An apolipoprotein B antisense oligonucleotide lowers LDL cholesterol in hyperlipidemic mice without causing hepatic steatosis

High levels of plasma apolipoprotein B-100 (apoB-100), the principal apolipoprotein of LDL, are associated with cardiovascular disease. We hypothesized that suppression of apoB-100 mRNA by an antisense oligonucleotide (ASO) would reduce LDL cholesterol (LDL-C). Because most of the plasma apoB is made in the liver, and antisense drugs distribute to that organ, we tested the effects of a mouse-specific apoB-100 ASO in several mouse models of hyperlipidemia, including C57BL/6 mice fed a high-fat diet, Apoe-deficient mice, and Ldlr-deficient mice. The lead apoB-100 antisense compound, ISIS 147764, reduced apoB-100 mRNA levels in the liver and serum apoB-100 levels in a dose- and time-dependent manner. Consistent with those findings, total cholesterol and LDL-C decreased by 25-55% and 40-88%, respectively. Unlike small-molecule inhibitors of microsomal triglyceride transfer protein, ISIS 147764 did not produce hepatic or intestinal steatosis and did not affect dietary fat absorption or elevate plasma transaminase levels. These findings, as well as those derived from interim phase I data with a human apoB-100 antisense drug, suggest that antisense inhibition of this target may be a safe and effective approach for the treatment of humans with hyperlipidemia.     

Inhibition of ACAT by avasimibe decreases both VLDL and LDL apolipoprotein B production in miniature pigs.

An orally bioavailable acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitor, avasimibe (CI-1011), was used to test the hypothesis that inhibition of cholesterol esterification, in vivo, would reduce hepatic very low density (VLDL) apolipoprotein (apo) B secretion into plasma. ApoB kinetic studies were carried out in 10 control miniature pigs, and in 10 animals treated with avasimibe (10 mg/kg/d, n = 6; 25 mg/kg/d, n = 4). Pigs were fed a diet containing fat (34% of calories) and cholesterol (400 mg/d; 0.1%). Avasimibe decreased the plasma concentrations of total triglyceride, VLDL triglyceride, and VLDL cholesterol by 31;-40% 39-48%, and 31;-35%, respectively. Significant reductions in plasma total cholesterol (35%) and low density lipoprotein (LDL) cholesterol (51%) concentrations were observed only with high dose avasimibe. Autologous 131I-labeled VLDL, 125I-labeled LDL, and [3H]leucine were injected simultaneously into each pig and apoB kinetic data were analyzed using multicompartmental analysis (SAAM II). Avasimibe decreased the VLDL apoB pool size by 40;-43% and the hepatic secretion rate of VLDL apoB by 38;-41%, but did not alter its fractional catabolism. Avasimibe decreased the LDL apoB pool size by 13;-57%, largely due to a dose-dependent 25;-63% in the LDL apoB production rate. Hepatic LDL receptor mRNA abundances were unchanged, consistent with a marginal decrease in LDL apoB FCRs. Hepatic ACAT activity was decreased by 51% (P = 0.050) and 68% (P = 0.087) by low and high dose avasimibe, respectively. The decrease in total apoB secretion correlated with the decrease in hepatic ACAT activity (r = 0.495; P = 0.026).We conclude that inhibition of hepatic ACAT by avasimibe reduces both plasma VLDL and LDL apoB concentrations, primarily by decreasing apoB secretion.     

The peroxisome proliferator-activated receptor alpha (PPARalpha) agonist ciprofibrate inhibits apolipoprotein B mRNA editing in low density lipoprotein receptor-deficient mice: effects on plasma lipoproteins and the development of atherosclerotic lesions

