Vascular Endothelial Growth Factor Governs Endothelial Nitric-oxide Synthase Expression via a KDR/Flk-1 Receptor and a Protein Kinase C Signaling Pathwayстатья из журнала
Аннотация: The mechanism by which vascular endothelial growth factor (VEGF) regulates endothelial nitric-oxide synthase (eNOS) expression is presently unclear. Here we report that VEGF treatment of bovine adrenal cortex endothelial cells resulted in a 5-fold increase in both eNOS protein and activity. Endothelial NOS expression was maximal following 2 days of constant VEGF exposure (500 pm) and declined to base-line levels by day 5. The elevated eNOS protein level was sustained over the time course if VEGF was co-incubated withl-N G-nitroarginine methyl ester, a competitive eNOS inhibitor. Addition ofS-nitroso-N-acetylpenicillamine, a nitric oxide donor, prevented VEGF-induced eNOS up-regulation. These data suggest that nitric oxide participates in a negative feedback mechanism regulating eNOS expression. Various approaches were used to investigate the role of the two high affinity VEGF receptors in eNOS up-regulation. A KDR receptor-selective mutant increased eNOS expression, whereas an Flt-1 receptor-selective mutant did not. Furthermore, VEGF treatment increased eNOS expression in a KDR but not in an Flt-1 receptor-transfected porcine aorta endothelial cell line. SU1498, a selective inhibitor of the KDR receptor tyrosine kinase, blocked eNOS up-regulation, thus providing further evidence that the KDR receptor signals for eNOS up-regulation. Finally, treatment of adrenal cortex endothelial cells with VEGF or phorbol ester resulted in protein kinase C activation and elevated eNOS expression, whereas inhibition of protein kinase C with isoform-specific inhibitors abolished VEGF-induced eNOS up-regulation. Taken together, these data demonstrate that VEGF increases eNOS expression via activation of the KDR receptor tyrosine kinase and a downstream protein kinase C signaling pathway. The mechanism by which vascular endothelial growth factor (VEGF) regulates endothelial nitric-oxide synthase (eNOS) expression is presently unclear. Here we report that VEGF treatment of bovine adrenal cortex endothelial cells resulted in a 5-fold increase in both eNOS protein and activity. Endothelial NOS expression was maximal following 2 days of constant VEGF exposure (500 pm) and declined to base-line levels by day 5. The elevated eNOS protein level was sustained over the time course if VEGF was co-incubated withl-N G-nitroarginine methyl ester, a competitive eNOS inhibitor. Addition ofS-nitroso-N-acetylpenicillamine, a nitric oxide donor, prevented VEGF-induced eNOS up-regulation. These data suggest that nitric oxide participates in a negative feedback mechanism regulating eNOS expression. Various approaches were used to investigate the role of the two high affinity VEGF receptors in eNOS up-regulation. A KDR receptor-selective mutant increased eNOS expression, whereas an Flt-1 receptor-selective mutant did not. Furthermore, VEGF treatment increased eNOS expression in a KDR but not in an Flt-1 receptor-transfected porcine aorta endothelial cell line. SU1498, a selective inhibitor of the KDR receptor tyrosine kinase, blocked eNOS up-regulation, thus providing further evidence that the KDR receptor signals for eNOS up-regulation. Finally, treatment of adrenal cortex endothelial cells with VEGF or phorbol ester resulted in protein kinase C activation and elevated eNOS expression, whereas inhibition of protein kinase C with isoform-specific inhibitors abolished VEGF-induced eNOS up-regulation. Taken together, these data demonstrate that VEGF increases eNOS expression