Padmini Malladi

Osteopontin is induced by hedgehog pathway activation and promotes fibrosis progression in nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a leading cause of cirrhosis. Recently, we showed that NASH‐related cirrhosis is associated with Hedgehog (Hh) pathway activation. The gene encoding osteopontin (OPN), a profibrogenic extracellular matrix protein and cytokine, is a direct transcriptional target of the Hh pathway. Thus, we hypothesize that Hh signaling induces OPN to promote liver fibrosis in NASH. Hepatic OPN expression and liver fibrosis were analyzed in wild‐type (WT) mice, Patched‐deficient (Ptc+/−) (overly active Hh signaling) mice, and OPN‐deficient mice before and after feeding methionine and choline–deficient (MCD) diets to induce NASH‐related fibrosis. Hepatic OPN was also quantified in human NASH and nondiseased livers. Hh signaling was manipulated in cultured liver cells to assess direct effects on OPN expression, and hepatic stellate cells (HSCs) were cultured in medium with different OPN activities to determine effects on HSC phenotype. When fed MCD diets, Ptc+/− mice expressed more OPN and developed worse liver fibrosis (P < 0.05) than WT mice, whereas OPN‐deficient mice exhibited reduced fibrosis (P < 0.05). In NASH patients, OPN was significantly up‐regulated and correlated with Hh pathway activity and fibrosis stage. During NASH, ductular cells strongly expressed OPN. In cultured HSCs, SAG (an Hh agonist) up‐regulated, whereas cyclopamine (an Hh antagonist) repressed OPN expression (P < 0.005). Cholangiocyte‐derived OPN and recombinant OPN promoted fibrogenic responses in HSCs (P < 0.05); neutralizing OPN with RNA aptamers attenuated this (P < 0.05). Conclusion: OPN is Hh‐regulated and directly promotes profibrogenic responses. OPN induction correlates with Hh pathway activity and fibrosis stage. Therefore, OPN inhibition may be beneficial in NASH (HEPATOLOGY 2011)

Novel mechanism of fetal hepatocyte injury in congenital alloimmune hepatitis involves the terminal complement cascade

Evidence suggests that most neonatal hemochromatosis (NH) is the phenotypic expression of gestational alloimmune fetal liver injury. Gestational alloimmune diseases are induced by the placental passage of specific reactive immunoglobulin G and often involve the activation of fetal complement by the classical pathway leading to the formation of membrane attack complex (MAC) as the effector of cell injury. We examined liver specimens from cases of NH, from cases of non‐NH liver disease, and from infants without liver disease to determine if they would provide evidence that MAC is involved in hepatocyte injury. Sections were immunostained with anti‐human C5b‐9 complex, the terminal complement cascade (TCC) neoantigen formed in the assembly of MAC. Fetal liver injury in NH cases is associated with a severe loss of hepatocytes. In all NH cases examined, most remaining hepatocytes showed intense staining for TCC neoantigen, whereas hepatocytes in non‐NH liver disease cases showed variable light staining. The percentage of hepatocytes containing TCC neoantigen in NH was much greater than that in non‐NH liver disease, and there was no overlap between the groups. Findings in both groups suggest that hepatocytes have mechanisms to protect against MAC, including a biliary pathway for its excretion. Conclusion: The finding that all cases of proven NH contained TCC neoantigen far in excess of cases of other neonatal liver diseases suggests that a single process, namely congenital alloimmune hepatitis, is the principal cause of NH. MAC‐mediated alloimmune injury in congenital alloimmune hepatitis is a novel mechanism of liver injury that results from an interplay of maternal adaptive immunity and fetal innate immunity. HEPATOLOGY 2010

Neonatal hemochromatosis: is it an alloimmune disease?

Neonatal hemochromatosis (NH) has been defined clinically as severe neonatal liver disease in association with extrahepatic siderosis in a distribution similar to that seen in HFE-associated hereditary hemochromatosis (1). Though it is generally considered a rare disease, it is one of the most commonly recognized causes of liver failure in the neonate and a frequent indication for liver transplantation in the first 3 months of life (2–4). Its etiology and pathogenesis are as yet unknown. Indeed, it has been considered to be a syndrome in which a number of primary etiologies, such as infection, genetic-metabolic disease and toxic insult, lead to a common phenotype. We have hypothesized that much, if not all, NH is a consequence of gestational alloimmune disease. This review presents the clinical evidence leading to this hypothesis, the data we have collected in its support, and the direction our investigations are taking to ultimately prove or disprove it.

