatic membrane of several cell types and is able to cleave a wide variety of cell surface proteins. It is somatically expressed in mammalian organisms and its proteolytic action influences several physiological and pathological processes. This review focuses on the structure of ADAM17, its signaling in the cardiovascular system and its participation in certain disorders involving the heart, blood vessels, and neural regulation of autonomic and cardiovascular modulation.

  1. A DISINTEGRIN AND METALLOPROTEASE 17 IN THE CARDIOVASCULAR AND CENTRAL NERVOUS SYSTEMS

    Directory of Open Access Journals (Sweden)

    Jiaxi Xu

    2016-10-01

    Full Text Available ADAM17 is a metalloprotease and disintegrin that lodges in the plasmatic membrane of several cell types and is able to cleave a wide variety of cell surface proteins. It is somatically expressed in mammalian organisms and its proteolytic action influences several physiological and pathological processes. This review focuses on the structure of ADAM17, its signaling in the cardiovascular system and its participation in certain disorders involving the heart, blood vessels and neural regulation of autonomic and cardiovascular modulation.

  2. Prostacyclin Suppresses Twist Expression in the Presence of Indomethacin in Bone Marrow-Derived Mesenchymal Stromal Cells

    Science.gov (United States)

    Kemper, Oliver; Herten, Monika; Fischer, Johannes; Haversath, Marcel; Beck, Sascha; Classen, Tim; Warwas, Sebastian; Tassemeier, Tjark; Landgraeber, Stefan; Lensing-Höhn, Sabine; Krauspe, Rüdiger; Jäger, Marcus

    2014-01-01

    Background Iloprost, a stable prostacyclin I2 analogue, seems to have an osteoblast-protective potential, whereas indomethacin suppresses new bone formation. The aim of this study was to investigate human bone marrow stromal cell (BMSC) proliferation and differentiation towards the osteoblastic lineage by administration of indomethacin and/or iloprost. Material/Methods Human bone marrow cells were obtained from 3 different donors (A=26 yrs/m; B=25 yrs/f, C=35 yrs/m) via vacuum aspiration of the iliac crest followed by density gradient centrifugation and flow cytometry with defined antigens (CD105+/73+/45−/14−). The cells were seeded and incubated as follows: without additives (Group 0; donor A/B/C), with 10−7 M iloprost only (Group 0+ilo; A/B), with indomethacin only in concentrations of 10−6 M (Group 1, A), 10−5 M (Group 2, B), 10−4 M (Group 3, A/B), and together with 10−7 M iloprost (Groups 4–6, A/B/C). On Day 10 and 28, UV/Vis spectrometric and immunocytochemical assays (4 samples per group and donor) were performed to investigate cell proliferation (cell count measurement) and differentiation towards the osteoblastic lineage (CD34−, CD45−, CD105+, type 1 collagen (Col1), osteocalcin (OC), alkaline phosphatase (ALP), Runx2, Twist, specific ALP-activity). Results Indomethacin alone suppressed BMSC differentiation towards the osteoblastic lineage by downregulation of Runx2, Col1, and ALP. In combination with indomethacin, iloprost increased cell proliferation and differentiation and it completely suppressed Twist expression at Day 10 and 28. Iloprost alone did not promote cell proliferation, but moderately enhanced Runx2 and Twist expression. However, the proliferative effects and the specific ALP-activity varied donor-dependently. Conclusions Iloprost partially antagonized the suppressing effects of indomethacin on BMSC differentiation towards the osteoblast lineage. It enhanced the expression of Runx2 and, only in the presence of indomethacin

  3. Characterization of an extensin-modifying metalloprotease: N-terminal processing and substrate cleavage pattern of Pectobacterium carotovorum Prt1

    DEFF Research Database (Denmark)

    Feng, Tao; Nyffenegger, Christian; Højrup, Peter

    2014-01-01

    Compared to other plant cell wall-degrading enzymes, proteases are less well understood. In this study, the extracellular metalloprotease Prt1 from Pectobacterium carotovorum (formerly Erwinia carotovora) was expressed in Escherichia coli and characterized with respect to N-terminal processing...

  4. Extragastrointestinal Stromal Tumor during Pregnacy

    Directory of Open Access Journals (Sweden)

    Ilay Gözükara

    2012-01-01

    Full Text Available Extragastrointestinal stromal tumors (EGISTs are mesenchymal neoplasms without connection to the gastrointestinal tract. Gastrointestinal stromal tumors (GISTs and EGIST are similar according to their clinicopathologic and histomorphologic features. Both of them most often express immunoreactivity for CD-117, a c-kit proto-oncogene protein. The coexistence of GIST and pregnancy is very rare, with only two cases reported in the literature. In this paper, we presented the first EGIST case during pregnancy in the literature.

  5. Abundant Fas expression by gastrointestinal stromal tumours may serve as a therapeutic target for MegaFasL

    NARCIS (Netherlands)

    Rikhof, B.; van der Graaf, W. T. A.; Meijer, C.; Le, P. T. K.; Meersma, G. J.; de Jong, S.; Fletcher, J. A.; Suurmeijer, A. J. H.

    2008-01-01

    Although the tyrosine kinase inhibitor imatinib has been shown to be an active agent in patients with gastrointestinal stromal tumours ( GIST), complete remissions are almost never seen and most patients finally experience disease progression during their course of treatment. An alternative

  6. Abundant Fas expression by gastrointestinal stromal tumours may serve as a therapeutic target for MegaFasL.

    NARCIS (Netherlands)

    Rikhof, B.; Graaf, W.T.A. van der; Meijer, C.; Le, P.T.; Meersma, G.J.; Jong, S. de; Fletcher, J.A.; Suurmeijer, A.J.H.

    2008-01-01

    Although the tyrosine kinase inhibitor imatinib has been shown to be an active agent in patients with gastrointestinal stromal tumours (GIST), complete remissions are almost never seen and most patients finally experience disease progression during their course of treatment. An alternative

  7. Aryl hydrocarbon receptor (AhR agonists suppress interleukin-6 expression by bone marrow stromal cells: an immunotoxicology study

    Directory of Open Access Journals (Sweden)

    Schlezinger Jennifer J

    2003-12-01

    Full Text Available Abstract Background Bone marrow stromal cells produce cytokines required for the normal growth and development of all eight hematopoietic cell lineages. Aberrant cytokine production by stromal cells contributes to blood cell dyscrasias. Consequently, factors that alter stromal cell cytokine production may significantly compromise the development of normal blood cells. We have shown that environmental chemicals, such as aromatic hydrocarbon receptor (AhR agonists, suppress B lymphopoiesis by modulating bone marrow stromal cell function. Here, we extend these studies to evaluate the potential for two prototypic AhR agonists, 7,12-dimethylbenz [a]anthracene (DMBA and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, to alter stromal cell cytokine responses. Methods Bone marrow stromal cells were treated with AhR agonists and bacterial lipopolysaccharide (LPS to mimic innate inflammatory cytokine responses and to study the effects of AhR ligands on those responses. Steady state cytokine RNA levels were screened by RNAse protection assays (RPA and quantified by real-time PCR. Cytokine (IL-6 protein production was measured by ELISA. NF-κB EMSAs were used to study IL-6 transcriptional regulation. Results RPAs indicated that AhR+ bone marrow stromal cells consistently up-regulated genes encoding IL-6 and LIF in response to LPS, presumably through activation of Toll-like receptor 4. Pre-treatment with low doses of DMBA or TCDD selectively abrogated IL-6 gene induction but had no effect on LIF mRNA. Real-time-PCR indicated a significant inhibition of IL-6 mRNA by AhR ligands within 1 hour of LPS challenge which was reflected in a profound down-regulation of IL-6 protein induction, with DMBA and TCDD suppressing IL-6 levels as much as 65% and 88%, respectively. This potent inhibitory effect persisted for at least 72 hours. EMSAs measuring NF-κB binding to IL-6 promoter sequences, an event known to induce IL-6 transcription, indicated a significant decrease in

  8. Characterization of the astacin family of metalloproteases in C. elegans

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    Zapf Richard

    2010-01-01

    Full Text Available Abstract Background Astacins are a large family of zinc metalloproteases found in bacteria and animals. They have diverse roles ranging from digestion of food to processing of extracellular matrix components. The C. elegans genome contains an unusually large number of astacins, of which the majority have not been functionally characterized yet. Results We analyzed the expression pattern of previously uncharacterized members of the astacin family to try and obtain clues to potential functions. Prominent sites of expression for many members of this family are the hypodermis, the alimentary system and several specialized cells including sensory sheath and sockets cells, which are located at openings in the body wall. We isolated mutants affecting representative members of the various subfamilies. Mutants in nas-5, nas-21 and nas-39 (the BMP-1/Tolloid homologue are viable and have no apparent phenotypic defects. Mutants in nas-6 and nas-6; nas-7 double mutants are slow growing and have defects in the grinder of the pharynx, a cuticular structure important for food processing. Conclusions Expression data and phenotypic characterization of selected family members suggest a diversity of functions for members of the astacin family in nematodes. In part this might be due to extracellular structures unique to nematodes.

  9. MS4A1 dysregulation in asbestos-related lung squamous cell carcinoma is due to CD20 stromal lymphocyte expression.

    Directory of Open Access Journals (Sweden)

    Casey M Wright

    Full Text Available Asbestos-related lung cancer accounts for 4-12% of lung cancers worldwide. We have previously identified ADAM28 as a putative oncogene involved in asbestos-related lung adenocarcinoma (ARLC-AC. We hypothesised that similarly gene expression profiling of asbestos-related lung squamous cell carcinomas (ARLC-SCC may identify candidate oncogenes for ARLC-SCC. We undertook a microarray gene expression study in 56 subjects; 26 ARLC-SCC (defined as lung asbestos body (AB counts >20AB/gram wet weight (gww and 30 non-asbestos related lung squamous cell carcinoma (NARLC-SCC; no detectable lung asbestos bodies; 0AB/gww. Microarray and bioinformatics analysis identified six candidate genes differentially expressed between ARLC-SCC and NARLC-SCC based on statistical significance (p2-fold. Two genes MS4A1 and CARD18, were technically replicated by qRT-PCR and showed consistent directional changes. As we also found MS4A1 to be overexpressed in ARLC-ACs, we selected this gene for biological validation in independent test sets (one internal, and one external dataset (2 primary tumor sets. MS4A1 RNA expression dysregulation was validated in the external dataset but not in our internal dataset, likely due to the small sample size in the test set as immunohistochemical (IHC staining for MS4A1 (CD20 showed that protein expression localized predominantly to stromal lymphocytes rather than tumor cells in ARLC-SCC. We conclude that differential expression of MS4A1 in this comparative gene expression study of ARLC-SCC versus NARLC-SCC is a stromal signal of uncertain significance, and an example of the rationale for tumor cell enrichment in preparation for gene expression studies where the aim is to identify markers of particular tumor phenotypes. Finally, our study failed to identify any strong gene candidates whose expression serves as a marker of asbestos etiology. Future research is required to determine the role of stromal lymphocyte MS4A1 dysregulation in

  10. Nitrogen-containing bisphosphonate, YM529/ONO-5920 (a novel minodronic acid), inhibits RANKL expression in a cultured bone marrow stromal cell line ST2

    International Nuclear Information System (INIS)

    Nishida, Shozo; Tsubaki, Masanobu; Hoshino, Mayumi; Namimatsu, Ayumi; Uji, Hiromi; Yoshioka, Shohei; Tanimori, Yoshihiro; Yanae, Masashi; Iwaki, Masahiro; Irimajiri, Kiyohiro

    2005-01-01

    Increase in bone resorption by osteoclasts can cause metabolic bone diseases, such as osteoporosis. Recent attention has been paid to the receptor activator of the NF-κB ligand (RANKL), an accelerator of osteoclast differentiation. RANKL is expressed on the bone marrow-derived stromal cell membrane and induces the differentiation of osteoclasts by binding to RANK expressed on the osteoclast precursor cell membrane. Since the inhibition of RANKL expression can lead to the inhibition of osteoclastic bone resorption, the clinical application of RANKL inhibition could be expected to have a major effect on metabolic bone disease therapy. In this study, we investigated whether or not YM529/ONO-5920, a nitrogen-containing bisphosphonate (a novel minodronic acid), inhibits RANKL expression in a bone marrow-derived stromal cell line (ST2 cells). Reverse transcription-polymerase chain reaction revealed that the administration of YM529/ONO-5920 to ST2 cells inhibited RANKL mRNA expression and reduced RANKL proteins as assessed by Western blot analysis. The inhibition of RANKL mRNA expression was reversed when geranylgeranyl pyrophosphate (GGPP), an intermediate in the mevalonate pathway, was used in combination. Furthermore, YM529/ONO-5920 reduced phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), and similarly, U0126, a mitogen-activated protein kinase kinase 1/2 inhibitor, inhibited RANKL expression. Pretreatment with GGPP reversed the YM529/ONO-5920-induced decrease in phosphorylation of ERK. Furthermore, YM529/ONO-5920 decreased TRAP-positive cells in co-culture of ST2 cells and an osteoclast cell line, C7 cells, and this decrease was inhibited by pretreatment with GGPP. This indicates that YM529/ONO-5920 inhibits GGPP biosynthesis in the mevalonate pathway and then signal transduction in the Ras-mitogen-activated protein kinase pathway, thereby inhibiting RANKL expression on ST2 cells. These results suggest a newly elucidated action of bisphosphonates in

  11. Modulation of the epithelial sodium channel (ENaC by bacterial metalloproteases and protease inhibitors.

    Directory of Open Access Journals (Sweden)

    Michael B Butterworth

    Full Text Available The serralysin family of metalloproteases is associated with the virulence of multiple gram-negative human pathogens, including Pseudomonas aeruginosa and Serratia marcescens. The serralysin proteases share highly conserved catalytic domains and show evolutionary similarity to the mammalian matrix metalloproteases. Our previous studies demonstrated that alkaline protease (AP from Pseudomonas aeruginosa is capable of activating the epithelial sodium channel (ENaC, leading to an increase in sodium absorption in airway epithelia. The serralysin proteases are often co-expressed with endogenous, intracellular or periplasmic inhibitors, which putatively protect the bacterium from unwanted or unregulated protease activities. To evaluate the potential use of these small protein inhibitors in regulating the serralysin induced activation of ENaC, proteases from Pseudomonas aeruginosa and Serratia marcescens were purified for characterization along with a high affinity inhibitor from Pseudomonas. Both proteases showed activity against in vitro substrates and could be blocked by near stoichiometric concentrations of the inhibitor. In addition, both proteases were capable of activating ENaC when added to the apical surfaces of multiple epithelial cells with similar slow activation kinetics. The high-affinity periplasmic inhibitor from Pseudomonas effectively blocked this activation. These data suggest that multiple metalloproteases are capable of activating ENaC. Further, the endogenous, periplasmic bacterial inhibitors may be useful for modulating the downstream effects of the serralysin virulence factors under physiological conditions.

  12. Early osteoinductive human bone marrow mesenchymal stromal/stem cells support an enhanced hematopoietic cell expansion with altered chemotaxis- and adhesion-related gene expression profiles

    Energy Technology Data Exchange (ETDEWEB)

    Sugino, Noriko [Department of Hematology/Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507 (Japan); Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Miura, Yasuo, E-mail: ym58f5@kuhp.kyoto-u.ac.jp [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Yao, Hisayuki [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Iwasa, Masaki; Fujishiro, Aya [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Division of Gastroenterology and Hematology, Shiga University of Medical Science, Shiga 520-2192 (Japan); Fujii, Sumie [Department of Hematology/Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507 (Japan); Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Hirai, Hideyo [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Takaori-Kondo, Akifumi [Department of Hematology/Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507 (Japan); Ichinohe, Tatsuo [Department of Hematology and Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553 (Japan); Maekawa, Taira [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan)

    2016-01-22

    Bone marrow (BM) microenvironment has a crucial role in supporting hematopoiesis. Here, by using a microarray analysis, we demonstrate that human BM mesenchymal stromal/stem cells (MSCs) in an early osteoinductive stage (e-MSCs) are characterized by unique hematopoiesis-associated gene expression with an enhanced hematopoiesis-supportive ability. In comparison to BM-MSCs without osteoinductive treatment, gene expression in e-MSCs was significantly altered in terms of their cell adhesion- and chemotaxis-related profiles, as identified with Gene Ontology and Gene Set Enrichment Analysis. Noteworthy, expression of the hematopoiesis-associated molecules CXCL12 and vascular cell adhesion molecule 1 was remarkably decreased in e-MSCs. e-MSCs supported an enhanced expansion of CD34{sup +} hematopoietic stem and progenitor cells, and generation of myeloid lineage cells in vitro. In addition, short-term osteoinductive treatment favored in vivo hematopoietic recovery in lethally irradiated mice that underwent BM transplantation. e-MSCs exhibited the absence of decreased stemness-associated gene expression, increased osteogenesis-associated gene expression, and apparent mineralization, thus maintaining the ability to differentiate into adipogenic cells. Our findings demonstrate the unique biological characteristics of e-MSCs as hematopoiesis-regulatory stromal cells at differentiation stage between MSCs and osteoprogenitor cells and have significant implications in developing new strategy for using pharmacological osteoinductive treatment to support hematopoiesis in hematopoietic stem and progenitor cell transplantation. - Highlights: • Human BM-MSCs in an early osteoinductive stage (e-MSCs) support hematopoiesis. • Adhesion- and chemotaxis-associated gene signatures are altered in e-MSCs. • Expression of CXCL12 and VCAM1 is remarkably decreased in e-MSCs. • e-MSCs are at differentiation stage between MSCs and osteoprogenitor cells. • Osteoinductive treatment

  13. mRNA-binding protein TIA-1 reduces cytokine expression in human endometrial stromal cells and is down-regulated in ectopic endometrium.

    Science.gov (United States)

    Karalok, Hakan Mete; Aydin, Ebru; Saglam, Ozlen; Torun, Aysenur; Guzeloglu-Kayisli, Ozlem; Lalioti, Maria D; Kristiansson, Helena; Duke, Cindy M P; Choe, Gina; Flannery, Clare; Kallen, Caleb B; Seli, Emre

    2014-12-01

    Cytokines and growth factors play important roles in endometrial function and the pathogenesis of endometriosis. mRNAs encoding cytokines and growth factors undergo rapid turnover; primarily mediated by adenosine- and uridine-rich elements (AREs) located in their 3'-untranslated regions. T-cell intracellular antigen (TIA-1), an mRNA-binding protein, binds to AREs in target transcripts, leading to decreased gene expression. The purpose of this article was to determine whether TIA-1 plays a role in the regulation of endometrial cytokine and growth factor expression during the normal menstrual cycle and whether TIA-1 expression is altered in women with endometriosis. Eutopic endometrial tissue obtained from women without endometriosis (n = 30) and eutopic and ectopic endometrial tissues from women with endometriosis (n = 17) were immunostained for TIA-1. Staining intensities were evaluated by histological scores (HSCOREs). The regulation of endometrial TIA-1 expression by immune factors and steroid hormones was studied by treating primary cultured human endometrial stromal cells (HESCs) with vehicle, lipopolysaccharide, TNF-α, IL-6, estradiol, or progesterone, followed by protein blot analyses. HESCs were engineered to over- or underexpress TIA-1 to test whether TIA-1 regulates IL-6 or TNF-α expression in these cells. We found that TIA-1 is expressed in endometrial stromal and glandular cells throughout the menstrual cycle and that this expression is significantly higher in the perimenstrual phase. In women with endometriosis, TIA-1 expression in eutopic and ectopic endometrium was reduced compared with TIA-1 expression in eutopic endometrium of unaffected control women. Lipopolysaccharide and TNF-α increased TIA-1 expression in HESCs in vitro, whereas IL-6 or steroid hormones had no effect. In HESCs, down-regulation of TIA-1 resulted in elevated IL-6 and TNF-α expression, whereas TIA-1 overexpression resulted in decreased IL-6 and TNF-α expression. Endometrial

  14. Count of splenic stromal precursor cells in mice and expression of cytokine genes in these cells in primary cultures during different periods after immunization of animals with S. typhimurium antigens.

    Science.gov (United States)

    Gorskaya, Yu F; Danilova, T A; Mezentseva, M V; Shapoval, I M; Narovlyanskii, A N; Nesterenko, V G

    2011-06-01

    Injection of S. typhimurium antigens significantly (9-fold) increased cloning efficiency and, hence, the content of stromal precursor cells in the spleen as soon as after 24 h. These parameters returned to normal by days 6-15 after immunization. Cultured splenocytes collected from immune (but not intact) animals expressed the genes of proinflammatory cytokines IL-1β (on days 1, 6, 15) and IL-6 (on days 1 and 6), TNF-α (on days 6 and 15), and of IFN-α and IL-18 (on days 6 and 15). The expression of IL-4 gene was suppressed on day 6 after immunization, of IL-10 gene on days 1 and 6, of IL-6 gene on day 15. Hence, no signs of immune response suppression by stromal cells were found in this system. The spectrum and dynamics of the expression of pro- and anti-inflammatory cytokine genes in stromal cell cultures from the spleen of immunized mice seemed to correspond to those needed for support of the immune response to S. typhimurium antigens, observed in immunized animals. The results indicate possible involvement of stromal cells in the realization of immune response in vivo. The increase of stromal precursor cells cloning efficiency in response to antigen injection could not be reproduced in vitro: the presence of S. typhimurium antigens in primary cultures of intact mouse bone marrow and spleen throughout the entire period of culturing ≈ 20-fold reduced cloning efficiency in cultures.

  15. Prostacyclin Suppresses Twist Expression in the Presence of Indomethacin in Bone Marrow-Derived Mesenchymal Stromal Cells

    OpenAIRE

    Kemper, Oliver; Herten, Monika; Fischer, Johannes; Haversath, Marcel; Beck, Sascha; Classen, Tim; Warwas, Sebastian; Tassemeier, Tjark; Landgraeber, Stefan; Lensing-Höhn, Sabine; Krauspe, Rüdiger; Jäger, Marcus

    2014-01-01

    Background Iloprost, a stable prostacyclin I2 analogue, seems to have an osteoblast-protective potential, whereas indomethacin suppresses new bone formation. The aim of this study was to investigate human bone marrow stromal cell (BMSC) proliferation and differentiation towards the osteoblastic lineage by administration of indomethacin and/or iloprost. Material/Methods Human bone marrow cells were obtained from 3 different donors (A=26 yrs/m; B=25 yrs/f, C=35 yrs/m) via vacuum aspiration of t...

  16. Human umbilical cord mesenchymal stromal cells suppress MHC class II expression on rat vascular endothelium and prolong survival time of cardiac allograft

    Science.gov (United States)

    Qiu, Ying; Yun, Mark M; Han, Xia; Zhao, Ruidong; Zhou, Erxia; Yun, Sheng

    2014-01-01

    Background: Human umbilical cord mesenchymal stromal cells (UC-MSCs) have low immunogenicity and immune regulation. To investigate immunomodulatory effects of human UC-MSCs on MHC class II expression and allograft, we transplanted heart of transgenic rats with MHC class II expression on vascular endothelium. Methods: UC-MSCs were obtained from human umbilical cords and confirmed with flow cytometry analysis. Transgenic rat line was established using the construct of human MHC class II transactivator gene (CIITA) under mouse ICAM-2 promoter control. The induced MHC class II expression on transgenic rat vascular endothelial cells (VECs) was assessed with immunohistological staining. And the survival time of cardiac allograft was compared between the recipients with and without UC-MSC transfusion. Results: Flow cytometry confirmed that the human UC-MSCs were positive for CD29, CD44, CD73, CD90, CD105, CD271, and negative for CD34 and HLA-DR. Repeated infusion of human UC-MSCs reduced MHC class II expression on vascular endothelia of transplanted hearts, and increased survival time of allograft. The UC-MSCs increased regulatory cytokines IL10, transforming growth factor (TGF)-β1 and suppressed proinflammatory cytokines IL2 and IFN-γ in vivo. The UC-MSC culture supernatant had similar effects on cytokine expression, and decreased lymphocyte proliferation in vitro. Conclusions: Repeated transfusion of the human UC-MSCs reduced MHC class II expression on vascular endothelia and prolonged the survival time of rat cardiac allograft. PMID:25126177

  17. Caspase-8 regulates the expression of pro- and anti-inflammatory cytokines in human bone marrow-derived mesenchymal stromal cells.

    Science.gov (United States)

    Moen, Siv H; Westhrin, Marita; Zahoor, Muhammad; Nørgaard, Nikolai N; Hella, Hanne; Størdal, Berit; Sundan, Anders; Nilsen, Nadra J; Sponaas, Anne-Marit; Standal, Therese

    2016-09-01

    Mesenchymal stem cells, also called mesenchymal stromal cells, MSCs, have great potential in stem cell therapy partly due to their immunosuppressive properties. How these cells respond to chronic inflammatory stimuli is therefore of importance. Toll-like receptors (TLR)s are innate immune receptors that mediate inflammatory signals in response to infection, stress, and damage. Caspase-8 is involved in activation of NF-kB downstream of TLRs in immune cells. Here we investigated the role of caspase-8 in regulating TLR-induced cytokine production from human bone marrow-derived mesenchymal stromal cells (hBMSCs). Cytokine expression in hBMCs in response to poly(I:C) and LPS was evaluated by PCR, multiplex cytokine assay, and ELISA. TLR3, TRIF, and caspase-8 were silenced using siRNA. Caspase-8 was also inhibited using a caspase-8 inhibitor, z-IEDT. We found that TLR3 agonist poly(I:C) and TLR4 agonist LPS induced secretion of several pro-inflammatory cytokines in a TLR-dependent manner which required the TLR signaling adaptor molecule TRIF. Further, poly(I:C) reduced the expression of anti-inflammatory cytokines HGF and TGFβ whereas LPS reduced HGF expression only. Notably, caspase-8 was involved in the induction of IL- IL-1β, IL-6, CXCL10, and in the inhibition of HGF and TGFβ. Caspase-8 appears to modulate hBMSCs into gaining a pro-inflammatory phenotype. Therefore, inhibiting caspase-8 in hBMSCs might promote an immunosuppressive phenotype which could be useful in clinical applications to treat inflammatory disorders.

  18. Caspase‐8 regulates the expression of pro‐ and anti‐inflammatory cytokines in human bone marrow‐derived mesenchymal stromal cells

    Science.gov (United States)

    Moen, Siv H.; Westhrin, Marita; Zahoor, Muhammad; Nørgaard, Nikolai N.; Hella, Hanne; Størdal, Berit; Sundan, Anders; Nilsen, Nadra J.; Sponaas, Anne‐Marit

    2016-01-01

    Abstract Introduction Mesenchymal stem cells, also called mesenchymal stromal cells, MSCs, have great potential in stem cell therapy partly due to their immunosuppressive properties. How these cells respond to chronic inflammatory stimuli is therefore of importance. Toll‐like receptors (TLR)s are innate immune receptors that mediate inflammatory signals in response to infection, stress, and damage. Caspase‐8 is involved in activation of NF‐kB downstream of TLRs in immune cells. Here we investigated the role of caspase‐8 in regulating TLR‐induced cytokine production from human bone marrow‐derived mesenchymal stromal cells (hBMSCs). Methods Cytokine expression in hBMCs in response to poly(I:C) and LPS was evaluated by PCR, multiplex cytokine assay, and ELISA. TLR3, TRIF, and caspase‐8 were silenced using siRNA. Caspase‐8 was also inhibited using a caspase‐8 inhibitor, z‐IEDT. Results We found that TLR3 agonist poly(I:C) and TLR4 agonist LPS induced secretion of several pro‐inflammatory cytokines in a TLR‐dependent manner which required the TLR signaling adaptor molecule TRIF. Further, poly(I:C) reduced the expression of anti‐inflammatory cytokines HGF and TGFβ whereas LPS reduced HGF expression only. Notably, caspase‐8 was involved in the induction of IL‐ IL‐1β, IL‐6, CXCL10, and in the inhibition of HGF and TGFβ. Conclusion Caspase‐8 appears to modulate hBMSCs into gaining a pro‐inflammatory phenotype. Therefore, inhibiting caspase‐8 in hBMSCs might promote an immunosuppressive phenotype which could be useful in clinical applications to treat inflammatory disorders. PMID:27621815

  19. Inducible indoleamine 2,3-dioxygenase 1 and programmed death ligand 1 expression as the potency marker for mesenchymal stromal cells.

    Science.gov (United States)

    Guan, Qingdong; Li, Yun; Shpiruk, Tanner; Bhagwat, Swaroop; Wall, Donna A

    2018-05-01

    Establishment of a potency assay in the manufacturing of clinical-grade mesenchymal stromal cells (MSCs) has been a challenge due to issues of relevance to function, timeline and variability of responder cells. In this study, we attempted to develop a potency assay for MSCs. Clinical-grade bone marrow-derived MSCs were manufactured. The phenotype and immunosuppressive functions of the MSCs were evaluated based on the International Society for Cellular Therapy guidelines. Resting MSCs licensed by interferon (IFN)-γ exposure overnight were evaluated for changes in immune suppression and immune-relevant proteins. The relationship of immune-relevant protein expression with immunosuppression of MSCs was analyzed. MSC supressed third-party T-lymphocyte proliferation with high inter-donor and inter-test variability. The suppression of T-lymphocyte proliferation by IFN-γ-licensed MSCs correlated with that by resting MSCs. Many cellular proteins were up-regulated after IFN-γ exposure, including indoleamine 2,3-dioxygenase 1 (IDO-1), programmed death ligand 1 (PD-L1), vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1) and bone marrow stromal antigen 2 (BST-2). The expression levels of IDO-1 and PD-L1 on licensed MSCs, not VCAM-1, ICAM-1 or BST-2 on licensed MSCs, correlated with MSC suppression of third-party T-cell proliferation. A flow cytometry-based assay of MSCs post-IFN-γ exposure measuring expression of intracellular protein IDO-1 and cell surface protein PD-L1 captures two mechanisms of suppression and offers the potential of a relevant, rapid assay for MSC-mediated immune suppression that would fit with the manufacturing process. Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  20. Rosiglitazone attenuates the metalloprotease/anti-metalloprotease imbalance in emphysema induced by cigarette smoke: involvement of extracellular signal-regulated kinase and NFκB signaling

    Directory of Open Access Journals (Sweden)

    Hou G

    2015-04-01

    Full Text Available Gang Hou, Yan Yin, Dan Han, Qiu-yue Wang, Jian Kang Department of Respiratory Medicine, the First Hospital of China Medical University, Shenyang, People’s Republic of China Objective: We investigated how rosiglitazone attenuated cigarette smoke (CS-induced emphysema in a rat model. In particular, we focused on its possible effects on the imbalance between metalloprotease (MMP and anti-MMP activity, mitogen-activated protein kinase (MAPK phosphorylation, and nuclear factor kappa-light-chain-enhancer of activated B cell (NFκB signaling pathway over-activation.Methods: A total of 36 Wistar rats were divided into three groups (n=12 each: animals were exposed to CS for 12 weeks in the absence (the CS group or presence of 30 mg/kg rosiglitazone (the rosiglitazone-CS [RCS] group; a control group was treated with the rosiglitazone vehicle only, without any CS exposure. Histopathology of lung tissue in all groups was evaluated to grade severity of the disease. Expression levels of peroxisome proliferator-activated receptor γ (PPARγ, MMP2, and MMP9 in lung tissue were determined and compared using Western blotting and immunohistochemistry. Activation of MAPKs, NFκB, and the nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor, alpha (IκBα phosphorylation in lung tissue was examined by Western blotting.Results: Emphysema-related pathology, based on inter-alveolar wall distance and alveolar density, was less severe in the RCS group than in the CS group. Compared with the CS group, levels of PPARγ were higher in the RCS group, and levels of MMP2 and MMP9 proteins were lower in the RCS rats. Levels of activated MAPKs and NFκB were also lower, while the IκBαphosphorylation was increased in the lung tissue of RCS rats.Conclusion: Our findings suggest that oral administration of rosiglitazone attenuates the metalloprotease activity induced by CS, and the underlying mechanism might involve the activation of signaling pathways

  1. GATA-4 and FOG-2 expression in pediatric ovarian sex cord-stromal tumors replicates embryonal gonadal phenotype: results from the TREP project.

    Science.gov (United States)

    Virgone, Calogero; Cecchetto, Giovanni; Ferrari, Andrea; Bisogno, Gianni; Donofrio, Vittoria; Boldrini, Renata; Collini, Paola; Dall'Igna, Patrizia; Alaggio, Rita

    2012-01-01

    GATA proteins are a family of zinc finger transcription factors regulating gene expression, differentiation and proliferation in various tissues. The expression of GATA-4 and FOG-2, one of its modulators, was studied in pediatric Sex Cord-Stromal tumors of the ovary, in order to evaluate their potential role as diagnostic markers and prognostic factors. Clinical and histological data of 15 patients, enrolled into the TREP Project since 2000 were evaluated. When available, immunostaines for FOG-2, GATA-4, α-Inhibin, Vimentin and Pancytokeratin were also analyzed. In our series there were 6 Juvenile Granulosa Cell Tumors (JGCT), 6 Sertoli-Leydig Cell Tumors (SLCT), 1 Cellular Fibroma, 1 Theca Cell Tumor and 1 Stromal Sclerosing Tumor (SST). Thirteen patients obtained a complete remission (CR), 1 reached a second CR after the removal of a metachronous tumor and 1 died of disease. Inhibin was detectable in 11/15, Vimentin in 13/15, Pancytokeratin in 6/15, GATA-4 in 5/13 and FOG-2 in 11/15. FOG-2 was highly expressed in 5/6 JGCT, while GATA-4 was weakly detectable only in 1 of the cases. SLCT expressed diffusely FOG-2 (4/6) and GATA-4 (3/5). GATA-4 and FOG-2 were detected in fibroma and thecoma but not in the SST. Pediatric granulosa tumors appear to express a FOG-2/GATA-4 phenotype in keeping with primordial ovarian follicles. High expression of GATA-4 does not correlate with aggressive behaviour as seen in adults, but it is probably involved in cell proliferation its absence can be associated with the better outcome of JGCT. SLCTs replicate the phenotype of Sertoli cells during embryogenesis in normal testis. In this group, the lack of expression of FOG-2 in tumors in advanced stages might reveal a hypothetical role in inhibiting GATA-4 cell proliferation pathway. In fibroma/thecoma group GATA-4 and FOG-2 point out the abnormal activation of GATA pathway and might be involved in the onset of these tumors.

  2. Progestins Upregulate FKBP51 Expression in Human Endometrial Stromal Cells to Induce Functional Progesterone and Glucocorticoid Withdrawal: Implications for Contraceptive- Associated Abnormal Uterine Bleeding.

    Directory of Open Access Journals (Sweden)

    Ozlem Guzeloglu Kayisli

    Full Text Available Use of long-acting progestin only contraceptives (LAPCs offers a discrete and highly effective family planning method. Abnormal uterine bleeding (AUB is the major side effect of, and cause for, discontinuation of LAPCs. The endometria of LAPC-treated women display abnormally enlarged, fragile blood vessels, decreased endometrial blood flow and oxidative stress. To understanding to mechanisms underlying AUB, we propose to identify LAPC-modulated unique gene cluster(s in human endometrial stromal cells (HESCs. Protein and RNA isolated from cultured HESCs treated 7 days with estradiol (E2 or E2+ medroxyprogesterone acetate (MPA or E2+ etonogestrel (ETO or E2+ progesterone (P4 were analyzed by quantitative Real-time (q-PCR and immunoblotting. HSCORES were determined for immunostained-paired endometria of pre-and 3 months post-Depot MPA (DMPA treated women and ovariectomized guinea pigs (GPs treated with placebo or E2 or MPA or E2+MPA for 21 days. In HESCs, whole genome analysis identified a 67 gene group regulated by all three progestins, whereas a 235 gene group was regulated by E2+ETO and E2+MPA, but not E2+P4. Ingenuity pathway analysis identified glucocorticoid receptor (GR activation as one of upstream regulators of the 235 MPA and ETO-specific genes. Among these, microarray results demonstrated significant enhancement of FKBP51, a repressor of PR/GR transcriptional activity, by both MPA and ETO. q-PCR and immunoblot analysis confirmed the microarray results. In endometria of post-DMPA versus pre-DMPA administered women, FKBP51 expression was significantly increased in endometrial stromal and glandular cells. In GPs, E2+MPA or MPA significantly increased FKBP51 immunoreactivity in endometrial stromal and glandular cells versus placebo- and E2-administered groups. MPA or ETO administration activates GR signaling and increases endometrial FKBP51 expression, which could be one of the mechanisms causing AUB by inhibiting PR and GR

  3. Progestins Upregulate FKBP51 Expression in Human Endometrial Stromal Cells to Induce Functional Progesterone and Glucocorticoid Withdrawal: Implications for Contraceptive- Associated Abnormal Uterine Bleeding.

    Science.gov (United States)

    Guzeloglu Kayisli, Ozlem; Kayisli, Umit A; Basar, Murat; Semerci, Nihan; Schatz, Frederick; Lockwood, Charles J

    2015-01-01

    Use of long-acting progestin only contraceptives (LAPCs) offers a discrete and highly effective family planning method. Abnormal uterine bleeding (AUB) is the major side effect of, and cause for, discontinuation of LAPCs. The endometria of LAPC-treated women display abnormally enlarged, fragile blood vessels, decreased endometrial blood flow and oxidative stress. To understanding to mechanisms underlying AUB, we propose to identify LAPC-modulated unique gene cluster(s) in human endometrial stromal cells (HESCs). Protein and RNA isolated from cultured HESCs treated 7 days with estradiol (E2) or E2+ medroxyprogesterone acetate (MPA) or E2+ etonogestrel (ETO) or E2+ progesterone (P4) were analyzed by quantitative Real-time (q)-PCR and immunoblotting. HSCORES were determined for immunostained-paired endometria of pre-and 3 months post-Depot MPA (DMPA) treated women and ovariectomized guinea pigs (GPs) treated with placebo or E2 or MPA or E2+MPA for 21 days. In HESCs, whole genome analysis identified a 67 gene group regulated by all three progestins, whereas a 235 gene group was regulated by E2+ETO and E2+MPA, but not E2+P4. Ingenuity pathway analysis identified glucocorticoid receptor (GR) activation as one of upstream regulators of the 235 MPA and ETO-specific genes. Among these, microarray results demonstrated significant enhancement of FKBP51, a repressor of PR/GR transcriptional activity, by both MPA and ETO. q-PCR and immunoblot analysis confirmed the microarray results. In endometria of post-DMPA versus pre-DMPA administered women, FKBP51 expression was significantly increased in endometrial stromal and glandular cells. In GPs, E2+MPA or MPA significantly increased FKBP51 immunoreactivity in endometrial stromal and glandular cells versus placebo- and E2-administered groups. MPA or ETO administration activates GR signaling and increases endometrial FKBP51 expression, which could be one of the mechanisms causing AUB by inhibiting PR and GR-mediated transcription

  4. The mitochondrial inner membrane AAA metalloprotease family in metazoans.

    Science.gov (United States)

    Juhola, M K; Shah, Z H; Grivell, L A; Jacobs, H T

    2000-09-15

    Three metalloproteases belonging to the AAA superfamily (Yme1p, Afg3p and Rca1p) are involved in protein turnover and respiratory chain complex assembly in the yeast inner mitochondrial membrane. Analysis of the completed genome sequences of Caenorhabditis elegans and Drosophila melanogaster indicates that this gene family typically comprises 3-4 members in metazoans. Phylogenetic analysis reveals three main branches represented, respectively, by Saccharomyces cerevisiae YME1, human SPG7 (paraplegin) and S. cerevisiae AFG3 and RCA1. mt-AAA metalloproteases are weak candidates for several previously studied Drosophila mutants. A full elucidation of the cellular and physiological roles of mt-AAA metalloproteases in metazoans will require the creation of targeted mutations.

  5. Transforming growth factor β-activated kinase 1 negatively regulates interleukin-1α-induced stromal-derived factor-1 expression in vascular smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Bin [Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huangzhong University of Science and Technology, Wuhan 430022 (China); Li, Wei [Department of Gerontology, Union Hospital, Tongji Medical College, Huangzhong University of Science and Technology, Wuhan 430022 (China); Zheng, Qichang [Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huangzhong University of Science and Technology, Wuhan 430022 (China); Qin, Tao [Department of Hepatobiliary Pancreatic Surgery, People' s Hospital of Zhengzhou University, School of Medicine, Zhengzhou University, Zhengzhou 450003 (China); Wang, Kun; Li, Jinjin; Guo, Bing; Yu, Qihong; Wu, Yuzhe; Gao, Yang; Cheng, Xiang; Hu, Shaobo; Kumar, Stanley Naveen [Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huangzhong University of Science and Technology, Wuhan 430022 (China); Liu, Sanguang, E-mail: sanguang1998@sina.com [Department of Hepatobiliary Surgery, The Second Hospital, Hebei Medical University, Shijiazhuang 050000 (China); Song, Zifang, E-mail: zsong@hust.edu.cn [Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huangzhong University of Science and Technology, Wuhan 430022 (China)

    2015-07-17

    Stromal-derived Factor-1 (SDF-1) derived from vascular smooth muscle cells (VSMCs) contributes to vascular repair and remodeling in various vascular diseases. In this study, the mechanism underlying regulation of SDF-1 expression by interleukin-1α (IL-1α) was investigated in primary rat VSMCs. We found IL-1α promotes SDF-1 expression by up-regulating CCAAT-enhancer-binding protein β (C/EBPβ) in an IκB kinase β (IKKβ) signaling-dependent manner. Moreover, IL-1α-induced expression of C/EBPβ and SDF-1 was significantly potentiated by knockdown of transforming growth factor β-activated kinase 1 (TAK1), an upstream activator of IKKβ signaling. In addition, we also demonstrated that TAK1/p38 mitogen-activated protein kinase (p38 MAPK) signaling exerted negative effect on IL-1α-induced expression of C/EBPβ and SDF-1 through counteracting ROS-dependent up-regulation of nuclear factor erythroid 2-related factor 2 (NRF2). In conclusion, TAK1 acts as an important regulator of IL-1α-induced SDF-1 expression in VSMCs, and modulating activity of TAK1 may serve as a potential strategy for modulating vascular repair and remodeling. - Highlights: • IL-1α induces IKKβ signaling-dependent SDF-1 expression by up-regulating C/EBPβ. • Activation of TAK1 by IL-1α negatively regulates C/EBPβ-dependent SDF-1 expression. • IL-1α-induced TAK1/p38 MAPK signaling counteracts ROS-dependent SDF-1 expression. • TAK1 counteracts IL-1α-induced SDF-1 expression by attenuating NRF2 up-regulation.

  6. Transforming growth factor β-activated kinase 1 negatively regulates interleukin-1α-induced stromal-derived factor-1 expression in vascular smooth muscle cells

    International Nuclear Information System (INIS)

    Yang, Bin; Li, Wei; Zheng, Qichang; Qin, Tao; Wang, Kun; Li, Jinjin; Guo, Bing; Yu, Qihong; Wu, Yuzhe; Gao, Yang; Cheng, Xiang; Hu, Shaobo; Kumar, Stanley Naveen; Liu, Sanguang; Song, Zifang

    2015-01-01

    Stromal-derived Factor-1 (SDF-1) derived from vascular smooth muscle cells (VSMCs) contributes to vascular repair and remodeling in various vascular diseases. In this study, the mechanism underlying regulation of SDF-1 expression by interleukin-1α (IL-1α) was investigated in primary rat VSMCs. We found IL-1α promotes SDF-1 expression by up-regulating CCAAT-enhancer-binding protein β (C/EBPβ) in an IκB kinase β (IKKβ) signaling-dependent manner. Moreover, IL-1α-induced expression of C/EBPβ and SDF-1 was significantly potentiated by knockdown of transforming growth factor β-activated kinase 1 (TAK1), an upstream activator of IKKβ signaling. In addition, we also demonstrated that TAK1/p38 mitogen-activated protein kinase (p38 MAPK) signaling exerted negative effect on IL-1α-induced expression of C/EBPβ and SDF-1 through counteracting ROS-dependent up-regulation of nuclear factor erythroid 2-related factor 2 (NRF2). In conclusion, TAK1 acts as an important regulator of IL-1α-induced SDF-1 expression in VSMCs, and modulating activity of TAK1 may serve as a potential strategy for modulating vascular repair and remodeling. - Highlights: • IL-1α induces IKKβ signaling-dependent SDF-1 expression by up-regulating C/EBPβ. • Activation of TAK1 by IL-1α negatively regulates C/EBPβ-dependent SDF-1 expression. • IL-1α-induced TAK1/p38 MAPK signaling counteracts ROS-dependent SDF-1 expression. • TAK1 counteracts IL-1α-induced SDF-1 expression by attenuating NRF2 up-regulation

  7. Evaluation of mRNA expression levels and electrophysiological function of neuron-like cells derived from canine bone marrow stromal cells.

    Science.gov (United States)

    Nakano, Rei; Edamura, Kazuya; Sugiya, Hiroshi; Narita, Takanori; Okabayashi, Ken; Moritomo, Tadaaki; Teshima, Kenji; Asano, Kazushi; Nakayama, Tomohiro

    2013-10-01

    To investigate the in vitro differentiation of canine bone marrow stromal cells (BMSCs) into functional, mature neurons. Bone marrow from 6 adult dogs. BMSCs were isolated from bone marrow and chemically induced to develop into neurons. The morphology of the BMSCs during neuronal induction was monitored, and immunocytochemical analyses for neuron markers were performed after the induction. Real-time PCR methods were used to evaluate the mRNA expression levels of markers for neural stem or progenitor cells, neurons, and ion channels, and western blotting was used to assess the expression of neuronal proteins before and after neuronal induction. The electrophysiological properties of the neuron-like cells induced from canine BMSCs were evaluated with fluorescent dye to monitor Ca(2)+ influx. Canine BMSCs developed a neuron-like morphology after neuronal induction. Immunocytochemical analysis revealed that these neuron-like cells were positive for neuron markers. After induction, the cells' mRNA expression levels of almost all neuron and ion channel markers increased, and the protein expression levels of nestin and neurofilament-L increased significantly. However, the neuron-like cells derived from canine BMSCs did not have the Ca(2)+ influx characteristic of spiking neurons. Although canine BMSCs had neuron-like morphological and biochemical properties after induction, they did not develop the electrophysiological characteristics of neurons. Thus, these results have suggested that canine BMSCs could have the capacity to differentiate into a neuronal lineage, but the differentiation protocol used may have been insufficient to induce development into functional neurons.

  8. Quantum Dots-Based Immunofluorescent Imaging of Stromal Fibroblasts Caveolin-1 and Light Chain 3B Expression and Identification of Their Clinical Significance in Human Gastric Cancer

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    Honglei Chen

    2012-10-01

    Full Text Available Caveolin-1 (Cav-1 expression deficiency and autophagy in tumor stromal fibroblasts (hereafter fibroblasts are involved in tumor proliferation and progression, particularly in breast and prostate cancer. The aim of this study was to detect the expression of fibroblastic Cav-1 and LC3B, markers of autophagy, in gastric cancer (GC and to analyze their clinical significances. Furthermore, because Epstein-Barr virus (EBV-associated GC (EBVaGC is a unique subtype of GC; we compared the differential expression of fibroblastic Cav-1 and LC3B in EBVaGC and non-EBVaGC. Quantum dots (QDs-based immunofluorescence histochemistry was used to examine the expression of fibroblastic Cav-1 and LC3B in 118 cases of GC with adequate stroma. QDs-based double immunofluorescence labeling was performed to detect the coexpression of Cav-1 and LC3B proteins. EBV-encoded small RNA was detected by QDs-based fluorescence in situ hybridization to identify EBVaGC. Multivariate analysis indicated that low fibroblastic Cav-1 level was an independent prognosticator (p = 0.029 that predicted poorer survival of GC patients. Positive fibroblastic LC3B was correlated with lower invasion (p = 0.032 and was positively associated with Cav-1 expression (r = 0.432, p < 0.001. EBV infection did not affect fibroblastic Cav-1 and LC3B expression. In conclusion, positive fibroblastic LC3B correlates with lower invasion, and low expression of fibroblastic Cav-1 is a novel predictor of poor GC prognosis.

  9. Pulsed Electromagnetic Field Regulates MicroRNA 21 Expression to Activate TGF-β Signaling in Human Bone Marrow Stromal Cells to Enhance Osteoblast Differentiation

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    Nagarajan Selvamurugan

    2017-01-01

    Full Text Available Pulsed electromagnetic fields (PEMFs have been documented to promote bone fracture healing in nonunions and increase lumbar spinal fusion rates. However, the molecular mechanisms by which PEMF stimulates differentiation of human bone marrow stromal cells (hBMSCs into osteoblasts are not well understood. In this study the PEMF effects on hBMSCs were studied by microarray analysis. PEMF stimulation of hBMSCs’ cell numbers mainly affected genes of cell cycle regulation, cell structure, and growth receptors or kinase pathways. In the differentiation and mineralization stages, PEMF regulated preosteoblast gene expression and notably, the transforming growth factor-beta (TGF-β signaling pathway and microRNA 21 (miR21 were most highly regulated. PEMF stimulated activation of Smad2 and miR21-5p expression in differentiated osteoblasts, and TGF-β signaling was essential for PEMF stimulation of alkaline phosphatase mRNA expression. Smad7, an antagonist of the TGF-β signaling pathway, was found to be miR21-5p’s putative target gene and PEMF caused a decrease in Smad7 expression. Expression of Runx2 was increased by PEMF treatment and the miR21-5p inhibitor prevented the PEMF stimulation of Runx2 expression in differentiating cells. Thus, PEMF could mediate its effects on bone metabolism by activation of the TGF-β signaling pathway and stimulation of expression of miR21-5p in hBMSCs.

  10. Degenerative Suspensory Ligament Desmitis (DSLD in Peruvian Paso Horses Is Characterized by Altered Expression of TGFβ Signaling Components in Adipose-Derived Stromal Fibroblasts.

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    Wei Luo

    Full Text Available Equine degenerative suspensory ligament desmitis (DSLD in Peruvian Paso horses typically presents at 7-15 years and is characterized by lameness, focal disorganization of collagen fibrils, and chondroid deposition in the body of the ligament. With the aim of developing a test for disease risk (that can be used to screen horses before breeding we have quantified the expression of 76 TGFβ-signaling target genes in adipose-derived stromal fibroblasts (ADSCs from six DSLD-affected and five unaffected Paso horses. Remarkably, 35 of the genes showed lower expression (p<0.05 in cells from DSLD-affected animals and this differential was largely eliminated by addition of exogenous TGFβ1. Moreover, TGFβ1-mediated effects on expression were prevented by the TGFβR1/2 inhibitor LY2109761, showing that the signaling was via a TGFβR1/2 complex. The genes affected by the pathology indicate that it is associated with a generalized metabolic disturbance, since some of those most markedly altered in DSLD cells (ATF3, MAPK14, ACVRL1 (ALK1, SMAD6, FOS, CREBBP, NFKBIA, and TGFBR2 represent master-regulators in a wide range of cellular metabolic responses.

  11. Anti-angiogenesis therapy based on the bone marrow-derived stromal cells genetically engineered to express sFlt-1 in mouse tumor model

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    Chen X-C

    2008-10-01

    Full Text Available Abstract Background Bone marrow-derived stromal cells (BMSCs are important for development, tissue cell replenishment, and wound healing in physiological and pathological conditions. BMSCs were found to preferably reach sites undergoing the process of cell proliferation, such as wound and tumor, suggesting that BMSCs may be used as a vehicle for gene therapy of tumor. Methods Mouse BMSCs were loaded with recombinant adenoviruses which express soluble Vascular Endothelial Growth Factor Receptor-1 (sFlt-1. The anti-angiogenesis of sFlt-1 in BMSCs was determined using endothelial cells proliferation inhibition assay and alginate encapsulation assay. The anti-tumor effects of BMSCs expressing sFlt-1 through tail-vein infusion were evaluated in two mouse tumor metastases models. Results BMSCs genetically modified with Adv-GFP-sFlt-1 could effectively express and secret sFlt-1. BMSCs loaded with sFlt-1 gene could preferentially home to tumor loci and decrease lung metastases and prolong lifespan in mouse tumor model through inducing anti-angiogenesis and apoptosis in tumors. Conclusion We demonstrated that BMSCs might be employed as a promising vehicle for tumor gene therapy which can effectively not only improve the concentration of anticancer therapeutics in tumors, but also modify the tumor microenvironment.

  12. Ultra-structural changes and expression of chondrogenic and hypertrophic genes during chondrogenic differentiation of mesenchymal stromal cells in alginate beads

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    Havva Dashtdar

    2016-03-01

    Full Text Available Chondrogenic differentiation of mesenchymal stromal cells (MSCs in the form of pellet culture and encapsulation in alginate beads has been widely used as conventional model for in vitro chondrogenesis. However, comparative characterization between differentiation, hypertrophic markers, cell adhesion molecule and ultrastructural changes during alginate and pellet culture has not been described. Hence, the present study was conducted comparing MSCs cultured in pellet and alginate beads with monolayer culture. qPCR was performed to assess the expression of chondrogenic, hypertrophic, and cell adhesion molecule genes, whereas transmission electron microscopy (TEM was used to assess the ultrastructural changes. In addition, immunocytochemistry for Collagen type II and aggrecan and glycosaminoglycan (GAG analysis were performed. Our results indicate that pellet and alginate bead cultures were necessary for chondrogenic differentiation of MSC. It also indicates that cultures using alginate bead demonstrated significantly higher (p < 0.05 chondrogenic but lower hypertrophic (p < 0.05 gene expressions as compared with pellet cultures. N-cadherin and N-CAM1 expression were up-regulated in second and third weeks of culture and were comparable between the alginate bead and pellet culture groups, respectively. TEM images demonstrated ultrastructural changes resembling cell death in pellet cultures. Our results indicate that using alginate beads, MSCs express higher chondrogenic but lower hypertrophic gene expression. Enhanced production of extracellular matrix and cell adhesion molecules was also observed in this group. These findings suggest that alginate bead culture may serve as a superior chondrogenic model, whereas pellet culture is more appropriate as a hypertrophic model of chondrogenesis.

  13. After repeated division, bone marrow stromal cells express inhibitory factors with osteogenic capabilities, and EphA5 is a primary candidate.

    Science.gov (United States)

    Yamada, Tsuyoshi; Yuasa, Masato; Masaoka, Tomokazu; Taniyama, Takashi; Maehara, Hidetsugu; Torigoe, Ichiro; Yoshii, Toshitaka; Shinomiya, Kenichi; Okawa, Atsushi; Sotome, Shinichi

    2013-12-01

    The differentiation capability of human bone marrow stromal cells (hBMSCs) is thought to deteriorate over multiple doubling processes. To clarify the deterioration mechanisms, the multilineage differentiation capabilities of short- and long-term passaged BMSCs were compared. Predictably, long-term passaged BMSCs showed reduced differentiation capacities compared to short-term passaged cells. Furthermore, a non-human primate heterotopic bone formation model demonstrated that long-term passaged BMSCs have bone formation capabilities but also exert inhibitory effects on bone formation. This finding indicated that long-term passaged BMSCs express higher levels of inhibitory factors than short-term passaged BMSCs do. Co-culture assays of short- and long-term passaged BMSCs suggested that the inhibitory signals required cell-cell contact and would therefore be expressed on the cell membrane. A microarray analysis of BMSCs identified ephrin type-A receptor 5 (EphA5) as an inhibitory factor candidate. Quantitative PCR revealed that among all members of the ephrin and Eph receptor families, only the expression of EphA5 was increased by BMSC proliferation. A gene knockdown analysis using siRNAs demonstrated that knockdown of EphA5 gene expression in long-term passaged BMSCs led to an increase in ALP mRNA expression. These results indicate that EphA5 may be a negative regulator of bone formation. A better understanding of the roles of the ephrin and Eph receptor families in hBMSCs may lead to alternative approaches for manipulating hBMSC fate. In addition, this avenue of discovery may provide new therapeutic targets and quality-control markers of the osteogenic differentiation capabilities of hBMSCs. © 2013.

  14. Lim Mineralization Protein 3 Induces the Osteogenic Differentiation of Human Amniotic Fluid Stromal Cells through Kruppel-Like Factor-4 Downregulation and Further Bone-Specific Gene Expression

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    Marta Barba

    2012-01-01

    Full Text Available Multipotent mesenchymal stem cells with extensive self-renewal properties can be easily isolated and rapidly expanded in culture from small volumes of amniotic fluid. These cells, namely, amniotic fluid-stromal cells (AFSCs, can be regarded as an attractive source for tissue engineering purposes, being phenotypically and genetically stable, plus overcoming all the safety and ethical issues related to the use of embryonic/fetal cells. LMP3 is a novel osteoinductive molecule acting upstream to the main osteogenic pathways. This study is aimed at delineating the basic molecular events underlying LMP3-induced osteogenesis, using AFSCs as a cellular model to focus on the molecular features underlying the multipotency/differentiation switch. For this purpose, AFSCs were isolated and characterized in vitro and transfected with a defective adenoviral vector expressing the human LMP3. LMP3 induced the successful osteogenic differentiation of AFSC by inducing the expression of osteogenic markers and osteospecific transcription factors. Moreover, LMP3 induced an early repression of the kruppel-like factor-4, implicated in MSC stemness maintenance. KLF4 repression was released upon LMP3 silencing, indicating that this event could be reasonably considered among the basic molecular events that govern the proliferation/differentiation switch during LMP3-induced osteogenic differentiation of AFSC.

  15. Naringin promotes osteogenic differentiation of bone marrow stromal cells by up-regulating Foxc2 expression via the IHH signaling pathway.

    Science.gov (United States)

    Lin, Fei-Xiang; Du, Shi-Xin; Liu, De-Zhong; Hu, Qin-Xiao; Yu, Guo-Yong; Wu, Chu-Cheng; Zheng, Gui-Zhou; Xie, Da; Li, Xue-Dong; Chang, Bo

    2016-01-01

    Naringin is an active compound extracted from Rhizoma Drynariae, and studies have revealed that naringin can promote proliferation and osteogenic differentiation of bone marrow stromal cells (BMSCs). In this study, we explored whether naringin could promote osteogenic differentiation of BMSCs by upregulating Foxc2 expression via the Indian hedgehog (IHH) signaling pathway. BMSCs were cultured in basal medium, basal medium with naringin, osteogenic induction medium, osteogenic induction medium with naringin and osteogenic induction medium with naringin in the presence of the IHH inhibitor cyclopamine (CPE). We examined cell proliferation by using a WST-8 assay, and differentiation by Alizarin Red S staining (for mineralization) and alkaline phosphatase (ALP) activity. In addition, we detected core-binding factor α1 (Cbfα1), osteocalcin (OCN), bone sialoprotein (BSP), peroxisome proliferation-activated receptor gamma 2 (PPARγ2) and Foxc2 expression by using RT-PCR. We also determined Foxc2 and IHH protein levels by western blotting. Naringin increased the mineralization of BMSCs, as shown by Alizarin red S assays, and induced ALP activity. In addition, naringin significantly increased the mRNA levels of Foxc2, Cbfα1, OCN, and BSP, while decreasing PPARγ2 mRNA levels. Furthermore, the IHH inhibitor CPE inhibited the osteogenesis-potentiating effects of naringin. Naringin increased Foxc2 and stimulated the activation of IHH, as evidenced by increased expression of proteins that were inhibited by CPE. Our findings indicate that naringin promotes osteogenic differentiation of BMSCs by up-regulating Foxc2 expression via the IHH signaling pathway.

  16. Gene expression profiling of low-grade endometrial stromal sarcoma indicates fusion protein-mediated activation of the Wnt signaling pathway.

    Science.gov (United States)

    Przybyl, Joanna; Kidzinski, Lukasz; Hastie, Trevor; Debiec-Rychter, Maria; Nusse, Roel; van de Rijn, Matt

    2018-05-01

    Low-grade endometrial stromal sarcomas (LGESS) harbor chromosomal translocations that affect proteins associated with chromatin remodeling Polycomb Repressive Complex 2 (PRC2), including SUZ12, PHF1 and EPC1. Roughly half of LGESS also demonstrate nuclear accumulation of β-catenin, which is a hallmark of Wnt signaling activation. However, the targets affected by the fusion proteins and the role of Wnt signaling in the pathogenesis of these tumors remain largely unknown. Here we report the results of a meta-analysis of three independent gene expression profiling studies on LGESS and immunohistochemical evaluation of nuclear expression of β-catenin and Lef1 in 112 uterine sarcoma specimens obtained from 20 LGESS and 89 LMS patients. Our results demonstrate that 143 out of 310 genes overexpressed in LGESS are known to be directly regulated by SUZ12. In addition, our gene expression meta-analysis shows activation of multiple genes implicated in Wnt signaling. We further emphasize the role of the Wnt signaling pathway by demonstrating concordant nuclear expression of β-catenin and Lef1 in 7/16 LGESS. Based on our findings, we suggest that LGESS-specific fusion proteins disrupt the repressive function of the PRC2 complex similar to the mechanism seen in synovial sarcoma, where the SS18-SSX fusion proteins disrupt the mSWI/SNF (BAF) chromatin remodeling complex. We propose that these fusion proteins in LGESS contribute to overexpression of Wnt ligands with subsequent activation of Wnt signaling pathway and formation of an active β-catenin/Lef1 transcriptional complex. These observations could lead to novel therapeutic approaches that focus on the Wnt pathway in LGESS. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. Plasmid-based genetic modification of human bone marrow-derived stromal cells: analysis of cell survival and transgene expression after transplantation in rat spinal cord.

