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Serum apolipoprotein C3 (APOC3) predicts incident cardiovascular events in people with type 1 diabetes and silencing of APOC3 prevents both lesion initiation and advanced lesion necrotic core expansion in a mouse model of type 1 diabetes. APOC3 acts by slowing the clearance of triglyceride-rich lipoproteins, but lipid-free APOC3 has recently been reported to activate an inflammasome pathway in monocytes. We therefore investigated the contribution of hematopoietic inflammasome pathways to atherosclerosis in mouse models of type 1 diabetes. LDL receptor-deficient diabetes mouse models were transplanted with bone marrow from donors deficient in NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), absent in melanoma 2 (AIM2), or gasdermin D (GSDMD), an inflammasome-induced executor of pyroptotic cell death. Mice with diabetes exhibited inflammasome activation, and consistently, increased plasma interleukin-1b (IL-1b) and IL-18. Hematopoietic deletions of NLRP3, AIM2 or GSDMD caused smaller atherosclerotic lesions in diabetic mice. The increased lesion necrotic core size in diabetic mice was independent of macrophage pyroptosis because hematopoietic GSDMD-deficiency failed to prevent necrotic core expansion in advanced lesions. Our findings demonstrate that AIM2 and NLRP3 inflammasomes contribute to atherogenesis in diabetes and suggest that necrotic core expansion is independent of macrophage pyroptosis.

This manuscript was sent to Joyce Bischoff, Guest Editor, for review by expert referees, editorial decision, and final disposition. Final decisions were approved by Jane Leopold, Guest Editor-in-Chief.

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.

Diabetes increases the risk of both cardiovascular disease and kidney disease. Notably, most of the excess cardiovascular risk in people with diabetes is in those with kidney disease. Apolipoprotein C3 (APOC3) is a small apolipoprotein that is elevated by insulin-insufficiency and regulates plasma triglyceride levels. To test if APOC3, and the dyslipidemia it represents, play a role in diabetic kidney disease (DKD) and associated atherosclerosis, we treated BTBR wildtype (WT) and leptin-deficient (OB; diabetic) mice with an antisense oligonucleotide (ASO) to APOC3 or a control ASO (cASO), all in the setting of human-like dyslipidemia (accomplished by administration of an ASO targeting the LDLR). APOC3 ASO treatment reduced triglycerides, triglyceride-rich lipoproteins. It prevented diabetes-accelerated atherosclerosis in the aortic sinus, brachiocephalic artery, and aorta (sinus lesion: 67926 ± 8486 μm 2 lesion in cASO-treated OB mice compared to 38589 ± 9507 μm 2 in APOC3 ASO-treated OB mice, p

ADAM17 (A Disintegrin-like And Metalloproteinase 17) is a transmembrane protease that cleaves its substrates at the cell surface. Cell type-specific substrates cleaved by ADAM17, such as TNFα, can function locally and systemically. When fed a diet enriched in saturated fat, sucrose, and added cholesterol (DDC), Ldlr -/- mice develop prediabetes characterized by elevated triglyceride-rich lipoproteins (TRLs), obesity, glucose intolerance and atherosclerosis. Our results demonstrate that when ADAM17 is deleted from hematopoietic cells, DDC-fed Adam17 H-/- Ldlr -/- bone marrow transplant chimeras (n=15-30) are less obese and more insulin sensitive than wildtype Ldlr -/- chimeras. Adam17 H-/- chimeras show decreased plasma cholesterol and triglycerides, primarily due to a reduction in plasma VLDL assayed by FPLC fractionation. Atherosclerotic lesions are significantly (p Adam17 M-/- Ldlr -/- bone marrow transplant chimeras recapitulate the phenotypes of Adam17 H-/- mice, suggesting that the phenotypes are mediated by myeloid cells. Mechanistic studies indicate that the reduced plasma VLDL levels in ADAM17-deficient chimeras are not due to reduced VLDL production or reduced intestinal uptake of triglycerides. Furthermore, analysis of pre- and post-heparin plasma demonstrates that lipoprotein lipase activity is not increased in the ADAM17-/- chimeras. Instead, characterization of the TRLs by proteomics and differential ion mobility analysis revealed that ADAM17-deficient chimeras have reduced levels of 25-30 nm TRL remnant particles (e.g., p=0.027 for 25 nm particles; n=8-10) and reduced levels of APOC1, APOC2 and APOC3 per particle. Together, these results are consistent with the proposal that deletion of ADAM17 in myeloid cells results in increased hepatic clearance of TRLs and their remnants. Our work suggests that myeloid cells play important roles in regulating hepatic TRL remnant clearance and associated atherosclerosis in a model of prediabetes.

