Atherosclerosis Regression and Cholesterol Efflux in Hypertriglyceridemic Mice.

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Blocking Lipid Uptake Pathways Does not Prevent Toxicity in Adipose Triglyceride Lipase (ATGL) Deficiency.

Lipid accumulation in nonadipose tissues can cause lipotoxicity, leading to cell death and severe organ dysfunction. Adipose triglyceride lipase (ATGL) deficiency causes human neutral lipid storage disease and leads to cardiomyopathy; ATGL deficiency has no current treatment. One possible approach to alleviate this disorder has been to alter the diet and reduce the supply of dietary lipids and, hence, myocardial lipid uptake. However, in this study, when we supplied cardiac Atgl KO mice a low- or high-fat diet, we found that heart lipid accumulation, heart dysfunction, and death were not altered. We next deleted lipid uptake pathways in the ATGL-deficient mice through the generation of double KO mice also deficient in either cardiac lipoprotein lipase or cluster of differentiation 36, which is involved in an lipoprotein lipase-independent pathway for FA uptake in the heart. We show that neither deletion ameliorated ATGL-deficient heart dysfunction. Similarly, we determined that non-lipid-containing media did not prevent lipid accumulation by cultured myocytes; rather, the cells switched to increased de novo FA synthesis. Thus, we conclude that pathological storage of lipids in ATGL deficiency cannot be corrected by reducing heart lipid uptake.

Novel Reversible Model of Atherosclerosis and Regression Using Oligonucleotide Regulation of the LDL Receptor.

RATIONALE: Animal models have been used to explore factors that regulate atherosclerosis. More recently, they have been used to study the factors that promote loss of macrophages and reduction in lesion size after lowering of plasma cholesterol levels. However, current animal models of atherosclerosis regression require challenging surgeries, time-consuming breeding strategies, and methods that block liver lipoprotein secretion. OBJECTIVE: We sought to develop a more direct or time-effective method to create and then reverse hypercholesterolemia and atherosclerosis via transient knockdown of the hepatic LDLR (low-density lipoprotein receptor) followed by its rapid restoration. METHODS AND RESULTS: We used antisense oligonucleotides directed to LDLR mRNA to create hypercholesterolemia in wild-type C57BL/6 mice fed an atherogenic diet. This led to the development of lesions in the aortic root, aortic arch, and brachiocephalic artery. Use of a sense oligonucleotide replicating the targeted sequence region of the LDLR mRNA rapidly reduced circulating cholesterol levels because of recovery of hepatic LDLR expression. This led to a decrease in macrophages within the aortic root plaques and brachiocephalic artery, that is, regression of inflammatory cell content, after a period of 2 to 3 weeks. CONCLUSIONS: We have developed an inducible and reversible hepatic LDLR knockdown mouse model of atherosclerosis regression. Although cholesterol reduction decreased early en face lesions in the aortic arches, macrophage area was reduced in both early and late lesions within the aortic sinus after reversal of hypercholesterolemia. Our model circumvents many of the challenges associated with current mouse models of regression. The use of this technology will potentially expedite studies of atherosclerosis and regression without use of mice with genetic defects in lipid metabolism.

Role of LpL (Lipoprotein Lipase) in Macrophage Polarization In Vitro and In Vivo.

OBJECTIVE: Fatty acid uptake and oxidation characterize the metabolism of alternatively activated macrophage polarization in vitro, but the in vivo biology is less clear. We assessed the roles of LpL (lipoprotein lipase)-mediated lipid uptake in macrophage polarization in vitro and in several important tissues in vivo. Approach and Results: We created mice with both global and myeloid-cell specific LpL deficiency. LpL deficiency in the presence of VLDL (very low-density lipoproteins) altered gene expression of bone marrow-derived macrophages and led to reduced lipid uptake but an increase in some anti- and some proinflammatory markers. However, LpL deficiency did not alter lipid accumulation or gene expression in circulating monocytes nor did it change the ratio of Ly6Chigh/Ly6Clow. In adipose tissue, less macrophage lipid accumulation was found with global but not myeloid-specific LpL deficiency. Neither deletion affected the expression of inflammatory genes. Global LpL deficiency also reduced the numbers of elicited peritoneal macrophages. Finally, we assessed gene expression in macrophages from atherosclerotic lesions during regression; LpL deficiency did not affect the polarity of plaque macrophages. CONCLUSIONS: The phenotypic changes observed in macrophages upon deletion of Lpl in vitro is not mimicked in tissue macrophages.

