Serotonin Signaling in Mitral Valve Homeostasis, Maintenance and Restoration

粘液瘤性瓣膜病引起的退行性二尖瓣返流（MR）影响数百万人。唯一的治疗方法是瓣膜修复或置换。这种竞争性的更新将促进对神经递质5-羟色胺（5-HT）在MR中的作用的理解。心脏瓣膜疾病与5-HT分泌型类癌肿瘤和药物（如减肥药Dexfenfluoramine）有关。基于我们在第一个项目期间的进展，我们将研究以下假设：涉及5-羟色胺转运蛋白（SERT）和5-羟色胺受体（HTR）信号传导的5-HT机制，在血小板和骨髓来源的二尖瓣间质细胞（MVIC）中，有助于病理生理学MR，并代表有前途的治疗靶点。具体目标1：剖析影响人类受试者和小鼠MV瓣叶病理性重塑的5 HT介导机制Subaim 1A：与正常二尖瓣（MV）瓣叶和人源MVIC相比，表征MR中5 HT介导的MV重塑。SERT拮抗剂和HTR抑制剂研究将剖析在TGFβ、BMP 4和5 HT刺激下与体外和离体MR进展相关的独特5 HT相关转录组和细胞表型。终点将是与暴露于5 HT相关机械询问的正常瓣叶相比，人类MVIC和MR组织中的转录组学、细胞外基质（ECM）重塑。Subaim 1B：研究SERT在MR发病机制中的作用：SERT缺失小鼠（SERT-/-）自发发生影响二尖瓣和主动脉瓣的瓣膜病。机制研究将比较SERT-/-与SERT+/+。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SERT相关瓣膜病的疾病进展。具体目标二：研究5-HT在MR发病机制中的作用，涉及血小板机制和同时表达CD 34和HTR 2B的骨髓源性血液生长内皮细胞（BOEC）。Subaim 2A：值得注意的是，MR导致血小板活化的慢性状态，因为血小板是血液中5 HT的主要载体，在活化时释放5 HT。我们将在人类受试者、患有MR的犬和小鼠中研究血小板在5 HT相关MR病理生理学中的作用。终点将包括5 HT水平、血小板活化标志物和血小板α颗粒释放导致的TGF-β 1水平差异。Subaim 2B：为了研究骨髓源性细胞在影响MR的5-HT机制的病理生理学中的作用，将在Subaim 2A的研究组中培养BOEC。实验设计将使用流式细胞术进行细胞表征，qRT-PCR研究评估基因表达模式，蛋白质组学方法评估HTR活性，并测量体外ECM产生。预计这些研究的机制结果将导致靶向血小板衍生的5 HT的MR的治疗方向，并利用使用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.