Arendshorst, William J. Ph.D: The long-range goal of our research is to gain a better understanding of mechanisms that control vascular reactivity in the renal microcirculation by hormonal, paracrine and autacoid agents in health and disease. Our studies focus on the regulation of vascular reactivity and receptor signaling pathways in the preglomerular vasculature of the kidney. A unique combination of coordinated in vivo with in vitro approaches of overlapping themes is designed to gain insight into regulatory mechanisms responsible for renal vasoconstriction in young spontaneously hypertensive rats (SHR) during the developmental phase of genetic hypertension. Our previous studies indicate that excessive renal vasoconstriction is associated with the pathogenesis of hypertension and is mediated by an abnormal balance of actions of vasoconstrictor angiotensin II (Ang II), vasopressin (AVP), and thromboxane (TxA2) and vasodilator systems (prostanoids, nitric oxide). In future studies, we will characterize other vasoconstrictor systems, such as a-adrenergic nervous system and endothelin (ET), and the mechanisms by which they produce enhanced renal vasomotor tone in young SHR. We will test the central hypothesis that exaggerated vasoconstriction is mediated by direct actions of the constrictor agents on vascular smooth muscle cells, either alone, due to enhanced receptor density or postreceptor signaling, or in combination with a deficiency in the buffering capacity of vasodilator prostanoids. We will evaluate key signal transduction steps from receptor mRNA expression and protein binding, and coupling with intracellular pathways to stimulate cytosolic calcium via mobilization and recruitment of entry channels. Our search for significant abnormalities in vascular actions should provide important new information that advances a more complete understanding of normal regulatory mechanisms and defects in controllers that may cause or contribute to the development of genetic hypertension.

Bankaitis, Vytas A. Ph.D: My laboratory is interested in the regulatory interfaces between novel lipid-mediated signal transduction pathways and important cellular functions. The focus of our work is the phosphatidylinositol/phosphatidylcholine transfer proteins (PITPs), a ubiquitous but enigmatic class of proteins, whose in vivo function interfaces with Golgi secretory function and cellular differentiation in yeast, signaling in the Drosophila visual and olfactory systems, growth factor signaling in mammalian cells, and pathways for the maintenance of neuronal viability in mammals. Our collective evidence indicates that PITPs coordinate key interfaces of lipid-driven metabolic reactions and intracellular signaling pathways in both yeast and mammals. Inappropriate regulation of these interfaces compromises membrane trafficking events, growth factor receptor function, cell growth control, and regulation of key developmental pathways. Because defects in any one of these pathways define recognized mechanisms of disease, PITPs represent essentially unstudied regulators whose dysfunction is likely to influence the activities of cellular processes required for cellular homeostasis.

Bautch, Victoria Ph.D: Visit Dr. Bautch's webpage. We study angiogenesis using both cell culture and in vivo mouse models, with a focus on developmental aspects of blood vessel formation, since this process is likely to be repeated during healing and disease processes. We have characterized the ability of mouse embryonic stem cells to form de novo blood vessels upon differentiation in culture, and we are currently investigating the effects of several targeted mutations in this system, including components of the VEGF signaling pathway. The culture system is also the basis for an imaging project designed to study the many dynamic aspects of blood vessel formation, and for a genetic screen using a retroviral trap strategy. We have set up a mouse-avian chimera model to study patterning of blood vessels in vivo, and we are using mouse mutations to investigate the role of specific molecules in vascular patterning.

Beck, Melinda Ph.D: Visit Dr. Beck's webpage. My laboratory is interested in studying the relationship between nutritionally induced oxidative stress and viral infection. Currently, we are investigating two model systems: coxsackievirus-induced myocarditis and influenza-induced pneumonia. We have found that a host deficiency in either selenium or vitamin E will result in increased pathogenesis of both of these viruses. This deficiency also induces profound changes in the host immune response against the virus. Because both selenium and vitamin E are antioxidants (although they work by two very different mechanisms) we propose that increased oxidative stress in the host lead to the change in pathogenicity. In addition, we have found that the virus itself develops mutations when replicating in an oxidatively stressed host. Once these viral gene changes occur, even hosts with normal nutritional status are susceptible to the virus's newly acquired virulence. Our laboratory is actively studying the mechanism(s) involved in the viral mutations and how the immune response of the host may play a role.

Burridge, Keith Ph.D: The Burridge lab has a longstanding interest in the structure and function of cell adhesions made to the extracellular matrix (focal adhesions), mediated by integrins, and made to other cells (adherens junctions), mediated by cadherins. We are interested in the links between adhesion molecules and the cytoskeleton, in the assembly and disassembly of these structures, and in the signaling pathways generated downstream from integrins and cadherins. Key regulators of the assembly of focal adhesions and adherens junctions are members of the Rho family of GTPases. Engagement of integrins and cadherins affects the activity of RhoA, Rac and Cdc42, and we are exploring the signaling pathways involved. In the context of vascular biology, we are investigating the adhesions made by endothelial cells, both to the underlying extracellular matrix and between adjacent endothelial cells. We are particularly interested in the interaction of leukocytes with endothelial cells that occurs as leukocytes extravasate through endothelial cell monolayers in response to inflammatory signals. This involves multiple cell adhesion molecules and reciprocal signaling between leukocytes and endothelial cells, that ultimately result in passage of leukocytes through endothelial cell junctions.

Caron, Kathleen Ph.D: Visit Dr. Caron's webpage. Adrenomedullin (Adm) is a newly identified 52 amino acid peptide vasodilator that has been implicated in a wide variety of normal physiological processes, including maintenance of basal vascular tone, regulation of salt and water appetite, cellular proliferation, angiogenesis and anti-microbial defense. Most noteworthy is the finding that plasma levels of Adm are elevated in human patients with many types of cardiovascular diseases, including essential hypertension and sepsis, suggesting that elevations in Adm are compensatory to other primary cardiovascular stresses. The mechanism of G-protein coupled receptor signaling through which Adm signals is an area of robust investigation because it involves a new class of signaling proteins (called RAMPs; receptor activity modifying proteins) and represents a novel paradigm in cell signaling. As a result, Adm’s signaling partners (the calcitonin receptor-like receptor, CRLR and RAMPs) have been explored as potential therapeutic targets for the pharmacological treatment of conditions such as migraine, peptic ulcers and bacterial gingivitis and in Japan, Adm peptide is currently being surveyed as a potentially beneficial renal and hemodynamic treatment for patients with congestive heart failure. Thus, Adm and its associated signaling partners are quickly becoming recognized as a broadly expressed, multi-functional peptidehormone system that can impact on many other systems under both normal and pathological conditions.
Our laboratory uses gene targeting approaches and state-of-the-art phenotyping methods to elucidate the physiological role of Adm and its signaling partners. Previous work in our lab has demonstrated an essential role for the Adm gene in development, since Adm knockout mice die at mid-gestation with extreme hydrops fetalis and unusual cardiovascular defects. Quite surprisingly, we have also found that genetic reduction of this potent vasodilator does not affect basal blood pressure. We have also identified a reproductive defect among the Adm heterozygote female mice that parallels the pathological manifestation of preeclampsia and fetal growth restriction in humans, suggesting an important role for this peptide in placental development and function. Current studies in our lab are focused on defining the role of Adm peptide in reproductive and cardiovascular physiology. We are also addressing the quantitative and tissue-specific function of Adm’s receptor and RAMPs as a model to characterize a new paradigm in G-protein coupled receptor signaling.

