Ginseng, Panax ginseng C.A. Meyer, a precious Chinese traditional medicinal herb, has been known clinically used in China for thousands of years. The genus name 'Panax' was derived from Greek. 'Pan' means 'all' and 'axos' means 'cure'. Literally 'Panax' can be translated as 'cure-all' or panacea. The herbal root is named ginseng because it is shaped like a man. Actually the term 'ginseng' represents two Chinese ideograms: 'gin' (pronounced ren) refers to 'man' and 'seng' (pronounced shen) refers to 'essence' It is believed to embody man's three mythical essences – body, mind and spirit. Thus it is also referred to as the lord or king of herbs 1. Its medicinal efficacy was first documented in Shengnong Bencao Jing and it was later summarized by Li Shizhen in Bencao Gangmu and Zhongyao Zhi (Chinese Materia Medica) by People's Health Publishing House, Beijing, published in 1596 and 1959 respectively 12. In the 18th century, the effectiveness of ginseng was recognized in the West, and subsequently, a large number of investigations were conducted on its botany, chemistry, pharmacology and therapeutic applications 34567. Ginseng has been used as a general tonic or adaptogen for promoting longevity especially in the Far East, especially China, Korea and Japan 8. Ginseng is now one of the most popular herbal medicines used nutraceutically with an annual sale of over USD 200 million.

Ginseng is a deciduous perennial plant that belongs to the Araliaceae family. Currently, twelve species have been identified in the genus Panax (Table 1) 9. Among them, Panax ginseng C. A. Meyer (Araliaceae), cultivated in China, Korea, Japan, Russia, and the US, P. quinquefolium L (American ginseng), grown in southern Canada and the US and P. notoginseng, cultivated in Yunnan and Guangxi provinces in China, represent the three most extensively investigated species. The pharmacological and therapeutic effects of ginseng have been demonstrated to affect the central nervous system (CNS), cardiovascular system, endocrine secretion, immune function, metabolism, biomodulating action, anti-stress, and anti-aging 5. Recently, there have been controversies concerning the usefulness of ginseng in cancer therapy. Most studies claimed that the pharmacological effects of ginseng are attributed to its bioactive constituents such as ginsenosides, saponins, phytosterols, peptides, polysaccharides, fatty acids, polyacetylenes, vitamins and minerals 10. In this review, we focus on the recent advances in the studies of ginsenosides on the modulation of angiogenesis (i.e. formation of blood vessels) which is a common denominator of many diseases, such as tumor and cardiovascular disorders (e.g. atherosclerosis).

The most prominent constituent of ginseng is a saponin glycoside known as ginsenosides (Rx) (Figure 1). Recent research indicates that most of the pharmacological effects of ginseng are attributed to ginsenosides 11. In general, the contents of ginsenosides vary widely ranging from 2 to 20% depending on the species, age and part of ginseng, and even vary with the preservation or extraction method 111213. More than 30 ginsenosides have been isolated, and characterized from various Panax species 1415. In terms of their chemical structural characteristics, ginsenosides can be classified into three major categories, namely protopanaxadiols (PPD) (e.g. Rb₁, Rb₂, Rc, Rd, Rg₃, Rh₂), protopanaxatriols (PPT) (e.g. Re, Rf, Rg₁, Rg₂, Rh₁) and the oleanolic acid derivatives. Ginsenosides have a steroid-like skeleton consisting of four trans-rings, with modifications from each other depending the type (e.g. glucose, maltose and fructose), number of sugar moieties and the sites of attachment of the hydroxyl group (e.g. C-3, C-6, or C-20) (Figure 1). Ginsenosides are amphipathic in nature. The hydroxyl (-OH) group of ginsenosides allows both interactions between the polar head of the membrane phospholipids and the β-OH group of cholesterol, while the hydrophobic steroid backbone can interact with the hydrophobic side chains of fatty acids and cholesterol. Indeed, these physiochemical interactions are greatly determined by the numbers and sites of polar hydroxyl groups on each ginsenoside. Moreover, ginsenosides have been shown to interact with numerous membrane proteins such as ion channels, transporters and receptors, which leads to a broad range of physiological activities 16.

