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Randomized controlled trials of cyclooxygenase (COX)-2 selective NSAIDs revealed that these drugs confer a cardiovascular hazard, resulting mainly from suppression of COX-2 derived prostacyclin (PGI2). Disruption of rodent COX-2-derived PGI2 in vascular cells and cardiomyocytes has recapitulated all elements of the cardiovascular hazard attributable to NSAIDs in humans. To investigate further the impact of COX-2 inhibition in the vasculature, we examined the effects of therapeutic concentrations of two selective COX-2 inhibitors, celecoxib and rofecoxib, on thrombosis and vascular injury. Furthermore, we used RNA sequencing (RNA-Seq) to assess whether celecoxib and rofecoxib might elicit distinct genomic effects in mouse aorta. Celecoxib (100 mg/Kg) and rofecoxib (50 mg/Kg) caused a selective and equipotent COX-2 inhibition as indicated by measuring thromboxane (TXB-M, an in vivo index of COX-1 activity) and prostacyclin (PGI-M, an in vivo index of COX-2 activity) urinary metabolites by mass spectrometry. Both celecoxib and rofecoxib augmented platelet deposition and prolonged platelet disaggregation after laser-induced injury in the cremaster arterioles. A similar effect was observed in mice lacking PGI2 receptor IP. There was no additional pro-thrombotic effect of the drugs on an IPKO background, consistent with suppression of COX-2 dependent PGI2 mediating the predisposition to thrombosis induced by celecoxib and rofecoxib. Celecoxib, but not rofecoxib, caused a reduction of neointimal hyperplasia and the intima/media ratio of injured femoral artery at 3 weeks after wire injury. RNA-seq analysis revealed 903 transcripts differentially expressed between celecoxib and control group and 3,127 transcripts between rofecoxib and control group (at a false discovery rate of 10%) in mouse aorta. Thus, both celecoxib and rofecoxib cause a similar predisposition to thrombosis in mice. Celecoxib, but not rofecoxib, reduces neointimal hyperplasia after vascular injury and COX-2 independent genetic actions might modulate this effect. The predisposition to thrombosis and attenuation of the response to vascular injury by celecoxib recapitulates what has been found in humans.

Cardiovascular complications from cyclooxygenase (COX)-2-selective nonsteroidal anti-inflammatory drugs (NSAIDs) prompted interest in microsomal prostaglandin synthase 1 (mPGES-1), downstream of COX-2, as an alternative drug target. Unlike COX-2, global deletion of mPGES-1 in mice suppresses prostaglandin E2 (PGE2) and augments prostacyclin PGI2 through substrate rediversion, and does not predispose to thrombogenesis and hypertension. However, after cell-specific deletions of mPGES-1, the predominant substrate rediversion product amongst the prostaglandins varies by cell type. We hypothesized that inhibition of mPGES-1 leads to changes in bioactive lipids beyond the prostaglandin pathway, and that substrate rediversion associated with a more favorable cardiovascular profile after mPGES-1 vs COX-2 inhibition can be translated from mouse to human studies. In this project, we aimed 1) to develop a mass spectrometry-based, broad-spectrum lipidomics approach to measure arachidonate-derived products additional to prostanoids; and 2) to work out an in vitro human whole blood assay (hWBA) to study substrate rediversion upon mPGES-1 vs COX-2 inhibition. We have developed a novel, reproducible analytical method to scan the impact of drugs on the bioactive lipidome. Using an in vitro hWBA and HPLC-MS/MS lipidomics analysis, we observed substrate rediversion in both COX and LOX pathways after mPGES-1 inhibition, which was distinct from inhibiting COX-1/2. We further optimized this assay to screen combinations of inhibitors from COX and lipoxygenase (LOX) cascades. Overall, we have shown that a clear signature of inhibiting diverse targets - mPGES-1, COX and LOX enzymes - can be readily detected from experiments of small samples sizes, and discovered novel aspects of drug effect due to substrate rediversion. Our findings contribute to the comparative cardiovascular safety of inhibiting mPGES-1 vs COX-2 and demonstrate the impact of combining mPGES-1 with LOX inhibitions on the hemostatic system

