Volume 117, Issue 2, Pages 368-377 (August 1999) Modulation of intestinal epithelial wound healing in vitro and in vivo by lysophosphatidic acid  Andreas Sturm, Thomas Sudermann, Klaus–Martin Schulte, Harald Goebell, Axel U. Dignass  Gastroenterology  Volume 117, Issue 2, Pages 368-377 (August 1999) DOI: 10.1053/gast.1999.0029900368

1 Fig. 1. Intestinal epithelial cell migration (restitution) in an in vitro model of epithelial restitution. Standard wounds were made with razor blades in confluent monolayers of IEC-6 cells as described in Materials and Methods. Wounded monolayers were cultured for 24 hours in (A) control medium or (B) medium supplemented with 50 μmol/L LPA. Scratch indicates the original margin of the wound (original magnification 200×). Gastroenterology 1999 117, 368-377DOI: (10.1053/gast.1999.0029900368)

1 Fig. 1. Intestinal epithelial cell migration (restitution) in an in vitro model of epithelial restitution. Standard wounds were made with razor blades in confluent monolayers of IEC-6 cells as described in Materials and Methods. Wounded monolayers were cultured for 24 hours in (A) control medium or (B) medium supplemented with 50 μmol/L LPA. Scratch indicates the original margin of the wound (original magnification 200×). Gastroenterology 1999 117, 368-377DOI: (10.1053/gast.1999.0029900368)

2 Fig. 2. Effect of LPA on intestinal epithelial cell restitution in vitro. Standard wounds were made with razor blades in confluent IEC-6 cell monolayers as described in Materials and Methods. Wounded monolayers were cultured for 24 hours in control medium or medium supplemented with 1-100 μmol/L LPA. Migration of IEC-6 cells was quantitated as described in Materials and Methods. Data represent means ± SD of at least 3 different experiments. +P < 0.05 vs. 0 μmol/L LPA (control); *P < 0.01 vs. 0 μmol/L LPA (control). Gastroenterology 1999 117, 368-377DOI: (10.1053/gast.1999.0029900368)

lished Fig. 3. Effects of bradykinin (BK; 1 μmol/L), phorbol ester (PE; 1 μmol/L), pertussis toxin (PTX; 50 ng/mL), suramin (SU; 0.5 mg/L), or anti–TGF-β (a-TGFβ; 30 μg/mL) on LPA-induced enhancement of intestinal epithelial cell migration in vitro. Standard wounds were established in confluent monolayers of IEC-6 cells as detailed in Materials and Methods, and wounded monolayers were cultured for 24 hours after addition of culture medium (DMEM with 5% FCS) with or without test substances. Cell migration (restitution) was quantitated as described in Materials and Methods. Data represent means ± SD of at least 3 different experiments. +P < 0.05 vs. 0 μmol/L LPA (control); *P < 0.01 vs. 0 μmol/L LPA (control). The phospholipid derivative phosphatidic acid (PA; 50 μmol/L), which was evaluated for control purposes, did not significantly modulate epithelial restitution. Gastroenterology 1999 117, 368-377DOI: (10.1053/gast.1999.0029900368)

4 Fig. 4. Effect of LPA on TGF-β peptide levels in supernatants of IEC-6 monolayers treated with LPA or control medium alone. TGF-β peptide levels were measured in the supernatants of IEC-6 monolayers treated with LPA or control medium alone as described in Materials and Methods. Supernatants were obtained after a 24-hour incubation period from both migration and proliferation studies. Data represent means ± SD of latent (■) and bioactive (2) TGF-β peptide levels obtained from 3 different experiments. Gastroenterology 1999 117, 368-377DOI: (10.1053/gast.1999.0029900368)

5 Fig. 5. Effect of LPA on the proliferation of intestinal cell epithelial cells (IEC-6) in vitro. IEC-6 cells were cultured in the presence of 0-100 μmol/L LPA, and cell proliferation was assessed by colorimetric MTT assays as described in Materials and Methods. ■, 5% FCS; 2, 0.1% FCS. Data represent means ± SD of absorbance values obtained for a typical experiment performed with quadruplicate assays. Comparable data were obtained in four independently performed experiments. *P < 0.01 vs. 0 μmol/L LPA (control). Gastroenterology 1999 117, 368-377DOI: (10.1053/gast.1999.0029900368)

6 Fig. 6. Effects of bradykinin (BK), phorbol ester (PE), pertussis toxin (PTX), suramin (SU), or anti–TGF-β (a-TGFβ) on LPA-induced inhibition of intestinal epithelial cell proliferation in vitro. IEC-6 cells (n = 2 × 104) were seeded into 24-well plates in DMEM containing 5% FCS. Simultaneously, LPA (50 μmol/L), bradykinin (1 μmol/L), phorbol ester (1 μmol/L), pertussis toxin (50 ng/mL), suramin (0.5 mg/mL), or a panspecific TGF-β antibody (30 μg/mL) were added to individual wells. After an incubation period of 72 hours, cell proliferation was assessed by colorimetric MTT assays as described in Materials and Methods. Data represent means ± SD of absorbance values obtained for a typical experiment performed with quadruplicate assays. Comparable data were obtained in at least 3 independently performed experiments. +P < 0.01 vs. 0 μmol/L LPA (control); *P < 0.01 vs. 50 μmol/L LPA. The phospholipid derivative phosphatidic acid (PA; 50 μmol/L), which was evaluated for control purposes, did not significantly modulate epithelial proliferation. Gastroenterology 1999 117, 368-377DOI: (10.1053/gast.1999.0029900368)

