Shinichi Oikawa

Generation of polyclonal antiserum against the growth hormone secretagogue receptor (GHS-R): evidence that the GHS-R exists in the hypothalamus, pituitary and stomach of rats.

Growth hormone (GH) secretagogues (GHSs), which stimulate GH secretion, are synthetic compounds that act through the GHS receptor (GHS-R) which has been recently cloned. We raised an antiserum in a rabbit against a synthetic peptide corresponding to amino acid residues 248-260 of the third intracellular loop of the rat GHS-R. A competitive immunoassay showed that the antiserum had a specific affinity for the target peptide. To confirm the specificity of the antiserum, the GHS-R cDNA was stably expressed in COS-7 cells. In Western blot analysis, the band was detected at 44 kDa in the extracts from COS-7 cells expressing GHS-R (COS-7/tf3-2) but not in those from wild-type COS-7 cells. Furthermore, while COS-7/tf3-2 cells were strongly immunostained for GHS-R, no GHS-R-like immunoreactivity was observed in wild-type COS-7 cells. Immunoreactive bands were also observed at approximately 46 kDa in the extracts from rat hypothalamus, pituitary and stomach by Western blot analysis. These studies are the first to show the existence of GHS-R protein in the stomach. The antiserum for the GHS-R is sensitive and specific, and it would be useful for clarifying the roles of GHS/ghrelin.

Lipoprotein Glomerulopathy: Renal Lipidosis Induced by Novel Apolipoprotein E Variants

Some systemic disorders induced by inherited abnormal lipid metabolism, e.g., Fabry disease, lecithin:cholesterol acyltransferase deficiency, and von Gierke disease, are associated with specific renal lipidoses [1, 2]. In addition, many investigations have found that hyperlipidemia secondary to nephrotic syndrome, hypertension, and an unbalanced diet may contribute to the development of glomerulosclerosis analogous to atherosclerosis which involves oxidized low-density lipoprotein (LDL) [3–5]. In contrast to these renal lipidoses, lipoprotein glomerulopathy (LPG) is a unique disease, characterized by a peculiar histology, in that it exhibits intraglomerular lipoprotein thrombi [6]. In 1995, we reviewed this disease and reported both its histological characteristics and its lipid and lipoprotein profiles [7]. Since then, however, new cases and findings of LPG have been reported [8–14]. In particular, our project team has clarified that several novel apolipoprotein (apo) E variants are associated with LPG [15–18]. Accordingly, it is postulated that lipoproteins containing apo E mutants may play a key role in the pathogenesis of LPG. In this review, we reexamine the characteristics of LPG and introduce recent studies which should help clarify the unique mechanism of LPG.

Increase of serum phosphatidylcholine hydroperoxide dependent on glycemic control in type 2 diabetic patients

In order to clarify the relationship between serum phosphatidylcholine hydroperoxide (PCOOH) levels and blood glucose control in type 2 diabetes patients (DM), DM (n = 61) and normal control (n = 11) were enrolled. High-density lipoprotein (HDL) was separated from serum by the addition of sodium phosphotungstate and magnesium chloride, and the precipitated fraction was prepared as non-HDL. Phospholipids were extracted from whole serum, non-HDL and HDL to estimate PCOOH level with chemiluminescence high performance liquid chromatography (CL-HPLC). PCOOH level (nmol/l, mean +/- S.D.) was higher in DM than in control (33.1 +/- 9.5 vs. 23.0 +/- 8.2 for serum; P < 0.01, 17.0 +/- 5.5 vs. 10.6 +/- 3.8 for non-HDL; P < 0.01, and 16.1 +/- 6.3 vs. 12.3 +/- 5.5 for HDL; not significant, respectively). DM was divided into five groups according to hemoglobin A(1c) (HbA(1c)) levels (%): (1) less than 6, (2) 6-6.4, (3) 6.5-6.9 (4) 7.0-7.4, and (5) over than 7.5. Increase of PCOOH levels was dependent on HbA(1c). We concluded that (1) serum and non-HDL PCOOH increased in DM, (2) the level was strongly correlated with diabetic control, and (3) approximately a half amount of serum PCOOH was present in HDL of both control and DM.

Lipoprotein glomerulopathy: A new aspect of lipid induced glomerular injury

Summary: Lipoprotein glomerulopathy is a new type of glomerular disease which is thought to be induced by an abnormality of lipoprotein metabolism. Until the end of 1994, 21 cases had been reported from Japan. However, this disease is not restricted to Japan because two cases with similar features have been described in France and the United States recently. Clinically, the presenting feature in all patients is proteinuria, resulting in a nephrotic syndrome in the majority. Renal biopsy specimens reveal that capillary lumina in the glomerulus are markedly dilated with pale‐stained and mesh‐like substances which are composed of fine granules. Under electron microscopy, it is observed that a combination of granules forms strata as fingerprints. Sudan or oil red‐O staining and immunofluorescence study on snap‐frozen sections show lipid droplets and apolipoproteins (apos) B and E in the capillary lumina, respectively. Accordingly, the intracapillary substances are thought to be lipoprotein thrombi. Lipid and lipoprotein profiles show type III hyperlipoproteinaemia and high level of apo E in plasma. However, the apo E phenotype is usually the heterozygous E2/3 or E2/4, different from the homozygous E2/2 in familial type III hyperlipoproteinaemia. Systemic manifestations characteristic of the previously‐reported lipidoses are not observed.

