A novel Src kinase inhibitor, M475271, inhibits VEGF-induced human umbilical vein endothelial cell proliferation and migration

Vascular endothelial growth factor (VEGF) was reported to be a potent proangiogenic factor that plays a pivotal role in both physiological and pathological angiogenesis. M475271, 4-quinazolinamine, N-(2-chloro-5-methoxyphenyl)-6-methoxy-7-[(1-methyl-4-piperidinyl) methoxy]-(9Cl), is a new anilinoquinazoline derivative that showed selective inhibition of Src kinase activity and tumor growth in vivo. Here, we examined the effect of M475271 on VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation and migration and their intracellular mechanisms. Our findings showed that M475271 pretreatment resulted in a significant inhibition of VEGF-induced HUVEC proliferation, [(3)H]thymidine incorporation, and migration. M475271 inhibited VEGF-induced Flk-1 and Src phosphorylation and their association. Confocal laser microscopic examination confirmed the inhibitory effect of M475271 on VEGF-induced Flk-1/Src association. M475271 inhibited VEGF-induced extracellular signal-regulated kinase1/2 (ERK1/2) and p38 but not Akt activation in a concentration-dependent manner. M475271, PI3-K inhibitor, and p38 inhibitor inhibited VEGF-induced HUVEC proliferation and migration. However, a MEK1/2 inhibitor inhibited VEGF-induced proliferation but not migration. These findings suggest that M475271 attenuates VEGF-induced HUVEC proliferation and migration through the inhibition of signaling pathways involving Src, ERK1/2, and/or p38. Taken together, these data indicate that M475271 may be a useful candidate for inhibition of endothelial cell proliferation and migration relevant to angiogenesis.     

Beta-eudesmol suppresses tumour growth through inhibition of tumour neovascularisation and tumour cell proliferation

In the present study, we investigated the potential anti-angiogenic mechanism and anti-tumour activity of beta-eudesmol using in vitro and in vivo experimental models. Proliferation of human umbilical vein endothelial cells (HUVEC) stimulated with vascular endothelial growth factor (VEGF, 30 ng/ml) and basic fibroblast growth factor (bFGF, 30 ng/ml) was significantly inhibited by beta-eudesmol (50-100 microM). Beta-eudesmol (100 microM) also blocked the phosphorylation of cAMP response element binding protein (CREB) induced by VEGF (30 ng/ml) in HUVEC. Beta-eudesmol (10-100 microM) inhibited proliferation of HeLa, SGC-7901, and BEL-7402 tumour cells in a time- and dose-dependent manner. Moreover, beta-eudesmol treatment (2.5-5 mg/kg) significantly inhibited growth of H(22) and S(180) mouse tumour in vivo. These results indicated that beta-eudesmol inhibited angiogenesis by suppressing CREB activation in growth factor signalling pathway. This is the first study to demonstrate that beta-eudesmol is an inhibitor of tumour growth.     

The microtubule binding drug laulimalide inhibits vascular endothelial growth factor-induced human endothelial cell migration and is synergistic when combined with docetaxel (taxotere)

Laulimalide, a natural product from marine sponges, is a microtubule-stabilizing agent that binds to tubulin at a site distinct from that of the taxoids. In the present study, we found that laulimalide inhibited human umbilical vein endothelial cell (HUVEC) tubule formation and vascular endothelial growth factor (VEGF)-induced HUVEC migration, key components of the angiogenic process. These occurred at concentrations substantially lower than that which inhibited HUVEC proliferation. When combined, laulimalide and docetaxel (Taxotere) synergistically inhibited migration and tubule formation, but their combined effect on proliferation was antagonistic. Possible mechanism(s) by which laulimalide inhibited VEGF-induced HUVEC migration were explored. Similar to docetaxel, laulimalide had no effect on the VEGF-induced tyrosine phosphorylation of the VEGF receptor Flk-1/KDR (VEGFR-2). Low concentrations of laulimalide substantially blocked subsequent VEGFR-2 downstream events, as did docetaxel, including the phosphorylation of the Tyr397 and Tyr407 residues of focal adhesion kinase (FAK), the association of VEGFR-2 with FAK and Hsp90, and the Tyr31 phosphorylation of paxillin. Laulimalide inhibited integrin activation; however, compared with docetaxel, it had a weaker inhibitory effect on the VEGF-induced association of VEGFR-2 with the alpha5beta1 integrin. Compared with docetaxel, laulimalide more potently caused a reduction in the constitutive levels (i.e., in the absence of VEGF) of phosphorylated paxillin and more potently inhibited the association of RhoA with the alpha5beta1 integrin. In conclusion, although both docetaxel and laulimalide inhibited integrin-associated signaling pathways that mediated VEGF-induced cell migration, their actions on the signaling cascade seemed not to be identical. These complementary actions could account for their synergistic effects on HUVEC.     

