Cardiomyocyte-specific RXFP1 overexpression protects against pressure overload-induced cardiac dysfunction independently of relaxin.

Heart failure (HF) prevalence is rising due to reduced early mortality and demographic change. Relaxin (RLN) mediates protective effects in the cardiovascular system through Relaxin-receptor 1 (RXFP1). Cardiac overexpression of RXFP1 with additional RLN supplementation attenuated HF in the pressure-overload transverse aortic constriction (TAC) model. Here, we hypothesized that robust transgenic RXFP1 overexpression in cardiomyocytes (CM) protects from TAC-induced HF even in the absence of RLN. Hence, transgenic mice with a CM-specific overexpression of human RXFP1 (hRXFP1tg) were generated. Receptor functionality was demonstrated by in vivo hemodynamics, where the administration of RLN induced positive inotropy strictly in hRXFP1tg. An increase in phospholamban-phosphorylation at serine 16 was identified as a molecular correlate. hRXFP1tg were protected from TAC without additional RLN administration, presenting not only less decline in systolic left ventricular (LV) function but also abrogated LV dilation and pulmonary congestion compared to WT mice. Molecularly, transgenic hearts exhibited not only a significantly attenuated fetal and fibrotic gene activation but also demonstrated less fibrotic tissue and CM hypertrophy in histological sections. These protective effects were evident in both sexes. Similar cardioprotective effects of hRXFP1tg were detectable in a RLN-knockout model, suggesting an alternative mechanism of receptor activation through intrinsic activity, alternative endogenous ligands or crosstalk with other receptors. In summary, CM-specific RXFP1 overexpression provides protection against TAC even in the absence of endogenous RLN. This suggests RXFP1 overexpression as a potential therapeutic approach for HF, offering baseline protection with optional RLN supplementation for specific activation.

[Nonautonomous effects of oncogenic YAP in hepatocarcinogenesis]. / Nichtautonome Effekte des Onkogens YAP in der Hepatokarzinogenese.

BACKGROUND: The transcriptional coactivator yes-associated protein (YAP) is a strong oncogene in liver cancer development. OBJECTIVES: To investigate if and how YAP-induced paracrine-acting factors are regulated in hepatocytes and liver cancer cells. MATERIAL AND METHODS: Transcriptome analysis and proteomics of murine wildtype and YAP-transgenic hepatocytes were performed to identify paracrine-acting proteins. Molecular and biochemical techniques were used to examine the mechanisms of YAP-dependent gene regulation. Gene expression data from HCC (hepatocellular carcinoma) patients was evaluated. RESULTS: Several YAP-dependent, secreted factors (e. g. CXCL10, GDF15, PDGFB) were identified. YAP regulates these factors through transcription factors of the TEAD (TEA domain) protein family. Moreover, the dysregulation of the YAP-target genes is often associated with poor HCC patient prognosis. CONCLUSIONS: YAP induces the expression of paracrine-acting factors that may affect the tumor microenvironment and therefore support carcinogenesis. This multicellular network could allow the development of novel and specific perturbation approaches.

Brain-specific Foxp1 deletion impairs neuronal development and causes autistic-like behaviour.

Neurodevelopmental disorders are multi-faceted and can lead to intellectual disability, autism spectrum disorder and language impairment. Mutations in the Forkhead box FOXP1 gene have been linked to all these disorders, suggesting that it may play a central role in various cognitive and social processes. To understand the role of Foxp1 in the context of neurodevelopment leading to alterations in cognition and behaviour, we generated mice with a brain-specific Foxp1 deletion (Nestin-Cre(Foxp1-/-)mice). The mutant mice were viable and allowed for the first time the analysis of pre- and postnatal neurodevelopmental phenotypes, which included a pronounced disruption of the developing striatum and more subtle alterations in the hippocampus. More detailed analysis in the CA1 region revealed abnormal neuronal morphogenesis that was associated with reduced excitability and an imbalance of excitatory to inhibitory input in CA1 hippocampal neurons in Nestin-Cre(Foxp1-/-) mice. Foxp1 ablation was also associated with various cognitive and social deficits, providing new insights into its behavioural importance.

Investigation of manic and euthymic episodes identifies state- and trait-specific gene expression and STAB1 as a new candidate gene for bipolar disorder.

