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The role of mast cells (MC) in tubulointerstitial damage in glomerulonephritis (GN) is not fully understood. The distribution of MC was compared in renal biopsies from 50 patients with different stages of rapidly progressive GN (RPGN) and in 20 control samples. The immunoreactivity of renal MC with anti-tryptase and anti-chymase antibodies was studied. Interstitial myofibroblasts were stained with anti-α-smooth muscle actin (α-SMA) antibody, and inflammatory cells were identified by anti-CD3, -CD20, and -CD68 monoclonal antibodies. Positively stained cells were counted, and the relative interstitial and fractional areas of anti-α-SMA-stained cells were measured. MC were rarely found in control samples. In contrast, samples showing crescentic GN contained numerous tryptase-positive MC (MC T ) (43.7 ± 4.65 versus 7.14 ± 1.3/mm 2 ) and fewer tryptase- and chymase-positive MC (MC TC ) (13.8 ± 1.86 versus 1.89 ± 0.86/mm 2 ) in the renal interstitium but never in the glomerulus. Double immunostaining demonstrated the presence of both phenotypes of MC. Accumulation of MC was significantly correlated with the numbers of T lymphocytes (MC T , r = 0.67) and interstitial macrophages (MC T , r = 0.455). There was also a significant correlation between the number of MC T and the relative interstitial area. The number of MC TC was well correlated with the fractional area of α-SMA-positive interstitium ( r = 0.749) and the percentage of the interstitial fibrotic area ( r = 0.598). There was also a significant negative correlation between interstitial MC TC accumulation and creatinine clearance ( r = 0.661). The density of MC TC was higher (1.4-fold) in advanced forms of GN associated with fibrocellular crescents and interstitial fibrosis. These results show the potential involvement of MC in the fibroproliferative process in the renal interstitium of patients with RPGN. The results indicate that these cells constitute part of the overall inflammatory cell accumulation in RPGN.

Probucol decreases and bezafibrate increases plasma high density lipoprotein-cholesterol (HDL-C) levels in humans. This study was performed to determine whether the HDL-C-lowering effects of probucol could be reversed by treatment with bezafibrate in hypercholesterolemic rabbits. Forty-nine normolipidemic Japanese White rabbits were divided into 5 groups [group 1: normal chow; group 2: 0.2% cholesterol (Ch) diet; group 3: 0.2% Ch and 1% probucol diet; group 4: 0.2% Ch and 1% bezafibrate diet; group 5: 0.2% Ch and 1% probucol plus 1% bezafibrate diet] and treated for 8 weeks. Plasma lipids, cholesteryl ester transfer protein (CETP) activity in the lipoprotein-deficient plasma fraction, CETP mRNA in liver tissue and plasma drug concentrations were investigated. Serum total cholesterol (TC) increased after the rabbits in groups 2, 3, 4 and 5 were fed Ch, but overall, no significant differences were observed in serum TC and triglyceride (TG) among these groups. Serum HDL-C levels increased (p

To clarify the relationships between clinical and renal structural findings in non-insulin-dependent diabetes mellitus (NIDDM), we studied 19 renal biopsy specimens from patients with overt diabetic nephropathy induced by NIDDM. By a conventional biopsy examination using light, immunofluorescent and electron microscopy, we excluded patients with any non-diabetic renal diseases which may have caused urinary abnormalities. Using standard stereological methods, the glomerular filtration surface area per nephron (S-Filt/neph), mesangial volume per glomerulus (V-Mes/glom), mesangial matrix volume per glomerulus (V-MM/glom) and the width of the glomerular basement membrane (GBM) were measured in nonoccluded glomeruli. The hyaline change in the arteriole was analyzed semiquantitatively using light microscopy as an index of arteriolar hyalinosis. Light-microscopic findings demonstrated minimal lesions in 4 cases, focal and/or segmental sclerotic lesions in 4 cases, and diffuse mesangial expansion in 11 cases (with and without nodular lesions in 9 and 2 cases, respectively). Mean glomerular volume of an open glomerulus in diabetic patients was 1.6 times the reference value obtained from nondiabetic patients with mild proteinuria and/or hematuria and normal renal function. Clinical parameters, including the duration of diabetes, urinary protein excretion and creatinine clearance were all related to S-Filt/neph, V-Mes/glom, V-MM/glom and the width of the GBM. In addition, V-MM/glom, the width of the GBM and the index of arteriolar hyalinosis were closely interrelated. Based on these findings, diabetic renal changes, including an increase in the mesangial matrix, thickening of the GBM and arteriolar hyalinosis, appeared to progress in parallel, and may reflect various clinical manifestations in patients with overt diabetic nephropathy induced by NIDDM without nondiabetic renal lesions.

A new monoclonal antibody against malondialdehyde (MDA)-treated low density lipoprotein (LDL) was raised using homogenate of human atheroma as immunogen. This antibody, DLH2, was obtained by selecting the clones which did not react to native LDL but did react to copper-induced oxidized LDL (OxLDL). DLH2 showed a greater reactivity to MDA-LDL than to OxLDL. When LDL was treated with various aldehyde containing reagents, treatment of LDL with glutaraldehyde or MDA greatly increased the reactivity to the antibody, while LDL treated with 2,4-hexadienal or 4-hydroxynonenal was not reactive. Among many proteins tested, high density lipoprotein, bovine serum albumin and hemoglobin showed significant reactivity to DLH2 after they were treated with MDA or glutaraldehyde. When low density and high density lipoproteins treated with MDA were subjected to immunoblot analysis, newly formed products larger than the original apolipoproteins were detected with the antibody, suggesting that this antibody recognizes aggregated proteins with divalent short chain cross linkers. The antigenic materials were shown by immunohistochemical analysis to be present in foamy macrophages in human atheromatous lesions. DLH2 antigen did not colocalize either with apolipoprotein B. Furthermore, we found a massive accumulation of the antigenic material in Kupffer cells in the liver of rats treated with alcohol and carbonyl iron, a model of hepatic fibrosis due to oxidative stress. These results suggest the presence of cross linked proteins in damaged tissues.

We investigated the mechanism of action of gemfibrozil on high-density lipoproteins (HDL) and apolipoprotein (apo) A-I metabolism and atherogenesis in homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model of familial hypercholesterolemia and HDL deficiency. Two-month-old WHHL rabbits were fed either a normal control diet or a diet containing 0.5% gemfibrozil for 12 months. In vivo apo A-I kinetics, the fractional rate of cholesterol esterification in HDL (FERHDL), which reflects the reactivity of HDL to lecithin:cholesterol acyltransferase, and a morphometrical analysis of atherosclerotic lesions in the descending thoracic aorta, were examined. At12 months, the mean levels of serum total cholesterol, LDL cholesterol (LDL-C), and HDL cholesterol (HDL-C) in both groups had decreased to approximately 53%, 57%, and 87% of the initial levels (at 0 month), respectively, which is characteristic of homozygous WHHL rabbits of the physiologic influence of aging, and no differences in the levels of serum LDL-C, HDL-C, and triglycerides were found between the two groups. Rabbits treated with gemfibrozil exhibited a decreased FERHDL (38% of the controls, P = 0.039). Gemfibrozil induced a significant increase in the total mass of apo A-I (1.7-fold, P < 0.05) and in the rate of apo A-I synthesis (1.6-fold, P< 0.05). The atherosclerotic intimal area was positively correlated with serum LDL-C (P = 0.02) in both groups, but gemfibrozil did not affect the atherosclerotic intimal area. These results indicate that 12 months of treatment with gemfibrozil did not protect against atherosclerosis despite a significant increase in apo A-I synthesis and enhanced HDL function through FERHDL. It is possible that both the qualitative and quantitative improvement in HDL by gemfibrozil cannot overcome the massive and long-term exposure of the vascular wall to LDL in these animals.

