Response Of Endothelial Cells Research Articles

Quercetin ameliorates atherosclerosis by inhibiting inflammation of vascular endothelial cells via Piezo1 channels

BackgroundNatural antioxidants, exemplified by quercetin (Qu), have been shown to exert a protective effect against atherosclerosis (AS). However, the precise pharmacological mechanisms of Qu also remain elusive. PurposeHere, we aimed to uncover the anti-atherosclerotic mechanisms of Qu. Methods/Study designsThe inflammatory cytokine expression, activity of NLRP3 inflammasome and NF-κB, as well as mechanically activated currents and intracellular calcium levels were measured in endothelial cells (ECs). In addition, to explore whether Qu inhibited atherosclerotic plaque formation via Piezo1 channels, Ldlr−/- and Piezo1 endothelial-specific knockout mice (Piezo1△EC) were established. ResultsOur findings revealed that Qu significantly inhibited Yoda1-evoked calcium response in human umbilical vein endothelial cells (HUVECs), underscoring its role as a selective modulator of Piezo1 channels. Additionally, Qu effectively reduced mechanically activated currents in HUVECs. Moreover, Qu exhibited a substantial inhibitory effect on inflammatory cytokine expression and reduced the activity of NF-κB/NLRP3 in ECs exposed to ox-LDL or mechanical stretch, and these effects remained unaffected after Piezo1 genetic depletion. Furthermore, our study demonstrated that Qu substantially reduced the formation of atherosclerotic plaques, and this effect remained consistent even after Piezo1 genetic depletion. ConclusionThese results collectively provide compelling evidence that Qu ameliorates atherosclerosis by inhibiting the inflammatory response in ECs by targeting Piezo1 channels. In addition, Qu modulated atherosclerosis via inhibiting Piezo1 mediated NFκB/IL-1β and NLRP3/caspase1/ IL-1β axis to suppress the inflammation. Overall, this study reveals the potential mechanisms by which natural antioxidants, such as Qu, protect against atherosclerosis.

Impact of uniaxial cyclic stretching on matrix-associated endothelial cell responses

Uniaxial cyclic stretching plays a pivotal role in the fields of tissue engineering and regenerative medicine, influencing cell behaviors and functionality based on physical properties, including matrix morphology and mechanical stimuli. This study delves into the response of endothelial cells to uniaxial cyclic strain within the geometric constraints of micro-nano fibers. Various structural scaffold forms of poly(l-lactide-co-caprolactone) (PLCL), such as flat membranes, randomly oriented fiber membranes, and aligned fiber membranes, were fabricated through solvent casting and electrospinning methods. Our investigation focuses on the morphological variation of endothelial cells under diverse geometric constraints and the mechanical-dependent release of nitric oxide (NO) on oriented fibrous membranes. Our results indicate that while uniaxial cyclic stretching promotes endothelial cell spreading, the anisotropy of the matrix morphology remains the primary driving factor for cell alignment. Additionally, uniaxial cyclic stretching significantly enhances NO release, with a notably stronger effect correlated to the increasing strain amplitude. Importantly, this study reveals that uniaxial cyclic stretching enhances the mRNA expression of key proteins, including talin, vinculin, rac, and nitric oxide synthase (eNOS).

Influence of Substrate Structure and Associated Properties on Endothelial Cell Behavior in the Context of Behaviors Associated with Laminar Flow Conditions.

