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. 2021 Mar 19;7(12):eabf6054.
doi: 10.1126/sciadv.abf6054. Print 2021 Mar.

Endothelial cyclooxygenase-1 paradoxically drives local vasoconstriction and atherogenesis despite underpinning prostacyclin generation

Jane A Mitchell  1 , Fisnik Shala  1 , Maria Elisa Lopes Pires  1 , Rachel Y Loy  1 , Andrew Ravendren  1 , Joshua Benson  1 , Paula Urquhart  2 , Anna Nicolaou  2 , Harvey R Herschman  3 , Nicholas S Kirkby  4
Affiliations

Endothelial cyclooxygenase-1 paradoxically drives local vasoconstriction and atherogenesis despite underpinning prostacyclin generation

Jane A Mitchell et al. Sci Adv. .

Abstract

Endothelial cyclooxygenase-1-derived prostanoids, including prostacyclin, have clear cardioprotective roles associated with their anti-thrombotic potential but have also been suggested to have paradoxical pathological activities within arteries. To date it has not been possible to test the importance of this because no models have been available that separate vascular cyclooxygenase-1 products from those generated elsewhere. Here, we have used unique endothelial-specific cyclooxygenase-1 knockout mice to show that endothelial cyclooxygenase-1 produces both protective and pathological products. Functionally, however, the overall effect of these was to drive pathological responses in the context of both vasoconstriction in vitro and the development of atherosclerosis and vascular inflammation in vivo. These data provide the first demonstration of a pathological role for the vascular cyclooxygenase-1 pathway, highlighting its potential as a therapeutic target. They also emphasize that, across biology, the role of prostanoids is not always predictable due to unique balances of context, products, and receptors.

