Monoacylglycerol Acyltransferase 1 Knockdown Exacerbates Hepatic Ischemia/Reperfusion Injury in Mice With Hepatic Steatosis.

Nonalcoholic fatty liver disease (NAFLD) is becoming the most common indication for liver transplantation. The growing prevalence of NAFLD not only increases the demand for liver transplantation, but it also limits the supply of available organs because steatosis predisposes grafts to ischemia/reperfusion injury (IRI) and many steatotic grafts are discarded. We have shown that monoacylglycerol acyltransferase (MGAT) 1, an enzyme that converts monoacylglycerol to diacylglycerol, is highly induced in animal models and patients with NAFLD and is an important mediator in NAFLD-related insulin resistance. Herein, we sought to determine whether Mogat1 (the gene encoding MGAT1) knockdown in mice with hepatic steatosis would reduce liver injury and improve liver regeneration following experimental IRI. Antisense oligonucleotides (ASO) were used to knockdown the expression of Mogat1 in a mouse model of NAFLD. Mice then underwent surgery to induce IRI. We found that Mogat1 knockdown reduced hepatic triacylglycerol accumulation, but it unexpectedly exacerbated liver injury and mortality following experimental ischemia/reperfusion surgery in mice on a high-fat diet. The increased liver injury was associated with robust effects on the hepatic transcriptome following IRI including enhanced expression of proinflammatory cytokines and chemokines and suppression of enzymes involved in intermediary metabolism. These transcriptional changes were accompanied by increased signs of oxidative stress and an impaired regenerative response. We have shown that Mogat1 knockdown in a mouse model of NAFLD exacerbates IRI and inflammation and prolongs injury resolution, suggesting that Mogat1 may be necessary for liver regeneration following IRI and that targeting this metabolic enzyme will not be an effective treatment to reduce steatosis-associated graft dysfunction or failure.

Hepatocyte-Macrophage Acetoacetate Shuttle Protects against Tissue Fibrosis.

Metabolic plasticity has been linked to polarized macrophage function, but mechanisms connecting specific fuels to tissue macrophage function remain unresolved. Here we apply a stable isotope tracing, mass spectrometry-based untargeted metabolomics approach to reveal the metabolome penetrated by hepatocyte-derived glucose and ketone bodies. In both classically and alternatively polarized macrophages, [13C]acetoacetate (AcAc) labeled ∼200 chemical features, but its reduced form D-[13C]ß-hydroxybutyrate (D-ßOHB) labeled almost none. [13C]glucose labeled ∼500 features, and while unlabeled AcAc competed with only ∼15% of them, the vast majority required the mitochondrial enzyme succinyl-coenzyme A-oxoacid transferase (SCOT). AcAc carbon labeled metabolites within the cytoplasmic glycosaminoglycan pathway, which regulates tissue fibrogenesis. Accordingly, livers of mice lacking SCOT in macrophages were predisposed to accelerated fibrogenesis. Exogenous AcAc, but not D-ßOHB, ameliorated diet-induced hepatic fibrosis. These data support a hepatocyte-macrophage ketone shuttle that segregates AcAc from D-ßOHB, coordinating the fibrogenic response to hepatic injury via mitochondrial metabolism in tissue macrophages.

γ-Secretase Inhibition Lowers Plasma Triglyceride-Rich Lipoproteins by Stabilizing the LDL Receptor.

Excess plasma triglycerides (TGs) are a key component of obesity-induced metabolic syndrome. We have shown that γ-secretase inhibitor (GSI) treatment improves glucose tolerance due to inhibition of hepatic Notch signaling but found additional Notch-independent reduction of plasma TG-rich lipoproteins (TRLs) in GSI-treated, as well as hepatocyte-specific, γ-secretase knockout (L-Ncst) mice, which suggested a primary effect on hepatocyte TRL uptake. Indeed, we found increased VLDL and LDL particle uptake in L-Ncst hepatocytes and Ncst-deficient hepatoma cells, in part through reduced γ-secretase-mediated low-density lipoprotein receptor (LDLR) cleavage and degradation. To exploit this novel finding, we generated a liver-selective Nicastrin ASO, which recapitulated glucose and lipid improvements of L-Ncst mice, with increased levels of hepatocyte LDLR. Collectively, these results identify the role of hepatic γ-secretase to regulate LDLR and suggest that liver-specific GSIs may simultaneously improve multiple aspects of the metabolic syndrome.

