Thrombin Causes Vascular Endothelial Growth Factor Expression in Vascular Smooth Muscle Cells
Role of Reactive Oxygen Species
Abstract—Vascular endothelial growth factor (VEGF) has been implicated in the reendothelialization of the vascular wall after balloon injury. This study investigated whether thrombin, which is formed during activation of the coagulation cascade at sites of vascular injury, upregulates VEGF expression in vascular smooth muscle cells (VSMCs). VEGF expression was assessed in native and cultured VSMCs by Northern blot analysis and reverse transcription–polymerase chain reaction and the release of VEGF protein by immunoassay. α-Thrombin time- and concentration-dependently increased VEGF mRNA levels, mainly that mRNA coding for the soluble splice variant VEGF164/165, and stimulated the release of VEGF protein. These effects required the proteolytic activity of thrombin and were mimicked by a thrombin receptor activating–peptide. Upregulation of VEGF expression was also induced by conditioned medium from α-thrombin–stimulated VSMCs. Both the early and the delayed α-thrombin–induced VEGF expressions were attenuated by antioxidants and by diphenyleneiodonium. α-Thrombin–induced VEGF release was significantly reduced by a platelet-derived growth factor (PDGF)–, a transforming growth factor (TGF)-β–, and a basic fibroblast growth factor (bFGF)–neutralizing antibody. Thrombin caused a redox-sensitive upregulation of expression of VEGF in VSMCs through a direct and an indirect effect, which was dependent on the endogenous formation of PDGF, TGF-β, and bFGF. Upregulation of VEGF expression may represent an important mechanism by which the coagulation cascade contributes to the regeneration of the endothelial lining at sites of balloon injury.
Therapeutic angioplasty to restore normal blood flow in stenosed arteries is associated with disruption of the protective endothelial lining and severe damage to the underlying smooth muscle cell (SMC) layers.1 After contact of the blood with the subendothelial matrix, an instantaneous activation of the coagulation cascade and of platelets occurs. As a consequence of the hemostatic and thrombotic response, potent mitogenic and vasoactive factors are generated by the coagulation cascade, such as thrombin, or are released from aggregating platelets, such as platelet-derived growth factor (PDGF) and serotonin. Moreover, the arterial wall itself responds to injury by generating proarteriosclerotic factors such as PDGF, basic fibroblast growth factor (bFGF), endothelin, and angiotensin II. These blood- and vascular wall–derived factors contribute to limit the success of angioplasty by causing local vasospasm and by stimulating the migration and proliferation of vascular smooth muscle cells (VSMCs), which ultimately lead to the development of intimal thickening.
Recent investigations have suggested that vascular protection can be restored at sites of balloon injury with therapeutic approaches that expedite the recovery of the endothelial lining. Among the angiogenic factors that are of potential interest is vascular endothelial growth factor (VEGF), because this factor is a specific mitogen for endothelial cells that is also able to stimulate endothelial cell migration.2,3 The local application of VEGF either as the recombinant protein or by gene transfer to balloon-injured arteries accelerated reendothelialization.4,5 Moreover, restoration of the endothelial lining was associated with a diminished neointimal thickening, a reduction in thrombogenicity, and a restoration of endothelium-dependent local control of vascular tone.4,5 Subsequently, it was recognized that the arterial wall itself responds to balloon injury by an upregulation of VEGF expression and that this endogenous formation of VEGF is necessary for the normal recovery of the endothelium.6 Although the endogenous stimulus responsible for smooth muscle VEGF expression remains unclear, its upregulation in the arterial wall shortly after balloon injury suggests that events occurring during the hemostatic and thrombotic response to injury are likely to be implicated.
The aim of the present study was to determine the role of the coagulation cascade as a potential endogenous regulator of VEGF expression in VSMCs. The present findings indicate that thrombin upregulates VEGF expression in VSMCs through the activation of the protease-activated receptor (PAR)-1. This response is dependent on the generation of reactive oxygen species by a flavoenzyme(s) and involves an immediate, direct and a delayed, indirect activation of signaling cascades by thrombin in VSMCs.
