Direct GPCR-EGFR interaction enables synergistic membrane-to-nucleus information transfer.

We addressed the heteromerization of the epidermal growth factor receptor (EGFR) with G-protein coupled receptors (GPCR) on the basis of angiotensin-II-receptor-subtype-1(AT1R)-EGFR interaction as proof-of-concept and show its functional relevance during synergistic nuclear information transfer, beyond ligand-dependent EGFR transactivation. Following in silico modelling, we generated EGFR-interaction deficient AT1R-mutants and compared them to AT1R-wildtype. Receptor interaction was assessed by co-immunoprecipitation (CoIP), Förster resonance energy transfer (FRET) and fluorescence-lifetime imaging microscopy (FLIM). Changes in cell morphology, ERK1/2-phosphorylation (ppERK1/2), serum response factor (SRF)-activation and cFOS protein expression were determined by digital high content microscopy at the single cell level. FRET, FLIM and CoIP confirmed the physical interaction of AT1R-wildtype with EGFR that was strongly reduced for the AT1R-mutants. Responsiveness of cells transfected with AT1R-WT or -mutants to angiotensin II or EGF was similar regarding changes in cell circularity, ppERK1/2 (direct and by ligand-dependent EGFR-transactivation), cFOS-expression and SRF-activity. By contrast, the EGFR-AT1R-synergism regarding these parameters was completely absent for in the interaction-deficient AT1R mutants. The results show that AT1R-EGFR heteromerisation enables AT1R-EGFR-synergism on downstream gene expression regulation, modulating the intensity and the temporal pattern of nuclear AT1R/EGFR-information transfer. Furthermore, remote EGFR transactivation, via ligand release or cytosolic tyrosine kinases, is not sufficient for the complete synergistic control of gene expression.

The synergism of cytosolic acidosis and reduced NAD+/NADH ratio is responsible for lactic acidosis-induced vascular smooth muscle cell impairment in sepsis.

BACKGROUND: During sepsis, serve vascular dysfunctions lead to life-threatening multiple organ failure, due to vascular smooth muscle cells (VSMC) impairments, resulting in vasoplegia, hypotension and hypoperfusion. In addition, septic patients have an altered cell metabolism that leads to lactic acidosis. Septic patients suffering from lactic acidosis have a high risk of mortality. In addition, septic survivors are at risk of secondary vascular disease. The underlying mechanisms of whether and how lactic acidosis leads to the changes in VSMCs is not well understood. The aim of this study was to comprehensively investigate the effect of lactic acidosis on VSMCs and additionally compare the effects with those induced by pure acidosis and sodium lactate. METHODS: Primary human aortic smooth muscle cells (HAoSMCs) were treated for 48 h with lactic acidosis (LA_pH 6.8), hydrochloric acid (HCl_pH 6.8), sodium lactate (Na+-lactate_pH 7.4) and the respective controls (ctrl._pH 7.4; hyperosmolarity control: mannitol_pH 7.4) and comparatively analyzed for changes in (i) transcriptome, (ii) energy metabolism, and (iii) phenotype. RESULTS: Both types of acidosis led to comparable and sustained intracellular acidification without affecting cell viability. RNA sequencing and detailed transcriptome analysis revealed more significant changes for lactic acidosis than for hydrochloric acidosis, with lactate being almost ineffective, suggesting qualitative and quantitative synergism of acidosis and lactate. Bioinformatic predictions in energy metabolism and phenotype were confirmed experimentally. Lactic acidosis resulted in strong inhibition of glycolysis, glutaminolysis, and altered mitochondrial respiration which reduced cellular ATP content, likely due to increased TXNIP expression and altered NAD+/NADH ratio. Hydrochloric acidosis induced significantly smaller effects without changing the NAD+/NADH ratio, with the ATP content remaining constant. These metabolic changes led to osteo-/chondrogenic/senescent transdifferentiation of VSMCs, with the effect being more pronounced in lactic acidosis than in pure acidosis. CONCLUSIONS: Overall, lactic acidosis exerted a much stronger effect on energy metabolism than pure acidosis, whereas lactate had almost no effect, reflecting the qualitative and quantitative synergism of acidosis and lactate. As a consequence, lactic acidosis may lead to acute functional impairments of VSMC, sustained perturbations of the transcriptome and cellular dedifferentiation. Moreover, these effects may contribute to the acute and prolonged vascular pathomechanisms in septic patients.