Bipolar disorder (BD) is a highly heritable psychiatric disease characterized by recurrent episodes of mania and depression. To identify new BD genes and pathways, the present study employed a three-step approach. First, gene-expression profiles of BD patients were assessed during both a manic and an euthymic phase. These profiles were compared intra-individually and with the gene-expression profiles of controls. Second, those differentially expressed genes that were considered potential trait markers of BD were validated using data from the Psychiatric Genomics Consortiums' genome-wide association study (GWAS) of BD. Third, the implicated molecular mechanisms were investigated using pathway analytical methods. In the present patients, this novel approach identified: (i) sets of differentially expressed genes specific to mania and euthymia; and (ii) a set of differentially expressed genes that were common to both mood states. In the GWAS data integration analysis, one gene (STAB1) remained significant (P=1.9 × 10(-4)) after adjustment for multiple testing. STAB1 is located in close proximity to PBMR1 and the NEK4-ITIH1-ITIH3-ITIH4 region, which are the top findings from GWAS meta-analyses of mood disorder, and a combined BD and schizophrenia data set. Pathway analyses in the mania versus control comparison revealed three distinct clusters of pathways tagging molecular mechanisms implicated in BD, for example, energy metabolism, inflammation and the ubiquitin proteasome system. The present findings suggest that STAB1 is a new and highly promising candidate gene in this region. The combining of gene expression and GWAS data may provide valuable insights into the biological mechanisms of BD.

Identification of the novel differentiation marker MS4A8B and its murine homolog MS4A8A in colonic epithelial cells lost during neoplastic transformation in human colon.

The CD20-homolog Ms4a8a has recently been shown to be a marker for alternatively activated macrophages but its expression is not restricted to hematopoietic cells. Here, MS4A8A/MS4A8B expression was detected in differentiated intestinal epithelium in mouse and human, respectively. Interestingly, no MS4A8B expression was found in human colon carcinoma. Forced overexpression of MS4A8A in the murine colon carcinoma cell line CT26 led to a reduced proliferation and migration rate. In addition, MS4A8A-expressing CT26 cells displayed an increased resistance to hydrogen peroxide-induced apoptosis, which translated in an increased end weight of subcutaneous MS4A8A+ CT26 tumors. Gene profiling of MS4A8A+ CT26 cells revealed a significant regulation of 225 genes, most of them involved in cytoskeletal organization, apoptosis, proliferation, transcriptional regulation and metabolic processes. Thereby, the highest upregulated gene was the intestinal differentiation marker cytokeratin 20. In conclusion, we show that MS4A8A/MS4A8B is a novel differentiation marker of the intestinal epithelium that supports the maintenance of a physiological barrier function in the gut by modulating the transcriptome and by conferring an increased resistance to reactive oxygen species. The absence of MS4A8B in human colonic adenocarcinomas shown in this study might be a helpful tool to differentiate between healthy and neoplastic tissue.

Gremlin 1, frizzled-related protein, and Dkk-1 are key regulators of human articular cartilage homeostasis.

OBJECTIVE: The development of osteoarthritis (OA) may be caused by activation of hypertrophic differentiation of articular chondrocytes. Healthy articular cartilage is highly resistant to hypertrophic differentiation, in contrast to other hyaline cartilage subtypes, such as growth plate cartilage. The purpose of this study was to elucidate the molecular mechanism responsible for the difference in the propensity of human articular cartilage and growth plate cartilage to undergo hypertrophic differentiation. METHODS: Whole-genome gene-expression microarray analysis of healthy human growth plate and articular cartilage derived from the same adolescent donors was performed. Candidate genes, which were enriched in the articular cartilage, were validated at the messenger RNA (mRNA) and protein levels and examined for their potential to inhibit hypertrophic differentiation in two models. In addition, we studied a possible genetic association with OA. RESULTS: Pathway analysis demonstrated decreased Wnt signaling in articular cartilage as compared to growth plate cartilage. This was at least partly due to increased expression of the bone morphogenetic protein and Wnt antagonists Gremlin 1, Frizzled-related protein (FRP), and Dkk-1 at the mRNA and protein levels in articular cartilage. Supplementation of these proteins diminished terminal hypertrophic differentiation without affecting chondrogenesis in long-bone explant cultures and chondrogenically differentiating human mesenchymal stem cells. Additionally, we found that single-nucleotide polymorphism rs12593365, which is located in a genomic control region of GREM1, was significantly associated with a 20% reduced risk of radiographic hip OA in 2 population-based cohorts. CONCLUSION: Taken together, our study identified Gremlin 1, FRP, and Dkk-1 as natural brakes on hypertrophic differentiation in articular cartilage. As hypertrophic differentiation of articular cartilage may contribute to the development of OA, our findings may open new avenues for therapeutic intervention.

