Uncovering Novel Atheroprotective Mechanisms

描述（由申请人提供）： 背景：动脉粥样硬化病变是退伍军人和发达国家死亡的主要原因。动脉粥样硬化由血管平滑肌细胞（VSMCs）、炎症细胞、脂质和细胞外基质（ECM）组成，其位于功能障碍的内皮细胞之下。到目前为止，压倒性的重点一直是研究脂质和炎症在动脉粥样硬化发病机制中的作用，留下了重要的差距，我们的VSMC的作用的理解。我们实验室的目标是更好地了解它们的作用和关键的分子调节剂，以确定新的治疗靶点来阻止疾病进展和斑块破裂。使用基因组筛选，我们确定了小脯氨酸丰富的重复蛋白3（SPRR 3）是专门表达的动脉粥样硬化病变的人类和小鼠主要在VSMC。SPRR家族由十几种首先在复层上皮中鉴定的小细胞质蛋白组成，但最近在心肌细胞、骨骼肌和平滑肌以及癌细胞中发现。到目前为止，它们在细胞功能中的作用尚不清楚。目的/假设：为了确定SPRR 3在动脉粥样硬化病理生理学中的作用，我们在致动脉粥样硬化apoE-/-背景下产生SPRR 3缺陷（KO）小鼠。初步数据显示，与没有增加的巨噬细胞密度或循环脂质的apoE-/-小鼠相比，SPRR 3-/-/apoE-/-小鼠的病变大小和数量显著增加。重要的是，该模型显示了晚期病变，纤维帽中的VSMC减少，管腔内血栓和下游壁内心肌梗死，可能是由于斑块不稳定性。从KO小鼠分离的原代VSMCs表现出凋亡增加，但ECM合成和PI 3 K/Akt活化减少。我们还发现SPRR与FAK共免疫沉淀。总之，我们的研究结果支持了中心假设，即SPRR 3是动脉粥样硬化进展和斑块稳定性的独特且重要的调节剂，其通过增强动脉粥样硬化微环境中Akt依赖性VSMC存活和ECM合成来实现。采用的具体目标/方法：我们将研究我们的假设与以下目标：1）SPRR 3调节动脉粥样硬化进展的细胞机制是什么？在这个目标中，我们将研究SPRR 3损失是否通过增加病变内的VSMC损失来驱动疾病进展。2)SPRR 3如何调节VSMC细胞活性？在这个目标中，我们将确定SPRR 3的细胞效应，以及SPRR 3是否通过与FAK相互作用来调节PI 3 K/Akt活化来调节这些效应。与退伍军人事务部使命的相关性：动脉粥样硬化及其并发症是我们最大的越南和越南后时期退伍军人死亡的主要原因。我们的研究已经确定了一种新的动脉粥样硬化保护因子，其缺失导致疾病进展和斑块破裂的临床后果。我们的研究将导致对这种具有极高发病率和死亡率的普遍疾病的新见解和治疗方法。

DESCRIPTION (provided by applicant): Background: Atherosclerotic lesions underlie the leading cause of death among Veterans and in the developed world. Atheromas consist of vascular smooth muscle cells (VSMCs), inflammatory cells, lipid and extracellular matrix (ECM) underlying a dysfunctional endothelium. Thus far the overwhelming emphasis has been on studying the role of lipids and inflammation in atheroma pathogenesis, leaving important gaps in our understanding of the role of VSMCs. The goal of our laboratory is to gain better understanding of their role and key molecular regulators in order to identify new therapeutic targets to halt disease progression and plaque rupture. Using a genomic screen we identified that the small proline rich repeat protein 3 (SPRR3) is exclusively expressed in atherosclerotic lesions of humans and mice primarily in VSMCs. The SPRR family consists of over a dozen small cytoplasmic proteins first identified in stratified epithelia but more recently found in cardiomyocytes, skeletal and smooth muscle and cancer cells. As yet, their role in cell function is unclear. Objective/hypotheses: To determine SPRR3's role in the pathophysiology of atherosclerosis, we generated the SPRR3-deficient (KO) mouse in the atherogenic apoE-/- background. Preliminary data showed a dramatic increase in lesion size and numbers in SPRR3-/-/apoE-/- mice as compared to apoE-/- mice without increased macrophage density or circulating lipids. Importantly, this model showed advanced lesions with reduced VSMCs in the fibrous cap, intraluminal thrombi and downstream intramural myocardial infarcts, likely due to plaque instability. Primary VSMCs isolated from KO mice demonstrated increased apoptosis but reduced ECM synthesis and PI3K/Akt activation. We also found that SPRR co-immunoprecipitates with FAK. Together, our findings support the central hypothesis that SPRR3 is a unique and significant modulator of atheroma progression and plaque stability, which it does by enhancing Akt-dependent VSMC survival and ECM synthesis within the atheroma microenvironment. Specific aims/methods employed: We will study our hypothesis with the following aims: 1) What is the cellular mechanism by which SPRR3 regulates atheroma progression? In this aim we will investigate whether SPRR3 loss drives disease progression by augmenting VSMC loss within lesions. 2) How does SPRR3 regulate VSMC cellular activity? In this aim, we will determine the cellular effects of SPRR3 and whether SPRR3 regulates these effects by interacting with FAK to modulate PI3K/Akt activation. Relevance to VA mission: Atherosclerosis and its complications underlie the leading cause of death in our largest population of Veterans from the Vietnam and post-Vietnam time period. Our studies have identified a novel atheroprotective factor whose loss results in aggressive disease progression and clinical consequences of plaque rupture. Our studies will result in new insights and therapeutic approaches to this pervasive disease with exceedingly high morbidity and mortality.