Volume 65, Issue 5, Pages 1927-1936 (May 2004) Morphologic changes of peritoneum and expression of VEGF in encapsulated peritoneal sclerosis rat models  Hiroaki Io, Chieko Hamada, Yuuki Ro, Yoshifumi Ito, Ichiro Hirahara, Yasuhiko Tomino  Kidney International  Volume 65, Issue 5, Pages 1927-1936 (May 2004) DOI: 10.1111/j.1523-1755.2004.00599.x Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 1 Histologic features of the anterior abdominal wall. (A) Control rat peritoneum. (B) At day 7, there is thickening of the peritoneum with edema. (C) At day 14, with increased cellularity and angiogenesis. (D) At day 21,these findings were marked. (E) At day 28, angiogenesis had decreased although collagen fibrils had accumulated. (F) At day 35, submesothelial region was covered by fibrous tissues with cell infiltration. (G) At day 14, with increased cellularity and edema. Angiogenesis was improved compared with chlorhexidine gluconate and ethanol dissolved in saline (CH) group at day 14. (H) At day 21, the thickness of the compact zone was improved compared with CH group. (I) At day 28, its finding was marked. Masson's trichrome staining (original magnification, ×200). Kidney International 2004 65, 1927-1936DOI: (10.1111/j.1523-1755.2004.00599.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 2 The thickness of the submesothelial compact zone. In the chlorhexidine gluconate and ethanol dissolved in saline (CH) group, the thickness of the submesothelial compact zone gradually increased. In CH with anti-vascular endothelial growth factor (VEGF) antibody group, the thickness of the compact zone was improved compared with CH group. There was a remarkable difference on thickness of peritoneum in each observation between CH group and CH with anti-VEGF antibody group. *P < 0.01. Kidney International 2004 65, 1927-1936DOI: (10.1111/j.1523-1755.2004.00599.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 3 Quantitative analysis for blood vessel assessment. (A) Sections stained for Griffonia simplicifolia 1 (GS1)-lectin, a marker for capillaries and the microcirculatory vessels, using a laser scanning confocal imaging system. Panels a to f are chlorhexidine gluconate and ethanol dissolved in saline (CH) group. Panel a is control on day 35, panel b is at day 7, panel c is at day 14, and panel d is at day 21. From day 14 to day 21, blood vessels were dilated with increased branches and numbers. From day 28 (panel e) to day 35 (panel f), the area of blood vessels decreased and the diameter of the vessels became variable. Panels g to I are CH with anti-vascular endotheial growth factor (VEGF) antibody group. Panel g is at day 14, panel h is at day 21, and panel I is at day 28. From day 14 to day 21, dilatation of blood vessels were improved compared with CH group noticeably (magnification, ×100). (B) In the CH group, panel a (vascular area) (mm2/field), panel b (vascular diameter) (μm/vessel), and panel c (vascular length) (mm/field) showed peak levels at day 21 and then decreased at day 28 and day 35. The vascular diameter and length at day 35 were slightly higher than those at day 28, but the difference was not significant. In the CH with anti-VEGF antibody group, the vascular area, diameter and length were gradually increased from days 14 to 28. These changes were milder than those in the CH group. From days 14 to 21, vascular area, diameter, and length were improved compared with the CH group #P < 0.001 vs. CH group; *P < 0.001 vs. day 0; **P < 0.001 vs. day 21. Kidney International 2004 65, 1927-1936DOI: (10.1111/j.1523-1755.2004.00599.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 4 Immunohistochemical analysis of the anterior abdominal wall injected daily with chlorhexidine gluconate and ethanol dissolved in saline (CH). (A) At day 14, angiopietin-1 (Ang-1) was expressed in the endothelial cells and infiltrating cells. (B) At day 14, angiopietin-1 (Ang-2) and vascular endothelial growth factor (VEGF) (C) were strongly expressed in the endothelial cells in deep parts of the peritoneal tissues. (D) At day 28, Ang-1 expression became weak compared with (A). At day 28, the vascular proliferation was diminished, but (E) Ang-2 and (F) VEGF were strongly detected in shallow parts of the peritoneum (magnification, ×200). Kidney International 2004 65, 1927-1936DOI: (10.1111/j.1523-1755.2004.00599.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 5 Immunohistochemical analysis of anterior abdominal wall injected daily with chlorhexidine gluconate and ethanol dissolved in saline (CH). (A) CD34 staining at day 21 (magnification, ×200). (B) CD34 staining at day 21 (magnification ×400). (C) Platelet-endothelial cell adhesion molecule-1 (PECAM-1) staining at day 21 (magnification ×200). (D) PECAM-1 staining at day 21(magnification ×400). CD34-positive and PECAM-1–negative cells were localized in shallow regions of the peritoneum. CD34- and PECAM-1–positive cells were clustered in the superficial peritoneal layer. Kidney International 2004 65, 1927-1936DOI: (10.1111/j.1523-1755.2004.00599.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 6 Gene expression of vascular endothelial growth factor (VEGF), Flt-1, angiopietin-1 (Ang-1), angtiopietin (Ang-2), Tie-1, Tie-2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) analyzed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Total RNA fractions extracted from isolated peritoneum in each stage of peritonitis. PCR products were resolved on 1.5% agarose gel containing ethidium bromide. (A) Gels shown are representative of three to four experiments. Lane 1, control; lanes 2 through 6, a daily intraperitoneal injection of chlorhexidine gluconate and ethanol dissolved in saline (CH), days 7, 14, 21, 28, and 35, respectively. Lanes 7 through 9, a daily intraperitoneal injection of CH with anti-VEGF antibody three times a week. (B) The bands are quantified by densitometric scanning and corrected with GAPDH expression (arbitrary unit). Panel a is VEGF; panel b is Flt-1; panel c is Ang-1; panel d is Ang-2; panel e is Tie-1 and panel f is Tie-2. In CH with anti-VEGF antibody group, the levels of VEGF, Flt-1, Ang-2, Tie-1, and Tie-2 mRNA expression were gradually decreased from day 14 to 28 and statistically significant compared with CH. However, Ang-1 mRNA expression was not statistically significant compared with CH. Data represent the mean ±SD of three to four different experiments. *P < 0.01 vs. day 0; #P < 0.01 vs. CH. Kidney International 2004 65, 1927-1936DOI: (10.1111/j.1523-1755.2004.00599.x) Copyright © 2004 International Society of Nephrology Terms and Conditions