GROWTH FACTOR EXPRESSION IN RENAL ENDOTHELIAL CELLS

肾内皮细胞中生长因子的表达

• 批准号: 2140559
• 负责人: TOM DANIEL
• 金额: $ 22.62万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-01 至 1995-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2140559
• 关键词: DNA binding protein; DNA footprinting; Xenopus; alternatives to animals in research; biopsy; crosslink; gel mobility shift assay; gene expression; genetic mapping; genetic promoter element; genetic transcription; genetic translation; human tissue; in situ hybridization; kidney cell; messenger RNA; nephrosclerosis; nucleic acid sequence; platelet derived growth factor; protein kinase; tissue /cell culture; ultraviolet radiation; vascular endothelium; wheat

Destructive proliferation and sclerosis at sites underlying endothelium in the kidney microvasculature irreversibly impair renal function through luminal narrowing, destruction of glomerular architecture and reduction of filtration surface. The objective of these studies is to define mechanisms regulating renal endothelial cell production of growth factors that are implicated in proliferation and sclerosis subjacent to the endothelial layer. Especially relevant in destructive glomerular diseases are proliferation of mesangial cells, fibroblasts and matrix deposition, all of which are stimulated by platelet-derived growth factor (PDGF). Our data, using primary cultured human renal microvascular endothelial cells, have shown PDGF B/c-sis messenger RNA (mRNA) expression and activity release are regulated by agents relevant at vascular sites. Transcription of PDGF B/c-sis mRNA is induced by platelet (transforming growth factor beta) and coagulation products (thrombin) and suppressed by hormones which elevate cAMP levels (catecholamines). New data has shown renal endothelial expression of distinct structural forms of PDGF B mRNA. The proposed studies will determine the relative contribution of different PDGF B mRNA's to translation of active PDGF protein product, using in vitro and xenopus oocyte translation of isolated and synthesized structural forms of each PDGF B/c-cic mRNA. Additional experiments will determine if expression of different PDGF B/c-sis mRNAs is differentially regulated by transcriptional and stabilization mechanisms. Genomic PDGF B/c-cic sequences participating in regulation of renal endothelial expression will be identified by DNA footprinting techniques and transient expression of a series of flanking sequence deletion constructs. DNA sequences participating in cAMP-mediated repression of PDGF B/c-sis transcription will be identified and characterized by gel shift assays, and DNA binding proteins identified by UV cross-linking and as substrates for cAMP dependent kinase. Intact tissue roles for PDGF B/c-sis expression will be identified using in situ hybridization of PDGF B/c-sis mRNA in renal biopsy samples. These studies will define molecular mechanisms regulating microvascular endothelial production of PDGF and may provide potential targets for intervention in destructive subendothlial proliferative processes.

内皮下方部位的破坏性增殖和硬化 肾脏微血管不可逆地损害肾功能 管腔变窄、肾小球结构破坏和肾小球功能减少 过滤表面。这些研究的目的是定义机制 调节肾内皮细胞生长因子的产生 与内皮细胞下方的增殖和硬化有关 层。 与破坏性肾小球疾病尤其相关的是增殖 系膜细胞、成纤维细胞和基质沉积，所有这些都是 受到血小板衍生生长因子（PDGF）的刺激。我们的数据，使用 原代培养的人肾微血管内皮细胞，显示 PDGF B/c-sis 信使 RNA (mRNA) 表达和活性释放 受血管部位相关药物的调节。 PDGF的转录 B/c-sis mRNA 由血小板（转化生长因子β）诱导， 凝血产物（凝血酶）并被激素抑制，从而升高 cAMP 水平（儿茶酚胺）。新数据显示肾内皮细胞 PDGF B mRNA 不同结构形式的表达。拟议的 研究将确定不同 PDGF B mRNA 的相对贡献 使用体外和爪蟾翻译活性 PDGF 蛋白产物 卵母细胞翻译每个分离和合成的结构形式 PDGF B/c-cic mRNA。额外的实验将确定是否表达 不同的 PDGF B/c-sis mRNA 受转录调控差异 和稳定机制。 基因组 PDGF B/c-cic 序列参与肾功能调节 内皮表达将通过 DNA 足迹技术进行鉴定 以及一系列侧翼序列缺失的瞬时表达 构造。参与 cAMP 介导的 PDGF 抑制的 DNA 序列 B/c-sis 转录将通过凝胶位移进行鉴定和表征 分析，以及通过 UV 交联鉴定的 DNA 结合蛋白，以及 cAMP 依赖性激酶的底物。 PDGF B/c-sis 的完整组织作用 将使用 PDGF B/c-sis 原位杂交来鉴定表达 肾活检样本中的 mRNA。 这些研究将定义调节微血管的分子机制 PDGF 的内皮生成，可能为以下方面提供潜在靶点： 干预破坏性内皮下增殖过程。