Low density lipoprotein receptor (LDLR)-deficient mice fed a chow diet have a mild hypercholesterolemia caused by the abnormal accumulation in the plasma of apolipoprotein B (apoB)-100- and apoB-48-carrying intermediate density lipoproteins (IDL) and low density lipoproteins (LDL). Treatment of LDLR-deficient mice with ciprofibrate caused a marked decrease in plasma apoB-48-carrying IDL and LDL but at the same time caused a large accumulation of triglyceride-depleted apoB-100-carrying IDL and LDL, resulting in a significant increase in plasma cholesterol levels. These plasma lipoprotein changes were associated with an increase in the hepatic secretion of apoB-100-carrying very low density lipoproteins (VLDL) and a decrease in the secretion of apoB-48-carrying VLDL, accompanied by a significant decrease in hepatic apoB mRNA editing. Hepatic apobec-1 complementation factor mRNA and protein abundance were significantly decreased, whereas apobec-1 mRNA and protein abundance remained unchanged. No changes in apoB mRNA editing occurred in the intestine of the treated animals. After 150 days of treatment with ciprofibrate, consistent with the increased plasma accumulation of apoB-100-carrying IDL and LDL, the LDLR-deficient mice displayed severe atherosclerotic lesions in the aorta. These findings demonstrate that ciprofibrate treatment decreases hepatic apoB mRNA editing and alters the pattern of hepatic lipoprotein secretion toward apoB-100-associated VLDL, changes that in turn lead to increased atherosclerosis.     

In vivo contribution of LCAT to apolipoprotein B lipoprotein cholesteryl esters in LDL receptor and apolipoprotein E knockout mice

Previous studies have indicated that LCAT may play a role in the generation of cholesteryl esters (CE) in plasma apolipoprotein B (apoB) lipoproteins. The purpose of the present study was to examine the quantitative importance of LCAT on apoB lipoprotein CE fatty acid (CEFA) composition. LCAT(-/-) mice were crossed into the LDL receptor (LDLr)(-/-) and apoE(-/-) background to retard the clearance and increase the concentration of apoB lipoprotein in plasma. Plasma free cholesterol was significantly elevated but total and esterified cholesterol concentrations were not significantly affected by removal of functioning LCAT in either the LDLr(-/-) or apoE(-/-) mice consuming a chow diet. However, when functional LCAT was removed from LDLr(-/-) mice, the CEFA ratio (saturated + monounsaturated/polyunsaturated) of plasma LDL increased 7-fold because of a 2-fold increase in saturated and monounsaturated CE, a 40% reduction in cholesteryl linoleate, and a complete absence of long chain (>18 carbon) polyunsaturated CE (20:4, 20:5n-3, and 22:6n-3), from 29.3% to 0%. Removal of functional LCAT from apoE(-/-) mice resulted in only a 1.6-fold increase in the CEFA ratio, due primarily to a complete elimination of long chain CE (7.7% to 0%).Our results demonstrate that LCAT contributes significantly to the CEFA pool of apoB lipoprotein and is the only source of plasma long chain polyunsaturated CE in these mice.     

Comparison of the pharmacological profiles of murine antisense oligonucleotides targeting apolipoprotein B and microsomal triglyceride transfer protein

Therapeutic agents that suppress apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) levels/activity are being developed in the clinic to benefit patients who are unable to reach target LDL-C levels with maximally tolerated lipid-lowering drugs. To compare and contrast the metabolic consequences of reducing these targets, murine-specific apoB or MTP antisense oligonucleotides (ASOs) were administered to chow-fed and high fat-fed C57BL/6 or to chow-fed and Western diet-fed LDLr^−/− mice for periods ranging from 2 to 12 weeks, and detailed analyses of various factors affecting fatty acid metabolism were performed. Administration of these drugs significantly reduced target hepatic mRNA and protein, leading to similar reductions in hepatic VLDL/triglyceride secretion. MTP ASO treatment consistently led to increases in hepatic triglyceride accumulation and biomarkers of hepatotoxicity relative to apoB ASO due in part to enhanced expression of peroxisome proliferator activated receptor γ target genes and the inability to reduce hepatic fatty acid synthesis. Thus, although both drugs effectively lowered LDL-C levels in mice, the apoB ASO produced a more positive liver safety profile.     

