EXPRESSION/FUNCTION OF RECOMBINANT TGF-BETA IN ARTERIES

动脉粥样硬化和再狭窄是两种心血管疾病 改变心脏功能，并产生相当大的发病率和死亡率。 这些疾病的基因疗法前景看好，但 实施它们的一个主要障碍是发展 合适的动物模型及分子机制研究 血管病理生理学。我的实验室的一个主要兴趣是 建立基于直接基因转移的血管疾病动物模型 重组生长因子基因，这将促进 了解心血管疾病的治疗方法。目的 本课题旨在研究生长因子基因的基础生物学。 表达并开发治疗心血管疾病的基因治疗方法 疾病。转化生长因子-β基因将被 因为这种生长因子调节细胞增殖和 血管损伤后结缔组织的产生。我们将使用 直接基因转染法将重组转化生长因子-β基因转入 活体猪动脉中的血管细胞。转化生长因子-β基因的表达 在正常和受损的动脉中将允许对基因进行表征 表达和功能，以及设计可能的基因产品 调节和修饰转化生长因子-β诱导的变化。我们之前已经 发展了细胞介导和直接基因转移的方法 重组基因进入血管系统。这些研究已经确立了 通过证明建议的实验的可行性 重组基因可以在血管系统中表达。基因表达 可以在DNA、mRNA和蛋白质水平上证实其作用 可以对血管细胞上的重组基因产物进行分析。在这 建议，我们计划建立重组转化生长因子-β基因的动物模型 转化生长因子-β1、-β2和-β3在正常猪动脉中的表达 在直接基因转移之后，确认基因表达，并 研究转化生长因子-β对这些动脉的影响。第二，我们将 重组转化生长因子-β对血管内皮细胞的调控机制研究 通过表征胶原合成和细胞在转导动脉中的生长 增殖，并释放生长因子和细胞因子。第三， 转化生长因子-β基因表达对血管内膜增殖及血管生成的影响 将对受损动脉中结缔组织的产生进行调查。 初步数据表明，转化生长因子-β的表达可能有助于改变 对受伤的反应。这些系统也可用于开发 可进一步定义和/或治疗血管的转化生长因子-β拮抗剂 疾病。这类药物包括显性负性抑制物、反义 寡核苷酸或其他拮抗剂，将作为未来分析的一部分 超出本提案范围的研究。总而言之，这些研究 将通过直接基因转移开发血管疾病的动物模型 将重组转化生长因子-β注入正常和受损的猪动脉。 将进行基因表达的基础生物学研究，并 将开发针对转化生长因子-β的分子遗传干预 治疗心血管疾病。

Atherosclerosis and restenosis are two cardiovascular diseases which alter cardiac function and produce considerable morbidity and mortality. Gene therapy treatments to these diseases hold considerable promise, but a major obstacle to their implementation is the development of appropriate animal models and the investigation of molecular mechanisms of vascular pathophysiology. A major interest of my laboratory is to develop animal models of vascular disease based upon direct gene transfer of recombinant growth factor genes which will facilitate the understanding the treatment of cardiovascular diseases. The purpose of this proposal is to study the basic biology of growth factor gene expression and to develop gene therapy approaches to treat cardiovascular diseases. Transforming growth factor beta (TGF-beta) genes will be examined since this growth factor regulates cellular proliferation and connective tissue production following vascular injury. We will use direct gene transfer to transfect recombinant TGF-beta genes into vascular cells in porcine arteries in vivo. Expression of TGF-beta genes in normal and injured arteries will permit characterization of gene expression and function, and the design of gene products which might regulate and modify TGF-beta induced changes. We have previously developed methods for cell-mediated and direct gene transfer of recombinant genes into the vasculature. These studies have established the feasibility for the proposed experiments by demonstrating that recombinant genes can be expressed in the vasculature. Gene expression can be confirmed at the DNA, mRNA and protein level, and the effect of the recombinant gene product on vascular cells can be analyzed. In this proposal, we plan to develop an animal model of recombinant TGF-beta gene expression (TGF -beta1, -beta2 and -beta3) in normal porcine arteries following direct gene transfer, to confirm gene expression, and to investigate the effects of TGF-beta on these arteries. Second, we will investigate the mechanism of recombinant TGF-beta regulation of vascular growth in transduced arteries by characterizing collagen synthesis, cell proliferation, and release of growth factors and cytokines. Third, the effect of TGF-beta gene expression on intimal proliferation and connective tissue production in injured arteries will be investigated. Preliminary data suggest that expression of TGF-beta could help to modify the response to injury. These systems can also be used to develop antagonists to TGF-beta which can further define and/or treat vascular diseases. Such agents, including dominant negative inhibitors, antisense oligonucleotides or other antagonists, will be analyzed as part of future studies beyond the scope of this proposal. In summary, these studies will develop animal models of vascular disease from direct gene transfer of recombinant TGF-beta into normal and injured porcine arteries. Studies of basic biology of gene expression will be conducted and molecular genetic interventions which target TGF-beta will be developed to treat cardiovascular diseases.