Calcific Atherosclerosis is Mediated by Macrophage Adhesion Signaling

描述（由申请人提供）： 由动脉粥样硬化引起的缺血性心脏病是美国退伍军人发病率和死亡率的主要原因。动脉粥样硬化是一种慢性炎症性疾病，其特征在于异质性、充满脂质的血管斑块。传统上，炎症斑块破裂的风险很高，导致凝块形成和随后的心脏病发作。识别钙化斑块在总体动脉粥样硬化负荷方面具有预测价值;然而，炎性高危斑块往往具有最小或“点状”水平的钙沉积。因此，一些斑块是最低限度的钙化和非常炎性的性质，而其他类型的斑块是严重钙化和出现较少的炎性和不太可能破裂。关于什么机制驱动斑块钙组成的演变知之甚少。巨噬细胞粘附信号传导对于增强巨噬细胞分泌的促炎因子（包括重塑细胞外基质的蛋白酶）至关重要。细胞骨架元件如Rac 2和肌球蛋白IIA似乎是基于粘附的炎症信号传导的关键调节剂，因此将成为抑制斑块炎症和降低心脏病发作风险的极好的潜在靶点。Rac 2基因缺失、易患动脉粥样硬化的小鼠模型的初步数据表明，严重钙化斑块的演变可能不太具有炎症性。本研究的目的是确定驱动这种重度钙化的机制，并量化重度钙化是否真正代表斑块内炎症活动的降低。斑块钙组成被认为是来自合成代谢（促进钙）成骨细胞和分解代谢（钙和基质重塑）破骨细胞之间的平衡。巨噬细胞亚群具有成为破骨细胞样细胞的能力。因此，主要假设是巨噬细胞中通过Rac 2-肌球蛋白IIA网络的粘附信号传导是巨噬细胞衍生的破骨细胞样细胞的关键调节剂，导致其有效分化和效应器功能，其用于使斑块中的钙化沉积最小化并维持炎性表型。这一假设导致形成实验设计的三个主要目标。第一个目标是确定破骨细胞样细胞分化和功能对Rac 2和肌球蛋白IIA的依赖性，使用破骨细胞分化和矿化钙吸收的标志物进行功能评估。第二个目标是使用分子和生物化学技术鉴定炎症因子，如基质重塑蛋白，其表达由Rac 2和肌球蛋白IIA通过破骨细胞样细胞粘附驱动。第三个也是最后一个目标是使用分子成像工具和动脉粥样硬化易感动物模型，确定钙化动脉粥样硬化对肌球蛋白IIA的依赖性，并量化体内钙化程度与炎症标志物表达之间的负相关性。定义调节动脉粥样硬化斑块钙化的炎症机制将导致识别新的治疗靶点，用于开发可以降低心脏病发作风险的新药，最终对美国退伍军人的生活产生深远影响。

DESCRIPTION (provided by applicant): Ischemic heart disease caused by atherosclerosis is the leading cause of morbidity and mortality among U.S. Veterans. Atherosclerosis is a chronic inflammatory disease characterized by heterogeneous, lipid-laden vascular plaques. Inflammatory plaques have traditionally been at high risk of rupture leading to clot formation and subsequent heart attack. Identifying calcified plaque has a predictive value in terms of overall atherosclerotic burden; however, inflammatory high-risk plaques tend to have minimal or "spotty" levels of calcium deposition. Thus some plaques are minimally calcified and very inflammatory in nature, whereas other types of plaque are heavily calcified and appear less inflammatory and less likely to rupture. Little is known about what mechanisms drive the evolution of plaque calcium composition. Macrophage adhesion signaling is critical to the augmentation pro-inflammatory factors secreted by macrophages, including proteases that remodel extracellular matrix. Cytoskeletal elements like Rac2 and Myosin IIA appear to be critical modulators of that adhesion-based inflammatory signaling and thus would make excellent potential targets for inhibiting plaque inflammation and reducing the risk of heart attack. Preliminary data in a Rac2 gene-deletion, atherosclerosis-prone mouse model demonstrates the evolution of heavily calcified plaques that are likely to be less inflammatory. The goal of this study is to define the mechanisms that drive this heavy calcification and to quantify whether heavy calcification truly represents decreased inflammatory activity within the plaque. Plaque calcium composition is thought to be derived from a balance between anabolic (calcium-promoting) osteoblasts and catabolic (calcium and matrix remodeling) osteoclasts. Macrophage subsets have the capacity to become osteoclast-like cells. Thus, the primary hypothesis is that adhesion signaling in macrophages, through Rac2- Myosin IIA networks, is a critical modulator of macrophage-derived osteoclast-like cell, leading to their effective differentiation and effector function, which serves to minimize calcific depositon in plaques and maintain an inflammatory phenotype. This hypothesis leads to three primary objectives that form the experimental design. The first objective is to define the dependence of osteoclast-like cell differentiation and function on Rac2 and Myosin IIA, using markers of osteoclast differentiation and the mineralized calcium resorption for function assessment. The second objective is to identify inflammatory factors, like matrix remodeling proteins, whose expression is driven by Rac2 and Myosin IIA through osteoclast-like cell adhesion, using molecular and biochemical techniques. The third and final aim is to define the dependence of calcific atherosclerosis on Myosin IIA and to quantify an inverse correlation between the degree of calcification and the expression of inflammatory markers in vivo, using molecular imaging tools and the atherosclerosis-prone animal model. Defining the inflammatory mechanisms that modulate calcification of atherosclerotic plaques will lead to identification of novel therapeutic targets for the development of new medications that can reduce the risk of heart attack, ultimately having a profound effect on the lives of U.S. Veterans.