Regulation of angiogenesis by nuclear receptors and cofactors

 描述(申请人提供)：外周动脉疾病(PAD)及其严重形式的严重肢体缺血(CLI)影响近800万美国人。大多数患有慢性代谢疾病和心血管疾病(如糖尿病)的患者也患有PAD/CLI。患有PAD/CLI的患者会出现虚弱的症状，如行动不便、溃疡、伤口愈合障碍和顽固性缺血。PAD/CLI的病理涉及骨骼肌血管退行性变，从而导致显著的肌病。治疗仅限于外科重建，这通常是昂贵和无效的，导致截肢。“治疗性血管生成”以改善肌肉血管供应是PAD/CLI研究的一个迫切领域。在这一领域，焦点一直集中在促血管生成调节因子，如血管内皮生长因子-α(VEGFA)和低氧诱导因子1(HIF1)，以及它们如何改善肌肉血管系统。不幸的是，到目前为止，单个血管生成因子甚至HIF1都不是PAD/CLI的有效靶点。值得注意的是，骨骼肌的血管供应和新生血管生成是由肌肉分泌的促血管生成因子和抗血管生成因子以旁分泌的方式控制的。虽然肌肉中促血管生成因子如Vegfa的分子调控已被定义，但令人惊讶的是，抗血管生成因子的分子调控尚不明确。我们已初步发现，转录辅因子过氧化物酶体增殖物激活受体γ共激活因子1β(Pgc1β)通常通过激活核激素受体发挥作用，它编码骨骼肌中抑制血管生成的基因程序。该程序包括诱导抗血管生成因子和抑制促血管生成因子，从而在缺血时净抑制骨骼肌血管重建。有趣的是，我们发现糖尿病肌肉中Pgc1β的表达与抗血管生成因子一起被诱导，这是以PAD/CLI的病理症状为特征的，如血管退缩、缺血和新生血管生成障碍。因此，本研究建议的第一个目标是破译内源性肌肉前列腺素C_1β在调节抑制性血管生成程序中的作用，以及它在缺血性肌肉血管重建和糖尿病肌肉血管病变中的作用。第二个目标是建立鸡卵蛋白上游启动子转录因子I(COUP-TFI)作为参与Pgc1β驱动的抑制血管生成的核受体。我们的初步数据表明，PgC1TFI的抗血管生成作用可能是通过激活核受体COUP-β介导的。我们的中心假设是，骨骼肌中的PGC1TFI通过激活COUP-TFI，启动旁分泌的抗血管生成基因程序，阻断缺血性新生血管的生成，从而促进糖尿病肌肉血管病变。验证这一假说的具体目的是：(1)研究内源性前列腺素C_1β在肌肉血管生成中的作用；(目的2)研究前列腺素C_1β和COUPTF_1是否相互作用调节肌肉血管生成。在目标1中，我们将利用肌肉特异性缺失的前列腺素C_1β来研究肌肉前列腺素C_1β的缺失对肌肉血管形成、缺血血管重建和血管生成因子表达的影响。在目标2中，我们将首先进行体外实验，研究PGC1TFI和COUP-TFI之间的相互作用，以及它们如何调控肌肉血管生成基因。其次，我们将使用肌肉特异的复合转基因小鼠来研究肌肉特异的PGC1TFI过表达是否会因为肌肉特异的COUP-TFI缺失而失去抗血管生成的作用。我们还将研究双肌肉特异性PGC1TFI和COUP-TFI基因敲除对肌肉新生血管生成和血管重建的影响。我们工作的意义在于，它旨在发现骨骼肌中抗血管生成基因程序是如何调节的，以及它如何影响肌肉缺血和糖尿病肌肉血管病变的血管重建。我们的研究具有创新性，因为我们将研究PAD/CLI治疗中抗血管生成基因编程的未知领域。这一发现将对PAD/CLI中的肌肉缺血以及其他疾病(如心肌梗死、中风和肿瘤血管生成)设计新的治疗方法具有重要意义。

 DESCRIPTION (provided by applicant): Peripheral arterial disease (PAD) and its severe form critical limb ischemia (CLI) affect nearly 8 million Americans. Most patients with chronic metabolic and cardiovascular diseases such as diabetes also suffer from PAD/CLI. Patients with PAD/CLI suffer from debilitating symptoms such as immobility, ulceration, impaired wound healing, and intractable ischemia. The pathology of PAD/CLI involves vascular regression in the skeletal muscle, which leads to significant myopathy. Treatment is limited to surgical reconstruction, which is often costly and ineffective leading to limb amputations. 'Therapeutic angiogenesis' to improve muscle vascular supply is an urgent area of research in PAD/CLI. Within this field, focus has been on pro-angiogenic regulators such as vascular endothelial growth factor-alpha (Vegfa) and hypoxia inducible factor 1 (HIF1), and how they can improve muscle vasculature. Unfortunately, individual angiogenic factors and even HIF1 have so far been ineffective targets in PAD/CLI. Notably, skeletal muscle vascular supply and neo angiogenesis is controlled in a paracrine fashion by both pro-angiogenic and anti-angiogenic factors secreted by the muscle. While molecular regulation of pro-angiogenic factors such as Vegfa in the muscle is defined, surprisingly, the molecular regulation of anti-angiogenic factors is poorly defined. We have preliminarily found that