Sprouting angiogenesis consists of several consecutive steps with extensive interactions between soluble factors, extracellular matrix (ECM) and cells. As shown in Figure 2, during the early stage of angiogenesis, angiogenic factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) emanate from conditioned cells (e.g. stromal cells, endothelial cells, blood) or surrounding tissues (e.g. ECM) to stimulate ECs 19. These activated ECs secrete various types of enzymes including matrix metalloproteinases (MMPs), plasminogen activators (PA), gelatinase, collagenases and urokinases which bring about the degradation of basement membrane and ECM surrounding the parental blood vessels. Subsequently, the ECs migrate towards the angiogenic factors with the help of surface adhesion molecules or integrin receptors such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and integrins α_vβ₃ 2122. Upon the induction of various angiogenic factors, ECs proliferate and align in a bipolar mode to form capillary sprouts. Finally, these ECs form a lumen and a capillary loop through which the blood can flow.

Intussusceptive microvascular growth, a novel mechanism of blood vessel formation and remodeling, occurs by internal division of the pre-existing capillary plexus without sprouting. It is initiated by protrusion of opposing capillary walls into the lumen where a contact is created between the ECs. Then the endothelial bilayer is perforated and numerous transcapillary tissue pillars with an interstitial core are formed. An extensive vascular tree is formed from the subsequent intussusceptive pillar formation and pillar fusions 232425.

Among the angiogenic regulators, VEGF and nitric oxide (NO) are the critical angiogenic stimulators. We have demonstrated that they are closely related to ginsenoside-mediated angiomodulation. Both VEGF and NO play a critical role in the regulation of physiological angiogenesis including embryogenesis, reproduction and wound healing. They have also been implicated in pathological angiogenesis associated with tumors or many cardiovascular disorders (such as atherosclerosis or ischemic injury). VEGF, which belongs to the VEGF family, is expressed in different tissues including the brain, liver, spleen and many cell types 26. In vitro, VEGF not only acts as a survival factor and mitogen for ECs, but also induces the expression of many angiogenesis-related factors, including ICAM-1, VCAM-1, uPA, PAI-1, uPAR and MMPs. All these gene products are related to the increase of ECM degradation, migration and tube formation of ECs, whereas their effects on intussusception remain to be investigated 27282930. The critical roles played by VEGF in embryonic vasculogenesis and angiogenesis have been demonstrated 3132. Inactivation of a single VEGF allele in mice resulted in embryonic lethality between days 11 and 12. The growth of VEGF+/- embryos was retarded and there were many developmental anomalies.

Endothelium-derived NO has been implicated in mediating a multiplicity of processes involved in angiogenesis. In fact, there are many angiogenic factors, such as VEGF, transforming growth factor β (TGF-β) and fibroblast growth factor (FGF), which can up-regulate the expression of endothelial NO synthase (eNOS), thereby inducing the release of NO 33343536. VEGF- or bFGF-activated human umbilical vein endothelial cells (HUVEC) have been found to secrete NO on Matrigel substratum and subsequently form a tube-like structure, whereas such tube formating action is abolished by eNOS antagonist, N^ω-nitro-L-arginine methylester (L-NAME) 373839. On the one hand, NO increases EC proliferation by acting as a survival factor and enhances migration by augmenting the expression of α_vβ₃ and matrix-ECs interaction through the up-regulation of uPA 4041424344. Moreover, its vasodilating activity is the most prominent effect shown in the modulation of cardiovascular physiology 45. On the other hand, disruption of the eNOS pathway can result in an impairment of normal angiogenesis, enhancement of tumor pathogenesis, and cardiovascular disorders 46474849.

