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Invasion of atheromatous plaques into tunica media causes coronary outward remodeling in WHHLMI rabbits
Atherosclerosis 198 (2008) 287–293
Invasion of atheromatous plaques into tunica media causes coronary outward remodeling in WHHLMI rabbits Masashi Shiomi a,∗ , Satoshi Yamada a , Akihiro Matsukawa b , Hiroyuki Itabe c , Takashi Ito a a
b
Institute for Experimental Animals, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentist and Pharmaceutical Science, Okayama University, Shikata-cho, Okayama 700-8558, Japan c Department of Biological Chemistry, School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo 140-8555, Japan Received 19 April 2007; received in revised form 3 February 2008; accepted 10 February 2008 Available online 16 February 2008
Abstract To clarify the mechanism of coronary outward remodeling, we examined atherosclerotic coronary arteries morphologically using WHHLMI rabbits that develop coronary atherosclerosis spontaneously. Perfusion-fixed coronary segments of WHHLMI rabbits were prepared at 500 m intervals. After immunohistochemical staining and histopathological staining, the areas and lengths of the arterial wall and the lesions were measured. Obvious outward remodeling was observed in coronary sections with greater than 40% cross-sectional narrowing. In coronary sections with greater than 40% cross-sectional narrowing, the tunica media was thick at the shoulder of atheromatous plaque and was thin beneath the atheromatous plaques. Macrophages infiltrated those attenuated tunica media expressed matrix metalloproteinases and oxidized LDL was accumulated in those areas. In those areas, collagen fibers and the internal elastic lamina had disappeared partly and apoptotic smooth muscle cells were observed. Proliferation of smooth muscle cells was observed at the attenuated tunica media and adjacent adventitia. The present results suggest that invasion of atheromatous plaques into the tunica media causes coronary outward remodeling. © 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Atherosclerosis; Coronary outward remodeling; Macrophage; Matrix metalloproteinase; Smooth muscle cells; WHHLMI rabbit
1. Introduction Enlargement of arteries suppresses the reduction of the lumen size in the progression of atherosclerosis and appears to prevent tissue ischemia. Arterial remodeling has been used to refer to changes in the cross-sectional area of arteries and consists of outward remodeling, enlargement of arterial size, and inward remodeling, reduction of arterial size. The first detailed study was reported by Glagov et al., who identified coronary outward remodeling as compensatory enlargement [1]. In response to atherosclerotic plaques, the arteries enlarge and the progression of luminal stenosis delays. Although several hypotheses for the mechanism have been proposed, for ∗
Corresponding author. Tel.: +81 78 382 5660; fax: +81 78 382 5679. E-mail address: [email protected] (M. Shiomi).
0021-9150/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2008.02.010
example, hemodynamic stimuli [2], degradation of fibrous elements in the arterial wall [3], high-density lipoprotein levels [4] and the relation of matrix metalloproteinase (MMP)-9 [5,6], no conclusive evidence has been reported to date. In our previous study about the relation between arterial size and coronary cross-sectional narrowing (CSN) [7] using coronary atherosclerosis-prone WHHL rabbits [8], findings similar to those in a previous human study [1] were observed if we did not take into account arterial tapering and individual variance. In the previous study, we observed that the tunica media at the lesion site showed thickening and/or attenuation compared to that at lesion-free sites, even in the same arterial sections. Therefore, we attempted to examine the mechanisms of coronary outward remodeling focusing on morphological changes of the tunica media of coronary arteries.
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M. Shiomi et al. / Atherosclerosis 198 (2008) 287–293
2. Methods 2.1. Animals We used myocardial infarction-prone WHHLMI rabbits [9]; 8 rabbits (four rabbits each were 9 months old and 12 months old) for analyses of the relation of arterial size to CSN of the coronary arteries and 22 rabbits (17 rabbits were 9 months old, two rabbits were 13 months old, and three rabbits were 14 months old) for counting proliferating or apoptotic SMCs in the tunica media. Rabbits were anesthetized by an intravenous injection of sodium pentobarbital at a dose of 25 mg/kg of body weight followed by perfusion with 10% buffered neutral formalin solution or periodate-lysine-paraformaldehyde fixative under a constant pressure of 80 mmHg for 30 min. This study was approved by the Committee for Animal Experimentation, Kobe University School of Medicine (Permission numbers: P-011009 and P-011009R) and carried out according to the guidelines of Animal Experimentation of Kobe University, the Law for the Humane Treatment and Management of Animals (Law No. 105, 1973, revised in 1999), and the Standards Relating to the Care and Management, etc. of Experimental Animals (Notification No. 6, 1980). 2.2. Preparation of histological sections After perfusion fixation, the hearts were immersion-fixed with the same fixative indicated above and embedded in paraffin. Serial coronary sections were prepared using segments of the left circumflex arteries, which were prepared at intervals of 500 m. Since the left circumflex artery is a major coronary artery in rabbits and atherosclerotic lesions are very prevalent in this artery of WHHLMI rabbits [10], we examined arterial remodeling of the left circumflex arteries. Each section of every segment was stained with elastic van Gieson stain, hematoxylin and eosin stain, picrosirius red stain, or
immunohistochemically using monoclonal antibodies specific for rabbit macrophages (RAM-11), smooth muscle cell (SMC) actin (1A4), MMP-1, MMP-12, oxidized low-density lipoprotein (LDL) (DLH3) [11], proliferating cell nuclear (Ki-67), or goat polyclonal antibody specific for interleukin1 (IL-1) [12]. Immunohistochemical staining was carried out using a DAKO Envision + kit or Nichirei Histofine Simple Stain kit, or the labeled streptavidin-biotin alkaline phosphatase method with fuchsin chromogen according to the manufacturer’s instructions accompanied by hematoxylin counter staining. Staining of sections incubated with mouse IgG2a (for MMP-1), IgG2b (for MMP-12), and IgM (for DLH3), or goat IgG (for IL-1) instead of specific antibodies were negligible (data not shown). To detect apoptotic cells in arterial walls, TUNEL staining was carried out using ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit. 2.3. Measurement of arterial size and counting cells According to our previous study [13,14], the length of the external elastic lamina (EEL) and the area of the tunica media, intimal lesions, and lumen were measured with computer assisted color image analysis (Image-Pro Plus, version 4.5, Media Cybernetics Inc., Silver Spring, MD, USA) using sections stained with elastic van Gieson stain. Tunica media was defined as layers of SMCs plus collagen fibers located inside the EEL if the internal elastic lamina had disappeared or the tunica media was partly degenerated by macrophage infiltration. Average thickness of the whole tunica media of each coronary section was calculated by dividing the tunica media area by the length of the EEL. CSN was calculated by following equation; [CSN (%)] = [lesion area]/([lesion area] + [lumen area]) × 100. The numbers of SMCs or macrophages in the tunica media were counted using sections stained immunohistochemically with 1A4 or RAM-11 followed by hematoxylin counter stain.
