Apolipoprotein Mimetic Peptides: Potential New Therapies for Cardiovascular Diseases.

Since the seminal breakthrough of treating diabetic patients with insulin in the 1920s, there has been great interest in developing other proteins and their peptide mimetics as therapies for a wide variety of other medical disorders. Currently, there are at least 60 different peptides that have been approved for human use and over 150 peptides that are in various stages of clinical development. Peptides mimetic of the major proteins on lipoproteins, namely apolipoproteins, have also been developed first as tools for understanding apolipoprotein structure and more recently as potential therapeutics. In this review, we discuss the biochemistry, peptide mimetics design and clinical trials for peptides based on apoA-I, apoE and apoC-II. We primarily focus on applications of peptide mimetics related to cardiovascular diseases. We conclude with a discussion on the limitations of peptides as therapeutic agents and the challenges that need to be overcome before apolipoprotein mimetic peptides can be developed into new drugs.

Effect of niacin monotherapy on high density lipoprotein composition and function.

BACKGROUND: Niacin has modest but overall favorable effects on plasma lipids by increasing high density lipoprotein cholesterol (HDL-C) and lowering triglycerides. Clinical trials, however, evaluating niacin therapy for prevention of cardiovascular outcomes have returned mixed results. Recent evidence suggests that the HDL proteome may be a better indicator of HDL's cardioprotective function than HDL-C. The objective of this study was to evaluate the effect of niacin monotherapy on HDL protein composition and function. METHODS: A 20-week investigational study was performed with 11 participants receiving extended-release niacin (target dose = 2 g/day) for 16-weeks followed by a 4-week washout period. HDL was isolated from participants at weeks: 0, 16, and 20. The HDL proteome was analyzed at each time point by mass spectrometry and relative protein quantification was performed by label-free precursor ion intensity measurement. RESULTS: In this cohort, niacin therapy had typical effects on routine clinical lipids (HDL-C + 16%, q < 0.01; LDL-C - 20%, q < 0.01; and triglyceride - 15%, q = 0.1). HDL proteomics revealed significant effects of niacin on 5 proteins: serum amyloid A (SAA), angiotensinogen (AGT), apolipoprotein A-II (APOA2), clusterin (CLUS), and apolipoprotein L1 (APOL1). SAA was the most prominently affected protein, increasing 3-fold in response to niacin (q = 0.008). Cholesterol efflux capacity was not significantly affected by niacin compared to baseline, however, stopping niacin resulted in a 9% increase in efflux (q < 0.05). Niacin did not impact HDL's ability to influence endothelial function. CONCLUSION: Extended-release niacin therapy, in the absence of other lipid-modifying medications, can increase HDL-associated SAA, an acute phase protein associated with HDL dysfunction.

LCAT protects against Lipoprotein-X formation in a murine model of drug-induced intrahepatic cholestasis.

Familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is a rare genetic disease characterized by low HDL-C levels, low plasma cholesterol esterification, and the formation of Lipoprotein-X (Lp-X), an abnormal cholesterol-rich lipoprotein particle. LCAT deficiency causes corneal opacities, normochromic normocytic anemia, and progressive renal disease due to Lp-X deposition in the glomeruli. Recombinant LCAT is being investigated as a potential therapy for this disorder. Several hepatic disorders, namely primary biliary cirrhosis, primary sclerosing cholangitis, cholestatic liver disease, and chronic alcoholism also develop Lp-X, which may contribute to the complications of these disorders. We aimed to test the hypothesis that an increase in plasma LCAT could prevent the formation of Lp-X in other diseases besides FLD. We generated a murine model of intrahepatic cholestasis in LCAT-deficient (KO), wild type (WT), and LCAT-transgenic (Tg) mice by gavaging mice with alpha-naphthylisothiocyanate (ANIT), a drug well known to induce intrahepatic cholestasis. Three days after the treatment, all mice developed hyperbilirubinemia and elevated liver function markers (ALT, AST, Alkaline Phosphatase). The presence of high levels of LCAT in the LCAT-Tg mice, however, prevented the formation of Lp-X and other plasma lipid abnormalities in WT and LCAT-KO mice. In addition, we demonstrated that multiple injections of recombinant human LCAT can prevent significant accumulation of Lp-X after ANIT treatment in WT mice. In summary, LCAT can protect against the formation of Lp-X in a murine model of cholestasis and thus recombinant LCAT could be a potential therapy to prevent the formation of Lp-X in other diseases besides FLD.