VEGF and NO are working closely with each other in the modulation of angiogenesis, whereas NO is both an upstream and a downstream mediator or an effector of VEGF-dependent angiogenesis. Upon binding to its receptors, VEGF initiates complicated signaling cascades resulting in NO production and subsequent activation of ECs and smooth muscle cells in vessels, keratinocytes and macrophages in wounds, and tumor cells. Furthermore, through the activation of the PI3K/Akt pathway, NO can increase VEGF synthesis in a positive feedback manner 465051525354.

In Asia, ginseng has been used as a medicinal herb for a long time to treat various ailments such as malignant diseases. Experimental studies from the past two decades have shown that both the anti-tumor activities and therapeutic effects of ginseng are mainly attributed to ginsenosides which induce cell death and inhibit metastasis.

Various ginsenosides including Rg₃, Rb₂ and compound K demonstrated anti-angiogenic activity in different tumor models. Among them, Rg₃ is the most extensively investigated. It exerts an anti-angiogenic action in different animal models when administered alone or in combination with other conventional chemicals. In those animal models which have been orthotopically implanted with different tumor cells such as human breast infiltrating duct carcinoma, ovarian carcinoma SKOV3 cells, B16-BL6 melanoma cells and colon 26-M3.1 carcinoma, Rg₃ or Rb₂ was found to inhibit angiogenesis in vivo. A significant reduction of intra-tumoral microvessel density (MVD), VEGF mRNA and VEGF protein levels in tumor tissues and sera of the tumor-bearing animals was observed 104105106122. Similar results were also reported when Rg₃ was used in combination with a low dose of cyclophosphamide in bearing mice Lewis lung carcinoma 109. These data indicate that one of the mechanisms of anti-metastatic effect of ginsenosides is probably related to suppression of tumor-induced angiogenesis 104.

In a recent study, we demonstrated that 20(R)-Rg₃ (1–10³ nM) was able to inhibit HUVEC proliferation, VEGF-induced chemoinvasion and tubulogenesis in vitro in a dose-dependent manner. In a Matrigel plug assay, 20(R)-Rg₃ (600 nM) was able to reduce the hemoglobin content by 5-fold when compared with the positive control containing bFGF and heparin. The extent of microvascular sprouting was inhibited dose-dependently by 20(R)-Rg₃ in the ex vivo organtypic cultures of rat aortic ring fragments (Figure 5). Basement membrane degrading enzymes, especially MMP-2 and -9, are secreted by activated ECs (or even tumor cells) throughout the angiogenic process. They have been demonstrated to play a critical role in cell invasion and migration, as well as angiogenesis. Our data further demonstrate that the anti-angiogenic effect of 20(R)-Rg₃ was probably related to reduction of expression and activities of MMP-2 and -9 124. By using DNA microarray, 20(R)-Rg₃ was found to inhibit the protein expression of angiogenic factors via several target genes (e.g. VEGF, bFGF and MMP-2) in both human lung adenocarcinoma cell line A549 and HUVEC304 cells 123. Current research findings, indicating that some PD type ginsenosides (e.g. Rg₃ and Rb₂) exhibit potent anti-tumor and anti-angiogenic activities in vitro and in vivo, have far-reaching implications for future development of ginsenosides as an anti-tumor medicine.

In contrast to the anti-angiogenic effects of ginsenosides such as Rb₁ and Rg₃, another group of ginsenosides, represented by the panaxatriols Rg₁ and Re, have been found to be angiogenic. Rg₁ increased HUVEC proliferation, migration and tube formation in a dose-dependent manner 125126127128. We further demonstrated its angiogenic effects using the in vivo Matrigel plug and ex vivo aortic ring sprouting models (Figure 5). Histological evaluation of the Matrigel implants indicated that functional neovessels were formed as induced by Rg₁. The in vivo data collected seven days after the implantation of genipin-fixed porous acellular bovine pericardium (same as extracellular matrices), which was dip-coated with Re or Rg₁, indicate that the density of neovessels and tissue hemoglobin content inside the matrices were significantly increased by both ginsenosides. Furthermore, vascularized neo-connective tissue fibrils were found to fill the pores in the matrices loaded with Re 125. Similar angiogenic activities were also observed in the above assays when Rg₁ was used 126. Interestingly, ginsenoside-induced angiogenesis was found to be comparable to or even better than bFGF-induced angiogenesis in vivo. These data suggest that both ginsenosides are very potent angiogenic agents and may potentially be useful in therapeutic angiogenesis in tissue-engineering strategies

