Search

Protein S (PROS1) is a vitamin K-dependent anticoagulant factor, which also acts as an agonist for the TYRO3, AXL, and MERTK (TAM) tyrosine kinase receptors. PROS1 is produced by the endothelium which also expresses TAM receptors, but little is known about its effects on vascular function and permeability. Transwell permeability assays as well as Western blotting and immunostaining analysis were used to monitor the possible effects of PROS1 on both endothelial cell permeability and on the phosphorylation state of specific signaling proteins. We show that human PROS1, at its circulating concentrations, substantially increases both the basal and VEGFA-induced permeability of endothelial cell (EC) monolayers. PROS1 induces p38 MAPK (Mitogen Activated Protein Kinase), Rho/ROCK (Rho-associated protein kinase) pathway activation, and actin filament remodeling, as well as substantial changes in Vascular Endothelial Cadherin (VEC) distribution and its phosphorylation on Ser665 and Tyr685. It also mediates c-Src and PAK-1 (p21-activated kinase 1) phosphorylation on Tyr416 and Ser144, respectively. Exposure of EC to human PROS1 induces VEC internalization as well as its cleavage into a released fragment of 100 kDa and an intracellular fragment of 35 kDa. Using anti-TAM neutralizing antibodies, we demonstrate that PROS1-induced VEC and c-Src phosphorylation are mediated by both the MERTK and TYRO3 receptors but do not involve the AXL receptor. MERTK and TYRO3 receptors are also responsible for mediating PROS1-induced MLC (Myosin Light Chain) phosphorylation on a site targeted by the Rho/ROCK pathway. Our report provides evidence for the activation of the c-Src/VEC and Rho/ROCK/MLC pathways by PROS1 for the first time and points to a new role for PROS1 as an endogenous vascular permeabilizing factor.

Background: In the present study, we measured the serum vascular endothelial cadherin (VEC) and vinculin (Vcn) concentrations in patients with type B acute aortic dissection (TBAD) to evaluate their diagnostic value for this condition. Methods: A total of 100 patients with TBAD and 90 matched controls were included in the study. The serum concentrations of VEC and Vcn were measured using enzyme-linked immunosorbent assays. Results: The serum VEC and Vcn concentrations were significantly higher in participants with TBAD than in healthy controls. Compared with patients with acute myocardial infarction (AMI), the serum concentrations of VEC and Vcn in patients with TBAD were higher, and the Vcn showed significant difference, with statistical significance. Receiver operating characteristic analysis generated areas under the curves for VEC and Vcn that were diagnostic for TBAD (0.599 and 0.655, respectively). The optimal cut-off values were 3.975 ng/μL and 128.1 pg/mL, the sensitivities were 43.0% and 35.0%, and the specificities were 73.3% and 90.0%, respectively. In addition, the use of a combination of serum VEC and Vcn increased the AUC to 0.661, with a sensitivity of 33.0% and a specificity of 93.33%. A high serum Vcn concentration was associated with a higher risk of visceral malperfusion in participants with TBAD (odds ratio (OR) = 1.007, 95% confidence interval [CI]: 1.001–1.013, p = 0.014). In participants with refractory pain, the adjusted OR for the serum VEC concentration increased to 1.172 (95% CI: 1.010–1.361; p = 0.036), compared with participants without refractory pain. Conclusion: This study is the first to show the diagnostic value of serum VEC and Vcn for AAD and their relationships with the clinical characteristics of patients with TBAD. Thus, VEC and Vcn are potential serum markers of TBAD. [ABSTRACT FROM AUTHOR]

To investigate the expression of serum vascular endothelial cadherin (VE CAD), angiopoietin-2 (Ang-2) and urinary renal injury molecule-1 (KIM-1) in patients with renal failure and their correlation with the severity of the disease. 76 patients with chronic renal failure treated in our hospital from February 2018 to February 2021 were selected as the research objects. They were divided into renal function compensation group (n=25), azotemia group (n=18), renal failure group (n=21) and uremia group (n=12) according to the severity of their condition. The expressions of ve-cad, Ang-2 and KIM-1 in the four groups were compared and analyzed Correlation between KIM-1 and the severity of chronic renal failure. All patients were followed up by telephone or reexamination. 76 patients with chronic renal failure were divided into two subgroups according to the prognosis, survival group (n=56) and death group (n=20). The clinical conditions and index levels of the two groups were compared, and the predictive value of ve-cad, Ang-2 and KIM-1 on the prognosis of chronic renal failure was analyzed by logistic regression analysis. The expression levels of ve-cad, Ang-2 and KIM-1 in uremia group were significantly higher than those in renal failure group, azotemia group and renal function compensation group (P<0.05); Spearman correlation analysis showed that VE CAD, Ang-2 and KIM-1 were positively correlated with the severity of chronic renal failure (P<0.05); There was no significant difference in gender, age, BMI, organ failure ≥ 2, oliguria and ve CAD between the survival group and the death group (P>0.05). There was significant difference in Apache Ⅱ score, CysC, Ang-2 and KIM-1 between the survival group and the death group (P<0.05); Logistic regression analysis showed that Apache Ⅱ score, CysC and KIM-1 were independent risk factors affecting the prognosis of chronic renal failure (P<0.05). Ve-cad, Ang-2 and KIM-1 are positively correlated with the severity of patients with chronic renal failure. It can be considered to evaluate the severity of patients with chronic renal failure by referring to the levels of the three. Among the three, only KIM-1 has a certain relationship with the prognosis of patients with renal failure. Therefore, it can be considered to add KIM-1 index to judge on the basis of Apache Ⅱ score and CysC prediction of prognosis, so as to improve the accuracy of prognosis prediction. [ABSTRACT FROM AUTHOR]

Vascular endothelial-cadherin (VE-cadherin), matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) have emerged as key-factors of atherogenesis. The aim of this study was to evaluate the effects of exercise training (ET) on those key-factors in relation to the progression of atherosclerotic lesions in hypercholesterolemic mice. Thirty male, apoE knockout (apoE−/−) mice were randomly assigned to the following equivalent groups: 1) CO-control: High-fat diet (HFD) administration for 12 weeks. 2) EX-exercise: HFD administration as in CO, and during the last 4 weeks (9th −12th week) ET on treadmill (5sessions/week, 60min/session). At the end of study, blood samples were obtained and all mice were sacrificed. Aortic roots were excised and analysed regarding the percentage of aortic stenosis, and the relative concentrations of collagen, elastin, VE-cadherin, MMP-8,-9 and TIMP-1,-2 within the atherosclerotic lesions. Aortic stenosis was significantly lower in the EX than the CO group (39.63 ± 7.22% vs 62.04 ± 8.55%; p < 0.001), along with considerable increase in fibrous cap thickness and of collagen and elastin contents within plaques (p < 0.05). Compared to controls, exercised-treated mice showed reduced intra-plaque relative concentrations of VE-cadherin (15.09 ± 1.89% vs 23.49 ± 3.01%, p < 0.001), MMP-8 (8.51 ± 2.24% vs 18.51 ± 4.08%, p < 0.001) and MMP-9 (12.1 ± 4.86% vs 18.88 ± 6.23%, p < 0.001). Inversely, the relative concentrations of TIMP-1 and TIMP-2 in the ET group were considerably higher by 62.5% and 31.2% than in the EX group (p < 0.05), respectively. Finally, body weight and lipids concentrations did not differ between groups at the end of the study (p > 0.05). ET treatment induced regression of established atherosclerotic lesions in apoE−/− mice and improved their stability. Those effects seemed to be mediated by favourable modification of VE-cadherin, MMPs and TIMPs. • Exercise training induced regression of established atherosclerotic lesions in apoE−/− mice and improved their stability. • Effective VE-cadherin reduction associated with stabilization of the atherosclerotic plaques after exercise training. • Shift of the MMP/TIMP balance towards less proteolytic and pro-inflammatory activity within the atherosclerotic plaques. [ABSTRACT FROM AUTHOR]

