Expression of Heparin-Binding Epidermal Growth Factor–Like Growth Factor in Neointimal Cells Induced by Balloon Injury in Rat Carotid Arteries
Balloon catheter injury of rat carotid arteries induces migration and proliferation of smooth muscle cells (SMCs), with subsequent neointimal formation. Several growth factors, such as platelet-derived growth factor and basic fibroblast growth factor, have been shown to be involved in this process, but the mechanisms that modulate the growth and/or migratory properties of SMCs remain unclear. In this study, we investigated whether heparin-binding epidermal growth factor–like growth factor (HB-EGF), which is known to be a potent SMC stimulator from in vitro study, is associated with the proliferative response of SMCs to arterial injury. Northern blot analysis showed that the transcript levels of HB-EGF increased rapidly approximately 12-fold within 2 hours after injury and declined by 2 days but remained 3-fold at 14 days. In situ hybridization analysis demonstrated that the transcript of HB-EGF remained strongly expressed in the neointima, especially near the luminal surface, at 14 days after injury. Immunohistochemical staining showed that HB-EGF protein was positive in the endothelium and only faintly visible in medial SMCs in uninjured vessels. In contrast, 2 days after injury, positive HB-EGF immunostaining was detected in the medial SMCs along the luminal surface. At 7 days, the neointimal SMCs exhibited strong immunostaining for HB-EGF, and at 14 days, they exhibited a gradient of HB-EGF expression with strong immunoreactivity in the most luminal cells. SMCs labeled with 5-bromo-2′-deoxyuridine in their nuclei showed strong immunostaining for HB-EGF protein. Furthermore, the epidermal growth factor receptor to which HB-EGF can bind was also immunostained positively in neointimal SMCs. These data suggest that HB-EGF may play an important role of the proliferation and migration of SMCs in the process of neointimal accumulation induced by arterial injury, probably in an autocrine, paracrine, and/or juxtacrine manner.
- heparin-binding epidermal growth factor
- epidermal growth factor receptor
- smooth muscle cells
- Received August 16, 1995.
- Revision received April 26, 1996.
The phenomenon of restenosis remains the major clinical problem following successful PTCA.1 2 Balloon catheter injury of rat common carotid arteries has been used as an experimental model of restenosis, because it induces the proliferation of medial SMCs, migration into the intima, and subsequent intimal thickening, which are consistent with histological features found in human restenotic lesions after PTCA.3 4
Previous studies showed that several endogenous growth factors synthesized by SMCs are thought to play an important role in the initiation of the SMC migration and proliferation following arterial injury. Majesky et al5 showed that increased expression of both PDGF and its receptor could be demonstrated by Northern blot analysis, in situ hybridization, and immunohistochemical studies following balloon injury. Therefore, PDGF may contribute to neointimal formation after balloon injury via an autocrine pathway. Another growth factor is bFGF, which is thought to be localized in endothelial cells, SMCs, and extracellular matrix.6 7 8 Using Northern blot and Western blot analyses and immunohistochemical study, Olson et al9 demonstrated that no increase in bFGF expression occurred in injured carotid arteries. Thus, balloon injury may disrupt the vessel wall and release stored bFGF, which then may stimulate SMC proliferation. However, using in situ hybridization on en face preparations, Lindner and Reidy10 demonstrated successful detection of mRNA of both bFGF and its receptor in replicating intimal SMCs that had not been observed by analysis of arterial cross sections.
HB-EGF is a member of the EGF family that was originally identified as a 20- to 22-kD glycoprotein secreted by the macrophage-like cell line, U-937.11 12 HB-EGF is synthesized as a membrane-anchored precursor (proHB-EGF). Mature HB-EGF includes a heparin-binding domain in the N-terminal region and an EGF-like domain in the C-terminal region.11 12 13 HB-EGF binds to the EGF-R of human14 and bovine SMCs15 and stimulates its phosphorylation. However, the activity of this protein in the migration and proliferation of bovine SMCs is much higher than that of EGF, being comparable to that of PDGF. This suggests that interactions of the heparin-binding domain of HB-EGF with cell surface HSPG may modulate its bioactivity.15
HB-EGF is not only a potent mitogen for SMCs but is also transcribed by cultured rat and human SMCs and is released into conditioned medium by human SMCs.14 16 17 This implies that HB-EGF may regulate the growth of SMCs in an autocrine, paracrine, and/or juxtacrine manner in vitro. However, whether HB-EGF is involved in the process of atherogenesis and restenosis after PTCA is unknown. Using immunohistochemical techniques, we have recently demonstrated that a large amount of HB-EGF protein can be detected in SMCs and macrophages in human atherosclerotic plaques.18 In the present study, we examined the expression and production of HB-EGF in rat carotid arteries following balloon injury using Northern hybridization, in situ hybridization, and immunohistochemical methods.
