SM22α promoter targets gene expression to vascular smooth muscle cells in vitro and in vivo
SM22α promoter targets gene expression to vascular smooth muscle cells in vitro and in vivo
复制标题
DOI：
10.1007/bf03401832
复制
发表时间：
2000-11-01
影响因子：
5.7
通讯作者：
Nabel, EG
中科院分区：
文献类型：
--
作者：
Akyürek, LM;Yang, ZY;Nabel, EG
背景：基因转移到血管平滑肌细胞（vsmcs）为研究动脉疾病的发病机制提供了希望。然而，带有异源启动子的载体的一个潜在限制是由不受限制的转基因表达引起的器官毒性。血管平滑肌细胞特异性基因的表达可提高血管疾病载体的安全性。材料和方法：为了构建靶向vsmcs基因表达的载体，我们构建了由小鼠SM22 α启动子（AdSM22 α -hpAP） 441 bp区域驱动的人胎盘碱性磷酸酶（hpAP）和氯霉素转移酶（CAT）载体。结果：AdSM22 α -hpAP转染血管细胞和非血管细胞可导致碱性磷酸酶（AP）在原代动脉和静脉smcs中表达，但在原代内皮细胞或美国国立卫生研究院（NIH） 3T3细胞中不表达。在感染多重感染数（MOI）为500的猪vsmcs原代感染的AdSM22 α - hpap中，有32.5 +/- 1.4%的人表达了AP；而感染AdCMV-hpAP导致100 +/- 0.0%的表达，MOI为250。在体外，与SM22 α启动子相比，来自异源巨细胞病毒（CMV）启动子的表达在vsmcs中大约高出10(3)倍。将AdSM22 α - hpap载体导入球囊损伤的猪动脉后，在新生内膜（2.23 +/- 1.14%）和内侧（0.56 +/- 0.21%）smcs中检测到AP重组蛋白，但在内皮细胞和外表皮细胞中未检测到AP重组蛋白。相比之下，AdCMV-hpAP载体导致AP在内膜内皮细胞和smcs细胞（39.14 +/- 10.09%）和内侧smcs细胞（2.84 +/- 1.05%）中表达。用缺乏E1的腺病毒载体（Ad δ E1）转染内皮细胞或vsmcs后，未观察到AP表达。结论：SM22 α启动子可在体外和体内对血管间充质干细胞特异性地表达重组基因。虽然表达水平低于异源启动子，但这些载体可能为血管疾病的基因治疗提供一种安全有效的工具。
Background: Gene transfer into vascular smooth muscle cells (vsmcs) holds promise for studying the pathogenesis of arterial disorders. However, a potential limitation of vectors with heterologous promoters is organ toxicity resulting from unrestricted transgene expression. Vascular smooth muscle cell-specific gene expression could increase the safety of vectors for vascular diseases.Materials and Methods: To develop vectors that target gene expression to vsmcs, we constructed vectors encoding, human placental alkaline phosphatase (hpAP) and chloramphenicol transferase (CAT) driven by a 441-bp region of the murine SM22 alpha promoter (AdSM22 alpha -hpAP).Results: Transfection of AdSM22 alpha -hpAP into vascular and nonvascular cells resulted in the expression of alkaline phosphatase (AP) in primary arterial and venous smcs, but not in primary endothelial cells or National Institutes of Health (NIH) 3T3 cells. Expression of AP was observed on 32.5 +/- 1.4% of primary pig vsmcs-infected AdSM22 alpha -hpAP at a multiplicity of infection (MOI) of 500; whereas, infection with AdCMV-hpAP resulted in 100 +/- 0.0% expression at a MOI of 250. In vitro, expression from the heterologous cytomegalovirus (CMV) promoter was approximately 10(3)-fold higher in vsmcs, compared with the SM22 alpha promoter. Following introduction of AdSM22 alpha -hpAP vectors into balloon-injured pig arteries, AP recombinant protein was detected in neointimal (2.23 +/- 1.14%) and medial (0.56 +/- 0.21%) smcs, but not in endothelial or adventitial cells. In contrast, AdCMV-hpAP vectors led to AP expression in intimal endothelial and smcs cells (39.14 +/- 10.09%) and medial smcs (2.84 +/- 1.05%). AP expression was not observed in endothelial or vsmcs following transfection with the control vector, adenoviral vector lacking E1 (Ad Delta E1).Conclusions: The SM22 alpha promoter programs recombinant gene expression exclusively to vascular smcs in vitro and in vivo. Although expression levels are lower than with heterologous promoters, these vectors may provide a safe and effective tool for gene therapy of vascular diseases.