© 1998 by European Society of Cardiology
Copyright © 1998, European Society of Cardiology
Enhanced expression of heparin-binding EGF-like growth factor and its receptor in hypertrophied left ventricle of spontaneously hypertensive rats
aFirst Department of Internal Medicine, Asahikawa Medical College, Nishikagura 4-5, Asahikawa, Hokkaido 078, Japan
bDepartment of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan
* Corresponding author. Tel.: +81 (166) 652111, ext. 2442; Fax: +81 (166) 659473.
Received 21 May 1997; accepted 29 December 1997
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Abstract |
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Objectives: Although heparin-binding epidermal growth factor-like growth factor (HB-EGF) is thought to produce hypertrophy in isolated cardiomyocytes via an autocrine mechanism, the pathophysiological role of HB-EGF, in myocardial hypertrophy in vivo, is not yet known. To investigate the involvement of HB-EGF in cardiac remodeling associated with hypertension in vivo, we assayed the expression of HB-EGF mRNA and protein in the left ventricle (LV) during the development of left ventricular hypertrophy in spontaneously hypertensive rats (SHR). Methods: Prior to sacrifice and assay of HB-EGF and EGF-receptor (EGF-R) mRNA, morphologic and hemodynamic variables were measured in SHR and in age-matched Wistar Kyoto rats (WKY). At 5, 9 and 12 weeks of age, rats were killed, their hearts were removed, and the expression of HB-EGF and EGF-R mRNA and protein were measured. In addition, SHR and WKY were treated with enalapril, atenolol, or both for 4 weeks. Results: In untreated SHR, double products (i.e. systolic blood pressure (sBP) multiplied by heart rate (HR)), an index of mechanical load, peaked at 9 weeks. Expression of HB-EGF mRNA was also observed to peak in these animals at 9 weeks, while expression of EGF-R mRNA increased from 5 to 9 weeks, but remained constant thereafter. In untreated WKY, double products and EGF-R mRNA expression did not change over time, whereas the level of HB-EGF message increased gradually. Antibody to HB-EGF reacted primarily with myocyte membranes in SHR, whereas antibody to EGF-R reacted mainly with interstitial cells in these animals. The angiotensin-converting enzyme inhibitor, enalapril, markedly decreased sBP in SHR, whereas the β1-adrenoreceptor antagonist, atenolol, significantly decreased HR. While neither alone affected the expression of HB-EGF mRNA, their combination significantly reduced the expression of HB-EGF mRNA, as well as double products, in these rats, but had no effect on expression of EGF-R mRNA. Conclusions: The enhanced expression of HB-EGF mRNA and protein in LV of SHR suggest that this growth factor may play an important role during the early development of LV hypertrophy and cardiac fibrosis in SHR. The association between double products and HB-EGF expression suggest that the latter may be induced by increased mechanical load and may contribute, in turn, to cardiac remodeling.
KEYWORDS Growth factor; Spontaneously hypertensive rat; Cardiac hypertrophy; Mechanical load
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1 Introduction |
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In patients with systemic hypertension, left ventricular hypertrophy (LVH) begins as an adaptive response to normalize the increased stress on the wall of ventricle. Subsequently, however, LVH leads to an increase in oxygen consumption, an impairment of coronary reserve, and a reduction in myocardial contractility, and eventually to heart failure. LVH is now known to be an independent risk factor for cardiovascular events [1].
The spontaneously hypertensive rat (SHR), which develops hypertension and LVH spontaneously in its life time, has been utilized as an animal model of human primary hypertension. While the mechanism of development of hypertension and LVH in SHR is not fully understood, several factors have been suggested to play a role in the induction of LVH. These include mechanical stress, alteration in the sympathetic nervous system, and activation of the renin–angiotensin system [2–4].
