Mark T. Nelson, Ph.D. - US grants

Calcium entry through voltage.dependent calcium channels into arterial smooth muscle cells regulates contraction and, therefore, blood vessel diameter, and blood flow. The overall goal of this research is to characterize the role that calcium entry through calcium channels plays in the regulation of contraction of smooth muscle cells in arteries. Calcium entry will be characterized by examining the physiological and pharmacological regulation of single calcium channels incorporated into planar lipid bilayers and in membrane patches from single smooth muscle cells isolated from cerebral (basilar) arteries. The results of this research will advance our understanding of vascular smooth muscle and calcium channel function.

[unreadable] DESCRIPTION (provided by applicant): The major objective of this proposed renewal is to elucidate the function of intracerebral arterioles in the context of the neurovascular unit - arterioles/astrocytes/neurons. In the brain, increased neuronal activity is accompanied by an increase in local cerebral blood flow that serves to satisfy enhanced glucose and oxygen demand. Although this process has been exploited clinically to map brain function, the mechanisms by which increased synaptic activity is communicated to the cerebral vasculature to cause vasodilation are poorly understood. Our central hypothesis is that Ca2+ signals arising in the astrocyte as the result of neuronal stimulation propagate to the endfeet (which encase the arterioles), leading to activation of large conductance, Ca2+-sensitive potassium (BK) channels in the endfeet and potassium (K+) release into the astrocyte-arteriolar space. This localized K+ elevation is proposed to act on arteriolar inward rectifier K (Kir) channels to hyperpolarize the smooth muscle cell (SMC) membrane, lower arteriolar Ca2+ and thereby cause vasodilation. A companion hypothesis posits a central role for SMC BK channels as a target of arachidonic acid (AA) metabolites (EETs, PGE2, 20-HETE), which are known vasomodulators, recently implicated in neurovascular coupling. An additional mechanism to be explored is that astrocytic AA metabolites may act on endfoot BK channels to modulate K+ release. To elucidate these mechanisms, Aim 1 will elucidate the roles of Ca2+ signaling, BK and Kir channels, and their modulation by external K+ and AA metabolites in isolated intracerebral arterioles and single arteriolar myocytes. Aim 2 will provide an unparalleled view of Ca2+ dynamics and BK channel modulation in astrocytic endfeet which encase the arterioles. Aim 3 will integrate the information from Aims 1 and 2 to elucidate key elements in signaling from active neurons, via astrocytes, to arterioles in brain slices. A novel combination of high speed calcium imaging, electrophysiology, and arteriolar diameter measurements in conjunction with unique mouse models lacking key elements of arteriolar smooth muscle and astrocytic BK and Kir channels will be exploited in myocytes, isolated arterioles, and the intact neurovascular unit. The cerebral microcirculation has a major role in a number of diseases including stroke, migraine, Parkinson's, Alzheimer's and early onset dementia. Thus, elucidating the mechanisms by which neuronal and astrocyte activity regulates intracerebral arterioles is critical to the development of new targets for effective therapies of pathological conditions associated with the cerebral vasculature. [unreadable] [unreadable] [unreadable]

Coronary heart disease (CHD) in women increases dramatically at menopause, as estrogen levels drop. Part of the beneficial effect of estrogen on CHD may be through effects on the coronary arteries in ways that protect the heart from ischemic injury. Coronary artery tone and reactivity are regulated by voltage-dependent Ca channels, which allow Ca2+ entry on depolarization, and K+ channels, which when activated cause hyperpolarization. We have provided the first direct measurements in smooth muscle of two types of K+ channels that are likely to play important roles in the response of coronary arteries to estrogen and ischemia: 1) The ATP-sensititive K+ (Katp) channel and 2) The inward rectifier K= (K) channel. Hypoxia, adenosine, and synthetic agents (e.g. lemakalim) activated K channels, and through vasodilation, may be involved in the increase in coronary blood flow to counteract periods of cardiac ischemia. Small increases in extracellular K+, which can occur during ischemia, may activate K channels which may also lead to a beneficial increase in coronary blood flow. Our preliminary results suggest the exciting possibility that estrogen, through alteration in the activity of coronary artery ion channels increases arterial diameter and response to vasodilating signals. We, therefore propose to test the hypothesis that gender and sex steroids, particularly estrogen, affect the reactivity of coronary arteries through their regulation of K+ and Ca channels. Our preliminary evidence to support this hypothesis forms the basis of our 4 Specific Aims: 1) We found that female coronary arteries constrict less to intravascular pressure than male or ovariectomized female coronary arteries from rat. In Specific Aim 1, we will investigate the role of membrane potential (K+ channels), Ca channels, and intracellular free Ca2+ in this response. 2) We found that estrogen but not progesterone dilates coronary arteries and inhibits calcium channels. In specific Aim 2, we shall explore the effects of acute estrogen and other sex steroids on arterial tone, membrane potential, an ion channels. 3) We found that small elevations in external K+ dilate female but not male coronary arteries, suggesting gender and sex steroids on: dilatins and hyperolarizations to external K+, adenosine and K+ channels play a more important role in females. In Specific Aim 3, we shall characterize the effects of gender and sex steroids on: dilations and hyperpolarization to external K+, adenosine and K+ channel openers, and K and K channels. 4) In Specific Aim 4, we will explore the role of the endothelium in all of these responses. We will use a combination of techniques to obtain an integrated picture of coronary artery function: diameter measurements of coronary arteries at different levels of transmural pressure, membrane potential recordings from smooth muscle cells in intact arteries, whole cell and single ion channel measurements in freshly dissociated smooth muscle cells from coronary arteries, and intracellular Ca2+ measurements. The proposed study should provide important insights into the control of coronary artery reactivity by estrogen, other sex steroids and gender. This study should also deepen our understanding of the regulation of coronary artery tone by ion channels, advancing our understanding of possible response mechanism of the female coronary vasculature to ischemia. Finally, the proposed project should suggest new strategies for treatment of coronary heart diseases in women.

