Angiogenesis is the development of new vessels from the pre-existing microvesseles, which plays a vital role in a variety of physiological and pathological processes, such as atherosclerosis, diabetic retinopathy, age-related macular degeneration, tumor growth, and metastasis, etc. (D'Alessio et al., 2015; Secomb and Pries, 2016). Angiogenesis is initiated by the degradation of basal membrane, which contributes to the enhanced vascular permeability and more dissolution of extracellular matrix, followed by endothelial cell migration, proliferation. All of these steps finally resulted in the formation of associated pericytes and leading to the increased new blood vessel formation.

Ginseng, a well-known traditional Chinese medicine, has been considered an “all healing” panacea for a long time. Most of the pharmacological activity of ginseng is attributed to its constituents, namely ginsenosides. On the basis of their aglycones, ginsenosides are classified structurally into protopanaxadiol (PPD)-type and protopanaxatriol (PPT)-type (Yue et al., 2007). Ginsenoside Rb1 and Rg1 are representative PPD- and PPT-type saponins, respectively. The “Yin and Yang” action of ginseng on angiomodulation is mainly decided by the compositional ratio between Rg1 and Rb1 (Yue et al., 2007). Rb1 is a representative component of ginseng possesses several properties, including anti-obesity, anti-oxidative stress, and anti-fatigue, anti-angiogenesis etc. (Sengupta et al., 2004; Cheng et al., 2005; Xiong et al., 2010). Additionally, Rb1 and its metabolite have been demonstrated to suppress angiogenesis in tumor cells (Sato et al., 1994; Shibata, 2001; Kim et al., 2004). Our previous findings had also suggested that Rb1 was able to inhibit angiogenesis through the up-regulation of pigment epithelial-derived factor (PEDF) in HUVECs (Leung et al., 2007).

PEDF, originally discovered from the retinal pigmented epithelium, has been recognized as the most potent natural angiogenesis inhibitor (Dawson et al., 1999). It plays a vital role in maintaining the avascular nature of the cornea and determining the balance between angiogenesis and anti-angiogenesis. Previous studies have found that PEDF exerted angiogenesis inhibitory effect in several disorders, such as ocular diseases, cardiovascular diseases, and cancer, etc. (Takenaka et al., 2008; Tombran-Tink, 2010; Becerra and Notario, 2013).

MicroRNAs (miRNAs) are tiny, endogenously expressed non-coding RNAs (18–25 nt long). They regulate crucial posttranscriptional mRNA expression, typically by binding to the 3′ untranslated region (3′-UTR) of the complementary mRNA sequence, thereby repressing translation and genes silencing (Wen, 2016). Recent studies have identified that miRNAs are highly expressed in endothelial cells (ECs), which are also believed to be novel regulators for the regulation of the vascular development and angiogenesis (Zhang, 2010). Moreover, modulation of miRNAs levels may also have potential therapeutic function for the treatment of angiogenesis related diseases (ARD), such as heart disease, cancer, and neurodegeneration, etc. (Andreou et al., 2015).

In the present study, the potential role of miR-33a in the Rb1-induced PEDF expression and the functional role of miRNA in anti-angiogenic action had been explored. This is the first study revealing that Rb1 exerted anti-angiogenic effects via the modulation of miR-33a and its target gene PEDF expression through PPAR-γ signaling.

Ginsenoside-Rb1 with ~98% purity was obtained from Fleton (Chengdu, China) (Kwok et al., 2012). Cell culture medium M199 was purchased from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS), penicillin–streptomycin (PS), phosphate-buffered saline (PBS), and trypan blue were purchased from Invitrogen (Carlsbad, CA, USA). Endothelial cell growth supplement (ECGS) was from Upstate (Waltham, MA, USA). Anti-PEDF antibody was obtained from R&D Systems (Minneapolis, MN, USA), anti-GAPDH antibody was from Santa Cruz Biotechnology (California, CA, USA). Horseradish peroxidase-conjugated (HRP)-conjugated secondary antibodies were purchased from Zymed (South San Francisco, CA, USA). Lipofectamine®2000 and Opti-MEM® reduced serum were purchased from Life Technologies (Carlsbad, CA, USA). MiRNA precursor and negative control were from Applied Biosystems (Foster City, CA, USA). All the inhibitors were bought from Tocris Bioscience (Atlantic Road, Bristol, UK). PPAR-α siRNA and PPAR-γ siRNA were obtained from Ambion (Austin, Texas, USA).

