Serotonin Signaling in Mitral Valve Homeostasis, Maintenance and Restoration

由黏液瘤性瓣膜疾病引起的退行性二尖瓣反流（MR）影响了数百万人。唯一的治疗方法是修复或更换阀门。这种竞争更新将促进对神经递质5 -羟色胺（5HT）在mr中的作用的理解。心脏瓣膜疾病与分泌5HT的类癌肿瘤和药物（如减肥药右芬氟胺）有关。基于我们在第一个项目期间的进展，我们将研究血小板和骨髓源二尖瓣间质细胞（MVIC）中涉及5 -羟色胺转运体（SERT）和5HT受体（HTR）信号传导的5HT机制的假设，这有助于病理生理MR，并代表有希望的治疗靶点。具体目的1：解剖5ht介导的影响人类受试者和小鼠二尖瓣小叶病理重塑的机制Subaim 1A：与正常二尖瓣小叶和人源性二尖瓣微血管相比，在MR中表征5ht介导的二尖瓣重塑。sert拮抗剂和HTR抑制剂研究将在体外和体外TGFb、BMP4和5HT刺激下解剖与MR进展相关的独特的5HT相关转录组和细胞表型。终点将是与暴露于5ht相关机制询问的正常小叶相比，MR中人类MVIC和组织中的转录组学、细胞外基质（ECM）重塑。Subaim 1B：为了研究SERT在MR发病机制中的作用：SERT缺失小鼠（SERT-/-）自发地发生影响二尖瓣和主动脉瓣的瓣膜病变。机制研究将比较SERT-/-和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。特异性目的2：探讨5HT在MR发病机制中的作用，包括血小板机制和表达CD34和HTR2B的骨髓源性血外生内皮细胞（BOEC）。Subaim 2A：值得注意的是，MR导致血小板激活的慢性状态，因为血小板是血液中5HT的主要载体，激活后释放5HT。我们将研究血小板在5HT相关MR病理生理中的作用，包括人类受试者、MR犬和小鼠。终点将包括5HT水平、血小板活化标志物和血小板α颗粒释放引起的TGF-b1水平差异。Subaim 2B：为了研究骨髓来源细胞在5HT影响MR. BOEC的病理生理机制中的作用，将从Subaim 2A的研究组中培养。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的产生。预计这些研究的机制结果将导致针对血小板衍生5HT的MR治疗方向，并利用BOEC来模拟病理生理的个性化方法。

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 TGFb, 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.