Correlation between serum levels of proprotein convertase subtilisin/kexin type 9 (PCSK9) and atherogenic lipoproteins in patients with coronary artery disease.

BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of serum low-density lipoprotein (LDL) cholesterol levels. Recently, PCSK9 has additionally been related to metabolic risk factors such as the levels of triglycerides, apolipoprotein B (apoB), insulin, and glucose, as well as body mass index. The purpose of this study was to investigate correlations between serum levels of PCSK9 and apoB-containing atherogenic lipoproteins in patients with coronary artery disease (CAD). METHODS: Serum levels of PCSK9 and lipoprotein(a) [Lp(a)]; small, dense LDL; and oxidized LDL were measured in 101 patients with CAD who were not receiving lipid-lowering therapy. RESULTS: Serum hetero-dimer PCSK9 levels were positively correlated with serum levels of Lp(a) (r = 0.195, p = 0.05); small, dense LDL (r = 0.336, p = 0.0006); and oxidized LDL (r = 0.268, p = 0.008). Multivariate regression analyses showed that serum hetero-dimer PCSK9 was a significant predictor of serum levels of Lp(a) (ß = 0.235, p = 0.01); small, dense LDL (ß = 0.143, p = 0.03); and oxidized LDL (ß = 0.268, p = 0.008). CONCLUSIONS: Serum PCSK9 levels were positively correlated with serum levels of Lp(a); small, dense LDL; and oxidized LDL in patients with CAD. This suggests that the interaction between serum PCSK9 and apoB-containing lipoproteins plays a role in establishing the atherosclerotic status of patients. TRIAL REGISTRATION: UMIN Clinical Trials Registry, UMIN ID: C000000311 .

Lipoprotein remodeling generates lipid-poor apolipoprotein A-I particles in human interstitial fluid.

Although much is known about the remodeling of high density lipoproteins (HDLs) in blood, there is no information on that in interstitial fluid, where it might have a major impact on the transport of cholesterol from cells. We incubated plasma and afferent (prenodal) peripheral lymph from 10 healthy men at 37°C in vitro and followed the changes in HDL subclasses by nondenaturing two-dimensional crossed immunoelectrophoresis and size-exclusion chromatography. In plasma, there was always initially a net conversion of small pre-ß-HDLs to cholesteryl ester (CE)-rich α-HDLs. By contrast, in lymph, there was only net production of pre-ß-HDLs from α-HDLs. Endogenous cholesterol esterification rate, cholesteryl ester transfer protein (CETP) concentration, CE transfer activity, phospholipid transfer protein (PLTP) concentration, and phospholipid transfer activity in lymph averaged 5.0, 10.4, 8.2, 25.0, and 82.0% of those in plasma, respectively (all P < 0.02). Lymph PLTP concentration, but not phospholipid transfer activity, was positively correlated with that in plasma (r = +0.63, P = 0.05). Mean PLTP-specific activity was 3.5-fold greater in lymph, reflecting a greater proportion of the high-activity form of PLTP. These findings suggest that cholesterol esterification rate and PLTP specific activity are differentially regulated in the two matrices in accordance with the requirements of reverse cholesterol transport, generating lipid-poor pre-ß-HDLs in the extracellular matrix for cholesterol uptake from neighboring cells and converting pre-ß-HDLs to α-HDLs in plasma for the delivery of cell-derived CEs to the liver.

Identification of three new autoantibodies associated with systemic lupus erythematosus using two proteomic approaches.

