Macrophage Lipoprotein Lipase Expression Research Articles

MicroRNA-590 Inhibits Lipoprotein Lipase Expression and Prevents Atherosclerosis in apoE Knockout Mice.

Recent studies have suggested that miR-590 may play critical roles in cardiovascular disease. This study was designed to determine the effects of miR-590 on lipoprotein lipase (LPL) expression and development of atherosclerosis in apolipoprotein E knockout (apoE−/−) mice and explore the potential mechanisms. En face analysis of the whole aorta revealed that miR-590 significantly decreased aortic atherosclerotic plaque size and lipid content in apoE−/− mice. Double immunofluorescence staining in cross-sections of the proximal aorta showed that miR-590 agomir reduced CD68 and LPL expression in macrophages in atherosclerotic lesions. MiR-590 agomir down-regulated LPL mRNA and protein expression as analyzed by RT-qPCR and western blotting analyses, respectively. Consistently, miR-590 decreased the expression of CD36 and scavenger receptor A1 (SRA1) mRNA and protein. High-performance liquid chromatography (HPLC)analysis confirmed that treatment with miR-590 agomir reduced lipid levels either in plasma orinabdominal cavity macrophages of apoE−/− mice. ELISA analysis showed that miR-590 agomir decreased plasma levels of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), interleukin-1β (IL-1β)and interleukin-6 (IL-6). In contrast, treatment with miR-590 antagomir prevented or reversed these effects. Taken together, these results reveal a novel mechanism of miR-590 effects, and may provide new insights into the development of strategies for attenuating lipid accumulation and pro-inflammatory cytokine secretion.

MicroRNA-467b targets LPL gene in RAW 264.7 macrophages and attenuates lipid accumulation and proinflammatory cytokine secretion

LPL (lipoprotein lipase) is a rate-limiting enzyme involved in the hydrolysis of triglycerides. Previous studies have shown that microRNA (miR)-467b regulates hepatic LPL expression and plays a role in the progression of steatosis or abnormal lipid retention in obese mice. Macrophage-derived LPL has been shown to promote atherosclerosis. However, if miR-476b influences macrophage LPL expression and the subsequent effects are unknown. Here, we utilized oxLDL-treatment RAW 264.7 macrophages that were transfected with miR-467b mimics or inhibitors to investigate the potential roles of macrophage miR-476b. We found that miR-467b significantly decreased lipid accumulation and IL-6, IL-1β, TNF-α and MCP-1 secretions. Furthermore, our studies suggested an additional explanation for the regulatory mechanism of miR-467b on its functional target, LPL in RAW 264.7 macrophages. Thus, our findings indicate that miR-467b may regulate lipid accumulation and proinflammatory cytokine secretion in oxLDL-stimulated RAW 264.7 macrophages by targeting the LPL gene.

CRP enhances soluble LOX-1 release from macrophages by activating TNF-α converting enzyme

Circulating levels of soluble lectin-like oxidized low-density lipoprotein receptor-1 (sLOX-1) play an important role in the development and progression of atherosclerosis. We hypothesized that the inflammatory marker C-reactive protein (CRP) might stimulate sLOX-1 release by activating tumor necrosis factor-α converting enzyme (TACE). Macrophages differentiated from THP-1 cells were stimulated with TNF-α and further treated with CRP in the absence or presence of specific inhibitors or small interfering RNA (siRNA). Our results showed that CRP increased sLOX-1 release from activated macrophages in a dose-dependent manner and that these effects were regulated by Fc γ receptor II (FcγRII)-mediated p47(phox) phosphorylation, reactive oxygen species (ROS) production, and TACE activation. CRP also enhanced sLOX-1 release from macrophages derived from peripheral blood mononuclear cells (PBMC) of patients with acute coronary syndrome (ACS). Pretreatment with antibody against FcγRII or with CD32 siRNA, p47(phox) siRNA, apocynin, N-acetylcysteine, tumor necrosis factor-α protease inhibitor 1 (TAPI-1) or TACE siRNA attenuated sLOX-1 release induced by CRP. CRP also elevated serum sLOX-1 levels in a rabbit model of atherosclerosis. Thus, CRP might stimulate sLOX-1 release, and the underlying mechanisms possibly involved FcγRII-mediated p47(phox) phosphorylation, ROS production, and TACE activation.