Cascio, Wayne MD: Visit the Cardiac Physiology and In Situ Confocal Microscopy Laboratory webpage. Our laboratory has Developed in situ fluorescent confocal microscopy techniques for 3D imaging and rendering of cardiac microvasculature. Confocal microscopy is a non-invasive method for imaging the distribution of fluorescence in thin 2-D optical sections of living tissue. Recently, we have developed imaging techniques with high temporal and spatial resolution to visualize the intracellular and interstitial spaces in compact ventricular tissue from small mammal hearts. To develop this technique, perfused rabbit papillary muscles were suspended in a lexan chamber and surrounded by humidified gas. The chamber was mounted on the stage of an epi-fluorescent confocal microscope. Di-8-ANEPPS was used to identify the endothelial cell membranes and image the microvasculature during perfusion. A cooled CCD camera recorded fluorescence images as the focal depth was increased in 2µm steps The intravascular space was also imaged with the fluoroprobe fluorescein isothiocyanate dextran. Optical slices, 5.5µm thick, were acquired and the intravascular space was rendered from 3-D images. These techniques allow us better understand the role of the microvasculature for impulse propagation in heart, and we are extending this technique to studies of vascularture of the mouse.. Funds were recently approved to support the implementation of a 2-photon fluorescence microscope. Such a microscope is expected to increase image resolution from the deeper intramural cell layers although the studies can be accomplished with existing confocal microscopes present in the Confocal Imaging Facility.
We have also developed a fiber-optic array and photo-diode imaging system with high spatial and temporal resolution for imaging voltage and calcium transients in confluent monolayers of cardiac and endothelial cell cultures. Studies are currently underway investigating the interaction of endothelial cells and ventricular myocytes on propagation.

Church, Frank Ph.D: The research area of Frank Church, Ph.D. is concerned with proteases and their inhibitors in human biology and in various disease processes (including thrombosis and tumor cell metastasis). They have an extensive interest in principles of proteolysis and they take a three-pronged approach to research: In the first approach, Dr. Church’s lab group performs structure to activity to function studies with heparin-binding serpins (serine protease inhibitors; heparin cofactor II, protein C inhibitor and antithrombin) and serine proteases (thrombin, activated protein C, and urokinase). They have made substantial progress in identifying specific residues in these serpins that are important for glycosaminoglycan binding and for protease recognition. In the second approach, they are applying basic biological techniques to investigate newly emerging principles of proteases in biological processes, especially cancer. This research is directed at delineating the in vivo localization and molecular regulation/expression of these proteins in human tissues and in human cancer cell lines, especially breast and ovary. They have made progress comparing and contrasting two different serpins, PAI-1 and PAI-3, in breast cancer cell lines in how they contribute to migration, invasion, adhesion, and apoptosis; and they are studying archived ovarian cancer tissue sections to localize these proteins. In the third approach, the Church lab has initiated work in the area of RNA aptamer technology to create novel antithrombotic reagents that bind to either thrombin or to heparin-binding serpins; ultimately, to characterize new substances that are potential therapeutic anticoagulants.

Clemmons, David MD:
Basic Research Interests
l. Mechanisms by which insulin-like growth factor binding proteins act to alter cellular replication and differentiation.
2. Control of fibroblast and smooth muscle cell replication by IGF's, their binding proteins and related peptides.
3. Structure and function analysis of insulin-like growth factor binding proteins.
4. Role of IGF's in atherosclerosis, arthritis and the response to injury.
Clinical Research Interests:
1. Interaction between growth hormone and nutritional status in controlling protein metabolism.
2. Metabolic responses to IGF-I administration in humans.
3. Use of IGF and IGFBP measurement to assess growth hormone and nutritional status.

Coleman, Rosalind MD: We are investigating the relationship between triacylglycerol synthesis and disorders like diabetes, atherosclerosis, and obesity. In order to understand how triacylglycerol synthesis is regulated during normal physiological states such as feeding, fasting, and the neonatal period, we have focused on the initial and committed step, the acylation of sn-glycerol-3-P by glycerol-3-phosphate acyltransferase (GPAT). We will determine 1) the structural aspects of mitochondrial GPAT that contribute to its specificity and regulation; 2) the phosphorylation sites that regulate mitochondrial GPAT activity; 3) the function and specificity of mitochondrial GPAT's lipid cofactors; 4) the role of mitochondrial GPAT in directing the fate of fatty acids during different nutritional and hormonal states; and 5) the phenotype of mice constructed to be deficient in mitochondrial GPAT. Answers to these questions will allow us to understand how GPAT functions in cells to partition acyl-CoAs towards glycerolipid synthesis and away from b-oxidation and how cells regulate their triacylglycerol content. The second interest is to understand how acyl-CoAs are channeled towards specific pathways within cellular compartments. Channeling of acyl-CoAs would aid in metabolic regulation and the partitioning of fatty acids within cells towards either synthetic or degradative pathways. To this end, we have cloned and expressed the three ACS isoforms that are expressed in liver and adipocytes, and examined their properties. We have found that ACS1, ACS4, and ACS5 proteins are located in different subcellular organelles and that their activity and mRNA expression change independently during different nutritional and physiological conditions in liver. We also discovered that ACS4 is specifically and potently inhibited by thiazolidinediones. This surprising finding suggests that the thiazolidinedione drugs, widely used to treat type 2 diabetes, may exert some of their beneficial effects through ACS4 in addition to known effects on the nuclear transcription factor PPARg.

Conlon, Frank Ph.D: The overall objective of my lab's research is to gain insight into the cellular and molecular pathways that establish the early vertebrate body plan. This work couples experimental embryology and molecular techniques in amphibians with genetic analysis in the mouse. The labs current research focuses on the patterning of the nascent mesoderm and how this initial patterning is eventually translated to the anterior-posterior patterning of the developing heart. To study how individual cell types are specified from the lateral and anterior mesoderm we have been addressing how cells of the cardiac region are determined and patterned during gastrulation. In amphibians the heart forming tissue is located in two regions at the anterior edge of the involuting mesoderm that flanks Spemann's organizer. These cells migrate laterally and anteriorly to give rise to a single heart tube comprised of an inner endocardium and an outer myocardium layer. The heart tube is further partitioned along the anterior-posterior axis in response to signals that may arise from the Spemann's organizer and the endoderm. However, the nature and extent of these tissues in heart patterning remain controversial. These signals ultimately lead to the differentiation of the heart tube along the anterior-posterior axis resulting in the formation of the presumptive atrium in the anterior region and the formation of the presumptive ventricle in the posterior region. One of the main focuses of the lab is to try and identify the molecular basis of the signals involved in heart formation and patterning. To this end we are using three approaches. First, we are characterizing the Xenopus homologues of TBX5, a gene that is mutated in the congenital human heart disease Holt-Oram, by constructing an allelic series of Xenopus TBX5 mutations in vivo. Second, we are identifying the direct downstream targets of TBX5. Third, we are conducting a mutagenesis screen in Xenopus to detect additional genes involved in anterior-posterior patterning of the early heart tube. Collectively, these approaches will provide insights as to the molecular basis of Holt-Oram disease and eventually lead to clues as the mechanisms of heart development.

Edgell, Cora-Jean S. Ph.D: My focus has been on vascular endothelial cells which have important roles in basic pathophysiologic processes including hemostasis, inflammation and tumor angiogenesis. I have developed a human endothelial cell line, EA.hy926, that is continuous, vigorous, and clonally pure, yet maintains differentiated properties including von Willebrand factor, Weibel-Palade bodies, and even forms capillary-like tubes on Matrigel. Continuous cell lines, especially human cell lines, that sustain differentiated functions are unusual and valuable. Currently our main interest is a recently defined gene product, testican, a proteoglycan that is differentially expressed in endothelial cells and certain other cell types. We have demonstrated testican in blood plasma, and shown that the purified proteoglycan inhibits attachment of cells in culture. Future experiment are aimed at defining testican function in vivo.