Ginseng contains two groups of ginsenosides with activity in the modulation of angiogenesis. These observed counteracting effects can be interpreted by the Yin/Yang theory in traditional Chinese medicine (TCM). In fact, we have, in our previous publications, demonstrated strong scientific evidence supporting the ancient Yin/Yang theory 129. Administration of Rg₁ or Rb₁ alone was shown to exert counteracting effects in angiogenesis. Rg₁ alone promoted functional neovascularization in the polymer scaffold in Matrigel implant model and the chemoinvasion of HUVEC. By contract, Rb₁ exerted inhibition in both. Moreover, we found that ginseng extract reconstituted with a defined ratio of Rg₁ and Rb₁ could alter the angiogenic outcome. In an in vivo scaffold implant neovascularization model, administration of an extract with a higher concentration of Rg₁ than Rb₁ (Rg₁:Rb₁ = 5:2) resulted in the induction of significant angiogenesis in the implant compared with control implants. In contrast, overabundance of Rb₁ (Rg₁:Rb₁ = 2:5) inhibited Rg₁-induced neovascularization 130. These counteracting actions manifested the logic of Yin/Yang theory of TCM, which advocates that everything has opposing Yin and Yang aspects, and these aspects are reciprocally regulated and inhibited by each other in such a way that a continuous state of dynamic balance is maintained. The contents of ginsenosides in P. ginseng, P. quinquefolius and P. notoginseng were then measured by an HPLC method. The data show that P. ginseng has a higher ratio of Rg₁ to Rb₁ (0.51 – 2.08), while P. quinquefolius has a lower ratio (0.07 – 1.4) 131132. These findings are also consistent with the TCM's attributes of 'hot' (i.e. stimulating) properties of P. ginseng and 'cool' (i.e. calming) properties of P. quinquefolius.

Functional genomics refers to the functions and interactions of genes towards a holistic view of the biological system in terms of gene expression and proteins. It focuses on the dynamics of gene transcription, translation and protein-protein interactions, instead of the static views of genomic information (e.g. gene sequences). It can be achieved by means of transcriptomics (differential expression of genes), proteomics (the study of total protein complement of a genome), phosphoproteomics and metabolomics studies (the study of the entire metabolic content of a cell or an organism). In general, the functional genomics approach is accompanied by high-throughput technology platforms such as the DNA microarray (i.e. DNA chip) technology and two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry for characterization of the abundant gene and protein products. The chip-based microarray allows a quantitative parallel assessment of gene expression and facilitates the study of complicated drug actions at the molecular level. This rapid increase in functional genomic-based drug studies enables an early and more accurate prediction and diagnosis of disease and disease progression. Understanding of the individual responses to drugs will have implications for their use and development by the pharmaceutical industry.

