Telmisartan inhibits methylglyoxal-mediated cell death in human vascular endothelium

Methylglyoxal (MGO) is a metabolite of glucose. Since serum MGO level is increased in diabetic patients, MGO is implicated in diabetic complications related to vascular injury. We have recently demonstrated that glucose metabolite is a more powerful stimulant for endothelial cells (ECs) injury rather than glucose or advanced glycation-end products. Recent clinical trials suggest that angiotensin receptor blockers are effective to prevent diabetes-associated cardiovascular disorders beyond blood pressure lowering effect. To explore the mechanisms, we examined effects of telmisartan on MGO-induced ECs injury. Treatment of human umbilical vein ECs with MGO (560 μM) induced time-dependent (0-24 h) cell death. MGO-induced cell death was apoptosis since MGO increased cleaved caspase-3 expression. Telmisartan (0.1-10 μM) inhibited MGO-induced cell death and caspase-3 activation. These results indicate that telmisartan prevents MGO-induced apoptosis by inhibiting caspase-3 activation, which might explain at least in part the beneficial effects of telimisartan against diabetes-related cardiovascular diseases.     

Glyoxal and methylglyoxal trigger distinct signals for map family kinases and caspase activation in human endothelial cells.

Carbonyl compounds with diverse carbon skeletons may be differentially related to the pathogenesis of vascular diseases. In this study, we compared intracellular signals delivered into cultured human umbilical vein endothelial cells (HUVECs) by glyoxal (GO) and methylglyoxal (MGO), which differ only by a methyl group. Depending on their concentrations, GO and MGO promoted phosphorylations of ERK1 and ERK2, which were blocked by the protein-tyrosine kinase (PTK) inhibitors herbimycin A and staurosporine, thereby being PTK-dependent. GO and MGO also induced phosphorylations of JNK, p38 MAPK, and c-Jun, either PTK-dependently (GO) or -independently (MGO). Next, we found that MGO, but not GO, induced degradation of poly(ADP-ribose) polymerase (PARP) as the intracellular substrate of caspase-3. Curcumin and SB203580, which inhibit JNK and p38 MAPK signaling pathways, but not herbimycin A/staurosporine, prevented the MGO-induced PARP degradation. We then found that MGO, but not GO, reduced the intracellular glutathione level, and that cysteine, but not cystine, inhibited the MGO-mediated activation of ERK, JNK, p38 MAPK, or c-Jun more extensively than did lysine or arginine. In addition, all the signals triggered by GO and MGO were blocked by amino guanidine (AG), which traps carbonyls. These results demonstrated that GO and MGO triggered two distinct signal cascades, one for PTK-dependent control of ERK and another for PTK-independent redox-linked activation of JNK/p38 MAPK and caspases in HUVECs, depending on the structure of the carbon skeleton of the chemicals.     

Glyoxal causes inflammatory injury in human vascular endothelial cells

To explore mechanisms of diabetes-associated vascular endothelial cells (ECs) injury, human umbilical vein ECs were treated for 24 h with high glucose (HG; 26 mM), advanced glycation end-products (AGEs; 100 μg/ml) or their intermediate, glyoxal (GO: 50-5000 μM). HG and AGEs had no effects on ECs morphology and inflammatory states as measured by vascular cell adhesion molecule (VCAM)-1 and cyclooxygenase (COX)-2 expressions. GO (500 μM, 24 h) induced cytotoxic morphological changes and protein expression of COX-2 but not VCAM-1. GO (500 μM, 24 h) activated ERK but not JNK, p38 or NF-κB. However, ERK inhibitor PD98059 was ineffective to GO-induced COX-2. While EUK134, synthetic combined superoxide dismutase/catalase mimetic, had no effect on GO-mediated inflammation, sodium nitroprusside inhibited it. The present results indicate that glyoxal, a metabolite of glucose might be a more powerful inducer for vascular ECs inflammatory injury. Nitric oxide but not anti-oxidant is preventive against GO-mediated inflammatory injury.     

