Andrew S. Plump

Increased Atherosclerosis in ApoE and LDL Receptor Gene Knock-Out Mice as a Result of Human Cholesteryl Ester Transfer Protein Transgene Expression

The plasma cholesteryl ester transfer protein (CETP) plays a major role in the catabolism of HDL cholesteryl ester (CE). CETP transgenic mice have decreased HDL cholesterol levels and have been reported to have either increased or decreased early atherosclerotic lesions. To evaluate the impact of CETP expression on more advanced forms of atherosclerosis, we have cross-bred the human CETP transgene into the apoE knock-out (apoE0) background with and without concomitant expression of the human apo A-I transgene. In this model the CETP transgene is induced to produce plasma CETP levels 5 to 10 times normal human levels. CETP expression resulted in moderately reduced HDL cholesterol (34%) in apoE0 mice and markedly reduced HDL cholesterol (76%) in apoE0/apoA1 transgenic mice. After injection of radiolabeled HDL CE, the CETP transgene significantly delayed the clearance of CE radioactivity from plasma in apoE0 mice, but accelerated the clearance in apoE0/apoA1 transgenic mice. ApoE0/CETP mice displayed an increase in mean atherosclerotic lesion area on the chow diet (approximately 2-fold after 2 to 4 months, and 1.4- to 1.6-fold after 7 months) compared with apoE0 mice (P<0.02). At 7 months apoA1 transgene expression resulted in a 3-fold reduction in mean lesion area in apoE0 mice (P<0.001). In the apoE0/apoA1 background, CETP produced an insignificant 1.3- to 1.7-fold increase in lesion area. In further studies the CETP transgene was bred onto the LDL receptor knock-out background (LDLR0). After 3 months on the Western diet, the mean lesion area was increased 1.8-fold (P<0.01) in LDLR0/CETP mice, compared with LDLR0 mice. These studies indicate that CETP expression leads to a moderate increase in atherosclerosis in apoE0 and LDLR0 mice, and suggest a proatherogenic effect of CETP activity in metabolic settings in which clearance of remnants or LDL is severely impaired. However, apoA1 overexpression has more dramatic protective effects on atherosclerosis in apoE0 mice, which are not significantly reversed by concomitant expression of CETP.

Niacin Lipid Efficacy Is Independent of Both the Niacin Receptor GPR109A and Free Fatty Acid Suppression

GPR109A is not the target mediating niacin’s lipid efficacy and the free fatty acid hypothesis does not explain niacin’s mechanism of action. Breaking Free of the “FFA Hypothesis” Free fatty acids (FFAs) appear in the blood plasma after a meal. Niacin—a vitamin that helps to regulate lipid levels in the body—is given to patients to reduce the amount of FFAs. It also works to raise “good” cholesterol [high-density lipoprotein (HDL)] and lower both “bad” cholesterol [low-density lipoprotein (LDL)] and triglycerides. The “FFA hypothesis” suggests that niacin works to exert these beneficial lipid effects by limiting the amount of FFAs available to synthesize triglycerides. Lauring, Taggart, and colleagues now challenge this long-standing theory. In studies in mice and humans, the authors debunk the hypothesis, showing that the effect on HDL, LDL, and triglycerides is not directly linked to FFAs. To study the lipid-modifying effects of niacin (nicotinic acid), Lauring et al. used a genetic, humanized mouse model lacking the LDL receptor. In these animals, niacin increased HDL cholesterol levels, as expected. Lack of GPR109A in these animals blocked the anti-lipolytic effect of nicotinic acid on FFAs but had no effect on drug-related changes in plasma HDL and LDL cholesterol or triglyceride levels. Treatment of the mice with a GPR109A agonist, MK-1903, also caused an anti-lipolytic effect but did not affect levels of triglyceride or LDL and HDL cholesterol. Together, these in vivo preclinical studies suggest that niacin works to lower FFAs through GPR109A but has an independent mechanism of action on other lipids. The authors addressed the role of GPR109A in humans by testing the effects of MK-1903 and of another synthetic GPR109A agonist in clinical trials. Both agonists affected FFA lipolysis but had only minor effects on HDL cholesterol and triglyceride levels in patients, thus mirroring results seen in animals and showing that niacin works independently of GPR109A to modify dyslipidemia. The studies by Lauring et al. point to a new, yet-uncovered mechanism of action for niacin’s beneficial effects on lipids in the blood. Despite overturning the FFA hypothesis and potentially redirecting drug development away from GPR109A agonists, niacin could still be useful for treating other diseases in patients, including atherosclerosis and inflammation, where GPR109A plays a major role in cell signaling. Nicotinic acid (niacin) induces beneficial changes in serum lipoproteins and has been associated with beneficial cardiovascular effects. Niacin reduces low-density lipoprotein, increases high-density lipoprotein, and decreases triglycerides. It is well established that activation of the seven-transmembrane Gi-coupled receptor GPR109A on Langerhans cells results in release of prostaglandin D2, which mediates the well-known flushing side effect of niacin. Niacin activation of GPR109A on adipocytes also mediates the transient reduction of plasma free fatty acid (FFA) levels characteristic of niacin, which has been long hypothesized to be the mechanism underlying the changes in the serum lipid profile. We tested this “FFA hypothesis” and the hypothesis that niacin lipid efficacy is mediated via GPR109A by dosing mice lacking GPR109A with niacin and testing two novel, full GPR109A agonists, MK-1903 and SCH900271, in three human clinical trials. In mice, the absence of GPR109A had no effect on niacin’s lipid efficacy despite complete abrogation of the anti-lipolytic effect. Both MK-1903 and SCH900271 lowered FFAs acutely in humans; however, neither had the expected effects on serum lipids. Chronic FFA suppression was not sustainable via GPR109A agonism with niacin, MK-1903, or SCH900271. We conclude that the GPR109A receptor does not mediate niacin’s lipid efficacy, challenging the long-standing FFA hypothesis.