Fig. 1. Photomicrographs of coronary arterial sections of a WHHLMI rabbit aged 9 months old showing typical outward remodeling with the growth of atherosclerotic lesions. These sections were stained with elastic van Gieson staining. Arrowheads indicate attenuation of the tunica media and arrows indicate thickening of the tunica media. CSN, cross-sectional narrowing; EEL, external elastic lamina.
M. Shiomi et al. / Atherosclerosis 198 (2008) 287–293
2.4. Statistical analysis Data are represented as the mean ± standard error. Statistical analyses were carried out with the Dunnett multiple comparison test for mean values and with the Cochran–Mantel–Haenszel test for frequency. Significance was set at P < 0.05.
3. Results 3.1. Typical outward remodeling of the circumflex artery Fig. 1 shows the typical outward remodeling observed in the serial segments of the left circumflex artery. The tunica media was thick at the shoulder of the lesions (sections 2 and 4) and thin under the large intimal plaque (section 3), although the thickness of the tunica media is relatively uniform at arterial sections without lesions (sections 1 and 5). The internal elastic lamina partly disappears under the intimal
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plaque of sections 2 and 3. Those findings suggest that both thickening and attenuation of tunica media may be associated with coronary outward remodeling. 3.2. Changes in arterial size and cell number in the tunica media In longitudinal observation from the proximal to distal segments of the coronary arteries of WHHLMI rabbits after maturation, atherosclerotic lesions show early lesion at the plaque edge and established lesion at the plaque center. We examined changes in the arterial size using 54 coronary segments from 9 atheromatous plaques of 8 rabbits aged 9–12 months old. In comparison with the proximal lesionfree coronary section, the frequency of coronary sections showing enlargement of the coronary periphery (enlargement degree greater than 1.1) increased with progression of CSN (P < 0.001, Fig. 2A). Regarding the thickness of the tunica media at the lesion site (Fig. 2B), the frequency of coronary sections showing thinning of the tunica
Fig. 2. Relation of coronary cross-sectional narrowing (CSN) to the arterial size and cell number in the tunica media. Panel-A shows enlargement of the coronary periphery with an increase of CSN compared to those in the proximal lesion-free section. The coronary periphery was evaluated in relation to the length of the external elastic lamina (EEL). The X-axis shows the ratio of the EEL length of each arterial sections/the EEL length of the proximal lesion-free coronary section. A value of 1.1 means the EEL length was 10% longer than that of the proximal lesion-free section. Panel-B shows the thickness of the tunica media at the lesion site with increasing CSN. Panel-C shows changes in SMC content throughout the whole tunica media with progression of CSN compared to that in the proximal lesion-free section. The X-axis shows the ratio of SMC content throughout the whole tunica media of each coronary section/that of the proximal lesion-free section. A value of 1.1 means the SMC content was 10% more than that of the proximal lesion-free section. Panel-D shows the number of SMC in the tunica media in relation to the length of the tunica media (EEL length) at the lesion site. Panel-E shows an increase in the number of macrophages that infiltrated the tunica media with an increase of CSN. The X-axis shows the number of macrophages that infiltrated the tunica media in relation to the length of tunica media (EEL length) with lesion. Statistical analysis was carried out for the frequency with Cochran–Mantel–Haenszel test. Gray bars, coronary segments showing less than 20% CSN; white bars, coronary segments showing 20 to <40% CSN; crossed bars, coronary segments showing 40 to <60% CSN; black bars, coronary segments showing 60 to 85% CSN.
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media (less than 40 m) increased with progression of CSN (P < 0.001). These findings indicate that with the growth of atheromatous plaques, the tunica media was elongated and attenuated. In comparison with the proximal lesion-free coronary section, the frequency of coronary sections showing an increase in SMC number throughout the whole tunica media (degree greater than 1.1) increased with progression of CSN
(P = 0.007, Fig. 2C). Fig. 2D shows that tunica media SMC content in relation to tunica media length (EEL length) was decreased (less than 100 cells/mm) at the lesion site (Fig. 2D) when CSN was greater than 60% although the frequency of coronary sections showing high SMC number (more than 200 cells/mm) was higher when the CSN was less than 40% (P < 0.001). Fig. 2E shows that the number of macrophages
Fig. 3. Photomicrographs of coronary sections of WHHLMI rabbits aged 9 months old showing attenuation of the tunica media. Elastic van Gieson staining (A, H, I), picrosirius red staining (B), immunohistochemical staining with 1A4 for smooth muscle cell (SMC) (C and J), RAM-11 for macrophage (D), matrix metalloproteinase-12 (MMP-12) (E), MMP-1 (F), DLH3 for oxidized-LDL (G and L), and IL-1 (M), and double staining with RAM-11 and TUNEL (K). Symbols in the photomicrographs indicate the following; arrowheads, internal elastic lamina; large arrows, macrophages (A–G), TUNEL-positive cells (K), and oxidized LDL (L); asterisks, tunica media; parallel cross, tunica adventitia.
