Elucidating the role of miRNA-126 via PI3K/AKT pathway in hypoxia induced human angiogenesis model treated with navitoclax

Abstract

Uncontrolled angiogenesis contributes to the progression and instability of atherosclerotic plaques, which may lead to the development of various cardiovascular diseases. Hypoxia, commonly present within atherosclerotic lesions, is a key trigger of pro-angiogenic signalling, including activation of the PI3K/AKT pathway. miRNA-126 is highly expressed in endothelial cells, has been reported to play a regulatory role in angiogenesis. This study aimed to determine the role of miRNA-126 in regulating angiogenesis via the PI3K/AKT pathway in hypoxia-induced in vitro (HUVECs) and ex vivo (human aortic punch) angiogenesis models treated with navitoclax. In the in vitro model, five experimental groups were established: 1) Normoxia, 2) Hypoxia, 3) Hypoxia + navitoclax, 4) Hypoxia + mimic miRNA-126-3p, and 5) Hypoxia + mimic miRNA-126-3p + recombinant PI3K protein. In contrast, the ex vivo model was limited to three groups: 1) Normoxia, 2) Hypoxia, and 3) Hypoxia + navitoclax. Expression of miRNA-126-3p was quantified using RT-qPCR, while VEGF-A and ANGPT-1 protein levels were measured via ELISA. Angiogenic activity was evaluated using tube formation assays in vitro and ex vivo. The results showed that hypoxia significantly upregulated miRNA-126-3p and VEGF-A levels in both models (p < 0.05) potentially through the activation of HIF-1α. In the in vitro model, transfection with mimic miRNA-126-3p enhanced VEGF-A expression, although not significantly compared to the hypoxia group (p = 0.225). Co-treatment with recombinant PI3K protein and miRNA-126-3p mimic led to a significant reduction in VEGF-A levels compared to hypoxia + mimic miRNA-126 (p < 0.05), suggesting a possible feedback regulatory effect. Navitoclax treatment under hypoxia reduced miRNA-126-3p expression and VEGF-A levels significantly (p < 0.05), indicating an inhibitory effect on proangiogenic signalling in both models. ANGPT-1 expression was decreased under hypoxia but restored by navitoclax treatment in both models. Functionally, navitoclax suppressed angiogenic activity in vitro and ex vivo, as evidenced by reduced tube formation and vessel sprouting. In conclusion, navitoclax exerts anti-angiogenic effects in hypoxia-induced angiogenesis through modulation of the miRNA-126/PI3K/AKT pathway. These findings provide mechanistic insight into the therapeutic potential of navitoclax for limiting pathological angiogenesis in cardiovascular diseases, particularly atherosclerosis.