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Cardiovascular Research 2005 67(4):714-722; doi:10.1016/j.cardiores.2005.04.017
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Copyright © 2005, European Society of Cardiology

Mitochondrial reactive oxygen species and c-Src play a critical role in hypoxic response in vascular smooth muscle cells

Hiroko Satoa,b, Mahito Satoa, Hiroyoshi Kanaia, Tsuyoshi Uchiyamaa, Tatsuya Isoa, Yoshio Ohyamaa, Hironosuke Sakamotob, Junichi Tamurab, Ryozo Nagaic and Masahiko Kurabayashia,*

aDepartment of Medicine and Biological Science, Gunma University Graduate School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371-8511, Japan
bDepartment of General Medicine, Gunma University Hospital, 3-39-15 Showa-machi, Maebashi, Gunma 371-8511, Japan
cCardiovascular Medicine, Graduate School of Medicine University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan

* Corresponding author. Tel.: +81 27 220 8140; fax: +81 27 220 8150. Email address: mkuraba{at}med.gunma-u.ac.jp

Received 2 September 2004; revised 14 April 2005; accepted 15 April 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 4.4. Mitochondrial ETC as...
 Acknowledgments
 References
 
Objective: Thickened atherosclerotic plaques are prone to be hypoxic because of poor perfusion. In this study, we tested (a) whether reactive oxygen species (ROS) and c-Src play roles in hypoxic induction of HIF-1{alpha} protein and PAI-1 gene expression in the rabbit aortic smooth muscle cell line C2/2 cells and primary cultures of rat aortic smooth muscle cells, and (b) how mitochondria act on the hypoxia-induced signaling mechanism.

Methods and results: Hypoxic exposure of C2/2 cells increased H2O2 generation, c-Src phosphorylation, HIF-1{alpha} protein expression, and PAI-1 gene expression. Catalase, a scavenger of H2O2, inhibited the hypoxia-induced ROS generation and PAI-1 gene expression. Src kinase inhibitors PP1 and PP2 inhibited hypoxia-induced HIF-1{alpha} protein and PAI-1 gene expression. Ablation of mitochondrial respiration by rotenone abolished hypoxia-induced ROS generation, c-Src phosphorylation, HIF-1{alpha} protein expression, and PAI-1 gene expression.

Conclusion: Induction of HIF-1{alpha} protein and PAI-1 gene expression in response to hypoxia was regulated by ROS production and c-Src activation in vascular smooth muscle cells. Mitochondria linked the hypoxic signal to c-Src, which in turn led to HIF-1{alpha} protein and PAI-1 gene expression. These results provide evidence that hypoxia induces the ROS-mediated and c-Src-dependent signaling cascades which are closely associated with angiogenesis and thrombosis in atherosclerotic vasculature.

KEYWORDS Atherosclerosis; Hypoxia/anoxia; Smooth muscle; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 4.4. Mitochondrial ETC as...
 Acknowledgments
 References
 
Hypoxia is an important regulatory stimulus for diverse biological processes such as angiogenesis [1] and thrombosis [2]. The development of atherosclerotic plaques is associated with neovascularization in the plaque [3]. For the neointimal angiogenesis, hypoxia of the vessel wall has been considered to be an important stimulus, because neovascularization develops in response to inadequate perfusion through the thickened intima [4].

The molecular mechanisms which control hypoxia-induced gene expression have been extensively studied. Under low oxygen, a variety of cells produce many vasoactive substances such as plasminogen activator inhibitor-1 (PAI-1) [5] and vascular endothelial growth factor (VEGF) [6]. Expression of these genes is regulated by hypoxia-inducible factor-1{alpha} (HIF-1{alpha}), a transcription factor whose levels are tightly regulated by oxygen levels. Under normoxic conditions, HIF-1{alpha} is ubiquitinated and proteosomally degraded. Once oxygen concentration is decreased, HIF-1{alpha} escapes the ubiquitination/degradation. Its protein level becomes sufficient to translocate into the nucleus along with its heterodimer partner HIF-1β, and then it binds to a hypoxia-response element, thus activating hypoxia-sensitive genes [7]. Although the molecular characterization of the HIF-1 complex has been performed, upstream signaling mechanisms regulating HIF-1{alpha} protein expression remain to be studied.

Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) and superoxide anion (O2–.), have been implicated in diverse pathophysiological responses of the cardiovascular system, including induction of vascular smooth muscle cell (VSMC) proliferation, release/activation of paracrine factors and endothelial dysfunction. An in vivo study demonstrated ROS were produced in human atherosclerotic plaques [8]. Moreover, the role of ROS in the proatherogenic actions of angiotensin II (Ang II) has been well documented in a number of experimental studies. For example, in rats, chronic Ang II infusion doubles O2–. production in aortic segments through an NAD(P)H oxidase-dependent mechanism [9]. In VSMC, in vitro, subnanomolar concentrations of Ang II activate NAD(P)H oxidase, in which the ROS produced serve as intracellular second messengers, mediating the mitogenic response [10]. Thus, an induction of vascular ROS tone is probably a major mechanism by which Ang II enhances atherosclerosis.

Because of these important activities of ROS, the mechanisms by which they are generated and by which they activate the intracellular signaling cascade have been extensively investigated. c-Src has been shown to be of foremost physiological importance among the signaling molecules responsible for ROS-induced cellular signaling cascades in VSMC. Changes in c-Src activation are tightly regulated by ROS generated by Ang II [10] and PDGF [11]. However, the role of ROS and c-Src in the hypoxic response remains to be determined.

In view of the potential role of ROS and c-Src in the hypoxic response, this study was designed to analyze the in vitro effects of hypoxia on generation of ROS and activation of c-Src in VSMCs. We demonstrated that these events are critical for the hypoxia-mediated increase in HIF-1{alpha} protein and PAI-1 gene expression. In addition, our study suggests that mitochondria play an important role in activating c-Src during hypoxia.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 4.4. Mitochondrial ETC as...
 Acknowledgments
 References
 
2.1. Chemicals
Catalase, 3-amino-1,2,4-triazole (aminotriazole), 2',7'-dichlorofluorescein diacetate (DCF-DA), dihydroethidium (DHE), antimycin A, rotenone, and potassium cyanide were purchased from Sigma-Aldorich (St. Louis, MO). PD98059, SB203580, calphostin C, wortmannin, PP2, and PP3 were from Calbiochem (San Diego, CA). PP1 was from Biomol (Plymouth Meeting, PA). Sodium azide (NaN3) and diphenyleneiodonium (DPI) were from WAKO (Osaka, Japan). Anti-phospho-tyrosine antibody (Cat. No. 05-320) was from Upstate Biotechnology (Charlottesville, VA). Anti-actin antibody (Cat. No. SC-1615) was from Santa Cruz Biotechnology (Santa Cruz, CA). Phosphospecific anti-c-Src (PY418) antibody (Cat. No. 44-660) was from Biosource (Netherland). Anti-HIF-1{alpha} antibody (Cat. No. 610958) was from BD Bio Science (Tokyo, Japan). An oxygen absorbent, Anaeropack, and hypoxic chamber (sealed case) were obtained from Mitsubishi Gas Chemistry (Tokyo, Japan).

2.2. Cell culture
C2/2 cells, an established cell line derived from rabbit aortic SMCs, were obtained from Life Science Center, Biochemical Research Lab., Asahi Chemical Industry [12]. Physiological and electronmicroscopic studies have demonstrated that C2/2 cells conserve a smooth muscle phenotype [12,13]. Cells were grown in DMEM supplemented with 10% calf serum, 100 units/mL penicillin, and 100 mg/mL streptomycin. Src++ (Src-overexpressing) cells and SYF(–) (Src, Yes, and Fyn are inactive) cells were obtained from American Type Culture Collection and were grown in DMEM supplemented with 10% calf serum, 100 units/mL penicillin, and 100 µg/mL streptomycin. Rat aortic smooth muscle cells were isolated from rat thoracic aortas by enzymatic digestion, as described previously [14], and were grown in M199 supplemented with 10% calf serum. When inhibitors were used, cells were pretreated for 1 h with inhibitors before exposure to hypoxia. C2/2 cells were pretreated for 12 h with catalase.

