ARAP3 protects from excessive formylated peptide-induced microvascular leakage by acting on endothelial cells and neutrophils.

Vascular permeability is temporarily heightened during inflammation, but excessive inflammation-associated microvascular leakage can be detrimental, as evidenced in the inflamed lung. Formylated peptides regulate vascular leakage indirectly via formylated peptide receptor-1 (FPR1)-mediated recruitment and activation of neutrophils. Here we identify how the GTPase-activating protein ARAP3 protects against formylated peptide-induced microvascular permeability via endothelial cells and neutrophils. In vitro, Arap3-/- endothelial monolayers were characterised by enhanced formylated peptide-induced permeability due to upregulated endothelial FPR1 and enhanced vascular endothelial cadherin internalisation. In vivo, enhanced inflammation-associated microvascular leakage was observed in Arap3-/- mice. Leakage of plasma protein into the lungs of Arap3-/- mice increased within hours of formylated peptide administration. Adoptive transfer experiments indicated this was dependent upon ARAP3 deficiency in both immune and non-immune cells. Bronchoalveolar lavages of formylated peptide-challenged Arap3-/- mice contained neutrophil extracellular traps (NETs). Pharmacological inhibition of NET formation abrogated excessive microvascular leakage, indicating a critical function of NETs in this context. The observation that Arap3-/- mice developed more severe influenza suggests these findings are pertinent to pathological situations characterised by abundant formylated peptides. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Control of endothelial cell function and arteriogenesis by MEG3:EZH2 epigenetic regulation of integrin expression.

Epigenetic processes involving long non-coding RNAs regulate endothelial gene expression. However, the underlying regulatory mechanisms causing endothelial dysfunction remain to be elucidated. Enhancer of zeste homolog 2 (EZH2) is an important rheostat of histone H3K27 trimethylation (H3K27me3) that represses endothelial targets, but EZH2 RNA binding capacity and EZH2:RNA functional interactions have not been explored in post-ischemic angiogenesis. We used formaldehyde/UV-assisted crosslinking ligation and sequencing of hybrids and identified a new role for maternally expressed gene 3 (MEG3). MEG3 formed the predominant RNA:RNA hybrid structures in endothelial cells. Moreover, MEG3:EZH2 assists recruitment onto chromatin. By EZH2-chromatin immunoprecipitation, following MEG3 depletion, we demonstrated that MEG3 controls recruitment of EZH2/H3K27me3 onto integrin subunit alpha4 (ITGA4) promoter. Both MEG3 knockdown or EZH2 inhibition (A-395) promoted ITGA4 expression and improved endothelial cell migration and adhesion to fibronectin in vitro. The A-395 inhibitor re-directed MEG3-assisted chromatin remodeling, offering a direct therapeutic benefit by increasing endothelial function and resilience. This approach subsequently increased the expression of ITGA4 in arterioles following ischemic injury in mice, thus promoting arteriogenesis. Our findings show a context-specific role for MEG3 in guiding EZH2 to repress ITGA4. Novel therapeutic strategies could antagonize MEG3:EZH2 interaction for pre-clinical studies.

Non-coding RNAs as therapeutic targets and biomarkers in ischaemic heart disease.

The adult heart is a complex, multicellular organ that is subjected to a series of regulatory stimuli and circuits and has poor reparative potential. Despite progress in our understanding of disease mechanisms and in the quality of health care, ischaemic heart disease remains the leading cause of death globally, owing to adverse cardiac remodelling, leading to ischaemic cardiomyopathy and heart failure. Therapeutic targets are urgently required for the protection and repair of the ischaemic heart. Moreover, personalized clinical biomarkers are necessary for clinical diagnosis, medical management and to inform the individual response to treatment. Non-coding RNAs (ncRNAs) deeply influence cardiovascular functions and contribute to communication between cells in the cardiac microenvironment and between the heart and other organs. As such, ncRNAs are candidates for translation into clinical practice. However, ncRNA biology has not yet been completely deciphered, given that classes and modes of action have emerged only in the past 5 years. In this Review, we discuss the latest discoveries from basic research on ncRNAs and highlight both the clinical value and the challenges underscoring the translation of these molecules as biomarkers and therapeutic regulators of the processes contributing to the initiation, progression and potentially the prevention or resolution of ischaemic heart disease and heart failure.

