This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nakata, A. Articles by Matsuzawa, Y. Search for Related Content PubMed PubMed Citation Articles by Nakata, A. Articles by Matsuzawa, Y.

(Circulation. 1996;94:2778-2786.)
© 1996 American Heart Association, Inc.

Articles

Localization of Heparin-Binding Epidermal Growth Factor–Like Growth Factor in Human Coronary Arteries

Possible Roles of HB-EGF in the Formation of Coronary Atherosclerosis

Atsuyuki Nakata, MD; Jun-ichiro Miyagawa, MD, PhD; Shizuya Yamashita, MD, PhD; Makoto Nishida, MD; Ritsu Tamura, MD; Katsumi Yamamori, BE; Tadashi Nakamura, MD, PhD; Shuichi Nozaki, MD, PhD; Kaoru Kameda-Takemura, MD, PhD; Sumio Kawata, MD, PhD; Naoyuki Taniguchi, MD, PhD; Shigeki Higashiyama, PhD; Yuji Matsuzawa, MD, PhD

the Second Department of Internal Medicine and the Department of Biochemistry (N.T., S.H.), Osaka (Japan) University Medical School.

Correspondence to Atsuyuki Nakata, MD, Second Department of Internal Medicine, Osaka University Medical School, 2-2, Yamadaoka, Suita, Osaka 565, Japan.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background Heparin-binding epidermal growth factor (EGF)–like growth factor (HB-EGF) is a newly identified member of the EGF family. Our previous in vitro studies showed that HB-EGF is a potent mitogen and chemoattractant for vascular smooth muscle cells (SMCs), suggesting the role of HB-EGF in the pathogenesis of atherosclerosis. The purposes of the present study were to investigate the localization of HB-EGF in both normal and atherosclerotic human coronary arteries and to elucidate the possible roles of this growth factor in the formation of atherosclerotic lesions.

Methods and Results The immunohistochemical localization of HB-EGF, SMCs, macrophages, and EGF receptors (EGFRs) was examined in human coronary arteries obtained at autopsy. The medial SMCs of coronary arteries in neonates, infants, and children consistently synthesized HB-EGF protein. In normal adults, however, the relative number of HB-EGF–positive medial SMCs decreased gradually with age after about 30 years of age. In nonatherosclerotic coronary arteries with diffuse intimal thickening, SMCs of the intima, especially those located in the area of the medial side of the intima, were strongly positive for HB-EGF protein. In atherosclerotic plaques of coronary arteries with eccentric intimal thickening, both SMCs and macrophages in and around the core lesions, in addition to the intimal and medial SMCs located adjacent to the plaque, produced HB-EGF protein. A strong immunostaining of EGFRs was observed in these SMCs, suggesting a close association of HB-EGF and EGFR expression.

Conclusions These data suggest that HB-EGF might play important roles in the migration of SMCs from the media to the intima, the proliferation of intimal SMCs, and the interaction between SMCs and macrophages in the process of coronary atherogenesis.

Key Words: atherosclerosis • coronary disease • growth substances • muscle, smooth

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Coronary atherosclerosis is a unique process in that the stenosis or occlusion occurs more easily compared with aortic atherosclerosis. Unlike the atherosclerotic process of the aorta, DIT of the coronary arteries can be found even in newborn babies.¹ DIT was reported to progress gradually with age,² and the lesion of EIT develops easily from youth.³ It is widely accepted that SMCs, macrophages, endothelial cells, and T lymphocytes are involved in the atherosclerotic process of the aorta, playing major roles through their proliferation and migration.^{4 5 6 7 8 9 10 11} These cells interact with each other by producing many cytokines, including PDGF,^{12 13 14 15} basic fibroblast growth factor,^{16 17 18 19 20} SMC-derived growth factor,²¹ and TGF-ß.^{22 23 24 25 26 27} Among these growth factors, PDGF and basic fibroblast growth factor have been shown to be potent chemoattractants for monocytes and SMCs and potent mitogens for SMCs. However, it is possible that other unknown mechanisms might be operating in the migration and proliferation of the cells involved in the process of atherosclerosis in vivo.

HB-EGF, a member of the EGF family, has been found to be a potent mitogen and chemoattractant for SMCs in in vitro studies.²⁸ Mature bioactive HB-EGF was originally purified from the conditioned medium of a macrophage-like cell line (U937) and is a polypeptide containing 86 amino acid residues. The mature HB-EGF, which spans amino acid residues 63 through 148 of proHB-EGF, has two characteristic domains: a highly hydrophilic domain with an affinity for heparin in the N-terminal region and an EGF-like domain with 35% homology to human EGF and TGF- in the C-terminal region.^{28 29 30} When HB-EGF binds to an EGFR, it induces EGFR autophosphorylation and exerts various biological activities such as cell proliferation and migration. We reported previously that HB-EGF was comparable to PDGF, which also was synthesized by macrophages and SMCs, in that it was a potent mitogen and chemoattractant for SMCs in vitro.³¹ We recently demonstrated the immunohistochemical localization of HB-EGF in human aortic walls and atherosclerotic plaques.³² In the present study, we investigated immunohistochemically the localization of HB-EGF and EGFR in human coronary arteries to determine whether HB-EGF is implicated in the process of coronary atherogenesis.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials
Coronary arteries (proximal portions of the left anterior descending coronary artery and right coronary artery) were obtained from 44 autopsied cases. Autopsies were performed within 5 hours of death with the informed consent of the families. To determine whether each coronary artery was atherosclerotically involved, we examined at least three different tissues (5 to 10 mm long and 5 mm distant from each specimen). Five different sections were taken from each tissue block, with each section being 1 mm away from the previous section. The Table gives the age, sex, and cause of death for all cases and indicates whether the coronary artery of each individual was atherosclerotic. From light microscopic observations, individuals with atherosclerosis were subdivided into two groups: those with atherosclerotic plaque (EIT) and those with EITadv. When both EIT and EITadv were observed in sections from the same subject, the case was designated as an individual with EITadv. The samples from patients with severe inflammatory disease that might alter or induce the expression of HB-EGF protein were excluded.