Lipoprotein Lipase Deficiency Impairs Bone Marrow Myelopoiesis and Reduces Circulating Monocyte Levels.

OBJECTIVE: Tissue macrophages induce and perpetuate proinflammatory responses, thereby promoting metabolic and cardiovascular disease. Lipoprotein lipase (LpL), the rate-limiting enzyme in blood triglyceride catabolism, is expressed by macrophages in atherosclerotic plaques. We questioned whether LpL, which is also expressed in the bone marrow (BM), affects circulating white blood cells and BM proliferation and modulates macrophage retention within the artery. APPROACH AND RESULTS: We characterized blood and tissue leukocytes and inflammatory molecules in transgenic LpL knockout mice rescued from lethal hypertriglyceridemia within 18 hours of life by muscle-specific LpL expression (MCKL0 mice). LpL-deficient mice had ≈40% reduction in blood white blood cell, neutrophils, and total and inflammatory monocytes (Ly6C/Ghi). LpL deficiency also significantly decreased expression of BM macrophage-associated markers (F4/80 and TNF-α [tumor necrosis factor α]), master transcription factors (PU.1 and C/EBPα), and colony-stimulating factors (CSFs) and their receptors, which are required for monocyte and monocyte precursor proliferation and differentiation. As a result, differentiation of macrophages from BM-derived monocyte progenitors and monocytes was decreased in MCKL0 mice. Furthermore, although LpL deficiency was associated with reduced BM uptake and accumulation of triglyceride-rich particles and macrophage CSF-macrophage CSF receptor binding, triglyceride lipolysis products (eg, linoleic acid) stimulated expression of macrophage CSF and macrophage CSF receptor in BM-derived macrophage precursor cells. Arterial macrophage numbers decreased after heparin-mediated LpL cell dissociation and by genetic knockout of arterial LpL. Reconstitution of LpL-expressing BM replenished aortic macrophage density. CONCLUSIONS: LpL regulates peripheral leukocyte levels and affects BM monocyte progenitor differentiation and aortic macrophage accumulation.

Cardiac Myocyte KLF5 Regulates Ppara Expression and Cardiac Function.

RATIONALE: Fatty acid oxidation is transcriptionally regulated by peroxisome proliferator-activated receptor (PPAR)α and under normal conditions accounts for 70% of cardiac ATP content. Reduced Ppara expression during sepsis and heart failure leads to reduced fatty acid oxidation and myocardial energy deficiency. Many of the transcriptional regulators of Ppara are unknown. OBJECTIVE: To determine the role of Krüppel-like factor 5 (KLF5) in transcriptional regulation of Ppara. METHODS AND RESULTS: We discovered that KLF5 activates Ppara gene expression via direct promoter binding. This is blocked in hearts of septic mice by c-Jun, which binds an overlapping site on the Ppara promoter and reduces transcription. We generated cardiac myocyte-specific Klf5 knockout mice that showed reduced expression of cardiac Ppara and its downstream fatty acid metabolism-related targets. These changes were associated with reduced cardiac fatty acid oxidation, ATP levels, increased triglyceride accumulation, and cardiac dysfunction. Diabetic mice showed parallel changes in cardiac Klf5 and Ppara expression levels. CONCLUSIONS: Cardiac myocyte KLF5 is a transcriptional regulator of Ppara and cardiac energetics.

Kidney triglyceride accumulation in the fasted mouse is dependent upon serum free fatty acids.

