Stage of fibrosis is not a predictive determinant of weight loss in patients undergoing bariatric surgery.

BACKGROUND: Obesity and nonalcoholic fatty liver disease (NAFLD) are an increasing health care burden worldwide. Weight loss is currently the best option to alleviate NAFLD and is efficiently achieved by bariatric surgery. Presence of NAFLD seems to be predictive for postoperative weight loss. To date, only few predictive factors for postbariatric weight loss (age, diabetes, psychiatric disorders) are established. OBJECTIVES: Since liver fibrosis is the pathogenic driver for the progression of liver disease, we investigated its role in predicting postoperative weight loss. This study focuses on the correlation between fibrosis stage and weight loss. SETTING: University and university-affiliated cooperation, Germany. METHODS: We used a prospective, single-center cohort study including 164 patients who underwent bariatric surgery with simultaneous liver biopsies. Liver fibrosis was determined histologically according to Kleiner score and noninvasively by APRI and FIB-4 score. Percentage of total body weight loss was calculated at 1-year follow up visit. RESULTS: Thirty-two patients were found without fibrosis, whereas 91 patients showed mild fibrosis (F1), 37 significant fibrosis (F2), and only 4 patients presented advanced fibrosis (F3) at the time of bariatric surgery. Weight loss was similar across different degrees of fibrosis stage. Accordingly, linear regression analysis did not identify predictors of weight loss among fibrosis scores. In multivariable analysis, age and presence of diabetes showed the strongest predictive value. CONCLUSIONS: Baseline presence of fibrosis was not associated with postoperative weight loss, while age and diabetes were independent predictors of weight loss. Bariatric surgery should be applied independently of the fibrosis stage.

CXCL10 deficiency limits macrophage infiltration, preserves lung matrix, and enables lung growth in bronchopulmonary dysplasia.

Preterm infants with oxygen supplementation are at high risk for bronchopulmonary dysplasia (BPD), a neonatal chronic lung disease. Inflammation with macrophage activation is central to the pathogenesis of BPD. CXCL10, a chemotactic and pro-inflammatory chemokine, is elevated in the lungs of infants evolving BPD and in hyperoxia-based BPD in mice. Here, we tested if CXCL10 deficiency preserves lung growth after neonatal hyperoxia by preventing macrophage activation. To this end, we exposed Cxcl10 knockout (Cxcl10-/-) and wild-type mice to an experimental model of hyperoxia (85% O2)-induced neonatal lung injury and subsequent regeneration. In addition, cultured primary human macrophages and murine macrophages (J744A.1) were treated with CXCL10 and/or CXCR3 antagonist. Our transcriptomic analysis identified CXCL10 as a central hub in the inflammatory network of neonatal mouse lungs after hyperoxia. Quantitative histomorphometric analysis revealed that Cxcl10-/- mice are in part protected from reduced alveolar. These findings were related to the preserved spatial distribution of elastic fibers, reduced collagen deposition, and protection from macrophage recruitment/infiltration to the lungs in Cxcl10-/- mice during acute injury and regeneration. Complimentary, studies with cultured human and murine macrophages showed that hyperoxia induces Cxcl10 expression that in turn triggers M1-like activation and migration of macrophages through CXCR3. Finally, we demonstrated a temporal increase of macrophage-related CXCL10 in the lungs of infants with BPD. In conclusion, our data demonstrate macrophage-derived CXCL10 in experimental and clinical BPD that drives macrophage chemotaxis through CXCR3, causing pro-fibrotic lung remodeling and arrest of alveolarization. Thus, targeting the CXCL10-CXCR3 axis could offer a new therapeutic avenue for BPD.

Vesicular Release and Uptake of Circular LSD1-RNAs from Non-Cancer and Cancer Lung Cells.

