BRN-103, a novel nicotinamide derivative, inhibits VEGF-induced angiogenesis and proliferation in human umbilical vein endothelial cells

Anti-angiogenesis is regarded as an effective strategy for cancer treatment, and vascular endothelial growth factor (VEGF) plays a key role in the regulations of angiogenesis and vasculogenesis. In the present study, the authors synthesized five novel nicotinamide derivatives which structurally mimic the receptor tyrosine kinase inhibitor sunitinib and evaluated their anti-angiogenic effects. Transwell migration assays revealed that 2-(1-benzylpiperidin-4-yl) amino-N-(3-chlorophenyl) nicotinamide (BRN-103), among the five derivatives most potently inhibited VEGF-induced human umbilical vein endothelial cells (HUVECs). In addition, BRN-103 dose-dependently inhibited VEGF-induced migration, proliferation, and capillary-like tube formation of HUVECs and vessel sprouting from mouse aortic rings. To understand the molecular mechanisms responsible for these activities, the authors examined the effect of BRN-103 on VEGF signaling pathways in HUVECs. BRN-103 was found to suppress the VEGF-induced phosphorylation of VEGF receptor 2 (VEGR2) and the activations of AKT and eNOS. Taken together, these results suggest that BRN-103 inhibits VEGF-mediated angiogenesis signaling in human endothelial cells.     

Scutellarin promotes in vitro angiogenesis in human umbilical vein endothelial cells

Angiogenesis is critical to a wide range of physiological and pathological processes. Scutellarin, a major flavonoid of a Chinese herbal medicine Erigeron breviscapus (Vant.) Hand. Mazz. has been shown to offer beneficial effects on cardiovascular and cerebrovascular functions. However, scutellarin’s effects on angiogenesis and underlying mechanisms are not fully elucidated. Here, we studied angiogenic effects of scutellarin on human umbilical vein endothelial cells (HUVECs) in vitro. Scutellarin was found by MTT assay to induce proliferation of HUVECs. In scutellarin-treated HUVECs, a dramatic increase in migration was measured by wound healing assay; Transwell chamber assay found significantly more invading cells in scutellarin-treated groups. Scutellarin also promoted capillary-like tube formation in HUVECs on Matrigel, and significantly upregulated platelet endothelial cell adhesion molecule-1 at both mRNA and protein levels. Scutellarin’s angiogenic mechanism was investigated in vitro by measuring expression of angiogenic factors associated with cell migration and invasion. Scutellarin strongly induced MMP-2 activation and mRNA expression in cultured HUVECs in a concentration-dependent manner. Taken together, these results suggest that scutellarin promotes angiogenesis and may form a basis for angiogenic therapy.     

Palm tocotrienols decrease levels of pro-angiogenic markers in human umbilical vein endothelial cells (HUVEC) and murine mammary cancer cells

Anti-angiogenic therapy is widely being used to halt tumour angiogenesis. In this study, the anti-angiogenic activity of palm tocotrienol-rich fraction (TRF) and its individual components (γ- and δ-tocotrienol) were first investigated in vitro in human umbilical vein endothelial cells (HUVEC) and 4T1 mouse mammary cancer cells. Results showed reduced levels of Interkeukin (IL)-8 and IL-6, two pro-angiogenic cytokines in HUVEC treated with palm tocotrienols compared with α-tocopherol (α-T) and control cells (P < 0.05). The production of IL-8 and IL-6 was lowest in δ-tocotrienol (δ-T3)-treated cells followed by γ-tocotrienol (γ-T3) and TRF. There was significant (P < 0.05) reduction in IL-8 and vascular endothelial growth factor (VEGF) production in 4T1 cells treated with TRF or δ-T3. There was decreased expression of VEGF and its receptors; VEGF-R1 (fms-like tyrosine kinase, Flt-1) and VEGF-R2 (Kinase-insert-domain-containing receptor, KDR/Flk-2) in tumour tissues excised from mice supplemented with TRF were observed. There was also decreased expression of VEGF-R2 in lung tissues of mice supplemented with TRF. These observations correlate with the smaller tumour size recorded in the tocotrienol-treated mice. This study confirms previous observations that palm tocotrienols exhibit anti-angiogenic properties that may inhibit tumour progression.     

