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Poult Sci 2007. 86:1166-1173
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MOLECULAR, CELLULAR, AND DEVELOPMENTAL BIOLOGY

Expression of Cell-Cycle-Regulating Genes in the Development of Atherosclerosis in Japanese Quail (Coturnix japonica)

M. Inafuku*, T. Toda{dagger},1, T. Okabe{ddagger}, A. Shinjo{ddagger}, H. Iwasaki§ and H. Oku§

* United Graduate School of Agricultural Sciences, Kagoshima University, Korimoto, 890-0065, Japan; and {dagger} Department of Clinical Laboratory Medicine, School of Medicine, {ddagger} Laboratory of Plant and Animal Bleeding, Department of Bioproduction, Faculty of Agriculture, and § Division of Molecular Biotechnology, Center of Molecular Bioscience, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan

1 Corresponding author: b985703{at}med.u-ryukyu.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The levels of mRNA expression in regulatory genes that are involved in the pathological changes of aortic atherosclerotic and fibroblastic intimal thickening was investigated in Japanese quail. The quail were divided into a control diet group and an atherogenic diet group. The quail were euthanized at 2, 4, 8, and 12 wk after consuming either a control diet or an atherogenic diet. Thereafter, both histological and immunohistochemical studies and mRNA expression analysis of the cell-cycle-regulating genes in aortic atherosclerotic lesions were performed on selected ascending aortas and their large branches. In the atherogenic diet group, aortic lipid-containing intimal and atheromatous lesions were seen mainly at 8 and 12 wk, respectively. Semiquantitative reverse-transcription PCR was used to analyze the alterations of mRNA expression on the development of atherosclerotic lesions. Messenger RNA expression of the c-fos and c-src genes showed peak levels at 8 wk in the atherogenic diet group. However, no significant alteration of c-jun mRNA expression was noted during the entire experimental period. According to the progression of aortic atherosclerotic lesions, c-myc mRNA expression in the atherogenic diet group increased chronologically, and the highest level was observed at 12 wk. Alterations in mRNA expression of proliferating cell nuclear antigen and the p27 gene were similar to that of c-myc. The levels of c-myc, proliferating cell nuclear antigen, and p27 mRNA expression was significantly correlated with the degree of aortic atherosclerotic lesion development at 12 wk in our experiment.

Key Words: cell-cycle-regulating gene • molecular genetics • atherosclerosis • quail


