Janet K. Sawyer

Anatomic correlates of aortic pulse wave velocity and carotid artery elasticity during atherosclerosis progression and regression in monkeys.

We noninvasively measured changes in average aortic stiffness in 79 cynomolgus monkeys being fed cholesterol progression, regression, and control diets by measuring pulse wave velocity (PWV) in 260 experiments during a 30-month period. Every 6 months, a group of monkeys was studied with invasive aortic PWV techniques and with ultrasonically determined pressure-strain elastic modulus (Ep) of the carotid artery, and then the group was killed so that morphometric evaluation of atherosclerosis severity could be made. After 6 months of a cholesterol progression diet, PWV decreased slightly from 6.2 +/- 0.1 to 5.7 +/- 0.1 m/sec, followed by an approximate linear increase to 8.8 +/- 1.2 m/sec after 30 months on the diet. The corresponding ratio of intimal (plaque) area to medial area (IA/MA) measured on perfusion-fixed cross-sections of the abdominal and thoracic aortas increased from 0.16 +/- 0.07 at 6 months to 1.23 +/- 0.22 at 30 months. Monkeys in the regression groups were fed the cholesterol progression diet for 18 months, followed by a chow diet for 6 or 12 months. In the first 6 months of the cholesterol regression diet, PWV continued to increase from 7.0 +/- 0.2 to 8.1 +/- 0.4 m/sec, and IA/MA was 1.24 +/- 0.18. However, after 12 months of the cholesterol regression diet, PWV decreased to 6.8 +/- 0.4 m/sec, and IA/MA was 0.90 +/- 0.18. The variability of the data demonstrates that PWV is not a simple function of atherosclerosis severity, and the best simple correlation was r = 0.69 (r2 = 0.48) between PWV and intimal area. However, multiple regression analysis of aortic PWV, systolic (SP) and diastolic (DP) blood pressures, and total plasma cholesterol concentration (TPC), all of which can be measured with minimally invasive techniques, improved the prediction of the IA/MA ratio through the following equation: IA/MA = 0.127 PWV-0.039 DP+0.023SP+0.0003TPC-0.292 (r = 0.81, r2 = 0.66). These data suggest that arterial stiffness in combination with minimally invasive parameters can be used to predict the severity of diffuse asymptomatic atherosclerosis in monkeys. However, more widespread application of these data to humans is uncertain because of biological variability and differences between animal models and human subjects.

Compared With Dietary Monounsaturated and Saturated Fat, Polyunsaturated Fat Protects African Green Monkeys From Coronary Artery Atherosclerosis

Atherogenic diets enriched in saturated, n-6 polyunsaturated, and monounsaturated fatty acids were fed to African green monkeys for 5 years to define effects on plasma lipoproteins and coronary artery atherosclerosis. The monkeys fed polyunsaturated and monounsaturated fat had similar plasma concentrations of LDL cholesterol, and these values were significantly lower than for LDL in the animals fed saturated fat. Plasma HDL cholesterol concentrations were comparable in animals fed saturated and monounsaturated fat and were significantly higher than in animals fed polyunsaturated fat. Thus, the monounsaturated fat group had the lowest LDL/HDL ratio. LDL particle size was largest in the saturated and monounsaturated fat groups, significantly larger than in the polyunsaturated fat group. LDL particle enrichment with cholesteryl oleate was the greatest in the animals fed monounsaturated fat, next greatest in the saturated fat-fed animals, and was least in the polyunsaturated fat-fed animals. Coronary artery atherosclerosis as measured by intimal area was less in the polyunsaturated fat compared with the saturated fat groups, was less in the animals fed polyunsaturated fat compared with the monounsaturated fat-fed animals, but did not differ between the monounsaturated and saturated fat groups. Cholesteryl ester, particularly cholesteryl oleate, accumulation in the coronary arteries was also similar between groups fed monounsaturated and saturated fat but was minimal in the animals fed polyunsaturated fat. In sum, the monkeys fed monounsaturated fat developed equivalent amounts of coronary artery atherosclerosis as those fed saturated fat, but monkeys fed polyunsaturated fat developed less. The beneficial effects of the lower LDL and higher HDL in the animals fed monounsaturated fat apparently were offset by the atherogenic shifts in LDL particle composition. Dietary polyunsaturated fat appears to result in the least amount of coronary artery atherosclerosis because it prevents cholesteryl oleate accumulation in LDL and the coronary arteries in these primates.