C-reactive protein enhances macrophage lipoprotein lipase expression

High serum levels of C-reactive protein (CRP), a strong predictor of cardiovascular events, are documented in patients with type 2 diabetes. Accumulating evidence suggests that CRP could directly promote arterial damage. To determine the role of CRP in diabetic atherosclerosis, we examined the effect of CRP on the expression of macrophage lipoprotein lipase (LPL), a proatherogenic molecule upregulated in type 2 diabetes. Treatment of human macrophages with native CRP increased, in a dose- and time-dependent manner, LPL protein expression and secretion. Modified CRP reproduced these effects. Preincubation of human macrophages with antioxidants, protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) inhibitors prevented CRP-induced LPL expression. Exposure of human macrophages to CRP further increased intracellular reactive oxygen species generation, classic PKC isozymes expression, and extracellular signal-regulated protein kinase 1/2 phosphorylation. In CRP-treated J774 macrophages, increased macrophage LPL mRNA levels and enhanced binding of nuclear proteins to the activated protein-1 (AP-1)-enhancing element were observed. These effects were prevented by antioxidants, as well as by PKC, MAPK, and AP-1 inhibitors. These data show for the first time that CRP directly increases macrophage LPL expression and secretion. Given the predominant role of macrophage LPL in atherogenesis, LPL might represent a novel factor underlying the adverse effect of CRP on the diabetic vasculature.

Upregulation of Macrophage Endothelial Lipase by Toll-Like Receptors 4 and 3 Modulates Macrophage Interleukin-10 and -12 Production

Limited data suggest that endothelial lipase (EL) is synthesized not only by endothelial cells but also by macrophages. Previous studies showed that proinflammatory cytokines upregulate EL in endothelial cells, but there are very few data regarding EL expression, regulation, and functional consequences in macrophages. In the present study, RAW cells and mouse peritoneal macrophages were treated with Toll-like receptor (TLR) ligands and EL expression and its consequences were assessed. We demonstrate that lipopolysaccharide, a TLR4 ligand; and polyinosinic:polycytidylic acid (poly I:C), a TLR3 ligand; but not lipoteichoic acid, a TLR2 ligand, upregulate macrophage EL expression both ex vivo and in vivo. In contrast, macrophage lipoprotein lipase expression is significantly repressed by lipopolysaccharide or poly I:C. Using C3HJ and TLR3 knockout mice, we further show that upregulation of macrophage EL expression by lipopolysaccharide or poly I:C is TLR4 or TLR3 dependent, respectively. Furthermore, we demonstrate that lipopolysaccharide induced interleukin (IL)-10 production was significantly reduced, whereas IL-12 production is significantly increased in J744 macrophages and mouse peritoneal macrophages overexpressing human EL. Conversely, significantly increased IL-10 and significantly decreased IL-12 expression were observed in mouse peritoneal macrophages isolated from EL knockout mice. Finally we show that the catalytic activity is required for EL to modulate the balance of macrophage IL-10 and IL-12 production. These results suggest that macrophage EL may play important roles in modulating the macrophage inflammatory response through local hydrolysis of HDL.

Suppression of endothelial or lipoprotein lipase in THP-1 macrophages attenuates proinflammatory cytokine secretion

LPL and endothelial lipase (EL) are associated with macrophages in human atherosclerotic lesions, and overexpression of LPL in mouse macrophages is associated with a greater extent of atherosclerosis. To investigate potential mechanisms by which macrophage-derived lipase expression may mediate proatherogenic effects, we used lentivirus-mediated RNA interference to suppress the expression of either LPL or EL within THP-1 macrophages. After suppression of either LPL or EL, significant decreases in the concentration of interleukin-1beta, interleukin-6, monocyte chemoattractant protein-1, and tumor necrosis factor-alpha were observed. Incubation of THP-1 macrophages with either mildly or extensively oxidized LDL consistently decreased cytokine expression, which was additive to that contributed by lipase suppression. Decreased lipase expression was also associated with an altered lipid composition, with reduced percentages of cholesterol (unesterified and esterified), triglycerides, and lysophosphatidylcholine. Microarray data indicated a decreased expression of proinflammatory genes, growth factors, and antiapoptotic genes. By contrast, there was an increased expression of lipoprotein receptors (scavenger receptor 1, low density lipoprotein receptor, scavenger receptor class B type I, and CD36). Thus, we conclude that the suppression of either LPL or EL decreases proinflammatory cytokine expression and influences the lipid composition of THP-1 macrophages. These results provide further insight into the specific metabolic and potential pathological roles of LPL and EL in human macrophages.