Several growth factors are reported to contribute to cardiac hypertrophy, including TGF-β, the FGFs [5], IGF-1 [6]. Levels of message encoding heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the EGF family [7], were recently shown to be markedly increased in isolated neonatal and adult myocytes by 
-adrenergic or continual pacing stimuli, and it was suggested that HB-EGF may be associated with an increase in total protein content in myocytes [8]. Unlike insulin and IGFs, however, EGF and other growth factors were found to be limited in their ability to induce hypertrophy in cultured adult rabbit heart cells [9]. Thus, it is unclear if HB-EGF and its signal transduction system participate in the process of myocyte hypertrophy, or to cardiac hypertrophy in vivo.
HB-EGF was originally reported to function as a mitogen for fibroblasts and vascular smooth muscle cells, and it has been shown to exert its activity through the EGF-receptor [10]. In inducing vascular smooth muscle cell proliferation, HB-EGF was observed to be more potent than EGF or TGF-, but to have the same potency as PDGF [10]. This enhanced activity may be due to the cell-surface heparan sulfate proteoglycans, which have been found to facilitate HB-EGF binding to the EGF-receptor [10]. HB-EGF is expressed by fetal human vascular smooth muscle cells [11, 12], which synthesize and secrete the active mitogen.
Myocytes share a number of phenotypic characteristics with vascular smooth muscle cells, including similar functional responses to neurohumoral agonists and peptide autacoids, such as the endothelins [13]and angiotensin II [14], and similar growth response to many peptide mitogens, including basic fibroblast growth factor [5]. Compared to aortic smooth muscle cells (SMC) from WKY, SMC from SHR were found to display an increased growth response to EGF [15], suggesting that the HB-EGF signal transduction system may play a significant role in the development of LVH in SHR.
In addition to its ability to induce myocyte hypertrophy, EGF has been shown to trigger proliferation of mesenchymal cells in chicken hearts by a mechanism similar to that of insulin [16]. HB-EGF has also been observed to function as a juxtacrine growth factor, promoting the growth of one cell while still tethered to another [17]. These findings suggest that HB-EGF may be important for the proliferation of mesenchymal cells, and may promote the development of cardiac fibrosis in SHR by a paracrine or juxtacrine mechanism.
In order to test these hypothesis, we assayed the levels of expression of HB-EGF and EGF-receptor mRNA and protein in the left ventricle during the development of LVH in SHR. In addition, to investigate the mechanisms that regulate the expression of HB-EGF and its receptor in LVH of SHR, we tested the effects of an angiotensin-converting enzyme inhibitor and of a β1-adrenoreceptor antagonist, which modulate the development of LVH in SHR [4, 18], on the expression of HB-EGF and EGF-receptor mRNA in these rats.
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2 Methods |
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2.1 Animals
Male Wistar Kyoto rat (WKY)/Izm and SHR/Izm were from inbred original strains established at Kyoto University and maintained at Shimane Medical University under strict genetic control. They were fed standard laboratory chow (Funabashi SP diet) and given tap water ad libitum, from 4 weeks of age in our laboratory.
2.2 Protocols
Beginning at 4 weeks of age, systolic blood pressure was measured in each rat at weekly intervals by the tail plethysmography method (TK-370C: UNICOM, Chiba, JAPAN). At 5, 9, 12 weeks of age, six animals in each group were killed by decapitation. Their hearts were rapidly removed, the atria and great vessels were trimmed away, and the remainder was rinsed in cold saline. Each heart was divided into the left ventricular free wall and the septal wall and right ventricle. Each portion was weighed and divided into two pieces. One portion, for RNA preparation, was rapidly frozen in liquid nitrogen and stored at –80°C. The second portion, for immunohistochemistry, was divided into small pieces, embedded into O.C.T compound, frozen in liquid nitrogen, and stored at –80°C.