From the Cover: Development and Application of a Dual Rat and Human AHR Activation Assay.

Significant prolonged aryl hydrocarbon receptor (AHR) activation, classically exhibited following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin, can cause a variety of undesirable toxicological effects. Novel pharmaceutical chemistries also have the potential to cause activation of AHR and consequent toxicities in pre-clinical species and man. Previous methods either employed relatively expensive and low-throughput primary hepatocyte dosing with PCR endpoint, or low resolution overexpressing reporter gene assays. We have developed, validated and applied an in vitro microtitre plate imaging-based medium throughput screening assay for the assessment of endogenous species-specific AHR activation potential via detection of induction of the surrogate transcriptional target Cytochrome P450 CYP1A1. Routine testing of pharmaceutical drug development candidate chemistries using this assay can influence the chemical design process and highlight AHR liabilities. This assay should be introduced such that human AHR activation liability is flagged early for confirmatory testing.

Antisense oligonucleotides targeting translation inhibitory elements in 5' UTRs can selectively increase protein levels.

A variety of diseases are caused by deficiencies in amounts or activity of key proteins. An approach that increases the amount of a specific protein might be of therapeutic benefit. We reasoned that translation could be specifically enhanced using trans-acting agents that counter the function of negative regulatory elements present in the 5' UTRs of some mRNAs. We recently showed that translation can be enhanced by antisense oligonucleotides (ASOs) that target upstream open reading frames. Here we report the amount of a protein can also be selectively increased using ASOs designed to hybridize to other translation inhibitory elements in 5' UTRs. Levels of human RNASEH1, LDLR, and ACP1 and of mouse ACP1 and ARF1 were increased up to 2.7-fold in different cell types and species upon treatment with chemically modified ASOs targeting 5' UTR inhibitory regions in the mRNAs encoding these proteins. The activities of ASOs in enhancing translation were sequence and position dependent and required helicase activity. The ASOs appear to improve the recruitment of translation initiation factors to the target mRNA. Importantly, ASOs targeting ACP1 mRNA significantly increased the level of ACP1 protein in mice, suggesting that this approach has therapeutic and research potentials.

Blood Pressure Lowering and Safety Improvements With Liver Angiotensinogen Inhibition in Models of Hypertension and Kidney Injury.

Uncontrolled hypertension is an important contributor to cardiovascular disease. Despite the armamentarium of antihypertensive treatments, there remains a need for novel agents effective in individuals who cannot reach acceptable blood pressure levels. Inhibitors targeting the renin-angiotensin-aldosterone system (RAAS) are widely used but may not optimally inhibit RAAS and demonstrate an acceptable safety profile. Experiments were conducted to characterize a series of AGT (angiotensinogen) antisense oligonucleotides (ASOs) and compare their efficacy and tolerability to traditional RAAS blockade. AGT ASOs which target multiple systemic sites of AGT versus an N-acetylgalactosamine-conjugated AGT ASO that targets the liver were compared with captopril and losartan. Spontaneously hypertensive rats fed an 8% NaCl diet, a model of malignant hypertension resistant to standard RAAS inhibitors, demonstrated robust and durable blood pressure reductions with AGT ASO treatments, which was not observed with standard RAAS blockade. Studies in rat models of acute kidney injury produced by salt deprivation revealed kidney injury with ASO treatment that reduced kidney-expressed AGT, but not in animals treated with the N-acetylgalactosamine AGT ASO despite comparable plasma AGT reductions. Administration of either captopril or losartan also produced acute kidney injury during salt deprivation. Thus, intrarenal RAAS derived from kidney AGT, and inhibited by the standard of care, contributes to the maintenance of renal function during severe RAAS challenge. Such improvements in efficacy and tolerability by a liver-selective AGT inhibitor could be desirable in individuals not at their blood pressure goal with existing RAAS blockade.

