Erika Tirotta

Ghrelin restores nitric oxide availability in resistance circulation of essential hypertensive patients: role of NAD(P)H oxidase

AIMS We assessed whether acute intra-arterial infusion of exogenous ghrelin can improve endothelial dysfunction by restoring nitric oxide (NO) availability in the forearm microcirculation of essential hypertensive patients. The effect of ghrelin on endothelial dysfunction (pressurized myograph), vascular oxidative stress generation (fluorescent dihydroethidium), and phosphorylation of p47phox (western blot), an index of NAD(P)H oxidase activation, in isolated small arteries taken from essential hypertensive patients (subcutaneous biopsy) were also investigated. METHODS AND RESULTS In 18 normotensive control subjects and 18 essential hypertensive patients, we studied the forearm blood flow (strain-gauge plethysmography) response to intra-arterial acetylcholine, repeated under NO synthase inhibitor N(G)-monomethyl-l-arginine (l-NMMA) or the antioxidant ascorbic acid. The protocol was repeated at the end of exogenous ghrelin intra-arterial infusion. In hypertensive patients, ghrelin normalized the blunted response to acetylcholine, restored the inhibiting effect of l-NMMA and abrogated the potentiating effect of ascorbic acid on acetylcholine. In controls, ghrelin failed to modify these vascular responses. In hypertensive patients, ghrelin decreased venous levels of malondialdehyde, lipoperoxide, and interleukin-6, and concomitantly increased endogenous antioxidant capacity. Small vessels from hypertensive patients showed an enhanced intravascular oxidative stress, which was strongly and similarly decreased by incubation with ghrelin, the NAD(P)H oxidase inhibitor gp91 ds-tat, or both. Ghrelin also normalized the overexpression of p47 phosphorylation and restored the NO availability in small vessels from hypertensive patients. CONCLUSIONS Exogenous ghrelin increases endothelial dysfunction by restoring NO availability in the forearm microcirculation of essential hypertensive patients, an effect ascribable to an antioxidant effect via inhibition of NAD(P)H oxidase activation.

Fibrotic and Vascular Remodelling of Colonic Wall in Patients with Active Ulcerative Colitis

BACKGROUND AND AIMS Intestinal fibrosis is a complication of inflammatory bowel disease [IBD]. Although fibrostenosis is a rare event in ulcerative colitis [UC], there is evidence that a fibrotic rearrangement of the colon occurs in the later stages. This is a retrospective study aimed at examining the histopathological features of the colonic wall in both short-lasting [SL] and long-lasting [LL] UC. METHODS Surgical samples of left colon from non-stenotic SL [≤ 3 years, n = 9] and LL [≥ 10 years, n = 10] UC patients with active disease were compared with control colonic tissues from cancer patients without UC [n = 12] to assess: collagen and elastic fibres by histochemistry; vascular networks [CD31/CD105/nestin] by immunofluorescence; parameters of fibrosis [types I and III collagen, fibronectin, RhoA, alpha-smooth muscle actin [α-SMA], desmin, vimentin], and proliferation [proliferating nuclear antigen [PCNA]] by western blot and/or immunolabelling. RESULTS Colonic tissue from both SL-UC and LL-UC showed tunica muscularis thickening and transmural activated neovessels [displaying both proliferating CD105-positive endothelial cells and activated nestin-positive pericytes], as compared with controls. In LL-UC, the increased collagen deposition was associated with an up-regulation of tissue fibrotic markers [collagen I and III, fibronectin, vimentin, RhoA], an enhancement of proliferation [PCNA] and, along with a loss of elastic fibres, a rearrangement of the tunica muscularis towards a fibrotic phenotype. CONCLUSIONS A significant transmural fibrotic thickening occurs in colonic tissue from LL-UC, together with a cellular fibrotic switch in the tunica muscularis. A full-thickness angiogenesis is also evident in both SL- and LL-UC with active disease, as compared with controls.

