Suseela Srinivasan

Critical Role of Macrophage 12/15-Lipoxygenase for Atherosclerosis in Apolipoprotein E–Deficient Mice

Background—Mice lacking leukocyte type 12/15-lipoxygenase (12/15-LO) show reduced atherosclerosis in several models. 12/15-LO is expressed in a variety of cells, including vascular cells, adipocytes, macrophages, and cardiomyocytes. The purpose of this study was to determine which cellular source of 12/15-LO is important for atherosclerosis. Methods and Results—Bone marrow from 12/15-LO−/−/apoE−/− mice was transplanted into apoE−/− mice and vice versa. Deficiency of 12/15-LO in bone marrow cells protected apoE−/− mice fed a Western diet from atherosclerosis to the same extent as complete absence of 12/15-LO, although plasma 8,12-iso-iPF2&agr;-IV, a measure of lipid peroxidation, remained elevated. 12/15-LO−/−/apoE−/− mice regained the severity of atherosclerotic lesion typical of apoE−/− mice after replacement of their bone marrow cells with bone marrow from apoE−/− mice. Peritoneal macrophages obtained from wild-type but not 12/15-LO−/− mice caused endothelial activation in the presence of native LDL. Absence of 12/15-LO decreased the ability of macrophages to form foam cells when exposed to LDL. Conclusions—We conclude that macrophage 12/15-LO plays a dominant role in the development of atherosclerosis by promoting endothelial inflammation and foam cell formation.

Sphingosine-1-Phosphate Induces an Antiinflammatory Phenotype in Macrophages

Activated macrophages acquire a proinflammatory (classic) or antiinflammatory (alternative) phenotype that influences atherosclerosis. The present study investigated whether sphingosine-1-phosphate (S1P), with its known antiinflammatory effects, could regulate the inflammatory phenotype of lipopolysaccharide (LPS)-stimulated mouse macrophages. Activation of macrophages by LPS significantly increases proinflammatory cytokine secretion. Pretreatment of macrophages with 500 nmol/L S1P markedly reduced LPS-mediated secretion of tumor necrosis factor-&agr;, monocyte chemoattractant protein-1, and interleukin-12. Such antiinflammatory actions were also evident in LPS-stimulated macrophages treated with the S1P1 receptor–specific agonist SEW2871. Pharmacological antagonism of the S1P1 receptor on macrophages using the S1P1-specific antagonist VPC44116 also blocked proinflammatory cytokine secretion in response to LPS. Studies using bone marrow–derived macrophages from S1P2-deficient mice revealed that the S1P2 receptor did not play a pivotal role in this process. Thus, activation of the S1P1 receptor in mouse macrophages limits the expression of proinflammatory cytokines. Furthermore, we demonstrated that S1P increased arginase I activity and inhibited LPS-induced inducible NO synthase activity in LPS-treated macrophages, again through S1P1 receptor activation on macrophages. Analysis of a 1.7-kb region of the murine inducible NO synthase promoter revealed the presence of putative nuclear factor &kgr;B, activator protein-1, and STAT-1 response elements. Using inducible NO synthase promoter-reporter constructs, we found that S1P significantly reduced the nuclear factor &kgr;B–mediated induction of inducible NO synthase. These findings demonstrate an important role for S1P in the regulation of macrophage phenotypic switching. Therefore, we conclude that S1P promotes the production of an alternative antiinflammatory macrophage phenotype through activation of the macrophage S1P1 receptor.

Sphingosine-1-Phosphate Prevents Tumor Necrosis Factor-α-Mediated Monocyte Adhesion to Aortic Endothelium in Mice