Our objective was to identify new serum autoantibodies associated with systemic lupus erythematosus (SLE), focusing on those found in patients with central nervous system (CNS) syndromes. Autoantigens in human brain proteins were screened by multiple proteomic analyses: two-dimensional polyacrylamide gel electrophoresis/Western blots followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis and immunoprecipitation followed by liquid chromatography-tandem mass spectrometry shotgun analysis. The presence of serum IgG autoantibodies against 11 selected recombinant antigens was assessed by Western blot and enzyme-linked immunosorbent assay (ELISA) in the sera of 106 SLE patients and 100 normal healthy controls. The O.D. values in sera from SLE patients were significantly higher than those of controls for the antigens crystallin αB (p = 0.0002), esterase D (p = 0.0002), APEX nuclease 1 (p < 0.0001), ribosomal protein P0 (p < 0.0001), and PA28γ (p = 0.0005); the first three are newly reported. The anti-esterase D antibody levels were significantly higher in the CNS group than in the non-CNS group (p = 0.016). Moreover, when the SLE patients were categorized using CNS manifestations indicating neurologic or psychiatric disorders, the anti-APEX nuclease 1 antibody levels were significantly elevated in SLE patients with psychiatric disorders (p = 0.037). In conclusion, the association of SLE with several new and previously reported autoantibodies has been demonstrated. Statistically significant associations between anti-esterase D antibodies and CNS syndromes as well as between anti-APEX nuclease 1 antibodies and psychiatric disorders in SLE were also demonstrated. The combined immunoproteomic approaches used in this study are reliable and effective methods for identifying SLE autoantigens.

Serum antibodies against the 70k polypeptides of the U1 ribonucleoprotein complex are associated with psychiatric syndromes in systemic lupus erythematosus: a retrospective study.

OBJECTIVES: We assessed the association between serum autoantibodies against the 70-kDa polypeptide of the U1-ribonucleoprotein (RNP) complex (U1-70k) and the central nervous system (CNS) syndromes in systemic lupus erythematosus (SLE) patients. METHODS: We studied 106 hospitalized patients with active SLE, comparing those with (n = 32) and without (n = 74) CNS syndromes. CNS syndromes were further classified into neurologic (n = 21) and psychiatric (n = 15) disorders. Immunoglobulin G (IgG) anti-U1-70k antibodies were measured by enzyme-linked immunosorbent assay (ELISA) using recombinant antigens. IgG antibodies against whole U1-RNP were measured using commercial ELISA kits. RESULTS: Although there was no significant difference in the levels of serum anti-U1-70k antibodies in SLE patients with or without CNS syndromes (p = 0.83), the levels were significantly elevated in SLE patients compared with patients without psychiatric syndromes (p = 0.030). In contrast, no significant difference was observed in the levels of serum anti-U1-RNP antibodies in SLE patients with or without psychiatric syndromes (p = 0.555). CONCLUSIONS: These results indicate that serum anti-U1-70k antibodies are associated with psychiatric syndromes in SLE but that they are not associated with CNS syndromes as a whole or with neurologic syndromes. The anti-U1-70k antibodies might be involved in the pathological mechanisms of psychiatric syndromes in SLE.

Species differences of macrophage very low-density-lipoprotein (VLDL) receptor protein expression.

Triglyceride-rich lipoproteins (TGRLs) and low-density-lipoprotein (LDL) cholesterol are independent risk factors for coronary artery disease. We have previously proposed that the very low-density-lipoprotein (VLDL) receptor is one of the receptors required for foam cell formation by TGRLs in human macrophages. However, the VLDL receptor proteins have not been detected in atherosclerotic lesions of several animal models. Here we showed no VLDL receptor protein was detected in mouse macrophage cell lines (Raw264.7 and J774.2) or in mouse peritoneal macrophages in vitro. Furthermore, no VLDL receptor protein was detected in macrophages in atherosclerotic lesions of chow-fed apolipoprotein E-deficient or cholesterol-fed LDL receptor-deficient mice in vivo. In contrast, macrophage VLDL receptor protein was clearly detected in human macrophages in vitro and in atherosclerotic lesions in myocardial infarction-prone Watanabe-heritable hyperlipidemic (WHHLMI) rabbits in vivo. There are species differences in the localization of VLDL receptor protein in vitro and in vivo. Since VLDL receptor is expressed on macrophages in atheromatous plaques of both rabbit and human but not in mouse models, the mechanisms of atherogenesis and/or growth of atherosclerotic lesions in mouse models may be partly different from those of humans and rabbits.

