William M. Chilian - US grants

The objective of this application is to request funding for an automated Germanium gamma well counter. The principal advantage of the Germanium counter is that the energy resolution is 50 times better than traditional sodium iodide counters, that are presently employed at the Cardiovascular Center at Iowa. Our experimental protocols are limited by the low energy resolution of sodium iodide counters: we presently can only separate 6 different nuclide labelled microspheres. As a result of such a few numbers of measurements of regional perfusion with microspheres, experimental costa are high and experimental design is hampered. An automated Germanium gamma counter would enable us to measure regional perfusion up to 20 times (using 20 different nuclide labelled microspheres) in a single preparation. Our experimental costs would then be reduced and our experimental design would be strengthened.

The myocardium is a continually working aerobic organ. It is essential for normal myocardial function that a continual supply of oxygen is maintained to ensure a proper balance between myocardial oxygen supply and myocardial oxygen demands. The goal of this proposal is to investigate neural and microcirculatory mechanisms involved in regulating oxygen balance in the myocardium. Project I, Coronary Microvascular Dynamics, examines the hypothesis that the coronary microcirculation will exhibit a hierarchy of control to common stimuli. To accomplish Project I, I use a computer-controlled system which compensates for cardiac motion to enable microvascular pressure and diameter measurements in the beating heart. This computer-controlled device can move a micro-pipette in three dimensions in concert with a moving microvessel on a beating heart. Furthermore, using stroboscopic illumination (1 flash per heart cycle) microvessels are always in focus and "appear" stationary. With this approach in the beating heart, there is little trauma to the coronary system and I can obtain continuous measurements of coronary microvascular pressures and diameters. The specific goals of the project are to answer the following questions: (1) What is the distribution of resistance in the coronary circulation of the left ventricle? (2) What are the changes in the distribution of resistance in the left ventricular coronary circulation during autoregulation? (3) What are the effects of neural stimulation or norepinephrine infusion on the distribution of resistance in the coronary circulation of the left ventricle? Project II, Adrenergic Control Mechanisms in the Coronary Circulation, tests the hypothesis that adrenergic vasoconstriction is augmented in models of coronary pathophysiology. This project entails measurement of myocardial perfusion with microspheres to innervated and sympathectomized regions of the left ventricle in conscious dogs. To accomplish this the posterior region of the left ventricular free wall is sympathectomized with phenol whereas the innervation of the anterior left ventricle remains intact. The specific goals of this project are to answer the following questions: (1) In the presence of a proximal coronary stenosis, what are the effects of the coronary sympathetic nerves on limiting coronary blood flow? (2) Are adrenergic coronary vasoconstrictor influences augmented in the coronary vascular system in animals with hypertension and left ventricular hypertrophy? (3) During exercise is perfusion to the right ventricle under greater sympathetic control than that to the left ventricle?

The myocardium is a continually working aerobic organ. It is essential for normal myocardial function that a continual supply of oxygen is maintained to ensure a proper balance between myocardial oxygen supply and myocardial oxygen demands. The goal of this proposal is to investigate neural and microcirculatory mechanisms involved in regulating oxygen balance in the myocardium. Project I, Coronary Microvascular Dynamics, examines the hypothesis that the coronary microcirculation will exhibit a hierarchy of control to common stimuli. To accomplish Project I, I use a computer-controlled system which compensates for cardiac motion to enable microvascular pressure and diameter measurements in the beating heart. This computer-controlled device can move a micro-pipette in three dimensions in concert with a moving microvessel on a beating heart. Furthermore, using stroboscopic illumination (1 flash per heart cycle) microvessels are always in focus and "appear" stationary. With this approach in the beating heart, there is little trauma to the coronary system and I can obtain continuous measurements of coronary microvascular pressures and diameters. The specific goals of the project are to answer the following questions: (1) What is the distribution of resistance in the coronary circulation of the left ventricle? (2) What are the changes in the distribution of resistance in the left ventricular coronary circulation during autoregulation? (3) What are the effects of neural stimulation or norepinephrine infusion on the distribution of resistance in the coronary circulation of the left ventricle? Project II, Adrenergic Control Mechanisms in the Coronary Circulation, tests the hypothesis that adrenergic vasoconstriction is augmented in models of coronary pathophysiology. This project entails measurement of myocardial perfusion with microspheres to innervated and sympathectomized regions of the left ventricle in conscious dogs. To accomplish this the posterior region of the left ventricular free wall is sympathectomized with phenol whereas the innervation of the anterior left ventricle remains intact. The specific goals of this project are to answer the following questions: (1) In the presence of a proximal coronary stenosis, what are the effects of the coronary sympathetic nerves on limiting coronary blood flow? (2) Are adrenergic coronary vasoconstrictor influences augmented in the coronary vascular system in animals with hypertension and left ventricular hypertrophy? (3) During exercise is perfusion to the right ventricle under greater sympathetic control than that to the left ventricle?