Cardiovascular disease (CVD) risk is strongly and inversely associated with plasma levels of high-density lipoprotein cholesterol (HDL-C). Recent clinical studies suggest that the association between HDL-C and CVD risk is indirect and that HDL’s cardioprotective function may be attributed to its ability to promote cholesterol efflux from macrophages. Apolipoprotein A-I (APOA1), the major protein in HDL, serves as a structural scaffold to facilitate protein binding and enzyme activation. Our previous work demonstrated that two or three molecules of APOA1 form antiparallel dimer structures (rotamers) that wrap around discoidal and spherical HDL. Human HDL contains both LL5/5 and LL5/4 rotamers. Because little is known about the impact of different rotamers on HDL function, we generated human APOA1 mutants with single cysteine mutations that produced reconstituted HDL (rHDL) locked in specific rotamer (K133C for LL5/5 and L122C for LL5/4). The LL5/5 rotamer of rHDL demonstrated four-fold higher LCAT activation efficiency than the LL5/4 rotamer. To investigate the roles of APOA1 rotamers in vivo , we used a liver-targeted adeno-associated virus (AAV) to express WT hAPOA1 and the two cysteine mutants in APOA1-deficient ( Apoa1 -/- ) mice. HDL particle concentration (HDL-P) in WT and LL5/5 mice were similar but were 250% higher (n=8; P

Diabetes accelerates atherosclerosis progression in part by increasing lesional necrotic core expansion, but the underlying mechanisms remain elusive. Previous studies have shown that systemic inhibition of the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome prevents atherosclerosis progression in a mouse model of diabetes. Gasdermin D (GSDMD), which acts downstream of inflammasome activation, contributes to pyroptosis and IL-1β and IL-18 release. To explore if hematopoietic inflammasome activation mediates diabetes-related necrotic core expansion, we first generated hematopoietic GSDMD-deficient chimeras in an LDL receptor-deficient ( Ldlr -/- ) virally-induced mouse model of type 1 diabetes (T1D)-accelerated atherosclerosis. Mice with T1D demonstrated elevated plasma glucose, triglycerides, cholesterol, and plasma IL-18 (non-diabetes [ND]=180.4 pg/ml, diabetes [D]=316.6, N=17-18 per group). However, diabetic mice with hematopoietic GSDMD-deficiency had no reduction in plasma IL-18 (D-KO=437.5 pg/ml, p=0.29, N=17). Moreover, we found little evidence of inflammasome activation in peritoneal and splenic macrophages from diabetic mice. A 4-week duration of diabetes significantly increased necrotic core area measured at three different levels in the aortic sinus (p Ldlr -/- hematopoietic NLRP3 and absent in melanoma 2 (AIM2) inflammasome double knockout (DKO) chimeras in a streptozotocin mouse model of diabetes. Mice with diabetes demonstrated hyperglycemia and hypercholesterolemia. However, diabetic hematopoietic DKO chimeras did not exhibit reduced aortic sinus necrotic core area, as compared with diabetic wildtype controls. Our findings suggest that the increased plasma IL-18 levels in diabetic mice are not dependent on hematopoietic GSDMD, and that diabetes-mediated necrotic core expansion is independent of hematopoietic NLRP3 and AIM2 inflammasome and pyroptosis pathways.

Despite the benefits of LDL-cholesterol-reducing medications, patients with type 1 diabetes (T1D) continue to have an increased risk of incident CVD. Preclinical and clinical observations indicate that triglyceride-rich lipoproteins (TRLs) and their remnants may contribute significantly to CVD risk in T1D. Recently, we have shown that diabetes increases levels of atherogenic TRL remnants and that improvement of remnant clearance halts atherosclerosis in a mouse model of T1D. Cholesteryl ester transfer protein (CETP), which is normally absent in mice, links metabolism of TRLs to that of HDL by mediating the transfer of cholesteryl ester from HDL to VLDL and other APOB-containing lipoproteins in exchange for triglycerides. We hypothesized that CETP impairs the atheroprotective effects of APOA1 by altering TRL and HDL metabolism in diabetes. To test this hypothesis, we developed a virally-induced T1D mouse model expressing the human APOA1 transgene ( APOA1 Tg , Ldlr –/– ). Cohorts of these mice were injected with a liver-targeted adeno-associated virus to express human CETP, and the effects on lipoproteins, plasma apolipoproteins, HDL particle concentrations (HDL-P), and atherosclerosis in the presence and absence of diabetes were determined. This mouse model exhibits an HDL profile similar to humans, with small, medium and large HDL-P, whereas wildtype ( Ldlr -/- ) littermates have only one sized HDL-P. Expression of the APOA1 transgene significantly suppressed diabetes-accelerated necrotic core expansion and accumulation of APOB and APOE in the aortic sinus (compared to wildtype Ldlr -/- mice), while CETP impaired the protective effect of APOA1 on these parameters in diabetic APOA1 Tg , Ldlr –/– mice (n= 22-30, p < 0.05). Further characterization of plasma lipoproteins and apolipoproteins by targeted mass spectrometry, FPLC and differential ion mobility analysis indicated that CETP leads to smaller TRL remnant particles, increases triglyceride content in HDL, lower levels of plasma APOA1 and reduction in large HDL-P under diabetic conditions (n= 8-10, p < 0.05). Our results are consistent with the proposal that inhibition of CETP might be cardioprotective in the setting of T1D and highlight the close links between TRL and HDL metabolism in diabetes.

