VEGF-stimulated Human Umbilical Vein Endothelial Cells Research Articles

CPD-002, a novel VEGFR2 inhibitor, relieves rheumatoid arthritis by reducing angiogenesis through the suppression of the VEGFR2/PI3K/AKT signaling pathway

Synovial angiogenesis is a key player in the development of rheumatoid arthritis (RA), and anti-angiogenic therapy is considered a promising approach for treating RA. CPD-002 has demonstrated efficacy in suppressing tumor angiogenesis as a VEGFR2 inhibitor, but its specific impacts on RA synovial angiogenesis and possible anti-RA effects need further study. We examined the influences of CPD-002 on the migration and invasion of human umbilical vein endothelial cells (HUVECs) and its impacts on HUVECs' tube formation and vessel sprouting ex vivo. The therapeutic potential of CPD-002 in adjuvant-induced arthritis (AIA) rats and its suppression of synovial angiogenesis were examined. The involvement of the VEGFR2/PI3K/AKT pathway was assessed both in HUVECs and AIA rat synovium. Here, CPD-002 inhibited the migration and invasion of VEGF-stimulated HUVECs, decreased their chemotactic response to RA fibroblast-like synoviocyte-released chemoattractants, and exhibited anti-angiogenic effects in vitro and ex vivo. CPD-002′s targeting of VEGFR2 was confirmed with molecular docking and cellular thermal shift assays, supported by the abolishment of CPD-002′s effects upon using VEGFR2 siRNA. CPD-002 relieved paw swelling, arthritis index, joint damage, and synovial angiogenesis, indicating its anti-arthritic and anti-angiogenic effects in AIA rats. Moreover, the anti-inflammatory effects in vivo and in vitro of CPD-002 contributed to its anti-angiogenic effects. Mechanistically, CPD-002 hindered the activation of VEGFR2/PI3K/AKT pathway in VEGF-induced HUVECs and AIA rat synovium, as evidenced by reduced p-VEGFR2, p-PI3K, and p-AKT protein levels alongside elevated PTEN protein levels. Totally, CPD-002 showed anti-rheumatoid effects via attenuating angiogenesis through the inhibition of the VEGFR2/PI3K/AKT pathway.

E7050 Suppresses the Growth of Multidrug-Resistant Human Uterine Sarcoma by Inhibiting Angiogenesis via Targeting of VEGFR2-Mediated Signaling Pathways.

E7050 is an inhibitor of VEGFR2 with anti-tumor activity; however, its therapeutic mechanism remains incompletely understood. In the present study, we aim to evaluate the anti-angiogenic activity of E7050 in vitro and in vivo and define the underlying molecular mechanism. It was observed that treatment with E7050 markedly inhibited proliferation, migration, and capillary-like tube formation in cultured human umbilical vein endothelial cells (HUVECs). E7050 exposure in the chick embryo chorioallantoic membrane (CAM) also reduced the amount of neovessel formation in chick embryos. To understand the molecular basis, E7050 was found to suppress the phosphorylation of VEGFR2 and its downstream signaling pathway components, including PLCγ1, FAK, Src, Akt, JNK, and p38 MAPK in VEGF-stimulated HUVECs. Moreover, E7050 suppressed the phosphorylation of VEGFR2, FAK, Src, Akt, JNK, and p38 MAPK in HUVECs exposed to MES-SA/Dx5 cells-derived conditioned medium (CM). The multidrug-resistant human uterine sarcoma xenograft study revealed that E7050 significantly attenuated the growth of MES-SA/Dx5 tumor xenografts, which was associated with inhibition of tumor angiogenesis. E7050 treatment also decreased the expression of CD31 and p-VEGFR2 in MES-SA/Dx5 tumor tissue sections in comparison with the vehicle control. Collectively, E7050 may serve as a potential agent for the treatment of cancer and angiogenesis-related disorders.

