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Objective: HDL (high-density lipoprotein) can exert both anti-inflammatory and proinflammatory effects in macrophages due to its ability to induce cholesterol depletion. Because cholesterol depletion also increases sheddase activity of the membrane protease ADAM17 (ADAM metallopeptidase domain 17) in other cells, we examined if ADAM17 plays a role in HDL’s effects on inflammatory processes in macrophages in vitro and in vivo. Approach and Results: Sorted peritoneal macrophages from human APOA1 (apolipoprotein A1) transgenic LDL (low-density lipoprotein) receptor-deficient ( APOA1 Tg ; Ldlr −/− ) mice with and without myeloid cell-targeted ADAM17-deficiency were studied in parallel with wildtype and ADAM17-deficient bone marrow-derived macrophages stimulated with HDL in vitro. HDL increased ADAM17 expression and activity in macrophages. Furthermore, ADAM17-deficient macrophages exhibited reduced expression of ABCA1 (ATP-binding cassette A1) and reduced cholesterol efflux and were cholesterol loaded. This was caused by the absence of shedding of TNFα, a major ADAM17 substrate. Sorted thioglycollate-elicited peritoneal macrophages freshly isolated from APOA1 Tg ; Ldlr −/− mice, which have higher HDL levels than Ldlr −/− controls, showed reduced expression of interferon-inducible genes in response to lipopolysaccharide or interferon β, but exacerbated proinflammatory responses to lipopolysaccharide for Tnfa , Cxcl1 , Ccl2 , and Il1b , phenocopying cells stimulated with HDL in vitro. These effects were all prevented in ADAM17-deficient macrophages and associated with lower concentrations of large HDL particles in APOA1 Tg ; Ldlr −/− mice with myeloid cell-targeted ADAM17-deficiency. Conclusions: The increased cholesterol loading of ADAM17-deficient macrophages prevents both anti-inflammatory and proinflammatory responses of HDL. Our findings demonstrate a novel role for ADAM17 in maintaining cholesterol efflux in macrophages, thereby regulating the immune functions of these cells.

Loss-of-function mutations in the transcription factor CREB3L3 (CREBH) associate with severe hypertriglyceridemia in humans. CREBH is believed to lower plasma triglycerides by augmenting the activity of lipoprotein lipase (LPL). However, by using a mouse model of type 1 diabetes mellitus (T1DM), we found that greater liver expression of active CREBH normalized both elevated plasma triglycerides and cholesterol. Residual triglyceride-rich lipoprotein (TRL) remnants were enriched in apolipoprotein E (APOE) and impoverished in APOC3, an apolipoprotein composition indicative of increased hepatic clearance. The underlying mechanism was independent of LPL, as CREBH reduced both triglycerides and cholesterol in LPL-deficient mice. Instead, APOE was critical for CREBH's ability to lower circulating remnant lipoproteins because it failed to reduce TRL cholesterol in Apoe-/- mice. Importantly, individuals with CREB3L3 loss-of-function mutations exhibited increased levels of remnant lipoproteins that were deprived of APOE. Recent evidence suggests that impaired clearance of TRL remnants promotes cardiovascular disease in patients with T1DM. Consistently, we found that hepatic expression of CREBH prevented the progression of diabetes-accelerated atherosclerosis. Our results support the proposal that CREBH acts through an APOE-dependent pathway to increase hepatic clearance of remnant lipoproteins. They also implicate elevated levels of remnants in the pathogenesis of atherosclerosis in T1DM.

