Figure 5. PAI-1 loss anticipates fibrosis and exacerbates muscle disease progression: Reversal by pharmacological and genetic interference with uPA in vivo. (A) Fibrosis quantification in diaphragms of PAI-1+/+ and PAI-1−/− mdx mice over time (*, P < 0.01 vs. age-matched PAI-1+/+ mdx; means ± SEM; n = 5 for each group). (B) Quantitative PCR analysis of fibrosis-associated markers in diaphragm muscles of 3-mo-old PAI-1+/+ and PAI-1−/− mdx mice (*, P < 0.05 vs. PAI-1+/+ mdx; means ± SEM; n = 5 for each group). (C) As in B, serum creatine kinase (CK) levels are shown (*, P < 0.01; means ± SEM; n = 5 for each group). (D) Representative Sirius red staining in diaphragm muscle sections of 3.5-mo-old PAI-1+/+ and PAI-1−/− mdx mice and PAI-1−/− mdx mice treated with amiloride (a uPA inhibitor; see Fig. S3 for additional information). Bar, 25 µm. (E) Quantitative PCR analysis of PAI-1 expression in diaphragm muscle of mdx mice at the indicated ages; values are expressed as fold induction over WT (*, P < 0.01 vs. 1 mo; means ± SEM; n = 5 for each group). (F) Increased TGF-β1 active protein and miR-21 expression in the diaphragm muscle of PAI-1−/− mdx mice compared with PAI-1+/+ mdx mice, as measured by ELISA and quantitative PCR, respectively (*, P < 0.05; **, P < 001, means ± SEM; n = 4 for each group). (G, left) As in F, muscle samples were analyzed by Western blotting using antibodies against the proteins P-Smad2, total Smad2/3, and β-actin. (right) Quantification of P-Smad2 versus total Smad2/3 and normalized for β-actin levels is shown (*, P < 0.01; means ± SEM; n = 5 for each group). P-Smad2 levels in basal muscle of WT and PAI-1−/− mice were undistinguishable (not depicted). (H) siRNA for uPA or a scrambled control oligomiR was delivered into gastrocnemius muscles of 3-mo-old PAI-1−/− mdx mice every other day for 3 wk, and, after the treatment, muscles were analyzed for TGF-β1 active protein levels, collagen content, miR-21 expression, and Smad2 activity (P-Smad2; *, P < 0.05; **, P < 001, means ± SEM; n = 4 for each group). (I) siRNA for uPA or Scramble was delivered into the gastrocnemius muscle of 24-mo-old mdx mice every other day for an additional 1 mo. Then, muscles were analyzed for TGF-β1 activity, collagen content, and miR-21 expression (*, P < 0.01; means ± SEM; n = 5 for each group). (J) As in I, H/E staining is shown. Bar, 50 µm.
Figure 5.

PAI-1 loss anticipates fibrosis and exacerbates muscle disease progression: Reversal by pharmacological and genetic interference with uPA in vivo. (A) Fibrosis quantification in diaphragms of PAI-1+/+ and PAI-1−/− mdx mice over time (*, P < 0.01 vs. age-matched PAI-1+/+ mdx; means ± SEM; n = 5 for each group). (B) Quantitative PCR analysis of fibrosis-associated markers in diaphragm muscles of 3-mo-old PAI-1+/+ and PAI-1−/− mdx mice (*, P < 0.05 vs. PAI-1+/+ mdx; means ± SEM; n = 5 for each group). (C) As in B, serum creatine kinase (CK) levels are shown (*, P < 0.01; means ± SEM; n = 5 for each group). (D) Representative Sirius red staining in diaphragm muscle sections of 3.5-mo-old PAI-1+/+ and PAI-1−/− mdx mice and PAI-1−/− mdx mice treated with amiloride (a uPA inhibitor; see Fig. S3 for additional information). Bar, 25 µm. (E) Quantitative PCR analysis of PAI-1 expression in diaphragm muscle of mdx mice at the indicated ages; values are expressed as fold induction over WT (*, P < 0.01 vs. 1 mo; means ± SEM; n = 5 for each group). (F) Increased TGF-β1 active protein and miR-21 expression in the diaphragm muscle of PAI-1−/− mdx mice compared with PAI-1+/+ mdx mice, as measured by ELISA and quantitative PCR, respectively (*, P < 0.05; **, P < 001, means ± SEM; n = 4 for each group). (G, left) As in F, muscle samples were analyzed by Western blotting using antibodies against the proteins P-Smad2, total Smad2/3, and β-actin. (right) Quantification of P-Smad2 versus total Smad2/3 and normalized for β-actin levels is shown (*, P < 0.01; means ± SEM; n = 5 for each group). P-Smad2 levels in basal muscle of WT and PAI-1−/− mice were undistinguishable (not depicted). (H) siRNA for uPA or a scrambled control oligomiR was delivered into gastrocnemius muscles of 3-mo-old PAI-1−/− mdx mice every other day for 3 wk, and, after the treatment, muscles were analyzed for TGF-β1 active protein levels, collagen content, miR-21 expression, and Smad2 activity (P-Smad2; *, P < 0.05; **, P < 001, means ± SEM; n = 4 for each group). (I) siRNA for uPA or Scramble was delivered into the gastrocnemius muscle of 24-mo-old mdx mice every other day for an additional 1 mo. Then, muscles were analyzed for TGF-β1 activity, collagen content, and miR-21 expression (*, P < 0.01; means ± SEM; n = 5 for each group). (J) As in I, H/E staining is shown. Bar, 50 µm.

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