Andy Chu

Lineage tracing demonstrates no evidence of cholangiocyte epithelial‐to‐mesenchymal transition in murine models of hepatic fibrosis

Whether or not cholangiocytes or their hepatic progenitors undergo an epithelial‐to‐mesenchymal transition (EMT) to become matrix‐producing myofibroblasts during biliary fibrosis is a significant ongoing controversy. To assess whether EMT is active during biliary fibrosis, we used Alfp‐Cre × Rosa26‐YFP mice, in which the epithelial cells of the liver (hepatocytes, cholangiocytes, and their bipotential progenitors) are heritably labeled at high efficiency with yellow fluorescent protein (YFP). Primary cholangiocytes isolated from our reporter strain were able to undergo EMT in vitro when treated with transforming growth factor‐β1 alone or in combination with tumor necrosis factor‐α, as indicated by adoption of fibroblastoid morphology, intracellular relocalization of E‐cadherin, and expression of α‐smooth muscle actin (α‐SMA). To determine whether EMT occurs in vivo, we induced liver fibrosis in Alfp‐Cre × Rosa26‐YFP mice using the bile duct ligation (BDL) (2, 4, and 8 weeks), carbon tetrachloride (CCl4) (3 weeks), and 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC; 2 and 3 weeks) models. In no case did we find evidence of colocalization of YFP with the mesenchymal markers S100A4, vimentin, α‐SMA, or procollagen 1α2, although these proteins were abundant in the peribiliary regions. Conclusion: Hepatocytes and cholangiocytes do not undergo EMT in murine models of hepatic fibrosis. (Hepatology 2011;)

Foxl1-Cre-marked adult hepatic progenitors have clonogenic and bilineage differentiation potential

Isolation of hepatic progenitor cells is a promising approach for cell replacement therapy of chronic liver disease. The winged helix transcription factor Foxl1 is a marker for progenitor cells and their descendants in the mouse liver in vivo. Here, we purify progenitor cells from Foxl1-Cre; RosaYFP mice and evaluate their proliferative and differentiation potential in vitro. Treatment of Foxl1-Cre; RosaYFP mice with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet led to an increase of the percentage of YFP-labeled Foxl1(+) cells. Clonogenic assays demonstrated that up to 3.6% of Foxl1(+) cells had proliferative potential. Foxl1(+) cells differentiated into cholangiocytes and hepatocytes in vitro, depending on the culture condition employed. Microarray analyses indicated that Foxl1(+) cells express stem cell markers such as Prom1 as well as differentiation markers such as Ck19 and Hnf4a. Thus, the Foxl1-Cre; RosaYFP model allows for easy isolation of adult hepatic progenitor cells that can be expanded and differentiated in culture.

Comprehensive miRNA sequence analysis reveals survival differences in diffuse large B-cell lymphoma patients

BackgroundDiffuse large B-cell lymphoma (DLBCL) is an aggressive disease, with 30% to 40% of patients failing to be cured with available primary therapy. microRNAs (miRNAs) are RNA molecules that attenuate expression of their mRNA targets. To characterize the DLBCL miRNome, we sequenced miRNAs from 92 DLBCL and 15 benign centroblast fresh frozen samples and from 140 DLBCL formalin-fixed, paraffin-embedded tissue samples for validation.ResultsWe identify known and candidate novel miRNAs, 25 of which are associated with survival independently of cell-of-origin and International Prognostic Index scores, which are established indicators of outcome. Of these 25 miRNAs, six miRNAs are significantly associated with survival in our validation cohort. Abundant expression of miR-28-5p, miR-214-5p, miR-339-3p, and miR-5586-5p is associated with superior outcome, while abundant expression of miR-324-5p and NOVELM00203M is associated with inferior outcome. Comparison of DLBCL miRNA-seq expression profiles with those from other cancer types identifies miRNAs that were more abundant in B-cell contexts. Unsupervised clustering of miRNAs identifies two clusters of patients that have distinct differences in their outcomes. Our integrative miRNA and mRNA expression analyses reveal that miRNAs increased in abundance in DLBCL appear to regulate the expression of genes involved in metabolism, cell cycle, and protein modification. Additionally, these miRNAs, including one candidate novel miRNA, miR-10393-3p, appear to target chromatin modification genes that are frequent targets of somatic mutation in non-Hodgkin lymphomas.ConclusionsOur comprehensive sequence analysis of the DLBCL miRNome identifies candidate novel miRNAs and miRNAs associated with survival, reinforces results from previous mutational analyses, and reveals regulatory networks of significance for lymphomagenesis.

