Hypertension Enhances Advanced Atherosclerosis and Induces Cardiac Death in Watanabe Heritable Hyperlipidemic Rabbits
2018; Elsevier BV; Volume: 188; Issue: 12 Linguagem: Inglês
10.1016/j.ajpath.2018.08.007
ISSN1525-2191
AutoresBo Ning, Yajie Chen, Ahmed Bilal Waqar, Haizhao Yan, Masashi Shiomi, Jifeng Zhang, Y. Eugene Chen, Yanli Wang, Hiroyuki Itabe, Jingyan Liang, Jianglin Fan,
Tópico(s)Blood Pressure and Hypertension Studies
ResumoHypertension is a major risk factor for the development of atherosclerosis. Cardiovascular risk has been reported to be significantly increased in hyperlipidemic patients with hypertension. However, it is not clear whether hypertension can directly destabilize plaques, thereby enhancing cardiovascular events. To examine whether hypertension enhances the development of atherosclerosis and increases plaque vulnerability, we generated hypertensive Watanabe heritable hyperlipidemic (WHHL) rabbits by surgical removal of one kidney and partial ligation of the other renal artery and compared the nature of aortic and coronary atherosclerosis in hypertensive WHHL rabbits with normotensive WHHL rabbits. All hypertensive WHHL rabbits died from 34 to 56 weeks after surgery, whereas no normotensive WHHL rabbits died. Pathologic examinations revealed that hypertensive WHHL rabbits showed different degrees of myocardial infarction caused by severe coronary stenosis along with myocardial hypertrophy. Furthermore, aortic lesions in hypertensive WHHL rabbits exhibited a higher frequency of intraplaque hemorrhage and vulnerable plaques than those in normotensive WHHL rabbits. These results indicate that hypertension induced by the surgical removal of one kidney and partial ligation of the other renal artery method in WHHL rabbits may not only enhance the development of atherosclerosis but also destabilize the plaques, increasing cardiac death. Hypertension is a major risk factor for the development of atherosclerosis. Cardiovascular risk has been reported to be significantly increased in hyperlipidemic patients with hypertension. However, it is not clear whether hypertension can directly destabilize plaques, thereby enhancing cardiovascular events. To examine whether hypertension enhances the development of atherosclerosis and increases plaque vulnerability, we generated hypertensive Watanabe heritable hyperlipidemic (WHHL) rabbits by surgical removal of one kidney and partial ligation of the other renal artery and compared the nature of aortic and coronary atherosclerosis in hypertensive WHHL rabbits with normotensive WHHL rabbits. All hypertensive WHHL rabbits died from 34 to 56 weeks after surgery, whereas no normotensive WHHL rabbits died. Pathologic examinations revealed that hypertensive WHHL rabbits showed different degrees of myocardial infarction caused by severe coronary stenosis along with myocardial hypertrophy. Furthermore, aortic lesions in hypertensive WHHL rabbits exhibited a higher frequency of intraplaque hemorrhage and vulnerable plaques than those in normotensive WHHL rabbits. These results indicate that hypertension induced by the surgical removal of one kidney and partial ligation of the other renal artery method in WHHL rabbits may not only enhance the development of atherosclerosis but also destabilize the plaques, increasing cardiac death. Hypertension has long been known to increase the prevalence of coronary heart disease1Solberg L.A. Strong J.P. Risk factors and atherosclerotic lesions. A review of autopsy studies.Arteriosclerosis. 1983; 3: 187-198Crossref PubMed Google Scholar and also to increase the extent and severity of atherosclerosis in both humans1Solberg L.A. Strong J.P. Risk factors and atherosclerotic lesions. A review of autopsy studies.Arteriosclerosis. 1983; 3: 187-198Crossref PubMed Google Scholar, 2Robertson W.B. Strong J.P. Atherosclerosis in persons with hypertension and diabetes mellitus.Lab Invest. 