Background & Aims: The pathophysiology of irritable bowel syndrome (IBS) is multifactorial and includes epithelial barrier dysfunction, a key element at the interface between the gut lumen and the deeper intestinal layers. Beneath the epithelial barrier there is the vascular one representing the last barrier to avoid luminal antigen dissemination The aims of this study were to correlate morpho-functional aspects of epithelial and vascular barriers with symptom perception in IBS., Methods: Seventy-eight healthy subjects (controls) and 223 patients with IBS were enrolled in the study and phenotyped according to validated questionnaires. Sugar test was used to evaluate in vivo permeability. Immunohistochemistry, western blot, and electron microscopy were used to characterize the vascular barrier. Vascular permeability was evaluated by assessing the mucosal expression of plasmalemma vesicle-associated protein-1 and vascular endothelial cadherin. Caco-2 or human umbilical vein endothelial cell monolayers were incubated with soluble mediators released by mucosal biopsies to highlight the mechanisms involved in permeability alteration. Correlation analyses have been performed among experimental and clinical data., Results: The intestinal epithelial barrier was compromised in patients with IBS throughout the gastrointestinal tract. IBS-soluble mediators increased Caco-2 permeability via a downregulation of tight junction gene expression. Blood vessel density and vascular permeability were increased in the IBS colonic mucosa. IBS mucosal mediators increased permeability in human umbilical vein endothelial cell monolayers through the activation of protease-activated receptor-2 and histone deacetylase 11, resulting in vascular endothelial cadherin downregulation. Permeability changes correlated with intestinal and behavioral symptoms and health-related quality of life of patients with IBS., Conclusions: Epithelial and vascular barriers are compromised in patients with IBS and contribute to clinical manifestations., (Copyright © 2024 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The aim of the present study was to investigate the protective effect of dexmedetomidine (Dex) on endothelial injury in a cecal ligation and puncture (CLP)-induced rat model of sepsis. A total of 36 male Sprague-Dawley rats were divided into three groups: Sham, CLP and CLP + Dex. The wet/dry (W/D) ratio of lung weight, hematoxylin and eosin (H&E) staining of lung tissue, plasma levels of angiopoietin (Ang)1 and 2, ratio of Ang2/1 and vascular endothelial (VE)-cadherin protein expression levels in lung tissue were determined. The W/D ratio of lung tissue in the CLP + Dex group was significantly lower than that in the CLP group (P<0.01). The H&E staining results indicated that Dex treatment reduced the levels of CLP-induced alveolar septum widening, infiltrating white blood cells and congestion, when compared with CLP alone. In addition, the expression levels of plasma Ang2 and the Ang2/1 ratio in the CLP + Dex group were significantly lower than those of the CLP rats (P<0.01). Furthermore, the level of VE-cadherin protein in lung tissue of the CLP + Dex group was higher than that of the CLP group (P<0.05). The results indicated that Dex had a protective effect against CLP-induced endothelial injury, through the ability to reduce expression of the endothelial injury factor Ang2 and increase the expression of the endothelial adhesion junction factor VE-cadherin in a septic rat model. These data suggest a potential application of Dex in the clinical treatment of sepsis. [ABSTRACT FROM AUTHOR]

Background: Lung ischemia reperfusion injury (LIRI) is a complex pathophysiological process activated by lung transplantation and acute lung injury. The p38 mitogen-activated protein kinase (MAPK) is involved in breakdown of the endothelial barrier during LIRI, but the mechanism is still unclear. Therefore, we investigated the function of p38 MAPK in LIRI in vivo and in vitro.Methods: Sprague–Dawley rats were subjected to ischemia reperfusion with or without pretreatment with a p38 MAPK inhibitor. Lung injury was assessed using hematoxylin and eosin staining, and pulmonary blood–air barrier permeability was evaluated using Evans blue staining. A rat pulmonary microvascular endothelial cell line was infected with lentiviral expressing short hairpin (sh)RNA targeting p38 MAPK and then cells were subjected to oxygen/glucose deprivation and reoxygenation (OGD/R). Markers of endothelial destruction were measured by western blot and immunofluorescence.Results:In vivo LIRI models showed structural changes indicative of lung injury and hyperpermeability of the blood–air barrier. Inhibiting p38 MAPK mitigated these effects. Oxygen/glucose deprivation and reoxygenation promoted hyperpermeability of the endothelial barrier in vitro, but knockdown of p38 MAPK attenuated cell injury; maintained endothelial barrier integrity; and partially reversed injury-induced downregulation of permeability protein AQP1, endothelial protective protein eNOS, and junction proteins ZO-1 and VE-cadherin while downregulating ICAM-1, a protein involved in destroying the endothelial barrier, and ET-1, a protein involved in endothelial dysfunction.Conclusion: Inhibition of p38 MAPK alleviates LIRI by decreasing blood–air hyperpermeability. Blocking p38 MAPK may be an effective treatment against acute lung injury.

