Autocrine motility factor enhances hepatoma cell invasion across the basement membrane through activation of beta1 integrins.

Autocrine motility factor/phosphohexose isomerase (AMF/PHI) is a cytokine that is linked to tumor invasion and metastasis. In hepatocellular carcinoma (HCC) tissues, hepatoma cells produce AMF/PHI and its receptor, Mr 78,000 glycoprotein (gp78), is strongly detected in hepatoma cells invading into the stroma and tumor thrombi in the portal vein. Here, we investigated the mechanism of hepatoma cell invasion through Matrigel induced by AMF/PHI using 3 hepatoma cell lines. Production of AMF/PHI, phosphorylation of MEK1/2, and Rho activity were investigated by immunoblotting. Expression of AMF/PHI and gp78 was observed by confocal fluorescence microscopy. The influence of AMF/PHI on activated integrin beta1 subunit expression was evaluated by flow cytometry. Changes in invasion, adhesion, and motility induced by AMF/PHI were evaluated using chemoinvasion, adhesion, and phagokinetic track motility assays. The effect of AMF/PHI on matrix metalloproteinase (MMP) secretion was evaluated by gelatin zymography. Hepatoma cells produced AMF/PHI and expressed gp78. Although AMF/PHI was ubiquitously detected, gp78 was strongly expressed in migrating cells. AMF/PHI induced up-regulation of activated integrin beta1 subunit expression. AMF/PHI stimulated hepatoma cell invasion through Matrigel, and stimulated the adhesion, motility, and MMP-2 secretion of hepatoma cells. The latter effects were suppressed by the function-blocking antibody for integrin beta1 subunit. AMF/PHI also enhanced Rho activity and the phosphorylation of MEK1 and MEK 2. Our results indicate that AMF/PHI enhances hepatoma cell invasion through Matrigel in an autocrine manner by stimulating the adhesion, motility, and MMP-2 secretion of these cells through activation of beta1 integrins.     

TGF-β effects on airway smooth muscle cell proliferation, VEGF release and signal transduction pathways

Background and objective:   Airway smooth muscle (ASM) cell hyperplasia is a key feature of airway remodelling. Mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) are key components in signal transduction associated with cell proliferation; MAPK consists of the extracellular signal-regulated kinase (ERK), p38MAPK and c-Jun NH₂-terminal kinase (JNK). The effect of transforming growth factor (TGF)-β on the proliferation of ASM cells, the release of vascular endothelial growth factor (VEGF) by ASM cells and relevant signal transduction pathways were investigated.
Methods:   ASM cells were growth-arrested for 48 h then stimulated with platelet-derived growth factor (PDGF), TGF-β and dexamethasone. ASM cells were also treated with specific inhibitors of MAPK (PD98059), PI3K (wortmannin) and JNK (SP600125). Cell proliferation and VEGF concentrations were measured.
Results:   TGF-β neither augmented ASM cell proliferation nor showed a synergistic effect on PDGF-mediated ASM cell proliferation. Dexamethasone did not suppress ASM cell proliferation. VEGF release was augmented by TGF-β stimulation in a time-dependent manner, and was further enhanced by co-stimulation with PDGF and TGF-β. Dexamethasone suppressed VEGF release significantly. TGF-β enhanced PI3K phosphorylation, while PDGF augmented both ERK and PI3K phosphorylation. Wortmannin inhibited both TGF-β- and PDGF-stimulated VEGF release.
Conclusions:   TGF-β may facilitate airway remodelling by promoting VEGF release through the PI3K pathway, rather than via ASM cell proliferation.     

Involvement of SAPK/JNK in prostaglandin E(1)-induced VEGF synthesis in osteoblast-like cells