COVID-19 plasma induces subcellular remodelling within the pulmonary microvascular endothelium.

BACKGROUND: COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can affect multiple organ systems, including the pulmonary vasculature. Endothelial cells (ECs) are thought to play a key role in the propagation of COVID-19, however, our understanding of the exact scale of dysregulation sustained by the pulmonary microvasculature (pMV) remains incomplete. Here we aim to identify transcriptional, phenotypic, and functional changes within the pMV induced by COVID-19. METHODS AND RESULTS: Human pulmonary microvascular endothelial cells (HPMVEC) treated with plasma acquired from patients hospitalised with severe COVID-19 were compared to HPMVEC treated with plasma from patients hospitalised without COVID-19 but with other severe illnesses. Exposure to COVID-19 plasma caused a significant functional decline in HPMVECs as seen by a decrease in both cell viability via the WST-1 cell-proliferation assay and cell-to-cell barrier function as measured by electric cell-substrate impedance sensing. High-content imaging using a Cell Painting image-based assay further quantified morphological variations within sub-cellular organelles to show phenotypic changes in the whole endothelial cell, nucleus, mitochondria, plasma membrane and nucleolus morphology. RNA-sequencing of HPMVECs treated with COVID-19 plasma suggests the observed phenotype may, in part, be regulated by genes such as SMAD7, BCOR, SFMBT1, IFIT5 and ZNF566 which are involved in transcriptional regulation, protein monoubiquitination and TGF-ß signalling. CONCLUSION AND IMPACT: During COVID-19, the pMV undergoes significant remodelling, which is evident based on the functional, phenotypic, and transcriptional changes seen following exposure to COVID-19 plasma. The observed morphological variation may be responsible for downstream complications, such as a decline in overall cellular function and cell-to-cell barrier integrity. Moreover, genes identified through bulk RNA sequencing may contribute to our understanding of the observed phenotype and assist in developing strategies that can inform the rescue of the dysregulated endothelium.

Extracellular vesicles from differentiated stem cells contain novel proangiogenic miRNAs and induce angiogenic responses at low doses.

Extracellular vesicles (EVs) released from healthy endothelial cells (ECs) have shown potential for promoting angiogenesis, but their therapeutic efficacy remains poorly understood. We have previously shown that transplantation of a human embryonic stem cell-derived endothelial cell product (hESC-ECP), promotes new vessel formation in acute ischemic disease in mice, likely via paracrine mechanism(s). Here, we demonstrated that EVs from hESC-ECPs (hESC-eEVs) significantly increased EC tube formation and wound closure in vitro at ultralow doses, whereas higher doses were ineffective. More important, EVs isolated from the mesodermal stage of the differentiation (hESC-mEVs) had no effect. Small RNA sequencing revealed that hESC-eEVs have a unique transcriptomic profile and are enriched in known proangiogenic microRNAs (miRNAs, miRs). Moreover, an in silico analysis identified three novel hESC-eEV-miRNAs with potential proangiogenic function. Differential expression analysis suggested that two of those, miR-4496 and miR-4691-5p, are highly enriched in hESC-eEVs. Overexpression of miR-4496 or miR-4691-5p resulted in increased EC tube formation and wound closure in vitro, validating the novel proangiogenic function of these miRNAs. In summary, we demonstrated that hESC-eEVs are potent inducers of EC angiogenic response at ultralow doses and contain a unique EV-associated miRNA repertoire, including miR-4496 and miR-4691-5p, with novel proangiogenic function.

The Non-Coding RNA Journal Club: Highlights on Recent Papers-13.

We are delighted to share with you our thirteenth Journal Club and highlight some of the most interesting papers published recently [...].

Targeting the Interplay of Independent Cellular Pathways and Immunity: A Challenge in Cancer Immunotherapy.