Wild-type PCSK9 inhibits LDL clearance but does not affect apoB-containing lipoprotein production in mouse and cultured cells

Mutations in Proprotein Convertase Subtilisin Kexin 9 (PCSK9) have been associated with autosomal dominant hypercholesterolemia. In vivo kinetic studies indicate that LDL catabolism was impaired and apolipoprotein B (apoB)-containing lipoprotein synthesis was enhanced in two patients presenting with the S127R mutation on PCSK9. To understand the physiological role of PCSK9, we overexpressed human PCSK9 in mouse and cellular models as well as attenuated the endogenous expression of PCSK9 in HuH7 hepatoma cells using RNA interference. Here, we show that PCSK9 dramatically impairs the expression of the low density lipoprotein receptor (LDLr) and, in turn, LDL cellular binding as well as LDL clearance from the plasma compartment in C57BL6/J mice but not in LDLr-deficient mice, establishing a definitive role for PCSK9 in the modulation of the LDLr metabolic pathway. In contrast to data obtained in S127R-PCSK9 patients presenting with increased apoB production, our study indicates that wild-type PCSK9 does not significantly alter the production and/or secretion of VLDL apoB in either cultured cells or mice. Finally, we show that unlike PCSK9 overexpression in mice, the S127R mutation in patients led to increased VLDL apoB levels, suggesting a potential gain of function for S127R-PCSK9 in humans.     

siRNA-induced liver ApoB knockdown lowers serum LDL-cholesterol in a mouse model with human-like serum lipids

Increased serum apolipoprotein (apo)B and associated LDL levels are well-correlated with an increased risk of coronary disease. ApoE^–/– and low density lipoprotein receptor (LDLr)^–/– mice have been extensively used for studies of coronary atherosclerosis. These animals show atherosclerotic lesions similar to those in humans, but their serum lipids are low in apoB-containing LDL particles. We describe the development of a new mouse model with a human-like lipid profile. Ldlr CETP^+/– hemizygous mice carry a single copy of the human CETP transgene and a single copy of a LDL receptor mutation. To evaluate the apoB pathways in this mouse model, we used novel short-interfering RNAs (siRNA) formulated in lipid nanoparticles (LNP). ApoB siRNAs induced up to 95% reduction of liver ApoB mRNA and serum apoB protein, and a significant lowering of serum LDL in Ldlr CETP^+/– mice. ApoB targeting is specific and dose-dependent, and it shows lipid-lowering effects for over three weeks. Although specific triglycerides (TG) were affected by ApoB mRNA knockdown (KD) and the total plasma lipid levels were decreased by 70%, the overall lipid distribution did not change. Results presented here demonstrate a new mouse model for investigating additional targets within the ApoB pathways using the siRNA modality.     

Upregulation of hepatic LDL transport by n-3 fatty acids in LDL receptor knockout mice

We determined the effects of dietary n-6 and n-3 polyunsaturated fatty acids (PUFA) on parameters of plasma lipoprotein and hepatic lipid metabolism in LDL receptor (LDLr) knockout mice. Dietary n-3 PUFA decreased the rate of appearance and increased the hepatic clearance of IDL/LDL resulting in a marked decrease in the plasma concentration of these particles. Dietary n-3 PUFA increased the hepatic clearance of IDL/LDL through a mechanism that appears to involve apolipoprotein (apo)E but is independent of the LDLr, the LDLr related protein (LRP), the scavenger receptor B1, and the VLDLr. The decreased rate of appearance of IDL/VLDL in the plasma of animals fed n-3 PUFA could be attributed to a marked decrease in the plasma concentration of precursor VLDL. Decreased plasma VLDL concentrations were due in part to decreased hepatic secretion of VLDL triglyceride and cholesteryl esters, which in turn was associated with decreased concentrations of these lipids in liver. Decreased hepatic triglyceride concentrations in animals fed n-3 PUFA were due in part to suppression of fatty acid synthesis as a result of a decrease in sterol regulatory element binding protein-1 (SREBP-1) expression and processing. In conclusion, these studies indicate that n-3 PUFA can markedly decrease the plasma concentration of apoB-containing lipoproteins and enhance hepatic LDL clearance through a mechanism that does not involve the LDLr pathway or LRP.     

Effects of dietary fats and cholesterol on liver lipid content and hepatic apolipoprotein A-I, B, and E and LDL receptor mRNA levels in cebus monkeys.