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Table 1 The number of TUNEL-positive or Ki-65-positive spindle-shaped cells in the coronary arterial walls of WHHLMI rabbits Cross-sectional narrowing
1–29% 30–59% 60–85%
n
11 12 14
TUNEL-positive cells
Ki-65-positive cells
Tunica media
Tunica adventitia
Total
Tunica media
Tunica adventitia
Total
0.00 ± 0.00 0.17 ± 0.11 0.64 ± 0.25*
0.09 ± 0.09 0.25 ± 0.13 0.57 ± 0.25
0.09 ± 0.09 0.42 ± 0.23 1.21 ± 0.46*
0.27 ± 0.27 0.08 ± 0.08 1.86 ± 0.64*
0.00 ± 0.00 0.58 ± 0.36 1.71 ± 0.55†
0.27 ± 0.27 0.67 ± 0.43 3.57 ± 1.06†
Data are represented as the mean ± S.E. Statistical analyses were carried out with Dunnett multiple comparison test in comparison with the coronary sections with cross-sectional narrowing 1–29% (* P < 0.05; † P < 0.01).
that infiltrated the tunica media increased with progression of CSN (P = 0.003). These findings demonstrate that although coronary arteries enlarged with an increase of SMCs throughout the whole tunica media, infiltration of macrophages into the tunica media increased and the number of SMC in the tunica media was decreased at the lesion sites in the coronary sections showing greater than 60% CSN. 3.3. Histopathological and immunohistochemical examination of the attenuation of coronary tunica media In Fig. 3A, the coronary tunica media was attenuated beneath the atheromatous plaque and the internal elastic lamina had partly disappeared. In the attenuated tunica media, collagen fibers (Panel-B) and SMCs (Panel-C) had partly decreased. At those areas, macrophages infiltrated the tunica media (Panel-D) or vacuoles (supposedly derived from collapsed macrophages) were observed (Panel-A). Those macrophages expressed MMP-12 (Panel-E) and MMP-1 (Panel-F). In addition, oxidized LDL was detected in those areas (Panel-G). The positive areas for oxidized LDL cor-
responded to the positive areas of RAM-11, MMP-12 and MMP-1. In the MMP-12-positive area, the internal elastic lamina disappeared (Panel-A–G) and the collagen layers of the tunica media were attenuated (Panel-B) at the MMP-1positive area. These observations suggest that macrophage infiltration into the tunica media strongly related to attenuation of the tunica media by secretion of MMP-1 and -12. In Fig. 3H and I, the internal elastic lamina had partly disappeared, 1A4-positive SMC decreased in the tunica media (Panel-J), and TUNEL-positive cells (PanelK) were observed in the intimal plaque (RAM-11-positive macrophages) and tunica media (RAM-11-negative SMCs). In this area, DLH3 monoclonal antibody for oxidized LDL (Panel-L) and IL-1 (Panel-M) were positive. In TUNELstaining of coronary arterial walls (Table 1), there were no TUNEL-positive spindle-shaped cells in the coronary tunica media with CSN less than 30% and the number increased significantly with progression of CSN. These findings suggest that apoptosis of SMCs in the tunica media probably related to attenuation of the coronary tunica media.
Fig. 4. Photomicrographs of SMC proliferation in coronary sections of WHHLMI rabbits aged 14 (A–C) and 9 (D–F) months old. Serial coronary sections were stained with elastic van Gieson stain (A and D) and immunohistochemically with 1A4 plus Ki-67 for proliferating cells (B, E and F), and RAM-11 for macrophage (C). Symbols in the photomicrographs indicate the following; arrowheads, internal elastic lamina; small arrows, external elastic lamina; large arrows, proliferating SMCs; asterisks, tunica media; parallel cross, tunica adventitia.
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3.4. Histopathological and immunohistochemical examination of proliferating SMCs Since the SMC number in the tunica media increased with progression of CSN (Fig. 2C), SMC proliferation in the coronary wall was examined immunohistochemically (Fig. 4). At the shoulder of atheromatous plaque, the tunica media was thick (Panel-A) and several Ki-67-positive cells (proliferating cells) were observed in the intimal lesion and the tunica media (Panel-B). Ki-67-positive cells in the tunica media were 1A4-positive (SMC) and those in the intimal lesions were 1A4-negative (macrophages). There were no RAM-11positive cells in the tunica media (Panel-C). At the bottom of a large intimal plaque (Panel-D), proliferating SMCs were observed in the tunica adventitia (Panel-E) and tunica media (Panel-F). There are two types of Ki-67-positive spindleshaped cells in the coronary adventitia. One is 1A4-positive (SMC) and the other is 1A4-negative. 1A4-negative spindleshaped cells may be proliferating fibroblasts. The number of Ki-67-positive spindle-shaped cells in the tunica media and adventitia increased significantly with progression of CSN (Table 1). These findings suggest that proliferation of SMCs and fibroblasts in the coronary arterial wall is probably related to coronary outward remodeling.
