Altered Esophageal Smooth Muscle Phenotype in Achalasia.

Background/aims: Achalasia is a disorder characterized by impairment in lower esophageal sphincter relaxation and esophageal aperistalsis, caused primarily by loss of inhibitory innervation. However, little is known about associated changes in esophageal smooth muscle. We examined the contractile phenotype and innervation of the circular smooth muscle, as well as inflammatory status, and correlated these with patient-specific parameters.

Nintedanib regulates intestinal smooth muscle hyperplasia and phenotype in vitro and in TNBS colitis in vivo.

Chronic inflammation of the human intestine in Crohn's disease (CD) causes bowel wall thickening, which typically progresses to stricturing and a recurrent need for surgery. Current therapies have limited success and CD remains idiopathic and incurable. Recent evidence shows a key role of intestinal smooth muscle cell (ISMC) hyperplasia in stricturing, which is not targeted by current anti-inflammatory therapeutics.

GDNF requires HIF-1α and RET activation for suppression of programmed cell death of enteric neurons by metabolic challenge.

Intestinal inflammation challenges both function and structure of the enteric nervous system (ENS). In the animal model of TNBS-induced colitis, an influx of immune cells causes early neuron death in the neuromuscular layers, followed by axonal outgrowth from surviving neurons associated with upregulation of the neurotrophin GDNF (glial cell line-derived neurotrophic factor). Inflammation could involve ischemia and metabolic inhibition leading to neuronal damage, which might be countered by a protective action of GDNF.

Obligatory Activation of SRC and JNK by GDNF for Survival and Axonal Outgrowth of Postnatal Intestinal Neurons.

The neurotrophin GDNF acts through its co-receptor RET to direct embryonic development of the intestinal nervous system. Since this continues in the post-natal intestine, co-cultures of rat enteric neurons and intestinal smooth muscle cells were used to examine how receptor activation mediates neuronal survival or axonal extension. GDNF-mediated activation of SRC was essential for neuronal survival and axon outgrowth and activated the major downstream signaling pathways.

MMP-9 Processing of Intestinal Smooth Muscle-derived GDNF is Required for Neurotrophic Action on Enteric Neurons.

The neurotrophin GDNF guides development of the enteric nervous system (ENS) in embryogenesis and directs survival and axon outgrowth in postnatal myenteric neurons in vitro. GDNF expression in intestinal smooth muscle cells is dynamic, with upregulation by inflammatory cytokines in vitro or intestinal inflammation in vivo, but the role of post-translational proteolytic cleavage is undefined. In a co-culture model of myenteric neurons, smooth muscle and glia, inhibition of serine or cysteine protease activity was ineffective against the >2-fold increase in axon density caused by TNFα.

M2 Macrophages and Phenotypic Modulation of Intestinal Smooth Muscle Cells Characterize Inflammatory Stricture Formation in Rats.

The progression of Crohn disease to intestinal stricture formation is poorly controlled, and the pathogenesis is unclear, although increased smooth muscle mass is present. A previously described rat model of trinitrobenzenesulfonic acid-induced colitis is re-examined here. Although inflammation of the mid-descending colon typically resolved, a subset showed characteristic stricturing by day 16, with an inflammatory infiltrate in the neuromuscular layers including eosinophils, CD3-positive T cells, and CD68-positive macrophages.

The impact of dietary fermentable carbohydrates on a postinflammatory model of irritable bowel syndrome.

Background: A low fermentable carbohydrate (FODMAP) diet is used in quiescent inflammatory bowel disease when irritable bowel syndrome-like symptoms occur. There is concern that the diet could exacerbate inflammation by modifying microbiota and short-chain fatty acid (SCFA) production. We examined the effect of altering dietary FODMAP content on inflammation in preclinical inflammatory models.

Epigenetic modification of intestinal smooth muscle cell phenotype during proliferation.

Inflammation causes proliferation of intestinal smooth muscle cells (ISMC), contributing to a thickened intestinal wall and to stricture formation in Crohn's disease. Proliferation of ISMC in vitro and in vivo caused decreased expression of marker proteins, but the underlying cause is unclear. Since epigenetic change is important in other systems, we used immunocytochemistry, immunoblotting, and quantitative PCR to examine epigenetic modification in cell lines from rat colon at low passage or after extended growth to evaluate phenotype.

Analgesia and mouse strain influence neuromuscular plasticity in inflamed intestine.