Crosstalk with renal proximal tubule cells drives acidosis-induced inflammatory response and dedifferentiation of fibroblasts via p38-singaling.

BACKGROUND: Tubulointerstitial kidney disease associated microenvironmental dysregulation, like acidification, inflammation and fibrosis, affects tubule cells and fibroblasts. Micromilieu homeostasis influences intracellular signaling and intercellular crosstalk. Cell-cell communication in turn modulates the interstitial microenvironment. We assessed the impact of acidosis on inflammatory and fibrotic responses in proximal tubule cells and fibroblasts as a function of cellular crosstalk. Furthermore, cellular signaling pathways involved were identified. METHODS: HK-2 (human proximal tubule) and CCD-1092Sk (human fibroblasts), in mono and coculture, were exposed to acidic or control media for 3 or 48 h. Protein expression of inflammation markers (TNF, TGF-ß and COX-2), dedifferentiation markers (N-cadherin, vinculin, ß-catenin and vimentin), fibrosis markers (collagen III and fibronectin) and phospho- as well as total MAPK levels were determined by western blot. Secreted collagen III and fibronectin were measured by ELISA. The impact of MAPK activation was assessed by pharmacological intervention. In addition, necrosis, apoptosis and epithelial permeability were determined. RESULTS: Independent of culture conditions, acidosis caused a decrease of COX-2, vimentin and fibronectin expression in proximal tubule cells. Only in monoculture, ß-Catenin expression decreased and collagen III expression increased in tubule cells during acidosis. By contrast, in coculture collagen III protein expression of tubule cells was reduced. In fibroblasts acidosis led to an increase of TNF, COX-2, vimentin, vinculin, N-cadherin protein expression and a decrease of TGF-ß expression exclusively in coculture. In monoculture, expression of COX-2 and fibronectin was reduced. Collagen III expression of fibroblasts was reduced by acidosis independent of culture conditions. In coculture, acidosis enhanced phosphorylation of ERK1/2, JNK1/2 and p38 transiently in proximal tubule cells. In fibroblasts, acidosis enhanced phosphorylation of p38 in a sustained and very strong manner. ERK1/2 and JNK1/2 were not affected in fibroblasts. Inhibition of JNK1/2 and p38 under coculture conditions reduced acidosis-induced changes in fibroblasts significantly. CONCLUSIONS: Our data show that the crosstalk between proximal tubule cells and fibroblasts is crucial for acidosis-induced dedifferentiation of fibroblasts into an inflammatory phenotype. This dedifferentiation is at least in part mediated by p38 and JNK1/2. Thus, cell-cell communication is essential for the pathophysiological impact of tubulointerstitial acidosis.

Liquid biomarkers of macrophage dysregulation and circulating spike protein illustrate the biological heterogeneity in patients with post-acute sequelae of COVID-19.

Post-acute sequelae of COVID-19 (PASC) are long-term consequences of SARS-CoV-2 infection that can substantially impair the quality of life. Underlying mechanisms ranging from persistent viruses to innate and adaptive immune dysregulation have been discussed. Here, we profiled the plasma of 181 individuals from the cohort study for digital health research in Germany (DigiHero), including individuals after mild to moderate COVID-19 with or without PASC and uninfected controls. We focused on soluble factors related to monocyte/macrophage biology and on circulating SARS-CoV-2 spike (S1) protein as a potential biomarker for persistent viral reservoirs. At a median time of 8 months after infection, we found pronounced dysregulation in almost all tested soluble factors, including both pro-inflammatory and pro-fibrotic cytokines. These immunological perturbations were remarkably independent of ongoing PASC symptoms per se, but further correlation and regression analyses suggested PASC-specific patterns involving CCL2/MCP-1 and IL-8 that either correlated with sCD162, sCD206/MMR, IFN-α2, IL-17A and IL-33, or IL-18 and IL-23. None of the analyzed factors correlated with the detectability or levels of circulating S1, indicating that this represents an independent subset of patients with PASC. These data confirm prior evidence of immune dysregulation and persistence of viral protein in PASC and illustrate its biological heterogeneity that still awaits correlation with clinically defined PASC subtypes.

Modulation of transcriptional mineralocorticoid receptor activity by casein kinase 1.

