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-1β, interleukin-6, monocyte chemoattractant protein-1, and tumor necrosis factor-α 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. 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-1β, interleukin-6, monocyte chemoattractant protein-1, and tumor necrosis factor-α 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. From early fatty streak lesions to advanced plaques, macrophages are integral to the development and progression of atherosclerosis. It is well recognized that a variety of proinflammatory cytokines are expressed in activated macrophages, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-8, IL-1β, and monocyte chemoattractant protein-1 (MCP-1). These cytokines have been shown to promote lesion progression by aggravating endothelial dysfunction, recruiting additional inflammatory cells, or stimulating smooth muscle proliferation (1Tedgui A. Mallat Z. Cytokines in atherosclerosis: pathogenic and regulatory pathways. Physiol. Rev. 2006; 86: 515-581Google Scholar). LPL primarily hydrolyzes triglycerides contained within chylomicrons and very low density lipoproteins, whereas endothelial lipase (EL) preferentially hydrolyzes phospholipids within HDL (2Ma K. Cilingiroglu M. Otvos J.D. Ballantyne C.M. Marian A.J. Chan L. Endothelial lipase is a major genetic determinant for high-density lipoprotein concentration, structure, and metabolism. Proc. Natl. Acad. Sci. USA. 2003; 100: 2748-2753Google Scholar). The expression of LPL and EL has been associated with macrophages within human atherosclerotic lesions (3Van Eck M. Zimmermann R. Groot P.H. Zechner R. Van Berkel T.J. Role of macrophage-derived lipoprotein lipase in lipoprotein metabolism and atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 2000; 20: E53-E62Crossref PubMed Google Scholar, 4O'Brien K.D. Gordon D. Deeb S. Ferguson M. Chait A. Lipoprotein lipase is synthesized by macrophage-derived foam cells in human coronary atherosclerotic plaques. J. Clin. Invest. 1992; 89: 1544-1550Google Scholar, 5Azumi H. Hirata K. Ishida T. Kojima Y. Rikitake Y. Takeuchi S. Inoue N. Kawashima S. Hayashi Y. Itoh H. Immunohistochemical localization of endothelial cell-derived lipase in atherosclerotic human coronary arteries. Cardiovasc. Res. 2003; 58 (et al.): 647-654Google Scholar). Although the specific roles of these lipases in atherosclerosis likely depend on their tissue localization, accumulating evidence supports a proatherogenic role of macrophage-derived lipases. LPL expression was observed to be increased in human macrophages isolated from patients with either heterozygous familial hypercholesterolemia or type II diabetes (6Beauchamp M.C. Letendre E. Renier G. Macrophage lipoprotein lipase expression is increased in patients with heterozygous familial hypercholesterolemia. J. Lipid Res. 2002; 43: 215-222Google Scholar, 7Sartippour M.R. Renier G. Upregulation of macrophage lipoprotein lipase in patients with type 2 diabetes: role of peripheral factors. Diabetes. 2000; 49: 597-602Google Scholar). These observations are substantiated by animal studies in which macrophage-specific expression of human LPL promotes the extent of aortic atherosclerosis in transgenic apolipoprotein E knockout mice and rabbits (8Wilson K. Fry G.L. Chappell D.A. Sigmund C.D. Medh J.D. Macrophage-specific expression of human lipoprotein lipase accelerates atherosclerosis in transgenic apolipoprotein E knockout mice but not in C57BL/6 mice. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1809-1815Google Scholar, 9Ichikawa T. Liang J. Kitajima S. Koike T. Wang X. Sun H. Morimoto M. Shikama H. Watanabe T. Yamada N. Macrophage-derived lipoprotein lipase increases aortic atherosclerosis in cholesterol-fed Tg rabbits. Atherosclerosis. 