The AMPK enzyme-complex: from the regulation of cellular energy homeostasis to a possible new molecular target in the management of chronic inflammatory disorders

Introduction: Adenosine monophosphate-activated protein kinase (AMPK), known as an enzymatic complex that regulates the energetic metabolism, is emerging as a pivotal enzyme and enzymatic pathway involved in the regulation of immune homeostatic networks. It is also involved in the molecular mechanisms underlying the pathophysiology of chronic inflammatory diseases. Areas covered: AMPK is expressed in several immune cell types including macrophages, lymphocytes, neutrophils and dendritic cells, and governs a broad array of cell functions, which include cytokine production, chemotaxis, cytotoxicity, apoptosis and proliferation. Based on its wide variety of immunoregulatory actions, the AMPK system has been targeted to reveal its impact on the course of immune-related diseases, such as atherosclerosis, psoriasis, joint inflammation and inflammatory bowel diseases. Expert opinion: The identification of AMPK subunits responsible for specific anti-inflammatory actions and the understanding of the underlying molecular mechanisms will promote the generation of novel AMPK activators, endowed with improved pharmacodynamic and pharmacokinetic profiles. These new tools will aid us to utilize AMPK pathway activation in the management of acute and chronic inflammatory diseases, while minimizing potential adverse reactions related to the effects of AMPK on metabolic energy.

Small bowel protection against NSAID-injury in rats: Effect of rifaximin, a poorly absorbed, GI targeted, antibiotic

Nonsteroidal anti-inflammatory drugs, besides exerting detrimental effects on the upper digestive tract, can also damage the small and large intestine. Although the underlying mechanisms remain unclear, there is evidence that enteric bacteria play a pivotal role. The present study examined the enteroprotective effects of a delayed-release formulation of rifaximin-EIR (R-EIR, 50mg/kg BID, i.g.), a poorly absorbed antibiotic with a broad spectrum of antibacterial activity, in a rat model of enteropathy induced by indomethacin (IND, 1.5mg/kg BID for 14 days) administration. R-EIR was administered starting 7 days before or in concomitance with IND administration. At the end of treatments, blood samples were collected to evaluate hemoglobin (Hb) concentration (as an index of digestive bleeding). Small intestine was processed for: (1) histological assessment of intestinal damage (percentage length of lesions over the total length examined); (2) assay of tissue myeloperoxidase (MPO) and TNF levels, as markers of inflammation; (3) assay of tissue malondialdehyde (MDA) and protein carbonyl concentrations, as an index of lipid and protein peroxidation, respectively; (4) evaluation of the major bacterial phyla. IND significantly decreased Hb levels, this effect being significantly blunted by R-EIR. IND also induced the occurrence of lesions in the jejunum and ileum. In both intestinal regions, R-EIR significantly reduced the percentage of lesions, as compared with rats receiving IND alone. Either the markers of inflammation and tissue peroxidation were significantly increased in jejunum and ileum from IND-treated rats. However, in rats treated with R-EIR, these parameters were not significantly different from those observed in controls. R-EIR was also able to counterbalance the increase in Proteobacteria and Firmicutes abundance induced by INDO. To summarize, R-EIR treatment significantly prevents IND-induced intestinal damage, this enteroprotective effect being associated with a decrease in tissue inflammation, oxidative stress and digestive bleeding as well as reversal of NSAID-induced alterations in bacterial population.

Effects of L-DOPA/benserazide co-treatment on colonic excitatory cholinergic motility and enteric inflammation following dopaminergic nigrostriatal neurodegeneration

