Endothelial Lipase Expression Research Articles

Targeted inactivation of endothelial lipase attenuates lung allergic inflammation through raising plasma HDL level and inhibiting eosinophil infiltration

Endothelial lipase (EL) is a novel phospholipase that determines plasma high-density lipoprotein cholesterol (HDL-C) levels. We have investigated the role of HDL-C in lung allergic inflammation by using EL knockout (EL-KO) mice that are high in HDL-C. EL-KO and wild-type control mice were sensitized and challenged with ovalbumin to evoke eosinophilic inflammation in the lung. EL was expressed in epithelial cells, alveolar type II cells, and endothelial cells in the lung, and its expression was upregulated during inflammation. Concomitant with attenuated hyperresponsiveness of the airway smooth muscles, the number of eosinophils in bronchoalveolar lavage and the expression of VCAM-1 were lower in EL-KO mice than in control mice. HDL reduced cytokine-induced VCAM-1 expression in cultured endothelial cells. When plasma HDL levels were decreased to similar levels in both mouse groups by adenovirus-mediated overexpression of EL, however, eosinophil infiltration was still lower in EL-KO mice. In vitro adhesion assays revealed that EL expression on the cell surface promoted the interaction of eosinophils through the ligand-binding function of EL. In summary, targeted inactivation of EL attenuated allergic inflammation in the lung, and the protective effects in EL-KO mice were associated with high plasma HDL levels, downregulation of VCAM-1, and loss of the direct ligand-binding function of EL. Thus EL is a novel modulator of the progression of allergic asthma.

Endothelial Lipase Promotes Apolipoprotein AI-Mediated Cholesterol Efflux in THP-1 Macrophages

Endothelial lipase (EL) is expressed by macrophages within atherosclerotic lesions. We investigated the influence of EL expression on cholesterol efflux in macrophages. The present study used lentivirus to introduce either EL shRNA for loss-of-function studies or EL cDNA for gain-of-function studies to investigate the role of EL in apoAI-mediated cholesterol efflux. ApoAI-mediated cholesterol efflux was decreased after EL suppression, but increased with EL overexpression in free cholesterol labeled and acLDL loaded THP-1 macrophages. Similar findings were observed in THP-1 macrophages after exogenous EL addition and in transfected 293 cells. EL-related apoAI-mediated cholesterol efflux decreased after treatment with heparin or catalytic inactivation (S149A mutation or tetrahydrolipstatin) alone, and completely inhibited in combination. Furthermore, EL expression did not change ABCA1 expression, but was positively correlated with apoAI binding to macrophages and 293 cells. This effect was mitigated after heparin treatment but not influenced by catalytic inactivation via tetrahydrolipstatin or the S149A mutation. Moreover, EL expression was positively associated with lysophosphatidylcholine production and inversely with phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin levels. Lysophosphatidylcholine treatment dose-dependently stimulated apoAI-mediated cholesterol efflux in THP-1 macrophages. EL appears to promote apoAI-mediated cholesterol efflux through catalytic and noncatalytic-dependent mechanisms.

Abstract 1693: Effect of Pitavastatin on Serum Concentrations of Endothelial Lipase and High-Density Lipoprotein Cholesterol in Humans

[Purpose] Endothelial lipase (EL) is a novel phospholipase which regulates serum high-density lipoprotein cholesterol (HDL-C) levels. In the present study, we have measured serum levels of EL in human subjects, and examined the effect of statins on serum HDL levels and EL mass. [Methods and Results] A sandwich ELISA for human EL was established using two monoclonal antibodies against amino- or carboxy-terminus of human EL. Serum EL concentrations were measured and compared with the serum lipid profile in 237 patients with cardiovascular diseases. There was no significant correlation between serum EL mass and age or gender. Serum EL levels were negatively associated with serum HDL-C levels, but not with serum LDL-C, total cholesterol, or triglyceride levels. Forty-eight patients with hypercholesterolemia were treated with pitavastatin for 6 months, and serum EL mass was evaluated before and after the pitavastatin treatment. Pitavastatin treatment significantly reduced serum EL levels by 15% and increased HDL-C levels by 12%. Complimentary cell culture experiments revealed that pitavastatin suppressed basal and cytokine-induced EL expression and phospholipase activities in endothelial cells, which is reversed by the concomitant treatment with mevalonate or geranylgeranylpyrophosphate. Also, overexpression of RhoA T19N, a dominant negative mutant of RhoA, decreased the EL expression. [Conclusion] EL is a determinant of serum HDL-C levels in humans. Pitavastatin reduced EL expression through the protein isoprenylation and inhibition of Rho activity, and raised serum HDL-C levels. Thus, EL would be a target molecule for the HDL-raising pharmaceutical interventions.