Dietary Monounsaturated Fatty Acids Promote Aortic Atherosclerosis in LDL Receptor–Null, Human ApoB100–Overexpressing Transgenic Mice

In mice with genetically engineered high levels of plasma low density lipoprotein (LDL), we tested the hypothesis that an increase in the dietary content of monounsaturated fatty acids but not of polyunsaturated fatty acids would promote atherosclerosis. The mouse model used was an LDL receptor-null, human apoB100-overexpressing strain. Six experimental groups of 19 to 38 mice of both sexes were established when the animals had reached 8 weeks of age. For the next 16 weeks, individual groups were fed either a commercial diet or prepared diets including fat as 10% of energy, with 5 different fatty acid enrichment patterns including the following: saturated (sat), cis and trans monounsaturated (mono), and n-3 and n-6 polyunsaturated (poly). Highly significant differences (ANOVA, P<0. 0001) in LDL cholesterol (in mg/dL) were found, with the rank order at 16 weeks being trans mono (mean, 1390)>sat (922)=cis mono (869)=n-6 poly (868)>n-3 poly (652)>commercial diet (526). Significant elevations in very low density lipoprotein cholesterol were also found in the trans and cis mono and sat groups, and triacylglycerol concentrations were also elevated in all groups. High density lipoprotein cholesterol concentrations were consistently low (20 to 50 mg/dL) in all groups. Highly significant differences (ANOVA, P<0.0001) in atherosclerosis, quantified by measurement of aortic cholesteryl ester concentration (mg/g protein) among dietary fatty acid groups were found, with the order being trans mono (mean, 50.4)>sat (35.6)=cis mono (34.6)>n-6 poly (18. 3)=n-3 poly (9.7)=commercial diet (7.8). Therefore, in this mouse model of hypercholesterolemia, dietary cis or trans monounsaturated fat did not protect against atherosclerosis development, whereas aortic atherosclerosis in either of the polyunsaturated fat groups was significantly less than in the saturated fat group.

Trans Fat Diet Induces Abdominal Obesity and Changes in Insulin Sensitivity in Monkeys

Objective: There is conflicting evidence about the propensity of trans fatty acids (TFAs) to cause obesity and insulin resistance. The effect of moderately high intake of dietary monounsaturated TFAs on body composition and indices of glucose metabolism was evaluated to determine any pro‐diabetic effect in the absence of weight gain.

Inhibition of Stearoyl-Coenzyme A Desaturase 1 Dissociates Insulin Resistance and Obesity From Atherosclerosis

Background— Stearoyl-coenzyme A desaturase 1 (SCD1) is a well-known enhancer of the metabolic syndrome. The purpose of the present study was to investigate the role of SCD1 in lipoprotein metabolism and atherosclerosis progression. Methods and Results— Antisense oligonucleotides were used to inhibit SCD1 in a mouse model of hyperlipidemia and atherosclerosis (LDLr−/−Apob100/100). In agreement with previous reports, inhibition of SCD1 protected against diet-induced obesity, insulin resistance, and hepatic steatosis. Unexpectedly, however, SCD1 inhibition strongly promoted aortic atherosclerosis, which could not be reversed by dietary oleate. Further analyses revealed that SCD1 inhibition promoted accumulation of saturated fatty acids in plasma and tissues and reduced plasma triglyceride, yet had little impact on low-density lipoprotein cholesterol. Because dietary saturated fatty acids have been shown to promote inflammation through toll-like receptor 4, we examined macrophage toll-like receptor 4 function. Interestingly, SCD1 inhibition resulted in alterations in macrophage membrane lipid composition and marked hypersensitivity to toll-like receptor 4 agonists. Conclusions— This study demonstrates that atherosclerosis can occur independently of obesity and insulin resistance and argues against SCD1 inhibition as a safe therapeutic target for the metabolic syndrome.

