Joey Barnett - US grants

Peptide growth factors, such as the Transforming Growth Factor beta (TGFbeta) family, play central roles in morphogenesis and organogenesis. The narrowly defined vertebrate TGFbeta family is composed of at least three 25 kilodalton homodimeric proteins, TGFbeta1, TGFbeta2, and TGFbeta3. TGFbeta1 and TGFbeta 3 share identical ligand binding and biological activities while TGFbeta2 has a unique requirement for the Type III TGFbeta receptor (TBRIII) and distinct biological activities. We have recently demonstrated a requirement for TBRIII in TGFbeta-mediated epithelial-mesenchymal cell transformation that occurs in the atrioventricular (AV) cushion of the developing heart. We are testing the hypothesis that a unique receptor signal transduction complex is responsible for AV cushion transformation. Since current models of TGFbeta signal transduction presuppose a requirement for a Type I receptor (TBRI) in the signal transduction complex, initial experiments will determine if a Type I receptor (TBRI) is a component of the receptor complex in the AV cushion. Specifically, we will determine whether the ALK2 or ALK5 TBRI is required for transformation. This will be determined both by misexpression of constituitively active ALK2 and ALK5 and antisense constructs. A well described pathway for downstream signal transduction from TGFbeta receptors includes the Smads family of transcription factors. We will test the hypothesis that specific Smads are necessary for AV cushion transformation by , determining if dominate negative inhibitors of Smads or constitutively active Smads alter transformation in AV cushion explants and ventricular explants. Preliminary data in our laboratory suggests a role for the ALK2, and not ALK5, in transformation. Therefore we will test the hypothesis that ALK2 associates with TBRIII to mediate transformation by immunoprecipitation of the TGFbeta receptor complex and identification of ALK2 and ALK5. We also will immunolocalize TBRI and TBRIII on both chick embryonic fibroblasts and AV cushion endothelial cells to determine whether they associate after the addition of TGFbeta. These experiments are part of a concerted strategy to determine whether TBRIII requires a TBRI and downstream Smad signaling. Our long-term goal to understand the role of TGFbeta in cushion transformation and the genesis of congenital heart defects due to abnormal cushion transformation.

The Morphology and Imaging Core is critical to the support of the studies proposed. The function of the Morphology and Imaging Core is briefly outlined in four areas: 1) To provide and process specimens for in situ hybridization and immunohistochemistry; 2) To provide a facility for imaging, data, data storage and retrieval; 3) To provide training and support for all aspects of morphological analysis; and 4) To assay selected protein and gene markers to provide a comprehensive analysis of coronary vessel development. The Morphology and Imaging Core will focus our resources to provide consistent, state-of-the-art tissue processing, imaging and analysis for each Project. The Core will also act as a mechanism to disseminate results between Projects and to develop, create and maintain a comprehensive data base for gene expressing in the developing and mature coronary vasculature. Given the reliance of each project on morphological analysis, each Project will use a significantg amount of the resources available in the Morphology and Imaging Core. The support of this Core is essential to completing the stated aims of each of the four individual projects.

Cells destined to form the coronary vessels reside in the pro-epicardial organ (PEO) prior to migrating toe the developing heart tube. These cells form an epithelial layer over the heart and undergo epithelial- mesenchymal transformation and subsequent migration into the heart, followed by vessel formation. Similarly, the migration of neural crest cells into the heart is required for proper cardiogenesis, including coronary artery patterning and the septation of the outflow tract. We have shown that the Type III TGFb receptor (TBRIII) is required for epithelial- mesenchymal transformation and mesenchymal cell migration in the atrioventricular (AV) cushion of the heart. We will test the overall hypotheses that TBRIII is a mediator of epithelial-mesenchymal migration. We will use experimental embryological approaches in chick and genetic approaches in the mouse to gain a comprehensive understanding of the role of TBRIII in these events as they relate to coronary vessel development. Preliminary data in the chick demonstrate that TBRIII is expressed by both migrating neural crest cells and cells of the PEO. In addition, anti-TBRIII antisera inhibits the migration of neural crest cells in in vitro explant assays. The experiments we propose will determine the function of TBRIII in coronary vessel formation. The determination of which TGFb signal transduction complexes mediate cell transformation and cell migration will give general insight into TGFb signal transduction. Coronary vessel disease is a major cause of death in humans. A clearer understanding of coronary vessel development may identify novel approaches to treating coronary disease.