The combined effects of pravastatin and probucol on high- density lipoprotein (HDL) apolipoprotein (apo) A-I kinetics in cholesterol (Ch)-fed rabbits were investigated. Japanese White rabbits were treated with 0.15% pravastatin and 0.5% Ch (group 1) or 0.15% pravastatin plus 1% probucol and 0.5% Ch (group 2) for 2 months. After treatment, the serum total cholesterol levels in groups 1 and 2 had significantly (p < 0.01) increased (37.4 +/- 6.7 mg/dl vs 117.1 +/- 46.4 mg/dl, and 31.4 +/- 4.9 mg/dl vs 143.0 +/- 84.5 mg /dl, respectively). The serum HDL-cholesterol levels in both groups decreased (18.2 +/- 2.8 mg/dl, vs 16.2 +/- 3.7 mg/dl p < 0.01 for group 1; 18.2 +/- 1.9 mg/dl vs 15.5 +/- 4.5 mg/dl, ns for group 2). Apo A-I kinetics were assessed by injecting (125)I-labeled HDL intravenously into both groups of rabbits, and taking blood samples periodically for 6 days. Kinetic parameters calculated from apo A-I specific radioactivity decay curves showed that the apo A-I fractional catabolic rates in rabbits fed pravastatin and Ch (group 1) were significantly less than those in rabbits fed pravastatin plus probucol and Ch (group 2) (0.546 +/- 0.017 /day vs 0.730 +/- 0.126 /day, p < 0.05), while the synthetic rate of apo A-I was lower in group 2 than in group 1 (14.76 +/- 1.71 mg/kg per day vs 11.21 +/- 2.38 mg/kg per day, respectively, p < 0.01). These data indicate that pravastatin and probucol have different effects on HDL-apo A-I kinetics in a diet which includes cholesterol.

To clarify the relationship between the dilatation of angiographically non-stenotic coronary artery segments in response to isosorbide dinitrate (ISDN), and serum lipids, 5 coronary segments in 7 patients with familial hypercholesterolemia (FH) and 43 patients with non-familial hypercholesterolemia (non-FH), who had either 1- or 2-vessel coronary heart disease, were investigated. The serum total cholesterol level was significantly greater in FH than in non-FH. Before and after the direct intracoronary injection of ISDN, coronary diameter was measured by a computer-assisted coronary angiography analysis system. The order of the dilative responses in coronary segments in non-FH patients (segments: #5 > #11 > #6 > #13 > #7) was exactly the same as the order of their original diameters, while the ratio of the increase in diameter (after/before ISDN injection) did not differ among any of the segments (mean: 1.08-fold increase). In the non-FH group, no correlation was found between the serum total cholesterol, triglyceride, low-density lipoprotein-cholesterol, or high-density lipoprotein-cholesterol, and the ratio of the coronary diameter after and before injection of ISDN. Moreover, hypercholesterolemia in non-FH did not affect the coronary dilative response to ISDN injection. In the FH group, although the original diameter of each segment did not differ from that in the non-FH group, the ratio of the diameter after and before injection of ISDN was significantly smaller in FH than in non-FH (p < 0.01). These results suggest that a non-stenotic coronary artery in FH has a lower capacity for vasodilation in response to ISDN. Although hypercholesterolemia was excluded as a factor which may suppress the capacity for coronary dilation in non-FH, spontaneous hypercholesterolemia in FH may affect medial smooth muscle functions of the coronary artery.

A practical high-performance liquid chromatography using a Cosmosil HILIC column and UV detection was developed for determining the concentrations of cytosine arabinoside (Ara-C) and uracil arabinoside (Ara-U), which is a major metabolite of Ara-C, in human plasma. This method was used to determine the plasma concentrations of Ara-C and Ara-U in a patient treated with high-dose Ara-C therapy for end-stage renal failure.

Resistance to gemtuzumab ozogamicin (GO) hampers the effective treatment of refractory acute myeloid leukemia (AML). To clarify the mechanism of resistance to GO, HL-60 cells were persistently exposed to GO in order to establish GO-resistant HL-60 (HL-60/GOR) cells. Multidrug resistance 1 (MDR-1) was strongly expressed in HL-60/GOR cells, but not in HL-60 cells. Although withdrawal of GO after the chronic exposure of HL-60/GOR cells to this compound gradually decreased MDR-1 expression to trace levels, reintroducing GO restored high MDR-1 expression in HL-60/GOR cells, but not in HL-60 cells. These results indicate that HL-60/GOR cells acquired the ability to induce MDR-1 expression in response to GO. U0126, a MEK1/2 inhibitor, prevented GO-inducible MDR-1 expression and abrogated GO resistance in HL-60/GOR cells. These results suggest that in the clinical use of GO, inducible MDR-1 expression in tumor cells should be investigated before treatment with GO. If the cells are positive then MEK1/2 inhibitors may be effective in overcoming resistance to GO.

A cell line derived from medial smooth muscle cells (SMC) was established from the porcine coronary artery by transfection with ori-defective simian virus 40 plasmid DNA (SV40 DNA). The characteristics of transfected cells (SV40-SMC) such as cell growth, collagen and non-collagen syntheses were investigated. SV40-SMC expressed SV40 large T antigen, c-myc and c-myb encoded proteins in the nuclei. SV40-SMC demonstrated a 'hills and valleys'-like arrangement in overconfluence and actin filaments upon immunofluorescent staining. Under electron microscopic observation, SV40-SMC had larger amounts of synthetic organelles and smaller amounts of filament bundles than those of SMC. SV40-SMC demonstrated three times higher growth activity and 4.4 times greater cellular density than SMC. Smooth muscle cells did not grow in media containing 5% plasma derived serum (PDS) instead of normal serum, whereas SV40-SMC proliferated in this medium. SV40-SMC did not grow in soft agar gel, while HeLa S3 cells, a cell line of human cervical carcinoma, formed colonies in this gel. By immunofluorescent (IF) staining, collagen phenotypes I, III, IV and V were detected in both SV40-SMC and SMC. However, protein synthesis including collagen and non-collagen was higher in SV40-SMC than in the control sample. It was concluded that SV40-SMC were a continuous cell line for vascular SMC regarding morphological characteristics, and demonstrated a higher growth activity, with increased collagen and non-collagen syntheses. This cell line is useful for the investigation of atherogenesis in relation to a proliferation of SMC and an accumulation of extracellular matrices in vascular intima.

A simple and sensitive HPLC method has been developed for the determination of methotrexate (MTX) and its major metabolites, 7-hydroxymethotrexate (7-OH-MTX) and 2,4-diamino-N(10-) methylpteroic acid (DAMPA), in human plasma. After deproteinization of the plasma with 5% aqueous acetonitrile solution containing 5% trichloroacetic acid, MTX, 7-OH-MTX, DAMPA and 2,4-diaminopteroic acid (DAPA) as an internal standard were separated on a reversed-phase column, and the eluent was subsequently irradiated with UV light (245 nm), producing fluorescent photolytic degradation products. The analytes were then detected spectrofluorometrically at 452 nm with excitation at 368 nm. The extraction efficiencies of MTX, 7-OH-MTX and DAMPA from plasma at 100 pmol/mL were 81.5±5.4, 82.5±5.3 and 56.2±7.0%, respectively. The limits of quantification for MTX, 7-OH-MTX and DAMPA in plasma were 5 pmol (2.3 ng), 0.8 pmol (0.38 ng) and 10 pmol (3.4 ng)/mL, respectively. The within- and between-day variations for MTX, 7-OH-MTX and DAMPA were reliable (each was lower than 6.3%). This method was also used to monitor the concentrations of MTX and its metabolites in a patient on MTX therapy.

