New insights into irritable bowel syndrome pathophysiological mechanisms: contribution of epigenetics.

Irritable bowel syndrome (IBS) is a complex multifactorial condition including alterations of the gut-brain axis, intestinal permeability, mucosal neuro-immune interactions, and microbiota imbalance. Recent advances proposed epigenetic factors as possible regulators of several mechanisms involved in IBS pathophysiology. These epigenetic factors include biomolecular mechanisms inducing chromosome-related and heritable changes in gene expression regardless of DNA coding sequence. Accordingly, altered gut microbiota may increase the production of metabolites such as sodium butyrate, a prominent inhibitor of histone deacetylases. Patients with IBS showed an increased amount of butyrate-producing microbial phila as well as an altered profile of methylated genes and micro-RNAs (miRNAs). Importantly, gene acetylation as well as specific miRNA profiles are involved in different IBS mechanisms and may be applied for future diagnostic purposes, especially to detect increased gut permeability and visceromotor dysfunctions. In this review, we summarize current knowledge of the role of epigenetics in IBS pathophysiology.

Ex vivo effect of vascular wall stromal cells secretome on enteric ganglia.

BACKGROUND: Mesenchymal stromal cell (MSC)-based therapy is currently under study to treat inflammatory bowel diseases. MSC bioactive products could represent a valid alternative to overcome issues associated with systemic whole-cell therapies. However, MSC anti-inflammatory mechanisms differ between rodents and humans, impairing the reliability of preclinical models. AIM: To evaluate the effect of conditioned medium (CM) derived from porcine vascular wall MSCs (pVW-MSCs) on survival and differentiation of porcine and guinea pig enteric ganglia exposed to lipopolysaccharide (LPS). METHODS: Primary cultures of enteric ganglia were obtained by mechanic and enzymatic digestion of ileum resections from guinea pigs (Cavia porcellus) (GPEG) and pigs (Suus scrofa) (PEG). pVW-MSCs were derived by enzymatic digestion from vascular wall resections of porcine aorta and tested by immunoflowcytometry for MSC immune profile. Enteric ganglia were treated with increasing concentrations of LPS, CM derived by pVW-MSCs or a combination of CM and LPS 1 µg/mL. Cell count and morphometric analysis of HuD positive neurons and glial fibrillary acidic protein positive glial cells were performed by immunofluorecent staining of cultured ganglia. RESULTS: PEG showed a higher number of neurons compared to GPEG. Overall, CM exerted a protective role on LPS-treated enteric ganglia. CM in combination with LPS increased the number of glial cells per ganglion in both cultures evoking glial cells differentiation in porcine cultures. CONCLUSION: These findings suggest an immunomodulating activity of pVW-MSCs mediators on the enteric nervous system in inflammatory conditions.

µ-opioid receptor, ß-endorphin, and cannabinoid receptor-2 are increased in the colonic mucosa of irritable bowel syndrome patients.

BACKGROUND AND AIMS: The gut immune, cannabinoid, and opioid systems constitute an integrated network contributing to visceral sensation and pain modulation. We aimed to assess the expression of the µ-opioid receptor (MOR), its ligand ß-endorphin (ß-END), and cannabinoid receptor-2 (CB2 ) in patients with irritable bowel syndrome (IBS) and asymptomatic controls (AC) and their correlation with sex and symptom perception. METHODS: Mucosal biopsies were obtained from the left colon of 31 IBS patients (45% women) with predominant constipation (IBS-C, 9) or diarrhea (IBS-D, 10) or with mixed bowel habits (IBS-M, 12) and 32 AC (44% women) and processed for qRT-PCR, Western blotting, and immunohistochemistry. KEY RESULTS: µ-opioid receptor and CB2 mRNA and protein expression and ß-END protein levels were increased in patients with IBS compared to AC (all Ps=0.021). A significant sex by IBS interaction was found in relation to CB2 mRNA expression (P = .003) with women showing a markedly higher expression to men (P = .035). In contrast, in AC, men had higher expression than women (P = .033). ß-END, MOR, and CB2 immunoreactivities (IR) were localized to CD4+T cells including EMR-1+ eosinophils and CD31+ T cells but not to mast cells. CONCLUSIONS: The increased expression of MOR, ß-END, and CB2 in the mucosa of IBS patients, where they are localized to immune cells, suggests that opioid and cannabinoid systems play an immune-related compensatory role in visceral pain in IBS patients. Further work is necessary to support this hypothesis.

Constitutive and LPS-stimulated secretome of porcine Vascular Wall-Mesenchymal Stem Cells exerts effects on in vitro endothelial angiogenesis.

