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A novel model of intimal hyperplasia with graded hypoosmotic damage
Cardiovascular Pathology 21 (2012) 490 – 498
Original Article
A novel model of intimal hyperplasia with graded hypoosmotic damage Mowei Songa,b,c,1 , Hong-tao Shend,1 , Jin-jin Cuic , Xin-gang Zhoub , Xin Zhonge , Cheng-hai Pengc , Hong-yu Liub,⁎, Ye Tiana,c,e,⁎ a
b
Department of Cardiology, First Affiliated Hospital, Harbin Medical University, Harbin, 150001, Heilongjiang, China Department of Cardiovascular Surgery, First Affiliated Hospital, Harbin Medical University, Harbin, 150001, Heilongjiang, China c Department of Cardiology, Second Affiliated Hospital, Harbin Medical University, Harbin, 150001, Heilongjiang, China d Second Department of Orthopedics, First Affiliated Hospital, Harbin Medical University, Harbin, 150001, Heilongjiang, China e Department of Pathophysiology, Bio-pharmaceutical Key Laboratory of Heilongjiang Province, Harbin Medical University, Harbin, 150086, Heilongjiang, China Received 2 December 2011; received in revised form 19 February 2012; accepted 20 February 2012
Abstract Background: The purpose was to develop a rabbit model of intimal hyperplasia with controllable lesion. Methods: Following 1 week of a 2% cholesterol diet, 32 New Zealand White male rabbits underwent right femoral arteries surgical perfusion with distilled water for 1, 3, 5, or 7 min (n=8/group). After a further 4 weeks of the same diet, serum total cholesterol, triglyceride, low-density lipoprotein, and high-density lipoprotein were measured in all rabbits. Intimal hyperplasia in histological sections of arteries were assessed by intimal proliferation ratio. Macrophage numbers and levels of proteins matrix metalloproteinase 9, tissue inhibitor of metalloproteinase 2, and alpha smooth muscle actin in lesions were analyzed by immunohistochemistry. Results: Serum lipids levels showed no statistical difference between experimental groups. Intimal proliferation ratio increased gradually with perfusion time, and a positive linear correlation was calculated between intimal proliferation ratio and duration of distilled water perfusion. Similarly, number of macrophages and levels of matrix metalloproteinase 9, tissue inhibitor of metalloproteinase 2, and alpha smooth muscle actin in lesions increased with perfusion time. Conclusions: A novel model of intimal hyperplasia was established by intravascular distilled water perfusion in high-cholesterol-fed rabbits. Importantly, this model exhibits time-dependent neointimal proliferation lesions that can be readily controlled in terms of extent, thus providing an avenue for further studies. © 2012 Elsevier Inc. All rights reserved. Keywords: Intimal hyperplasia; Hypoosmotic injury; Hyperlipidemia; Endothelial cells; Rabbit
1. Introduction Accelerated intimal hyperplasia is an important cause of morbidity and mortality in patients with atherosclerosis. Intimal proliferation and migration of smooth muscle cells have for a long time been emphasized in the development of atherosclerotic plaques [1,2] as well as in the process of There was no conflict of interest in the study. ⁎ Corresponding authors. Hong-yu Liu is to be contacted at Department of Cardiovascular Surgery, First Affiliated Hospital, Harbin Medical University, Harbin, 150001, Heilongjiang, China. Tel.: +86045185555817; fax: +86045185555817. Ye Tian Department of Cardiology, First Affiliated Hospital, Harbin Medical University, Harbin, 150001, Heilongjiang, China. Tel.: +86045185555943; fax: +86045185555943. E-mail addresses: [email protected] (H. Liu), [email protected] (Y. Tian). 1054-8807/12/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.carpath.2012.02.007
restenosis following angioplasty [3]. In the current vascular interventional environment, high restenosis rates after stents graft and veins graft have also increased awareness of the significance of intimal hyperplasia [4,5], a chronic structural lesion that develops after vessel wall injury and which can lead to luminal stenosis and occlusion [6,7]. Intravascular endothelial injury is central to intimal hyperplasia, and excessive accumulation of cells can be observed with a subsequent intimal thickening causing restenotic lesion formation [8]. However, endoluminal manipulation of small arteries is too technically difficult to develop different intimal injury. As excessive cholesterol can lead to atherosclerotic changes in the arterial intima, and lesions appear to develop as a result of repeated or continuous intimal injury, various intimal hyperplasia animal models have been developed based on
M. Song et al. / Cardiovascular Pathology 21 (2012) 490–498 Table 1 Lipid levels in experimental groups Group
TC
TG
LDL
HDL
1 min 3 min 5 min 7 min g
23.07±10.37 20.52±11.90 14.0±11.87 29.86±11.86
1.18±0.40 0.89±0.60 1.03±0.38 2.45±2.11
17.75±8.19 15.96±9.75 10.53±9.85 23.13±9.77
4.57±2.55 4.84±2.55 3.00±2.07 5.64±2.38
Values are expressed in millimoles as mean±S.E.M. Groups refer to the duration of exposure to distilled water perfusion.
high-cholesterol diet, arterial wall injury, or a combination of the two approaches. But, to date, there are no mature reproducible models with controllable intimal–proliferation lesions. Distilled water can cause hypoosmotic stress with resulting cell swelling, dysfunction, and even hypotonic shock [9–11]. As intravascular perfusion with distilled water can result in superficial intimal injury of rabbit artery [12], we hypothesized that this intimal injury would help to develop a rabbit model of intimal hyperplasia on a highcholesterol diet. Moreover, as such endothelial injury likely increases with distilled water perfusion time, this novel model may allow the time-dependent study of preatherosclerotic intimal hyperplasia development. This is to our knowledge the first report on the rabbit model with controllable lesions of intimal hyperplasia.
