DEVELOPMENTAL REGULATION OF ANGIOTENSIN II VASCULAR MITOGENESIS

血管紧张素II是一种有效的血管收缩药，它也可以诱导血管生长 血管平滑肌细胞(VSMC)。证据支持了一个重要的角色 在胎儿/新生儿血管生成中的作用。我们已经在成人中展示了 猪主动脉VSMC的血管有丝分裂作用是不完全的，仅限于 晚期细胞周期事件：AngII通过以下方式诱导细胞分裂完成 细胞可逆地滞留在G2期，但没有能力刺激或 蛋白质DNA合成。相比之下，根据我们的初步结果， 血管紧张素转换酶能有效地刺激DNA和蛋白质的合成并增加细胞 新生猪VSMC的增殖率。这一点，连同 已发表的证据表明，人类对血管紧张素转换酶的全面促有丝分裂反应 胎儿肾小球系膜细胞随着年龄的增长而丢失，提示我们的假设 血管紧张素转换酶促有丝分裂能力受发育调节。使用我们的 建立了猪VSMC模型，我们将在近亲繁殖的小型猪身上进行测试， 允许精确控制胎儿和新生儿组织，减少 非特异性遗传变异性，以及减少传代的数量 通过从性别匹配的窝产仔中收集VSMC进行培养。我们的三个 从胎儿、新生儿和产后获得的VSMC的具体目标 青春期小型猪，并在匹配的亚汇合流密度下进行研究 第五章。在SA#1中，我们将确定每个发育阶段的 血管紧张素转换酶促有丝分裂作用的早期细胞周期与晚期细胞周期基因座 (分别)使用新验证的G0或G2同步VSMC 同步协议。在SA#2中，我们将评估开发 血管紧张素Ⅱ受体数目、亚型及AT-1/AT-2比值的调节 确定每个亚型在促有丝分裂作用中的功能作用 Angii是在每个发育年龄定义的。在SA#3中，基于重要性 表皮生长因子受体在胎儿发育中的作用及其意义 增加AT和减少AT-2血管紧张素Ⅱ受体的能力，我们将 确定构造性EGF-R数量和激活状态在 血管紧张素转换酶促有丝分裂能力和意志的发育调节 慢性“启动”与转化生长因子的疗效比较 α，激活EGF受体的主要胎儿配体，在 生长、对血管紧张素Ⅱ的促有丝分裂反应以及两种血管紧张素Ⅱ受体(AT- 1和AT-2)和EGF-R在各发育阶段的VSMC中表达。因为一个 在成熟的VSMC中可以重新诱导出类似胎儿的表型 损伤，了解血管生长的发育生理学 监管不仅关系到正常增长，也关系到 对高危儿童和成人的预防/治疗干预 治疗血管疾病。

Angiotensin II is a potent vasoconstrictor which also induces growth in vascular smooth muscle cells (VSMC). Evidence supports an essential role for AngII in fetal/neonatal vasculogenesis. We have shown in adult porcine aortic VSMC the AngII mitogenic action is incomplete, confined to late cell-cycle events: AngII induces completion of cell division by cells reversibly arrested in G2 Phase but has no capacity to stimulate or protein DNA synthesis. In contrast, based on our preliminary results, AngII potently stimulated DNA and protein synthesis and increased cell proliferation rate in neonatal porcine VSMC. This, together with published evidence that the full mitogenic response to AngII in human fetal mesangial cells is lost with aging, prompts our HYPOTHESIS that AngII mitogenic capacity is developmentally regulated. Using our established porcine VSMC model, we will test this in inbreed microswine, permitting control of precisely-timed fetal and neonatal tissues, reducing nonspecific genetic variability, and reducing number of passages in culture by pooling of VSMC from sex-matched littermates. Our three SPECIFIC AIMS each address VSMC obtained from fetal, neonatal, vs. post- pubertal microswine and studied at matched subconfluent density in 3rd- 5th passage. In SA#1, we will determine for each developmental stage the early cell-cycle vs. late cell-cycle loci of AngII mitogenic actions in (respectively) G0- or G2-synchronized VSMC using a newly validated synchronization protocol. In SA#2, we will assess developmental regulation of AngII receptor number, subtype, and AT-1:AT-2 ratio and determine the functional role of each subtype in the mitogenic actions of AngII defined at each developmental age. In SA#3, based on the importance of the Epidermal Growth Factor (EGF) receptor in fetal development and its capacity to increase AT and decrease AT-2 AngII receptors, we will determine the role of constitutive EGF-R number and activation status in the developmental regulation of AngII mitogenic capacity and will and compare effects of chronic "priming" with Transforming Growth Factor (TGF) alpha, the dominant fetal ligand for EGF receptor activation, on basal growth, on mitogenic responses to AngII, and on both Ang II receptors (AT- 1 and AT-2) and EGF-R in VSMC from each developmental stage. Because a fetal-like phenotype can be re-induced in mature VSMC in response to injury, understanding the developmental physiology of vascular growth regulation has relevance not only to normal growth, but also to preventive/therapeutic interventions in both children and adults at risk for vascular disease.