    Science.gov (United States)

    Ronsyn, Mark W; Daans, Jasmijn; Spaepen, Gie; Chatterjee, Shyama; Vermeulen, Katrien; D'Haese, Patrick; Van Tendeloo, Viggo Fi; Van Marck, Eric; Ysebaert, Dirk; Berneman, Zwi N; Jorens, Philippe G; Ponsaerts, Peter

    2007-12-14

    Bone marrow-derived stromal cells (MSC) are attractive targets for ex vivo cell and gene therapy. In this context, we investigated the feasibility of a plasmid-based strategy for genetic modification of human (h)MSC with enhanced green fluorescent protein (EGFP) and neurotrophin (NT)3. Three genetically modified hMSC lines (EGFP, NT3, NT3-EGFP) were established and used to study cell survival and transgene expression following transplantation in rat spinal cord. First, we demonstrate long-term survival of transplanted hMSC-EGFP cells in rat spinal cord under, but not without, appropriate immune suppression. Next, we examined the stability of EGFP or NT3 transgene expression following transplantation of hMSC-EGFP, hMSC-NT3 and hMSC-NT3-EGFP in rat spinal cord. While in vivo EGFP mRNA and protein expression by transplanted hMSC-EGFP cells was readily detectable at different time points post-transplantation, in vivo NT3 mRNA expression by hMSC-NT3 cells and in vivo EGFP protein expression by hMSC-NT3-EGFP cells was, respectively, undetectable or declined rapidly between day 1 and 7 post-transplantation. Further investigation revealed that the observed in vivo decline of EGFP protein expression by hMSC-NT3-EGFP cells: (i) was associated with a decrease in transgenic NT3-EGFP mRNA expression as suggested following laser capture micro-dissection analysis of hMSC-NT3-EGFP cell transplants at day 1 and day 7 post-transplantation, (ii) did not occur when hMSC-NT3-EGFP cells were transplanted subcutaneously, and (iii) was reversed upon re-establishment of hMSC-NT3-EGFP cell cultures at 2 weeks post-transplantation. Finally, because we observed a slowly progressing tumour growth following transplantation of all our hMSC cell transplants, we here demonstrate that omitting immune suppressive therapy is sufficient to prevent further tumour growth and to eradicate malignant xenogeneic cell transplants. In this study, we demonstrate that genetically modified hMSC lines can survive

  18. Disseminated intravascular coagulation caused by moojenactivase, a procoagulant snake venom metalloprotease.

    Science.gov (United States)

    Sartim, Marco A; Cezarette, Gabriel N; Jacob-Ferreira, Anna L; Frantz, Fabiani G; Faccioli, Lucia H; Sampaio, Suely V

    2017-10-01

    Snake venom toxins that activate coagulation factors are key players in the process of venom-induced coagulopathy, and account for severe clinical manifestations. The present study applies a variety of biochemical, hematological, and histopathological approaches to broadly investigate the intravascular and systemic effects of moojenactivase (MooA), the first described PIIId subclass metalloprotease isolated from Bothrops sp. venom that activates coagulation factors. MooA induced consumption coagulopathy with high toxic potency, characterized by prolongation of prothrombin and activated partial thromboplastin time, consumption of fibrinogen and the plasma coagulation factors X and II, and thrombocytopenia. MooA promoted leukocytosis and expression of the proinflammatory cytokines interleukin-6 and tumor necrosis factor-α, accompanied by tissue factor-dependent procoagulant activity in peripheral blood mononuclear cells. This metalloprotease also caused intravascular hemolysis, elevated plasma levels of creatine kinase-MB, aspartate transaminase, and urea/creatinine, and induced morphopathological alterations in erythrocytes, heart, kidney, and lungs associated with thrombosis and hemorrhage. Diagnosis of MooA-induced disseminated intravascular coagulation represents an important approach to better understand the pathophysiology of Bothrops envenomation and develop novel therapeutic strategies targeting hemostatic disturbances. Copyright © 2017. Published by Elsevier B.V.

  19. Creatine Enhances Transdifferentiation of Bone Marrow Stromal Cell-Derived Neural Stem Cell Into GABAergic Neuron-Like Cells Characterized With Differential Gene Expression.

    Science.gov (United States)

    Darabi, Shahram; Tiraihi, Taki; Delshad, AliReza; Sadeghizadeh, Majid; Taheri, Taher; Hassoun, Hayder K

    2017-04-01

    Creatine was reported to induce bone marrow stromal cells (BMSC) into GABAergic neuron-like cells (GNLC). In a previous study, creatine was used as a single inducer for BMSC into GNLC with low yield. In this study, BMSC-derived neurospheres (NS) have been used in generating GABAergic phenotype. The BMSC were isolated from adult rats and used in generating neurospheres and used for producing neural stem cells (NSC). A combination of all-trans-retinoic acid (RA), the ciliary neurotrophic factor (CNTF), and creatine was used in order to improve the yield of GNLC. We also used other protocols for the transdifferentiation including RA alone; RA and creatine; RA and CNTF; and RA, CNTF, and creatine. The BMSC, NSC, and GNLC were characterized by specific markers. The activity of the GNLC was evaluated using FM1-43. The isolated BMSC expressed Oct4, fibronectin, and CD44. The NS were immunoreactive to nestin and SOX2, the NSC were immunoreactive to nestin, NF68 and NF160, while the GNLC were immunoreactive to GAD1/2, VGAT, GABA, and synaptophysin. Oct4 and c-MYC, pluripotency genes, were expressed in the BMSC, while SOX2 and c-MYC were expressed in the NSC. The activity of GNLC indicates that the synaptic vesicles were released upon stimulation. The conclusion is that the combination of RA, CNTF, and creatine induced differentiation of neurosphere-derived NSC into GNLC within 1 week. This protocol gives higher yield than the other protocols used in this study. The mechanism of induction was clearly associated with several differential pluripotent genes.

  20. Endometrial Stromal Sarcoma of the Uterus: Magnetic Resonance Imaging Findings Including Apparent Diffusion Coefficient Value and Its Correlation With Ki-67 Expression.

    Science.gov (United States)

    Li, Hai Ming; Liu, Jia; Qiang, Jin Wei; Gu, Wei Yong; Zhang, Guo Fu; Ma, Feng Hua

    2017-11-01

    This study aimed to investigate the conventional magnetic resonance imaging (MRI) and diffusion-weighted imaging (DWI) features of endometrial stromal sarcoma (ESS) including a preliminary investigation of the correlation between the apparent diffusion coefficient (ADC) value and Ki-67 expression. The clinical and MRI data of 15 patients with ESS confirmed by surgery and pathology were analyzed retrospectively. The conventional MR morphological features, signal intensity on DWI, ADC value (n = 14), and clinicopathological marker Ki-67 (n = 13) were evaluated. Of 15 patients with ESS, 13 tumors were low-grade ESS (LGESS), and the remaining 2 were high-grade ESS (HGESS); 9 tumors were located in the myometrium, 5 were located in the endometrium and/or cervical canal, and 1 was located in extrauterine. Thirteen (87%) of 15 tumors showed a homo- or heterogeneous isointensity on T1-weighted imaging and a heterogeneous hyperintensity on T2-weighted imaging. The hypointense bands were observed in 11 tumors (73%) on T2-weighted imaging. The degenerations (cystic/necrosis/hemorrhage) were observed in 7 LGESS tumors and 2 HGESS tumors. The DWI hyperintensity was observed in 13 tumors (93%) and isointensity in remaining 1. The mean ADC value of the solid components in 14 ESSs was (1.05 ± 0.20) × 10mm/s. The contrast-enhanced MRI showed an obvious enhancement in 14 tumors (93%) (heterogeneous in 7 LGESSs and 2 HGESSs; homogeneous in 5 LGESSs). The ADC value was inversely correlated with the Ki-67 expression (r = -0.613, P = 0.026). Patients with ESS showed some characteristics on conventional MRI and DWI, and there was an inverse correlation between the ADC value and Ki-67 expression.

  1. Syndecan-1 Acts as an Important Regulator of CXCL1 Expression and Cellular Interaction of Human Endometrial Stromal and Trophoblast Cells

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    Dunja Maria Baston-Buest

    2017-01-01

    Full Text Available Successful implantation of the embryo into the human receptive endometrium is substantial for the establishment of a healthy pregnancy. This study focusses on the role of Syndecan-1 at the embryo-maternal interface, the multitasking coreceptor influencing ligand concentration, release and receptor presentation, and cellular morphology. CXC motif ligand 1, being involved in chemotaxis and angiogenesis during implantation, is of special interest as a ligand of Syndecan-1. Human endometrial stromal cells with and without Syndecan-1 knock-down were decidualized and treated with specific inhibitors to evaluate signaling pathways regulating CXC ligand 1 expression. Western blot analyses of MAPK and Wnt members were performed, followed by analysis of spheroid interactions between human endometrial cells and extravillous trophoblast cells. By mimicking embryo contact using IL-1β, we showed less ERK and c-Jun activation by depletion of Syndecan-1 and less Frizzled 4 production as part of the canonical Wnt pathway. Additionally, more beta-catenin was phosphorylated and therefore degraded after depletion of Syndecan-1. Secretion of CXC motif ligand 1 depends on MEK-1 with respect to Syndecan-1. Regarding the interaction of endometrial and trophoblast cells, the spheroid center-to-center distances were smaller after depletion of Syndecan-1. Therefore, Syndecan-1 seems to affect signaling processes relevant to signaling and intercellular interaction at the trophoblast-decidual interface.

  2. Expression analysis and specific blockade of the receptor for human thymic stromal lymphopoietin (TSLP) by novel antibodies to the human TSLPRα receptor chain.

    Science.gov (United States)

    Borowski, Andreas; Vetter, Tina; Kuepper, Michael; Wohlmann, Andreas; Krause, Sebastian; Lorenzen, Thomas; Virchow, Johann Christian; Luttmann, Werner; Friedrich, Karlheinz

    2013-02-01

    Thymic stromal lymphopoietin (TSLP) is an interleukin-7 (IL-7)-like cytokine with a pivotal role in development and maintenance of atopic diseases such as allergic asthma and atopic dermatitis. Moreover, recent studies show an involvement of TSLP in the progression of various cancers. TSLP signaling is mediated by the TSLP receptor (TSLPR), a heterodimeric type I cytokine receptor. It consists of the IL-7 receptor alpha chain (IL-7Rα), which is shared with the IL-7 receptor, and the TSLPRα chain as a specific subunit. Blocking signal release by TSLP without affecting IL-7 function is a potentially interesting option for the treatment of atopic diseases or certain tumors. By employing the extracellular domain of human TSLPRα chain (hTSLPRα(ex)) as an antigen, we generated a set of monoclonal antibodies. Several binders to native and/or denatured receptor protein were identified and characterized by cytometry and Western blot analysis. A screen based on a STAT3-driven reporter gene assay in murine pro-B cells expressing a functional hTSLPR yielded two hybridoma clones with specific antagonistic properties towards hTSLP, but not IL-7. Kinetic studies measuring blockade of hTSLP-dependent STAT phosphorylation in a TSLP-responsive cell line revealed an inhibitory constant in the nanomolar range. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Relative contribution of matrix metalloprotease and cysteine protease activities to cytokine-stimulated articular cartilage degradation

    DEFF Research Database (Denmark)

    Sondergaard, B C; Henriksen, K; Wulf, H

    2006-01-01

    OBJECTIVE: Both matrix metalloprotease (MMP) activity and cathepsin K (CK) activity have been implicated in cartilage turnover. We investigated the relative contribution of MMP activity and CK activity in cartilage degradation using ex vivo and in vivo models. METHODS: Bovine articular cartilage...... explants were stimulated with oncostatin M (OSM) 10 ng/ml and tumor necrosis factor-alpha (TNF-alpha) 20 ng/ml in the presence or absence of the broad-spectrum MMP inhibitor GM6001 and the cysteine protease inhibitor, E64. Cartilage degradation was evaluated in the conditioned medium by glycosaminoglycans...... was measured from CK-deficient mice. RESULTS: OSM and TNF-alpha combined induced significant (Pcartilage degradation products measured by hydroxyproline and CTX-II compared to vehicle control. The cytokines potently induced MMP expression, assessed by zymography, and CK expression...

  4. GASTROINTESTINAL STROMAL TUMOR (GIST

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    Luigi eTornillo

    2014-11-01

    Full Text Available Gastrointestinal stromal tumors are the most frequent mesenchymal tumors of the gastrointestinal tract. The discovery that these tumors, formerly thought of smooth muscle origin, are indeed better characterized by specific activating mutation in genes coding for the receptor tyrosine kinases CKIT and PDGFRA and that these mutations are strongly predictive for the response to targeted therapy with receptor tyrosine kinase inhibitors has made GISTs the typical example of the integration of basic molecular knowledge in the daily clinical activity. The information on the mutational status of these tumors is essential to predict (and subsequently to plan the therapy. As resistant cases are frequently wild-type, other possible oncogenic events, defining other entities, have been discovered (e.g. succinil dehydrogenase mutation/dysregulation, insuline growth factor expression, mutations in the RAS-RAF-MAPK pathway. The classification of disease must nowadays rely on the integration of the clinico-morphological characteristics with the molecular data.

  5. Whole-Genome Expression Analysis of Human Mesenchymal Stromal Cells Exposed to Ultrasmooth Tantalum vs. Titanium Oxide Surfaces

    DEFF Research Database (Denmark)

    Stiehler, C.; Bunger, C.; Overall, R. W.

    2013-01-01

    to titanium (Ti) surface. The aim of this study was to extend the previous investigation of biocompatibility by monitoring temporal gene expression of MSCs on topographically comparable smooth Ta and Ti surfaces using whole-genome gene expression analysis. Total RNA samples from telomerase-immortalized human...... MSCs cultivated on plain sputter-coated surfaces of Ta or Ti for 1, 2, 4, and 8 days were hybridized to n = 16 U133 Plus 2.0 arrays (Affymetrix(A (R))). Functional annotation, cluster and pathway analyses were performed. The vast majority of genes were differentially regulated after 4 days...... of cultivation and genes upregulated by MSCs exposed to Ta and Ti were predominantly related to the processes of differentiation and transcription, respectively. Functional annotation analysis of the 1,000 temporally most significantly regulated genes suggests earlier cellular differentiation on Ta compared...

  6. Smooth Muscle-Like Cells Generated from Human Mesenchymal Stromal Cells Display Marker Gene Expression and Electrophysiological Competence Comparable to Bladder Smooth Muscle Cells.

    Directory of Open Access Journals (Sweden)

    Juliane Brun

    Full Text Available The use of mesenchymal stromal cells (MSCs differentiated toward a smooth muscle cell (SMC phenotype may provide an alternative for investigators interested in regenerating urinary tract organs such as the bladder where autologous smooth muscle cells cannot be used or are unavailable. In this study we measured the effects of good manufacturing practice (GMP-compliant expansion followed by myogenic differentiation of human MSCs on the expression of a range of contractile (from early to late myogenic markers in relation to the electrophysiological parameters to assess the functional role of the differentiated MSCs and found that differentiation of MSCs associated with electrophysiological competence comparable to bladder SMCs. Within 1-2 weeks of myogenic differentiation, differentiating MSCs significantly expressed alpha smooth muscle actin (αSMA; ACTA2, transgelin (TAGLN, calponin (CNN1, and smooth muscle myosin heavy chain (SM-MHC; MYH11 according to qRT-PCR and/or immunofluorescence and Western blot. Voltage-gated Na+ current levels also increased within the same time period following myogenic differentiation. In contrast to undifferentiated MSCs, differentiated MSCs and bladder SMCs exhibited elevated cytosolic Ca2+ transients in response to K+-induced depolarization and contracted in response to K+ indicating functional maturation of differentiated MSCs. Depolarization was suppressed by Cd2+, an inhibitor of voltage-gated Ca2+-channels. The expression of Na+-channels was pharmacologically identified as the Nav1.4 subtype, while the K+ and Ca2+ ion channels were identified by gene expression of KCNMA1, CACNA1C and CACNA1H which encode for the large conductance Ca2+-activated K+ channel BKCa channels, Cav1.2 L-type Ca2+ channels and Cav3.2 T-type Ca2+ channels, respectively. This protocol may be used to differentiate adult MSCs into smooth muscle-like cells with an intermediate-to-late SMC contractile phenotype exhibiting voltage-gated ion

  7. Smooth Muscle-Like Cells Generated from Human Mesenchymal Stromal Cells Display Marker Gene Expression and Electrophysiological Competence Comparable to Bladder Smooth Muscle Cells.

    Science.gov (United States)

    Brun, Juliane; Lutz, Katrin A; Neumayer, Katharina M H; Klein, Gerd; Seeger, Tanja; Uynuk-Ool, Tatiana; Wörgötter, Katharina; Schmid, Sandra; Kraushaar, Udo; Guenther, Elke; Rolauffs, Bernd; Aicher, Wilhelm K; Hart, Melanie L

    2015-01-01

    The use of mesenchymal stromal cells (MSCs) differentiated toward a smooth muscle cell (SMC) phenotype may provide an alternative for investigators interested in regenerating urinary tract organs such as the bladder where autologous smooth muscle cells cannot be used or are unavailable. In this study we measured the effects of good manufacturing practice (GMP)-compliant expansion followed by myogenic differentiation of human MSCs on the expression of a range of contractile (from early to late) myogenic markers in relation to the electrophysiological parameters to assess the functional role of the differentiated MSCs and found that differentiation of MSCs associated with electrophysiological competence comparable to bladder SMCs. Within 1-2 weeks of myogenic differentiation, differentiating MSCs significantly expressed alpha smooth muscle actin (αSMA; ACTA2), transgelin (TAGLN), calponin (CNN1), and smooth muscle myosin heavy chain (SM-MHC; MYH11) according to qRT-PCR and/or immunofluorescence and Western blot. Voltage-gated Na+ current levels also increased within the same time period following myogenic differentiation. In contrast to undifferentiated MSCs, differentiated MSCs and bladder SMCs exhibited elevated cytosolic Ca2+ transients in response to K+-induced depolarization and contracted in response to K+ indicating functional maturation of differentiated MSCs. Depolarization was suppressed by Cd2+, an inhibitor of voltage-gated Ca2+-channels. The expression of Na+-channels was pharmacologically identified as the Nav1.4 subtype, while the K+ and Ca2+ ion channels were identified by gene expression of KCNMA1, CACNA1C and CACNA1H which encode for the large conductance Ca2+-activated K+ channel BKCa channels, Cav1.2 L-type Ca2+ channels and Cav3.2 T-type Ca2+ channels, respectively. This protocol may be used to differentiate adult MSCs into smooth muscle-like cells with an intermediate-to-late SMC contractile phenotype exhibiting voltage-gated ion channel

  8. Dual delivery of rhPDGF-BB and bone marrow mesenchymal stromal cells expressing the BMP2 gene enhance bone formation in a critical-sized defect model.

    Science.gov (United States)

    Park, Shin-Young; Kim, Kyoung-Hwa; Shin, Seung-Yun; Koo, Ki-Tae; Lee, Yong-Moo; Seol, Yang-Jo

    2013-11-01

    Bone tissue healing is a dynamic, orchestrated process that relies on multiple growth factors and cell types. Platelet-derived growth factor-BB (PDGF-BB) is released from platelets at wound sites and induces cellular migration and proliferation necessary for bone regeneration in the early healing process. Bone morphogenetic protein-2 (BMP-2), the most potent osteogenic differentiation inducer, directs new bone formation at the sites of bone defects. This study evaluated a combinatorial treatment protocol of PDGF-BB and BMP-2 on bone healing in a critical-sized defect model. To mimic the bone tissue healing process, a dual delivery approach was designed to deliver the rhPDGF-BB protein transiently during the early healing phase, whereas BMP-2 was supplied by rat bone marrow stromal cells (BMSCs) transfected with an adenoviral vector containing the BMP2 gene (AdBMP2) for prolonged release throughout the healing process. In in vitro experiments, the dual delivery of rhPDGF-BB and BMP2 significantly enhanced cell proliferation. However, the osteogenic differentiation of BMSCs was significantly suppressed even though the amount of BMP-2 secreted by the AdBMP2-transfected BMSCs was not significantly affected by the rhPDGF-BB treatment. In addition, dual delivery inhibited the mRNA expression of BMP receptor type II and Noggin in BMSCs. In in vivo experiments, critical-sized calvarial defects in rats showed enhanced bone regeneration by dual delivery of autologous AdBMP2-transfected BMSCs and rhPDGF-BB in both the amount of new bone formed and the bone mineral density. These enhancements in bone regeneration were greater than those observed in the group treated with AdBMP2-transfected BMSCs alone. In conclusion, the dual delivery of rhPDGF-BB and AdBMP2-transfected BMSCs improved the quality of the regenerated bone, possibly due to the modulation of PDGF-BB on BMP-2-induced osteogenesis.

  9. Syndecan-1 knock-down in decidualized human endometrial stromal cells leads to significant changes in cytokine and angiogenic factor expression patterns

    Directory of Open Access Journals (Sweden)

    Krüssel Jan-Steffen

    2010-11-01

    Full Text Available Abstract Background Successful embryonic implantation depends on a synchronized embryo-maternal dialogue. Chemokines, such as chemokine ligand 1 (CXCL1, play essential roles in the maternal reproductive tract leading to morphological changes during decidualization, mediating maternal acceptance towards the semi-allograft embryo and induction of angiogenesis. Chemokine binding to their classical G-protein coupled receptors is essentially supported by the syndecan (Sdc family of heparan sulfate proteoglycans. The aim of this study was to identify the involvement of Sdc-1 at the embryo-maternal interface regarding changes of the chemokine and angiogenic profile of the decidua during the process of decidualization and implantation in human endometrium. Methods A stable Sdc-1 knock-down was generated in the immortalized human endometrial stromal cell line St-T1 and was named KdS1. The ability of KdS1 to decidualize was proven by Insulin-like growth factor binding 1 (IGFBP1 and prolactin (PRL confirmation on mRNA level before further experiments were carried out. Dot blot protein analyses of decidualized knock-down cells vs non-transfected controls were performed. In order to imitate embryonic implantation, decidualized KdS1 were then incubated with IL-1beta, an embryo secretion product, vs controls. Statistical analyses were performed applying the Student's t-test with p Results The induction of the Sdc-1 knock-down revealed significant changes in cytokine and angiogenic factor expression profiles of dKdS1 vs decidualized controls. Incubation with embryonic IL-1beta altered the expression patterns of KdS1 chemokines and angiogenic factors towards inflammatory-associated molecules and factors involved in matrix regulation. Conclusions Sdc-1 knock-down in human endometrial stroma cells led to fulminant changes regarding cytokine and angiogenic factor expression profiles upon decidualization and imitation of embryonic contact. Sdc-1 appears to play an

  10. Expression of chemokine receptor-4 in bone marrow mesenchymal stem cells on experimental rat abdominal aortic aneurysms and the migration of bone marrow mesenchymal stem cells with stromal-derived factor-1

    Directory of Open Access Journals (Sweden)

    Miao-Yun Long

    2014-05-01

    Full Text Available This study investigated the expression and role of chemokine receptor-4 (CXCR4 in bone marrow mesenchymal stem cells (BMSCs from experimental rats with abdominal aortic aneurysms (AAA for migration of BMSCs. Sprague–Dawley rats were divided into an experimental group and control group (n = 18 each. AAA was induced with 0.75 M solution infiltrate for 30 minutes, after which the abdomen was rinsed and closed. Saline was used in place of CaCl2 in the control group. CD34 and CD29 were detected by flow cytometry, the gene and protein expression of CXCR4 were detected by real-time polymerase chain reaction and western blot, respectively. The migration of BMSCs with stromal-derived factor-1 was detected by Transwell chamber. CD34 expression was negative and CD29 expression was positive. The gene and protein expression of CXCR4 were significantly higher in experimental group than them in control group (p < 0.05, the migration ability of BMSCs from the experimental group was significantly higher than that from the control group (p < 0.05. Stromal-derived factor -1/CXCR4 can enhance the migration of BMSCs in vitro in a rat AAA model.

  11. Activation of the epidermal growth factor receptor (EGFR) by a novel metalloprotease pathway.

    LENUS (Irish Health Repository)

    Bergin, David A

    2008-11-14

    Neutrophil Elastase (NE) is a pro-inflammatory protease present at higher than normal levels in the lung during inflammatory disease. NE regulates IL-8 production from airway epithelial cells and can activate both EGFR and TLR4. TACE\\/ADAM17 has been reported to trans-activate EGFR in response to NE. Here, using 16HBE14o-human bronchial epithelial cells we demonstrate a new mechanism by which NE regulates both of these events. A high molecular weight soluble metalloprotease activity detectable only in supernatants from NE-treated cells by gelatin and casein zymography was confirmed to be meprin alpha by Western immunoblotting. In vitro studies demonstrated the ability of NE to activate meprin alpha, which in turn could release soluble TGFalpha and induce IL-8 production from 16HBE14o- cells. These effects were abrogated by actinonin, a specific meprin inhibitor. NE-induced IL-8 expression was also inhibited by meprin alpha siRNA. Immunoprecipitation studies detected EGFR\\/TLR4 complexes in NE-stimulated cells overexpressing these receptors. Confocal studies confirmed colocalization of EGFR and TLR4 in 16HBE14o- cells stimulated with meprin alpha. NFkappaB was also activated via MyD88 in these cells by meprin alpha. In bronchoalveolar lavage fluid from NE knock-out mice infected intra-tracheally with Pseudomonas aeruginosa meprin alpha was significantly decreased compared with control mice, and was significantly increased and correlated with NE activity, in bronchoalveolar lavage fluid from individuals with cystic fibrosis but not healthy controls. The data describe a previously unidentified lung metalloprotease meprin alpha, and its role in NE-induced EGFR and TLR4 activation and IL-8 production.

  12. Reduction of the disintegrin and metalloprotease ADAM12 in preeclampsia

    DEFF Research Database (Denmark)

    Laigaard, Jennie; Sørensen, Tina; Placing, Sophie

    2005-01-01

    OBJECTIVES: The secreted form of ADAM12 is a metalloprotease that may be involved in placental and fetal growth. We examined whether the concentration of ADAM12 in first-trimester maternal serum could be used as a marker for preeclampsia. METHODS: We developed a semiautomated, time-resolved, immu......OBJECTIVES: The secreted form of ADAM12 is a metalloprotease that may be involved in placental and fetal growth. We examined whether the concentration of ADAM12 in first-trimester maternal serum could be used as a marker for preeclampsia. METHODS: We developed a semiautomated, time......-resolved, immunofluorometric assay for the quantification of ADAM12 in serum. The assay detected ADAM12 in a range of 78-1248 microg/L. Serum samples derived from women in the first trimester of a normal pregnancy (n = 324) and from women who later developed preeclampsia during pregnancy (n = 160) were obtained from the First...... Trimester Copenhagen Study. ADAM12 levels were assayed in these serum samples. Serum levels of ADAM12 were converted to multiples of the median (MoM) after log-linear regression of concentration versus gestational age. RESULTS: Serum ADAM12 levels in women who developed preeclampsia during pregnancy had...

  13. RpA, an extracellular protease similar to the metalloprotease of serralysin family, is required for pathogenicity of Ralstonia pickettii.

    Science.gov (United States)

    Chen, C-M; Liu, J-J; Chou, C-W; Lai, C-H; Wu, L-T

    2015-10-01

    To investigate the biochemical and functional properties of an extracellular protease, RpA, in Ralstonia pickettii WP1 isolated from water supply systems. An extracellular protease was identified and characterized from R. pickettii WP1. A mutant strain WP1M2 was created from strain WP1 by mini-Tn5 transposition. The culture filtrates from WP1M2 had a lower cytotoxic effect than the parental WP1 on several mammalian cell lines. Cloning and sequence analysis revealed the Tn5 transposon inserted at a protease gene (rpA) which is 81% homologous to prtA and aprX genes of Pseudomonas fluorescens. The rpA gene encodes a 482-residue protein showing sequence similarity to metalloproteases of the serralysin family. The RpA protein was expressed in Escherichia coli using a pET expression vector and purified as a 55 kDa molecular weight protein. Furthermore, the protease activity of RpA was inhibited by protease inhibitor and heat treatment. The in vitro cytotoxic activity of R. pickettii culture filtrates was attributed to RpA protease. An extracellular protease, RpA, was identified from R. pickettii WP1 isolated from water supply system. The RpA metalloproteases is required for the pathogenicity of R. pickettii to mammalian cell lines. © 2015 The Society for Applied Microbiology.

  14. Co-Culturing of Multipotent Mesenchymal Stromal Cells with Autological and Allogenic Lymphocytes.

    Science.gov (United States)

    Kapranov, N M; Davydova, Yu O; Gal'tseva, I V; Petinati, N A; Bakshinskaitė, M V; Drize, N I; Kuz'mina, L A; Parovichnikova, E N; Savchenko, V G

    2018-03-01

    We studied the effect of autologous and allogeneic lymphocytes on multipotent mesenchymal stromal cells in co-culture. It is shown that changes in multipotent mesenchymal stromal cells and in lymphocytes did not depend on the source of lymphocytes. Contact with lymphocytes triggers expression of HLA-DR molecules on multipotent mesenchymal stromal cells and these cells lose their immune privilege. In multipotent mesenchymal stromal cells, the relative level of expression of factors involved in immunomodulation (IDO1, PTGES, and IL-6) and expression of adhesion molecule ICAM1 increased, while expression of genes involved in the differentiation of multipotent mesenchymal stromal cells remained unchanged. Priming of multipotent mesenchymal stromal cells with IFN did not affect these changes. In turn, lymphocytes underwent activation, expression of HLA-DR increased, subpopulation composition of lymphocytes changed towards the increase in the content of naïve T cells. These findings are important for cell therapy.

  15. Structure and evolution of N-domains in AAA metalloproteases.

    Science.gov (United States)

    Scharfenberg, Franka; Serek-Heuberger, Justyna; Coles, Murray; Hartmann, Marcus D; Habeck, Michael; Martin, Jörg; Lupas, Andrei N; Alva, Vikram

    2015-02-27

    Metalloproteases of the AAA (ATPases associated with various cellular activities) family play a crucial role in protein quality control within the cytoplasmic membrane of bacteria and the inner membrane of eukaryotic organelles. These membrane-anchored hexameric enzymes are composed of an N-terminal domain with one or two transmembrane helices, a central AAA ATPase module, and a C-terminal Zn(2+)-dependent protease. While the latter two domains have been well studied, so far, little is known about the N-terminal regions. Here, in an extensive bioinformatic and structural analysis, we identified three major, non-homologous groups of N-domains in AAA metalloproteases. By far, the largest one is the FtsH-like group of bacteria and eukaryotic organelles. The other two groups are specific to Yme1: one found in plants, fungi, and basal metazoans and the other one found exclusively in animals. Using NMR and crystallography, we determined the subunit structure and hexameric assembly of Escherichia coli FtsH-N, exhibiting an unusual α+β fold, and the conserved part of fungal Yme1-N from Saccharomyces cerevisiae, revealing a tetratricopeptide repeat fold. Our bioinformatic analysis showed that, uniquely among these proteins, the N-domain of Yme1 from the cnidarian Hydra vulgaris contains both the tetratricopeptide repeat region seen in basal metazoans and a region of homology to the N-domains of animals. Thus, it is a modern-day representative of an intermediate in the evolution of animal Yme1 from basal eukaryotic precursors. Copyright © 2015. Published by Elsevier Ltd.

  16. Identification of metalloprotease/disintegrins in Xenopus laevis testis with a potential role in fertilization.

    Science.gov (United States)

    Shilling, F M; Krätzschmar, J; Cai, H; Weskamp, G; Gayko, U; Leibow, J; Myles, D G; Nuccitelli, R; Blobel, C P

    1997-06-15

    Proteins containing a membrane-anchored metalloprotease domain, a disintegrin domain, and a cysteine-rich region (MDC proteins) are thought to play an important role in mammalian fertilization, as well as in somatic cell-cell interactions. We have identified PCR sequence tags encoding the disintegrin domain of five distinct MDC proteins from Xenopus laevis testis cDNA. Four of these sequence tags (xMDC9, xMDC11.1, xMDC11.2, and xMDC13) showed strong similarity to known mammalian MDC proteins, whereas the fifth (xMDC16) apparently represents a novel family member. Northern blot analysis revealed that the mRNA for xMDC16 was only expressed in testis, and not in heart, muscle, liver, ovaries, or eggs, whereas the mRNAs corresponding to the four other PCR products were expressed in testis and in some or all somatic tissues tested. The xMDC16 protein sequence, as predicted from the full-length cDNA, contains a metalloprotease domain with the active-site sequence HEXXH, a disintegrin domain, a cysteine-rich region, an EGF repeat, a transmembrane domain, and a short cytoplasmic tail. To study a potential role for these xMDC proteins in fertilization, peptides corresponding to the predicted integrin-binding domain of each protein were tested for their ability to inhibit X. laevis fertilization. Cyclic and linear xMDC16 peptides inhibited fertilization in a concentration-dependent manner, whereas xMDC16 peptides that were scrambled or had certain amino acid replacements in the predicted integrin-binding domain did not affect fertilization. Cyclic and linear xMDC9 peptides and linear xMDC13 peptides also inhibited fertilization similarly to xMDC16 peptides, whereas peptides corresponding to the predicted integrin-binding site of xMDC11.1 and xMDC11.2 did not. These results are discussed in the context of a model in which multiple MDC protein-receptor interactions are necessary for fertilization to occur.

  17. Equine corneal stromal abscesses

    DEFF Research Database (Denmark)

    Henriksen, M. D. L.; Andersen, P. H.; Plummer, C. E.

    2013-01-01

    The last 30 years have seen many changes in the understanding of the pathogenesis and treatment of equine corneal stromal abscesses (SAs). Stromal abscesses were previously considered an eye problem related to corneal bacterial infection, equine recurrent uveitis, corneal microtrauma and corneal....... Medical and surgical treatments are now directed towards elimination of fungal and bacterial infections, reduction and replacement of diseased corneal stroma, and suppression of iridocyclitis. If the abscess and anterior uveitis do not respond satisfactorily to medical therapy, full thickness or split...

  18. Lipoxin A4 regulates expression of the estrogen receptor and inhibits 17β-estradiol induced p38 mitogen-activated protein kinase phosphorylation in human endometriotic stromal cells.

    Science.gov (United States)

    Chen, Shuo; Wu, Rong-Feng; Su, Lin; Zhou, Wei-Dong; Zhu, Mao-Bi; Chen, Qiong-Hua

    2014-07-01

    To study the role of lipoxin A4 (LXA4) in endometriosis. Molecular analysis in human samples and primary human endometriotic stromal cells (ESCs). University hospital. Forty-nine premenopausal women (30 patients with endometriosis and 19 controls). Normal and ectopic endometrial biopsies obtained during surgery performed during the proliferative phase of the menstrual cycle; ESCs used for in vitro studies. Levels of LXA4 measured by enzyme-linked immunosorbent assay (ELISA); mRNA levels of the estrogen receptor (ER), progestogen receptor (PR), tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) quantified by quantitative reverse-transcription polymerase chain reaction (qRT-PCR); and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation evaluated by Western blotting. The LXA4 expression level decreased in ectopic tissue as well as ERα and PR, although the expression of ERβ increased in ectopic endometrium compared with the controls. Investigations with correlation analysis revealed the expression of LXA4 was positively correlated with ERα and negatively correlated with ERβ in vivo. Moreover, administering LXA4 could augment ERβ expression in ESCs and inhibit the 17β-estradiol-induced phosphorylation of p38 MAPK very likely through ERβ. Our findings indicate that LXA4 regulates ERβ expression and inhibits 17β-estradiol-induced phosphorylation of p38 MAPK, very likely through ERβ in ESCs. Copyright © 2014. Published by Elsevier Inc.

  19. Embryonal carcinoma cell induction of miRNA and mRNA changes in co-cultured prostate stromal fibromuscular cells

    Science.gov (United States)

    VÊNCIO, ENEIDA F.; PASCAL, LAURA E.; PAGE, LAURA S.; DENYER, GARETH; WANG, AMY J.; RUOHOLA-BAKER, HANNELE; ZHANG, SHILE; WANG, KAI; GALAS, DAVID J.; LIU, ALVIN Y.

    2014-01-01

    The prostate stromal mesenchyme controls organ-specific development. In cancer, the stromal compartment shows altered gene expression compared to non-cancer. The lineage relationship between cancer-associated stromal cells and normal tissue stromal cells is not known. Nor is the cause underlying the expression difference. Previously, the embryonal carcinoma (EC) cell line, NCCIT, was used by us to study the stromal induction property. In the current study, stromal cells from non-cancer (NP) and cancer (CP) were isolated from tissue specimens and co-cultured with NCCIT cells in a trans-well format to preclude heterotypic cell contact. After 3 days, the stromal cells were analyzed by gene arrays for microRNA (miRNA) and mRNA expression. In co-culture, NCCIT cells were found to alter the miRNA and mRNA expression of NP stromal cells to one like that of CP stromal cells. In contrast, NCCIT had no significant effect on the gene expression of CP stromal cells. We conclude that the gene expression changes in stromal cells can be induced by diffusible factors synthesized by EC cells, and suggest that cancer-associated stromal cells represent a more primitive or less differentiated stromal cell type. PMID:20945389

  20. Novel Apigenin Based Small Molecule that Targets Snake Venom Metalloproteases

    Science.gov (United States)

    Anusha, Sebastian; Hemshekhar, Mahadevappa; Chandra Nayaka, Siddaiah; Kemparaju, Kempaiah; Basappa; Girish, Kesturu S.; Rangappa, Kanchugarakoppal S.

    2014-01-01

    The classical antivenom therapy has appreciably reduced snakebite mortality rate and thus is the only savior drug available. Unfortunately, it considerably fails to shield the viper bite complications like hemorrhage, local tissue degradation and necrosis responsible for severe morbidity. Moreover, the therapy is also tagged with limitations including anaphylaxis, serum sickness and poor availability. Over the last decade, snake venom metalloproteases (SVMPs) are reported to be the primary component responsible for hemorrhage and tissue degradation at bitten site. Thus, antivenom inability to offset viper venom-induced local toxicity has been a basis for an insistent search for SVMP inhibitors. Here we report the inhibitory effect of compound 5d, an apigenin based molecule against SVMPs both in silico and in vivo. Several apigenin analogues are synthesized using multicomponent Ugi reactions. Among them, compound 5d effectively abrogated Echis carinatus (EC) venom-induced local hemorrhage, tissue necrosis and myotoxicity in a dose dependant fashion. The histopathological study further conferred effective inhibition of basement membrane degradation, and accumulation of inflammatory leucocytes at the site of EC venom inoculation. The compound also protected EC venom-induced fibrin and fibrinogen degradation. The molecular docking of compound 5d and bothropasin demonstrated the direct interaction of hydroxyl group of compound with Glu146 present in hydrophobic pocket of active site and does not chelate Zn2+. Hence, it is concluded that compound 5d could be a potent agent in viper bite management. PMID:25184206

  1. Impact of Leishmania metalloprotease GP63 on macrophage signaling

    Science.gov (United States)

    Isnard, Amandine; Shio, Marina T.; Olivier, Martin

    2012-01-01

    The intramacrophage protozoan parasites of Leishmania genus have developed sophisticated ways to subvert the innate immune response permitting their infection and propagation within the macrophages of the mammalian host. Several Leishmania virulence factors have been identified and found to be of importance for the development of leishmaniasis. However, recent findings are now further reinforcing the critical role played by the zinc-metalloprotease GP63 as a virulence factor that greatly influence host cell signaling mechanisms and related functions. GP63 has been found to be involved not only in the cleavage and degradation of various kinases and transcription factors, but also to be the major molecule modulating host negative regulatory mechanisms involving for instance protein tyrosine phosphatases (PTPs). Those latter being well recognized for their pivotal role in the regulation of a great number of signaling pathways. In this review article, we are providing a complete overview about the role of Leishmania GP63 in the mechanisms underlying the subversion of macrophage signaling and functions. PMID:22919663

  2. A highly conserved metalloprotease effector enhances virulence in the maize anthracnose fungus Colletotrichum graminicola.

    Science.gov (United States)

    Sanz-Martín, José M; Pacheco-Arjona, José Ramón; Bello-Rico, Víctor; Vargas, Walter A; Monod, Michel; Díaz-Mínguez, José M; Thon, Michael R; Sukno, Serenella A

    2016-09-01

    Colletotrichum graminicola causes maize anthracnose, an agronomically important disease with a worldwide distribution. We have identified a fungalysin metalloprotease (Cgfl) with a role in virulence. Transcriptional profiling experiments and live cell imaging show that Cgfl is specifically expressed during the biotrophic stage of infection. To determine whether Cgfl has a role in virulence, we obtained null mutants lacking Cgfl and performed pathogenicity and live microscopy assays. The appressorium morphology of the null mutants is normal, but they exhibit delayed development during the infection process on maize leaves and roots, showing that Cgfl has a role in virulence. In vitro chitinase activity assays of leaves infected with wild-type and null mutant strains show that, in the absence of Cgfl, maize leaves exhibit increased chitinase activity. Phylogenetic analyses show that Cgfl is highly conserved in fungi. Similarity searches, phylogenetic analysis and transcriptional profiling show that C. graminicola encodes two LysM domain-containing homologues of Ecp6, suggesting that this fungus employs both Cgfl-mediated and LysM protein-mediated strategies to control chitin signalling. © 2015 BSPP and John Wiley & Sons Ltd.

  3. Expression of Programmed Death-Ligand 1 by Human Colonic CD90+ Stromal Cells Differs Between Ulcerative Colitis and Crohn’s Disease and Determines Their Capacity to Suppress Th1 Cells

    Directory of Open Access Journals (Sweden)

    Ellen J. Beswick

    2018-05-01

    Full Text Available Background and AimsThe role of programmed cell death protein 1 (PD-1 and its ligands in the dysregulation of T helper immune responses observed in the inflammatory bowel disease (IBD is unclear. Recently, a novel concept emerged that CD90+ colonic (myofibroblasts (CMFs, also known as stromal cells, act as immunosuppressors, and are among the key regulators of acute and chronic inflammation. The objective of this study was to determine if the level of the PD-1 ligands is changed in the IBD inflamed colonic mucosa and to test the hypothesis that changes in IBD-CMF-mediated PD-1 ligand-linked immunosuppression is a mechanism promoting the dysregulation of Th1 cell responses.MethodsTissues and cells derived from Crohn’s disease (CD, ulcerative colitis (UC, and healthy individuals (N were studied in situ, ex vivo, and in culture.ResultsA significant increase in programmed death-ligand 1 (PD-L1 was observed in the inflamed UC colonic mucosa when compared to the non-inflamed matched tissue samples, CD, and healthy controls. UC-CMFs were among the major populations in the colonic mucosa contributing to the enhanced PD-L1 expression. In contrast, PD-L1 expression was decreased in CD-CMFs. When compared to CD-CMFs and N-CMFs, UC-CMFs demonstrated stronger suppression of IL-2, Th1 transcriptional factor Tbet, and IFN-γ expression by CD3/CD28-activated CD4+ T cells, and this process was PD-L1 dependent. Similar observations were made when differentiated Th1 cells were cocultured with UC-CMFs. In contrast, CD-CMFs showed reduced capacity to suppress Th1 cell activity and addition of recombinant PD-L1 Fc to CD-CMF:T cell cocultures partially restored the suppression of the Th1 type responses.ConclusionWe present evidence showing that increased PD-L1 expression suppresses Th1 cell activity in UC. In contrast, loss of PD-L1 expression observed in CD contributes to the persistence of the Th1 inflammatory milieu in CD. Our data suggest that

  4. Gastrointestinal Stromal Tumor: Diagnosis and Prognosis

    International Nuclear Information System (INIS)

    Martin, M. T.; Olmedilla, P.; Gonzalez, S.; Oliver, J. M.

    2003-01-01

    Gastrointestinal stromal tumors (GIST) are mesenquimal tumors derived from cell precursors. They have the capacity for myogenic and neurogenic differentiation and are characterized by expression of KIT protein /tyrosine kinase growth factor). Clinically, they exhibit various biological behaviors. We present 8 cases of GIST, describing both their radiological manifestation through computerized tomography (CT) and most accepted criteria for benignity and malignancy. We also describe the response of one meta statically diagnosed tumor to tyrosine kinase inhibitor. (Author) 9 refs

  5. PHD-2 Suppression in Mesenchymal Stromal Cells Enhances Wound Healing.

    Science.gov (United States)

    Ko, Sae Hee; Nauta, Allison C; Morrison, Shane D; Hu, Michael S; Zimmermann, Andrew S; Chung, Michael T; Glotzbach, Jason P; Wong, Victor W; Walmsley, Graham G; Peter Lorenz, H; Chan, Denise A; Gurtner, Geoffrey C; Giaccia, Amato J; Longaker, Michael T

    2018-01-01

    Cell therapy with mesenchymal stromal cells is a promising strategy for tissue repair. Restoration of blood flow to ischemic tissues is a key step in wound repair, and mesenchymal stromal cells have been shown to be proangiogenic. Angiogenesis is critically regulated by the hypoxia-inducible factor (HIF) superfamily, consisting of transcription factors targeted for degradation by prolyl hydroxylase domain (PHD)-2. The aim of this study was to enhance the proangiogenic capability of mesenchymal stromal cells and to use these modified cells to promote wound healing. Mesenchymal stromal cells harvested from mouse bone marrow were transduced with short hairpin RNA (shRNA) against PHD-2; control cells were transduced with scrambled shRNA (shScramble) construct. Gene expression quantification, human umbilical vein endothelial cell tube formation assays, and wound healing assays were used to assess the effect of PHD knockdown mesenchymal stromal cells on wound healing dynamics. PHD-2 knockdown mesenchymal stromal cells overexpressed HIF-1α and multiple angiogenic factors compared to control (p cells treated with conditioned medium from PHD-2 knockdown mesenchymal stromal cells exhibited increased formation of capillary-like structures and enhanced migration compared with human umbilical vein endothelial cells treated with conditioned medium from shScramble-transduced mesenchymal stromal cells (p cells healed at a significantly accelerated rate compared with wounds treated with shScramble mesenchymal stromal cells (p cells (p cells augments their proangiogenic potential in wound healing therapy. This effect appears to be mediated by overexpression of HIF family transcription factors and up-regulation of multiple downstream angiogenic factors.

  6. PKC/ROS-Mediated NLRP3 Inflammasome Activation Is Attenuated by Leishmania Zinc-Metalloprotease during Infection

    Science.gov (United States)

    Jung, Jee Yong; Chang, Kwang-Poo; Olivier, Martin

    2015-01-01

    Parasites of the Leishmania genus infect and survive within macrophages by inhibiting several microbicidal molecules, such as nitric oxide and pro-inflammatory cytokines. In this context, various species of Leishmania have been reported to inhibit or reduce the production of IL-1β both in vitro and in vivo. However, the mechanism whereby Leishmania parasites are able to affect IL-1β production and secretion by macrophages is still not fully understood. Dependent on the stimulus at hand, the maturation of IL-1β is facilitated by different inflammasome complexes. The NLRP3 inflammasome has been shown to be of pivotal importance in the detection of danger molecules such as inorganic crystals like asbestos, silica and malarial hemozoin, (HZ) as well as infectious agents. In the present work, we investigated whether Leishmania parasites modulate NLRP3 inflammasome activation. Using PMA-differentiated THP-1 cells, we demonstrate that Leishmania infection effectively inhibits macrophage IL-1β production upon stimulation. In this context, the expression and activity of the metalloprotease GP63 - a critical virulence factor expressed by all infectious Leishmania species - is a prerequisite for a Leishmania-mediated reduction of IL-1β secretion. Accordingly, L. mexicana, purified GP63 and GP63-containing exosomes, caused the inhibition of macrophage IL-1β production. Leishmania-dependent suppression of IL-1β secretion is accompanied by an inhibition of reactive oxygen species (ROS) production that has previously been shown to be associated with NLRP3 inflammasome activation. The observed loss of ROS production was due to an impaired PKC-mediated protein phosphorylation. Furthermore, ROS-independent inflammasome activation was inhibited, possibly due to an observed GP63-dependent cleavage of inflammasome and inflammasome-related proteins. Collectively for the first time, we herein provide evidence that the protozoan parasite Leishmania, through its surface

  7. Skeletal (stromal) stem cells

    DEFF Research Database (Denmark)

    Abdallah, Basem M; Kermani, Abbas Jafari; Zaher, Walid

    2015-01-01

    Skeletal (marrow stromal) stem cells (BMSCs) are a group of multipotent cells that reside in the bone marrow stroma and can differentiate into osteoblasts, chondrocytes and adipocytes. Studying signaling pathways that regulate BMSC differentiation into osteoblastic cells is a strategy....../preadipocyte factor 1 (Dlk1/Pref-1), the Wnt co-receptor Lrp5 and intracellular kinases. This article is part of a Special Issue entitled: Stem Cells and Bone....

  8. Moojenactivase, a novel pro-coagulant PIIId metalloprotease isolated from Bothrops moojeni snake venom, activates coagulation factors II and X and induces tissue factor up-regulation in leukocytes.

    Science.gov (United States)

    Sartim, Marco A; Costa, Tassia R; Laure, Helen J; Espíndola, Milena S; Frantz, Fabiani G; Sorgi, Carlos A; Cintra, Adélia C O; Arantes, Eliane C; Faccioli, Lucia H; Rosa, José C; Sampaio, Suely V

    2016-05-01

    Coagulopathies following snakebite are triggered by pro-coagulant venom toxins, in which metalloproteases play a major role in envenomation-induced coagulation disorders by acting on coagulation cascade, platelet function and fibrinolysis. Considering this relevance, here we describe the isolation and biochemical characterization of moojenactivase (MooA), a metalloprotease from Bothrops moojeni snake venom, and investigate its involvement in hemostasis in vitro. MooA is a glycoprotein of 85,746.22 Da, member of the PIIId group of snake venom metalloproteases, composed of three linked disulfide-bonded chains: an N-glycosylated heavy chain, and two light chains. The venom protease induced human plasma clotting in vitro by activating on both blood coagulation factors II (prothrombin) and X, which in turn generated α-thrombin and factor Xa, respectively. Additionally, MooA induced expression of tissue factor (TF) on the membrane surface of peripheral blood mononuclear cells (PBMC), which led these cells to adopt pro-coagulant characteristics. MooA was also shown to be involved with production of the inflammatory mediators TNF-α, IL-8 and MCP-1, suggesting an association between MooA pro-inflammatory stimulation of PBMC and TF up-regulation. We also observed aggregation of washed platelets when in presence of MooA; however, the protease had no effect on fibrinolysis. Our findings show that MooA is a novel hemostatically active metalloprotease, which may lead to the development of coagulopathies during B. moojeni envenomation. Moreover, the metalloprotease may contribute to the development of new diagnostic tools and pharmacological approaches applied to hemostatic disorders.

  9. A disintegrin and metalloprotease-17 and galectin-9 are important regulators of local 4-1BB activity and disease outcome in rheumatoid arthritis

    DEFF Research Database (Denmark)

    Nielsen, Morten Aagaard; Andersen, Thomas; Etzerodt, Anders

    2016-01-01

    OBJECTIVE: Co-stimulatory T cell cytokines are important in the progression of RA. This study investigates the interplay between 4-1BB, a disintegrin and metalloprotease-17 (ADAM17) and galectin-9 (Gal-9) in RA. METHODS: Stimulated mononuclear cells from patients with chronic RA (n = 12) were co...... treatment outcome serially over a 2-year period. RESULTS: RA CD4(+) and CD8(+) synovial T cells express high levels of 4-1BB. The addition of TNF-α to cultured synovial mononuclear cells increased shedding of 4-1BB. 4-1BB ligand only increased TNF-α shedding in combination with Gal-9. RNA interference...

  10. Expression of matrix metalloproteases-2 and -9 in horse hoof laminae after intestinal obstruction, with or without Hydrocortisone treatment Expressão de metaloproteinases 2 e 9 no tecido laminar do casco de equinos após obstrução intestinal e tratamento com hidrocortisona

    Directory of Open Access Journals (Sweden)

    Luciane Maria Laskoski

    2013-01-01

    Full Text Available Twenty horses were used in the experiment, for composed control group, (Cg instrumented group, (Ig;without intestinal obstruction, treated group (Tg;submitted to intestinal obstruction and hydrocortisone treatment and non-treated group (Ntg;submitted to intestinal obstruction without treatment. Immunohistochemistry and zymography techniques were used for researches on MMPs 2 and 9 in horse hoof laminae. There was an increase in the expression of MMP-2 in animals of Tg and Ntg. MMP-9 increased on animals from groups Ntg and Ig, however there was no rise of this MMP on the Tg when compared to the other groups in the immunohistochemistry analysis. Based on the results, it was observed that the intestinal injury caused by enterotomy and intestinal obstruction raise the quantities of MMPs in the hoof laminae.Vinte cavalos foram usados no experimento: para compor o grupo controle (Cg, grupo instrumentado, Ig (sem obstrução intestinal, grupo tratado, Tg (submetidos à obstrução intestinal e tratamento com hidrocortisona e grupo não tratado, Ntg (submetidos à obstrução intestinal, sem tratamento. Técnicas de zimografia e imunoistoquímica foram utilizadas para pesquisa de MMP-2 e MMP-9 no tecido laminar do casco dos equinos. Houve um aumento na expressão de MMP-2 nos animais dos grupos Tg e Ntg. A MMP-9 aumentou nos animais dos grupos Ig e Ntg. Houve aumento desta MMP no Tg quando comparado aos demais grupos na análise por zimografia. Observou-se que a injúria intestinal, causada pela enterotomia e obstrução intestinal, eleva a quantidade de MMPs no tecido laminar do casco.

  11. Cell-surface metalloprotease ADAM12 is internalized by a clathrin- and Grb2-dependent mechanism

    DEFF Research Database (Denmark)

    Hansen, Dorte Stautz; Leyme, Anthony; Grandal, Michael Vibo

    2012-01-01

    ADAM12 (A Disintegrin And Metalloprotease 12), a member of the ADAMs family of transmembrane proteins, is involved in ectodomain shedding, cell-adhesion and signaling, with important implications in cancer. Therefore, mechanisms that regulate the levels and activity of ADAM12 at the cell-surface ......ADAM12 (A Disintegrin And Metalloprotease 12), a member of the ADAMs family of transmembrane proteins, is involved in ectodomain shedding, cell-adhesion and signaling, with important implications in cancer. Therefore, mechanisms that regulate the levels and activity of ADAM12 at the cell...

  12. Helicobacter pylori and gastritis: the role of extracellular matrix metalloproteases, their inhibitors, and the disintegrins and metalloproteases--a systematic literature review.

    Science.gov (United States)

    Sampieri, Clara L

    2013-10-01

    Helicobacter pylori (H. pylori) is the etiologic agent of gastritis; it has been estimated that 50 % of the world's population could be infected by this bacteria. Gastritis may progress to chronic atrophic gastritis, a condition associated with the development of gastric cancer (GC). Several matrix metalloproteases (MMP) and tissue inhibitors of MMPs (TIMP) as well as disintegrins and metalloproteases (ADAM) have been reported as being involved in gastritis. Among other processes, these protein families participate in remodeling the extracellular matrix, cell signaling, immune response, angiogenesis, inflammation and epithelial mesenchymal transition. This systematic review analyzes the scientific evidence surrounding the relationship between members of the MMP, TIMP and ADAM families and infection by H. pylori in gastritis, considering both in vitro and in vivo studies. Given the potential clinical value of certain members of the MMP, TIMP and ADAM families as molecular markers in gastritis and the association of gastritis with GC, the need for further study is highlighted.

  13. Electroacupuncture modulates stromal cell-derived factor-1α expression and mobilization of bone marrow endothelial progenitor cells in focal cerebral ischemia/reperfusion model rats.

    Science.gov (United States)

    Xie, Chenchen; Gao, Xiang; Luo, Yong; Pang, Yueshan; Li, Man

    2016-10-01

    Stromal cell-derived factor-1α(SDF-1α) plays a crucial role in regulating the mobilization, migration and homing of endothelial progenitor cells(EPCs). Electroacupuncture(EA), a modern version of Traditional Chinese Medicine, can improve neurological recovery and angiogenesis in cerebral ischemic area. This study aimed to investigate the effects of electroacupuncture(EA) on the mobilization and migration of bone marrow EPCs and neurological functional recovery in rats model after focal cerebral ischemia/reperfusion and the potentially involved mechanisms. Sprague-Dawley rats received filament occlusion of the right middle cerebral artery for 2h followed by reperfusion for 12h, 1d, 2d, 3d, 7d respectively. Rats were randomly divided into sham group, model group and EA group. After 2h of the reperfusion, EA was given at the "Baihui" (GV 20)/Siguan ("Hegu" (LI 4)/"Taichong" (LR 3)) acupoints in the EA group. Modified neurological severity score (mNSS) was used to assess the neurological functional recovery. EPCs number and SDF-1α level in bone marrow(BM) and peripheral blood(PB) were detected by using fluorescence-activated cell sorting (FACS) analysis and quantitative real time polymerase chain reaction (qRT-PCR) respectively. An mNSS test showed that EA treatment significantly improved the neurological functional outcome. EPCs number in PB and BM were obviously increased in the EA group. After cerebral ischemia, the SDF-1α level was decreased in BM while it was increased in PB, which implied a gradient of SDF-1α among BM and PB after ischemia. It suggested that the forming of SDF-1α concentration gradient can induce the mobilization and homing of EPCs. Eletroacupuncture as a treatment can accelerate and increase the forming of SDF-1α concentration gradient to further induce the mobilization of EPCs and angiogenesis in ischemic brain and improve the neurological function recovery. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Gastrointestinal stromal tumors

    International Nuclear Information System (INIS)

    Sufliarsky, J.

    2011-01-01

    Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumours of the digestive tract. Better understanding of the molecular characteristics of GISTs led to the clinical development of imatinib for treating patients with this disease. New immuno markers and mechanisms of primary and secondary resistance were discovered. Adjuvant imatinib in intermediate or high risk GIST has improved the recurrence-free survival. Sunitinib in patients with intolerance or progression on imatinib demonstrated significant improvements in progression-free and overall survival versus placebo. Second-generation tyrosine kinase inhibitors, such as sorafenib, dasatinib, and nilotinib, have shown activity in patients with imatinib- and sunitinib-resistant GIST. (author)

  15. Membrane-type matrix metalloproteases as diverse effectors of cancer progression.

    Science.gov (United States)

    Turunen, S Pauliina; Tatti-Bugaeva, Olga; Lehti, Kaisa

    2017-11-01

    Membrane-type matrix metalloproteases (MT-MMP) are pivotal regulators of cell invasion, growth and survival. Tethered to the cell membranes by a transmembrane domain or GPI-anchor, the six MT-MMPs can exert these functions via cell surface-associated extracellular matrix degradation or proteolytic protein processing, including shedding or release of signaling receptors, adhesion molecules, growth factors and other pericellular proteins. By interactions with signaling scaffold or cytoskeleton, the C-terminal cytoplasmic tail of the transmembrane MT-MMPs further extends their functionality to signaling or structural relay. MT-MMPs are differentially expressed in cancer. The most extensively studied MMP14/MT1-MMP is induced in various cancers along malignant transformation via pathways activated by mutations in tumor suppressors or proto-oncogenes and changes in tumor microenvironment including cellular heterogeneity, extracellular matrix composition, tissue oxygenation, and inflammation. Classically such induction involves transcriptional programs related to epithelial-to-mesenchymal transition. Besides inhibition by endogenous tissue inhibitors, MT-MMP activities are spatially and timely regulated at multiple levels by microtubular vesicular trafficking, dimerization/oligomerization, other interactions and localization in the actin-based invadosomes, in both tumor and the stroma. The functions of MT-MMPs are multifaceted within reciprocal cellular responses in the evolving tumor microenvironment, which poses the importance of these proteases beyond the central function as matrix scissors, and necessitates us to rethink MT-MMPs as dynamic signaling proteases of cancer. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Regulated expression of homeobox genes Msx-1 and Msx-2 in mouse mammary gland development suggests a role in hormone action and epithelial-stromal interactions.

