Cardiac Surgery Techniques to Treat Ventricular and Aortic Remodeling
Grant # U01HL088957 »
PI: Michael A. Acker »

We propose to study strategies for ameliorating adverse cardiovascular remodeling within the context of the Network for Cardiothoracic Surgical Interventions. This proposal consists of two separate prospective randomized studies:

1) "Prospective Randomized Evaluation of Mitral Valve Repair for Functional Mitral Insufficiency and Ventricular Dysfunction" (Dr. Joseph Woo, Co-investigator)
2) "Adjunct Delivery of Aortic Stent Graft in the Descending Thoracic Aorta in Acute Type A Dissection Repair Obliterates the Residual False Lumen and Prevents Aneurysmal Remodeling of the Distal Thoracoabdominal Aorta" (Dr. Joseph Bavaria, Co-investigator)

Each of the proposed studies addresses an important manifestation of adverse cardiovascular remodeling which currently confront cardiologists and cardiac surgeons on a routine basis. Both studies will be done in a multi-disciplinary fashion with full collaborative support from the Divisions of Cardiovascular Surgery, Cardiology, Vascular Surgery and Cardiac Anesthesiology.

ECM and Cell Cycle Control in Aortic Smooth Muscle Cells
Grant # P01HL062250 »
PI: Richard K. Assoian »

During atherogenesis, vascular smooth muscle cells are thought to undergo conversion from a contractile to a "synthetic" phenotype, acquiring the ability to proliferate. Our studies over the past several years have shown that the ECM and integrins have essential roles in activating the G1 phase cyclin-dependent kinases (cdks), the critical regulators of cell proliferation. New data described here shows that CD44, an adhesion receptor that binds to hyaluronic acid (HA), regulates integrin-dependent signaling events in aortic smooth muscle cells and fibroblasts, controlling both the actin cytoskeleton and G1 phase cell cycle progression. These results validate a long-standing assumption about the potential interaction between integrins and CD44. Genetic analysis in CD44-null mice suggest that the CD44-integrin interaction may explain, at least in part, the pro-atherosclerotic effect of CD44.

The Role of Sema4D / CD100 and its Receptors in Platelet Biology and Thrombosis
Grant # P50HL081012 »
PI: Lawrence Brass »

In addition to adhering to each other and to the vessel wall, platelets contribute to thrombotic events by releasing bioactive molecules such as ADP, TxA2 and CD40L. In studies that form the basis for this proposal, human and mouse platelets were found to express on their surface the class IV semaphorin, sema4D or CD 100, a protein best known for its role in B-cell/T-cell interactions. It was also found that activated platelets shed the exodomain of sema4D and the "sheddase" was identified as the TNFa cleaving enzyme, ADAM17. Based on these observations and preliminary studies on the effects of soluble sema4D on platelets, we have developed the following hypotheses: 1) sema4D, either as a soluble molecule or surfacebound, contributes to platelet activation by binding to receptors expressed on nearby platelets, 2) plateletderived sema4D can also affect cells other than platelets within the circulation and the vessel wall, and 3) plasma levels of soluble sema4D will increase when pathological platelet activation occurs.

Translational Studies in Heart Failure Gene Therapy
Grant # R01HL083078 »
PI: Charles R. Bridges »

This year, one million Americans will die of heart failure at a cost of 25 billion dollars. Yet, only two thousand Americans will receive heart transplants and even fewer will receive mechanical ventricular assist devices. Gene therapy is an important emerging technology with the potential to save thousands of lives. Correction of the heart failure phenotype has been convincingly demonstrated in transgenic mice, rabbit and rodent models of heart failure using the beta adreno-receptor kinase C-terminus (BARKct)) as a therapeutic transgene. Prior to our work, none of the delivery techniques utilized to date has been clinically translatable and had high global myocyte transduction efficiency. These observations galvanize our central hypothesis: the rate-limiter in the quest for clinically relevant heart failure gene therapy is the successful achievement of global vector-mediated gene delivery to a significant percentage of cardiac myocytes in situ in a translational animal model.

