Serotonin receptor contribution to inflammation-induced calcific aortic valve disease

项目摘要/摘要 目前还没有发现预防/治疗钙化性主动脉瓣疾病(CAVD)的药物方法，其 病理生理变化包括钙化和纤维化，导致主动脉狭窄，通常合并 反胃。有证据表明，促炎细胞因子是心血管疾病的有力刺激因子 钙化，而5-羟色胺能系统是纤维化的有力刺激因素。特别是，5-羟色胺受体2B 已知(SR-2B)的激活会导致主动脉瓣的结构和机械改变，如纤维化和 增厚，在某些情况下，致密的结节，但不钙化。临床研究还表明，5-羟色胺(5- 在主动脉狭窄患者中，羟色胺水平几乎是前者的两倍，并且与 主动脉狭窄的进展。这些发现表明，SR-2B的激活也有助于 炎症性CAVD的发病机制。有趣的是，正如我们的初步研究证实的那样， 在基础状态下，小鼠主动脉瓣间质细胞表达的主要受体亚型是SR-2A，而不是 而不是SR-2B。然而，在我们额外的初步研究中，我们发现SR-2B是戏剧性的 被肿瘤坏死因子-a上调(30倍)。这种诱导需要BMP-2信号，这可能解释了我们的发现 5-羟色胺虽能增强肿瘤坏死因子-a诱导的钙化，但单独作用不明显。我们还发现， 肿瘤坏死因子-a/5-羟色胺诱导的钙化是通过诱导质膜钙通道Orai1和 这种钙化可通过特定的抑制剂或仅敲除SR-2B来减弱。额外的初步报告 研究表明，高脂血症(APOE-/-)患者的外周循环5-羟色胺水平高于野生型 类型的小鼠，并且SR-2B在心脏瓣膜尖部的表达邻近胆固醇裂隙和钙化病变。 APOE-/-，但在野生型小鼠中不存在。根据集体证据，我们假设5-羟色胺在 高脂血症环境在CAVD发病中起关键作用。我们假设，高脂血症会增加循环中的 5-羟色胺和炎性细胞因子，后者诱导SR-2B和瓣膜尖部成骨，以及， 总而言之，这些事件导致了CAVD。在目标1中，我们将确定5-羟色胺的受体亚型特异性效应 非炎症性(SR-2A为主)下的基质力学以及炎症下的钙化 (SR-2B占优势)条件和介导SR-2B和 下游成骨细胞分化。在目标2中，我们将确定通过SR-2B的5-HT信号是否 使用1)5-羟色胺缺乏的炎性小鼠模型(TPH1-/-Ldlr-/-)对炎症诱导的CAVD至关重要 Apob100/100)，以及2)受体亚型特异性激动剂和拮抗剂。我们将应用尖端技术， 包括激光薄片荧光显微镜；活体系列融合18F-µPET-µCT成像；原子力 显微镜；三维水凝胶培养；受体亚型敲除细胞的植入。建议数 实验将阐明5-羟色胺受体亚型特异性信号如何改变瓣膜尖端的结构 以及靶向SR-2B是否为有效的基于药物的CAVD治疗开辟了道路。

PROJECT SUMMARY/ABSTRACT No pharmacological approach has been found to prevent/treat calcific aortic valve disease (CAVD), whose pathophysiological changes include calcification and fibrosis, causing aortic stenosis, often in combination with regurgitation. Evidence shows that pro-inflammatory cytokines are potent stimulators of cardiovascular calcification, while the serotonergic system is a potent stimulator of fibrosis. In particular, serotonin receptor 2B (SR-2B) activation is known to cause structural and mechanical changes in aortic valves, such as fibrosis and thickening, in some cases, dense nodules, though not calcification. Clinical studies also show that serotonin (5- HT) levels are almost twice as high in patients with aortic stenosis, and are significantly associated with progression of aortic stenosis. These findings suggest that SR-2B activation also contributes to the pathogenesis of inflammation-induced CAVD. Interestingly, as confirmed in our preliminary studies, the predominant receptor subtype expressed in murine aortic valve interstitial cells, at baseline, is SR-2A, rather than SR-2B. However, in our additional preliminary studies, we found that it was SR-2B that was dramatically upregulated (> 30-fold) by TNF-a. This induction required BMP-2 signaling and may account for our findings that, although 5-HT augmented TNF-a-induced calcification, it had no effect alone.. We also found that the TNF-a/5-HT-induced calcification occurred via induction of Orai1, a plasma membrane calcium channel, and this calcification was attenuated by specific inhibitors or knockdown of only SR-2B. Additional preliminary studies show that circulating peripheral 5-HT levels were higher in hyperlipidemic (Apoe-/-) compared with wild type mice, and SR-2B expression in the valve cusps was adjacent to cholesterol clefts and calcified lesions in Apoe-/- but not in wild type mice. Based on the collective evidence, we hypothesize that 5-HT in the hyperlipidemic milieu plays a key role in CAVD. We posit that hyperlipidemia increases circulating levels of both 5-HT and inflammatory cytokines, the latter of which induce SR-2B and osteogenesis in valve cusps, and, together, these events lead to CAVD. In Aim 1, we will determine receptor subtype-specific effects of 5-HT on matrix mechanics under non-inflammatory (SR-2A dominant) as well as on calcification under inflammatory (SR-2B dominant) conditions and on the signaling pathways mediating TNF-a induction of SR-2B and of downstream osteoblastic differentiation. In Aim 2, we will determine whether 5-HT signaling via SR-2B is critical for inflammation-induced CAVD using 1) a 5-HT-deficient, inflammatory mouse model (Tph1-/-Ldlr-/- Apob100/100), and 2) receptor subtype-specific agonists and antagonists. We will apply cutting edge technology, including laser light sheet fluorescence microscopy; serial in vivo fused 18F-µPET-µCT imaging; atomic force microscopy; 3-D hydrogel cultures; and implantation of receptor subtype knockdown cells. The proposed experiments will elucidate how 5-HT receptor subtype-specific signaling changes the structure of valve cusps and whether targeting SR-2B opens the path for effective pharmacological-based treatment of CAVD.