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的主要死亡原因。动脉粥样硬化瘤由血管平滑肌细胞(VSMCs)、炎性细胞、脂质和内皮功能障碍的细胞外基质(ECM)组成。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，在我们对血管平滑肌细胞的作用的理解上留下了重要的空白。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因子，以便确定新的治疗靶点，以阻止疾病进展和斑块破裂。通过基因组筛选，我们发现富含脯氨酸的小重复蛋白3(SPRR3)仅在人和小鼠的动脉粥样硬化病变中表达，主要在血管平滑肌细胞中表达。SPRR家族由十几种小的细胞质蛋白组成，最初发现于复层上皮细胞，但最近在心肌细胞、骨骼肌、平滑肌和癌细胞中发现。到目前为止，它们在细胞功能中的作用尚不清楚。目的/假设：本研究在致动脉粥样硬化的载脂蛋白E-/-背景中建立了SPRR3基因缺陷(KO)小鼠，以确定SPRR3基因S在动脉粥样硬化的病理生理机制中的作用。初步数据显示，与没有巨噬细胞密度或循环脂质增加的apoE-/-小鼠相比，SPRR3-/-/apoE-/-小鼠的病变大小和数量显著增加。重要的是，这个模型显示了晚期病变，纤维帽、腔内血栓和下游壁内心肌梗死的VSMCs减少，可能是由于斑块不稳定所致。原代培养的KO小鼠VSMCs细胞凋亡率增加，细胞外基质合成减少，PI3K/Akt活性降低。我们还发现SPRR与FAK共沉淀。综上所述，我们的发现支持了中心假设，即SPRR3是动脉粥样硬化进展和斑块稳定性的独特而重要的调节器，它通过促进动脉粥样硬化微环境中Akt依赖的VSMC存活和ECM合成来实现这一点。所采用的具体目标/方法：我们将以以下目标研究我们的假说：1)SPRR3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SPRR3缺失是否通过增加病变内的VSMC缺失来推动疾病的进展。2)SPRR3是如何调节VSMC细胞活动的？为此，我们将确定SPRR3的细胞效应，以及SPRR3是否通过与FAK相互作用调节PI3K/Akt的激活来调节这些效应。与退伍军人事务部任务相关：动脉粥样硬化及其并发症是越南和后越南时期最大的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，其缺失会导致侵袭性疾病的进展和斑块破裂的临床后果。我们的研究将为这种发病率和死亡率极高的普遍疾病带来新的见解和治疗方法。

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.