This Article Abstract Full Text (PDF) All Versions of this Article: 98/5/642    most recent 01.RES.0000207394.39249.fcv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Li, R. Articles by Berliner, J. A. Search for Related Content PubMed PubMed Citation Articles by Li, R. Articles by Berliner, J. A. Related Collections Cell signalling/signal transduction Gene expression Mechanism of atherosclerosis/growth factors Related Article

(Circulation Research. 2006;98:642.)
© 2006 American Heart Association, Inc.

Molecular Medicine

Identification of Prostaglandin E2 Receptor Subtype 2 As a Receptor Activated by OxPAPC

Rongsong Li, Kevin P. Mouillesseaux, Dennis Montoya, Daniel Cruz, Navid Gharavi, Martin Dun, Lukasz Koroniak, Judith A. Berliner

From the Departments of Pathology (R.L., N.G., M.D., J.A.B.), Medicine (R.L., K.P.M., D.M., D.C., J.A.B.), Chemistry and Biochemistry (L.K.), University of California, Los Angeles.

Correspondence to Judith A Berliner, PhD, UCLA-Department of Medicine, 10833 Le Conte Ave, CHS 13-229, Los Angeles, CA 90095. E-mail jberliner{at}mednet.ucla.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), which has been shown to accumulate in atherosclerotic lesions and other sites of chronic inflammation, activates endothelial cells (EC) to bind monocytes by activation of endothelial ß1 integrin and subsequent deposition of fibronectin on the apical surface. Our previous studies suggest this function of OxPAPC is mediated via a Gs protein–coupled receptor (GPCR). PEIPC (1-palmitoyl-2-epoxyisoprostane E2-sn-glycero-3-phosphorylcholine) is the most active lipid in OxPAPC that activates this pathway. We screened a number of candidate GPCRs for their interaction with OxPAPC and PEIPC, using a reporter gene assay; we identified prostaglandin E2 receptor EP2 and prostaglandin D2 receptor DP as responsive to OxPAPC. We focused on EP2, which is expressed in ECs, monocytes, and macrophages. OxPAPC component PEIPC, but not POVPC, activated EP2 with an EC₅₀ of 108.6 nmol/L. OxPAPC and PEIPC were also able to compete with PGE2 for binding to EP2 in a ligand-binding assay. The EP2 specific agonist butaprost was shown to mimic the effect of OxPAPC on the activation of ß1 integrin and the stimulation of monocyte binding to endothelial cells. Butaprost also mimicked the effect of OxPAPC on the regulation of tumor necrosis factor- and interleukin-10 in monocyte-derived cells. EP2 antagonist AH6809 blocked the activation of EP2 by OxPAPC in HEK293 cells and blocked the interleukin-10 response to PEIPC in monocytic THP-1 cells. These results suggest that EP2 functions as a receptor for OxPAPC and PEIPC, either as the phospholipid ester or the released fatty acid, in both endothelial cells and macrophages.

Key Words: OxPAPC • GPCR • EP2 • monocyte binding • receptor • atherosclerosis

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Important early events in atherogenesis involve the activation of endothelial cells to bind monocytes and the entry of these monocytes into the vessel wall where they take up lipid, forming foam cells. There is evidence that deposition and oxidative modification of low-density lipoprotein (LDL) in the artery wall plays an important role in atherogenesis.^1,2 Oxidized phospholipids have been demonstrated to accumulate in atherosclerotic lesions and other sites of chronic inflammation.^1,2 OxPAPC and its component phospholipids 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC) and 1-palmitoyl-2-epoxyisoprostane E2-sn-glycero-3-phosphorylcholine (PEIPC), like mildly modified LDL (MM-LDL), activate human aortic endothelial cells (HAEC) to bind monocytes.^3,4 This increase in binding induced by MM-LDL and OxPAPC was shown to be caused by a change in configuration of the 5ß1 integrin on the apical surface of the endothelial cells, resulting in the binding of fibronectin. This fibronectin contains connecting segment-1 (CS-1), which binds to 4ß1 integrin on the surface of monocytes and enables firm adhesion of monocyte to endothelial cells.⁵ Interestingly, the increase in monocyte binding was shown to be independent of vascular cell adhesion molecule (VCAM)-1, which was not increased in response to OxPAPC. We demonstrated that the alteration of 5ß1 integrin was mediated by a cAMP-dependent R-Ras/phosphatidylinositol 3-kinase pathway.³ Upregulation of CS-1 expression on the large vessel endothelium was demonstrated in vivo in inflammatory areas of human atherosclerotic lesions.⁵ Because of the potential importance of the cAMP/CS-1 pathway in human atherosclerosis, we have performed studies to identify the receptor for OxPAPC, which mediates this process.

Several lines of evidence support a role of a Gs protein–coupled receptor (GPCR) in OxPAPC and MM-LDL–stimulated monocyte binding. Treatment of endothelial cells with OxPAPC rapidly increases intracellular level of cAMP.^3,4,6,7 Dibutyryl cAMP, which can mimic the function of cAMP, also stimulated monocyte binding by increasing CS-1 on the apical surface of endothelial cells. RpcAMP (a competitive antagonist of cAMP) and H89 (an inhibitor of PKA) inhibit OxPAPC and MM-LDL–stimulated monocyte binding.^3,6 In the current studies, a number of GPCRs of the rhodopsin family, including known lipid receptors or orphans, were screened as candidate receptors with a reporter assay. Using this screening procedure, 2 members of the prostaglandin receptor family were identified as Gs-coupled OxPAPC–responsive receptors: EP2 and DP. Both receptors are known to couple to Gs and increase synthesis of cAMP. We present evidence here suggesting EP2 functions as a receptor for OxPAPC in endothelial cells and monocytes/macrophages.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials and Reagents
Cell culture media and reagents were obtained from Irvine Scientific Inc and Invitrogen Inc. FBS was obtained from Hyclone Inc. PAPC and POVPC were purchased from Avanti Polar Lipids Inc. OxPAPC and PEIPC were prepared and analyzed in our laboratory as previously described.⁸ GPCR constructs were either bought from NMR cDNA Resource Center (www.cdna.org) or cloned by ourselves. Six GPCR CHO stable cell lines were provided by Dr Kevin Lynch (University of Virginia). Prostaglandins, butaprost, AH6809, and anti-EP2 antibody were purchased from Cayman Chemical Inc. ³H-PGE2 was from PerkinElmer Inc. Antibody against active Integrin ß1 was obtained from Pharmingen/BD Biosciences. HRP-conjugated secondary antibodies were obtained from Cell Signaling Inc.

