Figure 5

The role of selective receptor tyrosine kinase activation in esophageal epithelial regeneration. (a) Immunoblot analyses of p-cMET, p-TrkA, p-EGFR, p-PDGFR and β-tubulin protein levels from neotissues treated with vehicle or inhibitors for 7 days of scaffold implantation. Vehicle and inhibitor comparisons were made on the same immunoblots and with equal amounts of protein loaded per lane (N = 3–4 animals per group). Targeted bands were cropped from original full length immunoblots displayed in Supplementary Fig. S2. (b–f) Representative photomicrographs of global and magnified esophageal cross-sections from nonsurgical controls (NSC) and untreated surgical controls as well as neotissues (NT) treated with vehicles or RTK inhibitors with or without pan-caspase inhibition for 7 days post-op. Samples were stained with Masson’s trichrome (top 2 rows in each panel) or subjected to immunohistochemical analysis for pan-cytokeratin (CK) expression (bottom row in each panel). Scale bars for top, middle, and bottom rows are respectively 1.25 mm, 500 μm, and 200 μm. (h) Immunohistochemical evaluations of Ki67 + pan-CK + epithelial cells in NSC and host-graft interface in specimens described in (b–f). Scale bars in (h) for all photomicrographs are 200 μm. For (b–h), respective pan-CK expression is displayed in red, Ki67 expression is displayed in green and blue represents DAPI nuclear counterstain. (g, i) Histomorphometric evaluations of pan-CK expression in controls and neotissues described in (b–f) as well as the percentage of Ki67 + pan-CK + epithelial cells at the host-graft interface in samples described in (h). N = 4–8 rats per group and 2–4 sections per animal were analyzed for each data point. For (g,i), multi-group (> 3 groups) comparisons were performed with the Kruskal Wallis test in combination with the post hoc Dunn’s test. Two group comparisons were performed with the Mann–Whitney U test. (*) = p < 0.05 in comparison to respective vehicle control. (#) = p < 0.05 in comparison to respective inhibitor group. (α) = p > 0.05 in comparison to respective vehicle cohort. (β) = p < 0.05 in comparison to NSC. Data are expressed as means ± standard deviations.