High postprandial triglyceridemia in patients with type 2 diabetes and microalbuminuria

Microalbuminuria (MA) is an independent risk factor for atherosclerosis in patients with type 2 diabetes mellitus (T2DM). Postprandial lipemia is also associated with excess cardiovascular risk. However, the association between MA and postprandial lipemia in diabetes has not been investigated. A total of 64 patients with T2DM, 30 with and 34 without MA, were examined. Plasma total triglycerides (TGs), triglycerides contained in chylomicrons (CM-TG), and TGs in CM-deficient plasma were measured at baseline and every 2 h for 6 h after a mixed meal. Postheparin LPL and HL activities were also determined. Plasma levels of apolipoprotein A-V (apoA-V), apoC-II, and apoC-III were measured in the fasting state and 2 h postprandially. Patients with MA had higher postprandial total TG levels than those without MA (P < 0.001); this increase been attributed mainly to CM-TG. LPL activity and fasting concentrations of the measured apolipoproteins were not different between the studied groups, whereas HL activity was higher in the patients with MA. ApoC-II and apoC-III levels did not change postprandially in either study group, whereas apoA-V increased more in the patients with MA. These data demonstrate for the first time that MA is characterized by increased postprandial lipemia in patients with T2DM and may explain in part the excess cardiovascular risk in these patients.

A critical role for the Sp1-binding sites in the transforming growth factor- -mediated inhibition of lipoprotein lipase gene expression in macrophages

Increasing evidence suggests that the cytokine transforming growth factor-β (TGF-β) inhibits the development of atherosclerosis. The lipoprotein lipase (LPL) enzyme expressed by macrophages has been implicated in the pathogenesis of atherosclerosis by stimulating the uptake of lipoprotein particles. Unfortunately, the action of TGF-β on the expression of LPL in macrophages remains largely unclear. We show that TGF-β inhibits LPL gene expression at the transcriptional level. Transient transfection assays reveal that the −31/+187 sequence contains the minimal TGF-β-responsive elements. Electrophoretic mobility shift assays show that Sp1 and Sp3 interact with two regions in the −31/+187 sequence. Mutations of these Sp1/Sp3 sites abolish the TGF-β-mediated suppression whereas multimers of the sequence impart the response to a heterologous promoter. TGF-β has no effect on the binding or steady-state polypeptide levels of Sp1 and Sp3. These results, therefore, suggest a novel mechanism for the TGF-β-mediated repression of LPL gene transcription that involves regulation of the action of Sp1 and Sp3.

Advanced glycation end products potentiate the stimulatory effect of glucose on macrophage lipoprotein lipase expression

Lipoprotein lipase (LPL) secreted by macrophages in the arterial wall promotes atherosclerosis. We have shown that macrophages of patients with type 2 diabetes overproduce LPL and that metabolic factors, including glucose, stimulate macrophage LPL secretion. In this study, we determined the effect of advanced glycation end products (AGEs) on LPL expression by macrophages cultured in a high-glucose environment and the molecular mechanisms underlying this effect. Our results demonstrate that AGEs potentiate the stimulatory effect of high glucose on murine and human macrophage LPL gene expression and secretion. Induction of macrophage LPL mRNA levels by AGEs was identical to that elicited by physiologically relevant modified albumin and was inhibited by anti-AGE receptor as well as by antioxidants. Treatment of macrophages with AGEs resulted in protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation. Inhibition of these kinases abolished the effect of AGEs on LPL mRNA levels. Finally, exposure of macrophages to AGEs increased the binding of nuclear proteins to the activated protein-1 consensus sequence of the LPL promoter. This effect was inhibited by PKC and MAPK inhibitors. These results demonstrate for the first time that AGEs potentiate the stimulatory effect of high glucose on macrophage LPL expression. This effect appears to involve oxidative stress and PKC/MAPK activation.

