title: Engineered endothelial progenitor cells that overexpress prostacyclin protect vascular cells.
abstract: Prostacyclin (PGI2) is a potent vasodilator and important mediator of vascular homeostasis; however, its clinical use is limited because of its short (<2-min) 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 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 (CM) from PGI2-EPCs promoted better tube formation than CM 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 ± 1.59 pA/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.
authors.affiliation: The Texas Heart Institute at St. Luke's Episcopal Hospital, Houston, TX, USA.
authors.forename: Qi
authors.initials: Q
authors.lastname: Liu
authors.forename: Yutao
authors.initials: Y
authors.lastname: Xi
authors.forename: Toya
authors.initials: T
authors.lastname: Terry
authors.forename: Shui-Ping
authors.initials: SP
authors.lastname: So
authors.forename: Anita
authors.initials: A
authors.lastname: Mohite
authors.forename: Jia
authors.initials: J
authors.lastname: Zhang
authors.forename: Geru
authors.initials: G
authors.lastname: Wu
authors.forename: Xiaobing
authors.initials: X
authors.lastname: Liu
authors.forename: Jie
authors.initials: J
authors.lastname: Cheng
authors.forename: Ke-He
authors.initials: KH
authors.lastname: Ruan
authors.forename: James T
authors.initials: JT
authors.lastname: Willerson
authors.forename: Richard A F
authors.initials: RA
authors.lastname: Dixon
mesh_terms: D015761:4-Aminopyridine / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000235:genetics; D060846:Cell Engineering / Q000379:methods*; D048708:Cell Growth Processes / Q000235:genetics; D017077:Culture Media, Conditioned / Q000378:metabolism; D051545:Cyclooxygenase 1 / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D011464:Epoprostenol / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D006207:Half-Life; D000860:Hypoxia / Q000235:genetics / Q000378:metabolism; D019746:Intramolecular Oxidoreductases / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D018919:Neovascularization, Physiologic; D010641:Phenotype; D015221:Potassium Channels / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013234:Stem Cells / Q000378:metabolism*; D014162:Transfection / Q000379:methods
chemical_list: D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D008565:Membrane Proteins; D015221:Potassium Channels; D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; D015761:4-Aminopyridine; D011464:Epoprostenol; D051545:Cyclooxygenase 1; C496513:Ptgs1 protein, rat; D019746:Intramolecular Oxidoreductases; C022461:prostacyclin synthetase