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Table 2 IPR; macrophage numbers; and expression levels of MMP9, TIMP2, and α-SMA in plaque samples Group
IPR
MMP9
TIMP2
Macrophage
α-SMA
1 3 5 7
0.80±0.20 0.82±0.13 0.93±0.06 0.94±0.04
0.05±0.03 0.08±0.02 0.09±0.03 0.13±0.02
0.10±0.06 0.11±0.03 0.13±0.04 0.18±0.08
0.04±0.02 0.07±0.02 0.09±0.03 0.20±0.04
0.11±0.02 0.15±0.04 0.15±0.04 0.21±0.05
min min min min
Values are expressed as median value±S.E.M.
2. Methods 2.1. Animal model Thirty-two adult male New Zealand White rabbits, weighing 2.5 to 3.0 kg, were randomly divided into four treatment groups (n=8, each). All received a standard laboratory diet supplemented with cholesterol (2%) and lard (2%) for 1 week prior to surgery and 4 weeks thereafter. A surgical level of anesthesia was induced by intramuscular injection of ketamine hydrochloride (40 mg/kg) and xylazine (5 mg/kg). The right femoral artery of each rabbit was surgically exposed, and a 3-cm segment was isolated using two artery clamps. A 28-ga needle was inserted into the proximal end of the segment, and the segment was perfused
Fig. 1. Histological sections demonstrating increasing intimal thickness correlated with increasing time of exposure to distilled water. The arrows highlight increasing plaque, and an increasing IPR can be readily appreciated. All images are of H&E-stained sections at 100×. (A) One minute, (B) 3 min, (C) 5 min, and (D) 7 min. (E) The positive linear relationship between the median value of IPR and perfusion time: y=0.75188+0.02988x; Sy=0.08023 and Sxy=0.02200.
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with distilled water. For the four treatment groups, perfusion times were 1, 3, 5, or 7 min. Following perfusion, the circulation was reestablished, hemostasis was ensured, and the surgical incision was closed. The contralateral femoral artery of each rabbit served as a control. All procedures were performed in compliance with the Principles of Laboratory Animal Care and Guide for the Care and Use of Laboratory Animals (NIH publication No. 80-23, revised 1985) and were approved by the Animal Care and Use Committee of Harbin Medical University. 2.2. Measurement of serum lipids Abdominal aorta blood samples were taken by catheter under anesthesia. Serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and highdensity lipoprotein (HDL) were measured using the Olympus AU5400 Clinical Chemistry System. 2.3. Histomorphometry and immunohistochemistry Four weeks after surgery, rabbits were euthanized by an intravenous overdose of pentobarbital. For harvesting of specimens, perfusion–fixation was done with 4% paraformaldehyde in phosphate-buffered saline at 100 mmHg for 15 min. Five-micrometer-thick sections, cut 100 μm apart from each other, were stained with hematoxylin and eosin (H&E)
and immunochemistry. Images of stained sections were taken with an Olympus IX70 microscope (magnification ×100) and analyzed by using computerized morphometry (Image-Pro Plus, version 6.0, Media Cybernetics, Silver Spring, MD, USA). Intimal proliferation ratio (IPR), also called intima-to-media ratio, was calculated to measure the percentage of luminal narrowing as previously described [13]. Arteries with occlusive thrombus were not included in the morphometric analysis. Numbers of macrophages and levels of expression of matrix metalloproteinase 9 (MMP9), tissue inhibitor of metalloproteinase 2 (TIMP2), and smooth muscle α-actin (α-SMA) in lesions, associated with degree of neointimal proliferation, were determined by immunohistochemistry. The primary monoclonal antibodies used were as follows: mouse anti-rabbit α-smooth muscle actin antibody (1:2000, Sigma-Aldrich, St. Louis, MO, USA) to identify smooth muscle cells (SMCs), mouse anti-rabbit macrophage antibody (anti-RAM-11, 1:1500, Dako North America, Carpinteria, CA, USA) to identify macrophage cells, mouse anti-rabbit MMP9 antibody (Millipore, Billerica, MA, USA) to identify MMP9, and mouse anti-rabbit TIMP2 antibody (Thermo Fisher Scientific, Waltham, MA, USA) to identify TIMP2. All immunohistochemistry slides were viewed with a Leica DM 2500 microscope, and Maikeaodi Instrument Company software was used for gray-scale image analysis.
Fig. 2. Immunohistochemical evidence for increased MMP9 expression in plaque of arteries exposed to distilled water for different time periods. (A) One minute, (B) 3 min, (C) 5 min, and (D) 7 min. Sections were examined at 100×. (E) The linear relationship between the median MMP9 levels and perfusion time: y=0.03744+0.01231x; Sy=0.03253 and Sxy=0.000687.
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2.4. Statistical analysis Values of serum lipid levels were expressed as mean± standard deviation (x±S.D.) and analyzed by the Newman– Keuls method. Values of IPR and all indicators above tested by immunohistochemistry are presented as median values. Comparative differences of each indicator among the four experimental groups were analyzed by the Kruskal–Wallis one-way analysis-of-variance-by-ranks (H) test, followed by the Nemenyi test and linear regression analysis. A value of Pb.05 was considered to be indicative of a statistically significant difference. 3. Results 3.1. Serum lipid levels Following 5 weeks of the high-cholesterol diet (1 week before and 4 weeks after surgery), the serum lipid levels in all experimental rabbit groups were significantly higher than the baseline [14]. Severe hypercholesterolemia; elevated levels of TC, TG, and LDL; and decreased HDL-cholesterol are detailed in Table 1. No differences in lipid levels were detected between the four experimental groups (Table 1).
