Endothelial progenitor cells in chronic obstructive pulmonary disease and emphysema

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Tracy, Russell P.; Parikh, Megha A.; Hoffman, Eric A.; Shimbo, Daichi; Austin, John H. M.; Smith, Benjamin M.; Hueper, Katja; Vogel-Claussen, Jens; Lima, Joao; Gomes, Antoinette; Watson, Karol; Kawut, Steven; Barr, R. Graham

2017-01-01

Endothelial injury is implicated in the pathogenesis of COPD and emphysema; however the role of endothelial progenitor cells (EPCs), a marker of endothelial cell repair, and circulating endothelial cells (CECs), a marker of endothelial cell injury, in COPD and its subphenotypes is unresolved. We hypothesized that endothelial progenitor cell populations would be decreased in COPD and emphysema and that circulating endothelial cells would be increased. Associations with other subphenotypes were examined. The Multi-Ethnic Study of Atherosclerosis COPD Study recruited smokers with COPD and controls age 50–79 years without clinical cardiovascular disease. Endothelial progenitor cell populations (CD34+KDR+ and CD34+KDR+CD133+ cells) and circulating endothelial cells (CD45dimCD31+CD146+CD133-) were measured by flow cytometry. COPD was defined by standard spirometric criteria. Emphysema was assessed qualitatively and quantitatively on CT. Full pulmonary function testing and expiratory CTs were measured in a subset. Among 257 participants, both endothelial progenitor cell populations, and particularly CD34+KDR+ endothelial progenitor cells, were reduced in COPD. The CD34+KDR+CD133+ endothelial progenitor cells were associated inversely with emphysema extent. Both endothelial progenitor cell populations were associated inversely with extent of panlobular emphysema and positively with diffusing capacity. Circulating endothelial cells were not significantly altered in COPD but were inversely associated with pulmonary microvascular blood flow on MRI. There was no consistent association of endothelial progenitor cells or circulating endothelial cells with measures of gas trapping. These data provide evidence that endothelial repair is impaired in COPD and suggest that this pathological process is specific to emphysema. PMID:28291826

Circulating Endothelial Cells in Patients with Heart Failure and Left Ventricular Dysfunction

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Martínez-Sales, Vicenta; Sánchez-Lázaro, Ignacio; Vila, Virtudes; Almenar, Luis; Contreras, Teresa; Reganon, Edelmiro

2011-01-01

Introduction and Aims: Acute and chronic heart failure may manifest different degrees of endothelial damage and angiogenesis. Circulating endothelial cells (CEC) have been identified as marker of vascular damage. The aim of our study was to evaluate the evolution of the CEC at different stages of patients with heart failure. We also investigated a potential correlation between CEC and markers of vascular damage and angiogenesis. Methods: We studied 32 heart failure patients at hospital admission (acute phase) and at revision after 3 months (stable phase) and 32 controls. Circulating markers of endothelial damage (CEC; von Willebrand factor, vWF and soluble E-selectin, sEsel) and angiogenesis (vascular endothelial growth factor, VEGF and thrombospondin-1) were quantified. Results: Levels of CEC, vWF, sEsel and VEGF are significantly higher in heart failure patients than in controls. Levels of CEC (36.9 ± 15.3 vs. 21.5 ± 10.0 cells/ml; p < 0.001), vWF (325 ± 101 vs. 231 ± 82%; p < 0.001) and VEGF (26.3 ± 15.2 vs. 21.9 ± 11.9 ng/ml; p < 0.001) are significantly higher in the acute phase than in the stable phase of heart failure. CEC levels correlate with vWF and VEGF. Results show than 100% of patients in acute phase and 37.5% in stable phase have levels of CEC higher than the 99th percentile of the distribution of controls (16 cells/ml). Therefore, increases in CEC represent a relative risk of 9.5 for heart failure patients suffering from acute phase. Conclusions: CEC, in addition to being elevated in heart failure, correlate with vWF levels, providing further support for CEC as markers of endothelial damage. Levels of CEC are associated with the acute phase of heart failure and could be used as a marker of the worsening in heart failure. PMID:21897001

The association between circulating endothelial progenitor cells and coronary collateral formation.

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Tokgözoğlu, Lale; Yorgun, Hikmet; Gürses, Kadri Murat; Canpolat, Uğur; Ateş, Ahmet Hakan; Tülümen, Erol; Kaya, Ergün Barış; Aytemir, Kudret; Kabakçı, Giray; Tuncer, Murat; Oto, Ali

2011-12-01

We investigated the relationship between coronary collateral formation and circulating endothelial progenitor cells (EPC) in patients undergoing coronary angiography. Circulating CD133(+)/34(+) and CD34(+)/KDR(+) EPCs were determined in 68 patients (normal coronary vessels in 24 patients and coronary artery disease (CAD) in 44 patients) (age: 58.7 ± 10.1, 64.7% male). Circulating EPCs were higher among patients with normal coronary vessels compared to patients with CAD for CD133(+)/34(+) (p collateral formation (p collateral formation after adjustment for other cardiovascular risk factors and extent of CAD (p = 0.037). In patients with severe coronary stenosis, those with increased circulating EPCs had better collateral formation compared to those with lower EPC counts. Our findings implicate that in addition to presence of critical stenosis, intact response of bone marrow is necessary for collateral formation in CAD. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

The level of circulating endothelial progenitor cells may be associated with the occurrence and recurrence of chronic subdural hematoma

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Yan Song

2013-01-01

Full Text Available OBJECTIVES: The onset of chronic subdural hematoma may be associated with direct or indirect minor injuries to the head or a poorly repaired vascular injury. Endothelial progenitor cells happen to be one of the key factors involved in hemostasis and vascular repair. This study was designed to observe the levels of endothelial progenitor cells, white blood cells, platelets, and other indicators in the peripheral blood of patients diagnosed with chronic subdural hematoma to determine the possible relationship between the endothelial progenitor cells and the occurrence, development, and outcomes of chronic subdural hematoma. METHOD: We enrolled 30 patients with diagnosed chronic subdural hematoma by computer tomography scanning and operating procedure at Tianjin Medical University General Hospital from July 2009 to July 2011. Meanwhile, we collected 30 cases of peripheral blood samples from healthy volunteers over the age of 50. Approximately 2 ml of blood was taken from veins of the elbow to test the peripheral blood routine and coagulation function. The content of endothelial progenitor cells in peripheral blood mononuclear cells was determined by flow cytometry. RESULTS: The level of endothelial progenitor cells in peripheral blood was significantly lower in preoperational patients with chronic subdural hematomas than in controls. There were no significant differences between the two groups regarding the blood routine and coagulation function. However, the levels of circulating endothelial progenitor cells were significantly different between the recurrent group and the non-recurrent group. CONCLUSIONS: The level of circulating endothelial progenitor cells in chronic subdural hematoma patients was significantly lower than the level in healthy controls. Meanwhile, the level of endothelial progenitor cells in recurrent patients was significantly lower than the level in patients without recurrence. Endothelial progenitor cells may be related to the

Circulating endothelial cells in patients with venous thromboembolism and myeloproliferative neoplasms.

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Cláudia Torres

Full Text Available BACKGROUND: Circulating endothelial cells (CEC may be a biomarker of vascular injury and pro-thrombotic tendency, while circulating endothelial progenitor cells (CEP may be an indicator for angiogenesis and vascular remodelling. However, there is not a universally accepted standardized protocol to identify and quantify these cells and its clinical relevancy remains to be established. OBJECTIVES: To quantify CEC and CEP in patients with venous thromboembolism (VTE and with myeloproliferative neoplasms (MPN, to characterize the CEC for the expression of activation (CD54, CD62E and procoagulant (CD142 markers and to investigate whether they correlate with other clinical and laboratory data. PATIENTS AND METHODS: Sixteen patients with VTE, 17 patients with MPN and 20 healthy individuals were studied. The CEC and CEP were quantified and characterized in the blood using flow cytometry, and the demographic, clinical and laboratory data were obtained from hospital records. RESULTS: We found the CEC counts were higher in both patient groups as compared to controls, whereas increased numbers of CEP were found only in patients with MPN. In addition, all disease groups had higher numbers of CD62E+ CEC as compared to controls, whereas only patients with VTE had increased numbers of CD142+ and CD54+ CEC. Moreover, the numbers of total and CD62+ CEC correlated positively with the white blood cells (WBC counts in both groups of patients, while the numbers of CEP correlated positively with the WBC counts only in patients with MPN. In addition, in patients with VTE a positive correlation was found between the numbers of CD54+ CEC and the antithrombin levels, as well as between the CD142+ CEC counts and the number of thrombotic events. CONCLUSIONS: Our study suggests that CEC counts may reveal endothelial injury in patients with VTE and MPN and that CEC may express different activation-related phenotypes depending on the disease status.

Circulating endothelial progenitor cells, Th1/Th2/Th17-related cytokines, and endothelial dysfunction in resistant hypertension.

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Magen, Eli; Feldman, Arie; Cohen, Ziona; Alon, Dora Ben; Minz, Evegeny; Chernyavsky, Alexey; Linov, Lina; Mishal, Joseph; Schlezinger, Menacham; Sthoeger, Zev

2010-02-01

A possible link between chronic vascular inflammation and arterial hypertension is now an object of intensive studies. To compare Th1/Th2/Th17 cells-related cytokines, circulating endothelial progenitor cells (EPC), and endothelial function in subjects with resistant arterial hypertension (RAH) and controlled arterial hypertension (CAH). Blood pressure was measured by electronic sphygmomanometer. EPC were identified as CD34+/CD133+/kinase insert domain receptor (KDR)+ cells by flow cytometry. Th1/Th2/Th17 cells-related cytokines were identified using the Human Th1/Th2/Th17 Cytokines MultiAnalyte ELISArray Kit. Endothelium-dependent (FMD) vasodilatation of brachial artery was measured by Doppler ultrasound scanning. RAH group (n = 20) and CAH group (n = 20) and 17 healthy individuals (control group) were recruited. In the RAH group, lower blood levels of EPC number (42.4 +/- 16.7 cells/mL) and EPC% (0.19 +/- 0.08%) were observed than in the CAH group (93.1 +/- 88.7 cells/mL; P = 0.017; 0.27 +/- 0.17; P = 0.036) and control group (68.5 +/- 63.6 cells/mL; P < 0.001; 0.28 +/- 0.17%; P = 0.003), respectively. Plasma transforming growth factor-beta1 levels were significantly higher in the RAH group (1767 +/- 364 pg/mL) than in the CAH group (1292 +/- 349; P < 0.001) and in control group (1203 +/- 419 pg/mL; P < 0.001). In the RAH group, statistically significant negative correlation was observed between systolic blood pressure and EPC% (r = -0.72, P < 0.01). FMD in the RAH group was significantly lower (5.5 +/- 0.8%) than in the CAH group (9.2 +/- 1.4; P < 0.001) and in healthy controls (10.1 +/- 1.1%; P < 0.001). RAH is characterized by reduced circulating EPC, substantial endothelial dysfunction, and increased plasma transforming growth factor-beta1 levels.

Key endothelial cell angiogenic mechanisms are stimulated by the circulating milieu in sickle cell disease and attenuated by hydroxyurea

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Lopes, Flavia C. M.; Traina, Fabiola; Almeida, Camila B.; Leonardo, Flavia C.; Franco-Penteado, Carla F.; Garrido, Vanessa T.; Colella, Marina P.; Soares, Raquel; Olalla-Saad, Sara T.; Costa, Fernando F.; Conran, Nicola

2015-01-01

As hypoxia-induced inflammatory angiogenesis may contribute to the manifestations of sickle cell disease, we compared the angiogenic molecular profiles of plasma from sickle cell disease individuals and correlated these with in vitro endothelial cell-mediated angiogenesis-stimulating activity and in vivo neovascularization. Bioplex demonstrated that plasma from patients with steady-state sickle cell anemia contained elevated concentrations of pro-angiogenic factors (angiopoietin-1, basic fibroblast growth factor, vascular endothelial growth factor, vascular endothelial growth factor-D and placental growth factor) and displayed potent pro-angiogenic activity, significantly increasing endothelial cell proliferation, migration and capillary-like structure formation. In vivo neovascularization of Matrigel plugs was significantly greater in sickle cell disease mice than in non-sickle cell disease mice, consistent with an up-regulation of angiogenesis in the disease. In plasma from patients with hemoglobin SC disease without proliferative retinopathy, anti-angiogenic endostatin and thrombospondin-2 were significantly elevated. In contrast, plasma from hemoglobin SC individuals with proliferative retinopathy had a pro-angiogenic profile and more significant effects on endothelial cell proliferation and capillary formation than plasma from patients without retinopathy. Hydroxyurea therapy was associated with significant reductions in plasma angiogenic factors and inhibition of endothelial cell-mediated angiogenic mechanisms and neovascularization. Thus, individuals with sickle cell anemia or hemoglobin SC disease with retinopathy present a highly angiogenic circulating milieu, capable of stimulating key endothelial cell-mediated angiogenic mechanisms. Combination anti-angiogenic therapy to prevent the progression of unregulated neovascularization and associated manifestations in sickle cell disease, such as pulmonary hypertension, may be indicated; furthermore, the

Analysis of circulating hem-endothelial marker RNA levels in preterm infants

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Kuint Jacob

2009-06-01

Full Text Available Abstract Background Circulating endothelial cells may serve as novel markers of angiogenesis. These include a subset of hem-endothelial progenitor cells that play a vital role in vascular growth and repair. The presence and clinical implications of circulating RNA levels as an expression for hematopoietic and endothelial-specific markers have not been previously evaluated in preterm infants. This study aims to determine circulating RNA levels of hem-endothelial marker genes in peripheral blood of preterm infants and begin to correlate these findings with prenatal complications. Methods Peripheral blood samples from seventeen preterm neonates were analyzed at three consecutive post-delivery time points (day 3–5, 10–15 and 30. Using quantitative reverse transcription-polymerase chain reaction we studied the expression patterns of previously established hem-endothelial-specific progenitor-associated genes (AC133, Tie-2, Flk-1 (VEGFR2 and Scl/Tal1 in association with characteristics of prematurity and preterm morbidity. Results Circulating Tie-2 and SCL/Tal1 RNA levels displayed an inverse correlation to gestational age (GA. We observed significantly elevated Tie-2 levels in preterm infants born to mothers with amnionitis, and in infants with sustained brain echogenicity on brain sonography. Other markers showed similar expression patterns yet we could not demonstrate statistically significant correlations. Conclusion These preliminary findings suggest that circulating RNA levels especially Tie2 and SCL decline with maturation and might relate to some preterm complication. Further prospective follow up of larger cohorts are required to establish this association.

Reversing resistance to vascular-disrupting agents by blocking late mobilization of circulating endothelial progenitor cells.

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Taylor, Melissa; Billiot, Fanny; Marty, Virginie; Rouffiac, Valérie; Cohen, Patrick; Tournay, Elodie; Opolon, Paule; Louache, Fawzia; Vassal, Gilles; Laplace-Builhé, Corinne; Vielh, Philippe; Soria, Jean-Charles; Farace, Françoise

2012-05-01

The prevailing concept is that immediate mobilization of bone marrow-derived circulating endothelial progenitor cells (CEP) is a key mechanism mediating tumor resistance to vascular-disrupting agents (VDA). Here, we show that administration of VDA to tumor-bearing mice induces 2 distinct peaks in CEPs: an early, unspecific CEP efflux followed by a late yet more dramatic tumor-specific CEP burst that infiltrates tumors and is recruited to vessels. Combination with antiangiogenic drugs could not disrupt the early peak but completely abrogated the late VDA-induced CEP burst, blunted bone marrow-derived cell recruitment to tumors, and resulted in striking antitumor efficacy, indicating that the late CEP burst might be crucial to tumor recovery after VDA therapy. CEP and circulating endothelial cell kinetics in VDA-treated patients with cancer were remarkably consistent with our preclinical data. These findings expand the current understanding of vasculogenic "rebounds" that may be targeted to improve VDA-based strategies. Our findings suggest that resistance to VDA therapy may be strongly mediated by late, rather than early, tumor-specific recruitment of CEPs, the suppression of which resulted in increased VDA-mediated antitumor efficacy. VDA-based therapy might thus be significantly enhanced by combination strategies targeting late CEP mobilization. © 2012 AACR

Late Release of Circulating Endothelial Cells and Endothelial Progenitor Cells after Chemotherapy Predicts Response and Survival in Cancer Patients

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Jeanine M. Roodhart

2010-01-01

Full Text Available We and others have previously demonstrated that the acute release of progenitor cells in response to chemotherapy actually reduces the efficacy of the chemotherapy. Here, we take these data further and investigate the clinical relevance of circulating endothelial (progenitor cells (CE(PCs and modulatory cytokines in patients after chemotherapy with relation to progression-free and overall survival (PFS/OS. Patients treated with various chemotherapeutics were included. Blood sampling was performed at baseline, 4 hours, and 7 and 21 days after chemotherapy. The mononuclear cell fraction was analyzed for CE(PC by FACS analysis. Plasma was analyzed for cytokines by ELISA or Luminex technique. CE(PCs were correlated with response and PFS/OS using Cox proportional hazard regression analysis. We measured CE(PCs and cytokines in 71 patients. Only patients treated with paclitaxel showed an immediate increase in endothelial progenitor cell 4 hours after start of treatment. These immediate changes did not correlate with response or survival. After 7 and 21 days of chemotherapy, a large and consistent increase in CE(PC was found (P < .01, independent of the type of chemotherapy. Changes in CE(PC levels at day 7 correlated with an increase in tumor volume after three cycles of chemotherapy and predicted PFS/OS, regardless of the tumor type or chemotherapy. These findings indicate that the late release of CE(PC is a common phenomenon after chemotherapeutic treatment. The correlation with a clinical response and survival provides further support for the biologic relevance of these cells in patients' prognosis and stresses their possible use as a therapeutic target.

Circulating endothelial cells (CECs and E-selectin: Predictors of preeclampsia

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Ferdous Mehrabian

2012-01-01

Full Text Available Background: Circulating endothelial cells (CECs and E-selectin are known as sensitive and specific markers of en-dothelial dysfunction. This study investigated whether CECs and E-selectin are surrogate biomarkers of preeclampsia and if measurement of CECs and E-selectin, early in the third trimester, could be a means of predicting preeclampsia. Methods: In this prospective, descriptive-analytic study, rollover test was performed on 523 pregnant women during 28-30 weeks of gestation. CECs were measured by anti-CD 146-driven immunomagnetic isolation in women with posi-tive rollover test. They were followed up prospectively until delivery without any active intervention. Women with and without preeclampsia were determined. The number of CECs and level of E-selectin were compared in the two studied groups. Results: From the 47 pregnant women with positive rollover test who were selected and followed up, 22 individuals were diagnosed with preeclampsia while the remainder were normotensive. Mean CEC numbers was significantly high-er in preeclamptic women than normal pregnancies (24.7 cells/mL vs. 13 cells/mL. The best cut-off point for CEC numbers was 6.5 with a sensitivity of 78.9% and a specificity of 69.1%. The level of E-selectin was significantly higher in mothers with preeclampsia (p < 0.05. Conclusions: Higher levels of CECs and E-selectin in women with positive rollover test who developed preeclampsia prior to onset of the complication were predictive of preeclampsia. However, larger studies are needed to confirm these findings.

Quantitative determination of circulating endothelial cells in persons with low dose radioactivity exposure

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Al-Massarani, Gh.; Najjar, F.

2013-04-01

The aim of this study was to determine the risk of occupational exposure to low doses of ionizing radiation on the endothelium detachment through the quantification of circulating endothelial cells (CEC) in the peripheral blood of 63 workers in the Atomic Energy Commission in Syria (AECS) using a Magnetic Immuno-separation technique (IMS) and compare the results with 28 healthy (controls) is not exposed during their careers for any type of ionizing radiation. Our study showed for the first time the significantly increasing in the circulating endothelial cells count (P <0.0001) when employees are exposed to low doses of radiation less than 50 mSv. This result with previous studies about the late effects of radiation, assuming the existence of impact late radiation exposure on the cohesion of the endothelium, despite the lack of correlation with radiation dose measured during the past four years of work in AECS (between 2006-2010). This is due to several reasons, including the small sample size and lack of commitment by some workers develop individual control films during some periods of their work. The prospective studies for such workers can allow us to know if the rise in the number of CEC will be considered an early indicator for the risk of a cardiovascular disease when workers exposed to low-doses of ionizing radiation( tens of millisievert) that up to date are considered harmless (author).

Plasma levels of stromal cell-derived factor-1 (CXCL12) and circulating endothelial progenitor cells in women with idiopathic heavy menstrual bleeding.

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Elsheikh, E; Andersson, E; Sylvén, C; Ericzon, B-G; Palmblad, J; Mints, M

2014-01-01

Do plasma levels of stromal cell-derived factor-1 (CXCL12, sometimes termed SDF-1) and the numbers of circulating endothelial progenitor cells (EPCs), EPC colony-forming units (EPC-CFU) and mature endothelial cells (ECs) differ between women with idiopathic heavy menstrual bleeding of endometrial origin (HMB-E) and controls and are they related to plasma levels of other angiogenic growth factors? Angiogenesis is altered in women with HMB-E, characterized by a reduction in mean plasma levels of CXCL12, a low number of EPCs-CFUs and a high level of circulating ECs. Plasma levels of CXCL12 are significantly higher during the proliferative than the secretory phase of the menstrual cycle in healthy women and exhibit a negative correlation with blood EPC-CFUs. A prospective cohort study in a university hospital setting. Between 2008 and 2009 10 HMB-E patients were recruited from Karolinska University Hospital. Ten healthy women were also included in the analysis. Ten healthy control women and 10 HMB-E patients, all with regular menstrual cycles, provided 4 blood samples during a single menstrual cycle: 2 in the proliferative phase, 1 at ovulation and 1 in the secretory phase. We assessed plasma levels of CXCL12, vascular endothelial growth factor A(165) (VEGFA), basic fibroblast growth factor (bFGF) and granulocyte and granulocyte-macrophage colony-stimulating factors by ELISA. We counted circulating EPC-CFUs by culture, and ECs and EPCs by flow cytometry and immunostaining for cell surface markers. Plasma levels of CXCL12 were significantly lower in HMB-E patients compared with control women (P Market Insurance. The authors have no conflict of interest to declare.

Circulating endothelial progenitor cells do not contribute to regeneration of endothelium after murine arterial injury

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Hagensen, Mette; Raarup, Merete Krog; Mortensen, Martin Bødtker

2012-01-01

into endothelial cells (ECs). We tested this theory in a murine arterial injury model using carotid artery transplants and fluorescent reporter mice. METHODS AND RESULTS: Wire-injured carotid artery segments from wild-type mice were transplanted into TIE2-GFP transgenic mice expressing green fluorescent protein...... (GFP) in ECs. We found that the endothelium regenerated with GFP(+) ECs as a function of time, evolving from the anastomosis sites towards the centre of the transplant. A migration front of ECs at Day 7 was verified by scanning electron microscopy and by bright-field microscopy using recipient TIE2-lac......Z mice with endothelial β-galactosidase expression. These experiments indicated migration of flanking ECs rather than homing of circulating cells as the underlying mechanism. To confirm this, we interposed non-injured wild-type carotid artery segments between the denuded transplant and the TIE2-GFP...

Impairment of circulating endothelial progenitors in Down syndrome

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Costa Valerio

2010-09-01

Full Text Available Abstract Background Pathological angiogenesis represents a critical issue in the progression of many diseases. Down syndrome is postulated to be a systemic anti-angiogenesis disease model, possibly due to increased expression of anti-angiogenic regulators on chromosome 21. The aim of our study was to elucidate some features of circulating endothelial progenitor cells in the context of this syndrome. Methods Circulating endothelial progenitors of Down syndrome affected individuals were isolated, in vitro cultured and analyzed by confocal and transmission electron microscopy. ELISA was performed to measure SDF-1α plasma levels in Down syndrome and euploid individuals. Moreover, qRT-PCR was used to quantify expression levels of CXCL12 gene and of its receptor in progenitor cells. The functional impairment of Down progenitors was evaluated through their susceptibility to hydroperoxide-induced oxidative stress with BODIPY assay and the major vulnerability to the infection with human pathogens. The differential expression of crucial genes in Down progenitor cells was evaluated by microarray analysis. Results We detected a marked decrease of progenitors' number in young Down individuals compared to euploid, cell size increase and some major detrimental morphological changes. Moreover, Down syndrome patients also exhibited decreased SDF-1α plasma levels and their progenitors had a reduced expression of SDF-1α encoding gene and of its membrane receptor. We further demonstrated that their progenitor cells are more susceptible to hydroperoxide-induced oxidative stress and infection with Bartonella henselae. Further, we observed that most of the differentially expressed genes belong to angiogenesis, immune response and inflammation pathways, and that infected progenitors with trisomy 21 have a more pronounced perturbation of immune response genes than infected euploid cells. Conclusions Our data provide evidences for a reduced number and altered

Kinetics of circulating endothelial progenitor cells in patients undergoing carotid artery surgery

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Kalender G

2016-12-01

Full Text Available G Kalender,1 A Kornberger,2 M Lisy,1 Andres Beiras-Fernandez,2 UA Stock2 1Deparment of General, Thoracic and Vascular Surgery, Hoechst Hospital, 2Department of Thoracic and Cardiovascular Surgery, University Hospital Frankfurt, Frankfurt am Main, Germany Aim: Endothelial progenitor cells (EPCs are primitive cells found in the bone marrow and peripheral blood (PB. In particular, the potential of EPCs to differentiate into mature endothelial cells remains of high interest for clinical applications such as bio-functionalized patches for autologous seeding after implantation. The objective of this study was to determine EPCs’ kinetics in patients undergoing carotid artery thromboendarterectomy (CTEA and patch angioplasty. Methods: Twenty CTEA patients were included (15 male, mean age 76 years. PB samples were taken at 1 day preoperatively, and at 1, 3, and 5 days postoperatively. Flow cytometric analysis was performed for CD34, CD133, KDR, and CD45. Expression of KDR, SDF-1α, and G-CSF was analyzed by means of enzyme-linked immunosorbent assay. Results: Fluorescence-activated cell sorting analysis revealed 0.031%±0.016% (% of PB mononuclear cells KDR+ cells and 0.052%±0.022% CD45-/CD34+/CD133+ cells, preoperatively. A 33% decrease of CD45–/CD34+/CD133+ cells was observed at day 1 after surgery. However, a relative number (compared to initial preoperative values of CD45-/CD34+/CD133+ cells was found on day 3 (82% and on day 5 (94% postoperatively. More profound upregulated levels of CD45–CD34+/CD133+ cells were observed for diabetic (+47% compared to nondiabetic and male (+38% compared to female patients. No significant postoperative time-dependent differences were found in numbers of KDR+ cells and the concentrations of the cytokines KDR and G-CSF. However, the SDF-1α levels decreased significantly on day 1 postoperatively but returned to preoperative levels by day 3. Conclusion: CTEA results in short-term downregulation of circulating

Data regarding association between serum osteoprotegerin level, numerous of circulating endothelial-derived and mononuclear-derived progenitor cells in patients with metabolic syndrome

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Alexander E. Berezin

2016-09-01

Full Text Available Metabolic syndrome (MetS is defined as cluster of multiple metabolic and cardiovascular (CV abnormalities included abdominal obesity, high-normal blood pressure, dyslipidaemia, and impaired fasting glucose tolerance that exhibits has a growing prevalence worldwide. We investigated whether an elevated level of osteoprotegerin (OPG predicts imbalance between different phenotypes of circulating endothelial (EPCs and mononuclear (MPCs progenitor cells in MetS patients. We have analyzed data regarding dysmetabolic disorder subjects without known CV disease, as well as with known type two diabetes mellitus. All patients have given their informed written consent for participation in the study. This article contains data on the independent predictors of depletion in numerous of circulating EPCs and MPCs in MetS patients. The data are supplemental to our original research article describing detailed associations of elevated OPG level in MetS patients with numerous of EPCs and MPCs beyond traditional CV risk factors. Keywords: Metabolic syndrome, Osteoprotegerin, Circulating endothelial derived progenitor cells, Mononuclear-derived progenitor cells

Leukocyte- and endothelial-derived microparticles: a circulating source for fibrinolysis

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Lacroix, Romaric; Plawinski, Laurent; Robert, Stéphane; Doeuvre, Loïc; Sabatier, Florence; Martinez de Lizarrondo, Sara; Mezzapesa, Anna; Anfosso, Francine; Leroyer, Aurelie S.; Poullin, Pascale; Jourde, Noémie; Njock, Makon-Sébastien; Boulanger, Chantal M.; Anglés-Cano, Eduardo; Dignat-George, Françoise

2012-01-01

Background We recently assigned a new fibrinolytic function to cell-derived microparticles in vitro. In this study we explored the relevance of this novel property of microparticles to the in vivo situation. Design and Methods Circulating microparticles were isolated from the plasma of patients with thrombotic thrombocytopenic purpura or cardiovascular disease and from healthy subjects. Microparticles were also obtained from purified human blood cell subpopulations. The plasminogen activators on microparticles were identified by flow cytometry and enzyme-linked immunosorbent assays; their capacity to generate plasmin was quantified with a chromogenic assay and their fibrinolytic activity was determined by zymography. Results Circulating microparticles isolated from patients generate a range of plasmin activity at their surface. This property was related to a variable content of urokinase-type plasminogen activator and/or tissue plasminogen activator. Using distinct microparticle subpopulations, we demonstrated that plasmin is generated on endothelial and leukocyte microparticles, but not on microparticles of platelet or erythrocyte origin. Leukocyte-derived microparticles bear urokinase-type plasminogen activator and its receptor whereas endothelial microparticles carry tissue plasminogen activator and tissue plasminogen activator/inhibitor complexes. Conclusions Endothelial and leukocyte microparticles, bearing respectively tissue plasminogen activator or urokinase-type plasminogen activator, support a part of the fibrinolytic activity in the circulation which is modulated in pathological settings. Awareness of this blood-borne fibrinolytic activity conveyed by microparticles provides a more comprehensive view of the role of microparticles in the hemostatic equilibrium. PMID:22733025

Endothelial glycocalyx on brain endothelial cells is lost in experimental cerebral malaria

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Hempel, Casper; Hyttel, Poul; Kurtzhals, Jørgen Al

2014-01-01

We hypothesized that the glycocalyx, which is important for endothelial integrity, is lost in severe malaria. C57BL/6 mice were infected with Plasmodium berghei ANKA, resulting in cerebral malaria, or P. chabaudi AS, resulting in uncomplicated malaria. We visualized the glycocalyx with transmission...... electron microscopy and measured circulating glycosaminoglycans by dot blot and ELISA. The glycocalyx was degraded in brain vasculature in cerebral and to a lesser degree uncomplicated malaria. It was affected on both intact and apoptotic endothelial cells. Circulating glycosaminoglycan levels suggested...

New insights into circulating FABP4: Interaction with cytokeratin 1 on endothelial cell membranes.

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Saavedra, Paula; Girona, Josefa; Bosquet, Alba; Guaita, Sandra; Canela, Núria; Aragonès, Gemma; Heras, Mercedes; Masana, Lluís

2015-11-01

Fatty acid-binding protein 4 (FABP4) is an adipose tissue-secreted adipokine that is involved in the regulation of energetic metabolism and inflammation. Increased levels of circulating FABP4 have been detected in individuals with cardiovascular risk factors. Recent studies have demonstrated that FABP4 has a direct effect on peripheral tissues, specifically promoting vascular dysfunction; however, its mechanism of action is unknown. The objective of this work was to assess the specific interactions between exogenous FABP4 and the plasma membranes of endothelial cells. Immunofluorescence assays showed that exogenous FABP4 localized along the plasma membranes of human umbilical vein endothelial cells (HUVECs), interacting specifically with plasma membrane proteins. Anti-FABP4 immunoblotting revealed two covalent protein complexes containing FABP4 and its putative receptor; these complexes were approximately 108 kDa and 77 kDa in size. Proteomics and mass spectrometry experiments revealed that cytokeratin 1 (CK1) was the FABP4-binding protein. An anti-CK1 immunoblot confirmed the presence of CK1. FABP4-CK1 complexes were also detected in HAECs, HCASMCs, HepG2 cells and THP-1 cells. Pharmacological FABP4 inhibition by BMS309403 results in a slight decrease in the formation of these complexes, indicating that fatty acids may play a role in FABP4 functionality. In addition, we demonstrated that exogenous FABP4 crosses the plasma membrane to enter the cytoplasm and nucleus in HUVECs. These findings indicate that exogenous FABP4 interacts with plasma membrane proteins, specifically CK1. These data contribute to our current knowledge regarding the mechanism of action of circulating FABP4.

Influence of depression and anxiety on circulating endothelial progenitor cells in patients with acute coronary syndromes.

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Felice, Francesca; Di Stefano, Rossella; Pini, Stefano; Mazzotta, Gianfranco; Bovenzi, Francesco M; Bertoli, Daniele; Abelli, Marianna; Borelli, Lucia; Cardini, Alessandra; Lari, Lisa; Gesi, Camilla; Michi, Paola; Morrone, Doralisa; Gnudi, Luigi; Balbarini, Alberto

2015-05-01

Circulating endothelial progenitor cells (EPCs) are related to endothelial function and progression of coronary artery disease. There is evidence of decreased numbers of circulating EPCs in patients with a current episode of major depression. We investigated the relationships between the level of circulating EPCs and depression and anxiety in patients with acute coronary syndrome (ACS). Patients with ACS admitted to three Cardiology Intensive Care Units were evaluated by the SCID-I to determine the presence of lifetime and/or current mood and anxiety disorders according to DSM-IV criteria. The EPCs were defined as CD133(+) CD34(+) KDR(+) and evaluated by flow cytometry. All patients underwent standardized cardiological and psychopathological evaluations. Parametric and nonparametric statistical tests were performed where appropriate. Out of 111 ACS patients, 57 were found to have a DSM-IV lifetime or current mood or anxiety disorder at the time of the inclusion in the study. The ACS group with mood or anxiety disorders showed a significant decrease in circulating EPC number compared with ACS patients without affective disorders. In addition, EPC levels correlated negatively with severity of depression and anxiety at index ACS episode. The current study indicates that EPCs circulate in decreased numbers in ACS patients with depression or anxiety and, therefore, contribute to explore new perspectives in the pathophysiology of the association between cardiovascular disorders and affective disorders. Copyright © 2015 John Wiley & Sons, Ltd.

Endothelial cell-derived microparticles induce plasmacytoid dendritic cell maturation: potential implications in inflammatory diseases.

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Angelot, Fanny; Seillès, Estelle; Biichlé, Sabeha; Berda, Yael; Gaugler, Béatrice; Plumas, Joel; Chaperot, Laurence; Dignat-George, Françoise; Tiberghien, Pierre; Saas, Philippe; Garnache-Ottou, Francine

2009-11-01

Increased circulating endothelial microparticles, resulting from vascular endothelium dysfunction, and plasmacytoid dendritic cell activation are both encountered in common inflammatory disorders. The aim of our study was to determine whether interactions between endothelial microparticles and plasmacytoid dendritic cells could contribute to such pathologies. Microparticles generated from endothelial cell lines, platelets or activated T cells were incubated with human plasmacytoid dendritic cells sorted from healthy donor blood or with monocyte-derived dendritic cells. Dendritic cell maturation was evaluated by flow cytometry, cytokine secretion as well as naive T-cell activation and polarization. Labeled microparticles were also used to study cellular interactions. Endothelial microparticles induced plasmacytoid dendritic cell maturation. In contrast, conventional dendritic cells were resistant to endothelial microparticle-induced maturation. In addition to upregulation of co-stimulatory molecules, endothelial microparticle-matured plasmacytoid dendritic cells secreted inflammatory cytokines (interleukins 6 and 8, but no interferon-alpha) and also induced allogeneic naive CD4(+) T cells to proliferate and to produce type 1 cytokines such as interferon-gamma and tumor necrosis factor-alpha. Endothelial microparticle endocytosis by plasmacytoid dendritic cells appeared to be required for plasmacytoid dendritic cell maturation. Importantly, the ability of endothelial microparticles to induce plasmacytoid dendritic cells to mature was specific as microparticles derived from activated T cells or platelets (the major source of circulating microparticules in healthy subjects) did not induce such plasmacytoid dendritic cell maturation. Our data show that endothelial microparticles specifically induce plasmacytoid dendritic cell maturation and production of inflammatory cytokines. This novel activation pathway may be implicated in various inflammatory disorders and

The dynamic change of endothelial cell, endothelin and 6-K-PGF1α in circulating blood of the patients with coronary heart disease

International Nuclear Information System (INIS)

Xia Zhiyun; Wang Linglin; Zou Songhai

1995-01-01

With the circulating endothelial cell (CEC) as an indicator of vessel endothelial cell (VEC) injury, and plasma endothelin (ET) and prostaglandin F 1α (PGI 2 ) reflecting the functional change of the VEC, a comparative study between 85 patients with coronary heart disease and 30 normal health, and also a dynamic observation of 50 patients with unstable angina pectoris and 20 patients with acute myocardial infarction (AMI) were reported. The result showed that in patients with coronary heart disease, peripheral blood circulating CEC and ET level was increased (P 2 decreased (P<0.01). All these were more significant in patients with unstable angina pectoris and myocardial infarction, and its magnitude correlated closely with the severity of the disease. All these showed that the VEC injury and the imbalance of its endothelial relaxing and contracting factors have played an important role in the pathogenesis of coronary heart disease

Kalirin and CHD7: novel endothelial dysfunction indicators in circulating extracellular vesicles from hypertensive patients with albuminuria

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de la Cuesta, Fernando; Baldan-Martin, Montserrat; Moreno-Luna, Rafael; Alvarez-Llamas, Gloria; Gonzalez-Calero, Laura; Mourino-Alvarez, Laura; Sastre-Oliva, Tamara; López, Juan A.; Vázquez, Jesús; Ruiz-Hurtado, Gema; Segura, Julian; Vivanco, Fernando; Ruilope, Luis M.; Barderas, Maria G.

2017-01-01

Despite of the great advances in anti-hypertensive therapies, many patients under Renin-Angiotensin- System (RAS) suppression develop albuminuria, which is a clear indicator of therapeutic inefficiency. Hence, indicators of vascular function are needed to assess patients’ condition and help deciding future therapies. Proteomic analysis of circulating extracellular vesicles (EVs) showed two proteins, kalirin and chromodomain-helicase-DNA-binding protein 7 (CHD7), increased in albuminuric patients. A positive correlation of both with the expression of the endothelial activation marker E-selectin was found in EVs. In vitro analysis using TNFα-treated adult human endothelial cells proved their involvement in endothelial cell activation. Hence, we propose protein levels of kalirin and CHD7 in circulating EVs as novel endothelial dysfunction markers to monitor vascular condition in hypertensive patients with albuminuria. PMID:28152519

Obesity suppresses circulating level and function of endothelial progenitor cells and heart function

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Tsai Tzu-Hsien

2012-07-01

Full Text Available Abstract Background and aim This study tested the hypothesis that obesity suppresses circulating number as well as the function of endothelial progenitor cells (EPCs and left ventricular ejection fraction (LVEF. Methods High fat diet (45 Kcal% fat was given to 8-week-old C57BL/6 J mice (n = 8 for 20 weeks to induce obesity (group 1. Another age-matched group (n = 8 were fed with control diet for 20 weeks as controls (group 2. The animals were sacrificed at the end of 20 weeks after obesity induction. Results By the end of study period, the heart weight, body weight, abdominal fat weight, serum levels of total cholesterol and fasting blood sugar were remarkably higher in group 1 than in group 2 (all p Conclusions Obesity diminished circulating EPC level, impaired the recovery of damaged endothelium, suppressed EPC angiogenesis ability and LVEF, and increased LV remodeling.

Endothelial microparticle-mediated transfer of MicroRNA-126 promotes vascular endothelial cell repair via SPRED1 and is abrogated in glucose-damaged endothelial microparticles.

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Jansen, Felix; Yang, Xiaoyan; Hoelscher, Marion; Cattelan, Arianna; Schmitz, Theresa; Proebsting, Sebastian; Wenzel, Daniela; Vosen, Sarah; Franklin, Bernardo S; Fleischmann, Bernd K; Nickenig, Georg; Werner, Nikos

2013-10-29

Repair of the endothelium after vascular injury is crucial for preserving endothelial integrity and preventing the development of vascular disease. The underlying mechanisms of endothelial cell repair are largely unknown. We sought to investigate whether endothelial microparticles (EMPs), released from apoptotic endothelial cells (ECs), influence EC repair. Systemic treatment of mice with EMPs after electric denudation of the endothelium accelerated reendothelialization in vivo. In vitro experiments revealed that EMP uptake in ECs promotes EC migration and proliferation, both critical steps in endothelial repair. To dissect the underlying mechanisms, Taqman microRNA array was performed, and microRNA (miR)-126 was identified as the predominantly expressed miR in EMPs. The following experiments demonstrated that miR-126 was transported into recipient human coronary artery endothelial cells by EMPs and functionally regulated the target protein sprouty-related, EVH1 domain-containing protein 1 (SPRED1). Knockdown of miR-126 in EMPs abrogated EMP-mediated effects on human coronary artery endothelial cell migration and proliferation in vitro and reendothelialization in vivo. Interestingly, after simulating diabetic conditions, EMPs derived from glucose-treated ECs contained significantly lower amounts of miR-126 and showed reduced endothelial repair capacity in vitro and in vivo. Finally, expression analysis of miR-126 in circulating microparticles from 176 patients with stable coronary artery disease with and without diabetes mellitus revealed a significantly reduced miR-126 expression in circulating microparticles from diabetic patients. Endothelial microparticles promote vascular endothelial repair by delivering functional miR-126 into recipient cells. In pathological hyperglycemic conditions, EMP-mediated miR-126-induced EC repair is altered.

Ticagrelor Improves Endothelial Function by Decreasing Circulating Epidermal Growth Factor (EGF

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Francesco Vieceli Dalla Sega

2018-04-01

Full Text Available Ticagrelor is one of the most powerful P2Y12 inhibitor. We have recently reported that, in patients with concomitant Stable Coronary Artery Disease (SCAD and Chronic Obstructive Pulmonary Disease (COPD undergoing percutaneous coronary intervention (PCI, treatment with ticagrelor, as compared to clopidogrel, is associated with an improvement of the endothelial function (Clinical Trial NCT02519608. In the present study, we showed that, in the same population, after 1 month treatment with ticagrelor, but not with clopidogrel, there is a decrease of the circulating levels of epidermal growth factor (EGF and that these changes in circulating levels of EGF correlate with on-treatment platelet reactivity. Furthermore, in human umbilical vein endothelial cells (HUVEC incubated with sera of the patients treated with ticagrelor, but not with clopidogrel there is an increase of p-eNOS levels. Finally, analyzing the changes in EGF and p-eNOS levels after treatment, we observed an inverse correlation between p-eNOS and EGF changes only in the ticagrelor group. Causality between EGF and eNOS activation was assessed in vitro in HUVEC where we showed that EGF decreases eNOS activity in a dose dependent manner. Taken together our data indicate that ticagrelor improves endothelial function by lowering circulating EGF that results in the activation of eNOS in the vascular endothelium.

Characterization of a distinct population of circulating human non-adherent endothelial forming cells and their recruitment via intercellular adhesion molecule-3.

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Sarah L Appleby

Full Text Available Circulating vascular progenitor cells contribute to the pathological vasculogenesis of cancer whilst on the other hand offer much promise in therapeutic revascularization in post-occlusion intervention in cardiovascular disease. However, their characterization has been hampered by the many variables to produce them as well as their described phenotypic and functional heterogeneity. Herein we have isolated, enriched for and then characterized a human umbilical cord blood derived CD133(+ population of non-adherent endothelial forming cells (naEFCs which expressed the hematopoietic progenitor cell markers (CD133, CD34, CD117, CD90 and CD38 together with mature endothelial cell markers (VEGFR2, CD144 and CD31. These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8 or myeloid markers (CD11b and CD14 which distinguishes them from 'early' endothelial progenitor cells (EPCs. Functional studies demonstrated that these naEFCs (i bound Ulex europaeus lectin, (ii demonstrated acetylated-low density lipoprotein uptake, (iii increased vascular cell adhesion molecule (VCAM-1 surface expression in response to tumor necrosis factor and (iv in co-culture with mature endothelial cells increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human endothelial cells (ECs. Extensive genomic and proteomic analyses of the naEFCs showed that intercellular adhesion molecule (ICAM-3 is expressed on their cell surface but not on mature endothelial cells. Furthermore, functional analysis demonstrated that ICAM-3 mediated the rolling and adhesive events of the naEFCs under shear stress. We suggest that the distinct population of naEFCs identified and characterized here represents a new valuable therapeutic target to control aberrant vasculogenesis.

Characterization of a Distinct Population of Circulating Human Non-Adherent Endothelial Forming Cells and Their Recruitment via Intercellular Adhesion Molecule-3

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Thompson, Emma J.; Barrett, Jeffrey M.; Tooley, Katie; Sen, Shaundeep; Sun, Wai Yan; Grose, Randall; Nicholson, Ian; Levina, Vitalina; Cooke, Ira; Talbo, Gert; Lopez, Angel F.; Bonder, Claudine S.

2012-01-01

Circulating vascular progenitor cells contribute to the pathological vasculogenesis of cancer whilst on the other hand offer much promise in therapeutic revascularization in post-occlusion intervention in cardiovascular disease. However, their characterization has been hampered by the many variables to produce them as well as their described phenotypic and functional heterogeneity. Herein we have isolated, enriched for and then characterized a human umbilical cord blood derived CD133+ population of non-adherent endothelial forming cells (naEFCs) which expressed the hematopoietic progenitor cell markers (CD133, CD34, CD117, CD90 and CD38) together with mature endothelial cell markers (VEGFR2, CD144 and CD31). These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8) or myeloid markers (CD11b and CD14) which distinguishes them from ‘early’ endothelial progenitor cells (EPCs). Functional studies demonstrated that these naEFCs (i) bound Ulex europaeus lectin, (ii) demonstrated acetylated-low density lipoprotein uptake, (iii) increased vascular cell adhesion molecule (VCAM-1) surface expression in response to tumor necrosis factor and (iv) in co-culture with mature endothelial cells increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human endothelial cells (ECs). Extensive genomic and proteomic analyses of the naEFCs showed that intercellular adhesion molecule (ICAM)-3 is expressed on their cell surface but not on mature endothelial cells. Furthermore, functional analysis demonstrated that ICAM-3 mediated the rolling and adhesive events of the naEFCs under shear stress. We suggest that the distinct population of naEFCs identified and characterized here represents a new valuable therapeutic target to control aberrant vasculogenesis. PMID:23144795

Human endothelial precursor cells express tumor endothelial marker 1/endosialin/CD248.

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Bagley, Rebecca G; Rouleau, Cecile; St Martin, Thia; Boutin, Paula; Weber, William; Ruzek, Melanie; Honma, Nakayuki; Nacht, Mariana; Shankara, Srinivas; Kataoka, Shiro; Ishida, Isao; Roberts, Bruce L; Teicher, Beverly A

2008-08-01

Angiogenesis occurs during normal physiologic processes as well as under pathologic conditions such as tumor growth. Serial analysis of gene expression profiling revealed genes [tumor endothelial markers (TEM)] that are overexpressed in tumor endothelial cells compared with normal adult endothelial cells. Because blood vessel development of malignant tumors under certain conditions may include endothelial precursor cells (EPC) recruited from bone marrow, we investigated TEM expression in EPC. The expression of TEM1 or endosialin (CD248) and other TEM has been discovered in a population of vascular endothelial growth factor receptor 2+/CD31+/CD45-/VE-cadherin+ EPC derived from human CD133+/CD34+ cells. EPC share some properties with fully differentiated endothelial cells from normal tissue, yet reverse transcription-PCR and flow cytometry reveal that EPC express higher levels of endosialin at the molecular and protein levels. The elevated expression of endosialin in EPC versus mature endothelial cells suggests that endosialin is involved in the earlier stages of tumor angiogenesis. Anti-endosialin antibodies inhibited EPC migration and tube formation in vitro. In vivo, immunohistochemistry indicated that human EPC continued to express endosialin protein in a Matrigel plug angiogenesis assay established in nude mice. Anti-endosialin antibodies delivered systemically at 25 mg/kg were also able to inhibit circulating murine EPC in nude mice bearing s.c. SKNAS tumors. EPC and bone marrow-derived cells have been shown previously to incorporate into malignant blood vessels in some instances, yet they remain controversial in the field. The data presented here on endothelial genes that are up-regulated in tumor vasculature and in EPC support the hypothesis that the angiogenesis process in cancer can involve EPC.

Fractalkine expression induces endothelial progenitor cell lysis by natural killer cells.

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Dilyana Todorova

Full Text Available BACKGROUND: Circulating CD34(+ cells, a population that includes endothelial progenitors, participate in the maintenance of endothelial integrity. Better understanding of the mechanisms that regulate their survival is crucial to improve their regenerative activity in cardiovascular and renal diseases. Chemokine-receptor cross talk is critical in regulating cell homeostasis. We hypothesized that cell surface expression of the chemokine fractalkine (FKN could target progenitor cell injury by Natural Killer (NK cells, thereby limiting their availability for vascular repair. METHODOLOGY/PRINCIPAL FINDINGS: We show that CD34(+-derived Endothelial Colony Forming Cells (ECFC can express FKN in response to TNF-α and IFN-γ inflammatory cytokines and that FKN expression by ECFC stimulates NK cell adhesion, NK cell-mediated ECFC lysis and microparticles release in vitro. The specific involvement of membrane FKN in these processes was demonstrated using FKN-transfected ECFC and anti-FKN blocking antibody. FKN expression was also evidenced on circulating CD34(+ progenitor cells and was detected at higher frequency in kidney transplant recipients, when compared to healthy controls. The proportion of CD34(+ cells expressing FKN was identified as an independent variable inversely correlated to CD34(+ progenitor cell count. We further showed that treatment of CD34(+ circulating cells isolated from adult blood donors with transplant serum or TNF-α/IFN-γ can induce FKN expression. CONCLUSIONS: Our data highlights a novel mechanism by which FKN expression on CD34(+ progenitor cells may target their NK cell mediated killing and participate to their immune depletion in transplant recipients. Considering the numerous diseased contexts shown to promote FKN expression, our data identify FKN as a hallmark of altered progenitor cell homeostasis with potential implications in better evaluation of vascular repair in patients.

Increased circulating endothelial apoptotic microparticle to endothelial progenitor cell ratio is associated with subsequent decline in glomerular filtration rate in hypertensive patients.

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Chien-Yi Hsu

Full Text Available BACKGROUND: Recent research indicates hypertensive patients with microalbuminuria have decreased endothelial progenitor cells (EPCs and increased levels of endothelial apoptotic microparticles (EMP. However, whether these changes are related to a subsequent decline in glomerular filtration rate (GFR remains unclear. METHODS AND RESULTS: We enrolled totally 100 hypertensive out-patients with eGFR ≥ 30 mL/min/1.73 m(2. The mean annual rate of GFR decline (△GFR/y was -1.49 ± 3.26 mL/min/1.73 m(2 per year during the follow-up period (34 ± 6 months. Flow cytometry was used to assess circulating EPC (CD34(+/KDR(+ and EMP levels (CD31(+/annexin V(+ in peripheral blood. The △GFR/y was correlated with the EMP to EPC ratio (r= -0.465, p<0.001, microalbuminuria (r= -0.329, p=0.001, and the Framingham risk score (r= -0.245, p=0.013. When we divided the patients into 4 groups according to the EMP to EPC ratio, there was an association between the EMP to EPC ratio and the ΔGFR/y (mean ΔGFR/y: 0.08 ± 3.04 vs. -0.50 ± 2.84 vs. -1.25 ± 2.49 vs. -4.42 ± 2.82, p<0.001. Multivariate analysis indicated that increased EMP to EPC ratio is an independent predictor of ΔeGFR/y. CONCLUSIONS: An increased circulating EMP to EPC ratio is associated with subsequent decline in GFR in hypertensive patients, which suggests endothelial damage with reduced vascular repair capacity may contribute to further deterioration of renal function in patients with hypertension.

The non-alcoholic fraction of beer increases stromal cell derived factor 1 and the number of circulating endothelial progenitor cells in high cardiovascular risk subjects: a randomized clinical trial.

