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Article

Thrombin Activates the Hypoxia-Inducible Factor-1 Signaling Pathway in Vascular Smooth Muscle Cells

Role of the p22^phox-Containing NADPH Oxidase

A. Görlach, I. Diebold, V. B. Schini-Kerth, U. Berchner-Pfannschmidt, U. Roth, R. P. Brandes, T. Kietzmann R. Busse

From the Institut für Kardiovaskuläre Physiologie (A.G., I.D., V.B.S.-K., R.P.B., R.B.), Klinikum der J.W. Goethe-Universität, Frankfurt/M.; Max-Planck-Institut für Molekulare Physiologie (U.B.-P.), Dortmund; and Institut für Biochemie und Molekulare Zellbiologie (U.R., T.K.), Universität Göttingen, Germany

Correspondence to Agnes Görlach, MD, Institut für Kardiovaskuläre Physiologie, Klinikum der J.W. Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt/M, Germany. E-mail A.Goerlach{at}em.uni-frankfurt.de

Abstract

Abstract—The heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1) is activated under hypoxic conditions, resulting in the upregulation of its target genes plasminogen activator inhibitor-1 (PAI-1) and vascular endothelial growth factor (VEGF). PAI-1 and VEGF are also induced in response to vascular injury, which is characterized by the activation of platelets and the coagulation cascade as well as the generation of reactive oxygen species (ROS). However, it is not known whether HIF-1 is also stimulated by thrombotic factors. We investigated the role of thrombin, platelet-associated growth factors, and ROS derived from the p22^phox-containing NADPH oxidase in the activation of HIF-1 and the induction of its target genes PAI-1 and VEGF in human vascular smooth muscle cells (VSMCs). Thrombin, platelet-derived growth factor-AB (PDGF-AB), and transforming growth factor-ß₁ (TGF-ß₁) upregulated HIF-1 protein in cultured and native VSMCs. This response was accompanied by nuclear accumulation of HIF-1 as well as by increased HIF-1 DNA-binding and reporter gene activity. The thrombin-induced expression of HIF-1, PAI-1, and VEGF was attenuated by antioxidant treatment as well as by transfection of p22^phox antisense oligonucleotides. Inhibition of p38 mitogen-activated protein kinase and phosphatidylinositol-3-kinase significantly decreased thrombin-induced HIF-1, PAI-1, and VEGF expression. These findings demonstrate that the HIF-1 signaling pathway can be stimulated by thrombin and platelet-associated growth factors and that a redox-sensitive cascade activated by ROS derived from the p22^phox-containing NADPH oxidase is crucially involved in this response.

Key Words: oxygen radicals • p22^phox • platelets • vascular endothelial growth factor • plasminogen activator inhibitor-1

The initial response to vessel-wall injury is characterized by the rapid deposition and activation of platelets and stimulation of the coagulation cascade as well as enhanced formation of reactive oxygen species (ROS) and precedes a more prolonged period of vascular repair.^{1 2} Growth factors released from activated platelets, such as platelet-derived growth factor-AB (PDGF-AB) and transforming growth factor-ß₁ (TGF-ß₁), as well as thrombin and other coagulation factors do not only contribute to thrombus formation but can also exert direct effects on the vessel wall and stimulate the formation of ROS.^{1 2 3 4 5 6}

Although in excess amounts ROS are cytotoxic, at low concentrations they serve as signaling molecules and second messengers affecting signal transduction and gene expression in a variety of processes including the response to injury.^{7 8} A p22^phox-containing NADPH oxidase has been identified in vascular smooth muscle cells (VSMCs) and shown to generate ROS on stimulation with various agonists, among them thrombin and activated platelets.^{4 5 6 9} Moreover, p22^phox-dependent generation of ROS contributes to upregulation of tissue factor by activated platelets in VSMCs,⁴ suggesting a role for this oxidase in the interaction between thrombotic factors and the vessel wall.

