MOLECULAR ATHERECTOMY USING FGF RECEPTORS

使用 FGF 受体的分子动脉粥样硬化切除术

• 批准号: 2226389
• 负责人: SAMUEL WARD CASSCELLS
• 金额: $ 19.18万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2226389
• 关键词: RNA directed DNA polymerase; cardiovascular disorder chemotherapy; cardiovascular pharmacology; carotid artery; cell migration; coronary artery; dosage; fibroblast growth factor; genetic manipulation; growth factor receptors; high performance liquid chromatography; human tissue; immunoaffinity chromatography; immunocytochemistry; in situ hybridization; intraluminal angioplasty; laboratory rat; messenger RNA; molecular cloning; monoclonal antibody; muscle cells; polymerase chain reaction; protein isoforms; radionuclide double label; stainings; vascular endothelium; vascular smooth muscle

The long-term objective of this proposal is to determine if receptors for fibroblast growth factors (FGF) can be used to develop therapeutic tools to prevent, or treat restenosis. The success of balloon coronary angioplasty remains compromised by a restenosis rate of 30-50%. Histologic studies indicate the neointimal thickening is largely due to the proliferation of smooth muscle cells (SMC). Studies in vitro have identified multiple mitogens for SMC, many of which are up-regulated after balloon injury, and no clinical therapies -- alone or in combination, including anti-coagulants, anti-platelet agents, calcium antagonists, lipid-lowering agents, or ACE inhibitors -- have markedly reduced restenosis rates. An alternative is to selectively ablate the proliferating subpopulation of SMC's using the recent observation that when these cells are proliferating they express more basic EGF receptors (FGFRs). When exposed to a conjugate of betaFGF and the ribosome- inactivating enzyme, saporin (SAP; which by itself enters mammalian cells very inefficiently), the proliferating smooth muscle cells (SMCs) are killed while the non-proliferating SMCs are spared because they have few FGFR. Endothelial cells, which have fewer FGFRs than SMCs, are killed only at higher doses and in fact are stimulated by doses that kill SMCs. This raises the possibility of using betaFGF, or an antibody to a FGFR, as a targeting vector for a single-dose, intravenous, cytocidal therapy. Simply put, the presence of multiple growth factors and their receptors make it difficult to inhibit SMCs with an antibody or other antagonist directed at betaFGF or any other growth factor or receptor. However, virtually all SMCs have FGFRs and so are killed by FGF-SAP; in other words, the cells are not protected by the presence of other growth factors. Because of the surprising finding that most of the seven FGF ligands bind most of the FGFRs, cell-specific targeting is likely to depend on which of the 6 FGFR genes (and which of their many alternatively-spliced forms) are up-regulated after vascular injury. The present proposal is 1) to determine if our recently expressed recombinant FGF-SAP can be used as a local or systemic therapy to prevent or reverse SMC accumulation in the balloon-injured rat carotid artery; 2) to evaluate the effect of FGF-SAP on ECs; 3) to screen for major toxicity; 4) to determine which FGFRs are up-regulated in injured vessels by analysis of PCR extension products of FGFR primers, using mRNA from ballooned rat carotid arteries and human coronary atherectomy specimens; 5) to determine the cell source of the receptor mRNAs by simultaneous in situ hybridization and cell type- specific immunocytochemistry; 6) determine whether there is a FGFR isoform specific for proliferating SMCs; 7) develop monoclonal antibodies to an extracellular domain of that isoform (for eventual use in diagnostic imaging and therapeutic targeting).

本提案的长期目标是确定是否存在 成纤维细胞生长因子（FGF）可用于开发治疗工具 以预防或治疗再狭窄。 球囊冠状动脉成形术的成功 血管成形术仍然受到30- 50%的再狭窄率的影响。 组织学研究表明，新生内膜增厚主要是由于 平滑肌细胞（SMC）增殖。 体外研究表明， 鉴定了SMC的多种有丝分裂原，其中许多是上调的 球囊损伤后，没有临床治疗-单独或联合 联合用药，包括抗凝剂、抗血小板剂、钙 拮抗剂，降脂剂，或ACE抑制剂-有显着 降低再狭窄率。 一种替代方案是选择性地消融 增殖的SMC亚群使用最近的观察， 当这些细胞增殖时，它们表达更基本的EGF受体。 （FGFRs）。 当暴露在β-FGF和核糖体的结合物中时- 失活酶，皂草素（SAP），其本身进入哺乳动物细胞 非常低效），增殖的平滑肌细胞（SMC） 而非增殖的SMC则幸免于难，因为它们几乎没有 FGFR。 与SMC相比，具有较少FGFR的内皮细胞被杀死， 只有在更高的剂量下，实际上是由杀死SMC的剂量刺激的。 这增加了使用β FGF或FGFR抗体的可能性， 作为单剂量静脉内杀细胞疗法的靶向载体。 简单地说，多种生长因子及其受体的存在 使得难以用抗体或其它拮抗剂抑制SMC 针对β FGF或任何其他生长因子或受体。 然而，在这方面， 几乎所有的SMC都具有FGFR，因此被FGF-SAP杀死;在其他研究中， 换句话说，细胞不受其他生长物的保护， 因素 由于令人惊讶的发现，七种FGF配体中的大多数结合 大多数FGFR，细胞特异性靶向可能取决于 6个FGFR基因（以及它们的许多选择性剪接形式） 在血管损伤后表达上调。 目前的建议是1） 确定我们最近表达的重组FGF-SAP是否可以用作 局部或全身治疗，以防止或逆转SMC在 2）评价FGF-SAP对球囊损伤大鼠颈动脉的影响 3）筛选主要毒性; 4）确定哪些FGFR是 通过PCR延伸产物分析， FGFR引物，使用来自气球样大鼠颈动脉和人的mRNA 冠状动脉粥样硬化切除术标本; 5）确定细胞来源， 受体mRNA的同时原位杂交和细胞类型- 特异性免疫细胞化学; 6）确定是否存在FGFR 增殖SMC特异性同种型; 7）开发单克隆抗体 连接到该同种型的胞外结构域（最终用于 诊断成像和治疗靶向）。