Pro-angiogenic actions of Salvianolic acids on in vitro cultured endothelial progenitor cells and chick embryo chorioallantoic membrane model

Abstract

Salvia miltiorrhiza (SM, also known as DanShen) is one of the well-known widely-used Chinese herbal medicines in clinical practice. In this study we aimed to demonstrate the pro-angiogenic effects of Salvianolic acids (SAs) to treat illnesses such as ischemic cardiovascular diseases, the main active components of aqueous extract of SM. To do this, new-born rat spleen mononuclear cells were isolated and endothelial progenitor cells (EPCs) were expanded (not more than 24h) SD. Then the pro-angiogenic activities of SAs were evaluated on in vitro cultured EPCs and chick embryo chorioallantoic membrane (CAM) model. And the adherent cells were stained with DiI complexed acetylated low-density lipoprotein (DiI-acLDL) and fluorescein Ulex Europaeus agglutinin-1 (FITC-UEA-1), and then viewed by laser scanning confocal microscope (LSCM) to confirm EPCs lineage. EPCs identification was also tested by ultrastructural analyses. EPCs proliferation, migration and in vitro vasculogenesis activity were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, transwell chamber assay and in vitro vasculogenesis kit, respectively. EPCs adhesion assay was performed by replating those on fibronectin-coated dishes, and then counting adherent cells. EPCs phenotype was confirmed by the presence of double positive cells for DiI-acLDL uptake and lectin binding and identification of Weibel-Palade body in cytoplasm by ultrastructural analyses. Incubation of EPCs with SAs increased the number of EPCs and promoted EPCs migratory, adhesive and in vitro vasculogenesis capacity. SAs also promoted angiogenesis as evidenced by CAM model. The results of the present study suggest that SAs may have utility for therapeutic postnatal vasculogenesis of ischemic tissue, contributing to the clinical benefit of SM therapy in patients with coronary artery disease.