Engineered endothelial progenitor cells that overexpress prostacyclin protect vascular cells.
Engineered endothelial progenitor cells that overexpress prostacyclin protect vascular cells.
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DOI：
10.1002/jcp.23035
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发表时间：
2012-07
影响因子：
5.6
通讯作者：
Dixon, Richard A. F.
中科院分区：
文献类型：
--
作者：
Liu, Qi;Xi, Yutao;Terry, Toya;So, Shui-Ping;Mohite, Anita;Zhang, Jia;Wu, Geru;Liu, Xiaobing;Cheng, Jie;Ruan, Ke-He;Willerson, James T.;Dixon, Richard A. F.
前列环素(PGI2)是一种有效的血管扩张剂和重要的血管动态平衡调节剂，但由于其半衰期短(2分钟)，其临床应用受到限制。因此，我们假设，使用结构性分泌高水平PGI2的工程化内皮祖细胞(EPC)可以克服PGI2治疗的这一局限性。将编码人环氧合酶-1(COX-1)和前列环素合成酶(PGIs)的环氧合酶-1(COX-1)-10aa-PGIs基因通过核重组技术导入大鼠骨髓单个核细胞来源的生长内皮祖细胞中。经G418筛选后连续传代获得PGI2分泌菌株(PGI2-EPC)。基因组聚合酶链式反应、逆转录聚合酶链式反应和Western印迹分析证实COX-1-10aa-PGIs在PGI2-EPC中高表达。PGI2-EPC体外分泌的PGI2水平显著高于天然EPC(P<；0.05)，并显示出更高的内在血管生成能力；PGI2-EPC的条件培养液比天然EPC的条件培养液更能促进管状的形成(P<；0.05)。当COX-1-10aa-PGIS蛋白表达下调时，细胞和旁分泌介导的体外血管生成被减弱。全细胞膜片钳实验显示，在低氧条件下，与PGI2-EPC共培养的大鼠血管内皮细胞4-氨基吡啶敏感性钾电流密度显著增加(7.50±1.59pA/pF，P<；0.05)。与天然内皮祖细胞共培养的rSMC(3.99±1.26 pA/pF)相比。总而言之，我们成功地创造了过表达一种活性新酶的EPC菌株，从而使PGI2持续分泌。PGI2-EPC表现出增强的内在促血管生成特性，并提供良好的旁分泌介导的细胞保护，包括促进天然EPC的体外血管生成和缺氧下SMC的超极化。
Prostacyclin (PGI2) is a potent vasodilator and important mediator of vascular homeostasis; however, its clinical use is limited because of its short (<2 minute) half-life. Thus, we hypothesize that the use of engineered endothelial progenitor cells (EPCs) that constitutively secrete high levels of PGI2 may overcome this limitation of PGI2 therapy. A cDNA encoding COX-1-10aa-PGIS, which links human cyclooxygenase-1(COX-1) to prostacyclin synthase (PGIS), was delivered via nucleofection into outgrowth endothelial progenitor cells (EPCs) derived from rat bone marrow mononuclear cells. PGI2-secreting strains (PGI2-EPCs) were established by continuous subculturing of transfected cells under G418 selection. Genomic PCR, RT-PCR, and Western blot analyses confirmed the overexpression of COX-1-10aa-PGIS in PGI2-EPCs. PGI2-EPCs secreted significantly higher levels of PGI2 in vitro than native EPCs (P<0.05) and showed higher intrinsic angiogenic capability; conditioned medium from PGI2-EPCs promoted better tube formation than conditioned medium from native EPCs (P<0.05). Cell- and paracrine-mediated in vitro angiogenesis was attenuated when COX-1-10aa-PGIS protein expression was knocked down. Whole-cell patch-clamp studies showed that 4-aminopyridine–sensitive K+ current density was increased significantly in rat smooth muscle cells (rSMCs) cocultured under hypoxia with PGI2-EPCs (7.50±1.59pA/pF, P<0.05.) compared with rSMCs cocultured with native EPCs (3.99±1.26 pA/pF.). In conclusion, we successfully created EPC strains that overexpress an active novel enzyme resulting in consistent secretion of PGI2. PGI2-EPCs showed enhanced intrinsic proangiogenic properties and provided favorable paracrine-mediated cellular protections, including promoting in-vitro angiogenesis of native EPCs and hyperpolarization of SMCs under hypoxia.
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影响因子：
56.9
作者：
Asahara, T;Murohara, T;Isner, JM
通讯作者：
Isner, JM
影响因子：
37.8
作者：
Liu, Q;Chen, ZQ;Zoldhelyi, P
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Zoldhelyi, P
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20.3
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Hammond, WP
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20.1
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Dimmeler, S
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37.8
作者：
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Zeiher, AM