Background: Mouse models of inflammatory bowel disease (IBD) identify an impact on the enteric nervous system (ENS) but do not distinguish between Crohn's disease and ulcerative colitis phenotypes. In these models, analgesia is required, but its influence on different strains and disease outcomes is unknown. Therefore, changes to the ENS and intestinal smooth muscle were studied in trinitrobenzene sulfonic acid (TNBS) and dextran sodium sulfate (DSS) induced colitis to identify the effects of analgesia, and compared between two mouse strains.

Stress increases descending inhibition in mouse and human colon.

Background: A relationship between stress and the symptoms of irritable bowel syndrome (IBS) has been well established but the cellular mechanisms are poorly understood. Therefore, we investigated effects of stress and stress hormones on colonic descending inhibition and transit in mouse models and human tissues.

Methods: Stress was applied using water avoidance stress (WAS) in the animal model or mimicked using stress hormones, adrenaline (5 nM), and corticosterone (1 μM).

Intestinal smooth muscle phenotype determines enteric neuronal survival via GDNF expression.

Intestinal inflammation causes initial axonal degeneration and neuronal death, as well as the proliferation of intestinal smooth muscle cells (ISMC), but subsequent axonal outgrowth leads to re-innervation. We recently showed that expression of glial cell-derived neurotrophic factor (GDNF), the critical neurotrophin for the post-natal enteric nervous system (ENS) is upregulated in ISMC by inflammatory cytokines, leading us to explore the relationship between ISMC growth and GDNF expression. In co-cultures of myenteric neurons and ISMC, GDNF or fetal calf serum (FCS) was equally effective in supporting neuronal survival, with neurons forming extensive axonal networks among the ISMC.

Inflammatory cytokines promote growth of intestinal smooth muscle cells by induced expression of PDGF-Rβ.

Thickening of the inflamed intestinal wall involves growth of smooth muscle cells (SMC), which contributes to stricture formation. Earlier, the growth factor platelet-derived growth factor (PDGF)-BB was identified as a key mitogen for SMC from the rat colon (CSMC), and CSMC growth in colitis was associated with both appearance of its receptor, PDGF-Rβ and modulation of phenotype. Here, we examined the role of inflammatory cytokines in inducing and modulating the growth response to PDGF-BB.

Impaired platelet-derived growth factor receptor expression and function in cultured lower esophageal sphincter circular smooth muscle cells from W/W(v) mutant mice.

We have previously demonstrated that lower esophageal sphincter (LES) circular smooth muscle (CSM) is functionally impaired in W/W(v) mutant mice that lack interstitial cells of Cajal, and speculated that this could be due to altered smooth muscle differentiation. Platelet-derived growth factor (PDGF) is involved in the maturation and differentiation of smooth muscle. To determine whether PDGF expression and (or) function is altered in W/W(v) mutant mice, PDGF-Rβ expression was measured using RT-PCR, qPCR, and immunocytochemistry, and Ca(2+) imaging and perforated patch clamp recordings performed in isolated LES CSM cells.

The pro-inflammatory cytokines IL-1β and TNFα are neurotrophic for enteric neurons.

Intestinal inflammation causes initial axonal degeneration and neuronal death but subsequent axon outgrowth from surviving neurons restores innervation density to the target smooth muscle cells. Elsewhere, the pro-inflammatory cytokines TNFα and IL-1β cause neurotoxicity, leading us to test their role in promoting enteric neuron death. In a rat coculture model, TNFα or IL-1β did not affect neuron number but did promote significant neurite outgrowth to twofold that of control by 48 h, while other cytokines (e.

Proliferation modulates intestinal smooth muscle phenotype in vitro and in colitis in vivo.

Intestinal inflammation causes an increased intestinal wall thickness, in part, due to the proliferation of smooth muscle cells, which impairs the contractile phenotype elsewhere. To study this, cells from the circular muscle layer of the rat colon (CSMC) were isolated and studied, both in primary culture and after extended passage, using quantitative PCR, Western blot analysis, and immunocytochemistry. By 4 days in vitro, both mRNA and protein for the smooth muscle marker proteins α-smooth muscle actin, desmin, and SM22-α were reduced by >50%, and mRNA for cyclin D1 was increased threefold, evidence for modulation to a proliferative phenotype.

Entamoeba histolytica infection and secreted proteins proteolytically damage enteric neurons.