Cell Culture
HAECs were cultured as previously described.³ HEK293 cells were obtained from the American Type Culture Collection and cultured in DMEM (Irvine Scientific) supplemented with 10% FBS. HEK293-EBNA-EP2 stable cells were generously provided by Dr John W. Regan (University of Arizona) and cultured with DMEM/10% FBS supplemented with G418 and Hygromycin B. THP-1 cells were obtained from American Type Culture Collection and cultured in RPMI-1640 (Invitrogen) supplemented with 10% FBS and 50 µmol/L ß-mercaptoethanol. Human monocytes were isolated and differentiated into macrophage in vitro as described previously.⁹

Reporter Gene Assay
HEK293 cells were plated in 48 wells and grown to more than 90% confluence the next day. The Pathdetect-CREB Trans-Reporter System (Stratagene) was used according to the protocol of the manufacturer. Plasmids pFR-Luc (250 ng), pFA2-CREB (25 ng), and test plasmid with GPCR coding sequence (125 ng) were cotransfected into each well of HEK293 cells. Transfection was performed with Lipofectamine 2000 (Invitrogen) following the instructions of the manufacturer. Twenty-four hours after transfection, cells were treated with or without OxPAPC or other reagents in DMEM/1% FBS. DBcAMP was used as positive control. Six hours after treatment, media were removed and the cells were lysed in 150 µL of Reporter Lysis Buffer (Promega). Luciferase activity was measured with Bright Glow substrate (Promega) with Sirius Luminometer.

RT-PCR and Quantitative Real-Time PCR
RNA was isolated with RNeasy mini kit from Qiagen following the instructions of the manufacturer. Potential genomic DNA contamination was removed with on-column DNase I digestion. Total RNA (0.5 to 1 µg) was reverse transcribed with the iScript cDNA synthesis kit (Bio-Rad). Primers were designed with PrimerQuest software (Integrated DNA Technologies, www.idtdna.com) and were blasted against GenBank sequences to ensure specificity. The following forward (F) and reverse (R) primers were used: EP2 (F, 5'-ATGGGCAATGCCTCCAATGACTCCC-3'; R, 5'-ACACCAGCTCGGTCACCAGCACGT-3'); tumor necrosis factor (TNF)- (F, 5'-ATGAGCACTGAAAGCATGATCCGGGA-3'; R, 5'-CGGGGTTCGAGAAGATGATCTGACTGC-3'); interleukin (IL)-10 (F, 5'-GTTTTACCTGGAGGAGGTGATGCCCCA-3'; R, 5'-GGCCTTGCTCTTGTTTTCACAGGGAAG-3'). End-point RT-PCR was performed with GeneAmp 2400 (PerkinElmer). Quantitative real time PCR was done using iCylcer and iQ SYBR green supermix (Bio-Rad). The expression of target genes was normalized to GAPDH.

Western Blot
Cells were lysed in RIPA buffer (50 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail) at 4°C for 30 minutes. Protein concentration was determined with Bio-Rad DC protein assay kit. Equal protein amount of lysates were run in 4% to 12% gradient SDS-PAGE gel. The proteins were then transferred to polyvinylidene fluoride membrane and blotted with indicated primary and secondary antibody. GAPDH was used as loading control.

Monocyte Binding Assay and Cell Surface ELISA
Monocyte binding assay was performed with HAEC and human monocytes as described previously.³ Cell surface ELISA of active form ß1 integrin was done essentially as previously described,⁵ except that PBS/5% nonfat dry milk was used as blocking solution instead of PBS/3% BSA.

EP2 Ligand Binding Assay
Membrane Preparation
HEK293-EBNA-EP2 cells were grown to confluence in 100-mm dishes. Cells were collected and resuspended in 1 mmol/L NaHCO3/1 mmol/L CaCl2 (pH 8.0) in the presence of protease inhibitors. Cells were frozen at –80°C and then completely thawed in ice for approximately 5 minutes. Cells were further disrupted with a narrow bore needle (27 gauge) by aspirating the cell lysate 12 times. The cell lysate was centrifuged at 2000g to remove debris. The supernatant was collected and centrifuged at 100 000g for 1 hour. The pellet was re-suspend in binding assay buffer (10 mmol/L MES/KOH, pH 6.2 containing 1 mmol/L EDTA, 10 mmol/L MgCl₂) and sonicated for 10x2 seconds at power setting 3 (Sonicator Model XL2020 from Misonix Inc, Farmingdale, NY). The resulting suspension was quantitated with Bio-Rad Dc Protein Assay kit, aliquoted, stored at –80°C, and used as membrane preparation for binding assay.

Binding Assay
³H-PGE₂ binding assay was essentially done as described by Abramovitz et al¹⁰ using 25 µg of membrane preparation and 1.0 nmol/L ³H-PGE₂. After 2 hours of incubation at 30°C, reaction was terminated by rapid filtration onto GF/F membrane with a 96-well vacuum manifold. After washing, the membrane was dried at 37°C for 1.5 hour and the radioactivity bound to the membrane was determined by scintillation counting. Nonspecific binding was determined in the presence of 10 µmol/L unlabeled PGE2. Specific binding was calculated by subtracting nonspecific binding from total binding. Specific binding in the absence of competitor was used as 100% binding control.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
OxPAPC Activates Prostaglandin E2 Receptor EP2
Three main strategies were used to screen these candidate receptors. First, the expression of candidate GPCR in HAEC was determined by RT-PCR with 35 amplification cycles. Those not expressed in HAEC were eliminated from further screening. The remaining candidates with known ligand were then analyzed to see whether the ligand itself increased monocyte binding by a VCAM-1 independent pathway or whether a receptor antagonist blocked the effect of OxPAPC on monocyte binding. The remaining receptors were screened for activation by OxPAPC using the Pathdetect CREB reporter gene assay system. For these studies, stable transfectants of orphan receptors in CHO-K1 cells or transient expression of ligand-known receptors in HEK293 cells were used. Cells expressing candidate receptors with reporter system were treated with OxPAPC, and luciferase activity was measured. Increased luciferase activity reflects the activation of Gs coupled GPCR by OxPAPC. The overall result of candidate screening is summarized in the Table.