Lipid accumulation is a pathological feature of every type of kidney injury. Despite this striking histological feature, physiological accumulation of lipids in the kidney is poorly understood. We studied whether the accumulation of lipids in the fasted kidney are derived from lipoproteins or NEFAs. With overnight fasting, kidneys accumulated triglyceride, but had reduced levels of ceramide and glycosphingolipid species. Fasting led to a nearly 5-fold increase in kidney uptake of plasma [14C]oleic acid. Increasing circulating NEFAs using a ß adrenergic receptor agonist caused a 15-fold greater accumulation of lipid in the kidney, while mice with reduced NEFAs due to adipose tissue deficiency of adipose triglyceride lipase had reduced triglycerides. Cluster of differentiation (Cd)36 mRNA increased 2-fold, and angiopoietin-like 4 (Angptl4), an LPL inhibitor, increased 10-fold. Fasting-induced kidney lipid accumulation was not affected by inhibition of LPL with poloxamer 407 or by use of mice with induced genetic LPL deletion. Despite the increase in CD36 expression with fasting, genetic loss of CD36 did not alter fatty acid uptake or triglyceride accumulation. Our data demonstrate that fasting-induced triglyceride accumulation in the kidney correlates with the plasma concentrations of NEFAs, but is not due to uptake of lipoprotein lipids and does not involve the fatty acid transporter, CD36.

Cardiomyocyte-specific loss of diacylglycerol acyltransferase 1 (DGAT1) reproduces the abnormalities in lipids found in severe heart failure.

Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final step in triglyceride synthesis, the conversion of diacylglycerol (DAG) to triglyceride. Dgat1(-/-) mice exhibit a number of beneficial metabolic effects including reduced obesity and improved insulin sensitivity and no known cardiac dysfunction. In contrast, failing human hearts have severely reduced DGAT1 expression associated with accumulation of DAGs and ceramides. To test whether DGAT1 loss alone affects heart function, we created cardiomyocyte-specific DGAT1 knock-out (hDgat1(-/-)) mice. hDgat1(-/-) mouse hearts had 95% increased DAG and 85% increased ceramides compared with floxed controls. 50% of these mice died by 9 months of age. The heart failure marker brain natriuretic peptide increased 5-fold in hDgat1(-/-) hearts, and fractional shortening (FS) was reduced. This was associated with increased expression of peroxisome proliferator-activated receptor α and cluster of differentiation 36. We crossed hDgat1(-/-) mice with previously described enterocyte-specific Dgat1 knock-out mice (hiDgat1(-/-)). This corrected the early mortality, improved FS, and reduced cardiac ceramide and DAG content. Treatment of hDgat1(-/-) mice with the glucagon-like peptide 1 receptor agonist exenatide also improved FS and reduced heart DAG and ceramide content. Increased fatty acid uptake into hDgat1(-/-) hearts was normalized by exenatide. Reduced activation of protein kinase Cα (PKCα), which is increased by DAG and ceramides, paralleled the reductions in these lipids. Our mouse studies show that loss of DGAT1 reproduces the lipid abnormalities seen in severe human heart failure.

Lipoprotein lipase activity is required for cardiac lipid droplet production.

The rodent heart accumulates TGs and lipid droplets during fasting. The sources of heart lipids could be either FFAs liberated from adipose tissue or FAs from lipoprotein-associated TGs via the action of lipoprotein lipase (LpL). Because circulating levels of FFAs increase during fasting, it has been assumed that albumin transported FFAs are the source of lipids within heart lipid droplets. We studied mice with three genetic mutations: peroxisomal proliferator-activated receptor α deficiency, cluster of differentiation 36 (CD36) deficiency, and heart-specific LpL deletion. All three genetically altered groups of mice had defective accumulation of lipid droplet TGs. Moreover, hearts from mice treated with poloxamer 407, an inhibitor of lipoprotein TG lipolysis, also failed to accumulate TGs, despite increased uptake of FFAs. TG storage did not impair maximal cardiac function as measured by stress echocardiography. Thus, LpL hydrolysis of circulating lipoproteins is required for the accumulation of lipids in the heart of fasting mice.

CD36-deficient mice are resistant to alcohol- and high-carbohydrate-induced hepatic steatosis.