1968; 18: 538-551PubMed Google Scholar and experimental animals.3Moses C. 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Indeed, hypertension alone in the absence of elevation of plasma cholesterol levels usually cannot induce the atherosclerosis commonly observed in large arteries,11Chobanian A.V. The influence of hypertension and other hemodynamic factors in atherogenesis.Prog Cardiovasc Dis. 1983; 26: 177-196Crossref PubMed Scopus (99) Google Scholar suggesting that hypercholesterolemia is essential for the initiation and formation of atherosclerosis, whereas hypertension is an enhancer for its progression. However, it is not clear whether hypertension affects atherosclerotic complications such as myocardial infarction (MI). Watanabe heritable hyperlipidemic (WHHL) rabbits are a unique model for the study of human hypercholesterolemia and atherosclerosis because they develop spontaneous hypercholesterolemia and atherosclerosis because of deficient LDL receptor functions.12Shiomi M. Fan J. Unstable coronary plaques and cardiac events in myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits: questions and quandaries.Curr Opin Lipidol. 2008; 19: 631-636Crossref PubMed Scopus (39) Google Scholar, 13Shiomi M. Ito T. Yamada S. Kawashima S. Fan J. Development of an animal model for spontaneous myocardial infarction (WHHLMI rabbits).Arterioscler Thromb Vasc Biol. 2003; 23: 1239-1244Crossref PubMed Scopus (149) Google Scholar Chobanian et al5Chobanian A.V. Lichtenstein A.H. Nilakhe V. Haudenschild C.C. Drago R. Nickerson C. Influence of hypertension on aortic atherosclerosis in the Watanabe rabbit.Hypertension. 1989; 14: 203-209Crossref PubMed Scopus (92) Google Scholar reported that when WHHL rabbits were subjected to a short period of hypertension, they developed greater aortic atherosclerosis than normotensive (NTN)-WHHL rabbits,14Nickerson C.J. Haudenschild C.C. Chobanian A.V. Effects of hypertension and hyperlipidemia on the myocardium and coronary vasculature of the WHHL rabbit.Exp Mol Pathol. 1992; 56: 173-185Crossref PubMed Scopus (10) Google Scholar whereas this enhancement of atherosclerosis can be alleviated by treatment with captopril, an angiotensin-conversing enzyme inhibitor.15Chobanian A.V. Haudenschild C.C. Nickerson C. Drago R. Antiatherogenic effect of captopril in the Watanabe heritable hyperlipidemic rabbit.Hypertension. 1990; 15: 327-331Crossref PubMed Scopus (416) Google Scholar Infusion of angiotensin II into WHHL rabbits by osmotic minipumps destabilizes the coronary plaques and causes MI.16Li S. Wang Y.N. Niimi M. Ning B. Chen Y. Kang D. Wang Z. Yu Q. Waqar A.B. Liu E. Zhang J. Shiomi M. Chen Y.E. Fan J. Angiotensin II destabilizes coronary plaques in Watanabe heritable hyperlipidemic rabbits.Arterioscler Thromb Vasc Biol. 2016; 36: 810-816Crossref PubMed Scopus (15) Google Scholar Although the short-term effects of blood pressure elevation on the development of atherosclerosis have been extensively studied in different experimental animals, it has not been confirmed whether long-term blood pressure elevation affects the nature of atherosclerosis, coronary atherosclerosis, or cardiac pathologic processes. This is an important issue because hypertension is a life-long condition in human patients if not properly treated. Because most animal models cannot sustain to the long period of cholesterol diet feeding and rarely develop coronary atherosclerosis or MI, this hypothesis has been difficult to test by using experimental animal models. It has been reported that WHHL rabbits are susceptible to coronary atherosclerosis and MI at an old age.13Shiomi M. Ito T. Yamada S. Kawashima S. Fan J. Development of an animal model for spontaneous myocardial infarction (WHHLMI rabbits).Arterioscler Thromb Vasc Biol. 2003; 23: 1239-1244Crossref PubMed Scopus (149) Google Scholar Therefore, we examined whether a prolonged period of hypertension affects coronary atherosclerosis or subsequent MI in WHHL rabbits without a cholesterol-rich diet. The aim of this study was to characterize and compare the nature of aortic and coronary atherosclerotic lesions in HTN- and NTN-WHHL rabbits and to evaluate changes in the myocardium possibly related to ischemia or hypertension. We were particularly interested in whether long-term hypertension has any effects on plaque stability and cardiac-related events. Our present study demonstrated that prolonged blood pressure elevation not only enhances advanced atherosclerosis but also induces cardiac death in WHHL rabbits. Male WHHL rabbits aged 8 months in which aortic and coronary atherosclerosis was well developed were used.13Shiomi M. Ito T. Yamada S. Kawashima S. Fan J. Development of an animal model for spontaneous myocardial infarction (WHHLMI rabbits).Arterioscler Thromb Vasc Biol. 2003; 23: 1239-1244Crossref PubMed Scopus (149) Google Scholar To investigate the influence of hypertension on the development of atherosclerosis, hypertension was induced by surgical removal of one kidney and partial ligation of the other renal artery (1K1C),5Chobanian A.V. Lichtenstein A.H. Nilakhe V. Haudenschild C.C. Drago R. Nickerson C. Influence of hypertension on aortic atherosclerosis in the Watanabe rabbit.Hypertension. 1989; 14: 203-209Crossref PubMed Scopus (92) Google Scholar and the NTN group underwent sham surgery as a control. In brief, rabbits were anesthetized with ketamine/medetomidine and placed in dorsal recumbence. The abdominal hair was then shaved, and the abdomen was sterilized with betadine. After opening the abdominal cavity, the left kidney was removed with the adrenal gland remaining, and the right renal artery was partially ligated by using a silver restriction clip with a gap of 0.508 mm.5Chobanian A.V. Lichtenstein A.H. Nilakhe V. Haudenschild C.C. Drago R. Nickerson C. Influence of hypertension on aortic atherosclerosis in the Watanabe rabbit.Hypertension. 1989; 14: 203-209Crossref PubMed Scopus (92) Google Scholar Rabbits were fed a standard chow diet (CLEA Japan Inc., Tokyo, Japan) throughout the experiment. Animal experiments were performed after approval from the Animal Care Committee of the University of Yamanashi and conformed to NIH's Guide for the Care and Use of Laboratory Animals.17Committee for the Update of the Guide for the Care and Use of Laboratory AnimalsNational Research CouncilGuide for the Care and Use of Laboratory Animals: Eighth Edition. National Academies Press, Washington, DC2011Crossref Google Scholar The blood pressure (BP) and heart rate were examined every month during the experiment as described previously.16Li S. Wang Y.N. Niimi M. Ning B. Chen Y. Kang D. Wang Z. Yu Q. Waqar A.B. Liu E. Zhang J. Shiomi M. Chen Y.E. Fan J. Angiotensin II destabilizes coronary plaques in Watanabe heritable hyperlipidemic rabbits.Arterioscler Thromb Vasc Biol. 2016; 36: 810-816Crossref PubMed Scopus (15) Google Scholar, 18Waqar A.B. Koike T. Yu Y. Inoue T. Aoki T. Liu E. Fan J. High-fat diet without excess calories induces metabolic disorders and enhances atherosclerosis in rabbits.Atherosclerosis. 2010; 213: 148-155Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar In brief, to avoid stress from the environment, rabbits were placed in a quiet room at least 30 minutes before BP measurement. The medial auricular artery was cannulated with a 23-gauge cannula, and BP and heart rate were recorded with a transducer. These data were collected from each rabbit for approximately 15 to 20 minutes by BP amplifier (AD Instruments, Tokyo, Japan). BP was calculated with Chart5 Pro software version 5.5 (AD Instruments). Blood was collected from rabbits after 16-hour fasting. Plasma levels of total cholesterol, triglycerides, and