The delivery of oxygen and nutrients to the myocardium is dependent on the regulation of tone in coronary resistance vessels. Another important aspect of nutrient delivery to the myocardial tissue is blood-tissue exchange, which is regulated by many factors including permeability of exchange vessels The primary goal of this grant is to further elucidate the regulation of coronary microvascular tone and solute exchange, and to delineate underlying mechanisms of the regulatory factors. We propose to test the following hypotheses: 1) the mechanism of flow-dependent dilation involves transduction of shear stress through integrins to activate specific enzymes in endothelial cells; 2) shear stress-induced vasodilation occurs in the microcirculation of the beating heart; 3) shear stress-induced release of nitric oxide blunts the vasoconstrictor action of pressor substances in the coronary microcirculation; 4) ischemic coronary vasodilation is partially caused by carbon monoxide; 5) vasoactive substances released or carried by venules can diffuse to adjacent arterioles and induce alterations in tone; 6) cyclic GMP increases permeability through stimulation of specific kinases; 7) integrins are involved in the signaling of flow-induced increases in permeability; 8) Beta-adrenergic activation antagonizes the increase in permeability induced by NO through activation of specific phosphodiesterases. To test these hypotheses we will utilize methodologies that enable studies of the microcirculation of the beating heart and of isolated resistance and exchange vessels. Studies of the beating heart will include interventions designed to examine the preferential microvascular location(s) of shear stress-induced vasodilation under conditions of high viscosity, constant flow perfusion. The role of nitric oxide as a modulator of the pressor action of vasoconstrictors will be delineated by assessing action of the agonists before and after inhibition of nitric oxide. We will test if carbon monoxide produces ischemic coronary vasodilation by inhibiting the enzyme that induces its production. In isolated arterioles we will examine the signaling pathways involved in flow-dependent dilation by using inhibitors of tyrosine kinase, phospholipase C, and protein kinase C. Examination of pairs of isolated venules and arterioles will allow determination of shunting of vasoactive metabolites in the heart. Finally we will elucidate the mechanism of flow-induced increases in permeability of isolated venules by performing studies similar to those discussed for arterioles. We will also probe mechanisms by which cGMP influences permeability through its actions on the cytoskeleton, and the interactions between catecholamine and factors that increase permeability, by using specific inhibitors of phosphodiesterases that hydrolyze cGMP. The solutions to these hypotheses will provide unique knowledge concerning regulation of coronary microvascular regulation of coronary microvascular tone and solute exchange.

biomedical equipment purchase;

