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Genes to Cells (2005) 10, 277-285. doi:10.1111/j.1365-2443.2005.00832.x
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Effects of genetic ablation of bach1 upon smooth muscle cell proliferation and atherosclerosis after cuff injury

Shinji Omura1, Hiroshi Suzuki1,a, Mamoru Toyofuku2, Ryoji Ozono3, Nobuoki Kohno2 and Kazuhiko Igarashi1,*

1 Department of Biomedical Chemistry,
2 Department of Molecular and Internal Medicine and 3 Department of Clinical Laboratory Medicine, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima 734-8551, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Bach1 is a transcriptional repressor of the cytoprotective enzyme heme oxygenase-1 (HO-1). Although HO-1 protects against atherosclerosis, the function of Bach1 in this process is poorly understood. We isolated peritoneal macrophages and aortic smooth muscle cells (SMC) from wild-type and bach1-deficient mice. bach1-deficient macrophages expressed increased levels of HO-1 and showed elevated phagocytic activity when incubated with 0.75 µm microspheres. In SMC, bach1-ablation resulted in increased expression of HO-1 and decreased proliferation in bromodeoxyuridine incorporation assay as compared with wild-type cells. The up-regulated phagocytic activity and reduced SMC proliferation of bach1-deficient cells were not restored by Zinc (II) protoporphyrin IX, an inhibitor of HO, suggesting that HO-independent mechanisms are also involved in the regulation of phagocytosis of macrophages and proliferation of SMC by Bach1. In wild-type mice, cuff placement around femoral artery caused pronounced intimal proliferation without affecting the media, thus resulting in intimal to medial (I/M) volume ratio of 65.6%. bach1-deficient mice had less degree of intimal growth (I/M ratio of 45.6%). These results indicate that Bach1 plays a critical role in the regulation of HO-1 expression, macrophage function, SMC proliferation and neointimal formation. Bach1 may regulate gene expression in these cells during inflammation and atherogenesis.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in heme degradation, generating ferrous iron, carbon monoxide and biliverdin that is reduced to bilirubin by biliverdin reductase (Tenhunen et al. 1968; Maines & Trakshel 1993; Maines 1997; Shibahara 2003). Carbon monoxide, biliverdin, as well as bilirubin, have anti-oxidant and anti-inflammatory activities in vivo (Otterbein et al. 2000). Thus, HO-1 is an anti-inflammatory defense enzyme that produces a series of metabolites from heme, hence contributing to the suppression of oxidative tissue injuries. Recent studies have suggested that HO-1 is important for inhibiting progression of atherosclerosis by modulating functions of macrophages and smooth muscle cells (Otterbein et al. 2000; Otterbein & Choi 2000; Togane et al. 2000; Tulis et al. 2001a). Gene therapy approach employing HO-1 has been shown effective to inhibit development of atherosclerosis in several model systems (Otterbein et al. 1999; Duckers et al. 2001; Tulis et al. 2001b). Therefore, HO-1 is considered to be a protective factor against atherosclerosis.

Transcription of hmox-1 that encodes HO-1 is robustly induced in mammalian cells by diverse cellular stresses including oxidative stress (Shibahara et al. 1987; Alam et al. 1989; Keyse & Tyrrell 1989). The induction of hmox-1 expression is directed by its critical enhancer elements including the stress responsive element (StRE) (Inamdar et al. 1996) or Maf recognition element (MARE) (Kataoka et al. 1994, 2001). The small Maf (MafF, -G, and –K) and Nrf2 proteins form heterodimers to activate StRE/MARE-dependent hmox-1 expression (Alam et al. 1999; Ishii et al. 2000; Kataoka et al. 2001). On the other hand, Bach1 forms heterodimers with the small Maf to repress hmox-1 (Sun et al. 2002). The induction of HO-1 in fibroblastic cells is achieved by the exchange of Bach1 and Nrf2 on the hmox-1 enhancers (Sun et al. 2002, 2004). In mice lacking bach1, the transcription of HO-1 is constitutively up-regulated, leading to increased levels of protein and enzymatic activity under normal conditions in several organs such as heart (Sun et al. 2002; our unpublished observation).

