The vessel wall as a paracrine engine in TGF beta-induced aortic aneurysm

描述（由申请人提供）：动脉瘤是动脉的异常扩张，如果不及时治疗，可能导致致命的撕裂或破裂。在遗传性动脉瘤疾病（如Loeys-Dietz综合征（LDS））患者的主动脉组织中观察到tgf - β信号上调，这种疾病是由编码tgf - β途径阳性调节因子的基因的杂合性功能丧失突变引起的。这些矛盾的发现引起了相当大的争议，并对TGFβ拮抗剂作为这些疾病的治疗提出了质疑。本提案的目标是确定导致LDS中tgf - β信号过度驱动的细胞和生化机制。我们提出测试一种非细胞自主机制，基于观察到编码TGFβ受体亚基的两个基因中的任意一个的LDS突变对血管壁相邻但来自不同胚胎祖细胞的主动脉细胞类型的信号传导能力有不一致的影响。具体来说，这些突变使“高反应”细胞，如心脏神经嵴源性血管平滑肌细胞（CNC-VSMCs）相对不受影响，而它们损害了更脆弱的“低反应”细胞，如第二心场源性血管平滑肌细胞（SHF-VSMCs）的tgf - β反应。该建议的总体假设是，旨在恢复“低反应”细胞中正常信号传导的代偿事件，如TGFβ配体表达的上调，将诱导“高反应”细胞中的TGFβ信号传导过度，最终导致动脉瘤的发展。为了验证这一假设，我将与我的主要导师，动脉瘤发病机理方面的专家Dietz博士，以及我的共同导师，临床蛋白质组学应用方面的专家Van Eyk博士合作，以实现前两个目标。在Aim1中，我将通过分析对照组和LDS SHF-VSMCs和CNC-VSMCs的tgf - β反应，以及这些细胞在体内和体外分泌的旁分泌因子，来定义tgf - β旁分泌过度驱动的细胞介质。在Aim 2中，我将通过以下方法对TGFβ旁分泌超速模型进行功能测试

DESCRIPTION(provided by applicant): An aneurysm is an abnormal dilation of an artery that, if left untreated, can lead to fatal tear or rupture. TGFβ signaling upregulation is observed in aortic tissue from patients affected by hereditary aneurysm disorders, such as Loeys-Dietz syndrome (LDS), that are caused by heterozygous loss-of-function mutations in genes encoding positive regulators of the TGFβ pathway. These paradoxical findings have caused considerable controversy and have called into question the use of TGFβ antagonists as therapy for these disorders. The goal of this proposal is to identify the cellular and biochemical mechanisms that cause TGFβ signaling overdrive in LDS. We propose to test a non-cell autonomous mechanism based on the observation that LDS mutations in either of the two genes that encode TGFβ receptor subunits have discordant effects on the signaling capacity of aortic cell types that are adjacent in the vessel wall but derived from different embryonic progenitors. Specifically, these mutations leave "high responder" cells such as cardiac neural crest-derived vascular smooth muscle cells (CNC-VSMCs) relatively unaffected, while they impair the TGFβ response of more vulnerable "low responder" cells, such as second heart field-derived VSMCs (SHF-VSMCs). The overall hypothesis of this proposal is that compensatory events aimed at restoring normal signaling in "low responder" cells, such as upregulation of TGFβ ligand expression, will induce TGFβ signaling overdrive in "high responder" cells, ultimately culminating in the development of aneurysm. In order to test this hypothesis, I will work with my primary mentor, Dr. Dietz, an expert on aneurysm pathogenesis, and with my co-mentor, Dr. Van Eyk, an expert in clinical proteomics applications, to address the first two aims. In Aim1, I will define the cellular mediators of TGFβ paracrine overdrive by analyzing the TGFβ response of control and LDS SHF-VSMCs and CNC-VSMCs, and the repertoire of paracrine factors secreted by these cells both in vivo and in vitro. In Aim 2, I will functionally test the TGFβ paracrine overdrive model by pharmacological and genetic manipulations. Specifically, I will assess whether global TGFβ antagonism or cellspecific reduction of TGFβ signaling in "high responder" cells, but not "low responder" cells, ameliorates aneurysm progression in LDS mice. During this phase of the award I will become proficient in the field of proteomics and bioinformatics by working with Dr. Van Eyk's lab and by participating in formal coursework. The new skills, techniques, and knowledge that I will gain will foster my transition to the R00 phase, in which I will independentl address Aim 3 and Aim 4. In Aim 3 I propose to identify the molecular determinants of lineagespecific TGFβ responses by using proteomics-based techniques and conventional biochemical methods to analyze, both in vitro and in vivo, the differential expression and/or post-translational modifications of modulators of TGFβ signaling in "high responder" and "low responder" cells. In Aim 4, I intend to elucidate the contribution of CD45+ immune cells to TGFβ signaling paracrine overdrive. I will characterize the phenotype of the CD45+ immune cells infiltrate that is present at the site of aneurysm in LDS mouse models and assess whether TGFβ ligand secreted by these cells contributes to exacerbation of pathology. I am confident that the training and career development plan described in this proposal will enable me to elucidate the mechanisms of excessive TGFβ signaling in LDS and will provide critical information for the understanding of cell-type specific TGFβ-responses. The studies described in this proposal have the potential to increase our understanding of other TGFβ-related disorders, such as fibrosis and cancer, in which signaling imbalances between interacting cell-types may drive pathology.