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3.2. Histological features of distilled water perfusion-induced intimal hyperplasia A complex lesion is formed with significant neointimal hyperplasia in sections of surgical femoral arteries. Foam cells as well as extracellular lipid are prominent in the neointima and, to a lesser degree, the media as well (Fig. 6G).The degree of intimal hyperplasia markedly developed from the 1-min group to the 7-min group in turn (Fig. 1A–D). The median values of IPR (MIPR) for the four groups increased with perfusion time (Table 2) and demonstrated an apparent linear relationship described by y=0.751882+(0.029882x); (y means MIPR, and x means distilled water perfusion time Sy=0.08023 and Sxy=0.02200; Fig. 1E). The MIPR of the four groups showed an overall statistical difference by the H test (H=10.72, n'=3, Pb.05), with a significant difference found between the 1-min and 7-min groups (Nemenyi test, r=0.961664, Pb.01). 3.3. Immunohistochemistry The intimal layer in sections of surgical femoral arteries shows increased cellularity, not only SMCs and macrophages but also deposition of MMP9 and TIMP2 without disruption of the internal elastic lamina (circles). And the
Fig. 3. Immunohistochemical evidence for increased a-SMA expression in plaque of arteries exposed to distilled water for different time periods. (A) One minute, (B) 3 min, (C) 5 min, and (D) 7 min. Sections were examined at 100×. (E) The linear relationship between the median MMP9 levels and perfusion time: y=0.09697+0.014x; Sy=0.08009 and Sxy=0.02898.
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Fig. 4. Increased TIMP2 expression in the plaque of four group samples: (A) One minute, (B) 3 min, (C) 5 min, and (D) 7 min (all immunohistochemistry, original magnification 100×). (E) The linear equation between the median M of the TIMP2 level in plaque and perfusion time: y=0.07943+0.01261x; Sy=0.03483 and Sxy=0.0123497.
number of macrophages and expression levels of MMP9, TIMP2, and α-SMA in neointimal proliferation lesions also increased with distilled water perfusion time (Table 2 and Figs. 5A–D, 2A–D, 3A–D, and 4A–D). Moreover, four equations were calculated to describe positive linear correlation between perfusion time and median value of MMP9 (MMMP9), median value of α-SMA (Mα-SMA), median value of TIMP2 (MTIMP2), and median value of macrophages (Mmacrophage), which are separately shown in Figs. 2E, 3E, 4E, and 5E. When comparing between four groups, differences of MMMP9, Mα-SMA, and Mmacrophage were significant with Pb.01, but difference of MTIMP2 was only with Pb.05. When comparing between each two groups, especially in the 1-min group and 7-min group, evident differences were also verified by Nemenyi testing. Difference of MMMP9 was obvious in the 1-min and 5-min, 3-min and 7-min (Pb.05), and 1-min and 7-min groups (Pb.01). Nemenyi testing confirmed significant differences of Mα-SMA between the 1-min and 7-min groups (Pb.01) and between the 1-min and the other two groups (Pb.05). MTIMP2 in proliferated intimal showed a significant difference between the 1-min and 7-min groups (Pb.05). Difference of Mmacrophage was statistical between the 1-min and 5-min groups (Pb.05) and was evident between the 7-min and other three groups (each Pb.01).
4. Discussion In this study, we have established a novel rabbit model of intimal hyperplasia by intravascular distilled water perfusion. Distilled water perfusion was used to induce arterial endothelium injury, and damage occurred as a result of hypotonic challenge and endothelial cell swelling which affected numerous regulators of cell metabolism and transport [15]. So, this model is based on physical injury caused by low osmotic pressure, which is mechanistically different to currently used models, such as mechanical balloon catheter-induced injury [16,17], cold-induced injury [18], chemical injury [19], air-drying [20], nitrogen bubble techniques [21], and the Watanabe heritable hyperlipidemic rabbits (WHHL rabbits) [22,23]. The balloon injury-induced model, as the most popular model, is based on mechanical injury caused by balloon dilation to the vascular intima and inner elastic lamina, which are always with vessel medial damage. As the injury caused by balloon dilation is susceptible to many factors, including balloon pressure and the matching relationship between balloon diameter and vessel lumen [24], the degree of mechanical lesions is difficult to control, and the damage is easy to be a partial shedding of endothelial cells but not consistent (shown in Fig. 6A). Contrast to this mechanical injury, this physical injury is
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Fig. 5. Increased numbers of macrophages in the plaque of four group samples: (A) One minute, (B) 3 min, (C) 5 min, and (D) 7 min (all immunohistochemistry, original magnification 100×). (E) The linear equation between the median M of macrophage in plaque and perfusion time: y=0.0015529+0.02446x; Sy=0.06871 and Sxy=0.027063.