It has been shown that HO-1 represses proliferation of smooth muscle cells (SMC) by producing carbon monoxide (Morita et al. 1997; Togane et al. 2000; Duckers et al. 2001; Peyton et al. 2002). Thus HO-1 appears to play an important role against progression of atherosclerosis by modulating proliferation of SMC (Soares et al. 1998; Duckers et al. 2001). While Nrf2 is a key activator of HO-1 in macrophages and SMC (Ishii et al. 2004), little is known regarding the role of Bach1 in these cells. In the present study, we examined whether Bach1 regulates the functions of macrophages and SMC by comparing these cells isolated from wild-type and bach1-deficient mice. To understand roles for Bach1 in atherosclerosis, we also compared neointimal formation in the cuff placement model. The results indicate that Bach1 plays critical roles in the regulation of not only macrophages and SMC functions but also pathogenesis of atherosclerosis.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Expression of HO-1 in macrophages and smooth muscle cells

It remains unclear whether Bach1 regulates HO-1 in macrophages or SMC. We examined expression levels of HO-1 mRNA in bach1-deficient macrophages using quantitative RT-PCR (Fig. 1A). The exposure of wild-type cells to various oxidative agents (10 µM CdCl2, 10 µM hemin, or 200 µg/mL oxidized LDL) resulted in induction of HO-1 (14.7-fold and P < 0.01; 18.3-fold and P < 0.05; 39.6-fold and P < 0.05, respectively) (Fig. 1B). In contrast, HO-1 mRNA was expressed at higher levels in bach1-deficient macrophages even under normal culture conditions as compared with the wild-type cells (18.3-fold and P < 0.01). Cadmium, hemin or oxidized LDL (ox-LDL) further enhanced expression of HO-1 (40.0-fold and P = 0.09; 39.1-fold and P = 0.14; 67.5-fold and P = 0.06, respectively, compared with untreated wild-type cells). HO-1 protein levels were higher in the bach1-deficient macrophages (Fig. 1C).



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  		Figure 1  Expression of HO-1 in bach1-deficient macrophages. (A) HO-1 (upper panel) and ß-actin (lower panel) mRNA levels with or without various stimulation were compared by RT-PCR. Macrophages from wild-type or bach1-deficient mice (WT and KO, respectively) were incubated with or without CdCl2, hemin or ox-LDL for 6 h. (B) Relative levels of HO-1 mRNA were determined and corrected for ß-actin mRNA levels using real time PCR. Results are the mean of at least three mice for each genotype, with the SEM indicated. *P < 0.01, #P < 0.05, compared with WT cells without stimulation, respectively. (C) HO-1 protein levels in WT or KO macrophages were analyzed by immunoblot assay (upper panel). The equivalency of loading and transfer of protein was confirmed by the expression of MafK (lower panel).

 
To investigate whether Bach1 regulates expression of HO-1 in SMC, we prepared and cultured aortic SMC from wild-type and bach1-deficient mice. As shown in Fig. 2A,B, bach1-deficient SMC expressed HO-1 mRNA at 14.8-fold higher levels as compared with wild-type cells under normal culture conditions (P < 0.01). Treatment of the wild-type cells with 10 µM CdCl2 resulted in 62.2-fold induction of HO-1 (P < 0.005). CdCl2 further enhanced expression of HO-1 mRNA in the bach1-deficient cells (90.5-fold and P < 0.01 compared with untreated wild-type cells). As shown in Fig. 2C, immunoblot analysis using anti-HO-1 antibody showed that exposure of wild-type SMC to hemin or ox-LDL resulted in induction of HO-1 protein. In contrast, HO-1 protein was expressed at higher levels in the bach1-deficient cells than in the wild-type cells irrespective of the absence or presence of hemin or ox-LDL. Taken together, these results establish that Bach1 is a key repressor of the HO-1 gene in macrophages and SMC and suggest that oxidative stressors such as heme and ox-LDL abrogate the Bach1-mediated transcription repression in these cells. Because the inducers examined here increased HO-1 mRNA expression in the bach1-deficient macrophage and SMC, we can not exclude the possibility that these inducers not only inhibit Bach1 but also enhance activator function such as Nrf2.



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  		Figure 2  Expression of HO-1 in bach1-deficient SMC. (A) HO-1 (upper panel) and ß-actin (lower panel) mRNA levels with or without stimulation with CdCl2 for 6 h were compared by RT-PCR. (B) Relative levels of HO-1 mRNA were determined and corrected for ß-actin mRNA levels using real time PCR. Results are the mean of three mice for each genotype. *P < 0.005, #P < 0.01, compared with WT cells without stimulation, respectively. **P < 0.01, compared with KO cells without stimulation. (C) HO-1 (upper panel) and MafK (lower panel) protein levels with or without various stimulations were compared by immunoblotting.

