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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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

The thromboxane A 2 receptor (TP) has been shown to play a role in angiotensin II (Ang II)-mediated hypertension and pathological vascular remodeling. To assess the impact of vascular TP on Ang II-induced hypertension, atherogenesis, and pathological aortic alterations, i.e. aneurysms, we analysed Western-type diet-fed and Ang II-infused TP VSMC KO /Ldlr KO, TP EC KO /Ldlr KO mice and their respective wild-type littermates (TP WT /Ldlr KO). These analyses showed that neither EC- nor VSMC-specific deletion of the TP significantly affected basal or Ang II-induced blood pressure or aortic atherosclerotic lesion area. In contrast, VSMC-specific TP deletion abolished and EC-specific TP deletion surprisingly reduced the ex vivo reactivity of aortic rings to the TP agonist U-46619, whereas VSMC-specific TP knockout also diminished the ex vivo response of aortic rings to Ang II. Furthermore, despite similar systemic blood pressure, there was a trend towards less atherogenesis in the aortic arch and a trend towards fewer pathological aortic alterations in Ang II-treated female TP VSMC KO /Ldlr KO mice. Survival was impaired in male mice after Ang II infusion and tended to be higher in TP VSMC KO /Ldlr KO mice than in TP WT /Ldlr KO littermates. Thus, our data may suggest a deleterious role of the TP expressed in VSMC in the pathogenesis of Ang II-induced aortic atherosclerosis in female mice, and a surprising role of the endothelial TP in TP-mediated aortic contraction. However, future studies are needed to substantiate and further elucidate the role of the vascular TP in the pathogenesis of Ang II-induced hypertension, aortic atherosclerosis and aneurysm formation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Endothelial cells (EC) are key players in vascular function, homeostasis and inflammation. EC show substantial heterogeneity due to inter-individual variability (e.g. sex-differences) and intra-individual differences as they originate from different organs or vessels. This variability may lead to different responsiveness to external stimuli. Here we compared the responsiveness of female human primary EC from the aorta (HAoEC) and coronary arteries (HCAEC) to Epidermal Growth Factor Receptor (EGFR) activation. EGFR is an important signal integration hub for vascular active substances with physiological and pathophysiological relevance. Our transcriptomic analysis suggested that EGFR activation differentially affects the inflammatory profiles of HAoEC and HCAEC, particularly by inducing a HCAEC-driven leukocyte attraction but a downregulation of adhesion molecule and chemoattractant expression in HAoEC. Experimental assessments of selected inflammation markers were performed to validate these predictions and the results confirmed a dual role of EGFR in these cells: its activation initiated an anti-inflammatory response in HAoEC but a pro-inflammatory one in HCAEC. Our study highlights that, although they are both arterial EC, female HAoEC and HCAEC are distinguishable with regard to the role of EGFR and its involvement in inflammation regulation, what may be relevant for vascular maintenance but also the pathogenesis of endothelial dysfunction., (© 2023. The Author(s).)

The epidermal growth factor receptor (EGFR) belongs to the ErbB-family of receptor tyrosine kinases that are of importance in oncology. During the last years, substantial evidence accumulated for a crucial role of EGFR concerning the action of the angiotensin II type 1 receptor (AT1R) in blood vessels, resulting form AT1R-induced EGFR transactivation. This transactivation occurs through the release of membrane-anchored EGFR-ligands, cytosolic tyrosine kinases, heterocomplex formation or enhanced ligand expression. AT1R-EGFR crosstalk amplifies the signaling response and enhances the biological effects of angiotensin II. Downstream signaling cascades include ERK1/2 and p38 MAPK, PLCγ and STAT. AT1R-induced EGFR activation contributes to vascular remodeling and hypertrophy via e.g. smooth muscle cell proliferation, migration and extracellular matrix production. EGFR transactivation results in increased vessel wall thickness and reduced vascular compliance. AT1R and EGFR signaling pathways are also implicated the induction of vascular inflammation. Again, EGFR transactivation exacerbates the effects, leading to endothelial dysfunction that contributes to vascular inflammation, dysfunction and remodeling. Dysregulation of the AT1R-EGFR axis has been implicated in the pathogenesis of various cardiovascular diseases and inhibition or prevention of EGFR signaling can attenuate part of the detrimental impact of enhanced renin-angiotensin-system (RAAS) activity, highlighting the importance of EGFR for the adverse consequences of AT1R activation. In summary, EGFR plays a critical role in vascular AT1R action, enhancing signaling, promoting remodeling, contributing to inflammation, and participating in the pathogenesis of cardiovascular diseases. Understanding the interplay between AT1R and EGFR will foster the development of effective therapeutic strategies of RAAS-induced disorders., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Vascular smooth muscle cells (VSMC) are critical for the vascular tone, but they can also drive the development of vascular diseases when they lose their contractile phenotype and de-differentiate. Previous studies showed that the epidermal growth factor receptor (EGFR) of VSMC is critical for vascular health, but most of the underlying mechanisms by which VSMC-EGFR controls vascular fate have remained unknown. We combined RNA-sequencing and bioinformatics analysis to characterize the effect of EGFR-activation on the transcriptome of human primary VSMC (from different female donors) and to identify potentially affected cellular processes. Our results indicate that the activation of human VSMC-EGFR is sufficient to trigger a phenotypical switch toward a proliferative and inflammatory phenotype. The extent of this effect is nonetheless partly donor-dependent. Our hypothesis-generating study thus provides a first insight into mechanisms that could partly explain variable susceptibilities to vascular diseases in between individuals., Competing Interests: The authors declare no conflict of interest., (© 2023 The Author(s).)