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.

Changes in cardiac lipid metabolism during sepsis: the essential role of very low-density lipoprotein receptors.

OBJECTIVE: Sepsis accompanies myocardial dysfunction and dynamic alterations of cardiac metabolism. We have recently demonstrated that the very low-density lipoprotein receptor (VLDL-R), which is abundantly expressed in the heart, plays a key role in energy metabolism of the fasting heart. However, little is known about the function and regulation of the VLDL-R during sepsis. In the present study, we explored lipid accumulation and VLDL-R expression in the lipopolysaccharide (LPS)-stimulated heart in vivo and regulation of VLDL-R expression in vitro. METHODS AND RESULTS: Electron microscopy and immunohistochemistry demonstrated that LPS significantly decreased both lipid accumulation and VLDL-R expression in the hearts of fasting mice. Treatment with LPS also downregulated VLDL-R in rat neonatal cardiac myocytes, and this downregulation was completely reversed by interleukin (IL)-1beta receptor antagonist. IL-1beta downregulated the expression of VLDL-R in a time- and dose-dependent manner and markedly reduced the uptake of DiI-labeled beta-VLDL but not DiI-labeled low-density lipoprotein (LDL). Use of specific pharmacologic inhibitors and short interference RNA revealed that Hsp90 was required for IL-1beta to downregulate VLDL-R expression. CONCLUSIONS: These findings suggest that IL-1beta is a principle mediator of changes in cardiac lipid and energy metabolism during sepsis through the downregulation of myocardial VLDL-R expression.

Serum apolipoprotein j in health, coronary heart disease and type 2 diabetes mellitus.

Apolipoprotein (apo) J, clusterin, is ubiquitously expressed in many tissues, and is a component of high-density lipoproteins (HDLs). There is experimental evidence that it may be anti-atherogenic through its effects on cholesterol transport, smooth muscle cell proliferation and lipid peroxidation. HDLs containing apo J and apo A-I carry paraoxonase (PON1), which protects low-density lipoproteins from oxidative modification; however, the extent to which apo J affects coronary heart disease (CHD) is not known. We have developed a sandwich ELISA that enables apo J to be assayed in the range of 13-200 microg/mL. Serum apo J was 52.8+/-0.8 microg/mL (mean+/-SEM; range, 36.0-84.3 microg/mL; n=92) in healthy Japanese men, and 49.3+/-0.5 microg/mL (34.5-72.8; n=241) in healthy Japanese women. Multiple regression of these data and results from 67 men with CHD showed that apo J concentration was unrelated to age, sex or body mass index, but was positively related to serum PON1 (p<0.001) and apo B (p<0.02) concentrations. In women, it was also positively related to blood glucose (p<0.02). After adjusting for its associations with covariates, serum apo J averaged 5.4 microg/mL, lower in CHD men than in controls (p<0.003). Type 2 diabetics had higher apo J concentrations (men, 83.1+/-3.4 microg/mL, n=64; women, 64.0+/-2.3 microg/mL, n=46) than healthy men and women (p<0.001). In these Type 2 diabetics, apo J concentration was unrelated to PON1 concentration, but was positively related to blood glucose (p<0.01). After adjustment for its relation to blood glucose, the mean apo J concentration was similar in diabetics and healthy subjects. These findings suggest that apo J may be anti-atherogenic in humans, and that its concentration is raised by Type 2 diabetes.

Effects of Statin Therapy on Plasma Proprotein Convertase Subtilisin/kexin Type 9 and Sortilin Levels in Statin-Naive Patients with Coronary Artery Disease.

