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治疗有相当大的希望，但 执行这些建议的一个主要障碍是， 适当的动物模型和分子机制的研究 血管病理生理学。 我实验室的一个主要兴趣是 开发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， 了解心血管疾病的治疗。 的目的 本研究拟对生长因子基因的基础生物学进行研究。 表达和开发基因治疗方法来治疗心血管疾病 疾病 转化生长因子β（TGF-β）基因将被 检查，因为这种生长因子调节细胞增殖， 血管损伤后的结缔组织生成。 我们将使用 直接基因转移以将重组TGF-β基因转染入 血管细胞在猪体内动脉中。 TGF-β基因的表达 在正常和受损动脉中， 表达和功能，以及基因产物的设计， 调节和修饰TGF-β诱导的变化。 我们先前已经 开发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。 这些研究建立了 通过证明所提出的实验的可行性， 重组基因可以在脉管系统中表达。 基因表达 可以在DNA、mRNA和蛋白质水平上得到证实， 可以分析血管细胞上的重组基因产物。 在这 在此基础上，我们计划建立一种重组TGF-β基因的动物模型 正常猪动脉中TGF -β 1、TGF-β 2和TGF-β 3的表达 在直接基因转移后，确认基因表达，并 研究TGF-β对这些动脉的影响。 二是 研究重组TGF-β调节血管内皮细胞增殖的机制， 通过表征胶原合成、细胞在转导的动脉中的生长 增殖以及生长因子和细胞因子的释放。 三是 TGF-β基因表达对血管内膜增殖及 将研究受损动脉中结缔组织的产生。 初步数据表明，TGF-β的表达可能有助于改变 对伤害的反应。 这些系统也可以用来开发 可进一步限定和/或治疗血管性疾病的TGF-β拮抗剂 疾病这些试剂，包括显性负抑制剂、反义 寡核苷酸或其他拮抗剂，将被分析作为未来的一部分， 研究超出了本建议的范围。 总之，这些研究 将通过直接基因转移来开发血管疾病的动物模型 将重组TGF-β导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， 将开发针对TGF-β的分子遗传干预措施 来治疗心血管疾病

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.