In order to treat most vascular diseases, arterial grafts are commonly employed for replacing small-diameter vessels, yet they often cause thrombosis. The growth of endothelial cells along the interior surfaces of these grafts (substrates) is critical to mitigate thrombosis. Typically, endothelial cells are cultured inside these grafts under laminar flow conditions to emulate the native environment of blood vessels and produce an endothelium. Alternatively, the substrate structure could have a similar influence on endothelial cell behavior as laminar flow conditions. In this study, we investigated whether substrates with aligned fiber structures could induce responses in human umbilical vein endothelial cells (HUVECs) akin to those elicited by laminar flow. Our observations revealed that HUVECs on aligned substrates displayed significant morphological changes, aligning parallel to the fibers, similar to effects reported under laminar flow conditions. Conversely, HUVECs on random substrates maintained their characteristic cobblestone appearance. Notably, cell migration was more significant on aligned substrates. Also, we observed that while vWF expression was similar between both substrates, the HUVECs on aligned substrates showed more expression of platelet/endothelial cell adhesion molecule-1 (PECAM-1/CD31), laminin, and collagen IV. Additionally, these cells exhibited increased gene expression related to critical functions such as proliferation, extracellular matrix production, cytoskeletal reorganization, autophagy, and antithrombotic activity. These findings indicated that aligned substrates enhanced endothelial growth and behavior compared to random substrates. These improvements are similar to the beneficial effects of laminar flow on endothelial cells, which are well-documented compared to static or turbulent flow conditions.

Exploring the underlying molecular mechanisms of acute myocardial infarction after SARS-CoV-2 infection

An increase in acute myocardial infarction (AMI)-related deaths has been reported during the COVID-19 pandemic. Despite evidence suggesting the association between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and AMI, the underlying mechanisms remain unclear. Here, we integrated mRNA and microRNA expression profiles related to SARS-CoV-2 infection and AMI from public databases. We then performed transcriptomic analysis using bioinformatics and systems biology approaches to explore the potential molecular mechanisms of SARS-CoV-2 infection affects AMI. First, twenty-one common differentially expressed genes (DEGs) were identified from SARS-CoV-2 infection and AMI patients in endothelial cells datasets and then we performed functional analysis to predict the roles of these DEGs. The functional analysis emphasized that the endothelial cell response to cytokine stimulus due to excessive inflammation was essential in these two diseases. Importantly, the tumor necrosis factor and interleukin-17 signaling pathways appeared to be integral factors in this mechanism. Interestingly, most of these common genes were also upregulated in transcriptomic datasets of SARS-CoV-2-infected cardiomyocytes, suggesting that these genes may be shared in cardiac- and vascular-related injuries. We subsequently built a protein-protein interaction network and extracted hub genes and essential modules from this network. At the transcriptional and post-transcriptional levels, regulatory networks with common DEGs were also constructed, and some key regulator signatures were further identified and validated. In summary, our research revealed that a highly activated inflammatory response in patients with COVID-19 might be a crucial factor for susceptibility to AMI and we identified some candidate genes and regulators that could be used as biomarkers or potential therapeutic targets.

A feedback loop driven by H3K9 lactylation and HDAC2 in endothelial cells regulates VEGF-induced angiogenesis

BackgroundVascular endothelial growth factor (VEGF) is one of the most powerful proangiogenic factors and plays an important role in multiple diseases. Increased glycolytic rates and lactate accumulation are associated with pathological angiogenesis.ResultsHere, we show that a feedback loop between H3K9 lactylation (H3K9la) and histone deacetylase 2 (HDAC2) in endothelial cells drives VEGF-induced angiogenesis. We find that the H3K9la levels are upregulated in endothelial cells in response to VEGF stimulation. Pharmacological inhibition of glycolysis decreases H3K9 lactylation and attenuates neovascularization. CUT& Tag analysis reveals that H3K9la is enriched at the promoters of a set of angiogenic genes and promotes their transcription. Interestingly, we find that hyperlactylation of H3K9 inhibits expression of the lactylation eraser HDAC2, whereas overexpression of HDAC2 decreases H3K9 lactylation and suppresses angiogenesis.ConclusionsCollectively, our study illustrates that H3K9la is important for VEGF-induced angiogenesis, and interruption of the H3K9la/HDAC2 feedback loop may represent a novel therapeutic method for treating pathological neovascularization.