DESCRIPTION (Adapted from the Applicant's Abstract): Urinary incontinence has devastating lifestyle effects on at least 13 million Americans, of whom 85% are women, and is a major financial burden on the health care system. Yet, the basic mechanism of the regulation urinary bladder smooth muscle (UBSM) contractility, the control of intracellular Ca2+ and membrane excitability, are poorly understood. The role of UBSM is unique in that it must regulate tone during filling, similar to arterial smooth muscle, and yet when full, contracts forcefully, similar to cardiac muscle. This proposal is based on several major advancements in understanding of Ca2+ regulation in arterial smooth muscle and cardiac muscle, including the measurement of elementary Ca2+ release events ("Ca2+ sparks") caused by opening of ryanodine-sensitive Ca2+ release (RyR) channels in the sarcoplasmic reticulum (SR). The application proposes that Ca2+ sparks have a novel antagonistic role in the regulation of UBSM excitability and contractility: Ca2+ sparks contribute Ca2+ for contraction during an action potential and may oppose contractility by limiting Ca2+ entry through activation of Kca channels, which decrease excitability. Specifically, the project will examine the nature of Ca2+ sparks and their subsequent activation of Ca2+-sensitive potassium (Kca) channels (Hypothesis 1), the role of Ca2+ sparks to augment global Ca2+ transients during an action potential to enhance contractility (Hypothesis 2A), the role of Ca2+ sparks to limit Ca2+ entry and SR Ca2+ release through shortening the action potential and hyperpolarizing the membrane potential by activation of Kca channels (Hypotheses 2B,C). Further, the proposal will explore the novel idea that frequency and amplitude modulation of Ca2+ sparks and Kca channel currents is a mechanism by which cholinergic stimulation affects UBSM excitability and contractility (Hypothesis 3). Finally, the effort will explore the idea that stretch of UBSM cells differentially affects the antagonistic actions of Ca2+ sparks on global and Kca channels (Hypothesis 4). The proposed study should significantly deepen our understanding of the regulation of UBSM function, and provide new therapeutic targets that may be unique for urinary bladder.

DESCRIPTION (Verbatim from the application): The cardioprotective effect of estrogen is complex, and incompletely understood. We have identified one potentially important action of physiological circulating estrogen on coronary arteries, which involved an elevation of basal nitric oxide release from the endothelium and an ensuing smooth muscle relaxation through activation of one type of calcium-sensitive (large conductance; BK) potassium channel in the smooth muscle. Our results indicate that estrogen causes fundamental alterations in Ca2+ signaling in the coronary endothelium, which leads to an elevation of nitric oxide production, which in turns alters Ca2+ signaling in the smooth muscle. This proposal focuses on novel, interlinked Ca2+ signaling mechanisms to explain the effects of estrogen and nitric oxide on coronary artery diameter. Specifically, Aim 1 will determine the mechanisms by which estrogen leads to an elevation of endothelial [Ca2+]i; Specific Aim 2 will explore positive feedback regulation of endothelial Ca2+ by local and global Ca2+ signaling from ryanodine receptors (RyRs) in the endoplasmic reticulum to small conductance calcium-sensitive (SK) and BK channels; Specific Aim 3 will determine the negative feedback mechanisms by which endogenously produced nitric oxide activates RyRs and BK channels in coronary artery smooth muscle, with a focus on the key roles of phospholamban, which regulates SR Ca2+-ATPase activity, and on the beta-subunit of the BK channel. To address these issues, we have developed techniques to measure global and local calcium in the endothelium and smooth muscle of intact pressurized coronary arteries, including coronary arteries from phospholamban and beta-subunit gene-ablated mice, and developed novel, selective peptide inhibitors of cGMP-dependent protein kinase. The proposed study should significantly deepen our understanding of the regulation of Ca2+ signaling in coronary arteries, and the important influences of physiological estrogen and nitric oxide on coronary artery function. This work should also suggest novel mechanisms for therapeutic interventions to mimic the beneficial effects of estrogen and nitric oxide on calcium signaling in coronary arteries.

