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Columbia, MO, United States

Kim J.-A.,University of Alabama at Birmingham | Jang H.-J.,University of Alabama at Birmingham | Martinez-Lemus L.A.,Dalton Cardiovascular Research Center | Sowers J.R.,Dalton Cardiovascular Research Center | Sowers J.R.,University of Missouri
American Journal of Physiology - Endocrinology and Metabolism | Year: 2012

Elevated tissue levels of angiotensin II (ANG II) are associated with impairment of insulin actions in metabolic and cardiovascular tissues. ANG II-stimulated activation of mammalian target of rapamycin (mTOR)/p70 S6 kinase (p70S6K) in cardiovascular tissues is implicated in cardiac hypertrophy and vascular remodeling. However, the role of ANG II-stimulated mTOR/p70S6K in vascular endothelium is poorly understood. In the present study, we observed that ANG II stimulated p70S6K in bovine aortic endothelial cells. ANG II increased phosphorylation of insulin receptor substrate-1 (IRS-1) at Ser 636/639 and inhibited the insulin-stimulated phosphorylation of endothelial nitric oxide synthase (eNOS). An inhibitor of mTOR, rapamycin, attenuated the ANG II-stimulated phosphorylation of p70S6K and phosphorylation of IRS-1 (Ser 636/639) and blocked the ability of ANG II to impair insulin-stimulated phosphorylation of eNOS, nitric oxide production, and mesenteric-arteriole vasodilation. Moreover, point mutations of IRS-1 at Ser 636/639 to Ala prevented the ANG II-mediated inhibition of insulin signaling. From these results, we conclude that activation of mTOR/p70S6K by ANG II in vascular endothelium may contribute to impairment of insulin-stimulated vasodilation through phosphorylation of IRS-1 at Ser 636/639. This ANG II-mediated impairment of vascular actions of insulin may help explain the role of ANG II as a link between insulin resistance and hypertension. © 2012 by the American Physiological Society.

Huang S.-Y.,Dalton Cardiovascular Research Center | Zou X.,University of Missouri
Proteins: Structure, Function and Bioinformatics | Year: 2010

A hierarchical approach has been developed for protein-protein docking. In the first step, a Fast Fourier Transform (FFT)-based docking algorithm is used to globally sample all putative binding modes, in which the protein is represented by a reduced model, that is, each side chain on the protein surface is represented by its center of mass. Compared to conventional FFT docking with all-atom models, the FFT docking method with a reduced model is expected to generate more hits because it allows larger side-chain flexibility. Next, the filtered binding modes (normally several thousands) are refined by an iteratively derived knowledge-based scoring function ITScorePP and by considering backbone/loop flexibility using an ensemble docking algorithm. The distance-dependent potentials of ITScorePP were extracted by a physics-based iterative method, which circumvents the long-standing reference state problem in the knowledge-based approaches. With this hierarchical protocol, we have participated in the CAPRI experiments for Rounds 15-19 of 11 targets (T32-T42). In the predictor experiments, we achieved correct binding modes for six targets: three are with high accuracy (T40 for both distinct binding modes, T41, and T42), two are with medium accuracy (T34 and T37), and one is acceptable (T32). In the scorer experiments, of the seven target complexes that contain at least one acceptable mode submitted by the CAPRI predictor groups, we obtained correct binding modes for four targets: three are with high accuracy (T37, T40, and T41) and one is with medium accuracy (T34), suggesting good accuracy and robustness of ITScorePP. © 2010 Wiley-Liss, Inc.

Here, we demonstrate a novel, directacting, and synergistic role for 3 hematopoietic stem cell cytokines: stem cell factor, interleukin-3, and stromal derived factor-1α, in controlling human endothelial cell (EC) tube morphogenesis, sprouting, and pericyte-induced tube maturation under defined serum-free conditions in 3-dimensional matrices. Angiogenic cytokines such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) alone or VEGF/FGF combinations do not support these responses. In contrast, VEGF and FGF prime EC responses to hematopoietic cytokines via up-regulation of c-Kit, IL-3Rα, and C-X-C chemokine receptor type 4 from either human ECs or embryonic quail vessel explants. In support of these findings, EC Runx1 is demonstrated to be critical in coordinating vascular morphogenic responses by controlling hematopoietic cytokine receptor expression. Combined blockade of hematopoietic cytokines or their receptors in vivo leads to blockade of developmental vascularization in quail embryos manifested by vascular hemorrhage and disrupted vascular remodeling events in multiple tissue beds. This work demonstrates a unique role for hematopoietic stem cell cytokines in vascular tube morphogenesis and sprouting and further demonstrates a novel upstream priming role for VEGF and FGF to facilitate the action of promorphogenic hematopoietic cytokines. © 2011 by The American Society of Hematology.