An Anti-PCSK9 Antibody Reduces LDL-Cholesterol On Top Of A Statin And Suppresses Hepatocyte SREBP-Regulated Genes

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a promising therapeutic target for treating coronary heart disease. We report a novel antibody 1B20 that binds to PCSK9 with sub-nanomolar affinity and antagonizes PCSK9 function in-vitro. In CETP/LDLR-hemi mice two successive doses of 1B20, administered 14 days apart at 3 or 10 mpk, induced dose dependent reductions in LDL-cholesterol (≥ 25% for 7-14 days) that correlated well with the extent of PCSK9 occupancy by the antibody. In addition, 1B20 induces increases in total plasma antibody-bound PCSK9 levels and decreases in liver mRNA levels of SREBP-regulated genes PCSK9 and LDLR, with a time course that parallels decreases in plasma LDL-cholesterol (LDL-C). Consistent with this observation in mice, in statin-responsive human primary hepatocytes, 1B20 lowers PCSK9 and LDLR mRNA levels and raises serum steady-state levels of antibody-bound PCSK9. In addition, mRNA levels of several SREBP regulated genes involved in cholesterol and fatty-acid synthesis including ACSS2, FDPS, IDI1, MVD, HMGCR, and CYP51A1 were decreased significantly with antibody treatment of primary human hepatocytes. In rhesus monkeys, subcutaneous (SC) dosing of 1B20 dose-dependently induces robust LDL-C lowering (maximal ~70%), which is correlated with increases in target engagement and total antibody-bound PCSK9 levels. Importantly, a combination of 1B20 and Simvastatin in dyslipidemic rhesus monkeys reduced LDL-C more than either agent alone, consistent with a mechanism of action that predicts additive effects of anti-PCSK9 agents with statins. Our results suggest that antibodies targeting PCSK9 could provide patients powerful LDL lowering efficacy on top of statins, and lower cardiovascular risk.

Genetic Associations for Activated Partial Thromboplastin Time and Prothrombin Time, their Gene Expression Profiles, and Risk of Coronary Artery Disease

