Nonhypoxic Pathway Mediates the Induction of Hypoxia-inducible Factor 1α in Vascular Smooth Muscle Cells

Hypoxia-inducible factor-1 (HIF-1) controls the expression of a number of genes such as vascular endothelial growth factor (VEGF) and Erythropoietin in low oxygen conditions (hypoxia). VEGF is strongly induced at both the mRNA and protein expression level by a number of hormones and growth factors in vascular smooth muscle cells (VSMC) independently of the oxygen environment. However, the role of HIF-1α in this induction has not been studied. We report here that HIF-1α protein levels are strongly increased by fetal calf serum in quiescent VSMC. More interestingly, Angiotensin II (Ang II), thrombin, platelet-derived growth factor, and other hormones can also increase HIF-1α in VSMC to levels that are substantially more elevated than the hypoxic treatment. HIF-1α induced by Ang II is located in the nucleus, binds to the hypoxic response element, and is transcriptionally active. The induction of HIF-1α by hormones is mediated through the production of reactive oxygen species (ROS), since it can be blocked by the ROS inhibitors, diphenyleneiodonium and catalase. Finally, strong induction of VEGF mRNA by Ang II can also be inhibited by these ROS inhibitors. These results implicate HIF-1α and HIF-1-dependent transcriptional activity in the induction of VEGF expression after agonist stimulation and define novel hypoxia-independent mechanisms that should play a major role in vascular remodeling. Hypoxia-inducible factor-1 (HIF-1) controls the expression of a number of genes such as vascular endothelial growth factor (VEGF) and Erythropoietin in low oxygen conditions (hypoxia). VEGF is strongly induced at both the mRNA and protein expression level by a number of hormones and growth factors in vascular smooth muscle cells (VSMC) independently of the oxygen environment. However, the role of HIF-1α in this induction has not been studied. We report here that HIF-1α protein levels are strongly increased by fetal calf serum in quiescent VSMC. More interestingly, Angiotensin II (Ang II), thrombin, platelet-derived growth factor, and other hormones can also increase HIF-1α in VSMC to levels that are substantially more elevated than the hypoxic treatment. HIF-1α induced by Ang II is located in the nucleus, binds to the hypoxic response element, and is transcriptionally active. The induction of HIF-1α by hormones is mediated through the production of reactive oxygen species (ROS), since it can be blocked by the ROS inhibitors, diphenyleneiodonium and catalase. Finally, strong induction of VEGF mRNA by Ang II can also be inhibited by these ROS inhibitors. These results implicate HIF-1α and HIF-1-dependent transcriptional activity in the induction of VEGF expression after agonist stimulation and define novel hypoxia-independent mechanisms that should play a major role in vascular remodeling. vascular endothelial growth factor hypoxia-inducible factor hypoxic response element vascular smooth muscle cell(s) angiotensin II fetal calf serum platelet-derived growth factor fibroblast growth factor-2 diphenyleneiodonium 5-hydroxytryptamine epidermal growth factor reactive oxygen species mitogen-activated protein kinase N-2-hydroxy-1,1-bis(hydroxymethyl)ethylglycine The growth of new blood vessels is termed angiogenesis. Angiogenesis occurs in wound and fracture healing, arthritis, cardiovascular and cerebral ischemia, and most types, if not every type, of cancer known in humans. These events share a common characteristic of occurring in a hypoxic environment. A major mediator of angiogenesis is vascular endothelial growth factor (VEGF)1 (1Ferrara N. J. Mol. Med. 1999; 77: 527-543Crossref PubMed Scopus (1077) Google Scholar, 2Hanahan D. Folkman J. Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (6068) Google Scholar, 3Mustonen T. Alitalo K. J. Cell Biol. 1995; 129: 895-898Crossref PubMed Scopus (475) Google Scholar, 4Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4835) Google Scholar). Transcriptional up-regulation has been shown to play a major role in the induction of the VEGF gene, an action mediated by the specific binding of the hypoxia-inducible factor-1 (HIF-1) to the hypoxic response element (HRE). The HIF-1 transcription factor is a heterodimer composed of HIF-1α and HIF-1β (5Wang G.L. Semenza G.L. J. Biol. Chem. 1995; 270: 1230-1237Abstract Full Text Full Text PDF PubMed Scopus (1717) Google Scholar). Each subunit contains an N-terminal basic helix-loop-helix domain, responsible for heterodimerization and DNA binding. Each subunit also contains a PAS (Per, ARNT, Sim) motif, which is found in a number of transcription factors including the Drosophila proteins Period, Single-minded, and Trachealess, as well as mammalian proteins such as AHR (aryl hydrocarbon receptor). HIF-1β was identified as being a previously described member of this family, the ARNT (aryl hydrocarbon receptornuclear translocator) protein (5Wang G.L. Semenza G.L. J. Biol. Chem. 1995; 270: 1230-1237Abstract Full Text Full Text PDF PubMed Scopus (1717) Google Scholar). C-terminal transactivation domains can be found on both HIF-1α and HIF-1β (6Pugh C.W. O'Rourke J.F. Nagao M. Gleadle J.M. Ratcliffe P.J. J. Biol. Chem. 1997; 272: 11205-11214Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 7Li H. Dong L. Whitlock Jr., J.P. J. Biol. 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Nature. 1998; 394: 485-490Crossref PubMed Scopus (2215) Google Scholar). HIF-1α−/− embryos showed certain lacks and abnormalities in vessel formation. Similar defects have also been observed in VEGF knockout mice (13Ferrara N. Carver-Moore K. Chen H. Dowd M. Lu L. O'Shea K.S. Powell-Braxton L. Hillan K.J. Moore M.W. Nature. 1996; 380: 439-442Crossref PubMed Scopus (3041) Google Scholar, 14Carmeliet P. Ferreira V. Breier G. Pollefeyt S. Kieckens L. Gertsenstein M. Fahrig M. Vandenhoeck A. Harpal K. Eberhardt C. Declercq C. Pawling J. Moons L. Collen D. Risau W. Nagy A. Nature. 1996; 380: 435-439Crossref PubMed Scopus (3447) Google Scholar). While the HIF-1β protein is readily found in all cells, HIF-1α is virtually undetectable in normal oxygen conditions. Studies have shown that, in these conditions, HIF-1α is rapidly degraded by the ubiquitin-proteasome system (15Salceda S. Caro J. J. Biol. Chem. 1997; 272: 22642-22647Abstract Full Text Full Text PDF PubMed Scopus (1403) Google Scholar, 16Huang L.E. Gu J. Schau M. Bunn H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7987-7992Crossref PubMed Scopus (1845) Google Scholar, 17Kallio P.J. Wilson W.J. O'Brien S. Makino Y. Poellinger L. J. Biol. Chem. 1999; 274: 6519-6525Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar). While hypoxia has been shown to be the ubiquitous inducer of HIF-1α in all cells tested, other stimuli, such as insulin, insulin-like growth factors 1 and 2, and EGF, have also been shown to increase HIF-1α protein levels in certain cell types (18Zhong 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, 19Feldser D. Agani F. Iyer N.V. Pak B. Ferreira G. Semenza G.L. Cancer Res. 1999; 59: 3915-3918PubMed Google Scholar, 20Zelzer E. Levy Y. Kahana C. Shilo B.Z. Rubinstein M. Cohen B. EMBO J. 1998; 17: 5085-5094Crossref PubMed Scopus (495) Google Scholar). These stimuli are also able to induce VEGF expression in an HIF-1-dependent manner. In vascular smooth muscle cells (VSMC), a range of different extracellular receptor agonists have been shown to induce VEGF expression, including angiotensin II (Ang II) and platelet-derived growth factor (PDGF) (21Brogi E. Wu T. Namiki A. Isner J.M. Circulation. 1994; 90: 649-652Crossref PubMed Scopus (536) Google Scholar, 22Williams B. Baker A.Q. Gallacher B. Lodwick D. Hypertension. 