Aspirin prevents heart attack and stroke by irreversibly acetylating serine-529 in human platelet cyclooxygenase-1 (COX-1) and thereby inhibiting the biosynthesis of thromboxane (Tx). We hypothesized that precise quantification of COX-1 acetylation would allow the direct detection of drug-drug interactions between reversible COX inhibitors, nonsteroidal anti-inflammatory drugs (NSAIDs), and aspirin, which may abrogate its cardio-protective effect. We developed a stable isotope dilution liquid chromatography multiple reaction monitoring/mass spectrometry (LC-MRM/MS) method for measurement of platelet COX-1 acetylation. Serine-529 acetylation reflected faithfully aspirin pharmacology determined with conventional markers (serum TxB2 formation, urinary excretion of 11-dehydro-TxB2, and arachidonic acid induced platelet aggregation), but showed lower technical (CV% 3.7±2.7) and inter-individual variability (CV% 3.7). Oral administration of 325 mg of plain aspirin resulted in complete acetylation of platelet COX-1 within 2 hours and sustained maximal acetylation (71 ± 3% of total COX-1 amount) for up to two days in health volunteers (n=8). Recovery from acetylation, which reflects platelet turn-over, occurred at a rate of 7.2 ± 0.2 % per day. We compared the potential of five NSAIDs to block aspirin from acetylating platelet COX-1 in healthy volunteers (n=7 for each NSAID). Single doses of naproxen (500 mg), ibuprofen (600 mg), and diclofenac (100 mg) but not celecoxib (200 mg) and acetaminophen (1000 mg) prevented complete COX-1 acetylation by 325 mg aspirin administered 2 hours after the NSAID. The competition of NSAIDs with aspirin was conditioned by NSAID pharmacokinetics, potency, and COX isoform selectivity, suggesting variability in the likelihood of the drug-drug interaction between compounds and between patients. Monitoring platelet COX-1 acetylation may have utility in the identification of drug interactions that may limit the cardio-protective effect of low dose aspirin.

Objectives— Apoptotic monocytes release membrane microparticles which may play a major role in thrombogenicity through a P-selectin glycoprotein ligand (PGSL-1)–mediated mechanism. We have studied systematically the regulation of PSGL-1 expression and function in apoptotic monocytic cells. Methods and Results— PSGL-1 expression (flow cytometry, immunofluorescence microscopy, immunoblot) was virtually abolished in apoptotic monocytes by proteolytic shedding. This was accompanied by a complete loss of PSGL-1–mediated platelet–leukocyte (flow cytometry) and leukocyte–endothelial cell (parallel plate flow chamber) interactions. Systematic screening of protease inhibitors combined with knock-out and siRNA experiments characterized the PSGL-1-cleaving enzyme as an N-ethylmaleimide-inhibitable metalloproteinase of the ADAM family. Conclusions— Downmodulation of PGSL-1 in apoptotic monocytes may prevent ectopic cell clearance in the peripheral vasculature to reduce local inflammatory and proliferative responses. Depletion of PSGL-1 expression on apoptotic microparticles may also act as a molecular switch to modulate their thrombogenic activity.

Objective— Prostaglandin E synthases (PGESs) are being explored as antiinflammtory drug targets as alternatives to cyclooxygenase (COX)-2. Located downstream of the cyclooxygenases, PGESs catalyze PGE 2 formation, and deletion of microsomal (m)-PGES-1 abrogates inflammation. We sought to characterize the developmental expression of COX and PGES in zebrafish. Methods and Results— We cloned zebrafish cytosolic (c) and m-PGES orthologs and mapped them to syntenic regions of chromosomes 23 and 5. cPGES was widely expressed during development and was coordinately regulated with zCOX-1 in the inner ear, the pronephros, and intestine. COX-2 and mPGES-1 exhibited restricted expression, dominantly in the vasculature of the aortic arch. However, the enzymes were anatomically segregated within the vessel wall. Experiments with antisense morpholinos and with nonsteroidal antiinflammatory drugs suggest that these genes may not be critical for development. Conclusions— mPGES-1 is developmentally coregulated with COX-2 in vasculature. Given the high fecundidity and translucency of the zebrafish, this model may afford a high throughput system for characterization of novel PGES inhibitors.