AIM: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of serum low-density lipoprotein (LDL) cholesterol levels, and sortilin is linked to lipoprotein metabolism. Although statin therapy increases PCSK9 levels, effects of this therapy on plasma sortilin levels have not been evaluated. The purpose of the present study was to examine the effects of statins on plasma PCSK9 and sortilin levels, and association of statin-induced increase in PCSK9 levels with sortilin. METHODS: Serum lipid levels and plasma PCSK9 and sortilin levels were measured at baseline and 8 months after statin therapy in 90 statin-naive patients with coronary artery disease (CAD). Pitavastatin 4 mg/day was used to treat 44 patients and pravastatin 20 mg/day to treat the remaining 46 patients. RESULTS: For both statin groups, significant increases in hetero-dimer PCSK9 levels (pitavastatin: 31%, p＜0.0001; pravastatin: 34%, p=0.03) and decreases in sortilin levels (pitavastatin: －8%, p=0.02; pravastatin: －16%, p=0.002) were observed. Although a reduction in LDL cholesterol was greater in the pitavastatin group than in the pravastatin group, no significant differences were observed in percentage changes in hetero-dimer PCSK9 and sortilin levels. A significant positive correlation was observed between percentage changes in hetero-dimer PCSK9 levels and those in sortilin levels (pitavastatin: r=0.359, p=0.02; pravastatin: r=0.276, p=0.06). CONCLUSIONS: Use of pitavastatin and pravastatin increased plasma PCSK9 and decreased sortilin levels. Statin-induced increases in PCSK9 were associated with changes in sortilin in statin-naive patients with CAD.

Soluble sortilin is released by activated platelets and its circulating levels are associated with cardiovascular risk factors.

OBJECTIVE: Sortilin is involved multilaterally in the development of atherosclerosis. Here, we examine the release of soluble sortilin (sSortilin) from platelets and assess the association between circulating levels of sSoritlin and atherothrombosis such as coronary artery disease (CAD). METHODS AND RESULTS: sSortilin levels measured in healthy subjects were higher in serum than in plasma (38.4 ± 8.7 vs. 15.8 ± 2.9 ng/mL; p < 0.0001). Platelets were shown to contain both membrane-bound sortilin and its soluble form lacking the cytoplasmic tail. Stimulation of platelet-rich plasma with collagen induced sSortilin release concomitantly with platelet aggregation, and the release was suppressed by aspirin. In clinical evaluation, plasma sSortilin was detected at significantly higher levels in cardiovascular risk patients with hypertension, dyslipidemia, and/or diabetes without CAD (non-CAD, 18.7 ± 3.3 ng/mL) than in patients with CAD under aspirin therapy (17.1 ± 3.6 ng/mL; p < 0.01) or in healthy controls (16.8 ± 2.9 ng/mL; p < 0.01). In these patients, plasma sSortilin levels were significantly correlated with platelet counts (rs = 0.33; p = 0.0085) and showed significant positive associations with cardiovascular risk factors: low-density lipoprotein cholesterol (rs = 0.37; p = 0.0023), triglycerides (rs = 0.28; p = 0.023), and serum uric acid (rs = 0.30; p = 0.017) in non-CAD, and γ-glutamyltransferase (rs = 0.43; p = 0.020) and high-sensitivity C-reactive protein (rs = 0.33, p = 0.0022) in CAD. CONCLUSION: Elevated plasma sSortilin levels may be associated with in vivo platelet activation and could be a risk factor for atherothrombosis.

Deficiency of the very low-density lipoprotein (VLDL) receptors in streptozotocin-induced diabetic rats: insulin dependency of the VLDL receptor.

