Tai-Ping Fan

Modulating Angiogenesis The Yin and the Yang in Ginseng

Background—Ginseng is a commonly used nutraceutical. Intriguingly, existing literature reports both wound-healing and antitumor effects of ginseng extract through opposing activities on the vascular system. To elucidate this perplexity, we merged a chemical fingerprinting approach with a deconstructional study of the effects of pure molecules from ginseng extract on angiogenesis. Methods and Results—A mass spectrometric compositional analysis of American, Chinese and Korean, and Sanqi ginseng revealed distinct “sterol ginsenoside” fingerprints, especially in the ratio between a triol, Rg1, and a diol, Rb1, the 2 most prevalent constituents. Using a Matrigel implant model and reconstituting the extracts using distinct ratios of the 2 ginsenosides, we demonstrate that the dominance of Rg1 leads to angiogenesis, whereas Rb1 exerts an opposing effect. Rg1 also promoted functional neovascularization into a polymer scaffold in vivo and the proliferation of, chemoinvasion of, and tubulogenesis by endothelial cells in vitro, an effect mediated through the expression of nitric oxide synthase and the phosphatidylinositol-3 kinase→Akt pathway. In contrast, Rb1 inhibited the earliest step in angiogenesis, the chemoinvasion of endothelial cells. Conclusions—The present study explains, for the first time, the ambiguity about the effects of ginseng in vascular pathophysiology based on the existence of opposing active principles in the extract. We also unraveled a speciogeographic variation impinging on the compositional fingerprint that may modulate the final phenotype. This emphasizes the need for regulations standardizing herbal therapy, currently under the Dietary Supplement and Health Education Act. Furthermore, we propose that Rg1 could be a prototype for a novel group of nonpeptide molecules that can induce therapeutic angiogenesis, such as in wound healing.

Controlling the vasculature: Angiogenesis, anti-angiogenesis and vascular targeting of gene therapy

Angiogenesis is the development of new blood vessels from an existing vascular bed. Normal vascular proliferation occurs only during embryonic development, the female reproductive cycle and wound repair. By contrast, many pathological conditions (for example, cancer, atherosclerosis and diabetic retinopathy), are characterized by persistent, unregulated angiogenesis. Conversely, inadequate angiogenesis can lead to failure of ulcers to heal and myocardial infarction. Control of vascular development could permit new therapeutic approaches to these disorders. For example, several anti-angiogenic drugs are currently undergoing clinical trials for the treatment of cancer, whereas enhancement of angiogenesis by exogenous growth factors can prevent or limit the damage in chronic wounds and duodenal ulcers. Here Tai-Ping Fan, Rhys Jaggar and Roy Bicknell highlight recent achievements and discuss the prospects of receptor antagonists, enzyme inhibitors, tumour suppressor genes and vascular targeted approaches, especially that of gene therapy, in the future development of angiotherapy.

Interleukin-8 stimulates angiogenesis in rats

To test the hypothesis that the cytokines interleukin-6 (IL-6) and IL-8 may play regulatory roles in the aberrant neovascularization in chronic inflammatory diseases, we examined their effects in a rat sponge model and compared their actions with those of IL-1 and lumor necrosis factor-α (TNF-α). Daily doses of 3 pmol IL-8, IL-1, TNF-α, but nol IL-6, significantly accelerated the sponge-induced angio-genesis. Although lower doses (0.3 pmol) of these cytokines were inactive, IL-1 acted synergistically with subthreshold daily doses (10 pmol) of substance P (SP) and bradykinin (BK) to produce an intense angiogenic response. In contrast, IL-8 only interacted positively with IL-1, but not TNF-α, SP, or BK. There was no synergism or antagonism between IL-6 and SP. These results demonstrate the discrete interactions between angiogenic factors and cytokines in chronic inflammation and suggest that the sponge model is a good means for the study of such interactions.