1995; 25: 913-917Crossref PubMed Scopus (266) Google Scholar). However, the status of HIF-1α in these conditions has not been studied. In this work, we show that HIF-1α levels in VSMC are strongly increased in normal oxygen conditions when cells are stimulated with the cell surface receptor agonists Ang II, thrombin, and PDGF. Induced HIF-1α is localized in the nucleus, binds to HRE, and is transcriptionally active. Our results suggest that the increase of HIF-1α protein levels is mediated through the production of reactive oxygen species (ROS). Our results clearly demonstrate that hypoxia is not the only major player in HIF-1α induction and that this pathway, for the moment specific to VSMC, should play a major role in vascular VEGF production and angiogenesis. Angiotensin II, thrombin, 5-hydroxytryptamine (5-HT), diphenyleneiodonium chloride, and catalase from Aspergillus niger were from Sigma. PDGF and FGF were from Pepro Tech Inc. Anti-HIF-1α antiserum 2087 was raised by our laboratory in rabbits immunized against the last 20 amino acids of the C termini of human HIF-1α. Monoclonal anti-HIF-1α antibody and polyclonal anti-HIF-1β antibody were from Novus Biologicals (Littleton, CO). Monoclonal anti-phospho-p44/p42 MAPK antibody and PD 98059 were from New England Biolabs or Sigma. U0126 and horseradish peroxidase-coupled anti-mouse and anti-rabbit antibody were from Promega. Ly294002 was obtained from Alexis Corp. The PRE-tk-LUC reporter construct was a kind gift from Steven L. McKnight (University of Texas). pcDNA3-HA-DN-HIF-1α was generated by internal digestion of pcDNA3-HA-HIF-1α (23Richard D.E. Berra E. Gothié E. Roux D. Pouysségur J. J. Biol. Chem. 1999; 274: 32631-32638Abstract Full Text Full Text PDF PubMed Scopus (717) Google Scholar) with EcoRI and subsequent religation. VSMC were isolated from the thoracic aortas of 6-week-old male Harlan Sprague-Dawley rats by enzymatic dissociation (24Owens G.K. Loeb A. Gordon D. Thompson M.M. J. Cell Biol. 1986; 102: 343-352Crossref PubMed Scopus (308) Google Scholar). Cells were cultured in Dulbecco's modified Eagle's medium containing 7.5% fetal calf serum (FCS), penicillin (50 units/ml), and streptomycin (50 μg/ml) (Life Technologies, Inc.) in a humid atmosphere (5% CO2, 95% air). Cells were serially passaged upon reaching confluence, and all experiments were performed on passages 3–10. Quiescent cells were obtained by total deprivation of FCS for 16–20 h. Pretreatment of cells with different compounds was performed 30 min prior to stimulation. Hypoxic conditions were obtained by placing the cells in a sealed "Bug-Box" anaerobic workstation (Ruskinn Technologies, Leeds, United Kingdom/Jouan, Saint Herblain, France). The oxygen levels in this workstation were maintained at 1–2% with the residual gas mixture containing 93–94% nitrogen and 5% carbon dioxide. Confluent cells were lysed in 2× Laemmli sample buffer. Protein concentration was determined with the use of the Lowry assay. 30 μg of whole cell extracts were resolved in SDS-polyacrylamide gels (7.5%) and electrophoretically transferred onto a polyvinylidene difluoride membrane (Immobilon-P, Millipore Corp.). Proteins of interest were revealed with specific antibodies as indicated (1:1000 dilution). The bands were visualized with the ECL system (Amersham Pharmacia Biotech). Cells grown on glass coverslips were fixed using 3% paraformaldehyde for 20 min followed by permeabilization with 0.1% Triton X-100 for 10 min. Coverslips were then incubated with anti-HIF-1α antiserum or preimmune serum from the same animal (1:3000). Steptavidin-coupled anti-rabbit antibodies were added for 45 min followed by biotin-coupled Texas Red. Immunofluorescence was then analyzed with a Leica DM-R microscope equipped with a DC-100 digital camera. Electrophoretic mobility shift assays were performed on VSMC nuclear extracts using a32P-labeled oligonucleotide containing the wild type HIF-1 binding site (25Semenza G.L. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Crossref PubMed Scopus (2191) Google Scholar). The sense stand sequence of the oligonucleotide is 5′-GCCCTACGTGCTGTCTCA-3′. 32P-Labeled oligonucleotides were generated by 5′ end labeling by T4 polynucleotide kinase (New England Biolabs) with γ-32PATP (Amersham Pharmacia Biotech). Preparation of nuclear extracts was performed as described previously (25Semenza G.L. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Crossref PubMed Scopus (2191) Google Scholar). Binding reactions were carried out in a total volume of 30 μl containing 10 μg of nuclear extracts and 0.1 μg of calf thymus DNA (Sigma) in 10 mm Tris-HCl (pH 7.5), 50 mm KCl, 50 mm NaCl, 1 mm MgCl2, 1 mm EDTA, 5 mm dithiothreitol, and 5% glycerol. Labeled oligonucleotide probe (40,000 cpm; 0.25 ng) was added and incubated for 30 min at 4 °C. For competition experiments, a 100-fold excess of unlabeled wild-type of mutant (sense stand sequence of mutant oligonucleotide: 5′-GCCCTAAAAGCTGTCTCA-3′) annealed oligonucleotide was added prior to the addition of the labeled probe. Supershift experiments were performed with either anti-HIF-1α antiserum 2087, preimmune antiserum from the same animal, monoclonal anti-HIF-1α antibody, or polyclonal anti-HIF-1β that was added after the addition of the labeled probe. DNA-protein complexes were resolved on native 6% polyacrylamide gels in 0.3× TBE (1× TBE: 89 mm Tris-HCl, 89 mm boric acid, and 5 mm EDTA) at 4 °C. Gels were then dried and analyzed by autoradiography. 1 μg/well of reporter plasmid was used along with 100 ng/well of cytomegalovirus β-galactosidase as a control for transfection efficiency. Transfection of VSMC was performed by using the Superfect transfection reagent (Qiagen) at a 1:5 DNA/reagent ratio. For dominant negative experiments, cells were transfected with 4 μg of the pcDNA3-HA-DN-HIF-1α construct, and in control cells, the same concentration of parental pcDNA3 vector was added. At 3 h post-transfection, cells were washed, and new medium was added. At 12 h post-transfection, cells were deprived of FCS for 16 h. Stimulation with Ang II and hypoxia was performed for 18 h. Cells were then washed twice with cold phosphate-buffered saline, and luciferase assays were performed as follows. Cells were lysed in a lysis buffer (25 mm Tris-phosphate (pH 7.8), 2 mm dithiothreitol, 2 mm1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, 10% glycerol, and 1% Triton X-100) for 15 min at room temperature, and the lysate was cleared by centrifugation. The luciferase assay was performed in a buffer containing 20 mmTricine, 1.07 mm(MgCO3)Mg(OH)2-5H2O (Sigma), 2.67 mm MgSO4, 3.1 mm EDTA, 33.3 mm dithiothreitol, 270 μm coenzyme A (Sigma), 470 μm beetle luciferin (Promega), and 530 μm ATP. β-Galactosidase activity was evaluated with the use of the Galacto-Light Chemiluminescent Reporter Assay kit from Tropix. Results were quantified with a MicroBeta TRILUX luminescence counter (Wallac). Results are expressed as a ratio of luciferase activity over β-galactosidase activity. Confluent cells were lysed, and RNA was isolated with RNA Instapure (Eurogentec). RNA was resolved on agarose/formaldehyde gels, was transferred to Hybond N+ nylon membrane (Amersham Pharmacia Biotech), and was hybridized with a radioactive cDNA probe comprising the total coding sequence of the mouse VEGF gene. Ethidium bromide staining was used as a control to verify gel loading. In all cell lines tested, exposure of cells to hypoxia rapidly increased HIF-1α cellular protein levels. This was also the case for VSMC, since exponentially growing cells showed a strong induction of HIF-1α when incubated for 4 h in 1% oxygen (Fig.1 A). However, the level of HIF-1α protein expression in VSMC under normal oxygen conditions appears to be elevated in comparison with other cell lines (23Richard D.E. Berra E. Gothié E. Roux D. Pouysségur J. J. Biol. Chem. 1999; 274: 32631-32638Abstract Full Text Full Text PDF PubMed Scopus (717) Google Scholar). In FCS-deprived cells, the expression of HIF-1α protein in normoxia was undetectable (Fig. 1 A). Interestingly, when FCS-deprived VSMC were restimulated with 10% FCS for 4 h, HIF-1α protein expression was strongly induced to a level that was equivalent to the increase seen in hypoxic conditions (Fig. 1 A). When VSMC were stimulated with 10% FCS in hypoxic conditions, the level of HIF-1α induction was additive for both stimuli. These results suggest that components of FCS are inducing HIF-1α and that hypoxia and FCS increase HIF-1α protein expression levels through different mechanisms. FCS contains a number of hormones and growth factors that can stimulate cell surface receptors. Receptor agonists such as Ang II, thrombin, and PDGF have been shown to induce