Immunotherapy is a cancer treatment that exploits the capacity of the body's immune system to prevent, control, and remove cancer. Immunotherapy has revolutionized cancer treatment and significantly improved patient outcomes for several tumor types. However, most patients have not benefited from such therapies yet. Within the field of cancer immunotherapy, an expansion of the combination strategy that targets independent cellular pathways that can work synergistically is predicted. Here, we review some consequences of tumor cell death and increased immune system engagement in the modulation of oxidative stress and ubiquitin ligase pathways. We also indicate combinations of cancer immunotherapies and immunomodulatory targets. Additionally, we discuss imaging techniques, which are crucial for monitoring tumor responses during treatment and the immunotherapy side effects. Finally, the major outstanding questions are also presented, and directions for future research are described.

Insights into Online microRNA Bioinformatics Tools.

MicroRNAs (miRNAs) are members of the small non-coding RNA family regulating gene expression at the post-transcriptional level. MiRNAs have been found to have critical roles in various biological and pathological processes. Research in this field has significantly progressed, with increased recognition of the importance of miRNA regulation. As a result of the vast data and information available regarding miRNAs, numerous online tools have emerged to address various biological questions related to their function and influence across essential cellular processes. This review includes a brief introduction to available resources for an investigation covering aspects such as miRNA sequences, target prediction/validation, miRNAs associated with disease, pathway analysis and genetic variants within miRNAs.

Metformin protects against vascular calcification through the selective degradation of Runx2 by the p62 autophagy receptor.

Vascular calcification is associated with aging, type 2 diabetes, and atherosclerosis, and increases the risk of cardiovascular morbidity and mortality. It is an active, highly regulated process that resembles physiological bone formation. It has previously been established that pharmacological doses of metformin alleviate arterial calcification through adenosine monophosphate-activated protein kinase (AMPK)-activated autophagy, however the specific pathway remains elusive. In the present study we hypothesized that metformin protects against arterial calcification through the direct autophagic degradation of runt-related transcription factor 2 (Runx2). Calcification was blunted in vascular smooth muscle cells (VSMCs) by metformin in a dose-dependent manner (0.5-1.5 mM) compared to control cells (p < 0.01). VSMCs cultured under high-phosphate (Pi) conditions in the presence of metformin (1 mM) showed a significant increase in LC3 puncta following bafilomycin-A1 (Baf-A; 5 nM) treatment compared to control cells (p < 0.001). Furthermore, reduced expression of Runx2 was observed in the nuclei of metformin-treated calcifying VSMCs (p < 0.0001). Evaluation of the functional role of autophagy through Atg3 knockdown in VSMCs showed aggravated Pi-induced calcification (p < 0.0001), failure to induce autophagy (punctate LC3) (p < 0.001) and increased nuclear Runx2 expression (p < 0.0001) in VSMCs cultured under high Pi conditions in the presence of metformin (1 mM). Mechanistic studies employing three-way coimmunoprecipitation with Runx2, p62, and LC3 revealed that p62 binds to both LC3 and Runx2 upon metformin treatment in VSMCs. Furthermore, immunoblotting with LC3 revealed that Runx2 specifically binds with p62 and LC3-II in metformin-treated calcified VSMCs. Lastly, we investigated the importance of the autophagy pathway in vascular calcification in a clinical setting. Ex vivo clinical analyses of calcified diabetic lower limb artery tissues highlighted a negative association between Runx2 and LC3 in the vascular calcification process. These studies suggest that exploitation of metformin and its analogues may represent a novel therapeutic strategy for clinical intervention through the induction of AMPK/Autophagy Related 3 (Atg3)-dependent autophagy and the subsequent p62-mediated autophagic degradation of Runx2.

Telomere Dysfunction Is Associated with Altered DNA Organization in Trichoplein/Tchp/Mitostatin (TpMs) Depleted Cells.