    Science.gov (United States)

    Friedmann, Y; Daniel, C W

    1996-07-10

    The murine homeobox genes Msx-1 and Msx-2 are related to the Drosophila msh gene and are expressed in a variety of tissues during mouse embryogenesis. We now report the developmentally regulated expression of Msx-1 and Msx-2 in the mouse mammary gland and show that their expression patterns point toward significant functional roles. Msx-1 and Msx-2 transcripts were present in glands of virgin mice and in glands of mice in early pregnancy, but transcripts decreased dramatically during late pregnancy. Low levels of Msx-1 transcripts were detected in glands from lactating animals and during the first days of involution, whereas Msx-2 expression was not detected during lactation or early involution. Expression of both genes increased gradually as involution progressed. Msx-2 but not Msx-1 expression was decreased following ovariectomy or following exposure to anti-estrogen implanted directly into the gland. Hormonal regulation of Msx-2 expression was confirmed when transcripts returned to normal levels after estrogen was administered to ovariectomized animals. In situ molecular hybridization for Msx-1 showed transcripts localized to the mammary epithelium, whereas Msx-2 expression was confined to the periductal stroma. Mammary stroma from which mammary epithelium had been removed did not transcribe detectable amounts of Msx-2, showing that expression is regulated by contiguous mammary epithelium, and indicating a role for these homeobox genes in mesenchymal-epithelial interactions during mammary development.

  17. Correlation of serum and urinary matrix metalloproteases/tissue inhibitors of metalloproteases with subclinical allograft fibrosis in renal transplantation.

    Science.gov (United States)

    Hirt-Minkowski, Patricia; Marti, Hans-Peter; Hönger, Gideon; Grandgirard, Denis; Leib, Stephen L; Amico, Patrizia; Schaub, Stefan

    2014-01-01

    Progressive interstitial fibrosis and tubular atrophy (IF/TA) is a leading cause of chronic allograft dysfunction. Increased extracellular matrix remodeling regulated by matrix metalloproteases (MMPs) and their inhibitors (TIMPs) has been implicated in the development of IF/TA. The aim of this study was to investigate whether urinary/serum MMPs/TIMPs correlate with subclinical IF/TA detected in surveillance biopsies within the first 6months post-transplant. We measured eight different MMPs/TIMPs simultaneously in urine and serum samples from patients classified as normal histology (n=15), IF/TA 1 (n=15) and IF/TA 2-3 (n=10). There was no difference in urinary MMPs/TIMPs among the three groups, and only 1/8 serum MMPs/TIMPs (i.e. MMP-1) was significantly elevated in biopsies with IF/TA 2-3 (p=0.01). In addition, urinary/serum MMPs/TIMPs were not different between surveillance biopsies demonstrating an early development of IF/TA (i.e. delta IF/TA≥1 compared to a previous biopsy obtained three months before; n=11) and stable grade of IF/TA (i.e. delta IF/TA=0; n=20). Next, we investigated whether urinary/serum MMP/TIMP levels are elevated during acute subclinical tubulitis in surveillance biopsies obtained within the first 6months post-transplant (n=25). Compared to biopsies with normal histology, serum MMPs/TIMPs were not different; however, all urinary MMP/TIMP levels were numerically higher during subclinical tubulitis (MMP-1, MMP-7, TIMP-1 with p≤0.04). We conclude that urinary/serum MMPs/TIMPs do hardly correlate with existing or early developing IF/TA in surveillance biopsies obtained within the first 6months post-transplant. This could be explained by the dynamic process of extracellular matrix remodeling, which seems to be active during acute tubulo-interstitial injury/inflammation, but not in quiescent IF/TA. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. Role of matrix metalloprotease-2 in oxidant activation of Ca ATPase ...

    Indian Academy of Sciences (India)

    Unknown

    We sought to determine the role of matrix metalloprotease-2 (MMP-2) ... Ca2+ overload in cells and tissues (Bennet and Williams. 1993; Ghosh et ..... tease balance in favour of the protease. ... This work was partly supported by the Life Science.

  19. Development of an integrated system for activity-based profiling of matrix metallo-proteases

    NARCIS (Netherlands)

    Freije, Jan Robert

    2006-01-01

    Matrix metallo-proteases constitute a family of extracellular zinc-dependent endopeptidases that are involved in degradation of extracellular matrix (ECM) components and other bioactive non-ECM molecules. A plethora of studies have implicated important roles for MMPs in many diseases (including

  20. All-trans retinoic acid inhibits KIT activity and induces apoptosis in gastrointestinal stromal tumor GIST-T1 cell line by affecting on the expression of survivin and Bax protein

    Directory of Open Access Journals (Sweden)

    Taguchi Takahiro

    2010-12-01

    Full Text Available Abstract Background Imatinib, a selective tyrosine kinase inhibitor, has been used as a standard first-line therapy for irresectable and metastasized gastrointestinal stromal tumor (GIST patients. Unfortunately, most patients responding to imatinib will eventually exhibit imatinib-resistance, the cause of which is not fully understood. The serious clinical problem of imatinib-resistance demands alternative therapeutic strategy. This study was conducted to investigate the effect of all-trans retinoic acid (ATRA on GIST cell lines. Methods Cell proliferation was determined by trypan blue dye exclusion test. Western blot analysis was performed to test the expression of activated KIT, its downstream proteins, and apoptosis associated proteins. The cytotoxic interactions of imatinib with ATRA were evaluated using the isobologram of Steel and Peckham. Results and conclusion In this work, for the first time we have demonstrated that ATRA affected on cell proliferation of GIST-T1 and GIST-882 cell line through inhibition of cell growth in a dose dependent manner and induced apoptosis. High dose of ATRA induced morphologic change in GIST-T1 cells, rounded-up cells, and activated the caspase-3 protein. In further examination, we found that the ATRA-induced apoptosis in GIST-T1 cells was accompanied by the down-regulated expression of survivin and up-regulated expression of Bax protein. Moreover, ATRA suppressed the activity of KIT protein in GIST-T1 cells and its downstream signal, AKT activity, but not MAPK activity. We also have demonstrated that combination of ATRA with imatinib showed additive effect by isobologram, suggesting that the combination of ATRA and imatinib may be a novel potential therapeutic option for GIST treatment. Furthermore, the scracht assay result suggested that ATRA was a potential reagent to prevent the invasion or metastasis of GIST cells.

  1. Targeting Stromal Androgen Receptor Suppresses Prolactin-Driven Benign Prostatic Hyperplasia (BPH)

    Science.gov (United States)

    Lai, Kuo-Pao; Huang, Chiung-Kuei; Fang, Lei-Ya; Izumi, Kouji; Lo, Chi-Wen; Wood, Ronald; Kindblom, Jon; Yeh, Shuyuan

    2013-01-01

    Stromal-epithelial interaction plays a pivotal role to mediate the normal prostate growth, the pathogenesis of benign prostatic hyperplasia (BPH), and prostate cancer development. Until now, the stromal androgen receptor (AR) functions in the BPH development, and the underlying mechanisms remain largely unknown. Here we used a genetic knockout approach to ablate stromal fibromuscular (fibroblasts and smooth muscle cells) AR in a probasin promoter-driven prolactin transgenic mouse model (Pb-PRL tg mice) that could spontaneously develop prostate hyperplasia to partially mimic human BPH development. We found Pb-PRL tg mice lacking stromal fibromuscular AR developed smaller prostates, with more marked changes in the dorsolateral prostate lobes with less proliferation index. Mechanistically, prolactin mediated hyperplastic prostate growth involved epithelial-stromal interaction through epithelial prolactin/prolactin receptor signals to regulate granulocyte macrophage-colony stimulating factor expression to facilitate stromal cell growth via sustaining signal transducer and activator of transcription-3 activity. Importantly, the stromal fibromuscular AR could modulate such epithelial-stromal interacting signals. Targeting stromal fibromuscular AR with the AR degradation enhancer, ASC-J9®, led to the reduction of prostate size, which could be used in future therapy. PMID:23893956

  2. Equine deep stromal abscesses (51 cases - 2004-2009) - Part 2

    DEFF Research Database (Denmark)

    Henriksen, Michala de Linde; Andersen, Pia Haubro; Mietelka, Kristy

    2014-01-01

    To investigate histopathologic and immunohistochemical aspects of equine deep stromal abscesses (DSA) with a focus on the histopathologic diagnosis, presumptive etiology, and the immunohistochemical expression of three angiogenesis-related factors: vascular endothelial growth factor-A (VEGF...

  3. Mesenchymal Stromal Cells for Antineoplastic Drug Loading and Delivery.

    Science.gov (United States)

    Petrella, Francesco; Rimoldi, Isabella; Rizzo, Stefania; Spaggiari, Lorenzo

    2017-11-23

    Mesenchymal stromal cells are a population of undifferentiated multipotent adult cells possessing extensive self-renewal properties and the potential to differentiate into a variety of mesenchymal lineage cells. They express broad anti-inflammatory and immunomodulatory activity on the immune system and after transplantation can interact with the surrounding microenvironment, promoting tissue healing and regeneration. For this reason, mesenchymal stromal cells have been widely used in regenerative medicine, both in preclinical and clinical settings. Another clinical application of mesenchymal stromal cells is the targeted delivery of chemotherapeutic agents to neoplastic cells, maximizing the cytotoxic activity against cancer cells and minimizing collateral damage to non-neoplastic tissues. Mesenchymal stem cells are home to the stroma of several primary and metastatic neoplasms and hence can be used as vectors for targeted delivery of antineoplastic drugs to the tumour microenvironment, thereby reducing systemic toxicity and maximizing antitumour effects. Paclitaxel and gemcitabine are the chemotherapeutic drugs best loaded by mesenchymal stromal cells and delivered to neoplastic cells, whereas other agents, like pemetrexed, are not internalized by mesenchymal stromal cells and therefore are not suitable for advanced antineoplastic therapy. This review focuses on the state of the art of advanced antineoplastic cell therapy and its future perspectives, emphasizing in vitro and in vivo preclinical results and future clinical applications.

  4. Molecular detection and analysis of a novel metalloprotease gene of entomopathogenic Serratia marcescens strains in infected Galleria mellonella.

    Science.gov (United States)

    Tambong, J T; Xu, R; Sadiku, A; Chen, Q; Badiss, A; Yu, Q

    2014-04-01

    Serratia marcescens strains isolated from entomopathogenic nematodes (Rhabditis sp.) were examined for their pathogenicity and establishment in wax moth (Galleria mellonella) larvae. All the Serratia strains were potently pathogenic to G. mellonella larvae, leading to death within 48 h. The strains were shown to possess a metalloprotease gene encoding for a novel serralysin-like protein. Rapid establishment of the bacteria in infected larvae was confirmed by specific polymerase chain reaction (PCR) detection of a DNA fragment encoding for this protein. Detection of the viable Serratia strains in infected larvae was validated using the SYBR Green reverse transcriptase real-time PCR assay targeting the metalloprotease gene. Nucleotide sequences of the metalloprotease gene obtained in our study showed 72 single nucleotide polymorphisms (SNP) and 3 insertions compared with the metalloprotease gene of S. marcescens E-15. The metalloprotease gene had 60 synonymous and 8 nonsynonymous substitutions relative to the closest GenBank entry, S. marcescens E-15. A comparison of the amino acid composition of the new serralysin-like protein with that of the serralysin protein of S. marcescens E-15 revealed differences at 11 positions and a new aspartic acid residue. Analysis of the effect of protein variation suggests that a new aspartic acid residue resulting from nonsynonymous nucleotide mutations in the protein structure could have the most significant effect on its biological function. The new metalloprotease gene and (or) its product could have applications in plant agricultural biotechnology.

  5. Bone marrow stromal cells spontaneously produce Flt3-ligand: influence of ionizing radiations and cytokine stimulation.

    Science.gov (United States)

    Bertho, Jean Marc; Demarquay, Christelle; Mouiseddine, Moubarak; Douenat, Noémie; Stefani, Johanna; Prat, Marie; Paquet, François

    2008-08-01

    To define the ability of human bone marrow (BM) stromal cells to produce fms-like tyrosine kinase 3 (Flt3)-ligand (FL), and the effect of irradiation, tumour necrosis factor-alpha (TNFalpha) or tumour growth factor beta (TGFbeta) on FL production. Primary BM stromal cell cultures were irradiated at 2-10 Gy or were stimulated with TNFalpha or TGFbeta1. The presence of FL was tested in culture supernatants and in cell lysate. The presence of a membrane-bound form of FL and the level of gene expression were also tested. Primary BM stromal cells spontaneously released FL. This production was increased by TNFalpha but not by TGFbeta1 or by irradiation. Chemical induction of osteoblastic differentiation from BM stromal cells also induced an increase in FL release. Our results suggest that the observed increase in FL concentration after in vivo irradiation is an indirect effect. The possible implication of BM stromal cells in these mechanisms is discussed.

  6. Involvement of Bmi-1 gene in the development of gastrointestinal stromal tumor by regulating p16Ink4A/p14ARF gene expressions: An in vivo and in vitro study.

    Science.gov (United States)

    Wang, Jiang-Li; Wu, Jiang-Hong; Hong, Cai; Wang, Ya-Nong; Zhou, Ye; Long, Zi-Wen; Zhou, Ying; Qin, Hai-Shu

    2017-12-01

    This study was conducted in order to explore the role that Bmi-1 plays during the development of a gastrointestinal stromal tumor (GIST) by regulation of the p16 Ink4A and p14 ARF expressions. Eighty-six patients diagnosed with GIST were selected to take part in this experiment. The Bmi-1 protein expressions in GIST and adjacent normal tissues were detected using immunohistochemistry and further analyzed by using photodensitometry. To monitor and track the progression of the GIST, a 3-year follow-up was conducted for all affected patients. After cell transfection, the GIST cells were assigned into the control group (without transfection), the negative control (NC) group (transfected with Bmi-1-Scramble plasmid), and the Bmi-1 shRNA group (transfected with the pcDNA3.1-Bmi-1 shRNA plasmid). Protein and mRNA expressions collected from Bmi-1, p16 lnk4A , P14 ARF , cyclin D1, and CDK4 were measured using both the RT-qPCR and western blotting methods Cell senescence was assessed and obtained by using the β-Galactosidase (β-Gal) activity assay. The use of a Soft agar colony formation assay and CCK-8 assay were performed in order to detect the cell growth and subsequent proliferation. Cell invasion and migration were analyzed using the Transwell assay and scratch test. Bmi-1 in the GIST tissues was found to be significantly higher and the p16 lnk4A and P14 ARF expressions were lower than those in the adjacent normal tissues. Bmi-1 was negatively correlated with p16 lnk4A and P14 ARF expressions according to the correlation analysis. Bmi-1 expression was associated with the TNM stage, postoperative recurrence, metastasis, tumor size, and the 5-year survival rate. Area under ROC curve was calculated at 0.884, and sensitivity, specificity, and accuracy of Bmi-1 predicting the GIST were 67.44%, 97.67%, and 65.12%, respectively. Patients exhibiting a high Bmi-1 expression in the GIST tissues had lower survival rates than those with low Bmi-1 expression. In comparison with

  7. The Propeptide of the Metalloprotease of Listeria monocytogenes Controls Compartmentalization of the Zymogen during Intracellular Infection▿

    OpenAIRE

    O'Neil, Heather S.; Forster, Brian M.; Roberts, Kari L.; Chambers, Andrew J.; Bitar, Alan Pavinski; Marquis, Hélène

    2009-01-01

    Integral to the virulence of the intracellular bacterial pathogen Listeria monocytogenes is its metalloprotease (Mpl). Mpl regulates the activity and compartmentalization of the bacterial broad-range phospholipase C (PC-PLC). Mpl is secreted as a proprotein that undergoes intramolecular autocatalysis to release its catalytic domain. In related proteases, the propeptide serves as a folding catalyst and can act either in cis or in trans. Propeptides can also influence protein compartmentalizati...

  8. The Metalloprotease of Listeria monocytogenes Is Activated by Intramolecular Autocatalysis▿ †

    OpenAIRE

    Bitar, Alan Pavinski; Cao, Min; Marquis, Hélène

    2007-01-01

    The metalloprotease (Mpl) of Listeria monocytogenes is a thermolysin-like protease that mediates the maturation of a broad-range phospholipase C, whose function contributes to the ability of this food-borne bacterial pathogen to survive intracellularly. Mpl is made as a proprotein that undergoes maturation by proteolytic cleavage of a large N-terminal prodomain. In this study, we identified the N terminus of mature Mpl and generated Mpl catalytic mutants to investigate the mechanism of Mpl ma...

  9. Comparison of the Internal Dynamics of Metalloproteases Provides New Insights on Their Function and Evolution.

    Directory of Open Access Journals (Sweden)

    Henrique F Carvalho

    Full Text Available Metalloproteases have evolved in a vast number of biological systems, being one of the most diverse types of proteases and presenting a wide range of folds and catalytic metal ions. Given the increasing understanding of protein internal dynamics and its role in enzyme function, we are interested in assessing how the structural heterogeneity of metalloproteases translates into their dynamics. Therefore, the dynamical profile of the clan MA type protein thermolysin, derived from an Elastic Network Model of protein structure, was evaluated against those obtained from a set of experimental structures and molecular dynamics simulation trajectories. A close correspondence was obtained between modes derived from the coarse-grained model and the subspace of functionally-relevant motions observed experimentally, the later being shown to be encoded in the internal dynamics of the protein. This prompted the use of dynamics-based comparison methods that employ such coarse-grained models in a representative set of clan members, allowing for its quantitative description in terms of structural and dynamical variability. Although members show structural similarity, they nonetheless present distinct dynamical profiles, with no apparent correlation between structural and dynamical relatedness. However, previously unnoticed dynamical similarity was found between the relevant members Carboxypeptidase Pfu, Leishmanolysin, and Botulinum Neurotoxin Type A, despite sharing no structural similarity. Inspection of the respective alignments shows that dynamical similarity has a functional basis, namely the need for maintaining proper intermolecular interactions with the respective substrates. These results suggest that distinct selective pressure mechanisms act on metalloproteases at structural and dynamical levels through the course of their evolution. This work shows how new insights on metalloprotease function and evolution can be assessed with comparison schemes that

  10. Potential Effect of CD271 on Human Mesenchymal Stromal Cell Proliferation and Differentiation

    Directory of Open Access Journals (Sweden)

    Giovanna Calabrese

    2015-07-01

    Full Text Available The Low-Affinity Nerve Growth Factor Receptor (LNGFR, also known as CD271, is a member of the tumor necrosis factor receptor superfamily. The CD271 cell surface marker defines a subset of multipotential mesenchymal stromal cells and may be used to isolate and enrich cells derived from bone marrow aspirate. In this study, we compare the proliferative and differentiation potentials of CD271+ and CD271− mesenchymal stromal cells. Mesenchymal stromal cells were isolated from bone marrow aspirate and adipose tissue by plastic adherence and positive selection. The proliferation and differentiation potentials of CD271+ and CD271− mesenchymal stromal cells were assessed by inducing osteogenic, adipogenic and chondrogenic in vitro differentiation. Compared to CD271+, CD271− mesenchymal stromal cells showed a lower proliferation rate and a decreased ability to give rise to osteocytes, adipocytes and chondrocytes. Furthermore, we observed that CD271+ mesenchymal stromal cells isolated from adipose tissue displayed a higher efficiency of proliferation and trilineage differentiation compared to CD271+ mesenchymal stromal cells isolated from bone marrow samples, although the CD271 expression levels were comparable. In conclusion, these data show that both the presence of CD271 antigen and the source of mesenchymal stromal cells represent important factors in determining the ability of the cells to proliferate and differentiate.

  11. Potential Effect of CD271 on Human Mesenchymal Stromal Cell Proliferation and Differentiation.

    Science.gov (United States)

    Calabrese, Giovanna; Giuffrida, Raffaella; Lo Furno, Debora; Parrinello, Nunziatina Laura; Forte, Stefano; Gulino, Rosario; Colarossi, Cristina; Schinocca, Luciana Rita; Giuffrida, Rosario; Cardile, Venera; Memeo, Lorenzo

    2015-07-09

    The Low-Affinity Nerve Growth Factor Receptor (LNGFR), also known as CD271, is a member of the tumor necrosis factor receptor superfamily. The CD271 cell surface marker defines a subset of multipotential mesenchymal stromal cells and may be used to isolate and enrich cells derived from bone marrow aspirate. In this study, we compare the proliferative and differentiation potentials of CD271+ and CD271- mesenchymal stromal cells. Mesenchymal stromal cells were isolated from bone marrow aspirate and adipose tissue by plastic adherence and positive selection. The proliferation and differentiation potentials of CD271+ and CD271- mesenchymal stromal cells were assessed by inducing osteogenic, adipogenic and chondrogenic in vitro differentiation. Compared to CD271+, CD271- mesenchymal stromal cells showed a lower proliferation rate and a decreased ability to give rise to osteocytes, adipocytes and chondrocytes. Furthermore, we observed that CD271+ mesenchymal stromal cells isolated from adipose tissue displayed a higher efficiency of proliferation and trilineage differentiation compared to CD271+ mesenchymal stromal cells isolated from bone marrow samples, although the CD271 expression levels were comparable. In conclusion, these data show that both the presence of CD271 antigen and the source of mesenchymal stromal cells represent important factors in determining the ability of the cells to proliferate and differentiate.

  12. The Disintegrin and Metalloprotease ADAM12 Is Associated with TGF-β-Induced Epithelial to Mesenchymal Transition.

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    Michaël Ruff

    Full Text Available The increased expression of the Disintegrin and Metalloprotease ADAM12 has been associated with human cancers, however its role remain unclear. We have previously reported that ADAM12 expression is induced by the transforming growth factor, TGF-β and promotes TGF-β-dependent signaling through interaction with the type II receptor of TGF-β. Here we explore the implication of ADAM12 in TGF-β-mediated epithelial to mesenchymal transition (EMT, a key process in cancer progression. We show that ADAM12 expression is correlated with EMT markers in human breast cancer cell lines and biopsies. Using a non-malignant breast epithelial cell line (MCF10A, we demonstrate that TGF-β-induced EMT increases expression of the membrane-anchored ADAM12L long form. Importantly, ADAM12L overexpression in MCF10A is sufficient to induce loss of cell-cell contact, reorganization of actin cytoskeleton, up-regulation of EMT markers and chemoresistance. These effects are independent of the proteolytic activity but require the cytoplasmic tail and are specific of ADAM12L since overexpression of ADAM12S failed to induce similar changes. We further demonstrate that ADAM12L-dependent EMT is associated with increased phosphorylation of Smad3, Akt and ERK proteins. Conversely, inhibition of TGF-β receptors or ERK activities reverses ADAM12L-induced mesenchymal phenotype. Together our data demonstrate that ADAM12L is associated with EMT and contributes to TGF-β-dependent EMT by favoring both Smad-dependent and Smad-independent pathways.

  13. Polymorphisms in stromal genes and susceptibility to serous epithelial ovarian cancer: a report from the Ovarian Cancer Association Consortium

    DEFF Research Database (Denmark)

    Amankwah, Ernest K; Wang, Qinggang; Schildkraut, Joellen M

    2011-01-01

    Alterations in stromal tissue components can inhibit or promote epithelial tumorigenesis. Decorin (DCN) and lumican (LUM) show reduced stromal expression in serous epithelial ovarian cancer (sEOC). We hypothesized that common variants in these genes associate with risk. Associations with sEOC among...

  14. Cooperative effects of aminopeptidase N (CD13) expressed by nonmalignant and cancer cells within the tumor microenvironment.

    Science.gov (United States)

    Guzman-Rojas, Liliana; Rangel, Roberto; Salameh, Ahmad; Edwards, Julianna K; Dondossola, Eleonora; Kim, Yun-Gon; Saghatelian, Alan; Giordano, Ricardo J; Kolonin, Mikhail G; Staquicini, Fernanda I; Koivunen, Erkki; Sidman, Richard L; Arap, Wadih; Pasqualini, Renata

    2012-01-31

    Processes that promote cancer progression such as angiogenesis require a functional interplay between malignant and nonmalignant cells in the tumor microenvironment. The metalloprotease aminopeptidase N (APN; CD13) is often overexpressed in tumor cells and has been implicated in angiogenesis and cancer progression. Our previous studies of APN-null mice revealed impaired neoangiogenesis in model systems without cancer cells and suggested the hypothesis that APN expressed by nonmalignant cells might promote tumor growth. We tested this hypothesis by comparing the effects of APN deficiency in allografted malignant (tumor) and nonmalignant (host) cells on tumor growth and metastasis in APN-null mice. In two independent tumor graft models, APN activity in both the tumors and the host cells cooperate to promote tumor vascularization and growth. Loss of APN expression by the host and/or the malignant cells also impaired lung metastasis in experimental mouse models. Thus, cooperation in APN expression by both cancer cells and nonmalignant stromal cells within the tumor microenvironment promotes angiogenesis, tumor growth, and metastasis.

  15. VanT, a homologue of Vibrio harveyi LuxR, regulates serine, metalloprotease, pigment, and biofilm production in Vibrio anguillarum.

    Science.gov (United States)

    Croxatto, Antony; Chalker, Victoria J; Lauritz, Johan; Jass, Jana; Hardman, Andrea; Williams, Paul; Cámara, Miguel; Milton, Debra L

    2002-03-01

    Vibrio anguillarum possesses at least two N-acylhomoserine lactone (AHL) quorum-sensing circuits, one of which is related to the luxMN system of Vibrio harveyi. In this study, we have cloned an additional gene of this circuit, vanT, encoding a V. harveyi LuxR-like transcriptional regulator. A V. anguillarum Delta vanT null mutation resulted in a significant decrease in total protease activity due to loss of expression of the metalloprotease EmpA, but no changes in either AHL production or virulence. Additional genes positively regulated by VanT were identified from a plasmid-based gene library fused to a promoterless lacZ. Three lacZ fusions (serA::lacZ, hpdA-hgdA::lacZ, and sat-vps73::lacZ) were identified which exhibited decreased expression in the Delta vanT strain. SerA is similar to 3-phosphoglycerate dehydrogenases and catalyzes the first step in the serine-glycine biosynthesis pathway. HgdA has identity with homogentisate dioxygenases, and HpdA is homologous to 4-hydroxyphenylpyruvate dioxygenases (HPPDs) involved in pigment production. V. anguillarum strains require an active VanT to produce high levels of an L-tyrosine-induced brown color via HPPD, suggesting that VanT controls pigment production. Vps73 and Sat are related to Vibrio cholerae proteins encoded within a DNA locus required for biofilm formation. A V. anguillarum Delta vanT mutant and a mutant carrying a polar mutation in the sat-vps73 DNA locus were shown to produce defective biofilms. Hence, a new member of the V. harveyi LuxR transcriptional activator family has been characterized in V. anguillarum that positively regulates serine, metalloprotease, pigment, and biofilm production.

  16. Cyclin G1 inhibits the proliferation of mouse endometrial stromal cell in decidualization

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    Xu Qian

    2017-01-01

    Full Text Available Uterine stromal cell decidualization is a dynamic physiological process in which cell proliferation, differentiation and apoptosis are orchestrated and occur in a temporal and cell-specific manner. This process is important for successful embryo implantation. Many cell-cycle regulators are involved in decidualization. The protein cyclin G1 is a unique regulator of the cell cycle with dual functions in cell proliferation. It was reported that cyclin G1 is expressed in mouse uterine stromal cells during the period of peri-implantation. To prove the function of cyclin G1 in mouse uterine stromal cells during this period, immunohistochemistry was used to stain mouse uterine tissues on days 4-8 of pregnancy. The results showed obvious spatial and temporal expression of cyclin G1 in uterine stromal cells, and that it is expressed in the cells of the primary decidual zone (PDZ on day 5 and secondary decidual zone (SDZ on days 6 and 7, when the stromal cells experienced active proliferation and differentiation was initiated. Applying the decidualization model of cultured primary stromal cells in vitro, we further revealed that the expression of cyclin G1 is associated with decidualization of stromal cells induced by medroxyprogesterone acetate (MPA and estradiol-17β (E2. RNA interference was used for the knockdown of cyclin G1 in the induced decidual cells. Flow cytometry analysis indicated that the proportion of cells in the S stage was increased, and decreased in the G2/M phase. Our study indicates that cyclin G1, as a negative regulator of the cell cycle, plays an important role in the process of decidualization in mouse uterine stromal cells by inhibiting cell-cycle progression.

  17. Gastrointestinal stromal tumor: a case report and review of the literature

    International Nuclear Information System (INIS)

    Macedo, Leonardo Lopes de; Torres, Lucas Rios; Faucz, Rafael Artigas; Tornin, Olger de Souza; Souza, Ricardo Pires de; Fonseca, Carlos Alberto Marcovechio

    2006-01-01

    Gastrointestinal stromal tumors are the most common mesenchymal tumors and are characterized by expression of KIT (CD117), a tyrosine-kinase growth factor receptor. They occur in individuals over 50 years of age and commonly arise in stomach or in the small intestine. To emphasize our paper we report a case of gastrointestinal stromal tumor that showed the typical image and pathologic findings of the primary lesion and its metastases. (author)

  18. A smallest 6 kda metalloprotease, mini-matrilysin, in living world: a revolutionary conserved zinc-dependent proteolytic domain- helix-loop-helix catalytic zinc binding domain (ZBD

    Directory of Open Access Journals (Sweden)

    Yu Wei-Hsuan

    2012-05-01

    Full Text Available Abstract Background The Aim of this study is to study the minimum zinc dependent metalloprotease catalytic folding motif, helix B Met loop-helix C, with proteolytic catalytic activities in metzincin super family. The metzincin super family share a catalytic domain consisting of a twisted five-stranded β sheet and three long α helices (A, B and C. The catalytic zinc is at the bottom of the cleft and is ligated by three His residues in the consensus sequence motif, HEXXHXXGXXH, which is located in helix B and part of the adjacent Met turn region. An interesting question is - what is the minimum portion of the enzyme that still possesses catalytic and inhibitor recognition?” Methods We have expressed a 60-residue truncated form of matrilysin which retains only the helix B-Met turn-helix C region and deletes helix A and the five-stranded β sheet which form the upper portion of the active cleft. This is only 1/4 of the full catalytic domain. The E. coli derived 6 kDa MMP-7 ZBD fragments were purified and refolded. The proteolytic activities were analyzed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay and CM-transferrin zymography analysis. SC44463, BB94 and Phosphoramidon were computationally docked into the 3day structure of the human MMP7 ZBD and TAD and thermolysin using the docking program GOLD. Results This minimal 6 kDa matrilysin has been refolded and shown to have proteolytic activity in the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay. Triton X-100 and heparin are important factors in the refolding environment for this mini-enzyme matrilysin. This minienzyme has the proteolytic activity towards peptide substrate, but the hexamer and octamer of the mini MMP-7 complex demonstrates the CM-transferrin proteolytic activities in zymographic analysis. Peptide digestion is inhibited by SC44463, specific MMP7 inhibitors, but not phosphorimadon. Interestingly, the mini MMP-7 can be processed by autolysis and producing ~ 6

  19. Cerebellar hemangioblastomas: A study of the immunoprofile of neoplastic stromal component

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    Tasić Desanka

    2004-01-01

    Full Text Available Background. Central nervous system hemangioblastomas (HBs are uncommon highly vascularized tumors that are predominantly found in the cerebellum. They occur sporadically or in association with von Hippel-Lindau (VHL disease. HBs are of unknown histogenesis, and the origin of stromal cells is still a subject of debate. The aim of this study was to investigate the immunoprofile of neoplastic stromal component, and to determine whether the profile of the expression of immunomarkers used can contribute to the elucidation of the histogenesis of HBs. Methods. A series of eight cerebellar HBs were histochemically examined for the detection of mast cells and immunohistochemically for the expression of factor VIII-related antigen (FVIII-RAg, CD34, vimentin, factor XIIIa (FXIIIa, S-100 protein, glial fibrillary acidic protein (GFAP, neuron-specific enolase (NSE neurofilaments (NF, synaptophysin, chromogranin, and somatostatin. Results. Mast cells were present in all hemangioblastomas, and were particularly abundant in one tumor. Immunohistochemically, intense reactivity for vimentin and NSE in the stromal cells was constantly seen. Immunoreactivity with S-100 protein and FXIIIa was variable, but generally many HBs stromal cells were negative for these markers. However, stromal cells were uniformly negative for FVIII-RAg in all HBs investigated. They were negative for CD34 GFAP, NF, synaptophysin, chromogranin, as well as somatostatin. GFAP-positivity of the occasional stromal type cells, located only peripherally, was interpreted as "pseudopositivity". Conclusion. The immunoprofile of neoplastic stromal component in this study suggested a possible origin from undifferentiated multipotential mesenchymal cells. High expression of NSE (glycolytic and hypoxia-inducible enzyme in the HBs stromal cells might be related to the loss of the VHL protein function.

  20. Insufficient stromal support in MDS results from molecular and functional deficits of mesenchymal stromal cells.

    Science.gov (United States)

    Geyh, S; Oz, S; Cadeddu, R-P; Fröbel, J; Brückner, B; Kündgen, A; Fenk, R; Bruns, I; Zilkens, C; Hermsen, D; Gattermann, N; Kobbe, G; Germing, U; Lyko, F; Haas, R; Schroeder, T

    2013-09-01

    Ineffective hematopoiesis is a major characteristic of myelodysplastic syndromes (MDS) causing relevant morbidity and mortality. Mesenchymal stromal cells (MSC) have been shown to physiologically support hematopoiesis, but their contribution to the pathogenesis of MDS remains elusive. We show that MSC from patients across all MDS subtypes (n=106) exhibit significantly reduced growth and proliferative capacities accompanied by premature replicative senescence. Osteogenic differentiation was significantly reduced in MDS-derived MSC, indicated by cytochemical stainings and reduced expressions of Osterix and Osteocalcin. This was associated with specific methylation patterns that clearly separated MDS-MSC from healthy controls and showed a strong enrichment for biological processes associated with cellular phenotypes and transcriptional regulation. Furthermore, in MDS-MSC, we detected altered expression of key molecules involved in the interaction with hematopoietic stem and progenitor cells (HSPC), in particular Osteopontin, Jagged1, Kit-ligand and Angiopoietin as well as several chemokines. Functionally, this translated into a significantly diminished ability of MDS-derived MSC to support CD34+ HSPC in long-term culture-initiating cell assays associated with a reduced cell cycle activity. Taken together, our comprehensive analysis shows that MSC from all MDS subtypes are structurally, epigenetically and functionally altered, which leads to impaired stromal support and seems to contribute to deficient hematopoiesis in MDS.

  1. Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo.

    Science.gov (United States)

    Seo, Tadahiko; Sakon, Taketo; Nakazawa, Shiori; Nishioka, Asuka; Watanabe, Kohei; Matsumoto, Kaori; Akasaka, Mari; Shioi, Narumi; Sawada, Hitoshi; Araki, Satohiko

    2017-06-01

    Snake venom metalloproteases (SVMPs) are members of the a disintegrin and metalloprotease (ADAM) family of proteins, as they possess similar domains. SVMPs are known to elicit snake venom-induced haemorrhage; however, the target proteins and cleavage sites are not known. In this work, we identified a target protein of vascular apoptosis-inducing protein 1 (VAP1), an SVMP, relevant to its ability to induce haemorrhage. VAP1 disrupted cell-cell adhesions by relocating VE-cadherin and γ-catenin from the cell-cell junction to the cytosol, without inducing proteolysis of VE-cadherin. The Wnt receptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) are known to promote catenin relocation, and are rendered constitutively active in Wnt signalling by truncation. Thus, we examined whether VAP1 cleaves LRP5/6 to induce catenin relocation. Indeed, we found that VAP1 cleaved the extracellular region of LRP6 and LRP5. This cleavage removes four inhibitory β-propeller structures, resulting in activation of LRP5/6. Recombinant human ADAM8 and ADAM12 also cleaved LRP6 at the same site. An antibody against a peptide including the LRP6-cleavage site inhibited VAP1-induced VE-cadherin relocation and disruption of cell-cell adhesions in cultured cells, and blocked haemorrhage in mice in vivo. Intriguingly, animals resistant to the effects of haemorrhagic snake venom express variants of LRP5/6 that lack the VAP1-cleavage site, or low-density lipoprotein receptor domain class A domains involved in formation of the constitutively active form. The results validate LRP5/6 as physiological targets of ADAMs. Furthermore, they indicate that SVMP-induced cleavage of LRP5/6 causes disruption of cell-cell adhesion and haemorrhage, potentially opening new avenues for the treatment of snake bites. © 2017 Federation of European Biochemical Societies.

  2. Secretome analysis to elucidate metalloprotease-dependent ectodomain shedding of glycoproteins during neuronal differentiation.

    Science.gov (United States)

    Tsumagari, Kazuya; Shirakabe, Kyoko; Ogura, Mayu; Sato, Fuminori; Ishihama, Yasushi; Sehara-Fujisawa, Atsuko

    2017-02-01

    Many membrane proteins are subjected to limited proteolyses at their juxtamembrane regions, processes referred to as ectodomain shedding. Shedding ectodomains of membrane-bound ligands results in activation of downstream signaling pathways, whereas shedding those of cell adhesion molecules causes loss of cell-cell contacts. Secreted proteomics (secretomics) using high-resolution mass spectrometry would be strong tools for both comprehensive identification and quantitative measurement of membrane proteins that undergo ectodomain shedding. In this study, to elucidate the ectodomain shedding events that occur during neuronal differentiation, we establish a strategy for quantitative secretomics of glycoproteins released from differentiating neuroblastoma cells into culture medium with or without GM6001, a broad-spectrum metalloprotease inhibitor. Considering that most of transmembrane and secreted proteins are N-glycosylated, we include a process of N-glycosylated peptides enrichment as well as isotope tagging in our secretomics workflow. Our results show that differentiating N1E-115 neurons secrete numerous glycosylated polypeptides in metalloprotease-dependent manners. They are derived from cell adhesion molecules such as NCAM1, CADM1, L1CAM, various transporters and receptor proteins. These results show the landscape of ectodomain shedding and other secretory events in differentiating neurons and/or during axon elongation, which should help elucidate the mechanism of neurogenesis and the pathogenesis of neurological disorders. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  3. Production, purification, and characterization of metalloprotease from Candida kefyr 41 PSB.

    Science.gov (United States)

    Yavuz, Sevgi; Kocabay, Samet; Çetinkaya, Serap; Akkaya, Birnur; Akkaya, Recep; Yenidunya, Ali Fazil; Bakıcı, Mustafa Zahir

    2017-01-01

    A thermostable metalloprotease, produced from an environmental strain of Candida kefyr 41 PSB, was purified 16 fold with a 60% yield by cold ethanol precipitation and affinity chromatography (bentonite-acrylamide-cysteine microcomposite). The purified enzyme appeared as a single protein band at 43kDa. Its optimum pH and temperature points were found to be 7.0 and 105°C, respectively. K m and V max values of the enzyme were determined to be 3.5mg/mL and 4.4μmolmL -1 min -1 , 1.65mg/mL and 6.1μmolmL -1 min -1 , using casein and gelatine as the substrates, respectively. The activity was inhibited by using ethylenediamine tetraacetic acid (EDTA), indicating that the enzyme was a metalloprotease. Stability of the enzyme was investigated by using thermodynamic and kinetic parameters. The thermal inactivation profile of the enzyme conformed to the first order kinetics. The half life of the enzyme at 95, 105, 115, 125 and 135°C was 1310, 610, 220, 150, and 86min, respectively. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Microarray Analysis on Gene Regulation by Estrogen, Progesterone and Tamoxifen in Human Endometrial Stromal Cells

    Directory of Open Access Journals (Sweden)

    Chun-E Ren

    2015-03-01

    Full Text Available Epithelial stromal cells represent a major cellular component of human uterine endometrium that is subject to tight hormonal regulation. Through cell-cell contacts and/or paracrine mechanisms, stromal cells play a significant role in the malignant transformation of epithelial cells. We isolated stromal cells from normal human endometrium and investigated the morphological and transcriptional changes induced by estrogen, progesterone and tamoxifen. We demonstrated that stromal cells express appreciable levels of estrogen and progesterone receptors and undergo different morphological changes upon hormonal stimulation. Microarray analysis indicated that both estrogen and progesterone induced dramatic alterations in a variety of genes associated with cell structure, transcription, cell cycle, and signaling. However, divergent patterns of changes, and in some genes opposite effects, were observed for the two hormones. A large number of genes are identified as novel targets for hormonal regulation. These hormone-responsive genes may be involved in normal uterine function and the development of endometrial malignancies.

  5. HOX and TALE signatures specify human stromal stem cell populations from different sources.

    Science.gov (United States)

    Picchi, Jacopo; Trombi, Luisa; Spugnesi, Laura; Barachini, Serena; Maroni, Giorgia; Brodano, Giovanni Barbanti; Boriani, Stefano; Valtieri, Mauro; Petrini, Mario; Magli, Maria Cristina

    2013-04-01

    Human stromal stem cell populations reside in different tissues and anatomical sites, however a critical question related to their efficient use in regenerative medicine is whether they exhibit equivalent biological properties. Here, we compared cellular and molecular characteristics of stromal stem cells derived from the bone marrow, at different body sites (iliac crest, sternum, and vertebrae) and other tissues (dental pulp and colon). In particular, we investigated whether homeobox genes of the HOX and TALE subfamilies might provide suitable markers to identify distinct stromal cell populations, as HOX proteins control cell positional identity and, together with their co-factors TALE, are involved in orchestrating differentiation of adult tissues. Our results show that stromal populations from different sources, although immunophenotypically similar, display distinct HOX and TALE signatures, as well as different growth and differentiation abilities. Stromal stem cells from different tissues are characterized by specific HOX profiles, differing in the number and type of active genes, as well as in their level of expression. Conversely, bone marrow-derived cell populations can be essentially distinguished for the expression levels of specific HOX members, strongly suggesting that quantitative differences in HOX activity may be crucial. Taken together, our data indicate that the HOX and TALE profiles provide positional, embryological and hierarchical identity of human stromal stem cells. Furthermore, our data suggest that cell populations derived from different body sites may not represent equivalent cell sources for cell-based therapeutical strategies for regeneration and repair of specific tissues. Copyright © 2012 Wiley Periodicals, Inc.

  6. YghJ, the secreted metalloprotease of pathogenic E. coli induces hemorrhagic fluid accumulation in mouse ileal loop.

    Science.gov (United States)

    Tapader, Rima; Bose, Dipro; Pal, Amit

    2017-04-01

    YghJ, also known as SslE (Secreted and surface associated lipoprotein) is a cell surface associated and secreted lipoprotein harbouring M60 metalloprotease domain. Though the gene is known to be conserved among both pathogenic and commensal Escherichia coli isolates, the expression and secretion of YghJ was found to be higher among diverse E. coli pathotypes. YghJ, secreted from intestinal pathogens such as enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC) has been demonstrated to possess mucinase activity and hence facilitates colonization of these enteric pathogens to intestinal epithelial cells. Importantly, YghJ is also reported to be secreted from extraintestinal pathogenic E. coli isolates. In our previous study we have shown that YghJ, purified from a neonatal septicemic E. coli isolate could trigger induction of various proinflammatory cytokines in vitro. This led us to investigate the role of YghJ in causing in vivo tissue hemorrhage. In the present study, we validate the earlier in vitro finding and have showed that YghJ can cause extensive tissue damage in mouse ileum and is also able to induce significant fluid accumulation in a dose dependent manner in a mouse ileal loop (MIL) assay. Hence, our present study not only confirms the pathogenic potential of YghJ in sepsis pathophysiology but also indicates the enterotoxic ability of YghJ which makes it an important virulence determinant of intestinal pathogenic E. coli. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Characterization of an extensin-modifying metalloprotease: N-terminal processing and substrate cleavage pattern of Pectobacterium carotovorum Prt1.

    Science.gov (United States)

    Feng, Tao; Nyffenegger, Christian; Højrup, Peter; Vidal-Melgosa, Silvia; Yan, Kok-Phen; Fangel, Jonatan Ulrik; Meyer, Anne S; Kirpekar, Finn; Willats, William G; Mikkelsen, Jørn D

    2014-12-01

    Compared to other plant cell wall-degrading enzymes, proteases are less well understood. In this study, the extracellular metalloprotease Prt1 from Pectobacterium carotovorum (formerly Erwinia carotovora) was expressed in Escherichia coli and characterized with respect to N-terminal processing, thermal stability, substrate targets, and cleavage patterns. Prt1 is an autoprocessing protease with an N-terminal signal pre-peptide and a pro-peptide which has to be removed in order to activate the protease. The sequential cleavage of the N-terminus was confirmed by mass spectrometry (MS) fingerprinting and N-terminus analysis. The optimal reaction conditions for the activity of Prt1 on azocasein were at pH 6.0, 50 °C. At these reaction conditions, K M was 1.81 mg/mL and k cat was 1.82 × 10(7) U M(-1). The enzyme was relatively stable at 50 °C with a half-life of 20 min. Ethylenediaminetetraacetic acid (EDTA) treatment abolished activity; Zn(2+) addition caused regain of the activity, but Zn(2+)addition decreased the thermal stability of the Prt1 enzyme presumably as a result of increased proteolytic autolysis. In addition to casein, the enzyme catalyzed degradation of collagen, potato lectin, and plant extensin. Analysis of the cleavage pattern of different substrates after treatment with Prt1 indicated that the protease had a substrate cleavage preference for proline in substrate residue position P1 followed by a hydrophobic residue in residue position P1' at the cleavage point. The activity of Prt1 against plant cell wall structural proteins suggests that this enzyme might become an important new addition to the toolbox of cell-wall-degrading enzymes for biomass processing.

  8. Lysyl oxidase activates cancer stromal cells and promotes gastric cancer progression: quantum dot-based identification of biomarkers in cancer stromal cells

    Directory of Open Access Journals (Sweden)

    Peng CW

    2017-12-01

    Full Text Available Chunwei Peng,1 Jiuyang Liu,1 Guifang Yang,2 Yan Li3 1Department of Gastrointestinal Surgery, Zhongnan Hospital of Wuhan University, Hubei Key Laboratory of Tumor Biological Behaviors & Hubei Cancer Clinical Study Center, 2Department of Pathology, Zhongnan Hospital of Wuhan University, Wuchang District, Wuhan, 3Department of Peritoneal Cancer Surgery, Cancer Center of Beijing Shijitan Hospital Affiliated to the Capital Medical University, Yangfangdian, Beijing, People’s Republic of China Purpose: Semiconductor quantum dots (QDs are a promising alternative to organic fluorescent dyes for multiplexed molecular imaging of cancer stroma, which have great advantages in holistically analyzing the complex interactions among cancer stromal components in situ.Patients and methods: A QD probe-based multiplexed spectral molecular imaging method was established for simultaneous imaging. Three tissue microarrays (TMAs including 184 gastric cancer (GC tissues were constructed for the study. Multispectral analyses were performed for quantifying stromal biomarkers, such as lysyl oxidase (LOX. The stromal status including infiltrating of immune cells (high density of macrophages, angiogenesis (high density of microvessel density [MVD], low neovessel maturation and extracellular matrix (ECM remodeling (low density of type IV collagen, intense expression of matrix metalloproteinase 9 [MMP-9] was evaluated.Results: This study compared the imaging features of the QD probe-based single molecular imaging method, immunohistochemistry, and organic dye-based immunofluorescent methods, and showed the advantages of the QD probe-based multiple molecular imaging method for simultaneously visualizing complex components of cancer stroma. The risk of macrophages in high density, high MVD, low neomicrovessel maturation, MMP-9 expression and low type IV collagen was significantly increased for the expression of LOX. With the advantages of the established QD probe

  9. Transcriptome analysis of epithelial and stromal contributions to mammogenesis in three week prepartum cows.

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    Theresa Casey

    Full Text Available Transcriptome analysis of bovine mammary development has provided insight into regulation of mammogenesis. However, previous studies primarily examined expression of epithelial and stromal tissues combined, and consequently did not account for tissue specific contribution to mammary development. Our objective was to identify differences in gene expression in epithelial and intralobular stromal compartments. Tissue was biopsied from non-lactating dairy cows 3 weeks prepartum, cut into explants and incubated for 2 hr with insulin and hydrocortisone. Epithelial and intralobular stromal tissues were isolated with laser capture microdissection. Global gene expression was measured with Bovine Affymetrix GeneChips, and data were preprocessed using RMA method. Moderated t-tests from gene-specific linear model analysis with cell type as a fixed effect showed more than 3,000 genes were differentially expressed between tissues (P<0.05; FDR<0.17. Analysis of epithelial and stromal transcriptomes using Database for Annotation, Visualization and Integrated Discovery (DAVID and Ingenuity Pathways Analysis (IPA showed that epithelial and stromal cells contributed distinct molecular signatures. Epithelial signatures were enriched with gene sets for protein synthesis, metabolism and secretion. Stromal signatures were enriched with genes that encoded molecules important to signaling, extracellular matrix composition and remodeling. Transcriptome differences also showed evidence for paracrine interactions between tissues in stimulation of IGF1 signaling pathway, stromal reaction, angiogenesis, neurogenesis, and immune response. Molecular signatures point to the dynamic role the stroma plays in prepartum mammogenesis and highlight the importance of examining the roles of cell types within the mammary gland when targeting therapies and studying mechanisms that affect milk production.

  10. Gut Mesenchymal Stromal Cells in Immunity

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    Valeria Messina

    2017-01-01

    Full Text Available Mesenchymal stromal cells (MSCs, first found in bone marrow (BM, are the structural architects of all organs, participating in most biological functions. MSCs possess tissue-specific signatures that allow their discrimination according to their origin and location. Among their multiple functions, MSCs closely interact with immune cells, orchestrating their activity to maintain overall homeostasis. The phenotype of tissue MSCs residing in the bowel overlaps with myofibroblasts, lining the bottom walls of intestinal crypts (pericryptal or interspersed within intestinal submucosa (intercryptal. In Crohn’s disease, intestinal MSCs are tightly stacked in a chronic inflammatory milieu, which causes their enforced expression of Class II major histocompatibility complex (MHC. The absence of Class II MHC is a hallmark for immune-modulator and tolerogenic properties of normal MSCs and, vice versa, the expression of HLA-DR is peculiar to antigen presenting cells, that is, immune-activator cells. Interferon gamma (IFNγ is responsible for induction of Class II MHC expression on intestinal MSCs. The reversal of myofibroblasts/MSCs from an immune-modulator to an activator phenotype in Crohn’s disease results in the formation of a fibrotic tube subverting the intestinal structure. Epithelial metaplastic areas in this context can progress to dysplasia and cancer.

  11. Ceramic hydroxyapatite coating on titanium implants drives selective bone marrow stromal cell adhesion.

    NARCIS (Netherlands)

    Torensma, R.; Brugge, P.J. ter; Jansen, J.A.; Figdor, C.G.

    2003-01-01

    The aim of this study was to determine the cell characteristics that regulate implant osseointegration. The heterogeneity of bone marrow stromal cells obtained from 11 donors was assessed by measuring the expression of a large panel of adhesion molecules. Large differences in expression of adhesion

  12. Extrauterine Low-Grade Endometrial Stromal Sarcoma

    Directory of Open Access Journals (Sweden)

    Yu-Ju Chen

    2005-12-01

    Conclusions: Low-grade endometrial stromal sarcoma typically has an indolent clinical course and favorable prognosis. Surgical resection is the primary therapeutic approach, and adjuvant therapy with radiotherapy, chemotherapy, or progesterone therapy should be considered for the management of residual or recurrent low-grade endometrial stromal sarcomas.

  13. Stromal gastrointestinal tumors (GIST)

    International Nuclear Information System (INIS)

    Balev, B.; Boykova, K.

    2015-01-01

    Full text: GIST are a heterogeneous group of mesenchymal tumors of the gastrointestinal tract with varying tumor grade and frequency of 1: 100 000 per year. Mazur and Clark introduced the term for the first time in 1983. GIST constitute approximately 2% of the tumors in the gastrointestinal tract. The average age is 60 years. The most common locations are the stomach (60%), small intestine (30%), esophagus (1%), and rectum (5%). Learning objective: to demonstrate the imaging characteristics of the disease according to the current ESMO guidelines and to present the diagnostic accuracy of different imaging modalitiesnbased on review of literature and on own observations. GIST originate from interstitial cells (of Cajal) in the GIT wall, belonging to the autonomic nervous system, which is responsible for motility. 90% of GIST show overexpression of the KIT receptor, also known as CD117 or stem cell factor receptor. those that do not express c-KIT mutations, activate mutations in PDGFRA gene. Tumor’s macromorphology determines the imaging features on different modalities. Most of these tumors are exophytic, subepithelial, reach large size and enhance inhomogeneous due to necrosis. They usually do not cause obstruction. Ultrasound as the initiation method shows low sensitivity and specificity in GIST detection, CT with intravenous contrast is the gold standard. MRI contributes with assessing the vascularisation, cellularity and pH. FDG-PET/CT registers the metabolism of intratumoral acidosis. CT is the method of choice in the early diagnosis and determination of resectability of GIST. MRI is an additional method. PET FDG-CT is useful for the monitoring of patients treated with Imatinib

  14. A novel synthetic quinolinone inhibitor presents proteolytic and hemorrhagic inhibitory activities against snake venom metalloproteases.

    Science.gov (United States)

    Baraldi, Patrícia T; Magro, Angelo J; Matioli, Fábio F; Marcussi, Silvana; Lemke, Ney; Calderon, Leonardo A; Stábeli, Rodrigo G; Soares, Andreimar M; Correa, Arlene G; Fontes, Marcos R M

    2016-02-01

    Metalloproteases play a fundamental role in snake venom envenomation inducing hemorrhagic, fibrigen(ogen)olytic and myotoxic effects in their victims. Several snake venoms, such as those from the Bothrops genus, present important local effects which are not efficiently neutralized by conventional serum therapy. Consequently, these accidents may result in permanent sequelae and disability, creating economic and social problems, especially in developing countries, leading the attention of the World Health Organization that considered ophidic envenomations a neglected tropical disease. Aiming to produce an efficient inhibitor against bothropic venoms, we synthesized different molecules classified as quinolinones - a group of low-toxic chemical compounds widely used as antibacterial and antimycobacterial drugs - and tested their inhibitory properties against hemorrhage caused by bothropic venoms. The results from this initial screening indicated the molecule 2-hydroxymethyl-6-methoxy-1,4-dihydro-4-quinolinone (Q8) was the most effective antihemorrhagic compound among all of the assayed synthetic quinolinones. Other in vitro and in vivo experiments showed this novel compound was able to inhibit significantly the hemorrhagic and/or proteolytic activities of bothropic crude venoms and isolated snake venom metalloproteases (SVMPs) even at lower concentrations. Docking and molecular dynamic simulations were also performed to get insights into the structural basis of Q8 inhibitory mechanism against proteolytic and hemorrhagic SVMPs. These structural studies demonstrated that Q8 may form a stable complex with SVMPs, impairing the access of substrates to the active sites of these toxins. Therefore, both experimental and structural data indicate that Q8 compound is an interesting candidate for antiophidic therapy, particularly for the treatment of the hemorrhagic and necrotic effects induced by bothropic venoms. Copyright © 2015 Elsevier B.V. and Société Française de

  15. The Metalloprotease Mpl Supports Listeria monocytogenes Dissemination through Resolution of Membrane Protrusions into Vacuoles.

    Science.gov (United States)

    Alvarez, Diego E; Agaisse, Hervé

    2016-06-01

    Listeria monocytogenes is an intracellular pathogen that disseminates within the intestinal epithelium through acquisition of actin-based motility and formation of plasma membrane protrusions that project into adjacent cells. The resolution of membrane protrusions into vacuoles from which the pathogen escapes results in bacterial spread from cell to cell. This dissemination process relies on the mlp-actA-plcB operon, which encodes ActA, a bacterial nucleation-promoting factor that mediates actin-based motility, and PlcB, a phospholipase that mediates vacuole escape. Here we investigated the role of the metalloprotease Mpl in the dissemination process. In agreement with previous findings showing that Mpl is required for PlcB activation, infection of epithelial cells with the ΔplcB or Δmpl strains resulted in the formation of small infection foci. As expected, the ΔplcB strain displayed a strong defect in vacuole escape. However, the Δmpl strain showed an unexpected defect in the resolution of protrusions into vacuoles, in addition to the expected but mild defect in vacuole escape. The Δmpl strain displayed increased levels of ActA on the bacterial surface in protrusions. We mapped an Mpl-dependent processing site in ActA between amino acid residues 207 to 238. Similar to the Δmpl strain, the ΔactA207-238 strain displayed increased levels of ActA on the bacterial surface in protrusions. Although the ΔactA207-238 strain displayed wild-type actin-based motility, it formed small infection foci and failed to resolve protrusions into vacuoles. We propose that, in addition to its role in PlcB processing and vacuole escape, the metalloprotease Mpl is required for ActA processing and protrusion resolution. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  16. Human stromal (mesenchymal) stem cells

    DEFF Research Database (Denmark)

    Aldahmash, Abdullah; Zaher, Walid; Al-Nbaheen, May

    2012-01-01

    Human stromal (mesenchymal) stem cells (hMSC) represent a group of non-hematopoietic stem cells present in the bone marrow stroma and the stroma of other organs including subcutaneous adipose tissue, placenta, and muscles. They exhibit the characteristics of somatic stem cells of self......-renewal and multi-lineage differentiation into mesoderm-type of cells, e.g., to osteoblasts, adipocytes, chondrocytes and possibly other cell types including hepatocytes and astrocytes. Due to their ease of culture and multipotentiality, hMSC are increasingly employed as a source for cells suitable for a number...

  17. Hematopoietic microenvironment. Origin, lineage, and transplantability of the stromal cells in long-term bone marrow cultures from chimeric mice

    International Nuclear Information System (INIS)

    Perkins, S.; Fleischman, R.A.

    1988-01-01

    Studies of bone marrow transplant patients have suggested that the stromal cells of the in vitro hematopoietic microenvironment are transplantable into conditioned recipients. Moreover, in patients with myeloproliferative disorders, all of the stromal cells, which include presumptive endothelial cells, appear to be derived from hematopoietic precursors. To confirm these findings, we have constructed two chimeric mouse models: (a) traditional radiation chimeras, and (b) fetal chimeras, produced by placental injection of bone marrow into genetically anemic Wx/Wv fetuses, a technique that essentially precludes engraftment of nonhematopoietic cells. Using two-color indirect immunofluorescence, the stromal cells in long-term bone marrow culture derived from these chimeras were analyzed for donor or host origin by strain-specific H-2 antigens, and for cell lineage by a variety of other specific markers. 75-95% of the stromal cells were shown to be hematopoietic cells of the monocyte-macrophage lineage, based upon donor origin, phagocytosis, and expression of specific hematopoietic surface antigens. The remaining 5-25% of the stromal cells were exclusively host in origin. Apart from occasional fat cells, these cells uniformly expressed collagen type IV, laminin, and a surface antigen associated with endothelial cells. Since these endothelial-like cells are not transplantable into radiation or fetal chimeras, they are not derived from hematopoietic stem cells. The contrast between our findings and human studies suggests either unexpected species differences in the origin of stromal lineages or limitations in the previous methodology used to detect nonhematopoietic stromal cells

  18. Virally and physically transgenized equine adipose-derived stromal cells as a cargo for paracrine secreted factors

    Directory of Open Access Journals (Sweden)

    Cavirani Sandro

    2010-09-01

    Full Text Available Abstract Background Adipose-Derived Stromal Cells have been shown to have multiple lineage differentiation properties and to be suitable for tissues regeneration in many degenerative processes. Their use has been proposed for the therapy of joint diseases and tendon injuries in the horse. In the present report the genetic manipulation of Equine Adipose-Derived Stromal Cells has been investigated. Results Equine Adipose-Derived Stromal Cells were successfully virally transduced as well as transiently and stably transfected with appropriate parameters, without detrimental effect on their differentiation properties. Moreover, green fluorescent protein alone, fused to neo gene, or co-expressed as bi-cistronic reporter constructs, driven by viral and house-keeping gene promoters, were tested. The better expressed cassette was employed to stably transfect Adipose-Derived Stromal Cells for cell therapy purposes. Stably transfected Equine Adipose-Derived Stromal Cells with a heterologous secreted viral antigen were able to immunize horses upon injection into the lateral wall of the neck. Conclusion This study provides the methods to successfully transgenize Adipose-Derived Stromal Cells both by lentiviral vector and by transfection using optimized constructs with suitable promoters and reporter genes. In conclusion these findings provide a working platform for the delivery of potentially therapeutic proteins to the site of cells injection via transgenized Equine Adipose-Derived Stromal Cells.