Intracellular methylglyoxal induces oxidative damage to pancreatic beta cell line INS-1 cell through Ire1α-JNK and mitochondrial apoptotic pathway

An increased intracellular methylglyoxal (MGO) under hyperglycemia led to pancreatic beta cell death. However, its mechanism in which way with MGO induced beta cell death remains unknown. We investigated both high glucose and MGO treatment significantly inclined intracellular MGO concentration and inhibited cell viability in vitro. MGO treatment also triggered intracellular advanced glycation end products (AGEs) formation, declined mitochondrial membrane potential (MMP), increased oxidative stress and the expression of ER stress mediators Grp78/Bip and p-PERK; activated mitochondrial apoptotic pathway, which could mimic by Glo1 knockdown. Aminoguanidine (AG), a MGO scavenger, however, prevented AGEs formation and MGO-induced cell death by inhibiting oxidative stress and ER stress. Furthermore, both antioxidant N-acetylcysteine (NAC) and ER stress inhibitor 4-phenylbutyrate (4-PBA) could attenuate MGO-induced cell death through ameliorating ER stress. MGO treatment down-regulated Ire1α, a key ER stress mediator, increased JNK phosphorylation and activated mitochondrial apoptosis; down-regulated Bcl-2 expression which could be attenuated by the JNK inhibitor SP600125 and further inhibited cytochrome c leakage from mitochondria and blocked the conversion of pro caspase 3 into cleaved caspase 3, all these might contribute to the inhibition of INS-1 cell apoptosis. Ire1α down-regulation by Ire1α siRNAs mimicked MGO-induced cytotoxicity by activating the JNK phosphorylation and mitochondrial apoptotic pathway. In summary, we demonstrated that increased intracellular MGO induced cytotoxicity in INS-1 cells primarily by activating oxidative stress and further triggering mitochondrial apoptotic pathway, and ER stress-mediated Ire1α-JNK pathway. These findings may have implication on new mechanism of glucotoxicity-mediated pancreatic beta-cell dysfunction.     

LR-90 prevents methylglyoxal-induced oxidative stress and apoptosis in human endothelial cells

Methylglyoxal (MGO) is a highly reactive dicarbonyl compound known to induce cellular injury and cytoxicity, including apoptosis in vascular cells. Vascular endothelial cell apoptosis has been implicated in the pathophysiology and progression of atherosclerosis. We investigated whether the advanced glycation end-product inhibitor LR-90 could prevent MGO-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). HUVECs were pre-treated with LR-90 and then stimulated with MGO. Cell morphology, cytotoxicity and apoptosis were evaluated by light microscopy, MTT assay, and Annexin V-FITC and propidium iodide double staining, respectively. Levels of Bax, Bcl-2, cytochrome c, mitogen-activated protein kinases (MAPKs) and caspase activities were assessed by Western blotting. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. LR-90 dose-dependently prevented MGO-associated HUVEC cytotoxicity and apoptotic biochemical changes such as loss of MMP, increased Bax/Bcl-2 protein ratio, mitochondrial cytochrome c release and activation of caspase-3 and 9. Additionally, LR-90 blocked intracellular ROS formation and MAPK (p44/p42, p38, JNK) activation, though the latter seem to be not directly involved in MGO-induced HUVEC apoptosis. LR-90 prevents MGO-induced HUVEC apoptosis by inhibiting ROS and associated mitochondrial-dependent apoptotic signaling cascades, suggesting that LR-90 possess cytoprotective ability which could be beneficial in prevention of diabetic related-atherosclerosis.     

Methylglyoxal inhibits the binding step of collagen phagocytosis

Bacterial infection-induced fibrosis affects a wide variety of tissues, including the periodontium, but the mechanisms that dysregulate matrix turnover and mediate fibrosis are not defined. Since collagen turnover by phagocytosis is an important pathway for matrix remodeling, we studied the effect of the bacterial and eukaryotic cell metabolite, methylglyoxal (MGO), on the binding step of phagocytosis by periodontal fibroblasts. Type 1 collagen was treated with various concentrations of methylglyoxal, an important glucose metabolite that modifies Arg and Lys residues. The extent of MGO-induced modifications was authenticated by amino acid analysis, solubility, and cross-linking. Cells were incubated with fluorescent beads coated with collagen, and the percentage of phagocytic cells was estimated by flow cytometry. MGO inhibited collagen binding (20% of control for 10 mm MGO) in a time- and concentration-dependent manner. MGO-induced inhibition of binding was prevented by aminoguanidine, which blocks the formation of collagen cross-links. MGO reduced collagen binding strength and blocked intracellular calcium signaling. MGO modified the Arg residue in the critical alpha2beta1 integrin-binding recognition sequence of triple helical collagen peptides, whereas MGO-induced cross-linking of Lys residues played only a small role in binding inhibition. Thus, MGO modifications of Arg residues in collagen could be a key factor in the impaired degradation of collagen that promotes fibrosis in chronic infections, such as periodontitis.     