Prevention of hepatic fibrosis with liver microsomal triglyceride transfer protein deletion in liver fatty acid binding protein null mice.

Blocking hepatic very low-density lipoprotein secretion through genetic or pharmacologic inhibition of microsomal triglyceride transfer protein (Mttp) causes hepatic steatosis, yet the risks for developing hepatic fibrosis are poorly understood. We report that liver-specific Mttp knockout mice (Mttp-LKO) exhibit both steatosis and fibrosis, which is exacerbated by a high-transfat/fructose diet. When crossed into germline liver fatty acid (FA) binding protein null mice (Mttp-LKO, i.e., double knockout mice) hepatic steatosis was greatly diminished and fibrosis prevented, on both low-fat and high-fat diets. The mechanisms underlying protection include reduced long chain FA uptake, shifts in FA distribution (lipidomic profiling), and metabolic turnover, specifically decreased hepatic 18:2 FA and triglyceride species and a shift in 18:2 FA use for oxidation versus incorporation into newly synthesized triglyceride. Double knockout mice were protected against fasting-induced hepatic steatosis (a model of enhanced exogenous FA delivery) yet developed steatosis upon induction of hepatic de novo lipogenesis with fructose feeding. Mttp-LKO mice, on either the liver FA binding protein null or Apobec-1 null background (i.e., apolipoprotein B100 only) exhibited only subtle increases in endoplasmic reticulum stress, suggesting that an altered unfolded protein response is unlikely to account for the attenuated phenotype in double knockout mice. Acute, antisense-mediated liver FA binding protein knockdown in Mttp-LKO mice also reduced FA uptake, increased oxidation versus incorporation of 18:2 species with complete reversal of hepatic steatosis, increased hepatic injury, and worsened fibrosis. CONCLUSION: Perturbing exogenous hepatic FA use modulates both hepatic steatosis and fibrosis in the setting of hepatic Mttp deletion, adding new insight into the pathophysiological mechanisms and consequences of defective very low-density lipoprotein secretion. (Hepatology 2017;65:836-852).

ApoC-III inhibits clearance of triglyceride-rich lipoproteins through LDL family receptors.

Hypertriglyceridemia is an independent risk factor for cardiovascular disease, and plasma triglycerides (TGs) correlate strongly with plasma apolipoprotein C-III (ApoC-III) levels. Antisense oligonucleotides (ASOs) for ApoC-III reduce plasma TGs in primates and mice, but the underlying mechanism of action remains controversial. We determined that a murine-specific ApoC-III-targeting ASO reduces fasting TG levels through a mechanism that is dependent on low-density lipoprotein receptors (LDLRs) and LDLR-related protein 1 (LRP1). ApoC-III ASO treatment lowered plasma TGs in mice lacking lipoprotein lipase (LPL), hepatic heparan sulfate proteoglycan (HSPG) receptors, LDLR, or LRP1 and in animals with combined deletion of the genes encoding HSPG receptors and LDLRs or LRP1. However, the ApoC-III ASO did not lower TG levels in mice lacking both LDLR and LRP1. LDLR and LRP1 were also required for ApoC-III ASO-induced reduction of plasma TGs in mice fed a high-fat diet, in postprandial clearance studies, and when ApoC-III-rich or ApoC-III-depleted lipoproteins were injected into mice. ASO reduction of ApoC-III had no effect on VLDL secretion, heparin-induced TG reduction, or uptake of lipids into heart and skeletal muscle. Our data indicate that ApoC-III inhibits turnover of TG-rich lipoproteins primarily through a hepatic clearance mechanism mediated by the LDLR/LRP1 axis.

Hypercholesterolemia Induced by a PCSK9 Gain-of-Function Mutation Augments Angiotensin II-Induced Abdominal Aortic Aneurysms in C57BL/6 Mice-Brief Report.