In a second experiment, twenty-four 4-week-old rats in each strain were divided into four groups of six rats each. Group 1 (the control group) was given vehicle alone (saline); group 2 was given 10 mg/kg/day enalapril (Sigma Chemical, St. Louis, MO); group 3 was given 100 mg/kg/day atenolol (Sigma); and group 4 was given both enalapril and atenolol, at the same dose as above. Reagents were administered by gastric tube once a day, in the morning, for 5 weeks. Systolic blood pressure was measured weekly 5 h after the administration of reagents. At 9 weeks of age, the rats were decapitated and the LV (free wall and septum) and RV were immediately excised, and the tissue samples treated as described above. The preliminary experiments with enalapril (5, 10, 20 mg/kg/day) showed that 10 mg/kg/day of enalapril from 4 to 9 weeks of age decrease sBP in SHR to a level similar to that in WKY, the preliminary experiments with atenolol (50, 100, 200 mg/kg/day) showed that the 100 mg/kg/day of atenolol had a similar effect on heart rate.
All procedures were performed in accordance with the guidelines for animal research of the Asahikawa Medical College.
2.3 RNA extraction, Northern blotting and hybridization
Ten-µg aliquots of poly(A)+ RNA, prepared as described [19], were electrophoresed through 1.0% agarose–formaldehyde gels, transferred to nitrocellulose filters (BA-S85, Schleicher and Schuell), and prehybridized for 12 h at 42°C with 50% formamide, 5xSSC, 5xDenhardt's solution, 25 mM sodium phosphate (pH 6.5), 0.1% SDS, 0.25 mg/ml denatured salmon sperm DNA. The filters were hybridized at 42°C overnight in the same solution, also containing a cDNA probe labeled with [32P]dCTP by random priming, and then washed under increasingly stringent conditions. Hybridization was visualized by autoradiography, and the radioactivity quantified with a bioimage analyzer and its accompanying software (BAStation: Fuji Film, Tokyo, Japan). For each blot, hybridization signal of a β-actin probe was used to correct for differences in loading and/or transfer.
2.4 cDNA probe
The probes utilized for Northern blot analysis included: HB-EGF, a 1.6-kb EcoRI–PstI rat cDNA fragment encompassing the entire coding region of HB-EGF [20], the kind gift from Dr. Judith A. Abraham; EGF-receptor, a 4.2-kb XhoI–SacII human cDNA fragment [21], the gift of the Riken Gene Bank; and β-actin, a 0.8-kb EcoRI canine cDNA fragment, the gift of Dr. Yoshihiro Ishikawa.
2.5 HB-EGF and EGF-receptor immunostaining
Rabbit antiserum to HB-EGF was produced using the synthetic peptide, H-1, consisting of HB-EGF precursor residues 185–208 [22]. A mouse monoclonal antibody to human EGF receptor, produced using trypsinized A431 cells, was shown to cross-react with rat EGF receptor (Transformation Research, Framingham, MA, USA). Mouse monoclonal antibodies to desmin and vimentin were purchased from Nichirei, Tokyo, Japan.
Immunohistochemistry was performed using the streptavidin–biotin complex method (Histofine, Nichirei, Tokyo, Japan). Frozen sections (about 5 µm thick) were fixed in acetone at room temperature for 10 min, washed in phosphate-buffered saline (PBS), and incubated with 10% normal goat serum for 10 min at room temperature. Tissue sections were incubated overnight at 4°C with a 1:120 dilution (in PBS +1% bovine serum albumin) of rabbit anti-HB-EGF antiserum (H-1) or a 1:100 dilution (in the same buffer) of mouse anti-EGF-receptor monoclonal antibody, or with the appropriate dilution of mouse anti-desmin and anti-vimentin monoclonal antibodies. After washing in PBS, the sections were incubated for 10 min with a 1:200 dilution of biotinylated goat anti-rabbit IgG or goat anti-mouse IgG and then with the labeled streptavidin–biotin immunoperoxidase reagent (Nichirei). Antigen–antibody complexes were visualized with 3,3'-diaminobenzidine tetrahydrochloride (Nichirei).