 
Migration and phagocytic activity of macrophages

We compared infiltration of macrophages into intraperitoneal cavity after intraperitoneal administration of thioglycollate. There was no significant difference in the numbers of recruited macrophages between wild-type and bach1-deficient mice (13.4 ± 4.8 x 106 cells/mouse vs. 11.3 ± 2.2 x 106 cells/mouse, respectively; n = 9, P = 0.25). Next we compared the chemotactic activity in vitro using chemotaxis chamber. We found no significant difference in the percentages of migrated macrophages between wild-type and bach1-deficient mice (1.61 ± 0.91% vs. 2.05 ± 0.44%, respectively; n = 4, P = 0.41). These results suggest that Bach1 is not involved in the regulation of chemotaxis of macrophages.

Next we compared the phagocytic activities of wild-type and bach1-deficient macrophages. The percentage of microspheres-positive macrophages was higher in bach1-deficient cells than in wild-type cells (Fig. 3A, left column set). Strongly phagocytosing macrophages were more evident in bach1-deficient cells than in wild-type cells (Fig. 3B, left column set). Furthermore, as shown in Fig. 3C, the numbers of phagocytosed microspheres per macrophage were obviously higher in the bach1-deficient cells than in the wild-type cells. We investigated whether the increased phagocytic activity in bach1-deficient macrophages was attributable to the increased levels of HO-1 using zinc (II) protoporhyrin IX (ZnPP), an inhibitor of both HO-1 and HO-2. Co-incubation of cells with 10 µM of ZnPP resulted in decreases in the phagocytic activities of both wild-type and bach1-deficient macrophages (Fig. 3A,B). Lower concentrations of ZnPP did not cause statistically significant effect upon phagocytic activities. To investigate whether carbon monoxide, one of the metabolites of HO reaction, is involved in the phagocytosis, we examined effect of hemoglobin. Hemoglobin is known to scavenge carbon monoxide (Peyton et al. 2002). Under the experimental conditions previously used for SMC analysis (Peyton et al. 2002), 50 µM hemoglobin efficiently inhibited phagocytosis by both wild-type and bach1-deficient macrophages (Fig. 3A,B). It is formally possible that hemoglobin induces HO-1 expression in macrophages upon phagocytosis. However, when taken together with the effects of ZnPP, these results suggest that HO-1 and its catalytic metabolite carbon monoxide are involved in the positive regulation of phagocytosis. The fact that ZnPP and hemoglobin did not completely attenuate the difference between wild-type and bach1-deficient cells suggests that additional downstream target genes of Bach1 are also involved in phagocytosis.



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  		Figure 3  Increased phagocytic activity in bach1-deficient macrophages. (A, B) Phagocytosing macrophages were viewed on fluorescence microscopy. The proportions of macrophages containing more than one microsphere (A) or containing more than eight microspheres (B) were determined. Where indicated, cells were preincubated with ZnPP (5 or 10 µM) or hemoglobin (50 µM). Results are the mean of at least three mice for each genotype. *P < 0.05, compared with WT cells without treatment. **P < 0.05, compared with WT cells with the same treatment. &P = 0.08, compared with WT cells with hemoglobin. #P < 0.05, compared with KO cells without treatment. (C) Images of phagocytosing macrophages are shown. The scale bars indicate as 10 µm.

 
Expression of MCP-1 mRNA in macrophages and smooth muscle cells

Monocytic chemoattractant protein-1 (MCP-1) plays important roles in the regulation of macrophage and SMC. Several investigators have reported that up-regulation of HO-1 accompanies induction of MCP-1 in various organs and cell-types, including SMC and macrophages (Ishii et al. 2000; Nath et al. 2001; Kadl et al. 2002). However, no difference was found in the levels of MCP-1 expression between wild-type and bach1-deficient macrophages (1.0-fold, Fig. 4A). In contrast, MCP-1 expression in SMC was significantly increased 4.3-fold (P < 0.05) in bach1-deficient cells (Fig. 4B,C). These results suggest that Bach1 may regulate MCP-1 expression in SMC, but not in macrophages.