CGI-58 knockdown in mice causes hepatic steatosis, but prevents diet-induced obesity and glucose intolerance

Mutations of Comparative Gene Identification-58 (CGI-58) in humans cause triglyceride (TG) accumulation in multiple tissues. Mice genetically lacking CGI-58 die shortly after birth due to a skin barrier defect. To study the role of CGI-58 in integrated lipid and energy metabolism, we utilized antisense oligonucleotides (ASOs) to inhibit CGI-58 expression in adult mice. Treatment with two distinct CGI-58-targeting ASOs resulted in ∼80–95% knockdown of CGI-58 protein expression in both liver and white adipose tissue. In chow-fed mice, ASO-mediated depletion of CGI-58 did not alter weight gain, plasma TG, or plasma glucose, yet raised hepatic TG levels ∼4-fold. When challenged with a high-fat diet (HFD), CGI-58 ASO-treated mice were protected against diet-induced obesity, but their hepatic contents of TG, diacylglycerols, and ceramides were all elevated, and intriguingly, their hepatic phosphatidylglycerol content was increased by 10-fold. These hepatic lipid alterations were associated with significant decreases in hepatic TG hydrolase activity, hepatic lipoprotein-TG secretion, and plasma concentrations of ketones, nonesterified fatty acids, and insulin. Additionally, HFD-fed CGI-58 ASO-treated mice were more glucose tolerant and insulin sensitive. Collectively, this work demonstrates that CGI-58 plays a critical role in limiting hepatic steatosis and maintaining hepatic glycerophospholipid homeostasis and has unmasked an unexpected role for CGI-58 in promoting HFD-induced obesity and insulin resistance.

Plasma Cholesteryl Esters Provided by Lecithin:Cholesterol Acyltransferase and Acyl-Coenzyme A:Cholesterol Acyltransferase 2 Have Opposite Atherosclerotic Potential

Evidence suggests that ACAT2 is a proatherogenic enzyme that contributes cholesteryl esters (CEs) to apoB-containing lipoproteins, whereas LCAT is an antiatherogenic enzyme that facilitates reverse cholesterol transport by esterifying free cholesterol on HDL particles. We hypothesized that deletion of LCAT and ACAT2 would lead to absence of plasma CEs and reduced atherosclerosis. To test this hypothesis, ACAT2−/− LCAT−/− LDLr−/−, ACAT2−/− LDLr−/−, and LCAT−/− LDLr−/− mice were fed a 0.15% cholesterol diet for 20 weeks. In comparison to LDLr−/− mice, the total plasma cholesterol (TPC) of ACAT2−/− LCAT−/− LDLr−/− mice was 67% lower because of the complete absence of plasma CEs, leading to 94% less CE accumulation in the aorta. In the LCAT−/− LDLr−/− mice, TPC and atherosclerosis were significantly higher because of increased accumulations of ACAT2-derived CE. In ACAT2−/− LDLr−/− mice, again compared with LDLr−/− mice, TPC was 19% lower, whereas atherosclerosis was 88% lower. Therefore, the absence of ACAT2 led to a significant reduction in TPC although benefits in reduction of atherosclerosis were much more pronounced. Overall, the data suggest that ACAT2-derived CE is the predominant atherogenic lipid in blood, and that an important goal for prevention of atherosclerosis is to limit ACAT2-derived CE accumulation in lipoproteins.