The mineralocorticoid receptor (MR) with its ligand aldosterone (aldo) physiologically regulates electrolyte homeostasis and blood pressure but it can also lead to pathophysiological effects in the cardiovascular system. Previous results show that posttranslational modifications (PTM) can influence MR signaling and function. Based on in silico and in vitro data, casein kinase 1 (CK1) was predicted as a candidate for MR phosphorylation. To gain a deeper mechanistic insight into MR activation, we investigated the influence of CK1 on MR function in HEK cells. Co-immunoprecipitation experiments indicated that the MR is located in a protein-protein complex with CK1α and CK1ε. Reporter gene assays with pharmacological inhibitors and MR constructs demonstrated that especially CK1ε acts as a positive modulator of GRE activity via the C-terminal MR domains CDEF. CK1 enhanced the binding affinity of aldosterone to the MR, facilitated nuclear translocation and DNA interaction of the MR, and led to expression changes of pathophysiologically relevant genes like Per-1 and Phlda1. By peptide microarray and site-directed mutagenesis experiments, we identified the highly conserved T800 as a direct CK1 phosphorylation site of the MR, which modulates the nuclear import and genomic activity of the receptor. Direct phosphorylation of the MR was unable to fully account for all of the CK1 effects on MR signaling, suggesting additional phosphorylation of MR co-regulators. By LC/MS/MS, we identified the MR-associated proteins NOLC1 and TCOF1 as candidates for such CK1-regulated co-factors. Overall, we found that CK1 acts as a co-activator of MR GRE activity through direct and indirect phosphorylation, which accelerates cytosolic-nuclear trafficking, facilitates nuclear accumulation and DNA binding of the MR, and increases the expression of pathologically relevant MR-target genes.

Synergy of epidermal growth factor (EGFR) and angiotensin II (AT1R) receptor determines composition and temporal pattern of transcriptome variation.

The tyrosine kinase receptor EGFR and the G-protein-coupled receptor AT1R induce essential cellular responses, in part via receptor crosstalk with an unknown role in nuclear information transfer and transcription regulation. We investigated whether this crosstalk results in linear, EGFR-mediated nuclear signalling or in parallel, synergistic information transfer leading to qualitative and temporal variations, relevant for gene expression and environment interaction. AT1R and EGFR synergistically activate SRF via the ERK1/2-TCF and actin-MRTF pathways. Synergism, comprised of switch-like and graded single cell response, converges on the transcription factors AP1 and EGR, resulting in synergistic transcriptome alterations, in qualitative (over-additive number of genes), quantitative (over-additive expression changes of individual genes) and temporal (more late onset and prolonged expressed genes) terms. Gene ontology and IPA® pathway analysis indicate prolonged cell stress (e.g. hypoxia-like) and dysregulated vascular biology. Synergism occurs during separate but simultaneous activation of both receptors and during AT1R-induced EGFR transactivation. EGFR and AT1R synergistically regulate gene expression in qualitative, quantitative and temporal terms with (patho)physiological relevance, extending the importance of EGFR-AT1R crosstalk beyond cytoplasmic signalling.

Identification and initial characterization of POLIII-driven transcripts by msRNA-sequencing.

Non-coding RNAs (ncRNAs) are powerful regulators of gene expression but medium-sized (50-300 nts in length) ncRNAs (msRNAs) are barely picked-up precisely by RNA-sequencing. Here we describe msRNA-sequencing (msRNAseq), a modified protocol that associated with a computational analyses pipeline identified about ~1800 msRNA loci, including over 300 putatively novel msRNAs, in human and murine cells. We focused on the identification and initial characterization of three POLIII-derived transcripts. The validation of these uncharacterized msRNAs identified an ncRNA in antisense orientation from the POLR3E locus transcribed by POLIII. This msRNA, termed POLAR (POLR3E Antisense RNA), has a strikingly short half-life, localizes to paraspeckles (PSPs) and associates with PSP-associated proteins indicating that msRNAseq identifies functional msRNAs. Thus, our analyses will pave the way for analysing the roles of msRNAs in cells, development and diseases.

miR-221 and -222 target CACNA1C and KCNJ5 leading to altered cardiac ion channel expression and current density.