2.3. Hypoxic stimulation
Cells were entered into the sealed box (hypoxic chamber) with an Anaeropack, a disposable oxygen-absorbing and CO2-generating agent, and incubated in 37 °C for the indicated times. The control cells were incubated at 37 °C in an atmosphere of 21% O2 and 5% CO2 for the same duration as hypoxic cells. The Anaeropack starts to absorb oxygen within 1 min; oxygen tension inside the box drops to 1 mm Hg within 1 h [15]. This method has been used for the culture of anaerobic bacteria [16] and cells [15]. We checked that the oxygen tension was 159.5 mm Hg in the 21% O2 and 5% CO2 incubator, and it decreased to 89.5 mm Hg within 15 min, and 32.5 mm Hg within 60 min of hypoxia.

2.4. Measurements of intracellular ROS levels
For detection of O2–., we used DHE, which is specifically oxidized to ethidium by O2–.. To detect H2O2, we used DCF-DA, which acts as an H2O2-sensitive fluorophore. Cells plated on the gelatin-coated slide glass were kept in hypoxic condition for 6 h. Cells were then moved into the dark with DHE and DCF-DA for 30 min at room temperature under normoxia. The cells were imaged by laser confocal microscopy (MRC-1000, Bio-Rad), using a laser intensity of 30, an iris setting of 3.5, and a gain of 1200. To quantify intracellular H2O2 levels, the cells treated with DCF-DA were rinsed twice with ice-cold PBS, and scraped. A 200 µL cell suspension was loaded into a 96-well plate and examined with a fluorescent plate reader at excitation and emission wavelengths of 475 and 525 nm, respectively [17].

2.5. RNA isolation and Northern blot analysis
Total cellular RNA was isolated from cells with Isogen (Nippon genes, Tokyo, Japan). Equal amounts of RNA were resolved by electrophoresis and were transferred onto Hybond N+ nylon membranes (Amersham Corp., Tokyo, Japan). The membranes were hybridized for 16 h at 42 °C with a specific cDNA probe that was radiolabeled with [a-32P] dCTP (Amersham Corp.), as described previously [5].

2.6. Protein isolation and immunoblotting
Protein isolation and Western blot analysis were performed as described previously [18]. Equal amounts of protein were separated using SDS-polyacrylamide gel electrophoresis. Blots were incubated with primary rabbit polyclonal phosphospecific c-Src antibody at 1:1000, and with primary mouse monoclonal anti-HIF-1{alpha} antibody at 1:1000.

2.7. Statistical analysis
Data are presented as the mean ± S.E.M. for {gtrsim} 3 separate experiments. To compare the data between normoxia and hypoxia, Student's t-test was used. For multiple comparisons, one-way ANOVA was used. All tests were considered to be statistically significant at p<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 4.4. Mitochondrial ETC as...
 Acknowledgments
 References
 
3.1. Mitochondrial O2–. and H2O2 production due to hypoxia in C2/2 cells
We determined if hypoxia stimulates ROS production in C2/2 cells by using DHE (10 µmol/L) and DCF-DA (10 µmol/L) as fluorescent probes of intracellular O2–. and H2O2, respectively. As shown in Fig. 1A, production of O2–. and H2O2 was detected in the cells exposed to hypoxia for 6 h. Hypoxia increased H2O2 generation as determined by DCF fluorescence in C2/2 cells (Fig. 1B,C), and in rat aortic smooth muscle cells (RASMCs) (Fig 1D). This suggests that ROS production due to hypoxia occurs not only in C2/2 cells but also in other lines of vascular smooth muscle cells.


