Differential effects of medroxyprogesterone acetate on thrombosis and atherosclerosis in mice

Abstract

Background and purpose: The risk for cardiovascular events including venous and arterial disease and stroke is elevated after treatment with estrogen and medroxyprogesterone acetate (MPA) in postmenopausal women. Here, we have investigated the effect of MPA on arterial thrombosis and atherosclerosis in a murine model of atherosclerosis.

Experimental approach: Apolipoprotein E (ApoE)-/- mice were bilaterally ovariectomized and treated with placebo, MPA (27.7 microg day(-1)) and MPA + 17-beta-oestradiol (E2; 1.1 microg day(-1)) for 90 days, on a Western-type diet. Thrombotic response was measured in a photothrombosis model, platelet activation by fluorescence activated cell sorting (FACS) analysis (CD62P) and thrombin generation by the endogenous thrombin potential (ETP). Furthermore, aortic plaque burden and aortic root plaque composition were determined.

Key results: MPA and MPA + E2-treated animals showed an aggravated thrombotic response shown by significantly reduced time to stable occlusion. The pro-thrombotic effect of MPA was paralleled by increased ETP whereas platelet activation was not affected. Furthermore, MPA + E2 reduced the number of cells positive for alpha-smooth muscle actin and increased hyaluronan in the plaque matrix. Interestingly, total plaque burden was reduced by MPA but unchanged by MPA + E2.

Conclusion and implications: Long-term treatment with MPA and MPA + E2 increased arterial thrombosis despite inhibitory effects of MPA on atherosclerosis in ApoE-deficient mice. Increased thrombin formation, reduced smooth muscle content and remodelling of non-collagenous plaque matrix may be involved in the pro-thrombotic effects. Thus, MPA exhibits differential effects on arterial thrombosis and on atherosclerosis.

Figure 1

Thrombotic response. (A) Experimental design. (B) Representative blood-flow measurement after initiation of photochemical injury at time 0. (C) Times to stable occlusion from mice receiving placebo, medroxyprogesterone acetate (MPA; 27.7 µg·day⁻¹) and MPA + E₂ (1.1 µg·day⁻¹). (D) Graph showing the frequency of flow-recovery as a measure of thrombus stability in mice treated with placebo, MPA and MPA + E₂. Data represent means ± standard error of the mean; n= 6–8, *, P < 0.05 versus placebo.

Figure 2

Platelet activation and endogenous thrombin potential (ETP) (A) Representative plots of ex vivo FACS analysis of CD62P-expression on platelets in platelet-rich plasma from mice treated with placebo, medroxyprogesterone acetate (MPA; 27.7 µg·day⁻¹ay) and MPA + E₂ (1.1 µg·day⁻¹). Left panel: basal CD62P expression; right panel: CD62P expression after stimulation with 5 µg/mL convulxin (B) Quantitative analysis of CD62P expression. (C) Representative curves for thrombin generation over time (ETP) in mice treated with placebo, MPA and MPA + E₂. (D) Quantitative analysis of ETP. Data represent means ± standard error of the mean; n= 6–7, *P < 0.05 versus placebo, #P < 0.05 versus MPA.

Figure 3

Plaque burden. (A) Oil-Red-O stained aortas from mice substituted with medroxyprogesterone acetate (MPA; 27.7 µg·day⁻¹) and MPA + E₂ (1.1 µg·day⁻¹). (B) Aortic plaque scores from mice treated with placebo, MPA and MPA + E₂. Data represent means ± standard error of the mean; n= 18–21; *P < 0.05 versus placebo.

Figure 4

ACh-dependent vasorelaxation of aortic rings. (A) Concentration-response curves in response to ACh of aortic rings derived from mice treated with placebo, medroxyprogesterone acetate (MPA; 27.7 µg·day⁻¹) and MPA + E₂ (1.1 µg·day⁻¹). (B) Half maximal effective concentration (EC₅₀) values for ACh-induced vasorelaxation. (C) Concentration-response curves in response to S-nitroso-N-acetyl-D,L-penicillamine (SNAP) in response to treatment with placebo, MPA and MPA + E₂. (D) EC₅₀ values for SNAP. EC₅₀ values for ACh calculated from concentration-response curves in (A). EC₅₀ values for SNAP calculated from concentration-response curves in (C). Data represent means ± standard error of the mean; n= 5–7, *P < 0.05 for placebo versus MPA, #P < 0.05 for placebo versus MPA + E₂.

Figure 6

Lipid content of aortic root plaques. (A–C) Aortic root sections stained for lipid deposits from mice treated with placebo, medroxyprogesterone acetate (MPA; 27.7 µg·day⁻¹) and MPA + E₂ (1.1 µg·day⁻¹). (D) Data represent fractions of positively stained areas as determined by digital image analysis; means ± standard error of the mean; n= 5–9.

Figure 5

Plaque composition. (A–C, G) Aortic root sections stained for α-SM-actin (red) from mice treated with placebo, medroxyprogesterone acetate (MPA; 27.7 µg·day⁻¹) and MPA + E₂ (1.1 µg·day⁻¹). (D–F, H–I). Aortic root sections stained for hyaluronan and the macrophage antigen Mac2. (G–I) Data represent area fractions of positively stained antigens as determined by digital image analysis; means ± standard error of the mean; n= 6–10; *P < 0.05 versus placebo.