Transcription factor cellular promoter 2 is required for upstream binding protein 1 -mediated angiogenesis

ObjectiveAngiogenesis is a key process of repairing tissue damage, and it is regulated by the delicate balance between anti-angiogenesis factors. In the present study, we investigate whether transcription factor cellular promoter 2 (TFCP2) is required for upstream binding protein 1 (UBP1)-mediated angiogenesis. MethodsLevels of UBP1 and TFCP2 in human umbilical vein endothelial cells (HUVECs) are detected by quantitative polymerase chain reaction (q-PCR) and Western blotting (WB). Effects of UBP1 on angiogenesis and migration are detected by tube-like network formation on matrigel assay and scratch assay. The interaction between UBP1 and TFCP2 is predicted and verified by STRING and Co-immunoprecipitation (Co-IP). ResultsFirstly, the UBP1 expression level was up-regulated in the stimuli of vascular endothelial growth factor (VEGF) in HUVECs, and the knockdown of UBP1 inhibited angiogenesis and migration of HUVECs. Then, UBP1 interacted with TFCP2. Besides, the TFCP2 expression level was up-regulated in VEGF-stimulated HUVECs. Furthermore, knockdown of TFCP2 inhibited angiogenesis and migration in VEGF-stimulated HUVECs, and down-regulation of UBP1 enhanced the inhibition. ConclusionTFCP2 also plays a key role in UBP1 mediated angiogenesis of HUVECs stimulated by VEGF. These findings will provide a new theoretical basis for the treatment of angiogenic diseases.

Platycodin D Inhibits Vascular Endothelial Growth Factor-Induced Angiogenesis by Blocking the Activation of Mitogen-Activated Protein Kinases and the Production of Interleukin-8.

Platycodin D is a major constituent in the root of Platycodon grandiflorum and has diverse pharmacologic activities, including anti-inflammatory, anti-allergic, and antitumor activities. Vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) are potent angiogenic factors and contribute to tumor angiogenesis by directly and indirectly promoting angiogenic processes, including the proliferation, adhesion, migration, and tube formation of endothelial cells. Here, we found that platycodin D at noncytotoxic concentrations inhibited VEGF-induced proliferation, adhesion to the extracellular matrix proteins fibronectin and vitronectin, chemotactic motility, and tube formation of human umbilical vein endothelial cells (HUVECs). Platycodin D reduced the phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) and the secretion of IL-8 in VEGF-stimulated HUVECs. Moreover, platycodin D inhibited tube formation and the phosphorylation of ERK and p38 in IL-8-stimulated HUVECs. The in vitro anti-angiogenic activity of platycodin D was confirmed by in vivo experimental models. Platycodin D inhibited the formation of new blood vessels into mouse Matrigel plugs with VEGF or IL-8. In mice injected with MDA-MB-231 human breast cancer cells, orally administered platycodin D inhibited tumor growth, the number of CD34 [Formula: see text]vessels, and the expression of VEGF and IL-8. Taken together, platycodin D directly and indirectly prevents VEGF-induced and IL-8-induced angiogenesis by blocking the activation of mitogen-activated protein kinases (MAPKs). Platycodin D may be beneficial for the prevention or treatment of tumor angiogenesis and angiogenesis-related human diseases.

Antofine, a natural phenanthroindolizidine alkaloid, suppresses angiogenesis via regulation of AKT/mTOR and AMPK pathway in endothelial cells and endothelial progenitor cells derived from mouse embryonic stem cells

Although antofine, a natural phenanthroindolizidine alkaloid, exerts potential biological activities, including anticancer effect and anti-angiogenic activity, the underlying mechanisms have not yet been investigated. In the present study, the inhibitory effect of antofine on angiogenesis was determined in cultured mouse embryonic stem (mES)/embryoid body (EB)-derived endothelial cells and vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cells (HUVECs). Antofine effectively inhibited VEGF-induced cell migration and tube formation of HUVECs. Antofine also significantly decreased ex vivo microvessel sprouting in cultured mouse aortic rings, and inhibited the vascular formation and platelet/endothelial cell adhesion molecule (PECAM) expression of mES/EB-derived cells in 3-D collagen gel. The underlying mechanism of anti-angiogenic activity of antofine was, in part, associated with the modulation of AKT/mTOR and AMP-activated protein kinase (AMPK) signaling in VEGF-stimulated HUVECs.

Two natural eudesmane-type sesquiterpenes from Laggera alata inhibit angiogenesis and suppress breast cancer cell migration through VEGF- and Angiopoietin 2-mediated signaling pathways.