Latrophilin-2 mediates fluid shear stress mechanotransduction at endothelial junctions

Endothelial cell responses to fluid shear stress from blood flow are crucial for vascular development, function, and disease. A complex of PECAM-1, VE-cadherin, VEGF receptors (VEGFRs), and Plexin D1 located at cell–cell junctions mediates many of these events. However, available evidence suggests that another mechanosensor upstream of PECAM-1 initiates signaling. Hypothesizing that GPCR and Gα proteins may serve this role, we performed siRNA screening of Gα subunits and found that Gαi2 and Gαq/11 are required for activation of the junctional complex. We then developed a new activation assay, which showed that these G proteins are activated by flow. We next mapped the Gα residues required for activation and developed an affinity purification method that used this information to identify latrophilin-2 (Lphn2/ADGRL2) as the upstream GPCR. Latrophilin-2 is required for all PECAM-1 downstream events tested. In both mice and zebrafish, latrophilin-2 is required for flow-dependent angiogenesis and artery remodeling. Furthermore, endothelial-specific knockout demonstrates that latrophilin plays a role in flow-dependent artery remodeling. Human genetic data reveal a correlation between the latrophilin-2-encoding Adgrl2 gene and cardiovascular disease. Together, these results define a pathway that connects latrophilin-dependent G protein activation to subsequent endothelial signaling, vascular physiology, and disease.

Under pressure: integrated endothelial cell response to hydrostatic and shear stresses.

Blood flow within the vasculature is a critical determinant of endothelial cell (EC) identity and functionality, yet the intricate interplay of various hemodynamic forces and their collective impact on endothelial and vascular responses are not fully understood. Specifically, the role of hydrostatic pressure in the EC flow response is understudied, despite its known significance in vascular development and disease. To address this gap, we developed in vitro models to investigate how pressure influences EC responses to flow. Our study demonstrates that elevated pressure conditions significantly modify shear-induced flow alignment and increase endothelial cell density. Bulk and single-cell RNA sequencing analyses revealed that, while shear stress remains the primary driver of flow-induced transcriptional changes, pressure modulates shear-induced signaling in a dose-dependent manner. These pressure-responsive transcriptional signatures identified in human ECs were conserved during the onset of circulation in early mouse embryonic vascular development, where pressure was notably associated with transcriptional programs essential to arterial and hemogenic EC fates. Our findings suggest that pressure plays a synergistic role with shear stress on ECs and emphasizes the need for an integrative approach to endothelial cell mechanotransduction, one that encompasses the effects induced by pressure alongside other hemodynamic forces.

Aging is associated with an insufficient early inflammatory response of lung endothelial cells in SARS-CoV-2 infection.

Advanced age is associated with an increased susceptibility to Coronavirus Disease (COVID)-19 and more severe outcomes, although the underlying mechanisms are understudied. The lung endothelium is located next to infected epithelial cells and bystander inflammation may contribute to thromboinflammation and COVID-19-associated coagulopathy. Here, we investigated age-associated SARS-CoV-2 pathogenesis and endothelial inflammatory responses using humanized K18-hACE2 mice. Survival was reduced to 20% in aged mice (85-112 weeks) versus 50% in young mice (12-15 weeks) at 10 days post infection (dpi). Bulk RNA-sequencing of endothelial cells from mock and infected mice at 2dpi of both age groups (aged: 72-85 weeks; young: 15 weeks) showed substantially lower significant differentially regulated genes in infected aged mice than in young mice (712 versus 2294 genes). Viral recognition and anti-viral pathways such as RIG-I-like receptor signaling, NOD-like receptor signaling and interferon signaling were regulated in response to SARS-CoV-2. Young mice showed several fold higher interferon responses (Ifitm3, Ifit1, Isg15, Stat1) and interferon-induced chemokines (Cxcl10 and Cxcl11) than aged mice. Endothelial cells from infected young mice displayed elevated expression of chemokines (Cxcl9, Ccl2) and leukocyte adhesion markers (Icam1) underscoring that inflammation of lung endothelium during infection could facilitate leukocyte adhesion and thromboinflammation. TREM1 and acute phase response signaling were particularly prominent in endothelial cells from infected young mice. Immunohistochemistry was unable to detect viral protein in pulmonary endothelium. In conclusion, our data demonstrate that the early host response of the endothelium to SARS-CoV-2 infection declines with aging, which could be a potential contributor to disease severity.

