Multidisciplinary approaches to gene therapy of pulmonary hypertension

为了寻求治疗顽固性肺动脉高压的新方法，我们进行了多学科的研究，取得了以下成果：a)血管重构的抑制：与血管重构密切相关的血管细胞凋亡。不同的他汀类药物通过不同的途径诱导血管平滑肌细胞凋亡。阐明了内皮素-1和血管紧张素II对心肌细胞的作用机制。b)血管再生：研究血管内皮细胞祖细胞体外集落形成能力，发现肺气肿和稳定型冠状动脉功能不全患者体外集落形成能力明显下降。然而，在主动脉瘤患者中，它明显增加。这一发现提示了祖细胞在修复血管内皮损伤中的重要作用。基因导入方法。a) vector的开发。携带外源VEGF基因的AAV载体在动物模型中成功地恢复了器官缺血，b)建立了肺特异性基因传递的动物模型：将外源基因转染到同基因的提母细胞中，通过静脉或气管内途径注入肺，实现了肺实质特异性基因的高水平表达。筛选与血管功能相关的未知基因。分别用前列环素和波生坦处理体外培养的人肺动脉内皮细胞和平滑肌细胞，并对其基因表达进行全面研究。两种药物共有的几个基因对治疗有反应，并通过实时PCR分析进行了验证。这些发现将有助于治疗难治性肺动脉高压的新策略。

To develop novel therapeutic methods for intractable pulmonary hypertension, multidisciplinary researches were conducted and the following findings were obtained.1.On known genes related to PH. a)Suppression of vascular remodeling : Apoptosis of vascular cells closely related to the vascular remodeling. Different statines worked via distinctively different pathways to induce apoptosis in vascular smooth muscles. Mechanisms of endothelin-1 and angiotensin II on myocardial cells were elucidated. Polymorphism of prostacyclin synthase gene resulted in varied production of 6-keto PG. b)Regeneration of vasculature : In vitro colony forming ability of vascular endothelial cell progenitors were investigated to find that the ability was significantly decreased in patients with pulmonary emphysema and stable coronary insufficiency. It was significantly increased, however, in patients with aortic aneurism. This findings suggested critical roles of the progenitor cells in repairing vascular endothelial damage.2.On gene introduction methods. a)Development of vector. An AAV vector carrying foreign VEGF gene was successful in recovering organ ischemia in an animal model, b)Establishment of an animal model which enabled gene delivciy specific to the lung : By transfecting a foreign gene to a syngeneic tibroblasts and injecting them to the lung via intra-venous or intra-tracheal routs, the gene expression specific to lung parenchyma was achieved in high levels.3.Screening of unknown genes related to vascular functions. Cultured human pulmonary arterial endothelial cells and smooth muscle cells were treated by prostacyclin or bosentan, and the gene expressions were exhaustively investigated. Several genes common to both agents responded to the treatment, and were validated by real-time PCR analysis. These findings together would contribute to new strategies for treating intractable pulmonary hypertension.