Mitochondrial Modulation of Endothelia Function

描述(申请人提供)：已知线粒体参与了许多细胞过程，如细胞凋亡、血红素代谢和能量平衡的调节。很大程度上是由于线粒体对能量状态的影响，大多数线粒体研究涉及肝脏、肌肉和胰腺细胞。由于内皮细胞主要是糖酵解的，所以线粒体在内皮细胞中的作用很少被研究，尽管人们最近认识到内皮细胞可以影响组织新陈代谢和动态平衡。在之前的资助期间，我们发现内皮PGC-1是许多线粒体基因的转录辅助激活因子，对内皮细胞应激适应至关重要。在此应用程序中， 我们发现解偶联蛋白-2(UCP2)是PGC-1的靶基因，对内皮细胞线粒体应激适应特别重要。在组织修复或燃料利用增加的情况下，内皮细胞通过解偶联蛋白-2(UCP2)严格调节其线粒体质子梯度，以防止“线粒体应激”表现为促进内皮功能障碍的线粒体网络断裂。这项研究计划的长期目标是了解线粒体如何调节内皮功能，以及我们如何将这些信息用于新的治疗。为了实现这一目标，我们提出了一个中心假设，即内源性UCP2具有防止“线粒体应激”的功能，因此，UCP2是内皮细胞功能和血管内稳态的主要决定因素。为了实现这一项目目标，我们将首先确定内皮UCP2在饮食诱导的胰岛素抵抗中的意义，这是一种已知的应激线粒体的情况。由于我们的初步数据表明，UCP2可以防止线粒体碎裂，这是患有2型(胰岛素抵抗)糖尿病的肥胖患者的已知特征，我们将测试UCP2如何在饮食诱导的肥胖和胰岛素抵抗中影响内皮功能。我们将利用我们新创建的具有内皮细胞特异性基因敲除(ECKOUCP2)或仅内皮细胞(ECUCP2)UCP2表达的UCP2模型来确定高脂肪饮食导致肥胖和胰岛素抵抗的内皮UCP2的功能、形态和分子意义。然后，我们将确定UCP2如何影响血管内皮细胞对应激反应的反应，如体内缺血血管重建和肿瘤血管生成。由于我们的数据表明UCP2基因缺失的小鼠在后肢缺血应激下血管生成受损，我们将在体内确定UCP2在内皮应激中的具体作用。因此，ECKOUCP2和ECUCP2小鼠将在两种内皮应激模型中进行测试：a)后肢缺血导致的缺血血管重建；b)肿瘤血管生成。对于后者，我们还将确定急性UCP2抑制是否具有限制或缩小实体瘤的治疗潜力。除了对血管形成的影响外，我们还将在每个模型中测试内皮UCP2对线粒体质量、形态和网络碎裂的影响。我们还将探讨P53在促进内皮细胞UCP2缺失表型中的作用，因为UCP2缺失的内皮细胞表现出P53依赖的过早衰老。最后，我们将确定UCP2在内皮细胞中的功能含义。由于我们的数据表明UCP2通过阻止线粒体碎裂来保护内皮功能，我们将确定UCP2如何影响线粒体的功能和形态。具有被操纵的UCP2状态的ECS将被测试线粒体网络断裂，并确定O2-介导的蛋白损伤、有丝分裂、线粒体生物发生和P53在这一过程中的作用。然后，我们将把线粒体网络断裂的机制(S)与血管生成相关的内皮功能联系起来，包括增殖、迁移、管子形成和NO生物活性。上面概述的实验应该为我们提供重要的洞察力，了解UCP2是如何控制线粒体动力学的，并因此控制内皮功能。这些见解将告诉我们线粒体如何影响血管内稳态，并为我们提供必要的知识来利用线粒体作为一种在体内操纵内皮的手段，这些知识可能对伤口愈合、肢体缺血和肿瘤转移有广泛的影响。

DESCRIPTION (provided by applicant): Mitochondria are known to participate in a host of cellular processes such as apoptosis, heme metabolism, and the regulation of energy balance. Largely due to the mitochondrial impact on energy status, the bulk of most mitochondrial research involved liver, muscle, and pancreatic cells. Since endothelial cells are predominantly glycolytic, little effort was applied to the role of mitochondria in the endothelium, despite recen appreciation that endothelial cells can impact tissue metabolism and homeostasis. In the previous funding period, we found that endothelial PGC-1¿, a transcriptional co-activator for many mitochondrial genes, was critical for endothelial cell stress adaptation. In this application, we present data that uncoupling protein-2 (UCP2), a PGC-1¿ target gene, is particularly important for mitochondrial stress adaptation in the endothelium. In settings of tissue repair or increased fuel utilization, endothelial cells tightly regulate their mitochondrial proton gradient (¿¿) via uncoupling protein-2 (UCP2), in order to prevent "mitochondrial stress" manifest as mitochondrial network fragmentation that promotes endothelial dysfunction. The long-term objective of this investigative program is to understand how mitochondria modulate endothelial function and how we can use this information for new therapies. In order to achieve this objective, we submit as a central hypothesis that endogenous UCP2 functions to prevent "mitochondrial stress" and, as a consequence, UCP2 is a major