Eudesmane-type sesquiterpenes are natural sesquiterpenes with anti-inflammatory properties, but their anti-angiogenic activities are not known. The present study demonstrated that 5α-hydroxycostic acid and hydroxyisocostic acid, two eudesmane-type sesquiterpenes(ETSs), isolated from the herb Laggera alata, possessed anti-angiogenic effects. Under non-toxic dosage, ETSs suppressed VEGF‑induced proliferation in human umbilical vein endothelial cells(HUVECs) and vessel formation in zebrafish embryos. Moreover, ETSs inhibited VEGF-stimulated HUVEC migration, stress fibers and tube formation. Results from real‑time PCR analysis involving invivo and invitro experiments indicated that pro-angiogenic-related mRNA levels were downregulated, including VEGFA, VEGFR2 and Tie2 genes after ETS treatments. Western blot analysis showed that ETSs suppressed VEGF-stimulated VEGFR2 phosphorylation and activation of its downstream molecules, such as Src/AKT/eNOS, FAK, PLCγ/ERK1/2 and p38. Moreover, the VEGF-stimulation of angiopoietin2(Ang2) mRNA level increase was significantly downregulated in the presence of ETSs. ETSs inhibited Ang2-induced phosphorylation of the receptor Tie2 in HUVECs, which indicated that ETSs not just suppressed VEGF/VEGFR2 axis, but also the Ang2/Tie2 one. Furthermore, the wound-healing assay revealed that ETSs reduced the migration of Ang2-stimulated human breast cancer (MCF-7) cells. Mechanistically, the anti-migration effect of ETSs correlated with the blockade of Ang2-induced E-cadherin loss and AKT activation. Collectively, the present study suggests that ETSs possess anti-angiogenic ability by interfering the VEGF- and Ang2-related pathways, and they may be good drug candidates.

A Novel Clerodane Diterpene from Vitex cofassus.

New clerodane diterpene, 16-hydroxy-pentandralactone (1) and known diterpene acuminolide (2) were isolated from the methanol extract of Vitex cofassus leaves. The chemical structure and the absolute configuration of 1 were determined by MS, NMR and electron circular dichroism (ECD) experiments. The isolated compounds were evaluated for their antiproliferative activities against a panel of human tumor cell lines, including a multidrug-resistant (MDR) cell line. Both compounds showed potent antiproliferative activities against all the tested cell lines with IC50 values of 5.4-11.4 µM. Their effects on cell viability were also tested using vascular endothelial growth factor (VEGF)-stimulated human umbilical vein endothelial cells (HUVECs). Compound 1 inhibited VEGF-stimulated HUVEC proliferation in a dose-dependent manner. Based on these results, compound 1 could be a candidate for antitumor agent and inhibitor of angiogenesis.

Daphnetin inhibits TNF-α and VEGF-induced angiogenesis through inhibition of the IKKs/IκBα/NF-κB, Src/FAK/ERK1/2 and Akt signalling pathways.

Coumarins, identified as plant secondary metabolites possess diverse biological activities including anti-angiogenic properties. Daphnetin (DAP), a plant derived dihydroxylated derivative of coumarin has shown significant pharmacological properties such as anticancer, anti-arthritic and anti-inflammatory. The present study was performed to investigate the anti-angiogenic potential of DAP, focusing on the mechanism of action. The in vivo anti-angiogenic potential of DAP was evaluated by vascular endothelial growth factor (VEGF)-induced rat aortic ring (RAR) assay and chick chorioallantoic membrane (CAM) assay. For in vitro evaluation, wounding migration, transwell invasion, tube formation and apoptosis assays were performed on VEGF (8ng/mL)-induced human umbilical vein endothelial cells (HUVECs). The cellular mechanism of DAP was examined on TNFα (10ng/mL) and VEGF-induced HUVECs by extracting the mRNA and protein levels using RT-qPCR and western blotting. Our data demonstrated that DAP inhibited the in vivo angiogenesis in the RAR and CAM assay. DAP also inhibited the different steps of angiogenesis, such as migration, invasion, and tube formation in HUVECs. DAP inhibited nuclear factor-κB signalling together including TNF-α induced IκBα degradation; phosphorylation of IκB kinase (IKKα/β) and translocation of the NF-κB-p65 protein. Furthermore, western blotting revealed that DAP significantly down-regulated the VEGF-induced signalling such as c-Src, FAK, ERK1/2 and the related phosphorylation of protein kinase B (Akt) and VEGFR2 expressions. DAP reduced the elevated mRNA expression of iNOS, MMP2 and also, induced apoptosis in VEGF-stimulated HUVECs by the caspase-3 dependent pathway. Taken together, this study reveals that DAP may have novel prospective as a new multi-targeted medication for the anti-angiogenesis and cancer therapy.