We attempted to clarify the effects of various purified extracellular components, including types I, III, IV, and V collagen and fibronectin on attachment, spread, growth, and DNA synthesis of porcine aortic smooth muscle cells (SMCs) in vitro. The number, area and shape index ( SI = 4π S L 2 ) of cells attached to different substrates were determined at various intervals of incubation. The cell number and [3H]thymidine incorporation into DNA were measured on the 1st and 6th days of culture. SMCs showed the largest number of attached cells on fibronectin, but the smallest number of attached cells on type V collagen. There was no evidence of effects of the serum in media on the attachment of SMCs to the substrates. The areas of attached SMCs were the largest on fibronectin and the smallest on type V collagen. The shape index of SMCs on fibronectin decreased relative to those on other substrates. On the 6th day in culture, the number and population doubling of SMCs on type V collagen were significantly fewer than those on other substrates. Both the incorporation rate of [3H]thymidine into DNA and the percentage of nuclei labeled with [3H]thymidine were significantly less in the SMCs on type V collagen on the 1st day than those on other substrates. SMCs on types I, III, and IV collagen showed intermediate levels of cell attachment, spread, and growth. These results suggest that attachment, spread, and growth of SMCs are affected mainly by solid phase purified extracellular components and are most strongly suppressed by type V collagen. When DNA synthesis of growth-arrested SMCs was reinitiated by the addition of serum, type V collagen most intensively inhibited the rate and amount of [3H]thymidine incorporation. Flow cytometric analysis demonstrated an increase in the proportion of cells in G 0 G 1 phase on type V collagen in comparison with that on other substrates. Thus, the antiproliferative effect of type V collagen may relate to inhibition of transition of SMCs from the G 0 G 1 into the S phase.

Cholesterol/egg phosphatidylcholine (PC) liposomes (1:1 or 4:1, M/M), in which the absolute amount of PC was adjusted to be the same, were incubated with cultured bovine arterial smooth muscle cells for up to 8 h at 37°C. The effect of increased cellular cholesterol on the accumulation of intracellular calcium in these cells was studied. The results indicate that the intracellular calcium content, measured by Fura-2/AM, was increased 2,3-fold by incubation with 4:1, cholesterol/PC liposomes. Kinetic analysis using 45 Ca 2+ indicated that the increased calcium influx was due to increase of pool size, not from a change of rate constant. (Ca 2+ +Mg 2+ )-ATPase activity was decreased by 4:1, cholesterol/PC liposomes. The molar ratio of cholesterol/phospholipids in the cell membranes was directly proportional to that in liposomes. No change in phospholipid composition was noted. We suggest that the accumulation of intracellular calcium was a composite result due to the altering effect of inserted cholesterol on surface area, and to direct interactions between cholesterol and the proteins of the Ca 2+ channel and (Ca 2+ + Mg 2+ )-ATPase.

Since little is known about the effects of reconstituted high-density lipoprotein (rHDL) in left ventricular (LV) remodeling, these effects were examined in rats after acute myocardial infraction (MI). Sixteen male Wistar rats were randomly divided into three groups: Sham-operated (n=6), and MI rats that received a permanent ligation around the proximal left coronary artery and infusions of placebo (MI group, n=5) or rHDL (containing as apolipoproteinA-I 6mg/kg) administered intravenously (MI+rHDL group, n=5). rHDL was infused once a week for 4 weeks. In addition, in vitro assays were performed to examine the effect of rHDL. The MI+rHDL group showed a significant increase in LV ejection fraction (EF) between weeks 1 and 4, a decrease in LV end-systolic diameter, compared with the progressive deterioration of LV size and function in the MI group. In addition, the MI+rHDL group showed a significant decrease in fibrotic area of MI in LV compared to that in the MI group, while there were no significant increases in capillary density or cell size in LV in the MI+rHDL group. Interestingly, the MI+rHDL group showed a significant activation of retinoblastoma and ERK (extracellular-signal-regulated kinase) but not cleaved caspase-3, p38 MAPK or Jun N-terminal kinase. rHDL suppressed H(2)O(2)-induced arrest of cell growth in myocytes. This effect was blocked by PD98059, an ERK inhibitor. In conclusions, rHDL-promoted cell survival has beneficial morphological effects that help to prevent LV remodeling and improve function after MI, and may prevent arrest of cell growth through ERK pathway in myocytes.

Retinoids are used for treatment of acute promyelocytic leukemia (APL). Am-80, Tamibarotene, binds to retinoic acid receptor alpha (RARalpha) more specifically than all-trans retinoic acid. We studied the tumor cell suppressive effects of Am-80, with respect to cytotoxicity and growth inhibition using eight myeloid and lymphoid malignant cells in culture (HL-60, HL-60R, K-562, Kasumi-1, MEG01, Raji, U266B1, and U937). The effects of Am-80 were examined during 9 days of incubation with 10(-7)-10(-5) M of Am-80 in culture medium, which was changed every 3 days. HL-60 were the only cells sensitive to Am-80-induced cytotoxicity; the latter reached more than 95% after 9 days of incubation, and death was primarily through apoptosis. The total mass of RARalpha in HL-60 was significantly greater (p

Retinoids are used for treatment of acute promyelocytic leukemia (APL). Am-80, Tamibarotene, binds to retinoic acid receptor alpha (RARalpha) more specifically than all-trans retinoic acid. We studied the tumor cell suppressive effects of Am-80, with respect to cytotoxicity and growth inhibition using eight myeloid and lymphoid malignant cells in culture (HL-60, HL-60R, K-562, Kasumi-1, MEG01, Raji, U266B1, and U937). The effects of Am-80 were examined during 9 days of incubation with 10(-7)-10(-5) M of Am-80 in culture medium, which was changed every 3 days. HL-60 were the only cells sensitive to Am-80-induced cytotoxicity; the latter reached more than 95% after 9 days of incubation, and death was primarily through apoptosis. The total mass of RARalpha in HL-60 was significantly greater (p0.006) than in ATRA-resistant HL-60 (HL-60R) as well as all of other cells tested. However, in all cells excluding HL-60, Am-80 induced time- and dose-dependent cell growth inhibition without noticeable cytotoxicity. TGF-beta2 was released into the media containing cells incubated with Am-80 for 3 days. A dose-dependent increment of phosphorylation of Smad-2 was also detected. The relative amount of secreted TGF-beta2 correlated with the growth inhibition rates in all cells tested excluding HL-60, and with the total mass of RARalpha in the cells (p=0.0137). Our results indicate that Am-80-induced cell-type non-specific growth inhibition is mediated by TGF-beta2, where the total mass of RARalpha could be an important regulatory factor in hematologic malignant cells.

Small globular protein (SGP) is an artificially designed protein that forms small pores on lipid bilayer membranes. The anti-tumor effect of SGP has been reported in several papers. However, the exact mechanism underlying SGP-induced cell death remains to be determined.The mechanisms of SGP-mediated cytotoxicity were examined using cultured malignant glioblastoma cells (U87-MG). Morphological changes were observed under a light microscope. Ultrastructural changes were examined by electron microscopy. Changes in membrane permeability were evaluated by measuring the extracellular release of lactate dehydrogenase (LDH) and intracellular calcium concentration ([Ca2+]i). A new cell viability assay was also conducted using DAPI/calcein fluorescent-double staining methods.SGP cytotoxic effects were strongly influenced by fetal bovine serum (FBS) concentration in culture media. After 30 min of incubation, cytotoxic effects were noted at SGP concentrations greater than 1.5 microM. [Ca2+]i increased immediately after SGP addition. Extracellular efflux of LDH increased linearly with SGP doses (r(2) =0.936), and occurred even at concentrations less than 1.5 microM. Non-viable and viable cells were simultaneously detected by fluorescent staining with DAPI and calcein-AM, respectively. After 30 min of incubation with SGP, DAPI-positive nuclei were evident at concentrations higher than 1.5 microM. Cultures were then maintained in 10% FBS-containing media for 24 h before addition of calcein-AM. Calcein-positive cells were found at SGP concentrations lower than 1.5 5M. Cells positive for both DAPI and calcein appeared at a concentration of 1.5 microM.Our results indicate that SGP induces rapid but transient cytotoxicity, which may be beneficial in local therapy of malignant brain tumors.

