Tomonari Koike

C-Reactive Protein in Atherosclerotic Lesions: Its Origin and Pathophysiological Significance

C-reactive protein (CRP) is frequently deposited in the lesions of the arterial intima; however, the origin and pathological significance of CRP in these lesions are not completely understood. In this study, we measured CRP levels in the plasma of hypercholesterolemic rabbits and investigated CRP expression at both the mRNA and protein levels using rabbit and human atherosclerotic specimens. CRP levels were significantly elevated in both cholesterol-fed and Watanabe heritable hyperlipidemic rabbits, and CRP levels were clearly correlated with aortic atherosclerotic lesion size. Immunohistochemical staining coupled with Western blotting analysis revealed that CRP-immunoreactive proteins were found at all stages of atherosclerosis from the early to advanced lesions. CRP was present extracellularly and co-localized with apolipoprotein B but was rarely associated with the cytoplasm of macrophages and foam cells. Real-time reverse transcriptase-polymerase chain reaction analysis revealed that CRP mRNA in atherosclerotic lesions was barely detectable, and isolated macrophages did not express CRP mRNA, suggesting that CRP proteins found in the lesions were essentially derived from the circulation rather than synthesized de novo by vascular cells. These results suggest that there is a link between plasma CRP and the degree of atherosclerosis and that inhibition of plasma CRP may represent a therapeutic modality for the treatment of cardiovascular disease.

Overexpression of Human Matrix Metalloproteinase-12 Enhances the Development of Inflammatory Arthritis in Transgenic Rabbits

Increased proteolytic activity of matrix metalloproteinases (MMPs) may promote articular destruction such as occurs in rheumatoid arthritis and osteoarthritis. Recently, we reported that synovial tissue and fluid obtained from patients with rheumatoid arthritis contained higher activity of macrophage elastase (MMP-12). To examine the hypothesis that MMP-12 may potentially enhance the progression of arthritis, we investigated the effects of overexpression of MMP-12 on inflammatory arthritis in transgenic rabbits that express the human MMP-12 transgene in the macrophage lineage. Inflammatory arthritis was produced by articular injection of carrageenan solution and the degree of inflammatory arthritis in transgenic rabbits was compared with that in control rabbits. We found that overexpression of MMP-12 in transgenic rabbits significantly enhanced the arthritic lesions, resulting in severe synovial thickening, pannus formation, and prominent macrophage infiltration at an early stage and a marked destruction of articular cartilage associated with loss of proteoglycan at a later stage. These results demonstrate that excessive MMP-12 expression exacerbates articular connective tissue and cartilage degradation and thus plays a critical role in the development of inflammatory joint disease.

A minipig model of high‐fat/high‐sucrose diet‐induced diabetes and atherosclerosis

Type 2 diabetes is a major risk factor of the development of atherosclerosis in humans. However, studies examining mechanisms underlying diabetes‐accelerated atherosclerosis have been limited by the lack of suitable humanoid animal models. Pigs have a cardiovascular system that is very similar to that of humans and is useful as a model for human physiology and pathophysiology. In this study, we established a new miniature pig model for studying dyslipidaemia and atherosclerosis in diabetes. Chinese Guizhou minipigs were fed a normal control diet or a high‐fat/high‐sucrose diet (HFSD) for 6 months. Plasma total cholesterol (TC), high‐density lipoprotein cholesterol, triglyceride (TG), insulin and glucose were quantified at monthly intervals. The induction of insulin resistance and dysfunction of the pancreatic β‐cell were assessed by oral glucose tolerance test and insulin sensitivity test. The aortic fatty streak lesions were quantified following lipid staining with Sudan IV. During the feeding period, mild high plasma TC and TG were induced. At the end of 6 months, in HFSD‐fed animals, the adipocytes were hypertrophic, fat deposit in the liver was observed, loss of pancreatic β‐cells was observed, and the aortic fatty streak lesions were clearly present in the animals aortas. Our study established that miniature pigs that were fed a HFSD without adding dietary cholesterol developed insulin resistance, mild diabetes and atherosclerotic lesions. HFSD‐fed miniature pigs may be good animal models for research on the treatment of diabetic dyslipidaemia complicated with atherosclerosis.

Overexpression of Lipoprotein Lipase in Transgenic Watanabe Heritable Hyperlipidemic Rabbits Improves Hyperlipidemia and Obesity

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.