View this table: [in this window] [in a new window]   Table 1. Results of Candidate Screening

With the reporter gene assay, we identified EP2 as activated by OxPAPC. Luciferase activity was significantly increased in HEK293 cells transfected with EP2 in response to OxPAPC treatment (Figure 1A). The extent of activation of EP2 by OxPAPC is comparable to the activation by PGE2, the native ligand of EP2 (Figure 1A). The closest homolog of EP2 is DP, the receptor for prostaglandin D2 (PGD2). DP is 41% identical to EP2. OxPAPC also activates DP at a comparable level to PGD2 (Figure 1B). Prostacyclin receptor IP has the next closest homology to EP2 (35% identical, 48% similar to EP2). However, IP does not respond to OxPAPC stimulation (Figure 1C). Thromboxane A2 receptor TP was not activated by OxPAPC (Figure 1D). Other PGE2 receptors (EP1, EP3, and EP4) do not significantly respond to OxPAPC stimulation (Figure 1E), although these receptors responded well to their native ligand (data not shown). These results indicate that OxPAPC activates EP2 and DP specifically and initiates downstream signal transduction that leads to CREB activation. The remaining studies focused on EP2 because of its expression in endothelial cells, monocytes, and macrophages.

View larger version (18K): [in this window] [in a new window]   Figure 1. Activation of prostaglandin receptors in response to OxPAPC. Prostaglandin receptors were transfected into HEK293 cells together with reporter plasmids and treated with indicated reagents. Activation of prostaglandin receptors was measured by luciferase activity. A, HEK293 cells were transfected with prostaglandin E2 (PGE2) receptor EP2 and then treated with or without PGE2 (1 µmol/L) or OxPAPC (10 µg/mL). B, HEK293 cells were transfected with prostaglandin D2 (PGD2) receptor DP and then treated with or without PGD2 (1 µmol/L) or OxPAPC (10 µg/mL). C, HEK293 cells were transfected with prostacyclin receptor IP and then treated with or without prostacyclin (I2, 1 µmol/L) or OxPAPC (10 µg/mL). D, HEK293 cells were transfected with thromboxane A2 (TxA2) receptor TP and then treated with or without TxA2 (1 µmol/L) or OxPAPC (10 µg/mL). E, HEK293 cells were transfected with empty plasmid (C) or prostaglandin E2 receptors EP1, EP2, EP3, and EP4. Cells were than treated with OxPAPC at 50 µg/mL. Native ligand was also tested (data not shown).

PEIPC, but Not POVPC, Activates EP2
PEIPC and POVPC are the 2 major components in OxPAPC that activate monocyte binding to endothelial cells via a cAMP dependent pathway. To see whether PEIPC and POVPC both activate EP2, HEK293 cells were transfected with EP2 and then treated with PEIPC or POVPC. PEIPC at 100 ng/mL significantly activated EP2 and increased reporter gene expression, whereas POVPC at 10 µg/mL had no effect (Figure 2A). PEIPC has previously been reported to be the most active phospholipid to induce monocyte binding. In the reporter gene assay, PEIPC even at a concentration of 10 ng/mL activated EP2. The EC₅₀ of OxPAPC and PEIPC in activating EP2 with the reporter assay were determined to be 1.8 µg/mL (Figure 2B, the molar concentration of 1.8 µg/mL OxPAPC is &2.15 µmol/L) and 108.6 nmol/L (Figure 2C), respectively. The EC₅₀ of PGE2 to activate EP2 was reported to be 31 nmol/L.¹¹

View larger version (12K): [in this window] [in a new window]   Figure 2. Effect of OxPAPC components on the activation of EP2. A, HEK293 cells were transfected with EP2 and reporter plasmids. Transfected cells then were treated with or without OxPAPC (10 µg/mL), PEIPC (0.1 µg/mL), and POVPC (10 µg/mL). Activation of EP2 was measured by luciferase activity. B and C, Dose response of OxPAPC and PEIPC on EP2 activation. Same procedure as A using different concentration of OxPAPC and PEIPC.

OxPAPC and PEIPC Inhibits the Binding of PGE2 to EP2
To gain insight into whether there is direct interaction of OxPAPC and its component PEIPC with EP2, we examined the ability of OxPAPC and PEIPC to compete for binding of PGE2 to EP2. For these studies, the binding of ³H-PGE2 to membrane prepared from HEK293-EBNA-EP2 stable cells was examined. OxPAPC at 0.1, 1, 10, and 50 µg/mL (which corresponds to molar concentration of 0.127, 1.27, 12.7, and 63.5 µmol/L) inhibited specific PGE2 binding to EP2 by 10%, 17%, 46%, and 92%, respectively (Figure 3C), whereas PEIPC at 80 nmol/L and 800 nmol/L inhibited PGE2 binding to EP2 by 22% and 57% respectively(Figure 3B). In comparison, unlabeled PGE2 at 10 nmol/L, 100 nmol/L, and 1 µmol/L inhibited ³H-PGE2 binding by 53%, 86%, and 95%, respectively (Figure 3A). Thus, the binding affinity of PEIPC to EP2 is approximately 80-fold less than PGE2. These results demonstrate that OxPAPC and PEIPC are able to specifically compete with PGE2 for binding to EP2.

View larger version (13K): [in this window] [in a new window]   Figure 3. Inhibition of binding of PGE2 to EP2 by OxPAPC. Binding of 3H-PGE2 was examined with EP2 membrane prepared from HEK293-EBNA-EP2 stable cells. A, Specific 3H-PGE2 binding to EP2 in the presence of different concentrations of unlabeled PGE2. B, Specific 3H-PGE2 binding to EP2 in the presence of different concentrations of PEIPC. C, Specific 3H-PGE2 binding to EP2 in the presence of different concentrations of OxPAPC.

