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Consolidated, monoclinic ZrO2 polycrystal was produced from undoped ZrO2 powders in air by flash sintering at the sintering temperature of 1350°C for 5 minutes or 3 hours under an applied DC electric field of 175 V/cm. When the ZrO2 was heated under the applied DC field, the electric current of the specimen steeply increased at the furnace temperature of 1335°C below the sintering temperature of 1350°C. When the furnace temperature was decreased from the sintering temperature of 1350°C to room temperature, volumetric expansion associated with tetragonal-to-monoclinic phase transformation gradually took place at the furnace temperature from 1000°C to 750°C, and monoclinic ZrO2 body was remained consolidated even at room temperature in both specimens. In contrast, conventionally sintered ZrO2 without applying DC field exhibited the abrupt volumetric expansion at about 1000°C, and shattered. SEM observation revealed the presence of grain-boundary second phase in the flash-sintered specimen for 3 hours, which is a possible origin of keeping a bulk body at room temperature. The thinner second phase is considered to be formed also in the flash-sintered specimen for 5 minutes, although the formation of the phase could not be observed clearly by SEM observation. On the other hand, XRD measurements showed that directions of the monoclinic ZrO2 grains were oriented along the applied DC field after the isothermal flash sintering for 3 hours while the grain alignment could not be observed in flash-sintered specimen for 5 minutes. The alignment of ZrO2 grains observed in the isothermal flash sintering is considered to be closely related to the preferential direction of oxygen ionic conduction and the second phase formed along grain boundaries.

Platelet-derived growth factor (PDGF) stimulates expression of matrix metalloproteinases (MMPs), including stromelysin-1 (MMP-3). Induction of these expressions is known to occur during the course of atherosclerosis, tumor invasion, and metastasis. We investigated PDGF-alpha receptor (alphaR)- and beta receptor (betaR)-mediated signaling pathways for the expression of MMP-3 and invasion activity using porcine aortic endothelial (PAE) cells with stable expression of normal or mutated PDGF receptors. RT-PCR and Western blot analyses revealed that PDGF-BB induces MMP-3 expression in PAE cells that exclusively express either the PDGF-alphaR or the -betaR, but not in non-transfected control cells. To identify the signals necessary for PDGF receptor-mediated induction of MMP-3 expression, several lines of PAE cells expressing mutant PDGF receptors were further analyzed. Cells expressing mutant PDGF receptors unable to associate with Src or PLCgamma, retained the ability to induce MMP-3 expression as a result of PDGF-BB stimulation. However, incubation with PDGF-BB did not induce MMP-3 expression in cells expressing a mutant PDGF-betaR unable to associate with phosphatidylinositol 3(')-kinase (PI3K). LY294002, a PI3K inhibitor, reduced PDGF-BB-stimulated MMP-3 expression in PAE cells expressing wild-type PDGF receptors. In contrast, PDGF-BB induced MMP-3 expression in the presence of U-73122, a PLCgamma inhibitor. Moreover, PDGF-BB enhanced the invasiveness of cells expressing wild type PDGF-beta receptors, but not of cells expressing mutant PDGF-betaRs impaired in their ability to associate with PI3K. In light of these results, it appears that PDGF-BB is capable of inducing MMP-3 expression through both the PDGF-alphaR and the -betaR, and the effects are contributed by the PI3K-mediated transduction pathways.

The authors investigated the binding of human plasma 125I-labelled chylomicrons to Chinese hamster ovary (CHO) cells, i.e. native CHO cells are mutant ldl-A7 cells lacking the low-density lipoproteins receptor, in the absence and presence of exogenous bovine milk lipoprotein lipase (LPL) in the culture medium. Only a small amount of binding to either cell was observed in the absence of added LPL. Exogenously added LPL increased the specific binding of chylomicrons to ldlA7 cells, as well as to native CHO cells. The enhanced binding of chylomicrons to ldl-A7 cells or native CHO cells by LPL was inhibited by heparinase and a monoclonal antibody against LPL (5D2) which recognizes the carboxyl terminal of LPL. However, the enhanced binding was not inhibited by 1M NaCl, which abolishes the enzymatic activity of LPL in either ldl-A7 cell or native CHO cells. These results suggest that LPL enhances the binding of chylomicrons to heparan sulphate proteoglycans of CHO cells, and that it is the carboxyl terminal of LPL but not the enzymatic activity of LPL that is essential for LPL to mediate the binding of chylomicrons to CHO cells.

In a previous study it was shown that postprandial lipid metabolism is delayed in individuals with intra-abdominal visceral fat accumulation. Population studies have shown that as compared with individuals with apolipoprotein (apo) E3/3, those with phenotype apo E3/4 phenotype have higher plasma and low density lipoprotein (LDL)-cholesterol (C) concentration and increased susceptibility to coronary heart disease. The aim of the present study is to determine how apo E4 affects postprandial lipid metabolism by comparing individuals with apo E3/4 to those with apo E3/3 phenotype matched for abdominal visceral fat. Sixty-two Japanese subjects (41 male, 21 female) [average age 48+/-14 years; mean body mass index (BMI) 25+/-5.6 kg/m2] were recruited for this study. The subjects were divided into two groups: those with apo E3/3 (n=43) and those with apo E3/4 phenotype (n=19), as determined by isoelectric focusing (IEF). Visceral fat accumulation was analyzed as area of fat deposition by computerized tomography at the umbilicus level. After a 12-h overnight fasting, an oral vitamin A and a fatty meal were administered to these subjects. The plasma triglyceride (TG) increased significantly hours after fat loading in both groups but the levels of TG were significantly higher in apo E3/4 than in apo E3/3 phenotype at 2, 4 and 6 h after fat loading. Plasma retinyl palmitate (RP) levels were also significantly higher in individuals with apo E3/4 than in those with apo E3/3 phenotype at 2, 4 and 6 h after fat loading. This investigation was then conducted in both genders separately, and found that these associations were statistically significant in men. Furthermore, after matching men for fasting TG levels, these associations did not persist for plasma TG levels at any time point, while plasma RP levels were still significantly higher in apo E3/4 group at 2 and 6 h after fat loading. These results indicate that in Japanese population especially for men apo E phenotype E3/4 is associated with an impaired postprandial TG-rich lipoprotein metabolism relative to apo E3/3 phenotype when matched for intra-abdominal visceral fat accumulation, which has a substantial effect on the metabolism of plasma TG-rich lipoproteins.