Hyperlipidemia is a common feature of diabetes and is related to cardiovascular disease. The very low-density lipoprotein receptor (VLDL-R) is a member of the low-density lipoprotein receptor (LDL-R) family. It binds and internalizes triglyceride-rich lipoproteins with high specificity. We examined the etiology of hyperlipidemia in the insulin-deficient state. VLDL-R expression in heart and skeletal muscle were measured in rats with streptozotocin (STZ)-induced diabetes. STZ rats showed severe hyperlipidemia on d 21 and 28, with a dramatic decline in VLDL-R protein in skeletal muscle (>90%), heart (approximately 50%) and a loss of adipose tissues itself on d 28. The reduction of VLDL-R protein in skeletal muscle could not be explained simply by a decrease at the transcriptional level, because a dissociation between VLDL-R protein and mRNA expression was observed. The expression of LDL-R and LDL-R-related protein in liver showed no consistent changes. Furthermore, no effect on VLDL-triglyceride production in liver was observed in STZ rats. A decrease in postheparin plasma lipoprotein lipase activity started on d 7 and continued to d 28 at the 50% level even though severe hyperlipidemia was detected only on d 21 and 28. In rat myoblast cells, serum deprivation for 24 h induced a reduction in VLDL-R proteins. Insulin (10(-6) m), but not IGF-I (10 ng/ml), restored the decreased VLDL-R proteins by serum deprivation. These results suggest that the combination of VLDL-R deficiency and reduced plasma lipoprotein lipase activity may be responsible for severe hyperlipidemia in insulin-deficient diabetes.

Effects of intravenous apolipoprotein A-I/phosphatidylcholine discs on paraoxonase and platelet-activating factor acetylhydrolase in human plasma and tissue fluid.

We have previously shown that intravenous apolipoprotein (apo) A-I/phosphatidylcholine (apo A-I/PC) discs increase plasma high-density lipoprotein (HDL) concentration in humans. We have now studied the associated changes in two enzymes, paraoxonase (PON) and platelet-activating factor acetylhydrolase (PAF-AH) that are carried in whole or in part by HDLs, and are thought to influence atherogenesis by hydrolyzing oxidized phospholipids in lipoproteins. Apo A-I/PC discs (40 mg/kg over 4 h) were infused into eight healthy males. Although plasma apo A-I and HDL cholesterol increased on average by 178 and 158%, respectively, plasma total PON and total PAF-AH concentrations did not rise. By the end of the infusion, HDL-associated PAF-AH had increased by 0.56 +/- 0.14 microg/mL (mean +/- S.D., P < 0.01), and nonHDL-associated PAF-AH had decreased by 0.84 +/- 0.11 microg/mL (P < 0.05). These changes were accompanied by an increase in the HDL-associated PAF-AH/apo A-I ratio from 0.19 to 0.35 (P < 0.05), and by a decrease in the nonHDL-associated PAF-AH/apo B ratio from 2.1 to 1.4 (P < 0.05). No changes in PON or PAF-AH concentrations were detected in prenodal lymph (tissue fluid), collected continuously from the leg. Our results show that the total concentrations of PON and PAF-AH in plasma are uninfluenced by plasma HDL concentration. PAF-AH transfers readily between HDLs and LDLs in vivo, and its distribution between them is determined partly by their relative concentrations and partly by HDL composition.

Apolipoprotein E receptor 2 is involved in neuritic plaque formation in APP sw mice.

Apolipoprotein E receptor 2 (apoER2) is a receptor for apolipoprotein E containing lipoprotein and also for Reelin. Apolipoprotein E-associated risk of developing Alzheimer's disease (AD) may be related to its binding to and clearance by cell surface receptors, including members of the low-density lipoprotein receptor family. Otherwise there is circumstantial evidence that the Reelin signaling pathway may contribute to neurodegeneration in AD. To investigate the role of apoER2 on amyloid deposition and neurodegeneration in vivo, we examined the presence of apoER2 in the brains of APP sw transgenic mice (Tg2576) using three apoER2 monoclonal antibodies. Our immunohistochemical study revealed that apoER2 was localized in fine granular structure and reactive astrocytes surrounding amyloid plaques. The double labeling immunohistochemistry revealed that this granular structure overlaps synaptophysin-positive dystrophic neurites. These findings indicate that neuronal apoER2 may play a role for amyloid deposition and neuronal degeneration in AD.

