Inhibition of mTOR mediates alleviation of intestinal fibrosis via suppression of myofibroblast proliferation *Archana V. Patel, MD1; *Jun Yang MD, PhD1, 2; James Cao, MD, PhD1; David Jones, MD3; David Conti, MD4 ; Catherine Bartholomew, MD1; Xinjun Zhu, MD1, 2

1Department of Medicine Division of Gastroenterology, 2Center for Cardiovascular Sciences, 3Department of Pathology, 4Department of Surgery, Albany Medical College, Albany, NY

Abstract: Background: Intestinal fibrosis is irreversible which happens in response to prolonged injury or inflammation. The pathophysiology of intestinal fibrosis in inflammatory bowel disease (IBD) is not fully understood. Rapamcyin, an inhibitor of mTOR, has been reported to alleviate IBD, presumably by improving the inflammatory response by down-regulating the immune system. The mTOR signaling pathway was reported to be involved in transforming growth factor β1(TGFβ1)-dependent fibrogenic processes in several organ systems. Low dose oral administration of rapamycin reduces fibrogenesis, improves liver function, and prolongs survival in mice with liver cirrhosis. We hypothesized that rapamycin ameliorates intestinal fibrosis via inhibition of submucosal myofibroblast proliferation. Methods: Mice were divided into four groups including control, TNBS alone, rapamyicn alone, and both rapamycin and TNBS. Rapamycin (5mg/kg/day) was administrated intraperitoneally for 6 weeks and TNBS enema weekly for 6 weeks to induce intestinal fibrosis. Mouse colon samples were collected for examination of submucosal collagen deposition by masson's trichrome blue and myofibroblast proliferation by alpha-SMA or HiC-5 (focal adhesion protein hydrogen peroxide inducible clone 5). Isolated primary intestinal cells or smooth muscle cell line were treated with TGFβ1 either with or without rapamycin. Results: Colon samples harvested from TNBS-treated mice displayed severe stricture over the distal colon while only mild, patchy erythema and edema observed in the rectum from rapamycin treated TNBS group. While being completely distorted in vehicle treated TNBS group, mucosal crypt architecture was largely preserved in rapamycin treated TNBS group. Colon thickness is an indirect measurement of intestinal inflammation and fibrosis. TNBS treatment induced overall thickening of the colon tissue, especially in the submucosa and muscularis layers, which was markedly prevented by rapamycin. Furthermore, we observed the presence of hyperproliferation of myofibroblasts along with significant collagen deposition in the intestine from TNBS treated group. In contrast, intestine from rapamycin group exhibited very mild proliferation of myofibroblasts and deposition of collagen. Direct effect of rapamyicn on myofibroblast proliferation was also examined in primary intestinal cells and smooth muscle cell line. We observed that rapamycin markedly reduced the number of alpha-SMA and Hic-5 positive cells in primary intestinal cell culture treated with TGFβ1. Conclusion: Rapamycin ameliorates intestinal fibrosis in TNBS mouse model. Our data suggest that Rapamycin exerts anti-fibrotic effect in part via direct suppression of myofibroblast proliferation. These findings may have preventive or therapeutic applications in fibrotic Crohn’s disease.

Introduction: Fibrosis is the formation of excess fibrous connective tissue in an organ or tissue in a reparative or reactive process. Scarring is confluent fibrosis that obliterates the architecture of the underlying organ or tissue.

Myofibroblasts

Fibrogenesis

(+) aSMA (+) HiC-5

Collagen deposition

The Mammalian Target of Rapamycin (mTOR) Pathway

Hypothesis: Rapamycin, a bona fide inhibitor of intestinal fibrosis?

Methods: I. Small bowel specimens were obtained from retrospective chart review of newly diagnosed Crohn’s patients and pateints with renal transplant received

rapamycin at Albany Medical College. The specimens were then used in immunohistochemistry assay for monitoring myofibroblast proliferation in the submucosa.

