Mitochondrial Modulation of Endothelia Function

内皮功能的线粒体调节

• 批准号: 8505559
• 负责人: John Francis Keaney
• 金额: $ 40.41万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-15 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8505559
• 关键词: Acute; Apoptosis; Atherosclerosis; Behavior; Behavior Control; Biogenesis; Blood Clot; Blood Vessels; Blood coagulation; Cell physiology; Cells; Cellular Stress; Characteristics; Communication; Data; Diabetes Mellitus; Diet; Disease; Endothelial Cells; Endothelium; Exhibits; Fatty acid glycerol esters; Functional disorder; Funding; Gene Targeting; Genes; Heme; Hindlimb; Homeostasis; Insulin Resistance; Ischemia; Knock-out; Knockout Mice; Knowledge; Limb structure; Link; Liver; Mediating; Metabolism; Mitochondria; Modeling; Molecular; Morphology; Mus; Muscle; Myocardial Infarction; Neoplasm Metastasis; Obesity; Pancreas; Patients; Phenotype; Principal Investigator; Process; Proteins; Protons; Regulation; Research; Respiration; Role; Signal Transduction; Solid Neoplasm; Stress; Stroke; Superoxides; Testing; Therapeutic; Tissues; Tube; Tumor Angiogenesis; UCP2 protein; Vascular Diseases; Work; Wound Healing; angiogenesis; cell behavior; design; energy balance; in vivo; insight; migration; premature; prevent; programs; public health relevance; research study; response; senescence; tissue repair; tool; tumor

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.

描述（由申请人提供）：众所周知，线粒体参与许多细胞过程，例如细胞凋亡、血红素代谢和能量平衡调节，这很大程度上是由于线粒体对能量状态的影响，大多数线粒体研究都涉及肝脏。由于内皮细胞主要是糖酵解细胞，因此尽管最近认识到内皮细胞可以影响组织代谢和内皮细胞中线粒体的作用，但人们很少研究线粒体的作用。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共激活因子，对于内皮细胞应激适应至关重要。 我们提供了解偶联蛋白 2 (UCP2)（PGC-1）的数据靶基因对于内皮细胞的线粒体应激适应特别重要，在组织修复或增加燃料利用率的情况下，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碎片。该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利用线粒体为了实现这一目标，我们提出了一个中心假设，即内源性 UCP2 具有预防“线粒体应激”的功能，因此，UCP2 是实现内皮细胞功能和血管稳态的主要决定因素。在这个项目目标中，我们将首先确定内皮 UCP2 在饮食引起的胰岛素抵抗中的影响，这是一种已知对线粒体造成压力的疾病，因为我们的初步数据表明 UCP2 可以防止线粒体断裂，这是一种已知的应激状态。鉴于 2 型（胰岛素抵抗）糖尿病肥胖患者的已知特征，我们将测试 UCP2 如何影响饮食引起的肥胖和胰岛素抵抗中的内皮功能。我们将利用我们新创建的 UCP2 模型与内皮细胞特异性敲除 (ECKOCP2) 或仅内皮细胞 (ECUCP2) UCP2 表达，以确定内皮 UCP2 与已知会诱发肥胖的高脂肪饮食的功能、形态和分子影响然后，我们将确定 UCP2 如何影响内皮对应激（如体内缺血血运重建和肿瘤血管生成）的反应，因为我们的数据表明，全球 UCP2 缺失小鼠的血管生成因后肢缺血的应激而受损，因此我们将确定具体的影响因素。 UCP2 对体内内皮应激的作用因此，将在两种内皮应激模型中测试 ECKOUCP2 和 ECUCP2 小鼠：a) 缺血性模型。后肢缺血的血运重建；b) 对于后者，除了对血管形成的影响之外，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。内皮 UCP2 对每个模型中线粒体质量、形态和网络碎片的影响我们还将探讨自 UCP2 缺失以来 p53 在促进内皮 UCP2 缺失表型中的作用。最后，我们将确定 UCP2 在内皮细胞中的功能意义，因为我们的数据表明 UCP2 通过防止线粒体断裂来保留内皮功能，因此我们将确定 UCP2 如何通过操纵 UCP2 影响线粒体功能和形态。将测试线粒体网络碎片的状态和 ¿在此过程中确定 O2 介导的蛋白质损伤、线粒体自噬、线粒体生物发生和 p53，然后我们将线粒体网络断裂的机制与与血管生成相关的内皮功能（包括增殖、迁移、管形成和 NO）联系起来。上述实验应该为我们提供有关 UCP2 如何控制线粒体动力学以及内皮功能的重要见解，这些见解将告诉我们线粒体如何影响血管稳态，并为我们提供必要的知识。线粒体作为体内操纵内皮的一种手段，这一知识可能对伤口愈合、肢体缺血和肿瘤转移产生广泛的影响。