Westcott E.B.,University of Missouri | Segal S.S.,University of Missouri | Segal S.S.,Dalton Cardiovascular Research Center
Microcirculation | Year: 2013

The control of vascular resistance and tissue perfusion reflect coordinated changes in the diameter of feed arteries and the arteriolar networks they supply. Against a background of myogenic tone and metabolic demand, vasoactive signals originating from perivascular sympathetic and sensory nerves are integrated with endothelium-derived signals to produce vasodilation or vasoconstriction. PVNs release adrenergic, cholinergic, peptidergic, purinergic, and nitrergic neurotransmitters that lead to SMC contraction or relaxation via their actions on SMCs, ECs, or other PVNs. ECs release autacoids that can have opposing actions on SMCs. Respective cell layers are connected directly to each other through GJs at discrete sites via MEJs projecting through holes in the IEL. Whereas studies of intercellular communication in the vascular wall have centered on endothelium-derived signals that govern SMC relaxation, attention has increasingly focused on signaling from SMCs to ECs. Thus, via MEJs, neurotransmission from PVNs can evoke distinct responses from ECs subsequent to acting on SMCs. To integrate this emerging area of investigation in light of vasomotor control, the present review synthesizes current understanding of signaling events that originate within SMCs in response to perivascular neurotransmission in light of EC feedback. Although often ignored in studies of the resistance vasculature, PVNs are integral to blood flow control and can provide a physiological stimulus for myoendothelial communication. Greater understanding of these underlying signaling events and how they may be affected by aging and disease will provide new approaches for selective therapeutic interventions. © 2013 John Wiley & Sons Ltd.

Behringer E.J.,University of Missouri | Segal S.S.,University of Missouri | Segal S.S.,Dalton Cardiovascular Research Center
Journal of Physiology | Year: 2015

Endothelial function in resistance vessels entails Ca2+ and electrical signalling to promote vasodilatation and increase tissue blood flow. Whether membrane potential (Vm) governs intracellular calcium concentration ([Ca2+]i) of the endothelium remains controversial. [Ca2+]i and Vm were evaluated simultaneously during intracellular current injection using intact endothelial tubes freshly isolated from mouse skeletal muscle resistance arteries. [Ca2+]i did not change during hyperpolarization or depolarization under resting conditions. However in the presence of 100 nM ACh (∼EC50), [Ca2+]i increased during hyperpolarization and decreased during depolarization. These responses required extracellular Ca2+ and were attenuated by half with genetic ablation of TRPV4 channels. In native microvascular endothelium, half-maximal stimulation of muscarinic receptors enables Vm to govern [Ca2+]i by activating Ca2+-permeable channels in the plasma membrane. This effect of Vm is absent at rest and can be masked during maximal receptor stimulation. In resistance arteries, coupling a rise of intracellular calcium concentration ([Ca2+]i) to endothelial cell hyperpolarization underlies smooth muscle cell relaxation and vasodilatation, thereby increasing tissue blood flow and oxygen delivery. A controversy persists as to whether changes in membrane potential (Vm) alter endothelial cell [Ca2+]i. We tested the hypothesis that Vm governs [Ca2+]i in endothelium of resistance arteries by performing Fura-2 photometry while recording and controlling Vm of intact endothelial tubes freshly isolated from superior epigastric arteries of C57BL/6 mice. Under resting conditions, [Ca2+]i did not change when Vm shifted from baseline (∼-40 mV) via exposure to 10 μM NS309 (hyperpolarization to ∼-80 mV), via equilibration with 145 mm [K+]o (depolarization to ∼-5 mV), or during intracellular current injection (±0.5 to 5 nA, 20 s pulses) while Vm changed linearly between ∼-80 mV and +10 mV. In contrast, during the plateau (i.e. Ca2+ influx) phase of the [Ca2+]i response to approximately half-maximal stimulation with 100 nm ACh (∼EC50), [Ca2+]i increased as Vm hyperpolarized below -40 mV and decreased as Vm depolarized above -40 mV. The magnitude of [Ca2+]i reduction during depolarizing current injections correlated with the amplitude of the plateau [Ca2+]i response to ACh. The effect of hyperpolarization on [Ca2+]i was abolished following removal of extracellular Ca2+, was enhanced subtly by raising extracellular [Ca2+] from 2 mm to 10 mm and was reduced by half in endothelium of TRPV4-/- mice. Thus, during submaximal activation of muscarinic receptors, Vm can modulate Ca2+ entry through the plasma membrane in accord with the electrochemical driving force. © 2015 The Physiological Society.

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