The enteric protozoan parasite Entamoeba histolytica causes amebic colitis through disruption of the mucus layer, followed by binding to and destruction of epithelial cells. However, it is not known whether ameba infections or ameba components can directly affect the enteric nervous system. Analysis of mucosal innervations in the mouse model of cecal amebiasis showed that axon density was diminished to less than 25% of control.

Mitogenic factors promoting intestinal smooth muscle cell proliferation.

Intestinal smooth muscle cells are normally quiescent, but in the widely studied model of trinitrobenzene sulfonic acid (TNBS)-induced colitis in the rat, the onset of inflammation causes proliferation that leads to increased cell number and an altered phenotype. The factors that drive this are unclear and were studied in primary cultures of circular smooth muscle cells (CSMC) from the rat colon. While platelet-derived growth factor (PDGF)-AA, fibroblast growth factor (FGF), and epidermal growth factor (EGF) were ineffective, PDGF-BB and insulin-like growth factor-1 (IGF-1) caused significant increase in [(3)H]thymidine incorporation, bromodeoxyuridine uptake, and increased CSMC number, with PDGF-BB (≥0.

Proteinase-activated receptor-2 activation evokes oesophageal longitudinal smooth muscle contraction via a capsaicin-sensitive and neurokinin-2 receptor-dependent pathway.

Background: Intraluminal acid evokes sustained oesophageal longitudinal smooth muscle (LSM) contraction and oesophageal shortening, which may play a role in oesophageal pain and the aetiology of hiatus hernia. In the opossum model, this reflex has been shown to involve mast cell activation and release of neurokinins from capsaicin-sensitive neurons. The aim of this study was to determine whether proteinase-activated receptor-2 (PAR-2) activation evokes reflex LSM contraction via similar mechanisms.

Discrete responses of myenteric neurons to structural and functional damage by neurotoxins in vitro.

Damage to the enteric nervous system is implicated in human disease and animal models of inflammatory bowel disease, diabetes, and Parkinson's disease, but the mechanism of death and the response of surviving neurons are poorly understood. We explored this in a coculture model of myenteric neurons, glia, and smooth muscle during exposure to the established or potential neurotoxins botulinum A, hydrogen peroxide, and acrylamide. Neuronal survival, axonal degeneration and regeneration, and neurotransmitter release were assessed during acute exposure (0-24 h) to neurotoxin and subsequent recovery (96-144 h).

Inflammation causes expression of NGF in epithelial cells of the rat colon.

Nerve growth factor (NGF) is a neurotrophin implicated in intestinal pathophysiology, such as impaired barrier function, altered motility and a lowered threshold to noxious stimuli in colitis. We evaluated the cellular source of NGF and determined the effect of inflammation on its expression in TNBS-induced colitis in the rat. Receptors for NGF were studied by immunocytochemistry, showing that submucosal neurons expressed both trkA and p75(NTR).

Impaired acetylcholine-induced smooth muscle contraction in colitis involves altered calcium mobilization and AKT phosphorylation.

In trinitrobenzene sulphonic acid (TNBS)-induced colitis in the rat, isolated circular smooth muscle cells (CSMC) show decreased contraction to acetylcholine (ACh) but the presence and contribution of altered intracellular signaling is poorly understood. To characterize ACh-induced signaling via calcium and the principal signaling kinases ERK1/2 and AKT in CSMC during colitis, isolated colonic CSMC from control, TNBS-inflamed (day 4) or recovered (day 36) rats were treated with ACh. Intracellular Ca2+ and contraction was determined by fluorescence video microscopy.

Intraluminal acid induces oesophageal shortening via capsaicin-sensitive neurokinin neurons.

Objective: Intraluminal acid evokes reflex contraction of oesophageal longitudinal smooth muscle (LSM) and consequent oesophageal shortening. This reflex may play a role in the pathophysiology of oesophageal pain syndromes and hiatus hernia formation. The aim of the current study was to elucidate further the mechanisms of acid-induced oesophageal shortening.

Differential responses of intrinsic and extrinsic innervation of smooth muscle cells in rat colitis.

Intestinal smooth muscle cells receive neural input from axons that originate within the intestine, as well as from axons of extrinsic origin. In the inflamed intestine, altered motility may arise from damage to the axon/smooth muscle cell relationship, but the extent of change is unknown. Western blotting, histology and immunocytochemistry were used in the TNBS model of colitis in the rat to evaluate intrinsic and extrinsic axon numbers, which were then correlated with circular smooth muscle cell (CSMC) number during the time course from the acute onset of colitis to apparent recovery, at Day 35 post TNBS.