EP2 mRNA and Protein Are Expressed in Endothelial Cells and Other Cells Involved in Atherogenesis
Endothelial cells, monocytes, macrophages, and smooth muscle cells all express detectable amounts of EP2 mRNA by end-point RT-PCR (Figure 4A). Real-time RT-PCR indicates macrophage (PM, differentiated from peripheral human monocytes in vitro) express highest level of EP2 mRNA (Figure 4B). Monocytes (THP-1) and aortic smooth muscle cells (ASMC) express median level of EP2 mRNA. Endothelial cells HAEC and HMEC express low level of EP2 mRNA. EP2 protein was reported to be a 53-kDa molecule. Antibody against EP2 (Cayman Chemical Inc) detected a protein at the appropriate size in HAEC, HMEC, PM, and THP-1 cells (Figure 4C). Although the mRNA level of EP2 varies significantly among these cells, there are similar levels of EP2 protein expression.

View larger version (34K): [in this window] [in a new window]   Figure 4. Expression of EP2 in different cell types. A, RNA was isolated from endothelial cells (HAEC, HMEC), macrophage (PM), monocyte (THP-1), and aortic smooth muscle cell (ASMC). EP2 mRNA expression measured by end-point RT-PCR. B, Comparison of EP2 mRNA expression in different cells by quantitative RT-PCR using the same RNA from A. EP2 cloned into pcDNA3.1 was used as standard to measure the copy number of EP2. C, Protein expression of EP2. Cell lysates from different cells with same amount of protein were loaded on to 4% to 12% gradient SDS-PAGE. EP2 expression was measured by Western blot with rabbit anti-EP2 polyclonal antibody. GAPDH was used as an indicator of loading. PM indicates peripheral monocyte differentiated into macrophage in vitro.

Activation of EP2 Results in the Activation of ß1 Integrin and the Stimulation of Monocyte Binding to HAEC
Butaprost, a specific agonist of EP2, like OxPAPC and PEIPC, increases the active form of ß1 integrin expression on endothelial cell surface as measured by cell surface ELISA (Figure 5A) and stimulates monocyte binding to HAEC (Figure 5B). The effect of OxPAPC and butaprost is not additive. These results suggest a role for EP2 in OxPAPC induced monocyte binding.

View larger version (19K): [in this window] [in a new window]   Figure 5. Activation of integrin ß1 and stimulation of monocyte binding by OxPAPC and butaprost. A, HAEC cells plated in 96-well plate were treated with vehicle control or MnCl2 (10 mmol/L, positive control), OxPAPC (50 µg/mL), butaprost (BTP) (1 µmol/L and 10 µmol/L), or DBcAMP (1 mmol/L) in M-199/1% FBS for 30 minutes. The level of activated ß1 integrin was measured by cell surface ELISA (*P<0.01). B, HAEC plated in 48-well plate were treated with vehicle control or OxPAPC (50 µg/mL), butaprost (1 µmol/L and 10 µmol/L), or butaprost (10 µmol/L) plus OxPAPC (50 µg/mL) in M-199/1% FBS for 4 hours. Monocytes binding to HAEC were measured. *P<0.05, **P<0.01.

Activation of EP2 by Butaprost and OxPAPC Modifies the Expression of TNF- and IL-10 in Monocytes and Macrophages
PGE2 has been reported to downregulate the expression of TNF- via EP2 and EP4.^12,13 We demonstrated that THP-1 cells expressed significantly lower level of TNF- when treated with OxPAPC. Butaprost, like OxPAPC, also downregulated TNF- expression (Figure 6A). POVPC, which does not activate EP2, had no effect on TNF- expression at 10 µg/mL (data not shown). OxPAPC, PEIPC, and butaprost also reduced TNF- expression in macrophages differentiated from human peripheral monocytes (Figure 6B). OxPAPC also inhibited TNF- expression in undifferentiated human peripheral monocytes (data not shown).

View larger version (14K): [in this window] [in a new window]   Figure 6. Regulation of TNF- and IL-10 expression by OxPAPC and butaprost. Total RNA was isolated from indicated cells and the expression of TNF- or IL-10 mRNA was measured by quantitative RT-PCR. A, THP-1 cells were treated with or without OxPAPC (50 µg/mL), butaprost (1 µmol/L), or DBcAMP (1 mmol/L) in RPMI medium 1640/1% FBS for 4 hours. The expression of TNF- was measured. B, Human peripheral monocytes were differentiated in vitro into macrophage and treated with or without OxPAPC (50 µg/mL), PEIPC (PEI, 0.1 µg/mL and 0.5 µg/mL), butaprost (BTP, 1 µmol/L), or DBcAMP (DB, 1 mmol/L) in RPMI medium 1640/1% FBS for 4 hours. The expression of TNF- was measured. C, THP-1 cells were treated with or without OxPAPC (25 µg/mL), PEIPC (0.1 µg/mL), butaprost (1 µmol/L), or DBcAMP (1 mmol/L) in RPMI medium 1640/1% FBS for 4 hours. The expression of IL-10 was measured. *P<0.05).

IL-10 is another cytokine regulated by PGE2 via EP2 and EP4. Unlike TNF-, IL-10 is upregulated by PGE2 in some cell types.^12,14 OxPAPC, PEIPC, and butaprost all stimulate IL-10 expression in THP-1 cells (Figure 6C).

EP2 Antagonist AH6809 Blocked the Activation of EP2 and Reduced IL-10 Stimulation by OxPAPC/PEIPC
To further confirm that OxPAPC functions via EP2, we tested the effect of EP/DP antagonist AH6809. AH6809 is a weak EP2 antagonist that inhibits the effects of low concentration of EP2 agonist.¹⁵ In the CREB reporter gene assay, 10 µmol/L AH6809 inhibited 71% of EP2 activation by 1 µg/mL (1.27 µmol/L) OxPAPC (Figure 7) and 51% by 10 µg/mL (12.7 µmol/L) OxPAPC. Furthermore, in THP-1 cells, PEIPC at 0.5 µg/mL stimulated IL-10 mRNA production by 6.1±0.7-fold. AH6809 at 10 µmol/L reduced the stimulation by 50% to 3.1±0.3-fold.