Type 1 diabetes mellitus (T1DM) increases the risk of atherosclerotic cardiovascular disease (CVD) in humans by poorly understood mechanisms. Using mouse models of T1DM-accelerated atherosclerosis, we found that relative insulin deficiency rather than hyperglycemia elevated levels of apolipoprotein C3 (APOC3), an apolipoprotein that prevents clearance of triglyceride-rich lipoproteins (TRLs) and their remnants. We then showed that serum APOC3 levels predict incident CVD events in subjects with T1DM in the Coronary Artery Calcification in Type 1 Diabetes (CACTI) study. To explore underlying mechanisms, we investigated the impact of Apoc3 antisense oligonucleotides (ASOs) on lipoprotein metabolism and atherosclerosis in a mouse model of T1DM. Apoc3 ASO treatment abolished the increased hepatic Apoc3 expression in diabetic mice - resulting in lower levels of TRLs - without improving glycemic control. APOC3 suppression also prevented arterial accumulation of APOC3-containing lipoprotein particles, macrophage foam cell formation, and the accelerated atherosclerosis in diabetic mice. Our observations demonstrate that relative insulin deficiency increases APOC3 and that this results in elevated levels of TRLs and accelerated atherosclerosis in a mouse model of T1DM. Because serum levels of APOC3 predicted incident CVD events in the CACTI study, inhibiting APOC3 might reduce CVD risk in T1DM patients.

Cardiovascular disease caused by atherosclerosis is the leading cause of mortality associated with type 2 diabetes and metabolic syndrome. Insulin therapy is often needed to improve glycemic control, but it does not clearly prevent atherosclerosis. Upon binding to the insulin receptor (IR), insulin activates distinct arms of downstream signaling. The IR-Akt arm is associated with blood glucose lowering and beneficial effects, whereas the IR-Erk arm might exert less desirable effects. We investigated whether selective activation of the IR-Akt arm, leaving the IR-Erk arm largely inactive, would result in protection from atherosclerosis in a mouse model of metabolic syndrome. The insulin mimetic peptide S597 lowered blood glucose and activated Akt in insulin target tissues, mimicking insulin’s effects, but only weakly activated Erk and even prevented insulin-induced Erk activation. Strikingly, S597 retarded atherosclerotic lesion progression through a process associated with protection from leukocytosis, thereby reducing lesional accumulation of inflammatory Ly6Chi monocytes. S597-mediated protection from leukocytosis was accompanied by reduced numbers of the earliest bone marrow hematopoietic stem cells and reduced IR-Erk activity in hematopoietic stem cells. This study provides a conceptually novel treatment strategy for advanced atherosclerosis associated with metabolic syndrome and type 2 diabetes.

In addition to increasing the risk of an initial myocardial infarction (MI), diabetes increases the risk of a recurrent MI. Previous work suggests that an experimental MI can accelerate atherosclerosis via monocytosis. To test whether diabetes and experimental MI synergize to accelerate atherosclerosis, we performed ligation of the left anterior descending coronary artery to induce experimental MI or sham surgery in nondiabetic and diabetic mice with preexisting atherosclerosis. All mice subjected to experimental MI had significantly reduced left ventricular function. In our model, in comparisons with nondiabetic sham mice, neither diabetes nor MI resulted in monocytosis. Neither diabetes nor MI led to increased atherosclerotic lesion size, but diabetes accelerated lesion progression, exemplified by necrotic core expansion. The necrotic core expansion was dependent on monocyte recruitment, as mice with myeloid cells deficient in the adhesion molecule integrin α4 were protected from necrotic core expansion. In summary, diabetes, but not MI, accelerates lesion progression, suggesting that the increased risk of recurrent MI in diabetes is due to a higher lesional burden and/or elevated risk factors rather than the acceleration of the underlying pathology from a previous MI.