Identification of a residue crucial for the angiostatic activity of human mini tryptophanyl-tRNA synthetase by focusing on its molecular evolution.

Human tryptophanyl-tRNA synthetase (TrpRS) exists in two forms: a full-length TrpRS and a mini TrpRS. We previously found that human mini, but not full-length, TrpRS is an angiostatic factor. Moreover, it was shown that the interaction between mini TrpRS and the extracellular domain of vascular endothelial (VE)-cadherin is crucial for its angiostatic activity. However, the molecular mechanism of the angiostatic activity of human mini TrpRS is only partly understood. In the present study, we investigated the effects of truncated (mini) form of TrpRS proteins from human, bovine, or zebrafish on vascular endothelial growth factor (VEGF)-stimulated chemotaxis of human umbilical vein endothelial cells (HUVECs). We show that both human and bovine mini TrpRSs inhibited VEGF-induced endothelial migration, whereas zebrafish mini TrpRS did not. Next, to identify residues crucial for the angiostatic activity of human mini TrpRS, we prepared several site-directed mutants based on amino acid sequence alignments among TrpRSs from various species and demonstrated that a human mini K153Q TrpRS mutant cannot inhibit VEGF-stimulated HUVEC migration and cannot bind to the extracellular domain of VE-cadherin. Taken together, we conclude that the Lys153 residue of human mini TrpRS is a VE-cadherin binding site and is therefore crucial for its angiostatic activity.

BET Bromodomain Suppression Inhibits VEGF-induced Angiogenesis and Vascular Permeability by Blocking VEGFR2-mediated Activation of PAK1 and eNOS.

The tyrosine kinase receptor vascular endothelial growth factor receptor 2 (VEGFR2) is a critical modulator of angiogenesis. Increasing evidence indicate the important role of bromodomain and extra-terminal domain (BET) of chromatin adaptors in regulating tumor growth and inflammatory response. However, whether BET proteins have a role in angiogenesis and endothelial permeability is unclear. In this study, we observed that treatment with JQ1, a specific BET inhibitor, suppressed in vitro tube formation of human umbilical vein endothelial cells (HUVECs) and in vivo angiogenesis in a Matrigel plug and oxygen-induced retinopathy neovascularization. JQ1 attenuated the VEGF-induced decrease in TEER in HUVECs and prevented Evans blue dye leakage in the VEGF-induced Miles assay in athymic Balb/c nude mice. BET inhibition with JQ1 or shRNA for Brd2 or Brd4 suppressed VEGF-induced migration, proliferation, and stress fiber formation of HUVECs. Furthermore, BET inhibition suppressed phosphorylation of VEGFR2 and PAK1, as well as eNOS activation in VEGF-stimulated HUVECs. Inhibition with VEGFR2 and PAK1 also reduced migration and proliferation, and attenuated the VEGF-induced decrease in TEER. Thus, our observations suggest the important role of BET bromodomain in regulating VEGF-induced angiogenesis. Strategies that target the BET bromodomain may provide a new therapeutic approach for angiogenesis-related diseases.

Clopidogrel inhibits angiogenesis of gastric ulcer healing via downregulation of vascular endothelial growth factor receptor 2.

Although clopidogrel does not cause gastric mucosal injury, it does not prevent peptic ulcer recurrence in high-risk patients. We explored whether clopidogrel delays gastric ulcer healing via inhibiting angiogenesis and to elucidate the possible mechanisms. Gastric ulcers were induced in Sprague Dawley rats, and ulcer healing and angiogenesis of ulcer margin were compared between clopidogrel-treated rats and controls. The expressions of the proangiogenic growth factors and their receptors including basic fibroblast growth factor (bFGF), bFGF receptor (FGFR), vascular endothelial growth factor (VEGF), VEGFR1, VEGFR2, platelet-derived growth factor (PDGF)A, PDGFB, PDGFR A, PDGFR B, and phosphorylated form of mitogenic activated protein kinase pathways over the ulcer margin were compared via western blot and reverse transcription polymerase chain reaction. Invitro, human umbilical vein endothelial cells (HUVECs) were used to elucidate how clopidogrel inhibited growth factors-stimulated HUVEC proliferation. The ulcer sizes were significantly larger and the angiogenesis of ulcer margin was significantly diminished in the clopidogrel (2 and 10mg/kg/d) treated groups. Ulcer induction markedly increased the expression of phosphorylated form of extracellular signal-regulated kinase (pERK), FGFR2, VEGF, VEGFR2, and PDGFRA when compared with those of normal mucosa. Clopidogrel treatment significantly decreased pERK, FGFR2, VEGF, VEGFR2, and PDGFRA expression at the ulcer margin when compared with those of the respective control group. Invitro, clopidogrel (10(-6)M) inhibited VEGF-stimulated (20ng/mL) HUVEC proliferation, at least, via downregulation of VEGFR2 and pERK. Clopidogrel inhibits the angiogenesis of gastric ulcer healing at least partially by the inhibition of the VEGF-VEGFR2-ERK signal transduction pathway.

