Identification of Molecular Mechanisms Underlying Early Atherosclerosis

文摘

Abstract This K01 proposal describes a research and career development plan for Genesio Karere, Ph.D., the goal of which is to prepare Dr. Karere for an independent career focused on identification of molecular and epigenetic mechanisms underlying initiation and progression of atherosclerosis using a baboon model of human atherosclerosis. Dr. Karere has experience in genetics, genomics and molecular biology, which will enable completion of Aim 1 of the study. However, to complete all Aims and be competitive as an independent research investigator, Dr. Karere will require additional research skills, including immunohistochemistry (IHC), in vitro reporter assays, CRISPR-Cas9 genome-editing, laser capture microdissection, proteomics and metabolomics techniques. In addition, Dr, Karere will receive didactic training through formal coursework in Cell Biology and Research Ethics at the University of Texas Health Science Center, San Antonio (UTHSCSA). Informal training will be provided in the areas of grant writing and preparation of effective presentations. The applicant will receive guidance from primary mentor, Dr. Laura Cox (Texas Biomedical Research Institute, TBRI), and co-mentors, Drs. Henry McGill (TBRI), Marcel Daadi (TBRI), Cun Li (University of Wyoming) and Michael Mahaney (University of Texas Rio Grande Valley). Drs. Edward Dick (TBRI), Michael Olivier (TBRI), Mark Labordoni (Southwest Research Institute, SWRI), Xiangle Sun (UNTHSC) and Alexander Pertsemlidis (UTHSCSA) will serve consultants to enable Dr. Karere to establish an independent research career and successful program. Over the past three decades, TRBI has developed a unique baboon resource for research on atherosclerosis. This baboon colony includes seven generations of pedigreed, genotyped baboons that have been phenotyped for more than 200 quantitative traits related to dyslipidemia, and genetically characterized in relation to a baboon linkage map. It is well known that cardiovascular disease (CVD), the leading cause of death in the United States, is caused by atherosclerosis, which begins in childhood as arterial intimal lipid deposits and progresses to plaques in adulthood. However, the genetic, epigenetics and molecular mechanisms that underlie initiation and progression of atherosclerotic lesions are not known. The research plan described in this application will begin to fill that gap. Since it is not tenable to obtain tissue samples from healthy individuals, the baboon model of atherosclerosis described here provides a suitable alternative. Recent advancements in science have identified regimes for lowering serum cholesterol and retarding clinical end-points. However, these therapies are limited due to side effects, intolerance and ineffectiveness for some individuals. The limitations underscore the need for novel therapies for these individuals. We predict that miRNA-mediated molecular mechanisms are involved in the initiation and progression of atherosclerotic lesions in baboons, which not only develop lesions similar to those in humans but also are physiologically and genetically similar. The outcome will be the identification of candidate miRNA-modulated functional mechanisms underpinning early atherosclerosis. The findings from this study will provide insights essential for the identification of novel therapeutic targets to treat early atherosclerosis prior to advancement to life- threatening unstable plaques. This effort has potential to save millions of lives and dollars. The applicant collected baboon right coronary arteries (RCA) and blood samples from adult baboons fed a high-cholesterol, high-fat (HCHF) diet for two years. Preliminary data indicate that baboons developed atherosclerotic lesions (flat and raised fatty streaks) in RCA. We assessed a subset of arterial samples and found that several human mRNAs associated with atherosclerosis are differentially expressed between diseased and healthy control tissue in baboons. In this study, we will: 1) Identify and quantify lesions in baboon RCA; 2) Identify RCA miRNA expression profiles that correlate with RCA phenotypes (healthy and diseased) using small RNA-Seq; 3) Identify differentially expressed miRNA target genes in diseased and neighboring healthy regions of RCA using RNA-Seq, and validate expression of the targets’ gene products using IHC; and 4) Identify functional mechanisms by which RCA miRNAs influence lesion initiation and progression by integrating in vitro transfection reporter assay and CRISPR-Case genome-editing technique. In future studies we will translate our findings to humans for development of therapeutic targets to prevent early atherosclerosis. In addition, outcomes from this study will provide Dr. Karere with the necessary research skills and foundation data required to generate a hypothesis-driven proposal and compete for an NIH R01. The overall outcome of the proposed study will be elucidation of miRNA-mediated molecular mechanisms underpinning development of atherosclerosis. The results could lead to the development of novel drugs to treat atherosclerosis and alleviate the socio-economic impact of the disease.