Introdução: A atresia biliar (AB) é uma doença que se inicia na infância e caracteriza-se por obstrução das vias biliares extra-hepáticas, e por colangiopatia progressiva, de etiologia desconhecida. Seu tratamento consiste numa portoenterostomia, cujo sucesso é variável. Fatores como a idade na portoenterostomia e normalização de bilirrubinas séricas influenciam a sobrevida do paciente com seu próprio fígado. Nosso grupo estuda o envolvimento de uma arteriopatia na etiologia da AB. O fator de crescimento vascular endotelial (VEGF) e seus receptores VEGFR1 e VEGFR2 participam da angiogênese em situações de distúrbio vascular. Objetivo: Quantificar a expressão gênica de VEGFA e seus receptores, relacionando com variáveis moleculares, morfométricas e clínico-laboratoriais associadas com a gravidade da AB e o prognóstico pós-portoenterostomia. Métodos: Estudo de amostras de biópsias em cunha coletadas na laparotomia exploradora de pacientes com AB, na sua forma isolada IgM- para citomegalovírus (n= 32), comparando com lactentes com colestase intra-hepática (CIH, n= 09), pareados por idade. Uma amostra foi ultracongelada (análise molecular) e outra, parafinizada (análises histológica e imunoistoquímica). A expressão dos genes angiogênicos (VEGFA, VEGFR1 e VEGFR2), fibrogênico (MCP1, proteína quimiotática de monócitos) e de reação ductular (CK19, citoqueratina 19), além do gene normalizador, ribossomal 18S, foi medida por PCRq com sondas TaqMan®. As análises morfométricas, a partir de material parafinado, incluindo extensão de fibrose e reação ductular foram mensuradas através do programa Image Pro Plus 6.0. Variáveis clínico-laboratoriais foram prospectivamente coletadas no banco de dados da Unidade de Hepatologia Pediátrica do HCPA. Resultados: VEGFR2 foi menos expresso na AB em comparação com CIH (P <0,001), enquanto não houve diferença significante na expressão de VEGFR1 (P= 0,086) e apenas uma tendência de menor expressão do VEGFA (P= 0,060) no grupo AB. Quanto à expressão de VEGFA e VEGFR2, dois subgrupos de AB foram observados: um com expressão levemente superior ou igual à CIH e outro com expressão 3 vezes menor que a mediana do grupo CIH. Os subgrupos com menor expressão foram denominados loVEGFA e loVEGFR2. Na AB, a expressão do VEGFA teve correlação positiva com a de seus receptores VEGFR1 e VEGFR2 (rs=0,8; P<0,001 e rs=0,5; P=0,001). A expressão de VEGFR2 teve uma forte correlação negativa com a idade na portoenterostomia (rs=-0,6;P=0,001). As bilirrubinas séricas total e direta foram negativamente correlacionadas com VEGFA (rs=-0,5;P=0,007 e rs=-0,4;P=0,033) e VEGFR2 (rs=-0,5;P=0;004 e rs=-0,6;P=0,001). No subgrupo loVEGFR2 a expressão de CK19 estava diminuída, e a de MCP1 não se associou à expressão gênica das moléculas angiogênicas. Conclusões: Na AB há um subgrupo com diminuição na expressão de VEGFA e VEGFR2. A expressão de ambas as moléculas correlacionou-se ao comportamento das variáveis associadas a gravidade da doença e obstrução do fluxo biliar na portoenterostomia, incluindo bilirrubinas séricas e idade. O subgrupo de baixa expressão do VEGFR2 apresentou diminuição da massa de colangiócitos, segundo a expressão do CK 19, mas foi independente da expressão do marcador de fibrose, MCP1. / Background: Biliary atresia (BA) is a disease that begins in infancy and is characterized by complete obstruction of extrahepatic biliary ducts and a progressive cholangiopathy of undefined etiology. Treatment of BA consists of a portoenterostomy, whose success is variable. Factors such as age at portoenterostomy and post-operative normalization of serum bilirubin influence the native liver survival. Our group studies the role of an arteriopathy, and thus of hypoxia, in the etiology of BA. Expression of vascular endothelial growth factor (VEGF) A and its receptors, VEGFR1 and VEGFR2, induce angiogenesis in response to hypoxia. Aim: To assess gene expression of VEGFA and its receptors in BA, correlating with molecular, morphometric and clinical-laboratory variables associated with the disease severity and the post-portoenterostomy prognosis of BA. Methods: Liver biopsy specimens collected at exploratory laparotomy of age-matched patients with isolated, cytomegalovirus IgM-negative BA (n=32) and intrahepatic cholestasis (IHC, n=9) were evaluated. A sample was ultrafrozen (for molecular analysis) and the other was paraffin-embedded (for histological and morphometric analyzes). The expression of genes involved in angiogenic (VEGFA, VEGFR1 and VEGFR2), biliary fibrosis (MCP1 - monocyte chemotaxis protein 1) and cholangiocyte phenotype (CK19 – cytokeratin 19), was measured by TaqMan® probes with qPCR, using ribosomal 18S as the normalizing gene. Extents of fibrosis and ductular reaction were morphometrically assessed using the Image Pro Plus 6.0. Clinical-laboratory variables were collected from the database of the Pediatric Hepatology Unit of Hospital de Clínicas de Porto Alegre. Results: VEGFR2 was less expressed in BA compared with IHC (P <0.001), whereas there was no significant difference in the expression of VEGFR1 (P=0.086) and only a trend of a lower expression of VEGFA (P=0.060) in the BA group. Regarding VEGFA and VEGFR2, two subgroups of BA were defined: low expression, with values 3 times lower than the median of the IHC group, and high expression. In BA, VEGFA expression was positively correlated with that of VEGFR1 and VEGFR2 (rs=0.8, P<0.001; rs=0.58, P=0.001). The expression of VEGFR2 had a strong negative correlation with age at portoenterostomy (rs=-0.6, P=0.001). Total and direct reacting bilirubins were both negatively correlated with VEGFA expression (rs=-0.5, P=0.007; rs=-0.4,P=0.033) and VEGFR2 (rs=-0.5,P=0.004; rs=-0.6,P=0.001). In the low VEGFR2 expression (loVEGFR2) subset, gene expression of CK19, marker of cholangiocyte phenotype, was decreased. MCP1, marker of fibrosis, was not associated with he expression of the angiogenic factors. Conclusion: In BA there is a subset of patients with decreased expression of VEGFA and VEGFR2. The expression of these molecules is correlated with variables associated to disease severity and bile flow obstruction at portoenterostomy, including age and serum bilirubin levels. The loVEGFR2 expression is associated with a decreased cholangiocyte mass as evaluated by CK19 expression, and is independent of the expression of the fibrosis marker MCP1.