Apolipoprotein C-II Mimetic Peptide Promotes the Plasma Clearance of Triglyceride-Rich Lipid Emulsion and the Incorporation of Fatty Acids into Peripheral Tissues of Mice.

AIM: Plasma apolipoprotein C-II (apoC-II) activates lipoprotein lipase (LPL) and thus lowers plasma triglycerides (TG). We previously reported that a human apoC-II mimetic peptide (C-II-a) decreased plasma TG in apoC-II mutant mice, as well as in apoE-knockout mice. Because it is unknown what tissues take up free fatty acids (FFAs) released from TG after C-II-a peptide administration, we investigated in mice TG plasma clearance and tissue incorporation, using 3H-triolein as a tracer, with and without C-II-a treatment. METHODS AND RESULTS: Intralipid® fat emulsion was labeled with 3H-triolein and then mixed with or without C-II-a. Addition of the peptide did not alter mean particle size of the lipid emulsion particles (298 nm) but accelerated their plasma clearance. After intravenous injection into C57BL/6N mice, the plasma half-life of the 3H-triolein for control and C-II-a treated emulsions was 18.3 ± 2.2 min and 14.8 ± 0.1 min, respectively. In apoC-II mutant mice, the plasma half-life of 3H-triolein for injected control and C-II-a treated emulsions was 30.1 ± 0.1 min and 14.8 ± 0.1 min, respectively. C57BL/6N and apoC-II mutant mice at 120 minutes after the injection showed increased tissue incorporation of radioactivity in white adipose tissue when C-II-a treated emulsion was used. Higher radiolabeled uptake of lipids from C-II-a treated emulsion was also observed in the skeletal muscle of C57BL/6N mice only. In case of apoC-II mutant mice, decreased uptake of radioactive lipids was observed in the liver and kidney after addition of C-II-a to the lipid emulsion. CONCLUSIONS: C-II-a peptide promotes the plasma clearance of TG-rich lipid emulsions in wild type and apoC-II mutant mice and promotes the incorporation of fatty acids from TG in the lipid emulsions into specific peripheral tissues.

Apolipoprotein C-II: New findings related to genetics, biochemistry, and role in triglyceride metabolism.

Apolipoprotein C-II (apoC-II) is a small exchangeable apolipoprotein found on triglyceride-rich lipoproteins (TRL), such as chylomicrons (CM) and very low-density lipoproteins (VLDL), and on high-density lipoproteins (HDL), particularly during fasting. ApoC-II plays a critical role in TRL metabolism by acting as a cofactor of lipoprotein lipase (LPL), the main enzyme that hydrolyses plasma triglycerides (TG) on TRL. Here, we present an overview of the role of apoC-II in TG metabolism, emphasizing recent novel findings regarding its transcriptional regulation and biochemistry. We also review the 24 genetic mutations in the APOC2 gene reported to date that cause hypertriglyceridemia (HTG). Finally, we describe the clinical presentation of apoC-II deficiency and assess the current therapeutic approaches, as well as potential novel emerging therapies.

Human SR-BII mediates SAA uptake and contributes to SAA pro-inflammatory signaling in vitro and in vivo.