Figure 1
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  		Fig. 1 Effect of hypoxia on ROS generation in VSMCs as detected by DHE and DCF-DA. (A) Cells were stained for O2–. (red) and H2O2 (green) as observed by confocal microscopy. Cells cultured under normoxic (a) or hypoxic (b) condition were treated with DHE (10 µmol/L). Cells cultured under normoxic (c) or hypoxic (d) condition were treated with DCF-DA (10 µmol/L). (B–D) Quantitative analysis of the effect of hypoxia on H2O2 generation in C2/2 cells. (B) Cells treated with or without catalase and aminotriazole were cultured under normoxia or hypoxia. (C) C2/2 cells pretreated with vehicle, rotenone, antimycin A, NaN3, and KCN were cultured under normoxia or hypoxia. (D) RASMCs were cultured under normoxia or hypoxia. *p<0.05 relative to normoxia.

 
As shown in Fig. 1B, pretreatment with 110 units/mL of H2O2 scavenger catalase abolished the hypoxia-induced H2O2 generation. Aminotriazole (30 mmol/L), a catalase inhibitor, enhanced H2O2 generation and negated the effect of catalase on the hypoxia-mediated increase in H2O2 generation, suggesting that the effect of catalase is specific in C2/2 cells.

We next tested the effect of various inhibitors of mitochondrial respiration on hypoxia-induced H2O2 generation. C2/2 cells were pretreated with or without 25 µmol/L of rotenone, 1 µg/L of antimycin A, 1 mmol/L of sodium azide (NaN3), and 1 mmol/L of potacium cyanide (KCN), and then exposed to hypoxia for 6 h. As shown in Fig. 1C, the hypoxia-induced H2O2 production was inhibited by rotenone and antimycin A, but enhanced by NaN3 and KCN under normoxia (Fig. 1C). These results suggest that ROS are generated at mitochondrial electron transport chain (ETC) under hypoxia.

3.2. Effects of protein kinase inhibitors on hypoxia-induced PAI-1 gene and HIF1-{alpha} protein expression
To determine the signaling mechanisms regulating the hypoxia-mediated PAI-1 mRNA expression, C2/2 cells were pretreated with or without various protein kinase inhibitors prior to hypoxic exposure. C2/2 cells were harvested 12 h after hypoxic exposure, when the induction of PAI-1 gene expression reached its peak (data not shown). As shown in Fig. 2A, PAI-1 gene expression increased 6.5-fold in hypoxic C2/2 cells. This induction was significantly inhibited by Src family tyrosine kinase inhibitor PP1 (10 µmol/L), but not by MEK1 inhibitor PD98059 (50 µmol/L), MAPK inhibitor SB203580 (10 µmol/L), PI3K inhibitor wortmannin (1 µmol/L), or protein kinase C inhibitor calphostin C (200 µmol/L). To exclude non-specific effects of PP1, we evaluated the effect of lower dose of PP1 and another Src family tyrosine kinase inhibitor PP2 on the hypoxic response [19]. As shown in Fig 2B, hypoxia-induced PAI-1 gene expression was clearly inhibited by 1 µmol/L of PP1 and 3 µmol/L of PP2 but not by PP3. PP3, a negative control for PP2, had no effects, suggesting that the inhibition of hypoxia-induced PAI-1 gene expression by PP2 is not due to the non-specific toxic effects of PP2. PP1 (1 µmol/L) inhibited the hypoxic induction of PAI-1 gene expression in RASMCs (Fig 2C), indicating that PAI-1 gene expression under hypoxic condition does not occur only in C2/2 cells.


Figure 2
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  		Fig. 2 Effect of protein kinase inhibitors on hypoxia-induced PAI-1 gene (A, B, C) and HIF-1{alpha} protein expression (D, E) in VSMCs. (A–C) Northern blot analysis for PAI-1. (A) C2/2 cells were pretreated with vehicle, PD98059, SB203580, PP1 (10 µmol/L), wortmannin, and calphostin C prior to hypoxic condition for 12 h. (B) C2/2 cells were exposed to hypoxia with vehicle, PP1 (1 µmol/L), PP2 (3 µmol/L), and PP3 (3 µmol/L). (C) RASMCs pretreated with or without PP1 (10 µmol/L) were cultured under normoxia or hypoxia. (D, E) Western blot analysis for HIF-1{alpha}. (D) C2/2 cells were exposed to hypoxia for the indicated times. (E) C2/2 cells were exposed to hypoxia for 3 h with vehicle, PP1 (1 µmol/L), PP2 (3 µmol/L), and PP3 (3 µmol/L). *p<0.05 relative to normoxia. *p<0.05 for increase in PAI-1 gene and HIF-1{alpha} protein expression due to hypoxia in the presence of inhibitor vs. vehicle.