high-density lipoprotein cholesterol were measured biweekly by using Wako assay kits (Wako Pure Chemical Industries, Osaka, Japan).19Wang C. Nishijima K. Kitajima S. Niimi M. Yan H. Chen Y. Ning B. Matsuhisa F. Liu E. Zhang J. Chen Y.E. Fan J. Increased hepatic expression of endothelial lipase inhibits cholesterol diet-induced hypercholesterolemia and atherosclerosis in transgenic rabbits.Arterioscler Thromb Vasc Biol. 2017; 37: 1282-1289Crossref PubMed Scopus (28) Google Scholar To evaluate the renal functions, plasma creatinine and urea were measured with a commercially available enzymatic assay kit (Cat-KGE005; R&D System Inc., Minneapolis, MN) and urea assay kit (DIUR-100) (BioAssay Systems, Hayward, CA), respectively. In addition, angiotensin II levels in plasma were determined with an enzyme-linked immunosorbent assay kit (Enzo Life Sciences, Inc., New York, NY) according to the manufacturer's instructions. At 56 weeks after surgery, all WHHL rabbits were sacrificed by an overdose injection of sodium pentobarbital (100 mg/kg). Aortic trees were isolated, and periaortic adipose tissue was removed. Aortas were opened and fixed on a corkboard with 10% neutral-buffered formalin solution. The hearts and brains from each animal were weighed, photographed, and then fixed in 10% formalin. After fixing, whole aortic trees were stained with Sudan IV and analyzed as described previously.20Koike T. Liang J. Wang X. Ichikawa T. Shiomi M. Sun H. Watanabe T. Liu G. Fan J. Enhanced aortic atherosclerosis in transgenic Watanabe heritable hyperlipidemic rabbits expressing lipoprotein lipase.Cardiovasc Res. 2005; 65: 524-534Crossref PubMed Scopus (30) Google Scholar For microscopic quantification of the aortic lesions, the aorta was divided into three parts: aortic arch and thoracic and abdominal aortas.20Koike T. Liang J. Wang X. Ichikawa T. Shiomi M. Sun H. Watanabe T. Liu G. Fan J. Enhanced aortic atherosclerosis in transgenic Watanabe heritable hyperlipidemic rabbits expressing lipoprotein lipase.Cardiovasc Res. 2005; 65: 524-534Crossref PubMed Scopus (30) Google Scholar Each part was further cut into several segments (8 for the arch, 20 for the thoracic aorta, and 30 for the abdominal aorta) and embedded in paraffin. Specimens were cut in 3-μm serial sections and used for hematoxylin and eosin, elastic van Gieson, and Masson's trichrome staining. Immunohistochemical staining was performed by using monoclonal antibodies against rabbit macrophage RAM11 (Dako Co., Carpinteria, CA) and smooth muscle α-actin (Dako Co.).21Li S. Liang J. Niimi M. Bilal Waqar A. Kang D. Koike T. Wang Y. Shiomi M. Fan J. Probucol suppresses macrophage infiltration and MMP expression in atherosclerotic plaques of WHHL rabbits.J Atheroscler Thromb. 2014; 21: 648-658Crossref PubMed Scopus (26) Google Scholar In addition, the lesion types were quantified as follows: fatty streaks (type II lesions: mainly composed of foam cells), fibrotic lesions [type III lesions: mainly composed of smooth muscle cells (SMCs) and extracellular matrix], and complicated or advanced lesions (type IV or V fibrous plaques combined with lipid/necrotic core or with calcification) according to the American Heart Association classification.22Stary H.C. Chandler A.B. Dinsmore R.E. Fuster V. Glagov S. Insull Jr., W. Rosenfeld M.E. Schwartz C.J. Wagner W.D. Wissler R.W. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association.Circulation. 1995; 92: 1355-1374Crossref PubMed Scopus (2273) Google Scholar The lengths of different lesions on each specimen were measured and averaged.21Li S. Liang J. Niimi M. Bilal Waqar A. Kang D. Koike T. Wang Y. Shiomi M. Fan J. Probucol suppresses macrophage infiltration and MMP expression in atherosclerotic plaques of WHHL rabbits.J Atheroscler Thromb. 