Notch1信号通路激活在低氧诱导人脐静脉内皮细胞血管形成中的作用

目的观察低氧条件下HIF-1α/VEGF/Notch信号通路在人脐静脉内皮细胞（HUVEC）血管生成中的作用。 方法将HUVEC进行常氧和低氧[二氯化钴（CoCl2）,200 μmol/L]诱导,再将常氧和低氧处理的HUVEC应用Notch1信号通路的抑制剂DAPT （30 μmol/L,24 h）和激活剂JAG-1 （30 μmol/L,24 h）干预。通过体外小管形成实验观察低氧对HUVEC血管生成能力的影响。应用RT-PCR和Western blot检测HUVEC中低氧诱导因子-1α （HIF-1α）、血管内皮生长因子（VEGF）、基质金属蛋白酶-9 （MMP-9）和Notch1信号分子（Notch1、Dell4和JAG-1）的mRNA和蛋白表达。通过Transwell迁移实验和伤口愈合实验观察低氧、DAPT、JAG-1对HUVEC迁移能力的影响。应用MTT法检测低氧及Notch1对HUVEC增殖的影响。两组间比较采用t检验,采用析因设计方差分析低氧和DAPT以及低氧和JAG-1对HUVEC迁移能力、距离、小管形成能力和细胞增殖的交互作用。 结果与常氧组比较,低氧组小管总长[（8.18±0.62）mm比（15.43±1.32）mm]增高,差异具有统计学意义（P < 0.05）。与常氧组比较,低氧组的HIF-1α、VEGF、MMP-9、Notch1、Dell4和JAG-1的mRNA相对表达量和蛋白相对表达量（1.01±0.03比4.43±0.35,1.02±0.03比3.55±0.28,0.98±0.04比3.24±0.25,1.01±0.03比3.22±0.25,0.99±0.02比2.89±0.22,1.02±0.04比2.43±0.19,0.98±0.01比3.13±0.24,0.98±0.02比2.67±0.21,0.97±0.03比2.45±0.19,1.01±0.03比2.44±0.19,1.00±0.04比2.30±0.18,1.03±0.05比2.27±0.18）均升高,差异有统计学意义（P均< 0.05）。Transwell迁移实验和伤口愈合实验显示,低氧条件下,DAPT干预使HUVEC的迁移能力降低,JAG-1干预使HUVEC的迁移能力升高（P均< 0.05）。小管形成和MTT法测定显示,低氧条件下,DAPT干预使HUVEC的小管形成能力和细胞增殖能力降低,JAG-1干预使HUVEC的小管形成能力和细胞增殖能力升高（P均< 0.05）。析因设计的方差分析结果显示,低氧和JAG-1对迁移细胞数、小管形成和细胞增殖能力交互作用具有协同作用（P < 0.05）。 结论低氧可通过激活HIF-1α/VEGF/Notch1信号通路提高HUVEC的血管生成能力、迁移能力和细胞增殖能力。     

VEGF,not VEGFR2, is associated with the angiogenesis effect of mini-TyrRS/mini-TrpRS in human umbilical vein endothelial cells in hypoxia

The purpose of this study was to determine the relationship between VEGF and mini-TyrRS/mini-TrpRS in angiogenesis in hypoxic culture and to begin to comprehend their mechanism in angiogenesis. We designed a VEGF gene silencing assay by using lentivirus vectors, and then western blotting was used to determine the protein expression of VEGF, VEGFR2 and pVEGFR2 in three groups in hypoxic culture at 3, 6, 12, or 24 h: (1) untransfected human umbilical vein endothelial cells (HUVECs) (Control); (2) pGCSIL-GFP lentivirus vector-transduced HUVECs (Mock); and (3) pGCSIL-shVEGF lentivirus vector-transduced HUVECs (Experimental). We also detected the effects of mini-TyrRS/mini-TrpRS peptides on HUVEC proliferation, migration and tube formation after lentivirus vector transfection and VEGFR2 antibody injection. The results indicated that expression of the mini-TyrRS protein was increased, whereas that of mini-TrpRS was specifically decreased in hypoxic culture both in control and mock groups. However, this trend in protein levels of mini-TyrRS and mini-TrpRS was lost in the experimental group after transduction with the pGCSIL-shVEGF lentivirus vector. The protein expression of VEGF was increased in hypoxic culture both in control and mock groups. After transduction with the pGCSIL-shVEGF lentivirus vector, the protein level of VEGF was noticeably decreased in the experimental group; however, for VEGFR2, the results showed no significant difference in VEGFR2 protein expression in any of the groups. For pVEGFR2, we found a distinct trend from that seen with VEGF. The protein expression of pVEGFR2 was sharply increased in hypoxic culture in the three groups. The addition of mini-TyrRS significantly promoted proliferation, migration and tube formation of HUVECs, while mini-TrpRS inhibited these processes in both control and mock groups in hypoxic culture. However, these effects disappeared after transduction with the pGCSIL-shVEGF lentivirus vector in the experimental group, but no significant difference was observed after VEGFR2 antibody injection. The protein expression of VEGF is similar to that of mini-TyrRS in hypoxic culture and plays an important role in the mini-TyrRS/mini-TrpRS-stimulated proliferation, migration and tube formation of HUVECs in hypoxia. These results also suggest that the change in mini-TyrRS and mini-TrpRS expression in hypoxic culture is not related to VEGFR2 and that some other possible mechanisms, are involved in the phosphorylation of VEGFR2.     