In addition to increasing the risk of an initial myocardial infarction (MI), diabetes increases the risk of a recurrent MI. Work by others suggests that an experimental MI can accelerate atherosclerosis via monocytosis. To test if diabetes and experimental myocardial infarction synergize to accelerate atherosclerosis, we induced atherosclerosis in Ldlr-/-; Gp+ mice, lethally irradiated them and transplanted with either wild type (WT) or LysM-Cre-mediated CD49D-deficient bone marrow. Following recovery, diabetes was induced using lymphocytic choriomeningitis virus, or saline was used as a control. The mice were subjected to ligation of the left anterior descending coronary artery to induce experimental MI or a sham surgery and then maintained for an additional 3 weeks. Diabetic (D) mice exhibited hyperglycemia and elevated plasma cholesterol and triglyceride levels compared to nondiabetic (ND) mice, but neither hematopoietic genotype or MI influenced that. All mice subjected to MI had significantly reduced left ventricular function. Neither diabetes nor MI resulted in monocytosis compared to ND sham mice. Despite not affecting total lesion size, the combination of diabetes and MI resulted in larger necrotic cores in the aortic sinus and in the brachiocephalic artery (9.8 ± 1% of total lesion in aortic sinus in ND sham, 11.9 ± 1% in ND MI, 15.4 ± 1% in D sham and 18.1 ± 2% in D MI, P In summary, diabetes and experimental MI destabilize the atherosclerotic lesion, potentially explaining why patients with diabetes are at an increased risk of recurrent MI. Disclosure F. Kramer: None. A.M. Martinson: None. T. Papayannopoulou: None. C.E. Murry: Other Relationship; Self; Cytocardia. J.E. Kanter: None. Funding American Diabetes Association (1-16-IBS-153 to J.E.K.)

The dietary fatty acid 10,12 conjugated linoleic acid (10,12 CLA) promotes weight loss by increasing fat oxidation, but its effects on atherosclerosis are less clear. We recently showed that weight loss induced by 10,12 CLA in an atherosclerosis-susceptible mouse model with characteristics similar to human metabolic syndrome is accompanied by accumulation of alternatively activated macrophages within subcutaneous adipose tissue. The objective of this study was to evaluate whether 10,12 CLA-mediated weight loss was associated with an atheroprotective phenotype. Male low-density lipoprotein receptor deficient (Ldlr&minus, /&minus, ) mice were made obese with 12 weeks of a high-fat, high-sucrose diet feeding (HFHS: 36% fat, 36% sucrose, 0.15% added cholesterol), then either continued on the HFHS diet with or without caloric restriction (CR), or switched to a diet with 1% of the lard replaced by either 9,11 CLA or 10,12 CLA for 8 weeks. Atherosclerosis and lipid levels were quantified at sacrifice. Weight loss in mice following 10,12 CLA supplementation or CR as a weight-matched control group had improved cholesterol and triglyceride levels, yet only the 10,12 CLA-treated mice had improved en face and aortic sinus atherosclerosis. 10,12 CLA-supplemented mice had increased lesion macrophage content, with enrichment of surrounding perivascular adipose tissue (PVAT) alternative macrophages, which may contribute to the anti-atherosclerotic effect of 10,12 CLA.

Introduction: The acute-phase protein serum amyloid A (SAA) exists as 4 different subtypes in mice. Levels of the SAA3 subtype increase in response to acute inflammatory stimuli, and its expression is modestly and chronically elevated in adipose tissue and macrophages in obese mice. Previously we showed that SAA3 deficiency protected C57Bl/6 female mice, but not males, from obesity, adipose tissue inflammation, and hyperlipidemia in response to a high fat high sucrose diet (HFHS: 36% calories from fat, 36% calories from sucrose with 0.15% added cholesterol). We therefore investigated whether SAA3 deficiency modulates atherosclerosis in mice deficient in the low-density lipoprotein receptor (LDLR). Methods: We used two models of LDLR-deficiency to promote atherosclerosis in Saa3 +/+ and Saa3 -/- male and female mice: (1) 16 weekly injections of an LDLR antisense oligonucleotide (LDLR-ASO), or (2) genetic deletion of LDLR ( Ldlr -/- ). All mice consumed the HFHS diet for 16 weeks. Results: While both models of LDLR deficiency promoted hypercholesterolemia, Ldlr -/- mice had nearly 1.5- and 2-fold higher cholesterol levels than LDLR-ASO mice (males: 753±109 vs. 560±62 mg/dL, n=8, pLdlr -/- mice ( Saa3 -/- Ldlr -/- : 623±26 vs. Saa3 +/+ Ldlr -/- : 519±20 mg/dL, n=10, pen face aortic atherosclerotic lesion area was 2-fold greater in female Saa3 -/- Ldlr -/- mice than in their wild type counterparts (n=10, pSaa3 -/- mice in both models of hypercholesterolemia (n=8). Summary: While both LDLR-ASO and Ldlr -/- promote hypercholesterolemia in male and female mice, SAA3 deficiency worsens hypercholesterolemia and atherosclerosis in female mice, and reduces atherosclerosis in male mice. Conclusion: In the setting of hypercholesterolemia, SAA3 may be pro-atherogenic in male and atheroprotective in female mice, solidifying the sexual dimorphic nature of SAA3.