Laminin and hyaluronan supplementation of collagen hydrogels enhances endothelial function and tight junction expression on three-dimensional cylindrical microvessel-on-a-chip

Vasculature-on-chip (VoC) models have become a prominent tool in the study of microvasculature functions because of their cost-effective and ethical production process. These models typically use a hydrogel in which the three-dimensional (3D) microvascular structure is embedded. Thus, VoCs are directly impacted by the physical and chemical cues of the supporting hydrogel. Endothelial cell (EC) response in VoCs is critical, especially in organ-specific vasculature models, in which ECs exhibit specific traits and behaviors that vary between organs. Many studies customize the stimuli ECs perceive in different ways; however, customizing the hydrogel composition accordingly to the target organ's extracellular matrix (ECM), which we believe has great potential, has been rarely investigated. We explored this approach to organ-specific VoCs by fabricating microvessels (MVs) with either human umbilical vein ECs or human brain microvascular ECs in a 3D cylindrical VoC using a collagen hydrogel alone or one supplemented with laminin and hyaluronan, components found in the brain ECM. We characterized the physical properties of these hydrogels and analyzed the barrier properties of the MVs. Barrier function and tight junction (ZO-1) expression improved with the addition of laminin and hyaluronan in the composite hydrogel.

Activating A1 adenosine receptor signaling boosts early pulmonary neutrophil recruitment in aged mice in response to Streptococcus pneumoniae infection

BackgroundStreptococcus pneumoniae (pneumococcus) is a leading cause of pneumonia in older adults. Successful control of pneumococci requires robust pulmonary neutrophil influx early in infection. However, aging is associated with aberrant neutrophil recruitment and the mechanisms behind that are not understood. Here we explored how neutrophil recruitment following pneumococcal infection changes with age and the host pathways regulating this.ResultsFollowing pneumococcal infection there was a significant delay in early neutrophil recruitment to the lungs of aged mice. Neutrophils from aged mice showed defects in trans-endothelial migration in vitro compared to young controls. To understand the pathways involved, we examined immune modulatory extracellular adenosine (EAD) signaling, that is activated upon cellular damage. Signaling through the lower affinity A2A and A2B adenosine receptors had no effect on neutrophil recruitment to infected lungs. In contrast, inhibition of the high affinity A1 receptor in young mice blunted neutrophil recruitment to the lungs following infection. A1 receptor inhibition decreased expression of CXCR2 on circulating neutrophils, which is required for trans-endothelial migration. Indeed, A1 receptor signaling on neutrophils was required for their ability to migrate across endothelial cells in response to infection. Aging was not associated with defects in EAD production or receptor expression on neutrophils. However, agonism of A1 receptor in aged mice rescued the early defect in neutrophil migration to the lungs and improved control of bacterial burden.ConclusionsThis study suggests age-driven defects in EAD damage signaling can be targeted to rescue the delay in pulmonary neutrophil migration in response to bacterial pneumonia.

Inositol-requiring enzyme 1 pathway and autophagy drive sequential response of endothelial cells to febrile range hyperthermia

Inositol-requiring enzyme 1 pathway and autophagy drive sequential response of endothelial cells to febrile range hyperthermia

Aortic endothelial cells response to pulsatile vs. continuous flow at high shear stress

Aortic endothelial cells response to pulsatile vs. continuous flow at high shear stress

Responses of Endothelial Progenitor Cells to Chronic and Acute Physical Activity in Healthy Individuals.

Endothelial progenitor cells (EPCs) are circulating cells of various origins that possess the capacity for renewing and regenerating the endothelial lining of blood vessels. During physical activity, in response to factors such as hypoxia, changes in osmotic pressure, and mechanical forces, endothelial cells undergo intense physiological stress that results in endothelial damage. Circulating EPCs participate in blood vessel repair and vascular healing mainly through paracrine signalling. Furthermore, physical activity may play an important role in mobilising this important cell population. In this narrative review, we summarise the current knowledge on the biology of EPCs, including their characteristics, assessment, and mobilisation in response to both chronic and acute physical activity in healthy individuals.

Early Injury Landscape in Vein Harvest by Single-Cell and Spatial Transcriptomics.