Extracellular pH is an important parameter influencing cell function and fate. Microenvironmental acidosis accompanies different pathological situations, including inflammation, hypoxia and ischemia. Research focussed mainly on acidification of the tumour micromilieu and the possible consequences on proliferation, migration and drug resistance. Much less is known regarding the impact of microenvironmental acidosis on the transcriptome of non-tumour cells, which are exposed to local acidosis during inflammation, hypoxia, ischemia or metabolic derailment. In the present hypothesis-generating study, we investigated the transcriptional impact of extracellular acidosis on five non-tumour cell types of human and rat origin, combining RNA-Sequencing and extensive bioinformatics analyses. For this purpose, cell type-dependent acidosis resiliences and acidosis-induced transcriptional changes within these resilience ranges were determined, using 56 biological samples. The RNA-Sequencing results were used for dual differential-expression analysis (DESeq and edgeR) and, after appropriate homology mapping, Gene Ontology enrichment analysis (g:Profiler), Ingenuity Pathway Analysis (IPA®), as well as functional enrichment analysis for predicted upstream regulators, were performed. Extracellular acidosis led to substantial, yet different, quantitative transcriptional alterations in all five cell types. Our results identify the regulator of the transcriptional activity NCOA5 as the only general acidosis-responsive gene. Although we observed a species- and cell type-dominated response regarding gene expression regulation, Gene Ontology enrichment analysis and upstream regulator analysis predicted a general acidosis response pattern. Indeed, they suggested the regulation of four general acidosis-responsive cellular networks, which comprised the integrated stress response (ISR), TGF-β signalling, NFE2L2 and TP53. Future studies will have to extend the results of our bioinformatics analyses to cell biological and cell physiological validation experiments, in order to test the refined working hypothesis here., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Dubourg et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Crosstalk of renal epithelial cells with interstitial fibroblasts plays an important role in kidney pathophysiology. A previous study showed that crosstalk between renal epithelial cells and renal fibroblasts protects against acidosis-induced damage. In order to gain further mechanistic insight into this crosstalk, we investigated the effect of acidosis on the transcriptome of renal epithelial cells (NRK-52E) and renal fibroblasts (NRK-49F) in co-culture by RNASeq, bioinformatics analysis and experimental validation. Cells were exposed to acidic media or control media for 48 h. RNA and protein from whole cell lysate were isolated. In addition, cells were fractionated into cytosol, nucleus and chromatin. RNASeq data were analyzed for differential expression and pathway enrichment (ingenuity pathway analysis, IPA, QIAGEN). Total and phosphorylated protein expression was assessed by Western blot (WB). Transcription factor activity was assessed by luciferase reporter assay. Bioinformatic analysis using differentially expressed genes according to RNASeq (7834 for NRK-52E and 3197 for NRK-49F) predicted the antioxidant and cell-protective Nrf2 pathway as acidosis-induced in NRK-52E and NRK-49F cells. Activation of Nrf2 comprises enhanced Nrf2 phosphorylation, nuclear translocation, DNA binding and initiation of a cell protective transcriptional program. Our data show that acidosis enhances chromatin-associated Nrf2 expression and the abundance of phosphorylated Nrf2 in the chromatin fraction of NRK-52E cells in co-culture but not in monoculture. Furthermore, acidosis enhances the activity of a reporter for Nrf2 (ARE-luciferase). Despite the bioinformatics prediction, NRK-49F cells did not respond with Nrf2 activation. Transketolase (TKT) is an important regulator of antioxidant and homeostatic responses in the kidney and a canonical Nrf2 target gene. We show that protein and mRNA expression of TKT is increased in NRK-52E cells under co-culture but not under monoculture conditions. In conclusion, our data show that extracellular acidosis activates the cytoprotective transcription factor Nrf2 in renal epithelial cells co-cultivated with renal fibroblasts, thereby enhancing the expression of cytoprotective TKT. This protective response is not observed in monoculture. Activation of the Nrf2 pathway represents a co-operative cellular strategy of protection against acidosis.

