Hidetoshi Kotake

Human Arterial Smooth Muscle Cell Proliferation in Diabetes

In the present study, we focus on the proliferation of human arterial smooth muscle cells (SMCs) from NIDDM patients (DM-SMCs) to clarify the reactivity to the growth factor(s) in fetal calf serum (FCS) and the factors) secreted by T-cells. The proliferation of DM-SMCs was significantly greater than SMCs from nondiabetic patients (nonDM-SMC). DM-SMC conditioned medium (DM-condMed) increased the growth of nonDM-SMCs. These results suggest that the growth factor is secreted from DM-SMCs as an autocrine system, which increases the proliferation of nonDM-SMCs. T-cells increased DNA synthesis of SMCs, and DM-SMCs strikingly reacted to T-cells. The present results support a function of T-cells in stimulating SMC growth. In conclusion, human arterial SMC proliferation is increased in diabetes in the same fashion as in experimentally induced diabetes in animals through responses to growth factors and an increased autocrine system. These results provide a mechanism for the increase in atherosclerotic disease in diabetes.

Eradication of insulin resistance

In July, 2007, an 84-year-old Japanese man, with a bodymass index of 23 kg/m2, presented in a cold sweat and shivering. Clinical examination was unremarkable. His blood glucose was low (3·1 mmol/L). He was not taking any medications. Platelet count (285×109 per L) and HbA1c (5·0%) were normal. His symptoms resolved and he was discharged. At follow-up 1 month later, his platelet count had fallen to 56×109 per L and his HbA1c had risen (fi gure). Despite treatment with several antidiabetic agents, including acarbose, pioglitazone, and metformin, HbA1c increased to 9·0% 9 months after the fi rst visit, and he was still experiencing frequent hypoglycaemic attacks. Antibodies against insulin were not detected, but antibodies against the insulin receptor were present. Our patient’s serum inhibited binding of iodine-125-labelled insulin to the insulin receptor of IM-9 cells by 69·1% at a 1 to 4 dilution. Fasting plasma insulin was very high (137·9 mU/L), indicating severe insulin resistance. Type B insulin resistance syndrome was diagnosed. In the mean time, thrombocytopenia also worsened, with the platelet count falling to 10×109 per L. Antibodies against platelets (PA IgG) were detected (302 fg/platelet). Leucocytes, erythrocytes, infl ammatory markers, and complement factors were all within normal limits. Bonemarrow aspirate showed no evidence of megakaryocyte hypoplasia. There were no fi ndings suggesting collagen diseases. CT showed no evidence of pancreatic tumour, liver cirrhosis, or splenomegaly. On the basis of these fi ndings, immune thrombocytopenic purpura (ITP) was diagnosed. Helicobacter pylori infection was detected by the carbon-14 urea breath test and eradication therapy (amoxicillin, lansoprazole, and clarithromycin) was given for 7 days. Following H pylori eradication (confi rmed by breath test) platelet count increased and HbA1c decreased. 6 months after H pylori eradication PA IgG decreased (80 fg/platelet), insulin receptor antibodies were not detectable, HbA1c normalised, and hypoglycaemic episodes no longer occurred. Notably, fasting plasma insulin decreased to 10·1 mU/L, confi rming striking improve ment of insulin resistance. When last seen in February, 2009, our patient was not taking glucoselowering drugs; anti-IR antibodies were still undetectable, and HbA1c was normal (4·8%). Our patient had not had any new hypoglycaemic symptoms. Type B insulin resistance syndrome is a rare cause of diabetes with severe insulin resistance and is caused by polyclonal immunoglobulin G antibodies directed against the insulin receptor. These antibodies block insulin binding to the receptor, resulting in hyperglycaemia. Paradoxically, hypoglycaemia, particularly while fasting, is occasionally associated with this disorder. Type B insulin resistance syndrome is frequently associated with other auto immune diseases. Our patient’s clinical course strongly suggests that type B insulin resistance syndrome and ITP developed simultaneously and that both improved with H pylori eradication, which is the recommended treatment for ITP. In this case, H pylori eradication also ameliorated type B insulin resistance syndrome. There is increasing evidence that H pylori infection is directly involved in modulating host immune responses. Furthermore, H pylori eradication reportedly ameliorates some immunological disorders, including anti phospholipid antibody syndrome and rheumatoid arthritis. Our case suggests an H pylori infection-related pathological mechanism underlying type B insulin resistance syndrome. There is no established eff ective therapy for type B insulin resistance syndrome. Indeed, it was very diffi cult to manage our patient’s diabetes, which was also associated with occasional hypoglycaemia; treatment was no longer necessary after H pylori eradication. In cases of type B insulin resistance syndrome, testing for H pylori infection may be worthwhile, with a view to treating the infection if present.