OBJECTIVE: Gain-of-function mutations of PCSK9 (proprotein convertase subtilisin/kexin type 9) lead to hypercholesterolemia. This study was to determine whether infection of normocholesterolemic mice with an adeno-associated viral (AAV) vector expressing a gain-of-function mutation of mouse PCSK9 increased angiotensin II (AngII)-induced abdominal aortic aneurysms. APPROACH AND RESULTS: In an initial study, male C57BL/6 mice were injected intraperitoneally with either an empty vector or PCSK9 gain-of-function mutation (D377Y). AAV at 3 doses and fed a saturated fat-enriched diet for 6 weeks. Two weeks after AAV injection, mice were infused with AngII for 4 weeks. Plasma PCSK9 concentrations were increased dose dependently in mice injected with AAV containing PCSK9D377Y mutation and positively associated with elevations of plasma cholesterol concentrations. Infection with intermediate and high doses of PCSK9D377Y.AAV led to equivalent increases of maximal width of abdominal aortas in C57BL/6 mice infused with AngII. Therefore, the intermediate dose was used in subsequent experiments. We then determined effects of PCSK9D377Y.AAV infection on 5 normolipidemic mouse strains, demonstrating that C57BL/6 mice were the most susceptible to this AAV infection. PCSK9D377Y.AAV infected male C57BL/6 mice were also compared with age-matched male low-density lipoprotein receptor(-/-) mice. Although plasma cholesterol concentrations were lower in mice infected with PCSK9D377Y.AAV, these mice had equivalent abdominal aortic aneurysmal formation, compared to low-density lipoprotein receptor(-/-) mice. In a separate study, reduced plasma PCSK9 concentrations by PCSK9 antisense oligonucleotides in male low-density lipoprotein receptor(-/-) mice did not influence AngII-induced abdominal aortic aneurysms. CONCLUSION: AAV-mediated infection with a mouse PCSK9 gain-of-function mutation is a rapid, easy, and efficient approach for inducing hypercholesterolemia and promoting abdominal aortic aneurysms in C57BL/6 mice infused with AngII.

Type 1 Deiodinase Regulates ApoA-I Gene Expression and ApoA-I Synthesis Independent of Thyroid Hormone Signaling.

OBJECTIVE: Plasma levels of high-density lipoprotein cholesterol (HDL-C) and apolipoprotein A-I (ApoA-I) are reduced in individuals with defective insulin signaling. Initial studies using liver-specific insulin receptor (InsR) knockout mice identified reduced expression of type 1 deiodinase (Dio1) as a potentially novel link between defective hepatic insulin signaling and reduced expression of the ApoA-I gene. Our objective was to examine the regulation of ApoA-I expression by Dio1. APPROACH AND RESULTS: Acute inactivation of InsR by adenoviral delivery of Cre recombinase to InsR floxed mice reduced HDL-C and expression of both ApoA-I and Dio1. Overexpression of Dio1 in InsR knockout mice restored HDL-C and ApoA-I levels and increased the expression of ApoA-I. Dio1 knockout mice had low expression of ApoA-I and reduced serum levels of HDL-C and ApoA-I. Treatment of C57BL/6J mice with antisense to Dio1 reduced ApoA-I mRNA, HDL-C, and serum ApoA-I. Hepatic 3,5,3'-triiodothyronine content was normal or elevated in InsR knockout mice or Dio1 knockout mice. Knockdown of either InsR or Dio1 by siRNA in HepG2 cells decreased the expression of ApoA-I and ApoA-I synthesis and secretion. siRNA knockdown of InsR or Dio1 decreased activity of a region of the ApoA-I promoter lacking thyroid hormone response elements (region B). Electrophoretic mobility shift assay demonstrated that reduced Dio1 expression decreased the binding of nuclear proteins to region B. CONCLUSIONS: Reductions in Dio1 expression reduce the expression of ApoA-I in a 3,5,3'-triiodothyronine-/thyroid hormone response element-independent manner.

Synergistic effect of phosphorothioate, 5'-vinylphosphonate and GalNAc modifications for enhancing activity of synthetic siRNA.

Chemical modifications are essential to improve metabolic stability and pharmacokinetic properties of siRNA to enable their systemic delivery. We investigated the effect of combing the phosphorothioate (PS) modification with metabolically stable phosphate analog (E)-5'-vinylphosphonate and GalNAc cluster conjugation on the activity of fully 2'-modified siRNA in cell culture and mice. Our data suggest that integrating multiple chemical approaches in one siRNA molecule improved potency 5-10 fold and provide a roadmap for developing more efficient siRNA drugs.