Purified human α-thrombin (specific clotting activity was 3261 U/mg) was obtained from Hemochrom Diagnostika GmbH; diphenyleneiodonium (DPI) was from Alexis Biochemicals; human recombinant bFGF and insulin-like growth factor-I (IGF-I) were from PreproTech Inc; thrombin receptor activating–peptide (TRAP; SFLLRNP) was from Bachem Biochemica GmbH; TGF-β–neutralizing antibody, bFGF-neutralizing antibody, and normal rabbit IgG and goat IgG were from R&D Systems; and PDGF-neutralizing antibody was from Upstate Biotechnology Inc. γ-Thrombin (6.65 U/mg), diisopropylfluorophosphate (DIP)–α-thrombin (0.18 U/mg), and d-phenylalanyl-l-propyl-l-arginyl-chloromethyl ketone (PPACK)–α-thrombin (0 U/mg) were kindly provided by Dr J.W. Fenton II, Albany, NY. All other chemicals were from Sigma. A 350-bp-long restriction fragment obtained from the cloned rat VEGF cDNA was kindly provided by Dr C. Frelin (Université de Nice, Nice, France). Deoxycytidine 5′-[α-32P]triphosphate (3000 Ci/mmol) was supplied by Hartmann Analytic.
VSMCs were isolated from the thoracic aortas of male Wistar rats (Charles River Wiga Deutschland GmbH) by the explant method. Rat VSMCs were cultured serially in minimum essential medium (Gibco BRL) containing 2 mmol/L l-glutamine, 100 U/mL penicillin, 50 μg/mL streptomycin, and 10% fetal calf serum (Biochrom). Human aortic SMCs (PromoCell GmbH or Clonetics) were cultured as recommended by the suppliers. All experiments were performed on rat (passages 8 to 19) and human (passages 8 to 10) VSMCs. When the VSMCs reached confluence, the culture medium was replaced by serum-free medium containing 0.1% fatty acid–free bovine serum albumin for 1 day before treatment.
Preparation of Human Renal Arteries
Human renal arteries free of atherosclerotic lesions were provided by the Department of Urology (Klinikum der JWG-Universität, Frankfurt am Main) and placed in HEPES-Tyrode’s solution. The adventitia and intima were removed mechanically, and the SMC layers were incubated in MCDB 131 containing 0.1% bovine serum albumin. After a 30-minute incubation period, the incubation medium was replaced again with serum-free MCDB 131 before the addition of thrombin or solvent for 24 hours.
Reverse Transcription–Polymerase Chain Reaction and Northern Blot Analyses
Total RNA was isolated by guanidinium isothiocyanate and phenol extraction. For reverse transcription (RT), 4 μg total RNA was incubated with 200 U reverse transcriptase (Gibco), dNTP (175 μmol/L), oligo(dT) (200 ng), dithiothreitol (1 mmol/L), and reaction buffer in a final volume of 20 μL at 37°C for 60 minutes. In some reaction mixtures, reverse transcriptase or total RNA was omitted to determine the amplification of contaminating genomic DNA or cDNA. After a final denaturation at 94°C for 7 minutes, 6 μL of cDNA was subjected to polymerase chain reaction (PCR) consisting of denaturation at 94°C for 1 minute followed by 90 seconds of annealing at 65°C and 2 minutes of elongation at 72°C for 30 cycles. The last cycle was ended by 7 minutes of elongation at 72°C. The oligonucleotide primers used for amplification of VEGF cDNAs were derived from the sequence of the cloned human cDNA (sense primer 5′-GGAGAGATGAGCTTCCTACAG-3′, antisense primer 5′-TCACCGCCTTGGCTTGTCACA-3′) and have previously been shown to amplify all reported VEGF splice variants.7 The PCR contained 0.4 μmol/L of each primer, dNTP (200 μmol/L), MgCl2 (1 mmol/L) reaction buffer, and 2.5 U Taq DNA polymerase (Promega) in a final volume of 50 μL. The amplified cDNAs were size fractionated on a 2% agarose gel, visualized under UV light after ethidium bromide staining, and transferred to nylon membranes (Porablot NY amp, Macherey-Nagel). VEGF PCR products were identified by Southern blot analysis with a rat 32P-labeled VEGF cDNA probe. Northern blotting was performed with 20 to 25 μg total RNA. RNA was electrophoresed on a 1.2% formaldehyde-denatured agarose gel, visualized with ethidium bromide, transferred to nylon membranes, and hybridized with either a rat 32P-labeled VEGF cDNA probe or a 32P-labeled 18S ribosomal RNA fragment. Autoradiography was performed with Fuji RX film with intensifying screens (DuPont de Nemours) at −70°C. The autoradiographs were analyzed by scanning densitometry. VEGF mRNA levels were normalized to their respective 18S ribosomal RNA levels and expressed in arbitrary units as the fold increase of signal obtained with untreated cells.
