Fig. 2.

Increased IGF-IEc and E peptide (MGF) in smooth muscle cells of stricturing Crohn's disease. A: transcript levels of IGF-IEc were low in normal non-Crohn's disease intestinal muscle. IGF-IEc transcripts specifically increase in muscle cells of strictures in Montreal B2 Crohn's disease even compared with the normal resection margin of the same patient. The opposite pattern was seen in muscle cells of Montreal B1 and B3 phenotype Crohn's disease in which IGF-IEc transcripts are decreased in the affected segments. IGF-IEc transcripts were measured by quantitative RT-PCR (qRT-PCR) with the 2^−ΔΔCt method. B: E peptide (MGF) protein levels increased in muscle cells of strictures in Crohn's disease compared with normal margin in the same patient. E peptide levels were measured by ELISA. C: representative immunofluorescent images of immunoreactive E peptide. Greater levels of immunoreactive E peptide were seen in the muscle cells of the muscularis propria of strictured intestine compared with normal resection margin in the same patient. D: exogenous TGF-β₁ (10 ng/ml) elicited time-dependent increase in IGF-IEc transcripts in primary cultures of normal muscle cells. E: the increase in IGF-IEc expression after 8 h incubation with 10 ng/ml TGF-β₁ in primary cultures of normal resection margin muscle cells is abolished by the Smad3 inhibitor SIS3 (10 μM). Immunoneutralization of endogenous TGF-β₁ (1 μg/ml TGF-β₁ antibody) in primary cultures of muscle cells from strictured intestine decreased basal IGF-IEc transcript levels compared with incubation with isotype control antibody. F: transcript levels of the smooth muscle-specific genes: SMTH (smoothelin), DES (desmin), ACTA (α-smooth muscle actin), and ACTG2 (γ-enteric muscle actin) were increased in strictured intestinal muscle compared with normal intestine in the same patient or to non-Crohn's patient intestine. Transcript levels were measured by quantitative RT-PCR using GAPDH as control. Results are means ± SE of 4–6 paired samples. *P < 0.05 vs. normal resection margin in the same patient or untreated cells. **P < 0.01 vs. control. ***P < 0.001 vs. control. CD, Crohn's disease; α-SMA, α-smooth muscle actin; H&E, hematoxylin and eosin; NA, neutralizing antibody.