We previously showed that an elevated content of fibrinogen (Fg) increased formation of filamentous actin and enhanced endothelial layer permeability. In the present work we tested the hypothesis that Fg binding to endothelial cells (ECs) alters expression of actin-associated endothelial tight junction proteins (TJP). Rat cardiac microvascular ECs were grown in gold plated chambers of an electrical cell-substrate impedance system, 8-well chambered, or in 12-well plates. Confluent ECs were treated with Fg (2 or 4 mg/ml), Fg (4 mg/ml) with mitogen-activated protein kinase (MEK) kinase inhibitors (PD98059 or U0126), Fg (4 mg/ml) with anti-ICAM-1 antibody or BQ788 (endothelin type B receptor blocker), endothelin-1, endothelin-1 with BQ788, or medium alone for 24 h. Fg induced a dose-dependent decrease in EC junction integrity as determined by transendothelial electrical resistance (TEER). Western blot analysis and RT-PCR data showed that the higher dose of Fg decreased the contents of TJPs, occludin, zona occluden-1 (ZO-1), and zona occluden-2 (ZO-2) in ECs. Fg-induced decreases in contents of the TJPs were blocked by PD98059, U0126, or anti-ICAM-1 antibody. While BQ788 inhibited endothelin-1-induced decrease in TEER, it did not affect Fg-induced decrease in TEER. These data suggest that Fg increases EC layer permeability via the MEK kinase signaling pathway by affecting occludin, ZO-1, and ZO-2, TJPs, which are bound to actin filaments. Therefore, increased binding of Fg to its major EC receptor, ICAM-1, during cardiovascular diseases may increase microvascular permeability by altering the content and possibly subcellular localization of endothelial TJPs.

WE, Tseng MT, Tyagi SC. Mitochondrial matrix metalloproteinase activation decreases myocyte contractility in hyperhomocysteinemia. myocyte N-methyl-D-aspartate receptor-1 (NMDA-R1) activation induces mitochondrial dysfunction. Matrix metalloproteinase protease (MMP) induction is a negative regulator of mitochondrial function. Elevated levels of homocysteine [hyperhomocysteinemia (HHCY)] activate latent MMPs and causes myocardial contractile abnormalities. HHCY is associated with mitochondrial dysfunction. We tested the hypothesis that HHCY activates myocyte mitochondrial MMP (mtMMP), induces mitochondrial permeability transition (MPT), and causes contractile dysfunction by agonizing NMDA-R1. The C57BL/6J mice were administered homocystinemia (1.8 g/l) in drinking water to induce HHCY. NMDA-R1 expression was detected by Western blot and confocal microscopy. Localization of MMP-9 in the mitochondria was determined using confocal microscopy. Ultrastructural analysis of the isolated myocyte was determined by electron microscopy. Mitochondrial permeability was measured by a decrease in light absorbance at 540 nm using the spectrophotometer. The effect of MK-801 (NMDA-R1 inhibitor), GM-6001 (MMP inhibitor), and cyclosporine A (MPT inhibitor) on myocyte contractility and calcium transients was evaluated using the IonOptix video edge track detection system and fura 2-AM. Our results demonstrate that HHCY activated the mtMMP-9 and caused MPT by agonizing NMDA-R1. A significant decrease in percent cell shortening, maximal rate of contraction (ϪdL/dt), and maximal rate of relaxation (ϩdL/dt) was observed in HHCY. The decay of calcium transient amplitude was faster in the wild type compared with HHCY. Furthermore, the HHCY-induced decrease in percent cell shortening, ϪdL/dt, and ϩdL/dt was attenuated in the mice treated with MK-801, GM-6001, and cyclosporin A. We conclude that HHCY activates mtMMP-9 and induces MPT, leading to myocyte mechanical dysfunction by agonizing NMDA-R1. myocyte; calcium; mitochondrial permeability; N-methyl-D-aspartate receptor-1; arrhythmogenesis THE PATHOPHYSIOLOGY of chronic heart failure (CHF) involves abnormalities in systolic and/or diastolic function and increases the propensity for reentry arrhythmias (30, 6). Continued elevation of cardiac sympathetic drive contributes to myocardial toxicity, leading to the decline in cardiac contractility (29). Recent observations suggest an increase in glutamatergic activity on sympathetic regulation, due to the upregulation of hypothalamic N-methyl-D-aspartate receptor-1 subunits (NMDA-R1) during CHF (16). Ischemia-and reperfusion-induced arrhythmias are sensitive to NMDA-R1 blockade (8).Hyperhomocysteinemia (HHCY) is a graded risk factor for CHF (12, 7) and for sudden cardiac death (SCD) resulting from coronary fibrous plaques (4, 1, 5). Homocysteinemia (HCY) induces interstitial cardiac fibrosis leading to systolic/diastolic dysfunction (13). The antagonist to the NMDA-R protects against HCY-induced oxidative damage in neurons (10) and protects against...