Objective—Endothelial activation and monocyte adhesion to endothelium are key events in inflammation. Sphingosine-1-phosphate (S1P) is a sphingolipid that binds to G protein-coupled receptors on endothelial cells (ECs). We examined the role of S1P in modulating endothelial activation and monocyte–EC interactions in vivo. Methods and Results—We injected C57BL/6J mice intravenously with tumor necrosis factor (TNF)-&agr; in the presence and absence of the S1P1 receptor agonist SEW2871 and examined monocyte adhesion. Aortas from TNF-&agr;–injected mice had a 4-fold increase in the number of monocytes bound, whereas aortas from TNF-&agr; plus SEW2871-treated mice had few monocytes bound (P<0.0001). Using siRNA, we found that inhibiting the S1P1 receptor in vascular ECs blocked the ability of S1P to prevent monocyte–EC interactions in response to TNF-&agr;. We examined signaling pathways downstream of S1P1 and found that 100 nM S1P increased phosphorylation of Akt and decreased activation of c-jun. Conclusions—Thus, we provide the first evidence that S1P signaling through the endothelial S1P1 receptor protects the vasculature against TNF-&agr;–mediated monocyte–EC interactions in vivo.

Sphingosine-1 Phosphate Prevents Monocyte/Endothelial Interactions in Type 1 Diabetic NOD Mice Through Activation of the S1P1 Receptor

Monocyte recruitment and adhesion to vascular endothelium are key early events in atherosclerosis. We examined the role of sphingosine-1-phosphate (S1P) on modulating monocyte/endothelial interactions in the NOD/LtJ (NOD) mouse model of type 1 diabetes. Aortas from nondiabetic and diabetic NOD mice were incubated in the absence or presence of 100 nmol/L S1P. Fluorescently labeled monocytes were incubated with the aortas. Aortas from NOD diabetic mice bound 7-fold more monocytes than nondiabetic littermates (10±1 monocytes bound/field for nondiabetic mice vs 74±12 monocytes bound/field for diabetic mice, P<0.0001). Incubation of diabetic aortas with 100 nmol/L S1P reduced monocyte adhesion to endothelium by 90%. We found expression of S1P1, S1P2, and S1P3 receptors on NOD aortic endothelial cells. The S1P1 receptor-specific agonist SEW2871 inhibited monocyte adhesion to diabetic aortas. Studies in diabetic S1P3-deficient mice revealed that the S1P3 receptor did not play a pivotal role in this process. S1P reduced endothelial VCAM-1 induction in type 1 diabetic NOD mice, most likely through inhibition of nuclear factor &kgr;B translocation to the nucleus. Thus, S1P activation of the S1P1 receptor functions in an antiinflammatory manner in type 1 diabetic vascular endothelium to prevent monocyte/endothelial interactions. S1P may play an important role in the prevention of vascular complications of type 1 diabetes.

12/15-Lipoxygenase Regulates Intercellular Adhesion Molecule-1 Expression and Monocyte Adhesion to Endothelium Through Activation of RhoA and Nuclear Factor-κB

Background—12/15-lipoxygenase (12/15-LO) activity leads to the production of the proinflammatory eicosanoids 12-S-hydroxyeicosatetraenoic acid (12SHETE) and 13-S-hydroxyoctadecadienoic acid. We have previously shown a 3.5-fold increase in endothelial intercellular adhesion molecule (ICAM)-1 expression in mice overexpressing the 12/15-LO gene. We examined whether 12/15-LO activity regulated endothelial ICAM-1 expression. Methods and Results—Freshly isolated aortic endothelial cells (EC) from 12/15-LO transgenic mice had significantly greater nuclear factor-&kgr;B (NF-&kgr;B) activation and ICAM mRNA expression compared with C57BL/6J control. 12/15-LO transgenic EC showed elevated RhoA activity, and inhibition of RhoA using either C3 toxin or the Rho-kinase inhibitor Y-27632 blocked NF-&kgr;B activation, ICAM-1 induction, and monocyte adhesion. Furthermore, we show that 12SHETE activates protein kinase C&agr;, which forms a complex with active RhoA and is required for NF-&kgr;B–dependent ICAM expression in response to 12SHETE. Conclusions—The 12/15-LO pathway stimulates ICAM-1 expression through the RhoA/protein kinase C&agr;-dependent activation of NF-&kgr;B. These findings identify a major signaling pathway in EC through which 12/15-LO contributes to vascular inflammation and atherosclerosis.

