Identification of Lineage-Specific Transcription Factors That Prevent Activation of Hepatic Stellate Cells and Promote Fibrosis Resolution.

BACKGROUND & AIMS: Development of liver fibrosis is associated with activation of quiescent hepatic stellate cells (HSCs) into collagen type I-producing myofibroblasts (activated HSCs). Cessation of liver injury often results in fibrosis resolution and inactivation of activated HSCs/myofibroblasts into a quiescent-like state (inactivated HSCs). We aimed to identify molecular features of phenotypes of HSCs from mice and humans. METHODS: We performed studies with LratCre, Ets1-floxed, Nf1-floxed, Pparγ-floxed, Gata6-floxed, Rag2-/-γc-/-, and C57/Bl6 (control) mice. Some mice were given carbon tetrachloride (CCl4) to induce liver fibrosis, with or without a peroxisome proliferator-activated receptor-γ (PPARγ) agonist. Livers from mice were analyzed by immunohistochemistry. Quiescent, activated, and inactivated HSCs were isolated from livers of Col1α1YFP mice and analyzed by chromatin immunoprecipitation and sequencing. Human HSCs were isolated from livers denied for transplantation. We compared changes in gene expression patterns and epigenetic modifications (histone H3 lysine 4 dimethylation and histone H3 lysine 27 acetylation) in primary mouse and human HSCs. Transcription factors were knocked down with small hairpin RNAs in mouse HSCs. RESULTS: Motif enrichment identified E26 transcription-specific transcription factors (ETS) 1, ETS2, GATA4, GATA6, interferon regulatory factor (IRF) 1, and IRF2 transcription factors as regulators of the mouse and human HSC lineage. Small hairpin RNA-knockdown of these transcription factors resulted in increased expression of genes that promote fibrogenesis and inflammation, and loss of HSC phenotype. Disruption of Gata6 or Ets1, or Nf1 or Pparγ (which are regulated by ETS1), increased the severity of CCl4-induced liver fibrosis in mice compared to control mice. Only mice with disruption of Gata6 or Pparγ had defects in fibrosis resolution after CCl4 administration was stopped, associated with persistent activation of HSCs. Administration of a PPARγ agonist accelerated regression of liver fibrosis after CCl4 administration in control mice but not in mice with disruption of Pparγ. CONCLUSIONS: Phenotypes of HSCs from humans and mice are regulated by transcription factors, including ETS1, ETS2, GATA4, GATA6, IRF1, and IRF2. Activated mouse and human HSCs can revert to a quiescent-like, inactivated phenotype. We found GATA6 and PPARγ to be required for inactivation of human HSCs and regression of liver fibrosis in mice.

Automated, high-throughput derivation, characterization and differentiation of induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are an essential tool for modeling how causal genetic variants impact cellular function in disease, as well as an emerging source of tissue for regenerative medicine. The preparation of somatic cells, their reprogramming and the subsequent verification of iPSC pluripotency are laborious, manual processes limiting the scale and reproducibility of this technology. Here we describe a modular, robotic platform for iPSC reprogramming enabling automated, high-throughput conversion of skin biopsies into iPSCs and differentiated cells with minimal manual intervention. We demonstrate that automated reprogramming and the pooled selection of polyclonal pluripotent cells results in high-quality, stable iPSCs. These lines display less line-to-line variation than either manually produced lines or lines produced through automation followed by single-colony subcloning. The robotic platform we describe will enable the application of iPSCs to population-scale biomedical problems including the study of complex genetic diseases and the development of personalized medicines.

De novo missense variants in PPP2R5D are associated with intellectual disability, macrocephaly, hypotonia, and autism.