Arterial Injury Model and Tissue Preparation
Wistar rats (350 g, 15 to 17 weeks old) (Clea) were anesthetized, and acute injury to the left common carotid artery was induced with a 2F balloon catheter (Baxter Healthcare Co) as described previously.19 All rats were anesthetized with sodium pentobarbital (50 mg/kg body weight IP), and the distal left common carotid and external carotid arteries were exposed through a midline wound in the neck. The catheter was passed three times with the balloon inflated with air to distend the common carotid arteries. The external carotid was ligated after removal of the catheter, and the wound was closed. The right carotid arteries were not damaged and served as controls.
For RNA isolation, animals were killed, and both the injured (left) and uninjured (right) common carotid arteries were retrieved and carefully stripped of periadventitial connective tissues at 2 hours and at 2, 7, and 14 days after injury. The endothelium of the right carotid was removed by gentle scraping of the luminal surface with a plastic cell scraper. The arteries were then snap-frozen in liquid nitrogen for subsequent RNA isolation.
For immunohistochemical study, the carotid arteries were fixed with 10% phosphate-buffered neutral formalin by perfusion at 120 mm Hg pressure via the left ventricle at 2, 4, 7, and 14 days after injury. Two hours before fixation, BrdU (50 mg/kg, Sigma) was administered intraperitoneally to three rats at each time point. Because the S phase of SMCs is estimated to last approximately 8 hours,20 21 a single dose 2 hours before killing should label SMCs that have undergone DNA synthesis within a 9-hour period before fixation. After 5 minutes of perfusion, the entire right and left arteries including the aortic arch were removed and then fixed for 4 hours by immersion in the same fixative at 4°C. The tissues were then washed twice with PBS. The central one third of the carotid arteries, in which the endothelial cell lining does not regenerate until 12 weeks after injury,19 was excised and dehydrated in ethanol series for paraffin embedding.
For in situ hybridization, the carotid arteries were fixed with 4% paraformaldehyde in 0.1 mol/L PB, pH 7.4, by perfusion, as described above. The tissues were removed then fixed for 4 hours by immersion in the same fixative at 4°C and then dehydrated in ethanol series. The tissues were embedded in paraffin.
RNA Isolation and Northern Hybridization
Total cellular RNA was extracted by acid guanidine thiocyanate extraction as described elsewhere.22 Twenty micrograms of total RNA was denatured and fractionated through 1% agarose-formaldehyde gel. After the transfer of RNA to nylon membranes (Hybond-N, Amersham) by a vacuum blotting system (VacuGene XL, Pharmacia Biotech AB), the RNA was cross-linked to the membranes using ultraviolet irradiation (XL-1000 UV cross-linker, Spectro Linker, Tomy Seiko Co). After prehybridization, the membranes were hybridized using cDNA probes labeled with [32P]dCTP by random primer extension (Amersham). Blots hybridized with labeled rat HB-EGF23 and 18S ribosome24 probes were exposed to XAR film (Kodak) for 14 days and 12 hours, respectively, at −70°C using intensifying screens. The intensity of hybridization signals on autoradiograms was determined by densitometry using a scanner (HP ScanJet IIcx scanner, Hewlett-Packard Co) with Adobe Photoshop software (Adobe Systems Inc) and Power Macintosh 8100/80 (Apple Computer Inc). Quantification of the autoradiogram from densitometry was performed using NIH Image software (Wayne Rasband, National Institutes of Health, Bethesda, Md).
Preparation of RNA Probe of HB-EGF
Digoxigenin-labeled single-strand RNA probes were prepared using a DIG RNA labeling kit (Boehringer Mannheim GmbH Biochemica) according to the manufacturer's instructions. For generation of rat HB-EGF probe, a 412-base fragment of rat cDNA (bases 1 to 412), which was obtained by digestion of a 1.8-kb fragment of rat HB-EGF with EcoRI and Sca I, was subcloned into Bluescript pKS(+) plasmid. This plasmid was either linearized with EcoRI and transcribed with T7 RNA polymerase to generate a 412-base-long antisense probe or linearized with BamHI and transcribed with T3 RNA polymerase to generate a sense probe.
