Influence of defunctionalization and mechanical forces on intestinal epithelial wound healing.

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.

ERK regulates strain-induced migration and proliferation from different subcellular locations.

Repetitive deformation like that engendered by peristalsis or villous motility stimulates intestinal epithelial proliferation on collagenous substrates and motility across fibronectin, each requiring ERK. We hypothesized that ERK acts differently at different intracellular sites. We stably transfected Caco-2 cells with ERK decoy expression vectors that permit ERK activation but interfere with its downstream signaling. Targeting sequences constrained the decoy inside or outside the nucleus. We assayed proliferation by cell counting and migration by circular wound closure with or without 10% repetitive deformation at 10 cycles/min. Confocal microscopy confirmed localization of the fusion proteins. Inhibition of phosphorylation of cytoplasmic RSK or nuclear Elk confirmed functionality. Both the nuclear-localized and cytosolic-localized ERK decoys prevented deformation-induced proliferation on collagen. Deformation-induced migration on fibronectin was prevented by constraining the decoy in the nucleus but not in the cytosol. Like the nuclear-localized ERK decoy, a Sef-overexpressing adenovirus that sequesters ERK in the cytoplasm also blocked the motogenic and mitogenic effects of strain. Inhibiting RSK or reducing Elk ablated both the mitogenic and motogenic effects of strain. RSK isoform reduction revealed isoform specificity. These results suggest that ERK must translocate to the nucleus to stimulate cell motility while ERK must act in both the cytosol and the nucleus to stimulate proliferation in response to strain. Selectively targeting ERK within different subcellular compartments may modulate or replace physical force effects on the intestinal mucosa to maintain the intestinal mucosal barrier in settings when peristalsis or villous motility are altered and fibronectin is deposited into injured tissue.

The effects of increased extracellular deformation, pressure, and integrin phosphorylation on fibroblast migration.

Wound healing requires fibroblast migration. Increased pressure slows migration and ulcer healing. Pressure also induces beta1 integrin phosphorylation. We hypothesized that beta1 phosphorylation influences cell adhesion and migration. We compared the effects of increased pressure on the adhesion and motility of GD25 beta1-integrin null fibroblasts transfected with wild-type beta1A-integrin, S785A or TT788/9AA (phosphorylation-deficient), or T788D (constitutively phosphomimetic) mutants. GD25 beta1 null cells adhered less than wild type beta1A cells, suggesting adherence by non-integrin mechanisms. Preventing Ser-785 or Thr 788/789 phosphorylation reduced adhesion, suggesting that phosphorylation regulates adhesiveness. Substituting Asp for Thr788 stimulated adhesion on both substrates. Pressure decreased migration in all lines and on all matrixes, the most in wild type beta1A integrin cells and only slightly in beta1A TT788/9AA cells. In comparison, another physical force, repetitive deformation, increased migration in the beta1A integrin T788D, S785A, and wild type cells on fibronectin, and decreased migration on collagen. Deformation did not affect the migration of GD25 beta1-integrin null or TT788/9AA cells. Extracellular signal-regulated kinase (ERK) blockade neither altered basal migration nor prevented pressure inhibition, while the cellular deformation response on fibronectin was altered. beta1-Integrin phosphorylation regulates cellular adhesion and the deformation effects on motility. The pressure-induced motility response is independently regulated.

Delineating the signals by which repetitive deformation stimulates intestinal epithelial migration across fibronectin.

Repetitive strain stimulates intestinal epithelial migration across fibronectin via focal adhesion kinase (FAK), Src, and extracellular signal-related kinase (ERK) although how these signals act and interact remains unclear. We hypothesized that PI3K is central to this pathway. We subjected Caco-2 and intestinal epithelial cell-6 cells to 10 cycles/min deformation on flexible fibronectin-coated membranes, assayed migration by wound closure, and signaling by immunoblots. Strain stimulated PI3K, AKT, glycogen synthase kinase (GSK), and p38 phosphorylation. Blocking each kinase prevented strain stimulation of migration. Blocking PI3K prevented strain-stimulated ERK and p38 phosphorylation. Blocking AKT did not. Downstream, blocking PI3K, AKT, or ERK inhibited strain-induced GSK-Ser9 phosphorylation. Upstream of AKT, reducing FAK or Rac1 by siRNA blocked strain-stimulated AKT phosphorylation, but inhibiting Src by PP2 or siRNA did not. Transfection with FAK point mutants at Tyr397, Tyr576/577, or Tyr925 demonstrated that only FAK925 phosphorylation is required for strain-stimulated AKT phosphorylation. Myosin light chain activation by strain required FAK, Rac1, PI3K, AKT, GSK, and ERK but not Src or p38. Finally, blebbistatin, a nonmuscle myosin II inhibitor, blocked the motogenic effect of strain downstream of myosin light chain. Thus strain stimulates intestinal epithelial migration across fibronectin by a complex pathway including Src, FAK, Rac1, PI3K, AKT, GSK, ERK, p38, myosin light chain, and myosin II.

Supraphysiologic extracellular pressure inhibits intestinal epithelial wound healing independently of luminal nutrient flow.

BACKGROUND: Luminal pressure may injure the gut mucosa in obstruction, ileus, or inflammatory bowel disease. METHODS: We formed Roux-en-Y anastomoses in 19 mice, creating proximal and defunctionalized partially obstructed limbs and a distal limb to vary luminal pressure and flow. We induced mucosal ulcers by serosal acetic acid, and assessed proliferation (proliferating cell nuclear antigen) and ERK (immunoblotting). Parallel studies compared Caco-2 enterocyte migration and proliferation after pressure and/or ERK blockade. RESULTS: At 3 days, anastomoses were probe-patent, proximal and distal limbs contained chyme, and defunctionalized limbs were empty. The proximal and defunctionalized limbs showed increased pressure and slower healing despite increased proliferation, ERK protein, and ERK activation. In vitro, pressure decreased Caco-2 migration across collagen or fibronectin, stimulated proliferation, and activated ERK. However, ERK blockade did not prevent pressure effects. CONCLUSIONS: Luminal pressure during obstruction or ileus may impair mucosal healing independently of luminal flow despite increased mitosis and ERK activation.

Complications are increased with the need for an abdominal-assisted Kraske procedure.

The Kraske procedure offers a sphincter-saving alternative for surgical correction of rectal disease. This study was performed to investigate the complication rate with the traditional (transsacral) Kraske procedure versus an abdominal-assisted Kraske approach (laparoscopic or open). We conducted a retrospective review of all patients undergoing the Kraske procedure at Harper University Hospital over a 10-year period. A total of 54 patients were identified. Indications for surgery included rectal carcinoma (43), large villous adenomas (6), and other (5). Average post-operative follow-up was 40 +/- 25 months (mean +/- SD). Complications included rectocutaneous fistulae (9), perineal infections (13), and incontinence (8). In patients requiring an abdominal-assisted approach for colorectal mobilization, the fistula rate was significantly higher (33% vs 3%; P = 0.007), as were the rates of perineal infections (33% vs 17%) and of initial incontinence (25% vs 7%). The laparoscopic-assisted approach significantly reduced the operating time (272 +/- 72 minutes) compared to the open-assisted approach (498 +/- 138 minutes) (P < 0.001). The traditional Kraske procedure can be utilized in a safe, effective manner for treatment of rectal disease. Knowledge of the increased rate of complications with the abdominal-assisted Kraske approach can guide the patient and physician considering sphincter salvage.