Columbia, MO, United States
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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.


Thyfault J.P.,University of Missouri | Booth F.W.,Dalton Cardiovascular Research Center | Booth F.W.,University of Missouri
Current Opinion in Clinical Nutrition and Metabolic Care | Year: 2011

Purpose of Review: To discuss the current data that acute periods of physical inactivity are harmful to health. Recent Findings: Bed rest prescribed for recovery from clinical conditions causes changes in thousands of mRNAs in leg muscles within days. Humans genetically more susceptible to metabolic disorders (low birth weight babies and type 2 diabetic offspring) are as, or more, susceptible to further metabolic dysfunction by the environmental perturbation of bed rest, as compared with healthy controls without these risk factors. High daily accumulations of sitting are not only associated with enhanced metabolic risk, but current findings report that increased sitting time leads to a reduction in insulin sensitivity. Reductions in walking or in ambulatory activity (lower step numbers taken by healthy humans) reduce insulin sensitivity and insulin signaling through Akt in skeletal muscle. Summary: New findings using human models of physical inactivity (bed rest, increased sitting time, and reduced daily ambulatory activity), extend pre-existing research showing that transitioning to physical inactivity rapidly reduced metabolic health. Modern technological advances that remove standing, walking, and major limb movement initiate metabolic dysfunctions that likely play a fundamental role in the development of obesity and type 2 diabetes. © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins.


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.


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.


Behringer E.J.,University of Missouri | Segal S.S.,University of Missouri | Segal S.S.,Dalton Cardiovascular Research Center
Journal of Physiology | Year: 2012

Blood flow control requires coordinated contraction and relaxation of smooth muscle cells (SMCs) along and among the arterioles and feed arteries that comprise vascular resistance networks. Whereas smooth muscle contraction of resistance vessels is enhanced by noradrenaline release along perivascular sympathetic nerves, the endothelium is integral to coordinating smooth muscle relaxation. Beyond producing nitric oxide in response to agonists and shear stress, endothelial cells (ECs) provide an effective conduit for conducting hyperpolarization along vessel branches and into surrounding SMCs through myoendothelial coupling. In turn, bidirectional signalling from SMCs into ECs enables the endothelium to moderate adrenergic vasoconstriction in response to sympathetic nerve activity. This review focuses on the endothelium as the cellular pathway that coordinates spreading vasodilatation. We discuss the nature and regulation of cell-to-cell coupling through gap junctions, bidirectional signalling between ECs and SMCs, and how oxidative stress during ageing may influence respective signalling pathways. Our recent findings illustrate the role of small (SKCa) and intermediate (IKCa) Ca2+ activated K+ channels as modulators of electrical conduction along the endothelium. Gaps in current understanding indicate the need to determine mechanisms that regulate intracellular Ca2+ homeostasis and ion channel activation in the resistance vasculature with advancing age. © 2012 The Physiological Society.


Behringer E.J.,University of Missouri | Segal S.S.,University of Missouri | Segal S.S.,Dalton Cardiovascular Research Center
Circulation Research | Year: 2012

Rationale: Electrical conduction through gap junction channels between endothelial cells of resistance vessels is integral to blood flow control. Small and intermediate-conductance Ca 2+-activated K channels (SK Ca/IK Ca) initiate electrical signals in endothelial cells, but it is unknown whether SK Ca/IK Ca activation alters signal transmission along the endothelium. Objective: We tested the hypothesis that SK Ca/IK Ca activity regulates electrical conduction along the endothelium of resistance vessels. Methods and Results: Freshly isolated endothelial cell tubes (60 μm wide; 1-3 mm long; cell length, ≈35 μm) from mouse skeletal muscle feed (superior epigastric) arteries were studied using dual intracellular microelectrodes. Current was injected (±0.1-3 nA) at site 1 while recording membrane potential (V m) at site 2 (separation distance=50-2000 μm). SK Ca/IK Ca activation (NS309, 1 μmol/L) reduced the change in V m along endothelial cell tubes by ≥50% and shortened the electrical length constant (λ) from 1380 to 850 μm (P<0.05) while intercellular dye transfer (propidium iodide) was maintained. Activating SK Ca/IK Ca with acetylcholine or SKA-31 also reduced electrical conduction. These effects of SK Ca/IK Ca activation persisted when hyperpolarization (>30 mV) was prevented with 60 mmol/L [K +] o. Conversely, blocking SK Ca/IK Ca (apamin+charybdotoxin) depolarized cells by ≈10 mV and enhanced electrical conduction (ie, changes in V m) by ≈30% (P<0.05). Conclusions: These findings illustrate a novel role for SK Ca/IK Caactivity in tuning electrical conduction along the endothelium of resistance vessels by governing signal dissipation through changes in membrane resistance. Voltage-insensitive ion channels can thereby tune intercellular electrical signaling independent from gap junction channels. © 2012 American Heart Association, Inc.