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Chiva-Blanch, Gemma; Condines, Ximena; Magraner, Emma; Roth, Irene; Valderas-Martínez, Palmira; Arranz, Sara; Casas, Rosa; Martínez-Huélamo, Miriam; Vallverdú-Queralt, Anna; Quifer-Rada, Paola; Lamuela-Raventos, Rosa M; Estruch, Ramon

2014-04-01

Moderate alcohol consumption is associated with a decrease in cardiovascular risk, but fermented beverages seem to confer greater cardiovascular protection due to their polyphenolic content. Circulating endothelial progenitor cells (EPC) are bone-marrow-derived stem cells with the ability to repair and maintain endothelial integrity and function and are considered as a surrogate marker of vascular function and cumulative cardiovascular risk. Nevertheless, no study has been carried out on the effects of moderate beer consumption on the number of circulating EPC in high cardiovascular risk patients. To compare the effects of moderate consumption of beer, non-alcoholic beer and gin on the number of circulating EPC and EPC-mobilizing factors. In this crossover trial, 33 men at high cardiovascular risk were randomized to receive beer (30 g alcohol/d), the equivalent amount of polyphenols in the form of non-alcoholic beer, or gin (30 g alcohol/d) for 4 weeks. Diet and physical exercise were carefully monitored. The number of circulating EPC and EPC-mobilizing factors were determined at baseline and after each intervention. After the beer and non-alcoholic beer interventions, the number of circulating EPC significantly increased by 8 and 5 units, respectively, while no significant differences were observed after the gin period. In correlation, stromal cell derived factor 1 increased significantly after the non-alcoholic and the beer interventions. The non-alcoholic fraction of beer increases the number of circulating EPC in peripheral blood from high cardiovascular risk subjects. http://www.controlled-trials.com/ISRCTN95345245 ISRCTN95345245. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

EPO Receptor Gain-of-Function Causes Hereditary Polycythemia, Alters CD34+ Cell Differentiation and Increases Circulating Endothelial Precursors

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Perrotta, Silverio; Cucciolla, Valeria; Ferraro, Marcella; Ronzoni, Luisa; Tramontano, Annunziata; Rossi, Francesca; Scudieri, Anna Chiara; Borriello, Adriana; Roberti, Domenico; Nobili, Bruno; Cappellini, Maria Domenica; Oliva, Adriana; Amendola, Giovanni; Migliaccio, Anna Rita; Mancuso, Patrizia; Martin-Padura, Ines; Bertolini, Francesco; Yoon, Donghoon; Prchal, Josef T.; Della Ragione, Fulvio

2010-01-01

Background Gain-of-function of erythropoietin receptor (EPOR) mutations represent the major cause of primary hereditary polycythemia. EPOR is also found in non-erythroid tissues, although its physiological role is still undefined. Methodology/Principal Findings We describe a family with polycythemia due to a heterozygous mutation of the EPOR gene that causes a G→T change at nucleotide 1251 of exon 8. The novel EPOR G1251T mutation results in the replacement of a glutamate residue by a stop codon at amino acid 393. Differently from polycythemia vera, EPOR G1251T CD34+ cells proliferate and differentiate towards the erythroid phenotype in the presence of minimal amounts of EPO. Moreover, the affected individuals show a 20-fold increase of circulating endothelial precursors. The analysis of erythroid precursor membranes demonstrates a heretofore undescribed accumulation of the truncated EPOR, probably due to the absence of residues involved in the EPO-dependent receptor internalization and degradation. Mutated receptor expression in EPOR-negative cells results in EPOR and Stat5 phosphorylation. Moreover, patient erythroid precursors present an increased activation of EPOR and its effectors, including Stat5 and Erk1/2 pathway. Conclusions/Significance Our data provide an unanticipated mechanism for autosomal dominant inherited polycythemia due to a heterozygous EPOR mutation and suggest a regulatory role of EPO/EPOR pathway in human circulating endothelial precursors homeostasis. PMID:20700488

Quantification of circulating endothelial progenitor cells using the modified ISHAGE protocol.

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Caroline Schmidt-Lucke

2010-11-01

Full Text Available Circulating endothelial progenitor cells (EPC, involved in endothelial regeneration, neovascularisation, and determination of prognosis in cardiovascular disease can be characterised with functional assays or using immunofluorescence and flow cytometry. Combinations of markers, including CD34+KDR+ or CD133+KDR+, are used. This approach, however may not consider all characteristics of EPC. The lack of a standardised protocol with regards to reagents and gating strategies may account for the widespread inter-laboratory variations in quantification of EPC. We, therefore developed a novel protocol adapted from the standardised so-called ISHAGE protocol for enumeration of haematopoietic stem cells to enable comparison of clinical and laboratory data.In 25 control subjects, 65 patients with coronary artery disease (CAD; 40 stable CAD, 25 acute coronary syndrome/acute myocardial infarction (ACS, EPC were quantified using the following approach: Whole blood was incubated with CD45, KDR, and CD34. The ISHAGE sequential strategy was used, and finally, CD45(dimCD34(+ cells were quantified for KDR. A minimum of 100 CD34(+ events were collected. For comparison, CD45(+CD34(+ and CD45(-CD34(+ were analysed simultaneously. The number of CD45(dimCD34(+KDR(+ cells only were significantly higher in healthy controls compared to patients with CAD or ACS (p = 0.005 each, p<0.001 for trend. An inverse correlation of CD45(dimCD34(+KDR(+ with disease activity (r = -0.475, p<0.001 was confirmed. Only CD45(dimCD34(+KDR(+ correlated inversely with the number of diseased coronaries (r = -0.344; p<0.005. In a second study, a 4-week de-novo treatment of atorvastatin in stable CAD evoked an increase only of CD45(dimCD34(+KDR(+ EPC (p<0.05. CD45(+CD34(+KDR(+ and CD45(-CD34(+KDR(+ were indifferent between the three groups.Our newly established protocol adopted from the standardised ISHAGE protocol achieved higher accuracy in EPC enumeration confirming previous

Magnetizable stent-grafts enable endothelial cell capture

Science.gov (United States)

Tefft, Brandon J.; Uthamaraj, Susheil; Harburn, J. Jonathan; Hlinomaz, Ota; Lerman, Amir; Dragomir-Daescu, Dan; Sandhu, Gurpreet S.

2017-04-01

Emerging nanotechnologies have enabled the use of magnetic forces to guide the movement of magnetically-labeled cells, drugs, and other therapeutic agents. Endothelial cells labeled with superparamagnetic iron oxide nanoparticles (SPION) have previously been captured on the surface of magnetizable 2205 duplex stainless steel stents in a porcine coronary implantation model. Recently, we have coated these stents with electrospun polyurethane nanofibers to fabricate prototype stent-grafts. Facilitated endothelialization may help improve the healing of arteries treated with stent-grafts, reduce the risk of thrombosis and restenosis, and enable small-caliber applications. When placed in a SPION-labeled endothelial cell suspension in the presence of an external magnetic field, magnetized stent-grafts successfully captured cells to the surface regions adjacent to the stent struts. Implantation within the coronary circulation of pigs (n=13) followed immediately by SPION-labeled autologous endothelial cell delivery resulted in widely patent devices with a thin, uniform neointima and no signs of thrombosis or inflammation at 7 days. Furthermore, the magnetized stent-grafts successfully captured and retained SPION-labeled endothelial cells to select regions adjacent to stent struts and between stent struts, whereas the non-magnetized control stent-grafts did not. Early results with these prototype devices are encouraging and further refinements will be necessary in order to achieve more uniform cell capture and complete endothelialization. Once optimized, this approach may lead to more rapid and complete healing of vascular stent-grafts with a concomitant improvement in long-term device performance.

Magnetizable stent-grafts enable endothelial cell capture

Energy Technology Data Exchange (ETDEWEB)

Tefft, Brandon J. [Department of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (United States); Uthamaraj, Susheil [Division of Engineering, Mayo Clinic, Rochester, MN (United States); Harburn, J. Jonathan [School of Medicine, Pharmacy and Health, Durham University, Stockton-on-Tees (United Kingdom); Hlinomaz, Ota [Department of Cardioangiology, St. Anne' s University Hospital, Brno (Czech Republic); Lerman, Amir [Department of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (United States); Dragomir-Daescu, Dan [Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN (United States); Sandhu, Gurpreet S., E-mail: sandhu.gurpreet@mayo.edu [Department of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (United States)

2017-04-01

Emerging nanotechnologies have enabled the use of magnetic forces to guide the movement of magnetically-labeled cells, drugs, and other therapeutic agents. Endothelial cells labeled with superparamagnetic iron oxide nanoparticles (SPION) have previously been captured on the surface of magnetizable 2205 duplex stainless steel stents in a porcine coronary implantation model. Recently, we have coated these stents with electrospun polyurethane nanofibers to fabricate prototype stent-grafts. Facilitated endothelialization may help improve the healing of arteries treated with stent-grafts, reduce the risk of thrombosis and restenosis, and enable small-caliber applications. When placed in a SPION-labeled endothelial cell suspension in the presence of an external magnetic field, magnetized stent-grafts successfully captured cells to the surface regions adjacent to the stent struts. Implantation within the coronary circulation of pigs (n=13) followed immediately by SPION-labeled autologous endothelial cell delivery resulted in widely patent devices with a thin, uniform neointima and no signs of thrombosis or inflammation at 7 days. Furthermore, the magnetized stent-grafts successfully captured and retained SPION-labeled endothelial cells to select regions adjacent to stent struts and between stent struts, whereas the non-magnetized control stent-grafts did not. Early results with these prototype devices are encouraging and further refinements will be necessary in order to achieve more uniform cell capture and complete endothelialization. Once optimized, this approach may lead to more rapid and complete healing of vascular stent-grafts with a concomitant improvement in long-term device performance. - Highlights: • Magnetic stent-grafts were made from 2205 steel stents and polyurethane nanofibers. • Stent-grafts remained patent and formed a thin and uniform neointima when implanted. • Stent-grafts captured endothelial cells labeled with magnetic nanoparticles. �

Functional activities of receptors for tumor necrosis factor-alpha on human vascular endothelial cells.

NARCIS (Netherlands)

Paleolog, E.M.; Delasalle, S.A.; Buurman, W.A.; Feldmann, M.

1994-01-01

Tumor necrosis factor-alpha (TNF-alpha) plays a critical role in the control of endothelial cell function and hence in regulating traffic of circulating cells into tissues in vivo. Stimulation of endothelial cells in vitro by TNF-alpha increases the surface expression of leukocyte adhesion

Angiogenesis gene expression in murine endothelial cells during post-pneumonectomy lung growth

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Konerding Moritz A

2011-07-01

Full Text Available Abstract Although blood vessel growth occurs readily in the systemic bronchial circulation, angiogenesis in the pulmonary circulation is rare. Compensatory lung growth after pneumonectomy is an experimental model with presumed alveolar capillary angiogenesis. To investigate the genes participating in murine neoalveolarization, we studied the expression of angiogenesis genes in lung endothelial cells. After left pneumonectomy, the remaining right lung was examined on days 3, 6, 14 and 21days after surgery and compared to both no surgery and sham thoracotomy controls. The lungs were enzymatically digested and CD31+ endothelial cells were isolated using flow cytometry cell sorting. The transcriptional profile of the CD31+ endothelial cells was assessed using quantitative real-time polymerase chain reaction (PCR arrays. Focusing on 84 angiogenesis-associated genes, we identified 22 genes with greater than 4-fold regulation and significantly enhanced transcription (p

Impaired endothelial progenitor cell mobilization and dysfunctional bone marrow stroma in diabetes mellitus.

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Westerweel, Peter E; Teraa, Martin; Rafii, Shahin; Jaspers, Janneke E; White, Ian A; Hooper, Andrea T; Doevendans, Pieter A; Verhaar, Marianne C

2013-01-01

Circulating Endothelial Progenitor Cell (EPC) levels are reduced in diabetes mellitus. This may be a consequence of impaired mobilization of EPC from the bone marrow. We hypothesized that under diabetic conditions, mobilization of EPC from the bone marrow to the circulation is impaired -at least partly- due to dysfunction of the bone marrow stromal compartment. Diabetes was induced in mice by streptozotocin injection. Circulating Sca-1(+)Flk-1(+) EPC were characterized and quantified by flow cytometry at baseline and after mobilization with G-CSF/SCF injections. In vivo hemangiogenic recovery was tested by 5-FU challenge. Interaction within the bone marrow environment between CD34(+) hematopoietic progenitor cells (HPC) and supporting stroma was assessed by co-cultures. To study progenitor cell-endothelial cell interaction under normoglycemic and hyperglycemic conditions, a co-culture model using E4Orf1-transfected human endothelial cells was employed. In diabetic mice, bone marrow EPC levels were unaffected. However, circulating EPC levels in blood were lower at baseline and mobilization was attenuated. Diabetic mice failed to recover and repopulate from 5-FU injection. In vitro, primary cultured bone marrow stroma from diabetic mice was impaired in its capacity to support human CFU-forming HPC. Finally, hyperglycemia hampered the HPC supportive function of endothelial cells in vitro. EPC mobilization is impaired under experimental diabetic conditions and our data suggest that diabetes induces alterations in the progenitor cell supportive capacity of the bone marrow stroma, which could be partially responsible for the attenuated EPC mobilization and reduced EPC levels observed in diabetic patients.

Impaired endothelial progenitor cell mobilization and dysfunctional bone marrow stroma in diabetes mellitus.

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Peter E Westerweel

Full Text Available Circulating Endothelial Progenitor Cell (EPC levels are reduced in diabetes mellitus. This may be a consequence of impaired mobilization of EPC from the bone marrow. We hypothesized that under diabetic conditions, mobilization of EPC from the bone marrow to the circulation is impaired -at least partly- due to dysfunction of the bone marrow stromal compartment.Diabetes was induced in mice by streptozotocin injection. Circulating Sca-1(+Flk-1(+ EPC were characterized and quantified by flow cytometry at baseline and after mobilization with G-CSF/SCF injections. In vivo hemangiogenic recovery was tested by 5-FU challenge. Interaction within the bone marrow environment between CD34(+ hematopoietic progenitor cells (HPC and supporting stroma was assessed by co-cultures. To study progenitor cell-endothelial cell interaction under normoglycemic and hyperglycemic conditions, a co-culture model using E4Orf1-transfected human endothelial cells was employed.In diabetic mice, bone marrow EPC levels were unaffected. However, circulating EPC levels in blood were lower at baseline and mobilization was attenuated. Diabetic mice failed to recover and repopulate from 5-FU injection. In vitro, primary cultured bone marrow stroma from diabetic mice was impaired in its capacity to support human CFU-forming HPC. Finally, hyperglycemia hampered the HPC supportive function of endothelial cells in vitro.EPC mobilization is impaired under experimental diabetic conditions and our data suggest that diabetes induces alterations in the progenitor cell supportive capacity of the bone marrow stroma, which could be partially responsible for the attenuated EPC mobilization and reduced EPC levels observed in diabetic patients.

Chronic congestive heart failure is associated with a phenotypic shift of intramyocardial endothelial cells

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Marijianowski, M. M.; van Laar, M.; Bras, J.; Becker, A. E.

1995-01-01

There is evidence that patients with chronic congestive heart failure have endothelial cell-related abnormalities of the peripheral circulation and the coronary microvasculature. For that reason, we have studied the phenotypic expression of endothelial cells in hearts of patients with congestive

High-intensity Interval training enhances mobilization/functionality of endothelial progenitor cells and depressed shedding of vascular endothelial cells undergoing hypoxia.

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Tsai, Hsing-Hua; Lin, Chin-Pu; Lin, Yi-Hui; Hsu, Chih-Chin; Wang, Jong-Shyan

2016-12-01

Exercise training improves endothelium-dependent vasodilation, whereas hypoxic stress causes vascular endothelial dysfunction. Monocyte-derived endothelial progenitor cells (Mon-EPCs) contribute to vascular repair process by differentiating into endothelial cells. This study investigates how high-intensity interval (HIT) and moderate-intensity continuous (MCT) exercise training affect circulating Mon-EPC levels and EPC functionality under hypoxic condition. Sixty healthy sedentary males were randomized to engage in either HIT (3-min intervals at 40 and 80 % VO 2max for five repetitions, n = 20) or MCT (sustained 60 % VO 2max , n = 20) for 30 min/day, 5 days/week for 6 weeks, or to a control group (CTL) that did not received exercise intervention (n = 20). Mon-EPC characteristics and EPC functionality under hypoxic exercise (HE, 100 W under 12 % O 2 ) were determined before and after HIT, MCT, and CTL. The results demonstrated that after the intervention, the HIT group exhibited larger improvements in VO 2peak , estimated peak cardiac output (Q C ), and estimated peak perfusions of frontal cerebral lobe (Q FC ) and vastus lateralis (Q VL ) than the MCT group. Furthermore, HIT (a) increased circulating CD14 ++ /CD16 - /CD34 + /KDR + (Mon-1 EPC) and CD14 ++ /CD16 + /CD34 + /KDR + (Mon-2 EPC) cell counts, (b) promoted the migration and tube formation of EPCs, (c) diminished the shedding of endothelial (CD34 - /KDR + /phosphatidylserine + ) cells, and (d) elevated plasma nitrite plus nitrate, stromal cell-derived factor-1, matrix metalloproteinase-9, and vascular endothelial growth factor-A concentrations at rest or following HE, compared to those of MCT. In addition, Mon-1 and -2 EPC counts were directly related to VO 2peak and estimated peak Q C , Q FC , and Q VL . HIT is superior to MCT for improving hemodynamic adaptation and Mon-EPC production. Moreover, HIT effectively enhances EPC functionality and suppresses endothelial injury undergoing hypoxia.

Endothelial cell proliferation in swine experimental aneurysm after coil embolization.

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Yumiko Mitome-Mishima

Full Text Available After coil embolization, recanalization in cerebral aneurysms adversely influences long-term prognosis. Proliferation of endothelial cells on the coil surface may reduce the incidence of recanalization and further improve outcomes after coil embolization. We aimed to map the expression of proliferating tissue over the aneurysmal orifice and define the temporal profile of tissue growth in a swine experimental aneurysm model. We compared the outcomes after spontaneous thrombosis with those of coil embolization using histological and morphological techniques. In aneurysms that we not coiled, spontaneous thrombosis was observed, and weak, easily detachable proliferating tissue was evident in the aneurysmal neck. In contrast, in the coil embolization group, histological analysis showed endothelial-like cells lining the aneurysmal opening. Moreover, immunohistochemical and morphological analysis suggested that these cells were immature endothelial cells. Our results indicated the existence of endothelial cell proliferation 1 week after coil embolization and showed immature endothelial cells in septal tissue between the systemic circulation and the aneurysm. These findings suggest that endothelial cells are lead to and proliferate in the former aneurysmal orifice. This is the first examination to evaluate the temporal change of proliferating tissue in a swine experimental aneurysm model.

Circulating endothelial progenitor cell numbers are not associated with donor organ age or allograft vasculopathy in cardiac transplant recipients.

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Thomas, H E; Parry, G; Dark, J H; Arthur, H M; Keavney, B D

2009-02-01

Increasing age is associated with reduced numbers of circulating endothelial progenitor cells (EPCs). It is unclear whether this relates to depletion or impairment of bone marrow progenitors, or to deficient mobilization signals from aging tissues. In cardiac transplant patients, one previous study has reported an association between circulating EPCs and the risk of cardiac allograft vasculopathy (CAV). We investigated whether increased donor heart age, a strong risk factor for CAV, was associated with reduced circulating EPC numbers in a group of cardiac transplant recipients matched for factors which influence EPC numbers, but with maximally discordant donor heart ages. We identified 32 patient pairs, matched for factors known to influence EPC numbers, but who had discordant donor heart ages by at least 20 years. EPCs were quantified using flow cytometry for absolute counts of cells expressing all the combinations of CD45, CD34, CD133 and the kinase domain receptor (KDR). There were no significant differences in the numbers of circulating EPCs between patients with old or young donor heart age. There was no association between the presence of CAV and circulating EPC numbers. We suggest that the increased susceptibility to CAV of older donor hearts is not mediated via circulating EPCs. Our results are consistent with the theory that the normal age-related decline in EPC numbers relates to bone marrow aging rather than failure of target tissues to induce EPC mobilization.

CD13 is a novel mediator of monocytic/endothelial cell adhesion

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Mina-Osorio, Paola; Winnicka, Beata; O'Conor, Catherine

2008-01-01

During inflammation, cell surface adhesion molecules guide the adhesion and migration of circulating leukocytes across the endothelial cells lining the blood vessels to access the site of injury. The transmembrane molecule CD13 is expressed on monocytes and endothelial cells and has been shown...... to mediate homotypic cell adhesion, which may imply a role for CD13 in inflammatory monocyte trafficking. Here, we show that ligation and clustering of CD13 by mAb or viral ligands potently induce myeloid cell/endothelial adhesion in a signal transduction-dependent manner involving monocytic cytoskeletal...... rearrangement and filopodia formation. Treatment with soluble recombinant (r)CD13 blocks this CD13-dependent adhesion, and CD13 molecules from monocytic and endothelial cells are present in the same immunocomplex, suggesting a direct participation of CD13 in the adhesive interaction. This concept...

Activation of Endothelial Nitric Oxide (eNOS Occurs through Different Membrane Domains in Endothelial Cells.

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Jason Tran

Full Text Available Endothelial cells respond to a large range of stimuli including circulating lipoproteins, growth factors and changes in haemodynamic mechanical forces to regulate the activity of endothelial nitric oxide synthase (eNOS and maintain blood pressure. While many signalling pathways have been mapped, the identities of membrane domains through which these signals are transmitted are less well characterized. Here, we manipulated bovine aortic endothelial cells (BAEC with cholesterol and the oxysterol 7-ketocholesterol (7KC. Using a range of microscopy techniques including confocal, 2-photon, super-resolution and electron microscopy, we found that sterol enrichment had differential effects on eNOS and caveolin-1 (Cav1 colocalisation, membrane order of the plasma membrane, caveolae numbers and Cav1 clustering. We found a correlation between cholesterol-induced condensation of the plasma membrane and enhanced high density lipoprotein (HDL-induced eNOS activity and phosphorylation suggesting that cholesterol domains, but not individual caveolae, mediate HDL stimulation of eNOS. Vascular endothelial growth factor (VEGF-induced and shear stress-induced eNOS activity was relatively independent of membrane order and may be predominantly controlled by the number of caveolae on the cell surface. Taken together, our data suggest that signals that activate and phosphorylate eNOS are transmitted through distinct membrane domains in endothelial cells.

Quantification of circulating mature endothelial cells using a whole blood four-color flow cytometric assay.

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Jacques, Nathalie; Vimond, Nadege; Conforti, Rosa; Griscelli, Franck; Lecluse, Yann; Laplanche, Agnes; Malka, David; Vielh, Philippe; Farace, Françoise

2008-09-15

Circulating endothelial cells (CEC) are currently proposed as a potential biomarker for measuring the impact of anti-angiogenic treatments in cancer. However, the lack of consensus on the appropriate method of CEC measurement has led to conflicting data in cancer patients. A validated assay adapted for evaluating the clinical utility of CEC in large cohorts of patients undergoing anti-angiogenic treatments is needed. We developed a four-color flow cytometric assay to measure CEC as CD31(+), CD146(+), CD45(-), 7-amino-actinomycin-D (7AAD)(-) events in whole blood. The distinctive features of the assay are: (1) staining of 1 ml whole blood, (2) use of a whole blood IgPE control to measure accurately background noise, (3) accumulation of a large number of events (almost 5 10(6)) to ensure statistical analysis, and (4) use of 10 microm fluorescent microbeads to evaluate the event size. Assay reproducibility was determined in duplicate aliquots of samples drawn from 20 metastatic cancer patients. Assay linearity was tested by spiking whole blood with low numbers of HUVEC. Five-color flow cytometric experiments with CD144 were performed to confirm the endothelial origin of the cells. CEC were measured in 20 healthy individuals and 125 patients with metastatic cancer. Reproducibility was good between duplicate aliquots (r(2)=0.948, mean difference between duplicates of 0.86 CEC/ml). Detected HUVEC correlated with spiked HUVEC (r(2)=0.916, mean recovery of 100.3%). Co-staining of CD31, CD146 and CD144 confirmed the endothelial nature of cells identified as CEC. Median CEC levels were 6.5/ml (range, 0-15) in healthy individuals and 15.0/ml (range, 0-179) in patients with metastatic carcinoma (p<0.001). The assay proposed here allows reproducible and sensitive measurement of CEC by flow cytometry and could help evaluate CEC as biomarkers of anti-angiogenic therapies in large cohorts of patients.

Impact of age and gender interaction on circulating endothelial progenitor cells in healthy subjects.

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Rousseau, Alexandra; Ayoubi, Fida; Deveaux, Christel; Charbit, Beny; Delmau, Catherine; Christin-Maitre, Sophie; Jaillon, Patrice; Uzan, Georges; Simon, Tabassome

2010-02-01

To assess the level of circulating endothelial progenitor cells (CEPC) in cycling women compared with men and menopausal women. Controlled clinical study. Healthy, nonsmoking volunteers. Twelve women, aged 18-40 years, with regular menstrual cycles, 12 menopausal women, and two groups of 12 age-matched men were recruited. Women did not receive any hormone therapy. Collection of 20 mL of peripheral blood. The number of CEPC, defined as (Lin-/7AAD-/CD34+/CD133+/KDR+) cells per 10(6) mononuclear cells (MNC), was measured by flow cytometry. The number of CEPC was significantly higher in cycling women than in age-matched men and menopausal women (26.5 per 10(6) MNC vs. 10.5 per 10(6) MNC vs. 10 per 10(6) MNC, respectively). The number of CEPC was similar in menopausal women, age-matched, and young men. The number of CEPC is influenced by an age-gender interaction. This phenomenon may explain in part the better vascular repair and relative cardiovascular protection in younger women as compared with age-matched men. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Improving the characterization of endothelial progenitor cell subsets by an optimized FACS protocol.

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Karin Huizer

Full Text Available The characterization of circulating endothelial progenitor cells (EPCs is fundamental to any study related to angiogenesis. Unfortunately, current literature lacks consistency in the definition of EPC subsets due to variations in isolation strategies and inconsistencies in the use of lineage markers. Here we address critical points in the identification of hematopoietic progenitor cells (HPCs, circulating endothelial cells (CECs, and culture-generated outgrowth endothelial cells (OECs from blood samples of healthy adults (AB and umbilical cord (UCB. Peripheral blood mononuclear cells (PBMCs were enriched using a Ficoll-based gradient followed by an optimized staining and gating strategy to enrich for the target cells. Sorted EPC populations were subjected to RT-PCR for tracing the expression of markers beyond the limits of cell surface-based immunophenotyping. Using CD34, CD133 and c-kit staining, combined with FSC and SSC, we succeeded in the accurate and reproducible identification of four HPC subgroups and found significant differences in the respective populations in AB vs. UCB. Co-expression analysis of endothelial markers on HPCs revealed a complex pattern characterized by various subpopulations. CECs were identified by using CD34, KDR, CD45, and additional endothelial markers, and were subdivided according to their apoptotic state and expression of c-kit. Comparison of UCB-CECs vs. AB-CECs revealed significant differences in CD34 and KDR levels. OECs were grown from PBMC-fractions We found that viable c-kit+ CECs are a candidate circulating precursor for CECs. RT-PCR to angiogenic factors and receptors revealed that all EPC subsets expressed angiogenesis-related molecules. Taken together, the improvements in immunophenotyping and gating strategies resulted in accurate identification and comparison of better defined cell populations in a single procedure.

Circulating Endothelial Microparticles: A Key Hallmark of Atherosclerosis Progression

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Keshav Raj Paudel

2016-01-01

Full Text Available The levels of circulating microparticles (MPs are raised in various cardiovascular diseases. Their increased level in plasma is regarded as a biomarker of alteration in vascular function. The prominent MPs present in blood are endothelial microparticles (EMPs described as complex submicron (0.1 to 1.0 μm vesicles like structure, released in response to endothelium cell activation or apoptosis. EMPs possess both physiological and pathological effects and may promote oxidative stress and vascular inflammation. EMPs release is triggered by inducer like angiotensin II, lipopolysaccharide, and hydrogen peroxide leading to the progression of atherosclerosis. However, there are multiple physiological pathways for EMPs generation like NADPH oxidase derived endothelial ROS formation, Rho kinase pathway, and mitogen-activated protein kinases. Endothelial dysfunction is a key initiating event in atherosclerotic plaque formation. Atheroemboli, resulting from ruptured carotid plaques, is a major cause of stroke. Increasing evidence suggests that EMPs play an important role in the pathogenesis of cardiovascular disease, acting as a marker of damage, either exacerbating disease progression or triggering a repair response. In this regard, it has been suggested that EMPs have the potential to act as biomarkers of disease status. This review aims to provide updated information of EMPs in relation to atherosclerosis pathogenesis.

The acute exposure effects of inhaled nickel nanoparticles on murine endothelial progenitor cells.

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Liberda, Eric N; Cuevas, Azita K; Qu, Qingshan; Chen, Lung Chi

2014-08-01

The discovery of endothelial progenitor cells (EPCs) may help to explain observed cardiovascular effects associated with inhaled nickel nanoparticle exposures, such as increases in vascular inflammation, generation of reactive oxygen species, altered vasomotor tone and potentiated atherosclerosis in murine species. Following an acute whole body inhalation exposure to 500 µg/m(3) of nickel nanoparticles for 5 h, bone marrow EPCs from C57BL/6 mice were isolated. EPCs were harvested for their RNA or used in a variety of assays including chemotaxis, tube formation and proliferation. Gene expression was assessed for important receptors involved in EPC mobilization and homing using RT-PCR methods. EPCs, circulating endothelial progenitor cells (CEPCs), circulating endothelial cells (CECs) and endothelial microparticles (EMPs) were quantified on a BD FACSCalibur to examine endothelial damage and repair associated with the exposure. Acute exposure to inhaled nickel nanoparticles significantly increased both bone marrow EPCs as well as their levels in circulation (CEPCs). CECs were significantly elevated indicating that endothelial damage occurred due to the exposure. There was no significant difference in EMPs between the two groups. Tube formation and chemotaxis, but not proliferation, of bone marrow EPCs was impaired in the nickel nanoparticle exposed group. These results coincided with a decrease in the mRNA of receptors involved in EPC mobilization and homing. These data provide new insight into how an acute nickel nanoparticle exposure to half of the current Occupational Safety & Health Administration (OSHA) permissible exposure limit may adversely affect EPCs and exacerbate cardiovascular disease states.

Physalis minima Leaves Extract Induces Re-Endothelialization in Deoxycorticosterone Acetate-Salt-Induced Endothelial Dysfunction in Rats

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Dian Nugrahenny

2018-02-01

Full Text Available The administration of deoxy-corticosterone acetate (DOCA-salt can induce oxidative stress leading to decrease the bioavailability of nitric oxide (NO, increase senescence of circulating endothelial progenitor cells (EPCs, thus contributing to endothelial dysfunction. This study was aimed to investigate the effects of Physalis minima L. leaves extract on serum NO levels, circulating EPCs number, and histopathology of tail artery endothelial cells in DOCA-salt-induced endothelial dysfunction in rats. Twenty-five male Wistar rats were randomly divided into five groups: rats without any treatment (normal, rats treated with DOCA (10 mg/kgBW s.c. twice weekly and given 0.9% NaCl to drink ad libitum for 6 weeks, and DOCA-salt-induced rats orally supplemented with P. minima leaves extract at doses of 500, 1500, or 2500 mg/kgBW for 4 weeks. Serum NO levels were measured by colorimetry. The number of circulating EPCs (CD34+/CD133+ cells was determined by flow cytometry. The tail artery sections were histologically processed with hematoxylin-eosin staining. DOCA-salt-induced rats showed significantly (p<0.05 decrease in serum NO levels and circulating EPCs number compared to the normal. There was also more detached tail artery endothelial cells in DOCA-salt-induced rats. P. minima leaves extract at a dose of 500 mg/kgBW significantly (p<0.05 increased serum NO level and circulating EPCs number, and also induced an optimal re-endothelialization in DOCA-salt-induced rats. P. minima leave extract dose-dependently increases NO bioavailability contributing to enhanced EPCs mobilization, thereby promoting re-endothelialization in DOCA-salt-induced endothelial dysfunction in rats.

Sustained apnea induces endothelial activation.

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Eichhorn, Lars; Dolscheid-Pommerich, Ramona; Erdfelder, Felix; Ayub, Muhammad Ajmal; Schmitz, Theresa; Werner, Nikos; Jansen, Felix

2017-09-01

Apnea diving has gained worldwide popularity, even though the pathophysiological consequences of this challenging sport on the human body are poorly investigated and understood. This study aims to assess the influence of sustained apnea in healthy volunteers on circulating microparticles (MPs) and microRNAs (miRs), which are established biomarkers reflecting vascular function. Short intermittent hypoxia due to voluntary breath-holding affects circulating levels of endothelial cell-derived MPs (EMPs) and endothelial cell-derived miRs. Under dry laboratory conditions, 10 trained apneic divers performed maximal breath-hold. Venous blood samples were taken, once before and at 4 defined points in time after apnea. Samples were analyzed for circulating EMPs and endothelial miRs. Average apnea time was 329 seconds (±103), and SpO 2 at the end of apnea was 79% (±12). Apnea was associated with a time-dependent increase of circulating endothelial cell-derived EMPs and endothelial miRs. Levels of circulating EMPs in the bloodstream reached a peak 4 hours after the apnea period and returned to baseline levels after 24 hours. Circulating miR-126 levels were elevated at all time points after a single voluntary maximal apnea, whereas miR-26 levels were elevated significantly only after 30 minutes and 4 hours. Also miR-21 and miR-92 levels increased, but did not reach the level of significance. Even a single maximal breath-hold induces acute endothelial activation and should be performed with great caution by subjects with preexisting vascular diseases. Voluntary apnea might be used as a model to simulate changes in endothelial function caused by hypoxia in humans. © 2017 Wiley Periodicals, Inc.

Endothelial progenitor cell subsets and preeclampsia: Findings and controversies

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Armin Attar

2017-10-01

Full Text Available Vascular remodeling is an essential component of gestation. Endothelial progenitor cells (EPCs play an important role in the regulation of vascular homeostasis. The results of studies measuring the number of EPCs in normal pregnancies and in preeclampsia have been highly controversial or even contradictory because of some variations in technical issues and different methodologies enumerating three distinct subsets of EPCs: circulating angiogenic cells (CAC, colony forming unit endothelial cells (CFU-ECs, and endothelial colony-forming cells (ECFCs. In general, most studies have shown an increase in the number of CACs in the maternal circulation with a progression in the gestational age in normal pregnancies, while functional capacities measured by CFU-ECs and ECFCs remain intact. In the case of preeclampsia, mobilization of CACs and ECFCs occurs in the peripheral blood of pregnant women, but the functional capacities shown by culture of the derived colony-forming assays (CFU-EC and ECFC assays are altered. Furthermore, the number of all EPC subsets will be reduced in umbilical cord blood in the case of preeclampsia. As EPCs play an important role in the homeostasis of vascular networks, the difference in their frequency and functionality in normal pregnancies and those with preeclampsia can be expected. In this review, there was an attempt to provide a justification for these controversies.

Vildagliptin, but not glibenclamide, increases circulating endothelial progenitor cell number: a 12-month randomized controlled trial in patients with type 2 diabetes.

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Dei Cas, Alessandra; Spigoni, Valentina; Cito, Monia; Aldigeri, Raffaella; Ridolfi, Valentina; Marchesi, Elisabetta; Marina, Michela; Derlindati, Eleonora; Aloe, Rosalia; Bonadonna, Riccardo C; Zavaroni, Ivana

2017-02-23

Fewer circulating endothelial progenitor cells (EPCs) and increased plasma (C-term) stromal cell-derived factor 1α (SDF-1α), a substrate of DPP-4, are biomarkers, and perhaps mediators, of cardiovascular risk and mortality. Short-term/acute treatment with DPP-4 inhibitors improve EPC bioavailability; however, long-term effects of DPP-4i on EPCs bioavailability/plasma (C-term) SDF-1α are unknown. Randomized (2:1) open-label trial to compare the effects of vildagliptin (V) (100 mg/day) vs glibenclamide (G) (2.5 mg bid to a maximal dose of 5 mg bid) on circulating EPC levels at 4 and 12 months of treatment in 64 patients with type 2 diabetes in metformin failure. At baseline, and after 4 and 12 months, main clinical/biohumoral parameters, inflammatory biomarkers, concomitant therapies, EPC number (CD34 + /CD133 + /KDR + /10 6 cytometric events) and plasma (C-term) SDF-1α (R&D system) were assessed. Baseline characteristics were comparable in the two groups. V and G similarly and significantly (p < 0.0001) improved glucose control. At 12 months, V significantly increased EPC number (p < 0.05) and significantly reduced (C-term) SDF-1α plasma levels (p < 0.01) compared to G, with no differences in inflammatory biomarkers. V exerts a long-term favorable effect on EPC and (C-term) SDF-1α levels at glucose equipoise, thereby implying a putative beneficial effect on vascular integrity. Trial registration Clinical Trials number: NCT01822548; name: Effect of Vildagliptin vs. Glibenclamide on Circulating Endothelial Progenitor Cell Number Type 2 Diabetes. Registered 28 March, 2013.

Circulating Endothelial-Derived Activated Microparticle: A Useful Biomarker for Predicting One-Year Mortality in Patients with Advanced Non-Small Cell Lung Cancer

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Chin-Chou Wang

2014-01-01

Full Text Available Background. This study tested the hypothesis that circulating microparticles (MPs are useful biomarkers for predicting one-year mortality in patients with end-stage non-small cell lung cancer (ES-NSCLC. Methods and Results. One hundred seven patients were prospectively enrolled into the study between April 2011 and February 2012, and each patient received regular follow-up after enrollment. Levels of four MPs in circulation, (1 platelet-derived activated MPs (PDAc-MPs, (2 platelet-derived apoptotic MPs (PDAp-MPs, (3 endothelial-derived activated MPs (EDAc-MPs, and (4 endothelial-derived apoptotic MPs (EDAp-MPs, were measured just after the patient was enrolled into the study using flow cytometry. Patients who survived for more than one year were categorized into group 1 (n=56 (one-year survivors and patients who survived less than one year were categorized into group 2 (n=51 (one-year nonsurvivors. Male gender, incidence of liver metastasis, progression of disease after first-line treatment, poor performance status, and the Charlson comorbidity index were significantly higher in group 2 than in group 1 (all P<0.05. Additionally, as measured by flow cytometry, only the circulating level of EDAc-MPs was found to be significantly higher in group 2 than in group 1 (P=0.006. Multivariate analysis demonstrated that circulating level of EDAc-MPs along with brain metastasis and male gender significantly and independently predictive of one-year mortality (all P<0.035. Conclusion. Circulating EDAc-MPs may be a useful biomarker predictive of one-year morality in ES-NSCLC patients.

Prognostic Value of CD109+ Circulating Endothelial Cells in Recurrent Glioblastomas Treated with Bevacizumab and Irinotecan

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Cuppini, Lucia; Calleri, Angelica; Bruzzone, Maria Grazia; Prodi, Elena; Anghileri, Elena; Pellegatta, Serena; Mancuso, Patrizia; Porrati, Paola; Di Stefano, Anna Luisa; Ceroni, Mauro; Bertolini, Francesco; Finocchiaro, Gaetano; Eoli, Marica

2013-01-01

Background Recent data suggest that circulating endothelial and progenitor cells (CECs and CEPs, respectively) may have predictive potential in cancer patients treated with bevacizumab, the antibody recognizing vascular endothelial growth factor (VEGF). Here we report on CECs and CEPs investigated in 68 patients affected by recurrent glioblastoma (rGBM) treated with bevacizumab and irinotecan and two Independent Datasets of rGBM patients respectively treated with bevacizumab alone (n=32, independent dataset A: IDA) and classical antiblastic chemotherapy (n=14, independent dataset B: IDB). Methods rGBM patients with KPS ≥50 were treated until progression, as defined by MRI with RANO criteria. CECs expressing CD109, a marker of tumor endothelial cells, as well as other CEC and CEP subtypes, were investigated by six-color flow cytometry. Results A baseline count of CD109+ CEC higher than 41.1/ml (1st quartile) was associated with increased progression free survival (PFS; 20 versus 9 weeks, P=0.008) and overall survival (OS; 32 versus 23 weeks, P=0.03). Longer PFS (25 versus 8 weeks, P=0.02) and OS (27 versus 17 weeks, P=0.03) were also confirmed in IDA with CD109+ CECs higher than 41.1/ml but not in IDB. Patients treated with bevacizumab with or without irinotecan that were free from MRI progression after two months of treatment had significant decrease of CD109+ CECs: median PFS was 19 weeks; median OS 29 weeks. The presence of two non-contiguous lesions (distant disease) at baseline was an independent predictor of shorter PFS and OS (P<0.001). Conclusions Data encourage further studies on the predictive potential of CD109+ CECs in GBM patients treated with bevacizumab. PMID:24069296

Acute myocardial infarction is associated with endothelial glycocalyx and cell damage and a parallel increase in circulating catecholamines

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Ostrowski, Sisse R; Pedersen, Sune H; Jensen, Jan S

2013-01-01

INTRODUCTION: Excessive sympathoadrenal activation in critical illness contributes directly to organ damage, and high concentrations of catecholamines damage the vascular endothelium. This study investigated associations between potential drivers of sympathoadrenal activation, circulating...... catecholamines and biomarkers of endothelial damage and outcome in ST segment elevation myocardial infarction (STEMI)-patients, hypothesizing that the catecholamine surge would reflect shock degree and correlate with biomarkers of endothelial damage. METHODS: This was a prospective study of 678 consecutive STEMI...

Triazole RGD antagonist reverts TGFβ1-induced endothelial-to-mesenchymal transition in endothelial precursor cells.

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Bianchini, Francesca; Peppicelli, Silvia; Fabbrizzi, Pierangelo; Biagioni, Alessio; Mazzanti, Benedetta; Menchi, Gloria; Calorini, Lido; Pupi, Alberto; Trabocchi, Andrea

2017-01-01

Fibrosis is the dramatic consequence of a dysregulated reparative process in which activated fibroblasts (myofibroblasts) and Transforming Growth Factor β1 (TGFβ1) play a central role. When exposed to TGFβ1, fibroblast and epithelial cells differentiate in myofibroblasts; in addition, endothelial cells may undergo endothelial-to-mesenchymal transition (EndoMT) and actively participate to the progression of fibrosis. Recently, the role of αv integrins, which recognize the Arg-Gly-Asp (RGD) tripeptide, in the release and signal transduction activation of TGFβ1 became evident. In this study, we present a class of triazole-derived RGD antagonists that interact with αvβ3 integrin. Above different compounds, the RGD-2 specifically interferes with integrin-dependent TGFβ1 EndoMT in Endothelial Colony-Forming Cells (ECPCs) derived from circulating Endothelial Precursor Cells (ECPCs). The RGD-2 decreases the amount of membrane-associated TGFβ1, and reduces both ALK5/TGFβ1 type I receptor expression and Smad2 phosphorylation in ECPCs. We found that RGD-2 antagonist reverts EndoMT, reducing α-smooth muscle actin (α-SMA) and vimentin expression in differentiated ECPCs. Our results outline the critical role of integrin in fibrosis progression and account for the opportunity of using integrins as target for anti-fibrotic therapeutic treatment.

Cancer cells remodel themselves and vasculature to overcome the endothelial barrier.

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Shenoy, Anitha K; Lu, Jianrong

2016-10-01

Metastasis refers to the spread of cancer cells from a primary tumor to distant organs mostly via the bloodstream. During the metastatic process, cancer cells invade blood vessels to enter circulation, and later exit the vasculature at a distant site. Endothelial cells that line blood vessels normally serve as a barrier to the movement of cells into or out of the blood. It is thus critical to understand how metastatic cancer cells overcome the endothelial barrier. Epithelial cancer cells acquire increased motility and invasiveness through epithelial-to-mesenchymal transition (EMT), which enables them to move toward vasculature. Cancer cells also express a variety of adhesion molecules that allow them to attach to vascular endothelium. Finally, cancer cells secrete or induce growth factors and cytokines to actively prompt vascular hyperpermeability that compromises endothelial barrier function and facilitates transmigration of cancer cells through the vascular wall. Elucidation of the mechanisms underlying metastatic dissemination may help develop new anti-metastasis therapeutics. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

The Effects of Inhaled Nickel Nanoparticles on Murine Endothelial Progenitor Cells

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Liberda, Eric N.

Introduction. Particulate air pollution, specifically nickel found on or in particulate matter, has been associated with an increased risk of mortality in human population studies and can cause increases in vascular inflammation, generate reactive oxygen species, alter vasomotor tone, and potentiate atherosclerosis in murine exposures. With the discovery of endothelial progenitor cells (EPCs), a door has been opened which may explain these observed cardiovascular effects associated with inhaled air particles and nickel exposure. In order to further quantify the effects of inhaled nickel nanoparticles and attempt to elucidate how the observed findings from other studies may occur, several whole body inhalation exposure experiments to nickel nanoparticles were performed. Methods. Following whole body exposure to approximately 500mug/m3 of nickel nanoparticles for 5 hrs, bone marrow EPCs from C57BL/6 mice were isolated. EPCs were harvested for their RNA or used in a variety of assays including chemotaxis, tube formation, and proliferation. Gene expression was assessed for important receptors involved in EPC mobilization and homing using RT-PCR methods. EPCs, circulating endothelial progenitor cells, circulating endothelial cells (CECs), and endothelial microparticles (EMPs) were quantified on a BD FACSCalibur to examine endothelial damage and repair associated with the inhalation exposure. Plasma proteins were assessed using the 2D DIGE proteomic approach and commercially available ELISAs. Results and Conclusions. Exposure to inhaled nickel nanoparticles significantly increased both bone marrow EPCs as well as their levels in circulation. CECs were significantly upregulated suggesting that endothelial damage occurred due to the exposure. There was no significant difference in EMPs between the two groups. Tube formation and chemotaxis, but not proliferation, of bone marrow EPCs was impaired in the nickel nanoparticle exposed group. This decrease in EPC function

Identification of a Monocyte Receptor on Herpesvirus-Infected Endothelial Cells

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Etingin, Orli R.; Silverstein, Roy L.; Hajjar, David P.

1991-08-01

The adhesion of circulating blood cells to vascular endothelium may be an initial step in atherosclerosis, inflammation, and wound healing. One mechanism for promoting cell-cell adhesion involves the expression of adhesion molecules on the surface of the target cell. Herpes simplex virus infection of endothelium induces arterial injury and has been implicated in the development of human atherosclerosis. We now demonstrate that HSV-infected endothelial cells express the adhesion molecule GMP140 and that this requires cell surface expression of HSV glycoprotein C and local thrombin generation. Monocyte adhesion to HSV-infected endothelial cells was completely inhibited by anti-GMP140 antibodies but not by antibodies to other adhesion molecules such as VCAM and ELAM-1. The induction of GMP140 expression on HSV-infected endothelium may be an important pathophysiological mechanism in virus-induced cell injury and inflammation.

Erythropoietin Receptor Positive Circulating Progenitor Cells and Endothelial Progenitor Cells in Patients with Different Stages of Diabetic Retinopathy

Institute of Scientific and Technical Information of China (English)

Liu-mei Hu; Guo-xu Xu; Guo-tong XU; Wei-ye Li; Xia Lei; Bo Ma; Yu Zhang; Yan Yan; Ya-lan Wu; Ge-zhi Xu; Wen Ye; Ling Wang

2011-01-01

Objective To investigate the possible involvement of erythropoietin (EPO)/erythropoietin receptor(EPOR) system in neovascularization and vascular regeneration in diabetic retinopathy (DR).Methods EPOR positive circulating progenitor cells (CPCs: CD34+) and endothelial progenitor cells (EPCs: CD34+KDR+) were assessed by flow cytometry in type 2 diabetic patients with different stages of DR. The cohort consisted of age- and sex-matched control patients without diabetes (n=7), non-prolif-erative DR (NPDR, n=7), proliferative DR (PDR, n=8), and PDR complicated with diabetic nephropathy (PDR-DN, n=7). Results The numbers of EPOR+ CPCs and EPOR+ EPCs were reduced remarkably in NPDR compared with the control group (both P<0.01), whereas rebounded in PDR and PDR-DN groups in varying degrees. Similar changes were observed in respect of the proportion of EPOR+ CPCs in CPCs (NPDR vs.control, P< 0.01) and that of EPOR+ EPCs in EPCs (NPDR vs. control, P< 0.05). Conclusion Exogenous EPO, mediated via the EPO/EPOR system of EPCs, may alleviate the im-paired vascular regeneration in NPDR, whereas it might aggravate retinal neovascularization in PDR due to a rebound of EPOR+ EPCs associated with ischemia.

Lysophosphatidic acid stimulates thrombomodulin lectin-like domain shedding in human endothelial cells

International Nuclear Information System (INIS)

Wu Hualin; Lin ChiIou; Huang Yuanli; Chen, Pin-Shern; Kuo, Cheng-Hsiang; Chen, Mei-Shing; Wu, G.C.-C.; Shi, G.-Y.; Yang, H.-Y.; Lee Hsinyu

2008-01-01

Thrombomodulin (TM) is an anticoagulant glycoprotein highly expressed on endothelial cell surfaces. Increased levels of soluble TM in circulation have been widely accepted as an indicator of endothelial damage or dysfunction. Previous studies indicated that various proinflammatory factors stimulate TM shedding in various cell types such as smooth muscle cells and epithelial cells. Lysophosphatidic acid (LPA) is a bioactive lipid mediator present in biological fluids during endothelial damage or injury. In the present study, we first observed that LPA triggered TM shedding in human umbilical vein endothelial cells (HUVECs). By Cyflow analysis, we showed that the LPA-induced accessibility of antibodies to the endothelial growth factor (EGF)-like domain of TM is independent of matrix metalloproteinases (MMPs), while LPA-induced TM lectin-like domain shedding is MMP-dependent. Furthermore, a stable cell line expressing TM without its lectin-like domain exhibited a higher cell proliferation rate than a stable cell line expressing full-length TM. These results imply that LPA induces TM lectin-like domain shedding, which might contribute to the exposure of its EGF-like domain for EGF receptor (EGFR) binding, thereby stimulating subsequent cell proliferation. Based on our findings, we propose a novel mechanism for the exposure of TM EGF-like domain, which possibly mediates LPA-induced EGFR transactivation

Detection and Quantification of Vascular Endothelial Growth Factor Receptor Tyrosine Kinases in Primary Human Endothelial Cells.

Science.gov (United States)

Fearnley, Gareth W; Wheatcroft, Stephen B; Ponnambalam, Sreenivasan

2015-01-01

Proteins differ widely in their pattern of expression depending on organism, tissue, and regulation in response to changing conditions. In the mammalian vasculature, the endothelium responds to vascular endothelial growth factors (VEGFs) via membrane-bound receptor tyrosine kinases (VEGFRs) to modulate many aspects of vascular physiology including vasculogenesis, angiogenesis, and blood pressure. Studies on VEGFR biology are thus dependent on detecting expression levels in different cell types and evaluating how changes in protein levels correlate with changing conditions including circulating VEGF levels. Here, we present a robust immunoblot-based protocol for detecting and quantifying VEGFRs in human endothelial cells. Using internal and external standards, we can rapidly evaluate receptor copy number and assess how this is altered in response to the cellular environment.

Transcellular transport of cobalamin in aortic endothelial cells.

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Hannibal, Luciana; Bolisetty, Keerthana; Axhemi, Armend; DiBello, Patricia M; Quadros, Edward V; Fedosov, Sergey; Jacobsen, Donald W

2018-05-09

Cobalamin [Cbl (or B 12 )] deficiency causes megaloblastic anemia and a variety of neuropathies. However, homeostatic mechanisms of cyanocobalamin (CNCbl) and other Cbls by vascular endothelial cells are poorly understood. Herein, we describe our investigation into whether cultured bovine aortic endothelial cells (BAECs) perform transcytosis of B 12 , namely, the complex formed between serum transcobalamin and B 12 , designated as holo-transcobalamin (holo-TC). We show that cultured BAECs endocytose [ 57 Co]-CNCbl-TC (source material) via the CD320 receptor. The bound Cbl is transported across the cell both via exocytosis in its free form, [ 57 Co]-CNCbl, and via transcytosis as [ 57 Co]-CNCbl-TC. Transcellular mobilization of Cbl occurred in a bidirectional manner. A portion of the endocytosed [ 57 Co]-CNCbl was enzymatically processed by methylmalonic aciduria combined with homocystinuria type C (cblC) with subsequent formation of hydroxocobalamin, methylcobalamin, and adenosylcobalamin, which were also transported across the cell in a bidirectional manner. This demonstrates that transport mechanisms for Cbl in vascular endothelial cells do not discriminate between various β-axial ligands of the vitamin. Competition studies with apoprotein- and holo-TC and holo-intrinsic factor showed that only holo-TC was effective at inhibiting transcellular transport of Cbl. Incubation of BAECs with a blocking antibody against the extracellular domain of the CD320 receptor inhibited uptake and transcytosis by ∼40%. This study reveals that endothelial cells recycle uncommitted intracellular Cbl for downstream usage by other cell types and suggests that the endothelium is self-sufficient for the specific acquisition and subsequent distribution of circulating B 12 via the CD320 receptor. We posit that the endothelial lining of the vasculature is an essential component for the maintenance of serum-tissue homeostasis of B 12 .-Hannibal, L., Bolisetty, K., Axhemi, A., DiBello, P

A PEDF-Derived Peptide Inhibits Retinal Neovascularization and Blocks Mobilization of Bone Marrow-Derived Endothelial Progenitor Cells

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Richard Longeras

2012-01-01

Full Text Available Proliferative diabetic retinopathy is characterized by pathological retinal neovascularization, mediated by both angiogenesis (involving mature endothelial cells and vasculogenesis (involving bone marrow-derived circulating endothelial progenitor cells (EPCs. Pigment epithelium-derived factor (PEDF contains an N-terminal 34-amino acid peptide (PEDF-34 that has antiangiogenic properties. Herein, we present a novel finding that PEDF-34 also possesses antivasculogenic activity. In the oxygen-induced retinopathy (OIR model using transgenic mice that have Tie2 promoter-driven GFP expression, we quantified Tie2GFP+ cells in bone marrow and peripheral blood by fluorescence-activated cell sorting (FACS. OIR significantly increased the number of circulating Tie2-GFP+ at P16, correlating with the peak progression of neovascularization. Daily intraperitoneal injections of PEDF-34 into OIR mice decreased the number of Tie2-GFP+ cells in the circulation at P16 by 65% but did not affect the number of Tie2-GFP+ cells in the bone marrow. These studies suggest that PEDF-34 attenuates EPC mobilization from the bone marrow into the blood circulation during retinal neovascularization.