2005; 179 (et al.): 87-95Google Scholar, 10Koike T. Liang J. Wang X. Ichikawa T. Shiomi M. Sun H. Watanabe T. Liu G. Fan J. Enhanced aortic atherosclerosis in transgenic Watanabe heritable hyperlipidemic rabbits expressing lipoprotein lipase. Cardiovasc. Res. 2005; 65: 524-534Google Scholar). Also, despite the presence of a proatherogenic lipid profile, EL deficiency was associated with an ∼70% decrease in atherosclerotic disease area in apolipoprotein E knockout mice compared with controls (11Ishida T. Choi S.Y. Kundu R.K. Spin J. Yamashita T. Hirata K. Kojima Y. Yokoyama M. Cooper A.D. Quertermous T. Endothelial lipase modulates susceptibility to atherosclerosis in apolipoprotein-E-deficient mice. J. Biol. Chem. 2004; 279: 45085-45092Google Scholar). More recently, in a cohort of healthy subjects with a family history of premature coronary heart disease, increased human plasma EL concentrations were significantly associated with the metabolic syndrome and subclinical coronary heart disease (12Badellino K.O. Wolfe M.L. Reilly M.P. Rader D.J. Endothelial lipase concentrations are increased in metabolic syndrome and associated with coronary atherosclerosis. PLoS Med. 2006; 3: e22Google Scholar). The proatherogenic nature of LPL and EL in macrophages has been largely ascribed to their noncatalytic or bridging function, which mediates the interaction between lipoproteins and cell surfaces. An exogenous source of LPL has been shown to facilitate the binding and uptake of LDL by THP-1 monocytes and macrophages, possibly by involving various proteoglycans and lipid rafts (13Olin K.L. Potter-Perigo S. Barrett P.H. Wight T.N. Chait A. Lipoprotein lipase enhances the binding of native and oxidized low density lipoproteins to versican and biglycan synthesized by cultured arterial smooth muscle cells. J. Biol. Chem. 1999; 274: 34629-34636Google Scholar, 14Kaplan M. Aviram M. Retention of oxidized LDL by extracellular matrix proteoglycans leads to its uptake by macrophages: an alternative approach to study lipoproteins cellular uptake. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 386-393Google Scholar, 15Makoveichuk E. Castel S. Vilaro S. Olivecrona G. Lipoprotein lipase-dependent binding and uptake of low density lipoproteins by THP-1 monocytes and macrophages: possible involvement of lipid rafts. Biochim. Biophys. Acta. 2004; 1686: 37-49Google Scholar). Similarly, endogenously produced EL in transfected Chinese hamster ovary cells enhances the binding and cellular processing of plasma lipoproteins via a heparan sulfate proteoglycan-mediated route (16Fuki I.V. Blanchard N. Jin W. Marchadier D.H. Millar J.S. Glick J.M. Rader D.J. Endogenously produced endothelial lipase enhances binding and cellular processing of plasma lipoproteins via heparan sulfate proteoglycan-mediated pathway. J. Biol. Chem. 2003; 278: 34331-34338Google Scholar). Moreover, EL and LPL can play a role as adhesion molecules through the interaction with heparan sulfate proteoglycans to facilitate the binding of monocytes onto the surface of endothelial cells (17Mamputu J.C. Desfaits A.C. Renier G. Lipoprotein lipase enhances human monocyte adhesion to aortic endothelial cells. J. Lipid Res. 1997; 38: 1722-1729Google Scholar, 18Obunike J.C. Paka S Pillarisetti S. Goldberg I.J. Lipoprotein lipase can function as a monocyte adhesion protein. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1414-1420Google Scholar, 19Kojma Y. Hirata K. Ishida T. Shimokawa Y. Inoue N. Kawashima S. Quertermous T. Yokoyama M. Endothelial lipase modulates monocyte adhesion to the vessel wall. A potential role in inflammation. J. Biol. Chem. 2004; 279: 54032-54038Google Scholar). There is little information on the effect of endogenous lipase expression on the secretion of proinflammatory cytokines in human macrophages. LPL expression in mouse macrophages and adipocytes is decreased by interferon-γ, TNF-α, lipopolysaccharide, IL-11, IL-1, and IL-6 (20Muhammad T.S. Hughes T.R. Cryer A. Ramji D.P. The suppression of lipoprotein lipase expression in J774.2 macrophages by IFN-gamma and TNF-alpha is mediated at the transcriptional level. Biochem. Soc. Trans. 1998; 26 (Suppl.):: 12Google Scholar, 21Tengku-Muhammad T.S. Hughes T.R. Cryer A. Ramji D.P. Differential regulation of lipoprotein lipase in the macrophage J774.2 cell line by cytokines. Cytokine. 