In vivo action of trefoil factor 2 (TFF2) to speed gastric repair is independent of cyclooxygenase

Abstract

Objective: Trefoil factor (TFF) peptides are expressed in gastric tissues, where they are part of the epithelial defences. To complement previous in vitro work, the goal of the present study was to examine directly if TFF2 was essential for gastric restitution in vivo during the recovery from microscopic damage.

Design: TFF2 mutant (KO) mice were examined to study the epithelial repair process in vivo after laser-induced photodamage (LPD). Using two-photon laser energy absorption (710 nm), LPD was imposed on an approximately 3-5 cell region of surface epithelium in anaesthetised mouse stomach. Responses to damage were evaluated during confocal time-lapse microscopy; including area of damage and the extracellular pH adjacent to the damaged surface (Cl-NERF pH sensor).

Results: In control (TFF2+/+ and TFF2+/-) mice, damaged cells were exfoliated and the damaged epithelium was repaired by indomethacin. The resting surface pH was similar between control and TFF2-KO animals, but the post-LPD alkalisation of surface pH observed in control mice (pH 0.3 + or - 0.05, n=21) was attenuated in the TFF2-KO stomach (pH -0.08 + or - 0.09, n=18). Recobinant rat TFF3 partially rescued the attenuated surface pH change in TFF2-KO stomach, in the presence or absence of indomethacin.

Conclusions: In the gastric epithelium in vivo, TFFs promote epithelial restitution via a mechanism that does not require cyclooxygenase activation. A novel role for TFFs to affect gastric surface pH is observed.

Figure 1

Data analysis procedures during two-photon laser-induced photodamage (LPD) of the mouse gastric epithelial surface. (A) Analysis of the damaged area. Left: sample image of NAD(P)H autofluorescence collected from the mouse gastric epithelial surface ~5 min post-LPD. Autofluorescence is reduced markedly after photodamage in the region bounded by the dotted line in the insert image, and the included area is used to estimate damage size. Right: the graph shows normalised representative data points measuring the damaged area over time in a control mouse. Time of maximal damage (T_max) and area of maximal damage (A_max) are estimated from individual data points. The rate constant of area recovery (κ, s⁻¹) is calculated by curve fitting the data between A_max and the following 10 min (black dots in trace) to a single exponential decay equation. Dotted lines in the graph show the 95% CI on the curve fit. (B) Analysis of the extracellular pH at the gastric surface. Left: Cl-NERF added to the perfusate allows sequential collection of confocal images of juxtamucosal fluorescence at 514 and 458 nm excitation. White circles show areas used for measuring the surface pH from 514/458 ratio images. Right: the graph shows representative surface pH measurements in control mouse stomach before and after LPD (arrow). Scale bar=50 μm.

Figure 2

Time course of changes in damaged area after laser-induced photodamage (LPD) in (A) control mice, (B) trefoil factor 2 knockout (TFF2-KO) mice and (C) TFF2-KO mice after topical addition of recombinant rat TFF3 (rTFF3), as described in the Methods section. In each panel, eight images from a representative time course experiment are shown on the left. In those images, the white rectangle in the first image of the series shows the gastric surface region exposed to LPD (70×4 μm²) at time zero, and all other images indicate the time of imaging the same tissue location after LPD. On the right of each panel, results are compiled for the indicated number of experiments (each collected from at least three animals) with values presented as mean±SEM.

Figure 3

Damage recovery parameters from control mice, trefoil factor 2 knockout (TFF2-KO) mice and TFF2-KO mice after topical addition of recombinant rat TFF3 (+rTFF3). As described in the Methods section and figure 1A, parameters were extracted from individual experiments performed as in figure 2 and then results were compiled. The parameters were (A) maximum damaged area (A_max), (B) the time to attain the maximal damaged area after laser-induced photodamage (LPD) at time zero (T_max), (C) the restitution rate constant (κ) and (D) the time required to repair half of the maximal damage (T_1/2). Results are presented as the mean±SEM of n=4–10 experiments (each condition performed in at least three animals). *p<0.05, **p<0.001 in unpaired t test versus values from the TFF2-KO animals. NS, no significance.

Figure 4

Gastric epithelial surface pH values from control mice, trefoil factor 2 knockout (TFF2-KO) mice and TFF2-KO mice + recombinant rat TFF3 (rTFF3). As described in the Methods section and figure 1B, extracellular pH was measured adjacent to the gastric surface (pHex) using confocal microscopy of Cl-NERF fluorescence. (A) Resting surface pH collected prior to laser-induced photodamage (LPD). Every pH value is the average value of 6–8 measurements from an individual animal. No significant difference of resting pH among conditions (p=0.87, one-way analysis of variance; SDs were not significantly different). (B) Representative time course of pHex following LPD in control mice (open circles), TFF2-KO mice (filled circles) and TFF2-KO mice after topical application of rTFF3 (open squares). The initial pHex values before LPD were 3.44, 3.52 and 3.61 in control, TFF2-KO and TFF2-KO+rTFF3, respectively. (C) The maximal surface pH change after LPD (ΔpH) was calculated relative to the pre-LPD surface pH in individual experiments, and then values were compiled. The results are presented as the mean±SEM for n experiments. *p<0.05, **p<0.001 in unpaired t test versus values from the TFF2-KO animals.

Figure 5

Role of cyclooxygenase (COX) in gastric restitution mediated by trefoil factor 2 knockout (TFF2-KO) mice. (A) Representative experiments examining the damaged area in response to laser-induced photodamage (LPD) imposed on TFF2-KO mice after 1 h indomethacin pretreatment (5 mg/kg subcutaneously), in either the absence (filled triangles) or presence (open squares) of recombinant rat TFF3 (rTFF3). (B) Values of the restitution rate compiled from experiments performed as in A (n=3, indo.; n=5, indo+rTFF3). In an unpaired t test, values in the presence of indomethacin were not different compared with rates in the absence of indomethacin in figure 3C. In the presence of indomethacin, rTFF3 significantly accelerated the restitution rate (p<0.05). (C) Prostaglandin E₂ (PGE₂) levels (measured by enzyme immunoassay; see the Methods section) in TFF2-KO animals in the absence or presence of rTFF3 (D) Upper panel: western blot analysis qualitatively reports COX-1 expression in wild type (+/+) and TFF2-KO mice gastric mucosa, using glyceraldehyde phosphate dehydrogenase (GAPDH) as loading control in the same blots. Lower panel: densitometry of COX-1 blots, normalised to GAPDH and then standardised to the response in wild-type (WT) animals, shows no significant difference in COX-1 protein between phenotypes (n=4).