[unreadable] DESCRIPTION (provided by applicant): Detrusor instability is a major component of urinary bladder dysfunction, including outflow obstruction associated with benign prostate hyperplasia. The goal of this project is to understand the normal physiological regulation of urinary bladder smooth muscle (UBSM) excitability and to apply an experimental model of partial urinary outflow obstruction to address pathophysiological aspects of UBSM function. This proposal focuses on the communication among four key ion channels involved in regulating the excitation-contraction (E-C) coupling process in UBSM: 1) Voltage-dependent calcium channels (VDCC), which mediate the upstroke of the UBSM action potential. 2) Ryanodine-sensitive calcium release channels in the sarcoplasmic reticulum (RyRs), which release Ca2+ in the form of Ca2+ sparks. 3) Large-conductance, voltage/calcium-activated potassium (BK) channels, which mediate membrane repolarization of an action potential. 4) Small-conductance, calcium-activated (SK) channels, which are responsible for the after-hyperpolarization. This work builds on our discovery of Ca2+ sparks and their communication to BK channels in smooth muscle, and elucidation of key molecular components of this process. In an important advancement, we have found that the beta 1-subunit of the BK channel plays a major role by tuning the voltage/Ca2+ -sensitivity of this channel. We provide novel evidence that the BK channel beta 1-subunit and SK channels have profound effects on bladder function; data that point to potassium channel dysfunction as a significant contributor to detrusor instability following obstruction. An integrated approach, combining molecular and electrophysiological studies with functional measures of bladder contractility and cystometric parameters, will be applied using wild-type and genetically engineered mouse models. Our specific objectives are to elucidate the functional communication among VDCCs, RyRs and BK channels in normal and outflow obstructed bladders (Aim 1), to characterize the role of the beta 1-subunit in tuning the Ca2~ and voltage sensitivity of the BK channel (Aim 2), and to defme the roles of SK channels in the regulation of UBSM function (Aim 3). The long-term goal is to develop novel approaches for regulating urinary bladder function, with the main focus being on the therapy of detrusor instability.

DESCRIPTION (provided by applicant): Partial support is requested for the funding of the 2003 FASEB Smooth Muscle Conference to be held from July 05-10th at Snowmass, Colorado. This conference is the 8th in a highly successful series of smooth muscle meetings that have been held every 3 years since 1982. The FASEB Smooth Muscle Conferences are widely regarded as the premier forum in the field of smooth muscle biology. The meetings have been extremely popular and attended by leading investigators from Australia, Asia, Europe and North America. An understanding of smooth muscle function is vital to those interested in cardiovascular, respiratory, reproductive, digestive and urinary tract physiology and pathophysiology. Thus, this conference regularly attracts both basic science and clinical investigators from several disciplines. Rapid progress has been made during the past 3 years using techniques such as molecular biology, cellular and sub-cellular confocal imaging, and patch clamping, and new insights have been gained about myofilament interactions, development and plasticity of smooth muscles, ion channel identity and regulation, second messenger systems, Ca2+ signaling, and cell-to-cell interactions. Because many smooth muscle investigators work on specific organ systems (i.e. cardiovascular system) and frequently attend society meetings related to the specifics of these organ systems, a comprehensive smooth muscle meeting where common issues related to the cell and tissue biology of smooth muscles are discussed is extremely important. The FASEB Smooth Muscle Conferences have served this critical need for over 2 decades. The format of the meeting is especially conducive to informal discussion and opportunities to form new collaborations. In addition to the programmed lectures described in the application and organized into 9 topical symposia, contributions from young investigators will be selected from the abstracts submitted and added to the symposia as oral presentations. The 2003 FASEB Smooth Muscle Conference will provide a comprehensive review of recent progress in smooth muscle biology and constitute an intensive short course for scientists who are beginning investigations in the field or clinical investigators with broader clinical interests in smooth muscle function. The extensive opportunities for informal interactions will allow investigators at all levels to meet new colleagues, explore new ideas and techniques, and develop new collaborations.

DESCRIPTION (provided by applicant): A central function of urinary bladder smooth muscle (UBSM) is the translation of neural inputs into a normal micturition response, a function that is dramatically altered with outlet obstruction. This proposal focuses on how these neural inputs through cholinergic and purinergic pathways differentially impact calcium (Ca2+) signal patterning in UBSM, and on electrical and contractile responses under normal conditions and with partial outlet obstruction. Despite the importance of these pathways, major gaps remain in our knowledge o1 the underlying nerve-evoked excitation-contraction (E-C) coupling mechanisms and the changes that occur with obstruction. In a major advance, we have been able to measure, with high spatial and temporal resolution, nerve excitation, and local Ca2+ signals in intact whole urinary bladders and bladder strips. We have identified two distinct local calcium transients in UBSM: 1) nerve-evoked calcium transients, mediated through purinergic receptors, and 2) the local release of calcium from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs). Aim 1 seeks to elucidate the novel mechanisms by which UBSM decodes cholinergic and purinergic stimulation into different calcium signals, which differentially depend on Ca2+ entry through voltage-dependent calcium channels (VDCC), inositol triphosphate receptors (IP3Rs) and RyRs in the SR. Aim 2 focuses on UBSM E-C coupling mechanisms in response to the unique calcium signals elicited by cholinergic and purinergic mechanisms. In Aim 3, we explore the interaction of VDCC and SR Ca2+ load in mediating cholinergic and purinergic excitation in normal and dysfunctional bladders. The hypothesis that SR dysfunction following outlet obstruction differentially affects cholinergic and purinergic stimulation by altered UBSM calcium signaling will be tested. Using state-of-the-art techniques, genetically altered mice, and our recently developed mouse model of partial bladder outlet obstruction, the proposed study will provide new insights into the differential impact of cholinergic and purinergic pathways on UBSM in normal and obstructed bladders. The novel findings of this study will be highly relevant to understanding fundamental mechanisms of nerve-evoked E-C coupling in UBSM, and identify key elements that underlie bladder dysfunction, and as such should be highly relevant to the understanding and treatment of bladder dysfunction.

