Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter. Targeted demethylation of the BDNF promoter IV or the MyoD distal enhancer by dCas9-Tet1 induced BDNF expression in post-mitotic neurons or activated MyoD facilitating reprogramming of fibroblasts into myoblasts, respectively. Targeted de novo methylation of a CTCF loop anchor site by dCas9-Dnmt3a blocked CTCF binding and interfered with DNA looping, causing altered gene expression in the neighboring loop. Finally, we show that these tools can edit DNA methylation in mice, demonstrating their wide utility for functional studies of epigenetic regulation.

Lung mucoepidermoid carcinoma (MEC) is a very poorly characterized rare subtype of non-small-cell lung cancer (NSCLC) associated with more favorable prognoses than other forms of intrathoracic malignancies. We have previously identified that heme oxygenase-1 (HO-1, encoded by HMOX1) inhibits MEC tumor growth and modulates the transcriptome of microRNAs. Here we investigate the role of a major upstream regulator of HO-1 and a master regulator of cellular antioxidant responses, transcription factor Nrf2, in MEC biology. Nrf2 overexpression in the NCI-H292 MEC cell line mimicked the phenotype of HO-1 overexpressing cells, leading to inhibition of cell proliferation and migration and down-regulation of oncogenic miR-378. HMOX1 silencing identified HO-1 as a major mediator of Nrf2 action. Nrf2- and HO-1 overexpressing cells exhibited strongly diminished expression of multiple matrix metalloproteinases and inflammatory cytokine interleukin-1β, which was confirmed in an NCI-HO-1 xenograft model. Overexpression of HO-1 altered not only human MMP levels in tumor cells but also murine MMP levels within tumor microenvironment and metastatic niche. This could possibly contribute to decreased metastasis to the lungs and inhibitory effects of HO-1 on MEC tumor growth. Our profound transcriptome analysis and molecular characterization of the mucoepidermoid lung carcinoma helps to understand the specific clinical presentations of these tumors, emphasizing a unique antitumoral role of the Nrf2-HO-1 axis.

Autoantibodies specific for malondialdehyde-modified LDL (MDA-LDL) represent potential biomarkers to predict cardiovascular risk. However, MDA-LDL is a high variability antigen with limited reproducibility. To identify peptide mimotopes of MDA-LDL, phage display libraries were screened with the MDA-LDL-specific IgM monoclonal Ab LRO4, and the specificity and antigenic properties of MDA mimotopes were assessed in vitro and in vivo. We identified one 12-mer linear (P1) and one 7-mer cyclic (P2) peptide carrying a consensus sequence, which bound specifically to murine and human anti-MDA monoclonal Abs. Furthermore, MDA mimotopes were found to mimic MDA epitopes on the surface of apoptotic cells. Immunization of mice with P2 resulted in the induction of MDA-LDL-specific Abs, which strongly immunostained human atherosclerotic lesions. We detected IgG and IgM autoAbs to both MDA mimotopes in sera of healthy subjects and patients with myocardial infarction and stable angina pectoris undergoing percutaneous coronary intervention, and the titers of autoAbs correlated significantly with respective Ab titers against MDA-LDL. In conclusion, we identified specific peptides that are immunological mimotopes of MDA. These mimotopes can serve as standardized and reproducible antigens that will be useful for diagnostic and therapeutic applications in cardiovascular disease.

Obesity causes DNA damage, which is causally related to several disorders including cancer, infertility, and cognitive dysfunctions. The aim of this study is to investigate whether weight loss improves the integrity of the genetic material.

Liver fibrosis represents the wound healing response to sustained hepatic injury with activation of hepatic stellate cells (HSCs). The I148M variant of the PNPLA3 gene represents a risk factor for development of severe liver fibrosis. Activated HSCs carrying the I148M variant display exacerbated pro-inflammatory and pro-fibrogenic features. We aimed to examine whether the I148M variant may impair Hedgehog and Yap signaling, as key pathways implicated in the control of energy expenditure and maintenance of myofibroblastic traits. First, we show that TGF-β rapidly up-regulated the PNPLA3 transcript and protein and Yap/Hedgehog target gene expression. In addition, HSCs overexpressing PNPLA3 I148M boosted anaerobic glycolysis, as supported by higher lactate release and decreased phosphorylation of the energy sensor AMPK. These cells displayed higher Yap and Hedgehog signaling, due to accumulation of total Yap protein, Yap promoter activity and increased downstream targets expression, compared to WT cells. HSCs exposed to TGF-β and leptin rapidly increased total Yap, together with a reduction in its inhibited form, phosphorylated Yap. In line, Yap-specific inhibitor Verteporfin strongly abolished Yap-mediated genes expression, at baseline as well as after TGF-β and leptin treatments in HSCs with I148M PNPLA3. Finally, Yap transcriptional activity was strongly reduced by a combination of Verteporfin and Rosiglitazone, a PPARγ synthetic agonist. In conclusion, HSCs carrying the PNPLA3 variant show activated Yap/Hedgehog pathways, resulting in altered anaerobic glycolysis and enhanced synthesis of Hedgehog markers and sustained Yap signaling. TGF-β and leptin exacerbate Yap/Hedgehog-related fibrogenic genes expression, while Yap inhibitors and PPARγ agonists abrogate these effects in PNPLA3 I148M carrying HSCs.