Objective To observe the changes of serum kallikrein binding protein（Kal）and vascular endothelial cadherin（VE-Cad）levels and to evaluate their predictive value for the prognosis of septic shock（SS). Methods Totally 201 patients with SS（observation group）were divided into the death group and survival group according to the 28-day prog⁃ nosis；another 200 healthy people who took physical examination in the same period were selected as the control group. The levels of serum VE-Cad and Kal, sepsis-related organ failure assessment（SOFA）score，and acute physiology and chronic health evaluation（APACHE）Ⅱ score in each group were compared between the observation group and the control group. On the 1st, 3rd and 5th days, the serum Kal and VE-Cad levels were compared between the death group and survival group. The correlation between the above indexes was analyzed by Pearson correlation. The risk factors for death of SS patients were analyzed by Logistic Regression analysis. The predictive value of serum VE-Cad and Kal levels for prognosis of patients with SS were analyzed by ROC. Results The levels of serum VE-Cad，SOFA，and APACHE Ⅱ scores in the observation group were higher than those in the control group, while the level of Kal in the observation group was lower than in the control group（all P<0. 05). On the 1st, 3rd and 5th days, the serum VE-Cad levels of the death group were higher than those of the survival group，and the serum Kal level was lower than that of the survival group（all P<0. 05)， and SOFA and APACHE Ⅱ scores of the death group were higher than those of the survival group（both P<0. 05）. Serum VE-Cad level was positively correlated with SOFA and APACHE Ⅱ scores in the death group on the 1st day after admission（both P<0. 05)，while serum Kal level was negatively correlated with SOFA and Apache Ⅱ scores（both P<0. 05）. Multivariate stepwise logistic regression analysis showed that VE-Cad（OR=1. 382，95%CI=1. 045-1. 786）was a risk fac⁃ tor for death of SS patients，and Kal（OR=0. 429，95%CI=0. 187-0. 968）was a protective factor for SS patients（P< 0. 05）. On the 1st day after admission，the AUC of serum VE-Cad combined with Kal in judging the prognosis of SS patients was significantly higher than that of VE-Cad or Kal alone（all P<0. 05), as well as the accuracy，sensitivity，and specificity（all P<0. 05). Conclusions The decrease of serum Kal level and the increase of VE-Cad level are the independent influencing factors for death of SS patients. The combined detection of serum Kal and VE-Cad can predict the prognosis of SS patients better than the single detection of each index. [ABSTRACT FROM AUTHOR]

Protein S (PROS1) is a vitamin K-dependent anticoagulant factor, which also acts as an agonist for the TYRO3, AXL, and MERTK (TAM) tyrosine kinase receptors. PROS1 is produced by the endothelium which also expresses TAM receptors, but little is known about its effects on vascular function and permeability. Transwell permeability assays as well as Western blotting and immunostaining analysis were used to monitor the possible effects of PROS1 on both endothelial cell permeability and on the phosphorylation state of specific signaling proteins. We show that human PROS1, at its circulating concentrations, substantially increases both the basal and VEGFA-induced permeability of endothelial cell (EC) monolayers. PROS1 induces p38 MAPK (Mitogen Activated Protein Kinase), Rho/ROCK (Rho-associated protein kinase) pathway activation, and actin filament remodeling, as well as substantial changes in Vascular Endothelial Cadherin (VEC) distribution and its phosphorylation on Ser 665 and Tyr 685 . It also mediates c-Src and PAK-1 (p21-activated kinase 1) phosphorylation on Tyr 416 and Ser 144 , respectively. Exposure of EC to human PROS1 induces VEC internalization as well as its cleavage into a released fragment of 100 kDa and an intracellular fragment of 35 kDa. Using anti-TAM neutralizing antibodies, we demonstrate that PROS1-induced VEC and c-Src phosphorylation are mediated by both the MERTK and TYRO3 receptors but do not involve the AXL receptor. MERTK and TYRO3 receptors are also responsible for mediating PROS1-induced MLC (Myosin Light Chain) phosphorylation on a site targeted by the Rho/ROCK pathway. Our report provides evidence for the activation of the c-Src/VEC and Rho/ROCK/MLC pathways by PROS1 for the first time and points to a new role for PROS1 as an endogenous vascular permeabilizing factor., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Objective: To study the correlation of serum monocyte chemotactant protein-1 (MCP-1) and vascular endothelia cadherin (VE-cadherin) levels in patients with acute cerebral infarction, and nerve injury molecules, interleukins and matrix metalloproteinases. Methods: A total of 86 patients with acute cerebral infarction treated in our hospital from April 2012 to October 2015 were selected as the observation group and 50 healthy subjects in the same period treated in our hospital were selected as the control group. The serums were collected and the contents of MCP-1, VE-cadherin, heart-type fatty acid binding protein (H-FABP), S100 calcium binding protein B (S100B), neuron-specific enolase (NSE), interleukin-lb (IL-1b), IL-6, IL-17, IL-18, matrix metalloproteinase-2 (MMP2), MMP3 and MMP9 were measured. Results: The serum contents of MCP-1, VE-cadherin, H-FABP, S100B, NSE, IL-1b, IL- 6, IL-17, IL-18, MMP2, MMP3 and MMP9 in observation group were significantly higher than those of control group. Carotid artery plaque formation and unstable plaque properties will increase the serum contents of MCP-1, VE-cadherin, H-FABP, S100B, NSE, IL-1b, IL-6, IL-17, IL-18, MMP2, MMP3 and MMP9 in patients with cerebral infarction. The serum levels of MCP-1, VE-cadherin and the contents of H-FABP, S100B, NSE, IL-1b, IL-6, IL-17, IL-18, MMP2, MMP3 and MMP9 were positively correlated. Conclusions: The serum levels of VE-cadherin and MCP-1 were significantly increased in patients with acute cerebral infarction. MCP-1 and VE-cadherin can increase the secretion of interleukins and matrix metalloproteinases, which can result in the carotid artery plaque formation, unstable plaque properties and the injury of nerve function.

Objective: The objective of our study was to determine levels of endothelial progenitor cells (EPCs) in preeclampsia and normotensive pregnant women.Study Design: Prospective cohort study of women with preeclampsia and normotensive pregnancies. EPCs were estimated by flow cytometry. Multiple linear regression was used to assess the association of EPCs with preeclampsia adjusting for maternal age, body mass index (BMI), gestation and ethnicity.Main Outcome Measure: Levels of EPCs in preeclampsia and normotensive pregnancies, with CD-34 and vascular endothelial (VE)-cadherin as markers of EPCs. VE-cadherin is an endothelial cell adhesion molecule used to delineate endothelial lineage of EPCs.Results: There were thirty women in the preeclampsia group and thirty-three in the normotensive group. The two groups were similar except for the BMI and blood pressures, which were higher in preeclampsia. On multiple linear regression, EPCs numbers were significantly higher by 29 (95% confidence interval 11.7-46.6, p = 0.001) in preeclampsia compared to the normotensive group. There was significant positive correlation between EPCs and systolic blood pressure in preeclampsia (Spearman correlation coefficient 0.39, p = 0.03).Conclusion: Although widely used in cardiovascular disease other than preeclampsia, this is the first study using VE-cadherin as a marker of endothelial lineage to define EPCs in preeclampsia. Our results suggest the higher number of EPCs in preeclampsia may be a response of the bone marrow to endothelial injury. [ABSTRACT FROM AUTHOR]