Serum amyloid A (SAA) is an acute phase protein with cytokine-like and chemotactic properties, that is markedly up-regulated during various inflammatory conditions. Several receptors, including FPRL-1, TLR2, TLR4, RAGE, class B scavenger receptors, SR-BI and CD36, have been identified as SAA receptors. This study provides new evidence that SR-BII, splice variant of SR-BI, could function as an SAA receptor mediating its uptake and pro-inflammatory signaling. The uptake of Alexa Fluor488 SAA was markedly (~3 fold) increased in hSR-BII-expressing HeLa cells when compared with mock-transfected cells. The levels of SAA-induced interleukin-8 secretion by hSR-BII-expressing HEK293 cells were also significantly (~3-3.5 fold) higher than those detected in control cells. Moderately enhanced levels of phosphorylation of all three mitogen-activated protein kinases, ERK1/2, and p38 and JNK, were observed in hSR-BII-expressing cells following SAA stimulation when compared with control wild type cells. Transgenic mice with pLiv-11-directed liver/kidney overexpression of hSR-BI or hSR-BII were used to assess the in vivo role of each receptor in SAA-induced pro-inflammatory response in these organs. Six hours after intraperitoneal SAA injection both groups of transgenic mice demonstrated markedly higher (~2-5-fold) expression levels of inflammatory mediators in the liver and kidney compared to wild type mice. Histological examinations of hepatic and renal tissue from SAA-treated mice revealed moderate level of damage in the liver of both transgenic but not in the wild type mice. Activities of plasma transaminases, biomarkers of liver injury, were also moderately higher in hSR-B transgenic mice when compared to wild type mice. Our findings identify hSR-BII as a functional SAA receptor that mediates SAA uptake and contributes to its pro-inflammatory signaling via the MAPKs-mediated signaling pathways.

Dietary α-cyclodextrin reduces atherosclerosis and modifies gut flora in apolipoprotein E-deficient mice.

SCOPE: α-Cyclodextrin (α-CD), a cyclic polymer of glucose, has been shown to lower plasma cholesterol in animals and humans; however, its effect on atherosclerosis has not been previously described. METHODS AND RESULTS: apoE-knockout mice were fed either low-fat diet (LFD; 5.2% fat, w/w), or Western high fat diet (21.2% fat) containing either no additions (WD), 1.5% α-CD (WDA); 1.5% ß-CD (WDB); or 1.5% oligofructose-enriched inulin (WDI). Although plasma lipids were similar after 11 weeks on the WD vs. WDA diets, aortic atherosclerotic lesions were 65% less in mice on WDA compared to WD (P < 0.05), and similar to mice fed the LFD. No effect on atherosclerosis was observed for the other WD supplemented diets. By RNA-seq analysis of 16S rRNA, addition of α-CD to the WD resulted in significantly decreased cecal bacterial counts in genera Clostridium and Turicibacterium, and significantly increased Dehalobacteriaceae. At family level, Comamonadaceae significantly increased and Peptostreptococcaceae showed a negative trend. Several of these bacterial count changes correlated negatively with % atherosclerotic lesion and were associated with increased cecum weight and decreased plasma cholesterol levels. CONCLUSION: Addition of α-CD to the diet of apoE-knockout mice decreases atherosclerosis and is associated with changes in the gut flora.

Randomized double blind clinical trial on the effect of oral α-cyclodextrin on serum lipids.

BACKGROUND: This single center, double-blinded, cross-over, placebo controlled clinical trial investigated the effect of oral α-cyclodextrin (α-CD), a soluble dietary fiber, on blood lipid and lipoprotein levels in healthy human subjects. α-CD, a cyclical polymer containing 6 glucose subunits, is currently sold as an over the counter food supplement and is also a common additive in many foods. α-CD forms a hydrophobic central cavity that binds lipids and has been shown in animal studies and in previous clinical trials to alter plasma lipid levels. METHODS: We screened for healthy subjects, males and females, between ages 18 to 75. Out of total 103 subjects interviewed, 75 subjects completed the study. Qualified individuals in each gender group were randomized into two groups in terms of which treatment arm they received first (placebo vs. α-CD, receiving 6 grams P.O. a day, for 12-14 weeks with a 7 day wash out between arms). The primary outcome variable, plasma total cholesterol, as well as other tests related to lipids and lipoprotein and glucose metabolism, were measured at baseline and at the end of each arm of the study. RESULTS: α-CD was well tolerated; no serious adverse events related to α-CD were observed. Approximately 8 % of the subjects on α-CD complained of minor gastrointestinal symptoms versus 3 % on placebo (p = 0.2). Small-LDL particle number decreased 10 % (p < 0.045) for subjects on α-CD versus placebo. Fasting plasma glucose (1.6 %, p < 0.05) and Insulin resistance index (11 %, p < 0.04) were also decreased when on α-CD versus placebo. CONCLUSION: α-CD treatment appears to be safe and well tolerated in healthy individuals and showed a modest reduction in small LDL particles, and an improvement in glucose related parameters. TRIAL REGISTRATION: NCT01131299.