uniform. It may be due to the same hypoosmotic environment caused by intravascular distilled water perfusion and then the same hypoosmotic injury to all endothelial cells. Moreover, this hypoosmotic damage is limited to the endothelium without disruption of the elastic lamina or overt medial changes [12], as proven in preliminary experiments (shown in Fig. 6B). Most importantly, this model of intimal hyperplasia exhibited an important advantage due to the perfusion timerelated characteristics. Corresponding to a loss of arterial
lumen, IPR is the standard for histological evaluation of the extent of neointimal proliferation [13]. In this study, we found that MIPR increased with distilled water perfusion time as a positive line. It is conceivable that a longer perfusion time causes greater hypoosmotic injury to the endothelial cells, resulting in more neointimal cell necrosis and more serious proliferation under hyperlipidemic conditions. Lipid accumulation is associated with decreased endothelial cells migration and intimal hyperplasia [25]. And in
Fig. 6. Representative light micrographs of femoral artery. (A–D) H&E-stained sections. (E and F) Masson's trichrome-stained sections. (G) Oil red O-stained section. (A) Balloon injury, 400×; (B) distilled water injury, 400×; (C) distilled water injury with high-fat feeding, 100×; (D) high-fat feeding only, 100×; (E) high-fat feeding only, 400×; (F) distilled water injury with high-fat feeding 200×; (G) disposition of lipid in intimal hyperplasia lesions, 100×.
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the absence of hypercholesterolemia, intimal injury and hyperplasia regress and resolve spontaneously, but in the presence of hypercholesterolemia, they are not only sustained but progress to larger lesions [26]. After being combined with hyperlipidemia, neointimal proliferation developed rapidly after hypoosmotic injury in each group. Although hypercholesterolemia can accelerate intimal hyperplasia [25,27], lesions were not apparent in the contralateral femoral artery without water perfusion (Fig. 6D and F). Maybe, 5 weeks of treatment with a 2% cholesterol diet was, by itself, insufficient to induce neoproliferation in the femoral artery of rabbits, or a 5-week time frame is too short. Progressive intimal hyperplasia lesions contained SMC and macrophages [28]. Proliferation and migration of smooth muscle cells have for a long time been emphasized in the development of atherosclerosis [29] as well as in the process of restenosis following angioplasty [3]. The accumulation of SMC, in response to wall injury, contributes to arterial wound repair and thickening of the intimal layer [30,31]. Recent studies have suggested that SMC-derived factors, such as Tissue factor (TF), also plays an important role in promoting arterial thrombosis and in mediating intimal hyperplasia in response to arterial injury [32]. Macrophages contribute to stimulating SMC migration [33,34] and proliferation [35]. Moreover, macrophages themselves also play key roles in the development of intimal hyperplasia [36–38]. Increased production of MMP9, as a necessary component for collagen organization by SMC, degrades extracellular matrix and facilitates the migration of SMC, which is a fundamental process in arterial intimal hyperplasia [39,40]. TIMP2 is expressed and secreted by neointimal SMC and has been implicated in the progress of neointimal hyperplasia [41,42]. In this study, not only increased numbers of macrophages but also increased expression level of α-SMA, MMP9, and TIMP2 were found in lesions of distilled-water-treated arteries. Furthermore, a positive linear relationship was also observed between these four indicators in lesions and the duration of distilled water perfusion. As we known, the underlying pathophysiology of venous graft disease and late bypass failure includes intimal hyperplasia and subsequent accelerated atherosclerosis [43]. In this study the four indicators, such as macrophage [44–47], α-SMA [48], MMP9 [49–53], and TIMP2 [54], all contributed to the severity of atherosclerosis and eventual plaque rupture. It means that with intimal hyperplasia accelerating, the lesions trend to be vulnerable plaques. Consistent with this inference, we have found the lesions in some sections, characterized by a lipidrich core, accumulation of macrophages, thin fibrous cap, and arterial neovascularization (Fig. 6C). In addition to the duration of distilled water exposure, a number of other factors in this model may affect the progression of intimal hyperplasia in the damaged arteries, including the area exposed to water perfusion, lumina diameter of artery, perfusion pressure, and so on. These may result in different extent of endothelial damage and intimal
hyperplasia. Another important consideration is that we examined the responses to injury at 4 weeks. It is uncertain whether a steady state had been achieved at that time, although it is assumed that, at a particular time point, endothelial injury and intimal hyperplasia cease developing. Future studies should be directed at studying these aspects of this new time-dependent atherosclerotic model. In summary, this model is a combination of hypoosmotic damage made by distilled water and hyperlipidemia. Importantly, intimal hyperplasia is positively correlated with perfusion time. This method is attractive because of its relative simplicity, short operative time, safe procedure, and bilateral operation. Acknowledgments The authors would like to thank Chao Gao for expert skill in performing all survival surgeries, Lei Shi for the use of her fluorescence microscope and camera system, Xue-feng Zhang for figures modification, and Medjaden Bioscience Limited for revisions to parts of the content of the article. References [1] 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;91:1469–80. [2] Hanke H, Strohschneider T, Oberhoff M, Betz E, Karsch KR. Time course of smooth muscle cell proliferation in the intima and media of arteries following experimental angioplasty. Circ Res 1990;67:651–9. [3] Kamenz J, Seibold W, Wohlfrom M, Hanke S, Heise N, Lenz C, et al. Incidence of intimal proliferation and apoptosis following balloon angioplasty in an atherosclerotic rabbit model. Cardiovasc Res 2000; 45:766–76. [4] Kwa AT, Yeo KK, Laird JR. The role of stent-grafts for prevention and treatment of restenosis. J Cardiovasc Surg (Torino) 2010;51:579–89. [5] Wallitt EJ, Jevon M, Hornick PI. Therapeutics