Angiotensinogen Exerts Effects Independent of Angiotensin II.

OBJECTIVE: This study determined whether angiotensinogen (AGT) has angiotensin II-independent effects using multiple genetic and pharmacological manipulations. APPROACH AND RESULTS: All study mice were in low-density lipoprotein receptor -/- background and fed a saturated fat-enriched diet. In mice with floxed alleles and a neomycin cassette in intron 2 of the AGT gene (hypoAGT mice), plasma AGT concentrations were >90% lower compared with their wild-type littermates. HypoAGT mice had lower systolic blood pressure, less atherosclerosis, and diminished body weight gain and liver steatosis. Low plasma AGT concentrations and all phenotypes were recapitulated in mice with hepatocyte-specific deficiency of AGT or pharmacological inhibition of AGT by antisense oligonucleotide administration. In contrast, inhibition of AGT cleavage by a renin inhibitor, aliskiren, failed to alter body weight gain and liver steatosis in low-density lipoprotein receptor -/- mice. In mice with established adiposity, administration of AGT antisense oligonucleotide versus aliskiren led to equivalent reductions of systolic blood pressure and atherosclerosis. AGT antisense oligonucleotide administration ceased body weight gain and further reduced body weight, whereas aliskiren did not affect body weight gain during continuous saturated fat-enriched diet feeding. Structural comparisons of AGT proteins in zebrafish, mouse, rat, and human revealed 4 highly conserved sequences within the des(angiotensin I)AGT domain. des(angiotensin I)AGT, through adeno-associated viral infection in hepatocyte-specific AGT-deficient mice, increased body weight gain and liver steatosis, but did not affect atherosclerosis. CONCLUSIONS: AGT contributes to body weight gain and liver steatosis through functions of the des(angiotensin I)AGT domain, which are independent of angiotensin II production.

Role of Insulin in the Regulation of Proprotein Convertase Subtilisin/Kexin Type 9.

OBJECTIVE: Proprotein convertase subtilisin/kexin type 9 (PCSK9), which binds the low-density lipoprotein receptor and targets it for degradation, has emerged as an important regulator of serum cholesterol levels and cardiovascular disease risk. Although much work is currently focused on developing therapies for inhibiting PCSK9, the endogenous regulation of PCSK9, particularly by insulin, remains unclear. The objective of these studies was to determine the effects of insulin on PCSK9 in vitro and in vivo. APPROACH AND RESULTS: Using rat hepatoma cells and primary rat hepatocytes, we found that insulin increased PCSK9 expression and increased low-density lipoprotein receptor degradation in a PCSK9-dependent manner. In parallel, hepatic Pcsk9 mRNA and plasma PCSK9 protein levels were reduced by 55% to 75% in mice with liver-specific knockout of the insulin receptor; 75% to 88% in mice made insulin-deficient with streptozotocin; and 65% in ob/ob mice treated with antisense oligonucleotides against the insulin receptor. However, antisense oligonucleotide-mediated knockdown of insulin receptor in lean, wild-type mice had little effect. In addition, we found that fasting was able to reduce PCSK9 expression by 80% even in mice that lack hepatic insulin signaling. CONCLUSIONS: Taken together, these data indicate that although insulin induces PCSK9 expression, it is not the sole or even dominant regulator of PCSK9 under all conditions.

Flavin-containing monooxygenase 3 as a potential player in diabetes-associated atherosclerosis.

Despite the well-documented association between insulin resistance and cardiovascular disease, the key targets of insulin relevant to the development of cardiovascular disease are not known. Here, using non-biased profiling methods, we identify the enzyme flavin-containing monooxygenase 3 (Fmo3) to be a target of insulin. FMO3 produces trimethylamine N-oxide (TMAO), which has recently been suggested to promote atherosclerosis in mice and humans. We show that FMO3 is suppressed by insulin in vitro, increased in obese/insulin resistant male mice and increased in obese/insulin-resistant humans. Knockdown of FMO3 in insulin-resistant mice suppresses FoxO1, a central node for metabolic control, and entirely prevents the development of hyperglycaemia, hyperlipidemia and atherosclerosis. Taken together, these data indicate that FMO3 is required for FoxO1 expression and the development of metabolic dysfunction.