Elevated plasma triglycerides (TG) and VLDL are often present in poorly controlled type 1 diabetes mellitus (T1DM). Furthermore, T1DM patients exhibit an increased risk of cardiovascular disease (CVD) despite normal or high HDL-cholesterol. There is a need for new mouse models to study T1DM lipid abnormalities because mice do not express cholesteryl ester transfer protein (CETP), which transfers cholesteryl ester (CE) from HDL to VLDL and other lipoproteins in exchange for TG, and because mice do not exhibit human-like HDL populations. To develop a physiologically relevant model, we crossed a virally-induced mouse model of T1DM ( Ldlr -/- ;Gp Tg ) deficient in the LDL receptor with human apolipoprotein A-I ( APOA1 ) transgenic mice. These mice (n=8-10) were injected with a liver-specific adeno-associated virus driving expression of human CETP, mimicking the human CETP activity/apoA-I ratio, and the 3 major human HDL subpopulations. Using this model ( APOA1 Tg ;Ldlr -/- ;Gp Tg ), we examined the impact of diabetes on dyslipidemia, HDL particle concentration (HDL-P), and serum HDL function. Induction of diabetes increased plasma TG and cholesterol, primarily due to an increase in VLDL, as compared with non-diabetic (ND) littermates. The dyslipidemia was associated with increases in hepatic TG content and hepatic TG production rate (2-fold over ND). Moreover, resident peritoneal macrophages from diabetic mice exhibited a 5-fold increase in CE loading, increased lipid raft content (20%), and increased Tnfa (3-fold) and Il1b mRNA (4-fold), as compared with ND littermates. In addition, T1DM mice had increased small, medium and large HDL-P (2-fold) concentrations compared with ND controls. Serum HDL from T1DM mice showed a significant increase in its cholesterol efflux capacity through both ABCA1 and ABCG1. Thus, in this humanized mouse model, poorly controlled T1DM leads to elevated plasma TG and VLDL due in part to increased hepatic TG production. This dyslipidemia is associated with higher HDL-P and an increase in serum HDL’s cholesterol efflux capacity, likely due to the increased plasma HDL-P. We have found similar changes in HDL concentration and function in humans with T1DM, suggesting that our mouse model might provide insights into CVD risk in T1DM patients.

Human genomic studies have highlighted the importance of arterial wall-specific inflammatory processes in cardiovascular disease (CVD) risk. Diabetes increases systemic inflammation, local arterial inflammation, and CVD risk. To clarify the relative contributions of systemic inflammation versus artery wall inflammatory processes in atherosclerosis, we studied LDL receptor-deficient mice with streptozotocin-induced diabetes. The damage-associated molecular pattern protein S100A9 and toll-like receptor 4 (TLR4) have both been implicated in diabetes-induced inflammation. S100A9-deficient bone marrow chimeras were used to inhibit systemic inflammation, 5-aminosalicylic acid (5-ASA) was used to inhibit intestinal inflammation, and TLR4-deficient bone marrow chimeras were used to inhibit artery wall inflammation. No model affected the severity of diabetes, plasma cholesterol or blood leukocyte numbers. Hematopoietic S100A9-deficiency, but not TLR4-deficiency, reduced diabetes-associated systemic inflammation to levels observed in non-diabetic mice. 5-ASA differentially altered measures of systemic inflammation. Thus, diabetes induced a 2-fold increase in circulating leukocyte Il1b mRNA, which was normalized by S100A9-deficiency (pen face Sudan IV staining (n=16-21). Finally, laser capture microdissection of CD68-positive lesional macrophages revealed that hematopoietic TLR4-deficiency prevents diabetes-induced inflammatory processes in the artery wall including expression of Il1b, Ccr2 , and S100a9 mRNA. Together, our data strongly suggest that although systemic inflammation is increased in diabetes, inhibition of inflammatory processes in the artery wall is required to prevent lesional macrophage accumulation.

