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

open16noIntestinal 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. Surgical samples of left colon from non-stenotic SL [a parts per thousand currency sign 3 years, n = 9] and LL [a parts per thousand yen 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 [alpha-SMA], desmin, vimentin], and proliferation [proliferating nuclear antigen [PCNA]] by western blot and/or immunolabelling. 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. 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.openIppolito, Chiara; Colucci, Rocchina; Segnani, Cristina; Errede, Mariella; Girolamo, Francesco; Virgintino, Daniela; Dolfi, Amelio; Tirotta, Erika; Buccianti, Piero; Di Candio, Giulio; Campani, Daniela; Castagna, Maura; Bassotti, Gabrio; Villanacci, Vincenzo; Blandizzi, Corrado; Bernardini, NunziaIppolito, Chiara; Colucci, ROCCHINA LUCIA; Segnani, Cristina; Errede, Mariella; Girolamo, Francesco; Virgintino, Daniela; Dolfi, Amelio; Tirotta, Erika; Buccianti, Piero; Di Candio, Giulio; Campani, Daniela; Castagna, Maura; Bassotti, Gabrio; Villanacci, Vincenzo; Blandizzi, Corrado; Bernardini, Nunzi

Effect of dipyridamole on gap junctions regulation in diseased myocardium

Gap junctions (GJ) mediate electrical coupling between cardiac myocytes, allowing the spreading of the electrical wave responsible for synchronized contraction [1]. GJ function can be regulated by modulation of connexon densities on membranes, connexin (Cx) phosphorylation, trafficking and degradation. Recent studies showed that adenosine involves Cx43 turn-over in A1 receptor-dependent manner [2], and dipyridamole increases GJ coupling and amount of Cx43 in endothelial cells [3]. As the abnormalities in GJ organization and regulation have been implicated in diseased myocardium [1], the aim of the present study was to assess the regional expression of molecules involved in GJ regulation in a model of left ventricular disfunction (LVD). For this purpose the distribution and quantitative expression of Cx43, its phosphorylated form pS368-Cx43, PKC phosphorylated substrates, RhoA and A receptors, were investigated in experimental models of right ventricular-pacing induced LVD, undergoing concomitant dipyridamole therapy or placebo, and compared with healty myocardium obtained from sham operated minipigs. Results demonstrates that an altered pattern of factors involved in Cx43-made GJ regulation is present in ventricular myocardium with left ventricular dysfunction. Moreover, the dipyridamole treatment, that results in an improvement of heart function, seems to act also modulating expression and activation of these factors

Dipyridamole increases Cx43 expression in heart muscle cells through Adenosine 2A receptor/PKC pathway

Cx43, a predominant connexin in the heart, forms gap junctions (GJs) that facilitate electrical cell-cell coupling and hemichannels that represent a pathway for the exchange of ions and metabolites between cytoplasm and the extracellular milieu. Our recent results (1) demonstrated that an altered distribution and quantitative expression of factors involved in Cx43-made GJ regulation as Cx43, its phosphorylated form pS368-Cx43, PKC phosphorylated substrates, and adenosine 2A receptor (A2AR) are present in ventricular myocardium with left ventricular dysfunction. Moreover, dipyridamole treatment, which shows a mild protective role on left ventricular function, seems to act through re-modulating the expression and activation of these factors. The role of these factors on signal transduction cascade triggered by dipyridamole was evaluated in this study by pharmacological and immunoistochemical experiments using the rat cardiomyoblast cell line H9c2. The treatment of H9c2 cells with dipyridamole enhanced the expression of Cx43, A2AR and PKC activity while induced a decrease of pS368-Cx43. Interestingly, we found that the A2AR activation was a prerequisite for the effects of dipyridamole, in fact, the pre-treatment with CSC, a selective A2AR receptor antagonist, abolished its effects on the expression of these factors