Mobilization of endothelial precursor cells: systemic vascular response to musculoskeletal trauma.

LENUS (Irish Health Repository)

Laing, A J

2012-02-03

Postnatal vasculogenesis, the process by which vascular committed bone marrow stem cells or endothelial precursor cells (EPC) migrate, differentiate, and incorporate into the nacent endothelium contributing to physiological and pathological neovascularization, has stimulated much interest. Its contribution to tumor nonvascularization, wound healing, and revascularization associated with skeletal and cardiac muscles ischaemia is established. We evaluated the mobilization of EPCs in response to musculoskeletal trauma. Blood from patients (n = 15) following AO type 42a1 closed diaphyseal tibial fractures was analyzed for CD34 and AC133 cell surface marker expression. Immunomagnetically enriched CD34+ mononuclear cell (MNC(CD34+)) populations were cultured and examined for phenotypic and functional vascular endothelial differentiation. Circulating MNC(CD34+) levels increased sevenfold by day 3 postinjury. Circulating MNC(AC133+) increased 2.5-fold. Enriched MNC(CD34+) populations from day 3 samples in culture exhibited cell cluster formation with sprouting spindles. These cells bound UEA-1 and incorporated fluorescent DiI-Ac-LDL intracellularily. Our findings suggest a systemic provascular response is initiated in response to musculoskeletal trauma. Its therapeutic manipulation may have implications for the potential enhancement of fracture healing.

Circulating vascular endothelial growth factor during the normal menstrual cycle

NARCIS (Netherlands)

Kusumanto, YH; Hospers, GAP; Sluiter, WJ; Dam, WA; Meijer, C; Mulder, NH

2004-01-01

Background: The purpose of the study was to investigate whether cycle-related variations in circulating Vascular Endothelial Growth Factor (VEGF) levels would increase the metastatic potential at specific times during the menstrual cycle. Materials and Methods: VEGF levels in serum and whole blood

Platelet-derived stromal cell-derived factor-1 is required for the transformation of circulating monocytes into multipotential cells.

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Noriyuki Seta

Full Text Available BACKGROUND: We previously described a primitive cell population derived from human circulating CD14(+ monocytes, named monocyte-derived multipotential cells (MOMCs, which are capable of differentiating into mesenchymal and endothelial lineages. To generate MOMCs in vitro, monocytes are required to bind to fibronectin and be exposed to soluble factor(s derived from circulating CD14(- cells. The present study was conducted to identify factors that induce MOMC differentiation. METHODS: We cultured CD14(+ monocytes on fibronectin in the presence or absence of platelets, CD14(- peripheral blood mononuclear cells, platelet-conditioned medium, or candidate MOMC differentiation factors. The transformation of monocytes into MOMCs was assessed by the presence of spindle-shaped adherent cells, CD34 expression, and the potential to differentiate in vitro into mesenchymal and endothelial lineages. RESULTS: The presence of platelets or platelet-conditioned medium was required to generate MOMCs from monocytes. A screening of candidate platelet-derived soluble factors identified stromal cell-derived factor (SDF-1 as a requirement for generating MOMCs. Blocking an interaction between SDF-1 and its receptor CXCR4 inhibited MOMC generation, further confirming SDF-1's critical role in this process. Finally, circulating MOMC precursors were found to reside in the CD14(+CXCR4(high cell population. CONCLUSION: The interaction of SDF-1 with CXCR4 is essential for the transformation of circulating monocytes into MOMCs.

Endothelial-regenerating cells: an expanding universe.

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Steinmetz, Martin; Nickenig, Georg; Werner, Nikos

2010-03-01

Atherosclerosis is the most common cause for cardiovascular diseases and is based on endothelial dysfunction. A growing body of evidence suggests the contribution of bone marrow-derived endothelial progenitor cells, monocytic cells, and mature endothelial cells to vessel formation and endothelial rejuvenation. To this day, various subsets of these endothelial-regenerating cells have been identified according to cellular origin, phenotype, and properties in vivo and in vitro. However, the definition and biology, especially of endothelial progenitor cells, is complex and under heavy debate. In this review, we focus on current definitions of endothelial progenitor cells, highlight the clinical relevance of endothelial-regenerating cells, and provide new insights into cell-cell interactions involved in endothelial cell rejuvenation.

Effect of Low Level Ionizing Radiation on Endothelial Progenitor Cells in Atherosclerotic Patients with Lower Limb Ischemia

International Nuclear Information System (INIS)

Taha, E.F.S.

2013-01-01

Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality throughout the developed world (Williamson et al., 2012). Coronary artery disease (CAD) or atherosclerotic heart disease is a chronic life-threatening disease, which characterized by reducing blood supply to the heart as a result of the accumulation of atheromatous plaques within the walls of the arteries supplying the myocardium. Progressive atherosclerosis in the coronary arteries may lead to intimal thickening and eventual artery occlusion. Coronary artery occlusion can cause acute myocardial ischemia as a result of reduced oxygen supply or increased oxygen demand (Luthje and Andreas, 2008). Convincing evidence indicates that atherosclerosis is associated with endothelial dysfunction at the early stage of the disease process (Chiang et al., 2012). The endothelium is a dynamic cell layer that represents a physiological barrier between circulating blood and the surrounding tissues. Impaired endothelial function is a critical event in the initiation of atherosclerotic plaque development and thus may lead to vasoconstriction, vascular smooth muscle proliferation, hypercoagulability, thrombosis, and eventually, adverse cardiovascular events (Berger and Lavie, 2011). Asahara et al., (1997) described endothelial progenitor cells (EPC) in human peripheral blood. EPC are immature endothelial circulating cells mobilized from the bone marrow. These cells are involved in Introduction and aim of the work repairing the damaged endothelium and in facilitating neovascularization after ischemia (Rouhl et al., 2008). The role of EPC in health and disease is not understood completely. Most studies of healthy subjects and patients with coronary artery disease (CAD) report that the number and function of circulating EPC decrease with age and with the presence of classical vascular risk factors (Fadini et al., 2007). Recent studies suggested that EPCs play an important role in the risk of vascular

RADIOAUTOGRAPHIC DEMONSTRATION OF 5-HYDROXYTRYPTAMINE-3H UPTAKE BY PULMONARY ENDOTHELIAL CELLS

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Strum, Judy M.; Junod, Alain F.

1972-01-01

The lung is able to rapidly remove 5-hydroxytryptamme (5-HT) from the circulation by a Na+-dependent transport mechanism. In order to identify the sites of uptake, radioautographic studies were done on rat lungs which had been isolated and perfused with 5-HT-3H and 0 5 mM iproniazid, a monoamine oxidase inhibitor. In control experiments 10-4 M imipramine was added to the perfusate to inhibit the membrane transport of 5-HT At the light microscope level, silver grains were seen concentrated near capillaries and in the endothelium of large vessels From electron microscope radioautographs a semiquantitative grain count was made and 90% of the silver grains were observed over capillary endothelial cells. The grains were found over the nucleus and cytoplasm of the cell and shewed no preferential association with any particular cytoplasmic inclusion bodies, organelles, or vesicles Other cell types were unlabeled except for a few mast cells, certain vascular smooth muscle cells, and one nerve ending. This radioautographic demonstration of the cell type responsible for the rapid removal of 5-HT from the lung circulation clearly establishes the existence of a new metabolic role for pulmonary endothelial cells. PMID:5044755

Citrus Polyphenol Hesperidin Stimulates Production of Nitric Oxide in Endothelial Cells while Improving Endothelial Function and Reducing Inflammatory Markers in Patients with Metabolic Syndrome

Science.gov (United States)

Rizza, Stefano; Muniyappa, Ranganath; Iantorno, Micaela; Kim, Jeong-a; Chen, Hui; Pullikotil, Philomena; Senese, Nicoletta; Tesauro, Manfredi; Lauro, Davide; Cardillo, Carmine

2011-01-01

Context: Hesperidin, a citrus flavonoid, and its metabolite hesperetin may have vascular actions relevant to their health benefits. Molecular and physiological mechanisms of hesperetin actions are unknown. Objective: We tested whether hesperetin stimulates production of nitric oxide (NO) from vascular endothelium and evaluated endothelial function in subjects with metabolic syndrome on oral hesperidin therapy. Design, Setting, and Interventions: Cellular mechanisms of action of hesperetin were evaluated in bovine aortic endothelial cells (BAEC) in primary culture. A randomized, placebo-controlled, double-blind, crossover trial examined whether oral hesperidin administration (500 mg once daily for 3 wk) improves endothelial function in individuals with metabolic syndrome (n = 24). Main Outcome Measure: We measured the difference in brachial artery flow-mediated dilation between placebo and hesperidin treatment periods. Results: Treatment of BAEC with hesperetin acutely stimulated phosphorylation of Src, Akt, AMP kinase, and endothelial NO synthase to produce NO; this required generation of H2O2. Increased adhesion of monocytes to BAEC and expression of vascular cell adhesion molecule-1 in response to TNF-α treatment was reduced by pretreatment with hesperetin. In the clinical study, when compared with placebo, hesperidin treatment increased flow-mediated dilation (10.26 ± 1.19 vs. 7.78 ± 0.76%; P = 0.02) and reduced concentrations of circulating inflammatory biomarkers (high-sensitivity C-reactive protein, serum amyloid A protein, soluble E-selectin). Conclusions: Novel mechanisms for hesperetin action in endothelial cells inform effects of oral hesperidin treatment to improve endothelial dysfunction and reduce circulating markers of inflammation in our exploratory clinical trial. Hesperetin has vasculoprotective actions that may explain beneficial cardiovascular effects of citrus consumption. PMID:21346065

Effect of onion peel extract on endothelial function and endothelial progenitor cells in overweight and obese individuals.

Science.gov (United States)

Choi, Eun-Yong; Lee, Hansongyi; Woo, Jong Shin; Jang, Hyun Hee; Hwang, Seung Joon; Kim, Hyun Soo; Kim, Woo-Sik; Kim, Young-Seol; Choue, Ryowon; Cha, Yong-Jun; Yim, Jung-Eun; Kim, Weon

2015-09-01

Acute or chronic intake of polyphenol-rich foods has been reported to improve endothelial function. Quercetin, found abundantly in onion, is a potent antioxidant flavonoid. The aim of this study was to investigate whether consumption of onion peel extract (OPE) improves endothelial function in healthy overweight and obese individuals. This was a randomized double-blind, placebo-controlled study. Seventy-two healthy overweight and obese participants were randomly assigned to receive a red, soft capsule of OPE (100 mg quercetin/d, 50 mg quercetin twice daily; n = 36 participants) or an identical placebo capsule (n = 36) for 12 wk. Endothelial function, defined by flow-mediated dilation (FMD), circulating endothelial progenitor cells (EPCs) by flow cytometry, and laboratory test were determined at baseline and after treatment. Baseline characteristics and laboratory findings did not significantly differ between the two groups. Compared with baseline values, the OPE group showed significantly improved FMD at 12 wk (from 12.5 ± 5.2 to 15.2 ± 6.1; P = 0.002), whereas the placebo group showed no difference. Nitroglycerin-mediated dilation did not change in either group. EPC counts (44.2 ± 25.6 versus 52.3 ± 18.6; P = 0.005) and the percentage of EPCs were significantly increased in the OPE group. When FMD was divided into quartiles, rate of patients with endothelial dysfunction defined as lowest quartile (cutoff value, 8.6%) of FMD improved from 26% to 9% by OPE. Medium-term administration of OPE an improvement in FMD and circulating EPCs. Copyright © 2015 Elsevier Inc. All rights reserved.

Cilostazol activates function of bone marrow-derived endothelial progenitor cell for re-endothelialization in a carotid balloon injury model.

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Rie Kawabe-Yako

Full Text Available BACKGROUND: Cilostazol(CLZ has been used as a vasodilating anti-platelet drug clinically and demonstrated to inhibit proliferation of smooth muscle cells and effect on endothelial cells. However, the effect of CLZ on re-endothelialization including bone marrow (BM-derived endothelial progenitor cell (EPC contribution is unclear. We have investigated the hypothesis that CLZ might accelerate re-endothelialization with EPCs. METHODOLOGY/PRINCIPAL FINDINGS: Balloon carotid denudation was performed in male Sprague-Dawley rats. CLZ group was given CLZ mixed feed from 2 weeks before carotid injury. Control group was fed normal diet. CLZ accelerated re-endothelialization at 2 weeks after surgery and resulted in a significant reduction of neointima formation 4 weeks after surgery compared with that in control group. CLZ also increased the number of circulating EPCs throughout the time course. We examined the contribution of BM-derived EPCs to re-endothelialization by BM transplantation from Tie2/lacZ mice to nude rats. The number of Tie2-regulated X-gal positive cells on injured arterial luminal surface was increased at 2 weeks after surgery in CLZ group compared with that in control group. In vitro, CLZ enhanced proliferation, adhesion and migration activity, and differentiation with mRNA upregulation of adhesion molecule integrin αvβ3, chemokine receptor CXCR4 and growth factor VEGF assessed by real-time RT-PCR in rat BM-derived cultured EPCs. In addition, CLZ markedly increased the expression of SDF-1α that is a ligand of CXCR4 receptor in EPCs, in the media following vascular injury. CONCLUSIONS/SIGNIFICANCE: CLZ promotes EPC mobilization from BM and EPC recruitment to sites of arterial injury, and thereby inhibited neointima formation with acceleration of re-endothelialization with EPCs as well as pre-existing endothelial cells in a rat carotid balloon injury model. CLZ could be not only an anti-platelet agent but also a promising tool for

Endothelial RIG-I activation impairs endothelial function

International Nuclear Information System (INIS)

Asdonk, Tobias; Motz, Inga; Werner, Nikos; Coch, Christoph; Barchet, Winfried; Hartmann, Gunther; Nickenig, Georg; Zimmer, Sebastian

2012-01-01

Highlights: ► RIG-I activation impairs endothelial function in vivo. ► RIG-I activation alters HCAEC biology in vitro. ► EPC function is affected by RIG-I stimulation in vitro. -- Abstract: Background: Endothelial dysfunction is a crucial part of the chronic inflammatory atherosclerotic process and is mediated by innate and acquired immune mechanisms. Recent studies suggest that pattern recognition receptors (PRR) specialized in immunorecognition of nucleic acids may play an important role in endothelial biology in a proatherogenic manner. Here, we analyzed the impact of endothelial retinoic acid inducible gene I (RIG-I) activation upon vascular endothelial biology. Methods and results: Wild type mice were injected intravenously with 32.5 μg of the RIG-ligand 3pRNA (RNA with triphosphate at the 5′end) or polyA control every other day for 7 days. In 3pRNA-treated mice, endothelium-depended vasodilation was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticle (EMP) numbers significantly elevated compared to controls. To gain further insight in RIG-I dependent endothelial biology, cultured human coronary endothelial cells (HCAEC) and endothelial progenitor cells (EPC) were stimulated in vitro with 3pRNA. Both cells types express RIG-I and react with receptor upregulation upon stimulation. Reactive oxygen species (ROS) formation is enhanced in both cell types, whereas apoptosis and proliferation is not significantly affected in HCAEC. Importantly, HCAEC release significant amounts of proinflammatory cytokines in response to RIG-I stimulation. Conclusion: This study shows that activation of the cytoplasmatic nucleic acid receptor RIG-I leads to endothelial dysfunction. RIG-I induced endothelial damage could therefore be an important pathway in atherogenesis.

Circulating CD34-positive cells, glomerular filtration rate and triglycerides in relation to hypertension.

Science.gov (United States)

Shimizu, Yuji; Sato, Shimpei; Koyamatsu, Jun; Yamanashi, Hirotomo; Nagayoshi, Mako; Kadota, Koichiro; Maeda, Takahiro

2015-11-01

Serum triglycerides have been reported to be independently associated with the development of chronic kidney disease (CKD), which is known to play a role in vascular disturbance. On the other hand, circulating CD34-positve cells, including endothelial progenitor cells, are reported to contribute to vascular repair. However, no studies have reported on the correlation between triglycerides and the number of CD34-positive cells. Since hypertension is well known factor for vascular impairment, the degree of correlation between serum triglycerides and circulating CD34-positve cells should account for hypertension status. We conducted a cross-sectional study of 274 elderly Japanese men aged ≥ 60 years (range 60-79 years) undergoing general health checkups. Multiple linear regression analysis of non-hypertensive subjects adjusting for classical cardiovascular risk factors showed that although triglyceride levels (1SD increments; 64 mg/dL) did not significantly correlate with glomerular filtration rate (GFR) (β = -2.06, p = 0.163), a significant positive correlation was seen between triglycerides and the number of circulating CD34-positive cells (β = 0.50, p = 0.004). In hypertensive subjects, a significant inverse correlation between triglycerides and GFR was observed (β = -2.66, p = 0.035), whereas no significant correlation between triglycerides and the number of circulating CD34-positive cells was noted (β = -0.004, p = 0.974). Since endothelial progenitor cells (CD34-positive cells) have been reported to contribute to vascular repair, our results indicate that in non-hypertensive subjects, triglycerides may stimulate an increase in circulating CD34-positive cells (vascular repair) by inducing vascular disturbance. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Ascorbic acid attenuates endothelial permeability triggered by cell-free hemoglobin.

Science.gov (United States)

Kuck, Jamie L; Bastarache, Julie A; Shaver, Ciara M; Fessel, Joshua P; Dikalov, Sergey I; May, James M; Ware, Lorraine B

2018-01-01

Increased endothelial permeability is central to shock and organ dysfunction in sepsis but therapeutics targeted to known mediators of increased endothelial permeability have been unsuccessful in patient studies. We previously reported that cell-free hemoglobin (CFH) is elevated in the majority of patients with sepsis and is associated with organ dysfunction, poor clinical outcomes and elevated markers of oxidant injury. Others have shown that Vitamin C (ascorbate) may have endothelial protective effects in sepsis. In this study, we tested the hypothesis that high levels of CFH, as seen in the circulation of patients with sepsis, disrupt endothelial barrier integrity. Human umbilical vein endothelial cells (HUVEC) were grown to confluence and treated with CFH with or without ascorbate. Monolayer permeability was measured by Electric Cell-substrate Impedance Sensing (ECIS) or transfer of 14 C-inulin. Viability was measured by trypan blue exclusion. Intracellular ascorbate was measured by HPLC. CFH increased permeability in a dose- and time-dependent manner with 1 mg/ml of CFH increasing inulin transfer by 50% without affecting cell viability. CFH (1 mg/ml) also caused a dramatic reduction in intracellular ascorbate in the same time frame (1.4 mM without CFH, 0.23 mM 18 h after 1 mg/ml CFH, p < 0.05). Pre-treatment of HUVECs with ascorbate attenuated CFH induced permeability. CFH increases endothelial permeability in part through depletion of intracellular ascorbate. Supplementation of ascorbate can attenuate increases in permeability mediated by CFH suggesting a possible therapeutic approach in sepsis. Copyright © 2017 Elsevier Inc. All rights reserved.

Production of soluble Neprilysin by endothelial cells

International Nuclear Information System (INIS)

Kuruppu, Sanjaya; Rajapakse, Niwanthi W.; Minond, Dmitriy; Smith, A. Ian

2014-01-01

Highlights: • A soluble full-length form of Neprilysin exists in media of endothelial cells. • Exosomal release is the key mechanism for the production of soluble Neprilysin. • Inhibition of ADAM-17 by specific inhibitors reduce Neprilysin release. • Exosome mediated release of Neprilysin is dependent on ADAM-17 activity. - Abstract: A non-membrane bound form of Neprilysin (NEP) with catalytic activity has the potential to cleave substrates throughout the circulation, thus leading to systemic effects of NEP. We used the endothelial cell line Ea.hy926 to identify the possible role of exosomes and A Disintegrin and Metalloprotease 17 (ADAM-17) in the production of non-membrane bound NEP. Using a bradykinin based quenched fluorescent substrate (40 μM) assay, we determined the activity of recombinant human NEP (rhNEP; 12 ng), and NEP in the media of endothelial cells (10% v/v; after 24 h incubation with cells) to be 9.35 ± 0.70 and 6.54 ± 0.41 μmols of substrate cleaved over 3 h, respectively. The presence of NEP in the media was also confirmed by Western blotting. At present there are no commercially available inhibitors specific for ADAM-17. We therefore synthesised two inhibitors TPI2155-14 and TPI2155-17, specific for ADAM-17 with IC 50 values of 5.36 and 4.32 μM, respectively. Treatment of cells with TPI2155-14 (15 μM) and TPI2155-17 (4.3 μM) resulted in a significant decrease in NEP activity in media (62.37 ± 1.43 and 38.30 ± 4.70, respectively as a % of control; P < 0.0001), implicating a possible role for ADAM-17 in NEP release. However, centrifuging media (100,000g for 1 h at 4 °C) removed all NEP activity from the supernatant indicating the likely role of exosomes in the release of NEP. Our data therefore indicated for the first time that NEP is released from endothelial cells via exosomes, and that this process is dependent on ADAM-17

Granulocyte colony-stimulating factor mobilizes functional endothelial progenitor cells in patients with coronary artery disease.

Science.gov (United States)

Powell, Tiffany M; Paul, Jonathan D; Hill, Jonathan M; Thompson, Michael; Benjamin, Moshe; Rodrigo, Maria; McCoy, J Philip; Read, Elizabeth J; Khuu, Hanh M; Leitman, Susan F; Finkel, Toren; Cannon, Richard O

2005-02-01

Endothelial progenitor cells (EPCs) that may repair vascular injury are reduced in patients with coronary artery disease (CAD). We reasoned that EPC number and function may be increased by granulocyte colony-stimulating factor (G-CSF) used to mobilize hematopoietic progenitor cells in healthy donors. Sixteen CAD patients had reduced CD34(+)/CD133(+) (0.0224+/-0.0063% versus 0.121+/-0.038% mononuclear cells [MNCs], P<0.01) and CD133(+)/VEGFR-2(+) cells, consistent with EPC phenotype (0.00033+/-0.00015% versus 0.0017+/-0.0006% MNCs, P<0.01), compared with 7 healthy controls. Patients also had fewer clusters of cells in culture, with out-growth consistent with mature endothelial phenotype (2+/-1/well) compared with 16 healthy subjects at high risk (13+/-4/well, P<0.05) or 14 at low risk (22+/-3/well, P<0.001) for CAD. G-CSF 10 microg/kg per day for 5 days increased CD34(+)/CD133(+) cells from 0.5+/-0.2/microL to 59.5+/-10.6/microL and CD133(+)/ VEGFR-2(+) cells from 0.007+/-0.004/microL to 1.9+/-0.6/microL (both P<0.001). Also increased were CD133(+) cells that coexpressed the homing receptor CXCR4 (30.4+/-8.3/microL, P<0.05). Endothelial cell-forming clusters in 10 patients increased to 27+/-9/well after treatment (P<0.05), with a decline to 9+/-4/well at 2 weeks (P=0.06). Despite reduced EPCs compared with healthy controls, patients with CAD respond to G-CSF with increases in EPC number and homing receptor expression in the circulation and endothelial out-growth in culture. Endothelial progenitor cells (EPCs) are reduced in coronary artery disease. Granulocyte colony-stimulating factor (CSF) administered to patients increased: (1) CD133+/VEGFR-2+ cells consistent with EPC phenotype; (2) CD133+ cells coexpressing the chemokine receptor CXCR4, important for homing of EPCs to ischemic tissue; and (3) endothelial cell-forming clusters in culture. Whether EPCs mobilized into the circulation will be useful for the purpose of initiating vascular growth and myocyte repair

Endothelial RIG-I activation impairs endothelial function

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Asdonk, Tobias, E-mail: tobias.asdonk@ukb.uni-bonn.de [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Motz, Inga; Werner, Nikos [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Coch, Christoph; Barchet, Winfried; Hartmann, Gunther [Institute for Clinical Chemistry and Clinical Pharmacology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Nickenig, Georg; Zimmer, Sebastian [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany)

2012-03-30

Highlights: Black-Right-Pointing-Pointer RIG-I activation impairs endothelial function in vivo. Black-Right-Pointing-Pointer RIG-I activation alters HCAEC biology in vitro. Black-Right-Pointing-Pointer EPC function is affected by RIG-I stimulation in vitro. -- Abstract: Background: Endothelial dysfunction is a crucial part of the chronic inflammatory atherosclerotic process and is mediated by innate and acquired immune mechanisms. Recent studies suggest that pattern recognition receptors (PRR) specialized in immunorecognition of nucleic acids may play an important role in endothelial biology in a proatherogenic manner. Here, we analyzed the impact of endothelial retinoic acid inducible gene I (RIG-I) activation upon vascular endothelial biology. Methods and results: Wild type mice were injected intravenously with 32.5 {mu}g of the RIG-ligand 3pRNA (RNA with triphosphate at the 5 Prime end) or polyA control every other day for 7 days. In 3pRNA-treated mice, endothelium-depended vasodilation was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticle (EMP) numbers significantly elevated compared to controls. To gain further insight in RIG-I dependent endothelial biology, cultured human coronary endothelial cells (HCAEC) and endothelial progenitor cells (EPC) were stimulated in vitro with 3pRNA. Both cells types express RIG-I and react with receptor upregulation upon stimulation. Reactive oxygen species (ROS) formation is enhanced in both cell types, whereas apoptosis and proliferation is not significantly affected in HCAEC. Importantly, HCAEC release significant amounts of proinflammatory cytokines in response to RIG-I stimulation. Conclusion: This study shows that activation of the cytoplasmatic nucleic acid receptor RIG-I leads to endothelial dysfunction. RIG-I induced endothelial damage could therefore be an important pathway in atherogenesis.

Impact of obesity control on circulating level of endothelial progenitor cells and angiogenesis in response to ischemic stimulation

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Chen Yung-Lung

2012-07-01

Full Text Available Abstract Background and aim We tested the hypothesis that obesity reduced circulating number of endothelial progenitor cells (EPCs, angiogenic ability, and blood flow in ischemic tissue that could be reversed after obesity control. Methods 8-week-old C57BL/6J mice (n = 27 were equally divided into group 1 (fed with 22-week control diet, group 2 (22-week high fat diet, and group 3 (14-week high fat diet, followed by 8-week control diet. Critical limb ischemia (CLI was induced at week 20 in groups 2 and 3. The animals were sacrificed at the end of 22 weeks. Results Heart weight, body weight, abdominal fat weight, serum total cholesterol level, and fasting blood sugar were highest in group 2 (all p  Conclusion Obesity suppressed abilities of angiogenesis and recovery from CLI that were reversed by obesity control.

Increased endothelial cell-leukocyte interaction in murine schistosomiasis: possible priming of endothelial cells by the disease.

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Suellen D S Oliveira

Full Text Available BACKGROUND AND AIMS: Schistosomiasis is an intravascular parasitic disease associated with inflammation. Endothelial cells control leukocyte transmigration and vascular permeability being modulated by pro-inflammatory mediators. Recent data have shown that endothelial cells primed in vivo in the course of a disease keep the information in culture. Herein, we evaluated the impact of schistosomiasis on endothelial cell-regulated events in vivo and in vitro. METHODOLOGY AND PRINCIPAL FINDINGS: The experimental groups consisted of Schistosoma mansoni-infected and age-matched control mice. In vivo infection caused a marked influx of leukocytes and an increased protein leakage in the peritoneal cavity, characterizing an inflamed vascular and cellular profile. In vitro leukocyte-mesenteric endothelial cell adhesion was higher in cultured cells from infected mice as compared to controls, either in the basal condition or after treatment with the pro-inflammatory cytokine tumor necrosis factor (TNF. Nitric oxide (NO donation reduced leukocyte adhesion to endothelial cells from control and infected groups; however, in the later group the effect was more pronounced, probably due to a reduced NO production. Inhibition of control endothelial NO synthase (eNOS increased leukocyte adhesion to a level similar to the one observed in the infected group. Besides, the adhesion of control leukocytes to endothelial cells from infected animals is similar to the result of infected animals, confirming that schistosomiasis alters endothelial cells function. Furthermore, NO production as well as the expression of eNOS were reduced in cultured endothelial cells from infected animals. On the other hand, the expression of its repressor protein, namely caveolin-1, was similar in both control and infected groups. CONCLUSION/SIGNIFICANCE: Schistosomiasis increases vascular permeability and endothelial cell-leukocyte interaction in vivo and in vitro. These effects are partially

Differential Gene Expression of Primary Cultured Lymphatic and Blood Vascular Endothelial Cells

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Gregory M. Nelson

2007-12-01

Full Text Available Blood vascular endothelial cells (BECs and the developmentally related lymphatic endothelial cells (LECs create complementary, yet distinct vascular networks. Each endothelial cell type interacts with flowing fluid and circulating cells, yet each vascular system has evolved specialized gene expression programs and thus both cell types display different phenotypes. BECs and LECs express distinct genes that are unique to their specific vascular microenvironment. Tumors also take advantage of the molecules that are expressed in these vascular systems to enhance their metastatic potential. We completed transcriptome analyses on primary cultured LECs and BECs, where each comparative set was isolated from the same individual. Differences were resolved in the expression of several major categories, such as cell adhesion molecules (CAMs, cytokines, cytokine receptors. We have identified new molecules that are associated with BECs (e.g., claudin-9, CXCL11, neurexin-1, neurexin-2, the neuronal growth factor regulator-1 and LECs (e.g., claudin-7, CD58, hyaluronan and proteoglycan link protein 1 (HAPLN1, the poliovirus receptor-related 3 molecule that may lead to novel therapeutic treatments for diseases of lymphatic or blood vessels, including metastasis of cancer to lymph nodes or distant organs.

Signaling hierarchy regulating human endothelial cell development.

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Kelly, Melissa A; Hirschi, Karen K

2009-05-01

Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these studies. Using human embryonic stem cells as a model system, we were able to reproducibly and robustly generate differentiated endothelial cells via coculture on OP9 marrow stromal cells. We found that, in contrast to studies in the mouse, bFGF and VEGF had no specific effects on the initiation of human vasculogenesis. However, exogenous Ihh promoted endothelial cell differentiation, as evidenced by increased production of cells with cobblestone morphology that coexpress multiple endothelial-specific genes and proteins, form lumens, and exhibit DiI-AcLDL uptake. Inhibition of BMP signaling using Noggin or BMP4, specifically, using neutralizing antibodies suppressed endothelial cell formation; whereas, addition of rhBMP4 to cells treated with the hedgehog inhibitor cyclopamine rescued endothelial cell development. Our studies revealed that Ihh promoted human endothelial cell differentiation from pluripotent hES cells via BMP signaling, providing novel insights applicable to modulating human endothelial cell formation and vascular regeneration for human clinical therapies.

Endothelial Progenitor Cells for Diagnosis and Prognosis in Cardiovascular Disease

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Caterina Oriana Aragona

2016-01-01

Full Text Available Objective. To identify, evaluate, and synthesize evidence on the predictive power of circulating endothelial progenitor cells (EPCs in cardiovascular disease, through a systematic review of quantitative studies. Data Sources. MEDLINE was searched using keywords related to “endothelial progenitor cells” and “endothelium” and, for the different categories, respectively, “smoking”; “blood pressure”; “diabetes mellitus” or “insulin resistance”; “dyslipidemia”; “aging” or “elderly”; “angina pectoris” or “myocardial infarction”; “stroke” or “cerebrovascular disease”; “homocysteine”; “C-reactive protein”; “vitamin D”. Study Selection. Database hits were evaluated against explicit inclusion criteria. From 927 database hits, 43 quantitative studies were included. Data Syntheses. EPC count has been suggested for cardiovascular risk estimation in the clinical practice, since it is currently accepted that EPCs can work as proangiogenic support cells, maintaining their importance as regenerative/reparative potential, and also as prognostic markers. Conclusions. EPCs showed an important role in identifying cardiovascular risk conditions, and to suggest their evaluation as predictor of outcomes appears to be reasonable in different defined clinical settings. Due to their capability of proliferation, circulation, and the development of functional progeny, great interest has been directed to therapeutic use of progenitor cells in atherosclerotic diseases. This trial is registered with registration number: Prospero CRD42015023717.

Cathepsin L is required for endothelial progenitor cell-induced neovascularization

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Urbich, Carmen; Heeschen, Christopher; Aicher, Alexandra; Sasaki, Ken-ichiro; Bruhl, Thomas; Hofmann, Wolf K.; Peters, Christoph; Reinheckel, Thomas; Pennacchio, Len A.; Abolmaali, Nasreddin D.; Chavakis, Emmanouil; Zeiher, Andreas M.; Dimmeler, Stefanie

2004-01-15

Infusion of endothelial progenitor cells (EPCs), but not of mature endothelial cells (ECs), promotes neovascularization after ischemia. We performed a gene expression profiling of EPCs and ECs to identify genes, which might be important for the neovascularization capacity of EPCs. Intriguingly, the protease cathepsin L (CathL) was highly expressed in EPCs as opposed to ECs and is essential for matrix degradation and invasion by EPCs in vitro. CathL deficient mice showed impaired functional recovery after hind limb ischemia supporting the concept for an important role of CathL in postnatal neovascularization. Infused CathL deficient progenitor cells failed to home to sites of ischemia and to augment neovascularization. In contrast, over expression of CathL in mature ECs significantly enhanced their invasive activity and induced their neovascularization capacity in vivo. Taken together, CathL plays a crucial role for the integration of circulating EPCs into the ischemic tissue and is required for neovascularization mediated by EPCs.

Optimized multiparametric flow cytometric analysis of circulating endothelial cells and their subpopulations in peripheral blood of patients with solid tumors: a technical analysis

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Zhou F

2018-03-01

Full Text Available Fangbin Zhou,1,2 Yaying Zhou,3 Ming Yang,1 Jinli Wen,3 Jun Dong,4 Wenyong Tan1 1Department of Oncology, The Second Clinical Medical College, Shenzhen People’s Hospital, Jinan University, Shenzhen, People’s Republic of China; 2Integrated Chinese and Western Medicine Postdoctoral Research Station, Jinan University, Guangzhou, People’s Republic of China; 3Clinical Medical Research Center, The Second Clinical Medical College, Shenzhen People’s Hospital, Jinan University, Shenzhen, People’s Republic of China; 4Department of Pathophysiology, Key Laboratory of the State Administration of Traditional Chinese Medicine, Medical College of Jinan University, Guangzhou, People’s Republic of China Background: Circulating endothelial cells (CECs and their subpopulations could be potential novel biomarkers for various malignancies. However, reliable enumerable methods are warranted to further improve their clinical utility. This study aimed to optimize a flow cytometric method (FCM assay for CECs and subpopulations in peripheral blood for patients with solid cancers.Patients and methods: An FCM assay was used to detect and identify CECs. A panel of 60 blood samples, including 44 metastatic cancer patients and 16 healthy controls, were used in this study. Some key issues of CEC enumeration, including sample material and anticoagulant selection, optimal titration of antibodies, lysis/wash procedures of blood sample preparation, conditions of sample storage, sufficient cell events to enhance the signal, fluorescence-minus-one controls instead of isotype controls to reduce background noise, optimal selection of cell surface markers, and evaluating the reproducibility of our method, were integrated and investigated. Wilcoxon and Mann–Whitney U tests were used to determine statistically significant differences.Results: In this validation study, we refined a five-color FCM method to detect CECs and their subpopulations in peripheral blood of patients

Reduced Ang2 expression in aging endothelial cells

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Hohensinner, P.J.; Ebenbauer, B.; Kaun, C.; Maurer, G.; Huber, K.; Wojta, J.

2016-01-01

Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. -- Highlights: •Endothelial cells display signs of aging before reaching proliferative senescence. •Aging endothelial cells express more angiopoietin 1 and less angiopoietin 2 than young endothelial cells. •Migratory capacity is reduced in aging endothelial cells.

Reduced Ang2 expression in aging endothelial cells

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Hohensinner, P.J., E-mail: philipp.hohensinner@meduniwien.ac.at [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ebenbauer, B. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); Kaun, C.; Maurer, G. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Huber, K. [Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); 3rd Medical Department, Wilhelminenhospital, Vienna (Austria); Sigmund Freud University, Medical Faculty, Vienna (Austria); Wojta, J. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); Core Facilities, Medical University of Vienna, Vienna (Austria)

2016-06-03

Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. -- Highlights: •Endothelial cells display signs of aging before reaching proliferative senescence. •Aging endothelial cells express more angiopoietin 1 and less angiopoietin 2 than young endothelial cells. •Migratory capacity is reduced in aging endothelial cells.

In Vitro Endothelial Cell Proliferation Assay Reveals Distinct Levels of Proangiogenic Cytokines Characterizing Sera of Healthy Subjects and of Patients with Heart Failure

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Rebecca Voltan

2014-01-01

Full Text Available Although myocardial angiogenesis is thought to play an important role in heart failure (HF, the involvement of circulating proinflammatory and proangiogenic cytokines in the pathogenesis and/or prognosis of HF has not been deeply investigated. By using a highly standardized proliferation assay with human endothelial cells, we first demonstrated that sera from older (mean age 52±7.6 years; n=46 healthy donors promoted endothelial cell proliferation to a significantly higher extent compared to sera obtained from younger healthy donors (mean age 29±8.6 years; n=20. The promotion of endothelial cell proliferation was accompanied by high serum levels of several proangiogenic cytokines. When we assessed endothelial cell proliferation in response to HF patients’ sera, we observed that a subset of sera (n=11 promoted cell proliferation to a significantly lesser extent compared to the majority of sera (n=18. Also, in this case, the difference between the patient groups in the ability to induce endothelial cell proliferation correlated to significant (P<0.05 differences in serum proangiogenic cytokine levels. Unexpectedly, HF patients associated to the highest endothelial proliferation index showed the worst prognosis as evaluated in terms of subsequent cardiovascular events in the follow-up, suggesting that high levels of circulating proangiogenic cytokines might be related to a worse prognosis.

High levels of circulating VEGFR2+ Bone marrow-derived progenitor cells correlate with metastatic disease in patients with pediatric solid malignancies.

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Taylor, Melissa; Rössler, Jochen; Geoerger, Birgit; Laplanche, Agnès; Hartmann, Olivier; Vassal, Gilles; Farace, Françoise

2009-07-15

Pediatric solid malignancies display important angiogenic potential, and blocking tumor angiogenesis represents a new therapeutic approach for these patients. Recent studies have evidenced rare circulating cells with endothelial features contributing to tumor neovascularization and have shown the pivotal role of bone marrow-derived (BMD) progenitor cells in metastatic disease progression. We measured these cells in patients with pediatric solid malignancies as a prerequisite to clinical trials with antiangiogenic therapy. Peripheral blood was drawn from 45 patients with localized (n = 23) or metastatic (n = 22) disease, and 20 healthy subjects. Subsets of circulating vascular endothelial growth factor receptor (VEGFR)2+-BMD progenitor cells, defined as CD45-CD34+VEGFR2(KDR)+7AAD- and CD45(dim)CD34+VEGFR2+7AAD- events, were measured in progenitor-enriched fractions by flow cytometry. Mature circulating endothelial cells (CEC) were measured in whole blood as CD31+CD146+CD45-7AAD- viable events. Data were correlated with VEGF and sVEGFR2 plasma levels. The CD45-CD34+VEGFR2(KDR)+7AAD- subset represented <0.003% of circulating BMD progenitor cells (< or =0.05 cells/mL). However, the median level (range) of the CD45(dim)CD34+VEGFR2+7AAD- subset was higher in patients compared with healthy subjects, 1.5% (0%-10.3%) versus 0.3% (0%-1.6%) of circulating BMD progenitors (P < 0.0001), and differed significantly between patients with localized and metastatic disease, 0.7% (0%-8.6%) versus 2.9% (0.6%-10.3%) of circulating BMD progenitors (P < 0.001). Median CEC value was 7 cells/mL (0-152 cells/mL) and similar in all groups. Unlike VEGFR2+-BMD progenitors, neither CECs, VEGF, or sVEGFR2 plasma levels correlated with disease status. High levels of circulating VEGFR2+-BMD progenitor cells correlated with metastatic disease. Our study provides novel insights for angiogenesis mechanisms in pediatric solid malignancies for which antiangiogenic targeting of VEGFR2+-BMD progenitors

Smoking decreases the level of circulating CD34+ progenitor cells in young healthy women - a pilot study

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Baumann Gert

2010-05-01

Full Text Available Abstract Background Decreased levels of circulating bone marrow-derived progenitor cells have been associated with risk factors and cardiovascular diseases. Smoking is the most important modifiable risk factor for atherosclerosis in young women. The aim of this pilot study was to assess in healthy premenopausal women without other risk factors for cardiovascular disease the influence of nicotine abuse on the number of circulating progenitor cells in relation to endothelial function. Methods The number of endothelial progenitor cells, measured as colony-forming units in a cell-culture assay (EPC-CFU and the number of circulating CD34 + and CD34 + /CD133 + cells, measured by flow cytometry, was estimated in 32 women at the menstrual phase of the menstrual cycle. In addition, flow-mediated dilation (FMD was assessed as a marker for vascular function. In a subgroup of these women (n = 20, progenitor cells were also investigated at the mid-follicular and luteal phases of the menstrual cycle. Results Compared to non-smokers, the abundance of circulating CD34 + cells was significantly lower in smoking women in the menstrual, mid-luteal, and mid-follicular phases of the menstrual cycle. The number of CD34 + progenitor cells was revealed to have significant positive correlation with FMD in young healthy women, whereas CD34 + /CD133 + progenitor cells and EPC-CFU showed no significant correlation. Conclusion The number of CD34 + progenitor cells positively correlates with FMD in young healthy women and is decreased by smoking.

Circulating cell-derived microparticles in severe preeclampsia and in fetal growth restriction.

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Alijotas-Reig, Jaume; Palacio-Garcia, Carles; Farran-Codina, Immaculada; Ruiz-Romance, Mar; Llurba, Elisa; Vilardell-Tarres, Miquel

2012-02-01

The behavior of the circulating microparticles (cMP) in severe preeclampsia (PE) and fetal growth restriction (FGR) is disputed. METHOD OF STUDY  Non-matched case-control study. Seventy cases of severe PE/HELLP/FGR were compared to 38 healthy pregnant women. Twenty healthy non-pregnant women acted as a control. cMP were analyzed using flow cytometry. Results are given as total (annexin-A5-ANXA5+), platelet (CD41+), leukocyte (CD45+), endothelial (CD144+CD31+//CD41-), and CD41-negative cMP/μL of plasma. Antiphospholipid antibodies (aPL) were analyzed through usual methods. Platelet and endothelial cMP increased in healthy pregnant women. PE whole group (PE±FGR) showed an increase in endothelial and CD41-negative, but not in platelet-derived, cMP. Comparing PE whole group versus healthy pregnant, we found cMP levels of endothelial and CD41- had increased. The cMP results obtained in PE group were similar to those of the PE whole group. Comparing PE group to isolated FGR, significant CD41-negative cMP increase was found in PE. According to its aPL positivity, a trend to decrease in leukocyte and endothelial-derived cMP was found in PE group. Normal pregnancy is accompanied by endothelial and platelet cell activation. Endothelial cell activation has been shown in PE but not in isolated FGR. In PE, aPL may contribute to endothelial and possibly to leukocyte cell activation. © 2011 John Wiley & Sons A/S.

Arachidonic acid metabolites and endothelial dysfunction of portal hypertension.

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Sacerdoti, David; Pesce, Paola; Di Pascoli, Marco; Brocco, Silvia; Cecchetto, Lara; Bolognesi, Massimo

2015-07-01

Increased resistance to portal flow and increased portal inflow due to mesenteric vasodilatation represent the main factors causing portal hypertension in cirrhosis. Endothelial cell dysfunction, defined as an imbalance between the synthesis, release, and effect of endothelial mediators of vascular tone, inflammation, thrombosis, and angiogenesis, plays a major role in the increase of resistance in portal circulation, in the decrease in the mesenteric one, in the development of collateral circulation. Reduced response to vasodilators in liver sinusoids and increased response in the mesenteric arterioles, and, viceversa, increased response to vasoconstrictors in the portal-sinusoidal circulation and decreased response in the mesenteric arterioles are also relevant to the pathophysiology of portal hypertension. Arachidonic acid (AA) metabolites through the three pathways, cyclooxygenase (COX), lipoxygenase, and cytochrome P450 monooxygenase and epoxygenase, are involved in endothelial dysfunction of portal hypertension. Increased thromboxane-A2 production by liver sinusoidal endothelial cells (LSECs) via increased COX-1 activity/expression, increased leukotriens, increased epoxyeicosatrienoic acids (EETs) (dilators of the peripheral arterial circulation, but vasoconstrictors of the portal-sinusoidal circulation), represent a major component in the increased portal resistance, in the decreased portal response to vasodilators and in the hyper-response to vasoconstrictors. Increased prostacyclin (PGI2) via COX-1 and COX-2 overexpression, and increased EETs/heme-oxygenase-1/K channels/gap junctions (endothelial derived hyperpolarizing factor system) play a major role in mesenteric vasodilatation, hyporeactivity to vasoconstrictors, and hyper-response to vasodilators. EETs, mediators of liver regeneration after hepatectomy and of angiogenesis, may play a role in the development of regenerative nodules and collateral circulation, through stimulation of vascular endothelial

The effect of angiotensin-2 receptor blocker valsartan on circulating level of endothelial progenitor cells in diabetic patients with asymptomatic coronary artery disease.

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Berezin, Alexander E; Kremzer, Alexander A; Martovitskaya, Yulia V; Samura, Tatyana A

2015-01-01

Decreased circulating endothelial progenitor cells (EPCs) are considered as strong and robust biomarkers for the prediction of cardiovascular outcomes in diabetic populations. The perspectives for modulating EPCs levels in T2DM with known coronary artery disease (CAD) with different drugs, affected mechanisms of improving mobilization of EPCs from tissue, are not still understood. To evaluate an effect of angiotensin-2 receptor blocker valsartan on circulating level of EPCs in diabetic patients with asymptomatic CAD. The study population was structured retrospectively after determining the CAD by contrast-enhanced spiral computed tomography angiography in 126 asymptomatic subjects. All subjects were distributed into two cohorts depending on daily doses of valsartan given. Low (80-160 mg daily orally) and high doses (240-320 mg daily orally) of valsartan were used and they were adjusted depending on achieving BP level less than 140/80 mmHg. The change from baseline in CD34(+) subset cells (frequencies and absolute values) was not significantly different between treatment cohorts. We found a significant increase of circulating level of CD14(+)CD309(+) cells in two patient cohorts. But more prominent change of CD14(+)CD309(+) cells was verified in subjects who were given valsartan in high daily doses when compared with persons who were included into cohort with low daily doses of the drug (1.96% versus 2.59%, respectively; Pvalsartan only. We found positive influence of angiotensin-2 receptor blocker valsartan in escalation doses on bone marrow-derived EPCs phenotyped as CD14(+)CD309(+) and CD14(+)CD309(+)Tie(2+) in T2DM patients with known asymptomatic CAD. Copyright © 2014 Diabetes India. Published by Elsevier Ltd. All rights reserved.

Human Brain Microvascular Endothelial Cells and Umbilical Vein Endothelial Cells Differentially Facilitate Leukocyte Recruitment and Utilize Chemokines for T Cell Migration

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Shumei Man

2008-01-01

Full Text Available Endothelial cells that functionally express blood brain barrier (BBB properties are useful surrogates for studying leukocyte-endothelial cell interactions at the BBB. In this study, we compared two different endothelial cellular models: transfected human brain microvascular endothelial cells (THBMECs and human umbilical vein endothelial cells (HUVECs. With each grow under optimal conditions, confluent THBMEC cultures showed continuous occludin and ZO-1 immunoreactivity, while HUVEC cultures exhibited punctate ZO-1 expression at sites of cell-cell contact only. Confluent THBMEC cultures on 24-well collagen-coated transwell inserts had significantly higher transendothelial electrical resistance (TEER and lower solute permeability than HUVECs. Confluent THBMECs were more restrictive for mononuclear cell migration than HUVECs. Only THBMECs utilized abluminal CCL5 to facilitate T-lymphocyte migration in vitro although both THBMECs and HUVECs employed CCL3 to facilitate T cell migration. These data establish baseline conditions for using THBMECs to develop in vitro BBB models for studying leukocyte-endothelial interactions during neuroinflammation.

Sphingosine kinase-2 maintains viral latency and survival for KSHV-infected endothelial cells.

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Lu Dai

Full Text Available Phosphorylation of sphingosine by sphingosine kinases (SphK1 and SphK2 generates sphingosine-1-phosphate (S1P, a bioactive sphingolipid which promotes cancer cell survival and tumor progression in vivo. We have recently reported that targeting SphK2 induces apoptosis for human primary effusion lymphoma (PEL cell lines infected by the Kaposi's sarcoma-associated herpesvirus (KSHV, and this occurs in part through inhibition of canonical NF-κB activation. In contrast, pharmacologic inhibition of SphK2 has minimal impact for uninfected B-cell lines or circulating human B cells from healthy donors. Therefore, we designed additional studies employing primary human endothelial cells to explore mechanisms responsible for the selective death observed for KSHV-infected cells during SphK2 targeting. Using RNA interference and a clinically relevant pharmacologic approach, we have found that targeting SphK2 induces apoptosis selectively for KSHV-infected endothelial cells through induction of viral lytic gene expression. Moreover, this effect occurs through repression of KSHV-microRNAs regulating viral latency and signal transduction, including miR-K12-1 which targets IκBα to facilitate activation of NF-κB, and ectopic expression of miR-K12-1 restores NF-κB activation and viability for KSHV-infected endothelial cells during SphK2 inhibition. These data illuminate a novel survival mechanism and potential therapeutic target for KSHV-infected endothelial cells: SphK2-associated maintenance of viral latency.

Protective effects on vascular endothelial cell in N'-nitro-L-arginine (L-NNA)-induced hypertensive rats from the combination of effective components of Uncaria rhynchophylla and Semen Raphani.

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Li, Yunlun; Yang, Wenqing; Zhu, Qingjun; Yang, Jinguo; Wang, Zhen

2015-08-01

Endothelial dysfunction is closely associated with hypertension. Protection of vascular endothelial cell is the key to prevention and treatment of hypertension. Uncaria rhynchophylla total alkaloids and Semen Raphani soluble alkaloid, isolated from traditional Chinese medicine Uncaria rbyncbopbylla and Semen Raphani respectively, exhibit properties of anti-hypertension and protection of blood vessels. In the present study, we observed the protective effect of the combined use of Uncaria rhynchophylla total alkaloids and Semen Raphani soluble alkaloid to the vascular endothelial cell in N'-nitro-L-arginine-induced hypertensive rats and investigate the preliminary mechanism. Blood pressure was detected by non-invasive rats tail method to observe the anti-hypertension effect of drugs. Scanning electron microscopy was used to observe the integrity or shedding state of vascular endothelial cell. The amount of circulating endothelial cells and CD54 and CD62P expression on circulating endothelial cells were tested to evaluate the endothelium function. In this study, we found that the Uncaria rhynchophylla total alkaloids and Semen Raphani soluble alkaloid compatibility can effectively lower the blood pressure, improve the structural integrity of vascular endothelium, and significantly reduce the number of circulating endothelial cells. Furthermore, the mean fluorescence intensity of CD54 and CD62P expressed showed decrease after the intervention of Uncaria rhynchophylla total alkaloids and Semen Raphani soluble alkaloid compatibility. In conclusion, the combination of effective components of the Uncaria rhynchophylla total alkaloids and Semen Raphani soluble alkaloid demonstrated good antihypertension effect and vascular endothelium protective effect. The preliminary mechanism of the protective effect may attribute to relieve the overall low-grade inflammation.

Endothelial juxtaposition of distinct adult stem cells activates angiogenesis signaling molecules in endothelial cells.