  19. Generation of mesenchymal stromal cells in the presence of platelet lysate: a phenotypic and functional comparison of umbilical cord blood- and bone marrow-derived progenitors

    Science.gov (United States)

    Avanzini, Maria Antonietta; Bernardo, Maria Ester; Cometa, Angela Maria; Perotti, Cesare; Zaffaroni, Nadia; Novara, Francesca; Visai, Livia; Moretta, Antonia; Del Fante, Claudia; Villa, Raffaella; Ball, Lynne M.; Fibbe, Willem E.; Maccario, Rita; Locatelli, Franco

    2009-01-01

    Background Mesenchymal stromal cells are employed in various different clinical settings in order to modulate immune response. However, relatively little is known about the mechanisms responsible for their immunomodulatory effects, which could be influenced by both the cell source and culture conditions. Design and Methods We tested the ability of a 5% platelet lysate-supplemented medium to support isolation and ex vivo expansion of mesenchymal stromal cells from full-term umbilical-cord blood. We also investigated the biological/functional properties of umbilical cord blood mesenchymal stromal cells, in comparison with platelet lysate-expanded bone marrow mesenchymal stromal cells. Results The success rate of isolation of mesenchymal stromal cells from umbilical cord blood was in the order of 20%. These cells exhibited typical morphology, immunophenotype and differentiation capacity. Although they have a low clonogenic efficiency, umbilical cord blood mesenchymal stromal cells may possess high proliferative potential. The genetic stability of these cells from umbilical cord blood was demonstrated by a normal molecular karyotype; in addition, these cells do not express hTERT and telomerase activity, do express p16ink4a protein and do not show anchorage-independent cell growth. Concerning alloantigen-specific immune responses, umbilical cord blood mesenchymal stromal cells were able to: (i) suppress T- and NK-lymphocyte proliferation, (ii) decrease cytotoxic activity and (iii) only slightly increase interleukin-10, while decreasing interferon-γ secretion, in mixed lymphocyte culture supernatants. While an indoleamine 2,3-dioxygenase-specific inhibitor did not reverse mesenchymal stromal cell-induced suppressive effects, a prostaglandin E2-specific inhibitor hampered the suppressive effect of both umbilical cord blood- and bone marrow-mesenchymal stromal cells on alloantigen-induced cytotoxic activity. Mesenchymal stromal cells from both sources expressed HLA

  20. Decidualized Human Endometrial Stromal Cells Mediate Hemostasis, Angiogenesis, and Abnormal Uterine Bleeding

    Science.gov (United States)

    Lockwood, Charles J.; Krikun, Graciela; Hickey, Martha; Huang, S. Joseph; Schatz, Frederick

    2011-01-01

    Factor VII binds trans-membrane tissue factor to initiate hemostasis by forming thrombin. Tissue factor expression is enhanced in decidualized human endometrial stromal cells during the luteal phase. Long-term progestin only contraceptives elicit: 1) abnormal uterine bleeding from fragile vessels at focal bleeding sites, 2) paradoxically high tissue factor expression at bleeding sites; 3) reduced endometrial blood flow promoting local hypoxia and enhancing reactive oxygen species levels; and 4) aberrant angiogenesis reflecting increased stromal cell-expressed vascular endothelial growth factor, decreased Angiopoietin-1 and increased endothelial cell-expressed Angiopoietin-2. Aberrantly high local vascular permeability enhances circulating factor VII to decidualized stromal cell-expressed tissue factor to generate excess thrombin. Hypoxia-thrombin interactions augment expression of vascular endothelial growth factor and interleukin-8 by stromal cells. Thrombin, vascular endothelial growth factor and interlerukin-8 synergis-tically augment angiogenesis in a milieu of reactive oxygen species-induced endothelial cell activation. The resulting enhanced vessel fragility promotes abnormal uterine bleeding. PMID:19208784

  1. Reproducible isolation of lymph node stromal cells reveals site-dependent differences in fibroblastic reticular cells

    Directory of Open Access Journals (Sweden)

    Anne L Fletcher

    2011-09-01

    Full Text Available Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.

  2. Reproducible isolation of lymph node stromal cells reveals site-dependent differences in fibroblastic reticular cells.

    Science.gov (United States)

    Fletcher, Anne L; Malhotra, Deepali; Acton, Sophie E; Lukacs-Kornek, Veronika; Bellemare-Pelletier, Angelique; Curry, Mark; Armant, Myriam; Turley, Shannon J

    2011-01-01

    Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs) between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.

  3. Inflammatory response of a prostate stromal cell line induced by Trichomonas vaginalis.

    Science.gov (United States)

    Im, S J; Han, I H; Kim, J H; Gu, N Y; Seo, M Y; Chung, Y H; Ryu, J S

    2016-04-01

    While Trichomonas vaginalis, a cause of sexually transmitted infection, is known as a surface-dwelling protozoa, trichomonads have been detected in prostatic tissue from benign prostatic hyperplasia and prostatitis by immunoperoxidase assay or PCR. However, the immune response of prostate stromal cells infected with T. vaginalis has not been investigated. Our objective was to investigate whether T. vaginalis could induce an inflammatory response in prostate stromal cells. Incubation of a human prostate stromal myofibroblast cells (WPMY-1) with live T. vaginalis T016 increased expression of the inflammatory chemokines CXCL8 and CCL2. In addition, TLR4, ROS, MAPK and NF-κB expression increased, while inhibitors of TLR4, ROS, MAPKs and NF-κB reduced CXCL8 and CCL2 production. Medium conditioned by incubation of WPMY-1 cells with T. vaginalis stimulated the migration of human neutrophils and monocytes (THP-1 cells). We conclude that T. vaginalis increases CXCL8 and CCL2 production by human prostate stromal cells by activating TLR4, ROS, MAPKs and NF-κB, and this in turn attracts neutrophils and monocytes and leads to an inflammatory response. This study is the first attempt to demonstrate an inflammatory reaction in prostate stromal cells caused by T. vaginalis. © 2016 John Wiley & Sons Ltd.

  4. Acute morphine activates satellite glial cells and up-regulates IL-1β in dorsal root ganglia in mice via matrix metalloprotease-9

    Directory of Open Access Journals (Sweden)

    Berta Temugin

    2012-03-01

    Full Text Available Abstract Background Activation of spinal cord glial cells such as microglia and astrocytes has been shown to regulate chronic opioid-induced antinociceptive tolerance and hyperalgesia, due to spinal up-regulation of the proinflammatory cytokines such as interleukin-1 beta (IL-1β. Matrix metalloprotease-9 (MMP-9 has been implicated in IL-1β activation in neuropathic pain. However, it is unclear whether acute opioid treatment can activate glial cells in the peripheral nervous system. We examined acute morphine-induced activation of satellite glial cells (SGCs and up-regulation of IL-1β in dorsal root ganglia (DRGs, and further investigated the involvement of MMP-9 in these opioid-induced peripheral changes. Results Subcutaneous morphine injection (10 mg/kg induced robust peripheral glial responses, as evidenced by increased GFAP expression in DRGs but not in spinal cords. The acute morphine-induced GFAP expression is transient, peaking at 2 h and declining after 3 h. Acute morphine treatment also increased IL-1β immunoreactivity in SGCs and IL-1β activation in DRGs. MMP-9 and GFAP are expressed in DRG neurons and SGCs, respectively. Confocal analysis revealed a close proximity of MMP-9 and GFAP immunostaining. Importantly, morphine-induced DRG up-regulation of GFAP expression and IL-1β activation was abolished after Mmp9 deletion or naloxone pre-treatment. Finally, intrathecal injections of IL-1β-selective siRNA not only reduced DRG IL-1β expression but also prolonged acute morphine-induced analgesia. Conclusions Acute morphine induces opioid receptors- and MMP-9-dependent up-regulation of GFAP expression and IL-1β activation in SGCs of DRGs. MMP-9 could mask and shorten morphine analgesia via peripheral neuron-glial interactions. Targeting peripheral glial activation might prolong acute opioid analgesia.

  5. Gastrointestinal Stromal Tumors: A Case Report

    Directory of Open Access Journals (Sweden)

    Palankezhe Sashidharan

    2014-03-01

    Full Text Available Advances in the identification of gastrointestinal stromal tumors, its molecular and immunohiostochemical basis, and its management have been a watershed in the treatment of gastrointestinal tumors. This paradigm shift occurred over the last two decades and gastrointestinal stromal tumors have now come to be understood as rare gastrointestinal tract tumors with predictable behavior and outcome, replacing the older terminologies like leiomyoma, schwannoma or leiomyosarcoma. This report presents a case of gastric gastrointestinal stromal tumor operated recently in a 47-year-old female patient and the outcome, as well as literature review of the pathological identification, sites of origin, and factors predicting its behavior, prognosis and treatment.

  6. Stromal and epithelial cells react differentially to c-kit in fibroepithelial tumors of the breast.

    Science.gov (United States)

    Logullo, Angela F; Nonogaki, Suely; Do Socorro Maciel, Maria; Mourão-Neto, Mário; Soares, Fernando Augusto

    2008-01-01

    The CD117 protein is a tyrosine-kinase receptor encoded by the c-kit gene that frequently bears activating mutations in gastrointestinal tumors. Conflicting findings regarding CD117 expression in other stromal tumors, including phyllodes tumors (PTs), have been reported in the literature. The purpose of this study was to evaluate c-kit expression in the stroma and epithelia of fibroepithelial breast tumors and its correlation with clinical pathological variables. Ninety-six fibroepithelial tumors of the breast, including 14 fibroadenomas (FAs), 12 juvenile FAs and 70 PTs, were classified according to stromal cellularity, atypia, epithelial hyperplasia, mitosis and borders into 45 benign (PTB), 17 borderline (PTBL) and 8 malignant (PTM) tumors. CD117 expression was identified in the stromal component in only two cases of PTBL. Overall, 38 cases (39.6%) showed positive CD117 in the epithelial component, including 20 FAs (10 regular, 10 juvenile) and 18 PTs (11 PTBs and 8 PTBLs). Other cases, including all PTMs, 6 FAs (4 regular, 2 juvenile), 34 PTBs and 10 PTBLs, showed no positivity in the epithelial component. Expression of c-kit did not correlate with diagnosis or malignancy (p>0.05). In conclusion, c-kit is expressed more often in the epithelial than in the stromal component in fibroepithelial tumors of the breast, and is associated with benign lesions.

  7. Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Wenyao; Li, Xuezhong; Xu, Tong; Ma, Mengru [College of Veterinary Medicine, Northwest A& F University, Yangling 712100, Shaanxi (China); Zhang, Yong, E-mail: zhangyong1956@nwsuaf.edu.cn [College of Veterinary Medicine, Northwest A& F University, Yangling 712100, Shaanxi (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100, Shaanxi (China); Gao, Ming-Qing, E-mail: gaomingqing@nwsuaf.edu.cn [College of Veterinary Medicine, Northwest A& F University, Yangling 712100, Shaanxi (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100, Shaanxi (China)

    2016-11-15

    Hypernomic secretion of epithelial cytokines has several effects on stromal cells. The contributions of inflammatory epithelial cells to stromal fibroblasts in bovine mammary glands with mastitis remain poorly understood. Here, we established an inflammatory epithelial cell model of bovine mastitis with gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. We characterized immune responses of mammary stromal fibroblasts induced by inflammatory epithelial cells. Our results showed that inflammatory epithelial cells affected stromal fibroblast characteristics by increasing inflammatory mediator expression, elevating extracellular matrix protein deposition, decreasing proliferation capacity, and enhancing migration ability. The changes in stromal fibroblast proliferation and migration abilities were mediated by signal molecules, such as WNT signal pathway components. LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. Thus, in mastitis, bovine mammary gland stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes, suggesting that fibroblasts play crucial roles in bovine mastitis. - Highlights: • Inflammatory BMEs affect the properties of BMFs during mastitis. • BMEs inhibited the proliferation and promoted the migration of BMFs. • BMEs enhanced secretion of inflammatory mediators and deposition of ECM in BMFs. • Changes of the properties of BMFs were mediated by specific signal molecules.

  8. Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis.

    Science.gov (United States)

    Zhang, Wenyao; Li, Xuezhong; Xu, Tong; Ma, Mengru; Zhang, Yong; Gao, Ming-Qing

    2016-11-15

    Hypernomic secretion of epithelial cytokines has several effects on stromal cells. The contributions of inflammatory epithelial cells to stromal fibroblasts in bovine mammary glands with mastitis remain poorly understood. Here, we established an inflammatory epithelial cell model of bovine mastitis with gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. We characterized immune responses of mammary stromal fibroblasts induced by inflammatory epithelial cells. Our results showed that inflammatory epithelial cells affected stromal fibroblast characteristics by increasing inflammatory mediator expression, elevating extracellular matrix protein deposition, decreasing proliferation capacity, and enhancing migration ability. The changes in stromal fibroblast proliferation and migration abilities were mediated by signal molecules, such as WNT signal pathway components. LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. Thus, in mastitis, bovine mammary gland stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes, suggesting that fibroblasts play crucial roles in bovine mastitis. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis

    International Nuclear Information System (INIS)

    Zhang, Wenyao; Li, Xuezhong; Xu, Tong; Ma, Mengru; Zhang, Yong; Gao, Ming-Qing

    2016-01-01

    Hypernomic secretion of epithelial cytokines has several effects on stromal cells. The contributions of inflammatory epithelial cells to stromal fibroblasts in bovine mammary glands with mastitis remain poorly understood. Here, we established an inflammatory epithelial cell model of bovine mastitis with gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. We characterized immune responses of mammary stromal fibroblasts induced by inflammatory epithelial cells. Our results showed that inflammatory epithelial cells affected stromal fibroblast characteristics by increasing inflammatory mediator expression, elevating extracellular matrix protein deposition, decreasing proliferation capacity, and enhancing migration ability. The changes in stromal fibroblast proliferation and migration abilities were mediated by signal molecules, such as WNT signal pathway components. LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. Thus, in mastitis, bovine mammary gland stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes, suggesting that fibroblasts play crucial roles in bovine mastitis. - Highlights: • Inflammatory BMEs affect the properties of BMFs during mastitis. • BMEs inhibited the proliferation and promoted the migration of BMFs. • BMEs enhanced secretion of inflammatory mediators and deposition of ECM in BMFs. • Changes of the properties of BMFs were mediated by specific signal molecules.

  10. SlpE is a calcium-dependent cytotoxic metalloprotease associated with clinical isolates of Serratia marcescens.

    Science.gov (United States)

    Stella, Nicholas A; Callaghan, Jake D; Zhang, Liang; Brothers, Kimberly M; Kowalski, Regis P; Huang, Jean J; Thibodeau, Patrick H; Shanks, Robert M Q

    Serralysin-like proteases are found in a wide variety of bacteria. These metalloproteases are frequently implicated in virulence and are members of the widely conserved RTX-toxin family. We identified a serralysin-like protease in the genome of a clinical isolate of Serratia marcescens that is highly similar to the canonical serralysin protein, PrtS. This gene was named serralysin-like protease E, SlpE, and was found in the majority (67%) of tested clinical isolates, but was absent from most tested non-clinical isolates including the insect pathogen and reference S. marcescens strain Db11. Purified recombinant SlpE exhibited calcium-dependent protease activity similar to metalloproteases PrtS and SlpB. Induction of slpE in the low-protease-producing S. marcescens strain PIC3611 highly elevated extracellular protease activity, and extracellular secretion required the lipD type 1 secretion system gene. Transcription of slpE was highly reduced in an eepR transcription factor mutant. Mutation of the slpE gene in a highly proteolytic clinical isolate reduced its protease activity, and evidence suggests that SlpE confers cytotoxicity of S. marcescens to the A549 airway carcinoma cell line. Together, these data reveal SlpE to be an EepR-regulated cytotoxic metalloprotease associated with clinical isolates of an important opportunistic pathogen. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  11. Overexpression of matrix metalloproteinase-7 and -9 in NSCLC tumor and stromal cells: correlation with a favorable clinical outcome.

    Science.gov (United States)

    Stenvold, Helge; Donnem, Tom; Andersen, Sigve; Al-Saad, Samer; Al-Shibli, Khalid; Busund, Lill-Tove; Bremnes, Roy M

    2012-02-01

    Matrix metalloproteinases (MMPs) are considered important players in angiogenesis and cancer progression. Several drugs developed for targeting MMPs have until now been without clinical efficacy. As both malignant cells and cells of the surrounding stroma contribute to tumor growth, we have explored the impact of MMP-2, -7 and -9 expression in both the tumor and stromal compartment of non-small-cell lung cancers (NSCLC). From 335 unselected stage I to IIIA NSCLC carcinomas, duplicate tumor and tumor-associated stromal cores were collected in tissue microarrays (TMAs). Immunohistochemistry was used to detect the expression of MMP-2, -7 and -9 in tumor and stromal cells. In univariate analyses, high tumor cell MMP-7 expression (P=0.029) and high stromal MMP-9 expression (P=0.001) were positive prognostic factors. In the multivariate analysis, high tumor cell MMP-7 expression (HR 1.58, CI 1.08-2.32, P=0.020) and high stromal MMP-9 expression (HR 1.92, CI 1.25-2.96, P=0.003) were independent positive prognostic factors for disease-specific survival. High levels of MMP-7 in tumor cells and high levels of MMP-9 in tumor associated stroma were independent positive prognostic factors in NSCLC patients. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  12. Drugs Approved for Gastrointestinal Stromal Tumors

    Science.gov (United States)

    This page lists cancer drugs approved by the Food and Drug Administration (FDA) for gastrointestinal stromal tumors (GIST). The list includes generic names and brand names. The drug names link to NCI's Cancer Drug Information summaries.

  13. COX-2 and Prostaglandin EP3/EP4 Signaling Regulate the Tumor Stromal Proangiogenic Microenvironment via CXCL12-CXCR4 Chemokine Systems

    Science.gov (United States)

    Katoh, Hiroshi; Hosono, Kanako; Ito, Yoshiya; Suzuki, Tatsunori; Ogawa, Yasufumi; Kubo, Hidefumi; Kamata, Hiroki; Mishima, Toshiaki; Tamaki, Hideaki; Sakagami, Hiroyuki; Sugimoto, Yukihiko; Narumiya, Shuh; Watanabe, Masahiko; Majima, Masataka

    2010-01-01

    Bone marrow (BM)–derived hematopoietic cells, which are major components of tumor stroma, determine the tumor microenvironment and regulate tumor phenotypes. Cyclooxygenase (COX)−2 and endogenous prostaglandins are important determinants for tumor growth and tumor-associated angiogenesis; however, their contributions to stromal formation and angiogenesis remain unclear. In this study, we observed that Lewis lung carcinoma cells implanted in wild-type mice formed a tumor mass with extensive stromal formation that was markedly suppressed by COX-2 inhibition, which reduced the recruitment of BM cells. Notably, COX-2 inhibition attenuated CXCL12/CXCR4 expression as well as expression of several other chemokines. Indeed, in a Matrigel model, prostaglandin (PG) E2 enhanced stromal formation and CXCL12/CXCR4 expression. In addition, a COX-2 inhibitor suppressed stromal formation and reduced expression of CXCL12/CXCR4 and a fibroblast marker (S100A4) in a micropore chamber model. Moreover, stromal formation after tumor implantation was suppressed in EP3−/− mice and EP4−/− mice, in which stromal expression of CXCL12/CXCR4 and S100A4 was reduced. The EP3 or EP4 knockout suppressed S100A4+ fibroblasts, CXCL12+, and/or CXCR4+ stromal cells as well. Immunofluorescent analyses revealed that CXCL12+CXCR4+S100A4+ fibroblasts mainly comprised stromal cells and most of these were recruited from the BM. Additionally, either EP3- or EP4-specific agonists stimulated CXCL12 expression by fibroblasts in vitro. The present results address the novel activities of COX-2/PGE2-EP3/EP4 signaling that modulate tumor biology and show that CXCL12/CXCR4 axis may play a crucial role in tumor stromal formation and angiogenesis under the control of prostaglandins. PMID:20110411

  14. Gastrointestinal Stromal Tumors: A Case Report

    OpenAIRE

    Sashidharan, Palankezhe; Matele, Apoorva; Matele, Usha; Al Felahi, Nowfel; Kassem, Khalid F.

    2014-01-01

    Advances in the identification of gastrointestinal stromal tumors, its molecular and immunohiostochemical basis, and its management have been a watershed in the treatment of gastrointestinal tumors. This paradigm shift occurred over the last two decades and gastrointestinal stromal tumors have now come to be understood as rare gastrointestinal tract tumors with predictable behavior and outcome, replacing the older terminologies like leiomyoma, schwannoma or leiomyosarcoma. This report present...

  15. Targeting Stromal Recruitment by Prostate Cancer Cells

    Science.gov (United States)

    2006-03-01

    Ensinger, C., Tumer , Z., Tommerup, N. et al.: Hedgehog signaling in small-cell lung cancer : frequent in vivo but a rare event in vitro. Lung Cancer , 52...W81XWH-04-1-0157 TITLE: Targeting Stromal Recruitment by Prostate Cancer Cells PRINCIPAL INVESTIGATOR: Jingxian Zhang, Ph.D...DATES COVERED (From - To) 15 Feb 2004 – 14 Feb 2006 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Targeting Stromal Recruitment by Prostate Cancer

  16. Heat-shock proteins in stromal joint tissues: innocent bystanders or disease-initiating proteins?

    Science.gov (United States)

    Lambrecht, Stijn; Juchtmans, Nele; Elewaut, Dirk

    2014-02-01

    Heat-shock proteins (HSPs) are molecular chaperones that are highly conserved between species. In recent decades it has become clear that these proteins play an important role in the pathogenesis of inflammatory and degenerative joint diseases by (dys)regulating the immune system and by direct effects on the stromal tissues of the joint. In this review we discuss current insights into the expression pattern of HSPs in connective tissues, the direct biological role of HSPs in stromal tissues and the potential clinical applications.

  17. A non-catalytic histidine residue influences the function of the metalloprotease of Listeria monocytogenes.

    Science.gov (United States)

    Forster, Brian M; Bitar, Alan Pavinski; Marquis, Hélène

    2014-01-01

    Mpl, a thermolysin-like metalloprotease, and PC-PLC, a phospholipase C, are synthesized as proenzymes by the intracellular bacterial pathogen Listeria monocytogenes. During intracellular growth, L. monocytogenes is temporarily confined in a membrane-bound vacuole whose acidification leads to Mpl autolysis and Mpl-mediated cleavage of the PC-PLC N-terminal propeptide. Mpl maturation also leads to the secretion of both Mpl and PC-PLC across the bacterial cell wall. Previously, we identified negatively charged and uncharged amino acid residues within the N terminus of the PC-PLC propeptide that influence the ability of Mpl to mediate the maturation of PC-PLC, suggesting that these residues promote the interaction of the PC-PLC propeptide with Mpl. In the present study, we identified a non-catalytic histidine residue (H226) that influences Mpl secretion across the cell wall and its ability to process PC-PLC. Our results suggest that a positive charge at position 226 is required for Mpl functions other than autolysis. Based on the charge requirement at this position, we hypothesize that this residue contributes to the interaction of Mpl with the PC-PLC propeptide.

  18. Phosphorylation of iRhom2 Controls Stimulated Proteolytic Shedding by the Metalloprotease ADAM17/TACE

    Directory of Open Access Journals (Sweden)

    Miguel Cavadas

    2017-10-01

    Full Text Available Cell surface metalloproteases coordinate signaling during development, tissue homeostasis, and disease. TACE (TNF-α-converting enzyme, is responsible for cleavage (“shedding” of membrane-tethered signaling molecules, including the cytokine TNF, and activating ligands of the EGFR. The trafficking of TACE within the secretory pathway requires its binding to iRhom2, which mediates the exit of TACE from the endoplasmic reticulum. An important, but mechanistically unclear, feature of TACE biology is its ability to be stimulated rapidly on the cell surface by numerous inflammatory and growth-promoting agents. Here, we report a role for iRhom2 in TACE stimulation on the cell surface. TACE shedding stimuli trigger MAP kinase-dependent phosphorylation of iRhom2 N-terminal cytoplasmic tail. This recruits 14-3-3 proteins, enforcing the dissociation of TACE from complexes with iRhom2, promoting the cleavage of TACE substrates. Our data reveal that iRhom2 controls multiple aspects of TACE biology, including stimulated shedding on the cell surface.

  19. Identification of a candidate proteomic signature to discriminate multipotent and non-multipotent stromal cells.

    Science.gov (United States)

    Rosu-Myles, Michael; She, Yi-Min; Fair, Joel; Muradia, Gauri; Mehic, Jelica; Menendez, Pablo; Prasad, Shiv S; Cyr, Terry D

    2012-01-01

    Bone marrow stromal cell cultures contain multipotent cells that may have therapeutic utility for tissue restoration; however, the identity of the cell that maintains this function remains poorly characterized. We have utilized a unique model of murine bone marrow stroma in combination with liquid chromatography mass spectrometry to compare the nuclear, cytoplasmic and membrane associated proteomes of multipotent (MSC) (CD105+) and non-multipotent (CD105-) stromal cells. Among the 25 most reliably identified proteins, 10 were verified by both real-time PCR and Western Blot to be highly enriched, in CD105+ cells and were members of distinct biological pathways and functional networks. Five of these proteins were also identified as potentially expressed in human MSC derived from both standard and serum free human stromal cultures. The quantitative amount of each protein identified in human stromal cells was only minimally affected by media conditions but varied highly between bone marrow donors. This study provides further evidence of heterogeneity among cultured bone marrow stromal cells and identifies potential candidate proteins that may prove useful for identifying and quantifying both murine and human MSC in vitro.

  20. Identification of a candidate proteomic signature to discriminate multipotent and non-multipotent stromal cells.

    Directory of Open Access Journals (Sweden)

    Michael Rosu-Myles

    Full Text Available Bone marrow stromal cell cultures contain multipotent cells that may have therapeutic utility for tissue restoration; however, the identity of the cell that maintains this function remains poorly characterized. We have utilized a unique model of murine bone marrow stroma in combination with liquid chromatography mass spectrometry to compare the nuclear, cytoplasmic and membrane associated proteomes of multipotent (MSC (CD105+ and non-multipotent (CD105- stromal cells. Among the 25 most reliably identified proteins, 10 were verified by both real-time PCR and Western Blot to be highly enriched, in CD105+ cells and were members of distinct biological pathways and functional networks. Five of these proteins were also identified as potentially expressed in human MSC derived from both standard and serum free human stromal cultures. The quantitative amount of each protein identified in human stromal cells was only minimally affected by media conditions but varied highly between bone marrow donors. This study provides further evidence of heterogeneity among cultured bone marrow stromal cells and identifies potential candidate proteins that may prove useful for identifying and quantifying both murine and human MSC in vitro.

  1. The cannabinoid receptor type 2 as mediator of mesenchymal stromal cell immunosuppressive properties.

    Directory of Open Access Journals (Sweden)

    Francesca Rossi

    Full Text Available Mesenchymal stromal cells are non-hematopoietic, multipotent progenitor cells producing cytokines, chemokines, and extracellular matrix proteins that support hematopoietic stem cell survival and engraftment, influence immune effector cell development, maturation, and function, and inhibit alloreactive T-cell responses. The immunosuppressive properties of human mesenchymal stromal cells have attracted much attention from immunologists, stem cell biologists and clinicians. Recently, the presence of the endocannabinoid system in hematopoietic and neural stem cells has been demonstrated. Endocannabinoids, mainly acting through the cannabinoid receptor subtype 2, are able to modulate cytokine release and to act as immunosuppressant when added to activated T lymphocytes. In the present study, we have investigated, through a multidisciplinary approach, the involvement of the endocannabinoids in migration, viability and cytokine release of human mesenchymal stromal cells. We show, for the first time, that cultures of human mesenchymal stromal cells express all of the components of the endocannabinoid system, suggesting a potential role for the cannabinoid CB2 receptor as a mediator of anti-inflammatory properties of human mesenchymal stromal cells, as well as of their survival pathways and their capability to home and migrate towards endocannabinoid sources.

  2. Knock out of the BASIGIN/CD147 chaperone of lactate/H+ symporters disproves its pro-tumour action via extracellular matrix metalloproteases (MMPs) induction.

    Science.gov (United States)

    Marchiq, Ibtissam; Albrengues, Jean; Granja, Sara; Gaggioli, Cédric; Pouysségur, Jacques; Simon, Marie-Pierre

    2015-09-22

    BASIGIN/CD147/EMMPRIN is a multifunctional transmembrane glycoprotein strongly expressed in tumours. BASIGIN controls tumour metabolism, particularly glycolysis by facilitating lactic acid export through the two monocarboxylate transporters MCT1 and hypoxia-inducible MCT4. However, before being recognized as a co-carrier of MCTs, BASIGIN was described as an inducer of extracellular matrix metalloproteases (MMPs). Early on, a model emerged in which, tumour cells use the extracellular domain of BASIGIN to recognize and stimulate neighbouring fibroblasts to produce MMPs. However, this model has remained hypothetical since a direct link between BASIGIN and MMPs production has not yet been clearly established. To validate the BASIGIN/MMP hypothesis, we developed BASIGIN knockouts in three human tumour cell lines derived from glioma, colon, and lung adenocarcinoma. By using co-culture experiments of either human or mouse fibroblasts and tumour cell lines we showed, contrary to what has been abundantly published, that the disruption of BASIGIN in tumour cells and in MEFs has no action on the production of MMPs. Our findings do not support the notion that the pro-tumoural action of BASIGIN is mediated via induction of MMPs. Therefore, we propose that to date, the strongest pro-tumoural action of BASIGIN is mediated through the control of fermentative glycolysis.

  3. A phage display selected 7-mer peptide inhibitor of the Tannerella forsythia metalloprotease-like enzyme Karilysin can be truncated to Ser-Trp-Phe-Pro.

    Science.gov (United States)

    Skottrup, Peter Durand; Sørensen, Grete; Ksiazek, Miroslaw; Potempa, Jan; Riise, Erik

    2012-01-01

    Tannerella forsythia is a gram-negative bacteria, which is strongly associated with the development of periodontal disease. Karilysin is a newly identified metalloprotease-like enzyme, that is secreted from T. forsythia. Karilysin modulates the host immune response and is therefore considered a likely drug target. In this study peptides were selected towards the catalytic domain from Karilysin (Kly18) by phage display. The peptides were linear with low micromolar binding affinities. The two best binders (peptide14 and peptide15), shared the consensus sequence XWFPXXXGGG. A peptide15 fusion with Maltose Binding protein (MBP) was produced with peptide15 fused to the N-terminus of MBP. The peptide15-MBP was expressed in E. coli and the purified fusion-protein was used to verify Kly18 specific binding. Chemically synthesised peptide15 (SWFPLRSGGG) could inhibit the enzymatic activity of both Kly18 and intact Karilysin (Kly48). Furthermore, peptide15 could slow down the autoprocessing of intact Kly48 to Kly18. The WFP motif was important for inhibition and a truncation study further demonstrated that the N-terminal serine was also essential for Kly18 inhibition. The SWFP peptide had a Ki value in the low micromolar range, which was similar to the intact peptide15. In conclusion SWFP is the first reported inhibitor of Karilysin and can be used as a valuable tool in structure-function studies of Karilysin.

  4. A phage display selected 7-mer peptide inhibitor of the Tannerella forsythia metalloprotease-like enzyme Karilysin can be truncated to Ser-Trp-Phe-Pro.

    Directory of Open Access Journals (Sweden)

    Peter Durand Skottrup

    Full Text Available Tannerella forsythia is a gram-negative bacteria, which is strongly associated with the development of periodontal disease. Karilysin is a newly identified metalloprotease-like enzyme, that is secreted from T. forsythia. Karilysin modulates the host immune response and is therefore considered a likely drug target. In this study peptides were selected towards the catalytic domain from Karilysin (Kly18 by phage display. The peptides were linear with low micromolar binding affinities. The two best binders (peptide14 and peptide15, shared the consensus sequence XWFPXXXGGG. A peptide15 fusion with Maltose Binding protein (MBP was produced with peptide15 fused to the N-terminus of MBP. The peptide15-MBP was expressed in E. coli and the purified fusion-protein was used to verify Kly18 specific binding. Chemically synthesised peptide15 (SWFPLRSGGG could inhibit the enzymatic activity of both Kly18 and intact Karilysin (Kly48. Furthermore, peptide15 could slow down the autoprocessing of intact Kly48 to Kly18. The WFP motif was important for inhibition and a truncation study further demonstrated that the N-terminal serine was also essential for Kly18 inhibition. The SWFP peptide had a Ki value in the low micromolar range, which was similar to the intact peptide15. In conclusion SWFP is the first reported inhibitor of Karilysin and can be used as a valuable tool in structure-function studies of Karilysin.

  5. Dissecting Tumor-Stromal Interactions in Breast Cancer Bone Metastasis

    Directory of Open Access Journals (Sweden)

    Yibin Kang

    2016-06-01

    Full Text Available Bone metastasis is a frequent occurrence in breast cancer, affecting more than 70% of late stage cancer patients with severe complications such as fracture, bone pain, and hypercalcemia. The pathogenesis of osteolytic bone metastasis depends on cross-communications between tumor cells and various stromal cells residing in the bone microenvironment. Several growth factor signaling pathways, secreted micro RNAs (miRNAs and exosomes are functional mediators of tumor-stromal interactions in bone metastasis. We developed a functional genomic approach to systemically identified molecular pathways utilized by breast cancer cells to engage the bone stroma in order to generate osteolytic bone metastasis. We showed that elevated expression of vascular cell adhesion molecule 1 (VCAM1 in disseminated breast tumor cells mediates the recruitment of pre-osteoclasts and promotes their differentiation to mature osteoclasts during the bone metastasis formation. Transforming growth factor β (TGF-β is released from bone matrix upon bone destruction, and signals to breast cancer to further enhance their malignancy in developing bone metastasis. We furthered identified Jagged1 as a TGF-β target genes in tumor cells that engaged bone stromal cells through the activation of Notch signaling to provide a positive feedback to promote tumor growth and to activate osteoclast differentiation. Substantially change in miRNA expression was observed in osteoclasts during their differentiation and maturation, which can be exploited as circulating biomarkers of emerging bone metastasis and therapeutic targets for the treatment of bone metastasis. Further research in this direction may lead to improved diagnosis and treatment strategies for bone metastasis.

  6. IL-1β-induced matrix metalloproteinase-13 is activated by a disintegrin and metalloprotease-28-regulated proliferation of human osteoblast-like cells

    International Nuclear Information System (INIS)

    Ozeki, Nobuaki; Kawai, Rie; Yamaguchi, Hideyuki; Hiyama, Taiki; Kinoshita, Katsue; Hase, Naoko; Nakata, Kazuhiko; Kondo, Ayami; Mogi, Makio; Nakamura, Hiroshi

    2014-01-01

    We reported previously that matrix metalloproteinase (MMP)-13 accelerates bone remodeling in oral periradicular lesions, and indicated a potentially unique role for MMP-13 in wound healing and regeneration of alveolar bone. The ADAM (a disintegrin and metalloprotease) family is a set of multifunctional cell surface and secreted glycoproteins, of which ADAM-28 has been localized in bone and bone-like tissues. In this study, we show that interleukin (IL)-1β induces the expression of MMP-13 and ADAM-28 in homogeneous α7 integrin-positive human skeletal muscle stem cell (α7 + hSMSC)-derived osteoblast-like (α7 + hSMSC-OB) cells, and promotes proliferation while inhibiting apoptosis in these cells. At higher concentrations, however, IL-1β failed to induce the expression of these genes and caused an increase in apoptosis. We further employed ADAM-28 small interfering RNA (siRNA) to investigate whether IL-1β-induced MMP-13 expression is linked to this IL-1β-mediated changes in cell proliferation and apoptosis. Silencing ADAM-28 expression potently suppressed IL-1β-induced MMP-13 expression and activity, decreased cell proliferation and increased apoptosis in α7 + hSMSC-OB cells. In contrast, MMP-13 siRNA had no effect on ADAM-28 expression, suggesting ADAM-28 regulates MMP-13. Exogenous MMP-13 induced α7 + hSMSC-OB cell proliferation and could rescue ADAM-28 siRNA-induced apoptosis, and we found that proMMP-13 is partially cleaved into its active form by ADAM-28 in vitro. Overall, our results suggest that IL-1β-induced MMP-13 expression and changes in cell proliferation and apoptosis in α7 + hSMSC-OB cells are regulated by ADAM-28. - Highlights: • IL-1β induces the MMP-13 and ADAM-28 expression in human osteoblast-like cells. • IL-1β-induced MMP-13 expression increases proliferation and decreased apoptosis. • MMP-13 expression induced by IL-1β is regulated by ADAM-28. • proMMP-13 appears to be cleaved into its active form via ADAM-28

  7. IL-1β-induced matrix metalloproteinase-13 is activated by a disintegrin and metalloprotease-28-regulated proliferation of human osteoblast-like cells

    Energy Technology Data Exchange (ETDEWEB)

    Ozeki, Nobuaki; Kawai, Rie; Yamaguchi, Hideyuki; Hiyama, Taiki; Kinoshita, Katsue; Hase, Naoko; Nakata, Kazuhiko [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi 464-8651 (Japan); Kondo, Ayami [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya, Aichi 464-8650 (Japan); Mogi, Makio, E-mail: makio@dpc.agu.ac.jp [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya, Aichi 464-8650 (Japan); Nakamura, Hiroshi [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi 464-8651 (Japan)

    2014-04-15

    We reported previously that matrix metalloproteinase (MMP)-13 accelerates bone remodeling in oral periradicular lesions, and indicated a potentially unique role for MMP-13 in wound healing and regeneration of alveolar bone. The ADAM (a disintegrin and metalloprotease) family is a set of multifunctional cell surface and secreted glycoproteins, of which ADAM-28 has been localized in bone and bone-like tissues. In this study, we show that interleukin (IL)-1β induces the expression of MMP-13 and ADAM-28 in homogeneous α7 integrin-positive human skeletal muscle stem cell (α7{sup +}hSMSC)-derived osteoblast-like (α7{sup +}hSMSC-OB) cells, and promotes proliferation while inhibiting apoptosis in these cells. At higher concentrations, however, IL-1β failed to induce the expression of these genes and caused an increase in apoptosis. We further employed ADAM-28 small interfering RNA (siRNA) to investigate whether IL-1β-induced MMP-13 expression is linked to this IL-1β-mediated changes in cell proliferation and apoptosis. Silencing ADAM-28 expression potently suppressed IL-1β-induced MMP-13 expression and activity, decreased cell proliferation and increased apoptosis in α7{sup +}hSMSC-OB cells. In contrast, MMP-13 siRNA had no effect on ADAM-28 expression, suggesting ADAM-28 regulates MMP-13. Exogenous MMP-13 induced α7{sup +}hSMSC-OB cell proliferation and could rescue ADAM-28 siRNA-induced apoptosis, and we found that proMMP-13 is partially cleaved into its active form by ADAM-28 in vitro. Overall, our results suggest that IL-1β-induced MMP-13 expression and changes in cell proliferation and apoptosis in α7{sup +}hSMSC-OB cells are regulated by ADAM-28. - Highlights: • IL-1β induces the MMP-13 and ADAM-28 expression in human osteoblast-like cells. • IL-1β-induced MMP-13 expression increases proliferation and decreased apoptosis. • MMP-13 expression induced by IL-1β is regulated by ADAM-28. • proMMP-13 appears to be cleaved into its active form via

  8. Changing the Properties of Multipotent Mesenchymal Stromal Cells by IFNγ Administration.

    Science.gov (United States)

    Petinati, N A; Kapranov, N M; Bigil'deev, A E; Popova, M D; Davydova, Yu O; Gal'tseva, I V; Drize, N I; Kuz'mina, L A; Parovichnikova, E N; Savchenko, V G

    2017-06-01

    We studied changes in the population of human multipotent mesenchymal stromal cells activated by IFNγ. The cells were cultured under standard conditions; IFNγ was added in various concentrations for 4 h or over 2 passages. It was shown that the total cell production significantly decreased after long-term culturing with IFNγ, but 4-h exposure did not affect this parameter. After 4-h culturing, the expression levels of IDO1, CSF1, and IL-6 increased by 300, 7, and 2.4 times, respectively, and this increase persisted 1 and 2 days after removal of IFNγ from the culture medium. The expression of class I and II MHC (HLA) on cell surface practically did not change immediately after exposure to IFNγ, but during further culturing, HLA-ABC (MHC I) and HLA-DR (MHC II) expression significantly increased, which abolished the immune privilege in these cells, the property allowing clinical use of allogenic multipotent mesenchymal stromal cells. Multipotent mesenchymal stromal cells can suppress proliferation of lymphocytes. The degree of this suppression depends on individual properties of multipotent mesenchymal stromal cell donor. Treatment with IFNγ did not significantly affect the intensity of inhibition of lymphocyte proliferation by these cells.

  9. Ghrelin and gastrointestinal stromal tumors.

    Science.gov (United States)

    Zhu, Chang-Zhen; Liu, Dong; Kang, Wei-Ming; Yu, Jian-Chun; Ma, Zhi-Qiang; Ye, Xin; Li, Kang

    2017-03-14

    Ghrelin, as a kind of multifunctional protein polypeptide, is mainly produced in the fundus of the stomach and can promote occurrence and development of many tumors, including gastrointestinal tumors, which has been proved by the relevant researches. Most gastrointestinal stromal tumors (GISTs, about 80%), as the most common mesenchymal tumor, also develop in the fundus. Scientific research has confirmed that ghrelin, its receptors and mRNA respectively can be found in GISTs, which demonstrated the existence of a ghrelin autocrine/paracrine loop in GIST tissues. However, no reports to date have specified the mechanism whether ghrelin can promote the occurrence and development of GISTs. Studies of pulmonary artery endothelial cells in a low-oxygen environment and cardiac muscle cells in an ischemic environment have shown that ghrelin can activate the phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway. Moreover, some studies of GISTs have confirmed that activation of the PI3K/AKT/mTOR pathway can indeed promote the growth and progression of GISTs. Whether ghrelin is involved in the development or progression of GISTs through certain pathways remains unknown. Can we find a new target for the treatment of GISTs? This review explores and summaries the relationship among ghrelin, the PI3K/AKT/mTOR pathway and the development of GISTs.

  10. Multicentric malignant gastrointestinal stromal tumor

    International Nuclear Information System (INIS)

    Shukla, Shailaja; Singh, Sanjeet K; Pujani, Mukta

    2009-01-01

    Malignant gastrointestinal stromal tumor (GIST) is a rare type of sarcoma that is found in the digestive system, most often in the wall of the stomach. Multiple GISTs are extremely rare and usually associated with type 1 neurofibromatosis and familial GIST. We report here a case of a 70-year-old woman who reported pain in the abdomen, loss of appetite, and weight loss for six months. Ultrasound examination showed a small bowel mass along with multiple peritoneal deposits and a mass within the liver. Barium studies were suggestive of a neoplastic pathology of the distal ileum. A differential diagnosis of adenocarcinoma/lymphoma with metastases was entertained. Perioperative findings showed two large growths arising from the jejunum and the distal ileum, along with multiple smaller nodules on the serosal surface and adjoining mesentery of the involved bowel segments. Segmental resection of the involved portions of the intestine was performed. Histopathological features were consistent with those of multicentric malignant GIST-not otherwise specified (GIST-NOS). Follow-up examination three months after surgery showed no evidence of recurrence. (author)

  11. Purification and characterization of a new neutral metalloprotease from marine Exiguobacterium sp. SWJS2.

    Science.gov (United States)

    Lei, Fenfen; Cui, Chun; Zhao, Haifeng; Tang, Xuelu; Zhao, Mouming

    2016-01-01

    Among the protease-producing bacterial strains isolated from deep-sea sediments, SWJS2 was finally selected and identified as genus Exiguobacterium. Plackett-Burman and orthogonal array designs were applied to optimize the fermentation conditions, and the results are as follows: Glucose 5g, yeast extract 15g, glycerin 2g and CaCl2 ⋅2H2 O 0.5 g dissolved in 1 L artificial seawater; temperature 25 °C, original pH 7, inoculum rate 2%, seed age 12 H, loading volume 25 mL (250-mL Erlenmeyer flask), shaking speed 150 rpm, and fermentation time 44 H. The protease activity production was improved from about 80 to 660 U/mL under the optimized parameters. The protease was purified fourfold with specificity activity of 30,654.1 U/mg protein and a total yield of 16.2%. The protease exhibited the maximum activity at 40-45 °C and pH 7. Moreover, the enzyme activity was found to be inhibited by Cu(2+) , Ba(2+) , Cd(2+) , Hg(2+) , and Al(3+) at 5 mM, whereas it can be increased by Mg(2+) , Mn(2+) , and Ca(2+) at 0.5-5 mM. The enzyme was totally inactivated by 1 or 5 mM ethylenediaminetetraacetic acid but not by phenylmethanesulfonyl fluoride, tyrpsin inhibitor from Glycine max (STI), benzamidine, 5,5'-dithio-bis-(2-nitro benzoic acid), or pepstatin A, suggesting that it belonged to metalloprotease. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

  12. Plasmatic ADAMTS-13 metalloprotease and von Willebrand factor in children with cyanotic congenital heart disease

    Energy Technology Data Exchange (ETDEWEB)

    Soares, R.P.S. [Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, SP (Brazil); Bydlowski, S.P.; Nascimento, N.M. [Laboratório de Investigação Médica-31, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil); Thomaz, A.M.; Bastos, E.N.M.; Lopes, A.A. [Faculdade de Medicina, Instituto do Coração, Universidade de São Paulo, São Paulo, SP (Brazil)

    2013-04-05

    Changes in plasma von Willebrand factor concentration (VWF:Ag) and ADAMTS-13 activity (the metalloprotease that cleaves VWF physiologically) have been reported in several cardiovascular disorders with prognostic implications. We therefore determined the level of these proteins in the plasma of children with cyanotic congenital heart disease (CCHD) undergoing surgical treatment. Forty-eight children were enrolled (age 0.83 to 7.58 years). Measurements were performed at baseline and 48 h after surgery. ELISA, collagen-binding assays and Western blotting were used to estimate antigenic and biological activities, and proteolysis of VWF multimers. Preoperatively, VWF:Ag and ADAMTS-13 activity were decreased (65 and 71% of normal levels considered as 113 (105-129) U/dL and 91 ± 24% respectively, P < 0.003) and correlated (r = 0.39, P = 0.0064). High molecular weight VWF multimers were not related, suggesting an interaction of VWF with cell membranes, followed by proteolytic cleavage. A low preoperative ADAMTS-13 activity, a longer activated partial thromboplastin time and the need for cardiopulmonary bypass correlated with postoperative bleeding (P < 0.05). Postoperatively, ADAMTS-13 activity increased but less extensively than VWF:Ag (respectively, 2.23 and 2.83 times baseline, P < 0.0001), resulting in an increased VWF:Ag/ADAMTS-13 activity ratio (1.20 to 1.54, respectively, pre- and postoperative median values, P = 0.0029). ADAMTS-13 consumption was further confirmed by decreased ADAMTS-13 antigenic concentration (0.91 ± 0.30 to 0.70 ± 0.25 µg/mL, P < 0.0001) and persistent proteolysis of VWF multimers. We conclude that, in pediatric CCHD, changes in circulating ADAMTS-13 suggest enzyme consumption, associated with abnormal structure and function of VWF.

  13. Production and Partial Purification of a Neutral Metalloprotease by Fungal Mixed Substrate Fermentation

    Directory of Open Access Journals (Sweden)

    Alagarsamy Sumantha

    2005-01-01

    Full Text Available Five strains of fungi belonging to Aspergillus sp. were evaluated by casein agar plate assay and a wheat bran-based solid-state fermentation for selecting a neutral protease-producing culture. Based on the results, A. oryzae NRRL 2217 was selected for further studies. Sixteen different agro-industrial residues were evaluated for their potential to serve as a substrate for neutral protease production by this fungal strain. Results showed that a combination of coconut oil cake and wheat bran in the mass ratio of 1:3 was the best substrate for enzyme production. Various process parameters influencing protease production including fermentation time, initial moisture content, and fermentation temperature were optimised. The medium was supplemented with different nutrients in the form of organic and inorganic nitrogen and carbon sources. Supplementation of chitin increased the enzyme production significantly. Ammonium nitrate as inorganic nitrogen supplement slightly enhanced enzyme production. No organic nitrogen supplement was effective enhancer of enzyme production. Fermentation was performed under optimised conditions (initial moisture content V/m = 50 %, temperature 30 °C, 48 h. Partial purification of the enzyme resulted in a 3-fold increase in the specific activity of the enzyme. The partially purified enzyme was characterised by various features that govern the enzyme activity such as assay temperature, assay pH and substrate concentration. The effect of various metal ions and known protease inhibitors on the enzyme activity was also studied. The enzyme was found to be stable in pH range 7.0–7.5, and at temperature of 50 °C for 35 min. By the activating effect of divalent cations (Mg2+, Ca2+, Fe2+ and inhibiting effect of certain chelating agents (EGTA, EDTA, the enzyme was found to be a metalloprotease.

  14. Gastrointestinal Stromal Tumor of the Esophagus: Report of a Case

    OpenAIRE

    Mehmet Erol

    2014-01-01

    Gastrointestinal stromal tumors are rare neoplasms to be thought to arise from mesenchymal cells of the gastrointestinal tract. Gastrointestinal stromal tumors (GIST) of the esophagus are well documented but are very much rarer than gastrointestinal stromal tumors of the stomach and small bowel. We describe a case of GIST of the esophagus that was resected with wide surgical resection.

  15. Stromal and Epithelial Caveolin-1 Both Confer a Protective Effect Against Mammary Hyperplasia and Tumorigenesis

    Science.gov (United States)

    Williams, Terence M.; Sotgia, Federica; Lee, Hyangkyu; Hassan, Ghada; Di Vizio, Dolores; Bonuccelli, Gloria; Capozza, Franco; Mercier, Isabelle; Rui, Hallgeir; Pestell, Richard G.; Lisanti, Michael P.

    2006-01-01

    Here, we investigate the role of caveolin-1 (Cav-1) in breast cancer onset and progression, with a focus on epithelial-stromal interactions, ie, the tumor microenvironment. Cav-1 is highly expressed in adipocytes and is abundant in mammary fat pads (stroma), but it remains unknown whether loss of Cav-1 within mammary stromal cells affects the differentiated state of mammary epithelia via paracrine signaling. To address this issue, we characterized the development of the mammary ductal system in Cav-1−/− mice and performed a series of mammary transplant studies, using both wild-type and Cav-1−/− mammary fat pads. Cav-1−/− mammary epithelia were hyperproliferative in vivo, with dramatic increases in terminal end bud area and mammary ductal thickness as well as increases in bromodeoxyuridine incorporation, extracellular signal-regulated kinase-1/2 hyperactivation, and up-regulation of STAT5a and cyclin D1. Consistent with these findings, loss of Cav-1 dramatically exacerbated mammary lobulo-alveolar hyperplasia in cyclin D1 Tg mice, whereas overexpression of Cav-1 caused reversion of this phenotype. Most importantly, Cav-1−/− mammary stromal cells (fat pads) promoted the growth of both normal mammary ductal epithelia and mammary tumor cells. Thus, Cav-1 expression in both epithelial and stromal cells provides a protective effect against mammary hyperplasia as well as mammary tumorigenesis. PMID:17071600

  16. Inhibition of Stromal PlGF Suppresses the Growth of Prostate Cancer Xenografts

    Directory of Open Access Journals (Sweden)

    Dietmar Abraham

    2013-09-01

    Full Text Available The growth and vascularization of prostate cancer is dependent on interactions between cancer cells and supporting stromal cells. The primary stromal cell type found in prostate tumors is the carcinoma-associated fibroblast, which produces placental growth factor (PlGF. PlGF is a member of the vascular endothelial growth factor (VEGF family of angiogenic molecules and PlGF mRNA levels increase after androgen deprivation therapy in prostate cancer. In this study, we show that PlGF has a direct dose-dependent proliferative effect on human PC-3 prostate cancer cells in vitro and fibroblast-derived PlGF increases PC-3 proliferation in co-culture. In xenograft tumor models, intratumoral administration of murine PlGF siRNA reduced stromal-derived PlGF expression, reduced tumor burden and decreased the number of Ki-67 positive proliferating cells associated with reduced vascular density. These data show that targeting stromal PlGF expression may represent a therapeutic target for the treatment of prostate cancer.

  17. Retrovirus-mediated gene transfer of a human c-fos cDNA into mouse bone marrow stromal cells.

    Science.gov (United States)

    Roux, P; Verrier, B; Klein, B; Niccolino, M; Marty, L; Alexandre, C; Piechaczyk, M

    1991-11-01

    A cDNA encoding a complete human c-fos protein was isolated and inserted into two different murine MoMuLV-derived recombinant retroviruses allowing expression of c-fos protein in different cell types. One c-fos-expressing retrovirus, chosen for its ability to express high levels of proteins in fibroblast-like cells, was shown to potentiate long-term cultures of mouse bone marrow stromal cells in vitro and therefore constitutes a potential tool for immortalizing such cells. Moreover, when tested in an in vitro differentiation assay, stromal cells constitutively expressing c-fos favor the granulocyte differentiation of hematopoietic precursors. Interestingly, retroviruses expressing v-src and v-abl oncogenes, included as controls in our experiments, do not produce any detectable effects, whereas those expressing polyoma virus middle T antigen facilitate long-term growth in vitro of stromal cells that favor the macrophage differentiation pathway of bone marrow stem cells. Our observation supports the idea that constitutive expression of some oncogenes, including c-fos and polyoma virus middle T antigen, may influence cytokine production by bone marrow stromal cells.

  18. Urokinase receptor-associated protein (uPARAP) is expressed in connection with malignant as well as benign lesions of the human breast and occurs in specific populations of stromal cells

    DEFF Research Database (Denmark)

    Schnack Nielsen, Boye; Rank, Fritz; Engelholm, Lars H

    2002-01-01

    recognized uPARAP in Western blotting and gave a strong signal in immunohistochemistry. The immunohistochemic localization pattern was found to be identical to that of uPARAP mRNA as determined in parallel by in situ hybridization. uPARAP expression was then studied in both benign and malignant breast...... conclude that expression of uPARAP is associated with the abnormal breast and that expression appears in myofibroblasts, macrophages and myoepithelium. We suggest that uPARAP is involved in the clearance of the uPA:uPAR complex as well as other possible ligands during benign and malignant tissue remodeling....

  19. The Bone Marrow-Derived Stromal Cells

    DEFF Research Database (Denmark)

    Tencerova, Michaela; Kassem, Moustapha

    2016-01-01

    Bone marrow (BM) microenvironment represents an important compartment of bone that regulates bone homeostasis and the balance between bone formation and bone resorption depending on the physiological needs of the organism. Abnormalities of BM microenvironmental dynamics can lead to metabolic bone...... diseases. BM stromal cells (also known as skeletal or mesenchymal stem cells) [bone marrow stromal stem cell (BMSC)] are multipotent stem cells located within BM stroma and give rise to osteoblasts and adipocytes. However, cellular and molecular mechanisms of BMSC lineage commitment to adipocytic lineage...

  20. Solution structure and dynamics of C-terminal regulatory domain of Vibrio vulnificus extracellular metalloprotease

    Energy Technology Data Exchange (ETDEWEB)

    Yun, Ji-Hye; Kim, Heeyoun [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Park, Jung Eun [Department of Biotechnology, College of Natural Sciences, Chosun University, Gwangju 501-759 (Korea, Republic of); Lee, Jung Sup, E-mail: jsplee@mail.chosun.ac.kr [Department of Biotechnology, College of Natural Sciences, Chosun University, Gwangju 501-759 (Korea, Republic of); Lee, Weontae, E-mail: wlee@spin.yonsei.ac.kr [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer We have determined solution structures of vEP C-terminal regulatory domain. Black-Right-Pointing-Pointer vEP C-ter100 has a compact {beta}-barrel structure with eight anti-parallel {beta}-strands. Black-Right-Pointing-Pointer Solution structure of vEP C-ter100 shares its molecular topology with that of the collagen-binding domain of collagenase. Black-Right-Pointing-Pointer Residues in the {beta}3 region of vEP C-ter100 might be important in putative ligand/receptor binding. Black-Right-Pointing-Pointer vEP C-ter100 interacts strongly with iron ion. -- Abstract: An extracellular metalloprotease (vEP) secreted by Vibrio vulnificus ATCC29307 is a 45-kDa proteolytic enzyme that has prothrombin activation and fibrinolytic activities during bacterial infection. The action of vEP could result in clotting that could serve to protect the bacteria from the host defense machinery. Very recently, we showed that the C-terminal propeptide (C-ter100), which is unique to vEP, is involved in regulation of vEP activity. To understand the structural basis of this function of vEP C-ter100, we have determined the solution structure and backbone dynamics using multidimensional nuclear magnetic resonance spectroscopy. The solution structure shows that vEP C-ter100 is composed of eight anti-parallel {beta}-strands with a unique fold that has a compact {beta}-barrel formation which stabilized by hydrophobic and hydrogen bonding networks. Protein dynamics shows that the overall structure, including loops, is very rigid and stabilized. By structural database analysis, we found that vEP C-ter100 shares its topology with that of the collagen-binding domain of collagenase, despite low sequence homology between the two domains. Fluorescence assay reveals that vEP C-ter100 interacts strongly with iron (Fe{sup 3+}). These findings suggest that vEP protease might recruit substrate molecules, such as collagen, by binding at C-ter100 and that vEP participates

  1. Purification of a toxic metalloprotease produced by the pathogenic Photobacterium damselae subsp. piscicida isolated from cobia (Rachycentron canadum).

    Science.gov (United States)

    Liu, Ping-Chung; Chuang, Wen-Hsiao; Lee, Kuo-Kau

    2011-01-01

    The aim of the present study was to purify and characterize a toxic protease secreted by the pathogenic Photobacterium damselae subsp. piscicida strain CP1 originally isolated from diseased cobia (Rachycentron canadum). The toxin isolated by anion exchange chromatography, was a metalloprotease, inhibited by L-cysteine, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA), 1,10-phenanthroline, N-tosyl-L-phenylalanine-chloromethyl ketone (TPCK), and N-alpha-p-tosyl-L-lysine-chloromethyl ketone (TLCK), and showed maximal activity at pH 6.0-8.0 and an apparent molecular mass of about 34.3 kDa. The toxin was also completely inhibited by HgCl2, and partially by sodium dodecyl sulfate (SDS) and CuCl2. The extracellular products and the partially purified protease were lethal to cobia with LD50 values of 1.26 and 6.8 microg protein/g body weight, respectively. The addition of EDTA completely inhibited the lethal toxicity of the purified protease, indicating that this metalloprotease was a lethal toxin produced by the bacterium.

  2. Origin of hemopoietic stromal progenitor cells in chimeras

    International Nuclear Information System (INIS)

    Chertkov, J.L.; Drize, N.J.; Gurevitch, O.A.; Samoylova, R.S.

    1985-01-01

    Intravenously injected bone marrow cells do not participate in the regeneration of hemopoietic stromal progenitors in irradiated mice, nor in the curetted parts of the recipient's marrow. The hemopoietic stromal progenitors in allogeneic chimeras are of recipient origin. The adherent cell layer (ACL) of long-term cultures of allogeneic chimera bone marrow contains only recipient hemopoietic stromal progenitors. However, in ectopic hemopoietic foci produced by marrow implantation under the renal capsule and repopulated by the recipient hemopoietic cells after irradiation and reconstitution by syngeneic hemopoietic cells, the stromal progenitors were of implant donor origin, as were stromal progenitors of the ACL in long-term cultures of hemopoietic cells from ectopic foci. Our results confirm that the stromal and hemopoietic progenitors differ in origin and that hemopoietic stromal progenitors are not transplantable by the intravenous route in mice

  3. Gastrointestinal stromal tumour presenting as gastroduodenal intussusception.

    LENUS (Irish Health Repository)

    Wilson, Mark H

    2012-08-01

    Gastroduodenal intussusception secondary to gastrointestinal stromal tumour is a very rare cause for intestinal obstruction. The diagnosis of this condition can be challenging, as symptoms are often non-specific and intermittent. This article reports a case where the diagnosis was made preoperatively with abdominal imaging and was treated by a combination of endoscopic reduction and laparoscopic resection.

  4. Construction of a human corneal stromal equivalent with non-transfected human corneal stromal cells and acellular porcine corneal stromata.

    Science.gov (United States)

    Diao, Jin-Mei; Pang, Xin; Qiu, Yue; Miao, Ying; Yu, Miao-Miao; Fan, Ting-Jun

    2015-03-01

    A tissue-engineered human corneal stroma (TE-HCS) has been developed as a promising equivalent to the native corneal stroma for replacement therapy. However, there is still a crucial need to improve the current approaches to render the TE-HCS equivalent more favorable for clinical applications. At the present study, we constructed a TE-HCS by incubating non-transfected human corneal stromal (HCS) cells in an acellular porcine corneal stromata (aPCS) scaffold in 20% fetal bovine serum supplemented DMEM/F12 (1:1) medium at 37 °C with 5% CO2in vitro. After 3 days of incubation, the constructed TE-HCS had a suitable tensile strength for transplantation, and a transparency that is comparable to native cornea. The TE-HCS had a normal histological structure which contained regularly aligned collagen fibers and differentiated HCS cells with positive expression of marker and functional proteins, mimicking a native HCS. After transplantation into rabbit models, the TE-HCS reconstructed normal corneal stroma in vivo and function well in maintaining corneal clarity and thickness, indicating that the completely biological TE-HCS could be used as a HCS equivalent. The constructed TE-HCS has promising potentials in regenerative medicine and treatment of diseases caused by corneal stromal disorders. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Stromal laminin chain distribution in normal, hyperplastic and malignant oral mucosa: relation to myofibroblast occurrence and vessel formation.