View larger version (8K): [in this window] [in a new window]   Figure 7. EP2 antagonist AH6809 inhibits activation of EP2 by OxPAPC. HEK293 cells were transfected with EP2 and CREB reporter plasmids. Transfected cells then were untreated (C) (control) or treated with AH6809 (10 µmol/L), OxPAPC (1 µg/mL), or OxPAPC plus AH6809. Activation of EP2 was measured by luciferase activity.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previous studies demonstrated that OxPAPC stimulated monocyte binding involves a GPCR. In this study, we identified EP2 and DP as Gs coupled receptors for OxPAPC and also demonstrated that the component phospholipid PEIPC, but not POVPC, activated EP2. This study also eliminated a number of candidate GPCRs as the endothelial receptors mediating the effect of OxPAPC on monocyte binding.

We present evidence that both OxPAPC and its component PEIPC activate DP and EP2. PEIPC was approximately 20-fold more active than OxPAPC, suggesting that it is the OxPAPC component responsible for activation of these receptors. The ability of PEIPC to activate these receptors relates to their known ligands. PEIPC used for these studies contains a mixture of E and D epoxyisoprostane (EI) isomers.¹⁶ The epoxyisoprostane structure is presumably the core moiety in PEIPC that activates EP2 and DP. The most active isomer (in inducing endothelial cells to bind monocyte binding) contains an E epoxyisoprostane, which would likely bind to EP2 receptor. The lack of a DP receptor on endothelial cells would explain why the D-epoxyisoprostanes were inactive in inducing monocyte binding in our previous study.¹⁶

This and our previous studies provide some insight into the activation of the EP2 receptor by OxPAPC components. We conducted experiments to determine whether OxPAPC and PEIPC could directly activate PGE2 or whether they caused the synthesis of PGE2 by treated cells, which then bound to the receptor. Our data showing that OxPAPC and PEIPC compete for binding of PGE2 to the EP2 receptor strongly support a direct interaction of OxPAPC and PEIPC with the receptor. Because OxPAPC only activates EP2 and not other PGE2 receptors, it is unlikely that it acts by increasing PGE2 synthesis. Furthermore, we previously published that cyclooxygenase inhibitor indomethacin had no effect on monocyte binding induced by MM-LDL (of which OxPAPC is the active component).¹⁷ At this time, we cannot determine how much of the activation of EP2 is contributed by esterified PEIPC and how much by EI. Previous studies have demonstrated that esterified butaprost interacted less effectively with EP2.¹¹ Our previous studies demonstrated that PLA2 treatment strongly decreased the activity of PEIPC on monocyte binding.¹⁸ However, those studies (though demonstrating 90% hydrolysis of PEIPC) were not able to determine levels of recoverable EI because of a lack of suitable standard. Thus, it is still possible that cell surface PLA2 can hydrolyze PEIPC resulting in a more active, receptor-accessible EI.

The failure of PEIPC to activate other prostaglandin receptors reveals important differences in specificity of these receptors. The prostacyclin receptor IP is the next closest homolog to EP2 and DP. It has been reported a single amino acid mutation on EP2 confers EP2 responsiveness to prostacyclin.¹⁹ However, IP does not respond to OxPAPC. Interestingly, EP4 has the same native ligand as EP2 and both EP2 and EP4 mediate some of the functions of PGE2 via cAMP.^20,21 Yet, OxPAPC does not significantly activate EP4 to increase cAMP, suggesting that the particular E prostaglandin structure is highly selective in receptor binding. Previous studies have demonstrated a difference in the binding pocket of the EP receptors.^10,11,22 The current studies demonstrate that this difference is recognized by PEIPC.

There are both similarities and differences in the action of isoprostane E and OxPAPC/PEIPC. Tintut et al has shown that Isoprostane E and OxPAPC, like PGE2, enhance osteoclast differentiation. EP2/DP receptors and cAMP were shown to be involved in isoprostane E–stimulated osteoclast differentiation.²³ Clarke et al demonstrated that isoprostane E regulates colony-stimulating factor in human airway smooth muscle cells by activating EP2.²⁴ Like PEIPC, isoprostane E also stimulates human umbilical vein endothelial cells to bind monocytes via increasing cAMP. However, this effect is mediated by TP.²⁵ The abovementioned studies were performed using receptor antagonists, which may show different specificities in different cell types. Our studies with PEIPC directly tested the activation of receptors by PEIPC using reporter constructs. Taken together, these studies suggest that the receptors mediating the action of isoprostane E may be cell-type specific and could also depend on the structure of the E prostaglandin.

Our studies present evidence that EP2 is involved in mediating the effects of OxPAPC on endothelial cells and macrophages. Both cell types express high levels of EP2 protein (Figure 4C). Butaprost, a known specific EP2 activator, activates similar pathways to those seen with OxPAPC, including activation of endothelial cell binding of monocytes by a VCAM-1–independent mechanism (Figure 5) and downregulation of TNF- transcription and upregulation of IL-10 transcription in monocytes/macrophages (Figure 6). Furthermore, the effects of OxPAPC and butaprost were not additive, indicating similar pathways were involved in the activation. Finally, the EP2 inhibitor AH6809 blocked the activation of EP2 (Figure 7) and the effect of PEIPC on IL-10 synthesis in THP-1 cells. In addition, previous studies demonstrate that cAMP-elevating agents and PGE2 activate the pathways regulated by OxPAPC. Our group previously demonstrated that monocyte binding mediated by OxPAPC required an increase in cAMP.^3,6 cAMP and cAMP-elevating agents have been shown to inhibit the expression of TNF-^12,13,26 and increase the expression of IL-10 at the transcriptional level.^26,27 The effects of PGE2 on TNF- and IL-10 have been reported to be mediated by EP2 and EP4.^{12–14,28,29} Taken together, this is strong evidence that EP2 is a mediator of OxPAPC action.