of vein graft intimal hyperplasia: 100 years on. Ann Thorac Surg 2007;84:317–23. [6] Davies MG, Hagen PO. Pathobiology of intimal hyperplasia. Br J Surg 1994;81:1254–69. [7] Desai M, Mirzay-Razzaz J, von Delft D, Sarkar S, Hamilton G, Seifalian AM. Inhibition of neointimal formation and hyperplasia in vein grafts by external stent/sheath. Vasc Med 2010;15:287–97. [8] O'Brien ER, Alpers CE, Stewart DK, Ferguson M, Tran N, Gordon D, et al. Proliferation in primary and restenotic coronary atherectomy tissue: implications for antiproliferative therapy. Circ 1993;73:223–31. [9] Fuse H, Ohta S, Sakamoto M, Kazama T, Katayama T. Hypoosmotic swelling test with a medium of distilled water. Arch Androl 1993;30: 111–6. [10] Sliwa L, Macura B. Evaluation of cell membrane integrity of spermatozoa by hypoosmotic swelling test—“water test” in mice after intraperitoneal daidzein administration. Arch Androl 2005;51:443–8. [11] Solenov EI, Baturina GS, Katkova LE. Role of water channels in the regulation of the volume of principal cells of rat kidney collecting ducts in hypoosmotic medium. Biofizika 2008;53:684–90. [12] Tanimura A, Tanaka S, Kitazono M. Superficial intimal injury of the rabbit carotid artery induced by distilled water. Virchows Arch B Cell Pathol Incl Mol Pathol 1986;51:197–205. [13] Gallo R, Padurean A, Toschi V, Bichler J, Fallon JT, Chesebro JH, et al. Prolonged thrombin inhibition reduces restenosis after balloon angioplasty in porcine coronary arteries. Circulation 1998;97:581–8.
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Original Article
A novel model of intimal hyperplasia with graded hypoosmotic damage Mowei Songa,b,c,1 , Hong-tao Shend,1 , Jin-jin Cuic , Xin-gang Zhoub , Xin Zhonge , Cheng-hai Pengc , Hong-yu Liub,⁎, Ye Tiana,c,e,⁎ a
b
Department of Cardiology, First Affiliated Hospital, Harbin Medical University, Harbin, 150001, Heilongjiang, China Department of Cardiovascular Surgery, First Affiliated Hospital, Harbin Medical University, Harbin, 150001, Heilongjiang, China c Department of Cardiology, Second Affiliated Hospital, Harbin Medical University, Harbin, 150001, Heilongjiang, China d Second Department of Orthopedics, First Affiliated Hospital, Harbin Medical University, Harbin, 150001, Heilongjiang, China e Department of Pathophysiology, Bio-pharmaceutical Key Laboratory of Heilongjiang Province, Harbin Medical University, Harbin, 150086, Heilongjiang, China Received 2 December 2011; received in revised form 19 February 2012; accepted 20 February 2012
Abstract Background: The purpose was to develop a rabbit model of intimal hyperplasia with controllable lesion. Methods: Following 1 week of a 2% cholesterol diet, 32 New Zealand White male rabbits underwent right femoral arteries surgical perfusion with distilled water for 1, 3, 5, or 7 min (n=8/group). After a further 4 weeks of the same diet, serum total cholesterol, triglyceride, low-density lipoprotein, and high-density lipoprotein were measured in all rabbits. Intimal hyperplasia in histological sections of arteries were assessed by intimal proliferation ratio. Macrophage numbers and levels of proteins matrix metalloproteinase 9, tissue inhibitor of metalloproteinase 2, and alpha smooth muscle actin in lesions were analyzed by immunohistochemistry. Results: Serum lipids levels showed no statistical difference between experimental groups. Intimal proliferation ratio increased gradually with perfusion time, and a positive linear correlation was calculated between intimal proliferation ratio and duration of distilled water perfusion. Similarly, number of macrophages and levels of matrix metalloproteinase 9, tissue inhibitor of metalloproteinase 2, and alpha smooth muscle actin in lesions increased with perfusion time. Conclusions: A novel model of intimal hyperplasia was established by intravascular distilled water perfusion in high-cholesterol-fed rabbits. Importantly, this model exhibits time-dependent neointimal proliferation lesions that can be readily controlled in terms of extent, thus providing an avenue for further studies. © 2012 Elsevier Inc. All rights reserved. Keywords: Intimal hyperplasia; Hypoosmotic injury; Hyperlipidemia; Endothelial cells; Rabbit
1. Introduction Accelerated intimal hyperplasia is an important cause of morbidity and mortality in patients with atherosclerosis. Intimal proliferation and migration of smooth muscle cells have for a long time been emphasized in the development of atherosclerotic plaques [1,2] as well as in the process of There was no conflict of interest in the study. ⁎ Corresponding authors. Hong-yu Liu is to be contacted at Department of Cardiovascular Surgery, First Affiliated Hospital, Harbin Medical University, Harbin, 150001, Heilongjiang, China. Tel.: +86045185555817; fax: +86045185555817. Ye Tian Department of Cardiology, First Affiliated Hospital, Harbin Medical University, Harbin, 150001, Heilongjiang, China. Tel.: +86045185555943; fax: +86045185555943. E-mail addresses: [email protected] (H. Liu), [email protected] (Y. Tian). 1054-8807/12/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.carpath.2012.02.007
restenosis following angioplasty [3]. In the current vascular interventional environment, high restenosis rates after stents graft and veins graft have also increased awareness of the significance of intimal hyperplasia [4,5], a chronic structural lesion that develops after vessel wall injury and which can lead to luminal stenosis and occlusion [6,7]. Intravascular endothelial injury is central to intimal hyperplasia, and excessive accumulation of cells can be observed with a subsequent intimal thickening causing restenotic lesion formation [8]. However, endoluminal manipulation of small arteries is too technically difficult to develop different intimal injury. As excessive cholesterol can lead to atherosclerotic changes in the arterial intima, and lesions appear to develop as a result of repeated or continuous intimal injury, various intimal hyperplasia animal models have been developed based on
M. Song et al. / Cardiovascular Pathology 21 (2012) 490–498 Table 1 Lipid levels in experimental groups Group
TC
TG
LDL
HDL
1 min 3 min 5 min 7 min g
23.07±10.37 20.52±11.90 14.0±11.87 29.86±11.86
1.18±0.40 0.89±0.60 1.03±0.38 2.45±2.11
17.75±8.19 15.96±9.75 10.53±9.85 23.13±9.77
4.57±2.55 4.84±2.55 3.00±2.07 5.64±2.38
Values are expressed in millimoles as mean±S.E.M. Groups refer to the duration of exposure to distilled water perfusion.
