LGMN在肺动脉高压发病中的促炎作用及其机制研究

Perivascular inflammation underlies the pathogenesis of pulmonary hypertension, of which the activation of macrophages plays a crucial role. Legumain (LGMN) is a new member of the cysteine protease family, and a possible association with pulmonary hypertension has not yet been reported. Our previous studies have demonstrated that levels of LGMN increased in serum of pulmonary hypertension patients, while LGMN-/- mice were protected against hypoxic pulmonary hypertension compared to wild-type mice. Interestingly, this protective effect of LGMN deficiency occurred concomitantly with the decreases in the perivascular macrophage infiltration, and in the macrophage-derived factors of VEGF and PDGF counts in alveolar lavage fluid，and in the concentrations of active TGF-β1 and level of Smad2/3 phosphorylation in lung tissue. Therefore, we speculate that macrophage-derived LGMN may hydrolyze and activate TGF-β1 precursor, and in turn mediate the release of macrophage factors ,ultimately lead to pulmonary hypertension. To test this, we propose in this study to use the LGMN-knockout mice and bone marrow-reconstructed mice (WT-KO and KO-KO) as the research subjects, and establish the models of hypoxia- and SU5416-induced pulmonary hypertension for analyzing the impact of LGMN deletion on pulmonary perivascular inflammation, pulmonary vascular structure and pulmonary hypertension. Thus, from animal phenotype to cellular and molecular levels, we will systematically explore the detailed proinflammatory activity of LGMN involved in pulmonary hypertension through the activation of macrophages. We believe that this study will provide mechanistic basis for designing the new LGMN-targeted therapy for pulmonary hypertension.

肺血管周围炎症在肺动脉高压(PH)的发病中发挥着核心作用，其中巨噬细胞扮演重要角色，但相关细胞和分子机制尚不清楚。我们前期研究发现：PH患者血清半胱氨酸蛋白酶家族的新成员Legumain(LGMN)显著升高，而在PH模型小鼠，LGMN敲除显著降低血管周围巨噬细胞浸润、肺组织中活化的TGF-β1含量与Smad2/3磷酸化水平和肺泡灌洗液巨噬细胞源性因子VEGF与PDGF的分泌，改善PH。据此，我们推测巨噬细胞来源LGMN通过水解TGF-β1前体激活TGF-β1信号通路以介导巨噬细胞源性因子释放，参与PH发生发展。为验证该假说，本研究拟以LGMN敲除小鼠、造血重构小鼠为研究对象，建多种PH模型，观察全身和造血特异性LGMN缺失对肺血管周围炎症、肺血管结构和PH的影响，从动物表型到细胞分子水平系统解析LGMN通过调节巨噬细胞活化参与PH发病的详细炎症机制，有望开发以LGMN为靶标的新PH治疗。

肺动脉平滑肌增生所致的管腔变窄和炎症细胞浸润导致的血管周围炎症是肺动脉高压(PH)最主要的病理特征，其中巨噬细胞在PH发生发展中起重要作用。本课题组前期发现，PH患者血清半胱氨酸蛋白酶家族的新成员Legumain(LGMN)显著升高，而在PH模型小鼠，LGMN敲除显著降低血管周围巨噬细胞浸润、肺组织中活化的TGF-β1含量与Smad2/3磷酸化水平和肺泡灌洗液巨噬细胞源性因子VEGF与PDGF的分泌，改善PH。据此，我们推测巨噬细胞来源LGMN通过水解TGF-β1前体激活TGF-β1信号通路以介导巨噬细胞源性因子释放，参与PH发生发展。在本研究中我们发现在低氧+SU5416诱导的PAH小鼠和MCT诱导的PAH大鼠的肺组织中Lgmn的含量升高，Lgmn敲除可以通过减少低氧+SU5416诱导小鼠的肺动脉细胞外基质蛋白的含量来抑制肺动脉高压的发展，药物抑制Lgmn可以缓解低氧+SU5416诱导的小鼠或MCT诱导的大鼠的肺动脉高压，巨噬细胞特异性Lgmn敲除可以缓解低氧+SU5416诱导小鼠的肺动脉高压。在体外实验中，TGF-β1信号通路参与了Lgmn促进肺动脉细胞外基质蛋白沉积的过程，在低氧+SU5416诱导的小鼠PASMCs中，Lgmn是通过激活MMP-2来上调TGF-β1信号通路的。在临床数据中我们发现特发性肺动脉高压患者血清Lgmn水平与疾病严重程度相关。这提示我们Lgmn可以作为一个潜在的PH治疗靶点。