Activated partial thromboplastin time (aPTT) and prothrombin time (PT) are clinical tests commonly used to screen for coagulation-factor deficiencies. One genome-wide association study (GWAS) has been reported previously for aPTT, but no GWAS has been reported for PT. We conducted a GWAS and meta-analysis to identify genetic loci for aPTT and PT. The GWAS for aPTT was conducted in 9,240 individuals of European ancestry from the Atherosclerosis Risk in Communities (ARIC) study, and the GWAS for PT was conducted in 2,583 participants from the Genetic Study of Three Population Microisolates in South Tyrol (MICROS) and the Lothian Birth Cohorts (LBC) of 1921 and 1936. Replication was assessed in 1,041 to 3,467 individuals. For aPTT, previously reported associations with KNG1, HRG, F11, F12, and ABO were confirmed. A second independent association in ABO was identified and replicated (rs8176704, p = 4.26 × 10(-24)). Pooling the ARIC and replication data yielded two additional loci in F5 (rs6028, p = 3.22 × 10(-9)) and AGBL1 (rs2469184, p = 3.61 × 10(-8)). For PT, significant associations were identified and confirmed in F7 (rs561241, p = 3.71 × 10(-56)) and PROCR/EDEM2 (rs2295888, p = 5.25 × 10(-13)). Assessment of existing gene expression and coronary artery disease (CAD) databases identified associations of five of the GWAS loci with altered gene expression and two with CAD. In summary, eight genetic loci that account for ∼29% of the variance in aPTT and two loci that account for ∼14% of the variance in PT were detected and supported by functional data.

Differential profiles of thrombin inhibitors (heparin, hirudin, bivalirudin, and dabigatran) in the thrombin generation assay and thromboelastography in vitro.

Thrombin is a central enzyme in hemostasis and thrombosis, and a proven target for anticoagulant therapies. We compared four marketed and representative thrombin inhibitors, heparin, hirudin, bivalirudin, and dabigatran, in in-vitro spike-in assays that covered their therapeutic ranges. The assays employed were low tissue factor (1 pmol/l)-triggered thrombin generation assay (TGA) with plasma and 1 : 8000 Recombiplastin-triggered thromboelastography (TEG) with whole blood, with or without tissue plasminogen activator (tPA)-induced fibrinolysis. The three direct thrombin inhibitors (DTIs) prolonged TGA lag time and TEG clotting time (R) with a potency stack-ranking of hirudin > dabigatran ≅ bivalirudin. Heparin had the most steep concentration–response curve for both parameters. In TGA, 1–2 &mgr;mol/l dabigatran or hirudin resulted in complete inhibition on peak, slope, and endogenous thrombin potential, whereas bivalirudin had no effect on these parameters up to 10 &mgr;mol/l. All three DTIs, but not heparin, displayed a paradoxical increase in peak and slope in the low concentration range. In TEG, whereas all four agents reduced clot strength (maximal amplitude) in synergy with tPA, hirudin was the only DTI that reduced maximal amplitude appreciably without tPA. Dabigatran had the strongest potentiating effect on tPA-induced fibrinolytic activity (Ly30). With regard to the effects on coagulation and clot strength (lag time, R, and maximal amplitude) in the respective therapeutic range, dabigatran elicited the most modest changes. In summary, our observations highlight the distinct features of each agent in thrombin generation, coagulation, and fibrinolysis. The contrasts between the agents are consistent with their known properties and are informative on efforts to define the optimal profiles of new anticoagulants.

Small molecule activation of lecithin cholesterol acyltransferase modulates lipoprotein metabolism in mice and hamsters.

The objective was to assess whether pharmacological activation of lecithin cholesterol acyltransferase (LCAT) could exert beneficial effects on lipoprotein metabolism. A putative small molecule activator (compound A) was used as a tool compound in in vitro and in vivo studies. Compound A increased LCAT activity in vitro in plasma from mouse, hamster, rhesus monkey, and human. To assess the acute pharmacodynamic effects of compound A, C57Bl/6 mice and hamsters received a single dose (20 mg/kg) of compound A. Both species displayed a significant increase in high-density lipoprotein cholesterol (HDLc) and a significant decrease in non-HDLc and triglycerides acutely after dosing; these changes tracked with ex vivo plasma LCAT activity. To examine compound As chronic effect on lipoprotein metabolism, hamsters received a daily dosing of vehicle or of 20 or 60 mg/kg of compound A for 2 weeks. At study termination, compound treatment resulted in a significant increase in HDLc, HDL particle size, plasma apolipoprotein A-I level, and plasma cholesteryl ester (CE) to free cholesterol ratio, and a significant reduction in very low-density lipoprotein cholesterol. The increase in plasma CE mirrored the increase in HDL CE. Triglycerides trended toward a dose-dependent decrease in very low-density lipoprotein and HDL, with multiple triglyceride species reaching statistical significance. Gallbladder bile acids content displayed a significant and more than 2-fold increase with the 60 mg/kg treatment. We characterized pharmacological activation of LCAT by a small molecule extensively for the first time, and our findings support the potential of this approach in treating dyslipidemia and atherosclerosis; our analyses also provide mechanistic insight on LCATs role in lipoprotein metabolism.

