Hyperlipidemias: Shiomi M

Shiomi M, Fan J. · Institute for Experimental Animals, Kobe University School of Medicine, Kobe, Hyogo, Japan. · Curr Opin Lipidol. · Pubmed #18957890 No free full text.

Abstract: PURPOSE OF REVIEW: Use of suitable animal models is essential for investigation of the mechanisms underlying cardiac events and development of the therapeutic strategies; however, ideal animal models that can recapitulate human coronary atherosclerosis and subsequent acute myocardial infarction are still lacking. In this article, we review the insights learned from myocardial infarction-prone Watanabe heritable hyperlipidemic (designated as WHHLMI) rabbits and discuss the possibility of using this model for the study of human acute coronary syndromes. RECENT FINDINGS: The vulnerable plaques of human coronary arteries are histologically characterized by a large lipid core and a thin fibrous cap with inflammatory cells. Recent studies have revealed that inflammatory cells and inflammatory mediators (such as cytokines and matrix metalloproteinases) play an important role in the plaque rupture. SUMMARY: We developed the WHHLMI rabbit that shows spontaneous myocardial infarction caused by coronary atherosclerosis. The coronary lesions of WHHLMI rabbits have features of fatty streaks, fibrous plaques, and fibroatheromatous plaques. Some plaques contain a lipid core and a thin fibrous cap similar to human vulnerable plaques. In spite of this, the plaque rupture is not observed in WHHLMI rabbits, suggesting that other additional factors such as mechanical stress are required to trigger the rupture. WHHLMI rabbits may become an important means for elucidating the possible mechanisms of plaque rupture by exposing the plaques to additional risk factors beyond hyperlipidemia.

Shiomi M, Ito T. · Institute for Experimental Animals, Kobe University School of Medicine. · Nippon Rinsho. · Pubmed #11351660 No free full text.

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Ishino S, Mukai T, Kuge Y, Kume N, Ogawa M, Takai N, Kamihashi J, Shiomi M, Minami M, Kita T, Saji H. · Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan. · J Nucl Med. · Pubmed #18794262 No free full text.

Abstract: Lectinlike oxidized low-density lipoprotein (LDL) receptor 1 (LOX-1), a cell surface receptor for oxidized LDL, has been implicated in vascular cell dysfunction related to plaque instability, which could be a potential target for an atherosclerosis imaging tracer. In this study, we designed and prepared (99m)Tc-labeled anti-LOX-1 monoclonal IgG and investigated its usefulness as an atherosclerosis imaging agent. METHODS: Anti-LOX-1 monoclonal IgG and control mouse IgG2a were labeled with (99m)Tc after derivatization with 6-hydrazinonicotinic acid to yield (99m)Tc-LOX-1-mAb and (99m)Tc-IgG2a, respectively. Myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits (atherosclerosis model) and control rabbits were injected intravenously with these probes, and in vivo planar imaging was performed. At 24 h after the injection, the aortas were removed, and radioactivity was measured. Autoradiography and histologic studies were performed with serial aortic sections. RESULTS: The level of (99m)Tc-LOX-1-mAb accumulation was 2.0-fold higher than the level of (99m)Tc-IgG2a accumulation in WHHLMI rabbit aortas, and the level of (99m)Tc-LOX-1-mAb accumulation in WHHLMI rabbit aortas was 10.0-fold higher than the level of (99m)Tc-LOX-1-mAb accumulation in control rabbit aortas. In vivo imaging clearly visualized the atherosclerotic aortas of WHHLMI rabbits. Autoradiography and histologic studies revealed that regional (99m)Tc-IgG2a accumulation was independent of the histologic grade of the lesions; however, regional (99m)Tc-LOX-1-mAb accumulation was significantly correlated with LOX-1 expression density and the vulnerability index. The highest level of (99m)Tc-LOX-1-mAb accumulation, expressed as {radioactivity in region of interest (Bq/mm(2))/[injected radioactivity (Bq)/animal body weight (g)]} x 10(2), was found in atheromatous lesions (3.8 +/- 1.1 [mean +/- SD]), followed in decreasing order by fibroatheromatous lesions (2.0 +/- 1.0), collagen-rich lesions (1.6 +/- 0.8), and neointimal lesions (1.4 +/- 0.7). CONCLUSION: The level of (99m)Tc-LOX-1-mAb accumulation in grade IV atheroma was higher than that in neointimal lesions or other, more stable lesions. Nuclear imaging of LOX-1 expression with (99m)Tc-LOX-1-mAb may be a useful means for predicting atheroma at high risk for rupture.

Imanishi T, Ikejima H, Tsujioka H, Tsujioka A, Kuroi A, Kobayashi K, Shiomi M, Muragaki Y, Mochizuki S, Goto M, Yoshida K, Akasaka T. · Department of Cardiovascular Medicine, Wakayama Medical University, Wakayama, Japan. · Hypertens Res. · Pubmed #18716369 No free full text.

