Jinya Suzuki

Pitavastatin-Induced Thrombomodulin Expression by Endothelial Cells Acts Via Inhibition of Small G Proteins of the Rho Family

Objective—3-Hydroxyl-3-methyl coenzyme A reductase inhibitors (statins) can function to protect the vasculature in a manner that is independent of their lipid-lowering activity. The main feature of the antithrombotic properties of endothelial cells is an increase in the expression of thrombomodulin (TM) without induction of tissue factor (TF) expression. We investigated the effect of statins on the expression of TM and TF by endothelial cells. Methods and Results—The incubation of endothelial cells with pitavastatin led to a concentration- and time-dependent increase in cellular TM antigen and mRNA levels. In contrast, the expression of TF mRNA was not induced under the same conditions. A nuclear run-on study revealed that pitavastatin accelerates TM transcription rate. The stimulation of TM expression by pitavastatin was prevented by either mevalonate or geranylgeranylpyrophosphate. Specific inhibition of geranylgeranyltransferase-I and Rac/Cdc42 by GGTI-286 and Clostridium sordellii lethal toxin, respectively, enhanced TM expression, whereas inactivation of Rho by Clostridium botulinum C3 exoenzyme was ineffective. Conclusions—Statins regulate TM expression via inhibition of small G proteins of the Rho family; Rac/Cdc42. A statin-mediated increase in TM expression by endothelial cells may contribute to the beneficial effects of statins on endothelial function.

Cardiac overexpression of hormone-sensitive lipase inhibits myocardial steatosis and fibrosis in streptozotocin diabetic mice.

Intracellular lipid accumulation (steatosis) and resultant lipotoxicity are key features of diabetic cardiomyopathy. Since cardiac hormone-sensitive lipase (HSL) is activated in diabetic mice, we sought to explore a pathophysiological function of cardiac HSL in the development of diabetic cardiomyopathy. Transgenic (Tg) mice with heart-specific HSL overexpression were generated, and cardiac histology, function, lipid profile, and gene expressions were analyzed after induction of diabetes by streptozotocin. Electron microscopy showed numerous lipid droplets in wild-type (Wt) hearts after 3 wk of diabetes, whereas Tg mice showed no lipid droplet accumulation. Cardiac content of acylglycerides was increased approximately 50% with diabetes in Wt mice, whereas this was blunted in Tg hearts. Cardiac lipid peroxide content was twofold lower in Tg hearts than in Wt hearts. The mRNA expressions for peroxisome proliferator-activated receptor-alpha, genes for triacylglycerol synthesis, and lipoprotein lipase were increased with diabetes in Wt hearts, whereas this induction was absent in Tg hearts. Expression of genes associated with lipoapoptosis was decreased, whereas antioxidant protein metallothioneins were increased in diabetic Tg hearts. Diabetic Wt hearts showed interstitial fibrosis and increased collagen content. However, Tg hearts displayed no overt fibrosis, concomitant with decreased expression of collagens, transforming growth factor-beta, and matrix metalloproteinase 2. Notably, mortality during the experimental period was approximately twofold lower in diabetic Tg mice compared with Wt mice. In conclusion, since HSL overexpression inhibits cardiac steatosis and fibrosis by apparently hydrolyzing toxic lipid metabolites, cardiac HSL could be a therapeutic target for regulating diabetic cardiomyopathy.

Very low density lipoprotein receptor binds apolipoprotein E2/2 as well as apolipoprotein E3/3

The VLDL receptor, a newly identified lipoprotein receptor, recognizes apoE containing lipoproteins. The human VLDL receptor was overexpressed in ldlA‐7, a mutant Chinese hamster ovary cells lacking LDL receptors. Each VLDL obtained from a normolipidemic subject with two ε3 or ε2 alleles similarly competed for the binding of radiolabeled rabbit β‐VLDL to the VLDL receptors. The anti‐apoE monoclonal antibody 1D7, which inhibited binding of apoE3 to the LDL receptors, failed to compete for the binding of VLDL (apoE3 or apoE2) to the VLDL receptors. Results indicate that the binding site of apoE on the VLDL receptor may differ from its binding site on the LDL receptor.