We previously reported that prostaglandin E₁ (PGE₁) activates both p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase via cAMP-dependent protein kinase in osteoblast-like MC3T3-E1 cells, and that p38 MAP kinase but not p42/p44 MAP kinase is involved in PGE₁-induced synthesis of vascular endothelial growth factor (VEGF). In the present study, we investigated the involvement of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in the PGE₁-induced VEGF synthesis in MC3T3-E1 cells. PGE₁ induced the phosphorylation of SAPK/JNK. SP600125, a specific inhibitor of SAPK/JNK, markedly reduced the PGE₁-induced VEGF synthesis. Forskolin, a direct activator of adenylyl cyclase, elicited the phosphorylation of SAPK/JNK, and 8bromo-cAMP, a plasma membrane-permeable cAMP analogue-stimulated VEGF synthesis was significantly reduced by SP600125. SP600125 suppressed the PGE₁-induced phosphorylation of SAPK/JNK without affecting the phosphorylation of p38 MAP kinase induced by PGE₁. The phosphorylation of c-Jun induced by PGE₁ was also inhibited by SP600125. SB203580, a p38 MAP kinase inhibitor, failed to reduce the PGE₁ induced phosphorylation of SAPK/JNK. A combination of SP600125 and SB203580 suppressed the PGE₁-stimulated VEGF synthesis in an additive manner. These results strongly suggest that PGE₁ activates SAPK/JNK in osteoblasts, and that SAPK/JNK plays a part in PGE₁-induced VEGF synthesis.     

The effect of insulin-like growth factor-I on production of vascular endothelial growth factor by amnion-derived (WISH) cells.

OBJECTIVE: Our objective was to clarify the modulation of vascular endothelial growth factor (VEGF) by amniotic cells. DESIGN: Amnion-derived (WISH) cells were cultured, and the effect of insulin-like growth factor (IGF), mitogen-activated protein (MAP) kinase kinase and/or extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors (U0126), and phosphatidylinositol (PI) 3-kinase inhibitors (wortmannin) on the production of VEGF was examined. VEGF was assayed using ELISA. The activations of MAP kinase and akt, which is phosphorylated by PI 3-kinase, were detected by Western blot analysis using anti-phosphorylated MAP kinase antibody and anti-phosphorylated akt antibody. RESULTS: In the time course of VEGF production following IGF-I treatment, VEGF production showed significant increases only at 16 and 32 h (p < 0.01). Also, IGF-I increased the production of VEGF by WISH cells in a dose-dependent manner. The MAP kinase and akt activities were recognized by treatment with IGF-I and suppressed by U0126 and wortmannin, respectively. When WISH cells were pretreated for 2 h with U0126 and wortmannin and then treated with IGF-I for 16 h, the production of VEGF was significantly decreased in a dose-dependent manner (p < 0.01, p < 0.01, respectively). CONCLUSIONS: WISH cells appeared to produce VEGF via a mechanism involving tyrosine kinase interaction with IGF-I receptor, resulting in MAP kinase and PI 3-kinase activation. It is suggested that VEGF may contribute to the neovascularization and proliferation of the placenta and gestational tissue, and IGF-I may play an important role in the modulation of VEGF production in the placenta.     

Platelet-activating factor enhances vascular endothelial growth factor-induced endothelial cell motility and neoangiogenesis in a murine matrigel model

We previously reported that platelet-activating factor (PAF) enhances the angiogenic activity of certain polypeptide mediators such as tumor necrosis factor and hepatocyte growth factor by promoting endothelial cell motility. The purpose of the present study was to evaluate whether the synthesis of PAF induced by vascular endothelial growth factor (VEGF) might affect endothelial cell motility, microvascular permeability, and angiogenesis. The neoangiogenesis and synthesis of PAF induced by VEGF were studied in vivo in a murine Matrigel model. Dermal permeability was studied in mice by injection of (125)I-albumin. The synthesis of PAF, cell motility, and the increased (125)I-albumin transfer across endothelial monolayers were studied in vitro by using cultures of human umbilical cord vein-derived endothelial cells (HUVECs). The results obtained demonstrate that the neoangiogenesis induced by VEGF in vivo was associated with a local synthesis of PAF and was inhibited by WEB2170 and CV3988, 2 chemically unrelated, specific PAF-receptor antagonists. In contrast, WEB2170 did not inhibit VEGF-enhanced dermal permeability, suggesting that the latter was independent of the synthesis of PAF. In vitro, it was found that VEGF induced the synthesis of PAF by HUVECs in a dose- and time-dependent manner. The cell motility induced by VEGF was inhibited by PAF-receptor antagonists. In contrast, VEGF-induced proliferation of HUVECs and albumin transfer through HUVEC monolayer were unaffected by PAF-receptor antagonists. These results suggest that the synthesis of PAF induced by VEGF enhances endothelial cell migration and contributes to the angiogenic effect of VEGF in the in vivo Matrigel model.     