In this study, we found that Rb1 treatment on human endothelial cells, through the interaction with PPAR-γ, decreased miR-33a level and increased PEDF expression, which resulted in the inhibition of angiogenesis. Overexpression of miR-33a significantly reverted the Rb1-induced PEDF expression and anti-angiogenic effect in vitro. Moreover, Rb1-mediated PEDF increment and miR-33a reduction were blocked by the pre-incubation with GW9662, an inhibitor of PPAR-γ, as well as PPAR-γ siRNA, which suggests that the action of Rb1 was PPAR-γ-dependent.

Ginseng, a traditional herbal medicine, has been used to treat various diseases for thousands of years. Accumulating evidences have revealed its biological activities of ginsenoside Rb1 in diverse pathological processes, including cardiovascular diseases, neurodegenerative diseases, diabetes, obesity, and cancer, etc. (Sato et al., 1994; Xiong et al., 2010; Cho, 2012; Lee and Kim, 2014). Our previous findings had also revealed that Rb1 exerted anti-angiogenic effects, which may have the therapeutic potential in cardiovascular disease (e.g., atherosclerosis, hypertension, restenosis, etc.), central nervous system diseases (e.g., Alzheimer's disease, stroke, diabetic retinopathy, etc.), chronic inflammatory diseases (e.g., obesity, diabetes, Crohn's disease), and the control cancer development and progression (D'Alessio et al., 2015; Jayakumar et al., 2017; Ramjiawan et al., 2017; Wu et al., 2017).

PEDF can block abnormal neovessels without overt harm to the established retinal vessels even at high doses, which provides the advantages for therapy of angiogenesis-related diseases (Stellmach et al., 2001; Bouck, 2002). In addition to the potent and selective anti-angiogenic function, PEDF has neuroprotective, antioxidant, anti-inflammation, and antithrombotic properties, and has been associated with therapy of atherosclerotic diseases, choroidal neovascularization, cancer, etc. (Takenaka et al., 2008; Rychli et al., 2009; Yamagishi and Matsui, 2010, 2014; Wen et al., 2017).

We had found that Rb1 exerted anti-angiogenic activity via the induction of PEDF, even at relatively low doses (5–20 nM). Also, PEDF was involved in Rb1-mediated inhibition of endothelial tube formation, which was consistent with our previous findings (Leung et al., 2007). Our study had demonstrated that Rb1 (250 nM) could induce both PEDF mRNA and protein expressions (Leung et al., 2007). Here, Rb1 (10 nM) was able to increase PEDF protein expression but without any change on the mRNA level, and the major difference between the present and the previous work is difference in concentration of Rb1. Thus, Rb1 at various concentrations exerted different effects on PEDF expression which contributed to distinct actions. We speculated that post-transcriptional modulation may be involved in this process.

MiRNAs, a novel class of endogenous, small, non-coding RNAs, can regulate gene expression by inhibiting translation and/or degradation of their target genes (Bartel, 2007). Recently, we studied the possible roles of miRNAs in ginsenoside-mediated angiogenesis (Chan et al., 2009, 2013). Expression profiling of various miRNAs in HUVECs confirmed that ginsenoside-Rb1 could modulate miRNA expression. We verified that Rb1 suppressed miR-33a mRNA expression in HUVECs in a time-dependent manner. Furthermore, pre-miR-33a indeed partially reversed the Rb1-increased PEDF expression. We further confirmed that miR-33a reduced PEDF 3′-UTR reporter activity, which suggested that miR-33a could affect the translation of PEDF by targeting its 3′-UTR region. All of these findings supported that Rb1 could promote PEDF protein expression by downregulating miR-33a. This is the first report demonstrated that miR-33a plays a key role in Rb1-mediated PEDF induction and that PEDF is one of the target genes of miR-33a.