    Science.gov (United States)

    Franz, Marcus; Wolheim, Anke; Richter, Petra; Umbreit, Claudia; Dahse, Regine; Driemel, Oliver; Hyckel, Peter; Virtanen, Ismo; Kosmehl, Hartwig; Berndt, Alexander

    2010-04-01

    The contribution of stromal laminin chain expression to malignant potential, tumour stroma reorganization and vessel formation in oral squamous cell carcinoma (OSCC) is not fully understood. Therefore, the expression of the laminin chains alpha2, alpha3, alpha4, alpha5 and gamma2 in the stromal compartment/vascular structures in OSCC was analysed. Frozen tissue of OSCC (9x G1, 24x G2, 8x G3) and normal (2x)/hyperplastic (11x) oral mucosa was subjected to laminin chain and alpha-smooth muscle actin (ASMA) immunohistochemistry. Results were correlated to tumour grade. The relation of laminin chain positive vessels to total vessel number was assessed by immunofluorescence double labelling with CD31. Stromal laminin alpha2 chain significantly decreases and alpha3, alpha4, alpha5 and gamma2 chains and also ASMA significantly increase with rising grade. The amount of stromal alpha3, alpha4 and gamma2 chains significantly increased with rising ASMA positivity. There is a significant decrease in alpha3 chain positive vessels with neoplastic transformation. Mediated by myofibroblasts, OSCC development is associated with a stromal up-regulation of laminin isoforms possibly contributing to a migration promoting microenvironment. A vascular basement membrane reorganization concerning alpha3 and gamma2 chain laminins during tumour angioneogenesis is suggested.

  6. CXC chemokine receptor 4 expression and stromal cell-derived factor-1alpha-induced chemotaxis in CD4+ T lymphocytes are regulated by interleukin-4 and interleukin-10

    DEFF Research Database (Denmark)

    Jinquan, T; Quan, S; Jacobi, H H

    2000-01-01

    -10. IL-4 and IL-10 up- or down-regulated CXCR4 mRNA expression in CD4+ T lymphocytes, respectively, as detected by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). Scatchard analysis revealed a type of CXCR4 with affinity (Kd approximately 6.3 nM), and approximately 70....... The regulation of CXCR4 expression in CD4+ T lymphocytes by IL-4 and IL-10 could be blocked by a selective inhibitor of protein kinase (staurosporine) or by a selective inhibitor of cAMP- and cGMP-dependent protein kinase (H-8), indicating that these cytokines regulate CXCR4 on CD4+ T lymphocytes via both c......AMP and cGMP signalling pathways. The fact that cyclosporin A or ionomycin were able to independently change the CXCR4 expression and block the effects of IL-4 and IL-10 on CXCR4 expression implied that the capacity of IL-4 and IL-10 to regulate CXCR4 on CD4+ T lymphocytes is not linked to calcium...

  7. Aging up-regulates ARA55 in stromal cells, inducing androgen-mediated prostate cancer cell proliferation and migration.

    Science.gov (United States)

    Zou, Qingsong; Cui, Di; Liang, Shengjie; Xia, Shujie; Jing, Yifeng; Han, Bangmin

    2016-06-01

    Stromal cells in the peripheral zone (PZ) of the prostate from older males (PZ-old) could significantly promote Prostate cancer (PCa) growth compared with stromal cells from young males (PZ-young). But the mechanism is still unknown. In the co-culture system with PZ-old cells, Pc3/Du145 cells showed advanced proliferation and migration after Dihydrotestosterone (DHT) incubation, but DHT didn't show the similar effect in PZ-young co-culture system. Also, higher androgen/AR signal pathway activity and AR-related cytokines secretion (FGF-2, KGF, IGF-1) were found in PZ-old cells. As AR exprssison was equivalent in PZ-old and PZ-young cells, we focused on Androgen receptor associated protein-55(ARA55), a stromal-specific androgen receptor (AR) coactivator. ARA55 expression was higher in PZ-old cells compared with PZ-young cells in vitro. After knocking down ARA55 expression in PZ-old cells, the PCa growth- promoting effect from the PZ-old cells was diminished, which may be explained by the decreased the progressive cytokines secretion (FGF-2, KGF, IGF-1) from PZ-old stromal cells. In vivo, the consistent results were also found: PZ-old cells promoted prostate cancer cells growth, but this effect receded when knocking down ARA55 expression in PZ-old cells. From our study, we found PZ stromal cells presented age-related effects in proliferation and migration of prostate cancer cells in the androgen/AR dependent manner. As aging increased, more ARA55 were expressed in PZ stromal cells, leading to more sensitive androgen/androgen receptor (AR) signal pathway, then constituting a more feasible environment to cancer cells.

  8. Isoform-specific interactions between meprin metalloproteases and the catalytic subunit of protein kinase A: significance in acute and chronic kidney injury

    Science.gov (United States)

    Niyitegeka, Jean-Marie V.; Bastidas, Adam C.; Newman, Robert H.; Taylor, Susan S.

    2014-01-01

    Meprin metalloproteases are abundantly expressed in the brush-border membranes of kidney proximal tubules. Meprins are implicated in ischemia-reperfusion (IR)-induced renal injury and diabetic nephropathy. The protein kinase A (PKA) signaling pathway modulates extracellular matrix metabolism in diabetic kidneys. The present study evaluated isoform-specific interactions between the catalytic subunit of PKA (PKA C) and meprins. To this end, cytosolic-enriched kidney proteins from meprin αβ double knockout mice, and purified forms of recombinant mouse PKA Cα, Cβ1, and Cβ2, were incubated with activated forms of either homomeric meprin A or meprin B. The cleaved protein products were subjected to SDS-PAGE and analyzed by Coomassie staining and Western blot analysis. While meprin A only cleaved PKA Cβ1, meprin B cleaved all three PKA C isoforms. Analysis of the proteolytic fragments by mass spectrometry revealed that meprin A and B cleave the PKA C isoforms at defined sites, resulting in unique cleavage products. Michaelis-Menten enzyme kinetics demonstrated that meprin B-mediated cleavage of PKA Cα occurs at a rate consistent with that of other physiologically relevant meprin substrates. Meprin cleavage decreased the kinase activity of PKA Cα, Cβ1, and Cβ2. PKA C levels were higher in diabetic kidneys, with evidence of in vivo fragmentation in wild-type diabetic kidneys. Confocal microscopy showed localization of meprin A in the glomeruli of diabetic kidneys. At 3 h post-IR, PKA C levels in proximal tubules decreased compared with distal tubules, which lack meprins. These data suggest that meprins may impact kidney injury, in part, via modulation of PKA signaling pathways. PMID:25354939

  9. Effect of pirfenidone on the proliferation of rat corneal stromal cells

    Directory of Open Access Journals (Sweden)

    Jun-Jie Chen

    2015-02-01

    Full Text Available AIM: To investigate the effects of pirfenidone(PFDon the proliferation and transfomring growth factor-β1(TGF-β1expression in vitro culture rat corneal stromal cells. METHODS: Corneal stromal cells from 8 to 10wk SD rats were isolated, cultured and treated with different concentrations of PFD 0mg/mL(control group, 0.15mg/mL(experimental group Ⅰ, 0.3mg/mL(experimental group Ⅱ, 1mg/mL(experimental group Ⅲfor 48h. CCK-8 assay was performed to assess cell proliferation, while immunocytochemistry and Western Blot were used to detect the expression of ki-67 and TGF-β1 expression, respectively. RESULTS: Compared with control group, PFD significantly inhibited the proliferation in a dose-dependent manner(all P1 in a dose-dependent manner(PCONCLUSION: Pirfenidone can significantly inhibit the proliferation of rat corneal stromal cell by down regulating TGF-β1 expression, therefore, it has potential prospect in lightening the corneal wound healing reaction.

  10. A phage display delected 7-mer peptide inhibitor of the tannerella forsythia metalloprotease-like enzyme karilysin can be truncated to ser-trp-phe-pro

    DEFF Research Database (Denmark)

    Skottrup, Peter Durand; Sørensen, Grete; Ksiazek, Miroslaw

    2012-01-01

    Tannerella forsythia is a gram-negative bacteria, which is strongly associated with the development of periodontal disease. Karilysin is a newly identified metalloprotease-like enzyme, that is secreted from T. forsythia. Karilysin modulates the host immune response and is therefore considered...

  11. Stromal Androgen Receptor in Prostate Cancer Development and Progression

    Science.gov (United States)

    Leach, Damien A.; Buchanan, Grant

    2017-01-01

    Prostate cancer development and progression is the result of complex interactions between epithelia cells and fibroblasts/myofibroblasts, in a series of dynamic process amenable to regulation by hormones. Whilst androgen action through the androgen receptor (AR) is a well-established component of prostate cancer biology, it has been becoming increasingly apparent that changes in AR signalling in the surrounding stroma can dramatically influence tumour cell behavior. This is reflected in the consistent finding of a strong association between stromal AR expression and patient outcomes. In this review, we explore the relationship between AR signalling in fibroblasts/myofibroblasts and prostate cancer cells in the primary site, and detail the known functions, actions, and mechanisms of fibroblast AR signaling. We conclude with an evidence-based summary of how androgen action in stroma dramatically influences disease progression. PMID:28117763

  12. Actin depolymerization enhances adipogenic differentiation in human stromal stem cells

    DEFF Research Database (Denmark)

    Chen, Li; Hu, Huimin; Qiu, Weimin

    2018-01-01

    Human stromal stem cells (hMSCs) differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined...... differentiation as evidenced by decreased number of mature adipocytes and decreased adipocyte specific gene expression (ADIPOQ, LPL, PPARG, FABP4). In contrast, disruption of actin cytoskeleton by Cytochalasin D enhanced adipocyte differentiation. Follow up studies revealed that the effects of CFL1 on adipocyte...... differentiation depended on the activity of LIM domain kinase 1 (LIMK1) which is the major upstream kinase of CFL1. Inhibiting LIMK by its specific chemical inhibitor LIMKi inhibited the phosphorylation of CFL1 and actin polymerization, and enhanced the adipocyte differentiation. Moreover, treating h...

  13. FGF7 supports hematopoietic stem and progenitor cells and niche-dependent myeloblastoma cells via autocrine action on bone marrow stromal cells in vitro

    International Nuclear Information System (INIS)

    Ishino, Ruri; Minami, Kaori; Tanaka, Satowa; Nagai, Mami; Matsui, Keiji; Hasegawa, Natsumi; Roeder, Robert G.; Asano, Shigetaka; Ito, Mitsuhiro

    2013-01-01

    Highlights: •FGF7 is downregulated in MED1-deficient mesenchymal cells. •FGF7 produced by mesenchymal stromal cells is a novel hematopoietic niche molecule. •FGF7 supports hematopoietic progenitor cells and niche-dependent leukemia cells. •FGF7 activates FGFR2IIIb of bone marrow stromal cells in an autocrine manner. •FGF7 indirectly acts on hematopoietic cells lacking FGFR2IIIb via stromal cells. -- Abstract: FGF1 and FGF2 support hematopoietic stem and progenitor cells (HSPCs) under stress conditions. In this study, we show that fibroblast growth factor (FGF7) may be a novel niche factor for HSPC support and leukemic growth. FGF7 expression was attenuated in mouse embryonic fibroblasts (MEFs) deficient for the MED1 subunit of the Mediator transcriptional coregulator complex. When normal mouse bone marrow (BM) cells were cocultured with Med1 +/+ MEFs or BM stromal cells in the presence of anti-FGF7 antibody, the growth of BM cells and the number of long-time culture-initiating cells (LTC-ICs) decreased significantly. Anti-FGF7 antibody also attenuated the proliferation and cobblestone formation of MB1 stromal cell-dependent myeloblastoma cells. The addition of recombinant FGF7 to the coculture of BM cells and Med1 −/− MEFs increased BM cells and LTC-ICs. FGF7 and its cognate receptor, FGFR2IIIb, were undetectable in BM cells, but MEFs and BM stromal cells expressed both. FGF7 activated downstream targets of FGFR2IIIb in Med1 +/+ and Med1 −/− MEFs and BM stromal cells. Taken together, we propose that FGF7 supports HSPCs and leukemia-initiating cells indirectly via FGFR2IIIb expressed on stromal cells

  14. FGF7 supports hematopoietic stem and progenitor cells and niche-dependent myeloblastoma cells via autocrine action on bone marrow stromal cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Ishino, Ruri; Minami, Kaori; Tanaka, Satowa [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Nagai, Mami [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 159-8555 (Japan); Matsui, Keiji; Hasegawa, Natsumi [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Roeder, Robert G. [Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Asano, Shigetaka [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 159-8555 (Japan); Ito, Mitsuhiro, E-mail: itomi@med.kobe-u.ac.jp [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 159-8555 (Japan); Department of Family and Community Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 654-0142 (Japan)

    2013-10-11

    Highlights: •FGF7 is downregulated in MED1-deficient mesenchymal cells. •FGF7 produced by mesenchymal stromal cells is a novel hematopoietic niche molecule. •FGF7 supports hematopoietic progenitor cells and niche-dependent leukemia cells. •FGF7 activates FGFR2IIIb of bone marrow stromal cells in an autocrine manner. •FGF7 indirectly acts on hematopoietic cells lacking FGFR2IIIb via stromal cells. -- Abstract: FGF1 and FGF2 support hematopoietic stem and progenitor cells (HSPCs) under stress conditions. In this study, we show that fibroblast growth factor (FGF7) may be a novel niche factor for HSPC support and leukemic growth. FGF7 expression was attenuated in mouse embryonic fibroblasts (MEFs) deficient for the MED1 subunit of the Mediator transcriptional coregulator complex. When normal mouse bone marrow (BM) cells were cocultured with Med1{sup +/+} MEFs or BM stromal cells in the presence of anti-FGF7 antibody, the growth of BM cells and the number of long-time culture-initiating cells (LTC-ICs) decreased significantly. Anti-FGF7 antibody also attenuated the proliferation and cobblestone formation of MB1 stromal cell-dependent myeloblastoma cells. The addition of recombinant FGF7 to the coculture of BM cells and Med1{sup −/−} MEFs increased BM cells and LTC-ICs. FGF7 and its cognate receptor, FGFR2IIIb, were undetectable in BM cells, but MEFs and BM stromal cells expressed both. FGF7 activated downstream targets of FGFR2IIIb in Med1{sup +/+} and Med1{sup −/−} MEFs and BM stromal cells. Taken together, we propose that FGF7 supports HSPCs and leukemia-initiating cells indirectly via FGFR2IIIb expressed on stromal cells.

  15. Gastrointestinal Stromal Tumor: Diagnosis and Prognosis; Tumor estromal gastrointestinal: diagnostico y pronostico

    Energy Technology Data Exchange (ETDEWEB)

    Martin, M. T.; Olmedilla, P.; Gonzalez, S.; Oliver, J. M. [Fundacion Hospital de Alcorcon. Madrid (Spain)

    2003-07-01

    Gastrointestinal stromal tumors (GIST) are mesenquimal tumors derived from cell precursors. They have the capacity for myogenic and neurogenic differentiation and are characterized by expression of KIT protein (tyrosine kinase growth factor). Clinically, they exhibit various biological behaviors. We present 8 cases of GIST, describing both their radiological manifestation through computerized tomography (CT) and most accepted criteria for benignity and malignancy. We also describe the response of one meta statically diagnosed tumor to tyrosine kinase inhibitor. (Author) 9 refs.

  16. Crosstalk between stromal cells and cancer cells in pancreatic cancer: New insights into stromal biology.

    Science.gov (United States)

    Zhan, Han-Xiang; Zhou, Bin; Cheng, Yu-Gang; Xu, Jian-Wei; Wang, Lei; Zhang, Guang-Yong; Hu, San-Yuan

    2017-04-28

    Pancreatic cancer (PC) remains one of the most lethal malignancies worldwide. Increasing evidence has confirmed the pivotal role of stromal components in the regulation of carcinogenesis, invasion, metastasis, and therapeutic resistance in PC. Interaction between neoplastic cells and stromal cells builds a specific microenvironment, which further modulates the malignant properties of cancer cells. Instead of being a "passive bystander", stroma may play a role as a "partner in crime" in PC. However, the role of stromal components in PC is complex and requires further investigation. In this article, we review recent advances regarding the regulatory roles and mechanisms of stroma biology, especially the cellular components such as pancreatic stellate cells, macrophages, neutrophils, adipocytes, epithelial cells, pericytes, mast cells, and lymphocytes, in PC. Crosstalk between stromal cells and cancer cells is thoroughly investigated. We also review the prognostic value and molecular therapeutic targets of stroma in PC. This review may help us further understand the molecular mechanisms of stromal biology and its role in PC development and therapeutic resistance. Moreover, targeting stroma components may provide new therapeutic strategies for this stubborn disease. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Tumor stromal vascular endothelial growth factor A is predictive of poor outcome in inflammatory breast cancer

    International Nuclear Information System (INIS)

    Arias-Pulido, Hugo; Chaher, Nabila; Gong, Yun; Qualls, Clifford; Vargas, Jake; Royce, Melanie

    2012-01-01

    Inflammatory breast cancer (IBC) is a highly angiogenic disease; thus, antiangiogenic therapy should result in a clinical response. However, clinical trials have demonstrated only modest responses, and the reasons for these outcomes remain unknown. Therefore, the purpose of this retrospective study was to determine the prognostic value of protein levels of vascular endothelial growth factor (VEGF-A), one of the main targets of antiangiogenic therapy, and its receptors (VEGF-R1 and -R2) in IBC tumor specimens. Specimens from IBC and normal breast tissues were obtained from Algerian patients. Tumor epithelial and stromal staining of VEGF-A, VEGF-R1, and VEGF-R2 was evaluated by immunohistochemical analysis in tumors and normal breast tissues; this expression was correlated with clinicopathological variables and breast cancer-specific survival (BCSS) and disease-free survival (DFS) duration. From a set of 117 IBC samples, we evaluated 103 ductal IBC tissues and 25 normal specimens. Significantly lower epithelial VEGF-A immunostaining was found in IBC tumor cells than in normal breast tissues (P <0.01), cytoplasmic VEGF-R1 and nuclear VEGF-R2 levels were slightly higher, and cytoplasmic VEGF-R2 levels were significantly higher (P = 0.04). Sixty-two percent of IBC tumors had high stromal VEGF-A expression. In univariate analysis, stromal VEGF-A levels predicted BCSS and DFS in IBC patients with estrogen receptor-positive (P <0.01 for both), progesterone receptor-positive (P = 0.04 and P = 0.03), HER2+ (P = 0.04 and P = 0.03), and lymph node involvement (P <0.01 for both). Strikingly, in a multivariate analysis, tumor stromal VEGF-A was identified as an independent predictor of poor BCSS (hazard ratio [HR]: 5.0; 95% CI: 2.0-12.3; P <0.01) and DFS (HR: 4.2; 95% CI: 1.7-10.3; P <0.01). To our knowledge, this is the first study to demonstrate that tumor stromal VEGF-A expression is a valuable prognostic indicator of BCSS and DFS at diagnosis and can therefore be used to

  18. Computed tomography in gastrointestinal stromal tumors

    International Nuclear Information System (INIS)

    Ghanem, Nadir; Altehoefer, Carsten; Winterer, Jan; Schaefer, Oliver; Springer, Oliver; Kotter, Elmar; Langer, Mathias; Furtwaengler, Alex

    2003-01-01

    The aim of this study was to define the imaging characteristics of primary and recurrent gastrointestinal stromal tumors (GIST) in computed tomography with respect to the tumor size. Computed tomography was performed in 35 patients with histologically confirmed gastrointestinal stromal tumors and analyzed retrospectively by two experienced and independent radiologist. The following morphologic tumor characteristics of primary (n=20) and (n=16) recurrent tumors were evaluated according to tumor size, shape, homogeneity, density compared with liver, contrast enhancement, presence of calcifications, ulcerations, fistula or distant metastases and the anatomical relationship to the intestinal wall, and the infiltration of adjacent visceral organs. Small GIST ( 5-10 cm) demonstrated an irregular shape, inhomogeneous density on unenhanced and contrast-enhanced images, a combined intra- and extraluminal tumor growth with aggressive findings, and infiltration of adjacent organs in 9 primary diagnosed and 2 recurrent tumors. Large GIST (>10 cm), which were observed in 8 primary tumors and 11 recurrent tumors, showed an irregular margin with inhomogeneous density and aggressive findings, and were characterized by signs of malignancy such as distant and peritoneal metastases. Small recurrent tumors had a similar appearance as compared with large primary tumors. Computed tomography gives additional information with respect to the relationship of gastrointestinal stromal tumor to the gastrointestinal wall and surrounding organs, and it detects distant metastasis. Primary and recurrent GIST demonstrate characteristic CT imaging features which are related to tumor size. Aggressive findings and signs of malignancy are found in larger tumors and in recurrent disease. Computed tomography is useful in detection and characterization of primary and recurrent tumors with regard to tumor growth pattern, tumor size, and varied appearances of gastrointestinal stromal tumors, and indirectly

  19. Innate lymphoid cells and their stromal microenvironments.

    Science.gov (United States)

    Kellermayer, Zoltán; Vojkovics, Dóra; Balogh, Péter

    2017-09-01

    In addition to the interaction between antigen presenting cells, T and B lymphocytes, recent studies have revealed important roles for a diverse set of auxiliary cells that profoundly influence the induction and regulation of immune responses against pathogens. Of these the stromal cells composed of various non-hematopoietic constituents are crucial for the creation and maintenance of specialized semi-static three-dimensional lymphoid tissue microenvironment, whereas the more recently described innate lymphoid cells are generated by the diversification of committed lymphoid precursor cells independently from clonally rearranged antigen receptor genes. Recent findings have revealed important contributions by innate lymphoid cells in inflammation and protection against pathogens in a tissue-specific manner. Importantly, lymphoid stromal cells also influence the onset of immune responses in tissue-specific fashion, raising the possibility of tissue-specific stromal - innate lymphoid cell collaboration. In this review we summarize the main features and interactions between these two cells types, with particular emphasis on ILC type 3 cells and their microenvironmental partners. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  20. VISLISI trial, a prospective clinical study allowing identification of a new metalloprotease and putative virulence factor from Staphylococcus lugdunensis.

    Science.gov (United States)

    Argemi, X; Prévost, G; Riegel, P; Keller, D; Meyer, N; Baldeyrou, M; Douiri, N; Lefebvre, N; Meghit, K; Ronde Oustau, C; Christmann, D; Cianférani, S; Strub, J M; Hansmann, Y

    2017-05-01

    Staphylococcus lugdunensis is a coagulase-negative staphylococcus that displays an unusually high virulence rate close to that of Staphylococcus aureus. It also shares phenotypic properties with S. aureus and several studies found putative virulence factors. The objective of the study was to describe the clinical manifestations of S. lugdunensis infections and investigate putative virulence factors. We conducted a prospective study from November 2013 to March 2016 at the University Hospital of Strasbourg. Putative virulence factors were investigated by clumping factor detection, screening for proteolytic activity, and sequence analysis using tandem nano-liquid chromatography-mass spectrometry. In total, 347 positive samples for S. lugdunensis were collected, of which 129 (37.2%) were from confirmed cases of S. lugdunensis infection. Eighty-one of these 129 patients were included in the study. Bone and prosthetic joints (PJI) were the most frequent sites of infection (n=28; 34.6%) followed by skin and soft tissues (n=23; 28.4%). We identified and purified a novel protease secreted by 50 samples (61.7%), most frequently associated with samples from deep infections and PJI (pr 0.97 and pr 0.91, respectively). Protease peptide sequencing by nano-liquid chromatography-mass spectrometry revealed a novel protease bearing 62.42% identity with ShpI, a metalloprotease secreted by Staphylococcus hyicus. This study confirms the pathogenicity of S. lugdunensis, particularly in bone and PJI. We also identified a novel metalloprotease called lugdulysin that may contribute to virulence. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  1. Metanephric stromal tumor: A novel pediatric renal neoplasm

    Directory of Open Access Journals (Sweden)

    Rajalakshmi V

    2009-07-01

    Full Text Available Metanephric stromal tumor of kidney is a novel pediatric benign stromal specific renal neoplasm. A few cases have been reported in adults also. This tumor is usually centered in the renal medulla with a characteristic microscopic appearance which differentiates this lesion from congenital mesoblastic nephroma and clear cell sarcoma of the kidney. In most cases complete excision alone is curative. The differentiation of metanephric stromal tumor from clear cell sarcoma of the kidney will spare the child from the ill effects of adjuvant chemotherapy. In this communication we describe the gross and microscopic features of metanephric stromal tumor in a one-month-old child with good prognosis.

  2. The application of PET-CT in gastrointestinal stromal tumor

    International Nuclear Information System (INIS)

    Xian Weijun; Feng Yanlin

    2009-01-01

    Gastrointestinal stromal tumor (GIST) is a mesenchymal neoplasm of uncertain malignant potential that arises predominantly in the gastrointestinal tract. Due to lack of specific physical signs, imagin g-x examination is an important auxiliary means in diagnosing gastrointestinal stromal tumor. Compared to other conventional imaging examinations, PET-CT has demonstrated unique superiority in staging, response evaluation and follow-up of gastrointestinal stromal tumor. And now it presents an overview of the application valuation of PET-CT and related imaging technology in gastrointestinal stromal tumor as follow. (authors)

  3. Sclerosing stromal tumor of the ovary: A case report

    Directory of Open Access Journals (Sweden)

    Navjot Kaur

    2014-01-01

    Full Text Available Sclerosing stromal tumors are benign ovarian neoplasms of the sex cord-stromal category, occurring predominantly in the second and third decades of life. Herein, we report a 23-year-old female who presented with pelvic pain, irregular menses but normal hormonal status and was diagnosed as having a right ovarian tumor. A right oophorectomy was performed, and microscopic examination revealed a sclerosing stromal tumor of the right ovary. We stress the importance of being familiar with sclerosing stromal tumors when evaluating ovarian neoplasms in young women, in order to contribute to the appropriate clinical management, preventing extensive and unnecessary surgery, and preserving fertility.

  4. Sclerosing stromal tumor of the ovary in a premenarchal female

    International Nuclear Information System (INIS)

    Fefferman, Nancy R.; Pinkney, Lynne P.; Rivera, Rafael; Popiolek, Dorota; Hummel-Levine, Pascale; Cosme, Jaqueline

    2003-01-01

    Sclerosing stromal tumor (SST) is a rare benign ovarian neoplasm of stromal origin with less than 100 cases reported in the literature. Unlike the other stromal tumors, thecomas and fibromas, which tend to occur in the fifth and sixth decades, sclerosing stromal tumors predominantly affect females in the second and third decades. Computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound findings have been described, but have not been reported previously in the pediatric literature. We present a case of SST of the ovary in a 10-year-old premenarchal female, the youngest patient to our knowledge reported in the literature, and describe the ultrasound and CT findings with pathologic correlation. (orig.)

  5. Sclerosing Stromal Tumor of Ovary: A Case Report

    Directory of Open Access Journals (Sweden)

    Menka Khanna

    2012-01-01

    Full Text Available Sclerosing stromal tumor (SST is an extremely rare and distinctive sex cord stromal tumor which occurs predominantly in the second and third decades of life. We report a case of a 32-year-old woman who developed a sclerosing stromal tumor of ovary and presented with irregular menstruation and pelvic pain. Her hormonal status was normal but CA-125 was raised. She was suspected to have a malignant tumor on computed tomography and underwent bilateral salpingo-oopherectomy. It is therefore necessary to keep in mind the possibility of sclerosing stromal tumor in a young woman.

  6. Tetraspanin CD9 participates in dysmegakaryopoiesis and stromal interactions in primary myelofibrosis.

    Science.gov (United States)

    Desterke, Christophe; Martinaud, Christophe; Guerton, Bernadette; Pieri, Lisa; Bogani, Costanza; Clay, Denis; Torossian, Frederic; Lataillade, Jean-Jacques; Hasselbach, Hans C; Gisslinger, Heinz; Demory, Jean-Loup; Dupriez, Brigitte; Boucheix, Claude; Rubinstein, Eric; Amsellem, Sophie; Vannucchi, Alessandro M; Le Bousse-Kerdilès, Marie-Caroline

    2015-06-01

    Primary myelofibrosis is characterized by clonal myeloproliferation, dysmegakaryopoiesis, extramedullary hematopoiesis associated with myelofibrosis and altered stroma in the bone marrow and spleen. The expression of CD9, a tetraspanin known to participate in megakaryopoiesis, platelet formation, cell migration and interaction with stroma, is deregulated in patients with primary myelofibrosis and is correlated with stage of myelofibrosis. We investigated whether CD9 participates in the dysmegakaryopoiesis observed in patients and whether it is involved in the altered interplay between megakaryocytes and stromal cells. We found that CD9 expression was modulated during megakaryocyte differentiation in primary myelofibrosis and that cell surface CD9 engagement by antibody ligation improved the dysmegakaryopoiesis by restoring the balance of MAPK and PI3K signaling. When co-cultured on bone marrow mesenchymal stromal cells from patients, megakaryocytes from patients with primary myelofibrosis displayed modified behaviors in terms of adhesion, cell survival and proliferation as compared to megakaryocytes from healthy donors. These modifications were reversed after antibody ligation of cell surface CD9, suggesting the participation of CD9 in the abnormal interplay between primary myelofibrosis megakaryocytes and stroma. Furthermore, silencing of CD9 reduced CXCL12 and CXCR4 expression in primary myelofibrosis megakaryocytes as well as their CXCL12-dependent migration. Collectively, our results indicate that CD9 plays a role in the dysmegakaryopoiesis that occurs in primary myelofibrosis and affects interactions between megakaryocytes and bone marrow stromal cells. These results strengthen the "bad seed in bad soil" hypothesis that we have previously proposed, in which alterations of reciprocal interactions between hematopoietic and stromal cells participate in the pathogenesis of primary myelofibrosis. Copyright© Ferrata Storti Foundation.

  7. Reciprocal upregulation of Notch signaling molecules in hematopoietic progenitor and mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Kikuchi Y

    2011-01-01

    Full Text Available Although mesenchymal stem cells (MSCs play pivotal supportive roles in hematopoiesis, how they interact with hematopoietic stem cells (HSCs is not well understood. We investigated the interaction between HSCs and surrogate MSCs (C3H10T1/2 stromal cells, focusing on the molecular events induced by cell contact of these bipartite populations. C3H10T1/2 is a mesenchymal stromal cell line that can be induced to differentiate into preadipocytes (A54 and myoblasts (M1601. The stromal cell derivatives were cocultured with murine HSCs (Lineage-Sca1+, and gene expression profiles in stromal cells and HSCs were compared before and after the coculture. HSCs gave rise to cobblestone areas only on A54 cells, with ninefold more progenitors than on M1601 or undifferentiated C3H10T1/2 cells. Microarray-based screening and a quantitative reverse transcriptase directed-polymerase chain reaction showed that the levels of Notch ligands (Jagged1 and Delta-like 3 were increased in A54 cells upon interaction with HSCs. On the other hand, the expression of Notch1 and Hes1 was upregulated in the HSCs cocultured with A54 cells. A transwell assay revealed that the reciprocal upregulation was dependent on cell-to-cell contact. The result suggested that in the hematopoietic niche, HSCs help MSCs to produce Notch ligands, and in turn, MSCs help HSCs to express Notch receptor. Such a reciprocal upregulation would reinforce the downstream signaling to determine the fate of hematopoietic cell lineage. Clarification of the initiating events on cell contact should lead to the identification of specific molecular targets to facilitate HSC engraftment in transplantation therapy.

  8. Expression

    Directory of Open Access Journals (Sweden)

    Wang-Xia Wang

    2014-02-01

    Full Text Available The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs, sharing a 5′ AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively. In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS. In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs. Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.

  9. Mirna biogenesis pathway is differentially regulated during adipose derived stromal/stem cell differentiation.

    Science.gov (United States)

    Martin, E C; Qureshi, A T; Llamas, C B; Burow, M E; King, A G; Lee, O C; Dasa, V; Freitas, M A; Forsberg, J A; Elster, E A; Davis, T A; Gimble, J M

    2018-02-07

    Stromal/stem cell differentiation is controlled by a vast array of regulatory mechanisms. Included within these are methods of mRNA gene regulation that occur at the level of epigenetic, transcriptional, and/or posttranscriptional modifications. Current studies that evaluate the posttranscriptional regulation of mRNA demonstrate microRNAs (miRNAs) as key mediators of stem cell differentiation through the inhibition of mRNA translation. miRNA expression is enhanced during both adipogenic and osteogenic differentiation; however, the mechanism by which miRNA expression is altered during stem cell differentiation is less understood. Here we demonstrate for the first time that adipose-derived stromal/stem cells (ASCs) induced to an adipogenic or osteogenic lineage have differences in strand preference (-3p and -5p) for miRNAs originating from the same primary transcript. Furthermore, evaluation of miRNA expression in ASCs demonstrates alterations in both miRNA strand preference and 5'seed site heterogeneity. Additionally, we show that during stem cell differentiation there are alterations in expression of genes associated with the miRNA biogenesis pathway. Quantitative RT-PCR demonstrated changes in the Argonautes (AGO1-4), Drosha, and Dicer at intervals of ASC adipogenic and osteogenic differentiation compared to untreated ASCs. Specifically, we demonstrated altered expression of the AGOs occurring during both adipogenesis and osteogenesis, with osteogenesis increasing AGO1-4 expression and adipogenesis decreasing AGO1 gene and protein expression. These data demonstrate changes to components of the miRNA biogenesis pathway during stromal/stem cell differentiation. Identifying regulatory mechanisms for miRNA processing during ASC differentiation may lead to novel mechanisms for the manipulation of lineage differentiation of the ASC through the global regulation of miRNA as opposed to singular regulatory mechanisms.

  10. Actin depolymerization enhances adipogenic differentiation in human stromal stem cells

    Directory of Open Access Journals (Sweden)

    Li Chen

    2018-05-01

    Full Text Available Human stromal stem cells (hMSCs differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined the effect of direct modulation of actin microfilament dynamics on adipocyte differentiation. Stabilizing actin filaments in hMSCs by siRNA-mediated knock down of the two main actin depolymerizing factors (ADFs: Cofilin 1 (CFL1 and Destrin (DSTN or treating the cells by Phalloidin reduced adipocyte differentiation as evidenced by decreased number of mature adipocytes and decreased adipocyte specific gene expression (ADIPOQ, LPL, PPARG, FABP4. In contrast, disruption of actin cytoskeleton by Cytochalasin D enhanced adipocyte differentiation. Follow up studies revealed that the effects of CFL1 on adipocyte differentiation depended on the activity of LIM domain kinase 1 (LIMK1 which is the major upstream kinase of CFL1. Inhibiting LIMK by its specific chemical inhibitor LIMKi inhibited the phosphorylation of CFL1 and actin polymerization, and enhanced the adipocyte differentiation. Moreover, treating hMSCs by Cytochalasin D inhibited ERK and Smad2 signaling and this was associated with enhanced adipocyte differentiation. On the other hand, Phalloidin enhanced ERK and Smad2 signaling, but inhibited adipocyte differentiation which was rescued by ERK specific chemical inhibitor U0126. Our data provide a link between restructuring of hMSCs cytoskeleton and hMSCs lineage commitment and differentiation. Keywords: Actin cytoskeleton, Actin depolymerizing factors, Adipocyte differentiation, Human stromal stem cells

  11. Endogenous collagen influences differentiation of human multipotent mesenchymal stromal cells.

    Science.gov (United States)

    Fernandes, Hugo; Mentink, Anouk; Bank, Ruud; Stoop, Reinout; van Blitterswijk, Clemens; de Boer, Jan

    2010-05-01

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix mainly composed of collagen type I. Here we assessed the potential role of endogenous collagen synthesis in hMSC differentiation and stem cell maintenance. We observed a sharp reduction in proliferation rate of hMSCs in the absence of ascorbic acid, concomitant with a reduction in osteogenesis in vitro and bone formation in vivo. In line with a positive role for collagen type I in osteogenesis, gene expression profiling of hMSCs cultured in the absence of ascorbic acid demonstrated increased expression of genes involved in adipogenesis and chondrogenesis and a reduction in expression of osteogenic genes. We also observed that matrix remodeling and anti-osteoclastogenic signals were high in the presence of ascorbic acid. The presence of collagen type I during the expansion phase of hMSCs did not affect their osteogenic and adipogenic differentiation potential. In conclusion, the collagenous matrix supports both proliferation and differentiation of osteogenic hMSCs but, on the other hand, presents signals stimulating matrix remodeling and inhibiting osteoclastogenesis.

  12. CD146/MCAM defines functionality of human bone marrow stromal stem cell populations

    DEFF Research Database (Denmark)

    Harkness, Linda; Zaher, Walid; Ditzel, Nicholas

    2016-01-01

    BACKGROUND: Identification of surface markers for prospective isolation of functionally homogenous populations of human skeletal (stromal, mesenchymal) stem cells (hMSCs) is highly relevant for cell therapy protocols. Thus, we examined the possible use of CD146 to subtype a heterogeneous hMSC...... population. METHODS: Using flow cytometry and cell sorting, we isolated two distinct hMSC-CD146(+) and hMSC-CD146(-) cell populations from the telomerized human bone marrow-derived stromal cell line (hMSC-TERT). Cells were examined for differences in their size, shape and texture by using high...... and adipocytes on the basis of gene expression and protein production of lineage-specific markers. In vivo, hMSC-CD146(+) and hMSC-CD146(-) cells formed bone and bone marrow organ when implanted subcutaneously in immune-deficient mice. Bone was enriched in hMSC-CD146(-) cells (12.6 % versus 8.1 %) and bone...

  13. The effect of LHRH antagonist cetrorelix in crossover conditioned media from epithelial (BPH-1) and stromal (WPMY-1) prostate cells.

    Science.gov (United States)

    Siejka, A; Schally, A V; Barabutis, N

    2014-01-01

    Stromal cells strictly modulate the differentiation of the normal prostate epithelium. In benign prostatic hyperplasia (BPH) tissue, the ratio of stromal to epithelial cells reaches a 5:1 ratio. In this study, we evaluated the effects of crossover conditioned media (CM) of stromal and epithelial prostate cells before and after treatment with LHRH antagonist Cetrorelix. WPMY-1 human prostate stromal cells and BPH-1 human benign prostatic hyperplasia cells were cultured in vitro and the effects of crossover conditioned media (CM) from those cells were studied. We evaluated the effect of Cetrorelix on the expression of PCNA and p53 in those cells. We then studied the effect of Cetrorelix on BPH-1 cells cultured with the CM from WPMY-1 cells, as well as the mechanisms which govern these interactions. CM from WPMY-1 cells strongly stimulated the proliferation of BPH-1 cells in a dose dependent manner, while CM from BPH-1 cells only slightly increased the proliferation of WPMY-1 cells. Cetrorelix inhibited the proliferation of both cell lines and the expression of PCNA, while the expression of p53 was increased. Cetrorelix also inhibited the proliferation of BPH-1 cells stimulated with the CM from WPMY-1 cells. In the crossover experiment, conditioned media from WPMY-1 and BPH-1 cells increased the expression of phosphorylated ERK1/2 and STAT3. Our results support previous observations on the bidirectional stromal-epithelial interactions in prostate gland and shed more light on the mechanistic action of those effects. Our study strongly supports the hypothesis that LHRH antagonists may be beneficial for BPH prevention and treatment. © Georg Thieme Verlag KG Stuttgart · New York.

  14. Normal development of fetal hepatic haematopoiesis during the second trimester of gestation is upregulated by fibronectin expression in the stromal cells of the portal triads El desarrollo normal de la hematopoyesis hepática fetal durante el segundo trimestre de embarazo está regulado al alza por la expresión de fibronectina en las células del estroma de las tríadas portales

    Directory of Open Access Journals (Sweden)

    D. Tamiolakis

    2007-10-01

    Full Text Available Objective: in midtrimester fetuses the principal site of hematopoiesis is the liver. In hematopoietic organs, stromal cells such as fibroblasts, epithelial cells, and macrophage-like cells develop networks to maintain hematopoiesis, i.e. hematopoietic stem cell self-renewal, proliferation, and growth, by interaction with hematopoietic progenitor cells. ECM glycoproteins produced by the stromal cells are known to play a critical role in the regulation of cell growth and differentiation. Numerous soluble and membrane-bound factors directly regulating haematopoiesis have been documented, but little is known about fetal hepatic stromal cell activity and stromal extracellular matrix protein-fibronectin, on fetal hepatic haematopoiesis. The binding of late stage erythroid cells to fibronectin has been well characterized and is believed to be critical for the terminal stages of erythroid differentiation. The intention of this article is to determine the role of fibronectin in fetal hepatic hematopoietic proliferation and differentiation in different stages of development. Material and method: we examined and compared the immunohistochemical expression of fibronectin in the hepatic stromal portal fields in the 1st, 2nd, and 3rd trimester of gestation respectively, in relation to the appearance of CD34 progenitor hematopoietic, stromal progenitor and vascular endothelial positive cells. Results: our results demonstrated a quantitative difference in the second trimester of gestation concerning the expression of fibronectin in the connective tissue stroma of the hepatic portal fields over the equivalent expression of the protein in the first (p Objetivo: en el segundo trimestre de la gestación, el principal foco de hematopoyesis del feto es el hígado. En los órganos hematopoyéticos, las células del estroma, como fibroblastos, células epiteliales y células de tipo macrófago, desarrollan redes para mantener la hematopoyesis, es decir, la auto

  15. Virus vector-mediated genetic modification of brain tumor stromal cells after intravenous delivery.

    Science.gov (United States)

    Volak, Adrienn; LeRoy, Stanley G; Natasan, Jeya Shree; Park, David J; Cheah, Pike See; Maus, Andreas; Fitzpatrick, Zachary; Hudry, Eloise; Pinkham, Kelsey; Gandhi, Sheetal; Hyman, Bradley T; Mu, Dakai; GuhaSarkar, Dwijit; Stemmer-Rachamimov, Anat O; Sena-Esteves, Miguel; Badr, Christian E; Maguire, Casey A

    2018-05-16

    The malignant primary brain tumor, glioblastoma (GBM) is generally incurable. New approaches are desperately needed. Adeno-associated virus (AAV) vector-mediated delivery of anti-tumor transgenes is a promising strategy, however direct injection leads to focal transgene spread in tumor and rapid tumor division dilutes out the extra-chromosomal AAV genome, limiting duration of transgene expression. Intravenous (IV) injection gives widespread distribution of AAV in normal brain, however poor transgene expression in tumor, and high expression in non-target cells which may lead to ineffective therapy and high toxicity, respectively. Delivery of transgenes encoding secreted, anti-tumor proteins to tumor stromal cells may provide a more stable and localized reservoir of therapy as they are more differentiated than fast-dividing tumor cells. Reactive astrocytes and tumor-associated macrophage/microglia (TAMs) are stromal cells that comprise a large portion of the tumor mass and are associated with tumorigenesis. In mouse models of GBM, we used IV delivery of exosome-associated AAV vectors driving green fluorescent protein expression by specific promoters (NF-κB-responsive promoter and a truncated glial fibrillary acidic protein promoter), to obtain targeted transduction of TAMs and reactive astrocytes, respectively, while avoiding transgene expression in the periphery. We used our approach to express the potent, yet toxic anti-tumor cytokine, interferon beta, in tumor stroma of a mouse model of GBM, and achieved a modest, yet significant enhancement in survival compared to controls. Noninvasive genetic modification of tumor microenvironment represents a promising approach for therapy against cancers. Additionally, the vectors described here may facilitate basic research in the study of tumor stromal cells in situ.

  16. A Color-coded Imageable Syngeneic Mouse Model of Stromal-cell Recruitment by Metastatic Lymphoma.

    Science.gov (United States)

    Matsumoto, Takuro; Suetsugu, Atsushi; Shibata, Yuhei; Nakamura, Nobuhiko; Aoki, Hitomi; Kunisada, Takahiro; Tsurumi, Hisashi; Shimizu, Masahito; Hoffman, Robert M

    2015-09-01

    A syngeneic color-coded imageable lymphoma model has been developed to visualize recruitment of host stromal cells by malignant lymphoma during metastasis. The EL4 cell line was previously derived from a lymphoma induced in a C57/BL6 mouse by 9,10-dimethyl-1,2-benzanthracene. EL4 lymphoma cells expressing red fluorescent protein (EL4-RFP) were initially established. EL4-RFP cells were subsequently injected into the tail vein of C57/BL6-GFP transgenic mice. EL4-RFP metastasis was observed in the lymph nodes of the upper mediastinum and in the liver 28 days after cell injection. Large EL4-RFP liver metastases in C57/BL6-GFP mice contained GFP-expressing stromal cells derived from the host. In addition, EL4-RFP lymphoma metastasis was formed in peri-gastric lymph nodes, which were also enriched in host GFP-expressing cells. Furthermore, EL4-RFP lymphoma cells were also observed in the peripheral blood and bone marrow of C57/BL6-GFP transgenic mice, where they were associated with GFP-expressing host cells. Lymph node, liver and bone marrow metastases were found approximately 4 weeks after transplantation and all RFP-expressing metastases were highly enriched in GFP-expressing host stromal cells. This model of malignant lymphoma can be used to study early tumor development, metastasis, and the role of the stroma, as well as for discovery and evaluation of novel therapeutics for this treatment-resistant disease. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  17. Color-Coded Imaging of Syngeneic Orthotopic Malignant Lymphoma Interacting with Host Stromal Cells During Metastasis.

    Science.gov (United States)

    Matsumoto, Takuro; Suetsugu, Atsushi; Hasegawa, Kosuke; Nakamura, Miki; Aoki, Hitomi; Kunisada, Takahiro; Tsurumi, Hisashi; Shimizu, Masahito; Hoffman, Robert M

    2016-04-01

    The EL4 cell line was previously derived from a lymphoma induced in a C57/BL6 mouse by 9,10-dimethyl-1,2-benzanthracene. In a previous study, EL4 lymphoma cells expressing red fluorescent protein (EL4-RFP) were established and injected into the tail vein of C57/BL6 green fluorescent protein (GFP) transgenic mice. Metastasis was observed at multiple sites which were also enriched with host GFP-expressing stromal cells. In the present study, our aim was to establish an orthotopic model of EL4-RFP. In the present study, EL4-RFP lymphoma cells were injected in the spleen of C57/BL6 GFP transgenic mice as an orthotopic model of lymphoma. Resultant primary tumor and metastases were imaged with the Olympus FV1000 scanning laser confocal microscope. EL4-RFP metastasis was observed 21 days later. EL4-RFP tumors in the spleen (primary injection site), liver, supra-mediastinum lymph nodes, abdominal lymph nodes, bone marrow, and lung were visualized by color-coded imaging. EL4-RFP metastases in the liver, lymph nodes, and bone marrow in C57/BL6 GFP mice were rich in GFP stromal cells such as macrophages, fibroblasts, dendritic cells, and normal lymphocytes derived from the host animal. Small tumors were observed in the spleen, which were rich in host stromal cells. In the lung, no mass formation of lymphoma cells occurred, but lymphoma cells circulated in lung peripheral blood vessels. Phagocytosis of EL4-RFP lymphoma cells by macrophages, as well as dendritic cells and fibroblasts, were observed in culture. Color-coded imaging of the lymphoma microenvironment suggests an important role of stromal cells in lymphoma progression and metastasis. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  18. Osteoactivin regulates head and neck squamous cell carcinoma invasion by modulating matrix metalloproteases.

    Science.gov (United States)

    Arosarena, Oneida A; Barr, Eric W; Thorpe, Ryan; Yankey, Hilary; Tarr, Joseph T; Safadi, Fayez F

    2018-01-01

    Nearly 60% of patients with head and neck squamous cell carcinoma (HNSCC) die of metastases or locoregional recurrence. Metastasis is mediated by cancer cell migration and invasion, which are in part dependent on extracellular matrix degradation by matrix metalloproteinases. Osteoactivin (OA) overexpression plays a role in metastases in several malignancies, and has been shown to upregulate matrix metalloproteinase (MMP) expression and activity. To determine how OA modulates MMP expression and activity in HNSCC, and to investigate OA effects on cell invasion, we assessed effects of OA treatment on MMP mRNA and protein expression, as well as gelatinase and caseinolytic activity in HNSCC cell lines. We assessed the effects of OA gene silencing on MMP expression, gelatinase and caseinolytic activity, and cell invasion. OA treatment had differential effects on MMP mRNA expression. OA treatment upregulated MMP-10 expression in UMSCC14a (p = 0.0431) and SCC15 (p < 0.0001) cells, but decreased MMP-9 expression in UMSCC14a cells (p = 0.0002). OA gene silencing decreased MMP-10 expression in UMSCC12 cells (p = 0.0001), and MMP-3 (p = 0.0005) and -9 (p = 0.0036) expression in SCC25 cells. In SCC15 and SCC25 cells, OA treatment increased MMP-2 (p = 0.0408) and MMP-9 gelatinase activity (p < 0.0001), respectively. OA depletion decreased MMP-2 (p = 0.0023) and -9 (p < 0.0001) activity in SCC25 cells. OA treatment increased 70 kDa caseinolytic activity in UMSCC12 cells consistent with tissue type plasminogen activator (p = 0.0078). OA depletion decreased invasive capacity of UMSCC12 cells (p < 0.0001). OA's effects on MMP expression in HNSCC are variable, and may promote cancer cell invasion. © 2017 Wiley Periodicals, Inc.

  19. Uterine endometrial stromal sarcoma located in uterine myometrium: MRI appearance

    Energy Technology Data Exchange (ETDEWEB)

    Ueda, M.; Otsuka, M.; Hatakenaka, M. [Dept. of Radiology, Medical Institute of Bioregulation, Kyushu University, Beppu (Japan); Torii, Y. [Dept. of Radiology, Saga Prefectural Hospital (Japan)

    2000-05-01

    Two cases of uterine endometrial stromal sarcoma whose main mass was located in uterine myometrium are reported. They mimicked uterine leiomyoma with cystic degeneration or uterine leiomyosarcoma. Endometrial stromal sarcoma should be suggested in the differential diagnosis of mass lesion in uterine myometrium. (orig.)

  20. An exceptional collision tumor: gastric calcified stromal tumor and ...

    African Journals Online (AJOL)

    The authors report an exceptional case of collision tumor comprised of a gastric calcified stromal tumor and a pancreatic adenocarcinoma. The pancreatic tumor was detected fortuitously on the histological exam of resection specimen. Key words: Collision tumor, stromal tumor, adenocarcinoma ...

  1. Compartmentalised expression of meprin in small intestinal mucosa: enhanced expression in lamina propria in coeliac disease.

    Science.gov (United States)

    Lottaz, Daniel; Buri, Caroline; Monteleone, Giovanni; Rösmann, Sandra; Macdonald, Thomas T; Sanderson, Ian R; Sterchi, Erwin E

    2007-03-01

    Epithelial cells in the human small intestine express meprin, an astacin-like metalloprotease, which accumulates normally at the brush border membrane and in the gut lumen. Therefore, meprin is targeted towards luminal components. In coeliac disease patients, peptides from ingested cereals trigger mucosal inflammation in the small intestine, disrupting epithelial cell differentiation and function. Using in situ hybridisation on duodenal tissue sections, we observed a marked shift of meprin mRNA expression from epithelial cells, the predominant expression site in normal mucosa, to lamina propria leukocytes in coeliac disease. Meprin thereby gains access to the substrate repertoire present beneath the epithelium.

  2. Mammary fibroadenoma with pleomorphic stromal cells.

    Science.gov (United States)

    Abid, Najla; Kallel, Rim; Ellouze, Sameh; Mellouli, Manel; Gouiaa, Naourez; Mnif, Héla; Boudawara, Tahia

    2015-01-01

    The presence of enlarged and pleomorphic nuclei is usually regarded as a feature of malignancy, but it may on occasion be seen in benign lesions such as mammary fibroadenomas. We present such a case of fibroadenoma occurring in a 37-year-old woman presenting with a self-palpable right breast mass. Histological examination of the tumor revealed the presence of multi and mononucleated giant cells with pleomorphic nuclei. The recognition of the benign nature of these cells is necessary for differential diagnosis from malignant lesions of the breast. fibroadenoma - pleomorphic stromal cells - atypia - breast.

  3. Hemangioblastomas: histogenesis of the stromal cell studied by immunocytochemistry.

    Science.gov (United States)

    Jurco, S; Nadji, M; Harvey, D G; Parker, J C; Font, R L; Morales, A R

    1982-01-01

    Twenty-one cases of hemangioblastoma from the cerebellum, spinal cord and retina were studied using the unlabeled antibody peroxidase-antiperoxidase technique with antibodies directed against glial fibrillary acidic protein (GFAP) and factor VIII related antigen (VIIIR:Ag). In 19 of 21 cases studied with anti-GFAP, astrocytes were identified peripherally, and in 13 cases they were found centrally within the tumor. In no instance did stromal cells react positively for GFAP. Sixteen cases with anti-VIIIR:Ag antibody were examined, and in all cases many stromal cells showed positive staining. It is concluded that the stromal cells were of endothelial origin. The occasional stromal cells that other investigators have identified as reacting positively for GFAP may represent stromal cells capable of ingesting extracellular GFAP derived from reactive astrocytes within the tumor, or they may be lipidized astrocytes.

  4. Decidualization and syndecan-1 knock down sensitize endometrial stromal cells to apoptosis induced by embryonic stimuli.

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    Sarah Jean Boeddeker

    Full Text Available Human embryo invasion and implantation into the inner wall of the maternal uterus, the endometrium, is the pivotal process for a successful pregnancy. Whereas disruption of the endometrial epithelial layer was already correlated with the programmed cell death, the role of apoptosis of the subjacent endometrial stromal cells during implantation is indistinct. The aim was to clarify whether apoptosis plays a role in the stromal invasion and to characterize if the apoptotic susceptibility of endometrial stromal cells to embryonic stimuli is influenced by decidualization and Syndecan-1. Therefore, the immortalized human endometrial stromal cell line St-T1 was used to first generate a new cell line with a stable Syndecan-1 knock down (KdS1, and second to further decidualize the cells with progesterone. As a replacement for the ethically inapplicable embryo all cells were treated with the embryonic factors and secretion products interleukin-1β, interferon-γ, tumor necrosis factor-α, transforming growth factor-β1 and anti-Fas antibody to mimic the embryo contact. Detection of apoptosis was verified via Caspase ELISAs, PARP cleavage and Annexin V staining. Apoptosis-related proteins were investigated via antibody arrays and underlying signaling pathways were analyzed by Western blot. Non-decidualized endometrial stromal cells showed a resistance towards apoptosis which was rescinded by decidualization and Syndecan-1 knock down independent of decidualization. This was correlated with an altered expression of several pro- and anti-apoptotic proteins and connected to a higher activation of pro-survival Akt in non-differentiated St-T1 as an upstream mediator of apoptotis-related proteins. This study provides insight into the largely elusive process of implantation, proposing an important role for stromal cell apoptosis to successfully establish a pregnancy. The impact of Syndecan-1 in attenuating the apoptotic signal is particularly interesting in the

  5. Role of Corneal Stromal Cells on Epithelial Cell Function during Wound Healing

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    Bhavani S. Kowtharapu

    2018-02-01

    Full Text Available Following injury, corneal stromal keratocytes transform into repair-phenotype of activated stromal fibroblasts (SFs and participate in wound repair. Simultaneously, ongoing bi-directional communications between corneal stromal-epithelial cells also play a vital role in mediating the process of wound healing. Factors produced by stromal cells are known to induce proliferation, differentiation, and motility of corneal epithelial cells, which are also subsequently the main processes that occur during wound healing. In this context, the present study aims to investigate the effect of SFs conditioned medium (SFCM on corneal epithelial cell function along with substance P (SP. Antibody microarrays were employed to profile differentially expressed cell surface markers and cytokines in the presence of SFCM and SP. Antibody microarray data revealed enhanced expression of the ITGB1 in corneal epithelial cells following stimulation with SP whereas SFCM induced abundant expression of IL-8, ITGB1, PD1L1, PECA1, IL-15, BDNF, ICAM1, CD8A, CD44 and NTF4. All these proteins have either direct or indirect roles in epithelial cell growth, movement and adhesion related signaling cascades during tissue regeneration. We also observed activation of MAPK signaling pathway along with increased expression of focal adhesion kinase (FAK, paxillin, vimentin, β-catenin and vasodilator-stimulated phosphoprotein (VASP phosphorylation. Additionally, epithelial-to-mesenchymal transition (EMT regulating transcription factors Slug and ZEB1 expression were enhanced in the presence of SFCM. SP enriched the expression of integrin subunits α4, α5, αV, β1 and β3 whereas SFCM increased α4, α5, αV, β1 and β5 integrin subunits. We also observed increased expression of Serpin E1 following SP and SFCM treatment. Wound healing scratch assay revealed enhanced migration of epithelial cells following the addition of SFCM. Taken together, we conclude that SFCM-mediated sustained

  6. Cytogenetic and molecular profile of endometrial stromal sarcoma.

    Science.gov (United States)

    Micci, Francesca; Gorunova, Ludmila; Agostini, Antonio; Johannessen, Lene E; Brunetti, Marta; Davidson, Ben; Heim, Sverre; Panagopoulos, Ioannis

    2016-11-01

    Recent cytogenetic and molecular investigations have improved our understanding of endometrial stromal tumors, including sarcomas (ESS), and helped redefine their classification into more pathogenetically meaningful categories. Because much more can be gained through such studies, we add information on another 22 ESS examined by karyotyping, PCR analysis, expression array analysis, and transcriptome sequencing. In spite of the known preference for certain pathogenetic pathways, we found considerable genetic heterogeneity in high-grade (HG) as well as in low-grade (LG) ESS. Not all HG tumors showed a YWHAE-NUTM chimeric transcript and as many as six LGESS showed no hitherto known ESS-related fusions. Among the transcripts identified by transcriptome sequencing and verified by Sanger sequencing, new variants of ZC3H7-BCOR and its reciprocal BCOR-ZC3H7 were identified as was involvement of the CREBBP and MLLT4 genes (both well known leukemia-related genes) in two new fusions. FISH analysis identified a known EPC1-PHF1 fusion which led to the identification of a new variant at the molecular level. The fact that around 70 genes were found differentially expressed, by microarray analysis, when comparing LGESS showing ESS-related fusions with LGESS without such transcripts, underscores the biochemical importance of the observed genetic heterogeneity and hints that new subgroups/entities in LGESS still remain undiscovered. © 2016 The Authors. Genes, Chromosomes & Cancer Published by Wiley Periodicals, Inc. © 2016 The Authors. Genes, Chromosomes & Cancer Published by Wiley Periodicals, Inc.

