Protein tyrosine phosphatase non-receptor 14 in vascular stability and remodeling

摘要 血管系统对生命至关重要，为身体的每个器官注入氧气和营养， 运输并与保护身体的免疫细胞相互作用。在成人中，维持一个完整的 血管内皮处于严格的稳态控制下以防止水肿或出血。伤口或组织 缺氧可导致血管生成和血管重塑。血管内稳态的过程是高度 调节，并涉及许多分子的球员在音乐会上表演。在疾病条件下， 这些分子过程可能会出错。这在罕见的孟德尔疾病中尤其如此， 这种机制的关键组成部分发生突变，如遗传性出血性毛细血管扩张症（HHT）， 由ENG、ACVRL 1或SMAD 4的功能缺失突变引起。了解分子 调节血管稳态的基础对于许多疾病是至关重要的，包括对 从急性肺损伤和COVID-19中恢复。在这里，我们将研究蛋白酪氨酸的作用， 磷酸酶非受体14型（PTPN 14）作为血液和淋巴调节的关键参与者 血管稳态我们先前表明PTPN 14基因内的遗传变异与 HHT患者的肺动静脉畸形（AVM）和人类遗传学研究表明， PTPN 14在淋巴发育和体内平衡中的作用。PTPN 14是雅普信号传导的拮抗剂， 已经表明它支持ALK 1（ACVRL 1）/SMAD 4信号传导。我们已经确定了几个顺式eQTL， PTPN 14基因与HHT中PTPN 14的表达和肺AVM的存在相关， 提示PTPN 14表达水平影响AVM发病率。我们还发现了两种罕见的非- 与AVM分离的同义PTPN 14 SNP，我们还将确定这些SNP如何影响PTPN 14 功能和与SMAD 4和雅普/TAZ的分子相互作用。我们将使用人类工程微血管 在流动条件下，研究PTPN 14敲低或突变的影响，有或没有ENG或 ACVRL 1敲低，对内皮细胞，大小，增殖，迁移，与血流对齐，和血管 不同流动条件下的渗透率。最后，我们将使用我们的Cre介导的Ptpn 14-loxp等位基因， 在内部，调查由于遗传缺失导致的血管和淋巴管畸形的发展， Ptpn 14在体内内皮细胞或实质细胞中的作用，并检查PTPN 14如何与BMP 9- 内皮素-ALK 1信号通路调节体内AVM形成。我们会产生他莫昔芬诱导的 细胞类型特异性Ptpn 14-/-，并研究这如何影响发育性血管生成，病理性 受伤角膜中的血管生成，以及成人肺、皮肤、肝、肠和脑的血管床。我们还将 研究Ptpn 14 DiEC对Eng+/-、EngDiEC表型的影响，以确定这些基因在细胞内如何相互作用。 vivo.肺内血流和潜在的动静脉畸形将使用我们的新的 Quantum GX 2微型CT成像仪通过S10赠款获得。

ABSTRACT The vascular system is critical to life, infusing each organ of the body with oxygen and nutrients, and transporting and interacting with immune cells that protect the body. In the adult, maintenance of an intact vascular endothelium is under strict homeostatic control to prevent edema or hemorrhage. Wounding or tissue hypoxia can result in angiogenesis and vascular remodeling. The process of vascular homeostasis is highly regulated and involves many molecular players acting in concert. Under disease conditions, orchestration of these molecular processes may go awry. This is especially true in rare Mendelian disorders that are caused by mutations in key components of this machinery, such as Hereditary Hemorrhagic Telangiectasia (HHT), which is caused by loss of function mutations in ENG, ACVRL1, or SMAD4. Understanding the molecular underpinnings that regulate vascular homeostasis is critical to many diseases, including susceptibility to, and recovery from, acute lung injury and COVID-19. Here, we will investigate the role of protein tyrosine phosphatase non-receptor, type 14 (PTPN14) as a critical player in regulation of both blood and lymphatic vessel homeostasis. We previously showed that genetic variation within the PTPN14 gene associates with pulmonary arteriovenous malformations (AVMs) in HHT patients, and human genetics studies suggest a role for PTPN14 in lymphatic development and homeostasis. PTPN14 is an antagonist of YAP signaling and we have shown that it supports ALK1(ACVRL1)/SMAD4 signaling. We have identified several cis-eQTL in the PTPN14 gene that associated with PTPN14 expression and with the presence of pulmonary AVM in HHT, suggesting that PTPN14 expression levels influence AVM incidence. We have also identified two rare non- synonymous PTPN14 SNPs that segregate with AVMs and we will also determine how these affect PTPN14 function and molecular interactions with SMAD4 and YAP/TAZ. We will use human engineered microvessels under flow conditions to investigate the effects of PTPN14 knockdown or mutation, with or without ENG or ACVRL1 knockdown, on endothelial cell, size, proliferation, migration, alignment with flow, and vascular permeability under differing flow conditions. Finally, we will use our Cre-mediated Ptpn14-loxp allele, generated in-house, to investigate development of vascular and lymphatic malformations that result from genetic loss of Ptpn14 in endothelial or parenchymal cells in vivo, and examine how PTPN14 interacts with the BMP9- endoglin-ALK1 signaling pathway to modulate formation of AVMs in vivo. We will generate tamoxifen-inducible cell type-specific Ptpn14-/- and investigate how this affects developmental angiogenesis, pathological angiogenesis in wounded cornea, and vascular beds of adult lung, skin, liver, gut and brain. We will also investigate the effects of Ptpn14DiEC on Eng+/-, EngDiEC phenotypes to determine how these genes interact in vivo. Blood flow in the lung and potential arteriovenous malformations will be assessed using our new Quantum GX2 micro-CT imager obtained through an S10 grant.