Abstract 1186: Overexpression Of Endothelial Lipase In The Liver Promotes The Clearance Of Plasma Lipids But Accelerates Liver Steatosis In A Mouse Model Of Metabolic Syndrome

The metabolic syndrome includes high triglyceride (TG) and low HDL-cholesterol (HDL-C) levels in the plasma, and often accompanies steatosis in the liver. Endothelial lipase (EL) is a phospholipase that regulates HDL metabolism. EL is expressed by hepatocytes, while the function of EL in the liver has not been identified. Here, we examined the role of EL in the liver using a mouse model of metabolic syndrome. The EL expression in the liver was analyzed by real-time PCR. It revealed that liver EL expression was significantly increased in obese and diabetic db/db mice compared to that of control mice. To examine the significance of the EL upregulation in the liver, we injected the recombinant adenovirus encoding human EL into mice. The EL overexpression in the liver resulted in a significant decrease in plasma HDL-C, TG, and free fatty acid levels. Interestingly, the EL overexpression in the liver increased liver weight and liver TG content both in wild-type and db/db mice. In db/db mice, particularly, EL overexression accelerated the formation of steatosis by increasing the mRNA level of fatty acid synthase. These findings indicate that EL expression is increased in the liver in the metabolic syndrome. The upregulation of EL promotes the uptake of plasma lipids by hepatocytes, and accelerates the progression of steatosis in db/db mice. Thus EL may play a role in the genesis of steatosis as well as dyslipidemia in the metabolic syndrome.

Atorvastatin decreases lipoprotein lipase and endothelial lipase expression in human THP-1 macrophages

Macrophage-derived lipases are associated with atherosclerosis in human and animal studies. Despite numerous non-lipid-lowering effects of statins, their effect on macrophage LPL and endothelial lipase (EL) expression has not been investigated. In the present study, atorvastatin and simvastatin dose-dependently decreased LPL and EL expression as well as Rho, liver X receptor alpha (LXRalpha), and nuclear factor kappaB (NF-kappaB) activation in THP-1 macrophages. Atorvastatin-reduced LPL and EL expression was only partially recovered by mevalonate cotreatment, indicating that mechanisms independent of reductase inhibition may be present. By contrast, Rho activation by lysophosphatidyl acid further decreased LPL and EL expression in the presence or absence of atorvastatin. Another Rho activator, farnysyl pyrophosphate, decreased EL expression only in the absence of atorvastatin. LXRalpha activation by T0901317 and 22(R)-hydroxycholesterol not only rescued but also significantly increased LPL expression in the presence and absence of atorvastatin, respectively, whereas LXRalpha inhibition by 22(S)-hydroxycholesterol decreased LPL expression. By contrast, EL expression was suppressed by LXRalpha activation in the presence or absence of atorvastatin. NF-kappaB inhibition by SN50 was associated with an approximately 30% reduction of EL expression. Furthermore, atorvastatin treatment significantly attenuated the lipid accumulation in macrophages treated with oxidized LDL. We conclude that atorvastatin reduces LPL and EL expression by reducing the activation of LXRalpha and NF-kappaB, respectively.

Endothelial lipase enhances low density lipoprotein binding and cell association in THP-1 macrophages