  7. Ultrasound-assisted liposuction provides a source for functional adipose-derived stromal cells.

    Science.gov (United States)

    Duscher, Dominik; Maan, Zeshaan N; Luan, Anna; Aitzetmüller, Matthias M; Brett, Elizabeth A; Atashroo, David; Whittam, Alexander J; Hu, Michael S; Walmsley, Graham G; Houschyar, Khosrow S; Schilling, Arndt F; Machens, Hans-Guenther; Gurtner, Geoffrey C; Longaker, Michael T; Wan, Derrick C

    2017-12-01

    Regenerative medicine employs human mesenchymal stromal cells (MSCs) for their multi-lineage plasticity and their pro-regenerative cytokine secretome. Adipose-derived mesenchymal stromal cells (ASCs) are concentrated in fat tissue, and the ease of harvest via liposuction makes them a particularly interesting cell source. However, there are various liposuction methods, and few have been assessed regarding their impact on ASC functionality. Here we study the impact of the two most popular ultrasound-assisted liposuction (UAL) devices currently in clinical use, VASER (Solta Medical) and Lysonix 3000 (Mentor) on ASCs. After lipoaspirate harvest and processing, we sorted for ASCs using fluorescent-assisted cell sorting based on an established surface marker profile (CD34 + CD31 - CD45 - ). ASC yield, viability, osteogenic and adipogenic differentiation capacity and in vivo regenerative performance were assessed. Both UAL samples demonstrated equivalent ASC yield and viability. VASER UAL ASCs showed higher osteogenic and adipogenic marker expression, but a comparable differentiation capacity was observed. Soft tissue healing and neovascularization were significantly enhanced via both UAL-derived ASCs in vivo, and there was no significant difference between the cell therapy groups. Taken together, our data suggest that UAL allows safe and efficient harvesting of the mesenchymal stromal cellular fraction of adipose tissue and that cells harvested via this approach are suitable for cell therapy and tissue engineering applications. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  8. Adipose derived stromal vascular fraction improves early tendon healing: an experimental study in rabbits

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    Mehdi Behfar

    2011-11-01

    Full Text Available Tendon never restores the complete biological and mechanical properties after healing. Bone marrow and recently adipose tissue have been used as the sources of mesenchymal stem cells, which have been proven to enhance tendon healing. Stromal vascular fraction (SVF, derived from adipose tissue by an enzymatic digestion, represents an alternative source of multipotent cells, which undergo differentiation into multiple lineages to be used in regenerative medicine. In the present study, we investigated potentials of this source on tendon healing. Twenty rabbits were divided into control and treatment groups. Five rabbits were used as donors of adipose tissue. The injury model was unilateral complete transection through the middle one third of deep digital flexor tendon. Immediately after suture repair, either fresh stromal vascular fraction from enzymatic digestion of adipose tissue or placebo was intratendinously injected into the suture site in treatments and controls, respectively. Cast immobilization was continued for two weeks after surgery. Animals were sacrificed at the third week and tendons underwent histological, immunohistochemical, and mechanical evaluations. By histology, improved fibrillar organization and remodeling of neotendon were observed in treatment group. Immunohistochemistry revealed an insignificant increase in collagen type III and I expression in treatments over controls. Mechanical testing showed significant increase in maximum load and energy absorption in SVF treated tendons. The present study showed that intratendinous injection of uncultured adipose derived stromal vascular fraction improved structural and mechanical properties of repaired tendon and it could be an effective modality for treating tendon laceration.

  9. Stromal fibroblasts derived from mammary gland of bovine with mastitis display inflammation-specific changes.

    Science.gov (United States)

    Chen, Qing; He, Guiliang; Zhang, Wenyao; Xu, Tong; Qi, Hongliang; Li, Jing; Zhang, Yong; Gao, Ming-Qing

    2016-06-07

    Fibroblasts are predominant components of mammary stromal cells and play crucial roles in the development and involution of bovine mammary gland; however, whether these cells contribute to mastitis has not been demonstrated. Thus, we have undertaken biological and molecular characterization of inflammation-associated fibroblasts (INFs) extracted from bovine mammary glands with clinical mastitis and normal fibroblasts (NFs) from slaughtered dairy cows because of fractured legs during lactation. The functional contributions of INFs to normal epithelial cells were also investigated by using an in vitro co-culture model. We present evidence that the INFs were activated fibroblasts and showed inflammation-related features. Moreover, INFs significantly inhibited the proliferation and β-casein secretion of epithelial cells, as well as upregulated the expression of tumor necrosis factor-α and interleukin-8 in epithelial cells. These findings indicate that functional alterations can occur in stromal fibroblasts within the bovine mammary gland during mastitis, demonstrating the importance of stromal fibroblasts in bovine mastitis and its treatment.

  10. Pulmonary stromal cells induce the generation of regulatory DC attenuating T-cell-mediated lung inflammation.

    Science.gov (United States)

    Li, Qian; Guo, Zhenhong; Xu, Xiongfei; Xia, Sheng; Cao, Xuetao

    2008-10-01

    The tissue microenvironment may affect the development and function of immune cells such as DC. Whether and how the pulmonary stromal microenvironment can affect the development and function of lung DC need to be investigated. Regulatory DC (DCreg) can regulate T-cell response. We wondered whether such regulatory DC exist in the lung and what is the effect of the pulmonary stromal microenvironment on the generation of DCreg. Here we demonstrate that murine pulmonary stromal cells can drive immature DC, which are regarded as being widely distributed in the lung, to proliferate and differentiate into a distinct subset of DCreg, which express high levels of CD11b but low levels of MHC class II (I-A), CD11c, secrete high amounts of IL-10, NO and prostaglandin E2 (PGE2) and suppress T-cell proliferation. The natural counterpart of DCreg in the lung with similar phenotype and regulatory function has been identified. Pulmonary stroma-derived TGF-beta is responsible for the differentiation of immature DC to DCreg, and DCreg-derived PGE2 contributes to their suppression of T-cell proliferation. Moreover, DCreg can induce the generation of CD4+CD25+Foxp3+ Treg. Importantly, infusion with DCreg attenuates T-cell-mediated eosinophilic airway inflammation in vivo. Therefore, the pulmonary microenvironment may drive the generation of DCreg, thus contributing to the maintenance of immune homoeostasis and the control of inflammation in the lung.

  11. Inorganic Arsenic–Related Changes in the Stromal Tumor Microenvironment in a Prostate Cancer Cell–Conditioned Media Model

    Science.gov (United States)

    Shearer, Joseph J.; Wold, Eric A.; Umbaugh, Charles S.; Lichti, Cheryl F.; Nilsson, Carol L.; Figueiredo, Marxa L.

    2015-01-01

    Background: The tumor microenvironment plays an important role in the progression of cancer by mediating stromal–epithelial paracrine signaling, which can aberrantly modulate cellular proliferation and tumorigenesis. Exposure to environmental toxicants, such as inorganic arsenic (iAs), has also been implicated in the progression of prostate cancer. Objective: The role of iAs exposure in stromal signaling in the tumor microenvironment has been largely unexplored. Our objective was to elucidate molecular mechanisms of iAs-induced changes to stromal signaling by an enriched prostate tumor microenvironment cell population, adipose-derived mesenchymal stem/stromal cells (ASCs). Results: ASC-conditioned media (CM) collected after 1 week of iAs exposure increased prostate cancer cell viability, whereas CM from ASCs that received no iAs exposure decreased cell viability. Cytokine array analysis suggested changes to cytokine signaling associated with iAs exposure. Subsequent proteomic analysis suggested a concentration-dependent alteration to the HMOX1/THBS1/TGFβ signaling pathway by iAs. These results were validated by quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) and Western blotting, confirming a concentration-dependent increase in HMOX1 and a decrease in THBS1 expression in ASC following iAs exposure. Subsequently, we used a TGFβ pathway reporter construct to confirm a decrease in stromal TGFβ signaling in ASC following iAs exposure. Conclusions: Our results suggest a concentration-dependent alteration of stromal signaling: specifically, attenuation of stromal-mediated TGFβ signaling following exposure to iAs. Our results indicate iAs may enhance prostate cancer cell viability through a previously unreported stromal-based mechanism. These findings indicate that the stroma may mediate the effects of iAs in tumor progression, which may have future therapeutic implications. Citation: Shearer JJ, Wold EA, Umbaugh CS, Lichti CF, Nilsson CL

  12. Non-multipotent stroma inhibit the proliferation and differentiation of mesenchymal stromal cells in vitro.

    Science.gov (United States)

    Rosu-Myles, Michael; Fair, Joel; Pearce, Nelson; Mehic, Jelica

    2010-10-01

    The ability to expand and maintain bone marrow (BM)-derived mesenchymal stem cells (MSC) in vitro is an important aspect of their therapeutic potential. Despite this, the exact composition of stromal cell types within these cultures and the potential effects of non-stem cells on the maintenance of MSC are poorly understood. C57BL/6J BM stroma was investigated as a model to determine the relationship between MSC and non-multipotent cells in vitro. Whole BM and single-cell derived cultures were characterized using flow cytometry and cell sorting combined with multipotent differentiation. Proliferation of individual stromal populations was evaluated using BrdU. At a single-cell level, MSC were distinguished from committed progenitors, and cells lacking differentiation ability, by the expression of CD105 (CD105+). A 3-fold reduction in the percentage of CD105+ cells was detected after prolonged culture and correlated with loss of MSC. Depletion of CD105+ cells coincided with a 10-20% increase in the frequency of proliferating CD105(-) cells. Removal of CD105(-) stroma caused increased proliferation in CD105+ cells, which could be diminished by conditioned media from parent cultures. Comparison of the multipotent differentiation potential in purified and non-purified CD105+ cells determined that MSC were detectable for at least 3 weeks longer when cultured in the absence of CD105(-) cells. This work identifies a simple model for characterizing the different cellular components present in BM stromal cultures and demonstrates that stromal cells lacking multipotent differentiating capacity greatly reduce the longevity of MSC.

  13. Interleukin-6 mediates epithelial-stromal interactions and promotes gastric tumorigenesis.

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    Hiroto Kinoshita

    Full Text Available Interleukin-6 (IL-6 is a pleiotropic cytokine that affects various functions, including tumor development. Although the importance of IL-6 in gastric cancer has been documented in experimental and clinical studies, the mechanism by which IL-6 promotes gastric cancer remains unclear. In this study, we investigated the role of IL-6 in the epithelial-stromal interaction in gastric tumorigenesis. Immunohistochemical analysis of human gastritis, gastric adenoma, and gastric cancer tissues revealed that IL-6 was frequently detected in the stroma. IL-6-positive cells in the stroma showed positive staining for the fibroblast marker α-smooth muscle actin, suggesting that stromal fibroblasts produce IL-6. We compared IL-6 knockout (IL-6(-/- mice with wild-type (WT mice in a model of gastric tumorigenesis induced by the chemical carcinogen N-methyl-N-nitrosourea. The stromal fibroblasts expressed IL-6 in tumors from WT mice. Gastric tumorigenesis was attenuated in IL-6(-/- mice, compared with WT mice. Impaired tumor development in IL-6(-/- mice was correlated with the decreased activation of STAT3, a factor associated with gastric cancer cell proliferation. In vitro, when gastric cancer cell line was co-cultured with primary human gastric fibroblast, STAT3-related genes including COX-2 and iNOS were induced in gastric cancer cells and this response was attenuated with neutralizing anti-IL-6 receptor antibody. IL-6 production from fibroblasts was increased when fibroblasts were cultured in the presence of gastric cancer cell-conditioned media. IL-6 production from fibroblasts was suppressed by an interleukin-1 (IL-1 receptor antagonist and siRNA inhibition of IL-1α in the fibroblasts. IL-1α mRNA and protein were increased in fibroblast lysate, suggesting that cell-associated IL-1α in fibroblasts may be involved. Our results suggest the importance of IL-6 mediated stromal-epithelial cell interaction in gastric tumorigenesis.

  14. Developpement of a photoaffinity probe for the sensitive detection of matrix metallo-protease active forms from complex biological systems

    International Nuclear Information System (INIS)

    Nury, Catherine

    2012-01-01

    A new activity-based probe able to covalently modify the active site of proteases belonging to the matrix metallo-protease family (MMPs) has been developed in this thesis project. The probe was shown to behave as potent inhibitor of several MMPs, with nanomolar Ki values. This probe was also able to modify specifically only the free active site of MMPs, with particular high yields of cross-linking varying from 50 % to 11 %, depending of the MMPs tested. Using radioactivity as means of detection, this probe was able to detect active form of MMPs with a threshold of 1 femto-mole. Applied to the study of bronchoalveolar fluids (BAL) from mice exposed to nanoparticles by a lung aspiration protocol, this probe revealed the presence of the catalytic domain of MMP-12 under its active form, but not in control animals. When used to detect active form of MMPs from extracts obtained from human arteries of patient suffering from atherosclerosis, the probe was not able to detect such MMP active forms. Despite this negative result, the detection of active form of MMP in pathological fluid like BAL has never been reported before this work. Having validated this novel MMP activity-based probe, it will be possible to use it now for detecting MMPs from other pathological fluids or tissues extracts in which MMPs can be good markers of the pathology. (author) [fr

  15. Intracellular Cleavage of the Cx43 C-Terminal Domain by Matrix-Metalloproteases: A Novel Contributor to Inflammation?

    Directory of Open Access Journals (Sweden)

    Marijke De Bock

    2015-01-01

    Full Text Available The coordination of tissue function is mediated by gap junctions (GJs that enable direct cell-cell transfer of metabolic and electric signals. GJs are formed by connexin (Cx proteins of which Cx43 is most widespread in the human body. Beyond its role in direct intercellular communication, Cx43 also forms nonjunctional hemichannels (HCs in the plasma membrane that mediate the release of paracrine signaling molecules in the extracellular environment. Both HC and GJ channel function are regulated by protein-protein interactions and posttranslational modifications that predominantly take place in the C-terminal domain of Cx43. Matrix metalloproteases (MMPs are a major group of zinc-dependent proteases, known to regulate not only extracellular matrix remodeling, but also processing of intracellular proteins. Together with Cx43 channels, both GJs and HCs, MMPs contribute to acute inflammation and a small number of studies reports on an MMP-Cx43 link. Here, we build further on these reports and present a novel hypothesis that describes proteolytic cleavage of the Cx43 C-terminal domain by MMPs and explores possibilities of how such cleavage events may affect Cx43 channel function. Finally, we set out how aberrant channel function resulting from cleavage can contribute to the acute inflammatory response during tissue injury.

  16. Cyclosporin A Impairs the Secretion and Activity of ADAMTS13 (A Disintegrin and Metalloprotease with Thrombospondin Type 1 Repeat)*

    Science.gov (United States)

    Hershko, Klilah; Simhadri, Vijaya L.; Blaisdell, Adam; Hunt, Ryan C.; Newell, Jordan; Tseng, Sandra C.; Hershko, Alon Y.; Choi, Jae Won; Sauna, Zuben E.; Wu, Andrew; Bram, Richard J.; Komar, Anton A.; Kimchi-Sarfaty, Chava

    2012-01-01

    The protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat) cleaves multimers of von Willebrand factor, thus regulating platelet aggregation. ADAMTS13 deficiency leads to the fatal disorder thrombotic thrombocytopenic purpura (TTP). It has been observed that cyclosporin A (CsA) treatment, particularly in transplant patients, may sometimes be linked to the development of TTP. Until now, the reason for such a link was unclear. Here we provide evidence demonstrating that cyclophilin B (CypB) activity plays an important role in the secretion of active ADAMTS13. We found that CsA, an inhibitor of CypB, reduces the secretion of ADAMTS13 and leads to conformational changes in the protein resulting in diminished ADAMTS13 proteolytic activity. A direct, functional interaction between CypB (which possesses peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone functions) and ADAMTS13 is demonstrated using immunoprecipitation and siRNA knockdown of CypB. Finally, CypB knock-out mice were found to have reduced ADAMTS13 levels. Taken together, our findings indicate that cyclophilin-mediated activity is an important factor affecting secretion and activity of ADAMTS13. The large number of proline residues in ADAMTS13 is consistent with the important role of cis-trans isomerization in the proper folding of this protein. These results altogether provide a novel mechanistic explanation for CsA-induced TTP in transplant patients. PMID:23144461

  17. Cyclosporin A impairs the secretion and activity of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat).

    Science.gov (United States)

    Hershko, Klilah; Simhadri, Vijaya L; Blaisdell, Adam; Hunt, Ryan C; Newell, Jordan; Tseng, Sandra C; Hershko, Alon Y; Choi, Jae Won; Sauna, Zuben E; Wu, Andrew; Bram, Richard J; Komar, Anton A; Kimchi-Sarfaty, Chava

    2012-12-28

    The protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat) cleaves multimers of von Willebrand factor, thus regulating platelet aggregation. ADAMTS13 deficiency leads to the fatal disorder thrombotic thrombocytopenic purpura (TTP). It has been observed that cyclosporin A (CsA) treatment, particularly in transplant patients, may sometimes be linked to the development of TTP. Until now, the reason for such a link was unclear. Here we provide evidence demonstrating that cyclophilin B (CypB) activity plays an important role in the secretion of active ADAMTS13. We found that CsA, an inhibitor of CypB, reduces the secretion of ADAMTS13 and leads to conformational changes in the protein resulting in diminished ADAMTS13 proteolytic activity. A direct, functional interaction between CypB (which possesses peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone functions) and ADAMTS13 is demonstrated using immunoprecipitation and siRNA knockdown of CypB. Finally, CypB knock-out mice were found to have reduced ADAMTS13 levels. Taken together, our findings indicate that cyclophilin-mediated activity is an important factor affecting secretion and activity of ADAMTS13. The large number of proline residues in ADAMTS13 is consistent with the important role of cis-trans isomerization in the proper folding of this protein. These results altogether provide a novel mechanistic explanation for CsA-induced TTP in transplant patients.

  18. The apoptogenic toxin AIP56 is a metalloprotease A-B toxin that cleaves NF-κb P65.

    Directory of Open Access Journals (Sweden)

    Daniela S Silva

    2013-02-01

    Full Text Available AIP56 (apoptosis-inducing protein of 56 kDa is a major virulence factor of Photobacterium damselae piscicida (Phdp, a Gram-negative pathogen that causes septicemic infections, which are among the most threatening diseases in mariculture. The toxin triggers apoptosis of host macrophages and neutrophils through a process that, in vivo, culminates with secondary necrosis of the apoptotic cells contributing to the necrotic lesions observed in the diseased animals. Here, we show that AIP56 is a NF-κB p65-cleaving zinc-metalloprotease whose catalytic activity is required for the apoptogenic effect. Most of the bacterial effectors known to target NF-κB are type III secreted effectors. In contrast, we demonstrate that AIP56 is an A-B toxin capable of acting at distance, without requiring contact of the bacteria with the target cell. We also show that the N-terminal domain cleaves NF-κB at the Cys(39-Glu(40 peptide bond and that the C-terminal domain is involved in binding and internalization into the cytosol.

  19. EXPRESS

    International Nuclear Information System (INIS)

    Ancelin, C.; Le, P.; DeSaint-Quentin, S.; Villatte, N.

    1987-01-01

    This paper presents EXPRESS, an expert system developed for the automation of reliability studies. The first part consists in the description of the method for static thermohydraulic systems. In this step, the authors define the knowledge representation based on the two inference engines - ALOUETTE and LCR developed by EDF. They explain all the process to construct a fault tree from a topological and functional description of the system. Numerous examples are exhibited in illustration of the method. This is followed by the lessons derived from the studies performed on some safety systems of the PALUEL nuclear plant. The development of the same approach for electric power systems is described, insisting on the difference resulting from the sequential nature of these systems. Finally, they show the main advantages identified during the studies

  20. Antigen Presenting Cells and Stromal Cells Trigger Human Natural Killer Lymphocytes to Autoreactivity: Evidence for the Involvement of Natural Cytotoxicity Receptors (NCR and NKG2D

    Directory of Open Access Journals (Sweden)

    Alessandro Poggi

    2006-01-01

    Full Text Available Human natural killer (NK lymphocytes should not damage autologous cells due to the engagement of inhibitory receptor superfamily (IRS members by HLA-I. Nevertheless, NK cells kill self cells expressing low levels or lacking HLA-I, as it may occur during viral infections (missing-self hypothesis. Herein, we show that human NK cells can be activated upon binding with self antigen presenting cells or stromal cells despite the expression of HLA-I. Indeed, NK cells can kill and produce pro-inflammatory and regulating cytokines as IFN-γ, TNF-α and IL10 during interaction with autologous dendritic cells or bone marrow stromal cells or skin fibroblasts. The killing of antigen presenting and stromal cells is dependent on LFA1/ICAM1 interaction. Further, the natural cytotoxicity receptors (NCR NKp30 and NKp46 are responsible for the delivery of lethal hit to DC, whereas NKG2D activating receptor, the ligand of the MHC-related molecule MIC-A and the UL16 binding protein, is involved in stromal cell killing. These findings indicate that different activating receptors are involved in cell to self cell interaction. Finally, NK cells can revert the veto effect of stromal cells on mixed lymphocyte reaction further supporting the idea that NK cells may alter the interaction between T lymphocytes and microenvironment leading to autoreactivity.

  1. Adipose-derived mesenchymal stromal cells for chronic myocardial ischemia (MyStromalCell Trial)

    DEFF Research Database (Denmark)

    Qayyum, Abbas Ali; Haack-Sørensen, Mandana; Mathiasen, Anders Bruun

    2012-01-01

    Adipose tissue represents an abundant, accessible source of multipotent adipose-derived stromal cells (ADSCs). Animal studies have suggested that ADSCs have the potential to differentiate in vivo into endothelial cells and cardiomyocytes. This makes ADSCs a promising new cell source...... for regenerative therapy to replace injured tissue by creating new blood vessels and cardiomyocytes in patients with chronic ischemic heart disease. The aim of this special report is to review the present preclinical data leading to clinical stem cell therapy using ADSCs in patients with ischemic heart disease....... In addition, we give an introduction to the first-in-man clinical trial, MyStromalCell Trial, which is a prospective, randomized, double-blind, placebo-controlled study using culture-expanded ADSCs obtained from adipose-derived cells from abdominal adipose tissue and stimulated with VEGF-A(165) the week...

  2. Sex cord-gonadal stromal tumor of the rete testis.

    Science.gov (United States)

    Sajadi, Kamran P; Dalton, Rory R; Brown, James A

    2009-01-01

    A 34-year-old tetraplegic patient with suppurative epididymitis was found on follow-up examination and ultrasonography to have a testicular mass. The radical orchiectomy specimen contained an undifferentiated spindled sex cord-stromal tumor arising in the rete testis. Testicular sex cord-stromal tumors are far less common than germ cell neoplasms and are usually benign. The close relationship between sex cords and ductules of the rete testis during development provides the opportunity for these uncommon tumors to arise anatomically within the rete tesis. This undifferentiated sex cord-stromal tumor, occurring in a previously unreported location, is an example of an unusual lesion mimicking an intratesticular malignant neoplasm.

  3. The Significance of the Stromal Response in Breast Cancer: An Immunohistochemical Study of Myofibroblasts in Primary and Metastatic Breast Cancer.

    Science.gov (United States)

    Roozdar, Alale; Hayes, Malcolm M; Pourseyedei, Bahram; Zeinalinejad, Hamid; Shamsi Meymandi, Manzumeh; Dabiri, Bahram; Dabiri, Shahriar

    2018-05-01

    Gene expression profiling of breast cancer has demonstrated the importance of stromal response in determining the prognosis of invasive breast cancer. The host response to breast cancer is of increasing interest to pathologists and may be a future focus for novel pharmacological treatments. This study describes the pattern of distribution of stromal myofibroblasts using immunostains for CD10 and smooth muscle actin (SMA) in 50 primary breast cancers and their matched nodal metastases (68.6% nodes positive and 31.4% nodes negative). The stroma within the tumor (intratumoral) and at the advancing tumor edge (peri-tumoral) was studied in both primary and nodal sites. A simple quantitative scoring system was employed for both immunostains. The correlation between expression of these markers by stromal cells and standard pathological prognostic factors of stage, grade, hormone receptor and Her-2 status was analysed. SMA-positive stromal cells were more abundant in peri-tumoral stroma compared with intratumoral stroma in both primary and metastatic lesions. SMA expression in the lymph node metastases showed a significant correlation with tumor stage. SMA expression in peri-tumoral stroma correlated with Her-2 status. The results of this study suggest that myofibroblasts, particularly those expressing SMA, might potentiate the progression of the carcinomatous process especially in nodal metastases. Thus these cells may be a potential therapeutic target. © 2018 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

  4. Cryopreservation and revival of mesenchymal stromal cells

    DEFF Research Database (Denmark)

    Haack-Sørensen, Mandana; Kastrup, Jens

    2011-01-01

    initiated. As there has been a precedent for the use of bone marrow stem cells in the treatment of hematological malignancies and ischemic heart diseases through randomized clinical safety and efficacy trials, the development of new therapies based on culture-expanded human mesenchymal stromal cells (MSCs......Over the past few years, the pace of preclinical stem cell research is astonishing and adult stem cells have become the subject of intense research. Due to the presence of promising supporting preclinical data, human clinical trials for stem cell regenerative treatment of various diseases have been......) opens up new possibilities for cell therapy. To facilitate these applications, cryopreservation and long-term storage of MSCs becomes an absolute necessity. As a result, optimization of this cryopreservation protocol is absolutely critical. The major challenge during cellular cryopreservation...

  5. Characteristics of Emergency Gastrointestinal Stromal Tumor (GIST).

    Science.gov (United States)

    Uçar, Ahmet Deniz; Oymaci, Erkan; Carti, Erdem Bariş; Yakan, Savaş; Vardar, Enver; Erkan, Nazif; Mehmet, Yildirim

    2015-05-01

    Gastrointestinal Stromal Tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract (GIT). Importance of GISTs is increasing while surgeons are facing with more frequent either in emergency setting of elective cases. Delineating the presentation and management of emergency GIST is important. From 2005 to 2014, emergency cases with final diagnosis of GIST were examined retrospectively. Total of 13 operated cases were evaluated by patients characteristics, clinical presentation, operational findings and postoperative prognosis. There were 9 male and 4 female with the mean age of 48.15 years. The most frequent presentations are ileus and GIT hemorrhage both covering the 84% of patients. Small bowel was the dominating site with ileus. Stomach was the second frequent site of the disease with the finding of hemorrhage. Emergency patients are more likely to come with small bowel GIST and obstruction symptoms. Hemorrhage is the most frequent symptom for emergency GIST of stomach and duodenum.

  6. Update on gastrointestinal stromal tumors for radiologists

    Energy Technology Data Exchange (ETDEWEB)

    Tirumani, Sree Harsha; O' Neill, Alibhe; Jagannathan, Jyothi P. [Dept. of Imaging, Dana-Farber Cancer Institute, Boston (United States); Baheti, Akahay D. [Dept. of Radiology, Tata Memorial Centre, Mumbai (India); Tirumani, Harika [Dept. of Radiology, University of Arkansas for Medical Sciences, Little Rock (United States)

    2017-01-15

    The management of gastrointestinal stromal tumors (GISTs) has evolved significantly in the last two decades due to better understanding of their biologic behavior as well as development of molecular targeted therapies. GISTs with exon 11 mutation respond to imatinib whereas GISTs with exon 9 or succinate dehydrogenase subunit mutations do not. Risk stratification models have enabled stratifying GISTs according to risk of recurrence and choosing patients who may benefit from adjuvant therapy. Assessing response to targeted therapies in GIST using conventional response criteria has several potential pitfalls leading to search for alternate response criteria based on changes in tumor attenuation, volume, metabolic and functional parameters. Surveillance of patients with GIST in the adjuvant setting is important for timely detection of recurrences.

  7. Imaging of gastrointestinal stromal tumour (GIST)

    International Nuclear Information System (INIS)

    Lau, S.; Tam, K.F.; Kam, C.K.; Lui, C.Y.; Siu, C.W.; Lam, H.S.; Mak, K.L.

    2004-01-01

    Gastrointestinal stromal tumour (GIST) represents the most common kind of mesenchymal tumour that arises from the alimentary tract. GIST is currently defined as a gastrointestinal tract mesenchymal tumour containing spindle cells (or less commonly epithelioid cells or rarely both) and showing CD117 (c-kit protein) positivity. Targeted molecular therapy of non-resectable GIST using imatinib, a specific tyrosine kinase receptor inhibitor, represents a real milestone in the management of solid malignancy. Imaging studies, both anatomical and functional, are playing an increasingly important role in management of patients with GIST. This review illustrates the radiological appearance of GISTs and the site-specific roles of each imaging tool. Clinical features and radiological differential diagnosis of GIST are also discussed

  8. Ectopic Overexpression of Sonic Hedgehog (Shh Induces Stromal Expansion and Metaplasia in the Adult Murine Pancreas

    Directory of Open Access Journals (Sweden)

    Volker Fendrich

    2011-10-01

    Full Text Available Ligand-dependent activation of the Hedgehog (Hh signaling pathway has been implicated in both tumor initiation and metastasis of pancreatic ductal adenocarcinoma (PDAC. Prior studies in genetically engineered mouse models (GEMMs have assessed the role of Hh signaling by cell autonomous expression of a constitutively active Gli2 within epithelial cells. On the contrary, aberrant pathway reactivation in the human exocrine pancreas occurs principally as a consequence of Sonic Hh ligand (Shh overexpression from epithelial cells. To recapitulate the cognate pathophysiology of Hh signaling observed in the human pancreas, we examined GEMM where Hh ligand is conditionally overexpressed within the mature exocrine pancreas using a tamoxifen-inducible Elastase-Cre promoter (Ela-CreERT2;LSL-mShh. We also facilitated potential cell autonomous epithelial responsiveness to secreted Hh ligand by generating compound transgenic mice with concomitant expression of the Hh receptor Smoothened (Ela-CreERT2;LSL-mShh;LSL-mSmo. Of interest, none of these mice developed intraductal precursor lesions or PDAC during the follow-up period of up to 12 months after tamoxifen induction. Instead, all animals demonstrated marked expansion of stromal cells, consistent with the previously described epithelial-to-stromal paracrine Hh signaling. Hh responsiveness was mirrored by the expression of primary cilia within the expanded mesenchymal compartment and the absence within mature acinar cells. In the absence of cooperating mutations, Hh ligand overexpression in the mature exocrine pancreas is insufficient to induce neoplasia, even when epithelial cells coexpress the Smo receptor. This autochthonous model serves as a platform for studying epithelial stromal interactions in pancreatic carcinogenesis.

  9. LIF Mediates Proinvasive Activation of Stromal Fibroblasts in Cancer

    Directory of Open Access Journals (Sweden)

    Jean Albrengues

    2014-06-01

    Full Text Available Signaling crosstalk between tumor cells and fibroblasts confers proinvasive properties to the tumor microenvironment. Here, we identify leukemia inhibitory factor (LIF as a tumor promoter that mediates proinvasive activation of stromal fibroblasts independent of alpha-smooth muscle actin (α-SMA expression. We demonstrate that a pulse of transforming growth factor β (TGF-β establishes stable proinvasive fibroblast activation by inducing LIF production in both fibroblasts and tumor cells. In fibroblasts, LIF mediates TGF-β-dependent actomyosin contractility and extracellular matrix remodeling, which results in collective carcinoma cell invasion in vitro and in vivo. Accordingly, carcinomas from multiple origins and melanomas display strong LIF upregulation, which correlates with dense collagen fiber organization, cancer cell collective invasion, and poor clinical outcome. Blockade of JAK activity by Ruxolitinib (JAK inhibitor counteracts fibroblast-dependent carcinoma cell invasion in vitro and in vivo. These findings establish LIF as a proinvasive fibroblast producer independent of α-SMA and may open novel therapeutic perspectives for patients with aggressive primary tumors.

  10. Fetal liver stromal cells promote hematopoietic cell expansion

    International Nuclear Information System (INIS)

    Zhou, Kun; Hu, Caihong; Zhou, Zhigang; Huang, Lifang; Liu, Wenli; Sun, Hanying

    2009-01-01

    Future application of hematopoietic stem and progenitor cells (HSPCs) in clinical therapies largely depends on their successful expansion in vitro. Fetal liver (FL) is a unique hematopoietic organ in which hematopoietic cells markedly expand in number, but the mechanisms involved remain unclear. Stromal cells (StroCs) have been suggested to provide a suitable cellular environment for in vitro expansion of HSPCs. In this study, murine StroCs derived from FL at E14.5, with a high level of Sonic hedgehog (Shh) and Wnt expression, were found to have an increased ability to support the proliferation of HSPCs. This effect was inhibited by blocking Shh signaling. Supplementation with soluble Shh-N promoted the proliferation of hematopoietic cells by activating Wnt signaling. Our findings suggest that FL-derived StroCs support proliferation of HSPCs via Shh inducing an autocrine Wnt signaling loop. The use of FL-derived StroCs and regulation of the Shh pathway might further enhance HPSC expansion.

  11. Actin depolymerization enhances adipogenic differentiation in human stromal stem cells.

    Science.gov (United States)

    Chen, Li; Hu, Huimin; Qiu, Weimin; Shi, Kaikai; Kassem, Moustapha

    2018-05-01

    Human stromal stem cells (hMSCs) differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined the effect of direct modulation of actin microfilament dynamics on adipocyte differentiation. Stabilizing actin filaments in hMSCs by siRNA-mediated knock down of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) or treating the cells by Phalloidin reduced adipocyte differentiation as evidenced by decreased number of mature adipocytes and decreased adipocyte specific gene expression (ADIPOQ, LPL, PPARG, FABP4). In contrast, disruption of actin cytoskeleton by Cytochalasin D enhanced adipocyte differentiation. Follow up studies revealed that the effects of CFL1 on adipocyte differentiation depended on the activity of LIM domain kinase 1 (LIMK1) which is the major upstream kinase of CFL1. Inhibiting LIMK by its specific chemical inhibitor LIMKi inhibited the phosphorylation of CFL1 and actin polymerization, and enhanced the adipocyte differentiation. Moreover, treating hMSCs by Cytochalasin D inhibited ERK and Smad2 signaling and this was associated with enhanced adipocyte differentiation. On the other hand, Phalloidin enhanced ERK and Smad2 signaling, but inhibited adipocyte differentiation which was rescued by ERK specific chemical inhibitor U0126. Our data provide a link between restructuring of hMSCs cytoskeleton and hMSCs lineage commitment and differentiation. Copyright © 2018 Elsevier B.V. All rights reserved.

  12. Signal transduction around thymic stromal lymphopoietin (TSLP in atopic asthma

    Directory of Open Access Journals (Sweden)

    Kuepper Michael

    2008-08-01

    Full Text Available Abstract Thymic stromal lymphopoietin (TSLP, a novel interleukin-7-like cytokine, triggers dendritic cell-mediated inflammatory responses ultimately executed by T helper cells of the Th2 subtype. TSLP emerged as a central player in the development of allergic symptoms, especially in the airways, and is a prime regulatory cytokine at the interface of virus- or antigen-exposed epithelial cells and dendritic cells (DCs. DCs activated by epithelium-derived TSLP can promote naïve CD4+ T cells to adopt a Th2 phenotype, which in turn recruite eosinophilic and basophilic granulocytes as well as mast cells into the airway mucosa. These different cells secrete inflammatory cytokines and chemokines operative in inducing an allergic inflammation and atopic asthma. TSLP is, thus, involved in the control of both an innate and an adaptive immune response. Since TSLP links contact of allergen with the airway epithelium to the onset and maintainance of the asthmatic syndrome, defining the signal transduction underlying TSLP expression and function is of profound interest for a better understandimg of the disease and for the development of new therapeutics.

  13. Osterix enhances proliferation and osteogenic potential of bone marrow stromal cells

    International Nuclear Information System (INIS)

    Tu Qisheng; Valverde, Paloma; Chen, Jake

    2006-01-01

    Osterix (Osx) is a zinc-finger-containing transcription factor that is expressed in osteoblasts of all endochondral and membranous bones. In Osx null mice osteoblast differentiation is impaired and bone formation is absent. In this study, we hypothesized that overexpression of Osx in murine bone marrow stromal cells (BMSC) would be able to enhance their osteoblastic differentiation and mineralization in vitro. Retroviral transduction of Osx in BMSC cultured in non-differentiating medium did not affect expression of Runx2/Cbfa1, another key transcription factor of osteoblast differentiation, but induced an increase in the expression of other markers associated with the osteoblastic lineage including alkaline phosphatase, bone sialoprotein, osteocalcin, and osteopontin. Retroviral transduction of Osx in BMSC also increased their proliferation, alkaline phosphatase activity, and ability to form bone nodules. These events occurred without significant changes in the expression of α1(II) procollagen or lipoprotein lipase, which are markers of chondrogenic and adipogenic differentiation, respectively

  14. Stromal infrastructure of the lymph node and coordination of immunity.

    Science.gov (United States)

    Chang, Jonathan E; Turley, Shannon J

    2015-01-01

    The initiation of adaptive immune responses depends upon the careful maneuvering of lymphocytes and antigen into and within strategically placed lymph nodes (LNs). Non-hematopoietic stromal cells form the cellular infrastructure that directs this process. Once regarded as merely structural features of lymphoid tissues, these cells are now appreciated as essential regulators of immune cell trafficking, fluid flow, and LN homeostasis. Recent advances in the identification and in vivo targeting of specific stromal populations have resulted in striking new insights to the function of stromal cells and reveal a level of complexity previously unrealized. We discuss here recent discoveries that highlight the pivotal role that stromal cells play in orchestrating immune cell homeostasis and adaptive immunity. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Intestinal stromal cells in mucosal immunity and homeostasis.

    Science.gov (United States)

    Owens, B M J; Simmons, A

    2013-03-01

    A growing body of evidence suggests that non-hematopoietic stromal cells of the intestine have multiple roles in immune responses and inflammation at this mucosal site. Despite this, many still consider gut stromal cells as passive structural entities, with past research focused heavily on their roles in fibrosis, tumor progression, and wound healing, rather than their contributions to immune function. In this review, we discuss our current knowledge of stromal cells in intestinal immunity, highlighting the many immunological axes in which stromal cells have a functional role. We also consider emerging data that broaden the potential scope of their contribution to immunity in the gut and argue that these so-called "non-immune" cells are reclassified in light of their diverse contributions to intestinal innate immunity and the maintenance of mucosal homeostasis.

  16. Management of hemorrhage in gastrointestinal stromal tumors: a review.

    Science.gov (United States)

    Liu, Qi; Kong, Fanmin; Zhou, Jianping; Dong, Ming; Dong, Qi

    2018-01-01

    Gastrointestinal stromal tumors (GISTs) are relatively common mesenchymal tumors. They originate from the wall of hollow viscera and may be found in any part of the digestive tract. The prognosis of patients with stromal tumors depends on various risk factors, including size, location, presence of mitotic figures, and tumor rupture. Emergency surgery is often required for stromal tumors with hemorrhage. The current literature suggests that stromal tumor hemorrhage indicates poor prognosis. Although the optimal treatment options for hemorrhagic GISTs are based on surgical experience, there remains controversy with regard to optimum postoperative management as well as the classification of malignant potential. This article reviews the biological characteristics, diagnostic features, prognostic factors, treatment, and postoperative management of GISTs with hemorrhage.

  17. Engineering epithelial-stromal interactions in vitro for toxicology assessment

    Science.gov (United States)

    Background: Crosstalk between epithelial and stromal cells drives the morphogenesis of ectodermal organs during development and promotes normal mature adult epithelial tissue function. Epithelial-mesenchymal interactions (EMIs) have been examined using mammalian models, ex vivo t...

  18. Cryopreservation and revival of human mesenchymal stromal cells

    DEFF Research Database (Denmark)

    Haack-Sørensen, Mandana; Ekblond, Annette; Kastrup, Jens

    2016-01-01

    Cell-based therapy is a promising and innovative new treatment for different degenerative and autoimmune diseases, and mesenchymal stromal cells (MSCs) from the bone marrow have demonstrated great therapeutic potential due to their immunosuppressive and regenerative capacities. The establishment ...

  19. Complex regulation of AprA metalloprotease in Pseudomonas fluorescens M114: evidence for the involvement of iron, the ECF sigma factor, PbrA and pseudobactin M114 siderophore.

    Science.gov (United States)

    Maunsell, Bláithín; Adams, Claire; O'Gara, Fergal

    2006-01-01

    In the soil bacterium Pseudomonas fluorescens M114, extracellular proteolytic activity and fluorescent siderophore (pseudobactin M114) production were previously shown to be co-ordinately negatively regulated in response to environmental iron levels. An iron-starvation extracytoplasmic function sigma factor, PbrA, required for the transcription of siderophore biosynthetic genes, was also implicated in M114 protease regulation. The current study centred on the characterization and genetic regulation of the gene(s) responsible for protease production in M114. A serralysin-type metalloprotease gene, aprA, was identified and found to encode the major, if not only, extracellular protease produced by this strain. The expression of aprA and its protein product were found to be subject to complex regulation. Transcription analysis confirmed that PbrA was required for full aprA transcription under low iron conditions, while the ferric uptake regulator, Fur, was implicated in aprA repression under high iron conditions. Interestingly, the iron regulation of AprA was dependent on culture conditions, with PbrA-independent AprA-mediated proteolytic activity observed on skim milk agar supplemented with yeast extract, when supplied with iron or purified pseudobactin M114. These effects were not observed on skim milk agar without yeast extract. PbrA-independent aprA expression was also observed from a truncated transcriptional fusion when grown in sucrose asparagine tryptone broth supplied with iron or purified pseudobactin M114. Thus, experimental evidence suggested that iron mediated its effects via transcriptional activation by PbrA under low iron conditions, while an as-yet-unidentified sigma factor(s) may be required for the PbrA-independent aprA expression and AprA proteolytic activity induced by siderophore and iron.

  20. MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo

    DEFF Research Database (Denmark)

    Eskildsen, Tilde; Taipaleenmäki, Hanna; Stenvang, Jan

    2011-01-01

    Elucidating the molecular mechanisms that regulate human stromal (mesenchymal) stem cell (hMSC) differentiation into osteogenic lineage is important for the development of anabolic therapies for treatment of osteoporosis. MicroRNAs (miRNAs) are short, noncoding RNAs that act as key regulators......-regulated during osteoblast differentiation of hMSCs. Overexpression of miR-138 inhibited osteoblast differentiation of hMSCs in vitro, whereas inhibition of miR-138 function by antimiR-138 promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Furthermore...

  1. A simple and efficient method for deriving neurospheres from bone marrow stromal cells

    International Nuclear Information System (INIS)

    Yang Qin; Mu Jun; Li Qi; Li Ao; Zeng Zhilei; Yang Jun; Zhang Xiaodong; Tang Jin; Xie Peng

    2008-01-01

    Bone marrow stromal cells (MSCs) can be differentiated into neuronal and glial-like cell types under appropriate experimental conditions. However, previously reported methods are complicated and involve the use of toxic reagents. Here, we present a simplified and nontoxic method for efficient conversion of rat MSCs into neurospheres that express the neuroectodermal marker nestin. These neurospheres can proliferate and differentiate into neuron, astrocyte, and oligodendrocyte phenotypes. We thus propose that MSCs are an emerging model cell for the treatment of a variety of neurological diseases

  2. DHT and testosterone, but not DHEA or E2, differentially modulate IGF-I, IGFBP-2, and IGFBP-3 in human prostatic stromal cells.

    Science.gov (United States)

    Le, Hanh; Arnold, Julia T; McFann, Kimberly K; Blackman, Marc R

    2006-05-01

    Prostate cancer is one of the four most common cancers in the United States, affecting one of six men. Increased serum levels of androgens and IGF-I are associated with an augmented risk of prostate cancer. Dihydrotestosterone (DHT) and testosterone (T) stimulate prostate cancer cell growth, development, and function, whereas the effects of DHT and T in prostate stromal cells, and of dehydroepiandrosterone (DHEA) in prostate cancer or stromal cells, are uncertain. We investigated the actions of DHT, T, DHEA, and estradiol (E2) on insulin-like growth factor (IGF)-I, IGF-II, IGF-I receptor (R), IGF-binding protein (IGFBP)-2, IGFBP-3, and IGFBP-5 in primary cultures of human prostatic stromal cells by assessing cell proliferation, mRNA expression, and protein secretion by MTT growth assay, quantitative real-time PCR, and ELISA, respectively. DHT and T each increased IGF-I (7-fold) and decreased IGFBP-3 (2-fold) mRNA expression and protein secretion in a dose- and time-dependent manner and increased IGFBP-2 (2-fold) mRNA in a dose- and time-dependent manner. DHEA and E2 did not significantly alter these measures. Flutamide abolished the DHT-modulated increases in IGF-I and IGFBP-2, suggesting that the influences of DHT and T on these measures were androgen receptor mediated. None of the four steroids significantly affected IGF-IR, IGF-II, or IGFBP-5 mRNA levels or stromal cell proliferation. The effects of DHT on IGF-I, IGFBP-2, and IGFBP-3 were more pronounced in stromal cultures that did not express desmin. These data suggest that DHT and T promote prostate growth partly via modulation of the stromal cell IGF axis, with potential paracrine effects on prostate epithelial cells.

  3. Seminal plasma induces global transcriptomic changes associated with cell migration, proliferation and viability in endometrial epithelial cells and stromal fibroblasts

    OpenAIRE

    Chen, Joseph C.; Johnson, Brittni A.; Erikson, David W.; Piltonen, Terhi T.; Barragan, Fatima; Chu, Simon; Kohgadai, Nargis; Irwin, Juan C.; Greene, Warner C.; Giudice, Linda C.; Roan, Nadia R.

    2014-01-01

    STUDY QUESTION How does seminal plasma (SP) affect the transcriptome of human primary endometrial epithelial cells (eEC) and stromal fibroblasts (eSF)? SUMMARY ANSWER Exposure of eEC and eSF to SP in vitro increases expression of genes and secreted proteins associated with cellular migration, proliferation, viability and inhibition of cell death. WHAT IS KNOWN ALREADY Studies in both humans and animals suggest that SP can access and induce physiological changes in the upper female reproductiv...

  4. The Stromal Contribution to the Development of Resistance to New-Generation Drugs by Castration-Resistant Prostate Cancer

    Science.gov (United States)

    2015-10-01

    abiraterone treatment by further increasing their expressions of steroidogenic genes and by further increasing their outputs of T and DHT . Finally, we...support (stromal) cells within a bone metastasis have a supportive role in the development of CRPC through their ability to synthesize T and DHT upon Hh...dihydrotestosterone ( DHT ) (3, 9-11). 4 After ADT, recurrence as castration-resistant disease is common (8). For PCa, the most common site for metastasis is bone

  5. Tissue non-specific alkaline phosphatase production by human dental pulp stromal cells is enhanced by high density cell culture.

    Science.gov (United States)

    Tomlinson, Matthew J; Dennis, Caitriona; Yang, Xuebin B; Kirkham, Jennifer

    2015-08-01

    The cell surface hydrolase tissue non-specific alkaline phosphatase (TNAP) (also known as MSCA-1) is used to identify a sub-population of bone marrow stromal cells (BMSCs) with high mineralising potential and is found on subsets of cells within the dental pulp. We aim to determine whether TNAP is co-expressed by human dental pulp stromal cells (hDPSCs) alongside a range of BMSC markers, whether this is an active form of the enzyme and the effects of culture duration and cell density on its expression. Cells from primary dental pulp and culture expanded hDPSCs expressed TNAP. Subsequent analyses revealed persistent TNAP expression and co-expression with BMSC markers such as CD73 and CD90. Flow cytometry and biochemical assays showed that increased culture durations and cell densities enhanced TNAP expression by hDPSCs. Arresting the hDPSC cell cycle also increased TNAP expression. These data confirm that TNAP is co-expressed by hDPSCs together with other BMSC markers and show that cell density affects TNAP expression levels. We conclude that TNAP is a potentially useful marker for hDPSC selection especially for uses in mineralised tissue regenerative therapies.

  6. Normal endometrial stromal cells regulate 17β-estradiol-induced epithelial-mesenchymal transition via slug and E-cadherin in endometrial adenocarcinoma cells in vitro.

    Science.gov (United States)

    Zhang, Hui; Li, Hongyan; Qi, Shasha; Liu, Zhao; Fu, Yibing; Li, Mingjiang; Zhao, Xingbo

    2017-01-01

    Stroma-tumor communication participates in the pathogenesis of endometrial carcinomas. In previous studies, we found that normal stromal cells inhibited the growth of endometrial carcinoma cells. Here, we investigated the role of normal stromal cells in the epithelial-mesenchymal transition (EMT) of endometrial carcinoma cells and explored the possible mechanism implied. We found that conditioned medium (CM) by normal endometrial stromal cells (NSC) reduced cell growth and induced cell apoptosis in Ishikawa cells. CM by NSC inhibited 17β-estradiol-induced cell growth and apoptosis decrease in Ishikawa cells. Moreover, CM by NSC inhibited the migration and invasion, and 17β-estradiol-induced migration and invasion in Ishikawa cells. Meanwhile, CM by NSC decreased Slug expression and 17β-estradiol-induced Slug expression, increased E-cadherin expression and abolished 17β-estradiol-induced E-cadherin reduction in Ishikawa cells. In conclusion, normal stromal factors can inhibit 17β-estradiol-induced cell proliferation and apoptosis inhibition, and abolished 17β-estradiol-induced EMT in endometrial cancer cell via regulating E-cadherin and Slug expression.

  7. Activation of non-canonical Wnt/JNK pathway by Wnt3a is associated with differentiation fate determination of human bone marrow stromal (mesenchymal) stem cells

    DEFF Research Database (Denmark)

    Qiu, Weimin; Chen, Li; Kassem, Moustapha

    2011-01-01

    The canonical Wnt signaling pathway can determine human bone marrow stromal (mesenchymal) stem cell (hMSC) differentiation fate into osteoblast or adipocyte lineages. However, its downstream targets in MSC are not well characterized. Thus, using DNA microarrays, we compared global gene expression...

  8. Modulation of hemopoiesis by novel stromal cell factors

    International Nuclear Information System (INIS)

    Zipori, D.

    1988-01-01

    The microenvironment of the bone marrow in mammals is a crucial site for the maintenance of a pluripotent hemopoietic stem cell pool. Our previous studies and present findings support the notion that both this function and the fine architecture of hemopoietic organs, i.e., the spatial arrangement of blood cells within the tissue, may be directed by stromal cells. Despite the ability of cloned stromal cells to support prolonged hempoiesis and maintenance in vitro of stem cells with high radioprotective ability, they are a poor source of colony stimulating factor-1 (CSF-1) and do not secrete the other species of CSF. Furthermore, cultured stromal cells antagonize the activity of CSF. It is proposed that stromal cell factors distinct from known CSFs, regulate stem cell renewal. An additional phenomenon that is mediated by stromal cells and can not be attributed to CSF, is their ability to specifically inhibit the accumulation of cells of particular lineage and stage of differentiation. A glycoprotein that inhibits the growth of plasmacytomas but not a variety of other cell types was isolated from one type of cloned stromal cells. Such specific inhibitors may account for the control of cell localization in the hemopoietic system

  9. Insecticidal activity of the metalloprotease AprA occurs through suppression of host cellular and humoral immunity.

    Science.gov (United States)

    Lee, Seung Ah; Jang, Seong Han; Kim, Byung Hyun; Shibata, Toshio; Yoo, Jinwook; Jung, Yunjin; Kawabata, Shun-Ichiro; Lee, Bok Luel

    2018-04-01

    The biochemical characterization of virulence factors from entomopathogenic bacteria is important to understand entomopathogen-insect molecular interactions. Pseudomonas entomophila is a typical entomopathogenic bacterium that harbors virulence factors against several insects. However, the molecular actions of these factors against host innate immune responses are not clearly elucidated. In this study, we observed that bean bugs (Riptortus pedestris) that were injected with P. entomophila were highly susceptible to this bacterium. To determine how P. entomophila counteracts the host innate immunity to survive within the insect, we purified a highly enriched protein with potential host insect-killing activity from the culture supernatant of P. entomophila. Then, a 45-kDa protein was purified to homogeneity and identified as AprA which is an alkaline zinc metalloprotease of the genus Pseudomonas by liquid chromatography mass spectrometry (LC-MS). Purified AprA showed a pronounced killing effect against host insects and suppressed both host cellular and humoral innate immunity. Furthermore, to show that AprA is an important insecticidal protein of P. entomophila, we used an aprA-deficient P. entomophila mutant strain (ΔaprA). When ΔaprA mutant cells were injected to host insects, this mutant exhibited extremely attenuated virulence. In addition, the cytotoxicity against host hemocytes and the antimicrobial peptide-degrading ability of the ΔaprA mutant were greatly decreased. These findings suggest that AprA functions as an important insecticidal protein of P. entomophila via suppression of host cellular and humoral innate immune responses. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Matrix metalloproteases as maestros for the dual role of LPS- and IL-10-stimulated macrophages in cancer cell behaviour

    International Nuclear Information System (INIS)

    Cardoso, Ana P.; Pinto, Marta L.; Pinto, Ana T.; Pinto, Marta T.; Monteiro, Cátia; Oliveira, Marta I.; Santos, Susana G.; Relvas, João B.; Seruca, Raquel; Mantovani, Alberto; Mareel, Marc; Barbosa, Mário A.; Oliveira, Maria J.

    2015-01-01

    The interactions established between macrophages and cancer cells are largely dependent on instructions from the tumour microenvironment. Macrophages may differentiate into populations with distinct inflammatory profiles, but knowledge on their role on cancer cell activities is still very scarce. In this work, we investigated the influence of pro-inflammatory (LPS-stimulated) and anti-inflammatory (IL-10-stimulated) macrophages on gastric and colorectal cancer cell invasion, motility/migration, angiogenesis and proteolysis, and the associated molecular mechanisms. Following exposure of gastric and colon cancer cell lines to LPS- and IL-10-stimulated human macrophages, either by indirect contact or conditioned media, we analyzed the effect of the different macrophage populations on cancer cell invasion, migration, motility and phosphorylation status of EGFR and several interacting partners. Cancer-cell induced angiogenesis upon the influence of conditioned media from both macrophage populations was assessed using the chick embryo chorioallantoic membrane assay. MMP activities were evaluated by gelatin zymograhy. Our results show that IL-10-stimulated macrophages are more efficient in promoting in vitro cancer cell invasion and migration. In addition, soluble factors produced by these macrophages enhanced in vivo cancer cell-induced angiogenesis, as opposed to their LPS-stimulated counterparts. We further demonstrate that differences in the ability of these macrophage populations to stimulate invasion or angiogenesis cannot be explained by the EGFR-mediated signalling, since both LPS- and IL-10-stimulated macrophages similarly induce the phosphorylation of cancer cell EGFR, c-Src, Akt, ERK1/2, and p38. Interestingly, both populations exert distinct proteolytic activities, being the IL-10-stimulated macrophages the most efficient in inducing matrix metalloprotease (MMP)-2 and MMP-9 activities. Using a broad-spectrum MMP inhibitor, we demonstrated that proteolysis was

  11. Ex Vivo and In Vivo Imaging and Biodistribution of Aptamers Targeting the Human Matrix MetalloProtease-9 in Melanomas.

    Directory of Open Access Journals (Sweden)

    David Kryza

    Full Text Available The human Matrix MetalloProtease-9 (hMMP-9 is overexpressed in tumors where it promotes the release of cancer cells thus contributing to tumor metastasis. We raised aptamers against hMMP-9, which constitutes a validated marker of malignant tumors, in order to design probes for imaging tumors in human beings. A chemically modified RNA aptamer (F3B, fully resistant to nucleases was previously described. This compound was subsequently used for the preparation of F3B-Cy5, F3B-S-acetylmercaptoacetyltriglycine (MAG and F3B-DOTA. The binding properties of these derivatives were determined by surface plasmon resonance and electrophoretic mobility shift assay. Optical fluorescence imaging confirmed the binding to hMMP-9 in A375 melanoma bearing mice. Quantitative biodistribution studies were performed at 30 min, 1h and 2 h post injection of 99mTc-MAG-aptamer and 111In-DOTA-F3B. 99mTc radiolabeled aptamer specifically detected hMMP-9 in A375 melanoma tumors but accumulation in digestive tract was very high. Following i.v. injection of 111In-DOTA-F3B, high level of radioactivity was observed in kidneys and bladder but digestive tract uptake was very limited. Tumor uptake was significantly (student t test, p<0.05 higher for 111In-DOTA-F3B with 2.0%ID/g than for the 111In-DOTA-control oligonucleotide (0.7%ID/g with tumor to muscle ratio of 4.0. Such difference in tumor accumulation has been confirmed by ex vivo scintigraphic images performed at 1h post injection and by autoradiography, which revealed the overexpression of hMMP-9 in sections of human melanomas. These results demonstrate that F3B aptamer is of interest for detecting hMMP-9 in melanoma tumor.

  12. Canonical hedgehog signaling augments tumor angiogenesis by induction of VEGF-A in stromal perivascular cells

    Science.gov (United States)

    Chen, Weiwei; Tang, Tracy; Eastham-Anderson, Jeff; Dunlap, Debra; Alicke, Bruno; Nannini, Michelle; Gould, Stephen; Yauch, Robert; Modrusan, Zora; DuPree, Kelly J.; Darbonne, Walter C.; Plowman, Greg; de Sauvage, Frederic J.; Callahan, Christopher A.

    2011-01-01

    Hedgehog (Hh) signaling is critical to the patterning and development of a variety of organ systems, and both ligand-dependent and ligand-independent Hh pathway activation are known to promote tumorigenesis. Recent studies have shown that in tumors promoted by Hh ligands, activation occurs within the stromal microenvironment. Testing whether ligand-driven Hh signaling promotes tumor angiogenesis, we found that Hh antagonism reduced the vascular density of Hh-producing LS180 and SW480 xenografts. In addition, ectopic expression of sonic hedgehog in low-Hh–expressing DLD-1 xenografts increased tumor vascular density, augmented angiogenesis, and was associated with canonical Hh signaling within perivascular tumor stromal cells. To better understand the molecular mechanisms underlying Hh-mediated tumor angiogenesis, we established an Hh-sensitive angiogenesis coculture assay and found that fibroblast cell lines derived from a variety of human tissues were Hh responsive and promoted angiogenesis in vitro through a secreted paracrine signal(s). Affymetrix array analyses of cultured fibroblasts identified VEGF-A, hepatocyte growth factor, and PDGF-C as candidate secreted proangiogenic factors induced by Hh stimulation. Expression studies of xenografts and angiogenesis assays using combinations of Hh and VEGF-A inhibitors showed that it is primarily Hh-induced VEGF-A that promotes angiogenesis in vitro and augments tumor-derived VEGF to promote angiogenesis in vivo. PMID:21597001

  13. Hypoxic stress simultaneously stimulates vascular endothelial growth factor via hypoxia-inducible factor-1α and inhibits stromal cell-derived factor-1 in human endometrial stromal cells.

    Science.gov (United States)

    Tsuzuki, Tomoko; Okada, Hidetaka; Cho, Hisayuu; Tsuji, Shoko; Nishigaki, Akemi; Yasuda, Katsuhiko; Kanzaki, Hideharu

    2012-02-01

    Hypoxia of the human endometrium is a physiologic event occurring during the perimenstrual period and the local stimulus for angiogenesis. The aim of this study was to investigate the effects of hypoxic stress on the regulation of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1/CXCL12), and the potential role of hypoxia-inducible factor-1α (HIF-1α) in the endometrium. Human endometrial stromal cells (ESCs, n= 22 samples) were studied in vitro. ESCs were cultured under hypoxic and normoxic conditions and treated with cobalt chloride (CoCl₂; a hypoxia-mimicking agent) and/or echinomycin, a small-molecule inhibitor of HIF-1α activity. The mRNA levels and production of VEGF and SDF-1 were assessed by real-time PCR and ELISA, respectively. The HIF-1α protein levels were measured using western blot analysis. Hypoxia simultaneously induced the expression of mRNA and production of VEGF and attenuated the expression and production of SDF-1 from ESCs in a time-dependent manner. Similar changes were observed in the ESCs after stimulation with CoCl₂ in a dose-dependent manner. CoCl₂ significantly induced the expression of HIF-1α protein, and its highest expression was observed at 6 h. Echinomycin inhibited hypoxia-induced VEGF production without affecting the HIF-1α protein level and cell toxicity and had no effect on SDF-1 secretion (P hypoxic conditions that could influence angiogenesis in the human endometrium.

  14. The globoseries glycosphingolipid SSEA-4 is a marker of bone marrow-derived clonal multipotent stromal cells in vitro and in vivo.

    Science.gov (United States)

    Rosu-Myles, Michael; McCully, Jennifer; Fair, Joel; Mehic, Jelica; Menendez, Pablo; Rodriguez, Rene; Westwood, Carole

    2013-05-01

    The therapeutic potential of multipotent stromal cells (MSC) may be enhanced by the identification of markers that allow their discrimination and enumeration both in vivo and in vitro. Here, we investigated the ability of embryonic stem cell-associated glycosphingolipids to isolate human MSC from both whole-bone-marrow (BM) and stromal cell cultures. Only SSEA-4 was consistently expressed on cells within the CD45loCD105hi marrow fraction and could be used to isolate cells with the capacity to give rise to stromal cultures containing MSC. Human stromal cultures, generated in either the presence or absence of serum, contained heterogeneous cell populations discriminated by the quantity of SSEA-4 epitopes detected on their surface. A low level of surface SSEA-4 (SSEA-4lo) correlated with undetectable levels of the α2,3-sialyltransferase-II enzyme required to synthesize SSEA-4; a reduced proliferative potential; and the loss of fat-, bone-, and cartilage-forming cells during long-term culture. In vitro, single cells with the capacity to generate multipotent stromal cultures were detected exclusively in the SSEA-4hi fraction. Our data demonstrate that a high level of surface epitopes for SSEA-4 provides a definitive marker of MSC from human BM.

