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

BACKGROUND: Gastroschisis (GS) is a congenital abdominal wall defect that results in the development of GS-related intestinal dysfunction (GRID). Transforming growth factor-ß, 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-ß3 on intestinal smooth muscle function and contractile gene expression. METHODS: Archived human intestinal tissue was analyzed using immunohistochemistry and RT-PCR for TGF-ß isoforms and markers of smooth muscle gene and micro-RNA contractile phenotype. Intestinal motility was measured in neonatal rats ± TGF-ß3 (0.2 and 1 mg/kg). Human intestinal smooth muscle cells (hiSMCs) were incubated with fetal bovine serum ± 100 ng/ml of TGF-ß 3 isoforms for 6, 24 and 72 h. The effects of TGF-ß3 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-ß3 and elevated levels of micro-RNA 143 & 145 in the intestinal smooth muscle. Rats had significantly decreased intestinal transit when exposed to TGF-ß3 in a dose-dependent manner compared with Sham animals. TGF-ß3 significantly increased hiSMC gel contraction and contractile protein gene and micro-RNA expression. CONCLUSION: TGF-ß3 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.

A murine model for the study of edema induced intestinal contractile dysfunction.

BACKGROUND: We have published extensively regarding the effects of edema on intestinal contractile function. However, we have found the need to expand our model to mice to take advantage of the much larger arsenal of research support, especially in terms of transgenic mouse availability and development. To that end, we have developed and validated a hydrostatic intestinal edema model in mice. METHODS: Male C57 Black 6 mice were subjected to a combination of high volume crystalloid resuscitation and mesenteric venous hypertension in an effort to induce hydrostatic intestinal edema. Wet to dry ratios, myeloperoxidase activity, mucosal injury scoring, STAT-3 nuclear activation, phosphorylated STAT-3 levels, NF-κB nuclear activation, myosin light chain phosphorylation, intestinal contractile activity, and intestinal transit were measured to evaluate the effects of the model. KEY RESULTS: High volume crystalloid resuscitation and mesenteric venous hypertension resulted in the development of significant intestinal edema without an increase in myeloperoxidase activity or mucosal injury. Edema development was associated with increases in STAT-3 and NF-κB nuclear activation as well as phosphorylated STAT-3. There was a decrease in myosin light chain phosphorylation, basal and maximally stimulated intestinal contractile activity, and intestinal transit. CONCLUSION & INFERENCES: Hydrostatic edema in mice results in activation of a signal transduction profile that culminates in intestinal contractile dysfunction. This novel model allows for advanced studies into the pathogenesis of hydrostatic edema induced intestinal contractile dysfunction.

Inhibition of intestinal transit by resuscitation-induced gut edema is reversed by L-NIL.

BACKGROUND: Post-resuscitation gut edema and associated gut dysfunction is a common and significant clinical problem that occurs after traumatic injury and shock. We have shown previously that gut edema without ischemia/reperfusion injury delays intestinal transit [1]. We hypothesized that gut edema increases expression of inducible nitric oxide synthase (iNOS) protein, and that selective iNOS inhibition using L-NIL reverses the delayed intestinal transit associated with gut edema. MATERIALS AND METHODS: One hour prior to laparotomy, rats were pretreated with 10 mg/kg body weight of intraperitoneal L-NIL or saline vehicle and underwent 80 ml/kg body weight of 0.9% saline + superior mesenteric venous pressure elevation (Edema) or sham surgery (Sham). A duodenal catheter was placed to allow injection of a fluorescent dye for the measurement of intestinal transit. At 6 h, the small bowel was divided and the mean geometric center (MGC) of fluorescent dye was measured to determine transit. Ileum was harvested for histological assessment of mucosal injury, evaluation of iNOS protein expression by Western blotting, and MPO activity. Tissue water was determined using the wet-to-dry weight ratio to assess gut edema. Data are expressed as mean +/- SEM, n = 3-6 and * = P <0.05 using ANOVA. RESULTS: Gut edema, expressed as increased wet-to-dry ratio, was associated with decreased intestinal transit and elevated iNOS protein expression. Pretreatment with l-NIL improved intestinal transit and decreased expression of iNOS protein without decreasing intestinal tissue water compared to edema animals. There was no difference in mucosal injury or MPO activity among groups. CONCLUSION: Gut edema delays intestinal transit via an iNOS-mediated mechanism.

Heme oxygenase-1 induction by hemin protects against gut ischemia/reperfusion injury.

BACKGROUND: We have shown that both intraischemic hypothermia and hypertonic saline resuscitation provide dramatic protection against gut ischemia/reperfusion (I/R) injury that is in part mediated by heme oxygenase-1 (HO-1). We therefore hypothesized that induction of HO-1 by hemin would lessen damage and improve function after gut I/R. MATERIALS AND METHODS: Male Sprague-Dawley rats were treated with 50 micromol/kg hemin (HO-1 inducer ferric protoporphyrin IX chloride) sq or vehicle 2 h before superior mesenteric artery occlusion for 60 min or sham laparotomy. After 6 h of reperfusion, transit was determined by quantitation of percentage of tracer in 10 equal segments of small intestine 30 min following injection into the duodenum (expressed as mean geometric center). Ileum was harvested for assessment of mucosal histologic injury (Chiu score 0-5 by blinded observer), myeloperoxidase activity (MPO, index of inflammation), and HO-1 protein expression. RESULTS: Hemin treatment was associated with increased HO-1 protein expression, lessened mucosal injury, decreased MPO activity, and improved intestinal transit following gut I/R. CONCLUSION: These data corroborate that HO-1 plays an important role in protecting the gut against I/R-induced injury.