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recently, the importance of in vivo studies related to atherogenesis has increased, and atherogenesis has become an interesting research topic in medical science because such studies may provide valuable information regarding the development of future remedies for this disease. Pearson et al. (1975) and Benditt (1977) suggested the possibility that atherosclerotic lesions may be benign tumors of vascular smooth muscle cells (VSMC) in the arterial wall. A series of molecular markers and gene pathways common in both atherosclerosis and cancer have recently been associated with the development and progression of the disease (Ross et al., 2001). The molecular basis of studies regarding atherogenesis in various animal experimental models is still important because cultured cells tend to alter their phenotype (Thyberg et al., 1983). Among various animal models used in studying atherogenesis, we have chosen the Japanese quail (Coturnix japonica) for the following reasons: They are economical, with a small body size and short life span, and they are highly susceptible to atherosclerosis (Toda and Oku, 1995). One of the major cell types of proliferating intimal cells in aortic atherosclerotic lesions in humans and animals is the VSMC (Ross, 1993), whereas in avian species such as the chicken (Moss and Benditt, 1970) and quail (Toda and Oku, 1995), the major cell types are fibroblasts and VSMC in the thoracic and abdominal aortas, respectively. Recent molecular pathological studies have indicated that numerous genes regulating the cell cycle and cell growth are also involved in the initiation and development of atherosclerotic lesions in humans and animals (Doevendans et al., 2001). In comparison with the number of studies carried out in humans and other animals such as rats and mice, few molecular pathological studies of atherosclerosis have been carried out in birds. Therefore, to obtain more insight into the molecular mechanism of atherosclerosis induction in the quail animal model, we investigated gene mRNA expression during the development of atherosclerotic lesions in the Japanese quail. The c-fos and c-jun protooncogenes were originally found in mutated forms in murine and avian oncogenic retroviruses (Jariel-Encontre et al., 1997). Basic region-Leu zipper proteins such as c-fos and c-jun constitute the homodimers or heterodimers that make up the nuclear transcription factor activator protein-1. c-Myc is a transcription factor involved in the G1- to S-phase transition of the cell cycle, and has been observed in a high percentage of tumors (Hurlin and Dezfouli, 2004). Messenger RNA expression of the protooncogenes c-fos, c-jun, and c-myc has been suggested to play a key role in cell proliferation, differentiation, and neoplastic transformation (van Dam and Castellazzi, 2001; Hurlin and Dezfouli, 2004). In mammals, activator protein-1 has also been clearly shown to be related to the various cellular events involved in cell proliferation, transformation, differentiation, and apoptosis (Ameyar et al., 2003). The c-fos, c-jun, and c-myc genes have also been reported to be involved in the development of atherosclerosis (Miano et al., 1990; Goetze et al., 2001; Khachigian et al., 2002). c-Src, a nonreceptor tyrosine kinase, has been reported to be activated in numerous types of human cancers, and to be a critical component of multiple signaling pathways that regulate cell differentiation, motility, proliferation, survival, metastasis, and angiogenesis (Thomas and Brugge, 1997; Courtneidge, 2002). The signal pathways of src are also reportedly involved in glucose uptake in the VSMC and the formation of macrophage-derived foam cells (Kanda and Watanabe, 2005; Watanabe et al., 2005). Proliferating cell nuclear antigen (PCNA), one of the cell-cycle-regulating genes, also plays an essential role in the progression of the cell cycle from the G1-phase to the S-phase, and in chromosomal replication and repair (Kelman, 1997). Proliferating cell nuclear antigen is known to be required in VSMC proliferation because antisense oligonucleotides of mRNA encoding PCNA inhibit the proliferation of various cells (Simons et al., 1994; Maeshima et al., 1996). p27, a member of the CDK interacting protein/kinase inhibitory protein family of cyclin-dependent kinase inhibitors, is known to be a negative regulator of cell proliferation; its expression is high in the G1-phase and is down-regulated when cells pass through the S-phase (Adachi et al., 2003). Dellas et al. (1998) suggested that p27 plays a complex role in carcinogenesis that is not simply related to its inhibitory effects on cell proliferation. Several authors (Tanner et al., 1998; Andres et al., 2001) have demonstrated the enhanced expression of p27 mRNA in balloon-injured arteries. Sato et al. (2000) reported VSMC proliferation to be inhibited via the up-regulation of p27, but not via the apoptotic pathway. In the present study on the development of atherosclerotic lesions in the Japanese quail, we examined mRNA expression of the genes shown above, which have been reported to be involved in both tumorigenesis and atherogenesis in humans and rodents. Special emphasis was placed on the correlation between gene expression and anatomical changes in the atherosclerotic lesions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Experiments
A total of 60 Japanese male quail (Coturnix japonica, 2 mo old) were purchased from a commercial supplier (Toukaiyuki Co., Ltd., Aichi, Japan) and were housed individually in stainless-steel cages under controlled room temperature (25°C) with a 12-h light-dark cycle. Experiments were conducted in accordance with the guidelines for animal experiments of the University of the Ryukyus. As shown in Table 1Go, the birds were randomly divided into 2 groups: a control diet (CD) group (fed a basal commercial diet) and an atherogenic diet AD) group (fed an atherogenic diet of 15% corn oil and 2% cholesterol in the basal commercial diet). The basal commercial diet was purchased from Kyoei Co., Ltd. (Okinawa, Japan). Experimental diets and water were given ad libitum. All birds were euthanized at 2, 4, 8, and 12 wk of the feeding period. The entire aorta (ascending aorta and aortic arch) and its large branches along with the heart were excised from each bird to examine aortic lesions and mRNA expression of each gene. The levels of serum total cholesterol and triglycerides were also measured using a commercially available enzymatic kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan).