Several single-nucleotide polymorphisms have been identified to be disadvantageous or protective in regard to disease severity in patients with non-alcoholic fatty liver disease (NAFLD). However, it is unclear, whether including genetic risk factor(s) either alone or combined into risk stratification algorithms for NAFLD actually provides incremental benefit over clinical risk factors.

Obesity causes genetic instability, which plays a key-role in the etiology of cancer and aging. We investigated the impact of bariatric surgery (BS) on DNA repair, oxidative DNA damage, telomere lengths, alterations of antioxidant enzymes and, selected proteins which reflect inflammation. The study was realized with BS patients (n = 35). DNA damage, base oxidation, BER, and NER were measured before and 1 month and 6 months after surgery with the single-cell gel electrophoresis technique. SOD and GPx were quantified spectrophotometrically, malondealdehyde (MDA) was quantified by HPLC. Telomere lengths were determined with qPCR, and plasma proteome profiling was performed with high-resolution mass spectrophotometry. Six months after the operations, reduction of body weight by 27.5% was observed. DNA damage decreased after this period, this effect was paralleled by reduced formation of oxidized DNA bases, a decline in the MDA levels and of BER and NER, and an increase in the telomere lengths. The activities of antioxidant enzymes were not altered. Clear downregulation of certain proteins (CRP, SAA1) which reflect inflammation and cancer risks was observed. Our findings show that BS causes reduced oxidative damage of DNA bases, possibly as a consequence of reduction of inflammation and lipid peroxidation, and indicate that the surgery has beneficial long-term health effects.

Obesity and diabetes affect more than half a billion individuals worldwide. Interestingly, the two conditions do not always coincide and the molecular determinants of "healthy" versus "unhealthy" obesity remain ill-defined. Chronic metabolic inflammation (metaflammation) is believed to be pivotal. Here, we tested a hypothesized anti-inflammatory role for heme oxygenase-1 (HO-1) in the development of metabolic disease. Surprisingly, in matched biopsies from "healthy" versus insulin-resistant obese subjects we find HO-1 to be among the strongest positive predictors of metabolic disease in humans. We find that hepatocyte and macrophage conditional HO-1 deletion in mice evokes resistance to diet-induced insulin resistance and inflammation, dramatically reducing secondary disease such as steatosis and liver toxicity. Intriguingly, cellular assays show that HO-1 defines prestimulation thresholds for inflammatory skewing and NF-κB amplification in macrophages and for insulin signaling in hepatocytes. These findings identify HO-1 inhibition as a potential therapeutic strategy for metabolic disease.

Bariatric patients often suffer from vitamin D deficiency (VDD), and both, morbid obesity and VDD, are related to non-alcoholic fatty liver disease. However, limited data are available regarding best strategies for treating VDD, particularly, in bariatric patients undergoing omega-loop gastric bypass (OLGB). Therefore, we examined the efficacy and safety of a forced vitamin D dosing regimen and intervention effects in liver fibrotic patients.

T cells and level of the cytokine interferon-γ (IFN-γ) are increased in adipose tissue in obesity. Hedgehog (Hh) signaling has been shown to potently inhibit white adipocyte differentiation. In light of recent findings in neurons that IFN-γ and Hh signaling cross-talk, we examined their potential interaction in the context of adipogenesis.