The in vitro effects of OxPAPC mediated by the EP2 receptor, presented in this article, could play an important role in the early stages of atherosclerosis by stimulating monocyte entry into vessel wall and regulating the phenotype of lesion macrophages. Stimulation of CS-1 expression on endothelial cells has been demonstrated to contribute to monocyte entry into the vessel wall.³⁰ Based on previous studies, the effect of EP2 activation on macrophages would increase foam cell formation. IL-10 has been shown to enhance Ox-LDL–induced foam cell formation by inhibiting apoptosis of the foam cells both in vivo and in vitro.^31,32 Decreased levels of TNF- would also be expected to result in lower foam cell apoptosis. Macrophages overexpressing IL-10 also show a decreased ability to present antigens and mediate T-cell activation.³³ Supporting this decreased activation, butaprost, like PGE2, inhibited CD1a expression in dendritic cells differentiated from monocytes (D.C. and D.M., unpublished data, 2005). Thus, OxPAPC activation of EP2 can play an important role in monocyte entry and foam cell formation.

In summary, our results demonstrate that EP2 and DP receptors are activated in response to OxPAPC and specifically PEIPC-inducing CREB activation. The EP2 receptor is expressed in endothelial cells, smooth muscle cells, and macrophages. EP2-specific agonist butaprost mimics the effects of OxPAPC on several inflammatory functions in endothelial cells and macrophages. Although EP2 is likely not the only receptor responding to OxPAPC, these studies suggest that EP2 has an important role in OxPAPC action and may be an important mediator of monocyte entry into the vessel wall and foam cell formation.

	Acknowledgments

 
This project was funded by United States Public Health Service grants HL30568 and HL 064731.

	Footnotes

 
Original received July 8, 2005; resubmission received December 5, 2005; revised resubmission received January 6, 2006; accepted January 20, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Berliner JA, Subbanagounder G, Leitinger N, Watson AD, Vora D. Evidence for a role of phospholipid oxidation products in atherogenesis. Trends Cardiovasc Med. 2001; 11: 142–147.[CrossRef][Medline] [Order article via Infotrieve]

2. Leitinger N. Oxidized phospholipids as modulators of inflammation in atherosclerosis. Curr Opin Lipidol. 2003; 14: 421–430.[CrossRef][Medline] [Order article via Infotrieve]

3. Cole AL, Subbanagounder G, Mukhopadhyay S, Berliner JA, Vora DK. Oxidized phospholipid-induced endothelial cell/monocyte interaction is mediated by a cAMP-dependent R-Ras/PI3-kinase pathway. Arterioscler Thromb Vasc Biol. 2003; 23: 1384–1390.[Abstract/Free Full Text]

4. Leitinger N, Tyner TR, Oslund L, Rizza C, Subbanagounder G, Lee H, Shih PT, Mackman N, Tigyi G, Territo MC, Berliner JA, Vora DK. Structurally similar oxidized phospholipids differentially regulate endothelial binding of monocytes and neutrophils. Proc Natl Acad Sci U S A. 1999; 96: 12010–12015.[Abstract/Free Full Text]

5. Shih PT, Elices MJ, Fang ZT, Ugarova TP, Strahl D, Territo MC, Frank JS, Kovach NL, Cabanas C, Berliner JA, Vora DK. Minimally modified low-density lipoprotein induces monocyte adhesion to endothelial connecting segment-1 by activating beta1 integrin. J Clin Invest. 1999; 103: 613–625.[Medline] [Order article via Infotrieve]

6. Parhami F, Fang ZT, Fogelman AM, Andalibi A, Territo MC, Berliner JA. Minimally modified low density lipoprotein-induced inflammatory responses in endothelial cells are mediated by cyclic adenosine monophosphate. J Clin Invest. 1993; 92: 471–478.[Medline] [Order article via Infotrieve]

7. Parhami F, Fang ZT, Yang B, Fogelman AM, Berliner JA. Stimulation of Gs and inhibition of Gi protein functions by minimally oxidized LDL. Arterioscler Thromb Vasc Biol. 1995; 15: 2019–2024.[Abstract/Free Full Text]

8. Watson AD, Subbanagounder G, Welsbie DS, Faull KF, Navab M, Jung ME, Fogelman AM, Berliner JA. Structural identification of a novel pro-inflammatory epoxyisoprostane phospholipid in mildly oxidized low density lipoprotein. J Biol Chem. 1999; 274: 24787–24798.[Abstract/Free Full Text]

9. Seager Danciger J, Lutz M, Hama S, Cruz D, Castrillo A, Lazaro J, Phillips R, Premack B, Berliner J. Method for large scale isolation, culture and cryopreservation of human monocytes suitable for chemotaxis, cellular adhesion assays, macrophage and dendritic cell differentiation. J Immunol Methods. 2004; 288: 123–134.[CrossRef][Medline] [Order article via Infotrieve]

10. Abramovitz M, Adam M, Boie Y, Carriere M, Denis D, Godbout C, Lamontagne S, Rochette C, Sawyer N, Tremblay NM, Belley M, Gallant M, Dufresne C, Gareau Y, Ruel R, Juteau H, Labelle M, Ouimet N, Metters KM. The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs. Biochim Biophys Acta. 2000; 1483: 285–293.[Medline] [Order article via Infotrieve]

11. Wilson RJ, Rhodes SA, Wood RL, Shield VJ, Noel LS, Gray DW, Giles H. Functional pharmacology of human prostanoid EP2 and EP4 receptors. Eur J Pharmacol. 2004; 501: 49–58.[Medline] [Order article via Infotrieve]

12. Treffkorn L, Scheibe R, Maruyama T, Dieter P. PGE2 exerts its effect on the LPS-induced release of TNF-alpha, ET-1, IL-1alpha, IL-6 and IL-10 via the EP2 and EP4 receptor in rat liver macrophages. Prostaglandins Other Lipid Mediat. 2004; 74: 113–123.[CrossRef][Medline] [Order article via Infotrieve]

13. Vassiliou E, Jing H, Ganea D. Prostaglandin E2 inhibits TNF production in murine bone marrow-derived dendritic cells. Cell Immunol. 2003; 223: 120–132.[CrossRef][Medline] [Order article via Infotrieve]

14. Shinomiya S, Naraba H, Ueno A, Utsunomiya I, Maruyama T, Ohuchida S, Ushikubi F, Yuki K, Narumiya S, Sugimoto Y, Ichikawa A, Oh-ishi S. Regulation of TNFalpha and interleukin-10 production by prostaglandins I(2) and E(2): studies with prostaglandin receptor-deficient mice and prostaglandin E-receptor subtype-selective synthetic agonists. Biochem Pharmacol. 2001; 61: 1153–1160.[CrossRef][Medline] [Order article via Infotrieve]