Objective The blood-brain barrier (BBB) of patients with moyamoya angiopathy (MMA) is unstable, which may contribute to transient neurologic symptoms (TNS) after direct bypass surgery. However, BBB-related proteins have never been investigated. The purpose of this study was to evaluate the perioperative serum levels of biomarkers representing BBB function in MMA patients based on the hypothesis that postoperative hemodynamic change may disrupt the BBB. Methods A total of 12 hemispheres in 11 patients with MMA were prospectively examined. Direct revascularization surgery was performed for all cases. The serum levels of tight junction (occludin and claudin 5), adherens junction (vascular endothelial−cadherin) proteins, and matrix metalloproteinase (MMP)-2 and MMP-9 were measured quantitatively 1 day before surgery and on postoperative days 1, 4, and 7. Results Successful patency of the direct bypass was achieved in all. The serum level of occludin was significantly increased on postoperative day 1, and the levels in 2 cases with TNS were markedly elevated over 10-fold higher than baseline. Furthermore, the postoperative MMP-9 levels were significantly elevated on each day. On the other hand, there was no significant fluctuation in claudin 5, vascular endothelial-cadherin, and MMP-2 level. Conclusions Marked changes in biomarkers representing the tight junction of the BBB were observed. These preliminary results suggest that marked hemodynamic change and TNS in some patients are associated with disruption of the BBB after direct bypass surgery for MMA. Highlights • Blood-brain barrier (BBB) of patients with moyamoya angiopathy (MMA) is unstable. • Direct bypass surgery was performed for MMA. • The serum levels of BBB-related proteins were measured before and after surgery. • The serum levels of OCLN and MMP-9 were significantly increased postoperatively. • Postoperative marked hemodynamic change may induce the disruption of the BBB. [ABSTRACT FROM AUTHOR]

AIM: To explore the effects and mechanism of vascular endothelial cadherin (VE-cadherin) on experimental corneal neovascularization (CRNV). METHODS: Mouse corneas were burned with sodium hydroxide to build a CRNV model. The burned corneas were locally administrated with anti-mouse VE-cadherin neutralizing antibody. Annexin V and cluster of differentiation 31 (CD31) double staining was used to measure vascular endothelial cell apoptosis with the use of flow cytometry (FCM). The protein expression of NADPH oxidase 2 (Nox2), caspase-3, and protein kinase C (PKC) in the burned corneas were examined by Western blot. Human retinal endothelial cell (HREC) proliferation was detected using a Cell Counting Kit 8 (CCK-8) assay in vitro. RESULTS: The amount of CRNV peaked two weeks after the alkali burn. FCM confirmed that VE-cadherin neutralizing antibody treatment increased CD31 positive cell apoptosis. Western blot revealed that the intracorneal protein expression of Nox2 and caspase-3 were up-regulated, while PKC was down-regulated in the VE-cadherin neutralizing antibody administrated group. CCK-8 assay showed that VE-cadherin neutralizing antibody markedly inhibited HREC proliferation. CONCLUSION: VE-cadherin exhibited an anti-apoptosis effect through enhanced PKC signaling and an enhanced cell proliferation pathway.

Angiotensin II (Ang II) is reported to be involved in the development of various cardiovascular diseases by disrupting microvessel permeability, however, the underlying mechanism remains to be elucidated. The present study aimed to investigate the mechanism by which Ang II disrupts microvascular permeability. Rat endothelial cells were subjected to primary culture and identification. Cells in passages 4–7 were then used for the following experiments. The cells were divided into control, Ang II, and Ang II + valsartan groups, and reverse transcription-quantitative polymerase chain reaction and western blot analyses were perform to evaluate the expression of zonula occludens-1 (ZO-1) and vascular endothelial (VE)-cadherin in the cells. The distribution of ZO-1 protein was also detected using immunofluorescence assays. It was found that, compared with the control group, lower expression levels of ZO-1 and VE-cadherin were present in the Ang II group (P<0.01). ZO-1 was also irregularly distributed at the periphery of the cells. In addition, the overexpression of VE-cadherin reversed the effect of Ang II on the expression and distribution of ZO-1 in endothelial cells. Together, these results suggested that Ang II inhibited the protein expression of ZO-1 in vascular endothelial cells by downregulating VE-cadherin, thus destroying the tight junctions between endothelial cells, which may also be the mechanism by which Ang II is involved in the development of cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Per- and polyfluoroalkyl substances (PFASs) are ubiquitous and high persistent in human blood, thus potentially inducing a myriad of deleterious consequences. Plasma kallikrein-kinin system (KKS), which physiologically regulates vascular permeability, is vulnerable to exogenous stimulators, like PFASs with long-chain alkyl backbone substituted by electronegative fluorine. The study on the interactions of PFASs with the KKS and the subsequent effects on vascular permeability would be helpful to illustrate how the chemicals penetrate the biological vascular barriers to reach different tissues. In present study, three representative PFASs, including perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA) and perfluorohexadecanoic acid (PFHxDA), were investigated for their effects on the activation of the KKS, paracellular permeability in human retina endothelial cells (HRECs) and integrity of the adherens junctions. In contrast to either PFOS or PFOA, PFHxDA efficiently triggered KKS activation in a concentration-dependent manner based on protease activity assays. The plasma activated by PFHxDA significantly increased paracellular permeability of HRECs through the degradation of adherens junctions. As evidenced by the antagonistic effect of aprotinin, PFHxDA-involved effects on vascular permeability were mediated by KKS activation. The results herein firstly revealed the mechanistic pathway for PFHxDA induced effects on vascular endothelial cells. Regarding the possible structure-related activities of the chemicals, this finding would be of great help in the risk assessment of PFASs. [ABSTRACT FROM AUTHOR]

Background: The diagnosis of non-alcoholic fatty liver disease (NAFLD) is usually based on liver ultrasonography and serum alanine aminotransferase (ALT) levels. However, the serum ALT level is not sensitive for detecting NAFLD. If more serum markers are available, serum analysis may play a more important role in the diagnosis of NAFLD. Methods: Here, we have investigated whether vascular endothelial cadherin (VE-cad) and thrombomodulin (TM) are markers of NAFLD in children. After an examination of liver ultrasonography, 90 children were divided into a lean control group (n = 32), an overweight/obese NAFLD group (group-NAFLD, n = 34) and an overweight/obese non-NAFLD group (group-SOO, n = 24). Results: Two overweight/obese groups had similar obesity. However, serum VE-cad and TM levels were increased in group-NAFLD but not group-SOO. When data from all children were pooled, serum VE-cad and TM levels were positively correlated to body-mass index (BMI) and serum ALT levels. Conclusions: In conclusion, VE-cad and TM are markers of pediatric NAFLD. [ABSTRACT FROM AUTHOR]

