Induction of Prostacyclin by Steady Laminar Shear Stress Suppresses Tumor Necrosis Factor-α Biosynthesis via Heme Oxygenase-1 in Human Endothelial Cells

Abstract

Abstract-Cyclooxygenase (COX)-2 is among the endothelial genes upregulated by uniform laminar shear stress (LSS), characteristically associated with atherosclerotic lesion-protected areas. We have addressed whether the induction of COX-2-dependent prostanoids in endothelial cells by LSS plays a role in restraining endothelial tumor necrosis factor (TNF)-␣ generation, a proatherogenic cytokine, through the induction of heme oxygenase-1 (HO)-1, an antioxidant enzyme. In human umbilical vein endothelial cells (HUVECs) exposed to steady LSS of 10 dyn/cm 2 for 6 hours, COX-2 protein was significantly induced, whereas COX-1 and the downstream synthases were not significantly modulated. This was associated with significant (PϽ0.05) increase of 6-keto-prostaglandin (PG)F 1␣ (the hydrolysis product of prostacyclin), PGE 2 , and PGD 2 . In contrast, TNF-␣ released in the medium in 6 hours (3633Ϯ882 pg) or detected in cells lysates (1091Ϯ270 pg) was significantly (PϽ0.05) reduced versus static condition (9100Ϯ2158 and 2208Ϯ300 pg, respectively). Coincident induction of HO-1 was detected. The finding that LSS-dependent reduction of TNF-␣ generation and HO-1 induction were abrogated by the selective inhibitor of COX-2 NS-398, the nonselective COX inhibitor aspirin, or the specific prostacyclin receptor (IP) antagonist RO3244794 illuminates the central role played by LSS-induced COX-2-dependent prostacyclin in restraining endothelial inflammation. Carbacyclin, an agonist of IP, induced HO-1. Similarly to inhibition of prostacyclin biosynthesis or activity, the novel imidazole-based HO-1 inhibitor QC15 reversed TNF-␣ reduction by LSS. These findings suggest that inhibition of COX-2-dependent prostacyclin might contribute to acceleration of atherogenesis in patients taking traditional nonsteroidal antiinflammatory drugs (NSAIDs) and NSAIDs selective for COX-2 through downregulation of HO-1, which halts TNF-␣ generation in human endothelial cells. Key Words: cyclooxygenase Ⅲ endothelial cells Ⅲ prostaglandins Ⅲ prostanoids Ⅲ shear stress Ⅲ tumor necrosis factor-␣ P rostanoids, including prostaglandin (PG)E 2 , PGF 2␣ , PGD 2 , prostacyclin (PGI 2 ), and thromboxane (TX)A 2 , are lipid autacoids, immediately released outside the cell after intracellular biosynthesis, that modulate a wide variety of physiological and pathological processes. 1 They are generated by 3 sequential enzymatic steps involving: (1) phospholipase A 2 enzymes, (2) cyclooxygenase (COX) enzymes (ie, COX-1 and COX-2), 2 and (3) [3][4][5][6][7][8][9] Two isoforms of COX (COX-1 and COX-2) have been cloned and characterized. 2 COX-1 is considered a "housekeeping gene" by virtue of constitutive low levels of expression in most cell types.Differently, the gene for COX-2 is a primary response gene with many regulatory sites; thus, COX-2 expression can be rapidly induced by bacterial endotoxin (lipopolysaccharide), cytokines, such as interleukin (IL)-1␤, and tumor necrosis factor (TNF)-␣, growth factors, and tumor promoter phorbol myristate acetate. 10 However, COX-2...

Statins as Regulators of Redox State in the Vascular Endothelium: Beyond Lipid Lowering

“…It has been recently reported that vascular PGI 2 may be involved in the regulation of eNOS expression (122). Importantly, Di Francesco et al showed that cyclooxygenase-2-dependent PGI 2 induced by steady laminar shear stress upregulates heme-oxygenase 1, which halts the effects of TNFa in human ECs (31). Given that fluvastatin has been shown to increase expression of PGI 2 synthase and PGI 2 in human ECs (100), this pathway may also participate in the vasoprotective effects of statins.…”

Enlarging the NSAIDs Family: Ether, Ester and Acid Derivatives of the 1,5-Diarylpyrrole Scaffold as Novel Anti-Inflammatory and Analgesic Agents

“…The exception is performed by the fluorinated series of compounds, whose ethyl ester derivatives seem to be more selective than the bulkier analogues (i-propyl and n-butyl derivatives). Since the hydrolysis of the ester can occur at least to some extent in vivo, ester compounds 26-32 and 59-66 were evaluated in comparison with the corresponding acid (19)(20)(21)(22)(23)(24)(25) [25g]. In general the ester derivatives proved to be more effective and selective toward COX-2 with respect to acid derivatives and only in the case of 3-F phenyl substituted derivatives the acid compound 23 and the esters compounds 30, 61 and 62 displayed closer potencies.…”

“…COX-2 is among the endothelial genes upregulated by uniform laminar shear stress (LSS), characteristically associated with atherosclerotic lesion-protected areas [10c]. We have recently enlightened a central role played by LSS-induced COX-2-dependent prostacyclin in restraining endothelial inflammation [20]. Thus, inhibition of COX-2-dependent prostacyclin might contribute to acceleration of atherogenesis in patients taking tNSAIDs and NSAIDs selective for COX-2 [20].…”

Effects of AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide], a novel inhibitor of human microsomal prostaglandin E synthase-1, on prostanoid biosynthesis in human monocytes in vitro

“…Two clones were sequenced in comparison with a sequence from EMBL Databank (accession number AF027740) and a conservative mutation in [19] and for PGE 2 production by a validated radioimmunoassay [17]. Clone D8L was selected for its mPGES-1 high expression level.…”

“…This compound was dissolved in DMSO and 2 l of vehicle or stock solution was added to 1ml of cell suspension. PGE 2 , 10 TXB 2 (the stable hydrolysis product of TXA 2 ), PGF 2 and 6-keto-PGF 1 (the hydrolysis product of PGI 2 ) levels were measured in cell culture media by previously described and validated radioimmunoassay techniques [17,19,23], while COX-2, COX-1, mPGES-1, mPGES-2, cPGES, TXS and PGIS protein levels were evaluated in cell lysates by previously described Western blot techniques [17,19].…”