a transcriptional cofactor peroxisome proliferator-activated receptor gamma co-activator 1 beta (PGC1β), which typically functions by activating nuclear hormone receptors, encodes an inhibitory angiogenesis gene program in the skeletal muscles. This program constitutes induction of anti-angiogenic factors and repression of proangiogenic factors, resulting in a net inhibition of skeletal muscle revascularization in ischemia. Interestingly, we found that PGC1β expression is induced along with the anti-angiogenic factors in diabetic muscles, which are characterized by the pathological symptoms of PAD/CLI such as vascular regression, ischemia and impaired neo-angiogenesis. Therefore, the first objective of this research proposal is to decipher the role of endogenous muscle PGC1β in regulating inhibitory angiogenic program and its effect on ischemic muscle revascularization and diabetic muscle angiopathy. The second objective is to establish COUP-TFI (chicken ovalbumin upstream promoter transcription factor I) as the nuclear receptor involved in PGC1β-driven inhibitory angiogenesis. Our preliminary data indicates that the anti-angiogenic effects of PGC1β may be mediated by activating nuclear receptor COUP-TFI. Our central hypothesis is that PGC1β in the skeletal muscle instigates a paracrine anti-angiogenic gene program by activating COUP-TFI, blocks ischemic neo angiogenesis, and contributes to diabetic muscle angiopathy. The specific aims to test this hypothesis are to: (Aim 1) Investigate the role of endogenous PGC1β in muscle angiogenesis and (Aim 2) Investigate whether PGC1β and COUPTFI interact to regulate muscle angiogenesis. In Aim 1, we will use muscle-specific deletion of PGC1β in mice to investigate how the loss of muscle PGC1β affects muscle vasculature, ischemic revascularization and the expression of angiogenic factors. In Aim 2, we will first perform in vitro experiments to study the interaction between PGC1β and COUP-TFI, and how they regulate muscle angiogenic genes. Second, we will use muscle-specific compound transgenic mice to investigate whether the anti-angiogenic effects of musclespecific PGC1β over-expression is lost on muscle-specific COUP-TFI deletion. We will also investigate the impact of double muscle-specific PGC1β and COUP-TFI knockout on muscle neo-angiogenesis and revascularization. The significance of our work is that it aims to discover how anti-angiogenic gene program is regulated in the skeletal muscle, and how it affects revascularization in muscle ischemia and diabetic muscle angiopathy. Our research is innovative as we will study the unexplored area of anti-angiogenic gene programming in the treatment of PAD/CLI. The findings will have implications for devising new treatment for muscle ischemia in PAD/CLI, and broadly in other diseases such as cardiac infarction, stroke and tumor angiogenesis.