Abstract: We investigated the effects of co-administration of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor and angiotensin II type 1 receptor blocker (ARB) on nitric oxide (NO) bioavailability in genetically hyperlipidemic rabbits with our newly developed NO sensor. A total of 36 myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits equally derived (n=6 per group) were treated with 1) vehicle (control), 2) hydralazine (15 mg/kg/d), 3) the HMG-CoA reductase inhibitor pitavastatin (P: 0.5 mg/kg/d), 4) the ARB valsartan (V: 5 mg/kg/d), and 5) pitavastatin+valsartan (P+V) together without or 6) with N(G)-nitro-L-arginine methyl ester (L-NAME) for 8 weeks. After treatment, acetylcholine (ACh)-induced NO production was measured as a surrogate for endothelium protective function, and vascular peroxynitrite (a product of superoxide and NO) was measured for assessing dysfunctional endothelial NO synthase activity. Plaque area was quantified by histology as well as optical coherence tomography (OCT). Intra-aortic infusion of ACh produced an increase in plasma NO concentration, which was significantly greater with all drug treatments than with the control. P+V increased ACh-induced NO by 4.1 nmol/L significantly more than either P or V singly. The vascular peroxynitrite concentration was 1.6 pmol/mg protein in the control group and significantly less than those in the P- and V-monotherapy-groups. The lowest peroxynitrite concentration was observed in the P+V group (0.4 pmol/mg protein), which was significantly lower than those in the P- and the V-monotherapy-groups. OCT and histology of the thoracic aorta revealed that the plaque area decreased significantly more with the combination than with the monotherapy. In conclusion, the combined treatment with an HMG-CoA reductase inhibitor and an ARB may have additive protective effects on endothelial function as well as atherosclerotic change.

Kuge Y, Kume N, Ishino S, Takai N, Ogawa Y, Mukai T, Minami M, Shiomi M, Saji H. · Department of Patho-functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan. · Biol Pharm Bull. · Pubmed #18670075 links to  free full text

Abstract: BACKGROUND: Despite increasing in vitro evidence that lectin-like oxidized low density lipoprotein (LDL) receptor-1 (LOX-1), a cell-surface receptor for oxidized LDL, is implicated in the atherogenesis and thrombus formation, its in vivo participation to the atherosclerotic plaque destabilization, rupture and thrombus formation remains unclear. Here, we compared the in vivo expression of LOX-1, with tissue factor (TF) expression and cell apoptosis, in atherosclerotic lesions of myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits. METHODS AND RESULTS: We prepared sixty series of cross sections in the aortic arch and the thoracic aorta from four WHHLMI rabbits. LOX-1 and TF expression, as well as apoptotic events were determined by immunohistochemical staining and TUNEL methods, respectively. LOX-1 expression was mainly observed in the macrophage-rich lipid areas of vulnerable plaque-like atheromatous lesions where TF expression and apoptotic events were prominent. LOX-1 expression was positively correlated with TF expression (r=0.53, p<0.0001), apoptotic events (r=0.52, p<0.0001) and morphological vulnerability (r=0.63, p<0.0001). CONCLUSIONS: LOX-1 expression appears to be closely associated with TF expression, apoptotic events and the morphological vulnerability, suggesting the in vivo involvement of LOX-1 in the destabilization and rupture of atherosclerotic lesions and the subsequent thrombus formation. The present findings in hypercholesterolemic rabbits should help advance our understanding of the pathophysiology of atherosclerosis.

Shiomi M, Yamada S, Amano Y, Nishimoto T, Ito T. · Institute for Experimental Animals, Kobe University School of Medicine, Kobe, Japan. · Br J Pharmacol. · Pubmed #18587443 links to  free full text

Abstract: BACKGROUND AND PURPOSE: Inhibition of squalene synthesis could transform unstable, macrophage/lipid-rich coronary plaques into stable, fibromuscular plaques. We have here treated WHHLMI rabbits, a model for coronary atherosclerosis and myocardial infarction, with a novel squalene synthase inhibitor, lapaquistat acetate (TAK-475). EXPERIMENTAL APPROACH: Young male WHHLMI rabbits were fed a diet supplemented with lapaquistat acetate (100 or 200 mg per kg body weight per day) for 32 weeks. Serum lipid levels were monitored every 4 weeks. After the treatment, lipoprotein lipid and coenzyme Q10 levels were assayed, and coronary atherosclerosis and xanthomas were examined histopathologically or immunohistochemically. From histopathological and immunohistochemical sections, the composition of the plaque was analysed quantitatively with computer-assisted image analysis. Xanthoma was evaluated grossly. KEY RESULTS: Lapaquistat acetate decreased plasma cholesterol and triglyceride levels, by lowering lipoproteins containing apoB100. Development of atherosclerosis and xanthomatosis was suppressed. Accumulation of oxidized lipoproteins, macrophages and extracellular lipid was decreased in coronary plaques of treated animals. Treatment with lapaquistat acetate increased collagen concentration and transformed coronary plaques into fibromuscular plaques. Lapaquistat acetate also suppressed the expression of matrix metalloproteinase-1 and plasminogen activator inhibitor-1 in the plaque and increased peripheral coenzyme Q10 levels. Increased coenzyme Q10 levels and decreased very low-density lipoprotein cholesterol levels were correlated with improvement of coronary plaque composition. CONCLUSION AND IMPLICATIONS: Inhibition of squalene synthase by lapaquistat acetate delayed progression of coronary atherosclerosis and changed coronary atheromatous plaques from unstable, macrophage/lipid accumulation-rich, lesions to stable fibromuscular lesions.