In Situ Hybridization of HB-EGF
Hybridization was carried out as described previously,25 with some minor modifications. Sections were deparaffinized and hydrated. After washing in 0.1 mol/L PB twice for 5 minutes, these sections were incubated at 37°C for 30 minutes in 1× TE (0.1 mol/L Tris, pH 8.0, and 50 mmol/L EDTA, pH 8.0, containing 1 μg/mL proteinase K). They were fixed again with 4% paraformaldehyde in 0.1 mol/L PB for 20 minutes, washed with 0.1 mol/L PB for 5 minutes twice, and treated with 0.2 mol/L HCl to inactivate endogenous alkaline phosphatase. The sections were then washed twice with 0.1 mol/L PB for 5 minutes and acetylated with 0.25% acetic anhydride in 0.1 mol/L triethanolamine, pH 8.0, for 10 minutes. The hybridization solution contained 50% deionized formamide, 10% dextran sulfate, 1× Denhardt's solution, 4× SSC, 50 mmol/L dithiothreitol, 150 μg/mL of Escherichia coli tRNA, and approximately 0.5 mg/mL of RNA probe. Sections were covered with paraform sheet (American National Can) and hybridized at 50°C for 16 hours in a moist chamber. After hybridization, paraform sheets were removed, and slides were washed in 5× SSC at 50°C briefly and treated in 5× SSC at 50°C for 30 minutes. They were then incubated with 50% formamide in 2× SSC at 50°C for 30 minutes. After rinsing in 1× TES (10 mmol/L Tris-HCl, pH 7.6, 1 mmol/L EDTA, and 0.5 mol/L NaCl) at 37°C for 15 minutes, the slides were treated with RNase A (10 mg/mL 1× TES) at 37°C for 30 minutes and then rinsed again in 1× TES at 37°C for 15 minutes. They were incubated with 2× SSC for 15 minutes twice at 50°C, followed by 0.2× SSC in the same manner. Positive hybridization signal was detected immunohistochemically using a nucleic acid detection kit (Boehringer Mannheim) according the manufacturer's instructions. The slides were incubated with DIG buffer 1 (100 mmol/L Tris-HCl, pH 7.5, and 150 mmol/L NaCl) for 2 minutes and then 1.5% blocking reagent in DIG buffer 1 for 60 minutes at room temperature. Next, 100 μL/cm2 solution of diluted polyclonal sheep anti-digoxigenin Fab fragment in DIG buffer 1 was mounted on the sections and incubated for 30 minutes at room temperature. After immunoreaction, slides were washed twice with DIG buffer 1 for 15 minutes and equilibrated with DIG buffer 3 (100 mmol/L Tris-HCl, pH 9.5, 100 mmol/L NaCl, and 50 mmol/L MgCl2) for 3 minutes. Coloring solution containing nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate in DIG buffer 3 was mounted on the sections, which were incubated at room temperature. The color reaction was stopped with 10 mmol/L Tris-HCl, pH 8.0, and 1 mmol/L EDTA. The specificity of the signal was confirmed as follows: (1) hybridization with the sense mRNA, (2) RNase digestion before hybridization, and (3) use of neither an antisense RNA probe nor the anti-digoxigenin antibody. All three experiments described above showed no positive signals.
Preparation of Antibodies Against ProHB-EGF
The polyclonal antibodies against proHB-EGF were produced by immunizing female New Zealand White rabbits with the synthetic peptide H1, localized in the cytoplasmic tail (HB-EGF precursor residues 185 to 208), or H6 (HB-EGF precursor residues 54 to 73), localized in the extracellular domain, as described previously.18 Neither H1 nor H6 has any amino acid sequence homology with other members of the EGF family, such as EGF, TGF-α, betacellulin, or amphiregulin.17 Antibody H1 does not cross-react with mature HB-EGF purified from U-937 cell–conditioned medium by Western blotting and immunoprecipitates 35S-labeled proHB-EGF. Antibody H6 cross-reacts with mature HB-EGF.12 Neither antibody detects EGF, TGF-α, or amphiregulin by Western blotting (S.H. and N.T., unpublished data, 1996). In the present study, we used the antibodies H1 and H6, and the same staining pattern was observed with these antibodies.
For the immunohistostaining of HB-EGF, paraffin sections (about 4 μm thick) were deparaffinized, treated with 3% H2O2 solution for 10 minutes, and washed in 0.05 mol/L Tris-HCl buffer (pH 7.6) three times for 3 minutes each. After incubation with normal swine serum for 20 minutes at room temperature, the three-step immunoperoxidase procedure was performed with rabbit anti-HB-EGF antiserum (antibody H1 or H6) diluted 1:100 in Tris buffer containing 1% BSA for 20 minutes at room temperature, swine anti-rabbit immunoglobulins (DAKO) diluted 1: 200 in Tris buffer containing 1% BSA for 20 minutes at room temperature, and peroxidase-labeled rabbit anti-peroxidase immunoglobulin complexes (DAKO) for 20 minutes at room temperature. Tissue staining was visualized with 3-amino-9-ethylcarbazole (DAKO) in 0.1 mol/L acetate buffer (pH 5.2) in the presence of 0.3% H2O2. For a negative control, the primary antiserum (antibody H1 or H6) was replaced by normal rabbit serum (DAKO) or preabsorbed with an excess amount of the peptide antigen. To quantify the HB-EGF immunoreactive intensity in the carotid arteries, the sections were photographed, and images were generated from color photographs. Scanning was performed with a scanner (Hewlett-Packard Co) at 400 dots per inch, and image manipulation was performed using Adobe Photoshop software (Adobe Systems Inc) and Power Macintosh 8100/80 (Apple Computer Inc). To selectively measure HB-EGF immunoreactive image intensity, a red-brown color was selected preferentially from the color images, and then the images were converted to gray scale. Each vessel was divided into four quadrants, and the gray scale density was obtained for the randomly chosen area within the first one third of the neointimal layers of the luminal side and within one third of the neointima of the internal side close to the IEL. Quantification of gray scale densities was performed using NIH Image software, and then the relative value of HB-EGF immunoreactivity per cell was obtained from dividing the gray scale density of a field by the number of cells counted in the field.