Angiopoietin-1 regulates endothelial cell survival through the phosphatidylinositol 3'-Kinase/Akt signal transduction pathway

Angiopoietin-1 (Ang1) is a strong apoptosis survival factor for endothelial cells. In this study, the receptor/second messenger signal transduction pathway for the antiapoptotic effect of Ang1 on human umbilical vein endothelial cells was examined. Pretreatment with soluble Tie2 receptor, but not Tie1 receptor, blocked the Ang1-induced antiapoptotic effect. Ang1 induced phosphorylation of Tie2 and the p85 subunit of phosphatidylinositol 3'-kinase (PI 3'-kinase) and increased PI 3'-kinase activity in a dose-dependent manner. The PI 3'-kinase-specific inhibitors wortmannin and LY294002 blocked the Ang1-induced antiapoptotic effect. Ang1 induced phosphorylation of the serine-threonine kinase Akt at Ser473 in a PI 3'-kinase-dependent manner. Expression of a dominant-negative form of Akt reversed the Ang1-induced antiapoptotic effect. Ang1 mRNA and protein were present in vascular smooth muscle cells but not in endothelial cells. Cultured vascular smooth muscle cells, but not human umbilical vein endothelial cells, secreted Ang1. These findings indicate that the Tie2 receptor, PI 3'-kinase, and Akt are crucial elements in the signal transduction pathway leading to endothelial cell survival induced by the paracrine activity of Ang1.     

Natriuretic peptides suppress vascular endothelial cell growth factor signaling to angiogenesis

Vascular endothelial cell growth factor (VEGF) is essential for angiogenesis. Atrial natriuretic peptide (ANP) inhibits the production of VEGF, but whether this important vascular peptide also inter- rupts VEGF signaling to angiogenesis is unknown. In cultured bovine aortic endothelial cells, VEGF significantly stimulated extracellular signal-regulated protein kinase activity and phosphorylation, which was inhibited 60% by coincubation with ANP or a natriuretic peptide clearance receptor specific ligand (NPRC), C-type NAP-(4-23) [C-ANP-(4-23)]. VEGF also stimulated c-Jun N-terminal kinase (JNK) and p38 activities/phosphorylation that were prevented by the two natriuretic peptides (NP). A specific NP guanylate cyclase (GC) receptor antagonist, HS-142-1, blocked the actions of ANP [but not those of C-ANP-(4-23)], supporting the involvement of both GC and NPRC receptors. VEGF and expression of constituitively active JNK each stimulated the synthesis of cyclin D1 and increased the activity of the cyclin-dependent kinase-4, which was inhibited 55% by ANP. VEGF induced endothelial cell proliferation and migration, which was significantly blocked by NP or by expressing a dominant negative JNK-1. VEGF stimulated human microvascular endothelial cells to form capillary tubes, which was significantly inhibited by expressing dominant negative JNK-1 and by NP. Therefore, VEGF induction of critical steps in angiogenesis is enhanced through JNK activation. The actions are significantly prevented by NP, which act through both the NPRC and GC receptors to block growth factor signaling. Thus, NP are candidate antiangiogenesis factors that inhibit both the synthesis and function of VEGF.     

Caveolin‐1 and Rac regulate endothelial capillary‐like tubular formation and fenestral contraction in sinusoidal endothelial cells

Background/Aims: Rho guanidine triphosphatases (GTPases) are major regulators of cell migration. We investigated the cytoskeleton and Rho GTPases during cell migration and morphogenesis processes in isolated rat liver sinusoidal endothelial cells (LSECs) cultured on Matrigel while stimulated by the vascular endothelial growth factor (VEGF). Methods: To obtain primary monolayers, LSECs were cultured on Matrigel for 5–17 h with or without VEGF. Sinusoidal endothelial fenestrae (SEF) morphology was observed using scanning electron microscopy and transmission electron microscopy. RhoA, Rac1 and phosphorylated myosin light‐chain kinase, Rho‐binding domain of Rhotekin and the p21‐binding domain of p21‐activated protein kinase were analysed using Western blotting. Results: The LSECs showed cellular protrusions and or cords of aligned cells resembling primitive capillary‐like structures, with SEF contraction. Time course analyses of Rac1 activation matched specific morphological changes. Rac1 activity increased progressively to 17 h in cells cultured without VEGF, but markedly increased at 7 h in the presence of VEGF. RhoA activity was slightly elevated at 5 h. The levels of endogenous caveolin‐1 (CAV‐1) expression increased in a time‐dependent manner, reaching a peak at 7 h. CAV‐1 expression occurred immediately before the formation of the capillary‐like tube. Moreover, treatment with VEGF regulated CAV‐1 expression in LSECs. Conclusions: Spatial activation of Rac1 is involved in the formation of a capillary‐like tubular network accompanying SEF contraction in LSECs, implying that endothelial migration and adhesion are necessary for LSECs tubular formation in the liver. CAV‐1 might play an important positive role in the regulation of LSEC tubular formation.     