View this table: [in this window] [in a new window]   Table 1. Clinical Status or Cause of Death

HB-EGF Antibodies
Polyclonal antibodies recognizing mature and proform of HB-EGF were produced by immunization of female White New Zealand rabbits with synthetic peptide H-1 (HB-EGF precursor C-terminal residues 185 through 208) and H-6 (HB-EGF precursor residues 54 through 73). The method for the preparation of these antibodies was described in detail elsewhere.^{29 32} Neither peptide H-1 nor H-6 had amino acid sequence homology with other members of the EGF family proteins such as EGF, TGF-, betacellulin, and amphiregulin.³³ Antibodies against peptide H-1 did not cross-react with mature HB-EGF purified from U-937 cell–conditioned medium by Western blotting but immunoprecipitated ³⁵S-labeled proform of HB-EGF. Antibodies against peptide H-6 cross-reacted with purified U-937–derived mature HB-EGF. Neither antibody detected EGF, TGF-, or amphiregulin by Western blotting. In the present study, we used both H-1 and H-6 antibodies.

Immunohistochemical Detection of HB-EGF Protein
For immunohistochemical detection of HB-EGF, tissues from coronary arteries obtained at autopsy were fixed with 10% phosphate-buffered formalin for 4 to 6 hours at 4°C. After being washed in 0.01 mol/L PBS, the tissues were decalcified in decalcifying solution B (Wako Pure Chemical Industries, Ltd) for 3 days at 4°C. Paraffin sections (about 4 µm thick) were deparaffinized, treated with 3% H₂O₂ solution for 5 minutes, and washed in distilled water for 5 minutes and in 0.05 mol/L Tris-HCl buffer (pH 7.6) for 5 minutes. After incubation with 10% normal swine serum for 20 minutes at room temperature, a three-step immunoperoxidase method was used to detect HB-EGF with rabbit anti–HB-EGF immunoglobulins (H-1 or H-6) diluted 1/200 in Tris-HCl buffer containing 1% BSA (for 30 minutes at room temperature); swine anti-rabbit immunoglobulins (DAKO) diluted 1/500 in Tris buffer containing 1% BSA (for 20 minutes at room temperature); and peroxidase-labeled, rabbit anti-peroxidase immunoglobulins (PAP) complexes (DAKO) (for 20 minutes at room temperature). Positive reaction on the tissue sections was visualized with 3-amino-9-ethylcarbazol (DAKO) in 0.1 mol/L acetate buffer (pH 5.2) in the presence of 0.3% H₂O₂. As a negative control, the primary antiserum (H-1 or H-6) was replaced by normal rabbit immunoglobulins (DAKO) or preabsorbed with an excess amount of the peptide antigen.

Identification of Macrophages and SMCs and Detection of EGFR
For the identification of macrophages and SMCs and for the detection of EGFR in the coronary artery, deparaffinized tissue sections were incubated with 0.3% H₂O₂ in methanol and washed in PBS for 20 minutes. Sections were then incubated for 20 minutes at room temperature with 1.5% normal goat serum diluted in PBS containing 1% BSA for macrophages or 1.5% normal horse serum diluted in PBS containing 1% BSA for SMCs and EGFR. This was followed by incubation with mouse monoclonal antibodies against human macrophage (HAM56, Biomeda) diluted 1/50 in PBS containing 1% BSA for macrophages or mouse monoclonal antibodies against an -isoform of SMC actin (HISTOFINE, Nichirei Co Ltd) and mouse monoclonal antibodies against human EGFR (Cambridge Research Biochemicals) for SMCs and EGFR, respectively. After the sections were washed in PBS, the avidin-biotin complex method was applied to detect macrophages by use of biotinylated goat anti-mouse IgM diluted 1/2000 or to detect SMCs and EGFR by use of biotinylated horse anti-mouse IgG diluted 1/2000 in PBS (Vector Laboratories Inc) and the VECSTATIN avidin-biotin complex Reagent (Vector Laboratories Inc). Positive reaction was visualized by incubation for 5 to 20 minutes at room temperature in peroxidase substrate solution containing 3,3'-diaminobenzidine or 3,3'-diaminobenzidine/nickel chloride (Zymed Laboratories, Inc).

For double immunohistostainings, sections were immunostained for HB-EGF by use of the peroxidase-antiperoxidase method as described above and washed in 0.1 mol/L glycine hydrochloride buffer (pH 2.2) for 2 hours. After further washing in PBS, the sections were incubated for 20 minutes at room temperature with 1.5% normal goat serum diluted in PBS containing 1% BSA for macrophages or 1.5% normal sheep serum diluted in PBS containing 1% BSA for SMCs. This was followed by incubation with mouse monoclonal antibodies against human macrophage (HAM56) diluted 1/50 in PBS containing 1% BSA for macrophages or mouse monoclonal antibodies against an -isoform of SMC actin for SMCs. After being washed in PBS for 20 minutes, these sections were incubated for 1 hour at room temperature with FITC-conjugated goat anti-mouse IgM F(ab') fragment (Jackson ImmunoResearch Laboratories, Inc) diluted 1/100 in PBS containing 1% BSA for macrophages or FITC-conjugated sheep anti-mouse IgG (Organon Teknika Corp) diluted 1/100 in PBS containing 1% BSA for SMCs. The sections were washed in PBS for 30 minutes, mounted with Perma Fluor Aqueous Mounting Medium (Immunon), and observed with a fluorescence microscope with epi-illumination (Olympus).