For the detection of EGF-R, deparaffinized tissue sections were incubated in 0.3% H2O2 in methanol for 10 minutes and washed in PBS three times for 3 minutes at room temperature. Sections were incubated with 0.5% normal rabbit serum for 20 minutes at room temperature, followed by incubation with sheep anti-human EGF-R polyclonal antibody (Upstate Biotechnology, Inc) diluted 1:200 in PBS containing 1% BSA for 60 minutes at room temperature. After they were washed in PBS, sections were incubated with biotinylated rabbit anti-sheep IgG (Vector Laboratories, Inc) for 30 minutes at room temperature, then washed in PBS, and incubated with Vectastain ABC Reagent (Vector Laboratories) for 30 minutes at room temperature. Tissue staining was visualized by incubation in a peroxidase substrate solution containing 3,3′-diaminobenzidine (Zymed Laboratories, Inc) for 1 to 2 minutes at room temperature. For a negative control, the primary antiserum (EGF-R) was replaced by normal sheep IgG (Organon Teknica Co).
Detection of BrdU-Labeled Cells
We used peroxidase-conjugated antibody to mouse immunoglobulin (Amersham) to detect BrdU incorporation into cellular DNA. After deparaffinization, the sections were incubated in 0.1% trypsin in PBS for 30 minutes at 37°C and incubated with 0.3% H2O2 in methanol for 30 minutes at room temperature. After incubation with normal sheep serum for 20 minutes, a mouse anti-BrdU monoclonal antibody diluted 1:100 in distilled water (for 60 minutes at 37°C) and peroxidase-labeled anti-mouse IgG containing BSA (all from Amersham) (for 30 minutes at 37°C) were applied. Positive reactions were visualized by incubation in the presence of peroxidase substrate solution containing 3,3′-diaminobenzidine/nickel chloride (Amersham) for 2 to 3 minutes at room temperature. Counterstaining for the nucleus was carried out by Mayer's hematoxylin. The nuclei with positive immunostaining for BrdU were counted, and the labeling index was determined as the ratio of stained nuclei to the total number of nuclei in the cross sections from two separate segments of the central portion of the artery.
Identification of Cell Types
To identify macrophages and SMCs in the arterial wall, deparaffinized tissue sections were incubated with 0.3% H2O2 in methanol, washed in PBS three times for 3 minutes each, and incubated with 0.1% trypsin in PBS for 30 minutes at room temperature. Sections then were incubated with 1.5% normal horse serum for 20 minutes at room temperature and then with a mouse monoclonal antibody against rat macrophages (BMA Ki-M2R, BMA Biomedicals AG) diluted 1:50 in PBS containing 1% BSA or with a mouse monoclonal antibody against an α isoform of SMC actin (Histofine, Nichirei Co). After they were washed in PBS, macrophages and SMCs were detected using biotinylated horse anti-mouse IgG diluted 1:200 in PBS (Vector) for 30 minutes at room temperature and Vecstatin ABC reagent (Vector) for 30 minutes at room temperature. Positive reactions were visualized as described for detecting EGF-R.
Gene Expression of HB-EGF in Injured Carotid Arteries
A single 2.5-kb transcript for HB-EGF was found at low levels in the uninjured carotid arteries (time zero), similar to previous reports for cultured rat aortic SMCs16 (Fig 1a⇓). However, the HB-EGF transcript levels increased rapidly approximately 12-fold within 2 hours after injury. The levels of HB-EGF mRNA declined by 2 days but remained elevated at 3-fold above levels for the uninjured carotids at every time point studied (Fig 1b⇓). After injury, the regenerating endothelium could affect the level of HB-EGF mRNA. However, the removal of endothelial cells did not change the level of HB-EGF mRNA in uninjured vessels.
Localization of HB-EGF mRNA in the Neointima
We used in situ hybridization analysis to examine the cells expressing HB-EGF after arterial injury. At 2 hours after injury, HB-EGF mRNA was markedly induced throughout the media. At 14 days after injury, HB-EGF mRNA was still expressed strongly in neointimal cells, especially those localized near the luminal surface, whereas only a small amount of HB-EGF mRNA could be detected in underlying intimal cells localized near the IEL (Fig 2⇓).