via activation of the KDR receptor tyrosine kinase and a downstream protein kinase C signaling pathway. vascular endothelial growth factor endothelial nitric-oxide synthase adrenal cortex endothelial cells l-N G-nitroarginine methyl ester S-nitroso-N-acetylpenicillamine nitric oxide protein kinase C phorbol 12-myristate 13-acetate porcine aorta endothelial endothelial cell placental growth factor-2 phospholipase C phosphatidylinositol 3-kinase hepatocyte growth factor fibroblast growth factor transforming growth factor epidermal growth factor Western blot Vascular endothelial growth factor (VEGF)1 is a potent vascular endothelial cell (EC)-specific mitogen that stimulates EC proliferation, microvascular permeability, vasodilation, and angiogenesis (1Neufeld G. Cohen T. Gengrinovitch S. Poltorak Z. FASEB J. 1999; 13: 9-22Crossref PubMed Scopus (3148) Google Scholar, 2Ferrara N. Davis-Smyth T. Endocr. Rev. 1997; 18: 4-25Crossref PubMed Scopus (3668) Google Scholar). Recently, VEGF has also been shown to improve EC function and survival in vitro and vascular reactivityin vivo (3Spyridopoulos I. Brogi E. Kearney M. Sullivan A.B. Cetrulo C. Isner J.M. Losordo D.W. J. Mol. Cell Cardiol. 1997; 29: 1321-1330Abstract Full Text PDF PubMed Scopus (193) Google Scholar, 4Gerber H.P. Dixit V. Ferrara N. J. Biol. Chem. 1998; 273: 13313-13316Abstract Full Text Full Text PDF PubMed Scopus (835) Google Scholar, 5Couffinhal T. Kearney M. Witzenbichler B. Chen D. Murohara T. Losordo D.W. Symes J. Isner J.M. Am. J. Pathol. 1997; 150: 1673-1685PubMed Google Scholar, 6Katoh O. Takahashi T. Oguri T. Kuramoto K. Mihara K. Kobayashi M. Hirata S. Watanabe H. Cancer Res. 1998; 58: 5565-5569PubMed Google Scholar, 7Mitchell C.A. Risau W. Drexler H.C. Dev. Dyn. 1998; 213: 322-333Crossref PubMed Scopus (98) Google Scholar). Several VEGF isoforms including VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206 have been identified (8Houck K.A. Ferrara N. Winer J. Cachianes G. Li B. Leung D.W. Mol. Endocrinol. 1991; 5: 1806-1814Crossref PubMed Scopus (1236) Google Scholar, 9Poltorak Z. Cohen T. Sivan R. Kandelis Y. Spira G. Vlodavsky I. Keshet E. Neufeld G. J. Biol. Chem. 1997; 272: 7151-7158Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar). All isoforms except VEGF121 have a heparin-binding domain that interacts with cell-associated heparan sulfate proteoglycans (10Athanassiades A. Hamilton G.S. Lala P.K. Biol. Reprod. 1998; 59: 643-654Crossref PubMed Scopus (119) Google Scholar, 11Mathews M.K. Merges C. McLeod D.S. Lutty G.A. Invest. Ophthalmol. & Visual Sci. 1997; 38: 2729-2741PubMed Google Scholar). VEGF165 is the most predominant form secreted by various cell types and some tumor cells (2Ferrara N. Davis-Smyth T. Endocr. Rev. 1997; 18: 4-25Crossref PubMed Scopus (3668) Google Scholar). VEGF has two high affinity tyrosine kinase receptors, the Fms-like tyrosine kinase (Flt-1) and kinase insert domain-containing receptor (KDR/Flk-1), which are predominantly expressed on endothelial cells (12Terman B.I. Carrion M.E. Kovacs E. Rasmussen B.A. Eddy R.L. Shows T.B. Oncogene. 1991; 6: 1677-1683PubMed Google Scholar). Recently, Soker et al. (13Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2076) Google Scholar) identified a novel VEGF receptor that has a sequence identical to that of neuropilin. This receptor binds to VEGF165 and placental growth factor-2 (PlGF-2) but not to VEGF121 (13Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2076) Google Scholar, 14Migdal M. Huppertz B. Tessler S. Comforti A. Shibuya M. Reich R. Baumann H. Neufeld G. J. Biol. Chem. 1998; 273: 22272-22278Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Recent studies have demonstrated that eNOS/NO plays an important role in many VEGF-induced actions. VEGF has been shown to induce the release of NO from rabbit, pig, bovine, and human vascular endothelial cells (15van der Zee R. Murohara T. Luo Z. Zollmann F. Passeri J. Lekutat C. Isner J.M. Circulation. 