Insulin resistance and its associated compensatory hyperinsulinemia are considered to be etiologically related to the onset of NIDDM and diabetic pathophysiological states such as hypertension, obesity and atherosclerosis. The present study was designed to investigate the relationship between carotid arterial wall thickness and the degree of insulin resistance measured by the minimal model analysis.
We demonstrated that the advance of carotid arterial wall thickness (intimal plus medial thickness, IMT; plaque score, PS) of NIDDM patients is intense compared to that of non-diabetic group. Furthermore, negative correlation are significantly recognized between ΔIMT (progression of IMT per year) and SI (insulin sensitivity index.) The systolic blood pressure in patients with highly advanced IMT is significantly elevated than less advanced patients. These results suggest that advance of IMT is influenced by the degree of insulin resistance. The improvement of insulin resistance is necessary for the protection of advance in IMT.

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It has been well known that epidemiologically, there is clear correlation between insulin resistance and atherogenesis. Among several etiological factors for insulin resistance, hyperinsulinemia has been considered the major cause of the atherosclerosis. However, there has been no direct experimental evidence supportive of this hypothesis. In an attempt to prove such hypothesis, we have created a novel animal model for chronic hyperinsulinemia by means of the continuous insulin infusion (2u/kg body weight/day) into CSK miniature swine fed with a high cholesterol/high fat diet for 6 months. The serum insulin levels at fast in those animals were raised (ranging from 38.6 to 55.4μu/ml) and this hyperinsulinemia was maintained throughout the 6 months experiment. In this animal model, marked exacerbation of hypercholesterolemia (1348 vs. 883mg/dl) and atherosclerosis, especially coronary atherosclerosis were observed. These experimental data suggest that hyperinsulinemia may accelerate atherogenesis in hypercholesterolemic subjects. This animal model should also prove useful for further investigation of the mechanisms of atherosclerosis in hyperinsulinism.

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Insulin resistance and hyperinsulinemia may play a role in the development of atherosclerosis. To clarify this, rats with transplanted pancreas and insulin substrate-1 (IRS-1) knockout mice were examined. Syngeneic pancreatico duodenal transplantation with systemic venous drainage was performed in Wistar Shionogi (WS) and Spontaneous Hypertensive Rat (SHR) rats. Blood insulin levels of transplanted group are 1.5 to 2.5-fold higher than non-transplanted group. They revealed increased accumulation of cholesterol ester in aorta and slight body weight gain but neither hypertriglycemia nor hypertension. After a targeted disruption of the IRS-1 gene locus, mice show insulin resistance. They revealed both increased systolic blood pressure and increased serum triglyceride. Our results indicate that insulin resistance is important for the development of hypertension and hypertriglycemia, and hyperinsulinemia contributes to the acceleration of cholesterol ester accumulation in aorta.

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In recent years, multiple risk factor syndromes, including Syndrome X, the deadly quartet syndrome, and visceral fat syndrome have been noted as common condition for the development of coronary artery disease (CAD). Although these syndromes are often accompanied by insulin resistance, its pathogenesis have not been elucidated.
We have investigated the relationship between body fat composition and obesity-linked disorders and found the accumulation of intra-abdominal visceral fat is closely related to glucose intolerance, hypertension, hyperlipidemia, and insulin resistance determined by steady-state plasma glucose level in obese and even in non-obese people. Thus, visceral fat accumulation is the feasible condition for the development of insulin resistance and multiple coronary risks.
Although it is speculated that the multiple features in “multiple risk factor syndrome” are caused by insulin resistance, its molecular pathophysilogy has not been clarified. Recently it has been revealed that adipose tissue secretes various biologically active molecules. To clarify the molecular basis of hyperlipidemia, we analyzed expression of the genes for VLDL synthesis in animal model of visceral fat syndrome. Portal FFAs were elevated according to visceral fat accumulation even at early stage when insulin resistance has not manifested. Hepatic acyl-CoA synthetase and microsomal triglyceride transfer protein mRNA levels were upregulated compared to their lean littermates. These results suggested that enhancement of expression of these genes may be one of the mechanisms for hyperlipidemia in visceral fat accumulation. Type 1 plasminogen activator inhibitor (PAI-1) has a regulatory role in fibrinolytic process. Plasma PAI-1 levels were significantly correlated with the visceral fat area in obese (r=0.32, p<.02) and even in non-obese subjects (r=0.38, p<.02) but not with the subcutaneous fat area. PAI-1mRNA was detected in human and rodent adipose tissues and increased to 10-fold in visceral fat after making of VMH lesions, remaining unchanged in the subcutaneous fat, suggesting that secretion of PAI-1 from visceral fat may be responsible for elevated plasma PAI-1 and have a role in the development of vascular diseases in visceral fat syndrome.
These results suggested that visceral fat accumulation causes multiple coronary risks through the insulin resistance or independently by the molecules secreted from accumulated visceral fat.