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  		Table 1. Body and liver weights, and serum lipid profiles in each experimental group1
 
Histological and Immunohistochemical Examinations
The heart and a 1-cm-long proximal portion of ascending aorta and its large branches were excised and fixed in 10% buffered formalin for histological and immunohistochemical examinations. The rest of the aortas were frozen in liquid nitrogen and stored at –80°C until total RNA extraction. For histological examination, formalin-fixed paraffin-embedded aortic tissue samples were sectioned into 4-µm thicknesses and were then stained with hematoxylin eosin, Mallory azan, and elastica van Gieson. To evaluate the degree of atherosclerosis, the total intimal thickness to medial thickness (I:M) ratio was obtained by averaging of the maximum I:M ratio of 3 aortic segments from each bird (Figure 1Go). Immunohistochemical study was also carried out on tissue sections using the Envision system (Dako, Kyoto, Japan). The antibody for {alpha}-smooth muscle actin (Dako), which was previously confirmed to react with the quail VSMC (Sadi et al., 1994), was used in this study. To reduce the nonspecific background staining, endogenous peroxidase activity was blocked with 3% hydrogen peroxidase. The sections were reacted with primary antibodies and were then reacted with labeled dextran polymer. The sections were developed with activated 3,3'-diaminobenzidine-tetrahydrochloride, followed by counterstaining with hematoxylin. Washing was performed with Tris-buffered saline 3 times after each step. The sections were then studied by light microscopy.


Figure 1
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  		Figure 1. Histological findings in the aortic lesions from the control diet (CD) and atherogenic diet (AD) groups (elastica van Gieson stain). No intimal thickening in the ascending aortas was seen in the CD group at 12 wk (panel A). A lipid-containing intimal lesion (panel C) and atheromatous lesion (panel E) in the aortas were seen in the AD group at 8 and 12 wk, respectively. Panels B, D, and F show a higher magnification of panels A, C, and E, respectively. Note the lamellar structure of elastic fibers in the aortic tunica media (M). I = intimal lesion thickness; M = medial wall thickness. A star indicates the thickened intimal lesion.

 
Total RNA Extraction and cDNA Synthesis
The rest of the aortas stored at –80°C were powdered in liquid nitrogen using an SK mill manufactured to process small sample specimens (Funakoshi, Tokyo, Japan). Total RNA was extracted from each powdered aorta with Isogen (Nippon Gene Co., Ltd., Tokyo, Japan) according to the manufacturer’s protocol. To remove genomic DNA, extracted total RNA was treated with DNase I (RNase free, Takara Bio Inc., Shiga, Japan), followed by a phenol-chloroform treatment and ethanol precipitation to remove the DNase I. The first-strand cDNA was synthesized in a 20-µL reaction mixture containing 5 µg of total RNA, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 5 mM MgCl2, 0.5 mM each deoxy nucleotide 5'-triphosphate, 2.5 µM oligo(dT)20, 10 mM dithiothreitol, 40 units of RNaseOUT, and 200 units of Superscript III reverse transcription (In-vitrogen, Carlsbad, CA), according to the manufacturer’s protocol. After first-strand cDNA synthesis, cDNA was treated with 2 units of RNase H to remove the RNA.

mRNA Expression Analysis
Semiquantitative PCR was used for the analysis of mRNA expression in this study. The PCR primers were designed based on genetic information on chickens or quail from previous reports: glyceraldehyde-3-phosphate dehydrogenase (Weiskirchen et al., 1993), c-jun (Brun et al., 1989), c-myc (Watson et al., 1983), c-src (Yatsula et al., 1994), and the online National Center for Biotechnology Information nucleotide database (http://www.ncbi.nlm.-nih.gov/entrez/query.fcgi?db=Nucleotide; Table 2Go).


