Influence of defunctionalization and mechanical forces on intestinal epithelial wound healing

Abstract

The influence on mucosal healing of luminal nutrient flow and the forces it creates are poorly understood. We hypothesized that altered deformation and extracellular pressure mediate, in part, the effects of defunctionalization on mucosal healing. We created patent or partially obstructing defunctionalizing jejunal Roux-en-Y anastomoses in rats to investigate mucosal healing in the absence or presence of luminal nutrient flow and measured luminal pressures to document partial obstruction. We used serosal acetic acid to induce ulcers in the proximal, distal, and defunctionalized intestinal segments. After 3 days, we assessed ulcer area, proliferation, and phosphorylated ERK. In vitro, we measured proliferation and migration in Caco-2 and IEC-6 intestinal epithelial cells subjected to cyclic strain, increased extracellular pressure, or strain and pressure together. Defunctionalization of intestine without obstruction reduced phosphorylated ERK, slowed ulcer healing, and inhibited mucosal proliferation. This outcome was blocked by PD-98059. Partial obstruction delayed ulcer healing but stimulated proliferation independently of ERK. In vitro, strain increased Caco-2 and IEC-6 proliferation and reduced migration across collagen but reduced proliferation and increased migration across fibronectin. In contrast, increased pressure and the combination of pressure and strain increased proliferation and reduced migration independently of substrate. PD-98059 reduced basal migration but increased migration under pressure. These results suggest that loss of the repetitive distension may decrease mucosal healing in defunctionalized bowel, while increased luminal pressure above anastomoses or in spastic bowel disease could further inhibit mucosal healing, despite peristaltic repetitive strain. ERK may mediate the effects of repetitive deformation but not the effects of pressure.

Fig. 1.

Impaired ulcer healing in defunctionalized rat intestinal limb. A: contraction amplitude in intact intestine of control animals and proximal, defunctionalized (Roux), and distal limbs of the anastomosis group (n = 4). *P < 0.05. B: representative images of 3-day ulcers in corresponding areas of control animals and animals subjected to Roux-en-Y anastomosis. Ulcer size was significantly greater in the Roux limb. C: proliferation in intestinal mucosa, assessed as percentage of proliferating cell nuclear antigen (PCNA)-positive cells, was lowest in the Roux limb (n = 5). D: healing was also impaired in the Roux limb in ulcers induced 30 days after Roux-en-Y anastomosis (n = 12). Bars without a common letter are significantly different at P < 0.05.

Fig. 2.

Partial obstruction of the Roux-en-Y anastomosis increases intraluminal pressures and impairs upstream ulcer healing, despite increased proliferation. A: intestinal diameter of proximal and Roux limbs was increased in partially obstructed anastomosis 3 days after surgery (n = 6). B: incidence of pressures exceeding 99% of the pressure of normal nonoperated animals was higher in proximal and Roux limbs of partially obstructed rats (n = 4). *P < 0.05. C: representative images of ulcers (top) and histogram showing ulcer size (bottom) in proximal, Roux, and distal limbs of partially obstructed animals. Ulcer size was greatest in the proximal limb experiencing the highest incidence of increased pressure (n = 9). D: proliferation was elevated in proximal and Roux limbs compared with distal limb in partially obstructed rats (n = 4). Bars without a common letter are significantly different at P < 0.05.

Fig. 3.

Effects of pressure, strain, and the combination of pressure and strain on migration of Caco-2 (A and C) and IEC-6 (B, D, and E) cells grown on a collagen I (A, B, and E) or tissue fibronectin (C and D) matrix. A and B: pressure, strain, or the combination of pressure and strain inhibits migration of Caco-2 (A) and IEC-6 (B) cells grown on collagen I (n = 15). C and D: strain enhances and pressure decreases migration of Caco-2 (C) and IEC-6 (D) cells grown on tissue fibronectin. Pressure effect predominates when cells are exposed to the combination of pressure and strain (n = 15). E: pretreatment with the MEK inhibitor PD-98059 lowers basal migration of IEC-6 cells grown on a collagen I matrix but has no effect in cells exposed to increased pressure for 12 h (n = 15). Bars without a common letter are significantly different at P < 0.05. F: migration of Caco-2 and IEC-6 cells on collagen I after exposure to constant strain for 24 and 12 h, respectively (n = 6). Constant strain decreases migration in each cell type. *P < 0.05 (by Student's t-test).

Fig. 4.

Proliferative effects of pressure, strain, and the combination of pressure and strain on Caco-2 (A and C) and IEC-6 (B and D) cells grown on collagen I or tissue fibronectin matrix. A and B: pressure, strain, and the combination of pressure and strain significantly enhance proliferation of Caco-2 (A) and IEC-6 (B) cells grown on collagen I (n = 12). C and D: pressure decreases and strain increases proliferation of Caco-2 (C) and IEC-6 (D) cells grown on tissue fibronectin. Proliferation is significantly higher in both cell types after exposure to pressure + strain (n = 12). *P < 0.05.

Fig. 5.

ERK inhibition improves ulcer healing in the defunctionalized (Roux) limb but has no effect on ulcer healing in partially obstructed animals. A: representative Western blots (top) and densitometric analysis (bottom) of phosphorylated ERK (pERK), total ERK (tERK), and β-actin in proximal (Prox), Roux, and distal limbs of the anastomosis after treatment with vehicle (0.1% DMSO) or the MEK inhibitor PD-98059 (26.75 μg·kg⁻¹·day⁻¹) (n = 6–7). pERK was significantly decreased in the Roux limb of vehicle-treated animals; treatment was effective, as PD-98059 lowered pERK in all limbs. B: PD-98059 significantly improved ulcer healing in the Roux limb of nonobstructed animals (n = 10). C: ERK inhibition by PD-98059 had no effect on ulcer healing in partially obstructed animals (n = 10). Bars without a common letter are significantly different at P < 0.05.

Fig. 6.

Reduced proliferation in the defunctionalized Roux limb can be reversed by ERK inhibition or increased pressure as a result of partial obstruction. A: in nonobstructed animals, PD-98059 normalized proliferation rate in the defunctionalized Roux limb. B: PD-98059 had no further effect on the already high proliferation rate in proximal and Roux limbs of partially obstructed animals. Bars without a common letter are significantly different at P < 0.05.