The subject was a 57-year-old Japanese woman with a body mass index of 21.2 kgm(-2). Her serum total cholesterol (TC), triglycerides (TG) and HDL-cholesterol levels were 7.11 mmoll(-1), 0.53 mmoll(-1) and 2.05 mmoll(-1), respectively. She had a marked increase of serum apolipoprotein (Apo) E concentration of 25 mgdl(-1) with normal concentrations of serum Apo A-I, A-II, B, C-II and C-III. Polymerase chain reaction-restriction fragments length polymorphism analysis of the cholesteryl ester transfer protein (CETP) gene from this subject revealed the heterozygous nucleotide change causing a Asp442 to Gly substitution (D442G) in the CETP protein. For comparison, 11 unrelated female subjects with this mutation (age, 57+/-5.1 years; BMI, 22+/-1.5 kgm(-2); TC, 7.23+/-1.16 mmoll(-1); TG, 1.44+/-0.80 mmoll(-1); HDL-C, 2.47+/-0.53 mmoll(-1)) were found to have a serum Apo E concentration of 7+/-1.5 mgdl(-1), about a third of the patient's concentration. The lipoprotein profile of the proband's serum analyzed by disk polyacrylamide gel electrophoresis showed a trace amount of VLDL. A vitamin A fat-loading test showed little increase in serum triglycerides and retinyl palmitate levels compared with control subjects at 2, 4 and 6 h after fat loading. Ultracentrifugation analysis of her serum revealed no detectable Apo E in the VLDL fraction but showed a large amount of Apo E in the HDL fraction, in contrast to a normal control, who had Apo E in the VLDL fraction as well as in the HDL fraction. Sequence analysis of the Apo E gene from the subject showed no nucleotide changes in exon 3 and exon 4, which code the mature Apo E protein, indicating there is no structural abnormality in the Apo E protein. Direct sequence analysis of the LDL receptor gene also did not show any nucleotide change. Based on these findings, it was hypothesized that the marked increase of Apo E in the patient's serum was caused by a decreased transfer of Apo E from HDL particles to TG-rich lipoproteins or impaired uptake of Apo E-containing HDL by LDL receptor or remnant receptor, due presumably to a dysfunction of these receptors in the patient.

Leptin exerts its effects by interacting with specific membrane receptors (Ob-R). We studied the exact localization of long intracellular domain form (Ob-Rb) in human brain. In addition, we analyzed the regulatory features of Ob-Rb expression in two neuroblastoma cell lines. The Ob-Rb mRNAs were abundant in putamen, frontal lobe, medulla, cerebral cortex, cerebellum, thalamus, hippocampus, corpus callosum, caudate nucleus, and amygdala, indicating that Ob-Rb transcripts are expressed differently from that of other Ob-R isoforms. In SK-N-MC cells, the expression of Ob-Rb mRNA was induced by increasing doses of insulin, and the maximum amount of mRNA expression was 9.4-fold higher in the presence of insulin (100 nM for 24 h), compared to the absence of insulin. In IMR32 cells, the transcripts were increased 4.0-fold when cells were incubated with 1 nM of insulin for 48 h. In contrast, Ob-Rb expression in IMR32 cells decreased to 18% of control following a 24-h incubation period with 50 ng/mL of leptin, compared to incubation in the absence of leptin. These results indicate that expression of Ob-Rb is differentially regulated by inhibitory signals of energy balance in neuroblastoma cells. The identification of the novel regulatory mechanisms involving the Ob-Rb isoform by insulin and leptin now makes it possible to elucidate the underlying mechanisms involving increased food intake and uncontrolled energy balance associated with leptin resistance in obese individuals.

Intimal hyperplasia following arterial endothelial denudation results in large part from the proliferation of vascular smooth muscle cells (SMCs) and matrix accumulation. Procollagen COOH-terminal proteinase enhancer (PCPE) binds procollagen COOH-propeptides and potentiates procollagen COOH-proteinase activity to cleave COOH-propeptides of procollagens I-III. Here we report the enhanced expression of PCPE in cultured SMCs and in intimal thickening induced by arterial injury. The levels of PCPE mRNA in parallel with the level of p21(Cip1) mRNA, as a negative regulator of cellular proliferation, increased under serum deprivation or reduced cellular proliferation in cultured SMCs. In contrast, rapidly proliferating cells show the decreased levels of PCPE mRNA. In vivo, the marked induction of PCPE in injured rat arteries occurred at 14 days after endothelial denudation. The induced expression levels of PCPE as well as p21(Cip1) were maintained until 42 days, although cyclin E expression declined. Furthermore, transforming growth factor beta1 (TGF-beta1), an important regulator of cellular proliferation in atheroma, increased the levels of the PCPE mRNA in cultured SMCs. Thus, the regulatory expression of PCPE dependent on cellular proliferation, and particularly contact inhibition, may play a key role in the proliferation of SMCs and matrix production during the process of atheroma formation.

To clarify how troglitazone, an insulin-sensitizing agent, affects lipid metabolism and post-heparin plasma lipoprotein lipase(LPL), fifteen patients ( 3 male, 12 female) [ the average age 62±7 y; the mean body mass index ( BMI) 25 ± 3 kg/m2] were recruited and the serum lipids and postheparin plasma lipoprotein lipase(LPL) mass before and 4 weeks after oral administration of troglitazone ( 200 mg per day ) were measured. Mouse preadipocyte cell line, 3T3-L1 cells were treated with this compound and LPL enzyme protein mass in the culture media was measured by an enzyme linked immunosorbent assay. A reverse transcription polymerase chain reaction (RT-PCR) and Northern blot analysis was conducted to investigate the effect of this compound on the expression of LPL.The average levels before treatment of fasting serum total cholesterol, triglycerides and high density lipoprotein-cholesterol, plasma glucose and glycohemoglobin Alc were 216 ± 34, 160±84, 57± 19, 145±30 mg/dl and 7.8 ± 1.6 %. Four weeks after treatment, those levels were 209 ±36, 105 ±27 ( p=0.004 ), 63 ± 19 ( p=0.02 ) mmol/l, 139±41 mg/dl and 7.3 ±0.6 % ( p=0.01), respectively. The postheparin plasma LPL mass increased from 226 ± 39 to 257 ± 68 ng/ml( p = 0.03 ) during that period. RT-PCR and Northern blot analysis revealed that in the cultured 3T3-L1 cells, the expression of LPL was enhanced in the presence of troglitazone. These results suggests that troglitazone improves plasma triglyceride-rich lipoproteins metabolism by enhancing the expression of LPL in adipocytes.