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  		Table 2. Sequences of the semiquantitative reverse-transcription PCR primers
 
The DNA sequence of the PCR products was determined. Semiquantitative PCR consisted of 25 cycles of amplification with 95°C denaturation for 30 s, 55°C annealing for 30 s, and 72°C extension for 30 s using TaKaRa Ex Taq Hot Start Version (Takara Bio Inc.). The PCR products were electrophoresed in 3% agarose gel containing ethidium bromide and were quantified fluorometrically by Scion Imaging software (Scion Corporation, Frederick, MD) as described elsewhere (Perl et al., 2003). To measure the cDNA concentrations, a standard curve was constructed by performing PCR with serially diluted known concentrations of template DNA, and by plotting the concentrations of template DNA vs. fluorescence intensities of the amplified DNA. The fluorescence intensities of the amplicon were converted into concentrations of cDNA based on this standard curve. The experiments were performed in triplicate. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control to normalize the PCR for the amount of template cDNA.

Statistical Analysis
The Mann-Whitney U-test was performed for statistical analysis to compare the liver weights and serum lipid profiles between the CD and AD groups. Time-dependent changes in mRNA expression within the same dietary group were evaluated by the Kruskal-Wallis test. The Spearman rank correlation test was also performed to evaluate the relationships between the ratio of the level of mRNA expression of each gene and the degree of atherosclerotic lesions (I:M ratio) from all quail in the AD groups. All statistical analyses were performed using the SAS statistical software program (SAS Institute, Inc., Tokyo, Japan).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Body and Liver Weights, and Serum Lipid Profile in Each Experimental Group
No significant difference in BW was noted between the CD and AD groups. The liver weights of the AD group were significantly heavier than those of the CD group at 4, 8, and 12 wk of the experimental period. The serum total cholesterol levels in the AD group were also significantly higher than those in the CD group at 4, 8, and 12 wk in this study. No significant difference was seen in the serum triglyceride level between the CD and AD groups.

Histological and Immunohistochemical Studies
No significant intimal thickening was found in the aortas from the CD group (Figure 1A and 1BGo). Two types of aortic atherosclerotic lesions, lipid-containing intimal lesions (LL; Figure 1C and 1DGo) and atheromatous lesions (AL; Figure 1E and 1FGo), were seen in the AD group. The LL were mainly composed of round lipid-containing cells. The superficial and deeper layers of the AL contained round and spindle-shaped lipid-containing cells, respectively (Figure 2A and 2BGo). Newly formed slender elastic fibers were seen only in the deeper layer of the advanced AL of the AD group (Figure 2BGo). The tunica media (M) consisted of smooth muscle cells and fibroblasts. No lipid deposition was seen, and the lamellar structure of elastic fibers was preserved in the aortic M of the CD group. The antibody for {alpha}-smooth muscle actin labeled the smooth muscle cells of the aortic M, but not the thickened intimal cells such as lipid-containing cells (Figure 3A and 3BGo). Figure 4Go showed the degree of atherosclerotic lesions in the AD group. No significant aortic intimal thickening was seen in any of the birds of the CD group throughout the entire experimental period, nor was aortic intimal thickening seen in birds of the AD group at 2 and 4 wk of the study. Slight aortic intimal thickening lesions were found in some quail (6 of 10 quail) of the AD group at 8 wk of the experimental period. Those intimal thickening lesions were LL, and the total I:M ratio derived from all birds in the AD group was 0.07. Most quail (8 of 10 quail) had aortic intimal thickening lesions such as LL and AL at 12 wk of the experimental period. The total I:M ratio derived from all birds in the AD group was 0.41 at 12 wk.