Transcriptional Modules in Human Heart Failure
Grant # R21HL092379 »
PI: Thomas P. Cappola »

Heart failure has become the most common reason for adult hospitalization in the industrialized world. Basic research has shown that pathologic stresses promote heart failure via activation of cardiac transcription factors (TFs). These TFs interact with each other and with co-activators to form "transcriptional modules" (TMs) that alter cardiac gene expression to cause myocyte hypertrophy and failure. In light of their critical role in heart failure pathogenesis, TMs have been the subject of intensive research in animal models over the past decade. By contrast, the role of TMs in human heart failure remains largely unexplored because of limited methods for studying TFs in human subjects. We have piloted computational approaches that enable assessment of TF function in the failing human heart by integrating cardiac gene expression data from microarray experiments with readily available genome sequence data. However, these do not sufficiently address the complexity of the underlying biology, and more rigorous methods are needed. The purpose of this exploratory/developmental research proposal is to determine transcriptional modules (TMs) associated with human heart failure using a refined computational approach and to experimentally validate the most promising TM using standard in vitro techniques.

Genomics of Myocardial Transcription Factors in Cardiac Remodeling
Grant # R01HL088577 »
PI: Thomas P. Cappola »

Cardiac remodeling is a central feature of human heart failure and shows substantial variation in human subjects. A decade of research in murine models and research in humans performed by the Principal Investigator show that a discreet set of cardiac transcription factors integrate stress signals to cause cardiac remodeling. Our central hypothesis is that common genetic variation in a core set of cardiac transcription factors (MEF2, NKX, NFAT, GATA, FOX) is in large part responsible for the variable course of cardiac remodeling in humans. We will address this hypothesis by performing SNP- and haplotype-based association studies of candidate transcription factors in two existing cohort studies that capture the common phenotypes of remodeling encountered in clinical practice. Aim 1 tests the hypothesis that variation in candidate transcription factors is associated with concentric cardiac remodeling in the Chronic Renal Insufficiency Cohort study (CRIC), a large cohort with a high prevalence of concentric remodeling. Aim 2 similar analyses is performed in the Penn Heart Failure Study, a large single-center cohort initiated by the applicant with a high prevalence of eccentric remodeling. In Aim 3 we collaborate with Dr. Edward Morrisey, an expert molecular biologist, to determine the mechanisms by which the observed risk variants alter transcription factor function using in vitro techniques.

Inflammatory Response to Sleep Apnea in Obese Subjects
Grant # R01HL080076 »
PI: Julio Chirinos »

Obstructive sleep apnea (OSA) is associated with an increased risk for cardiovascular events. Whether the mechanism for this association is directly attributable to pro-atherosclerotic effects of OSA or primarily related to obesity is not known. Obesity is common in patients with OSA, and is itself associated with an inflammatory state and insulin resistance. In individuals with both obesity and OSA, the repetitive hypoxia associated with OSA may promote inflammation and insulin resistance. Our primary aim is to test the hypothesis that OSA plays a primary role in the inflammatory state of patients with obesity and moderate-severe OSA, and that therefore the elimination of apnea by continuous positive airway pressure (CPAP) therapy in these patients will more greatly reduce inflammation (measured by C-reactive protein or CRP) than weight loss alone, and that combining these two therapies will have additive effects on reducing inflammation.

Mechanisms of Cardiopulmonary Gene Transfer
Grant # R01HL066565 »
PI: Scott L. Diamond »

Achieving the full potential of intraarterial, intravenous, or pulmonary gene therapy requires quantitative bioengineering analysis of extracellular and intracellular barriers. The central focus of this proposal is to achieve high expression of transgenes delivered to cardiovascular and cardiopulmonary cells such as arterial endothelium and airway epithelium. We will bioengineer and investigate sterol, peptide, and polymer- based transfection vehicles for their ability to enhance gene transfer.

Genetic Determinants of Hypertensive Heart Disease in Chronic Renal Insufficiency
Grant # R01HL091663 »
PI: Daniel L. Dries »

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in patients with chromic renal insufficiency (CRI). CVD remains the leading cause of death in dialysis patients and in patients undergoing renal transplantation. There is accumulating evidence that the increase in CVD burden is present in the pre-dialysis population. Left-ventricular hypertrophy (LVH) is perhaps the best studied marker of CVD in the chronic kidney disease (CKD) population. The central hypothesis of this project is that genetic variation in corin and related NPS pathway genes contributes to the progression of hypertensive heart disease in the setting of chronic renal insufficiency (CRI). The candidate genes of interest are: corin, furin, ANP, BNP, CNP, NPR-A, NPR-B, NPR-C, PKG-I, and NEP. We will test these hypotheses in the Chronic Renal Insufficiency Cohort (CRIC): an NIH-sponsored, multi-center, prospective cohort study of cardiovascular disease in subjects (N=3000) with chronic renal insufficiency.