1997; 95: 1030-1037Crossref PubMed Scopus (372) Google Scholar, 16Parenti A. Morbidelli L. Cui X.-L. Douglas J.G. Hood J.D. Granger H.J. Ledda F. Ziche M. J. Biol. Chem. 1998; 273: 4220-4226Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 17Morbidelli L. Chang C.-H. Douglas J.G. Am. J. Physiol. 1996; 270: H411-H415Crossref PubMed Google Scholar). In vitro, VEGF stimulated human EC to grow in a NO-dependent manner and promoted the NO-dependent formation of vessel-like structures in the three-dimensional collagen gel model (18Papapetropoulos A. Garcı̂a-Cardeña G. Madri J.A. Sessa W.C. J. Clin. Invest. 1997; 100: 3131-3139Crossref PubMed Scopus (1018) Google Scholar). Conversely, inhibition of NO production by eNOS inhibitors significantly inhibited VEGF-induced mitogenic and angiogenic effects (18Papapetropoulos A. Garcı̂a-Cardeña G. Madri J.A. Sessa W.C. J. Clin. Invest. 1997; 100: 3131-3139Crossref PubMed Scopus (1018) Google Scholar). Similar results have been obtained in vivo where inactivation of eNOS expression significantly impaired VEGF-induced angiogenesis in an eNOS knockout mouse model (19Murohara T. Asahara T. Silver M. Bauters C. Masuda H. Kalka C. Kearney M. Chen D. Chen D. Symes J.F. Fishman M.C. Huang P.L. Isner J.M. J. Clin. Invest. 1998; 101: 2567-2578Crossref PubMed Scopus (1085) Google Scholar, 20Ziche M. Morbidelli L. Choudhuri R. Zhang H.-T. Donnini S. Granger H.J. J. Clin. Invest. 1997; 99: 2625-2634Crossref PubMed Google Scholar, 21Rudic R.D. Shesely E.G. Maeda N. Smithies O. Segal S.S. Sessa W.C. J. Clin. Invest. 1998; 101: 731-736Crossref PubMed Scopus (704) Google Scholar). Endothelial NOS/NO has also been implicated as one of the important mediators for VEGF-induced hemodynamic changes and microvascular permeability. For example, Yang et al. (22Yang R. Thomas R. Bunting S. Ko A. Ferrara N. Keyt B.A. Jin H. J. Cardiovasc. Pharmacol. 1996; 27: 838-844Crossref PubMed Scopus (218) Google Scholar) and Hariawala et al. (23Hariawala M.D. Horowitz J.R. Esakof D. Sheriff D.D. Walter D.H. Keyt B.A. Isner J.M. Symes J.F. J. Surg. Res. 1996; 63: 77-82Abstract Full Text PDF PubMed Scopus (238) Google Scholar) reported that pretreatment of rats with an eNOS inhibitor significantly attenuated the hypotensive effects of an intravenous bolus dose of VEGF. Wu et al. (24Wu H.M. Huang Q.B. Yuan Y. Granger H.J. Am. J. Physiol. 1996; 271: H2735-H2739Crossref PubMed Google Scholar) showed that topical application of VEGF resulted in a transient and dose-dependent increase in albumin permeability in isolated coronary venules. Inhibition of NO synthesis with an eNOS inhibitor completely abolished VEGF-induced venular hyperpermeability. These observations highlight the importance of understanding the underlying mechanisms involved in VEGF regulation of eNOS. Although eNOS was originally described as a constitutive enzyme, recent studies indicated that a variety of stimuli including hypoxia, shear stress, inflammatory cytokines, high glucose, and injury could modulate eNOS expression and activity (25Sase K. Michel T. Trends Cardiovasc. Med. 1997; 7: 28-37Crossref PubMed Scopus (84) Google Scholar, 26Le Cras T.D. Xue C. Rengasamy A. Johns R.A. Am. J. Physiol. 1996; 270: L164-L170PubMed Google Scholar, 27Ziegler T. Silacci P. Harrison V.J. Hayoz D. Hypertension. 