Phosphodiesterase 3B (PDE3B) regulates NLRP3 inflammasome in adipose tissue.

Activation of inflammation in white adipose tissue (WAT), includes infiltration/expansion of WAT macrophages, contributes pathogenesis of obesity, insulin resistance, and metabolic syndrome. The inflammasome comprises an intracellular sensor (NLR), caspase-1 and the adaptor ASC. Inflammasome activation leads to maturation of caspase-1 and processing of IL1ß, contributing to many metabolic disorders and directing adipocytes to a more insulin-resistant phenotype. Ablation of PDE3B in WAT prevents inflammasome activation by reducing expression of NLRP3, caspase-1, ASC, AIM2, TNFα, IL1ß and proinflammatory genes. Following IP injection of lipopolysaccharide (LPS), serum levels of IL1ß and TNFα were reduced in PDE3B(-/-)mice compared to WT. Activation of signaling cascades, which mediate inflammasome responses, were modulated in PDE3B(-/-)mice WAT, including smad, NFAT, NFkB, and MAP kinases. Moreover, expression of chemokine CCL2, MCP-1 and its receptor CCR2, which play an important role in macrophage chemotaxis, were reduced in WAT of PDE3B(-/-)mice. In addition, atherosclerotic plaque formation was significantly reduced in the aorta of apoE(-/-)/PDE3B(-/-)and LDL-R(-/-)/PDE3B(-/-)mice compared to apoE(-/-)and LDL-R(-/-)mice, respectively. Obesity-induced changes in serum-cholesterol were blocked in PDE3B(-/-)mice. Collectively, these data establish a role for PDE3B in modulating inflammatory response, which may contribute to a reduced inflammatory state in adipose tissue.

Familial lecithin:cholesterol acyltransferase deficiency: First-in-human treatment with enzyme replacement.

BACKGROUND: Humans with familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) have extremely low or undetectable high-density lipoprotein cholesterol (HDL-C) levels and by early adulthood develop many manifestations of the disorder, including corneal opacities, anemia, and renal disease. OBJECTIVE: To determine if infusions of recombinant human LCAT (rhLCAT) could reverse the anemia, halt progression of renal disease, and normalize HDL in FLD. METHODS: rhLCAT (ACP-501) was infused intravenously over 1 hour on 3 occasions in a dose optimization phase (0.3, 3.0, and 9.0 mg/kg), then 3.0 or 9.0 mg/kg every 1 to 2 weeks for 7 months in a maintenance phase. Plasma lipoproteins, lipids, LCAT levels, and several measures of renal function and other clinical labs were monitored. RESULTS: LCAT concentration peaked at the end of each infusion and decreased to near baseline over 7 days. Renal function generally stabilized or improved and the anemia improved. After infusion, HDL-C rapidly increased, peaking near normal in 8 to 12 hours; analysis of HDL particles by various methods all revealed rapid sequential disappearance of preß-HDL and small α-4 HDL and appearance of normal α-HDL. Low-density lipoprotein cholesterol increased more slowly than HDL-C. Of note, triglyceride routinely decreased after meals after infusion, in contrast to the usual postprandial increase in the absence of rhLCAT infusion. CONCLUSIONS: rhLCAT infusions were well tolerated in this first-in-human study in FLD; the anemia improved, as did most parameters related to renal function in spite of advanced disease. Plasma lipids transiently normalized, and there was rapid sequential conversion of small preß-HDL particles to mature spherical α-HDL particles.

Human SR-BI and SR-BII Potentiate Lipopolysaccharide-Induced Inflammation and Acute Liver and Kidney Injury in Mice.

