Genetic engineering with endothelial nitric oxide synthase improves functional properties of endothelial progenitor cells from patients with coronary artery disease: an in vitro study

• Published in: Basic research in cardiology Vol. 104; no. 6; pp. 739 - 749
• Main Authors: Kaur, Savneet, Kumar, T. R. Santhosh, Uruno, Akira, Sugawara, Akira, Jayakumar, Karunakaran, Kartha, Chandrasekharan Cheranellore
• Format: Journal Article
• Language: English
• Published: Heidelberg D. Steinkopff-Verlag 01.11.2009; Springer Nature B.V
• Subjects: Adult; Aged; Cardiology; Cell Differentiation; Cells, Cultured; Coronary Artery Disease - enzymology; Coronary Artery Disease - pathology; Endothelial Cells - cytology; Endothelial Cells - metabolism; Female; Flow Cytometry; Gene Transfer Techniques; Genetic Therapy - methods; Humans; Immunohistochemistry; In Vitro Techniques; Male; Medicine; Medicine & Public Health; Middle Aged; Neovascularization, Physiologic - physiology; Nitric Oxide Synthase Type III - biosynthesis; Nitric Oxide Synthase Type III - genetics; Original Contribution; Protein Engineering - methods; Reverse Transcriptase Polymerase Chain Reaction; Stem Cells - cytology; Stem Cells - metabolism; Transfection; Angiogenesis; Endothelial nitric oxide synthase; Coronary artery disease; Endothelial progenitor cells
• ISSN: 0300-8428; 1435-1803
• DOI: 10.1007/s00395-009-0039-x

Recent studies have reported a marked impairment in the number and functions of endothelial progenitor cells (EPCs) in patients with coronary artery disease (CAD). In view of an important role of eNOS in angiogenesis, in the present study, we evaluated the effects of eNOS gene transfer in ex vivo expanded EPCs isolated from patients with CAD. The expanded EPCs were transfected with mammalian expression vector pcDNA3.1-eNOS containing the full-length human eNOS gene using lipofectamine. About 35–40% of the eNOS–EPCs had higher expression of eNOS as compared to untransfected EPCs. EPCs transfected with pcDNA3.0-EGFP, the plasmid vector expressing green fluorescent protein (GFP) were used as control. The untransfected, GFP-transfected and eNOS-transfected EPCs were compared in terms of important functional attributes of angiogenesis such as proliferation, migration, differentiation and adhesion/integration into tube-like structures in vitro. Functional studies revealed that in the presence of defined growth conditions, compared to the untransfected and GFP-transfected cells, eNOS–EPCs from patients with CAD have a significant increase in [ 3 H] thymidine-labeled DNA ( P  < 0.01), migration (14.6 ± 1.8 and 16.5 ± 1.9 vs. 23.5 ± 3.4 cells/field, P  < 0.01), ability to differentiate into endothelial-like spindle-shaped cells (46 ± 4.5 and 56.5 ± 2.1 vs. 93.2 ± 6.6 cells/field, P  < 0.001) and also incorporation into tube-like structures on the matrigel (GFP-EPCs: 21.25 ± 2.9 vs. GFP-eNOS-EPCs: 34.5 ± 5.5 cells/field, P  < 0.05). We conclude that eNOS gene transfection is a valuable approach to augment angiogenic properties of ex vivo expanded EPCs and eNOS-modified EPCs may offer significant advantages than EPCs alone in terms of their clinical use in patients with myocardial ischemia.