Induction of Hypoxia-inducible Factor 1α Gene Expression by Vascular Endothelial Growth Factor

Abstract

Transcriptional regulation of vascular endothelial growth factor (VEGF) is critically dependent on hypoxia-inducible factor 1 (HIF-1). However, not only hypoxia, but selected growth factors can induce HIF-1. High levels of both VEGF and HIF-1 coexist in certain conditions, e.g. tumors. Nonetheless, the possibility that the stimulatory relationship between HIF-1 and VEGF may be bi-directional has not been addressed up to date. The present study in endothelial cells analyzed whether HIF-1 is regulated by a product of its own transcriptionally activated genes, namely, VEGF. As a main finding, VEGF-A165 induced the increase of HIF-1α mRNA and HIF-1α protein and nuclear translocation. Autologous endothelial cell VEGF mRNA and protein were also increased upon exposure to exogenous VEGF. The signaling implication of reactive oxygen species was examined by comparison with H2O2 and hypoxanthine/xanthine oxidase and by the superoxide dismutase mimetic, MnTMPyP, the Rac1-NAD(P)H oxidase complex inhibitor, apocynin, transfection of a dominant negative Rac1 mutant, and transfection of a p67phox antisense oligonucleotide. Superoxide anion, largely dependent on Rac1-NAD(P)H oxidase complex activity, was the critical signaling element. The transductional functionality of the pathway was confirmed by means of a reporter gene flanked by a transcription site-related VEGF sequence and by quantitative PCR. In summary, the present results reveal a previously undescribed action of VEGF on the expression of its own transcription factor, HIF-1, and on VEGF itself. This effect is principally mediated by superoxide anion, therefore identifying a new, potentially relevant role of reactive oxygen species in VEGF signaling. Transcriptional regulation of vascular endothelial growth factor (VEGF) is critically dependent on hypoxia-inducible factor 1 (HIF-1). However, not only hypoxia, but selected growth factors can induce HIF-1. High levels of both VEGF and HIF-1 coexist in certain conditions, e.g. tumors. Nonetheless, the possibility that the stimulatory relationship between HIF-1 and VEGF may be bi-directional has not been addressed up to date. The present study in endothelial cells analyzed whether HIF-1 is regulated by a product of its own transcriptionally activated genes, namely, VEGF. As a main finding, VEGF-A165 induced the increase of HIF-1α mRNA and HIF-1α protein and nuclear translocation. Autologous endothelial cell VEGF mRNA and protein were also increased upon exposure to exogenous VEGF. The signaling implication of reactive oxygen species was examined by comparison with H2O2 and hypoxanthine/xanthine oxidase and by the superoxide dismutase mimetic, MnTMPyP, the Rac1-NAD(P)H oxidase complex inhibitor, apocynin, transfection of a dominant negative Rac1 mutant, and transfection of a p67phox antisense oligonucleotide. Superoxide anion, largely dependent on Rac1-NAD(P)H oxidase complex activity, was the critical signaling element. The transductional functionality of the pathway was confirmed by means of a reporter gene flanked by a transcription site-related VEGF sequence and by quantitative PCR. In summary, the present results reveal a previously undescribed action of VEGF on the expression of its own transcription factor, HIF-1, and on VEGF itself. This effect is principally mediated by superoxide anion, therefore identifying a new, potentially relevant role of reactive oxygen species in VEGF signaling. Angiogenesis is a principal component of physiologic vascular remodeling. However, it is also a major mechanism in diseases as diverse as tumor neovascularization, proliferative retinopathies, or chronic ischemia. VEGF 2The abbreviations used are: VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; MAPK, mitogen-activated protein kinase; ROS, reactive oxygen species; JNK, c-Jun NH2-terminal kinase; CHX, cycloheximide; HIF-1, hypoxia-inducible factor 1; EC, endothelial cell(s); PlGF, placental growth factor; ESA, erythropoietic-stimulating