Introdução: A atresia biliar (AB) é uma doença que se inicia na infância e caracteriza-se por obstrução das vias biliares extra-hepáticas, e por colangiopatia progressiva, de etiologia desconhecida. Seu tratamento consiste numa portoenterostomia, cujo sucesso é variável. Fatores como a idade na portoenterostomia e normalização de bilirrubinas séricas influenciam a sobrevida do paciente com seu próprio fígado. Nosso grupo estuda o envolvimento de uma arteriopatia na etiologia da AB. O fator de crescimento vascular endotelial (VEGF) e seus receptores VEGFR1 e VEGFR2 participam da angiogênese em situações de distúrbio vascular. Objetivo: Quantificar a expressão gênica de VEGFA e seus receptores, relacionando com variáveis moleculares, morfométricas e clínico-laboratoriais associadas com a gravidade da AB e o prognóstico pós-portoenterostomia. Métodos: Estudo de amostras de biópsias em cunha coletadas na laparotomia exploradora de pacientes com AB, na sua forma isolada IgM- para citomegalovírus (n= 32), comparando com lactentes com colestase intra-hepática (CIH, n= 09), pareados por idade. Uma amostra foi ultracongelada (análise molecular) e outra, parafinizada (análises histológica e imunoistoquímica). A expressão dos genes angiogênicos (VEGFA, VEGFR1 e VEGFR2), fibrogênico (MCP1, proteína quimiotática de monócitos) e de reação ductular (CK19, citoqueratina 19), além do gene normalizador, ribossomal 18S, foi medida por PCRq com sondas TaqMan®. As análises morfométricas, a partir de material parafinado, incluindo extensão de fibrose e reação ductular foram mensuradas através do programa Image Pro Plus 6.0. Variáveis clínico-laboratoriais foram prospectivamente coletadas no banco de dados da Unidade de Hepatologia Pediátrica do HCPA. Resultados: VEGFR2 foi menos expresso na AB em comparação com CIH (P <0,001), enquanto não houve diferença significante na expressão de VEGFR1 (P= 0,086) e apenas uma tendência de menor expressão do VEGFA (P= 0,060) no grupo AB. Quanto à expressão de VEGFA e VEGFR2, dois subgrupos de AB foram observados: um com expressão levemente superior ou igual à CIH e outro com expressão 3 vezes menor que a mediana do grupo CIH. Os subgrupos com menor expressão foram denominados loVEGFA e loVEGFR2. Na AB, a expressão do VEGFA teve correlação positiva com a de seus receptores VEGFR1 e VEGFR2 (rs=0,8; P<0,001 e rs=0,5; P=0,001). A expressão de VEGFR2 teve uma forte correlação negativa com a idade na portoenterostomia (rs=-0,6;P=0,001). As bilirrubinas séricas total e direta foram negativamente correlacionadas com VEGFA (rs=-0,5;P=0,007 e rs=-0,4;P=0,033) e VEGFR2 (rs=-0,5;P=0;004 e rs=-0,6;P=0,001). No subgrupo loVEGFR2 a expressão de CK19 estava diminuída, e a de MCP1 não se associou à expressão gênica das moléculas angiogênicas. Conclusões: Na AB há um subgrupo com diminuição na expressão de VEGFA e VEGFR2. A expressão de ambas as moléculas correlacionou-se ao comportamento das variáveis associadas a gravidade da doença e obstrução do fluxo biliar na portoenterostomia, incluindo bilirrubinas séricas e idade. O subgrupo de baixa expressão do VEGFR2 apresentou diminuição da massa de colangiócitos, segundo a expressão do CK 19, mas foi independente da expressão do marcador de fibrose, MCP1. / Background: Biliary atresia (BA) is a disease that begins in infancy and is characterized by complete obstruction of extrahepatic biliary ducts and a progressive cholangiopathy of undefined etiology. Treatment of BA consists of a portoenterostomy, whose success is variable. Factors such as age at portoenterostomy and post-operative normalization of serum bilirubin influence the native liver survival. Our group studies the role of an arteriopathy, and thus of hypoxia, in the etiology of BA. Expression of vascular endothelial growth factor (VEGF) A and its receptors, VEGFR1 and VEGFR2, induce angiogenesis in response to hypoxia. Aim: To assess gene expression of VEGFA and its receptors in BA, correlating with molecular, morphometric and clinical-laboratory variables associated with the disease severity and the post-portoenterostomy prognosis of BA. Methods: Liver biopsy specimens collected at exploratory laparotomy of age-matched patients with isolated, cytomegalovirus IgM-negative BA (n=32) and intrahepatic cholestasis (IHC, n=9) were evaluated. A sample was ultrafrozen (for molecular analysis) and the other was paraffin-embedded (for histological and morphometric analyzes). The expression of genes involved in angiogenic (VEGFA, VEGFR1 and VEGFR2), biliary fibrosis (MCP1 - monocyte chemotaxis protein 1) and cholangiocyte phenotype (CK19 – cytokeratin 19), was measured by TaqMan® probes with qPCR, using ribosomal 18S as the normalizing gene. Extents of fibrosis and ductular reaction were morphometrically assessed using the Image Pro Plus 6.0. Clinical-laboratory variables were collected from the database of the Pediatric Hepatology Unit of Hospital de Clínicas de Porto Alegre. Results: VEGFR2 was less expressed in BA compared with IHC (P <0.001), whereas there was no significant difference in the expression of VEGFR1 (P=0.086) and only a trend of a lower expression of VEGFA (P=0.060) in the BA group. Regarding VEGFA and VEGFR2, two subgroups of BA were defined: low expression, with values 3 times lower than the median of the IHC group, and high expression. In BA, VEGFA expression was positively correlated with that of VEGFR1 and VEGFR2 (rs=0.8, P<0.001; rs=0.58, P=0.001). The expression of VEGFR2 had a strong negative correlation with age at portoenterostomy (rs=-0.6, P=0.001). Total and direct reacting bilirubins were both negatively correlated with VEGFA expression (rs=-0.5, P=0.007; rs=-0.4,P=0.033) and VEGFR2 (rs=-0.5,P=0.004; rs=-0.6,P=0.001). In the low VEGFR2 expression (loVEGFR2) subset, gene expression of CK19, marker of cholangiocyte phenotype, was decreased. MCP1, marker of fibrosis, was not associated with he expression of the angiogenic factors. Conclusion: In BA there is a subset of patients with decreased expression of VEGFA and VEGFR2. The expression of these molecules is correlated with variables associated to disease severity and bile flow obstruction at portoenterostomy, including age and serum bilirubin levels. The loVEGFR2 expression is associated with a decreased cholangiocyte mass as evaluated by CK19 expression, and is independent of the expression of the fibrosis marker MCP1.

Introdução: A atresia biliar (AB) é uma doença que se inicia na infância e caracteriza-se por obstrução das vias biliares extra-hepáticas, e por colangiopatia progressiva, de etiologia desconhecida. Seu tratamento consiste numa portoenterostomia, cujo sucesso é variável. Fatores como a idade na portoenterostomia e normalização de bilirrubinas séricas influenciam a sobrevida do paciente com seu próprio fígado. Nosso grupo estuda o envolvimento de uma arteriopatia na etiologia da AB. O fator de crescimento vascular endotelial (VEGF) e seus receptores VEGFR1 e VEGFR2 participam da angiogênese em situações de distúrbio vascular. Objetivo: Quantificar a expressão gênica de VEGFA e seus receptores, relacionando com variáveis moleculares, morfométricas e clínico-laboratoriais associadas com a gravidade da AB e o prognóstico pós-portoenterostomia. Métodos: Estudo de amostras de biópsias em cunha coletadas na laparotomia exploradora de pacientes com AB, na sua forma isolada IgM- para citomegalovírus (n= 32), comparando com lactentes com colestase intra-hepática (CIH, n= 09), pareados por idade. Uma amostra foi ultracongelada (análise molecular) e outra, parafinizada (análises histológica e imunoistoquímica). A expressão dos genes angiogênicos (VEGFA, VEGFR1 e VEGFR2), fibrogênico (MCP1, proteína quimiotática de monócitos) e de reação ductular (CK19, citoqueratina 19), além do gene normalizador, ribossomal 18S, foi medida por PCRq com sondas TaqMan®. As análises morfométricas, a partir de material parafinado, incluindo extensão de fibrose e reação ductular foram mensuradas através do programa Image Pro Plus 6.0. Variáveis clínico-laboratoriais foram prospectivamente coletadas no banco de dados da Unidade de Hepatologia Pediátrica do HCPA. Resultados: VEGFR2 foi menos expresso na AB em comparação com CIH (P <0,001), enquanto não houve diferença significante na expressão de VEGFR1 (P= 0,086) e apenas uma tendência de menor expressão do VEGFA (P= 0,060) no grupo AB. Quanto à expressão de VEGFA e VEGFR2, dois subgrupos de AB foram observados: um com expressão levemente superior ou igual à CIH e outro com expressão 3 vezes menor que a mediana do grupo CIH. Os subgrupos com menor expressão foram denominados loVEGFA e loVEGFR2. Na AB, a expressão do VEGFA teve correlação positiva com a de seus receptores VEGFR1 e VEGFR2 (rs=0,8; P<0,001 e rs=0,5; P=0,001). A expressão de VEGFR2 teve uma forte correlação negativa com a idade na portoenterostomia (rs=-0,6;P=0,001). As bilirrubinas séricas total e direta foram negativamente correlacionadas com VEGFA (rs=-0,5;P=0,007 e rs=-0,4;P=0,033) e VEGFR2 (rs=-0,5;P=0;004 e rs=-0,6;P=0,001). No subgrupo loVEGFR2 a expressão de CK19 estava diminuída, e a de MCP1 não se associou à expressão gênica das moléculas angiogênicas. Conclusões: Na AB há um subgrupo com diminuição na expressão de VEGFA e VEGFR2. A expressão de ambas as moléculas correlacionou-se ao comportamento das variáveis associadas a gravidade da doença e obstrução do fluxo biliar na portoenterostomia, incluindo bilirrubinas séricas e idade. O subgrupo de baixa expressão do VEGFR2 apresentou diminuição da massa de colangiócitos, segundo a expressão do CK 19, mas foi independente da expressão do marcador de fibrose, MCP1. / Background: Biliary atresia (BA) is a disease that begins in infancy and is characterized by complete obstruction of extrahepatic biliary ducts and a progressive cholangiopathy of undefined etiology. Treatment of BA consists of a portoenterostomy, whose success is variable. Factors such as age at portoenterostomy and post-operative normalization of serum bilirubin influence the native liver survival. Our group studies the role of an arteriopathy, and thus of hypoxia, in the etiology of BA. Expression of vascular endothelial growth factor (VEGF) A and its receptors, VEGFR1 and VEGFR2, induce angiogenesis in response to hypoxia. Aim: To assess gene expression of VEGFA and its receptors in BA, correlating with molecular, morphometric and clinical-laboratory variables associated with the disease severity and the post-portoenterostomy prognosis of BA. Methods: Liver biopsy specimens collected at exploratory laparotomy of age-matched patients with isolated, cytomegalovirus IgM-negative BA (n=32) and intrahepatic cholestasis (IHC, n=9) were evaluated. A sample was ultrafrozen (for molecular analysis) and the other was paraffin-embedded (for histological and morphometric analyzes). The expression of genes involved in angiogenic (VEGFA, VEGFR1 and VEGFR2), biliary fibrosis (MCP1 - monocyte chemotaxis protein 1) and cholangiocyte phenotype (CK19 – cytokeratin 19), was measured by TaqMan® probes with qPCR, using ribosomal 18S as the normalizing gene. Extents of fibrosis and ductular reaction were morphometrically assessed using the Image Pro Plus 6.0. Clinical-laboratory variables were collected from the database of the Pediatric Hepatology Unit of Hospital de Clínicas de Porto Alegre. Results: VEGFR2 was less expressed in BA compared with IHC (P <0.001), whereas there was no significant difference in the expression of VEGFR1 (P=0.086) and only a trend of a lower expression of VEGFA (P=0.060) in the BA group. Regarding VEGFA and VEGFR2, two subgroups of BA were defined: low expression, with values 3 times lower than the median of the IHC group, and high expression. In BA, VEGFA expression was positively correlated with that of VEGFR1 and VEGFR2 (rs=0.8, P<0.001; rs=0.58, P=0.001). The expression of VEGFR2 had a strong negative correlation with age at portoenterostomy (rs=-0.6, P=0.001). Total and direct reacting bilirubins were both negatively correlated with VEGFA expression (rs=-0.5, P=0.007; rs=-0.4,P=0.033) and VEGFR2 (rs=-0.5,P=0.004; rs=-0.6,P=0.001). In the low VEGFR2 expression (loVEGFR2) subset, gene expression of CK19, marker of cholangiocyte phenotype, was decreased. MCP1, marker of fibrosis, was not associated with he expression of the angiogenic factors. Conclusion: In BA there is a subset of patients with decreased expression of VEGFA and VEGFR2. The expression of these molecules is correlated with variables associated to disease severity and bile flow obstruction at portoenterostomy, including age and serum bilirubin levels. The loVEGFR2 expression is associated with a decreased cholangiocyte mass as evaluated by CK19 expression, and is independent of the expression of the fibrosis marker MCP1.

No description available.

ΣΚΟΠΟΣ: Σήμερα είναι πλέον γνωστό ότι το χολοστατικό ήπαρ είναι πιο ευαίσθητο στην ισχαιμία από το φυσιολογικό ήπαρ. Η ηπατική αναγέννηση μετά μερική ηπατεκτομή (ΡΗχ) ρυθμίζεται από διάφορους παράγοντες οι οποίοι ενεργοποιούν ή αναστέλλουν τον πολλαπλασιασμό των ηπατοκυττάρων. Ένα από τα κυτταρικά στοιχεία που συμβάλλει στην αναγέννηση του ήπατος είναι το ηπατοκύτταρο με χολαγγειακή μορφολογία (DΗ). Από την άλλη μεριά, η απόπτωση φαίνεται να παίζει ένα σημαντικό ρόλο στον κυτταρικό πολλαπλασιασμό και στην ηπατική αναγέννηση. Στην παρούσα μελέτη, μετά από Ρηχ σε φυσιολογικούς ποντικούς και σε άλλους με ίκτερο, με ή χωρίς επιπλέον ισχαιμία, ερευνήσαμε: α) τα ιστοπαθολογικά χαρακτηριστικά της ισχαιμικής ηπατικής βλάβης και αναγέννησης, β) το δυνητικό ρόλο των ηπατοκυττάρων με χολαγγειακή μορφολογία κατά τη διάρκεια της ηπατικής αναγέννησης και γ)την έκφραση των γονιδίων bcl-2 και bax, τα οποία σχετίζονται με την απόπτωση, καθώς και την παρουσία της απόπτωσης. ΥΛΙΚΑ ΚΑΙ ΜΕΘΟΔΟΙ: Η μελέτη συμπεριέλαβε 140 ποντικούς Wistar οι οποίοι χωρίστηκαν σε 14 ομάδες: Ι –control, II - εγχείρηση sham. III-IV-V-παροδικός αγγειακός αποκλεισμός ηπατικής αρτηρίας και πυλαίας φλέβας (ολική ηπατική ισχαιμία) (TLI) για 15-30-60 λεπτά αντίστοιχα. VI - απολίνωση του κοινού χοληδόχου πόρου (BDL) για 10 ημέρες, VII-VIII-ΙΧ-BDL-ΤLI-ΡΗχ. Χ-μερική ηπατεκτομή (ΡΗχ 68%}, XI- ΡΗχ + TLI (30 min), XII-BDL+ΡΗχ, XIII-BDL+ ΡΗχ+TLI (30 min) και XIV- BDL+TLI (30 min) + ΡΗχ. Τα ζώα θυσιάστηκαν 24 και 48 ώρες μετά από Ρηχ, με εξαίρεση τις περιπτώσεις όπου η κατάσταση υγείας ήταν κρίσιμη. Η μελέτη του ηπατικού ιστού περιελάμβανε: (α) Η&Α stain, (β) υβριδισμό in situ (ανίχνευση mRΝΑ των bcl-2 και bax σε παραφίνη, (γ) ανάλυση Western Blot για τη μελέτη των πρυτεϊνικών επιπέδων των bcl-2 και bax, (δ) in situ υβριδισμός (TUNEL) για την ανίχνευση παραγόντων απόπτωσης, (ε) ανοσοϊστοχημικά stains σε παραφίνη (μέθοδος στρεπταβιδίνης-βιοτίνης) με σκοπό να μελετηθεί η έκφραση των ακόλουθων αντιγόνων: κυτοκερατίνη 7 και 19 (χολικός φαινότυπος), ηπατοκυτταρίκή και α1-σντιθρυψίνη (ηπατοκυτταρικός φαινότυπος), βιμεντίνη, CD34, α-φετοπρωτείνη, GST-π(γλουταθειονιν-S-τρανσφεράση-π). Κι67 (κυτταρικός πολλαπλασιασμός), bcl-2 και bax (γονίδια απόπτωσης). ΑΠΟΤΕΛΕΣΜΑΤΑ: Ήπαρ από ποντικούς α)με TLI ανέπτυξε βλάβες ηπατικής ισχαιμίας, β)με BDL βλάβες ηπατικής απόφραξης, γ)με BDL+TLI ανέπτυξαν βλάβες ηπατικής ισχαιμίας μαζί με αλλαγές λόγω απόφραξης των χοληφόρων. Το ποσοστό του επηρεασμένου ηπατικού