Protein phosphatase 2A (PP2A) is a heterotrimeric protein serine/threonine phosphatase and is involved in a broad range of cellular processes. PPP2R5D is a regulatory B subunit of PP2A and plays an important role in regulating key neuronal and developmental regulation processes such as PI3K/AKT and glycogen synthase kinase 3 beta (GSK3ß)-mediated cell growth, chromatin remodeling, and gene transcriptional regulation. Using whole-exome sequencing (WES), we identified four de novo variants in PPP2R5D in a total of seven unrelated individuals with intellectual disability (ID) and other shared clinical characteristics, including autism spectrum disorder, macrocephaly, hypotonia, seizures, and dysmorphic features. Among the four variants, two have been previously reported and two are novel. All four amino acids are highly conserved among the PP2A subunit family, and all change a negatively charged acidic glutamic acid (E) to a positively charged basic lysine (K) and are predicted to disrupt the PP2A subunit binding and impair the dephosphorylation capacity. Our data provides further support for PPP2R5D as a genetic cause of ID.

Engineering bone tissue substitutes from human induced pluripotent stem cells.

Congenital defects, trauma, and disease can compromise the integrity and functionality of the skeletal system to the extent requiring implantation of bone grafts. Engineering of viable bone substitutes that can be personalized to meet specific clinical needs represents a promising therapeutic alternative. The aim of our study was to evaluate the utility of human-induced pluripotent stem cells (hiPSCs) for bone tissue engineering. We first induced three hiPSC lines with different tissue and reprogramming backgrounds into the mesenchymal lineages and used a combination of differentiation assays, surface antigen profiling, and global gene expression analysis to identify the lines exhibiting strong osteogenic differentiation potential. We then engineered functional bone substitutes by culturing hiPSC-derived mesenchymal progenitors on osteoconductive scaffolds in perfusion bioreactors and confirmed their phenotype stability in a subcutaneous implantation model for 12 wk. Molecular analysis confirmed that the maturation of bone substitutes in perfusion bioreactors results in global repression of cell proliferation and an increased expression of lineage-specific genes. These results pave the way for growing patient-specific bone substitutes for reconstructive treatments of the skeletal system and for constructing qualified experimental models of development and disease.

Mutations in ARID2 are associated with intellectual disabilities.

The etiology of intellectual disabilities (ID) remains unknown for the majority of patients. Due to reduced reproductive fitness in many individuals with ID, de novo mutations account for a significant portion of severe ID. The ATP-dependent SWI/SNF chromatin modifier has been linked with neurodevelopmental disorders including ID and autism. ARID2 is an intrinsic component of polybromo-associated BAF (PBAF), the SWI/SNF subcomplex. In this study, we used clinical whole exome sequencing (WES) in proband-parent-trios to identify the etiology of ID. We identified four independent, novel, loss of function variants in ARID2 gene in four patients, three of which were confirmed to be de novo. The patients all have ID and share other clinical characteristics including attention deficit hyperactivity disorder, short stature, dysmorphic facial features, and Wormian bones. All four novel variants are predicted to lead to a premature termination with the loss of the two conservative zinc finger motifs. This is the first report of mutations in ARID2 associated with developmental delay and ID.

Aldose reductase modulates cardiac glycogen synthase kinase-3ß phosphorylation during ischemia-reperfusion.

Earlier studies have demonstrated that aldose reductase (AR) plays a key role in mediating ischemia-reperfusion (I/R) injury. Our objective was to investigate if AR mediates I/R injury by influencing phosphorylation of glycogen synthase kinase-3ß (p-GSK3ß). To investigate this issue, we used three separate models to study the effects of stress injury on the heart. Hearts isolated from wild-type (WT), human expressing AR transgenic (ARTg), and AR knockout (ARKO) mice were perfused with/without GSK3ß inhibitors (SB-216763 and LiCl) and subjected to I/R. Ad-human AR (Ad-hAR)-expressing HL-1 cardiac cells were exposed to hypoxia (0.5% O(2)) and reoxygenation (20.9% O(2)) conditions. I/R in a murine model of transient occlusion and reperfusion of the left anterior descending coronary artery (LAD) was used to study if p-GSK3ß was affected through increased AR flux. Lactate dehydrogenase (LDH) release and left ventricular developed pressure (LVDP) were measured. LVDP was decreased in hearts from ARTg mice compared with WT and ARKO after I/R, whereas LDH release and apoptotic markers were increased (P < 0.05). p-GSK3ß was decreased in ARTg hearts compared with WT and ARKO (P < 0.05). In ARKO, p-GSK3ß and apoptotic markers were decreased compared with WT (P < 0.05). WT and ARTg hearts perfused with GSK3ß inhibitors improved p-GSK3ß expression and LVDP and exhibited decreased LDH release, apoptosis, and mitochondrial pore opening (P < 0.05). Ad-hAR-expressing HL-1 cardiac cells, exposed to hypoxia (0.5% O(2)) and reoxygenation (20.9% O(2)), had greater LDH release compared with control HL-1 cells (P < 0.05). p-GSK3ß was decreased and correlated with increased apoptotic markers in Ad-hAR HL-1 cells (P < 0.05). Treatment with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) inhibitor increased injury demonstrated by increased LDH release in ARTg, WT, and ARKO hearts and in Ad-hAR-expressing HL-1 cells. Cells treated with protein kinase C (PKC) α/ß inhibitor displayed significant increases in p-Akt and p-GSK3ß expression, and resulted in decreased LDH release. In summary, AR mediates changes in p-GSK3ß, in part, via PKCα/ß and Akt during I/R.