Activation of the coagulation system leads to fibrin formation, which stabilizes the hemostatic plug but also is required for wound healing and vessel repair.¹⁰ Plasminogen activator inhibitor-1 (PAI-1), which regulates tissue fibrinolysis, is markedly upregulated in endothelial cells and VSMCs within hours of vascular wounding.¹¹ Microvascular permeability, which allows local fibrin formation, is enhanced by the angiogenic peptide vascular endothelial growth factor (VEGF) in addition to its stimulatory effect on endothelial cell proliferation,¹⁰ and elevated levels of VEGF have been found in the vessel wall after injury.^{12 13} Overexpression of PAI-1 or application of recombinant VEGF enhanced the regeneration of the endothelial layer and decreased intima formation, suggesting a role for these factors in vascular repair.^{14 15} Increased expression of VEGF and PAI-1, on the other hand, has been observed in atherosclerotic lesions.^{16 17} The mechanisms underlying these apparently controversial findings, however, have not been elucidated yet.

PAI-1 as well as VEGF are target genes of the transcription factor hypoxia-inducible factor-1 (HIF-1).^{18 19} This heterodimer consists of the constitutively expressed HIF-1ß and the inducible protein HIF-1.²⁰ Although hypoxic activation of HIF-1 has been considered to play a prominent role in the upregulation of PAI-1 and VEGF, no clear evidence for a significant tissue hypoxia has been observed at 1 day after injury.²¹ These findings suggest that factors other than hypoxia may be responsible for VEGF and PAI-1 production after vascular injury. Indeed, thrombotic factors, including activated platelets, platelet-associated growth factors, and thrombin, can upregulate these genes in VSMCs.^{22 23 24} Moreover, exposure to ROS enhanced VEGF expression in VSMCs¹³ and induced PAI-1 accumulation in rat heart microvessels.²⁵

We therefore hypothesized that thrombotic factors can activate the HIF-1 signaling pathway and that the generation of ROS may contribute to this response. In the present study, we investigated whether thrombin, platelet-associated growth factors, and the p22^phox-containing NADPH oxidase can activate the HIF-1 pathway and subsequently induce the expression of PAI-1 and VEGF in human VSMCs.

Materials and Methods

Reagents
SB203580 was from Alexis. Wortmannin, LY294002, PD98059, SB202190, SB220025, U0126, trolox, vitamin E succinate, and recombinant hirudin were from Calbiochem. Recombinant human PDGF-AB, TGF-ß₁, and factor Xa were from R&D Systems; FCS was from Biochrom; and deoxycytidine 5'-³²P-triphosphate (3000 Ci/mmol) was from Hartmann Analytic. Human -thrombin (thrombin-specific clotting activity 3261 U/mg) was from Hämochrom Diagnostika. -thrombin (6.65 U/mg), DIP--thrombin (0.18 U/mg), and PPACK--thrombin were prepared as described elsewhere.²⁶ All other chemicals were from Sigma.

Cell Culture and Preparation of Human Renal Arteries and Platelet-Derived Products
Human aortic VSMCs were from Clonetics and cultured in MCDB131 with 8% FCS and were used from passages 3 to 13. VSMCs were serum-deprived in MCDB131 with 0.1% BSA for 24 to 48 hours before stimulation. The human carcinoma cell line ECV304 was cultivated in M199 with 10% FCS. Hypoxic stimulation was performed in an incubator with 3% O₂, 87% N₂, and 5% CO₂. The PO₂ values were measured using an oxygen electrode.²⁷

The adventitia and intima from human renal arteries (kindly provided by the Department of Urology, Klinikum der JWG-Universität, Frankfurt/M.) were mechanically removed, and the smooth muscle cell layers were washed twice and then incubated in MCDB131 containing 0.1% BSA for 30 minutes before stimulation.

Platelet-derived products (PDPs) were prepared from human washed platelets stimulated with -thrombin (1 U/mL) for 2 minutes before inactivation with hirudin (10 thrombin-inactivating U/mL).^{4 26}

Dichlorofluorescein Assay
Serum-deprived, confluent VSMCs were washed with HEPES-modified Tyrode’s solution containing (in mmol/L) CaCl₂ 1.8, KCl 2.6, MgCl₂ 0.49, NaCl 137, NaH₂PO₄ 0.36, and glucose 5.6 at pH 7.4 and loaded with dichlorodihydrofluorescein diacetate 5 µmol/L (Molecular Probes) for 20 minutes at 37°C. Dichlorofluorescein (DCF) fluorescence was measured over the whole field of vision using a fluorescence microscope (Zeiss) connected to an imaging system (Improvision).