Objective To study the correlation of serum monocyte chemotactant protein-1 (MCP-1) and vascular endothelia cadherin (VE-cadherin) levels in patients with acute cerebral infarction, and nerve injury molecules, interleukins and matrix metalloproteinases. Methods A total of 86 patients with acute cerebral infarction treated in our hospital from April 2012 to October 2015 were selected as the observation group and 50 healthy subjects in the same period treated in our hospital were selected as the control group. The serums were collected and the contents of MCP-1, VE-cadherin, heart-type fatty acid binding protein (H-FABP), S100 calcium binding protein B (S100B), neuron-specific enolase (NSE), interleukin-lβ (IL-1β), IL-6, IL-17, IL-18, matrix metalloproteinase-2 (MMP2), MMP3 and MMP9 were measured. Results The serum contents of MCP-1, VE-cadherin, H-FABP, S100B, NSE, IL-1β, IL-6, IL-17, IL-18, MMP2, MMP3 and MMP9 in observation group were significantly higher than those of control group. Carotid artery plaque formation and unstable plaque properties will increase the serum contents of MCP-1, VE-cadherin, H-FABP, S100B, NSE, IL-1β, IL-6, IL-17, IL-18, MMP2, MMP3 and MMP9 in patients with cerebral infarction. The serum levels of MCP-1, VE-cadherin and the contents of H-FABP, S100B, NSE, IL-1β, IL-6, IL-17, IL-18, MMP2, MMP3 and MMP9 were positively correlated. Conclusions The serum levels of VE-cadherin and MCP-1 were significantly increased in patients with acute cerebral infarction. MCP-1 and VE-cadherin can increase the secretion of interleukins and matrix metalloproteinases, which can result in the carotid artery plaque formation, unstable plaque properties and the injury of nerve function. [ABSTRACT FROM AUTHOR]

Dense monolayers of living cells display intriguing relaxation dynamics, reminiscent of soft and glassy materials close to the jamming transition, and migrate collectively when space is available, as in wound healing or in cancer invasion. Here we show that collective cell migration occurs in bursts that are similar to those recorded in the propagation of cracks, fluid fronts in porous media, and ferromagnetic domain walls. In analogy with these systems, the distribution of activity bursts displays scaling laws that are universal in different cell types and for cells moving on different substrates. The main features of the invasion dynamics are quantitatively captured by a model of interacting active particles moving in a disordered landscape. Our results illustrate that collective motion of living cells is analogous to the corresponding dynamics in driven, but inanimate, systems. [ABSTRACT FROM AUTHOR]

Endothelial barrier restoration reverses microvessel hyperpermeability and facilitates recovery from lung injury. Because inhibiting connexin 43 (Cx43)-dependent interendothelial communication blunts hyperpermeability in single microvessels, we determined whether endothelial Cx43 levels correlate with changes in microvessel permeability during recovery from lung injury. Toward this, bacterial endotoxin was instilled intratracheally into rat lungs, and at different durations postinstillation the lungs were isolated and blood perfused. Microvessel Cx43 expression was quantified by in situ immunofluorescence and microvessel permeability via a fluorescence method. To supplement the immunofluorescence data, protein levels were determined by immunoblots of lung tissue from endotoxin-instilled rats. Immunofluorescence and immunoblot together revealed that both Cx43 expression and microvessel permeability increased above baseline within a few hours after endotoxin instillation but declined progressively over the next few days. On day 5 postendotoxin, microvessel Cx43 declined to negligible levels, resulting in complete absence of intermicrovessel communication determined by photolytic uncaging of Ca2+. However, by day 14, both Cx43 expression and microvessel permeability returned to baseline levels. In contrast to Cx43, expression of microvessel vascular endothelial (VE)-cadherin, a critical determinant of vascular barrier integrity, exhibited an inverse trend by initially declining below baseline and then returning to baseline at a longer duration. Knockdown of vascular Cx43 by tail vein injection of Cx43 shRNA increased VE-cadherin expression, suggesting that reduction in Cx43 levels may modulate VE-cadherin levels in lung microvessels. Together, the data suggest that endotoxin challenge initiates interrelated changes in microvessel Cx43, VE-cadherin, and microvessel permeability, with changes in Cx43 temporally leading the other responses. [ABSTRACT FROM AUTHOR]

Vascular endothelial cadherin and β-catenin play a key role in establishment and maintenance of the endothelial monolayer integrity, regulation of vascular barrier function, and initiation of angiogenesis. The cadherin-catenin complex has been shown to be reduced in type 1 diabetic placenta, but the exact relationship between histopathologic findings and clinical data is not known. Immunohistochemistry of placental tissue from type 1, type 2, and gestational diabetes showed that diabetes per se might be compatible with normal levels of vascular endothelial (VE)-cadherin and β-catenin in fetoplacental vessels as long as the patient has not been treated with insulin. Immunoreactivity of VE-cadherin did correlate poorly with maternal glycemic control, as was investigated in this study, by birth weight, body mass index, and hemoglobin A1c (HbA1c). There was no correlation found between the immunoreactivity of β-catenin and birth weight, body mass index, or HbA1c. However our data did show a strong correlation between immunoreactivity and whether or not the patient had been treated with insulin. Patients diagnosed with gestational diabetes who had not been treated with insulin had similar levels of VE-cadherin and β-catenin to the control group, thus indicating that diabetes per se must not necessarily lead to a reduction. Our study suggests that therapeutic intervention using insulin in pregnancies complicated by diabetes might have potentially harmful effects on placental morphology. Future studies should further investigate these findings. [ABSTRACT FROM AUTHOR]

Vascular endothelium is a target of cadmium (Cd), which is a global pollutant of the environment. However, the detailed effects and underlying mechanisms remain to be elucidated. In the present study, human umbilical vein endothelial cells (HUVECs) were treated with 0.1, 1, 5, 10, 50 µM cadmium chloride (CdCl2) for 12 h. It was found that vascular endothelial (VE)-cadherin mRNA and protein expression was upregulated by Cd in HUVECs in a dose-dependent manner. Higher levels of VE-cadherin were detected at cell-to-cell junctions in HUVECs treated with 10 µM CdCl2 compared with normal condition. The phosphorylation level of myosin-binding subunit of myosin phosphatase, a downstream substrate of Rho-associated protein kinase (ROCK), was reduced by 10 µM CdCl2, suggesting that Cd inhibited the Rho/ROCK pathway. Activation of ROCK by narciclasine reversed the Cd-induced increase of VE-cadherin expression. By contrast, ROCK pathway inhibitor Y27632 increased VE-cadherin expression in HUVECs. Following inhibition of the ROCK pathway, Cd did not significantly alter the level of VE-cadherin. Taken together, the results suggested that Cd exposure enhanced VE-cadherin expression in endothelial cells via suppression of ROCK signaling. [ABSTRACT FROM AUTHOR]

Interaction between platelets and artificial materials within cardiovascular devices triggers blood coagulation and represents a frequent adverse response to implant deployment. Avoidance of this interaction is obtained through the generation and sustenance under flow of a confluent and stable endothelial monolayer covering the luminal device surface, altogether defined as the process of endothelialization. Supraphysiological wall shear stress (WSS) levels generated within vascular assist devices (VADs) constitute a major challenge toward endothelialization. Here we report the experimental demonstration that stable endothelialization can be achieved at supraphysiological WSS levels by pure means of appropriate surface micro-structuring. Using a custom-designed flow bioreactor we exposed endothelial monolayers to physiological and supraphysiological WSS levels and investigated the resulting integrity of cell-to-cell junctions, the cell density and the cell polarization. At physiological WSS levels, optimal endothelialization was obtained independently from surface topography. However, at higher WSS levels, only monolayers grown on appropriately micro-structured surfaces preserved optimal integrity. Under these flow conditions, endothelial cells polarized by the contact with the micro-structure and, interestingly, oriented themselves in the direction perpendicular to flow. Such endothelial layers withstood WSS levels exceeding of 100% or more the thresholds detected on flat substrates. [ABSTRACT FROM AUTHOR]

