Yuichi Chikaraishi

10-Hydroxy-2-decenoic Acid, a Major Fatty Acid from Royal Jelly, Inhibits VEGF-induced Angiogenesis in Human Umbilical Vein Endothelial Cells

Vascular endothelial growth factor (VEGF) is reported to be a potent pro-angiogenic factor that plays a pivotal role in both physiological and pathological angiogenesis. Royal jelly (RJ) is a honeybee product containing various proteins, sugars, lipids, vitamins and free amino acids. 10-Hydroxy-2-decenoic acid (10HDA), a major fatty acid component of RJ, is known to have various pharmacological effects; its antitumor activity being especially noteworthy. However, the mechanism underlying this effect is unclear. We examined the effect of 10HDA on VEGF-induced proliferation, migration and tube formation in human umbilical vein endothelial cells (HUVECs). Our findings showed that, 10HDA at 20 µM or more significantly inhibited such proliferation, migration and tube formation. Similarly, 10 µM GM6001, a matrix metalloprotease inhibitor, prevented VEGF-induced migration and tube formation. These findings indicate that 10HDA exerts an inhibitory effect on VEGF-induced angiogenesis, partly by inhibiting both cell proliferation and migration. Further experiments will be needed to clarify the detailed mechanism.

Role of Soluble Vascular Endothelial Growth Factor Receptor-1 in the Vitreous in Proliferative Diabetic Retinopathy

PURPOSE To determine the vitreous level of soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) in patients with proliferative diabetic retinopathy (PDR) or idiopathic macular hole (MH). Furthermore, to investigate the relationships among sVEGFR-1, vascular endothelial growth factor (VEGF), and pigment epithelium-derived factor (PEDF). DESIGN Retrospective case-control study. PARTICIPANTS Thirty-eight patients who underwent vitrectomy (PDR [27 eyes in 26 patients] or MH [12 eyes in 12 patients]). METHODS In vitreous fluid samples obtained during vitreoretinal surgery, sVEGFR-1, VEGF, and PEDF levels were measured by enzyme-linked immunosorbent assay. Effects of sVEGFR-1 on VEGF-A-induced tube formation were investigated using human umbilical vein endothelial cells co-cultured with fibroblasts. MAIN OUTCOME MEASURES Levels of sVEGFR-1 and the relationships among sVEGFR-1, VEGF, and PEDF in vitreous fluids from patients with PDR or MH. RESULTS In PDR (vs MH), there were significantly higher vitreous concentrations of both sVEGFR-1 (3949.4+/-608.9 pg/mL [mean +/- standard error, n = 27] vs 1568.8+/-595.0 pg/mL [n = 12, P = 0.009]) and VEGF (1316.2+/-404.6 pg/mL [n = 27] vs 11.7+/-8.1 pg/mL [n = 12, P = 0.003]), whereas the vitreous concentration of PEDF was significantly lower (2.1+/-1.1 ng/mL [n = 27] vs 41.6+/-17.0 ng/mL [n = 12, P = 0.041]). In PDR, there was a significant positive correlation between the sVEGFR-1 and VEGF vitreous concentrations (r = 0.414, P = 0.032), but not between PEDF and VEGF (r = 0.196, P = 0.328) or between sVEGFR-1 and PEDF (r = 0.167, P = 0.406). In vitro, sVEGFR-1 (0.1-1000 ng/mL) concentration-dependently inhibited VEGF-A-induced tube formation, its effect being significant at 100 to 1000 ng/mL on the tube area, length, and path. CONCLUSIONS In the vitreous fluids of patients with PDR, the sVEGFR-1 level was increased (vs that in patients with MH), and sVEGFR-1 correlated significantly with VEGF. In vitro, sVEGFR-1 reduced VEGF-induced angiogenesis. Thus, sVEGFR-1 may play a pivotal antiangiogenic role in PDR.

Ruboxistaurin, a PKCβ inhibitor, inhibits retinal neovascularization via suppression of phosphorylation of ERK1/2 and Akt

Ruboxistaurin, a protein kinase C (PKC) beta inhibitor, exhibits significant anti-angiogenic activity that reduces the response of vascular endothelial cells to stimulation by vascular endothelial growth factor (VEGF). In addition, the activation of PKC, specifically PKCbeta, plays a key role in the retinal neovascularization. However, the effect of ruboxistaurin on oxygen-induced retinopathy (OIR), an experimental murine model of proliferative retinopathy, has not yet been investigated. In this study, we assessed the efficacy of ruboxistaurin both in vitro and in vivo and also evaluated its anti-angiogenic mechanisms. Ruboxistaurin inhibited formation, proliferation, and migration of VEGF-induced human umbilical vein endothelial cells (HUVECs) in a concentration-dependent manner. It also inhibited the VEGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and serine/threonine protein kinase family protein kinase B (Akt). In in vivo retinal neovascularization experiments, induced in neonatal mice by returning the retina to normoxia (21% O2) after exposure to hyperoxia (75% O2), ruboxistaurin given subcutaneously significantly reduced pathologic vascular changes. No effect was seen on revascularization of the capillary-free area. These findings indicate that ruboxistaurin has anti-angiogenic effects both in vitro and in vivo that are exerted partly via suppressing the phosphorylation of ERK1/2 and Akt. Ruboxistaurin may be a candidate for treatment of angiogenesis in retinal neovascularization diseases.

