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 Cardio Training Grant | Postdoctoral Fellowships | Past Postdoctoral Fellows

Past Postdoctoral Fellows

Lauren E. Ball, Ph.D.
Mentor: Maria G. Buse, Ph.D., Department of Medicine, Division of Endocrinology

Micro- and macro-vascular complications associated with Type 1 and Type 2 diabetes mellitus (T1DM, T2DM) cause substantial morbidity and mortality in diabetic patients and contribute to cardiovascular disease, the leading cause of premature death among these patients. Resistance to insulin action, observed predominately in T2DM patients, and hyperglycemia are both risk factors for the development of cardiovascular disease. Increased flux through the hexosamine biosynthetic pathway (HBP) has been postulated to contribute to the pathogenic effects of hyperglycemia. Excess glucose entering the HBP is converted to UDP-GlcNAc, a sugar donor for the posttranslational glycosylation of Ser/Thr residues by O-linked N-acetylglucosamine monosaccharide (GlcNAc). The dynamic and reversible nature of this modification has prompted studies investigating the role of this modification in providing an alternative mechanism of protein regulation to phosphorylation. O-GlcNAc modification has been proposed to regulate protein function in response to changes in the nutrient supply. The aim of this project is to determine the contribution of protein O-GlcNAc modification to glucose-induced insulin resistance and to the devlopment of complications associated with hyperglycemia. Overall, the goal of this work is to elucidate potential therapeutic targets for the prevention or reversal of insulin resistance and complications of diabetes.

During the past year and a half we investigated the temporal relationship of hyperglycemia induced O-GlcNAc modification of hepatic proteins with the development of insulin resistance. We have demonstrated for the first time that mild hyperglycemia, modeling the degree of hyperglycemia seen in T2DM patients, induces a change in protein O-GlcNAc modification in vivo. Furthermore, the increase in protein O-GlcNAc modification in hepatic proteins occurs within 3 hrs of glucose infusion and precedes the development of insulin resistance. We are in the process of preparing a manuscript describing these observations.

In parallel studies, we have focused our attention on mapping the sites of O-GlcNAc modification of the insulin receptor substrate 1 (IRS-1) with the goal of determining how this modification may impact insulin signaling at the level of IRS-1. The methodology for mapping the sites of protein O-GlcNAc modification by mass spectrometry has been optimized. So far we have identified one peptide of IRS-1 that is O-GlcNAc modified and fully anticipate that within the very near future the exact site of modification will be determined and other putative sites of O-GlcNAc modification will be identified. We are in the process of preparing a manuscript describing the technological advances enabling the detection and identification of sites of O-GlcNAc modification and the sites of modification identified in IRS-1.

Marion Cooley, Ph.D.
Mentor: Scott Argraves, Ph.D., Department of Cell Biology

Dr. Cooley studied focal adhesion kinase (FAK) function to obtain her Ph.D. from the Department of Cellular and Molecular Biology at University of South Carolina in Columbia, South Carolina. In Dr. Argrave's laboratory she studied fibulin-lD, an extracellular matrix protein implicated in cardiac morphogenesis. In endocardial cushion and epicardium, fibulin-lD expression is associated with migrating cells, suggesting a role of fibulin-1D -in regulating cell motility. To better understand the function of fibulin-lD in heart development, she used yeast two-hybrid analysis to identify novel fibulin-lD binding proteins. She has identified several potential binding partners of fibulin-lD, including bone morphogenetic protein-1 (BNT-1) and novel serine protease-1 (NSP-1). She also investigated the interaction of BNT-1 and NSP-1 with extracellular matrix proteins.

