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Am J Physiol Heart Circ Physiol 288: H1836-H1843, 2005. First published November 18, 2004; doi:10.1152/ajpheart.00679.2004
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Iron chelation and a free radical scavenger suppress angiotensin II-induced upregulation of TGF-{beta}1 in the heart

Kan Saito,1 Nobukazu Ishizaka,1 Toru Aizawa,1 Masataka Sata,1 Naoyuki Iso-o,2 Eisei Noiri,3 Ichiro Mori,4 Minoru Ohno,1 and Ryozo Nagai1

Departments of 1Cardiovascular Medicine and 2Metabolic Disease and 3Nephrology and Endocrinology, University of Tokyo Graduate School of Medicine, and 4Department of Pathology, Wakayama Medical College, Tokyo, Japan

Submitted 8 July 2004 ; accepted in final form 16 November 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Long-term administration of angiotensin II causes myocardial loss and cardiac fibrosis. We previously found iron deposition in the heart of the angiotensin II-infused rat, which may promote angiotensin II-induced cardiac damage. In the present study, we have investigated whether an iron chelator (deferoxamine) and a free radical scavenger (T-0970) affect the angiotensin II-induced upregulation of transforming growth factor-{beta}1 (TGF-{beta}1). Angiotensin II infusion for 7 days caused a robust increase in TGF-{beta}1 mRNA expression in vascular smooth muscle cells, myofibroblast-like cells, and migrated monocytes/macrophages. T-0970 and deferoxamine suppressed the upregulation of TGF-{beta}1 mRNA and reduced the extent of cardiac fibrosis in the heart of rats treated with angiotensin II. These agents blocked the angiotensin II-induced upregulation of heme oxygenase-1, a potent oxidative and cellular stress-responsive gene, but they did not significantly affect systolic blood pressure or plasma levels of aldosterone. In addition, T-0970 and deferoxamine suppressed the angiotensin II-induced upregulation of monocyte chemoattractant protein-1 in the heart. These results collectively suggest that iron and the iron-mediated generation of reactive oxygen species may contribute to angiotensin II-induced upregulation of profibrotic and proinflammatory genes, such as TGF-{beta}1 and monocyte chemoattractant protein-1.

fibrosis; iron chelator; oxidative stress; aldosterone

TRANSFORMING GROWTH FACTOR (TGF)-{beta} is one of the key mediators that induce fibrotic and inflammatory changes in various organs. The crucial role of TGF-{beta} in the development of cardiac fibrosis has also been demonstrated by in vitro and in vivo experiments (12, 13, 29). A body of evidence indicates that activation of the angiotensin II-AT1 receptor axis stimulates cardiac fibrosis. In addition, TGF-{beta} is thought to play a crucial role in mediating the links between the AT1 receptor and cardiac fibrosis, because blockade of the AT1 receptor suppresses upregulation of TGF-{beta} expression in the heart of animal models of myocardial infarction (37) and hypertension (19), and, conversely, administration of angiotensin II increases expression of cardiac TGF-{beta} (36).

We previously showed that long-term administration of angiotensin II to rats causes accumulation of iron in the heart, which may act to promote angiotensin II-induced cardiac fibrosis (6, 7). Iron and, presumably, the iron-mediated generation of hydroxyl radicals may have a role in TGF-{beta}1 upregulation and tissue fibrosis in the liver and kidney (5, 28, 38). Thus we have examined the effects of iron chelation and free radical scavenging on the angiotensin II-induced upregulation of TGF-{beta}1 expression in rat heart. We have also investigated localization of TGF-{beta}1 mRNA histologically to identify the types of cells with increased levels of TGF-{beta}1 mRNA expression after angiotensin II infusion.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal models. The experiments were performed in accordance with the guidelines for animal experimentation approved by the Animal Center for Biomedical Research, Faculty of Medicine, University of Tokyo. Male Sprague-Dawley rats were prepared as described previously (9). Briefly, Val5-angiotensin II and norepinephrine (Sigma) were infused at 0.7 and 2.8 mg·kg–1·day–1, respectively, by subcutaneously implanted osmotic minipumps (Alza) for 7 days to exert comparable hypertensive effects: 192 ± 4 mmHg for angiotensin II (n = 10) and 196 ± 6 mmHg for norepinephrine (n = 9) vs. 131 ± 3 mmHg for control rats (n = 6, P < 0.01). The iron chelator deferoxamine (DFO; a kind gift from Novartis) was injected at 200 mg·kg–1·day–1 sc. Iron dextran (a kind gift from Teikoku Hormone) was injected at 240 mg of elemental iron/kg ip on days 0, 2, 4, and 6 of angiotensin II infusion. A free radical scavenger, T-0970 (33) (a kind gift from Tanabe Seiyaku), was given at 10 mg·kg–1·day–1 po. Neither DFO nor T-0970 significantly affected the blood pressure of angiotensin II-treated and untreated rats, as described elsewhere (30). Some rats were subjected to daily injections of the heme oxygenase (HO)-1 inhibitor zinc protoporphyrin (ZnPP, 50 µmol·kg–1·day–1 ip; Porphyrin Products), which were started 2 days before pump implantation and continued until death. The plasma aldosterone levels of rats in each group were measured by solid-phase radioimmunoassay.