1998; 32: 351-355Crossref PubMed Scopus (208) Google Scholar, 28Xiao Z. Zhang Z. Ranjan V. Diamond S.L. J. Cell. Physiol. 1997; 171: 205-211Crossref PubMed Scopus (96) Google Scholar). More recent studies indicate that VEGF also regulates eNOS expression (18Papapetropoulos A. Garcı̂a-Cardeña G. Madri J.A. Sessa W.C. J. Clin. Invest. 1997; 100: 3131-3139Crossref PubMed Scopus (1018) Google Scholar, 29Kroll J. Waltenberger J. Biochem. Biophys. Res. Commun. 1998; 252: 743-746Crossref PubMed Scopus (307) Google Scholar, 30Hood J.D. Meininger C.J. Ziche M. Granger H.J. Am. J. Physiol. 1998; 274: H1054-H1058PubMed Google Scholar). Here we provide a detailed characterization of the mechanism governing VEGF-induced eNOS up-regulation and demonstrate that activation of the KDR receptor tyrosine kinase and a downstream PKC pathway are required. Furthermore, our data indicate that NO inhibits the VEGF-induced increase in eNOS expression, suggesting a physiological role for NO in feedback inhibiting eNOS expression. These results provide important insights into our understanding of the mechanism(s) by which VEGF regulates eNOS and endothelial function. Recombinant human VEGF165(rhVEGF165) was produced in Escherichia coli(Genentech). The VEGF110 heparin-binding domain-deficient variant was made from VEGF165 by limited proteolytic digestion with plasmin as described previously (31Keyt B. Berleau L.T. Nguyen H.V. Chen H. Heinsohn H. Vandlen R. Ferrara N. J. Biol. Chem. 1996; 271: 7788-7795Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar). VEGF receptor-selective mutants Flt-sel (R82E/K84E/H86E, deficient in the KDR binding) and KDR-sel (D63A/E64A/E67A, deficient in Flt-1 binding) were prepared using the Muta-Gene Phagemid in vitromutagenesis kit as described previously (32Keyt B.A. Nguyen H.V. Berleau L.T. Duarte C.M. Park J. Chen H. Ferrara N. J. Biol. Chem. 1996; 271: 5638-5646Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar). The heterodimeric form of recombinant human hepatocyte growth factor (HGF) was produced in and isolated from Chinese hamster ovary cells as described previously (33Shen B.-Q. Panos R.J. Hansen-Guzman K. Widdicombe J.H. Mrsny R.J. Am. J. Physiol. 1997; 272: L1115-L1120PubMed Google Scholar). Recombinant human basic fibroblast growth factor (FGF), recombinant human placental growth factor (PlGF), recombinant human transforming growth factor (TGF)-β1, TGF-β2, and recombinant human epidermal growth factor (EGF) were purchased from R & D Systems, Inc. (Minneapolis, MN). Monoclonal anti-eNOS antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-PKC (α, β, γ, δ, ε, θ, ι, λ) antibodies were purchased from Transduction Laboratories (Lexington, KY). (±)-S-Nitroso-N-acetylpenicillamine (SNAP), PP1, staurosporine, herbimycin A, wortmannin, phorbol 12-myristate 13-acetate (PMA), l-N G-nitroarginine methyl ester (l-NAME), GF 109203X, rottlerin, and chelerythrine chloride were obtained from Biomol (Plymouth Meeting, PA). SU1498, calphostin C, Go 6976, H-8, and H-89 were purchased from Calbiochem. The PKC assay kit was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY), the NOSdetect assay kit was purchased from Stratagene (La Jolla, CA), and the enhanced chemiluminescent kit, l-[2,3,4,5-3H]arginine monohydrochloride and [γ-32P]ATP(∼3000 Ci/mmol) were purchased from Amersham Pharmacia Biotech. Geneticin (G480) was obtained from Life Technologies, Inc. All reagents were prepared as 1000× stock solutions unless otherwise specified. Bovine adrenal cortex capillary endothelial cells (ACE) were prepared and maintained as described previously (34Ferrara N. Henzel W.J. Biochem. Biophys. Res. Commun. 