Erin Eaton, Ph.D.
Mentor: Steven A. Rosenzweig, Ph.D., Department of Cell and Molecular Pharmacology

Dysregulation of the insulin-like growth factor (IGF) system is implicated in a number of human cancers, including breast, prostate and colon cancer. Aberrations in the natural balance of the system include up-regulation of the IGF-1 receptor, as well as increased concentrations of the IGF-1 receptor ligands, IGF-1 and IGF-2. Our lab is interested in utilizing the third component of the IGF system, the IGF binding proteins (IGFBPs), to design a small-molecule inhibitor of IGF action. The six IGFBPs (IGFBP-1 through 6) are natural antagonists to IGF action, acting by binding IGF-1 and IGF-2 and sequestering the growth factors away from the receptor. Currently, we are characterizing the action of IGFBP-2 upon breast cancer cell lines; we have demonstrated that addition of IGFBP-2 inhibits cell proliferation of MCF-7 breast cancer cells. The mechanism of this inhibition appears to be, in part, due to inhibition of IGF-1 stimulated S-phase entry. We are examining downstream effectors of proliferation, such as ERK-1/-2 to determine which pathways are blocked by IGFBP-2 addition. As IGF-1 is a potent anti-apoptotic factor, as well as a mitogenic factor, we are also investigating the effects of IGF-1 blockade upon apoptosis of these cells.

Interestingly, we have observed that IGFBP-2 added concomitantly with IGF-1 is degraded after 24 hours in culture with MCF-7 cells; this degradation does not occur when IGFBP-2 is added alone. This implies that an as yet unidentified protease is cleaving the exogenously added IGFBP-2 in an IGF-dependent manner. We are undertaking steps to characterize and identify this protease; its existence underscores the importance of developing protease-resistant mimetics of IGFBP action. To this end, our laboratory will perform cell growth and apoptosis assays utilizing recombinant fragments of IGFBP-2 which bind IGF-1 to determine the biological efficacy of these peptides in inhibiting IGF action.

Lisa Ervin, Ph.D.
Mentor: Kevin L. Schey, Ph.D., Department of Cell and Molecular Pharmacology

My scientific training over the past year has involved the utilization of mass spectrometry to study post-translational modifications of proteins. New skills I have gained include the techniques in isolating and preparing membrane proteins, utilizing MALDI-TOF and LC-MS/MS, and interpreting mass spectrometric data. Specific projects include studying post-translational modifications of the major intrinsic protein (MIP), an aquaporin, in the lenses of children with cataracts and studying modifications of bovine and human membrane protein 20 (MP20). Preliminary data indicates there is an unusual type of glycosylation of MP20. Future research directions include studying protein modifications of MIP in lenses with senile or diabetic cataracts, determining other sites and types of modifications of MP20, and developing methods to characterize glycosylated MP20. Studying the post-translational modifications of MIP that occur during aging or cataract formation may give important information that applies to other diseases involving aquaporins such as hyponatremia in advanced heart failure and congenital nephrogenic diabetes insipidus. By utilizing mass spectrometric techniques such as precursor ion scanning, we will be able to determine other sites of the unusual type of glycosyation of MP20 which may also be used to find this type of glycosylation in other proteins.

Maria Hamilton, Ph.D.
Mentor: John Hildebrandt, Ph.D., Department of Pharmacology

Dr. Hamilton received her Ph.D. from the Department of Pharmacology and Cancer Biology at Duke University for her work on the characterization of a novel ubiquitin E3 ligase, hRPF1/Nedd4, and the identification of its substrates. She joined Dr. Hildebrandt's laboratory to apply her knowledge of the ubiquitinylation pathway to a problem involving an important cell signaling system, the heterotrimeric G proteins. She will follow up on work generated by another fellow, Dr. Lana Cook, implicating ubiquitin-mediated pathways in determining the turnover and trafficking of the heterotrimeric G proteins. From these studies she will complement her knowledge of cell biology and molecular biology, with biochemistry and protein chemistry, and their application to a new field of study, signal transduction. Particularly important to her work and her future research potential will be her training in the use of mass spectrometry applications to the characterization of posttranslational modifications of proteins.