Selective adapter recruitment and differential signaling networks by VEGF vs. shear stress

Vascular endothelial cells are continuously exposed to mechanical and chemical stimuli, such as shear stress and VEGF, respectively. It is still not clear how cells perceive these stimuli and orchestrate their responses. Studying the molecular mechanism by which shear stress and VEGF regulate the signaling pathways in bovine endothelial aortic cells, we found that VEGF induced a rapid association of VEGF receptor 2 (Flk-1) with Nck beta, but shear stress did not have such an effect. SU1498 (a specific inhibitor of Flk-1) and Nck beta(nm) (a negative mutant of Nck beta) blocked the VEGF-induced ERK and JNK activities. Only SU1498, but not Nck beta(nm), inhibited the shear-induced ERK activity. Furthermore, neither SU1498 nor Nck beta(nm) had significant effects on the shear-induced JNK activity, which can be blocked by inhibitors of Src family kinase and ROCK kinase. Therefore, mechanical (shear stress) and chemical (VEGF) stimuli diverge at the receptor Flk-1 in terms of the recruitment of the adapter protein Nck beta, and they employ different components of the complex signaling network in regulating downstream molecules, e.g., ERK and JNK.     

Involvement of SAPK/JNK in basic fibroblast growth factor-induced vascular endothelial growth factor release in osteoblasts

We previously reported that basic fibroblast growth factor (FGF-2) activates p44/p42 mitogen-activated protein (MAP) kinase resulting in the stimulation of vascular endothelial growth factor (VEGF) release in osteoblast-like MC3T3-E1 cells and that FGF-2-activated p38 MAP kinase negatively regulates VEGF release. In the present study, we investigated the involvement of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in FGF-2-induced VEGF release in these cells. FGF-2 markedly induced the phosphorylation of SAPK/JNK. SP600125, an inhibitor of SAPK/JNK, markedly reduced the FGF-2-induced VEGF release. SP600125 suppressed the FGF-2-induced phosphorylation of SAPK/JNK without affecting the phosphorylation of p44/p42 MAP kinase or p38 MAP kinase induced by FGF-2. PD98059, an inhibitor of upstream kinase of p44/p42 MAP kinase, or SB203580, an inhibitor of p38 MAP kinase, failed to affect the FGF-2-induced phosphorylation of SAPK/JNK. A combination of SP600125 and SB203580 suppressed the FGF-2-stimulated VEGF release in an additive manner. These results strongly suggest that FGF-2 activates SAPK/JNK in osteoblasts, and that SAPK/JNK plays a part in FGF-2-induced VEGF release.     

Rho and ROCK signaling in VEGF-induced microvascular endothelial hyperpermeability