We examined the histochemical characteristics of soleus muscle in the angiotensin-converting enzyme (ACE) gene (Ace in mice, ACE in humans) knockout mice. Serial sections of soleus muscle of wild-type (Ace+/+, n=20) and heterozygous mutant (Ace+/-, n=24) mice were stained for myosin adenosine triphosphatase activity to identify different muscle fiber types. Capillaries were visualized by amylase-periodic acid-Schiff staining. ACE activity in the serum and gastrocnemius muscle was higher in male mice than in female mice. Female and male Ace+/- mice had markedly lower ACE activity in the serum and the gastrocnemius muscle than did female and male Ace+/+ mice, respectively. In both male and female mice, the composition of fiber types (type I and IIa) did not differ significantly between Ace+/+ and Ace+/- mice. There was no significant gender difference in capillary density. Ace+/- mice had significantly more capillaries around type IIa fibers (5.44 +/- 0.18 vs. 5.01 +/- 0.13, p0.05) than Ace+/+ mice. The differences in the number of capillaries around type I fibers and in the number of capillaries around per fiber (capillary:fiber ratio) between Ace+/- and Ace+/+ mice were not significant (p0.1). There was no significant difference in the mean cross-sectional area occupied by one capillary and the number of capillaries per fiber area between Ace+/+ and Ace+/- mice. In conclusion, knockout of the Ace gene in mice increased capillary density, as expressed by the mean number of capillaries around type IIa fibers. This finding suggests a possible mechanism for the cardioprotective effects of ACE inhibitors.

BACKGROUND: As perlecan contains a low-density lipoprotein (LDL) receptor-like repeats in the second domain of its core protein, LDL may be bound to perlecan, which is rich in granulation tissues. We wanted to study if this is the case in the cyst wall of radicular cysts, which are often associated with cholesterol granuloma. METHODS: Thirty-three specimens of radicular cyst with cholesterol granulomas were immunohistochemically examined for comparative localizations of perlecan, apoprotein B (apo B), and oxidized LDL (Ox-LDL), and for mRNA expression levels for perlecan. RESULTS: Myxoid or edematous stroma of immature granulation tissues was strongly positive for perlecan and simultaneously for apo B and Ox-LDL. Macrophages including foamy cells scattered in the granulation tissues were also immunopositive for Ox-LDL and occasionally for apo B. In situ hybridization showed that fibroblasts, endothelial cells, and pericytes had strong signals for perlecan, which was also confirmed by RT-PCR. CONCLUSION: These results suggest that perlecan, which is abundantly produced and accumulated in the cyst wall of immature granulation tissue, traps Ox-LDL locally, and that Ox-LDL is phagocytosed by macrophages. Thus, LDL-laden foamy macrophages are aggregated in the granulation tissue, and free cholesterol from ruptured macrophages may be concentrated locally to be crystallized, which may induce foreign body granulomas in the cyst wall.

Long-term exposure to cadmium (Cd) induces perturbation of kidney proximal tubular epithelial cells. Mitochondrial dysfunction in renal cortical cells may contribute to the pathogenesis of Cd-induced nephropathy. In this study, we examined the accumulation of mitochondrial DNA (mtDNA) with a large deletion and cellular senescence in the renal cortex. Wistar rats at 8 weeks of age were intraperitoneally injected with 1 mL of 1 mM CdCl(2) or saline, 3 times/week for 5, 20, 40, or 80 weeks. Mitochondrial Cd content in the renal cortex was quantified by atomic absorption analysis. Cytochrome c oxidase (CCO) and senescence-associated beta-galactosidase (SA-beta-gal) activity were determined in renal cortex by enzyme-histochemistry. mtDNA in total DNA extracted from the renal cortex was amplified by PCR, and mtDNA deletions, including 4,834-bp (nt8118-nt12937) deletion, were determined and semiquantified. After 40 weeks of Cd injection, Cd levels in the renal cortex reached a saturation level, and 30% of the level of the whole-cell fraction was found in the mitochondria. CCO activity in the renal cortex, which was predominantly found in proximal tubular cells, decreased after 40 weeks of Cd exposure. Expression of SA-beta-gal was detected primarily in the proximal tubular cells and significantly increased after 80 weeks of Cd exposure. After 40 weeks of study, accumulation of 4,834-bp deletion in mtDNA was evident in both groups of rats; however, the amount of the deletion was significantly greater in Cd-treated rats than in control rats. Our results indicate that long-term Cd exposure induced a post-regenerative state of proximal tubular cells, which accelerated accumulation of 4,834-bp mtDNA deletions in the renal cortex, suggesting that Cd may be a senescence acceleration factor for kidney proximal tubular epithelial cells, which results in Cd-induced nephropathy.

Type 2 diabetes mellitus results from a complex interaction between nutritional excess and multiple genes. Whereas pancreatic beta-cells normally respond to glucose challenge by rapid insulin release (first phase insulin secretion), there is a loss of this acute response in virtually all of the type 2 diabetes patients with significant fasting hyperglycemia. Our previous studies demonstrated that irreversible intracellular accumulation of a glucose metabolite, protein O-linked N-acetylglucosamine modification (O-GlcNAc), is associated with pancreatic beta-cell apoptosis. In the present study, we show that streptozotocin (STZ), a non-competitive chemical blocker of O-GlcNAcase, induces an insulin secretory defect in isolated rat islet cells. In contrast, transgenic mice with down-regulated glucose to glucosamine metabolism in beta-cells exhibited an enhanced insulin secretion capacity. Interestingly, the STZ blockade of O-GlcNAcase activity is also associated with a growth hormone secretory defect and impairment of intracellular secretory vesicle trafficking. These results provide evidence for the roles of O-GlcNAc in the insulin secretion and possible involvement of O-GlcNAc in general glucose-regulated hormone secretion pathways.

Background: Lenalidomide (LND) is an oral immunomodulatory drug which is directly eliminated in the urine. LND has been used for the treatment of anemia associating chromosomal 5q deleted low- or intermediate-risk myelodysplastic syndromes (MDS) patients and recurrent multiple myeloma (MM) patients. If patients have renal failures, LND clearance could decline, resulting in thrombocytopenia as a side effect. To optimize LND doses for such patients, we developed a simple method analyzing LND concentrations in the plasma. Methods: The mixture of 150 µL plasma added 3-aminobenzyl alcohol (IS), saturated ammonium sulfate, sodium chloride and 3mL ethyl acetate/acetonitrile solution (95:5, v/v) was centrifuged. The supernatant solution was evaporated and the residue was dissolved in water. After the reaction with fluorescamine, the reaction mixture was injected into a HPLC. HPLC conditions: Capcell Pak C18 UG120 (150 × 4.6mm, 5 µm); mobile phase: 50mM phosphate buffer (pH4.0) /methanol / tetrahydrofurane (70:10:20, v/v); flow rate: 0.7mL/min; detection: excitation 382nm, emission 495nm. Results: The lower limits of quantifying the concentrations of LND in the plasma were 5ng/mL. The within- and between-day variations for LND were reliable (5.9 ∼ 13.4% and 5.9 ∼ 15.1%, respectively). This method was used to determine the plasma concentrations of LND in patients treated with LND therapy. Conclusions: This HPLC-based method is very simple, rapid and sensitive for plasma concentrations of LND, and may therefore be useful as a routine analysis for the therapeutic drug monitoring in LND-treated patients with renal failures.

Patients with vesicoureteral reflux (VUR) often develop reflux nephropathy with focal segmental glomerular sclerosis (FSGS), although the exact mechanisms leading to the development of this complication are unknown. To determine the early changes in glomeruli of VUR patients that ultimately cause poor renal outcome, we examined morphometrically renal biopsies of 16 young patients (age 10-20 years) with VUR at baseline pre-operatively. Patients were divided into two groups, those who subsequently showed good prognosis and those with poor renal prognosis at the end of a 10-year follow-up period. Patients with poor prognosis had worse proteinuria and lower creatinine at baseline than those with good prognosis. We also examined 40 age-matched control cases with previous temporal microhematuria and/or proteinuria but normal renal function and histology. Although the mean diameter of glomerular capillary did not change in VUR cases irrespective of prognosis, glomerular capillary length increased by 125% in cases with good prognosis, and 335% in cases with poor prognosis (P0.01). Cystically expanded capillaries, with diameteror = 95% of that in age-matched control, were detected in five of eight patients with poor prognosis, but only in one of eight patients with good prognosis. In VUR, the number of podocytes/capillary diminished with increased length of the capillaries. Tuft adhesion to Bowman's capsule and podocyte detachment were primarily found in patients with poor prognosis. Our results suggest that lengthening of glomerular capillaries in young patients with VUR is a compensatory reaction to hyperfiltration. The appearance of cystic capillary expansion, podocyte detachment and/or tuft adhesion to Bowman's capsule in such glomeruli may be important indicators of renal prognosis in patients with VUR. These changes may lead to FSGS due to podocyte injury in patients with VUR, with subsequent deterioration of renal function.