DESCRIPTION (provided by applicant): A number of diseases and pathologies are associated with both urinary incontinence and erectile dysfunction (ED), including diabetes, benign prostate hyperplasia, hypertension, and hyperlipidemia. The co-occurrence of urinary bladder and erectile dysfunction suggests the existence of mechanistic commonalities. One such point of convergence is the large conductance Ca2+-activated K+ (BK) channel, as evidenced by the fact that targeted disruption of the pore-forming BKa-subunit leads to overactive detrusor, urinary incontinence, and ED. A second potential common denominator is the NO/cGMP/PKG pathway, which regulates BK channel function directly as well as indirectly through calcium signaling elements that modulate BK channel function. The NO/cGMP/PKG pathway is nearly universally associated with smooth muscle (SM) relaxation. Yet, in contrast to corpus cavernosum (CC) SM, where nitrergic signaling through the PKG pathway is a well-established mediator of SM relaxation and erectile function, the role of NO in urinary bladder (UB) SM is an unsettled issue. In Aim 1, UBSM and CCSM will be investigated at the molecular and cellular levels to establish the potential of NO/cGMP/PKG signaling to regulate SM function, with an emphasis on the role of the BK channel pathway. BK channel function and PKG regulatory mechanisms in intact bladder and corpus tissue will be addressed in Aim 2, which will also consider the role of the cGMP-specific phosphodiesterase, PDE5, and Ca2+-sensitive chloride currents (ClCa) as potential contributors to tissue-specific differences in NO sensitivity. Finally, Aim 3 seeks to integrate the findings of Aim 1 and 2 and address the roles of the BK channel and cGMP/PKG pathways in regulating function during bladder filling using an ex vivo whole bladder model. Additionally, the role of these pathways in regulating intracavernous pressure and urodynamic properties during bladder filling will be addressed in vivo.The clinical success of the cGMP-specific phosphodiesterase-5 (PDE5) inhibitor sildenafil (Viagra) and related compounds in treating erectile dysfunction demonstrates a clear role for the cGMP-dependent protein kinase (PKG) pathway in regulating corpus cavernosum smooth muscle relaxation and erectile function. Despite the existence of both neuronal and urothelial sources of NO in the urinary bladder, and evidence for the expression of NO-responsive cGMP/PKG signaling pathway components in myocytes, the significance of the cGMP/PKG pathway in bladder physiology remains an unsettled question. The large conductance, calcium activated potassium (BK) channel, which has a central role in relaxing smooth muscle in the lower urinary tract, is a target of the PKG pathway, and represents a promising therapeutic target in the treatment of sildenafil-resistant ED and urinary incontinence.

DESCRIPTION (provided by applicant): The vascular endothelium in intracerebral (parenchymal) arterioles is a critical mediator of normal cerebral function, serving as both a physical barrier and a modulator of blood flow within the brain. Ca2+ signaling and the Ca2+-sensitive K+ channels, IK and SK, and TRPV4 channels in endothelial cells (ECs) activate pathways that transmit vasoregulatory signals to adjacent smooth muscle (SM) and along the endothelial lining of blood vessels. These signals may also communicate to nearby astrocytes and neurons to modulate neurovascular coupling (NVC). Despite the importance of parenchymal arteriolar (PA) endothelium, little is known about its control of vascular tone or potential influence on NVC in the brain. To support the Project, we provide novel data that EC SK, IK and TRPV4 channels have a profound effect on PA tone and cortical cerebral blood flow. Aim 1 will elucidate the properties and roles of endothelial Ca2+ signaling modalities, and IK, SK and TRPV4 channels in PA ECs using a novel mouse model that expresses a Ca2+ biosensor (GCaMP2) in the endothelium. Exploiting these GCaMP2 mice, we have recently discovered a localized, stationary IP3- mediated Ca2+ signal in endothelial projections to the SM. This signal, termed a "pulsar", activates co-localized IK channels to modulate vascular tone. Aim 2 will determine the impact of endothelial function on the SM of PAs, exploring Ca2+ signaling, membrane potential and vascular diameter. Aim 3 builds on Aims 1 and 2 to explore the role of the endothelium in the context of the brain, evaluating its effects on NVC and blood flow using a novel approach based on simultaneous measurement of astrocytic endfoot Ca2+ and vascular responses. The proposed project will provide significant new insight into PA endothelial function and communication to SM. This project should reveal novel targets involved in modulating blood flow in the brain and suggest therapeutic agents that do not require passage through the blood brain barrier. PUBLIC HEALTH RELEVANCE: The cells (endothelial) that line the small blood vessels (arterioles) in the brain are critical mediators of normal cerebral function, serving as both a physical barrier and a modulator of blood flow. Endothelial cell dysfunction is a common feature of blood vessel diseases (hypertension, atherosclerosis) and a likely contributor to neurological disorders (Alzheimer's). This project will elucidate key control mechanisms by which endothelial cells in cerebral arterioles regulate blood flow in the brain.