Lysine-specific demethylase 1 (LSD1) is highly expressed in many cancer types and strongly associated with cancer progression and metastasis. Circular RNAs (circRNAs) are produced by back-splicing and influence the interactive RNA network by microRNA and protein sponging. In the present study, we aimedto identify circRNAs that derive from the LSD1-encoding KDM1A gene, and to investigate their potential to be released and uptaken by lung cancer versus non-cancer epithelial cells. We identified four circLSD1-RNAs by RT-PCR with divergent primers, followed by sequencing. The expression level of circLSD1-RNAs was then studied by quantitative PCR on cellular and extracellular fractions of lung cancer (PC9) and non-cancer primary small airway epithelial (PSAE) cells. Moreover, we established the transgenic overexpression of circLSD1-RNAs. We show that circLSD1-RNAs are primarily located in the cytoplasm, but are packaged and released from lung cancer and non-cancer cells by extracellular vesicles (EVs) and ribonucleoprotein (RNP) complexes, respectively. Proteomics demonstrated a different protein pattern of EV fractions released from PC9 versus PSAE cells. Importantly, released circLSD1-RNAs were differently taken up by PSAE and PC9 cells. In conclusion, our findings provide primary evidence that circLSD1-RNAs participate in the intercellular communication of lung cancer cells with the tumor environment.

E4 ubiquitin ligase promotes mitofusin turnover and mitochondrial stress response.

Ubiquitin-dependent control of mitochondrial dynamics is important for protein quality and neuronal integrity. Mitofusins, mitochondrial fusion factors, can integrate cellular stress through their ubiquitylation, which is carried out by multiple E3 enzymes in response to many different stimuli. However, the molecular mechanisms that enable coordinated responses are largely unknown. Here we show that yeast Ufd2, a conserved ubiquitin chain-elongating E4 enzyme, is required for mitochondrial shape adjustments. Under various stresses, Ufd2 translocates to mitochondria and triggers mitofusin ubiquitylation. This elongates ubiquitin chains on mitofusin and promotes its proteasomal degradation, leading to mitochondrial fragmentation. Ufd2 and its human homologue UBE4B also target mitofusin mutants associated with Charcot-Marie-Tooth disease, a hereditary sensory and motor neuropathy characterized by progressive loss of the peripheral nerves. This underscores the pathophysiological importance of E4-mediated ubiquitylation in neurodegeneration. In summary, we identify E4-dependent mitochondrial stress adaptation by linking various metabolic processes to mitochondrial fusion and fission dynamics.

Differential Role of Aldosterone and Transforming Growth Factor Beta-1 in Cardiac Remodeling.

Angiotensin II, a major culprit in cardiovascular disease, activates mediators that are also involved in pathological cardiac remodeling. In this context, we aimed at investigating the effects of two of them: aldosterone (Ald) and transforming growth factor beta-1 (TGF-ß1) in an in vivo model. Six-week-old male wild-type (WT) and TGF-ß1-overexpressing transgenic (TGF-ß1-TG) mice were infused with subhypertensive doses of Ald for 2 weeks and/or treated orally with eplerenone from postnatal day 21. Thehearts' ventricles were examined by morphometry, immunoblotting to assess the intracellular signaling pathways and RT qPCR to determine hypertrophy and fibrosis marker genes. The TGF-ß1-TG mice spontaneously developed cardiac hypertrophy and interstitial fibrosis and exhibited a higher baseline phosphorylation of p44/42 and p38 kinases, fibronectin and ANP mRNA expression. Ald induced a comparable increase in the ventricular-heart-weight-to-body-weight ratio and cardiomyocyte diameter in both strains, but a less pronounced increase in interstitial fibrosis in the transgenic compared to the WT mice (23.6% vs. 80.9%, p < 0.005). Ald increased the phosphorylation of p44/42 and p38 in the WT but not the TGF-ß1-TG mice. While the eplerenone-enriched chow partially prevented Ald-induced cardiac hypertrophy in both genotypes and interstitial fibrosis in the WT controls, it completely protected against additional fibrosis in transgenic mice. Ald appears to induce cardiac hypertrophy independently of TGF-ß1, while in the case of fibrosis, the downstream signaling pathways of these two factors probably converge.

Circulating miRNA-192 and miR-29a as Disease Progression Biomarkers in Hepatitis C Patients with a Prevalence of HCV Genotype 3.