In order to identify the cell types expressing HB-EGF, sections were stained with monoclonal antibodies against macrophage- and SMC-specific antigens. As shown in previous reports demonstrating that the neointima is composed homogeneously of SMCs,19 26 most of the neointimal cells as well as medial cells exhibited positive immunostaining for the SMC antigen, but these cells did not show any immunoreactivity specific for macrophages at any time point studied. Thus, most of the cells expressing HB-EGF mRNA were considered to be SMCs.
Immunohistochemical Detection of HB-EGF in Injured Carotid Arteries
Uninjured vessels exhibited positive immunoreactivity for HB-EGF in the endothelial cells and faint immunoreactivity in the medial SMCs (Fig 3a⇓). Two days after injury, medial SMCs just below the IEL showed positive immunostaining for HB-EGF protein (Fig 3b⇓). At 7 days, there was a significant increase in HB-EGF immunoreactivity in neointimal cells (Fig 3c⇓). At 14 days, neointimal cells still showed strong HB-EGF immunoreactivity, particularly along the luminal surface. To quantify this spatial distribution of HB-EGF, the sections (n=3) were scanned, and image analysis was carried out to determine the relative levels of immunoreactivity for HB-EGF. In the neointimal cells, this immunoreactivity localized in one third of the layer of the luminal side was 5.4-fold greater than that localized in one third of the layer of the medial side close to the IEL. However, the value might be an overestimation, because in the cross section, the cytoplasmic size of SMCs localized in the neointima near the IEL was smaller than that of SMCs near the luminal surface.
Immunostaining for BrdU
As previously reported,19 the BrdU-labeling index reached a maximum at 2 days in the media and at 7 days in the intima. The BrdU-labeling index in the intima and media declined to the control level at 14 days after injury (Table⇓).
Double immunostaining using antibodies for HB-EGF (antibody H1) and BrdU revealed that at 4 days after injury, the SMCs that had migrated across the IEL into the luminal surface took up BrdU into their nuclei and also showed strong immunostaining for HB-EGF (Fig 4a⇓). At 7 days, neointimal cells were extensively labeled (Fig 4b⇓) (labeling index, 17±6%; mean±SD), and most of the intimal cells exhibited strong immunoreactivities for HB-EGF. At 14 days after injury, BrdU-positive cells were limited to the neointimal SMCs localized close to the luminal surface, and the BrdU labeling index declined to nearly the control level (2.1±0.6%), while BrdU-positive cells continued to produce much HB-EGF protein (Fig 4c⇓).
Detection of EGF-R
Since HB-EGF binds to EGF-R to cause its various biological effects, the expression of EGF-R was examined by immunohistochemistry. Uninjured vessels exhibited faint immunoreactivity for EGF-R in the medial SMCs. The immunostaining pattern of EGF-R in the injured carotid arteries was similar to that of HB-EGF at all time points during 14 days after injury. Fig 5⇓ shows the results at 14 days after injury. Consecutive sections of carotid arteries revealed that positive immunostaining for EGF-R in the intimal cells showed a slight gradient of the staining, with more intense immunoreactivity in cells of the intima near the lumen and faint expression in the medial SMCs, similar to the result of HB-EGF immunostaining. Thus, it appears that the neointimal SMCs producing HB-EGF protein also synthesize EGF-R.
In the present study, we investigated the expression and localization of HB-EGF in rat balloon-injured carotid arteries during vascular remodeling. As shown in Fig 1⇑, HB-EGF transcripts, which are expressed in the uninjured carotid artery, increased rapidly within 2 hours after injury. Separate experiments performed in parallel with the present study showed that the expression of the HB-EGF gene increased 1 hour after injury and reached a maximum at 6 hours (data not shown). The HB-EGF gene is defined as an immediate-early gene in a variety of cells and tissues. In vitro, HB-EGF mRNA is induced rapidly in SMCs by phorbol ester, thrombin, angiotensin II, PDGF, EGF, and HB-EGF itself16 17 27 ; in endothelial cells by tumor necrosis factor-α, interleukin-1β, and lysophosphatidylcholine and in response to shear stress28 29 30 ; and in monocytes/macrophages by phorbol ester, lysophosphatidylcholine, and platelet-activating factor.31 32 In vivo, similar induction of HB-EGF mRNA is reported after liver injury33 and renal injury.34 However, the regulatory mechanism for transcription of the HB-EGF gene is still unclear. Recently, the promoter region of both mouse and human HB-EGF has been reported to contain both transcription factor activator protein-1 and κB binding sites.35 36 Thus, growth factors, cytokines, shear stress, stretching,37 and pressure,38 all of which appear to be involved in the formation of atheromatous tissue, may induce HB-EGF mRNA expression by activating AP-1 and nuclear factor-κB, and whatever the regulatory mechanisms of the HB-EGF expression, this growth factor may be implicated in the development of atherosclerotic and restenotic lesions in an autocrine, paracrine, and juxtacrine fashion.