BACKGROUND: MSCs secretome is under investigation as an alternative to whole-cell-based therapies, since it is enriched of bioactive molecules: growth factors, cytokines and chemokines. Taking into account the translational value of the pig model, the leading aim of the present paper was to characterize the secretome of porcine Vascular Wall-Mesenchymal Stem Cells (pVW-MSCs) and its change in presence of LPS stimulation. Moreover, considering the importance of angiogenesis in regenerative mechanisms, we analysed the effect of pVW-MSCs secretome on in vitro angiogenesis. RESULTS: Our results demonstrated that conditioned medium from unstimulated pVW-MSCs contained high levels of IL-8, GM-CSF, IFN-γ and other immunomodulatory proteins: IL-6 IL-18 IL-4 IL-2 IL-10. LPS modulates pVW-MSCs gene expression and secretome composition, in particular a significant increase of IL-6 and IL-8 was observed; conversely, the amount of GM-CSF, IFN-γ, IL-2, IL-4, IL-10 and IL-18 showed a significant transient decrease with the LPS stimulation. Conditioned medium from unstimulated pVW-MSCs induced in vitro endothelial angiogenesis, which is more evident when the conditioned medium was from LPS stimulated pVW-MSCs. CONCLUSIONS: The lines of evidence here presented shed a light on possible future application of secretome derived by pVW-MSCs on research studies in translational regenerative medicine.

Altered globotriaosylceramide accumulation and mucosal neuronal fiber density in the colon of the Fabry disease mouse model.

BACKGROUND: Fabry disease (FD) is a hereditary X-linked metabolic storage disorder characterized by deficient or absent lysosomal α-galactosidase A (α-Gal A) activity. This deficiency causes progressive accumulation of glycosphingolipids, primarily globotriaosylceramide (Gb3), in nearly all organ systems. Gastrointestinal (GI) symptoms can be very debilitating and are among the most frequent and earliest of the disease. As the pathophysiology of these symptoms is poorly understood, we carried out a morphological and molecular characterization of the GI tract in α-Gal A knockout mice colon in order to reveal the underlying mechanisms. METHODS: Here, we performed the first morphological and biomolecular characterization of the colon wall structure in the GI tract of the α-Gal A knock-out mouse (α-Gal A -/0), a murine model of FD. KEY RESULTS: Our data show a greater thickness of the gastrointestinal wall in α-Gal A (-/0) mice due to enlarged myenteric plexus' ganglia. This change is paralleled by a marked Gb3 accumulation in the gastrointestinal wall and a decreased and scattered pattern of mucosal nerve fibers. CONCLUSIONS AND INFERENCES: The observed alterations are likely to be a leading cause of gut motor dysfunctions experienced by FD patients and imply that the α-Gal A (-/0) male mouse represents a reliable model for translational studies on enteropathic pain and GI symptoms in FD.

Advancements in drug development for diarrhea-predominant irritable bowel syndrome.

INTRODUCTION: Diarrhea-predominant irritable bowel syndrome (IBS-D) is a common disorder characterized by a complex pathophysiology hampering optimal targeted drug development. Recent advances in our understanding of key underlying mechanisms prompted novel therapeutics including novel pharmacological approaches. AREAS COVERED: This review summarizes the latest advancements in the pipeline of IBS-D drugs focusing on new pharmacological targets, efficacy and safety of medicinal products considering the recent harmonization of regulatory requirements by the FDA and the EMA. EXPERT OPINION: The new 5-HT3 receptor antagonist ramosetron appears a promising therapeutic approach devoid of significant adverse events, although it is presently unavailable in Western countries, most likely because of the precautionary approach taken by regulatory agencies with this drug class. New pharmacological concepts on full agonists/antagonists, mixed-receptor activity and novel drug targets may streamline the present drug pipeline along with the adherence on new regulatory guidelines on outcome measures. Eluxadoline can be taken as an example of this paradigm shift. It has now been granted marketing authorization for IBS-D on both sides of the Atlantic, but it is still considered as a second-line agent by the NICE. There is still much work to be done to fully cover clinical needs of patients with IBS-D.

Mesenchymal stromal cell-based therapy: Regulatory and translational aspects in gastroenterology.

The past decade has witnessed an outstanding scientific production focused towards the possible clinical applications of mesenchymal stromal cells (MSCs) in autoimmune and chronic inflammatory diseases. This raised the need of novel standards to adequately address quality, efficacy and safety issues of this advanced therapy. The development of a streamlined regulation is currently hampered by the complexity of analyzing dynamic biological entities rather than chemicals. Although numerous pieces of evidence show efficacy in reducing intestinal inflammation, some inconsistencies between the mechanisms of action of rodent vs human MSCs suggest caution before assigning translational value to preclinical studies. Preliminary evidence from clinical trials showed efficacy of MSCs in the treatment of fistulizing Crohn's disease (CD), and preparations of heterologous MSCs for CD treatment are currently tested in ongoing clinical trials. However, safety issues, especially in long-term treatment, still require solid clinical data. In this regard, standardized guidelines for appropriate dosing and methods of infusion could enhance the likelihood to predict more accurately the number of responders and the duration of remission periods. In addition, elucidating MSC mechanisms of action could lead to novel and more reliable formulations such as those derived from the MSCs themselves (e.g., supernatants).