We previously demonstrated that fibrinogen (Fg) binding to the vascular endothelial intercellular adhesion molecule-1 (ICAM-1) leads to microvascular constriction in vivo and in vitro. Although a role of endothelin-1 (ET-1) in this Fg-induced vasoconstriction was suggested, the mechanism of action was not clear. In the current study, we tested the hypothesis that Fg-induced vasoconstriction results from ET-1 production by vascular endothelial cells (EC) and is mediated by activation of extracellular signal-regulated kinase -1/2 (ERK-1/2). Confluent, rat heart microvascular endothelial cells (RHMECs) were treated with one of the following: Fg (2 or 4 mg/ml), Fg (4 mg/ml) with ERK-1/2 kinase inhibitors (PD-98059 or U-0126), Fg (4 mg/ml) with an antibody against ICAM-1, or medium alone for 45 min. The amount of ET-1 formed and the concentration of released von Willebrand factor (vWF) in the cell culture medium were measured by ELISAs. Fg-induced exocytosis of Weibel-Palade bodies (WPBs) was assessed by immunocytochemistry. Phosphorylation of ERK-1/2 was detected by Western blot analysis. Fg caused a dose-dependent increase in ET-1 formation and release of vWF from the RHMECs. This Fg-induced increase in ET-1 production was inhibited by specific ERK-1/2 kinase inhibitors and by anti-ICAM-1 antibody. Immunocytochemical staining showed that an increase in Fg concentration enhanced exocytosis of WPBs in ECs. A specific endothelin type B receptor blocker, BQ-788, attenuated the enhanced phosphorylation of ERK-1/2 in ECs caused by increased Fg content in the culture medium. The presence of an endothelin converting enzyme inhibitor, SM-19712, slightly decreased Fg-induced phosphorylation of ERK-1/2, but inhibited production of Fg-induced ET-1 production. These results suggest that Fg-induced vasoconstriction may be mediated, in part, by activation of ERK-1/2 signaling and increased production of ET-1 that further increases EC ERK-1/2 signaling. Thus, an increased content of Fg may enhance vasoconstriction through increased production of ET-1.

Background: Susceptibility/resistance to Plasmodium falciparum malaria has been correlated with polymorphisms in more than 30 human genes with most association analyses having been carried out on patients from Africa and south-east Asia. The aim of this study was to examine the possible contribution of genetic variants in the TNF and FCGR2A genes in determining severity/resistance to P. falciparum malaria in Indian subjects.

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Type 2 diabetes significantly elevates the risk of cardiovascular disease. This can be largely attributed to the adverse effects of hyperglycemic conditions on normal endothelial cell (EC) function. ECs in both large and small vessels are influenced by hyperglycemic conditions, which increase susceptibility to EC dysfunction and atherosclerotic lesion formation. Fluid shear stress and flow patterns play an essential role in atherogenesis: lesions form only at locations where fluid flow behavior can be classified as "disturbed flow" (i.e., low shear stress recirculation and/or retrograde flow). Since regions of disturbed flow are the focal points of atherosclerotic cardiovascular disease, we hypothesized that the combinatorial effects of high glucose and disturbed flow conditions elicit significantly different responses from ECs than high glucose alone. To validate our hypothesis, we used our endothelial cell culture model (ECCM) to establish vascular niches associated with "normal" and "disturbed" flow conditions typically seen in vivo along with physiological pressure and stretch. We subjected human aortic endothelial cells (HAECs) to hyperglycemic conditions under both "normal" and "disturbed" flow. Our results confirm significant and quantifiable differences in phenotypic and functional markers between cells cultured under conditions of "normal" and "disturbed flow" under hyperglycemic conditions suggesting that elevated glucose in conjunction with "disturbed" flow conditions results in significantly higher level of EC dysfunction. The ECCM can therefore be used as a physiologically relevant model to study early stage hyperglycemia induced atherosclerosis for basic research, drug discovery, and screening and toxicity studies.

Copper deficiency inactivates Cu/Zn-SOD and promotes accumulation of reactive oxygen species. This process likely impairs nitric oxide (NO)-mediated relaxation as well as triggers vascular inflammation. The current study was designed to determine whether COX-2, a proinflammatory protein, expression and activity are upregulated in the oxidative environment associated with inadequate Cu. Weanling male Sprague Dawley rats were fed purified diets which were either Cu-adequate (Cu-A); Cu-marginal (Cu-M), Cu-deficient (Cu-D), or the Cu-D diet combined with the SOD mimetic Tempol (Cu-D/T; 1 mM in drinking water) for 4 weeks. COX-2 protein, PGE(2) (COX-2 metabolite) and isoprostanes (index of oxidative stress) were all higher in the Cu-D group vs Cu-A group, but no significant differences occurred between the Cu-M and Cu-A groups. Tempol protected against an attenuation of NO-mediated vasodilation in the Cu-D rats but did not prevent the elevation of PGE(2) or isoprostanes. Our data suggest a role for copper as a modulator of oxidative stress and inflammation independent of SOD activity or NO-derived oxidants.

Background Excessive complement activation is an integral part of ischemia and reperfusion (IR) injury (IRI) of organs. In kidney transplantation the pathological consequence of IRI and complement activation can lead to delayed graft function which in turn is associated with acute rejection. Previous strategies to reduce complement induced IRI required systemic administration of agents, which can lead to increased susceptibility to infections/immune diseases. The objective of this study was to determine whether an increase in complement control defenses of rat kidney endothelium reduces IRI. We hypothesized that increased complement control on the endothelial barrier reduces IR-mediated complement activation and reduces kidney dysfunction. Materials and methods Fisher 344 rats underwent left kidney ischemia for 45 min. and treatment with a novel fusogenic lipid vesicle (FLVs) delivery system to decorate endothelial cells with Vaccinia virus complement control protein (VCP), followed by reperfusion for 24h. Assessment included renal function by serum creatinine and urea, myeloperoxidase assay for neutrophil infiltration, histopathology, and quantification of C3 production in kidneys. Results Animals in which the kidney endothelium was bolstered by FLVs+VCP treatment had better renal function with a significant reduction in serum creatinine as compared to vehicle controls. C3 production was significantly reduced (p<0.05) in treated animals compared to vehicle controls. Conclusion Increasing complement control at the endothelial barrier with FLVs+VCP modulates complement activation/production during the first 24h, reducing renal dysfunction following IRI.