Recently, we highlighted a novel role for the protein Trichoplein/TCHP/Mitostatin (TpMs), both as mitotic checkpoint regulator and guardian of chromosomal stability. TpMs-depleted cells show numerical and structural chromosome alterations that lead to genomic instability. This condition is a major driving force in malignant transformation as it allows for the cells acquiring new functional capabilities to proliferate and disseminate. Here, the effect of TpMs depletion was investigated in different TpMs-depleted cell lines by means of 3D imaging and 3D Structured illumination Microscopy. We show that TpMs depletion causes alterations in the 3D architecture of telomeres in colon cancer HCT116 cells. These findings are consistent with chromosome alterations that lead to genomic instability. Furthermore, TpMs depletion changes the spatial arrangement of chromosomes and other nuclear components. Modified nuclear architecture and organization potentially induce variations that precede the onset of genomic instability and are considered as markers of malignant transformation. Our present observations connect the tumor suppression ability of TpMs with its novel functions in maintaining the proper chromosomal segregation as well as the proper telomere and nuclear architecture. Further investigations will investigate the connection between alterations in telomeres and nuclear architecture with the progression of human tumors with the aim of developing personalized therapeutic interventions.

Cardiac pericyte reprogramming by MEK inhibition promotes arteriologenesis and angiogenesis of the ischemic heart.

Pericytes (PCs) are abundant yet remain the most enigmatic and ill-defined cell population in the heart. Here, we investigated whether PCs can be reprogrammed to aid neovascularization. Primary PCs from human and mouse hearts acquired cytoskeletal proteins typical of vascular smooth muscle cells (VSMCs) upon exclusion of EGF/bFGF, which signal through ERK1/2, or upon exposure to the MEK inhibitor PD0325901. Differentiated PCs became more proangiogenic, more responsive to vasoactive agents, and insensitive to chemoattractants. RNA sequencing revealed transcripts marking the PD0325901-induced transition into proangiogenic, stationary VSMC-like cells, including the unique expression of 2 angiogenesis-related markers, aquaporin 1 (AQP1) and cellular retinoic acid-binding protein 2 (CRABP2), which were further verified at the protein level. This enabled us to trace PCs during in vivo studies. In mice, implantation of Matrigel plugs containing human PCs plus PD0325901 promoted the formation of αSMA+ neovessels compared with PC only. Two-week oral administration of PD0325901 to mice increased the heart arteriolar density, total vascular area, arteriole coverage by PDGFRß+AQP1+CRABP2+ PCs, and myocardial perfusion. Short-duration PD0325901 treatment of mice after myocardial infarction enhanced the peri-infarct vascularization, reduced the scar, and improved systolic function. In conclusion, myocardial PCs have intrinsic plasticity that can be pharmacologically modulated to promote reparative vascularization of the ischemic heart.

Nox2 underpins microvascular inflammation and vascular contributions to cognitive decline.

Chronic microvascular inflammation and oxidative stress are inter-related mechanisms underpinning white matter disease and vascular cognitive impairment (VCI). A proposed mediator is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (Nox2), a major source of reactive oxygen species (ROS) in the brain. To assess the role of Nox2 in VCI, we studied a tractable model with white matter pathology and cognitive impairment induced by bilateral carotid artery stenosis (BCAS). Mice with genetic deletion of Nox2 (Nox2 KO) were compared to wild-type (WT) following BCAS. Sustained BCAS over 12 weeks in WT mice induced Nox2 expression, indices of microvascular inflammation and oxidative damage, along with white matter pathology culminating in a marked cognitive impairment, which were all protected by Nox2 genetic deletion. Neurovascular coupling was impaired in WT mice post-BCAS and restored in Nox2 KO mice. Increased vascular expression of chemoattractant mediators, cell-adhesion molecules and endothelial activation factors in WT mice post-BCAS were ameliorated by Nox2 deficiency. The clinical relevance was confirmed by increased vascular Nox2 and indices of microvascular inflammation in human post-mortem subjects with cerebral vascular disease. Our results support Nox2 activity as a critical determinant of VCI, whose targeting may be of therapeutic benefit in cerebral vascular disease.

The Non-Coding RNA Journal Club: Highlights on Recent Papers-10.

We are delighted to share with you our seventh Journal Club and highlight some of the most interesting papers published recently [...].

Autophagy at the interface of endothelial cell homeostasis and vascular disease.

