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Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

ABSTRACT: AIM: In vascular strips, the adjacent endothelial cells modulate the contraction of vascular smooth muscle cells (VSMCs) induced by sphingosylphosphorylcholine (SPC) through nitric oxide (NO). The aim of this study was to elucidate the mechanisms by which vascular endothelial cells (VECs) reduce the SPC-induced contraction of VSMCs in a co-culture system. METHODS: Human umbilical VECs and VSMCs were co-cultured. The VECs were transfected with integrin β4- or Fyn-specific siRNA. The areas of VSMCs that are involved in cell contractility were quantified using the Leica confocal software and collagen contractility assay. The production of NO in VECs was measured in the cell supernatants using NO Detection Kit. The levels of integrin β4 and Fyn in VECs and the levels of Rho kinase (ROCK) in VSMC were detected using immunofluorescence assays or Western blots. RESULTS: Co-culture with VECs reduced the contraction of VSMCs induced by SPC (30 μmol/L). The down-regulation of integrin β4 or Fyn in VECs by the specific siRNA (20 nmol/L) was able to counteract the effects of VECs on the SPC-induced VSMC contractions. Furthermore, the integrin β4-specific siRNA (20 and 40 nmol/L) significantly reduced the level of Fyn protein and the production of NO in VECs, while increased the level of ROCK in VSMCs that had been stimulated by SPC. CONCLUSION: The VECs reduced the SPC-induced contraction of VSMCs in the co-culture system through integrin β4 and Fyn proteins. In this process, NO may be the factor downstream of integrin β4 in VECs, while ROCK may be the key protein regulating the contraction of VSMCs.

SUBMITTER: Ge D

PROVIDER: S-EPMC4010278 | biostudies-other | 2012 Jan

REPOSITORIES: biostudies-other

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Project description:Vascular smooth muscle (VSM) plays an important role in the regulation of vascular function. Identifying the mechanisms of VSM contraction has been a major research goal in order to determine the causes of vascular dysfunction and exaggerated vasoconstriction in vascular disease. Major discoveries over several decades have helped to better understand the mechanisms of VSM contraction. Ca2+ has been established as a major regulator of VSM contraction, and its sources, cytosolic levels, homeostatic mechanisms and subcellular distribution have been defined. Biochemical studies have also suggested that stimulation of Gq protein-coupled membrane receptors activates phospholipase C and promotes the hydrolysis of membrane phospholipids into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 stimulates initial Ca2+ release from the sarcoplasmic reticulum, and is buttressed by Ca2+ influx through voltage-dependent, receptor-operated, transient receptor potential and store-operated channels. In order to prevent large increases in cytosolic Ca2+ concentration ([Ca2+]c), Ca2+ removal mechanisms promote Ca2+ extrusion via the plasmalemmal Ca2+ pump and Na+/Ca2+ exchanger, and Ca2+ uptake by the sarcoplasmic reticulum and mitochondria, and the coordinated activities of these Ca2+ handling mechanisms help to create subplasmalemmal Ca2+ domains. Threshold increases in [Ca2+]c form a Ca2+-calmodulin complex, which activates myosin light chain (MLC) kinase, and causes MLC phosphorylation, actin-myosin interaction, and VSM contraction. Dissociations in the relationships between [Ca2+]c, MLC phosphorylation, and force have suggested additional Ca2+ sensitization mechanisms. DAG activates protein kinase C (PKC) isoforms, which directly or indirectly via mitogen-activated protein kinase phosphorylate the actin-binding proteins calponin and caldesmon and thereby enhance the myofilaments force sensitivity to Ca2+. PKC-mediated phosphorylation of PKC-potentiated phosphatase inhibitor protein-17 (CPI-17), and RhoA-mediated activation of Rho-kinase (ROCK) inhibit MLC phosphatase and in turn increase MLC phosphorylation and VSM contraction. Abnormalities in the Ca2+ handling mechanisms and PKC and ROCK activity have been associated with vascular dysfunction in multiple vascular disorders. Modulators of [Ca2+]c, PKC and ROCK activity could be useful in mitigating the increased vasoconstriction associated with vascular disease.

Project description:Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme in the pentose phosphate pathway and a major source of nicotinamide adenine dinucleotide phosphate reduced (NADPH), which regulates numerous enzymatic (including glutathione reductase and NADPH oxidase that, respectively, generates reduced glutathione and reactive oxygen species) reactions involved in various cellular actions, yet its physiological function is seldom investigated. We, however, recently showed that inhibiting G6PD causes precontracted coronary artery (CA) to relax in an endothelium-derived relaxing factor- and second messenger-independent manner. Here we assessed the role of G6PD in regulating CA contractility. Treating bovine CAs for 20 min with potassium chloride (KCl; 30 mM), amphotericin B (50 ?M), or U46619 (100 nM) significantly (p < 0.05) increased both G6PD activity and glucose flux through the pentose phosphate pathway. The effect was Ca(2+) independent, and there was a corresponding increase in protein kinase C (PKC) activity. Activation of G6PD by KCl was blocked by the PKC? inhibitor rottlerin (10 ?M) or by knocking down PKC? expression using siRNA. Phorbol 12, 13-dibutyrate (10 ?M), a PKC activator, significantly increased G6PD phosphorylation and activity, whereas single (S210A, T266A) and double (S210A/T266A) mutations at sites flanking the G6PD active site significantly inhibited phosphorylation, shifted the isoelectric point, and reduced enzyme activity. Knocking down G6PD decreased NADPH and reactive oxygen species generation, and reduced KCl-evoked increases in [Ca(2+)](i) and myosin light chain phosphorylation, thereby reducing CA contractility. Similarly, aortas from G6PD-deficient mice developed less KCl/phorbol 12, 13-dibutyrate-evoked force than those from their wild-type littermates. Conversely, overexpression of G6PD augmented KCl-evoked increases in [Ca(2+)](i), thereby augmenting CA contraction. Our findings demonstrate that G6PD activity and NADPH is increased in activated CA in a PKC?-dependent manner and that G6PD modulates Ca(2+) entry and CA contractions evoked by membrane depolarization.

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Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

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