Shuji Kitajima

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.

Macrophage Metalloelastase Accelerates the Progression of Atherosclerosis in Transgenic Rabbits

Background— Macrophage metalloelastase (matrix metalloproteinase [MMP]-12) is upregulated in atherosclerotic lesions and aneurysm; thus, increased MMP-12 activity may play an important role in the pathogenesis of atherosclerosis. However, the pathological roles of MMP-12 in the initiation and progression of atherosclerosis have not been defined. Methods and Results— We compared the susceptibility of MMP-12 transgenic (Tg) rabbits to cholesterol-rich diet–induced atherosclerosis with that of non-Tg littermate rabbits. The rabbits were maintained at either relatively lower levels of hypercholesterolemia for shorter periods or higher levels of hypercholesterolemia for longer periods through a diet containing different amounts of cholesterol. We found no significant difference in the aortic atherosclerotic lesion size or quality between Tg and non-Tg rabbits at lower hypercholesterolemia. At higher hypercholesterolemia for longer periods, however, Tg rabbits developed more extensive atherosclerosis in the aortas and coronary arteries than did non-Tg rabbits. Histological examinations revealed that atherosclerotic lesions of Tg rabbits contained prominent macrophage infiltration associated with marked disruption of the elastic lamina in the tunica media with occasional formation of aneurysm-like lesions. Furthermore, increased expression of MMP-12 derived from macrophages was associated with elevated expression of MMP-3, suggesting that MMP-12 may play a pivotal role in the cascade activation of other MMPs, thereby exacerbating extracellular matrix degradation during the progression of atherosclerosis. Conclusions— Overexpression of MMP-12 causes accelerated atherosclerosis in Tg rabbits. These results suggest that macrophage-derived MMP-12 participates in the progression of atherosclerosis.

Increased Expression of Vascular Endothelial Growth Factor in Kidney Leads to Progressive Impairment of Glomerular Functions

Vascular endothelial growth factor (VEGF) is an important mediator in maintaining normal kidney functions. In addition, several lines of evidence suggest that upregulation of VEGF in glomeruli may be associated with or cause renal dysfunction such as diabetic nephropathy. For elucidation of the pathologic consequences of high levels of VEGF in glomeruli, transgenic (Tg) rabbits that express human VEGF(165) isoform in both kidney and liver under the control of the human alpha-1-antitrypsin promoter were generated and characterized. With the use of heterozygous Tg rabbits and their littermates aged 8 to 55 wk, renal functions and structures were investigated. Compared with control rabbits, Tg rabbits exhibited progressive proteinuria with increased GFR at the early stage and decreased GFR at the later stage. Histologic examinations revealed that Tg rabbit kidneys were characterized by considerable glomerular hypertrophy as a result of increased proliferation of both glomerular capillaries and mesangial cells accompanied by prominent podocyte hypertrophy. With increasing age starting from 20 wk, Tg rabbit kidneys showed prominent formation of microaneurysms and capillary proliferation at the vascular pole area. At a later stage (55 wk), many glomeruli showed sclerosis and tuft collapse with the formation of glomerular cysts on a background of tubular atrophy and interstitial fibrosis. This study provides the first evidence that increased expression of VEGF in glomeruli directly causes the glomerular hypertrophy that is associated with proteinuria, suggesting that VEGF exerts multiple effects on the glomerular pathophysiologic processes.

Human C-Reactive Protein Does Not Promote Atherosclerosis in Transgenic Rabbits

Background— Although there is a statistically significant association between modestly raised baseline plasma C-reactive protein (CRP) values and future cardiovascular events, the debate is still unsettled in regard to whether CRP plays a causal role in the pathogenesis of atherosclerosis. Methods and Results— We generated 2 lines of transgenic (Tg) rabbits expressing human CRP (hCRP). The plasma levels of hCRP in hCRP-Tg-1 and hCRP-Tg-2 rabbits were 0.4±0.13 (n=14) and 57.8±20.6 mg/L (n=12), respectively. In addition, hCRP isolated from Tg rabbit plasma exhibited the ability to activate the rabbit complement. To define the role of hCRP in atherosclerosis, we compared the susceptibility of hCRP-Tg rabbits to cholesterol-rich diet-induced aortic and coronary atherosclerosis with that of non-Tg rabbits. After being fed with a cholesterol-rich diet for 16 weeks, Tg and non-Tg rabbits developed similar hypercholesterolemia and lesion sizes in both aortic and coronary arteries. Immunohistochemical staining and Western blotting revealed that hCRP was indeed present in the lesions but did not affect macrophage accumulation and smooth muscle cell proliferation of the lesions. Conclusions— Neither high nor low plasma concentrations of hCRP affected aortic or coronary atherosclerosis lesion formation in hCRP-Tg rabbits.