MicroRNAs miR-29a and miR-192 are involved in inflammatory and fibrotic processes of chronic liver disease, and circulating miR-29a is suggested to diagnose fibrosis progression due to hepatitis C virus (HCV) infection. This study aimed to evaluate the expression profile of circulating miR-192 and 29a in a patient cohort with a high frequency of HCV genotype-3. A total of 222 HCV blood samples were collected and serum were separated. Patients were classified into mild, moderate, and severe liver injury based on their Child-Turcotte-Pugh CTP score. RNA was isolated from the serum and used for quantitative real-time PCR. The HCV genotype-3 (62%) was the predominant HCV genotype. In HCV patients, the serum miR-192 and miR-29a levels were significantly upregulated in comparison to healthy controls (p = 0.0017 and p = 0.0001, respectively). The progression rate of miR-192 and 29a in the patient group with mild was highly upregulated compared to patients with moderate and severe hepatitis infection. The ROC curve of miR-192 and miR-29a of moderate liver disease had a significant diagnostic performance compared to the other HCV-infected groups. The increase in miR-29a and miR-192 serum levels was even slightly higher in patients with HCV genotype-3 than in non-genotype-3 patients. In conclusion, serum miR-192 and miR-29a levels significantly increased during the progression of chronic HCV infection. The marked upregulation in patients with HCV genotype-3 suggests them as potential biomarkers for hepatic disease, independently of the HCV genotype.

Oncogenic role and target properties of the lysine-specific demethylase KDM1A in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), epigenetic alterations are considered to centrally shape the transcriptional signatures that drive disease evolution and underlie its biological and clinical subsets. Characterizations of epigenetic regulators, particularly histone-modifying enzymes, are very rudimentary in CLL. In efforts to establish effectors of the CLL-associated oncogene T-cell leukemia 1A (TCL1A), we identified here the lysine-specific histone demethylase KDM1A to interact with the TCL1A protein in B cells in conjunction with an increased catalytic activity of KDM1A. We demonstrate that KDM1A is upregulated in malignant B cells. Elevated KDM1A and associated gene expression signatures correlated with aggressive disease features and adverse clinical outcomes in a large prospective CLL trial cohort. Genetic Kdm1a knockdown in Eµ-TCL1A mice reduced leukemic burden and prolonged animal survival, accompanied by upregulated p53 and proapoptotic pathways. Genetic KDM1A depletion also affected milieu components (T, stromal, and monocytic cells), resulting in significant reductions in their capacity to support CLL-cell survival and proliferation. Integrated analyses of differential global transcriptomes (RNA sequencing) and H3K4me3 marks (chromatin immunoprecipitation sequencing) in Eµ-TCL1A vs iKdm1aKD;Eµ-TCL1A mice (confirmed in human CLL) implicate KDM1A as an oncogenic transcriptional repressor in CLL which alters histone methylation patterns with pronounced effects on defined cell death and motility pathways. Finally, pharmacologic KDM1A inhibition altered H3K4/9 target methylation and revealed marked anti-B-cell leukemic synergisms. Overall, we established the pathogenic role and effector networks of KDM1A in CLL via tumor-cell intrinsic mechanisms and its impacts in cells of the microenvironment. Our data also provide rationales to further investigate therapeutic KDM1A targeting in CLL.

Hepatitis C virus replication requires integrity of mitochondria-associated ER membranes.

