Serotonin receptor contribution to inflammation-induced calcific aortic valve disease

项目总结/摘要 尚未发现预防/治疗钙化性主动脉瓣疾病（CAVD）的药理学方法， 病理生理变化包括钙化和纤维化，导致主动脉瓣狭窄，通常与 返流程度有证据表明，促炎细胞因子是心血管疾病的有效刺激因子， 钙化，而肾上腺素能系统是纤维化的有效刺激物。特别是血清素受体2B 已知SR-2B激活引起主动脉瓣的结构和机械变化，例如纤维化和纤维化。 增厚，在某些情况下，致密的结节，虽然没有钙化。临床研究还表明，血清素（5- HT）水平几乎是主动脉瓣狭窄患者的两倍，并且与 主动脉瓣狭窄的进展。这些发现表明，SR-2B激活也有助于 炎症诱导的CAVD的发病机制。有趣的是，正如我们的初步研究所证实的那样， 在基线时，鼠主动脉瓣间质细胞中表达的主要受体亚型是SR-2A，而 比SR-2B。然而，在我们额外的初步研究中，我们发现SR-2B是一种显著的 TNF-α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 尽管5-HT增强了TNF-α诱导的钙化，但它本身没有作用。我们还发现 TNF-α/5-HT诱导的钙化通过诱导质膜钙通道Orai 1发生， 这种钙化通过特异性抑制剂或仅SR-2B的敲低而减弱。补充初步 研究表明，高脂血症（ApoE-/-）患者外周血5-HT水平高于正常对照组（P <0.05）。 SR-2B在瓣膜尖的表达与胆固醇裂隙和钙化病变相邻， ApoE-/-，但在野生型小鼠中没有。基于集体证据，我们假设，5-HT在 高脂血症环境在CAVD中起关键作用。我们认为高脂血症会增加血液循环中的 5-HT和炎性细胞因子，后者诱导SR-2B和瓣尖中的骨生成，以及， 这些事件一起导致CAVD。在目的1中，我们将确定5-HT的受体亚型特异性作用 在非炎症条件下（SR-2A占主导地位）对基质力学的影响以及在炎症条件下对钙化的影响 （SR-2B显性）条件和介导TNF-α诱导SR-2B和SR-2B的信号传导途径。 下游成骨细胞分化。在目标2中，我们将确定经由SR-2B的5-HT信号传导是否是有效的。 使用1）5-HT缺陷的炎性小鼠模型（Tph 1-/-Ldlr-/- Apob 100/100），和2）受体亚型特异性激动剂和拮抗剂。我们将应用尖端技术， 包括激光光片荧光显微镜;系列体内融合18F-µPET-µCT成像;原子力 显微镜; 3-D水凝胶培养物;和受体亚型敲低细胞的植入。拟议 实验将阐明5-HT受体亚型特异性信号传导如何改变瓣膜尖的结构 以及靶向SR-2B是否为CAVD的有效药物治疗开辟了道路。

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.