agents; HX, hypoxanthine; XO, xanthine oxidase; PBS, phosphate-buffered saline; mAb, monoclonal antibody; L-NAME, NG-nitro-l-arginine methyl ester. family members are key mediators of the angiogenic process. In EC, VEGF-A165 binds to two main tyrosine kinase receptors, i.e. VEGF receptor 1 (VEGFR1) and VEGF receptor 2 (VEGFR2), activating a host of mechanisms, including proliferation, chemotaxis, and permeability and increasing the expression of numerous proteins (1Ferrara N. Endocr. Rev. 2004; 25: 581-611Crossref PubMed Scopus (3009) Google Scholar). In addition to its function as a mitogen, VEGF has EC protective properties which result in enhanced survival and resistance (2Zachary I. Am. J. Physiol. Cell Physiol. 2001; 280: 1375-1386Crossref PubMed Google Scholar, 3Gonzalez-Pacheco F.R. Deudero J.J. Castellanos M.C. Castilla M.A. Alvarez-Arroyo M.V. Yague S. Caramelo C. Am. J. Physiol. Heart Circ. Physiol. 2006; 291: 1395-1401Crossref PubMed Scopus (88) Google Scholar). This VEGF-induced protection is effective against different noxious stimuli, including serum deprivation, oxidative challenge, calcineurin binding drugs, and cytoskeletal disruption (4Alvarez Arroyo M.V. Suzuki Y. Yague S. Lorz C. Jimenez S. Soto C. Barat A. Belda E. Gonzalez-Pacheco F.R. Deudero J.J. Castilla M.A. Egido J. Ortiz A. Caramelo C. Transplantation. 2002; 74: 1618-1624Crossref PubMed Scopus (40) Google Scholar). Even though VEGF expression is regulated by a number of stimuli, hypoxia is the principal trigger to increase VEGF gene transcription. This increment is mainly mediated by a specialized transcription factor, HIF-1 (5Forsythe J.A. Jiang B.H. Iyer N.V. Agani F. Leung S.W. Koos R.D. Semenza G.L. Mol. Cell. Biol. 1996; 16: 4604-4613Crossref PubMed Scopus (3234) Google Scholar). HIF-1 is a heterodimer composed by two subunits (HIF-1α and HIF-1β) with apparent molecular masses of 120–130 and 91–94 kDa, respectively (6Wang G.L. Semenza G.L. J. Biol. Chem. 1995; 270: 1230-1237Abstract Full Text Full Text PDF PubMed Scopus (1737) Google Scholar). The mRNA of both subunits is expressed constitutively, albeit in variable amounts. However, HIF-1α protein and HIF-1 activity are only detectable in cells exposed to hypoxia (7Wang G.L. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4304-4308Crossref PubMed Scopus (1212) Google Scholar). Recently, several growth factors and cytokines have also been reported to induce HIF-1α protein synthesis and HIF-1 activity in normoxia (8Zelzer E. Levy Y. Kahana C. Shilo B.Z. Rubinstein M. Cohen B. EMBO J. 1998; 17: 5085-5094Crossref PubMed Scopus (498) Google Scholar, 9Feldser D. Agani F. Iyer N.V. Pak B. Ferreira G. Semenza G.L. Cancer Res. 1999; 59: 3915-3918PubMed Google Scholar, 10Hellwig-Burgel T. Rutkowski K. Metzen E. Fandrey J. Jelkmann W. Blood. 1999; 94: 1561-1567Crossref PubMed Google Scholar, 11Zhong H. Chiles K. Feldser D. Laughner E. Hanrahan C. Georgescu M.M. Simons J.W. Semenza G.L. Cancer Res. 2000; 60: 1541-1545PubMed Google Scholar, 12Gorlach A. Diebold I. Schini-Kerth V.B. Berchner-Pfannschmidt U. Roth U. Brandes R.P. Kietzmann T. Busse R. Circ. Res. 2001; 89: 47-54Crossref PubMed Scopus (357) Google Scholar, 13Spinella F. Rosano L. Di C.V. Natali P.G. Bagnato A. J. Biol. Chem. 2002; 277: 27850-27855Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 14Laughner E. Taghavi P. Chiles K. Mahon P.C. Semenza G.L. Mol. Cell. Biol. 2001; 21: 3995-4004Crossref PubMed Scopus (1133) Google Scholar). The major signaling pathways identified as contributing to the HIF-1α induction by these growth factors include phosphatidylinositol 3-kinase/Akt (protein kinase B) and Ras/MAPK/phosphatidylinositol 3-kinase (MEK) (10Hellwig-Burgel T. Rutkowski K. Metzen E. Fandrey J. Jelkmann W. Blood. 