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Mohammadi, Elham; Nassiri, Seyed Mahdi; Rahbarghazi, Reza; Siavashi, Vahid; Araghi, Atefeh

2015-12-01

Efficacy of therapeutic angiogenesis needs a comprehensive understanding of endothelial cell (EC) function and biological factors and cells that interplay with ECs. Stem cells are considered the key components of pro- and anti-angiogenic milieu in a wide variety of physiopathological states, and interactions of EC-stem cells have been the subject of controversy in recent years. In this study, the potential effects of three tissue-specific adult stem cells, namely rat marrow-derived mesenchymal stem cells (rBMSCs), rat adipose-derived stem cells (rADSCs) and rat muscle-derived satellite cells (rSCs), on the endothelial activation of key angiogenic signaling molecules, including VEGF, Ang-2, VEGFR-2, Tie-2, and Tie2-pho, were investigated. Human umbilical vein endothelial cells (HUVECs) and rat lung microvascular endothelial cells (RLMECs) were cocultured with the stem cells or incubated with the stem cell-derived conditioned media on Matrigel. Following HUVEC-stem cell coculture, CD31-positive ECs were flow sorted and subjected to western blotting to analyze potential changes in the expression of the pro-angiogenic signaling molecules. Elongation and co-alignment of the stem cells were seen along the EC tubes in the EC-stem cell cocultures on Matrigel, with cell-to-cell dye communication in the EC-rBMSC cocultures. Moreover, rBMSCs and rADSCs significantly improved endothelial tubulogenesis in both juxtacrine and paracrine manners. These two latter stem cells dynamically up-regulated VEGF, Ang-2, VREGR-2, and Tie-2 but down-regulated Tie2-pho and the Tie2-pho/Tie-2 ratio in HUVECs. Induction of pro-angiogenic signaling in ECs by marrow- and adipose-derived MSCs further indicates the significance of stem cell milieu in angiogenesis dynamics.

MicroRNA-939 governs vascular integrity and angiogenesis through targeting γ-catenin in endothelial cells

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Hou, Shiqiang; Fang, Ming; Zhu, Qian; Liu, Ying; Liu, Liang; Li, Xinming

2017-01-01

Coronary collateral circulation (CCC) functions as a natural bypass in the event of coronary obstruction, which markedly improves prognosis in patients with coronary artery disease (CAD). MicroRNAs (miRNAs) have been implicated in multiple physiological and pathological processes, including angiogenesis involved in CCC growth. The roles that miRNA-939 (miR-939) plays in angiogenesis remain largely unknown. We conducted this study to explore the expression of miR-939 in CAD patients and its role in angiogenesis. For the first time, our results indicated that the expression of circulating miR-939 was down-regulated in patients with sufficient CCC compared with patients with poor CCC. Overexpression of miR-939 in primary human umbilical vein endothelial cells (HUVECs) significantly inhibited the proliferation, adhesion and tube formation, but promoted the migration of cells. In contrast, miR-939 knockdown exerted reverse effects. We further identified that γ-catenin was a novel target of miR-939 by translational repression, which could rescue the effects of miR-939 in HUVECs. In summary, this study revealed that the expression of circulating miR-939 was down-regulated in CAD patients with sufficient CCC. MiR-939 abolished vascular integrity and repressed angiogenesis through directly targeting γ-catenin. It provided a potential biomarker and a therapeutic target for CAD. - Highlights: • Circulating miR-939 is decreased in sufficient coronary collateral circulation. • MiR-939 abolishes vascular integrity in endothelial cells. • MiR-939 represses angiogenesis. • γ-catenin is a novel target of miR-939.

Autoantigens targeted in scleroderma patients with vascular disease are enriched in endothelial lineage cells

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McMahan, Zsuzsanna H.; Cottrell, Tricia R.; Wigley, Fredrick M.; Antiochos, Brendan; Zambidis, Elias T.; Park, Tea Soon; Halushka, Marc K.; Gutierrez-Alamillo, Laura; Cimbro, Raffaello; Rosen, Antony; Casciola-Rosen, Livia

2016-01-01

Objective Scleroderma patients with autoantibodies to centromere proteins (CENPs) and/or interferon-inducible protein 16 (IFI16) are at increased risk of severe vascular complications. We set out to define whether these autoantigens are enriched in cells of the vasculature. Methods Successive stages of embryoid bodies (EBs) as well as vascular progenitors were used to evaluate the expression of scleroderma autoantigens IFI16 and CENP by immunoblotting. CD31 was included to mark early blood vessels. IFI16 and CD31 expression were defined in skin paraffin sections from scleroderma patients and from healthy controls. IFI16 expression was determined by flow cytometry in circulating endothelial cells (CECs) and circulating progenitor cells (CPCs). Results Expression of CENP-A, IFI16 and CD31 was enriched in EBs at days 10 and 12 of differentiation, and particularly in cultures enriched in vascular progenitors (IFI16, CD31, CENPs A and-B). This pattern was distinct from that of comparator autoantigens. Immunohistochemical staining of skin paraffin sections showed enrichment of IFI16 in CD31-positive vascular endothelial cells in biopsies from scleroderma patients and normal controls. Flow cytometry analysis revealed IFI16 expression in CPCs, but minimal expression in CECs. Conclusion Expression of scleroderma autoantigens IFI16 and CENPs, which are associated with severe vascular disease, is increased in vascular progenitors and mature endothelial cells. High level, lineage-enriched expression of autoantigens may explain the striking association between clinical phenotypes and the immune targeting of specific autoantigens. PMID:27159521

Endothelial MMP14 is required for endothelial-dependent growth support of human airway basal cells

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Ding, Bi-Sen; Gomi, Kazunori; Rafii, Shahin; Crystal, Ronald G.; Walters, Matthew S.

2015-01-01

ABSTRACT Human airway basal cells are the stem (or progenitor) population of the airway epithelium, and play a central role in anchoring the epithelium to the basement membrane. The anatomic position of basal cells allows for potential paracrine signaling between them and the underlying non-epithelial stromal cells. In support of this, we have previously demonstrated that endothelial cells support growth of basal cells during co-culture through vascular endothelial growth factor A (VEGFA)-mediated signaling. Building on these findings, we found, by RNA sequencing analysis, that basal cells expressed multiple fibroblast growth factor (FGF) ligands (FGF2, FGF5, FGF11 and FGF13) and that only FGF2 and FGF5 were capable of functioning in a paracrine manner to activate classical FGF receptor (FGFR) signaling. Antibody-mediated blocking of FGFR1 during basal-cell–endothelial-cell co-culture significantly reduced the endothelial-cell-dependent basal cell growth. Stimulation of endothelial cells with basal-cell-derived growth factors induced endothelial cell expression of matrix metallopeptidase 14 (MMP14), and short hairpin RNA (shRNA)-mediated knockdown of endothelial cell MMP14 significantly reduced the endothelial-cell-dependent growth of basal cells. Overall, these data characterize a new growth-factor-mediated reciprocal ‘crosstalk’ between human airway basal cells and endothelial cells that regulates proliferation of basal cells. PMID:26116571

The effects of smoking on levels of endothelial progenitor cells and microparticles in the blood of healthy volunteers.

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Fariborz Mobarrez

Full Text Available BACKGROUND: Cigarette smoking, both active and passive, is one of the leading causes of morbidity and mortality in cardiovascular disease. To assess the impact of brief smoking on the vasculature, we determined levels of circulating endothelial progenitor cells (EPCs and circulating microparticles (MPs following the smoking of one cigarette by young, healthy intermittent smokers. MATERIALS AND METHODS: 12 healthy volunteers were randomized to either smoking or not smoking in a crossover fashion. Blood sampling was performed at baseline, 1, 4 and 24 hours following smoking/not smoking. The numbers of EPCs and MPs were determined by flow cytometry. MPs were measured from platelets, leukocytes and endothelial cells. Moreover, MPs were also labelled with anti-HMGB1 and SYTO 13 to assess the content of nuclear molecules. RESULTS: Active smoking of one cigarette caused an immediate and significant increase in the numbers of circulating EPCs and MPs of platelet-, endothelial- and leukocyte origin. Levels of MPs containing nuclear molecules were increased, of which the majority were positive for CD41 and CD45 (platelet- and leukocyte origin. CD144 (VE-cadherin or HMGB1 release did not significantly change during active smoking. CONCLUSION: Brief active smoking of one cigarette generated an acute release of EPC and MPs, of which the latter contained nuclear matter. Together, these results demonstrate acute effects of cigarette smoke on endothelial, platelet and leukocyte function as well as injury to the vascular wall.

Optimized multiparametric flow cytometric analysis of circulating endothelial cells and their subpopulations in peripheral blood of patients with solid tumors: a technical analysis.

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Zhou, Fangbin; Zhou, Yaying; Yang, Ming; Wen, Jinli; Dong, Jun; Tan, Wenyong

2018-01-01

Circulating endothelial cells (CECs) and their subpopulations could be potential novel biomarkers for various malignancies. However, reliable enumerable methods are warranted to further improve their clinical utility. This study aimed to optimize a flow cytometric method (FCM) assay for CECs and subpopulations in peripheral blood for patients with solid cancers. An FCM assay was used to detect and identify CECs. A panel of 60 blood samples, including 44 metastatic cancer patients and 16 healthy controls, were used in this study. Some key issues of CEC enumeration, including sample material and anticoagulant selection, optimal titration of antibodies, lysis/wash procedures of blood sample preparation, conditions of sample storage, sufficient cell events to enhance the signal, fluorescence-minus-one controls instead of isotype controls to reduce background noise, optimal selection of cell surface markers, and evaluating the reproducibility of our method, were integrated and investigated. Wilcoxon and Mann-Whitney U tests were used to determine statistically significant differences. In this validation study, we refined a five-color FCM method to detect CECs and their subpopulations in peripheral blood of patients with solid tumors. Several key technical issues regarding preanalytical elements, FCM data acquisition, and analysis were addressed. Furthermore, we clinically validated the utility of our method. The baseline levels of mature CECs, endothelial progenitor cells, and activated CECs were higher in cancer patients than healthy subjects ( P technical issues found in previously published assays and validated the reproducibility and sensitivity of our proposed method. Future work is required to explore the potential of our optimized method in clinical oncologic applications.

Exposure to ultrafine particles, intracellular production of reactive oxygen species in leukocytes and altered levels of endothelial progenitor cells

International Nuclear Information System (INIS)

Jantzen, Kim; Møller, Peter; Karottki, Dorina Gabriela; Olsen, Yulia; Bekö, Gabriel; Clausen, Geo; Hersoug, Lars-Georg; Loft, Steffen

2016-01-01

Exposure to particles in the fine and ultrafine size range has been linked to induction of low-grade systemic inflammation, oxidative stress and development of cardiovascular diseases. Declining levels of endothelial progenitor cells within systemic circulation have likewise been linked to progression of cardiovascular diseases. The objective was to determine if exposure to fine and ultrafine particles from indoor and outdoor sources, assessed by personal and residential indoor monitoring, is associated with altered levels of endothelial progenitor cells, and whether such effects are related to leukocyte-mediated oxidative stress. The study utilized a cross sectional design performed in 58 study participants from a larger cohort. Levels of circulating endothelial progenitor cells, defined as either late (CD34 + KDR + cells) or early (CD34 + CD133 + KDR + cells) subsets were measured using polychromatic flow cytometry. We additionally measured production of reactive oxygen species in leukocyte subsets (lymphocytes, monocytes and granulocytes) by flow cytometry using intracellular 2′,7′-dichlorofluoroscein. The measurements encompassed both basal levels of reactive oxygen species production and capacity for reactive oxygen species production for each leukocyte subset. We found that the late endothelial progenitor subset was negatively associated with levels of ultrafine particles measured within the participant residences and with reactive oxygen species production capacity in lymphocytes. Additionally, the early endothelial progenitor cell levels were positively associated with a personalised measure of ultrafine particle exposure and negatively associated with both basal and capacity for reactive oxygen species production in lymphocytes and granulocytes, respectively. Our results indicate that exposure to fine and ultrafine particles derived from indoor sources may have adverse effects on human vascular health.

Probing the mechanical properties of TNF-α stimulated endothelial cell with atomic force microscopy

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Sei-Young Lee

2011-01-01

Full Text Available Sei-Young Lee1,2, Ana-Maria Zaske3, Tommaso Novellino1,4*, Delia Danila3, Mauro Ferrari1,5*, Jodie Conyers3, Paolo Decuzzi1,6*1Department of Nanomedicine and Biomedical Engineering, The University of Texas Medical School at Houston, Houston, TX, USA; 2Department of Mechanical Engineering, The University of Texas at Austin, Austin, TX, USA; 3CeTIR – Center for Translational Injury Research, The University of Texas Health Science Center at Houston, Houston, TX, USA; 4Department of Biomedical Engineering, Biomedical Campus University of Rome, Italy; 5MD Anderson Cancer Center, Houston, TX, USA; 6BioNEM – Center of Bio-Nanotechnology and Engineering for Medicine, University of Magna Graecia, Catanzaro, Italy; *Currently at Department of Nanomedicine and Biomedical Engineering, The Methodist Hospital Research Institute, Houston, TX, USAAbstract: TNF-α (tumor necrosis factor-α is a potent pro-inflammatory cytokine that regulates the permeability of blood and lymphatic vessels. The plasma concentration of TNF-α is elevated (> 1 pg/mL in several pathologies, including rheumatoid arthritis, atherosclerosis, cancer, pre-eclampsia; in obese individuals; and in trauma patients. To test whether circulating TNF-α could induce similar alterations in different districts along the vascular system, three endothelial cell lines, namely HUVEC, HPMEC, and HCAEC, were characterized in terms of 1 mechanical properties, employing atomic force microscopy; 2 cytoskeletal organization, through fluorescence microscopy; and 3 membrane overexpression of adhesion molecules, employing ELISA and immunostaining. Upon stimulation with TNF-α (10 ng/mL for 20 h, for all three endothelial cells, the mechanical stiffness increased by about 50% with a mean apparent elastic modulus of E ~5 ± 0.5 kPa (~3.3 ± 0.35 kPa for the control cells; the density of F-actin filaments increased in the apical and median planes; and the ICAM-1 receptors were overexpressed compared with

Mesenchymal Stem/Multipotent Stromal Cells from Human Decidua Basalis Reduce Endothelial Cell Activation.

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Alshabibi, Manal A; Al Huqail, Al Joharah; Khatlani, Tanvir; Abomaray, Fawaz M; Alaskar, Ahmed S; Alawad, Abdullah O; Kalionis, Bill; Abumaree, Mohamed Hassan

2017-09-15

Recently, we reported the isolation and characterization of mesenchymal stem cells from the decidua basalis of human placenta (DBMSCs). These cells express a unique combination of molecules involved in many important cellular functions, which make them good candidates for cell-based therapies. The endothelium is a highly specialized, metabolically active interface between blood and the underlying tissues. Inflammatory factors stimulate the endothelium to undergo a change to a proinflammatory and procoagulant state (ie, endothelial cell activation). An initial response to endothelial cell activation is monocyte adhesion. Activation typically involves increased proliferation and enhanced expression of adhesion and inflammatory markers by endothelial cells. Sustained endothelial cell activation leads to a type of damage to the body associated with inflammatory diseases, such as atherosclerosis. In this study, we examined the ability of DBMSCs to protect endothelial cells from activation through monocyte adhesion, by modulating endothelial proliferation, migration, adhesion, and inflammatory marker expression. Endothelial cells were cocultured with DBMSCs, monocytes, monocyte-pretreated with DBMSCs and DBMSC-pretreated with monocytes were also evaluated. Monocyte adhesion to endothelial cells was examined following treatment with DBMSCs. Expression of endothelial cell adhesion and inflammatory markers was also analyzed. The interaction between DBMSCs and monocytes reduced endothelial cell proliferation and monocyte adhesion to endothelial cells. In contrast, endothelial cell migration increased in response to DBMSCs and monocytes. Endothelial cell expression of adhesion and inflammatory molecules was reduced by DBMSCs and DBMSC-pretreated with monocytes. The mechanism of reduced endothelial proliferation involved enhanced phosphorylation of the tumor suppressor protein p53. Our study shows for the first time that DBMSCs protect endothelial cells from activation by

Endothelial cell repopulation after stenting determines in-stent neointima formation: effects of bare-metal vs. drug-eluting stents and genetic endothelial cell modification.

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Douglas, Gillian; Van Kampen, Erik; Hale, Ashley B; McNeill, Eileen; Patel, Jyoti; Crabtree, Mark J; Ali, Ziad; Hoerr, Robert A; Alp, Nicholas J; Channon, Keith M

2013-11-01

Understanding endothelial cell repopulation post-stenting and how this modulates in-stent restenosis is critical to improving arterial healing post-stenting. We used a novel murine stent model to investigate endothelial cell repopulation post-stenting, comparing the response of drug-eluting stents with a primary genetic modification to improve endothelial cell function. Endothelial cell repopulation was assessed en face in stented arteries in ApoE(-/-) mice with endothelial-specific LacZ expression. Stent deployment resulted in near-complete denudation of endothelium, but was followed by endothelial cell repopulation, by cells originating from both bone marrow-derived endothelial progenitor cells and from the adjacent vasculature. Paclitaxel-eluting stents reduced neointima formation (0.423 ± 0.065 vs. 0.240 ± 0.040 mm(2), P = 0.038), but decreased endothelial cell repopulation (238 ± 17 vs. 154 ± 22 nuclei/mm(2), P = 0.018), despite complete strut coverage. To test the effects of selectively improving endothelial cell function, we used transgenic mice with endothelial-specific overexpression of GTP-cyclohydrolase 1 (GCH-Tg) as a model of enhanced endothelial cell function and increased NO production. GCH-Tg ApoE(-/-) mice had less neointima formation compared with ApoE(-/-) littermates (0.52 ± 0.08 vs. 0.26 ± 0.09 mm(2), P = 0.039). In contrast to paclitaxel-eluting stents, reduced neointima formation in GCH-Tg mice was accompanied by increased endothelial cell coverage (156 ± 17 vs. 209 ± 23 nuclei/mm(2), P = 0.043). Drug-eluting stents reduce not only neointima formation but also endothelial cell repopulation, independent of strut coverage. In contrast, selective targeting of endothelial cell function is sufficient to improve endothelial cell repopulation and reduce neointima formation. Targeting endothelial cell function is a rational therapeutic strategy to improve vascular healing and decrease neointima formation after stenting.

Effect of colorectal cancer on the number of normal stem cells circulating in peripheral blood.

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Marlicz, Wojciech; Sielatycka, Katarzyna; Serwin, Karol; Kubis, Ewa; Tkacz, Marta; Głuszko, Rafał; Białek, Andrzej; Starzyńska, Teresa; Ratajczak, Mariusz Z

2016-12-01

Bone marrow (BM) residing stem cells are mobilized from their BM niches into peripheral blood (PB) in several pathological situations including tissue organ injury and systemic inflammation. We recently reported that the number of BM-derived stem cells (SCs) increases in patients with pancreatic and stomach cancer. Accordingly, we observed higher numbers of circulating very small embryonic/epiblast‑like stem cells (VSELs) and mesenchymal stem cells (MSCs) that were associated with the activation of pro-mobilizing complement cascade and an elevated level of sphingosine-1 phosphate (S1P) in PB plasma. We wondered if a similar correlation occurs in patients with colorectal cancer (CRC). A total of 46 patients were enrolled in this study: 17 with CRC, 18 with benign colonic adenomas (BCA) and 11 healthy individuals. By employing fluorescence-activated cell sorting (FACS) we evaluated the number of BM-derived SCs circulating in PB: i) CD34+/Lin-/CD45- and CD133-/Lin-/CD45- VSELs; ii) CD45-/CD105+/CD90+/CD29+ MSCs; iii) CD45-/CD34+/CD133+/KDR+ endothelial progenitor cells (EPCs); and iv) CD133+/Lin-/CD45+ or CD34+/Lin-/CD45+ cells enriched for hematopoietic stem/progenitor cells (HSPCs). In parallel, we measured in the PB parameters regulating the egress of SCs from BM into PB. In contrast to pancreatic and gastric cancer patients, CRC subjects presented neither an increase in the number of circulating SCs nor the activation of pro-mobilizing factors such as complement, coagulation and fibrinolytic cascade, circulating stromal derived factor 1 (SDF‑1), vascular endothelial growth factor (VEGF) and intestinal permeability marker (zonulin). In conclusion, mobilization of SCs in cancer patients depends on the type of malignancy and its ability to activate pro-mobilization cascades.

Exposure to ultrafine particles, intracellular production of reactive oxygen species in leukocytes and altered levels of endothelial progenitor cells

DEFF Research Database (Denmark)

Jantzen, Kim; Møller, Peter Horn; Karottki, Dorina Gabriela

2016-01-01

. Additionally, the early endothelial progenitor cell levels were positively associated with a personalised measure of ultrafine particle exposure and negatively associated with both basal and capacity for reactive oxygen species production in lymphocytes and granulocytes, respectively. Our results indicate......Exposure to particles in the fine and ultrafine size range has been linked to induction of low-grade systemic inflammation, oxidative stress and development of cardiovascular diseases. Declining levels of endothelial progenitor cells within systemic circulation have likewise been linked...... to progression of cardiovascular diseases. The objective was to determine if exposure to fine and ultrafine particles from indoor and outdoor sources, assessed by personal and residential indoor monitoring, is associated with altered levels of endothelial progenitor cells, and whether such effects are related...

Sickle erythrocytes inhibit human endothelial cell DNA synthesis

International Nuclear Information System (INIS)

Weinstein, R.; Zhou, M.A.; Bartlett-Pandite, A.; Wenc, K.

1990-01-01

Patients with sickle cell anemia experience severe vascular occlusive phenomena including acute pain crisis and cerebral infarction. Obstruction occurs at both the microvascular and the arterial level, and the clinical presentation of vascular events is heterogeneous, suggesting a complex etiology. Interaction between sickle erythrocytes and the endothelium may contribute to vascular occlusion due to alteration of endothelial function. To investigate this hypothesis, human vascular endothelial cells were overlaid with sickle or normal erythrocytes and stimulated to synthesize DNA. The erythrocytes were sedimented onto replicate monolayers by centrifugation for 10 minutes at 17 g to insure contact with the endothelial cells. Incorporation of 3H-thymidine into endothelial cell DNA was markedly inhibited during contact with sickle erythrocytes. This inhibitory effect was enhanced more than twofold when autologous sickle plasma was present during endothelial cell labeling. Normal erythrocytes, with or without autologous plasma, had a modest effect on endothelial cell DNA synthesis. When sickle erythrocytes in autologous sickle plasma were applied to endothelial monolayers for 1 minute, 10 minutes, or 1 hour and then removed, subsequent DNA synthesis by the endothelial cells was inhibited by 30% to 40%. Although adherence of sickle erythrocytes to the endothelial monolayers was observed under these experimental conditions, the effect of sickle erythrocytes on endothelial DNA synthesis occurred in the absence of significant adherence. Hence, human endothelial cell DNA synthesis is partially inhibited by contact with sickle erythrocytes. The inhibitory effect of sickle erythrocytes occurs during a brief (1 minute) contact with the endothelial monolayers, and persists for at least 6 hours of 3H-thymidine labeling

Histones Induce the Procoagulant Phenotype of Endothelial Cells through Tissue Factor Up-Regulation and Thrombomodulin Down-Regulation.

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Kim, Ji Eun; Yoo, Hyun Ju; Gu, Ja Yoon; Kim, Hyun Kyung

2016-01-01

The high circulating levels of histones found in various thrombotic diseases may compromise the anticoagulant barrier of endothelial cells. We determined how histones affect endothelial procoagulant tissue factor (TF) and anticoagulant thrombomodulin (TM). Surface antigens, soluble forms, and mRNA levels of TF and TM were measured by flow cytometry, ELISA, and real-time RT-PCR, respectively. TF and TM activity were measured using procoagulant activity, thrombin generation, or chromogenic assays. Involvement of the toll-like receptor (TLR) was assessed using the neutralizing antibodies. Histones dose-dependently induced surface antigens, activity and mRNA levels of endothelial TF. Histone-treated endothelial cells significantly shortened the lag time and enhanced the endogenous thrombin potential of normal plasma, which was normalized by a TF neutralizing antibody. Histones induced phosphatidylserine and protein-disulfide isomerase expression in endothelial cells. Histones also reduced the surface antigen, activity, and mRNA levels of endothelial TM. Polysialic acid and heparin reversed the histone-induced TF up-regulation and TM down-regulation. Activated protein C did not affect the TF up-regulation, but interrupted TM down-regulation. TLR2, and TLR4 inhibitors partially blocked the TF up-regulation. Histones induced the endothelial procoagulant phenotype through TF up-regulation and TM down-regulation. The effects of histones were partly mediated by TLR2, TLR4. Strategies to inhibit the harmful effects of histones in endothelial cells may be required in order to prevent a thrombotic environment.

Glucosamine exposure reduces proteoglycan synthesis in primary human endothelial cells in vitro

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Trine M. Reine

2016-09-01

Full Text Available Purpose: Glucosamine (GlcN supplements are promoted for medical reasons, for example, for patients with arthritis and other joint-related diseases. Oral intake of GlcN is followed by uptake in the intestine, transport in the circulation and thereafter delivery to chondrocytes. Here, it is postulated to have an effect on synthesis and turnover of extracellular matrix constituents expressed by these cells. Following uptake in the intestine, serum levels are transiently increased, and the endothelium is exposed to increased levels of GlcN. We investigated the possible effects of GlcN on synthesis of proteoglycans (PGs, an important matrix component, in primary human endothelial cells. Methods: Primary human endothelial cells were cultured in vitro in medium with 5 mM glucose and 0–10 mM GlcN. PGs were recovered and analysed by western blotting, or by SDS-PAGE, gel chromatography or ion-exchange chromatography of 35S-PGs after 35S-sulphate labelling of the cells. Results: The synthesis and secretion of 35S-PGs from cultured endothelial cells were reduced in a dose- and time-dependent manner after exposure to GlcN. PGs are substituted with sulphated glycosaminoglycan (GAG chains, vital for PG function. The reduction in 35S-PGs was not related to an effect on GAG chain length, number or sulphation, but rather to the total expression of PGs. Conclusion: Exposure of endothelial cells to GlcN leads to a general decrease in 35S-PG synthesis. These results suggest that exposure to high levels of GlcN can lead to decreased matrix synthesis, contrary to what has been claimed by supporters of such supplements.

Verocytotoxin-induced apoptosis of human microvascular endothelial cells.

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Pijpers, A H; van Setten, P A; van den Heuvel, L P; Assmann, K J; Dijkman, H B; Pennings, A H; Monnens, L A; van Hinsbergh, V W

2001-04-01

The pathogenesis of the epidemic form of hemolytic uremic syndrome is characterized by endothelial cell damage. In this study, the role of apoptosis in verocytotoxin (VT)-mediated endothelial cell death in human glomerular microvascular endothelial cells (GMVEC), human umbilical vein endothelial cells, and foreskin microvascular endothelial cells (FMVEC) was investigated. VT induced apoptosis in GMVEC and human umbilical vein endothelial cells when the cells were prestimulated with the inflammatory mediator tumor necrosis factor-alpha (TNF-alpha). FMVEC displayed strong binding of VT and high susceptibility to VT under basal conditions, which made them suitable for the study of VT-induced apoptosis without TNF-alpha interference. On the basis of functional (flow cytometry and immunofluorescence microscopy using FITC-conjugated annexin V and propidium iodide), morphologic (transmission electron microscopy), and molecular (agarose gel electrophoresis of cellular DNA fragments) criteria, it was documented that VT induced programmed cell death in microvascular endothelial cells in a dose- and time-dependent manner. Furthermore, whereas partial inhibition of protein synthesis by VT was associated with a considerable number of apoptotic cells, comparable inhibition of protein synthesis by cycloheximide was not. This suggests that additional pathways, independent of protein synthesis inhibition, may be involved in VT-mediated apoptosis in microvascular endothelial cells. Specific inhibition of caspases by Ac-Asp-Glu-Val-Asp-CHO, but not by Ac-Tyr-Val-Ala-Asp-CHO, was accompanied by inhibition of VT-induced apoptosis in FMVEC and TNF-alpha-treated GMVEC. These data indicate that VT can induce apoptosis in human microvascular endothelial cells.

Influence of sex on the number of circulating endothelial microparticles and microRNA expression in middle-aged adults.

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Bammert, Tyler D; Hijmans, Jamie G; Kavlich, Philip J; Lincenberg, Grace M; Reiakvam, Whitney R; Fay, Ryan T; Greiner, Jared J; Stauffer, Brian L; DeSouza, Christopher A

2017-08-01

What is the central question of this study? Are there sex-related differences in the number of circulating endothelial microparticles (EMPs) and microparticle microRNA expression in middle-aged adult humans? What is the main finding and its importance? Although the numbers of circulating endothelial microparticles do not differ between middle-aged men and women, there are sex-related differences in the expression of miR-125a in activation-derived EMPs and miR-34a in apoptosis-derived EMPs. Differences in circulating endothelial microparticle microRNA content may provide new insight into the sex-related disparity in the risk and prevalence of vascular disease in middle-aged adults. The aims of this study were to determine: (i) whether circulating concentrations of endothelial microparticles (EMPs) differ in middle-aged men compared with women; and (ii) whether there are sex-related differences in microRNA expression in EMPs. Peripheral blood was collected from 30 sedentary adults: 15 men (56 ± 6 years old) and 15 women (56 ± 5 years old). Endothelial microparticles were defined by markers of activation (CD62e + ) or apoptosis (CD31 + /CD42b - ) by flow cytometry. Expression of microRNA (miR-34a, 92a, 125a and 126) in activation- and apoptosis-derived EMPs was measured by RT-PCR. Circulating activation- (33 ± 31 versus 39 ± 35 microparticles μl -1 ) and apoptosis-derived EMPs (49 ± 54 versus 42 ± 43 microparticles μl -1 ) were not significantly different between men and women. Expression of miR-125a (2.23 ± 2.01 versus 6.95 ± 3.99 a.u.) was lower (∼215%; P < 0.05) in activation-derived EMPs, whereas expression of miR-34a (1.17 ± 1.43 versus 0.38 ± 0.35 a.u.) was higher (∼210%; P < 0.05) in apoptosis-derived EMPs from men compared with women. Expression of microRNA in circulating EMPs may provide new insight into sex-related differences in cardiovascular disease. © 2017 The Authors. Experimental Physiology © 2017 The

Reduced Ang2 expression in aging endothelial cells.

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Hohensinner, P J; Ebenbauer, B; Kaun, C; Maurer, G; Huber, K; Wojta, J

2016-06-03

Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. Copyright © 2016 Elsevier Inc. All rights reserved.

Strategies to reverse endothelial progenitor cell dysfunction in diabetes.

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Petrelli, Alessandra; Di Fenza, Raffaele; Carvello, Michele; Gatti, Francesca; Secchi, Antonio; Fiorina, Paolo

2012-01-01

Bone-marrow-derived cells-mediated postnatal vasculogenesis has been reported as the main responsible for the regulation of vascular homeostasis in adults. Since their discovery, endothelial progenitor cells have been depicted as mediators of postnatal vasculogenesis for their peculiar phenotype (partially staminal and partially endothelial), their ability to differentiate in endothelial cell line and to be incorporated into the vessels wall during ischemia/damage. Diabetes mellitus, a condition characterized by cardiovascular disease, nephropathy, and micro- and macroangiopathy, showed a dysfunction of endothelial progenitor cells. Herein, we review the mechanisms involved in diabetes-related dysfunction of endothelial progenitor cells, highlighting how hyperglycemia affects the different steps of endothelial progenitor cells lifetime (i.e., bone marrow mobilization, trafficking into the bloodstream, differentiation in endothelial cells, and homing in damaged tissues/organs). Finally, we review preclinical and clinical strategies that aim to revert diabetes-induced dysfunction of endothelial progenitor cells as a means of finding new strategies to prevent diabetic complications.

Strategies to Reverse Endothelial Progenitor Cell Dysfunction in Diabetes

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Alessandra Petrelli

2012-01-01

Full Text Available Bone-marrow-derived cells-mediated postnatal vasculogenesis has been reported as the main responsible for the regulation of vascular homeostasis in adults. Since their discovery, endothelial progenitor cells have been depicted as mediators of postnatal vasculogenesis for their peculiar phenotype (partially staminal and partially endothelial, their ability to differentiate in endothelial cell line and to be incorporated into the vessels wall during ischemia/damage. Diabetes mellitus, a condition characterized by cardiovascular disease, nephropathy, and micro- and macroangiopathy, showed a dysfunction of endothelial progenitor cells. Herein, we review the mechanisms involved in diabetes-related dysfunction of endothelial progenitor cells, highlighting how hyperglycemia affects the different steps of endothelial progenitor cells lifetime (i.e., bone marrow mobilization, trafficking into the bloodstream, differentiation in endothelial cells, and homing in damaged tissues/organs. Finally, we review preclinical and clinical strategies that aim to revert diabetes-induced dysfunction of endothelial progenitor cells as a means of finding new strategies to prevent diabetic complications.

Endothelial induced EMT in breast epithelial cells with stem cell properties

DEFF Research Database (Denmark)

Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla

2011-01-01

endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression...... of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D......492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close...

Endothelial cells in the eyes of an immunologist.

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Young, M Rita

2012-10-01

Endothelial cell activation in the process of tumor angiogenesis and in various aspects of vascular biology has been extensively studied. However, endothelial cells also function in other capacities, including in immune regulation. Compared to the more traditional immune regulatory populations (Th1, Th2, Treg, etc.), endothelial cells have received far less credit as being immune regulators. Their regulatory capacity is multifaceted. They are critical in both limiting and facilitating the trafficking of various immune cell populations, including T cells and dendritic cells, out of the vasculature and into tissue. They also can be induced to stimulate immune reactivity or to be immune inhibitory. In each of these parameters (trafficking, immune stimulation and immune inhibition), their role can be physiological, whereby they have an active role in maintaining health. Alternatively, their role can be pathological, whereby they contribute to disease. In theory, endothelial cells are in an ideal location to recruit cells that can mediate immune reactivity to tumor tissue. Furthermore, they can activate the immune cells as they transmigrate across the endothelium into the tumor. However, what is seen is the absence of these protective effects of endothelial cells and, instead, the endothelial cells succumb to the defense mechanisms of the tumor, resulting in their acquisition of a tumor-protective role. To understand the immune regulatory potential of endothelial cells in protecting the host versus the tumor, it is useful to better understand the other circumstances in which endothelial cells modulate immune reactivities. Which of the multitude of immune regulatory roles that endothelial cells can take on seems to rely on the type of stimulus that they are encountering. It also depends on the extent to which they can be manipulated by potential dangers to succumb and contribute toward attack on the host. This review will explore the physiological and pathological roles

Endothelial cell capture of heparin-binding growth factors under flow.

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Bing Zhao

2010-10-01

Full Text Available Circulation is an important delivery method for both natural and synthetic molecules, but microenvironment interactions, regulated by endothelial cells and critical to the molecule's fate, are difficult to interpret using traditional approaches. In this work, we analyzed and predicted growth factor capture under flow using computer modeling and a three-dimensional experimental approach that includes pertinent circulation characteristics such as pulsatile flow, competing binding interactions, and limited bioavailability. An understanding of the controlling features of this process was desired. The experimental module consisted of a bioreactor with synthetic endothelial-lined hollow fibers under flow. The physical design of the system was incorporated into the model parameters. The heparin-binding growth factor fibroblast growth factor-2 (FGF-2 was used for both the experiments and simulations. Our computational model was composed of three parts: (1 media flow equations, (2 mass transport equations and (3 cell surface reaction equations. The model is based on the flow and reactions within a single hollow fiber and was scaled linearly by the total number of fibers for comparison with experimental results. Our model predicted, and experiments confirmed, that removal of heparan sulfate (HS from the system would result in a dramatic loss of binding by heparin-binding proteins, but not by proteins that do not bind heparin. The model further predicted a significant loss of bound protein at flow rates only slightly higher than average capillary flow rates, corroborated experimentally, suggesting that the probability of capture in a single pass at high flow rates is extremely low. Several other key parameters were investigated with the coupling between receptors and proteoglycans shown to have a critical impact on successful capture. The combined system offers opportunities to examine circulation capture in a straightforward quantitative manner that

Shear stress-induced mitochondrial biogenesis decreases the release of microparticles from endothelial cells.

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Kim, Ji-Seok; Kim, Boa; Lee, Hojun; Thakkar, Sunny; Babbitt, Dianne M; Eguchi, Satoru; Brown, Michael D; Park, Joon-Young

2015-08-01

The concept of enhancing structural integrity of mitochondria has emerged as a novel therapeutic option for cardiovascular disease. Flow-induced increase in laminar shear stress is a potent physiological stimulant associated with exercise, which exerts atheroprotective effects in the vasculature. However, the effect of laminar shear stress on mitochondrial remodeling within the vascular endothelium and its related functional consequences remain largely unknown. Using in vitro and in vivo complementary studies, here, we report that aerobic exercise alleviates the release of endothelial microparticles in prehypertensive individuals and that these salutary effects are, in part, mediated by shear stress-induced mitochondrial biogenesis. Circulating levels of total (CD31(+)/CD42a(-)) and activated (CD62E(+)) microparticles released by endothelial cells were significantly decreased (∼40% for both) after a 6-mo supervised aerobic exercise training program in individuals with prehypertension. In cultured human endothelial cells, laminar shear stress reduced the release of endothelial microparticles, which was accompanied by an increase in mitochondrial biogenesis through a sirtuin 1 (SIRT1)-dependent mechanism. Resveratrol, a SIRT1 activator, treatment showed similar effects. SIRT1 knockdown using small-interfering RNA completely abolished the protective effect of shear stress. Disruption of mitochondrial integrity by either antimycin A or peroxisome proliferator-activated receptor-γ coactivator-1α small-interfering RNA significantly increased the number of total, and activated, released endothelial microparticles, and shear stress restored these back to basal levels. Collectively, these data demonstrate a critical role of endothelial mitochondrial integrity in preserving endothelial homeostasis. Moreover, prolonged laminar shear stress, which is systemically elevated during aerobic exercise in the vessel wall, mitigates endothelial dysfunction by promoting

Mechanisms of Endothelial Dysfunction in Hypertensive Pregnancy and Preeclampsia

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Possomato-Vieira, José S.; Khalil, Raouf A.

2016-01-01

Preeclampsia is a pregnancy-related disorder characterized by hypertension, and could lead to maternal and fetal morbidity and mortality. Although the causative factors and pathophysiological mechanisms are unclear, endothelial dysfunction is a major hallmark of preeclampsia. Clinical tests and experimental research have suggested that generalized endotheliosis in the systemic, renal, cerebral and hepatic circulation could decrease endothelium-derived vasodilators such as nitric oxide, prostacyclin and hyperpolarization factor and increase vasoconstrictors such as endothelin-1 and thromboxane A2, leading to increased vasoconstriction, hypertension and other manifestation of preeclampsia. In search for the upstream mechanisms that could cause endothelial dysfunction, certain genetic, demographic and environmental risk factors have been suggested to cause abnormal expression of uteroplacental integrins, cytokines and matrix metalloproteinases, leading to decreased maternal tolerance, apoptosis of invasive trophoblast cells, inadequate spiral arteries remodeling, reduced uterine perfusion pressure (RUPP), and placental ischemia/hypoxia. RUPP may cause imbalance between the anti-angiogenic factors soluble fms-like tyrosine kinase-1 and soluble endoglin and the pro-angiogenic factors vascular endothelial growth factor and placental growth factor, or stimulate the release of other circulating bioactive factors such as inflammatory cytokines, hypoxia-inducible factor-1, reactive oxygen species, and angiotensin AT1 receptor agonistic autoantibodies. These circulating factors could then target endothelial cells and cause generalized endothelial dysfunction. Therapeutic options are currently limited, but understanding the factors involved in endothelial dysfunction could help design new approaches for prediction and management of preeclampsia. PMID:27451103

Hypoxia Mediated Release of Endothelial Microparticles and Increased Association of S100A12 with Circulating Neutrophils

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Rebecca V. Vince

2009-01-01

Full Text Available Microparticles are released from the endothelium under normal homeostatic conditions and have been shown elevated in disease states, most notably those characterised by endothelial dysfunction. The endothelium is sensitive to oxidative stress/status and vascular cell adhesion molecule-1 (VCAM-1 expression is upregulated upon activated endothelium, furthermore the presence of VCAM-1 on microparticles is known. S100A12, a calcium binding protein part of the S100 family, is shown to be present on circulating leukocytes and is thought a sensitive marker to local inflammatory process, which may be driven by oxidative stress. Eight healthy males were subjected to breathing hypoxic air (15% O2, approximately equivalent to 3000 metres altitude for 80 minutes in a temperature controlled laboratory and venous blood samples were processed immediately for VCAM-1 microparticles (VCAM-1 MP and S100A12 association with leukocytes by flow cytometry. A pre-hypoxic blood sample was used for comparison. Both VCAM-1 MP and S100A12 association with neutrophils were significantly elevated post hypoxic breathing later declining to levels observed in the pre-test samples. A similar trend was observed in both cases and a correlation may exist between these two markers in response to hypoxia. These data offer evidence using novel markers of endothelial and circulating blood responses to hypoxia.

Endothelial progenitor cells (EPCs) in ageing and age-related diseases: How currently available treatment modalities affect EPC biology, atherosclerosis, and cardiovascular outcomes.

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Altabas, Velimir; Altabas, Karmela; Kirigin, Lora

2016-10-01

Endothelial progenitor cells (EPCs) are mononuclear cells that circulate in the blood and are derived from different tissues, expressing cell surface markers that are similar to mature endothelial cells. The discovery of EPCs has lead to new insights in vascular repair and atherosclerosis and also a new theory for ageing. EPCs from the bone marrow and some other organs aid in vascular repair by migrating to distant vessels where they differentiate into mature endothelial cells and replace old and injured endothelial cells. The ability of EPCs to repair vascular damage depends on their number and functionality. Currently marketed drugs used in a variety of diseases can modulate these characteristics. In this review, the effect of currently available treatment options for cardiovascular and metabolic disorders on EPC biology will be discussed. The various EPC-based therapies that will be discussed include lipid-lowering agents, antihypertensive agents, antidiabetic drugs, phosphodiesteraze inhibitors, hormones, as well as EPC capturing stents. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Endothelial actions of atrial and B-type natriuretic peptides.

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Kuhn, Michaela

2012-05-01

The cardiac hormone atrial natriuretic peptide (ANP) is critically involved in the maintenance of arterial blood pressure and intravascular volume homeostasis. Its cGMP-producing GC-A receptor is densely expressed in the microvascular endothelium of the lung and systemic circulation, but the functional relevance is controversial. Some studies reported that ANP stimulates endothelial cell permeability, whereas others described that the peptide attenuates endothelial barrier dysfunction provoked by inflammatory agents such as thrombin or histamine. Many studies in vitro addressed the effects of ANP on endothelial proliferation and migration. Again, both pro- and anti-angiogenic properties were described. To unravel the role of the endothelial actions of ANP in vivo, we inactivated the murine GC-A gene selectively in endothelial cells by homologous loxP/Cre-mediated recombination. Our studies in these mice indicate that ANP, via endothelial GC-A, increases endothelial albumin permeability in the microcirculation of the skin and skeletal muscle. This effect is critically involved in the endocrine hypovolaemic, hypotensive actions of the cardiac hormone. On the other hand the homologous GC-A-activating B-type NP (BNP), which is produced by cardiac myocytes and many other cell types in response to stressors such as hypoxia, possibly exerts more paracrine than endocrine actions. For instance, within the ischaemic skeletal muscle BNP released from activated satellite cells can improve the regeneration of neighbouring endothelia. This review will focus on recent advancements in our understanding of endothelial NP/GC-A signalling in the pulmonary versus systemic circulation. It will discuss possible mechanisms accounting for the discrepant observations made for the endothelial actions of this hormone-receptor system and distinguish between (patho)physiological and pharmacological actions. Lastly it will emphasize the potential therapeutical implications derived from the

Nipah virus infection and glycoprotein targeting in endothelial cells

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Maisner Andrea

2010-11-01

Full Text Available Abstract Background The highly pathogenic Nipah virus (NiV causes fatal respiratory and brain infections in animals and humans. The major hallmark of the infection is a systemic endothelial infection, predominantly in the CNS. Infection of brain endothelial cells allows the virus to overcome the blood-brain-barrier (BBB and to subsequently infect the brain parenchyma. However, the mechanisms of NiV replication in endothelial cells are poorly elucidated. We have shown recently that the bipolar or basolateral expression of the NiV surface glycoproteins F and G in polarized epithelial cell layers is involved in lateral virus spread via cell-to-cell fusion and that correct sorting depends on tyrosine-dependent targeting signals in the cytoplasmic tails of the glycoproteins. Since endothelial cells share many characteristics with epithelial cells in terms of polarization and protein sorting, we wanted to elucidate the role of the NiV glycoprotein targeting signals in endothelial cells. Results As observed in vivo, NiV infection of endothelial cells induced syncytia formation. The further finding that infection increased the transendothelial permeability supports the idea of spread of infection via cell-to-cell fusion and endothelial cell damage as a mechanism to overcome the BBB. We then revealed that both glycoproteins are expressed at lateral cell junctions (bipolar, not only in NiV-infected primary endothelial cells but also upon stable expression in immortalized endothelial cells. Interestingly, mutation of tyrosines 525 and 542/543 in the cytoplasmic tail of the F protein led to an apical redistribution of the protein in endothelial cells whereas tyrosine mutations in the G protein had no effect at all. This fully contrasts the previous results in epithelial cells where tyrosine 525 in the F, and tyrosines 28/29 in the G protein were required for correct targeting. Conclusion We conclude that the NiV glycoprotein distribution is responsible for

Radioprotection of mouse CNS endothelial cells in vivo

International Nuclear Information System (INIS)

Lyubimova, N.; Coultas, P.; Martin, R.

1996-01-01

Full text: Radioprotection using the minor groove binding DNA ligand Hoechst 33342 has been demonstrated in vitro, and more recently in vivo, in mouse lung. Intravenous administration was used for the lung studies, and both endothelial and alveolar epithelial cells-showed good up-take. Radiation damage to the endothelial cell population has also been postulated as important in late developing radionecrosis of spinal cord and brain. Endothelial cell density in brain can be readily determined by a fluorescent-histochemical technique. Treatment with a monoamine oxidase inhibitor and subsequent injection with L-DOPA results in an accumulation of dopamine (DA) in CNS endothelial cells. DA is converted to a fluorophore by exposure to paraformaldehyde, and cell numbers assayed by fluorescence microscopy. Earlier studies used this technique to monitor post-irradiation changes in endothelial cell density in rodent brain and showed the loss, within 24 hours, of a sensitive subpopulation comprising about 15% of the endothelial cells. Ten minutes after intravenous injection of Hoechst 33342 (80mg/kg) the ligand is confined by its limited penetration to the endothelial cells in mouse brain. When we irradiated at this time, there was protection against early endothelial cell loss. Ablation of the sensitive subpopulation in unprotected mice takes place over a dose range of 1 to 3 Gy γ-rays, but doses between 12 to 20 Gy are required in the presence of ligand. This protection equates to a very high dose modification factor of about 7 and possibly reflects a suppression of apoptosis in the sensitive endothelial subpopulation. The extent to which there is enhanced survival in the endothelial population as a whole and how the observed protection affects late CNS necrosis development has yet to be determined. However present results clearly show potential for the use of DNA-binding radioprotectors with limited penetration for investigations into the relative significance of

Endothelial microparticles: Pathogenic or passive players in endothelial dysfunction in autoimmune rheumatic diseases?

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McCarthy, E M; Wilkinson, F L; Parker, B; Alexander, M Y

2016-11-01

Autoimmune rheumatic diseases are characterised by systemic inflammation and complex immunopathology, with an increased risk of cardiovascular disease, initiated by endothelial dysfunction in a chronic inflammatory environment. Endothelial microparticles (EMPs) are released into the circulation from activated endothelial cells and may therefore, reflect disease severity, vascular and endothelial dysfunction, that could influence disease pathogenesis via autocrine/paracrine signalling. The exact function of EMPs in rheumatic disease remains unknown, and this has initiated research to elucidate EMP composition and function, which may be determined by the mode of endothelial activation and the micro environment. To date, EMPs are thought to play a role in angiogenesis, thrombosis and inflammation by transferring specific proteins and microRNAs (miRs) to target cells. Here, we review the mechanisms underlying the generation and composition of EMPs and the clinical and experimental studies describing the involvement of EMPs in rheumatic diseases, since we have previously shown endothelial dysfunction and an elevated risk of cardiovascular disease are characteristics in systemic lupus erythematosus. We will also discuss the potential of EMPs as future biomarkers of cardiovascular risk in these diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

Adhesion and endothelialization of endothelial cells on the surface of endovascular stents by the novel rotational culture of cells

International Nuclear Information System (INIS)

Tang Chaojun; Wang Guixue; Cao Yi; Wu Xue; Xie Xiang; Xiao Li

2008-01-01

Recent researches indicate that the initial event in the implantation of endovascular stents involves mechanical injury to the vessel wall. Confluent endothelialization of vascular grafts in vitro before implantation has been suggested as a way to reduce injury of the blood vessel. The purpose of this study is to establish a useful way to improve the adhesion of endothelial cells and accelerate endothelialization on the surface of endovascular stents by a novel rotational culture device. Numerical simulation was used to predict the shear stress on the surface of stents. The number of cellular adhesion was calculated by cell counting, the cell growth was observed by scanning electron microscope and fluorescence microscope. Numerical simulation results showed that the stents was exposed to shear stress of 2.66 x 10 -3 to 8.88 x 10 -2 Pa. Rotational culture of human umbilical vein endothelial cells could enhance the adhesion of cells and accelerate endothelialization on the surface of stents when the culture conditions for EC adhesion were intermediate rotation speed, higher dynamic incubation times, lower cell densities

Differentiation state determines neural effects on microvascular endothelial cells

International Nuclear Information System (INIS)

Muffley, Lara A.; Pan, Shin-Chen; Smith, Andria N.; Ga, Maricar; Hocking, Anne M.; Gibran, Nicole S.

2012-01-01

Growing evidence indicates that nerves and capillaries interact paracrinely in uninjured skin and cutaneous wounds. Although mature neurons are the predominant neural cell in the skin, neural progenitor cells have also been detected in uninjured adult skin. The aim of this study was to characterize differential paracrine effects of neural progenitor cells and mature sensory neurons on dermal microvascular endothelial cells. Our results suggest that neural progenitor cells and mature sensory neurons have unique secretory profiles and distinct effects on dermal microvascular endothelial cell proliferation, migration, and nitric oxide production. Neural progenitor cells and dorsal root ganglion neurons secrete different proteins related to angiogenesis. Specific to neural progenitor cells were dipeptidyl peptidase-4, IGFBP-2, pentraxin-3, serpin f1, TIMP-1, TIMP-4 and VEGF. In contrast, endostatin, FGF-1, MCP-1 and thrombospondin-2 were specific to dorsal root ganglion neurons. Microvascular endothelial cell proliferation was inhibited by dorsal root ganglion neurons but unaffected by neural progenitor cells. In contrast, microvascular endothelial cell migration in a scratch wound assay was inhibited by neural progenitor cells and unaffected by dorsal root ganglion neurons. In addition, nitric oxide production by microvascular endothelial cells was increased by dorsal root ganglion neurons but unaffected by neural progenitor cells. -- Highlights: ► Dorsal root ganglion neurons, not neural progenitor cells, regulate microvascular endothelial cell proliferation. ► Neural progenitor cells, not dorsal root ganglion neurons, regulate microvascular endothelial cell migration. ► Neural progenitor cells and dorsal root ganglion neurons do not effect microvascular endothelial tube formation. ► Dorsal root ganglion neurons, not neural progenitor cells, regulate microvascular endothelial cell production of nitric oxide. ► Neural progenitor cells and dorsal root

Tumor and Endothelial Cell Hybrids Participate in Glioblastoma Vasculature

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Soufiane El Hallani

2014-01-01

Full Text Available Background. Recently antiangiogenic therapy with bevacizumab has shown a high but transient efficacy in glioblastoma (GBM. Indeed, GBM is one of the most angiogenic human tumors and endothelial proliferation is a hallmark of the disease. We therefore hypothesized that tumor cells may participate in endothelial proliferation of GBM. Materials and Methods. We used EGFR FISH Probe to detect EGFR amplification and anti-CD31, CD105, VE-cadherin, and vWF to identify endothelial cells. Endothelial and GBM cells were grown separately, labeled with GFP and DsRed lentiviruses, and then cocultured with or without contact. Results. In a subset of GBM tissues, we found that several tumor endothelial cells carry EGFR amplification, characteristic of GBM tumor cells. This observation was reproduced in vitro: when tumor stem cells derived from GBM were grown in the presence of human endothelial cells, a fraction of them acquired endothelial markers (CD31, CD105, VE-cadherin, and vWF. By transduction with GFP and DsRed expressing lentiviral vectors, we demonstrate that this phenomenon is due to cell fusion and not transdifferentiation. Conclusion. A fraction of GBM stem cells thus has the capacity to fuse with endothelial cells and the resulting hybrids may participate in tumor microvascular proliferation and in treatment resistance.

Syncytin is involved in breast cancer-endothelial cell fusions

DEFF Research Database (Denmark)

Bjerregaard, Bolette; Holck, S.; Christensen, I.J.

2006-01-01

Cancer cells can fuse spontaneously with normal host cells, including endothelial cells, and such fusions may strongly modulate the biological behaviour of tumors. However, the underlying mechanisms are unknown. We now show that human breast cancer cell lines and 63 out of 165 (38%) breast cancer...... specimens express syncytin, an endogenous retroviral envelope protein, previously implicated in fusions between placental trophoblast cells. Additionally, endothelial and cancer cells are shown to express ASCT-2, a receptor for syncytin. Syncytin antisense treatment decreases syncytin expression...... and inhibits fusions between breast cancer cells and endothelial cells. Moreover, a syncytin inhibitory peptide also inhibits fusions between cancer and endothelial cells. These results are the first to show that syncytin is expressed by human cancer cells and is involved in cancer-endothelial cell fusions....