Statistical Analysis
Statistical analyses were performed with SAS software to examine the correlation between the percentage of HB-EGF–positive cells and aging. Multiple regression analysis was used to investigate the effect of atherosclerosis on the percentage of HB-EGF–positive cells.

	Results

Top Abstract Introduction Methods Results Discussion References

 
In human coronary arteries, HB-EGF protein could be detected in several cell types, including SMCs, macrophages, and endothelial cells. However, the distribution pattern and the staining intensity of HB-EGF in these cells were characteristically different with age and the presence or absence of atherosclerotic lesions.

In the coronary artery of a 6-month-old baby, a positive immunostaining of HB-EGF was detected in almost all medial SMCs of coronary artery (Fig 1a, 1b, and 1d). The intima consisted of an endothelial cell lining and SMCs scattering in extracellular matrices of subendothelial space. In addition to the medial SMCs, the intimal SMCs that migrated from the media and endothelial cells (Fig 1b) were positive for this protein. Macrophages were rarely observed in the intima of the coronary arteries in the baby and infants. Both antibodies H-1 and H-6, which recognize the cytoplasmic domain (proform of HB-EGF) and extracellular domain (proform and mature form of HB-EGF), respectively, showed the same staining pattern, and the positive immunohistochemical reaction was completely abolished by the H-1 or H-6 antibody preincubated with each synthetic peptide antigen (Fig 1c).

View larger version (174K): [in this window] [in a new window]   Figure 1. Immunohistochemical localization of HB-EGF and identification of SMCs in sections from the left anterior descending coronary artery of a 6-month-old baby (male, case 1). a, Low-power view of HB-EGF localization with antibody H-1 (x50; bar=200 µm). b, Part of a higher magnification of the section in a (x250; bar=40 µm). c, Control section for a and b incubated with a preabsorbed antibody solution by a sufficient amount of antigen peptide (H-1) (x50; bar=200 µm). d, Part of the consecutive section from a immunostained for SMCs with anti–-SMC actin monoclonal antibodies (x250; bar=40 µm). Intima (I) showed a very mild diffuse intimal thickening (a, b), and intimal cells consisted of an endothelial cell lining and SMCs (d). Most SMCs in both the media (M) and intima were positive for HB-EGF immunostaining (b, d). Note that endothelial cells (arrowhead) were stained positively for HB-EGF protein. When antibodies were preabsorbed by antigen peptide (H-1), positive immunoreaction was totally abolished (c).

In children to young adults (<30 years of age), the intima became diffusely thickened with an increase in age. SMCs that occupied the major part of intimal cells were HB-EGF positive, and the number of intimal SMCs increased gradually with the progress of DIT (Fig 2a and 2b). Except for endothelial cells, which have been shown to have an immunoreactive cellular component to the antibody HAM56,³⁴ cells with a positive immunoreactivity to this antibody were rarely found in the intima and media around this age (Fig 2c). The distribution pattern of HB-EGF immunostaining was essentially the same as those in the baby and infants.

View larger version (101K): [in this window] [in a new window]   Figure 2. Immunohistochemical localization of HB-EGF–producing cells, SMCs, and macrophages in sections from a normal left anterior descending coronary artery (28-year-old woman, case 6). a, Immunostaining for HB-EGF (antibody H-1). b and c, Immunostaining for SMCs with mouse anti–-SMC actin monoclonal antibody and macrophages with mouse anti-macrophage antibody (HAM56), respectively (a, b, and c, x220; bar=50 µm). Intimal cells appeared to be increased in number compared with those in the coronary artery of the baby shown in Fig 1. Almost all the intimal cells and medial SMCs showed positive HB-EGF immunostaining (a). In the intima (I), SMCs occupied most of the cell population (b), whereas macrophages (arrow) were rarely found in the intima, although endothelial cells showed a positive immunoreactivity to this antibody (c). M indicates media.

In coronary arteries of young adults and middle-aged individuals, the thickness of intima was further increased with age, and the major cell population in the intima was SMCs even at this age, although a small number of macrophages with positive HB-EGF immunostaining could be recognized. From about 30 to 40 years of age, the intima of coronary arteries with DIT of some individuals began to show an irregular luminal surface, and some individuals had typical atherosclerotic plaques (EIT lesion), but the appearance of arteries with advanced atherosclerotic plaques (EITadv lesion) was still rare. At this stage of atherosclerotic process, the relative number of SMCs with strongly positive HB-EGF staining was increased in the intimal side area of the medial wall (Fig 3a and 3b). Furthermore, almost all the intimal SMCs of the coronary artery at this stage showed a strongly positive immunostaining of HB-EGF, particularly those located at the medial side of intima (Figs 3b, 3c, and 4a). As to the expression of EGFR, SMCs strongly positive for EGFR immunostaining appeared to be localized in the limited area around the border between the intima and the media, and the distribution pattern of EGFR-positive SMCs was very similar to that of SMCs strongly positive for HB-EGF immunostaining (Fig 4a and 4b).