The purpose of this grant is to delineate the effects of sex hormones and gender on coronary vascular smooth muscle. We will determine if the sex hormones, 17beta-estradiol and dihydrotestosterone, influence reactivity of coronary arterioles and arteries, and also influence vascular smooth muscle proliferation. The experimental plan is to study five different groups of animals: intact males, intact females, neutered, neutered supplemented with 17beta-estradiol, and neutered supplemented with dihydrotestosterone. We will also determine if the effects of the sex hormones are influenced by diet, specifically comparing effects of a normal diet versus that of an atherosclerotic diet. We propose to test two major hypotheses. First, 17beta-estradiol exerts a "protective" role in large coronary arteries when compared to the effects of dihydrotestosterone. This hypothesis will be tested utilizing physiological studies of coronary artery reactivity, molecular studies of gene expression in the arterial wall following an angioplasty procedure (in situ hybridization), and studies of vascular cells in culture. In culture we plan to determine if the sex hormones modulate the proliferative response of vascular smooth muscle to various mitogens, or if the hormones influence endothelial production of substances, which also then modulate the proliferative response of vascular smooth muscle. The second general hypothesis that we propose to test is that testosterone will exert a "protective" role in coronary arterioles as opposed to the effects of estrogen. We propose that the protective effect of dihydrotestosterone will be exhibited by enhanced production of endothelium-derived nitric oxide and enhanced vasodilatory responses to endothelium-dependent stimuli with lessened responses to vasoconstrictors. In contrast, we also speculate that 17beta-estradiol will exert effects opposite to that of testosterone. Our experimental plan is to compare effects of the hormone-treated animals to that of the neutered group and intact males and females, which will mimic results for the dihydrotestosterone - or 17beta-estradiol-treated animals, respectively. The results of these studies would provide novel information regarding the effects of sex hormones on reactivity of coronary arteries, and effects of the hormones on the development of atherosclerotic lesions in the coronary arteries. These results should offer insight into mechanisms by which the incidence of coronary artery disease is greater in men than in pre-menopausal women, and why women more frequently suffer from Syndrome X (microvascular spasm in the heart) than men.

DESCRIPTION: (Adapted from the Investigator's Abstract) The goal of this proposal is to delineate the signals involved in coronary collateral growth or non-budding angiogenesis. The general thesis is that collateral growth is critically dependent on the time-dependent expression of specific growth factors and their receptors. The PI hypothesizes that: At the onset of ischemia: a. angiogenic factors regulated by hypoxia (insulin-like growth factor-2 [IGF-2] and vascular endothelial growth factor [VEGF] and the cellular redox state (transforming growth factor beta-1 [TGF-beta 1] and basic fibroblast growth factor [beta FGF]) are expressed. b. receptors for these angiogenic factors will be expressed (IGF-II receptor, flk-1 receptor for VEGF, TGF receptor Types I and II, FGF-receptor-1). During angiogenesis, and establishment of sufficient perfusion to ameliorate ischemia in the occluded territory: a. angiogenic factors regulated by shear stress (platelet derived growth factor-B [PDGF-B] and its receptors (PDGF-alpha and -beta receptor), and endothelial nitric oxide synthase [eNOS]) will be expressed. b. angiogenic factors and their receptors regulated by hypoxia and redox state will have their expression return toward baseline. After achievement of normalized perfusion and coronary vasodilator reserve to the occluded territory: a. proteins and genes that are markers of endothelial and vascular smooth muscle cell quiescence (gax, gas-1) will be expressed. b. growth factors and their receptors associated with angiogenesis and/or cellular proliferation will have their expression return to baseline. Coronary angiogenesis will be induced in chronically-instrumented dogs by 2 minute repetitive coronary artery occlusions: 8 occlusions/day, 7 days/week. Angiogenesis will be evaluated at several different times: 1. Early response: 2, 24, and 48 hours after initiation of the occlusions; 2. Rapid growth phase: 7, 10, and 14 days after initiation of the occlusions; and 3. Maintenance phase: 18 and 21 days after initiation of the occlusions when collateral conductance enables flow to reach levels comparable that in the normal zone; and 4. Final growth phase: 28 and 35 days after initiation of occlusions when collateral conductance allows for vasodilator reserve and growth ceases. Sham control groups will be studied in addition to the animals receiving the repetitive ischemic stimuli. Collateral conductance will be evaluated by measurements of myocardial function in the occluded territory, collateral flow (radioactive microspheres), and diminution of reactive hyperemic responses following the occlusions. Mitogens associated with angiogenesis will be assayed from myocardial interstitial dialysate of the normal and ischemic vascular regions (cell proliferation, tube formation, Western analysis). Northern analysis, and reverse transcriptase polymerase chain reaction will be used to evaluate expression of the specific transcripts in the myocardium and vasculature. This approach which integrates a range of techniques from the molecular to physiological levels will facilitate a complete understanding of the signals associated with coronary angiogenesis and the impact of hypertension on these responses.