1989; 161: 851-858Crossref PubMed Scopus (2011) Google Scholar). Briefly, cells were plated onto 6-well tissue culture plates (Costar) and grown in low glucose Dulbecco's modified Eagle's medium, supplemented with 2 mml-glutamate (Life Technologies, Inc.), 10% bovine calf serum (HyClone), 100 μg/ml penicillin/streptomycin (Life Technologies, Inc.). ACE cells were used between passages 4 and 8. Porcine aorta endothelial (PAE) cells and receptor-transfected PAE cells (PAE/KDR and PAE/Flt-1) were obtained from Dr. Napoleone Ferrara (Genentech) and cultured in Ham's F-12 medium containing 10% fetal bovine serum (for PAE) or plus 250 μg/ml G480 (for PAE/KDR and PAE/Flt-1). For drug treatment, cells were incubated in the medium containing 10% fetal bovine serum supplemented with VEGF or other drugs as specified. Complete medium was replenished every 24 h. The methods for cell lysis and Western blot (WB) analysis have been previously described (35Shen B.-Q. Lee D.Y. Gerber H.-P. Keyt B.A. Ferrara N. Zioncheck T.F. J. Biol. Chem. 1998; 273: 29979-29985Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Monoclonal anti-eNOS or anti-PKC antibodies were used at the manufacturers' suggested concentration. A secondary antibody conjugated with horseradish peroxidase (Zymed Laboratories Inc., South San Francisco, CA) and an enhanced chemiluminescent kit was used to visualize the immunoreactive bands. Multiple exposures of films were obtained to determine the optimal exposure time. The protein bands were scanned by a densitometer, and the relative intensities were quantified using ImageQuant software (Molecular Dynamics). The eNOS activity in ACE cells treated with or without rhVEGF for 2 days was determined by measuring the formation of [3H]citrulline from [3H]arginine. Briefly, ACE cells were homogenized in a buffer containing 25 mm Tris-HCl, pH 7.4, 1 mmEDTA, and 1 mm EGTA and then subjected to microcentrifugation at 14,000 rpm for 5 min. Fifty μg of protein from the supernatants was incubated at 37 °C for 45 min with 1 mm NADPH, 25 mm Tris-HCl, pH 7.4, 3 μm tetrahydrobiopterin, 1 μm flavin adenine dinucleotide, 1 μm flavin adenine mononucleotide, 0.1 μCi/ml [3H]arginine, and other cofactors (calcium and calmodulin) provided in the assay kit. The reaction was stopped with 50 mm HEPES, pH 5.5, 5 mm EDTA. Equilibrated resin, which binds to the arginine, was added to the reactions and then pipetted into spin cups. [3H]Citrulline, which is ionically neutral at pH 5.5, flowed through the cups and was quantitated by scintillation counting. Confluent ACE cells were incubated with VEGF for 0, 2.5, 5, 10, 15, 30, and 60 min. The cells were washed once with cold phosphate-buffered saline, harvested, and homogenized by sonication in a buffer containing 20 mm Tris-HCl, pH 7.5, 2 mm EDTA, 0.5 mm EGTA, 330 mmsucrose, 1 mm phenylmethylsulfonyl fluoride, 1 mm sodium orthovanadate, 0.025% leupeptin, and aprotonin. Total PKC activity in equal amount of homogenates was determined by measuring phosphatidylserine/diacylglyceroldependent phosphorylation of exogenous PKC-specific peptide substrate (QKRPSQRSKYL) in the presence of Ca2+, Mg2+, protein kinase A inhibitor peptide, compound R 24571, and [γ-32P]ATP. The kinase reaction was terminated after 10 min at 30 °C. Endogenous phosphorylation of proteins in the sample extract was determined by substituting assay dilution buffer for substrate. PKC activity (pmol/min/mg) was calculated using the following equation: (A − B) ×F/S.A. × 10 min/mg, where A is total radioactivity (cpm), B is background protein phosphorylation (cpm), F is a dilution factor, and S.A. is [γ-32P]ATP-specific radioactivity (∼2500 CPM/pmole ATP). To determine the redistribution of PKC isoforms, cell homogenates following 10 min of VEGF treatment were fractionated into cytosolic and membrane fractions according to the method described by Xia et al. (36Xia P. Aiello L.P. Ishii H. Jiang Z.Y. Park D.J. Robinson G.S. Takagi H. Newsome W.P. Jirousek M.R. King G.L. J. Clin. Invest. 