In situ hybridization experiments demonstrated the localization of the HB-EGF transcripts in the injured vessels (Fig 2⇑). At 2 hours after injury, the HB-EGF gene was induced markedly in the medial SMCs, whereas at 14 days after injury, expression of this gene is relatively strong in a subpopulation of neointimal SMCs localized near the lumen. The distribution of HB-EGF–expressing SMCs at later time points after injury was similar to the immunostaining for HB-EGF. However, there appears to be a time discordance between the peak level of HB-EGF gene expression judged by Northern blotting and the peak of immunohistochemical staining for HB-EGF protein after injury, since the level of HB-EGF transcript was induced markedly within 2 hours (Fig 1⇑), but the immunoreactivities to HB-EGF appeared to increase gradually (Fig 3⇑). These results may be explained as follows: the level of HB-EGF transcript at early time points after injury is due to global activation of the HB-EGF gene in the injured medial SMCs, whereas at later time points HB-EGF gene expression is still strong but much more restricted in the neointimal SMCs located near the luminal surface, so that Northern blots of RNA from the whole vessel do not appear to contain much HB-EGF mRNA relative to the total extracted RNA.
BrdU-labeled SMCs were scattered among neointimal SMCs 7 days after injury and in intimal SMCs localized at or near the luminal surface on day 14 (Fig 4b and 4c⇑⇑). These SMCs with positive BrdU labeling were also producing HB-EGF protein, suggesting that HB-EGF might be involved in SMC proliferation in this model. In addition, neointimal SMCs at 4 days (Fig 4a⇑) and the medial SMCs just beneath the neointima at 7 days (Fig 3c⇑) exhibited strong immunoreactivity for HB-EGF. These results, together with our previous findings that HB-EGF-positive SMCs in the human aortic media increase in number with increasing age in adults and that intimal SMCs just above the media express HB-EGF strongly in atherosclerotic plaques,18 suggest that HB-EGF protein is implicated not only in SMC proliferation but also in SMC migration.
The neointimal SMCs producing HB-EGF protein also expressed EGF-R (Fig 5⇑), suggesting that HB-EGF may regulate SMC growth in an autocrine and/or paracrine manner. Furthermore, the expression of EGF-R mRNA slightly increased 7 days after injury, something that was not observed with the uninjured vessels (data not shown). Earp et al39 demonstrated that EGF-R mRNA levels and protein synthesis were induced by activation of EGF-R in rat liver epithelial cells, and recently, Epstein et al40 demonstrated that proliferating SMCs expressed a larger number of binding sites for EGF in culture than did nonproliferating SMCs. These findings suggest that EGF-R may be overexpressed in neointimal SMCs and may enhance HB-EGF signal transduction. Similar to EGF-R, HSPG binds to HB-EGF and modulates its biological activity in vitro. Therefore, HB-EGF may be regulated in part by both extracellular matrix–associated and cell-associated HSPG after arterial injury. However, the present study offered no data to confirm whether such a modulation is important for HB-EGF to act as an effective growth factor during vascular remodeling.
Various degrees of SMC proliferation after this type of arterial injury have been reported from previous rat studies. Fingerle et al41 showed that gentle denudation of the endothelium from rat carotid arteries without medial injury induced minimal SMC proliferation, compared with that observed after balloon denudation, but similar PDGF-A gene expression. Recently, Indolfi et al42 demonstrated that proportional increase in neointimal formation and c-fos expression is related to the degree of injury. In the present study, a balloon inflated with 0.2 mL air was used to induce injury. We had confirmed with Evans blue staining that the endothelium was denuded completely throughout the entire common carotid artery and that the endothelium had not regenerated in the central one-third portion at 14 days after injury, as reported previously.19 Although we did not measure the pressure of the balloon inflation, the cross-sectional neointimal area and neointimal-to-medial area ratio at 14 days reached 0.23±0.07 mm2 and 1.5±0.5, respectively. These findings were comparable to the data obtained by Indolfi et al with the maximal pressure of the balloon inflation at 2.0 atm. HB-EGF expression may be related to the degree of injury, but we did not vary this in the present study.
Ferns et al43 have demonstrated that antibody to PDGF partially inhibited the neointimal accumulation that occurred 3 to 6 days after injury, which may be mainly due to the suppression of migration. In addition, Lindner and Reidy44 showed that antibody against bFGF decreased the proliferation of the medial SMCs that occurred 24 to 48 hours after injury, without diminution of neointimal accumulation. Thus, further studies in which the action of HB-EGF is blocked are needed to ascertain whether HB-EGF actually contributes to neointimal formation after arterial injury.
