PAI-1 inhibition improves HL-ischemic tissue regeneration. (A-E) C57BL/6 mice were HL treated and gastrocnemius muscles were analyzed on day 1. (A-C) Quantitative RT-PCR analysis of the mRNA expression of PAI-1 (A), uPA (B), and tPA (C) in nonischemic and HL-ischemic muscle tissue using β-actin as an internal control (n = 3/group for all experiments). (D-E) Homogenates of ischemic and nonischemic tissues that were harvested on day 1 after HL-ischemia induction in 3 different C57BL/6 mice were assayed for murine PAI-1 protein by Western blot analysis (D) or for PAI activity by reverse fibrin zymography (E). Densitometric analysis is shown (bottom). (F-N) HL ischemia was induced in C57BL/6 mice, followed by oral administration of the PAI-1 inhibitor or vehicle given daily from days 0-6. Arrows indicate when the PAI inhibitor was administered. Plasma levels of active PAI-1 (F), tPA (G), and plasmin (H) were assayed in PAI-1– or vehicle-treated HL-ischemic mice by ELISA (n = 7/group for PAI-1 and tPA; n = 6/group for plasmin). (I-J) Representative macroscopic images (I) and the limb perfusion ratio (ischemic/nonischemic; J) of ischemic limbs after HL-ischemia induction. Macroscopic evaluation of the limbs on day 21 (I, bottom) shows foot-digit necrosis only in HL + vehicle-treated animals (n = 5/group). (K-N) Muscle sections of a nonischemic limb and of gastrocnemius (K-L) and hamstring muscles (M-N) of the ischemic limbs from treated mice were stained with H&E (after 21 days; scale bars, 200 mm; K,M) and necrotic areas were evaluated (n = 4 for vehicle group; n = 3 for PAI-1 inhibitor group; L,N). Data represent means ± SEM. *P < .05; **P < .001.