In order to test whether a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor has an anti-atherogenic activity, the effects of carvastatin, a newly developed potent inhibitor, and pravastatin were examined on the intimal thickening of the artery after the endothelial denudation induced by balloon catheter injury. Rabbits were divided into four groups; control, pravastatin-treated (20 mg kg(-1) day(-1)) and two of carvastatin-treated groups (10 or 20 mg kg(-1) day(-1)). Two weeks after balloon catheter injury, the areas of intima and media of the injured carotid arteries were determined, and the ratios of intima to media (I/M) were calculated as an index of intimal thickening. Average I/M ratios of the injured artery were 0.42+/-0.05 for control, 0.49+/-0.07 for pravastatin, 0.19+/-0.03 (10 mg kg(-1) day(-1)) and 0.20+/-0.04 (20 mg kg(-1) day(-1)) for carvastatin-treated rabbits, respectively. Thus, carvastatin reduced I/M ratio of the injured artery to approximately half versus control, but pravastatin failed to suppress the intimal thickening. For in vitro study, vascular smooth muscle cells (SMC) from rabbit aorta were explanted, then cultured, and the effects of carvastatin on SMC migration and SMC proliferation were also examined. Carvastatin inhibited dose-dependently SMC migration and SMC proliferation with IC50 values of 0.5 microM and 1 microM, respectively. These inhibitory effects of carvastatin were cancelled by the coexistence of mevalonate, a metabolite of cholesterol synthesis. Our results suggest that carvastatin may be useful in rabbits as an anti-atherogenic drug by means of the inhibition of SMC migaration or SMC proliferation.

1. The effects of saponin from Ginseng Radix rubra on extracellular matrix metabolism, the activation and synthesis of TGF-beta1, and the modification of TGF-beta receptor in fibroblasts were examined to elucidate the contribution of the TGF-beta pathway to the mechanism of wound healing by saponin. 2. Fibronectin synthesis was analysed by the immunoprecipitation method. Activation and synthesis of TGF-beta1 were measured by ELISA. The expressions of TGF-beta receptors in fibroblasts were examined at protein and mRNA levels by the cross-linking method and Northern blot analysis, respectively. 3. The fibronectin synthesis increased 2.3- and 3.9-fold at fibroblasts treated with 1 and 10 microg ml(-1) of saponin, respectively, compared with that in non-treated cells. Fibronectin synthesis stimulated with 10 microg ml(-1) of saponin was inhibited with 69% by 5 microg ml(-1) of an anti-TGF-beta1 antibody. mRNA of TGF-beta type I receptor increased 4.8- and 4.4-fold at fibroblasts treated with 1 and 10 microg ml(-1) of saponin, respectively, and that of TGF-beta type II receptor also increased 3.4- and 3.2-fold at fibroblasts treated with 1 and 10 microg ml(-1) of saponin, respectively. The significant increases of TGF-beta type I and II receptors and of fibronectin synthesis were observed at the same concentrations of saponin. TGF-beta content increased 1.74- and 1.87-fold at conditioned medium of fibroblasts treated with 100 and 250 microg ml(-1) of saponin, respectively, higher concentrations than those which accelerated fibronectin synthesis. Furthermore, the active TGF-beta content was below 10% of total TGF-beta at each concentration of saponin. 4. These results indicate that saponin stimulates fibronectin synthesis through the changes of TGF-beta receptor expressions in fibroblasts.

BACKGROUND In mutations of the low-density lipoprotein (LDL) receptor gene, the defect of internalization is caused by a mutation in the cytoplasmic domain of the receptor linked with exons 17 and 18, and the O-linked sugar domain linked with exon 15 has been speculated not to affect the function of the receptor. Here, we describe a novel mutation of the O-linked sugar domain of the LDL receptor gene, designated familial hypercholesterolaemia (FH)-Mishima with Japanese pedigree, which resembles but still differs from classical defective internalization cases. METHODS LDL metabolism was examined in cultured skin fibroblasts from patients. Immunoprecipitation and immunohistochemical techniques were applied for the detection of the receptor protein size and distribution. Screening of the mutant exon(s) of the LDL receptor gene was performed using the polymerase chain reaction-single-strand conformation polymorphism technique (PCR-SSCP), and sequencing of the mutated alleles was carried out using the dideoxy chain termination method. RESULTS LDL-binding activity at 4 degrees C in skin fibroblasts from patients was similar to normal, but that at 37 degrees C with the ligand decreased time dependently and was lost at 6 h, resulting in the defect of internalization and degradation of LDL. The receptor protein on the cell surface was detected at 4 degrees C by IgG-C7, an anti-LDL receptor antibody, but was not detected after incubation with LDL at 37 degrees C. The size of the receptor was 112 kD as determined by immunoprecipitation analysis. A deletion of two nucleotides in exon 15 was detected in the DNA sequence of the LDL receptor gene. The deletion results in a shift of the reading frame after Thr-713 of the mutant and makes a stop codon at amino acid 759. CONCLUSION Deletion of the two nucleotides caused novel amino acid sequences after the O-linked sugar domain, which has the ability of sorting on the cell membrane at 4 degrees C, but not at 37 degrees C in vivo, resulting in the complete cessation of activity of the LDL receptor.

Receptors belonging to the low density lipoprotein receptor (LDLR) superfamily play important biological roles in addition to mediating lipoprotein metabolism. The recent discovery of a novel mosaic LDLR family member by us (Yamazaki H., Bujo, H., Kusunoki, J., Seimiya, K., Kanaki, T., Morisaki, N., Schneider, W.J., and Saito, Y. (1996) J. Biol. Chem. 271, 24761-24768) and others, which we termed LR11, offers the opportunity to gain new insights into receptor multifunctionality. The predominant expression of LR11 in brain and the presence of elements found in neural adhesion molecules suggested a function(s) in the central nervous system (CNS). In order to gain information about this complex receptor in an accessible system, we have molecularly characterized the murine LR11 and report on its detailed localization and developmental expression pattern. The primary sequence of the murine protein further establishes that LRlls are among the closest relatives within the LDLR family and that brain is the predominant site of expression. In situ hybridization showed that neuronal bodies such as Purkinje cells in the cerebellum and other neurons in the hippocampal formations and the cerebral cortex are particularly rich in LR11 transcripts. The developmental pattern of LR11 expression in brain, which peaks at 2 weeks, is in contrast to those of two other LDLR family members, the very low density lipoprotein receptor and the LDLR. During early development, murine LR11 expression levels are highly dependent on neural cell types. These findings are compatible with function(s) of LR11 in neural organization and, possibly, pathogenesis of degenerative brain diseases. In addition, detailed knowledge of LR11 biology will help to elucidate the roles of other mosaic proteins that share with LR11 elements whose function is not yet known.

