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Summary Understanding epigenetic mechanisms regulating embryonic stem cell (ESC) differentiation to endothelial cells may lead to increased efficiency of generation of vessel wall endothelial cells needed for vascular engineering. Here we demonstrated that the histone demethylases KDM4A and KDM4C played an indispensable but independent role in mediating the expression of fetal liver kinase (Flk)1 and VE-cadherin, respectively, and thereby the transition of mouse ESCs (mESCs) to endothelial cells. KDM4A was shown to bind to histones associated with the Flk1 promoter and KDM4C to bind to histones associated with the VE-cadherin promoter. KDM4A and KDM4C were also both required for capillary tube formation and vasculogenesis in mice. We observed in zebrafish that KDM4A depletion induced more severe vasculogenesis defects than KDM4C depletion, reflecting the early involvement of KDM4A in specifying endothelial cell fate. These findings together demonstrate the essential role of KDM4A and KDM4C in orchestrating mESC differentiation to endothelial cells through the activation of Flk1 and VE-cadherin promoters, respectively., Highlights • Histone demethylases KDM4A and KDM4C are upregulated in endothelial differentiation • KDM4A and KDM4A bind to histones associated with Flk1 and VE-cadherin promoters • Depletion of either KDM4A or KDM4C in mESCs prevented endothelial differentiation • Depletion of KDM4A and KDM4C prevented blood vessel formation in zebrafish, In this article, Malik, Rehman, and colleagues show that the histone demethylases KDM4A and KDM4C are upregulated during the differentiation of mouse embryonic stem cells into vascular endothelial cells. Depletion of these two histone demethylases suppressed endothelial differentiation and angiogenesis in vitro as well as vascular development in a zebrafish model.

Blood neutrophils perform an essential host-defense function by directly migrating to bacterial invasion sites to kill bacteria. The mechanisms mediating the transition from the migratory to bactericidal phenotype remain elusive. Here, we demonstrate that TRPM2, a trp superfamily member, senses neutrophil-generated reactive oxygen species and restrains neutrophil migration. The inhibitory function of oxidant sensing by TRPM2 requires the oxidation of Cys549, which then induces TRMP2 binding to formyl peptide receptor 1 (FPR1) and subsequent FPR1 internalization and signaling inhibition. The oxidant sensing-induced termination of neutrophil migration at the site of infection permits a smooth transition to the subsequent microbial killing phase.

Endothelial cells exhibit regulated exocytosis in response to inflammatory mediators such as thrombin and histamine. The exocytosis of Weibel-Palade bodies (WPBs) containing von Willebrand factor, P-selectin, and interleukin-8 within minutes after stimulation is important for vascular homeostasis. SNARE proteins are key components of the exocytic machinery in neurons and some secretory cells, but their role in regulating exocytosis in endothelial cells is not well understood. We examined the function of SNARE proteins in mediating exocytosis of WPBs in endothelial cells. We identified the presence of syntaxin 4, syntaxin 3, and the high affinity syntaxin 4-regulatory protein Munc18c in human lung microvascular endothelial cells. Small interfering RNA-induced knockdown of syntaxin 4 (but not of syntaxin 3) inhibited exocytosis of WPBs as detected by the reduction in thrombin-induced cell surface P-selectin expression. Thrombin ligation of protease-activated receptor-1 activated the phosphorylation of syntaxin 4 and Munc18c, which, in turn, disrupted the interaction between syntaxin 4 and Munc18. Protein kinase Cα activation was required for the phosphorylation of syntaxin 4 and Munc18c as well as the cell surface expression of P-selectin. We also observed that syntaxin 4 knockdown inhibited the adhesion of neutrophils to thrombin-activated endothelial cells, demonstrating the functional role of syntaxin 4 in promoting endothelial adhesivity. Thus, protease-activated receptor-1-induced protein kinase Cα activation and phosphorylation of syntaxin 4 and Munc18c are required for the cell surface expression of P-selectin and the consequent binding of neutrophils to endothelial cells.