high-cholesterol diet, arterial wall injury, or a combination of the two approaches. But, to date, there are no mature reproducible models with controllable intimal–proliferation lesions. Distilled water can cause hypoosmotic stress with resulting cell swelling, dysfunction, and even hypotonic shock [9–11]. As intravascular perfusion with distilled water can result in superficial intimal injury of rabbit artery [12], we hypothesized that this intimal injury would help to develop a rabbit model of intimal hyperplasia on a highcholesterol diet. Moreover, as such endothelial injury likely increases with distilled water perfusion time, this novel model may allow the time-dependent study of preatherosclerotic intimal hyperplasia development. This is to our knowledge the first report on the rabbit model with controllable lesions of intimal hyperplasia.
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Table 2 IPR; macrophage numbers; and expression levels of MMP9, TIMP2, and α-SMA in plaque samples Group
IPR
MMP9
TIMP2
Macrophage
α-SMA
1 3 5 7
0.80±0.20 0.82±0.13 0.93±0.06 0.94±0.04
0.05±0.03 0.08±0.02 0.09±0.03 0.13±0.02
0.10±0.06 0.11±0.03 0.13±0.04 0.18±0.08
0.04±0.02 0.07±0.02 0.09±0.03 0.20±0.04
0.11±0.02 0.15±0.04 0.15±0.04 0.21±0.05
min min min min
Values are expressed as median value±S.E.M.
2. Methods 2.1. Animal model Thirty-two adult male New Zealand White rabbits, weighing 2.5 to 3.0 kg, were randomly divided into four treatment groups (n=8, each). All received a standard laboratory diet supplemented with cholesterol (2%) and lard (2%) for 1 week prior to surgery and 4 weeks thereafter. A surgical level of anesthesia was induced by intramuscular injection of ketamine hydrochloride (40 mg/kg) and xylazine (5 mg/kg). The right femoral artery of each rabbit was surgically exposed, and a 3-cm segment was isolated using two artery clamps. A 28-ga needle was inserted into the proximal end of the segment, and the segment was perfused
Fig. 1. Histological sections demonstrating increasing intimal thickness correlated with increasing time of exposure to distilled water. The arrows highlight increasing plaque, and an increasing IPR can be readily appreciated. All images are of H&E-stained sections at 100×. (A) One minute, (B) 3 min, (C) 5 min, and (D) 7 min. (E) The positive linear relationship between the median value of IPR and perfusion time: y=0.75188+0.02988x; Sy=0.08023 and Sxy=0.02200.
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with distilled water. For the four treatment groups, perfusion times were 1, 3, 5, or 7 min. Following perfusion, the circulation was reestablished, hemostasis was ensured, and the surgical incision was closed. The contralateral femoral artery of each rabbit served as a control. All procedures were performed in compliance with the Principles of Laboratory Animal Care and Guide for the Care and Use of Laboratory Animals (NIH publication No. 80-23, revised 1985) and were approved by the Animal Care and Use Committee of Harbin Medical University. 2.2. Measurement of serum lipids Abdominal aorta blood samples were taken by catheter under anesthesia. Serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and highdensity lipoprotein (HDL) were measured using the Olympus AU5400 Clinical Chemistry System. 2.3. Histomorphometry and immunohistochemistry Four weeks after surgery, rabbits were euthanized by an intravenous overdose of pentobarbital. For harvesting of specimens, perfusion–fixation was done with 4% paraformaldehyde in phosphate-buffered saline at 100 mmHg for 15 min. Five-micrometer-thick sections, cut 100 μm apart from each other, were stained with hematoxylin and eosin (H&E)
and immunochemistry. Images of stained sections were taken with an Olympus IX70 microscope (magnification ×100) and analyzed by using computerized morphometry (Image-Pro Plus, version 6.0, Media Cybernetics, Silver Spring, MD, USA). Intimal proliferation ratio (IPR), also called intima-to-media ratio, was calculated to measure the percentage of luminal narrowing as previously described [13]. Arteries with occlusive thrombus were not included in the morphometric analysis. Numbers of macrophages and levels of expression of matrix metalloproteinase 9 (MMP9), tissue inhibitor of metalloproteinase 2 (TIMP2), and smooth muscle α-actin (α-SMA) in lesions, associated with degree of neointimal proliferation, were determined by immunohistochemistry. The primary monoclonal antibodies used were as follows: mouse anti-rabbit α-smooth muscle actin antibody (1:2000, Sigma-Aldrich, St. Louis, MO, USA) to identify smooth muscle cells (SMCs), mouse anti-rabbit macrophage antibody (anti-RAM-11, 1:1500, Dako North America, Carpinteria, CA, USA) to identify macrophage cells, mouse anti-rabbit MMP9 antibody (Millipore, Billerica, MA, USA) to identify MMP9, and mouse anti-rabbit TIMP2 antibody (Thermo Fisher Scientific, Waltham, MA, USA) to identify TIMP2. All immunohistochemistry slides were viewed with a Leica DM 2500 microscope, and Maikeaodi Instrument Company software was used for gray-scale image analysis.