Plasmids, Oligonucleotides, and Transfections
Phosphorothioate-modified p22^phox antisense and scrambled oligonucleotides have been described.⁴ pGLhVEGFHRE contains a HindIII-ApaI fragment (-1124 to -417 bp) from the human VEGF promoter cloned into the plasmid pGL3 promoter (Promega). pGLEPOHRE harbors three copies of the erythropoietin hypoxia-responsive element (HRE) in front of the SV40 promoter.²⁸ pGLhPAI-796 contains the human PAI-1 promoter from -796 to +13 bp.¹⁸ Transfection efficiency was controlled by the pRL-TK Renilla luciferase plasmid (Promega). Transfections were performed as described elsewhere.⁴

Northern Blot
Total RNA from VSMCs was subjected to Northern blot analysis as described elsewhere.⁴ Hybridizations were carried out with ³²P-labeled rat p22^phox,⁴ rat VEGF,²² or digoxigenin-labeled rat PAI-1 and ß-actin cDNA probes.¹⁸

Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assay
Nuclear extracts from VSMCs were prepared according to standard protocols, and electrophoretic mobility shift assay (EMSA) was performed as described¹⁸ using the oligonucleotide W18 containing the erythropoietin HRE. For supershift analysis, 1 µL HIF-1 antibody was added to the EMSA reaction.

Western Blot and Immunofluorescence
VSMCs were lysed in 1% SDS. Protein was dissolved in Laemmli buffer, subjected to SDS-PAGE, and detected with an antibody against human HIF-1 (1:1000 dilution) (Transduction Laboratories) using the ECL system (Pierce). Immunofluorescence was performed in VSMCs fixed with ice-cold methanol/acetone (1:1 dilution) for 10 minutes at -20°C using a monoclonal antibody against human HIF-1 (1:200 dilution) (Alexis) and an alexa 546-coupled secondary anti-mouse antibody (1:400 dilution) (Molecular Probes). Visualization was by confocal microscopy (Zeiss).

Statistical Analysis
Values presented are mean±SEM. Results were compared by ANOVA for repeated measurements followed by the Newman-Keuls test. P<0.05 was accepted as significant.

Results

Thrombin and Platelet-Associated Growth Factors Induce HIF-1 Protein in VSMCs
Incubation of cultured human VSMCs with thrombin (-thrombin, 2 U/mL) resulted in a transient time- and concentration-dependent elevation of HIF-1 protein levels (Figures 1A and 1B). HIF-1 protein was also increased in response to proteolytically active -thrombin and factor Xa (1 U/mL), the activator of thrombin generation. DIP--thrombin, which has strongly reduced proteolytic activity, and PPACK--thrombin, which is proteolytically inactive, had only a mild or no effect (Figure 1C). Whereas conditioned medium from thrombin-stimulated VSMCs increased HIF-1, this response was abrogated by the thrombin inhibitor hirudin (5 thrombin-inhibiting U/mL) (data not shown), additionally indicating that HIF-1 protein levels were specifically induced by thrombin.

View larger version (44K): [in this window] [in a new window]   Figure 1. Thrombin increases HIF-1 protein. Human VSMCs were incubated for different time periods with thrombin (2 U/mL) (A) or with different concentrations of thrombin for 4 hours (B). C, VSMCs were exposed to -thrombin (Thr) (3 U/mL), PPACK--thrombin (PPACK), and DIP--thrombin (DIP) (74.2 nmol/L each), -thrombin (-Thr) (3 U/mL), or factor Xa (Xa) (1 U/mL) for 4 hours.

HIF-1 protein expression was also stimulated by PDGF-AB and TGF-ß₁ to a similar extent as by the iron chelator and HIF-1 activator desferrioxamine (DFO) (150 µmol/L),²⁰ which was used as a positive control (Figure 2A). Moreover, thrombin and TGF-ß₁ also induced HIF-1 protein levels in native VSMCs derived from human renal arteries (Figure 2B).