1996; 98: 2018-2026Crossref PubMed Scopus (525) Google Scholar). Equivalent amounts of protein from both cytosolic and membrane fractions were electrophoresed by SDS-polyacrylamide gel electrophoresis, blotted, and probed with monoclonal anti-PKC-isoform peptide antibodies as described above. The results are reported as mean ± S.D. An unpaired Student's t test was used to determine statistical significance. A value of p < 0.05 was considered significant. To study whether VEGF regulates eNOS expression, ACE cells were incubated with rhVEGF for 0–5 days. At the end of incubation total cell lysates were prepared, and eNOS protein levels were determined by Western blot as described under "Experimental Procedures." Fig.1 A shows the results of a time course experiment, indicating that prolonged VEGF treatment induced a transient increase in eNOS expression. The peak expression (5.5-fold) was observed 2 days post-exposure to 500 pm VEGF. The eNOS protein levels returned to baseline by day 5 (Fig. 1 B). VEGF also induced eNOS up-regulation in a dose-dependent manner with the maximal increase occurring following incubation with 500 pm VEGF (data not shown). To investigate whether VEGF treatment increases eNOS activity, an equal amount of cell lysate from cells treated with or without VEGF for 2 days was used to run a NOS activity assay. Fig. 1 C shows that NOS activity in VEGF-treated cells was ∼5-fold greater than that in untreated control cells (p < 0.05), which was proportional to the increase in eNOS protein level (5.5-fold). The total NOS activity measured likely represents eNOS activity because only Ca2+-dependent NOS activity was measured, and inducible NOS protein was not detected in ACE cells by WB (data not shown). Taken together, these data demonstrate that VEGF increases both eNOS expression and activity in cultured endothelial cells. The role of NO in VEGF-induced eNOS expression was investigated by co-incubation of VEGF with either l-NAME, an eNOS inhibitor, or SNAP, a stable NO donor. Fig.2 shows that VEGF alone induced a transient increase in eNOS expression, whereas co-incubation of VEGF with 2 mml-NAME resulted in a sustained increase in eNOS expression. Addition of 100 μm SNAP to the media blocked both transient and sustained up-regulation of eNOS induced by VEGF (Fig. 2) or VEGF/l-NAME, respectively (data not shown). Neither l-NAME nor SNAP alone had a significant effect on eNOS expression (Fig. 2). To rule-out the possibility thatl-NAME or SNAP affected VEGF stability, VEGF-containing medium was replaced every 24 h, and culture medium was assayed in a VEGF enzyme-linked immunosorbent assay. There was no difference in VEGF levels among these groups (data not shown). These results suggest that endogenous NO produced by endothelial cells may negatively regulate VEGF-induced eNOS expression. Several approaches were used to determine which VEGF receptor(s) was involved in signaling for increasing eNOS expression. First, VEGF receptor-selective mutants, which bind preferentially to KDR or Flt-1, were incubated with ACE cells. Fig.3 A shows that VEGF165 and VEGF110, a heparin-binding domain-deficient mutant with normal binding to KDR and Flt-1, induced a similar degree (5-fold) of eNOS up-regulation. The KDR-selective mutant (KDR-sel) that binds to KDR receptor normally but with