παρεγχύματος (%ηπατικής νέκρωσης) ήταν υψηλότερο στις ομάδες με TLI+BDL. Σε αυτές τις ομάδες, ο ηπατοκυτταρικός δείκτης (δείκτης πολλαπλασιασμού) του Κi67 ήταν μικρότερος από τον αντίστοιχο της ομάδας χωρίς TLI. Το εύρημα αυτό υποδηλώνει ότι το χολοστστικό ήπαρ είναι πιο ευαίσθητο στην ισχαιμία κι εκδηλώνει μειωμένη αναγεννητική ικανότητα, συγκρινόμενο με μη χολοστατικό ήπαρ. Βιοψίες ήπατος έδειξαν χαρακτήρες ηπατικής αναγέννησης προερχόμενης από τη ζώνη 2, η οποία επεκτεινόταν στη ζώνη 1 και κάποιες φορές στη ζώνη 3. Ανοσοϊστοχημικές αναλύσεις ανέδειξαν κύτταρα θετικά στο ΑΕ1 και κύτταρα θετικά στο ΗΕΡΡΑΡ. Σε όλες τις ομάδες με TLI τα ηπατοκύτταρα χολαγγειακής μορφολογίας εμφανίζονταν κυρίως σε περιοχές περι-πυλαίες. Εξέφραζαν κυτοκερατίνες 19 και 7, ηπατοκυτταρίκή και α1-αντιθρυψίνη ενώ εμφάνιζαν και συνδυασμένη έκφραση CΚ7/CΚ19 και ηπατοκυτταρική ή ΑΕ1/CΚ19 και α1-αντιθρυψϊνη. Το ποσοστό των κυττάρων αυτών ήταν ευθέως ανάλογο με το χρόνο επιβίωσης των ζώων (r=0,354, ρ<0,05). Όσον αφορά την απόπτωση, προ ηπατεκτομής, τα επίπεδα του bcl-2 ( πρωτεΐνη ή mRΝΑ) ήταν υψηλότερα σε ποντικούς με ίκτερο απ' ό,τι στους ποντικούς controls. Επιπλέον τα επίπεδα του bax (πρωτεΐνη ή mRΝΑ) και ο σωματικός δείκτης απόπτωσης (ΑΒΙ) ήταν υψηλότερα σε χολοστατικό ήπαρ. Μετά ηπατεκτομή, παρατηρήθηκε πρώιμη ελάττωση των επιπέδων πρωτεϊνών και mRΝΑ του γονιδίου bcl-2 και μια ύστερη αύξηση του προ-αποπτωτικού γονιδίου bax και του ΑΒΙ, σε σύγκριση με τα controls. ΣΥΜΠΕΡΑΣΜΑΤΑ: Σε περιπτώσεις αποφρακτικού ίκτερου (χολαγγειοπάθεια), η ηπατική ισχαιμία καθιστά το όργανο πιο ευαίσθητο σε ενδεχόμενη βλάβη, καθώς προκαλεί ευρύτερη νέκρωση παρεγχύματος και ελαττώνει το ρυθμό πολλαπλασιασμού των ηπατοκυττάρων, Η διεργασία αναγέννησης του ήπατος επιτελείται κυρίως μέσω του πολλαπλασιασμού μη νεκρωτικών κυττάρων τα οποία εκφράζουν ηπατοκυτταρικά ή χολαγγειακά επιθηλιακά χαρακτηριστικά. Σε τέτοιες περιπτώσεις, το κύριο στοιχείο της ηπατικής αναγέννησης αποτελεί η παρουσία των ηπατοκυττάρων με χολαγγειακή μορφολογία (κύτταρα που εκδηλώνουν φαινότυπο ενδιάμεσο μεταξύ κυττάρων χολαγγειακής και ηπατικής προέλευσης) και η διαφοροποίηση τους σε ώριμα ηπατοκύτταρα. Τέλος, η απόπτωση έχει ρόλο στο χολοστατικό ήπαρ με ή χωρίς επιπλέον ισχαιμία και πιστεύεται ότι μπορεί να συμβάλλει στην παρουσία κατεσταλμένης αναγεννητικής απόκρισης που παρατηρήθηκε σε ήπαρ ποντικών με ίκτερο μετά από μερική ηπατεκτομή. / AIM: Today, it is known that cholestatic liver is more susceptible to ischemia than normal liver. Liver regeneration after partial hepatectomy (PHx) is regulated by several factors that activate or inhibit hepatocyte proliferation. One of the cellular elements that contributes to liver regeneration is ductular hepatocyte (DH). On the other hand, apoptosis seems to play an important role in cellular proliferation and liver regeneration. In this study and. after PHx. in normal and jaundiced rats with or without superimposed ischemia, we assessed a) the histopathologic features of hepatic Ischemic damage and liver regeneration, b) the potential role of ductular hepatocytes during liver regeneration and c) the expression apoptosis-asscciated genes bcl-2 and bax. and the presence of apoptosis, MATERIALS £ METHODS: The study comprised 140 male Wistar rats, assigned randomly in 14-groups: l-controls. ll-sham. Ill-IV-V-cIamping of hepatic artery and portal vein (total liver Ischemia-TLI) for 15-30-60 min respectively. Vl-common bile duct ligation (BDL) for lOdays. VII-VIII-IX-BDL-TLI-X-partial hepatectomy(PHx-68%). XI-PHx+TLI(30min). XU-BDL+PHx. XIII-BDL+PHx+TLI(30min) and XIV-BDL+TLI (30min)+PHx. Animals were sacrificed 24 and 48hrs after PHx unless their condition was critical. Liver tissue evaluation Included: (a) H&E stain, (b) in situ hybridization (detection of bcl-2 and bax mRNA) in paraffin sections, (c) Western blot analysis for the evaluation of bcl-2 and bax protein levels, (d) in situ hybridization (TUNEL) for the detection of apoptotic bodies, and (e) immunohistochemical stains (streptavidin-biotin method) in paraffin sections, in order to evaluate the expression of the following antigens: cytokeratins 7 and 16 (biliary phenotype), Hepatocyte and a 1-antitrypsin (hepatocytic phenotype), vimentin, CD34, alpha-fetoprotein, GST-pi (glutahione-S-trarsferase-pi), Ki67 cell proliferation), bcl-2 and bax (apoptotlc genes). RESULTS: Sections from rats a) with TLI developed changes of liver ischemia, b) with BDL changes of liver obstruction and c) with BDL+TLI developed changes of ischemia together with changes of biliary obstruction. The total liver parenchymal area affected (% liver necrosis} was higher in groups with TLI+BDL. In these groups the Ki67 hepatocytic index (proliferation index) was lower compared to the groups without TLI. This finding implies that cholestatic livers are more vulnerable to ischemia and also display impaired generative capabilities, compared to non-cholestatic ones. Liver biopsies exhibited features of liver regeneration that originated from zone 2, extended to zone 1 and occasionally to zone 3. Immunonistochemical stains revealed cells