OBJECTIVES: Vascular endothelial growth factor (VEGF) plays an important role in the regulation of microvascular permeability under various physiological and pathological conditions. The authors tested the hypothesis that the small GTPase Rho and its downstream effector ROCK (Rho-associated coiled-coil-containing protein kinase) mediate VEGF-induced increases in venular permeability. They also investigated myosin light chain (MLC) phosphorylation and actin polymerization, two well-characterized targets of the Rho-ROCK pathway that are implicated in the regulation of endothelial barrier function. METHODS: The apparent permeability coefficient of albumin (P(a)) was measured in intact isolated porcine coronary venules and in cultured coronary venular endothelial cell (CVEC) monolayers. RhoA activation was determined using a Rhotekin-agarose pull down assay. MLC phosphorylation was evaluated by immunoblotting with phospho-specific antibodies, and endothelial cellular F-actin was viewed using fluorescence microscopy. RESULTS: VEGF increased P(a) in both isolated coronary venules and CVEC monolayers. The hyperpermeability response occurred in a similar time course to that of Rho activation, MLC phosphorylation, and actin stress fiber formation. Selective blockage of ROCK with Y27632 dose-dependently inhibited VEGF-induced venular hyperpermeability. Moreover, inhibition of either Rho with exoenzyme C3 or ROCK with Y-27632 attenuated VEGF-induced increases in permeability, MLC phosphorylation, and actin-stress fiber formation in CVEC monolayers. CONCLUSIONS: Collectively, these findings suggest that the Rho-ROCK signal pathway contributes to VEGF-induced hyperpermeability. Myosin light-chain phosphorylation and actin stress fiber formation occur concomitantly with the increase in permeability upon VEGF stimulation.     

IP-10 blocks vascular endothelial growth factor-induced endothelial cell motility and tube formation via inhibition of calpain

Angiogenesis plays a critical role in wound repair. Endothelial cells present CXC receptor 3 (CXCR3) for chemokines expressed late in wound regeneration. To understand the physiological role CXCR3 plays in regulating endothelial function, we analyzed the ability of a CXCR3 ligand, IP-10 (CXCL10), to influence endothelial cell tube formation. Treatment of endothelial cells with IP-10 in the presence of vascular endothelial growth factor (VEGF) inhibited tube formation on growth factor-reduced Matrigel and in a subcutaneous Matrigel plug. Furthermore, IP-10 significantly inhibited VEGF-induced endothelial motility, a response critical for angiogenesis. Previous work showed that CXCR3 ligandation initiates protein kinase A (PKA) phosphorylation-dependent inhibition of m-calpain, required for induced cell motility, in fibroblasts but not epithelial cells. Here we show that CXCR3 activation in endothelial cells induces an increase in cAMP and PKA activation. Treatment of endothelial cells with Rp-8-Br-cAMP, an inhibitor of PKA, or small interference RNA to PKA was able to reverse the inhibitory effects of IP-10 on VEGF-mediated tube formation and motility. Importantly, treatment of endothelial cells with VEGF induced the activation of m-calpain, but costimulation with IP-10 significantly decreased this activity. Using Rp-8-Br-cAMP, we show blocking PKA reversed the IP-10 inhibition of VEGF-induced m-calpain activity. These data indicate that the activation of CXCR3 inhibits endothelial tube formation through a PKA mediated inhibition of m-calpain. This provides a means by which late wound repair signals limit the angiogenesis driven early in the wound response process.     

Angiotensin II impairs the insulin signaling pathway promoting production of nitric oxide by inducing phosphorylation of insulin receptor substrate-1 on Ser312 and Ser616 in human umbilical vein endothelial cells

It has been suggested that serine (Ser) phosphorylation of insulin receptor substrate-1 (IRS-1) decreases the ability of IRS-1 to be phosphorylated on tyrosine, thereby attenuating insulin signaling. There is evidence that angiotensin II (AII) may impair insulin signaling to the IRS-1/phosphatydilinositol 3-kinase (PI 3-kinase) pathway by enhancing Ser phosphorylation. Insulin stimulates NO production by a pathway involving IRS-1/PI3-kinase/Akt/endothelial NO synthase (eNOS). We addressed the question of whether AII affects insulin signaling involved in NO production in human umbilical vein endothelial cells and tested the hypothesis that the inhibitory effect of AII on insulin signaling was caused by increased site-specific Ser phosphorylation in IRS-1. Exposure of human umbilical vein endothelial cells to AII resulted in inhibition of insulin-stimulated production of NO. This event was associated with impaired IRS-1 phosphorylation at Tyr612 and Tyr632, two sites essential for engaging the p85 subunit of PI3-kinase, resulting in defective activation of PI 3-kinase, Akt, and eNOS. This inhibitory effect of AII was reversed by the type 1 receptor antagonist losartan. AII increased c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 activity, which was associated with a concomitant increase in IRS-1 phosphorylation at Ser312 and Ser616, respectively. Inhibition of JNK and ERK1/2 activity reversed the negative effects of AII on insulin-stimulated NO production. Our data suggest that AII, acting via the type 1 receptor, increases IRS-1 phosphorylation at Ser312 and Ser616 via JNK and ERK1/2, respectively, thus impairing the vasodilator effects of insulin mediated by the IRS-1/PI 3-kinase/Akt/eNOS pathway.     