Ikejima H, Imanishi T, Tsujioka H, Kuroi A, Kobayashi K, Shiomi M, Muragaki Y, Mochizuki S, Goto M, Yoshida K, Akasaka T. · Department of Cardiovascular Medicine, Wakayama Medical University Japan. · J Hypertens. · Pubmed #18398339 No free full text.

Abstract: OBJECTIVE: Telmisartan is a unique angiotensin II (Ang II) receptor blocker (ARB) with selective peroxisome proliferator-activated receptor-gamma (PPAR gamma). We therefore investigated the effects of telmisartan on endothelial function and atherosclerotic change in genetically hyperlipidemic rabbits, compared with candesartan, an ARB without PPAR gamma activity. METHODS: A total of 30 Watanabe heritable hyperlipidemic (WHHL) rabbits equally derived (n = 6 each) were treated with (1) vehicle (control), (2) GW9662, a PPAR gamma antagonist (0.5 mg/kg per day), (3) telmisartan (5 mg/kg per day), (4) telmisartan + GW9662, (5) candesartan (5 mg/kg per day) for 8 weeks. After treatment, acetylcholine (ACh)-induced nitric oxide production was measured as a surrogate for endothelium protective function, and vascular nitrotyrosine (a product of superoxide and nitric oxide) was measured for assessing dysfunctional endothelial nitric oxide synthase activity. Plaque area was quantified by histology. RESULTS: Telmisartan increased ACh-induced nitric oxide by 5.5 nmol/l, significantly more than control. Interestingly, cotreatment with GW9662 significantly attenuated telmisartan-induced ACh-induced nitric oxide almost to the levels observed with candesartan. Vascular nitrotyrosine concentration was 1.4 pmol/mg protein in the control group and significantly higher than that in the telmisartan or candesartan group. The lowest nitrotyrosine concentration was observed in the telmisartan group, which was significantly lower than that in the candesartan or telmisartan + GW9662 group. Histology of the thoracic aorta revealed that the plaque area was more significantly decreased in the telmisartan group than in the candesartan or telmisartan + GW9662 group. CONCLUSION: In addition to a class effect of ARBs, telmisartan may have additional effects on nitric oxide bioavailability and atherosclerotic change through its PPAR gamma-mediated effects in genetically hyperlipidemic rabbits.

Ishino S, Kuge Y, Takai N, Tamaki N, Strauss HW, Blankenberg FG, Shiomi M, Saji H. · Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto, 606-8501, Japan. · Eur J Nucl Med Mol Imaging. · Pubmed #17216472 No free full text.

Abstract: PURPOSE: Apoptosis is commonly observed in advanced atherosclerotic lesions. 99mTc-annexin A5 (99mTc-annexin V) has been proposed as a potential tracer for imaging apoptosis in atherosclerotic plaques. Accordingly, we determined the usefulness of 99mTc-annexin A5 as an atherosclerosis imaging tracer in a rabbit model (myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits; WHHLMI rabbits) of spontaneous atherosclerosis. METHODS: The WHHLMI and control rabbits were injected intravenously with 99mTc-annexin A5. After in vivo planar imaging, the radioactivity in the aorta was measured. Autoradiography, TUNEL staining, Azan-Mallory staining and immunohistological studies were performed serially throughout the aorta. RESULTS: 99mTc-Annexin A5 accumulation in the aorta of the WHHLMI rabbits was 5.6-fold higher than in that of control rabbits. Autoradiography showed heterogeneous multifocal accumulation of 99mTc-annexin A5 in WHHLMI rabbits. 99mTc-Annexin A5 accumulation was highest in the atheromatous lesions (6.2+/-2.5, %IDxBW/mm2x10(3)), followed in decreasing order by neointimal (4.9+/-1.3), fibroatheromatous (4.5+/-1.9), and collagen-rich lesions (3.3+/-1.4). The regional 99mTc-annexin A5 accumulation was significantly correlated with the TUNEL-positive cell density, macrophage density and "vulnerability index," an index of the morphological destabilized characteristics. The in vivo imaging clearly visualized the atherosclerotic lesions in WHHLMI rabbits. CONCLUSION: The present study in WHHLMI rabbits showed higher 99mTc-annexin A5 accumulation in grade IV atheroma than in other more stable lesions. 99mTc-Annexin A5 may be useful in identifying atheroma that is at higher risk for rupture and possibly in assessing the response to anti-atherosclerotic therapy.

Kitajima S, Jin Y, Koike T, Yu Y, Liu E, Shiomi M, Marcovina SM, Morimoto M, Watanabe T, Fan J. · Analytical Research Center for Experimental Sciences, Saga University, Saga 849-8501, Japan. · Atherosclerosis. · Pubmed #17045271 No free full text.

Abstract: Elevated plasma levels of LDL and lipoprotein (a) [Lp(a)] are associated with an increased risk of atherosclerosis and coronary heart disease. However, it is not known whether Lp(a) would enhance the atherogenic effect of LDL on coronary atherosclerosis and myocardial infarction. To address this issue, we cross-bred human Lp(a) transgenic (Tg) rabbits with Watanabe heritable hyperlipidemic (WHHL) rabbits and evaluated the long-term (at the age of 2 years) effects of Lp(a) on the development of coronary atherosclerosis. Compared to non-Tg WHHL rabbits, Tg WHHL rabbits did not show significant changes in plasma total cholesterol, triglycerides, or HDL-C. However, Tg WHHL rabbits showed significantly larger lesions in the right coronary arteries (p<0.05). Immunohistochemical staining revealed that the lesions of Tg WHHL rabbits were enriched in the extracellular matrix contents whereas the cellular components were not different from those in non-Tg WHHL rabbits. Increased atherosclerosis in the coronary arteries in Tg WHHL rabbit hearts was also associated with a higher incidence of chronic ischemia and myocardial infarction. These results suggest that increased plasma levels of Lp(a) enhance coronary atherosclerosis and myocardial infarction in the setting of hypercholesterolemia.

