Cell-based gene transfer to the pulmonary vasculature - Endothelial nitric oxide synthase overexpression inhibits monocrotaline-induced pulmonary hypertension
Cell-based gene transfer to the pulmonary vasculature - Endothelial nitric oxide synthase overexpression inhibits monocrotaline-induced pulmonary hypertension
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DOI：
10.1165/ajrcmb.21.5.3640
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发表时间：
1999-11-01
影响因子：
6.4
通讯作者：
Stewart, DJ
中科院分区：
文献类型：
--
作者：
Campbell, AIM;Kuliszewski, MA;Stewart, DJ
为了规避体内转染的问题并避免使用病毒载体或蛋白质，我们试图确定离体转染的平滑肌细胞（SMC）是否可以通过体静脉循环递送到肺床中以实现肺中的局部转基因表达。将Fisher 344大鼠肺动脉SMC的原代培养物用荧光、膜不可渗透染料氯甲基三甲基罗丹明标记或在巨细胞病毒（CMV）增强子/启动子（pCMV-β）控制下用β-半乳糖苷酶（β Gal）报告基因转染。通过颈静脉注射将转染或标记的SMC（5 × 10（5）细胞/动物）递送至同系受体大鼠;每隔15 min至2 wk处死动物;切除肺、脾、肾和骨骼肌并进行检查。在移植后15分钟，主要在小肺动脉和小动脉的管腔中检测到注射的细胞，通常在三个或更多个细胞的组中。24 h后，标记的SMC被发现纳入肺小动脉的血管壁，转基因表达持续原位14 d，没有免疫反应的证据。使用简单的几何假设，计算出15 min后，在肺中可识别出约57 +/- 5%的重新引入静脉循环的标记细胞，48 h时为34 +/- 7%，1周时为16 +/- 3%，2周时为15 +/- 5%。用报告基因β Gal转染的细胞观察到类似的结果。为了确定这种方法的基因转移是否可以证明有效地抑制肺血管疾病的发展，肺动脉平滑肌细胞转染的全长编码序列的内皮型一氧化氮合酶（NOS）的CMV增强子/启动子的控制下，或与对照载体（pcDNA3.1），并同时注射肺内皮毒素野百合碱。在注射后28天，右心室收缩压从注射空转染细胞的动物的50 ± 4 mm Hg显著降低到注射NOS转染细胞的动物的33 ± 3 mm Hg（P < 0.01）。这些结果表明，一个基于细胞的策略，离体转染可能提供一个有效的非病毒的方法，选择性递送外源转基因到肺微血管在治疗肺血管疾病。
To circumvent the problems of in vivo transfection and avoid the use of viral vectors or proteins, we sought to establish whether smooth-muscle cells (SMCs) transfected ex vivo could be delivered via the systemic venous circulation into the pulmonary bed to achieve local transgene expression in the lung. Primary cultures of pulmonary artery SMCs from Fisher 344 rats were labeled with a fluorescent, membrane-impermeable dye chloromethyl trimethyl rhodamine or transfected with the beta-galactosidase (beta Gal) reporter gene under the control of the cytomegalovirus (CMV) enhancer/promoter (pCMV-beta). Transfected or labeled SMCs (5 x 10(5) cells/animal) were delivered to syngeneic recipient rats by injection into the jugular vein; the animals were killed at intervals between 15 min and 2 wk; and the lungs, spleens, kidneys, and skeletal muscle were excised and examined. At 15 min after transplantation, injected cells were detected mainly in the lumen of small pulmonary arteries and arterioles, often in groups of three or more cells. After 24 h, labeled SMCs were found incorporated into the vascular wall of pulmonary arterioles, and transgene expression persisted in situ for 14 d with no evidence of immune response. Using simple geometric assumptions, it was calculated that approximately 57 +/- 5% of the labeled cells reintroduced into the venous circulation could be identified in the lungs after 15 min, 34 +/- 7% at 48 h, 16 +/- 3% at 1 wk, and 15 +/- 5% at 2 wk. Similiar results were observed using cells transfected with the reporter gene beta Gal. To determine whether this method of gene transfer could prove effective in inhibiting the development of pulmonary vascular disease, pulmonary artery SMCs were transfected with either the full-length coding sequence of endothelial nitric oxide synthase (NOS) under the control of the CMV enhancer/promoter or with the control vector (pcDNA3.1) and injected simultaneously with the pulmonary endothelial toxin monocrotaline. At 28 d after injection the right ventricular systolic pressure was significantly decreased from 50 +/- 4 mm Hg in animals injected with the null-transfected cells to 33 +/- 3 mm Hg in animals injected with the NOS-transfected cells (P < 0.01). These results suggest that a cell-based strategy of ex vivo transfection may provide an effective nonviral approach for the selective delivery of foreign transgenes to pulmonary microvessels in the treatment of pulmonary vascular disease.