Cyclooxygenases, microsomal prostaglandin E synthase-1, and cardiovascular function

Abstract

We investigated the mechanisms by which inhibitors of prostaglandin G/H synthase-2 (PGHS-2; known colloquially as COX-2) increase the incidence of myocardial infarction and stroke. These inhibitors are believed to exert both their beneficial and their adverse effects by suppression of PGHS-2-derived prostacyclin (PGI(2)) and PGE(2). Therefore, the challenge remains to identify a mechanism whereby PGI(2) and PGE(2) expression can be suppressed while avoiding adverse cardiovascular events. Here, selective inhibition, knockout, or mutation of PGHS-2, or deletion of the receptor for PGHS-2-derived PGI(2), was shown to accelerate thrombogenesis and elevate blood pressure in mice. These responses were attenuated by COX-1 knock down, which mimics the beneficial effects of low-dose aspirin. PGE(2) biosynthesis is catalyzed by the coordinate actions of COX enzymes and microsomal PGE synthase-1 (mPGES-1). We show that deletion of mPGES-1 depressed PGE(2) expression, augmented PGI(2) expression, and had no effect on thromboxane biosynthesis in vivo. Most importantly, mPGES-1 deletion affected neither thrombogenesis nor blood pressure. These results suggest that inhibitors of mPGES-1 may retain their antiinflammatory efficacy by depressing PGE(2), while avoiding the adverse cardiovascular consequences associated with PGHS-2-mediated PGI(2) suppression.

Figure 1. PGHS enzymes and eicosanoid biosynthesis.

(A) Urinary excretion of TXM was decreased significantly (n = 6; *P < 0.001) from values in WT mice by PGHS-1 deletion (KO) or knock down (KD), but not in PGHS-2 KO or PGHS-2^Y385F mice or those treated with 100 mg/kg/d of the PGHS-2 inhibitor celecoxib for 30 days on a mixed C57BL/6 × 129/Sv genetic background. (B) Urinary PGIM was depressed significantly in PGHS-2 KO and PGHS-2^Y385F mice and by celecoxib, but not in PGHS-1 KO or KD mice.

Figure 2. Suppressive effects of IP deletion on vascular reactivity, platelet aggregation, and thrombogenesis are gene/dose dependent.

(A) The maximum decline (P < 0.0001) and duration (P < 0.005) of hypotension evoked in mean arterial pressure (MAP) by 1 μg/kg of IP agonist, cicaprost (Cica), was greater in WT than in IP^+/– or IP^–/– mice on a C57BL/6 background. (B) Platelet aggregation was initiated with 2 μg/ml collagen with (+) or without (–) pretreatment with 10 nM Cica. Inhibition of aggregation was not evident in IP^–/– mice and averaged 86% of WT in IP^+/– mice (P < 0.0001). (C) Carotid artery blood flow after photochemical injury. The time to complete occlusion after rose bengal dye injection fell from 66.3 ± 5.1 minutes in WT to 44.4 ± 7 minutes in IP^+/– and to 29.7 ± 7.6 minutes in IP^–/– mice (P < 0.006). The mean impact of the COX-2 inhibitor, DFU (10 mg/kg for 3 days), on time to occlusion (56.2% of WT value) was intermediate between that of IP^+/– (68.1%) and IP^–/– (45.5%) mice.

Figure 3. PGHS-2 disruption or inhibition promotes thrombogenesis and hypertension and modulation effect of PGHS-1 KD.

(A) Circulating platelets before and 2 minutes after injection of collagen (12.5 and 25 μg/kg) plus epinephrine (15 μg/ml, 100 μl) into WT, PGHS-2^Y385F, and PGHS-2 KO mice on a mixed C57BL/6 × 129/Sv background. Thrombocytopenia was more pronounced (**P < 0.01) in PGHS-2–disrupted mice. Sudden death was induced more commonly (**P < 0.01) within 15 minutes of U46619 injection in PHGS-2–deleted or –mutated mice. (B) The time to thrombotic carotid artery occlusion after photochemical injury was delayed in PGHS-1 KD mice (**P < 0.01) but accelerated by DFU treatment (^#P < 0.05). The time to occlusion in DFU-treated animals was delayed in the PGHS-1 KD group compared with WT controls (**P < 0.01), while the time to occlusion in DFU-treated PGHS-1 KD mice did not differ significantly from that in vehicle-treated WT controls on a mixed C57BL/6 × 129/Sv background. (C) Systolic blood pressure, as measured by the tail cuff method, was elevated significantly in 3-month-old PGHS-2 KO, PGHS^Y385F, and celecoxib-treated (100 mg/kg/d for 30 days) mice as compared with WT mice on a mixed C57BL/6 × 129/Sv background (*P < 0.05; **P < 0.01). The hypertensive effect of celecoxib was attenuated in PGHS-1 KD mice compared with that in WT (^##P < 0.01) mice.

Figure 4. The major urinary metabolite of PGE₂ and the suppressive effect of PGHS-1 disruption or KD and PGHS-2 disruption or mutation on PGE₂ biosynthesis.

(A) A selected ion-monitoring trace of the methoxime derivative of endogenous PGEM (9,15-dioxo-11α-hydroxy-2,3,4,5-tetranor-prostane-1,20-dioic-17,17,18,18,19,19-d₆ acid) (bottom panel) and its hexadeuterated internal standard (top panel). (B) Urinary PGEM decreased significantly in both male and female PGHS-2 KO or PGHS-2^Y385F mice compared with WT controls on a mixed C57BL/6 × 129/Sv genetic background (n = 5–6; *P < 0.05; **P < 0.001). PGEM was also significantly lower in PGHS-1 KD and PGHS-1 KO groups compared with WT mice of mixed C57BL/6 × 129/Sv genetic background (n = 5–6; *P < 0.05; **P < 0.001). PGEM was significantly higher in PGHS-2^Y385F mice compared with PGHS-2 KO mice (^#P < 0.05) on the same genetic background.

Figure 5. Impact of mPGES-1 disruption on thrombogenesis, blood pressure regulation, and eicosanoid biosynthesis.

(A) Deletion of mPGES-1 failed to alter the time to thrombotic carotid artery occlusion after photochemical injury, while it was accelerated by the PGHS-2 inhibitor DFU in mice on a DBA/11acJ background (*P < 0.05). (B) MAP exhibited diurnal variation in mPGES-1 KO and WT littermates on a mixed DBA/11acJ × C57BL/6 background. MAP was averaged over 4 days for 12 hours dark (active phase) and light (resting phase) periods. MAP was higher during active phase, and a high-salt diet elevated pressure similarly, a mean 6% in both groups (**P < 0.01). Oral DFU administration (10 mg/kg/d) for 21 days increased MAP in both the active (^##P < 0.01) and resting (^#P < 0.05) phases compared with vehicle-treated animals. (C) Urinary PGEM was lower (**P < 0.01) and PGIM was higher (*P < 0.05), while TXM was unaltered in male mPGES-1 KO mice compared with gender-matched WT littermates on a DBA/11acJ background.