Type V collagen expression during hepatic fibrosis progression in biliary atresia

Abstract

Biliary atresia (BA) is a specific cholestatic liver disease of the child, with etiology and physiopathology not completely established. Its precocious and gradual evolution for hepatic fibrosis, despite of the accomplishment of an effective bile derivation, is responsible for positioning it as the main indication of pediatric liver transplantation. The understanding of the mechanisms of fibrogenesis perpetuation is the key to establish effective treatments for chronic hepatopathies. During the progression of hepatic fibrosis, there is great interaction among cellular elements (myofibroblasts, inflammatory cells), the cytokines involved in its activation and modulation (mainly TGF beta) and the proper components of the extracellular matrix (different types of collagens, molecules of adhesion and chemotaxis). Recent studies approaching the importance of the extracellular matrix in the promotion and perpetuation of chronic inflammatory processes with development of tissue fibrosis have demonstrated the importance of the participation of type V collagen as a regulator of fibrogenesis and as a self-antigen in these models. Our project aims to study the function of type V collagen in pathogenesis of liver fibrosis in patients with BA submitted to bile derivation by Kasai procedure, to establish deposition patterns of type V collagen in the initial and advanced phases of the illness, to correlate the deposition of type V collagen to total hepatic fibrosis, to the deposition of other fibril (types I) and non-fibrils (type IV) collagens in the liver, to the tissue expression of pro-fibrotic citocina TGF beta and to the postoperative clinical results. Patients with definitive diagnosis of BA who have been submitted to liver biopsies, surgical treatment (Kasai procedure and liver transplantation) and clinical follow-up in the Pediatric Surgery Department of the Medical School of São Paulo University (HCFMUSP) in the last 20 years will be classified by their postoperative outcome (success of the bile derivation and time interval to hepatic dysfunction and transplantation). For the histopathological analysis, biopsies fragments carried through previously at three sequential moments will be used: in the initial diagnostic investigation, in the Kasai operation and in the liver transplantation. Biopsies specimens in children with normal hepatic histology (corresponding to the free edge in resections of hepatic tumors) will be selected for control-group. The Commission of Ethics in Research of the HCFMUSP has approved the study project. Indirect immunofluorescence assays with type-specific antibodies will be used for evaluation of the tissue distribution and quantification of type V, type IV and type I collagens, as previously standardized. For quantification of total hepatic fibrosis area, picrosirius stain method will be carried through. The quantitative parameters will be obtained by digital morphometric analysis, using photographic digitalization of the images of microscopy and computational calculation of the percentage of area marked specifically in relation to the delimited total area. For marking of the tissue expression of pro-fibrotic citocina TGF beta, it will be used immunohistochemic assay and quantitative analysis for point-counting technique. Descriptive anatomopathological datas regarding the compartimental distribution of the collagens type V, IV and I in the patients with BA will be related to the group-control (normal histological pattern) and to the evaluative phases of hepatic fibrosis according to half-quantitative classification. The quantitative data will be expressed as the average and shunting line-standard of the fractions of area calculated for each collagen marker and as the average and shunting line standard of the density of expression of the TGF, allowing to correlate the markers between themselves, to the progressive evolution of liver fibrosis and to the patient clinical outcome. (AU)