View larger version (144K): [in this window] [in a new window]   Figure 3. Immunohistochemical localization of HB-EGF protein in sections from the left anterior descending coronary artery with a mild EIT (63-year-old woman, case 28). a, Low-power view of transverse section (x60; bar=200 µm). b and c, Two different patterns of HB-EGF localization from a (b and c, x290; bar=40 µm). In a, HB-EGF–positive cells were observed mainly in the intima (I), especially in the area just above the media (M; arrow). In contrast, medial SMCs with positive HB-EGF staining were markedly decreased in number, although HB-EGF–positive SMCs were recognized in the restricted area of the media (double arrow). In some areas of the border in b between the media and intima (the area indicated by double arrows in a), medial SMCs showed positive HB-EGF staining, and intimal SMCs located just above such a medial wall also showed a positive immunostaining for HB-EGF. c, In contrast to the feature in b, the medial SMCs were virtually negative for HB-EGF staining in many places (the area indicated by an arrow in a), whereas the intimal SMCs that probably migrated from such media were strongly positive for this protein.

View larger version (88K): [in this window] [in a new window]   Figure 4. Immunohistochemical localization of HB-EGF and EGFR in sections from the left anterior descending coronary artery with an irregularly thickened intima (63-year-old woman, case 28). a, Localization of HB-EGF (antibody H-1) in the area of the border between the intima (I) and media (M). b, Localization of the cells expressing EGFR in the section consecutive to that in a. c, Control section for EGFR immunostaining using normal mouse IgG (a, b, and c, x270; bar=40 µm). In a, HB-EGF protein was detected in SMCs localized in the area just below the intima (arrowheads). Many of these HB-EGF–positive cells had lost their normal arrangement with their long axis directed to the vertical direction. The intimal cells localized above such a region also showed a strong HB-EGF immunostaining. In the consecutive section in b, EGFR protein was detected in both medial and intimal SMCs. However, the distribution of the cells expressing EGFR was restricted to the area in which HB-EGF was produced (arrowheads). c, No EGFR staining was detected in the negative control section.

In coronary arteries with typical atherosclerotic lesions, the thicknesses of the media appeared to be decreased. In contrast, the intima of EIT lesions showed a marked increase in thickness, and a number of HB-EGF–positive cells were observed (Fig 5a and 5c). In the medial walls of the coronary arteries with EIT, the relative number of HB-EGF–positive medial SMCs appeared to be increased compared with that in the coronary arteries with only DIT. In addition to SMCs, many macrophages and foam cells could be observed primarily in the center of the atherosclerotic lesions, although macrophages were also observed scattering in the so-called cap and shoulder regions of the plaque. These macrophages that often aggregated in the center of atherosclerotic lesions were also stained positively for this protein (Fig 5c and 5d). Foamed macrophages tended to be stained more weakly for HB-EGF than nonfoamed macrophages, possibly because of the loss of this antigen accompanied by the decrease of intracellular lipids in the foamed macrophages during the processing of tissues. Nonetheless, the cell densities of HB-EGF–positive SMCs and macrophages accumulating in the atherosclerotic plaque were markedly higher than those in the media and intima of noninvolved regions in the coronary arteries with EIT.

View larger version (141K): [in this window] [in a new window]   Figure 5. Immunohistochemical localization of HB-EGF protein and immunofluorescent localization of SMCs or macrophages by double immunohistostaining in sections from the left anterior descending coronary artery with atherosclerotic plaque (53-year-old woman, case 16). a, Immunohistochemical localization of HB-EGF in the atherosclerotic plaque (antibody H-6). b, Localization of SMCs detected by anti–-SMC actin monoclonal antibodies in the same section. c, Immunohistochemical localization of HB-EGF in the atherosclerotic plaque (antibody H-1). d, Detection of macrophages in the plaque by HAM56 in the same section as in c (a, b, c, and d, x140; bar=70 µm). Many HB-EGF–positive cells were observed in the intima of this atherosclerotic plaque (a, c). Relatively large cells with a granular staining pattern of the cytoplasm were identified as macrophages; small and round or spindle-shaped cells were identified as SMCs (b, d). Cells indicated by arrows in a and b are the same SMCs; cells indicated by arrows in c and d are the same macrophages.

In lesions with EITadv, however, the intimal cell density became rather sparse, whereas the extracellular matrices were markedly increased. Intimal cells in such lesions often showed degenerative changes. The percentage of HB-EGF–producing intimal cells, including SMCs and macrophages, also was decreased, and such cells were usually observed in the subendothelial and basal regions in the plaque of advanced stage (data not shown).

To examine the age-dependent difference and/or the difference between the presence and absence of atherosclerosis in the production of HB-EGF protein, we analyzed the relative number of medial SMCs with positive HB-EGF immunostaining in noninvolved regions (not plaque) of the coronary arteries of individuals with or without atherosclerotic plaques. As Fig 6 shows, the percentage of HB-EGF–positive medial SMCs was correlated with aging on the polynomial, nonlinear, and square root models. From the results of the above examinations, the percentage of HB-EGF–positive cells was considered to correlate with the first power of aging. In individuals with EIT (from mild to typical lesions of atherosclerotic plaque), the percentage of HB-EGF–producing medial SMCs appeared to be higher than that in individuals with only DIT (physiological DIT). However, the percentage of HB-EGF–positive medial SMCs in individuals with EITadv (advanced or late-stage lesions of atherosclerotic plaque) became lower compared with that in individuals with EIT. After adjustment for the difference of aging, the effect of atherosclerosis on the percentage of HB-EGF–positive cells was statistically significant (P=.0019) when individuals with DIT were compared with those with EIT, but there was no significant difference between individuals with DIT and those with EIT and EITadv.