Introduction: The mainstay therapy for Parkinsons disease (PD) relies on L‐3,4‐dihydroxyphenylalanine (L‐DOPA) plus a DOPA‐decarboxylase inhibitor. However, their effects on colonic dysmotility and inflammation observed in PD are undetermined. This study examined the effects of L‐DOPA plus benserazide (BE) on colonic motility and inflammation in rats with central nigrostriatal dopaminergic denervation. Methods: Neurodegeneration was induced by 6‐hydroxydopamine (6‐OHDA) injection into the medial forebrain bundle (MFB). 6‐OHDA animals were treated orally with L‐DOPA/BE for 28 days, starting 28 days after 6‐OHDA injection. At the end of treatment, in vivo colonic transit was evaluated by a radiologic assay. Electrically stimulated (ES) cholinergic contractions were recorded in vitro from colonic preparations, while acetylcholine release was measured in the incubation medium. Choline acetyltransferase (ChAT) and glial fibrillary acidic protein (GFAP) expression as well as eosinophil and mast cell density were examined in the colonic wall by immunohistochemistry. Colonic TNF and IL‐1&bgr; levels were also assayed. Results: 6‐OHDA animals displayed: 1) decrease in in vivo colonic transit; 2) impairment of ES‐stimulated cholinergic contractions; 3) decreased acetylcholine release from myenteric nerves; 4) decrease in ChAT and increase in GFAP myenteric immunopositivity; 5) increase in eosinophil and mast cell density; 6) increase in TNF and IL‐1&bgr; levels. Treatment with L‐DOPA/BE elicited an improvement of in vivo and in vitro colonic motor activity, a normalization of acetylcholine release, ChAT immunopositivity, as well as pro‐inflammatory cytokine patterns, ganglionic GFAP levels, eosinophil and mast cell density. Conclusion: Under dopaminergic nigrostriatal denervation, treatment with L‐DOPA/BE ameliorated colonic motility through a normalization of myenteric cholinergic neurotransmission, along with an improvement of colonic inflammation. HighlightsL‐DOPA effects on colonic dysfunctions in 6‐OHDA rats are evaluated.L‐DOPA administration restores colonic in vivo transit and in vitro contractility.L‐DOPA treatment normalizes colonic cholinergic neurotransmission in 6‐OHDA rats.L‐DOPA improves colonic inflammation associated with central dopaminergic denervation.

Colonic Dysmotility Associated with High Fat Diet-Induced Obesity: Role of the Enteric Glia

Interplay between colonic inflammation and tachykininergic pathways in the onset of colonic dysmotility in a mouse model of diet-induced obesity Antonioli, Luca; Caputi, Valentina; Fornai, Matteo; Pellegrini, Carolina; Gentile, Daniela; Giron, Maria Cecilia; Orso, Genny; Bernardini, Nunzia; Segnani, Cristina; Ippolito, Chiara; Csóka, Balázs; Haskó, György; Németh, Zoltán H.; Scarpignato, Carmelo; Blandizzi, Corrado; Colucci, Rocchina 2019

The flavonoid compound apigenin prevents colonic inflammation and motor dysfunctions associated with high fat diet-induced obesity

Background and purpose Apigenin can exert beneficial actions in the prevention of obesity. However, its putative action on obesity-associated bowel motor dysfunctions is unknown. This study examined the effects of apigenin on colonic inflammatory and motor abnormalities in a mouse model of diet-induced obesity. Experimental approach Male C57BL/6J mice were fed with standard diet (SD) or high-fat diet (HFD). SD or HFD mice were treated with apigenin (10 mg/Kg/day). After 8 weeks, body and epididymal fat weight, as well as cholesterol, triglycerides and glucose levels were evaluated. Malondialdehyde (MDA), IL-1β and IL-6 levels, and let-7f expression were also examined. Colonic infiltration by eosinophils, as well as substance P (SP) and inducible nitric oxide synthase (iNOS) expressions were evaluated. Motor responses elicited under blockade of NOS and tachykininergic contractions were recorded in vitro from colonic longitudinal muscle preparations. Key results When compared to SD mice, HFD animals displayed increased body weight, epididymal fat weight and metabolic indexes. HFD mice showed increments in colonic MDA, IL-1β and IL-6 levels, as well as a decrease in let-7f expression in both colonic and epididymal tissues. HFD mice displayed an increase in colonic eosinophil infiltration. Immunohistochemistry revealed an increase in SP and iNOS expression in myenteric ganglia of HFD mice. In preparations from HFD mice, electrically evoked contractions upon NOS blockade or mediated by tachykininergic stimulation were enhanced. In HFD mice, Apigenin counteracted the increase in body and epididymal fat weight, as well as the alterations of metabolic indexes. Apigenin reduced also MDA, IL-1β and IL-6 colonic levels as well as eosinophil infiltration, SP and iNOS expression, along with a normalization of electrically evoked tachykininergic and nitrergic contractions. In addition, apigenin normalized let-7f expression in epididymal fat tissues, but not in colonic specimens. Conclusions and implications Apigenin prevents systemic metabolic alterations, counteracts enteric inflammation and normalizes colonic dysmotility associated with obesity.