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Insulin sensitivity has been determined in nonobese, non-diabetic, non hypertensive active for daily life subjects with either occlusive (OAP) or vasospastic AP (YAP). In both groups, SSPG which reflects insulin resistance increased significantly together with hyperinsulinemia. The resistance correlated with the degree of stenosis as well as lipoprotein abnormality (LDL-chol/LDL-apoB↓). In ASO insulin resistance is also present, but with less degree of hyperinsulinemia. In atherothrombotic cerebral infarction, insulin resistance together with compensated hyperinsulinemia has been also demonstrated, but not in lacunar infarction. The results indicate that subjects with the above atherosclerotic lesion tend to develop diabetes during the course of the diseases. In essential hypertension (HT) without obesity and diabetes, insulin resistance was noted in 70∼80% together with hyperinsulinemia in 25∼35%. Insulin resistance was correlated with smaller size of LDL particle and low HDL-chol. In essential HT, the resistance was inversely correlated with the degree of eduction of plasma K⁺ during insulin sensitivity test. Collagen stimulated intraplatelet Ca⁺⁺ also decreased during insulin infusion. In addition, antihypertensive drugs improved insulin sensitivity.
These results indicate that under or inadequate action of insulin rather than excessive insulin action plays a major role in the pathophysiology of insulin resistance syndrome. In order to correct the above vicious cycle, efforts to maintain normal insulin sensitivity is crucial for the prevention and treatment of atherosclerosis.

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Lipoprotein glomerulopathy (LPG) is a rare renal disease characterized by proteinuria, lipoprotein thrombi in the glomeruli and increased concentration of plasma apolipoprotein E. We analyzed apo E abnormality in 6 patients of LPG from 5 families. We found a new variant of apolipoprotein E (E Sendai) with a proline residue substituted for an arginine residue at position 145. All patients were heterozygous for this variant. The 4 carriers of apo E Sendai were found in KO and MI families. One of them was treated for chronic renal failure. The histological examination had not been done, and the diagnosis was not clarified. Our results suggest that apo E Sendai may be etiologically related to lipoprotein glomerulopathy.

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We examined the expression of bone matrix protein mRNAs both in rat cultured aortic rings and in human aortic tissues to investigate the roles of bone matrix proteins in injured aortic tissues. In rat cultured aortic rings from 3-month-old rats, the expression of OPN mRNA was strong before incubation and increased during 72-hour incubation. The expression of OPN mRNA was first detected after a 5-hour incubation and increased thereafter. On the other hand, the expression of ON mRNA was strong before the incubation and decreased during the incubation. The expression of ON mRNA was decreased age-dependently. The cell types expressing OPN and ON mRNAs were vascular smooth muscle cells. In human aortic tissues, the expression of OPN mRNA increased when atherosclerosis developed. In contrast, the expression of ON mRNA decreased when atherosclerosis developed. The combination of in situ hybridization and immunohistochemistry revealed that the OPN mRNAexpressing cells were macrophages around the atheromas, and that the ON mRNA-expressing cells were synthetic form of vascular smooth muscle cells which invaded into intima. Moreover, the localization of OPN protein was consistent with that of calcium phosphate. These results suggest that the bone matrix proteins, OPN and ON, may play important roles in vascular injury.

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Recently, it has been reported that calciumbinding proteins such as osteopontin and matrix Gla protein were expressed in calcified atherosclerotic lesion. In order to determine the role of osteopontin (OPN) in vascular calcification, we developed an in vitro model of vascular calcification by using bovine vascular smooth muscle cells (BVSMC) and demonstrated that alkaline phosphatase (ALP) plays an crucial role in BVSMC calcification and that the expression of OPN mRNA increases during this calcification. However, the mechanism by which OPN is induced in the development of vascular calcification still remains unknown. Therefore, we investigated the regulation of osteopontin expression during vascular calcification by using the above-mentioned model. Since β-glycerophosphate (β-GP) was used to induce calcification, we hypothesized that increased oncentrations of inorganic phosphate (Pi) resulting from the degradation of β-GP by ALP may modulate the expression of OPN in BVSMC. β-GP (3-10mM) dose-dependently increased the expression of OPN gene. Levamisole, a specific inhibitor of ALP, inhibited the induction of OPN gene by β-GP in a dose-dependent manner (10^-5-10^-3M). Furthermore, 2.5mM Pi restored the levels of OPN mRNA inhibited by levamisole, suggesting that Pi may directly increase OPN expression. Pi induced OPN mRNA in time-and dose-dependent manners. Since it is likely that calcium deposition resulting from raising the concentrations of Pi may induce OPN gene, we finally examined the effect of Pi on OPN expression under uncalcified condition [in the presence of etidronate (EHDP)]. Even in the presence of etidronate, OPN mRNA was upregulated by Pi. These data suggest that Pi may regulate OPN expression under calcifying microenvironment.