Background & Aims: Chronic HCV infection causes cellular stress, fibrosis and predisposes to hepatocarcinogenesis. Mitochondria play key roles in orchestrating stress responses by regulating bioenergetics, inflammation and apoptosis. To better understand the role of mitochondria in the viral life cycle and disease progression of chronic hepatitis C, we studied morphological and functional mitochondrial alterations induced by HCV using productively infected hepatoma cells and patient livers. Methods: Biochemical and imaging assays were used to assess localization of cellular and viral proteins and mitochondrial functions in cell cultures and liver biopsies. Cyclophilin D (CypD) knockout was performed using CRISPR/Cas9 technology. Viral replication was quantified by quantitative reverse-transcription PCR and western blotting. Results: Several HCV proteins were found to associate with mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), the points of contact between the ER and mitochondria. Downregulation of CypD, which is known to disrupt MAM integrity, reduced viral replication, suggesting that MAMs play an important role in the viral life cycle. This process was rescued by ectopic CypD expression. Furthermore, HCV proteins were found to associate with voltage dependent anion channel 1 (VDAC1) at MAMs and to reduce VDAC1 protein levels at MAMs in vitro and in patient biopsies. This association did not affect MAM-associated functions in glucose homeostasis and Ca2+ signaling. Conclusions: HCV proteins associate specifically with MAMs and MAMs play an important role in viral replication. The association between viral proteins and MAMs did not impact Ca2+ signaling between the ER and mitochondria or glucose homeostasis. Whether additional functions of MAMs and/or VDAC are impacted by HCV and contribute to the associated pathology remains to be assessed. Impact and implications: Hepatitis C virus infects the liver, where it causes inflammation, cell damage and increases the long-term risk of liver cancer. We show that several HCV proteins interact with mitochondria in liver cells and alter the composition of mitochondrial subdomains. Importantly, HCV requires the architecture of these mitochondrial subdomains to remain intact for efficient viral replication.

Adeno-associated virus serotype 2 capsid variants for improved liver-directed gene therapy.

BACKGROUND AND AIMS: Current liver-directed gene therapies look for adeno-associated virus (AAV) vectors with improved efficacy. With this background, capsid engineering is explored. Whereas shuffled capsid library screenings have resulted in potent liver targeting variants with one first vector in human clinical trials, modifying natural serotypes by peptide insertion has so far been less successful. Here, we now report on two capsid variants, MLIV.K and MLIV.A, both derived from a high-throughput in vivo AAV peptide display selection screen in mice. APPROACH AND RESULTS: The variants transduce primary murine and human hepatocytes at comparable efficiencies, a valuable feature in clinical development, and show significantly improved liver transduction efficacy, thereby allowing a dose reduction, and outperform parental AAV2 and AAV8 in targeting human hepatocytes in humanized mice. The natural heparan sulfate proteoglycan binding ability is markedly reduced, a feature that correlates with improved hepatocyte transduction. A further property that might contribute to the improved transduction efficacy is the lower capsid melting temperature. Peptide insertion also caused a moderate change in sensitivity to human sera containing anti-AAV2 neutralizing antibodies, revealing the impact of epitopes located at the basis of the AAV capsid protrusions. CONCLUSIONS: In conclusion, MLIV.K and MLIV.A are AAV peptide display variants selected in immunocompetent mice with improved hepatocyte tropism and transduction efficiency. Because these features are maintained across species, MLIV variants provide remarkable potential for translation of therapeutic approaches from mice to men.

Differentially expressed messenger RNA/proteins can distinguish teratoma from necrosis in postchemotherapy retroperitoneal lymph node dissection tissue.

BACKGROUND: Before postchemotherapy retroperitoneal lymph node dissection (pcRPLND), in patients with metastasized germ cell tumors (GCTs), those harboring necrosis (NEC) cannot be distinguished from those who have teratoma (TER), resulting in relevant overtreatment, whereas microRNA-371a-3p may be predictive for viable GCT. The purpose of this study was to explore messenger RNA (mRNA) and proteins to distinguish TER from NEC in pcRPLND tissue. METHODS: The discovery cohort consisted in total of 48 patients, including 16 each with TER, viable GCT, and NEC. Representative areas were microdissected. A NanoString panel and proteomics were used to analyze 770 genes and >5000 proteins. The most significantly and differentially expressed combination of both parameters, mRNA and its associated protein, between TER and NEC was validated using immunohistochemistry (IHC) in an independent validation cohort comprising 66 patients who were not part of the discovery cohort. RESULTS: The authors observed that anterior gradient protein 2 homolog (AGR2) and keratin, type I cytoskeletal 19 (KRT19) were significantly differentially expressed in TER versus NEC in mRNA and protein analyses (proteomics). The technical validation using IHC was successful in the same patients. These proteins were further validated by IHC in the independent patient cohort and exhibited significantly higher levels in TER versus NEC (p < .0001; area under the curve, 1.0; sensitivity and specificity, 100% each). CONCLUSIONS: The current study demonstrated that KRT19 and AGR2 mRNA and protein are overexpressed in TER versus NEC in pcRPLND tissue and might serve as a future diagnostic target to detect TER, for instance, by functional imaging, to avoid overtreatment. PLAIN LANGUAGE SUMMARY: The proteins and the corresponding genes called AGR2 and KRT19 can differentiate between teratoma and necrosis in remaining tumor masses after chemotherapy in patients who have metastasized testicular cancer. This may be a way to improve presurgical diagnostics and to reduce the current overtreatment of patients with necrosis only, who could be treated sufficiently by surveillance.

