Deletion of Calponin 2 Reduces the Formation of Postoperative Peritoneal Adhesions

Abstract

Aim of the study: Postoperative peritoneal adhesions are a common cause of morbidity after surgery, resulting in multiple complications. Macrophage-mediated inflammation and myofibroblast differentiation after tissue injury play central roles in the pathogenesis and progression of adhesion formation. Calponin 2 is an actin cytoskeleton regulatory protein in endothelial cells, macrophages and fibroblasts that are key players in the development of fibrosis. Deletion of calponin 2 has been shown to attenuate inflammatory arthritis, atherosclerosis and fibrocalcification of the aortic valves. The present study investigated the effect of calponin 2 deletion on attenuating the formation of peritoneal adhesions in a mouse model for potential use as a new therapeutic target.Materials and methods: Sterile surgical procedures under general anesthesia were used on paired wild type (WT) and calponin 2 knockout (KO) mice to generate mild injury on the cecal and abdominal wall peritonea. Three and seven days post-operation, the mice were compared postmortem for the formation of peritoneal adhesions. Tissues at the adhesion sites were examined with histology and immunofluorescent studies for macrophage and myofibroblast activations.Results: Quantitative scoring demonstrated that calponin 2 KO mice developed significantly less postoperative peritoneal adhesions than that in WT mice. Calponin 2 deletion resulted in less infiltration of F4/80⁺ macrophages at the adhesion sites with less myofibroblast differentiation and collagen deposition than WT controls.Conclusions: The data show that deletion of calponin 2 effectively reduces postoperative peritoneal adhesion, presenting a novel molecular target for clinical prevention.

Keywords: Calponin 2; inflammation; myofibroblast; peritoneal adhesions.

Figure 1.. Western blot conformation of Cnn2 KO mice.

12% SDS-polyacrylamide gel of total protein extract from spleen tissue and Western blot of a replica gel using an anti-calponin2 antibody RAH2 confirmed the complete deletion of calponin 2 in Cnn2^−/− mice. Wild type (WT) mouse spleen was used as control.

Figure 2.. Surgical induction of peritoneal adhesions.

A. An area defined using an underneath 9 mm × 9 mm metal plate was abraded, producing a rough surface with mild petechiae without bleeding or oozing. B. The entirely abraded cecum showing a rough surface of the visceral peritoneum was folded to make contact between the injured surfaces before replacement into the abdominal cavity.

Figure 3.. Examples of scored cecal adhesions.

A. Score 1 represents minimal adhesions causing cecal wall contractures such as that pointed by the arrow. B. Score 2 corresponds to adhesions formed in less than 50% of the cecum length such as that between the two arrowheads. C. Score 3 is for adhesions formed in more than 50% of the cecum length such as the region outlined by the three arrowheads corresponding to ~90% of the cecum length. D. Score 4 indicates additional adhesions between cecum and abdominal wall or other organs. The arrows indicate adhesions formed between cecum and abdominal wall.

Figure 4:. Deletion of calponin 2 minimizes the formation of proliferating mesothelial layer and collagen deposition in postoperative Day 7 peritoneal adhesions.

(A) A cross section of a representative Day 7 WT cecal adhesion site showing proliferating mesothelial cells between muscle layers (indicated by the stars) of the cecal walls was used to illustrate the calculation of A/L ratio in the tissue sections. The adhesion image was divided geometrically and the total area containing mesothelial cells measured using a computer software was divided by the total length of adhesion. (B) Quantitative analysis of the A/L ratio showed that Cnn2 KO mice had a significantly lower A/L ratio than WT control. (C) Cross sections of a Day 7 abdominal wall abrasive site revealed that collagen deposition in Masson’s Trichrome stain mainly located in the deeper layer of the proliferating mesothelial layer, which was less in calponin 2 KO than that in WT control (the proliferating mesothelial cell layer was marked by the two + symbols).

Figure 5:. Calponin 2 KO mice exhibit attenuated macrophage infiltration and myofibroblast differentiation during the healing of operational injuries.

(A) Immunofluorescence micrographs of thin sections of cecal adhesion sites at postoperative Day 3 showed significantly less infiltration of F4/80⁺ macrophages (green) in calponin 2 KO mice in comparison with that of wild type controls. (B) At postoperative Day 7, immunofluorescence microscopy further showed significantly less α-SMA-positive (red) myofibroblasts in sections of cecal adhesion sites from calponin 2 KO mice than that in WT control. DAPI nucleus stain (blue) was used to identify the total number of cells.