Orally Administered Mucolytic Drug l-Carbocisteine Inhibits Angiogenesis and Tumor Growth in Mice.

Angiogenesis, the formation of new blood vessels from pre-existing vessels, is essential for the growth and metastasis of tumors. In this study, we found that l-carbocisteine, a widely used expectorant, potently inhibits angiogenesis in vitro and in vivo. An in vivo Matrigel plug assay revealed that l-carbocisteine (2.5 mg/kg i.p. twice daily) significantly inhibited vascular endothelial growth factor (VEGF)-induced angiogenesis. l-Carbocisteine also suppressed VEGF-stimulated proliferation, migration, and formation of capillary-like structures of human umbilical vein endothelial cells (HUVECs). We examined the signaling pathways affected in VEGF-stimulated HUVECs, and found that l-carbocisteine significantly inhibited VEGF-induced phosphorylation of phospholipase C (PLC) γ, protein kinase C (PKC) μ, and extracellular signal-related kinases (ERK) 1/2, which have been shown to be essential for angiogenesis. However, these inhibitory effects of l-carbocisteine were not observed in the HeLa human cervical cancer cell line. An in vivo study of Colon-26 tumor-bearing mice found that tumor volumes were significantly smaller in mice treated with l-carbocisteine (150 mg/kg administered orally twice daily) in comparison with vehicle-treated mice. However, l-carbocisteine had no direct effect on Colon-26 cell proliferation or ERK activation. Collectively, our results suggest that l-carbocisteine inhibits tumor angiogenesis by suppressing PLCγ/PKC/ERK signaling.

Tumour growth inhibition and anti-angiogenic effects using curcumin correspond to combined PDE2 and PDE4 inhibition.

Vascular endothelial growth factor (VEGF) plays a major role in angiogenesis by stimulating endothelial cells. Increase in cyclic AMP (cAMP) level inhibits VEGF-induced endothelial cell proliferation and migration. Cyclic nucleotide phosphodiesterases (PDEs), which specifically hydrolyse cyclic nucleotides, are critical in the regulation of this signal transduction. We have previously reported that PDE2 and PDE4 up-regulations in human umbilical vein endothelial cells (HUVECs) are implicated in VEGF-induced angiogenesis and that inhibition of PDE2 and PDE4 activities prevents the development of the in vitro angiogenesis by increasing cAMP level, as well as the in vivo chicken embryo angiogenesis. We have also shown that polyphenols are able to inhibit PDEs. The curcumin having anti-cancer properties, the present study investigated whether PDE2 and PDE4 inhibitors and curcumin could have similar in vivo anti-tumour properties and whether the anti-angiogenic effects of curcumin are mediated by PDEs. Both PDE2/PDE4 inhibitor association and curcumin significantly inhibited in vivo tumour growth in C57BL/6N mice. In vitro, curcumin inhibited basal and VEGF-stimulated HUVEC proliferation and migration and delayed cell cycle progression at G0/G1, similarly to the combination of selective PDE2 and PDE4 inhibitors. cAMP levels in HUVECs were significantly increased by curcumin, similarly to rolipram (PDE4 inhibitor) and BAY-60-550 (PDE2 inhibitor) association, indicating cAMP-PDE inhibitions. Moreover, curcumin was able to inhibit VEGF-induced cAMP-PDE activity without acting on cGMP-PDE activity and to modulate PDE2 and PDE4 expressions in HUVECs. The present results suggest that curcumin exerts its in vitro anti-angiogenic and in vivo anti-tumour properties through combined PDE2 and PDE4 inhibition.