Renal interstitial fibrosis is the final common pathway leading to end-stage renal disease in various nephropathies including renal amyloidosis. However, the role of mast cells (MCs) in the fibrotic process of renal amyloidosis is not fully understood. We compared the distribution of MCs in renal biopsies from 30 patients with AA type renal amyloidosis and 20 control cases. Immunoreactivity of renal MCs to anti-tryptase and anti-chymase was studied. Interstitial myofibroblasts were stained with anti-alpha-smooth muscle actin (alpha-SMA) antibody, and inflammatory cells were identified by anti-CD45, -CD20, and -CD68 mAbs. Positively stained cells were counted, and the relative interstitial and fractional areas of anti-alpha-SMA stained cells were measured. Anti-CD29 mAb was used to detect beta1 integrin and anti-basic fibroblast growth factor (bFGF) mAb for the growth factor on MCs. MCs were rarely found in control samples. In contrast, samples showing amyloid deposition contained numerous tryptase-positive (MCT) (940.17 +/- 5.4 versus 6.74 +/- 1.1/mm2) but fewer chymase-positive (MCTC) cells (20.7 +/- 2.86 versus 1.7 +/- 0.76/mm2) in the renal interstitium. There was a significant relationship between interstitial MCT and creatinine clearance (r = -0.72), and between interstitial MCT and glomerular amyloid-index (GAI) (r = 0.723) and interstitial amyloid area (r = 0.824). Accumulation of MCs correlated significantly with the number of T lymphocytes (MCT: r = 0.694). There was also a significant relationship between mast cell (MC) number and the fractional area of alpha-SMA positive interstitium (r = 0.733) and interstitial fibrotic area (r = 0.6). Double immunostaining demonstrated intracytoplasmic presence of beta1 integrin on 87% of MCT and correlated significantly with the interstitial amyloid area (r = 0.818, P = .001) and T-cell number (r = 0.639, P = .002). bFGF was also detected on 85.5% of MCTC correlating well with the interstitial alpha-SMA-area (r = 0.789). Our results indicate that MCs constitute an integral part of the overall inflammatory process and play a crucial role in interstitial fibrosis in renal amyloidosis.

Summary Osteomalacia of cadmium (Cd) poisoning (Itai-Itai disease) is induced by renal tubular dysfunction; however, the precise pathological changes and mechanisms have not been adequately elucidated. Of the 25 inhabitants in a Cd-polluted area who developed chronic tubular proteinuria, 22 individuals died over a 16-year period. Autopsies were performed in 11 cases and osteomalacia was detected in 9 cases (mean age at death 82.2 ± 7.8 years; I man and 8 women). Histologically, osteomalacia occurred coincidentally with diffuse atrophy of the proximal tubules, moderate thickening of the tubular basement membrane and mild interstitial fibrosis in the renal cortex. Ultrastructurally, mitochondria in the proximal tubules were decreased in number and showed abnormal structure, while membrane enzymes, such as 5′-nucleotidase and ALPase, were still well preserved in their brush border. Glomeruli and distal tubules were minimally damaged. Severity of osteomalacia correlated with the damage of the proximal tubules as well as reduced serum calcium (Ca), serum Ca×phosphorus (P) and hematocrit, increased urine P2-microglobulin, lysozymes, N-acetyl-b-D-glucosaminidase, retinol binding protein, creatinine, and reduced percent tubular reabsorption of phosphate. Multiple regression analysis showed that among these factors, serum Ca×P was an independent factor for predicting the severity of osteomalacia. Our findings suggest that osteomalacia by Cd poisoning causes degenerative changes in the proximal tubules, especially in mitochondria, which might affect the disturbance of the intracellular active transport energy system for calcium and phosphorus, resulting in osteomalacia.

To investigate the possible role of mast cells (MC) in the angiogenic process in cutaneous melanoma, we examined tissue samples from 35 adult patients with primary malignant melanoma and compared with 20 intradermal benign nevi. MC were identified by anti-tryptase, microvessels by anti-CD34, and vascular endothelial growth factor (VEGF) expression by standard immunohistochemical methods. Tryptase-positive MC expressing VEGF were identified by double immunostaining. The numbers of MC and microvessels around the tumor were determined by the point counting method. MC density was significantly greater in melanoma compared with benign nevi (197.6 +/- 19.4 v 95.7 +/- 5.0/mm2, P < .001). Vascular density was also significantly higher in melanoma than in benign lesions (3.6-fold, P < .001). Double immunostaining showed the presence of VEGF in the cytoplasm of tryptase-positive peritumoral MC. The percentage of this MC-subtype was significantly higher in melanoma than in nevus tissues (71.9 +/- 2.4% v 30.6 +/- 2.5%, P < .001). A strong significant correlation was shown between the number of VEGF+ MC and microvessel density (r = .811, P < .001). MC count and VEGF+ MC count, as well as microvessel density were significantly higher in aggressive (metastasizing) melanomas (P < .001). Our results suggest that peritumoral accumulation of MC and the subsequent release of potent angiogenic factor such as VEGF may thus represent a tumor-host interaction that may favor progression of this tumor.

Abstract — N,N -Dipalmitylglycyl–apolipoprotein E (129–169) peptide (dpGapoE) is an efficient gene delivery system for both plasmids and antisense oligodeoxynucleotides (ODNs). To develop a new and efficient approach to the regulation of cholesteryl ester transfer protein (CETP) expression, we used dpGapoE to transfect phosphorothioate antisense ODNs against nucleotides 329 to 349 of human CETP cDNA into a human CETP–stably transfected Chinese hamster ovary (CHO) cell line (hCETP-CHO). After transfection, translocation to the nuclei and concentration in nuclear structures were observed in >95% of the cells at 6 and 12 hours by fluorescence microscopy. No membrane disruption was observed after transfection of ODNs by dpGapoE. Although the translocation stability of phosphorothioate ODNs in the nuclei continued for >48 hours, it had weakened after 24 hours. Cellular CETP mRNA levels gradually declined, and the maximum reduction in the mRNA level (>50%) was observed at 36 hours, after which the mRNA level started to recover. CETP activity in the culture medium declined over 72 hours. The maximum reduction in CETP activity was observed at 36 hours (53.8% of control). Neither CETP mRNA nor CETP activities changed throughout the experiment after the transfection of sense phosphorothioate ODNs delivered by dpGapoE complex or naked antisense ODNs. We conclude that (1) the novel synthetic dpGapoE was a highly effective and nontoxic vehicle for the nuclear delivery of antisense ODNs into hCETP-CHO cells and (2) antisense ODNs selectively inhibited both CETP expression and activity in an hCETP-CHO cell line. This approach may enable gene regulation in vivo and could possibly be used as an antiatherosclerotic agent to alter high density lipoprotein metabolism.

The authors report five patients with a missense mutation of the endothelial nitric oxide synthase (eNOS) gene (Glu298Asp) who have angiographically proven coronary artery disease (CAD). They compare their clinical findings and coronary arteriographic charac teristics. They conclude that these case reports show that this mutation is not solely responsible for development of CAD. Diabetes mellitus, smoking, and hyperlipidemia are other risk factors.