View larger version (14K): [in this window] [in a new window]   Figure 6. Percentage of HB-EGF–positive medial SMCs in 44 subjects of various ages in noninvolved regions (not plaques) of the coronary arteries of the individuals with or without atherosclerotic plaques. After the immunostaining for HB-EGF protein with antibodies against peptide (H-1), sections were counterstained with Mayer's hematoxylin. The number of nuclei of HB-EGF–positive SMCs that showed red or red-brown immunohistochemical reaction in the cytoplasm and the total number of nuclei of SMCs were counted with a tablet measure unit for micromeasurement (Krypton-40, Flovel) in the medial wall. Each point is the mean of the percentage of HB-EGF–positive SMCs in five different areas of the medial wall of coronary arteries in individuals without atherosclerosis or with only DIT (), individuals with an EIT of atherosclerotic plaque (), and individuals with an EITadv (). Solid line represents the correlation between the percentage of HB-EGF–positive cells and aging in individuals with DIT; dashed line, the correlation between the percentage of HB-EGF–positive cells and aging in individuals with EIT. The results of statistical analyses are given in the text.

Taken together, HB-EGF protein was consistently produced in the medial SMCs of human coronary arteries until middle age; then the relative number of HB-EGF–positive SMCs in the media was gradually decreased with age. However, HB-EGF–producing medial SMCs appeared to be increased in the relative number in coronary arteries with mild to typical atherosclerotic lesions. In the intima showing DIT, SMCs that migrated and/or proliferated from the media were strongly positive for HB-EGF protein, especially those localized in the medial side of the intima. In the lesions of atherosclerotic plaques, in addition to the neointimal SMCs, macrophages or foam cells were also positive for this protein. A strong EGFR immunostaining was detected not only in the intimal cells, most of which were SMCs that migrated from the media, but also in the medial SMCs of some part close to the intima where the SMCs were also strongly positive for HB-EGF immunostaining.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
HB-EGF protein was discovered in 1991 to be a member of the EGF family. This protein, like other members of the EGF family, binds to EGFR on EGFR-expressing cells and activates EGFR tyrosine kinase to induce various biological effects. From the functional viewpoint of this growth factor, it has been shown that HB-EGF is mitogenic for some types of cells such as fibroblasts, hepatocytes, keratinocytes, and vascular SMCs.^{35 36 37} Because of its highly potent mitogenic and chemoattractant activities to SMCs in in vitro studies,²⁸ HB-EGF has been implicated to be involved in the pathogenesis of atherosclerosis.

We have demonstrated that in coronary arteries of neonates or babies, almost all the medial SMCs produced HB-EGF protein, and the relative number of HB-EGF–producing SMCs was not decreased with age until about 30 years of age. However, the relative number of HB-EGF–positive SMCs gradually decreased after 30 years of age, suggesting that HB-EGF may have developmentally important roles not only in the growth of vascular wall but also in the functional and/or structural maintenance of coronary arteries. In our previous study on the localization of HB-EGF in the human aorta,³² HB-EGF–producing medial SMCs were markedly decreased in the relative number of young adults. Interestingly, this does not appear to be the case with coronary arteries, and such a difference might be due to the difference in the size, the structure of the vessel wall, or the intensity of mechanical stress between the two types of arteries.

Our previous study on the localization of HB-EGF in the human aorta also demonstrated that HB-EGF–producing medial SMCs gradually increased again with age, especially in individuals with atherosclerotic lesions.³² In the coronary arteries, there was a similar tendency for the relative number of HB-EGF–positive medial SMCs in coronary arteries with EIT of early to typical atherosclerosis to be higher than that of coronary arteries with only DIT. We demonstrated that the percentage of HB-EGF–positive SMCs was generally elevated in noninvolved regions of coronary arteries in individuals with atherosclerotic plaques (EIT). However, the cell density of HB-EGF–positive SMCs and macrophages accumulating in the atherosclerotic plaques appeared to be much higher than in noninvolved regions. These observations suggest that HB-EGF may play crucial roles in the initiation and progression of atherosclerotic lesions. In the lesion of more advanced or late-stage atherosclerosis, medial SMCs with positive HB-EGF staining were often markedly decreased. It might thus be speculated that medial SMCs in coronary arteries, when activated for migration and/or proliferation, begin to produce HB-EGF protein, which further accelerates the migration and/or proliferation of the medial SMCs. However, once the process of atherosclerosis is completed or the trigger of atherosclerotic changes subsides, the percentage of SMCs producing HB-EGF may be decreased and the cell density itself may be decreased in many cases. Thus, it is of interest that strong EGFR expression in the coronary artery was detected in both intimal and medial SMCs and that the distribution pattern of the SMCs with a strongly positive EGFR staining was very similar to that of the SMCs strongly positive for HB-EGF immunostaining. Aviezer and Yayon³⁸ showed in vitro the heparin-dependent binding of HB-EGF and autophosphorylation of EGFR by HB-EGF in SMCs. This implies that HB-EGF secreted by SMCs and macrophages could bind to EGFR expressed on SMCs in the intima and media of coronary arteries probably in an autocrine, a paracrine, and/or a juxtacrine manner, by which various biological effects such as the migration and proliferation of SMCs may be induced.