This Program Project consists of four individual related research projects and three cores. This core oversees the administrative needs of the program. The Program Project director (Dr. Nelson) will maintain a staff who are responsible for financial records, progress reports, internal documents necessary for the functioning of the program project, and basic computer hardware and software support. Given that two subcontracts (Project 2 Subcontract with the University of Washington and Core B Subcontract with Cornell University) are off-site, it will be crucial that communication between the projects and Core members and these external individuals be at its maximum level. Therefore, the Administrative Core will be responsible for fostering electronic means of inter-Project/Core communication and scheduling semi-annual retreats that will assemble all the Pis to discuss the progress of the Program Project. At the University of Vermont, the Administrative Core will schedule the monthly Program Project meeting as well as arrange for the scheduled Internal and External Advisory Committee meetings. The Core will also schedule at least 6 research seminars from internationally recognized scientists outside of the University, whose expertise complements the research interests of the Program Project. The Core will provide secretarial and administrative assistance to the Internal and External Advisory Committee. Manuscripts, progress reports, budgets, personnel recruitment, and assistance with other matters and documents intrinsic to the program will be handled by personnel within the Core. Individual principal investigators are responsible for the scientific progress of the functional units they direct and for utilizing the budgets involved. The Program Project Director will critically review, for approval or disapproval, all significant personnel changes (changes in rank, percent effort, persons involved, etc.), all major shifts of money from one listed in the budget of this proposal.

ABSTRACT: The vascular endothelium in parenchymal (intracerebral) arterioles is a critical mediator of normal cerebral function, serving as both a physical barrier and a modulator of blood flow within the brain. Ca2+ signaling and the Ca2+sensitive K+ channels, IK and SK, and TRPV4 in endothelial cells (ECs) activate pathways that transmit vasoregulatory signals to adjacent smooth muscle (SM) and along the endothelial lining of blood vessels. These signals may also communicate to nearby astrocytes and neurons to modulate neurovascular coupling (NVC). Despite the importance of parenchymal arteriolar (PA) endothelium, little is known about its control of vascular tone or potential influence on NVC in the brain. Aim 1 will elucidate the properties and roles of endothelial Ca2+ signaling modalities and IK, SK and TRPV4 channels in PA ECs using a novel mouse model that expresses a Ca2+ biosensor (GCaMP2) in the endothelium. Exploiting these GCaMP2 mice, we have recently discovered a localized, stationary IPs-mediated Ca2+ signal in endothelial projections to the SM. This signal, termed a pulsar, activates co-localized IK channels to modulate vascular tone. Aim 2 will determine the impact of endothelial function on the SM of PAs, exploring Ca2+ signaling, membrane potential and vascular diameter. Aim 3 builds on Aims 1 & 2 to explore the role of the endothelium in the context of the brain, evaluating its effects on NVC and blood flow using a novel approach based on simultaneous measurement of astrocytic endfoot Ca2+ and vascular responses. The proposed project will provide signiflcant new insight into endothelial function and communication to SM in PAs. Close collaboration between Projects 1 and 2 will assure appropriate consideration of the physiological interactions and communication between endothelium and smooth muscle in PAs. In conjunction with Projects 3 and 4, Project 1 will help illuminate the role of the endothelium under the clinically important pathological conditions of ischemia/reperfusion injury and subarachnoid hemorrhage. This project should reveal novel targets involved in modulating blood flow in the brain and suggest therapeutic agents that do not require passage through the blood brain barrier.