Inhibition of vascular endothelial growth factor-induced angiogenesis by resveratrol through interruption of Src-dependent vascular endothelial cadherin tyrosine phosphorylation

Resveratrol, a polyphenolic compound found in grapes and other fruits, has been reported to inhibit angiogenesis with an as yet elusive mechanism. Here, we investigate the detailed mechanism by which resveratrol inhibits vascular endothelial growth factor (VEGF)-induced angiogenic effects in human umbilical endothelial cells (HUVECs). Exposure of HUVECs to 1 to 2.5 muM resveratrol significantly blocked VEGF-mediated migration and tube formation but not cell proliferation. Under the same concentrations, resveratrol failed to affect VEGF-stimulated activation of VEGF receptor, extracellular signal-regulated protein kinase 1/2, p38 mitogen-activated protein kinase, and Akt. Of interest, resveratrol, at the dose of 1 or 2.5 muM, effectively abrogated VEGF-mediated tyrosine phosphorylation of vascular endothelial (VE)-cadherin and its complex partner, beta-catenin. This inhibitory effect of resveratrol reflected on the retention of VE-cadherin at cell-cell contacts as demonstrated by immunofluorescence. Src kinase assay showed that VEGF-induced endogenous Src kinase activation was strongly inhibited by 1 and 2.5 muM resveratrol. Supportively, inhibition of Src activity by overexpression of Csk resulted in attenuation of the tyrosine phosphorylation of VE-cadherin and endothelial cell (EC) tube formation. Again, transfection with v-Src, an active form of Src, could reverse resveratrol inhibition of VE-cadherin tyrosine phosphorylation and EC tube formation. Reactive oxygen species (ROS) has been shown to be involved in VE-cadherin phosphorylation and its related functions. Flow cytometric analysis showed that VEGF stimulated an evident increase of peroxide, which was strongly attenuated by resveratrol. In addition, antioxidant N-acetyl-cysteine was demonstrated to strongly inhibit VEGF-mediated Src activation, VE-cadherin tyrosine phosphorylation, and HUVEC tube formation. Together, our data suggest that resveratrol inhibition of VEGF-induced angiogenesis was mediated by disruption of ROS-dependent Src kinase activation and the subsequent VE-cadherin tyrosine phosphorylation.     

The angiosuppressive effects of 20(R)- ginsenoside Rg3

Aberrant angiogenesis is an essential step for the progression of solid tumors. Thus anti-angiogenic therapy is one of the most promising approaches to control tumor growth. In this study, we examined the ability of 20(R)-ginsenoside Rg3 (Rg3), one of the active compounds present in ginseng root, to interfere with the various steps of angiogenesis. Rg3 was found to inhibit the proliferation of human umbilical vein endothelial cells (HUVEC) with an IC50 of 10 nM in Trypan blue exclusion assay. Rg3 (1-10(3) nM) also dose dependently suppressed the capillary tube formation of HUVEC on the Matrigel in the presence or absence of 20 ng/ml vascular endothelial growth factor (VEGF). The VEGF-induced chemoinvasion of HUVEC and ex vivo microvascular sprouting in rat aortic ring assay were both significantly attenuated by Rg3. In addition, Rg3 (150 and 600 nM) remarkably abolished the basic fibroblast growth factor (bFGF)-induced angiogenesis in an in vivo Matrigel plug assay. The Matrix metalloproteinases (MMPs), such as MMP-2 and MMP-9, which play an important role in the degradation of basement membrane in angiogenesis and tumor metastasis present in the culture supernatant of Rg3-treated aortic ring culture were found to decrease in their gelatinolytic activities. Taken together, these data underpin the anti-tumor property of Rg3 through its angiosuppressive activity.     