View larger version (29K): [in this window] [in a new window]   Figure 2. Enhanced HIF-1 protein in response to thrombin and platelet-associated growth factors. A, Human VSMCs were incubated for 4 hours under control conditions (Ctr) or with thrombin (Thr) (2 U/mL), PDGF-AB (PDGF) (30 ng/mL), TGF-ß1 (TGF) (10 ng/mL), or desferrioxamine (DFO) (150 µmol/L). Western blot analysis was performed as in Figure 1 (n=4; *P<0.05 treatment vs control). B, Segments of human renal arteries were incubated under control conditions or with thrombin (3 U/mL) or TGF-ß1 (10 ng/mL) for 8 hours. Western blot analysis was performed subsequently as in Figure 1.

Thrombin Stimulates HIF-1 Transcriptional Activity and the Expression of HIF-1 Target Genes
VSMCs were exposed to 2 U/mL thrombin or 3% oxygen (hypoxia), and DNA-binding activity was detected in nuclear fractions using a 18-bp oligonucleotide probe containing the HRE with the HIF-1–binding site of the erythropoietin gene (Figure 3A). Similar to hypoxia, thrombin induced DNA-binding complexes, which could be supershifted by a specific HIF-1 antibody, indicating the presence of this protein in these complexes (Figure 3B).

View larger version (38K): [in this window] [in a new window]   Figure 3. Thrombin stimulates HIF-1 DNA binding and expression of HIF-1–dependent reporter gene constructs. A, EMSA was performed using an oligonucleotide containing the HRE with the HIF-1–binding sequence (underlined) from the erythropoietin gene (EPO-HRE). B, Human VSMCs were incubated for 4 hours under control conditions (16% O2), hypoxic conditions (3% O2), or in the presence of thrombin (2 U/mL) before EMSA. For supershift analysis, an antibody against human HIF-1 was used. S indicates supershift; I, induced bands; C, constitutive band; and U, unspecific band. The gel shift is representative for 3 independent experiments. C, VSMCs were transiently transfected with luciferase reporter genes containing either three copies of the HRE of the erythropoietin gene (pGLEPOHRE), a promoter element of the VEGF gene harboring the HRE (pGLhVEGFHRE), or the human PAI-1 promoter with the HRE (pGLhPAI-796). Transfected cells were stimulated for 18 hours with thrombin (THR) (2 U/mL), desferrioxamine (DFO) (150 µmol/L), or thrombin in the presence of 100 µmol/L vitamin C (THR+VitC) or incubated for this time under hypoxic (3% O2) conditions (Hyp). The increase in luciferase activity was related to induction of the control vector pGL3Promoter (Ctr) (n=3 to 8; *P<0.05 vs control). Northern blot analysis was performed from VSMCs stimulated with DFO (150 µmol/L) or thrombin (2 U/mL) for 4 hours using a rat VEGF cDNA probe (n=4; *P<0.05 vs control) (D) or from VSMCs incubated for 4 hours under hypoxic (3% O2) conditions or in the presence of thrombin (2 U/mL) using a rat PAI-1 cDNA probe (n=4; *P<0.05 vs control) (E).

Thrombin and DFO activated reporter gene activity of a luciferase reporter gene construct containing three HREs from the erythropoietin gene in front of the SV40 promoter (pGLEPOHRE) by 170±12% and 225±32%, respectively (Figure 3C). Moreover, thrombin and DFO induced reporter gene activity of a VEGF reporter gene harboring a 707-bp fragment of the VEGF promoter by 290±38% and 260±25%, respectively. This fragment contained the HRE with the HIF-1–binding site¹⁹ but lacked a region of Sp1/AP2 sites known to be sensitive to ERK1/2 stimulation by TGF-ß₁ or serum^{29 30} (Figure 3C). Concomitantly, VEGF mRNA expression paralleled reporter gene activity in response to thrombin (230±40%) and DFO (243±20%) (Figure 3D). Moreover, thrombin and hypoxia enhanced reporter gene activity of a PAI-1 promoter plasmid harboring a HRE with a functional HIF-1–binding site¹⁸ by 168±6% and 170±14.7%, respectively (Figure 3C), and PAI-1 mRNA levels by 256±33% and 306±28%, respectively (Figure 3E). The relatively low PAI-1 reporter gene activity may be attributable to different kinetics between PAI-1 mRNA and luciferase protein induction. Alternatively, cell type–specific elements required for full activation in VSMCs may lack in this construct.

