Inhibition of Angiopoietin-Like Protein 3 With a Monoclonal Antibody Reduces Triglycerides in Hypertriglyceridemia.

BACKGROUND: Hypertriglyceridemia is associated with increased cardiovascular risk and may be caused by impaired lipoprotein clearance. Angiopoietin-like protein 3 (ANGPTL3) inhibits lipoprotein lipase activity, increasing triglycerides and other lipids. Evinacumab, an ANGPTL3 inhibitor, reduced triglycerides in healthy human volunteers and in homozygous familial hypercholesterolemic individuals. Results from 2 Phase 1 studies in hypertriglyceridemic subjects are reported here. METHODS: Subjects with triglycerides >150 but ≤450 mg/dL and low-density lipoprotein cholesterol ≥100 mg/dL (n=83 for single ascending dose study [SAD]; n=56 for multiple ascending dose study [MAD]) were randomized 3:1 to evinacumab:placebo. SAD subjects received evinacumab subcutaneously at 75/150/250 mg, or intravenously at 5/10/20 mg/kg, monitored up to day 126. MAD subjects received evinacumab subcutaneously at 150/300/450 mg once weekly, 300/450 mg every 2 weeks, or intravenously at 20 mg/kg once every 4 weeks up to day 56 with 6 months of follow-up. The primary outcomes were incidence and severity of treatment-emergent adverse events. Efficacy analyses included changes in triglycerides and other lipids over time. RESULTS: In the SAD, 32 (51.6%) versus 9 (42.9%) subjects on evinacumab versus placebo reported treatment-emergent adverse events. In the MAD, 21 (67.7%) versus 9 (75.0%) subjects on subcutaneously evinacumab versus placebo and 6 (85.7%) versus 1 (50.0%) on intravenously evinacumab versus placebo reported treatment-emergent adverse events. No serious treatment-emergent adverse events or events leading to death or treatment discontinuation were reported. Elevations in alanine aminotransferase (7 [11.3%] SAD), aspartate aminotransferase (4 [6.5%] SAD), and creatinine phosphokinase (2 [3.2%) SAD, 1 [14.3%] MAD) were observed with evinacumab (none in the placebo groups), which were single elevations and were not dose-related. Dose-dependent reductions in triglycerides were observed in both studies, with maximum reduction of 76.9% at day 3 with 10 mg/kg intravenously (P<0.0001) in the SAD and of 83.1% at day 2 with 20 mg/kg intravenously once every 4 weeks (P=0.0003) in the MAD. Significant reductions in other lipids were observed with most evinacumab doses versus placebo. CONCLUSION: Evinacumab was well-tolerated in 2 Phase 1 studies. Lipid changes in hypertriglyceridemic subjects were similar to those observed with ANGPTL3 loss-of-function mutations. Because the latter is associated with reduced cardiovascular risk, ANGPTL3 inhibition may improve clinical outcomes. CLINICAL TRIAL REGISTRATION: https://www.clinicaltrials.gov. Unique identifiers: NCT01749878 and NCT02107872.

Genetic and Pharmacologic Inactivation of ANGPTL3 and Cardiovascular Disease.

BACKGROUND: Loss-of-function variants in the angiopoietin-like 3 gene (ANGPTL3) have been associated with decreased plasma levels of triglycerides, low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol. It is not known whether such variants or therapeutic antagonism of ANGPTL3 are associated with a reduced risk of atherosclerotic cardiovascular disease. METHODS: We sequenced the exons of ANGPTL3 in 58,335 participants in the DiscovEHR human genetics study. We performed tests of association for loss-of-function variants in ANGPTL3 with lipid levels and with coronary artery disease in 13,102 case patients and 40,430 controls from the DiscovEHR study, with follow-up studies involving 23,317 case patients and 107,166 controls from four population studies. We also tested the effects of a human monoclonal antibody, evinacumab, against Angptl3 in dyslipidemic mice and against ANGPTL3 in healthy human volunteers with elevated levels of triglycerides or LDL cholesterol. RESULTS: In the DiscovEHR study, participants with heterozygous loss-of-function variants in ANGPTL3 had significantly lower serum levels of triglycerides, HDL cholesterol, and LDL cholesterol than participants without these variants. Loss-of-function variants were found in 0.33% of case patients with coronary artery disease and in 0.45% of controls (adjusted odds ratio, 0.59; 95% confidence interval, 0.41 to 0.85; P=0.004). These results were confirmed in the follow-up studies. In dyslipidemic mice, inhibition of Angptl3 with evinacumab resulted in a greater decrease in atherosclerotic lesion area and necrotic content than a control antibody. In humans, evinacumab caused a dose-dependent placebo-adjusted reduction in fasting triglyceride levels of up to 76% and LDL cholesterol levels of up to 23%. CONCLUSIONS: Genetic and therapeutic antagonism of ANGPTL3 in humans and of Angptl3 in mice was associated with decreased levels of all three major lipid fractions and decreased odds of atherosclerotic cardiovascular disease. (Funded by Regeneron Pharmaceuticals and others; ClinicalTrials.gov number, NCT01749878 .).