Vein graft failure following cardiovascular bypass surgery results in significant patient morbidity and cost to the healthcare system. Vein graft injury can occur during autogenous vein harvest and preparation, as well as after implantation into the arterial system, leading to the development of intimal hyperplasia, vein graft stenosis, and, ultimately, bypass graft failure. Although previous studies have identified maladaptive pathways that occur shortly after implantation, the specific signaling pathways that occur during vein graft preparation are not well defined and may result in a cumulative impact on vein graft failure. We, therefore, aimed to elucidate the response of the vein conduit wall during harvest and following implantation, probing the key maladaptive pathways driving graft failure with the overarching goal of identifying therapeutic targets for biologic intervention to minimize these natural responses to surgical vein graft injury. Employing a novel approach to investigating vascular pathologies, we harnessed both single-nuclei RNA-sequencing and spatial transcriptomics analyses to profile the genomic effects of vein grafts after harvest and distension, then compared these findings to vein grafts obtained 24 hours after carotid-carotid vein bypass implantation in a canine model (n=4). Spatial transcriptomic analysis of canine cephalic vein after initial conduit harvest and distention revealed significant enrichment of pathways (P<0.05) involved in the activation of endothelial cells (ECs), fibroblasts, and vascular smooth muscle cells, namely pathways responsible for cellular proliferation and migration and platelet activation across the intimal and medial layers, cytokine signaling within the adventitial layer, and ECM (extracellular matrix) remodeling throughout the vein wall. Subsequent single-nuclei RNA-sequencing analysis supported these findings and further unveiled distinct EC and fibroblast subpopulations with significant upregulation (P<0.05) of markers related to endothelial injury response and cellular activation of ECs, fibroblasts, and vascular smooth muscle cells. Similarly, in vein grafts obtained 24 hours after arterial bypass, there was an increase in myeloid cell, protomyofibroblast, injury response EC, and mesenchymal-transitioning EC subpopulations with a concomitant decrease in homeostatic ECs and fibroblasts. Among these markers were genes previously implicated in vein graft injury, including VCAN, FBN1, and VEGFC, in addition to novel genes of interest, such as GLIS3 and EPHA3. These genes were further noted to be driving the expression of genes implicated in vascular remodeling and graft failure, such as IL-6, TGFBR1, SMAD4, and ADAMTS9. By integrating the spatial transcriptomics and single-nuclei RNA-sequencing data sets, we highlighted the spatial architecture of the vein graft following distension, wherein activated and mesenchymal-transitioning ECs, myeloid cells, and fibroblasts were notably enriched in the intima and media of distended veins. Finally, intercellular communication network analysis unveiled the critical roles of activated ECs, mesenchymal-transitioning ECs, protomyofibroblasts, and vascular smooth muscle cells in upregulating signaling pathways associated with cellular proliferation (MDK [midkine], PDGF [platelet-derived growth factor], VEGF [vascular endothelial growth factor]), transdifferentiation (Notch), migration (ephrin, semaphorin), ECM remodeling (collagen, laminin, fibronectin), and inflammation (thrombospondin), following distension. Vein conduit harvest and distension elicit a prompt genomic response facilitated by distinct cellular subpopulations heterogeneously distributed throughout the vein wall. This response was found to be further exacerbated following vein graft implantation, resulting in a cascade of maladaptive gene regulatory networks. Together, these results suggest that distension initiates the upregulation of pathological pathways that may ultimately contribute to bypass graft failure and presents potential early targets warranting investigation for targeted therapies. This work highlights the first applications of single-nuclei and spatial transcriptomic analyses to investigate venous pathologies, underscoring the utility of these methodologies and providing a foundation for future investigations.

Correction to: ORAI1 Activates Proliferation of Lymphatic Endothelial Cells in Response to Laminar Flow Through Krüppel-Like Factors 2 and 4.

Correction to: ORAI1 Activates Proliferation of Lymphatic Endothelial Cells in Response to Laminar Flow Through Krüppel-Like Factors 2 and 4.