This approach was applied in the mechanistic study of ginsenoside Rg₁ on HUVEC 127133. Our microarray data indicated that ginsenoside Rg₁ could up-regulate a set of genes related to cell adhesion, migration and cytoskeleton such as RhoA, RhoB, IQ-motif-containing GTPase-activating protein 1 (IQGAP1), calmodulin (CALM2), Vav2 and laminin-α4 (LAMA4) in HUVEC. These proteins interact with one another in a hierarchical cascade pattern in modulating cell architectural dynamics. As we observed in the tube formation assay, HUVEC undergo rapid migration and morphological differentiation to form tubular structures by involving a dynamic assembly and disassembly of cytoskeletons. RhoA and RhoB, which belong to Rho family GTPases, have been found to regulate cell-cell adhesion and hence the motility through interfering with membrane ruffling, stress fiber formation and E-cadherin-mediated cell-cell adhesions 134135136137138139140141142. The activities of GTPases are regulated by means of a molecular switch; cycling of the GDP-bound form (inactive state) and GTP-bound form (active state). As demonstrated by Bustelo et al., such transformation can be activated by guanine nucleotide exchange factors (Rho-GEFs) 143144. Vav2, one of Rho-GEFs, can increase the exchange of bound-GDP for a GTP molecule and translocate the Rho GTPases to the plasma membrane for interaction with its Rho effectors such as IQGAP1 145. Moreover, Vav2 can also affect cadherin-mediated cell-cell adhesion by binding with p120 catenin leading to formation of lamellipodia and membrane ruffling 146147148. IQGAP1 acts as a negative regulator in E-cadherin-mediated cell-cell adhesion by interacting directly with β-catenin and the cytoplasmic domain of cadherin 149150. Over-expression of IQGAP1 is believed to compete with α-catenin for the same binding site on β-catenin and displace α-catenin from the β-catenin-α-catenin complex which disrupts the association of the complex with F-actin. Such cytomechanical modifications can subsequently cause the reduction of E-cadherin-mediated cell-cell adhesion that accounts for the changes in cell morphology and migration 151. Moreover, laminins, which are the trimeric basement membrane glycoproteins, have been found to participate in the formation and differentiation of tubular structures of HUVEC in vitro and to provide a structural link between the ECM and the actin-based cytoskeletal system of cells 152153154155156. Undoubtedly, microarray technology provides a high throughput platform for the elucidation of drug mechanism in study models.

On a different front, a proteomic study was carried out by Ma et al. to elucidate the cardiovascular protective role of Rg₁ on ECs 133. The data show that protein expression of MEKK-3, reticulocalbin, phosphoglycerate, 6-phosphogluconolactonase, zinc finger protein, NSAP1 protein, recombination-activating protein was increased, while that of eNOS, and mineralocorticoid receptor (MR) was decreased upon tumor necrosis factor-α (TNF-α) stimulation of HUVEC. However, Rg₁ restored the expression of these proteins to the normal level. Western blotting and RT-PCR further validated that NO production in such TNF-α induced condition was correlated to the increase of eNOS mRNA and protein expression. They suggest that the increase of NO is related to the protective role of Rg₁ on TNF-α stimulated HUVEC.

A critical function of the vasculature is to provide a nutritional supply to tissues and organs. In general, proper vascular homeostasis is controlled by capillary permeability modulation, vasodilation and angiogenesis 157158159. All these activities can govern the rate of material exchange, transportation and the site of distribution so as to meet the physiological needs of the human body. Steroid hormones, such as glucocorticoids, estrogens, progestagens, mineralocorticoids and androgens, play an important role in such vascular modulation at the molecular, cellular and even systematic levels 160161162163164.

Among the various steroid hormones, estrogen is the most promising example used to elaborate the maintaining and protective roles of steroid hormones on vascular homeostasis and cardiovascular disorders. The growing evidence implicated that estrogen reduces the risk of atherosclerosis by (1) decreasing the serum levels of both total and low-density-lipoprotein (LDL) cholesterol, while raising those of high-density-lipoprotein (HDL) cholesterol and triglycerides; (2) interfering with the inflammatory processes (e.g. monocyte adherence and trans-endothelial migration on the endothelium) in the vasculature; and (3) acting as an antioxidant 165166167168. Furthermore, estrogen is also a potent vasodilator, and hypotensive agents that can induce vascular relaxation by producing an increase of endothelium-derived vasodilators (e.g. NO). Mechanistic studies clearly indicate that while estrogen binds with its corresponding steroid hormone receptor – estrogen receptor (ERα, and ERβ), it may undergo either or both genomic and non-genomic actions inside the cells. Through the conventional pathway, activated-ER induces the gene transcription of eNOS, thereby increasing NO generation. Alternatively, through triggering the intracellular signaling (non-genomic) pathway, phosphorylated estrogen-bound ER (ligand-activated ER) has been found to induce rapid endothelial NO release via the PI3K-Akt-dependent pathway in HUVEC 169170. Various ginsenosides have been shown to exhibit an estrogen-like activity by acting as a functional ligand of ER 171172173174.