determinant of endothelial cell function and vascular homeostasis. In order to achieve this project objective, we will first determine the implications of endothelial UCP2 in diet- induced insulin resistance, a condition known to stress mitochondria. Since our preliminary data indicate that UCP2 prevents mitochondrial fragmentation, a known characteristic of obese patients with type 2 (insulin- resistant) diabetes, we will test how UCP2 impacts endothelial function in diet-induced obesity and insulin resistance. We will utilize our newly created UCP2 models with Endothelial Cell-specific KnockOut (ECKOUCP2) or Endothelial Cell-only (ECUCP2) UCP2 expression to determine the functional, morphologic, and molecular implications of endothelial UCP2 with a high-fat diet known to induce obesity and insulin resistance. We will then determine how UCP2 impacts the endothelial responses to stresses such as ischemic revascularization and tumor angiogenesis in vivo. Since our data indicate global UCP2-null mice have impaired angiogenesis with the stress of hindlimb ischemia, we will determine the specific role of UCP2 for endothelial stress in vivo. Accordingly, ECKOUCP2 and ECUCP2 mice will be tested in two models of endothelial stress: a) ischemic revascularization from hind limb ischemia and; b) tumor angiogenesis. With regards to the latter, we will also determine if acute UCP2 inhibition has the therapeutic potential to limit or shrink solid tumors. In addition to the impact on blood vessel formation, we will test endothelial UCP2 for its implications on mitochondrial mass, morphology, and network fragmentation in each model. We will also explore the role of p53 in promoting the endothelial UCP2-null phenotype since UCP2-null endothelium exhibits premature p53-dependent senescence. Finally, we will determine the functional implications of UCP2 in the endothelium. Since our data indicate UCP2 preserves endothelial function by preventing mitochondrial fragmentation, we will determine how UCP2 impacts mitochondrial function and morphology. ECs with manipulated UCP2 status will be tested mitochondrial network fragmentation and the roles of ¿O2--mediated protein damage, mitophagy, mitochondrial biogenesis, and p53 determined in this process. We will then link the mechanism(s) of mitochondrial network fragmentation to endothelial functions relevant to angiogenesis including proliferation, migration, tube formation, and NO¿ bioactivity. The experiments outlined above should provide us with important insight as to how UCP2 controls mitochondrial dynamics and, as a consequence, endothelial function. These insights will inform us as to how the mitochondria impact vascular homeostasis and provide us with the requisite knowledge to utilize mitochondria as a means of manipulating the endothelium in vivo and this knowledge could have wide ranging implications for wound healing, limb ischemia, and tumor metastasis.