CD36 is a scavenger receptor with multiple ligands and cellular functions, including facilitating cellular uptake of free fatty acids (FFAs). Chronic alcohol consumption increases hepatic CD36 expression, leading to the hypothesis that this promotes uptake of circulating FFAs, which then serve as a substrate for triglyceride (TG) synthesis and the development of alcoholic steatosis. We investigated this hypothesis in alcohol-fed wild-type and Cd36-deficient (Cd36(-/-)) mice using low-fat/high-carbohydrate Lieber-DeCarli liquid diets, positing that Cd36(-/-) mice would be resistant to alcoholic steatosis. Our data show that the livers of Cd36(-/-) mice are resistant to the lipogenic effect of consuming high-carbohydrate liquid diets. These mice also do not further develop alcoholic steatosis when chronically fed alcohol. Surprisingly, we did not detect an effect of alcohol or CD36 deficiency on hepatic FFA uptake; however, the lower baseline levels of hepatic TG in Cd36(-/-) mice fed a liquid diet were associated with decreased expression of genes in the de novo lipogenesis pathway and a lower rate of hepatic de novo lipogenesis. In conclusion, Cd36(-/-) mice are resistant to hepatic steatosis when fed a high-carbohydrate liquid diet, and they are also resistant to alcoholic steatosis. These studies highlight an important role for CD36 in hepatic lipid homeostasis that is not associated with hepatic fatty acid uptake.

Adipose-specific lipoprotein lipase deficiency more profoundly affects brown than white fat biology.

Adipose fat storage is thought to require uptake of circulating triglyceride (TG)-derived fatty acids via lipoprotein lipase (LpL). To determine how LpL affects the biology of adipose tissue, we created adipose-specific LpL knock-out (ATLO) mice, and we compared them with whole body LpL knock-out mice rescued with muscle LpL expression (MCK/L0) and wild type (WT) mice. ATLO LpL mRNA and activity were reduced, respectively, 75 and 70% in gonadal adipose tissue (GAT), 90 and 80% in subcutaneous tissue, and 84 and 85% in brown adipose tissue (BAT). ATLO mice had increased plasma TG levels associated with reduced chylomicron TG uptake into BAT and lung. ATLO BAT, but not GAT, had altered TG composition. GAT from MCK/L0 was smaller and contained less polyunsaturated fatty acids in TG, although GAT from ATLO was normal unless LpL was overexpressed in muscle. High fat diet feeding led to less adipose in MCK/L0 mice but TG acyl composition in subcutaneous tissue and BAT reverted to that of WT. Therefore, adipocyte LpL in BAT modulates plasma lipoprotein clearance, and the greater metabolic activity of this depot makes its lipid composition more dependent on LpL-mediated uptake. Loss of adipose LpL reduces fat accumulation only if accompanied by greater LpL activity in muscle. These data support the role of LpL as the "gatekeeper" for tissue lipid distribution.

Cardiomyocyte specific deficiency of serine palmitoyltransferase subunit 2 reduces ceramide but leads to cardiac dysfunction.

The role of serine palmitoyltransferase (SPT) and de novo ceramide biosynthesis in cardiac ceramide and sphingomyelin metabolism is unclear. To determine whether the de novo synthetic pathways, rather than ceramide uptake from circulating lipoproteins, is important for heart ceramide levels, we created cardiomyocyte-specific deficiency of Sptlc2, a subunit of SPT. Heart-specific Sptlc2-deficient (hSptlc2 KO) mice had a >35% reduction in ceramide, which was limited to C18:0 and very long chain ceramides. Sphingomyelinase expression, and levels of sphingomyelin and diacylglycerol were unchanged. But surprisingly phospholipids and acyl CoAs contained increased saturated long chain fatty acids. hSptlc2 KO mice had decreased fractional shortening and thinning of the cardiac wall. While the genes regulating glucose and fatty acid metabolism were not changed, expression of cardiac failure markers and the genes involved in the formation of extracellular matrices were up-regulated in hSptlc2 KO hearts. In addition, ER-stress markers were up-regulated leading to increased apoptosis. These results suggest that Sptlc2-mediated de novo ceramide synthesis is an essential source of C18:0 and very long chain, but not of shorter chain, ceramides in the heart. Changes in heart lipids other than ceramide levels lead to cardiac toxicity.