OBJECTIVES The purpose of this study was to investigate the possibility of impaired lipolysis of triglyceride-rich lipoproteins in patients with abdominal visceral fat accumulation by assessing two major lipolytic enzymes in the plasma, lipoprotein lipase (LPL) and hepatic lipase (HL). DESIGN AND PATIENTS A total of 31 patients [20 men, 11 women, age 50 ± 7 years old, body mass index (BMI) 26 ± 2 kg/m2 (mean ± sd)] were analyzed. Visceral fat and subcutaneous fat areas were evaluated using a computerized tomographic (CT) method at the level of the umbilicus. Total lipolytic activity in the postheparin plasma (PHP) was measured using Triton X-100-emulsified triolein and LPL activity was calculated as the activity in whole plasma inhibited by the 5D2 monoclonal antibody for LPL. LPL enzyme mass was determined by a sandwich enzyme immunoassay. RESULTS The visceral fat area was found to be negatively correlated with LPL mass (V vs LPL mass, r = − 0.37, P = 0.04) in PHP and had a tendency toward negative correlation with the LPL activity in the PHP (V vs LPL activity, r = − 0.29, P = 0.12). Subcutaneous fat area, on the other hand, did not show any correlation with LPL activity (r = 0.13, P = 0.49) or mass (r = 0.22, P = 0.25) in the PHP. The visceral fat area was found to be positively correlated with fasting serum insulin levels (r = 0.67, P

Effects of fatty acids on intimal thickening induced by a balloon catheter injury model were investigated by feeding rabbits a small amount of either lard [L] or fish oil [F]. Serum lipids of these groups were not different from those of basal diet-fed rabbits [controls] after 4 weeks of feeding. Serum saturated fatty acids such as 14:0, 16:0, and 18.0 were significantly greater in the L-fed rabbits compared with controls, but those of the aorta were not significantly different. Fatty acid composition of the F-fed rabbits was only different from that of the controls in that n-3 fatty acids slightly increased. The mean and maximum intimal thickening 2 weeks after ballooning, carried out 2 weeks after feeding, were significantly higher in the carotid arteries of the L-fed rabbits than in the controls. The intimal thickening of the F-fed rabbits did not significantly differ from that of the controls. These results suggest that lard promotes the formation of the smooth muscle cell dominant type of arteriosclerosis without affecting serum lipid levels.

Abstract Certain receptors belonging to the LDL receptor (LDLR) gene family appear to constitute a newly identified branch whose members are expressed in brain, in addition to other tissues. In support of this concept, we have now discovered the expression and delineated the molecular structures of a representative of this emerging branch from two such diverse species as human and chicken. This membrane receptor, called LR11 and thus far only known to exist in the rabbit, is a complex seven-domain mosaic protein containing, among other structural elements, a cluster of 11 LDLR ligand-binding repeats and a domain with homology to VPS10, a yeast receptor for vacuolar protein sorting. Cytoplasmic signature sequences define the receptor as competent for endocytosis. The most striking properties of LR11s are their (1) high degree of structural conservation (>80% identity among mammals and birds), with 100% identity in the membrane-spanning and cytoplasmic domains of rabbit and human; (2) lack of regulation by cholesterol and estrogen; and (3) expression in brain. The features of LR11 suggest important roles in intercellular and intracellular ligand transport processes, some of which it may share with other brain-specific LDLR family members.

Interaction between cultured endothelial cells (EC) and pericytes (PC) was studied in vitro to clarify the mechanism of diabetic proliferative retinopathy. Conditioned medium (CM) from retinal PC strongly increased the proliferation and moderately stimulated migration of retinal EC. Moreover, CM from PC caused stimulation of angiogenesis of retinal EC and umbilical cord vein EC in vitro at the same extent as basic fibroblast growth factor (bFGF). PC also stimulated angiogenesis by EC in mixed cultures. The angiogenic, proliferative and migration activities in CM from PC were inhibited by an antibody to bFGF. These data suggest that PC play an important role in angiogenesis through secretion of an FGF-like molecule.

Smooth muscle cells in arteries of diabetic rats and rabbits have unique properties including the overexpression of platelet-derived growth factor (PDGF) beta-receptor compared with controls. Fibronectin, one of the increased components of extra-cellular matrices in diabetic arteries, plays an important role in the phenotypic change of smooth muscle cells from the contractile to the synthetic type with the expression of the PDGF beta-receptor. Moreover, fibronectin synthesis is regulated by transforming growth factor-beta (TGF-beta). In this study, we report on the expression of TGF-beta receptors in diabetic smooth muscle cells, by immunohistochemistry, cross-linking of 125I-TGF-beta 1 to cells and quantitative reverse transcription-polymerase chain reaction. We also report on the effects of TGF-beta 1 on fibronectin synthesis of diabetic smooth muscle cells by use of ELISA and immunoprecipitation, in order to clarify the role of TGF-beta-fibronectin pathway in forming characteristic changes of diabetic smooth muscle cells. Cultured aortic smooth muscle cells of diabetic rats expressed TGF-beta type II receptor about 8.7 times that of controls at the protein level and 5.7 times at the mRNA level, whereas the expression of the type I receptor did not differ between the two types of smooth muscle cells. These changes were accompanied by increased fibronectin synthesis in diabetic smooth muscle cells in response to TGF-beta 1. Furthermore, protein expression of fibronectin, and mRNA and protein of TGF-beta type II receptor were increased in the diabetic aorta compared with the control aorta in vivo, implying the importance of the TGF-beta-fibronectin pathway for the unique biology of smooth muscle cells in the diabetic artery.

Long-term incubation of proteins with glucose leads to advanced glycation end products (AGEs) with fluorescence and a brown color. We recently demonstrated immunologically the intracellular AGE accumulation in smooth muscle cell (SMC)-derived foam cells in advanced atherosclerotic lesions. To understand the mechanism of AGE accumulation in these foam cells, we have now characterized the interaction of AGE proteins with rabbit-cultured arterial SMCs. In experiments at 4 degrees C, 125I-labeled AGE-bovine serum albumin (AGE-BSA) showed a dose-dependent saturable binding to SMCs with an apparent dissociation constant (Kd) of 4.0 microg/ml. In experiments at 37 degrees C, AGE-BSA underwent receptor-mediated endocytosis and subsequent lysosomal degradation. The endocytic uptake of 125I-AGE-BSA was effectively inhibited by unlabeled AGE proteins such as AGE-BSA and AGE-hemoglobin, but not by acetylated LDL and oxidized LDL, well-known ligands for the macrophage scavenger receptor (MSR). Moreover, the binding of 125I-AGE-BSA to SMCs was affected neither by amphoterin, a ligand for one type of the AGE receptor, named RAGE, nor by 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole-hexanoic acid-BSA, a ligand for the other AGE receptors, p60 and p90. This indicates that the endocytic uptake of AGE proteins by SMCs is mediated by an AGE receptor distinct from MSR, RAGE, p60, and p90. To examine the functional role of this AGE receptor, the migratory effects of AGE-BSA on these SMCs were tested. Incubation with 1-50 microg/ml of AGE-BSA for 14 h resulted in significant dose-dependent cell migration. The AGE-BSA-induced SMC migration was chemotactic in nature and was significantly inhibited (approximately 80%) by an antibody against transforming growth factor-beta (TGF-beta), and the amount of TGF-beta secreted into the culture medium from SMC by AGE-BSA was sevenfold higher than that of control, indicating that TGF-beta is involved in the AGE-induced SMC chemotaxis. These data suggest that AGE may play a role in SMC migration in advanced atherosclerotic lesions.