reduced binding to Flt-1 also up-regulated eNOS expression, whereas the Flt-1-selective binding mutant (Flt-sel) failed to do so. PlGF, which is known to only bind to Flt-1 receptor, had no effect on eNOS expression. These data suggest that the KDR receptor was the dominant receptor involved in VEGF-induced increases in eNOS expression. The involvement of KDR receptor in eNOS up-regulation was further confirmed by using VEGF receptor-transfected PAE cells. Fig.3 B demonstrates that PAE/KDR cells were able to increase eNOS expression in response to VEGF, whereas PAE and PAE/Flt cells were not. The base-line expression of eNOS in PAE/Flt was lower than that in PAE and PAE/KDR cells, although equal amount of protein was used for eNOS detection (Fig. 3 B). The reason for this phenomenon is presently unclear. The critical role of KDR receptor tyrosine kinase in eNOS up-regulation was further demonstrated by co-incubation of VEGF with a selective KDR tyrosine kinase inhibitor, SU-1498. Fig. 3 C reveals that co-incubation of SU-1498 with VEGF resulted in a dose-dependent inhibition of eNOS expression. At high concentration (100 μm), SU-1498 completely blocked VEGF-induced eNOS up-regulation. Taken together, these data demonstrate that KDR receptor activation is required for VEGF-induced eNOS expression. Various inhibitors were also tested to investigate the role of downstream signaling molecules in VEGF-induced eNOS expression. Fig.4 shows that inhibition of tyrosine kinase activity with herbimycin A, an irreversible and selective inhibitor of protein tyrosine kinases, blocked VEGF-induced eNOS expression. Furthermore, PP1 inhibition of PLC-γ, an upstream signaling molecule of PKC and PI3-K, also blocked eNOS expression (Fig.4). However, inhibition of PI3-K with wortmannin had no significant effect on eNOS levels (data not shown). These data indicate that PKC signaling was the predominant pathway. The role of PKC in VEGF-induced eNOS up-regulation was further characterized by examining specific PKC isoform expression, inhibition, and activation. First, WB analysis with eight PKC isoform-specific antibodies (anti-PKC-α, -β, -γ, -δ, -ε, -θ, -ι, and -λ) showed that ACE cells expressed multiple PKC isoforms, mainly, PKC-α, -γ, -δ, -ε, and -ι (data not shown). Inhibition of PKC with both broad (staurosporine, calphostin C, and chelerythrine chloride) and isoform-specific (GF 109203X, Go 6976, and rottlerin) inhibitors prevented eNOS up-regulation, whereas inhibition of protein kinase A or cyclic nucleotide kinases with H8 and H89, respectively, had no effect on VEGF-induced eNOS expression (Fig.4). Conversely, Fig. 5 Afurther shows that VEGF treatment only induced a rapid redistribution of PKC-α, -γ, and -ε from cytosolic to membrane fractions, indicating that VEGF specifically activated these three PKC isoforms. A PKC activity assay further showed that VEGF treatment (2.5–60 min) induced a time-dependent increase in total PKC activity (Fig. 5 B). Finally, direct activation of PKC with the phorbol ester PMA also resulted in a time- (Fig. 5 C) and dose-dependent (data not shown) increase in eNOS levels. These data imply an important role for PKC, and more specifically PKC-α, -γ, and -ε in the activation and signaling for VEGF-induced eNOS expression.Figure 5A PKC-dependent pathway is required for VEGF-induced eNOS up-regulation. A, a representative WB result showing that VEGF treatment resulted in a rapid redistribution of PKC-α, -γ, and -ε from cytosolic to membrane fractions. ACE cells were treated with VEGF for 10 min. Cytosolic (C) and membrane (M) fractions were prepared as described under "Experimental Procedures."C, cytosolic