 
We next tested whether hypoxic induction of HIF-1{alpha} protein expression was mediated by activation of Src family tyrosine kinases. Incubation of C2/2 cells under hypoxic condition resulted in a time-dependent elevation of hypoxia-induced HIF-1{alpha} protein expression (Fig. 2D), with a marked increase at 1.5 h after hypoxic stimulation. Such an induction was inhibited by 1 µmol/L of PP1 and 3 µmol/L of PP2, but not by 3 µmol/L of PP3 (Fig. 2E). Similar results were observed in Northern blot analysis of PAI-1 gene expression and suggest that Src family tyrosine kinases play an important role in hypoxic induction of HIF-1{alpha} protein and PAI-1 gene expression in VSMCs.

3.3. Phosphorylation of c-Src in hypoxic cells
To clarify whether hypoxia causes tyrosine kinases to be phosphorylated, growth arrested C2/2 cells were exposed to hypoxic condition for specified durations. Protein extracts were immunoblotted using monoclonal phospho-tyrosine antibody. As shown in Fig. 3A, hypoxia induced tyrosine phosphorylation in a time-dependent manner. Pretreatment of C2/2 cells with 10 µmol/L of PP1 significantly inhibited this phosphorylation, suggesting that the major tyrosine kinases mediating hypoxic response are of the Src family.


Figure 3
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  		Fig. 3 Role of c-Src in the hypoxic response of VSMCs. (A) Western bolt analysis for tyrosine phosphorylation. C2/2 cells were pre-incubated with vehicle or PP1 (10 µmol/L) prior to the hypoxic exposure for the indicated times. (B) Northern blot analysis for PAI-1. Src++ cells and SYF(–) cells were exposed to hypoxia for indicated times. (C) Western bolt analysis for c-Src phosphorylation. RASMCs were cultured in hypoxic condition for the indicated times. Cellular lysates were immunoblotted with anti-phospho-c-Src (PY418). *p<0.05 relative to normoxia.

 
To confirm the role of c-Src, we performed Northern blot analysis on Src++ cells and SYF(–) cells. PAI-1 gene expression was markedly induced at 6 h after hypoxic stimulation in Src++ cells. In SYF(–) cells, in which c-Src, Yes, and Fyn are inactive, hypoxic induction of PAI-1 was, however, significantly attenuated (Fig 3B). The measurable induction that occurred at 12 h may be due to the residual actions by members of the Src family kinases.

We examined whether hypoxia phosphorylates c-Src by Western blot analysis using rabbit polyclonal phospho-specific c-Src (PY418) antibody. This antibody recognizes auto-phosphorylation sites of c-Src and reflects c-Src activity. As shown in Fig. 3C, hypoxia induced c-Src phosphorylation within 15 min in RASMCs. We could not detect c-Src phosphorylation in C2/2 cells possibly because the antibody that we used was rabbit polyclonal antibody, and gave rise to high background signals when blotted against rabbit cell lysates. These results indicate a specific involvement of c-Src in the hypoxic response of VSMCs.

3.4. Hypoxia-induced PAI-1 gene and HIF-1{alpha} protein expression were mediated through H2O2
We further examined the role of ROS in the hypoxic response. C2/2 cells were pretreated with catalase (110 units/mL) and DPI (an inhibitor of flavin-containing oxidase such as the NAD(P)H oxidase, 10 µmol/L) and then exposed to hypoxia for 12 h to detect PAI-1 gene expression. As shown in Fig. 4A, hypoxia-induced PAI-1 gene expression was significantly inhibited by catalase and DPI. As shown in Fig. 4B, aminotriazole (30 mmol/L) not only cancelled the catalase-inhibition of PAI-1 gene expression, but also augmented the PAI-1 gene expression even in the presence of oxygen. This suggests that aminotriazole increased the intracellular H2O2 levels via inhibition of both native and exogenous catalase, and the effect of catalase is specific for the degradation of H2O2.