Antioxidants Prevent Induction and Activation of HIF-1 in VSMCs
When applied to VSMCs, thrombin stimulated DCF fluorescence within minutes, indicating the rapid onset of intracellular ROS production (Figure 4A). This response was prevented by the antioxidant vitamin C (100 µmol/L). Furthermore, ROS production was significantly enhanced in VSMCs stimulated with PDGF-AB and TGF-ß₁ for 2 hours (Figure 4B).

View larger version (17K): [in this window] [in a new window]   Figure 4. Production of ROS in response to thrombin, PDGF-AB, and TGF-ß1. A, Human VSMCs were incubated under control conditions (Ctr) or in the presence of vitamin C (VitC) (100 µmol/L) for 30 minutes, and ROS production in response to thrombin (2 U/mL) was measured over 10 minutes using the DCF fluorescence (n=3; *P<0.05 vs thrombin). B, VSMCs were incubated with PDGF-AB (PDGF) (30 ng/mL) or TGF-ß1 (TGF) (10 ng/mL) for 2 hours, and DCF fluorescence was measured subsequently (n=6; *P<0.05 vs control).

The antioxidants N-acetylcysteine (NAC) (10 mmol/L) or pyrrolidinedithiocarbamate (PDTC) (100 µmol/L) attenuated the thrombin-induced HIF-1 accumulation (Figures 5A and 5B). Moreover, pretreatment with vitamin C (100 µmol/L), vitamin E succinate (20 mmol/L), or the vitamin E derivative trolox (100 µmol/L) (alone or in combination) completely prevented thrombin-induced HIF-1 accumulation (Figure 5A). In addition, vitamin C inhibited HIF-1 protein levels elicited by PDGF-AB, TGF-ß₁, and PDPs (Figure 5C). Exposure to H₂O₂ (10 to 100 µmol/L) increased HIF-1 protein expression in VSMCs but not in the human carcinoma cell line ECV304 (Figure 5D).

View larger version (36K): [in this window] [in a new window]   Figure 5. Antioxidants inhibit HIF-1 protein expression induced by thrombin and platelet-associated growth factors. A, Human VSMCs were preincubated for 30 minutes with vitamin C (C) (100 µmol/L), vitamin E succinate (E) (20 mmol/L), trolox (Tr) (100 µmol/L), NAC (10 mmol/L), or PDTC (100 µmol/L) and stimulated with thrombin (2 U/mL) for 4 hours. Western blot analysis was performed as in Figure 1 (n=3 to 5; *P<0.05 vs thrombin). B, Representative Western blots from antioxidant-treated VSMCs. C, VSMCs were preincubated with vitamin C (VitC) (100 µmol/L) and stimulated for 4 hours with TGF-ß1 (TGF) (10 ng/mL), PDGF-AB (PDGF) (30 ng/mL) or platelet-derived products (PDPs) (20 µg protein/mL). The Western blot (performed as in Figure 1) is representative of 3 independent experiments. D, VSMCs and human carcinoma cells (ECV304) were exposed to increasing concentrations of H2O2 or to desferrioxamine (DFO) (150 µmol/L) for 4 hours. The Western blot (performed as in Figure 1) is representative of 3 independent experiments.

Consistent with the DNA-binding and reporter gene assays, immunofluorescence studies demonstrated nuclear translocation and accumulation of HIF-1 in response to thrombin and PDGF-AB (Figure 6A through 6C) as well as to TGF-ß₁ (data not shown). Vitamin C treatment completely prevented this response (Figure 6D through 6F) as well as thrombin-induced PAI-1 reporter gene activity (Figure 3C).