MAFG is a transcriptional repressor of bile acid synthesis and metabolism.

Specific bile acids are potent signaling molecules that modulate metabolic pathways affecting lipid, glucose and bile acid homeostasis, and the microbiota. Bile acids are synthesized from cholesterol in the liver, and the key enzymes involved in bile acid synthesis (Cyp7a1, Cyp8b1) are regulated transcriptionally by the nuclear receptor FXR. We have identified an FXR-regulated pathway upstream of a transcriptional repressor that controls multiple bile acid metabolism genes. We identify MafG as an FXR target gene and show that hepatic MAFG overexpression represses genes of the bile acid synthetic pathway and modifies the biliary bile acid composition. In contrast, loss-of-function studies using MafG(+/-) mice causes de-repression of the same genes with concordant changes in biliary bile acid levels. Finally, we identify functional MafG response elements in bile acid metabolism genes using ChIP-seq analysis. Our studies identify a molecular mechanism for the complex feedback regulation of bile acid synthesis controlled by FXR.

Ketogenesis prevents diet-induced fatty liver injury and hyperglycemia.

Nonalcoholic fatty liver disease (NAFLD) spectrum disorders affect approximately 1 billion individuals worldwide. However, the drivers of progressive steatohepatitis remain incompletely defined. Ketogenesis can dispose of much of the fat that enters the liver, and dysfunction in this pathway could promote the development of NAFLD. Here, we evaluated mice lacking mitochondrial 3-hydroxymethylglutaryl CoA synthase (HMGCS2) to determine the role of ketogenesis in preventing diet-induced steatohepatitis. Antisense oligonucleotide-induced loss of HMGCS2 in chow-fed adult mice caused mild hyperglycemia, increased hepatic gluconeogenesis from pyruvate, and augmented production of hundreds of hepatic metabolites, a suite of which indicated activation of the de novo lipogenesis pathway. High-fat diet feeding of mice with insufficient ketogenesis resulted in extensive hepatocyte injury and inflammation, decreased glycemia, deranged hepatic TCA cycle intermediate concentrations, and impaired hepatic gluconeogenesis due to sequestration of free coenzyme A (CoASH). Supplementation of the CoASH precursors pantothenic acid and cysteine normalized TCA intermediates and gluconeogenesis in the livers of ketogenesis-insufficient animals. Together, these findings indicate that ketogenesis is a critical regulator of hepatic acyl-CoA metabolism, glucose metabolism, and TCA cycle function in the absorptive state and suggest that ketogenesis may modulate fatty liver disease.

Effects of an antisense oligonucleotide inhibitor of C-reactive protein synthesis on the endotoxin challenge response in healthy human male volunteers.

BACKGROUND: C-reactive protein (CRP) binds to damaged cells, activates the classical complement pathway, is elevated in multiple inflammatory conditions, and provides prognostic information on risk of future atherosclerotic events. It is controversial, however, as to whether inhibiting CRP synthesis would have any direct anti-inflammatory effects in humans. METHODS AND RESULTS: A placebo-controlled study was used to evaluate the effects of ISIS 329993 (ISIS-CRPR x) on the acute-phase response after endotoxin challenge in 30 evaluable subjects. Healthy adult males were randomly allocated to receive 6 injections over a 22-day period of placebo or active therapy with ISIS 329993 at 400- or 600-mg doses. Eligible subjects were subsequently challenged with a bolus of endotoxin (2 ng/kg). Inflammatory and hematological biomarkers were measured before and serially after the challenge. ISIS-CRPR x was well tolerated with no serious adverse events. Median CRP levels increased more than 50-fold from baseline 24 hours after endotoxin challenge in the placebo group. In contrast, the median increase in CRP levels was attenuated by 37% (400 mg) and 69% (600 mg) in subjects pretreated with ISIS-CRPR x (P<0.05 vs. placebo). All other aspects of the acute inflammatory response were similar between treatment groups. CONCLUSION: Pretreatment of subjects with ISIS-CRPR x selectively reduced the endotoxin-induced increase in CRP levels in a dose-dependent manner, without affecting other components of the acute-phase response. These data demonstrate the specificity of antisense oligonucleotides and provide an investigative tool to further define the role of CRP in human pathological conditions.

