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相关性。主要针对肿瘤生长和器官发育的研究已经确定了血管生成的两种主要细胞机制：发芽血管生成(SA)和肠套叠血管生成(IA)。SA包括从现有血管中萌发和增殖新的毛细血管，而IA涉及分离和分裂现有血管，细胞增殖有限。SA和IA在成人心脏血管生成中的相对作用尚不清楚，这主要是由于缺乏良好的动物模型。在证明了甲状腺切除术或丙基硫氧尿嘧啶(PTU)引起的甲状腺功能减退症(甲减)会在6周内导致心肌小动脉显著丢失后，我们在甲状腺功能低下动物中发现，在开始T3治疗后1.5天内，心肌小动脉显著增加。细胞增殖在1.5d时最低，但在3d时急剧增加。这表明小动脉的IA起早期作用，其次是SA。这个模型将被用来研究成人心脏毛细血管和小动脉生长的细胞和分子机制。这一提议将检验甲状腺激素通过Akt依赖的信号机制萌发血管生成和肠套叠血管生成来促进心肌血管生成的总体假设。周细胞是成人心脏中知之甚少但非常常见的细胞，可能在血管生成过程中发挥关键作用，将进行详细研究。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。我们假设成年小鼠的PTU+T3可以触发心肌毛细血管和小动脉的快速而有力的增殖，从而可以对生长过程进行详细的检查。将使用共聚焦显微镜、扫描显微镜和透射显微镜来实现这一目标。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所致血管丢失中的作用。我们假设Akt信号在T3介导的血管生成过程中增加，并且Akt抑制阻止了相关的血管生成。相反，甲状腺功能减退症患者Akt-enos-NO减少会导致血管丢失。目的3探讨Akt信号在T3诱导的血管重塑和体外血管生成过程中的作用。我们将使用一种新的模型，允许直接观察从AIMS 1和2中使用的心脏培养组织块中萌发的血管生成，以验证Akt参与甲状腺激素介导的血管生成的假设。培养的内皮细胞也将被用来研究T3介导的Akt信号在增殖、迁移和管状形成中的作用。我们将在体外抑制Akt信号，以研究该途径在上述血管生成过程中的作用。因此，这项拟议的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。与公共健康相关：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尚不清楚，将在拟议的工作中进行研究。

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.