Evaluation of CETP activity in vivo under non-steady-state conditions: influence of anacetrapib on HDL-TG flux.

Studies in lipoprotein kinetics almost exclusively rely on steady-state approaches to modeling. Herein, we have used a non-steady-state experimental design to examine the role of cholesteryl ester transfer protein (CETP) in mediating HDL-TG flux in vivo in rhesus macaques, and therefore, we developed an alternative strategy to model the data. Two isotopomers ([(2)H11] and [(13)C18]) of oleic acid were administered (orally and intravenously, respectively) to serve as precursors for labeling TGs in apoB-containing lipoproteins. The flux of a specific TG (52:2) from these donor lipoproteins to HDL was used as the measure of CETP activity; calculations are also presented to estimate total HDL-TG flux. Based on our data, we estimate that the peak total postprandial TG flux to HDL via CETP is ∼ 13 mg · h(-1) · kg(-1) and show that this transfer was inhibited by 97% following anacetrapib treatment. Collectively, these data demonstrate that HDL TG flux can be used as a measure of CETP activity in vivo. The fact that the donor lipoproteins can be labeled in situ using well-established stable isotope tracer techniques suggests ways to measure this activity for native lipoproteins in free-living subjects under any physiological conditions.

Measurement of LDL-C after treatment with the CETP inhibitor anacetrapib.

Estimation of low-density lipoprotein cholesterol (LDL-C) using the Friedewald (FR) formula is often inaccurate when triglycerides are elevated or VLDL particle composition is altered. We hypothesized that LDL-C estimation by the FR formula and other measurement methods might also be inaccurate in individuals treated with a cholesteryl ester transfer protein (CETP) inhibitor. An assay comparison study was conducted using pre and posttreatment serum samples from 280 of the 811 patients treated with the CETP inhibitor anacetrapib in the DEFINE study (determining the efficacy and tolerability of CETP inhibition with anacetrapib). After 24 weeks of treatment with anacetrapib, mean LDL-C values by FR formula, Roche direct method (RDM) and Genzyme direct method (GDM) deviated from that measured by the ß-quantification (BQ) reference method by -12.2 ± 7.5, -10.2 ± 6.6, -10.8 ± 8.8 mg/dl, respectively. After treatment with anacetrapib, the FR formula and detergent-based direct methods provided lower LDL-C values than those obtained by the BQ reference method. The bias by the FR formula appeared to be due to an overestimation of VLDL-C by the TG/5 component of the formula. Evaluation of the clinical significance of these findings awaits comprehensive lipid and cardiovascular outcome data from ongoing Phase III clinical studies of anacetrapib.

Safety of anacetrapib in patients with or at high risk for coronary heart disease.