Nakano A, Kinoshita M, Okuda R, Yasuda T, Abe M, Shiomi M. · Department of Orthopedic Surgery, Osaka Medical College, 2-7 Daigaku-Machi, Takatsuki, Osaka, 569-8686, Japan. · J Orthop Sci. · Pubmed #16437353 No free full text.

Abstract: BACKGROUND: Tendinous xanthomas associated with heritable hyperlipidemia are clinically well known. Nevertheless, there have been few basic investigations of the pathogenesis of these xanthomas. To clarify the pathogenesis of these xanthomas, we examined the localization and histopathological features of xanthomatous tissues in the extremities of Watanabe heritable hyperlipidemic (WHHL) rabbits. METHODS: Twenty-six WHHL rabbits at 1-31 months of age were dissected to observe the localization of xanthomas. In the histopathological study, tendons and ligaments that included xanthomatous tissues were sectioned and stained with hematoxylin and eosin, Masson's trichrome, and toluidine blue. Immunohistochemical staining was performed with RAM-11, a monoclonal antibody specific for rabbit macrophages, and CD31, a monoclonal antibody specific for endothelial cells. RESULTS: At necropsy examination, spontaneous development of xanthomas was observed in the plantar side of the plantaris tendon, the flexor retinaculum of the carpus, and around the digital flexor tendons of each joint level. Xanthoma formation was observed from 10 months of age and progressed with advancing age. The histomorphological study revealed that xanthomas had developed in superficial paratenon of the tendons that wrap around bony or fibrous pulleys. Many fibrocartilage cells were observed in the deep side of affected tendons. A large number of blood vessels were seen in the xanthomatous tissues of these WHHL rabbits. Immunohistochemical evaluation revealed that the xanthoma plaques contained endothelial cells and macrophages. CONCLUSIONS: It is likely that mechanical stress and extensive vascularization are essential factors for xanthoma formation. Moreover, endothelial cells and macrophages cells are principal contributors to the pathogenesis of tendinous xanthomas and to atherogenesis.

Koike T, Liang J, Wang X, Ichikawa T, Shiomi M, Sun H, Watanabe T, Liu G, Fan J. · Cardiovascular Disease Laboratory, Department of Pathology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba 305-8575, Japan. · Cardiovasc Res. · Pubmed #15639492 links to  free full text

Abstract: OBJECTIVE: This study was designed to address the effects of increased lipoprotein lipase (LPL) activity on atherosclerosis in the setting of LDL receptor deficiency. METHODS: We generated transgenic (Tg) Watanabe heritable hyperlipidemic (WHHL) rabbits overexpressing human LPL and compared their plasma lipids and aortic atherosclerosis with non-Tg WHHL rabbits. RESULTS: Increased expression of LPL significantly ameliorated hypertriglyceridemia and hypercholesterolemia in Tg WHHL rabbits [64% reduction in total cholesterol (TC) and 91% reduction in triglycerides (TG) vs. non-Tg]. In spite of this beneficial effect of LPL, Tg WHHL rabbits had two-fold greater aortic atherosclerosis than non-Tg WHHL rabbits. Analysis of plasma lipoprotein profiles revealed that increased LPL activity in Tg WHHL rabbits resulted in the dramatic reduction of large TG-rich lipoproteins (VLDL, d<1.006 g/ml and IDL, d=1.006-1.02) but concomitant increases in LDL fractions, especially those of small and dense LDL particles (d=1.04-1.06, 2.6-fold over non-Tg). Using apoB-containing lipoproteins, we found that small-sized LDL from Tg WHHL rabbits contained more oxidizable substrate and exhibited higher affinity to biglycan than large TG-rich LDL of non-Tg WHHL rabbits. CONCLUSIONS: We conclude that in the absence of LDL receptor function, increased LPL activity accelerates the catabolism of large TG-rich VLDL (possibly via the LRP pathway) and subsequently improves hyperlipidemia. However, LPL may also enhance the generation and accumulation of small dense LDLs, which are more atherogenic.