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Objectives Sulfasalazine is an anti-inflammatory and immune modulating drug. Its mode of action has remained elusive, however recent evidence suggests it induces potent antioxidant enzyme heme-oxygenase1 (HO1) via nuclear erythroid-2-related factor 2 (Nrf2). Given its anti-oxidant capacity and that it is safe in pregnancy, we examined the potential for sulfasalazine as a novel therapeutic for preeclampsia. In particular, we examined its ability to quench anti-angiogenic factors sFlt1 and soluble endoglin (sEng) and reverse endothelial dysfunction in vitro. Methods Increasing doses of sulfasalazine were administered to primary human umbilical vein endothelial cells (HUVECs) and sFlt1 and sEng release assessed. To induce endothelial dysfunction, HUVECs were treated with TNF α and the effect of sulfasalazine on monocyte adhesion determined. Finally the effect of sulfasalazine on HUVEC migration and proliferation was assessed. Results Excitingly, we observed a significant dose dependent reduction in both sFlt1 and sEng release. Importantly, mRNA expression of newly described human and placental specific variant sFlt1-e15a mRNA was significantly decreased, as was MMP14 mRNA expression (MMP14 is the protease that produces sEng). As expected, sulfasalazine also significantly increased mRNA expression of HO-1. Treatment of HUVECs with TNF α induced significant upregulation of VCAM which was potently reversed by Sulfasalazine, indicating its ability to quench endothelial dysfunction. Furthermore, sulfalsazine significantly reduced monocyte adhesion to HUVECs treated with TNF α , again supporting its anti-inflammatory properties. Finally we demonstrated that sulfasalazine could reverse the negative effects of sFlt1 on VEGF-stimulated HUVEC migration, and enhance HUVEC proliferation. Conclusions Sulfasalazine is a novel agent, safe in pregnancy that significantly quenches sFlt1 and sEng release from human endothelial cells. In addition, it significantly reduces endothelial dysfunction and enhances endothelial cell migration and proliferation. This provides strong evidence to suggest that sulfasalazine may be able to quench the endothelial dysfunction of preeclampsia and could be an effective treatment for preeclampsia. Disclosures F.C. Brownfoot: None. S. Tong: None. N. Hannan: None. R. Hastie: None. P. Cannon: None. T.J. Kaitu’u-Lino: None.

Abstract 117: RhoC Regulates VEGF-induced Signaling in Endothelial Cells

Vascular permeability factor/vascular endothelial growth factor A (VEGF) is a central regulator of angiogenesis and potently promotes vascular permeability. VEGF plays a key role in the pathologies of heart disease, stroke, and cancer. Therefore, understanding the molecular regulation of VEGF signaling is an important pursuit. Rho GTPase proteins play various roles in vasculogenesis and angiogenesis. While the functions of RhoA and RhoB in these processes have been well defined, little is known about the role of RhoC in VEGF-mediated signaling in endothelial cells and vascular development. Here, we describe how RhoC modulates VEGF signaling to regulate endothelial cell proliferation, migration and permeability. We found VEGF stimulation activates RhoC in human umbilical vein endothelial cells (HUVECs), which was completely blocked after VEGF receptor 2 (VEGFR-2) knockdown indicating that VEGF activates RhoC through VEGFR-2 signaling. Interestingly, RhoC knockdown delayed the degradation of VEGFR-2 compared to control siRNA treated HUVECs, thus implicating RhoC in VEGFR-2 trafficking. In light of our results suggesting VEGF activates RhoC through VEGFR-2, we sought to determine whether RhoC regulates vascular permeability through the VEGFR-2/phospholipase Cγ (PLCγ) /Ca 2+ /eNOS cascade. We found RhoC knockdown in VEGF-stimulated HUVECs significantly increased PLC-γ1 phosphorylation at tyrosine 783, promoted basal and VEGF-stimulated eNOS phophorylation at serine 1177, and increased calcium flux compared with control siRNA transfected HUVECs. Taken together, our findings suggest RhoC negatively regulates VEGF-induced vascular permeability. We confirmed this finding through a VEGF-inducible zebrafish model of vascular permeability by observing significantly greater vascular permeability in RhoC morpholino (MO)-injected zebrafish than control MO-injected zebrafish. Furthermore, we showed that RhoC promotes endothelial cell proliferation and negatively regulates endothelial cell migration. Our data suggests a scenario in which RhoC promotes proliferation by upregulating -catenin in a Wnt signaling-independent manner, which in turn, promotes Cyclin D1 expression and subsequently drives cell cycle progression.