Determination of VEGF, PDGF, TGF-β, and bFGF
Commercially available immunoassays (R&D Systems) were used for the determination of growth factor content in conditioned medium from cultured human VSMCs or SMC layers from human renal arteries.
Results are shown as mean±SEM. Statistical analyses were performed with Student’s paired t test (2-tailed) or an ANOVA followed by Fisher’s protected least significant difference test to compare 2 treatments. A value of P<0.05 was considered statistically significant.
Expression of VEGF mRNA in VSMCs
VEGF mRNA levels in serum-deprived quiescent rat and human VSMCs were low and sometimes even barely detectable (Figures 1 and 2⇓). Steady-state levels of the transcript were markedly increased after exposure of rat and human VSMCs to α-thrombin (Figures 1 and 2⇓). α-Thrombin induced VEGF mRNA expression in a biphasic manner, with a first peak at ≈0.5 to 1 hour, followed by a second, higher peak at 24 hours (Figure 1A). The stimulatory effect of α-thrombin (examined after 1 hour) was concentration dependent, with an ≈6-fold increase induced by 10 U/mL α-thrombin (Figure 1B).
To determine the role of the proteolytic activity of thrombin, the effect of proteolytically active (α-thrombin, γ-thrombin) and inactive (DIP–α-thrombin, PPACK–α-thrombin) forms of thrombin on the early phase of VEGF expression was investigated. Exposure of rat and human VSMCs to α-thrombin or γ-thrombin significantly increased VEGF mRNA levels, whereas DIP–α-thrombin and PPACK–α-thrombin had only minor effects (Figure 2). In addition, the stimulatory effect of thrombin was mimicked by the exposure of rat and human VSMCs to the direct activator of PAR-1, TRAP (Figure 2).
Previous studies have shown that α-thrombin induces the generation of reactive oxygen species by a flavoenzyme(s)8 and that reactive oxygen species are potent inducers of VEGF expression in VSMCs.9 Therefore, the role of reactive oxygen species in mediating the α-thrombin–induced expression of VEGF mRNA (after 1 hour) was assessed. Exposure of VSMCs to either N-acetylcysteine or vitamin C, 2 antioxidants, significantly reduced the stimulatory effect of α-thrombin (Figure 3, and from 3.04±0.49- to 1.52±0.53-fold of the basal level in the presence of 200 μmol/L vitamin C; n=5). Next, the putative reactive oxygen species–generating system involved in the stimulatory effect of α-thrombin was characterized by using DPI, an inhibitor of flavoprotein-containing enzymes such as NAD(P)H oxidase; allopurinol, an inhibitor of xanthine oxidase; and indomethacin, an inhibitor of cyclooxygenase. DPI significantly reduced the stimulatory effect of α-thrombin, whereas allopurinol and indomethacin had only minor effects (Figure 3, and the stimulatory effect of α-thrombin on the VEGF mRNA level was 3.6±1.0-fold of basal levels and in the presence of allopurinol or indomethacin, 3.1±0.8- or 5.5±3.4-fold, respectively, of basal levels; n=4). Exposure of VSMCs to N-acetylcysteine, vitamin C, DPI, allopurinol, or indomethacin alone had no significant effect (Figure 3 and authors’ unpublished data, 2001).
Thrombin can activate vascular cells to release several growth factors, including PDGF, TGF-β, bFGF, IGF-I, and endothelin-1, all of which have been shown to upregulate VEGF expression in VSMCs.10–17 To determine whether the delayed (after a 24-hour incubation period) response to α-thrombin was dependent on the release of such factors, which in turn act in an autocrine manner to stimulate VEGF expression, conditioned medium from α-thrombin (3 U/mL for 24 hours)–stimulated VSMCs was removed and transferred (after addition of thrombin-inactivating hirudin to prevent the direct effect of α-thrombin) to new cultures of VSMCs, and VEGF mRNA expression was determined after 1 hour. The conditioned medium from α-thrombin–stimulated VSMCs significantly increased VEGF mRNA levels, whereas the conditioned medium from control cells had no effect (Figure 4A). The stimulatory effect of conditioned medium from α-thrombin–stimulated VSMCs was significantly reduced by N-acetylcysteine, vitamin C, or DPI (Figure 4B). Next, the possibility that the released stimulatory factors are dependent on protein synthesis was assessed with cycloheximide (an inhibitor of protein synthesis). However, exposure of VSMCs to cycloheximide (5 μg/mL) alone for 24 hours caused a marked upregulation of VEGF mRNA (5.22±2.44, n=3), which precluded its study on the delayed α-thrombin–induced expression of VEGF mRNA.