Glucose Regulates Monocyte Adhesion Through Endothelial Production of Interleukin-8

Abstract— We have shown that glucose increases monocyte adhesion to human aortic endothelial cells (HAECs) in vitro. 1 In the present study, we examined mechanisms by which glucose stimulates monocyte:endothelial interactions. HAECs cultured for 7 days in 25 mmol/L glucose had a 2-fold elevation in interleukin-8 (IL-8) secretion over control cells cultured in 5.5 mmol/L glucose (P <0.001). Use of a neutralizing antibody to IL-8 prevented glucose-mediated monocyte adhesion. Both glucose and IL-8 activated &bgr;1 integrin on the HAEC surface, suggesting that both activate the &agr;5&bgr;1 integrin complex on the endothelial surface. The &agr;5&bgr;1 integrin complex is important for anchoring connecting segment-1 fibronectin on the HAEC surface for monocyte adhesion. Analysis of the human IL-8 promoter revealed binding sites for NF-&kgr;B and AP-1 as well as several aligned carbohydrate response elements (also known as E-boxes). Glucose dramatically stimulated IL-8 promoter activity. Using mutated IL-8 promoter constructs and EMSA, we found that the AP-1 element and the glucose-response element were responsible for much of the glucose-mediated activation of IL-8 transcription. Interestingly, inhibition of reactive oxygen species (ROS) production through use of pharmacological uncouplers of the mitochondrial electron transport chain significantly reduced glucose-mediated induction of IL-8 expression. These data indicate that glucose regulates monocyte:endothelial interactions through stimulation of IL-8 and ROS production and activation of the &agr;5&bgr;1 integrin complex on HAECs.

Reduced Expression of ATP-Binding Cassette Transporter G1 Increases Cholesterol Accumulation in Macrophages of Patients With Type 2 Diabetes Mellitus

Background— Patients with type 2 diabetes mellitus are at increased risk for the development of atherosclerosis. A pivotal event in the development of atherosclerosis is macrophage foam cell formation. The ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 regulate macrophage cholesterol efflux and hence play a vital role in macrophage foam cell formation. We have previously found that chronic elevated glucose reduces ABCG1 expression. In the present study, we examined whether patients with type 2 diabetes mellitus had decreased ABCG1 and/or ABCA1, impaired cholesterol efflux, and increased macrophage foam cell formation. Methods and Results— Blood was collected from patients with and without type 2 diabetes mellitus. Peripheral blood monocytes were differentiated into macrophages, and cholesterol efflux assays, immunoblots, histological analysis, and intracellular cholesteryl ester measurements were performed. Macrophages from patients with type 2 diabetes mellitus had a 30% reduction in cholesterol efflux with a corresponding 60% increase in cholesterol accumulation relative to control subjects. ABCG1 was present in macrophages from control subjects but was undetectable in macrophages from patients with type 2 diabetes mellitus. In contrast, ABCA1 expression in macrophages was similar in both control subjects and patients with type 2 diabetes mellitus. Macrophage expression of ABCG1 in both patients and control subjects was induced by treatment with the liver X receptor agonist TO-901317. Upregulation of liver X receptor dramatically reduced foam cell formation in macrophages from patients with type 2 diabetes mellitus. Conclusions— ABCG1 expression and cholesterol efflux are reduced in patients with type 2 diabetes mellitus. This impaired ABCG1-mediated cholesterol efflux significantly correlates with increased intracellular cholesterol accumulation. Strategies to upregulate ABCG1 expression and function in type 2 diabetes mellitus could have therapeutic potential for limiting the accelerated vascular disease observed in patients with type 2 diabetes mellitus.

Reduction in ABCG1 in Type 2 Diabetic Mice Increases Macrophage Foam Cell Formation