Etiological Significance of Apolipoprotein E Mutations in Lipoprotein Glomerulopathy

Lipoprotein glomerulopathy (LPG) is a newly recognized renal disease characterized by thrombus-like lipoproteins in the glomerular capillaries and abnormal lipoprotein profiles similar to those in type III hyperlipoproteinemia. Recently, these conditions have been shown to be associated with some apolipoprotein E (apoE) mutations. We found an apoE mutation (designated apoE-Sendai) that substitutes arginine 145 with proline. This mutation occurs most frequently in Japanese patients with LPG. To elucidate the etiological role of this mutation in the apoE gene, we established an experimental model for LPG by transducing apoE-Sendai in apoE knockout mice with the use of an adenovirus vector. Based on the findings in patients with LPG and its animal model, we suggest that the glomerular lesions are not only caused by hyperlipidemia, but also by in situ interaction between lipoprotein-containing mutant apoE with the glomerulus. In this review, we outline the clinical features of LPG and discuss the relationship between apoE mutations and LPG.

Design and baseline characteristics of a cohort study in Japanese patients with hypercholesterolemia: the Japan lipid intervention trial (J-LIT)

Abstract Objectives: The Japan Lipid Intervention Trial is a cohort study to (1) ascertain the optimal cholesterol levels to be maintained in preventing coronary heart disease (CHD) and other cardiovascular diseases and (2) assess the effect of simvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, on the serum lipid profile of Japanese patients with hypercholesterolemia. This report summarizes the study protocol and baseline characteristics of the study patients. Methods: Between November 1992 and June 1993, Japanese patients from all 43 prefectures and 3 specially administered cities were screened based on relative population statistics. Of 54,203 patients screened at 5289 institutions by 6511 investigators, 52,421 patients (96.7%; 17,424 men and 34,997 women) with a serum total cholesterol (TC) level ≥220 mg/dL were enrolled in this study and treated with open-labeled simvastatin 5 to 10 mg/d for 6 years. Patients were assigned as follows: 5127 patients (9.8%; 2194 men and 2933 women) with documented CHD to the secondary prevention cohort; the remaining 47,294 patients (90.2%; 15,230 men and 32,064 women) formed the primary prevention cohort. Results: Baseline lipid levels for the primary and secondary prevention cohorts, respectively, were: TC, 270 ± 34 mg/dL and 265 ± 30 mg/dL; high-density lipoprotein cholesterol (HDL-C), 52.9 ± 15.1 mg/dL and 50.5 ± 15.0 mg/dL; triglycerides (TG), 196 ± 171 mg/dL and 196 ± 144 mg/dL; and low-density lipoprotein cholesterol, 182 ± 34 mg/dL and 179 ± 32 mg/dL. Hypertension (45.0% and 47.0%, respectively) and diabetes mellitus (15.1% and 18.6%, respectively) were prevalent in both cohorts. In addition to hypercholesterolemia, 3 or more risk factors were detected in 12.5% and 32.8% of the primary and secondary prevention cohorts, respectively. In both cohorts serum TC and HDL-C levels were higher in women than in men. In contrast, the serum TG level was higher im men than in women. Serum TC and TG levels were higher in younger patients than in elderly patients. The serum HDL-C level was also higher in younger patients, except in women in the primary prevention cohort. Obesity was more common in patients with low serum HDL-C. Patients with high serum TG levels had a high frequency of hypertension, diabetes mellitus, and obesity. No significant correlation was noted between any of the lipid levels and blood pressure. Conclusions: The male:female and CHD:non-CHD distributions were similar to those found in a cross-sectional survey conducted in Japan, suggesting that the study population provides an appropriate model for investigating hypercholesterolemia in Japanese patients. Final analysis of the results of this study will clarify the relationship between the incidence of CHD and other cardiovascular diseases in Japanese hypercholesterolemic patients and cholesterol levels in a large number of subjects receiving simvastatin. It will also indicate the optimal cholesterol levels to be maintained to prevent CHD and other cardiovascular disease and assess the safety of long-term simvastatin therapy. These results will be reported at the end of 2000.

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.

Effect of low density lipoprotein on DNA synthesis of cultured human arterial smooth muscle cells

Increased concentration of plasma low-density lipoprotein (LDL) is one of the risk factors in atherosclerosis. We studied how DNA synthesis of human arterial smooth muscle cells (SMC) was influenced in the culture added with or without small dose (25 micrograms of protein/ml) of LDL. LDL were ultracentrifugally obtained from normal subjects and diabetics. DNA synthesis was investigated at 6-h intervals during 36 h in the culture with or without LDL using [methyl-3H]thymidine. We found that DNA synthesis reached a maximum value at 24 h after addition of LDL. On the other hand sequential changes were not detectable in the culture without LDL addition. This effect of diabetic LDL was significantly (P less than 0.001) greater than that of normal LDL. These results suggested that LDL induces synchronization of the cultured SMC to synthesize DNA and diabetic LDL may play an atherogenic role more strongly than normal LDL in the arterial wall even in the normal range of LDL concentration.

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.