2.5. Directed In Vivo Angiogenesis Assay (DIVAA)

Angiogenic responses of EPCs of each patient were quantitatively assessed by directed in vivo angiogenesis assay (DIVAA) in a nude mouse according to the protocol of the manufacturer (Trevigen, MD, USA). Briefly, female nude mice, 6 to 8 weeks of age, were anesthetized with 2% isoflurane for the implantation of the angioreactor. Two incisions were made on the dorsal-lateral skin of a nude mouse, one on each side, approximately 1 cm in length. Two angioreactors containing growth factors with (i.e., right side) and two without (i.e., left side) cultured EPCs (4 × 10⁵ EPCs per angioreactor) from one patient were subcutaneously implanted under the surgical incisions in one nude mouse which were closed with interrupted 4-0 nylon sutures (Figures 2(a) and 2(b)). After 14 days, the 30 nude mice carrying EPCs from 30 patients were anesthetized with 2% isoflurane before careful removal of the implanted angioreactors. Cells in the angioreactor were collected for incubation with fluorescein isothiocyanate- (FITC-) conjugated lectin at 4°C overnight. Vascularization in the angioreactor was assessed with a spectrophotometer (Thermo Scientific™ Fluoroskan Ascent™ FL Microplate Fluorometer, Vantaa, Finland) at 485 nm based on the degree of specific binding between lectin and endothelial cells as reflected in the intensity of FITC-lectin fluorescence signals (i.e., relative fluorescent units, RFU). After a 14-day implantation, the level of angiogenesis and neovascularization as reflected in the mean fluorescence intensity in the two angioreactors on the right side (i.e., EPCs) to that in the other two on the left side (i.e., controls) was compared using the DIVAA ratio (i.e., mean fluorescent units in angioreactors with EPCs/mean fluorescent units in angioreactors without EPCs) for each animal. All animal experimental procedures were approved by the Institute of Animal Care and Use Committee at Kaohsiung Chang Gung Memorial Hospital (Affidavit of Approval of Animal Use Protocol No. 2014121814) and performed in accordance with the National Research Council of the National Academies Guide for the Care and Use of Laboratory Animals (Eighth Edition, 2011).

Evaluation of endothelial progenitor cell- (EPC-) mediated angiogenesis/neovascularization by directed in vivo angiogenesis assay (DIVAA) in nude mice. (a) Angioreactors implanted subcutaneously on each flank of a nude mouse. (b) Two angioreactors containing growth factors with (i.e., right side, EPCs) and two without (i.e., left side, controls) cultured EPCs (4 × 10⁵ EPCs/angioreactor) from one patient subcutaneously implanted under the surgical incisions. After a 14-day implantation, retrieved angioreactors displayed on the right panel. (c) Comparison of relative fluorescent units (RFUs) between controls and EPCs with the paired t-test. (d) Comparison of the DIVAA ratio between the low-score group (grades 0-1) and the high-score group (grades 2-3), using the independent t-test; each error bar represents the mean with SD, ^∗∗∗p < 0.0001 and ^∗p < 0.05.