BACKGROUND: Anacetrapib is a cholesteryl ester transfer protein inhibitor that raises high-density lipoprotein (HDL) cholesterol and reduces low-density lipoprotein (LDL) cholesterol. METHODS: We conducted a randomized, double-blind, placebo-controlled trial to assess the efficacy and safety profile of anacetrapib in patients with coronary heart disease or at high risk for coronary heart disease. Eligible patients who were taking a statin and who had an LDL cholesterol level that was consistent with that recommended in guidelines were assigned to receive 100 mg of anacetrapib or placebo daily for 18 months. The primary end points were the percent change from baseline in LDL cholesterol at 24 weeks (HDL cholesterol level was a secondary end point) and the safety and side-effect profile of anacetrapib through 76 weeks. Cardiovascular events and deaths were prospectively adjudicated. RESULTS: A total of 1623 patients underwent randomization. By 24 weeks, the LDL cholesterol level had been reduced from 81 mg per deciliter (2.1 mmol per liter) to 45 mg per deciliter (1.2 mmol per liter) in the anacetrapib group, as compared with a reduction from 82 mg per deciliter (2.1 mmol per liter) to 77 mg per deciliter (2.0 mmol per liter) in the placebo group (P<0.001)--a 39.8% reduction with anacetrapib beyond that seen with placebo. In addition, the HDL cholesterol level increased from 41 mg per deciliter (1.0 mmol per liter) to 101 mg per deciliter (2.6 mmol per liter) in the anacetrapib group, as compared with an increase from 40 mg per deciliter (1.0 mmol per liter) to 46 mg per deciliter (1.2 mmol per liter) in the placebo group (P<0.001)--a 138.1% increase with anacetrapib beyond that seen with placebo. Through 76 weeks, no changes were noted in blood pressure or electrolyte or aldosterone levels with anacetrapib as compared with placebo. Prespecified adjudicated cardiovascular events occurred in 16 patients treated with anacetrapib (2.0%) and 21 patients receiving placebo (2.6%) (P = 0.40). The prespecified Bayesian analysis indicated that this event distribution provided a predictive probability (confidence) of 94% that anacetrapib would not be associated with a 25% increase in cardiovascular events, as seen with torcetrapib. CONCLUSIONS: Treatment with anacetrapib had robust effects on LDL and HDL cholesterol, had an acceptable side-effect profile, and, within the limits of the power of this study, did not result in the adverse cardiovascular effects observed with torcetrapib. (Funded by Merck Research Laboratories; ClinicalTrials.gov number, NCT00685776.).

Preclinical, randomized phase 1, and compassionate use evaluation of REGN4461, a leptin receptor agonist antibody for leptin deficiency.

Deficiency in the adipose-derived hormone leptin or leptin receptor signaling causes class 3 obesity in individuals with genetic loss-of-function mutations in leptin or its receptor LEPR and metabolic and liver disease in individuals with hypoleptinemia secondary to lipoatrophy such as in individuals with generalized lipodystrophy. Therapies that restore leptin-LEPR signaling may resolve these metabolic sequelae. We developed a fully human monoclonal antibody (mAb), REGN4461 (mibavademab), that activates the human LEPR in the absence or presence of leptin. In obese leptin knockout mice, REGN4461 normalized body weight, food intake, blood glucose, and insulin sensitivity. In a mouse model of generalized lipodystrophy, REGN4461 alleviated hyperphagia, hyperglycemia, insulin resistance, dyslipidemia, and hepatic steatosis. In a phase 1, randomized, double-blind, placebo-controlled two-part study, REGN4461 was well tolerated with an acceptable safety profile. Treatment of individuals with overweight or obesity with REGN4461 decreased body weight over 12 weeks in those with low circulating leptin concentrations (<8 ng/ml) but had no effect on body weight in individuals with higher baseline leptin. Furthermore, compassionate-use treatment of a single patient with atypical partial lipodystrophy and a history of undetectable leptin concentrations associated with neutralizing antibodies to metreleptin was associated with noteable improvements in circulating triglycerides and hepatic steatosis. Collectively, these translational data unveil an agonist LEPR mAb that may provide clinical benefit in disorders associated with relatively low leptin concentrations.

Changes in lipoprotein subfraction concentration and composition in healthy individuals treated with the CETP inhibitor anacetrapib.

We investigated the effects of the cholesteryl ester (CE) transfer protein inhibitor anacetrapib (ANA) on plasma lipids, lipoprotein subfraction concentrations, and lipoprotein composition in 30 healthy individuals. Participants (n = 30) were randomized to ANA 20 mg/day, 150 mg/day, or placebo for 2 weeks. Changes in concentration of lipoprotein subfractions were assessed using ion mobility, and compositional analyses were performed on fractions separated by density gradient ultracentrifugation. ANA 150 mg/day versus placebo resulted in significant decreases in LDL-cholesterol (26%) and apo B (29%) and increases in HDL-cholesterol (82%). Concentrations of medium and small VLDL, large intermediate density lipoprotein (IDL), and medium and small LDL (LDL2a, 2b, and 3a) decreased whereas levels of very small and dense LDL4b were increased. There was enrichment of triglycerides and reduction of CE in VLDL, IDL, and the densest LDL fraction. Levels of large buoyant HDL particles were substantially increased, and there was enrichment of CE, apo AI, and apoCIII, but not apoAII or apoE, in the mid-HDL density range. Changes in lipoprotein subfraction concentrations and composition with ANA 20 mg/day were similar to those for ANA 150 mg/day but were generally smaller in magnitude. The impact of these changes on cardiovascular risk remains to be determined.

