Identification of molecular mechanisms underlying early atherosclerosis

鉴定早期动脉粥样硬化的分子机制

• 批准号: 9166939
• 负责人: Genesio Karere
• 金额: $ 12.2万
• 依托单位: TEXAS BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9166939
• 关键词: Address; Adult; Adverse effects; Animal Model; Area; Arterial Fatty Streak; Atherosclerosis; Biological Assay; Biomedical Research; Blood specimen; CRISPR/Cas technology; Candidate Disease Gene; Cardiovascular Diseases; Cause of Death; Cellular biology; Childhood; Cholesterol; Chromosome Mapping; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Coronary; Coronary Arteriosclerosis; Coronary artery; Data; Deposition; Development; Development Plans; Diet; Dietary Cholesterol; Disease; Doctor of Philosophy; Dyslipidemias; Early Diagnosis; Epigenetic Process; Foundations; Future; Gene Proteins; Gene Targeting; Generations; Genes; Genetic; Genomics; Genotype; Goals; Grant; Health Sciences; High Fat Diet; Histological Techniques; Histology; Human; Human Development; Imaging Techniques; Immunohistochemistry; In Vitro; Individual; Knowledge; Lead; Lesion; Life; Lipids; Mediating; Mentors; Messenger RNA; MicroRNAs; Modeling; Molecular; Molecular Biology; Molecular Profiling; Operative Surgical Procedures; Outcome; Outcome Study; Papio; Phenotype; Physiological; Plasma; Population; Preparation; Prevention; Progressive Disease; Proteomics; Reporter; Research; Research Ethics; Research Institute; Research Personnel; Resources; Sampling; Science; Serum; Severities; Small RNA; Social Impacts; Staging; Stroke; Techniques; Testing; Texas; The Sun; Time; Tissue Sample; Tissues; Training; Transfection; Translating; United States; United States National Institutes of Health; Universities; Validation; Variant; Writing; Wyoming; abstracting; candidate identification; career; career development; cellular targeting; cohort; differential expression; digital imaging; early onset; economic impact; experience; feeding; gene product; genome editing; human subject; insight; laser capture microdissection; metabolomics; miRNA expression profiling; mouse model; new therapeutic target; nonhuman primate; novel strategies; novel therapeutics; phenotypic data; prevent; programs; research and development; response; skills; socioeconomics; success; therapeutic target; trait; transcriptome sequencing

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.

摘要 本K 01提案描述了Genesio Karere博士的研究和职业发展计划，的目标 这是准备博士Karere一个独立的职业生涯，重点是鉴定分子和表观遗传 使用狒狒模型研究动脉粥样硬化的发生和发展机制 动脉粥样硬化Karere博士在遗传学、基因组学和分子生物学方面拥有丰富的经验， 完成研究目标1。然而，为了完成所有目标，并作为一个独立的竞争力 研究者Karere博士将需要额外的研究技能，包括免疫组织化学（IHC）， 体外报告基因测定、CRISPR-Cas9基因组编辑、激光捕获显微切割、蛋白质组学和 代谢组学技术。此外，Karere博士将通过以下正式课程接受教学培训： 细胞生物学和研究伦理学，德克萨斯大学健康科学中心，圣安东尼奥（UTHSCSA）。 将在赠款的撰写和有效的演讲准备方面提供非正式培训。的 申请人将接受主要导师Laura考克斯博士（德克萨斯生物医学研究所， TBRI），以及共同导师亨利麦吉尔博士（TBRI），马塞尔达迪（TBRI），李存（怀俄明州大学）和 Michael Mahaney（德克萨斯大学格兰德河谷分校）。Edward Dick博士（TBRI），Michael Olivier博士（TBRI）， 马克·阿利多尼（西南研究所）、孙翔乐（联合国人类住区中心）和亚历山大·佩尔特塞姆利迪斯 （UTHSCSA）将提供顾问服务，使Karere博士能够建立独立的研究生涯， 成功的方案。在过去的三十年里，TRBI开发了一种独特的狒狒资源用于研究 关于动脉粥样硬化。这个狒狒群体包括七代纯种狒狒，基因型狒狒， 已经对200多个与血脂异常相关的数量性状进行了表型分析， 与狒狒连锁图谱相关的特征。 众所周知，心血管疾病（CVD）是美国的主要死亡原因， 动脉粥样硬化，始于儿童时期的动脉内膜脂质沉积， 成年然而，遗传学，表观遗传学和分子机制，基础的启动和 动脉粥样硬化病变的进展是未知的。本申请中所述的研究计划将开始 来填补这个空白。由于从健康个体获得组织样本是不可行的， 这里描述的动脉粥样硬化提供了合适的替代方案。 最近的科学进步已经确定了降低血清胆固醇和延缓临床症状的方案。 终点然而，这些疗法是有限的，由于副作用，不耐受和无效的一些 个体这些局限性强调了对这些个体的新疗法的需求。我们预测 miRNA介导的分子机制参与了动脉粥样硬化的发生和发展 狒狒的病变，不仅发展出与人类相似的病变，而且在生理上和 基因相似结果将是鉴定候选的miRNA调节的功能性 早期动脉粥样硬化的机制这项研究的结果将提供重要的见解， 确定新的治疗靶点，在生命进展之前治疗早期动脉粥样硬化- 威胁着不稳定的斑块这项工作有可能挽救数百万人的生命和资金。 申请人收集了狒狒右冠状动脉（RCA），并从成年狒狒中采集了血液样本， 高胆固醇、高脂肪（HCHF）饮食两年。初步数据显示狒狒进化出了 RCA中的动脉粥样硬化病变（平坦和凸起的脂肪条纹）。我们评估了一部分动脉样本， 发现与动脉粥样硬化相关的几种人类mRNA在 患病和健康的对照组织。在本研究中，我们将：1）识别和量化狒狒的病变 2）鉴定与RCA表型（健康和患病）相关的RCA miRNA表达谱 使用小RNA-Seq; 3）鉴定病变和邻近组织中差异表达的miRNA靶基因 使用RNA-Seq检测RCA的健康区域，并使用IHC验证靶基因产物的表达;以及 4)确定RCA miRNA影响病变起始和进展的功能机制， 整合体外转染报告基因分析和CRISPR-Case基因组编辑技术。 在未来的研究中，我们将把我们的发现应用于人类，以开发治疗靶点， 早期动脉粥样硬化此外，本研究的结果将为Karere博士提供必要的研究 技能和基础数据需要生成一个假设驱动的建议，并为NIH R 01竞争。 这项研究的总体结果将是阐明miRNA介导的分子机制 支持动脉粥样硬化的发展。这些结果可能会导致开发新的药物来治疗 动脉粥样硬化和减轻疾病的社会经济影响。