SQSTM1/p62 promotes miR-198 loading into extracellular vesicles and its autophagy-related secretion.

MicroRNA dysregulation is a hallmark of hepatocellular carcinoma (HCC), leading to tumor growth and metastasis. Previous screening on patient specimens identified miR-198 as the most downregulated miRNA in HCC. Here, we show that miR-198 compensation leads to self-release into extracellular vesicles (EVs). Importantly, the vesicular secretion is mediated by autophagy-related pathway, initiated by sequestration of p62/miR-198 complexes in autophagosome-associated vesicle fractions. miR-198 is selectively recognized and loaded by p62 into autophagosomal fractions, whereas mutated miR-198 forms neither induce autophagy and nor interact with p62. Gain and loss of function experiments, using a CRIPR/Cas knockout (KO) and transgenic site-specific p62 mutants, identified p62 as an essential repressor of cellular miR-198 abundancy. Notably, EVs, harboring miR-198/p62 protein complexes, can be uptaken by cells in the close vicinity, leading to change of gene expression in recipient cells. In conclusion, miR-198 enhances autophagy; conversely autophagic protein p62 reduces the miR-198 levels by sorting into extracellular space. miR-198 is at first transcribed as primary miRNA, after being processed into single stranded mature miR-198 form, it is transported into cytoplasm ①. By interaction with p62 protein, miR-198 conglomerates and forms a binding complex ②. Since LC3 protein is an interaction partner of p62 protein, hence miR-198 is included into autophagosomes ③. By fusion with multivesicular bodies (MVB), miR-198-binding complex was recruited into amphisomes ④, the latter of which quickly turns into secretory MVB containing intraluminal vesicles⑤. By fusion with cell membrane, intraluminal vesicles were released into extracellular space as EVs ⑥.

EGFR Inhibition Strongly Modulates the Tumour Immune Microenvironment in EGFR-Driven Non-Small-Cell Lung Cancer.

EGFR-driven non-small-cell lung cancer (NSCLC) patients are currently treated with TKIs targeting EGFR, such as erlotinib or osimertinib. Despite a promising initial response to TKI treatment, most patients gain resistance to oncogene-targeted therapy, and tumours progress. With the development of inhibitors against immune checkpoints, such as PD-1, that mediate an immunosuppressive microenvironment, immunotherapy approaches attempt to restore a proinflammatory immune response in tumours. However, this strategy has shown only limited benefits in EGFR-driven NSCLC. Approaches combining EGFR inhibition with immunotherapy to stimulate the immune response and overcome resistance to therapy have been limited due to insufficient understanding about the effect of EGFR-targeting treatment on the immune cells in the TME. Here, we investigate the impact of EGFR inhibition by erlotinib on the TME and its effect on the antitumour response of the immune cell infiltrate. For this purpose, we used a transgenic conditional mouse model to study the immunological profile in EGFR-driven NSCLC tumours. We found that EGFR inhibition mediated a higher infiltration of immune cells and increased local proliferation of T-cells in the tumours. Moreover, inhibiting EGFR signalling led to increased activation of immune cells in the TME. Most strikingly, combined simultaneous blockade of EGFR and anti-PD-1 (aPD-1) enhanced tumour treatment response in a transgenic mouse model of EGFR-driven NSCLC. Thus, our findings show that EGFR inhibition promotes an active and proinflammatory immune cell infiltrate in the TME while improving response to immune checkpoint inhibitors in EGFR-driven NSCLC.

Maternal and perinatal obesity induce bronchial obstruction and pulmonary hypertension via IL-6-FoxO1-axis in later life.