Faber, James Ph.D: The catecholamines (CATs), norepinephrine (noradrenalin) and epinephrine (adrenaline), circulate in blood and are released by nerve endings in blood vessel walls. Blood levels and amounts released by nerves are increased by stress and many other factors, and plasma levels progressively with age, especially in association with hypertension, arteriosclerosis, and atherosclerosis. Although elevated levels are known to be indirect risk factors for cardiac and vascular wall diseases, the nature of this assocation is not at all understood. CATs are well-known to bind to a certain a-adrenergic receptor (AR) subtype (s) on smooth muscle cells (SMCs) to cause constriction for the control of blood flow and arterial pressure. This laboratory is interested in adrenergic regulation of the circulation, with specific focus on defining the distribution and function of the different a-ARs expressed by SMCs and adventitial fibroblasts (AFBs). One of our recent unique findings is that a specific aAR subtype on these SMCs and AFBs (different receptor types on these two cells), when stimulated for prolonged periods (hours-to-many days), induces growth of the cells (increased number and size), and increases secretion of extracellular matrix, including collagen. We have found that this growth-promoting action of CATs is greatly augmented in blood vessels that are either diseased themselves or injured by surgical or pathophysiological processes that themselves promote growth and fibrosis of the vascular wall. We find that chronic stimulation of the receptors worsens intimal lesion growth, fibrosis, structural narrowing of the vessel lumen, and shrinks outer vessel diameter; blocking aARs attenuates intimal growth and lumen loss induced by vascular wall injury. These findings have been obtained in vessels and cells of laboratory rats, and in mice with targeted deletion of aAR subtypes or the rate-limiting enzyme responsible for CAT synthesis. The receptors mediating these growth-promoting actions of CATs on SMCs and AFBs are different subtypes from the one responsible for constriction of the SMCs in most vascular beds. Based on this information, we have proposed the hypothesis that these growth-promoting receptors may be blocked pharmacologically to lessen diseases and complications characterized by wall fibrosis, intimal lesion growth, vessel remodeling, and lumen loss. Furthermore, blockade of these trophic receptors may be accomplished with minimal side effects on vascular hemodynamics and other processes regulated by different aARs.

Falk, Ronald MD: Our research program consists of two cores focusing on anti-neutrophil cytoplasmic autoantibody (ANCA) necrotizing and crescentic glomerulonephritis (GN) and small vessel vasculitits (SVV). The scope of the investigations and the diversity of the investigators allow for an integrated evaluation of basic molecular and clinical immunological and epidemiological studies. Experimental research projects include the consideration of the derivation of ANCA autoantibodies using basic molecular immunologic techniques and using transgenic mice. In parallel, we are testing the hypothesis that ANCA directly participate in the pathogenesis of the ANCA immune response. We are exploring the mechanism by which ANCA activate neutrophils and monocytes leading to the release of ANCA antigens MPO and PR3 and vascular damage. Leukocytes from patients during various stages of disease, including onset, remission, relapse have been analyzed using state of the art techniques (Affymetrix chip and Real-time PCR) to create patient profiles of gene expression. The clinical research program provides information on clinical presentation, disease associations, and evaluates the role of environmental factors in renal disease progression. Epidemiological studies on a large population of ANCA GN patients are underway to acertain those environmental factors, such as silica exposure, that predispose to the development and exacerbation of the ANCA immune response.

Goy, Michael Ph.D: The Goy lab is interested in cellular signaling via cyclic GMP. In a broadly-based research program, we are identifying the agonists that stimulate increases in cGMP, defining the guanylate cyclase iso-forms that are activated, exploring the physiological processes mediated by this signaling pathway, and determining the pathophysiological consequences of pharmacologic and genetic interventions that com-promise the function of the pathway. Two projects related to vascular biology are under investigation in the laboratory.

1) Peptides that elevate cyclic GMP and control epithelial ion transport in the intestine and kidney. Twenty years ago, it was shown that the heat-stable bacteria toxin (STa) causes diarrhea by activating a cyclic GMP-dependent epithelial ion transport pathway. Over the past 10 years, we and others have shown that this toxin opportunistically activates a guanylate cyclase that actually serves as a receptor for a family of endogenous gut peptides. My lab has been investigating the structural interrelationships, tis-sue distributions, and physiological roles of the members of this peptide family. We have demonstrated that one member of the family, guanylin, is an exocrine peptide produced by intestinal goblet cells that acts within the lumen of the intestine to promote luminal entry of water and electrolytes. Our current hy-pothesis is that this plays a key physiological role in the hydration of intestinal mucus. In contrast, a sec-ond member of the family, uroguanylin, is produced by a specific type of enteroendocrine cell, known as the enterochromaffin cell. Current evidence indicates that uroguanylin is probably released both into the intestinal lumen, where it performs an exocrine function and into the general circulation, where it per-forms an endocrine function. Of particular interest is the proposal that uroguanylin is the primary hor-monal agent in a novel endocrine pathway that allows the intestine (a physiological site of salt ingestion) to communicate with the kidney (a physiological sight of salt excretion). In this regard, our most recent studies have revealed the unexpected presence of uroguanylin within the kidney itself, and we are cur-rently investigating the function and expression patterns of this renally-expressed uroguanylin. Studies of this intriguing new family of peptide ligands will help us understand diseases that affect epithelial ion transport, including infectious diarrhea, cystic fibrosis, and essential hypertension.

2) Peptides that elevate cyclic GMP and control blood pressure. NPRA is a receptor guanylate cy-clase (rGC) that synthesizes cGMP in response to ANP and a related peptide, called brain natriuretic peptide (BNP). The family of rGCs is rapidly expanding, and it seems plausible that there might be addi-tional, as-yet undiscovered rGCs whose function is to provide alternative signaling pathways for one or both of these peptides -- particularly given the low affinity of NPRA for BNP. We have investigated this hypothesis, using a knockout mouse in which the gene encoding NPRA has been ablated. We first evaluated the tissue distribution of NPRA protein and its associated GC activity in wild-type mice. These studies confirmed that immunoreactive NPRA is not detectable in tissues isolated from NPRA knockout animals and showed that ANP- and BNP-stimulatable guanylate cyclase activity is markedly reduced in all mutant tissues. However, testis, kidney, and adrenal retained statistically-significant, high-affinity responses to BNP that could not be accounted for by any known mammalian rGC, suggesting the presence in these tissues of a novel, BNP-preferring receptor. We are currently investigating ways to establish the identity of this previously-undetected natriuretic peptide receptor.

Heiss, Gerardo MD, Ph.D: Dr. Heiss' research interests are in the epidemiology of atherosclerosis ascertained through non-invasive measures, genetic epidemiology, ethnicity and disease, the role of inflammation in atherogenesis and atherothrombotic events, and community surveillance. He currently teaches Cardiovascular Disease Epidemiology, and Research Methods in Cardiovascular Disease Epidemiology. He also participates in the Cardiovascular Disease Epidemiology Seminars, and as guest lecturer in various departmental courses.

Jennette, J.Charles MD: Dr. Jennette's research focuses on understanding the pathogenesis of inflammatory vascular diseases, especially immune mediated vasculitis and glomerulonephritis. A major current emphasis is on the pathogenic role of ANCA (antineutrophil cytoplasmic autoantibodies) in vascular inflammation, including the establishment of animal models to test the hypothesis that ANCA cause inflammatory vascular disease by synergistic interactions with other proinflammatory stimuli, such as circulating cytokines. Mice with circulating anti-MPO antibodies (MPO-ANCA) have been generated by passive transfer of MPO-specific B-lymphocytes and T-lymphocytes derived from MPO-KO mice immunized with murine MPO. Wild type mice with circulating anti-MPO, MPO-knockout mice with circulating anti-MPO, and normal control mice are being challenged with proinflammatory stimuli to preferentially induce glomerulonephritis and vasculitis in mice with circulating anti-MPO. The pathogenesis of the inflammatory injury caused by anti-MPO in mice will be evaluated not only by standard histologic and immunopathologic examination but also by evaluation of differential gene expression using quantitative RT-PCR, laser capture microdisection, and mouse gene chip analysis.