Comparison of effects of pitavastatin versus pravastatin on serum proprotein convertase subtilisin/kexin type 9 levels in statin-naive patients with coronary artery disease.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of serum low-density lipoprotein cholesterol levels. Although statins increase serum PCSK9 levels, the effects of different types of statins on the serum PCSK9 levels have not been examined in detail. The purpose of the present study was to compare the effects of pitavastatin versus pravastatin on the serum PCSK9 levels. A total of 164 patients with coronary artery disease who were not receiving lipid-lowering therapy were randomly assigned to receive either 4 mg/day of pitavastatin (intensive lipid-lowering therapy) or 20 mg/day of pravastatin (moderate lipid-lowering therapy). The serum PCSK9 levels were measured before statin treatment and 8 months after therapy. A significantly greater reduction in low-density lipoprotein cholesterol was observed in the pitavastatin group (-41% vs -28%, p = 0.0001). The serum levels of total PCSK9 and heterodimer PCSK9 significantly increased from 192 to 249 ng/ml (37%, p <0.0001) and 147 to 206 ng/ml (78%, p <0.0001) in the pitavastatin group and from 192 to 249 ng/ml (39%, p <0.0001) and 143 to 201 ng/ml (65%, p <0.0001) in the pravastatin group, respectively. The increase in total and heterodimer PSCK9 did not differ between the 2 groups. No significant correlations were found between the percentage of changes in heterodimer PCSK9 and changes in the various lipid parameters in either group. In conclusion, significant increases in the total and heterodimer PSCK9 levels were observed at 8 months after treatment with pitavastatin and pravastatin; however, these increases did not differ between the 2 statins.

Clinical significance of plasma apolipoprotein F in Japanese healthy and hypertriglyceridemic subjects.

AIM: Apolipoprotein F (apo F), also known as lipid transfer inhibitory protein (LTIP), is a protein component of plasma lipoprotein classes including HDL and functions to inhibit lipid transfer between lipoproteins in vitro. To study the role of plasma apo F, a reliable and sensitive tool for the quantification would be needed. METHODS: We have developed a sandwich ELISA using two monoclonal antibodies for human plasma apo F, and analyzed apo F concentration in 397 Japanese healthy and 221 hypertriglyceridemic subjects. RESULTS: Our ELISA enables apo F to be assayed in the range of 0.6-25 µg/mL with intra- and inter-assay coefficients of variation less than 3.8% and 7.8%, respectively. In healthy subjects, plasma apo F concentration was 12.5±2.9 µg/mL (mean±SD), and was significantly higher in females than in males (p<0.05). By linear regression analysis in healthy subjects, plasma apo F concentration correlated positively with HDL cholesterol and apo A-I levels, and in males but not in females, negatively with apo B and triglyceride levels. It also correlated negatively with intrinsic CETP activity measured using intrinsic apo B-containing lipoprotein as an acceptor, and positively with PLTP mass and apo J levels. Apo F concentration in hypertriglyceridemic patients (10.3±3.1 µg/mL) was lower than in healthy controls (p<0.0001) and correlated positively with PLTP mass. CONCLUSIONS: Our ELISA is reliable and sensitive for the quantification of plasma apo F concentration. This system can be applicable for clinical significance in lipoprotein metabolism and reverse cholesterol transport.

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 ß-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, ß-VLDL and LDL all bound to LDL receptor. Cold RLP competed efficiently with DiI-ß-VLDL; however, cold LDL competed weakly. In VLDL receptor transfectants, RLP and ß-VLDL bound to VLDL receptor, but not LDL. RLP bound to VLDL receptor with higher affinity than ß-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.

Functional protein delivery into neurons using polymeric nanoparticles.