Stimulation of angiogenesis by substance P and interleukin-1 in the rat and its inhibition by NK1 or interleukin-1 receptor antagonists

1 Daily administration of 1 nmol substance P or 3 pmol recombinant human interleukin‐1 alpha (IL‐1α) caused intense neovascularization in a rat sponge model of angiogenesis. Lower doses of substance P (10 pmol) or IL‐1α (0.3 pmol) were ineffective when given alone. When combined at these low doses, substance P and IL‐1α interacted to produce an enhanced neovascular response. 2 By use of selective tachykinin NK1, NK2 and NK3 receptor agonists, ([Sar9,Met(O2)11]substance P, [β‐Ala8]neurokinin A(4–10), Succ‐[Asp6,MePhe8]substance P(6–11) (senktide), respectively), it was established that the activation of NK1 receptors is most likely to mediate the angiogenic response to substance P in this model. 3 The angiogenic activity of substance P and IL‐1α (10 pmol and 0.3 pmol day−1, respectively) was abolished by co‐administration of (i) the selective peptide NK1 receptor antagonist, L‐668,169 (1 nmol day−1), (ii) the selective non‐peptide NK1 receptor antagonists, RP 67580 and (±)‐CP‐96,345 (both at 1 nmol day−1) or (iii) the IL‐1 receptor antagonist, IL‐1ra, (50 μg day−1). In contrast, the selective NK2 receptor antagonist, L‐659,874 (1 nmol day−1) was ineffective. 4 The angiogenic action of substance P and IL‐1α was resistant to modification by mepyramine (1 nmol day−1) and/or cimetidine (10 nmol day−1), indomethacin (7 nmol day−1) or the platelet‐activating factor (PAF) antagonist, WEB‐2086 (22 nmol day−1), indicating that histamine, prostaglandins and PAF are not likely to be involved in this neovascular response. 5 The inhibition of the substance P/IL‐1 angiogenic response by selective NK1 receptor antagonists or by an IL‐1 receptor antagonist demonstrates that angiosuppression can be achieved by blocking the activity of angiogenic factors at the receptor level.

Diverse and potent activities of HGF/SF in skin wound repair†

Genetic studies in the mouse have highlighted essential roles for several growth factors in skin repair and have offered a rationale for their use in therapy. The present study shows that the plasminogen‐related growth factor HGF/SF (hepatocyte growth factor/scatter factor) promotes wound repair in homozygous diabetic db/db mice by recruiting neutrophils, monocytes, and mast cells to the wound; by promoting the migration of endothelial cells to the injured area; and by enhancing keratinocyte migration and proliferation. As a result, granulation tissue formation, wound angiogenesis, and re‐epithelialization are all increased. The results demonstrate that HGF/SF affects and sustains all key cellular processes responsible for wound repair and point to a unique potential of this molecule for the therapy of chronic skin wounds. Copyright

Hepatocyte Growth Factor/Scatter Factor Can Induce Angiogenesis Independently of Vascular Endothelial Growth Factor

Objective—Hepatocyte growth factor/scatter factor (HGF/SF) promotes vascular endothelial growth factor (VEGF) expression and induces angiogenesis in multiple pathological conditions. The present study was designed to delineate the HGF/SF and VEGF signaling cascades during angiogenesis by using PTK787, a selective VEGF receptor antagonist. Methods and Results—PTK787 produced a concentration-dependent (10−8 to 10−6 mol/L) inhibition of VEGF-induced angiogenesis, without altering the basal or HGF/SF-induced response in vitro. In contrast, the nonspecific kinase inhibitor genistein blocked the HGF/SF-induced effect. Both VEGF and HGF/SF induced a rapid phosphorylation of extracellular receptor kinases-1 and -2 (ERKs) and Akt. PTK787 inhibited the VEGF-induced activation of Akt and ERKs, without affecting the HGF/SF-induced phosphorylation. Treatment with VEGF and HGF/SF increased total neovascularization in a murine scaffold granuloma model, but no additive or synergistic interactions were observed. PTK787 (50 mg/kg) blocked the VEGF-induced response without altering the basal or HGF/SF-induced neovascularization. Conclusions—We demonstrate that HGF/SF can induce angiogenesis independently of VEGF, possibly through the direct activation of the Akt and ERKs. These results demonstrate the necessity of a multitargeted approach for the rational design of newer therapies to inhibit pathophysiological angiogenesis.

Sequential development of angiotensin receptors and angiotensin I converting enzyme during angiogenesis in the rat subcutaneous sponge granuloma.