Diabetic kidney disease and atherosclerotic disease are major causes of morbidity and mortality associated with type 2 diabetes (T2D), and diabetic kidney disease is a major cardiovascular risk factor. The black and tan, brachyury (BTBR) mouse strain with leptin deficiency (Lepob) has emerged as one of the best models of human diabetic kidney disease. However, no T2D mouse model of combined diabetic kidney disease and atherosclerosis exists. Our goal was to generate such a model. To this end, the low-density lipoprotein (LDL) receptor was targeted for degradation via inducible degrader of the LDL receptor (IDOL) overexpression, using liver-targeted adenoassociated virus serotype DJ/8 (AAV-DJ/8) in BTBR wild-type and BTBR Lepob mice. Liver-targeted IDOL-AAV-DJ/8 increased plasma LDL cholesterol compared with the control enhanced green fluorescent protein AAV-DJ/8. IDOL-induced dyslipidemia caused formation of atherosclerotic lesions of an intermediate stage, which contained both macrophages and smooth muscle cells. BTBR Lepob mice exhibited diabetic kidney disease. IDOL-induced dyslipidemia worsened albuminuria and glomerular macrophage accumulation but had no effect on mesangial expansion or podocyte numbers. Thus, by inducing hepatic degradation of the LDL receptor, we generated a T2D model of combined kidney disease and atherosclerosis. This model provides a new tool to study mechanisms, interactions, and treatment strategies of kidney disease and atherosclerosis in T2D.

Type 1 diabetes mellitus (T1DM) is associated with cardiovascular complications induced by atherosclerosis. Prostaglandin E2 (PGE2) is often raised in states of inflammation, including diabetes, and regulates inflammatory processes. In myeloid cells, a key cell type in atherosclerosis, PGE2 acts predominately through its Prostaglandin E Receptor 4 (EP4; Ptger4) to modulate inflammation. The effect of PGE2-mediated EP4 signaling specifically in myeloid cells on atherosclerosis in the presence and absence of diabetes is unknown. Because diabetes promotes atherosclerosis through increased arterial myeloid cell accumulation, we generated a myeloid cell-targeted EP4-deficient mouse model (EP4M-/-) of T1DM-accelerated atherogenesis to investigate the relationship between myeloid cell EP4, inflammatory phenotypes of myeloid cells, and atherogenesis. Diabetic mice exhibited elevated plasma PGE metabolite levels and elevated Ptger4 mRNA in macrophages, as compared with non-diabetic littermates. PGE2 increased Il6, Il1b, Il23 and Ccr7 mRNA while reducing Tnfa mRNA through EP4 in isolated myeloid cells. Consistently, the stimulatory effect of diabetes on peritoneal macrophage Il6 was mediated by PGE2-EP4, while PGE2-EP4 suppressed the effect of diabetes on Tnfa in these cells. In addition, diabetes exerted effects independent of myeloid cell EP4, including a reduction in macrophage Ccr7 levels and increased early atherogenesis characterized by relative lesional macrophage accumulation. These studies suggest that this mouse model of T1DM is associated with increased myeloid cell PGE2-EP4 signaling, which is required for the stimulatory effect of diabetes on IL-6, markedly blunts the effect of diabetes on TNF-α and does not modulate diabetes-accelerated atherogenesis.