Vascular endothelial growth factor protects hepatoma cells against oxidative stress-induced cell death

BACKGROUND: The aim of the present study was to examine coordination of the vascular endothelial growth factor (VEGF) and VEGF receptor (Flk-1) system and to study control of VEGF expression by oxidative stress, which is considered a model for chronic liver disease. METHODS: Cell viability was determined by test method with 3-[4, 5-dimethylthiazol-2-yl]-2, 5-dephenyl tetrazolium bromide (MTT). Expressions of cellular proteins were evaluated by western blot analysis. RESULTS: The c-Met tyrosine phosphorylation in PLC/PRF/5 hepatoma cells was increased by treatment with 20 ng/mL hepatocyte growth factor (HGF), and extracellular signal-regulated kinase (ERK) was also activated. Although Flk-1 was phosphorylated in response to VEGF (>50 ng/mL), phosphorylated ERK was not detected at these concentrations. A total of 5.0 and 10 micromol/L hydrogen peroxide (H(2)O(2)) caused cell death in a dose-dependent manner after 24 h. On western blot analysis at 1 h with H(2)O(2), rapid phosphorylation of both ERK1/2 and c-Jun NH(2)-terminal kinase (JNK) was observed. In the first 6 h, H(2)O(2) induced cell death for 58.4 +/- 6.8%, whereas the presence of 100 ng/mL VEGF improved the survival rate to 77.2 +/- 4.2%. The VEGF significantly decreased H(2)O(2)-induced cell death after 12 h, whereas HGF (20 ng/mL) did not have a similar effect. When cells were incubated with 5 micromol/L H(2)O(2), expression of VEGF protein was detected. Furthermore, H(2)O(2)-induced phosphorylation of ERK and JNK was also reduced by VEGF (100 ng/mL). In contrast, HGF did not induce phosphorylation of ERK and JNK. CONCLUSION: Hepatoma cells might be able to survive under continuous oxidative stress through expression of VEGF.     

Role of RhoA and Rho kinase in lysophosphatidic acid-induced endothelial barrier dysfunction

In the present study, the roles of the small GTPase RhoA and its target Rho kinase in endothelial permeability were investigated in vitro. We have shown previously that, in addition to a rise in the intracellular Ca(2+) concentration ([Ca(2+)](i)), RhoA is involved in the prolonged thrombin-induced barrier dysfunction. To study the role of RhoA and Rho kinase more specifically, endothelial cells were stimulated with lysophosphatidic acid (LPA), a commonly used RhoA activator. LPA induced a 2- to 3-fold increase in the passage of horseradish peroxidase (HRP) across endothelial monolayers that lasted for several hours, whereas thrombin induced a 5- to 10-fold increase. Comparable to the thrombin-induced barrier dysfunction, the LPA-induced barrier dysfunction was accompanied by a reorganization of the F-actin cytoskeleton and the formation of focal attachment sites. LPA induced only a transient increase in myosin light-chain (MLC) phosphorylation, which returned to basal level within 10 minutes. In endothelial cells, [Ca(2+)](i) was not elevated by LPA. Chelation of Ca(2+)(i) ions by 1, 2-bis(2-aminophenoxy)ethane-N:,N:,N:',N:'-tetraacetic acid did not prevent the LPA-induced passage of HRP. Apparently, a low degree of MLC kinase activation occurred, because the MLC kinase inhibitor KT5926 reduced the levels of both basal and LPA-stimulated HRP passage. Inhibition of RhoA by the C3 transferase from Clostridium botulinum inhibited the LPA-induced cytoskeletal changes and prevented the LPA-induced HRP passage. Inhibition of Rho kinase by Y-27632 completely prevented the LPA-induced increase in HRP passage without affecting basal permeability. These data indicate that LPA-induced endothelial hyperpermeability occurs without a change in [Ca(2+)](i) and requires activation of RhoA and Rho kinase.     