We determined the role of vascular endothelial (VE)-cadherin complex in regulating the permeability of pulmonary microvessels. Studies were made in mouse lungs perfused with albumin-Krebs containing EDTA, a Ca2+ chelator, added to study the VE-cadherin junctional disassembly. We then repleted the perfusate with Ca2+ to restore VE-cadherin integrity. Confocal microscopy showed a disappearance of VE-cadherin immunostaining in a time- and dose-dependent manner after Ca2+ chelation and reassembly of the VE-cadherin complex within 5 min after Ca2+ repletion. We determined the125I-labeled albumin permeability-surface area product and capillary filtration coefficient ( K fc) to quantify alterations in the pulmonary microvessel barrier. The addition of EDTA increased 125I-albumin permeability-surface area product and K fc in a concentration-dependent manner within 5 min. The permeability response was reversed within 5 min after repletion of Ca2+. An anti-VE-cadherin monoclonal antibody against epitopes responsible for homotypic adhesion augmented the increase in K fc induced by Ca2+chelation and prevented reversal of the response. We conclude that the disassembled VE-cadherins in endothelial cells are mobilized at the junctional plasmalemmal membrane such that VE-cadherins can rapidly form adhesive contact and restore microvessel permeability by reannealing the adherens junctions.

IQGAP1 is a scaffold protein that associates with the endothelial adherens junction (AJs) protein beta-catenin and with the active form of monomeric GTPase Cdc42 to play an important role in mainta...

We addressed the role of class 1B phosphatidylinositol 3-kinase (PI3K) isoform PI3Kgamma in mediating NADPH oxidase activation and reactive oxidant species (ROS) generation in endothelial cells (ECs) and of PI3Kgamma-mediated oxidant signaling in the mechanism of NF-kappaB activation and intercellular adhesion molecule (ICAM)-1 expression. We used lung microvascular ECs isolated from mice with targeted deletion of the p110gamma catalytic subunit of PI3Kgamma. Tumor necrosis factor (TNF) alpha challenge of wild type ECs caused p110gamma translocation to the plasma membrane and phosphatidylinositol 1,4,5-trisphosphate production coupled to ROS production; however, this response was blocked in p110gamma-/- ECs. ROS production was the result of TNFalpha activation of Ser phosphorylation of NADPH oxidase subunit p47(phox) and its translocation to EC membranes. NADPH oxidase activation failed to occur in p110gamma-/- ECs. Additionally, the TNFalpha-activated NF-kappaB binding to the ICAM-1 promoter, ICAM-1 protein expression, and PMN adhesion to ECs required functional PI3Kgamma. TNFalpha challenge of p110gamma-/- ECs failed to induce phosphorylation of PDK1 and activation of the atypical PKC isoform, PKCzeta. Thus, PI3Kgamma lies upstream of PKCzeta in the endothelium, and its activation is crucial in signaling NADPH oxidase-dependent oxidant production and subsequent NF-kappaB activation and ICAM-1 expression.

Thromboxane A2 (TXA2)-mediated platelet secretion and aggregation are important in thrombosis. Here, we present a novel finding that the stable TXA2 analogue, U46619, induces two waves of platelet secretion, each of which precedes a distinct wave of platelet aggregation. ADP released from platelets during the first wave of secretion played a major role in augmenting the first wave of platelet aggregation. The second wave of platelet secretion and aggregation required the first wave of both ADP secretion and aggregation and were blocked by either the integrin inhibitor RGDS or a P2Y12 receptor antagonist, indicating a requirement for both the integrin outside-in signal and ADP-activated Gi pathway. U46619 stimulated phosphoinositide 3-kinase (PI3K)-dependent phosphorylation of Akt, which was augmented by ADP but did not require integrin outside-in signaling. Platelets from PI3Kgamma knock-out mice or PI3K inhibitor-treated platelets showed an impaired second wave of platelet secretion and aggregation. However, the second wave of platelet aggregation was restored by addition of exogenous ADP to PI3Kgamma deficient or PI3K inhibitor-treated platelets. Thus, our data indicate that PI3K, together with the integrin outside-in signaling, play a central role in inducing the second wave of platelet secretion, which leads to the second wave of irreversible platelet aggregation.