The long-term goal of this project is to advance our understanding of the mechanisms that are involved in stroke. Stroke is a leading cause of brain disease in the United States, and involves abnormal regulation of vascular tone during ischemia and subsequent reperfusion. It is known that free radicals and reactive oxidants, including nitric oxide (NO) and superoxide (O2-), are produced during ischemia/reperfusion. Peroxynitrite (ONOO-) is a highly reactive oxidant formed by the reaction of NO and O2-. Preliminary evidence from our own laboratory and from others indicates that ONOO- may be a key modulator of vascular tone. In the proposed experiments, we will use freshly isolated cells and vessels from the rat brain Circle of Willis arteries. Cell-imaging amd quantitative videomicroscopy will be used to define the contraction responses of single cells and small arteries to ONOO. Ion channels are major determinants of vascular tone through their influence on resting membrane potential in endothelial and vascular smooth muscle cells. Our preliminary data indicate that ONOO-activates a cation current in vascular endothelial cells and inhibits calcium-activated K+ current in cerebrovascular smooth muscle cells. The patch-clamp technique will be used to (1) identify the endothelial cell ion channels that are activated by ONOO- and that are responsible for membrane depolarization, and (2) define the effect of ONOO- on K+ channels in vascular smooth muscle cells. Fluxes of O2 and NO will be varied and titrated against each other to define the underlying chemical basis for the electrical and contractile responses to ONOO. We will test whether the modulation of channel activity and cell contraction by ONOO- is thiol-dependent and involves glutathione. By bringing together free radical chemistry, ion channel electrophysiology and whole-vessel contractile responses, these studies are expected to reveal important mechanistic information regarding the effects of NO, O2- and ONOO on membrane potential, cellular-ionic signaling and vascular tone within the cerebral vasculature. This information will provide new insight into the regulation of vascular homeostasis in the brain and could help identify new measures to reduce the impact of stroke.

The SCOR in Ischemic Heart Disease in Blacks at the Medical College of Wisconsin represents a multi-disciplinary proposal that focuses on solving basic (physiological, molecular, and genetic) and clinical (physiological and genetic) problems underlying this pathology. We have focused our efforts on diabetic heart disease, because this disease is of near epidemic proportions in the Black population, especially those with proven coronary artery disease, but they may also abrogate collateral growth in patients and augment the deleterious consequences of myocardial ischemia. Our program is composed of 4 Projects and 2 Core Facilities. Our strategy is to use experimental approaches that deviate from traditional methods to investigate the problems and to utilize corroborative experimental tools to test hypotheses at one level (documentation of physiological or pathophysiological responses) and then elucidate fundamental mechanisms at cellular, ion challenge molecular, and/or genetic levels. Projects 1 and 2 study mechanisms of coronary collateralization. Project 1, "Integrative Analysis of Coronary Adaptations to Ischemia" will delineate the temporal sequence of expression of growth factors, and their respective receptors from the initiation to the final stages of collateralization in dogs. This project also elucidate the mechanisms by which diabetes compromises coronary collateral growth. Project 2 (Genetic Basis of Coronary Artery Disease and Coronary Collateralization) will define the genetic basis of coronary artery disease and coronary collateralization in Black and White patients. This project will also examine the familial transmission of alleles involved in coronary artery disease. In Project 3, "Oxidant Stress and Human Coronary Microvascular Function," the effects of diabetes on vascular function, vessels on vascular cells obtained from human hearts of Black and White patients. We will also utilize molecular strains of rats: Brown Norway, which are resistant to ischemia and Dah, which are sensitive to ischemia and Dahl, which are sensitive to ischemia (Project 4: "Molecular Genetics of Cardioprotection"). The Dahl strain is also insulin resistant and demonstrates salt sensitive hypertension. The two cores will be involved as repositories for data and administration, and data analysis. The Administration Core will contain a centralized file server for all results and will be accessible to all investigators. The Biostatistics Core will analyze all experimental results, and perform the association analyses of phenotype with genotype. We believe the science generated by this program will provide the template for rational pharmacological therapies to treat ischemic heart disease and will elucidate mechanisms contributing to ischemic heart disease in Blacks.

