Salvianolic acid B and ferulic acid synergistically promote angiogenesis in HUVECs and zebrafish via regulating VEGF signaling

Induced vascular growth in the myocardium has been widely acknowledged as a promising intervention strategy for patients with ischemic coronary artery disease. Yet despite long-term efforts on gene, protein or cell-based pro-angiogenic therapies, the clinical translation remains challenging. Noticeably, multiple medicinal herbs have long-term documented effects in promoting blood circulation. Salvia miltiorrhiza and Ligusticum stratum are two representative traditional Chinese medicine herbs with suggested roles in enhancing organ blood supply, and Guanxinning Tablet (GXNT), a botanical drug which is formulated with these two herbs, exhibited significant efficacy against angina pectoris in clinical practices.This study aimed to examine the pro-angiogenic activity of GXNT and its major components, as well as to explore their pharmacological mechanism in promoting angiogenesis.In vitro, the pro-angiogenic effects of GXNT and its major components were examined on human umbilical vein endothelial cells by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), scratch assay, and endothelial cell tube formation assay. In vivo, the pro-angiogenic effects were examined on the ponatinib-induced angiogenesis defective zebrafish model. The active compounds were identified through phenotype-based screening in zebrafish, and their pharmacological mechanism was explored in both in vitro and in vivo models by immunofluorescent staining, cell cycle analysis, quantitative PCR and whole embryo in-situ hybridization.We demonstrated strong pro-angiogenic effects of GXNT in both human umbilical vein endothelial cells and zebrafish model. Moreover, through phenotype-based screening in zebrafish for active compounds, pro-angiogenic effects was discovered for salvianolic acid B (Sal B), a major component of Salvia miltiorrhiza, and its activity was further enhanced when co-administered with ferulic acid (FA), which is contained in Ligusticum stratum. On the cellular level, Sal B and FA cotreatment increased endothelial cell proliferation of sprouting arterial intersomitic vessels in zebrafish, as well as largely restored G1-S cell cycle progression and cyclin D1 expression in angiogenic defective HUVECs. Through quantitative transcriptional analysis, increased expression of vegfr2 (kdr, kdrl) and vegfr1 was detected after GXNT or SalB/FA treatment, together with upregulated transcription of their ligands including vegf-a, vegf-b, and pgfb. Bevacizumab, an anti-human VEGF-A monoclonal antibody, was able to significantly, but not completely, block the pro-angiogenic effects of GXNT or SalB/FA, suggesting their multi-targeting properties.In conclusion, from a traditional Chinese medicine with effects in enhancing blood circulation, we demonstrated the synergistic pro-angiogenic effects of Sal B and FA via both in vitro and in vivo models, which function at least partially through regulating the expression of VEGF receptors and ligands. Future studies are warranted to further elaborate the molecular interaction between these two compounds and the key regulators in the process of neovascularization.