Koike T, Liang J, Wang X, Ichikawa T, Shiomi M, Liu G, Sun H, Kitajima S, Morimoto M, Watanabe T, Yamada N, Fan J. · Cardiovascular Disease Laboratory, Department of Pathology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba 305-8575, Japan. · J Biol Chem. · Pubmed #14660566 links to  free full text

Abstract: Lipoprotein lipase (LPL) is the rate-limiting enzyme for the hydrolysis of the triglyceride-rich lipoproteins and plays a critical role in lipoprotein and free fatty acid metabolism. Genetic manipulation of LPL may be beneficial in the treatment of hypertriglyceridemias, but it is unknown whether increased LPL activity may be effective in lowering plasma cholesterol and improving insulin resistance in familial hypercholesterolemic patients. To test the hypothesis that stimulation of LPL expression may be used as an adjunctive therapy for treatment of homozygous familial hypercholesterolemia, we have generated transgenic (Tg) Watanabe heritable hyperlipidemic (WHHL) rabbits that overexpress the human LPL transgene and compared their plasma lipid levels, glucose metabolism, and body fat accumulation with those of non-Tg WHHL rabbits. Overexpression of LPL dramatically ameliorated hypertriglyceridemia in Tg WHHL rabbits. Furthermore, increased LPL activity in male Tg WHHL rabbits also corrected hypercholesterolemia (544 +/- 52 in non-Tg versus 227 +/- 29 mg/dl in Tg, p < 0.01) and reduced body fat accumulation by 61% (323 +/- 27 in non-Tg versus 125 +/- 21ginTg, p < 0.01), suggesting that LPL plays an important role in mediating plasma cholesterol homeostasis and adipose accumulation. In addition, overexpression of LPL significantly suppressed high fat diet-induced obesity and insulin resistance in Tg WHHL rabbits. These results imply that systemic elevation of LPL expression may be potentially useful for the treatment of hyperlipidemias, obesity, and insulin resistance.

Shiomi M, Ito T, Yamada S, Kawashima S, Fan J. · Institute for Experimental Animals, Kobe University School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. · Arterioscler Thromb Vasc Biol. · Pubmed #12738683 links to  free full text

Abstract: OBJECTIVE: Coronary heart disease is the most common cause of death in developed countries. However, there are no suitable animal models that mimic spontaneous myocardial infarction in humans. In this study, we attempted to obtain a rabbit strain with spontaneous myocardial infarction by selective breeding of coronary atherosclerosis-prone Watanabe heritable hyperlipidemic (WHHL) rabbits, designated as WHHLMI rabbits. METHODS AND RESULTS: WHHLMI rabbits were characterized by the high incidence of fatal myocardial infarction at ages 11 to 35 months, being increased from 23% to 97% after the selective breeding. The ECG on WHHLMI rabbits showed a typical feature of myocardial infarction. Histological examination of hearts from suddenly deceased WHHLMI rabbits revealed old myocardial infarction accompanied by fresh myocardial lesions. The culprit coronary arteries exhibited severe atheromatous plaques (>90% lumen area stenosis), suggesting that coronary atherosclerosis is responsible for myocardial infarction observed in WHHLMI rabbits. In addition, the coronary plaques showed vulnerable features including macrophage-rich thin cap and large necrotic core. CONCLUSIONS: To the best of our knowledge, this is the first report of spontaneous myocardial infarction in rabbits, and it is suggested that this WHHLMI rabbit strain will be a useful animal model to study human myocardial infarction.

Yamada S, Ito T, Adachi J, Ueno Y, Shiomi M. · Institute for Experimental Animals, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. · Exp Anim. · Pubmed #12451710 links to  free full text

Abstract: We examined changes in blood pressure and blood flow of the arteries of WHHL and Japanese white rabbits after intravenous bolus injections of acetylcholine (3.0 micrograms/kg), bradykinin (0.5 microgram/kg), and sodium nitroprusside (3.0 micrograms/kg) under a condition of anesthesia. These vasodilators lowered the blood pressure and increased the blood flow in WHHL and Japanese white rabbits. The changes in the hemodynamic parameters of WHHL rabbits after injection of sodium nitroprusside were similar to those of Japanese white rabbits. This suggests that the relaxation response of the tunica media was not diminished in WHHL rabbits. In contrast, the changes in the hemodynamic parameters of WHHL rabbits after injection of acetylcholine or bradykinin were significantly lower than those in Japanese white rabbits. In the histopathological and immunohistological examination, atherosclerotic lesions were observed in the ascending aortas of WHHL rabbits. In the surface of the atheromatous plaques, CD31-positive endothelial cells disappeared partly and the accumulation of RAM-11-positive macrophages was observed in these regions. In addition, plasma NO2- and NO3- levels of WHHL rabbits were significantly lower than those of Japanese white rabbits. These findings suggest that relaxation responses derived from arterial endothelial cells were probably depressed in WHHL rabbits due to dysfunction or denudation of the arterial endothelial cells.

Sun H, Usui S, Shiomi M, Watanabe T, Fan J. · Department of Pathology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Japan. · J Atheroscler Thromb. · Pubmed #12226556 links to  free full text

Abstract: WHHL rabbits are a valuable model for the study of human familial hypercholesterolemia and atherosclerosis. To use this animal model, it is often necessary to confirm LDL receptor status in WHHL rabbits. Here, we described a simple and rapid PCR method to detect LDL mutations in WHHL rabbits.

