Serotonin Signaling in Mitral Valve Homeostasis, Maintenance and Restoration

粘液瘤瓣膜病引起的退行性二尖瓣反流(MR)影响数百万人。唯一的治疗方法是瓣膜修复或替换。这种相互竞争的更新将促进对神经递质作用的理解，心脏瓣膜病中的5-羟色胺(5-羟色胺)一直与分泌5-羟色胺的类癌和药物有关，如减肥药右旋苯氟胺。根据我们在第一个计划阶段的进展，我们将探讨5-羟色胺转运体(SERT)和5-羟色胺受体(HTR)信号机制在骨髓来源的血小板和二尖瓣间质细胞(MVIC)中参与MR的病理生理机制的假设，并代表有希望的治疗靶点。具体目的1：剖析5-羟色胺(5-HT)介导的影响人和小鼠二尖瓣叶病理重塑的机制：以正常二尖瓣(MV)叶和人源性MVICs为对照，研究5-羟色胺(5-羟色胺)介导的二尖瓣叶病变重塑的特征。在转化生长因子β、骨形成蛋白4和5羟色胺的刺激下，SERT拮抗剂和hTR抑制剂的研究将剖析与体外和体外MR进展相关的独特的5羟色胺相关转录组和细胞表型。在人类MVIC和MR组织中，与暴露于5HT相关机械询问的正常小叶相比，终点将是转录、细胞外基质(ECM)重塑。Subaim 1B：研究SERT在MR发病机制中的作用：SERT缺失的小鼠(SERT-/-)自发地发展成影响二尖瓣和主动脉瓣的瓣膜病变。机械论研究将SERT-/-与SERT+/+进行比较。终点将包括瓣膜厚度、心肌纤维化和MV转录组的变化，以了解这种SERT相关瓣膜病的疾病进展。特定目的2：探讨5-羟色胺在MR发病机制中的作用，包括血小板机制和同时表达CD34和HTR2B的骨髓来源的血液生长内皮细胞(BOEC)。Subaim 2A：值得注意的是，MR会导致血小板处于慢性激活状态，因为血小板是血液中5-羟色胺的主要载体，激活后释放5-羟色胺。我们将在人类受试者、患有MR的犬和小鼠中研究血小板在5HT相关MR病理生理学中的作用。终点将包括5-羟色胺水平、血小板激活标志物和由于血小板α颗粒释放而导致的转化生长因子-β1水平的差异。Subaim 2B：为了研究骨髓来源细胞在影响BOEC先生的5HT机制的病理生理学中的作用，将从Subaim 2A的研究组进行培养。实验设计将使用流式细胞术进行细胞鉴定，qRT-PCR研究来评估基因表达模式，蛋白质组学方法来评估HTR活性，并测量体外细胞外基质的产生。预计这些研究的机制结果将为MR提供靶向5-羟色胺的治疗方向，并利用BOEC的个性化方法来模拟病理生理学。

Degenerative mitral regurgitation (MR) due to myxomatous valve disease affects millions. The only treatment is either valve repair or replacement. This competing renewal will advance understanding of the role of the neurotransmitter, serotonin (5HT) in MR. Heart valve disease has been associated with both 5HT-secreting carcinoid tumors and medications, such as the diet drug, Dexfenfluoramine. Based on our progress in the 1ST program period, we will investigate the hypothesis that 5HT-mechanisms involving the serotonin transporter (SERT), and 5HT receptor (HTR) signaling, both in platelets and mitral valve interstitial cells (MVIC) of bone marrow origin, contribute to the pathophysiology MR, and represent promising therapeutic targets. Specific Aim 1: To dissect 5HT-mediated mechanisms that influence pathologic remodeling of MV leaflets in human subjects and mice Subaim 1A: To characterize 5HT-mediated MV remodeling in MR compared to normal mitral valve (MV) leaflets and human-derived MVICs. SERT-antagonists, and HTR inhibitor studies will dissect unique 5HT related transcriptome and cellular phenotypes associated with MR progression in vitro and ex vivo under TGFβ, BMP4, and 5HT stimulation. Endpoints will be transcriptomic, extracellular matrix (ECM) remodeling in human MVIC and tissue from MR compared to normal leaflets exposed to 5HT-related mechanistic interrogation. Subaim 1B: To investigate the role of SERT in the pathogenesis of MR: The SERT deleted mouse (SERT-/-) spontaneously develops a valvulopathy affecting the mitral and aortic valves. Mechanistic studies will compare SERT-/- to SERT+/+ . Endpoints will include valve thickness, myocardial fibrosis, and changes in the MV transcriptome, to understand disease progression in this SERT-related valvulopathy. Specific Aim 2: To investigate the role of 5HT in the pathogenesis of MR involving platelet mechanisms and bone marrow derived blood outgrowth endothelial cells (BOEC) that express both CD34 and HTR2B. Subaim 2A: It is noteworthy that MR results in a chronic state of platelet activation, since platelets are the primary carriers of 5HT in blood, releasing 5HT upon activation. We will study the role of platelets in 5HT related MR pathophysiology in human subjects, canines with MR, and mice. Endpoints will include 5HT levels, platelet activation markers, and differences in TGF-b1 levels resulting from platelet alpha granule release. Subaim 2B: To investigate the role of bone marrow derived cells in the pathophysiology of 5HT mechanisms that affect MR. BOEC will be cultivated from the study groups in Subaim 2A. The experimental designs will use flow cytometry for cell characterization, qRT-PCR studies to assess gene expression patterns, and proteomic approaches to assess HTR activity, and measurements of ECM production in vitro. It is expected that the mechanistic results of these studies will lead to therapeutic directions for MR that target platelet derived 5HT and leverage a personalized approach using BOEC to model the pathophysiology.