MicroRNAs (miRs) contribute to different aspects of cardiovascular pathology, among others cardiac hypertrophy and atrial fibrillation. The aim of our study was to evaluate the impact of miR-221/222 on cardiac electrical remodeling. Cardiac miR expression was analyzed in a mouse model with altered electrocardiography parameters and severe heart hypertrophy. Next generation sequencing revealed 14 differentially expressed miRs in hypertrophic hearts, with miR-221 and -222 being the strongest regulated miR-cluster. This increase was restricted to cardiomyocytes and not observed in cardiac fibroblasts. Additionally, we evaluated the change of miR-221/222 in vivo in two models of pharmacologically induced heart hypertrophy (angiotensin II, isoprenaline), thereby demonstrating a stimulus-induced increase in miR-221/222 in vivo by angiotensin II but not by isoprenaline. Whole transcriptome analysis by RNA-seq and qRT-PCR validation revealed an enriched number of downregulated mRNAs coding for proteins located in the T-tubule, which are also predicted targets for miR-221/222. Among those, mRNAs were the L-type Ca2+ channel subunits as well as potassium channel subunits. We confirmed that both miRs target the 3'-untranslated regions of Cacna1c and Kcnj5. Furthermore, enhanced expression of these miRs reduced L-type Ca2+ channel and Kcnj5 channel abundance and function, which was analyzed by whole-cell patch clamp recordings or Western blot and flux measurements, respectively. miR-221 and -222 contribute to the regulation of L-type Ca2+ channels as well as Kcnj5 channels and, therefore, potentially contribute to disturbed cardiac excitation generation and propagation. Future studies will have to evaluate the pathophysiological and clinical relevance of aberrant miR-221/222 expression for electrical remodeling.

Knockout of vascular smooth muscle EGF receptor in a mouse model prevents obesity-induced vascular dysfunction and renal damage in vivo.

AIMS/HYPOTHESIS: Obesity causes type 2 diabetes leading to vascular dysfunction and finally renal end-organ damage. Vascular smooth muscle (VSM) EGF receptor (EGFR) modulates vascular wall homeostasis in part via serum response factor (SRF), a major regulator of VSM differentiation and a sensor for glucose. We investigated the role of VSM-EGFR during obesity-induced renovascular dysfunction, as well as EGFR-hyperglycaemia crosstalk. METHODS: The role of VSM-EGFR during high-fat diet (HFD)-induced type 2 diabetes was investigated in a mouse model with inducible, VSM-specific EGFR-knockout (KO). Various structural and functional variables as well as transcriptome changes, in vivo and ex vivo, were assessed. The impact of hyperglycaemia on EGFR-induced signalling and SRF transcriptional activity and the underlying mechanisms were investigated at the cellular level. RESULTS: We show that VSM-EGFR mediates obesity/type 2 diabetes-induced vascular dysfunction, remodelling and transcriptome dysregulation preceding renal damage and identify an EGFR-glucose synergism in terms of SRF activation, matrix dysregulation and mitochondrial function. EGFR deletion protects the animals from HFD-induced endothelial dysfunction, creatininaemia and albuminuria. Furthermore, we show that HFD leads to marked changes of the aortic transcriptome in wild-type but not in KO animals, indicative of EGFR-dependent SRF activation, matrix dysregulation and mitochondrial dysfunction, the latter confirmed at the cellular level. Studies at the cellular level revealed that high glucose potentiated EGFR/EGF receptor 2 (ErbB2)-induced stimulation of SRF activity, enhancing the graded signalling responses to EGF, via the EGFR/ErbB2-ROCK-actin-MRTF pathway and promoted mitochondrial dysfunction. CONCLUSIONS/INTERPRETATION: VSM-EGFR contributes to HFD-induced vascular and subsequent renal alterations. We propose that a potentiated EGFR/ErbB2-ROCK-MRTF-SRF signalling axis and mitochondrial dysfunction underlie the role of EGFR. This advanced working hypothesis will be investigated in mechanistic depth in future studies. VSM-EGFR may be a therapeutic target in cases of type 2 diabetes-induced renovascular disease. DATA AVAILABILITY: The datasets generated during and/or analysed during the current study are available in: (1) share_it, the data repository of the academic libraries of Saxony-Anhalt ( https://doi.org/10.25673/32049.2 ); and (2) in the gene expression omnibus database with the study identity GSE144838 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE144838 ). Graphical abstract.

A hydraulic model of cardiovascular physiology and pathophysiology embedded into a computer-based teaching system for student training in laboratory courses.

