Leading causes of death, such as heart disease, stroke, and diabetes, and are all associated with vascular dysfunction. Thus, understanding the physiologic mechanisms that control vascular function is vital for understanding the pathogenesis of these conditions and for developing new treatments. Many important classes of vasomodulators work by binding to G-protein coupled receptors (GPCRs) that initiate signaling cascades that converge on the small GTPase, RhoA. RhoA-GTP activates Rho-associated kinase (ROK), which regulates contraction of smooth muscle through inhibition of myosin light chain phosphatase (MLCP) and is also involved in pathophysiological responses of arteries; vascular remodeling, smooth muscle cell proliferation, and recruitment of inflammatory cells. RhoA can therefore be regarded as an integrative control point that translates diverse GPCR signaling to numerous artery functions. The fraction of RhoA molecules that are bound to GTP constitutes the 'fractional activation' of RhoA, and is a quantitative measure of the potential activation of ROK. In preliminary work we have constructed a high performance FRET-based RhoA activation sensor molecule, RhoA.v2. RhoA.v2 utilizes mCerulean3 and mCitrine to provide outstanding characteristics for quantitative FRET measurements, particularly with two-photon excitation. Two-photon excitation also provides the ability to image RhoA.v2 within cells of intact tissues of the living mouse and even entirely non-invasively, through the skin. The major Aims of this proposal are to 1) develop a novel transgenic mouse model that expresses RhoA.v2 specifically in smooth muscle cells, 2) develop methods, utilizing two- photon imaging, that unlock the full quantitative power inherent to the design of the RhoA.v2, such that the fractional activation of RhoA can be quantified in arteries in vivo, and 3) pursue a preliminary investigation into the role of RhoA in control of contraction of smooth muscle cells in arteries by the sympathetic nervous system (SNS) activity. SNS hyperactivity, which can exist only in living animals, is a key factor in hypertension, metabolic syndrome, heart failure and other conditions. We will test the hypothesis that RhoA is a critical effector of SNS in certain arteries in vivo. Ths work will be accomplished by a team of investigators with complimentary expertise in optical probe development/FRET imaging (Dr. Rizzo) and vascular biology and in vivo imaging (Dr. Wier). In summary, a novel RhoA biosensor mice will be created and methods, utilizing two-photon imaging, will be developed for quantification of RhoA activation in vivo, with subcellular resolution. The model and methods developed by this proposal will be broadly impactful to hypertension, diabetes, many areas of vascular biology (including stroke), and areas of general smooth muscle involvement, such as gastrointestinal and bladder function.
Vascular dysfunctions directly participate in the pathogenesis of many of the leading causes of death in the United States, including heart disease, diabetes and stroke. Modulation of vascular function by cell surface receptors converges on the activation of the RhoA enzyme and these receptors are the target of many existing and emerging therapies, yet key questions remain about the true nature of their actual potency and function in vivo, such as, for example, whether particular receptors principally exert control locally or systemically through the central nervous system. To answer this and related questions important to human diseases and their therapies, a novel mouse model and quantitative methodology will be developed to enable measurement of integrative receptor signaling in living animals using fluorescence microscopy.
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