1999; 94: 1561-1567Crossref PubMed Google Scholar, 11Zhong H. Chiles K. Feldser D. Laughner E. Hanrahan C. Georgescu M.M. Simons J.W. Semenza G.L. Cancer Res. 2000; 60: 1541-1545PubMed Google Scholar, 14Laughner E. Taghavi P. Chiles K. Mahon P.C. Semenza G.L. Mol. Cell. Biol. 2001; 21: 3995-4004Crossref PubMed Scopus (1133) Google Scholar, 15Zundel W. Schindler C. Haas-Kogan D. Koong A. Kaper F. Chen E. Gottschalk A.R. Ryan H.E. Johnson R.S. Jefferson A.B. Stokoe D. Giaccia A.J. Genes Dev. 2000; 14: 391-396Crossref PubMed Google Scholar, 16Fukuda R. Hirota K. Fan F. Jung Y.D. Ellis L.M. Semenza G.L. J. Biol. Chem. 2002; 277: 38205-38211Abstract Full Text Full Text PDF PubMed Scopus (708) Google Scholar). In these circumstances the transactivated HIF-1 leads to increased VEGF synthesis and/or protein stabilization, therefore amplifying the angiogenic signal. The limits and potential importance of this type of HIF-1 activation have not yet been accurately appraised. Among their numerous effects, ROS participate in activation pathways for redox-sensitive transcription factors mainly through protein tyrosine phosphorylation (17Monteiro H.P. Stern A. Free Radic. Biol. Med. 1996; 21: 323-333Crossref PubMed Scopus (219) Google Scholar, 18Sun Y. Oberley L.W. Free Radic. Biol. Med. 1996; 21: 335-348Crossref PubMed Scopus (628) Google Scholar, 19Gonzalez-Pacheco F.R. Caramelo C. Castilla M.A. Deudero J.J. Arias J. Yague S. Jimenez S. Bragado R. Alvarez-Arroyo M.V. Nephrol. Dial. Transplant. 2002; 17: 392-398Crossref PubMed Scopus (43) Google Scholar). Among these activations, that of HIF-1 is singularly relevant. In addition, the interaction of certain growth factors with their tyrosine kinase receptors leads to ROS production. ROS act as ubiquitous intracellular messengers, e.g. (O2·¯), and H2O2 in mitogenic and cytoprotective responses (20Maulik N. Antioxid. Redox Signal. 2002; 4: 805-815Crossref PubMed Scopus (137) Google Scholar, 21Mikkelsen R.B. Wardman P. Oncogene. 2003; 22: 5734-5754Crossref PubMed Scopus (488) Google Scholar, 22Millar T.M. Phan V. Tibbles L.A. Free Radic. Biol. Med. 2007; 42: 1165-1177Crossref PubMed Scopus (79) Google Scholar, 23Lu Q. Jourd'heuil F.L. Jourd'heuil D. J. Cell. Physiol. 2007; 212: 827-839Crossref PubMed Scopus (20) Google Scholar). In terms of mechanism, the main pathway of receptor tyrosine kinase-mediated ROS production involves the Rac1-NAD(P)H oxidase-like membrane complex (24Sundaresan M. Yu Z.X. Ferrans V.J. Sulciner D.J. Gutkind J.S. Irani K. Goldschmidt-Clermont P.J. Finkel T. Biochem. J. 1996; 318: 379-382Crossref PubMed Scopus (441) Google Scholar, 25Ushio-Fukai M. Alexander R.W. Mol. Cell. Biochem. 2004; 264: 85-97Crossref PubMed Scopus (401) Google Scholar, 26Ushio-Fukai M. Antioxid. Redox Signal. 2007; 9: 731-739Crossref PubMed Scopus (203) Google Scholar, 27Hordijk P.L. Circ. Res. 2006; 98: 453-462Crossref PubMed Scopus (434) Google Scholar). Colavitti et al. (28Colavitti R. Pani G. Bedogni B. Anzevino R. S. J. T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google have the effect of VEGF-induced ROS production as a angiogenic which is through the M. Res. 2006; PubMed Scopus Google Scholar). the role of Rac1-NAD(P)H oxidase complex as a main of ROS in vascular D. M. Circ. Res. 2000; PubMed Scopus Google as as the of VEGF for signaling through ROS and the effect of ROS in HIF-1 the was that the of genes, e.g. VEGF or may act on the expression of HIF-1 itself. of has been to this and to the signaling The potential importance of the pathway examined can be to of and to Cell EC were up to and as previously M.A. Arroyo M.V. E. P. Gonzalez-Pacheco F.R. E. Bragado R. Caramelo C. Circ. Res. 1999; PubMed Scopus Google Scholar). as EC the were used between the and these conditions, EC has been by growth in the of VEGF-A165 as M.A. Arroyo M.V. E. P. Gonzalez-Pacheco F.R. E. Bragado R. Caramelo C. Circ. Res. 1999; PubMed Scopus Google Scholar, Arroyo M.V. Castilla M.A. F.R. D. A. S. Caramelo C. J. Am. Nephrol. 