Appearance of multidisperse low density lipoprotein and altered lipoprotein composition in non-insulin-dependent diabetes with type IIa hyperlipoproteinemia.

The purpose of the present study is to elucidate the characteristics of lipoprotein disorders in diabetes mellitus. By analytical ultracentrifugation, non-insulin-dependent diabetic patients (NIDDM) with type IIa hyperlipoproteinemia (HLP) showed significantly higher incidence of multidisperse low density lipoprotein (LDL) than non-diabetics with type IIa HLP. Furthermore, LDL multidispersity in diabetic subjects seemed to be directly related to neither triglyceride (TG) levels in very low density lipoprotein (VLDL) nor the degree of glycemic control. Diabetics with multidisperse LDL had a lipoprotein profile that was different from the subjects with paucidisperse LDL as follows: (1) enrichment in the cholesterol content of VLDL, (2) TG-rich LDL with small flotation coefficient, (3) low cholesterol levels in high density lipoprotein2 along with enrichment in TG, and (4) high plasma concentrations of apoprotein B. Although the underlying mechanism behind the prevalence of multidisperse LDL in NIDDM with type IIa HLP remains unknown, it seems important that lipoprotein disorders in diabetics with multidisperse LDL were potentially atherogenic.

Anti-angiogenic effect of TGFβ in aqueous humor

Abstract Neovascularization is mediated by various factors in ocular tissues. Recent studies have emphasized the role of vascular endothelial growth factor in the induction of angiogenesis. We have previously reported that aqueous humor (AH) suppressed vascular endothelial cell growth and angiogenesis. We speculated that the anti-angiogenic effect of AH is mediated by transforming growth factor beta (TGFβ). In order to clarify the presence of TGFβ in bovine AH, we applied it on the heparin-sepharose affinity column and prepared two fractions (bound and unbound fractions). We measured TGFβ concentration in each fraction and examined how the anti-TGFβ antibody decreased the inhibitory effect of AH on human umbilical vein endothelial cell growth and on in vitro angiogenesis. We found the presence of TGFβ2, but not TGFβ1, in the heparin bound fraction, and the inhibitory effect was detected in the heparin-bound fraction. Anti-TGFβ antibody completely and dose-dependently extinguished the inhibitory effect of AH. We propose that the inhibitory effect of AH on endothelial cell growth and in vitro angiogenesis are both mediated by TGFβ2. Our results indicate TGFβ2 is normally present in AH and protects the eye tissue against abnormal neovascularization.

T lymphocytes increase the synthesis of esterified cholesterol in human monocyte-derived macrophages by activation of the scavenger pathway