Cardiovascular disease, largely because of disruption of atherosclerotic lesions, accounts for the majority of deaths in people with type 1 diabetes. Recent mouse models have provided insights into the accelerated atherosclerotic lesion initiation in diabetes, but it is unknown whether diabetes directly worsens more clinically relevant advanced lesions. We therefore used an LDL receptor-deficient mouse model, in which type 1 diabetes can be induced at will, to investigate the effects of diabetes on preexisting lesions. Advanced lesions were induced by feeding mice a high-fat diet for 16 weeks before induction of diabetes. Diabetes, independently of lesion size, increased intraplaque hemorrhage and plaque disruption in the brachiocephalic artery of mice fed low-fat or high-fat diets for an additional 14 weeks. Hyperglycemia was not sufficient to induce plaque disruption. Furthermore, diabetes resulted in increased accumulation of monocytic cells positive for S100A9, a proinflammatory biomarker for cardiovascular events, and for a macrophage marker protein, without increasing lesion macrophage content. S100A9 immunoreactivity correlated with intraplaque hemorrhage. Aggressive lowering primarily of triglyceride-rich lipoproteins prevented both plaque disruption and the increased S100A9 in diabetic atherosclerotic lesions. Conversely, oleate promoted macrophage differentiation into an S100A9-positive population in vitro , thereby mimicking the effects of diabetes. Thus, diabetes increases plaque disruption, independently of effects on plaque initiation, through a mechanism that requires triglyceride-rich lipoproteins and is associated with an increased accumulation of S100A9-positive monocytic cells. These findings indicate an important link between diabetes, plaque disruption, and the innate immune system.

Very low-density lipoprotein (VLDL) and LDL plasma levels are associated with cardiovascular mortality. Whereas VLDL/LDL lowering causes regression of early atherosclerotic lesions, less is known about the effects of aggressive lipid lowering on regression of advanced complex lesions. We therefore investigated the effect of VLDL/LDL lowering on pre-existing lesions in LDL receptor-deficient mice. Mice fed a high-fat diet for 16 weeks developed advanced lesions with fibrous caps, necrotic cores, and cholesterol clefts in the brachiocephalic artery. After an additional 14 weeks on a low-fat diet, plasma cholesterol levels decreased from 21.0 +/- 2.6 to 8.4 +/- 0.6 mmol/L, but lesions did not regress. Levels of VLDL/LDL were further lowered by using a helper-dependent adenovirus encoding the VLDL receptor (HD-Ad-VLDLR) under control of a liver-selective promoter. Treatment with HD-Ad-VLDLR together with a low-fat diet regimen resulted in reduced lesion size (cross-sectional area decreased from 146,272 +/- 19,359 to 91,557 +/- 15,738 microm2) and an 89% reduction in the cross-sectional lesion area occupied by macrophages compared to controls. These results show that aggressive VLDL/LDL lowering achieved by hepatic overexpression of VLDLR combined with a low-fat diet regimen induces regression of advanced plaques in the brachiocephalic artery of LDL receptor-deficient mice.

Hyperglycemia and hyperlipidemia are important risk factors for diabetes-accelerated atherosclerosis. Macrophage proliferation has been implicated in the progression of atherosclerosis. We therefore investigated the effects of hyperglycemia and hyperlipidemia on macrophage proliferation in murine atherosclerotic lesions and isolated primary macrophages. Hyperglycemic LDL receptor-deficient mice that were fed a cholesterol-free diet for 12 weeks did not have elevated cholesterol levels compared with nondiabetic mice, and there was no evidence of increased macrophage proliferation in atherosclerotic lesions. Moreover, elevated glucose levels did not increase proliferation of isolated mouse peritoneal macrophages. In contrast, hyperglycemic LDL receptor-deficient mice that were fed a cholesterol-rich diet showed increased cholesterol levels concomitant with macrophage proliferation in atherosclerotic lesions. Glucose promoted lipid and protein oxidation of LDL in vitro. Glucose-oxidized LDL resulted in phosphorylation of extracellular signal-regulated kinase and protein kinase B/Akt and stimulated proliferation of isolated macrophages. The mitogenic effect of glucose-oxidized LDL was mediated by CD36 and by extracellular signal-regulated kinase activation induced by protein kinase C-dependent and phosphatidylinositol 3-kinase-dependent pathways. Thus, hyperglycemia is not sufficient to stimulate macrophage proliferation in lesions of atherosclerosis or in isolated macrophages. A combination of hyperglycemia and hyperlipidemia, however, stimulates macrophage proliferation by a pathway that may involve the glucose-dependent oxidation of LDL.