Sun H, Unoki H, Wang X, Liang J, Ichikawa T, Arai Y, Shiomi M, Marcovina SM, Watanabe T, Fan J. · Laboratory of Cardiovascular Disease, Department of Pathology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba 305-8575, Japan. · J Biol Chem. · Pubmed #12196525 links to  free full text

Abstract: High lipoprotein(a) (Lp(a)) levels are a major risk factor for the development of atherosclerosis. The risk of elevated Lp(a) concentration is increased significantly in patients who also have high levels of low density lipoprotein (LDL) cholesterol. To test the hypothesis that increased plasma levels of Lp(a) may enhance the development of atherosclerosis in the setting of hypercholesterolemia, we generated Watanabe heritable hyperlipidemic (WHHL) transgenic (Tg) rabbits expressing human apolipoprotein(a) (apo(a)). We report here that Tg WHHL rabbits developed more extensive advanced atherosclerotic lesions than did non-Tg WHHL rabbits. In particular, the advanced atherosclerotic lesions in Tg WHHL rabbits were frequently associated with calcification, which was barely evident in non-Tg WHHL rabbits. To investigate the molecular mechanism of Lp(a)-induced vascular calcification, we examined the effect of human Lp(a) on cultured rabbit aortic smooth muscle cells and found that smooth muscle cells treated with Lp(a) showed increased alkaline phosphatase activity and enhanced calcium accumulation. These results demonstrate for the first time that Lp(a) accelerates advanced atherosclerotic lesion formation and may play an important role in vascular calcification.

Shiomi M, Ito T. · Institute for Experimental Animals, Kobe University School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, 650-0017, Kobe, Japan. · Eur J Pharmacol. · Pubmed #11716851 No free full text.

Abstract: To examine whether a microsomal triglyceride transfer protein (MTP)-inhibitor is effective in patients with homozygous familial hypercholesterolemia, we administered (2S)-2-cyclopentyl-2-[4-[(2,4-dimethyl-9H-pyrido[2,3-b]indol-9-yl)methyl]phenyl]-N-[(1S)-2-hydroxy-1-phenylethyl]ethanamide (Implitapide), a new MTP inhibitor, to low-density lipoprotein (LDL)-receptor-deficient Watanabe heritable hyperlipidemic (WHHL) rabbits at doses of 3, 6, and 12 mg/kg for 4 weeks. In the 12 mg/kg group, the plasma cholesterol and triglyceride levels were decreased by 70% and 45%, respectively, and the very low-density lipoprotein (VLDL) secretion rate was decreased by 80%. The composition of newly secreted VLDL was similar in each group. This suggests that Implitapide diminished the number of VLDL particles secreted from the liver. Although the ratio of vitamin E/LDL was not altered by Implitapide, triglyceride accumulation and a decrease in vitamin E were observed in the liver. In conclusion, an inhibition of VLDL secretion led to a decrease of plasma LDL in WHHL rabbits, and MTP inhibitors should have hypolipidemic effects against homozygous familial hypercholesterolemia.

Mori M, Itabe H, Higashi Y, Fujimoto Y, Shiomi M, Yoshizumi M, Ouchi Y, Takano T. · Department of Molecular Pathology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Tsukui, Kanagawa 199-0195, Japan. · J Lipid Res. · Pubmed #11714846 links to  free full text

Abstract: A monoclonal antibody, ASH1a/256C (256C), which binds to atherosclerotic lesions in Watanabe heritable hyperlipidemic rabbit (WHHL) aorta in vivo, recognizes complex structures of phosphatidylcholine mixed with neutral lipids. In the present study, a cell culture system is described in which foam cells express 256C-positive lipid droplets. J774.1 macrophages were incubated in the presence of a small volume of WHHL serum for 24 h to produce foam cells, which were then incubated without the WHHL serum for 3 days. Oil red O-positive lipid droplets appeared on day 1, and were present in the cells during the whole incubation period. The lipid droplets in the cells were positively immunostained with antibody 256C on day 4, although they were negative on day 1. Expression of the antigenic lipid droplets was also induced by the addition of acetylated LDL or sera from patients with hyperlipidemia. When foam cells were induced by the addition of WHHL serum, cellular content of cholesteryl ester was greatly increased but then decreased to near basal levels by day 4. Concomitantly, cellular free cholesterol increased during the culture period, indicating that the cholesteryl ester changes to free cholesterol by day 4. The lipid droplets in the foam cells on day 4 were positively stained with filipin, a fluorescent probe for free cholesterol, as well as with 256C antibody, indicating that free cholesterol is enriched in antigenic lipid droplets. These observations suggest that hydrolysis and rearrangement of cellular cholesterol take place in foam cells to form complex structures of phosphatidylcholine and free cholesterol in lipid droplets.

Zhang B, Shiomi M, Tanaka H, Mei J, Fan P, Tsujita Y, Horikoshi H, Saku K. · Department of Internal Medicine, Fukuoka University, Japan. · Eur J Drug Metab Pharmacokinet. · Pubmed #11695719 No free full text.

Abstract: To clarify the dose-response effects of troglitazone on insulin sensitivity and beta-cell function, we examined the effects of high-dose troglitazone (100 mg/day per animal, administered as a food admixture) on glucose and insulin metabolism in hyperinsulinemic Watanabe heritable hyperlipidemic (WHHL) rabbits, and compared the results with our previous results with low-dose troglitazone (10 mg /day per animal). MATERIALS AND METHODS: Glucose and insulin metabolism were quantitatively characterized by a minimal model technique as reported previously. RESULTS: When troglitazone was administrated at a high dose for 6 months, it reduced hyperinsulinemia as reflected by a reduced basal (steady-state) insulin concentration lb and the insulin response to a glucose load, improved beta-cell function as reflected by decreased second-phase post-hepatic insulin delivery to glucose phi2, and reduced insulin resistance as reflected by increased insulin sensitivity to glucose disposal Si, without affecting glucose tolerance as reflected by an unchanged rate of glucose utilization Kg or insulin-independent glucose disposal Sg. The reductions in Ib and phi2 and the increases in Si in WHHL rabbits treated with a high dose of troglitazone were greater (p<0.05) than those observed in WHHL rabbits treated with a low dose of troglitazone, as assessed by a two-way repeated measures analysis of variance and the Wilcoxon-Mann-Whitney test. CONCLUSION: In WHHL rabbits, troglitazone dose-dependently reduced hyperinsulinemia, improved beta-cell function, and increased insulin sensitivity.

