Shared Mechanisms of Valvular and Vascular Calcification

说明(申请人提供)：钙化性主动脉瓣病(CAVD)是一种退行性疾病，唯一的治疗方法是外科瓣膜置换术。CAVD影响所有社会经济水平的个人，尤其与年龄、男性、高血压、高血脂、吸烟、II型糖尿病和肥胖症有关。CAVD的许多方面类似于动脉粥样硬化和血管钙化。一个以前没有在瓣膜中被研究过的病理特征，但是为治疗的发展提供了重要的机会，是糖胺多聚糖，碳水化合物为基础的细胞外基质分子的组织滞留脂质。一种特殊的修饰脂质，溶血磷脂酰胆碱(LPC)，被广泛认为是导致动脉粥样硬化细胞对居留的氧化低密度脂蛋白的各种反应的原因，最近被证明促进血管平滑肌细胞成骨基因的表达。然而，目前还没有关于LPC存在于病变的主动脉瓣硬化区和钙化区的研究，也没有关于LPC对瓣膜间质细胞(VICs)的影响的研究。同样不清楚的是，为什么主动脉瓣比二尖瓣早大约10年出现脂质和钙积聚。此外，最近的研究表明，原发性主动脉瓣疾病(钙化性)和原发性二尖瓣疾病(粘液瘤)涉及软骨生成基因的上调，但进展为成骨只发生在主动脉瓣，而不是二尖瓣。因此，这项R21提案召集了两名研究人员，利用最先进的技术来研究这一问题，以表征细胞表型，分析糖胺多糖的结构，并评估糖胺多糖与载脂蛋白的结合。这项研究的全球假设是，导致钙化的成骨蛋白在主动脉瓣中比二尖瓣中更丰富。为了测试主动脉VIC是否易于钙化，我们将首先使用激光捕获显微解剖和猪瓣膜组织的成骨基因分析来区分二尖瓣和主动脉VIC(目标1)的钙化潜力，并通过LPC处理培养的猪VIC。接下来，我们将确定从移植的人主动脉瓣膜钙化和非钙化区域分离的细胞中LPC水平和钙化促进蛋白之间是否存在相关性(目标2)。最后，我们将鉴定从人的钙化主动脉瓣分离的糖胺多糖，并评估从猪主动脉瓣、猪二尖瓣和人钙化的主动脉瓣不同区域分离的糖胺多糖结合血浆脂蛋白的能力(目标3)。这项对CAVD的关键早期阶段--脂类的滞留和作用机制的研究，将为开发新的CAVD药物疗法提供机会，并将导致对心脏瓣膜生物学的更清晰的理解。 公共卫生相关性：在钙化性主动脉瓣疾病中，瓣膜组织内形成骨质结节，阻止瓣膜正常打开和关闭，并导致心脏更努力地向前泵血。这种疾病的唯一治疗方法是用人工瓣膜对患病的瓣膜进行外科置换。我们认为，有可能通过开发干预两个关键早期事件的药物来逆转CAVD，这两个关键早期事件是复杂的碳水化合物分子与脂蛋白的结合，以及修饰的脂蛋白对心脏瓣膜细胞行为的负面影响。

DESCRIPTION (provided by applicant): Calcific aortic valve disease (CAVD) is a degenerative disease for which the only treatment is surgical valve replacement. CAVD afflicts individuals at all socioeconomic levels and is particularly associated with age, male gender, high blood pressure, high lipid levels, smoking, type II diabetes, and obesity. Many aspects of CAVD resemble atherosclerosis and vascular calcification. One pathological feature that has not been previously investigated in valves, but offers significant opportunity for therapeutic development, is the tissue retention of lipids by glycosaminoglycans, carbohydrate-based extracellular matrix molecules. One particular modified lipid, lyso-phosphatidylcholine (LPC), is widely considered to be responsible for various responses by atherosclerotic cells to residential oxidized LDL, and has recently been shown to promote osteogenic gene expression in vascular smooth muscle cells. There has been no investigation, however, regarding the presence of LPC within sclerotic and calcific regions of diseased aortic valves nor the effect of this agent on valvular interstitial cells (VICs). It is also unknown why aortic valves suffer from lipid and calcium accumulation roughly 10 years earlier than do mitral valves. Moreover, it has recently been shown that the primary aortic valve disease (calcific) and primary mitral valve disease (myxomatous) involve upregulation of chondrogenic genes, but progression to osteogenesis only occurs in the aortic valve, not in the mitral valve. Thus, this R21 proposal brings together two investigators to examine this problem using state of the art technologies for characterizing cell phenotype, analyzing the structure of glycosaminoglycans, and evaluating glycosaminoglycan-apolipoprotein binding. The global hypothesis of this research is that the osteogenic proteins leading to calcification are more abundant in aortic valves than mitral valves. To test whether aortic VICs are predisposed towards calcification, we will first differentiate between the potential for calcification by mitral and aortic VICs (Aim 1) using laser capture micro-dissection and osteogenic gene analysis of porcine valve tissues and by treating cultured porcine VICs with LPC. Next, we will determine if there is a correlation between the levels of LPC and calcification promoting proteins in cells isolated from calcified and non-calcified regions of explanted diseased human aortic valves (Aim 2). Finally, we will characterize the glycosaminoglycans isolated from human calcified aortic valves and evaluate the ability of glycosaminoglycans isolated from porcine aortic valves, porcine mitral valves, and various regions of human calcified aortic valves to bind plasma lipoproteins (Aim 3). This proposed research into the mechanisms behind a pivotal early stage of CAVD, the retention and action of lipids, will offer opportunities to develop new pharmacological therapies for CAVD, and will also lead to a clearer understanding of heart valve biology. PUBLIC HEALTH RELEVANCE: In calcific aortic valve disease, bony nodules form within the valve tissues, prevent the valve from opening and closing normally, and cause the heart to work harder to pump blood forward. The only treatment for this disease is surgical replacement of the diseased valve with an artificial valve. We propose that it might be possible to reverse CAVD by developing medications that interfere with two critical early events, the binding of lipoproteins by complex carbohydrate-based molecules, and the negative impact of modified lipoproteins on the behavior of heart valve cells.