In addition to SMCs, many macrophages expressed HB-EGF protein in the atherosclerotic plaque, suggesting that macrophage-derived HB-EGF production is most prominent in the region of atherosclerotic plaque. In the aorta, macrophages infiltrating into intima were observed even frequently in young adults; in the coronary artery, they were observed less frequently until middle age, at least in our study. However, infiltration of macrophages into the intima appeared to be closely associated with the lesion of pathological thickening or EIT, suggesting that macrophage-derived HB-EGF may play a crucial role in the process of coronary atherogenesis at a time when macrophages would potentially become a more dominant cell type than SMCs. Although the regulatory mechanism of HB-EGF production in macrophages has not been clarified, Nakano et al³⁹ showed that lysophosphatidylcholine, one of the endogenous atherogenic substances, upregulates the level of HB-EGF mRNA expression in human monocytes. The regulation or modulation of HB-EGF protein synthesis in macrophages might be related to the metabolism of oxidized LDL in the intima.

In view of the initial event of endothelial injury in the "response to injury hypothesis" of atherosclerosis by Ross,⁷ it is noteworthy that endothelial cells in human coronary arteries were HB-EGF positive by immunohistostaining through all generations. Morita et al⁴⁰ have reported that shear stress increases HB-EGF mRNA levels in human vascular endothelial cells. This leads to the possibility that more HB-EGF protein might be induced and/or produced in the shear-stressed endothelial cells and that HB-EGF secreted by endothelial cells might stimulate the migration and proliferation of SMCs in the intima and media. Kume and Gimbrone⁴¹ showed that lysophosphatidylcholine induced HB-EGF mRNA in human umbilical vein endothelial cells, suggesting that endothelial cells may also be related to the metabolism of oxidized LDL. Yoshizumi et al⁴² found that tumor necrosis factor- increased the transcription of HB-EGF gene in vascular endothelial cells. It is therefore speculated that some other triggers that induce the production of tumor necrosis factor- in endothelial cells may also be indirectly associated with the regulation of HB-EGF production by endothelial cells.

In conclusion, the present study showed, for the first time, the existence of HB-EGF protein in human coronary arteries, and its localization was closely associated with the progression of coronary atherosclerosis. It is suggested that HB-EGF protein produced by SMCs and macrophages may be involved in atherosclerotic process in human coronary arteries, especially in the migration and proliferation of SMCs and in the interaction between SMCs and macrophages through the EGFR-mediated signaling pathway.

	Selected Abbreviations and Acronyms

 

DIT = diffuse intimal thickening EGF = epidermal growth factor EGFR = EGF receptor EIT = eccentric intimal thickening EITadv = advanced atherosclerotic plaque accompanied by calcification HB-EGF = heparin-binding EGF-like growth factor PDGF = platelet-derived growth factor SMC = smooth muscle cell TGF = transforming growth factor

	Acknowledgments

 
This study was supported in part by a grant-in-aid for Dr Matsuzawa (grant 04404085) from the Ministry of Education, Science, Culture and Sports of Japan and by a grant-in-aid for cancer research for Drs Higashiyama and Taniguchi (grant 05151047). Dr Higashiyama is the recipient of a Searle Scientific Research Fellowship. We thank T. Oh-ito in the Department of Pathology and Prof C. Wakasugi and Dr H. Kuroki in the Department of Legal Medicine for providing some of the tissue materials.

Received January 23, 1996; revision received June 22, 1996; accepted July 2, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Rapola J, Pesonen E. Coronary artery changes in newborn babies. Acta Path Microbiol Scand. 1977;85(Sect A):286-296.

2. Stary HC. Macrophages, macrophage foam cells, and eccentric intimal thickening in the coronary arteries of young children. Atherosclerosis. 1987;64:91-108.[Medline] [Order article via Infotrieve]

3. Joseph A, Ackerman D, Talley JD, Johnstone J, Kupersmith J. Manifestations of coronary atherosclerosis in young trauma victims: an autopsy study. J Am Coll Cardiol. 1993;22:459-467.[Abstract]

4. Rosenfeld ME, Ross R. Macrophage and smooth muscle cell proliferation in atherosclerotic lesions of WHHL and comparably hypercholesterolemic fat-fed rabbits. Arteriosclerosis. 1990;10:680-687.[Abstract/Free Full Text]

5. Katsuda S, Coltrera MD, Ross R, Gown AM. Human atherosclerosis, IV: immunocytochemical analysis of cell activation and proliferation in lesions of young adults. Am J Pathol. 1993;142:1787-1793.[Abstract]

6. Pickering JG, Weir L, Jekanowski J, Kearney MA, Isner JM. Proliferative activity in peripheral and coronary atherosclerotic plaque among patients undergoing percutaneous revascularization. J Clin Invest. 1993;91:1469-1480.

7. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801-809.[Medline] [Order article via Infotrieve]

8. DiCorleto PE. Cellular mechanisms of atherogenesis. Am J Hypertens. 1993;6:314S-318S.[Medline] [Order article via Infotrieve]

9. Kishikawa H, Shimokawa T, Watanabe T. Localization of T lymphocytes and macrophages expressing IL-1, IL-2 receptor, IL-6 and TNF in human aortic intima: role of cell-mediated immunity in human atherogenesis. Virchows Arch. 1993;423:433-442.

10. Badimon JJ, Fuster V, Chesebro JH, Badimon L. Coronary atherosclerosis: a multifactorial disease. Circulation. 1993;87(suppl II):II-3-II-16.

11. Jonasson L, Holm J, Skalli O, Bondjers G, Hansson GK. Regional accumulations of T cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque. Arteriosclerosis. 1986;6:131-138.[Abstract/Free Full Text]

12. Ross R, Rains EW, Bowen-Pope DF. The biology of platelet-derived growth factor. Cell. 1986;46:155-169.[Medline] [Order article via Infotrieve]

13. Ross R, Masuda J, Raines EW, Gown AM, Katsuda S, Sasahara M, Malden LT, Masuko H, Saito H. Localization of PDGF-B protein in macrophages in all phases of atherosclerosis. Science. 1990;248:1009-1012.[Abstract/Free Full Text]

14. Wilcox JN, Smith KM, Williams LT, Schwartz SM, Gordon D. Platelet-derived growth factor mRNA detection in human atherosclerotic plaques by in situ hybridization. J Clin Invest. 1988;82:1134-1143.