Background Intracranial pressure changes during head impact cause brain injuries such as vasogenic edema and cerebral contusion. However, the influence of impulsive pressure on endothelial function has not yet been fully studied in vitro. In this study, we developed a pressure loading device that produced positive and negative pressures by modifying an in vitro fluid percussion model and examined the effects of the amplitude and duration of the pressures on endothelial permeability. Methods Human umbilical vein endothelial cells were subjected to three types of positive pressure (average amplitude/average duration of 352 kPa/23 ms, 73 kPa/27 ms, and 70 kPa/44 ms) and three types of negative pressure (-72 kPa/41 ms, -67 kPa/104 ms, and -91 kPa/108 ms), and the transendothelial electrical resistance (TEER) was measured between 15 min and 24 h after pressure loading for quantifying the formation of an integral monolayer of endothelial cells. After loading, vascular endothelial- (VE-) cadherin, an endothelium-specific cell-cell adhesion molecule involved in endothelial barrier function, was stained and observed using fluorescence microscopy. Results The pressure loading device could produce positive pressure pulses with amplitudes of 53- 1348 kPa and durations of 9-29.1 ms and negative pressure pulses with amplitudes of -52-93 kPa and durations of 42.9-179.5 ms. The impulsive pressure reduced the TEER associated with the change in VE-cadherin localization. Additionally, TEER decreased considerably at 15 min and 6 h post-loading, with these changes being significant in positive pressure with larger amplitude and shorter duration and in all types of negative pressures compared to pre-loading. Conclusions The changes in intracranial pressure during head impact impair endothelial barrier function by the disruption of the integrity of endothelial cell-cell junctions, and the degree of increase in endothelial permeability depends on the amplitude, duration, and direction (compressive and tensile) of the impulsive pressure. [ABSTRACT FROM AUTHOR]

Abstract: Understanding and accelerating the mechanisms of endothelial wound healing is of fundamental interest for biotechnology and of significant medical utility in repairing pathologic changes to the vasculature induced by invasive medical interventions. We report the fundamental mechanisms that determine the influence of substrate topography and flow on the efficiency of endothelial regeneration. We exposed endothelial monolayers, grown on topographically engineered substrates (gratings), to controlled levels of flow-induced shear stress. The wound healing dynamics were recorded and analyzed in various configurations, defined by the relative orientation of an inflicted wound, the topography and the flow direction. Under flow perpendicular to the wound, the speed of endothelial regeneration was significantly increased on substrates with gratings oriented in the direction of the flow when compared to flat substrates. This behavior is linked to the dynamic state of cell-to-cell adhesions in the monolayer. In particular, interactions with the substrate topography counteract Vascular Endothelial Cadherin phosphorylation induced by the flow and the wounding. This effect contributes to modulating the mechanical connection between migrating cells to an optimal level, increasing their coordination and resulting in coherent cell motility and preservation of the monolayer integrity, thus accelerating wound healing. We further demonstrate that the reduction of vascular endothelial cadherin phosphorylation, through specific inhibition of Src activity, enhances endothelial wound healing in flows over flat substrates. [Copyright &y& Elsevier]

Background/Aims: Renal interstitial fibrosis is a final common pathway of all chronic, progressive kidney diseases. Peritubular capillary rarefaction is strongly correlated with fibrosis. The adherens junction protein vascular endothelial cadherin (VE-cadherin) is thought to play a critical role in vascular integrity. We hypothesized that VE-cadherin modulates the renal microcirculation during fibrogenesis and ultimately affects renal fibrosis. Methods: Unilateral ureteral obstruction (UUO) was used as a renal fibrosis model in VE-cadherin heterozygote (VE+/-) and wild-type (WT) mice, and the kidneys were harvested at days 3, 7, and 14. Peritubular capillary changes and fibrogenesis were investigated. Results: VE+/- mice had lower levels of VE-cadherin protein than WT mice at 3 and 7, but not 14 days after UUO. Vascular permeability was significantly greater in VE+/- mice 7 days after UUO, while peritubular capillary density was not significantly different in VE+/- and WT mice. Interstitial myofibroblast numbers and collagen I and III mRNA levels were significantly higher in VE+/- mice, consistent with a stronger early fibrogenic response. Expression of the pericyte marker neuron-glial antigen 2 was upregulated after UUO, but was not greater in VE+/- mice compared to the WT mice. Conclusion: Our data suggest that VE-cadherin controls vascular permeability and limits fibrogenesis after UUO. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Aims Transendothelial migration (TEM) of monocytes is a crucial step in inflammatory processes such as atherogenesis. Tyrosine phosphorylation of vascular endothelial cadherin (VE-cad) has been implicated in the dissociation of adherens junctions and the increased paracellular permeability of endothelial cells (ECs) that occur during TEM of monocytes. However, the underlying molecular mechanism has not been determined. We tested the hypothesis that the phosphorylation of myosin light chain (MLC) in ECs is crucial for the dissociation of adherens junctions during TEM of monocytes. Methods and results Using a combination of biochemical and cellular techniques, we provide evidence for the signal transduction pathways that regulate tyrosine phosphorylation of VE-cad in ECs after the attachment of monocytes. Our findings indicate that after interaction of integrins on THP-1 cells with adhesion molecules on ECs, the induction of the HRas\Raf\MEK\ERK signalling cascade leads to the phosphorylation of MLC. This results in the recruitment of Src to the VE-cad complex and tyrosine phosphorylation of VE-cad, which leads to dissociation of β-catenin from the VE-cad complex, formation of gaps between ECs, and enhancement of THP-1 cell TEM. Conclusion Our studies suggest that monocyte-induced phosphorylation of MLC in ECs enhances TEM of monocytes through dissociation of EC adherens junctions. [ABSTRACT FROM AUTHOR]

Abstract: Many inflammatory diseases are associated with elevated blood concentration of fibrinogen (Fg) leading to vascular dysfunction. We showed that pathologically high (4mg/ml) content of Fg disrupts integrity of endothelial cell (EC) layer and causes macromolecular leakage affecting tight junction proteins. However, role of adherence junction proteins, particularly vascular endothelial cadherin (VE-cadherin) and matrix metalloproteinase-9 (MMP-9) in this process is not clear. We tested the hypothesis that at high levels Fg affects integrity of mouse brain endothelial cell (MBEC) monolayer through activation of MMP-9 and downregulation of VE-cadherin expression and in part its translocation to the cytosol. The effect of Fg on cultured MBEC layer integrity was assessed by measuring transendothelial electrical resistance. Cellular expression and translocation of VE-cadherin were assessed by Western blot and immunohistochemical analyses, (respectively). Our results suggest that high content of Fg decreased VE-cadherin expression at protein and mRNA levels. Fg induced translocation of VE-cadherin to cytosol, which led to disruption of cell-to-cell interaction and cell to subendothelial matrix attachment. Fg-induced alterations in cell layer integrity and their attachment were diminished during inhibition of MMP-9 activity. Thus Fg compromises EC layer integrity causing downregulation and translocation of VE-cadherin and through MMP-9 activation. These results suggest that increased level of Fg could play a significant role in vascular dysfunction and remodeling. [Copyright &y& Elsevier]