Korey R. Johnson, Ph.D.
Mentor: Lina M. Obeid, M.D., Department of Medicine, Division of General Internal Medicine

Our research goals center around studying the enzymatic regulation of sphingosine-1-phosphate (S1P) levels. With the emergence of S1P as a ligand for the endothelial differentiation gene (EDG) G-coupled receptor, it has become evident that this lipid is involved in such biological processes as cell growth, differentiation, migration, angiogenesis, and apoptosis. This sphingolipid has been implicated to play an important role in both neoplasia and atherosclerosis. Sphingosine-1-phosphate production occurs in response to a variety of stimuli, including growth factors, cytokines, and G-protein-coupled receptor agonists. Sphingosine kinase (SK1) catalyzes the production of this sphingolipid, while sphingosine-1-phosphate phosphatase (SPPase) dephosphorylates the molecule. Having recently cloned these two enzymes, we are currently in the process of elucidating the mechanisms regulating their respective activities. Dr. Johnson received his Ph.D. from the Medical University of South Carolina.

Eric Kilpatrick, Ph.D.
Mentor: John D. Hildebrandt, Ph.D., Department of Cell and Molecular Pharmacology

Many of the external factors regulating the normal and pathological states of cardiac cellular function are mediated by the activation of G-proteins. G-proteins provide transduction of extracellular signals to intracellular effectors and are composed of three separate subunits, α, β and γ, each with multiple isoforms. My research is attempting to understand at a fundamental level the role of G-protein isoform heterogeneity on signaling specificity. One of the first questions I am examining is the relative preference of endogenous α subunits for different exogenously expressed FLAG-labeled βγ isoforms. A second question of importance is how certain factors important to cardiac function, such as acetylcholine, adenosine or opioid agonists, can have different functions even though they are thought to act through common G-proteins. My research will attempt to determine whether in fact these agents use common pathways or are instead utilizing different G-protein isoforms to achieve distinct functions.

Eric Klett, M.D.
Mentor: Shailesh Patel, M.D., DPhil, FRCP, Department of Medicine, Division of Endocrinology

Sitosterolemia (also known as phytosterolemia) is a rare autosomal recessively inherited metabolic disorder that was first described in two sisters in 1974. The clinical presentation includes tendon xanthomas (usually involving the Achilles tendon), hemolytic episodes, arthritis/arthralgias and most strikingly accelerated atherosclerosis. Demonstrating increased plasma and tissue levels of plant sterol makes the diagnosis of sitosterolemia.

Previous studies have suggested that the defect in sitosterolemia may involve a sterol transporter expressed solely in the liver and intestine. By using linkage analysis and positional cloning of sitosterolemia pedigrees our lab isolated two genes named ABCG5 and ABCG8 to chromosome 2p21. These genes encode half-transporters characteristic of the ABC family of proteins termed ABCG5/sterolin-1 and ABCG8/sterolin-2 respectively. Specific aims now are to characterize the function of the proteins and how they relate to the development of premature atherosclerosis.

Over the past year, I have worked extensively on a knockout mouse model deficient in Abcg8/sterolin-2. Specifically, I have characterized this mouse model by looking at: 1) gene expression by real time quantitative RT-PCR of genes essential in cholesterol homeostasis, 2) effects of deficiency of Abcg8/sterolin-2 on biliary sterol secretion (absorption studies are difficult in animals that non-selectively absorb dietary sterols, so thus have not been done at this point), 3) the cellular localization of these proteins in the liver and intestine. This work has culminated in a manuscript submitted to BMC Gastroenterology. This work has led to more questions and currently more studies are underway to characterize these proteins' role in cholesterol homeostasis and development of atherosclerosis.

Amanda LaRue, Ph.D.
Mentor: Makio Ogawa, M.D., Ph.D., Department of Medicine, Division of Experimental Hematology

The overall focus of our laboratory is the plasticity of adult murine hematopoietic stem cells (HSCs); studies that are critically dependant on establishing methods for high efficiency purification and transplantation of a single HSC. To date, my efforts in the laboratory have been directed at learning the basic concepts and techniques of the field, the development of new methods for high efficiency transplantation of a single murine HSC and examination of the differentiation potential of the HSC.