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  		Figure 4  Expression of MCP-1 in bach1-deficient cells. (A) Relative levels of MCP-1 mRNA in macrophages were determined and corrected for ß-actin mRNA levels. Results are the mean of seven independent experiments. (B) MCP-1 (upper panel) and ß-actin (lower panel) mRNA levels in SMC were compared by RT-PCR. (C) Relative levels of MCP-1 mRNA in SMC were determined and corrected for ß-actin mRNA levels. Results are the mean of three independent experiments. *P < 0.05, compared with WT cells.

 
Decreased proliferation of bach1-deficient smooth muscle cells

We investigated the effect of bach1-ablation on the proliferation of SMC in vitro using the bromodeoxyuridine (BrdU) labeling assay (Fig. 5A). Serum- or platelet-derived growth factor (PDGF)-induced proliferation was significantly reduced in bach1-deficient SMC as compared with wild-type cells. Interestingly, co-incubation with ZnPP did not attenuate the proliferative difference between wild-type and bach1-deficient SMC. These results indicate that the reduced proliferation of bach1-deficient SMC is not fully attributable to the over-expression of HO-1. Rather, Bach1 appears to function as a positive regulator of proliferation in SMC through both HO-dependent and HO-independent mechanisms.



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  		Figure 5  Proliferation of bach1-deficient smooth muscle cells and responses to cuff injury. (A) SMC proliferation was compared using BrdU labeling assay. Cells were incubated with ZnPP (5 or 10 µM) or PDGF (10 ng/mL). Results are the mean of three independent experiments. *P < 0.05, **P = 0.05, #P = 0.08, compared with WT cells with the same treatment, respectively. (B) Histology and immunochemical staining of femoral arteries from WT (left) and KO (right) mice are shown. Adjacent sections from sham-operated (a, b) or cuffed vessels (c–f) were processed for Elastica van Gieson-staining (a–d) or {alpha}-smooth muscle actin staining (e, f). The intima is seen within the internal elastic lamina. KO mice show substantially less neointimal formation in response to cuff injury than WT mice. In sham operation (a, b), the vessels collapsed due to not placing a cuff. The scale bars, 50 µm.

 
Vessel response to external vascular cuff placement

To assess the role of Bach1 in the pathogenesis of atherosclerosis, we evaluated the effects of bach1-deficiency on external vascular cuff-induced neointima formation (Moroi et al. 1998, 2003). The intima was thin in the control femoral arteries of both wild-type and bach1-deficient mice (Fig. 5B;a,b). When cuffed, we observed a significant neointima formation in the wild-type mice with an intimal to medial volume ratio (I/M) of 65.6% (Fig. 5B;c). In contrast, bach1-deficient mice showed significantly less neointima formation (I/M was 45.6%) compared with wild-type mice (P = 0.02, Table 1; Fig. 5B;d). Medial thickness after cuff placement was not significantly different between the two groups. The majority of cells in neointima regions in wild-type mice expressed {alpha}-actin, suggesting proliferation of smooth muscle cells (Fig. 5B; compare e and f).


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  		Table 1  Effect of bach1-ablation on response to vessel injury
 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
The induction of HO-1 appears to be one of the critical gene responses in atherosclerosis. While Nrf2 is obviously involved in HO-1 activation in various types of cells including macrophages and SMC (Ishii et al. 2000, 2004), the relevance of Bach1-mediated transcriptional repression in these cells or in the process of atherosclerosis remains unclear. In this study, we addressed this issue using bach1-deficient mice and found that Bach1 is inhibitory for HO-1 expression in both macrophages and SMC as well as for the phagocytosis activity of macrophages. Interestingly, proliferation of SMC was reduced in bach1-deficient SMC. Consistent with the increased expression of HO-1 and reduced proliferation of SMC, neointimal formation after cuff injury was significantly reduced in bach1-deficient mice. In wild-type mice, most cells in the neointima were predominantly SMC, as reported previously (Moroi et al. 1998,, 2003; Lardenoye et al. 2000; Egashira et al. 2002). This indicates that invasion and/or proliferation of medial SMC into the intimal region is reduced in bach1-deficient mice. These results place Bach1 as one of the critical regulators of gene expression in atherosclerosis. However, they also raise several issues that may be important for further understanding of atherosclerosis.