The novel Src kinase inhibitor M475271 inhibits VEGF-induced vascular endothelial-cadherin and beta-catenin phosphorylation but increases their association

M475271, 4-quinazolinamine, N-(2-chloro-5-methoxyphenyl)-6-methoxy-7-[(1-methyl-4-piperidinyl) methoxy]-(9Cl), is a new anilinoquinazoline derivative that displays selective inhibition of Src kinase activity and tumor growth in vivo. Vascular endothelial growth factor (VEGF)-induced angiogenesis plays a pivotal role in tumor growth and metastasis. Vascular endothelial (VE)-cadherin is an endothelial cell-specific adhesion molecule that can interact with the cytoskeleton via several anchoring molecules such as beta-catenin. Here, we examined the effect of M475271 on VE-cadherin and beta-catenin phosphorylation and association. We also examined its effect on VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation, migration, and tube formation. The findings reveal pretreatment with M475271 significantly inhibits VEGF-induced VE-cadherin and beta-catenin phosphorylation. However, M475271 significantly increases VE-cadherin and beta-catenin association compared to the VEGF-treated group. Confocal laser microscopic examination confirmed the augmentation effect of M475271 on VE-cadherin and beta-catenin association. Finally, M475271 was shown to have inhibitory effects comparable to those of PP2 and Herbimycin A on VEGF-induced HUVEC proliferation, migration, and tube formation. These findings suggest that M475271 attenuates VEGF-induced angiogenesis by maintaining cell-cell junction stability. Although the involvement of other signaling molecules cannot be ruled out, M475271 has potential as a drug for the inhibition of the angiogenesis needed for tumor growth and metastasis.     

槲皮素抑制血管生成作用的实验研究

目的　研究槲皮素 (Quercetin)对血管生成和培养的人脐静脉内皮细胞 (HUVEC)的影响。方法　采用生长因子 (血管内皮细胞生长因子VEGF、碱性成纤维细胞生长因子bFGF)诱导的鸡胚绒毛尿囊膜 (CAM)血管增生模型观察槲皮素对血管生成的影响 ;利用培养的HUVEC ,用MTT法观察槲皮素抑制内皮细胞增殖的作用 ;流式细胞仪观察槲皮素对HUVEC细胞周期的影响。结果　槲皮素 (0 1、0 0 5和 0 0 2 5mmol·L-1)能明显抑制VEGF诱导的CAM小血管生成 ;槲皮素 (0 1和 0 0 5mmol·L-1)能明显抑制bFGF诱导的CAM小血管生成 ;槲皮素 (2 4 0、12 0 μmol·L-1和 6 0 μmol·L-1)对内皮细胞增殖有抑制作用 ,抑制率分别为 6 7 0 %、5 8 1%和39 7% ;槲皮素 (2 4 0、12 0 μmol·L-1)能显著导致HUVEC的S、G2 期阻滞。结论　槲皮素能抑制VEGF和bFGF诱导的血管生成 ,且对内皮细胞增殖具有抑制作用。     

Pseudolarix acid B inhibits angiogenesis by antagonizing the vascular endothelial growth factor-mediated anti-apoptotic effect

