Shared Mechanisms of Valvular and Vascular Calcification

描述（由申请方提供）：钙化性主动脉瓣疾病（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困扰着所有社会经济水平的个体，尤其与年龄、男性、高血压、高血脂水平、吸烟、II型糖尿病和肥胖相关。CAVD的许多方面类似于动脉粥样硬化和血管钙化。一种病理学特征是糖胺聚糖（基于碳水化合物的细胞外基质分子）对脂质的组织滞留，这一特征以前未在瓣膜中进行研究，但为治疗开发提供了重要机会。一种特殊的改性脂质，溶血磷脂酰胆碱（LPC），被广泛认为是动脉粥样硬化细胞对住宅氧化LDL的各种反应的原因，最近已被证明可以促进血管平滑肌细胞中的成骨基因表达。然而，目前还没有关于LPC在病变主动脉瓣的钙化和钙化区域内的存在的研究，也没有这种药物对瓣膜间质细胞（VIC）的影响。也不知道为什么主动脉瓣比二尖瓣早大约10年发生脂质和钙积聚。此外，最近已经表明，原发性主动脉瓣疾病（钙化性）和原发性二尖瓣疾病（粘液瘤性）涉及软骨形成基因的上调，但骨形成的进展仅发生在主动脉瓣中，而不是二尖瓣中。因此，这项R21提案汇集了两名研究人员，使用最先进的技术来表征细胞表型，分析糖胺聚糖的结构，并评估糖胺聚糖-载脂蛋白结合来研究这个问题。这项研究的总体假设是，导致钙化的成骨蛋白在主动脉瓣中比在二尖瓣中更丰富。为了测试主动脉VIC是否倾向于钙化，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以及通过用LPC处理培养的猪VIC来区分二尖瓣和主动脉VIC的钙化潜力（目标1）。接下来，我们将确定从钙化和非钙化区域分离的细胞中LPC和钙化促进蛋白的水平之间是否存在相关性（目的2）。最后，我们将表征从人钙化主动脉瓣分离的糖胺聚糖，并评价从猪主动脉瓣、猪二尖瓣和人钙化主动脉瓣的各个区域分离的糖胺聚糖结合血浆脂蛋白的能力（目的3）。这项对CAVD关键早期阶段（脂质的保留和作用）背后机制的拟议研究将为开发CAVD的新药物疗法提供机会，也将导致对心脏瓣膜生物学的更清晰理解。 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送血液。这种疾病的唯一治疗方法是用人工瓣膜手术置换患病的瓣膜。我们提出，通过开发干扰两个关键早期事件的药物来逆转CAVD是可能的，这两个关键早期事件是脂蛋白与复杂碳水化合物分子的结合，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.