2014; 21: 648-658Crossref PubMed Scopus (26) Google Scholar HTN-WHHL rabbits that died at night during the experiment were autopsied in the morning. Autopsy included gross and microscopic examinations such as body appearance examination, thoracic and abdominal cavity examination, and all important organs (liver, adrenal, spleen, kidneys, stomach and intestines, heart and lung, and brain). After autopsy, all organs collected were fixed in 10% neutral-buffered formalin solution, and specimens were routinely stained with hematoxylin and eosin for light microscopy examinations. In the aortas of HTN-WHHL rabbits, many aortic lesions that contained hemorrhagic sites that varied in size from approximately 3 μm were observed (Analysis of Heart and Coronary Atherosclerosis). To quantify these hemorrhagic lesions, the hemorrhagic number was calculated and plotted on the sections by using hematoxylin and eosin–stained specimens under a light microscope.23Yamada S. Wang K.Y. Tanimoto A. Fan J. Shimajiri S. Kitajima S. Morimoto M. Tsutsui M. Watanabe T. Yasumoto K. Sasaguri Y. Matrix metalloproteinase 12 accelerates the initiation of atherosclerosis and stimulates the progression of fatty streaks to fibrous plaques in transgenic rabbits.Am J Pathol. 2008; 172: 1419-1429Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar In addition, immunohistochemical staining of these hemorrhagic areas was performed with two monoclonal antibodies against either copper-oxidized LDL (a gift from Dr. Hiroyuki Itabe, Showa University, Tokyo, Japan) or malondialdehyde-oxidized LDL (Ab63975; Abcam plc., Cambridge, UK) (Table 1).Table 1Monoclonal Antibodies Used for Immunohistochemical StainingAntibodiesDilutionCatalog numberManufacturerMacrophage (RAM)1:400M0633Dako Co., Carpinteria, CAMuscle actin (HHF35)1:300M0635Dako Co.MDA-oxidized LDL1:400Ab63975Abcam plc., Cambridge, UKCopper-oxidized LDL1:400020418Division of Biological Chemistry, Showa University, JapanLDL, low-density lipoprotein; MDA, malondialdehyde. Open table in a new tab LDL, low-density lipoprotein; MDA, malondialdehyde. The heart appearance was first examined and the weight was measured. The hearts were then dissected by using the methods for analysis of coronary atherosclerosis as described previously.16Li S. Wang Y.N. Niimi M. Ning B. Chen Y. Kang D. Wang Z. Yu Q. Waqar A.B. Liu E. Zhang J. Shiomi M. Chen Y.E. Fan J. Angiotensin II destabilizes coronary plaques in Watanabe heritable hyperlipidemic rabbits.Arterioscler Thromb Vasc Biol. 2016; 36: 810-816Crossref PubMed Scopus (15) Google Scholar, 24Fan J. Kitajima S. Watanabe T. Xu J. Zhang J. Liu E. Chen Y.E. Rabbit models for the study of human atherosclerosis: from pathophysiological mechanisms to translational medicine.Pharmacol Ther. 2015; 146: 104-119Crossref PubMed Scopus (222) Google Scholar In brief, the hearts were dissected into five blocks according to the distribution of coronary arteries. Blocks I and II contained the main trunks of the left and right coronary arteries, whereas blocks III to V contained the small branches of the left circumflex, left descending artery, and obtuse marginal arteries along with arterioles with a diameter <200 μm. The stenosis rate (%) was defined as atherosclerotic lesions area/lumen area. Block V included the maximal vertical surface of both right and left ventricles and was thus used for histologic and morphometric analyses of the myocardium, including MI, cardiac fibrosis, and myocardial hypertrophy. The thickness of the left ventricular wall and interventricular septum was measured and averaged. Cardiac fibrosis defined by Masson's trichrome staining was calculated by using an image analysis system. All values were expressed as means ± SEM. The Kaplan-Meier estimator and the log-rank test were used to evaluate the survival rate after surgery-induced hypertension in WHHL rabbits. All other data were examined by the Shapiro-Wilk test to verify normal distribution. The