Lipoprotein-a, Lp(a), is a variant form of low density lipoprotein (LDL) that contains apolipoprotein-a, whose structure has 75-85% homology with plasminogen. Elevated plasma levels of Lp(a) are considered to be one of the independent risk factors for cardiovascular disease. We studied the effects of niceritrol, a nicotinic acid derivative, on plasma Lp(a) levels in 72 patients with hypercholesterolaemia. The dose of niceritrol was increased every 4 weeks, from 750 to 1500 and then to 2250 mg day-1. The final dose was adjusted to obtain a plasma cholesterol level less than 5.69 mmol l-1. Niceritrol led to significant decreases in plasma levels of median Lp(a), from 16.1 mg dl-1 (interquartile intervals, 8.7 to 32.8) to 11.1 mg dl-1 (interquartile intervals, 6.6 to 21), the mean reduction rate being 17.6%. In the group with pretreatment Lp(a) levels of over 20 mg dl-1, Lp(a) decreased by 10.0, 22.0 and 31.8% at the doses of 750, 1500, and 2250 mg day-1, respectively. In the group with levels less than 20 mg dl-1, only the dose of 2250 mg day-1 was effective in the reduction of Lp(a). The results suggest that the reduction of Lp(a) was dependent on the dose of niceritrol and on the pretreatment level of Lp(a). In conclusion, niceritrol is effective, in a dose-dependent manner, for reducing Lp(a) levels.

The effect of phospholipids on cholesteryl ester hydrolysis by neutral cholesterol esterase in alveolar macrophages was studied. Among the phospholipids used as emulsifiers, those with a negative charge, such as phosphatidylserine, phosphatidic acid, phosphatidylinositol, and cardiolipin, gave a higher level of hydrolysis by neutral cholesterol esterase than other less negatively charged phospholipids, such as phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, and sphingomyelin. Phospholipase D treatment of liposomes emulsified with phosphatidylcholine produced phosphatidic acid and enhanced cholesteryl ester hydrolysis. Phospholipase A2 treatment produced lysophosphatidylcholine and decreased the hydrolysis. The hydrolysis of cholesteryl ester in lipid droplets obtained from cholesterol-laden macrophages elicited by thioglycollate in the rat peritoneal cavity was low compared to artificial liposomes emulsified with phosphatidylcholine. The reason for this was speculated to be that lipid droplets were low in total phospholipids and poor in phospholipids with strong negative charges but rich in phosphatidylethanolamine and sphingomyelin. These results suggest that the polar heads of phospholipids may play an important role in cholesteryl ester hydrolysis by neutral cholesterolesterase.

1. The effects of saponin from Ginseng Radix rubra on angiogenesis (tube formation) and its key steps (protease secretion, proliferation and migration) in human umbilical vein endothelial cells (HUVEC) were examined to elucidate the mechanism of the tissue repairing effects of Ginseng Radix rubra. The effect on a wound healing model was also studied. 2. Tube formation was measured by an in vitro system. The activity and immunoreactivity of tissue-type plasminogen activator (tPA) as a protease for angiogenesis and the immunoreactivity of its inhibitor, plasminogen activator inhibitor-1 (PAI-1), were measured in conditioned medium of HUVEC stimulated for 24 h with saponin. Cell proliferation was measured by counting the cell numbers at 2-7 days after seeding. Migration was measured by Boyden's chamber method. The effect on wound healing was studied in the skin of diabetic rats. 3. Saponin at 10-100 micrograms ml-1 significantly stimulated tube formation by HUVEC in a dose-dependent manner. Saponin in a similar concentration-range increased the secretion of tPA from HUVEC as estimated by immunoreactivity and enzyme activity. On the other hand, PAI-1 immunoreactivity was slightly increased at 10 micrograms ml-1 of saponin, but then was significantly decreased at 50 and 100 micrograms ml-1. Cell proliferation was only slightly enhanced by 1-100 micrograms ml-1 of saponin, but migration was significantly enhanced by 10-100 micrograms ml-1 in a dose-dependent manner. Moreover, saponin stimulated wound healing with enhanced angiogenesis in vivo. 4. These results indicate that saponin stimulates tube formation mainly by modifying the balance of protease/protease inhibitor secretion from HUVEC and enhancing the migration of HUVEC, and that it is effective in vivo.

Abstract Cholesterol metabolism in macrophages from atherosclerosis-prone C57BL/6J mice was compared with that in macrophages from atherosclerosis-resistant C3H/HeN mice. Plasma total cholesterol levels of both types of mice were significantly increased, but HDL cholesterol level was increased only in C3H/HeN mice when a high-cholesterol diet (1% cholesterol) was fed for 5 weeks. After incubation of macrophages from male and female mice on the high-cholesterol diet with β-VLDL for 24 hours, cholesterol content in macrophages from C57BL/6J was approximately 1.5- to 2.0-fold higher than in those from C3H/HeN mice. [ 3 H]Cholesterol oleate–β-VLDL incorporation into macrophages from C57BL/6J mice on the high-cholesterol diet was greater than incorporation into those from C3H/HeN mice. The release of [ 3 H]cholesterol from macrophages from C57BL/6J mice on the high-cholesterol diet was one seventh that from macrophages from C57BL/6J mice on the basal diet or that from macrophages from C3H/HeN mice on the basal or high-cholesterol diet. Acid cholesterol esterase activity was almost the same in macrophages from any group. Acyl CoA:cholesterol acyltransferase activity in macrophages from C57BL/6J mice on the high-cholesterol diet increased compared with that from macrophages from C57BL/6J mice on the normal diet. Neutral cholesterol esterase activity in macrophages from C57BL/6J mice was about half of that in macrophages from C3H/HeN mice independent of the type of diet. There were no sex differences in these metabolisms. Considered with our previous data, these results suggested that a high-cholesterol diet may cause metabolic changes to accumulate cholesterol ester in macrophages from C57BL/6J mice in accordance with genetic abnormalities.

We synthesized zirconium oxynitride from yttria-stabilized zirconia (YSZ) in air by applying DC electric fields that produced a controlled electric current in the specimen. When YSZ was heated under an applied DC electric field, the electric current of the specimen steeply increased at a critical temperature, called a flash event, during flash sintering. By keeping the electric current of the specimen constant during the flash event and then holding the specimen at the critical temperature, YSZ was transformed into zirconium oxynitride under the optimal conditions of 50 V/cm, 500 mA, and 1000 °C. We confirmed that zirconium oxynitride formed using high-resolution transmission electron microscopy, electron energy-loss spectroscopy, and energy-dispersive spectrometry. To convert oxides to nitrides, reducing conditions are necessary to form excess oxygen vacancies. Our technique produced the strong reducing conditions necessary to form nitrides from the oxides by delivering a controlled electric current to t...