RT-PCR analysis performed to characterize the VEGF isoforms expressed in rat VSMCs revealed low levels of a single 224-bp VEGF cDNA in quiescent VSMCs, corresponding to amplification of the VEGF164 transcript (data not shown). In contrast, in RNA samples from α-thrombin (3 U/mL for 1 hour)– or TRAP (100 μmol/L for 1 hour)-treated VSMCs, levels of the 224-bp VEGF transcript were markedly increased, and additional low levels of 295- and 93-bp VEGF cDNAs corresponding to VEGF188 and VEGF120 were observed (data not shown).
Release of VEGF, PDGF, TGF-β, and bFGF Protein
To prove that the increased VEGF mRNA after stimulation of VSMCs by α-thrombin was accompanied by increases in VEGF secretion, VEGF concentration in the conditioned medium from VSMCs was quantified by immunoassay. In conditioned medium from unstimulated human VSMCs, detectable amounts of VEGF were found after 24 hours (Figure 5). Exposure of VSMCs to either α-thrombin or TRAP but not to PPACK–α-thrombin significantly increased the release of VEGF (Figure 5A). In addition, VEGF release was also significantly enhanced by exposure of VSMCs to factor Xa (1 U/mL, from 1971±89 to 2416±114 pg/mL; n=7). To identify putative mediators of the stimulatory effect of α-thrombin, the effect of several exogenous growth factors was investigated at concentrations previously shown to evoke maximal activation of VSMCs. Exposure of VSMCs to bFGF (30 ng/mL for 24 hours) markedly increased the release of VEGF from 694.8±120.7 to 1327.9±132.6 pg/mL, whereas endothelin-1 (100 nmol/L) only slightly increased the release of VEGF, to 813.8±120.4 pg/mL (n=6). In addition, we have previously shown an upregulation of VEGF expression in VSMCs by TGF-β and PDGF but not by IGF-I.17 Moreover, exposure of VSMCs to α-thrombin (1 U/mL) for 24 hours significantly increased the release of PDGF, TGF-β, and bFGF (see Pircher et al18 and the Table).
To identify the role of VSMC-derived bFGF, PDGF, and TGF-β on the stimulatory effect of α-thrombin, selective neutralizing antibodies against these 3 factors were used. Treatment of VSMCs with α-thrombin in the presence of a bFGF-, PDGF-, or TGF-β–neutralizing antibody significantly reduced the stimulatory effect of the serine protease by 48%, 49%, and 59%, respectively, whereas a neutralizing antibody alone had only minor effects (Figure 5B). Control IgGs (0.2 mg/mL) as well as a nonrelevant neutralizing antibody directed against thrombomodulin (0.2 mg/mL) did not affect the stimulatory effect of α-thrombin (data not shown).
Finally, experiments were performed to confirm that the stimulatory effect of α-thrombin occurs not only with cultured VSMCs but also with native human VSMCs. Freshly excised SMC layers from human renal arteries, kept in culture medium for 24 hours, released detectable amounts of VEGF, which were significantly increased after incubation with α-thrombin (from 3.9±0.9 to 8.7±1.1 pg/mg wet weight; n=5).
Both immunohistochemical staining and in situ hybridization have shown that the expression of VEGF is upregulated in mechanically injured arteries removed from healthy baboons and rats.6,9 VEGF is expressed at low levels primarily in endothelial cells. However, after balloon angioplasty, VEGF protein appears in medial VSMCs of rat carotid arteries within hours, and thereafter VEGF levels in VSMCs remain elevated for at least 7 days.6 Increased VEGF expression is also found in the neointima and media of balloon-injured brachial arteries of baboons 7 days after injury.9 These findings indicate that upregulation of VEGF expression, predominantly in VSM, is a consequence of mechanical vascular injury. Moreover, VEGF has been identified as a key factor contributing to the rapid regeneration of the endothelium and the restoration of protective endothelial function at sites of balloon injury.6 The rapid induction (within 8 hours) of VEGF protein expression in balloon-injured arteries6 suggests that hemostatic and thrombotic events occurring during the early phase of the vascular response to injury might be involved. The present findings indicating that α-thrombin markedly stimulated the expression of VEGF mRNA and protein in cultured and native VSMCs are consistent with the involvement of the coagulation cascade.
