Figure 1.
Morphological changes in peritoneal mesothelial cells (PMCs) after fibrin application.
The morphology of the PMCs changed from a polygonal cobblestone-like appearance (A) to a spindle-shaped form (B) after fibrin overlay for 24 h. (C) Morphological changes were attenuated by treatment with 0.3 mg/ml of pentoxifylline.
Figure 2.
Changes in cell markers after application of fibrin to peritoneal mesothelial cells (PMCs).
Expression of α-smooth muscle actin (α-SMA), fibronectin, fibroblast specific protein-1 (FSP-1), and β3 integrin were detected by immunofluorescence staining with FITC-labeled secondary antibodies (green). Nuclei were counterstained with PI (red). PMCs overlaid with fibrin for 4 h (+Fibrin) expressed higher levels of α-SMA, fibronectin, FSP-1, and β3 integrin than untreated PMCs (-Fibrin). Original magnification ×400.
Figure 3.
Western blots of cell markers in fibrin-covered peritoneal mesothelial cells (PMCs).
Equal amounts of protein (20 µg per lane) from untreated or fibrin-covered PMCs were resolved, transferred, and blotted for α-SMA, fibronectin, FSP-1, αvβ3 integrin, cytokeratin 18, E-cadherin, and GAPDH (A). The relative levels of α-SMA/GAPDH (B), αvβ3 integrin/GAPDH (C and D), fibronectin/GAPDH (E), FSP-1/GAPDH (F), cytokeratin 18/GAPDH (G), and E-cadherin/GAPDH (H) were measured by densitometry. C, PMCs without fibrin; F1, fibrin covered for 1 h; P1, fibrin covered and treated with pentoxifylline (PTX) 0.3 mg/ml for 1 h; F4, fibrin covered for 4 h, P4, fibrin covered and treated with PTX 0.3 mg/ml for 4 h; A4, fibrin covered and treated with αvβ3 integrin antibody for 4 h. *P<0.05 vs. C, # P<0.05 between groups, n = 3.
Figure 4.
Western blots of integrin-activated kinases.
Equal amounts of protein (20 µg per lane) from untreated or fibrin-covered PMCs were resolved, transferred, and probed for phosphorylated FAK (focal adhesion kinase) and Src (A). The relative levels of Src/GAPDH (B) and FAK/GAPDH (C) were measured by densitometry. C, PMCs without fibrin; F10, fibrin covered for 10 min; P10, fibrin covered and treated with pentoxifylline 0.3 mg/ml for 10 min; A10, fibrin covered and treated with αvβ3 integrin antibody for 10 min; F20, fibrin covered for 20 min; F30, fibrin covered for 30 min. *p<0.05 vs. Control.
Figure 5.
Group 4 had a significantly higher score than Group 1. Group 5 had a significantly lower score than Group 4. *p<0.05 vs. Group 1, #p<0.05 vs. Group 4.
Figure 6.
Histological analysis of the submesothelial compact zones.
The compact zones were thicker in Group 4 than in Group 1 on both the parietal peritoneum (A) and liver surface (B). Rats treated with PTX (Group 5) had significantly less submesothelial matrix formation than Group 4 on both the parietal peritoneum and liver surface. *p<0.05 vs. Group 1 (Control), #p<0.05 vs. Group 4.
Table 1.
Peritoneal equilibrium tests.
Table 2.
Cytokine concentrations in peritoneal dialysates.
Figure 7.
TGFβ1 gene expression in the parietal peritoneum.
TGFβ1 mRNA was measured by RT-PCR. mRNA levels increased in Groups 2 and 4 compared to Group 1 but expression in Groups 2 and 5 was significantly lower than in Group 4. GAPDH expression served as a loading control. *p<0.05 vs. Group 1, #p<0.05 vs. Group 4.
Figure 8.
Immunohistochemical staining of cytokeratin.
(A) Cytokeratin-positive cells were present not only on the surface mesothelium, but also in the submesothelial fibrosis on the liver surface, parietal peritoneum, and omentum of Group 4 rats. Cytokeratin-positive cells were present only on the surface mesothelium of Group 1 rats. Original magnification ×400. (B) Percentage of cytokeratin-positive cells in the fibrotic tissue of the omentum. *p<0.05 vs. Group 1, #p<0.05.
Figure 9.
Western blots of cell markers in the peritoneum of animals with EPS.
Equal amounts of protein (30 µg per lane) from the peritoneum of rats in Group 1 to 5 were resolved, transferred, and probed for α-SMA, αvβ3 integrin, fibronectin, FSP-1, and GAPDH (A). The relative levels of α-SMA/GAPDH (B), αvβ3 integrin/GAPDH (C and D), fibronectin/GAPDH (E), and FSP-1/GAPDH (F) were measured by densitometry.*p<0.05 vs. Group 1, #p<0.05 vs. Group 4.
Figure 10.
Protocol for the animal study.
Thirty male Wistar rats were randomly divided into 5 groups of 6 rats per group. Rats received intraperitoneal injections of PBS (Group 1), S. aureus (6×108 colony forming units (CFU); Group 2), 15 ml fibrinogen (10 mg/ml; Group 3), or both S. aureus and fibrinogen (Group 4). Group 5 animals received intraperitoneal injections of S. aureus and fibrinogen and also received daily intravenous injections of PTX (100 mg/kg) for 7 days. Animals were then subjected to the peritoneal equilibration test (PET) and sacrificed. The details of procedures are shown in the figure. ▾: Catheter placement in the internal jugular vein. ○: PBS intraperitoneally. •: 1 ml S. aureus (6×108 CFU/ml) intraperitoneally. ◊: 15 ml PBS intraperitoneally. ⧫: 15 ml fibrinogen (10 mg/ml) intraperitoneally. ▪ ▪ ▪ ▪ ▪: 1.5 ml intravenous PBS daily for 7 days. ▪▪▪▪▪▪: 100 mg/kg intravenous pentoxifylline (20 mg/ml; approximately 1.5 ml) daily for 7 days. ×: Performed PET and then sacrificed.
Table 3.
Gross adhesion scoring system.