Juliano, Rudy L. Ph.D: Our laboratory works on two distinct avenues of research . One research theme, concerning cell adhesion molecules, is very basic in orientation, and deals with fundamental cell biological processes. The other theme, macromolecular therapeutics, has a more pragmatic orientation, with the long term goal of developing new therapeutic agents.
Adhesive interactions between a cell and its neighbors, or between cells and the extracellular matrix, play a major role in the regulation of normal cellular growth and differentiation. Aberrations in cell interactions are a hallmark characteristic of the invasive and metastatic behavior of malignant cells. Research in this laboratory centers around the biology, biochemistry and molecular biology of membrane receptors that mediate cell interactions. Our efforts, along with those of many other laboratories around the world, have resulted in the discovery of a new superfamily of membrane proteins, the integrins, which seem to be the key receptors for many forms of cell interactions. Recently it has become clear that integrins and other families of adhesion receptors are also signal transducing molecules. Our laboratory was the first to demonstrate that integrins can trigger tyrosine phosphorylation in cells. This has led to the identification of intracellular tyrosine kinases, including FAK and Syk, that are activated via integrins and that contribute to subsequent signaling events. We have also shown that the integrin signaling pathway intersects with pathways activated by peptide mitogens, impinging on some of the same downstream components, such as mitogen activated protein kinases (MAP kinases). Ultimately, integrin mediated signals help to regulate gene expression and can influence cell differentiation, traverse through the cell cycle, or apoptosis.
Perhaps the most fundamental approach to the control of cancer would be to be able to regulate the aberrant expression of key genes involved in cancer progression. We have adopted two strategies in pursuit of this goal. In one strategy we use "antisense" oligonucleotides to inhibit messenger RNA from cancer-related genes. In another approach we have sought to identify novel peptides that can interact directly with the DNA of cancer-related genes. In this second approach we make use of a so called "combinatorial library" strategy to select active DNA-binding peptides from a vast number of possibilities. This is an exciting approach that could give rise to new insights into protein-DNA interactions as well as yielding new therapeutic entities.

Knisley, Stephen B. Ph.D: Dr. Knisley is attempting to optimize the lifesaving technique known as cardiac electrical defibrillation. To accomplish this he is studying the initial effects that lead to defibrillation, which are the transmembrane voltage changes that occur during the brief time in which the defibrillation shock is turned on. The research, which involves bioelectric measurements as well as theoretical modeling with finite element, Monte Carlo and bidomain simulations, indicates that both the heart's fiber structure and the shock-induced electric field play crucial roles in these initial effects. With this knowledge, Dr. Knisley is developing new methods of defibrillation that take advantage of the heart's natural fiber structure and linear shock electrodes in order to increase effectiveness and safety of defibrillation. Another research aim is to discover how fibrillation starts during heart attacks. It is hoped that this knowledge will help in developing ways to prevent the fibrillation so that eventually no defibrillation shocks will be needed. Also, in order to perform the research, new measurement techniques and hardware have been developed including ratiometric optical methods with a laser scanner to sense the transmembrane voltage and transparent electrodes to simultaneously sense the electric field without blocking the laser light.

Koller, Beverly H. Ph.D:Our research focuses on the role of lipid metabolites and nucleotides as local mediators important both for maintaining homeostasis and for coordinating the inflammatory response initiated in many cases when homeostasis is disturbed. One particularly complex group of lipid mediators is those generated by the metabolism of arachidonic acid by cyclooxygenases and lipoxygenases. This includes the prostaglandins, prostacyclin, thromboxane, and the leukotrienes. ATP and UTP are released by many different cells in response to a broad spectrum of stimuli, including physical disturbances such as shear stress. Both nucleotides and lipid mediators modify the physiology of the organism in part by binding to G coupled membrane receptors. To study the role of these lipids and nucleotides, we have generated a series of mice deficient in the enzymes necessary for the production of the mediators and/or the receptors for these mediators. Using these we have been able to determine the contribution of these to the development of inflammatory responses in a number of models of acute and chronic inflammation. In addition, we have identified a role for one of the prostaglandin E2 receptors, the EP4 receptor, in the remodeling of the cardiovascular system that occurs at birth. We are currently using this model to identify the mechanism by which synthesis and metabolism of PGE2 and activation of PGE2 receptors can contribute to the various morphological changes that take place during the remodeling of the ductus arteriosus, a shunt that directs blood flow in the fetus away from the lungs and toward the placenta where oxygenation takes place.
We have also established a mouse model of pulmonary hypertension in our lab. Mice are housed for three to six weeks in chambers with reduced oxygen tension, after which time right ventricle pressures are determined, as well as changes in ventricle weight and the extent of remodeling of the small arterioles. Using this model we have identified a role for nucleotides in the vascular changes that accompany the development of pulmonary hypertension. We are currently exploring the mechanisms by which these ligands and their receptors contribute to the pathogenesis of this disease as well as their relationship to prostacyclin and thromboxane, lipid mediators that have been shown to be involved in the development of this disease.

Lin, Weili Ph.D: Dr. Lin's research interest is in quantitative estimatation of brain water using MRI. Absolute measurements of in vivo brain water content are of critical importance for the management of elevated brain water content resulting from acute cerebral injury such as head trauma and cerebral ischemia. Although it has been demonstrated that a linear relationship exists between relaxation times and water content, only relative measurements are available. The main thrust of this project is to develop imaging methods for obtaining quantitative estimates of brain water in vivo. We have successfully demonstrated that an absolute measurement of brain water in vivo can be obtained. With a three-dimensional approach and focal cerebral ischemic rat models, a highly linear relationship is obtained between MR estimated brain water and that obtained from wet/dry measurement.

Lentz, Barry Ph.D: In general, my research has applied the techniques and concepts of physical chemistry to the solution of biologically relevant problems. Since completing my formal training, I have been engaged in experimental studies that aim to determine the molecular basis for the physical and biological properties of increasingly complex membrane systems. In these studies, I have used fluorescence spectroscopy, nuclear magnetic resonance spectroscopy, Fourier transform infrared and CD spectroscopy, microcalorimetry, electron microscopy, enzyme kinetics, molecular biological protein manipulations, standard biochemical techniques, and the concepts of statistical thermodynamics to define the relationship of membrane and protein structure to biological function. In one project, my laboratory seeks to define the mechanisms by which platelet-membrane phosphatidylserine (PS) regulates thrombin formation during blood coagulation. Currently, our work focuses on biophysical studies of the membrane assembly and lipid-regulation of the enzyme-cofactor complex that activates prothrombin to thrombin. Another project aims to establish in model membranes the molecular mechanism by which poly(ethylene glycol) (PEG) induces membrane fusion. We have found that PEG-mediated of pure lipid synthetic membranes is an excellent model for much more complex, protein-mediated cellular fusion events. We plan to extend these studies to examine the mechanism by which the simplest and best understood cellular fusion machine, the influenza virus protein hemagglutinin, induces fusion of viral membrane to the endosome membrane during viral infection. We will also examine the effect of the basic machinery of exocytotic fusion (the "core complex") on fusion of our model membranes. Ultimately, we hope to use this information to find methods for better delivering drug-laden liposomes to and inducing their fusion with target cells.

Lord, Susan Ph.D: Research in our laboratory is focused on the role of fibrinogen in cardiovascular disease. When blood vessels are injured, fibrinogen is converted to an insoluble fibrin matrix that acts as the scaffold for blood clot formation. During wound healing, fibrin is dissolved and the clot is removed. Cardiovascular disease can arise when either clot formation or clot dissolution is abnormal. Biochemical data demonstrate that fibrinogen affects both these processes. Further, epidemiological data indicate that elevated plasma fibrinogen is associated with an increased risk of cardiovascular disease. We study these multiple roles of fibrinogen using three approaches. We examine patients with cardiovascular disease to determine whether fibrinogen abnormalities are linked to clinical symptoms. Using protein engineering, we prepare variant fibrinogens, and test the functional effects of the changes in biochemical assays. Using transgenic techniques, we generated mice with elevated plasma fibrinogen; we use these mice to examine the link between elevated fibrinogen and cardiovascular disease.