Large-scale profiling of microRNAs for The Cancer Genome Atlas

The comprehensive multiplatform genomics data generated by The Cancer Genome Atlas (TCGA) Research Network is an enabling resource for cancer research. It includes an unprecedented amount of microRNA sequence data: ∼11 000 libraries across 33 cancer types. Combined with initiatives like the National Cancer Institute Genomics Cloud Pilots, such data resources will make intensive analysis of large-scale cancer genomics data widely accessible. To support such initiatives, and to enable comparison of TCGA microRNA data to data from other projects, we describe the process that we developed and used to generate the microRNA sequence data, from library construction through to submission of data to repositories. In the context of this process, we describe the computational pipeline that we used to characterize microRNA expression across large patient cohorts.

Mysteries of α1-antitrypsin deficiency: emerging therapeutic strategies for a challenging disease

The classical form of α1-antitrypsin deficiency (ATD) is an autosomal co-dominant disorder that affects ~1 in 3000 live births and is an important genetic cause of lung and liver disease. The protein affected, α1-antitrypsin (AT), is predominantly derived from the liver and has the function of inhibiting neutrophil elastase and several other destructive neutrophil proteinases. The genetic defect is a point mutation that leads to misfolding of the mutant protein, which is referred to as α1-antitrypsin Z (ATZ). Because of its misfolding, ATZ is unable to efficiently traverse the secretory pathway. Accumulation of ATZ in the endoplasmic reticulum of liver cells has a gain-of-function proteotoxic effect on the liver, resulting in fibrosis, cirrhosis and/or hepatocellular carcinoma in some individuals. Moreover, because of reduced secretion, there is a lack of anti-proteinase activity in the lung, which allows neutrophil proteases to destroy the connective tissue matrix and cause chronic obstructive pulmonary disease (COPD) by loss of function. Wide variation in the incidence and severity of liver and lung disease among individuals with ATD has made this disease one of the most challenging of the rare genetic disorders to diagnose and treat. Other than cigarette smoking, which worsens COPD in ATD, genetic and environmental modifiers that determine this phenotypic variability are unknown. A limited number of therapeutic strategies are currently available, and liver transplantation is the only treatment for severe liver disease. Although replacement therapy with purified AT corrects the loss of anti-proteinase function, COPD progresses in a substantial number of individuals with ATD and some undergo lung transplantation. Nevertheless, advances in understanding the variability in clinical phenotype and in developing novel therapeutic concepts is beginning to address the major clinical challenges of this mysterious disorder.

A role for microRNA in cystic liver and kidney diseases

The polycystic liver and kidney diseases are a family of disorders with heterogeneous etiologies. Proposed mechanisms of disease include ciliary dysfunction, excess cell proliferation, and altered cell-cell or cell-matrix interactions. In this issue of the JCI, Lee and colleagues provide data to support a novel mechanism for cystogenesis involving microRNA (miRNA) (see the related article beginning on page 3714). They demonstrate that levels of the miRNA miR15a are decreased in livers of patients with autosomal recessive and autosomal dominant polycystic kidney disease (ARPKD and ADPKD, respectively) and congenital hepatic fibrosis as well as in the PKC rat model of ARPKD. This results in increased expression of the cell-cycle regulator Cdc25A, which is a direct target of miR15a, and increased cellular proliferation and cystogenesis in vitro. These findings suggest that other miRNAs may also participate in the molecular pathogenesis of cystic liver and kidney diseases.

Cholangiocyte cilia are abnormal in syndromic and non- syndromic biliary atresia

Biliary atresia (BA) is a neonatal disorder characterized by aggressive fibroinflammatory obliteration of the biliary tract. Approximately 20 percent of BA patients demonstrate left–right laterality defects (syndromic BA). Cilia participate in important physiological functions in cholangiocytes, and as some ciliopathies have been associated with both laterality defects and hepatic fibrosis, we hypothesized that patients with syndromic BA exhibit abnormalities of cholangiocyte cilia that disrupt cholangiocyte homeostasis. Nine BA specimens were studied, including pre-Kasai diagnostic biopsies (n=7) and liver explants (n=2). Five specimens were from patients with laterality defects. These were compared with normal pediatric livers, as well as livers affected by primary sclerosing cholangitis, Wilsons disease, and cardiac cirrhosis. Biopsy sections were stained with antibodies against keratin 19 (a cholangiocyte marker) and acetylated α-tubulin (a cilia marker) and were visualized by confocal microscopy. Computer-assisted relative quantification was used to compare staining of cilia within bile ducts among samples. Surprisingly, cilia in BA specimens were significantly shorter, abnormal in their orientation, and less abundant compared with normal liver and disease controls regardless of the presence of a laterality defect. There are significant abnormalities of cholangiocyte cilia in both syndromic and non-syndromic BA livers compared with normal livers and livers affected by other cholestatic diseases. Although this may result from severe cholestasis or inflammation, it may also reflect common mechanistic pathways in different forms of BA and may have important implications for understanding the progression of the disease.