Ginsenosides could exhibit their actions through plasma membrane, cytosol, or even in the nucleus. They can initiate their mode of actions by binding to the membrane receptors (e.g. ATPase pump, ion transporters and channels, voltage gated channels and G-proteins) and subsequently activating the associated downstream signaling cascades. As described in the previous section, ginsenosides are amphipathic in nature, they can intercalate into the plasma membrane resulting in the alteration of membrane fluidity and trigger a series of cellular responses. Binding with the intracellular steroid hormone receptors including glucocorticoid receptor (GR), estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR) and mineralocorticoid receptor (MR), using their hydrophobic 'steroid-like' backbone is another alternative to trigger cellular responses. Like other steroids, ginsenosides can gain access to the nuclear receptors, where they regulate gene transcription by binding with the specific gene response elements to activate the so called 'genomic pathway' 175176 (Figure 6).

A previous study indeed demonstrated that Rg₁ can trans-activate glucocorticoid response element (GRE)-luciferase activity by acting as a functional ligand of the GR 177. Glucocorticoids (GCs) have been reported to activate the phosphatidylinositol-3 kinase (PI3K)/Akt pathway after binding with the GR 178. Interestingly, our recent findings also indicate that Rg₁ can act as GCs to increase the phosphorylation of GR, PI3K, Akt/PKB and eNOS, leading to an enhancement of NO production in HUVEC (Figure 7). Activation of eNOS was abolished by differential inhibition using RU486 (GR antagonist), LY294002 (PI3K inhibitor) and SH-6 (Akt inhibitor), indicating that Rg₁ induced NO production from eNOS via the PI3K/Akt pathway. Moreover, knockout of GR by siRNA completely eliminated such reaction. Taken together, we concluded that such NO generation pathway was mediated through the activation of GR by ginsenoside Rg₁ 179. This nuclear receptor mediated response, which occurs within seconds or minutes upon stimulation and is independent of any transcriptional activity, is classified as the 'non-genomic' pathway 175176 (Figure 6).

In a later study, we discovered that the angiogenic Rg₁ was able to enhance VEGF expression in ECs by increasing the level of β-catenin within the nucleus. We concluded that Rg₁ induced increase of VEGF in HUVEC through activation of a PI3K/Akt → GSK3β→β-catenin/TCF pathway via GR 180. In addition, the PI3K/Akt-mediated NO generation has been found in androgen-responsive LNCaP and estrogen-responsive MCF-7 cells by others 181. Re was found to bind with steroid hormone receptors (including AR, ERα and PR). Lee et al., however, showed that ginsenoside Rh₁ could only activate ER in the CV-1 cell model 173.

The estrogen-like activity of various ginsenosides including Rg₁, Rh₁ and Rb₁ has been reported 170171172. In fact, ginseng has been used in TCM for alleviation of the symptoms of menopause, which means ginseng, probably ginsenosides, can act as phytoestrogens to elicit an ER-mediated pathway for protecting the cardiovascular system. These recent research findings clearly indicate that ginsenosides may act as steroid hormones to activate various steroid hormone receptors to exert these pleiotropic responses. Although many mechanisms behind the physiological effects induced by ginsenosides are still not clear, their ability to interact with steroid hormone receptors may hold the key to finally elucidate these diverse actions of ginseng.