Analysis of a Novel Homeobox Gene in Cardiovascular Development
Grant # R01HL071546 »
PI: Jonathan A. Epstein »

This project focuses on the causes of congestive heart failure, and on finding new therapeutic targets. Heart failure is a leading cause of death and disability in the United States, and the incidence is rising. We have found that HDAC inhibitors, which are drugs currently in clinical trials for cancer therapy, have beneficial effects in terms of heart failure prevention in animal models. This project explores the possibility that an enzyme called HDAC2, expressed in the heart, is the molecular target for HDAC inhibitors, and that it functions by regulating a novel phosphatase in the heart called INPP5F. We hope to determine if HDAC2 and INPP5F represent new targets for the development of specific therapies for heart disease.

Pax3 Regulation of Cardiac Conotruncal Development
Grant # R01HL061475 »
PI: Jonathan A. Epstein »

Neural crest plays important roles in cardiovascular development since derivatives contribute to the outflow tract of the heart and to the great vessels. Mutations in neural crest genes, including Pax3, lead to congenital heart disease. This competitive renewal application builds upon significant data and resources that we have developed during the first granting cycle in order to elucidate the molecular pathways responsible for regulating Pax3 expression in neural crest.

Molecular Basis of Vascular Heterogenity and Function
Grant # P01HL075215 »
PI: Jonathan A. Epstein »

The overall goal of this program project proposal is to elucidate the signaling and transcriptional pathways involved in specifying unique functions of sub-populations of vascular smooth muscle and endothelial cells. Recent molecular data confirms prior clinical observations that different parts of the vasculature are inherently distinct. For instance, pulmonary vascular smooth muscle and arterial smooth muscle react differently to disease states and pharmacological intervention. Even within the arterial tree, smooth muscle arises from distinct precursors variably affecting congenital vascular disorders. Vascular and lymphatic endothelial cells are specified in such a way as to express distinct molecular markers and adopt divergent functions. Despite these observations, little is known about how these distinctions are orchestrated or how they contribute to functional differences within the vasculature. The overlapping approaches and techniques used in this program will be relevant for understanding congenital and acquired vascular disease and for development of interventions that target specific populations of vascular cells.

Strain Induced Myopathy in Post Infarction Heart Failure
Grant # R01HL076560 »
PI: Joseph H. Gorman »

Five million Americans suffer from congestive heart failure (CHF). Despite 30 years of advances in revascularization techniques coronary disease still accounts for 70% of all cases of CHF. Once the diagnosis is made the 5-year mortality is 50%, regardless of treatment. We hypothesize that following acute myocardial infarction, infarct expansion occurs, which stretches adjacent perfused myocardium. This stretching induces reactive oxygen species (ROS) mediated myocyte apoptosis, which results in progressive non-ischemic myopathic process that recruits additional contiguous, fully perfused, remodeled myocardium to cause dilated cardiomyopathy, CHF and death. This project uses sonomicrometry array localization (SAL) to study a sheep model of post infarction left ventricular (LV) remodeling.

Regulation of vascular development and function by LKLF
Grant # R01HL081654 »
PI: Mark L. Kahn »

During vascular development a primitive vascular plexus differentiates into a diverse network of arteries, veins and capillaries. Concurrent with vascular differentiation is the onset of circulating blood pumped by the heart and the generation of hemodynamic forces that vary with vessel type. Although these processes are temporally linked, the extent to which vascular differentiation and genetic responses to hemodynamic forces are connected is unknown. Lung Kruppel-Like Factor (LKLF, KLF2) is a transcription factor required for cardiovascular development. LKLF mRNA expression in endothelial cells is rapidly induced by pulsatile shear stress and during development and in adult life LKLF is expressed in endothelial cells in proportion to their predicted exposure to fluid shear forces. Although LKLF expression is restricted to the endothelium, LKLF-deficient animals experience non-endothelial cardiovascular defects. Understanding the mechanism by which LKLF regulates genetic responses in the developing and mature vasculature is expected to provide new insights into the pathogenesis and treatment of human vascular diseases.

