Immune cells and STAT3-mediated inflammation in calcific aortic valve disease

项目摘要 主动脉瓣疾病是美国第三大常见的心血管疾病。钙化性主动脉瓣 疾病（CAVD）是迄今为止最常见的原因，但它没有有效的药物治疗。目前所有 病理生理学的理论集中在主动脉瓣间质细胞和内皮细胞的贡献上 （分别为AVIC和AVEC）。然而，巨噬细胞占天然主动脉的很大一部分， 瓣膜，病理学研究和钙化主动脉瓣的无偏转录组学观察到 富集巨噬细胞和T和B淋巴细胞。此外，转录因子STAT 3及其关键基因 已经发现活化剂IL-6 --Th 17炎症的两种标志物--在体外和体内增加 钙化认识到免疫细胞功能与内皮和心血管生物学的相互作用，我们 已经开始研究免疫细胞对CAVD病理生理学的贡献。我们的体外研究 STAT 3阻断显示了营养不良的机械和转录水平特征的废除。 钙化此外，培养主动脉瓣细胞与巨噬细胞增加钙化表型。 这些背景信息和初步数据表明免疫细胞对免疫系统的作用机制。 CAVD的病理生理学具体地说，我们怀疑已知的钙化刺激会诱导炎症反应。 造血细胞表型，导致T淋巴细胞浸润和STAT 3依赖性钙化结节 阵在目标1中，我们将利用骨髓移植和通过Rag 1抑制淋巴细胞成熟 敲除以测试造血细胞对整体和淋巴细胞的作用，分别在Notch 1 +/- CAVD小鼠模型。为了测试体外钙化模型对免疫细胞的影响，在目标2中，我们将 利用AVIC和巨噬细胞的体外共培养模型，并探测对STAT 3信号传导的影响， AVIC钙化。目的2将询问瓣膜细胞的钙化结果和免疫学结果。 巨噬细胞的结果。为了测试该模型的临床转化，在目标3中，我们将评估 服用甲氨蝶呤（一种抑制T淋巴细胞成熟的药物）的患者的主动脉瓣狭窄进展， 从而获得适应性免疫系统活性。通过这些调查，该项目将（a）成为第一个 机械地评估免疫细胞在CAVD中的影响，（B）直接有助于临床知识 基于主动脉瓣疾病，和（c）推进免疫细胞生理学的基础科学理解 在机械应力下。最后，该项目不仅将促进对主动脉瓣疾病的科学认识， 但也进一步成为我成为一名独立的物理学家和科学家的基础。

PROJECT SUMMARY Aortic valve disease is the third most common cardiovascular disease in the United States. Calcific aortic valve disease (CAVD) is by far the most common cause, yet it has no effective pharmacologic therapy. Currently, all theories of pathophysiology focus on the contribution of resident aortic valve interstitial and endothelial cells (AVICs and AVECs respectively). However, macrophages make up a significant fraction of the native aortic valve, and pathology studies and unbiased transcriptomics of the calcified aortic valve have observed enrichment of macrophages and T and B lymphocytes. In addition, transcription factor STAT3 and its key activator IL-6 –both markers of Th17 inflammation – have been found to be increased in in vitro and in vivo calcification. Recognizing the interplay of immune cell function with endothelial and cardiovascular biology, we have begun to investigate the contribution of immune cells to CAVD pathophysiology. Our in vitro studies of STAT3 blockade show an abrogation of mechanical and transcript-level characteristics of dystrophic calcification. In addition, culture of aortic valve cells with macrophages augments the calcification phenotype. This background information and preliminary data suggest a mechanism for immune cell contribution to the pathophysiology of CAVD. Specifically, we suspect that known calcification stimuli induce an inflammatory phenotype in hematopoietic cells, leading to T lymphocyte infiltration and STAT3-dependent calcific nodule formation. In Aim 1 we will utilize bone marrow transplants and inhibition of lymphocyte maturation via Rag1 knockout to test the role of hematopoietic cells on the whole and lymphocytes, respectively, in the Notch1+/- mouse model of CAVD. To test the impact of in vitro models of calcification on immune cells, in Aim 2 we will utilize in vitro co-culture models of AVICs and macrophages, and probe the effects on STAT3 signaling and AVIC calcification. Aim 2 will interrogate both the calcification outcomes of valvular cells and the immunological outcomes of macrophages. To test the clinical translation of this model, in Aim 3 we will assess the progression of aortic stenosis in patients taking methotrexate, a drug that inhibits T lymphocyte maturation and thus adaptive immune system activity. With these investigations, this project will (a) be the first to mechanistically assess the impact of immune cells in CAVD, (b) contribute directly to the clinical knowledge base on aortic valve disease, and (c) advance the basic science understanding of immune cell physiology under mechanical strain. Finally, this project will not only advance scientific knowledge on aortic valve disease, but further serve as the foundation of my training to be an independent physician-scientist.