Expression pattern differences between osteoarthritic chondrocytes and mesenchymal stem cells during chondrogenic differentiation.

OBJECTIVE: The use of mesenchymal stem cells (MSCs) for cartilage regeneration is hampered by lack of knowledge about the underlying molecular differences between chondrogenically stimulated chondrocytes and MSCs. The aim of this study was to evaluate differences in phenotype and gene expression between primary human chondrocytes and MSCs during chondrogenic differentiation in three-dimensional (3D) pellet culture (PC). MATERIALS AND METHODS: Chondrocytes isolated from cartilage samples obtained during total knee alloarthroplastic procedure (N=8) and MSCs, purified from bone marrow aspirates of healthy donors (N=8), were cultivated in PC under chondrogenic conditions. Immunohistology and quantitative reverse transcribing PCR (RT-PCR) were performed for chondrogenic-specific markers (i.e., Sox9, Collagen II). Global gene expression of the so-cultivated chondrocytes and MSCs was assessed by a novel approach of microarray-based pathway analysis. Refinement of data was done by hypothesis-driven gene expression omnibus (GEO) dataset comparison. Validation was performed with separate samples in transforming growth factor (TGF)ß+ or TGFß- conditions by use of quantitative real-time RT-PCR. RESULTS/CONCLUSIONS: Chondrogenic commitment of both cell types was observed. Interestingly, chondrocytes demonstrated an upregulated fatty acid/cholesterol metabolism which may give hints for future optimization of culture conditions. The novel microarray-based pathway analysis applied in this study seems suitable for the evaluation of whole-genome based array datasets in case when hypotheses can be backed with already existing GEO datasets. Future experiments should further explore the different metabolic behaviour of chondrocytes and MSC.

Modulation of brain dead induced inflammation by vagus nerve stimulation.

Because the vagus nerve is implicated in control of inflammation, we investigated if brain death (BD) causes impairment of the parasympathetic nervous system, thereby contributing to inflammation. BD was induced in rats. Anaesthetised ventilated rats (NBD) served as control. Heart rate variability (HRV) was assessed by ECG. The vagus nerve was electrically stimulated (BD + STIM) during BD. Intestine, kidney, heart and liver were recovered after 6 hours. Affymetrix chip-analysis was performed on intestinal RNA. Quantitative PCR was performed on all organs. Serum was collected to assess TNFalpha concentrations. Renal transplantations were performed to address the influence of vagus nerve stimulation on graft outcome. HRV was significantly lower in BD animals. Vagus nerve stimulation inhibited the increase in serum TNFalpha concentrations and resulted in down-regulation of a multiplicity of pro-inflammatory genes in intestinal tissue. In renal tissue vagal stimulation significantly decreased the expression of E-selectin, IL1beta and ITGA6. Renal function was significantly better in recipients that received a graft from a BD + STIM donor. Our study demonstrates impairment of the parasympathetic nervous system during BD and inhibition of serum TNFalpha through vagal stimulation. Vagus nerve stimulation variably affected gene expression in donor organs and improved renal function in recipients.

Amplification of Cyclin L1 is associated with lymph node metastases in head and neck squamous cell carcinoma (HNSCC).

Overrepresentation of chromosomal bands 3q25-q29 has been associated with shortened disease-specific survival in head and neck squamous cell carcinoma (HNSCC). To assess the prevalence of copy number gains (>4 signals per cell) and high-level amplifications (>8 signals per cell) from putative oncogenes in this chromosomal region (CCNL1, SNO, PIK3CA, TP73L), tissue microarray analysis was applied on 280 HNSCCs by fluorescence in situ hybridization. Overall frequency of additional copy numbers was 34.3% for CCNL1, 31.8% for SNO, 39.0% for PIK3CA and 38.3% for TP73L, respectively. In general, gains were more frequently detected in stage IV compared to stage I-III tumours. Performing multivariate logistic regression analysis, a significant association of CCNL1 gains and the presence of lymph node metastases was found, which was independent of anatomical site and T-stage of the primary tumour (P=0.049). Site-specific subgroup analysis further showed that copy number gains of CCNL1 and SNO occurred more frequently in oral carcinomas in advanced clinical stages as compared to N0 oral lesions (CCNL1: P=0.03; SNO: P=0.03). Finally, Kaplan-Meier analysis revealed that high-level amplifications of CCNL1 correlated with shorter overall survival of the patients. Our results indicate that CCNL1 plays a critical role in the loco-regional progression of HNSCC and may serve as an indicator for occult advanced tumour stages.