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Background: Angiogenesis and regenerative wound healing rely on the promotion of distinct endothelial cell phenotypes exhibiting increased migratory capacity. Monitoring of these hallmark events in vitro is invaluable for discovering novel therapeutics. However, respective methods often lack a high-throughput character or accurate analysis tools, which are essential for effective screening suitability. Methods and results: We stained nuclei of confluent human umbilical vein endothelial cells with Hoechst33342 prior to induction of an artificial scratch wound. Treatments with various growth factors and several concentrations of nintedanib were performed to microscopically evaluate impacts on wound closure. We developed 2 tools for automated analysis of wound closure image sets. Utilizing cell-free area measuring or cellular density evaluation, respectively, migration behavior was assessed well-wise for each time point. We identified pro-migratory effects of interleukin 1β as well as inhibitory actions of nintedanib. Hoechst33342 staining allowed for cell counting which was excluded as a contributing factor to wound closure in our assay. Conclusion: We developed a cost-effective, high-throughput pipeline for monitoring cell migration in vitro. We believe that our protocol will accelerate pre-clinical screenings not only for medications targeting endothelial wound closure but also drug discovery research in a broad range of diseases involving cellular migration.

Abstract Beneficial effects of sodium glucose co-transporter 2 inhibitors (SGLT2is) in cardiovascular diseases have been extensively reported leading to the inclusion of these drugs in the treatment guidelines for heart failure. However, molecular actions especially on non-myocyte cells remain uncertain. We observed dose-dependent inhibitory effects of two SGLT2is, dapagliflozin (DAPA) and empagliflozin (EMPA), on inflammatory signaling in human umbilical vein endothelial cells. Proteomic analyses and subsequent enrichment analyses discovered profound effects of these SGLT2is on proteins involved in mitochondrial respiration and actin cytoskeleton. Validation in functional oxygen consumption measurements as well as tube formation and migration assays revealed strong impacts of DAPA. Considering that most influenced parameters played central roles in endothelial to mesenchymal transition (EndMT), we performed in vitro EndMT assays and identified substantial reduction of mesenchymal and fibrosis marker expression as well as changes in cellular morphology upon treatment with SGLT2is. In line, human cardiac fibroblasts exposed to DAPA showed less proliferation, reduced ATP production, and decelerated migration capacity while less extensive impacts were observed upon EMPA. Mechanistically, sodium proton exchanger 1 (NHE1) as well as sodium-myoinositol cotransporter (SMIT) and sodium-multivitamin cotransporter (SMVT) could be identified as relevant targets of SGLT2is in non-myocyte cardiovascular cells as validated by individual siRNA-knockdown experiments. In summary, we found comprehensive beneficial effects of SGLT2is on human endothelial cells and cardiac fibroblasts. The results of this study therefore support a distinct effect of selected SGLT2i on non-myocyte cardiovascular cells and grant further insights into potential molecular mode of action of these drugs.

This study aimed to demonstrate the behavior of different complexes using IR spectroelectrochemistry (SEC), a technique that combines IR spectroscopy with electrochemistry. Four different Mn and Re catalysts for electrochemical CO2 reduction were studied in dry acetonitrile. In the case of Mn(apbpy)(CO)3Br (apbpy = 4(4-aminophenyl)-2,2′-bipyridine), SEC suggested that a very slow catalytic reduction of CO2 also occurs in acetonitrile in the absence of proton donors, but at rather negative potentials. In contrast, the corresponding Re(apbpy)(CO)3Br clearly demonstrated slow catalytic conversion at the first reduction potential. Switching to saturated CO2 solutions in a mixture of acetonitrile and 5% water as a proton donor, the SEC of Mn(apbpy)(CO)3Br displayed a faster catalytic behavior.

ObjectiveMajor depressive disorder (MDD) constitutes a main risk factor for suicide. Suicide risk in psychiatric patients is primarily determined by often unreliable, self-reported information. We assessed serum levels of three microRNAs (miRNAs), previously demonstrated to be dysregulated in post-mortem brain samples of suicide victims, as potential peripheral biomarkers for suicidality.MethodsAll study participants were diagnosed with MDD according to Diagnostic and Statistical Manual of Mental Disorders, 5th edition criteria. Suicidality, defined as acute suicide risk or suicide attempt within one week prior to study entry, was assessed by clinical interview. Relative serum levels of miR-30a, miR-30e, and miR-200a, normalized to U6, were measured by quantitative real-time PCR in MDD inpatients with (MDD/SI, N = 19) and without (MDD, N = 31) acute suicide risk. Median age and gender distribution were comparable in both groups.ResultsLevels of miR-30a, miR-30e, and miR-200a were significantly elevated in MDD/SI compared to MDD. Subgroup analysis of the MDD/SI group showed that levels of miR-30e and miR-200a were significantly higher and miR-30a was increased by trend in patients admitted following a suicide attempt (N = 7) compared to patients with acute suicide risk but without recent suicide attempt (N = 12). Additionally, use of two databases for in silico transcription factor–miRNA interaction prediction indicated early growth response protein (EGR) 1 as potential transcriptional regulator for all three miRNAs.ConclusionThis study demonstrates suicide risk in MDD patients to be associated with increased levels of miR-30a, miR-30e, and miR-200a. Thus, these miRNAs might constitute potential biomarkers to predict suicidal behavior in MDD patients.

BackgroundConstant supply of oxygen is crucial for multicellular tissue homeostasis and energy metabolism in cardiac tissue. As a first response to acute hypoxia, endothelial cells (ECs) promote recruitment and adherence of immune cells to the dysbalanced EC barrier by releasing inflammatory mediators and growth factors, whereas chronic hypoxia leads to the activation of a transcription factor (TF) battery, that potently induces expression of growth factors and cytokines including platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). We report a hypoxia-minded, targeted bioinformatics approach aiming to identify and validate TFs that regulate angiogenic signaling.ResultsA comprehensive RNA-Seq dataset derived from human ECs subjected to normoxic or hypoxic conditions was selected to identify significantly regulated genes based on (i) fold change (normoxia vs. hypoxia) and (ii) relative abundancy. Transcriptional regulation of this gene set was confirmed via qPCR in validation experiments where HUVECs were subjected to hypoxic conditions for 24 h. Screening the promoter and upstream regulatory elements of these genes identified two TFs, KLF5 and SP1, both with a potential binding site within these regions of selected target genes. In vitro, siRNA experiments confirmed SP1- and KLF5-mediated regulation of identified hypoxia-sensitive endothelial genes. Next to angiogenic signaling, we also validated the impact of TFs on inflammatory signaling, both key events in hypoxic sensing. Both TFs impacted on inflammatory signaling since endogenous repression led to increased NF-κB signaling. Additionally, SP1 silencing eventuated decreased angiogenic properties in terms of proliferation and tube formation.ConclusionBy detailed in silico analysis of promoter region and upstream regulatory elements for a list of hypoxia-sensitive genes, our bioinformatics approach identified putative binding sites for TFs of SP or KLF family in vitro. This strategy helped to identify TFs functionally involved in human angiogenic signaling and therefore serves as a base for identifying novel RNA-based drug entities in a therapeutic setting of vascularization.