Involvement of COX-2 in VEGF-induced angiogenesis via P38 and JNK pathways in vascular endothelial cells

OBJECTIVE: Cyclooxygenase-2 (COX-2) is induced by hypoxic stimuli and is also involved in the process of angiogenesis. We previously demonstrated that vascular endothelial growth factor (VEGF) is one of the principal factors produced by hypoxic myocytes and is responsible for the induction of COX-2 expression in endothelial cells. Yet the signaling pathways by which VEGF modulates COX-2 gene expression are still less well defined. We therefore examined the regulation of VEGF-induced COX-2 expression by the mitogen-activated protein kinase (MAPK) family in endothelial cells. METHODS AND RESULTS: Human umbilical vascular endothelial cells (HUVECs) were incubated with U0126 (ERK1/2 inhibitor, 10 microM), SB203580 (p38 inhibitor, 20 microM), and SP600125 (JNK inhibitor, 20 microM), as well as the COX-2 selective inhibitor, NS398, for 1 h before treating with VEGF (20 ng/ml). COX-2 expression induced by VEGF at both mRNA and protein levels was significantly inhibited by selective p38 and JNK inhibitors but not by the ERK1/2 inhibitor. The phosphorylation of p38 and JNK kinases was observed as early as 5 min in HUVECs after VEGF stimulation. Furthermore, the biological significance of the COX-2 gene in endothelial cells was examined by over-expressing or knocking down COX-2 gene expression. (3)H-Thymidine incorporation and Matrigel techniques were used to determine cell proliferation and vascular structure formation. VEGF-induced cell proliferation was significantly reduced when HUVECs were either pre-treated with NS398 (21.52+/-3.6%) or transfected with COX-2 siRNA (34.12+/-5.81%). In contrast, in HUVECs with over-expression of COX-2, VEGF-induced cell proliferation was increased 42.56+/-7.69%. Moreover, the formation of vascular structure assayed by Matrigel demonstrated that VEGF-induced vascular structure formation was accelerated in COX-2 over-expressing cells but attenuated in COX-2 siRNA-transfected cells. CONCLUSION: COX-2 plays an important role in VEGF-induced angiogenesis via p38 and JNK kinase activation pathways. These findings suggest that the cardioprotective role of COX-2 may be, at least in part, through its angiogenic activity.     

Visfatin induces human endothelial VEGF and MMP-2/9 production via MAPK and PI3K/Akt signalling pathways: novel insights into visfatin-induced angiogenesis

AIMS: Visfatin is a novel adipokine whose plasma concentrations are altered in obesity and obesity-related disorders; these states are associated with an increased incidence of cardiovascular disease. We therefore investigated the effect of visfatin on vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP-2, MMP-9) production and the potential signalling cascades. METHODS AND RESULTS: In human umbilical vein endothelial cells (HUVECs), visfatin significantly and dose-dependently up-regulated gene expression and protein production of VEGF and MMPs and down-regulated expression of tissue inhibitors of MMPs (TIMP-1 and TIMP-2). The gelatinolytic activity of MMPs (analysed by zymography) correlated with mRNA and western blot findings. Interestingly, visfatin significantly up-regulated VEGF receptor 2 expression. Inhibition of VEGFR2 and VEGF [by soluble FMS-like tyrosine kinase-1 (sFlt1)] down-regulated visfatin-induced MMP induction. Visfatin induced dose- and time-dependent proliferation and capillary-like tube formation. Importantly, visfatin was noted to have anti-apoptotic effects. In HUVECs, visfatin dose-dependently activated PI3K/Akt (phosphatidylinositol 3-kinase/Akt) and ERK(1/2) (extracellular signal-regulated kinase) pathways. The functional effects and MMP/VEGF induction were shown to be dependent on the MAPK/PI3K-Akt/VEGF signalling pathways. Inhibition of PI3K/Akt and ERK(1/2) pathways led to significant decrease of visfatin-induced MMP and VEGF production and activation, along with significant reduction in endothelial proliferation and capillary tube formation. CONCLUSION: Our data provide the first evidence of visfatin-induced endothelial VEGF and MMP production and activity. Further, we show for the first time the involvement of the MAPK and PI3K/Akt signalling pathways in mediating these actions, as well as endothelial cell proliferation. Collectively, our findings provide novel insights into visfatin-induced endothelial angiogenesis.