1996; 8: 525-533Google Scholar, 22Greenberg A.S. Nordan R.P. McIntosh J. Calvo J.C. Scow R.O. Jablons D. Interleukin 6 reduces lipoprotein lipase activity in adipose tissue of mice in vivo and in 3T3-L1 adipocytes: a possible role for interleukin 6 in cancer cachexia. Cancer Res. 1992; 52: 4113-4116Google Scholar). Regarding EL, IL-1β and TNF-α have been shown to stimulate its expression in endothelial cells (23Jin W. Sun G.S. Marchadier D. Octtaviani E. Glick J.M. Rader D.J. Endothelial cells secrete triglyceride lipase and phospholipase activities in response to cytokines as a result of endothelial lipase. Circ. Res. 2003; 92: 644-650Google Scholar). However, very few studies have addressed how lipase expression may affect cytokine expression. Exogenous LPL has been shown to induce TNF-α by increasing TNF-α mRNA transcription and stability (24Reiner G. Oliver M. Skamene E. Radzioch D. Induction of tumor necrosis factor alpha gene expression by lipoprotein lipase requires protein kinase C activation. J. Lipid Res. 1994; 35: 1413-1421Google Scholar, 25Renier G. Skamene E. DeSanctis J.B. Radzioch D. Induction of tumor necrosis factor alpha gene expression by lipoprotein lipase. J. Lipid Res. 1994; 35: 271-278Google Scholar). To there are on the of EL expression on cytokine expression. we have lentivirus-mediated RNA interference to suppress the expression of either LPL or EL in macrophages. observed that the expression of LPL and EL in THP-1 macrophages is with proinflammatory cytokine expression in THP-1 macrophages. THP-1 monocytes were from the and in with and cells were cultured in with and cells were in a at and with THP-1 monocytes were used within to either EL or LPL were A with to human as a were in and into of the from were by The with the from of was the the from the via the were by was produced by with and from into cells at a of were for and by a of was on cells with of the The was as cell at the of of of for EL, and endothelial lipase. in a EL, endothelial lipase. LDL was with for 2 or at for mildly and extensively oxidized low density lipoprotein The was by to a concentration of LDL was a The protein concentration of the LDL was a protein The extent of of LDL was by of was to a of by the of of After and at for the were at for and the of the was at a The of was by with a of An LDL with a of was as mildly whereas of were as extensively THP-1 monocytes were onto a and by at of of on the was to the cells to a concentration of at 2 to stimulate the of the monocytes into macrophages. was for cytokine for was to the macrophage activity was a as J.S. D. J. H. of lipase and lipoprotein lipase. of and J. Biol. Chem. 1998; Scholar). An phospholipase was used to the phospholipase the phospholipase was in and to cell to a concentration of 2 After were for the of at and of and which was for cell protein. RNA was isolated the The was the with and of and EL was on the were as for for for to and of for and for A was from from as an were in with for of protein were onto a and for at were onto for at After of with were with a at and with for were by The of proinflammatory cytokines was by IL-1β, IL-8, and TNF-α were from of from THP-1 macrophages to the of were to a of and for at were of was for were with of at for A was and in the for was at of macrophages were in and in a After a cells were in of and an additional of and After of at the was at for and the was into a The were a in of onto an with of and The was in in for Lipid composition was the by and of from fatty is at the of J. Lipid Res. 2002; 43: Scholar). To gene expression lipase an was to the a RNA and an RNA was from with the and for RNA was synthesized into and the was into RNA with the of was in an and the was and with the 2 The expression of was by and gene is by on the of with THP-1 monocytes was by a of a of was whereas was at a of not were with a of with with a a LPL and EL in and decreases in mRNA LPL suppression in a decrease in activity whereas EL suppression was associated with a in phospholipase a of of phospholipase activity There was suppression or for the lipase in as by mRNA and and phospholipase activity not Also, of EL and LPL from indicated decreases for lipase protein The concentrations of proinflammatory cytokines were in THP-1 macrophages suppression of either LPL or EL there were increases in and TNF-α the observed However, were observed for IL-1β and decreases were observed for IL-1β for LPL suppression and at the and for EL suppression in decreases in cytokine lipase suppression were observed for and TNF-α decreases in were observed were in the presence of various oxidized of decreases in cytokine were observed There were decreases for cytokines for at for and macrophages with mildly effect was macrophages were with extensively with the of THP-1 macrophages with mildly and extensively of was associated with cytokine of IL-1β, and TNF-α, with observed