Celecoxib alleviates pathological cardiac hypertrophy and fibrosis via M1-like macrophage infiltration in neonatal mice

Figures

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  Figure 1. Cardiac hypertrophy and fibrosis were induced in non-regenerative mice (P8) following CI (A) Experimental design of cryoinfarction (cryoinjury [CI]) injury model and analysis in (B–F). (B) Heart weight/body weight (HW/BW, mg/g) in sham, regenerating mice (P3, at postnatal day 3), and non-regenerating mice (P8) at 4 weeks after injury. (C) Heart tissue of sham, P3 injury, and P8 injury was cross-sectioned and stained with hematoxylin and eosin (H&E) for analysis; scale bars: 300 μm (40× objective) and 50 μm (400× objective); (D) Quantification of sham, P3, and P8 cardiomyocytes at 4 weeks after injury (n = 3 per group, independent experiments [n = 2]). (E) Heart tissue of P3 or P8 after injury (copper wire area: 1 mm2, ICR mother mice: 4–6 weeks) was stained with Masson's trichrome; scale bar, 2,000 μm. (F) Quantification of fibrosis coverage (%, 100 × scar perimeter/total perimeter) in P3 and P8 left ventricle at 4 weeks following CI. Data are representative of two independent experiments (n = 4–6 per group, mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001)

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  Figure 2. Pro-inflammatory cytokines, chemokines, and pro-fibrotic gene expression increased in P3 and P8 groups following CI (A) Protein levels of COX-2, TGF-β2, and Snail in sham, P3, and P8 heart were measured using western blot at 7 days post CI, respectively; (B–E) (B) mRNA level of COX-2 in Sham, P3, and P8 heart was measured using real-time PCR at 7 days and 4 weeks after injury; mRNA levels of (C) cytokines, (D) chemokines (MCP-1), adhesion molecular genes (ICAM-I and VCAM-I), and (E) pro-fibrotic genes (TGF-βs and TGFBR1) were measured using real-time PCR in Sham, P3, and P8 heart at 4 weeks after injury. Data are representative of two independent experiments (n = 4–6 per group, mean ± SD, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

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  Figure 3. Celecoxib administration inhibited cardiac hypertrophy and fibrosis to repair injured neonatal heart in non-regenerative mice (A) Experimental design of DMSO treated- and celecoxib-treated heart following CI and analysis for 4 weeks in (B–E). (B) Neonatal ICR mice were intraperitoneally (i.p.) injected with DMSO and celecoxib at 50 mg/kg per mouse 0, 1, and 2 days after CI; scale bar, 2,000 μm. (C and D) (C) Heart weight/body weight (mg/g) and (D) size of cardiomyocytes (μm2) were measured by H&E and wheat germ agglutinin (WGA) staining in sham, DMSO treated-, and celecoxib-treated P8 mice at 4 weeks after injury; scale bars: 50 μm (400× objective), 100 μm (200× objective), and 200 μm (100× objective). (E) Fibrosis coverage (%, 100 × scar perimeter/total perimeter) of heart tissue was measured using Masson's trichrome staining in DMSO treated- and celecoxib-treated P8 mice at 4 weeks after injury; scale bar, 1,000 μm. Data are representative of two independent experiments (n = 4–6 per group, mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

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  Figure 4. Celecoxib administration reduced cardiac hypertrophic and fibrotic gene expression as well as inflammatory-related gene expression in non-regenerative mice following injury (A and B) (A) mRNA levels of hypertrophic genes and (B) pro-fibrotic genes were analyzed in sham, P3, P8, and DMSO-treated and celecoxib-treated P8 mice at 7 days after injury; (C) Protein levels of pro-fibrotic genes and Snail in DMSO treated- and celecoxib-treated P8 mice were measured by western blot at 7 days after injury, respectively. Data are representative of two independent experiments (n = 5–6 per group, mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

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  Figure 5. Celecoxib administration inhibited inflammatory-related gene expression in non-regenerative mice following injury (A–D) (A) Experimental design of DMSO treated- and celecoxib-treated heart following CI and analysis for 1, 3, and 7 days in (B–D). mRNA levels (B) or protein levels (C) of MCP-1 were measured by real-time PCR or ELISA in both groups; protein levels of (D) chemokines and adhesion molecular genes were measured by western blot at 7 days after injury in DMSO treated- and celecoxib-treated P8 mice. Data are representative of two independent experiments (n = 5–6 per group, mean ± SD; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

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  Figure 6. Celecoxib administration increased M1-like macrophage infiltration after injury (A) Experimental design of DMSO treated- and celecoxib-treated P8 heart following CI and analysis for 7 days in (B–H). (B–E) (B and C) Percentage of total F4/80+ macrophages of infarct/border zones was analyzed by flow cytometry at 7 days after injury; percentage and quantification of (D) M1-like macrophages (F480 + Ly6C high M1) and (E) M2-like macrophages (F480+ CD206+ Ly6C low M2). (F–H) (F and G) Percentage and quantification of M1-like macrophages and (H) M2-like macrophages from the spleen were analyzed by flow cytometry in both groups the same as those in the heart. Data are representative of three independent experiments (n = 3–5 per group, mean ± SD, ∗p < 0.05, ∗∗∗p < 0.001).

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  Figure 7. Celecoxib improved cardiac function by inhibiting inflammatory response after injury (A and B) (A) The mRNA levels of cytokines and (B) chemokines as well as adhesion molecular genes were measured by real-time PCR at 4 weeks after injury in DMSO treated- and celecoxib-treated P8 mice. (C–F) (C and D) Internal diameters (LVID), posterior wall thickness (LVPW), and intraventricular septal thickness (IVS) at the end diastole (C) or at the end systole (D) of the left ventricle and end-diastolic/systolic volume (EDVs and ESVs) were analyzed by a digital ultrasound system (E) at 4 weeks following cryoinjury. (F) Ejection fraction (EF%) and fractional shortening (FS%) were measured by a digital ultrasound system, the same as that in (E). Data are representative of mean ± SD (n = 5 per group, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).