Obesity is a pre-disposing condition for chronic obstructive pulmonary disease, asthma, and pulmonary arterial hypertension. Accumulating evidence suggests that metabolic influences during development can determine chronic lung diseases (CLD). We demonstrate that maternal obesity causes early metabolic disorder in the offspring. Here, interleukin-6 induced bronchial and microvascular smooth muscle cell (SMC) hyperproliferation and increased airway and pulmonary vascular resistance. The key anti-proliferative transcription factor FoxO1 was inactivated via nuclear exclusion. These findings were confirmed using primary SMC treated with interleukin-6 and pharmacological FoxO1 inhibition as well as genetic FoxO1 ablation and constitutive activation. In vivo, we reproduced the structural and functional alterations in offspring of obese dams via the SMC-specific ablation of FoxO1. The reconstitution of FoxO1 using IL-6-deficient mice and pharmacological treatment did not protect against metabolic disorder but prevented SMC hyperproliferation. In human observational studies, childhood obesity was associated with reduced forced expiratory volume in 1 s/forced vital capacity ratio Z-score (used as proxy for lung function) and asthma. We conclude that the interleukin-6-FoxO1 pathway in SMC is a molecular mechanism by which perinatal obesity programs the bronchial and vascular structure and function, thereby driving CLD development. Thus, FoxO1 reconstitution provides a potential therapeutic option for preventing this metabolic programming of CLD.

AAV capsid engineering identified two novel variants with improved in vivo tropism for cardiomyocytes.

AAV vectors are promising delivery tools for human gene therapy. However, broad tissue tropism and pre-existing immunity against natural serotypes limit their clinical use. We identified two AAV capsid variants, AAV2-THGTPAD and AAV2-NLPGSGD, by in vivo AAV2 peptide display library screening in a murine model of pressure overload-induced cardiac hypertrophy. Both variants showed significantly improved efficacy in in vivo cardiomyocyte transduction compared with the parental serotype AAV2 as indicated by a higher number of AAV vector episomes in the nucleus and significant improved transduction efficiency. Both variants also outcompeted the reference serotype AAV9 regarding cardiomyocyte tropism, reaching comparable cardiac transduction efficiencies accompanied with liver de-targeting and decreased transduction efficiency of non-cardiac cells. Capsid modification influenced immunogenicity as sera of mice treated with AAV2-THGTPAD and AAV2-NLPGSGD demonstrated a poor neutralization capacity for the parental serotype and the novel variants. In a therapeutic setting, using the long non-coding RNA H19 in low vector dose conditions, novel AAV variants mediated superior anti-hypertrophic effects and revealed a further improved target-to-noise ratio, i.e., cardiomyocyte tropism. In conclusion, AAV2-THGTPAD and AAV2-NLPGSGD are promising novel tools for cardiac-directed gene therapy outperforming AAV9 regarding the specificity and therapeutic efficiency of in vivo cardiomyocyte transduction.

Circulating microRNA-122 in HCV cirrhotic patients with high frequency of genotype 3.

MicroRNA-122 (miR-122) is a liver abundant microRNA that is released upon liver injury. In the present study, we investigated the circulating miR-122 profiles in a Pakistani patients´ cohort with HCV chronic liver disease that was mainly based on HCV genotype 3 infections. From 222 patients with chronic HCV liver disease, classified as mild, moderate, or severe, serum samples were collected. Cell-free RNA was isolated and used for miR-122 quantification by qPCR. More than 60% of 222 patients were infected with HCV genotype 3. ALT values and HCV viral load showed no correlation with the HCV genotype. Circulating miR-122 levels were significantly upregulated in patients with cirrhosis. Notably, HCV patients with mild cirrhosis showed the most marked increase in serum miR-122 levels (p = 0.0001). Furthermore, we proved a positive correlation (r = 0.46) of miR-122 with the ALT values in patients with mild cirrhosis. Importantly, our data of increased miR-122 levels in serum samples obtained from a patient cohort with a high prevalence of chronic genotype 3 HCV infection confirmed the previous findings collected from cohorts with a high prevalence of genotype 1. Therefore, we suggest that miR-122 increase after HCV infection does not depend on the HCV genotype. In conclusion, our findings confirm that serum miR-122 levels are significantly upregulated in the HCV cirrhotic patients serving in particular as a biomarker for the non-advanced stages of cirrhosis, independently of the HCV genotype.

