Microvascular Control -A Role for Integrins

描述(申请人提供)：机械力是许多血管平滑肌细胞(VSMC)功能的重要刺激因素，包括收缩过程、增殖、迁移和附着。这些功能定义了血管系统的生理特性，如控制血流、毛细血管压力和外周血管阻力，并在病理生理过程中发挥作用。整合素是细胞外基质(ECM)蛋白的重要受体，既介导力传递又介导信号转导。因此，整合素被认为是VSMC中的机械感受器，并在机械转导和肌源性反应中发挥核心作用。然而，我们关于整合素如何感知物理力量并将其转化为细胞信号以及涉及哪些整合素的知识是不完整的。因此，重要的问题涉及VSMC中整合素介导的信号的性质和来源，以及整合素和肌源性反应之间的联系。我们的中心假设是，整合素(α5beta1、αVbeta3、alpha4beta1)在机械诱导的VSMC中启动细胞信号，涉及整合素的键形成、键压力和/或整合素与ECM蛋白(纤维连接蛋白(FN)和玻璃体连接蛋白(VN))之间的键解离。本研究的具体目的是：1.测量VSMCα5bbeta1、αVbeta3或alpha4beta1整合素与ECM蛋白Fn和Vn之间的受体-配体解离或解离力。2.观察VSMC膜上整合素-ECM(FN和VN)键的形成、键应力或强迫解离是否导致[Ca~(2+)]_i的变化，进而确定[Ca~(2+)]_i的变化是否可引起皮质VSMC硬度/弹性的变化。3.探讨粘着斑蛋白在α5beta1、αVbeta3或alpha4beta1整合素调节VSMC钙离子中的作用。4.研究α5beta1、αvbeta3和α4beta1整合素受体拮抗剂对小动脉压力依赖性肌源性反应和钙信号的影响。为了达到这些目的，将使用培养的小动脉VSMC和从大鼠骨骼肌分离的小动脉。FN和VN与其整合素受体之间的结合力、细胞皮质硬度的测量以及VSMC[钙]i的测量将使用独特的混合原子力显微镜(AFM)/荧光显微镜系统进行。对分离小动脉的研究将为从分子尺度到完整组织水平的数据集成提供一种手段。我们预测，所采用的创新方法将为理解VSMC的机械传感和转导机制提供新的信息。

DESCRIPTION (provided by applicant): Mechanical force is an important stimulus for many vascular smooth muscle cell (VSMC) functions including the contractile process, proliferation, migration, and attachment. These functions define physiological properties of the vasculature like control of blood flow, capillary pressure and peripheral vascular resistance and play a role in pathophysiological processes. Integrins are important receptors for extracellular matrix (ECM) proteins that mediate both force transmission and signal transduction. Consequently, integrins have been hypothesized to be the mechanosensor in VSMC and play a central role in mechanotransduction and the myogenic response. Our knowledge, however, concerning how integrins sense and transduce physical forces into cellular signals and which integrins are involved is incomplete. Thus, important questions concern the nature and origin of integrin-mediated signaling in VSMC and the link between integrins and the myogenic response. Our central hypothesis is that initiation of mechanically induced cell signaling in VSMC by integrins (alpha5beta1, alphaVbeta3, alpha4beta1) involves either integrin bond formation, bond stressing and/or bond dissociation between integrins and ECM proteins (fibronectin (FN) and vitronectin (VN)). The specific aims of this proposal are: 1. To measure the receptor-ligand unbinding or dissociation force between VSMC alpha5bbeta1, alphaVbeta3 or alpha4beta1 integrins with the ECM proteins FN and VN. 2. To determine if integrin-ECM (FN and VN) bond formation, bond stressing or forced bond dissociation on the VSMC membrane result in changes of [Ca2+]i and further determine whether the [Ca2+]I changes result in detectable alterations of cortical VSMC stiffness/elasticity. 3. To determine the role of focal adhesion proteins in regulation of VSMC calcium by the alpha5beta1, alphaVbeta3, or alpha4beta1 integrins. 4. To determine the effect of alpha5beta1, alphavbeta3 or alpha4beta1 integrin receptor antagonism on the pressure-dependent myogenic response and Ca 2+ signaling of isolated arterioles. To accomplish these aims, cultured arteriole VSMC and isolated arterioles from rat skeletal muscle will be used. The binding forces between FN and VN and their integrin receptors, measurements cell cortical stiffness and measurements of VSMC [Ca2+]i will be performed using a unique hybrid Atomic Force Microscope (AFM)/Fluorescence Microscope System. Studies in the isolated arteriole will provide a means for integration of data from molecular scale to the intact tissue level. We predict that the innovative approaches used will provide new information for understanding the mechanism of VSMC mechanosensation and -transduction.