Figure 4
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  		Fig. 4 Effect of antioxidants on the hypoxic response of VSMCs. (A) Northern blot analysis for PAI-1. C2/2 cells were pretreated with catalase and DPI, and then were exposed to hypoxia for 12 h. (B) Northern blot analysis for PAI-1. C2/2 cells were pretreated with catalase and/or aminotriazole and then were exposed to hypoxia for 12 h. (C) Western blot analysis for HIF-1{alpha}. C2/2 cells were treated with H2O2 under normoxia for indicated times and concentrations. *p<0.05 relative to normoxia. *p<0.05 for increase in HIF-1{alpha} protein expression due to hypoxia in the presence of inhibitor vs. vehicle.

 
Next we evaluated whether H2O2 plays a role in HIF-1{alpha} protein expression. C2/2 cells were treated with 1 µmol/L and 100 µmol/L of H2O2 for the indicated times and then harvested for Western blot analysis. As shown in Fig. 4C, 100 µmol/L of H2O2 transiently induced HIF-1{alpha} protein expression, although 1 µmol/L of H2O2 had no effect. This transient increase in HIF-1{alpha} protein expression might be due to the short life of H2O2 and vulnerability of HIF-1{alpha} protein. These results suggest that H2O2 serves as an upstream mediator of hypoxic induction of HIF-1{alpha} expression and PAI-1 gene expression.

3.5. Role of mitochondria in hypoxia-induced PAI-1 gene, HIF-1{alpha} protein expression, and c-Src phosphorylation
The role of mitochondria in hypoxia-mediated gene expression and protein activation was examined in C2/2 cells. Pretreatment with the complex I inhibitor, rotenone (25 µmol/L), inhibited the hypoxic induction of PAI-1 mRNA and HIF-1{alpha} protein expression (Fig. 5A,B). Rotenone and DPI (10 µmol/L) blunted the c-Src phosphorylation (Fig. 5C). Complex III inhibitor, antimycin A (1 µg/mL), had no effects on hypoxia-induced-PAI-1 gene and HIF-1{alpha} protein expression. Furthermore, antimycin A did not inhibit hypoxia-induced c-Src phosphorylation, but augmented the phosphorylation level under normoxia. NaN3 (10 mmol/L) only minimally decreased HIF-1{alpha} protein expression, and had no effect on PAI-1 gene expression. KCN (10 mmol/L) had no effect on HIF-1{alpha} protein and PAI-1 gene expression. Taken together, these results suggest that rotenone-sensitive complex I plays a critical role in hypoxia-mediated responses. Although antimycin A blocked the hypoxia-induced H2O2 level, an induction of c-Src phosphorylation, HIF-1{alpha} and PAI-1 gene expression does not seem to be solely dependent upon ROS generated downstream of complex III.


Figure 5
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  		Fig. 5 Effect of mitochondrial respiration inhibitors on hypoxia-mediated gene and protein expression. (A) Northern blot analysis for PAI-1. C2/2 cells were pretreated with vehicle, rotenone, antimycin A, NaN3, and KCN and then were exposed to hypoxia for 12 h. (B) Western blot analysis for HIF-1{alpha}. C2/2 cells were pretreated with vehicle, rotenone, antimycin A, NaN3, and KCN prior to 3 h of hypoxia. (C) Western blot analysis for c-Src phosphorylation. RASMCs were cultured under hypoxic condition for 15 min with vehicle, rotenone, antimycin A, and DP1. *p<0.05 relative to normoxia. *p<0.05 for increase in PAI-1 gene and HIF-1{alpha} protein expression due to hypoxia in the presence of inhibitor vs. vehicle.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
4.4. Mitochondrial ETC as...
 Acknowledgments
 References
 