Leptin Increases Lipoprotein Lipase Secretion by Macrophages: Involvement of Oxidative Stress and Protein Kinase C

Recent data suggest that plasma leptin may represent a cardiovascular risk factor in diabetic patients. To gain further insight into the role of leptin in atherogenesis associated with diabetes, we investigated in the present study the role of this hormone in the regulation of macrophage lipoprotein lipase (LPL), a proatherogenic cytokine overexpressed in patients with type 2 diabetes. Treatment of human macrophages with leptin (1-10 nmol/l) increased LPL expression, at both the mRNA and protein levels. Pretreatment of these cells with anti-leptin receptor (Ob-R) antibody, protein kinase C (PKC) inhibitors, calphostin C, and GF109203X, or the antioxidant N-acetylcysteine (NAC) blocked the effects of leptin. Similar results were observed in leptin-treated J774 macrophages. In these cells, leptin increased the membrane expression of conventional PKC isoforms and downregulation of endogenous PKC expression abolished the effects of leptin on macrophage LPL expression. In leptin-treated J774 cells, enhanced LPL synthetic rate and increased binding of nuclear proteins to the activated protein-1 (AP-1) consensus sequence of the LPL gene promoter were also observed. This latter effect was abrogated by GF109203X. Overall, these data demonstrate that binding of leptin at the macrophage cell surface increases, through oxidative stress- and PKC-dependent pathways, LPL expression. This effect appears to be exerted at the transcriptional level and to involve AP-1 activation.

VLDL-induced triglyceride accumulation in human macrophages is mediated by modulation of LPL lipolytic activity in the absence of change in LPL mass

Mixed dyslipidemia of phenotype IIB is characterized by elevated levels of very low density lipoprotein (VLDL)-1 and VLDL-2 subfractions and of low density lipoprotein (LDL), which are associated with premature formation of atherosclerotic plaques, characterized by the presence of lipid-rich macrophage foam cells. Lipoprotein lipase (LPL) is a key factor in mediating macrophage lipid accumulation and foam-cell formation from native VLDL particles. The action of macrophage-derived LPL in the induction of intracellular lipid accumulation from triglyceride-rich lipoprotein (TRL) subfractions (VLDL-1, VLDL-2) is, however, indeterminate, as is the potential role of VLDL-1 and VLDL-2 in modulating macrophage LPL expression. We evaluated the role of LPL in the interaction of type IIB VLDL-1 and VLDL-2 with human macrophages. Both VLDL-1 and VLDL-2 subfractions induced significant accumulation of triglyceride (9.8-fold, P<0.0001, and 4.8-fold, P<0.0001, respectively) and of free cholesterol content (1.4-fold, P<0.001, and 1.2-fold, P=0.02, respectively). Specific inhibition (90%) of the lipolytic activity of endogenous LPL by tetrahydrolipstatin (THL) in the presence of VLDL-1 or VLDL-2 resulted in marked reduction in cellular loading of both triglycerides (−89%, P=0.008, and −89%, P=0.015, respectively) and free cholesterol (−76%, P=0.02, and −55%, P=0.06 respectively). Furthermore, VLDL-1 and VLDL-2 induced marked increase in macrophage-derived LPL enzyme activity (+81%, P=0.002, and +45%, P=0.02), but did not modulate macrophage-derived LPL mRNA and protein expression; consequently, LPL specific activity was significantly increased from 1.6 mU/μg at baseline to 4.1 mU/μg ( P=0.01) and 3.1 mU/μg ( P=0.05), in the presence of VLDL-1 and VLDL-2, respectively. We conclude that type IIB VLDL-1 and VLDL-2 induce triglyceride accumulation in human monocyte-macrophages primarily via the lipolytic action of LPL, which may involve stabilization and activation of the macrophage-secreted enzyme, rather than via modulation of enzyme production.