Effects of sphondin,isolated from Heracleum laciniatum,on IL-1beta-induced cyclooxygenase-2 expression in human pulmonary epithelial cells

Recently, under large-scale screening experiments, we found that sphondin, a furanocoumarin derivative isolated from Heracleum laciniatum, possessed an inhibitory effect on IL-1beta-induced increase in the level of COX-2 protein and PGE(2) release in A549 cells. Accordingly, we examined in the present study the action mechanism of sphondin on the inhibition of IL-1beta-induced COX-2 protein expression and PGE(2) release in a human pulmonary epithelial cell line (A549). Pretreatment of cells with sphondin (10-50 microM) concentration-dependently attenuated IL-1beta-induced COX-2 protein expression and PGE(2) release. The IL-1beta-induced increase in COX-2 mRNA expression was also attenuated by sphondin (50 microM). The selective COX-2 inhibitor, NS-398 (0.01-1 microM), inhibited the activity of the COX-2 enzyme in a concentration-dependent manner, while sphondin (10-50 microM) had no effect. Sphondin (50 microM) did not affect the IL-1beta-induced activations of p44/42 MAPK, p38 MAPK, and JNK. Treatment of cells with sphondin (50 microM) or the NF-kappaB inhibitor, PDTC (50 microM) partially inhibited IL-1beta-induced degradation of IkappaB-alpha in the cytosol and translocation of p65 NF-kappaB from the cytosol to the nucleus. Furthermore, IL-1beta-induced NF-kappaB-specific DNA-protein complex formation in the nucleus was partially inhibited by sphondin (50 microM) or PDTC (50 microM). Taken together, we demonstrate that sphondin inhibits IL-1beta-induced PGE(2) release in A549 cells; this inhibition is mediated by suppressing of COX-2 expression, rather than by inhibiting COX-2 enzyme activity. The inhibitory mechanism of sphondin on IL-1beta-induced COX-2 expression may be, at least in part, through suppression of NF-kappaB activity. We conclude that sphondin may have the therapeutic potential as an anti-inflammatory drug on airway inflammation.     

Streptococcus pneumoniae induced p38 MAPK- and NF-kappaB-dependent COX-2 expression in human lung epithelium

Streptococcus pneumoniae is a major cause of community-acquired pneumonia and death from infectious diseases in industrialized countries. Lung airway and alveolar epithelial cells comprise an important barrier against airborne pathogens. Cyclooxygenase (COX)-derived prostaglandins, such as PGE(2), are considered to be important regulators of lung function. Herein, we tested the hypothesis that pneumococci induced COX-2-dependent PGE(2) production in pulmonary epithelial cells. Pneumococci-infected human pulmonary epithelial BEAS-2B cells released PGE(2). Expression of COX-2 but not COX-1 was dose and time dependently increased in S. pneumoniae-infected BEAS-2B cells as well as in lungs of mice with pneumococcal pneumonia. S. pneumoniae induced degradation of IkappaBalpha and DNA binding of NF-kappaB. A specific peptide inhibitor of the IkappaBalpha kinase complex blocked pneumococci-induced PGE(2) release and COX-2 expression. In addition, we noted activation of p38 MAPK and JNK in pneumococci-infected BEAS-2B cells. PGE(2) release and COX-2 expression were reduced by p38 MAPK inhibitor SB-202190 but not by JNK inhibitor SP-600125. We analyzed interaction of kinase pathways and NF-kappaB activation: dominant-negative mutants of p38 MAPK isoforms alpha, beta(2), gamma, and delta blocked S. pneumoniae-induced NF-kappaB activation. In addition, recruitment of NF-kappaB subunit p65/RelA and RNA polymerase II to the cox2 promoter depended on p38 MAPK but not on JNK activity. In summary, p38 MAPK- and NF-kappaB-controlled COX-2 expression and subsequent PGE(2) release by lung epithelial cells may contribute significantly to the host response in pneumococcal pneumonia.     