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.

Chronic inflammation contributes to cardiovascular disease. Increased levels of the inflammatory cytokine, TNF-α, are often present in conditions associated with cardiovascular disease risk, and TNF-α induces a number of pro-atherogenic effects in macrovascular endothelial cells, including expression of adhesion molecules and chemokines, and lipoprotein uptake and transcytosis to the subendothelial tissue. However, little is known about the roles of acyl-CoA synthetases (ACSLs), enzymes that esterify free fatty acids into their acyl-CoA derivatives, or about the effects of TNF-α on ACSLs in endothelial cells. Therefore, we investigated the effects of TNF-α on ACSLs and downstream lipids in cultured human coronary artery endothelial cells and human umbilical vein endothelial cells. We demonstrated that TNF-α induces ACSL1, ACSL3, and ACSL5, but not ACSL4, in both cell types. TNF-α also increased oleoyl-CoA levels, consistent with the increased ACSL3 expression. RNA-sequencing demonstrated that knockdown of ACSL3 had no marked effects on the TNF-α transcriptome. Instead, ACSL3 was required for TNF-α-induced lipid droplet formation in cells exposed to oleic acid. These results demonstrate that increased acyl-CoA synthesis as a result of ACSL3 induction is part of the TNF-α response in human macrovascular endothelial cells.

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.

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 if diabetes and experimental myocardial infarction synergize to accelerate atherosclerosis, we performed ligation of the left anterior descending coronary artery to induce experimental MI or sham surgery in non-diabetic and diabetic mice with pre-existing atherosclerosis. All mice subjected to experimental MI had significantly reduced left ventricular function. In our model, neither diabetes nor MI resulted in monocytosis compared to non-diabetic sham mice. 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 because mice with myeloid cells deficient in the adhesion molecule alpha 4 integrin 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 the acceleration of the underlying pathology from a previous MI.

The molecular mechanisms of prostate inflammation are unclear. We hypothesized that heme oxygenase 1 (HMOX1; HO-1), an enzyme responsible for degradation of heme to carbon monoxide, bilirubin, and iron, is an important regulator of inflammation and epithelial responses in the prostate. Injection of non-uropathogenic Escherichia coli (MG1655 strain) or phosphate-buffered saline into the urethra of mice led to increased numbers of CD45(+) leukocytes and mitotic markers (phosphorylated histone H(3) and phosphorylated ERK1/2) in the prostate glands. Leukocyte infiltration was elevated in the prostates harvested from mice lacking HO-1 in myeloid compartment. Conversely, exogenous carbon monoxide (250 ppm) increased IL-1β levels and suppressed cell proliferation in the prostates. Carbon monoxide did not affect the number of infiltrating CD45(+) cells in the prostates of E. coli– or phosphate-buffered saline–treated mice. Interestingly, immunomodulatory effects of HO-1 and/or carbon monoxide correlated with early induction of the long-chain acyl-CoA synthetase 1 (ACSL1). ACSL1 levels were elevated in response to E. coli treatment, and macrophage-expressed ACSL1 was in part required for controlling of IL-1β expression and prostate cancer cell colony growth in soft agar. These results suggest that HO-1 and/or carbon monoxide might play a distinctive role in modulating prostate inflammation, cell proliferation, and IL-1β levels in part via an ACSL1-mediated pathway.

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.