Ma, Alice MD: Work on my laboratory focuses on signal transduction pathways that lead to the reorganization of the actin cytoskeleton, with an emphasis on pleckstrin and other proteins which have pleckstrin homology (PH) domains. Pleckstrin is a 40 kDa hematopoietic protein with homology between its first and last 100 residues, regions now known as the amino- and carboxyl pleckstrin homology (PH) domains. PH domains are sequences of approximately 100 amino acids which form "modules" that have been proposed to target their respective proteins to membranes by interacting with inositol phospholipids. PH domains are now recognized to be present in more than 100 signaling and structural proteins, among which are proteins important in involved in almost every known signaling pathway. We are interested in the role of the PH domains of pleckstrin and Vav, as well as those from other proteins, in the regulation of the actin cytoskeleton.

Mack, Christopher P. Ph.D: Smooth muscle cell (SMC) differentiation is an important process during blood vessel development, and alterations in SMC phenotype play a prominent role in several cardiovascular disease states including atherosclerosis. My research goals are to identify mechanisms by which environmental factors regulate SMC phenotype and to define the transcriptional pathways that regulate SMC-specific gene expression. We have used a variety of cell culture and in vivo model systems to show that the transcription factor, serum response factor (SRF), is an important regulator of SMC differentiation marker gene expression. Because SRF is a ubiquitously expressed protein, we are currently trying to identify additional transcription factors that, perhaps in concert with SRF, regulate SMC-specific gene expression. SRF activity is also affected by signaling through the small GTPase, RhoA, and we have begun to study this signaling pathway in regard to its effects on SMC differentiation. Our initial data indicate that activation of Rho leads to increases in SMC-specific gene expression probably through activation of SRF. Since many of the environmental factors that regulate SMC phenotype have also been shown to activate Rho, we hypothesize that this signaling pathway may be important for integrating a large number of the environmental cues that regulate SMC differentiation. We are currently using SMC-specific expression and knockout technologies to alter Rho function specifically in SMC in vivo. These studies should lead more complete understanding of how Rho signaling affects SMC differentiation.

Maeda, Nobuyo Ph.D: The research in my laboratory is centered on developing an understanding of the genetic basis of atherosclerosis. This common but complex multifactorial disease is the leading cause of death in modern societies. We use gene targeting experiments in mice to introduce specific alterations in various genes that are involved in lipid metabolism or in the maintenance of vascular integrity. Using the spontaneous atherosclerosis developing in apolipoprotein E-deficient mice as a basis, we investigate how combinations of genetic factors modulate the development of vascular lesions and/or how specific environmental factors such as diet and drug treatment interact with the gene defects. The mouse mutants will soon be used in a genome-wide screen to identify genes that respond when cardiovascular homeostatic mechanisms are altered.

Malouf, Nadia MD: My research interests have recently focused on two projects. The first is understanding the role of voltage dependent calcium channels in platelet function. We have found that the blood platelets express two isoforms of the L-type channel major subunits. The a1B previously described in neuroendocrine tissues and the a1S previously reported in skeletal muscle. Extrapolating from the function of the channels in these tissues we hypothesize that they have a role in secretion and contraction of platelets respectively. The second project is understanding the mechanisms that lead cells from a line derived stem cell line to acquire cardiac phenotypes when engrafted in the heart in vivo. We have recently demonstrated that these cells differentiate into a phenotype controlled by the cardiac niche in which they engraft. They become mature myocardial cells in the myocardial wall, endocardial or epicardial cells that line these respective surfaces at these sites.

Milgram, Sharon Ph D: The Milgram lab is interested in identifying mechanisms that target hormone receptors to the apical cell surface and in identifying protein interactions that coordinate signaling from these receptors. Specifically, we want to understand how apical membrane receptors regulate cell function, including changes in gene expression, and changes in the activity of apical membrane ion channels (for example, the cystic fibrosis transmembrane conductance regulator chloride channel). Although much of our recent work focused on the airway epithelium, we speculate that the pathways and mechanisms we identify in airway will play important roles in other epithelia and we are now developing other epithelial model systems in the lab.

Morris, Andrew J. Ph.D: Research in my laboratory concerns the roles of phospholipids in cell regulation. We have a particular focus on the receptor active lysophospholipid, lysophosphatidic acid (LPA). The vasoactive actions of LPA were first decribed over 30 years ago but our understanding of the complex roles of this compound in regulation of cells of the vascular system has only advanced significantly in recent years. Platelets are a major source of LPA in plasma. LPA is a potent platelet activator and autocrine actions of LPA are therefore thought to play an important role in agonist-dependent platelet activation and thrombus formation. LPA is a potent mitogen for vascular smooth muscle cells and endothelial cells. Production of LPA by platelets has been suggested to promote the accumulation of smooth muscle cells and extracellular matrix at the site of vascular injury (intimal hyperplasia). Finally, LPA is a potent chemotactic agent that may serve to recruit circulating monocytes to areas of damaged endothelium. In turn, this action of LPA may promote monocytic extravasation into the subendothelial space and the development of atherosclerosis. The recent molecular identification of G-protein linked LPA receptors promises to provide pharmacologists with an essential tool for the development of LPA receptor selective agonists and antagonists. We have identified biochemical pathways and enzymes involved in the generation and metabolism of LPA. We expect that our work in this area will lead to the definition of new molecular targets for pharmacological modulation of the biological actions of LPA.

Nichols, Timothy MD: The focus of our vascular biology research is on the role of von Willebrand factor in thrombosis and atherosclerosis. Our laboratory is currently working on the molecular biology of porcine von Willebrand factor, the role of shear in the control of vWF production, the influence of atherosclerosis on vWF content in the vessel wall. A new direction in our vascular biology research is on the role of activation of the transcription factor NF-kappaB in the atherosclerotic plaque. This research addresses the recent finding of the role of oral inflammation in atherosclerosis and is being done with Al Baldwin in the LCCC and with researchers in the P60 grant entitled Center for Inflammatory Diseases. The focus of our hemophilia work is on developing improved treatments for hemophilia A hemophilia B, and von Willebrand disease. This includes collaborative gene therapy and protein replacment studies. I also work with Drs. Read and Fischer whose research goals are directed towards production and characterization of dried platelets and platelet substitutes for use in transfusion medicine. The same technology is being used to the develop and characterization dried red blood cells. These studies are directed towards providing long-term stored blood products in the dried state.

Offenbacher, Steven DDS, Ph.D: Our research has focused on the biochemical mediators of inflammation. Studies have examined the role of prostaglandins, thromboxanes, leukotrienes and the cytokines IL-1b, TNFa on periodontal inflammation, connective tissue destruction and bone loss. Research models include non-human primates, dogs, rodents and humans. The emphasis has been in identifying those key mediators which are associated with progressive bone loss. Studies have been conducted in animal and human models of periodontal disease to elucidate mechanisms of pathogenesis and to determine the effects of anti-inflammatory drugs. Particular emphasis has been placed on the use of non-steroidal anti-inflammatory agents and our laboratory has been involved in testing, developing new formulations and in multicenter clinical trials.
Other research areas focus on rodent infection models using periodontal Gram-negative pathogens. These involve examining pathogen-specific (e.g. Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans) inflammatory cytokine pathways, as well as phagocytic cell responses. These infection models have been used to study not only periodontal diseases but also Gram-negative abnormal pregnancy outcomes. The role of Gram-negative infection of oral origin inducing preterm labor, premature rupture of membranes and low birth weight is a major focus of our research team. We are studying the role of maternal infection-induced cytokines in eliciting these abnormal pregnancy outcomes. Furthermore, in collaboration with OB/GYN, we are conducting human case/control epidemiology studies to examine the role of periodontal infection in preterm delivery. Other molecular epidemiology studies of periodontal disease include collaboration with Jim Beck and Gerardo Heiss where we are examining the role of periodontal disease and inflammatory biomarkers on cardiovascular disease.
Finally, we are examining the molecular basis of the monocytic response to bacterial endotoxin and disease wound healing. We are studying CD14 receptor density, membrane transduction, and the genetic regulation of cyclooxygenase levels as well as the levels of IL-1b, TNFa, PDGF and TGFb1 transcripts. This work includes regeneration growth factors and animal models of repair.