4. Discussion The present observations suggest that the primary factor in coronary outward remodeling due to atherosclerosis is macrophage infiltration into the tunica media. Those macrophages expressed MMPs. Consequently, in the coronary tunica media, the internal elastic lamina and collagen fibers disappeared, probably due to degradation by MMPs and the SMC density decreased due to apoptosis. Finally, the coronary tunica media was attenuated and became fragile. However, proliferation of SMCs in the coronary tunica media is also probably related to outward remodeling. Proliferating SMCs were observed mainly at the attenuated tunica media and the adjacent adventitia in addition to that at the shoulder of atheromatous plaque. We consider that findings of the coronary tunica media observed in Fig. 3A–G demonstrate the process of attenuation of the coronary tunica media by macrophage infiltration. Lesion-free coronary tunica media consists of SMCs with a small amount of collagen fibers (Panel-B and C). At the shoulder of atheromatous plaque, collagen fibers partly replaced SMCs in the thick tunica media (Panel-B and C) adjacent to the site of macrophage infiltration. At the area showing macrophage infiltration where the tunica media was attenuated (Panel-B–D), both SMCs and collagen fibers were decreased. In those areas, macrophages expressed MMP-12 (Fig. 3E) and MMP-1 (Fig. 3F). Since MMP-12 is an elastase, disappearance of the internal elastic lamina (Fig. 3A) was probably due to degradation by MMP-12. Since MMP-1 is an interstitial collagenase, disappearance of the collagen layers
in the tunica media was probably due to degradation by MMP1. Similarly, in studies of murine models, MMP-9 expression was observed throughout the entire plaque in carotid arteries that showed outward remodeling [5,6]. The present findings suggest a mechanism underlying atrophy of the tunica media associated with outward arterial remodeling in human coronary arteries [15]. In addition, since oxidized LDL stimulates the synthesis of MMPs in activated monocytes [16], accumulation of oxidized LDL in the atheromatous plaque (Fig. 3G) probably relates to expression of MMP-1 and -12 in macrophages that infiltrated the tunica media. Number of SMC in the tunica media in relation to EEL length at lesion site decreased with progression of atheromatous plaques (Fig. 2D). Regarding a decreased number of SMC in the tunica media, TUNEL-positive SMC in the tunica media and adventitia increased with progression of CSN (Table 1) and those areas were positive for oxidized LDL and IL-1 (Fig. 3L–M). Since in vitro studies have demonstrated that oxidized LDL [17], IL-1 [18], and other cytokines relate to SMC apoptosis [19,20], accumulation of oxidized-LDL and IL-1 in the coronary tunica media probably relate to SMC apoptosis in the tunica media. Therefore, we consider that the tunica media was attenuated beneath the large atheromatous plaques due to the combined degradation of the internal elastic lamina and collagen fibers and decrease in SMCs. Consequently, arterial walls became fragile and probably enlarged easily. In the present study, SMCs increased in the tunica media (Fig. 2C) but the SMC density decreased at the attenuated tunica media (Fig. 3C). These findings appear to be contradictory. Since Ki-67-positive SMCs were observed at the attenuated tunica media where the SMC density was low (Fig. 4), proliferation and decrease of SMCs are observed simultaneously in attenuated tunica media. Those proliferating cells consisted of 1A4-positive and negative spindle-shaped cells. These findings may suggest that those proliferating SMCs and 1A4-negative spindle-shaped cells (probably fibroblasts) play a role in the repair of the attenuated tunica media. Shi et al. [21] reported the involvement of the adventitia in the vascular repair process after the tunica media injury in porcine coronary arteries. Therefore, proliferating cells in the adventitia observed in the present study may relate to the repair process of attenuated coronary arterial wall. Further examination is required. The present observation suggests that attenuation of the tunica media by macrophages and SMC proliferation at the attenuated coronary wall were probably repeated during the growth of atheromatous lesions and then coronary arteries enlarged. These observations suggest that atheromatous lesions expand in the circumference direction of coronary arteries (outward) as well as in the lumen direction (inward). Therefore, we consider that invasion of atheromatous plaques into the tunica media causes coronary outward remodeling in WHHLMI rabbits. In conclusion, the present findings demonstrated that coronary arteries enlarged and were attenuated in the coronary
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sections showing greater than 40% CSN mainly due to macrophage infiltration into the tunica media. Degradation of the internal elastic lamina and collagen fibers in the tunica media due to MMPs secreted by those macrophages and reduction of SMCs in the tunica media due to apoptosis probably related to the attenuation and weakening of the coronary wall. These findings suggest that coronary outward remodeling was due to invasion of atheromatous plaques into the tunica media.
Acknowledgements This work was supported in part by unrestricted research grants from Sankyo Co. Ltd. We would like to express our appreciation of Mr. Toshiaki Tamura of the Institute for Experimental Animals, Kobe University School of Medicine for the preparation of histological sections. We are also grateful to Miss Kaori Yamane and Miss Haruka Adachi of ASK Co. Ltd. for animal care.
References [1] Glagov S, Weisenberg E, Zains CK, Stankunavicius R, Kolettis GJ. Compensatory enlargement of human atherosclerotic coronary arteries. N Engl J Med 1987;316:1371–5. [2] Lamontagne D, Pohl U, Busse R. Mechanical deformation of vessel wall and shear stress determine the basal release of endothelium-derived relaxing factor in the intact rabbit coronary vascular bed. Circ Res 1992;70:123–30. [3] Zarins CK, Runyon-Hass A, Zatina MA, Lu CT, Glagov S. Increased collagenase activity in early aneurismal dilation. J Vasc Surge 1986;3:238–48. [4] Taylor AJ, Burke AP, Farb A, et al. Arterial remodeling in the left coronary system. J Am Coll Cardiol 1999;34:760–7. [5] Godin D, Ivan E, Hohnson C, Magid R, Galis ZS. Remodeling of carotid artery is associated with increased expression of matrix metalloproteinase in mouse blood flow cessation model. Circulation 2000;102:2861–6. [6] Ivan E, Khatri JJ, Johnson C, et al. Expansive arterial remodeling is associated with increased neointimal macrophage foam cell content, the murine model of macrophage-rich carotid artery lesions. Circulation 2002;105:2686–91. [7] Shiomi M, Ito T, Hasegawa M, Yoshida K, Gould KL. Novel insights into coronary lumen preservation during progression of coronary atherosclerosis in coronary atherosclerosis-prone rabbits. Corn Artery Dis 2004;15:419–26.