Protein purification and Western blot analysis. Protein was isolated after samples were homogenized, as described previously (9). Polyclonal antibodies against rat ferritin (Panapharm), HO-1 (StressGen), and phosphorylated epidermal growth factor receptor (pEGFR; Santa Cruz Biotechnology) were used at dilutions of 1:3,000, 1:3,000, and 1:1,000, respectively (7). The enhanced chemiluminescence Western blotting system (Amersham Life Sciences) was used for detection. Bands were visualized by a Lumino-analyzer (Fuji Photo Film). Band intensity is expressed as a percentage of the control value.

In situ hybridization and immunohistochemical and histological analyses. A rat TGF-{beta}1 cDNA probe (a kind gift from Dr. Shiow-Shih Tang, Harvard Medical School, Massachusetts General Hospital, and Dr. John S. D. Chan, Centre hospitalier de l'Université de Montréal-Hôtel-Dieu) was subcloned into a pGEM-T vector in sense and antisense orientations by standard methodology. In situ hybridization was performed using In Situ Hybridization Reagents (Nippongene). Immunohistochemistry was performed as described previously (7). Primary antibodies against rat macrophage/monocyte (ED-1; Chemicon), human {alpha}-smooth muscle actin (Sigma), and rat ferritin were used at dilutions of 1:200, 1:800, and 1:200, respectively. For immunofluorescence staining, rhodamine-conjugated anti-mouse (Chemicon) and fluorescein-conjugated anti-rabbit (Sigma) antibodies were used at a dilution of 1:100. Laser scanning confocal fluorescence microscopy combined with differential interference contrast imaging was performed using FLUOVIEW FV300 (Olympus).

Quantification of cardiac fibrosis. Quantification of the fibrous areas was performed by the operator without knowledge of the treatment group. After Masson trichrome staining, heart sections were photographed and digitalized, and the number of pixels of blue color was counted using a photoimaging system (Canon). The ratios of the area affected by fibrosis to the total cardiac area in the samples were expressed as percent fibrosis (6).

RNA extraction and Northern blot analysis. Probes for Northern blot analysis were obtained by polymerase chain reaction with reverse transcription. Sense and antisense primers were 5'-TGCCGTGACCTCAAGATGTG-3' and 5'-CACAAGCGTGCTGTAGGTGA-3', respectively, for collagen type I, 5'-AGATCATGTCTTCACTCAAGTC-3' and 5'-TTTACATTGCCATTGGCCTGA-3', respectively, for collagen type III, and 5'-CAGGTCTCTGTCACGCTTCT-3' and 5'-AGTATTCATGGAAGGGAATAG-3', respectively, for monocyte chemoattractant protein-1 (MCP-1). The cDNAs were confirmed as target cDNAs by direct sequencing. mRNA was isolated and Northern blot analysis was performed as described previously (16).

Statistical analysis. Values are means ± SE. We used ANOVA followed by a multiple comparison test to compare raw data before expressing the results as a percentage of the control value using the statistical analysis software Statistica version 5.1 J for Windows (StatSoft). P < 0.05 was considered to be statistically significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Expression and localization of TGF-{beta}1 after angiotensin II infusion in the heart. Administration of angiotensin II, but not norepinephrine, increased mRNA expression of TGF-{beta}1 and collagen types I and III (Fig. 1), which may be related to the previous finding that angiotensin II is far more potent in generating granulation regions in the rat heart observed in the previous studies (6). In situ hybridization showed that TGF-{beta}1 was only weakly expressed in the heart of untreated rats (Fig. 2, A and D), and this expression markedly increased in the interstitial cells (Fig. 2, B and C) and vascular smooth muscle cells (Fig. 2, E and F) after angiotensin II infusion. In granulation regions, some, but not all, of the TGF-{beta}1-positive inflammatory cells were positive for iron (Fig. 2, G–I). These TGF-{beta}1-positive inflammatory cells were found to be ED-1 positive and, thus, were identified as monocytes/macrophages (Fig. 3, A–C). Some of the {alpha}-smooth muscle actin-positive myofibroblast-like cells were also positive for TGF-{beta}1 mRNA (Fig. 3, D–F, arrows).



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  		Fig. 1. Expression of transforming growth factor-{beta}1 (TGF-{beta}1) and collagen (Coll) types I and III after administration of pressor agents. Left: representative Northern blots. Right: data from 5 to 7 rats in each group. NorEpi, norepinephrine. {dagger}P < 0.01 vs. untreated control (–NorEpi/–ANG II).