t-test was used. In all cases, a P value of 90%) (Figure 3A). The lesions in the main trunks were fibrotic with few macrophages and SMCs (Figure 3A). Moreover, small coronary arteries (with a diameter ranging from 200 to 500 μm) or arterioles ( 80% among all arterioles that ranged from 100 to 200 μm on block V), it was found that occlusive lesions in HTN-WHHL rabbits accounted for 40% (range, 30% to 73%) of all vessels compared with 5% (range, 0% to 12%) in NTN-WHHL rabbits (Figure 4B). Furthermore, these coronary lesions were accompanied by an increased number of macrophages and SMCs compared with NTN-WHHL rabbits (Figure 4C).Figure 4Quantitation of coronary stenosis in each block (A), percentage of high-risk stenosis in blocks III to V (B), and macrophages (Mϕ) and smooth muscle cells (SMCs) in the lesions (C). For the analysis of coronary stenosis, specimens were stained with elastica van Gieson and measured by using the described methods. In blocks III to V, there were many occlusive lesions (>80%) defined as high-risk stenosis. High-risk stenosis (%) in blocks III to V was calculated as the number of occlusive arteries divided by the total number of coronary arteries in each section. Immunohistochemical staining was performed by using monoclonal antibodies against either Mϕ or α-smooth muscle actin for SMCs, and cellular content in all blocks was quantified with an image analysis system. Data are expressed as means ± SEM. n = 7 normotensive Watanabe heritable hyperlipidemic (WHHL) rabbits; n = 8 hypertensive (HTN)-WHHL rabbits. ∗P < 0.05, ∗∗P < 0.01 versus normotensive WHHL rabbits.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The aortic lesions in HTN-WHHL rabbits were studied. Both HTN- and NTN-WHHL rabbits developed prominent aortic atherosclerosis, and the aortic surface was almost entirely stained by Sudan IV staining (Figure 5A). However, the lesions in the thoracic aorta (1.1-fold increase over NTN; P < 0.05) and abdominal aorta (1.3-fold increase over NTN; P < 0.05) were significantly increased in HTN-WHHL rabbits compared with NTN-WHHL rabbits Aortic lesions were fibrotic and more common on the extracellular matrix, but there were fewer macrophages and SMCs in both groups on histologic examinations (Figure 5B). Microscopically, the intimal lesions of HTN-WHHL rabbits were thick, and the microscopic lesion area was significantly increased in all parts of the aorta: 1.6-fold increase in the aortic arch, 1.4-fold increase in the thoracic aorta, and 1.4-fold increase in the abdominal aorta compared with the NTN-WHHL group. The increased aortic lesion area in each part was also associated with increased macrophages in HTN-WHHL rabbits: 2.5-fold increase (P < 0.01) in the aortic arch, 2.2-fold increase (P = 0.05) in the thoracic aorta, and 8.5-fold increase (P < 0.01) in the abdominal aorta compared with the NTN-WHHL group. Lesional SMCs in the HTN-WHHL group were slightly increased even though not significantly (Figure 5B). Because the 1.7–year-old WHHL rabbits (at the time of analysis) developed severe atherosclerosis in the aorta, the proportion of early-stage lesions (fatty streaks) and complicated lesions (fibrous plaques), which contained lipid cores, calcification, and hemorrhage, were further quantified by using hematoxylin and eosin–stained specimens. Compared with NTN-WHHL rabbits, advanced lesions were increased in all parts of the aorta: 1.7-fold increase in the aortic arch, 2.4-fold increase in the thoracic aorta, and 2.1-fold increase in the abdominal aorta in HTN-WHHL rabbits, although early-stage lesions in the aortic arch (1.7-fold increase) were also increased in HTN-WHHL rabbits (Figure 5C). The fibrotic plaques were typically covered by a fibrous cap and contained lipid cores in the center, and/or were often associated with c
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