Ishikawa K, Sugawara D, Goto J, Watanabe Y, Kawamura K, Shiomi M, Itabe H, Maruyama Y. · First Department of Internal Medicine, Fukushima Medical University, Fukushima, Japan. · Circulation. · Pubmed #11591622 links to  free full text

Abstract: BACKGROUND: Heme oxygenase-1 (HO-1) is proposed to have a variety of adaptive responses against oxidative stress. To examine the function of HO-1 against atherogenesis in vivo, we observed the effects of HO-1 inhibition on atherosclerotic lesion formation in Watanabe heritable hyperlipidemic rabbits (WHHL). Methods and Results- During 4 weeks of a 1% cholesterol diet, intravenous injections of Sn-protoporphyrin IX to inhibit HO-1 (S group, n=10) and saline as a control (C group, n=10) were given to 3-month-old WHHL rabbits. The percentages of en face atherosclerotic lesion areas in total descending aorta by Sudan IV staining (EFA) and the ratio of intima to media in microscopic atherosclerotic lesions in the ascending aortas (I/M) were calculated. Two different quantitative methods revealed significantly greater atherosclerotic lesions in the S group than the C group (EFA, P<0.001; I/M, P<0.005). HO-1 expression in atherosclerotic lesions was confirmed by Northern blot and immunohistochemical analyses. The dominant cell types expressing HO-1 were macrophages and foam cells, in which oxidized phospholipids were also accumulated. HO inhibition increased plasma and tissue lipid peroxide levels without affecting plasma lipid co osition. CONCLUSIONS: These results suggest the possibilities that HO-1 has antiatherogenic properties in vivo and that the antiatherogenic properties of HO-1 are conducted through the prevention of lipid peroxidation.

Takahashi S, Ohsugi K, Yamamoto T, Shiomi M, Sakuragawa N. · Department of Inherited Metabolic Disease, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan. · Tohoku J Exp Med. · Pubmed #11453536 links to  free full text

Abstract: This study has demonstrated the potential of human amniotic epithelial cells (HAEC) as a transgene carrier to treat patients with familial hypercholesterolemia (FH). One approach to liver-directed gene therapy is represented by transplantation of autologous hepatocytes that have been genetically modified in vitro. However, the hepatocytes must be isolated from surgically resected tissue and it is difficult to expand the hepatocytes in culture. In contrast, the advantages for using HAEC are the higher availability and the nonimmunogenicity after allotransplantation. Our strategy involved isolating HAEC from an amnion, transducing a human low-density lipoprotein receptor (LDLR) gene into these cells with a recombinant adenovirus, and transplanting the genetically modified cells into the liver of an animal model of FH. Each animal, treated with the LDLR-transduced HAEC, exhibited a substantial decrease in serum cholesterol with an eventual return to pretreatment level. Moreover, the transplanted HAEC migrated out of the sinusoids into the hepatic parenchyma and expressed the LDLRs until at least 20 days after transplantation. However, the transplanted HAEC markedly decreased in number after 10 days post-transplant with an increase of inflammatory cells. The temporary nature of the metabolic improvement may be associated with xenograft rejection and transient function of the adenoviral vector.

Ito T, Shiomi M. · Institute for Experimental Animals, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. · Atherosclerosis. · Pubmed #11368997 No free full text.

Abstract: We demonstrated that selectively bred homozygous WHHL rabbits known to show hypercholesterolemia and severe coronary atherosclerosis also spontaneously develop cerebral atherosclerosis beginning at 9 months of age. These intracranial lesions occurred in the absence of hypertension in 24 of 25 animals at various sites, mainly along arteries at the base of the brain. No lesions were observed in penetrating arteries. Lesions were rich in smooth muscle cells and fibrous tissue, showing only rare fragmentation or disappearance of the internal elastic lamina, and only limited lipid deposition. Few macrophages were observed in these lesions. No significant correlation was seen between severity of cerebral atherosclerosis and age, systolic blood pressure (BP), serum total cholesterol, or triglyceride concentration. Xanthomas of the pia mater were observed in all 25 rabbits. Arterial findings were similar to those in human cerebral atherosclerosis, indicating that the coronary atherosclerosis-prone homozygous WHHL rabbit represents the first animal model for spontaneous cerebral atherosclerosis.