Modulation of lipoprotein metabolism by antisense technology: preclinical drug discovery methodology.

Antisense oligonucleotides (ASOs) are a new class of specific therapeutic agents that alter the intermediary metabolism of mRNA, resulting in the suppression of disease-associated gene products. ASOs exert their pharmacological effects after hybridizing, via Watson-Crick base pairing, to a specific target RNA. If appropriately designed, this event results in the recruitment of RNase H, the degradation of targeted mRNA or pre-mRNA, and subsequent inhibition of the synthesis of a specific protein. A key advantage of the technology is the ability to selectively inhibit targets that cannot be modulated by traditional therapeutics such as structural proteins, transcription factors, and, of topical interest, lipoproteins. In this chapter, we will first provide an overview of antisense technology, then more specifically describe the status of lipoprotein-related genes that have been studied using the antisense platform, and finally, outline the general methodology required to design and evaluate the in vitro and in vivo efficacy of those drugs.

Antisense oligonucleotide inhibition of cholesteryl ester transfer protein enhances RCT in hyperlipidemic, CETP transgenic, LDLr-/- mice.

Due to their ability to promote positive effects across all of the lipoprotein classes, cholesteryl ester transfer protein (CETP) inhibitors are currently being developed as therapeutic agents for cardiovascular disease. In these studies, we compared an antisense oligonucleotide (ASO) inhibitor of CETP to the CETP small molecule inhibitor anacetrapib. In hyperlipidemic CETP transgenic (tg) mice, both drugs provided comparable reductions in total plasma cholesterol, decreases in CETP activity, and increases in HDL cholesterol. However, only mice treated with the antisense inhibitor showed an enhanced effect on macrophage reverse cholesterol transport, presumably due to differences in HDL apolipoprotein composition and decreases in plasma triglyceride. Additionally, the ASO-mediated reductions in CETP mRNA were associated with less accumulation of aortic cholesterol. These preliminary findings suggest that CETP ASOs may represent an alternative means to inhibit that target and to support their continued development as a treatment for cardiovascular disease in man.

Endothelial lipase mediates HDL levels in normal and hyperlipidemic rabbits.

AIM: Existing evidence suggests that endothelial lipase (EL) plays an important role in high-densitylipoprotein (HDL) metabolism. Because rabbits are a useful animal model for the study of human lipid metabolism and atherosclerosis, we characterized rabbit EL (rEL) expression and investigated its relationship with plasma HDL levels in normal and hyperlipidemic rabbits. METHODS: We cloned the rEL cDNA and analyzed the EL tissue expression using Northern blotting, real-time RT-PCR, Western blotting, and in situ hybridization. We evaluated the effects of rEL antisense on plasma HDL levels. RESULTS: We found that rEL mRNA was highly expressed in cholesterol synthesis-related organs, including the liver, testis, and adrenal along with its expression in the lung, kidney, bone marrow, and small intestine. Interestingly, Watanabe heritable hyperlipidemic (WHHL) rabbits, a model of human familial hypercholesterolemia, had lower plasma levels of HDLs than normal rabbits. The plasma HDL levels in WHHL rabbits were inversely associated with high levels of plasma rEL proteins and hepatic expression of rEL mRNA. Injection of rEL-specific antisense oligonucleotides into rabbits resulted in the elevation of plasma large HDLs. Furthermore, we demonstrated that rEL mRNA was expressed by both endothelial cells and macrophages in the lesions of aortic atherosclerosis of WHHL rabbits. CONCLUSIONS: rEL is expressed in multiple tissues and may have many physiological and pathophysiological functions, such as in the regulation of cholesterol metabolism and atherosclerosis. Our results suggest that EL is an important regulator of plasma HDL levels in rabbits.