decreases of and and and and and 2 A was observed for the additional However, significant were observed for The of decreased cytokine was also observed for cells but was of EL suppression To lipase expression, LPL and EL were the and the of mildly and extensively consistently and significantly decreased EL mRNA expression by monocyte whereas LPL mRNA not to be of on lipase expression in THP-1 macrophages. was into cell and mRNA was for Macrophage LPL expression Macrophage EL expression are as The lipid composition of THP-1 macrophages was altered significantly After of by decreases in cholesterol by and in by and in cholesterol by and and in triglycerides by and were observed in and macrophages, After LPL and EL the concentrations were decreased by and the of were increased by and for LPL and EL including and also to LPL and EL suppression. Microarray of was on and macrophages. The suppression of either LPL or EL in macrophages the gene expression or or of of the contained within the The and are in 2 of the data indicated that proinflammatory including IL-8, and TNF-α, were by and for LPL suppression and by and for EL suppression. growth factor of the to the inflammatory was by and in and macrophages, of of the in cell adhesion and the extracellular including and adhesion and and was by to whereas the expression of and increased to to of the data also indicated decreases in the expression of growth and antiapoptotic and The expression of receptors and transcription was also altered in and cells, as with and decreases for receptor and and increases for Also, low density lipoprotein receptor, scavenger receptor 1, and scavenger receptor class B type were increased in macrophages and and cells and was also in the presence of EL suppression and in human atherosclerosis for of of necrosis growth factor necrosis protein growth response receptor receptor factor factor density lipoprotein growth factor density lipoprotein interaction receptor class B type factor factor adhesion receptor receptor that are expressed in but in cells are not in a that are expressed in but in cells are not LPL and EL have been shown to have significant on lipid metabolism and to the development of lesions in mouse of atherosclerosis. we used a lentivirus-mediated RNA interference approach to investigate the effect of LPL and EL suppression in cytokine secretion and cellular lipid To IL-1β, IL-8, and TNF-α were to be decreased in the presence of endogenous lipase suppression. suppression was also by decreases in the concentrations of cellular triglycerides, and study that lipase suppression the decreased expression of proinflammatory cytokines as IL-1β, and TNF-α as well as the decreased of Decreased expression of proinflammatory cytokines in macrophages be ascribed to to lipase suppression. LPL and EL are of EL has been shown to phospholipids contained within M. G. A. G. E. W. S. Endothelial lipase and fatty of density lipoprotein J. Lipid Res. 2005; Scholar). The of with LPL with for at the of D.A. J.D. Lipoprotein and phospholipase of with an of Biochim. Biophys. Acta. Scholar). on cell was increased in patients of decreased of phospholipids by LPL M.R. in lipid composition and in lipoprotein lipase Biochim. Biophys. Acta. 1992; PubMed Google Scholar). The of which has been shown to induce the expression of IL-8, and by human endothelial cells and smooth muscle cells and to promote the of and G. M.R. K. human endothelial cell expression of J. 2003; 35: PubMed Google Scholar, monocyte chemoattractant protein-1 gene expression in aortic smooth muscle cells. Arterioscler. Thromb. Vasc. Biol. 2002; Scholar). promotes proinflammatory in monocytes through stimulating L. M. W. S. S. of protein from the 2003; Scholar). IL-1β expression was also in macrophages by which on a receptor for Y. E. the of by human Atherosclerosis. 