  15. Isolation of Mesenchymal Stromal Cells (MSCs from Human Adenoid Tissue

    Directory of Open Access Journals (Sweden)

    Yoon Se Lee

    2013-04-01

    Full Text Available Background: Mesenchymal stromal cells (MSCs are multipotent progenitor cells that originally derived from bone marrow. Clinical use of bone marrow-derived MSC is difficult due to morbidity and low MSC abundance and isolation efficiency. Recently, MSCs have been isolated from various adult tissues. Here we report the isolation of adenoid tissue-derived MSCs (A-MSCs and their characteristics. Methods: We compared the surface markers, morphologies, and differentiation and proliferation capacities of previously established tonsil-derived MSCs (T-MSCs and bone marrow-derived MSCs (BM-MSCs with cells isolated from adenoid tissue. The immunophenotype of A-MSCs was investigated upon interferon (IFN-γ stimulation. Results: A-MSCs, T-MSCs, and BM-MSCs showed negative CD45, CD31 HLA-DR, CD34, CD14, CD19 and positive CD 90, CD44, CD73, CD105 expression. A-MSCs were fibroblast-like, spindle-shaped non-adherent cells, similar to T-MSCs and BM-MSCs. Adipogenesis was observed in A-MSCs by the formation of lipid droplets after Oil Red O staining. Osteogenesis was observed by the formation of the matrix mineralization in Alizarin Red staining. Chondrogenesis was observed by the accumulation of sulfated glycosaminoglycan-rich matrix in collagen type II staining. These data were similar to those of T-MSCs and BM-MSCs. Expression of marker genes (i.e., adipogenesis; lipoprotein lipase, proliferator-activator receptor-gamma, osteogenesis; osteocalcin, alkaline phasphatase, chondrogenesis; aggrecan, collagen type II α1 in A-MSCs were not different from those in T-MSCs and BM-MSCs. Conclusions: A-MSCs possess the characteristics of MSCs in terms of morphology, multipotent differentiation capacity, cell surface markers, and immunogeneity. Therefore, A-MSCs fulfill the definition of MSCs and represent an alternate source of MSCs.

  16. Cooperation of two ADAMTS metalloproteases in closure of the mouse palate identifies a requirement for versican proteolysis in regulating palatal mesenchyme proliferation

    OpenAIRE

    Enomoto, Hiroyuki; Nelson, Courtney M.; Somerville, Robert P. T.; Mielke, Katrina; Dixon, Laura J.; Powell, Kimerly; Apte, Suneel S.

    2010-01-01

    We have identified a role for two evolutionarily related, secreted metalloproteases of the ADAMTS family, ADAMTS20 and ADAMTS9, in palatogenesis. Adamts20 mutations cause the mouse white-spotting mutant belted (bt), whereas Adamts9 is essential for survival beyond 7.5 days gestation (E7.5). Functional overlap of Adamts9 with Adamts20 was identified using Adamts9+/–;bt/bt mice, which have a fully penetrant cleft palate. Palate closure was delayed, although eventually completed, in both Adamts9...

  17. Processing of the major autolysin of E. faecalis, AtlA, by the zinc-metalloprotease, GelE, impacts AtlA septal localization and cell separation

    OpenAIRE

    Stinemetz, Emily K.; Gao, Peng; Pinkston, Kenneth L.; Montealegre, Maria Camila; Murray, Barbara E.; Harvey, Barrett R.

    2017-01-01

    AtlA is the major peptidoglycan hydrolase of Enterococcus faecalis involved in cell division and cellular autolysis. The secreted zinc metalloprotease, gelatinase (GelE), has been identified as an important regulator of cellular function through post-translational modification of protein substrates. AtlA is a known target of GelE, and their interplay has been proposed to regulate AtlA function. To study the protease-mediated post-translational modification of AtlA, monoclonal antibodies were ...

  18. Bone marrow-derived stromal cells are more beneficial cell sources for tooth regeneration compared with adipose-derived stromal cells.

    Science.gov (United States)

    Ye, Lanfeng; Chen, Lin; Feng, Fan; Cui, Junhui; Li, Kaide; Li, Zhiyong; Liu, Lei

    2015-10-01

    Tooth loss is presently a global epidemic and tooth regeneration is thought to be a feasible and ideal treatment approach. Choice of cell source is a primary concern in tooth regeneration. In this study, the odontogenic differentiation potential of two non-dental-derived stem cells, adipose-derived stromal cells (ADSCs) and bone marrow-derived stromal cells (BMSCs), were evaluated both in vitro and in vivo. ADSCs and BMSCs were induced in vitro in the presence of tooth germ cell-conditioned medium (TGC-CM) prior to implantation into the omentum majus of rats, in combination with inactivated dentin matrix (IDM). Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the mRNA expression levels of odontogenic-related genes. Immunofluorescence and immunohistochemical assays were used to detect the protein levels of odontogenic-specific genes, such as DSP and DMP-1 both in vitro and in vivo. The results suggest that both ADSCs and BMSCs have odontogenic differentiation potential. However, the odontogenic potential of BMSCs was greater compared with ADSCs, showing that BMSCs are a more appropriate cell source for tooth regeneration. © 2015 International Federation for Cell Biology.

  19. Proliferation of Prostate Stromal Cell Induced by Benign Prostatic Hyperplasia Epithelial Cell Stimulated With Trichomonas vaginalis via Crosstalk With Mast Cell.

    Science.gov (United States)

    Kim, Jung-Hyun; Kim, Sang-Su; Han, Ik-Hwan; Sim, Seobo; Ahn, Myoung-Hee; Ryu, Jae-Sook

    2016-11-01

    Chronic inflammation has a role in the pathogenesis of benign prostatic hyperplasia (BPH) and prostate cancer. Mast cells have been detected in chronic inflammatory infiltrate of the prostate, and it is possible that the interaction between prostate epithelial cells and Trichomonas vaginalis influences the activity of mast cells in the prostate stroma. Activated mast cells might influence the biological functions of nearby tissues and cells. In this study, we investigated whether mast cells reacted with the culture supernatant of BPH epithelial cells infected with T. vaginalis may induce the proliferation of prostate stromal cells. To measure the proliferation of prostate stromal cells in response to chronic inflammation caused by the infection of BPH-1 cells with T. vaginalis, the CCK-8 assay and wound healing assay were used. ELISAs, quantitative real-time PCR, western blotting and immunofluorescence were used to measure the production and expression of inflammatory cytokine and cytokine receptor. BPH-1 cells incubated with live trichomonads produced increased levels of CCL2, IL-1β, IL-6, and CXCL8, and induced the migration of mast cells and monocytes. When the culture supernatant of BPH-1 cells stimulated with trichomonads (TCM) was added to mast cells, they became activated, as confirmed by release of β-hexosaminidase and CXCL8. Prostate stromal cells incubated with the culture supernatant of mast cells activated with TCM (M-TCM) proliferated and expressed increased levels of CXCL8, CCL2, and the cytokine receptors CXCR1 and CCR2. Blocking the chemokine receptors reduced the proliferation of stromal cells and also decreased the production of CXCL8 and CCL2. Moreover, the expression of FGF2, cyclin D1, and Bcl-2 was increased in the proliferated stromal cells stimulated with M-TCM. Additionally, the M-TCM-treated stromal cells were more invasive than control cells. The inflammatory mediators released by BPH epithelial cells in response to infection by

  20. [Risk factors for malignant evolution of gastrointestinal stromal tumors].

    Science.gov (United States)

    Andrei, S; Andrei, Adriana; Tonea, A; Andronesi, D; Becheanu, G; Dumbravă, Mona; Pechianu, C; Herlea, V; Popescu, I

    2007-01-01

    Gastrointestinal stromal tumors are the most frequent non-epithelial digestive tumors, being classified in the group of primitive mesenchymal tumors of the digestive tract. These tumors have a non predictable evolution and where stratified regarding the risk for malignant behavior in 4 categories: very low risk, low risk, intermediate risk and high risk. We performed a retrospective non randomised study including the patients with gastrointestinal stromal tumors treated in the Department of General Surgery and Liver Transplantation of Fundeni Clinical Institute in the period January 2002 - June 2007, to define the epidemiological, clinico-paraclinical, histological and especially evolutive features of the gastrointestinal stromal tumors from this group, with a special regard to the risk factors for their malignant behavior. The most important risk factors in gastrointestinal stromal tumors are the tumor size and the mitotic index, based on them being realised the classification of Fletcher in the 4 risk categories mentioned above. In our group all the local advanced or metastatic gastrointestinal stromal tumors, regardless of their location, were classified in the group of high risk for the malignant behavior. The gastric location and the epithelioid type were positive prognostic factors, and the complete resection of the tumor, an other important positive prognostic feature, was possible in about 80% of the cases, probably because the gastrointestinal stromal tumors in our study were diagnosed in less advanced evolutive situations, only about one third being metastatic and about 14% being locally advanced at the time of diagnose. The association with other neoplasias was in our cases insignificant, only 5% of the patients presenting concomitant malignant digestive tumors and 7.6% intraabdominal benign tumors. Gastrointestinal stromal tumors remain a challenge for the medical staff, regarding their diagnose and therapeutical management, the stratification of the

  1. The proteasomal Rpn11 metalloprotease suppresses tombusvirus RNA recombination and promotes viral replication via facilitating assembly of the viral replicase complex.

    Science.gov (United States)

    Prasanth, K Reddisiva; Barajas, Daniel; Nagy, Peter D

    2015-03-01

    RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a "matchmaker" that brings the viral p92(pol) replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs. RNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, although the role

  2. Malignant gastroduodenal stromal tumor imaging diagnosis

    International Nuclear Information System (INIS)

    Guo Qiang; Wen Feng; Zhao Zhenguo

    2010-01-01

    Objective: To assess the imaging features of malignant gastroduodenal stromal tumor (mGDST)as an aid to its diagnosis. Methods: The unenhanced and multi-phasic contrast-enhanced CT scans of 24 patients with pathologically proven mGDST and air-contrast upper gastrointestinal studies(15 patients) were reviewed by two radiologists. The tumor location, size, contour, margin, growth type, contrast enhancement pattern and presence of ulcer were recorded. Results: The mGDST was located in the gastric fundus (15), gastric body(3), pylorus(2) and duodenum(4). The pathological types were submucosal(9), intramuscular(9) and subserosal(6). CT findings of mGDST included lobular shape(17), tumor size>5cm(14), central necrosis(15), large and deep ulcer(6), heterogeneous contrast enhancement(1), metastasis(1). The diagnostic accuracy of air-contrast upper gastrointestinal studies and CT for location of mGDST was 93.3% and 100% respectively, for malignant features was both 75.0%. Conclusion: Most mGDST have some characteristic appearances including large tumor size greater than 5 cm, lobular shape, central necrosis, large and deep ulcer, heterogeneous contrast enhancement and metastasis. Lymph node enlargement was uncommon. The diagnostic accuracy can be improved by CT scan combined with upper gastrointestinal barium examination. (authors)

  3. Pancreatic cancer stromal biology and therapy

    Science.gov (United States)

    Xie, Dacheng; Xie, Keping

    2015-01-01

    Pancreatic cancer is one of the most lethal malignancies. Significant progresses have been made in understanding of pancreatic cancer pathogenesis, including appreciation of precursor lesions or premalignant pancreatic intraepithelial neoplasia (PanINs), description of sequential transformation from normal pancreatic tissue to invasive pancreatic cancer and identification of major genetic and epigenetic events and the biological impact of those events on malignant behavior. However, the currently used therapeutic strategies targeting tumor epithelial cells, which are potent in cell culture and animal models, have not been successful in the clinic. Presumably, therapeutic resistance of pancreatic cancer is at least in part due to its drastic desmoplasis, which is a defining hallmark for and circumstantially contributes to pancreatic cancer development and progression. Improved understanding of the dynamic interaction between cancer cells and the stroma is important to better understanding pancreatic cancer biology and to designing effective intervention strategies. This review focuses on the origination, evolution and disruption of stromal molecular and cellular components in pancreatic cancer, and their biological effects on pancreatic cancer pathogenesis. PMID:26114155

  4. Primary observation on adherent function of bone marrow stromal cells in mice post combined radiation-burn injury

    International Nuclear Information System (INIS)

    Chen Xinghua; Luo Chengji; Guo Chaohua; Wang Ping; Deng Xuecai

    1999-01-01

    Objective: To investigate the adherent function of bone marrow stromal cells in hematopoietic inductive microenvironment post combined radiation-burn injury. Methods: The expression of cell adhesion molecules including vascular cell adhesion molecule-1 (VCAM-1), fibro-connection (Fn), laminin (Ln) and collagen type IV (Col IV) on bone marrow stromal cells cultured in vitro was detected by flow cytometry and the binding capacity of bone marrow mononuclear cells to stromal cell adherence layer was tested by cell binding assay and cell binding blocking assay respectively from mice treated with 5.0 Gy γ-ray 15% of total body surface area (TBSA), third-degree burn injury and combined irradiation-burn injury, respectively. Results: 1. The expression levels of molecules mentioned above in burn-injured mice were the highest. The molecules levels in control mice were greater than those in radiation-injured mice, which were lower than those in mice with combined radiation-burn injury. 2. The binding capacity of stromal cell adherence layer in burn-injured mice was greater than that in control mice, and significantly increased from 3 to 7 days post injury as compared with that in controls, radiation-injured mice and combined radiation-burn-injured mice, respectively (P < 0.05-0.01). Contrarily, the capacity of binding in the radiation-injured and combined radiation-burn-injured mice was the lowest from 3 to 7 days post injury. 3. The binding rate of bone marrow mononuclear cells to stromal cell adherence layer descended in different degrees after pre-treatment with monoclonal antibodies directed to VCAM-1, Fn, Ln, or Col IV respectively or VCAM-1 combined with anti-Fn, anti-Ln or anti-Col IV, respectively, in stromal cell adherence layer. Conclusion: The damage of cell adherent function for bone marrow hematopoietic inductive microenvironment post combined radiation-burn injury might be one of the important factors in hematopoietic disorder in combined radiation-burn injury

  5. Overexpression of COL11A1 by cancer-associated fibroblasts: clinical relevance of a stromal marker in pancreatic cancer.

    Directory of Open Access Journals (Sweden)

    Carmen García-Pravia

    Full Text Available BACKGROUND: The collagen11A1 (COL11A1 gene is overexpressed in pancreatic cancer. The expression of COL11A1 protein could be involved in desmoplastic events in pancreatic cancer, but an antibody that specifically stains the COL11A1 protein is not currently available. METHODS AND FINDINGS: A total of 54 pancreatic ductal adenocarcinomas (PDAC, 23 chronic pancreatitis (CP samples, and cultured peritumoral stromal cells of PDAC (passages 3-6 were studied. Normal human pancreas tissue samples were obtained through a cadaveric organ donation program. 1 Validation of COL11A1 gene overexpression by q-RT-PCR. FINDINGS: the expression of COL11A1 gene is significantly increased in PDAC samples vs. normal and CP samples. 2 Analysis of COL11A1 by immunohistochemistry using highly specific anti-proCOL11A1 antibodies. FINDINGS: anti-proCOL11A1 stains stromal cells/cancer-associated fibroblasts (CAFs of PDAC but it does not stain chronic benign condition (chronic pancreatitis stromal cells, epithelial cells, or normal fibroblasts. 3 Evaluation of the discrimination ability of the antibody. FINDINGS: anti-proCOL11A1 immunostaining accurately discriminates between PDAC and CP (AUC 0.936, 95% CI 0.851, 0.981. 4 Phenotypic characterization of proCOL11A1+ stromal cells co-staining with mesenchymal, epithelial and stellate cell markers on pancreatic tissue samples and cultured peritumoral pancreatic cancer stromal cells. FINDINGS: ProCOL11A1+ cells present co-staining with mesenchymal, stellate and epithelial markers (EMT phenotype in different proportions. CONCLUSIONS/SIGNIFICANCE: Detection of proCOL11A1 through immunostaining with this newly-developed antibody allows for a highly accurate distinction between PDAC and CP. Unlike other available antibodies commonly used to detect CAFs, anti-proCOL11A1 is negative in stromal cells of the normal pancreas and almost absent in benign inflammation. These results strongly suggest that proCOL11A1 is a specific marker for

  6. Regulation of mesenchymal stromal cells through fine tuning of canonical Wnt signaling

    Directory of Open Access Journals (Sweden)

    Jin-A Kim

    2015-05-01

    Full Text Available Mesenchymal stromal cells (MSCs have been extensively utilized for various cell therapeutic trials, but the signals regulating their stromal function remain largely unclear. Here, we show that canonical Wnt signals distinctively regulate MSCs in a biphasic manner depending on signal intensity, i.e., MSCs exhibit proliferation and progenitor self-renewal under low Wnt/β-catenin signaling, whereas they exhibit enhanced osteogenic differentiation with priming to osteoblast-like lineages under high Wnt/β-catenin signaling. Moreover, low or high levels of β-catenin in MSCs distinctly regulated the hematopoietic support of MSCs to promote proliferation or the undifferentiated state of hematopoietic progenitors, respectively. A gene expression study demonstrated that different intracellular levels of β-catenin in MSCs induce distinct transcriptomic changes in subsets of genes belonging to different gene function categories. Different β-catenin levels also induced differences in intracellular levels of the β-catenin co-factors, Tcf-1 and Lef-1. Moreover, nano-scale mass spectrometry of proteins that co-precipitated with β-catenin revealed distinctive spectra of proteins selectively interacting with β-catenin at specific expression levels. Together, these results show that Wnt/β-catenin signals can coax distinct transcription milieu to induce different transcription profiles in MSCs depending on the signal intensity and that fine-tuning of the canonical Wnt signaling intensity can regulate the phase-specific functionality of MSCs.

  7. Immune suppressor factor confers stromal cell line with enhanced supporting activity for hematopoietic stem cells

    International Nuclear Information System (INIS)

    Nakajima, Hideaki; Shibata, Fumi; Fukuchi, Yumi; Goto-Koshino, Yuko; Ito, Miyuki; Urano, Atsushi; Nakahata, Tatsutoshi; Aburatani, Hiroyuki; Kitamura, Toshio

    2006-01-01

    Immune suppressor factor (ISF) is a subunit of the vacuolar ATPase proton pump. We earlier identified a short form of ISF (ShIF) as a stroma-derived factor that supports cytokine-independent growth of mutant Ba/F3 cells. Here, we report that ISF/ShIF supports self-renewal and expansion of primary hematopoietic stem cells (HSCs). Co-culture of murine bone marrow cells with a stromal cell line overexpressing ISF or ShIF (MS10/ISF or MS10/ShIF) not only enhanced their colony-forming activity and the numbers of long-term culture initiating cells, but also maintained the competitive repopulating activity of HSC. This stem cell supporting activity depended on the proton-transfer function of ISF/ShIF. Gene expression analysis of ISF/ShIF-transfected cell lines revealed down-regulation of secreted frizzled-related protein-1 and tissue inhibitor of metalloproteinase-3, and the restoration of their expressions in MS10/ISF cells partially reversed its enhanced LTC-IC supporting activity to a normal level. These results suggest that ISF/ShIF confers stromal cells with enhanced supporting activities for HSCs by modulating Wnt-activity and the extracellular matrix

  8. Epithelial-stromal interaction via Notch signaling is essential for the full maturation of gut-associated lymphoid tissues.

    Science.gov (United States)

    Obata, Yuuki; Kimura, Shunsuke; Nakato, Gaku; Iizuka, Keito; Miyagawa, Yurika; Nakamura, Yutaka; Furusawa, Yukihiro; Sugiyama, Machiko; Suzuki, Keiichiro; Ebisawa, Masashi; Fujimura, Yumiko; Yoshida, Hisahiro; Iwanaga, Toshihiko; Hase, Koji; Ohno, Hiroshi

    2014-12-01

    Intrinsic Notch signaling in intestinal epithelial cells restricts secretory cell differentiation. In gut-associated lymphoid tissue (GALT), stromal cells located beneath the follicle-associated epithelium (FAE) abundantly express the Notch ligand delta-like 1 (Dll1). Here, we show that mice lacking Rbpj-a gene encoding a transcription factor implicated in Notch signaling-in intestinal epithelial cells have defective GALT maturation. This defect can be attributed to the expansion of goblet cells, which leads to the down-regulation of CCL20 in FAE. These data demonstrate that epithelial Notch signaling maintained by stromal cells contributes to the full maturation of GALT by restricting secretory cell differentiation in FAE. © 2014 The Authors.

  9. Epithelial–stromal interaction via Notch signaling is essential for the full maturation of gut-associated lymphoid tissues

    Science.gov (United States)

    Obata, Yuuki; Kimura, Shunsuke; Nakato, Gaku; Iizuka, Keito; Miyagawa, Yurika; Nakamura, Yutaka; Furusawa, Yukihiro; Sugiyama, Machiko; Suzuki, Keiichiro; Ebisawa, Masashi; Fujimura, Yumiko; Yoshida, Hisahiro; Iwanaga, Toshihiko; Hase, Koji; Ohno, Hiroshi

    2014-01-01

    Intrinsic Notch signaling in intestinal epithelial cells restricts secretory cell differentiation. In gut-associated lymphoid tissue (GALT), stromal cells located beneath the follicle-associated epithelium (FAE) abundantly express the Notch ligand delta-like 1 (Dll1). Here, we show that mice lacking Rbpj—a gene encoding a transcription factor implicated in Notch signaling—in intestinal epithelial cells have defective GALT maturation. This defect can be attributed to the expansion of goblet cells, which leads to the down-regulation of CCL20 in FAE. These data demonstrate that epithelial Notch signaling maintained by stromal cells contributes to the full maturation of GALT by restricting secretory cell differentiation in FAE. Subject Categories Development & Differentiation; Immunology; Signal Transduction PMID:25378482

  10. Mouse endometrial stromal cells produce basement-membrane components

    DEFF Research Database (Denmark)

    Wewer, U M; Damjanov, A; Weiss, J

    1986-01-01

    During mouse pregnancy, uterine stromal cells transform into morphologically distinct decidual cells under the influence of the implanting embryo and a proper hormonal environment. Mechanical stimulation of hormonally primed uterine stromal cells leads to the same morphologic alterations. The dec......During mouse pregnancy, uterine stromal cells transform into morphologically distinct decidual cells under the influence of the implanting embryo and a proper hormonal environment. Mechanical stimulation of hormonally primed uterine stromal cells leads to the same morphologic alterations....... Mouse decidual cells isolated from 6- to 7-day pregnant uteri explanted in vitro continue to synthesize basement-membrane-like extracellular matrix. Using immunohistochemistry and metabolic labeling followed by immunoprecipitation, SDS-PAGE, and fluorography, it was shown that the decidual cells...... to undergo pseudodecidualization. We thus showed that stromal cells from pregnant and nonpregnant mouse uteri synthesize significant amounts of basement-membrane components in vitro, and hence could serve as a good model for the study of normal basement-membrane components....

  11. H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen

    International Nuclear Information System (INIS)

    Collier, I.E.; Wilhelm, S.M.; Eisen, A.Z.

    1988-01-01

    H-ras transformed human bronchial epithelial cells (TBE-1) secrete a single major extracellular matrix metalloprotease which is not found in the normal parental cells. The enzyme is secreted in a latent form which can be activated to catalyze the cleavage of the basement membrane macromolecule type IV collagen. The substrates in their order of preference are: gelatin, type IV collagen, type V collagen, fibronectin, and type VII collagen; but the enzyme does not cleave the interstitial collagens or laminin. This protease is identical to gelatinase isolated from normal human skin explants, normal human skin fibroblasts, and SV40-transformed human lung fibroblasts. Based on this ability to initiate the degradation of type IV collagen in a pepsin-resistant portion of the molecule, it will be referred to as type IV collagenase. This enzyme is most likely the human analog of type IV collagenase detected in several rodent tumors. Type IV collagenase consists of three domains. Type IV collagenase represents the third member of a newly recognized gene family coding for secreted extracellular matrix metalloproteases, which includes interstitial fibroblast collagenase and stromelysin

  12. Hypoxia impedes hypertrophic chondrogenesis of human multipotent stromal cells.

    Science.gov (United States)

    Gawlitta, Debby; van Rijen, Mattie H P; Schrijver, Edmée J M; Alblas, Jacqueline; Dhert, Wouter J A

    2012-10-01

    Within the field of bone tissue engineering, the endochondral approach to forming bone substitutes represents a novel concept, where cartilage will undergo hypertrophic differentiation before its conversion into bone. For this purpose, clinically relevant multipotent stromal cells (MSCs), MSCs, can be differentiated into the chondrogenic lineage before stimulating hypertrophy. Controversy exists in literature on the oxygen tensions naturally present during this transition in, for example, the growth plate. Therefore, the present study focused on the effects of different oxygen tensions on the progression of the hypertrophic differentiation of MSCs. Bone marrow-derived MSCs of four human donors were expanded, and differentiation was induced in aggregate cultures. Normoxic (20% oxygen) and hypoxic (5%) conditions were imposed on the cultures in chondrogenic or hypertrophic differentiation media. After 4 weeks, the cultures were histologically examined and by real-time polymerase chain reaction. Morphological assessment showed the chondrogenic differentiation of cultures from all donors under normoxic chondrogenic conditions. In addition, hypertrophic differentiation was observed in cultures derived from all but one donor. The deposition of collagen type X was evidenced in both chondrogenically and hypertrophically stimulated cultures. However, mineralization was exclusively observed in hypertrophically stimulated, normoxic cultures. Overall, the progression of hypertrophy was delayed in hypoxic compared with normoxic groups. The observed delay was supported by the gene expression patterns, especially showing the up-regulation of the late hypertrophic markers osteopontin and osteocalcin under normoxic hypertrophic conditions. Concluding, normoxic conditions are more beneficial for hypertrophic differentiation of MSCs than are hypoxic conditions, as long as the MSCs possess hypertrophic potential. This finding has implications for cartilage tissue engineering as well

  13. Equine Mesenchymal Stromal Cells Retain a Pericyte-Like Phenotype.

    Science.gov (United States)

    Esteves, Cristina L; Sheldrake, Tara A; Dawson, Lucy; Menghini, Timothy; Rink, Burgunde Elisabeth; Amilon, Karin; Khan, Nusrat; Péault, Bruno; Donadeu, Francesc Xavier

    2017-07-01

    Mesenchymal stem/stromal cells (MSCs) have been used in human and equine regenerative medicine, and interest in exploiting their potential has increased dramatically over the years. Despite significant effort to characterize equine MSCs, the actual origin of these cells and how much of their native phenotype is maintained in culture have not been determined. In this study, we investigated the relationship between MSCs, derived from adipose tissue (AT) and bone marrow (BM), and pericytes in the horse. Both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD90, and CD73) markers were detected in equine AT and colocalized around blood vessels. Importantly, as assessed by flow cytometry, both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD44, CD90, and CD105) markers were present in a majority (≥90%) of cells in cultures of AT-MSCs and BM-MSCs; however, levels of pericyte markers were variable within each of those populations. Moreover, the expression of pericyte markers was maintained for at least eight passages in both AT-MSCs and BM-MSCs. Hematopoietic (CD45) and endothelial (CD144) markers were also detected at low levels in MSCs by quantitative polymerase chain reaction (qPCR). Finally, in coculture experiments, AT-MSCs closely associated with networks produced by endothelial cells, resembling the natural perivascular location of pericytes in vivo. Our results indicate that equine MSCs originate from perivascular cells and moreover maintain a pericyte-like phenotype in culture. Therefore, we suggest that, in addition to classical MSC markers, pericyte markers such as CD146 could be used when assessing and characterizing equine MSCs.

  14. CD34+ Testicular Stromal Cells Support Long-Term Expansion of Embryonic and Adult Stem and Progenitor Cells

    Science.gov (United States)

    Kim, Jiyeon; Seandel, Marco; Falciatori, Ilaria; Wen, Duancheng; Rafii, Shahin

    2010-01-01

    Stem cells reside in specialized microenvironments created by supporting stromal cells that orchestrate self-renewal and lineage-specific differentiation. However, the precise identity of the cellular and molecular pathways that support self-renewal of stem cells is not known. For example, long-term culture of prototypical stem cells, such as adult spermatogonial stem and progenitor cells (SPCs), in vitro has been impeded by the lack of an optimal stromal cell line that initiates and sustains proliferation of these cells. Indeed, current methods, including the use of mouse embryonic fibroblasts (MEFs), have not been efficient and have generally led to inconsistent results. Here, we report the establishment of a novel CD34-positive cell line, referred to as JK1, derived from mouse testicular stromal cells that not only facilitated long-term SPC culture but also allowed faithful generation of SPCs and multipotent stem cells. SPCs generated on JK1 maintained key features of germ line stem cells, including expression of PLZF, DAZL, and GCNA. Furthermore, these feeders also promoted the long-term cultivation of other types of primitive cells including multi-potent adult spermatogonial-derived stem cells, pluripotent murine embryonic stem cells, and embryonic germ cells derived from primordial germ cells. Stem cells could be passaged serially and still maintained expression of characteristic markers such as OCT4 and NANOG in vitro, as well as the ability to generate all three germ layers in vivo. These results indicate that the JK1 cell line is capable of promoting long-term culture of primitive cells. As such, this cell line allows for identification of stromal-derived factors that support long-term proliferation of various types of stem cells and constitutes a convenient alternative to other types of feeder layers. PMID:18669907

  15. Columnar cell lesions and pseudoangiomatous hyperplasia like stroma: is there an epithelial-stromal interaction?

    Science.gov (United States)

    Recavarren, Rosemary A; Chivukula, Mamatha; Carter, Gloria; Dabbs, David J

    2009-10-10

    The significance of association between cancer and its microenvironment has been increasingly recognized. It has been shown in animal models that interaction between neoplastic epithelial cells and adjacent stroma can modulate tumor behavior. Carcinoma associated stromal cells can transform normal epithelial cells into neoplastic cells. In breast, columnar cell lesions are non-obligate precursors of low grade ductal carcinoma in situ. Columnar cell lesions can be seen intimately associated with PASH-like-stroma, a lesion we termed as CCPLS. Our aim is to investigate epithelial-stromal interactions in CCPLS and compare them to PASH without columnar cell lesions in breast core needle biopsies. Normal terminal duct lobular unit (TDLU) epithelium was seen in association with columnar cell lesions as well as PASH. Eight (8) cases of each category were examined by a panel of immunostains: CD117 (C-kit), CD34, CD105, bFGF, AR, ER-beta, MIB-1. We observed a markedly decreased expression of c-kit in columnar cell lesions compared to TDLU-epithelium. CD105 showed a quantitative increase in activated vessels in CCPLS compared to PASH. A subset of CCPLS and PASH were androgen receptor positive. A strong nuclear positivity for ER-beta is observed in the epithelium and stroma of all CCPLS cases. We conclude that (1) activated blood vessels predominate in CCPLS; (2) A molecular alteration is signified by c-kit loss in columnar cell lesions; (3) ER-beta and androgen receptor positivity indicate CCPLS are hormonally responsive lesions. Our study suggests an intimate vascular and hormone dependent epithelial-stromal interaction exists in CCPLS lesions.

  16. Aging- and Senescence-associated Changes of Mesenchymal Stromal Cells in Myelodysplastic Syndromes.

    Science.gov (United States)

    Mattiucci, Domenico; Maurizi, Giulia; Leoni, Pietro; Poloni, Antonella

    2018-01-01

    Hematopoietic stem and progenitor cells reside within the bone marrow (BM) microenvironment. By a well-balanced interplay between self-renewal and differentiation, they ensure a lifelong supply of mature blood cells. Physiologically, multiple different cell types contribute to the regulation of stem and progenitor cells in the BM microenvironment by cell-extrinsic and cell-intrinsic mechanisms. During the last decades, mesenchymal stromal cells (MSCs) have been identified as one of the main cellular components of the BM microenvironment holding an indispensable role for normal hematopoiesis. During aging, MSCs diminish their functional and regenerative capacities and in some cases encounter replicative senescence, promoting inflammation and cancer progression. It is now evident that alterations in specific stromal cells that comprise the BM microenvironment can contribute to hematologic malignancies, and there is growing interest regarding the contribution of MSCs to the pathogenesis of myelodysplastic syndromes (MDSs), a clonal hematological disorder, occurring mostly in the elderly, characterized by ineffective hematopoiesis and increased tendency to acute myeloid leukemia evolution. The pathogenesis of MDS has been associated with specific genetic and epigenetic events occurring both in hematopoietic stem cells (HSCs) and in the whole BM microenvironment with an aberrant cross talk between hematopoietic elements and stromal compartment. This review highlights the role of MSCs in MDS showing functional and molecular alterations such as altered cell-cycle regulation with impaired proliferative potential, dysregulated cytokine secretion, and an abnormal gene expression profile. Here, the current knowledge of impaired functional properties of both aged MSCs and MSCs in MDS have been described with a special focus on inflammation and senescence induced changes in the BM microenvironment. Furthermore, a better understanding of aberrant BM microenvironment could

  17. Aging enhances the vulnerability of mesenchymal stromal cells to uniaxial tensile strain-induced apoptosis.

    Science.gov (United States)

    McKayed, Katey; Prendergast, Patrick J; Campbell, Veronica A

    2016-02-08

    Mechanical priming can be employed in tissue engineering strategies to control the fate and differentiation pattern of mesenchymal stromal cells. This is relevant to regenerative medicine whereby mechanical cues can promote the regeneration of a specific tissue type from mesenchymal precursors. The ability of cells to respond to mechanical forces is dependent upon mechanotransduction pathways that involve membrane-associated proteins, such as integrins. During the aging process changes in the mechanotransduction machinery may influence how cells from aged individuals respond to mechanical priming. In this study mesenchymal stromal cells were prepared from young adult and aged rats and exposed to uniaxial tensile strain at 5% and 10% for 3 days, or 2.5% for 7 days. Application of 5% tensile strain had no impact on cell viability. In contrast, application of 10% tensile strain evoked apoptosis and the strain-induced apoptosis was significantly higher in the mesenchymal stromal cells prepared from the aged rats. In parallel to the age-related difference in cellular responsiveness to strain, an age-related decrease in expression of α2 integrin and actin, and enhanced lipid peroxidation was observed. This study demonstrates that mesenchymal stem cells from aged animals have an altered membrane environment, are more vulnerable to the pro-apoptotic effects of 10% tensile strain and less responsive to the pro-osteogenic effects of 2.5% tensile strain. Thus, it is essential to consider how aged cells respond to mechanical stimuli in order to identify optimal mechanical priming strategies that minimise cell loss, particularly if this approach is to be applied to an aged population. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Epimorphin Regulates the Intestinal Stem Cell Niche via Effects on the Stromal Microenvironment.

    Science.gov (United States)

    Vishy, Courtney E; Swietlicki, Elzbieta A; Gazit, Vered; Amara, Suneetha; Heslop, Gabriela; Lu, Jianyun; Levin, Marc S; Rubin, Deborah C

    2018-04-06

    Stem cell therapy is a potential therapeutic approach for disorders characterized by intestinal injury or loss of functional surface area. Stem cell function and proliferation are mediated by the stem cell niche. Stromal cells such as intestinal subepithelial myofibroblasts (ISEMFs) are important but poorly studied components of the stem cell niche. To examine the role of ISEMFs, we have previously generated mice with deletion of epimorphin (Epim), an ISEMF protein and member of the syntaxin family of intracellular vesicle docking proteins that regulate cell secretion. Herein we explore the mechanisms for previous observations that Epim deletion increases gut crypt cell proliferation, crypt fission and small bowel length in vivo. Stem cell derived crypt culture techniques were used to explore the interaction between enteroids and myofibroblasts from Epim -/- and WT mice. Enteroids co-cultured with ISEMFS had increased growth and crypt-like budding compared to enteroids cultured without stromal support. Epim deletion in ISEMFs resulted in increased enteroid budding and surface area compared to co-cultures with WT ISEMFs. In primary crypt cultures, Epim -/- enteroids had significantly increased surface area and budding compared WTs. However stem cell assays comparing the number of Epim -/- vs WT colony forming units after first passage showed no differences in the absence of ISEMF support. Epim -/- vs. WT ISEMFs had increased Wnt4 expression and addition of Wnt4 to WT co-cultures enhanced budding. We conclude that ISEMFs play an important role in the stem cell niche. Epim regulates stem cell proliferation and differentiation via stromal contributions to the niche microenvironment.

  19. In vitro inhibitory effects of imatinib mesylate on stromal cells and hematopoietic progenitors from bone marrow

    Directory of Open Access Journals (Sweden)

    P.B. Soares

    2013-01-01

    Full Text Available Imatinib mesylate (IM is used to treat chronic myeloid leukemia (CML because it selectively inhibits tyrosine kinase, which is a hallmark of CML oncogenesis. Recent studies have shown that IM inhibits the growth of several non-malignant hematopoietic and fibroblast cells from bone marrow (BM. The aim of the present study was to evaluate the effects of IM on stromal and hematopoietic progenitor cells, specifically in the colony-forming units of granulocyte/macrophage (CFU-GM, using BM cultures from 108 1.5- to 2-month-old healthy Swiss mice. The results showed that low concentrations of IM (1.25 µM reduced the growth of CFU-GM in clonogenic assays. In culture assays with stromal cells, fibroblast proliferation and α-SMA expression by immunocytochemistry analysis were also reduced in a concentration-dependent manner, with a survival rate of approximately 50% with a dose of 2.5 µM. Cell viability and morphology were analyzed using MTT and staining with acrydine orange/ethidium bromide. Most cells were found to be viable after treatment with 5 µM IM, although there was gradual growth inhibition of fibroblastic cells while the number of round cells (macrophage-like cells increased. At higher concentrations (15 µM, the majority of cells were apoptotic and cell growth ceased completely. Oil red staining revealed the presence of adipocytes only in untreated cells (control. Cell cycle analysis of stromal cells by flow cytometry showed a blockade at the G0/G1 phases in groups treated with 5-15 µM. These results suggest that IM differentially inhibits the survival of different types of BM cells since toxic effects were achieved.

  20. Human endometrial stromal stem cells differentiate into megakaryocytes with the ability to produce functional platelets.

    Directory of Open Access Journals (Sweden)

    Jinju Wang

    Full Text Available Human endometrium is a high dynamic tissue that contains endometrial stromal stem cells (hESSCs. The hESSCs have been differentiated into a number of cell lineages. However, differentiation of hESSCs into megakaryocytes (MKs has not yet been investigated. The aim of this study was to investigate the feasibility of MK generation from hESSCs and subsequent production of functional platelets (PLTs. In our study, hESSCs were cultured from endometrial stromal cells as confirmed by positive stromal cell specific markers (CD90 and CD29 and negative hematopoietic stem cell markers (CD45 and CD34 expression. Then, hESSCs were differentiated in a medium supplemented with thrombopoietin (TPO for 18 days. The MK differentiation was analyzed by flow cytometry and confocal microscopy. The differentiation medium was collected for PLT production analysis by flow cytometry, transmission electron microscopy and functional measurements. Our results show: 1 MKs were successfully generated from hESSCs as identified by expression of specific markers (CD41a: 1 ± 0.09% and 39 ± 3.0%; CD42b: 1.2 ± 0.06% and 28 ± 2.0%, control vs. differentiation accompanied with reduction of pluripotent transcription factors (Oct4 and Sox2 expression; 2 The level of PLTs in the differentiation medium was 16 ± 1 number/µl as determined by size (2-4 µm and CD41a expression (CD41a: 1 ± 0.4% and 90±2.0%, control vs. differentiation; 3 Generated PLTs were functional as evidenced by the up-regulation of CD62p expression and fibrinogen binding following thrombin stimulation; 4 Released PLTs showed similar ultra-structure characteristics (alpha granules, vacuoles and dense tubular system as PLTs from peripheral blood determined by electron microscopic analysis. Data demonstrate the feasibility of generating MKs from hESSCs, and that the generated MKs release functional PLTs. Therefore, hESSCs could be a potential new stem cell source for in vitro MK/PLT production.

  1. Endometrial stromal tumors with sex cord-like elements: a case report

    African Journals Online (AJOL)

    Endometrial stromal nodules are rare. They represent less than a quarter of endometrial stromal tumors. Clement and Scully described as variants of endometrial stromal nodules two types of tumor ressembling ovarian sex cord tumors. Type I is tumor that resembles focally an ovarian sex cord tumor which can be ...

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  1. File list: Pol.Utr.20.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

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  2. A gastrointestinal stromal tumor (GIST masquerading as an ovarian mass

    Directory of Open Access Journals (Sweden)

    Beneduce Pasquale

    2004-05-01

    Full Text Available Abstract Background Malignant gastrointestinal stromal tumors (GIST are rare mesenchymal tumors originating in the wall of the gastrointestinal tract. Myogenic gastrointestinal stromal tumor, a distinctive morphologic variant is characterized by an unusually prominent myxoid stromal background. Case presentation We report a case of myxoid variant of GIST in a 42 years old woman presenting as an epigastric mass associated to an ovarian cyst and elevated CA-125. Histologically, the lesions was composed of a proliferation of spindle cells in an abundant myxoid stroma, without evidence of atypia or anaplasia. Immunohistochemical stains showed strong positive staining with muscle actin, positive staining with CD34 and weak positive staining with CD117, while showed negative for S-100. Conclusion At surgery every effort should be made to identify the origin of the tumor. A complete surgical removal of the tumor should be obtained, as this is the only established treatment that offers long term survival.

  3. Sex Cord-Gonadal Stromal Tumor of the Rete Testis

    Directory of Open Access Journals (Sweden)

    Kamran P. Sajadi

    2009-01-01

    Full Text Available A 34-year-old tetraplegic patient with suppurative epididymitis was found on follow-up examination and ultrasonography to have a testicular mass. The radical orchiectomy specimen contained an undifferentiated spindled sex cord-stromal tumor arising in the rete testis. Testicular sex cord-stromal tumors are far less common than germ cell neoplasms and are usually benign. The close relationship between sex cords and ductules of the rete testis during development provides the opportunity for these uncommon tumors to arise anatomically within the rete tesis. This undifferentiated sex cord-stromal tumor, occurring in a previously unreported location, is an example of an unusual lesion mimicking an intratesticular malignant neoplasm.

  4. Sclerosing stromal tumor of the ovary: a case report

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Hyun Koo; Koh, Byung Hee; Rhim, Hyun Chul; Cho, On Koo; Kim, Yong Soo; Hahm, Chang Kok [School of Medicine, Hanyang Univ., Seoul (Korea, Republic of)

    2002-07-01

    Sclerosing stromal tumor of the ovary is a rare benign neoplasm, with distinctive clinical and pathologic features. It occurs predominantly in females during the second and third decades of life. Histologically, it is composed of cellular and acellular collagenized areas, and edematous stromal areas, and at ultrasonography and computed tomography is seen as a distinctive mixed solid and cystic mass lesion. We report a case of sclerosing stromal tumor of the ovary in a 15-year-old girl with a history of menorrhagia since menarche. Ultrasonography revealed the tumor as a well-defined, lobulated, heterogenous echogenic pelvic mass, while at CT, a huge pelvic mass 9 x 9 x 10 cm in size, was seen. This comprised a well-enhanced internal solid portion, a capsule, septa, and a non-enhanced cystic portion.

  5. Stromal cell regulation of homeostatic and inflammatory lymphoid organogenesis

    Science.gov (United States)

    Kain, Matthew J W; Owens, Benjamin M J

    2013-01-01

    Summary Secondary lymphoid organs function to increase the efficiency of interactions between rare, antigen-specific lymphocytes and antigen presenting cells, concentrating antigen and lymphocytes in a supportive environment that facilitates the initiation of an adaptive immune response. Homeostatic lymphoid tissue organogenesis proceeds via exquisitely controlled spatiotemporal interactions between haematopoietic lymphoid tissue inducer populations and multiple subsets of non-haematopoietic stromal cells. However, it is becoming clear that in a range of inflammatory contexts, ectopic or tertiary lymphoid tissues can develop inappropriately under pathological stress. Here we summarize the role of stromal cells in the development of homeostatic lymphoid tissue, and assess emerging evidence that suggests a critical role for stromal involvement in the tertiary lymphoid tissue development associated with chronic infections and inflammation. PMID:23621403

  6. Epithelial-stromal interaction 1 (EPSTI1) substitutes for peritumoral fibroblasts in the tumor microenvironment

    DEFF Research Database (Denmark)

    De Neergaard, Michala; Kim, Jiyoung; Villadsen, René

    2010-01-01

    Tumor cells can activate stroma, yet the implication of this activation in terms of reciprocal induction of gene expression in tumor cells is poorly understood. Epithelial Stromal Interaction 1 (EPSTI1) is an interferon response gene originally isolated from heterotypic recombinant cultures...... of human breast cancer cells and activated breast myofibroblasts. Here we describe the first immunolocalization of EPSTI1 in normal and cancerous breast tissue, and we provide evidence for a role of this molecule in the regulation of tumor cell properties and epithelial-mesenchymal transition. In general...... cell line and silenced endogenous EPSTI1 by RNA interference in another. Irrespective of the experimental approach, EPSTI1 expression led to an increase in tumorsphere formation-a property associated with breast stem/progenitor cells. Most remarkably, we show that EPSTI1, by conveying spread of tumor...

  7. Legumain Regulates Differentiation Fate of Human Bone Marrow Stromal Cells and Is Altered in Postmenopausal Osteoporosis

    Directory of Open Access Journals (Sweden)

    Abbas Jafari

    2017-02-01

    Full Text Available Secreted factors are a key component of stem cell niche and their dysregulation compromises stem cell function. Legumain is a secreted cysteine protease involved in diverse biological processes. Here, we demonstrate that legumain regulates lineage commitment of human bone marrow stromal cells and that its expression level and cellular localization are altered in postmenopausal osteoporotic patients. As shown by genetic and pharmacological manipulation, legumain inhibited osteoblast (OB differentiation and in vivo bone formation through degradation of the bone matrix protein fibronectin. In addition, genetic ablation or pharmacological inhibition of legumain activity led to precocious OB differentiation and increased vertebral mineralization in zebrafish. Finally, we show that localized increased expression of legumain in bone marrow adipocytes was inversely correlated with adjacent trabecular bone mass in a cohort of patients with postmenopausal osteoporosis. Our data suggest that altered proteolytic activity of legumain in the bone microenvironment contributes to decreased bone mass in postmenopausal osteoporosis.

  8. Nonviral transfection of adipose tissue stromal cells: an experimental study.

    Science.gov (United States)

    Lopatina, T V; Kalinina, N I; Parfyonova, E V

    2009-04-01

    Delivery of plasmid DNA and interfering RNA into adipose tissue stromal cells was carried out by the methods of lipofection, calcium phosphate method, and by electroporation. The percent of transfected cells after delivery of plasmid DNA by the calcium phosphate method and lipofection was 0 and 15%, respectively, vs. more than 50% after electroporation. Similar results were obtained for delivery of short-strand RNA into cells. These data indicate that electroporation is the most effective method of nonviral transfection of adipose tissue stromal cells.

  9. Esophageal Gastrointestinal Stromal Tumor: Diagnostic Complexity and Management Pitfalls

    Directory of Open Access Journals (Sweden)

    Charalampos G. Markakis

    2013-01-01

    Full Text Available Introduction. Gastrointestinal stromal tumors of the esophagus are rare. Case Presentation. This is a case of a 50-year-old male patient who was referred to our department complaining of atypical chest pain. A chest computed tomographic scan and endoscopic ultrasound revealed a submucosal esophageal tumor measuring 5 cm in its largest diameter. Suspecting a leiomyoma, we performed enucleation via right thoracotomy. The pathology report yielded a diagnosis of an esophageal gastrointestinal stromal tumor. The patient has shown no evidence of recurrence one year postoperatively. Conclusions. This report illustrates the complexity and dilemmas inherent in diagnosing and treating esophageal GISTs.

  10. Analysis for apoptosis and necrosis on adipocytes, stromal vascular fraction, and adipose-derived stem cells in human lipoaspirates after liposuction.

    Science.gov (United States)

    Wang, Wei Z; Fang, Xin-Hua; Williams, Shelley J; Stephenson, Linda L; Baynosa, Richard C; Wong, Nancy; Khiabani, Kayvan T; Zamboni, William A

    2013-01-01

    Adipose-derived stem cells have become the most studied adult stem cells. The authors examined the apoptosis and necrosis rates for adipocyte, stromal vascular fraction, and adipose-derived stem cells in fresh human lipoaspirates. Human lipoaspirate (n = 8) was harvested using a standard liposuction technique. Stromal vascular fraction cells were separated from adipocytes and cultured to obtain purified adipose-derived stem cells. A panel of stem cell markers was used to identify the surface phenotypes of cultured adipose-derived stem cells. Three distinct stem cell subpopulations (CD90/CD45, CD105/CD45, and CD34/CD31) were selected from the stromal vascular fraction. Apoptosis and necrosis were determined by annexin V/propidium iodide assay and analyzed by flow cytometry. The cultured adipose-derived stem cells demonstrated long-term proliferation and differentiation evidenced by cell doubling time and positive staining with oil red O and alkaline phosphatase. Isolated from lipoaspirates, adipocytes exhibited 19.7 ± 3.7 percent apoptosis and 1.1 ± 0.3 percent necrosis; stromal vascular fraction cells revealed 22.0 ± 6.3 percent of apoptosis and 11.2 ± 1.9 percent of necrosis; stromal vascular fraction cells had a higher rate of necrosis than adipocytes (p vascular fraction cells, 51.1 ± 3.7 percent expressed CD90/CD45, 7.5 ± 1.0 percent expressed CD105/CD45, and 26.4 ± 3.8 percent expressed CD34/CD31. CD34/CD31 adipose-derived stem cells had lower rates of apoptosis and necrosis compared with CD105/CD45 adipose-derived stem cells (p necrosis than adipocytes. However, the extent of apoptosis and necrosis was significantly different among adipose-derived stem cell subpopulations.

  11. Pleiotrophin commits human bone marrow mesenchymal stromal cells towards hypertrophy during chondrogenesis.

    Science.gov (United States)

    Bouderlique, Thibault; Henault, Emilie; Lebouvier, Angelique; Frescaline, Guilhem; Bierling, Phillipe; Rouard, Helene; Courty, José; Albanese, Patricia; Chevallier, Nathalie

    2014-01-01

    Pleiotrophin (PTN) is a growth factor present in the extracellular matrix of the growth plate during bone development and in the callus during bone healing. Bone healing is a complicated process that recapitulates endochondral bone development and involves many cell types. Among those cells, mesenchymal stromal cells (MSC) are able to differentiate toward chondrogenic and osteoblastic lineages. We aimed to determine PTN effects on differentiation properties of human bone marrow stromal cells (hBMSC) under chondrogenic induction using histological analysis and quantitative reverse transcription polymerase chain reaction. PTN dramatically potentiated chondrogenic differentiation as indicated by a strong increase of collagen 2 protein, and cartilage-related gene expression. Moreover, PTN increased transcription of hypertrophic chondrocyte markers such as MMP13, collagen 10 and alkaline phosphatase and enhanced calcification and the content of collagen 10 protein. These effects are dependent on PTN receptors signaling and PI3 K pathway activation. These data suggest a new role of PTN in bone regeneration as an inducer of hypertrophy during chondrogenic differentiation of hBMSC.

  12. Pleiotrophin commits human bone marrow mesenchymal stromal cells towards hypertrophy during chondrogenesis.

    Directory of Open Access Journals (Sweden)

    Thibault Bouderlique

    Full Text Available Pleiotrophin (PTN is a growth factor present in the extracellular matrix of the growth plate during bone development and in the callus during bone healing. Bone healing is a complicated process that recapitulates endochondral bone development and involves many cell types. Among those cells, mesenchymal stromal cells (MSC are able to differentiate toward chondrogenic and osteoblastic lineages. We aimed to determine PTN effects on differentiation properties of human bone marrow stromal cells (hBMSC under chondrogenic induction using histological analysis and quantitative reverse transcription polymerase chain reaction. PTN dramatically potentiated chondrogenic differentiation as indicated by a strong increase of collagen 2 protein, and cartilage-related gene expression. Moreover, PTN increased transcription of hypertrophic chondrocyte markers such as MMP13, collagen 10 and alkaline phosphatase and enhanced calcification and the content of collagen 10 protein. These effects are dependent on PTN receptors signaling and PI3 K pathway activation. These data suggest a new role of PTN in bone regeneration as an inducer of hypertrophy during chondrogenic differentiation of hBMSC.

  13. Transcriptomic comparisons between cultured human adipose tissue-derived pericytes and mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Lindolfo da Silva Meirelles

    2016-03-01

    Full Text Available Mesenchymal stromal cells (MSCs, sometimes called mesenchymal stem cells, are cultured cells able to give rise to mature mesenchymal cells such as adipocytes, osteoblasts, and chondrocytes, and to secrete a wide range of trophic and immunomodulatory molecules. Evidence indicates that pericytes, cells that surround and maintain physical connections with endothelial cells in blood vessels, can give rise to MSCs (da Silva Meirelles et al., 2008 [1]; Caplan and Correa, 2011 [2]. We have compared the transcriptomes of highly purified, human adipose tissue pericytes subjected to culture-expansion in pericyte medium or MSC medium, with that of human adipose tissue MSCs isolated with traditional methods to test the hypothesis that their transcriptomes are similar (da Silva Meirelles et al., 2015 [3]. Here, we provide further information and analyses of microarray data from three pericyte populations cultured in pericyte medium, three pericyte populations cultured in MSC medium, and three adipose tissue MSC populations deposited in the Gene Expression Omnibus under accession number GSE67747. Keywords: Mesenchymal stromal cells, Mesenchymal stem cells, Pericytes, Microarrays

  14. Targeting of Mesenchymal Stromal Cells by Cre-Recombinase Transgenes Commonly Used to Target Osteoblast Lineage Cells.

    Science.gov (United States)

    Zhang, Jingzhu; Link, Daniel C

    2016-11-01

    The targeting specificity of tissue-specific Cre-recombinase transgenes is a key to interpreting phenotypes associated with their use. The Ocn-Cre and Dmp1-Cre transgenes are widely used to target osteoblasts and osteocytes, respectively. Here, we used high-resolution microscopy of bone sections and flow cytometry to carefully define the targeting specificity of these transgenes. These transgenes were crossed with Cxcl12 gfp mice to identify Cxcl12-abundant reticular (CAR) cells, which are a perivascular mesenchymal stromal population implicated in hematopoietic stem/progenitor cell maintenance. We show that in addition to osteoblasts, Ocn-Cre targets a majority of CAR cells and arteriolar pericytes. Surprisingly, Dmp1-Cre also targets a subset of CAR cells, in which expression of osteoblast-lineage genes is enriched. Finally, we introduce a new tissue-specific Cre-recombinase, Tagln-Cre, which efficiently targets osteoblasts, a majority of CAR cells, and both venous sinusoidal and arteriolar pericytes. These data show that Ocn-Cre and Dmp1-Cre target broader stromal cell populations than previously appreciated and may aid in the design of future studies. Moreover, these data highlight the heterogeneity of mesenchymal stromal cells in the bone marrow and provide tools to interrogate this heterogeneity. © 2016 American Society for Bone and Mineral Research. © 2016 American Society for Bone and Mineral Research.

  15. Melanoma Cells Can Adopt the Phenotype of Stromal Fibroblasts and Macrophages by Spontaneous Cell Fusion in Vitro.

    Science.gov (United States)

    Kemény, Lajos V; Kurgyis, Zsuzsanna; Buknicz, Tünde; Groma, Gergely; Jakab, Ádám; Zänker, Kurt; Dittmar, Thomas; Kemény, Lajos; Németh, István B

    2016-06-02

    After the removal of primary cutaneous melanoma some patients develop local recurrences, even after having histologically tumor-free re-excision. A potential explanation behind this phenomenon is that tumor cells switch their phenotype, making their recognition via standard histopathological assessments extremely difficult. Tumor-stromal cell fusion has been proposed as a potential mechanism for tumor cells to acquire mesenchymal traits; therefore, we hypothesized that melanoma cells could acquire fibroblast- and macrophage-like phenotypes via cell fusion. We show that melanoma cells spontaneously fuse with human dermal fibroblasts and human peripheral blood monocytes in vitro. The hybrid cells' nuclei contain chromosomes from both parental cells and are indistinguishable from the parental fibroblasts or macrophages based on their morphology and immunophenotype, as they could lose the melanoma specific MART1 marker, but express the fibroblast marker smooth muscle actin or the macrophage marker CD68. Our results suggest that, by spontaneous cell fusion in vitro, tumor cells can adopt the morphology and immunophenotype of stromal cells while still carrying oncogenic, tumor-derived genetic information. Therefore, melanoma-stromal cell fusion might play a role in missing tumor cells by routine histopathological assessments.

  16. [Markers of stromal invasion during background and precancerous changes of the glandular epithelium and in adenocarcinoma of the cervix uteri].

    Science.gov (United States)

    Danilova, N V; Andreeva, Iu Iu; Zavalishina, L É; Mal'kov, P G

    2012-01-01

    It is very difficult to identify stromal invasion when the glandular epithelium of the cervix uteri is involved. It is necessary to draw a clear distinction between its glandular structures and adenocarcinoma in situ, involving the preexisting crypts and invasive glands. An attempt was made to assess the possibilities of using as markers of invasion the following stromal proteins and adhesion molecules: CD44, E-cadherin, beta-catenin, tenascin, and laminin. Fifty-three cases of benign glandular changes, 66 cases of dysplasias and adenocarcinomas in situ, and 47 cases of invasive adenocarcinoma were examined. An immunohistochemical study was performed according to the standard protocol using the antibodies to CD44, laminin, tenascin, E-cadherin, and beta-catenin and a semiquantitative assessment of results was made. CD44 was found to be redistributed from the cells to the tumor stroma. CD44 was not detected in the stroma surrounding the intact glands, so were benign epithelial changes. In the tumor environment, there was, on the contrary, a reaction with CD44 in 74.5% of invasive adenocarcinomas cases (p 0.05). CD44 and tenascin are of great diagnostic value in examining invasive and microinvasive adenocarcinomas of the cervix uteri. E-cadherin and beta-catenin are of no diagnostic value in the study groups of pathological processes. Laminin is a potential marker of stromal invasion; however, its expression calls for further investigation.

  17. Stromal Activation Associated with Development of Prostate Cancer in Prostate-Targeted Fibroblast Growth Factor 8b Transgenic Mice

    Directory of Open Access Journals (Sweden)

    Teresa D. Elo

    2010-11-01

    Full Text Available Expression of fibroblast growth factor 8 (FGF-8 is commonly increased in prostate cancer. Experimental studies have provided evidence that it plays a role in prostate tumorigenesis and tumor progression. To study how increased FGF-8 affects the prostate, we generated and analyzed transgenic (TG mice expressing FGF-8b under the probasin promoter that targets expression to prostate epithelium. Prostates of the TG mice showed an increased size and changes in stromal and epithelialmorphology progressing fromatypia and prostatic intraepithelial neoplasia (mouse PIN, mPIN lesions to tumors with highly variable phenotype bearing features of adenocarcinoma, carcinosarcoma, and sarcoma. The development of mPIN lesions was preceded by formation of activated stroma containing increased proportion of fibroblastic cells, rich vasculature, and inflammation. The association between advancing stromal and epithelial alterations was statistically significant. Microarray analysis and validation with quantitative polymerase chain reaction revealed that expression of osteopontin and connective tissue growth factor was markedly upregulated in TG mouse prostates compared with wild type prostates. Androgen receptor staining was decreased in transformed epithelium and in hypercellular stroma but strongly increased in the sarcoma-like lesions. In conclusion, our data demonstrate that disruption of FGF signaling pathways by increased epithelial production of FGF-8b leads to strongly activated and atypical stroma, which precedes development of mPIN lesions and prostate cancer with mixed features of adenocarcinoma and sarcoma in the prostates of TG mice. The results suggest that increased FGF-8 in human prostate may also contribute to prostate tumorigenesis by stromal activation.

  18. Immunocytochemical characterization of explant cultures of human prostatic stromal cells

    NARCIS (Netherlands)

    A. Kooistra (Anko); A.M.J. Elissen (Arianne ); J.J. Konig (Josee); M. Vermey; Th.H. van der Kwast (Theo); J.C. Romijn (Johannes); F.H. Schröder (Fritz)

    1995-01-01

    textabstractThe study of stromal-epithelial interactions greatly depends on the ability to culture both cell types separately, in order to permit analysis of their interactions under defined conditions in reconstitution experiments. Here we report the establishment of explant cultures of human

  19. Stromal cell-derived factor 1α (SDF-1α)

    DEFF Research Database (Denmark)

    Li, Dana; Bjørnager, Louise; Langkilde, Anne

    2016-01-01

    OBJECTIVES: Stromal cell-derived factor 1a (SDF-1α), is a chemokine and is able to home hematopoietic progenitor cells to injured areas of heart tissue for structural repair. Previous studies have found increased levels of SDF-1α in several cardiac diseases, but only few studies have investigated...

  20. POLYCYSTIC OVARIES WITH STROMAL HYPERTHECOSIS: A RARE PRESENTATION

    Directory of Open Access Journals (Sweden)

    Shaila

    2016-05-01

    Full Text Available Presenting a 47-year-old peri-menopausal lady with hypomenorrhoea, temporal baldness, alopecia, hirsutism. The histopathology was polycystic ovaries with stromal hyperthecosis. Hughesdon described hyperthecosis as a severe form of PCOS. Hyperthecosis is rare in young women, with clinical features similar to PCOS. However, these women are usually more virilised.