Mitochondrial Reactive Oxygen Species Signal Hepatocyte Steatosis by Regulating the Phosphatidylinositol 3-Kinase Cell Survival Pathway

Abnormal dietary intake of macronutrients is implicated in the development of obesity and fatty liver disease. Steatosis develops in cultured hepatocytes exposed to medium containing either a high concentration of long chain free fatty acids (HFFA) or medium deficient in methionine and choline (MCD). This study examined the mitochondrial reactive oxygen species (ROS)-dependent regulation of the phosphoinositol (PI) 3-kinase pathway in steatosis induced by exposure of AML-12 mouse hepatocytes to MCD or HFFA medium. Exposure to either MCD or HFFA medium resulted in increased production of superoxide anions and H2O2, transduction of the PI 3-kinase pathway and steatosis. Inhibition of PI 3-kinase with LY294002 prevented steatosis. Pharmacologically inhibiting electron transport chain complex III production of ROS prevented activation of PI 3-kinase during macronutrient perturbation, whereas pharmacologically promoting electron transport chain complex III ROS production activated PI 3-kinase independent of nutrient input. The data suggest that H2O2 is the ROS species involved in signal transduction; promoting the rapid conversion of superoxide to H2O2 does not inhibit PI 3-kinase pathway activation during nutrient perturbation, and exogenous H2O2 activates it independent of nutrient input. In addition to transducing PI 3-kinase, the ROS-dependent signal cascade amplifies the PI 3-kinase signal by maintaining phosphatase and tensin homolog in its inactive phosphorylated state. Knockdown of phosphatase and tensin homolog by small interfering RNA independently activated the PI 3-kinase pathway. Our findings suggest a common path for response to altered nutrition involving mitochondrial ROS-dependent PI 3-kinase pathway regulation, leading to steatosis.

Expression of Osteopontin Correlates with Portal Biliary Proliferation and Fibrosis in Biliary Atresia

The acquired or perinatal form of biliary atresia is a Th1 fibro-inflammatory disease affecting both the extrahepatic and intrahepatic bile ducts. Osteopontin (OPN) is a Th1 cytokine implicated in several fibro-inflammatory and autoimmune diseases. We examined the expression of OPN in acquired biliary atresia in comparison to normal liver and several pediatric cholestatic liver diseases. We also assessed OPN expression by cultured human bile duct epithelial cells. We found that liver OPN mRNA and protein expression were significantly increased in biliary atresia versus normal and other cholestatic diseases. OPN expression in biliary atresia was localized to epithelium of proliferating biliary structures (ductules and/or ducts) and bile plugs contained therein. No portal biliary OPN expression could be demonstrated in normal liver, syndromic biliary atresia, biliary obstruction not due to biliary atresia, and idiopathic neonatal hepatitis. OPN expression by human bile duct epithelial cells in culture was responsive to IL-2 and TNF-α. Our results demonstrate an up-regulation of OPN expression by interlobular biliary epithelium in biliary atresia, which correlates with biliary proliferation and portal fibrosis. These findings suggest a role for OPN in the pathogenesis of biliary atresia.

Gestational Alloimmune Liver Disease in Cases of Fetal Death

OBJECTIVE To determine whether alloimmune liver disease can be identified as a cause of fetal death. STUDY DESIGN This is a retrospective examination of the autopsy tissue of 6 stillborn fetuses and 2 extreme preterm infants (gestational age, 20 to 34 weeks) drawn from families referred for suspected neonatal hemochromatosis. Thirteen appropriate nondisease controls and 8 cases of neonatal acute liver failure with known etiology were also examined. Liver sections were immunostained using anti-human C5b-9 complex. RESULTS All of the study cases had died with no preceding evidence of fetal distress. Histopathology showed findings of acute liver injury, including global hepatocyte necrosis with minimal reticulum collapse and no fibrosis. Hepatocytes in cases stained strongly positively for C5b-9 complex, suggesting premortem lgG complement-mediated liver injury. Hepatocytes in acute liver failure case controls did not demonstrate a similar mechanism of liver injury. CONCLUSIONS Alloimmune liver disease is sometimes associated with fetal death.

DETECTION OF A NOVEL DNA POLYMORPHISM IN THE β-GLOBIN CLUSTER AND EVIDENCE FOR SITE-SPECIFIC RECOMBINATION

Within the εγδβ-globin gene region a non-random association of polymorphic restriction sites 5′ and 3′ to the δ globin gene has been observed. Between these two clusters lies a 9 kb region including the δ-globin gene which may contain sites of increased recombination. Analysis of DNA from an Albanian family revealed a novel Rsa I site 550 bp 5′ to the β-globin gene within the 9 kb region. One maternal and one paternal chromosome were identical at 9 polymorphic sites but differed at the Rsa site. This finding suggests that this polymorphism is randomly associated with previously defined haplotypes. Population screening showed the presence of this site in people of northern European, Mediterranean, Middle Eastern, African, Southeast Asian and Asian Indian descent, with an overall frequency of 0.39. DNA regions with βA, βS,βE and β-thalassemia alleles carrying and lacking the Rsa site were also identified. In one individual sequence analysis showed 28 alternating purine-pyrimidine dinucleotides in the region containing the Rsa site, rather than the 26 previously found in other individuals, suggesting that unequal crossing-over in this immediate region may lead to randomization of 5′ and 3′ polymorphic clusters.