Endothelial lipase (EL) is expressed in macrophages in human atherosclerotic lesions. However, its specific metabolic role in human macrophages has not been fully explored. The present study used lentivirus containing either shRNA or cDNA for EL to decrease or increase EL expression, respectively in THP-1 macrophages to investigate the consequence on LDL binding and cell association. EL suppression significantly decreased the binding and cell association of native LDL (52% and 33%) and mildly oxLDL (43% and 36%) as well as extensively oxLDL binding (36%) in THP-1 macrophages. EL overexpression markedly increased the binding and cell association of native LDL (3.1- and 2.2-fold), mildly oxLDL (1.9- and 1.4-fold), and extensively oxLDL (1.5- and 1.5-fold). An inactive mutant EL compromised EL-mediated cell association of native and mildly oxLDL but not extensively oxLDL. Heparinase treatment almost completely eliminated EL-mediated native and oxLDL binding and cell association in THP-1 macrophages. LDL receptor blocking by antibodies decreased EL-mediated native LDL binding and cell association by 24% and 54%, respectively. Neither receptor associated protein or CD36 antibody treatment led to changes in EL-mediated lipoprotein binding and cell association. Furthermore, wild-type and the catalytically inactive mutant EL increased lipid accumulation in THP-1 macrophages. EL expression promotes the binding and uptake of native and oxidized LDL in THP-1 macrophages in a heparan sulfate proteoglycan-dependent manner, and the LDL receptor was partly responsible for the EL-enhanced uptake of native LDL.

Hepatic Proprotein Convertases Modulate HDL Metabolism

The risk of atherosclerosis is inversely associated with plasma levels of high-density lipoprotein cholesterol (HDL-C). However, HDL metabolism is incompletely understood, and there are few effective approaches to modulate HDL-C levels. Here we show that inhibition in the liver of the classical proprotein convertases (PCs), but not the atypical PCs S1P and PCSK9, decreases plasma HDL-C levels. This metabolic effect of hepatic PCs is critically dependent on expression of endothelial lipase (EL), an enzyme that directly hydrolyzes HDL phospholipids and promotes its catabolism. Hepatic PCs reduce EL function through direct inactivating cleavage of EL as well as through activating cleavage of angiopoietin-like protein 3 (ANGPTL3), an endogenous inhibitor of EL. Thus, inhibition of hepatic PCs results in increased EL activity, leading to reduced HDL-C as well as impaired reverse cholesterol transport. The hepatic PC-ANGPTL3-EL-HDL pathway is therefore a novel mechanism controlling HDL metabolism and cholesterol homeostasis.

Endothelial lipase is highly expressed in macrophages in advanced human atherosclerotic lesions

Endothelial lipase (EL) is expressed in endothelial cells, and affects plasma lipoprotein metabolism by hydrolyzing phospholipids in HDL. To determine the cellular expression of EL mRNA and protein in human atherosclerotic lesions, we performed in situ hybridization and immunohistochemical studies on sections of carotid endarterectomy specimens from patients with symptomatic cerebrovascular disease. In each of eight patients, EL mRNA and/or protein were seen in areas between the necrotic core and the fibrotic cap where they colocalized with LPL and macrophage-specific CD68. Moreover, there was a positive association between the expression of EL mRNA and CD68 mRNA in plaques from 26 patients. The impact of differentiation from monocytes into macrophages, and subsequently foam cells (by incubation with acetylated LDL) on expression was studied using THP-1 monocytes and primary human monocytes. EL mRNA expression increased markedly when either type of monocytes was differentiated into macrophages. Upon further differentiation into foam cells EL mRNA decreased whereas protein levels remained high compared to monocytes. In conclusion, macrophages in advanced human atherosclerotic lesions display high levels of EL expression, and the level of EL expression varies greatly during transformation of blood monocytes into foam cells.

Glycosylation of endothelial lipase at asparagine-116 reduces activity and the hydrolysis of native lipoproteins in vitro and in vivo

We previously identified that four of five putative N-linked glycosylation sites of human endothelial lipase (EL) are utilized and suggested that the substitution of asparagine-116 (Asn-116) with alanine (Ala) (N116A) increased the hydrolytic activity of EL. The current study demonstrates that mutagenesis of either Asn-116 to threonine (Thr) or Thr-118 to Ala also disrupted the glycosylation of EL and enhanced catalytic activity toward synthetic substrates by 3-fold versus wild-type EL. Furthermore, we assessed the hydrolysis of native lipoprotein lipids by EL-N116A. EL-N116A exhibited a 5-fold increase in LDL hydrolysis and a 1.8-fold increase in HDL2 hydrolysis. Consistent with these observations, adenovirus-mediated expression of EL-N116A in mice significantly reduced the levels of both LDL and HDL cholesterol beyond the reductions observed by the expression of wild-type EL alone. Finally, we introduced Asn-116 of EL into the analogous positions within LPL and HL, resulting in N-linked glycosylation at this site. Glycosylation at this site suppressed the LPL hydrolysis of synthetic substrates, LDL, HDL2, and HDL3 but had little effect on HL activity. These data suggest that N-linked glycosylation at Asn-116 reduces the ability of EL to hydrolyze lipids in LDL and HDL2.