II. Mice were divided into four groups including control, TNBS alone, rapamyicn alone, and both rapamycin and TNBS. Rapamycin (5mg/kg/day) was administrated intraperitoneally for 6 weeks and TNBS enema weekly for 6 weeks to induce intestinal fibrosis. Mouse colon samples were collected for examination of submucosal collagen deposition by masson's trichrome blue and myofibroblast proliferation by either aSMA or HiC-5.

III. Isolated primary intestinal cells or smooth muscle cell line was treated with TGFb1 for 72 hours either with or without rapamycin treatment.

Results: Fig 1. Increased myofibroblasts in the submucosa of small intestine from patients with newly diagnosed Crohn’s disease

Small bowel specimens from controls (left) and Crohn’s (right) were stained with anti-aSMA (in brown). Hyperproliferation of aSMA positive cells were present in the newly diagnosed Crohn’s small bowel.

Control Crohn’s

aSMA (+) cells

Submucosa Submucosa

Fig 2. Rapamycin (Rapa) reversed TNBS induced rectal wall thickening/ stiffness and colon length.

Contl Rapa TNBS TNBS+Rapa

Gross appearance of mouse large bowel from mice in control, rapamycin alone, TNBS and both Rapamycin and TNBS groups. Co-administration of rapamycin decreased rectal inflammation and wall thickening, as well as the reversed the shortened colon.

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Mice were divided into four groups including control, rapamyicn alone, TNBS alone, and both rapamycin and TNBS. Rapamycin (5mg/kg/day) was administrated intraperitoneally for 6 weeks and TNBS enema weekly for 6 weeks to induce intestinal fibrosis. Mouse colon samples were collected for examination of myofibroblast proliferation by aSMA (left) and submucosal collagen deposition by masson's trichrome blue (right). * represents submucosa

Fig 3. Rapamycin reversed TNBS induced hyperproliferation of myofibroblasts and fibrosis in the submucosal layer in micecolon.

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Fig 4. Rapamycin reduces TGFb1-induced proliferation of myofibroblasts from isolated intestinal cells and primary smooth muscle cells.

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Primary smooth muscle cells treated with TGFb1 as controls and additional rapamycin for 72 hours. DAPI stains nuclei (blue) and aSMA stains myofibroblast cells (green). *p<0.01

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Fig 5. Rapamycin reduces myofibroblasts in normal mice small intestine

Control

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Small bowel specimens from controls (left) and Rapamycin treated mice (right) were stained with anti-Hic5 (in brown). Decreased myofibroblasts in the mucosa of normal small intestine in mice treated with rapamycin.

Normal Treated with rapamycin

aSMA (+) cells

Fig 6. The effect of rapamycin on submucosal myofibroblasts in human small intestine.

Small bowel specimens from controls (left) and transplant patients received rapamycin (right) were stained with anti-aSMA (in brown). Decreased myofibroblasts in the submucosa of normal small intestine from transplant patients who were treated with rapamycin.

Fibrogenesis

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mTOR signaling

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Fig 7. Working model: Rapamycin reduces TGFb1induced proliferation of myofibroblasts in mucosa and submucosa, and collagen deposition in submucosa of intestine.

Conclusions: 1. Hyperproliferation of myofibroblasts occurs in active, but yet stricturing, early Crohn’s disease. 2. Rapamycin suppresses proliferation of myofibroblasts in small intestine from mice and humans. 3. Rapamycin ameliorates intestinal fibrosis inTNBS mouse model. 4. Our data suggest that rapamycin exerts anti-fibrotic effect in part via direct suppression of myofibroblast proliferation. 5. These findings may have preventive or therapeutic applications in fibrotic Crohn disease.

Clinical Relevance: 1. Once hyperproliferation of myofibroblasts is detected on small intestinal biopsy, it can be used as a tool to identify

patients at risk for developing fibrosing Crohn’s disease. 2. Rapamycin can be used as a novel therapy to prevent Crohn’s induced intestinal fibrosis.

This work was supported in whole by National Institutes of Health Grants NIDDK 08DK088950 and The GI IBD Research Fund by Michael Kerr. DISCLOSURE There are no disclosures to report.