In the present study, we have provided several lines of evidence that indicate ROS and c-Src are critical mediators of the hypoxic induction of PAI-1 mRNA and HIF-1{alpha} protein expression in C2/2 cells and RASMCs. Firstly, we demonstrated that hypoxia increased O2–. and H2O2 generation. Secondly, hypoxic induction of PAI-1 mRNA and HIF-1{alpha} protein expression was completely inhibited by PP1 and PP2, specific inhibitors of c-Src. Thirdly, hypoxia rapidly induced tyrosine phosphorylation of the autophosphorylation site of c-Src. Finally, hypoxia induced PAI-1 gene expression more markedly in Src++ cells than in SYF(–) cells. These results strongly suggest a role of c-Src in the hypoxic response of VSMCs. In addition, this study is the first to show that mitochondria are linked to the hypoxic signal to c-Src in VSMCs. Although it has been described that ROS are released from mitochondria during hypoxia in cardiac myocytes [20] and endothelial cells [21], the participation of individual mitochondrial complexes in the ROS production and the involvement of c-Src in the genetic response to hypoxia in VSMCs have not yet been documented.

4.1. Role of c-Src in hypoxic induction of HIF-1{alpha} protein and PAI-1 gene expression
This study has shown for the first time that c-Src is required for hypoxia-induced HIF-1{alpha} protein expression in VSMCs. Our results are consistent with those of Mukhopadhyay et al., who showed that overexpression of v-Src or growth hormone-activated c-Src increased HIF-1{alpha} protein expression in U87 glioma cells [22].

Our study raised the important question as to the mechanisms by which c-Src increases HIF-1{alpha} protein expression. Recently, much has been learned about the mechanisms of stabilization of HIF-1{alpha} during hypoxia. Under normoxic conditions, prolyl residues within the oxygen-dependent degradation domain are hydroxylated by prolyl hydoxylase (PHD), allowing von Hippel–Lindaw (VHL) to bind and polyubiquitinate HIF-1{alpha}. This in turn leads to proteasomal degradation of HIF-1{alpha}. Under hypoxic conditions, the PHD cannot hydroxylate HIF-1{alpha}, and therefore HIF-1{alpha} is not recognized by VHL. As a result, HIF-1{alpha} accumulates in the cell and is available to activate transcription [23]. From this model, it is speculated that phosphorylated c-Src somehow inhibits PHD activity under hypoxic condition. Indeed, Chan et al. demonstrated that HIF-1{alpha} could be stabilized by oncogenic stimuli including v-Src expression via inhibition of a prolyl hydroxylation event [24]. Evidence of a direct interaction between c-Src and HIF-1{alpha}, however, remains unknown, as the induction of HIF-1{alpha} was not detected until 1.5 h after hypoxic exposure while c-Src phosphorylation occurred 15 min after hypoxia.

4.2. An increase in H2O2 generation induces HIF-1{alpha} protein expression
In contrast to the present findings, previous studies have shown that exogenous H2O2 did not induce, but rather inhibited HIF-1{alpha} activation [25] and erythropoietin secretion [26] during hypoxia. One possible reason for such a discrepancy would be a difference in experimental condition. While we tested the effects of 100 µmol/L of H2O2 on HIF-1{alpha} expression under normoxia, Huang et al. examined the effects of 100 µmol/L of H2O2 on the DNA binding activity of HIF-1{alpha} under hypoxia [25]. One could expect that H2O2 plus hypoxia disturb the redox-state more markedly than H2O2 alone.

4.3. Mitochondria as an O2 sensor
Mitochondria generate ROS in many pathological conditions which constrain respiratory chain components. Mitochondria are proposed to be a metabolic sensor that adapts the fate of the cell to the metabolic environment. They also act as components of the O2 sensing mechanisms. Paddenberg et al. presented data favoring an important role of complex II in the hypoxic response of pulmonary vasoconstriction [27]. Chen et al. showed that respiration-deficient cells exhibited impaired oxidative stress-induced signaling and mitochondria-targeted antioxidants inhibited H2O2-induced cell signaling. They proposed that mitochondria function as not only a source of ROS but also as a proximal sensor for oxidative stress [28]. This process, which is known as ROS-induced ROS release, may result from differential activation of the mitochondrial ROS generating system; the initial low-level release induces a signaling cascade leading to the generation of additional ROS generation [29]. Our observations appear to fit well with this phenomenon because a large increase in H2O2 generation was detected 6 h after hypoxic stimulation although the effect of rotenone on c-Src was observed 15 min after hypoxic stimulation. The failure to detect an increase in H2O2 generation shortly after hypoxic stimulation was probably due to the subtle increase in mitochondrial ROS which were less than the detectable range.