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Figures

Fig. 1
Fig. 1. Contribution of endothelial cyclooxygenase isoforms to vascular eicosanoid production.
Eicosanoid and related mediator formation from wild-type (A), global cyclooxygenase-1 knockout (B; COX1 KO), and global cyclooxygenase-2 knockout (C; COX2 KO) mouse aorta stimulated with Ca2+ ionophore (n = 4). Production of prostacyclin (D; n = 6 to 8), PGE2 (E; n = 6 to 8), and thromboxane (F; n = 6 to 8) from aorta of endothelial cyclooxygenase-1 or cyclooxygenase-2 knockout mice (EC COX1/COX2 KO) versus respective floxed, Cre-negative littermate controls (Flox COX1/COX2 Ctrl). Prostacyclin and thromboxane were measured as their stable breakdown products, 6-keto-PGF1α and thromboxane B2, respectively. Data are means ± SEM. *P < 0.05 versus respective control strain by two-way analysis of variance (ANOVA) with Dunnett’s post-test (A to C) or unpaired t test (D to F).
Fig. 2
Fig. 2. Effect of endothelial cyclooxygenase-1 deletion on contractile and dilator responses in isolated arteries.
Prostacyclin (as 6-keto-PGF1α) formation from acetylcholine-stimulated arteries (A; n = 6 to 8) from endothelial cyclooxygenase-1 knockout mice (EC COX1 KO) and floxed littermate controls (Flox COX1 Ctrl). Response to the prostacyclin analog treprostinil in arteries from Flox COX1 Ctrl mice (B; n = 4 to 5). Vasomotor responses to phenylephrine and acetylcholine in isolated aortae (C and F; n = 6 to 11), mesenteric arteries (D and G; n = 3 to 8), and carotid arteries (E and H; n = 6 to 13) from EC COX1 and respective Flox COX1 Ctrl mice. Responses to phenylephrine (I; n = 9 to 12) and acetylcholine (J; n = 10 to 12) in carotid arteries from endothelial cyclooxygenase-2 (EC COX2 KO) mice and respective Flox COX2 Ctrl mice with and without the nonspecific cyclooxygenase-1/2 inhibitor diclofenac (1 μM). Data are means ± SEM. *P < 0.05 by unpaired t test (A) or two-way repeated-measures ANOVA with Holm-Sidak post-test (C to J).
Fig. 3
Fig. 3. Effect of endothelial cyclooxygenase-1 and cyclooxygenase-2 deletion on hemodynamic responses in vivo.
Mean arterial blood pressure (MABP) (A; n = 17 to 18) and carotid artery blood flow (B; n = 11 to 14) in anesthetized endothelial cyclooxygenase-1 knockout mice (EC COX1 KO) versus floxed littermate controls (Flox COX1 Ctrl). Cumulative dose-blood pressure response curves to intravenous phenylephrine (C; n = 4 to 10) and L-NAME (D; n = 7 to 12) in EC COX1 KO and Flox COX1 Ctrl mice. Mean arterial blood pressure (E; n = 11) and carotid artery blood flow (F; n = 14 to 16) in anesthetized endothelial cyclooxygenase-2 knockout mice (EC COX2 KO) and floxed littermate controls (Flox COX2 Ctrl). Cumulative dose-blood pressure response curves to intravenous phenylephrine (G; n = 3) and L-NAME (H; n = 5) in EC COX2 KO and Flox COX2 Ctrl mice. Data are means ± SEM. All P > 0.05 by unpaired t test (A, B, E, and F) or two-way repeated-measures ANOVA (C, D, G, and H).
Fig. 4
Fig. 4. Effect of endothelial cyclooxygenase-1 deletion on prostanoid formation and vascular function in atherosclerotic animals.
Prostacyclin (A; n = 6 to 12; as 6-keto-PGF1α) and PGE2 formation (B; n = 6 to 10) from A23187-stimulated carotid arteries from healthy and atherosclerotic [ApoE−/−; 12-week high-fat diet (HFD)/high-cholesterol diet] endothelial cyclooxygenase-1 knockout mice (EC COX1 KO) and floxed littermate controls (Flox COX1 Ctrl). Vasomotor responses to acetylcholine in isolated carotid arteries ex vivo (C; n = 6 to 8) and carotid artery blood flow under anesthesia in vivo (D; n = 5 to 7) in atherosclerotic EC COX1 KO and Flox COX1 Ctrl mice. Data are means ± SEM. *P < 0.05 by (A and B) one-way ANOVA with Holm-Sidak post-test, two-way repeated-measures ANOVA (C) or unpaired t test (D).
Fig. 5
Fig. 5. Effect of endothelial cyclooxygenase-1 deletion on aortic and circulating lipid accumulation in atherosclerotic animals.
Aortic lipid accumulation in the aortic arch (A; n = 12 to 14), the brachiocephalic artery (B; n = 12 to 14), and carotid artery (C; n = 12 to 14) from atherosclerotic (ApoE−/−; 12-week high-fat/cholesterol diet) endothelial cyclooxygenase-1 knockout mice (EC COX1 KO) and floxed littermate control mice (Flox COX1 Ctrl) and representative photographs of sudan IV–stained aortic arches from each strain (D). Plasma total cholesterol levels from healthy and atherosclerotic EC COX1 KO and Flox COX1 Ctrl mice (E; n = 8 to 21). Data are means ± SEM. *P < 0.05 by unpaired t test (A to C) or one-way ANOVA with Holm-Sidak post-test (E).
Fig. 6
Fig. 6. Effect of endothelial cyclooxygenase-1 deletion on inflammatory and platelet function in atherosclerotic animals.
Levels of IL-6 in plasma (A; n = 4 to 10) and released from isolated carotid artery (B; n = 4 to 8) from healthy and atherosclerotic (ApoE−/−; 12-week high-fat/cholesterol diet) endothelial cyclooxygenase-1 knockout mice (EC COX1 KO) and floxed littermate control mice (Flox COX1 Ctrl). Expression of mRNA for ICAM-1 (C), VCAM-1 (D), and P-selectin (E) in aortic arches from healthy and atherosclerotic EC COX1 KO and Flox COX1 Ctrl mice. Macrophage (Mac2+ cell) immunoreactivity in brachiocephalic artery sections from atherosclerotic EC COX1 KO and Flox COX1 Ctrl mice (F) and representative micrographs of staining in each genotype (G). Thromboxane generation from A23187-stimulated whole blood from atherosclerotic EC COX1 KO and Flox COX1 Ctrl mice (H). AYGKPF (a thrombin mimetic peptide)–induced platelet GPIIb/IIIa activation (I; n = 5 to 10) and basal circulating platelet-leukocyte binding (J; n = 4 to 9) in whole blood from healthy and atherosclerotic EC COX1 KO and Flox COX1 Ctrl animals. Thrombosis in vivo measured as time to occlusion after FeCl3-induced injury to the carotid artery in atherosclerotic EC COX1 KO and Flox COX1 Ctrl mice (K; n = 5 to 7) and representative flow traces from each genotype (L). Data are means ± SEM. *P < 0.05 by one-way ANOVA with Holm-Sidak post-test (A to E and J), unpaired t test (F, H, and K), or two-way repeated-measures ANOVA (I).
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