Rescue of heart lipoprotein lipase-knockout mice confirms a role for triglyceride in optimal heart metabolism and function.

Hearts utilize fatty acids as a primary source of energy. The sources of those lipids include free fatty acids and lipoprotein triglycerides. Deletion of the primary triglyceride-hydrolyzing enzyme lipoprotein lipase (LPL) leads to cardiac dysfunction. Whether heart LPL-knockout (hLPL0) mice are compromised due a deficiency in energetic substrates is unknown. To test whether alternative sources of energy will prevent cardiac dysfunction in hLPL0 mice, two different models were used to supply nonlipid energy. 1) hLPL0 mice were crossed with mice transgenically expressing GLUT1 in cardiomyocytes to increase glucose uptake into the heart; this cross-corrected cardiac dysfunction, reduced cardiac hypertrophy, and increased myocardial ATP. 2) Mice were randomly assigned to a sedentary or training group (swimming) at 3 mo of age, which leads to increased skeletal muscle production of lactate. hLPL0 mice had greater expression of the lactate transporter monocarboxylate transporter-1 (MCT-1) and increased cardiac lactate uptake. Compared with hearts from sedentary hLPL0 mice, hearts from trained hLPL0 mice had adaptive hypertrophy and improved cardiac function. We conclude that defective energy intake and not the reduced uptake of fat-soluble vitamins or cholesterol is responsible for cardiac dysfunction in hLPL0 mice. In addition, our studies suggest that adaptations in cardiac metabolism contribute to the beneficial effects of exercise on the myocardium of patients with heart failure.

Human aldose reductase expression accelerates atherosclerosis in diabetic apolipoprotein E-/- mice.

OBJECTIVE: There are several pathways that mediate the aberrant metabolism of glucose and that might induce greater vascular damage in the setting of diabetes. The polyol pathway mediated by aldose reductase (AR) has been postulated to be one such pathway. However, it has been reported that AR reduces toxic lipid aldehydes and, under some circumstances, might be antiatherogenic. METHODS AND RESULTS: Atherosclerosis development was quantified in 2 lines of transgenic mice expressing human AR (hAR) crossed on the apolipoprotein E knockout background. The transgenes were used to increase the normally low levels of this enzyme in wild-type mice. Both generalized hAR overexpression and hAR expression via the Tie 2 promoter increased lesion size in streptozotocin diabetic mice. In addition, pharmacological inhibition of AR reduced lesion size. CONCLUSIONS: Although in some settings AR expression might reduce levels of toxic aldehydes, transgenic expression of this enzyme within the artery wall leads to greater atherosclerosis.

DGAT1 deficiency decreases PPAR expression and does not lead to lipotoxicity in cardiac and skeletal muscle.

Diacylglycerol (DAG) acyl transferase 1 (Dgat1) knockout ((-/-)) mice are resistant to high-fat-induced obesity and insulin resistance, but the reasons are unclear. Dgat1(-/-) mice had reduced mRNA levels of all three Ppar genes and genes involved in fatty acid oxidation in the myocardium of Dgat1(-/-) mice. Although DGAT1 converts DAG to triglyceride (TG), tissue levels of DAG were not increased in Dgat1(-/-) mice. Hearts of chow-diet Dgat1(-/-) mice were larger than those of wild-type (WT) mice, but cardiac function was normal. Skeletal muscles from Dgat1(-/-) mice were also larger. Muscle hypertrophy factors phospho-AKT and phospho-mTOR were increased in Dgat1(-/-) cardiac and skeletal muscle. In contrast to muscle, liver from Dgat1(-/-) mice had no reduction in mRNA levels of genes mediating fatty acid oxidation. Glucose uptake was increased in cardiac and skeletal muscle in Dgat1(-/-) mice. Treatment with an inhibitor specific for DGAT1 led to similarly striking reductions in mRNA levels of genes mediating fatty acid oxidation in cardiac and skeletal muscle. These changes were reproduced in cultured myocytes with the DGAT1 inhibitor, which also blocked the increase in mRNA levels of Ppar genes and their targets induced by palmitic acid. Thus, loss of DGAT1 activity in muscles decreases mRNA levels of genes involved in lipid uptake and oxidation.

