Figure 1.
PAI-1 genetic deficiency is associated with reduced acute and late radiation-induced intestinal injury.
Global radiation injury score (RIS) in Wt and PAI-1−/− mice 3, 14 and 42 days after irradiation (A). Detailed spider histograms of RIS 3 and 14 days after irradiation (n = 8 to 10 mice/group) (B). RIS includes A: mucosal ulceration, B: epithelial atypia, C: thickening of subserosa, D: vascular sclerosis, E: intestinal wall fibrosis, F: ileitis cystica profunda, G: lymph congestion. Representative images of intestinal damage 3 days after 19 Gy in Wt and PAI-1 −/− mice (C). Quantitative assessment of mucosal integrity in mice 3 days after 19 Gy (D). (n = 8 to 10 mice/group) # p<0.05 versus the three other groups.
Figure 2.
PAI-1 contributes slightly to radiation-induced intestinal epithelial cell apoptosis in crypts.
High magnification of double TUNEL/E-cadherin staining in crypts in Wt mice 5 hours after 19 Gy. Arrows indicate apoptotic epithelial cells. Nuclei were counterstained with DAPI (blue) (A). Representative double TUNEL/E-cadherin staining in Sham (A–C) and irradiated (B–D) Wt (A–B) and PAI-1 −/− (C–D) mice 5 hours after 19 Gy. (B). Quantitative assessment of TUNEL+/E-cadherin+ cells in crypts in Wt and PAI-1 −/− mice 4, 5 and 24 hours after irradiation (C). Radiation-induced epithelial cell apoptosis in crypts was stimulated in both types of mice. The number of apoptotic epithelial cells was higher in Wt mice than in PAI-1 −/− mice only 5 hours after irradiation. (n = 6 mice/group) # p<0.05 versus sham mice with the same genotype. * p<0.05 between irradiated Wt and PAI-1 −/− mice.
Figure 3.
PAI-1 contributes strongly to radiation-induced intestinal endothelial apoptosis.
High magnification of double TUNEL/CD31 staining in the villus lamina propria in Wt mice 4 hours after 19 Gy. Arrows indicate apoptotic endothelial cells. Nuclei were counterstained with DAPI (blue) (A). Representative double TUNEL/CD31 staining in Sham (A–C) and irradiated (B–D) Wt (A–B) and PAI-1 −/− (C–D) mice 5 hours after 19 Gy (B). Radiation-induced endothelial intestinal apoptosis was stronger in irradiated Wt mice than in irradiated PAI-1 −/− mice 4 hours after irradiation. Nuclei were counterstained with DAPI (blue). Quantitative assessment of TUNEL+/CD31+ cells in the villus lamina propria in Wt and PAI-1 −/− mice (C). Percentage of apoptotic endothelial cells/total endothelial cells in the villus lamina propria 4 and 5 hours after irradiation in Wt and PAI-1 −/− mice (D). Frequency of apoptotic endothelial cells in the villus lamina propria in Wt and PAI-1 −/− mice 4 and 5 hours after irradiation (E). (n = 6 mice/group) # p<0.05 versus sham mice with the same genotype. * p<0.05 between irradiated Wt and PAI-1 −/− mice.
Figure 4.
PAI-1 genetic deficiency is associated with reduced acute radiation-induced intestinal vascular injury.
Intestinal vasculature was visualized by CD31 staining (red) and computerized with confocal microscopy imaging. Nuclei were counterstained with Sytox Green (A). Representative images of intestinal vasculature 24 hours after irradiation in Wt and PAI-1 −/− mice (B). Quantitative assessment of vasculature integrity 24 hours after 19 Gy (C). (n = 6 mice/group) # p<0.05.
Figure 5.
PAI-1 genetic deficiency in ECs is associated with increased survival after irradiation.
In vitro Matrigel endothelial tube formation assay (A). Quantification was from three independent experiments performed in triplicate.* P<0.05. Percentage of living ECs 24 and 48 hours after 20 Gy. Wt and PAI-1 −/− ECs were plated on vitronectin, poly-L-ornithine/laminin, poly-D-lysine or poly-L-lysine (B). For each coating, results are the mean +/− SEM for two independent experiments performed in triplicate. * P<0.05 versus unirradiated cells for each genotype. # P<0.05 versus Wt irradiated cells.
Figure 6.
PAI-1 overexpression is associated with increased radiation sensitivity of endothelial cells.
Representative western blot (A) and quantification of PAI-1 protein expression of five clones of HUVECs that stably overexpressed PAI-1 (B). Clonogenic assay in control HUVECs and clones 1, 3 and 5 (C). Surviving fraction after 2 Gy (D). Results are the mean +/− SEM (n = 6 per conditions) * p<0.05 versus control HUVECs.
Figure 7.
PAI-1 knockdown in human endothelial cells is associated with increased survival after irradiation.
Representative western blots and quantification of PAI-1 protein expression 8 hours (A) and 24 hours (B) after irradiation in the absence or presence of siPAI-1. Percentage of living cells 24 hours after irradiation in the absence or presence of siPAI-1 (C). Results are the mean +/− SEM of two independent experiments performed in triplicate. * p<0.05 versus unirradiated HUVECs. Representative images of HUVECs 24 hours after 20 Gy in the absence or presence of siPAI-1 (D).
Figure 8.
PAI-1 influences the pro-survival Akt pathway in murine endothelial cells.
Representative western blots of phospho and total Akt in Wt and PAI-1 −/− ECs (A) and in Wt and PAI-1 −/− ECs platted on different coatings 24 hours after 20 Gy (B). Representative western blots of Phospho p38 MAPK, Phospho ERK1/2, Phospho PDK-1, Phospho PTEN and total PTEN in Wt and PAI-1 −/− ECs 24 hours after 20 Gy (C). Percentage of active PTEN in Wt and PAI-1 −/− ECs 24 hours after 20 Gy (D). All experiments were realized in triplicates.
Figure 9.
PAI-1 influences the pro-survival Akt pathway in human endothelial cells by PTEN inactivation.
Representative western blots of phospho-Akt, total Akt, phospho-PTEN, total PTEN, phospho-p38MAPK, p65/RelA, phospho-ERK1/2, phospho-PDK-1, Bcl-2 and Bcl-XL in HUVECs transfected or not with siRNA PAI-1 24 hours after 20 Gy (A). Representative western blots of phospho and total Akt in HUVECs that stably overexpress PAI-1. Akt activation is reduced in human ECs that overexpress PAI-1 (B). Silencing efficiency determined by western blot in HUVECs transfected with siRNA PTEN or siRNA PDK-1 (C). Representative images of HUVECs knocked-down for PTEN, PDK-1, PAI-1, PTEN/PAI-1, or PDK-1/PAI-1 24 hours after 20 Gy (D). Representative western blots of phospho-Akt, total Akt, p65/RelA, phospho-ERK1/2, PTEN, BCL-2 and BCL-XL in HUVECs transfected with siRNA PTEN, PDK-1 and/or siRNA PAI-1 24 hours after 20 Gy(E) .