During the last year, we have focused on the development of new methods for high efficiency transplantation of a single murine HSC based on Hoechst 33342 staining and Side Population (SP) cell identification. Recent works involving HSC purification have taken advantage of the ability of HSCs to efficiently efflux dyes such as Hoechst 33342 (Goodell et al., 1996; Goodell et al., 1997). This population of cells, referred to as the Side Population, has been shown to have hematopoietic reconstituting ability in vivo. In the last year, remarkable advances have been made in the purification of mouse HSCs based on sorting of cells which are from the “Tip” subpopulation of SP cells (cells which showed strongest dye efflux) and have the Lin- c-kit+ Sca-1+ CD34- phenotype (Matsuzaki et al., 2004). In light of these findings, our laboratory has begun to supplement our original methods for purification of HSCs based on cell surface antigens with a purification scheme based cell surface antigens in combination with Hoechst dye efflux. Although methods for isolating potential HSCs based on dye exclusion had previously been published, adapting this method has been very time consuming, as key information in the published studies was not adequately described. Within the last year, we have achieved success in identifying “Tip” cells from the side population. This method, combined with our methods for sorting based on cell surface antigens, has allowed isolation and transplantation of a single “Tip” cell that is phenotypically CD34-Sca1+ and has resulted in a marked increase in the number of transplanted mice that exhibit high levels of engraftment. In addition to these advances, we have recently obtained a MoFlo cell sorter (DakoCytomation), which allows for high efficiency purification of low incidence cells (Benveniste et al., 2003; Parmar et al., 2003; Olmstead-Davis et al., 2003), and are currently establishing protocols for its use.

Using these new purification and transplantation methods, we have demonstrated that HSCs can give rise to fibroblasts and fibroblast-like cells in various tissues including heart valves, infarcted myocardium and solid tumors (manuscripts in preparation). The direction of my future studies are three pronged: 1) to characterize HSC-derived cells within these tissues, 2) to determine their transitional phenotype from the bone marrow to the tissue site, and 3) evaluate the mechanisms by which these cells home to and incorporate these tissues.

Margaret Markiewicz, M.D.
Mentor: Maria Trojanowska, Ph.D., Department of Medicine, Division of Rheumatology and Immunology

The main area of research in this laboratory is regulation of extracellular matrix (ECM) deposition in healthy tissues and its dysregulation in diseases. There are two interrelated areas of investigation. The first is analysis of gene regulation of collagen type I and tenascin in healthy and fibrotic cells. These studies include analyses of cis-acting elements and cognate transcription factors in collagen and tenascin promotors regulating basal levels and responses to cytokines. Current emphasis is on the role of post-translational modifications of selected transcription factors, which are involved in the formation of collagen promoter-specific multiprotein complexes. The second area of investigation is the analysis of the signal transduction mechanisms involved in ECM turnover. Specifically, we focus on the interaction of the TGF-beta receptor signaling pathway with other signaling pathways involved in abnormal ECM turnover in fibrosis and cancer. These studies utilize adenoviral-mediated delivery of wild-type and mutated TGF-beta receptors and other signaling molecules, as well as transcription factors. A variety of other techniques are used to pursue these studies, including transient transfections, gel shifts and supershifts analyses, DNA footprinting and yeast two-hybrid screens.

Dr. Markiewicz, who received her M.D. from Wroclaw Medical University, is currently working on the role of human cytomegalovirus (hCMV) in pathogenesis of scleroderma. Her research is based on our novel observation indicating that gene products of the hCMV major immediate early locus (MIE), termed IE1 and IE2, are capable of inducing the fibrotic program in human dermal fibroblasts.

Daniel Shegogue, Ph.D.
Mentor: Maria Trojanowska, Ph.D., Department of Medicine, Division of Rheumatology

Dr. Shegogue received his Ph.D. from the Department of Microbiology and Immunology at the Medical University of South Carolina where he studied type I collagen synthesis. This laboratory has recently published data showing an interaction between transforming growth factor beta (TGF beta) and sphingolipid signaling pathways. TGF beta and sphingosine kinase are recognized as important contributors in angiogenesis and cancer. Current work involves extending our original observations to define the interaction between sphingosine kinase and the TGF beta signaling pathway.