Carbon monoxide (CO) dampens pro-inflammatory responses in a peroxisome proliferator-activated receptor-γ (PPARγ) and p38 mitogen-activated protein kinase (MAPK) dependent manner. Previously, we demonstrated that CO inhibits lipopolysaccharide (LPS)-induced expression of the proinflammatory early growth response-1 (Egr-1) transcription factor in macrophages via activation of PPARγ. Here, we further characterize the molecular mechanisms by which CO modulates the activity of PPARγ and Egr-1 repression. We demonstrate that CO enhances SUMOylation of PPARγ which we find was attributed to mitochondrial ROS generation. Ectopic expression of a SUMOylation-defective PPARγ-K365R mutant partially abolished CO-mediated suppression of LPS-induced Egr-1 promoter activity. Expression of a PPARγ-K77R mutant did not impair the effect of CO. In addition to PPARγ SUMOylation, CO-activated p38 MAPK was responsible for Egr-1 repression. Blocking both CO-induced PPARγ SUMOylation and p38 activation, completely reversed the effects of CO on inflammatory gene expression. In primary macrophages isolated form C57/BL6 male mice, we identify mitochondrial ROS formation by CO as the upstream trigger for the observed effects on Egr-1 in part through uncoupling protein 2 (UCP2). Macrophages derived from bone marrow isolated from Ucp2 gene Knock-Out C57/BL6 mice (Ucp2(-/-)), produced significantly less ROS with CO exposure versus wild-type macrophages. Moreover, absence of UCP2 resulted in a complete loss of CO mediated Egr-1 repression. Collectively, these results indentify p38 activation, PPARγ-SUMOylation and ROS formation via UCP2 as a cooperative system by which CO impacts the inflammatory response.

Mutations in the transcription factor SOX18 are responsible for specific cardiovascular defects in humans and mice. In order to gain insight into the molecular basis of its action, we identified target genes of SOX18 and analyzed one, MMP7, in detail.

Obesity is one of the most important health-related problems of the twenty-first century. Data on its prevalence in Austria remain scarce. Aim of this study was to determine current trends of overweight and obesity, associated comorbidities and socioeconomic status in all 18-year-old male Austrian citizens, and its potential impact on the demand for bariatric surgery in the future.

Obesity and body fat distribution are important risk factors for the development of type 2 diabetes and metabolic syndrome. Evidence has accumulated that this risk is related to intrinsic differences in behavior of adipocytes in different fat depots. We recently identified LIM domain only 3 (LMO3) in human mature visceral adipocytes; however, its function in these cells is currently unknown. The aim of this study was to determine the potential involvement of LMO3-dependent pathways in the modulation of key functions of mature adipocytes during obesity. Based on a recently engineered hybrid rAAV serotype Rec2 shown to efficiently transduce both brown adipose tissue (BAT) and white adipose tissue (WAT), we delivered YFP or Lmo3 to epididymal WAT (eWAT) of C57Bl6/J mice on a high-fat diet (HFD). The effects of eWAT transduction on metabolic parameters were evaluated 10 weeks later. To further define the role of LMO3 in insulin-stimulated glucose uptake, insulin signaling, adipocyte bioenergetics, as well as endocrine function, experiments were conducted in 3T3-L1 adipocytes and newly differentiated human primary mature adipocytes, engineered for transient gain or loss of LMO3 expression, respectively. AAV transduction of eWAT results in strong and stable Lmo3 expression specifically in the adipocyte fraction over a course of 10 weeks with HFD feeding. LMO3 expression in eWAT significantly improved insulin sensitivity and healthy visceral adipose tissue expansion in diet-induced obesity, paralleled by increased serum adiponectin. In vitro, LMO3 expression in 3T3-L1 adipocytes increased PPARγ transcriptional activity, insulin-stimulated GLUT4 translocation and glucose uptake, as well as mitochondrial oxidative capacity in addition to fatty acid oxidation. Mechanistically, LMO3 induced the PPARγ coregulator Ncoa1, which was required for LMO3 to enhance glucose uptake and mitochondrial oxidative gene expression. In human mature adipocytes, LMO3 overexpression promoted, while silencing of LMO3 suppressed mitochondrial oxidative capacity. LMO3 expression in visceral adipose tissue regulates multiple genes that preserve adipose tissue functionality during obesity, such as glucose metabolism, insulin sensitivity, mitochondrial function, and adiponectin secretion. Together with increased PPARγ activity and Ncoa1 expression, these gene expression changes promote insulin-induced GLUT4 translocation, glucose uptake in addition to increased mitochondrial oxidative capacity, limiting HFD-induced adipose dysfunction. These data add LMO3 as a novel regulator improving visceral adipose tissue function during obesity. KEY MESSAGES: LMO3 increases beneficial visceral adipose tissue expansion and insulin sensitivity in vivo. LMO3 increases glucose uptake and oxidative mitochondrial activity in adipocytes. LMO3 increases nuclear coactivator 1 (Ncoa1). LMO3-enhanced glucose uptake and mitochondrial gene expression requires Ncoa1.

Bariatric surgery reduces mortality in patients with severe obesity and is predominantly performed in women. Therefore, an analysis of sex-specific differences after bariatric surgery in a population-based dataset from Austria was performed. The focus was on deceased patients after bariatric surgery.