Immunohistochemical analyses have shown the presence of T lymphocytes (T-cells) in atherosclerotic places in addition to macrophages and smooth muscle cells. To elucidate the role of T-cells in the formation of atherosclerotic lesions, we studied whether T-cells can stimulate the scavenger pathway and promote esterified cholesterol (EC) synthesis by [14C]oleate incorporation in macrophages. Macrophages and T-cells were co-cultured in two ways. In one culture, macrophages were in direct contact with T-cells (direct contact form). In the other, macrophages and T-cells were separated by Transwell membrane, but shared the same culture medium via the membrane (indirect contact form). Based on the incorporation of [14C]oleate into EC, macrophages strikingly increased EC synthesis in both forms of co-culture. This increase was proportional to the number of T-cells present and was inhibited by cyclosporin A. When macrophages were co-cultured indirectly in contact with T-cells in the presence of AcLDL for 24 h, and the T-cells were subsequently removed, EC synthesis in macrophages increased. However, this increase was not observed in macrophages that were rinsed twice with PBS. When macrophages, previously incubated with AcLDL for 24 h, were co-cultured indirectly in contact with T-cells for 24 h, the medium were prepared as activated T-cell-conditioned medium (aTCM). EC synthesis in macrophages cultured with aTCM increased. The ability of aTCM to increase EC synthesis disappeared upon repeated freezing/thawing, boiling and trypsin treatment. T-cells (indirect contact form) and aTCM similarly increased AcLDL-binding and -degradation in macrophages. These results indicated that T-cells secreted an active substance(s), protein in nature, which could activate the scavenger pathway and increase EC synthesis in macrophages. These observations suggest that T-cells can promote the uptake of modified lipoproteins by macrophages to induce foam cell-formation.

Biliary lipid composition in heterozygous familial hypercholesterolemia and influence of treatment with probucol

The lipid composition of fasting duodenal bile was determined in 11 healthy subjects with normolipidemia and 15 patients with heterozygous familial hypercholesterolemia (FH) (12 with type IIa, three with type IIb). The age distribution among the groups of subjects was similar. In the patients with heterozygous FH type IIa, the mean value for molar percentage of cholesterol and lithogenic index (LI) of bile were significantly higher than those of controls (8.4±1.0%, 1.47±0.18 calculated by Hegard, Dam, and Holzbach vs 4.3±0.4%, 0.81±0.07, respectively). The value of LI in the patients with FH type IIb was also found to be significantly higher than that of the controls. In the patients with heterozygous FH type IIa, we observed both a significant decrease in the molar percentages of glycochenodeoxycholic acid, glycoursodeoxycholic acid, and glycolithocholic acid, and a significant increase of taurochenodeoxycholic acid compared to the corresponding values in the controls. Bile analysis of six patients was reexamined during probucol treatment after 16 weeks. Probucol significantly lowered serum cholesterol levels. However, biliary lipid composition and individual bile acid proportions was not altered by the treatment. The results suggest that most of the patients with heterozygous FH have supersaturated bile and are predisposed to cholesterol gallstone formation. In addition, the mechanism by which probucol lowers serum cholesterol appears to be independent of any change in the metabolism of biliary lipid.

DIABETES AND ATHEROSCLEROSIS

We investigated the effects of high glucose environment on the synthesis of cholesterol ester (CE) and intracellular cholesterol accumulation in cultured THP-1 cells, macrophage-like human monocytic cell line. THP-1 cells were grown in control (200 mg/dl of glucose) or high glucose concentrations (400, 600, 800, or 1600 mg/dl) medium for 6 days. The cells were incubated with 50 μg/ml of native, glycated, acetylated, or oxidized low-density lipoproteins (LDL). CE synthesis was the highest in the culture with 400 mg/dl of glucose, irrespective of the type of LDL compared with cells grown in 200 mg/dl of glucose. No increase of CE synthesis was observed in the culture with 600, 800 or 1600 mg/dl of glucose. CE accumulation in the culture with oxidized LDL (50 μ;g/ml) was also found in cells grown in the presence of 400 mg/dl of glucose. compared with that in cells grown 200 mg/dl of glucose (5.2±0.1 and 3.2±0.6 ng/mg cell protein, respectively; p<0.02). The amount of oxidized LDL associated to cells grown in 400 mg/dl of glucose was markedly increased compared with the cells grown in 200 mg/dl of glucose. Our results suggesetd that an suitable glucose concentration (400 mg/dl) associated with increased number of certain scavenger receptors (receptors for oxidized LDL) expressed on cells, which ultimately results in stimulation of CE synthesis and intracellular accumulation of CE in macrophages. Our results suggest that high blood glucose concentration may alter the metabolism of arterial wall cells, thus contributing to the pathogenesis of vascular complications of diabetes mellitus.