Efficacy and safety after cessation of treatment with the cholesteryl ester transfer protein inhibitor anacetrapib (MK-0859) in patients with primary hypercholesterolemia or mixed hyperlipidemia.

This report describes the lipid and safety data collected during an off-drug period that followed 8 weeks of treatment with the cholesteryl ester transfer protein inhibitor, anacetrapib (ANA). A total of 589 patients with primary hypercholesterolemia or mixed hyperlipidemia were randomized to placebo, atorvastatin (ATV) 20 mg, and varying doses of ANA, provided as monotherapy or coadministered with ATV 20 mg daily. Patients were treated for 8 weeks, followed by an 8-week follow-up period, during which ANA was switched to placebo. At week 16 (8 weeks after ANA was stopped), persistent reductions in low-density lipoprotein cholesterol (LDL-C) were evident for the monotherapy groups receiving ANA 150 and 300 mg (-9.3% and -15.3%, respectively), and residual increases in high-density lipoprotein cholesterol (HDL-C) were observed for the monotherapy groups receiving ANA 40 mg (18.6%), 150 mg (40.5%), and 300 mg (43.4%). The effects on apolipoprotein B and apolipoprotein A-I were consistent with the changes observed for LDL-C and HDL-C, respectively. Corresponding residual changes in LDL-C and HDL-C were also noted in the ATV coadministration groups at the similar doses of ANA compared with ATV 20 mg alone. Residual plasma drug levels accompanied by reductions in cholesteryl ester transfer protein activity were observed at week 16 and may account for the alterations in plasma lipids 8 weeks after cessation of ANA.

Leptin-enhanced neointimal hyperplasia is reduced by mTOR and PI3K inhibitors.

Despite the use of the sirolimus (rapamycin) drug-eluting coronary stent, diabetics are at increased risk of developing in-stent restenosis for unclear reasons. Hyperleptinemia, which often coexists with diabetes and metabolic syndrome, is an independent risk factor for progression of coronary artery disease. It has not been determined whether elevated circulating leptin decreases the efficacy of the sirolimus drug-eluting stent in inhibiting neointimal hyperplasia, the process underlying restenosis after stenting. Here we show that leptin activates the mammalian target of rapamycin (mTOR) signaling pathway in primary murine vascular smooth muscle cells (VSMC) and stimulates VSMC proliferation in a PI3K-dependent fashion. Exogenous leptin, administered at levels comparable to those found in obese humans, promotes neointimal VSMC hyperplasia in a murine femoral artery wire injury model. Leptin significantly increases the dose of the mTOR inhibitor sirolimus that is required for effective inhibition of neointimal formation. Combination therapy with LY294002, a PI3K inhibitor, and sirolimus effectively inhibits leptin-enhanced neointimal hyperplasia. These data show that, in the setting of hyperleptinemia, higher doses of an mTOR inhibitor, or combination therapy with mTOR and PI3K inhibitors, inhibits neointimal hyperplasia after arterial injury. These studies may explain the higher rates of restenosis observed in diabetics treated with a sirolimus-eluting coronary stent and suggest a potential novel therapeutic approach for inhibiting in-stent restenosis in such patients.

Novel strategy using F1-congenic mice for validation of QTLs: studies at the proximal chromosome 10 atherosclerosis susceptibility locus.

