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// Suowen Xu 1 , Bin Liu 1 , Meimei Yin 1 , Marina Koroleva 1 , Michael Mastrangelo 1 , Sara Ture 1 , Craig N. Morrell 1 , David X. Zhang 2 , Edward A. Fisher 3 and Zheng Gen Jin 1 1 Aab Cardiovascular Research Institute and Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA 2 Department of Medicine, Medical College of Wisconsin, Milwaukee, WI, USA 3 Department of Medicine, Division of Cardiology, and The Marc and Ruti Bell Program in Vascular Biology, New York University School of Medicine, New York, NY, USA Correspondence to: Zheng Gen Jin, email: // Keywords : AMPK, atherosclerosis, GSK1016790A, shear stress, TRPV4 Received : December 29, 2015 Accepted : April 29, 2016 Published : May 14, 2016 Abstract TRPV4 ion channel mediates vascular mechanosensitivity and vasodilation. Here, we sought to explore whether non-mechanical activation of TRPV4 could limit vascular inflammation and atherosclerosis. We found that GSK1016790A, a potent and specific small-molecule agonist of TRPV4, induces the phosphorylation and activation of eNOS partially through the AMPK pathway. Moreover, GSK1016790A inhibited TNF-α-induced monocyte adhesion to human endothelial cells. Mice given GSK1016790A showed increased phosphorylation of eNOS and AMPK in the aorta and decreased leukocyte adhesion to TNF-α-inflamed endothelium. Importantly, oral administration of GSK1016790A reduced atherosclerotic plaque formation in ApoE deficient mice fed a Western-type diet. Together, the present study suggests that pharmacological activation of TRPV4 may serve as a potential therapeutic approach to treat atherosclerosis.

Metabolic syndrome accelerates the atherosclerotic process, and the earliest event of which is endothelial dysfunction. Ghrelin, a newly discovered gastric peptide, improves endothelial function and inhibits proatherogenic changes. In particular, low ghrelin concentration has been associated with several features of metabolic syndrome, including obesity, insulin resistance, and high blood pressure. However, the molecular mechanisms underlying ghrelin vascular actions remain largely unclear. Here, we showed that ghrelin activated endothelial nitric oxide (NO) synthase (eNOS) in cultured endothelial cells (ECs) and in intact vessels. Specifically, ghrelin rapidly induced phosphorylation of eNOS on an activation site and production of NO in human umbilical vein ECs and bovine aortic ECs. The eNOS phosphorylation was also observed in mouse aortas ex vivo perfused with ghrelin and in aortic tissues isolated from mice injected with ghrelin. Mechanistically, ghrelin stimulated AMP-activated protein kinase (AMPK) and Akt activation in cultured ECs and intact vessels. Inhibiting AMPK and Akt with their pharmacological inhibitors, small interference RNA and adenoviruses carried dominant-negative mutants, markedly attenuated ghrelin-induced eNOS activation, and NO production. Furthermore, ghrelin receptor/Gq protein/calcium-dependent pathway mediates activation of AMPK, Akt, and eNOS, and calmodulin-dependent kinase kinase is a potential convergent point to regulate Akt and AMPK activation in ghrelin signaling. Importantly, eNOS activation is critical for ghrelin inhibition of vascular inflammation. Together, both in vitro and in vivo data demonstrate a new role of ghrelin signaling for eNOS activation, and highlight the therapeutic potential for ghrelin to correct endothelial dysfunction associated with atherosclerotic vascular diseases and metabolic syndrome.

Endothelial dysfunction, characterized by a decrease of nitric oxide (NO) bioavailability in the vessel wall, plays a crucial role in the pathogenesis of atherosclerosis. Oxidative stress due to increased reactive oxygen species (ROS) is implicated in endothelial dysfunction associated with diabetes. However the molecular mechanisms by which oxidative stress causes endothelial dysfunction remain incompletely understood. Blood flow, which generates fluid shear stress acting on endothelium, is the most potent physiological stimulus for NO production through endothelial NO synthase (eNOS) activation. Here we report that hyperglycemia and oxidative stress impairs fluid shear stress signal transduction and eNOS activation in endothelium. We found that the exposure of endothelial cells (ECs) to ROS generators such as menadione and xanthine/xanthine oxidase inhibited laminar flow-mediated Akt and eNOS phosphorylation and activation in human endothelial cells. Moreover, ECs pre-exposed to high glucose that generates ROS production in ECs, failed to respond to laminar flow for Akt/eNOS phosphorylation and activation. Consequently NO production from ECs in response to laminar flow was attenuated by high glucose treatment. Mechanistically, we observed that hyperglycemia and oxidative stress altered endothelial adherens junction integrity, manifested by the alternation of the localization of cell-cell junction molecules vascular endothelial cadherin (VE-cadherin) and beta-catenin. Silencing VE-cadherin or beta-catenin with small interference RNA also inhibited laminar flow-mediated signaling for eNOS activation, which mimics the effects of oxidative stress, suggesting that cell-cell junction integrity is critical for fluid shear stress signal transduction and eNOS activation. Collectively, our results demonstrate that hyperglycemia and oxidative stress impair laminar flow-mediated eNOS activation through the alternation of endothelial junction integrity and fluid shear stress signal transduction. Our findings suggest a novel mechanism whereby oxidative stress induces diabetes-associated endothelial dysfunction.