Infantile Cirrhosis, Growth Impairment, and Neurodevelopmental Anomalies Associated with Deficiency of PPP1R15B.

OBJECTIVE To assess the utility of whole-exome sequencing (WES) in a sibling pair with undetermined liver disease and describe the phenotype associated with mutations discovered therein. STUDY DESIGN Next-generation WES was performed on 2 siblings (S1 and S2) who were born to nonconsanguineous parents of European extraction. Both siblings developed cirrhosis of indeterminate etiology and required liver transplantation; S1 at 7 months and S2 at 22 months. RESULTS Sequencing of germline DNA identified compound heterozygous mutations in PPP1R15B resulting in increased levels of phosphorylated eukaryotic translation initiation factor 2α. CONCLUSIONS The first demonstration of PPP1R15B associated with liver disease expands the phenotypic spectrum of PPP1R15B related diseases. Our findings validate the application of WES in the diagnosis of children with undetermined liver disease. Understanding the genetic basis of liver disease may allow the development of targeted therapies for treatment and adequate counseling of families.

Elaboration of tubules with active hedgehog drives parenchymal fibrogenesis in gestational alloimmune liver disease

Gestational alloimmune liver disease (GALD) produces severe neonatal liver disease that is notable for paucity of hepatocytes, large numbers of parenchymal tubules, and extensive fibrosis. Liver specimens from 19 GALD cases were studied in comparison with 14 infants without liver disease (normal newborn liver; NNL) to better understand the pathophysiology that would produce this characteristic histopathology. GALD liver parenchyma contained large numbers of tubules comprising epithelium expressing KRT7/19, EPCAM, and SOX9, suggesting biliary progenitor status. Quantitative morphometry demonstrated that in GALD, the area density of KRT19+ tubules was 16.4 ± 6.2 versus 2.0 ± 2.6 area% in NNL (P < .0001). Functional hepatocyte mass was markedly reduced in GALD, 16.3 ± 6.2 versus 61.9 ± 11.0 area% of CPS1+ cells in NNL (P < .0001). A strong inverse correlation was established between CPS1+ area density and KRT19+ area density (r(2) = 0.66, P < .0001). Tubules showed active hedgehog signaling as determined by SHH and nuclear GLI2 expression and expressed the profibrogenic cytokine SPP1. SPP1 protein content and SPP1 expression were greater in GALD than NNL (15- and 13-fold respectively; P = .002). GALD liver contained large numbers of activated myofibroblasts and showed greater than 10-fold more fibrosis than NNL. The extent of fibrosis correlated with the area density of KRT19+ tubules (r(2) = 0.387, P = .001). The data support a pathogenic model in which immune injury to fetal hepatocytes provides a stimulus for expansion of parenchymal tubules, which, by way of Hh activation, produce fibrogenic signals leading to vibrant fibrosis.

Gestational autoimmune disease in newborns with an indeterminate cause of death following a complete autopsy

Gestational alloimmune liver disease (GALD) is the result of neonatal complement-mediated severe liver injury mediated by maternal alloantibodies, which is detected by immunohistochemistry staining for the complement C5b-9 complex. GALD leads to the neonatal hemochromatosis (NH) phenotype, which also shows extrahepatic siderosis, and can result in neonatal death. At autopsy, the histologic damage of the liver in GALD may be subtle and misinterpreted as non-specific post-mortem changes, resulting in the cause of death classified as indeterminate. We reviewed the pathologic diagnoses from autopsy material from 1996 to 2011 of infants 0-90 days of age from our institution. Liver samples were stained with H&E, trichrome and for C5b-9. 13 cases originally diagnosed as indeterminate cause of death were identified and divided in 3 groups: (1) No clinical or autopsy-derived diagnoses (n = 7), (2) Defined clinical diagnoses but no cause of death determined at autopsy (n = 2), and (3) Liver disease, but no clinical or autopsy diagnoses to establish the cause of the liver injury (n = 4). On reexamination, all group 1 and 3 cases were reclassified as GALD, based on a positive C5b-9 stain. Group 2 cases were not GALD, retaining the original, clinically-based cause of death. We conclude that, in cases of indeterminate cause of neonatal death, very careful examination for hepatocyte injury/necrosis, extrahepatic siderosis, liver fibrosis and/or C5b-9 stain should be considered.