Platelet-Specific Deletion of Cyclooxygenase-1 Ameliorates Dextran Sulfate Sodium-Induced Colitis in Mice.

Inflammatory bowel disease (IBD) is associated with an increased risk for thromboembolism, platelet activation, and abnormalities in platelet number and size. In colitis, platelets can extravasate into the colonic interstitium. We generated a mouse with a specific deletion of cyclooxygenase (COX)-1 in megakaryocytes/platelets [(COX-1 conditional knockout (cKO)] to clarify the role of platelet activation in the development of inflammation and fibrosis in dextran sodium sulfate (DSS)-induced colitis. The disease activity index was assessed, and colonic specimens were evaluated for histologic features of epithelial barrier damage, inflammation, and fibrosis. Cocultures of platelets and myofibroblasts were performed. We found that the specific deletion of COX-1 in platelets, which recapitulated the human pharmacodynamics of low-dose aspirin, that is, suppression of platelet thromboxane (TX)A2 production associated with substantial sparing of the systemic production of prostacyclin, resulted in milder symptoms of colitis, in the acute phase, and almost complete recovery from the disease after DSS withdrawal. Reduced colonic accumulation of macrophages and myofibroblasts and collagen deposition was found. Platelet-derived TXA2 enhanced the ability of myofibroblasts to proliferate and migrate in vitro, and these effects were prevented by platelet COX-1 inhibition or antagonism of the TXA2 receptor. Our findings allow a significant advance in the knowledge of the role of platelet-derived TXA2 in the development of colitis and fibrosis in response to intestinal damage and provide the rationale to investigate the potential efficacy of the antiplatelet agent low-dose aspirin in limiting the inflammatory response and fibrosis associated with IBD. SIGNIFICANCE STATEMENT: Inflammatory bowel disease (IBD) is characterized by the development of a chronic inflammatory response, which can lead to intestinal fibrosis for which currently there is no medical treatment. Through the generation of a mouse with specific deletion of cyclooxygenase-1 in megakaryocytes/platelets, which recapitulates the human pharmacodynamics of low-dose aspirin, we demonstrate the important role of platelet-derived thromboxane A2 in the development of experimental colitis and fibrosis, thus providing the rationale to investigate the potential efficacy of low-dose aspirin in limiting the inflammation and tissue damage associated with IBD.

NCX 4040, a nitric oxide-donating aspirin, exerts anti-inflammatory effects through inhibition of I kappa B-alpha degradation in human monocytes.

NO-donating aspirins consist of aspirin to which a NO-donating group is covalently linked via a spacer molecule. NCX 4040 and NCX 4016 are positional isomers with respect to the -CH(2)ONO(2) group (para and meta, respectively) on the benzene ring of the spacer. Because positional isomerism is critical for antitumor properties of NO-donating aspirins, we aimed to compare their anti-inflammatory effects with those of aspirin in vitro. Thus, we assessed their impacts on cyclooxygenase-2 activity (by measuring PGE(2) levels), protein expression, and cytokine generation(IL-1beta, IL-18, TNF-alpha, and IL-10) in human whole blood and isolated human monocytes stimulated with LPS. Interestingly, we found that micromolar concentrations of NCX 4040, but not NCX 4016 or aspirin, affected cyclooxygenase-2 expression and cytokine generation. We compared the effects of NCX 4040 with those of NCX 4016 or aspirin on IkappaB-alpha stabilization and proteasome activity in the LPS-stimulated human monocytic cell line THP1. Differently from aspirin and NCX 4016, NCX 4040, at a micromolar concentration range, inhibited IkappaB-alpha degradation. In fact, NCX 4040 caused concentration-dependent accumulation of IkappaB-alpha and its phosphorylated form. This effect was not reversed by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of guanylyl cyclase, thus excluding the contribution of NO-dependent cGMP generation. In contrast, IkappaB-alpha accumulation by NCX 4040 may involve an inhibitory effect on proteasome functions. Indeed, NCX 4040 inhibited 20S proteasome activity when incubated with intact cells but not in the presence of cell lysate supernatants, thus suggesting an indirect inhibitory effect. In conclusion, NCX 4040 is an inhibitor of IkappaB-alpha degradation and proteasome function, and it should be taken into consideration for the development of novel anti-inflammatory and chemopreventive agents.

Induction of prostacyclin by steady laminar shear stress suppresses tumor necrosis factor-alpha biosynthesis via heme oxygenase-1 in human endothelial cells.

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)-alpha 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 (P<0.05) increase of 6-keto-prostaglandin (PG)F(1alpha) (the hydrolysis product of prostacyclin), PGE(2), and PGD(2). In contrast, TNF-alpha released in the medium in 6 hours (3633+/-882 pg) or detected in cells lysates (1091+/-270 pg) was significantly (P<0.05) reduced versus static condition (9100+/-2158 and 2208+/-300 pg, respectively). Coincident induction of HO-1 was detected. The finding that LSS-dependent reduction of TNF-alpha 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-alpha 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-alpha generation in human endothelial cells.