p38 MAPK and NF-kappaB mediate COX-2 expression in human airway myocytes

We have previously demonstrated that p38 and extracellular signal-regulated protein kinase (ERK) mitogen-activated protein kinases (MAPK) are components of proinflammatory induced cytokine expression in human airway myocytes. The experiments described here further these studies by examining p38 MAPK and NF-kappaB regulation of cyclooxygenase-2 (COX-2) expression in response to a complex inflammatory stimulus consisting of 10 ng/ml interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF-alpha), and interferon (IFN)-gamma. COX-2 expression was induced with this stimulus in a time-dependent manner, with maximal expression seen 12-20 h after treatment. Semiquantitative RT-PCR and immunoblotting experiments demonstrate decreased COX-2 expression following treatment with the p38 MAPK inhibitor SB-203580 (25 microM) or the proteosome inhibitor MG-132 (1 microM). SB-203580 did not affect cytokine-stimulated IkappaBalpha degradation, NF-kappaB nuclear binding activity, or NF-kappaB-dependent signaling from the COX-2 promoter, indicating that p38 MAPK and NF-kappaB may affect COX-2 expression via separate signaling pathways. SB-203580, but not MG-132, also increased the initial rate of COX-2 mRNA decay, indicating p38 MAPK, but not NF-kappaB, participates in the regulation of COX-2 mRNA stability. These findings suggest that although p38 MAPK and NF-kappaB signaling regulate steady-state levels of COX-2 expression, p38 MAPK additionally affects stability of COX-2 mRNA in cytokine-stimulated human airway myocytes.     

Vascular endothelial growth factor receptor-2 couples cyclo-oxygenase-2 with pro-angiogenic actions of leptin on human endothelial cells

Background

The adipocyte-derived hormone leptin influences the behaviour of a wide range of cell types and is now recognised as a pro-angiogenic and pro-inflammatory factor. In the vasculature, these effects are mediated in part through its direct leptin receptor (ObRb)-driven actions on endothelial cells (ECs) but the mechanisms responsible for these activities have not been established. In this study we sought to more fully define the molecular links between inflammatory and angiogenic responses of leptin-stimulated human ECs.

Methodology/Principal Findings

Immunoblotting studies showed that leptin increased cyclo-oxygenase-2 (COX-2) expression (but not COX-1) in cultured human umbilical vein ECs (HUVEC) through pathways that depend upon activation of both p38 mitogen-activated protein kinase (p38^MAPK) and Akt, and stimulated rapid phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) on Tyr¹¹⁷⁵. Phosphorylation of VEGFR2, p38^MAPK and Akt, and COX-2 induction in cells challenged with leptin were blocked by a specific leptin peptide receptor antagonist. Pharmacological inhibitors of COX-2, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and p38^MAPK abrogated leptin-induced EC proliferation (assessed by quantifying 5-bromo-2′-deoxyuridine incorporation, calcein fluorescence and propidium iodide staining), slowed the increased migration rate of leptin-stimulated cells (in vitro wound healing assay) and inhibited leptin-induced capillary-like tube formation by HUVEC on Matrigel. Inhibition of VEGFR2 tyrosine kinase activity reduced leptin-stimulated p38^MAPK and Akt activation, COX-2 induction, and pro-angiogenic EC responses, and blockade of VEGFR2 or COX-2 activities abolished leptin-driven neo-angiogenesis in a chick chorioallantoic membrane vascularisation assay in vivo.

Conclusions/Significance

We conclude that a functional endothelial p38^MAPK/Akt/COX-2 signalling axis is required for leptin''s pro-angiogenic actions and that this is regulated upstream by ObRb-dependent activation of VEGFR2. These studies identify a new function for VEGFR2 as a mediator of leptin-stimulated COX-2 expression and angiogenesis and have implications for understanding leptin''s regulation of the vasculature in both non-obese and obese individuals.     