View larger version (123K): [in this window] [in a new window]   Figure 6. HIF-1 immunofluorescence in response to agonists and vitamin C. Human VSMCs were seeded on coverslips and incubated under control conditions (A) or in the presence of vitamin C (100 µmol/L) (D), exposed to thrombin (2 U/mL) under control conditions (B) or in the presence of vitamin C (E), or stimulated with PDGF-AB (30 ng/mL) in the absence (C) or presence (F) of vitamin C. Immunostaining was performed with an antibody against human HIF-1, and an alexa 546-coupled anti-mouse secondary antibody was used for visualization by confocal microscopy.

p22^phox-Containing NADPH Oxidase Is Involved in Thrombin-Induced HIF-1 Protein and VEGF and PAI-1 mRNA Expression
VSMCs were mock-transfected or transfected with p22^phox scrambled or antisense oligonucleotides, and ROS production was subsequently determined by DCF fluorescence (Figure 7A). Thrombin-stimulated ROS generation was significantly decreased in p22^phox antisense–transfected VSMCs compared with mock-transfected cells or cells transfected with p22^phox scrambled oligonucleotides, whereas basal ROS production remained unchanged. These findings are consistent with our previous observations that the p22^phox-containing NADPH oxidase can be rapidly activated by thrombin.⁶

View larger version (36K): [in this window] [in a new window]   Figure 7. p22phox contributes to ROS production, HIF-1, VEGF, and PAI-1 expression in response to thrombin. Human VSMCs were transfected with p22phox scrambled (SC) or antisense (AS) oligonucleotides or were mock transfected (M). A, DCF fluorescence was measured over 10 minutes after stimulation with 2 U/mL thrombin (n=4; *P<0.05 vs stimulus). B, Western blot analysis was performed as in Figure 1 (n=3; *P<0.05 vs stimulus). C, Northern blot analysis was performed with cDNA probes for rat p22phox, VEGF, PAI-1, and ß-actin. D, Statistical analysis of VEGF mRNA expression in VSMCs (n=3; *P<0.05 vs stimulus). E, Statistical analysis of PAI-1 mRNA expression in VSMCs (n=3; *P<0.05 vs stimulus).

Compared with VSMCs transfected with p22^phox scrambled oligonucleotides, thrombin-induced HIF-1 expression was significantly inhibited in p22^phox antisense–transfected cells (Figure 7B). Concomitantly, p22^phox mRNA levels were substantially decreased in p22^phox antisense–transfected cells compared with cells transfected with scrambled oligonucleotides (Figure 7C). In addition, p22^phox antisense treatment inhibited thrombin-induced VEGF and PAI-1 mRNA expression (Figures 7C through 7E), indicating that the NADPH oxidase is involved in the induction of HIF-1 and in the upregulation of the HIF-1 target genes VEGF and PAI-1.

p38 MAP Kinase and Phosphatidylinositol-3-Kinase Contribute to Upregulation of HIF-1 Protein and VEGF and PAI-1 mRNA Levels by Thrombin
We have previously shown that thrombin stimulates p38 MAP kinase and ERK1/2 phosphorylation in VSMCs and that thrombin-induced activation of p38 MAP kinase, but not of ERK1/2, is abrogated in p22^phox antisense–transfected VSMCs as well as by antioxidant treatment.⁶ To determine whether a similar pathway is involved in thrombin-induced HIF-1 expression, VSMCs were treated with the p38 MAP kinase inhibitor SB202190 (20 µmol/L) or with PD98059 (50 µmol/L), which prevents phosphorylation of ERK1/2 by MEK1. Inhibition of the p38 MAP kinase, but not of the ERK1/2 pathway, significantly decreased thrombin-induced HIF-1 expression (Figure 8A). Similarly, the p38 MAP kinase inhibitors SB203580 (20 µmol/L) and SB220025 (20 µmol/L), but not the nonfunctional control substance SB202474 (20 µmol/L) or the MEK1 inhibitor U0126, attenuated thrombin-induced HIF-1 expression (data not shown). In addition, thrombin-induced VEGF and PAI-1 mRNA expression was significantly reduced by p38 MAP kinase inhibitors, whereas inhibition of ERK1/2 only mildly decreased VEGF and PAI-1 mRNA levels (Figures 8B through 8D), additionally indicating that p38 MAP kinase is part of the (p22^phox-dependent) signaling cascade leading to the stimulation of HIF-1 and its target genes VEGF and PAI-1 by thrombin.