Peroxisome proliferator-activated receptor α and γ agonists upregulate human macrophage lipoprotein lipase expression

Peroxisome proliferator-activated receptors (PPARs) are transcriptional factors which mediate pleiotropic effects including regulation of genes involved in lipid metabolism and control of inflammation. In the present study, we measured the in vitro effects of PPARα and γ ligands on macrophage lipoprotein lipase (LPL) expression. Human monocyte-derived macrophages (MDM) were cultured for 1–3 days in the presence of PPARα and γ ligands. At the end of these incubation periods, extracellular LPL immunoreactive mass/activity and LPL mRNA levels were measured. Incubation of human MDM with PPARα and γ ligands stimulated, in a time- and dose-dependent manner, human MDM LPL mass and activity. These agents also significantly increased macrophage LPL mRNA expression. In THP-1 cells treated with PPARα and γ ligands, enhanced nuclear protein binding to the peroxisome proliferator responsive element (PPRE) of the human LPL promoter was observed. Furthermore, in these cells, a decreased rate of decay of LPL mRNA was documented. Overall, these results demonstrate that PPARα and γ activators increase macrophage LPL secretion. Given the proatherogenic effect of vascular wall LPL, better understanding of the role of PPARs in the regulation of macrophage LPL expression could lead to the development of new approaches in the prevention and treatment of atherosclerosis.

Homocysteine induces protein kinase C activation and stimulates c-Fos and lipoprotein lipase expression in macrophages.

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease in human diabetes. Among the multiple factors that may account for the atherogenicity of homocysteine (Hcys) in patients with diabetes, macrophage (Mo) lipoprotein lipase (LPL) has unique features in that it is increased in human diabetes and acts as a proatherogenic factor in the arterial wall. In the present study, we determined the direct regulatory effect of Hcys on Mo LPL gene expression and secretion. Incubation of J774 Mo with Hcys increased, in a time- and dose-dependent manner, LPL mRNA expression and secretion. Induction of LPL gene expression was biphasic, peaking at 1 and 6 h. Whereas Hcys treatment increased protein kinase C (PKC) activity in Mo, pretreatment of Mo with PKC inhibitors totally suppressed Hcys-induced LPL mRNA expression. Hcys also increases the levels of c-fos mRNA in Mo and enhanced nuclear protein binding to the AP-1 sequence of the LPL gene promoter. Overall, these results demonstrate that Hcys stimulates Mo LPL at both the gene and protein levels and that Hcys-induced LPL mRNA expression requires PKC activation. They also suggest a possible role of c-fos in the stimulatory effect of Hcys on Mo LPL mRNA expression. These observations suggest a new mechanism by which Hcys may exert its proatherogenic effects in human diabetes.

A Novel Role of Sp1 and Sp3 in the Interferon-γ-mediated Suppression of Macrophage Lipoprotein Lipase Gene Transcription

The regulation of macrophage lipoprotein lipase by cytokines is of potentially crucial importance in the pathogenesis of atherosclerosis. We have shown previously that macrophage lipoprotein lipase expression is suppressed by interferon-gamma (IFN-gamma) at the transcriptional level. We investigated the regulatory sequence elements and the transcription factors that are involved in this response. We demonstrated that the -31/+187 sequence contains the minimal IFN-gamma-responsive elements. Electrophoretic mobility shift assays showed that the binding of proteins to two regions in the -31/+187 sequence was reduced dramatically when the cells were exposed to IFN-gamma. Both competition electrophoretic mobility shift assays and antibody supershift assays showed that the interacting proteins were composed of Sp1 and Sp3. Mutations of the Sp1/Sp3-binding sites in the minimal IFN-gamma-responsive elements abolished the IFN-gamma-mediated suppression of promoter activity, whereas multimers of the sequence were able to impart the response to a heterologous promoter. Western blot analysis showed that IFN-gamma reduced the steady state levels of Sp3 protein. In contrast, the cytokine decreased the DNA binding activity of Sp1 without affecting the protein levels. These studies therefore reveal a novel mechanism for IFN-gamma-mediated regulation of macrophage gene transcription.