Autophagy is an essential intracellular process for cellular quality control. It enables cell homeostasis through the selective degradation of harmful protein aggregates and damaged organelles. Autophagy is essential for recycling nutrients, generating energy to maintain cell viability in most tissues and during adverse conditions such as hypoxia/ischaemia. The progressive understanding of the mechanisms modulating autophagy in the vasculature has recently led numerous studies to link intact autophagic responses with endothelial cell (EC) homeostasis and function. Preserved autophagic flux within the ECs has an essential role in maintaining their physiological characteristics, whereas defective autophagy can promote endothelial pro-inflammatory and atherogenic phenotype. However, we still lack a good knowledge of the complete molecular repertoire controlling various aspects of endothelial autophagy and how this is associated with vascular diseases. Here, we provide an overview of the current state of the art of autophagy in ECs. We review the discoveries that have so far defined autophagy as an essential mechanism in vascular biology and analyse how autophagy influences ECs behaviour in vascular disease. Finally, we emphasise opportunities for compounds to regulate autophagy in ECs and discuss the challenges of exploiting them to resolve vascular disease.

Long Non-Coding RNA Regulation of Epigenetics in Vascular Cells.

The vascular endothelium comprises the interface between the circulation and the vessel wall and, as such, is under the dynamic regulation of vascular signalling, nutrients, and hypoxia. Understanding the molecular drivers behind endothelial cell (EC) and vascular smooth muscle cell (VSMC) function and dysfunction remains a pivotal task for further clinical progress in tackling vascular disease. A newly emerging era in vascular biology with landmark deep sequencing approaches has provided us with the means to profile diverse layers of transcriptional regulation at a single cell, chromatin, and epigenetic level. This review describes the roles of major vascular long non-coding RNA (lncRNAs) in the epigenetic regulation of EC and VSMC function and discusses the recent progress in their discovery, detection, and functional characterisation. We summarise new findings regarding lncRNA-mediated epigenetic mechanisms-often regulated by hypoxia-within the vascular endothelium and smooth muscle to control vascular homeostasis in health and disease. Furthermore, we outline novel molecular techniques being used in the field to delineate the lncRNA subcellular localisation and interaction with proteins to unravel their biological roles in the epigenetic regulation of vascular genes.

Protective Role of Mytilus edulis Hydrolysate in Lipopolysaccharide-Galactosamine Acute Liver Injury.

Acute liver injury in its terminal phase trigger systemic inflammatory response syndrome with multiple organ failure. An uncontrolled inflammatory reaction is difficult to treat and contributes to high mortality. Therefore, to solve this problem a search for new therapeutic approaches remains urgent. This study aimed to explore the protective effects of M. edulis hydrolysate (N2-01) against Lipopolysaccharide-D-Galactosamine (LPS/D-GalN)-induced murine acute liver injure and the underlying mechanisms. N2-01 analysis, using Liquid Chromatography Mass Spectrometry (LCMS) metabolomic and proteomic platforms, confirmed composition, molecular-weight distribution, and high reproducibility between M. edulis hydrolysate manufactured batches. N2-01 efficiently protected mice against LPS/D-GalN-induced acute liver injury. The most prominent result (100% survival rate) was obtained by the constant subcutaneous administration of small doses of the drug. N2-01 decreased Vascular Cell Adhesion Molecule-1 (VCAM-1) expression from 4.648 ± 0.445 to 1.503 ± 0.091 Mean Fluorescence Intensity (MFI) and Interleukin-6 (IL-6) production in activated Human Umbilical Vein Endothelial Cells (HUVECs) from 7.473 ± 0.666 to 2.980 ± 0.130 ng/ml in vitro. The drug increased Nitric Oxide (NO) production by HUVECs from 27.203 ± 2.890 to 69.200 ± 4.716 MFI but significantly decreased inducible Nitric Oxide Synthase (iNOS) expression from 24.030 ± 2.776 to 15.300 ± 1.290 MFI and NO production by murine peritoneal lavage cells from 6.777 ± 0.373 µm to 2.175 ± 0.279 µm. The capability of the preparation to enhance the endothelium barrier function and to reduce vascular permeability was confirmed in Electrical Cell-substrate Impedance Sensor (ECIS) test in vitro and Miles assay in vivo. These results suggest N2-01 as a promising agent for treating a wide range of conditions associated with uncontrolled inflammation and endothelial dysfunction.