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[8] Shiomi M, Ito T, Shiraishi M, Watanabe Y. Inheritability of atherosclerosis and the role of lipoproteins as risk factors in the development of atherosclerosis in WHHL rabbits: risk factors related to coronary atherosclerosis are different from those related to aortic atherosclerosis. Atherosclerosis 1992;96:43–52. [9] Shiomi M, Ito T, Yamada S, Kawashima S, Fan J. Development of an animal model for spontaneous myocardial infarction (WHHLMI rabbit). Arterioscler Thromb Vasc Biol 2003;23:1239– 44. [10] Ito T, Yamada S, Shiomi M. Progression of coronary atherosclerosis relates to the onset of myocardial infarction in an animal model of spontaneous myocardial infarction (WHHLMI rabbits). Exp Anim 2004;53:339–46. [11] Itabe H, Takeshima E, Iwasaki H, et al. A monoclonal antibody against oxidized lipoprotein recognizes foam cells in atherosclerotic lesions. Complex formation of oxidized phosphatidylcholins and polypeptides. J Biol Chem 1994;269:15274–9. [12] Matsukawa A, Ohkawara S, Maeda T, Takagi K, Yoshinaga M. Production of IL-1 and IL-1 receptor antagonist and the pathological significance in lipopolysaccharide-induced arthritis in rabbits. Clin Exp Immunol 1993;93:206–11. [13] Shiomi M, Ito T, Watanabe Y, et al. Suppression of established atherosclerosis and xanthomas in mature WHHL rabbits by keeping their serum cholesterol levels extremely low: effect of pravastatin sodium in combination with cholestyramine. Atherosclerosis 1990;83:69–80. [14] Shiomi M, Ito T, Tsukada T, Yata T, Ueda M. Cell compositions of coronary and aortic atherosclerotic lesions differ; an immunohistochemical study. Arterioscler Thromb 1994;14:931–7. [15] Nurke AP, Kolodgie FD, Farb A, Weber D, Virmani R. Morphological predictors of arterial remodeling in coronary atherosclerosis. Circulation 2002;105:297–303. [16] Ardans JA, Economou AP, Martinson Jr JM, Zhou M, Wahl LM. Oxidized low-density and high-density lipoproteins regulate the production of matrix metalloproteinase-1 and -9 by activated monocytes. J Leukoc Biol 2002;71:1012–8. [17] Bjorkerud B, Bjorkerud S. Contrary effects of lightly and strongly oxidized LDL with potent promotion of growth versus apoptosis on arterial smooth muscle cells, macrophages, and fibrblasts. Arterioscler Thromb Vasc Biol 1996;156:416–24. [18] Niemann-Jonsson A, Ares MPS, Yan Z-Q, et al. Increased rate of apoptosis in intimal arterial smooth muscle cells through endogenous activation of TNF receptors. Arterioscler Thromb Vasc Biol 2001;21:1909–14. [19] Libby P. Molecular bases of the acute coronary syndromes. Circulation 1995;91:2844–50. [20] Gengy J, Libby P. Evidence for apoptosis in advanced: colocalization with interleukin-1 beta converting enzyme. Am J Pathol 1995;147:251–66. [21] Shi Y, Pieniek M, Fard A, et al. Adventitial remodeling after coronary artery injury. Circulation 1996;93:340–8.
Invasion of atheromatous plaques into tunica media causes coronary outward remodeling in WHHLMI rabbits Masashi Shiomi a,∗ , Satoshi Yamada a , Akihiro Matsukawa b , Hiroyuki Itabe c , Takashi Ito a a
b
Institute for Experimental Animals, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentist and Pharmaceutical Science, Okayama University, Shikata-cho, Okayama 700-8558, Japan c Department of Biological Chemistry, School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo 140-8555, Japan Received 19 April 2007; received in revised form 3 February 2008; accepted 10 February 2008 Available online 16 February 2008
Abstract To clarify the mechanism of coronary outward remodeling, we examined atherosclerotic coronary arteries morphologically using WHHLMI rabbits that develop coronary atherosclerosis spontaneously. Perfusion-fixed coronary segments of WHHLMI rabbits were prepared at 500 m intervals. After immunohistochemical staining and histopathological staining, the areas and lengths of the arterial wall and the lesions were measured. Obvious outward remodeling was observed in coronary sections with greater than 40% cross-sectional narrowing. In coronary sections with greater than 40% cross-sectional narrowing, the tunica media was thick at the shoulder of atheromatous plaque and was thin beneath the atheromatous plaques. Macrophages infiltrated those attenuated tunica media expressed matrix metalloproteinases and oxidized LDL was accumulated in those areas. In those areas, collagen fibers and the internal elastic lamina had disappeared partly and apoptotic smooth muscle cells were observed. Proliferation of smooth muscle cells was observed at the attenuated tunica media and adjacent adventitia. The present results suggest that invasion of atheromatous plaques into the tunica media causes coronary outward remodeling. © 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Atherosclerosis; Coronary outward remodeling; Macrophage; Matrix metalloproteinase; Smooth muscle cells; WHHLMI rabbit
1. Introduction Enlargement of arteries suppresses the reduction of the lumen size in the progression of atherosclerosis and appears to prevent tissue ischemia. Arterial remodeling has been used to refer to changes in the cross-sectional area of arteries and consists of outward remodeling, enlargement of arterial size, and inward remodeling, reduction of arterial size. The first detailed study was reported by Glagov et al., who identified coronary outward remodeling as compensatory enlargement [1]. In response to atherosclerotic plaques, the arteries enlarge and the progression of luminal stenosis delays. Although several hypotheses for the mechanism have been proposed, for ∗
Corresponding author. Tel.: +81 78 382 5660; fax: +81 78 382 5679. E-mail address: [email protected] (M. Shiomi).
0021-9150/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2008.02.010
example, hemodynamic stimuli [2], degradation of fibrous elements in the arterial wall [3], high-density lipoprotein levels [4] and the relation of matrix metalloproteinase (MMP)-9 [5,6], no conclusive evidence has been reported to date. In our previous study about the relation between arterial size and coronary cross-sectional narrowing (CSN) [7] using coronary atherosclerosis-prone WHHL rabbits [8], findings similar to those in a previous human study [1] were observed if we did not take into account arterial tapering and individual variance. In the previous study, we observed that the tunica media at the lesion site showed thickening and/or attenuation compared to that at lesion-free sites, even in the same arterial sections. Therefore, we attempted to examine the mechanisms of coronary outward remodeling focusing on morphological changes of the tunica media of coronary arteries.