Chylomicron- and VLDL-derived lipids enter the heart through different pathways: in vivo evidence for receptor- and non-receptor-mediated fatty acid uptake.

Lipids circulate in the blood in association with plasma lipoproteins and enter the tissues either after hydrolysis or as non-hydrolyzable lipid esters. We studied cardiac lipids, lipoprotein lipid uptake, and gene expression in heart-specific lipoprotein lipase (LpL) knock-out (hLpL0), CD36 knock-out (Cd36(-/-)), and double knock-out (hLpL0/Cd36(-/-)-DKO) mice. Loss of either LpL or CD36 led to a significant reduction in heart total fatty acyl-CoA (control, 99.5 ± 3.8; hLpL0, 36.2 ± 3.5; Cd36(-/-), 57.7 ± 5.5 nmol/g, p < 0.05) and an additive effect was observed in the DKO (20.2 ± 1.4 nmol/g, p < 0.05). Myocardial VLDL-triglyceride (TG) uptake was reduced in the hLpL0 (31 ± 6%) and Cd36(-/-) (47 ± 4%) mice with an additive reduction in the DKO (64 ± 5%) compared with control. However, LpL but not CD36 deficiency decreased VLDL-cholesteryl ester uptake. Endogenously labeled mouse chylomicrons were produced by tamoxifen treatment of ß-actin-MerCreMer/LpL(flox/flox) mice. Induced loss of LpL increased TG levels >10-fold and reduced HDL by >50%. After injection of these labeled chylomicrons in the different mice, chylomicron TG uptake was reduced by ∼70% and retinyl ester by ∼50% in hLpL0 hearts. Loss of CD36 did not alter either chylomicron TG or retinyl ester uptake. LpL loss did not affect uptake of remnant lipoproteins from ApoE knock-out mice. Our data are consistent with two pathways for fatty acid uptake; a CD36 process for VLDL-derived fatty acid and a non-CD36 process for chylomicron-derived fatty acid uptake. In addition, our data show that lipolysis is involved in uptake of core lipids from TG-rich lipoproteins.

Cardiomyocyte lipids impair ß-adrenergic receptor function via PKC activation.

Normal hearts have increased contractility in response to catecholamines. Because several lipids activate PKCs, we hypothesized that excess cellular lipids would inhibit cardiomyocyte responsiveness to adrenergic stimuli. Cardiomyocytes treated with saturated free fatty acids, ceramide, and diacylglycerol had reduced cellular cAMP response to isoproterenol. This was associated with increased PKC activation and reduction of ß-adrenergic receptor (ß-AR) density. Pharmacological and genetic PKC inhibition prevented both palmitate-induced ß-AR insensitivity and the accompanying reduction in cell surface ß-ARs. Mice with excess lipid uptake due to either cardiac-specific overexpression of anchored lipoprotein lipase, PPARγ, or acyl-CoA synthetase-1 or high-fat diet showed reduced inotropic responsiveness to dobutamine. This was associated with activation of protein kinase C (PKC)α or PKCδ. Thus, several lipids that are increased in the setting of lipotoxicity can produce abnormalities in ß-AR responsiveness. This can be attributed to PKC activation and reduced ß-AR levels.

Regulation of plasma fructose and mortality in mice by the aldose reductase inhibitor lidorestat.