Aims We investigated the mechanisms of action of lysophosphatidic acid (LPA) to regulate vascular endothelial (VE)-cadherin dynamics and cell–cell contact. Methods and results While a low concentration of LPA stimulated VE-cadherin internalization and subsequent cell–cell dissociation, a high concentration of LPA masked the disruptive actions on VE-cadherin and protected the barrier function in human vascular endothelial cells. Knockdown experiments of major LPA receptor subtypes, i.e. LPA1 and p2y5 (also termed LPA6), with their specific small interfering RNAs, showed that LPA1 and LPA6 mediate the LPA-induced disruptive and protective actions on barrier integrity, respectively. LPA6-mediated tube formation, reflecting stabilization of barrier integrity, was confirmed by in vitro angiogenesis assay. The LPA1-mediated disruptive actions were inhibited by pertussis toxin, dominant-negative Rac1, and inhibitors for c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK), but not by dominant-negative RhoA. In contrast, the LPA6-mediated protective actions were associated with activation of Src and Rap1 and attenuated by abrogation of their activities. Further characterization showed that Rap1 is located downstream of Src and dependent on C3G, a Rap1 guanine nucleotide exchange factor. Finally, an LPA antagonist significantly inhibited lactic acid-induced limb lesions in vivo, which may be attributed to dysfunction of endothelial cells. Conclusion LPA induced disruption and protection of VE-cadherin integrity through LPA1–Gi protein–Rac1–JNK/p38MAPK and LPA6–G12/13 protein–Src–C3G–Rap1 pathways, respectively. [ABSTRACT FROM PUBLISHER]

A hallmark of many, sometimes life-threatening, inflammatory diseases and disorders is vascular leakage. The extent and severity of vascular leakage is broadly mediated by the integrity of the endothelial cell (EC) monolayer, which is in turn governed by three major interactions: cell-cell and cell-substrate contacts, soluble mediators, and biomechanical forces. A potentially critical but essentially uninvestigated component mediating these interactions is the stiffness of the substrate to which the endothelial monolayer is adherent. Accordingly, we investigated the extent to which substrate stiffening influences endothelial monolayer disruption and the role of cell-cell and cell-substrate contacts, soluble mediators, and physical forces in that process. Traction force microscopy showed that forces between cell and cell and between cell and substrate were greater on stiffer substrates. On stiffer substrates, these forces were substantially enhanced by a hyperpermeability stimulus (thrombin, 1 U/ml), and gaps formed between cells. On softer substrates, by contrast, these forces were increased far less by thrombin, and gaps did not form between cells. This stiffness-dependent force enhancement was associated with increased Rho kinase activity, whereas inhibition of Rho kinase attenuated baseline forces and lessened thrombin-induced inter-EC gap formation. Our findings demonstrate a central role of physical forces in EC gap formation and highlight a novel physiological mechanism. Integrity of the endothelial monolayer is governed by its physical microenvironment, which in normal circumstances is compliant but during pathology becomes stiffer. [ABSTRACT FROM AUTHOR]

Abstract: Vascular endothelial (VE)-cadherin is an essential protein of adherens junctions of endothelial cells and plays a pivotal role in vascular homeostasis. Mammalian target of rapamycin complex 2 (mTORC2) deficient mice display defects in fetal vascular development. Blocking mTOR or the upstream kinase phosphoinositide 3-kinase (PI3K) led to a dose-dependently decrease of the VE-cadherin mRNA and protein expression. Immunofluorescent staining showed a strongly decreased expression of VE-cadherin at the interface of human umbilical endothelial cells (HUVECs) followed by intercellular gap formation. Herewith, we demonstrated that the expression of VE-cadherin is dependent on mTOR and PI3K signaling. [Copyright &y& Elsevier]

Splenic sinus endothelial cells, which adhere through tight and adherens junctions, regulate the passage of blood cells through the splenic cord. The objective of this study was to assess the localization of tight junctional proteins, claudin-5 and ZO-1 in the sinus endothelial cells of rat spleen and to characterize spatial and functional relationships between tight and adherens junctions. Immunofluorescence microscopy of tissue cryosections demonstrated that claudin-5, ZO-1, and α-catenin were distinctly localized in the junctional regions of adjacent endothelial cells. Immunogold electron microscopy demonstrated claudin-5 localized in the tight-junctional fused membranes of adjacent endothelial cells. Immunogold labeling for ZO-1 was localized not only in the tight-junctional-fused membranes of endothelial cells but also in the junctional membrane. α-Catenin was intermittently localized along the juxtaposed junctional membranes of adjacent endothelial cells. Double-staining immunogold microscopy for claudin-5 and ZO-1, claudin-5 and VE-cadherin, ZO-1 and VE-cadherin, and ZO-1 and α-catenin demonstrated that ZO-1 was closely localized to VE-cadherin and α-catenin in their juxtaposed membranes of endothelial cells. Thus, ZO-1 might play an important role in regulating the cell–cell junctions of sinus endothelial cells for blood–cell passage through splenic cords. [ABSTRACT FROM AUTHOR]

Lung inflammatory disease is characterized by increased polymorphonuclear leukocyte (PMN) infiltration and vascular permeability. PMN infiltration into tissue involves signaling between endothelial cells and migrating PMNs, which leads to alterations in the organization of adherens junctions (AJs). We addressed the possible role of the protein constituents of AJs, endothelium-specific vascular-endothelial (VE)-cadherin, in the migration of PMNs. Studies were made using VE-cadherin mutant constructs lacking the extracellular domain (ΔEXD) or, additionally, lacking the COOH-terminus β-catenin-binding domain (ΔEXDΔβ). Either construct was transduced in pulmonary microvessel endothelia of mice using cationic liposome-encapuslated cDNA constructs injected intravenously. Optimal expression of constructs was seen by Western blot analysis within 24 h. Vessel wall liquid permeability measured as the lung microvessel capillary filtration coefficient increased threefold in ΔEXD-transduced lungs, indicating patency of interendothelial junctions, whereas the control ΔEXDΔβ construct was ineffective. To study lung tissue PMN recruitment, we challenged mice intraperitoneally with LPS (3 mg/kg) for 6 h and measured PMN numbers by bronchoalveolar lavage and their accumulation morphometrically in lung tissue. ΔEXD expression markedly reduced the PMN sequestration and migration seen in nontransfected (control wild type) or ΔEXDΔβ-transfected (negative control) mice challenged with LPS. In addition, ΔEXD transfection suppressed LPS-induced activation of NF-κB and consequent ICAM-1 expression. These results suggest that disassembly of VE-cadherin junctions serves as a negative signal for limiting transendothelial PMN migration secondary to decreased ICAM-1 expression in The mouse model of LPS-induced sepsis. [ABSTRACT FROM AUTHOR]