Endothelial induced EMT in breast epithelial cells with stem cell properties.

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Valgardur Sigurdsson

Full Text Available Epithelial to mesenchymal transition (EMT is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad to N-Cadherin (N-Cad and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high/CD24(low ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.

Endothelial induced EMT in breast epithelial cells with stem cell properties.

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Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla; Fridriksdottir, Agla J R; Ringnér, Markus; Villadsen, Rene; Borg, Ake; Agnarsson, Bjarni A; Petersen, Ole William; Magnusson, Magnus K; Gudjonsson, Thorarinn

2011-01-01

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.

Endothelial dysfunction in the regulation of portal hypertension

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Iwakiri, Yasuko

2013-01-01

Portal hypertension is caused by an increased intrahepatic resistance, a major consequence of cirrhosis. Endothelial dysfunction in liver sinusoidal endothelial cells (LSECs) decreases the production of vasodilators, such as nitric oxide (NO) and favors vasoconstriction. This contributes to an increased vascular resistance in the intrahepatic/sinusoidal microcirculation. Portal hypertension, once developed, causes endothelial cell (EC) dysfunction in the extrahepatic, i.e. splanchnic and systemic, circulation. Unlike LSEC dysfunction, EC dysfunction in the splanchnic and systemic circulation overproduces vasodilator molecules, leading to arterial vasodilatation. In addition, portal hypertension leads to the formation of portosystemic collateral vessels. Both arterial vasodilatation and portosystemic collateral vessel formation exacerbate portal hypertension by increasing the blood flow through the portal vein. Pathologic consequences, such as esophageal varices and ascites, result. While the sequence of pathological vascular events in cirrhosis and portal hypertension have been elucidated, the underlying cellular and molecular mechanisms causing EC dysfunctions are not yet fully understood. This review article summarizes the current cellular and molecular studies on EC dysfunctions found during the development of cirrhosis and portal hypertension with a focus on intra- and extrahepatic circulation. The article ends by discussing future directions of study for EC dysfunctions. PMID:21745318

Oxidative stress induced pulmonary endothelial cell proliferation is ...

African Journals Online (AJOL)

Cellular hyper-proliferation, endothelial dysfunction and oxidative stress are hallmarks of the pathobiology of pulmonary hypertension. Indeed, pulmonary endothelial cells proliferation is susceptible to redox state modulation. Some studies suggest that superoxide stimulates endothelial cell proliferation while others have ...

Dysregulation of endothelial colony-forming cell function by a negative feedback loop of circulating miR-146a and -146b in cardiovascular disease patients.

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Ting-Yu Chang

Full Text Available Functional impairment of endothelial colony-forming cells (ECFCs, a specific cell lineage of endothelial progenitor cells (EPCs is highly associated with the severity of coronary artery disease (CAD, the most common type of cardiovascular disease (CVD. Emerging evidence show that circulating microRNAs (miRNAs in CAD patients' body fluid hold a great potential as biomarkers. However, our knowledge of the role of circulating miRNA in regulating the function of ECFCs and the progression of CAD is still in its infancy. We showed that when ECFCs from healthy volunteers were incubated with conditioned medium or purified exosomes of cultured CAD ECFCs, the secretory factors from CAD ECFCs dysregulated migration and tube formation ability of healthy ECFCs. It is known that exosomes influence the physiology of recipient cells by introducing RNAs including miRNAs. By using small RNA sequencing (smRNA-seq, we deciphered the circulating miRNome in the plasma of healthy individual and CAD patients, and found that the plasma miRNA spectrum from CAD patients was significantly different from that of healthy control. Interestingly, smRNA-seq of both healthy and CAD ECFCs showed that twelve miRNAs that had a higher expression in the plasma of CAD patients also showed higher expression in CAD ECFCs when compared with healthy control. This result suggests that these miRNAs may be involved in the regulation of ECFC functions. For identification of potential mRNA targets of the differentially expressed miRNA in CAD patients, cDNA microarray analysis was performed to identify the angiogenesis-related genes that were down-regulated in CAD ECFCs and Pearson's correlation were used to identify miRNAs that were negatively correlated with the identified angiogenesis-related genes. RT-qPCR analysis of the five miRNAs that negatively correlated with the down-regulated angiogenesis-related genes in plasma and ECFC of CAD patients showed miR-146a-5p and miR-146b-5p up

Clinical value of circulating endothelial cell levels in metastatic colorectal cancer patients treated with first-line chemotherapy and bevacizumab.

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Malka, D; Boige, V; Jacques, N; Vimond, N; Adenis, A; Boucher, E; Pierga, J Y; Conroy, T; Chauffert, B; François, E; Guichard, P; Galais, M P; Cvitkovic, F; Ducreux, M; Farace, F

2012-04-01

We investigated whether circulating endothelial cells (CECs) predict clinical outcome of first-line chemotherapy and bevacizumab in metastatic colorectal cancer (mCRC) patients. In a substudy of the randomized phase II FNCLCC ACCORD 13/0503 trial, CECs (CD45- CD31+ CD146+ 7-amino-actinomycin- cells) were enumerated in 99 patients by four-color flow cytometry at baseline and after one cycle of treatment. We correlated CEC levels with objective response rate (ORR), 6-month progression-free survival (PFS) rate (primary end point of the trial), PFS, and overall survival (OS). Multivariate analyses of potential prognostic factors, including CEC counts and Köhne score, were carried out. By multivariate analysis, high baseline CEC levels were the only independent prognostic factor for 6-month PFS rate (P < 0.01) and were independently associated with worse PFS (P = 0.02). High CEC levels after one cycle were the only independent prognostic factor for ORR (P = 0.03). High CEC levels at both time points independently predicted worse ORR (P = 0.025), 6-month PFS rate (P = 0.007), and PFS (P = 0.02). Köhne score was the only variable associated with OS. CEC levels at baseline and after one treatment cycle may independently predict ORR and PFS in mCRC patients starting first-line bevacizumab and chemotherapy.

Curcumin modulates endothelial permeability and monocyte transendothelial migration by affecting endothelial cell dynamics.

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Monfoulet, Laurent-Emmanuel; Mercier, Sylvie; Bayle, Dominique; Tamaian, Radu; Barber-Chamoux, Nicolas; Morand, Christine; Milenkovic, Dragan

2017-11-01

Curcumin is a phenolic compound that exhibits beneficial properties for cardiometabolic health. We previously showed that curcumin reduced the infiltration of immune cells into the vascular wall and prevented atherosclerosis development in mice. This study aimed to investigate the effect of curcumin on monocyte adhesion and transendothelial migration (TEM) and to decipher the underlying mechanisms of these actions. Human umbilical vein endothelial cells (HUVECs) were exposed to curcumin (0.5-1μM) for 3h prior to their activation by Tumor Necrosis Factor alpha (TNF-α). Endothelial permeability, monocyte adhesion and transendothelial migration assays were conducted under static condition and shear stress that mimics blood flow. We further investigated the impact of curcumin on signaling pathways and on the expression of genes using macroarrays. Pre-exposure of endothelial cells to curcumin reduced monocyte adhesion and their transendothelial migration in both static and shear stress conditions. Curcumin also prevented changes in both endothelial permeability and the area of HUVECs when induced by TNF-α. We showed that curcumin modulated the expression of 15 genes involved in the control of cytoskeleton and endothelial junction dynamic. Finally, we showed that curcumin inhibited NF-κB signaling likely through an antagonist interplay with several kinases as suggested by molecular docking analysis. Our findings demonstrate the ability of curcumin to reduce monocyte TEM through a multimodal regulation of the endothelial cell dynamics with a potential benefit on the vascular endothelial function barrier. Copyright © 2017 Elsevier Inc. All rights reserved.

Qidantongmai Protects Endothelial Cells Against Hypoxia-Induced ...

African Journals Online (AJOL)

induced damage. The ability of QDTM to modulate the serum VEGF-A level may play an important role in its effects on endothelial cells. Key words: Traditional Chinese Medicine, human umbilical vein endothelial cells, hypoxia, VEGF ...

Infection of endothelial cells by common human viruses.

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Friedman, H M

1989-01-01

Common human viruses were evaluated for their ability to replicate in the endothelial cells of human umbilical vein and bovine thoracic aorta in vitro. Infection occurred with most viruses. The susceptibilities of endothelial cells derived from bovine aorta, pulmonary artery, and vena cava were compared. Among the viruses studied, no differences were noted in the ability to grow in endothelial cells from these three large vessels. One virus, herpes simplex virus type 1, was evaluated for its ability to produce persistent infection of endothelial cells. Infection developed and persisted for up to 3 months. After the first week, productive infection was found in less than 1% of cells. Nevertheless, the infection markedly affected the growth and morphology of the endothelial monolayer. Infection with any of several different viruses was noted to alter endothelial cell functions, including adherence of granulocytes, production of colony-stimulating factor, and synthesis of matrix protein. In addition, herpes simplex virus type 1 induced receptors for the Fc portion of IgG and for complement component C3b. These findings indicate that common human viruses can profoundly affect the biology of the endothelium.

Activation of endothelial cells after exposure to ambient ultrafine particles: The role of NADPH oxidase

International Nuclear Information System (INIS)

Mo Yiqun; Wan Rong; Chien Sufan; Tollerud, David J.; Zhang Qunwei

2009-01-01

Several studies have shown that ultrafine particles (UFPs) may pass from the lungs to the circulation because of their very small diameter, and induce lung oxidative stress with a resultant increase in lung epithelial permeability. The direct effects of UFPs on vascular endothelium remain unknown. We hypothesized that exposure to UFPs leads to endothelial cell O 2 ·- generation via NADPH oxidase and results in activation of endothelial cells. Our results showed that UFPs, at a non-toxic dose, induced reactive oxygen species (ROS) generation in mouse pulmonary microvascular endothelial cells (MPMVEC) that was inhibited by pre-treatment with the ROS scavengers or inhibitors, but not with the mitochondrial inhibitor, rotenone. UFP-induced ROS generation in MPMVEC was abolished by p67 phox siRNA transfection and UFPs did not cause ROS generation in MPMVEC isolated from gp91 phox knock-out mice. UFP-induced ROS generation in endothelial cells was also determined in vivo by using a perfused lung model with imaging. Moreover, Western blot and immunofluorescence staining results showed that MPMVEC treated with UFPs resulted in the translocation of cytosolic proteins of NADPH oxidase, p47 phox , p67 phox and rac 1, to the plasma membrane. These results demonstrate that NADPH oxidase in the pulmonary endothelium is involved in ROS generation following exposure to UFPs. To investigate the activation of endothelial cells by UFP-induced oxidative stress, we determined the activation of the mitogen-activated protein kinases (MAPKs) in MPMVEC. Our results showed that exposure of MPMVEC to UFPs caused increased phosphorylation of p38 and ERK1/2 MAPKs that was blocked by pre-treatment with DPI or p67 phox siRNA. Exposure of MPMVEC obtained from gp91 phox knock-out mice to UFPs did not cause increased phosphorylation of p38 and ERK1/2 MAPKs. These findings confirm that UFPs can cause endothelial cells to generate ROS directly via activation of NADPH oxidase. UFP-induced ROS lead to

Black Tea Increases Circulating Endothelial Progenitor Cells and Improves Flow Mediated Dilatation Counteracting Deleterious Effects from a Fat Load in Hypertensive Patients: A Randomized Controlled Study

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Grassi, Davide; Draijer, Richard; Schalkwijk, Casper; Desideri, Giovambattista; D’Angeli, Anatolia; Francavilla, Sandro; Mulder, Theo; Ferri, Claudio

2016-01-01

(1) Background: Endothelial dysfunction predicts cardiovascular events. Circulating angiogenic cells (CACs) maintain and repair the endothelium regulating its function. Tea flavonoids reduce cardiovascular risk. We investigated the effects of black tea on the number of CACs and on flow-mediated dilation (FMD) before and after an oral fat in hypertensives; (2) Methods: In a randomized, double-blind, controlled, cross-over study, 19 patients were assigned to black tea (150 mg polyphenols) or a placebo twice a day for eight days. Measurements were obtained in a fasted state and after consuming whipping cream, and FMD was measured at baseline and after consumption of the products; (3) Results: Compared with the placebo, black tea ingestion increased functionally active CACs (36 ± 22 vs. 56 ± 21 cells per high-power field; p = 0.006) and FMD (5.0% ± 0.3% vs. 6.6% ± 0.3%, p FMD 1, 2, 3, and 4 h after consumption, with maximal response 2 h after intake (p FMD, while tea consumption counteracted FMD impairment (p < 0.0001); (4) Conclusions: We demonstrated the vascular protective properties of black tea by increasing the number of CACs and preventing endothelial dysfunction induced by acute oral fat load in hypertensive patients. Considering that tea is the most consumed beverage after water, our findings are of clinical relevance and interest. PMID:27854314

Modeling Group B Streptococcus and Blood-Brain Barrier Interaction by Using Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells

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Kim, Brandon J.; Bee, Olivia B.; McDonagh, Maura A.; Stebbins, Matthew J.; Palecek, Sean P.; Doran, Kelly S.; Shusta, Eric V.

2017-01-01

ABSTRACT Bacterial meningitis is a serious infection of the central nervous system (CNS) that occurs after bacteria interact with and penetrate the blood-brain barrier (BBB). The BBB is comprised of highly specialized brain microvascular endothelial cells (BMECs) that function to separate the circulation from the CNS and act as a formidable barrier for toxins and pathogens. Certain bacteria, such as Streptococcus agalactiae (group B Streptococcus [GBS]), possess the ability to interact with a...

Interleukin-3 greatly expands non-adherent endothelial forming cells with pro-angiogenic properties

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Lachlan M. Moldenhauer

2015-05-01

Full Text Available Circulating endothelial progenitor cells (EPCs provide revascularisation for cardiovascular disease and the expansion of these cells opens up the possibility of their use as a cell therapy. Herein we show that interleukin-3 (IL3 strongly expands a population of human non-adherent endothelial forming cells (EXnaEFCs with low immunogenicity as well as pro-angiogenic capabilities in vivo, making their therapeutic utilisation a realistic option. Non-adherent CD133+ EFCs isolated from human umbilical cord blood and cultured under different conditions were maximally expanded by day 12 in the presence of IL3 at which time a 350-fold increase in cell number was obtained. Cell surface marker phenotyping confirmed expression of the hematopoietic progenitor cell markers CD133, CD117 and CD34, vascular cell markers VEGFR2 and CD31, dim expression of CD45 and absence of myeloid markers CD14 and CD11b. Functional experiments revealed that EXnaEFCs exhibited classical properties of endothelial cells (ECs, namely binding of Ulex europaeus lectin, up-take of acetylated-low density lipoprotein and contribution to EC tube formation in vitro. These EXnaEFCs demonstrated a pro-angiogenic phenotype within two independent in vivo rodent models. Firstly, a Matrigel plug assay showed increased vascularisation in mice. Secondly, a rat model of acute myocardial infarction demonstrated reduced heart damage as determined by lower levels of serum creatinine and a modest increase in heart functionality. Taken together, these studies show IL3 as a potent growth factor for human CD133+ cell expansion with clear pro-angiogenic properties (in vitro and in vivo and thus may provide clinical utility for humans in the future.

Signaling hierarchy regulating human endothelial cell development

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Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these stud...

Radiation Effects on the Cytoskeleton of Endothelial Cells and Endothelial Monolayer Permeability

International Nuclear Information System (INIS)

Gabrys, Dorota; Greco, Olga; Patel, Gaurang; Prise, Kevin M.; Tozer, Gillian M.; Kanthou, Chryso

2007-01-01

Purpose: To investigate the effects of radiation on the endothelial cytoskeleton and endothelial monolayer permeability and to evaluate associated signaling pathways, which could reveal potential mechanisms of known vascular effects of radiation. Methods and Materials: Cultured endothelial cells were X-ray irradiated, and actin filaments, microtubules, intermediate filaments, and vascular endothelial (VE)-cadherin junctions were examined by immunofluorescence. Permeability was determined by the passage of fluorescent dextran through cell monolayers. Signal transduction pathways were analyzed using RhoA, Rho kinase, and stress-activated protein kinase-p38 (SAPK2/p38) inhibitors by guanosine triphosphate-RhoA activation assay and transfection with RhoAT19N. The levels of junction protein expression and phosphorylation of myosin light chain and SAPK2/p38 were assessed by Western blotting. The radiation effects on cell death were verified by clonogenic assays. Results: Radiation induced rapid and persistent actin stress fiber formation and redistribution of VE-cadherin junctions in microvascular, but not umbilical vein endothelial cells, and microtubules and intermediate filaments remained unaffected. Radiation also caused a rapid and persistent increase in microvascular permeability. RhoA-guanosine triphosphatase and Rho kinase were activated by radiation and caused phosphorylation of downstream myosin light chain and the observed cytoskeletal and permeability changes. SAPK2/p38 was activated by radiation but did not influence either the cytoskeleton or permeability. Conclusion: This study is the first to show rapid activation of the RhoA/Rho kinase by radiation in endothelial cells and has demonstrated a link between this pathway and cytoskeletal remodeling and permeability. The results also suggest that the RhoA pathway might be a useful target for modulating the permeability and other effects of radiation for therapeutic gain

Sun Ginseng Protects Endothelial Progenitor Cells From Senescence Associated Apoptosis

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Im, Wooseok; Chung, Jin-Young; Bhan, Jaejun; Lim, Jiyeon; Lee, Soon-Tae; Chu, Kon; Kim, Manho

2012-01-01

Endothelial progenitor cells (EPC) are a population of cells that circulate in the blood stream. They play a role in angiogenesis and, therefore, can be prognostic markers of vascular repair. Ginsenoside Rg3 prevents endothelial cell apoptosis through the inhibition of the mitochondrial caspase pathway. It also affects estrogen activity, which reduces EPC senescence. Sun ginseng (SG), which is heat-processed ginseng, has a high content of ginsenosides. The purpose of this study was to investigate the protective effects of SG on senescence-associated apoptosis in EPCs. In order to isolate EPCs, mononuclear cells of human blood buffy coats were cultured and characterized by their uptake of acetylated low-density lipoprotein (acLDL) and their binding of Ulex europaeus agglutinin I (ulex-lectin). Flow cytometry with annexin-V staining was performed in order to assess early and late apoptosis. Senescence was determined by β-galactosidase (β-gal) staining. Staining with 4′-6-Diamidino-2-phenylindole verified that most adherent cells (93±2.7%) were acLDL-positive and ulex-lectin-positive. The percentage of β-gal-positive EPCs was decreased from 93.8±2.0% to 62.5±3.6% by SG treatment. A fluorescence-activated cell sorter (FACS) analysis showed that 4.9% of EPCs were late apoptotic in controls. Sun ginseng decreased the apoptotic cell population by 39% in the late stage of apoptosis from control baseline levels. In conclusion, these results show antisenescent and antiapoptotic effects of SG in human-derived EPCs, indicating that SG can enhance EPC-mediated repair mechanisms. PMID:23717107

Animal study on transplantation of human umbilical vein endothelial cells for corneal endothelial decompensation

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Li Cui

2014-06-01

Full Text Available AIM: To explore the feasibility of culturing human umbilical vein endothelial cells(HUVECon acellular corneal stroma and performing the posterior lamellar endothelial keratoplasty(PLEKtreating corneal endothelial decompensation.METHODS: Thirty New-Zealand rabbits were divided into three groups randomly, 10 rabbits for experimental group, 10 for stroma group and 10 for control group. Corneal endothelial cells were removed to establish animal model of corneal endothelial failure. PLEK was performed on the rabbits of experimental group and stroma group, and nothing was transplantated onto the rabbits of control group with the deep layer excised only. Postoperative observation was taken for 3mo. The degree of corneal edema and central corneal thickness were recorded for statistical analysis.RESULTS: Corneas in experimental group were relieved in edema obviously compared with that in stroma group and the control group, and showed increased transparency 7d after the operation. The average density of endothelial cells was 2 026.4±129.3cells/mm2, and average central corneal thickness was 505.2±25.4μm in experimental group, while 1 535.6±114.5μm in stroma group and 1 493.5±70.2μm in control group 3mo after operation.CONCLUSION:We achieved preliminary success in our study that culturing HUVEC on acellular corneal stroma and performing PLEK for corneal endothelial decompensation. HUVEC transplanted could survive in vivo, and have normal biological function of keeping cornea transparent. This study provides a new idea and a new way clinically for the treatment of corneal endothelial diseases.

Endothelial progenitor cell mobilization and increased intravascular nitric oxide in patients undergoing cardiac rehabilitation.

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Paul, Jonathan D; Powell, Tiffany M; Thompson, Michael; Benjamin, Moshe; Rodrigo, Maria; Carlow, Andrea; Annavajjhala, Vidhya; Shiva, Sruti; Dejam, Andre; Gladwin, Mark T; McCoy, J Philip; Zalos, Gloria; Press, Beverly; Murphy, Mandy; Hill, Jonathan M; Csako, Gyorgy; Waclawiw, Myron A; Cannon, Richard O

2007-01-01

We investigated whether cardiac rehabilitation participation increases circulating endothelial progenitor cells (EPCs) and benefits vasculature in patients already on stable therapy previously shown to augment EPCs and improve endothelial function. Forty-six of 50 patients with coronary artery disease completed a 36-session cardiac rehabilitation program: 45 were treated with HMG-CoA reductase inhibitor (statin) therapy > or = 1 month (average baseline low-density lipoprotein cholesterol = 81 mg/dL). Mononuclear cells isolated from blood were quantified for EPCs by flow cytometry (CD133/VEGFR-2 cells) and assayed in culture for EPC colony-forming units (CFUs). In 23 patients, EPCs were stained for annexin-V as a marker of apoptosis, and nitrite was measured in blood as an indicator of intravascular nitric oxide. Endothelial progenitor cells increased from 35 +/- 5 to 63 +/- 10 cells/mL, and EPC-CFUs increased from 0.9 +/- 0.2 to 3.1 +/- 0.6 per well (both P < .01), but 11 patients had no increase in either measure. Those patients whose EPCs increased from baseline showed significant increases in nitrite and reduction in annexin-V staining (both P < .01) versus no change in patients without increase in EPCs. Over the course of the program, EPCs increased prior to increase in nitrite in the blood. Cardiac rehabilitation in patients receiving stable statin therapy and with low-density lipoprotein cholesterol at goal increases EPC number, EPC survival, and endothelial differentiation potential, associated with increased nitric oxide in the blood. Although this response was observed in most patients, a significant minority showed neither EPC mobilization nor increased nitric oxide in the blood.

Transport of lipoprotein lipase across endothelial cells

International Nuclear Information System (INIS)

Saxena, U.; Klein, M.G.; Goldberg, I.J.

1991-01-01

Lipoprotein lipase (LPL), synthesized in muscle and fat, hydrolyzes plasma triglycerides primarily while bound to luminal endothelial cell surfaces. To obtain information about the movement of LPL from the basal to the luminal endothelial cell surface, the authors studied the transport of purified bovine milk LPL across bovine aortic endothelial cell monolayers. 125 I-labeled LPL ( 125 I-LPL) added to the basal surface of the monolayers was detected on the apical side of the cells in two compartments: (1) in the medium of the upper chamber, and (2) bound to the apical cell surface. The amount of 125 I-LPL on the cell surface, but not in the medium, reached saturation with time and LPL dose. Catalytically active LPL was transported to the apical surface but very little LPL activity appeared in the medium. Heparinase treatment of the basal cell surface and addition of dextran sulfate to the lower chamber decreased the amount of 125 I-LPL appearing on the apical surface. Similarly, the presence of increasing molar ratios of oleic acid/bovine serum albumin at the basal surface decreased the transport of active LPL across the monolayer. Thus, a saturable transport system, which requires haparan sulfate proteoglycans and is inhibited by high concentrations of free fatty acids on the basal side of the cells, appears to exist for passage of enzymatically active LPL across endothelial cells. They postulate that regulation of LPL transport to the endothelial luminal surface modulates the physiologically active pool of LPL in vivo

A novel podoplanin-GFPCre mouse strain for gene deletion in lymphatic endothelial cells.

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Gil, Hyea Jin; Ma, Wanshu; Oliver, Guillermo

2018-04-01

The lymphatic vascular system is a one-direction network of thin-walled capillaries and larger vessels covered by a continuous layer of endothelial cells responsible for maintaining fluid homeostasis. Some of the main functions of the lymphatic vasculature are to drain fluid from the extracellular spaces and return it back to the blood circulation, lipid absorption from the intestinal tract, and transport of immune cells to lymphoid organs. A number of genes controlling the development of the mammalian lymphatic vasculature have been identified in the last few years, and their functional roles started to be characterized using gene inactivation approaches in mice. Unfortunately, only few mouse Cre strains relatively specific for lymphatic endothelial cells (LECs) are currently available. In this article, we report the generation of a novel Podoplanin (Pdpn) GFPCre transgenic mouse strain using its 5' regulatory region. Pdpn encodes a transmembrane mucin-type O-glycoprotein that is expressed on the surface of embryonic and postnatal LECs, in addition to few other cell types. Our detailed characterization of this novel strain indicates that it will be a valuable additional genetic tool for the analysis of gene function in LECs. © 2018 Wiley Periodicals, Inc.

Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture

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Magnusson Magnus K

2010-07-01

Full Text Available Abstract Background Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D co-culture assay. Methods Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy. Results In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors. Conclusion Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.

Aging impairs transcriptional regulation of vascular endothelial growth factor in human microvascular endothelial cells: implications for angiogenesis and cell survival.

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Ahluwalia, A; Jones, M K; Szabo, S; Tarnawski, A S

2014-04-01

In some tissues, aging impairs angiogenesis and reduces expression of vascular endothelial growth factor A (VEGF), a fundamental regulator of angiogenesis. We previously examined angiogenesis in aging and young gastric mucosa in vivo and in vitro and showed that an imbalance between expressions of VEGF (pro-angiogenic factor) and endostatin (anti-angiogenic protein) results in an aging-related impairment of angiogenesis in rats. However, the human relevance of these findings, and whether these mechanisms apply to endothelial cells derived from other tissues, is not clear. Since P-STAT3 and P-CREB are transcription factors that, in association with HIF-1α, can activate VEGF gene expression in some cells (e.g., liver cancer cells, vascular smooth muscle cells), we examined the expression of these two proteins in human dermal microvascular endothelial cells (HMVECs) derived from aging and neonatal individuals. We examined and quantified in vitro angiogenesis, expression of VEGF, P-STAT3, P-CREB and importin-α in HMVECs isolated from neonates (neonatal) and a 66 year old subject (aging). We also examined the effects of treatment with exogenous VEGF and endostatin on in vitro angiogenesis in these cells. Endothelial cells isolated from aging individuals had impaired angiogenesis (vs. neonatal endothelial cells) and reduced expression of VEGF mRNA and protein. Aged HMVECs also had reduced importin-α expression, and reduced expression and nuclear translocation of P-STAT3 and P-CREB. Reduced VEGF gene expression in aged HMVECs strongly correlated with the decreased levels of P-STAT3, P-CREB and importin-α in these cells. Our study clearly demonstrates that endothelial cells from aging individuals have impaired angiogenesis and reduced expression of VEGF likely due to impaired nuclear transport of P-STAT3 and P-CREB transcription factors in these cells.

Circulating histones are mediators of trauma-associated lung injury.

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Abrams, Simon T; Zhang, Nan; Manson, Joanna; Liu, Tingting; Dart, Caroline; Baluwa, Florence; Wang, Susan Siyu; Brohi, Karim; Kipar, Anja; Yu, Weiping; Wang, Guozheng; Toh, Cheng-Hock

2013-01-15

Acute lung injury is a common complication after severe trauma, which predisposes patients to multiple organ failure. This syndrome largely accounts for the late mortality that arises and despite many theories, the pathological mechanism is not fully understood. Discovery of histone-induced toxicity in mice presents a new dimension for elucidating the underlying pathophysiology. To investigate the pathological roles of circulating histones in trauma-induced lung injury. Circulating histone levels in patients with severe trauma were determined and correlated with respiratory failure and Sequential Organ Failure Assessment (SOFA) scores. Their cause-effect relationship was studied using cells and mouse models. In a cohort of 52 patients with severe nonthoracic blunt trauma, circulating histones surged immediately after trauma to levels that were toxic to cultured endothelial cells. The high levels were significantly associated with the incidence of acute lung injury and SOFA scores, as well as markers of endothelial damage and coagulation activation. In in vitro systems, histones damaged endothelial cells, stimulated cytokine release, and induced neutrophil extracellular trap formation and myeloperoxidase release. Cellular toxicity resulted from their direct membrane interaction and resultant calcium influx. In mouse models, cytokines and markers for endothelial damage and coagulation activation significantly increased immediately after trauma or histone infusion. Pathological examinations showed that lungs were the predominantly affected organ with edema, hemorrhage, microvascular thrombosis, and neutrophil congestion. An anti-histone antibody could reduce these changes and protect mice from histone-induced lethality. This study elucidates a new mechanism for acute lung injury after severe trauma and proposes that circulating histones are viable therapeutic targets for improving survival outcomes in patients.

Circulating Histones Are Mediators of Trauma-associated Lung Injury

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Abrams, Simon T.; Zhang, Nan; Manson, Joanna; Liu, Tingting; Dart, Caroline; Baluwa, Florence; Wang, Susan Siyu; Brohi, Karim; Kipar, Anja; Yu, Weiping

2013-01-01

Rationale: Acute lung injury is a common complication after severe trauma, which predisposes patients to multiple organ failure. This syndrome largely accounts for the late mortality that arises and despite many theories, the pathological mechanism is not fully understood. Discovery of histone-induced toxicity in mice presents a new dimension for elucidating the underlying pathophysiology. Objectives: To investigate the pathological roles of circulating histones in trauma-induced lung injury. Methods: Circulating histone levels in patients with severe trauma were determined and correlated with respiratory failure and Sequential Organ Failure Assessment (SOFA) scores. Their cause–effect relationship was studied using cells and mouse models. Measurements and Main Results: In a cohort of 52 patients with severe nonthoracic blunt trauma, circulating histones surged immediately after trauma to levels that were toxic to cultured endothelial cells. The high levels were significantly associated with the incidence of acute lung injury and SOFA scores, as well as markers of endothelial damage and coagulation activation. In in vitro systems, histones damaged endothelial cells, stimulated cytokine release, and induced neutrophil extracellular trap formation and myeloperoxidase release. Cellular toxicity resulted from their direct membrane interaction and resultant calcium influx. In mouse models, cytokines and markers for endothelial damage and coagulation activation significantly increased immediately after trauma or histone infusion. Pathological examinations showed that lungs were the predominantly affected organ with edema, hemorrhage, microvascular thrombosis, and neutrophil congestion. An anti-histone antibody could reduce these changes and protect mice from histone-induced lethality. Conclusions: This study elucidates a new mechanism for acute lung injury after severe trauma and proposes that circulating histones are viable therapeutic targets for improving survival

Adenine nucleotide depletion from endothelial cells exposed to xanthine oxidase

International Nuclear Information System (INIS)

Aalto, T.K.; Raivio, K.O.

1990-01-01

Hypoxia causes breakdown of cellular nucleotides, accumulation of hypoxanthine (HX), and conversion of xanthine dehydrogenase into xanthine oxidase (XO). Upon reoxygenation, the HX-XO reaction generates free radicals, one potential mechanism of tissue damage. Because endothelial cells contain XO and are exposed to circulating HX, they are a likely target for damage. We studied the effect of XO and/or HX at physiologically relevant concentrations on nucleotide metabolism of cultured endothelial cells from human umbilical veins. Cells were labeled with [14C]adenine and incubated for up to 6 h with HX, XO, or both, in the absence or presence of serum. Adenine nucleotides from cell extracts and nucleotide breakdown products (HX, xanthine, and urate) from the medium were separated and counted. HX alone had no effect. XO (80 mU/ml) alone caused a 70% (no serum) or 40% (with serum) fall in adenine nucleotides and an equivalent increase of xanthine and urate. The combination of HX and XO caused a 90% (no serum) or 70% (with serum) decrease in nucleotides, decrease in energy charge, and detachment of cells from the culture plate. Nucleotide depletion was not accounted for by proteolytic activity in the XO preparation. Albumin was only half as effective as serum in preventing nucleotide loss. Thus exogenous XO, in the presence of endogenous HX, triggers adenine nucleotide catabolism, but endogenous XO activity is too low to influence nucleotide levels even at high exogenous HX concentrations. Serum limits the catabolic effect of XO and thus protects cells from free radical damage

Comparison of endothelial progenitor cells in Parkinson's disease patients treated with levodopa and levodopa/COMT inhibitor.

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Phil Hyu Lee

Full Text Available BACKGROUND: Levodopa treatment in Parkinson's disease (PD increases in serum homocysteine levels due to its metabolism via catechol O-methyltransferase. Endothelial progenitor cells (EPCs have the capacity to differentiate into mature endothelial cells and are markers for endothelial functions and cardiovascular risks. Along with traditional vascular risk factors, hyperhomocysteinemia is known to decrease the level of EPCs. In the present study, we hypothesized that that levodopa-induced hyperhomocysteinemia leads to a change in EPC levels. METHODOLOGY/PRINCIPAL FINDINGS: We prospectively enrolled PD patients who had been prescribed either levodopa/carbidopa (PD-L group, n = 28 or levodopa/carbidopa/COMT inhibitor (PD-LC group, n = 25 for more than 1 year. The number of circulating EPCs was measured by flow cytometry using dual staining of anti-CD34 and anti-KDR antibodies. The EPCs were divided into tertiles based on their distributions and a logistic regression analysis was used to estimate independent predictors of the highest tertile of EPCs. The number of endothelial progenitor cells was significantly decreased in PD-L patients (118±99/mL compared with either PD-LC patients (269±258/mL, p = 0.007 or controls (206±204/mL, p = 0.012. The level of homocysteine was significantly increased in PD-L patients (14.9±5.3 µmol/L compared with either PD-LC patients (11.9±3.0 µmol/L, p = 0.028 or controls (11.1±2.5 µmol/L, p = 0.012. The level of homocysteine was negatively correlated with endothelial progenitor cell levels (r = -0.252, p = 0.028 and was an independent predictor of the highest tertile of endothelial progenitor cell levels (OR; 0.749 [95% CI: 0.584-0.961]. CONCLUSIONS/SIGNIFICANCE: These data indicate that a higher consumption of EPC for restoration of endothelial damage may be associated with chronic levodopa treatment in PD patients.

Identification and functional analysis of endothelial tip cell-enriched genes.

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del Toro, Raquel; Prahst, Claudia; Mathivet, Thomas; Siegfried, Geraldine; Kaminker, Joshua S; Larrivee, Bruno; Breant, Christiane; Duarte, Antonio; Takakura, Nobuyuki; Fukamizu, Akiyoshi; Penninger, Josef; Eichmann, Anne

2010-11-11

Sprouting of developing blood vessels is mediated by specialized motile endothelial cells localized at the tips of growing capillaries. Following behind the tip cells, endothelial stalk cells form the capillary lumen and proliferate. Expression of the Notch ligand Delta-like-4 (Dll4) in tip cells suppresses tip cell fate in neighboring stalk cells via Notch signaling. In DLL4(+/-) mouse mutants, most retinal endothelial cells display morphologic features of tip cells. We hypothesized that these mouse mutants could be used to isolate tip cells and so to determine their genetic repertoire. Using transcriptome analysis of retinal endothelial cells isolated from DLL4(+/-) and wild-type mice, we identified 3 clusters of tip cell-enriched genes, encoding extracellular matrix degrading enzymes, basement membrane components, and secreted molecules. Secreted molecules endothelial-specific molecule 1, angiopoietin 2, and apelin bind to cognate receptors on endothelial stalk cells. Knockout mice and zebrafish morpholino knockdown of apelin showed delayed angiogenesis and reduced proliferation of stalk cells expressing the apelin receptor APJ. Thus, tip cells may regulate angiogenesis via matrix remodeling, production of basement membrane, and release of secreted molecules, some of which regulate stalk cell behavior.

Altered decorin leads to disrupted endothelial cell function: a possible mechanism in the pathogenesis of fetal growth restriction?

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Chui, A; Murthi, P; Gunatillake, T; Brennecke, S P; Ignjatovic, V; Monagle, P T; Whitelock, J M; Said, J M

2014-08-01

Fetal growth restriction (FGR) is a key cause of adverse pregnancy outcome where maternal and fetal factors are identified as contributing to this condition. Idiopathic FGR is associated with altered vascular endothelial cell functions. Decorin (DCN) has important roles in the regulation of endothelial cell functions in vascular environments. DCN expression is reduced in FGR. The objectives were to determine the functional consequences of reduced DCN in a human microvascular endothelial cell line model (HMVEC), and to determine downstream targets of DCN and their expression in primary placental microvascular endothelial cells (PLECs) from control and FGR-affected placentae. Short-interference RNA was used to reduce DCN expression in HMVECs and the effect on proliferation, angiogenesis and thrombin generation was determined. A Growth Factor PCR Array was used to identify downstream targets of DCN. The expression of target genes in control and FGR PLECs was performed. DCN reduction decreased proliferation and angiogenesis but increased thrombin generation with no effect on apoptosis. The array identified three targets of DCN: FGF17, IL18 and MSTN. Validation of target genes confirmed decreased expression of VEGFA, MMP9, EGFR1, IGFR1 and PLGF in HMVECs and PLECs from control and FGR pregnancies. Reduction of DCN in vascular endothelial cells leads to disrupted cell functions. The targets of DCN include genes that play important roles in angiogenesis and cellular growth. Therefore, differential expression of these may contribute to the pathogenesis of FGR and disease states in other microvascular circulations. Copyright © 2014 Elsevier Ltd. All rights reserved.

Age-related changes in the endocytic capacity of rat liver Kupffer and endothelial cells

International Nuclear Information System (INIS)

Brouwer, A.; Barelds, R.J.; Knook, D.L.

1985-01-01

There are many indications that the functional capacity of the reticuloendothelial system (RES) declines with age. The aim of this study was to investigate the cellular basis of age-related changes in the clearance function of the RES. The experiments were focused mainly on Kupffer and endothelial cells of the liver which represent a major part of the RES and are primarily responsible for clearance of colloidal material from the circulation. The clearance capacity of the RES was tested clinically and experimentally by intravenous injection of colloids, such as radiolabeled heat-aggregated colloidal albumin. Age-related changes in the endocytosis of 125 I-labeled colloidal albumin (CA) in rats were determined by clearance and organ distribution of different doses of intravenously injected CA, uptake of CA by Kupffer and endothelial liver cells in vivo as determined after isolation of the cells from injected rats and kinetic studies on CA uptake by Kupffer cells in culture. The results show that, at a low dose, the clearance of CA is primarily determined by liver blood flow. At a higher saturating dose, plasma clearance and uptake by the liver are not significantly decreased with age. Endocytosis by endothelial cells, which accounts for about 60% of that of the whole liver, is also unchanged with age. In contrast, a significant decrease in endocytic capacity was observed for Kupffer cells in vivo. This age-related functional decline was also observed in Kupffer cells which were isolated from rats of different ages and maintained in culture

Insulin resistance in vascular endothelial cells promotes intestinal tumour formation

DEFF Research Database (Denmark)

Wang, X; Häring, M-F; Rathjen, Thomas

2017-01-01

in vascular endothelial cells. Strikingly, these mice had 42% more intestinal tumours than controls, no change in tumour angiogenesis, but increased expression of vascular cell adhesion molecule-1 (VCAM-1) in primary culture of tumour endothelial cells. Insulin decreased VCAM-1 expression and leukocyte...... adhesion in quiescent tumour endothelial cells with intact insulin receptors and partly prevented increases in VCAM-1 and leukocyte adhesion after treatment with tumour necrosis factor-α. Knockout of insulin receptors in endothelial cells also increased leukocyte adhesion in mesenteric venules...

Endothelial Cells Control Pancreatic Cell Fate at Defined Stages through EGFL7 Signaling

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Der-I Kao

2015-02-01

Full Text Available Although endothelial cells have been shown to affect mouse pancreatic development, their precise function in human development remains unclear. Using a coculture system containing human embryonic stem cell (hESC-derived progenitors and endothelial cells, we found that endothelial cells play a stage-dependent role in pancreatic development, in which they maintain pancreatic progenitor (PP self-renewal and impair further differentiation into hormone-expressing cells. The mechanistic studies suggest that the endothelial cells act through the secretion of EGFL7. Consistently, endothelial overexpression of EGFL7 in vivo using a transgenic mouse model resulted in an increase of PP proliferation rate and a decrease of differentiation toward endocrine cells. These studies not only identified the role of EGFL7 as the molecular handle involved in the crosstalk between endothelium and pancreatic epithelium, but also provide a paradigm for using hESC stepwise differentiation to dissect the stage-dependent roles of signals controlling organogenesis.

Black Tea Increases Circulating Endothelial Progenitor Cells and Improves Flow Mediated Dilatation Counteracting Deleterious Effects from a Fat Load in Hypertensive Patients: A Randomized Controlled Study

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Davide Grassi

2016-11-01

Full Text Available (1 Background: Endothelial dysfunction predicts cardiovascular events. Circulating angiogenic cells (CACs maintain and repair the endothelium regulating its function. Tea flavonoids reduce cardiovascular risk. We investigated the effects of black tea on the number of CACs and on flow-mediated dilation (FMD before and after an oral fat in hypertensives; (2 Methods: In a randomized, double-blind, controlled, cross-over study, 19 patients were assigned to black tea (150 mg polyphenols or a placebo twice a day for eight days. Measurements were obtained in a fasted state and after consuming whipping cream, and FMD was measured at baseline and after consumption of the products; (3 Results: Compared with the placebo, black tea ingestion increased functionally active CACs (36 ± 22 vs. 56 ± 21 cells per high-power field; p = 0.006 and FMD (5.0% ± 0.3% vs. 6.6% ± 0.3%, p < 0.0001. Tea further increased FMD 1, 2, 3, and 4 h after consumption, with maximal response 2 h after intake (p < 0.0001. Fat challenge decreased FMD, while tea consumption counteracted FMD impairment (p < 0.0001; (4 Conclusions: We demonstrated the vascular protective properties of black tea by increasing the number of CACs and preventing endothelial dysfunction induced by acute oral fat load in hypertensive patients. Considering that tea is the most consumed beverage after water, our findings are of clinical relevance and interest.

Bevacizumab inhibits proliferation of choroidal endothelial cells by regulation of the cell cycle.

Science.gov (United States)

Rusovici, Raluca; Patel, Chirag J; Chalam, Kakarla V

2013-01-01

The purpose of this study was to evaluate cell cycle changes in choroidal endothelial cells treated with varying doses of bevacizumab in the presence of a range of concentrations of vascular endothelial growth factor (VEGF). Bevacizumab, a drug widely used in the treatment of neovascular age-related macular degeneration, choroidal neovascularization, and proliferative diabetic retinopathy, neutralizes all isoforms of VEGF. However, the effect of intravitreal administration of bevacizumab on the choroidal endothelial cell cycle has not been established. Monkey choroidal endothelial (RF/6A) cells were treated with VEGF 50 ng/mL and escalating doses of bevacizumab 0.1-2 mg/mL for 72 hours. Cell cycle changes in response to bevacizumab were analyzed by flow cytometry and propidium iodide staining. Cell proliferation was measured using the WST-1 assay. Morphological changes were recorded by bright field cell microscopy. Bevacizumab inhibited proliferation of choroidal endothelial cells by stabilization of the cell cycle in G0/G1 phase. Cell cycle analysis of VEGF-enriched choroidal endothelial cells revealed a predominant increase in the G2/M population (21.84%, P, 0.01) and a decrease in the G0/G1 phase population (55.08%, P, 0.01). Addition of escalating doses of bevacizumab stabilized VEGF-enriched cells in the G0/G1 phase (55.08%, 54.49%, 56.3%, and 64% [P, 0.01]) and arrested proliferation by inhibiting the G2/M phase (21.84%, 21.46%, 20.59%, 20.94%, and 16.1% [P, 0.01]). The increase in G0/G1 subpopulation in VEGF-enriched and bevacizumab-treated cells compared with VEGF-enriched cells alone was dose-dependent. Bevacizumab arrests proliferation of VEGF-enriched choroidal endothelial cells by stabilizing the cell cycle in the G0/G1 phase and inhibiting the G2/M phase in a dose-dependent fashion.

Differential Effects of Leptin and Adiponectin in Endothelial Angiogenesis

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Raghu Adya

2015-01-01

Full Text Available Obesity is a major health burden with an increased risk of cardiovascular morbidity and mortality. Endothelial dysfunction is pivotal to the development of cardiovascular disease (CVD. In relation to this, adipose tissue secreted factors termed “adipokines” have been reported to modulate endothelial dysfunction. In this review, we focus on two of the most abundant circulating adipokines, that is, leptin and adiponectin, in the development of endothelial dysfunction. Leptin has been documented to influence a multitude of organ systems, that is, central nervous system (appetite regulation, satiety factor and cardiovascular system (endothelial dysfunction leading to atherosclerosis. Adiponectin, circulating at a much higher concentration, exists in different molecular weight forms, essentially made up of the collagenous fraction and a globular domain, the latter being investigated minimally for its involvement in proinflammatory processes including activation of NF-κβ and endothelial adhesion molecules. The opposing actions of the two forms of adiponectin in endothelial cells have been recently demonstrated. Additionally, a local and systemic change to multimeric forms of adiponectin has gained importance. Thus detailed investigations on the potential interplay between these adipokines would likely result in better understanding of the missing links connecting CVD, adipokines, and obesity.

Functional and gene expression analysis of hTERT overexpressed endothelial cells

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Haruna Takano

2008-09-01

Full Text Available Haruna Takano1, Satoshi Murasawa1,2, Takayuki Asahara1,2,31Institute of Biomedical Research and Innovation, Kobe, Japan; 2RIKEN Center for Developmental Biology, Kobe 650-0047, Japan; 3Tokai University of School of Medicine, Tokai, JapanAbstract: Telomerase dysfunction contributes to cellular senescence. Recent advances indicate the importance of senescence in maintaining vascular cell function in vitro. Human telomerase reverse transcriptase (hTERT overexpression is thought to lead to resistance to apoptosis and oxidative stress. However, the mechanism in endothelial lineage cells is unclear. We tried to generate an immortal endothelial cell line from human umbilical vein endothelial cells using a no-virus system and examine the functional mechanisms of hTERT overexpressed endothelial cell senescence in vitro. High levels of hTERT genes and endothelial cell-specific markers were expressed during long-term culture. Also, angiogenic responses were observed in hTERT overexpressed endothelial cell. These cells showed a delay in senescence and appeared more resistant to stressed conditions. PI3K/Akt-related gene levels were enhanced in hTERT overexpressed endothelial cells. An up-regulated PI3K/Akt pathway caused by hTERT overexpression might contribute to anti-apoptosis and survival effects in endothelial lineage cells.Keywords: endothelial, telomerase, senescence, oxidative stress, anti-apoptosis, PI3K/Akt pathway

5-Hydroxytryptamine 4 Receptor in the Endothelial Cells

DEFF Research Database (Denmark)

Profirovic, Jasmina; Vardya, Irina; Voyno-Yasenetskaya, Tatyana

2006-01-01

39 5-HYDROXYTRYPTAMINE 4 RECEPTOR IN THE ENDOTHELIAL CELLS. J. Profirovic, I. Vardya, T. Voyno-Yasenetskaya, Department of Pharmacology, University of Illinois at Chicago, Chicago, IL. Serotonin (5-hydroxytryptamine [5-HT]) is an important neurotransmitter that regulates multiple events in the ce......39 5-HYDROXYTRYPTAMINE 4 RECEPTOR IN THE ENDOTHELIAL CELLS. J. Profirovic, I. Vardya, T. Voyno-Yasenetskaya, Department of Pharmacology, University of Illinois at Chicago, Chicago, IL. Serotonin (5-hydroxytryptamine [5-HT]) is an important neurotransmitter that regulates multiple events...... gap formation in HUVECs. We are currently investigating the mechanism underlying 5-HT4 receptor-induced actin cytoskeleton changes in the endothelial cells. These data suggest that by activating 5-HT4 receptor, serotonin could be involved in regulation of actin cytoskeleton dynamics in the endothelial...

Vascular endothelial growth factor modified macrophages transdifferentiate into endothelial-like cells and decrease foam cell formation.

Science.gov (United States)

Yan, Dan; He, Yujuan; Dai, Jun; Yang, Lili; Wang, Xiaoyan; Ruan, Qiurong

2017-06-30

Macrophages are largely involved in the whole process of atherosclerosis from an initiation lesion to an advanced lesion. Endothelial disruption is the initial step and macrophage-derived foam cells are the hallmark of atherosclerosis. Promotion of vascular integrity and inhibition of foam cell formation are two important strategies for preventing atherosclerosis. How can we inhibit even the reverse negative role of macrophages in atherosclerosis? The present study was performed to investigate if overexpressing endogenous human vascular endothelial growth factor (VEGF) could facilitate transdifferentiation of macrophages into endothelial-like cells (ELCs) and inhibit foam cell formation. We demonstrated that VEGF-modified macrophages which stably overexpressed human VEGF (hVEGF 165 ) displayed a high capability to alter their phenotype and function into ELCs in vitro Exogenous VEGF could not replace endogenous VEGF to induce the transdifferentiation of macrophages into ELCs in vitro We further showed that VEGF-modified macrophages significantly decreased cytoplasmic lipid accumulation after treatment with oxidized LDL (ox-LDL). Moreover, down-regulation of CD36 expression in these cells was probably one of the mechanisms of reduction in foam cell formation. Our results provided the in vitro proof of VEGF-modified macrophages as atheroprotective therapeutic cells by both promotion of vascular repair and inhibition of foam cell formation. © 2017 The Author(s).

PECAM-1 polymorphism affects monocyte adhesion to endothelial cells.

Science.gov (United States)

Goodman, Reyna S; Kirton, Christopher M; Oostingh, Gertie J; Schön, Michael P; Clark, Michael R; Bradley, J Andrew; Taylor, Craig J

2008-02-15

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) plays an important role in leukocyte-endothelial cell adhesion and transmigration. Single nucleotide polymorphisms of PECAM-1 encoding amino acid substitutions at positions 98 leucine/valine (L/V), 536 serine/asparagine (S/N), and 643 arginine/glycine (R/G) occur in strong genetic linkage resulting in two common haplotypes (LSR and VNG). These PECAM-1 polymorphisms are associated with graft-versus-host disease after hematopoietic stem cell transplantation and with cardiovascular disease, but whether they influence PECAM-1 function is unknown. We examined the effect of homozygous and heterozygous expression of the PECAM-1 LSR and VNG genotypes on the adhesive interactions of peripheral blood monocytes and activated endothelial cell monolayers under shear stress in a flow-based cell adhesion assay. There was no difference in monocyte adhesion between the two homozygous genotypes of PECAM-1 but when monocytes expressed both alleles in heterozygous form, firm adhesion of monocytes to endothelial cells was markedly increased. PECAM-1 polymorphism expressed in homozygous or heterozygous form by endothelial cells did not influence monocyte adhesion. This is, to our knowledge, the first demonstration that PECAM-1 genotype can alter the level of monocyte binding to endothelial cells and a demonstration that heterozygous expression of a polymorphic protein may lead to altered function.

[Interaction of FABP4 with plasma membrane proteins of endothelial cells].

Science.gov (United States)

Saavedra, Paula; Girona, Josefa; Aragonès, Gemma; Cabré, Anna; Guaita, Sandra; Heras, Mercedes; Masana, Lluís

2015-01-01

Fatty acid binding protein (FABP4) is an adipose tissue-secreted adipokine implicated in the regulation of the energetic metabolism and inflammation. High levels of circulating FABP4 have been described in people with obesity, atherogenic dyslipidemia, diabetes and metabolic syndrome. Recent studies have demonstrated that FABP4 could have a direct effect on peripheral tissues and, specifically, on vascular function. It is still unknown how the interaction between FABP4 and the endothelial cells is produced to prompt these effects on vascular function. The objective of this work is studying the interaction between FABP4 and the plasma membrane proteins of endothelial cells. HUVEC cells were incubated with and without FABP4 (100 ng/ml) for 5 minutes. Immunolocalization of FABP4 was studied by confocal microscopy. The results showed that FABP4 colocalizates with CD31, a membrane protein marker. A strategy which combines 6XHistidine-tag FABP4 (FABP4-His), incubations with or without FABP4-His (100 ng/ml), formaldehyde cross-linking, cellular membrane protein extraction and western blot, was designed to study the FABP4 interactions with membrane proteins of HUVECs. The results showed different western blot profiles depending of the incubation with or without FABP4-His. The immunoblot revelead three covalent protein complexes of about 108, 77 and 33 kDa containing FAPB4 and its putative receptor. The existence of a specific binding protein complex able to bind FABP4 to endothelial cells is supported by these results. The obtained results will permit us advance in the molecular knowledge of FABP4 effects as well as use this protein and its receptor as therapeutic target to prevent cardiovascular. Copyright © 2014 Sociedad Española de Arteriosclerosis. Published by Elsevier España. All rights reserved.