Abstract —To clarify the causes of renal dysfunction in familial lecithin:cholesterol acyltransferase (LCAT) deficiency, kidney samples from 4 patients with LCAT deficiency (3 homozygotes and 1 heterozygote) were examined immunohistochemically. All of the patients exhibited corneal opacities, anemia, renal dysfunction, deficiencies in plasma high density lipoprotein and LCAT activity and mass, and an increase in the ratio of plasma unesterified cholesterol to esterified cholesterol. Renal lesions began with the deposition of lipidlike structures in the glomerular basement membrane, and these structures accumulated in the mesangium and capillary subendothelium. By electron microscopy, 2 types of distinctive structure were found in glomerular lesions: vacuole structures and cross-striated, membranelike structures. The plasma oxidized phosphatidylcholine (oxPC) –modified low density lipoprotein (LDL) levels in LCAT-deficient subjects were significantly ( P P

We previously showed that thyroid carcinoma distinctively expresses intracellular fibronectin (FN) compared to other carcinomas. To determine the persistency of such FN accumulation in metastasis, we immunohistochemically examined the accumulation of intracellular FN in 92 cases of different carcinomas originating from the thyroid gland, lung and kidney, 44 of which showed metastasis to other organs. In all of the cases, normal epithelial cells adjacent to carcinomas did not show intracellular FN. Almost all of the cases (31/32) of thyroid carcinoma with/without metastasis to the lung and/or kidney showed intracellular FN in both the primary and metastatic lesions. Few cases (2/38) of lung carcinoma and none of the 22 cases of kidney carcinoma showed intracellular FN in the primary and metastatic lesions. In conclusion, the intracellular accumulation of FN acquired after carcinogenic transformation is not a common phenomenon in carcinomas, but rather is distinctive for thyroid carcinoma, even when it metastasizes to other organs. The immunohistochemical detection of intracellular FN may be useful for diagnosing thyroid carcinoma, even in metastatic lesions.

BACKGROUND Coronary stenosis involves lipid accumulation, fibrosis and cell proliferation. OBJECTIVE To clarify the role of oxidized low-density lipoprotein (LDL) in coronary stenosis by examining atherectomized coronary lesions from patients with primary stenosis and restenosis after percutaneous transluminal coronary angioplasty (PTCA). METHODS Atherectomized coronary tissue from 28 patients with primary stenosis and restenosis at 4.3 +/- 1.0 months after PTCA were examined using morphometrical and immunohistochemical techniques. RESULTS Serum lipids in all of the patients were within the normal range and no differences were noted between the two groups. There were no differences in the mean cross-sectional areas of whole specimens obtained from each group, and sclerotic lesions with atheroma or calcification were found to a similar extent in both groups. However, the restenosis group had a significantly greater area (6-fold) of immature smooth-muscle-rich lesions than the primary stenosis group, although there was no difference in lipid-laden foam-cell containing lesions. In foam-cell-containing lesions, apolipoprotein B was accumulated extracellularly, while oxidized LDL was primarily deposited intracellularly in lipid-laden foam cells. However, no deposition of apolipoprotein B, oxidized LDL or lipids was noted in smooth-muscle-rich lesions. Proline hydroxylase, a key enzyme for collagen synthesis, was detected in most of the foam-cell-containing lesions, but not in smooth-muscle-rich lesions. CONCLUSIONS Atherectomized lesions from patients with coronary stenosis contained smooth-muscle-rich lesions in restenosis and lipid-laden cellular lesions in both stenosis and restenosis, in which the deposition of oxidized LDL and increased collagen synthesis occur coincidentally. Therefore, the mechanism of atherogenesis may involve coronary stenosis regardless of the occurrence of restenosis after PTCA therapy.

Fibronectin is a multifunctional protein that plays a role in tumor invasion. We immunohistochemically examined the in vivo expression of fibronectin in thyroid carcinoma in comparison with other carcinomas, such as hepatocellular carcinoma, transitional cell carcinoma and gastric adenocarcinoma. Intracellular localization of fibronectin was found in almost all cases of thyroid carcinoma. In contrast, hepatocellular carcinoma showed a lower expression rate and the other carcinomas were all negative. These results indicate that the intracellular expression of fibronectin is not a common phenomenon in carcinoma, but rather is distinctive for thyroid carcinoma.

We studied the quantitative and qualitative characteristics of lipoprotein(a) [Lp(a)] as a function of apolipoprotein(a) [apo(a)] phenotype in 87 members (42 males, 45 females) of 20 diabetic families, 26 of whom were diagnosed with non-insulin-dependent diabetes mellitus (NIDDM) with moderate glycaemic control (HbA1c 7.1 +/- 1.2%). Apo(a) phenotyping was performed by a sensitive, high-resolution technique using SDS-agarose/gradient PAGE (3-6%). To date, 26 different apo(a) phenotypes, including a null type, have been identified. Serum Lp(a) levels of NIDDM patients and non-diabetic members of the same family who had the same apo(a) phenotypes were compared, while case control subjects were chosen from high-Lp(a) non-diabetic and low-Lp(a) nondiabetic groups with the same apo(a) phenotypes in the same family. Serum Lp(a) levels were significantly higher in NIDDM patients than in non-diabetic subjects (39.8 +/- 33.3 vs 22.3 +/- 19.5 mg/dl, p0.05). The difference in the mean Lp(a) level between the diabetic and non-diabetic groups was significantly (p0.05) greater than that between the high-Lp(a) non-diabetic and low-Lp(a) non-diabetic groups. An analysis of covariance and a least square means comparison indicated that the regression line between serum Lp(a) levels [log Lp(a)] and apo(a) phenotypes in the diabetic patient group was significantly (p0.01) elevated for each apo(a) phenotype, compared to the regression line of the control group. These data together with our previous findings that serum Lp(a) levels are genetically controlled by apo(a) phenotypes, suggest that Lp(a) levels in diabetic patients are not regulated by smaller apo(a) isoforms, and that serum Lp(a) levels are greater in diabetic patients than in non-diabetic family members, even when they share the same apo(a) phenotypes.

Abstract We report a 39-year-old Japanese man with HDL and apoA-I deficiency as well as data from members of his family. Corneal opacity and a stomatocyte were found but not tonsillar hypertrophy, xanthomas, or splenomegaly. His serum HDL cholesterol, apoA-I, apoA-II, and LDL cholesterol levels were 6 mg/dL, Sty I) were consistent with an autosomal codominant trait. Coronary angiography was performed to evaluate coronary atherosclerosis, but no significant luminal narrowing was detected. An intracoronary ultrasound study showed mild intimal hyperplasia in segment 6. In summary, this is a case of apoA-I deficiency without evidence of coronary heart disease.

The short- and long-term effects of the angiotensin-converting enzyme inhibitor lisinopril and the cardioselective beta-blocker bisoprolol on serum lipids, lipoproteins, apolipoproteins, and lipoprotein(a) (Lp[a]) levels were investigated in patients with mild-to-moderate essential hypertension. Fifty-two patients completed the 12-month, randomized, multicenter trial. After administration of lisinopril (10 to 20 mg/d; n = 24) and bisoprolol (2.5 to 10 mg/d; n = 28), systolic and diastolic blood pressures decreased significantly (P0.01) from baseline in both groups at 3, 6, and 12 months. The reduction in diastolic blood pressure was significantly (P0.05) greater in the lisinopril group than in the bisoprolol group only at 6 months. Heart rates dropped significantly in the bisoprolol group but not in the lisinopril group. No significant changes in lipids, lipoproteins, apolipoproteins, or Lp(a) levels were observed with either drug at 3, 6, or 12 months, and no significant differences were noted between the two drugs for these parameters. We conclude that lisinopril and bisoprolol are effective as antihypertensive drugs without adverse metabolic effects after short- and long-term treatment in patients with mild-to-moderate essential hypertension.

We examined the interactive effect of oxidized low density lipoprotein (LDL) and ascorbic acid on collagen production in cultured smooth muscle cells (SMCs). Porcine aortic SMCs were incubated with 50-200 micrograms/ml of human LDL with/without 5 microM Cu2+ for 24 h. Collagen production was assayed by successive salt precipitation at acidic and neutral pH after pepsin digestion of 3H-proline-labeled collagenous protein. Oxidation of LDL was evaluated by electrophoresis and by the level of thiobarbituric acid reactive substances (TBARS). Ascorbic acid reduced the oxidation of LDL + Cu2+ (53% reduction). In the presence of ascorbic acid, no differences were noted in collagen production between LDL and LDL + Cu2+. Without ascorbic acid, collagen production with LDL + Cu2+ was increased dose-dependently up to 6-fold with 150 micrograms/ml LDL, while no such effects were observed at any doses of native LDL. The addition of butylated hydroxytoluene to LDL + Cu2+ strongly suppressed oxidation (88% reduction), and significantly reduced collagen production close to that seen with native LDL. These results indicate that oxidized LDL stimulates collagen production in SMCs, while native LDL does not. Therefore, oxidized LDL may play a direct role in stimulating collagen production in SMCs, which could lead to collagenosis in atherosclerosis.