1998; 9: PubMed Google Scholar). of in cells were for different with VEGF a of or a of EC were with or their for used were the oxidase inhibitor, the superoxide dismutase mimetic, the inhibitor, the inhibitor, the inhibitor, and the phosphatidylinositol 3-kinase inhibitor, The was used to study and synthesis was by was used as and were used as of The of H2O2 exposure in this has been previously by of the different the EC were and in 1 of and nuclear proteins for were the by et al. E. P. M.M. W. Res. 17: PubMed Scopus Google and by 1 and and were by and to and were by with were with or with and the and exposed to of was of and expression a dominant negative of Rac1 was and to superoxide production dependent Rac1-NAD(P)H This was by was used as the for or for the Rac1-NAD(P)H oxidase complex p67phox were to EC a 2 and for with VEGF for was used as of by of serum deprivation, EC in were with VEGF or for 1 and with EC were with and with the of EC with the as were in the of the of the of i.e. was were a of was reporter was by Semenza This the VEGF sequence to to the transcription (5Forsythe J.A. Jiang B.H. Iyer N.V. Agani F. Leung S.W. Koos R.D. Semenza G.L. Mol. Cell. Biol. 1996; 16: 4604-4613Crossref PubMed Scopus (3234) Google the of a reporter were with reporter and and the with or VEGF or for and with to the The results were expressed as expression of EC was the EC were to The transfection was a to of to the reporter the were and for and The complex was in The transfection of cells were with and growth was Cell were as transfection were also was in and was the to the was and on in of was to of were used as the for with the the were in a and were analyzed with the for of the analyzed was and of the product were used to a The number of of the gene of in was the by the were and the gene number was by the of The used in this study are reported S. Castellanos M.C. M. A. L. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). were and on and were with and with cells were with in and in for 1 with serum were with were and with to for 2 was for of the cell were a of was of are expressed as the to a of by or of were used to and for were used to the which was The were with the VEGF HIF-1α in a and were different conditions, and and nuclear were to protein As can be in only levels of HIF-1α be in the nuclear of VEGF increased nuclear HIF-1α protein in a the the HIF-1α protein was in the HIF-1α increased in the of VEGF in a was to whether a of i.e. placental growth factor was of increase of As can be in increased nuclear HIF-1α the of was with to that of VEGF VEGF binds to both of receptors, binds only to the between the of both that the two of VEGF receptor are in the whether the with VEGF were by the product of HIF-1 transcriptionally activated were erythropoietic-stimulating namely, and to and of of the on HIF-1α protein levels Superoxide and Rac1 for VEGF-induced HIF-1α a and on the regulation of HIF-1α of the VEGF-induced signaling pathways as a of HIF-1α However, two were to the M. Y. T. Y. M. P.J. Johnson C. Alexander R.W. Circ. Res. 2002; PubMed Scopus Google and own J. P. C. M. C. F. R. S. and F. R. VEGF is of ROS production and HIF-1α synthesis can by a signaling pathway A. Diebold I. Schini-Kerth V.B. Berchner-Pfannschmidt U. Roth U. Brandes R.P. Kietzmann T. Busse R. Circ. Res. 2001; 89: 47-54Crossref PubMed Scopus (357) Google Scholar, E. J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Google Scholar, J.J. 2001; PubMed Scopus Google Scholar). was on the of whether ROS were in the increased VEGF-induced HIF-1α this examined the effect of both H2O2 and for As in effect on HIF-1α protein was the of the a effect of was with the In these conditions, HIF-1α protein levels were to production. a was by the was dependent on on this the of in VEGF-induced HIF-1α The main of in EC in of are the of the Rac1-NAD(P)H complex M. Y. T. Y. M. P.J. Johnson C. Alexander R.W. Circ. Res. 2002; PubMed Scopus Google Scholar). EC were with VEGF or in the or in the of the Rac1-NAD(P)H oxidase complex inhibitor, and the superoxide dismutase mimetic, As in but not HIF-1α both whether or were in the and HIF-1α protein this EC were also with the inhibitor, effect of was in EC, but a be with The of the of to a of its role in HIF-1α protein EC were with a dominant negative of As in expression of a negative of Rac1 is to the VEGF effect on HIF-1α the of the albeit possibility that the effect of Rac1 have been of EC were with a p67phox antisense oligonucleotide. This antisense also the VEGF