a robust increase in VEGF mRNA levels in VSMC. Since HIF-1α is a major mediator of VEGF up-regulation, we wanted to evaluate the effect of the agonists on HIF-1α induction. When FCS-deprived VSMC were treated for 4 h with these compounds, a strong induction of HIF-1α protein expression could be observed (Fig. 1 B). Stimulation of cells with serotonin (5-HT) also strongly increased HIF-1α protein in this cell model (see Fig. 8 B), and a weak but detectable increase could be observed with FGF-2. To confirm the specificity of the 2087 antiserum against HIF-1α, we hybridized the same samples with preimmune serum from the same animal. As seen in the right panel of Fig. 1 B, preimmune serum detected no band above 103 kDa and only detected a nonspecific band at 85 kDa. As a control, receptor activity was verified by the evaluation of a downstream target, the phosphorylation of the mitogen-activated kinases, p42/p44 MAPK. These kinases are strongly activated in this cell system following the addition of the previously mentioned receptor agonists. The strongest inducer of HIF-1α protein expression assayed here was Ang II. A 4-h stimulation with Ang II (100 nm) increased HIF-1α protein expression to levels that were more elevated than cells incubated in hypoxic conditions. As was the case with FCS and hypoxia, the combination of Ang II and hypoxia caused an additive effect on HIF-1α induction (results not shown). The effect of Ang II was at a concentration of 100 However, a induction could be observed at of Ang II as low as 1 a concentration for this (Fig. 2 A). This induction was mediated through the of the receptor since the could the induction of HIF-1α, PD an no effect on or Ang levels of HIF-1α (Fig. 2 B). these results suggest that hormones such as Ang II can strongly induce HIF-1α in a normal an action mediated the receptor HIF-1α induction. Quiescent VSMC were maintained under control conditions under hypoxic conditions 1% or in the of either Ang II (100 or for 4 h. For inhibitors, cells were for 15 min with or catalase prior to stimulation. cell extracts were resolved by and using an anti-HIF-1α antiserum or an anti-phospho-p44/p42 MAPK monoclonal induction by Ang II in VSMC. quiescent VSMC were maintained under control conditions under hypoxic conditions 1% or in the of the indicated of Ang II for 4 h. B, quiescent cells were for 15 min with or PD and maintained under control conditions, under hypoxic conditions or in the of Ang II (100 nm) for 4 h. cell extracts were resolved by and using an anti-HIF-1α antiserum or an anti-phospho-p44/p42 MAPK monoclonal of HIF-1α has been shown to be for HIF-1 activity P.J. K. O'Brien S. P. Makino Y. H. Poellinger L. EMBO J. 1998; 17: PubMed Google Scholar). We wanted to HIF-1α is located in the As seen in quiescent cells in normal oxygen conditions showed HIF-1α When cells are incubated in hypoxic conditions, an increase in HIF-1α protein expression can be seen in the (Fig. 3 B). When cells are stimulated with Ang II, a number of cells show a strong nuclear (Fig. 3 A is seen after stimulation with (Fig. 3 These results show that HIF-1α is localized to the To be HIF-1α then the HIF-1 transcription with HIF-1β and to the specific DNA we evaluated HIF-1 DNA binding activity with mobility shift assay When an oligonucleotide probe containing the HIF-1 binding site was incubated with nuclear extracts from Ang VSMC, a number of complexes were increased 1 and The of HIF-1 in these complexes was by competition experiments and the use of specific 100-fold excess of unlabeled wild-type oligonucleotide with the probe for the binding of HIF-1 a 100-fold excess of an oligonucleotide containing a in the HIF-1 binding site not for binding and antibodies against HIF-1α and HIF-1β the probe and antiserum 2087 raised against HIF-1α also the probe the preimmune antiserum no effect (results not shown). These results demonstrate that HIF-1α protein induced by Ang II can the HIF-1 with HIF-1β and the We then evaluated HIF-1 induced by Ang II is transcriptionally with the use of a luciferase reporter assay. VSMC were transfected with a luciferase