As a negative control, the primary antibody was replaced by normal rabbit or mouse serum (Nichirei).
2.6 Statistical analysis
Data are reported as mean±s.e.m., and were analyzed by the unpaired Student's t-test. A level of P<0.05 was considered statistically significant.
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3 Result |
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3.1 Morphologic and hemodynamic changes in untreated SHR and WKY
In both strains of rats, we observed an increase in systolic blood pressure as a function of age; the increase, however, was greater in SHR (P<0.05) (Table 1). In contrast, heart rate, which was significantly higher in SHR than in WKY at 5 and 9 weeks of age (P<0.05), was the same in both strains at 12 weeks of age (Table 1). The LV/BW ratio, an index of left ventricular hypertrophy, was also significantly higher in SHR than in age-matched WKY at all time points tested (P<0.05) (Table 1). All of these data are in good agreement with previous reports [23–25]. When we calculated double products (i.e. systolic blood pressure multiplied by heart rate), which is an index of mechanical load, we noted that this parameter was significantly higher in SHR than in WKY at all time points (P<0.01 vs. age-matched WKY at 5, 9, 12 weeks) (Fig. 1A). In SHR, double products peaked at 9 weeks of age, and were significantly higher than at previous or subsequent time points (P<0.01 vs. 5-week SHR, P<0.05 vs. 12-week SHR) (Fig. 1A). In contrast, the changes in double products over time were minor in WKY (Fig. 1A).
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3.2 Expression of HB-EGF and EGF-receptor mRNA in untreated SHR and WKY
While a single 2.37-kb HB-EGF transcript was detected in the left ventricle (LV) of SHR and WKY at all time points tested (Fig. 2A), the pattern of expression was clearly different in the two strains (Fig. 2B). In WKY, the expression of HB-EGF mRNA gradually increased overtime, whereas, in SHR, there was a burst of expression at 9 weeks of age, with a decline thereafter (Fig. 2B). Expression of EGF-receptor mRNA was clearly detected as a marked 9.5-kb band in LV of SHR (Fig. 3A), increasing from 5 to 9 weeks of age, but not changing thereafter (Fig. 3B). In contrast, expression of EGF-receptor mRNA was only faintly detected in WKY at all ages examined, and did not change with age (Fig. 3B).
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P<0.01 vs. age-matched WKY.
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3.3 Immunohistochemical staining for HB-EGF and EGF receptor
To elucidate the localization and the changes in protein level of HB-EGF and EGF-receptor, we immunohistochemically assayed rat heart tissue sections with antibodies to these two proteins. In addition, to discriminate myocytes from interstitial cells, we also assayed these samples with antibodies to desmin and vimentin. Antiserum to HB-EGF reacted primarily with myocyte membranes in 9-week-old SHR (Fig. 4A); this antiserum also reacted weakly with vascular smooth muscle in the coronary arteries of these rats (data not shown). The pattern of immunostaining of HB-EGF in myocyte membranes was similar to that of desmin, a cytoskeletal protein specific for myocytes in mature hearts (Fig. 4C). In contrast, EGF-receptor protein was localized mainly in interstitial cells and faintly in myocyte membranes (Fig. 5A). The pattern of staining of EGF-receptor was similar to that of vimentin, a protein present mainly in myocardial fibroblasts in mature hearts (Fig. 5C). Immunohistochemical staining for both HB-EGF and EGF receptor was more pronounced in left ventricular tissue of 9-week-old SHR than in similarly aged WKY (Fig. 4A,B, and Fig. 5A,B), whereas the negative controls (normal rabbit and mouse serum) did not react with any rat tissue sample (Fig. 4D and Fig. 5D).