Dysregulation of Placental Endothelial Lipase and Lipoprotein Lipase in Intrauterine Growth-Restricted Pregnancies

Fetal supply of maternally derived fatty acids requires lipase-mediated hydrolysis of lipoprotein-borne triglycerides and phospholipids at the placental surface. The objective of the study was to test the hypothesis that members of the triglyceride lipase gene (TLG) family are expressed in the human placenta at the maternoplacental (syncytiotrophoblast) and fetoplacental (endothelial cells) interface and that their expression is altered in pregnancy pathologies. Expression of TLG family members in primary placental cells (trophoblast and endothelial cells) and tissues of first-trimester and term human placenta was analyzed by microarrays, RT-PCR, Western blotting, and immunohistochemistry. Their expression was compared between normal pregnancies and those complicated with intrauterine growth restriction (IUGR). Participants included women with uncomplicated pregnancies and pregnancies complicated by IUGR. Endothelial lipase (EL) and lipoprotein lipase (LPL) were the only lipases among the TLG family expressed in key cells of the human placenta. In first trimester, EL and LPL were expressed in trophoblasts. At term, EL was detected in trophoblasts and endothelial cells, whereas LPL was absent in these cells. Both lipases were found at placental blood vessels, EL in vascular endothelial cells and LPL in the surrounding smooth muscle cells. In total placental tissue EL expression prevails in first trimester and at term. Compared with normal placentas, EL mRNA was decreased (30%; P < 0.02), whereas LPL mRNA expression was increased (2.4-fold; P < 0.015) in IUGR. EL is the predominant TLG family member in the human placenta present at both interfaces. EL and LPL are dysregulated in IUGR.

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.

Endothelial Lipase is Increased by Inflammation and Promotes LDL Uptake in Macrophages

Endothelial lipase (EL) is a member of the lipoprotein lipase family that regulates HDL metabolism. EL is known to act as a bridging molecule for monocytes or lipoproteins in vascular endothelial cells. We investigated the role and regulatory mechanisms of EL expression in macrophages. Macrophages originating from wild-type (EL+/+) and EL-deficient (EL-/-) mice were stimulated with lipopolysaccharide (LPS). The expression of EL mRNA was evaluated by northern blotting. DiI-LDL was used to measure the uptake of native low-density lipoprotein (nLDL). LPS increased EL mRNA levels by increasing intracellular oxidative stress in the macrophages. LPS did not affect EL expression in macrophages derived from Toll-like receptor 4 (TLR4) gene mutant mice, C3H/HeJ. The uptake of nLDL after LPS-treatment was significantly lower in macrophages from EL-/- mice than those from EL+/+ mice. Simvastatin suppressed the LPS-induced upregulation of EL expression and uptake of nLDL. EL expression is upregulated by LPS via TLR4 and promotes the uptake of nLDL by macrophages. Simvastatin inhibits the LPS-induced up-regulation and uptake in macrophages. Thus, our findings provide a novel role for EL in lipoprotein metabolism and would expand the range of anti-atherogenic effects of statins.

Endothelial Lipase Gene Polymorphism is Associated With Acute Myocardial Infarction, Independently of High-Density Lipoprotein-Cholesterol Levels

Endothelial lipase (EL) is a major determinant of high-density lipoprotein-cholesterol (HDL-C) metabolism and promotes monocytes recruitment. The local expression of EL could influence atherogenesis directly, in addition to its systemic role in HDL metabolism. The EL gene has a common 584C/T polymorphism, but it is unclear whether this polymorphism is associated with HDL-C levels or acute myocardial infarction (AMI). A case-control study of 107 AMI patients and 107 control subjects was conducted. T allele frequency was lower in the AMI group than in controls (0.18 vs 0.26, p<0.05). No significant association was found between the 584C/T polymorphism and HDL-C levels. Multivariate regression analyses showed that the association of the T allele with AMI was statistically significant and independent of other risk factors when age, sex, hypertension, hypercholesterolemia, and diabetes mellitus were included in the analyses (odds ratio (OR), 0.52; 95% confidence interval (95% CI) 0.28-0.98; p=0.04). However, when smoking status was included, the association of the T allele with AMI did not remain statistically significant (OR, 0.61; 95% CI 0.32-1.18; p=0.14). The 584C/T polymorphism of the EL gene was associated with AMI independently of HDL-C levels and thus may be involved in the pathogenesis of AMI.