View larger version (49K): [in this window] [in a new window]   Figure 8. Effects of inhibitors of MAP kinases and PI3-kinase on thrombin-induced HIF-1, VEGF, and PAI-1 levels. Human VSMCs were incubated with PD98059 (PD) (50 µmol/L), SB202190 (SB) (20 µmol/L), or Wortmannin (Wo) (20 nmol/L) for 30 minutes before stimulation with 2 U/mL thrombin for 4 hours. A, Western blot analysis was performed as in Figure 1 (n=3 to 12; *P<0.05 vs stimulus). B, Northern blot analysis was carried out with cDNA probes for rat VEGF, PAI-1, or ß-actin. C, Statistical analysis of VEGF mRNA expression in VSMCs (n=3 to 7; *P<0.05 vs stimulus). D, Statistical analysis of PAI-1 mRNA expression in VSMCs (n=3 to 8; *P<0.05 vs stimulus).

Furthermore, pretreatment of VSMCs with Wortmannin (20 nmol/L) or LY 294002 (50 µmol/L, data not shown), which selectively inhibit phosphatidylinositol-3-kinase (PI3-kinase), prevented stimulation of HIF-1 protein as well as induction of VEGF and PAI-1 mRNA expression by thrombin (Figure 8), suggesting a role for this enzyme in activation of the HIF-1 pathway by thrombin.

Discussion

In this study, we demonstrate that the hypoxia-inducible transcription factor HIF-1 is activated by thrombotic factors in VSMCs. This is supported by (1) increased levels of the inducible subunit HIF-1 of this heterodimeric transcription factor after stimulation with thrombin, factor Xa, PDPs, PDGF-AB, and TGF-ß₁; (2) nuclear accumulation of HIF-1 in response to thrombin and PDGF-AB; (3) increased DNA-binding activity and enhanced transactivation of the luciferase gene driven by the HRE on stimulation with thrombin; and (4) enhanced reporter gene activity of constructs containing regions of the VEGF or PAI-1 promoter harboring functional HREs. Because thrombin, activated platelets, and platelet-associated growth factors enhance the mRNA expression of the HIF-1 target genes VEGF and PAI-1 in VSMCs,^{22 23 24} activation of HIF-1 may represent a novel mechanism by which thrombotic factors regulate gene expression in the vascular wall.

Inhibition of thrombin-induced expression, nuclear accumulation, and transactivation of HIF-1 by the antioxidants vitamin C, vitamin E, trolox, NAC, and PDTC indicated redox-sensitive mechanisms involved in this signaling pathway. Because thrombin induced ROS production in VSMCs and vitamin C attenuated this response, it is tempting to speculate that ROS contributed to activation of HIF-1 by thrombin. This is supported by findings demonstrating inhibition of ROS production in VSMCs by NAC and PDTC.³¹

Transfection of antisense oligonucleotides against the NADPH oxidase subunit p22^phox abrogated p22^phox mRNA expression, thrombin-stimulated ROS production, and thrombin-induced upregulation of HIF-1 and its target genes VEGF and PAI-1, indicating that this enzyme is involved in this signaling cascade. Furthermore, transfection of p22^phox antisense cDNA also inhibited p22^phox mRNA expression as well as ROS production and gene expression in response to activated platelets or thrombin to a similar extent than p22^phox antisense oligonucleotides.^{4 6} In addition, electroporation of an inhibitory p22^phox antibody prevented thrombin-stimulated ROS production comparable with the effects of p22^phox antisense oligonucleotides,^{4 6} underlining the efficacy of this approach in inhibiting the NADPH oxidase in VSMCs. Thus, the VSMC isoform of the NADPH oxidase, which expresses the p22^phox subunit and probably the gp91^phox homologue Nox1,⁹ may represent an important source for ROS, which may mediate activation of the HIF-1 pathway by thrombotic factors in VSMCs.

Interestingly, low doses of H₂O₂ increased HIF-1 protein levels in VSMCs but not in ECV304 cells, implicating that induction of HIF-1 by ROS may be specific to VSMCs. The complexity of HIF-1 regulation by ROS-dependent or other redox-sensitive pathways in different cell types is additionally underlined by studies in fetal alveolar epithelial cells where, in contrast to VSMCs, PDTC and NAC increased HIF-1 expression under nonhypoxic conditions³² as well as by a report demonstrating that redox mechanisms can target discrete regions of the HIF-1 protein.³³ Possibly, cell type–dependent and stimulus-dependent factors may control ROS dependency or redox-sensitivity of HIF-1 and thus HIF-1 activation either directly or by induction of specific signaling cascades.

