高表达ACE2基因的内皮祖细胞对肺动脉高压的干预作用及机制

Many studies suggest that angiotension converting enzyme 2 (ACE2) and endothelial progenitor cells (EPCs) maintain endothelial integrity and vascular homestasis and participate in angiogenesis. Our previous data revealed that lentivirius transduction of ACE2 gene into EPCs resulted in a 4-fold increase of ACE2 expression and enhanced significantly the abilities of EPCs migration and tube formation, and reduced the hypoxic-ischemic damage after transplantation. It is generally accepted that extended lesions of lung microvessels exist in PAH. However, the contribution of ACE2 and EPCs to the development of PAH is still elusive. Based on the data herein, this program will be completed by accomplishing experiments as below: (1) The dynamics of the transplanted cells will be investigated by fluorescent dye labeling assay and，the frequency, functionality and paracrine secretion features of EPCs in the peripheral blood and bone marrow from rats that have received different regimens will be analyzed. (2) By using a standard monocrotaline(MCT)-induced PAH rat model, the therapeutic effects of inhibitors and agonists of ACE2, EPCs and ACE2-transfected EPCs will be observed. The expression levels of ACE2 and its associated downstream molecules will be detected in the lungs, aiming to clarify the association of ACE2 over-expression and the amelioration of PAH and reveal the possible underlying mechanisms. (3) The frequency and the migration capacity of EPCs in peripheral blood from PAH and healthy controls are to be detected, and the expression levels of ACE2 and its associated molecules including Ang1-7 in lung biopsy will be assessed to identify the association of EPCs and ACE2 with the development and clinical progression of PAH. The investigations will clarify the contribution of ACE2 and endothelial progenitors to the development and post-therapy progression of pulmonary artery hypertension, aiming to provide novel targets for PAH.

我们既往研究表明，与内皮祖细胞（EPCs）比较，高表达血管紧张素转换酶2（ACE2）后，其体外成管及促血管新生能力更强，从而减轻缺血缺氧性损害。微血管损伤是肺动脉高压（PAH）的发病基础，然而，ACE2和EPCs与PAH具体的关系尚不清楚。基于此，本课题将：（1）探究移植高表达ACE2的EPCs体内动力学分布，观察其量和功能改变，并通过内皮细胞和平滑肌细胞进行信号通路研究，明确ACE2-EPCs的治疗机制。（2）利用野百合碱诱导PAH大鼠模型，探讨ACE2-EPCs的治疗效果，并通过测定肺组织中ACE2、Ang1-7等表达水平，阐明其与ACE2的关联性；（3）检测PAH患者外周血EPCs水平及迁移功能，测定肺组织中ACE2及其下游分子含量，以揭示ACE2和EPCs在PAH发生发展中的机制。研究将阐明ACE2与EPCs在PAH发病和治疗中的意义，为治疗PAH提供新的干预靶点奠定基础。

肺动脉高压（PAH）是一组以肺血管阻力进行性增加为特征的临床病理生理综合征，临床上只能眼看患者渐进至死而束手无策，为探索逆转病程的方法，我们从新近发现的血管紧张素转化酶2(ACE2)通过MAS受体发挥血管保护作用，可能成为PAH治疗的新靶点得到启发，开展了“高表达ACE2基因的内皮祖细胞（EPCs）对PAH大鼠的干预作用及机制研究”，但是在实验过程中发现培养EPCs难度高而纯度低，结合本课题组前期研究成果：间充质干细胞(MSCs）及其膜微粒（MPs）显著逆转肺小血管重构，提高生存质量与远期生存率，效用远非现在临床药物可企及，且国际上尚未见类似报道，创新性先进性较好，故将研究的主要方向调整高表达ACE2的EPCs、MSCs及MSC-MPs体内外促血管生成、逆转血管重构的作用与机制，为PAH的优化治疗奠定基础。本研究通过：（1）ACE2-EPCs和ACE2-MSCs对PAH大鼠的干预作用及机制研究，结果显示二者强强联合，明显更有效的逆转了血管重构，其机制可能与ACE2表达水平增高，改善了血管收缩状态、抑制平滑肌细胞增殖相关；（2）探讨常氧或低氧状态下人脐带间充质干细胞（huMSCs）及huMSC-MPs促血管新生的机制研究显示：众多促血管生成的蛋白在huMSCs与huMSC-MPs有丰富表达，且MPs中VEGF等表达量高于MSCs；（3）MSCs与MSC-MPs对PAH大鼠的干预作用及机制研究显示：MSCs与MSC-MPs可起到与ACE2激动剂类似作用，证实ACE2/Ang1-7/Mas参与MSCs、MSC-MPs改善PAH的机制；同时，改变巨噬细胞极化状态与抑制T淋巴细胞释放TNF-a均可能参与了血管重构的逆转；（4）通过PAH患者临床标本的收集与实验，探讨ACE2能否成为PAH病情的预测指标。本项目研究证实了MSCs、MSC-MPs、ACE2-MSCs、ACE2-EPCs治疗PAH的有效性，尤其是逆转血管重构与促进血管新生的能力，给PAH的临床治疗带来了新的希望。并从分子、细胞及动物水平揭示了其机制可能主要是通过上调ACE2/Ang1-7/Mas轴且抑制ACE/AngII/AT1R轴、以及免疫调控发挥作用。上述研究为MSCs、MSC-MPs以及ACE2-EPCs、ACE2-MSCs治疗PAH早日进入临床提供了一定的理论依据与实验基础，为治疗PAH开辟了一条新的途径。