Functional understanding of the different parts of the cardiovascular system is essential for an insight into pathomechanisms of numerous diseases. During training cardiovascular physiology, students and early-stage medical personnel should understand the role of different functional parameters for systolic and diastolic blood pressure, as well as for blood flow. The impact of isolated parameters can only be studied in models. Here physical hydraulic models are an advantage in which the students have a direct contact to the mechanical properties of the circulatory system. But these models are often difficult to handle. The aim of the present study was to develop a comprehensive model of the cardiovascular system, including a mechanical heart with valves, an elastic aorta, a more rigid peripheral artery system, a total peripheral resistance, and a venous reservoir representing the variable cardiac preload. This model allows one to vary systematically several functional parameters and to continuously record their impact on pressure and flow. This model is embedded into a computer-based teaching system (LabTutor) in which the students are guided through the handling of the model (as well as the systematic variation of parameters), and the measured data can be analyzed. This hybrid teaching system, which is routinely integrated in physiology laboratory courses of medical students, allows students to work with a complex hydraulic model of the cardiovascular system and to analyze systematically the impact of influencing variables (e.g., increased peripheral resistance or changed cardiac preload) as well as pathophysiological dysfunctions (e.g., reduced aortic compliance).

Identification of a novel lncRNA induced by the nephrotoxin ochratoxin A and expressed in human renal tumor tissue.

Long non-coding RNAs represent a fraction of the transcriptome that is being increasingly recognized. For most of them no function has been allocated so far. Here, we describe the nature and function of a novel non-protein-coding transcript, named WISP1-AS1, discovered in human renal proximal tubule cells exposed to the carcinogenic nephrotoxin ochratoxin A. WISP1-AS1 overlaps parts of the fourth intron and fifth exon of the Wnt1-inducible signaling pathway protein 1 (WISP1) gene. The transcript is 2922 nucleotides long, transcribed in antisense direction and predominantly localized in the nucleus. WISP1-AS1 is expressed in all 20 samples of a human tissue RNA panel with the highest expression levels detected in uterus, kidney and adrenal gland. Its expression was confirmed in primary tissues of human kidneys. In addition, WISP1-AS1 is expressed at higher levels in renal cell carcinoma (RCC) cell lines compared to primary proximal tubule cells as well as in RCC lesions than in the adjacent healthy control tissue from the same patient. Using specific gapmer antisense oligonucleotides to prevent its upregulation, we show that WISP1-AS1 (1) does not influence the mRNA expression of WISP1, (2) affects transcriptional regulation by Egr-1 and E2F as revealed by RNA-sequencing, enrichment analysis and reporter assays, and (3) modulates the apoptosis-necrosis balance. In summary, WISP1-AS1 is a novel lncRNA with modulatory transcriptional function and the potential to alter the cellular phenotype in situations of stress or oncogenic transformation. However, its precise mode of action and impact on cellular functions require further investigations.

Substance-specific importance of EGFR for vascular smooth muscle cells motility in primary culture.

Besides their importance for the vascular tone, vascular smooth muscle cells (VSMC) also contribute to pathophysiological vessel alterations. Various G-protein coupled receptor ligands involved in vascular dysfunction and remodeling can transactivate the epidermal growth factor receptor (EGFR) of VSMC, yet the importance of EGFR transactivation for the VSMC phenotype is incompletely understood. The aims of this study were (i) to characterize further the importance of the VSMC-EGFR for proliferation, migration and marker gene expression for inflammation, fibrosis and reactive oxygen species (ROS) homeostasis and (ii) to test the hypothesis that vasoactive substances (endothelin-1, phenylephrine, thrombin, vasopressin and ATP) rely differentially on the EGFR with respect to the abovementioned phenotypic alterations. In primary, aortic VSMC from mice without conditional deletion of the EGFR, proliferation, migration, marker gene expression (inflammation, fibrosis and ROS homeostasis) and cell signaling (ERK 1/2, intracellular calcium) were analyzed. VSMC-EGFR loss reduced collective cell migration and single cell migration probability, while no difference between the genotypes in single cell velocity, chemotaxis or marker gene expression could be observed under control conditions. EGF promoted proliferation, collective cell migration, chemokinesis and chemotaxis and leads to a proinflammatory gene expression profile in wildtype but not in knockout VSMC. Comparing the impact of five vasoactive substances (all reported to transactivate EGFR and all leading to an EGFR dependent increase in ERK1/2 phosphorylation), we demonstrate that the importance of EGFR for their action is substance-dependent and most apparent for crowd migration but plays a minor role for gene expression regulation.

Activated mineralocorticoid receptor regulates micro-RNA-29b in vascular smooth muscle cells.

