Thyroid hormone regulation of vasculature in adult myocardium

描述（由申请人提供）：可能刺激或调节健康心脏血管生长的干预措施具有相当大的临床意义。主要针对肿瘤生长和器官发育进行的研究已经确定了血管生成的两种主要细胞机制：萌芽血管生成（SA）和侵袭性血管生成（IA）。SA涉及新毛细血管从现有血管发芽和增殖，IA涉及具有有限细胞增殖的现有血管的分隔和分裂。SA和IA对成人心脏中诱导的血管生成的相对贡献是未知的，主要是由于缺乏良好的动物模型。在证明甲状腺切除术或丙基硫脲嘧啶（PTU）诱导的甲状腺功能减退症导致6周内心肌小动脉的急剧损失后，我们在甲状腺功能减退症动物中开始T3治疗后1.5天内显示心肌小动脉的稳健增加。细胞增殖在1.5天时最小，但在第3天时显著增加。提示小动脉IA起早期作用，SA次之。该模型将用于研究成年心脏毛细血管和小动脉生长的细胞和分子机制。该提议将检验甲状腺激素通过Akt依赖性信号传导机制通过萌芽血管生成和内分泌血管生成促进心肌血管生成的总体假设。周细胞，了解不多，但在成人心脏中非常常见的细胞，可能在血管生成过程中发挥关键作用，并将详细检查。目的1利用PTU+T3模型研究成人心脏快速血管生成的细胞机制。我们假设PTU+T3在成年小鼠中触发心肌毛细血管和小动脉的快速和稳健的增殖，允许详细检查生长过程。共聚焦，扫描和透射显微镜将用于实现这一目标的结果。细胞类型的免疫标记物和增殖标记物将用于共聚焦分析。目的2将研究Akt在T3诱导的成人心脏快速血管生成反应和甲状腺功能减退症血管损失中的作用。我们假设Akt信号在T3介导的血管生成过程中增加，并且Akt抑制阻止相关的血管生成。相反，甲状腺功能减退症中Akt-eNOS-NO减少导致血管损失。目的3探讨Akt信号通路在T3诱导的血管重建和血管完整性中的作用。我们将使用一种新的模型，允许直接观察从目标1和2中使用的心脏获得的培养组织块的萌芽血管生成，以检验Akt参与甲状腺癌介导的血管生成的假设。培养的内皮细胞也将用于检查T3介导的Akt信号传导在增殖、迁移和管形成中的作用。我们将在体外抑制Akt信号通路，以研究该通路在上述血管生成过程中的作用。因此，这项工作将为成人心脏血管生长的细胞和分子机制提供重要的新信息。公共卫生相关性：甲状腺功能低下，导致心脏和大脑血管损伤，据信影响5%的美国人口和10%的绝经后妇女。甲状腺激素调节心脏血管生长的细胞和分子机制知之甚少，将在拟议的工作进行调查。

DESCRIPTION (provided by applicant): Interventions that may stimulate or regulate healthy cardiac vessel growth are of considerable clinical relevance. Research conducted primarily on tumor growth and organ development has identified two major cellular mechanisms of angiogenesis: sprouting angiogenesis (SA) and intussusceptive angiogenesis (IA). SA involves sprouting and proliferation of new capillaries from existing vessels and IA involves septation and division of existing vessels with limited cell proliferation. The relative contribution of SA and IA to angiogenesis induced in adult heart is unknown primarily due to the lack of a good animal model. After demonstrating that hypothyroidism induced by either thyroidectomy or propylthioruacil (PTU) leads to a dramatic loss of myocardial arterioles within 6 weeks, we have shown a robust increase in myocardial arterioles within 1.5 days after initiating T3 treatment in hypothyroid animals. Cell proliferation was minimal at 1.5 days but increased dramatically by day 3. This suggests that IA of arterioles plays an early role and is followed by SA. This model will be exploited to study the cellular and molecular mechanisms of capillary and arteriolar growth in adult hearts. This proposal will test the overall hypothesis that thyroid hormones promote myocardial angiogenesis by sprouting angiogenesis and intussusceptive angiogenesis via an Akt dependent signaling mechanism. Pericytes, poorly understood but very common cells in adult heart, likely play a key role in the angiogenic process and will be examined in detail. Aim 1 will use the PTU+T3 model to examine the cellular mechanisms of rapid angiogenesis in adult heart. We hypothesize that PTU+T3 in adult mice triggers rapid and robust proliferation of myocardial capillaries and arterioles, allowing detailed examination of the growth process. Confocal, scanning, and transmission microscopy will be used to achieve the results in this aim. Immuno-markers for cell type and markers of proliferation will be used in the confocal analyses. Aim 2 will examine the role of Akt in the rapid angiogenic response induced in adult heart by T3 and in vessel loss from hypothyroidism. We hypothesize that Akt signaling is increased during T3 mediated angiogenesis and Akt inhibition prevents the associated angiogenesis. Conversely, reduced Akt-eNOS-NO in hypothyroidism leads to vessel loss. Aim 3 will explore the role of Akt signaling in T3 induced vascular remodeling and vessel integrity during the angiogenic process in vitro. We will use a novel model allowing direct observation of sprouting angiogenesis from cultured tissue pieces obtained from hearts used in Aims 1 and 2 to test the hypothesis that Akt is involved in thyroid hormone-mediated angiogenesis. Cultured endothelial cells will also be used to examine the role of T3 mediated Akt signaling in proliferation, migration, and tube formation. We will inhibit Akt signaling in vitro to investigate the role of this pathway in the above-mentioned angiogenic processes. Thus, the proposed work will provide important new information about the cellular and molecular mechanisms of vessel growth in adult heart. PUBLIC HEALTH RELEVANCE: Low thyroid function, which leads to vascular impairment in heart and brain, is believed to affect 5% of the US population and 10% of postmenopausal women. The cellular and molecular mechanisms by which thyroid hormones regulate cardiac vascular growth are poorly understood and will be investigated in the proposed work.