Fig. 2. Immunohistochemical evidence for increased MMP9 expression in plaque of arteries exposed to distilled water for different time periods. (A) One minute, (B) 3 min, (C) 5 min, and (D) 7 min. Sections were examined at 100×. (E) The linear relationship between the median MMP9 levels and perfusion time: y=0.03744+0.01231x; Sy=0.03253 and Sxy=0.000687.
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2.4. Statistical analysis Values of serum lipid levels were expressed as mean± standard deviation (x±S.D.) and analyzed by the Newman– Keuls method. Values of IPR and all indicators above tested by immunohistochemistry are presented as median values. Comparative differences of each indicator among the four experimental groups were analyzed by the Kruskal–Wallis one-way analysis-of-variance-by-ranks (H) test, followed by the Nemenyi test and linear regression analysis. A value of Pb.05 was considered to be indicative of a statistically significant difference. 3. Results 3.1. Serum lipid levels Following 5 weeks of the high-cholesterol diet (1 week before and 4 weeks after surgery), the serum lipid levels in all experimental rabbit groups were significantly higher than the baseline [14]. Severe hypercholesterolemia; elevated levels of TC, TG, and LDL; and decreased HDL-cholesterol are detailed in Table 1. No differences in lipid levels were detected between the four experimental groups (Table 1).
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3.2. Histological features of distilled water perfusion-induced intimal hyperplasia A complex lesion is formed with significant neointimal hyperplasia in sections of surgical femoral arteries. Foam cells as well as extracellular lipid are prominent in the neointima and, to a lesser degree, the media as well (Fig. 6G).The degree of intimal hyperplasia markedly developed from the 1-min group to the 7-min group in turn (Fig. 1A–D). The median values of IPR (MIPR) for the four groups increased with perfusion time (Table 2) and demonstrated an apparent linear relationship described by y=0.751882+(0.029882x); (y means MIPR, and x means distilled water perfusion time Sy=0.08023 and Sxy=0.02200; Fig. 1E). The MIPR of the four groups showed an overall statistical difference by the H test (H=10.72, n'=3, Pb.05), with a significant difference found between the 1-min and 7-min groups (Nemenyi test, r=0.961664, Pb.01). 3.3. Immunohistochemistry The intimal layer in sections of surgical femoral arteries shows increased cellularity, not only SMCs and macrophages but also deposition of MMP9 and TIMP2 without disruption of the internal elastic lamina (circles). And the
Fig. 3. Immunohistochemical evidence for increased a-SMA expression in plaque of arteries exposed to distilled water for different time periods. (A) One minute, (B) 3 min, (C) 5 min, and (D) 7 min. Sections were examined at 100×. (E) The linear relationship between the median MMP9 levels and perfusion time: y=0.09697+0.014x; Sy=0.08009 and Sxy=0.02898.
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Fig. 4. Increased TIMP2 expression in the plaque of four group samples: (A) One minute, (B) 3 min, (C) 5 min, and (D) 7 min (all immunohistochemistry, original magnification 100×). (E) The linear equation between the median M of the TIMP2 level in plaque and perfusion time: y=0.07943+0.01261x; Sy=0.03483 and Sxy=0.0123497.
number of macrophages and expression levels of MMP9, TIMP2, and α-SMA in neointimal proliferation lesions also increased with distilled water perfusion time (Table 2 and Figs. 5A–D, 2A–D, 3A–D, and 4A–D). Moreover, four equations were calculated to describe positive linear correlation between perfusion time and median value of MMP9 (MMMP9), median value of α-SMA (Mα-SMA), median value of TIMP2 (MTIMP2), and median value of macrophages (Mmacrophage), which are separately shown in Figs. 2E, 3E, 4E, and 5E. When comparing between four groups, differences of MMMP9, Mα-SMA, and Mmacrophage were significant with Pb.01, but difference of MTIMP2 was only with Pb.05. When comparing between each two groups, especially in the 1-min group and 7-min group, evident differences were also verified by Nemenyi testing. Difference of MMMP9 was obvious in the 1-min and 5-min, 3-min and 7-min (Pb.05), and 1-min and 7-min groups (Pb.01). Nemenyi testing confirmed significant differences of Mα-SMA between the 1-min and 7-min groups (Pb.01) and between the 1-min and the other two groups (Pb.05). MTIMP2 in proliferated intimal showed a significant difference between the 1-min and 7-min groups (Pb.05). Difference of Mmacrophage was statistical between the 1-min and 5-min groups (Pb.05) and was evident between the 7-min and other three groups (each Pb.01).