Modulation of cyclooxygenase in endothelial cells by fibronectin: relevance to angiogenesis

Cyclooxygenases (COX), which catalyze the formation of prostaglandins (PGs), have been implicated in angiogenesis. Adhesion of endothelial cells (ECs) to extracellular matrix (ECM) induces the expression of COX-2 and PG production. The present study was carried out to analyze the influence of the adhesive ECM protein, fibronectin (FN), in modulating COX expression and its implications to angiogenesis using in vitro cultures of human umbilical vein ECs. RT-PCR analysis showed that the level of COX-2 mRNA was significantly high while that of COX-1 decreased in ECs maintained on FN. On treatment with p38 MAPK inhibitor and anti-alpha(5)beta(1) integrin antibody, FN dependent effect on COX expression was not observed. Analysis by ELISA and immunoblotting confirmed FN-dependent upregulation of COX-2 protein. The ratio of PG E(2):PG D(2) was significantly high in cells maintained on FN and on treatment with p38 MAPK inhibitor, the relative level of PG D(2) increased and that of PG E(2) decreased. Concomitant with the modulation of COX-2 and changes in PGs, ECs maintained on FN showed angiogenic response in an alpha(5)beta(1) integrin/p38 MAPK dependent manner as evidenced by the expression of angiogenic markers, CD 31 and E-selectin. These results suggest a FN-alpha(5)beta(1)/FAK/p38 MAPK dependent upregulation of COX-2 causing a shift in the relative levels of PGs in HUVECs which contributes to the angiogenic effect of FN.     

Myricetin ameliorates high glucose-induced endothelial dysfunction in human umbilical vein endothelial cells

Endothelial dysfunction is recognized as the initial detectable stage of cardiovascular disease, a serious complication of diabetes. In this study, we evaluated effects of myricetin on high glucose (HG)-elicited oxidative damage in human umbilical vein endothelial cells (HUVECs). The cells were pre-incubated with myricetin and then treated with HG to induce apoptosis. The effect of myricetin on viability was investigated by MTT assay. The levels of lipid peroxidation (LPO) were determined by thiobarbituric acid (TBA) method. The protein expression of Bax, Bcl-2 and caspase-3 was measured by western blot analysis. Moreover, the effect of myricetin on total antioxidant capacity (TAC) and total thiol molecules was also determined. Our results showed that myricetin was able to markedly restore the viability of endothelial cells under oxidative stress. Myricetin reduced HG-caused increase in LPO levels. Also, TAC and total thiol molecules were notably elevated by myricetin. Incubation with myricetin decreased the protein expression levels of Bax, whereas it increased the expression levels of the Bcl-2, compared with HG treatment alone. Furthermore, myricetin significantly decreased cleaved caspase-3 protein expression. It is concluded that myricetin may protect HUVECs from oxidative stress induced by HG via increasing cell TAC and reducing Bax/Bcl-2 protein ratio, and caspase-3 expression.     

Effect of FGF-1 and FGF-2 on VEGF binding to human umbilical vein endothelial cells

When FGF-1 or FGF-2 and VEGF were added together, the mitogenic effect of FGF-1 or FGF-2 and VEGF on HUVEC was additive. However, when HUVECs were preincubated for 2 days with 10 ng/ml FGF-1 in the absence of VEGF, the Scatchard plot of [125I]VEGF binding sites was shifted to the right: both affinity classes of VEGF binding sites were equally affected, such that the total number of sites increased twofold. It is suggested that this type of interaction may be related to tumor angiogenesis and wound repair.     