In conclusion, we have demonstrated the expression and localization of HB-EGF in rat balloon-injured carotids and have shown that HB-EGF is likely to be associated with SMC proliferation and migration after injury. Our findings imply that HB-EGF, like PDGF and bFGF, could play an important role in vascular remodeling in response to arterial injury by an autocrine and/or paracrine mechanism.
Selected Abbreviations and Acronyms
|bFGF||=||basic fibroblast growth factor|
|BSA||=||bovine serum albumin|
|EGF||=||epidermal growth factor|
|EGF-R||=||epidermal growth factor receptor|
|HB-EGF||=||heparin-binding epidermal growth factor–like growth factor|
|HSPG||=||heparan sulfate proteoglycans|
|IEL||=||internal elastic lamina|
|PDGF||=||platelet-derived growth factor|
|PTCA||=||percutaneous transluminal coronary angioplasty|
|SMC||=||smooth muscle cell|
|SSC||=||standard saline citrate|
|TGF-α||=||transforming growth factor-α|
This study was supported in part by a grant-in-aid to Dr Matsuzawa (No. 04404085) and to Dr Kawata (No. 06557058) from the Ministry of Education, Science, Culture, and Sports of Japan. We are highly indebted to Dr Morishita (Medicine of Geriatrics, Osaka University Medical School) for his helpful advice.
Liu MW, Roubin GS, King SB III. Restenosis after coronary angioplasty: potential biologic determinants and role of intimal hyperplasia. Circulation. 1989;79:1374-1387.
Popma JJ, Califf RM, Topol EJ. Clinical trials of restenosis after coronary angioplasty. Circulation. 1991;84:1426-1436. Editorial.
Ueda M, Becker AE, Tsukada T, Numano F, Fujimoto T. Fibrocellular tissue response after percutaneous transluminal coronary angioplasty: an immunocytochemical analysis of the cellular composition. Circulation. 1991;83:1327-1332.
Majesky MW, Reidy MA, Bowen PD, Hart CE, Wilcox JN, Schwartz SM. PDGF ligand and receptor gene expression during repair of arterial injury. J Cell Biol. 1990;11:2149-2158.
Klagsbrun M, Edelman ER. Biological and biochemical properties of fibroblast growth factors: implications for the pathogenesis of atherosclerosis. Arteriosclerosis. 1989;9:269-278.
Lindner V, Reidy MA. Expression of basic fibroblast growth factor and its receptor by smooth muscle cells and endothelium in injured rat arteries: an en face study. Circ Res. 1993;73:589-595.
Higashiyama S, Abraham JA, Miller J, Fiddes JC, Klagsbrun M. A heparin-binding growth factor secreted by macrophage-like cells that is related to EGF. Science. 1991;251:936-939.
Higashiyama S, Lau K, Besner GE, Abraham JA, Klagsbrun M. Structure of heparin-binding EGF-like growth factor: multiple forms, primary structure, and glycosylation of the mature protein. J Biol Chem. 1992;267:6205-6212.
Thompson SA, Higashiyama S, Wood K, Pollitt NS, Damm D, McEnroe G, Garrick B, Ashton N, Lau K, Hancock N. Characterization of sequences within heparin-binding EGF-like growth factor that mediate interaction with heparin. J Biol Chem. 1994;269:2541-2549.
Higashiyama S, Abraham JA, Klagsbrun M. Heparin-binding EGF-like growth factor stimulation of smooth muscle cell migration: dependence on interactions with cell surface heparan sulfate. J Cell Biol. 1993;122:933-940.
Temizer DH, Yoshizumi M, Perrella MA, Susanni EE, Quertermous T, Lee ME. Induction of heparin-binding epidermal growth factor-like growth factor mRNA by phorbol ester and angiotensin II in rat aortic smooth muscle cells. J Biol Chem. 1992;267:24892-24896.
Dluz SM, Higashiyama S, Damm D, Abraham JA, Klagsbrun M. Heparin-binding epidermal growth factor-like growth factor expression in cultured fetal human vascular smooth muscle cells: induction of mRNA levels and secretion of active mitogen. J Biol Chem. 1993;268:18330-18334.
Miyagawa J, Higashiyama S, Kawata S, Inui Y, Tamura S, Yamamoto K, Nishida M, Nakamura T, Yamashita S, Matsuzawa Y. Localization of heparin-binding EGF-like growth factor in the smooth muscle cells and macrophages of human atherosclerotic plaques. J Clin Invest. 1995;95:404-411.
Abraham JA, Damm D, Bajardi A, Miller J, Klagsbrun M, Ezekowitz RA. Heparin-binding EGF-like growth factor: characterization of rat and mouse cDNA clones, protein domain conservation across species, and transcript expression in tissues. Biochem Biophys Res Commun. 1993;190:125-133.