Cholesterol in human atherosclerotic plaque is a marker for underlying disease state and plaque vulnerability

BackgroundCholesterol deposition in arterial wall drives atherosclerosis. The key goal of this study was to examine the relationship between plaque cholesterol content and patient characteristics that typically associate with disease state and lesion vulnerability. Quantitative assays for free cholesterol, cholesteryl ester, triglyceride, and protein markers in atherosclerotic plaque were established and applied to plaque samples from multiple patients and arterial beds (Carotid and peripheral arteries; 98 lesions in total).ResultsWe observed a lower cholesterol level in restenotic than primary peripheral plaque. We observed a trend toward a higher level in symptomatic than asymptomatic carotid plaque. Peripheral plaque from a group of well-managed diabetic patients displayed a weak trend of more free cholesterol deposition than plaque from non-diabetic patients. Plaque triglyceride content exhibited less difference in the same comparisons. We also measured cholesterol in multiple segments within one carotid plaque sample, and found that cholesterol content positively correlated with markers of plaque vulnerability, and negatively correlated with stability markers.ConclusionsOur results offer important biological validation of cholesterol as a key lipid marker for plaque severity. Results also suggest cholesterol is a more sensitive plaque marker than routine histological staining for neutral lipids.

11β-HSD1 inhibition reduces atherosclerosis in mice by altering proinflammatory gene expression in the vasculature

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is implicated in the etiology of metabolic syndrome. We previously showed that pharmacological inhibition of 11β-HSD1 ameliorated multiple facets of metabolic syndrome and attenuated atherosclerosis in ApoE-/- mice. However, the molecular mechanism underlying the atheroprotective effect was not clear. In this study, we tested whether and how 11β-HSD1 inhibition affects vascular inflammation, a major culprit for atherosclerosis and its associated complications. ApoE-/- mice were treated with an 11β-HSD1 inhibitor for various periods of time. Plasma lipids and aortic cholesterol accumulation were quantified. Several microarray studies were carried out to examine the effect of 11β-HSD1 inhibition on gene expression in atherosclerotic tissues. Our data suggest 11β-HSD1 inhibition can directly modulate atherosclerotic plaques and attenuate atherosclerosis independently of lipid lowering effects. We identified immune response genes as the category of mRNA most significantly suppressed by 11β-HSD1 inhibition. This anti-inflammatory effect was further confirmed in plaque macrophages and smooth muscle cells procured by laser capture microdissection. These findings in the vascular wall were corroborated by reduction in circulating MCP1 levels after 11β-HSD1 inhibition. Taken together, our data suggest 11β-HSD1 inhibition regulates proinflammatory gene expression in atherosclerotic tissues of ApoE-/- mice, and this effect may contribute to the attenuation of atherosclerosis in these animals.

Identification of four novel genes contributing to familial elevated plasma HDL cholesterol in humans

While genetic determinants strongly influence HDL cholesterol (HDLc) levels, most genetic causes underlying variation in HDLc remain unknown. We aimed to identify novel rare mutations with large effects in candidate genes contributing to extreme HDLc in humans, utilizing family-based Mendelian genetics. We performed next-generation sequencing of 456 candidate HDLc-regulating genes in 200 unrelated probands with extremely low (≤10th percentile) or high (≥90th percentile) HDLc. Probands were excluded if known mutations existed in the established HDLc-regulating genes ABCA1, APOA1, LCAT, cholesteryl ester transfer protein (CETP), endothelial lipase (LIPG), and UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2 (GALNT2). We identified 93 novel coding or splice-site variants in 72 candidate genes. Each variant was genotyped in the proband’s family. Family-based association analyses were performed for variants with sufficient power to detect significance at P < 0.05 with a total of 627 family members being assessed. Mutations in the genes glucokinase regulatory protein (GCKR), RNase L (RNASEL), leukocyte immunoglobulin-like receptor 3 (LILRA3), and dynein axonemal heavy chain 10 (DNAH10) segregated with elevated HDLc levels in families, while no mutations associated with low HDLc. Taken together, we have identified mutations in four novel genes that may play a role in regulating HDLc levels in humans.

Accelerating the pulse of cardiovascular R&D.

Renewed commitment to cardiovascular drug development is needed to combat what is likely to be the leading cause of death for the next two decades. How might this be achieved?