An essential role for EROS in redox-dependent endothelial signal transduction

The chaperone protein EROS (“Essential for Reactive Oxygen Species”) was recently discovered in phagocytes. EROS was shown to regulate the abundance of the ROS-producing enzyme NADPH oxidase isoform 2 (NOX2) and to control ROS-mediated cell killing. Reactive oxygen species are important not only in immune surveillance, but also modulate physiological signaling responses in multiple tissues. The roles of EROS have not been previously explored in the context of oxidant-modulated cell signaling. Here we show that EROS plays a key role in ROS-dependent signal transduction in vascular endothelial cells. We used siRNA-mediated knockdown and developed CRISPR/Cas9 knockout of EROS in human umbilical vein endothelial cells (HUVEC), both of which cause a significant decrease in the abundance of NOX2 protein, associated with a marked decrease in RAC1, a small G protein that activates NOX2. Loss of EROS also attenuates receptor-mediated hydrogen peroxide (H2O2) and Ca2+ signaling, disrupts cytoskeleton organization, decreases cell migration, and promotes cellular senescence. EROS knockdown blocks agonist-modulated eNOS phosphorylation and nitric oxide (NO●) generation. These effects of EROS knockdown are strikingly similar to the alterations in endothelial cell responses that we previously observed following RAC1 knockdown. Proteomic analyses following EROS or RAC1 knockdown in endothelial cells showed that reduced abundance of these two distinct proteins led to largely overlapping effects on endothelial biological processes, including oxidoreductase, protein phosphorylation, and endothelial nitric oxide synthase (eNOS) pathways. These studies demonstrate that EROS plays a central role in oxidant-modulated endothelial cell signaling by modulating NOX2 and RAC1.

ZO-1 interacts with YB-1 in endothelial cells to regulate stress granule formation during angiogenesis

Zonula occludens-1 (ZO-1) is involved in the regulation of cell-cell junctions between endothelial cells (ECs). Here we identify the ZO-1 protein interactome and uncover ZO-1 interactions with RNA-binding proteins that are part of stress granules (SGs). Downregulation of ZO-1 increased SG formation in response to stress and protected ECs from cellular insults. The ZO-1 interactome uncovered an association between ZO-1 and Y-box binding protein 1 (YB-1), a constituent of SGs. Arsenite treatment of ECs decreased the interaction between ZO-1 and YB-1, and drove SG assembly. YB-1 expression is essential for SG formation and for the cytoprotective effects induced by ZO-1 downregulation. In the developing retinal vascular plexus of newborn mice, ECs at the front of growing vessels express less ZO-1 but display more YB-1-positive granules than ECs located in the vascular plexus. Endothelial-specific deletion of ZO-1 in mice at post-natal day 7 markedly increased the presence of YB-1-positive granules in ECs of retinal blood vessels, altered tip EC morphology and vascular patterning, resulting in aberrant endothelial proliferation, and arrest in the expansion of the retinal vasculature. Our findings suggest that, through its interaction with YB-1, ZO-1 controls SG formation and the response of ECs to stress during angiogenesis.

Shear stress and pathophysiological PI3K involvement in vascular malformations.

Molecular characterization of vascular anomalies has revealed that affected endothelial cells (ECs) harbor gain-of-function (GOF) mutations in the gene encoding the catalytic α subunit of PI3Kα (PIK3CA). These PIK3CA mutations are known to cause solid cancers when occurring in other tissues. PIK3CA-related vascular anomalies, or "PIKopathies," range from simple, i.e., restricted to a particular form of malformation, to complex, i.e., presenting with a range of hyperplasia phenotypes, including the PIK3CA-related overgrowth spectrum. Interestingly, development of PIKopathies is affected by fluid shear stress (FSS), a physiological stimulus caused by blood or lymph flow. These findings implicate PI3K in mediating physiological EC responses to FSS conditions characteristic of lymphatic and capillary vessel beds. Consistent with this hypothesis, increased PI3K signaling also contributes to cerebral cavernous malformations, a vascular disorder that affects low-perfused brain venous capillaries. Because the GOF activity of PI3K and its signaling partners are excellent drug targets, understanding PIK3CA's role in the development of vascular anomalies may inform therapeutic strategies to normalize EC responses in the diseased state. This Review focuses on PIK3CA's role in mediating EC responses to FSS and discusses current understanding of PIK3CA dysregulation in a range of vascular anomalies that particularly affect low-perfused regions of the vasculature. We also discuss recent surprising findings linking increased PI3K signaling to fast-flow arteriovenous malformations in hereditary hemorrhagic telangiectasias.