15. Woodward DF, Pepperl DJ, Burkey TH, Regan JW. 6-Isopropoxy-9-oxoxanthene-2-carboxylic acid (AH 6809), a human EP2 receptor antagonist. Biochem Pharmacol. 1995; 50: 1731–1733.[CrossRef][Medline] [Order article via Infotrieve]

16. Subbanagounder G, Wong JW, Lee H, Faull KF, Miller E, Witztum JL, Berliner JA. Epoxyisoprostane and epoxycyclopentenone phospholipids regulate monocyte chemotactic protein-1 and interleukin-8 synthesis. Formation of these oxidized phospholipids in response to interleukin-1beta. J Biol Chem. 2002; 277: 7271–7281.[Abstract/Free Full Text]

17. Honda HM, Leitinger N, Frankel M, Goldhaber JI, Natarajan R, Nadler JL, Weiss JN, Berliner JA. Induction of monocyte binding to endothelial cells by MM-LDL: role of lipoxygenase metabolites. Arterioscler Thromb Vasc Biol. 1999; 19: 680–686.[Abstract/Free Full Text]

18. Subbanagounder G, Leitinger N, Schwenke DC, Wong JW, Lee H, Rizza C, Watson AD, Faull KF, Fogelman AM, Berliner JA. Determinants of bioactivity of oxidized phospholipids. Specific oxidized fatty acyl groups at the sn-2 position. Arterioscler Thromb Vasc Biol. 2000; 20: 2248–2254.[Abstract/Free Full Text]

19. Kedzie KM, Donello JE, Krauss HA, Regan JW, Gil DW. A single amino-acid substitution in the EP2 prostaglandin receptor confers responsiveness to prostacyclin analogs. Mol Pharmacol. 1998; 54: 584–590.[Abstract/Free Full Text]

20. Fennekohl A, Sugimoto Y, Segi E, Maruyama T, Ichikawa A, Puschel GP. Contribution of the two Gs-coupled PGE2-receptors EP2-receptor and EP4-receptor to the inhibition by PGE2 of the LPS-induced TNFalpha-formation in Kupffer cells from EP2-or EP4-receptor-deficient mice. Pivotal role for the EP4-receptor in wild type Kupffer cells. J Hepatol. 2002; 36: 328–334.[CrossRef][Medline] [Order article via Infotrieve]

21. Harizi H, Grosset C, Gualde N. Prostaglandin E2 modulates dendritic cell function via EP2 and EP4 receptor subtypes. J Leukoc Biol. 2003; 73: 756–763.[Abstract/Free Full Text]

22. Stillman BA, Breyer MD, Breyer RM. Importance of the extracellular domain for prostaglandin EP(2) receptor function. Mol Pharmacol. 1999; 56: 545–551.[Abstract/Free Full Text]

23. Tintut Y, Parhami F, Tsingotjidou A, Tetradis S, Territo M, Demer LL. 8-Isoprostaglandin E2 enhances receptor-activated NFkappa B ligand (RANKL)-dependent osteoclastic potential of marrow hematopoietic precursors via the cAMP pathway. J Biol Chem. 2002; 277: 14221–14226.[Abstract/Free Full Text]

24. Clarke DL, Belvisi MG, Hardaker E, Newton R, Giembycz MA. E-Ring 8-isoprostanes are agonists at EP2- and EP4-prostanoid receptors on human airway smooth muscle cells and regulate the release of colony-stimulating factors by activating cAMP-dependent protein kinase. Mol Pharmacol. 2005; 67: 383–393.[Abstract/Free Full Text]

25. Huber J, Bochkov VN, Binder BR, Leitinger N. The isoprostane 8-iso-PGE2 stimulates endothelial cells to bind monocytes via cyclic AMP- and p38 MAP kinase-dependent signaling pathways. Antioxid Redox Signal. 2003; 5: 163–169.[CrossRef][Medline] [Order article via Infotrieve]

26. Foey AD, Field S, Ahmed S, Jain A, Feldmann M, Brennan FM, Williams R. Impact of VIP and cAMP on the regulation of TNF-alpha and IL-10 production: implications for rheumatoid arthritis. Arthritis Res Ther. 2003; 5: R317–328.[CrossRef][Medline] [Order article via Infotrieve]

27. Platzer C, Meisel C, Vogt K, Platzer M, Volk HD. Up-regulation of monocytic IL-10 by tumor necrosis factor-alpha and cAMP elevating drugs. Int Immunol. 1995; 7: 517–523.[Abstract/Free Full Text]

28. Noguchi K, Iwasaki K, Shitashige M, Ishikawa I. Prostaglandin E2 receptors of the EP2 and EP4 subtypes downregulate tumor necrosis factor alpha-induced intercellular adhesion molecule-1 expression in human gingival fibroblasts. J Periodontal Res. 1999; 34: 478–485.[Medline] [Order article via Infotrieve]

29. Yamane H, Sugimoto Y, Tanaka S, Ichikawa A. Prostaglandin E(2) receptors, EP2 and EP4, differentially modulate TNF-alpha and IL-6 production induced by lipopolysaccharide in mouse peritoneal neutrophils. Biochem Biophys Res Commun. 2000; 278: 224–228.[CrossRef][Medline] [Order article via Infotrieve]

30. Huo Y, Hafezi-Moghadam A, Ley K. Role of vascular cell adhesion molecule-1 and fibronectin connecting segment-1 in monocyte rolling and adhesion on early atherosclerotic lesions. Circ Res. 2000; 87: 153–159.[Abstract/Free Full Text]

31. Halvorsen B, Waehre T, Scholz H, Clausen OP, von der Thusen JH, Muller F, Heimli H, Tonstad S, Hall C, Froland SS, Biessen EA, Damas JK, Aukrust P. Interleukin-10 enhances the oxidized LDL-induced foam cell formation of macrophages by antiapoptotic mechanisms. J Lipid Res. 2005; 46: 211–219.[Abstract/Free Full Text]