Resveratrol induces mitochondrial biogenesis in endothelial cells.

Science.gov (United States)

Csiszar, Anna; Labinskyy, Nazar; Pinto, John T; Ballabh, Praveen; Zhang, Hanrui; Losonczy, Gyorgy; Pearson, Kevin; de Cabo, Rafael; Pacher, Pal; Zhang, Cuihua; Ungvari, Zoltan

2009-07-01

Pathways that regulate mitochondrial biogenesis are potential therapeutic targets for the amelioration of endothelial dysfunction and vascular disease. Resveratrol was shown to impact mitochondrial function in skeletal muscle and the liver, but its role in mitochondrial biogenesis in endothelial cells remains poorly defined. The present study determined whether resveratrol induces mitochondrial biogenesis in cultured human coronary arterial endothelial cells (CAECs). In CAECs resveratrol increased mitochondrial mass and mitochondrial DNA content, upregulated protein expression of electron transport chain constituents, and induced mitochondrial biogenesis factors (proliferator-activated receptor-coactivator-1alpha, nuclear respiratory factor-1, mitochondrial transcription factor A). Sirtuin 1 (SIRT1) was induced, and endothelial nitric oxide (NO) synthase (eNOS) was upregulated in a SIRT1-dependent manner. Knockdown of SIRT1 (small interfering RNA) or inhibition of NO synthesis prevented resveratrol-induced mitochondrial biogenesis. In aortas of type 2 diabetic (db/db) mice impaired mitochondrial biogenesis was normalized by chronic resveratrol treatment, showing the in vivo relevance of our findings. Resveratrol increases mitochondrial content in endothelial cells via activating SIRT1. We propose that SIRT1, via a pathway that involves the upregulation of eNOS, induces mitochondrial biogenesis. Resveratrol induced mitochondrial biogenesis in the aortas of type 2 diabetic mice, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases.

Ionizing radiation activates vascular endothelial growth factor-A transcription in human umbilical vein endothelial cells

Energy Technology Data Exchange (ETDEWEB)

Lee, Hyounji; Kim, Kwang Seok; Jeong, Jae Hoon; Lim, Young Bin [Radiation Cancer Biology Team, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

2016-12-15

Vascular endothelial growth factor (VEGF) is an essential paracrine factor for developmental and pathological angiogenesis. VEGF also exerts its effects in an autocrine manner in VEGF-producing cells. For instance, autocrine VEGF signaling occurs in tumor cells and contributes to key aspects of tumorigenesis, such as in the function of cancer stem cells and tumor initiation, which are independent of angiogenesis. In addition to tumors cells, non-transformed cells also express VEGF. For example, a VEGF dependent intracellular autocrine mechanism is crucial for the survival of hematopoietic stem cells and hematopoiesis. Stereotactic body radiation therapy (SBRT) is a novel treatment modality for early primary cancer and oligometastatic disease. SBRT delivers high-dose hypofractionated radiation, such as 20-60 Gy, to tumors in a single fraction or 2-5 fractions. As VEGF is a critical regulator of functional integrity and viability of vascular endothelial cells, we examined whether high-dose irradiation alters VEGF signaling by measuring the expression levels of VEGFA transcript. It is generally believed that endothelial cells do not produce VEGF in response to radiation. In present study, however, we provide the first demonstration of transcriptional regulation of VEGFA in human vascular endothelial cells by IR treatment. Irradiation with doses higher than 10 Gy in a single exposure triggers up-regulation of VEGFA transcription within 2 hours in HUVECs, whereas irradiation with 10 Gy does not alter VEGFA levels. Our data have shown that high-dose irradiation triggers immediate transactivation of VEGFA in human vascular endothelial cells.

Efficient Generation of Human Embryonic Stem Cell-Derived Corneal Endothelial Cells by Directed Differentiation.

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Kathryn L McCabe

Full Text Available To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs for transplantation in patients with corneal endothelial dystrophies.Feeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology, expression of corneal endothelial markers, and microarray analysis of gene expression.hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1 and Na+/K+ATPaseα1 (ATPA1 on the apical surface in monolayer culture, and produced the key proteins of Descemet's membrane, Collagen VIIIα1 and VIIIα2 (COL8A1 and 8A2. Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis.hESC-CECs are morphologically similar, express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium.

Lipoprotein receptors in cultured bovine endothelial cells

International Nuclear Information System (INIS)

Struempfer, A.E.M.

1983-07-01

In this study, receptors that may be involved in the uptake of low density lipoproteins (LDL) and low density lipoproteins which have been modified by acetylation (AcLDL), were characterized. Aortic epithelial cells were used and a cell culture system which closely resembled the in vivo monolayer was established. Endothelial cell and lipoprotein interactions were examined by incubating the cells with 125 l-labelled lipoproteins under various conditions. The receptor affinity of bovine aortic endothelial cells was higher for AcLDL than that for LDL. Competition studies demonstrated that there were two distinct receptors for LDL and AcLDL on the endothelial cells. AcLDL did not compete with LDL for the LDL receptor, and conversely LDL did not compete with AcLDL for the AcLDL receptor. The receptor activities for LDL and AcLDL were examined as a function of culture age. Whereas the LDL receptor could be regulated, the AcLDL receptor was not as susceptible to regulation. Upon exposing endothelial cells for 72 h to either LDL or AcLDL, it was found that the total amount of cellular cholesterol increased by about 50%. However, the increase of total cholesterol was largely in the form of free cholesterol. This is in contrast to macrophages, where the increase in total cholesterol upon exposure to AcLDL is largely in the form cholesteryl esters

Characterization of fetal cells from the maternal circulation by microarray gene expression analysis - Could the extravillous trophoblasts be a target for future cell-based non-invasive prenatal diagnosis?

DEFF Research Database (Denmark)

Hatt, Lotte; Brinch, Marie; Singh, Ripudaman

2014-01-01

stem cell microarray analysis. Results: 39 genes were identified as candidates for unique fetal cell markers. More than half of these are genes known to be expressed in the placenta, especially in extravillous trophoblasts (EVTs). Immunohistochemical staining of placental tissue confirmed CD105......Introduction: Circulating fetal cells in maternal blood provide a tool for risk-free, non-invasive prenatal diagnosis. However, fetal cells in the maternal circulation are scarce, and to effectively isolate enough of them for reliable diagnostics, it is crucial to know which fetal cell type......(s) should be targeted. Materials and Methods: Fetal cells were enriched from maternal blood by magnetic-activated cell sorting using the endothelial cell marker CD105 and identified by XY fluorescence in situ hybridization. Expression pattern was compared between fetal cells and maternal blood cells using...

The Expression Profiles of Lysophospholipid Receptors (LPLRs in Different Endothelial Cells

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Yu-Wei Lee

2006-03-01

Full Text Available Sphingosine-1-phosphate (S1P and lysophosphatidic acid (LPA are two bioactive lysophospholipids (LPLs, stored primarily in platelets and released during platelet activation. Both LPLs are capable of regulating endothelial cell functions. The physiological functions of S1P and LPA are mediated by interacting with eight different G-protein coupled receptors: S1P1 through 5 and LPA1 through 3, which activate three different heterotrimeric GTP proteins－including Gi、Gq and G(12/13. The expression of LPL receptors in endothelial cells would affect the responses of S1P and LPA to these cells. There is no previous report discussing the expression profiles of LPL receptors in different endothelial cells from various species. In this study, we aim to investigate the expression profiles of S1P and LPA receptors in different endothelial cells isolated from human, rat, mouse and bovine origin. We used RT-PCR to determine LPLs receptors expression profiles in different endothelial cells. Our results indicated that endothelial cells from various species express different LPL receptors. Endothelial cells isolated from the same source of different species also had different LPLs receptors expression profiles. Therefore, different endothelial cells should respond to LPLs in different manners.

Fabrication of endothelial progenitor cell capture surface via DNA aptamer modifying dopamine/polyethyleneimine copolymer film

Energy Technology Data Exchange (ETDEWEB)

Li, Xin; Deng, Jinchuan; Yuan, Shuheng; Wang, Juan; Luo, Rifang; Chen, Si [Key Lab. of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China); School of Material Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Wang, Jin, E-mail: jinxxwang@263.net [Key Lab. of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China); Huang, Nan [Key Lab. of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China); School of Material Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China)

2016-11-15

Highlights: • The dopamine/PEI film with controlled amine density was successfully prepared. • The DNA aptamer was assembled onto the film via electrostatic incorporation. • The A@DPfilmscanspecificallyandeffectivelycaptureEPCs. • The A@DP film can support the survival of ECs, control the hyperplasia of SMCs. • The dynamic/co-culture models are useful for studying cells competitive adhesion. - Abstract: Endothelial progenitor cells (EPCs) are mainly located in bone marrow and circulate, and play a crucial role in repairmen of injury endothelium. One of the most promising strategies of stents designs were considered to make in-situ endothelialization in vivo via EPC-capture biomolecules on a vascular graft to capture EPCs directly from circulatory blood. In this work, an EPC specific aptamer with a 34 bases single strand DNA sequence was conjugated onto the stent surface via dopamine/polyethyleneimine copolymer film as a platform and linker. The assembled density of DNA aptamer could be regulated by controlling dopamine percentage in this copolymer film. X-ray photoelectron spectroscopy (XPS), water contact angle (WCA) and fluorescence test confirmed the successful immobilization of DNA aptamer. To confirm its biofunctionality and cytocompatibility, the capturing cells ability of the aptamer modified surface and the effects on the growth behavior of human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) were investigated. The aptamer functionalized sample revealed a good EPC-capture ability, and had a cellular friendly feature for both EPC and EC growth, while not stimulated the hyperplasia of SMCs. And, the co-culture experiment of three types of cells confirmed the specificity capturing of EPCs to aptamer modified surface, rather than ECs and SMCs. These data suggested that this aptamer functionalized surface may have a large potentiality for the application of vascular grafts with targeted endothelialization.

An analysis of endothelial microparticles as a function of cell surface antibodies and centrifugation techniques.

Science.gov (United States)

Venable, Adam S; Williams, Randall R; Haviland, David L; McFarlin, Brian K

2014-04-01

Chronic vascular disease is partially characterized by the presence of lesions along the vascular endothelial wall. Current FDA-approved clinical techniques lack the ability to measure very early changes in endothelial cell health. When endothelial cells are damaged, they release endothelial microparticles (EMPs) into circulation. Thus, blood EMP concentration may represent a useful cardiovascular disease biomarker. Despite the potential value of EMPs, current flow cytometry techniques may not consistently distinguish EMPs from other small cell particles. The purpose of this study was to use imaging flow cytometry to modify existing methods of identifying EMPs based on cell-surface receptor expression and visual morphology. Platelet poor plasma (PPP) was isolated using four different techniques, each utilizing a two-step serial centrifugation process. The cell-surface markers used in this study were selected based on those that are commonly reported in the literature. PPP (100μL) was labeled with CD31, CD42a, CD45, CD51, CD66b, and CD144 for 30-min in dark on ice. Based on replicated experiments, EMPs were best identified by cell-surface CD144 expression relative to other commonly reported EMP markers (CD31 & CD51). It is important to note that contaminating LMPs, GMPs, and PMPs were thought to be removed in the preparation of PPP. However, upon analysis of prepared samples staining CD31 against CD51 revealed a double-positive population that was less than 1% EMPs. In contrast, when using CD144 to identify EMPs, ~87% of observed particles were free of contaminating microparticles. Using a counterstain of CD42a, this purity can be improved to over 99%. More research is needed to understand how our improved EMP measurement method can be used in experimental models measuring acute vascular responses or chronic vascular diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

Fabrication of endothelial progenitor cell capture surface via DNA aptamer modifying dopamine/polyethyleneimine copolymer film

International Nuclear Information System (INIS)

Li, Xin; Deng, Jinchuan; Yuan, Shuheng; Wang, Juan; Luo, Rifang; Chen, Si; Wang, Jin; Huang, Nan

2016-01-01

Highlights: • The dopamine/PEI film with controlled amine density was successfully prepared. • The DNA aptamer was assembled onto the film via electrostatic incorporation. • The A@DPfilmscanspecificallyandeffectivelycaptureEPCs. • The A@DP film can support the survival of ECs, control the hyperplasia of SMCs. • The dynamic/co-culture models are useful for studying cells competitive adhesion. - Abstract: Endothelial progenitor cells (EPCs) are mainly located in bone marrow and circulate, and play a crucial role in repairmen of injury endothelium. One of the most promising strategies of stents designs were considered to make in-situ endothelialization in vivo via EPC-capture biomolecules on a vascular graft to capture EPCs directly from circulatory blood. In this work, an EPC specific aptamer with a 34 bases single strand DNA sequence was conjugated onto the stent surface via dopamine/polyethyleneimine copolymer film as a platform and linker. The assembled density of DNA aptamer could be regulated by controlling dopamine percentage in this copolymer film. X-ray photoelectron spectroscopy (XPS), water contact angle (WCA) and fluorescence test confirmed the successful immobilization of DNA aptamer. To confirm its biofunctionality and cytocompatibility, the capturing cells ability of the aptamer modified surface and the effects on the growth behavior of human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) were investigated. The aptamer functionalized sample revealed a good EPC-capture ability, and had a cellular friendly feature for both EPC and EC growth, while not stimulated the hyperplasia of SMCs. And, the co-culture experiment of three types of cells confirmed the specificity capturing of EPCs to aptamer modified surface, rather than ECs and SMCs. These data suggested that this aptamer functionalized surface may have a large potentiality for the application of vascular grafts with targeted endothelialization.

Endothelial Induced EMT in Breast Epithelial Cells with Stem Cell Properties

OpenAIRE

Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla; Fridriksdottir, Agla J. R.; Ringnér, Markus; Villadsen, Rene; Borg, Ake; Agnarsson, Bjarni A.; Petersen, Ole William; Magnusson, Magnus K.; Gudjonsson, Thorarinn

2011-01-01

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell proper...

A Cell Culture Platform to Maintain Long-term Phenotype of Primary Human Hepatocytes and Endothelial Cells.

Science.gov (United States)

Ware, Brenton R; Durham, Mitchell J; Monckton, Chase P; Khetani, Salman R

2018-03-01

Modeling interactions between primary human hepatocytes (PHHs) and primary human liver sinusoidal endothelial cells (LSECs) in vitro can help elucidate human-specific mechanisms underlying liver physiology/disease and drug responses; however, existing hepatocyte/endothelial coculture models are suboptimal because of their use of rodent cells, cancerous cell lines, and/or nonliver endothelial cells. Hence, we sought to develop a platform that could maintain the long-term phenotype of PHHs and primary human LSECs. Primary human LSECs or human umbilical vein endothelial cells as the nonliver control were cocultivated with micropatterned PHH colonies (to control homotypic interactions) followed by an assessment of PHH morphology and functions (albumin and urea secretion, and cytochrome P-450 2A6 and 3A4 enzyme activities) over 3 weeks. Endothelial phenotype was assessed via gene expression patterns and scanning electron microscopy to visualize fenestrations. Hepatic responses in PHH/endothelial cocultures were benchmarked against responses in previously developed PHH/3T3-J2 fibroblast cocultures. Finally, PHH/fibroblast/endothelial cell tricultures were created and characterized as described previously. LSECs, but not human umbilical vein endothelial cells, induced PHH albumin secretion for ∼11 days; however, neither endothelial cell type could maintain PHH morphology and functions to the same magnitude/longevity as the fibroblasts. In contrast, both PHHs and endothelial cells displayed stable phenotype for 3 weeks in PHH/fibroblast/endothelial cell tricultures; furthermore, layered tricultures in which PHHs and endothelial cells were separated by a protein gel to mimic the space of Disse displayed similar functional levels as the coplanar tricultures. PHH/fibroblast/endothelial tricultures constitute a robust platform to elucidate reciprocal interactions between PHHs and endothelial cells in physiology, disease, and after drug exposure.

Effect of bFGF on radiation-induced apoptosis of vascular endothelial cells

International Nuclear Information System (INIS)

Gu Qingyang; Wang Dewen; Li Yuejuan; Peng Ruiyun; Dong Bo; Wang Zhaohai; Liu Jie; Deng Hua; Jiang Tao

2003-01-01

Objective: To study the effect of bFGF on radiation-induced apoptosis vascular endothelial cells. Methods: A cell line PAE (porcine aortic endothelial cells) and primary cultured HUVEC (human umbilical vein endothelial cells) were irradiated with 60 Co γ-rays to establish cell apoptosis models. Flow cytometry with annexin-V-FITC + PI labeling was used to evaluate cell apoptosis. Different amounts of bFGF were used to study their effects on radiation-induced endothelial cell apoptosis. Results and Conclusions: It is found that bFGF could inhibit radiation-induced endothelial cell apoptosis in a considerable degree

Radiation-induced inhibition of human endothelial cells replicating in culture

International Nuclear Information System (INIS)

DeGowin, R.L.; Lewis, L.J.; Mason, R.E.; Borke, M.K.; Hoak, J.C.

1976-01-01

The radiosensitivity of some tumors may depend upon the sensitivity of their microvasculature to radiation. Heretofore, the dose-response of human endothelial cells replicating in tissue culture has not been published. In studies reported here, we exposed flasks containing 4 to 7 x 10 4 genetically identical human endothelial cells to doses of x irradiation from 125 to 1000 rad. During the phase of logarithmic growth, cell counts were compared to those of an unirradiated control to construct a dose--response curve. Similar studies were performed with normal fibroblasts. We found that 160 rad suppressed endothelial cell replication by 37 percent. Although recovery was evident with doses of 500 rad, no net increase in cell number occurred in 3 weeks in flasks of endothelial cells that received 750 or 1000 rad. Fibroblasts were slightly less sensitive under these conditions. To our knowledge, this is the first report of a radiation dose--response curve for human endothelial cells replicating in culture

The influence of biomaterials on endothelial cell thrombogenicity

Science.gov (United States)

McGuigan, Alison P.; Sefton, Michael V.

2007-01-01

Driven by tissue engineering and regenerative medicine, endothelial cells are being used in combination with biomaterials in a number of applications for the purpose of improving blood compatibility and host integration. Endothelialized vascular grafts are beginning to be used clinically with some success in some centers, while endothelial seeding is being explored as a means of creating a vasculature within engineered tissues. The underlying assumption of this strategy is that when cultured on artificial biomaterials, a confluent layer of endothelial cells maintain their non-thrombogenic phenotype. In this review the existing knowledge base of endothelial cell thrombogenicity cultured on a number of different biomaterials is summarized. The importance of selecting appropriate endpoint measures that are most reflective of overall surface thrombogenicity is the focus of this review. Endothelial cells inhibit thrombosis through three interconnected regulatory systems (1) the coagulation cascade (2) the cellular components of the blood such as leukocytes and platelets and (3) the complement cascade, and also through effects on fibrinolysis and vascular tone, the latter which influences blood flow. Thus, in order to demonstrate the thromobgenic benefit of seeding a biomaterial with EC, the conditions under which EC surfaces are more likely to exhibit lower thrombogenicity than unseeded biomaterial surfaces need to be consistent with the experimental context. The endpoints selected should be appropriate for the dominant thrombotic process that occurs under the given experimental conditions. PMID:17316788

Plasma factors in severe early-onset preeclampsia do not substantially alter endothelial gene expression in vitro

NARCIS (Netherlands)

Donker, RB; Asgeirsdottir, SA; Gerbens, F; van Pamus, MG; Kallenberg, CGM; Meerman, GJT; Aarnoudse, JG; Molema, G

OBJECTIVE: Systemic endothelial dysfunction is a central feature in the pathophysiology of preeclampsia. Its cell biologic and molecular basis is poorly understood. One leading hypothesis argues that endothelial dysfunction is caused by (at present largely unknown) circulating factors released from

Effects of Hypoglycemia on Circulating Stem and Progenitor Cells in Diabetic Patients.

Science.gov (United States)

Fadini, Gian Paolo; Boscari, Federico; Cappellari, Roberta; Galasso, Silvia; Rigato, Mauro; Bonora, Benedetta Maria; D'Anna, Marianna; Bruttomesso, Daniela; Avogaro, Angelo

2018-03-01

Iatrogenic hypoglycemia is the most common acute diabetic complication, and it significantly increases morbidity. In people with diabetes, reduction in the levels of circulating stem and progenitor cells predicts adverse outcomes. To evaluate whether hypoglycemia in diabetes affects circulating stem cells and endothelial progenitor cells (EPCs). We performed an experimental hypoglycemia study (Study 1) and a case-control study (Study 2). Tertiary referral inpatient clinic. Type 1 diabetic patients (Study 1, n = 19); diabetic patients hospitalized for severe iatrogenic hypoglycemia, matched inpatient and outpatient controls (Study 2, n = 22/group). Type 1 diabetic patients underwent two in-hospital sessions of glucose monitoring during a breakfast meal with or without induction of hypoglycemia in random order. In Study 2, patients hospitalized for hypoglycemia and matched controls were compared. Circulating stem cells and EPCs were measured by flow cytometry based on the expression of CD34 and kinase insert domain receptor (KDR). In Study 1, the physiologic decline of CD34+KDR+ EPCs from 8 am to 2 pm was abolished by insulin-induced hypoglycemia in type 1 diabetic patients. In Study 2, diabetic patients hospitalized for severe iatrogenic hypoglycemia had significantly lower levels of CD34+ stem cells and CD34+KDR+ EPCs compared with diabetic inpatients or outpatient controls. In diabetic patients, a single mild hypoglycemic episode can compromise the physiologic EPC fluctuation, whereas severe hypoglycemia is associated with a marked reduction in stem cells and EPCs. These data provide a possible link between hypoglycemia and adverse outcomes of diabetes.

Molecular and functional characterization of riboflavin specific transport system in rat brain capillary endothelial cells

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Patel, Mitesh; Vadlapatla, Ramya Krishna; Pal, Dhananjay; Mitra, Ashim K.

2012-01-01

Riboflavin is an important water soluble vitamin (B2) required for metabolic reactions, normal cellular growth, differentiation and function. Mammalian brain cells cannot synthesize riboflavin and must import from systemic circulation. However, the uptake mechanism, cellular translocation and intracellular trafficking of riboflavin in brain capillary endothelial cells are poorly understood. The primary objective of this study is to investigate the existence of riboflavin-specific transport system and delineate the uptake and intracellular regulation of riboflavin in immortalized rat brain capillary endothelial cells (RBE4). The uptake of [3H]-Riboflavin is sodium, temperature and energy dependent but pH independent. [3H]-Riboflavin uptake is saturable with Km and Vmax values of 19 ± 3 µM and 0.235 ± 0.012 picomoles/min/mg protein, respectively. The uptake process is inhibited by unlabelled structural analogs (lumiflavin, lumichrome) but not by structurally unrelated vitamins. Ca++/calmodulin and protein kinase A (PKA) pathways are found to play an important role in the intracellular regulation of [3H]-Riboflavin. Apical and baso-lateral uptake of [3H]-Riboflavin clearly indicate that riboflavin specific transport system is predominantly localized on the apical side of RBE4 cells. A 628 bp band corresponding to riboflavin transporter is revealed in RT-PCR analysis. These findings, for the first time report the existence of a specialized and high affinity transport system for riboflavin in RBE4 cells. Blood-brain barrier (BBB) is a major obstacle limiting drug transport inside the brain as it regulates drug permeation from systemic circulation. This transporter can be utilized for targeted delivery in enhancing brain permeation of highly potent drugs on systemic administration. PMID:22683359

Successful in vitro expansion and differentiation of cord blood derived CD34+ cells into early endothelial progenitor cells reveals highly differential gene expression.

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Ingo Ahrens

Full Text Available Endothelial progenitor cells (EPCs can be purified from peripheral blood, bone marrow or cord blood and are typically defined by a limited number of cell surface markers and a few functional tests. A detailed in vitro characterization is often restricted by the low cell numbers of circulating EPCs. Therefore in vitro culturing and expansion methods are applied, which allow at least distinguishing two different types of EPCs, early and late EPCs. Herein, we describe an in vitro culture technique with the aim to generate high numbers of phenotypically, functionally and genetically defined early EPCs from human cord blood. Characterization of EPCs was done by flow cytometry, immunofluorescence microscopy, colony forming unit (CFU assay and endothelial tube formation assay. There was an average 48-fold increase in EPC numbers. EPCs expressed VEGFR-2, CD144, CD18, and CD61, and were positive for acetylated LDL uptake and ulex lectin binding. The cells stimulated endothelial tube formation only in co-cultures with mature endothelial cells and formed CFUs. Microarray analysis revealed highly up-regulated genes, including LL-37 (CAMP, PDK4, and alpha-2-macroglobulin. In addition, genes known to be associated with cardioprotective (GDF15 or pro-angiogenic (galectin-3 properties were also significantly up-regulated after a 72 h differentiation period on fibronectin. We present a novel method that allows to generate high numbers of phenotypically, functionally and genetically characterized early EPCs. Furthermore, we identified several genes newly linked to EPC differentiation, among them LL-37 (CAMP was the most up-regulated gene.

CORNEAL ENDOTHELIAL CELL DENSITY IN ACUTE ANGLE CLOSURE GLAUCOMA

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Nishat Sultana K

2016-09-01

Full Text Available BACKGROUND Angle closure is characterised by apposition of the peripheral iris against the trabecular meshwork resulting in obstruction of aqueous outflow. Acute angle-closure glaucoma is characterised by pain, redness and blurred vision. The pain is typically a severe deep ache that follows the trigeminal distribution and maybe associated with nausea, vomiting, bradycardia and profuse sweating. The blurred vision, which is typically marked maybe caused by stretching of the corneal lamellae initially and later oedema of the cornea as well as a direct effect of the IOP on the optic nerve head. The modifications in corneal endothelial cell density after a crisis of angle-closure glaucoma is being evaluated. AIMS AND OBJECTIVES The objective of the study is to assess the corneal endothelial cell count (density by specular microscopy in patients presenting with acute angle-closure glaucoma. METHODS Corneal endothelial cell counts of 20 eyes of patients with PACG with an earlier documented symptomatic acute attack unilaterally were compared with 20 fellow eyes. Evaluation of patient included visual acuity, intraocular pressure, gonioscopy, disc findings and specular microscopy. RESULTS The mean endothelial cell density was 2104 cells/mm2 in the eye with acute attack and 2615 cells/mm2 in the fellow eye. The average endothelial cell count when the duration of attack lasted more than 72 hours was 1861 cells/mm2 . CONCLUSION Corneal endothelial cell density was found to be significantly reduced in eyes following an acute attack of primary angle closure glaucoma.

Assessing Clinical Outcomes in Colorectal Cancer with Assays for Invasive Circulating Tumor Cells.

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Zhang, Yue; Zarrabi, Kevin; Hou, Wei; Madajewicz, Stefan; Choi, Minsig; Zucker, Stanley; Chen, Wen-Tien

2018-06-06

Colorectal carcinoma (CRC) is the second leading cause of cancer-related mortality. The goals of this study are to evaluate the association between levels of invasive circulating tumor cells (iCTCs) with CRC outcomes and to explore the molecular characteristics of iCTCs. Peripheral blood from 93 patients with Stage I⁻IV CRC was obtained and assessed for the detection and characterization of iCTCs using a functional collagen-based adhesion matrix (CAM) invasion assay. Patients were followed and assessed for overall survival. Tumor cells isolated by CAM were characterized using cell culture and microarray analyses. Of 93 patients, 88 (95%) had detectable iCTCs, ranging over 0⁻470 iCTCs/mL. Patients with Stage I⁻IV disease exhibited median counts of 0.0 iCTCs/mL ( n = 6), 13.0 iCTCs/mL ( n = 12), 41.0 iCTCs/mL ( n = 12), and 133.0 iCTCs/mL ( n = 58), respectively ( p < 0.001). Kaplan⁻Meier curve analysis demonstrated a significant survival benefit in patients with low iCTC counts compared with in patients with high iCTC counts (log-rank p < 0.001). Multivariable Cox model analysis revealed that iCTC count was an independent prognostic factor of overall survival ( p = 0.009). Disease stage ( p = 0.01, hazard ratio 1.66; 95% confidence interval: 1.12⁻2.47) and surgical intervention ( p = 0.03, HR 0.37; 95% CI: 0.15⁻0.92) were also independent prognostic factors. Gene expression analysis demonstrated the expression of both endothelial and tumor progenitor cell biomarkers in iCTCs. CAM-based invasion assay shows a high detection sensitivity of iCTCs that inversely correlated with overall survival in CRC patients. Functional and gene expression analyses showed the phenotypic mosaics of iCTCs, mimicking the survival capability of circulating endothelial cells in the blood stream.

Extracellular histones disarrange vasoactive mediators release through a COX-NOS interaction in human endothelial cells.

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Pérez-Cremades, Daniel; Bueno-Betí, Carlos; García-Giménez, José Luis; Ibañez-Cabellos, José Santiago; Hermenegildo, Carlos; Pallardó, Federico V; Novella, Susana

2017-08-01

Extracellular histones are mediators of inflammation, tissue injury and organ dysfunction. Interactions between circulating histones and vascular endothelial cells are key events in histone-mediated pathologies. Our aim was to investigate the implication of extracellular histones in the production of the major vasoactive compounds released by human endothelial cells (HUVECs), prostanoids and nitric oxide (NO). HUVEC exposed to increasing concentrations of histones (0.001 to 100 μg/ml) for 4 hrs induced prostacyclin (PGI2) production in a dose-dependent manner and decreased thromboxane A2 (TXA2) release at 100 μg/ml. Extracellular histones raised cyclooxygenase-2 (COX-2) and prostacyclin synthase (PGIS) mRNA and protein expression, decreased COX-1 mRNA levels and did not change thromboxane A2 synthase (TXAS) expression. Moreover, extracellular histones decreased both, eNOS expression and NO production in HUVEC. The impaired NO production was related to COX-2 activity and superoxide production since was reversed after celecoxib (10 μmol/l) and tempol (100 μmol/l) treatments, respectively. In conclusion, our findings suggest that extracellular histones stimulate the release of endothelial-dependent mediators through an up-regulation in COX-2-PGIS-PGI2 pathway which involves a COX-2-dependent superoxide production that decreases the activity of eNOS and the NO production. These effects may contribute to the endothelial cell dysfunction observed in histone-mediated pathologies. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

The interaction between circulating complement proteins and cutaneous microvascular endothelial cells in the development of childhood Henoch-Schonlein Purpura.

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Yao-Hsu Yang

Full Text Available In addition to IgA, the deposition of complement (C3 in dermal vessels is commonly found in Henoch-Schönlein purpura (HSP. The aim of this study is to elucidate the role of circulating complement proteins in the pathogenesis of childhood HSP.Plasma levels of C3a, C4a, C5a, and Bb in 30 HSP patients and 30 healthy controls were detected by enzyme-linked immunosorbent assay (ELISA. The expression of C3a receptor (C3aR, C5a receptor (CD88, E-selectin, intercellular adhesion molecule 1 (ICAM-1, C3, C5, interleukin (IL-8, monocyte chemotactic protein (MCP-1, and RANTES by human dermal microvascular endothelial cells (HMVEC-d was evaluated either by flow cytometry or by ELISA.At the acute stage, HSP patients had higher plasma levels of C3a (359.5 ± 115.3 vs. 183.3 ± 94.1 ng/ml, p < 0.0001, C5a (181.4 ± 86.1 vs. 33.7 ± 26.3 ng/ml, p < 0.0001, and Bb (3.7 ± 2.6 vs. 1.0 ± 0.6 μg/ml, p < 0.0001, but not C4a than healthy controls. Although HSP patient-derived acute phase plasma did not alter the presentation of C3aR and CD88 on HMVEC-d, it enhanced the production of endothelial C3 and C5. Moreover, C5a was shown in vitro to up-regulate the expression of IL-8, MCP-1, E-selectin, and ICAM-1 by HMVEC-d with a dose-dependent manner.In HSP, the activation of the complement system in part through the alternative pathway may have resulted in increased plasma levels of C3a and C5a, which, especially C5a, may play a role in the disease pathogenesis by activating endothelium of cutaneous small vessels.

Blood on the tracks: hematopoietic stem cell-endothelial cell interactions in homing and engraftment.

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Perlin, Julie R; Sporrij, Audrey; Zon, Leonard I

2017-08-01

Cells of the hematopoietic system undergo rapid turnover. Each day, humans require the production of about one hundred billion new blood cells for proper function. Hematopoietic stem cells (HSCs) are rare cells that reside in specialized niches and are required throughout life to produce specific progenitor cells that will replenish all blood lineages. There is, however, an incomplete understanding of the molecular and physical properties that regulate HSC migration, homing, engraftment, and maintenance in the niche. Endothelial cells (ECs) are intimately associated with HSCs throughout the life of the stem cell, from the specialized endothelial cells that give rise to HSCs, to the perivascular niche endothelial cells that regulate HSC homeostasis. Recent studies have dissected the unique molecular and physical properties of the endothelial cells in the HSC vascular niche and their role in HSC biology, which may be manipulated to enhance hematopoietic stem cell transplantation therapies.

Circulating levels of cell-derived microparticles are reduced by mild hypobaric hypoxia: data from a randomised controlled trial.

Science.gov (United States)

Ayers, Lisa; Stoewhas, Anne-Christin; Ferry, Berne; Latshang, Tsogyal D; Lo Cascio, Christian M; Sadler, Ross; Stadelmann, Katrin; Tesler, Noemi; Huber, Reto; Achermann, Peter; Bloch, Konrad E; Kohler, Malcolm

2014-05-01

Hypoxia is known to induce the release of microparticles in vitro. However, few publications have addressed the role of hypoxia in vivo on circulating levels of microparticles. This randomised, controlled, crossover trial aimed to determine the effect of mild hypoxia on in vivo levels of circulating microparticles in healthy individuals. Blood was obtained from 51 healthy male volunteers (mean age of 26.9 years) at baseline altitude (490 m) and after 24 and 48 h at moderate altitude (2,590 m). The order of altitude exposure was randomised. Flow cytometry was used to assess platelet-poor plasma for levels of circulating microparticles derived from platelets, endothelial cells, leucocytes, granulocytes, monocytes, red blood cells and procoagulant microparticles. Mean (standard deviation) oxygen saturation was significantly lower on the first and second day after arrival at 2,590 m, 91.0 (2.0) and 92.0 (2.0) %, respectively, compared to 490 m, 96 (1.0) %, p microparticles (annexin V+ -221/μl 95 % CI -370.8/-119.0, lactadherin+ -202/μl 95 % CI -372.2/-93.1), platelet-derived microparticles (-114/μl 95 % CI -189.9/-51.0) and red blood cell-derived microparticles (-81.4 μl 95 % CI -109.9/-57.7) after 48 h at moderate altitude was found. Microparticles derived from endothelial cells, granulocytes, monocytes and leucocytes were not significantly altered by exposure to moderate altitude. In healthy male individuals, mild hypobaric hypoxia, induced by a short-term stay at moderate altitude, is associated with lower levels of procoagulant microparticles, platelet-derived microparticles and red blood cell-derived microparticles, suggesting a reduction in thrombotic potential.

Endothelial biocompatibility and accumulation of SPION under flow conditions

International Nuclear Information System (INIS)

Matuszak, Jasmin; Zaloga, Jan; Friedrich, Ralf P.; Lyer, Stefan; Nowak, Johannes; Odenbach, Stefan; Alexiou, Christoph; Cicha, Iwona

2015-01-01

Magnetic targeting is considered a promising method to accumulate the nanoparticles at the sites of atherosclerotic lesions, but little is known about the biological effects of magnetic nanoparticles on the vascular wall. Here, we investigated endothelial cell growth and vitality upon treatment with SPION (0–60 µg/mL) using two complementing methods: real-time cell analysis and live-cell microscopy. Moreover, the uptake of circulating superparamagnetic iron oxide nanoparticles (SPIONs) was assessed in an in vitro model of arterial bifurcations. At the tested concentrations, SPIONs were well tolerated and had no major influence on endothelial cell growth. Our results further showed a uniform distribution of endothelial SPION uptake independent of channel geometry or hemodynamic conditions: In the absence of magnetic force, no increase in accumulation of SPIONs at non-uniform shear stress region at the outer walls of bifurcation was observed. Application of external magnet allowed enhanced accumulation of SPIONs at the regions of non-uniform shear stress. Increased uptake of SPIONs at non-uniform shear stress region was well tolerated by endothelial cells (ECs) and did not affect endothelial cell viability or attachment. These findings indicate that magnetic targeting can constitute a promising and safe technique for the delivery of imaging and therapeutic nanoparticles to atherosclerotic lesions

Endothelial biocompatibility and accumulation of SPION under flow conditions

Energy Technology Data Exchange (ETDEWEB)

Matuszak, Jasmin; Zaloga, Jan; Friedrich, Ralf P.; Lyer, Stefan [Section of Experimental Oncology and Nanomedicine (SEON), Else Kröner-Fresenius Stiftungsprofessur for Nanomedicine, University Hospital Erlangen, Erlangen (Germany); Nowak, Johannes; Odenbach, Stefan [Chair of Magnetofluiddynamics, Measuring and Automation Technology, Technische Universität Dresden, Dresden (Germany); Alexiou, Christoph [Section of Experimental Oncology and Nanomedicine (SEON), Else Kröner-Fresenius Stiftungsprofessur for Nanomedicine, University Hospital Erlangen, Erlangen (Germany); Cicha, Iwona, E-mail: Iwona_Cicha@yahoo.com [Section of Experimental Oncology and Nanomedicine (SEON), Else Kröner-Fresenius Stiftungsprofessur for Nanomedicine, University Hospital Erlangen, Erlangen (Germany)

2015-04-15

Magnetic targeting is considered a promising method to accumulate the nanoparticles at the sites of atherosclerotic lesions, but little is known about the biological effects of magnetic nanoparticles on the vascular wall. Here, we investigated endothelial cell growth and vitality upon treatment with SPION (0–60 µg/mL) using two complementing methods: real-time cell analysis and live-cell microscopy. Moreover, the uptake of circulating superparamagnetic iron oxide nanoparticles (SPIONs) was assessed in an in vitro model of arterial bifurcations. At the tested concentrations, SPIONs were well tolerated and had no major influence on endothelial cell growth. Our results further showed a uniform distribution of endothelial SPION uptake independent of channel geometry or hemodynamic conditions: In the absence of magnetic force, no increase in accumulation of SPIONs at non-uniform shear stress region at the outer walls of bifurcation was observed. Application of external magnet allowed enhanced accumulation of SPIONs at the regions of non-uniform shear stress. Increased uptake of SPIONs at non-uniform shear stress region was well tolerated by endothelial cells (ECs) and did not affect endothelial cell viability or attachment. These findings indicate that magnetic targeting can constitute a promising and safe technique for the delivery of imaging and therapeutic nanoparticles to atherosclerotic lesions.

A fully automated cell segmentation and morphometric parameter system for quantifying corneal endothelial cell morphology.

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Al-Fahdawi, Shumoos; Qahwaji, Rami; Al-Waisy, Alaa S; Ipson, Stanley; Ferdousi, Maryam; Malik, Rayaz A; Brahma, Arun

2018-07-01

Corneal endothelial cell abnormalities may be associated with a number of corneal and systemic diseases. Damage to the endothelial cells can significantly affect corneal transparency by altering hydration of the corneal stroma, which can lead to irreversible endothelial cell pathology requiring corneal transplantation. To date, quantitative analysis of endothelial cell abnormalities has been manually performed by ophthalmologists using time consuming and highly subjective semi-automatic tools, which require an operator interaction. We developed and applied a fully-automated and real-time system, termed the Corneal Endothelium Analysis System (CEAS) for the segmentation and computation of endothelial cells in images of the human cornea obtained by in vivo corneal confocal microscopy. First, a Fast Fourier Transform (FFT) Band-pass filter is applied to reduce noise and enhance the image quality to make the cells more visible. Secondly, endothelial cell boundaries are detected using watershed transformations and Voronoi tessellations to accurately quantify the morphological parameters of the human corneal endothelial cells. The performance of the automated segmentation system was tested against manually traced ground-truth images based on a database consisting of 40 corneal confocal endothelial cell images in terms of segmentation accuracy and obtained clinical features. In addition, the robustness and efficiency of the proposed CEAS system were compared with manually obtained cell densities using a separate database of 40 images from controls (n = 11), obese subjects (n = 16) and patients with diabetes (n = 13). The Pearson correlation coefficient between automated and manual endothelial cell densities is 0.9 (p system, and the possibility of utilizing it in a real world clinical setting to enable rapid diagnosis and for patient follow-up, with an execution time of only 6 seconds per image. Copyright © 2018 Elsevier B.V. All rights reserved.

Effects of simvastatin/ezetimibe on microparticles, endothelial progenitor cells and platelet aggregation in subjects with coronary heart disease under antiplatelet therapy

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Camargo, L.M.; França, C.N.; Izar, M.C.; Bianco, H.T.; Lins, L.S.; Barbosa, S.P.; Pinheiro, L.F.; Fonseca, F.A.H. [Universidade Federal de São Paulo, Escola Paulista de Medicina, Departamento de Medicina, São Paulo, SP, Brasil, Departamento de Medicina, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP (Brazil)

2014-04-15

It is not known whether the addition of ezetimibe to statins adds cardiovascular protection beyond the expected changes in lipid levels. Subjects with coronary heart disease were treated with four consecutive 1-week courses of therapy (T) and evaluations. The courses were: T1, 100 mg aspirin alone; T2, 100 mg aspirin and 40 mg simvastatin/10 mg ezetimibe; T3, 40 mg simvastatin/10 mg ezetimibe, and 75 mg clopidogrel (300 mg initial loading dose); T4, 75 mg clopidogrel alone. Platelet aggregation was examined in whole blood. Endothelial microparticles (CD51), platelet microparticles (CD42/CD31), and endothelial progenitor cells (CD34/CD133; CDKDR/CD133, or CD34/KDR) were quantified by flow cytometry. Endothelial function was examined by flow-mediated dilation. Comparisons between therapies revealed differences in lipids (T2 and T3endothelial function (T2>T1 and T4, P=0.001). Decreased platelet aggregation was observed after aspirin (arachidonic acid, T1circulating endothelial and platelet microparticles, or endothelial progenitor cells. Cardiovascular protection following therapy with simvastatin/ezetimibe seems restricted to lipid changes and improvement of endothelial function not affecting the release of microparticles, mobilization of endothelial progenitor cells or decreased platelet aggregation.

Effects of simvastatin/ezetimibe on microparticles, endothelial progenitor cells and platelet aggregation in subjects with coronary heart disease under antiplatelet therapy

International Nuclear Information System (INIS)

Camargo, L.M.; França, C.N.; Izar, M.C.; Bianco, H.T.; Lins, L.S.; Barbosa, S.P.; Pinheiro, L.F.; Fonseca, F.A.H.

2014-01-01

It is not known whether the addition of ezetimibe to statins adds cardiovascular protection beyond the expected changes in lipid levels. Subjects with coronary heart disease were treated with four consecutive 1-week courses of therapy (T) and evaluations. The courses were: T1, 100 mg aspirin alone; T2, 100 mg aspirin and 40 mg simvastatin/10 mg ezetimibe; T3, 40 mg simvastatin/10 mg ezetimibe, and 75 mg clopidogrel (300 mg initial loading dose); T4, 75 mg clopidogrel alone. Platelet aggregation was examined in whole blood. Endothelial microparticles (CD51), platelet microparticles (CD42/CD31), and endothelial progenitor cells (CD34/CD133; CDKDR/CD133, or CD34/KDR) were quantified by flow cytometry. Endothelial function was examined by flow-mediated dilation. Comparisons between therapies revealed differences in lipids (T2 and T3endothelial function (T2>T1 and T4, P=0.001). Decreased platelet aggregation was observed after aspirin (arachidonic acid, T1circulating endothelial and platelet microparticles, or endothelial progenitor cells. Cardiovascular protection following therapy with simvastatin/ezetimibe seems restricted to lipid changes and improvement of endothelial function not affecting the release of microparticles, mobilization of endothelial progenitor cells or decreased platelet aggregation

Effect of strenuous physical exercise on circulating cell-derived microparticles.

Science.gov (United States)

Chaar, Vicky; Romana, Marc; Tripette, Julien; Broquere, Cédric; Huisse, Marie-Geneviève; Hue, Olivier; Hardy-Dessources, Marie-Dominique; Connes, Philippe

2011-01-01

Strenuous exercise is associated with an inflammatory response involving the activation of several types of blood cells. In order to document the specific activation of these cell types, we studied the effect of three maximal exercise tests conducted to exhaustion on the quantitative and qualitative pattern of circulating cell-derived microparticles and inflammatory molecules in healthy subjects. This study mainly indicated that the plasma concentration of microparticles from platelets and polymorphonuclear neutrophils (PMN) was increased immediately after the strenuous exercise. In addition, the increase in plasma concentration of microparticles from PMN and platelets was still observed after 2 hours of recovery. A similar pattern was observed for the IL-6 plasma level. In contrast, no change was observed for either soluble selectins or plasma concentration of microparticles from red blood cells, monocytes and endothelial cells. In agreement, sVCAM-1 and sICAM-1 levels were not changed by the exercise. We conclude that a strenuous exercise is accompanied by platelet- and PMN-derived microparticle production that probably reflects the activation of these two cell types.

Endothelial Protein C–Targeting Liposomes Show Enhanced Uptake and Improved Therapeutic Efficacy in Human Retinal Endothelial Cells

DEFF Research Database (Denmark)

Arta, Anthoula; Eriksen, Anne Z.; Melander, Fredrik

2018-01-01

PURPOSE. To determine whether human retinal endothelial cells (HRECs) express the endothelial cell protein C receptor (EPCR) and to realize its potential as a targeting moiety by developing novel single and dual corticosteroid–loaded functionalized liposomes that exhibit both enhanced uptake by H...... of cell tube formations in contrast to nontargeting liposomes. CONCLUSIONS. We show that HRECs express EPCR and this receptor could be a promising nanomedicine target in ocular diseases where the endothelial barrier of the retina is compromised....

Arecoline inhibits endothelial cell growth and migration and the attachment to mononuclear cells

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Shuei-Kuen Tseng

2014-09-01

Conclusion: Arecoline impaired vascular endothelial cells by inhibiting their growth and migration and their adhesion to U937 mononuclear cells. These results reveal that arecoline may contribute to the pathogenesis of oral submucous fibrosis and cardiovascular diseases by affecting endothelial cell function in BQ chewers.

Cell cycle progression in irradiated endothelial cells cultured from bovine aorta

International Nuclear Information System (INIS)

Rubin, D.B.; Drab, E.A.; Ward, W.F.; Bauer, K.D.

1988-01-01

Logarithmically growing endothelial cells from bovine aortas were exposed to single doses of 0-10 Gy of 60Co gamma rays, and cell cycle phase distribution and progression were examined by flow cytometry and autoradiography. In some experiments, cells were synchronized in the cell cycle with hydroxyurea (1 mM). Cell number in sham-irradiated control cultures doubled in approximately 24 h. Estimated cycle stage times for control cells were 14.4 h for G1 phase, 7.2 h for S phase, and 2.4 h for G2 + M phase. Irradiated cells demonstrated a reduced distribution at the G1/S phase border at 4 h, and an increased distribution in G2 + M phase at 24 h postirradiation. Autoradiographs of irradiated cells after continuous [3H]thymidine labeling indicated a block in G1 phase or at the G1/S-phase border. The duration of the block was dose dependent (2-3 min/cGy). Progression of the endothelial cells through S phase after removal of the hydroxyurea block also was retarded by irradiation, as demonstrated by increased distribution in early S phase and decreased distribution in late S phase. These results indicate that progression of asynchronous cultured bovine aortic endothelial cells through the DNA synthetic cycle is susceptible to radiation inhibition at specific sites in the cycle, resulting in redistribution and partial synchronization of the population. Thus aortic endothelial cells, diploid cells from a normal tissue, resemble many immortal cell types that have been examined in this regard in vitro

MicroRNAs in Hyperglycemia Induced Endothelial Cell Dysfunction

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Maskomani Silambarasan

2016-04-01

Full Text Available Hyperglycemia is closely associated with prediabetes and Type 2 Diabetes Mellitus. Hyperglycemia increases the risk of vascular complications such as diabetic retinopathy, diabetic nephropathy, peripheral vascular disease and cerebro/cardiovascular diseases. Under hyperglycemic conditions, the endothelial cells become dysfunctional. In this study, we investigated the miRNA expression changes in human umbilical vein endothelial cells exposed to different glucose concentrations (5, 10, 25 and 40 mM glucose and at various time intervals (6, 12, 24 and 48 h. miRNA microarray analyses showed that there is a correlation between hyperglycemia induced endothelial dysfunction and miRNA expression. In silico pathways analyses on the altered miRNA expression showed that the majority of the affected biological pathways appeared to be associated to endothelial cell dysfunction and apoptosis. We found the expression of ten miRNAs (miR-26a-5p, -26b-5p, 29b-3p, -29c-3p, -125b-1-3p, -130b-3p, -140-5p, -192-5p, -221-3p and -320a to increase gradually with increasing concentration of glucose. These miRNAs were also found to be involved in endothelial dysfunction. At least seven of them, miR-29b-3p, -29c-3p, -125b-1-3p, -130b-3p, -221-3p, -320a and -192-5p, can be correlated to endothelial cell apoptosis.

Endothelial adhesion molecules and leukocyte integrins in preeclamptic patients.

Science.gov (United States)

Haller, H; Ziegler, E M; Homuth, V; Drab, M; Eichhorn, J; Nagy, Z; Busjahn, A; Vetter, K; Luft, F C

1997-01-01

Endothelial cell activation is important in the pathogenesis of preeclampsia; however, the nature of the activation is unknown. We investigated 22 patients with preeclampsia. 29 normotensive pregnancies, and 18 nonpregnant women to test the hypothesis that serum from preeclamptic patients induces expression of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) and stimulates intracellular free calcium concentrations [Ca2+]i in cultured endothelial cells. We then asked whether the corresponding integrin adhesive counter receptors lymphocyte function-associated antigen-1 (CD11a/CD18), macrophage-1 antigen (CD11b/CD18), p150,95 (CD11c/CD18), and very late activation antigen-4 (CD49/CD29) are increased in patients with preeclampsia. In the pregnant women, the measurements were conducted both before and after delivery. Integrin expression was measured by fluorescent antibody cell sorting analysis using monoclonal antibodies. ICAM-1 and VCAM-1 were analyzed on endothelial cells by enzyme-linked immunosorbent assay. [Ca2+]i was measured with fura 2. Serum from preeclamptic patients increased endothelial cell ICAM-1 expression but not VCAM-1 expression. Preeclamptic patients' serum also increased [Ca2+]i in endothelial cells compared with serum from normal nonpregnant or normal pregnant women. Endothelial cell [Ca2+]i concentrations were correlated with the ICAM-1 expression in preeclamptic patients (r = .80, P preclampsia and normal pregnancy compared with the nonpregnant state. The expression decreased significantly after delivery in both groups. Our results demonstrate that serum from preeclamptic women induces increased ICAM-1 surface expression on endothelial cells, while the expression of the integrin counterreceptors was not different. The effect on endothelial cells may be related to an increase in [Ca2+]i. The effect on cultured endothelial cells and the rapid decrease after delivery suggests the presence of a circulating serum

Inhibition of microparticle release triggers endothelial cell apoptosis and detachment

NARCIS (Netherlands)

Abid Hussein, Mohammed N.; Böing, Anita N.; Sturk, Augueste; Hau, Chi M.; Nieuwland, Rienk

2007-01-01

Endothelial cell cultures contain caspase 3-containing microparticles (EMP), which are reported to form during or after cell detachment. We hypothesize that also adherent endothelial cells release EMP, thus protecting these cells from caspase 3 accumulation, detachment and apoptosis. Human umbilical

CXCL10 can inhibit endothelial cell proliferation independently of CXCR3.

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Gabriele S V Campanella

2010-09-01

Full Text Available CXCL10 (or Interferon-inducible protein of 10 kDa, IP-10 is an interferon-inducible chemokine with potent chemotactic activity on activated effector T cells and other leukocytes expressing its high affinity G protein-coupled receptor CXCR3. CXCL10 is also active on other cell types, including endothelial cells and fibroblasts. The mechanisms through which CXCL10 mediates its effects on non-leukocytes is not fully understood. In this study, we focus on the anti-proliferative effect of CXCL10 on endothelial cells, and demonstrate that CXCL10 can inhibit endothelial cell proliferation in vitro independently of CXCR3. Four main findings support this conclusion. First, primary mouse endothelial cells isolated from CXCR3-deficient mice were inhibited by CXCL10 as efficiently as wildtype endothelial cells. We also note that the proposed alternative splice form CXCR3-B, which is thought to mediate CXCL10's angiostatic activity, does not exist in mice based on published mouse CXCR3 genomic sequences as an in-frame stop codon would terminate the proposed CXCR3-B splice variant in mice. Second, we demonstrate that human umbilical vein endothelial cells and human lung microvascular endothelial cells that were inhibited by CXL10 did not express CXCR3 by FACS analysis. Third, two different neutralizing CXCR3 antibodies did not inhibit the anti-proliferative effect of CXCL10. Finally, fourth, utilizing a panel of CXCL10 mutants, we show that the ability to inhibit endothelial cell proliferation correlates with CXCL10's glycosaminoglycan binding affinity and not with its CXCR3 binding and signaling. Thus, using a very defined system, we show that CXCL10 can inhibit endothelial cell proliferation through a CXCR3-independent mechanism.