Isolated and combined impact of dietary olive oil and exercise on markers of health and energy metabolism in female mice.

An olive oil (OO) rich diet or high-intensity interval training (HIIT) independently improve markers of health and energy metabolism, but it is unknown if combining OO and HIIT synergize to improve these markers. This study characterized the isolated and combined impact of OO and HIIT on markers of health and energy metabolism in various tissues in C57BL/6J female mice. Nine-week-old mice were divided into four groups for a 12-week diet and/or exercise intervention including: (1) Control Diet without HIIT (CD), (2) Control Diet with HIIT (CD+HIIT), (3) OO diet (10% kcal from olive oil) without HIIT, and (4) OO diet with HIIT (OO+HIIT). Neither dietary OO or HIIT altered body weight, glucose tolerance, or serum lipids. HIIT, regardless of diet, increased aerobic capacity and HDL cholesterol levels. In liver and heart tissue, OO resulted in similar adaptations as HIIT including increased mitochondrial content and fatty acid oxidation but combining OO with HIIT did not augment these effects. In skeletal muscle, HIIT increased mitochondrial content in type II fibers similarly between diets. An RNA sequencing analysis on type I fibers revealed OO reduced muscle regeneration and lipid metabolism gene abundance, whereas HIIT increased the abundance of these genes, independent of diet. HIIT training, independent of diet, induced subcutaneous white adipose tissue (sWAT) hypertrophy, whereas OO induced gonadal white adipose tissue (gWAT) hypertrophy, an effect that was augmented with HIIT. These data highlight the pleiotropic effects of OO and HIIT, although their combination does not synergize to further improve most markers of health and energy metabolism.

Systemic lipolysis promotes physiological fitness in Drosophila melanogaster.

Since interventions such as caloric restriction or fasting robustly promote lipid catabolism and improve aging-related phenotypical markers, we investigated the direct effect of increased lipid catabolism via overexpression of bmm (brummer, FBgn0036449), the major triglyceride hydrolase in Drosophila, on lifespan and physiological fitness. Comprehensive characterization was carried out using RNA-seq, lipidomics and metabolomics analysis. Global overexpression of bmm strongly promoted numerous markers of physiological fitness, including increased female fecundity, fertility maintenance, preserved locomotion activity, increased mitochondrial biogenesis and oxidative metabolism. Increased bmm robustly upregulated the heat shock protein 70 (Hsp70) family of proteins, which equipped the flies with higher resistance to heat, cold, and ER stress via improved proteostasis. Despite improved physiological fitness, bmm overexpression did not extend lifespan. Taken together, these data show that bmm overexpression has broad beneficial effects on physiological fitness, but these effects did not impact lifespan.

Nonalcoholic Steatohepatitis and HCC in a Hyperphagic Mouse Accelerated by Western Diet.