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3.4 Morphologic and hemodynamic changes in treated SHR and WKY
Beginning at 4 weeks of age, SHR were treated for 5 weeks with enalapril, atenolol, or both. When compared with rats treated with vehicle alone, enalapril and enalapril+atenolol significantly reduced the systolic blood pressure (P<0.01 vs. vehicle SHR), whereas atenolol was not effective (Table 2). In contrast, the heart rate was suppressed by atenolol and enalapril+atenolol (P<0.01 vs. vehicle SHR), but not by enalapril alone (Table 2). While the body weight was similar in these groups of rats, enalapril+atenolol decreased LV/BW more than either alone (P<0.01 vs. vehicle SHR) (Table 2). When we calculated double products in these treated rats, we observed that this parameter was moderately reduced by either enalapril or atenolol, but more markedly by their combination (9-week vehicle WKY, 474±24x102; 9-week vehicle SHR, 931±44x102; 9-week enalapril SHR, 627±37x102; 9-week atenolol SHR, 646±10x102; 9-week enalapril+atenolol SHR, 468±13x102) (Fig. 1B). In particular, treatment with both drugs was more effective in reducing the mechanical load and LVH than treatment with either drug alone.
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3.5 Effects of drug treatment on expression of HB-EGF and EGF- receptor mRNA
The overexpression of HB-EGF mRNA, previously observed in 9-week-old SHR (Fig. 2), was suppressed slightly by treatment with either enalapril or atenolol, and moderately by both reagents together (Fig. 6). Although enalapril+atenolol reduced the expression of HB-EGF mRNA to 79% of that seen with vehicle-treated SHR (P<0.05), this level was still twice that seen in WKY (P<0.01) (Fig. 6A,B). We observed no significant change in the expression of EGF-receptor mRNA in rats treated with atenolol, enalapril, or both (data not shown).
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4 Discussion |
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Our finding, that the expression of HB-EGF mRNA and protein are enhanced in the left ventricle of SHR, suggests that this growth factor may play an important role during the early development of LVH and cardiac fibrosis in SHR.
Indeed, we showed that the overexpression of HB-EGF mRNA was transient, peaking at 9 weeks of age and then declining. This transient enhancement in the early stage of cardiac hypertrophy may indicate that HB-EGF is especially important for the initiation of the hypertrophic process, but less important for the maintenance of hypertrophy in SHR. Although it is still not fully understood what kind of factors promote cardiac hypertrophy in SHR, we speculate HB-EGF is a possible candidate for one of those factors.
The discrepancy between the transient enhancement of HB-EGF mRNA expression and the continual enhancement of EGF-receptor mRNA expression in SHR remains to be clarified. It has been reported that EGF itself regulates the expression of EGF-receptor mRNA [26], suggesting that the enhanced expression of HB-EGF mRNA in the early stages of LVH induces increased expression of EGF-receptor mRNA. Since the induction of proliferation by EGF has been found to be proportional to EGF-receptor density in SMC of SHR [15], it raises the possibility that the expression of EGF-receptor in SHR is regulated by a genetic factor. Other members of the EGF subfamily, such as EGF, TGF-, amphiregulin, and betacellulin, which also activate the EGF-receptor, may also play a role in the maintenance of LVH in SHR by continually stimulating the enhanced expression of the EGF-receptor.
It is also possible that the increased steady-state levels of HB-EGF and EGF-receptor mRNA that we observed in SHR were due to decreased degradation of these transcripts rather than increased synthesis. In primary cultures of rat neonatal and adult cardiac myocytes, however, increased levels of HB-EGF mRNA induced by the -adrenoreceptor agonist, phenylephrine, were shown to be caused by increased gene transcription, not by altered stability of this message [8]. In addition, our immunohistochemical experiments demonstrated that the increased levels of HB-EGF and EGF-receptor transcripts in SHR were mirrored by increased levels of their respective proteins. We believe that the enhanced expression of HB-EGF and its receptor mRNA leads to the increases in their protein level.