Induced Pluripotent Stem Cell-Derived Endothelial Cells in Insulin Resistance and Metabolic Syndrome.

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Carcamo-Orive, Ivan; Huang, Ngan F; Quertermous, Thomas; Knowles, Joshua W

2017-11-01

Insulin resistance leads to a number of metabolic and cellular abnormalities including endothelial dysfunction that increase the risk of vascular disease. Although it has been particularly challenging to study the genetic determinants that predispose to abnormal function of the endothelium in insulin-resistant states, the possibility of deriving endothelial cells from induced pluripotent stem cells generated from individuals with detailed clinical phenotyping, including accurate measurements of insulin resistance accompanied by multilevel omic data (eg, genetic and genomic characterization), has opened new avenues to study this relationship. Unfortunately, several technical barriers have hampered these efforts. In the present review, we summarize the current status of induced pluripotent stem cell-derived endothelial cells for modeling endothelial dysfunction associated with insulin resistance and discuss the challenges to overcoming these limitations. © 2017 American Heart Association, Inc.

Effects of blood products on inflammatory response in endothelial cells in vitro.

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Martin Urner

Full Text Available BACKGROUND: Transfusing blood products may induce inflammatory reactions within the vascular compartment potentially leading to a systemic inflammatory response. Experiments were designed to assess the inflammatory potential of different blood products in an endothelial cell-based in vitro model and to compare baseline levels of potentially activating substances in transfusion products. METHODS: The inflammatory response from pre-activated (endotoxin-stimulated and non-activated endothelial cells as well as neutrophil endothelial transmigration in response to packed red blood cells (PRBC, platelet concentrates (PC and fresh frozen plasma (FFP was determined. Baseline inflammatory mediator and lipid concentrations in blood products were evaluated. RESULTS: Following incubation with all blood products, an increased inflammatory mediator release from endothelial cells was observed. Platelet concentrates, and to a lesser extent also FFP, caused the most pronounced response, which was accentuated in already pre-stimulated endothelial cells. Inflammatory response of endothelial cells as well as blood product-induced migration of neutrophils through the endothelium was in good agreement with the lipid content of the according blood product. CONCLUSION: Within the group of different blood transfusion products both PC and FFP have a high inflammatory potential with regard to activation of endothelial cells. Inflammation upon blood product exposure is strongly accentuated when endothelial cells are pre-injured. High lipid contents in the respective blood products goes along with an accentuated inflammatory reaction from endothelial cells.

Circulating cell-derived microparticles in patients with minimally symptomatic obstructive sleep apnoea.

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Ayers, L; Ferry, B; Craig, S; Nicoll, D; Stradling, J R; Kohler, M

2009-03-01

Moderate-severe obstructive sleep apnoea (OSA) has been associated with several pro-atherogenic mechanisms and increased cardiovascular risk, but it is not known if minimally symptomatic OSA has similar effects. Circulating cell-derived microparticles have been shown to have pro-inflammatory, pro-coagulant and endothelial function-impairing effects, as well as to predict subclinical atherosclerosis and cardiovascular risk. In 57 patients with minimally symptomatic OSA, and 15 closely matched control subjects without OSA, AnnexinV-positive, platelet-, leukocyte- and endothelial cell-derived microparticles were measured by flow cytometry. In patients with OSA, median (interquartile range) levels of AnnexinV-positive microparticles were significantly elevated compared with control subjects: 2,586 (1,566-3,964) microL(-1) versus 1,206 (474-2,501) microL(-1), respectively. Levels of platelet-derived and leukocyte-derived microparticles were also significantly higher in patients with OSA (2,267 (1,102-3,592) microL(-1) and 20 (14-31) microL(-1), respectively) compared with control subjects (925 (328-2,068) microL(-1) and 15 (5-23) microL(-1), respectively). Endothelial cell-derived microparticle levels were similar in patients with OSA compared with control subjects (13 (8-25) microL(-1) versus 11 (6-17) microL(-1)). In patients with minimally symptomatic obstructive sleep apnoea, levels of AnnexinV-positive, platelet- and leukocyte-derived microparticles are elevated when compared with closely matched control subjects without obstructive sleep apnoea. These findings suggest that these patients may be at increased cardiovascular risk, despite being minimally symptomatic.

Infections and endothelial cells

NARCIS (Netherlands)

Keller, Tymen T.; Mairuhu, Albert T. A.; de Kruif, Martijn D.; Klein, Saskia K.; Gerdes, Victor E. A.; ten Cate, Hugo; Brandjes, Dees P. M.; Levi, Marcel; van Gorp, Eric C. M.

2003-01-01

Systemic infection by various pathogens interacts with the endothelium and may result in altered coagulation, vasculitis and atherosclerosis. Endothelium plays a role in the initiation and regulation of both coagulation and fibrinolysis. Exposure of endothelial cells may lead to rapid activation of

Endothelial cell energy metabolism, proliferation, and apoptosis in pulmonary hypertension.

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Xu, Weiling; Erzurum, Serpil C

2011-01-01

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by impaired regulation of pulmonary hemodynamics and excessive growth and dysfunction of the endothelial cells that line the arteries in PAH lungs. Establishment of methods for culture of pulmonary artery endothelial cells from PAH lungs has provided the groundwork for mechanistic translational studies that confirm and extend findings from model systems and spontaneous pulmonary hypertension in animals. Endothelial cell hyperproliferation, survival, and alterations of biochemical-metabolic pathways are the unifying endothelial pathobiology of the disease. The hyperproliferative and apoptosis-resistant phenotype of PAH endothelial cells is dependent upon the activation of signal transducer and activator of transcription (STAT) 3, a fundamental regulator of cell survival and angiogenesis. Animal models of PAH, patients with PAH, and human PAH endothelial cells produce low nitric oxide (NO). In association with the low level of NO, endothelial cells have reduced mitochondrial numbers and cellular respiration, which is associated with more than a threefold increase in glycolysis for energy production. The shift to glycolysis is related to low levels of NO and likely to the pathologic expression of the prosurvival and proangiogenic signal transducer, hypoxia-inducible factor (HIF)-1, and the reduced mitochondrial antioxidant manganese superoxide dismutase (MnSOD). In this article, we review the phenotypic changes of the endothelium in PAH and the biochemical mechanisms accounting for the proliferative, glycolytic, and strongly proangiogenic phenotype of these dysfunctional cells, which consequently foster the panvascular progressive pulmonary remodeling in PAH. © 2011 American Physiological Society.

Tumor endothelial marker 5 expression in endothelial cells during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of cell proliferation

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Vallon, Mario, E-mail: m.vallon@arcor.de [Nuklearmedizinische Klinik und Poliklinik, Technische Universitaet Muenchen, Ismaninger Strasse 22, 81675 Munich (Germany); Rohde, Franziska; Janssen, Klaus-Peter [Chirurgische Klinik und Poliklinik, Technische Universitaet Muenchen, Munich (Germany); Essler, Markus [Nuklearmedizinische Klinik und Poliklinik, Technische Universitaet Muenchen, Ismaninger Strasse 22, 81675 Munich (Germany)

2010-02-01

Tumor endothelial marker (TEM) 5 is an adhesion G-protein-coupled receptor upregulated in endothelial cells during tumor and physiologic angiogenesis. So far, the mechanisms leading to upregulation of TEM5 and its function during angiogenesis have not been identified. Here, we report that TEM5 expression in endothelial cells is induced during capillary-like network formation on Matrigel, during capillary morphogenesis in a three-dimensional collagen I matrix, and upon confluence on a two-dimensional matrix. TEM5 expression was not induced by a variety of soluble angiogenic factors, including VEGF and bFGF, in subconfluent endothelial cells. TEM5 upregulation was blocked by toxin B from Clostridium difficile, an inhibitor of the small GTPases Rho, Rac, and Cdc42. The Rho inhibitor C3 transferase from Clostridium botulinum did not affect TEM5 expression, whereas the Rac inhibitor NSC23766 suppressed TEM5 upregulation. An excess of the soluble TEM5 extracellular domain or an inhibitory monoclonal TEM5 antibody blocked contact inhibition of endothelial cell proliferation resulting in multilayered islands within the endothelial monolayer and increased vessel density during capillary formation. Based on our results we conclude that TEM5 expression during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of proliferation in endothelial cells.

Selective Deletion of Leptin Signaling in Endothelial Cells Enhances Neointima Formation and Phenocopies the Vascular Effects of Diet-Induced Obesity in Mice.

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Hubert, Astrid; Bochenek, Magdalena L; Schütz, Eva; Gogiraju, Rajinikanth; Münzel, Thomas; Schäfer, Katrin

2017-09-01

Obesity is associated with elevated circulating leptin levels and hypothalamic leptin resistance. Leptin receptors (LepRs) are expressed on endothelial cells, and leptin promotes neointima formation in a receptor-dependent manner. Our aim was to examine the importance of endothelial LepR (End.LepR) signaling during vascular remodeling and to determine whether the cardiovascular consequences of obesity are because of hyperleptinemia or endothelial leptin resistance. Mice with loxP-flanked LepR alleles were mated with mice expressing Cre recombinase controlled by the inducible endothelial receptor tyrosine kinase promoter. Obesity was induced with high-fat diet. Neointima formation was examined after chemical carotid artery injury. Morphometric quantification revealed significantly greater intimal hyperplasia, neointimal cellularity, and proliferation in End.LepR knockout mice, and similar findings were obtained in obese, hyperleptinemic End.LepR wild-type animals. Analysis of primary endothelial cells confirmed abrogated signal transducer and activator of transcription-3 phosphorylation in response to leptin in LepR knockout and obese LepR wild-type mice. Quantitative PCR, ELISA, and immunofluorescence analyses revealed increased expression and release of endothelin-1 in End.LepR-deficient and LepR-resistant cells, and ET receptor A/B antagonists abrogated their paracrine effects on murine aortic smooth muscle cell proliferation. Reduced expression of peroxisome proliferator-activated receptor-γ and increased nuclear activator protein-1 staining was observed in End.LepR-deficient and LepR-resistant cells, and peroxisome proliferator-activated receptor-γ antagonization increased endothelial endothelin-1 expression. Our findings suggest that intact endothelial leptin signaling limits neointima formation and that obesity represents a state of endothelial leptin resistance. These observations and the identification of endothelin-1 as soluble mediator of the

Sphingosine 1-Phosphate Induces Platelet/Endothelial Cell Adhesion Molecule-1 Tyrosine Phosphorylation in Bovine Aortic Endothelial Cells through a PP2-Inhibitable Mechanism

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Yu-Ting Huang

2007-12-01

Full Text Available Sphingosine-1-phosphate (S1P is a low-molecular-weight phospholipid derivative released by activated platelets. S1P transduces signals through a family of G protein-coupled receptors to modulate various physiological behaviors of endothelial cells. Platelet/endothelial cell adhesion molecule-1 (PECAM-1; CD31 is a 130-kDa protein expressed on the surfaces of leukocytes, platelets, and endothelial cells. Upon PECAM-1 activation, its cytoplasmic tyrosine residues become phosphorylated and bind with SH2 domain-containing proteins, thus leading to the downstream functions mediated by PECAM-1. In the present study, we found that S1P induced PECAM-1 tyrosine phosphorylation and SHP-2 association in bovine aortic endothelial cells (BAECs by immunoprecipitation and western blotting. The pretreatment of BAECs with a series of chemical inhibitors to determine the signaling pathway showed that the PECAM-1 phosphorylation was inhibited by PP2, indicating the participation of Src family kinases. These results demonstrated that S1P induced PECAM-1 tyrosine phosphorylation in BAECs through mediation of Src family kinases, and this may regulate the physiological behaviors of endothelial cells.

A novel minimally-invasive method to sample human endothelial cells for molecular profiling.

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Stephen W Waldo

Full Text Available The endothelium is a key mediator of vascular homeostasis and cardiovascular health. Molecular research on the human endothelium may provide insight into the mechanisms underlying cardiovascular disease. Prior methodology used to isolate human endothelial cells has suffered from poor yields and contamination with other cell types. We thus sought to develop a minimally invasive technique to obtain endothelial cells derived from human subjects with higher yields and purity.Nine healthy volunteers underwent endothelial cell harvesting from antecubital veins using guidewires. Fluorescence-activated cell sorting (FACS was subsequently used to purify endothelial cells from contaminating cells using endothelial surface markers (CD34/CD105/CD146 with the concomitant absence of leukocyte and platelet specific markers (CD11b/CD45. Endothelial lineage in the purified cell population was confirmed by expression of endothelial specific genes and microRNA using quantitative polymerase chain reaction (PCR.A median of 4,212 (IQR: 2161-6583 endothelial cells were isolated from each subject. Quantitative PCR demonstrated higher expression of von Willebrand Factor (vWF, P<0.001, nitric oxide synthase 3 (NOS3, P<0.001 and vascular cell adhesion molecule 1 (VCAM-1, P<0.003 in the endothelial population compared to similarly isolated leukocytes. Similarly, the level of endothelial specific microRNA-126 was higher in the purified endothelial cells (P<0.001.This state-of-the-art technique isolates human endothelial cells for molecular analysis in higher purity and greater numbers than previously possible. This approach will expedite research on the molecular mechanisms of human cardiovascular disease, elucidating its pathophysiology and potential therapeutic targets.

Intravenous Lipid Infusion Induces Endoplasmic Reticulum Stress in Endothelial Cells and Blood Mononuclear Cells of Healthy Adults.

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Tampakakis, Emmanouil; Tabit, Corey E; Holbrook, Monika; Linder, Erika A; Berk, Brittany D; Frame, Alissa A; Bretón-Romero, Rosa; Fetterman, Jessica L; Gokce, Noyan; Vita, Joseph A; Hamburg, Naomi M

2016-01-11

Endoplasmic reticulum (ER) stress and the subsequent unfolded protein response may initially be protective, but when prolonged, have been implicated in atherogenesis in diabetic conditions. Triglycerides and free fatty acids (FFAs) are elevated in patients with diabetes and may contribute to ER stress. We sought to evaluate the effect of acute FFA elevation on ER stress in endothelial and circulating white cells. Twenty-one healthy subjects were treated with intralipid (20%; 45 mL/h) plus heparin (12 U/kg/h) infusion for 5 hours. Along with increased triglyceride and FFA levels, intralipid/heparin infusion reduced the calf reactive hyperemic response without a change in conduit artery flow-mediated dilation consistent with microvascular dysfunction. To investigate the short-term effects of elevated triglycerides and FFA, we measured markers of ER stress in peripheral blood mononuclear cells (PBMCs) and vascular endothelial cells (VECs). In VECs, activating transcription factor 6 (ATF6) and phospho-inositol requiring kinase 1 (pIRE1) proteins were elevated after infusion (both P<0.05). In PBMCs, ATF6 and spliced X-box-binding protein 1 (XBP-1) gene expression increased by 2.0- and 2.5-fold, respectively (both P<0.05), whereas CHOP and GADD34 decreased by ≈67% and 74%, respectively (both P<0.01). ATF6 and pIRE1 protein levels also increased (both P<0.05), and confocal microscopy revealed the nuclear localization of ATF6 after infusion, suggesting activation. Along with microvascular dysfunction, intralipid infusion induced an early protective ER stress response evidenced by activation of ATF6 and IRE1 in both leukocytes and endothelial cells. Our results suggest a potential link between metabolic disturbances and ER stress that may be relevant to vascular disease. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Low-Intensity Pulsed Ultrasound Prevents the Oxidative Stress Induced Endothelial-Mesenchymal Transition in Human Aortic Endothelial Cells

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Jiamin Li

2018-02-01

Full Text Available Background/Aims: Endothelial-mesenchymal transition (EndMT has been shown to take part in the generation and progression of diverse diseases, involving a series of changes leading to a loss of their endothelial characteristics and an acquirement of properties typical of mesenchymal cells. Low-intensity pulsed ultrasound (LIPUS is a new therapeutic option that has been successfully used in fracture healing. However, whether LIPUS can inhibit oxidative stress-induced endothelial cell damages through inhibiting EndMT remained unknown. This study aimed to investigate the protective effects of LIPUS against oxidative stress-induced endothelial cell damages and the underlying mechanisms. Methods: EndMT was induced by H2O2 (100 µm for seven days. Human aortic endothelial cells (HAECs were exposed to H2O2 with or without LIPUS treatment for seven days. The expression of EndMT markers (CD31, VE-cadherin, FSP1 and α-SMA were analyzed. The levels of total and phosphorylated PI3K and AKT proteins were detected by Western Blot analysis. Cell chemotaxis was determined by wound healing and transwell assay. Results: LIPUS relieved EndMT by decreasing ROS accumulation and increasing activation of the PI3K signaling cascade. LIPUS alleviated the migration of EndMT-derived mesenchymal-like cells through reducing extracellular matrix (ECM deposition that is associated with matrix metallopeptidase (MMP proteolytic activity and collagen production. Conclusion: LIPUS produces cytoprotective effects against oxidative injuries to endothelial cells through suppressing the oxidative stress-induced EndMT, activating the PI3K/AKT pathway under oxidative stress, and limiting cell migration and excessive ECM deposition.

RCAN1.4 regulates VEGFR-2 internalisation, cell polarity and migration in human microvascular endothelial cells.

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Alghanem, Ahmad F; Wilkinson, Emma L; Emmett, Maxine S; Aljasir, Mohammad A; Holmes, Katherine; Rothermel, Beverley A; Simms, Victoria A; Heath, Victoria L; Cross, Michael J

2017-08-01

Regulator of calcineurin 1 (RCAN1) is an endogenous inhibitor of the calcineurin pathway in cells. It is expressed as two isoforms in vertebrates: RCAN1.1 is constitutively expressed in most tissues, whereas transcription of RCAN1.4 is induced by several stimuli that activate the calcineurin-NFAT pathway. RCAN1.4 is highly upregulated in response to VEGF in human endothelial cells in contrast to RCAN1.1 and is essential for efficient endothelial cell migration and tubular morphogenesis. Here, we show that RCAN1.4 has a role in the regulation of agonist-stimulated VEGFR-2 internalisation and establishment of endothelial cell polarity. siRNA-mediated gene silencing revealed that RCAN1 plays a vital role in regulating VEGF-mediated cytoskeletal reorganisation and directed cell migration and sprouting angiogenesis. Adenoviral-mediated overexpression of RCAN1.4 resulted in increased endothelial cell migration. Antisense-mediated morpholino silencing of the zebrafish RCAN1.4 orthologue revealed a disrupted vascular development further confirming a role for the RCAN1.4 isoform in regulating vascular endothelial cell physiology. Our data suggest that RCAN1.4 plays a novel role in regulating endothelial cell migration by establishing endothelial cell polarity in response to VEGF.

Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells

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Kobayashi, Hideki; Butler, Jason M.; O'Donnell, Rebekah; Kobayashi, Mariko; Ding, Bi-Sen; Bonner, Bryant; Chiu, Vi K.; Nolan, Daniel J.; Shido, Koji; Benjamin, Laura; Rafii, Shahin

2010-01-01

Endothelial cells establish an instructive vascular niche that reconstitutes haematopoietic stem and progenitor cells (HSPCs) through release of specific paracrine growth factors, known as angiocrine factors. However, the mechanism by which endothelial cells balance the rate of proliferation and lineage-specific differentiation of HSPCs is unknown. Here, we demonstrate that Akt activation in endothelial cells, through recruitment of mTOR, but not the FoxO pathway, upregulates specific angiocrine factors that support expansion of CD34−Flt3− KLS HSPCs with long-term haematopoietic stem cell (LT-HSC) repopulation capacity. Conversely, co-activation of Akt-stimulated endothelial cells with p42/44 MAPK shifts the balance towards maintenance and differentiation of the HSPCs. Selective activation of Akt1 in the endothelial cells of adult mice increased the number of colony forming units in the spleen and CD34−Flt3− KLS HSPCs with LT-HSC activity in the bone marrow, accelerating haematopoietic recovery. Therefore, the activation state of endothelial cells modulates reconstitution of HSPCs through the upregulation of angiocrine factors, with Akt–mTOR-activated endothelial cells supporting the self-renewal of LT-HSCs and expansion of HSPCs, whereas MAPK co-activation favours maintenance and lineage-specific differentiation of HSPCs. PMID:20972423

Effect of tributyltin on mammalian endothelial cell integrity.

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Botelho, G; Bernardini, C; Zannoni, A; Ventrella, V; Bacci, M L; Forni, M

2015-01-01

Tributyltin (TBT), is a man-made pollutants, known to accumulate along the food chain, acting as an endocrine disruptor in marine organisms, with toxic and adverse effects in many tissues including vascular system. Based on the absence of specific studies of TBT effects on endothelial cells, we aimed to evaluate the toxicity of TBT on primary culture of porcine aortic endothelial cells (pAECs), pig being an excellent model to study human cardiovascular disease. pAECs were exposed for 24h to TBT (100, 250, 500, 750 and 1000nM) showing a dose dependent decrease in cell viability through both apoptosis and necrosis. Moreover the ability of TBT (100 and 500nM) to influence endothelial gene expression was investigated at 1, 7 and 15h of treatment. Gene expression of tight junction molecules, occludin (OCLN) and tight junction protein-1 (ZO-1) was reduced while monocyte adhesion and adhesion molecules ICAM-1 and VCAM-1 (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) levels increased significantly at 1h. IL-6 and estrogen receptors 1 and 2 (ESR-1 and ESR-2) mRNAs, after a transient decrease, reached the maximum levels after 15h of exposure. Finally, we demonstrated that TBT altered endothelial functionality greatly increasing monocyte adhesion. These findings indicate that TBT deeply alters endothelial profile, disrupting their structure and interfering with their ability to interact with molecules and other cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Therapeutic assessment of cytochrome C for the prevention of obesity through endothelial cell-targeted nanoparticulate system.

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Hossen, Md Nazir; Kajimoto, Kazuaki; Akita, Hidetaka; Hyodo, Mamoru; Ishitsuka, Taichi; Harashima, Hideyoshi

2013-03-01

Because the functional apoptosis-initiating protein, cytochrome C (CytC) is rapidly cleared from the circulation (t1/2 (half-life): 4 minutes), it cannot be used for in vivo therapy. We report herein on a hitherto unreported strategy for delivering exogenous CytC as a potential and safe antiobesity drug for preventing diet-induced obesity, the most common type of obesity in humans. The functional activity of CytC encapsulated in prohibitin (a white fat vessel-specific receptor)-targeted nanoparticles (PTNP) was evaluated quantitatively, as evidenced by the observations that CytC-loaded PTNP causes apoptosis in primary adipose endothelial cells in a dose-dependent manner, whereas CytC alone did not. The delivery of a single dose of CytC through PTNP into the circulation disrupted the vascular structure by the targeted apoptosis of adipose endothelial cells in vivo. Intravenous treatment of CytC-loaded PTNP resulted in a substantial reduction in obesity in high-fat diet (HFD) fed wild-type (wt) mice, as evidenced by the dose-dependent prevention of the percentage of increase in body weight and decrease in serum leptin levels. In addition, no detectable hepatotoxicity was found to be associated with this prevention. Thus, the finding highlights the promising potential of CytC for use as an antiobesity drug, when delivered through a nanosystem.

CD133 antibody conjugation to decellularized human heart valves intended for circulating cell capture.

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Vossler, John D; Min Ju, Young; Williams, J Koudy; Goldstein, Steven; Hamlin, James; Lee, Sang Jin; Yoo, James J; Atala, Anthony

2015-09-03

The long term efficacy of tissue based heart valve grafts may be limited by progressive degeneration characterized by immune mediated inflammation and calcification. To avoid this degeneration, decellularized heart valves with functionalized surfaces capable of rapid in vivo endothelialization have been developed. The aim of this study is to examine the capacity of CD133 antibody-conjugated valve tissue to capture circulating endothelial progenitor cells (EPCs). Decellularized human pulmonary valve tissue was conjugated with CD133 antibody at varying concentrations and exposed to CD133 expressing NTERA-2 cl.D1 (NT2) cells in a microflow chamber. The amount of CD133 antibody conjugated on the valve tissue surface and the number of NT2 cells captured in the presence of shear stress was measured. Both the amount of CD133 antibody conjugated to the valve leaflet surface and the number of adherent NT2 cells increased as the concentration of CD133 antibody present in the surface immobilization procedure increased. The data presented in this study support the hypothesis that the rate of CD133(+) cell adhesion in the presence of shear stress to decellularized heart valve tissue functionalized by CD133 antibody conjugation increases as the quantity of CD133 antibody conjugated to the tissue surface increases.

Involvement of the lysophosphatidic acid-generating enzyme autotaxin in lymphocyte-endothelial cell interactions.

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Nakasaki, Tae; Tanaka, Toshiyuki; Okudaira, Shinichi; Hirosawa, Michi; Umemoto, Eiji; Otani, Kazuhiro; Jin, Soojung; Bai, Zhongbin; Hayasaka, Haruko; Fukui, Yoshinori; Aozasa, Katsuyuki; Fujita, Naoya; Tsuruo, Takashi; Ozono, Keiichi; Aoki, Junken; Miyasaka, Masayuki

2008-11-01

Autotaxin (ATX) is a secreted protein with lysophospholipase D activity that generates lysophosphatidic acid (LPA) from lysophosphatidylcholine. Here we report that functional ATX is selectively expressed in high endothelial venules (HEVs) of both lymph nodes and Peyer's patches. ATX expression was developmentally regulated and coincided with lymphocyte recruitment to the lymph nodes. In adults, ATX expression was independent of HEV-expressed chemokines such as CCL21 and CXCL13, innate immunity signals including those via TLR4 or MyD88, and of the extent of lymphocyte trafficking across the HEVs. ATX expression was induced in venules at sites of chronic inflammation. Receptors for the ATX enzyme product LPA were constitutively expressed in HEV endothelial cells (ECs). In vitro, LPA induced strong morphological changes in HEV ECs. Forced ATX expression caused cultured ECs to respond to lysophosphatidylcholine, up-regulating lymphocyte binding to the ECs in a LPA receptor-dependent manner under both static and flow conditions. Although in vivo depletion of circulating ATX did not affect lymphocyte trafficking into the lymph nodes, we surmise, based on the above data, that ATX expressed by HEVs acts on HEVs in situ to facilitate lymphocyte binding to ECs and that ATX in the general circulation does not play a major role in this process. Tissue-specific inactivation of ATX will verify this hypothesis in future studies of its mechanism of action.

Endothelial cell cultures as a tool in biomaterial research

NARCIS (Netherlands)

Kirkpatrick, CJ; Otto, M; van Kooten, T; Krump, [No Value; Kriegsmann, J; Bittinger, F

1999-01-01

Progress in biocompatibility and tissue engineering would today be inconceivable without the aid of in vitro techniques. Endothelial cell cultures represent a valuable tool not just in haemocompatibility testing, but also in the concept of designing hybrid organs. In the past endothelial cells (EC)

Endothelial network formed with human dermal microvascular endothelial cells in autologous multicellular skin substitutes.

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Ponec, Maria; El Ghalbzouri, Abdoelwaheb; Dijkman, Remco; Kempenaar, Johanna; van der Pluijm, Gabri; Koolwijk, Pieter

2004-01-01

A human skin equivalent from a single skin biopsy harboring keratinocytes and melanocytes in the epidermal compartment, and fibroblasts and microvascular dermal endothelial cells in the dermal compartment was developed. The results of the study revealed that the nature of the extracellular matrix of the dermal compartments plays an important role in establishment of endothelial network in vitro. With rat-tail type I collagen matrices only lateral but not vertical expansion of endothelial networks was observed. In contrast, the presence of extracellular matrix of entirely human origin facilitated proper spatial organization of the endothelial network. Namely, when human dermal fibroblasts and microvascular endothelial cells were seeded on the bottom of an inert filter and subsequently epidermal cells were seeded on top of it, fibroblasts produced extracellular matrix throughout which numerous branched tubes were spreading three-dimensionally. Fibroblasts also facilitated the formation of basement membrane at the epidermal/matrix interface. Under all culture conditions, fully differentiated epidermis was formed with numerous melanocytes present in the basal epidermal cell layer. The results of the competitive RT-PCR revealed that both keratinocytes and fibroblasts expressed VEGF-A, -B, -C, aFGF and bFGF mRNA, whereas fibroblasts also expressed VEGF-D mRNA. At protein level, keratinocytes produced 10 times higher amounts of VEGF-A than fibroblasts did. The generation of multicellular skin equivalent from a single human skin biopsy will stimulate further developments for its application in the treatment of full-thickness skin defects. The potential development of biodegradable, biocompatible material suitable for these purposes is a great challenge for future research.

Apoptosis of Endothelial Cells by 13-HPODE Contributes to Impairment of Endothelial Barrier Integrity

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Valerie E. Ryman

2016-01-01

Full Text Available Inflammation is an essential host response during bacterial infections such as bovine mastitis. Endothelial cells are critical for an appropriate inflammatory response and loss of vascular barrier integrity is implicated in the pathogenesis of Streptococcus uberis-induced mastitis. Previous studies suggested that accumulation of linoleic acid (LA oxygenation products derived from 15-lipoxygenase-1 (15-LOX-1 metabolism could regulate vascular functions. The initial LA derivative from the 15-LOX-1 pathway, 13-hydroperoxyoctadecadienoic acid (HPODE, can induce endothelial death, whereas the reduced hydroxyl product, 13-hydroxyoctadecadienoic acid (HODE, is abundantly produced during vascular activation. However, the relative contribution of specific LA-derived metabolites on impairment of mammary endothelial integrity is unknown. Our hypothesis was that S. uberis-induced LA-derived 15-LOX-1 oxygenation products impair mammary endothelial barrier integrity by apoptosis. Exposure of bovine mammary endothelial cells (BMEC to S. uberis did not increase 15-LOX-1 LA metabolism. However, S. uberis challenge of bovine monocytes demonstrated that monocytes may be a significant source of both 13-HPODE and 13-HODE during mastitis. Exposure of BMEC to 13-HPODE, but not 13-HODE, significantly reduced endothelial barrier integrity and increased apoptosis. Changing oxidant status by coexposure to an antioxidant during 13-HPODE treatment prevented adverse effects of 13-HPODE, including amelioration of apoptosis. A better understanding of how the oxidant status of the vascular microenvironment impacts endothelial barrier properties could lead to more efficacious treatments for S. uberis mastitis.

Changes of junctions of endothelial cells in coronary sclerosis: A review

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Li-Zi Zhang

2016-03-01

Full Text Available Atherosclerosis, the major cause of cardiovascular diseases, has been a leading contributor to morbidity and mortality in the United States and it has been on the rise globally. Endothelial cell–cell junctions are critical for vascular integrity and maintenance of vascular function. Endothelial cell junctions dysfunction is the onset step of future coronary events and coronary artery disease. Keywords: Coronary atherosclerosis, Junctions, Endothelial cells

Young endothelial cells revive aging blood.

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Chang, Vivian Y; Termini, Christina M; Chute, John P

2017-11-01

The hematopoietic system declines with age, resulting in decreased hematopoietic stem cell (HSC) self-renewal capacity, myeloid skewing, and immune cell depletion. Aging of the hematopoietic system is associated with an increased incidence of myeloid malignancies and a decline in adaptive immunity. Therefore, strategies to rejuvenate the hematopoietic system have important clinical implications. In this issue of the JCI, Poulos and colleagues demonstrate that infusions of bone marrow (BM) endothelial cells (ECs) from young mice promoted HSC self-renewal and restored immune cell content in aged mice. Additionally, delivery of young BM ECs along with HSCs following total body irradiation improved HSC engraftment and enhanced survival. These results suggest an important role for BM endothelial cells (ECs) in regulating hematopoietic aging and support further research to identify the rejuvenating factors elaborated by BM ECs that restore HSC function and the immune repertoire in aged mice.

Cyclosporine Induces Endothelial Cell Release of Complement-Activating Microparticles

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Renner, Brandon; Klawitter, Jelena; Goldberg, Ryan; McCullough, James W.; Ferreira, Viviana P.; Cooper, James E.; Christians, Uwe

2013-01-01

Defective control of the alternative pathway of complement is an important risk factor for several renal diseases, including atypical hemolytic uremic syndrome. Infections, drugs, pregnancy, and hemodynamic insults can trigger episodes of atypical hemolytic uremic syndrome in susceptible patients. Although the mechanisms linking these clinical events with disease flares are unknown, recent work has revealed that each of these clinical conditions causes cells to release microparticles. We hypothesized that microparticles released from injured endothelial cells promote intrarenal complement activation. Calcineurin inhibitors cause vascular and renal injury and can trigger hemolytic uremic syndrome. Here, we show that endothelial cells exposed to cyclosporine in vitro and in vivo release microparticles that activate the alternative pathway of complement. Cyclosporine-induced microparticles caused injury to bystander endothelial cells and are associated with complement-mediated injury of the kidneys and vasculature in cyclosporine-treated mice. Cyclosporine-induced microparticles did not bind factor H, an alternative pathway regulatory protein present in plasma, explaining their complement-activating phenotype. Finally, we found that in renal transplant patients, the number of endothelial microparticles in plasma increases 2 weeks after starting tacrolimus, and treatment with tacrolimus associated with increased C3 deposition on endothelial microparticles in the plasma of some patients. These results suggest that injury-associated release of endothelial microparticles is an important mechanism by which systemic insults trigger intravascular complement activation and complement-dependent renal diseases. PMID:24092930

Characterization of vascular endothelial progenitor cells from chicken bone marrow

Directory of Open Access Journals (Sweden)

Bai Chunyu

2012-05-01

Full Text Available Abstract Background Endothelial progenitor cells (EPC are a type of stem cell used in the treatment of atherosclerosis, vascular injury and regeneration. At present, most of the EPCs studied are from human and mouse, whereas the study of poultry-derived EPCs has rarely been reported. In the present study, chicken bone marrow-derived EPCs were isolated and studied at the cellular level using immunofluorescence and RT-PCR. Results We found that the majority of chicken EPCs were spindle shaped. The growth-curves of chicken EPCs at passages (P 1, -5 and -9 were typically “S”-shaped. The viability of chicken EPCs, before and after cryopreservation was 92.2% and 81.1%, respectively. Thus, cryopreservation had no obvious effects on the viability of chicken EPCs. Dil-ac-LDL and FITC-UAE-1 uptake assays and immunofluorescent detection of the cell surface markers CD34, CD133, VEGFR-2 confirmed that the cells obtained in vitro were EPCs. Observation of endothelial-specific Weibel-Palade bodies using transmission electron microscopy further confirmed that the cells were of endothelial lineage. In addition, chicken EPCs differentiated into endothelial cells and smooth muscle cells upon induction with VEGF and PDGF-BB, respectively, suggesting that the chicken EPCs retained multipotency in vitro. Conclusions These results suggest that chicken EPCs not only have strong self-renewal capacity, but also the potential to differentiate into endothelial and smooth muscle cells. This research provides theoretical basis and experimental evidence for potential therapeutic application of endothelial progenitor cells in the treatment of atherosclerosis, vascular injury and diabetic complications.

Drug-induced in vitro inhibition of neutrophil-endothelial cell adhesion.

Science.gov (United States)

Pellegatta, F.; Lu, Y.; Radaelli, A.; Zocchi, M. R.; Ferrero, E.; Chierchia, S.; Gaja, G.; Ferrero, M. E.

1996-01-01

1. Leukocyte-endothelial cell interactions play an important role during ischaemia-reperfusion events. Adhesion molecules are specifically implicated in this interaction process. 2. Since defibrotide has been shown to be an efficient drug in reducing damage due to ischaemia-reperfusion in many experimental models, we analysed the effect of defibrotide in vitro on leukocyte adhesion to endothelial cells in basal conditions and after their stimulation. 3. In basal conditions, defibrotide (1000 micrograms ml-1) partially inhibited leukocyte adhesion to endothelial cells by 17.3% +/- 3.6 (P defibrotide. 5. This result was confirmed in NIH/3T3-ICAM-1 transfected cells. 6. We conclude that defibrotide is able to interfere with leukocyte adhesion to endothelial cells mainly in activated conditions and that the ICAM-1/LFA-1 adhesion system is involved in the defibrotide mechanism of action. PMID:8762067

Catalase and superoxide dismutase conjugated with platelet-endothelial cell adhesion molecule antibody distinctly alleviate abnormal endothelial permeability caused by exogenous reactive oxygen species and vascular endothelial growth factor.

Science.gov (United States)

Han, Jingyan; Shuvaev, Vladimir V; Muzykantov, Vladimir R

2011-07-01

Reactive oxygen species (ROS) superoxide anion (O(2)()) and hydrogen peroxide (H(2)O(2)) produced by activated leukocytes and endothelial cells in sites of inflammation or ischemia cause endothelial barrier dysfunction that may lead to tissue edema. Antioxidant enzymes (AOEs) catalase and superoxide dismutase (SOD) conjugated with antibodies to platelet-endothelial cell adhesion molecule-1 (PECAM-1) specifically bind to endothelium, quench the corresponding ROS, and alleviate vascular oxidative stress and inflammation. In the present work, we studied the effects of anti-PECAM/catalase and anti-PECAM/SOD conjugates on the abnormal permeability manifested by transendothelial electrical resistance decline, increased fluorescein isothiocyanate-dextran influx, and redistribution of vascular endothelial-cadherin in human umbilical vein endothelial cell (HUVEC) monolayers. Anti-PECAM/catalase protected HUVEC monolayers against H(2)O(2)-induced endothelial barrier dysfunction. Polyethylene glycol-conjugated catalase exerted orders of magnitude lower endothelial uptake and no protective effect, similarly to IgG/catalase. Anti-PECAM/catalase, but not anti-PECAM/SOD, alleviated endothelial hyperpermeability caused by exposure to hypoxanthine/xanthine oxidase, implicating primarily H(2)O(2) in the disruption of the endothelial barrier in this model. Thrombin-induced endothelial permeability was not affected by treatment with anti-PECAM/AOEs or the NADPH oxidase inhibitor apocynin or overexpression of AOEs, indicating that the endogenous ROS play no key role in thrombin-mediated endothelial barrier dysfunction. In contrast, anti-PECAM/SOD, but not anti-PECAM/catalase, inhibited a vascular endothelial growth factor (VEGF)-induced increase in endothelial permeability, identifying a key role of endogenous O(2)() in the VEGF-mediated regulation of endothelial barrier function. Therefore, AOEs targeted to endothelial cells provide versatile molecular tools for testing the roles of

The Bony Side of Endothelial Cells in Prostate Cancer.

Science.gov (United States)

Peng, Jia; Kang, Yibin

2017-06-05

Prostate cancer bone metastases are primarily osteoblastic, but the source of bone-forming cells in these lesions remains poorly defined. In this issue of Developmental Cell, Lin et al. (2017) demonstrate that tumor-associated endothelial cells can give rise to osteoblasts in prostate cancer through endothelial-to-osteoblast (EC-to-OSB) conversion. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of sunitinib combined with ionizing radiation on endothelial cells

International Nuclear Information System (INIS)

Zhang Haiping; Jiao Xiaodong; Li Rui; Wang Jiejun; Takayama, Koichi; Su Bo

2011-01-01

The aims of present study were to evaluate the efficacy of combining sunitinib with ionizing radiation (IR) on endothelial cells in vitro and in vivo. Human umbilical vein endothelial cells (HUVECs) were exposed to IR with or without sunitinib pretreatment. Apoptosis assay and cell cycle distribution were analyzed by flow cytometry. Clonogenic survival assay at 3 Gy dose with or without sunitinib was performed. The activity of phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway was detected by Western immunoblot. Lewis lung carcinoma mouse model was built to examine the effect of combination therapy on endothelial cells in vivo. Microvasculature changes were detected by immunohistochemistry using anti-CD31 antibody. Our results showed combination therapy of sunitinib and IR significantly increased apoptosis of endothelial cells and inhibited colony formation compared to sunitinib or radiotherapy alone. It also resulted in cell cycle redistribution (decreasing cells in S phase and increasing cells in G2/M phase). The activity of PI3K/Akt signal pathway was inhibited, which could be the potential mechanisms that account for the enhanced radiation response induced by sunitinib. In vivo analysis showed that combination therapy significantly decreased microvasculature formation. The results demonstrated that combination therapy of sunitinib and IR has the potential to increase the cytotoxic effects on endothelial cells. (author)

Apelin is a novel angiogenic factor in retinal endothelial cells

International Nuclear Information System (INIS)

Kasai, Atsushi; Shintani, Norihito; Oda, Maki; Kakuda, Michiya; Hashimoto, Hitoshi; Matsuda, Toshio; Hinuma, Shuji; Baba, Akemichi

2004-01-01

There has been much focus recently on the possible functions of apelin, an endogenous ligand for the orphan G-protein-coupled receptor APJ, in cardiovascular and central nervous systems. We report a new function of apelin as a novel angiogenic factor in retinal endothelial cells. The retinal endothelial cell line RF/6A highly expressed both apelin and APJ transcripts, while human umbilical venous endothelial cells (HUVECs) only expressed apelin mRNA. In accordance with these observations, apelin at concentrations of 1 pM-1 μM significantly enhanced migration, proliferation, and capillary-like tube formation of RF/6A cells, but not those of HUVECs, whereas VEGF stimulates those parameters of both cell types. In vivo Matrigel plug assay for angiogenesis, the inclusion of 1 nM apelin in the Matrigel resulted in clear capillary-like formations with an increase of hemoglobin content in the plug. This is the first report showing that apelin is an angiogenic factor in retinal endothelial cells

Isolation and characterization of conditionally immortalized mouse glomerular endothelial cell lines.

Science.gov (United States)

Rops, Angelique L; van der Vlag, Johan; Jacobs, Cor W; Dijkman, Henry B; Lensen, Joost F; Wijnhoven, Tessa J; van den Heuvel, Lambert P; van Kuppevelt, Toin H; Berden, Jo H

2004-12-01

The culture and establishment of glomerular cell lines has proven to be an important tool for the understanding of glomerular cell functions in glomerular physiology and pathology. Especially, the recent establishment of a conditionally immortalized visceral epithelial cell line has greatly boosted the research on podocyte biology. Glomeruli were isolated from H-2Kb-tsA58 transgenic mice that contain a gene encoding a temperature-sensitive variant of the SV40 large tumor antigen, facilitating proliferative growth at 33 degrees C and differentiation at 37 degrees C. Glomerular endothelial cells were isolated from glomerular outgrowth by magnetic beads loaded with CD31, CD105, GSL I-B4, and ULEX. Clonal cell lines were characterized by immunofluorescence staining with antibodies/lectins specific for markers of endothelial cells, podocytes, and mesangial cells. Putative glomerular endothelial cell lines were analyzed for (1) cytokine-induced expression of adhesion molecules; (2) tube formation on Matrigel coating; and (3) the presence of fenestrae. As judged by immunostaining for Wilms tumor-1, smooth muscle actin (SMA), podocalyxin, and von Willebrand factor (vWF), we obtained putative endothelial, podocyte and mesangial cell lines. The mouse glomerular endothelial cell clone #1 (mGEnC-1) was positive for vWF, podocalyxin, CD31, CD105, VE-cadherin, GSL I-B4, and ULEX, internalized acetylated-low-density lipoprotein (LDL), and showed increased expression of adhesion molecules after activation with proinflammatory cytokines. Furthermore, mGEnC-1 formed tubes and contained nondiaphragmed fenestrae. The mGEnC-1 represents a conditionally immortalized cell line with various characteristics of differentiated glomerular endothelial cells when cultured at 37 degrees C. Most important, mGEnC-1 contains nondiaphragmed fenestrae, which is a unique feature of glomerular endothelial cells.

An ibuprofen-antagonized plasmin inhibitor released by human endothelial cells.

Science.gov (United States)

Rockwell, W B; Ehrlich, H P

1991-02-01

Serum-free culture medium harvested from endothelial cell monolayer cultures derived from human scars and dermis was examined for inhibition of fibrinolysis using a fibrin plate assay. Human cultured fibroblasts and smooth muscle cells did not produce any detectable inhibitory activity. The inhibitor is spontaneously released from the cultured endothelial cells over time. In the fibrin plate assay of plasmin-induced fibrinolysis, one nonsteroidal antiinflammatory (NSAI) drug, ibuprofen, was demonstrated to antagonize the inhibition of fibrinolysis. The antagonistic activity of ibuprofen appears unrelated to its NSAI drug activity because other NSAI drugs such as indomethacin and tolmetin have minimal antagonistic activity. Heating the cultured endothelial cells to 42 degrees C stimulates greater release of the inhibitor in a shorter period of time. This plasmin inhibitor, which is produced by endothelial cells, may contribute to postburn vascular occlusion, leading to secondary progressive necrosis in burn-traumatized patients.

Enhanced adhesion of early endothelial progenitor cells to radiation-induced senescence-like vascular endothelial cells in vitro

International Nuclear Information System (INIS)

Sermsathanasawadi, N.; Inoue, Yoshinori; Iwai, Takehisa; Ishii, Hideto; Yoshida, Masayuki; Igarashi, Kaori; Miura, Masahiko

2009-01-01

The effects of ionizing radiation (IR) on tumor neovascularization are still unclear. We previously reported that vascular endothelial cells (ECs) expressing the IR-induced senescence-like (IRSL) phenotype exhibit a significant decrease in angiogenic activity in vitro. In this study, we examined the effects of the IRSL phenotype on adhesion to early endothelial progenitor cells (early EPCs). Adhesion of human peripheral blood-derived early EPCs to human umbilical vein endothelial cells (HUVECs) expressing the IRSL phenotype was evaluated by an adhesion assay under static conditions. It was revealed that the IRSL HUVECs supported significantly more adhesion of early EPCs than normal HUVECs. Expressions of ICAM-1, VCAM-1 and E-selectin were up-regulated in IRSL HUVECs. Pre-treatment of IRSL HUVECs with adhesion-blocking monoclonal antibodies against E-selectin and VCAM-1 significantly reduced early EPC adhesion to IRSL HUVECs, suggesting a potential role for the E-selectin and VCAM-1 in the adhesion between IRSL ECs and early EPCs. Therefore, the IRSL phenotype expressed in ECs may enhance neovascularization via increased homing of early EPCs. Our findings are first to implicate the complex effects of this phenotype on tumor neovascularization following irradiation. (author)

Probing Leader Cells in Endothelial Collective Migration by Plasma Lithography Geometric Confinement

OpenAIRE

Yongliang Yang; Nima Jamilpour; Baoyin Yao; Zachary S. Dean; Reza Riahi; Pak Kin Wong

2016-01-01

When blood vessels are injured, leader cells emerge in the endothelium to heal the wound and restore the vasculature integrity. The characteristics of leader cells during endothelial collective migration under diverse physiological conditions, however, are poorly understood. Here we investigate the regulation and function of endothelial leader cells by plasma lithography geometric confinement generated. Endothelial leader cells display an aggressive phenotype, connect to follower cells via pe...

Vascular endothelial growth factor A-stimulated signaling from endosomes in primary endothelial cells.

Science.gov (United States)

Fearnley, Gareth W; Smith, Gina A; Odell, Adam F; Latham, Antony M; Wheatcroft, Stephen B; Harrison, Michael A; Tomlinson, Darren C; Ponnambalam, Sreenivasan

2014-01-01

The vascular endothelial growth factor A (VEGF-A) is a multifunctional cytokine that stimulates blood vessel sprouting, vascular repair, and regeneration. VEGF-A binds to VEGF receptor tyrosine kinases (VEGFRs) and stimulates intracellular signaling leading to changes in vascular physiology. An important aspect of this phenomenon is the spatiotemporal coordination of VEGFR trafficking and intracellular signaling to ensure that VEGFR residence in different organelles is linked to downstream cellular outputs. Here, we describe a series of assays to evaluate the effects of VEGF-A-stimulated intracellular signaling from intracellular compartments such as the endosome-lysosome system. These assays include the initial isolation and characterization of primary human endothelial cells, performing reverse genetics for analyzing protein function; methods used to study receptor trafficking, signaling, and proteolysis; and assays used to measure changes in cell migration, proliferation, and tubulogenesis. Each of these assays has been exemplified with studies performed in our laboratories. In conclusion, we describe necessary techniques for studying the role of VEGF-A in endothelial cell function. © 2014 Elsevier Inc. All rights reserved.

Endothelial cell chimerism associated with graft rejection after human lung transplantation.

OpenAIRE

Ratajczak , Philippe; Murata , Hideyuki; Meignin , Véronique; Groussard , Odile; Fournier , Michel; Socié , Gérard; Mal , Hervé; Janin , Anne

2008-01-01

International audience; Endotheliitis is a major sign of graft rejection. Recipient-derived endothelial cells found in two series of liver and kidney transplants were related to graft rejection. Here, we assessed the presence and the number of chimeric endothelial cells in lung transplants, and their relation with graft rejection. In six males grafted with female lungs out of 193 lung transplantations, endothelial chimerism was studied by combined XY-fluorescent in situ hybridization with CD3...

Differentiation of Human Pluripotent Stem Cells into Functional Endothelial Cells in Scalable Suspension Culture

Directory of Open Access Journals (Sweden)

Ruth Olmer

2018-05-01

Full Text Available Summary: Endothelial cells (ECs are involved in a variety of cellular responses. As multifunctional components of vascular structures, endothelial (progenitor cells have been utilized in cellular therapies and are required as an important cellular component of engineered tissue constructs and in vitro disease models. Although primary ECs from different sources are readily isolated and expanded, cell quantity and quality in terms of functionality and karyotype stability is limited. ECs derived from human induced pluripotent stem cells (hiPSCs represent an alternative and potentially superior cell source, but traditional culture approaches and 2D differentiation protocols hardly allow for production of large cell numbers. Aiming at the production of ECs, we have developed a robust approach for efficient endothelial differentiation of hiPSCs in scalable suspension culture. The established protocol results in relevant numbers of ECs for regenerative approaches and industrial applications that show in vitro proliferation capacity and a high degree of chromosomal stability. : In this article, U. Martin and colleagues show the generation of hiPSC endothelial cells in scalable cultures in up to 100 mL culture volume. The generated ECs show in vitro proliferation capacity and a high degree of chromosomal stability after in vitro expansion. The established protocol allows to generate hiPSC-derived ECs in relevant numbers for regenerative approaches. Keywords: hiPSC differentiation, endothelial cells, scalable culture

Nanofiber density determines endothelial cell behavior on hydrogel matrix

Energy Technology Data Exchange (ETDEWEB)

Berti, Fernanda V., E-mail: fernanda@intelab.ufsc.br [Department of Chemical and Food Engineering, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil); Rambo, Carlos R. [Department of Electrical Engineering, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil); Dias, Paulo F. [Department of Cell Biology, Embryology and Genetics, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil); Porto, Luismar M. [Department of Chemical and Food Engineering, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil)

2013-12-01

When cultured under static conditions, bacterial cellulose pellicles, by the nature of the polymer synthesis that involves molecular oxygen, are characterized by two distinct surface sides. The upper surface is denser in fibers (entangled) than the lower surface that shows greater surface porosity. Human umbilical vein endothelial cells (HUVECs) were used to exploit how the microarchitecture (i.e., surface porosity, fiber network structure, surface topology, and fiber density) of bacterial cellulose pellicle surfaces influence cell–biomaterial interaction and therefore cell behavior. Adhesion, cell ingrowth, proliferation, viability and cell death mechanisms were evaluated on the two pellicle surface sides. Cell behavior, including secondary necrosis, is influenced only by the microarchitecture of the surface, since the biomaterial is extremely pure (constituted of cellulose and water only). Cell–cellulose fiber interaction is the determinant signal in the cell–biomaterial responses, isolated from other frequently present interferences such as protein and other chemical traces usually present in cell culture matrices. Our results suggest that microarchitecture of hydrogel materials might determine the performance of biomedical products, such as bacterial cellulose tissue engineering constructs (BCTECs). - Highlights: • Topography of BC pellicle is relevant to determine endothelial cells' fate. • Cell–biomaterial response is affected by the topography of BC-pellicle surface. • Endothelial cells exhibit different behavior depending on the BC topography. • Apoptosis and necrosis of endothelial cells were affected by the BC topography.

Nanofiber density determines endothelial cell behavior on hydrogel matrix

International Nuclear Information System (INIS)

Berti, Fernanda V.; Rambo, Carlos R.; Dias, Paulo F.; Porto, Luismar M.