BACKGROUND & AIMS: How benign liver steatosis progresses to nonalcoholic steatohepatitis (NASH), fibrosis, and hepatocellular carcinoma (HCC) remains elusive. NASH progression entails diverse pathogenic mechanisms and relies on complex cross-talk between multiple tissues such as the gut, adipose tissues, liver, and the brain. Using a hyperphagic mouse fed with a Western diet (WD), we aimed to elucidate the cross-talk and kinetics of hepatic and extrahepatic alterations during NASH-HCC progression, as well as regression. METHODS: Hyperphagic mice lacking a functional Alms1 gene (Foz/Foz) and wild-type littermates were fed WD or standard chow for 12 weeks for NASH/fibrosis and for 24 weeks for HCC development. NASH regression was modeled by switching back to normal chow after NASH development. RESULTS: Foz+WD mice were steatotic within 1 to 2 weeks, developed NASH by 4 weeks, and grade 3 fibrosis by 12 weeks, accompanied by chronic kidney injury. Foz+WD mice that continued on WD progressed to cirrhosis and HCC within 24 weeks and had reduced survival as a result of cardiac dysfunction. However, NASH mice that were switched to normal chow showed NASH regression, improved survival, and did not develop HCC. Transcriptomic and histologic analyses of Foz/Foz NASH liver showed strong concordance with human NASH. NASH was preceded by an early disruption of gut barrier, microbial dysbiosis, lipopolysaccharide leakage, and intestinal inflammation. This led to acute-phase liver inflammation in Foz+WD mice, characterized by neutrophil infiltration and increased levels of several chemokines/cytokines. The liver cytokine/chemokine profile evolved as NASH progressed, with subsequent predominance by monocyte recruitment. CONCLUSIONS: The Foz+WD model closely mimics the pathobiology and gene signature of human NASH with fibrosis and subsequent HCC. Foz+WD mice provide a robust and relevant preclinical model of NASH, NASH-associated HCC, chronic kidney injury, and heart failure.

Bardet-Biedl syndrome proteins regulate intracellular signaling and neuronal function in patient-specific iPSC-derived neurons.

Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disorder caused by mutations in genes encoding components of the primary cilium and is characterized by hyperphagic obesity. To investigate the molecular basis of obesity in human BBS, we developed a cellular model of BBS using induced pluripotent stem cell-derived (iPSC-derived) hypothalamic arcuate-like neurons. BBS mutations BBS1M390R and BBS10C91fsX95 did not affect neuronal differentiation efficiency but caused morphological defects, including impaired neurite outgrowth and longer primary cilia. Single-cell RNA sequencing of BBS1M390R hypothalamic neurons identified several downregulated pathways, including insulin and cAMP signaling and axon guidance. Additional studies demonstrated that BBS1M390R and BBS10C91fsX95 mutations impaired insulin signaling in both human fibroblasts and iPSC-derived neurons. Overexpression of intact BBS10 fully restored insulin signaling by restoring insulin receptor tyrosine phosphorylation in BBS10C91fsX95 neurons. Moreover, mutations in BBS1 and BBS10 impaired leptin-mediated p-STAT3 activation in iPSC-derived hypothalamic neurons. Correction of the BBS mutation by CRISPR rescued leptin signaling. POMC expression and neuropeptide production were decreased in BBS1M390R and BBS10C91fsX95 iPSC-derived hypothalamic neurons. In the aggregate, these data provide insights into the anatomic and functional mechanisms by which components of the BBSome in CNS primary cilia mediate effects on energy homeostasis.

RETRACTED: Resveratrol augments the canonical Wnt signaling pathway in promoting osteoblastic differentiation of multipotent mesenchymal cells.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the authors. The authors were alerted about the loading control in a Western blot analysis of ERK phosphorylation shown in Figure 6B, and they were unable to locate the original scan. Although the authors stand by the conclusion based on this figure, and the conclusion of the entire article, they wish to retract this article.

Blockade of IL-17 signaling reverses alcohol-induced liver injury and excessive alcohol drinking in mice.