   4.4. Mitochondrial ETC as a source of ROS
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 4.4. Mitochondrial ETC as...
Acknowledgments
 References
 
An increasing number of reports describe enhanced mitochondrial ROS production when oxygen supply is reduced. Liu et al. demonstrated the role of complex I in free radical formation and flow mediated dilation in coronary resistance arteries [30]. In consistent with such previous findings, we found that rotenone, which inhibits entry of an electron from complex I to the distal site of ETC, inhibits ROS production under hypoxic condition. Thus, our data suggest that an inhibition of c-Src activation and HIF-1{alpha} protein expression by rotenone may be attributed to the inhibition of ROS generation from mitochondrial ETC at the distal site of complex I. In addition, we observed that antimycin A inhibited ROS production under hypoxic condition, suggesting that major sources of ROS were not at mitochondrial complex III. These results were not consistent with the previous study which indicated antimycin A enhanced ROS production under normoxia and hypoxia [31]. According to our data, antimycin A enhanced the phosphorylation of c-Src under normoxia and hypoxia. Since we could not detect ROS production immediately after hypoxic exposure, the exact mechanisms underlying these discrepant results remain unclear. An increase in ROS production by KCN and NaN3 suggested that ROS are generated via release of cytochrome c under hypoxia, because these agents are known to inhibit cytochrome oxidase and in turn increase ROS generation in mitochondria [32]. However, it has been known for a long time that KCN and NaN3 also exert their effects as inhibitors of heme-containing proteins including peroxidase, xanthine oxidase, and catalase, which generate H2O2 independently in mitochondria. Furthermore, cyanide increases ROS generation via NO synthesis [33]. Thus, we cannot conclude that release of cytochrome c is a main cause of ROS generation under hypoxia.

4.5. Are there the alternative sources for ROS?
In our study, apocynin and 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), selective inhibitors for the NAD(P)H oxidase, abolished the HIF-1{alpha} induction by hypoxia. These reagents, however, did not exert consistent effects on the hypoxic induction of PAI-1 expression (data not shown). These results suggest that NAD(P)H oxidase is partially involved in the hypoxic response and that the hypoxic induction of PAI-1 is partly regulated by HIF-1{alpha}-independent mechanisms.

There is a growing body of evidence showing that c-Src regulates activation of NAD(P)H oxidase. Recently, Touyz et al. demonstrated in human VSMC that c-Src enhanced NAD(P)H oxidase activity by inducing phosphorylation and translocation of p47phox [34]. These data are consistent with another report showing c-Src activates NAD(P)H oxidase in the rat VSMCs [35]. Thus, we propose that c-Src activation is responsible for NAD(P)H oxidase activation in the hypoxic response.

4.6. Summary
In summary, we demonstrated that hypoxia stimulates the production of superoxide and H2O2, and the phosphorylation of c-Src in VSMCs. ROS generation and c-Src activation were critical in the process of inducing HIF-1{alpha} protein and PAI-1 gene expression. This study also showed that mitochondria play a major role in ROS generation and c-Src phosphorylation during hypoxia. Hypoxia is the major cause of thrombosis and angiogenesis during atherosclerosis, so an understanding of the role of mitochondria and of c-Src in hypoxia-induced HIF-1{alpha} protein expression may provide new insights into the molecular mechanisms of plaque thrombosis and angiogenesis, which may influence late complications of atherosclerosis including plaque rupture.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 4.4. Mitochondrial ETC as...
 Acknowledgments
References
 
This study was supported in part by a Grant-in-Aid for Scientific Research (C) (KAKENHI 13832002) from the Japan Society for the Promotion of Science (JSPS). We thank Ms. M.Yamazaki for her excellent technical support.


   Notes
 
Time for primary review 24 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 4.4. Mitochondrial ETC as...
 Acknowledgments
 References
 

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