Diabetes accelerates cardiovascular disease caused by atherosclerosis. Accordingly, diabetes accelerates atherosclerotic lesion progression and increases arterial smooth muscle cell proliferation. We hypothesized that diabetes can exert growth-promoting effects on smooth muscle cells via increased advanced glycation end-products or by dyslipidaemia.Primary human arterial smooth muscle cells were stimulated with advanced glycation end-products, other ligands of the receptor for advanced glycation end-products or fatty acids common in triglycerides. Cell proliferation was measured as DNA synthesis, cell cycle distribution and cell number. Effects of oleate on cellular phospholipids, diacylglycerol, triglycerides and cholesterol esters were analyzed by thin-layer chromatography, and oleate accumulation into diacylglycerol was confirmed by gas chromatography.Human arterial smooth muscle cells express the receptor for advanced glycation end-products, but its ligands N(epsilon)-(carboxymethyl)lysine-modified proteins, methylglyoxal-modified proteins, S100B polypeptide and amyloid-beta (1-40) peptide, exert no mitogenic action. Instead, oleate, one of the most common fatty acids in triglycerides, enhances platelet-derived growth factor-BB-mediated proliferation and oleate-containing 1,2-diacylglycerol formation in smooth muscle cells. This mitogenic effect of oleate depends on phospholipase D activity and is associated with an increased formation of oleate-enriched 1,2-diacylglycerol.Oleate, not ligands of the receptor for advanced glycation end-products, acts as an enhancer of human smooth muscle cell proliferation. Thus, lipid abnormalities, rather than hyperglycaemia, could be a major factor promoting proliferation of smooth muscle cells in atherosclerotic lesions.

Diabetes causes accelerated atherosclerosis and subsequent cardiovascular disease through mechanisms that are poorly understood. We have previously shown, using a porcine model of diabetes-accelerated atherosclerosis, that diabetes leads to an increased accumulation and proliferation of arterial smooth muscle cells in atherosclerotic lesions and that this is associated with elevated levels of plasma triglycerides. We therefore used the same model to investigate the mechanism whereby diabetes may stimulate smooth muscle cell proliferation. We show that lesions from diabetic pigs fed a cholesterol-rich diet contain abundant insulin-like growth factor-I (IGF-I), in contrast to lesions from non-diabetic pigs. Furthermore, two fatty acids common in triglycerides, oleate and linoleate, enhance the growth-promoting effects of IGF-I in smooth muscle cells isolated from these animals. These fatty acids accumulate predominantly in the membrane phospholipid pool; oleate accumulates preferentially in phosphatidylcholine and phosphatidylethanolamine, whereas linoleate is found mainly in phosphatidylethanolamine. The growth-promoting effects of oleate and linoleate depend on phospholipid hydrolysis by phospholipase D and subsequent generation of diacylglycerol. Thus, concurrent increases in levels of IGF-I and triglyceride-derived oleate and linoleate in lesions may contribute to accumulation and proliferation of smooth muscle cells and lesion progression in diabetes-accelerated atherosclerosis.

The mechanisms that promote an inflammatory environment and accelerated atherosclerosis in diabetes are poorly understood. We show that macrophages isolated from two different mouse models of type 1 diabetes exhibit an inflammatory phenotype. This inflammatory phenotype associates with increased expression of long-chain acyl-CoA synthetase 1 (ACSL1), an enzyme that catalyzes the thioesterification of fatty acids. Monocytes from humans and mice with type 1 diabetes also exhibit increased ACSL1. Furthermore, myeloid-selective deletion of ACSL1 protects monocytes and macrophages from the inflammatory effects of diabetes. Strikingly, myeloid-selective deletion of ACSL1 also prevents accelerated atherosclerosis in diabetic mice without affecting lesions in nondiabetic mice. Our observations indicate that ACSL1 plays a critical role by promoting the inflammatory phenotype of macrophages associated with type 1 diabetes; they also raise the possibilities that diabetic atherosclerosis has an etiology that is, at least in part, distinct from the etiology of nondiabetic vascular disease and that this difference is because of increased monocyte and macrophage ACSL1 expression.