  1. A stromal myoid cell line provokes thymic erythropoiesis between ...

    African Journals Online (AJOL)

    Background: The thymus provides an optimal cellular and humoral microenvironment for cell line committed differentiation of haematopoietic stem cells. The immigration process requires the secretion of at least one peptide called thymotaxine by cells of the reticulo-epithelial (RE) network of the thymic stromal cellular ...

  2. Endogenous collagen influences differentiation of human multipotent mesenchymal stromal cells

    NARCIS (Netherlands)

    Fernandes, H.; Mentink, A.; Bank, R.; Stoop, R.; Blitterswijk, C. van; Boer, J. de

    2010-01-01

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix

  3. Endogenous Collagen Influences Differentiation of Human Multipotent Mesenchymal Stromal Cells

    NARCIS (Netherlands)

    Fernandes, Hugo; Mentink, Anouk; Bank, Ruud; Stoop, Reinout; van Blitterswijk, Clemens; de Boer, Jan

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix

  4. Endogenous Collagen Influences Differentiation of Human Multipotent Mesenchymal Stromal Cells

    NARCIS (Netherlands)

    Fernandes, H.A.M.; Mentink-Leusink, Anouk; Bank, Ruud; Stoop, Reinout; van Blitterswijk, Clemens; de Boer, Jan

    2010-01-01

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix

  5. Human mesenchymal stromal cells : biological characterization and clinical application

    NARCIS (Netherlands)

    Bernardo, Maria Ester

    2010-01-01

    This thesis focuses on the characterization of the biological and functional properties of human mesenchymal stromal cells (MSCs), isolated from different tissue sources. The differentiation capacity of MSCs from fetal and adult tissues has been tested and compared. Umbilical cord blood (UCB) has

  6. Senescence and quiescence in adipose-derived stromal cells

    DEFF Research Database (Denmark)

    Søndergaard, Rebekka Harary; Follin, Bjarke; Lund, Lisbeth Drozd

    2017-01-01

    Background aims. Adipose-derived stromal cells (ASCs) are attractive sources for cell-based therapies. The hypoxic niche of ASCs in vivo implies that cells will benefit from hypoxia during in vitro expansion. Human platelet lysate (hPL) enhances ASC proliferation rates, compared with fetal bovine...

  7. Bone marrow stromal cell : mediated neuroprotection for spinal cord repair

    NARCIS (Netherlands)

    Ritfeld, Gaby Jane

    2014-01-01

    Currently, there is no treatment available that restores anatomy and function after spinal cord injury. This thesis explores transplantation of bone marrow-derived mesenchymal stem cells (bone marrow stromal cells; BMSCs) as a therapeutic approach for spinal cord repair. BMSCs secrete neurotrophic

  8. IL-33 and Thymic Stromal Lymphopoietin in mast cell functions

    NARCIS (Netherlands)

    Saluja, Rohit; Zoltowska, Anna; Ketelaar, Maria Elizabeth; Nilsson, Gunnar

    2016-01-01

    Thymic Stromal Lymphopoietin (TSLP) and Interleukin 33 (IL-33) are two cytokines released by cells that are in proximity to our environment, e.g., keratinocytes of the skin and epithelial cells of the airways. Pathogens, allergens, chemicals and other agents induce the release of TSLP and IL-33,

  9. High Aldehyde Dehydrogenase Activity Identifies a Subset of Human Mesenchymal Stromal Cells with Vascular Regenerative Potential.

    Science.gov (United States)

    Sherman, Stephen E; Kuljanin, Miljan; Cooper, Tyler T; Putman, David M; Lajoie, Gilles A; Hess, David A

    2017-06-01

    During culture expansion, multipotent mesenchymal stromal cells (MSCs) differentially express aldehyde dehydrogenase (ALDH), an intracellular detoxification enzyme that protects long-lived cells against oxidative stress. Thus, MSC selection based on ALDH-activity may be used to reduce heterogeneity and distinguish MSC subsets with improved regenerative potency. After expansion of human bone marrow-derived MSCs, cell progeny was purified based on low versus high ALDH-activity (ALDH hi ) by fluorescence-activated cell sorting, and each subset was compared for multipotent stromal and provascular regenerative functions. Both ALDH l ° and ALDH hi MSC subsets demonstrated similar expression of stromal cell (>95% CD73 + , CD90 + , CD105 + ) and pericyte (>95% CD146 + ) surface markers and showed multipotent differentiation into bone, cartilage, and adipose cells in vitro. Conditioned media (CDM) generated by ALDH hi MSCs demonstrated a potent proliferative and prosurvival effect on human microvascular endothelial cells (HMVECs) under serum-free conditions and augmented HMVEC tube-forming capacity in growth factor-reduced matrices. After subcutaneous transplantation within directed in vivo angiogenesis assay implants into immunodeficient mice, ALDH hi MSC or CDM produced by ALDH hi MSC significantly augmented murine vascular cell recruitment and perfused vessel infiltration compared with ALDH l ° MSC. Although both subsets demonstrated strikingly similar mRNA expression patterns, quantitative proteomic analyses performed on subset-specific CDM revealed the ALDH hi MSC subset uniquely secreted multiple proangiogenic cytokines (vascular endothelial growth factor beta, platelet derived growth factor alpha, and angiogenin) and actively produced multiple factors with chemoattractant (transforming growth factor-β, C-X-C motif chemokine ligand 1, 2, and 3 (GRO), C-C motif chemokine ligand 5 (RANTES), monocyte chemotactic protein 1 (MCP-1), interleukin [IL]-6, IL-8) and matrix

  10. In vitro differentiation of human skin-derived multipotent stromal cells into putative endothelial-like cells

    Directory of Open Access Journals (Sweden)

    Vishnubalaji Radhakrishnan

    2012-01-01

    Full Text Available Abstract Background Multipotent stem cells have been successfully isolated from various tissues and are currently utilized for tissue-engineering and cell-based therapies. Among the many sources, skin has recently emerged as an attractive source for multipotent cells because of its abundance. Recent literature showed that skin stromal cells (SSCs possess mesoderm lineage differentiation potential; however, the endothelial differentiation and angiogenic potential of SSC remains elusive. In our study, SSCs were isolated from human neonatal foreskin (hNFSSCs and adult dermal skin (hADSSCs using explants cultures and were compared with bone marrow (hMSC-TERT and adipose tissue-derived mesenchymal stem cells (hADMSCs for their potential differentiation into osteoblasts, adipocytes, and endothelial cells. Results Concordant with previous studies, both MSCs and SSCs showed similar morphology, surface protein expression, and were able to differentiate into osteoblasts and adipocytes. Using an endothelial induction culture system combined with an in vitro matrigel angiogenesis assay, hNFSSCs and hADSSCs exhibited the highest tube-forming capability, which was similar to those formed by human umbilical vein endothelial cells (HUVEC, with hNFSSCs forming the most tightly packed, longest, and largest diameter tubules among the three cell types. CD146 was highly expressed on hNFSSCs and HUVEC followed by hADSSCs, and hMSC-TERT, while its expression was almost absent on hADMSCs. Similarly, higher vascular density (based on the expression of CD31, CD34, vWF, CD146 and SMA was observed in neonatal skin, followed by adult dermal skin and adipose tissue. Thus, our preliminary data indicated a plausible relationship between vascular densities, and the expression of CD146 on multipotent cells derived from those tissues. Conclusions Our data is the first to demonstrate that human dermal skin stromal cells can be differentiated into endothelial lineage. Hence, SSCs

  11. Production of interleukin-1alpha by human endometrial stromal cells is triggered during menses and dysfunctional bleeding and is induced in culture by epithelial interleukin-1alpha released upon ovarian steroids withdrawal.

    Science.gov (United States)

    Pretto, Chrystel M; Gaide Chevronnay, Héloïse P; Cornet, Patricia B; Galant, Christine; Delvaux, Denis; Courtoy, Pierre J; Marbaix, Etienne; Henriet, Patrick

    2008-10-01

    Endometrial breakdown during menstruation and dysfunctional bleeding is triggered by the abrupt expression of matrix metalloproteinases (MMPs), including interstitial collagenase (MMP-1). The paracrine induction of MMP-1 in stromal cells via epithelium-derived IL-1alpha is repressed by ovarian steroids. However, the control by estradiol (E) and progesterone (P) of endometrial IL-1alpha expression and bioactivity remains unknown. Variations of endometrial IL-1alpha mRNA and protein along the menstrual cycle and during dysfunctional bleeding were determined using RT-PCR, in situ hybridization, and immunolabeling. The mechanism of EP control was analyzed using culture of explants, laser capture microdissection, and purified cells. Data were compared with expression changes of IL-1beta and IL-1 receptor antagonist. IL-1alpha is synthesized by epithelial cells throughout the cycle but E and/or P prevents its release. In contrast, endometrial stromal cells produce IL-1alpha only at menses and during irregular bleeding in areas of tissue breakdown. Stromal expression of IL-1alpha, like that of MMP-1, is repressed by P (alone or with E) but triggered by epithelium-derived IL-1alpha released upon EP withdrawal. Our experiments in cultured endometrium suggest that IL-1alpha released by epithelial cells triggers the production of IL-1alpha by stromal cells in a paracrine amplification loop to induce MMP-1 expression during menstruation and dysfunctional bleeding. All three steps of this amplification cascade are repressed by EP.

  12. Kallikrein-related peptidase 4 induces cancer-associated fibroblast features in prostate-derived stromal cells.

    Science.gov (United States)

    Kryza, Thomas; Silva, Lakmali M; Bock, Nathalie; Fuhrman-Luck, Ruth A; Stephens, Carson R; Gao, Jin; Samaratunga, Hema; Lawrence, Mitchell G; Hooper, John D; Dong, Ying; Risbridger, Gail P; Clements, Judith A

    2017-10-01

    The reciprocal communication between cancer cells and their microenvironment is critical in cancer progression. Although involvement of cancer-associated fibroblasts (CAF) in cancer progression is long established, the molecular mechanisms leading to differentiation of CAFs from normal fibroblasts are poorly understood. Here, we report that kallikrein-related peptidase-4 (KLK4) promotes CAF differentiation. KLK4 is highly expressed in prostate epithelial cells of premalignant (prostatic intraepithelial neoplasia) and malignant lesions compared to normal prostate epithelia, especially at the peristromal interface. KLK4 induced CAF-like features in the prostate-derived WPMY1 normal stromal cell line, including increased expression of alpha-smooth muscle actin, ESR1 and SFRP1. KLK4 activated protease-activated receptor-1 in WPMY1 cells increasing expression of several factors (FGF1, TAGLN, LOX, IL8, VEGFA) involved in prostate cancer progression. In addition, KLK4 induced WPMY1 cell proliferation and secretome changes, which in turn stimulated HUVEC cell proliferation that could be blocked by a VEGFA antibody. Importantly, the genes dysregulated by KLK4 treatment of WPMY1 cells were also differentially expressed between patient-derived CAFs compared to matched nonmalignant fibroblasts and were further increased by KLK4 treatment. Taken together, we propose that epithelial-derived KLK4 promotes tumour progression by actively promoting CAF differentiation in the prostate stromal microenvironment. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

  13. High Harvest Yield, High Expansion, and Phenotype Stability of CD146 Mesenchymal Stromal Cells from Whole Primitive Human Umbilical Cord Tissue

    Directory of Open Access Journals (Sweden)

    Rebecca C. Schugar

    2009-01-01

    Full Text Available Human umbilical cord blood is an excellent primitive source of noncontroversial stem cells for treatment of hematologic disorders; meanwhile, new stem cell candidates in the umbilical cord (UC tissue could provide therapeutic cells for nonhematologic disorders. We show novel in situ characterization to identify and localize a panel of some markers expressed by mesenchymal stromal cells (MSCs; CD44, CD105, CD73, CD90 and CD146 in the UC. We describe enzymatic isolation and purification methods of different UC cell populations that do not require manual separation of the vessels and stroma of the coiled, helical-like UC tissue. Unique quantitation of in situ cell frequency and stromal cell counts upon harvest illustrate the potential to obtain high numerical yields with these methods. UC stromal cells can differentiate to the osteogenic and chondrogenic lineages and, under specific culturing conditions, they exhibit high expandability with unique long-term stability of their phenotype. The remarkable stability of the phenotype represents a novel finding for human MSCs, from any source, and supports the use of these cells as highly accessible stromal cells for both basic studies and potentially therapeutic applications such as allogeneic clinical use for musculoskeletal disorders.

  14. Immunohistochemical expression of platelet-derived growth factor receptors in ovarian cancer patients with long-term follow-up

    DEFF Research Database (Denmark)

    Madsen, Christine Vestergaard; Dahl Steffensen, Karina; Waldstrøm, Marianne

    2012-01-01

    relation to histopathological parameters and long-term overall survival. Methods. The immunohistochemical expression of PDGFR-α and PDGFR-β was investigated in tumor and stromal cells in 170 patients with histologically verified epithelial ovarian cancer. Results. Almost half of the tumor specimens showed......Introduction. The well-documented role of the PDGF system in tumor growth and angiogenesis has prompted the development of new biological agents targeting the PDGF system. The aim of the present study was to analyze the expression of the PDGF-receptors in ovarian cancer and to investigate its...... high expression of PDGFR-α and PDGFR-β in tumor cells (43% and 41%) and in stromal compartments (32% and 44%). There was a significant association between high expression of PDGFR-α and high expression of PDGFR-β in both tumor and stromal cells. Coexpression of PDGFR-α and PDGFR-β in stromal cells...

  15. Influence of vascular endothelial growth factor stimulation and serum deprivation on gene activation patterns of human adipose tissue-derived stromal cells

    DEFF Research Database (Denmark)

    Tratwal, Josefine; Mathiasen, Anders Bruun; Juhl, Morten

    2015-01-01

    INTRODUCTION: Stimulation of mesenchymal stromal cells and adipose tissue-derived stromal cells (ASCs) with vascular endothelial growth factor (VEGF) has been used in multiple animal studies and clinical trials for regenerative purposes. VEGF stimulation is believed to promote angiogenesis and VEGF...... stimulation is usually performed under serum deprivation. Potential regenerative molecular mechanisms are numerous and the role of contributing factors is uncertain. The aim of the current study was to investigate the effect of in vitro serum deprivation and VEGF stimulation on gene expression patterns...... of ASCs. METHODS: Gene expressions of ASCs cultured in complete medium, ASCs cultured in serum-deprived medium and ASCs stimulated with VEGF in serum-deprived medium were compared. ASC characteristics according to criteria set by the International Society of Cellular Therapy were confirmed by flow...

  16. Inhibiting actin depolymerization enhances osteoblast differentiation and bone formation in human stromal stem cells

    DEFF Research Database (Denmark)

    Chen, Li; Shi, Kaikai; Frary, Charles

    2015-01-01

    Remodeling of the actin cytoskeleton through actin dynamics is involved in a number of biological processes, but its role in human stromal (skeletal) stem cells (hMSCs) differentiation is poorly understood. In the present study, we demonstrated that stabilizing actin filaments by inhibiting gene...... expression of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) in hMSCs, enhanced cell viability and differentiation into osteoblastic cells (OB) in vitro, as well as heterotopic bone formation in vivo. Similarly, treating hMSC with Phalloidin, which is known to stabilize...... polymerized actin filaments, increased hMSCs viability and OB differentiation. Conversely, Cytocholasin D, an inhibitor of actin polymerization, reduced cell viability and inhibited OB differentiation of hMSC. At a molecular level, preventing Cofilin phosphorylation through inhibition of LIM domain kinase 1...

  17. Comparison of different culture conditions for human mesenchymal stromal cells for clinical stem cell therapy

    DEFF Research Database (Denmark)

    Haack-Sorensen, M.; Friis, T.; Bindslev, L.

    2008-01-01

    OBJECTIVE: Mesenchymal stromal cells (MSCs) from adult bone marrow (BM) are considered potential candidates for therapeutic neovascularization in cardiovascular disease. When implementing results from animal trials in clinical treatment, it is essential to isolate and expand the MSCs under...... conditions following good manufacturing practice (GMP). The aims of the study were first to establish culture conditions following GMP quality demands for human MSC expansion and differentiation for use in clinical trials, and second to compare these MSCs with MSCs derived from culture in four media commonly...... analysis showed that the plastic-adherent MSCs cultured in EMEA medium or in the other four media were identically negative for the haematopoietic surface markers CD45 and CD34 and positive for CD105, CD73, CD90, CD166 and CD13, which in combined expression is characteristic of MSCs. MSC stimulation...

  18. Induction of quiescence (G0) in bone marrow stromal stem cells enhances their stem cell characteristics

    DEFF Research Database (Denmark)

    Rumman, Mohammad; Majumder, Abhijit; Harkness, Linda

    2018-01-01

    Several studies have suggested that bone marrow stromal steam cells (BMSC) exist in a quiescent state (G0) within the in vivo niche; however, an explicit analysis of the biology of G0 state-BMSC has not been reported. We hypothesized that induction of G0 in BMSC might enhance their stem cell...... properties. Thus, we induced quiescence in BMSC in vitro by (a) suspension culture in a viscous medium or (b) culture on soft polyacrylamide substrate; and examined their molecular and functional phenotype. Induction of G0 was confirmed by bromo-deoxyuridine (BrdU) labelling and analysis of cell cycle gene...... expression. Upon reactivation and re-entry into cell cycle, G0 state-BMSC exhibited enhanced clonogenic self-renewal, preferential differentiation into osteoblastic rather than adipocytic cells and increased ectopic bone formation when implanted subcutaneously in vivo in immune-deficient mice, compared...

  19. Clinical-grade production of human mesenchymal stromal cells: occurrence of aneuploidy without transformation.

    Science.gov (United States)

    Tarte, Karin; Gaillard, Julien; Lataillade, Jean-Jacques; Fouillard, Loic; Becker, Martine; Mossafa, Hossein; Tchirkov, Andrei; Rouard, Hélène; Henry, Catherine; Splingard, Marie; Dulong, Joelle; Monnier, Delphine; Gourmelon, Patrick; Gorin, Norbert-Claude; Sensebé, Luc

    2010-02-25

    Clinical-grade human mesenchymal stromal cells (MSCs) have been expanded in vitro for tissue engineering or immunoregulatory purposes without standardized culture conditions or release criteria. Although human MSCs show poor susceptibility for oncogenic transformation, 2 recent studies described their capacity to accumulate chromosomal instability and to give rise to carcinoma in immunocompromised mice after long-term culture. We thus investigated the immunologic and genetic features of MSCs expanded with fetal calf serum and fibroblast growth factor or with platelet lysate in 4 cell-therapy facilities during 2 multicenter clinical trials. Cultured MSCs showed a moderate expression of human leukocyte antigen-DR without alteration of their low immunogenicity or their immunomodulatory capacity. Moreover, some transient and donor-dependent recurring aneuploidy was detected in vitro, independently of the culture process. However, MSCs with or without chromosomal alterations showed progressive growth arrest and entered senescence without evidence of transformation either in vitro or in vivo.

  20. Multiple intracellular signaling pathways orchestrate adipocytic differentiation of human bone marrow stromal stem cells

    DEFF Research Database (Denmark)

    Ayesh Hafez Ali, Dalia; Abuelreich, Sarah; Alkeraishan, Nora

    2018-01-01

    during adipocyte differentiation of human bone marrow stromal (mesenchymal) stem cells (hMSCs) and identified 2,589 up-regulated and 2,583 down-regulated mRNA transcripts. Pathway analysis on the up-regulated gene list untraveled enrichment in multiple signaling pathways including insulin receptor......Bone marrow adipocyte formation plays a role in bone homeostasis and whole body energy metabolism. However, the transcriptional landscape and signaling pathways associated with adipocyte lineage commitment and maturation are not fully delineated. Thus, we performed global gene expression profiling...... signaling, focal Adhesion, metapathway biotransformation, a number of metabolic pathways e.g. selenium metabolism, Benzo(a)pyrene metabolism, fatty acid, triacylglycerol, ketone body metabolism, tryptophan metabolism, and catalytic cycle of mammalian flavin-containing monooxygenase (FMOs). On the other hand...

  1. Transcriptional profiling of mesenchymal stromal cells from young and old rats in response to Dexamethasone

    Directory of Open Access Journals (Sweden)

    Rechavi Gideon

    2006-04-01

    Full Text Available Abstract Background Marrow-derived stromal cells (MSCs maintain the capability of self-renewal and differentiation into multiple lineages in adult life. Age-related changes are recognized by a decline in the stemness potential that result in reduced regeneration potential of the skeleton. To explore the molecular events that underline skeletal physiology during aging we catalogued the profile of gene expression in ex vivo cultured MSCs derived from 3 and 15 month old rats. The ex vivo cultured cells were analyzed following challenge with or without Dexamethasone (Dex. RNA retrieved from these cells was analyzed using Affymetrix Gene Chips to compare the effect of Dex on gene expression in both age groups. Results The molecular mechanisms that underline skeletal senescence were studied by gene expression analysis of RNA harvested from MSCs. The analysis resulted in complex profiles of gene expression of various differentiation pathways. We revealed changes of lineage-specific gene expression; in general the pattern of expression included repression of proliferation and induction of differentiation. The functional analysis of genes clustered were related to major pathways; an increase in bone remodeling, osteogenesis and muscle formation, coupled with a decrease in adipogenesis. We demonstrated a Dex-related decrease in immune response and in genes that regulate bone resorption and an increase in osteoblastic differentiation. Myogenic-related genes and genes that regulate cell cycle were induced by Dex. While Dex repressed genes related to adipogenesis and catabolism, this decrease was complementary to an increase in expression of genes related to osteogenesis. Conclusion This study summarizes the genes expressed in the ex vivo cultured mesenchymal cells and their response to Dex. Functional clustering highlights the complexity of gene expression in MSCs and will advance the understanding of major pathways that trigger the natural changes

  2. Epithelialization and stromalization of porcine follicular granulosa cells during real-time proliferation - a primary cell culture approach.

    Science.gov (United States)

    Ciesiółka, S; Bryja, A; Budna, J; Kranc, W; Chachuła, A; Bukowska, D; Piotrowska, H; Porowski, L; Antosik, P; Bruska, M; Brüssow, K P; Nowicki, M; Zabel, M; Kempisty, B

    2016-01-01

    The process of oocyte growth and development takes place during long stages of folliculogenesis and oogenesis. This is accompanied by biochemical and morphological changes, occurring from the preantral to antral stages during ovarian follicle differentiation. It is well known that the process of follicle growth is associated with morphological modifications of theca (TCs) and granulosa cells (GCs). However, the relationship between proliferation and/or differentiation of porcine GCs during long-term in vitro culture requires further investigation. Moreover, the expression of cytokeratins and vimentin in porcine GCs, in relation to real-time cell proliferation, has yet to be explored. Utilizing confocal microscopy, we analyzed cytokeratin 18 (CK18), cytokeratin 8 + 18 + 19 (panCK), and vimentin (Vim) expression, as well as their protein distribution, within GCs isolated from slaughtered ovarian follicles. The cells were cultured for 168 h with protein expression and cell proliferation index analyzed at 24-h intervals. We found the highest expression of CK18, panCK, and Vim occurred at 120 h of in vitro culture (IVC) as compared with other experimental time intervals. All of the investigated proteins displayed cytoplasmic distribution. Analysis of real-time cell proliferation revealed an increased cell index after the first 24 h of IVC. Additionally, during each period between 24-168 h of IVC, a significant difference in the proliferation profile, expressed as the cell index, was also observed. We concluded that higher expression of vimentin at 120 h of in vitro proliferation might explain the culmination of the stromalization process associated with growth and domination of stromal cells in GC culture. Cytokeratin expression within GC cytoplasm confirms the presence of epithelial cells as well as epithelial-related GC development during IVC. Moreover, expression of both cytokeratins and vimentin during short-term culture suggests that the process of GC proliferation

  3. Impaired Angiogenic Potential of Human Placental Mesenchymal Stromal Cells in Intrauterine Growth Restriction.

    Science.gov (United States)

    Mandò, Chiara; Razini, Paola; Novielli, Chiara; Anelli, Gaia Maria; Belicchi, Marzia; Erratico, Silvia; Banfi, Stefania; Meregalli, Mirella; Tavelli, Alessandro; Baccarin, Marco; Rolfo, Alessandro; Motta, Silvia; Torrente, Yvan; Cetin, Irene

    2016-04-01

    Human placental mesenchymal stromal cells (pMSCs) have never been investigated in intrauterine growth restriction (IUGR). We characterized cells isolated from placental membranes and the basal disc of six IUGR and five physiological placentas. Cell viability and proliferation were assessed every 7 days during a 6-week culture. Expression of hematopoietic, stem, endothelial, and mesenchymal markers was evaluated by flow cytometry. We characterized the multipotency of pMSCs and the expression of genes involved in mitochondrial content and function. Cell viability was high in all samples, and proliferation rate was lower in IUGR compared with control cells. All samples presented a starting heterogeneous population, shifting during culture toward homogeneity for mesenchymal markers and occurring earlier in IUGR than in controls. In vitro multipotency of IUGR-derived pMSCs was restricted because their capacity for adipocyte differentiation was increased, whereas their ability to differentiate toward endothelial cell lineage was decreased. Mitochondrial content and function were higher in IUGR pMSCs than controls, possibly indicating a shift from anaerobic to aerobic metabolism, with the loss of the metabolic characteristics that are typical of undifferentiated multipotent cells. This study demonstrates that the loss of endothelial differentiation potential and the increase of adipogenic ability are likely to play a significant role in the vicious cycle of abnormal placental development in intrauterine growth restriction (IUGR). This is the first observation of a potential role for placental mesenchymal stromal cells in intrauterine growth restriction, thus leading to new perspectives for the treatment of IUGR. ©AlphaMed Press.

  4. miRNA signature and Dicer requirement during human endometrial stromal decidualization in vitro.

    Directory of Open Access Journals (Sweden)

    Carlos Estella

    Full Text Available Decidualization is a morphological and biochemical transformation of endometrial stromal fibroblast into differentiated decidual cells, which is critical for embryo implantation and pregnancy establishment. The complex regulatory networks have been elucidated at both the transcriptome and the proteome levels, however very little is known about the post-transcriptional regulation of this process. miRNAs regulate multiple physiological pathways and their de-regulation is associated with human disorders including gynaecological conditions such as endometriosis and preeclampsia. In this study we profile the miRNAs expression throughout human endometrial stromal (hESCs decidualization and analyze the requirement of the miRNA biogenesis enzyme Dicer during this process. A total of 26 miRNAs were upregulated and 17 miRNAs downregulated in decidualized hESCs compared to non-decidualized hESCs. Three miRNAs families, miR-181, miR-183 and miR-200, are down-regulated during the decidualization process. Using miRNAs target prediction algorithms we have identified the potential targets and pathways regulated by these miRNAs. The knockdown of Dicer has a minor effect on hESCs during in vitro decidualization. We have analyzed a battery of decidualization markers such as cell morphology, Prolactin, IGFBP-1, MPIF-1 and TIMP-3 secretion as well as HOXA10, COX2, SP1, C/EBPß and FOXO1 expression in decidualized hESCs with decreased Dicer function. We found decreased levels of HOXA10 and altered intracellular organization of actin filaments in Dicer knockdown decidualized hESCs compared to control. Our results provide the miRNA signature of hESC during the decidualization process in vitro. We also provide the first functional characterization of Dicer during human endometrial decidualization although surprisingly we found that Dicer plays a minor role regulating this process suggesting that alternative biogenesis miRNAs pathways must be involved in human

  5. Potential of iPSC-Derived Mesenchymal Stromal Cells for Treating Periodontal Disease

    Directory of Open Access Journals (Sweden)

    K. Hynes

    2018-01-01

    Full Text Available Mesenchymal stromal cell-like populations have been derived from mouse-induced pluripotent stem cells (miPSC-MSC with the capability for tissue regeneration. In this study, murine iPSC underwent differentiation towards an MSC-like immunophenotype. Stable miPSC-MSC cultures expressed the MSC-associated markers, CD73, CD105, and Sca-1, but lacked expression of the pluripotency marker, SSEA1, and hematopoietic markers, CD34 and CD45. Functionally, miPSC-MSC exhibited the potential for trilineage differentiation into osteoblasts, adipocytes, and chondrocytes and the capacity to suppress the proliferation of mitogen-activated splenocytes. The efficacy of miPSC-MSC was assessed in an acute inflammation model following systemic or local delivery into mice with subcutaneous implants containing heat-inactivated P. gingivalis. Histological analysis revealed less inflammatory cellular infiltrate within the sponges in mice treated with miPSC-MSC cells delivered locally rather than systemically. Assessment of proinflammatory cytokines in mouse spleens found that CXCL1 transcripts and protein were reduced in mice treated with miPSC-MSC. In a periodontitis model, mice subjected to oral inoculation with P. gingivalis revealed less bone tissue destruction and inflammation within the jaws when treated with miPSC-MSC compared to PBS alone. Our results demonstrated that miPSC-MSC derived from iPSC have the capacity to control acute and chronic inflammatory responses associated with the destruction of periodontal tissue. Therefore, miPSC-MSC present a promising novel source of stromal cells which could be used in the treatment of periodontal disease and other inflammatory systemic diseases such as rheumatoid arthritis.

  6. Identification and validation of multiple cell surface markers of clinical-grade adipose-derived mesenchymal stromal cells as novel release criteria for good manufacturing practice-compliant production

    NARCIS (Netherlands)

    Camilleri, Emily T.; Gustafson, Michael P.; Dudakovic, Amel; Riester, Scott M.; Garces, Catalina Galeano; Paradise, Christopher R.; Takai, Hideki; Karperien, Marcel; Cool, Simon; Sampen, Hee Jeong Im; Larson, A. Noelle; Qu, Wenchun; Smith, Jay; Dietz, Allan B.; van Wijnen, Andre J.

    2016-01-01

    Background: Clinical translation of mesenchymal stromal cells (MSCs) necessitates basic characterization of the cell product since variability in biological source and processing of MSCs may impact therapeutic outcomes. Although expression of classical cell surface markers (e.g., CD90, CD73, CD105,

  7. Comprehensive analysis of copy number aberrations in microsatellite stable colon cancer in view of stromal component.

    Science.gov (United States)

    Alonso, M Henar; Aussó, Susanna; Lopez-Doriga, Adriana; Cordero, David; Guinó, Elisabet; Solé, Xavier; Barenys, Mercè; de Oca, Javier; Capella, Gabriel; Salazar, Ramón; Sanz-Pamplona, Rebeca; Moreno, Victor

    2017-07-25

    Somatic copy number aberrations (CNAs) are common acquired changes in cancer cells having an important role in the progression of colon cancer (colorectal cancer, CRC). This study aimed to perform a characterisation of CNA and their impact in gene expression. Copy number aberrations were inferred from SNP array data in a series of 99 CRC. Copy number aberration events were calculated and used to assess the association between copy number dosage, clinical and molecular characteristics of the tumours, and gene expression changes. All analyses were adjusted for the quantity of stroma in each sample, which was inferred from gene expression data. High heterogeneity among samples was observed; the proportion of altered genome ranged between 0.04 and 26.6%. Recurrent CNA regions with gains were frequent in chromosomes 7p, 8q, 13q, and 20, whereas 8p, 17p, and 18 cumulated losses. A significant positive correlation was observed between the number of somatic mutations and total CNA (Spearman's r=0.42, P=0.006). Approximately 37% of genes located in CNA regions changed their level of expression and the average partial correlation (adjusted for stromal content) with copy number was 0.54 (interquartile range 0.20 to 0.81). Altered genes showed enrichment in pathways relevant for CRC. Tumours classified as CMS2 and CMS4 by the consensus molecular subtyping showed higher frequency of CNA. Losses of one small region in 1p36.33, with gene CDK11B, were associated with poor prognosis. More than 66% of the recurrent CNA were validated in the The Cancer Genome Atlas (TCGA) data when analysed with the same procedure. Furthermore, 79% of the genes with altered expression in our data were validated in the TCGA. Although CNA are frequent events in microsatellite stable CRC, few focal recurrent regions were found. These aberrations have strong effects on gene expression and contribute to deregulate relevant cancer pathways. Owing to the diploid nature of stromal cells, it is important to

  8. PI3K/Akt inhibitor LY294002 potentiates homoharringtonine antimyeloma activity in myeloma cells adhered to stromal cells and in SCID mouse xenograft.

    Science.gov (United States)

    Chen, Ping; Wen, Xiaofang; Wang, Bin; Hou, Diyu; Zou, Hong; Yuan, Qin; Yang, Hui; Xie, Jieqiong; Huang, Huifang

    2018-05-01

    Homoharringtonine (HHT) is a known anti-leukemia drug that inhibits multiple myeloma (MM) cells both in vitro and in vivo. Our prior study demonstrated that the potency of HHT in MM cells was compromised significantly when myeloma cells were co-cultured with BM stromal cells. This study aimed to investigate whether PI3K/Akt inhibitor LY294002 could potentiate the antimyeloma activity of HHT against MM cells adhered to BM stromal cells and in vivo xenograft models. A co-culture system composed of MM cells and human stromal cells was employed to mimic MM cells in bone marrow niche. The inhibitory and pro-apoptotic effect of HHT and LY294002 was determined by CCK-8 assay or flow cytometry. Expression of PI3K/Akt signaling molecules and anti-apoptotic protein myeloid cell leukemia-1 (Mcl-1) was assessed by western blot analysis and/or reverse transcription real-time quantitative PCR (RT-qPCR). MM xenografts were used to evaluate antitumor effect of combined therapy with HHT and LY294002. Adhesion to BM stromal cells rendered MM cells resistant to HHT whereas silencing Mcl-1 partly reversed the resistance. LY294002 induced apoptosis in MM cells and potentiated the antimyeloma effects of HHT by inhibiting the PI3K/Akt signal pathway which was abnormally activated during adhesion. LY294002 also enhanced the antimyeloma effect of HHT in in vivo xenograft models. These findings suggest that activation of PI3K/Akt signal pathway was responsible for the resistance to HHT in MM cells adhered to stromal cells. LY294002 can potentiate the antimyeloma activity of HHT both in vitro and in vivo, which may represent a new clinical treatment in MM.

  9. Adult Stromal (Skeletal, Mesenchymal) Stem Cells: Advances Towards Clinical Applications

    DEFF Research Database (Denmark)

    Kermani, Abbas Jafari; Harkness, Linda; Zaher, Walid

    2014-01-01

    Mesenchymal Stem Cells (MSC) are non-hematopoietic adult stromal cells that reside in a perivascular niche in close association with pericytes and endothelial cells and possess self-renewal and multi-lineage differentiation capacity. The origin, unique properties, and therapeutic benefits of MSC ...... the translation of MSC into clinic: Generation of MSC-like cells from human pluripotent stem cells, strategies to enhance homing of MSC to injured tissues, and targeting of MSC in vivo.......Mesenchymal Stem Cells (MSC) are non-hematopoietic adult stromal cells that reside in a perivascular niche in close association with pericytes and endothelial cells and possess self-renewal and multi-lineage differentiation capacity. The origin, unique properties, and therapeutic benefits of MSC...

  10. Current management and prognostic features for gastrointestinal stromal tumor (GIST

    Directory of Open Access Journals (Sweden)

    Lamba Gurpreet

    2012-06-01

    Full Text Available Abstract Stromal or mesenchymal neoplasms affecting the gastrointestinal (GI tract have undergone a remarkable evolution in how they are perceived, classified, approached, diagnosed and managed over the last 30 years. Gastrointestinal stromal tumors (GIST account for approximately 1% to 3% of all malignant GI tumors. The clinical features can vary depending on the anatomic location, size and aggressiveness of the tumor. Metastatic GIST represents a successful example of molecular targeted therapy. In this comprehensive review, we discuss the epidemiology, clinical features and diagnostic modalities for GIST. We also describe treatment options for early stage, locally advanced and metastatic GIST. Indications for neoadjuvant and adjuvant therapy along with duration of therapy are also explained. A brief discussion of latest biomarkers and updates from recent meetings is also provided.

  11. [Effects of nicotine on bone marrow stromal cells proliferation and differentiation of chondrocyte in vitro].

    Science.gov (United States)

    Ying, Xiao-zhou; Peng, Lei; Cheng, Shao-wen; Chen, Qing-yu; Zhang, Wei; Kou, Dong-quan; Shen, Yue

    2011-11-01

    To examine the effects of various concentration of nicotine on bone marrow stromal cells (BMSCs) proliferation and differentiation of cartilaginous in vitro. BMSCs was obtained from femoral bone and tibia of New-Zealand albino rabbit. The cells of the 3rd generation were used in study. Different concentration of nicotine (0, 1 x 10(-7), 1 x 10(-6), 1 x 10(-5) M) were added into BMSCs. BMSCs proliferation was analyzed by MTT assay at the 1, 4, 7, 14 days. The expression of collagen type II and aggrecan as the marker genes of cartilaginous differentiation from BMSCs were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Microscope showed that BMSCs transformed from round to fusiform shape. The concentration of nicotine in 1 x 10(-7), 1 x 10(-6) M had a significant positive effect on cell proliferation and the expression of type II collagen in a time-dependent manner when supplemented in commonly used induction media (P<0.05). Concentrations of nicotine in 1 x 10(-7) can promote the expression of aggrecan at the 7th day after induction,and in 1 x 10(-5) M may inhibit the expression of type II collagen and aggrecan. It was implied that local application of nicotine at an appropriate concentration may be a promising approach for enhancing cartilaginous differentiation capacity of BMSCs in cartilage tissue engineering.

  12. [Long-term expansion of multipotent mesenchymal stromal cells under reduced oxygen tension].

    Science.gov (United States)

    Rylova, Iu V; Buravkova, L B

    2013-01-01

    We have shown that the decrease in oxygen tension in the culture medium of multipotent mesenchymal stromal cells (MMSCs) results in a short-term reduction in the proportion of CD73(+)-cells in the population, without effecting the number of cells expressing other constitutive surface markers (CD90 and CD105). In this case, the heterogeneity of the cell population declined: large spread cells disappeared. The proliferative activity of MMSCs significantly increased and remained stable in conditions in which the oxygen content was close to the tissue oxygen levels (5% O2). At lower oxygen concentration, proliferative activity of the cells gradually reduced from passages 3-4. The increase in proliferative activity was not accompanied by increased expression of telomerase gene indicateding the alsance of cell transformation. However, genome-wide analysis of MMSC gene expression level revealed changes in expression of cyclins (CCND2 and PCNA), regulatory subunit cyclin-dependent kinase (CKS2) and an inhibitor of cyclin-dependent kinase (CDKN2C), regulating the cell cycle, which is obviously facilitated the increase in the proliferative capacity of cells at lower oxygen tension.

  13. Esophageal Gastrointestinal Stromal Tumors Presenting as Mediastinal Mass

    Directory of Open Access Journals (Sweden)

    M. Kafeel

    2013-11-01

    Full Text Available Gastrointestinal stromal tumors (GISTs are the most common mesenchymal tumors of the gastrointestinal tract and are predominant in the stomach and intestine but rare in the esophagus. Here, we report a case of esophageal GIST which presented as a mediastinal mass on chest X-ray and dyspnea. The case was initially diagnosed as leiomyosarcoma, which could create a diagnostic dilemma. Therefore, recognizing this uncommon presentation as a mediastinal mass with esophageal GIST is important in the differential diagnosis.

  14. Effects of SO2 and sulfite on stromal metabolism

    International Nuclear Information System (INIS)

    Anderson, L.E.; Muschinek, G.; Marques, I.

    1986-01-01

    SO 2 appears to have multiple effects on chloroplast stromal metabolism. What is unique about metabolism in the chloroplast is reductive modulation of enzyme activity. The evidence summarized here implicates both the components of the modulation process and the light modulated enzymes and ribulosebisphosphate carboxylase in SO 2 -sensitivity. Interference with electron transport, acidification of the stroma, and depletion of phosphates will further complicate metabolism in the photosynthesizing chloroplast when sensitive plants are exposed to SO 2 . 35 refs., 6 figs

  15. Rare case of gastrointestinal stromal tumor of the anal canal

    Directory of Open Access Journals (Sweden)

    Madhu Kumar

    2013-01-01

    Full Text Available Gastrointestinal stromal tumor (GIST is a rare mesenchymal neoplasm of the gastrointestinal tract. GIST of anal canal is very rare representing only 3% of all anorectal mesenchymal tumors. We report an extremely rare case of GIST of the anal canal in 60-years-old man with history of irregular bowel habits with dark colored stool mixed with blood and constipation from 6 month. Diagnosis was made on the basis of histomorphological and immunohistochemical examination.

  16. PRL-3 Is Involved in Estrogen- and IL-6-Induced Migration of Endometrial Stromal Cells From Ectopic Endometrium.

    Science.gov (United States)

    Ren, Shifan; Zhou, Yefang; Fang, Xiaoling; She, Xiaoling; Wu, Yilin; Wu, Xianqing

    2016-05-24

    To investigate the role of phosphatase of regenerating liver-3 (PRL-3) in the 17β-estradiol (E2)- and interleukin 6 (IL-6)-induced migration of endometrial stromal cells (ESCs) from ectopic endometrium. Ectopic endometrial tissues were collected from patients with endometriosis, and PRL-3 expression in ectopic and eutopic endometrium was examined by immunohistochemistry. Endometrial stromal cells isolated from ectopic endometrium were treated with E2, progesterone (P), IL-6, or sodium orthovanadate (Sov) to inhibit PRL-3. Total RNA and protein were extracted from ESCs after treatment for quantitative real-time polymerase chain reaction and Western blot analyses. Cell migration was assessed using a scratch wound assay. Phosphatase of regenerating liver 3 protein was highly expressed in the endometrial glandular cells (EGCs) and ESCs in ectopic endometrium, whereas its weak expression was observed only in EGCs in eutopic endometrium. Both E2 and IL-6 treatment significantly increased PRL-3 messenger RNA and protein expression, and P treatment significantly inhibited PRL-3 expression. However, E2-induced PRL-3 expression in ESCs from ectopic endometrium was significantly blocked by IL-6 antibody. Moreover, E2- and IL-6-enhanced cell migration was completely abrogated by Sov treatment. Furthermore, Sov treatment could significantly promote PTEN expression but inhibit E2- and IL-6-induced p-AKT activation. Phosphatase of regenerating liver 3 plays a key role in the E2- and IL-6-induced migration of ESCs from ectopic endometrium, a process that is involved in the PTEN-AKT signaling pathway. © The Author(s) 2016.

  17. Minocycline enhances the mesenchymal stromal/stem cell pro-healing phenotype in triple antimicrobial-loaded hydrogels.

    Science.gov (United States)

    Guerra, Alberto Daniel; Rose, Warren E; Hematti, Peiman; Kao, W John

    2017-03-15

    Mesenchymal stromal/stem cells (MSCs) have demonstrated pro-healing properties including an anti-inflammatory cytokine profile and the promotion of angiogenesis via expression of growth factors in pre-clinical models. MSCs encapsulated in poly(ethylene glycol) diacrylate (PEGdA) and thiolated gelatin poly(ethylene glycol) (Gel-PEG-Cys) crosslinked hydrogels have led to controlled cellular presentation at wound sites with favorable wound healing outcomes. However, the therapeutic potential of MSC-loaded hydrogels may be limited by non-specific protein adsorption on the delivery matrix that could facilitate the initial adhesion of microorganisms and subsequent virulent biofilm formation. Antimicrobials loaded concurrently in the hydrogels with MSCs could reduce microbial bioburden and promote healing, but the antimicrobial effect on the MSC wound healing capacity and the antibacterial efficacy of the hydrogels is unknown. We demonstrate that minocycline specifically induces a favorable change in MSC migration capacity, proliferation, gene expression, extracellular matrix (ECM) attachment, and adhesion molecule and growth factor release with subsequent increased angiogenesis. We then demonstrate that hydrogels loaded with MSCs, minocycline, vancomycin, and linezolid can significantly decrease bacterial bioburden. Our study suggests that minocycline can serve as a dual mechanism for the regenerative capacity of MSCs and the reduction of bioburden in triple antimicrobial-loaded hydrogels. Wound healing is a complex biological process that can be hindered by bacterial infection, excessive inflammation, and inadequate microvasculature. In this study, we develop a new formulation of poly(ethylene glycol) diacrylate and thiolated gelatin poly(ethylene glycol) crosslinked hydrogels loaded with minocycline, vancomycin, linezolid, and mesenchymal stromal/stem cells that induces a favorable wound healing phenotype in mesenchymal stromal/stem cells and prevents bacterial

  18. Stromal-cell and cancer-cell exosomes leading the metastatic exodus for the promised niche

    OpenAIRE

    Hoffman, Robert M

    2013-01-01

    Exosomes are thought to play an important role in metastasis. Luga and colleagues have described the production of exosomes by stromal cells such as cancer-associated fibroblasts that are taken up by breast cancer cells and are then loaded with Wnt 11, which is associated with stimulation of the invasiveness and metastasis of the breast cancer cells. Previous studies have shown that exosomes produced by breast cancer cells are taken up by stromal fibroblasts and other stromal cells, suggestin...

  19. Therapeutic effect of mesenchymal multipotent stromal cells on memory in animals with Alzheimer-type neurodegeneration.

    Science.gov (United States)

    Bobkova, N V; Poltavtseva, R A; Samokhin, A N; Sukhikh, G T

    2013-11-01

    Transplantation of human mesenchymal multipotent stromal cells improved spatial memory in bulbectomized mice with Alzheimer-type neurodegeneration. The positive effect was observed in 1 month after intracerebral transplantation and in 3 months after systemic injection of mesenchymal multipotent stromal cells. No cases of malignant transformation were noted. These findings indicate prospects of using mesenchymal multipotent stromal cells for the therapy of Alzheimer disease and the possibility of their systemic administration for attaining the therapeutic effect.

  20. Terrien's marginal degeneration accompanied by latticed stromal opacities.

    Science.gov (United States)

    Zhang, Yibing; Jia, Hui

    2014-05-01

    We report a case of Terrien's marginal degeneration (TMD) with a unilaterally typical narrow band of peripheral corneal stroma thinning, accompanied by the presence of an unusual network of opacities diffusing throughout the anterior stroma layers. A 43-year-old woman presented with superior nasal peripheral corneal thinning and an unusual network of polygonal stromal opacities in the anterior corneal stroma of the right eye. Latticed corneal changes were unusually extensive and distributed diffusely in the stroma. No abnormalities were found in the corneal epithelium and in the basal epithelial cells. No noticeable changes were found in the left eye. Because of a progressively worse ocular irritation of the right eye, a diagnosis of TMD was made for this patient. This case of TMD accompanied by keratopathy was unusual. The branching stromal lattice pattern of the corneal opacities was difficult to distinguish from lattice corneal dystrophy. In this case, the polygonal stromal opacities were located in the anterior corneal stroma and therefore were distinguished from a similar manifestation in posterior crocodile shagreen.

  1. Terrien’s Marginal Degeneration Accompanied by Latticed Stromal Opacities

    Science.gov (United States)

    Zhang, Yibing; Jia, Hui

    2014-01-01

    ABSTRACT Purpose We report a case of Terrien’s marginal degeneration (TMD) with a unilaterally typical narrow band of peripheral corneal stroma thinning, accompanied by the presence of an unusual network of opacities diffusing throughout the anterior stroma layers. Case Report A 43-year-old woman presented with superior nasal peripheral corneal thinning and an unusual network of polygonal stromal opacities in the anterior corneal stroma of the right eye. Latticed corneal changes were unusually extensive and distributed diffusely in the stroma. No abnormalities were found in the corneal epithelium and in the basal epithelial cells. No noticeable changes were found in the left eye. Because of a progressively worse ocular irritation of the right eye, a diagnosis of TMD was made for this patient. Conclusions This case of TMD accompanied by keratopathy was unusual. The branching stromal lattice pattern of the corneal opacities was difficult to distinguish from lattice corneal dystrophy. In this case, the polygonal stromal opacities were located in the anterior corneal stroma and therefore were distinguished from a similar manifestation in posterior crocodile shagreen. PMID:24681833

  2. Comparison of stromal corneal nerves between normal and keratoconus patients using confocal microscopy.

    Science.gov (United States)

    Ramírez Fernández, M; Hernández Quintela, E; Naranjo Tackman, R

    2014-08-01

    To evaluate the differences in stromal corneal nerves between normal patients and keratoconus patients. A total of 140 eyes of 70 normal patients (group A) and 122 eyes of 87 keratoconus patients (group B) were examined with the confocal microscope, with a central scan of the total corneal thickness being taken. The morphology and thickness of the corneal stromal nerves were evaluated by using the Navis v. 3.5.0. software. Nerve thickness was obtained from the mean between the widest and the narrowest portions of each stromal nerve. Corneal stromal nerves were observed as irregular linear hyper-reflective structures with wide and narrow portions in all cases. Mean corneal stromal nerves thickness in group A was 5.7±1.7 (range from 3.3 to 10.4 μ), mean corneal stromal nerves thickness in group B was 7.2±1.9 (range from 3.5 to 12.0 μ). There was a statistical significant difference (P<.05) in stromal corneal nerves thickness between group A and group B. Stromal corneal nerves morphology was similar in both groups, but stromal nerves were thicker in keratoconus patients. Copyright © 2013 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

  3. Adipose-Derived Stromal Cells for Treatment of Patients with Chronic Ischemic Heart Disease (MyStromalCell Trial)

    DEFF Research Database (Denmark)

    Qayyum, Abbas Ali; Mathiasen, Anders Bruun; Mygind, Naja Dam

    2017-01-01

    We aimed to evaluate the effect of intramyocardial injections of autologous VEGF-A165-stimulated adipose-derived stromal cells (ASCs) in patients with refractory angina. MyStromalCell trial is a randomized double-blind placebo-controlled study including sixty patients with CCS/NYHA class II...... to 54) (P= 0.41), and in METs 0.1 (95%CI -1.7 to 1.9) (P= 0.757). The difference between the groups was not significant (P= 0.680,P= 0.608, andP= 0.720 for time duration, watt, and METs, resp.). Intramyocardial delivered VEGF-A165-stimulated ASC treatment was safe but did not improve exercise capacity...

  4. Stromal haze, myofibroblasts, and surface irregularity after PRK.

    Science.gov (United States)

    Netto, Marcelo V; Mohan, Rajiv R; Sinha, Sunilima; Sharma, Ajay; Dupps, William; Wilson, Steven E

    2006-05-01

    The aim of this study was to investigate the relationship between the level of stromal surface irregularity after photorefractive keratectomy (PRK) and myofibroblast generation along with the development of corneal haze. Variable levels of stromal surface irregularity were generated in rabbit corneas by positioning a fine mesh screen in the path of excimer laser during ablation for a variable percentage of the terminal pulses of the treatment for myopia that does not otherwise generate significant opacity. Ninety-six rabbits were divided into eight groups: [see table in text]. Slit lamp analysis and haze grading were performed in all groups. Rabbits were sacrificed at 4 hr or 4 weeks after surgery and histochemical analysis was performed on corneas for apoptosis (TUNEL assay), myofibroblast marker alpha-smooth muscle actin (SMA), and integrin alpha4 to delineate the epithelial basement membrane. Slit-lamp grading revealed severe haze formation in corneas in groups IV and VI, with significantly less haze in groups II, III, and VII and insignificant haze compared with the unwounded control in groups I and V. Analysis of SMA staining at 4 weeks after surgery, the approximate peak of haze formation in rabbits, revealed low myofibroblast formation in group I (1.2+/-0.2 cells/400x field) and group V (1.8+/-0.4), with significantly more in groups II (3.5+/-1.8), III (6.8+/-1.6), VII (7.9+/-3.8), IV (12.4+/-4.2) and VI (14.6+/-5.1). The screened groups were significantly different from each other (p PRK groups. The -9.0 diopter PRK group VI had significantly more myofibroblast generation than the -9.0 diopter PRK with PTK-smoothing group VII (p PRK and the level of stromal surface irregularity. PTK-smoothing with methylcellulose was an effective method to reduce stromal surface irregularity and decreased both haze and associated myofibroblast density. We hypothesize that stromal surface irregularity after PRK for high myopia results in defective basement membrane

  5. Quantitative analysis of corneal stromal riboflavin concentration without epithelial removal.

    Science.gov (United States)

    Rubinfeld, Roy S; Stulting, R Doyle; Gum, Glenwood G; Talamo, Jonathan H

    2018-02-01

    To compare the corneal stromal riboflavin concentration and distribution using 2 transepithelial corneal crosslinking (CXL) systems. Absorption Systems, San Diego, California, USA. Experimental study. The stromal riboflavin concentration of 2 transepithelial CXL systems was compared in rabbit eyes in vivo. The systems were the Paracel/Vibex Xtra, comprising riboflavin 0.25% solution containing TRIS and ethylenediaminetetraacetic acid and an isotonic solution of riboflavin 0.25%, (Group 1) and the CXLO system (Group 2). Manufacturers' Instructions For Use were followed. The intensity of riboflavin fluorescence by slitlamp observation 10, 15, and 20 minutes after instillation was graded on a scale of 0 to 5. The animals were humanely killed and the corneal stromal samples analyzed with liquid chromatography and mass spectrometry. The mean riboflavin fluorescence intensity grades in Group 1 (4 eyes) were 3.8, 4.8, and 4.8 at 10, 15, and 20 minutes, respectively. The mean grades in Group 2 (3 eyes) were 2.0, 2.3, and 2.0, respectively. The riboflavin distribution was uniform in Group 1 but not in Group 2. The mean riboflavin concentration by liquid chromatography and mass spectrometry was 27.0 μg/g stromal tissue in Group 1 and 6.7 μg/g in Group 2. A stromal riboflavin concentration theoretically adequate for CXL, 15 μg/g, was achieved in all eyes in Group 1 and no eyes in Group 2. Slitlamp grading correlated well with liquid chromatography and mass spectrometry concentration (R 2  = 0.940). The system used in Group 1 produced corneal riboflavin concentrations that were theoretically adequate for effective transepithelial CXL (≥15 μg/g), while the system in Group 2 did not. Slitlamp grading successfully estimated the corneal riboflavin concentration and can be used to ensure an adequate concentration of riboflavin in the cornea for transepithelial CXL. Copyright © 2018 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

  6. Interaction between TNF and BmooMP-Alpha-I, a Zinc Metalloprotease Derived from Bothrops moojeni Snake Venom, Promotes Direct Proteolysis of This Cytokine: Molecular Modeling and Docking at a Glance

    Directory of Open Access Journals (Sweden)

    Maraisa Cristina Silva

    2016-07-01

    Full Text Available Tumor necrosis factor (TNF is a major cytokine in inflammatory processes and its deregulation plays a pivotal role in several diseases. Here, we report that a zinc metalloprotease extracted from Bothrops moojeni venom (BmooMP-alpha-I inhibits TNF directly by promoting its degradation. This inhibition was demonstrated by both in vitro and in vivo assays, using known TLR ligands. These findings are supported by molecular docking results, which reveal interaction between BmooMP-alpha-I and TNF. The major cluster of interaction between BmooMP-alpha-I and TNF was confirmed by the structural alignment presenting Ligand Root Mean Square Deviation LRMS = 1.05 Å and Interactive Root Mean Square Deviation IRMS = 1.01 Å, this result being compatible with an accurate complex. Additionally, we demonstrated that the effect of this metalloprotease on TNF is independent of cell cytotoxicity and it does not affect other TLR-triggered cytokines, such as IL-12. Together, these results indicate that this zinc metalloprotease is a potential tool to be further investigated for the treatment of inflammatory disorders involving TNF deregulation.

  7. Titanium biomaterials with complex surfaces induced aberrant peripheral circadian rhythms in bone marrow mesenchymal stromal cells.

    Science.gov (United States)

    Hassan, Nathaniel; McCarville, Kirstin; Morinaga, Kenzo; Mengatto, Cristiane M; Langfelder, Peter; Hokugo, Akishige; Tahara, Yu; Colwell, Christopher S; Nishimura, Ichiro

    2017-01-01

    Circadian rhythms maintain a high level of homeostasis through internal feed-forward and -backward regulation by core molecules. In this study, we report the highly unusual peripheral circadian rhythm of bone marrow mesenchymal stromal cells (BMSCs) induced by titanium-based biomaterials with complex surface modifications (Ti biomaterial) commonly used for dental and orthopedic implants. When cultured on Ti biomaterials, human BMSCs suppressed circadian PER1 expression patterns, while NPAS2 was uniquely upregulated. The Ti biomaterials, which reduced Per1 expression and upregulated Npas2, were further examined with BMSCs harvested from Per1::luc transgenic rats. Next, we addressed the regulatory relationship between Per1 and Npas2 using BMSCs from Npas2 knockout mice. The Npas2 knockout mutation did not rescue the Ti biomaterial-induced Per1 suppression and did not affect Per2, Per3, Bmal1 and Clock expression, suggesting that the Ti biomaterial-induced Npas2 overexpression was likely an independent phenomenon. Previously, vitamin D deficiency was reported to interfere with Ti biomaterial osseointegration. The present study demonstrated that vitamin D supplementation significantly increased Per1::luc expression in BMSCs, though the presence of Ti biomaterials only moderately affected the suppressed Per1::luc expression. Available in vivo microarray data from femurs exposed to Ti biomaterials in vitamin D-deficient rats were evaluated by weighted gene co-expression network analysis. A large co-expression network containing Npas2, Bmal1, and Vdr was observed to form with the Ti biomaterials, which was disintegrated by vitamin D deficiency. Thus, the aberrant BMSC peripheral circadian rhythm may be essential for the integration of Ti biomaterials into bone.

  8. Cigarette Smoking Impairs Adipose Stromal Cell Vasculogenic Activity and Abrogates Potency to Ameliorate Ischemia.

    Science.gov (United States)

    Barwinska, Daria; Traktuev, Dmitry O; Merfeld-Clauss, Stephanie; Cook, Todd G; Lu, Hongyan; Petrache, Irina; March, Keith L

    2018-06-01

    Cigarette smoking (CS) adversely affects the physiologic function of endothelial progenitor, hematopoietic stem and progenitor cells. However, the effect of CS on the ability of adipose stem/stromal cells (ASC) to promote vasculogenesis and rescue perfusion in the context of ischemia is unknown. To evaluate this, ASC from nonsmokers (nCS-ASC) and smokers (CS-ASC), and their activity to promote perfusion in hindlimb ischemia models, as well as endothelial cell (EC) survival and vascular morphogenesis in vitro were assessed. While nCS-ASC improved perfusion in ischemic limbs, CS-ASC completely lost this therapeutic effect. In vitro vasculogenesis assays revealed that human CS-ASC and ASC from CS-exposed mice showed compromised support of EC morphogenesis into vascular tubes, and the CS-ASC secretome was less potent in supporting EC survival/proliferation. Comparative secretome analysis revealed that CS-ASC produced lower amounts of hepatocyte growth factor (HGF) and stromal cell-derived growth factor 1 (SDF-1). Conversely, CS-ASC secreted the angiostatic/pro-inflammatory factor Activin A, which was not detected in nCS-ASC conditioned media (CM). Furthermore, higher Activin A levels were measured in EC/CS-ASC cocultures than in EC/nCS-ASC cocultures. CS-ASC also responded to inflammatory cytokines with 5.2-fold increase in Activin A secretion, whereas nCS-ASC showed minimal Activin A induction. Supplementation of EC/CS-ASC cocultures with nCS-ASC CM or with recombinant vascular endothelial growth factor, HGF, or SDF-1 did not rescue vasculogenesis, whereas inhibition of Activin A expression or activity improved network formation up to the level found in EC/nCS-ASC cocultures. In conclusion, ASC of CS individuals manifest compromised in vitro vasculogenic activity as well as in vivo therapeutic activity. Stem Cells 2018;36:856-867. © 2018 AlphaMed Press.

  9. In vitro differentiation of neural cells from human adipose tissue derived stromal cells.

    Science.gov (United States)

    Dave, Shruti D; Patel, Chetan N; Vanikar, Aruna V; Trivedi, Hargovind L

    2018-01-01

    Stem cells, including neural stem cells (NSCs), are endowed with self-renewal capability and hence hold great opportunity for the institution of replacement/protective therapy. We propose a method for in vitro generation of stromal cells from human adipose tissue and their differentiation into neural cells. Ten grams of donor adipose tissue was surgically resected from the abdominal wall of the human donor after the participants' informed consents. The resected adipose tissue was minced and incubated for 1 hour in the presence of an enzyme (collagenase-type I) at 37 0 C followed by its centrifugation. After centrifugation, the supernatant and pellets were separated and cultured in a medium for proliferation at 37 0 C with 5% CO2 for 9-10 days in separate tissue culture dishes for generation of mesenchymal stromal cells (MSC). At the end of the culture, MSC were harvested and analyzed. The harvested MSC were subjected for further culture for their differentiation into neural c