Parise, Leslie V. Ph.D: Cell-cell adhesion is fundamental to the organization and survival of multicellular organisms. Cell adhesion is mediated by specific transmembrane proteins or receptors exposed on the cell surface. These receptors transmit signals into the cell in response to the cellular environment to regulate cellular behavior. Cells also transmit internal signals to the adhesion receptors to regulate or alter receptor function. Alterations in the function, expression or signaling through adhesion receptors contribute to major diseases such as heart disease and cancer. Thus, it is important to understand how these receptors receive and transmit cellular signals. In this laboratory, we are studying adhesion receptors and signaling in the contexts of heart disease, which involves the study of platelets, cancer, which involves breast epithelial cells, and sickle cell disease, which involves sickle red blood cells and endothelial cells. In each case we are mapping signal transduction pathways sent to and via adhesion receptors. The types of adhesion receptors that we study most often are called integrins, which are present on most cell types. Techniques used include an array of molecular and biochemical approaches. For example, we are using the yeast two-hybrid system to identify proteins and protein motifs that bind to cytoplasmic domains of integrins. Using this approach, we have identified a number of novel proteins potentially involved in integrin signaling that we are now in the process of characterizing. We have also identified novel signaling pathways in platelets, sickle cells and breast epithelial cells. To gain functional information, we express native and mutant proteins in cells, construct chimeras, and are initiating mouse knock-out and knock-in studies. We also use an extensive array of protein biochemical and biophysical approaches to characterize relevant proteins.

Patterson, Cam MD: The Patterson laboratory uses molecular, genetic, and physiologic approaches to ask questions regarding events that underlie the processes of angiogenesis, vascular development, cardiac failure, and atherosclerosis. Our laboratory employs a wide range of methods, including standard molecular techniques, gene discovery applications, genetically modified animals, and microphysiologic techniques. We have a particular interest in understanding the genes that regulate angiogenesis, identifying stress-responsive genes that modify cardiac function, and characterizing oxidative pathways in atherogenesis.

Roberts, Harold MD: Our primary work involves control mechanisms for coagulation. We have established a cell based model system that uses a tissue factor bearing cell (monocytes, fibroblasts, or endothelial cells), unactivated platelets, plasma levels of prothrombin, factor X, factor IX, factor V, factor VIII, and plasma levels of the inhibitors antithrombin III and tissue factor pathway inhibitor. The model system is initiated by factor VIIa. We have measured platelet activation and thrombin generation. We have used this system and variations of it to establish that the cellular location of coagulation factor activation is critically important to regulating thrombin generation. We have also used this system to show that the physiologic mechanism of activation of factor XI involves thrombin catalysis on a platelet surface. Currently we are investigating the consequences of varying each of the individual coagulation factors. We are also looking at the way in way cell surfaces - platelet vs endothelial cell vs monocyte - respond to activated protein C. These studies have led us to propose that all lipid containing surfaces are not equivalent and that the surface of endothelial cells is programmed to be sensitive to activated protein C.
Our other work is in the area of coagulation factors with a special emphasis on coagulation factor IX (the absence of which causes hemophilia B). We have been studying a number of aspects of factor IX trying to relate functional activity with specific structural regions of factor IX. We have worked on mutations in the epidermal growth factor (EGF) like domains and in the trypsin like serine protease domain. We have been especially interested in mutations in the amino terminal Gla domain. The Gla domain contains 12 glutamic acid residues that are posttranslationally modified to gamma-carboxy glutamic acid (Gla) residues by a vitamin K dependent carboxylase. These residues are critical to calcium ion binding to factor IX (and the other Gla containing coagulation factors); calcium ion binding

Rosamond, Wayne Ph.D: Dr. Rosamond participates in the teaching of the General Epidemiologic Methods course, contributes to the teaching of Cardiovascular Disease Epidemiology, and participates in the teaching of Clinical Trials. Dr. Rosamond is developing a focal point for this program in the area of the epidemiology and medical care of cerebral vascular diseases. He also provides opportunities for tutored field work experience and study implementation through a CDC-funded study conducted under his direction, linking community screening programs in cancer and cardiovascular diseases. Dr. Rosamond's research area is in chronic disease epidemiology, specializing in cardiovascular disease surveillance. He currently works with the Atherosclerosis Risk in Communities (ARIC) and the Delay in Accessing Stroke Healthcare (DASH) projects and has particular interest in the study of medical care issues in cardiovascular disease.

Rosenberg, Robert Ph.D: One project is focused on the regulation of cardiac L-type Ca channels by eicosanoid metabolites of arachidonic acid and other lipid metabolites. We study the Ca channels in reconstituted systems, either by incorporating the channels in artificial membranes or by expressing cloned channels in Xenopus oocytes. Our goals are to understand the direct and signaling-dependent mechanisms of Ca channel regulation.
Another project is focused on the modulation of ?7 neuronal nicotinic acetylcholine receptors by permeant and impermeant divalent cations. We express the receptors in Xenopus oocytes, and use site-directed mutations to identify amino acids that are essential for divalent ion permeation and modulation.

Runge, Marschall MD, Ph.D: There are 2 general areas of research in my lab. 1) To gain an understanding of how thrombin, a growth factor and key molecule in thrombosis, functions in atherosclerotic blood vessels. At present, our focus in this area is on understanding the mechanisms by which thrombin induces intracellular oxidative stress. In these studies, we are examining the regulation of key signaling proteins - protein tyrosine kinases and phosphatases - by thrombin and other pro-atherosclerotic mediators, and the intracellular signals that result in the generation of oxidative stress. Additional efforts in this area are in determining the mechanisms responsible for modulating thrombin's effects. To study this question, we have generated transgenic mice that express a reporter gene driven by thrombin receptor promoter sequences. By mating these mice with mice that are either pro-atherosclerotic or deficient in antioxidant mechanisms, we will be able to elucidate thrombin receptor expression in development - in mouse embryos, and in atherosclerosis.
2) A second major focus in my lab is to explore the relationship between oxidative damage, mitochondrial dysfunction, DNA damage, and atherosclerosis. We are currently exploring this relationship in several ways: a) use of genetically altered mice which have an increased susceptibility to atherosclerosis: By using a variety of knock-out mice as models, all of which lack genes critical for proper OXPHOS function, we are measuring DNA damage, mitochondrial-DNA (mtDNA) mutation, antioxidant gene expression, and the extent of atherosclerosis formation; b) development of transgenic mice to study specific gene expression and regulation as it relates to oxidative stress: Particularly, the thrombin receptor promoter transgenic mice, described above, will be crossed with existing genetically altered mice deficient in OXPHOS function. The resulting crosses will then be examined for effects of oxidative stress on specific gene expression and regulation important in the development of atherosclerosis; c) use of both healthy and atherosclerotic human aortic tissues to measure mtDNA damage and mutation and their relationship with markers of oxidative stress and atherogenic risk factors in vivo.

Samulski, R.Jude Ph.D: 1. Based on our assessment of our own human gene therapy clinical trials, and a general survey of the field, the UNC Gene therapy Center has elected to focus on expanding our knowledge of the molecular mechanisms involved with the delivery and permanent expression of the therapeutic transgenes. The long range objective is to provide novel therapeutic modalities for treating monogenetic diseases such as Hemophilia and Cystic Fibrosis. A challenge that that has emerged from the analysis of clinical gene therapy trials is the need for vectors capable for higher transduction efficiency than those currently employed. Drawing on the experience of the two gene transfer studies in cystic fibrosis at UNC, the Gene Therapy Center has established the following objectives; (1) translational research with defined clinical endpoints, that provide a basic understanding of efficient gene delivery (i.e. efficiency of transducing stem cells for Fanconi anemia, airway epithelial for CF, and liver cells for hemophilia); (2) the development of high titer viral vectors that offer safe, efficient long-term transgene expression; and (3) the development of novel animal models to help us better understand rate limiting steps in target cell transduction .
2. Current approaches to transfer genes in vivo employ either recombinant viral vectors or non-viral delivery systems. We are engaged in studying the molecular biology of the human parvovirus adeno-associated virus (AAV) with the intent to using the virion shell as a platform for developing a novel, safe, and efficient delivery system for human gene therapy. Our research pioneered the use of recombinant AAV (rAAV) as a gene delivery system for central nervous system, and muscle cells, with vector expression for over 1.5 years without immune consequences or vector toxicity.. The long-term objective is to develop novel delivery systems that exploit the advantages of AAV viral infectivity without the disadvantages of packaging constraints, rate limiting steps involved in second-strand synthesis, or the inability to target specific cell types.