Role of SYK in Lymphatic Vascular Development
Grant # P01HL075215 »
PI: Mark L. Kahn »

We have recently identified a novel signaling pathway containing the tyrosine kinase Syk and the adaptor SLP-76 that is required for separation of blood and lymphatic vessels during lymphatic vascular development. Loss of this signaling pathway in mice results in blood-filled lymphatics and the creation of arterio-venous shunts mediated by aberrant lymphatic connections. How Syk signaling regulates separation of blood and lymphatic vessels is unknown and is the focus of this proposal. During lymphatic vascular development Syk is expressed exclusively in hematopoietic cells and reconstitution of lethally irradiated wild-type mice with Syk-deficient fetal liver is sufficient to confer the vascular phenotype, suggesting that Syk signaling may influence vascular development indirectly through circulating cells. These studies will determine if Syk signaling defines a pathway required by hematopoietic progenitors to contribute to vascular development in the embryo and vascular repair in the adult. Identification of such a pathway has broad implications for our understanding of vascular development and for angiogenic therapies utilizing hematopioetic endothelial precursors.

Regulation of Angiogenesis by SLP-76 Signaling
Grant # R01HL072798 »
PI: Mark L. Kahn »

Vascular development consists of two phases: vasculogenic creation of vessels by endothelial progenitor cells and subsequent angiogenic remodeling of existing vessels. Mice lacking the hematopoietic signaling protein Slp-76 develop a vascular phenotype in which nascent lymphatic vessels connect to preexisting blood vessels. Our studies of the cell type responsible for this vascular phenotype suggest that Slp-76 is required in hematopoietic endothelial progenitor cells and not in mature endothelial cells. Remarkably, blood-lymphatic connections are corrected during later lymphatic remodeling, leaving surviving animals with arterio-venous shunts in which mesenteric lymphatics carry blood. These congenital lymphatics subsequently lose lymphatic identity, suggesting that fluid flow forces are sufficient for the molecular reprogramming of lymphatic vessel identity in vivo.

Mechanisms of Improved Diastolic Function in Human Heart
Grant # R01AG017022 »
PI: Kenneth Margulies »

As an endpoint for many different types of cardiovascular disease, heart failure (HF) is a leading cause of mortality and morbidity in the U.S. Though some patients with HF have intact systolic function, virtually all patients with HF have abnormal diastolic function and an impaired ability to increase cardiac performance in response to physiologic stress, including exercise. During exercise, myocardial responses to increased heart rate and adrenergic stimulation normally involve augmentation of cardiac filling (requiring relaxation reserve) and enhanced ejection (requiring contractility reserve). At the cellular level, relaxation reserve, the focus of this application, requires faster decay of the intracellular calcium (Ca) transient and a decrease in myofilament Ca sensitivity. Ordinarily, both processes are enhanced by beta- adrenergic stimulation triggering PKA-mediated phosphorylation of key Ca regulatory and myofilament proteins. However, both relaxation reserved and adrenergic modulation of relaxation reserve are abnormal in failing hearts. Recognizing that Ca cycling dynamics are themselves abnormal in failing myocardium, the broad objective of the proposed studies is to examine beta-adrenergic/PKA-mediated modulation of relaxation reserve in failing human hearts in a manner that accounts for the defects in Ca cycling present in these hearts.

Regulation of Airway Morphogenesis and Differentiation by Wnt Signaling
Grant # R01HL087825 »
PI: Edward E. Morrisey »

The lung airways form through a reiterative process of branching morphogenesis with concomitant cellular differentiation that is regulated, in part, through the activity of multiple signal transduction pathways. Accumulating evidence points to a role for both positive and negative signals from these pathways, which are regulated in a spatial and temporal manner during lung morphogenesis. The action of these pathways results in the distinct proximal-distal patterning observed in the lung airways, which is essential for the proper differentiation of airway epithelia into the active gas exchange interface in mammals. We have recently demonstrated a critical role for the Wnt signaling pathway in lung airway development and differentiation. Our studies show that inactivation of Wnt7b results in severe lung hypoplasia resulting from inhibited airway branching as well as defects in mesenchymal proliferation and type I cell differentiation.