Aims Vascular endothelial dysfunction and inflammation are hallmarks of atherosclerosis. Kruppel-like factor 2 (KLF2) is a key mediator of anti-inflammatory and anti-atherosclerotic properties of the endothelium. However, little is known of the molecular mechanisms for regulating KLF2 transcriptional activation. Methods and results Here, we found that histone deacetylase 5 (HDAC5) associates with KLF2 and represses KLF2 transcriptional activation. HDAC5 resided with KLF2 in the nuclei of human umbilical cord vein endothelial cells (HUVECs). Steady laminar flow attenuated the association of HDAC5 with KLF2 via stimulating HDAC5 phosphorylation-dependent nuclear export in HUVEC. We also mapped the KLF2–HDAC5-interacting domains and found that the N-terminal region of HDAC5 interacts with the C-terminal domain of KLF2. Chromatin immunoprecipitation and luciferase reporter assays showed that HDAC5 through a direct association with KLF2 suppressed KLF2 transcriptional activation. HDAC5 overexpression inhibited KLF2-dependent endothelial nitric oxide synthesis (eNOS) promoter activity in COS7 cell and gene expression in both HUVECs and bovine aortic endothelial cells (BAECs). Conversely, HDAC5 silencing enhanced KLF2 transcription and hence eNOS expression in HUVEC. Moreover, we observed that the level of eNOS protein in the thoracic aorta isolated from HDAC5 knockout mice was higher, whereas expression of pro-inflammatory vascular cell adhesion molecule 1 was lower, compared with those of HDAC5 wild-type mice. Conclusions We reveal a novel role of HDAC5 in modulating the KLF2 transcriptional activation and eNOS expression. These findings suggest that HDAC5, a binding partner and modulator of KLF2, could be a new therapeutic target to prevent vascular endothelial dysfunction associated with cardiovascular diseases.

Vascular endothelium–derived nitric oxide (NO), originally identified as endothelium-derived relaxing factor,1–3 plays a pivotal role in regulation of vascular homeostasis.4 NO is a major regulator of vascular tone and blood pressure, and has multiple antiatherogenic roles including antiinflammatory, antithrombotic, antiproliferative, and antioxidant effects.4–6 Loss of the bioavailability of endothelium-derived NO is the hallmark of endothelial dysfunction and is implicated in the pathogenesis of cardiovascular disease such as hypertension and atherosclerosis.7,8 Therefore, it is of great interest to understand the molecular mechanisms regulating NO production by endothelium, which is likely to provide new insight into endothelial function in health and disease. See page 1015 Endothelial NO synthase (eNOS) is a highly regulated, calcium (Ca2+)/calmodulin (CaM)-dependent enzyme responsible for the physiological production of NO in the vasculature.9,10 In endothelial cells (ECs), NO is formed from its precursor L-arginine via the enzymatic activation of eNOS with cofactors such as tetrahydrobiopterin (BH4). eNOS activation and subsequent NO production is stimulated by a variety of physical stimuli such as fluid shear stress generated by blood flow and by many humoral factors including acetylcholine, vascular endothelial growth factor (VEGF), bradykinin, estrogen, insulin, and angiopoietin.10–12 Increasing evidence suggest that eNOS is regulated by subcellular localization,9,13,14 posttranslational modifications such as phosphorylation at serine 1179 (S1179) by Akt,15–17 and interactions with several regulatory proteins such as heat shock protein 90 (HSP90) and caveolin-1.18,19 In particular, subcellular localization of eNOS is critical for optimal coupling of extracellular stimulation to NO production.10 In ECs, eNOS appears to localize at peripheral aspects of the Golgi complex and cholesterol-rich microdomains of the plasma membrane (PM) such as caveolae. Cotranslational N-myristoylation and posttranslational cysteine palmitoylation of eNOS determine its membrane targeting.9,13 It has been proposed that eNOS membrane localization may bring eNOS …

Fluid shear stress generated by blood flowing over the endothelium is a major determinant of arterial tone, vascular remodeling, and atherogenesis. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an essential role in regulation of vascular function and structure by blood flow, but the molecular mechanisms that transduce mechanical force to eNOS activation are not well understood. In this study, we found that laminar flow (shear stress=12 dyne/cm 2 ) rapidly activates vascular endothelial growth factor receptor 2 (VEGFR2) in a ligand-independent manner and leads to eNOS activation in cultured endothelial cells. Flow-stimulated VEGFR2 recruits phosphoinositide 3-kinase and mediates activation of Akt and eNOS. Inhibiting VEGFR2 kinase with selective inhibitors blocks flow-induced activation of Akt and eNOS and production of NO. Decreasing VEGFR2 expression with antisense VEGFR2 oligonucleotides significantly attenuates activation of Akt and eNOS. Furthermore, Src kinases are involved in flow-stimulated VEGFR2 because inhibiting Src kinases by PP2, a selective inhibitor for Src kinases, abolishes flow-induced VEGFR2 tyrosine phosphorylation and downstream signaling. Finally, we show that inhibiting VEGFR2 kinase significantly reduces flow-mediated NO-dependent arteriolar dilation in vivo. These data identify VEGFR2 as a key mechanotransducer that activates eNOS in response to blood flow.