Aldose reductase (AR), an enzyme widely believed to be involved in the aberrant metabolism of glucose and development of diabetic complications, is expressed at low levels in the mouse. We studied whether expression of human AR (hAR), its inhibition with lidorestat, which is an AR inhibitor (ARI), and the presence of streptozotocin (STZ)-induced diabetes altered plasma fructose, mortality, and/or vascular lesions in low-density lipoprotein (LDL) receptor-deficient [Ldlr(-/-)] mice. Mice were made diabetic at 12 weeks of age with low-dose STZ treatment. Four weeks later, the diabetic animals (glucose > 20 mM) were blindly assigned to a 0.15% cholesterol diet with or without ARI. After 4 and 6 weeks, there were no significant differences in body weights or plasma cholesterol, triglyceride, and glucose levels between the groups. Diabetic Ldlr(-/-) mice receiving ARI had plasma fructose levels of 5.2 +/- 2.3 microg/ml; placebo-treated mice had plasma fructose levels of 12.08 +/- 7.4 microg/ml, p < 0.01, despite the induction of fructose-metabolizing enzymes, fructose kinase and adolase B. After 6 weeks, hAR/Ldlr(-/-) mice on the placebo-containing diet had greater mortality (31%, n = 9/26 versus 6%, n = 1/21, p < 0.05). The mortality rate in the ARI-treated group was similar to that in non-hAR-expressing mice. Therefore, diabetic hAR-expressing mice had increased fructose and greater mortality that was corrected by inclusion of lidorestat, an ARI, in the diet. If similar effects are found in humans, such treatment could improve clinical outcome in diabetic patients.

In vivo arterial lipoprotein lipase expression augments inflammatory responses and impairs vascular dilatation.

OBJECTIVE: Although epidemiologic data suggest that hypertriglyceridemia and elevated plasma levels of fatty acids are toxic to arteries, in vitro correlates have been inconsistent. To investigate whether increased endothelial cell expression of lipoprotein lipase (LpL), the primary enzyme creating free fatty acids from circulating triglycerides (TG), affects vascular function, we created transgenic mice that express human LpL (hLpL) driven by the promoter and enhancer of the Tie2 receptor. METHODS AND RESULTS: Mice expressing this transgene, denoted EC-hLpL and L for low and H for high expression, had decreased plasma TG levels compared with wild-type mice (WT): 106+/-31 in WT, 37+/-17 (line H), and 63+/-31 mg/dL (line L) because of a reduction in VLDL TG; plasma cholesterol and HDL levels were unaltered. Crossing a high expressing EC-hLpL transgene onto the LpL knockout background allowed for survival of the pups; TG in these mice was approximately equal to that of heterozygous LpL knockout mice. Surprisingly, under control conditions the EC-hLpL transgene did not alter arterial function or endothelial cell gene expression; however, after tumor necrosis factor (TNF)-alpha treatment, arterial vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and endogenous TNF-alpha mRNA levels were increased and arteries had impaired endothelium-dependent vasodilatation. This was associated with reduced eNOS dimers. CONCLUSIONS: Therefore, we hypothesize that excess vascular wall LpL augments vascular dysfunction in the setting of inflammation.

Human aldose reductase expression accelerates diabetic atherosclerosis in transgenic mice.

Direct evidence that hyperglycemia, rather than concomitant increases in known risk factors, induces atherosclerosis is lacking. Most diabetic mice do not exhibit a higher degree of atherosclerosis unless the development of diabetes is associated with more severe hyperlipidemia. We hypothesized that normal mice were deficient in a gene that accelerated atherosclerosis with diabetes. The gene encoding aldose reductase (AR), an enzyme that mediates the generation of toxic products from glucose, is expressed at low levels in murine compared with human tissues. Mice in which diabetes was induced through streptozotocin (STZ) treatment, but not nondiabetic mice, expressing human AR (hAR) crossed with LDL receptor-deficient (Ldlr-/-) C57BL/6 male mice had increased aortic atherosclerosis. Diabetic hAR-expressing heterozygous LDL receptor-knockout mice (Ldlr+/-) fed a cholesterol/cholic acid-containing diet also had increased aortic lesion size. Lesion area at the aortic root was increased by STZ treatment alone but was further increased by hAR expression. Macrophages from hAR-transgenic mice expressed more scavenger receptors and had greater accumulation of modified lipoproteins than macrophages from nontransgenic mice. Expression of genes that regulate regeneration of glutathione was reduced in the hAR-expressing aortas. Thus, hAR increases atherosclerosis in diabetic mice. Inhibitors of AR or other enzymes that mediate glucose toxicity could be useful in the treatment of diabetic atherosclerosis.