Behringer E.J.,University of Missouri | Shaw R.L.,University of Missouri | Westcott E.B.,University of Missouri | Socha M.J.,University of Missouri | And 2 more authors.
Arteriosclerosis, Thrombosis, and Vascular Biology | Year: 2013

OBJECTIVE - : Intercellular conduction of electrical signals underlies spreading vasodilation of resistance arteries. Small- and intermediate- conductance Ca2+-activated K+ channels of endothelial cells serve a dual function by initiating hyperpolarization and modulating electrical conduction. We tested the hypothesis that regulation of electrical signaling by small- and intermediate-conductance Ca2+-activated K+ channels is altered with advancing age. APPROACH AND RESULTS - : Intact endothelial tubes (60 μm wide; 1-3 mm long) were freshly isolated from male C57BL/6 mouse (Young: 4-6 months; Intermediate: 12-14 months; Old: 24-26 months) superior epigastric arteries. Using dual intracellular microelectrodes, current was injected (±0.1-3 nA) at site 1 while recording membrane potential (Vm) at site 2 (separation distance: 50-2000 μm). Across age groups, greatest differences were observed between Young and Old. Resting Vm in Old (-38±1 mV) was more negative (P<0.05) than Young (-30±1 mV). Maximal hyperpolarization to both direct (NS309) and indirect (acetylcholine) activation of small- and intermediate-conductance Ca 2+-activated K+ channels was sustained (ΔV m ≈-40 mV) with age. The length constant (λ) for electrical conduction was reduced (P<0.05) from 1630±80 μm (Young) to 1320±80 μm (Old). Inhibiting small- and intermediate-conductance Ca2+-activated K+ channels with apamin+charybdotoxin or scavenging hydrogen peroxide (H2O2) with catalase improved electrical conduction (P<0.05) in Old. Exogenous H2O2 (200 μmol/L) in Young evoked hyperpolarization and impaired electrical conduction; these effects were blocked by apamin+charybdotoxin. CONCLUSIONS - : Enhanced current loss through Ca2+-activated K+ channel activation impairs electrical conduction along the endothelium of resistance arteries with aging. Attenuating the spatial domain of electrical signaling will restrict the spread of vasodilation and thereby contribute to blood flow limitations associated with advanced age. © 2013 American Heart Association, Inc.


Moore A.W.,University of Missouri | Bearden S.E.,Idaho State University | Segal S.S.,University of Missouri | Segal S.S.,Dalton Cardiovascular Research Center
Journal of Physiology | Year: 2010

Exercise onset entails motor unit recruitment and the initiation of vasodilatation. Dilatation can ascend the arteriolar network to encompass proximal feed arteries but is opposed by sympathetic nerve activity, which promotes vasoconstriction and inhibits ascending vasodilatation through activating α-adrenoreceptors. Whereas contractile activity can antagonize sympathetic vasoconstriction, more subtle aspects of this interaction remain to be defined. We tested the hypothesis that constitutive activation of α-adrenoreceptors governs blood flow distribution within individual muscles. The mouse gluteus maximus muscle (GM) consists of Inferior and Superior regions. Each muscle region is supplied by its own motor nerve and feed artery with an anastomotic arteriole (resting diameter ∼25 μm) that spans both muscle regions. In anaesthetized male C57BL/6J mice (3-5 months old), the GM was exposed and superfused with physiological saline solution (35°C; pH 7.4). Stimulating the inferior gluteal motor nerve (0.1 ms pulse, 100 Hz for 500 ms) evoked a brief tetanic contraction and produced rapid (<1 s) onset vasodilatation (ROV; diameter change, 10 ± 1 μm) of the anastomotic arteriole along the active (Inferior) muscle region but not along the inactive (Superior) region (n= 8). In contrast, microiontophoresis of acetylcholine (1 μm micropipette tip, 1 μA, 500 ms) initiated dilatation that travelled along the anastomotic arteriole from the Inferior into the Superior muscle region (diameter change, 5 ± 2 μm). Topical phentolamine (1 μ m) had no effect on resting diameter but this inhibition of α-adrenoreceptors enabled ROV to spread along the anastomotic arteriole into the inactive muscle region (dilatation, 7 ± 1 μm; P < 0.05), where remote dilatation to acetylcholine then doubled (P < 0.05). These findings indicate that constitutive activation of α-adrenoreceptors in skeletal muscle can restrict the spread of dilatation within microvascular resistance networks and thereby increase blood flow to active muscle regions. © 2010 The Authors. Journal compilation © 2010 The 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.

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