F. R. Demehri1, Y. Feng1, R. Sueyoshi1, H. K. Yoon1, J. Guan2, D. H. Teitelbaum1 1University Of Michigan,Surgery,Ann Arbor, MI, USA 2University Of Michigan,Internal Medicine,Ann Arbor, MI, USA
Introduction: Distraction-mediated enterogenesis (DME) is a novel therapeutic strategy for short bowel syndrome (SBS), wherein distractive force leads to substantial lengthening of functional bowel. The mechanism(s) by which this occurs is incompletely understood. Using a mouse model of DME, we recently identified increased expression of phosphorylated focal adhesion kinase (p-FAK) – a mediator of mechanotransduction in other tissues. We hypothesized that DME is dependent upon a FAK phosphorylation pathway.
Methods: C57BL/6 mice underwent creation of an isolated 3-cm jejunal segment with instillation of 300 µl of high molecular weight polyethylene glycol (PEG, 3350 kD) to induce osmotic stretch (and growth) of the segment. PF-562,271, a selective FAK inhibitor, was administered via oral gavage. Control mice received vehicle alone. A second group of tamoxifen-induced intestine-specific FAK knockout(KO) mice (Villin-Cre-ER-FAKf/f) underwent the same procedure, and were compared to non-induced and wild-type controls. After 5 days, gross morphology of the distracted segment was evaluated, volume measured via CT-imaging, and intestinal epithelial cell (IEC) proliferation measured via PCNA immunostaining. Results (mean±SEM) were analyzed for significance by t-test.
Results: Chemical inhibition of FAK significantly prevented DME, resulting in a decrease in length of 23.1±9.2%, vs an increase in length of 11.7±1.4% in control DME mice (p<0.05; Fig A, C). Volume gain was limited to 37.5±2.4% with FAK-inhibition, vs 154.0±8.3% in control mice (p<0.01). FAK inhibition with PF-562,271 also prevented the increase in IEC proliferation seen with DME (26.9±2.2% vs 52.9±3.3% PCNA positive cells; p<0.0001; Fig B, D). The proliferative index with FAK inhibition was similar to that of non-distracted controls (30.3±1.2% PCNA positive; p=0.34). Inhibition of FAK phosphorylation was confirmed via Western blot, with a blunted increase in p-FAK expression in treated mice vs controls. We next examined enterogenesis in tamoxifen-induced FAK-KO mice. A similar prevention in DME was noted, with a decrease in length of 9.0±7.1%, versus an increase in length of 19.5±3.5% in controls (p<0.05). Interestingly, FAK-KO led to a reduction in IEC proliferation during distraction (14.5±1.3% PCNA positive) relative to non-distracted FAK-KO (25.2±1.9% PCNA positive; p<0.001) and FAK-competent bowel (24.2±1.5% PCNA positive; p<0.001).
Conclusion: FAK activation is required for DME, with p-FAK inhibition restricting overall volumetric growth and IEC proliferation. FAK may represent a potential target to enhance DME as a novel treatment for SBS.