OBJECTIVE: We have previously identified a quantitative trait locus (QTL) for atherosclerosis susceptibility on proximal chromosome 10 (Chr10) (Ath11) in independent crosses of FVB and C57BL/6 (B6) mice on the apolipoprotein E (ApoE-/-) and LDL receptor (LDLR-/-) deficient backgrounds. The aims of the current study were to (1) test a novel strategy for validating QTLs using interval-specific congenic strains that were heterozygous (F1) across the genome, (2) validate the Chr10 QTL, and (3) to assess whether the phenotype is transferable by bone marrow transplantation. METHODS AND RESULTS: We generated Chr10 (0 to 21 cM) interval-specific mice on the F1.ApoE-/- background by crossing congenic FVB.ApoE-/-Chr10(B6/FVB) with B6.ApoE-/-, and B6.ApoE-/-Chr10(B6/FVB) with FVB.ApoE-/- mice. Lesion size was significantly larger in the resultant F1.ApoE-/-Chr10(FVB/FVB) mice compared to F1.ApoE-/-Chr10(B6/FVB) and F1.ApoE-/-Chr10(B6/B6) mice, validating the Chr10 QTL. The effect of the congenic interval was more robust on the F1.ApoE-/- than on the FVB.ApoE-/- and B6.ApoE-/- backgrounds. Bone marrow transplantation in congenic mice showed that the effect of the proximal Chr10 interval was not transferable by bone marrow-derived cells. CONCLUSIONS: A novel strategy of congenic strains on an F1 background proved useful to validate an atherosclerosis susceptibility QTL on mouse proximal Chr10.

Found in translation.

For those who wish to marry research with clinical applications, finding the right environment can be hard.

Symptoms and Dietary Impact in Hypertriglyceridemia-Associated Pancreatitis: Development and Content Validity of Two New Measures.

BACKGROUND: Severe hypertriglyceridemia (sHTG) is a rare condition, complicated by episodes of acute pancreatitis (AP), which can cause pain and/or life-threatening multi-organ dysfunction. Currently, there are no disease-specific patient-reported outcome (PRO) measures evaluating symptoms or dietary impact for this condition. OBJECTIVE: The objective of this study was to explore patient-reported symptoms and impacts of sHTG and AP and develop new measures to capture the symptoms and dietary impacts of this condition using patient language. METHODS: In-depth, semi-structured concept elicitation interviews were conducted with 12 US-based participants to explore their experience and identify key symptoms and impact on dietary behavior, both during and between episodes of AP. Participants had a range of AP severity with a previous triglyceride reading > 1000 mg/dL, and at least one attack of AP within the last 12 months. Transcripts were coded using thematic analysis. RESULTS: Qualitative data analysis revealed the substantial burden of AP associated with sHTG. Participants reported experiencing symptoms, especially abdominal pain, both during and between attacks of AP, and discussed considerable diet changes to prevent or minimize future attacks. A conceptual model was refined, based on patient input, and reviewed by clinical experts to determine key concepts for inclusion within two PRO measures, one evaluating symptoms and another evaluating impact on dietary behavior. Items were drafted using patient-derived language. A 19-item symptoms measure [Hypertriglyceridemia and Acute Pancreatitis Symptom Scale (HAP-SS)] and a 6-item dietary impact measure (Hypertriglyceridemia and Acute Pancreatitis Dietary Behavior (HAP-DB) measure) were developed, both with a 24-h recall period. CONCLUSIONS: The qualitative analysis confirmed the substantial burden of AP associated with sHTG. This research resulted in development of two disease-specific PRO measures for use during and between attacks of AP. These measures are being utilized in a clinical trial, which will confirm content, structure, and psychometric properties.

Design of the DEFINE trial: determining the EFficacy and tolerability of CETP INhibition with AnacEtrapib.

BACKGROUND: Residual cardiovascular (CV) risk often remains high despite statin therapy to lower low-density lipoprotein cholesterol (LDL-C). New therapies to raise high-density lipoprotein cholesterol (HDL-C) are currently being investigated. Anacetrapib is a cholesteryl ester transfer protein (CETP) inhibitor that raises HDL-C and reduces LDL-C when administered alone or with a statin. Adverse effects on blood pressure, electrolytes, and aldosterone levels, seen with another drug in this class, have not been noted in studies of anacetrapib to date. METHODS: Determining the EFficacy and Tolerability of CETP INhibition with AnacEtrapib (DEFINE) is a randomized, double-blind, placebo-controlled trial to assess the efficacy and safety profile of anacetrapib in patients with coronary heart disease (CHD) or CHD risk equivalents (clinical trials.gov NCT00685776). Eligible patients at National Cholesterol Education Program-Adult Treatment Panel III LDL-C treatment goal on a statin, with or without other lipid-modifying medications, are treated with anacetrapib, 100 mg, or placebo for 18 months, followed by a 3-month, poststudy follow-up. The primary end points are percent change from baseline in LDL-C and the safety and tolerability of anacetrapib. Comprehensive preplanned interim safety analyses will be performed at the 6- and 12-month time points to examine treatment effects on key safety end points, including blood pressure and electrolytes. A preplanned Bayesian analysis will be performed to interpret the CV event distribution, given the limited number of events expected in this study. RESULTS: A total of 2,757 patients were screened at 153 centers in 20 countries, and 1,623 patients were randomized into the trial. Lipid results, clinical CV events, and safety outcomes from this trial are anticipated in 2010.