Chronic alcohol abuse has a detrimental effect on the brain and liver. There is no effective treatment for these patients, and the mechanism underlying alcohol addiction and consequent alcohol-induced damage of the liver/brain axis remains unresolved. We compared experimental models of alcoholic liver disease (ALD) and alcohol dependence in mice and demonstrated that genetic ablation of IL-17 receptor A (IL-17ra-/-) or pharmacological blockade of IL-17 signaling effectively suppressed the increased voluntary alcohol drinking in alcohol-dependent mice and blocked alcohol-induced hepatocellular and neurological damage. The level of circulating IL-17A positively correlated with the alcohol use in excessive drinkers and was further increased in patients with ALD as compared with healthy individuals. Our data suggest that IL-17A is a common mediator of excessive alcohol consumption and alcohol-induced liver/brain injury, and targeting IL-17A may provide a novel strategy for treatment of alcohol-induced pathology.

Serum withdrawal up-regulates human SIRT1 gene expression in a p53-dependent manner.

SIRT1, a nicotinamide adenine dinucleotide (NAD(+))-dependent histone/protein deacetylase, has been extensively studied recently for its critical role in the regulation of physiology, calorie restriction and aging. Studies on laboratory mice showed that expression of SIRT1 can be induced by starvation in a p53-dependent manner and requires the p53-binding sites present in the Sirt1 promoter. However, it remains to be determined whether these findings based on rodents apply to human beings. In this paper, we characterized a putative p53-binding element in the human SIRT1 promoter that might be required for the up-regulation of SIRT1 in response to nutritional stress. The p53-binding site in the promoter of human SIRT1 is more deviant from the consensus sequence than the corresponding sequence in the mouse Sirt1. There is a C to A change at the second half site in human SIRT1, thus disrupting the core-binding element CWWG in the canonical RRRCWWGYYY. To test whether such sequence change would affect its binding with p53 and the SIRT1 expression under stress, we studied various human cell lines with different p53 status and cells with ectopic expression of functionally distinct p53. We found that serum withdrawal also up-regulates human SIRT1 gene expression in a p53-dependent manner and that the p53-binding element in SIRT1 is required for the up-regulation. Thus, the mechanism responsible for the regulation of SIRT1 expression by p53 is conserved between mice and human beings.

Transgenic substitution with Greater Amberjack Seriola dumerili fish insulin 2 in NOD mice reduces beta cell immunogenicity.

Type I diabetes (T1D) is caused by immune-mediated destruction of pancreatic beta cells. This process is triggered, in part, by specific (aa 9-23) epitopes of the insulin Β chain. Previously, fish insulins were used clinically in patients allergic to bovine or porcine insulin. Fish and human insulin differ by two amino acids in the critical immunogenic region (aa 9-23) of the B chain. We hypothesized that ß cells synthesizing fish insulin would be less immunogenic in a mouse model of T1D. Transgenic NOD mice in which Greater Amberjack fish (Seriola dumerili) insulin was substituted for the insulin 2 gene were generated (mouse Ins1-/- mouse Ins2-/- fish Ins2+/+). In these mice, pancreatic islets remained free of autoimmune attack. To determine whether such reduction in immunogenicity is sufficient to protect ß cells from autoimmunity upon transplantation, we transplanted fish Ins2 transgenic (expressing solely Seriola dumerili Ins2), NOD, or B16:A-dKO islets under the kidney capsules of 5 weeks old female NOD wildtype mice. The B:Y16A Β chain substitution has been previously shown to be protective of T1D in NOD mice. NOD mice receiving Seriola dumerili transgenic islet transplants showed a significant (p = 0.004) prolongation of their euglycemic period (by 6 weeks; up to 18 weeks of age) compared to un-manipulated female NOD (diabetes onset at 12 weeks of age) and those receiving B16:A-dKO islet transplants (diabetes onset at 12 weeks of age). These data support the concept that specific amino acid sequence modifications can reduce insulin immunogenicity. Additionally, our study shows that alteration of a single epitope is not sufficient to halt an ongoing autoimmune response. Which, and how many, T cell epitopes are required and suffice to perpetuate autoimmunity is currently unknown. Such studies may be useful to achieve host tolerance to ß cells by inactivating key immunogenic epitopes of stem cell-derived ß cells intended for transplantation.

Sp1 and CREB regulate basal transcription of the human SNF2L gene.

