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Ginsenosides are considered the major constituents that are responsible for most of the pharmacological actions of ginseng. However, some ginsenosides exist as stereoisomeric pairs, detailed and molecular exposition based on the structural differences of ginsenoside stereoisomers has not been emphasized in most studies. Here we explore the functional differences of ginsenoside Rg₃ stereoisomers on angiogenesis. In this study, we demonstrated the distinctive differential angiogenic activities of 20(S)-Rg₃ and 20(R)-Rg₃ stereoisomers. 20(S)-Rg₃ at micromolar concentration promotes human endothelial cells proliferation, migration and tube formation in vitro, as well as ex vivo endothelial sprouting. The effects induced by 20(S)-Rg₃ are significantly more potent than 20(R)-Rg₃. These effects are partially mediated through the activation of AKT/ERK-eNOS signaling pathways. Moreover, knockdown of peroxisome proliferator-activated receptor-gamma (PPARγ) by specific small interference RNA abolished the 20(S)-Rg₃-induced angiogenesis, indicating that PPARγ is responsible for mediating the angiogenic activity of Rg₃. Using reporter gene assay, the PPARγ agonist activity of 20(S)-Rg₃ has been found 10-fold higher than that of 20(R)-Rg₃. Computer modeling also revealed the differential binding is due to the chiral center of 20(S)-Rg₃ can form a critical hydrogen bond with Tyr473 of PPARγ ligand binding domain. The present study elucidated the differential angiogenic effects of Rg₃ stereoisomers by acting as agonist of PPARγ. The results shed light on the structural difference between two ginsenoside stereoisomers that can lead to significant differential physiological outcomes which should be carefully considered in the future development of ginsenoside-based therapeutics.

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Angiogenesis is the formation of new blood vessels from the existing vasculature, which is involved in multiple biological processes, including atherosclerosis, ischemic heart disease, and cancer. Ginsenoside-Rb1 (Rb1), the most abundant ginsenoside isolated form Panax ginseng, has been identified as a promising anti-angiogenic agent via the up-regulation of PEDF. However, the underlying molecular mechanisms still unknown. In the present study, human umbilical vein endothelial cells (HUVECs) were selected to perform in vitro assays. Rb1 (0–20 nM) treatment induced pigment epithelial-derived factor (PEDF) protein expression in concentration and time-dependent manners. Interestingly, it was also demonstrated that the exposure of Rb1 (10 nM) could increase PEDF protein expression without any alteration on mRNA level, suggesting the involvement of posttranscriptional regulation. Furthermore, bioinformatics predictions indicated the regulation of miR-33a on PEDF mRNA 3′-UTR, which was further confirmed by luciferase reporter gene assay and real-time PCR. Over-expression of pre-miR-33a was found to regress partly Rb1-mediated PEDF increment and anti-angiogenic effect in HUVECs. Additionally, Rb1-reduced miR-33a and increased PEDF expression was prevented by pre-incubation with peroxisome proliferator-activated receptor-γ (PPAR-γ) antagonist (GW9662) or transfection with PPAR-γ siRNA in HUVECs. Taken together, our findings demonstrated that Rb1 exerted anti-angiogenic effects through PPAR-γ signaling pathway via modulating miR-33a and PEDF expressions. Thus, Rb1 may have the potential of being developed as an anti-angiogenic agent, however, further appropriate studies are warranted to evaluate the effect in vivo.