The class B scavenger receptors BI (SR-BI) and BII (SR-BII) are high-density lipoprotein receptors that recognize various pathogens, including bacteria and their products. It has been reported that SR-BI/II null mice are more sensitive than normal mice to endotoxin-induced inflammation and sepsis. Because the SR-BI/II knockout model demonstrates multiple immune and metabolic disorders, we investigated the role of each receptor in the LPS-induced inflammatory response and tissue damage using transgenic mice with pLiv-11-directed expression of human SR-BI (hSR-BI) or human SR-BII (hSR-BII). At 6 h after i.p. LPS injection, transgenic hSR-BI and hSR-BII mice demonstrated markedly higher serum levels of proinflammatory cytokines and 2- to 3-fold increased expression levels of inflammatory mediators in the liver and kidney, compared with wild-type (WT) mice. LPS-stimulated inducible NO synthase expression was 3- to 6-fold higher in the liver and kidney of both transgenic strains, although serum NO levels were similar in all mice. Despite the lower high-density lipoprotein plasma levels, both transgenic strains responded to LPS by a 5-fold increase of plasma corticosterone levels, which were only moderately lower than in WT animals. LPS treatment resulted in MAPK activation in tissues of all mice; however, the strongest response was detected for hepatic extracellular signal-regulated protein kinase 1 and 2 and kidney JNK of both transgenic mice. Histological examination of hepatic and renal tissue from LPS-challenged mice revealed more injury in hSR-BII, but not hSR-BI, transgenic mice versus WT controls. Our findings demonstrate that hSR-BII, and to a lesser extent hSR-BI, significantly increase LPS-induced inflammation and contribute to LPS-induced tissue injury in the liver and kidney, two major organs susceptible to LPS toxicity.

Low ambient oxygen prevents atherosclerosis.

Large population studies have shown that living at higher altitudes, which lowers ambient oxygen exposure, is associated with reduced cardiovascular disease mortality. However, hypoxia has also been reported to promote atherosclerosis by worsening lipid metabolism and inflammation. We sought to address these disparate reports by reducing the ambient oxygen exposure of ApoE-/- mice. We observed that long-term adaptation to 10% O2 (equivalent to oxygen content at ∼5000 m), compared to 21% O2 (room air at sea level), resulted in a marked decrease in aortic atherosclerosis in ApoE-/- mice. This effect was associated with increased expression of the anti-inflammatory cytokine interleukin-10 (IL-10), known to be anti-atherogenic and regulated by hypoxia-inducible transcription factor-1α (HIF-1α). Supporting these observations, ApoE-/- mice that were deficient in IL-10 (IL10-/- ApoE-/- double knockout) failed to show reduced atherosclerosis in 10% oxygen. Our study reveals a specific mechanism that can help explain the decreased prevalence of ischemic heart disease in populations living at high altitudes and identifies ambient oxygen exposure as a potential factor that could be modulated to alter pathogenesis. Key messages: Chronic low ambient oxygen exposure decreases atherosclerosis in mice. Anti-inflammatory cytokine IL-10 levels are increased by low ambient O2. This is consistent with the established role of HIF-1α in IL10 transactivation. Absence of IL-10 results in the loss of the anti-atherosclerosis effect of low O2. This mechanism may contribute to decreased atherosclerosis at high altitudes.

Dietary Intake Patterns Are Consistent Across Seasons in a Cohort of Healthy Adults in a Metropolitan Population.

BACKGROUND: Current literature provides conflicting data regarding seasonal variability in dietary intake. OBJECTIVE: Our aim was to examine seasonal variation in dietary intake in healthy adults from the metropolitan Washington, DC, area. DESIGN: This study utilized an observational cohort design. PARTICIPANTS/SETTING: Male and female healthy volunteers (n=103) between the ages of 18 and 75 years were recruited from the metropolitan Washington, DC, area to participate in a clinical study at the National Institutes of Health Clinical Center from February 2011 to June 2014. MAIN OUTCOME MEASURES: Three- to seven-day food records were collected from subjects (n=76) at three time points (12 to 15 weeks apart). Subjects were excluded from analysis (n=27) if they completed less than three time points. Food records were reviewed by nutrition staff, assigned to a season, and coded in Nutrient Data System for Research for energy, macronutrient, micronutrient, and food-group serving analysis. STATISTICAL ANALYSES: Multivariate general linear models were run on energy, macronutrient, micronutrient, and food-group intakes, while being adjusted for age, sex, race, and body mass index (calculated as kg/m(2)). RESULTS: Subjects had a mean±standard deviation body mass index of 25±3.9 and age of 34±12.4 years. Subject demographics were 71.1% white, 9.2% black/African American, 13.2% Asian, and 6.6% unknown race, with 44.7% males and 55.3% females. Mean intake of energy across seasons was 2,214.6±623.4 kcal with 17.3%±4.1%, 33.6%±5.5%, 46.6%±8.0%, and 2.7%±3.2% of calories from protein, fat, carbohydrate, and alcohol, respectively. Intakes of energy, macronutrients, micronutrients, and food groups did not differ between seasons. CONCLUSIONS: People living in the metropolitan Washington, DC, area did not exhibit seasonal variation in dietary intake. Therefore, when designing studies of nutrient intake in a metropolitan population, these findings suggest that investigators do not need to consider the season during which diet is examined.