The vasoconstrictor peptide antiotensin II (AII) can stimulate angiogenesis, an important process in wound healing, tumour growth and chronic inflammation. To elucidate mechanisms underlying AII‐enhanced angiogenesis, we have studied a subcutaneous sponge granuloma model in the rat by use of 133Xe clearance, morphometry and quantitative in vitro autoradiography. When injected directly into the sponge, AII (1 nmol day−1) increased 133Xe clearance from, and fibrovascular growth in sponge granulomas, indicating enhanced angiogenesis 6 to 12 days after implantation. This AII‐enhanced angiogenesis was inhibited by daily doses (100 nmol/sponge) of the specific but subtype non‐selective AII receptor antagonist (Sar1, Ile8)AII, and by the selective non‐peptide AT1 receptor antagonists losartan and DuP 532. In contrast, AII‐enhanced neovascularization was not inhibited by the AT2 receptor antagonist PD123319, nor was it mimicked by the AT2 receptor agonist CGP42112A (each at 100 nmol/sponge day−1). AI (1 nmol/sponge day−1), the angiotensin converting enzyme (ACE) inhibitors captopril (up to 100 μg/sponge day−1) and lisinopril (40 μg/sponge day−1), or AII receptor antagonists did not affect angiogenesis in the absence of exogenous AII. [125I]‐(Sar1, Ile8)AII binding sites with characteristics of AT1 receptors were localized to microvessels and to non‐vascular cells within the sponge stroma from 4 days after implantation, and were at higher density than in skin throughout the study. [125I]‐(Sar1, Ile8)AII binding sites with characteristics of AT2 receptors were localized to non‐vascular stromal cells, were of lower density and appeared later than did AT1 sites. The ACE inhibitor [125I]‐351A bound to sites with characteristics of ACE, 14 days after sponge implantation. [125I]‐351A bound less densely to sponge stroma than to skin. We propose that AII can stimulate angiogenesis, acting via AT1 receptors within the sponge granuloma. AT1 and AT2 receptors and ACE develop sequentially during microvascular maturation, and the role of the endogenous angiotensin system in angiogenesis will depend on the balanced local expression of its various components. Pharmacological modulation of this balance may provide novel therapeutic approaches in angiogenesis‐dependent diseases.

Differential effects of angiostatic steroids and dexamethasone on angiogenesis and cytokine levels in rat sponge implants

1 . Subcutaneous implantation of sterile polyether sponges elicited a reproducible neovascular response in rats, as determined by blood flow measurement with a 133Xe clearance technique and confirmed histologically. This model was used to monitor the levels of two cytokines during angiogenesis and to compare the activities of angiostatic steroids and anti‐inflammatory steroids. 2 . Initial experiments followed the neovascular development over a 20‐day period. Daily local injection of hydrocortisone caused a dose‐dependent (0.5, 5 and 50 μg per sponge) inhibition of the basal sponge‐induced angiogenesis. However, daily systemic treatment of hydrocortisone (2, 10 and 50 mg kg−1, s.c.) was less effective at inhibiting angiogenesis, and this inhibition was not sustained by day 20 after sponge implantation. 3 . To investigate the involvement of cytokines during the course of angiogenesis, we measured the endogenous levels of tumour necrosis factor‐α (TNF‐α) and interleukin 6 (IL‐6) in sponge implants. Levels of IL‐6 and TNF‐α peaked at day 7 and day 11 after implantation, respectively. These cytokine levels subsided through the completion of angiogenesis by day 20. 4 . Subsequent experiments were carried out over a 14‐day period. Among the three angiostatic steroids tested, U‐24067 (6α‐fluoro‐17,21‐dihydroxy‐16α‐methylpregna‐4,9(11)‐diene‐3,20‐dione‐21‐acetate) showed a dose‐dependent inhibition (0.5, 5 and 50 μg per sponge per day) of sponge‐induced angiogenesis. Tetrahydro‐S was also effective at 5 μg doses, but medroxyprogesterone failed to affect the angiogenic response. None of these steroids caused atrophies of the spleen and thymus. 5 . Daily local injection of dexamethasone (0.5 μg per sponge) inhibited the basal sponge‐induced angiogenesis almost completely. Although higher doses of dexamethasone (5 and 50 μg per sponge) did not produce further inhibition of angiogenesis, they caused severe spleen and thymus weight losses, indicative of immunosuppression. 6 . At the daily dose of 5 μg per sponge, dexamethasone inhibited angiogenesis and produced a marked reduction in the levels of TNF‐α and IL‐6 at day 14. In contrast, hydrocortisone, U‐24067 and tetrahydro‐S did not influence the levels of TNF‐α and IL‐6. 7 . We concluded that the anti‐angiogenic activity of angiostatic steroids and anti‐inflammatory steroids in the rat sponge model is independent of their ability to reduce the production of TNF‐α and IL‐6. The differential effects of angiostatic and anti‐inflammatory steroids suggest that U‐24067 and its derivatives may have therapeutic potential in the management of angiogenic diseases such as rheumatoid arthritis.