effect and this of results that the Rac1-NAD(P)H oxidase complex is and in VEGF signaling on HIF-1α and that as a of VEGF in the induction of HIF-1α protein This effect to be to a action of by means of in production by VEGF, EC with the different were with this both on the of to with (O2·¯), which has different properties of the i.e. by means of and the analyzed production by a the cells were VEGF on the was in EC were on In both VEGF exposure increased and the superoxide dismutase this In the of a by VEGF and HIF-1α JNK, and 3-kinase/Akt number of proteins be in VEGF signaling on the of the main pathways in the the role of phosphatidylinositol JNK, and this EC were with of phosphatidylinositol 3-kinase/Akt and namely, and and with VEGF or As in of the the of both VEGF and signaling on HIF-1α on these analyzed VEGF to JNK, and by the of their VEGF As in VEGF increased and phosphorylation of with apparent phosphorylation was in the but increase was the effect of VEGF VEGF HIF-1α but HIF-1α mRNA as to study the mechanism of HIF-1α protein HIF-1α was by HIF-1α as induced by or VEGF in the of the protein synthesis inhibitor, was used as a as a of HIF-1α P. K. Cancer Biol. 2004; PubMed Scopus (199) Google Scholar). In EC with for HIF-1α levels protein synthesis with In VEGF HIF-1α protein levels were to in were to the mechanism of HIF-1α increase by VEGF. the HIF-1α mRNA production increased VEGF or and on the were with The by EC with VEGF or for that a VEGF and HIF-1α mRNA increase was in the of or VEGF and VEGF the of the transductional functionality of the whether exogenous VEGF and its intracellular (O2·¯), are of increasing VEGF levels in As in exogenous VEGF induced VEGF This induction was or by the Rac1-NAD(P)H oxidase complex and the superoxide dismutase mimetic, In the both VEGF and increased VEGF mRNA expression in quantitative This expression its of VEGF and was in the of or In with this finding, VEGF protein was by increase was of VEGF this increase was in the of and be that the of VEGF in cells exogenous VEGF in the of and and of the possibility that the VEGF in e.g. was to exogenous of VEGF by EC were with VEGF with or were to with The were by Cell are in with and VEGF is in by a to with VEGF VEGF VEGF to of of of to VEGF of in A. these used of was in its and the was of cells The to VEGF protein of has been with to cells signal. with VEGF VEGF VEGF has been with to cells The main of the present study is the of a by VEGF on its factor, HIF-1, and also on its own Even though of growth factors on HIF-1 activation have been previously to the of the present is the this type of effect with VEGF. of the present is that VEGF its action on HIF-1 in the of results the VEGF effect on HIF-1α to of reveal the of a of VEGF on HIF-1, which can in and VEGF production conditions, namely, tumors. The of VEGF to be through both and receptors, as by the of and VEGF. The study the that VEGF mRNA and VEGF protein are induced EC exposure to exogenous VEGF the that the increase in HIF-1α is in terms of of the VEGF However, the possibility albeit with of This is the of the present and be on the by which VEGF HIF-1 The effect of VEGF on HIF-1 not a of HIF-1α but increase in its This increment is to increased HIF-1α therefore this effect to of HIF-1α gene As the have to for the increase of HIF-1α induced by growth results increased HIF-1α gene expression upon VEGF this of to the of in HIF-1 activation pathways by growth factors or hypoxia H. Chiles K. Feldser D. Laughner E. Hanrahan C. Georgescu M.M. Simons J.W. Semenza G.L. Cancer Res. 2000; 60: 1541-1545PubMed Google Scholar, 12Gorlach A. Diebold I. Schini-Kerth V.B. Berchner-Pfannschmidt U. Roth U. Brandes R.P. Kietzmann T. Busse R. Circ. Res. 2001; 89: 47-54Crossref PubMed Scopus (357) Google Scholar, 14Laughner E. Taghavi P. Chiles K. Mahon P.C. Semenza G.L. Mol. Cell. Biol. 2001; 21: 3995-4004Crossref PubMed Scopus (1133) Google Scholar, 16Fukuda R. Hirota K. Fan F. Jung Y.D. Ellis L.M. Semenza G.L. J. Biol. Chem. 2002; 277: 38205-38211Abstract Full Text Full Text PDF PubMed Scopus (708) Google Scholar, L. P. E. M.A. 2001; 22: PubMed Scopus Google Scholar). The that has a critical role as a to HIF-1α increase by VEGF, a of VEGF signaling process. of superoxide production by VEGF has been previously by M. Antioxid. Redox Signal. 