 


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  		Fig. 2. In situ hybridization of TGF-{beta}1 mRNA after angiotensin II infusion. A and D: control rats. B, C, and E–I: angiotensin II-treated rats. B and C, E and F, and G–I are serial specimens. A, B, D, E, and G were treated with a TGF-{beta}1 antisense (AS) probe, and C, F, and H were treated with a TGF-{beta}1 sense (S) probe. I shows Prussian blue (iron) staining. TGF-{beta}1 mRNA was only weakly expressed in the heart of control rats (A and D); after angiotensin II infusion, TGF-{beta}1 expression increased in interstitial cells (B and G) and vascular smooth muscle cells (E). Some TGF-{beta}1-positive infiltrated inflammatory cells were positive for iron (I). Original magnifications: x800 (A–C and G–I) and x1,600 (D–F).

 


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  		Fig. 3. Identification of the types of cells with increased levels of TGF-{beta}1 mRNA. A–C and D–F are the same sections. A: ED-1 staining (red fluorescence). B and E: Nomarski differential interference contrast (DIC) image showing TGF-{beta}1 mRNA signals detected by in situ hybridization. C: overlay of ED-1 staining and DIC image. D: {alpha}-smooth muscle actin (SMA) staining (green fluorescence). F: overlay of {alpha}-SMA staining and DIC image. Increased expression of TGF-{beta}1 mRNA was detected in some ED-1-positive monocytes/macrophages (A–C), vascular smooth muscle cells (D–F), and {alpha}-SMA-positive myofibroblast-like cells (arrows in D–F). Scale bars, 20 µm. Original magnification, x800.

 
Effects of T-0970 and DFO on angiotensin II-induced upregulation of TGF-{beta}1, ferritin, and HO-1. We previously reported that DFO suppresses angiotensin II-induced cardiac fibrosis. Thus we next examined whether DFO inhibits the angiotensin II-mediated upregulation of TGF-{beta}1 expression. DFO inhibited angiotensin II-induced upregulation of TGF-{beta}1 and collagen types I and III (Fig. 4). Conversely, iron dextran upregulated TGF-{beta}1 mRNA (Fig. 4). In addition, T-0970 inhibited the angiotensin II-induced upregulation of TGF-{beta}1 and collagen types I and III. Western blot showed that angiotensin II-induced ferritin induction was blocked completely by DFO but partially by T-0970. T-0970 and DFO suppressed angiotensin II-induced HO-1 induction (Fig. 5).



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  		Fig. 4. Effects of iron chelation, iron overload, and free radical scavenging on angiotensin II-induced upregulation of TGF-{beta}1 and collagen types I and III. Top: representative Northern blots. Bottom: data from 5 to 7 rats in each group. DFO, deferoxamine. *P < 0.05; {dagger}P < 0.01 vs. untreated control. #P < 0.05 vs. angiotensin II-treated rats.

 


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  		Fig. 5. Effect of iron chelation and free radical scavenging on induction of ferritin and heme oxygenase-1 (HO-1) proteins by angiotensin II. Top: representative Western blots. Bottom: data from 4 to 6 rats in each group. *P < 0.05; {dagger}P < 0.01 vs. untreated control. #P < 0.05 vs. angiotensin II-treated rats.

 
Cardiac fibrosis and plasma levels of aldosterone. We previously showed that DFO reduces the extent of angiotensin II-induced cardiac fibrosis (10). In the present study, T-0970 treatment reduced the area of angiotensin II-induced cardiac fibrosis in the right ventricle [RV; from 7.6 ± 2.1% (n = 6) to 3.3 ± 0.4% (n = 7), P < 0.001] and the left ventricle [LV; from 2.0 ± 0.6% (n = 6) to 1.4 ± 0.2% (n = 7), P < 0.001]. Neither T-0970 nor DFO significantly affected plasma aldosterone levels in angiotensin II-treated or untreated rats (Fig. 6). In addition, iron did not significantly affect plasma aldosterone levels.



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  		Fig. 6. Plasma levels of aldosterone. Number in parentheses represents number of rats. NS, not significant. {dagger}P < 0.01 vs. untreated control.

 
Effects of iron chelation and free radical scavenging on angiotensin II-induced upregulation of MCP-1 mRNA. We next examined whether iron chelation and free radical scavenging also affect cardiac expression of the proinflammatory chemokine MCP-1, inasmuch as recent studies suggested that angiotensin II induces cardiac expression of MCP-1 via a pressor-independent mechanism (34). Angiotensin II infusion increased MCP-1 expression ~2.4-fold in the heart. Treatment of angiotensin II-infused rats with DFO or T-0970 suppressed, although not completely, angiotensin II-induced MCP-1 upregulation (Fig. 7).



