Cyclooxygenase-2-selective nonsteroidal anti-inflammatory drugs inhibit hepatocyte growth factor/scatter factor-induced angiogenesis.

Epidemiological studies have indicated a reduced risk of malignancies with the use of nonsteroidal anti-inflammatory drugs (NSAIDs), although the exact mechanisms are debated. NSAIDs inhibit angiogenesis, which is a key step for tumor growth. Hepatocyte growth factor/scatter factor (HGF/SF), a potent and independent angiogenic factor, has been implicated in tumorigenesis, but limited knowledge exists on the potential targets for inhibiting HGF/SF-induced pathological angiogenesis. The current study was designed to elucidate the possible role of cyclooxygenase (COX) downstream of HGF/SF during angiogenesis and to evaluate the potential for harnessing NSAIDs as a therapeutic strategy. Known NSAIDs were classified as COX-1 or COX-2 selective based on their activity in a platelet aggregation experiment. The inhibitors were administered into a polyether polyurethane scaffold implant in mice at the selected doses, and the total neovascularization after the administration of HGF/SF was quantified using a (133)Xenon clearance technique, vessel counts, and immunohistochemistry. Angiogenesis was also quantized into chemoinvasion, migration, proliferation, and tube formation events in vitro, and the effects of the NSAIDs were evaluated on HGF/SF-induced activity of human umbilical vein endothelial cells (HUVECs). HGF/SF accelerated the angiogenic process in the murine implant, and this activity was inhibited by COX-2-selective meloxicam and NS398. The COX-1 inhibitors ketoprofen and SC560 failed to inhibit the HGF/SF-induced angiogenic events in vitro and in vivo. A COX-2 blockade inhibited the HGF/SF-induced chemoinvasion and migration of human umbilical vein endothelial cells, without affecting the proliferative or tubulogenic responses. Western blots revealed the induction COX-2 expression after HGF/SF treatment, and the pharmacological inhibition of COX-2 executed a temporal inhibition of phosphorylation of the mitogen-activated protein kinases. The current study, for the first time, implicates COX-2 as a downstream signal during HGF/SF-induced angiogenesis, temporally impinging on the mitogen-activated protein kinase signaling. However, the mediation is restricted to only the early events of the angiogenic process, emphasizing the chemopreventive role for NSAIDs. Few therapeutic options currently exist for HGF/SF-induced pathological angiogenesis, and the vast knowledge on COX-2 inhibitors can be harnessed to design a newer therapeutic approach.

Targeting of mitogen-activated protein kinases and phosphatidylinositol 3 kinase inhibits hepatocyte growth factor/scatter factor-induced angiogenesis.

BACKGROUND: Hepatocyte growth factor/scatter factor (HGF/SF) can sufficiently and independently induce pathophysiological angiogenesis. However, the treatment strategies have mostly been unsuccessful. The present study is the first to evaluate the possible targeting of downstream signals for the inhibition of HGF/SF-induced angiogenesis. METHODS AND RESULTS: In a multichannel scratch assay with human endothelial cells (ECs), HGF/SF induced a strong and prolonged activation of MAPK and cell proliferation that was inhibited by PD98059 and LY294002/wortmannin, selective inhibitors of MAPK and PI3K signaling modules, respectively. Western blotting demonstrated a temporal relation between the activation of the two pathways. Chemical inhibition of the PI3K and MAPK signals inhibited HGF/SF-induced chemoinvasion of ECs in vitro and blocked the HGF/SF-induced neovascularization into a polymer scaffold in vivo, as quantified by vessel counts and the clearance of radioactive 133Xe. CONCLUSIONS: These data indicate that MEK and PI3K inhibitors represent a promising approach to the clinical management of pathological conditions characterized by overt HGF/SF-induced angiogenesis.

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.

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.

Antiangiogenic agents are effective inhibitors of endometriosis.

Endometriosis is a disease in which the lining of the uterus (endometrium), shed at the time of menstruation, becomes established at sites such as the peritoneum and ovaries. These explants develop a rich blood supply that enables them to survive and grow. We hypothesized that inhibitors of angiogenesis would prevent this growth by disrupting sensitive vessels supplying endometriotic lesions. Vessels sensitive to angiogenic antagonism have few associations with pericyte cells. The vessels supplying human endometriotic lesions were immunohistochemically characterized and found to be predominantly pericyte free. A model in which human endometrium is implanted into nude mice was used to test the effects of two antagonists of the angiogenic growth factor, vascular endothelial cell growth factor A. Soluble truncated receptor (flt-1; P = 0.002) and an affinity-purified antibody to human vascular endothelial cell growth factor A (P = 0.03) significantly inhibited the growth of nude mouse explants. Pericyte-free vessels were shown to supply endometrial lesions in nude mice and were disrupted in lesions taken from soluble flt-1-treated mice. In summary, antiangiogenic agents inhibited the growth of explants in an in vivo model of endometriosis by disrupting the vascular supply, and this effect is likely to apply to the human disease. These findings suggest that antiangiogenic agents may provide a novel therapeutic approach for the treatment of endometriosis.

