DEVELOPMENTAL REGULATION OF ANGIOTENSIN II VASCULAR MITOGENESIS

血管紧张素 II 是一种有效的血管收缩剂，还可诱导血管生长 血管平滑肌细胞（VSMC）。 证据支持重要作用 AngII 在胎儿/新生儿血管发生中的作用。 我们已经在成人中展示了 猪主动脉VSMC AngII促有丝分裂作用不完全，仅限于 晚期细胞周期事件：AngII 通过以下方式诱导细胞分裂的完成 细胞可逆地停滞在 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， AngII 有效刺激 DNA 和蛋白质合成并增加细胞 新生猪 VSMC 的增殖率。 这，连同 发表的证据表明人类对 AngII 的完全促有丝分裂反应 胎儿系膜细胞随着年龄的增长而丢失，提示我们的假设 AngII 的有丝分裂能力受到发育调节。 使用我们的 建立了猪VSMC模型，我们将在近交系微型猪中进行测试， 允许精确控制胎儿和新生儿组织，减少 非特异性遗传变异，并减少传代次数 通过汇集性别匹配同窝仔鼠的 VSMC 进行培养。 我们三个 具体目标 每个地址均从胎儿、新生儿和产后获得的 VSMC 青春期微型猪，并在第 3 代以匹配的亚汇合密度进行研究 第5段。 在 SA#1 中，我们将为每个发展阶段确定 AngII 促有丝分裂作用的早期细胞周期与晚期细胞周期位点 （分别）使用新验证的 G0 或 G2 同步 VSMC 同步协议。 在 SA#2 中，我们将评估发育情况 AngII 受体数量、亚型和 AT-1:AT-2 比例的调节 确定每个亚型在促有丝分裂作用中的功能作用 AngII 在每个发育年龄都有定义。 在SA#3中，基于重要性 表皮生长因子（EGF）受体在胎儿发育中的作用及其作用 增加 AT 和减少 AT-2 AngII 受体的能力，我们将 确定组成型 EGF-R 数量和激活状态的作用 AngII 有丝分裂能力和意志的发育调节 比较慢性“启动”与转化生长因子 (TGF) 的效果 α，EGF 受体激活的主要胎儿配体，在基础上 生长、对 AngII 的有丝分裂反应以及两种 Ang II 受体（AT- 1 和 AT-2) 和每个发育阶段 VSMC 中的 EGF-R。 因为一个 胎儿样表型可以在成熟的 VSMC 中重新诱导，以响应 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 对处于危险中的儿童和成人进行预防/治疗干预 用于血管疾病。

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.