The relationship between the extractability of collagen by enzymatic digestion and the degree of nonenzymatic glycation of collagen was examined in the aorta and skin from 38 subjects without diabetes mellitus (mean age: 62.3 +/- 20.2 years). Samples were obtained from the aortic media (M), lesion-free intima (I), atherosclerotic intima (A) and dermis of the skin (S). Collagen was extracted first by incubation with 1/50 (enzyme/substrate weight ratio) pepsin at 4 degrees C for 24 h (P-fraction) and then by incubation with 1/10 (enzyme/substrate weight ratio) pepsin at room temperature for 24 h (EP-fraction). The pepsin-insoluble precipitates were digested by incubation with 270 units of bacterial collagenase at 37 degrees C for 24 h (PIS-fraction). Collagen contents, ketoamines and collagen-linked fluorescence (CLF) were measured in each fraction. The amount of ketoamines and the level of CLF correlated inversely with the susceptibility of collagen to pepsin digestion in various tissues, including M, I, A and S. These values were highest in both the P- and EP-fractions of M, which contained the least amount of collagen extracted by pepsin digestion. In contrast, they were lowest in S, where the concentration of collagen extracted by pepsin digestion was greatest among all of the tissue samples. Atherosclerotic intima (A) and aortic media (M) showed an age-related increase in the total amount of collagen digested with pepsin and collagenase, which depended mainly on an increase in the content of pepsin-insoluble collagen. Although the total amount of collagen did not increase with advancing age in I or S, collagen in I and S became progressingly resistant to pepsin digestion. These results suggest that the age-related decrease in the susceptibility of collagen to pepsin digestion may be due to nonenzymatic glycation in atherosclerotic lesions as well as normal tissues, including the aortic media, lesion-free intima and skin. The level of CLF significantly increased with age in the P-fraction and/or EP fraction of M, I and S. However, there was no relationship between the level of CLF and the subject's age in A. Thus, the accumulation of advanced glycation endproducts (AGEs) on collagen fibers may be partially responsible for the increase in collagen matrix in atherosclerotic lesions of subjects without diabetes mellitus.

The effects of the administration of slow-release bezafibrate to hypercholesterolaemic patients who were already receiving long-term probucol treatment (mean 865 days, 500–1000 mg·day−1) were investigated. Bezafibrate was administered at either 200 mg·day−1 (13 males, 13 females, mean age 55.2 years) or 400 mg·day−1 (11 males, 14 females, mean age 57.2 years), and blood was taken at 0, 3, 6 and 12 months after the beginning of combination therapy. Overall, serum total cholesterol (TC), triglyceride (TG), very low density lipoprotein (VLDL)-TC, high-density lipoprotein (HDL)-TG, VLDL-TG, VLDL-phospholipid (PL), lipoprotein (a) [Lp(a)], apolipoprotein (apo) C-III, apo E levels and LCAT activity decreased significantly with this combination therapy, while HDL cholesterol (C), HDL3-C, HDL-PL, apo A-I and apo A-II levels significantly increased, as assessed by analysis of variance (ANOVA). Five patients (one receiving 200 mg·day−1, four receiving 400 mg·day−1 bezafibrate) showed drastic reductions in HDL-C (HDL-C levels were reduced by a mean of 46.2%, 59.3% and 61.6% at 3, 6 and 12 months, respectively) after beginning combination therapy. These HDL-C reductions were maintained for the 1 year of combination therapy, but then returned to pre-combination treatment levels 1 month after discontinuation of bezafibrate. Serum probucol concentrations and cholesteryl ester transfer protein (CETP) mass were assayed at 6 months, and the probucol concentration was higher in the HDL-deficient group (56.2 vs 26.5 μg/ml). In contrast, CETP mass was significantly lower in HDL-deficient patients than in non-HDL-deficient patients (2.08 vs 2.87 mg·l−1). When the patients in the non-HDL-deficient group were divided into two groups, receiving low (200 mg·day−1, n−25) and high (400 mg·day−1, n−21) doses of bezafibrate, the former group showed a significant increase in probucol-lowered HDL-C and apo A-I, although these levels did not return to pre-probucol treatment levels, while the latter group showed no changes in HDL. These data suggest that the addition of a low dose of bezafibrate to probucol tended to reverse probucol-induced HDL lowering, while 9.8% (5 of 51 patients) of the patients exhibited a severe HDL deficiency. Since it is unclear whether or not such an extreme HDL reduction is harmful, HDL deficiency should be carefully monitored with this combination therapy.

The accumulation of low density lipoprotein (LDL) in the arterial intima is an important characteristic of atherosclerosis. We investigated the mechanisms by which LDL binds to different types of collagen. The binding activities of 125I-labeled human native LDL (nLDL) and copper-oxidized LDL (oxLDL) with different collagen gels prepared in type I collagen-based mixtures with types I, III, IV and V (I+I, I+III, I+IV and I+V, respectively) were examined. A concentration of 20 micrograms LDL protein/150 micrograms collagen/well was used. The diffusion of both nLDL and oxLDL into the collagen gels reached an equilibrium after 48 h. All of the collagen gels showed the same rates of diffusion with both LDLs. The binding activities of oxLDL were significantly greater than those of nLDL (P0.001%), while the binding activities for both LDLs followed the order I+I and I+IIII+VI+IV. However, the increased binding rate of oxLDL compared to nLDL was 1.66 for I+IV, 1.50 for I+V, 1.33 for I+I and 1.19 for I+III. When a 10-fold higher dose of NaCl (1 M) was added to the oxLDL medium, the binding rate of oxLDL was reduced (rate of reduction: 52% (I+I), 48% (I+III), 35% (I+IV), 13% (I+V)). These results suggest that oxLDL binds more to type I and III collagens by negative charge-dependent mechanisms than to type IV and V collagens. Therefore, types I and III collagens may play an important role in trapping LDL, especially oxLDL. Therefore, oxidatively modified LDL may contribute to atherogenesis due to its longer retention in the arterial wall.

We investigated the growth characteristics of smooth muscle cells transformed by oridefective simian virus 40 plasmid DNA (TSMCs). TSMCs demonstrated a significantly higher proliferative activity when compared with nontransformed smooth muscle cells (SMCs). The SMCs were not able to proliferate in media with 5% plasma-derived serum (PDS) instead of fetal bovine serum, while TSMCs grew under this condition. When the DNA synthesis of quiescent SMCs was reinitiated by the addition of serum or platelet-derived growth factor (PDGF), the amount of [3H]thymidine incorporation into DNA varied with the concentrations of serum or PDGF and decreased on type V collagen compared with the other collagen substrates. In contrast, the TSMCs showed no differences in the amount of [3H]thymidine incorporation into DNA among the various concentrations of serum or PDGF and different types of collagen. When quiescent SMCs were cocultured with TSMCs, they showed a remarkable increase in the amount of [3H]thymidine incorporation into DNA, which was most intensively inhibited by type V collagen. These results suggest that TSMCs can promote the growth of TSMCs and SMCs in either an autocrine or paracrine manner and that they proliferate independently of the collagen matrices. Trapidil, a pyrimidine derivative that may inhibit the action of PDGF, decreased the number and cAMC levels of TSMCs in the media with 5% PDS in a concentration-dependent manner. The addition of polyclonal anti-PDGF-B antibody induced up to 59.5% inhibition of enhancement of DNA synthesis of SMCs cocultured with TSMCs. The level of cyclic adenosine monophosphate (cAMP) increased in the TSMCs compared with the SMCs. There was, however, no difference in the cAMP levels of either cells between the various types of collagen. Thus, the release of PDGF-like growth factor and an accumulation of cellular cAMP may be related to an increased DNA synthesis of TSMCs.