The effects of the long-term administration of the dietary fats coconut oil and corn oil at 31% of calories with or without 0.1% (wt/wt) dietary cholesterol on plasma lipoproteins, apolipoproteins (apo), hepatic lipid content, and hepatic apoA-I, apoB, apoE, and low density lipoprotein (LDL) receptor mRNA abundance were examined in 27 cebus monkeys. Relative to the corn oil-fed animals, no significant differences were noted in any of the parameters of the corn oil plus cholesterol-fed group. In animals fed coconut oil without cholesterol, significantly higher (P less than 0.05) plasma total cholesterol (145%), very low density lipoprotein (VLDL) + LDL (201%) and high density lipoprotein (HDL) (123%) cholesterol, apoA-I (103%), apoB (61%), and liver cholesteryl ester (263%) and triglyceride (325%) levels were noted, with no significant differences in mRNA levels relative to the corn oil only group. In animals fed coconut oil plus cholesterol, all plasma parameters were significantly higher (P less than 0.05), as were hepatic triglyceride (563%) and liver apoA-I (123%) and apoB (87%) mRNA levels relative to the corn oil only group, while hepatic LDL receptor mRNA (-29%) levels were significantly lower (P less than 0.05). Correlation coefficient analyses performed on pooled data demonstrated that liver triglyceride content was positively associated (P less than 0.05) with liver apoA-I and apoB mRNA levels and negatively associated (P less than 0.01) with hepatic LDL receptor mRNA levels. Liver free and esterified cholesterol levels were positively correlated (P less than 0.05) with liver apoE mRNA levels and negatively correlated (P less than 0.025) with liver LDL receptor mRNA levels. Interestingly, while a significant correlation (P less than 0.01) was noted between hepatic apoA-I mRNA abundance and plasma apoA-I levels, no such relationship was observed between liver apoB mRNA and plasma apoB levels, suggesting that the hepatic mRNA of apoA-I, but not that of apoB, is a major determinant of the circulating levels of the respective apolipoprotein. Our data indicate that a diet high in saturated fat and cholesterol may increase the accumulation of triglyceride and cholesterol in the liver, each resulting in the suppression of hepatic LDL receptor mRNA levels. We hypothesize that such elevations in hepatic lipid content differentially alter hepatic apoprotein mRNA levels, with triglyceride increasing hepatic mRNA concentrations for apoA-I and B and cholesterol elevating hepatic apoE mRNA abundance.     

Peroxisome proliferator-activated receptor-α selective ligand reduces adiposity, improves insulin sensitivity and inhibits atherosclerosis in LDL receptor-deficient mice

Fenofibrate, a selective ¹PPAR-α activator, is prescribed to treat human dyslipidemia. The aim of this study was to delineate the mechanism of fenofibrate-mediated reductions in adiposity, improvements in insulin sensitivity, and lowering of triglycerides (TG) and free fatty acids (FFA) and to investigate if these favorable changes are related to the inhibition of lipid deposition in the aorta. To test this hypothesis we used male LDLr deficient mice that exhibit the clinical features of metabolic syndrome X when fed a high fat high cholesterol (HF) diet. LDLr deficient mice fed HF diet and simultaneously treated with fenofibrate (100 mg/kg body weight) prevented development of obesity, lowered serum triglycerides and cholesterol, improved insulin sensitivity, and prevented accumulation of lipids in the aorta. Lowering of circulating lipids occurred via down-regulation of lipogenic genes, including fatty acid synthase, acetyl CoA carboxylase and diacyl glycerol acyl transferase-2, concomitant with decreased liver TG and cholesterol, and TG output rate. Fenofibrate also suppressed liver apoCIII mRNA levels and markedly increased lipoprotein lipase mRNA levels, known to enhance serum TG catabolism. In addition, fenofibrate profoundly reduced epididymal fat and mesenteric fat mass to the levels seen in lean mice. The reductions in body weight were associated with elevation of hepatic uncoupling protein 2 (UCP2) mRNA, a concomitant increase in the ketone body formation, and improved insulin sensitivity associated with tumor necrosis factor-α reductions and phosphoenol pyruvate carboxykinase down-regulation. These results demonstrate that fenofibrate improves lipid abnormalities partly via inhibition of TG production and partly via clearance of TG-rich apoB particles by elevating LPL and reduced apoCIII. The prevention of obesity development occurred via energy expenditure. Fenofibrate-mediated hypolipidemic effects together with improved insulin sensitivity and loss of adiposity led to the reductions in the aortic lipid deposition by inhibiting early stages of atherosclerosis possibly via vascular cell adhesion molecule-1 (VCAM-1) modulation. These results suggest that potent PPAR-α activators may be useful in the treatment of syndrome X. (Mol Cell Biochem xxx: 1–16, 2005)     