Placental triglyceride accumulation in maternal type 1 diabetes is associated with increased lipase gene expression

Maternal diabetes can cause fetal macrosomia and increased risk of obesity, diabetes, and cardiovascular disease in adulthood of the offspring. Although increased transplacental lipid transport could be involved, the impact of maternal type 1 diabetes on molecular mechanisms for lipid transport in placenta is largely unknown. To examine whether maternal type 1 diabetes affects placental lipid metabolism, we measured lipids and mRNA expression of lipase-encoding genes in placentas from women with type 1 diabetes (n = 27) and a control group (n = 21). The placental triglyceride (TG) concentration and mRNA expression of endothelial lipase (EL) and hormone-sensitive lipase (HSL) were increased in placentas from women with diabetes. The differences were more pronounced in women with diabetes and suboptimal metabolic control than in women with diabetes and good metabolic control. Placental mRNA expression of lipoprotein lipase and lysosomal lipase were similar in women with diabetes and the control group. Immunohistochemistry showed EL protein in syncytiotrophoblasts facing the maternal blood and endothelial cells facing the fetal blood in placentas from both normal women and women with diabetes. These results suggest that maternal type 1 diabetes is associated with TG accumulation and increased EL and HSL gene expression in placenta and that optimal metabolic control reduces these effects.

Endothelial lipase is less effective at influencing HDL metabolism in vivo in mice expressing apoA-II

Endothelial lipase (EL) plays an important physiological role in modulating HDL metabolism. Data suggest that plasma contains an inhibitor of EL, and previous studies have suggested that apolipoprotein A-II (apoA-II) inhibits the activity of several enzymes involved in HDL metabolism. Therefore, we hypothesized that apoA-II may reduce the ability of EL to influence HDL metabolism. To test this hypothesis, we determined the effect of EL expression on plasma phospholipase activity and HDL metabolism in human apoA-I and human apoA-I/A-II transgenic mice. Expression of EL in vivo resulted in lower plasma phospholipase activity and significantly less reduction of HDL-cholesterol, phospholipid, and apoA-I levels in apoA-I/A-II double transgenic mice compared with apoA-I single transgenic mice. We conclude that the presence of apoA-II on HDL particles inhibits the ability of EL to influence the metabolism of HDL in vivo.

Endothelial and lipoprotein lipases in human and mouse placenta

Placenta expresses various lipase activities. However, a detailed characterization of the involved genes and proteins is lacking. In this study, we compared the expression of endothelial lipase (EL) and LPL in human term placenta. When placental protein extracts were separated by heparin-Sepharose affinity chromatography, the EL protein eluted as a single peak without detectable phospholipid or triglyceride (TG) lipase activity. The major portion of LPL protein eluted slightly after EL. This peak also had no lipase activity and most likely contained monomeric LPL. Fractions eluting at a higher NaCl concentration contained small amounts of LPL protein (most likely dimeric LPL) and had substantial TG lipase activity. In situ hybridization studies showed EL mRNA expression in syncytiotrophoblasts and endothelial cells and LPL mRNA in syncytiotrophoblasts. In contrast, immunohistochemistry showed EL and LPL protein associated with both cell types. In mouse placentas, lack of LPL expression resulted in increased EL mRNA expression. These results suggest that the cellular expression of EL and LPL in human placenta is different. Nevertheless, the two lipases might have overlapping functions in the mouse placenta. Our data also suggest that the major portions of both proteins are stored in an inactive form in human term placenta.