DESCRIPTION (provided by applicant): The integration of our scientific knowledge of cardiovascular function at the molecular, cellular, and organismal levels is critical to impacting cardiovascular disease in humans. As our insight into molecular and cellular processes has grown, emphasis in many Ph.D. training programs has shifted away from integrated (whole animal or systems) biology. The lack of an available scientific workforce with an understanding of integrated biology, including drug metabolism and pharmacokinetics, and an appreciation of the unique challenges of working in intact organisms will delay the application of discoveries at the molecular and cellular levels to human disease. This training program addresses a critical need for scientists trained in the integrated biology of the cardiovascular system. Our program uses existing strengths in the Department of Pharmacology at Vanderbilt University and key collaborators who direct well established research programs in cardiovascular pharmacology and biology. The primary activity of trainees is research training on issues related to cardiovascular function and disease under the direction of an individual faculty mentor. The usual duration of training is 2-3 years and fellows with Ph.D. and M.D. degrees will be supported. Research training is supplemented with focused didactic lectures on drug metabolism and pharmacokinetics, cardiovascular physiology, and cardiovascular pharmacology (all part of the existing core curriculum of the Predoctoral Pharmacology Training Program). Fellows will be required to participate in Responsibility in Research training and weekly seminar series in Experimental Therapeutics, Cardiovascular Medicine, and Pharmacology. Trainees may elect to participate in the Vanderbilt Master of Science in Clinical Investigation Program. Our specific goals are to provide trainees with expertise in the integrated biology of the cardiovascular system, encourage collaboration between laboratories using whole animal and molecular approaches, and develop scientists trained to participate in drug discovery and drug development in the cardiovascular system. Our overall goal is to train scientists who will be leaders in academia, industry, or regulatory affairs.

DESCRIPTION (provided by applicant): The integration of our scientific knowledge at the molecular, cellular, and organismal levels is critical to impacting disease in humans. As our insight into molecular and cellular processes has grown, emphasis in many Ph.D. training programs has shifted away from integrated (whole animal or systems) biology, including the biology of humans. The lack of an available scientific workforce with an understanding of integrated or human biology, including drug metabolism and pharmacokinetics, and an appreciation of the unique challenges of converting discoveries into therapies will delay the application of discoveries at the molecular and cellular levels to human disease. Scientists trained in the disciplines of Pharmacology and Physiology form an important part of this necessary workforce. This meeting addresses critical issues related to training Pharmacologists and Physiologists and has been planned in response to a perceived and real decline in training programs in these disciplines. The goals of this meeting are to identify common problems and opportunities that face Ph.D. training programs in Pharmacology and Physiology and how best to provide training in these disciplines that are key to translational research. The target audienc for this meeting is Directors of Graduate Training Programs in Pharmacology, Physiology, or other biomedical sciences. This meeting will provide a unique opportunity for these scientists to exchange information and to interact with leaders in industry, government, and academia who help shape these disciplines. A continuing dialogue among Directors of training programs and other scientists with an interest in Ph.D. training is identified as a desired major outcome.

PROJECT SUMMARY This is a continuing application for a Ph.D. (only) program in the Pharmacological Sciences. Graduate student trainees from Vanderbilt University or Meharry Medical College may be supported for up to two years of graduate training, generally years 2 and 3 of training. The first year of training in biomedical sciences at Vanderbilt University is Interdisciplinary, and involves participation in an academic year long core curriculum. Students select preceptors in the Pharmacological Sciences Training Program in May at the end of the first year of graduate school before beginning the required course work for this graduate program. Program specific curriculum includes: Receptor Theory Module (short course); Fundamentals of Pharmacology: Receptor Theory & Signal Transduction Pathways; Drug Metabolism & Pharmacokinetics; Scientific Communication Skills I & II; Targets, Systems, and Drug Action; Experimental Design and training in Responsible Conduct of Research. In addition, students take at least six additional hours of elective courses to accommodate individual interests and needs. The curriculum for Meharry trainees is similar and cross registration agreements between Vanderbilt and Meharry results in Meharry trainees completing at least some of the above courses with Vanderbilt trainees. Research opportunities are available in several areas at both institutions: Receptor Mechanisms and Signal Transduction, Molecular mechanisms for Cellular Regulation, Neuropharmacology, Cardiovascular Pharmacology, Cancer Pharmacology, Pharmacology of Anti-Infective Agents, Clinical Pharmacology, Drug Discovery, and Drug Disposition and Pharmacokinetics. Course work is complemented by other important didactic experiences: weekly Journal Club; a student-invited annual Pharmacology Forum; an annual Department of Pharmacology Retreat for presenting upcoming research goals; and exchange programs between Vanderbilt and Meharry Medical College to strengthen the broad-based education of our students as well as to foster the careers of under-represented minorities in research. The training environment is further strengthened by partnerships with the Lipscomb College of Pharmacy, the ability of trainees to participate in a Program in Molecular Medicine, and the presence of a highly successful, in-house Drug Discovery Program. Comprehensive qualifying examinations and defense of the dissertation proposal precedes dissertation research. After admission to candidacy for degree, dissertation committee meetings occur frequently to facilitate mentoring. The breadth of interest of the faculty, their international recognition and success in acquiring extramural peer-reviewed support, their understanding of the scientific process, and an emphasis on scientific integrity make our program an outstanding environment for the training of independent scientists in the area of pharmacological sciences.