Angiogenesis is controlled by a number of growth factors, including vascular endothelial growth factor (VEGF). In this study, pseudolarix acid B, isolated from the traditional Chinese medicinal plant Pseudolarix kaempferi and originally identified as an early pregnancy-terminating agent, was evaluated for its potential as an angiogenesis inhibitor, using in vitro and in vivo models. After exposure to pseudolarix acid B 0.625-5 microM for 72 h, the proliferation of human umbilical vein endothelial cells was significantly inhibited. Pseudolarix acid B 0.313-2.5 microM for 24 h potently blocked the VEGF-induced tube formation of human umbilical vein endothelial cells in a dose-dependent manner. Matrigel plug assays disclosed that pseudolarix acid B reduced angiogenesis induced by VEGF in vivo. In addition, pseudolarix acid B antagonized VEGF-mediated anti-apoptotic effects on serum-deprived human umbilical vein endothelial cells and increased apoptosis of endothelial cells induced by VEGF in Matrigel plug assays. Moreover, pseudolarix acid B significantly inhibited VEGF-induced tyrosine phosphorylation of kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/flk-1), in correlation with a marked decrease in the phosphorylation of Akt and extracellular signal-regulated kinases (ERK). These findings collectively suggest that pseudolarix acid B possesses anti-angiogenic activity. One of the main anti-angiogenesis mechanisms of pseudolarix acid B may involve antagonism of the VEGF-mediated anti-apoptosis effect via inhibition of KDR/flk-1, ERK1/2, and Akt phosphorylation in endothelial cells.     

Phosphatidylinositol inhibits vascular endothelial growth factor-A--induced migration of human umbilical vein endothelial cells

Phosphatidylinositol (PI), a phospholipid in component of cell membranes, is widely distributed in animals, plants, and microorganisms. Here, we examined in vitro whether PI inhibits the angiogenesis induced by vascular endothelial growth factor-A (VEGF-A). PI concentration-relatedly and significantly (at 10 and 30 microg/ml) inhibited VEGF-A-induced tube formation in a co-culture of human umbilical vein endothelial cells (HUVECs) and fibroblasts. PI also inhibited the migration, but not proliferation, induced in HUVECs by VEGF-A. Furthermore, PI at 30 microg/ml inhibited the VEGF-A-induced phosphorylation of serine/threonine protein kinase family protein kinase B (Akt) and p38 mitogen activate kinase (p38MAPK), key molecules in cell migration, but not phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), a key molecule in cell proliferation. These findings indicate that PI inhibits VEGF-induced angiogenesis by inhibiting HUVECs migration and that inhibition of phosphorylated-Akt and -p38MAPK may be involved in the mechanism. Therefore, PI may be expected to prevent some diseases caused by angiogenesis.     

Inhibitory effects of artesunate on angiogenesis and on expressions of vascular endothelial growth factor and VEGF receptor KDR/flk-1

Artesunate (ART) is a semi-synthetic derivative of artemisinin extracted from the plant Artemisia annua is a safe and effective antimalarial drug. In the present investigation, ART was found also to inhibit angiogenesis in vivo and in vitro. The anti-angiogenic effect in vivo was evaluated in nude mice by means of human ovarian cancer HO-8910 implantation and immunohistochemical stainings for microvessel (CD(31)), vascular endothelial growth factor (VEGF) and VEGF receptor KDR/flk-1. Tumor growth was decreased and microvessel density was reduced following drug treatment with no apparent toxicity to the animals. ART also remarkably lowered VEGF expression on tumor cells and KDR/flk-1 expression on endothelial cells as well as tumor cells. The in vitro effect of ART was tested on models of angiogenesis, namely, proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVEC). The results showed that ART significantly inhibited angiogenesis in a dose-dependent form in the range of 0.5 approximately 50 micromol/l. Additionally, the inhibitory effect of ART on HVUEC proliferation was stronger than that on Hela, JAR, HO-8910 cancer cells, NIH-3T3 fibroblast cells and human endometrial cells, indicating that ART was selectively against HUVEC. These findings and the known low toxicity of ART are clues that ART may be a promising angiogenesis inhibitor.     