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One of the earliest systemic changes in response to tissue damage and inflammation caused by acute insults such as trauma or infection is the rapid synthesis by hepatocytes of a diverse group of blood proteins termed “acute phase reactants”. Among them, the major ones are C-reactive protein (CRP), serum amyloid A protein (SAA) and serum amyloid p component (SAP). In humans and most mammals, the concentrations of CRP and SAA may increase by several thousand-fold in the circulating blood and selectively deposit at sites of damaged tissue. A major function of CRP appears to be the recognition both of infectious agents and of damaged cells and their products. SAA is a member of HDL3 apolipoproteins and thought to inhibit reverse cholesterol transport. SAP is found in all forms of amyloid deposits. SAP responds to acute insults in mice but not in humans. It is a normal constituent of human plasma and tissues. Its association with many ligands and its conservation during evolution imply important functions. However, its physiological role is unclear.
It has been considered that atherosclerosis may be the result of a specialized chronic inflammatory fibroproliferative process; endothelial cells, macrophages, lymphocytes, smooth muscle cells and their cytokines participate in atherosclerotic process, which has become excessive and in its excess this protective response has become the disease state. This hypothesis led us to investigate if the three major acute phase reactants partcipate in human atherosclerosis. Recently, we have reported the presence of CRP and SAP in human aortic atherosclerotic lesions. The aim of this study is to investigate the localization of SAA in the lesions.
Serial sections of paraffin-embedded specimens of atherosclerotic abdominal aorta from autopsied patients, among whom amyloidosis was ruled out, were dewaxed. The one sections were stained with Congo-red to detect amyloid deposit but proved to be negative. The other ones were immunostained by the Streptavidin-Biotin method using a monoclonal anti-human SAA antibody (mcl; Dako) as the primary antibody. As a result, positive staining was observed extracellularly in the necrotic cores of atheromatous plaques, and intracellularly in foam cells. No staining was obtained when the primary antibody was substituted with non-immune mouse serum.
The present study demonstrates that SAA antigen is present in aortic atherosclerotic lesions of patients without amyloidosis. Together with our recent reports, this suggests that the three major acute phase reactants participate in atherosclerosis.

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The nonenzymatic glycation of body proteins, including extracellular matrix and plasma proteins, is thought to contribute to the development of atherosclerosis. The present study was performed to clarify the mechanism by which the glycated extracellular matrix accelerates the development of atherosclerotic lesions. First, we found the different localization of different epitopes of advanced glycation end products (AGEs) in atherosclerotic lesions. Namely, N^ε-(Carboxymethyl) lysine (CML), one of epitopes of AGEs, was localized mainly in macrophages or their related foam cells. In contrast, non-CML, another epitope of AGEs, was accumulated mainly in extracellular matrices and atheroma of intima. Second, when acid-soluble type I collagen was incubated with D-glucose in phosphate buffer, pH 7.4, at 37°C for 8 weeks, AGE-related cross-links were formed between collagen molecules. AGE contents were significanly greater in pepsin-insoluble collagen of rat aorta than pepsin-soluble collagen. In diabetic rats, AGEs were increased in pepsin-insoluble collagen, but not in pepsin-soluble collagen. Third, Glycated bovine serum albumin (BSA) showed a concentration-dependent cytotoxicity in smooth muscle cells in the presence of copper ion, but not in endothelial cells. When the cells were incubated with glycated BSA in the presence of copper ion, the extracellular generation of hydrogen peroxide was significantly less in endothelial cells than in smooth muscle cells. These results suggest that nonenzymatic glycation of extracellular matrix may contribute to the development of atheroslcerotic lesions throught the accumulation of lipid and collagen and smooth muscle cell injury.

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Recently several studies reported that proteoglycans modulate the function of cytokines. In particular, decorin and biglycan are known to bind to TGF-β and may participate in regulating extracellular matrix accumulation. In this study, we have analyzed the spatial and chronological distribution of these proteoglycans, cellular components and two cytokines, TGF-β and IL-1β in the stent-induced neointima formation to clarify their interactions in the process of neointima formation after vascular injury.
We implanted Gianturco's Z type stents in cholesterol-loaded or normal rbbbit aortas and harvested the stent-implanted aortas for immunohistochemical and in situ hybridization analysis. In normal aorta, biglycan and TGF-β mRNAs were expressed in the proliferating smooth muscle cells (SMCs) around the scent wire. These expressions gradually reduced as the intimal SMC proliferation ceased On the other hand, decorin and IL-1β mRNAs were rarely detected throughout the experimental period.
In cholesterol-loaded rabbit aortas, stent implantation injured the fibrofatty lesion, and induced more advanced neointima formation than that induced in normal aorta. TGF-β and biglycan mRNAs were expressed diffusely in the newly formed intima and their expressions were detectable throughout the experimental period. Decorin protein and mRNA were expressed in close proximity to accumulated macrophages expressing IL-1β mRNA around the stent wire at 2 months after the stent implantation.
In summary, the stent implantation in aorta with the pre-existing fibrofatty lesion enhanced neointima formation containing by further accumulation of biglycan and decorin as compared to normal aorta. Colocalization of ecorin and macrophages expressing IL-1β mRNA in the process of neointima formation provides a suggestive evidence that decorin synthesis and deposition are accelerated by IL-1β secreted from macrophages accumulating in the injured areas in the late stage after vascular injury.

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