Inappropriately activated mineralocorticoid receptor (MR) is a risk factor for vascular remodeling with unclear molecular mechanism. Recent findings suggest that post-transcriptional regulation by micro-RNAs (miRs) may be involved. Our aim was to search for MR-dependent miRs in vascular smooth muscle cells (VSMCs) and to explore the underlying molecular mechanism and the pathologic relevance. We detected that aldosteroneviathe MR reduces miR-29bin vivoin murine aorta and in human primary and cultured VSMCs (ED50= 0.07 nM) but not in endothelial cells [quantitative PCR (qPCR), luciferase assays]. This effect was mediated by an increased decay of miR-29b in the cytoplasm with unchanged miR-29 family member or primary-miR levels. Decreased miR-29b led to an increase in extracellular matrix measured by ELISA and qPCR and enhanced VSMC migration in single cell-tracking experiments. Additionally, cell proliferation and the apoptosis/necrosis ratio (caspase/lactate dehydrogenase assay) was modulated by miR-29b. Enhanced VSMC migration by aldosterone required miR-29b regulation. Control experiments were performed with scrambled RNA and empty plasmids, by comparing aldosterone-stimulated with vehicle-incubated cells. Overall, our findings provide novel insights into the molecular mechanism of aldosterone-mediated vascular pathogenesis by identifying miR-29b as a pathophysiologic relevant target of activated MR in VSMCs and by highlighting the importance of miR processing for miR regulation.-Bretschneider, M., Busch, B., Mueller, D., Nolze, A., Schreier, B., Gekle, M., Grossmann, C. Activated mineralocorticoid receptor regulates micro-RNA-29b in vascular smooth muscle cells.

Structure-activity relationship of ochratoxin A and synthesized derivatives: importance of amino acid and halogen moiety for cytotoxicity.

The enigma why the mycotoxin ochratoxin A (OTA) impairs cell and organ function is still not solved. However, an interaction with target molecules is a prerequisite for any observed adverse effect. This interaction depends on characteristics of the target molecule as well as on the OTA molecule itself. OTA has different structural moieties which may be relevant for these interrelations including a halogen (chlorine) and an amino acid group (phenylalanine). To test their importance for the impact of OTA, detailed structure-activity studies with various OTA derivatives were performed. For this, 23 OTA derivatives were available, which were modified by either an exchange of the halogen moiety against another halogen (fluorine, iodine or bromine) or by the amino acid moiety against another one (tyrosine or alanine) or a combination of both. Additionally, the configuration of the 3R carbon atom was changed to 3S. These derivatives were tested in human renal cells for their ability to induce cell death (cytotoxicity, apoptosis, necrosis), their impact on collagen protein secretion and for their influence on gene expression. It turned out that the substitution of the amino acid moiety against tyrosine or alanine almost completely prevented the adverse effects of OTA. The exchange of the halogen moiety had minor effects and the inversion of the stereochemistry at C3 did not prevent the effects of OTA. Therefore, we conclude that the amino acid moiety of OTA is indispensable for the interaction of OTA with its target molecules.

Consequences of postnatal vascular smooth muscle EGFR deletion on acute angiotensin II action.

Epi dermal growth factor (EGF) receptor (EGFR) is activated by its canonical ligands and transactivated by various vasoactive substances, e.g. angiotensin II (Ang II). Vascular EGFR has been proposed to be involved in vascular tissue homoeostasis and remodelling. Thus, most studies have focused on its role during long-term vascular changes whereas the relevance for acute regulation of vascular function in vivo and ex vivo is insufficiently understood. To investigate the postnatal role of VSMCs (vascular smooth muscle cells) EGFR in vivo and ex vivo, we generated a mouse model with cell-specific and inducible deletion of VSMC EGFR and studied the effect on basal blood pressure, acute pressure response to, among others, Ang II in vivo as well as ex vivo, cardiovascular tissue homoeostasis and vessel morphometry in male mice. In knockout (KO) animals, systolic, diastolic and mean blood pressures were reduced compared with wild-type (WT). Furthermore, Ang II-induced pressure load was lower in KO animals, as was Ang II-induced force development and extracellular-signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in aortic rings from KO animals. By contrast, we observed no difference in force development during application of serotonin, KCl, endothelin-1 or endothelin-1-induced pressure load in KO animals. In addition, nitric oxide (NO)-mediated vasodilation was not affected. Heart weight (HW) increase and up-regulation of aortic and cardiac expression of Ccl2 (chemoattractant protein-2) and serpinE1 (plasminogen activator inhibitor 1) during the transition from 4- to 10-months of age were prevented by VSMC EGFR KO. We conclude that VSMC EGFR is involved in basal blood pressure homoeostasis and acute pressure response to Ang II, and thereby contributes to maturation-related remodelling.

