In vivo blockade of PAI-1 augments neoangiogenesis and growth factor release. (A) Macroscopic images of the lower limb region of nonischemic and PAI-1 inhibitor- or vehicle-treated wild-type mice were captured on day 14 after HL ischemia induction (magnification, 25×; scale bars, 2000 mm). The insert box depicts areas of neoangiogenesis. (B-G) HL ischemia was induced in C57BL/6, tPA^+/+, and tPA^−/− mice, and the mice were then treated with or without PAI-1 inhibitor daily from days 0-6. (B-C) Capillary density was measured in sections of the hamstring (B) and adductor muscles (C) based on immunohistochemical staining of VWF per high power field (HPF). (B,D) Representative images of anti-VWF mAb immunohistochemical staining of ischemic muscle sections from HL-ischemia–induced C57BL/6, tPA^+/+, and tPA^−/− mice either left untreated or treated with or without the PAI-1 inhibitor (n = 6/group) analyzed on day 14 after the procedure (scale bars, 200 mm). Arrows depict VWF⁺ capillaries. (E-G) Plasma levels of VEGF-A (E) and KitL (F) and serum levels of G-CSF (G) in HL-ischemia–induced tPA^+/+ and tPA^−/− mice treated with or without PAI-1 inhibitor were determined by ELISA (for VEGF-A, n = 9 for tPA^+/+ mice and n = 3 for tPA^−/− mice; for KitL and G-CSF, n = 7 for tPA^+/+ mice and n = 3 for tPA^−/− mice; for KitL, n = 3 for G-CSF). (H) G-CSF serum levels were analyzed by ELISA in HL-ischemia–induced MMP-9^+/+ and MMP-9^−/− mice treated with or without PAI-1 inhibitor (H) and in C57BL/6 mice treated with a serpin-resistant tPA mutant (n = 4-5/group). Values represent the means ± SEM. *P < .05; **P < .001.