In vivo, cells are simultaneously exposed to multiple stimuli whose effects are difficult to distinguish. Therefore, they are often investigated in experimental cell culture conditions where stimuli are applied separately. However, it cannot be presumed that their individual effects simply add up. As a proof-of-principle to address the relevance of transcriptional signaling synergy, we investigated the interplay of the Epidermal Growth Factor Receptor (EGFR) with the Angiotensin-II (AT1R) or the Thromboxane-A2 (TP) receptors in murine primary aortic vascular smooth muscle cells. Transcriptome analysis revealed that EGFR-AT1R or EGFR-TP simultaneous activations led to different patterns of regulated genes compared to individual receptor activations (qualitative synergy). Combined EGFR-TP activation also caused a variation of amplitude regulation for a defined set of genes (quantitative synergy), including vascular injury-relevant ones ( Klf15 and Spp1 ). Moreover, Gene Ontology enrichment suggested that EGFR and TP-induced gene expression changes altered processes critical for vascular integrity, such as cell cycle and senescence. These bioinformatics predictions regarding the functional relevance of signaling synergy were experimentally confirmed. Therefore, by showing that the activation of more than one receptor can trigger a synergistic regulation of gene expression, our results epitomize the necessity to perform comprehensive network investigations, as the study of individual receptors may not be sufficient to understand their physiological or pathological impact.

The F2-isoprostane 8-iso-PGF 2α (also known as 15-F 2t -isoprostane, iPF 2α -III, 8-epi PGF 2α , 15(S)-8-iso-PGF 2α , or 8-Isoprostane), a thromboxane A 2 receptor (TP) agonist, stable biomarker of oxidative stress, and risk marker of cardiovascular disease, has been proposed to aggravate atherogenesis in genetic mouse models of atherosclerotic vascular disease. Moreover, the TP plays an eminent role in the pathophysiology of endothelial dysfunction, atherogenesis, and cardiovascular disease. Yet it is unknown, how the TP expressed by vascular cells affects atherogenesis or 8-iso-PGF 2α -related effects in mouse models of atherosclerosis. We studied Ldlr-deficient vascular endothelial-specific (EC) and vascular smooth muscle cell (VSMC)-specific TP knockout mice (TP EC KO /Ldlr KO; TP VSMC KO /Ldlr KO) and corresponding wild-type littermates (TP WT /Ldlr KO). The mice were fed a Western-type diet for eight weeks and received either 8-iso-PGF 2α or vehicle infusions via osmotic pumps. Subsequently, arterial blood pressure, atherosclerotic lesion formation, and lipid profiles were analyzed. We found that VSMC-, but not EC-specific TP deletion, attenuated atherogenesis without affecting blood pressure or plasma lipid profiles of the mice. In contrast to a previous report, 8-iso-PGF 2α tended to reduce atherogenesis in TP WT /Ldlr KO and TP EC KO /Ldlr KO mice, again without significantly affecting blood pressure or lipid profiles of these mice. However, no further reduction in atherogenesis was observed in 8-iso-PGF 2α -treated TP VSMC KO /Ldlr KO mice. Our work suggests that the TP expressed in VSMC but not the TP expressed in EC is involved in atherosclerotic lesion formation in Ldlr-deficient mice. Furthermore, we report an inhibitory effect of 8-iso-PGF 2α on atherogenesis in this experimental atherosclerosis model, which paradoxically appears to be related to the presence of the TP in VSMC., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2021. The Author(s).)

