Interactions between vascular integrins and extracellular matrix (ECM) components of the blood vessel wall are important determinants of arteriolar tone and blood flow control. Importantly, arteriolar responsiveness to pressure and to agonists is compromised in diabetes, atherosclerosis, and other inflammatory / injury conditions in which the composition of the vascular wall is altered. In this application, we focus on two aspects of vascular function in which ECM-integrin interactions acutely regulate vascular tone: 1) mechanotransduction of intravascular pressure through the voltage-gated, L-type Ca2+ channel (CaL), which is the primary Ca2+ entry pathway in VSM, and 2) the effects of biologically active matricryptins (i.e., proteolytic fragments of ECM proteins with vasoactive properties). Our interest centers on how these regulatory mechanisms converge on vascular smooth muscle (VSM) ion channels to regulate intracellular Ca2+ and control constriction/dilation. Our previous studies show that the regulation of CaL current is a key mechanism whereby at least two VSM integrins, 1521 and 1v23, control myogenic vascular tone. The central hypothesis of this proposal is that 1521 integrin plays a critical role in mediating the transduction of physiological stretch to VSM CaL channels to enhance myogenic tone, while 1v23 integrin functions as a matricryptin receptor to inhibit myogenic tone and initiate vasodilation in response to vessel wall injury. The hypothesis will be tested using patch-clamp methods to record CaL and BK (large-conductance calcium-activated K+ channel) currents in single rat or mouse VSM cells and diameter responses of isolated arterioles in conjunction with adenoviral methods and transgenic animals to manipulate the expression of selected proteins in arteriolar smooth muscle. There are two aims.
Aim A will determine how longitudinal cell stretch is transduced through integrins to potentiate VSM CaL channels and myogenic tone. We predict that: 1) 1521 integrin but not 1v23 integrin transduces mechanical force to regulate VSM CaL channels, 2) the intrinsic stretch-sensitivity of CaL is unimportant compared to integrin-mediated force transduction to CaL, and 3) talin-1, paxillin, 1-actinin and p130Cas are critical focal adhesion proteins required for force transmission through integrins to CaL channels in VSM.
Aim B will determine how 1v23 integrin functions as a matricryptin receptor to inhibit myogenic tone through VSM CaL and BK channels. We predict that: 1) fibronectin, osteopontin and collagen contain matricryptic sites that induce arteriolar dilatation;2) matricryptins act by inhibition of CaL channels and/or activation of BK channels;3) matricryptins inhibit CaL through PKG phosphorylation of an inhibitory site on CaL or activation of a tyrosine phosphatase to inhibit Src-induced CaL phosphorylation;and 4) matricryptins will not evoke dilatation in arterioles from 23 integrin-/- mice or modulate BK CaL channels in 23 integrin-/- VSM cells. These studies are important for understanding the mechanisms by which normal blood vessel function is regulated by ECM-integrin interactions and how these processes become impaired in diseases such as atherosclerosis and diabetes, where substantial changes in vessel wall composition and reactivity occur.
Blood vessels normally respond to physical forces, like blood pressure, to control and distribute blood flow to every tissue. One focus of this proposal is to determine how changes in pressure are detected by ion channels in the walls of blood vessels. Ion channels are proteins that control the flux of potassium and calcium across the cell membranes and their activity strongly influences the control of vessel diameter and blood flow. A second focus of the proposal relates to the fact that the protein composition of the vascular wall changes as the wall is remodeled in chronic inflammatory diseases such as diabetes and atherosclerosis. Part of the disease process involves breakdown of the existing proteins that hold the vessel wall together as well as infiltration of new proteins not normally found in the wall. We will determine how these proteins are modified by the same processes as found in the diseases states and then test the effects they have on vascular ion channels and blood vessel function.
