Growth of new blood vessels (angiogenesis), required for all tumor growth, is stimulated by the expression of vascular endothelial growth factor (VEGF). VEGF is up-regulated in all known solid tumors but also in atherosclerosis, diabetic retinopathy, arthritis, and many other conditions. Conventional VEGF isoforms have been universally described as proangiogenic cytokines. Here, we show that an endogenous splice variant, VEGF 165 b, is expressed as protein in normal cells and tissues and is circulating in human plasma. We also present evidence for a sister family of presumably inhibitory splice variants. Moreover, these isoforms are down-regulated in prostate cancer. We also show that VEGF 165 b binds VEGF receptor 2 with the same affinity as VEGF 165

Microvascular barrier injury has been implicated in the initiation and progress of end organ complications of diabetic mellitus. Plasma leakage and fluid retention are seen in various tissues of diabetic patients or animals at the early stages of the disease before structural microangiopathy can be detected. Clinical and experimental evidence suggests that hyperglycemia, often accompanied with insulin deficiency or insulin resistance, causes impaired autoregulation and increased permeability in microvessels. Multiple molecular pathways have been identified as contributors to the altered fluid homeostasis, including increased polyol flux that promotes oxidative stress, advanced glycation that leads to carbonyl stress, and excessive glucose metabolism that results in protein kinase C activation. These abnormal metabolic activities are associated with the production of pro-inflammatory cytokines and growth factors, which can stimulate an array of signaling reactions and structural changes at the endothelial barrier and ultimately cause microvascular leakage. Interventions that manipulate these metabolic and inflammatory pathways have demonstrated efficacy in delaying the progress of diabetic microvascular complications; however, their direct effects and mechanisms of action on the microcirculation remain elusive. A deeper understanding of the molecular basis of diabetes-induced endothelial barrier dysfunction will provide a framework for the development of new therapeutic targets to treat this chronic and debilitating disease process.

Microvascular leakage has been implicated in the pathogenesis of multiple organ dysfunction during trauma. Previous studies suggest the involvement of myosin light chain (MLC) phosphorylation-triggered endothelial contraction in the development of microvascular hyperpermeability. Myosin light chain kinase (MLCK) plays a key role in the control of MLC-phosphorylation status; thus, it is thought to modulate barrier function through its regulation of intracellular contractile machinery. The aim of this study was to further investigate the endothelial mechanism of MLC-dependent barrier injury in burns, focusing on the long isoform of MLCK (MLCK-210) that has recently been identified as the predominant isoform expressed in vascular endothelial cells. An MLCK-210 knockout mouse model was subjected to third-degree scald burn covering 25% total body surface area. The mesenteric microcirculation was observed using intravital microscopy, and the microvascular permeability was assessed by measuring the transvenular flux of fluorescein isothiocyanate-albumin. In a separate experiment, in vivo mesenteric hydraulic conductivity (Lp) was measured using the modified Landis technique. The injury caused a profound microvascular leakage, as indicated by a 2-fold increase in albumin flux and 4-fold increase in Lp at the early stages, which was associated with a high mortality within the 24-h period. Compared with wild-type control, the MLCK-210-deficient mice displayed a significantly improved survival with a greatly attenuated microvascular hyperpermeability response to albumin and fluid. These results provide direct evidence for a role of MLCK-210 in mediating burn-induced microvascular barrier injury and validate MLCK-210 as a potential therapeutic target in the treatment of burn edema.

Diabetic angiopathy is a major cause of morbidity and mortality in diabetes mellitus. Endothelial dysfunction and associated alterations in blood flow, pressure and permeability are widely accepted phenomena in the diabetic milieu and are understood to lead to microangiopathy. Despite the clinical importance of diabetic microangiopathy, the mechanisms of pathogenesis remain elusive. In particular, much is yet to be understood about the nature of the putative increased permeability with respect to diabetes. Microvessel permeability is intrinsically difficult to measure and a surrogate (solute or solvent flux) is usually reported, the measurement of which is hampered by haemodynamic factors, such as flow rate, hydrostatic pressure gradient, solute concentration and surface area available for exchange. Very few studies describing the measurement of permeability with respect to diabetes have controlled for all these factors. As a result, the nature of the increased microvessel permeability in diabetes mellitus and indeed its causes are poorly understood. Recent studies have shown that hyperglycaemia can alter the glycocalyx structure, and parallel findings have shown that the apparent increase in permeability demonstrated in hyperglycaemia may be due to an increase in the permeability of the vessels to water, and not an increase in protein permeability, an effect attributable to altered glycocalyx. This review focuses on the current understanding of microvascular permeability in terms of the endothelial glycocalyxfibre-matrix theory, those methods used to determine permeability in the context of diabetes, and the more recent developments in our understanding of elevated microvascular permeability in the diabetic circulation.

Objectives-The purposes of this study were to characterize the direct effect of the C-terminal fragment of fibrinogen ␥ chain (␥C) on microvascular endothelial permeability and to examine its molecular mechanism of action. Methods and Results-Intravital microscopy was performed to measure albumin extravasation in intact mesenteric microvasculature, followed by quantification of hydraulic conductivity in single perfused microvessels. Transendothelial electric resistance was measured in microvascular endothelial cells in combination with immunoblotting and immunocytochemistry. The results show that ␥C induced time-and concentration-dependent increases in protein transvascular flux and water permeability and decreases in endothelial barrier function, coupled with Rho GTPase activation, myosin light chain phosphorylation, and stress fiber formation. Depletion of RhoA via siRNA knockdown or pharmacological inhibition of RhoA signaling attenuated ␥C-induced barrier dysfunction. Imaging analyses demonstrated binding of ␥C to endothelial cells; the interaction was inhibited during blockage of the ␣v␤3 integrin. Furthermore, in vivo experiments showed that the microvascular leak response to ␥C was attenuated in integrin ␤3 Key Words: fibrinogen degradation products Ⅲ microvascular permeability Ⅲ signal transduction Ⅲ Rho-GTPase Ⅲ thrombosis F ibrinogen comprises double polypeptides termed ␣, ␤, and ␥ chains, which form an elongated structure with two outer globular D domains connected to central E domains through coiled-coil segments. [1][2][3] As essential steps of the coagulation cascade, thrombin cleavage of fibrinogen and conversion to cross-linked fibrin occur in response to activation of intrinsic or extrinsic factors, followed by fibrinolysis that generates fibrin degradation products (FDPs; supplemental Figure I, available online at http://atvb.ahajournals.org). Typical FDPs include D-dimer fragments and soluble monomers containing the C termini of fibrinogen-␣, ␤ and ␥ chains, clinically measured as markers of coagulation disorder. 4 -7 The fibrinolysis cascade exerts physiological functions and pathogenic impact well beyond its role in hemostasis. Increased plasma levels of fibrinogen have been correlated with atherosclerosis, stroke, myocardium infarction, and peripheral vascular disease. 5,6 Some fibrinogen proteolytic products have been shown to participate in cell proliferation, migration, and adhesion. 6 -8 An effect on endothelial barrier properties has been documented 9 -12 ; however, consensus has not been reached as to whether or not fibrinogen as intact protein increases vascular permeability, and even less is known regarding the receptor mechanism and signal transduction that mediates the endothelial response to specific subunits or degradation products, which are known to act distinctively based on their unique chemical structures and molecular conformations. 1,3,8 Investigation is often confounded by the multi-domain structure of fibrinogen and its diverse cellular targets ranging from platelets ...