Transforming growth factor-beta 3 alters intestinal smooth muscle function: implications for gastroschisis-related intestinal dysfunction

Abstract

Background: Gastroschisis (GS) is a congenital abdominal wall defect that results in the development of GS-related intestinal dysfunction (GRID). Transforming growth factor-β, a pro-inflammatory cytokine, has been shown to cause organ dysfunction through alterations in vascular and airway smooth muscle. The purpose of this study was to evaluate the effects of TGF-β3 on intestinal smooth muscle function and contractile gene expression.

Methods: Archived human intestinal tissue was analyzed using immunohistochemistry and RT-PCR for TGF-β isoforms and markers of smooth muscle gene and micro-RNA contractile phenotype. Intestinal motility was measured in neonatal rats ± TGF-β3 (0.2 and 1 mg/kg). Human intestinal smooth muscle cells (hiSMCs) were incubated with fetal bovine serum ± 100 ng/ml of TGF-β 3 isoforms for 6, 24 and 72 h. The effects of TGF-β3 on motility, hiSMC contractility and hiSMC contractile phenotype gene and micro-RNA expression were measured using transit, collagen gel contraction assay and RT-PCR analysis. Data are expressed as mean ± SEM, ANOVA (n = 6-7/group).

Results: GS infants had increased immunostaining of TGF-β3 and elevated levels of micro-RNA 143 & 145 in the intestinal smooth muscle. Rats had significantly decreased intestinal transit when exposed to TGF-β3 in a dose-dependent manner compared with Sham animals. TGF-β3 significantly increased hiSMC gel contraction and contractile protein gene and micro-RNA expression.

Conclusion: TGF-β3 contributed to intestinal dysfunction at the organ level, increased contraction at the cellular level and elevated contractile gene expression at the molecular level. A hyper-contractile response may play a role in the persistent intestinal dysfunction seen in GRID.

Fig. 1

Expression of TGF-β isoforms in the intestinal smooth muscle. a Calculated mean pixel units of TGF-β1, 2 and 3 from intestinal tissue sections of human infants with gastroschisis (GS) and premature infant controls, expressed as mean ± SEM. Groups were compared by a Student’s t test for each TGF-β group. b Representative photomicrographs (magnification ×20) from intestinal tissue sections of human infants with gastroschisis (GS) and premature infant controls are depicted and show the immunoreactivity of TGF-β3 (green) and α-actin (red) in the intestinal smooth muscle layers of the intestine. Co-localization of TGF-β3 and α-actin (yellow) is also seen in the intestinal smooth muscle layer of GS infant intestine (n = 7)

Fig. 2

TGF-β3 promotes increased contraction of the collagen gel matrix. a Line diagrams show cell contraction in a collagen gel matrix (mean ± SEM) with FBS ± TGF-β1, 2 or 3 over 6, 24 and 72 h, respectively. Intestinal smooth muscle cell contraction is significantly increased in a time-dependent manner on exposure to TGF-β3. Groups were compared by ANOVA for each TGF-β group at each time point. b Line diagrams show percentage of cell contraction in a collagen gel matrix (mean ± SEM) with FBS ± TGF-β3 over 6, 24 and 72 h. Intestinal smooth muscle cells exposed to TGF-β3 became more contracted as time increased. Experimental groups were compared by a Student’s t test

Fig. 3

Administration of TGF-β3 into the peritoneal cavity decreases intestinal motility. Bar diagrams show intestinal transit as the mean geometric center in the small intestine (mean ± SEM). At 12 h after i.p. injection of TGF-β3, intestinal transit is significantly impaired in a dose–response manner in the animals given i.p. TGF-β3. Experimental groups were compared by ANOVA with a Tukey–Kramer test

Fig. 4

Expression of contractile micro-RNA markers in human intestinal smooth muscle. Calculated mean relative miRNA levels of miRNA-143 (a) and miRNA-145 (b) from intestinal tissue sections of human infants with gastroschisis (GS), intestinal atresia (IA), necrotizing enterocolitis (NEC) and age-matched premature infant control tissue, expressed as mean ± SEM. Groups were compared by ANOVA. Intestinal tissue from the GS patients had significantly elevated levels of miRNA 143 & 145 compared with the other groups

Fig. 5

TGF-β3 promotes the expression of contractile micro-RNA markers in human intestinal smooth muscle. a Line diagrams show the contractile phenotype gene expression (mean ± standard error of the mean [SEM])) with FBS (a) and FBS ± TGF-β3 (b) over 6, 24 and 72 h. Contractile marker: ϒ-smooth muscle actin (ACTG2), calponin (CNN1), smooth muscle myosin heavy chain (MYH11) and smooth muscle-22α (TAGLN). Intestinal smooth muscles exposed to TGF-β3 have a significantly higher expression of contractile genes at all-time points. These data support that hISMCs exposed to TGF-β3 have a contractile phenotype

Fig. 6

TGF-β3 does not exhibit a synthetic protein gene expression profile. Line diagrams show the synthetic phenotype gene expression (mean ± standard error of the mean [SEM])) with FBS (a) and FBS ± TGF-β3 (b) over 6, 24 and 72 h. Synthetic markers: L-caldesmon (CALD1), non-muscle myosin heavy chain IIB (MYH10) and vimentin (VIM). There is no significant difference in the synthetic phenotypic gene expression when hISMCs are exposed to TGF-β3