Rick Visconti, Ph.D.
Mentor: Christopher Drake, Ph.D., Department of Cell Biology and Anatomy

Dr. Visconti comes to MUSC from a postdoctoral position in the Department of Cell Biology at University of Texas Medical Center at Dallas. He received his Ph.D. from Temple University and is joining the laboratory of Dr. Drake to pursue an academic research career directed at elucidating the cell biological and molecular mechanisms regulating vasculogenesis and angiogenesis. He will use a mouse vasculogenesis assay recently developed by Dr. Drake, in conjunction with microarray analysis, to analyze patterns of gene expression that distinguish pre-endothelial cells (angioblasts) from definitive endothelial cells. A long term goal of this project is to identify novel genes and pathways that control neovascular processes.

Jenny Walgren, Ph.D.
Mentor: Maria Buse, M.D., Department of Medicine, Division of Endocrinology

Dr. Walgren received her Ph.D. from the Department of Pharmacology at the Medical University of South Carolina where she studied trichorolethylene metabolism and its effects on peroxisome proliferation in hepatocytes. She had to remain in the Charleston area after the Ph.D. while her spouse finished the MSTP program. During this time she became particularly interested in pursuing research with a clinical basis and development of a career in drug development. She is currently working in the laboratory of Dr. Buse, who is studying the molecular mechanisms of insulin resistance as well as the mechanisms resulting in the complications associated with diabetes. The research of Dr. Buse focuses on alterations in metabolism, gene expression, signal transduction, and protein transport in diabetic conditions. Dr. Walgren is studying the molecular mechanisms of insulin resistance, as well as mechanisms associated with diabetic complications. She is using cell culture and animal models to investigate protein modifications (glycosylation of proteins) that may contribute to the pathology associated with diabetes, including cardiovascular complications.

Kimberly J. Worsencroft, Ph.D.
Mentors: Perry V. Halushka, M.D., Ph.D., Department of Pharmacology
G. Patrick Meier, Ph.D., Department of Pharmaceutical Sciences

Dr. Worsencroft came from Emory University where she obtained a Ph.D. degree from the Department of Organic Chemistry studying the mechanism and synthetic utility of ammonium and carbonylylides. In coming to MUSC to work with Drs. Halushka and Meier, she had the opportunity to apply her chemistry background to an important fundamental problem while acquiring a biological perspective for future research. The focus of their work has been the study the interactions necessary for optimal binding between thromboxane A2 (TxA2) and its receptor. Their efforts are directed towards the synthesis of a series of TxA2 receptor ligands that can affinity-label the TxA2 receptor and subsequently identify the regions of interaction between TxA2 ligands and the receptor. Elucidation of these interactions would aid in the development of better therapeutic agents in the treatment of cardiovascular diseases and as well as provide a better understanding of signal transduction in G-protein coupled receptors.

William Yarbrough, M.D.
Mentor: Francis G. Spinale, M.D., Ph.D., Department of Surgery, Division of Cardiothoracic Surgery

Cardiovascular disease is a major cause of morbidity and mortality and unlike other diseases, the relative incidence is increasing. A common cardiovascular disorder is coronary artery disease which can result in myocardial infarction (MI). It is now recognized that structural changes occur within the left ventricular (LV) wall following MI which lead to alterations in LV geometry and function. This process has been termed Òmyocardial remodeling. Our laboratory is actively investigating the cellular and molecular basis for post-MI remodeling. Specifically, a family of proteolytic enzymes called the matrix metalloproteinases (MMPs) have been implicated to contribute to myocardial remodeling following MI. In order to more closely examine the mechanistic relationship between MMPs and the post-MI remodeling process, several experimental systems have been developed by this laboratory. These model systems will allow for identifying the temporal expression of myocardial MMPs post-MI, which species of MMPs are expressed within the myocardium post-MI, and finally interventional strategies to interrupt MMP activation post-MI. The central hypothesis to be tested in these studies is that a specific portfolio of MMPs are expressed within the myocardium following MI and directly contribute to the progression of LV dilation and dysfunction. The results from these studies will likely yield new therapeutic targets for reducing the degree of pathological remodeling which occurs post-MI and thereby reduce the progression to LV dysfunction. Dr. Yarbrough received his M.D. from the University of South Carolina.

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