It is noteworthy that the peak of HB-EGF mRNA expression coincided with the peak of double products (sBPxHR), a hemodynamic indicator of mechanical stress, in SHR. In addition, treatment of these rats with both atenolol and enalapril reduced double products more effectively than treatment with either alone, and only treatment with both drugs significantly reduced the expression of HB-EGF mRNA. These intriguing observations suggest that mechanical stress may be associated with the enhanced expression of HB-EGF mRNA. This is supported by our experiments in which SHR were treated with hydralazine. This agent reduced sBP as markedly as did enalapril, and affected HB-EGF mRNA expression to the same degree as either enalapril or atenolol. When administered together with atenolol, however, hydralazine significantly reduced the double products and suppressed the expression of HB-EGF mRNA (data not shown).
Increased mechanical stress has been shown to stimulate protein synthesis and to induce the expression of several fetal and immediate early genes in isolated perfused hearts [27]as well as in isolated cardiomyocytes [28, 29]. Opening of the stretch-activated cation channel is one proposed mechanism by which stress-protein synthesis is activated [30]. Interestingly, EGF has been reported to affect the cardiac contractile frequency that operates through the voltage-dependent calcium channels and the stimulation of Na+/H+ and Na+/Ca2+ exchange [31]. In addition, overexpression of HB-EGF mRNA has been found to be induced by chronotrophic stimuli activated by electric pacing in isolated cultured myocytes [8], and by shear stress in human vascular endothelial cells [32]. These findings, along with the results reported here, suggest a possible link between increased myocardial stress and enhanced expression of HB-EGF mRNA, leading to a hypertrophic response in the myocardium, at least in the early phases of hypertension and cardiac hypertrophy in SHR.
Since both enalapril, an angiotensin-converting enzyme inhibitor, and atenolol, a β1-adrenoreceptor antagonist, reduced LV/BW ratios, but neither drug alone significantly affected the expression of HB-EGF mRNA, it is unlikely that the enhanced expression of HB-EGF message observed in untreated SHR is the result of cardiac hypertrophy. Although we could not exclude the role of the renin–angiotensin system, which is modulated by enalapril, or the sympathetic signal transduction system, which is modulated by atenolol, in the enhancement of HB-EGF mRNA expression, we believe it more likely that HB-EGF expression is independent of these humoral or neural mechanisms, and is related more to mechanical stress.
Another possible role of HB-EGF in LVH of SHR may be to promote the proliferation of interstitial cells, especially the fibroblasts. An increased synthesis of collagen, as well as a subsequent increase in total collagen, have been observed at 4–8 weeks of age, and later at 24 weeks in SHR [33]. It has been suggested that the expression of collagen gene isoforms, as well as the expression of ANP and skeletal -actin mRNA, are enhanced in 9-week-old SHR compared to WKY [34], and that a direct interaction between HB-EGF and adhesion molecules may regulate cell proliferation in the cell-to-cell adhesion process [35]. We detected HB-EGF primarily in myocyte membranes, whereas EGF receptor was observed mainly in interstitial area, primarily in the fibroblasts. These results suggest that HB-EGF may regulate fibroblast proliferation in the hypertrophied hearts of SHR by a paracrine or juxtacrine mechanism.
In summary, the results of the present investigation demonstrate, for the first time, an enhanced expression of HB-EGF and its receptor, particularly during early development of LVH in SHR. The mechanism seems to be operated, at least in part, through the enhanced mechanical stress during this particular stage of SHR, and might, in turn, contribute to the remodeling of hypertensive hypertrophied heart.
Time for primary review 28 days.
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Acknowledgements |
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The authors are grateful to Dr. M. Ono, the Third Department of Internal Medicine, Asahikawa Medical College, for meaningful advice; to Dr. Judith A. Abraham, Scios Nova, Mountain View, CA for rat HB-EGF cDNA; to Riken Gene Bank, Ibaragi, Japan, for human EGF-receptor cDNA; and to Dr. Yoshihiro Ishikawa, Harvard University, Cambridge, MA, for canine β-actin cDNA.
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