Vascular EGF receptors (EGFR) influence function and structure of arterial vessels. In genetic mouse models we described the role of vascular smooth muscle (VSMC) EGFR for proper physiological function and structure as well as for pathophysiological alterations by obesity or angiotensin II. As the importance of endothelial (EC) EGFR in vivo is unknown, we analyzed the impact of EC-EGFR knockout in a conditional mouse model on vascular and renal function under control condition as well as in obesity and in comparison to VSMC-KO. Heart and lung weight, blood pressure and aortic transcriptome (determined by RNA-seq) were not affected by EC-EGFR-KO. Aortic reactivity to α1-adrenergic stimulation was not affected by EC-EGFR-KO contrary to VSMC-EGFR-KO. Endothelial-induced relaxation was reduced in abdominal aorta of EC-EGFR-KO animals, whereas it was enhanced in VSMC-EGFR-KO animals. Mesenteric arteries of EC-EGFR-KO animals showed enhanced sensitivity to α1-adrenergic stimulation, whereas endothelial-induced relaxation and vessel morphology were not affected. Renal weight, histomorphology, function (albumin excretion, serum creatinine, fractional water excretion) or transcriptome were not affected by EC-EGFR-KO, likewise in VSMC-EGFR-KO. High fat diet (HFD) over 18 weeks induced arterial wall thickening, renal weight increase, creatininemia, renal and aortic transcriptome alterations with a similar pattern in EC-EGFR-WT and EC-EGFR-KO animals by contrast to the previously reported impact of VSMC-EGFR-KO. HFD induced endothelial dysfunction in abdominal aortae of EC-EGFR-WT, which was not additive to the EC-EGFR-KO-induced endothelial dysfunction. As shown before, VSMC-EGFR-KO prevented HFD-induced endothelial dysfunction. HFD-induced albuminuria was less pronounced in EC-EGFR-KO animals and abrogated in VSMC-EGFR-KO animals. Our results indicate that EC-EGFR, in comparison to VSMC-EGFR, is of minor and opposite importance for basal renovascular function as well as for high fat diet-induced vascular remodeling and renal end organ damage.

Environmental food contaminants constitute a threat to human health. For instance, the globally spread mycotoxin Ochratoxin A (OTA) contributes to chronic kidney damage by affecting proximal tubule cells via unknown mechanisms. We applied a top-down approach to identify relevant toxicological mechanisms of OTA using RNA-sequencing followed by in-depth bioinformatics analysis and experimental validation. Differential expression analyses revealed that OTA led to the regulation of gene expression in kidney human cell lines, including for genes enriched in cell cycle-related pathways, and OTA-induced gap 1 and 2 (G1 and G2) cell-cycle arrests were observed. Weighted correlation network analysis highlighted cyclin dependent kinase 2 (CDK2) as a putative key regulator of this effect. CDK2 was downregulated by OTA exposure, and its overexpression partially blocked the OTA-induced G1 but not G2 cell-cycle arrest. We, therefore, propose CDK2 as one of the key regulators of the G1 cell-cycle arrest induced by low nanomolar concentrations of OTA., Competing Interests: The authors declare no conflicts of interest.

Background: Prenatal and early postnatal exposures to environmental factors are considered responsible for the increasing prevalence of allergic diseases. Although there is some evidence for allergy-promoting effects in children because of exposure to plasticizers, such as phthalates, findings of previous studies are inconsistent and lack mechanistic information., Objective: We investigated the effect of maternal phthalate exposure on asthma development in subsequent generations and their underlying mechanisms, including epigenetic alterations., Methods: Phthalate metabolites were measured within the prospective mother-child cohort Lifestyle and Environmental Factors and Their Influence on Newborns Allergy Risk (LINA) and correlated with asthma development in the children. A murine transgenerational asthma model was used to identify involved pathways., Results: In LINA maternal urinary concentrations of mono-n-butyl phthalate, a metabolite of butyl benzyl phthalate (BBP), were associated with an increased asthma risk in the children. Using a murine transgenerational asthma model, we demonstrate a direct effect of BBP on asthma severity in the offspring with a persistently increased airway inflammation up to the F2 generation. This disease-promoting effect was mediated by BBP-induced global DNA hypermethylation in CD4 + T cells of the offspring because treatment with a DNA-demethylating agent alleviated exacerbation of allergic airway inflammation. Thirteen transcriptionally downregulated genes linked to promoter or enhancer hypermethylation were identified. Among these, the GATA-3 repressor zinc finger protein 1 (Zfpm1) emerged as a potential mediator of the enhanced susceptibility for T H 2-driven allergic asthma., Conclusion: These data provide strong evidence that maternal BBP exposure increases the risk for allergic airway inflammation in the offspring by modulating the expression of genes involved in T H 2 differentiation through epigenetic alterations., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)