Microvascular leakage due to endothelial barrier dysfunction is a prominent feature of T helper 2 (Th2) cytokine mediated allergic inflammation. Interleukin-4 (IL-4) is a potent Th2 cytokine, known to impair the barrier function of endothelial cells. However, the effectors mediating IL-4 induced cytoskeleton remodeling and consequent endothelial barrier dysfunction remain poorly defined. Here we have used whole genome transcriptome profiling and gene ontology analyses to identify the genes and processes regulated by IL-4 signaling in human coronary artery endothelial cells (HCAEC). The study revealed Wnt5A as an effector that can mediate actin cytoskeleton remodeling in IL-4 activated HCAEC through the regulation of LIM kinase (LIMK) and Cofilin (CFL). Following IL-4 treatment, LIMK and CFL were phosphorylated, thereby indicating the possibility of actin stress fiber formation. Imaging of actin showed the formation of stress fibers in IL-4 treated live HCAEC. Stress fiber formation was notably decreased in the presence of Wnt inhibitory factor 1 (WIF1). Non-invasive impedance measurements demonstrated that IL-4 increased the permeability and impaired the barrier function of HCAEC monolayers. Silencing Wnt5A significantly reduced permeability and improved the barrier function of HCAEC monolayers upon IL-4 treatment. Our study identifies Wnt5A as a novel marker of IL-4 activated vascular endothelium and demonstrates a critical role for Wnt5A in mediating IL-4 induced endothelial barrier dysfunction. Wnt5A could be a potential therapeutic target for reducing microvascular leakage and edema formation in Th2 driven inflammatory diseases.

Satisfactory therapeutic strategies for septic shock are still missing. Previously we found elevated levels of Wnt5A in patients with severe sepsis and septic shock. Wnt5A is released by activated macrophages but knowledge of its effects in the vascular system remains scant. Here we investigate the response of human coronary artery endothelial cells (HCAEC) to Wnt5A. We used a genome-wide differential expression approach to define novel targets regulated by Wnt5A. Gene ontology analysis of expression profiles revealed clusters of genes involved in actin cytoskeleton remodeling as the predominant targets of Wnt5A. Wnt5A targeted Rho-associated protein serine/threonine kinase (ROCK), leading to phosphorylation of LIM kinase-2 (LIMK2) and inactivation of the actin depolymerization factor cofilin-1 (CFL1). Functional experiments recording cytoskeletal rearrangements in living cells showed that Wnt5A enhanced stress fiber formation as a consequence of reduced actin depolymerization. The antagonist Wnt inhibitory factor 1 (WIF1) that specifically interferes with the WIF domain of Ryk receptors prevented actin polymerization. Wnt5A disrupted β-catenin and VE-cadherin adherens junctions forming inter-endothelial gaps. Functional experiments targeting the endothelial monolayer integrity and live recording of trans-endothelial resistance revealed enhanced permeability of Wnt5A-treated HCAEC. Ryk silencing completely prevented Wnt5A-induced endothelial hyperpermeability. Wnt5A decreased wound healing capacity of HCAEC monolayers; this was restored by the ROCK inhibitor Y-27632. Here we show that Wnt5A acts on the vascular endothelium causing enhanced permeability through Ryk interaction and downstream ROCK/LIMK2/CFL1 signaling. Wnt5A/Ryk signaling might provide novel therapeutic strategies to prevent capillary leakage in systemic inflammation and septic shock.

Rationale: αCGRP (α calcitonin gene-related peptide), one of the strongest vasodilators, is cardioprotective in hypertension by reducing the elevated blood pressure. Objective: However, we hypothesize that endogenous, physical activity–induced αCGRP has blood pressure–independent cardioprotective effects in chronic hypertension. Methods and Results: Chronically hypertensive (one-kidney-one-clip surgery) wild-type and αCGRP −/− sedentary or voluntary wheel running mice were treated with vehicle, αCGRP, or the αCGRP receptor antagonist CGRP8-37. Cardiac function and myocardial phenotype were evaluated echocardiographically and by molecular, cellular, and histological analysis, respectively. Blood pressure was similar among all hypertensive experimental groups. Endogenous αCGRP limited pathological remodeling and heart failure in sedentary, chronically hypertensive wild-type mice. In these mice, voluntary wheel running significantly improved myocardial phenotype and function, which was abolished by CGRP8-37 treatment. In αCGRP −/− mice, αCGRP treatment, in contrast to voluntary wheel running, improved myocardial phenotype and function. Specific inhibition of proliferation and myofibroblast differentiation of primary, murine cardiac fibroblasts by αCGRP suggests involvement of these cells in αCGRP-dependent blunting of pathological cardiac remodeling. Conclusions: Endogenous, physical activity–induced αCGRP has blood pressure–independent cardioprotective effects and is crucial for maintaining cardiac function in chronic hypertension. Consequently, inhibiting endogenous αCGRP signaling, as currently approved for migraine prophylaxis, could endanger patients with hypertension.

Switching of cellular energy production from oxidative phosphorylation (OXPHOS) by mitochondria to aerobic glycolysis occurs in many types of tumors. However, the significance of this switching for the development of gastric carcinoma and what connection it may have to Helicobacter pylori infection of the gut, a primary cause of gastric cancer, are poorly understood. Therefore, we investigated the expression of OXPHOS complexes in two types of human gastric carcinomas (“intestinal” and “diffuse”), bacterial gastritis with and without metaplasia, and chemically induced gastritis by using immunohistochemistry. Furthermore, we analyzed the effect of HP infection on several key mitochondrial proteins. Complex I expression was significantly reduced in intestinal type (but not diffuse) gastric carcinomas compared to adjacent control tissue, and the reduction was independent of HP infection. Significantly, higher complex I and complex II expression was present in large tumors. Furthermore, higher complex II and complex III protein levels were also obvious in grade 3 versus grade 2. No differences of OXPHOS complexes and markers of mitochondrial biogenesis were found between bacterially caused and chemically induced gastritis. Thus, intestinal gastric carcinomas, but not precancerous stages, are frequently characterized by loss of complex I, and this pathophysiology occurs independently of HP infection.