reporter gene by containing the from the gene H. McKnight Genes Dev. 1997; PubMed Scopus Google Scholar) and then stimulated with hypoxia Ang II. As shown in the panel of a induction of reporter activity was after an in 1% For the same Ang II increased reporter activity to a level than that by hypoxia over As was the case in experiments (Fig. the combination of both stimuli was additive increase in luciferase These results demonstrate that HIF-1α induced by Ang II is and suggest that hypoxia and Ang II induce HIF-1α through mechanisms. Since Ang II has been shown to induce VEGF the activity of the VEGF in the same conditions was also To these experiments, we used the previously described luciferase reporter plasmid by a and p42/p44 VEGF that to HIF-1 J. F. Pouysségur J. G. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The transfection of this construct in VSMC followed by 18 h of hypoxia increased reporter gene activity by (Fig. As with the construct, Ang II increased reporter activity to a level that hypoxia over both stimuli an additive effect To evaluate the of HIF-1 in Ang transcriptional of reporter genes containing the VEGF hypoxia response element, we used a dominant negative of HIF-1α. The pcDNA3-HA-DN-HIF-1α a of HIF-1α the C-terminal transactivation can with HIF-1β and reporter genes B.H. E. Wang G.L. Roe R. Semenza G.L. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Gothié and J. VSMC were with the VEGF reporter plasmid and the pcDNA3-HA-DN-HIF-1α construct followed by of cells in hypoxic conditions or in the of Ang II. A low concentration of Ang II nm) was since this concentration HIF-1α protein at levels to hypoxia (Fig. 2 A). As seen in Fig. of activity is hypoxic and Ang II stimulation and When cells were transfected with dominant negative HIF-1α, a strong of reporter activity could be observed when cells were incubated in hypoxic conditions of in the of 1 Ang II (Fig. all of these results demonstrate that Ang II VEGF expression by HIF-1α and the HIF-1 transcription II an VSMC were incubated in the or the of Ang II (100 nm) for 4 h. extracts were as indicated under protein was incubated in the of a oligonucleotide containing the HIF-1 binding and antibodies were also added to the binding assays to evaluate the specificity of the For competition unlabeled oligonucleotide (25 ng) was in the binding 1 and no 2 and wild type 3 and For monoclonal anti-HIF-1α 4 and and polyclonal anti-HIF-1β 5 antibodies were also added to the binding II HIF-1 transcriptional activity. VSMC were transfected with 1 μg of either the PRE-tk-LUC construct or reporter plasmid and 100 of an expression vector coding for β-galactosidase in to for transfection efficiency. 12 h after cells were deprived of FCS for 16 h. Cells were then maintained under control or hypoxic conditions in the or of Ang II (100 nm) for 18 h. At this VSMC were lysed, and luciferase and β-galactosidase activity were as described under Results are expressed as a ratio of luciferase activity over β-galactosidase activity and are of experiments performed in dominant negative of HIF-1α Ang VEGF activity. VSMC were transfected with 1 μg of reporter 4 μg of pcDNA3 4 μg of pcDNA3-HA-DN-HIF-1α and 100 of an expression vector coding for β-galactosidase in to for transfection efficiency. 12 h after cells were deprived of FCS for 16 h. Cells were then maintained under control conditions, under hypoxic conditions or in the of Ang II nm) for 18 h. At this VSMC were lysed, and luciferase and β-galactosidase activity were as described under Results are expressed as a ratio of luciferase activity over β-galactosidase activity and are of experiments performed in We wanted to the by which HIF-1α was induced by Ang II. Since Ang II strongly p42/p44 MAPK activity in VSMC, we to evaluate this was in the induction of HIF-1α protein expression by Ang II. PD 98059 and U0126 are specific and of an p42/p44 MAPK We VSMC with these compounds and evaluated HIF-1α induction after Ang II stimulation. a in the Ang levels of HIF-1α could be seen after this (Fig. p42/p44 MAPK activity was This is in with that obtained in model