OP08. SPARC modifies colonic tissue healing and inflammation by regulating collagen and MMP expression
Y.‑L. Ng1, B. Klopcic2, S.K. Fu2, F. Lloyd2, I. Lawrance2
1School of Veterinary and Biomedical Sciences, Murdoch University, Perth, Australia; 2Centre for Inflammatory Bowel diseases, School of Medicine and Pharmacology, Fremantle, Australia
Background: Secreted Protein Acidic and Rich in Cysteine (SPARC) is a matricellular protein expressed during tissue repair. It binds to, and interacts with, collagen and regulates matrix metalloproteinase (MMP) expression. The aim was to determine if SPARC modifies intestinal healing.
Methods: Wild-type (WT) and SPARC-null (KO) 129/SvJ × C57/BL6 mice received 7 days of 3% dextran sodium sulphate (DSS) in the drinking water and were sacrificed on days 7, 14, 21 and 35. Colonic tissue was assessed histologically and also stained with Sirius red, RNA isolated and the collagen bundles examined by transmission electronmicroscopy (TE). Colonic myofibroblasts were isolated from WT and KO mice, stimulated with phorbol 12-myristate 13-acetate (PMA) and the RNA isolated. Expression of collagen (Col)1α1, Col3α1, MMP13, MMP3, TIMP1, TIMP2, TGFβ1 and TGFβ3 in colonic tissue and the myofibroblasts were analysed by real time PCR.
Results: KO mice had significantly less endoscopic inflammation. By day 35, KO colonic mucosa had completely regenerated unlike WT mice and at days 14, 21 and 35 KO mice had lower histological inflammation and damage. At day 7, when intestinal inflammation was maximal, sirius red staining was greater in DSS-treated animals than controls (KO vs control p < 0.01) with no differences at other time points. Col1α1, Col3α1 MMP13 and MMP3 expression was reduced in treated WT colons at day 7 and these were significantly lower than in KO colons (p < 0.01). TIMP1, TIMP2 and TGFβ1, TGFβ3 were not different at any time point. The collagen fibre diameters by TE in KO colons were smaller than WTs (40 nm vs 30 nm p < 0.0001) suggesting that SPARC modifies collagen bundling. Compared to unstimulated WT fibroblasts, KO cells had lower Col1α1 and Col3α1 expression (p < 0.001). PMA reduced Col1α1 and Col3α1 (p < 0.05) and increased MMP13 (p < 0.01) and TIMP1 (p < 0.05) expression in the isolated cells but WT and KO cells were not different. PMA had no effect on MMP3, TIMP2, TGFβ1 and TGFβ3 expression.
Conclusions: SPARC appears to modify tissue healing by regulating collagen expression, bundling and its degradation by MMPs resulting in faster tissue turnover and a faster healing rate. Differences between the WT and KO fibroblasts, however, did not account for these changes suggesting that interaction between cells is important.