TRAIL negatively regulates VEGF-induced angiogenesis via caspase-8-mediated enzymatic and non-enzymatic functions

Solid tumors supply oxygen and nutrients required for angiogenesis by producing vascular endothelial growth factor (VEGF). Thus, inhibitors of VEGF signaling abrogate tumor angiogenesis, resulting in the suppression of tumor growth and metastasis. We here investigated the effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on VEGF-induced angiogenesis. TRAIL inhibited VEGF-induced in vitro angiogenesis of human umbilical vein endothelial cells (HUVECs) and in vivo neovascularization in chicken embryos and mice. TRAIL blocked VEGF-induced angiogenic signaling by inhibiting ERK, Src, FAK, paxillin, Akt, and eNOS. Further, TRAIL blocked intracellular Ca(2+) elevation and actin reorganization in HUVECs stimulated with VEGF, without inhibiting VEGF receptor-2 tyrosine phosphorylation. TRAIL increased caspase-8 activity, without inducing caspase-9/-3 activation and apoptosis. Moreover, TRAIL resulted in cleavage of FAK into FAK-related non-kinase-like fragments in VEGF-stimulated HUVECs, which was blocked by a caspase-8 inhibitor and cellular caspase-8-like inhibitory protein. Biochemical and pharmacological inhibition of caspase-8 and FAK blocked the inhibitory effects of TRAIL on VEGF-stimulated anti-angiogenic signaling and events. In addition, caspase-8 knockdown also suppressed VEGF-mediated signaling and angiogenesis, suggesting that procaspase-8 plays a role of a non-apoptotic modulator in VEGF-induced angiogenic signaling. These results suggest that TRAIL inhibits VEGF-induced angiogenesis by increasing caspase-8 activity and subsequently decreasing non-apoptotic signaling functions of procaspase-8, without inducing caspase-3 activation and endothelial cell cytotoxicity. These data indicate that caspase-8 may be used as an anti-angiogenic drug for solid tumors resistant to TRAIL and anti-tumor drugs.

Mechanisms in endovascular differentiation of the trophoblast and its regulation by decorin

Although antofine, a natural phenanthroindolizidine alkaloid, exerts potential biological activities, including anticancer effect and anti-angiogenic activity, the underlying mechanisms have not yet been investigated. In the present study, the inhibitory effect of antofine on angiogenesis was determined in cultured mouse embryonic stem (mES)/embryoid body (EB)-derived endothelial cells and vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cells (HUVECs). Antofine effectively inhibited VEGF-induced cell migration and tube formation of HUVECs. Antofine also significantly decreased ex vivo microvessel sprouting in cultured mouse aortic rings, and inhibited the vascular formation and platelet/endothelial cell adhesion molecule (PECAM) expression of mES/EB-derived cells in 3-D collagen gel. The underlying mechanism of anti-angiogenic activity of antofine was, in part, associated with the modulation of AKT/mTOR and AMP-activated protein kinase (AMPK) signaling in VEGF-stimulated HUVECs.

H-RN, a peptide derived from hepatocyte growth factor, inhibits corneal neovascularization by inducing endothelial apoptosis and arresting the cell cycle.

BackgroundThe goal of this study was to investigate the anti-angiogenic activity of a novel peptide H-RN, derived from the hepatocyte growth factor kringle 1 domain (HGF K1), in a mouse model of corneal neovascularization. The anti-angiogenic effect of H-RN on vascular endothelial growth factor (VEGF)-stimulated cell proliferation, cell migration and endothelial cell tube formation was assessed in vitro using Human Umbilical Vein Endothelial Cells (HUVECs) and in vivo using a mouse cornea micropocket assay. Apoptosis and cell cycle arrest were assessed by flow cytometry. A scrambled peptide was used as a negative control.ResultsH-RN effectively inhibited VEGF-stimulated HUVEC proliferation, migration and tube formation on Matrigel, while a scrambled peptide exerted no effect. In the mouse model of corneal angiogenesis, VEGF-stimulated angiogenesis was significantly inhibited by H-RN compared to a scrambled peptide that had no such activity. VEGF protected HUVECs from apoptosis, while H-RN inhibited this protective effect of VEGF. VEGF significantly increased the proportion of cells in the S phase compared to control treated cells (p<0.05). Treatment with H-RN (1.5 mM) induced the accumulation of cells in G0/G1 phase, while the proportion of cells in the S phase and G2/M phase decreased significantly compared to control group (p<0.05).ConclusionsH-RN has anti-angiogenic activity in HUVECs and in a mouse model of VEGF-induced corneal neovascularization. The anti-angiogenic activity of H-RN was related to apoptosis and cell cycle arrest, indicating a potential strategy for anti-angiogenic treatment in the cornea.