Autophagy-Related Activation of Hepatic Stellate Cells Reduces Cellular miR-29a by Promoting Its Vesicular Secretion.

BACKGROUND & AIMS: Liver fibrosis arises from long-term chronic liver injury, accompanied by an accelerated wound healing response with interstitial accumulation of extracellular matrix (ECM). Activated hepatic stellate cells (HSC) are the main source for ECM production. MicroRNA29a (miR-29a) is a crucial antifibrotic miRNA that is repressed during fibrosis, resulting in up-regulation of collagen synthesis. METHODS: Intracellular and extracellular miRNA levels of primary and immortalized myofibroblastic HSC in response to profibrogenic stimulation by transforming growth factor ß (TGFß) or platelet-derived growth factor-BB (PDGF-BB) or upon inhibition of vesicular transport and autophagy processes were determined by quantitative polymerase chain reaction. Autophagy flux was studied by electron microscopy, flow cytometry, immunoblotting, and immunocytochemistry. Hepatic and serum miR-29a levels were quantified by using both liver tissue and serum samples from a cohort of chronic hepatitis C virus patients and a murine CCl4 induced liver fibrosis model. RESULTS: In our study, we show that TGFß and PDGF-BB resulted in decrease of intracellular miR-29a and a pronounced increase of vesicular miR-29a release into the supernatant. Strikingly, miR-29a vesicular release was accompanied by enhanced autophagic activity and up-regulation of the autophagy marker protein LC3. Moreover, autophagy inhibition strongly prevented miR-29a secretion and repressed its targets' expression such as Col1A1. Consistently, hepatic miR-29a loss and increased LC3 expression in myofibroblastic HSC were associated with increased serum miR-29a levels in CCl4-treated murine liver fibrosis and specimens of hepatitis C virus patients with chronic liver disease. CONCLUSIONS: We provide evidence that activation-associated autophagy in HSC induces release of miR-29a, whereas inhibition of autophagy represses fibrogenic gene expression in part through attenuated miR-29a secretion.

Hepatocellular Carcinoma Is a Natural Target for Adeno-Associated Virus (AAV) 2 Vectors.

Although therapeutic options are gradually improving, the overall prognosis for patients with hepatocellular carcinoma (HCC) is still poor. Gene therapy-based strategies are developed to complement the therapeutic armamentarium, both in early and late-stage disease. For efficient delivery of transgenes with antitumor activity, vectors demonstrating preferred tumor tropism are required. Here, we report on the natural tropism of adeno-associated virus (AAV) serotype 2 vectors for HCC. When applied intravenously in transgenic HCC mouse models, similar amounts of vectors were detected in the liver and liver tumor tissue. In contrast, transduction efficiency, as indicated by the level of transgene product, was moderate in the liver but was elevated up to 19-fold in mouse tumor tissue. Preferred transduction of HCC compared to hepatocytes was confirmed in precision-cut liver slices from human patient samples. Our mechanistic studies revealed that this preference is due to the improved intracellular processing of AAV2 vectors in HCC, resulting, for example, in nearly 4-fold more AAV vector episomes that serve as templates for gene transcription. Given this background, AAV2 vectors ought to be considered to strengthen current-or develop novel-strategies for treating HCC.

Transcriptome analysis reveals upregulation of immune response pathways at the invasive tumour front of metastatic seminoma germ cell tumours.

