RNA-Binding Proteins Regulate Post-Transcriptional Responses to TGF-ß to Coordinate Function and Mesenchymal Activation of Murine Endothelial Cells.

BACKGROUND: Endothelial cells (ECs) are primed to respond to various signaling cues. For example, TGF (transforming growth factor)-ß has major effects on EC function and phenotype by driving ECs towards a more mesenchymal state (ie, triggering endothelial to mesenchymal activation), a dynamic process associated with cardiovascular diseases. Although transcriptional regulation triggered by TGF-ß in ECs is well characterized, post-transcriptional regulatory mechanisms induced by TGF-ß remain largely unknown. METHODS: Using RNA interactome capture, we identified global TGF-ß driven changes in RNA-binding proteins in ECs. We investigated specific changes in the RNA-binding patterns of hnRNP H1 (heterogeneous nuclear ribonucleoprotein H1) and Csde1 (cold shock domain containing E1) using RNA immunoprecipitation and overlapped this with RNA-sequencing data after knockdown of either protein for functional insight. Using a modified proximity ligation assay, we visualized the specific interactions between hnRNP H1 and Csde1 and target RNAs in situ both in vitro and in mouse heart sections. RESULTS: Characterization of TGF-ß-regulated RBPs (RNA-binding proteins) revealed hnRNP H1 and Csde1 as key regulators of the cellular response to TGF-ß at the post-transcriptional level, with loss of either protein-promoting mesenchymal activation in ECs. We found that TGF-ß drives an increase in binding of hnRNP H1 to its target RNAs, offsetting mesenchymal activation, but a decrease in Csde1 RNA-binding, facilitating this process. Both, hnRNP H1 and Csde1, dynamically bind and regulate specific subsets of mRNAs related to mesenchymal activation and endothelial function. CONCLUSIONS: Together, we show that RBPs play a key role in the endothelial response to TGF-ß stimulation at the post-transcriptional level and that the RBPs hnRNP H1 and Csde1 serve to maintain EC function and counteract mesenchymal activation. We propose that TGF-ß profoundly modifies RNA-protein interaction entailing feedback and feed-forward control at the post-transcriptional level, to fine-tune mesenchymal activation in ECs.

Fibroblast GATA-4 and GATA-6 promote myocardial adaptation to pressure overload by enhancing cardiac angiogenesis.

Heart failure due to high blood pressure or ischemic injury remains a major problem for millions of patients worldwide. Despite enormous advances in deciphering the molecular mechanisms underlying heart failure progression, the cell-type specific adaptations and especially intercellular signaling remain poorly understood. Cardiac fibroblasts express high levels of cardiogenic transcription factors such as GATA-4 and GATA-6, but their role in fibroblasts during stress is not known. Here, we show that fibroblast GATA-4 and GATA-6 promote adaptive remodeling in pressure overload induced cardiac hypertrophy. Using a mouse model with specific single or double deletion of Gata4 and Gata6 in stress activated fibroblasts, we found a reduced myocardial capillarization in mice with Gata4/6 double deletion following pressure overload, while single deletion of Gata4 or Gata6 had no effect. Importantly, we confirmed the reduced angiogenic response using an in vitro co-culture system with Gata4/6 deleted cardiac fibroblasts and endothelial cells. A comprehensive RNA-sequencing analysis revealed an upregulation of anti-angiogenic genes upon Gata4/6 deletion in fibroblasts, and siRNA mediated downregulation of these genes restored endothelial cell growth. In conclusion, we identified a novel role for the cardiogenic transcription factors GATA-4 and GATA-6 in heart fibroblasts, where both proteins act in concert to promote myocardial capillarization and heart function by directing intercellular crosstalk.

Endothelial cells drive organ fibrosis in mice by inducing expression of the transcription factor SOX9.

Fibrosis is a hallmark of chronic disease. Although fibroblasts are involved, it is unclear to what extent endothelial cells also might contribute. We detected increased expression of the transcription factor Sox9 in endothelial cells in several different mouse fibrosis models. These models included systolic heart failure induced by pressure overload, diastolic heart failure induced by high-fat diet and nitric oxide synthase inhibition, pulmonary fibrosis induced by bleomycin treatment, and liver fibrosis due to a choline-deficient diet. We also observed up-regulation of endothelial SOX9 in cardiac tissue from patients with heart failure. To test whether SOX9 induction was sufficient to cause disease, we generated mice with endothelial cell-specific overexpression of Sox9, which promoted fibrosis in multiple organs and resulted in signs of heart failure. Endothelial Sox9 deletion prevented fibrosis and organ dysfunction in the two mouse models of heart failure as well as in the lung and liver fibrosis mouse models. Bulk and single-cell RNA sequencing of mouse endothelial cells across multiple vascular beds revealed that SOX9 induced extracellular matrix, growth factor, and inflammatory gene expression, leading to matrix deposition by endothelial cells. Moreover, mouse endothelial cells activated neighboring fibroblasts that then migrated and deposited matrix in response to SOX9, a process partly mediated by the secreted growth factor CCN2, a direct SOX9 target; endothelial cell-specific Sox9 deletion reversed these changes. These findings suggest a role for endothelial SOX9 as a fibrosis-promoting factor in different mouse organs during disease and imply that endothelial cells are an important regulator of fibrosis.