The ratio of VEGF/PEDF expression in bone marrow mesenchymal stem cells regulates neovascularization

Angiogenesis, or neovascularization, is a finely balanced process controlled by pro- and anti-angiogenic factors. Vascular endothelial growth factor (VEGF) is a major pro-angiogenic factor, whereas pigment epithelial-derived factor (PEDF) is the most potent natural angiogenesis inhibitor. In this study, the regulatory role of bone marrow stromal cells (BMSCs) during angiogenesis was assessed by the endothelial differentiation potential, VEGF/PEDF production and responses to pro-angiogenic and hypoxic conditions. The in vivo regulation of blood vessel formation by BMSCs was also explored in a SCID mouse model. Results showed that PEDF was expressed more prominently in BMSCs compared to VEGF. This contrasted with human umbilical vein endothelial cells (HUVECs) where the expression of VEGF was higher than that of PEDF. The ratio of VEGF/PEDF gene expression in BMSCs increased when VEGF concentration reached 40ng/ml in the culture medium, but decreased at 80ng/ml. Under CoCl(2)-induced hypoxic conditions, the VEGF/PEDF ratio of BMSCs increased significantly in both normal and angiogenic culture media. There was no expression of endothelial cell markers in BMSCs cultured in either pro-angiogenic or hypoxia culture conditions when compared with HUVECs. The in vivo study showed that VEGF/PEDF expression closely correlated with the degree of neovascularization, and that hypoxia significantly induced pro-angiogenic activity in BMSCs. These results indicate that, rather than being progenitors of endothelial cells, BMSCs play an important role in regulating the neovascularization process, and that the ratio of VEGF and PEDF may, in effect, be an indicator of the pro- or anti-angiogenic activities of BMSCs.     

Regulation of factors controlling angiogenesis in liver development: a role for PEDF in the formation and maintenance of normal vasculature

PEDF and VEGF are important inhibitors and promoters of angiogenesis, and the ratio between the two is an important indicator in many neovascular diseases. In mouse liver PEDF and VEGF(165) were co-expressed at very early stages of liver development and their expression increased as liver embryogenesis progressed, suggesting that PEDF and VEGF are both crucial to vasculogenesis as well. VEGF(189) only appears at the P0 stage in liver organogenesis and is maintained at high levels thereafter. PEDF and the two VEGF isoforms are synthesized by fresh and cultured hepatocytes. Expression of VEGF(121) and overexpression of VEGF(165) were only seen in HepG2, a well-characterized hepatocellular carcinoma line. The results suggest that hepatic vascular architecture is under the control of both PEDF and VEGF, and that VEGF(165) and VEGF(189) have distinct functions in normal vascular development of the liver. The VEGF isoforms 121 and 189 may be key regulators of increased vascularity and progression of hepatocellular carcinoma, one of the most common malignant tumors, and may be of prognostic significance for this tumor.     

Vascular endothelial growth factor upregulates follistatin in human umbilical vein endothelial cells

Vascular endothelial growth factor (VEGF), plays a key role in angiogenesis. Many endogenous factors can affect angiogenesis in endothelial cells. VEGF is known to be a strong migration, sprouting, survival, and proliferation factor for endothelial cells during angiogenesis in endothelial cells. Searching for novel genes, involved in VEGF signaling during angiogenesis, we carried out differential display polymerase chain reaction on RNA from VEGF-stimulated human umbilical vein endothelial cells (HUVECs). In this study, follistatin (FS) differentially expressed in VEGF-treated HUVECs, compared with controls. Addition of VEGF (10 ng/mL) produced an approximately 11.8-fold increase of FS mRNA. FS or VEGF produced approximately 1.8- or 2.9-fold increases, respectively, in matrix metalloproteinase-2 (MMP-2) secretion for 12 h, compared to the addition of a control buffer. We suggest that VEGF may affect the angiogenic effect of HUVECs, through a combination of the direct effects of VEGF itself, and the indirect effects mediated via induction of FSin vitro.     