Tissue remodelling in the colonic wall of patients with ulcerative colitis

Inflammation-driven tissue remodelling may develop to fibrotic rearrangement. With regard to inflammatory bowel diseases, fibrotic remodelling has been evaluated in Crohn’s disease, while little attention to such processes has been paid to ulcerative colitis (UC) [1]. The present study evaluated the distribution of connective tissue and angiogenesis in colon of patients with UC, paying particular attention to the tonaca muscularis, which is poorly considered in histopathological studies. Full-thickness left colonic samples were obtained from 10 patients with established, severe and pharmacologically unresponsive UC, who underwent bowel resection. Routine histology, histochemistry and immunohistochemistry were conducted in paraffin cross-sections. The distribution of collagen and elastic fibers was evaluated and quantified by both histochemical (Van Gieson, orcein, Verroheff staining) and immunohistochemical (anti-collagen I and III, anti-elastin) assays. The vascular network pattern was studied by anti-CD31 and nestin immunostaining. For comparison, the same evaluations were performed in healthy colonic control samples obtained from 10 subjects, who underwent surgery for uncomplicated colon cancer. A significant increase in collagen fibers and a decrease in elastin content were detected in the UC inflamed colon, as compared with controls. In particular, enhanced collagen deposition (mainly collagen type III) were found at level of submucosa, and tonaca muscularis within the longitudinal muscle (mainly along the serosal side) and circular muscle layer, and in perivascular connective tissue. By contrast, elastic fibers were significantly reduced throughout the whole muscle compartment, with particular regard for the myenteric ridge. Microvessel density was significantly higher in both submucosa and tonaca muscularis of colonic samples from UC patients compared with those from healthy control individuals. The present findings indicate that a significant tissue remodelling occurs in the inflamed colonic wall in patients with UC, also at the level of muscle layers. This rearrangement of the connective fibers and vascular network, together with the known alterations affecting the myenteric neurons and interstitial cells of Cajal, may contribute to the development of enteric dysmotility, and, accordingly, to the serious digestive symptoms which afflict patients with UC

Deranged expression of smooth muscle molecules in colonic tunica muscularis of DD patients

The pathogenesis of diverticular disease (DD) seems to be a result of a complex interaction between exposure to a low-fibre diet, possible genetic influence, coexistence of other bowel disease and impact of medicine use. These conditions may lead to alterations in colonic pressure and motility [1]. To date, histopatological studies in the field of disturbances in intestinal motility have been mainly focused on the enteric nervous system and interstitial cells of Cajal, which have been found to be both variously affected in different conditions of gut dysmotility. By contrast, although smooth muscle cells (SMCs) are well recognized as the final effectors of the enteric neuromuscular units and have been extensively studied by electrophysiological experiments in colonic motility dysfunction, scarce attention has been paid to the molecular abnormalities of enteric musculature in gut dysmotility, and, in particular, data on SMCs in DD are fairly lacking. Accordingly, the aim of the present study was to evaluate the expression patterns of molecules involved in the contractile functions of SMCs within the colonic tunica muscularis from patients with DD. In particular, we examined the expression of the following molecules by immunohistochemistry and image analysis: Cx26 and Cx43, which are prominent components of gap junctions in human colonic SMCs [2], and an array of signaling molecules, which are known to regulate the functions of gap junctions and the contractile activity of SMCs [3,4,5]. The immunohistochemical analysis revealed significant abnormalities in DD samples, concerning both the expression and distribution patterns of most of the investigated molecular factors. Indeed, Cx26, Cx43, PKCps and RhoA appeared to be markedly deranged in all DD colonic samples compared to normal ones, except for α-SMA and pS368-Cx43. The present results provide the first evidence of an altered pattern of factors involved in smooth muscle contractility at level of tunica muscularis SMCs of DD patients. These abnormalities may contribute to the altered motility function, which characterize the colonic tunica muscularis of patients with DD

Gastrointestinal neuromuscular disease: methodological and ontological issues in histopathological analysis

The term gastrointestinal neuromuscular disease comprises a heterogeneous group of chronic conditions associated with impaired bowel motility. Gastrointestinal motor dysfunctions, differening for etiopathogenic mechanisms, pathologic lesions, and region of gut involvement (e.g., irritable bowel syndrome, slow transit constipation, inflammatory bowel disease, diverticular disease), represent a relevant matter for public health: in fact, they are very common, can be disabling, and induce major social and economic burdens. These motor disturbances are presumed or proven to arise as a result of dysfunctional enteric neuromuscular apparatus set up by myenteric ganglionic cells, interstitial cells of Cajal and smooth muscle cells of the muscularis propria. Despite the presence of intestinal dysmotility in the clinical phenotype of these patients, scarce attention has been paid to the morphological arrangements of the enteric neuromuscular apparatus. Furthermore, standards for histopathological reports remain relatively neglected resulting in significant differences in applied methodologies which confound the reliable delineation of normality and, as a consequence, the specificity of particular pathological changes for disease. Thus, in order to overcome the lack or heterogeneity of current data, to get normative data and delineation of abnormality, careful morphological examinations and development of standardized procedures are particularly required in the field of gastrointestinal neuromuscular pathology, as recently suggested