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  		Fig. 7. Effects of iron chelation, iron overload, and free radical scavenging on angiotensin II-induced upregulation of monocyte chemoattractant protein-1 (MCP-1). Top: representative Northern blots. Bottom: data from 5–7 rats in each group. *P < 0.05; {dagger}P < 0.01 vs. untreated control. #P < 0.01 vs. angiotensin II-treated rats.

 
Cardiac damage and iron deposition after treatment with HO inhibitor. Although the possible relation between iron and profibrotic gene regulation has been suggested in the experiments described above, it was not clear whether HO-1 induction in the heart played a causal role or was a result of cardiac iron deposition in the angiotensin II-infused rat. Therefore, to examine whether HO-1 upregulation played a causal role in cardiac iron deposition, we treated the angiotensin II-infused rats with the HO inhibitor ZnPP. ZnPP treatment did not significantly alter the blood pressure of angiotensin II-treated rats [201 ± 4 mmHg for angiotensin II alone (n = 8) vs. 200 ± 12 mmHg for angiotensin II + ZnPP (n = 6), not significant]. ZnPP treatment resulted in an increase in the area of granulation regions in the LV but not in the RV (Fig. 8A). Prussian blue staining showed iron deposition in granulation regions, which suggested that iron deposition could be induced by angiotensin II infusion, at least in part, via an HO-independent mechanism (Fig. 8, B–D).



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  		Fig. 8. Effect of zinc protoporphyrin (ZnPP) on iron deposition and granulation region formation in the heart of an angiotensin II-infused rat. A: Masson trichrome staining of a heart of a rat treated with angiotensin II and ZnPP. Marked granulation region formation, cardiac fibrosis, and fibrinoid necrosis (arrowhead) of the vessel wall can be observed. B and D: Prussian blue staining of the serial specimen of A. C: higher magnification image of bracketed region of B. Original magnifications: x100 (A and B) and x400 (C). D: semiquantitative analysis of granulation tissue area in both ventricles. Data are from 5 or 6 animals in each group. *P < 0.01 vs. sham-operated control. #P < 0.01 vs. angiotensin II-treated rats.

 
Activation of epidermal growth factor receptor by angiotensin II infusion. It has been reported that transactivation of epidermal growth factor receptor (EGFR) may have a role in the angiotensin II-induced upregulation of TGF-{beta} (17, 18). Therefore, we also examined whether EGFR is activated by angiotensin II treatment. Western blot showed that levels of pEGFR were not significantly different between the hearts of untreated and angiotensin II-treated rats (Fig. 9).



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  		Fig. 9. Expression of phosphorylated epidermal growth factor (pEGFR) in the heart of untreated and angiotensin II-treated rats. Top: representative Western blots. Bottom: data from 3 control animals and 5 angiotensin II-treated animals.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, angiotensin II, but not norepinephrine, increased the expression of TGF-{beta}1 and collagen types I and III in the heart. In situ hybridization showed a robust increase in TGF-{beta}1 mRNA in the infiltrated monocytes/macrophages, vascular smooth muscle, and myofibroblast-like cells. An iron chelator (DFO) and a free radical scavenger (T-0970), both of which have been shown to effectively reduce the extent of in vivo oxidative stress induced by angiotensin II (30), suppressed the angiotensin II-induced upregulation of TGF-{beta}1. These data collectively suggest that iron and iron-mediated generation of free radicals may have a crucial role in the angiotensin II-induced upregulation of TGF-{beta}1 in the heart.

The possible link between iron, tissue fibrosis, and upregulation of TGF-{beta} is best known in the liver in humans and animal models (5, 32, 35). Recent studies have also suggested that iron may have a role in the TGF-{beta} upregulation and tissue fibrosis in other organs, including the kidney (21, 38) and the heart (27). Very recently, Oudit et al. (23) showed that cardiac fibrosis occurs in the rodent model of iron overload. They reported that taurine, which possesses antioxidant properties and can inhibit L-type Ca2+ channels (22), reduced the interstitial fibrosis (23), which may further support the idea that iron and/or iron-mediated oxidative stress acts to promote cardiac fibrosis. We previously reported that long-term administration of angiotensin II to rats causes iron deposition in the heart and that iron chelation ameliorates the cardiac damage induced by angiotensin II (10). These findings led us to investigate the possible role of iron in the regulation of TGF-{beta}1 expression in the heart of the angiotensin II-infused rat.