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.

Rabbit models for the study of human atherosclerosis: from pathophysiological mechanisms to translational medicine

Laboratory animal models play an important role in the study of human diseases. Using appropriate animals is critical not only for basic research but also for the development of therapeutics and diagnostic tools. Rabbits are widely used for the study of human atherosclerosis. Because rabbits have a unique feature of lipoprotein metabolism (like humans but unlike rodents) and are sensitive to a cholesterol diet, rabbit models have not only provided many insights into the pathogenesis and development of human atherosclerosis but also made a great contribution to translational research. In fact, rabbit was the first animal model used for studying human atherosclerosis, more than a century ago. Currently, three types of rabbit model are commonly used for the study of human atherosclerosis and lipid metabolism: (1) cholesterol-fed rabbits, (2) Watanabe heritable hyperlipidemic rabbits, analogous to human familial hypercholesterolemia due to genetic deficiency of LDL receptors, and (3) genetically modified (transgenic and knock-out) rabbits. Despite their importance, compared with the mouse, the most widely used laboratory animal model nowadays, the use of rabbit models is still limited. In this review, we focus on the features of rabbit lipoprotein metabolism and pathology of atherosclerotic lesions that make it the optimal model for human atherosclerotic disease, especially for the translational medicine. For the sake of clarity, the review is not an attempt to be completely inclusive, but instead attempts to summarize substantial information concisely and provide a guideline for experiments using rabbits.

Overexpression of peroxiredoxin 4 protects against high-dose streptozotocin-induced diabetes by suppressing oxidative stress and cytokines in transgenic mice.

Peroxiredoxin 4 (PRDX4) is one of a newly discovered family of antioxidative proteins. We generated human PRDX4 (hPRDX4) transgenic (Tg) mice, displaying a high level of hPRDX4 expression in the pancreatic islets, and then focused on the functions of PRDX4 in a type 1 diabetes mellitus (T1DM) model using a single high dose of streptozotocin (SHDS). After SHDS-injection, Tg mice showed significantly less hyperglycemia and hypoinsulinemia and a much faster response on glucose tolerance test than wild-type (WT) mice. Morphologic and immunohistochemical observation revealed that the pancreatic islet areas of Tg mice were larger along with less CD3-positive lymphocyte infiltration compared with WT mice. Upon comparison between these two mouse models, β-cell apoptosis was also repressed, and reversely, β-cell proliferation was enhanced in Tg mice. Real-time RT-PCR demonstrated that the expression of many inflammatory-related molecules and their receptors and transcription factors were significantly downregulated in Tg mice. These data indicate that PRDX4 can protect pancreatic islet β-cells against injury caused by SHDS-induced insulitis, which strongly suggests that oxidative stress plays an essential role in SHDS-induced diabetes. This study, for the first time, implicates that PRDX4 has a pivotal protective function against diabetes progression in this T1DM model.

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.

Human Apolipoprotein A-II Protects Against Diet-Induced Atherosclerosis in Transgenic Rabbits

Objective—Apolipoprotein (apo) A-II is the second major apo of high-density lipoproteins, yet its pathophysiological roles in the development of atherosclerosis remain unknown. We aimed to examine whether apo A-II plays any role in atherogenesis and, if so, to elucidate the mechanism involved. Methods and Results—We compared the susceptibility of human apo A-II transgenic (Tg) rabbits to cholesterol diet-induced atherosclerosis with non-Tg littermate rabbits. Tg rabbits developed significantly less aortic and coronary atherosclerosis than their non-Tg littermates, while total plasma cholesterol levels were similar. Atherosclerotic lesions of Tg rabbits were characterized by reduced macrophages and smooth muscle cells, and apo A-II immunoreactive proteins were frequently detected in the lesions. Tg rabbits exhibited low levels of plasma C-reactive protein and blood leukocytes compared with non-Tg rabbits, and high-density lipoproteins of Tg rabbit plasma exerted stronger cholesterol efflux activity and inhibitory effects on the inflammatory cytokine expression by macrophages in vitro than high-density lipoproteins isolated from non-Tg rabbits. In addition, &bgr;-very-low-density lipoproteins of Tg rabbits were less sensitive to copper-induced oxidation than &bgr;-very-low-density lipoproteins of non-Tg rabbits. Conclusion—These results suggest that enrichment of apo A-II in high-density lipoprotein particles has atheroprotective effects and apo A-II may become a target for the treatment of atherosclerosis.

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.