BACKGROUND: Testicular germ cell tumours (TGCTs) have a high metastasis rate. However, the mechanisms related to their invasion, progression and metastasis are unclear. Therefore, we investigated gene expression changes that might be linked to metastasis in seminomatous testicular germ cell tumour (STGCT) patients. METHODS: Defined areas [invasive tumour front (TF) and tumour centre (TC)] of non-metastatic (with surveillance and recurrence-free follow-up >2 years) and metastatic STGCTs were collected separately using laser capture microdissection. The expression of 760 genes related to tumour progression and metastasis was analysed using nCounter technology and validated with quantitative real-time PCR and enzyme-linked immunosorbent assay. RESULTS: Distinct gene expression patterns were observed in metastatic and non-metastatic seminomas with respect to both the TF and TC. Comprehensive pathway analysis showed enrichment of genes related to tumour functions such as inflammation, angiogenesis and metabolism at the TF compared to the TC. Remarkably, prominent inflammatory and cancer-related pathways, such as interleukin-6 (IL-6) signalling, integrin signalling and nuclear factor-κB signalling, were significantly upregulated in the TF of metastatic vs non-metastatic tumours. CONCLUSIONS: IL-6 signalling was the most significantly upregulated pathway in metastatic vs non-metastatic tumours and therefore could constitute a therapeutic target for future personalised therapy. In addition, this is the first study showing intra- and inter-tumour heterogeneity in STGCT.

Diffusion kernel-based predictive modeling of KRAS dependency in KRAS wild type cancer cell lines.

Recent progress in clinical development of KRAS inhibitors has raised interest in predicting the tumor dependency on frequently mutated RAS-pathway oncogenes. However, even without such activating mutations, RAS proteins represent core components in signal integration of several membrane-bound kinases. This raises the question of applications of specific inhibitors independent from the mutational status. Here, we examined CRISPR/RNAi data from over 700 cancer cell lines and identified a subset of cell lines without KRAS gain-of-function mutations (KRASwt) which are dependent on KRAS expression. Combining machine learning-based modeling and whole transcriptome data with prior variable selection through protein-protein interaction network analysis by a diffusion kernel successfully predicted KRAS dependency in the KRASwt subgroup and in all investigated cancer cell lines. In contrast, modeling by RAS activating events (RAE) or previously published RAS RNA-signatures did not provide reliable results, highlighting the heterogeneous distribution of RAE in KRASwt cell lines and the importance of methodological references for expression signature modeling. Furthermore, we show that predictors of KRASwt models contain non-substitutable information signals, indicating a KRAS dependency phenotype in the KRASwt subgroup. Our data suggest that KRAS dependent cancers harboring KRAS wild type status could be targeted by directed therapeutic approaches. RNA-based machine learning models could help in identifying responsive and non-responsive tumors.

Macrophage-derived IL-6 trans-signalling as a novel target in the pathogenesis of bronchopulmonary dysplasia.

RATIONALE: Premature infants exposed to oxygen are at risk for bronchopulmonary dysplasia (BPD), which is characterised by lung growth arrest. Inflammation is important, but the mechanisms remain elusive. Here, we investigated inflammatory pathways and therapeutic targets in severe clinical and experimental BPD. METHODS AND RESULTS: First, transcriptomic analysis with in silico cellular deconvolution identified a lung-intrinsic M1-like-driven cytokine pattern in newborn mice after hyperoxia. These findings were confirmed by gene expression of macrophage-regulating chemokines (Ccl2, Ccl7, Cxcl5) and markers (Il6, Il17A, Mmp12). Secondly, hyperoxia-activated interleukin 6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signalling was measured in vivo and related to loss of alveolar epithelial type II cells (ATII) as well as increased mesenchymal marker. Il6 null mice exhibited preserved ATII survival, reduced myofibroblasts and improved elastic fibre assembly, thus enabling lung growth and protecting lung function. Pharmacological inhibition of global IL-6 signalling and IL-6 trans-signalling promoted alveolarisation and ATII survival after hyperoxia. Third, hyperoxia triggered M1-like polarisation, possibly via Krüppel-like factor 4; hyperoxia-conditioned medium of macrophages and IL-6-impaired ATII proliferation. Finally, clinical data demonstrated elevated macrophage-related plasma cytokines as potential biomarkers that identify infants receiving oxygen at increased risk of developing BPD. Moreover, macrophage-derived IL6 and active STAT3 were related to loss of epithelial cells in BPD lungs. CONCLUSION: We present a novel IL-6-mediated mechanism by which hyperoxia activates macrophages in immature lungs, impairs ATII homeostasis and disrupts elastic fibre formation, thereby inhibiting lung growth. The data provide evidence that IL-6 trans-signalling could offer an innovative pharmacological target to enable lung growth in severe neonatal chronic lung disease.