Increase in neutral cholesteryl ester hydrolase activity produced by extralysosomal hydrolysis of high-density lipoprotein cholesteryl esters in rat hepatoma cells (H-35)

The metabolism of high-density lipoprotein-associated cholesteryl esters (HDL-CE) in liver cells is not well understood. We studied the possible role of lysosomal and extralysosomal pathways on such metabolism by measuring the uptake and hydrolysis of HDL-CE in H-35 rat hepatoma cells. Incubation of cells with [3H]cholesteryl ester-labeled HDL led to the intracellular accumulation of both 3H-free cholesterol and [3H]cholesteryl ester. The ratio of 3H-free cholesterol/[3H]cholesteryl ester increased with an increase in incubation time even in the presence of chloroquine. Because chloroquine did not inhibit the conversion of cholesteryl ester to free cholesterol, the hydrolysis of HDL-CE may have been catalyzed by an extralysosomal enzyme, perhaps by neutral cholesteryl ester hydrolase (NCEH). When we incubated cells with increasing concentrations of HDL, NCEH activity increased. This increase in enzyme activity was not inhibited by the addition of chloroquine. A complex of dimyristoylphosphatidylcholine (DMPC)/apo HDL/cholesteryl ester enhanced the activity as well as native HDL. Neither the DMPC/apo HDL nor the DMPC/cholesteryl ester complex affected the activity, suggesting that apo HDL may be required for the uptake of HDL-CE. The present study demonstrated that the extralysosomal hydrolysis by NCEH is operating in the metabolism of HDL-CE in hepatoma cells.

Remnant lipoprotein particles are taken up into myocardium through VLDL receptor--a possible mechanism for cardiac fatty acid metabolism.

The VLDL (very low-density lipoprotein) receptor is a peripheral lipoprotein receptor expressing in fatty acid active tissues abundantly. In the Balb/c fasting mice, VLDL receptor as well as LPL (lipoprotein lipase), FAT (fatty acid translocase)/CD36, H-FABP (heart-type fatty acid-binding protein), ACS (acyl-CoA synthetase) and LCAD (long-chain acyl-CoA dehydrogenase) expressions increased. An electron microscopic examination indicated the lipid droplets that accumulated in the hearts of fasting Balb/c mice. During the development of SD (Sprague-Dawley) rats, VLDL receptor, LPL, FAT/CD36, H-FABP, ACS, and LCAD mRNAs concomitantly increased with growth. However, PK (pyruvate kinase) mRNA expression was negligible. In cultured neonatal rat cardiomyocytes, VLDL receptor expression increased with days in culture. Oil red-O staining showed that cardiomyocytes after 7 days in culture (when the VLDL receptor protein is present) accumulated beta-migrating VLDL. Thereby, we showed that the cardiac VLDL receptor pathway for delivery of remnant lipoprotein particles might be part of a cardiac fatty acid metabolism.

Glucose deprivation accelerates VLDL receptor-mediated TG-rich lipoprotein uptake by AMPK activation in skeletal muscle cells

Glucose and fatty acids are major energy sources in skeletal muscle. Very low-density lipoprotein receptor (VLDL-R), which is highly expressed in heart, skeletal muscle and adipose tissue, plays a crucial role in metabolism of triglyceride (TG)-rich lipoproteins. To explore energy switching between glucose and fatty acids, we studied expression of VLDL-R and lipoprotein uptake in rat L6 myoblasts. l-Glucose or d-glucose deprivation in the medium noticeably induced the AMPK (AMP-activated protein kinase) activation and VLDL-R expression. Dose-dependent induction of VLDL-R expression was observed when d-glucose was less than 4.2mM. The same phenomenon was also observed in rat primary skeletal myoblasts and cultured vascular smooth muscle cells. The uptake of beta-VLDL but not LDL was accompanied by induction of VLDL-R expression. Our study suggests that the VLDL-R-mediated uptake of TG-rich lipoproteins might compensate for glucose shortfall through AMPK activation in skeletal muscle.

Thrombomodulin expression by THP-1 but not by vascular endothelial cells is upregulated by pioglitazone

Thrombomodulin-protein C pathway is a major anti-thrombotic mechanism present in endothelial cells (EC), and an important modulator of inflammation. Peroxisomal proliferator activated receptor-gamma (PPARgamma) expressed in monocytes/macrophages may have a role in cell differentiation. Since the expression of thrombomodulin (TM) by monocytes is upregulated during differentiation into macrophages, we investigated the effect of pioglitazone, a thiazolidinedione (TZD) that is a synthetic ligand of PPARgamma, on the expression of TM by a human monocyte/macrophage cell line; human acute monocytic leukemia (THP-1) cells. Pioglitazone dose-dependently upregulated TM antigen expression by THP-1 cells accompanied by an upregulation of TM cofactor activity for thrombin-dependent protein C activation. Thrombomodulin mRNA expression in THP-1 cells was also upregulated by pioglitazone, whereas tissue factor (TF) mRNA expression was not induced at all. Treatment cells with a natural PPARgamma ligand, 15-deoxy-delta12,14-prostaglandin J(2) (PGJ2), also enhanced TM protein expression. PGF(2alpha) an agent known to inactivate PPARgamma, diminished the stimulatory effect of pioglitazone and PGJ2 on TM protein expression. In contrast, pioglitazone had no effect on TM antigen expression by human umbilical vein ECs. These results suggest that PPARgamma activation in macrophages may counteract potentially prothrombotic and putative inflammatory properties in activated macrophages.