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2. Methods 2.1. Animals We used myocardial infarction-prone WHHLMI rabbits [9]; 8 rabbits (four rabbits each were 9 months old and 12 months old) for analyses of the relation of arterial size to CSN of the coronary arteries and 22 rabbits (17 rabbits were 9 months old, two rabbits were 13 months old, and three rabbits were 14 months old) for counting proliferating or apoptotic SMCs in the tunica media. Rabbits were anesthetized by an intravenous injection of sodium pentobarbital at a dose of 25 mg/kg of body weight followed by perfusion with 10% buffered neutral formalin solution or periodate-lysine-paraformaldehyde fixative under a constant pressure of 80 mmHg for 30 min. This study was approved by the Committee for Animal Experimentation, Kobe University School of Medicine (Permission numbers: P-011009 and P-011009R) and carried out according to the guidelines of Animal Experimentation of Kobe University, the Law for the Humane Treatment and Management of Animals (Law No. 105, 1973, revised in 1999), and the Standards Relating to the Care and Management, etc. of Experimental Animals (Notification No. 6, 1980). 2.2. Preparation of histological sections After perfusion fixation, the hearts were immersion-fixed with the same fixative indicated above and embedded in paraffin. Serial coronary sections were prepared using segments of the left circumflex arteries, which were prepared at intervals of 500 m. Since the left circumflex artery is a major coronary artery in rabbits and atherosclerotic lesions are very prevalent in this artery of WHHLMI rabbits [10], we examined arterial remodeling of the left circumflex arteries. Each section of every segment was stained with elastic van Gieson stain, hematoxylin and eosin stain, picrosirius red stain, or
immunohistochemically using monoclonal antibodies specific for rabbit macrophages (RAM-11), smooth muscle cell (SMC) actin (1A4), MMP-1, MMP-12, oxidized low-density lipoprotein (LDL) (DLH3) [11], proliferating cell nuclear (Ki-67), or goat polyclonal antibody specific for interleukin1 (IL-1) [12]. Immunohistochemical staining was carried out using a DAKO Envision + kit or Nichirei Histofine Simple Stain kit, or the labeled streptavidin-biotin alkaline phosphatase method with fuchsin chromogen according to the manufacturer’s instructions accompanied by hematoxylin counter staining. Staining of sections incubated with mouse IgG2a (for MMP-1), IgG2b (for MMP-12), and IgM (for DLH3), or goat IgG (for IL-1) instead of specific antibodies were negligible (data not shown). To detect apoptotic cells in arterial walls, TUNEL staining was carried out using ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit. 2.3. Measurement of arterial size and counting cells According to our previous study [13,14], the length of the external elastic lamina (EEL) and the area of the tunica media, intimal lesions, and lumen were measured with computer assisted color image analysis (Image-Pro Plus, version 4.5, Media Cybernetics Inc., Silver Spring, MD, USA) using sections stained with elastic van Gieson stain. Tunica media was defined as layers of SMCs plus collagen fibers located inside the EEL if the internal elastic lamina had disappeared or the tunica media was partly degenerated by macrophage infiltration. Average thickness of the whole tunica media of each coronary section was calculated by dividing the tunica media area by the length of the EEL. CSN was calculated by following equation; [CSN (%)] = [lesion area]/([lesion area] + [lumen area]) × 100. The numbers of SMCs or macrophages in the tunica media were counted using sections stained immunohistochemically with 1A4 or RAM-11 followed by hematoxylin counter stain.
Fig. 1. Photomicrographs of coronary arterial sections of a WHHLMI rabbit aged 9 months old showing typical outward remodeling with the growth of atherosclerotic lesions. These sections were stained with elastic van Gieson staining. Arrowheads indicate attenuation of the tunica media and arrows indicate thickening of the tunica media. CSN, cross-sectional narrowing; EEL, external elastic lamina.
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2.4. Statistical analysis Data are represented as the mean ± standard error. Statistical analyses were carried out with the Dunnett multiple comparison test for mean values and with the Cochran–Mantel–Haenszel test for frequency. Significance was set at P < 0.05.
3. Results 3.1. Typical outward remodeling of the circumflex artery Fig. 1 shows the typical outward remodeling observed in the serial segments of the left circumflex artery. The tunica media was thick at the shoulder of the lesions (sections 2 and 4) and thin under the large intimal plaque (section 3), although the thickness of the tunica media is relatively uniform at arterial sections without lesions (sections 1 and 5). The internal elastic lamina partly disappears under the intimal
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plaque of sections 2 and 3. Those findings suggest that both thickening and attenuation of tunica media may be associated with coronary outward remodeling. 3.2. Changes in arterial size and cell number in the tunica media In longitudinal observation from the proximal to distal segments of the coronary arteries of WHHLMI rabbits after maturation, atherosclerotic lesions show early lesion at the plaque edge and established lesion at the plaque center. We examined changes in the arterial size using 54 coronary segments from 9 atheromatous plaques of 8 rabbits aged 9–12 months old. In comparison with the proximal lesionfree coronary section, the frequency of coronary sections showing enlargement of the coronary periphery (enlargement degree greater than 1.1) increased with progression of CSN (P < 0.001, Fig. 2A). Regarding the thickness of the tunica media at the lesion site (Fig. 2B), the frequency of coronary sections showing thinning of the tunica
Fig. 2. Relation of coronary cross-sectional narrowing (CSN) to the arterial size and cell number in the tunica media. Panel-A shows enlargement of the coronary periphery with an increase of CSN compared to those in the proximal lesion-free section. The coronary periphery was evaluated in relation to the length of the external elastic lamina (EEL). The X-axis shows the ratio of the EEL length of each arterial sections/the EEL length of the proximal lesion-free coronary section. A value of 1.1 means the EEL length was 10% longer than that of the proximal lesion-free section. Panel-B shows the thickness of the tunica media at the lesion site with increasing CSN. Panel-C shows changes in SMC content throughout the whole tunica media with progression of CSN compared to that in the proximal lesion-free section. The X-axis shows the ratio of SMC content throughout the whole tunica media of each coronary section/that of the proximal lesion-free section. A value of 1.1 means the SMC content was 10% more than that of the proximal lesion-free section. Panel-D shows the number of SMC in the tunica media in relation to the length of the tunica media (EEL length) at the lesion site. Panel-E shows an increase in the number of macrophages that infiltrated the tunica media with an increase of CSN. The X-axis shows the number of macrophages that infiltrated the tunica media in relation to the length of tunica media (EEL length) with lesion. Statistical analysis was carried out for the frequency with Cochran–Mantel–Haenszel test. Gray bars, coronary segments showing less than 20% CSN; white bars, coronary segments showing 20 to <40% CSN; crossed bars, coronary segments showing 40 to <60% CSN; black bars, coronary segments showing 60 to 85% CSN.