Smithies, Oliver D.Phil: Work in my laboratory over the past 10 years has focused on developing animal models of human genetic diseases. Homologous recombination (gene targeting) is used to alter a chosen gene in a pre-planned manner in mouse embryonic stem cells (ES cells) while they are in tissue culture. The genetically altered ES cells are then injected into normal mouse blastocysts which are introduced into pseudo-pregnant mice to complete their development. Chimeric mice are born which transmit the altered gene to their offspring. By the use of this procedure, we have made mouse models of cystic fibrosis (one of the most frequent single gene defects in Caucasians) and of b-thalassemia and b-thalassemia (among the most frequent world-wide single gene defects).
More recently we have been working towards understanding the genetics of essential hypertension - a complex disease with strong multigenetic and environmental components. Currently we have shown that genetic changes which affect the level of expression of the genes coding for angiotensinogen (AGT), or for renin, or the type 1a receptor for angiotensin II (Atr1a), or the endothelial form of nitric oxide synthase (eNOS), or the atrial natriuretic factor (ANF) or two of its receptors (NPRA and NPRC) affect blood pressures in the mouse. Surprisingly, comparable changes in the gene coding for the angiotensin converting enzyme (ACE) do not alter blood pressures. These several findings are of considerable help in understanding how genetic factors influence blood pressure in humans. The mouse system is particularly valuable because the effects of combinations of genetic changes can be studied, and because environmental influences (such as salt intake) can be varied in a controlled.

Susan S. Smyth MD, Ph.D: The broad goal of my research is to study the molecular basis of vascular disease and then, through creative and innovative approaches, apply that knowledge to improve the diagnosis and therapy of cardiovascular disorders. Arterial damage, such as that which occurs after the rupture of an atherosclerotic plaque or following percutaneous coronary interventions, triggers a stereotypic response that begins with platelet deposition and leukocyte recruitment. Acutely, the damage may elicit the formation of an occlusive platelet-rich thrombus or, over time, may stimulate smooth muscle cell proliferation, migration, matrix deposition, and the development of intimal hyperplasia. The focus of my work is on soluble mediators and cell adhesion receptors that promote interactions between platelets, leukocytes, and the vessel wall. A main approach of my laboratory is to develop and exploit mouse models of cardiovascular disease in which genetic and pharmacologic strategies can be used to identify and define roles for specific molecules in vascular (patho)physiology. My work also has broad implications for the treatment and prevention of tumor angiogenesis, osteoporosis, and other disorders that are mediated by specific adhesion molecules.

Stafford, Darrel Ph.D: My present research is focused in the area of hematology. In the first project we are working on the mechanism of action of the vitamin K dependent carboxylase. We have purified and cloned the carboxylase and shown that it has five transmembrane domains. The substrates of the carboxylase bind via a propeptide and remain bound until all the glutamates are modified. We have also shown that he relative affinities of the propeptides of the different vitamin K-dependent proteins vary at least one hundredfold. Factor X's propeptide has a kd of approximately 2.5 nM while that of prothrombin and proteins C are approximately 250 nM. This knowledge has had practical consequences as it has enabled us, in collaboration with Dr. Kathy High, to show that one can increase the production of factor X in cell culture by exchanging its propeptide for that of prothrombin thus increasing its turnover rate. Furthermore it has provided a rationale for explaining the bleeding problems of patients who were normal in terms of coagulation until a regimen of warfarin was initiated. At this point the patients became phenocopies of hemophilia B. These patients have mutations in their propeptide that reduces their affinity for the carboxylase thus allowing their release before carboxylation is complete. Additional studies have shown that the off rate of the propeptide is higher in the absence of glutamate substrates. This may offer one possibility for the release of the propeptide wants carboxylation is complete.
Our second project has to do with creating more active factor IX molecules for therapy and Gene therapy. For example we have created a hemophilia B. mouse which serves as a model for human hemophilia B. would have also shown that factor IX binds to collagen IV through residues in its Gla domain. This observation has helped explain why factor IX expressed in muscle cells is sequestered in the general area where it is secreted. We have made a mutant Factor IX that does not bind to collagen IV and could therefore be more readily available for coagulation. We have also made a mouse containing factor IX incapable of bind collagen IV. At this point we see no obvious deleterious effects and the mice with this mutant factor IX do not bleed. We have also made a factor IX molecule with approximately four times greater activity than wild type factor IX. It is conceivable that lower levels of this molecule would ameliorate bleeding.

Sulik, Kathy Ph.D: The Sulik laboratory's research has focused on the pathogenesis and mechanisms underlying birth defects induced by a number of teratogens including alcohol, retinoic acid, methotrexate and Ochratoxin. Additionally the genesis of genetically-based syndromes has been explored. Of particular interest has been identification of selectively vulnerable cell populations and mechanistic studies directed toward determining the basis for their sensitivity to teratogenesis. Emphasis has been placed on free radical damage and apoptosis. One research area is focused toward understanding the teratogenesis of alcohol utilizing an animal model in which the craniofacial features typical of Fetal Alcohol Syndrome are induced by acute ethanol exposure at a time corresponding to the third week of human gestation. This work has highlighted the danger of alcohol abuse at a time prior to recognition of pregnancy by most women. Current work is directed toward identification of genes that confer sensitivity to selected embryonic cell populations, examination of the effects of ethanol on membrane lipid rafts, examination of ethanol-induced abnormalities of the developing brain and peripheral nervous system, and identification of agents that may ameliorate ethanol-induced cell death. Our work is also currently directed toward analyses of CNS abnormalities resulting from prenatal cholesterol deficiency. This work utilizes a knockout mouse model for a human mental retardation syndrome that has a significant autism component.

Taylor, Joan M. Ph.D: The long-term goal of my research is to identify signaling mechanisms that contribute to normal and pathophysiological cell growth in the cardiovascular system. I am interested in studying cardiac and vascular development as well as mechanisms involved in heart failure and atherosclerosis. Studies from our laboratory have shown that adhesive interactions with extracellular matrix (ECM) components play a critical role in regulating a variety of intracellular signaling pathways that control cardiomyocyte hypertrophy and smooth muscle cell growth and migration. Each of these processes are critical for proper cardiovascular development and are altered under various pathophysiological stresses such as hypertension, valvular disease and myocardial infarction. We have shown that the protein tyrosine kinase focal adhesion kinase (FAK) is a key regulator of ECM signaling in these cell types and that activation of this kinase is tightly regulated by a unique mechanism in smooth muscle. Furthermore, we have shown that FAK may serve as a link between growth factor- and ECM-induced signaling pathways in these muscle cells. We are currently using gene-targeting approaches to determine the precise role for FAK signaling in cardiovascular development and disease. In addition, we continue to address specific signaling questions using cultured cardiomyocytes and smooth muscle cells in an attempt to identify the point of convergence between integrin- and growth factor- signaling. Understanding the precise mechanisms that govern cardiomyocyte hypertrophy and SMC growth may aid in the development of pharmacologic therapies for several major cardiovascular diseases.

Wang, Da-Zhi Ph.D: Research in our lab aims at understanding the genetic pathways for the formation and function of cardiac and vascular smooth muscle cell type. In particular, we are interested in the transcriptional control of mammalian heart growth and differentiation, vascular smooth muscle differentiation as well as cell proliferation and differentiation-related human cardiovascular disorders, such as cardiac hypertrophy and heart failure. We apply a variety of molecular, cellular, and genetic approaches, including transgenic and knock-out mice, to investigate the in vitro and in vivo functions of myocardin family of transcription factors during mouse development and function.