Wnt Signaling and Lung Vascular Development
Grant # P01HL075215 »
PI: Edward E. Morrisey »

The lung arises from the foregut endoderm as an epithelial bud surrounded by mesodermal mesenchyme. Lung mesenchyme gives rise to the various lineages of pulmonary smooth muscle, including vascular smooth muscle cells (VSMCs) which are essential for blood vessel integrity. However, little is known of the molecular signaling processes that are involved in the specification and/or differentiation of VSMCs in the lung, although paracrine signaling from the endoderm has been shown to play an important rote. Proper development of the pulmonary vasculature is important in the development of the lung as a functional organ and defects in this process can lead to several human diseases including pulmonary hypertension. We have shown that inactivation of the Wnt7b gene, which is expressed in the developing airway epithelium, results in loss of vascular smooth muscle integrity in the lung, leading to perinatal hemorrhage and death. Since Wnt7b expression in the lung decreases in late gestation, we hypothesize that Wnt7b is required for proper development of VSMCs from lung mesenchyme through epithelial-mesenchymal signaling occurring early in lung development. In support of this hypothesis, we have found that the winged-helix transcription factor Foxf2, which is expressed in lung mesenchyme, is specifically down regulated in the lungs of Wnt7b/lacz-/- embryos. These results suggest that vascular smooth muscle development and integrity requires Wnt7b signaling, possibly by regulating genes such as Foxf2, which are required for proper development of VSCMs from lung mesenchyme. The goal of this proposal is to characterize the molecular mechanisms underlying Wnt7b regulation of VSMC development in the lung by addressing three questions: 1) Does Wnt7b act on definitive VSMCs or their precursors during lung vascular development?, 2) Does Wnt7b signal via canonical or non-canonical Wnt pathways in lung VMSC development and what are the global roles for these pathways in this process?, and 3) Wh at are the down-stream effector pathways that Wnt7b influences to regulate VSMC differentiation and development? We expect that the results of these studies will lead to a better understanding of the mechanisms underlying vascular heterogeneity and, in particular, how Writ signaling regulates these developmental events.

The Role of GATA6 in Lung Development
Grant # R01HL064632 »
PI: Edward E. Morrisey »

Transcription factors are crucial in both directing and responding to developmental cues during organogenesis. The GATA family of zinc finger transcription factors are critical regulators of cell lineage specific gene expression and development. Previous studies from our laboratory have demonstrated that GATA6 regulates several aspects of lung development and gene expression including airway epithelial cell differentiation, surfactant protein expression, and branching morphogenesis. Transgenic inhibition of GATA6 activity in mice results in decreased SP-B and SP-C expression as well as decreased alveolar type 1 cell differentiation. Lungs from these mice also display dilated distal airspaces, due to decreased branching morphogenesis. Moreover, we have demonstrated that GATA6 binds to Nkx2.1 and synergistically regulates lung specific gene expression. Similarities between the down-stream targets of Nkx2.1 and GATA6 (i.e. SP-B and SP-C) have lead us to hypothesize that regulation of lung epithelial gene transcription by GATA6 occurs, at least in part, through protein-protein interactions with other transcriptional regulators, including Nkx2.1. Together, these protein-protein interactions likely regulate specific target genes in the lung epithelium that are important for cell differentiation and development, in a temporal specific manner. To characterize the importance of GATA6-Nkx2.1 interactions and the temporal requirement of GATA6 expression during lung development, we propose to 1) Define the functional interaction between GATA6 and Nkx2.1 on target promoters and during lung development, 2) Define the minimal peptide sequence in GATA6 that mediates GATA6 and Nkx2.1 interactions and determine its importance in vitro, and 3) Determine the temporal requirement of GATA6 in the lung epithelium using a floxed GATA6 allele and a temporally inducible transgenic cre mouse line.

Forkhead Repressors and Lung Development
Grant # R01HL071589 »
PI: Edward E. Morrisey »

