Role of endothelial Twist1 in vascular remodeling in pulmonary hypertension

项目摘要 肺动脉高压（PH）是一种多因素危及生命的肺血管疾病，其特征在于 正常非肌化远端肺小动脉（PA）的异常肌化。功能障碍 肺动脉内皮细胞（EC）在肺动脉平滑肌细胞（PASMCs）的聚集中起关键作用 远端肺动脉转录因子Twist 1的表达在PH患者的肺中上调。然而，在这方面， 内皮Twist 1在PH发病机制中的作用尚未完全了解。这项提案的目的是 探讨内皮Twist 1调控PASMCs向远端PA聚集的机制。我们 新的初步数据表明，Twist 1过表达增加了血小板源性生长的水平， 因子B（PDGFB）在人肺动脉内皮（HPAE）细胞中的表达并诱导增殖和迁移 的PASMC。缺氧增加HPAE细胞中Twist 1 Ser 42磷酸化和PDGFB表达， 来自这些细胞的条件培养基刺激PASMC的迁移，而EC中的Twist 1敲低抑制PASMC的迁移。 影响。当我们将补充有荧光标记EC的纤维蛋白凝胶植入小鼠肺时， Twist 1在这些内皮细胞中的过表达增加了PDGFB的表达，并促进了α-平滑肌细胞的募集。 肌肉肌动蛋白（SMA）阳性细胞在凝胶中。缺氧诱导α SMA阳性细胞向血液中蓄积 血管形成的凝胶，而Twist 1敲低EC减弱的影响。缺氧也会刺激 α SMA阳性细胞在小鼠肺中积累为小PA，这在Tie 2特异性 Twist 1基因敲除小鼠。Twist 1和PDGFB在从2型糖尿病小鼠分离的肺EC中的水平较高， 骨形态发生蛋白受体（Bmpr 2）突变，其影响人类PH的严重程度。 突变还刺激α SMA阳性细胞在植入缺氧的凝胶中积聚成EC- 处理的小鼠肺。我们假设内皮细胞Twist 1介导缺氧诱导的SMC积聚， 远端PA通过PDGFB旁分泌信号传导。在目标1中，我们将研究内皮Twist 1或其 在体外，磷酸化通过PDGFB旁分泌信号传导控制PASMC的行为。在目标2中，我们将 确定内皮Twist 1是否介导缺氧诱导的SMC向血管的积聚， 使用小鼠肺纤维蛋白凝胶植入系统的小鼠肺。我们还将探讨内皮细胞是否 Twist 1基因敲除可诱导VE-钙粘蛋白特异性调控低氧诱导的血管重构 小鼠在目标3中，我们将研究内皮Twist 1是否介导Bmpr 2突变诱导的血管内皮细胞凋亡。 在常氧或缺氧条件下的体外EC和凝胶植入模型中的重构。我们专注于探索 使用小鼠肺凝胶研究内皮Twist 1在缺氧诱导的SMC向远端PA聚集中的作用 植入系统是独特的和相关的进展，这将进一步我们了解的发病机制 药典

Project Summary Pulmonary hypertension (PH) is a multifactorial life threatening pulmonary vascular disease characterized by aberrant muscularization of the normally non-muscularized distal pulmonary arterioles (PAs). Dysfunction of PA endothelial cells (ECs) plays key roles in accumulation of pulmonary artery smooth muscle cells (PASMCs) to distal PAs. Expression of the transcription factor Twist1 is upregulated in the lungs of PH patients. However, the role of endothelial Twist1 in pathogenesis of PH has not been fully understood. The goal of this proposal is to explore the mechanism by which endothelial Twist1 controls accumulation of PASMCs to distal PAs. Our new preliminary data demonstrate that Twist1 overexpression increases the levels of platelet-derived growth factor B (PDGFB) in human pulmonary arterial endothelial (HPAE) cells and induces proliferation and migration of PASMCs. Hypoxia increases Twist1 Ser42 phosphorylation and PDGFB expression in HPAE cells and conditioned medium from these cells stimulates migration of PASMCs, while Twist1 knockdown in ECs inhibits the effects. When we implanted fibrin gel supplemented with fluorescently labeled ECs on the mouse lung, Twist1 overexpression in these ECs increases PDGFB expression and promotes the recruitment of α-smooth muscle actin (SMA)-positive cells in the gel. Hypoxia induces accumulation of αSMA-positive cells to blood vessels formed in the gel, while Twist1 knockdown in ECs attenuates the effects. Hypoxia also stimulates accumulation of αSMA-positive cells to small PAs in the mouse lungs, which is suppressed in Tie2-specific Twist1 knockout mice. The levels of Twist1 and PDGFB are higher in lung ECs isolated from mice with type-2 bone morphogenetic protein receptor (Bmpr2) mutation that influences the severity of human PH. The Bmpr2 mutation also stimulates accumulation of αSMA-positive cells to ECs in the gel implanted on the hypoxia- treated mouse lungs. We hypothesize that endothelial Twist1 mediates hypoxia-induced SMC accumulation to distal PAs through PDGFB paracrine signaling. In Aim 1, we will investigate whether endothelial Twist1 or its phosphorylation controls PASMC behaviors through PDGFB paracrine signaling in vitro. In Aim 2, we will determine whether endothelial Twist1 mediates hypoxia-induced SMC accumulation to blood vessels in the mouse lung using the mouse lung fibrin gel implantation system. We will also explore whether endothelial Twist1 mediates hypoxia-induced vascular remodeling using inducible VE-cadherin-specific Twist1 knockout mice. In Aim 3, we will investigate whether endothelial Twist1 mediates Bmpr2 mutations-induced vascular remodeling in ECs under normoxia or hypoxia in vitro and in the gel implantation model. Our focus on exploring the role of endothelial Twist1 in hypoxia-induced SMC accumulation to distal PAs using the mouse lung gel implantation system is unique and relevant advances, which will further our understanding of the pathogenesis of PH.