Endothelial lipase releases saturated and unsaturated fatty acids of high density lipoprotein phosphatidylcholine

We assessed the ability of endothelial lipase (EL) to hydrolyze the sn-1 and sn-2 fatty acids (FAs) from HDL phosphatidylcholine. For this purpose, reconstituted discoidal HDLs (rHDLs) that contained free cholesterol, apolipoprotein A-I, and either 1-palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-linoleoylphosphatidylcholine, or 1-palmitoyl-2-arachidonylphosphatidylcholine were incubated with EL- and control (LacZ)-conditioned media. Gas chromatography analysis of the reaction mixtures revealed that both the sn-1 (16:0) and sn-2 (18:1, 18:2, and 20:4) FAs were liberated by EL. The higher rate of sn-1 FA cleavage compared with sn-2 FA release generated corresponding sn-2 acyl lyso-species as determined by MS analysis. EL failed to release sn-2 FA from rHDLs containing 1-O-1'-hexadecenyl-2-arachidonoylphosphatidylcholine, whose sn-1 position contained a nonhydrolyzable alkyl ether linkage. The lack of phospholipase A(2) activity of EL and its ability to liberate [(14)C]FA from [(14)C]lysophosphatidylcholine (lyso-PC) led us to conclude that EL-mediated deacylation of phosphatidylcholine (PC) is initiated at the sn-1 position, followed by the release of the remaining FA from the lyso-PC intermediate. Thin-layer chromatography analysis of cellular lipids obtained from EL-overexpressing cells revealed a pronounced accumulation of [(14)C]phospholipid and [(14)C]triglyceride upon incubation with 1-palmitoyl-2-[1-(14)C]linoleoyl-PC-labeled HDL(3), indicating the ability of EL to supply cells with unsaturated FAs.

Regulated expression of endothelial lipase by porcine brain capillary endothelial cells constituting the blood–brain barrier

Normal neurological function depends on a constant supply of polyunsaturated fatty acids to the brain. A considerable proportion of essential fatty acids originates from lipoprotein-associated lipids that undergo uptake and/or catabolism at the blood-brain barrier (BBB). This study aimed at identifying expression and regulation of endothelial lipase (EL) in brain capillary endothelial cells (BCEC), major constituents of the BBB. Our results revealed that BCEC are capable of EL synthesis and secretion. Overexpression of EL resulted in enhanced hydrolysis of extracellular high-density lipoprotein (HDL)-associated sn-2-labeled [(14)C]20 : 4 phosphatidylcholine. [(14)C]20 : 4 was recovered in cellular lipids, indicating re-uptake and intracellular re-esterification. To investigate local regulation of EL in the cerebrovasculature, BCEC were cultured in the presence of peroxisome-proliferator activated receptor (PPAR)- and liver X receptor (LXR)-agonists, known to regulate HDL levels. These experiments revealed that 24(S)OH-cholesterol (a LXR agonist), bezafibrate (a PPARalpha agonist), or pioglitazone (a PPARgamma agonist) resulted in down-regulation of EL mRNA and protein levels. Our findings implicate that EL could generate fatty acids at the BBB for transport to deeper regions of the brain as building blocks for membrane phospholipids. In addition PPAR and LXR agonists appear to contribute to HDL homeostasis at the BBB by regulating EL expression.

Increased expression of endothelial lipase in rat models of hypertension

To gain a better understanding of the involvement of endothelial lipase (EL) in vascular disease, we examined whether the EL expression is regulated in animal models of hypertension. The rat cDNA homologue of EL was identified using reverse transcription-polymerase chain reaction. Cultured rat aortic smooth muscle cells were stimulated with angiotensin II (Ang II) and phorbol 12-myristate 13-acetate (PMA), and EL mRNA expression was analyzed by Northern blotting. EL mRNA levels in tissues from stroke-prone spontaneously hypertensive rats (SHR-SP) and Ang II-induced hypertensive rats were evaluated using RNase protection assays. Rat EL cDNA encoded a protein containing 493 amino acid residues including a signal peptide, and shares 91.9% and 80.9% sequence homology with murine and human EL, respectively. Northern blotting revealed that EL was expressed in a wide range of rat tissues. In cultured rat aortic smooth muscle cells, Ang II and PMA increased EL mRNA levels by 2.9- and 3.3-fold, respectively. In Ang II-induced hypertensive rats, EL expression was upregulated in the aorta, heart, and lung. In SHR-SP, EL expression was upregulated in the aorta and heart. EL expression is increased in rat models of hypertension. Thus, EL might have a role in the local pathophysiology of vascular diseases.