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The roles of secreted protein acidic and rich in cystiene (Sparc) in intestinal inflammation, healing and fibrosis

Ng, Yoke Leng (2012) The roles of secreted protein acidic and rich in cystiene (Sparc) in intestinal inflammation, healing and fibrosis. PhD thesis, Murdoch University.

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Secreted Protein Acidic and Rich in Cysteine (SPARC) is a matricellular protein expressed during tissue repair and regulates cell proliferation and migration. It binds to, and interacts with collagen and regulates matrix metalloproteinase (MMP) expression. The aim was to determine if SPARC modifies intestinal inflammation, healing and fibrogenesis.

Intestinal disease was investigated using SPARC null (KO) and wild-type (WT) mice in which inflammation was induced by 3% dextran sodium sulphate (DSS) in the drinking water for 7 days. Inflammation was assessed endoscopically, clinically and histologically on days 7, 14, 21 and 35 after initiation of DSS treatment. Systemic and colonic cytokines and chemokines were quantitated by ELISA and CBA. Colon, mesenteric lymph node and spleen were analysed by flow cytometry and immunofluorescence for inflammatory cell infiltrates. To determine the effect of SPARC on the extracellular matrix (ECM) genes regulation, RNA from colonic tissue, and colonic myofibroblasts from WT and KO mice, were analysed by real time PCR for expression of ECM related genes.

KO animals had significantly lower endoscopic scores of inflammation, suffered less weight loss, diarrhoea, faecal blood and had lower spleen/body weight ratios compared to WT animals consistent with less colonic and systemic inflammation. WT mice had higher levels of histological inflammation and damage when compared to KO animals and in the majority of KO animals the colonic mucosa had completely regenerated by day 35 in contrast to the WT mice. Compared to WT mice, in KO animal colons at day 7 there was significantly less IL1β and MIG expression while TGFβ1 levels were higher. Flow cytometric analysis identified a significantly greater percentage of FoxP3+ regulatory T cells in the spleen and draining lymph nodes of KO animals. KO mice also had fewer of cells, such as CD68+ macrophages and Ly6G+ neutrophils, of the innate immune e system infiltrating the inflamed colon.

Collagen (Col) 1α1, Col3α1 MMP13 and MMP3 expression levels were reduced in DSS-treated WT colons at day 7 and these were significantly lower than those observed in the KO colons. TIMP1 expression was significantly lower in KO mice at day 35 when healing was complete in this group. TIMP2 and TGFβ1, TGFβ3 were not different between the groups at any time point. The observation by others that collagen fiber diameters in KO colons were noted to be significant smaller than in WT animals suggesting that SPARC modifies the collagen bundling. Compared to unstimulated WT fibroblasts, KO cells had lower Col1α1 and Col3α1 expression. Stimulation with PMA reduced Col1α1 and Col3α1 and increased MMP13 and TIMP1 expression in all the isolated cells, but PMA had no effect on MMP3, TIMP2, TGFβ1 and TGFβ3 expression.

DSS induced less colonic and systemic inflammation in KO compared to WT mice and the inflammation appeared to resolve faster. This may be secondary to increased numbers of regulatory T cells and increased colonic TGF-β levels which may inhibit effector cell activity, including cytokine and chemokine expression and aid in the more rapid resolution of inflammation and restoration of the intestinal mucosa. SPARC is able to modify tissue healing potentially through the regulation of collagen expression, bundling and its degradation by MMPs, which impacts on tissue turnover rate and thus delays healing. Hence, SPARC might represent a promising therapeutic target in clinical management of inflammatory bowel diseases (IBD).

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Veterinary and Biomedical Sciences
Supervisor(s): Lawrance, Ian, Greene, Wayne and Klopcic, Borut
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