The effects of platelet-derived growth factor (PDGF) on the expression of intercellular adhesion molecule-1(ICAM-1) as an indicator of cell activation were investigated in cultured human arterial smooth muscle cells(SMC). PDGF-BB and -AB but not -AA at 2-10 ng/ml stimulated ICAM-1 expression at a subconfluent but not a confluent state in a dose-dependent manner. ICAM-1 expression was induced at 2h, reached a plateau at 4h, and continued for at least 24h after stimulation with PDGF. The maximal stimulatory effect of PDGF-BB at 10 ng/ml was comparable to that by optimal concentrations of other cytokines and inflammatory agents. These data suggested that PDGF was a potent stimulator of ICAM-1.

Objective: To assess whether control of diabetes mellitus is as important in the elderly as in young and middle-aged diabetic patients in terms of progression of retinopathy. Design: A 5-year longitudinal cohort study. Setting: Outpatient diabetic clinic. Patients: One hundred fourteen non-insulin-dependent diabetic patients (30 males, 84 females) ≥ 60 years of age. Measurements: Retinopathy was checked at the beginning and end of the follow-up period. During the 5-year follow-up period, demographic variables, body mass index, HbA***1c, blood pressure, and plasma lipids were monitored. Retinopathy was classified as follows: grade 0, no lesion; grade 1, non-proliferative retinopathy; grade 2, pre-proliferative retinopathy; grade 3, proliferative retinopathy. Progression of retinopathy during the 5-year follow-up was defined as an increase in its grade. Results: At the start of the study, 13% of the patients already had retinopathy, all of grade 1. The 5-year follow-up study showed that progression of retinopathy was 23.6% in all cases, 22.2% in those with grade 0 initially, and 33.3% in those with grade 1 initially. The progression rates of retinopathy as a function of the mean HbA***1c during the follow-up were as follows: lower than 7%, 2%; 7–8%, 20%; 8–9%, 40%; more than 9%, 61%. Multiple logistic regression analysis showed that, of the parameters examined, only HbA***1c was a significant risk factor for progression of retinopathy. Conclusions: Control of diabetes mellitus is the most important factor associated with prevention of progression of retinopathy in elderly patients.

Angiogenesis is a crucial event in the progression of diabetic retinopathy. Migration and proliferation of endothelial cells (EC) are important steps in angiogenesis and are caused by angiogenic factors such as basic fibroblast growth factor (bFGF). In this work, capillary EC were isolated from rabbit retinal tissues and rabbit retinal EC (RREC) were found to secrete a migration factor for RREC in conditioned medium (CM). The activity was inhibited by an anti-platelet-derived growth factor (PDGF) antibody, but not by an anti-bFGF antibody. We also found that RREC showed a migratory response to PDGF. The response was induced by PDGF-BB and PDGF-AB dose dependently, but not by PDGF-AA, indicating that it was mediated by PDGF-beta receptor-dependent pathways, and that the PDGF-like factor was PDGF-BB or -AB. In addition, PDGF-BB induced the proliferation of RREC as well as bFGF. These data indicate that RREC have an autocrine pathway of PDGF by the secretion of and the response to PDGF. PDGF may play significant parts in angiogenesis in the progression of diabetic retinopathy.

Mononuclear cell invasion into the vascular-vessel wall is a very important initial step in the development of atherosclerotic lesions. Hypercholesterolemia leads to a marked adhesion of circulating blood monocytes to arterial endothelial cells in vivo, and minimally oxidized low-density lipoprotein enhances monocyte adhesion to endothelial cells in vitro. The activation of phospholipase A2 (PLA2) is also important in the oxidation of low-density lipoprotein by endothelial cells. In this study, we investigated the role of PLA2 activation in the adhesion of a leukemic monocyte cell line (THP-1 cells) to endothelial cells in vitro using an adhesion assay and a cell-ELISA technique. The treatment of human umbilical-cord-vein endothelial cells with PLA2 stimulators such as interleukin-1 beta, tumor necrosis factor and lipopolysaccharide all increased the adhesion of THP-1 cells to endothelial cells. Exogenous PLA2 also increased the adhesion of these cell types. The increased adhesion induced by these PLA2 stimulators, as well as PLA2 itself, was reversed by various inhibitors of the PLA2 reaction. A product of the PLA2 reaction, lysophosphatidylcholine, also increased cell adhesion. A cell-ELISA technique showed the enhanced expression of vascular-cell-adhesion-molecule 1 and intercellular-adhesion-molecule 1 to endothelial cells after treatment with PLA2 stimulators, PLA2 or lysophosphatidylcholine. These results suggest that the PLA2 reaction enhances monocyte adhesion to endothelial cells through the expression of cellular adhesion molecules.

Migration of medial smooth muscle cells (SMC) into the intima is a key step in intimal thickening of atherosclerotic tissues. We previously reported that cultured SMC secrete a potent migration factor for SMC, named SMC-derived migration factor (SDMF). We purified this factor to homogeneity from 20 liters of serum-free conditioned medium of cultured rat aortic SMC by sequential heparin-Sepharose column, red-Sepharose column, TSK-heparin high performance liquid chromatography (HPLC) column, and Superose 6 HPLC column chromatographies. SDMF was found to be a 58-kDa polypeptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Reduction by mercaptoethanol caused only a slight decrease in its molecular mass to 53 kDa. Preparative isoelectric focusing revealed that SDMF is a basic protein with a pI of approximately 10. Purified SDMF enhanced the migration of rat SMC dose dependently, its maximal activity being 4 times that of platelet-derived growth factor-BB. In contrast, SDMF did not enhance the migration of endothelial cells from either human umbilical cord vein or rabbit retinal tissue. SDMF had no effect on the proliferation of SMC. These findings suggest that SDMF enhances SMC migration in vascular walls and that the autocrine system of SMC migration contributes to the formation of intimal thickening in atheroma formation.

The mechanism of diabetic macroangiopathy was studied from the view point of phenotypic change of aortic smooth muscle cells (SMC). The growth rates of cultured SMC of diabetic rats or rabbits were higher than those of non-diabetic animals (controls). This difference of the growth responses was observed specifically with platelet-derived growth factor (PDGF). Of the three PDGF dimers, PDGF-AB heterodimer (PDGF-AB) and PDGF-BB homodimer (PDGF-BB) stimulated growth of diabetic SMC more than that of control SMC but PDGF-AA homodimer (PDGF-AA) did not. The binding of 125I-PDGF to the diabetic SMC was greater than that to control SMC. This was due to increase in the number of cell surface receptors for PDGF. On in vitro culture, SMC from diabetic rats expressed more PDGF beta-receptor mRNA than SMC from non-diabetic rats. Moreover, in vivo, the aortic media of diabetic rabbits expressed PDGF beta-receptor mRNA, but that from non-diabetic rabbits did not. Thus diabetic SMC over-react on PDGF stimulation through over-expression of the PDGF beta-receptor gene. The significance of this fact in development of diabetic macroangiopathy is discussed.