Biliary Sterol Secretion Is Not Required for Macrophage Reverse Cholesterol Transport

Recent evidence suggests that the intestine may play a direct facilitative role in reverse cholesterol transport (RCT), independent of hepatobiliary secretion. In order to understand the nonbiliary pathway for RCT, we created both genetic and surgical models of biliary cholesterol insufficiency. To genetically inhibit biliary cholesterol secretion, we generated mice in which Niemann-Pick C1-Like 1 (NPC1L1) was overexpressed in the liver. Compared to controls, NPC1L1(Liver-Tg) mice exhibit a >90% decrease in biliary cholesterol secretion, yet mass fecal sterol loss and macrophage RCT are normal. To surgically inhibit biliary emptying into the intestine, we have established an acute biliary diversion model. Strikingly, macrophage RCT persists in mice surgically lacking the ability to secrete bile into the intestine. Collectively, these studies demonstrate that mass fecal sterol loss and macrophage RCT can proceed in the absence of biliary sterol secretion, challenging the obligate role of bile in RCT.

Targeted Depletion of Hepatic ACAT2-driven Cholesterol Esterification Reveals a Non-biliary Route for Fecal Neutral Sterol Loss

Deletion of acyl-CoA:cholesterol O-acyltransferase 2 (ACAT2) in mice results in resistance to diet-induced hypercholesterolemia and protection against atherosclerosis. Recently, our group has shown that liver-specific inhibition of ACAT2 via antisense oligonucleotide (ASO)-mediated targeting likewise limits atherosclerosis. However, whether this atheroprotective effect was mediated by: 1) prevention of packaging of cholesterol into apoB-containing lipoproteins, 2) augmentation of nascent HDL cholesterol secretion, or 3) increased hepatobiliary sterol secretion was not examined. Therefore, the purpose of these studies was to determine whether hepatic ACAT2 is rate-limiting in all three of these important routes of cholesterol homeostasis. Liver-specific depletion of ACAT2 resulted in reduced packaging of cholesterol into apoB-containing lipoproteins (very low density lipoprotein, intermediate density lipoprotein, and low density lipoprotein), whereas high density lipoprotein cholesterol levels remained unchanged. In the liver of ACAT2 ASO-treated mice, cholesterol ester accumulation was dramatically reduced, yet there was no reciprocal accumulation of unesterified cholesterol. Paradoxically, ASO-mediated depletion of hepatic ACAT2 promoted fecal neutral sterol excretion without altering biliary sterol secretion. Interestingly, during isolated liver perfusion, ACAT2 ASO-treated livers had augmented secretion rates of unesterified cholesterol and phospholipid. Furthermore, we demonstrate that liver-derived cholesterol from ACAT2 ASO-treated mice is preferentially delivered to the proximal small intestine as a precursor to fecal excretion. Collectively, these studies provide the first insight into the hepatic itinerary of cholesterol when cholesterol esterification is inhibited only in the liver, and provide evidence for a novel non-biliary route of fecal sterol loss.

Effects of peroxisome proliferator-activated receptor alpha/delta agonists on HDL-cholesterol in vervet monkeys.

The objective of this study was to demonstrate the efficacy of a novel peroxisome proliferator-activated receptor (PPAR) agonist and known PPARα and PPARδ agonists to increase HDL-cholesterol (HDL-C) in the St. Kitts vervet, a nonhuman primate model of atherosclerosis. Four groups (n = 6) were studied and each group was assigned one of the following “treatments”: a) vehicle only (vehicle); b) the PPARδ selective agonist GW501516 (GW); c) the PPARα/δ agonist T913659 (T659); and d) the PPARα agonist TriCor® (fenofibrate). No statistically significant changes were seen in body weight, total plasma cholesterol, plasma triglycerides, VLDL-C, LDL-C, or apolipoprotein B (apoB) concentrations. Each of the PPARα and PPARδ agonists investigated in this study increased plasma HDL-C, apoA-I, and apoA-II concentrations and increased HDL particle size in St. Kitts vervets. The maximum percentage increase in HDL-C from baseline for each group was as follows: vehicle, 5%; GW, 43%; T659, 43%; and fenofibrate, 20%. Treatment with GW and T659 resulted in an increase in medium-sized HDL particles, whereas fenofibrate showed increases in large HDL particles. These data provide additional evidence that PPARα and PPARδ agonists (both mixed and selective) have beneficial effects on HDL-C in these experimental primates.