1998; Google Scholar, Y. Hirata K. Yamashita T. K. S. Itoh H. M. J. Shiomi M. Inoue N. of a receptor for by macrophages in and human atherosclerotic plaques. Arterioscler. Thromb. Vasc. Biol. 2002; (et al.): Scholar). Thus, is possible that lipase suppression results in decreased of phospholipids and which results in decreased proinflammatory cytokine expression. It is also possible that a in the noncatalytic function of these lipases influences cytokine was that lipases are of lipid uptake and by cells, including macrophages E. Castel S. Vilaro S. Olivecrona G. Lipoprotein lipase-dependent binding and uptake of low density lipoproteins by THP-1 monocytes and macrophages: possible involvement of lipid rafts. Biochim. Biophys. Acta. 2004; 1686: 37-49Google Scholar, D. A. S. G. J. triglyceride in human macrophages is mediated by of LPL activity in the of in LPL Biochim. Biophys. Acta. 2003; Scholar). observed in the of cholesterol and triglycerides were decreased in macrophages. Moreover, studies from have indicated decreased binding of native LDL and lipase suppression that may be lipase suppression as a result of a reduced uptake of extracellular cholesterol is used for a of cellular including the of cell cytokine and can cytokines at the gene expression level. Lipid increases as a IL-1β is in foam cells G.S. in macrophages. 2006; Scholar). macrophages are to be an source of TNF-α, and Y. T. M.C. R. D.A. macrophages are an source of tumor necrosis and of and in advanced atherosclerosis. J. Biol. Chem. 2005; Scholar, N. R. S. A. Interleukin is by cholesterol of macrophages and expressed by macrophage foam cells in human J. Biol. Chem. 1996; Scholar). Thus, a decreased concentration of lipase suppression may also mediate decreases in cytokine expression. Microarray also indicated decreased expression of proinflammatory cytokines with the cytokine Although protein not to be altered by LPL decreases in mRNA were observed. It is possible that further regulation at or may have we that factors, as and were It is that of and lipid are the of and of LPL with LDL or increases activity in to fatty and transgenic mice LPL increased proliferation S. L. J. K.L. Rader D.J. A. Zechner R. G. of lipoproteins evidence for an role for lipoprotein lipase. Proc. Natl. Acad. Sci. USA. 2003; (et al.): Scholar). is observed LPL is or by in vivo S. L. J. K.L. Rader D.J. A. Zechner R. G. of lipoproteins evidence for an role for lipoprotein lipase. Proc. Natl. Acad. Sci. USA. 2003; (et al.): Scholar). EL an alternative for which is further into cells and D. Zimmermann R. Jin W. G. Rader D.J. Zechner R. Endothelial lipase an alternative for uptake in lipoprotein mouse adipose J. Clin. Invest. 2005; Scholar, W. G. L. Rader D.J. 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Also, a of may cell proliferation and lipoproteins have been well recognized as proatherogenic of their by cells, macrophages in atherosclerotic mildly and extensively consistently decreased IL-1β, and TNF-α from to However, the role of oxidized lipoproteins on cytokine expression is not expression of including IL-1β, and TNF-α and and interferon-γ, was observed with M. Yamashita S. K. N. H. K. Z. S. Y. B and expression of proinflammatory are in macrophages from Arterioscler. Thromb. Vasc. Biol. 2000; 20: Scholar, A.S. M. of and from macrophages by lipoprotein and its oxidized 2001; Scholar). However, with either mildly or extensively oxidized lipoproteins for consistently decreased proinflammatory cytokine expression in macrophages in and low density lipoprotein inflammatory in through of inflammation. Biochem. Biophys. Res. 2006; have that the of proinflammatory including and and TNF-α, was in an of the in were with either or the expression of interferon-γ, IL-1β, and IL-6 was mouse macrophages were or with extensively A. T. low density lipoprotein in human macrophages by with activation. J. Biol. Chem. 2004; 279: Scholar, D. G. T.S. low density lipoprotein in mouse macrophages via between and J. Biol. Chem. 2000; Scholar, Cooper A.D. of mRNA expression in mouse macrophages by oxidized low density J. Lipid Res. Scholar). EL expression in the reduced cytokine concentrations may in be mediated by decreased EL expression. The of the of on cytokine expression may be to the extent of of lipoproteins as well as the and we have observed that the suppression of LPL or EL decreased the expression of proinflammatory cytokines in human macrophages and reduced lipid suppression also altered associated with atherosclerotic their role in the development of atherosclerosis. was by from the of the and of and and the is a of the of The the of for lipid with endothelial lipase interleukin monocyte chemoattractant protein-1 of oxidized low density lipoprotein receptor tumor necrosis factor-α

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