Lung epithelial development is transcriptionally controlled through both positive and negative regulators. Amongst these regulators, the forkhead/winged helix family or Fox family of DMA binding proteins play a central role. Our previous studies have demonstrated that Foxp1/2/4 are potent transcriptional repressors of lung gene transcription and each gene is expressed in overlapping patterns in lung epithelia. We have shown that mouse knock-out models of each of these genes demonstrate unique roles in lung, cardiac, and neural development. In the lung, Foxp2 regulates postnatal alveolarization in part through direct regulation of the alveolar epithelial type 1 cell (AEC-1) gene T1 alpha. In addition to their individual roles in development, recent evidence from our laboratory has demonstrated that Foxp1 and Foxp2 regulate lung airway morphogenesis in a compensatory manner. Foxp1/2/4 are transcriptional repressors and we have demonstrated that these factors link chromatin remodeling to target promoters through interactions with p66, a component of the NuRD (nucleosome remodeling histone deacetylase) complex. Transcriptional repression through complexes such as NuRD and their components including HDAC2 (histone deacetylase 2) are important for surfactant protein gene expression and lung epithelial maturation as our recent data on HDAC2 and the interacting homeodomain only protein (HOP) indicate. These findings demonstrate that Foxp1/2/4 play critical roles in regulation of lung epithelial specific genes, which are required for airway development and postnatal lung homeostasis. However, little is known about whether these factors act redundantly/cooperatively to regulate transcriptional targets in the lung, the mechanism of how Foxp1/2/4 repress lung gene transcription and the effect that loss of these factors has on activation and differentiation of airway progenitor cells including bronchioalveolar stem cells (BASCs) after lung injury. These questions will be addressed in the specific aims of this proposal.

Targeting Carriers with Controlled Geometry to Endothelium
Grant # R01HL087036 »
PI: Vladimir Muzykantov »

Drug targeting to endothelial cells (EC) will help to attain more specific and effective treatment of a plethora of maladies including inflammation. We found that targeting prototype nanocarriers and cargoes to the endothelial Cell Adhesion Molecules (CAMs) ICAM-1 and VCAM-1, differentially expressed by resting vs pathologically altered EC, provides prophylactic and therapeutic drug delivery into EC. Further, ICAM-targeted nanoparticles transfer across EC without damage to the cells. Flow conditions, affinity and geometry of anti-CAM carriers govern their biological behavior, EC targeting and sub-cellular destination. Targeted delivery of drugs to endothelial cells lining vasculature will advance treatment of many health maladies. In this grant, we will design and test a novel drug delivery platform based on targeted recognition of endothelial surface determinants by polymer nanocarriers loaded by anti-inflammatory and other therapeutic agents.

Myocardin and Vascular Smooth Muscle
Grant # P01HL075215 »
PI: Michael Parmacek »

The differential patterning of smooth muscle cells (SMCs) and pericytes within specific blood vessels ultimately defines and distinguishes the functional properties of the arteries, veins and capillaries. SMC phenotype and patterning, in turn, are determined via transcriptional programs that respond to developmental and environmental signals and cues. We have used transgenic and gene targeting strategies to elucidate the transcriptional programs that regulate vascular SMC differentiation. Our group and others have reported recently that the SAP domain transcription factor, myocardin, plays a critical role in SMC differentiation. Preliminary studies presented herein demonstrate that: i) myocardin is expressed in a precise developmentally regulated pattern in vascular and visceral SMCs, ii) forced expression of myocardin in non-SMCs activates multiple SMC-specific transcriptional regulatory elements, iii) forced expression of myocardin activates SMC-restricted genes in undifferentiated embryonic stem (ES) cells, and iv) expression of adominant-negative myocardin mutant protein or myocardin siRNA in SMCs represses activity of the SMC-specific SM22alpha-promoter.

Arrhythmia Mechanisms of the Metabolic Sensor AMP Kinase
Grant # K08HL074108 »
PI: Vickas Patel »

A 5-year training program to develop an academic career in molecular cardiac electrophysiology. The principal investigator has extensive training in clinical cardiac electrophysiology, basic electrophysiology, biophysics, and electrical engineering and will expand his scientific skills through a unique integration of interdisciplinary resources. This program will promote the command of molecular developmental biology, as applied to the development of cardiac arrhythmias and conduction disorders. Dr. Michael Parmacek will mentor the principal investigator's scientific development. He is a recognized leader in the field of molecular cardiac development and has trained numerous postdoctoral fellows and graduate students. To enhance the training, an advisory committee of highly regarded medical scientists will provide scientific and career guidance. The research will focus on mechanisms of arrhythmogenesis produced by mutations in the metabolic sensor AMP-activated protein kinase.

Ectopic F.VIII Expression in Megakaryocytes: Treatment of Hemophilia A
Grant # P01HL064190 »
PI: Mortimer Poncz »

Platelets adhere, form a platelet plug and degranulate at sites of vascular injury. We are interested in testing whether ectopically expressed proteins during megakaryopoiesis would be stored within platelets and released at sites of injury, modulating the thrombotic process. Initial transgenic studies suggest that we can accomplish this process, delivering platelet-released (p) Factor VIII (FVIII) to a site of injury to improve the bleeding diathesis in FVIII null mice. We propose to better understand and improve on the transgenic pFVIII model, and to extend these studies to a gene therapy approach. We believe that successful application of these ideas may provide new approaches for the management of Hemophilia A and perhaps other diseases affecting thrombus formation.