Vaccinium myrtillus (Bilberry) Extracts Reduce Angiogenesis In Vitro and In Vivo

Vaccinium myrtillus (Bilberry) extracts (VME) were tested for effects on angiogenesis in vitro and in vivo. VME (0.3–30 µg ml−1) and GM6001 (0.1–100 µM; a matrix metalloproteinase inhibitor) concentration-dependently inhibited both tube formation and migration of human umbilical vein endothelial cells (HUVECs) induced by vascular endothelial growth factor-A (VEGF-A). In addition, VME inhibited VEGF-A-induced proliferation of HUVECs. VME inhibited VEGF-A-induced phosphorylations of extracellular signal-regulated kinase 1/2 (ERK 1/2) and serine/threonine protein kinase family protein kinase B (Akt), but not that of phospholipase Cγ (PLCγ). In an in vivo assay, intravitreal administration of VME inhibited the formation of neovascular tufts during oxygen-induced retinopathy in mice. Thus, VME inhibited angiogenesis both in vitro and in vivo, presumably by inhibiting the phosphorylations of ERK 1/2 and Akt. These findings indicate that VME may be effective against retinal diseases involving angiogenesis, providing it can reach the retina after its administration. Further investigations will be needed to clarify the major angiogenesis-modulating constituent(s) of VME.

Angiostatic effects of Brazilian green propolis and its chemical constituents

Propolis, a resinous substance collected by honeybees from various plant sources, has several pharmacological actions, such as anti-tumor and anti-inflammatory effects. The aim of this study was to evaluate the anti-angiogenic effects of a water extract of Brazilian green propolis (WEP) and its constituents, caffeoylquinic acid derivatives, against angiogenic processes in human umbilical vein endothelial cells (HUVECs) in vitro. We also examined the anti-angiogenic effects of WEP against retinal neovascularization in a murine oxygen-induced retinopathy model in vivo. WEP and its constituents significantly suppressed vascular endothelial growth factor (VEGF)-induced HUVEC proliferation, migration, and tube formation in vitro. WEP and its caffeoylquinic acid derivatives suppressed VEGF-stimulated phosphorylation of mitogen-activated protein kinase in HUVECs (versus VEGF alone). Moreover, WEP (300 mg/kg/day, subcutaneously for 5 days) significantly suppressed retinal neovascularization in the murine oxygen-induced retinopathy model. These data indicate that (i) WEP has angiostatic effects against angiogenic processes in vitro and in an in vivo model of murine oxygen-induced retinopathy and (ii) the inhibitory effects of WEP against in vitro angiogenesis are chiefly derived from its caffeoylquinic acid derivatives. Judging from these findings, WEP and its caffeoylquinic acid derivatives may represent candidates for preventive or therapeutic agents against diseases caused by angiogenesis.

CB-12181, a New Azasugar-Based Matrix Metalloproteinase/Tumor Necrosis Factor-α Converting Enzyme Inhibitor, Inhibits Vascular Endothelial Growth Factor-Induced Angiogenesis in Vitro and Retinal Neovascularization in Vivo

To evaluate the anti-angiogenic efficacy of CB-12181 [an azasugar derivative that has inhibitory actions against matrix metalloproteinases (MMPs) and tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE)], we investigated the suppressing ability on in vitro (tube formation by endothelial cells) and in vivo (retinal neovascularization on murine ischemia-induced proliferative retinopathy) models of angiogenesis. For in vitro analysis, a capillary-like tube formation model using human umbilical vein endothelial cells (HUVECs) and fibroblasts co-culture assay was employed. Tube formation of HUVECs was stimulated by vascular endothelial growth factor (VEGF) and incubated with different concentrations of CB-12181 (0.1-100 microM) for 11 days. For in vivo analysis, mice were exposed to 75% oxygen between postnatal days 7 and 12 (P7 to P12). Then, the mice were removed from the oxygen treatment and treated with CB-12181 (1, 15, or 50 mg/kg) by daily subcutaneous injection from the time of reintroduction to room air at P12 until P16. At P17, pathological and physiological angiogenesis was quantified using retinal flat-mounts visualized by fluorescent angiography. In the in vitro angiogenesis model, CB-12181 significantly suppressed VEGF-induced HUVEC tube formation. Furthermore, in the in vivo angiogenesis model, administration of CB-12181 significantly suppressed retinal neovascularization without any apparent side effects on physiological revascularization to the oxygen-induced obliteration area. These results suggest that CB-12181 might be useful in the treatment of various diseases that depend on pathologic angiogenesis, and especially valuable for the treatment of diabetic retinopathy and retinopathy of prematurity.