Northern blot showed that angiotensin II infusion increased TGF-{beta}1 mRNA expression in the whole heart. First, we determined whether TGF-{beta}1 induction occurred in the cells with iron accumulation. Iron staining showed that iron was positive in the migrated inflammatory cells (Fig. 2I), the majority of which were monocytes/macrophages (Fig. 3A) (7). On the other hand, upregulation of TGF-{beta}1 mRNA could be observed not only in iron-positive cells but also in vascular smooth muscle (Fig. 2E) and fibroblast-like cells (Fig. 3E), which were negative for stainable iron. One may question why iron chelation suppresses the upregulation of TGF-{beta}1 expression. It is possible that, in vascular smooth muscle cells and fibroblast-like cells, iron accumulation might be less extensive than in monocytes/macrophages to the extent that it was not histologically evident. This idea may be supported by the previous finding that concomitant administration of angiotensin II and iron causes apparent deposition of iron in the vascular smooth muscle and myofibroblast-like cells, as well as in monocytes/macrophages (10). Another possibility is that some factors, such as 4-hydroxy-2,3-nonenal (15), that are released from iron-positive cells might act to induce TGF-{beta}1 expression in a paracrine manner. These possibilities should be explored in future studies.

Western blot analysis showed that DFO and T-0970 blocked the angiotensin II-induced upregulation of HO-1 protein induction in the heart. Because iron is one of the breakdown products of heme through the enzymatic reaction of HO, we considered whether the angiotensin II-induced upregulation of HO-1 expression plays a causal role in, or is merely a result of, cardiac iron deposition. To address this question, we treated angiotensin II-infused rats with an HO inhibitor, ZnPP. We found that iron deposition could still be observed in the granulation regions of the heart, indicating that HO activity may not be essential for iron accumulation in the heart. In addition, we found that treatment with the HO inhibitor exacerbated cardiac damage in the angiotensin II-infused rat, suggesting that the HO system may act protectively against angiotensin II-induced cardiac damage. This notion of a cardioprotective effect of HO-1 is consistent with the findings obtained from HO-1 knockout and HO-1 transgenic mice (2, 4).

In addition, Masson trichrome staining showed that inhibition of HO activity by ZnPP increased the area of granulation regions in the LV but not in the RV. We do not know why the LV was more susceptible to ZnPP treatment; however, it is possible that the HO system acts more protectively in the LV than in the RV in the heart of the angiotensin II-infused rat, which might explain why histological damage was more prominent in the RV than in the LV in these animals (6). This possibility should be examined in future studies.

We did not investigate how iron accumulation occurs in the heart of the angiotensin II-treated rat. Very recently, Larkin et al. (14) reported that acute or chronic stimulation of angiotensin II strongly upregulates expression of the transferrin receptor, an iron-transporting cell membrane protein, in the heart of mice. Therefore, it is possible that angiotensin II directly alters the expression of genes that are involved in intracellular iron homeostasis. Interestingly, Panchenko et al. (25) reported the similar observation that iron chelation and free radical scavenging suppress HO-1 induction in cultured fibroblasts under certain conditions. Together with our previous findings that DFO and T-0970 reduce the extent of in vivo oxidative stress induced by angiotensin II, as assessed by plasma levels of prostaglandin F2{alpha} (1, 30), we speculate that both agents attenuate the angiotensin II-stimulated induction of HO-1 by reducing the extent of oxidative stress in the heart.

In the present study, neither DFO nor T-0970 affected plasma levels of aldosterone in the angiotensin II-infused rat, although both of these drugs suppressed angiotensin II-induced cardiac fibrosis. A relation between aldosterone and cardiac fibrosis has been shown in a rat model by Parkes et al. (27). It is possible that DFO and T-0970 suppress cardiac fibrosis by blocking an aldosterone-independent pathway or by reducing downstream fibrogenic signaling initiated by aldosterone, which might include reactive oxygen species (26). These possibilities remain to be clarified in further investigations.

Inasmuch as iron (heme) overload upregulates the expression of the proinflammatory cytokine MCP-1 in the kidney (11), we also tested the effects of DFO and T-0970 on cardiac MCP-1 expression and found that both of these agents suppressed the angiotensin II-induced upregulation of MCP-1 mRNA. Chen et al. (3) reported that an iron chelator and a hydroxyl radical scavenger block upregulation of MCP-1 induced by TNF-{alpha} in cultured vascular endothelial cells. Inasmuch as H2O2 by itself is a poorly reactive oxidant, they speculated that reaction of H2O2 and superoxide with intracellular iron to form hydroxyl radicals may have a crucial role in TNF-{alpha}-induced inflammatory gene expression (3). In vitro and in vivo experiments demonstrated that angiotensin II stimulation activates the cardiac NADH oxidase system (20, 24, 31); therefore, it is possible that iron enhances oxidant-induced damage in the heart of angiotensin II-infused animals by converting less harmful reactive oxygen species, H2O2 and superoxide, to highly toxic hydroxyl radicals, resulting in the upregulation of MCP-1.

