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. 2022 Aug 20;119(5):1218–1233. doi: 10.1093/cvr/cvac137

Figure 2.

Figure 2

Effect of the COX-2/mPGES-1 axis on Treg accumulation in ischaemic muscle tissue. (A) Foxp3 mRNA expression in ischaemic muscle tissue. Aspirin- and celecoxib-treated mice decreased mRNA expression of Foxp3 compared with PBS. Data are means ± SD of the n = 5 mice/group. *P < 0.05 vs. vehicle, one-way ANOVA. (B) Foxp3 staining of ischaemic limbs in aspirin- and celecoxib-treated mice 28 days after ligation. Triangles indicate FoxP3+ cells. Scale bar, 50 μm. (C) Number of FoxP3+ cells in ischaemic muscle tissue 28 days after ligation. Data are means ± SD of the n = 5 mice/group. *P < 0.05 vs. PBS, one-way ANOVA. (D) Mobilized CD4+CD25+ Tregs isolated from the peripheral blood of WT and mPges-1−/− mice 7 days after induction of hindlimb ischaemia were analysed by two-colour flow cytometry. Mobilization of CD4+CD25+ Tregs was impaired in mPges-1−/− mice compared with WT mice (upper panel). (E) Percentages of CD4+CD25+ Tregs in WT mice and CD4+CD25+ Tregs (lower panel). Data are means ± SD of the n = 5 mice/group. *P < 0.05 vs. mPges-1−/−, one-way ANOVA. (F) Foxp3 mRNA expression in ischaemic muscle tissue. mPges-1−/− mice decreased mRNA expression of Foxp3 relative to WT mice. Data are means ± SD of the n = 5 mice/group. *P < 0.05, one-way ANOVA. (G) FoxP3 staining of the ischaemic limb in WT and mPges-1−/− mice 28 days after ligation. Triangles indicate FoxP3+ cells. Scale bar, 50 μm. (H) Number of FoxP3+ cells in ischaemic muscle tissue 7 days after ligation. P < 0.05 vs. WT mice, Student’s t-test. (I) Number of accumulated CD4+CD25+Tregs in ischaemic muscle tissue 7 days after ligation. Data are means ± SD of the n = 3­–5 mice/group. *P < 0.05 vs. WT mice, Student’s t-test. (J) WT FR4 antibody-treated mice impaired blood flow recovery from ischaemia *P < 0.05, repeated-measures ANOVA (upper panel). (K) FoxP3 staining of ischaemic limb in FR4 antibody-treated WT mice 28 days after ligation. Triangles indicate FoxP3+ cells. Scale bar, 50 μm. (L) Number of FoxP3+ cells in ischaemic muscle tissue 28 days after ligation. P < 0.05 vs. IgG, Student’s t-test. (M) In mPges-1−/− mice, FR4 antibody treatment did not affect blood flow recovery from ischaemia. *P < 0.05 vs. IgG, repeated-measures ANOVA (upper panel). (N) FoxP3 staining of ischaemic limb in FR4 antibody-treated mPges-1−/− mice 28 days after ligation. Triangles indicate FoxP3+ cells. Scale bar, 50 μm. (O) Number of FoxP3+ cells in ischaemic muscle tissue 28 days after ligation. P < 0.05 vs. IgG, Student’s t-test.