Although deletion of bach1 caused marked up-regulation of HO-1, ZnPP (an inhibitor of HO activity) or hemoglobin (a scavenger of carbon monoxide) did not attenuate completely the difference in phagocytic activities or SMC proliferation between wild-type and bach1-deficient cells. These results suggest that phenotypic changes are not solely explained by the increased expression of HO-1. A more possible interpretation for this phenomenon could be that other critical genes are also regulated by Bach1 in macrophages and SMC. Identification of such genes will be important in promoting our understanding of pathogenesis of atherosclerosis.

bach1-ablation resulted in the higher levels of MCP-1 expression in SMC in vitro but not in macrophages. These results suggest that MCP-1 expression is regulated in a cell-type specific manner. Although up-regulation of MCP-1 is considered to deteriorate atherosclerosis (Aiello et al. 1999), bach1-deficient mice showed reduced neointimal formation after cuff injury. It is possible that changes in expression of other genes may mask MCP-1-mediated responses. The functional or pathogenic significance of the MCP-1 up-regulation in bach1-deficient SMC remains to be determined.

It has been reported that deletion of nrf2 causes reduction in HO-1 expression (Ishii et al. 2000, 2004). Deletion of bach1 induces an increase in HO-1 expression, hence establishing that HO-1 gene expression in macrophages and SMC is regulated by the balance between the activator Nrf2 and the repressor Bach1. Macrophages lacking nrf2 show exaggerated cell death upon oxidative stress (Ishii et al. 2000), suggesting that HO-1 protects activated macrophages from free radicals that are produced abundantly in the area of inflammation. Our present observations suggest that Bach1 is inhibitory for both phagocytosis and HO-1 induction. Because Bach1 is rapidly inactivated upon oxidative stress (Suzuki et al. 2003, 2004; Sun et al. 2004), thereby turning on the cytoprotective machinery including HO-1, Bach1 may play a role as a molecular switch to activate macrophage in response to oxidative stress. In this model, Bach1 precludes premature expression of genes such as HO-1 and those required for phagocytosis before activation. Upon encountering oxidative stress, Bach1 may be inactivated, allowing expression of HO-1 and other genes by activators such as Nrf2, thus leading to increased resistance to oxidative stress and up-regulation of phagocytic activity. This model predicts that a fluctuation in the balance of Nrf2 and Bach1 may affect the process of atherosclerosis. The exposure of wild-type cells to various atherogenic stresses such as ox-LDL resulted in higher expression of Bach1 mRNA (unpublished). These observations suggest the presence of a negative spiral transcription network that deteriorates atherosclerosis. Taken together with the fact that HO-1 has anti-inflammatory and anti-atherosclerotic properties, we can conclude that Bach1 may represent a novel molecular target in anti-inflammatory and anti-atherosclerotic therapy.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Mice

The bach1-deficient mice have been reported previously (Sun et al. 2002). Heterozygous mice were intercrossed to obtain homozygous bach1-deficent mice as well as the control wild-type mice. Congenic bach1-deficent mice were obtained by repeatedly backcrossing with C57BL/6 J mice at least 12 generations.

Macrophages and SMC

Peritoneal macrophages were isolated as previously described (Ishii et al. 2000) from littermates or congenic mice (6- to 10-week-old) after intraperitoneal administration of 1.5 mL of 4% thioglycollate (Nissui). Cells were re-suspended at 1 x 106 cells/mL in Dulbecco's modified Eagle's medium (DMEM, Sigma) with 10% foetal bovine serum (FBS, JRH BioSciences), and seeded in culture plates (Falcon). After incubation at 37 °C for 3 h, cells were washed three times with PBS to remove nonadherent cells. SMC were prepared from aorta of 8- to 12-week-old littermates of bach1-deficient and wild-type mice as previously described (Berk et al. 1989; Ishida et al. 1998) and maintained in 10% FBS/DMEM. Analysis of the SMC with antibody against smooth muscle–specific {alpha}-actin (Clone 1A4; Sigma) revealed that all cells expressed this antigen. Macrophages and SMC were treated with 10 µM CdCl2 (Sigma), 10 µM hemin (Sigma), or 100 or 200 µg/mL of ox-LDL. ox-LDL was prepared from human LDL (d = 1.019–1.063 g/mL) as previously described (Sakai et al. 1999) or purchased from Biomedical Technologies.

RT-PCR analysis

Total RNA was extracted using Trizol, and cDNA was prepared as previously described (Sun et al. 2002). The sequences of the primers for RT-PCR are available upon request. Real time PCR and conventional semiquantitative PCR were carried out using the LightCycler system (Roche) and BioAnalyzer 2100 (Agilent), respectively, as previously described (Sun et al. 2002, 2004).