Imitation Switch (ISWI) is a member of the SWI2/SNF2 superfamily of ATP-dependent chromatin remodelers, which are involved in multiple nuclear functions, including transcriptional regulation, replication, and chromatin assembly. Mammalian genomes encode two ISWI orthologs, SNF2H and SNF2L. In order to clarify the molecular mechanisms governing the expression of human SNF2L gene, we functionally examined the transcriptional regulation of human SNF2L promoter. Reporter gene assays demonstrated that the minimal SNF2L promoter was located between positions -152 to -86 relative to the transcription start site. In this region we have identified a cAMP-response element (CRE) located at -99 to -92 and a Sp1-binding site at -145 to -135 that play a critical role in regulating basal activity of human SNF2L gene, which were proven by deletion and mutation of specific binding sites, EMSA, and down-regulating Sp1 and CREB via RNAi. This study provides the first insight into the mechanisms that control basal expression of human SNF2L gene.

Human hepatic stellate cell isolation and characterization.

The hepatic stellate cells (HSCs) localize at the space of Disse in the liver and have multiple functions. They are identified as the major contributor to hepatic fibrosis. Significant understanding of HSCs has been achieved using rodent models and isolated murine HSCs; as well as investigating human liver tissues and human HSCs. There is growing interest and need of translating rodent study findings to human HSCs and human liver diseases. However, species-related differences impose challenges on the translational research. In this review, we focus on the current information on human HSCs isolation methods, human HSCs markers, and established human HSC cell lines.

A functional polymorphism in the TIM-1 gene is associated with asthma in a Chinese Han population.

BACKGROUND: TIM-1, a member of the T cell immunoglobulin and mucin domain (TIM) gene family was implicated as an asthma susceptibility gene in previous studies. TIM-1 is selectively expressed on activated CD4+ T cells and its expression is sustained preferentially on T helper type 2 (Th2) cells, which suggests that TIM-1 is associated with T cell differentiation and the development of Th2-biased immune response. METHODS: In order to evaluate the effects of the promoter polymorphism in the TIM-1 gene on asthma susceptibility in a Chinese Han population, 2 promoter polymorphisms, -416G>C and -1454G>A, from 2 alternative promoter regions, were genotyped in 409 unrelated asthma patients and 305 healthy controls by using polymerase chain reaction and restriction fragment length polymorphism. Also, we analyzed the functional significance of -416G>C using the luciferase reporter gene assay. RESULTS: We found that -416G>C was associated with asthma susceptibility in our study population (chi2 = 9.88, p = 0.002, odds ratio 1.41, 95% confidence interval 1.14-1.75). No statistically significant difference in the distribution of genotype and allele frequency of the -1454G>A site was observed. The -416G>C substitution increased the transcriptional activity of the TIM-1 gene. CONCLUSION: Our findings suggest that the -416G>C variation site in the human TIM-1 promoter region is associated with asthma susceptibility in a Chinese Han population.

Congenital diaphragmatic hernias: from genes to mechanisms to therapies.

Congenital diaphragmatic hernias (CDHs) and structural anomalies of the diaphragm are a common class of congenital birth defects that are associated with significant morbidity and mortality due to associated pulmonary hypoplasia, pulmonary hypertension and heart failure. In ∼30% of CDH patients, genomic analyses have identified a range of genetic defects, including chromosomal anomalies, copy number variants and sequence variants. The affected genes identified in CDH patients include transcription factors, such as GATA4, ZFPM2, NR2F2 and WT1, and signaling pathway components, including members of the retinoic acid pathway. Mutations in these genes affect diaphragm development and can have pleiotropic effects on pulmonary and cardiac development. New therapies, including fetal endoscopic tracheal occlusion and prenatal transplacental fetal treatments, aim to normalize lung development and pulmonary vascular tone to prevent and treat lung hypoplasia and pulmonary hypertension, respectively. Studies of the association between particular genetic mutations and clinical outcomes should allow us to better understand the origin of this birth defect and to improve our ability to predict and identify patients most likely to benefit from specialized treatment strategies.