This Program, consisting of four Projects and three Cores, focuses on parenchymal (intracerebral) arterioles, which distribute blood within the brain and exhibit unique, albeit poorly understood, properties. The overall goal Is to elucidate the molecular mechanisms by which the endothelium and smooth muscle (SM) of parenchymal arterioles (PAs) regulate vascular tone and neurovascular coupling (NVC) normally, and following ischemia/reperfusion (l/R) injury and subarachnoid hemorrhage (SAH). The central unifying theme is that the precise control of Ca^* signaling in cells composing the cerebrovascular unit¿ endothelium, SM and astrocytes¿determines normal brain function; by extension, dysfunction of Ca^* signaling dynamics contributes to cerebrovascular disorders. Each project studies overlapping elements of the biological system to form a whole. Project 1 will provide the first information on properties and function of PA endothelial cells (ECs), exploring Ca^* signaling and Ca^*-sensitive SK and IK potassium channels and their impact on SM function and NVC. Project 2 will focus on parenchymal arteriolar SM, with an emphasis on voltage-dependent Ca^* channels (Cav) and transient receptor potential (TRP) channels. Projects 1 and 2 provide the platform for Projects 3 and 4. Project 3 will interact with Projects 1 and 2 to explore the underlying mechanistic basis for the profound changes in parenchymal arteriolar reactivity that develop following i/R, focusing on EC-SM changes in ion channel functionality. Project 4, which focuses on PAs and NVC following SAH, intersects with Project 2 on the effects of SAH on SM Cav and TRP channels, and with Project 1 on endothelial function and NVC. Because the endothelium and SM function physiologically as one entity, Projects 1 and 2 are inherently highly interdependent. Projects 3 and 4, which focus on dysregulation of cerebrovascular unit function under two divergent, but clinically relevant, pathological conditions, depend on information derived from Projects 1 and 2. The Imaging Core will provide state-of-the-art approaches for Ca^* measurements in complex systems. The Animal and Instrumentation Core will consolidate and coordinate all animal and in vivo work, including the development of novel genetically encoded, ratiometric Ca^* biosensors. The long-term objective is to understand blood flow in the brain in health and disease, and by doing so, to reveal exciting novel targets that can be exploited in the treatment of cerebrovascular disease.

DESCRIPTION (provided by applicant): Endothelial cells (ECs) lining blood vessels are pivotal regulators of vascular tone. Their function is disrupted in cardiovascular diseases, including hypertension. Although some of the molecular players involved in mediating endothelial-dependent vascular regulation have been identified, key aspects of their signaling linkages remain poorly understood. Importantly, how these molecular circuits are spatially organized to enable efficient signaling is largely unknown. In this proposal, we test the novel hypothesis that EC A-kinase anchoring protein (AKAP150) and transient receptor potential vanilloid 4 (TRPV4) channels form the core of a dynamic integrator of endothelial and smooth muscle cell (SMC) signaling that is localized at myendothelial projections (MEPs)-specialized projections through the internal elastic lamina that connect ECs with adjacent SMCs through gap junctions. In support of this, we provide novel data that AKAP150, which binds protein kinase C (PKC), protein kinase A (PKA) and calcineurin (PP2B), is required for Gq-protein coupled receptor (GqPCR) activation of TRPV4 channels exclusively at MEPs. In contrast, shear stress preferentially stimulates non-MEP TRPV4 channels. Moreover, AKAP150 promotes cooperative gating of TRPV4 channels in a 4- channel metastructure but, surprisingly, is not a determining factor of TRPV4 channel agonist sensitivity, which is dramatically different between cerebral and mesenteric resistance arteries. Importantly, our data demonstrate that this signaling network is disrupted in hypertension through changes in local coupling caused by the loss of MEP AKAP150. In Aim 1, we investigate the roles of AKAP150-bound PKC, PKA and PP2B as well as caveolin-1 in the regulation of MEP TRPV4 activity and cooperativity using a genetically encoded, EC- specific Ca2+ biosensor (GCaMP2), an optogenetic technique for controlling spatial production of IP3/diacyl glycerol, and genetic mouse models of major network elements. We also explore the basis for the striking difference in TRPV4 agonist sensitivity between cerebral and systemic (mesenteric) arteries. In Aim 2, we use a variety of approaches, including multi-photolysis of caged IP3 and Ca2+, to define mechanisms of myoendothelial feedback to MEPs and shear stress-induced vasodilation via activation of non-MEP TRPV4 channels. In Aim 3, we use insights gained from Aims 1 and 2 to unravel the nature of the dysfunction of the MEP signaling network in hypertension using two mouse models. Taken together, these experiments will provide an unparalleled view of the bidirectional signaling network in MEPs and represent the first detailed exploration of the defects in local connections that likely contribute to endothelial dysfunction in hypertension.