In the last part of our study, we examined whether angiotensin II infusion transactivates EGFR, inasmuch as it has been shown that angiotensin II may transactivate EGFR, which in turn may play a role in the induction of fibrosis-related genes in vitro (17, 18). In contrast to those studies, we found that expression of pEGFR, an activated form of EGFR, did not increase after angiotensin II infusion. The discrepancy between these in vitro and in vivo systems may be partly due to the difference in the concentrations of angiotensin II used. In the in vitro system, angiotensin II was used at a concentration of 100 nmol/l (17). By contrast, the plasma concentration of angiotensin II was ~1/1,000th of this value after angiotensin II infusion for 7 days (147 ± 24 pmol/l) (8). Nevertheless, because blocking the EGFR may reduce angiotensin II-induced cardiac damage, whether inhibition of EGFR activity blocks iron accumulation in the heart of angiotensin II-infused animals should be examined in future studies.

In conclusion, an iron chelator and a radical scavenger suppressed the angiotensin II-induced upregulation of TGF-{beta}1 and cardiac fibrosis without significantly affecting plasma levels of aldosterone. Thus iron and the iron-mediated generation of reactive oxygen species may be involved in the angiotensin II-induced upregulation of profibrotic and proinflammatory genes, such as TGF-{beta}1 and MCP-1.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Ministry of Education, Science, and Culture of Japan Grant-in-Aid for Scientific Research 13671098 and grants from the Novartis Foundation for Gerontological Research and the Takeda Medical Research Foundation.