Colonic wall remodeling in patients with short- and long-lasting ulcerative colitis

Inflammatory bowel disease (IBD) are chronic and progressive pathologies associated with invalidating abdominal symptoms and increasing incidence in Western countries. Although fibrostenosis is a rare event in ulcerative colitis [UC], it may evolve to fibrosis in the later stages (1, 2). In the present study we examined the histopathological remodeling of the colonic wall from short- and long-lasting (SL and LL) UC patients. Full-thickness left colonic surgical samples were obtained from non-stenotic SL (≤ 3 years) and LL (≥ 10 years) UC patients with a severe exacerbation of colitis and active disease, without clinical signs of fibrostenosis. Collagen and elastic fibres, vascular networks and parameters of fibrosis have been evaluated by histochemistry, immunohistochemistry, confocal immunofluorescence and/or western blot. For comparison, normal colonic control samples from subjects who underwent surgery for uncomplicated colon cancer and without IBD and diverticular disease, were considered.Both SL- and LL-UC showed a thickening in tunica muscularis and activation of transmural neovessels, with proliferating CD105-positive endothelial cells and activated nestin-positive pericytes, as compared with controls. The colonic wall of LL-UC displayed a significant increase in collagen deposition and fibrotic markers (type I and III collagen, fibronectin, vimentin, RhoA), an enhancement of proliferation (PCNA), a decrease in elastic fibres, together with a fibrotic rearrangement of the tunica muscularis smooth muscle cells. In conclusion, a significant full-thickness remodeling of the colonic wall has been documented in the colonic tissues from both SL-UC and LL-UC, as compared to controls: transmural fibrotic thickening and angiogenesis were found in both UC groups, together with a cellular fibrotic/proliferative switch in the tunica muscularis which occurs in the later stages. These histopathological remodeling may contribute to the reduced elastic properties and tissue stretch capability of the wall, thereby impairing the occurrence of a normal motor activity in these subjects.

Alterations of colonic cholinergic and tachykininergic motility in a rat model of Parkinson’s Disease

Objective: In gastrointestinal diseases, drug discovery targeting 5-HT receptors has been developed focusing on antiemetic and treatment for irritable bowel syndrome targeting 5-HT3 receptors and on gastrointestinal motility activation targeting 5-HT4 receptors. Recently, we found a new pharmacological activity mediated 5-HT receptors showing actions of neural regeneration and neurogenesis. Methods: In this study, we are focusing on promoting effect of 5-HT4 receptor-activation on in vivo neural regeneration and neurogenesis in rodent rectal transection and anastomosis (RTA) and intestinal transection and anastomosis (ITA) models. Results: In rat and guinea-pig RTA models, local and oral treatment with mosapride citrate (10–100 l mol L) promoted enteric neural regeneration and neurogenesis at the anastomotic site from neural stem cells for 2 weeks after the surgery. In mouse ITA model, oral treatment with mosapride citrate (100 l mol L) promoted enteric neural regeneration and neurogenesis at the anastomotic site for 1 week after the surgery. Especially, in guinea pig model, the recovery of rectal distension-induced internal anal sphincter (IAS) relaxation reflex response was found for 2 weeks after the surgery. 5-HT4 receptor antagonists, GR113808 (10 l mol L) and SB 207266 (10–50 l mol L) suppressed 5-HT4 receptor activation induced neural regeneration and neurogenesis, and the recovery of IAS relaxation reflex response. In addition, we found mosapride citrate enhanced mobilization of neural stem cells by subcutaneous transplantation of gel sponge experiments in rats. This effect contributes to in vivo neural regeneration and neurogenesis. Furthermore, we confirmed transplanted embryonic neural stem cells of central nervous system from tail vein mobilized at the anastomotic site in mouse ITA model with oral application of mosapride for 1 week. Conclusion: Drug discovery based on the evidence of the present study will propose the possibility contributing to pharmacotherapy for defecation dysfunction after surgery and gastrointestinal motility dysfunction due to decreased enteric neurons in diabetic patients, pseudo-Hirschsprung’s disease patients and elders. 002 Mechanosensitive myenteric neurons in the guinea pig gastric corpus G. MAZZUOLI and M. SCHEMANN Techn. Universität München, Inst. für Human Biologie, Freising, Germany, and TU Munich, Human Biology, Freising, Germany