Shimada K, Yoshida K, Tadokoro H, Ueda M, Shiomi M, Kitsukawa S, Takami A, Komatsu R, Suzuki K, Tanada S, Masuda Y. · Third Department of Internal Medicine, Chiba University School of Medicine, Japan. · Jpn Circ J. · Pubmed #11194293 links to  free full text

Abstract: The Watanabe heritable hyperlipidemic (WHHL) rabbit develops coronary atherosclerosis and hypercholesterolemia because of a genetic deficiency of low-density lipoprotein receptors and is therefore a good animal model for studying the relationships of coronary atherosclerosis, hypercholesterolemia and coronary flow reserve. The aim of the present study was to assess myocardial perfusion at baseline and during adenosine infusion (0.2 mg x kg(-1) x min(-1)) in 8 WHHL rabbits (13.8+/-0.5 months) with 13N-ammonia, small-animal positron emission tomography (PET) and colored microspheres. Results were compared with those from 6 age-matched Japanese white rabbits. Plaque distribution was also examined in the extramural coronary arteries. All 8 WHHL rabbits had coronary plaques, with 6 showing multiple plaques. Mean global myocardial blood flow (ml x min(-1) x g(-1)) did not differ significantly between control and WHHL groups both at baseline (3.67+/-0.72 vs 4.26+/-1.12 ml x min(-1) x g(-1), p=NS) and with adenosine (7.92+/-2.00 vs 9.27+/-2.91 ml x min(-1) x g(-1), p=NS), nor did coronary flow reserve (2.16+/-0.37 vs 2.18+/-0.41, p=NS). None showed evidence of regional perfusion abnormalities by visual and semiquantitative analyses of PET images. It was concluded that WHHL rabbits preserve adenosine-induced coronary flow reserve despite coronary atherosclerosis and hypercholesterolemia, suggesting that a compensatory mechanism develops in this animal model.

Fukumoto Y, Libby P, Rabkin E, Hill CC, Enomoto M, Hirouchi Y, Shiomi M, Aikawa M. · Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA. · Circulation. · Pubmed #11181475 links to  free full text

Abstract: BACKGROUND: Acute coronary syndromes often result from rupture of vulnerable plaques. The collagen content of plaques probably regulates their stability. This study tested whether HMG-CoA reductase inhibitors (statins) alter interstitial collagen gene expression or matrix metalloproteinase (MMP) levels in rabbit atheroma. METHODS AND RESULTS: We administered equihypocholesterolemic doses of pravastatin (a hydrophilic statin, 50 mg. kg(-1). d(-1), n=9), fluvastatin (a cell-permeant lipophilic statin, 20 mg. kg(-1). d(-1), n=10), or placebo (n=10) to mature Watanabe heritable hyperlipidemic rabbits for 52 weeks. The fluvastatin group achieved a much higher peak plasma concentration (23.7 micromol/L) than did the pravastatin group (1.3 micromol/L) under these conditions. Immunohistochemistry revealed that MMP-1, MMP-3, and MMP-9 expression by macrophages in the intima was lower in both the pravastatin and fluvastatin groups than in the placebo group, whereas there was no difference in macrophage numbers. Numbers of intimal smooth muscle cells (SMCs) (identified by immunohistochemistry) and expression of type I procollagen mRNA (detected by in situ hybridization), however, were significantly higher in the pravastatin group than in the fluvastatin group. Treatment with pravastatin, but not fluvastatin, preserved interstitial collagen content in vivo (detected by picrosirius red polarization). In vitro, fluvastatin, but not pravastatin, decreased numbers of rabbit and human aortic SMCs without altering procollagen I mRNA expression. CONCLUSIONS: This study showed that statins can reduce MMP expression in atheroma and that cell-permeant statins can decrease SMC number and collagen gene expression in vivo.

Ayajiki K, Ozaki M, Shiomi M, Okamura T, Toda N. · Department of Pharmacology, Shiga University of Medical Science, Seta, Ohtsu, Japan. · J Cardiovasc Pharmacol. · Pubmed #11065223 No free full text.

Abstract: Modifications by atherosclerosis of endothelium-dependent and -independent relaxations were evaluated in carotid arteries isolated from Watanabe heritable hyperlipidemic (WHHL; age 20-29 months) and age-matched Japanese white (JW) rabbits. Marked, patchy atherosclerotic lesions were observed in all WHHL rabbit arteries. Endothelium-dependent relaxations induced by acetylcholine, partly depressed by N(G)-nitro-L-arginine (L-NA), were significantly inhibited in the WHHL rabbit arteries with atherosclerosis, compared with those in the arteries without atherosclerotic lesions from JW and WHHL rabbits. No difference was observed in the relaxation caused by superoxide dismutase in these arteries. Conversely, endothelium-dependent relaxations by substance P were greater in the arteries with and without atherosclerosis from WHHL rabbits than in the arteries from JW rabbits. Endothelium-independent relaxations elicited by sodium nitroprusside and 2,2-(hydroxynitrosohydrazino)bis-ethanamine (NOC18) did not differ in the arteries from JW and WHHL rabbits. The responses to acetylcholine and substance P of JW rabbit arteries with the endothelium were not attenuated by treatment with pertussis toxin. L-NA-resistant, endothelium-dependent relaxations by substance P were almost abolished by charybdotoxin, and atherosclerosis did not alter the response. It is concluded that endothelial functions, evaluated by substance P, in rabbit carotid arteries are not impaired by atherosclerosis and by long exposure to hyperlipidemia in vivo. Dysfunction of muscarinic receptors may be involved in the depressed response to acetylcholine. As far as the arteries used in the present study are concerned, responses mediated possibly by endothelium-derived hyperpolarizing factor (EDHF) are unlikely to be modulated by atherosclerosis.