Obesity is a major risk factor for several diseases including diabetes, heart disease, and some forms of cancer and due to its rapidly increasing prevalence it has become one of the biggest problems medicine is facing today. All the more surprising, a substantial percentage of obese patients are metabolically healthy when classified based on insulin resistance and systemic inflammation. Oxysterols are naturally occurring molecules that play important role in various metabolic and inflammatory processes and their levels are elevated in patients suffering from obesity and diabetes. 25-Hydroxycholesterol (25-OHC) is produced in cells from cholesterol by the enzyme cholesterol 25-hydroxylase (Ch25h) and is involved in lipid metabolism, inflammatory processes, and cell proliferation. Here, we investigated the role of hepatic Ch25h in the transition from metabolically healthy obesity to insulin resistance and diabetes. Using several different experimental approaches, we demonstrated the significance of Ch25h on the border of "healthy" and "diseased" states of obesity. Adenovirus-mediated Ch25h overexpression in mice improved glucose tolerance and insulin sensitivity and lowered HOMA-IR. Our data suggest that low hepatic Ch25h levels could be considered a risk marker for unhealthy obesity.

Over the past years, plenty of evidence has emerged illustrating how metabolism supports many aspects of cellular function and how metabolic reprogramming can drive cell differentiation and fate. Here, we present a method to assess the metabolic configuration of single cells within their native tissue microenvironment via the visualization and quantification of multiple enzymatic activities measured at saturating substrate conditions combined with subsequent cell type identification. After careful validation of the approach and to demonstrate its potential, we assessed the intracellular metabolic configuration of different human immune cell populations in healthy and tumor colon tissue. Additionally, we analyzed the intercellular metabolic relationship between cancer cells and cancer-associated fibroblasts in a breast cancer tissue array. This study demonstrates that the determination of metabolic configurations in single cells could be a powerful complementary tool for every researcher interested to study metabolic networks in situ.

Synovial fibroblasts are key cells orchestrating the inflammatory response in arthritis. Here we demonstrate that loss of miR-146a, a key epigenetic regulator of the innate immune response, leads to increased joint destruction in a TNF-driven model of arthritis by specifically regulating the behavior of synovial fibroblasts. Absence of miR-146a in synovial fibroblasts display a highly deregulated gene expression pattern and enhanced proliferation in vitro and in vivo. Deficiency of miR-146a induces deregulation of tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6) in synovial fibroblasts, leading to increased proliferation. In addition, loss of miR-146a shifts the metabolic state of fibroblasts towards glycolysis and augments the ability of synovial fibroblasts to support the generation of osteoclasts by controlling the balance of osteoclastogenic regulatory factors receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG). Bone marrow transplantation experiments confirmed the importance of miR-146a in the radioresistant mesenchymal compartment for the control of arthritis severity, in particular for inflammatory joint destruction. This study therefore identifies microRNA-146a as an important local epigenetic regulator of the inflammatory response in arthritis. It is a central element of an anti-inflammatory feedback loop in resident synovial fibroblasts, who are orchestrating the inflammatory response in chronic arthritis. MiR-146a restricts their activation, thereby preventing excessive tissue damage during arthritis.

A substantial number of patients undergoing bariatric surgery are prescribed psychopharmacological medication. However, the impact of concomitant psychopharmacological medication on the frequency of relevant vitamin deficiencies in postoperative follow-up is not known.

Excessive accumulation of white adipose tissue (WAT) is a hallmark of obesity. The expansion of WAT in obesity involves proliferation and differentiation of adipose precursors, however, the underlying molecular mechanisms remain unclear. Here, we used an unbiased transcriptomics approach to identify the earliest molecular underpinnings occuring in adipose precursors following a brief HFD in mice. Our analysis identifies Heme Oxygenase-1 (HO-1) as strongly and selectively being upregulated in the adipose precursor fraction of WAT, upon high-fat diet (HFD) feeding. Specific deletion of HO-1 in adipose precursors of Hmox1fl/flPdgfraCre mice enhanced HFD-dependent visceral adipose precursor proliferation and differentiation. Mechanistically, HO-1 reduces HFD-induced AKT2 phosphorylation via ROS thresholding in mitochondria to reduce visceral adipose precursor proliferation. HO-1 influences adipogenesis in a cell-autonomous way by regulating events early in adipogenesis, during the process of mitotic clonal expansion, upstream of Cebpα and PPARγ. Similar effects on human preadipocyte proliferation and differentiation in vitro were observed upon modulation of HO-1 expression. This collectively renders HO-1 as an essential factor linking extrinsic factors (HFD) with inhibition of specific downstream molecular mediators (ROS &AKT2), resulting in diminished adipogenesis that may contribute to hyperplastic adipose tissue expansion.