Creation of Apolipoprotein C-II (ApoC-II) Mutant Mice and Correction of Their Hypertriglyceridemia with an ApoC-II Mimetic Peptide.

Apolipoprotein C-II (apoC-II) is a cofactor for lipoprotein lipase, a plasma enzyme that hydrolyzes triglycerides (TGs). ApoC-II deficiency in humans results in hypertriglyceridemia. We used zinc finger nucleases to create Apoc2 mutant mice to investigate the use of C-II-a, a short apoC-II mimetic peptide, as a therapy for apoC-II deficiency. Mutant mice produced a form of apoC-II with an uncleaved signal peptide that preferentially binds high-density lipoproteins (HDLs) due to a 3-amino acid deletion at the signal peptide cleavage site. Homozygous Apoc2 mutant mice had increased plasma TG (757.5 ± 281.2 mg/dl) and low HDL cholesterol (31.4 ± 14.7 mg/dl) compared with wild-type mice (TG, 55.9 ± 13.3 mg/dl; HDL cholesterol, 55.9 ± 14.3 mg/dl). TGs were found in light (density < 1.063 g/ml) lipoproteins in the size range of very-low-density lipoprotein and chylomicron remnants (40-200 nm). Intravenous injection of C-II-a (0.2, 1, and 5 µmol/kg) reduced plasma TG in a dose-dependent manner, with a maximum decrease of 90% occurring 30 minutes after the high dose. Plasma TG did not return to baseline until 48 hours later. Similar results were found with subcutaneous or intramuscular injections. Plasma half-life of C-II-a is 1.33 ± 0.72 hours, indicating that C-II-a only acutely activates lipolysis, and the sustained TG reduction is due to the relatively slow rate of new TG-rich lipoprotein synthesis. In summary, we describe a novel mouse model of apoC-II deficiency and show that an apoC-II mimetic peptide can reverse the hypertriglyceridemia in these mice, and thus could be a potential new therapy for apoC-II deficiency.

Safety and Tolerability of ACP-501, a Recombinant Human Lecithin:Cholesterol Acyltransferase, in a Phase 1 Single-Dose Escalation Study.

RATIONALE: Low high-density lipoprotein-cholesterol (HDL-C) in patients with coronary heart disease (CHD) may be caused by rate-limiting amounts of lecithin:cholesterol acyltransferase (LCAT). Raising LCAT may be beneficial for CHD, as well as for familial LCAT deficiency, a rare disorder of low HDL-C. OBJECTIVE: To determine safety and tolerability of recombinant human LCAT infusion in subjects with stable CHD and low HDL-C and its effect on plasma lipoproteins. METHODS AND RESULTS: A phase 1b, open-label, single-dose escalation study was conducted to evaluate safety, tolerability, pharmacokinetics, and pharmacodynamics of recombinant human LCAT (ACP-501). Four cohorts with stable CHD and low HDL-C were dosed (0.9, 3.0, 9.0, and 13.5 mg/kg, single 1-hour infusions) and followed up for 28 days. ACP-501 was well tolerated, and there were no serious adverse events. Plasma LCAT concentrations were dose-proportional, increased rapidly, and declined with an apparent terminal half-life of 42 hours. The 0.9-mg/kg dose did not significantly change HDL-C; however, 6 hours after doses of 3.0, 9.0, and 13.5 mg/kg, HDL-C was elevated by 6%, 36%, and 42%, respectively, and remained above baseline ≤4 days. Plasma cholesteryl esters followed a similar time course as HDL-C. ACP-501 infusion rapidly decreased small- and intermediate-sized HDL, whereas large HDL increased. Pre-ß-HDL also rapidly decreased and was undetectable ≤12 hours post ACP-501 infusion. CONCLUSIONS: ACP-501 has an acceptable safety profile after a single intravenous infusion. Lipid and lipoprotein changes indicate that recombinant human LCAT favorably alters HDL metabolism and support recombinant human LCAT use in future clinical trials in CHD and familial LCAT deficiency patients. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01554800.