positive to AE1 and cells positive to HEPPAR. In all groups with TLI, DHs appeared mainly in periportal areas. They expressed cytokeratirs-19 and 7. hepatocyte and al-antrtrypsm and co-expressed CK7/CK1S and Hepatocyte or AE1/CK1S and a 1-antitrypsin The percentage of DHs was directly correlated with the time that animals survived (r=0,354, p<0,05). Regarding apoptosis, before hepatectomy, bcl-2 (protein or mRNA} levels were higher in jaundiced rats vs controls. Furthermore, bax (protein or mRNA) levels and apoptotic body index (ABl) were higher in cholestatic livers. After hepatectomy, there was an early decease in the protein and mRNA levels of antiapoptotic gene bcl-2 and a late increase of proapoptotic gene bax and the A8I. compared to controls. CONCLUSIONS: In cases with obstructive jaundice (cholangiopathy), liver ischemia makes the organ more vulnerable to damage because it causes greater parenchymal necrosis and decreases the degree of hepatocyles proliferation. Liver regenerative process is mediated mainly by proliferation of non-necrotic cells that express hepatocellular or ductular epithelial features. In such cases a main element of liver regeneration is the presence of DHs, cells that display a phenotype intermediate between ductular and hepatocytic origin} and their differentiation to mature hepatocytes. Finally, apoptosis takes place in cholestatic livers with or without superimposed ischemia and may contribute in the impaired regenerative response observed in livers of jaundiced rats after partial hepatectomy.

A atresia biliar (AB) é uma colangiopatia de etiologia indeterminada que leva à necessidade de transplante hepático, mesmo com a realização da Portoenterostomia em tempo hábil. O espessamento da camada média da artéria hepática sugere o envolvimento de um distúrbio angiogênico. Este estudo objetivou avaliar a expressão imunoistoquímica do VEGF A e seus receptores nas estruturas hepatobiliares de pacientes com AB. Nós avaliamos, por método semiquantitativo, a positividade do VEGF A, VEGFR1 e VEGFR2 em biópsia de fígados obtidas por ocasião da Portoenterostomia de crianças com AB (n=52),com (n=14) e sem (n=38) malformação extra-hepáticas. Seviram como controles, pacientes com colestase intra-hepática (CI)\ (n=7). A positividade do VEGFA foi também avaliada em explantes (n=33) e porta hepatis (n=16) de pacientes com AB. Avaliamos morfometricamente as variáveis positividade de CK7 (PCK7) em biópsia de pacientes com AB e a relação espessura da camada média arterial/ diâmetro luminal (REMD) em ramos da artéria hepática de pacientes com AB e de pacientes com CI. Nós encontramos que a positividade do VEGF A foi maior em pacientes com AB por ocasião da Portoenterostomia (p=0, 006) que nos outros grupos, enquanto que nos explantes, a positividade de VEGF A foi maior no parênquima (P<0.001). A positividade do VEGFR2 em ducto biliares e hepatócitos foi menor em pacientes com AB que nos casos de CI (P=0.023 e P=0.011, respectivamente). Maior positividade de CK7 ocorreu em pacientes com artérias e estruturas biliares positivas para VEGF A (P<0.001 e P=0.040, respectivamente). Nos pacientes com AB por ocasião da Portoenterostomia a positividade do VEGF A em estruturas biliares, artérias e hepatócitos correlacionou-se com PCK7 (P=0.031, P=0.031 e P=0.032, respectivamente). O VEGF A foi expresso no porta hepatis de pacientes com AB em artérias e em ductos biliares, principalmente nos pacientes sem malformações extra-hepáticas (P=0.013). A positividade do VEGF A associou-se com a maior REMD (P=0.016 e P=0.044, respectivamente). Nossos achados sugerem que a colangiopatia isquêmica, agravada pela proliferação biliar, ocorre na AB por ocasião da Portoenterostomia, começando no porta hepatis. O espessamento da camada média arterial está associada à expressão do VEGF A. / In Biliary atresia (BA) a cholangiopathy of elusive etiology leads to the need of liver transplantation regardless of timely performance of Portoenterostomy; hepatic arterial medial thickening suggests involvement of a disturbed angiogenesis. This study aimed to evaluate the immunohistochemical expression of VEGF A and its receptors in hepatobiliary structures in BA. We semiquantitatively analyzed the positivity rate of VEGF A, VEGF-R1 and -R2 in liver biopsies obtained at Portoenterostomy from infants with BA (n=52), with (n=14) and without (n=38) extrahepatic malformations. Controls were infants with intrahepatic cholestasis (IC, n=7). VEGF A positivity was also evaluated in explants (n=33) and at porta hepatis (n=16) from patients with BA. We morphometrically assessed the variables percentage of CK-7 positivity (PCK7) in biopsies from patients with BA, and the ratio medial layer thickness/luminal diameter (RMED) in hepatic arterial branches from infants with BA and IC. We found that arterial VEGF A positivity was higher in patients with BA at the time of Portoenterostomy (P=0.006) than in other groups, while explants’parenchyma presented the highest VEGF A positivity (P<0.001). Biliary and hepatocytic VEGFR2 positivity was lower in BA than IC (P=0.023 and P=0.011, respectively). Higher PCK7 occurred in arterial and biliary VEGF A-positive patients (P<0.001 and P=0.040, respectively). Biliary, arterial and hepatocytic VEGF A positivity in BA at Portoenterostomy was correlated with PCK7 (P=0.031, P=0.031 and P=0.032, respectively). VEGF A was expressed at porta hepatis from BA patients in arteries, and, in bile ducts mainly in patients without extrahepatic malformations (P=0.013). Biliary and arterial VEGF A positivity was associated with higher RMED (P=0.016 and P=0.044, respectively). Our findings suggest that an ischemic cholangiopathy, aggravated by biliary proliferation, exists in BA at the time of Portoenterostomy, beginning at porta hepatis. Medial layer thickening is associated with VEGF A expression.

A atresia biliar (AB) é uma colangiopatia de etiologia indeterminada que leva à necessidade de transplante hepático, mesmo com a realização da Portoenterostomia em tempo hábil. O espessamento da camada média da artéria hepática sugere o envolvimento de um distúrbio angiogênico. Este estudo objetivou avaliar a expressão imunoistoquímica do VEGF A e seus receptores nas estruturas hepatobiliares de pacientes com AB. Nós avaliamos, por método semiquantitativo, a positividade do VEGF A, VEGFR1 e VEGFR2 em biópsia de fígados obtidas por ocasião da Portoenterostomia de crianças com AB (n=52),com (n=14) e sem (n=38) malformação extra-hepáticas. Seviram como controles, pacientes com colestase intra-hepática (CI)\ (n=7). A positividade do VEGFA foi também avaliada em explantes (n=33) e porta hepatis (n=16) de pacientes com AB. Avaliamos morfometricamente as variáveis positividade de CK7 (PCK7) em biópsia de pacientes com AB e a relação espessura da camada média arterial/ diâmetro luminal (REMD) em ramos da artéria hepática de pacientes com AB e de pacientes com CI. Nós encontramos que a positividade do VEGF A foi maior em pacientes com AB por ocasião da Portoenterostomia (p=0, 006) que nos outros grupos, enquanto que nos explantes, a positividade de VEGF A foi maior no parênquima (P<0.001). A positividade do VEGFR2 em ducto biliares e hepatócitos foi menor em pacientes com AB que nos casos de CI (P=0.023 e P=0.011, respectivamente). Maior positividade de CK7 ocorreu em pacientes com artérias e estruturas biliares positivas para VEGF A (P<0.001 e P=0.040, respectivamente). Nos pacientes com AB por ocasião da Portoenterostomia a positividade do VEGF A em estruturas biliares, artérias e hepatócitos correlacionou-se com PCK7 (P=0.031, P=0.031 e P=0.032, respectivamente). O VEGF A foi expresso no porta hepatis de pacientes com AB em artérias e em ductos biliares, principalmente nos pacientes sem malformações extra-hepáticas (P=0.013). A positividade do VEGF A associou-se com a maior REMD (P=0.016 e P=0.044, respectivamente). Nossos achados sugerem que a colangiopatia isquêmica, agravada pela proliferação biliar, ocorre na AB por ocasião da Portoenterostomia, começando no porta hepatis. O espessamento da camada média arterial está associada à expressão do VEGF A. / In Biliary atresia (BA) a cholangiopathy of elusive etiology leads to the need of liver transplantation regardless of timely performance of Portoenterostomy; hepatic arterial medial thickening suggests involvement of a disturbed angiogenesis. This study aimed to evaluate the immunohistochemical expression of VEGF A and its receptors in hepatobiliary structures in BA. We semiquantitatively analyzed the positivity rate of VEGF A, VEGF-R1 and -R2 in liver biopsies obtained at Portoenterostomy from infants with BA (n=52), with (n=14) and without (n=38) extrahepatic malformations. Controls were infants with intrahepatic cholestasis (IC, n=7). VEGF A positivity was also evaluated in explants (n=33) and at porta hepatis (n=16) from patients with BA. We morphometrically assessed the variables percentage of CK-7 positivity (PCK7) in biopsies from patients with BA, and the ratio medial layer thickness/luminal diameter (RMED) in hepatic arterial branches from infants with BA and IC. We found that arterial VEGF A positivity was higher in patients with BA at the time of Portoenterostomy (P=0.006) than in other groups, while explants’parenchyma presented the highest VEGF A positivity (P<0.001). Biliary and hepatocytic VEGFR2 positivity was lower in BA than IC (P=0.023 and P=0.011, respectively). Higher PCK7 occurred in arterial and biliary VEGF A-positive patients (P<0.001 and P=0.040, respectively). Biliary, arterial and hepatocytic VEGF A positivity in BA at Portoenterostomy was correlated with PCK7 (P=0.031, P=0.031 and P=0.032, respectively). VEGF A was expressed at porta hepatis from BA patients in arteries, and, in bile ducts mainly in patients without extrahepatic malformations (P=0.013). Biliary and arterial VEGF A positivity was associated with higher RMED (P=0.016 and P=0.044, respectively). Our findings suggest that an ischemic cholangiopathy, aggravated by biliary proliferation, exists in BA at the time of Portoenterostomy, beginning at porta hepatis. Medial layer thickening is associated with VEGF A expression.