Figure 2
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  		Figure 2. Lipid-containing intimal lesion (LL; panel A) and atheromatous lesion (AL; panel B) lesions in the atherogenic diet (AD) group (elastica van Gieson stain). Aortic intimal lesions LL in panel A and AL in panel B were seen in the AD group at 8 and 12 wk, respectively. Solid arrowhead: round lipid-containing cells; open arrow head: spindle-shaped lipid-containing cells. EL = elastosis.

 

Figure 3
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  		Figure 3. Immunohistochemical findings in the aortic lesions from the atherogenic diet group. Anti-{alpha}-smooth muscle actin antibodies were reacted with smooth muscle cells of the aortic tunica media, but not with intimal cells of thickened intima in lipid-containing intimal lesion (panel A) and atheromatous lesion (panel B) at 8 and 12 wk, respectively.

 

Figure 4
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  		Figure 4. The chronological change of the degree of aortic atherosclerosis (intimal thickness to medial thickness ratio, I:M ratio) in the atherogenic diet group. The vertical bars and error bars indicate mean ± SE for 10, 9, 10, and 10 quail at 2, 4, 8, and 12 wk.

 
mRNA Expression in the Cell-Cycle-Regulating Genes
All products showed a high homology of more than 95% (Table 2Go). Throughout the entire experimental period, the mRNA expression levels of all examined genes showed few alterations in the CD group (Figure 5AGo), whereas all genes except for c-jun showed a significant alteration in the AD group (Figure 5BGo). The levels of c-fos mRNA expression at 4, 8, and 12 wk of the study were significantly higher than that of birds at 2 wk, and it reached its highest level at 8 wk of the study. c-Jun mRNA expression levels showed no significant difference between the 2 experimental groups. c-Myc mRNA expression was observed to increase chronologically throughout the entire experimental period. c-Myc mRNA expression levels at 8 and 12 wk were significantly higher than that at 2 wk. Messenger RNA expression of the c-src gene at 8 wk of the study was significantly increased compared with its expression at 2 and 4 wk. c-Src gene mRNA expression peaked at 8 wk and then declined up to 12 wk. Expression levels of PCNA mRNA at 4, 8, and 12 wk of the study were significantly higher than at 2 wk of the study. Messenger RNA expression levels of the p27 gene at 8 and 12 wk of the study were significantly higher than those at 2 and 4 wk.


Figure 5
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  		Figure 5. Chronological alterations of mRNA expression of the examined genes in each experimental group. The vertical bars and error bars indicate mean ± SE, which were obtained by calculating the average of all quail samples. Expression of mRNA at 2 wk was set at 1. The asterisk (*) indicates a statistically significant difference (*P < 0.05; **P < 0.01).

 
Correlation Between the Degree of Atherosclerosis (I:M Ratio) and Expression of the Cell-Cycle-Regulating Genes
Spearman’s rank-correlation coefficient (R) and the significance probability (P) between the I:M ratio and each mRNA expression level from all quail in the AD group are shown in Table 3Go. The level of expression of each gene examined in this study had no correlation with the I:M ratio at 8 wk of the experimental period. The levels of c-fos, c-jun, and c-src mRNA expression showed no correlation with the I:M ratio. A positive correlation was detected between the I:M ratio and mRNA expression of c-myc (P = 0.018), PCNA (P = 0.008), and p27 (P = 0.043) at 12 wk in the study.