PEDF蛋白在子痫前期患者胎盘上的表达及意义

目的:研究子痫前期患者胎盘组织中色素上皮衍生因子(pigment epithelium-derived factor,PEDF)的表达,探讨PEDF在子痫前期发病中的作用。方法:选取2012年3月至2013年3月在我院产科住院剖宫产的20例子痫前期孕妇作为研究对象,另选取同期正常分娩的孕妇20例作为对照组。采用Western blot、免疫荧光组织化学方法检测子痫前期患者和正常对照组妇女胎盘组织中PEDF和血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达,并通过免疫荧光方法计数胎盘微血管密度(MVD)。结果:同正常对照组相比,子痫前期患者胎盘组织中PEDF表达升高,而VEGF表达则减少。PEDF与VEGF组织表达位置大致相同。子痫前期患者胎盘组织中PEDF表达与24h尿蛋白定量呈正相关,与VEGF表达及MVD计数呈负相关;VEGF表达与MVD计数呈正相关。结论:PEDF与子痫前期疾病发生发展及病情轻重程度有关;PEDF可能是通过影响胎盘血管的重铸,而参与子痫前期疾病的发生发展。     

Upregulation of fibroblast growth factor-2 by visfatin that promotes endothelial angiogenesis

Adipokines have been known to act as angiogenic regulators in the process of angiogenesis. Recently, we have demonstrated that visfatin, a novel adipokine, has angiogenic activity. However, little has been reported on the underlying mechanism of visfatin-induced angiogenesis. In this study, we report that visfatin-induced angiogenesis is mediated by endothelial fibroblast growth factor-2 (FGF-2). Visfatin increased the levels of FGF-2 mRNA and protein in human endothelial cells. The enhancement in FGF-2 expression was prevented by an inhibitor of the extracellular signal-regulated kinase 1/2 (Erk1/2) pathway. Furthermore, visfatin-induced angiogenesis was reduced by inhibition of FGF-2 receptor kinase or by neutralization of FGF-2 function. Taken together, our results indicate that visfatin-induced endothelial angiogenesis is composed largely of two sequential steps: the induction of Erk1/2-dependent FGF-2 gene expression by visfatin and the subsequent FGF-2-induced angiogenesis. These data further suggest an integral role for visfatin-FGF-2 signaling axis in modulating endothelial angiogenesis.     

Beyond membrane integrity: Assessing the functionality of human umbilical vein endothelial cells after cryopreservation

Assessment of cell membrane integrity is one of the most widely used methods to measure post-cryopreservation viability of cells such as human umbilical vein endothelial cells (HUVECs). However, an evaluation of cell function provides a better measure of cell quality following cryopreservation. The tube formation assay mimics angiogenesis in vitro and can be used to quantitate the ability of endothelial cells to form capillary-like tubular structures when cultured on reconstituted basement membrane (Matrigel). We compared the membrane integrity (measured by flow cytometry) and tube forming ability of HUVEC suspensions exposed to 10% dimethyl sulfoxide (Me₂SO), cooled at 1 °C/min to various sub-zero temperatures, plunged directly into liquid nitrogen, stored for an hour, and thawed rapidly. We found that as membrane integrity increased so did the various parameters associated with the extent of in vitro angiogenesis; however, in comparison to fresh cells with a similar percentage of membrane-intact cells, the extent of tube formation, expressed as total tube length, is significantly lower in previously frozen cells for the lower range of post-thaw membrane integrities. Our findings underscore the value of an assay that quantifies a specific function that a cell is known to perform in vivo to measure the success of cryopreservation protocols.     

Galectin-3 protein modulates cell surface expression and activation of vascular endothelial growth factor receptor 2 in human endothelial cells

Angiogenesis is heavily influenced by VEGF-A and its family of receptors, particularly VEGF receptor 2 (VEGF-R2). Like most cell surface proteins, VEGF-R2 is glycosylated, although the function of VEGF-R2 with respect to its glycosylation pattern is poorly characterized. Galectin-3, a glycan binding protein, interacts with the EGF and TGFβ receptors, retaining them on the plasma membrane and altering their signal transduction. Because VEGF-R2 is glycosylated and both galectin-3 and VEGF-R2 are involved with angiogenesis, we hypothesized that galectin-3 binds VEGF-R2 and modulates its signal transduction as well. Employing a Western blot analysis approach, we found that galectin-3 induces phosphorylation of VEGF-R2 in endothelial cells. Knockdown of galectin-3 and Mgat5, an enzyme that synthesizes high-affinity glycan ligands of galectin-3, reduced VEGF-A mediated angiogenesis in vitro. A direct interaction on the plasma membrane was detected between galectin-3 and VEGF-R2, and this interaction was dependent on the expression of Mgat5. Using immunofluorescence and cell surface labeling, we found an increase in the level of internalized VEGF-R2 in both Mgat5 and galectin-3 knockdown cells, suggesting that galectin-3 retains the receptor on the plasma membrane. Finally, we observed reduced suture-induced neovascularization in the corneas of Gal3(-/-) and Mgat5(-/-) mice. These findings are consistent with the hypothesis that, like its role with the EGF and TGFβ receptors, galectin-3 contributes to the plasma membrane retention and proangiogenic function of VEGF-R2.     