Novel role of p66Shc in ROS-dependent VEGF signaling and angiogenesis in endothelial cells

p66Shc, a longevity adaptor protein, is demonstrated as a key regulator of reactive oxygen species (ROS) metabolism involved in aging and cardiovascular diseases. Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration and proliferation primarily through the VEGF receptor-2 (VEGFR2). We have shown that ROS derived from Rac1-dependent NADPH oxidase are involved in VEGFR2 autophosphorylation and angiogenic-related responses in ECs. However, a role of p66Shc in VEGF signaling and physiological responses in ECs is unknown. Here we show that VEGF promotes p66Shc phosphorylation at Ser36 through the JNK/ERK or PKC pathway as well as Rac1 binding to a nonphosphorylated form of p66Shc in ECs. Depletion of endogenous p66Shc with short interfering RNA inhibits VEGF-induced Rac1 activity and ROS production. Fractionation of caveolin-enriched lipid raft demonstrates that p66Shc plays a critical role in VEGFR2 phosphorylation in caveolae/lipid rafts as well as downstream p38MAP kinase activation. This in turn stimulates VEGF-induced EC migration, proliferation, and capillary-like tube formation. These studies uncover a novel role of p66Shc as a positive regulator for ROS-dependent VEGFR2 signaling linked to angiogenesis in ECs and suggest p66Shc as a potential therapeutic target for various angiogenesis-dependent diseases.     

Pigment epithelium-derived factor and its phosphomimetic mutant induce JNK-dependent apoptosis and p38-mediated migration arrest

Pigment epithelium-derived factor (PEDF) is a potent endogenous inhibitor of angiogenesis and a promising anticancer agent. We have previously shown that PEDF can be phosphorylated and that distinct phosphorylations differentially regulate its physiological functions. We also demonstrated that triple phosphomimetic mutant (EEE-PEDF), has significantly increased antiangiogenic activity and is much more efficient than WT-PEDF in inhibiting neovascularization and tumor growth. The enhanced antiangiogenic effect was associated with a direct ability to facilitate apoptosis of tumor-residing endothelial cells (ECs), and subsequently, disruption of intratumoral vascularization. In the present report, we elucidated the molecular mechanism by which EEE-PEDF exerts more profound effects at the cellular level. We found that EEE-PEDF suppresses EC proliferation due to caspase-3-dependent apoptosis and also inhibits migration of the EC much better than WT-PEDF. Although WT-PEDF and EEE-PEDF did not affect proliferation and did not induce apoptosis of cancer cells, these agents efficiently inhibited cancer cell motility, with EEE-PEDF showing a stronger effect. The stronger activity of EEE-PEDF was correlated with a better binding to laminin receptors. Furthermore, the proapoptotic and antimigratory activities of WT-PEDF and EEE-PEDF were found regulated by differential activation of two distinct MAPK pathways, namely JNK and p38, respectively. We show that JNK and p38 phosphorylation is much higher in cells treated with EEE-PEDF. JNK leads to apoptosis of ECs, whereas p38 leads to anti-migratory effect in both EC and cancer cells. These results reveal the molecular signaling mechanism by which the phosphorylated PEDF exerts its stronger antiangiogenic, antitumor activities.     

VEGF and thrombin induce MKP-1 through distinct signaling pathways: role for MKP-1 in endothelial cell migration

We have previously reported that MAPK phosphatase-1 (MKP-1/CL100) is a thrombin-responsive gene in endothelial cells (ECs). We now show that VEGF is another efficacious activator of MKP-1 expression in human umbilical vein ECs. VEGF-A and VEGF-E maximally induced MKP-1 expression in ECs; however, the other VEGF subtypes had no effect. Using specific neutralizing antibodies, we determined that VEGF induced MKP-1 specifically through VEGF receptor 2 (VEGFR-2), leading to the downstream activation of JNK. The VEGF-A(165) isoform stimulated MKP-1 expression, whereas the VEGF-A(162) isoform induced the gene to a lesser extent, and the VEGF-A(121) isoform had no effect. Furthermore, specific blocking antibodies against neuropilins, VEGFR-2 coreceptors, blocked MKP-1 induction. A Src kinase inhibitor (PP1) completely blocked both VEGF- and thrombin-induced MKP-1 expression. A dominant negative approach revealed that Src kinase was required for VEGF-induced MKP-1 expression, whereas Fyn kinase was critical for thrombin-induced MKP-1 expression. Moreover, VEGF-induced MKP-1 expression required JNK, whereas ERK was critical for thrombin-induced MKP-1 expression. In ECs treated with short interfering (si)RNA targeting MKP-1, JNK, ERK, and p38 phosphorylation were prolonged following VEGF stimulation. An ex vivo aortic angiogenesis assay revealed a reduction in VEGF- and thrombin-induced sprout outgrowth in segments from MKP-1-null mice versus wild-type controls. MKP-1 siRNA also significantly reduced VEGF-induced EC migration using a transwell assay system. Overall, these results demonstrate distinct MAPK signaling pathways for thrombin versus VEGF induction of MKP-1 in ECs and point to the importance of MKP-1 induction in VEGF-stimulated EC migration.     