Transforming growth factor-beta down-regulates apolipoprotein M in HepG2 cells

Apolipoprotein M (apoM) is a novel apolipoprotein presented mostly in high-density lipoprotein (HDL) in human plasma, and is exclusively expressed in liver and in kidney. The pathophysiological function of apoM has not yet been elucidated. Apolipoprotein B (apoB), the characteristic apolipoprotein of low-density lipoprotein (LDL), is like apoM, a very hydrophobic protein, and thereafter they both must co-circulate with lipoprotein particles in plasma. The cytokine, transforming growth factor-beta (TGF-beta), has been shown to decreased apoB secretion in HepG2 cells, and we hypothesized that TGF-beta may have the same effects on apoM expression in HepG2 cells. In the present study, we used real-time RT-PCR to analyze apoM and apoB mRNA levels during administration of TGF-beta, as well as TGF-alpha, epidermal growth factor (EGF) and hepatic growth factor (HGF). TGF-beta significantly inhibited both apoM and apoB mRNA expression in HepG2 cells. The inhibitory effects of TGF-beta were dose-dependent, i.e. 1 ng/ml of TGF-beta decreased apoM mRNA levels by 30%, and 10 or 100 ng/ml of TGF-beta decreased apoM mRNA levels more than 65%. The effect of TGF-beta on apoB mRNA expression was slightly weaker than that of apoM, with a maximum effect at 10 or 100 ng/ml TGF-beta where apoB mRNA levels decreased about 55%. The inhibitory effects of TGF-beta on apoM and apoB mRNA levels also increased with increasing incubation time, where the maximum effect was obtained at 24 h. Moreover TGF-alpha, EGF and HGF all decreased both apoM and apoB mRNA levels, but to a less extent than TGF-beta. Further, all four cytokines had more pronounced effects on apoM mRNA expression than apoB mRNA expression. The present study suggested that apoM, like apoB, may be involved in the hepatic lipoprotein assembly in vivo.     

Inhibition of both the apical sodium-dependent bile acid transporter and HMG-CoA reductase markedly enhances the clearance of LDL apoB

Discovery of the ileal apical sodium-dependent bile acid transporter (ASBT) permitted development of specific inhibitors of bile acid reabsorption, potentially a new class of cholesterol-lowering agents. In the present study, we tested the hypothesis that combining the novel ASBT inhibitor, SC-435, with the HMG-CoA reductase inhibitor, atorvastatin, would potentiate reductions in LDL cholesterol (LDL-C) and LDL apolipoprotein B (apoB). ApoB kinetic studies were performed in miniature pigs fed a typical human diet and treated with the combination of SC-435 (5 mg/kg/day) plus atorvastatin (3 mg/kg/day) (SC-435+A) or a placebo. SC-435+A decreased plasma total cholesterol by 23% and LDL-C by 40%. Multicompartmental analysis (SAAM II) demonstrated that LDL apoB significantly decreased by 35% due primarily to a 45% increase in the LDL apoB fractional catabolic rate (FCR). SC-435+A significantly decreased hepatic concentrations of free cholesterol and cholesteryl ester, and increased hepatic LDL receptor mRNA consequent to increased cholesterol 7alpha-hydroxylase expression and activity. In comparison, SC-435 (10 mg/kg/day) monotherapy decreased LDL apoB by 10% due entirely to an 18% increase in LDL apoB FCR, whereas atorvastatin monotherapy (3 mg/kg/day) decreased LDL apoB by 30% due primarily to a 22% reduction in LDL apoB production. We conclude that SC-435+A potentiates the reduction of LDL-C and LDL apoB due to complementary mechanisms of action.     