15. Nilsson J. Cytokines and smooth muscle cells in atherosclerosis. Cardiovasc Res. 1993;27:1184-1190.[Free Full Text]

16. Klagsbrun M, Edelman ER. Biological and biochemical properties of fibroblast growth factors: implications for the pathogenesis of atherosclerosis. Arteriosclerosis. 1989;9:269-278.[Free Full Text]

17. Gospodarowicz D, Ferrara N, Haaparanta T, Neufeld G. Basic fibroblast growth factor: expression in cultured bovine vascular smooth muscle cells. Eur J Cell Biol. 1988;46:144-151.[Medline] [Order article via Infotrieve]

18. Lindner V, Lappi DA, Baird A, Majack RA, Reidy MA. Role of basic fibroblast growth factor in vascular lesion formation. Circ Res. 1991;68:106-113.[Abstract/Free Full Text]

19. Hughes SE, Crossman D, Hall PA. Expression of basic and acidic fibroblast growth factors and their receptor in normal and atherosclerotic human arteries. Cardiovasc Res. 1993;27:1214-1219.[Abstract/Free Full Text]

20. Brogi E, Winkles JA, Underwood R, Clinton SK, Alberts GF, Libby P. Distinct patterns of expression of fibroblast growth factors and their receptors in human atheroma and nonatherosclerotic arteries: association of acidic FGF with plaque microvessels and macrophages. J Clin Invest. 1993;92:2408-2418.

21. Morisaki N, Koyama N, Mori S, Kanzaki T, Koshikawa T, Saito Y, Yoshida S. Effects of smooth muscle cell derived growth factor (SDGF) in combination with other growth factors on smooth muscle cells. Atherosclerosis. 1989;78:61-67.[Medline] [Order article via Infotrieve]

22. Majesky MW, Lindner V, Twardzik DR, Schwartz SM, Reidy MA. Production of transforming growth factor b1 during repair of arterial injury. J Clin Invest. 1991;88:904-910.

23. Sporn MB, Roberts AB, Wakefield LM, Crombrugghe B. Some recent advances in the chemistry and biology of transforming growth factor-beta. J Cell Biol. 1987;105:1039-1045.[Free Full Text]

24. Moses HL, Yang EY, Pietenpol JA. TGF-ß stimulation and inhibition of cell proliferation: new mechanistic insight. Cell. 1990;63:245-247.[Medline] [Order article via Infotrieve]

25. McCaffrey TA, Falcone DJ, Brayton CF, Agarwal LA, Welt FGP, Weksler BB. Transforming growth factor-ß activity is potentiated by heparin via dissociation of the transforming growth factor-ß/2-macroglobulin inactive complex. J Cell Biol. 1989;109:441-448.[Abstract/Free Full Text]

26. Battegay EJ, Rains EW, Seifert RA, Bowen-Pope DF, Ross R. TGF-ß induces bimodal proliferation of connective tissue cells via complex control of an autocrine PDGF loop. Cell. 1990;63:515-524.[Medline] [Order article via Infotrieve]

27. Nabel EG, Shum L, Pompili VJ, Yang Z, San H, Shu HB, Liptay S, Gold L, Gordon D, Derynck R. Direct transfer of transforming growth factor ß1 gene into arteries stimulates fibrocellular hyperplasia. Proc Natl Acad Sci U S A. 1993;90:10759-10763.[Abstract/Free Full Text]

28. 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.[Abstract/Free Full Text]

29. Higashiyama S, Lau K, Besner GE, Abraham JA, Klagsbrun M. Structure of heparin-binding EGF-like growth factor. J Biol Chem. 1992;267:6205-6212.[Abstract/Free Full Text]

30. Thompson SA, Higashiyama S, Wood K, Pollitt S, Damm D, McEnroe G, Garrick B, Ashton N, Lau K, Hancock N, Klagsbrun M, Abraham JA. Characterization of sequences within heparin-binding EGF-like growth factor that mediate interaction with heparin. J Biol Chem. 1994;269:2541-2549.[Abstract/Free Full Text]

31. 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.[Abstract/Free Full Text]

32. Miyagawa J, Higashiyama S, Kawata S, Inui Y, Tamura S, Yamamoto K, Nishida M, Nakamura T, Yamashita S, Matsuzawa Y, Taniguchi N. 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.

33. Plowman GD, Green JM, McDonald VL, Neubauer MG, Disteche CM, Todaro GJ, Shoyab M. The amphiregulin gene encodes a novel epidermal growth factor-related protein with tumor-inhibitory activity. Mol Cell Biol. 1990;10:1969-1981.[Abstract/Free Full Text]

34. Gown AM, Tsukada T, Ross R. Human atherosclerosis, II: immunocytochemical analysis of the cellular composition of human atherosclerotic lesions. Am J Pathol. 1986;125:191-207.[Abstract]

35. Ito N, Kawata S, Tamura S, Kiso S, Tsushima H, Damm D, Abraham JA, Higashiyama S, Taniguchi N, Matsuzawa Y. Heparin-binding EGF-like growth factor is a potent mitogen for rat hepatocytes. Biochem Biophys Res Commun. 1994;198:25-31.[Medline] [Order article via Infotrieve]