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media (less than 40 m) increased with progression of CSN (P < 0.001). These findings indicate that with the growth of atheromatous plaques, the tunica media was elongated and attenuated. In comparison with the proximal lesion-free coronary section, the frequency of coronary sections showing an increase in SMC number throughout the whole tunica media (degree greater than 1.1) increased with progression of CSN
(P = 0.007, Fig. 2C). Fig. 2D shows that tunica media SMC content in relation to tunica media length (EEL length) was decreased (less than 100 cells/mm) at the lesion site (Fig. 2D) when CSN was greater than 60% although the frequency of coronary sections showing high SMC number (more than 200 cells/mm) was higher when the CSN was less than 40% (P < 0.001). Fig. 2E shows that the number of macrophages
Fig. 3. Photomicrographs of coronary sections of WHHLMI rabbits aged 9 months old showing attenuation of the tunica media. Elastic van Gieson staining (A, H, I), picrosirius red staining (B), immunohistochemical staining with 1A4 for smooth muscle cell (SMC) (C and J), RAM-11 for macrophage (D), matrix metalloproteinase-12 (MMP-12) (E), MMP-1 (F), DLH3 for oxidized-LDL (G and L), and IL-1 (M), and double staining with RAM-11 and TUNEL (K). Symbols in the photomicrographs indicate the following; arrowheads, internal elastic lamina; large arrows, macrophages (A–G), TUNEL-positive cells (K), and oxidized LDL (L); asterisks, tunica media; parallel cross, tunica adventitia.
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Table 1 The number of TUNEL-positive or Ki-65-positive spindle-shaped cells in the coronary arterial walls of WHHLMI rabbits Cross-sectional narrowing
1–29% 30–59% 60–85%
n
11 12 14
TUNEL-positive cells
Ki-65-positive cells
Tunica media
Tunica adventitia
Total
Tunica media
Tunica adventitia
Total
0.00 ± 0.00 0.17 ± 0.11 0.64 ± 0.25*
0.09 ± 0.09 0.25 ± 0.13 0.57 ± 0.25
0.09 ± 0.09 0.42 ± 0.23 1.21 ± 0.46*
0.27 ± 0.27 0.08 ± 0.08 1.86 ± 0.64*
0.00 ± 0.00 0.58 ± 0.36 1.71 ± 0.55†
0.27 ± 0.27 0.67 ± 0.43 3.57 ± 1.06†
Data are represented as the mean ± S.E. Statistical analyses were carried out with Dunnett multiple comparison test in comparison with the coronary sections with cross-sectional narrowing 1–29% (* P < 0.05; † P < 0.01).
that infiltrated the tunica media increased with progression of CSN (P = 0.003). These findings demonstrate that although coronary arteries enlarged with an increase of SMCs throughout the whole tunica media, infiltration of macrophages into the tunica media increased and the number of SMC in the tunica media was decreased at the lesion sites in the coronary sections showing greater than 60% CSN. 3.3. Histopathological and immunohistochemical examination of the attenuation of coronary tunica media In Fig. 3A, the coronary tunica media was attenuated beneath the atheromatous plaque and the internal elastic lamina had partly disappeared. In the attenuated tunica media, collagen fibers (Panel-B) and SMCs (Panel-C) had partly decreased. At those areas, macrophages infiltrated the tunica media (Panel-D) or vacuoles (supposedly derived from collapsed macrophages) were observed (Panel-A). Those macrophages expressed MMP-12 (Panel-E) and MMP-1 (Panel-F). In addition, oxidized LDL was detected in those areas (Panel-G). The positive areas for oxidized LDL cor-
responded to the positive areas of RAM-11, MMP-12 and MMP-1. In the MMP-12-positive area, the internal elastic lamina disappeared (Panel-A–G) and the collagen layers of the tunica media were attenuated (Panel-B) at the MMP-1positive area. These observations suggest that macrophage infiltration into the tunica media strongly related to attenuation of the tunica media by secretion of MMP-1 and -12. In Fig. 3H and I, the internal elastic lamina had partly disappeared, 1A4-positive SMC decreased in the tunica media (Panel-J), and TUNEL-positive cells (PanelK) were observed in the intimal plaque (RAM-11-positive macrophages) and tunica media (RAM-11-negative SMCs). In this area, DLH3 monoclonal antibody for oxidized LDL (Panel-L) and IL-1 (Panel-M) were positive. In TUNELstaining of coronary arterial walls (Table 1), there were no TUNEL-positive spindle-shaped cells in the coronary tunica media with CSN less than 30% and the number increased significantly with progression of CSN. These findings suggest that apoptosis of SMCs in the tunica media probably related to attenuation of the coronary tunica media.
Fig. 4. Photomicrographs of SMC proliferation in coronary sections of WHHLMI rabbits aged 14 (A–C) and 9 (D–F) months old. Serial coronary sections were stained with elastic van Gieson stain (A and D) and immunohistochemically with 1A4 plus Ki-67 for proliferating cells (B, E and F), and RAM-11 for macrophage (C). Symbols in the photomicrographs indicate the following; arrowheads, internal elastic lamina; small arrows, external elastic lamina; large arrows, proliferating SMCs; asterisks, tunica media; parallel cross, tunica adventitia.