Inhibition of vascular endothelial growth factor activity by transfection with the soluble FLT-1 gene

Vascular endothelial growth factor (VEGF)-induced angiogenesis is involved in the etiology of some cardiovascular diseases. The soluble form of VEGF receptor, FLT-1 (sFLT-1), is a potent antagonist of VEGF. Therefore, we investigated whether transfection with the sFLT-1 gene could inhibit VEGF-induced angiogenesis. Human embryonic kidney (HEK)-293 cells were transfected with plasmids containing VEGF and sFLT-1 (pCMV-VEGF and pCMV-sFLT-1) by the calcium-phosphate co-precipitation method. VEGF- and/or sFLT-1-transfected HEK-293 cells were incubated for 24 h, and then conditioned medium was collected. The effects of conditioned medium on angiogenesis were tested by incorporation of [3H]thymidine into human umbilical vein endothelial cells (HUVECs). Expression of VEGF protein was determined by Western blotting. The conditioned medium from sFLT-1 gene-transfected HEK-293 cells significantly inhibited recombinant VEGF-induced increase in [3H]thymidine incorporation by HUVECs. VEGF gene-transfected HEK-293 cells secreted VEGF protein into conditioned medium. This conditioned medium increased [3H]thymidine incorporation by HUVECs, which was significantly inhibited by co-transfection of sFLT-1 gene with VEGF gene. These observations suggested that sFLT-1 gene transfer could inhibit VEGF-induced DNA synthesis of vascular endothelial cells.     

β-Eudesmol suppresses tumour growth through inhibition of tumour neovascularisation and tumour cell proliferation

In the present study, we investigated the potential anti-angiogenic mechanism and anti-tumour activity of β-eudesmol using in vitro and in vivo experimental models. Proliferation of human umbilical vein endothelial cells (HUVEC) stimulated with vascular endothelial growth factor (VEGF, 30 ng/ml) and basic fibroblast growth factor (bFGF, 30 ng/ml) was significantly inhibited by β-eudesmol (50-100 μM). β-Eudesmol (100 μM) also blocked the phosphorylation of cAMP response element binding protein (CREB) induced by VEGF (30 ng/ml) in HUVEC. β-Eudesmol (10-100 μM) inhibited proliferation of HeLa, SGC-7901, and BEL-7402 tumour cells in a time- and dose-dependent manner. Moreover, β-eudesmol treatment (2.5-5 mg/kg) significantly inhibited growth of H₂₂ and S₁₈₀ mouse tumour in vivo. These results indicated that β-eudesmol inhibited angiogenesis by suppressing CREB activation in growth factor signalling pathway. This is the first study to demonstrate that β-eudesmol is an inhibitor of tumour growth.     

木瓜酶促进大鼠血管内皮细胞成血管的功能研究

目的　通过体外鼠细胞模型实验,检测木瓜酶在体外培养环境下对血管内皮细胞成血管向分化的促进功能。方法　采用P3代大鼠血管内皮细胞,将木瓜酶溶解于完全培养基内,按照10 μg/ml、50 μg/ml、100 μg/ml浓度配置含木瓜酶的完全培养基,与空白对照组共同培养0、7、14 d,通过逆转录聚合酶链反应（qRT-PCR）法检测成血管分化相关基因VEGF（血管内皮生长因子）、CD31（血小板-内皮细胞粘附分子）的表达水平,通过CCK-8法检测不同组细胞的增殖活力。统计学方法为方差分析。结果　经14 d培养,添加木瓜酶的细胞VEGF、CD31表达水平及增殖能力显著高于空白对照组,木瓜酶浓度达到50 μg/ml时VEGF表达水平达到最高,为空白对照组的3.28倍,CD31表达水平随着木瓜酶浓度升高而升高,最高达到空白对照组的3.66倍。结论　适宜浓度的木瓜酶能够在体外促进内皮细胞的成血管分化及增殖,表明木瓜酶对血管组织形成具有一定的促进作用。     

Insulin-like growth factor binding protein-7 (IGFBP7) blocks vascular endothelial cell growth factor (VEGF)-induced angiogenesis in human vascular endothelial cells

Insulin-like growth factor binding protein-7 (IGFBP7) and vascular endothelial growth factor (VEGF) are expressed in vascular endothelial cells in several tumor types. In this study, we examined the effect of IGFBP7 on VEGF-induced tube formation in cultured human umbilical vein endothelial cells (HUVECs) and its potential action in the modulation of VEGF signaling in vascular cells. IGFBP7 treatment suppressed VEGF-induced tube formation, proliferation, and the phosphorylation of mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) 1/2 in HUVECs. IGFBP7 attenuated VEGF-enhanced cyclooxygenase (COX)-2 and VEGF mRNA expression, and prostaglandin E₂ secretion. Knocking down endogenous IGFBP7 enhanced COX-2 and VEGF mRNA expression. A significant increase in IGFBP7-induced caspases was not observed in the presence of VEGF. These findings indicate that IGFBP7 can modulate the stimulatory effect of VEGF on angiogenesis by interfering with VEGF expression as well as VEGF signaling and not by inducing apoptosis.     

