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. Author manuscript; available in PMC: 2016 May 1.
Published in final edited form as: Cell Signal. 2015 Jan 22;27(5):923–933. doi: 10.1016/j.cellsig.2015.01.009

Figure 5. Elastase-cleaved PAI-1 promotes VSMC apoptosis while tiplaxtinin and CL-PAI-1 attenuate neointima formation.

Figure 5

(A) Generation of CL-PAI-1 by incubation with human neutrophil elastase. Solid arrow indicates FL-PAI-1; dashed arrow denotes CL-PAI-1. (B) HuCASMCs treated with a final concentration of 10 nM FL-PAI-1 or CL-PAI-1 and a molar equivalent of elastase (as control) for 24 h were stained with ethidium homodimer and calcein AM to determine apoptotic index. Cells positive for ethidium homodimer were scored as apoptotic and normalized against calcein AM (total cells). Data are presented as mean ± SEM; asterisks=p<0.05, n=3. (C) HuCASMCs were treated with CL-PAI-1 (40 nM) and/or staruosporine (5 μM) as indicated for 24 h. Caspase-3 and actin (loading control) were assessed in whole cell lysates by western blot. (D) H&E stained cross-sections of contralateral control and ligated carotid arteries isolated from mice treated once daily with vehicle control (Con), 3 mg/kg tiplaxtinin (via oral gavage), 2 mg/kg FL-PAI-1 or CL-PAI-1 (both by intraperitoneal injection) for 14 days following ligation. Arrowheads indicate the internal elastic lamina. (E) Quantitation of the intima-to-media ration for the indicated ligated carotid artery groups. Data plotted represent the mean ± SEM; asterisks=p<0.05, n=4. (F) Time line of carotid ligation experiments; arrows indicate days of treatment with the indicated reagents.