2007; 9: 731-739Crossref PubMed Scopus (203) Google Scholar, L.A. Biol. 25: PubMed Scopus Google Scholar, C. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. L. M. Biol. 25: PubMed Scopus Google Scholar). However, the of this in the present to be In the of superoxide on HIF-1 are not by by the Rac1-NAD(P)H oxidase complex inhibitor, apocynin, that Rac1-NAD(P)H oxidase complex is the main of in this signaling is used in M. E. M. I. S. S. C. PubMed Scopus Google Scholar, M. M. A.B. S. M.A. R.W. R.B. Am. J. Full Text Full Text PDF PubMed Scopus Google therefore the possibility of a of the present of the of the results was by means of with transfection of the Rac1 negative In this Rac1 may be in pathways E. Circ. Res. 2006; 98: PubMed Scopus Google a was which by of by a p67phox antisense that the by Rac1 can be to of the oxidase The increase of HIF-1α by superoxide is not a as hypoxia is the role of superoxide in induction of HIF-1α is for the The of effect of a different growth factor, namely, on HIF-1α that in the present conditions, the on HIF-1α for VEGF is not a mechanism factors in their e.g. The of effect of on HIF-1α increase is of importance to their This may be in terms of the potential of in angiogenic and cytoprotective Full Text Full Text PDF PubMed Scopus Google Scholar). In results not a of as amplifying on the HIF-1 The between the of VEGF and on HIF-1 can largely be to their different intracellular signaling (2Zachary I. Am. J. Physiol. Cell Physiol. 2001; 280: 1375-1386Crossref PubMed Google Scholar, J.W. Biol. Med. 2003; PubMed Scopus Google but in on signaling may be to In a the of a between Rac1-NAD(P)H oxidase complex and VEGF receptors is in the to the of this interaction as as the of the superoxide is by in the a 26Ushio-Fukai M. Antioxid. Redox Signal. 2007; 9: 731-739Crossref PubMed Scopus (203) Google and M. Res. 2006; PubMed Scopus Google Scholar). Even though that the main signaling of the mechanism is (O2·¯), a role of or by the increase of J.S. Am. J. Physiol. 1996; PubMed Google and in not be a The were with this possibility in and their negative that the interaction between and on the HIF-1α increase by VEGF is not a relevant The have in that VEGF production. number of pathways between VEGF receptor and the increase in HIF-1α protein were The results with a of have a and that phosphatidylinositol JNK, and are in the VEGF and is This of activation is with in the L. P. E. M.A. 2001; 22: PubMed Scopus Google Scholar). the results that phosphatidylinositol JNK, and are for activation in the of VEGF-induced production and in the increased HIF-1α protein The that a previously effect of VEGF (1Ferrara N. Endocr. Rev. 2004; 25: 581-611Crossref PubMed Scopus (3009) Google was not was with the of VEGF-induced increase of HIF-1 by This result can be to the in the used of phosphorylation the as can be in This may the of the to increase in phosphorylation by the action of VEGF. may be that phosphorylation the Nonetheless, the of this signaling sequence be the present and is to this In summary, study the of a of the main angiogenic growth factor, VEGF, on its principal HIF-1. results also that VEGF is of activating its own effect are in to this as as which are the transcriptionally In has been However, in this the results can be by the possibility that a critical of HIF-1 is for VEGF HIF-1 can be to act as the of the In of levels of HIF-1 be for the of the of the present pathway can be but the of the of VEGF in the of hypoxia by means of the of is the that can be In this the gene activated by HIF-1 is by that activated by VEGF In be in the of in which a increase of both VEGF and HIF-1 D. K. Cancer Biol. 2004; 14: PubMed Scopus Google Scholar, Rev. 2002; PubMed Scopus Google Scholar). the effect in conditions, e.g. hypoxia or to be to the of this type of activation Semenza and for their of