4. Discussion In this study, we have established a novel rabbit model of intimal hyperplasia by intravascular distilled water perfusion. Distilled water perfusion was used to induce arterial endothelium injury, and damage occurred as a result of hypotonic challenge and endothelial cell swelling which affected numerous regulators of cell metabolism and transport [15]. So, this model is based on physical injury caused by low osmotic pressure, which is mechanistically different to currently used models, such as mechanical balloon catheter-induced injury [16,17], cold-induced injury [18], chemical injury [19], air-drying [20], nitrogen bubble techniques [21], and the Watanabe heritable hyperlipidemic rabbits (WHHL rabbits) [22,23]. The balloon injury-induced model, as the most popular model, is based on mechanical injury caused by balloon dilation to the vascular intima and inner elastic lamina, which are always with vessel medial damage. As the injury caused by balloon dilation is susceptible to many factors, including balloon pressure and the matching relationship between balloon diameter and vessel lumen [24], the degree of mechanical lesions is difficult to control, and the damage is easy to be a partial shedding of endothelial cells but not consistent (shown in Fig. 6A). Contrast to this mechanical injury, this physical injury is
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Fig. 5. Increased numbers of macrophages in the plaque of four group samples: (A) One minute, (B) 3 min, (C) 5 min, and (D) 7 min (all immunohistochemistry, original magnification 100×). (E) The linear equation between the median M of macrophage in plaque and perfusion time: y=0.0015529+0.02446x; Sy=0.06871 and Sxy=0.027063.
uniform. It may be due to the same hypoosmotic environment caused by intravascular distilled water perfusion and then the same hypoosmotic injury to all endothelial cells. Moreover, this hypoosmotic damage is limited to the endothelium without disruption of the elastic lamina or overt medial changes [12], as proven in preliminary experiments (shown in Fig. 6B). Most importantly, this model of intimal hyperplasia exhibited an important advantage due to the perfusion timerelated characteristics. Corresponding to a loss of arterial
lumen, IPR is the standard for histological evaluation of the extent of neointimal proliferation [13]. In this study, we found that MIPR increased with distilled water perfusion time as a positive line. It is conceivable that a longer perfusion time causes greater hypoosmotic injury to the endothelial cells, resulting in more neointimal cell necrosis and more serious proliferation under hyperlipidemic conditions. Lipid accumulation is associated with decreased endothelial cells migration and intimal hyperplasia [25]. And in
Fig. 6. Representative light micrographs of femoral artery. (A–D) H&E-stained sections. (E and F) Masson's trichrome-stained sections. (G) Oil red O-stained section. (A) Balloon injury, 400×; (B) distilled water injury, 400×; (C) distilled water injury with high-fat feeding, 100×; (D) high-fat feeding only, 100×; (E) high-fat feeding only, 400×; (F) distilled water injury with high-fat feeding 200×; (G) disposition of lipid in intimal hyperplasia lesions, 100×.
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the absence of hypercholesterolemia, intimal injury and hyperplasia regress and resolve spontaneously, but in the presence of hypercholesterolemia, they are not only sustained but progress to larger lesions [26]. After being combined with hyperlipidemia, neointimal proliferation developed rapidly after hypoosmotic injury in each group. Although hypercholesterolemia can accelerate intimal hyperplasia [25,27], lesions were not apparent in the contralateral femoral artery without water perfusion (Fig. 6D and F). Maybe, 5 weeks of treatment with a 2% cholesterol diet was, by itself, insufficient to induce neoproliferation in the femoral artery of rabbits, or a 5-week time frame is too short. Progressive intimal hyperplasia lesions contained SMC and macrophages [28]. Proliferation and migration of smooth muscle cells have for a long time been emphasized in the development of atherosclerosis [29] as well as in the process of restenosis following angioplasty [3]. The accumulation of SMC, in response to wall injury, contributes to arterial wound repair and thickening of the intimal layer [30,31]. Recent studies have suggested that SMC-derived factors, such as Tissue factor (TF), also plays an important role in promoting arterial thrombosis and in mediating intimal hyperplasia in response to arterial injury [32]. Macrophages contribute to stimulating SMC migration [33,34] and proliferation [35]. Moreover, macrophages themselves also play key roles in the development of intimal hyperplasia [36–38]. Increased production of MMP9, as a necessary component for collagen organization by SMC, degrades extracellular matrix and facilitates the migration of SMC, which is a fundamental process in arterial intimal hyperplasia [39,40]. TIMP2 is expressed and secreted by neointimal SMC and has been implicated in the progress of neointimal hyperplasia [41,42]. In this study, not only increased numbers of macrophages but also increased expression level of α-SMA, MMP9, and TIMP2 were found in lesions of distilled-water-treated arteries. Furthermore, a positive linear relationship was also observed between these four indicators in lesions and the duration of distilled water perfusion. As we known, the underlying pathophysiology of venous graft disease and late bypass failure includes intimal hyperplasia and subsequent accelerated atherosclerosis [43]. In this study the four indicators, such as macrophage [44–47], α-SMA [48], MMP9 [49–53], and TIMP2 [54], all contributed to the severity of atherosclerosis and eventual plaque rupture. It means that with intimal hyperplasia accelerating, the lesions trend to be vulnerable plaques. Consistent with this inference, we have found the lesions in some sections, characterized by a lipidrich core, accumulation of macrophages, thin fibrous cap, and arterial neovascularization (Fig. 6C). In addition to the duration of distilled water exposure, a number of other factors in this model may affect the progression of intimal hyperplasia in the damaged arteries, including the area exposed to water perfusion, lumina diameter of artery, perfusion pressure, and so on. These may result in different extent of endothelial damage and intimal
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