Overexpression of lipoprotein lipase improves insulin resistance induced by a high-fat diet in transgenic rabbits

Aims/hypothesisDysfunctions of lipoprotein lipase (LPL) have been found to be associated with dyslipidaemias, atherosclerosis, obesity and insulin resistance. There are two conflicting hypotheses regarding the roles of LPL in glucose metabolism and insulin resistance. Whether systemically increased LPL activity would be beneficial or detrimental to insulin sensitivity is yet to be resolved. To address this issue, we studied transgenic rabbits overexpressing human LPL transgene.MethodsLPL transgenic and control rabbits were fed a 10% high-fat diet (HFD) for 16 weeks. To evaluate glucose metabolism, we compared plasma levels of glucose and insulin in transgenic rabbits with control rabbits and performed an intravenous glucose tolerance test. In addition, we measured adipose tissue accumulation in HFD-fed rabbits.ResultsIncreased LPL activity in transgenic rabbits resulted in a significant reduction of plasma triglycerides and non-esterified fatty acids, but not in basal levels of glucose and insulin. HFD feeding induced an elevation of plasma glucose levels accompanied by hyperinsulinaemia in control rabbits, but was significantly inhibited in transgenic rabbits. The intravenous glucose tolerance test showed that transgenic rabbits had faster glucose clearance associated with lower levels of insulin secretion than control rabbits. In addition, there was a significant reduction of body adipose tissue in transgenic rabbits compared with in control rabbits fed an HFD. Scanning electron microscopic examination revealed that adipocytes in transgenic rabbits were predominately small cells.Conclusions/interpretationOur results showed that systemically increased LPL activity improves insulin resistance and reduces adipose accumulation in transgenic rabbits, indicating that systemic elevation of LPL may have potential benefits for the treatment of insulin resistance and obesity.

Overexpression of lipoprotein lipase in transgenic rabbits leads to increased small dense LDL in plasma and promotes atherosclerosis

Lipoprotein lipase (LPL) is a key enzyme in the hydrolysis of triglyceride-rich lipoproteins. Previous studies using transgenic mice and rabbits have demonstrated that high level of LPL activity in adipose and skeletal muscle protects against diet-induced hypercholesterolemia and subsequently prevents aortic atherosclerosis. However, it is unknown, per se, whether increased LPL activity itself is antiatherogenic, or whether the antiatherogenic effect of LPL is dependent upon the LPL lipid-lowering effect. To address this issue, we fed LPL transgenic and littermate rabbits diets containing different amounts of cholesterol (0.3–0.6%) adjusted to maintain their plasma cholesterol concentrations at similarly high levels for 16 weeks. We analyzed their lipoprotein profiles and compared their susceptibility to atherosclerosis. The results showed that the overexpression of LPL in transgenic rabbits reduced remnant lipoproteins (β-VLDL, d<1.006u2009g/ml) but concomitantly led to a significant increase of the large (d=1.02–1.04u2009g/ml) and small LDLs (d=1.04–1.06u2009g/ml) compared to the amounts in control rabbits. Furthermore, we found that with equally high hypercholesterolemia, transgenic rabbits developed 1.8-fold more extensive aortic atherosclerosis than control rabbits. To examine the hypothesis that altered lipoprotein profiles may be responsible for the enhanced atherosclerosis in transgenic rabbits, we studied the atherogenic properties of apoB-containing lipoproteins in vitro. These studies revealed that small-sized LDLs of transgenic rabbits were more susceptible to copper-induced oxidation and had higher affinity to biglycan than large remnant lipoproteins. We conclude, therefore, that LPL exerts a dual function in terms of its atherogenicity, namely antiatherogenicity, through enhancing receptor-mediated remnant lipoprotein catabolism and proatherogenicity via the generation of a large amount of small-sized LDLs. At an equal atherogenic-cholesterol level, small and dense LDLs are more atherogenic than large remnant lipoproteins.

Transgenic rabbits with increased VEGF expression develop hemangiomas in the liver: a new model for Kasabach-Merritt syndrome.

Clinical studies have provided ample evidence that high (either systemic or local) levels of vascular endothelial growth factor (VEGF) are associated with several pathophysiological disorders, including hemangiomas. To investigate whether elevated VEGF expression could directly affect these disorders, we created a transgenic (Tg) rabbit model with increased hepatic expression of the human VEGF165 transgene under the control of the human α-antitrypsin promoter. Tg rabbits exhibited marked hepatomegaly, with livers 2.5-fold heavier than those of control rabbits. Histological analysis revealed that the livers of Tg rabbits showed prominent dilation of the sinusoids and formed various-sized blood vessel networks, a feature of diffuse hemangiomas. Immunohistochemical staining revealed that the hepatocytes produced VEGF165, whereas plasma VEGF165 was not detected. Furthermore, Tg rabbits suffered from hemolytic anemia, thrombocytopenia and splenomegaly, which was associated with marked extramedullary hematopoiesis. The manifestations of Tg rabbits mimic many of the features of hemangiomatous disorders in humans such as the Kasabach–Merritt syndrome, and therefore this model may be potentially useful for the study of the pathogenesis and complications of hemangiomas as well as the investigation of angiogenesis inhibitors.