Whereas HIF-1 protein remains elevated as long as hypoxia is maintained,²⁰ thrombin stimulation transiently elevated HIF-1 protein levels, peaking at 1 to 4 hours, consistent with the idea that a rapid increase in ROS such as with thrombin may transiently activate a signaling cascade, leading to induction of HIF-1. Indeed, thrombin transiently activated the p38 MAP kinase after the induction of ROS production, and thrombin-induced p38 MAP kinase activation was sensitive to antioxidants and prevented in p22^phox antisense–transfected VSMCs.⁶ In addition, thrombin-induced HIF-1 protein and VEGF and PAI-1 mRNA expression was significantly decreased by inhibitors of p38 MAP kinase. On the other hand, ERK1/2, another MAP kinase, which is also activated by thrombin but is insensitive to antioxidants or p22^phox antisense treatment,⁶ did not seem to play an important role in thrombin-induced HIF-1 signaling, because inhibitors of ERK1/2 did not affect thrombin-induced HIF-1 protein levels and only mildly reduced VEGF and PAI-1 mRNA expression.

In line with our results, p22^phox also mediated activation of p38 MAP kinase, but not of ERK1/2, in response to angiotensin II, a known activator of the NADPH oxidase.³⁴ Furthermore, HIF-1 expression, stimulated by angiotensin II, was insensitive to inhibition of ERK1/2 in VSMCs.³⁵ However, in fibroblasts, overexpression of ERK1/2, but not of p38 MAP kinase, increased in vitro phosphorylation of HIF-1 under normoxic conditions,³⁶ and ERK1/2 phosphorylated HIF-1 in hypoxic endothelial cells.³⁷ These findings additionally suggest that activation of the NADPH oxidase and, subsequently, of p38 MAP kinase may be part of a specific pathway allowing induction of HIF-1 and its target genes by thrombotic factors under normoxic conditions.

In addition to inhibitors of p38 MAP kinase, PI3-kinase inhibitors also significantly reduced thrombin-stimulated HIF-1, VEGF, and PAI-1 expression. Similarly, in several tumor-cell lines, HIF-1 activation in response to hypoxia or growth factors, including EGF and IGF-1, was impaired by PI3-kinase inhibitors.^{38 39} Moreover, dominant-negative mutants of the PI3-kinase effector kinase Akt as well as rapamycin, an inhibitor of the Akt effector FRAP, attenuated HIF-1 activation by hypoxia or growth factors in these cells.^{38 39} Thrombin has been shown to activate PI3-kinase and Akt in smooth muscle cells,⁴⁰ and rapamycin inhibited thrombin-induced HIF-1 protein and VEGF mRNA expression (A. Görlach, unpublished data, 2000). In addition, the nonspecific NADPH oxidase inhibitor diphenyleneiodonium prevented activation of Akt in response to angiotensin II in VSMCs.⁴¹ These findings suggest that, in addition to p38 MAP kinase, PI3-kinase/Akt may contribute to activation of the HIF-1 cascade in response to thrombin in VSMCs.

Taken together, the present study favors a model in which thrombin-induced generation of ROS and subsequent stimulation of p38 MAP kinase or PI3-kinase leads to the activation of HIF-1 and induction of its target genes VEGF and PAI-1 in VSMCs. The finding that HIF-1 is not only activated by hypoxia but also by thrombotic factors via an ROS-sensitive, p22^phox-dependent mechanism points toward a more general role of this transcription factor in the vascular response to injury.

Acknowledgments

This research was supported in part by grants of the Deutsche Forschungsgemeinschaft (to R.P.B. and T.K.). We would like to thank Olaf Herkert, Dr Steffen Bassus, and Isabel Winter for their support.

Footnotes

Original received December 27, 2000; resubmission received April 9, 2001; revised resubmission received May 8, 2001; accepted May 8, 2001.

This manuscript was sent to Donald D. Heistad, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

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