   ACKNOWLEDGMENTS
 
We appreciate the excellent technical assistance of Naoko Amitani and the editorial assistance of Kyoko Furuta.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. Ishizaka, Dept. of Cardiovascular Medicine, Univ. of Tokyo, Graduate School of Medicine, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan (E-mail: nobuishizka-tky{at}umin.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Aizawa T, Ishizaka N, Usui S, Ohashi N, Ohno M, and Nagai R. Angiotensin II and catecholamines increase plasma levels of 8-epi-prostaglandin F2{alpha} with different pressor dependencies in rats. Hypertension 39: 149–154, 2002.[Abstract/Free Full Text]
  2. Bak I, Szendrei L, Turoczi T, Papp G, Joo F, Das DK, de Leiris J, Der P, Juhasz B, Varga E, Bacskay I, Balla J, Kovacs P, and Tosaki A. Heme oxygenase-1-related carbon monoxide production and ventricular fibrillation in isolated ischemic/reperfused mouse myocardium. FASEB J 17: 2133–2135, 2003.[Abstract/Free Full Text]
  3. Chen XL, Zhang Q, Zhao R, and Medford RM. Superoxide, H2O2 and iron are required for TNF-{alpha}-induced MCP-1 gene expression in endothelial cells: role of Rac1 and NADPH oxidase. Am J Physiol Heart Circ Physiol 286: H1001–H1007, 2004.[Abstract/Free Full Text]
  4. Chen YH, Yet SF, and Perrella MA. Role of heme oxygenase-1 in the regulation of blood pressure and cardiac function. Exp Biol Med (Maywood) 228: 447–453, 2003.[Abstract/Free Full Text]
  5. Houglum K, Ramm GA, Crawford DH, Witztum JL, Powell LW, and Chojkier M. Excess iron induces hepatic oxidative stress and transforming growth factor-{beta}1 in genetic hemochromatosis. Hepatology 26: 605–610, 1997.[CrossRef][ISI][Medline]
  6. Ishizaka N, Aizawa T, Mori I, Taguchi J, Yazaki Y, Nagai R, and Ohno M. Heme oxygenase-1 is upregulated in the rat heart in response to chronic administration of angiotensin II. Am J Physiol Heart Circ Physiol 279: H672–H678, 2000.[Abstract/Free Full Text]
  7. Ishizaka N, Aizawa T, Yamazaki I, Usui S, Mori I, Kurokawa K, Tang SS, Ingelfinger JR, Ohno M, and Nagai R. Abnormal iron deposition in renal cells in the rat with chronic angiotensin II administration. Lab Invest 82: 87–96, 2002.[ISI][Medline]
  8. Ishizaka N, Alexander RW, Laursen JB, Kai H, Fukui T, Oppermann M, Lefkowitz RJ, Lyons PR, and Griendling KK. G protein-coupled receptor kinase 5 in cultured vascular smooth muscle cells and rat aorta. Regulation by angiotensin II and hypertension. J Biol Chem 272: 32482–32488, 1997.[Abstract/Free Full Text]
  9. Ishizaka N, de Leon H, Laursen JB, Fukui T, Wilcox JN, De Keulenaer G, Griendling KK, and Alexander RW. Angiotensin II-induced hypertension increases heme oxygenase-1 expression in rat aorta. Circulation 96: 1923–1929, 1997.[Abstract/Free Full Text]
  10. Ishizaka N, Saito K, Mitani H, Yamazaki I, Sata M, Usui S, Mori I, Ohno M, and Nagai R. Iron overload augments angiotensin II-induced cardiac fibrosis and promotes neointima formation. Circulation 106: 1840–1846, 2002.[Abstract/Free Full Text]
  11. Kanakiriya SK, Croatt AJ, Haggard JJ, Ingelfinger JR, Tang SS, Alam J, and Nath KA. Heme: a novel inducer of MCP-1 through HO-dependent and HO-independent mechanisms. Am J Physiol Renal Physiol 284: F546–F554, 2003.[Abstract/Free Full Text]
  12. Koyanagi M, Egashira K, Kubo-Inoue M, Usui M, Kitamoto S, Tomita H, Shimokawa H, and Takeshita A. Role of transforming growth factor-{beta}1 in cardiovascular inflammatory changes induced by chronic inhibition of nitric oxide synthesis. Hypertension 35: 86–90, 2000.[Abstract/Free Full Text]
  13. Kuwahara F, Kai H, Tokuda K, Kai M, Takeshita A, Egashira K, and Imaizumi T. Transforming growth factor-{beta} function blocking prevents myocardial fibrosis and diastolic dysfunction in pressure-overloaded rats. Circulation 106: 130–135, 2002.[Abstract/Free Full Text]
  14. Larkin JE, Frank BC, Gaspard RM, Duka I, Gavras H, and Quackenbush J. Cardiac transcriptional response to acute and chronic angiotensin II treatments. Physiol Genomics 18: 152–166, 2004.[Abstract/Free Full Text]
  15. Leonarduzzi G, Scavazza A, Biasi F, Chiarpotto E, Camandola S, Vogel S, Dargel R, and Poli G. The lipid peroxidation end product 4-hydroxy-2,3-nonenal up-regulates transforming growth factor {beta}1 expression in the macrophage lineage: a link between oxidative injury and fibrosclerosis. FASEB J 11: 851–857, 1997.[Abstract]
  16. Mitani H, Ishizaka N, Aizawa T, Ohno M, Usui S, Suzuki T, Amaki T, Mori I, Nakamura Y, Sato M, Nangaku M, Hirata Y, and Nagai R. In vivo klotho gene transfer ameliorates angiotensin II-induced renal damage. Hypertension 39: 838–843, 2002.[Abstract/Free Full Text]
  17. Moriguchi Y, Matsubara H, Mori Y, Murasawa S, Masaki H, Maruyama K, Tsutsumi Y, Shibasaki Y, Tanaka Y, Nakajima T, Oda K, and Iwasaka T. Angiotensin II-induced transactivation of epidermal growth factor receptor regulates fibronectin and transforming growth factor-{beta} synthesis via transcriptional and posttranscriptional mechanisms. Circ Res 84: 1073–1084, 1999.[Abstract/Free Full Text]
  18. Murasawa S, Mori Y, Nozawa Y, Gotoh N, Shibuya M, Masaki H, Maruyama K, Tsutsumi Y, Moriguchi Y, Shibazaki Y, Tanaka Y, Iwasaka T, Inada M, and Matsubara H. Angiotensin II type 1 receptor-induced extracellular signal-regulated protein kinase activation is mediated by Ca2+/calmodulin-dependent transactivation of epidermal growth factor receptor. Circ Res 82: 1338–1348, 1998.[Abstract/Free Full Text]