Contribution of the WHHL rabbit, an animal model of familial hypercholesterolemia, to elucidation of the anti-atherosclerotic effects of statins.

This year marks the 40th year since the discovery of a mutant rabbit showing spontaneous hyperlipidemia, which is the proband of the Watanabe heritable hyperlipidemic (WHHL) rabbit strain, an animal model of familial hypercholesterolemia, and the first statin, a general term for inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase, a rate limiting enzyme in cholesterol biosynthesis. Nowadays, statins are the primary drug of choice for treating cardiovascular disease. Although several reviews have described clinical trials and in vitro studies of statins, the anti-atherosclerotic effects of statins on animal models have not been comprehensively reviewed. This review summarized the contribution of WHHL rabbits to elucidating the anti-atherosclerotic effects of statins in vivo. Studies using WHHL rabbits verified that statins suppress plaque destabilization by reducing unstable components (foam cells derived from macrophages, foam cell debris, and extracellular lipid accumulation), preventing smooth muscle cell reductions, and increasing the collagen content of plaques. In addition, the expression of matrix metalloproteinases and tissue factor are decreased in intimal macrophages by statin treatment. Lipid-lowering effects of statins alter plaque biology by reducing the proliferation and activation of macrophages, a prominent source of the molecules responsible for plaque instability and thrombogenicity. Although statins remain the standard treatment for cardiovascular disease, new therapeutics are eagerly awaited. WHHL rabbits will continue to contribute to the development of therapeutics.

Lipidomic analysis of plasma lipoprotein fractions in myocardial infarction-prone rabbits

Lipids play important roles in the body and are transported to various tissues via lipoproteins. It is commonly assumed that alteration of lipid levels in lipoproteins leads to dyslipidemia and serious diseases such as coronary artery disease (CAD). However, lipid compositions in each lipoprotein fraction induced by lipoprotein metabolism are poorly understood. Lipidomics, which involves the comprehensive and quantitative analysis of lipids, is expected to provide valuable information regarding the pathogenic mechanism of CAD. Here, we performed a lipidomic analysis of plasma and its lipoprotein fractions in myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits. In total, 172 lipids in plasma obtained from normal and WHHLMI rabbits were quantified with high throughput and accuracy using supercritical fluid chromatography hybrid quadrupole-Orbitrap mass spectrometry (SFC/Q-Orbitrap-MS). Plasma levels of each lipid class (i.e., phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, lysophosphatidylcholine, lysophosphatidylethanolamine, sphingomyelin, ceramide, triacylglycerol, diacylglycerol, and cholesterol ester, except for free fatty acids) in 21-month-old WHHLMI rabbits were significantly higher than those in normal rabbits. High levels of functional lipids, such as alkyl-phosphatidylcholines, phospholipids including ω-6 fatty acids, and plasmalogens, were also observed in WHHLMI rabbit plasma. In addition, high-resolution lipidomic analysis using very low density lipoprotein (VLDL) and low density lipoprotein (LDL) provided information on the specific molecular species of lipids in each lipoprotein fraction. In particular, higher levels of phosphatidylethanolamine plasmalogens were detected in LDL than in VLDL. Our lipidomics approach for plasma lipoprotein fractions will be useful for in-depth studies on the pathogenesis of CAD.

Macrophage-Specific Overexpression of Human Matrix Metalloproteinase-12 in Transgenic Rabbits

Increased matrix metalloproteinase-12 (MMP-12) has been implicated in atherosclerosis and many other inflammatory processes. To define MMP-12 functions in vivo, we generated transgenic rabbits that expressed human (h) MMP-12 gene under the control of a macrophage-specific promoter, the human scavenger receptor promoter. Two transgenic founder rabbits were found to have hMMP-12 transgene integration by Southern blot analysis. hMMP-12 mRNA was expressed in peritoneal and alveolar macrophages, and in tissues enriched in macrophages in transgenic rabbits. High levels of hMMP-12 protein were detected in the conditioned media of cultured peritoneal and alveolar macrophages from transgenic rabbits. Zymography showed that hMMP-12 secreted from macrophages possessed enzymatic activity toward β-casein. To evaluate the expression of hMMP-12 in inflammatory sites, we used carrageenan-induced granulomas as an in vivo model for tissue macrophages and foam cells. Granuloma size in transgenic rabbits was significantly increased compared to that in control rabbits, and histological examination revealed that granulomas of transgenic rabbits were enriched in macrophages associated with increased hMMP-12 expression. We believe that this transgenic rabbit model with increased expression of hMMP-12 may become a useful model for further mechanistic studies of MMP-12 in inflammatory diseases and cancer invasion; it is also an ideal model for testing the in vivo action of MMP-12 inhibitors.