Recipes for creating animal models of diabetic cardiovascular disease.

For more than 50 years, investigators have unsuccessfully tried to recreate in experimental animals the cardiovascular complications of diabetes seen in humans. In particular, accelerated atherosclerosis and dilated cardiomyopathy, the major causes of mortality in patients with diabetes, have been conspicuously absent in many mouse models of the disease. Under the auspices of the NIH, the Animal Models of Diabetic Complications Consortium has worked to address this issue. This effort has focused on the development of mouse models because of the high level of genomic information available and the many well-developed genetic manipulations that may be performed in mice. Importantly, the consortium has also worked to standardize many methods to assess metabolic and cardiovascular end points for measurement of the diabetic state and its macrovascular complications. Finally, for maximum benefits from these animal models in the study of atherosclerosis and of other diabetic complications, the consortium has created a system for sharing both the animal models and the accumulated phenotypic data with the greater scientific community.

Increased neointimal formation after surgical vein grafting in a murine model of type 2 diabetes.

BACKGROUND: Diabetes is an independent risk factor for the development of neointimal hyperplasia and subsequent vein graft failure after coronary or peripheral artery bypass grafting. We evaluate a new mouse model of surgical vein grafting to investigate the mechanisms of neointimal formation in the setting of type 2 diabetes. METHODS AND RESULTS: Surgical vein grafts were created by inserting vein segments from age-matched C57BL/KsJ wild-type mice into the infra-renal aorta of lepr(db/db) diabetic and C57BL/KsJ wild-type mice. Mice were euthanized &4 weeks later, and vein grafts were analyzed using morphometric and immunohistochemical techniques. A significant increase in neointimal formation was noted in lepr(db/db) mice (139+/-64 versus 109+/-62 mm2; P=0.008) after 4 weeks. This difference was mainly secondary to an increase in collagen formation within the lesion in the vein grafts from lepr(db/db) mice (0.53+/-0.4 versus 0.44+/-0.05; P<0.001), whereas only slight increases (P=not significant) in alpha actin-stained smooth muscle cells were noted in the lepr(db/db) mice. CONCLUSIONS: We established a new physiologically relevant model of surgical vein grafting in mice. In this report, type 2 diabetes was associated with significant increase in extracellular matrix deposition in addition to increased smooth muscle cell deposition. This new model may allow mechanistic studies of cellular and molecular pathways of increased neointimal formation in the setting of diabetes.

Diabetes induces endothelial dysfunction but does not increase neointimal formation in high-fat diet fed C57BL/6J mice.

Studies of diabetic vascular disease have traditionally used murine models of type 1 diabetes and genetic models of type 2 diabetes. Because the majority of patients with type 2 diabetes have diet induced obesity, we sought to study the effect of diabetes on arterial disease in a mouse model of diet induced obesity/diabetes. C57Bl/6 mice fed a high-fat diet for 9 weeks developed type 2 diabetes characterized by elevated body weight, hyperglycemia, and hyperinsulinemia. Arteries from diabetic mice exhibited a marked decrease in endothelium-dependent vasodilation, a modest decrease in endothelium independent vasodilation, and an increase in sensitivity to adrenergic vasoconstricting agents. Insulin stimulated protein kinase B (akt) and endothelial nitric oxide synthase (eNOS) phosphorylation were preserved in arteries from diabetic mice; however, eNOS protein dimers were markedly diminished. Arterial nitrotyrosine staining indicated that increased levels of peroxynitrite contributed to eNOS dimer disruption in the diabetic mice. The abnormal vasomotion was not an acute response to the high-fat diet, as short term high-fat diet feeding had no effect on endothelium dependent dilation. A trend toward smaller neointimal lesions was noted in high-fat diet fed mice after femoral artery wire denudation injury. In summary, disrupted eNOS dimer formation rather than impaired insulin mediated eNOS phosphorylation contributed to the endothelial dysfunction in diet induced obese/diabetic mice. The lack of an increase in neointimal formation indicates that additional diabetes associated parameters (such as hyperlipidemia and atherosclerotic vascular disease) may need to be present to increase neointimal formation in this model.