7 Fig. 7. Effects of topical LPA treatment on weight loss and macroscopic mucosal damage in experimentally induced colitis in rats. Colitis was induced by intracolonic administration of 2,4,6-trinitrobenzene sulfonic acid as described in Materials and Methods. Rats were subsequently treated with rectal application of PBS (■; 18 animals) or LPA (▩; 18 animals), and weight loss and mucosal damage were assessed after 3 days of treatment as described in Materials and Methods. Data represent means ± SD. *P < 0.01 vs. control; **P < 0.005 vs. control. Gastroenterology 1999 117, 368-377DOI: (10.1053/gast.1999.0029900368)

8 Fig. 8. Effects of topical LPA treatment on mucosal damage in experimentally induced colitis in rats. Colitis was induced by intracolonic administration of 2,4,6-trinitrobenzene sulfonic acid as described in Materials and Methods. Rats were subsequently treated with rectal application of PBS or LPA. After 3 days of treatment, rats were killed and tissue samples were obtained from the distal 10 cm of the colon and prepared for histological evaluation as described in Materials and Methods. (A, C, and E) Sections of distal colon treated with PBS as control (H&E). (B, D, and F) Sections of distal colon treated with LPA (H&E; original magnifications: A, B, 80×; C, D, 120×; E, F, 200×). Gastroenterology 1999 117, 368-377DOI: (10.1053/gast.1999.0029900368)

8 Fig. 8. Effects of topical LPA treatment on mucosal damage in experimentally induced colitis in rats. Colitis was induced by intracolonic administration of 2,4,6-trinitrobenzene sulfonic acid as described in Materials and Methods. Rats were subsequently treated with rectal application of PBS or LPA. After 3 days of treatment, rats were killed and tissue samples were obtained from the distal 10 cm of the colon and prepared for histological evaluation as described in Materials and Methods. (A, C, and E) Sections of distal colon treated with PBS as control (H&E). (B, D, and F) Sections of distal colon treated with LPA (H&E; original magnifications: A, B, 80×; C, D, 120×; E, F, 200×). Gastroenterology 1999 117, 368-377DOI: (10.1053/gast.1999.0029900368)

8 Fig. 8. Effects of topical LPA treatment on mucosal damage in experimentally induced colitis in rats. Colitis was induced by intracolonic administration of 2,4,6-trinitrobenzene sulfonic acid as described in Materials and Methods. Rats were subsequently treated with rectal application of PBS or LPA. After 3 days of treatment, rats were killed and tissue samples were obtained from the distal 10 cm of the colon and prepared for histological evaluation as described in Materials and Methods. (A, C, and E) Sections of distal colon treated with PBS as control (H&E). (B, D, and F) Sections of distal colon treated with LPA (H&E; original magnifications: A, B, 80×; C, D, 120×; E, F, 200×). Gastroenterology 1999 117, 368-377DOI: (10.1053/gast.1999.0029900368)

8 Fig. 8. Effects of topical LPA treatment on mucosal damage in experimentally induced colitis in rats. Colitis was induced by intracolonic administration of 2,4,6-trinitrobenzene sulfonic acid as described in Materials and Methods. Rats were subsequently treated with rectal application of PBS or LPA. After 3 days of treatment, rats were killed and tissue samples were obtained from the distal 10 cm of the colon and prepared for histological evaluation as described in Materials and Methods. (A, C, and E) Sections of distal colon treated with PBS as control (H&E). (B, D, and F) Sections of distal colon treated with LPA (H&E; original magnifications: A, B, 80×; C, D, 120×; E, F, 200×). Gastroenterology 1999 117, 368-377DOI: (10.1053/gast.1999.0029900368)

8 Fig. 8. Effects of topical LPA treatment on mucosal damage in experimentally induced colitis in rats. Colitis was induced by intracolonic administration of 2,4,6-trinitrobenzene sulfonic acid as described in Materials and Methods. Rats were subsequently treated with rectal application of PBS or LPA. After 3 days of treatment, rats were killed and tissue samples were obtained from the distal 10 cm of the colon and prepared for histological evaluation as described in Materials and Methods. (A, C, and E) Sections of distal colon treated with PBS as control (H&E). (B, D, and F) Sections of distal colon treated with LPA (H&E; original magnifications: A, B, 80×; C, D, 120×; E, F, 200×). Gastroenterology 1999 117, 368-377DOI: (10.1053/gast.1999.0029900368)

8 Fig. 8. Effects of topical LPA treatment on mucosal damage in experimentally induced colitis in rats. Colitis was induced by intracolonic administration of 2,4,6-trinitrobenzene sulfonic acid as described in Materials and Methods. Rats were subsequently treated with rectal application of PBS or LPA. After 3 days of treatment, rats were killed and tissue samples were obtained from the distal 10 cm of the colon and prepared for histological evaluation as described in Materials and Methods. (A, C, and E) Sections of distal colon treated with PBS as control (H&E). (B, D, and F) Sections of distal colon treated with LPA (H&E; original magnifications: A, B, 80×; C, D, 120×; E, F, 200×). Gastroenterology 1999 117, 368-377DOI: (10.1053/gast.1999.0029900368)