An efficient route for delivering specific proteins and peptides into neurons could greatly accelerate the development of therapies for various diseases, especially those involving intracellular defects such as Parkinson disease. Here we report the novel use of polybutylcyanoacrylate nanoparticles for delivery of intact, functional proteins into neurons and neuronal cell lines. Uptake of these particles is primarily dependent on endocytosis via the low density lipoprotein receptor. The nanoparticles are rapidly turned over and display minimal toxicity to cultured neurons. Delivery of three different functional cargo proteins is demonstrated. When primary neuronal cultures are treated with recombinant Escherichia coli beta-galactosidase as nanoparticle cargo, persistent enzyme activity is measured beyond the period of nanoparticle degradation. Delivery of the small GTPase rhoG induces neurite outgrowth and differentiation in PC12 cells. Finally, a monoclonal antibody directed against synuclein is capable of interacting with endogenous alpha-synuclein in cultured neurons following delivery via nanoparticles. Polybutylcyanoacrylate nanoparticles are thus useful for intracellular protein delivery in vitro and have potential as carriers of therapeutic proteins for treatment of neuronal disorders in vivo.

Selective uptake and efflux of cholesteryl linoleate in LDL by macrophages expressing 12/15-lipoxygenase.

Oxidation of low density lipoprotein (LDL) is a critical step for atherogenesis, and the role of the 12/15-lipoxygenase (12/15-LOX) as well as LDL receptor-related protein (LRP) expressed in macrophages in this process has been suggested. The oxygenation of cholesteryl linoleate in LDL by mouse macrophage-like J774A.1 cells overexpressing 12/15-LOX was inhibited by an anti-LRP antibody but not by an anti-LDL receptor antibody. When the cells were incubated with LDL double-labeled by [3H]cholesteryl linoleate and [125I]apoB, association with the cells of [3H]cholesteryl linoleate expressed as LDL protein equivalent exceeded that of [125I]apoB, indicating selective uptake of [3H]cholesteryl linoleate from LDL to these cells. An anti-LRP antibody inhibited the selective uptake of [3H]cholesteryl ester by 62% and 81% with the 12/15-LOX-expressing cells and macrophages, respectively. Furthermore, addition of LDL to the culture medium of the [3H]cholesteryl linoleate-labeled 12/15-LOX-expressing cells increased the release of [3H]cholesteryl linoleate to the medium in LDL concentration- and time-dependent manners. The transport of [3H]cholesteryl linoleate from the cells to LDL was also inhibited by an anti-LRP antibody by 75%. These results strongly suggest that LRP contributes to the LDL oxidation by 12/15-LOX in macrophages by selective uptake and efflux of cholesteryl ester in the LDL particle.

Interleukin-1beta attenuates beta-very low-density lipoprotein uptake and its receptor expression in vascular smooth muscle cells.

The very low-density lipoprotein (VLDL) receptor is a member of the low-density lipoprotein (LDL) receptor gene family with distinct tissue distribution and function. VLDL receptors are also expressed in vascular smooth muscle cells (VSMCs) and have been shown to be upregulated in atherosclerotic lesions. In the present study, we examined the effects of interleukin-1beta (IL-1beta) on the uptake of betaVLDL and its receptor expression in rat VSMCs. IL-1beta downregulated expression of the VLDL receptor in a time and dose-dependent manner as shown by Western blotting, Northern blotting, and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Treatment with IL-1beta significantly reduced the uptake of beta-VLDL but not LDL in VSMCs. Use of specific pharmacologic inhibitors indicated that the tyrosine kinase inhibitors, herbimycin A and geldanamycin, completely reversed IL-1beta-induced downregulation of the VLDL receptor expression. Another tyrosine kinase inhibitor, genistein, the protein kinase C inhibitors, GF109203X and H7, the mitogen-activated protein (MAP) kinase inhibitors (MEK inhibitor PD098059 for [MEK] and SB203580 for p38-MAP kinase), and the protein kinase A inhibitor, KT5270 all had no effect on receptor expression. In addition, the c-Src specific inhibitor PP2 or adenoviral-mediated gene transfer of kinase inactive (KI)-c-Src failed to reverse IL-1beta-induced downregulation of VLDL receptor expression. These results indicate that IL-1beta attenuates uptake of VLDL through downregulation of its receptor in VSMCs, and that this downregulation is mediated through a benzoquinone ansamycin-dependent but c-Src-independent pathway.