Stimulation of the in vivo production of very low density lipoproteins by apolipoprotein E is independent of the presence of the low density lipoprotein receptor

Apolipoprotein (apo) E stimulates the secretion of very low density lipoproteins (VLDLs) by an as yet unknown mechanism. Recently, a working mechanism for apoE was proposed (Twisk, J., Gillian-Daniel, D. L., Tebon, A., Wang, L., Barrett, P. H., and Attie, A. D. (2000) J. Clin. Invest. 105, 521-532) in which apoE prevents the inhibitory action of the low density lipoprotein receptor (LDLr) by binding to it. We have first tested whether this newly described effect of the LDLr on VLDL secretion, obtained in vitro, is also observed in vivo. In LDLr knockout mice (LDLr-/-), the production of VLDL triglycerides and apoB was 30% higher than that in controls. Also the ratio of apoB100:apoB48 secretion was increased in the LDLr-/- mice. The composition of nascent VLDL was similar in both strains. To test whether the action of apoE depends on the presence of the LDLr, VLDL production was measured in LDLr-/- and apoE-/- LDLr-/- mice. Deletion of apoE on a LDLr-/- background still caused a 50% decrease of VLDL triglycerides and apoB production. The composition of nascent VLDL was again similar for both strains. We conclude that the effect of apoE on hepatic VLDL production is independent of the presence of the LDLr.     

Studies on the expression of genes encoding apolipoproteins B100 and B48 and the low density lipoprotein receptor in nonhuman primates. Comparison of dietary fat and cholesterol

African green monkeys were fed diets containing low and moderate cholesterol concentrations with either polyunsaturated or unsaturated fat as 40% of calories. Plasma total cholesterol, low density lipoprotein (LDL) cholesterol, and apoB concentrations generally were higher in animals fed (a) the higher dietary cholesterol concentration and (b) saturated fat. At necropsy, liver and intestine were removed, and measurement of mRNAs for LDL receptors (liver) and for apolipoprotein B (liver and intestine) was done. Monkey small intestine mucosa made exclusively apoB48 while the liver made only apoB100, although apoB mRNA in both tissues was the same size (14 kilobases). No dietary cholesterol or fat effects were found for apoB mRNA abundance in the liver, while the animals fed the higher dietary cholesterol level had 50% lower levels of hepatic LDL receptor mRNA. In a separate group of animals, livers were perfused and the rate of apoB secretion was measured. No dietary fat effect on apoB secretion rate was found, and no relationship between plasma LDL cholesterol concentration and the rate of hepatic apoB production existed. These findings support the idea that the dietary factors that increase LDL concentrations act by reducing clearance of apoB-containing particles rather than by increasing production of these lipoproteins. Hepatic LDL receptor mRNA was similar in abundance in polyunsaturated fat and saturated fat-fed animals, suggesting that the difference in plasma cholesterol concentration between these groups is not mediated via effects on LDL receptor mRNA abundance. The level of intestinal apoB mRNA was about 30% higher in animals fed the moderate dietary cholesterol concentration. Earlier studies have shown that more cholesterol is transported in chylomicrons from the intestine when dietary cholesterol levels are higher, and the increased intestinal apoB mRNA abundance may reflect increased intestinal cholesterol transport and chylomicron apoB48 production.     

Human hepatic low-density lipoprotein receptors: associations of receptor activities in vitro with plasma lipid and apolipoprotein concentrations in vivo