? DESCRIPTION (provided by applicant): Neurons in the brain have limited energy reserves and thus rely on a just-in-time delivery strategy in which active neurons signal to the brain microvasculature to locally dilate and increase regional cerebral blood flow (CBF), resupplying nutrients and oxygen as well as removing toxic metabolites. Despite extensive study, the mechanisms underlying the functional linkage between neuronal demand and vascular supply, termed neurovascular coupling (NVC), remain poorly understood. CBF within the brain is mediated by parenchymal arterioles (PAs) and hundreds of miles of capillaries, which enormously extend the territory of perfusion. Capillaries are the proximate vascular elements for the vast majority of neurons; thus, the dense capillary network can be viewed as a sensory web, detecting the rhythm of neural activity. Notably, neuronal activity- dependent increases in CBF at the surface of the brain are conducted at a rate that can only be explained by an electrical signal. We propose that strong inward-rectifier K+ (Kir2) channels in the vascular endothelium of capillaries, as well as arterioles, serve as exquisite sensors of local external K ([K ]e)-a byproduct of neuronal activity-that transmit an electrical signal to cause upstream dilation of PAs and surface arteries, and coordinate blood flow within the brain. Our extensive preliminary data, including the discovery that capillary endothelial cells (ECs) express robust Kir currents, as do both smooth muscle cells (SMC) and ECs of PAs, support this. We further propose that Kir-dependent electrical signals are augmented/sustained by slower retrograde Ca signals mediated by EC TRPV4 channels and G protein-coupled receptors of the Gq/11 type. We deploy a wide variety of novel, state-of-the-art experimental approaches using intact animals, native tissue and freshly isolated cells, complemented by sophisticated computational modeling, to test these ideas. Aim 1 will explore the impact of dual expression of Kir channels in EC and SMC compartments on K+-sensing, vascular dynamics, and the efficacy of EC-dependent vasodilators. Aim 2 will provide the first insights into ion channel properties and electrical and Ca2+-signaling in brain capillary endothelium using our newly developed pressurized arteriole-capillary ex vivo preparation. Building on Aims 1 and 2, Aim 3 will use in vivo approaches to explore inside-out electrical and Ca2+ signaling from capillaries to upstream arterioles and surface arteries in the context of NVC. The proposed work has the potential to revolutionize our understanding of communication within the brain microcirculation, and as such will provide the foundation for understanding small vessel diseases of the brain.

PROJECT SUMMARY Cerebral blood flow (CBF) is exquisitely controlled to meet the diverse and ever-changing demands of active neurons. Blood flow into the brain is mediated by penetrating/parenchymal arterioles and hundreds of miles of capillaries, which enormously extend the territory of perfusion. Blood delivery to active neurons (functional hyperemia) is rapidly and precisely controlled through a process termed neurovascular coupling (NVC). We recently provided compelling evidence that brain capillaries act as a neural activity-sensing network, and therefore are much more than simple conduits for blood. This concept explains the rapid and coordinated delivery of blood to active neurons, demonstrating that brain capillary endothelial cells (cECs) are capable of initiating an electrical (hyperpolarizing) signal in response to neural activity that rapidly propagates upstream to cause dilation of feeding arterioles and locally increase blood flow. We have established the mechanistic basis for this electrical signal, showing that neuron- and/or astrocyte-derived potassium (K+) is the critical mediator and identifying the strong inward rectifier K+ channel, Kir2.1, as the key molecular player. We have recently discovered a second fundamental NVC mechanism based on calcium (Ca2+) signaling, which is initiated by Gq-protein coupled receptor signaling and is partly mediated by TRPV4 channels. Dynamic changes in membrane phosphatidylinositol 4,5-bisphosphate (PIP2) levels appear to control the balance between electrical and Ca2+ signaling. A major focus of our laboratory has been on the pathogenesis of Small Vessel Disease (SVD) of the brain, which is a major cause of stroke and dementia. Using a monogenic model of SVD (CADASIL) and our mechanistic insights into NVC, we discovered that SVD precipitates early defects in functional hyperemia, which we propose involve extracellular matrix changes and a loss of PIP2 activation of cEC Kir2.1 channels and suppression of TRPV4 channels. Importantly, we are able to rescue functional hyperemia in CADASIL through exogenous application of PIP2, suggesting a broad-spectrum approach for improving CBF control in disease. We have further found that hypertension, the major driver of sporadic SVDs, also leads to age-dependent deterioration of this major functional hyperemia mechanism. We propose to elucidate mechanisms for defective functional hyperemia in CADASIL (Aim 1) and hypertension (Aim 2), including common molecular intersections. A goal of this proposal is to create an integrated view of the impact of SVD on CBF regulation at molecular, biophysical, and computational-modeling levels by examining their operation in increasingly complex segments of the brain vasculature ex vivo, in vivo, and in silico.

PROJECT SUMMARY Neurons in the brain have limited energy reserves and thus rely on a ?just-in-time? delivery strategy in which active neurons signal to the brain microvasculature to increase regional cerebral blood flow (CBF), resupplying nutrients and oxygen as well as removing toxic metabolites. Despite extensive study, the mechanisms underlying the functional linkage between neuronal metabolic demand and vascular supply, termed neurovascular coupling (NVC), remain poorly understood. Blood flow to the brain is mediated by parenchymal arterioles and hundreds of miles of capillaries, which enormously extend the territory of perfusion. We recently presented evidence supporting the concept that brain capillaries act as a neuronal activity-sensing network, demonstrating that brain capillary endothelial cells (cECs) are capable of initiating an electrical (hyperpolarizing) signal in response to neuronal activity that propagates upstream to cause dilation of feeding arterioles and increase blood flow locally at the site of signal initiation. We have established the mechanistic basis for this electrical signal, showing that neuron- and/or astrocyte-derived potassium (K+) is the critical mediator and identifying the strong inward rectifier K+ channel, Kir2.1, as the key molecular player. We have recently discovered that a second fundamental NVC mechanism based on calcium (Ca2+) signaling, with distinct kinetics and regulatory features, also operates in brain capillaries, and can be initiated by the putative NVC mediator prostaglandin E2 (PGE2). We have further found that a mechanism initiated by Gq-protein coupled receptor signaling and mediated by dynamic changes in membrane phosphatidylinositol 4,5- bisphosphate (PIP2) levels controls the balance between electrical and Ca signaling. Additional preliminary 2+ data support a role for gasotransmission via Ca2+-dependent endothelial nitric oxide signaling and pericyte- mediated regulation of capillary blood flow in modulating NVC. The immediate goals of this proposal are to create an integrated view of electrical, Ca2+ and related regulatory signaling mechanisms at molecular, biophysical, and computational-modeling levels by examining their operation in increasingly complex segments of the brain vasculature ex vivo, in vivo, and in silico. Ultimately, we propose to weave these research threads together to create a systems-level view of physiological capillary-to-arteriole/pial artery signaling in the brain, and test the concept that gradual degradation of this sensory web and the attendant progressive decay of cerebrovascular function contributes to small vessel diseases of the brain.