DESCRIPTION (provided by applicant): Coronary artery disease accounts for 54% of all cardiovascular disease in the United States [1]. Understanding how coronary vessels develop is likely to uncover novel drug targets and therapeutic strategies useful in directing the repair or remodeling of coronary vessels in adults. Recent data from our laboratory has demonstrated that targeted deletion of the Type III Transforming Growth Factor 2 receptor (TGF2R3) results in embryonic death associated with failure of the formation or persistence of the coronary vessels while other vascular beds appear to develop normally. Experiments are proposed to identify how the loss of TGF2R3 results in the failure of coronary vessel development and to reveal the TGF2 signaling pathways that regulate epicardial cell differentiation. We will test 3 specific hypotheses. Hypothesis 1: TGF2R3 is required for proper epicardial differentiation. This will be tested by in vivo analysis of the epicardium and epicardial derivatives in wildtype, heterozygous Tgfbr3 null, and homozygous Tgfbr3 null animals. Hypothesis 2: TGF2R3 is required in the epicardium or myocardium for proper coronary vessel development. This will be tested by conditional deletion of Tgfbr3 in the epicardium and myocardium. Hypothesis 3: Specific TGF2 signaling pathways regulate epicardial cell differentiation. This will be tested in vitro in both explants and immortalized cells from wildtype, heterozygous Tgfbr3 null, and homozygous Tgfbr3 null animals The successful completion of the proposed experiments will yield significant insight into the role of Transforming Growth Factor 2 (TGF2) in coronary vessel development. The unique nature of the derivation of the coronary vessels and the significance of coronary vessel disease in humans makes the determination of the mechanisms behind TGF2R3 regulation of coronary vasculogenesis of high significance from both a basic science and clinical perspective.

DESCRIPTION (provided by applicant): This is a continuing application for a Ph.D. (only) program in the Pharmacological Sciences. Graduate student trainees from Vanderbilt University or Meharry Medical College may be supported for up to two years of graduate training, generally years 2 and 3 of training. The first year of training in biomedical sciences at Vanderbilt University Medical Center is Interdisciplinary, and involves participation in an academic year long core curriculum. Students who select preceptors in the Pharmacological Sciences Training Program in May of their first year then begin the required course work for this graduate program. Program specific curriculum includes: Systems, Targets, and Drug Action (includes in vivo physiology, drug disposition, and principles of drug action); Receptors and Signal Transduction; Scientific Communications; and Biostatistics. In addition, students take at least six additional hours of elective courses to accommodate individual interests and needs. The curriculum for Meharry trainees is similar and cross registration agreements between Vanderbilt and Meharry results in Meharry trainees completing at least some of the above courses with Vanderbilt trainees. Research opportunities are available in several areas at both institutions: Receptor Mechanisms and Signal Transduction, Molecular mechanisms for Cellular Regulation, Neuropharmacology, Cardiovascular Pharmacology, Pharmacology of Arachidonic Acid Metabolites, and Drug Disposition and Pharmacokinetics. Course work is complemented by other important didactic experiences: weekly Journal Club; a student-invited annual Pharmacology Forum; an annual Department of Pharmacology Retreat for presenting upcoming research goals; and exchange programs between Vanderbilt and Meharry Medical College to strengthen the broad-based education of our students as well as to foster the careers of under-represented minorities in research. The training environment is further strengthened by emerging partnerships with the Lipscomb College of Pharmacy, the ability of trainees to participate in a Howard Hughes Medical Institute-funded Program in Molecular Medicine, and the presence of a highly successful, in-house Drug Discovery Program. Comprehensive qualifying examinations and defense of the dissertation proposal precedes dissertation research. After admission to candidacy for degree, dissertation committee meetings occur frequently to facilitate mentoring. The breadth of interest of the faculty, their international recognition and success in acquiring extramural peer-reviewed support, their understanding of the scientific process and an emphasis on scientific integrity make our program an outstanding environment for the training of independent scientists in the area of pharmacological sciences.