Interleukin-6 promotes destabilized angiogenesis by modulating angiopoietin expression in rheumatoid arthritis

To examine whether IL-6 promotes angiogenesis by modulating angiopoietin (Ang) expression in RA. Synovial fibroblasts derived from RA patients (RASFs) and human umbilical vein endothelial cells (HUVECs) were co-cultured for 6 days with or without recombinant IL-6, VEGF or Ang-1. HUVECs were stained with anti-CD31 antibody and their growth was determined by quantifying the CD31-positive area. SFs were collected from RA (n = 25) and OA (n = 7) patients. In the co-culture system, IL-6 and VEGF significantly enhanced HUVEC growth to a similar extent. However, the morphology of proliferating cells was distinct between IL-6- and VEGF-stimulated HUVEC. HUVEC stimulated with IL-6 exhibited small, loose clusters surrounded by dispersed single cells, suggesting destabilized angiogenesis by IL-6. In the supernatants, IL-6 up-regulated VEGF compared with controls and Ang-2, while it down-regulated Ang-1. In contrast, down-regulation of Ang-1 was not observed with VEGF stimulation. Consistent with the destabilized morphology, stimulation with IL-6 decreased cell surface expression of vascular endothelial cadherin (VE-cadherin) on HUVEC, presumably by inducing internalization. Interestingly, adding recombinant Ang-1 partially inhibited IL-6-induced morphological changes in HUVEC including a destabilized morphology with small, loose clusters and internalization of VE-cadherin. In SFs from RA patients, VEGF was negatively correlated with Ang-1 (r = -0.559, P=0.004). IL-6 not only enhances VEGF expression but also inhibits Ang-1 signalling by directly down-regulating Ang-1 expression and up-regulating Ang-2, an antagonist of Ang-1. These synergistic effects may play a critical role in destabilized angiogenesis in RA.

Potent inhibition of angiogenesis by the IGF-1 receptor-targeting antibody SCH717454 is reversed by IGF-2.

Previously, we reported that a predominant action of a type-1 insulin-like growth factor receptor (IGF-1R)-targeted antibody was through inhibiting tumor-derived VEGF, and indirectly, angiogenesis. Here, we examined the direct antiangiogenic activity of the IGF-1R-targeted antibody SCH717454 that inhibits ligand-receptor binding and the mechanism by which tumors circumvent its antiangiogenic activity. Inhibition of ligand-stimulated activation of IGF-1R, insulin receptor (IN-R), or downstream signaling [phosphorylation of Akt (Ser473)] was determined by receptor-specific immunoprecipitation and immunoblotting. Inhibition of angiogenesis was determined by proliferation and tube formation using human umbilical vein endothelial cells (HUVEC) in vitro and in Matrigel plugs implanted in mice. SCH717454 blocked IGF-1-stimulated but not IGF-2-stimulated phosphorylation of Akt in sarcoma cells. Immunoprecipitation using anti-IGF-1R and anti-IN-R antibodies revealed that SCH717454 equally blocked IGF-1-stimulated and IGF-2-stimulated IGF-1R phosphorylation, but not IGF-2-stimulated phosphorylation of IN-R. SCH717454 completely blocked VEGF-stimulated proliferation and tube formation of HUVECs, but exogenous IGF-2 and insulin circumvented these inhibitory effects. Coculture of HUVECs with IGF-2-secreting tumor cells completely abrogated SCH717454 inhibition of VEGF-stimulated HUVEC tube formation. In mice, SCH717454 inhibited angiogenesis in VEGF-infused Matrigel plugs, but had no inhibitory activity when plugs contained both VEGF + IGF-2. These results reveal for the first time, a role for IGF-1R signaling in VEGF-mediated angiogenesis in vitro and indicate direct antiangiogenic activity of SCH717454. Both in vitro and in vivo IGF-2 circumvented these effects through IN-R signaling. Many childhood cancers secrete IGF-2, suggesting that tumor-derived IGF-2 in the microenvironment maintains angiogenesis in the presence of IGF-1R-targeted antibodies allowing tumor progression.