Diabetic vascular disease: an experimental objective.

It is well known that humans with diabetes have more atherosclerosis and its complications. The causes of this relationship are, however, unclear. Although recent data show that improved glycemic control reduces arterial disease in type 1 diabetes, other studies have shown that subjects with "prediabetes" have more cardiovascular disease before the development of hyperglycemia. Thus, either hyperglycemia and/or lack of insulin actions are toxic to arteries, or metabolic derangements exclusive of hyperglycemia are atherogenic. For >50 years animal models of diabetes and atherosclerosis have been used to uncover potential mechanisms underlying diabetes associated cardiovascular disease. Surprisingly, diabetes alone increases vascular disease in only a few select animal models. Increased atherosclerosis has been found in several animals and lines of genetically modified mice; however, diabetes often also leads to greater hyperlipidemia. This makes it difficult to separate the toxic effects of insulin lack and/or hyperglycemia from those caused by the lipids. These studies are reviewed, as well as more recent investigations using new methods to create diabetic-atherosclerotic models.

A phenotype-sensitizing Apoe-deficient genetic background reveals novel atherosclerosis predisposition loci in the mouse.

Therapeutic intervention for atherosclerosis has predominantly concentrated on regulating cholesterol levels; however, these therapeutics are not efficacious for all patients, suggesting that other factors are involved. This study was initiated to identify mechanisms that regulate atherosclerosis predisposition in mice other than cholesterol level regulation. To do so we performed quantitative trait locus analysis using two inbred strains that each carry the atherosclerosis phenotype-sensitizing Apoe deficiency and that have been shown to have widely disparate predilection to atherosclerotic lesion formation. One highly significant locus on chromosome 10 (LOD = 7.8) accounted for 19% of the variance in lesion area independent of cholesterol. Two additional suggestive loci were identified on chromosomes 14 (LOD = 3.2) and 19 (LOD = 3.2), each accounting for 7-8% of the lesion variance. In all, five statistically significant and suggestive loci affecting lesion size but not lipoprotein levels were identified. Many of these were recapitulated in an independent confirmatory cross. In summary, two independently performed crosses between C57BL/6 and FVB/N Apoe-deficient mice have revealed several previously unreported atherosclerosis susceptibility loci that are distinct from loci linked to lipoprotein levels.

Differential effects of angiotensin II on atherogenesis at the aortic sinus and descending aorta of apolipoprotein-E-deficient mice.

Angiotensin (Ang) II infusion increases atherosclerosis and leads to the formation of abdominal aortic aneurysms in apolipoprotein E-deficient (ApoE-/-) mice. Conversely, blockade of the renin-angiotensin system (RAS) decreases atherosclerosis in this model. However, there are conflicting data in the literature concerning responses to both Ang II infusion and RAS blockade which may depend on age, sex, dose, duration of treatment, and the site at which lesion area was measured. In the present study we examined the effects of Ang II infusion on lesion formation in male ApoE-/- mice both at the aortic sinus and in the descending aorta, starting at different ages, and varying in duration. We also tested the effects of the Ang II receptor antagonist losartan at different doses in both males and females. Blood pressure and plasma renin concentration (PRC) were measured as indicators of the hemodynamic and neurohormonal effects of these treatments. Administration of Ang II increased lesion area much more in the descending aorta than at the aortic sinus. However, spontaneous lesion development at the aortic sinus was much greater than in more distal regions of the aorta. Aneurysms were observed in all treatment groups but were less severe in animals treated from 4 weeks age, possibly because of protective remodeling. Losartan treatment reduced lesion area at the aortic sinus, although differences were only significant in female mice. These findings demonstrate regional and temporal differences in the sensitivity of the aorta to the effects of RAS stimulation and blockade, and may help to explain some of the discrepancies between previous reports from other laboratories.