Effect of heparin affin regulatory peptide on the expression of vascular endothelial growth factor receptors in endothelial cells

BACKGROUND: Heparin affin regulatory peptide (HARP) is an 18-kDa secreted protein that has been implicated in tumor growth and angiogenesis, although the mechanisms involved remain largely unknown. In the present work, the effect of human recombinant HARP on the expression of the vascular endothelial growth factor (VEGF) receptors KDR, Flt-1 and neuropilin-1 was studied in cultured human umbilical vein endothelial cells (HUVEC). MATERIALS AND METHODS: The mRNA and protein levels of VEGF receptors were estimated by semi-quantitative RT-PCR and Western blot, respectively. Cell proliferation and migration were measured by MTT, direct counting of the cells and modified Boyden chamber assays. RESULTS: HARP decreased the expression of KDR but increased the expression of Flt-1 and neuropilin-1 at both the mRNA and protein level. The effect reached a maximum 4 h after the addition of HARP into the cell culture medium and was reversed at later time-points. When HARP was added to the culture medium 4 h before the addition of VEGF165, it inhibited VEGF165-induced proliferation and migration of HUVEC. CONCLUSION: These data suggest that HARP affects the expression of VEGF receptors and inhibits VEGF165-induced activation of HUVEC.     

Design, synthesis, and evaluation of orally active benzimidazoles and benzoxazoles as vascular endothelial growth factor-2 receptor tyrosine kinase inhibitors

Inhibition of the VEGF signaling pathway has become a valuable approach in the treatment of cancers. Guided by X-ray crystallography and molecular modeling, a series of 2-aminobenzimidazoles and 2-aminobenzoxazoles were identified as potent inhibitors of VEGFR-2 (KDR) in both enzymatic and HUVEC cellular proliferation assays. In this report we describe the synthesis and structure-activity relationship of a series of 2-aminobenzimidazoles and benzoxazoles, culminating in the identification of benzoxazole 22 as a potent and selective VEGFR-2 inhibitor displaying a good pharmacokinetic profile. Compound 22 demonstrated efficacy in both the murine matrigel model for vascular permeability (79% inhibition observed at 100 mg/kg) and the rat corneal angiogenesis model (ED(50) = 16.3 mg/kg).     

芸香宁碱抑制血管生成作用的研究

目的：研究芸香宁碱对血管生成的抑制作用,并研究其初步的作用机制。方法用人脐静脉内皮细胞（HUVEC）的生长、迁移实验及体内动物模型－鸡胚绒毛尿囊膜法（CAM 法）研究化合物的体内外抗血管生成作用;用酶联免疫吸附法检测芸香宁碱在非凋亡剂量时对肿瘤细胞培养上清液中 VEGF 蛋白分泌量的影响。结果芸香宁碱对血管内皮细胞具有优先抑制作用,对 HUVEC 的半数抑制剂量为（33．2±0．4）μmol·L －1,而对其他肿瘤细胞的 IC50值均大于这一数值;芸香宁碱在体外能够明显抑制内皮细胞的迁移和对细胞外基质黏附作用,并呈剂量依赖性,体内实验显示30μmol·L －1剂量浓度时显著抑制 CAM新生血管形成;芸香宁碱在15μmol·L －1和30μmol·L －1的浓度剂量时显著抑制人肝癌 HepG2细胞培养上清液中VEGF 蛋白的分泌,具有显著性差异。结论芸香宁碱在非凋亡浓度剂量具有抑制新生血管形成作用,其机制与抑制血管内皮细胞增殖以及抑制肿瘤细胞表达 VEGF 有关。     