36. Hashimoto K, Higashiyama S, Asada H, Hashimura E, Kobayashi T, Sudo K, Nakagawa T, Damm D, Yoshikawa K, Taniguchi N. Heparin-binding epidermal growth factor-like growth factor is an autocrine growth factor for human keratinocytes. J Biol Chem. 1994;269:20060-20066.[Abstract/Free Full Text]

37. 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. J Biol Chem. 1993;268:18330-18334.[Abstract/Free Full Text]

38. Aviezer D, Yayon A. Heparin-dependent binding and autophosphorylation of epidermal growth factor (EGF) receptor by heparin-binding EGF-like growth factor but not by EGF. Proc Natl Acad Sci U S A. 1994;91:12173-12177.[Abstract/Free Full Text]

39. 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.[Abstract/Free Full Text]

40. Morita T, Yoshizumi M, Kurihara H, Maemura K, Nagai R, Yazaki Y. Shear stress increases heparin-binding epidermal growth factor-like growth factor mRNA levels in human vascular endothelial cells. Biochem Biophys Res Commun. 1993;197:256-262.[Medline] [Order article via Infotrieve]

41. Kume N, Gimbrone MA. Lysophosphatidylcholine transcriptionally induces growth factor gene expression in cultured human endothelial cells. J Clin Invest. 1994;93:907-911.

42. Yoshizumi M, Kourembanas S, Temizer DH, Cambria RP, Quertermous T, Lee M. 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.[Abstract/Free Full Text]

This article has been cited by other articles:

				E Matsuura and L. Lopez Autoimmune-mediated atherothrombosis Lupus, October 1, 2008; 17(10): 879 - 888. [Abstract] [PDF]

				Y. Nakashima, T. N. Wight, and K. Sueishi Early atherosclerosis in humans: role of diffuse intimal thickening and extracellular matrix proteoglycans Cardiovasc Res, July 1, 2008; 79(1): 14 - 23. [Abstract] [Full Text] [PDF]

				H. Zhang, S. W. Sunnarborg, K. K. McNaughton, T. G. Johns, D. C. Lee, and J. E. Faber Heparin-Binding Epidermal Growth Factor-Like Growth Factor Signaling in Flow-Induced Arterial Remodeling Circ. Res., May 23, 2008; 102(10): 1275 - 1285. [Abstract] [Full Text] [PDF]

				R. Khurana, M. Simons, J. F. Martin, and I. C. Zachary Role of Angiogenesis in Cardiovascular Disease: A Critical Appraisal Circulation, September 20, 2005; 112(12): 1813 - 1824. [Abstract] [Full Text] [PDF]

				H. Zhang, D. Chalothorn, L. F. Jackson, D. C. Lee, and J. E. Faber Transactivation of Epidermal Growth Factor Receptor Mediates Catecholamine-Induced Growth of Vascular Smooth Muscle Circ. Res., November 12, 2004; 95(10): 989 - 997. [Abstract] [Full Text] [PDF]

				C. M. Reynolds, S. Eguchi, G. D. Frank, and E. D. Motley Signaling Mechanisms of Heparin-Binding Epidermal Growth Factor-Like Growth Factor in Vascular Smooth Muscle Cells Hypertension, February 1, 2002; 39(2): 525 - 529. [Abstract] [Full Text] [PDF]

				B. C. Berk Vascular Smooth Muscle Growth: Autocrine Growth Mechanisms Physiol Rev, July 1, 2001; 81(3): 999 - 1030. [Abstract] [Full Text] [PDF]

				A. Kalmes, B. R. Vesti, G. Daum, J. A. Abraham, and A. W. Clowes Heparin Blockade of Thrombin-Induced Smooth Muscle Cell Migration Involves Inhibition of Epidermal Growth Factor (EGF) Receptor Transactivation by Heparin-Binding EGF-Like Growth Factor Circ. Res., July 21, 2000; 87(2): 92 - 98. [Abstract] [Full Text] [PDF]

				M. Nishida, J.-i. Miyagawa, S. Yamashita, S. Higashiyama, A. Nakata, N. Ouchi, R. Tamura, K. Yamamori, S. Kihara, N. Taniguchi, et al. Localization of CD9, an Enhancer Protein for Proheparin-Binding Epidermal Growth Factor-Like Growth Factor, in Human Atherosclerotic Plaques : Possible Involvement of Juxtacrine Growth Mechanism on Smooth Muscle Cell Proliferation Arterioscler Thromb Vasc Biol, May 1, 2000; 20(5): 1236 - 1243. [Abstract] [Full Text] [PDF]

				N. Vacaresse, I. Lajoie-Mazenc, N. Auge, I. Suc, M.-F. Frisach, R. Salvayre, and A. Negre-Salvayre Activation of Epithelial Growth Factor Receptor Pathway by Unsaturated Fatty Acids Circ. Res., November 12, 1999; 85(10): 892 - 899. [Abstract] [Full Text] [PDF]

				L. R. Bonin, K. Madden, K. Shera, J. Ihle, C. Matthews, S. Aziz, N. Perez-Reyes, J. K. McDougall, and S. C. Conroy Generation and Characterization of Human Smooth Muscle Cell Lines Derived From Atherosclerotic Plaque Arterioscler Thromb Vasc Biol, March 1, 1999; 19(3): 575 - 587. [Abstract] [Full Text] [PDF]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nakata, A. Articles by Matsuzawa, Y. Search for Related Content PubMed PubMed Citation Articles by Nakata, A. Articles by Matsuzawa, Y.