Hypoxic preconditioning as a key enhancer of angiogenesis: A novel in vitro strategy utilizing hucb-msc secretome and dapagliflozin to optimize endothelial progenitor cell function in stable coronary artery disease

Abstract Background and Aims Novel treatment for coronary artery disease(CAD) using angiogenesis approach requires an optimal microenvironment. The human umbilical cord blood mesenchymal stem cells secretome (hUCB-MSCs) contains paracrine factors that promote new vessel formation, while hypoxic preconditioning enhances endothelial progenitor cell(EPC) responsiveness. This study evaluates hypoxia preconditioning to optimize the secretome and dapagliflozin combination in inducing angiogenesis in vitro. Methods EPCs were isolated from stable CAD patients and cultured under normoxic or 48-hour hypoxic conditions. Each group was further stratified into control, 20% secretome, and secretome with 50 μM dapagliflozin combination group. Angiogenesis was assessed by tube formation assay using ImageJ software and functional analysis by measurement of endothelial nitric oxide synthase (eNOS) and von Willebrand factor (VWF) using ELISA. Statistical analysis was done by SPSS using ANOVA with post-hoc study. Results Secretome alone increased tube formation by 42.3 ± 8.7% compared to control (p<0.01). The addition of dapagliflozin further enhanced angiogenesis to 78.5 ± 10.2% under normoxia (p<0.01). Under hypoxic conditions, the combined therapy has the highest effect, with a 96.1 ± 12.4% increase (p<0.001). eNOS expression was significantly upregulated, showing a 1.9-fold rise in normoxia and 2.4-fold rise in hypoxia (p<0.01), while VWF levels remained unchanged (p=0.41). Hypoxic preconditioning markedly amplified angiogenic responses with nearly double that of control and consistently superior to all normoxic groups. Conclusion Hypoxic preconditioning is a critical determinant that optimizes the pro-angiogenic efficacy of this novel hUCB-MSC secretome-based combination therapy. This strategy provides a promising novel regenerative treatment option for stable CAD patients.Figure 1.Tube assay analysis of angiogenesis development in the hypoxia-preconditioned group.