DESCRIPTION (provided by applicant): Results from our and other laboratories demonstrate that a critical amount of ROS and a redox state within a certain boundary is critical for coronary collateral growth; however, oxidative stress, i.e., a shift in the redox state to a more oxidative environment, impairs coronary collateral growth. Previous investigations fall short of ascertaining the cell type (or types), in which alterations of redox state matter, and where redox signaling is critical. The overarching goal of this proposal is to determine the cell type or types in the heart responsible for redox signaling in the growth of the coronary collateral circulation in response to repetitive ischemia. A corollary to this aim is that we will also determine the cell type or types in which oxidative stress confers negative influences on coronary collateral growth. To solve these problems we propose the following specific aims: Aim 1. Determine in which cell type (or types) does oxidative stress corrupt coronary collateral growth. We will induce oxidative stress in the coronary endothelium, smooth muscle cells and cardiac myocytes using the cell-specific promoters VE-Cadherin, SM22, and cardiac myosin heavy chain (CMHC), respectively. Cells will be transfected with a plasmid (using in vivo electroporation) or transduced with an adenovirus expressing an iNOS mutant (E371A) that does not bind arginine, and therefore, only produces O2. Aim 2. Determine in which cell type (or types) the redox sensitive p38 MAPK is critical for coronary collateral growth. We will transfect or transduce coronary endothelium, smooth muscle cells and cardiac myocytes using cell-specific promoters described for Aim 1 using a vector expressing a dominant/negative p38 MAPK (DNp38). After determining the particular cell type in the heart most sensitive to the effects of oxidative stress and redox signaling in coronary collateral growth, we will extend our findings to an animal model of vascular pathology (the JCR rat: a model of the metabolic syndrome), which demonstrates poor coronary collateral growth. Specifically in the final two aims we will: Aim 3. Determine the cell type where reducing oxidative stress by over expressing Nrf2 in the JCR rat (a model of reduced coronary collateral growth and oxidative stress) will restore collateral growth. Aim 4. Determine the cell type where expression of a constitutively active p38 MAP kinase will restore collateral growth in the JCR rat. The proposed studies employ a multifaceted approach to solving cell-specific signaling in vivo by employing techniques to determine cell specific changes in protein expression, thiol oxidation and ROS production and ultimately linking these measurements to coronary collateral growth. These studies will provide insight into the cell-specific locations where ROS and redox signaling modulate coronary collateral growth. PUBLIC HEALTH RELEVANCE: Ischemic heart disease (IHD) continues to be one of the leading causes of death in the United States, and represents a major cost in the US healthcare system. The presence of a well developed collateral circulation in the heart has a tremendous impact on the morbidity and mortality of IHD. Patients with well developed collaterals show a lower incidence of sudden death, have smaller infarcts in the event of an occlusion, and demonstrate a better prognosis than patients with poor collaterals (low conductance or evidence of collateral growth). It is also worth noting that about 40% of patients with IHD demonstrate very little to no coronary collaterals. It is also recognized that many risk factors for IHD, e.g., hypertension, obesity, dyslipidemia, which also produce oxidative stress, have a negative influence on coronary collateral growth. However, the cell type where the corruption occurs, e.g., does oxidative stress corrupt the production of growth factors by cardiac myocytes, or does it corrupt endothelial responsiveness to growth factors, remains unknown. The goal of this proposal is to delineate the cell type in the heart where redox signaling and oxidative stress matter for coronary collateral growth. The ultimate goal of this project is to provide the foundation for more directed therapies to stimulate the growth of coronary collaterals in patients with IHD.