Macrophage lipoprotein lipase expression is increased in patients with heterozygous familial hypercholesterolemia

FH is associated with accelerated atherosclerosis. Based on the crucial role of macrophage LPL in atherogenesis, we determined in the present study macrophage LPL expression in patients with FH. Monocytes isolated from 13 FH patients and 13 control subjects were differentiated into macrophages by culturing the cells for 9 days in 20% autologous or heterologous serum. Macrophages of patients with FH cultured in their own sera showed a significant increase in LPL mRNA levels, extracellular LPL mass, and activity compared with macrophages of control subjects. Although these alterations positively correlated with the levels of serum platelet-derived growth factor-BB (PDGF-BB) in FH subjects, increased LPL secretion by cultured FH macrophages was reduced neither by immunoneutralizing FH serum with an anti-PDGF-BB antibody, nor by culturing these cells in sera from control subjects. With the exception of LPL, levels of other cytokines and 8-isoprostane were not increased in the supernatants of macrophages of FH patients. Serum from FH patients also enhances the levels of LPL secreted by macrophages from control subjects. Immunoneutralization of FH serum with an anti-PDGF-BB antibody totally reversed this alteration. Overall, this study demonstrates that macrophages from FH subjects overproduce LPL and that PDGF present in the serum from FH patients stimulates LPL secretion by control macrophages. These findings suggest that macrophage LPL induction in patients with FH might be related to the increased atherogenesis observed in these subjects.—Beauchamp, M-C., É. Letendre, and G. Renier. Macrophage lipoprotein lipase expression is increased in patients with heterozygous familial hypercholesterolemia.

Macrophage Lipoprotein Lipase Promotes Foam Cell Formation and Atherosclerosis in Low Density Lipoprotein Receptor-deficient Mice

The role of macrophage lipoprotein lipase (LPL) expression in atherosclerotic lesion formation was examined in low density lipoprotein receptor (LDLR(-/-)) mice using dietary conditions designed to induce either fatty streak lesions or complex atherosclerotic lesions. First, LDLR(-/-) mice chimeric for macrophage LPL expression were created by transplantation of lethally irradiated female LDLR(-/-) mice with LPL(-/-) (n = 12) or LPL(+/+) (n = 14) fetal liver cells as a source of hematopoietic cells. To induce fatty streak lesions, these mice were fed a Western diet for 8 weeks, resulting in severe hypercholesterolemia. There were no differences in plasma post-heparin LPL activity, serum lipid levels, or lipoprotein distribution between these two groups. The mean lesion area in the proximal aorta in LPL(-/-) --> LDLR(-/-) mice was significantly reduced by 33% compared with LPL(+/+) --> LDLR(-/-) mice, and a similar reduction (38%) in lesion area was found by en face analysis of the aortae. To induce complex atherosclerotic lesions, female LDLR(-/-) mice were lethally irradiated, transplanted with LPL(-/-) (n = 14), LPL(+/-) (n = 13), or LPL(+/+) (n = 14) fetal liver cells, and fed the Western diet for 19 weeks. Serum cholesterol and triglyceride levels did not differ between the three groups. After 19 weeks of diet, the lesions in the proximal aorta were complex with relatively few macrophages expressing LPL protein and mRNA in LPL(+/+) --> LDLR(-/-) mice. Analysis of cross-sections of the proximal aorta demonstrated no differences in the extent of lesion area between the groups, whereas en face analysis of the aortae revealed a dose-dependent effect of macrophage LPL on mean aortic lesion area in LPL(-/-) --> LDLR(-/-), LPL(-/+) --> LDLR(-/-), and LPL(+/+) --> LDLR(-/-) mice (1.8 +/- 0. 2%, 3.5 +/- 0.5% and 5.9 +/- 0.8%, respectively). Taken together, these data indicate that macrophage LPL expression in the artery wall promotes atherogenesis during foam cell lesion formation, but this impact may be limited to macrophage-rich lesions.

