Determine exogenous human DDAH2 gene function in rabbit bone marrow–derived endothelial progenitor cells in vitro

Abstract

The in vitro amplification of endothelial progenitor cells (EPCs) is an important method because of its role in gene transferring and regenerative medicine. In this study, we isolated rabbit bone marrow-derived EPCs to further manipulation and overexpression of dimethylarginine dimethylaminohydrolase (DDAH) in EPCs. Isolated EPCs were cultured, expanded in endothelial basal medium. Morphology of EPCs and expression levels of surface markers detected using immunocytochemistry staining and through the use of flow cytometery. Endothelial progenitor cells were transfected with plasmid vectors expressing human DDAH2 (DDAH2-EPCs). Three days after gene transfer, positive transfected-EPCs proliferation and DDAH activity were assayed. We observed colonies conformation and endothelium-like morphology gradually in the third week of culture. Characterization results revealed positive expression of EPC surface markers CD106, Flk-1, vWF, and CD34 using few identification techniques. Overexpression of DDAH2 increased citrulline production after 96 hours of transfection, 235.34 ± 0.69 vs 95.26 ± 5.76 ng/mL; P = .023. These results suggest that cell population with EPC characteristics can be simply isolated from rabbit bone marrow and successfully engineered to overexpress exogenous gene. In this study, we offer a feasible method to isolate and identify EPCs from bone marrow. In addition, an efficient transfection with a plasmid vector (without risk of interference) can be constructed a hybrid structure with EPC and DDAH2 gene to examine their function in vitro.

MiR-92a regulates endothelial progenitor cells (EPCs) by targeting GDF11 via activate SMAD2/3/FAK/Akt/eNOS pathway

“…Increased levels of FLK-1 positive EPCs have been associated with improved reendothelialization (28). With the EPCs passaged, EPCs gradually differentiated into ECs who mainly expressed the EC-specific markers CD31 and vWF (27). in this study, FLK-1 and vWF in late EPCs were detected.…”

“…Vascular endothelial growth factor (VEGF) receptor type 2 (FLK-1, VEGFR2) and von Willebrand Factor (vWF) both expressed on the late EPCs (27). Increased levels of FLK-1 positive EPCs have been associated with improved reendothelialization (28).…”

Dimethyloxalylglycine Attenuates Steroid-Associated Endothelial Progenitor Cell Impairment and Osteonecrosis of the Femoral Head by Regulating the HIF-1α Signaling Pathway

“…Rabbit EPCs were isolated and cultured as described previously [ 37 ]. In brief, New Zealand white rabbits were anesthetized via ear vein injection of 10% chloral hydrate (3.5 mL/kg), and approximately 5 mL of bone marrow was withdrawn from the bilateral femur and tibia per rabbit using an aspiration needle containing 0.2 mL of heparin (3000 U/mL).…”