Abstract 4812 Gemtuzumab ozogamicin (GO) is an immunoconjugate of an anti-CD33 antibody and a toxic calicheamicin-γ1 derivative that facilitates cellular uptake of the latter drug. CD33 is selectively expressed on myeloid cells, especially on acute myeloid leukemia (AML) cells, but usually not on non-hematopoietic cells, and therefore the clinical use is confined to AML. Despite its predominant expression on AML cells, GO induces complete remission only in 30% of recurrent AML. The major side-effects of GO include not only myelosuppression but also hepatotoxicity, although CD33 is not expressed on hepatobiliary cells. It suggests that the efficacy of GO is not strictly regulated by CD33 expression. We investigated relationship between CD33 expression and GO cytotoxicity by using leukemia cell lines, and classified them into four groups; group A defined as CD33(+) and cytotoxicity (+), group B as CD33(+) and cytotoxicity (-), group C as CD33(-) and cytotoxicity (+), and group D as CD33(-) and cytotoxicity (-). We selected four cell lines, HL-60, MEG-01, MOLT-3 and K562 which belong to group A, B, C and D, respectively, and were used for further experiment. CD33 was expressed on HL-60 but not on MOLT-3. However, GO-induced cytotoxicity was seen in both cells. We therefore compared effects of GO in HL-60 and MOLT-3. When incubated with GO for 72 hours, viability of HL-60 significantly decreased at 10 ng/mL of GO, while cytotoxic effect of GO against MOLT-3 became apparent when GO concentration was raised to 100 ng/mL. Viability of HL-60 markedly decreased when treated with GO for 30 minutes and then cultured in medium without containing GO for 72 hours, but no cytotoxic effect was observed in MOLT-3 unless culture with GO was prolonged to more than 6 hours. GO-induced apoptosis was detected in both HL-60 and MOLT-3 by Annexin V assay and TUNEL assay. The pretreatment with unconjugated anti-CD33 antibody (hP67.6) resulted in reduced cytotoxic effects of GO in HL-60, but not in MOLT-3. It indicates that GO induces apoptosis in CD33-non-expressing MOLT-3. Higher doses and longer exposure of GO treatment are necessary to induce cytotoxicity against MOLT-3 when compared with HL-60. GO might cause cytotoxicity against MOLT-3 through drug absorption pathway other than CD33-mediated endocytosis. Although HL-60 and MEG-01 expressed CD33, GO induced cytotoxicity on HL-60, but not on MEG-01. We then investigated internalization of antibody-bound CD33. After incubated with hP67.6 on ice, cells were cultured in antibody-free medium, and remaining cell surface-associated CD33 was measured. The loss of cell surface CD33 was observed in MEG-01 as well as HL-60, showing that internalization of CD33 following antibody binding occurred in MEG-01. It indicates that resistance to GO-induced cytotoxicity in MEG-01 is not associated with internalization property. Drug transport pathway other than receptor-mediated endocytosis was investigated. Cells were cultured in rhodamine 123 (Rh123)-containing medium for 30 minutes and the influx of Rh123 was assessed by the fluorescence activity of Rh123-containing cells. Cells were then cultured in medium without containing Rh123 for further 3 hours, and the efflux of Rh123 was analyzed by measuring the reduction curve of fluorescence activity. Rh123 accumulated in MEG-01 twice higher than HL-60. However, fluorescence activity of Rh123 decreased to 20% after 3 hours, while it remained unchanged in HL-60, indicating that drug efflux was activated in MEG-01. Multidrug resistance (MDR)-1 protein was strongly expressed in MEG-01. When cyclosporin-A, a modulator of MDR-1, was given with GO to MEG-01, GO-induced cytotoxicity was significantly increased, while no such effect was observed in HL-60. It suggests that p-glycoprotein-mediated efflux of GO is one of the resistance mechanisms in MEG-01. Disclosures: No relevant conflicts of interest to declare.

Liposomes prepared with 25-hydroxycholesterol and egg phosphatidylcholine (PC) were incubated with bovine arterial smooth muscle cells for 8 h at 37 degrees C. Cells incubated in the absence of liposomes or with liposomes containing cholesterol and PC were used as controls. The results indicated that calcium accumulated in the smooth muscle cells incubated in the presence of 25-hydroxycholesterol containing liposomes in an amount proportional to the time of incubation. The calcium accumulation, as indicated by kinetic analysis, resulted from an increased compartment size. (Ca(2+)+Mg2+)-ATPase exhibited decreased activity after pretreatment with 25-hydroxycholesterol containing liposomes and the increased intracellular calcium content was directly proportional to the decreased (Ca(2+) + Mg2+)-ATPase activity. When lipids in the cell membrane were examined, a failure to change the cholesterol/phospholipids ratio in the membrane was noted. The 25-hydroxycholesterol content in the membrane determined by HPLC did not increase. An increase in sphingomyelin and a decrease in phosphatidylethanolamine and acidic phospholipids in the membrane was noted. We suggest that the accumulation of intracellular calcium comes from both an increase of calcium influx and a decrease of (Ca(2+) + Mg2+)-ATPase activity, which may be the consequence of changes in membrane phospholipid composition.

We investigated the effects of 0.5, 2.5, and 10 micrograms/ml of cholesterol or 26-hydroxycholesterol on bovine aortic ECs and SMCs. Suppression of viable cell density and cytotoxic changes in both cells were induced by 2.5 and 10 micrograms/ml 26-hydroxycholesterol. ECs were more severely damaged than SMCs in the presence of 26-hydroxycholesterol. Levels of up to 10 micrograms/ml cholesterol had no effect on ECs or SMCs growth or cytotoxicity. Confluent ECs exposed to 2.5 or 10 micrograms/ml of 26-hydroxycholesterol secreted significant amounts of 6-ketoprostaglandin F1 alpha after a 24-hr incubation. An equivalent concentration of cholesterol had no such effect. SMCs cocultured with ECs exposed to 10 micrograms/ml 26-hydroxycholesterol, when compared with an equivalent level of cholesterol, synthesized DNA in significantly greater amounts during a 24-hr incubation. The cocultured ECs incubated in the presence of 2.5 and 10 micrograms/ml 26-hydroxycholesterol were partially detached due to cell death. However, no difference was observed in the DNA content of SMCs cultured without ECs in the presence of cholesterol or 26-hydroxycholesterol. The results suggest that 26-hydroxycholesterol produced not only cytotoxicity to ECs and SMCs but also stimulated DNA synthesis in SMCs through endothelial injury.

Gemtuzumab ozogamicin (GO) is a humanized anti-CD33 antibody conjugated to a derivative of a tumoricidal antibiotic calicheamicin. GO alone induces complete remission in 26% of AML patients in first relapse. One way to improve the response rate is to combine GO with other agents. A quantitative relationship has been shown between rates and degrees of CD33 expression and GO-induced cytotoxicity in vitro by using gene transfer to manipulate CD33 expression in myeloid cell lines. We first studied CD33 expression on myeloid leukemia cells by stimulating with several agents in vitro, and found that ubenimex, but not G-CSF, M-CSF, or ATRA, increased CD33 expression on both HL-60 and K562 cells. Ubenimex is an aminopeptidase inhibitor isolated from Streptomyces olivoreticuli and commercially available as an oral agent for clinical use for AML treatment. To investigate whether ubenimex enhances the cytotoxicity of GO, HL-60 and K562 cells were cultured with GO and/or ubenimex for 3 days. When GO was administered, cell viabilities of HL-60 and K562 were reduced to 31.5% and 90.3% as compared with control, respectively. Treatment with ubenimex alone did not influence viabilities of either HL-60 or K562. However, when cells were preincubated with ubenimex and then cultured with GO, cell viabilities decreased to 18.5% and 81.4% for HL-60 and K562, respectively, indicating that pretreatment with ubenimex enhanced GO-induced myeloid leukemia cell death in vitro. We next assessed the mechanism of cell death. The treatment with GO alone and ubenimex alone induced apoptosis in 39.2% and 2.9% of HL-60 cells, respectively. When HL-60 cells were preincubated with ubenimex and then cultured with GO, the number of apoptotic cells increased to 62.9%, demonstrating that ubenimex augments GO-induced apoptosis. Our data suggest that the priming of AML cells with ubenimex should improve the clinical efficacy of GO.