C1q and Tumor Necrosis Factor Related Protein 9 Protects from Diabetic Cardiomyopathy by Alleviating Cardiac Insulin Resistance and Inflammation.

(1) Background: Diabetic cardiomyopathy is a major health problem worldwide. CTRP9, a secreted glycoprotein, is mainly expressed in cardiac endothelial cells and becomes downregulated in mouse models of diabetes mellitus; (2) Methods: In this study, we investigated the impact of CTRP9 on early stages of diabetic cardiomyopathy induced by 12 weeks of high-fat diet; (3) Results: While the lack of CTRP9 in knock-out mice aggravated insulin resistance and triggered diastolic left ventricular dysfunction, AAV9-mediated cardiac CTRP9 overexpression ameliorated cardiomyopathy under these conditions. At this early disease state upon high-fat diet, no fibrosis, no oxidative damage and no lipid deposition were identified in the myocardium of any of the experimental groups. Mechanistically, we found that CTRP9 is required for insulin-dependent signaling, cardiac glucose uptake in vivo and oxidative energy production in cardiomyocytes. Extensive RNA sequencing from myocardial tissue of CTRP9-overexpressing and knock-out as well as respective control mice revealed that CTRP9 acts as an anti-inflammatory mediator in the myocardium. Hence, CTRP9 knock-out exerted more, while CTRP9-overexpressing mice showed less leukocytes accumulation in the heart during high-fat diet; (4) Conclusions: In summary, endothelial-derived CTRP9 plays a prominent paracrine role to protect against diabetic cardiomyopathy and might constitute a therapeutic target.

Bridging the gap between GRACE and GRACE-FO missions with deep learning aided water storage simulations.

The monthly high-resolution terrestrial water storage anomalies (TWSA) during the 11-months of gap between GRACE (Gravity Recovery And Climate Experiment) and its successor GRACE-FO (-Follow On) missions are missing. The continuity of the GRACE-like TWSA series with commensurate accuracy is of great importance for the improvement of hydrologic models both at global and regional scales. While previous efforts to bridge this gap, though without achieving GRACE-like spatial resolutions and/or accuracy have been performed, high-quality TWSA simulations at global scale are still lacking. Here, we use a suite of deep learning (DL) architectures, convolutional neural networks (CNN), deep convolutional autoencoders (DCAE), and Bayesian convolutional neural networks (BCNN), with training datasets including GRACE/-FO mascon and Swarm gravimetry, ECMWF Reanalysis-5 data, normalized time tag information to reconstruct global land TWSA maps, at a much higher resolution (100 km full wavelength) than that of GRACE/-FO, and effectively bridge the 11-month data gap globally. Contrary to previous studies, we applied no prior de-trending or de-seasoning to avoid biasing/aliasing the simulations induced by interannual or longer climate signals and extreme weather episodes. We show the contribution of Swarm and time inputs which significantly improved the TWSA simulations in particular for correct prediction of the trend component. Our results also show that external validation with independent data when filling large data gaps within spatio-temporal time series of geophysical signals is mandatory to maintain the robustness of the simulation results. The results and comparisons with previous studies and the adopted DL methods demonstrate the superior performance of DCAE. Validations of our DCAE-based TWSA simulations with independent datasets, including in situ groundwater level, Interferometric Synthetic Aperture Radar measured land subsidence rate (e.g. Central Valley), occurrence/timing of severe flash flood (e.g. South Asian Floods) and drought (e.g. Northern Great Plain, North America) events occurred within the gap, reveal excellent agreements.

Identification of watermelon heat shock protein members and tissue-specific gene expression analysis under combined drought and heat stresses.

Heat shock protein (Hsp) gene family members in the watermelon genome were identified and characterized by bioinformatics analysis. In addition, expression profiles of genes under combined drought and heat stress conditions were experimentally analyzed. In the watermelon genome, 39 genes belonging to the sHsp family, 101 genes belonging to the Hsp40 family, 23 genes belonging to the Hsp60 family, 12 genes belonging to the Hsp70 family, 6 genes belonging to the Hsp90 family, and 102 genes belonging to the Hsp100 family were found. It was also observed that the proteins in the same cluster in the phylogenetic trees had similar motif patterns. When the estimated 3-dimensional structures of the Hsp proteins were examined, it was determined that the α-helical structure was dominant in almost all families. The most orthologous relationship appeared to be between watermelon, soybean, and poplar in the ClaHsp gene families. For tissue-specific gene expression analysis under combined stress conditions, expression analysis of one representative Hsp gene each from root, stem, leaf, and shoot tissues was performed by real-time PCR. A significant increase was detected usually at 30 min in almost all tissues. This study provides extensive information for watermelon Hsps, and can enhance our knowledge about the relationships between Hsp genes and combined stresses.