Role of MAPK in the regulation of double-stranded RNA- and encephalomyocarditis virus-induced cyclooxygenase-2 expression by macrophages

In response to virus infection or treatment with dsRNA, macrophages express the inducible form of cyclooxygenase-2 (COX-2) and produce proinflammatory prostaglandins. Recently, we have shown that NF-kappaB is required for encephalomyocarditis virus (EMCV)- and dsRNA-stimulated COX-2 expression in mouse macrophages. The dsRNA-dependent protein kinase R is not required for EMCV-stimulated COX-2 expression, suggesting the presence of protein kinase R-independent pathways in the regulation of this antiviral gene. In this study, the role of MAPK in the regulation of macrophage expression of cyclooxygenase-2 (COX)-2 in response to EMCV infection was examined. Treatment of mouse macrophages or RAW-264.7 cells with dsRNA or infection with EMCV stimulates the rapid activation of the MAPKs p38, JNK, and ERK. Inhibition of p38 and JNK activity results in attenuation while ERK inhibition does not modulate dsRNA- and EMCV-induced COX-2 expression and PGE2 production by macrophages. JNK and p38 appear to selectively regulate COX-2 expression, as inhibition of either kinase fails to prevent dsRNA- or EMCV-stimulated inducible NO synthase expression by macrophages. Using macrophages isolated from TLR3-deficient mice, we show that p38 and JNK activation and COX-2 expression in response to EMCV or poly(IC) does not require the presence the dsRNA receptor TLR3. These findings support a role for p38 and JNK in the selective regulation of COX-2 expression by macrophages in response to virus infection.     

Regulation of the membrane-localized prostaglandin E synthases mPGES-1 and mPGES-2 in cardiac myocytes and fibroblasts

The proinflammatory mediator cyclooxygenase (COX)-2 and its product PGE(2) are induced in the ischemic heart, contributing to inflammatory cell infiltration, fibroblast proliferation, and cardiac hypertrophy. PGE(2) synthesis coupled to COX-2 involves two membrane-localized PGE synthases, mPGES-1 and mPGES-2; however, it is not clear how these synthases are regulated in cardiac myocytes and fibroblasts. To study this, we used primary cultures of neonatal ventricular myocytes (VM) and fibroblasts (VF) treated with IL-1beta for 24 h. To test for involvement of MAPKs in IL-1beta regulation of mPGES-1 and-2, cells were pretreated with the pharmacological inhibitors of p42/44 MAPK, p38 MAPK, and c-Jun kinase (JNK). mRNA was analyzed by RT-PCR. Protein was analyzed by densitometry of Western blots. mPGES-1 was undetectable in untreated VF but induced by IL-1beta; inhibition of either p42/44 MAPK or JNK, but not p38 MAPK, was almost completely inhibitory. In VM, inhibition of the three MAPKs reduced IL-1beta-stimulated mPGES-1 protein by 70-90%. mPGES-2 was constitutively synthesized in both VM and VF and was not regulated by IL-1beta or MAPKs. Confocal microscopy revealed colocalization of both mPGES-1 and mPGES-2 with COX-2 in the perinuclear area of both VF and VM. Finally, PGE(2) production was higher in VM than VF. Our data show that 1) mPGES-1 is induced in both VF and VM, 2) regulation of mPGES-1 by MAPK family members is different in the two cell types, 3) mPGES-2 is constitutively synthesized in both VM and VF and is not regulated, and 4) mPGES-1 and mPGES-2 are colocalized with COX-2 in both cells. Thus differences in activity of mPGES-1 and COX-2 or coupling of COX-2 with mPGES-1 may contribute to differences in PGE(2) production by myocytes and fibroblasts.