VEGF, a disulfide-linked dimeric glycoprotein of 34 to 42 kDa, is a potent and specific mitogen for endothelial cells and also an effective stimulus for endothelial cell migration.19 Five different human VEGF isoforms have been identified, which are generated as a result of alternative splicing from a single VEGF gene (VEGF121, 145, 165, 189, and 206). Nonhuman VEGFs are expected to be shorter by 1 amino acid.20 All VEGF isoforms are biologically active, but they are distinguished by their ability to bind to heparin and heparan sulfate proteoglygans on cell surfaces. VEGF121 does not bind to heparin or extracellular matrix, VEGF145 and VEGF165 bind with moderate affinity, and VEGF189 and VEGF206 bind with high affinity.19 Consistent with previous studies, quiescent, cultured VSMCs expressed low levels of a single VEGF mRNA transcript corresponding to VEGF165/164.17,21 In contrast, 3 distinct transcripts were found in VSMCs exposed to α-thrombin, demonstrating a marked upregulation of VEGF165/164 mRNA and the appearance of VEGF mRNA signals corresponding to VEGF189/188 and VEGF121/120. These findings indicate that thrombin upregulates predominantly the secreted isoforms of VEGF, mostly VEGF165/164, but also the cell-associated VEGF isoform VEGF189/188.
The kinetics of VEGF expression suggest that α-thrombin stimulates directly as indicated by the first transient increase in VEGF mRNA (peak after 1 hour) followed by an autocrine pathway as indicated by the second, transient increase in VEGF mRNA levels (peak after 24 hours). The finding that conditioned medium (24 hours) from α-thrombin–treated VSMCs strongly induced VEGF mRNA expression after inactivation of α-thrombin with an excess of hirudin provides additional evidence for an autocrine pathway. bFGF, PDGF, and TGF-β seem to be involved as major mediators of the thrombin-induced, delayed VEGF expression, because selective bFGF-neutralizing, PDGF-neutralizing, and TGF-β–neutralizing antibodies significantly prevented the stimulatory effect of α-thrombin. Moreover, increased levels of basic FGF, PDGF, and TGF-β were detected in conditioned media of VSMCs after α-thrombin stimulation. These findings are consistent with previous reports indicating that thrombin induces PDGF-A chain, bFGF, and TGF-β expression in cultured VSMCs10–12 and that all 3 growth factors stimulate VEGF expression in VSMCs.14,17
Thrombin exerts most if not all of its actions, including mitogenesis, on vascular cells by activation of a G protein–coupled PAR-1. Thrombin activates PAR-1 by binding to and cleaving its amino-terminal exodomain to unmask a new amino terminus.22,23 This new amino terminus then serves as a tethered peptide ligand, binding intramolecularly to the body of the receptor to effect transmembrane signaling. The present findings indicate that the thrombin-induced VEGF expression in VSMCs is mediated by PAR-1, because this effect is strictly dependent on the protease activity of thrombin and is mimicked by a TRAP. Both the early and the delayed thrombin-induced expressions of VEGF are critically dependent on the generation of reactive oxygen species by a flavoprotein-containing enzyme(s), because both effects were inhibited by the antioxidants N-acetylcysteine and vitamin C and by DPI. These findings are consistent with previous data showing that thrombin and PDGF induce the generation of O2− and H2O2 by a flavoenzyme(s) and that H2O2 is a strong inducer of VEGF expression in cultured VSMCs.8,9,24
Besides thrombin, coagulation cascade–derived factor Xa also significantly increased the release of VEGF from VSMCs (present findings). Tissue factor, the prime initiator of the coagulation cascade, has been shown to upregulate VEGF expression in human tumor cells.25 Thus, the balloon injury–induced VEGF expression in the arterial wall may reflect activation of the coagulation cascade through the generation of several inducers that strongly upregulate VEGF expression. A synergistic action of PDGF and TGF-β released from aggregating platelets may also contribute to local VEGF production.17 Although the hemostatic and the thrombotic responses may be involved in the initial upregulation of VEGF expression shortly after injury, both responses gradually decline after several days. Therefore, the sustained VEGF expression in the injured arterial wall is likely to be due to the generation of VEGF inducers by the arterial wall itself, such as endothelin, angiotensin II, TGF-β, bFGF, and PDGF.16,17,26,27
In conclusion, both the hemostatic and the thrombotic responses appear to be crucial events in the healing process of mechanically injured arteries by stimulating the local formation of VEGF. VEGF then may act in a paracrine manner on endothelial cells and stimulates their migration and proliferation, thereby contributing to the regeneration of the endothelial lining.
The authors thank G. Römer, M. Stächele, and I. Winter for technical assistance.
Received January 10, 2001; revision accepted June 14, 2001.
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