fraction; M, membrane fraction.B, VEGF increased total PKC activity. ACE cells were treated with VEGF for 2.5 to 60 min. PKC activities in cell homogenates were measured as described under "Experimental Procedures." Data reflect the mean ± S.D., n = 3. C, the PKC activator, PMA, also increased eNOS expression. A representative WB result showing that activation of PKC with PMA time-dependently increased eNOS expression. The data are representative of 3 independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The relative effect of several other angiogenic factors including FGF, HGF, EGF, and TGF-β on eNOS expression was also studied. ACE cells were treated with equimolar concentration of various growth factors for 2 days, and eNOS was detected by WB. Fig.6 shows that in addition to VEGF, HGF was also capable of increasing eNOS expression, whereas neither FGF nor EGF had a significant effect. TGF-β1 and TGF-β2 trended toward a slight reduction in eNOS expression. These data indicate that VEGF and HGF are distinct from the other angiogenic growth factors in their ability to increase eNOS expression in vitro. Exogenous NO and endothelium-derived NO have been shown to have several putative anti-atherosclerotic properties such as improved vascular homeostasis and decreased platelet aggregation. Others have shown a critical role for NO in VEGF-induced angiogenesis (18Papapetropoulos A. Garcı̂a-Cardeña G. Madri J.A. Sessa W.C. J. Clin. Invest. 1997; 100: 3131-3139Crossref PubMed Scopus (1018) Google Scholar, 25Sase K. Michel T. Trends Cardiovasc. Med. 1997; 7: 28-37Crossref PubMed Scopus (84) Google Scholar,37Kelly R.A. Balligand J.-L. Smith T.W. Circ. Res. 1996; 79: 363-380Crossref PubMed Scopus (629) Google Scholar, 38Ohashi Y. Kawashima S. Hirata K. Yamashita T. Ishida T. Inoue N. Sakoda T. Kurihara H. Yazaki Y. Yokoyama M. J. Clin. Invest. 1998; 102: 2061-2071Crossref PubMed Scopus (228) Google Scholar, 39Moncada S. Palmer R.M.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar). In vivo, inhibition of eNOS by l-NAME resulted in a dramatic increase in mean arterial pressure (22Yang R. Thomas R. Bunting S. Ko A. Ferrara N. Keyt B.A. Jin H. J. Cardiovasc. Pharmacol. 1996; 27: 838-844Crossref PubMed Scopus (218) Google Scholar, 25Sase K. Michel T. Trends Cardiovasc. Med. 1997; 7: 28-37Crossref PubMed Scopus (84) Google Scholar) and a decrease in VEGF-induced angiogenesis (18Papapetropoulos A. Garcı̂a-Cardeña G. Madri J.A. Sessa W.C. J. Clin. Invest. 1997; 100: 3131-3139Crossref PubMed Scopus (1018) Google Scholar). Genetically engineered mice lacking the eNOS gene have impaired endothelium-dependent vasodilation, angiogenesis, and hypertension (19Murohara T. Asahara T. Silver M. Bauters C. Masuda H. Kalka C. Kearney M. Chen D. Chen D. Symes J.F. Fishman M.C. Huang P.L. Isner J.M. J. Clin. Invest. 1998; 101: 2567-2578Crossref PubMed Scopus (1085) Google Scholar, 25Sase K. Michel T. Trends Cardiovasc. Med. 1997; 7: 28-37Crossref PubMed Scopus (84) Google Scholar). Overexpression of eNOS in mice by gene transfer increased NO production, significantly attenuated mean arterial pressure, and neointima formation (25Sase K. Michel T. Trends Cardiovasc. Med. 1997; 7: 28-37Crossref PubMed Scopus (84) Google Scholar, 40Drummond G.R. 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Год издания: 1999
Авторы: Ben‐Quan Shen, David Y. Lee, Thomas F. Zioncheck
Издательство: Elsevier BV
Источник: Journal of Biological Chemistry
Ключевые слова: Nitric Oxide and Endothelin Effects, Angiogenesis and VEGF in Cancer, Hormonal Regulation and Hypertension
Открытый доступ: hybrid
Том: 274
Выпуск: 46
Страницы: 33057–33063