The relationships of plasma lipid and apolipoprotein (apo) concentrations to hepatic low-density lipoprotein (LDL) receptor activity were examined in 21 subjects (16 females, 5 males), who were undergoing laparotomy for non-neoplastic disease (cholecystectomy in 16). None had familial hypercholesterolemia, or renal, endocrine or hepatic disease. Ages were 37-77 years (mean, 58 years), plasma cholesterol concentrations 4.09-6.72 mmol/l (5.38) and plasma triacylglycerol concentrations 0.75-2.35 mmol/l (1.36). Receptor activity was quantified in vitro as the total saturable binding and EDTA-suppressible binding (representing apoB,E receptors) of 125I-labelled human LDL (15 micrograms protein/ml) by liver homogenate at 37 degrees C. There were no significant differences between men and women in 125I-labeled LDL binding. In the pooled data, EDTA-suppressible binding averaged 50 ng 125I-LDL protein/mg cell protein (S.D., 15). Total saturable binding averaged 2-fold greater (mean, 101 ng/mg; S.D., 32). Plasma cholesterol, LDL cholesterol and apoB concentrations were negative functions of both EDTA-suppressible binding and total saturable binding, but the correlations with EDTA-suppressible binding were stronger (cholesterol: r = -0.59, P less than 0.01; LDL cholesterol: r = -0.48, P less than 0.05; apoB: r = -0.61, P less than 0.01). Plasma triacylglycerol, high-density lipoprotein cholesterol and apoA-I concentrations were not related to either measure of receptor activity. These results provide evidence that the activity of apoB,E receptors in the liver is a major determinant of the plasma LDL concentration in middle-aged and elderly humans.     

A PCSK9-binding antibody that structurally mimics the EGF(A) domain of LDL-receptor reduces LDL cholesterol in vivo

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) regulates LDL cholesterol levels by inhibiting LDL receptor (LDLr)-mediated cellular LDL uptake. We have identified a fragment antigen-binding (Fab) 1D05 which binds PCSK9 with nanomolar affinity. The fully human antibody 1D05-IgG2 completely blocks the inhibitory effects of wild-type PCSK9 and two gain-of-function human PCSK9 mutants, S127R and D374Y. The crystal structure of 1D05-Fab bound to PCSK9 reveals that 1D05-Fab binds to an epitope on the PCSK9 catalytic domain which includes the entire LDLr EGF(A) binding site. Notably, the 1D05-Fab CDR-H3 and CDR-H2 loops structurally mimic the EGF(A) domain of LDLr. In a transgenic mouse model (CETP/LDLr-hemi), in which plasma lipid and PCSK9 profiles are comparable to those of humans, 1D05-IgG2 reduces plasma LDL cholesterol to 40% and raises hepatic LDLr protein levels approximately fivefold. Similarly, in healthy rhesus monkeys, 1D05-IgG2 effectively reduced LDL cholesterol 20%-50% for over 2 weeks, despite its relatively short terminal half-life (t(1/2) = 3.2 days). Importantly, the decrease in circulating LDL cholesterol corresponds closely to the reduction in free PCSK9 levels. Together these results clearly demonstrate that the LDL-lowering effect of the neutralizing anti-PCSK9 1D05-IgG2 antibody is mediated by reducing the amount of PCSK9 that can bind to the LDLr.     

Effects of antisense-mediated inhibition of 11β-hydroxysteroid dehydrogenase type 1 on hepatic lipid metabolism

11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) converts inactive 11-keto derivatives to active glucocorticoids within tissues and may play a role in the metabolic syndrome (MS). We used an antisense oligonucleotide (ASO) to knock down 11β-HSD1 in livers of C57BL/6J mice consuming a Western-type diet (WTD). 11β-HSD1 ASO-treated mice consumed less food, so we compared them to ad libitum-fed mice and to food-matched mice receiving control ASO. Knockdown of 11β-HSD1 directly protected mice from WTD-induced steatosis and dyslipidemia by reducing synthesis and secretion of triglyceride (TG) and increasing hepatic fatty acid oxidation. These changes in hepatic and plasma lipids were not associated with reductions in genes involved in de novo lipogenesis. However, protein levels of both sterol regulatory element-binding protein (SREBP) 1 and fatty acid synthase were significantly reduced in mice treated with 11β-HSD1 ASO. There was no change in hepatic secretion of apolipoprotein (apo)B, indicating assembly and secretion of smaller apoB-containing lipoproteins by the liver in the 11β-HSD1-treated mice. Our results indicate that inhibition of 11β-HSD1 by ASO treatment of WTD-fed mice resulted in improved plasma and hepatic lipid levels, reduced lipogenesis by posttranslational regulation, and secretion of similar numbers of apoB-containing lipoproteins containing less TG per particle.