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  		Table 3. Correlations between mRNA expression and the degree of atherosclerosis in Japanese quail1
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study showed that the c-fos, c-jun, c-myc, c-src, PCNA, and p27 genes were expressed in atherosclerotic lesions in the Japanese quail. Messenger RNA expression levels of each examined gene in the AD group changed appreciably, but those in the CD group were hardly changed (Figure 5Go). Therefore, aortic mRNA expression of all examined genes did not appear to be affected by aging in the present study. In our previous electron microscopy and immunohistochemical study of quail atherosclerotic lesions (Sadi et al., 1994), we found that the superficial and deeper layers of AL contained round lipid-containing cells of macrophage (M{phi}) origin and spindle-shaped cells of fibroblast origin, respectively (Figure 2Go). Likewise, LL and AL were found to consist chiefly of M{phi} and fibroblasts, respectively, in this study. Several studies have revealed that the c-fos, c-jun, and c-myc protooncogenes show early mRNA expression in response to various stimuli (Lau and Nathans, 1987). In rat proliferating VSMC after vascular balloon injury, c-fos and c-jun mRNA levels increase immediately, followed by a decline in those mRNA levels and an increase in the c-myc mRNA levels (Miano et al., 1990). A large body of evidence has indicated that the c-myc gene is also responsible for VSMC proliferation in atherosclerotic lesions of animals and humans (Parkes et al., 1991) as well as apoptosis of VSMC (Macdonald and Bennett, 1999). Simons et al. (1994) showed that PCNA is required in VSMC proliferation. In addition, Lavezzi et al. (2003, 2005) suggested that PCNA expression is preceded by c-fos activation and stimulate VSMC division in human atherosclerotic lesions. In this study, mRNA expression of c-jun did not change among the experimental data, whereas expression levels of c-fos, c-myc, and PCNA were elevated after 2 wk and peaked at 8, 12, and 12 wk, respectively. Messenger RNA expression levels of c-myc and PCNA were positively correlated with the degree of aortic lesions at 12 wk in this study. Therefore, we suggest that alterations of c-fos, c-myc, and PCNA mRNA expression are involved in aortic fibroblast proliferation in the development of atherosclerotic lesions in the Japanese quail. The present results suggest that the proliferation of fibroblasts stimulated by c-fos was followed by their proliferation stimulated by c-myc and PCNA. Watanabe et al. (2005) reported that the src signal pathway was associated with the formation of M{phi}-derived foam cells induced by urotensin II. Thrombin induced the proliferation of VSMC and the uptake of glucose (which is metabolic energy for cell proliferation) in VSMC via the src signal pathway (Kanda and Watanabe, 2005). Cho et al. (2005) also indicated that the src signal pathway is involved in the proliferation of VSMC induced by glycated low-density lipoproteins. Our study showed that c-src mRNA expression significantly increased only at 8 wk. We speculate that c-src mRNA expression is involved in the foam cell formation of M{phi} or the preparation of fibroblast proliferation or both. We plan to examine whether c-src is activated in the development of quail atherosclerotic lesions, because several authors (Ishizawar and Parsons, 2004; Roskoski, 2005) have reported that protein phosphorylation plays a key regulatory role in src activation. Díez-Juan and Andres (2001) reported that global p27 inactivation induced the proliferation of VSMC and M{phi} and accelerated diet-induced atherosclerosis in apolipoprotein E-null mice. Ihling et al. (1999) suggested that the transforming growth factor-ß1 growth-suppressive function may be mediated by p27 blocking the activity of cyclin E/cyclin-dependent kinase 2 complexes. In addition, they suggested that p27 may play an important role in the processes associated with chronic inflammation and cell turnover in advanced human atherosclerotic plaque. Sato et al. (2000) reported VSMC proliferation to be inhibited via the up-regulation of p27, but not by the apoptotic pathway. The amount of p27 in the cell is regulated primarily at the level of translation and protein turnover (Sanz-Gonzalez et al., 2006). The present study showed that p27 mRNA expression significantly increased in advanced atherosclerotic lesions. Accordingly, we speculated that p27 mRNA expression increased reactively against c-myc and PCNA activation. It is also possible that the translation, degradation, or both of p27 protein may deteriorate in the proliferating fibroblasts on the development of atherosclerotic lesions in the quail. Finally, further study is required to examine which type of intimal cells, such as M{phi}, fibroblasts, or VSMC, are involved in the expression of various cell-cycle-regulating genes using specific antibodies to react with quail aortic VSMC and M{phi} to identify exact cell types in quail atherosclerotic lesions.

Received for publication October 17, 2006. Accepted for publication February 14, 2007.


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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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