Acyl-CoA thioesterase 7 (ACOT7) is an intracellular enzyme that converts acyl-CoAs to FFAs. ACOT7 is induced by lipopolysaccharide (LPS); thus, we investigated downstream effects of LPS-induced induction of ACOT7 and its role in inflammatory settings in myeloid cells. Enzymatic thioesterase activity assays in WT and ACOT7-deficient macrophage lysates indicated that endogenous ACOT7 contributes a significant fraction of total acyl-CoA thioesterase activity toward C20:4-, C20:5-, and C22:6-CoA, but contributes little activity toward shorter acyl-CoA species. Lipidomic analyses revealed that LPS causes a dramatic increase, primarily in bis(monoacylglycero)phosphate species containing long (≥C20) polyunsaturated acyl-chains in macrophages, and that the limited effect observed by ACOT7 deficiency is restricted to glycerophospholipids containing 20-carbon unsaturated acyl-chains. Furthermore, ACOT7 deficiency did not detectably alter the ability of LPS to induce cytokines or prostaglandin E2 production in macrophages. Consistently, although ACOT7 was induced in macrophages from diabetic mice, hematopoietic ACOT7 deficiency did not alter the stimulatory effect of diabetes on systemic inflammation or atherosclerosis in LDL receptor-deficient mice. Thus, inflammatory stimuli induce ACOT7 and remodeling of phospholipids containing unsaturated long (≥C20)-acyl chains in macrophages, and, although ACOT7 has preferential thioesterase activity toward these lipid species, loss of ACOT7 has no major detrimental effect on macrophage inflammatory phenotypes.≥

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.

Metabolic syndrome contributes to cardiovascular disease partly through systemic risk factors. However, local processes in the artery wall are becoming increasingly recognized to exacerbate atherosclerosis both in mice and humans. We show that arterial smooth muscle cell (SMC) glucose metabolism markedly synergizes with metabolic syndrome in accelerating atherosclerosis progression, using a low-density lipoprotein receptor–deficient mouse model. SMCs in proximity to atherosclerotic lesions express increased levels of the glucose transporter GLUT1. Cytokines, such as TNF-α produced by lesioned arteries, promote GLUT1 expression in SMCs, which in turn increases expression of the chemokine CCL2 through increased glycolysis and the polyol pathway. Furthermore, overexpression of GLUT1 in SMCs, but not in myeloid cells, accelerates development of larger, more advanced lesions in a mouse model of metabolic syndrome, which also exhibits elevated levels of circulating Ly6Chi monocytes expressing the CCL2 receptor CCR2. Accordingly, monocyte tracing experiments demonstrate that targeted SMC GLUT1 overexpression promotes Ly6Chi monocyte recruitment to lesions. Strikingly, SMC-targeted GLUT1 overexpression fails to accelerate atherosclerosis in mice that do not exhibit the metabolic syndrome phenotype or monocytosis. These results reveal a potentially novel mechanism whereby arterial smooth muscle glucose metabolism synergizes with metabolic syndrome to accelerate monocyte recruitment and atherosclerosis progression.

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.

Long-chain acyl-CoA synthetases (ACSLs) catalyze the thioesterification of long-chain FAs into their acyl-CoA derivatives. Purified ACSL4 is an arachidonic acid (20:4)-preferring ACSL isoform, and ACSL4 is therefore a probable regulator of lipid mediator production in intact cells. Eicosanoids play important roles in vascular homeostasis and disease, yet the role of ACSL4 in vascular cells is largely unknown. In the present study, the ACSL4 splice variant expressed in human arterial smooth muscle cells (SMCs) was identified as variant 1. To investigate the function of ACSL4 in SMCs, ACSL4 variant 1 was overexpressed, knocked-down by small interfering RNA, or its enzymatic activity acutely inhibited in these cells. Overexpression of ACSL4 resulted in a markedly increased synthesis of arachidonoyl-CoA, increased 20:4 incorporation into phosphatidylethanolamine, phosphatidylinositol, and triacylglycerol, and reduced cellular levels of unesterified 20:4. Accordingly, secretion of prostaglandin E₂ (PGE₂) was blunted in ACSL4-overexpressing SMCs compared with controls. Conversely, acute pharmacological inhibition of ACSL4 activity resulted in increased release of PGE₂. However, long-term downregulation of ACSL4 resulted in markedly reduced PGE₂ secretion. Thus, ACSL4 modulates PGE₂ release from human SMCs. ACSL4 may regulate a number of processes dependent on the release of arachidonic acid-derived lipid mediators in the arterial wall.

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.