Immunoblotting analysis

Ten micrograms of whole cell extracts, prepared from macrophages or SMC as previously described (Muto et al. 1998) and processed for immunoblotting using antisera against HO-1 (kind gift from Prof Shigeru Taketani, Kyoto Institute of Technology, Japan) and MafK (Igarashi et al. 1995). Immune complexes were detected using the enhanced chemiluminescence system (Amersham).

Cell migration assay

Transwell cell migration assays were performed as previously described (Shimonaka & Yamaguchi 1994) using a 96-well chemotaxis chambers (Neuroprobe) and 10–8 M N-Formyl-L-methionyl-L-leucyl-L-phenylalanine (Sigma) as an inducer of macrophage chemotaxis. 1 x 106 macrophages suspended in 200 µL DMEM were pipetted into each upper chamber. After incubation for 24 h at 37 °C in a humidified atmosphere of 95% air/5% CO2, portions of migrated cells were counted with the colorimetric MTT assay kit (Roche). All experiments were performed in triplicate or quadruplicate and repeated three times. Migration was expressed as a percentage of basal cells.

Assay for phagocytosis of microspheres

After incubation of the macrophages with ZnPP (Calbiochem) for 6 h (Kanakiriya et al. 2003) or bovine hemoglobin (Sigma) for 12 h, macrophages were cultured with 0.025% of 0.75 µm fluorescent polystyrene latex microspheres (PolySciences) in DMEM for 30 min. Cells were washed with PBS to remove non-phagocytosed beads and fixed with 4% paraformaldehyde in PBS. The images were taken with a Leica epifluorescence microscope equipped with a charge-coupled device camera controlled by QFluoro software (Leica). At least 200 cells were counted in random high-power fields.

BrdU labeling assay for cell proliferation

SMC were seeded into 96-well plates (1 x 104 cells/well). Twenty-four hours later, indicated concentrations of ZnPP were added to wells. After 4 h, 10 µM BrdU was added and the cells were incubated for additional four hours. To examine the effect of PDGF, SMC were made quiescent by incubating in 0.5% FBS/DMEM for 48 h. Then cells were cultured with 10 ng/mL of rat PDGF (Sigma) for 20 h before BrdU labeling. Amounts of BrdU incorporated were determined using enzyme-linked immuno-sorbent assay kit (Roche).

Femoral artery cuff placement model

The external vascular cuff-induced intima formation was examined as reported previously (Moroi et al. 1998, 2003) using (8- to 10-week-old) congenic mice. The femoral artery was isolated from surrounding tissues, loosely sheathed with a 2.0-mm polyethylene cuff made of PE-50 tubing (inner diameter, 0.56 mm; outer diameter, 0.965 mm; Becton Dickinson) and tied in place with an 8–0 suture. In sham operation, femoral arteries were dissected from surrounding tissues without placing a cuff. Vessels were isolated and processed for histological analyses (hematoxylin and eosin or elastica van Gieson staining) after two weeks. Parallel sections were examined by immunohistochemical staining using antibodies against smooth muscle-specific {alpha}-actin. Morphometric analyses were performed on elastica van Gieson-stained tissue as previously described (Moroi et al. 1998, 2003) using an image analysis computer program (NIH Image; National Institutes of Health). For each artery section, the thickness of the intima and media were measured. For area/volume calculations, four measurements were made using: luminal circumference, luminal area, area inside the inner elastic lamina, and area inside the outer elastic lamina. Mean vascular diameter was calculated as luminal circumference/{pi}. The volumes of intima and media were calculated by integrating the areas over the length of the cuffed region.

Statistical analysis

Data were expressed as mean ± SEM. The unpaired t-test or paired t-test (for the littermates) were used for statistical analysis of the results. Values of P < 0.05 were considered statistically significant.


   Acknowledgements
 
We thank Drs Satoshi Tashiro, Takafumi Ishida and Shigeru Taketani for discussion and reagents and Dr Toshisuke Morita for advice on the cuff injury model. Further thanks go to Ms. Maria Makri for critical reading of the manuscript. This work was supported by Grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Footnotes
 
Communicated by: Masayuki M. Yamamoto

aPresent address: Division of Advanced Surgical Science and Technology, Tohoku University Graduate School of Medicine, Sendai 980–8575 Japan Back

* Correspondence: E-mail: igarak{at}hiroshima-u.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 18 October 2004
Accepted: 5 December 2004




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