Atherosclerosis development is accelerated severalfold in patients with Type 2 diabetes. In the initial stages of disease, monocytes transmigrate into the subendothelial space and differentiate into foam cells. Scavenger receptors and ATP binding cassette (ABC) Transporters play an important role in foam cell formation as they regulate the influx and efflux of oxidized lipids. Here, we show that peritoneal macrophages isolated from Type 2 diabetic db/db mice have decreased expression of the ABC transporter ABCG1 and increased expression of the scavenger receptor CD36. We found a 2-fold increase in accumulation of esterified cholesterol in diabetic db/db macrophages compared with wild-type control macrophages. Diabetic db/db macrophages also had impaired cholesterol efflux to high density lipoprotein but not to lipid-free apo A-I, suggesting that the increased esterified cholesterol in diabetic db/db macrophages was due to a selective loss of ABCG1-mediated efflux to high density lipoprotein. Additionally, we were able to confirm down-regulation of ABCG1 using C57BL/6J peritoneal macrophages cultured in elevated glucose in vitro (25 mm glucose for 7 days), suggesting that ABCG1 expression in diabetic macrophages is regulated by chronic exposure to elevated glucose. Diabetic KKay mice were also studied and were found to have decreased ABCG1 expression without an increase in CD36. These observations demonstrate that ABCG1 plays a major role in macrophage cholesterol efflux and that decreased ABCG1 function can facilitate foam cell formation in Type 2 diabetic mice.

A Critical Role for ABCG1 in Macrophage Inflammation and Lung Homeostasis

ATP-binding cassette transporter G1 (ABCG1) effluxes cholesterol from macrophages and plays an important role in pulmonary lipid homeostasis. We hypothesize that macrophages from Abcg1−/− mice have increased inflammatory activity, thereby promoting acceleration of pulmonary disease. We herein demonstrate increased numbers of inflammatory cytokines and infiltrating neutrophils, eosinophils, dendritic cells, T cells, and B cells into lungs of Abcg1−/− mice before the onset of severe lipidosis. We further investigated the role of macrophages in causing pulmonary disease by performing bone marrow transplantations using B6 and Abcg1−/− bone marrow. We found that it was the macrophage, and not pneumocyte type II cells or other nonhematopoietic cells in the lung, that appeared to be the primary cell type involved in the onset of both pulmonary lipidosis and inflammation in the Abcg1−/− mice. Additionally, our results demonstrate that Abcg1−/− macrophages had elevated proinflammatory cytokine production, increased apoptotic cell clearance, and were themselves more prone to apoptosis and necrosis. However, they were quickly repopulated by monocytes that were recruited to Abcg1−/− lungs. In conclusion, we have shown that ABCG1 deletion in macrophages causes a striking inflammatory phenotype and initiates onset of pulmonary lipidosis in mice. Thus, our studies reveal a critical role for macrophage ABCG1 in lung inflammation and homeostasis.

12/15 Lipoxygenase Mediates Monocyte Adhesion to Aortic Endothelium in Apolipoprotein E–Deficient Mice Through Activation of RhoA and NF-κB

Objectives—12/15 lipoxygenase (12/15LO) has been implicated as a mediator of inflammation and atherosclerosis. In the current study, we identified mechanisms through which 12/15LO mediates monocyte:endothelial interactions in vivo in apolipoprotein E-deficient mice (apoEKO), a well-characterized mouse model of atherosclerosis. Methods and Results—In apoEKO mice that are also deficient in 12/15LO (doubleKO), monocyte adhesion to aorta in vivo was reduced by 95% in doubleKO mice compared with apoEKO mice. Inhibition of 12/15LO in apoEKO mice in vivo using CDC (Cinnamyl-3,4-Dihydroxy-a-Cyanocinnamate) prevented monocyte adhesion to aortic endothelium in apoEKO mice. Aortic endothelium of apoEKO mice had significant activation of rhoA compared with doubleKO aortic endothelium. Further, apoEKO aorta displayed significant activation of NF-&kgr;B. DoubleKO aorta displayed little nuclear localization of NF-&kgr;B. Finally, we found significant upregulation of intercellular adhesion molecule-1 (ICAM-1) on apoEKO aortic endothelium compared with doubleKO endothelium. Inhibition of rhoA and PKC&agr; significantly reduced NF-&kgr;B activation, ICAM-1 induction, and monocyte adhesion to aorta. Conclusions—We conclude that 12/15LO products activate endothelial rhoA and PKC&agr;. Activation of rhoA and PKC&agr; cause activation and translocation of NF-&kgr;B to the nucleus, which, in turn, results in induction of ICAM-1. Induction of ICAM-1 on aortic endothelium stimulates monocyte:endothelial adhesion in vivo in apoEKO mice.