Genome‐wide microRNA and messenger RNA profiling in rodent liver development implicates mir302b and mir20a in repressing transforming growth factor‐beta signaling

MicroRNAs (miRNAs) are recently discovered small RNA molecules that regulate developmental processes, such as proliferation, differentiation, and apoptosis; however, the identity of miRNAs and their functions during liver development are largely unknown. Here we investigated the miRNA and gene expression profiles for embryonic day (E)8.5 endoderm, E14.5 Dlk1+ liver cells (hepatoblasts), and adult liver by employing Illumina sequencing. We found that miRNAs were abundantly expressed at all three stages. Using K‐means clustering analysis, 13 miRNA clusters with distinct temporal expression patterns were identified. mir302b, an endoderm‐enriched miRNA, was identified as an miRNA whose predicted targets are expressed highly in E14.5 hepatoblasts but low in the endoderm. We validated the expression of mir302b in the endoderm by whole‐mount in situ hybridization. Interestingly, mir20a, the most highly expressed miRNA in the endoderm library, was also predicted to regulate some of the same targets as mir302b. We found that through targeting Tgfbr2, mir302b and mir20a are able to regulate transforming growth factor beta (TGFβ) signal transduction. Moreover, mir302b can repress liver markers in an embryonic stem cell differentiation model. Collectively, we uncovered dynamic patterns of individual miRNAs during liver development, as well as miRNA networks that could be essential for the specification and differentiation of liver progenitors. (HEPATOLOGY 2013)

Is severe progressive liver disease caused by alpha-1-antitrypsin deficiency more common in children or adults?

The classical form of alpha‐1‐antitrypsin deficiency (A1ATD) is known to cause liver disease in children and adults, but there is relatively little information about the risk of severe, progressive liver disease and the need for liver transplantation. To better understand how newly evolving pharmacological, genetic, and cellular therapies may be targeted according to risk for progressive liver disease, we sought to determine the age distribution of A1ATD as a cause of severe liver disease, as defined by the need for liver transplantation. Using 3 US liver transplantation databases for the period 1991‐2012, we found 77.2% of 1677 liver transplants with a reported diagnosis of A1ATD were adults. The peak age range was 50‐64 years. Using 2 of the databases which included specific A1AT phenotypes, we found that many of these adults who undergo liver transplantation with A1ATD as the diagnosis are heterozygotes and have other potential causes of liver disease, most notably obesity and ethanol abuse. However, even when these cases are excluded and only ZZ and SZ phenotypes are considered, severe liver disease requiring transplantation is more than 2.5 times as likely in adults. The analysis also showed a markedly increased risk for males. In the pediatric group, almost all of the transplants are done in children less than 5 years of age. In conclusion, A1ATD causes progressive liver disease most commonly in adults with males in the highest risk category. In the pediatric group, children less than 5 years of age are highest in risk. These results suggest that A1ATD most commonly causes liver disease by mechanisms similar to age‐dependent degenerative diseases and more rarely in children by powerful modifiers. Liver Transplantation 22 886–894 2016 AASLD

Capitalizing on the Autophagic Response for Treatment of Liver Disease Caused by Alpha-1-Antitrypsin Deficiency and Other Genetic Diseases

Alpha-1-antitrypsin deficiency (ATD) is one of the most common genetic causes of liver disease and is a prototype of liver diseases caused by the pathologic accumulation of aggregated mutant alpha-1-antitrypsin Z (ATZ) within liver cells. In the case of ATD-associated liver disease, the resulting “gain-of-function” toxicity can lead to serious clinical manifestations, including cirrhosis and hepatocellular carcinoma. Currently, the only definitive therapy for ATD-associated liver disease is liver transplantation, but recent efforts have demonstrated the exciting potential for novel therapies that target disposal of the mutant protein aggregates by harnessing a cellular homeostasis mechanism called autophagy. In this review, we will summarize research advances on autophagy and genetic liver diseases. We will discuss autophagy enhancer strategies for liver disease due to ATD and another genetic liver disease, inherited hypofibrinogenemia, caused by the proteotoxic effects of a misfolded protein. On the basis of recent evidence that autophagy plays a role in cellular lipid degradation, we also speculate about autophagy enhancer strategies for treatment of hepatic lipid storage diseases such as cholesterol ester storage disease.