1α,25-dihydroxyvitamin D3 induces very low density lipoprotein receptor mRNA expression in HL-60 cells in association with monocytic differentiation

Expression of VLDL receptor mRNA during differentiation of HL-60 cells was investigated by Northern analysis. The expression induced in 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3)-treated cells was 3 times that in untreated cells, while LDL receptor mRNA expression was unchanged. VLDL receptor mRNA levels were not changed in macrophages caused to differentiate from HL-60 cells by treatment with phorbol 12-myristate 13-acetate (PMA). Treatment of sarcoma cells which possess the vitamin D receptor (MG-63 cell line) with 1 alpha,25(OH)2D3 did not affect VLDL receptor mRNA levels. Therefore, 1 alpha,25(OH)2D3 induces VLDL receptor mRNA in HL-60 cells through differentiation-dependent mechanisms.

Comparative reactivity of remnant-like lipoprotein particles (RLP) and low-density lipoprotein (LDL) to LDL receptor and VLDL receptor: Effect of a high-dose statin on VLDL receptor expression

BACKGROUND Comparison of the reactivity of remnant-like lipoprotein particles (RLP) and LDL particles to LDL receptor and VLDL receptor has not been investigated. METHODS LDL receptor- or VLDL receptor-transfected ldlA-7, HepG2 and L6 cells were used. Human LDL and rabbit β-VLDL were isolated by ultracentrifugation. Human RLP was isolated using an immunoaffinity mixed gel. The effect of statin on lipoprotein receptors was examined. RESULTS Both LDL receptor and VLDL receptor recognized RLP. In LDL receptor transfectants, RLP, β-VLDL and LDL all bound to LDL receptor. Cold RLP competed efficiently with DiI-β-VLDL; however, cold LDL competed weakly. In VLDL receptor transfectants, RLP and β-VLDL bound to VLDL receptor, but not LDL. RLP bound to VLDL receptor with higher affinity than β-VLDL because of higher apolipoprotein E in RLP. LDL receptor expression was induced in HepG2 by the low concentration of statin while VLDL receptor expression was induced in L6 myoblasts at higher concentration. CONCLUSIONS RLP are bound to hepatic LDL receptor more efficiently than LDL, which may explain the mechanism by which statins prevent cardiovascular risk by primarily reducing plasma RLP rather than by reducing LDL. Additionally, a high-dose of statins also may reduce plasma RLP through muscular VLDL receptor.

Effects of hormone-sensitive lipase disruption on cardiac energy metabolism in response to fasting and refeeding

Increased fatty acid (FA) flux and intracellular lipid accumulation (steatosis) give rise to cardiac lipotoxicity in both pathological and physiological conditions. Since hormone-sensitive lipase (HSL) contributes to intracellular lipolysis in adipose tissue and heart, we investigated the impact of HSL disruption on cardiac energy metabolism in response to fasting and refeeding. HSL-knockout (KO) mice and wild-type (WT) littermates were fasted for 24 h, followed by ∼6 h of refeeding. Plasma FA concentration in WT mice was elevated twofold with fasting, whereas KO mice lacked this elevation, resulting in twofold lower cardiac FA uptake compared with WT mice. Echocardiography showed that fractional shortening was 15% decreased during fasting in WT mice and was associated with steatosis, whereas both of these changes were absent in KO mice. Compared with Langendorff-perfused hearts isolated from fasted WT mice, the isolated KO hearts also displayed higher contractile function and a blunted response to FA. Although cardiac glucose uptake in KO mice was comparable with WT mice under all conditions tested, cardiac VLDL uptake and lipoprotein lipase (LPL) activity were twofold higher in KO mice during fasting. The KO hearts showed undetectable activity of neutral cholesteryl esterase and 40% lower non-LPL triglyceride lipase activity compared with WT hearts in refed conditions accompanied by overt steatosis, normal cardiac function, and increased mRNA expression of adipose differentiation-related protein. Thus, the dissociation between cardiac steatosis and functional sequelae observed in HSL-KO mice suggests that excess FA influx, rather than steatosis per se, appears to play an important role in the pathogenesis of cardiac lipotoxicity.