  19. Nagata K, Somura F, Obata K, Odashima M, Izawa H, Ichihara S, Nagasaka T, Iwase M, Yamada Y, Nakashima N, and Yokota M. AT1 receptor blockade reduces cardiac calcineurin activity in hypertensive rats. Hypertension 40: 168–174, 2002.[Abstract/Free Full Text]
  20. Nakagami H, Takemoto M, and Liao JK. NADPH oxidase-derived superoxide anion mediates angiotensin II-induced cardiac hypertrophy. J Mol Cell Cardiol 35: 851–859, 2003.[CrossRef][ISI][Medline]
  21. Nath KA, Croatt AJ, Haggard JJ, and Grande JP. Renal response to repetitive exposure to heme proteins: chronic injury induced by an acute insult. Kidney Int 57: 2423–2433, 2000.[CrossRef][ISI][Medline]
  22. Oudit GY, Sun H, Trivieri MG, Koch SE, Dawood F, Ackerley C, Yazdanpanah M, Wilson GJ, Schwartz A, Liu PP, and Backx PH. L-type Ca2+ channels provide a major pathway for iron entry into cardiomyocytes in iron-overload cardiomyopathy. Nat Med 9: 1187–1194, 2003.[CrossRef][ISI][Medline]
  23. Oudit GY, Trivieri MG, Khaper N, Husain T, Wilson GJ, Liu P, Sole MJ, and Backx PH. Taurine supplementation reduces oxidative stress and improves cardiovascular function in an iron-overload murine model. Circulation 109: 1877–1885, 2004.[Abstract/Free Full Text]
  24. Oudot A, Vergely C, Ecarnot-Laubriet A, and Rochette L. Angiotensin II activates NADPH oxidase in isolated rat hearts subjected to ischaemia-reperfusion. Eur J Pharmacol 462: 145–154, 2003.[CrossRef][ISI][Medline]
  25. Panchenko MV, Farber HW, and Korn JH. Induction of heme oxygenase-1 by hypoxia and free radicals in human dermal fibroblasts. Am J Physiol Cell Physiol 278: C92–C101, 2000.[Abstract/Free Full Text]
  26. Park YM, Park MY, Suh YL, and Park JB. NAD(P)H oxidase inhibitor prevents blood pressure elevation and cardiovascular hypertrophy in aldosterone-infused rats. Biochem Biophys Res Commun 313: 812–817, 2004.[CrossRef][ISI][Medline]
  27. Parkes JG, Liu Y, Sirna JB, and Templeton DM. Changes in gene expression with iron loading and chelation in cardiac myocytes and non-myocytic fibroblasts. J Mol Cell Cardiol 32: 233–246, 2000.[CrossRef][ISI][Medline]
  28. Parkes JG and Templeton DM. Modulation of stellate cell proliferation and gene expression by rat hepatocytes: effect of toxic iron overload. Toxicol Lett 144: 225–233, 2003.[CrossRef][ISI][Medline]
  29. Petrov VV, Fagard RH, and Lijnen PJ. Stimulation of collagen production by transforming growth factor-{beta}1 during differentiation of cardiac fibroblasts to myofibroblasts. Hypertension 39: 258–263, 2002.[Abstract/Free Full Text]
  30. Saito K, Ishizaka N, Mitani H, Ohno M, and Nagai R. Iron chelation and a free radical scavenger suppress angiotensin II-induced downregulation of klotho, an anti-aging gene, in rat. FEBS Lett 551: 58–62, 2003.[CrossRef][ISI][Medline]
  31. Sano M, Fukuda K, Sato T, Kawaguchi H, Suematsu M, Matsuda S, Koyasu S, Matsui H, Yamauchi-Takihara K, Harada M, Saito Y, and Ogawa S. ERK and p38 MAPK, but not NF-{kappa}B, are critically involved in reactive oxygen species-mediated induction of IL-6 by angiotensin II in cardiac fibroblasts. Circ Res 89: 661–669, 2001.[Abstract/Free Full Text]
  32. Schafer AI, Cheron RG, Dluhy R, Cooper B, Gleason RE, Soeldner JS, and Bunn HF. Clinical consequences of acquired transfusional iron overload in adults. N Engl J Med 304: 319–324, 1981.[Abstract]
  33. Suzumura K, Hashimura Y, Kubota H, Ohmizu H, and Suzuki T. Antioxidative property of T-0970, a new ureidophenol derivative. Free Radic Res 32: 255–264, 2000.[CrossRef][ISI][Medline]
  34. Tokuda K, Kai H, Kuwahara F, Yasukawa H, Tahara N, Kudo H, Takemiya K, Koga M, Yamamoto T, and Imaizumi T. Pressure-independent effects of angiotensin II on hypertensive myocardial fibrosis. Hypertension 43: 499–503, 2004.[Abstract/Free Full Text]
  35. Tsukamoto H, Horne W, Kamimura S, Niemela O, Parkkila S, Yla-Herttuala S, and Brittenham GM. Experimental liver cirrhosis induced by alcohol and iron. J Clin Invest 96: 620–630, 1995.[ISI][Medline]
  36. Yoo KH, Thornhill BA, Wolstenholme JT, and Chevalier RL. Tissue-specific regulation of growth factors and clusterin by angiotensin II. Am J Hypertens 11: 715–722, 1998.[CrossRef][ISI][Medline]
  37. Yu CM, Tipoe GL, Wing-Hon Lai K, and Lau CP. Effects of combination of angiotensin-converting enzyme inhibitor and angiotensin receptor antagonist on inflammatory cellular infiltration and myocardial interstitial fibrosis after acute myocardial infarction. J Am Coll Cardiol 38: 1207–1215, 2001.[Abstract/Free Full Text]
  38. Zhou XJ, Laszik Z, Wang XQ, Silva FG, and Vaziri ND. Association of renal injury with increased oxygen free radical activity and altered nitric oxide metabolism in chronic experimental hemosiderosis. Lab Invest 80: 1905–1914, 2000.[ISI][Medline]



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N. Ishizaka, K. Saito, I. Mori, G. Matsuzaki, M. Ohno, and R. Nagai
Iron Chelation Suppresses Ferritin Upregulation and Attenuates Vascular Dysfunction in the Aorta of Angiotensin II-Infused Rats
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