Acidosis-Induced Changes in Proteome Patterns of the Prostate Cancer-Derived Tumor Cell Line AT-1.

Under various pathological conditions, such as inflammation, ischemia and in solid tumors, physiological parameters (local oxygen tension or extracellular pH) show distinct tissue abnormalities (hypoxia and acidosis). For tumors, the prevailing microenvironment exerts a strong influence on the phenotype with respect to proliferation, invasion, and metastasis formation and therefore influences prognosis. In this study, we investigate the impact of extracellular metabolic acidosis (pH 7.4 versus 6.6) on the proteome patterns of a prostate cancer-derived tumor cell type (AT-1) using isobaric labeling and LC-MS/MS analysis. In total, 2710 proteins were identified and quantified across four biological replicates, of which seven were significantly affected with changes >50% and used for validation. Glucose transporter 1 and farnesyl pyrophosphatase were found to be down-regulated after 48 h of acidic treatment, and metallothionein 2A was reduced after 24 h and returned to control values after 48 h. After 24 and 48 h at pH 6.6, glutathione S transferase A3 and NAD(P)H dehydrogenase 1, cellular retinoic acid-binding protein 2, and Na-bicarbonate transporter 3 levels were found to be increased. The changes in protein levels were confirmed by transcriptome and functional analyses. In addition to the experimental in-depth investigation of proteins with changes >50%, functional profiling (statistical enrichment analysis) including proteins with changes >20% revealed that acidosis upregulates GSH metabolic processes, citric acid cycle, and respiratory electron transport. Metabolism of lipids and cholesterol biosynthesis were downregulated. Our data comprise the first comprehensive report on acidosis-induced changes in proteome patterns of a tumor cell line.

Regulation of Expression of Renal Organic Anion Transporters OAT1 and OAT3 in a Model of Ischemia/Reperfusion Injury.

BACKGROUND: Recently, we gained evidence that impairment of rOat1 and rOat3 expression induced by ischemic acute kidney injury (AKI) is mediated by COX metabolites and this suppression might be critically involved in renal damage. METHODS: (i) Basolateral organic anion uptake into proximal tubular cells after model ischemia and reperfusion (I/R) was investigated by fluorescein uptake. The putative promoter sequences from hOAT1 (SLC22A6) and hOAT3 (SCL22A8) were cloned into a reporter plasmid, transfected into HEK cells and (ii) transcriptional activity was determined after model ischemia and reperfusion as a SEAP reporter gen assay. Inhibitors or antagonists were applied with the beginning of reperfusion. RESULTS: By using inhibitors of PKA (H89) and PLC (U73122), antagonists of E prostanoid receptor type 2 (AH6809) and type 4 (L161,982), we gained evidence that I/R induced down regulation of organic anion transport is mediated by COX1 metabolites via E prostanoid receptor type 4. The latter signaling was confirmed by application of butaprost (EP2 agonist) or TCS2510 (EP4 agonist) to control cells. In brief, the latter signaling was verified for the transcriptional activity in the reporter gen assay established. Therein, selective inhibitors for COX1 (SC58125) and COX2 (SC560) were also applied. CONCLUSION: Our data show (a) that COX1 metabolites are involved in the regulation of renal organic anion transport(ers) after I/R via the EP4 receptor and (b) that this is due to transcriptional regulation of the respective transporters. As the promoter sequences cloned were of human origin and expressed in a human renal epithelial cell line we (c) hypothesize that the regulatory mechanisms described after I/R is meaningful for humans as well.

NOS1 induces NADPH oxidases and impairs contraction kinetics in aged murine ventricular myocytes.

