Xpressed as mean 6 SD (n = 4). *, P,0.05 versus WT. doi:10.1371/journal.pone.0050094.gFoxM1 Promotes TLK199 cost endothelial RepairFigure 2. Accelerated resolution of lung inflammation in FoxM1 Tg mice. (A) MPO activities in lung tissues. Lung tissues at indicated times post-CLP challenge were collected for MPO activity determination. Lung tissues from sham-operated mice at 24 h post-surgery were collected as controls. Data are expressed as mean 6 SD (n = 3?). *, P,0.001 versus WT; **, P,0.05 versus WT. (B) Representative micrographs of H E staining of lung sections. At 24 h post-surgery, lungs were fixed for sectioning and H E staining. Arrows indicate perivascular leukocyte infiltration. Scale bar, 50 mm. doi:10.1371/journal.pone.0050094.gWe next determined whether FoxM1 expression in EC is indispensable for endothelial repair. As shown in Fig. 8A, both WT and FoxM1 CKO mice exhibited similar increases of lung vascular permeability at 18 h post-CLP challenge. At late time points (48 h and 72 h), lung vascular permeability in WT mice was markedly reduced whereas it remained elevated in FoxM1 CKO mice at levels similar to peak injury. Similarly, we observed a sustained increase of MPO activity in FoxM1 CKO lungs at 48 h and 72 h post-CLP challenge whereas MPO activity in WT lungs was drastically decreased at 48 h post-CLP challenge and returned to levels similar to baseline seen in sham-operated controls at 72 h post-CLP challenge (Fig. 8B).DiscussionWe have identified the necessary and sufficient role of FoxM1 in promoting endothelial repair and resolution of lung inflammation in a clinically relevant model of sepsis. 1480666 We showed that BCX-1777 transgenicexpression of FoxM1 resulted in rapid recovery of vascular integrity and resolution of lung inflammation following CLPinduced polymicrobial sepsis. FoxM1 Tg mice exhibited a marked increase of survival. Mechanistically, we observed early induction of FoxM1 target genes essential for cell cycle progression and resulting endothelial proliferation in FoxM1 Tg lungs following CLP challenge. Additionally, selective deletion of FoxM1 in EC resulted in defective endothelial repair in FoxM1 CKO mice following CLP-induced lung vascular injury. Together, these data demonstrate the critical role of FoxM1 in mediating endothelial repair following lung vascular injury induced by sepsis. Endothelial repair requires endothelial regeneration and subsequent re-annealing of the endothelial cell-cell contacts to restore the characteristic restrictive endothelial barrier function [2,8,29]. Our previous studies have demonstrated the essential 1407003 role of FoxM1 in regulating endothelial proliferation and re-annealing of the endothelial adherens junctions complexes employing the FoxM1 CKO mice [18,19]. This study further demonstrated theFigure 3. Normalized expression of proinflammatory cytokines and adhesion molecule in FoxM1 Tg lungs at 24 h post-CLP. RNA were isolated from lungs collected at 24 h post-surgery and QRT-PCR analysis were employed to assess the expression levels. Data are expressed as mean 6 SD (n = 3?). *, P,0.05 versus WT-sham. doi:10.1371/journal.pone.0050094.gFoxM1 Promotes Endothelial RepairFigure 4. Increased survival of FoxM1 Tg mice following CLP challenge. 3 month old mice were monitored for 7 days to determine the survival rate following CLP challenge (n = 13 WT and 14 FoxM1 Tg). Sham-operated mice (n = 5 WT or FoxM1 Tg) were also monitored for survival. *, P,0.001 versus CLP-WT. Tg, FoxM1 Tg. doi:10.1371/.Xpressed as mean 6 SD (n = 4). *, P,0.05 versus WT. doi:10.1371/journal.pone.0050094.gFoxM1 Promotes Endothelial RepairFigure 2. Accelerated resolution of lung inflammation in FoxM1 Tg mice. (A) MPO activities in lung tissues. Lung tissues at indicated times post-CLP challenge were collected for MPO activity determination. Lung tissues from sham-operated mice at 24 h post-surgery were collected as controls. Data are expressed as mean 6 SD (n = 3?). *, P,0.001 versus WT; **, P,0.05 versus WT. (B) Representative micrographs of H E staining of lung sections. At 24 h post-surgery, lungs were fixed for sectioning and H E staining. Arrows indicate perivascular leukocyte infiltration. Scale bar, 50 mm. doi:10.1371/journal.pone.0050094.gWe next determined whether FoxM1 expression in EC is indispensable for endothelial repair. As shown in Fig. 8A, both WT and FoxM1 CKO mice exhibited similar increases of lung vascular permeability at 18 h post-CLP challenge. At late time points (48 h and 72 h), lung vascular permeability in WT mice was markedly reduced whereas it remained elevated in FoxM1 CKO mice at levels similar to peak injury. Similarly, we observed a sustained increase of MPO activity in FoxM1 CKO lungs at 48 h and 72 h post-CLP challenge whereas MPO activity in WT lungs was drastically decreased at 48 h post-CLP challenge and returned to levels similar to baseline seen in sham-operated controls at 72 h post-CLP challenge (Fig. 8B).DiscussionWe have identified the necessary and sufficient role of FoxM1 in promoting endothelial repair and resolution of lung inflammation in a clinically relevant model of sepsis. 1480666 We showed that transgenicexpression of FoxM1 resulted in rapid recovery of vascular integrity and resolution of lung inflammation following CLPinduced polymicrobial sepsis. FoxM1 Tg mice exhibited a marked increase of survival. Mechanistically, we observed early induction of FoxM1 target genes essential for cell cycle progression and resulting endothelial proliferation in FoxM1 Tg lungs following CLP challenge. Additionally, selective deletion of FoxM1 in EC resulted in defective endothelial repair in FoxM1 CKO mice following CLP-induced lung vascular injury. Together, these data demonstrate the critical role of FoxM1 in mediating endothelial repair following lung vascular injury induced by sepsis. Endothelial repair requires endothelial regeneration and subsequent re-annealing of the endothelial cell-cell contacts to restore the characteristic restrictive endothelial barrier function [2,8,29]. Our previous studies have demonstrated the essential 1407003 role of FoxM1 in regulating endothelial proliferation and re-annealing of the endothelial adherens junctions complexes employing the FoxM1 CKO mice [18,19]. This study further demonstrated theFigure 3. Normalized expression of proinflammatory cytokines and adhesion molecule in FoxM1 Tg lungs at 24 h post-CLP. RNA were isolated from lungs collected at 24 h post-surgery and QRT-PCR analysis were employed to assess the expression levels. Data are expressed as mean 6 SD (n = 3?). *, P,0.05 versus WT-sham. doi:10.1371/journal.pone.0050094.gFoxM1 Promotes Endothelial RepairFigure 4. Increased survival of FoxM1 Tg mice following CLP challenge. 3 month old mice were monitored for 7 days to determine the survival rate following CLP challenge (n = 13 WT and 14 FoxM1 Tg). Sham-operated mice (n = 5 WT or FoxM1 Tg) were also monitored for survival. *, P,0.001 versus CLP-WT. Tg, FoxM1 Tg. doi:10.1371/.