Inui Y, Hausman AM, Nanthakumar N, Henning SJ, Davidson NO. Apolipoprotein B messenger RNA editing in rat liver: developmental and hormonal modulation is divergent from apolipoprotein A-IV gene expression despite increased hepatic lipogenesis. J Lipid Res. 1992;33:1843-1856.
Nomura S, Wills AJ, Edwards DR, Heath JK, Hogan BL. Developmental expression of 2ar (osteopontin) and SPARC (osteonectin) RNA as revealed by in situ hybridization. J Cell Biol. 1988;106:441-450.
Nakano T, Raines EW, Abraham JA, Wenzel FT, Higashiyama S, Klagsbrun M, Ross R. Glucocorticoid inhibits thrombin-induced expression of platelet-derived growth factor A-chain and heparin-binding epidermal growth factor-like growth factor in human aortic smooth muscle cells. J Biol Chem. 1993;268:22941-22947.
Yoshizumi M, Kourembanas S, Temizer DH, Cambria RP, Quertermous T, Lee ME. Tumor necrosis factor increases transcription of the heparin-binding epidermal growth factor-like growth factor gene in vascular endothelial cells. J Biol Chem. 1992;267:9467-9469.
Kume N, Gimbrone MJ. Lysophosphatidylcholine transcriptionally induces growth factor gene expression in cultured human endothelial cells. J Clin Invest. 1994;93:907-911.
Nakano T, Raines EW, Abraham JA, Klagsbrun M, Ross R. Lysophosphatidylcholine upregulates the level of heparin-binding epidermal growth factor-like growth factor mRNA in human monocytes. Proc Natl Acad Sci U S A. 1994;91:1069-1073.
Pan Z, Kravchenko VV, Ye RD. Platelet-activating factor stimulates transcription of the heparin-binding epidermal growth factor-like growth factor in monocytes: correlation with an increased kappa B binding activity. J Biol Chem. 1995;270:7787-7790.
Homma T, Sakai M, Cheng HF, Yasuda T, Coffey RJ, Harris RC. Induction of heparin-binding epidermal growth factor-like growth factor mRNA in rat kidney after acute injury. J Clin Invest. 1995;96:1018-1025.
Fen Z, Dhadly MS, Yoshizumi M, Hilkert RJ, Quertermous T, Eddy RL, Shows TB, Lee ME. Structural organization and chromosomal assignment of the gene encoding the human heparin-binding epidermal growth factor-like growth factor/diphtheria toxin receptor. Biochemistry. 1993;32:7932-7938.
Chen X, Raab G, Deutsch U, Zhang J, Ezzell RM, Klagsbrun M. Induction of heparin-binding EGF-like growth factor expression during myogenesis: activation of the gene by MyoD and localization of the transmembrane form of the protein on the myotube surface. J Biol Chem. 1995;270:18285-18294.
Komuro I, Kaida T, Shibazaki Y, Kurabayashi M, Katoh Y, Hoh E, Takaku F, Yazaki Y. Stretching cardiac myocytes stimulates protooncogene expression. J Biol Chem. 1990;265:3595-3598.
Hishikawa K, Nakaki T, Marumo T, Hayashi M, Suzuki H, Kato R, Saruta T. Pressure promotes DNA synthesis in rat cultured vascular smooth muscle cells. J Clin Invest. 1994;93:1975-1980.
Earp HS, Hepler JR, Petch LA, Miller A, Berry AR, Harris J, Raymond VW, McCune BK, Lee LW, Grisham JW. Epidermal growth factor (EGF) and hormones stimulate phosphoinositide hydrolysis and increase EGF receptor protein synthesis and mRNA levels in rat liver epithelial cells: evidence for protein kinase C-dependent and -independent pathways. J Biol Chem. 1988;263:13868-13874.
Epstein SE, Siegall CB, Biro S, Fu YM, FitzGerald D, Pastan I. Cytotoxic effects of a recombinant chimeric toxin on rapidly proliferating vascular smooth muscle cells. Circulation. 1991;84:778-787.
Fingerle J, Au YP, Clowes AW, Reidy MA. Intimal lesion formation in rat carotid arteries after endothelial denudation in absence of medial injury. Arteriosclerosis. 1990;10:1082-1087.
Indolfi C, Esposito G, Di Lorenzo E, Rapacciuolo A, Feliciello A, Porcellini A, Avvedimento VE, Condorelli M, Chiariello M. Smooth muscle cell proliferation is proportional to the degree of balloon injury in a rat model of angioplasty. Circulation. 1995;92:1230-1235.
Ferns GA, Raines EW, Sprugel KH, Motani AS, Reidy MA, Ross R. Inhibition of neointimal smooth muscle accumulation after angioplasty by an antibody to PDGF. Science. 1991;253:1129-1132.
Lindner V, Reidy MA. Proliferation of smooth muscle cells after vascular injury is inhibited by an antibody against basic fibroblast growth factor. Proc Natl Acad Sci U S A. 1991;88:3739-3743.