Oxidized low-density lipoprotein inhibits vascular endothelial growth factor-induced endothelial progenitor cell differentiation

1. Bone marrow-derived endothelial progenitor cells (EPC) in the peripheral blood of adult animals and humans have been shown to be incorporated into neovascularization. In contrast, hypercholesterolaemia impairs angiogenesis and collateral vessel formation in response to regional tissue ischaemia. We investigated whether oxidized LDL (oxLDL) affected human EPC differentiation. 2. When isolated human mononuclear cells (MNC) were incubated with vascular endothelial growth factor (VEGF), the number of differentiated, adherent EPC, as assessed by an in vitro culture assay, was increased in a dose-dependent manner (P < 0.01). When MNC were incubated with oxLDL at 1, 5 and 10 microg/mL in the presence of 100 ng/mL VEGF for 24 h, oxLDL dose-dependently reduced the number of differentiated, adherent EPC. 3. Vascular endothelial growth factor-induced EPC differentiation was significantly inhibited by pharmacological phosphatidylinositol 3-kinase blockers (either 10 nmol/L wortmannin or 10 micromol/L LY294002). Interestingly, immunoblotting analysis revealed that oxLDL dose-dependently led to dephosphorylation and, thus, deactivation of Akt in the presence of VEGF. Finally, these inhibitory effects induced by oxLDL were abolished by pretreatment with 1 micromol/L atorvastatin (P < 0.01). 4. Our data indicate that oxLDL inhibits VEGF-induced EPC differentiation through the dephosphorylation of Akt.     

Role of nitric oxide, prostaglandins and tyrosine kinase in vascular endothelial growth factor-induced increase in vascular permeability in mouse skin

We investigated role of nitric oxide (NO), prostaglandins (PG) and tyrosine kinase in vascular endothelial growth factor (VEGF)-induced increase in vascular permeability in mouse skin. Subcutaneous injection of VEGF (0.5–2.0 ng/site) induced dose- and time-dependent increase in vascular permeability at the injection site determined by a leakage of Pontamine sky blue. VEGF (1 ng/site)-induced dye leakage was partially inhibited by N^G-nitro-l-arginine methyl ester (an inhibitor for both constitutive and inducible NO synthase) (5 and 10 mg/kg, i.v.) and by aminoguanidine (a selective inducible NO synthase inhibitor) (5–20 mg/kg, i.v.), but not by an inactive enantiomer, N^G-nitro-d-arginine methyl ester (10 mg/kg, i.v.). Pretreatment with an intraperitoneal injection of indomethacin (a nonselective cyclooxygenase inhibitor) (5 mg/kg) or N-(2-cyclohexyloxy-4-nitrophenyl) methanesulphonamide (a cyclooxygenase-2 selective inhibitor) (1–100 μg/kg) almost completely inhibited the effect of VEGF (1 ng/site). Coadministration of PGE₂ (3 and 30 nmol/site) with VEGF did not restore the inhibitory effect of indomethacin on VEGF (1 ng/site)-induced increase in vascular permeability. Lavendustin A (a selective tyrosine kinase inhibitor) (10 and 50 μg/kg, s.c.) dose-relatedly inhibited the VEGF (1 ng/site)-induced increase in dye leakage, whereas its negative control, lavendustin B (10 μg/kg, s.c.) had no effect. Another tyrosine kinase inhibitor, genistein (2.5 mg/kg, s.c.) also inhibited the response. Cycloheximide (a protein biosynthesis inhibitor) (35 mg/kg, s.c.) suppressed the response of VEGF (1 ng/site). Histologically, no cellular infiltration was observed in the area of VEGF injection. These results suggest that increased vascular permeability induced by VEGF is mediated by local production of NO and arachidonic acid metabolites other than PGE₂, which are most probably produced by inducible NO synthase and cyclooxygenase-2, respectively. Protein tyrosine kinase-mediated phosphorylation and synthesis of any new proteins are likely to be required in this effect of VEGF in mouse skin. Received: 18 November 1996 / Accepted: 30 May 1997