Nerve fiber outgrowth is increased in the intestinal mucosa of patients with irritable bowel syndrome.

BACKGROUND & AIMS: Mediators released by the intestinal mucosa of patients with irritable bowel syndrome (IBS) affect the function of enteric and extrinsic sensory nerves, which can contribute to the development of symptoms. Little is known about the effects of mucosal mediators on intestinal neuroplasticity. We investigated how these mediators affect the phenotypes of colonic mucosa nerve fibers, neuron differentiation, and fiber outgrowth. METHODS: We analyzed mucosal biopsy samples collected from 101 patients with IBS and 23 asymptomatic healthy individuals (controls). We measured levels of neuronal-specific enolase, growth-associated protein 43, nerve growth factor (NGF), and tyrosine kinase receptor A (NTRK1) by immunohistochemistry and enzyme-linked immunosorbent assay. Primary rat enteric neurons and human SH-SY5Y cells were incubated with supernatants from the mucosal biopsies and analyzed by morphometric and polymerase chain reaction analyses. RESULTS: Compared with mucosal tissues of controls, mucosa from patients with IBS had a significant increase in the area of lamina propria occupied by neuronal-specific enolase-positive (57.7% increase) and growth-associated protein 43-positive fibers (56.1% increase) and staining density of NGF (89.3% increase) (P < .05 for all). Levels of NGF protein were also increased in tissues from patients with IBS vs controls (18% increase; P = .16) along with levels of NTRK1 (64% increase; P < .05). Mucosal supernatants from tissues of patients with IBS induced higher levels of neuritogenesis in primary culture of enteric neurons, compared with controls, and more NGF-dependent neuronal sprouting in SH-SY5Y cells. CONCLUSIONS: Nerve fiber density and sprouting, as well as expression of NGF and NTRK1, are significantly increased in mucosal tissues of patients with IBS. Mucosal mediators participate to these neuroplastic changes.

Animal models of chemically induced intestinal inflammation: predictivity and ethical issues.

The debate about the ethical and scientific issues regarding the use of animals in research is mainly focused on these questions: a) whether preclinical studies in animals are still ethically acceptable; b) whether it is possible to establish more soundly their predictivity; c) what measures should be taken to reduce the clinical attrition often due to biased preclinical assessment of potential efficacy of new drugs. This review aims at a critical revision of animal models of chemically induced intestinal inflammation in drug development. These models, notwithstanding differences among species, still represent a major source of information about biological systems and can have undisputable translational value, provided that appropriate measures are taken to ensure that experiments are both scientifically and ethically justified. These measures include: a) more stringent application to preclinical experiments of standards used in clinical studies (such as sample size, randomization, inclusion/exclusion criteria, blinding); b) selection of the animal model after careful pathophysiological scrutiny bearing in mind inherent limitations of each model (e.g. acute self-limiting vs chronic disease, animal species, role of the intestinal immune system and microbiome); and c) experimental design duly considering the specific pharmacological profile of each agent to be screened (such as bioavailability, route of administration, full consideration of the pharmacological spectrum). In this perspective, the new European legislation is an opportunity to fully apply these standards so that in vivo animal models can provide an invaluable mean to study complex physiological and biochemical interactions, which cannot be completely simulated in silico and/or in vitro.

Mechanisms underlying visceral hypersensitivity in irritable bowel syndrome.

Visceral hypersensitivity is currently considered a key pathophysiological mechanism involved in pain perception in large subgroups of patients with functional gastrointestinal disorders, including irritable bowel syndrome (IBS). In IBS, visceral hypersensitivity has been described in 20%-90% of patients. The contribution of the central nervous system and psychological factors to visceral hypersensitivity in patients with IBS may be significant, although still debated. Peripheral factors have gained increasing attention following the recognition that infectious enteritis may trigger the development of persistent IBS symptoms, and the identification of mucosal immune, neural, endocrine, microbiological, and intestinal permeability abnormalities. Growing evidence suggests that these factors play an important role in pain transmission from the periphery to the brain via sensory nerve pathways in large subsets of patients with IBS. In this review, we will report on recent data on mechanisms involved in visceral hypersensitivity in IBS, with particular attention paid to peripheral mechanisms.