Nitric oxide (NO) modulates calcium transients and contraction of cardiomyocytes. However, it is largely unknown whether NO contributes also to alterations in the contractile function of cardiomyocytes during aging. Therefore, we analyzed the putative role of nitric oxide synthases and NO for the age-related alterations of cardiomyocyte contraction. We used C57BL/6 mice, nitric oxide synthase 1 (NOS1)-deficient mice (NOS1(-/-)) and mice with cardiomyocyte-specific NOS1-overexpression to analyze contractions, calcium transients (Indo-1 fluorescence), acto-myosin ATPase activity (malachite green assay), NADPH oxidase activity (lucigenin chemiluminescence) of isolated ventricular myocytes and cardiac gene expression (Western blots, qPCR). In C57BL/6 mice, cardiac expression of NOS1 was upregulated by aging. Since we found a negative regulation of NOS1 expression by cAMP in isolated cardiomyocytes, we suggest that reduced efficacy of ß-adrenergic signaling that is evident in aged hearts promotes upregulation of NOS1. Shortening and relengthening of cardiomyocytes from aged C57BL/6 mice were decelerated, but were normalized by pharmacological inhibition of NOS1/NO. Cardiomyocytes from NOS1(-/-) mice displayed no age-related changes in contraction, calcium transients or acto-myosin ATPase activity. Aging increased cardiac expression of NADPH oxidase subunits NOX2 and NOX4 in C57BL/6 mice, but not in NOS1(-/-) mice. Similarly, cardiac expression of NOX2 and NOX4 was upregulated in a murine model with cardiomyocyte-specific overexpression of NOS1. We conclude that age-dependently upregulated NOS1, putatively via reduced efficacy of ß-adrenergic signaling, induces NADPH oxidases. By increasing nitrosative and oxidative stress, both enzyme systems act synergistically to decelerate contraction of aged cardiomyocytes.

Direct inhibition, but indirect sensitization of pacemaker activity to sympathetic tone by the interaction of endotoxin with HCN-channels.

In critically ill patients regulation of heart-rate is often severely disturbed. Interaction of bacterial endotoxin (lipopolysaccharide, LPS) with hyperpolarization-activated cyclic nucleotide-gated cation-(HCN)-channels may interfere with heart-rate regulation. This study analyzes the effect of LPS, the HCN-channel blocker ivabradine or Ca(2+) -channel blockers (nifedipine, verapamil) on pacemaking in spontaneously beating neonatal rat cardiomyocytes (CM) in vitro. In vivo, the effect of LPS on the heart-rate of adult CD1-mice with and without autonomic blockade is analyzed telemetrically. LPS (100 ng/mL) and ivabradine (5 µg/mL) reduced the beating-rate of CM by 20.1% and 24.6%, respectively. Coincubation of CM with both, LPS and ivabradine, did not further reduce the beating-rate, indicating interaction of both compounds with HCN-channels, while coincubation with Ca(2+) -channel blockers and LPS caused additive beating-rate reduction. In CD1-mice (containing an active autonomic-nervous-system), injection of LPS (0.4 mg/kg) expectedly resulted in increased heart-rate. However, if the autonomic nervous system was blocked by propranolol and atropine, in line with the in vitro data, LPS induced a significant reduction of heart-rate, which was not additive to ivabradine. The in vivo and in vitro results indicate that LPS interacts with HCN-channels of cardiomyocytes. Thus, LPS indirectly sensitizes HCN-channels for sympathetic activation (tachycardic-effect), and in parallel directly inhibits channel activity (bradycardic-effect). Both effects may contribute to the detrimental effects of septic cardiomyopathy and septic autonomic dysfunction.

Mineralocorticoid receptor interaction with SP1 generates a new response element for pathophysiologically relevant gene expression.

The mineralocorticoid receptor (MR) is a ligand-induced transcription factor belonging to the steroid receptor family and involved in water-electrolyte homeostasis, blood pressure regulation, inflammation and fibrosis in the renocardiovascular system. The MR shares a common hormone-response-element with the glucocorticoid receptor but nevertheless elicits MR-specific effects including enhanced epidermal growth factor receptor (EGFR) expression via unknown mechanisms. The EGFR is a receptor tyrosine kinase that leads to activation of MAP kinases, but that can also function as a signal transducer for other signaling pathways. In the present study, we mechanistically investigate the interaction between a newly discovered MR- but not glucocorticoid receptor- responsive-element (=MRE1) of the EGFR promoter, specificity protein 1 (SP1) and MR to gain general insights into MR-specificity. Biological relevance of the interaction for EGFR expression and consequently for different signaling pathways in general is demonstrated in human, rat and murine vascular smooth muscle cells and cells of EGFR knockout mice. A genome-wide promoter search for identical binding regions followed by quantitative PCR validation suggests that the identified MR-SP1-MRE1 interaction might be applicable to other genes. Overall, a novel principle of MR-specific gene expression is explored that applies to the pathophysiologically relevant expression of the EGFR and potentially also to other genes.