cAMP enhances endothelial barrier properties and is protective against various inflammatory mediators both in vivo and in vitro. However, the mechanisms whereby cAMP stabilizes the endothelial barrier are largely unknown. Recently we demonstrated that the Rho family GTPase Rac-1 is required for maintenance of endothelial barrier functions in vivo and in vitro. Therefore, in the present study we investigated the effect of forskolin (5 µM)- and rolipram (10 µM)-induced cAMP increase on reduction of barrier functions in response to Rac-1 inhibition by Clostridium sordellii lethal toxin (LT). Forskolin and rolipram treatment blocked LT (200 ng/ml)induced hydraulic conductivity (Lp) increase in mesenteric microvessels in vivo. Likewise, LT-induced intercellular gap formation in monolayers of cultured microvascular myocardial endothelial (MyEnd) cells and LT-induced loss of adhesion of vascular endothelial cadherin-coated microbeads were abolished. Inhibition of PKA by myristoylated inhibitor peptide (14-22) of PKA (100 µM) reduced the protective effect of cAMP on LT-induced Lp increase in vivo and gap formation in vitro, indicating that the effect of cAMP on Rac-1 inhibition was PKA dependent. Glucosylation assays demonstrated that cAMP prevents inhibitory Rac-1 glucosylation by LT, indicating that one way that cAMP enhances endothelial barrier functions may be by regulating Rac-1 signaling. Our study suggests that cAMP may provide its well-established protective effects at least in part by regulation of Rho proteins. [ABSTRACT FROM AUTHOR]

Interaction of p120 with juxtamembrane domain (JMD) of VE-cadherin has been implicated in regulation of endothelial cell-cell adhesion. We used a number of approaches to alter the level of p120 available for binding to VE-cadherin as a means to investigate the role of p120-VE-cadherin interaction in regulation of barrier function in confluent endothelial monolayers. Expression of an epitope-tagged fragment corresponding to JMD of VE-cadberin resulted in a decrease in endothelial barrier function as assessed by changes in albumin clearance and electrical resistance. Binding of JMD-Flag to p120 resulted in a decreased level of p120. In addition to decreasing pl20 level, expression of JMD also decreased level of VE-cadherin. Expression of JMD also caused an increase in MLC phosphorylation and rearrangement of actin cytoskeleton, which, coupled with decreased cadherin, can contribute to loss of barrier function. Reducing p120 by siRNA resulted in a decrease in VE-cadherin, whereas increasing the level of p120 increased the level of VE-cadherin, demonstrating that p120 regulates the level of VE-cadherin. Overexpression of p120 was, however, associated with decreased barrier function and rearrangement of the actin cytoskeleton. Interestingly, expression of p120 was able to inhibit thrombin-induced increases in MLC phosphorylation, suggesting that p120 inhibits activation of Rho/Rho kinase pathway in endothelial cells. Excess p120 also prevented JMD-induced increases in MLC phosphorylation, correlating this phospborylation with Rho/ Rho kinase pathway. These findings show p120 plays a major role in regulating endothelial barrier function, as either a decrease or increase of p120 resulted in disruption of permeability across cell monolayers. [ABSTRACT FROM AUTHOR]

Aims/hypothesis. Increased angiogenesis of fetoplacental vessels is a feature of pregnancies complicated by Type 1 diabetes mellitus, but the underlying molecular mechanisms are unknown. This investigation tests whether the diabetic maternal environment alters the phenotypic expression of placental vascular endothelial cadherin and β-catenin, which have been implicated as key molecules in barrier formation and angiogenesis in the endothelium. Methods. Term placental microvessels from normal pregnancies (n=8) and from those complicated by Type 1 diabetes (n=8) were perfused with 76-Mr dextran tracers (1 mg/ml) and subjected to immunocytochemistry, immunoblotting and microscopy. Junctional integrity, localisation and phosphorylation were investigated along with total protein levels of vascular endothelial cadherin, β-catenin and vascular endothelial growth factor. Stereological sampling and estimation tools were used to quantify aspects of angiogenesis and endothelial proliferation. Results. In the Type 1 diabetic placentae, junctional localisations of vascular endothelial cadherin and β-catenin altered significantly, with more than 50% of microvessels showing complete loss of immunoreactivity and with no overall loss of total protein. Tracer leakage was associated with these vessels. There was a two- to three-fold increase in vessels showing junctional phospho-tyrosine immunoreactivity and hyperphosphorylated β-catenin. Vascular endothelial growth factor levels were higher in these placentae. A four-fold increase in endothelial proliferation was observed, along with an increase in total length of capillaries without any change in luminal diameter. Conclusions/interpretation. Molecular perturbations of vascular endothelial cadherin and β-catenin occur in fetoplacental vessels of pregnancies complicated by Type 1 diabetes. Phosphorylation and loss of these molecules from the adherens junctional domains may be influenced in part by the elevated levels of vascular endothelial growth factor in the placenta. Perturbations of the junctional proteins may explain the observed breach in barrier integrity and may contribute to the mechanisms that drive proliferation and increases in capillary length. [ABSTRACT FROM AUTHOR]

Vasculogenesis and angiogenesis are regulated by the capacity of endothelial cells to adhere to each other and form new tubes. The presence and role of junctional adhesion molecules during physiological vasculogenesis is unknown. Using ultrastructural and immunocytochemical approaches, we compared the junctional phenotype of developing vessels of the first-trimester human placenta with vessels in the last trimester; the latter include newly formed terminal capillaries and the quiescent vascular bed. First-trimester placental vessels contained the adherens junctional molecules, vascular endothelial cadherin and α- and β-catenin but lacked plakoglobin, the component of fully differentiated adherens junctions. Furthermore, these vessels did not contain the transmembrane tight junctional molecules occludin and claudin-1 and -2. This profile reflects the phenotype of terminal capillaries but differs from large vessels of the full-term placenta. Electron microscopic studies revealed that endothelial tight junctions are present in the first-trimester placenta. Thus, occludin and claudin-1 appear to play no part in the formation of endothelial tight junctions, but are a later requirement. In the early placenta, the predominant growth factor appears to be vascular endothelial growth factor (VEGF), whilst at term, angiopoietin-1 was present in large vessels, with intense angiopoietin-2 immunofluorescence (and VEGF) located in terminal villous capillaries. Thus, endothelial junctions in the human placenta possess two distinct molecular phenotypes, i.e. stable or dynamic, dependent on maturity and plasticity. These distinct phenotypes may be influenced by the angiopoietins/VEGF present in the placenta.Copyright © 2002 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]