Genetics of Elevated High Density Lipoprotein Cholesterol
Grant # R01HL089309 »
PI: Daniel J. Rader »

The syndrome of "familial hyperalphalipoproteinemia," or elevated high density lipoprotein cholesterol (HDL-C), is known to be associated with longevity and protection from atherosclerotic cardiovascular disease. The only proven molecular etiology of familial hyperalphalipoproteinemia is homozygous cholesteryl ester transfer protein (CETP) deficiency, which is found almost exclusively in Japan and led to the concept of pharmacologic CETP inhibition as a strategy to raise HDL-C levels. We have recruited what we believe to be the largest existing cohort of non-Japanese probands with high HDL-C cholesterol levels and their nuclear and extended families in order to determine novel genetic factors that cause elevated HDL-C. We will perform a high density genome-wide SNP scan using the Illumina 550K chip and utilize these data for family-based tests of association to search for HDL-C QTL.

ApoE and Regulation of COX-2 in VSMCs
Grant # P01HL062250 »
PI: Daniel J. Rader »

Apolipoprotein E (apoE) is is highly inhibitory toward atherosclerosis, but the mechanisms of this inhibition remain poorly understood. In the first cycle of this P01, we showed that human apoE3 inhibited S-phase entry of primary murine vascular smooth muscle cells (VSMCs) through upregulation of COX-2 and promotion of prostacyclin production with ligation of the prostacyclin receptor (IP). The experiments in this new project in this P01 will continue to explore the mechanisms by which apoE signals to upregulate COX-2 and ultimately to inhibit VSMC proliferation, and we will explore the in vivo consequences of this novel effect of apoE.

Gene Therapy for Dyslipidemia and Atherosclerosis
Grant # P01HL059407 »
PI: Daniel J. Rader »

Dyslipidemia is a major risk factor for premature atherosclerotic cardiovascular disease (ASCVD). Although many patients with dyslipidemia can be treated effectively with existing drugs, others are not effectively treated and remain at exceptionally high risk of premature ASCVD; the classic example is homozygous familial hypercholesterolemia (FH). Therefore, understanding of the regulation of the secretion and catabolism of apoB-containing lipoproteins by the liver and the complex pathways of HDL metabolism is of major importance to the development of new therapies targeted toward these pathways. Liver-directed somatic gene transfer is a useful biological tool for addressing hypotheses regarding the physiological effects of expressing specific genes in the liver on apoB lipoprotein and HDL metabolism; furthermore, it could be a strategy for treating severe dyslipidemia. In this project, we will utilize liver-directed gene transfer using vectors based on novel adeno-associated virus (AAV) pseudotypes to address questions related to the impact of specific gene products and their interactions on the regulation of hepatic secretion of apoB-containing lipoproteins.

Genetics of Atherosclerosis in Renal Disease
Grant # R01DK071224 »
PI: Muredach P. Reilly »

Atherosclerotic cardiovascular disease (CVD) is an inflammatory disorder with a complex genetic basis. Chronic kidney disease (CKD), a major risk factor for CVD, is rapidly increasing in the U.S. The mechanistic link between these diseases remains largely undetermined. Recent studies suggest that insulin resistance, kidney dysfunction and CVD share common genetic bases. Innate immunity and insulin resistance converge in atherosclerosis and are prominent features of CKD. We hypothesize that, in this setting, genetic variation in innate immune and insulin resistance pathway genes will contribute individually and through multi-locus effects, to clinically important risk of CVD in the CKD population beyond traditional risk factors. The Chronic Renal Insufficiency Cohort (CRIC) Study, a prospective NIH sponsored multi-center study (n=3,000) of renal and CVD complications of CKD, provides a unique opportunity to examine these hypotheses.

Aging, Insulin Resistance, and Dilated Cardiomyopathy
Grant # R01AG023125 »
PI: Richard P. Shannon »

Congestive heart failure is a leading cause of morbidity and mortality in the elderly, although the mechanisms to explain the enhanced proclivity are poorly understood. It remains debatable as to whether the age-associated propensity to cardiovascular dysfunction is attributable to aging per se or the accumulation of cardiovascular risk factors that accrue over time. In particular, aging has been closely associated with the development of increased visceral adiposity that has been implicated in the pathogenesis of age associated insulin resistance. Whether age associated insulin resistance contributes to the progression of cardiac dysfunction following myocardial injury has not been explored systematically.

