Endothelial Injury, BMPR2 Dysfunction and Macrophage Activation Cause EndMT and PAH

大量的知识表明，炎症和免疫失调在 肺动脉高压(PAH)的发展现在已经导致了第一次免疫治疗试验 这种情况。然而，这种方法的成功需要我们确定异常是如何 肺血管内皮细胞(PAEC)损伤的免疫/炎症反应 以细胞凋亡、细胞凋亡抵抗为特征的闭塞性动脉病的演变 内皮细胞间充质转化(EndMT)。同样重要的是，将这些特征与脑功能障碍联系起来 PAH的关键遗传修饰物，骨形态发生蛋白受体(BMPR)2。这一建议带来了独特的 关注血管周围巨噬细胞(M？S)高表达的两种关键先天免疫介质 患有PAH并对肺血管系统发起协同攻击的患者：白三烯、LTB4、 而内源性逆转录病毒HERV-K是越来越多人认识到的自身免疫损伤的原因。整体而言 假说是BMPR2信号异常的PAECs募集并激活放大LTb4的M？S 和Herv-K；这些免疫因子协同作用，通过以下方式维持炎症并促进严重的PAH 细胞凋亡与EndMT。AIM 1评估了损伤的PAEC如何自主产生LTB4，这在 转而激活M？S进一步刺激LTB_4生物合成，导致肺血管内皮细胞凋亡和EndMT 信号受损。这一目标还探索了内源性Herv-K的扩增如何进一步刺激 LTB4的生物合成及其在单核细胞中如何诱导Herv-K的表达。单细胞RNA序列将 对LTB4反应中注定要发生凋亡或EndMT的PAEC人群进行基因分型 标志着向凋亡抵抗和EndMT过渡的表达变化。AIM 2评估是否 PAH患者的PAEC，包括BMPR2功能减退的患者，分泌因子反应 氧化剂或炎症性损伤，使单核细胞HERV-K表达增强。这一目标还考虑到 单核细胞BMPR2缺失对HERV-K扩增的相加效应 翻译机制参与了这一过程。同样需要调查的是，分泌的Herv-K 产物dUTPase促进伴有BMPR2功能障碍的PAEC的EndMT 转型。单细胞RNA序列分析将阐明Herv-K诱导的基因表达变化 PAEC向EndMT进化。目的3研究BMPR2缺陷动物在发育过程中的脆弱性 利用Fate定位的BMPR2缺失小鼠和新的转基因大鼠进行重度PH和EndMT BMPR2单倍体缺失模型。动物经气管内滴注Ad-5LO治疗。 产生高水平的LTB4或静脉注射重组HERV-K dUTPase。逆转EndMT的策略 将进行与PAH中致命性闭塞性血管病变有翻译相关性的重度PH的测试 病人。

A considerable body of knowledge indicating that inflammation and immune dysregulation play a pivotal role in the development of pulmonary arterial hypertension (PAH) have now led to the first immunotherapy trials for this condition. However the success of this approach requires that we determine how the aberrant immune/inflammatory response to pulmonary artery endothelial cell (PAEC) injury develops and contributes to the evolution of the occlusive arteriopathy, that is characterized by apoptosis, apoptosis resistance and endothelial mesenchymal transition (EndMT). It is also important to relate these features to dysfunction of a key genetic modifier of PAH, bone morphogenetic protein receptor (BMPR)2. This proposal brings unique focus to two key innate immune mediators that are highly expressed in perivascular macrophages (MØs) from patients with PAH and that mount a coordinated attack on the pulmonary vasculature: the leukotriene, LTB4, and the endogenous retrovirus, HERV-K, an increasingly recognized cause of autoimmune injury. The overall hypothesis is injured PAECs with dysfunctional BMPR2 signaling recruit and activate MØs that amplify LTB4 and HERV-K; these immune factors work in concert to sustain inflammation and promote severe PAH by apoptosis and EndMT. Aim 1 evaluates how LTB4 may be autonomously produced by injured PAECs, which in turn activate MØs to stimulate further LTB4 biosynthesis to cause PAEC apoptosis and EndMT when BMPR2 signaling is impaired. This aim also explores how amplification of endogenous HERV-K may further stimulate LTB4 biosynthesis and how LTB4 can induce HERV-K expression in monocytes. Single cell RNA Seq will genotype PAEC populations that are fated for apoptosis or EndMT in response to LTB4 to delineate gene expression changes that mark the transition to apoptosis resistance and EndMT. Aim 2 evaluates whether PAECs from PAH patients including those with reduced BMPR2 function, secrete factors in response to oxidant or inflammatory injury, that amplify HERV-K expression in monocytes. This aim also considers the additive effects of loss of BMPR2 in monocytes, on amplification of HERV-K and whether transcriptional and translational mechanisms are involved in this process. Also to be investigated is whether the secreted HERV-K product, dUTPase, promotes EndMT in PAEC with BMPR2 dysfunction that are primed to undergo transformation. Single cell RNA Seq analyses will elucidate gene expression changes induced by HERV-K in the PAEC evolution to EndMT. Aim 3 investigates the vulnerability of BMPR2-deficient animals to develop severe PH and EndMT using a mouse with loss of BMPR2 in fate mapped EC, and a new transgenic rat PH model with BMPR2 haploinsufficiency. The animals are treated with endotracheal instillation of Ad-5LO to generate high LTB4 levels or recombinant HERV-K dUTPase given intravenously. Strategies to reverse EndMT and severe PH will be tested that have translational relevance to the fatal obliterative vasculopathy in PAH patients.