DESCRIPTION (provided by applicant): This application addresses the broad Challenge Area, (11) Regenerative Medicine, and specific Challenge Topic, "11-HL-101, Develop cell-based therapies for cardiovascular, lung, and blood diseases." Cell-based therapies have spawned the emerging discipline of regenerative medicine and offer new paradigms for advancing patient care. Despite this potential, to date the outcomes of cell- based therapies to treat myocardial infarction are controversial with some positive, some negative, and even some detrimental results. We opine that treatment of acute myocardial infarction with stem cells may not be the most effective regenerative therapy rather stimulation of coronary collateral growth is the most likely strategy to produce an immediate benefit in the treatment of ischemic heart disease. The growth of coronary collaterals could prevent sudden death and myocardial infarction. Moreover, stimulation of collateral growth is a simpler goal than rebuilding myocardium in an infarct zone, where a complete arterial, capillary, and venous vasculature must be rebuilt along with myocytes and a conductible system working as a functional and electrical synctium. Notwithstanding this issue, in some regards the clinical studies may be premature insofar as several details, e.g., the most promising target patient population, the best cell type and number of cells to use, the optimal methods and timing of delivery have not yet been optimized. Within the context of this Challenge Area, in this application we propose to expand on practical aspects of stem cell therapies by determining which type of stem cell best promotes coronary collateral growth in the heart, the conditions in the heart that are conducive to promote collateral growth, and finally, a method to render stem cells resistant to oxidative stress to promote their survival and enhance their biological effect in models of vascular disease with existing oxidative stress. Specifically we propose 3 aims: 1) To determine which type of adult stem cell best stimulates coronary collateral growth. 2) To determine the conditions of the heart that are conducive for stem cell stimulation of collateral growth. 3) To determine if stem cells selected for resistance to oxidative stress will better stimulate coronary collateral growth in vascular disease than non-selected cells. These aims are built upon an interdisciplinary team with experience ranging from virology, study of embryonic stem cells, molecular biology, biochemistry, physiology, and vascular biology. PUBLIC HEALTH RELEVANCE: The outcomes of cell-based therapies to treat myocardial infarction are controversial with some positive, some negative, and even some detrimental results. However, we opine that treatment of acute myocardial infarction with stem cells may not be the most effective use for regenerative therapies, rather stimulation of coronary collateral growth is likely the most likely strategy to produce an immediate benefit in the treatment of ischemic heart disease using regenerative therapies. Our goals are to determine which adult stem cell, and under what specific conditions, best stimulates coronary collateral growth in the heart.

DESCRIPTION (provided by applicant): The overarching aim of this proposal is to elucidate vascular effectors that transduce metabolic signals that enable the connection of flow to cardiac metabolism-metabolic dilation. The heart is dependent on metabolic dilation for aerobic energy production because anaerobic reserve is virtually non-existent; however, the effectors responsible for coupling flow to metabolism in the heart remain unidentified. The matching of flow to metabolism is important and may play a role in microvascular diseases in the heart. We have suggested that Kv channels transduce the H2O2 metabolic signal into redox- mediated coronary metabolic vasodilation. Because certain members of the Kv1 family of channels are redox sensitive (e.g., Kv1.2, 1.3 and 1.5), our first goal will determine, which redox sensing Kv channels transduce metabolic signals to flow in the heart. This aim will be tested using loss and gain of function approaches. Loss of function will use mice null for Kv1.5 and 1.3 channels, and heterozygous null for Kv1.2 channels (Kv1.2-/- is lethal), and gain of function will study of expression of the specific ion channel using a smooth muscle specific Tet On system driving expression of the Kv channel. We have found that metabolic dilation in the diabetic db/db mouse is impaired and that expression of Kv1.2, 1.3, and 1.5 channels is substantially decreased in arteries of these mice. Our goal in the second aim is to perform gain of function studies in db/db mice by expressing Kv1.5, 1.2 and/or Kv1.3 channels using the Tet inducible system in smooth muscle. Our overall strategy is to measure the relationship between cardiac work, and myocardial blood flow and tissue oxygenation along with evaluating measures of cardiac function and myocardial ischemia. We also will perform in vitro studies to determine the production of vasoactive metabolites from cardiac myocytes, vascular reactivity of isolated arterioles and electrophysiological parameters in smooth muscle. This integrated approach-from electrophysiology to in vivo flow regulation should enable answers regarding basic coronary physiology and flow regulation, as well as potential therapies for diabetic cardiomyopathy.