In culture, rabbit aortic smooth muscle cells (SMC) from an atheroma differed phenotypically from SMC from normal media (M-SMC) in their growth rate, secretion of SMC-derived growth factor (SDGF), and metabolism of acetylated low density lipoproteins (a-LDL). The factor responsible for this in vivo phenotypic change of SMC was investigated in vitro. After preincubation of M-SMC with 0.1-10 U ml-1 of tumour necrosis factor-alpha (TNF) for 1-3 days, the cells grew faster than control cells and secreted a substantial amount of SDGF. The population doubling time and secretion of SDGF were inversely correlated. Moreover, after preincubation with TNF, the SMC metabolized [125I]a-LDL, unlike control M-SMC. These findings show that TNF can modulate the phenotype of SMC and suggest that it is important in the pathogenesis of atherosclerosis.

Hyaluronate in cultured skin fibroblasts derived from patients with Werner's syndrome, who excrete large amounts of urinary hyaluronate, was investigated. The amount of hyaluronate sectreted into the medium by Werner's fibroblasts was 2–3 times that of normal fibroblasts, whereas no difference in enzyme activities related to the degradation of hyaluronate was found. Werner's fibloblasts were then cultured in the presence of [ 3 H]glucosamine, and the amount of [ 3 H]hyaluronate and its chain lengths in the medium and matrix (trypsinate) fractions were compared with those of normal cells. No significant difference in the chain lenght of hyaluronate was observed between normal and Werner's fibroblasts. On the other hand, a significant increase of hyaluronate was found in the matrix fraction of Werner's fibroblasts when the cells reached confluency. In addition, a hyaluronate of small chain length was found in thematrix fraction of Werner's fibroblasts, although this was absent from that of normal cells. It was concluded that the constituents of the extracellular matrix of Werner's fibroblasts differed from those of normal cells, characterized by the presence of a large amount of hyaluronate and a relatively small hyaluronate chain.

Migration of arterial smooth muscle cells (SMC) in the arterial wall plays an important role in the formation of intimal thickening of atherosclerotic lesions. In this study, we examined the effect of ginsenosides on SMC migration induced by platelet-derived growth factor (PDGF) and SMC-derived migration factor (SDMF). Ginsenosides had inhibitory effects on SMC migration and the striking effects were observed with ginsenoside-Rb2 and -Rc in a dose-dependent manner. These results suggest that the administration of ginsenosides on the patients may prevent intimal thickening, in part, by inhibiting SMC migration in the arterial wall.

The patient was a 74-year-old woman who had been obese since age 18. Her obesity was refractory to dietary manipulation. She had been suffering from increasing dyspnea for several months and eventually could not even move. She was admitted to a hospital and diagnosed as having heart failure. Although her cardiac function recovered with medical treatment, her symptoms did not improve. The patient was then sent to our hospital. On admission, her height and weight were 149 cm and 81.9 kg, respectively, yielding a body mass index (BMI) of 36.6 kg/m2. Arterial blood gas analysis in room air revealed hypoxemia and an apnea index of 27 per hour. She was given a daily 500-1000 kcal diet. After four months of treatment, her weight decreased to 65 kg with a BMI of 29.3 kg/m2. Weight reduction together with the usage of progesterone-derivatives resulted in marked improvement of sleep apnea. The apnea index decreased to 3/h and arterial blood gas values normalized. This patient seemed to have suffered from both obesity hypoventilation syndrome and sleep apnea syndrome. Improvement of respiratory function was achieved through relief of airway obstruction and weight reduction, with activation of the respiratory center due to progesterone treatment.

Migration of smooth muscle cells (SMC) in the arterial wall is important in the formation of intimal thickening. In this work, cultured SMC from the rat and rabbit aortic media at 2nd to 12th passages were found to secrete a potent migration factor for SMC which was named SMC-derived migration factor (SDMF). This factor stimulated the migration of SMC dose-dependently and its maximum activity was 2-8 times that of PDGF. Checker board analysis showed that SDMF was chemotactic, but not chemokinetic. In further studies, SDMF was found to be inactivated at 100 degrees C for 10 min or by trypsinization, but not inactivated by mercaptoethanol. This factor was not dialyzable. Molecular weight was approximately 500 kDa by a gel filtration. The activity was not inhibited by an anti-PDGF antibody or a fibronectin antiserum. These data suggest that SDMF is a potent migration factor for SMC and that SDMF is distinct from PDGF, fibronectin or other known migration factors. This autocrine system of secretion of SDMF by SMC and its induction of SMC migration may contribute to intimal thickening of the arterial wall in atherosclerosis.

The effect of a novel antiatheromatous agent, N-{3-[4′-(2″,6″-dimethylhelptyl)phenyl]butanoyl}ethanolamine (E5050), on the proliferation of porcine aortic smooth muscle cells was studied in vitro. E5050 dose-dependently inhibited DNA synthesis as well as proliferation of cells stimulated with 10% fetal calf serum with no cytotoxic effects. An inhibitory effect of E5050 on DNA synthesis was also confirmed in cells stimulated with human platelet extract and with a combination of platelet-derived growth factor and human plasma-derived serum. DNA synthesis in smooth muscle cells stimulated with other mitogens, such as fibroblast growth factor and insulin, was inhibited by E5050 and this inhibitory effect was positively correlated with the E5050 uptake into smooth muscle cells. These results indicate that E5050 inhibits smooth muscle cells proliferation stimulated by various mitogenic factors. It is suggested that E5050 prevents atherogenesis and inhibits the progression of fibromuscular lesions by interfering with the proliferation of arterial smooth muscle cells.

The effect of probucol in lowering serum lipoprotein in young and middle-aged (YM) and elderly (E) patients with familial hypercholesterolemia were compared. Probucol at 1000 mg/day was administered orally to 37 YM patients and 14 E patients for an average of 10 months. Probucol treatment for this period caused significant reductions in the serum levels of total cholesterol, low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol, and apoprotein AI, AII, B, and CIII in both groups. The decreases in the levels of total cholesterol, LDL-C, and apoprotein B were greater in the E group than in the YM group (total cholesterol: YM, -19.3%, E, -31.3% [P less than .001]; LDL-C: YM, -17.0%, E, -35.4% [P less than .001]; apoprotein B: YM, -12.3%, E, -28.1% [P less than .01]). The decreases in other parameters in the two groups were not significantly different. The serum probucol concentrations in the YM and E groups were not significantly different. No significant side effects were observed in any patient. Thus probucol reduced the serum level of LDL more in the E group than in the YM group, and did so without any increase in the serum concentration of the drug or in side effects, suggesting that probucol is safe and beneficial for use in elderly patients.