miR-181a/b-5p regulates human umbilical vein endothelial cell angiogenesis by targeting PDGFRA

Type 2 diabetes mellitus (T2DM) is a growing burden in low-and middle-income countries. Changing lifestyles and lack of physical activity are some of the reasons contributing to this epidemic increase. Co-morbidities associated with T2DM are largely due to the complications which arise as a consequence of endothelial dysfunction. Platelet derived growth factor-alpha (PDGFRA) is a protein responsible for cell proliferation, angiogenesis, migration and invasion. Increased levels of PDGFRA have been reported in T2DM. This study assessed the epigenetic regulation of PDGFRA through microRNAs (miR-181a/b-5p).Using a bioinformatics-based approach, we assessed the binding of miR-181a/b-5p to PDGFRA. Experimentally, this binding was confirmed using a dual luciferase reporter assay. Further, we overexpressed miR-181a/b-5p in Human umbilical vein endothelial cells (HUVECs) and the influence of over-expression on cell proliferation, migration and angiogenesis was assessed using in-vitro approaches. The influence of miR-181a/b-5p over expression on cellular apoptosis was ascertained using a TUNEL assay with concomitant changes being observed in the levels of Bcl-2 and cleaved Caspase-3.In HUVECs, PDGFRA is a direct target for miR-181a/b-5p. Over expression of miR-181a/b-5p decreased cellular proliferation, migration, invasion, and tube formation—a surrogate marker for angiogenesis. miR-181a/b-5p may be used as a therapeutic intervention to restrict uncontrolled levels of PDGFRA and thereby rescue the phenotypes of increased cell proliferation, migration, invasion and tube formation. miR-181a/b negatively regulates PDGFRA levels. Significance of the study : T2DM and its associated complications emerge from endothelial dysfunction. The associated phenotypes are regulated by a number of proteins, one such member being, PDGFRA. PDGFRA is in turn regulated by miR-181a/b-5p. Complementation with miR-181a/b-5p resulted in reversion of phenotypes. Thus, miR-181a/b-5p-mediated suppression of PDGFRA may be used as a therapeutic intervention in the management of type 2 diabetes.     

Visfatin exerts angiogenic effects on human umbilical vein endothelial cells through the mTOR signaling pathway

The biologically active factors known as adipocytokines are secreted primarily by adipose tissues and can act as modulators of angiogenesis. Visfatin, an adipocytokine that has recently been reported to have angiogenic properties, is upregulated in diabetes, cancer, and inflammatory diseases. Because maintenance of an angiogenic balance is critically important in the management of these diseases, understanding the molecular mechanism by which visfatin promotes angiogenesis is very important. In this report, we describe our findings demonstrating that visfatin stimulates the mammalian target of the rapamycin (mTOR) pathway, which plays important roles in angiogenesis. Visfatin induced the expression of hypoxia-inducible factor 1α (HIF1α) and vascular endothelial growth factor (VEGF) in human endothelial cells. Inhibition of the mTOR pathway by rapamycin eliminated the angiogenic and proliferative effects of visfatin. The visfatin-induced increase in VEGF expression was also eliminated by RNA interference-mediated knockdown of the 70-kDa ribosomal protein S6 kinase (p70S6K), a downstream target of mTOR. Visfatin inactivated glycogen synthase kinase 3β (GSK3β) by phosphorylating it at Ser-9, leading to the nuclear translocation of β-catenin. Both rapamycin co-treatment and p70S6K knockdown inhibited visfatin-induced GSK3β phosphorylation at Ser-9 and nuclear translocation of β-catenin. Taken together, these results indicate that mTOR signaling is involved in visfatin-induced angiogenesis, and that this signaling leads to visfatin-induced VEGF expression and nuclear translocation of β-catenin.