Thymidine phosphorylase induces angiogenesis in vivo and in vitro: an evaluation of possible mechanisms.

1 Thymidine phosphorylase (TP) is elevated in the plasma of cancer patients, and has been implicated in pathophysiological angiogenesis. However, the downstream signals underlying this implication remain obscure. The purpose of the present study was to examine the effects of TP on the neovascularisation response in vitro and in vivo. 2 Both TP and its catalytic product, 2-deoxy-D-ribose-1-phosphate, and downstream 2-deoxy-D-ribose (2-DDR) promoted endothelial tubulogenesis in vitro, and the regeneration of a wounded monolayer of endothelial cells without exerting any mitogenic effect. In vivo, both TP and 2-DDR promoted the development of functional vasculature into an avascular sponge. A TP inhibitor, 6-amino-5-chlorouracil, was able to partially reverse the effects of TP, but had no effect on the 2-DDR-induced angiogenesis. 3 Enhanced monolayer regeneration was observed with TP-cDNA-transfected bladder carcinoma cells. The transfection of TP-cDNA, however, did not confer any proliferative advantage. The regeneration of TP overexpressing cells was associated with a time-dependent expression of the enzyme haeme-oxygenase (HO-1). 4 The present study demonstrates that both TP and its ribose-sugar metabolites induce angiogenesis by mediating a cohesive interplay between carcinoma and endothelial cells. The induction of HO-1 in TP-transfected cells suggests that it could be a possible downstream signal for the angiogenic effects of TP. Furthermore, reducing sugars have been shown to induce oxidative stress, and ribose could be a possible cause for the upregulation of HO-1, which has been implicated in the release of angiogenic factors. Therefore, we postulate that 2-DDR could be mediating the angiogenic effects of TP possibly through an oxidative stress mechanism and additionally getting integrated in the endothelial metabolic machinery.

Nitric oxide modulates hepatocyte growth factor/scatter factor-induced angiogenesis.

OBJECTIVE: There is limited knowledge about potential therapeutic targets in Hepatocyte growth factor/scatter factor (HGF)-induced pathophysiological angiogenesis. Recent candidates have included phosphatidylinositol-3-kinase, which is an upstream activator for endothelial nitric oxide (NO) synthase (NOS III). The current study is the first to evaluate the possible involvement of NOS-NO cascade in HGF-induced angiogenesis. METHODS AND RESULTS: NOS III inhibitors blocked the HGF-induced functional neovascularization in vivo, as quantified using vessel counts, 133Xe-clearance, and immunohistology. This was reversed by L-arginine. Western blot analysis of HGF-treated cells also revealed a temporal increase in HGF-induced phosphorylation. In a deconstructional approach, HGF induced the proliferation and chemokinesis of human endothelial cells. These phenotypic effects were inhibited by NOS inhibitors, L-NAME and L-NIO, and the NO scavenger, carboxy PTIO, but unaltered by 1400W, a NOS II inhibitor. This inhibition was reversed by spermine NONOate, a NO donor, which independently exerted a biphasic effect on endothelial cell proliferation. The modulation of NO did not alter HGF-induced chemoinvasion of endothelial cells, while spermine-NONOate destabilized HGF-induced tubulogenesis, suggesting that a single assay is not sufficient for predicting the final phenotypic outcome on angiogenesis. CONCLUSIONS: The study is the first to demonstrate that the NOS III nitric oxide is a key signal cascade in HGF-induced angiogenesis, and represents a promising target for the clinical management of pathological conditions characterized by overt HGF signaling.

Polymer-assisted, multi-step solution phase synthesis and biological screening of histone deacetylase inhibitors.

The polymer-assisted solution phase synthesis (PASP) of an array of histone deacetylase (HDAc) inhibitors is described. HDAc inhibitors have considerable potential as new anti-proliferative agents. Selected compounds were shown to inhibit both human endothelial cell proliferation, and the formation of tubules (neovascularisation) in an in vitro model of angiogenesis.