MiR-33a was primarily identified as a key regulator of metabolic programs including cholesterol and fatty acid homeostasis (Rayner et al., 2010). It is an intronic miRNA located with the sterol response element-binding protein (SREBP)-2 gene, which codes for transcription factors that coordinate cholesterol, fatty acid synthesis, and HDL metabolism (Marquart et al., 2010). Antagonism of miR-33 upregulates ATP-binding cassette transporter A1 (ABCA1) expression and promotes cholesterol efflux to ApoA-I, a key step in regulating reverse cholesterol transport (Gerin et al., 2010; Rayner et al., 2010). Cholesterol is a structural component of the cell and is indispensable for normal cellular function (Yvan-Charvet et al., 2010). Also, ApoA-I binding protein (AIBP)-targeted cholesterol efflux from ECs could disrupt lipid rafts and inhibit angiogenesis (Fang et al., 2013). Lipid rafts has the ability to support vascular endothelial growth factor (VEGF)-induced dimerization of VEGF receptor 2 (VEGFR2), the major pro-angiogenic receptor in ECs. It was also reported that AIBP removes cholesterol from ECs for reduced lipid raft and VEGFR2 dimerization, which further inhibits VEGFR2 signaling and angiogenesis in vitro (Fang and Miller, 2013). Thus, miR-33a reduces cholesterol efflux, but cholesterol efflux is critical for proper angiogenesis (Fang et al., 2013; Sene et al., 2013). However, there is no direct evidence to confirm the role of miR-33a in angiogenesis so far. In order to test the effect of miR-33a on angiogenesis, tube formation assay was performed. Pre-miR-33a transfection could significantly induce the formation of a capillary-like network in vitro, which was also able to reverse the inhibitory effects of Rb1 on tubulogenesis. However, miR-33a overexpression could also decrease Rb1-induced PEDF level, which highlights the key role of miR-33a in the PEDF-mediated angiogenic pathway.

Ginsenosides have a four trans-ring steroid-like skeleton structure so they can bind and activate different steroid hormone receptors to regulate gene expression (Attele et al., 1999; Kwok et al., 2012). Intracellular steroid binding proteins could be attractive targets for ginsenosides. We identified that Rb1 acted though PPAR-γ to downregulate miR-27b in 3T3-L1 cells and though PPAR-δ to decrease miR-25 in fibroblast cells (Chan et al., 2012; Kwok et al., 2012). To determine whether steroid hormone receptors are involved in regulation of Rb1 on miR-33a and PEDF, different specific antagonists were used. Western blot results demonstrated that Rb1-increased PEDF expression was abolished by both PPAR-γ and PPAR-α antagonists. However, only PPAR-γ was involved in the Rb1-downregulated miR-33a level. These results highlight the indispensable role of PPAR-γ in Rb1-induced miR-33a and reduced PEDF expression.

It had been suggested that PEDF could interact with PPAR-α for the regulation of angiogenesis and lipid metabolism in hepatocellular carcinoma (Chung et al., 2008). Our previous finding demonstrated that Rb1 was an anti-angiogenic compound in suppressing the formation of endothelial tube-like structures by modulating PEDF via estrogen receptor (ER) (Leung et al., 2007). PEDF could induce apoptosis by activating p53 and PPAR-γ signaling in HUVECs as well (Ho et al., 2008). In the present study, Rb1-induced PEDF expression was mediated by PPAR-γ activation and leading to the anti-angiogenic effect. Recently, multiple findings indicated the association of PPAR-γ with several miRNAs, which were believed to regulate many biological processes through post-transcriptional modulation. MiR-27a could directly target the 3′-UTR of PPAR-γ mRNA in 3T3-L1, thereby inhibiting adipogenesis (Chan et al., 2012). Our current findings suggest that the downregulation of miR-33a in Rb1-exposed HUVECs cells was blocked by the inhibition of PPAR-γ, so several processes may be involved. Specifically, PPAR-γ may bind directly to the miR-33a transcription regulatory sequence or interact with other transcription factors that bind to the miR-33a promoter. On the other hand, Drosha is responsible for the cleavage of pri-miRNAs into pre-miRNAs, and then further processed by Dicer to produce mature miR-33a (Krol et al., 2010). Direct interaction of PPAR-γ with Drosha may thus decrease mature miR-33a biogenesis (Ottaviani et al., 2014).

In our study, we first elaborated the tight link between miR-33a modulation and function by PPAR-γ signaling. We also provide the direct evidence of Rb1 exerted anti-angiogenic effect which involves the activation of PPAR-γ and downregulation of miR-33, thereby stimulating PEDF. Here, a schematic diagram was drawn to describe the protective role of Rb1 in the inhibition of angiogenesis and the underlying mechanisms (Figure 5), which deepens our understanding of Rb1 in treating ARD.

HL designed and performed the experiments. XZ performed the experiments. HL and XZ wrote the manuscript. H-HK and P-YP helped with the preformation of the experiments and collection of samples. MD and XL participated in data analyses. ZL contributed to the discussion of the manuscript. RN-SW participated in the design and revision of the manuscript. All authors read and approved the final manuscript.