[Leu8]des-Arg9-bradykinin inhibits the angiogenic effect of bradykinin and interleukin-1 in rats.

1 Subcutaneous implantation of sterile polyether sponges in rats elicited a reproducible neovascular response over 14 days, as determined by measurements of relative sponge blood flow by a 133Xe clearance technique. The angiogenic response was verified by quantitation of haemoglobin contents and histological evaluation of vascularized sponges. 2 Daily administration of 1 nmol of bradykinin (BK) into the implants significantly enhanced the basal sponge‐induced neovascularization, leading to higher 133Xe clearance values, increased haemoglobin contents, cellularity and vascularity. 3 When given alone, lower doses of BK (10 pmol) or recombinant human interleukin‐1 α (IL‐1α, 0.3 pmol) produced no apparent effects on the basal sponge‐induced angiogenesis. However, co‐administration of these two peptides produced an angiogenic response similar to that elicited by 1 nmol of BK. 4 The BK/IL‐1α‐induced neovascularization was abolished by the bradykinin B1 receptor antagonist, [Leu8]des‐Arg9‐BK (1 nmol day−1), but not by the B2 receptor antagonist Ac‐d‐Arg‐[Hyp3,d‐Phe7,Leu8]‐BK (1 nmol day−1). 5 Thus, if such interaction between BK and IL‐1α contributes to the excessive neovascularization in chronic inflammatory diseases, the blockade of B1 receptors may provide an effective treatment.

Comparative studies of the angiogenic activity of vasoactive intestinal peptide, endothelins-1 and -3 and angiotensin II in a rat sponge model.

1 The angiogenic activity of four vasoactive peptides with a range of vasodilator and vasoconstrictor properties, i.e. vasoactive intestinal peptide (VIP), endothelin‐1, endothelin‐3 and angiotensin II, were investigated in a rat sponge model. Neovascularization was assessed by the 133Xe clearance technique and confirmed by histological studies. 2 Daily doses of the vasodilator peptide, VIP (1000 pmol), caused intense neovascularization, but a lower dose (10 pmol) produced no apparent effect. However, the lower dose of VIP, when given with a subthreshold dose of interleukin‐1α (0.3 pmol), produced an angiogenic response similar to that seen with the higher dose of VIP. The neovascular response induced by co‐administration of VIP and interleukin‐1α was inhibited by simultaneous administration of 100 pmol VIP (10–28), a specific VIP receptor antagonist. 3 In contrast, daily doses of 10, 100 or 1000 pmol endothelin‐3 (a mixed vasoconstrictor and vasodilator with more marked vasodilator activity) or of 100 or 1000 pmol endothelin‐1 (also with mixed activity but with much more pronounced vasoconstrictor response) produced no apparent effect on sponge‐induced angiogenesis. 4 The vasoconstrictor peptide, angiotensin II, in daily doses of 1000 pmol, caused an intense neovascularization like VIP but lower doses of angiotensin II (10 or 100 pmol) produced no apparent effect. The lowest dose of angiotensin II (10 pmol) when administered with the subthreshold dose of interleukin‐1α (0.3 pmol) had no effect on the basal neovascular response in the sponges. The angiotensin II‐induced neovascular response was inhibited by co‐administration of 100 nmol of the specific AT1 receptor antagonist, losartan, but not by the AT2 receptor antagonist, PD 123319. 5 These data show that VIP and angiotensin II possess angiogenic activity. However, endothelin‐1 and endothelin‐3 had no activity at the doses used. Thus the angiogenic response is not related to local vasoconstriction or vasodilatation in the sponges. The blockade of VIP‐ and angiotensin II‐induced angiogenesis at the receptor level suggests that receptor modulation could provide a strategy for the management of angiogenic diseases.