Cerebral blood flow is exquisitely controlled to satisfy neuronal metabolic demands. An essential feature of this control is the on-demand increase in local blood flow triggered by neural activity; a process termed functional hyperemia (FH) that is coordinated by multiple neurovascular coupling mechanisms. Importantly, the increases in shear stress in response to enhanced blood flow constitute mechanical forces with the potential to impact vascular behavior. A growing body of evidence indicates that hypertension attenuates FH. Notably, uncontrolled elevation of blood pressure is associated with dramatic alterations in the hemodynamic forces imposed on the vasculature. However, the extent to which mechanosensitive properties of the cerebral circulation are impacted by hypertension has not been explored. Piezo1, a Ca2+/Na+-permeable, mechanosensitive channel expressed in vascular endothelial cells, is the major mechanosensor in brain capillaries. Intriguingly, a gain-of-function mutation in the PIEZO1 gene has been reported in African Americans populations, which show the highest prevalence of hypertension in the world (>40%). Based on the unique properties of Piezo1 channels and the essential role of endothelial Ca2+ signaling in FH, I will evaluate the following hypotheses: (1) brain capillary Piezo1 channel activity is altered during hypertension; and (2) this change in Piezo1 function is implicated in the deficits in FH that occur during hypertension. These hypotheses will be tested by directly measuring Piezo1 channel activity and by measuring cerebral blood flow in the context of normal and high blood pressure. We will use technical innovations introduced by our laboratory that include cutting-edge genetically engineered mouse models with increased or decreased Piezo1 activity and mice with endothelial-specific genetically encoded Ca2+ indicators. By viewing hyperemic 'responses' through a new lens as mechanical 'stimuli', the proposed studies envision normal and perturbed (i.e., hypertension) cerebral blood flow from a completely fresh perspective. This project has the potential to profoundly alter our understanding of cerebral blood flow dysregulation during hypertension and may reveal sorely needed new paths to treatment.

PROJECT SUMMARY Cardiovascular disease is the #1 cause of mortality in the United States and stroke is the #5 cause. Stroke is also the #1 cause of disability, and it, along with covert neurovascular disease including small vessel disease of the brain, leads to Vascular Cognitive Impairment and Dementia (VCID). Each year 800,000 Americans have a stroke and 720,000 Americans have myocardial infarction. There will be 75 million people with dementia by 2030 and 131 million by 2050. Direct annual costs of cardiovascular disease and stroke are $318 billion and dementia $277 billion. The American Heart Association issued a Presidential Advisory in 2017 highlighting the central role of cardiovascular health to brain health and cognitive function. Much of VCID is driven by increases in covert stroke and small vessel disease of the brain, research areas of expertise for the PIs of this application. To address these issues, this University of Vermont (UVM) Center on Cardiovascular and Brain Health (VCCBH) will leverage NIH funding and institutional commitment to convene multidisciplinary researchers across career stages to determine causes and suggest optimal treatments for cardiovascular and neurovascular disease, including stroke and VCID. We have selected four junior faculty (Project Directors, PDs), from three departments and two UVM colleges, through a rigorous selection process. The VCCBH will support the PDs' transition to independence using a Strategic Mentoring Initiative (SMI), Faculty Development Program (FDP), and assistance and education from two new Core facilities. The SMI will employ multidisciplinary Mentor Teams, including Peer Mentors. The FDP will comprise several educational programs highlighted by an annual 2-Day Research Symposium, and an impactful Pilot Grant program (supported by institutional commitments) that will provide $200,000 to four interdisciplinary research teams of junior faculty. We will establish two new Core facilities: Study Design and Molecular Epidemiology, and Customized Physiology and Imaging. A pipeline of new project directors will be developed from a pool of 19 current candidates and five new junior faculty, who will be recruited with committed institutional support. Capstones of the Center will include continual quality evaluation, clear communication and commitment to diversity. By the completion of Phase I of funding, the Center will be on a strong path to sustainability in research excellence.