Activated factor X stimulates atrial endothelial cells and tissues to promote remodelling responses through AT1R/NADPH oxidases/SGLT1/2.

Atrial fibrillation (AF), the most common cardiac arrhythmia favouring ischemic stroke and heart failure involves left atrial remodelling, fibrosis and a complex interplay between cardiovascular risk factors. This study examined whether activated factor X (FXa) induces pro-remodelling and pro-fibrotic responses in atrial endothelial cells (AECs) and human atrial tissues and determined the underlying mechanisms. AECs collected from porcine hearts and human right atrial appendages (RAA) from patients undergoing heart surgery. Protein expression levels were assessed by Western blot and immunofluorescence staining, mRNA levels by RT-qPCR, formation of reactive oxygen species (ROS) and NO using fluorescent probes, thrombin and angiotensin II generation by specific assays, fibrosis by Sirius red staining and senescence by senescence-associated beta-galactosidase (SA-β-gal) activity. In AECs, FXa increased ROS formation, senescence (SA-β-gal activity, p53, p21), angiotensin II generation and the expression of pro-inflammatory (VCAM-1, MCP-1), pro-thrombotic (tissue factor), pro-fibrotic (TGF-β and collagen-1/3a) and pro-remodelling (MMP-2/9) markers whereas eNOS levels and NO formation were reduced. These effects were prevented by inhibitors of FXa but not thrombin, protease-activated receptors antagonists (PAR-1/2) and inhibitors of NADPH oxidases, ACE, AT1R, SGLT1/SGLT2. FXa also increased expression levels of ACE1, AT1R, SGLT1/2 proteins which were prevented by SGLT1/2 inhibitors. Human RAA showed tissue factor mRNA levels that correlated with markers of endothelial activation, pro-remodelling and pro-fibrotic responses and SGLT1/2 mRNA levels. They also showed protein expression levels of ACE1, AT1R, p22phox, SGLT1/2, and immunofluorescence signals of nitrotyrosine and SGLT1/2 colocalized with those of CD31. FXa increased oxidative stress levels which were prevented by inhibitors of the AT1R/NADPH oxidases/SGLT1/2 pathway. FXa promotes oxidative stress triggering premature endothelial senescence and dysfunction associated with pro-thrombotic, pro-remodelling and pro-fibrotic responses in AECs and human RAA involving the AT1R/NADPH oxidases/SGLT1/2 pro-oxidant pathway. Targeting this pathway may be of interest to prevent atrial remodelling and the progression of atrial fibrillation substrate.

Targeting Neutrophil Extracellular Trap Formation: Exploring Promising Pharmacological Strategies for the Treatment of Preeclampsia.

Neutrophils, which constitute the most abundant leukocytes in human blood, emerge as crucial players in the induction of endothelial cell death and the modulation of endothelial cell responses under both physiological and pathological conditions. The hallmark of preeclampsia is endothelial dysfunction induced by systemic inflammation, in which neutrophils, particularly through the formation of neutrophil extracellular traps (NETs), play a pivotal role in the development and perpetuation of endothelial dysfunction and the hypertensive state. Considering the potential of numerous pharmaceutical agents to attenuate NET formation (NETosis) in preeclampsia, a comprehensive assessment of the extensively studied candidates becomes imperative. This review aims to identify mechanisms associated with the induction and negative regulation of NETs in the context of preeclampsia. We discuss potential drugs to modulate NETosis, such as NF-κβ inhibitors, vitamin D, and aspirin, and their association with mutagenicity and genotoxicity. Strong evidence supports the notion that molecules involved in the activation of NETs could serve as promising targets for the treatment of preeclampsia.