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.
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