32. Pinderski LJ, Fischbein MP, Subbanagounder G, Fishbein MC, Kubo N, Cheroutre H, Curtiss LK, Berliner JA, Boisvert WA. Overexpression of interleukin-10 by activated T lymphocytes inhibits atherosclerosis in LDL receptor-deficient Mice by altering lymphocyte and macrophage phenotypes. Circ Res. 2002; 90: 1064–1071.[Abstract/Free Full Text]

33. Lang R, Rutschman RL, Greaves DR, Murray PJ. Autocrine deactivation of macrophages in transgenic mice constitutively overexpressing IL-10 under control of the human CD68 promoter. J Immunol. 2002; 168: 3402–3411.[Abstract/Free Full Text]

Related Article:

A Rancid Culprit in Vascular Inflammation Acts on the Prostaglandin Receptor EP2

Norbert Leitinger
Circ. Res. 2006 98: 587-589. [Extract] [Full Text] [PDF]

This article has been cited by other articles:

				R. Chen, X. Chen, R. G. Salomon, and T. M. McIntyre Platelet Activation by Low Concentrations of Intact Oxidized LDL Particles Involves the PAF Receptor Arterioscler Thromb Vasc Biol, March 1, 2009; 29(3): 363 - 371. [Abstract] [Full Text] [PDF]

				S. Bluml, B. Rosc, A. Lorincz, M. Seyerl, S. Kirchberger, O. Oskolkova, V. N. Bochkov, O. Majdic, E. Ligeti, and J. Stockl The Oxidation State of Phospholipids Controls the Oxidative Burst in Neutrophil Granulocytes J. Immunol., September 15, 2008; 181(6): 4347 - 4353. [Abstract] [Full Text] [PDF]

				O. V. Oskolkova, T. Afonyushkin, A. Leitner, E. von Schlieffen, P. S. Gargalovic, A. J. Lusis, B. R. Binder, and V. N. Bochkov ATF4-dependent transcription is a key mechanism in VEGF up-regulation by oxidized phospholipids: critical role of oxidized sn-2 residues in activation of unfolded protein response Blood, July 15, 2008; 112(2): 330 - 339. [Abstract] [Full Text] [PDF]

				H.-K. Jyrkkanen, E. Kansanen, M. Inkala, A. M. Kivela, H. Hurttila, S. E. Heinonen, G. Goldsteins, S. Jauhiainen, S. Tiainen, H. Makkonen, et al. Nrf2 Regulates Antioxidant Gene Expression Evoked by Oxidized Phospholipids in Endothelial Cells and Murine Arteries In Vivo Circ. Res., July 3, 2008; 103(1): e1 - e9. [Abstract] [Full Text] [PDF]

				B. G. Gugiu, K. Mouillesseaux, V. Duong, T. Herzog, A. Hekimian, L. Koroniak, T. M. Vondriska, and A. D. Watson Protein targets of oxidized phospholipids in endothelial cells J. Lipid Res., March 1, 2008; 49(3): 510 - 520. [Abstract] [Full Text] [PDF]

				F. Gruber, O. Oskolkova, A. Leitner, M. Mildner, V. Mlitz, B. Lengauer, A. Kadl, P. Mrass, G. Kronke, B. R. Binder, et al. Photooxidation Generates Biologically Active Phospholipids That Induce Heme Oxygenase-1 in Skin Cells J. Biol. Chem., June 8, 2007; 282(23): 16934 - 16941. [Abstract] [Full Text] [PDF]

				A. A. Birukova, P. Fu, S. Chatchavalvanich, D. Burdette, O. Oskolkova, V. N. Bochkov, and K. G. Birukov Polar head groups are important for barrier-protective effects of oxidized phospholipids on pulmonary endothelium Am J Physiol Lung Cell Mol Physiol, April 1, 2007; 292(4): L924 - L935. [Abstract] [Full Text] [PDF]

				R. Li, W. Chen, R. Yanes, S. Lee, and J. A. Berliner OKL38 is an oxidative stress response gene stimulated by oxidized phospholipids J. Lipid Res., March 1, 2007; 48(3): 709 - 715. [Abstract] [Full Text] [PDF]

				S. Gross, P. Tilly, D. Hentsch, J.-L. Vonesch, and J.-E. Fabre Vascular wall-produced prostaglandin E2 exacerbates arterial thrombosis and atherothrombosis through platelet EP3 receptors J. Exp. Med., February 19, 2007; 204(2): 311 - 320. [Abstract] [Full Text] [PDF]

				A. Zimman, K. P. Mouillesseaux, T. Le, N. M. Gharavi, A. Ryvkin, T. G. Graeber, T. T. Chen, A. D. Watson, and J. A. Berliner Vascular Endothelial Growth Factor Receptor 2 Plays a Role in the Activation of Aortic Endothelial Cells by Oxidized Phospholipids Arterioscler Thromb Vasc Biol, February 1, 2007; 27(2): 332 - 338. [Abstract] [Full Text] [PDF]

				N. Clavreul, M. M. Bachschmid, X. Hou, C. Shi, A. Idrizovic, Y. Ido, D. Pimentel, and R. A. Cohen S-Glutathiolation of p21ras by Peroxynitrite Mediates Endothelial Insulin Resistance Caused by Oxidized Low-Density Lipoprotein Arterioscler Thromb Vasc Biol, November 1, 2006; 26(11): 2454 - 2461. [Abstract] [Full Text] [PDF]

				P. S. Gargalovic, N. M. Gharavi, M. J. Clark, J. Pagnon, W.-P. Yang, A. He, A. Truong, T. Baruch-Oren, J. A. Berliner, T. G. Kirchgessner, et al. The Unfolded Protein Response Is an Important Regulator of Inflammatory Genes in Endothelial Cells Arterioscler Thromb Vasc Biol, November 1, 2006; 26(11): 2490 - 2496. [Abstract] [Full Text] [PDF]

				N. Leitinger A Rancid Culprit in Vascular Inflammation Acts on the Prostaglandin Receptor EP2 Circ. Res., March 17, 2006; 98(5): 587 - 589. [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 98/5/642    most recent 01.RES.0000207394.39249.fcv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Li, R. Articles by Berliner, J. A. Search for Related Content PubMed PubMed Citation Articles by Li, R. Articles by Berliner, J. A. Related Collections Cell signalling/signal transduction Gene expression Mechanism of atherosclerosis/growth factors Related Article