We recently demonstrated immunologically the intraccllular accumulation of advanced glycation end products (AGEs) in foam cells derived from smooth muscle cells (SMCs) in advanced atherosclerotic lesions. To understand the mechanism of AGE-accumulation in these foam cells, the interaction of AGE-proteins with rabbit cultured arterial SMCs was studied in the present study. In experiments at 4°C, 125I-AGE-bovine serum albumin (AGE-BSA) showed dose-dependent saturable binding to SMCs with an apparent dissociation constant (Kd) of 4.0 μ/mL. In experiments at 37°C, AGE-BSA underwent receptor-mediated endocytosis and subsequent lysosomal degradation. The endocytic uptake of 125I-AGE-BSA was effectively inhibited by unlabeled AGE-proteins, but not by acetylated low density lipoprotein (LDL) and oxidized LDL, well-known ligands for the macrophage scavenger receptor (MSR). Moreover, the binding of 125I-AGE-BSA to SMCs was affected neither by amphoterin, a ligand for one type of the AGE receptor named RAGE, nor by 2-(2-Furoyl)-4(5)-(2-furanyl)-1 H-imidazolc-hexanoic acid-BSA (FFI-BSA), a ligand for the other AGE receptors called p60 and p90, indicating that the endocytic uptake of AGE-proteins by SMCs is mediated by an AGE receptor distinct either from MSR, RAGE, p60 or p90. To examine the functional role of (his AGE receptor, the effects of AGE-BSA on the migration of SMCs were tested. Incubation with 1-50 μg/mL of AGE-BSA resulted in significant dose-dependent cell migration. The AGE-BSA-induced SMCs migration was chemotactic in nature, and was significantly inhibited (−80%) by an antibody against transforming growth factor-β (TGF-β), and the amount of TGF-β secreted into the culture medium from SMCs by AGE-BSA was 7-fold higher than that of control, indicating that TGF-β is involved in the AGE-induced SMCs chemotaxis.

OBJECTIVE: To assess whether serum lipoprotein(a) is a risk factor for diabetic retinopathy in the elderly. DESIGN: A cross-sectional study. SETTING: Outpatient diabetic clinic. PATIENTS: One hundred four noninsulin-dependent diabetic patients (35 males, 69 females). Twenty-three were less than 60 years of age (middle-aged), and 81 were 60 years or older (elderly). MEASUREMENT: Levels of lipoprotein(a) (Lp(a)) and lipids were measured in fasting serum. HbA1c was also measured as an indicator of diabetic control. Other indicators possibly related to retinopathy were also checked. Retinopathy was estimated by photographs of fundi. RESULTS: Significantly higher indicators in the group with retinopathy than in the group without were: HbA1c, Lp(a), duration of diabetes, and systolic blood pressure (BP) in the total cases; HbA1c, duration of diabetes, and Lp(a) in the middle-aged; HbA1c, systolic BP, and Lp(a) in the elderly. Multiple logistic regression analysis showed that only HbA1c and Lp(a) were independent risk factors for retinopathy in all cases and in the elderly. The incidence of retinopathy was positively correlated to serum Lp(a) levels. CONCLUSION: Lp(a) is an independent risk factor for diabetic retinopathy.

Patients with type 2 diabetes are known to have abnormalities in their remnant metabolism and low density lipoprotein (LDL) subfraction pattern, with a preponderance of small dense LDL. The effects of pitavastatin, a newly synthesized 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, on lipoprotein profiles in patients with type 2 diabetes were determined. Thirty-three patients were treated with pitavastatin with a daily dose of 2 mg for 8 weeks. After treatment, triglyceride, total and LDL cholesterol were significantly reduced by 28.7 +/- 36.7%, 25.2 +/- 14.3% and 36.1 +/- 14.3%, respectively. Remnant-like particle cholesterol (RLP-C), an independent risk factor for CAD which is known to be elevated in diabetic patients, was also significantly reduced (-30.9 +/- 30.5%) by the treatment and this decrease correlated well with the decrease in triglyceride level. The proportion of small dense LDL, which is known for its atherogenisity, decreased from 29.9 +/- 26.2% to 19.7 +/- 22.7% and the mean LDL particle size significantly increased from 26.36 +/- 1.13 nm to 27.10 +/- 1.36 nm. Pitavastatin, which is known to improve triglyceride levels and cholesterol levels, also improves RLP-C level and LDL subfraction profiles, and this in turn may reduce the cardiovascular risk in patients with type 2 diabetes and dyslipidemia.

High density lipoprotein-cholesterol (HDL-C) levels are inversely related to the incidence of coronary artery disease. We studied the influence of a G(-75)--A transition in the promoter of the apolipoprotein (apo) A-I gene, a major protein component of HDL, on serum HDL-C levels in hyperlipidemic subjects. Seventy three hyperlipidemic subjects with serum levels of high HDL-C (HDL-Cor = 70 mg/dl, Group H) were compared with hyperlipidemic subjects with levels of HDL-C between 40 and 70 mg/dl (Group N) and those with HDL-C40 mg/dl (Group L). Group H showed a higher incidence (45.2%) of low plasma cholesteryl ester transfer protein (CETP) activity than Groups N (9.1%) and L (5.3%) (p0.001). Group H had a higher incidence of the G(-75)--A transition (0.275) than Groups N (0.117, p0.05) and L (0.056, p0.01), among subjects with normal CETP activities. The HDL-C levels in subjects with the transition (84 +/- 16 mg/dl) were higher than those in subjects without the transition (56 +/- 12 mg/dl) (p0.05). These data suggest that a G(-75)--A transition of the apo A-I gene promoter, in addition to the common mutation of CETP gene, contributes to high HDL-C levels among hyperlipidemic patients in Japan.

Vascular smooth muscle cells (SMCs) undergo phenotype change with the development of atherosclerosis. The phenotype changes of SMCs have been observed in various culture conditions, such as collagen-coated dishes. Here, we report the morphological and functional features of SMCs in a novel culture system using type I-collagen in a characteristic three-dimensional structure designated as honeycombs. The number of ribosome and mitochondria in SMCs cultured in honeycombs was one half or third of those cultured on collagen-coated plastic plates. DNA and protein synthesis of SMCs cultured in honeycombs were less than 1 and 30-40%, respectively, of those cultured on plastic plates. In addition, PDGF-BB did not increase the amount of DNA synthesis in SMCs in honeycombs. SMCs in honeycombs were shown to express several proteins, which are known to express in SMCs in medial layers of arteries. Particularly, caldesmon heavy chain was expressed in SMCs cultured in honeycombs, whereas not in those on plastic plates. Although focal adhesion kinase (FAK) was clearly detected in SMCs in honeycomb, the phosphotyrosine content of focal adhesion kin ase decreased in the process of culture. Immunoblot analysis showed dear different expression of ERK1 and ERK2 of mitogen-activated protein kinase in SMCs. SMCs in honeycombs expressed ERK2, more abundantly compared to ERK1, whereas SMCs in plates show the same levels of expressions for both proteins. Thus, the histological and functional feature of SMCs in the novel culture system is different from SMCs in plastic plates. The three-dimensional culture system described here may be indicating that cultured SMCs are able to express different proteins responding to the surrounding structures.