2013-01-01

When cultured under static conditions, bacterial cellulose pellicles, by the nature of the polymer synthesis that involves molecular oxygen, are characterized by two distinct surface sides. The upper surface is denser in fibers (entangled) than the lower surface that shows greater surface porosity. Human umbilical vein endothelial cells (HUVECs) were used to exploit how the microarchitecture (i.e., surface porosity, fiber network structure, surface topology, and fiber density) of bacterial cellulose pellicle surfaces influence cell–biomaterial interaction and therefore cell behavior. Adhesion, cell ingrowth, proliferation, viability and cell death mechanisms were evaluated on the two pellicle surface sides. Cell behavior, including secondary necrosis, is influenced only by the microarchitecture of the surface, since the biomaterial is extremely pure (constituted of cellulose and water only). Cell–cellulose fiber interaction is the determinant signal in the cell–biomaterial responses, isolated from other frequently present interferences such as protein and other chemical traces usually present in cell culture matrices. Our results suggest that microarchitecture of hydrogel materials might determine the performance of biomedical products, such as bacterial cellulose tissue engineering constructs (BCTECs). - Highlights: • Topography of BC pellicle is relevant to determine endothelial cells' fate. • Cell–biomaterial response is affected by the topography of BC-pellicle surface. • Endothelial cells exhibit different behavior depending on the BC topography. • Apoptosis and necrosis of endothelial cells were affected by the BC topography

Microparticles generated during chronic cerebral ischemia deliver proapoptotic signals to cultured endothelial cells

International Nuclear Information System (INIS)

Schock, Sarah C.; Edrissi, Hamidreza; Burger, Dylan; Cadonic, Robert; Hakim, Antoine; Thompson, Charlie

2014-01-01

Highlights: • Microparticles are elevated in the plasma in a rodent model of chronic cerebral ischemia. • These microparticles initiate apoptosis in cultured cells. • Microparticles contain caspase 3 and they activate receptors for TNF-α and TRAIL. - Abstract: Circulating microparticles (MPs) are involved in many physiological processes and numbers are increased in a variety of cardiovascular disorders. The present aims were to characterize levels of MPs in a rodent model of chronic cerebral hypoperfusion (CCH) and to determine their signaling properties. MPs were isolated from the plasma of rats exposed to CCH and quantified by flow cytometry. When MPs were added to cultured endothelial cells or normal rat kidney cells they induced cell death in a time and dose dependent manner. Analysis of pellets by electron microscopy indicates that cell death signals are carried by particles in the range of 400 nm in diameter or less. Cell death involved the activation of caspase 3 and was not a consequence of oxidative stress. Inhibition of the Fas/FasL signaling pathway also did not improve cell survival. MPs were found to contain caspase 3 and treating the MPs with a caspase 3 inhibitor significantly reduced cell death. A TNF-α receptor blocker and a TRAIL neutralizing antibody also significantly reduced cell death. Levels of circulating MPs are elevated in a rodent model of chronic cerebral ischemia. MPs with a diameter of 400 nm or less activate the TNF-α and TRAIL signaling pathways and may deliver caspase 3 to cultured cells

Microparticles generated during chronic cerebral ischemia deliver proapoptotic signals to cultured endothelial cells

Energy Technology Data Exchange (ETDEWEB)

Schock, Sarah C. [Ottawa Hospital Research Institute, Neuroscience, 451 Smyth Road, Ottawa, ON K1H 8M5 (Canada); Edrissi, Hamidreza [University of Ottawa, Neuroscience Graduate Program, 451 Smyth Road, Ottawa, ON K1H 8M5 (Canada); Burger, Dylan [Ottawa Hospital Research Institute, Kidney Centre, 451 Smyth Road, Ottawa, ON K1H 8M5 (Canada); Cadonic, Robert; Hakim, Antoine [Ottawa Hospital Research Institute, Neuroscience, 451 Smyth Road, Ottawa, ON K1H 8M5 (Canada); Thompson, Charlie, E-mail: charliet@uottawa.ca [Ottawa Hospital Research Institute, Neuroscience, 451 Smyth Road, Ottawa, ON K1H 8M5 (Canada)

2014-07-18

Highlights: • Microparticles are elevated in the plasma in a rodent model of chronic cerebral ischemia. • These microparticles initiate apoptosis in cultured cells. • Microparticles contain caspase 3 and they activate receptors for TNF-α and TRAIL. - Abstract: Circulating microparticles (MPs) are involved in many physiological processes and numbers are increased in a variety of cardiovascular disorders. The present aims were to characterize levels of MPs in a rodent model of chronic cerebral hypoperfusion (CCH) and to determine their signaling properties. MPs were isolated from the plasma of rats exposed to CCH and quantified by flow cytometry. When MPs were added to cultured endothelial cells or normal rat kidney cells they induced cell death in a time and dose dependent manner. Analysis of pellets by electron microscopy indicates that cell death signals are carried by particles in the range of 400 nm in diameter or less. Cell death involved the activation of caspase 3 and was not a consequence of oxidative stress. Inhibition of the Fas/FasL signaling pathway also did not improve cell survival. MPs were found to contain caspase 3 and treating the MPs with a caspase 3 inhibitor significantly reduced cell death. A TNF-α receptor blocker and a TRAIL neutralizing antibody also significantly reduced cell death. Levels of circulating MPs are elevated in a rodent model of chronic cerebral ischemia. MPs with a diameter of 400 nm or less activate the TNF-α and TRAIL signaling pathways and may deliver caspase 3 to cultured cells.

Lymphatic endothelial cell line (CH3) from a recurrent retroperitoneal lymphangioma.

Science.gov (United States)

Way, D; Hendrix, M; Witte, M; Witte, C; Nagle, R; Davis, J

1987-09-01

An endothelial cell line derived from a massive recurrent chyle-containing retroperitoneal lymphangioma was isolated in monolayer culture. Scanning and transmission electron microscopy and immunohistochemistry confirmed a close resemblance to blood vascular endothelium with typical cobblestone morphology, positive immunofluorescence staining for endothelial marker Factor VIII-associated antigen and fibronectin, and prominent Weibel-Palade bodies. The endothelial cells also exhibited other ultrastructural features characteristic of lymphatic endothelium, including sparse microvillous surface projections, overlapping intercellular junctions, and abundant intermediate filaments. This endothelial cell line represents a new source of proliferating lymphatic endothelium for future study, including structural and functional comparison to blood vascular endothelium.

Astrocytes Can Adopt Endothelial Cell Fates in a p53-Dependent Manner.

Science.gov (United States)

Brumm, Andrew J; Nunez, Stefanie; Doroudchi, Mehdi M; Kawaguchi, Riki; Duan, Jinhzu; Pellegrini, Matteo; Lam, Larry; Carmichael, S Thomas; Deb, Arjun; Hinman, Jason D

2017-08-01

Astrocytes respond to a variety of CNS injuries by cellular enlargement, process outgrowth, and upregulation of extracellular matrix proteins that function to prevent expansion of the injured region. This astrocytic response, though critical to the acute injury response, results in the formation of a glial scar that inhibits neural repair. Scar-forming cells (fibroblasts) in the heart can undergo mesenchymal-endothelial transition into endothelial cell fates following cardiac injury in a process dependent on p53 that can be modulated to augment cardiac repair. Here, we sought to determine whether astrocytes, as the primary scar-forming cell of the CNS, are able to undergo a similar cellular phenotypic transition and adopt endothelial cell fates. Serum deprivation of differentiated astrocytes resulted in a change in cellular morphology and upregulation of endothelial cell marker genes. In a tube formation assay, serum-deprived astrocytes showed a substantial increase in vessel-like morphology that was comparable to human umbilical vein endothelial cells and dependent on p53. RNA sequencing of serum-deprived astrocytes demonstrated an expression profile that mimicked an endothelial rather than astrocyte transcriptome and identified p53 and angiogenic pathways as specifically upregulated. Inhibition of p53 with genetic or pharmacologic strategies inhibited astrocyte-endothelial transition. Astrocyte-endothelial cell transition could also be modulated by miR-194, a microRNA downstream of p53 that affects expression of genes regulating angiogenesis. Together, these studies demonstrate that differentiated astrocytes retain a stimulus-dependent mechanism for cellular transition into an endothelial phenotype that may modulate formation of the glial scar and promote injury-induced angiogenesis.

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Interleukin 1 is an autocrine regulator of human endothelial cell growth

International Nuclear Information System (INIS)

Cozzolino, F.; Torcia, M.; Aldinucci, D.; Ziche, M.; Bani, D.; Almerigogna, F.; Stern, D.M.

1990-01-01

Proliferation of endothelial cells is regulated through the autocrine production of growth factors and the expression of cognate surface receptors. In this study, the authors demonstrate that interleukin 1 (IL-1) is an inhibitor of endothelial growth in vitro and in vivo. IL-1 arrested growing, cultured endothelial cells in G 1 phase; inhibition of proliferation was dose dependent and occurred in parallel with occupancy of endothelial surface IL-1 receptors. In an angiogenesis model, IL-1 could inhibit fibroblast growth factor-induced vessel formation. The autocrine nature of the IL-1 effect on endothelial proliferation was demonstrated by the observation that occupancy of cell-surface receptors by endogenous IL-1 depressed cell growth. The potential significance of this finding was emphasized by the detection of IL-1 in the native endothelium of human umbilical veins. A mechanism by which IL-1 may exert its inhibitory effect on endothelial cell growth was suggested by studies showing that IL-1 decreased the expression of high-affinity fibroblast growth factor binding sites on endothelium. These results point to a potentially important role of IL-1 in regulating blood vessel growth the suggest that autocrine production of inhibitory factors may be a mechanism controlling proliferation of normal cells

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Resveratrol combined with total flavones of hawthorn alleviate the endothelial cells injury after coronary bypass graft surgery.

Science.gov (United States)

Zhu, Ying; Feng, Bing; He, Songmin; Su, Zuqing; Zheng, Guangjuan

2018-02-01

To explore the preventive and therapeutic effects of Resveratrol combined with total flavones of hawthorn, compatibility of traditional Chinese medicines, on the endothelial cells injury after artery bypass graft surgery. The animal model of coronary artery bypass grafting (CABG) was prepared by transplanting a segment of autologous jugular vein onto the transected common carotid artery in rabbits. After CABG surgery, the rabbits were administrated with saline (model group), aspirin (Aspirin group), resveratrol (Res group), total flavones of hawthorn (Haw group) and resveratrol combined with total flavones of hawthorn (Res+Haw group) once a day for eight weeks, respectively. Eight weeks later, the grafting arteries from all group were obtained for the pathomorphism observation, peripheral blood was collected to detect circulating endothelial cells (CECs) by flow cytometry. And the concentration of albumen and mRNA of ICAM-1 in the serum were measured by western blot and quantitative real-time polymerase chain reaction, respectively. Compared with the model group, the level of CECs density and the expressions of albumen and mRNA of ICAM-1 were significantly decreased in the aspirin,resveratrol,total flavones of hawthorn and resveratrol combined with total flavones of hawthorn groups (P Resveratrol combined with total flavones of hawthorn could protect the endothelial cells after coronary artery bypass graft. Copyright © 2018 Elsevier GmbH. All rights reserved.

A role for activated endothelial cells in red blood cell clearance: implications for vasopathology

DEFF Research Database (Denmark)

Fens, Marcel H A M; van Wijk, Richard; Andringa, Grietje

2012-01-01

with endothelial cells, marked erythrophagocytosis occurred in the presence of lactadherin under both static and flow conditions. As a consequence, intracellular organization was disturbed and endothelial cells were seen to change shape (‘rounding up’). Increased expression of apoptotic markers indicated...

Human Endothelial Cells: Use of Heparin in Cloning and Long-Term Serial Cultivation

Science.gov (United States)

Thornton, Susan C.; Mueller, Stephen N.; Levine, Elliot M.

1983-11-01

Endothelial cells from human blood vessels were cultured in vitro, with doubling times of 17 to 21 hours for 42 to 79 population doublings. Cloned human endothelial cell strains were established for the first time and had similar proliferative capacities. This vigorous cell growth was achieved by addition of heparin to culture medium containing reduced concentrations of endothelial cell growth factor. The routine cloning and long-term culture of human endothelial cells will facilitate studying the human endothelium in vitro.

Biochemical and microscopic evidence for the internalization and degradation of heparin-containing mast cell granules by bovine endothelial cells

International Nuclear Information System (INIS)

Atkins, F.M.; Friedman, M.M.; Metcalfe, D.D.

1985-01-01

Incubation of [ 35 S]heparin-containing mast cell granules with cultured bovine endothelial cells was followed by the appearance of 35 S-granule-associated radioactivity within the endothelial cells and a decrease in radioactivity in the extracellular fluid. These changes occurred during the first 24 hours of incubation and suggested ingestion of the mast cell granules by the endothelial cells. Periodic electron microscopic examination of the monolayers confirmed this hypothesis by demonstrating apposition of the granules to the plasmalemma of endothelial cells, which was followed by the engulfment of the granules by cytoplasmic projections. Under light microscopic examination, mast cell granules within endothelial cells then appeared to undergo degradation. The degradation of [ 35 S]heparin in mast cell granules was demonstrated by a decrease in the amount of intracellular [ 35 S]heparin proteoglycan after 24 hours and the appearance of free [ 35 S]sulfate in the extracellular compartment. Intact endothelial cells were more efficient at degrading [ 35 S]heparin than were cell lysates or cell supernatants. These data provide evidence of the ability of endothelial cells to ingest mast cell granules and degrade native heparin that is presented as a part of the mast cell granule

Recovery of Corneal Endothelial Cells from Periphery after Injury.

Directory of Open Access Journals (Sweden)

Sang Ouk Choi

Full Text Available Wound healing of the endothelium occurs through cell enlargement and migration. However, the peripheral corneal endothelium may act as a cell resource for the recovery of corneal endothelium in endothelial injury.To investigate the recovery process of corneal endothelial cells (CECs from corneal endothelial injury.Three patients with unilateral chemical eye injuries, and 15 rabbit eyes with corneal endothelial chemical injuries were studied. Slit lamp examination, specular microscopy, and ultrasound pachymetry were performed immediately after chemical injury and 1, 3, 6, and 9 months later. The anterior chambers of eyes from New Zealand white rabbits were injected with 0.1 mL of 0.05 N NaOH for 10 min (NaOH group. Corneal edema was evaluated at day 1, 7, and 14. Vital staining was performed using alizarin red and trypan blue.Specular microscopy did not reveal any corneal endothelial cells immediately after injury. Corneal edema subsided from the periphery to the center, CEC density increased, and central corneal thickness decreased over time. In the animal study, corneal edema was greater in the NaOH group compared to the control at both day 1 and day 7. At day 1, no CECs were detected at the center and periphery of the corneas in the NaOH group. Two weeks after injury, small, hexagonal CECs were detected in peripheral cornea, while CECs in mid-periphery were large and non-hexagonal.CECs migrated from the periphery to the center of the cornea after endothelial injury. The peripheral corneal endothelium may act as a cell resource for the recovery of corneal endothelium.

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Maternal biomarkers of endothelial dysfunction and preterm delivery.

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Xinhua Chen

Full Text Available Endothelial dysfunction is key to the development of atherosclerosis. Preterm delivery foreshadows later maternal cardiovascular disease (CVD, but it is not known if endothelial dysfunction also occurs. We prospectively measured circulating biomarkers of endothelial dysfunction in pregnant women with preterm or term delivery.We conducted a case-control study nested within a large prospective epidemiological study of young, generally healthy pregnant women. Women who delivered preterm (<37 completed weeks gestation, n = 240 and controls who delivered at term (n = 439 were included. Pregnancies complicated by preeclampsia were analyzed separately. Circulating endothelial dysfunction biomarkers included soluble intercellular adhesion molecule-1 (sICAM-1, vascular cell adhesion molecule-1 (sVCAM-1 and soluble E-selectin (sE-selectin.Elevated levels of sICAM-1 and sVCAM-1 were positively associated with preterm delivery independent of usual risk factors. At entry (∼16 wks, the adjusted odds ratio (AOR was 1.73 (95% confidence interval (CI 1.09-2.74 for the highest quartile of sICAM-1 versus the lowest quartile and for sVCAM-1 the AOR was 2.17 (95% CI 1.36-3.46. When analysis was limited to cases with a spontaneous preterm delivery, the results were unchanged. Similar results were obtained for the 3rd trimester (∼30 wks. Elevated sE-selectin was increased only in preterm delivery complicated by preeclampsia; risk was increased at entry (AOR 2.32, 95% CI 1.22-4.40 and in the 3rd trimester (AOR 3.37, 95% CI 1.78-6.39.Impaired endothelial function as indicated by increased levels of soluble molecules commonly secreted by endothelial cells is a pathogenic precursor to CVD that is also present in women with preterm delivery. Our findings underscore the need for follow-up studies to determine if improving endothelial function prevents later CVD risk in women.

Matrix production and organization by endothelial colony forming cells in mechanically strained engineered tissue constructs.

Directory of Open Access Journals (Sweden)

Nicky de Jonge

Full Text Available AIMS: Tissue engineering is an innovative method to restore cardiovascular tissue function by implanting either an in vitro cultured tissue or a degradable, mechanically functional scaffold that gradually transforms into a living neo-tissue by recruiting tissue forming cells at the site of implantation. Circulating endothelial colony forming cells (ECFCs are capable of differentiating into endothelial cells as well as a mesenchymal ECM-producing phenotype, undergoing Endothelial-to-Mesenchymal-transition (EndoMT. We investigated the potential of ECFCs to produce and organize ECM under the influence of static and cyclic mechanical strain, as well as stimulation with transforming growth factor β1 (TGFβ1. METHODS AND RESULTS: A fibrin-based 3D tissue model was used to simulate neo-tissue formation. Extracellular matrix organization was monitored using confocal laser-scanning microscopy. ECFCs produced collagen and also elastin, but did not form an organized matrix, except when cultured with TGFβ1 under static strain. Here, collagen was aligned more parallel to the strain direction, similar to Human Vena Saphena Cell-seeded controls. Priming ECFC with TGFβ1 before exposing them to strain led to more homogenous matrix production. CONCLUSIONS: Biochemical and mechanical cues can induce extracellular matrix formation by ECFCs in tissue models that mimic early tissue formation. Our findings suggest that priming with bioactives may be required to optimize neo-tissue development with ECFCs and has important consequences for the timing of stimuli applied to scaffold designs for both in vitro and in situ cardiovascular tissue engineering. The results obtained with ECFCs differ from those obtained with other cell sources, such as vena saphena-derived myofibroblasts, underlining the need for experimental models like ours to test novel cell sources for cardiovascular tissue engineering.

Endothelial remodelling and intracellular calcium machinery.

Science.gov (United States)

Moccia, F; Tanzi, F; Munaron, L

2014-05-01

Rather being an inert barrier between vessel lumen and surrounding tissues, vascular endothelium plays a key role in the maintenance of cardiovascular homeostasis. The de-endothelialization of blood vessels is regarded as the early event that results in the onset of severe vascular disorders, including atherosclerosis, acute myocardial infarction, brain stroke, and aortic aneurysm. Restoration of the endothelial lining may be accomplished by the activation of neighbouring endothelial cells (ECs) freed by contact inhibition and by circulating endothelial progenitor cells (EPCs). Intracellular Ca(2+) signalling is essential to promote wound healing: however, the molecular underpinnings of the Ca(2+) response to injury are yet to be fully elucidated. Similarly, the components of the Ca(2+) toolkit that drive EPC incorporation into denuded vessels are far from being fully elucidated. The present review will survey the current knowledge on the role of Ca(2+) signalling in endothelial repair and in EPC activation. We propose that endothelial regeneration might be boosted by intraluminal release of specific Ca(2+) channel agonists or by gene transfer strategies aiming to enhance the expression of the most suitable Ca(2+) channels at the wound site. In this view, connexin (Cx) channels/hemichannels and store-operated Ca(2+) entry (SOCE) stand amid the most proper routes to therapeutically induce the regrowth of denuded vessels. Cx stimulation might trigger the proliferative and migratory behaviour of ECs facing the lesion site, whereas activation of SOCE is likely to favour EPC homing to the wounded vessel.

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Prognostic value of circulating VEGFR2+ bone marrow-derived progenitor cells in patients with advanced cancer.

Science.gov (United States)

Massard, Christophe; Borget, Isabelle; Le Deley, Marie Cécile; Taylor, Melissa; Gomez-Roca, Carlos; Soria, Jean Charles; Farace, Françoise

2012-06-01

We hypothesised that host-related markers, possibly reflecting tumour aggressiveness, such as circulating endothelial cells (CEC) and circulating VEGFR2(+) bone marrow-derived (BMD) progenitor cells, could have prognostic value in patients with advanced cancer enrolled in early anticancer drug development trials. Baseline CECs (CD45(-)CD31(+)CD146(+)7AAD(-) cells) and circulating VEGFR2(+)-BMD progenitor cells (defined as CD45(dim)CD34(+)VEGFR2(+)7AAD(-) cells) were measured by flow-cytometry in 71 and 58 patients included in phase 1 trials testing novel anti-vascular or anti-angiogenic agents. Correlations between levels of CECs, circulating VEGFR2(+)-BMD progenitor cells, clinical and biological prognostic factors (i.e. the Royal Marsden Hospital (RMH) score), and overall survival (OS) were studied. The median value of CECs was 12 CEC/ml (range 0-154/ml). The median level of VEGFR2(+)-BMD progenitor cells was 1.3% (range 0-32.5%) of circulating BMD-CD34(+) progenitors. While OS was not correlated with CEC levels, it was significantly worse in patients with high VEGFR2(+)-BMD progenitor levels (>1%) (median OS 9.0 versus 17.0 months), and with a RMH prognostic score >0 (median OS 9.0 versus 24.2 months). The prognostic value of VEGFR2(+)-BMD progenitor levels remained significant (hazard ratio (HR) = 2.3, 95% confidence interval (CI), 1.1-4.6, p = 0.02) after multivariate analysis. A composite VEGFR2(+)-BMD progenitor level/RHM score ≥ 2 was significantly associated with an increased risk of death compared to scores of 0 or 1 (median OS 9.0 versus 18.4 months, HR = 2.6 (95%CI, 1.2-5.8, p = 0.02)). High circulating VEGFR2(+)-BMD progenitor levels are associated with poor prognostics and when combined to classical clinical and biological parameters could provide a new tool for patient selection in early anticancer drug trials. Copyright © 2012 Elsevier Ltd. All rights reserved.

The adaptor CRADD/RAIDD controls activation of endothelial cells by proinflammatory stimuli.

Science.gov (United States)

Qiao, Huan; Liu, Yan; Veach, Ruth A; Wylezinski, Lukasz; Hawiger, Jacek

2014-08-08

A hallmark of inflammation, increased vascular permeability, is induced in endothelial cells by multiple agonists through stimulus-coupled assembly of the CARMA3 signalosome, which contains the adaptor protein BCL10. Previously, we reported that BCL10 in immune cells is targeted by the "death" adaptor CRADD/RAIDD (CRADD), which negatively regulates nuclear factor κB (NFκB)-dependent cytokine and chemokine expression in T cells (Lin, Q., Liu, Y., Moore, D. J., Elizer, S. K., Veach, R. A., Hawiger, J., and Ruley, H. E. (2012) J. Immunol. 188, 2493-2497). This novel anti-inflammatory CRADD-BCL10 axis prompted us to analyze CRADD expression and its potential anti-inflammatory action in non-immune cells. We focused our study on microvascular endothelial cells because they play a key role in inflammation. We found that CRADD-deficient murine endothelial cells display heightened BCL10-mediated expression of the pleotropic proinflammatory cytokine IL-6 and chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) in response to LPS and thrombin. Moreover, these agonists also induce significantly increased permeability in cradd(-/-), as compared with cradd(+/+), primary murine endothelial cells. CRADD-deficient cells displayed more F-actin polymerization with concomitant disruption of adherens junctions. In turn, increasing intracellular CRADD by delivery of a novel recombinant cell-penetrating CRADD protein (CP-CRADD) restored endothelial barrier function and suppressed the induction of IL-6 and MCP-1 evoked by LPS and thrombin. Likewise, CP-CRADD enhanced barrier function in CRADD-sufficient endothelial cells. These results indicate that depletion of endogenous CRADD compromises endothelial barrier function in response to inflammatory signals. Thus, we define a novel function for CRADD in endothelial cells as an inducible suppressor of BCL10, a key mediator of responses to proinflammatory agonists. © 2014 by The American Society for Biochemistry and Molecular Biology

Tumor necrosis factor-α enhanced fusions between oral squamous cell carcinoma cells and endothelial cells via VCAM-1/VLA-4 pathway

International Nuclear Information System (INIS)

Song, Kai; Zhu, Fei; Zhang, Han-zhong; Shang, Zheng-jun

2012-01-01

Fusion between cancer cells and host cells, including endothelial cells, may strongly modulate the biological behavior of tumors. However, no one is sure about the driving factors and underlying mechanism involved in such fusion. We hypothesized in this study that inflammation, one of the main characteristics in tumor microenvironment, serves as a prominent catalyst for fusion events. Our results showed that oral cancer cells can fuse spontaneously with endothelial cells in co-culture and inflammatory cytokine tumor necrosis factor-α (TNF-α) increased fusion of human umbilical vein endothelium cells and oral cancer cells by up to 3-fold in vitro. Additionally, human oral squamous cell carcinoma cell lines and 35 out of 50 (70%) oral squamous carcinoma specimens express VLA-4, an integrin, previously implicated in fusions between human peripheral blood CD34-positive cells and murine cardiomyocytes. Expression of VCAM-1, a ligand for VLA-4, was evident on vascular endothelium of oral squamous cell carcinoma. Moreover, immunocytochemistry and flow cytometry analysis revealed that expression of VCAM-1 increased obviously in TNF-α-stimulated endothelial cells. Anti-VLA-4 or anti-VCAM-1 treatment can decrease significantly cancer–endothelial adhesion and block such fusion. Collectively, our results suggested that TNF-α could enhance cancer–endothelial cell adhesion and fusion through VCAM-1/VLA-4 pathway. This study provides insights into regulatory mechanism of cancer–endothelial cell fusion, and has important implications for the development of novel therapeutic strategies for prevention of metastasis. -- Highlights: ► Spontaneous oral cancer–endothelial cell fusion. ► TNF-α enhanced cell fusions. ► VCAM-1/VLA-4 expressed in oral cancer. ► TNF-α increased expression of VCAM-1 on endothelial cells. ► VCAM-1/VLA-4 mediated TNF-α-enhanced cell fusions.

Tumor necrosis factor-{alpha} enhanced fusions between oral squamous cell carcinoma cells and endothelial cells via VCAM-1/VLA-4 pathway

Energy Technology Data Exchange (ETDEWEB)

Song, Kai; Zhu, Fei; Zhang, Han-zhong [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST), Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); Shang, Zheng-jun, E-mail: shangzhengjun@hotmail.com [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST), Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); First Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Wuhan University, Wuhan (China)

2012-08-15

Fusion between cancer cells and host cells, including endothelial cells, may strongly modulate the biological behavior of tumors. However, no one is sure about the driving factors and underlying mechanism involved in such fusion. We hypothesized in this study that inflammation, one of the main characteristics in tumor microenvironment, serves as a prominent catalyst for fusion events. Our results showed that oral cancer cells can fuse spontaneously with endothelial cells in co-culture and inflammatory cytokine tumor necrosis factor-{alpha} (TNF-{alpha}) increased fusion of human umbilical vein endothelium cells and oral cancer cells by up to 3-fold in vitro. Additionally, human oral squamous cell carcinoma cell lines and 35 out of 50 (70%) oral squamous carcinoma specimens express VLA-4, an integrin, previously implicated in fusions between human peripheral blood CD34-positive cells and murine cardiomyocytes. Expression of VCAM-1, a ligand for VLA-4, was evident on vascular endothelium of oral squamous cell carcinoma. Moreover, immunocytochemistry and flow cytometry analysis revealed that expression of VCAM-1 increased obviously in TNF-{alpha}-stimulated endothelial cells. Anti-VLA-4 or anti-VCAM-1 treatment can decrease significantly cancer-endothelial adhesion and block such fusion. Collectively, our results suggested that TNF-{alpha} could enhance cancer-endothelial cell adhesion and fusion through VCAM-1/VLA-4 pathway. This study provides insights into regulatory mechanism of cancer-endothelial cell fusion, and has important implications for the development of novel therapeutic strategies for prevention of metastasis. -- Highlights: Black-Right-Pointing-Pointer Spontaneous oral cancer-endothelial cell fusion. Black-Right-Pointing-Pointer TNF-{alpha} enhanced cell fusions. Black-Right-Pointing-Pointer VCAM-1/VLA-4 expressed in oral cancer. Black-Right-Pointing-Pointer TNF-{alpha} increased expression of VCAM-1 on endothelial cells. Black

Hypoxia, leukocytes, and the pulmonary circulation.

Science.gov (United States)

Stenmark, Kurt R; Davie, Neil J; Reeves, John T; Frid, Maria G

2005-02-01

Data are rapidly accumulating in support of the idea that circulating monocytes and/or mononuclear fibrocytes are recruited to the pulmonary circulation of chronically hypoxic animals and that these cells play an important role in the pulmonary hypertensive process. Hypoxic induction of monocyte chemoattractant protein-1, stromal cell-derived factor-1, vascular endothelial growth factor-A, endothelin-1, and tumor growth factor-beta(1) in pulmonary vessel wall cells, either directly or indirectly via signals from hypoxic lung epithelial cells, may be a critical first step in the recruitment of circulating leukocytes to the pulmonary circulation. In addition, hypoxic stress appears to induce release of increased numbers of monocytic progenitor cells from the bone marrow, and these cells may have upregulated expression of receptors for the chemokines produced by the lung circulation, which thus facilitates their specific recruitment to the pulmonary site. Once present, macrophages/fibrocytes may exert paracrine effects on resident pulmonary vessel wall cells stimulating proliferation, phenotypic modulation, and migration of resident fibroblasts and smooth muscle cells. They may also contribute directly to the remodeling process through increased production of collagen and/or differentiation into myofibroblasts. In addition, they could play a critical role in initiating and/or supporting neovascularization of the pulmonary artery vasa vasorum. The expanded vasa network may then act as a conduit for further delivery of circulating mononuclear cells to the pulmonary arterial wall, creating a feedforward loop of pathological remodeling. Future studies will need to determine the mechanisms that selectively induce leukocyte/fibrocyte recruitment to the lung circulation under hypoxic conditions, their direct role in the remodeling process via production of extracellular matrix and/or differentiation into myofibroblasts, their impact on the phenotype of resident smooth muscle

Association of endothelial progenitor cells and peptic ulcer treatment in patients with type 2 diabetes mellitus.

Science.gov (United States)

Nie, Zhihong; Xu, Limin; Li, Chuanyuan; Tian, Tao; Xie, Pingping; Chen, Xia; Li, Bojing

2016-05-01

The present study aimed to investigate the association between endothelial progenitor cells (EPCs) and peptic ulcers in patients with or without type 2 diabetes mellitus (T2DM), in association with the efficiency of peptic ulcer treatment. The study recruited healthy subjects and peptic ulcer patients with or without T2DM. All the ulcer patients, including those with and without T2DM, were administered omeprazole for 8 weeks. Peptic ulcer patients with T2DM were additionally treated with glipizide and novolin. Blood samples were then obtained from the three groups following ulcer treatment. CD133 + cells were isolated from the blood samples using magnetic bead selection, and cultured in complete medium 199. Morphological and quantity changes in EPCs were observed by light and fluorescence microscopy. In addition, flow cytometric analysis was used to quantify the number of vascular endothelial cells. The treatment was partially effective in 7 of the 32 peptic ulcer patients without T2DM and 12 of the 32 peptic ulcer patients with T2DM. However, this treatment was ineffective in 20 of the 32 peptic ulcer patients with T2DM. Notably, 25 peptic ulcer patients without T2DM were defined as completely recovered following treatment. In addition, the number of circulating EPCs as well as their colony forming ability was significantly reduced (Ppeptic ulcer patients with T2DM following ulcer treatment, compared with the other groups. Circulating EPC counts were significantly increased in peptic ulcer patients without T2DM, as compared with the healthy controls. With regards to colony formation, peptic ulcer patients without T2DM did not exhibit improved colony formation ability. In conclusion, the number of circulating EPCs and their colony-forming ability was significantly reduced in peptic ulcer patients with T2DM following ulcer treatment when compared with the other groups. This suggests that the poor curative effect of peptic ulcer treatment in these patients is

Viability and proliferation of endothelial cells upon exposure to GaN nanoparticles.

Science.gov (United States)

Braniste, Tudor; Tiginyanu, Ion; Horvath, Tibor; Raevschi, Simion; Cebotari, Serghei; Lux, Marco; Haverich, Axel; Hilfiker, Andres

2016-01-01

Nanotechnology is a rapidly growing and promising field of interest in medicine; however, nanoparticle-cell interactions are not yet fully understood. The goal of this work was to examine the interaction between endothelial cells and gallium nitride (GaN) semiconductor nanoparticles. Cellular viability, adhesion, proliferation, and uptake of nanoparticles by endothelial cells were investigated. The effect of free GaN nanoparticles versus the effect of growing endothelial cells on GaN functionalized surfaces was examined. To functionalize surfaces with GaN, GaN nanoparticles were synthesized on a sacrificial layer of zinc oxide (ZnO) nanoparticles using hydride vapor phase epitaxy. The uptake of GaN nanoparticles by porcine endothelial cells was strongly dependent upon whether they were fixed to the substrate surface or free floating in the medium. The endothelial cells grown on surfaces functionalized with GaN nanoparticles demonstrated excellent adhesion and proliferation, suggesting good biocompatibility of the nanostructured GaN.

Radiosensitization of human endothelial cells by IL-24

International Nuclear Information System (INIS)

Meyn, R.E.

2003-01-01

Radiation therapy remains an important cancer treatment modality but despite improvements in dose delivery many patients still fail at their primary tumor site. Therefore, new strategies designed to improve local control are needed. Protocols combining radiation with anti-angiogenic agents might be of particular advantage based on their documented low toxicity. In this regard, we have been conducting preclinical investigations of a novel cytokine, mda7/IL-24. Our collaborators have shown that mda7/IL-24 protein targets the endothelial cells of the tumor microvascular system and has potent anti-angiogenic properties in both in vitro and in vivo assays. Recently, we have demonstrated that recombinant mda7/IL-24 protein radiosensitizes human endothelial cells in vitro. Specifically, 10 ng/ml of recombinant human IL-24 protein for 12 hrs reduced the survival at 2 Gy for human umbilical vein endothelial cells (HUVECs) from 0.33 to 0.12. We are also working on understanding the molecular basis for this radiosensitizing effect. Preliminary data suggest a model whereby mda7/IL-24 engages a specific receptor on the surface of endothelial cells and initiates a signal transduction pathway that modulates the cell's propensity for radiation-induced apoptosis and capacity for repairing radiation-induced DNA double strand breaks. Mechanistic insight gained from these studies may have implications for the actions of other anti-angiogenic agents and may generally explain the regulation of radiosensitivity imparted by growth factors and cytokines

Hyaluronan protection of corneal endothelial cells against extracellular histones after phacoemulsification.

Science.gov (United States)

Kawano, Hiroki; Sakamoto, Taiji; Ito, Takashi; Miyata, Kazunori; Hashiguchi, Teruto; Maruyama, Ikuro

2014-11-01

To determine the effect of histones on corneal endothelial cells generated during cataract surgery. Kagoshima University Hospital, Kagoshima, Japan. Experimental study. Standard phacoemulsification was performed on enucleated pig eyes. Histones in the anterior segment of the eye were determined by immunohistochemistry. Cultured human corneal endothelial cells were exposed to histones for 18 hours, and cell viability was determined by 2-(2-methoxy-4-nitrophenyl)-3-(4-nitro-phenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt assay. The concentration of interleukin-6 (IL-6) in the culture medium of human corneal endothelial cells was measured using enzyme-linked immunosorbent assay. The effects of signal inhibitors U0126, SB203580, and SP600125 were evaluated. The protective effect of hyaluronan against histones was evaluated in human corneal endothelial cells with and without hyaluronan. Cellular debris containing histones was observed in the anterior chamber of pig eyes after phacoemulsification. Exposure of human corneal endothelial cells to 50 μg/mL of histones or more led to cytotoxic effects. The IL-6 concentration was significantly increased dose dependently after exposure of human corneal endothelial cells to histones (Phistone-induced IL-6 production was significantly decreased by extracellular signal-regulated kinases 1/2 and p-38 mitogen-activated protein kinase inhibitors (Phistones caused formation of histone aggregates, decreased the cytotoxic effects of the histones, and blocked the increase in IL-6 (PHistones were released extracellularly during phacoemulsification and exposure of human corneal endothelial cells to histones increased the IL-6 secretion. The intraoperative use of hyaluronan may decrease the cytotoxic effects of histones released during cataract surgery. No author has a financial or proprietary interest in any material or method mentioned. Copyright © 2014 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

Upcyte® Microvascular Endothelial Cells Repopulate Decellularized Scaffold

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Dally, Iris; Hartmann, Nadja; Münst, Bernhard; Braspenning, Joris; Walles, Heike

2013-01-01

A general problem in tissue engineering is the poor and insufficient blood supply to guarantee tissue cell survival as well as physiological tissue function. To address this limitation, we have developed an in vitro vascularization model in which a decellularized porcine small bowl segment, representing a capillary network within a collagen matrix (biological vascularized scaffold [BioVaSc]), is reseeded with microvascular endothelial cells (mvECs). However, since the supply of mvECs is limited, in general, and as these cells rapidly dedifferentiate, we have applied a novel technology, which allows the generation of large batches of quasi-primary cells with the ability to proliferate, whilst maintaining their differentiated functionality. These so called upcyte mvECs grew for an additional 15 population doublings (PDs) compared to primary cells. Upcyte mvECs retained endothelial characteristics, such as von Willebrandt Factor (vWF), CD31 and endothelial nitric oxide synthase (eNOS) expression, as well as positive Ulex europaeus agglutinin I staining. Upcyte mvECs also retained biological functionality such as tube formation, cell migration, and low density lipoprotein (LDL) uptake, which were still evident after PD27. Initial experiments using MTT and Live/Dead staining indicate that upcyte mvECs repopulate the BioVaSc Scaffold. As with conventional cultures, these cells also express key endothelial molecules (vWF, CD31, and eNOS) in a custom-made bioreactor system even after a prolonged period of 14 days. The combination of upcyte mvECs and the BioVaSc represents a novel and promising approach toward vascularizing bioreactor models which can better reflect organs, such as the liver. PMID:22799502

The targeting expression of the vascular endothelial growth factor gene in endothelial cells regulated by HRE.ppET-1.

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Zheng, Xiangrong; Zhang, Shangshang; Yang, Yujia; Wang, Xia; Zhong, Le; Yu, Xiaohe

2008-11-01

The success of gene therapy depends largely on the efficacy of gene delivery vector systems that can deliver genes to target organs or cells selectively and efficiently with minimal toxicity. Here, we show that by using the HRE.ppET-1 regulatory element, we were able to restrict expression of the transgene of vascular endothelial growth factor (VEGF) to endothelial cells exclusively in hypoxic conditions. Eukaryotic expression vectors such as pEGFP-HRE.ppET-1, pcDNA3.1-VEGF+Pa, pcDNA3.1-ppET-1+ EGF+Pa, and pcDNA3.1-HRE.ppET-1+VEGF+Pa were constructed by using a series of nuclear molecule handling methods like PCR, enzyme digestion. The recombinant vectors were transfected into HUVEC cells and HL7702 cells by the lipofectin method. GFP expression was observed with a fluorescence microscope to validate the specificity of expression in endothelial cells under the regulation of HRE.ppET-1 element. Cobalt chloride (final concentration 100 mumol/L) was added to the medium to mimic hypoxia in vitro. After transfection of vectors, the expression of VEGF mRNA was detected by RT-PCR, and the expression of VEGF was detected by Western blotting and ELISA methods under normoxia and hypoxia, respectively. The cell proliferation rate was detected by the MTT test. The expression of GFP revealed that the exterior gene was transcripted effectively in endothelial cells regulated by the HRE.ppET-1 element, while the expression of GFP was very weak in nonendothelial cells. The results of RT-PCR, Western blotting and ELISA showed that VEGF gene expression in the pcDNA3.1-HRE.ppET-1+VEGF+Pa group and in the pcDNA3.1-ppET-1+VEGF+Pa group was higher in hypoxia than it was in normoxia (PHRE.ppET-1 element was expressed specifically in endothelial cells, and can increase the expression of VEGF in hypoxia and stimulate proliferation of endothelial cells. Taking advantage of these facts could greatly improve the efficiency of gene therapy. The vector would be valuable for various gene transfer

Towards a Biohybrid Lung: Endothelial Cells Promote Oxygen Transfer through Gas Permeable Membranes.

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Menzel, Sarah; Finocchiaro, Nicole; Donay, Christine; Thiebes, Anja Lena; Hesselmann, Felix; Arens, Jutta; Djeljadini, Suzana; Wessling, Matthias; Schmitz-Rode, Thomas; Jockenhoevel, Stefan; Cornelissen, Christian Gabriel

2017-01-01

In patients with respiratory failure, extracorporeal lung support can ensure the vital gas exchange via gas permeable membranes but its application is restricted by limited long-term stability and hemocompatibility of the gas permeable membranes, which are in contact with the blood. Endothelial cells lining these membranes promise physiological hemocompatibility and should enable prolonged application. However, the endothelial cells increase the diffusion barrier of the blood-gas interface and thus affect gas transfer. In this study, we evaluated how the endothelial cells affect the gas exchange to optimize performance while maintaining an integral cell layer. Human umbilical vein endothelial cells were seeded on gas permeable cell culture membranes and cultivated in a custom-made bioreactor. Oxygen transfer rates of blank and endothelialized membranes in endothelial culture medium were determined. Cell morphology was assessed by microscopy and immunohistochemistry. Both setups provided oxygenation of the test fluid featuring small standard deviations of the measurements. Throughout the measuring range, the endothelial cells seem to promote gas transfer to a certain extent exceeding the blank membranes gas transfer performance by up to 120%. Although the underlying principles hereof still need to be clarified, the results represent a significant step towards the development of a biohybrid lung.

Characterization of Endothelial Progenitor Cell Interactions with Human Tropoelastin.

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Young Yu

Full Text Available The deployment of endovascular implants such as stents in the treatment of cardiovascular disease damages the vascular endothelium, increasing the risk of thrombosis and promoting neointimal hyperplasia. The rapid restoration of a functional endothelium is known to reduce these complications. Circulating endothelial progenitor cells (EPCs are increasingly recognized as important contributors to device re-endothelialization. Extracellular matrix proteins prominent in the vessel wall may enhance EPC-directed re-endothelialization. We examined attachment, spreading and proliferation on recombinant human tropoelastin (rhTE and investigated the mechanism and site of interaction. EPCs attached and spread on rhTE in a dose dependent manner, reaching a maximal level of 56±3% and 54±3%, respectively. EPC proliferation on rhTE was comparable to vitronectin, fibronectin and collagen. EDTA, but not heparan sulfate or lactose, reduced EPC attachment by 81±3%, while full attachment was recovered after add-back of manganese, inferring a classical integrin-mediated interaction. Integrin αVβ3 blocking antibodies decreased EPC adhesion and spreading on rhTE by 39±3% and 56±10% respectively, demonstrating a large contribution from this specific integrin. Attachment of EPCs on N-terminal rhTE constructs N25 and N18 accounted for most of this interaction, accompanied by comparable spreading. In contrast, attachment and spreading on N10 was negligible. αVβ3 blocking antibodies reduced EPC spreading on both N25 and N18 by 45±4% and 42±14%, respectively. In conclusion, rhTE supports EPC binding via an integrin mechanism involving αVβ3. N25 and N18, but not N10 constructs of rhTE contribute to EPC binding. The regulation of EPC activity by rhTE may have implications for modulation of the vascular biocompatibility of endovascular implants.

Cytomegalovirus-Induced Effector T Cells Cause Endothelial Cell Damage

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van de Berg, Pablo J. E. J.; Yong, Si-La; Remmerswaal, Ester B. M.; van Lier, René A. W.; ten Berge, Ineke J. M.

2012-01-01

Human cytomegalovirus (CMV) infection has been linked to inflammatory diseases that involve vascular endothelial cell damage, but definitive proof for a direct cytopathic effect of CMV in these diseases is lacking. CMV infection is associated with a strong increase in both CD4(+) and CD8(+) T cells

Iron oxide nanoparticles induce human microvascular endothelial cell permeability through reactive oxygen species production and microtubule remodeling

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Shi Xianglin

2009-01-01

Full Text Available Abstract Background Engineered iron nanoparticles are being explored for the development of biomedical applications and many other industry purposes. However, to date little is known concerning the precise mechanisms of translocation of iron nanoparticles into targeted tissues and organs from blood circulation, as well as the underlying implications of potential harmful health effects in human. Results The confocal microscopy imaging analysis demonstrates that exposure to engineered iron nanoparticles induces an increase in cell permeability in human microvascular endothelial cells. Our studies further reveal iron nanoparticles enhance the permeability through the production of reactive oxygen species (ROS and the stabilization of microtubules. We also showed Akt/GSK-3β signaling pathways are involved in iron nanoparticle-induced cell permeability. The inhibition of ROS demonstrate ROS play a major role in regulating Akt/GSK-3β – mediated cell permeability upon iron nanoparticle exposure. These results provide new insights into the bioreactivity of engineered iron nanoparticles which can inform potential applications in medical imaging or drug delivery. Conclusion Our results indicate that exposure to iron nanoparticles induces an increase in endothelial cell permeability through ROS oxidative stress-modulated microtubule remodeling. The findings from this study provide new understandings on the effects of nanoparticles on vascular transport of macromolecules and drugs.

Endothelial cell adhesion to ion implanted polymers

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Suzuki, Y; Kusakabe, M [SONY Corp., Tokyo (Japan); Lee, J S; Kaibara, M; Iwaki, M; Sasabe, H [RIKEN (Inst. of Physical and Chemical Research), Saitama (Japan)

1992-03-01

The biocompatibility of ion implanted polymers has been studied by means of adhesion measurements of bovine aorta endothelial cells in vitro. The specimens used were polystyrene (PS) and segmented polyurethane (SPU). Na{sup +}, N{sub 2}{sup +}, O{sub 2}{sup +} and Kr{sup +} ion implantations were performed at an energy of 150 keV with fluences ranging from 1x10{sup 15} to 3x10{sup 17} ions/cm{sup 2} at room temperature. The chemical and physical structures of ion-implanted polymers have been investigated in order to analyze their tissue compatibility such as improvement of endothelial cell adhesion. The ion implanted SPU have been found to exhibit remarkably higher adhesion and spreading of endothelial cells than unimplanted specimens. By contrast, ion implanted PS demonstrated a little improvement of adhesion of cells in this assay. Results of FT-IR-ATR showed that ion implantation broke the original chemical bond to form new radicals such as OH, ....C=O, SiH and condensed rings. The results of Raman spectroscopy showed that ion implantation always produced a peak near 1500 cm{sup -1}, which indicated that these ion implanted PS and SPU had the same carbon structure. This structure is considered to bring the dramatic increase in the extent of cell adhesion and spreading to these ion implanted PS and SPU. (orig.).

Expression of a retinoic acid signature in circulating CD34 cells from coronary artery disease patients

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van der Laan Anja M

2010-06-01

Full Text Available Abstract Background Circulating CD34+ progenitor cells have the potential to differentiate into a variety of cells, including endothelial cells. Knowledge is still scarce about the transcriptional programs used by CD34+ cells from peripheral blood, and how these are affected in coronary artery disease (CAD patients. Results We performed a whole genome transcriptome analysis of CD34+ cells, CD4+ T cells, CD14+ monocytes, and macrophages from 12 patients with CAD and 11 matched controls. CD34+ cells, compared to other mononuclear cells from the same individuals, showed high levels of KRAB box transcription factors, known to be involved in gene silencing. This correlated with high expression levels in CD34+ cells for the progenitor markers HOXA5 and HOXA9, which are known to control expression of KRAB factor genes. The comparison of expression profiles of CD34+ cells from CAD patients and controls revealed a less naïve phenotype in patients' CD34+ cells, with increased expression of genes from the Mitogen Activated Kinase network and a lowered expression of a panel of histone genes, reaching levels comparable to that in more differentiated circulating cells. Furthermore, we observed a reduced expression of several genes involved in CXCR4-signaling and migration to SDF1/CXCL12. Conclusions The altered gene expression profile of CD34+ cells in CAD patients was related to activation/differentiation by a retinoic acid-induced differentiation program. These results suggest that circulating CD34+ cells in CAD patients are programmed by retinoic acid, leading to a reduced capacity to migrate to ischemic tissues.

A small population of liver endothelial cells undergoes endothelial-to-mesenchymal transition in response to chronic liver injury.

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Ribera, Jordi; Pauta, Montse; Melgar-Lesmes, Pedro; Córdoba, Bernat; Bosch, Anna; Calvo, Maria; Rodrigo-Torres, Daniel; Sancho-Bru, Pau; Mira, Aurea; Jiménez, Wladimiro; Morales-Ruiz, Manuel

2017-11-01

Rising evidence points to endothelial-to-mesenchymal transition (EndMT) as a significant source of the mesenchymal cell population in fibrotic diseases. In this context, we hypothesized that liver endothelial cells undergo EndMT during fibrosis progression. Cirrhosis in mice was induced by CCl 4 A transgenic mouse expressing a red fluorescent protein reporter under the control of Tie2 promoter (Tie2-tdTomato) was used to trace the acquisition of EndMT. Sinusoidal vascular connectivity was evaluated by intravital microscopy and high-resolution three-dimensional confocal microscopy. A modest but significant fraction of liver endothelial cells from both cirrhotic patients and CCl 4 -treated Tie2-tdTomato mice acquired an EndMT phenotype characterized by the coexpression of CD31 and α-smooth muscle actin, compared with noncirrhotic livers. Bone morphogenetic protein-7 (BMP-7) inhibited the acquisition of EndMT induced by transforming growth factor-β1 (TGF-β1) treatment in cultured primary mouse liver endothelial cells from control mice. EndMT was also reduced significantly in vivo in cirrhotic Tie2-tdTomato mice treated intraperitoneally with BMP-7 compared with untreated mice (1.9 ± 0.2 vs. 3.8 ± 0.3%, respectively; P livers correlated with a significant decrease in liver fibrosis ( P livers in both animal models and patients. BMP-7 treatment decreases the occurrence of the EndMT phenotype and has a positive impact on the severity of disease by reducing fibrosis and sinusoidal vascular disorganization. NEW & NOTEWORTHY A subpopulation of liver endothelial cells from cirrhotic patients and mice with liver fibrosis undergoes endothelial-to-mesenchymal transition. Liver endothelial cells from healthy mice could transition into a mesenchymal phenotype in culture in response to TGF-β1 treatment. Fibrotic livers treated chronically with BMP-7 showed lower EndMT acquisition, reduced fibrosis, and improved vascular organization. Copyright © 2017 the American

Tolerogenic properties of lymphatic endothelial cells are controlled by the lymph node microenvironment.

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Jarish N Cohen

Full Text Available Peripheral self-tolerance eliminates lymphocytes specific for tissue-specific antigens not encountered in the thymus. Recently, we demonstrated that lymphatic endothelial cells in mice directly express peripheral tissue antigens, including tyrosinase, and induce deletion of specific CD8 T cells via Programmed Death Ligand-1 (PD-L1. Here, we demonstrate that high-level expression of peripheral tissue antigens and PD-L1 is confined to lymphatic endothelial cells in lymph nodes, as opposed to tissue (diaphragm and colon lymphatics. Lymphatic endothelial cells in the lymph node medullary sinus express the highest levels of peripheral tissue antigens and PD-L1, and are the only subpopulation that expresses tyrosinase epitope. The representation of lymphatic endothelial cells in the medullary sinus expressing high-level PD-L1, which is necessary for normal CD8 T cell deletion kinetics, is controlled by lymphotoxin-β receptor signaling and B cells. Lymphatic endothelial cells from neonatal mice do not express high-level PD-L1 or present tyrosinase epitope. This work uncovers a critical role for the lymph node microenvironment in endowing lymphatic endothelial cells with potent tolerogenic properties.

Coniferyl aldehyde attenuates radiation enteropathy by inhibiting cell death and promoting endothelial cell function.

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Jeong, Ye-Ji; Jung, Myung Gu; Son, Yeonghoon; Jang, Jun-Ho; Lee, Yoon-Jin; Kim, Sung-Ho; Ko, Young-Gyo; Lee, Yun-Sil; Lee, Hae-June

2015-01-01

Radiation enteropathy is a common complication in cancer patients. The aim of this study was to investigate whether radiation-induced intestinal injury could be alleviated by coniferyl aldehyde (CA), an HSF1-inducing agent that increases cellular HSP70 expression. We systemically administered CA to mice with radiation enteropathy following abdominal irradiation (IR) to demonstrate the protective effects of CA against radiation-induced gastrointestinal injury. CA clearly alleviated acute radiation-induced intestinal damage, as reflected by the histopathological data and it also attenuated sub-acute enteritis. CA prevented intestinal crypt cell death and protected the microvasculature in the lamina propria during the acute and sub-acute phases of damage. CA induced HSF1 and HSP70 expression in both intestinal epithelial cells and endothelial cells in vitro. Additionally, CA protected against not only the apoptotic cell death of both endothelial and epithelial cells but also the loss of endothelial cell function following IR, indicating that CA has beneficial effects on the intestine. Our results provide novel insight into the effects of CA and suggest its role as a therapeutic candidate for radiation-induced enteropathy due to its ability to promote rapid re-proliferation of the intestinal epithelium by the synergic effects of the inhibition of cell death and the promotion of endothelial cell function.

Organizational behavior of human umbilical vein endothelial cells