Cardiac Gene Therapy
Grant # P01HL059407 »
PI: Lee Sweeney »

The proposed research seeks to delineate the mechanisms underlying the progressive development of dilated cardiomyopathy and failure following myocardial infarction. We also will examine resetting of the calcium set point for contractility as a potential strategy to blunt hypertrophic drive resulting from pressure overload. In doing so, we will evaluate AAV-gene transfer approaches for the treatment of heart failure. First we will evaluate the hypothesis that an apoptotic component drives the dilated cardiomyopathy that follows focal infarction. Lastly, we will alter the calcium sensitivity of the cardiac troponin complex in an attempt to blunt pressure overload hypertrophy and the subsequent progression to failure.

Myosin Isozymes
Grant # R01AR035661 »
PI: Lee Sweeney »

The major questions remaining to be answered as to how myosin functions as a molecular motor surround the actin-activated product release steps. We propose that we now have high-resolution structures that show us the starting (transition state) and ending (myosin V closed cleft or rigor-like state) structures of myosin as it goes through its force-producing cycle. What we are lacking is any insight as to how the interaction with actin leads to the sequential release of phosphate and MgADP that is coupled to movement. There are two major goals of this study. Our goal is to characterize key structural elements of myosin that are involved in the product release mechanisms. We will make use of the fact that myosin V, non-muscle myosin MB, smooth muscle myosin II and Dictyostelium myosin II have kinetics that are fundamentally different from each other.

Modulation of Muscle Growth for the Muscular Dystrophies
Grant # U54AR052646 »
PI: Lee Sweeney »

The muscular dystrophies are characterized by progressive loss of strength over time. Stimulating muscle growth may delay the time before significant disability and death. The overall theme of this center is to study mechanisms to modulate muscle growth and breakdown for treatment of a variety of muscular dystrophies. IGF-1 is a potent stimulator and myostatin a specific inhibitor of muscle growth. Modulation of both pathways has been shown to ameliorate the mdx model of muscular dystrophy. Recently, protease inhibition with Bowman Birk Inhibitor Concentrate (BBIC) has been found to have similar effects. The center is composed of three sites, Johns Hopkins, University of Pennsylvania and intramural NINDS and includes investigators who are leaders in the field of myostatin and IGF-1 as well as clinical experts in muscular dystrophy.

DNA Virus as Vectors for Cardiovascular Diseases
Grant # P01HL059407 »
PI: James M. Wilson »

This competing renewal application builds on substantial progress that was made in the initial cycle of this grant in the development of gene transfer to heart and liver for treating heart disease and atherosclerosis. The structure of the grant has not changed in the renewal with includes projects by Drs. Wilson, Sweeney and Rader as well as Vector, Cell Morphology and Administrative Cores. The goals of the current cycle of this P01 have been realized resulting in 63 publications. The renewal application builds on this progress. The overall goal of the renewal grant is the development of effective gene therapy for cardiovascular disease, which can be achieved by targeting the heart and liver. An important theme in the renewal is that one needs to understand the biology and pathogenesis of the vectors systems used and the target diseases in order to realize this goal.

Angiogenic Tissue Engineering to Limit Post-infarction Ventricular Remodeling
Grant # R01HL089315 »
PI: Y. Joseph Woo »

Myocardial ischemia and infarction with resultant adverse ventricular remodeling and heart failure form an increasingly prevalent global health problem for which medical and surgical treatments are limited. Innovative therapies are greatly needed. This proposal seeks to investigate and develop a novel acute endogenous revascularization therapy which upregulates endothelial progenitor cells (EPCs) and specifically targets them to ischemic myocardium. This post- infarction angiogenic therapy to augment myocardial microcirculation will study three specific aims: 1) Mobilization and targeted chemokinesis of EPCs to revascularize ischemic myocardium. 2) Angiogenic reengineering of regional myocardial biomechanical properties to attenuate adverse ventricular remodeling and improve cardiac function. 3) Translational preclinical large animal model testing of endogenous revascularization therapy utilizing progressively less invasive, clinically-available technologies.