INVOLVEMENT OF BOTH THE TYROSINE KINASE AND THE PHOSPHATIDYLINOSITOL-3′ KINASE SIGNAL TRANSDUCTION PATHWAYS IN THE REGULATION OF LIPOPROTEIN LIPASE EXPRESSION IN J774.2 MACROPHAGES BY CYTOKINES AND LIPOPOLYSACCHARIDE

The regulation of macrophage lipoprotein lipase (LPL) by cytokines and lipopolysaccharide (LPS) is of potentially crucial importance in the pathogenesis of atherosclerosis and in the responses to endotoxin challenge. We show here that the reduction of LPL activity in J774.2 macrophages observed in the presence of interleukin (IL-1) and IL-11 was sensitive to herbimycin A, with the effect of LPS, interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) on LPL activity being sensitive to both herbimycin A and wortmannin. The action of the inhibitors on the IFN-γ-dependent reduction of LPL activity was mediated at the level of LPL mRNA metabolism, with translational and/or post-translational levels of regulation being involved in the action of all the other mediators tested. These observations suggest that both the tyrosine kinase and the phosphatidylinositol-3′-kinase signalling pathways are involved in the suppression of macrophage LPL expression by LPS and cytokines.

Macrophage lipoprotein lipase promotes foam cell formation and atherosclerosis in vivo.

Expression of lipoprotein lipase (LPL) by the macrophage has been proposed to promote foam cell formation and atherosclerosis, primarily on the basis of in vitro studies. LPL-deficient mice might provide a model for testing the role of LPL secretion by the macrophage in an in vivo system. Unfortunately, homozygous deficiency of LPL in the mouse is lethal shortly after birth. Because the fetal liver is the major site of hematopoiesis in the developing fetus, transplantation of C57BL/6 mice with LPL-/- fetal liver cells (FLCs) was used to investigate the physiologic role of macrophage LPL expression in vivo. Thirty-four female C57BL/6 mice were lethally irradiated and reconstituted with FLCs from day 14 LPL+/+, LPL+/-, and LPL-/- donors. No significant differences were detected in plasma levels of post-heparin LPL activity or in serum cholesterol or triglyceride levels between the 3 groups on either a chow diet or an atherogenic diet. After 19 weeks on the atherogenic diet, aortae were collected for quantitative analysis of the extent of aortic atherosclerosis. LPL expression was detected by immunocytochemistry and in situ hybridization in macrophages of aortic atherosclerotic lesions of LPL+/+-->C57BL/6 and LPL+/--->C57BL/6 mice, but not in LPL-/--->C57BL/6 mice, whereas myocardial cells expressed LPL in all groups. The mean aortic lesion area was reduced by 55% in LPL-/--->C57BL/6 mice compared with LPL+/+-->C57BL/6 mice and by 45% compared with LPL+/--->C57BL/6 mice, respectively. These data demonstrate in vivo that LPL expression by macrophages in the artery wall promotes foam cell formation and atherosclerosis. off

SYNERGISM BETWEEN LIPOPOLYSACCHARIDE AND INTERFERON γ IN THE REGULATION OF LIPOPROTEIN LIPASE IN MACROPHAGES

The regulation of macrophage lipoprotein lipase (LPL) by cytokines is potentially of crucial importance in the pathogenesis of atherosclerosis and in septic shock. The effect of combinations of lipopolysaccharide (LPS) and cytokines on the expression of LPL in macrophages was studied using the murine J774.2 cell line. The suppression of heparin-releasable LPL activity produced by combinations of LPS and interleukin 1 (IL-1), IL-11 or tumour necrosis factor α(TNF-α) was substantially less than that expected from the simple additive action of the corresponding two effectors. By contrast, co-exposure of the cells to LPS and interferon γ(IFN-γ) resulted in a more than additive, synergistic, suppression of LPL activity which was, additionally, also observed when the rat alveolar macrophage NR8383 cell line was studied. This synergistic action was also observed when J774.2 macrophages were exposed initially to IFN-γ (priming), washed and then treated with LPS. A comparison of the LPL activity and mRNA levels produced by the synergistic action of LPS and IFN-γand the priming action of IFN-γ indicated that a combination of mRNA metabolism (transcription or RNA stability), translation and post-translational mechanisms were responsible for the observed changes in LPL activity. These data, therefore, suggest that combinations of LPS and cytokines may be more important than the presence or absence of any given single effector in the modulation of LPL function during infection.

Regulation of macrophage lipoprotein lipase expression by activators of peroxisome proliferator-activated receptors

Regulation of macrophage lipoprotein lipase expression by activators of peroxisome proliferator-activated receptors