Endothelial Expression of Scavenger Receptor Class B, Type I Protects against Development of Atherosclerosis in Mice.

The role of scavenger receptor class B, type I (SR-BI) in endothelial cells (EC) was examined in several novel transgenic mouse models expressing SR-BI in endothelium of mice with normal C57Bl6/N, apoE-KO, or Scarb1-KO backgrounds. Mice were also created expressing SR-BI exclusively in endothelium and liver. Endothelial expression of the Tie2-Scarb1 transgene had no significant effect on plasma lipoprotein levels in mice on a normal chow diet but on an atherogenic diet, significantly decreased plasma cholesterol levels, increased plasma HDL cholesterol (HDL-C) levels, and protected mice against atherosclerosis. In 8-month-old apoE-KO mice fed a normal chow diet, the Tie2-Scarb1 transgene decreased aortic lesions by 24%. Mice expressing SR-BI only in EC and liver had a 1.5 ± 0.1-fold increase in plasma cholesterol compared to mice synthesizing SR-BI only in liver. This elevation was due mostly to increased HDL-C. In EC culture studies, SR-BI was found to be present in both basolateral and apical membranes but greater cellular uptake of cholesterol from HDL was found in the basolateral compartment. In summary, enhanced expression of SR-BI in EC resulted in a less atherogenic lipoprotein profile and decreased atherosclerosis, suggesting a possible role for endothelial SR-BI in the flux of cholesterol across EC.

A cholesterol-lowering VLP vaccine that targets PCSK9.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secretory protein that controls cholesterol homeostasis by enhancing endosomal and lysosomal degradation of the low-density lipoprotein receptor (LDL-R). Mutations that cause increased activity of PCSK9 are associated with hypercholesterolemia, atherosclerosis and early cardiovascular disease (CVD), whereas individuals with loss-of-function mutations in PCSK9 are apparently healthy but are hypocholesterolemic and have a dramatically decreased risk of CVD. In this study, we generated virus-like particle (VLP)-based vaccines targeting PCSK9. Mice and macaques vaccinated with bacteriophage VLPs displaying PCSK9-derived peptides developed high titer IgG antibodies that bound to circulating PCSK9. Vaccination was associated with significant reductions in total cholesterol, free cholesterol, phospholipids, and triglycerides. A vaccine targeting PCSK9 may, therefore, be an attractive alternative to monoclonal antibody-based therapies.

Apoc2 loss-of-function zebrafish mutant as a genetic model of hyperlipidemia.

Apolipoprotein C-II (APOC2) is an obligatory activator of lipoprotein lipase. Human patients with APOC2 deficiency display severe hypertriglyceridemia while consuming a normal diet, often manifesting xanthomas, lipemia retinalis and pancreatitis. Hypertriglyceridemia is also an important risk factor for development of cardiovascular disease. Animal models to study hypertriglyceridemia are limited, with no Apoc2-knockout mouse reported. To develop a genetic model of hypertriglyceridemia, we generated an apoc2 mutant zebrafish characterized by the loss of Apoc2 function. apoc2 mutants show decreased plasma lipase activity and display chylomicronemia and severe hypertriglyceridemia, which closely resemble the phenotype observed in human patients with APOC2 deficiency. The hypertriglyceridemia in apoc2 mutants is rescued by injection of plasma from wild-type zebrafish or by injection of a human APOC2 mimetic peptide. Consistent with a previous report of a transient apoc2 knockdown, apoc2 mutant larvae have a minor delay in yolk consumption and angiogenesis. Furthermore, apoc2 mutants fed a normal diet accumulate lipid and lipid-laden macrophages in the vasculature, which resemble early events in the development of human atherosclerotic lesions. In addition, apoc2 mutant embryos show ectopic overgrowth of pancreas. Taken together, our data suggest that the apoc2 mutant zebrafish is a robust and versatile animal model to study hypertriglyceridemia and the mechanisms involved in the pathogenesis of associated human diseases.