Network signature of low-flow endothelial dysfunction

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PROJECT SUMMARY/ABSTRACT The endothelium is a crucial regulator of vascular homeostasis and endothelial dysfunction is a hallmark of cardiovascular disease. The challenge in searching for new therapies is finding early control points that prevent the shift to broad pathologic signaling profiles and disrupt the endothelial network. Employing novel imaging and analysis approaches, we have identified discrete patterns of dynamic Ca2+ signalling along the vascular intima that underlie vascular function and direct the specificity, sensitivity and intensity of prevailing vascular responses. These patterns, defined by profiles of dynamic event parameters (frequency, amplitude, duration and spatial spread), form distinct signatures along the endothelial network. The complex spectrum of endothelial Ca2+ events (from isolated brief transients to broad multicellular waves) result from positive feedback interaction between plasma membrane TRP channels (Ca2+ entry) and endoplasmic reticulum IP3Rs (Ca2+ release). Small conductance Ca2+-activated K+ channels (KCa) play a key role in this signaling by exerting Ca2+-dependent hyperpolarization and amplifying Ca2+ influx through TRP channels (particularly fluid shear stress (FSS)- activated TRPV4 channels). In flow-deprived distal arteries from patients with peripheral artery disease, the endothelium exhibits a distinctive truncated Ca2+ signature characterized by spatially restricted small amplitude transients. This anomalous Ca2+ profile appears early in a low-flow carotid ligation mouse model, giving rise to endothelial dysfunction and vascular remodelling. These low-flow adaptations involve progressive loss of endothelial KCa2.3 channels and suggest an early loss of cooperative KCa/TRPV4 action. We hypothesize that disruption of TRPV4-KCa2.3 signaling under conditions of low FSS causes a progressive, highly restricted endothelial Ca2+ signature that promotes endothelial dysfunction and vascular remodeling. Aim 1 will characterize the role of TRPV4-KCa2.3 signaling in physiologic Ca2+ signatures along the arterial endothelium. We will conduct confocal imaging (with novel high-content analysis) and employ endothelium- specific knockout mice (ecKCa2.3-/- and ecTRPV4-/-) as well as human peripheral arteries to elucidate cooperative channel impacts under differential FSS. Aim 2 will determine whether low/oscillatory FSS causes truncation of the TRPV4-KCa2.3-dependent endothelial Ca2+ signature that leads to endothelial dysfunction and vascular remodeling. We will employ a partial ligation mouse model to assess the magnitude and time course of TRPV4- KCa2.3-specific impacts on Ca2+ signaling, vasoreactivity and vascular wall thickening. Aim 3 will determine whether preservation of endothelial TRPV4-KCa2.3 Ca2+ signaling ameliorates development of functional and structural vascular changes resulting from chronic low flow. We will also assess whether interventions to preserve the Ca2+ signature directly abate pathologic impacts of low flow.