White, II, Gilbert C. MD: The overall aim of work in Dr. White's laboratory is to understand the role of blood platelets in hemostasis and atherothrombosis. Platelets play an prominent role in the the initial phase of hemostasis and are important in mediating arterial thrombosis that underlies coronary and cerebral ischemic events. Understanding the role of platelets in hemostasis and atherothrombosis should provide new methods for influencing platelet function and for the treatment of atherothrombotic syndromes. Work is going on in two areas: (I) Integrin signaling pathways and (II) cAMP/G protein mediated signaling pathways.
Integrins are platelet surface glycoproteins which mediate platelet adhesion to the vessel wall and aggregation of one platelet with another. The major integrin on the platelet surface, integrin aIIbb3, is an activatable receptor for fibrinogen and the interaction of aIIbb3 with its fibrinogen ligand mediates platelet-platelet aggregation. Studies are aimed at understanding the sequences in the extracellular domains of aIIbb3 which mediate the interaction with fibrinogen. We are also interested in understanding the intracellular signals that activate aIIbb3 and the signals that are generated by the binding of fibrinogen and have shown that aIIbb3 interacts with cytoskeletal proteins, including -actinin and talin, and that signaling to mitogen-activated protein kinase pathways occurs. We are now using high-throughput assays to identify other pathways involved in the activation of aIIbb3.
cAMP is a powerful inhibitor of platelet function, but the mechanism of cAMP action is not well understood. Studies in numerous laboratories have documented inhibition of calcium metabolism, contractile proteins, phosphoinositide metabolism, and other pathways. In order to understand the molecular mechanisms involved in these effects of cAMP, we are examining the downstream pathways in the cAMP cascade. In previous work, we have identified rap1b, a 21 kDa low molecular weight GTP binding protein, as a prominent substrate for cAMP-dependent kinase in platelets. Rap1b is 85% homologous with Ras but is the only low molecular weight G protein which is a substrate for cAMP dependent protein kinase. To study this and other signaling proteins, we are developing a method for platelet specific knockout of signaling proteins which can be used to examine the role of any signaling protein in platelets. These studies should clarify the role of cAMP and G proteins in platelet biology and may provide new ways to inhibit platelet function that may be of use in the treatment of atherothrombotic disorders.

Anderson, Page A. W. MD: Our laboratory focuses on development, cardiovascular diseases, and the heart. This broad scope is reflected by the range of ongoing research projects that are NIH funded. An example of a disease related process that affects vascular integrity is the syndrome that follows cardiopulmonary bypass in the infant. No specific therapy is available for the multi-organ damage that results from inflammation and abnormal vascular permeability. Our investigations are based on our finding that inhibition of the complement cascades markedly decreases organ damage and edema. The experimental protocols make use of an intact piglet model, recombinant proteins, replacement of endogenous myofilament proteins, assessment of calcium regulation, and ventricular and pulmonary function. Other studies focus on the effects of development and heart failure on isoform expression of a thin filament regulatory contractile protein, cardiac troponin T. We have identified in the human and in other species multiple isoforms of cardiac troponin T, sequenced them to identify the exons that are combinatorially and alternatively spliced, generated transgenic animals using these cDNA, and have studied the physiological consequences of altered isoform expression in the intact mouse using echocardiography and cardiac catheterization, isolated intact myocardial preparations, and isolated single cardiac myocytes. Our cross-breeding of these transgenic animals with mouse models of cardiomyopathy will further test the biochemical, physiological, and pathological relevance of these isoforms. Our most recently developed project is a collaboration with Dr. Nadia Malouf at the University of North Carolina at Chapel Hill. These studies are based on our finding that adult-derived clonal stem cells differentiate into endothelial and cardiac myocytes in the mouse and rat in vivo. Our studies are focusing on the biology of engraftment and differentiation and the consequences of these processes on organ function in the normal animal and animal models of human diseases in vivo.

Coffman, Thomas MD: The general focus of our work has been hormone systems that mediate kidney injury. We primarily use transgenic mouse systems for these studies and we have focused primarily on two systems: the renin-angiotensin system and lipid mediators that are derived from arachidonic acid. In general, we have been interested in understanding the role of these systems in regulating blood pressure and kidney function in health and disease. We have been particularly interested in kidney transplant rejection as a model of inflammatory kidney disease and the goal of our studies in this area have been to develop novel targets for therapy.
Our experimental approach has been to produce transgenic mouse lines with targeted disruption of key genes that are involved in the renin-angiotensin or prostaglandin-thromboxane systems. By studying the renal and cardiovascular phenotypes of these animals, we can understand the role of these systems in normal physiology. In addition, by using these animals as donors or recipients of kidney transplants, by inducing disease, or by crossing these mutant mice with other lines that spontaneously develop genetic diseases, we can identify the contributions of the targeted genes to the pathogenesis of transplant rejection and kidney diseases.

Parks, John S. Ph.D: Dr. Parks's laboratory is involved with several projects that focus on high density lipoprotein (HDL) metabolism and atherosclerosis development. HDL concentrations are inversely associated with the development of coronary heart disease. In one project (PO1 HL49373-09, Project 3) we investigate the effect of dietary fat type on HDL subfraction metabolism, using non-human primate and transgenic mouse models of atherosclerosis. Studies are designed to elucidate the pathways of synthesis, intravascular metabolism, and tissue uptake of small, medium, and large HDL subfractions that contain two, three, or four molecules of apoA-I (the major apolipoprotein of HDL) per particle, respectively. We also study the intravascular metabolism of nascent liver perfusate particles to establish the metabolic relationship between newly secreted hepatic HDL and plasma HDL particles. In another project (RO1 HL54176-06) we are investigating the effect of a point mutation in the plasma cholesteryl ester synthesizing enzyme, lecithin:cholesterol acyltransferase (LCAT), that activates the enzyme towards long chain (>18 carbon) polyunsaturated fatty acids. We have used transgenic mice over-expressing the wild-type or mutant form of the enzyme as well as knock-in mice, in which the endogenous mouse gene has been replace by homologous recombination with the human wild type or mutant LCAT, to study the influence of dietary fat type on atherosclerosis development. We have hypothesized that the mice expressing the mutant form of LCAT will have relatively more polyunsaturated CE in plasma, resulting in less atherosclerosis regardless of the source of dietary fat. Finally, in collaboration with Dr. Maeda's lab, we are studying the mechanisms of HDL formation using tissue specific expression of the Abc A1 transporter, which is involved in the formation of nascent HDL particles. We are currently producing Abc A1 conditional knockout mice for the tissue specific expression of Abc A1. These studies will increase our basic knowledge of HDL formation, HDL subfraction metabolism and the interrelationships between HDL metabolism and atherosclerosis development.

Rockman, Howard MD: The major focus of this laboratory is to understand the molecular mechanisms of hypertrophy and heart failure. To achieve this goal, my laboratory uses a strategy that combines state of the art molecular techniques to generate transgenic and gene targeted mouse models, combined with sophisticated physiologic measures of in vivo cardiac function. In this manner, candidate molecules are either selectively overexpressed in the mouse heart or ablated by homologous recombination, which is followed by an in-depth analysis of the physiological phenotype. To model human cardiac disease, we have created several models of cardiac overload in the mouse using both microsurgical techniques and genetic models of cardiac dysfunction.
Specific areas of current research include:1) Signaling: G protein-coupled receptor signaling in hypertrophy and heart failure focusing on the interaction of Phosphoinositide-3 Kinase with ?-Adrenergic Receptors; 2) Identification of Strain Specific Modifiers: Genome mapping of microsatellite markers by SSLP to identify gene modifiers of the heart failure phenotype using mouse models of disease; 3) Molecular physiology. In depth physiological analysis of cardiac function in genetically altered mice to understand the role of G protein-coupled receptor signaling pathways on the development of heart failure in vivo.