Diabetes is associated with an increased risk of cardiovascular disease, largely due to increased atherosclerosis. Our studies have suggested myeloid cell prostaglandin E 2 (PGE 2 ) production as a possible mediator of diabetes-accelerated atherosclerosis in a virally-induced mouse model of type 1 diabetes. Prostaglandin E Receptor 4 (EP4; Ptger4 ) is a major PGE 2 receptor in myeloid cells. We hypothesized that generation of a mouse model of myeloid cell-targeted EP4-deficiency would allow us to test the role of myeloid EP4 in diabetes-accelerated atherosclerosis. Thus, we generated a Ptger4 flox/flox LysM-Cre tg/tg mouse model. Peritoneal macrophages isolated from these myeloid cell EP4-deficient (EP4 M-/- ) mice expressed Ptger4 mRNA compared to LysM-Cre tg/tg controls (n=10; pM-/- mice and littermate controls into lethally irradiated Ldlr -/- RIP-LCMV mice (the model of type 1 diabetes) and, after 7 weeks of recovery, induced diabetes by viral infection and fed the mice a low-fat semi-purified diet for an additional 12 weeks. Diabetic EP4 M-/- mice had similar blood glucose (568 ± 15 vs. 569 ± 15 mg/dl), blood cholesterol (531 ± 29 vs. 510 ± 37 mg/dl), and plasma triglycerides (249 ± 49 vs. 247 ± 44 mg/dl) as diabetic controls (n=15 all groups; mean ± SEM). At the endpoint, aortas were harvested for lesion area quantification. Diabetic EP4 M-/- and diabetic wild type mice had similar lesion area (1.9% ± 0.2 vs. 1.7% ± 0.2), which were both increased (p < 0.01; n=9-15) as compared to their non-diabetic controls. Additionally, we analyzed the role of EP4 in inflammatory activation of myeloid cells ex vivo. EP4-deficiency had no significant effect on basal or lipopolysaccharide (LPS)-induced inflammatory gene expression in the absence of PGE 2 . Pretreatment of the cells with PGE 2 (10 nM) followed by LPS stimulation resulted in a significant reduction of Tnfa and Il6 mRNA compared to LPS alone, and this anti-inflammatory effect of PGE 2 was completely blocked in EP4-deficient cells. These results suggest that myeloid cell EP4 mediates anti-inflammatory actions of PGE 2 but that it is not involved in diabetes-accelerated atherosclerosis.

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.

Objective— Saturated fatty acids, such as palmitic and stearic acid, cause detrimental effects in endothelial cells and have been suggested to contribute to macrophage accumulation in adipose tissue and the vascular wall, in states of obesity and insulin resistance. Long-chain fatty acids are believed to require conversion into acyl-CoA derivatives to exert most of their detrimental effects, a reaction catalyzed by acyl-CoA synthetases (ACSLs). The objective of this study was to investigate the role of ACSL1, an ACSL isoform previously shown to mediate inflammatory effects in myeloid cells, in regulating endothelial cell responses to a saturated fatty acid-rich environment in vitro and in vivo. Methods and Results— Saturated fatty acids caused increased inflammatory activation, endoplasmic reticulum stress, and apoptosis in mouse microvascular endothelial cells. Forced ACSL1 overexpression exacerbated the effects of saturated fatty acids on apoptosis and endoplasmic reticulum stress. However, endothelial ACSL1 deficiency did not protect against the effects of saturated fatty acids in vitro, nor did it protect insulin-resistant mice fed a saturated fatty acid-rich diet from macrophage adipose tissue accumulation or increased aortic adhesion molecule expression. Conclusion— Endothelial ACSL1 is not required for inflammatory and apoptotic effects of a saturated fatty acid-rich environment.

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.

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 in humans accelerates cardiovascular disease caused by atherosclerosis. The relative contributions of hyperglycemia and dyslipidemia to atherosclerosis in patients with diabetes are not clear, largely because there is a lack of suitable animal models. We therefore have developed a transgenic mouse model that closely mimics atherosclerosis in humans with type 1 diabetes by breeding low-density lipoprotein receptor-deficient mice with transgenic mice in which type 1 diabetes can be induced at will. These mice express a viral protein under control of the insulin promoter and, when infected by the virus, develop an autoimmune attack on the insulin-producing beta cells and subsequently develop type 1 diabetes. When these mice are fed a cholesterol-free diet, diabetes, in the absence of associated lipid abnormalities, causes both accelerated lesion initiation and increased arterial macrophage accumulation. When diabetic mice are fed cholesterol-rich diets, on the other hand, they develop severe hypertriglyceridemia and advanced lesions, characterized by extensive intralesional hemorrhage. This progression to advanced lesions is largely dependent on diabetes-induced dyslipidemia, because hyperlipidemic diabetic and nondiabetic mice with similar plasma cholesterol levels show a similar extent of atherosclerosis. Thus, diabetes and diabetes-associated lipid abnormalities have distinct effects on initiation and progression of atherosclerotic lesions.