A atresia biliar (AB) é uma colangiopatia de etiologia indeterminada que leva à necessidade de transplante hepático, mesmo com a realização da Portoenterostomia em tempo hábil. O espessamento da camada média da artéria hepática sugere o envolvimento de um distúrbio angiogênico. Este estudo objetivou avaliar a expressão imunoistoquímica do VEGF A e seus receptores nas estruturas hepatobiliares de pacientes com AB. Nós avaliamos, por método semiquantitativo, a positividade do VEGF A, VEGFR1 e VEGFR2 em biópsia de fígados obtidas por ocasião da Portoenterostomia de crianças com AB (n=52),com (n=14) e sem (n=38) malformação extra-hepáticas. Seviram como controles, pacientes com colestase intra-hepática (CI)\ (n=7). A positividade do VEGFA foi também avaliada em explantes (n=33) e porta hepatis (n=16) de pacientes com AB. Avaliamos morfometricamente as variáveis positividade de CK7 (PCK7) em biópsia de pacientes com AB e a relação espessura da camada média arterial/ diâmetro luminal (REMD) em ramos da artéria hepática de pacientes com AB e de pacientes com CI. Nós encontramos que a positividade do VEGF A foi maior em pacientes com AB por ocasião da Portoenterostomia (p=0, 006) que nos outros grupos, enquanto que nos explantes, a positividade de VEGF A foi maior no parênquima (P<0.001). A positividade do VEGFR2 em ducto biliares e hepatócitos foi menor em pacientes com AB que nos casos de CI (P=0.023 e P=0.011, respectivamente). Maior positividade de CK7 ocorreu em pacientes com artérias e estruturas biliares positivas para VEGF A (P<0.001 e P=0.040, respectivamente). Nos pacientes com AB por ocasião da Portoenterostomia a positividade do VEGF A em estruturas biliares, artérias e hepatócitos correlacionou-se com PCK7 (P=0.031, P=0.031 e P=0.032, respectivamente). O VEGF A foi expresso no porta hepatis de pacientes com AB em artérias e em ductos biliares, principalmente nos pacientes sem malformações extra-hepáticas (P=0.013). A positividade do VEGF A associou-se com a maior REMD (P=0.016 e P=0.044, respectivamente). Nossos achados sugerem que a colangiopatia isquêmica, agravada pela proliferação biliar, ocorre na AB por ocasião da Portoenterostomia, começando no porta hepatis. O espessamento da camada média arterial está associada à expressão do VEGF A. / In Biliary atresia (BA) a cholangiopathy of elusive etiology leads to the need of liver transplantation regardless of timely performance of Portoenterostomy; hepatic arterial medial thickening suggests involvement of a disturbed angiogenesis. This study aimed to evaluate the immunohistochemical expression of VEGF A and its receptors in hepatobiliary structures in BA. We semiquantitatively analyzed the positivity rate of VEGF A, VEGF-R1 and -R2 in liver biopsies obtained at Portoenterostomy from infants with BA (n=52), with (n=14) and without (n=38) extrahepatic malformations. Controls were infants with intrahepatic cholestasis (IC, n=7). VEGF A positivity was also evaluated in explants (n=33) and at porta hepatis (n=16) from patients with BA. We morphometrically assessed the variables percentage of CK-7 positivity (PCK7) in biopsies from patients with BA, and the ratio medial layer thickness/luminal diameter (RMED) in hepatic arterial branches from infants with BA and IC. We found that arterial VEGF A positivity was higher in patients with BA at the time of Portoenterostomy (P=0.006) than in other groups, while explants’parenchyma presented the highest VEGF A positivity (P<0.001). Biliary and hepatocytic VEGFR2 positivity was lower in BA than IC (P=0.023 and P=0.011, respectively). Higher PCK7 occurred in arterial and biliary VEGF A-positive patients (P<0.001 and P=0.040, respectively). Biliary, arterial and hepatocytic VEGF A positivity in BA at Portoenterostomy was correlated with PCK7 (P=0.031, P=0.031 and P=0.032, respectively). VEGF A was expressed at porta hepatis from BA patients in arteries, and, in bile ducts mainly in patients without extrahepatic malformations (P=0.013). Biliary and arterial VEGF A positivity was associated with higher RMED (P=0.016 and P=0.044, respectively). Our findings suggest that an ischemic cholangiopathy, aggravated by biliary proliferation, exists in BA at the time of Portoenterostomy, beginning at porta hepatis. Medial layer thickening is associated with VEGF A expression.