The goal of this proposal is to test the hypothesis that impaired regulation of the coronary microcirculation? more specifically, an inadequate connection between coronary blood flow and the metabolic needs of the myocardium?underlies the development of some types of heart failure (HF). The sequence we propose is that impaired coupling between coronary blood flow and cardiac work results in minute areas of hypoxia in cardiac myocytes, which induces apoptosis in small numbers of cardiac myocytes. Over time these minute areas of tissue death accumulate to an extent that cardiac function is compromised. Current treatments for heart failure are designed to reduce cardiac work, but not directly produce coronary vasodilation. None of these therapies stop or reverse the progression of the disease?progression is only slowed. We speculate that the reason these treatments do not stop or reverse the progression of the disease is that they are not targeting the causal problem of insufficient blood flow to the heart. We propose that by correcting the myocardial perfusion deficiencies in heart failure, we will stop and potentially reverse the progression of heart failure. We propose two aims. Aim 1 will determine if impaired regulation of myocardial blood flow, i.e., inadequate coupling of flow to metabolism, plays a critical role in the development of heart failure. In this aim we will measure myocardial perfusion, plasma BNP and cardiac troponin I, tissue oxygenation, cardiac metabolism and cardiac myocyte hypoxia (using hypoxia fate mapping) and myocyte apoptosis in the murine transaortic constriction model of heart failure. Measurements will be made at various points during the progression of the disease and in wild type mice and mice with an impairment in myocardial blood flow (Kv1.5 null mice). These measurements will enable precise evaluation of failure and also whether myocardial ischemia occurs during heart failure. Aim 2 will analyze if the progression of heart failure can be stopped or reversed by increasing blood flow to the heart. In this aim we will increase blood flow to the heart by increasing expression of Kv1.5 channels in smooth muscle or will administer a pharmacological vasodilator at varying times during the progression of the disease. We will compare these interventions to one of the current standards of care for heart failure (beta1-adrenergic antagonist). We will establish if increases in myocardial blood flow to the heart can stop, and/or reverse, the progression of heart failure resulting in a better outcome than the conventional therapy. This application builds upon the expertise of the Principal Investigator, and that of additional laboratories via subcontracts enabling corroborative, interdisciplinary measurements to definitely test whether subtle levels of myocardial ischemia lead to heart failure and if therapies that increase flow to the heart hold promise as a cure.

The purpose of this grant is to define the role of endogenous stem cells in coronary collateral growth. This problem is significant because patients with ischemic heart disease, who have well developed collaterals show a lower incidence of sudden death, have smaller infarcts in the event of an occlusion, and demonstrate a better prognosis than patients with poorly developed collaterals. Currently, there is no evidence (pro or con) that endogenous stem cells are even involved in coronary collateral growth. One goal of the present application is to rectify this deficiency by establishing if stem cells participate in coronary collateral growth and then the mechanisms underlying this effect. Within this context we propose three aims. In the first aim, we will determine the factors produced by the myocardium and coronary vasculature that induce homing and reprogramming of bone marrow stem cells. In this aim, we will define which factors that are produced by the heart and/or growing vasculature activate stem cell homing to the heart and coronary collateral vasculature. Studies will be performed to examine gain and loss of function of 4 key factors to establish their roles in this adaptive, important process. We also will determine if these factors also induce reprogramming of bone marrow in addition to their effects of homing. Reprogramming is used in this context to suggest that these factors will increase the population of sub-classes of bone marrow cells that are committed to a vascular differentiation program. In the second aim, we will determine if stem cells that are recruited to the heart or to the growing vasculature are essential for the process of collateral growth. We will first establish the sub-types of stem cells that home to the heart and coronary vasculature. Then will we study if depletion or enrichment of these cells (during the creation of the chimeric model) will blunt or magnify, respectively, coronary collateral growth. After we have established the type of stem cells that are critical for coronary collateral growth, our studies will focus on the third aim to delineate the fate of the stem cells in the heart by addressing the question: Do they engraft in collateral arteries and differentiate into smooth muscle or endothelium? The overarching goal of these studies is to fill the current void in our understanding about the role of endogenous stem cells in the growth of coronary collaterals. If coronary collateral growth could be stimulated it would theoretically obviate the need for a bypass as a collateral is ?nature?s bypass.? Our overall approach is to use the experience of diverse research team in many disciplines?physiology, vascular biology, in vivo imaging, biochemistry, molecular biology?to address this problem.