Project Summary This is a new application for a short term (summer, 10 week) training program in cardiovascular science for under-represented, undergraduate scholars. We propose a unique training experience that builds upon existing collaboration with a core group of Historically Black Colleges and Universities (HBCUs) and the American Heart Association/American Stroke Association (AHA/ASA) that seeks to create a new paradigm promoting the pathway to health science careers and aspirational life choices for a cadre of under-represented undergraduate scholars. The proposed program provides an intellectually, socially and culturally rewarding experience to engage scholars at multiple levels. We leverage the expertise Vanderbilt University to cultivate academic knowledge and research skills and AHA/ASA's public health leadership position to provide an integrated training program against a social determinants of health backdrop. We will provide a 10 week research intensive immersion for a cadre of 10 students, expanding to 15 in years 4 and 5. We have a pool of distinguished mentors with diverse interests related to cardiovascular science and disease. The leadership team is composed of senior individuals with overlapping expertise in cardiovascular disease, mentoring, and community engagement. In addition to an intense, mentored research experience in the area of cardiovascular science, we will offer unique perspectives and resources, provide a productive framework to fully understand health inequities, and foster invaluable connections among scholars and their mentors and communities. We posit that students on the path to professions in science, medicine, healthcare or public policy must also be trained on real-life community engagement and cultural competency. They must be aware and knowledgeable of the forces shaping the health disparities so prevalent in the nation, in addition to the social determinants of health that determine the nature and severity of those disparities. Our overall goal is ambitious and innovative; we seek to create a cadre of students that will ultimately become the integrators needed to connect science and systems of health care with underrepresented communities through their academic and professional credentials, ability to build and retain trust, and cultural competence skills. We will fulfill this vision through: an intensive academic research and training immersion experience, with mentoring as a central component; regular opportunities to interact with their peers and acquire the core values of collaboration, teamwork and personal enrichment; and exposure to AHA/ASA initiatives targeted to address health inequities through public awareness, education, policy and social change. Together, we will thus provide an exciting and comprehensive experience to minority scholars poised to make career-defining, life-changing decisions with future implications for the biological, behavioral, and social determinants of health across the nation.

Project Summary In this new application we propose a unique training experience that builds upon Vanderbilt University's (VU) extraordinary record of research, training, and fostering diversity in biomedical science and that seeks to create a new paradigm promoting the pathway to biomedical science careers and aspirational life choices for a cadre of underrepresented, postbaccalaureate scholars. This program provides an intellectually, socially and culturally rewarding experience to engage scholars at multiple levels throughout a 12 month training program. We will leverage VU's training expertise to cultivate knowledge and research skills and provide an integrated training program that supports resilience by fostering connections among program participants and their research mentors, peers, and respective communities. A strong multiple-PI leadership team is composed of senior individuals with overlapping expertise in scientific research, mentoring, inclusion, skill development, trainee engagement, and fostering a community of diverse scholars. This team is supported by internal and external board members with demonstrated commitment to training and diversity. We will provide a research intensive immersion for carefully-selected scholars each year. Our pool of distinguished mentors have a diversity of research interests that are aligned with the mission of NIGMS. In addition to an intense, mentored research experience, we will offer unique perspectives and resources, provide a productive framework to fully understand the promise and process of scientific discovery, and foster invaluable connections among scholars and their mentors and communities. We posit that scholars, most especially those from underrepresented groups, must also be trained in how to relate their work to the larger community. They must be aware and knowledgeable of the forces shaping the public's perception of science and how to shape the scientific narrative in both underrepresented and majority communities. Our overall goal is ambitious and innovative; we seek to create scholars that will become the integrators needed to connect science with communities through their academic credentials, ability to build and retain trust, and cultural competence skills. We will fulfill this vision through: an intensive research and training immersion experience, with mentoring as a central component; regular opportunities to interact with their peers and acquire the core values of collaboration, teamwork and personal enrichment; and exposure to the public awareness of science, education, policy and social change. Together, we will thus provide an exciting and comprehensive experience to minority scholars poised to make career-defining, life- changing decisions with future implications for the scientific enterprise across the nation.