Thrombospondin-1 Type 1 Repeats in a Model of Inflammatory Bowel Disease: Transcript Profile and Therapeutic Effects

Thrombospondin-1 (TSP-1) is a matricellular protein with regulatory functions in inflammation and cancer. The type 1 repeats (TSR) domains of TSP-1 have been shown to interact with a wide range of proteins that result in the anti-angiogenic and anti-tumor properties of TSP-1. To ascertain possible functions and evaluate potential therapeutic effects of TSRs in inflammatory bowel disease, we conducted clinical, histological and microarray analyses on a mouse model of induced colitis. We used dextran sulfate sodium (DSS) to induce colitis in wild-type (WT) mice for 7 days. Simultaneously, mice were injected with either saline or one form of TSP-1 derived recombinant proteins, containing either (1) the three type 1 repeats of the TSP-1 (3TSR), (2) the second type 1 repeat (TSR2), or (3) TSR2 with the RFK sequence (TSR2+RFK). Total RNA isolated from the mice colons were processed and hybridized to mouse arrays. Array data were validated by real-time qPCR and immunohistochemistry. Histological and disease indices reveal that the mice treated with the TSRs show different patterns of leukocytic infiltration and that 3TSR treatment was the most effective in decreasing inflammation in DSS-induced colitis. Transcriptional profiling revealed differentially expressed (DE) genes, with the 3TSR-treated mice showing the least deviation from the WT-water controls. In conclusion, this study shows that 3TSR treatment is effective in attenuating the inflammatory response to DSS injury. In addition, the transcriptomics work unveils novel genetic data that suggest beneficial application of the TSR domains in inflammatory bowel disease

Expression of WIF-1 in inflammatory bowel disease

The WNT/β-catenin cellular network has been extensively studied in numerous diseases including inflammatory bowel disease (IBD). IBD is a condition that increases the risk of developing colorectal cancer. WIF-1 is an inhibitory protein that acts by blocking the interactions of WNT with its receptor complex, thus leading to downregulation of end products of this pathway. While WIF-1 has been characterized in several cancers, its relationship with IBD has yet to be elucidated. In this study, the expression of WIF-1 in patients with IBD was analyzed in order to provide insights into the pathophysiology and rationale for alternative therapies. Biopsies of both normal and inflamed colonic mucosa from patients with Crohn’s disease or ulcerative colitis were histologically examined for the degree of morphologic changes, immune cell infiltration and presence of WIF-1 through immunohistochemistry. No differences were observed in WIF-1 expression linked to a particular condition, but WIF-1 stain was significantly enhanced in the crypts and lamina propria as inflammation increased in biopsies from patients with both, ulcerative colitis and Crohn’s disease. These findings could give guidance to new therapeutic applications of the WNT/β-catenin system and WIF-1 in IBD

Major pathways activated and genes enriched in the DSS- and TSR-treated mice.

<p>Major pathways activated and genes enriched in the DSS- and TSR-treated mice.</p

Differentially expressed genes in paired sets of comparison between the treatment and the control groups.

<p>Comparison of treatment groups with WT-water (A, upper panel of table) and WT-DSS saline controls (A, lower panel). Total DE genes refer to all genes that are up- or down-regulated by >1.5-fold at 95% confidence level. The 3TSR-treatment group clearly shows the least transcriptional perturbation among the TSR treatment groups. (B) A heatmap of the top 12 DE genes in the comparison that uses WT-water as the control. <i>Capsl</i>, <i>Cbr3</i>, <i>Abhd3</i> and <i>0610011F06Rik</i> are down-regulated in the TSR treatment groups. (C) A heatmap of the top DE genes in the 3TSR-treated group. The number of up- and down-regulated genes in the TSR treatment groups (when compared to the WT-water control) are presented in (D); (E) shows the breakdown of the DE genes in comparison to the WT-DSS saline.</p

Top 12 differentially expressed genes (8 up- and 4 down-regulated) in the treatment groups compared to WT-water.

<p>Top 12 differentially expressed genes (8 up- and 4 down-regulated) in the treatment groups compared to WT-water.</p

Immunohistochemistry (IHC) with the Ly-6G/Ly-6C antibody for detection of granulocytes and monocytes in colitic sections.

<p>Counts of Ly-6G/Ly-6C positive cells in TSR2 and 3TSR were considerably lower than in saline (n = 5 in each group) and TSR2+RFK-treated (n = 9) colons (A). However, only TSR2 showed lower counts that were statistically significant, p = 0.047 (B). IHC against Mac/CD68 (C) showed fewer CD68 positive cells (macrophages) in TSR2+RFK (n = 24) compared to TSR2 (n = 14) and saline (n = 18). These differences were statistically significant, p = 0.0006, when compared with 3TSR-treated (n = 18) colons (D). 400× magnification.</p

Top 14 differentially-expressed genes in the 3TSR group.

*<p>The gene ontology is given, enclosed in brackets, where pathway has not been annotated in the microarray datasheet.</p

IHC of MMP3, IL-6 and MRP8.

<p>Protein expression of MMP3 was localized in colonocytes, leukocytes and endothelial cells. Staining was particularly intense in the submucosal stroma of colitic areas. IL-6 was detected in the leukocytic infiltrate and endothelial cells. Calgranulin or MRP8 also showed selective staining in the leukocytic infiltrate.</p