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3.4. Histopathological and immunohistochemical examination of proliferating SMCs Since the SMC number in the tunica media increased with progression of CSN (Fig. 2C), SMC proliferation in the coronary wall was examined immunohistochemically (Fig. 4). At the shoulder of atheromatous plaque, the tunica media was thick (Panel-A) and several Ki-67-positive cells (proliferating cells) were observed in the intimal lesion and the tunica media (Panel-B). Ki-67-positive cells in the tunica media were 1A4-positive (SMC) and those in the intimal lesions were 1A4-negative (macrophages). There were no RAM-11positive cells in the tunica media (Panel-C). At the bottom of a large intimal plaque (Panel-D), proliferating SMCs were observed in the tunica adventitia (Panel-E) and tunica media (Panel-F). There are two types of Ki-67-positive spindleshaped cells in the coronary adventitia. One is 1A4-positive (SMC) and the other is 1A4-negative. 1A4-negative spindleshaped cells may be proliferating fibroblasts. The number of Ki-67-positive spindle-shaped cells in the tunica media and adventitia increased significantly with progression of CSN (Table 1). These findings suggest that proliferation of SMCs and fibroblasts in the coronary arterial wall is probably related to coronary outward remodeling.
4. Discussion The present observations suggest that the primary factor in coronary outward remodeling due to atherosclerosis is macrophage infiltration into the tunica media. Those macrophages expressed MMPs. Consequently, in the coronary tunica media, the internal elastic lamina and collagen fibers disappeared, probably due to degradation by MMPs and the SMC density decreased due to apoptosis. Finally, the coronary tunica media was attenuated and became fragile. However, proliferation of SMCs in the coronary tunica media is also probably related to outward remodeling. Proliferating SMCs were observed mainly at the attenuated tunica media and the adjacent adventitia in addition to that at the shoulder of atheromatous plaque. We consider that findings of the coronary tunica media observed in Fig. 3A–G demonstrate the process of attenuation of the coronary tunica media by macrophage infiltration. Lesion-free coronary tunica media consists of SMCs with a small amount of collagen fibers (Panel-B and C). At the shoulder of atheromatous plaque, collagen fibers partly replaced SMCs in the thick tunica media (Panel-B and C) adjacent to the site of macrophage infiltration. At the area showing macrophage infiltration where the tunica media was attenuated (Panel-B–D), both SMCs and collagen fibers were decreased. In those areas, macrophages expressed MMP-12 (Fig. 3E) and MMP-1 (Fig. 3F). Since MMP-12 is an elastase, disappearance of the internal elastic lamina (Fig. 3A) was probably due to degradation by MMP-12. Since MMP-1 is an interstitial collagenase, disappearance of the collagen layers
in the tunica media was probably due to degradation by MMP1. Similarly, in studies of murine models, MMP-9 expression was observed throughout the entire plaque in carotid arteries that showed outward remodeling [5,6]. The present findings suggest a mechanism underlying atrophy of the tunica media associated with outward arterial remodeling in human coronary arteries [15]. In addition, since oxidized LDL stimulates the synthesis of MMPs in activated monocytes [16], accumulation of oxidized LDL in the atheromatous plaque (Fig. 3G) probably relates to expression of MMP-1 and -12 in macrophages that infiltrated the tunica media. Number of SMC in the tunica media in relation to EEL length at lesion site decreased with progression of atheromatous plaques (Fig. 2D). Regarding a decreased number of SMC in the tunica media, TUNEL-positive SMC in the tunica media and adventitia increased with progression of CSN (Table 1) and those areas were positive for oxidized LDL and IL-1 (Fig. 3L–M). Since in vitro studies have demonstrated that oxidized LDL [17], IL-1 [18], and other cytokines relate to SMC apoptosis [19,20], accumulation of oxidized-LDL and IL-1 in the coronary tunica media probably relate to SMC apoptosis in the tunica media. Therefore, we consider that the tunica media was attenuated beneath the large atheromatous plaques due to the combined degradation of the internal elastic lamina and collagen fibers and decrease in SMCs. Consequently, arterial walls became fragile and probably enlarged easily. In the present study, SMCs increased in the tunica media (Fig. 2C) but the SMC density decreased at the attenuated tunica media (Fig. 3C). These findings appear to be contradictory. Since Ki-67-positive SMCs were observed at the attenuated tunica media where the SMC density was low (Fig. 4), proliferation and decrease of SMCs are observed simultaneously in attenuated tunica media. Those proliferating cells consisted of 1A4-positive and negative spindle-shaped cells. These findings may suggest that those proliferating SMCs and 1A4-negative spindle-shaped cells (probably fibroblasts) play a role in the repair of the attenuated tunica media. Shi et al. [21] reported the involvement of the adventitia in the vascular repair process after the tunica media injury in porcine coronary arteries. Therefore, proliferating cells in the adventitia observed in the present study may relate to the repair process of attenuated coronary arterial wall. Further examination is required. The present observation suggests that attenuation of the tunica media by macrophages and SMC proliferation at the attenuated coronary wall were probably repeated during the growth of atheromatous lesions and then coronary arteries enlarged. These observations suggest that atheromatous lesions expand in the circumference direction of coronary arteries (outward) as well as in the lumen direction (inward). Therefore, we consider that invasion of atheromatous plaques into the tunica media causes coronary outward remodeling in WHHLMI rabbits. In conclusion, the present findings demonstrated that coronary arteries enlarged and were attenuated in the coronary
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sections showing greater than 40% CSN mainly due to macrophage infiltration into the tunica media. Degradation of the internal elastic lamina and collagen fibers in the tunica media due to MMPs secreted by those macrophages and reduction of SMCs in the tunica media due to apoptosis probably related to the attenuation and weakening of the coronary wall. These findings suggest that coronary outward remodeling was due to invasion of atheromatous plaques into the tunica media.
Acknowledgements This work was supported in part by unrestricted research grants from Sankyo Co. Ltd. We would like to express our appreciation of Mr. Toshiaki Tamura of the Institute for Experimental Animals, Kobe University School of Medicine for the preparation of histological sections. We are also grateful to Miss Kaori Yamane and Miss Haruka Adachi of ASK Co. Ltd. for animal care.
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