Endothelial Cell Phenotype Research Articles

Abstract 13859: The Omics Signature of Human Pericardial Fluid Exosomes Reveals the Therapeutic Potential of APAF-1 Inhibition in Ischemic Heart Disease

Background: The human pericardial fluid (PF) contains heart-derived small extracellular vesicles (sEVs) able to induce vascular responses via microRNA (miR) transfer. People with diabetes (DM) are particularly at risk of severe ischemic heart disease (IHD) and might require personalized therapeutic treatment. Hypothesis: The PF-sEVs molecular profiling supports the identification of clinical-relevant therapeutic targets. Aims: 1) To characterize PF-sEVs of IHD patients with/out type-2 DM (T2DM) in comparison to controls; 2) to validate the emerging therapeutic target candidates. Methods: PF samples (n=12/group) were collected during cardiac surgeries, namely CABG (for IHD with/out DM groups) and mitral valve repair (non-IHD, non-DM control group). sEVs were prepared (SEC), quality controlled (Nanosight and Exoview) and submitted to untargeted proteomics and microRNA array (Exiqon) or validation experiments. miRNA-target prediction methods and integration of proteomics and transcriptomics via networks identified differentially expressed pathogenic proteins and their putative endogenous miRNA regulators. The emerging dysregulated miRNA/protein axis was investigated in vitro and in mice with myocardial infarction (MI) and with/out diet-induced T2DM. Results: the PF-EVs of IHD patients with DM showed increased APAF-1 and reduced let-7b-5p, a miR which we have demonstrated to repress APAF-1 by direct 3’-UTR-binding. Exposure of endothelial cells (ECs) to DM and IHD-mimicking culture conditions increases APAF-1 level, triggering apoptosis and reducing angiogenesis. The phenotype of ECs kept under disease-mimicking conditions was rescued by APAF-1 silencing (siRNAs) and by supplementation with either healthy PF-EV or LNPs carrying let-7b-5p as sole miR cargo. The synthetic APAF-1 inhibitor ZYZ-488 improved post-MI cardiac function (Vevo3100 echocardiography), increased capillary density and reduced EC apoptosis in the peri-infarct area of diabetic and non-diabetic mice of both sexes. Conclusions: PF-EVs represent a novel and privileged source for the discovery of clinical-relevant therapeutic targets. APAF-1 inhibition holds value for post-ischemic cardiac protection and repair, including in diabetic people.

Brain microvascular endothelial cells possess a second cilium that arises from the daughter centriole.

Primary cilia from the brain microvascular endothelial cells (ECs) are specialized cell-surface organelles involved in mediating sensory perception, cell signaling, and vascular stability. Immunofluorescence (IF) analysis of human primary brain microvascular ECs reveals two cilia per cell. To confirm the in vitro observation of the two-cilia phenotype in human primary brain ECs, ECs isolated from mouse brain were cultured and stained for cilium. Indeed, brain ECs from a ciliopathic mouse (polycystic kidney disease or Pkd2 -/-) also possess more than one cilium. Primary cilium emerges from the mother centriole. Centriole analysis by IF suggests that in brain ECs, markers for the mother and daughter centrioles stain both cilia, suggesting that the second cilium in brain ECs arises from the daughter centriole. Further quantification of cilia size in brain ECs revealed that cilia arising from the mother centriole are bigger in size compared with cilia from the daughter centriole. Cell cycle analyses using immunoblotting and flow cytometry suggest that the ciliary proteins ARL13B and IFT88 involved in brain EC ciliogenesis are highly expressed only in the G0/G1 and S phases of the cell cycle. The IF analyses of cells arrested at different cell cycle stages indicate that the two-cilia phenotype is highly specific to the G0/G1 phase. Our findings suggest that in addition to the mother centriole, the daughter centriole also plays a role in ciliogenesis in primary cultured ECs.

Senescent Endothelial Cells Sustain Their Senescence-Associated Secretory Phenotype (SASP) through Enhanced Fatty Acid Oxidation.

Cellular senescence is closely linked to endothelial dysfunction, a key factor in age-related vascular diseases. Senescent endothelial cells exhibit a proinflammatory phenotype known as SASP, leading to chronic inflammation (inflammaging) and vascular impairments. Albeit in a state of permanent growth arrest, senescent cells paradoxically display a high metabolic activity. The relationship between metabolism and inflammation is complex and varies across cell types and senescence inductions. While some cell types shift towards glycolysis during senescence, others favor oxidative phosphorylation (OXPHOS). Despite the high availability of oxygen, quiescent endothelial cells (ECs) tend to rely on glycolysis for their bioenergetic needs. However, there are limited data on the metabolic behavior of senescent ECs. Here, we characterized the metabolic profiles of young and senescent human umbilical vein endothelial cells (HUVECs) to establish a possible link between the metabolic status and the proinflammatory phenotype of senescent ECs. Senescent ECs internalize a smaller amount of glucose, have a lower glycolytic rate, and produce/release less lactate than younger cells. On the other hand, an increased fatty acid oxidation activity was observed in senescent HUVECs, together with a greater intracellular content of ATP. Interestingly, blockade of glycolysis with 2-deoxy-D-glucose in young cells resulted in enhanced production of proinflammatory cytokines, while the inhibition of carnitine palmitoyltransferase 1 (CPT1), a key rate-limiting enzyme of fatty acid oxidation, ameliorated the SASP in senescent ECs. In summary, metabolic changes in senescent ECs are complex, and this research seeks to uncover potential strategies for modulating these metabolic pathways to influence the SASP.

ApoSEVs-Mediated Modulation of Versatile Target Cells Promotes Diabetic Wound Healing: Unveiling a Promising Strategy.

Diabetic chronic wounds present a formidable challenge in clinical management, lacking effective treatment options. Mesenchymal stem cell (MSC) transplantation has emerged as a promising therapy for tissue repair and regeneration. However, transplanted MSCs often undergo rapid apoptosis, giving rise to heterogeneous extracellular vesicles (EVs), including apoptotic bodies (apoBDs) and apoptotic small extracellular vesicles (apoSEVs). The potential stimulatory role of these EVs in diabetic wound healing remains unknown. In this study, we investigated the effects of apoSEVs derived from adipose-derived mesenchymal/stromal cells (ADSCs) on the recovery of diabetic wounds by modulating the function of versatile target cells. First, we characterized the apoSEVs and apoBDs derived from apoptotic ADSCs. Subsequently, we evaluated the effects of apoSEVs and apoBDs on macrophages, endothelial cells, and fibroblasts, three essential cell types in wound healing, under high-glucose conditions. Furthermore, we developed a gelatin methacryloyl (GelMA) hydrogel for the sustained release of apoSEVs and investigated its therapeutic effects on wound healing in type 2 diabetic mice in vivo. apoSEVs facilitated the polarization of M1 phenotype macrophages to M2 phenotype, promoted proliferation, migration, and tube formation of endothelial cells, and enhanced fibroblast proliferation and migration. However, apoBDs failed to improve the function of endothelial cells and fibroblasts. In vivo, the apoSEVs-loaded GelMA effectively promoted wound healing by facilitating collagen fiber deposition, angiogenesis, and immune regulation. Our study elucidates the beneficial effects of apoSEVs on wound recovery in diabetes and introduces a novel strategy for diabetic wound treatment based on apoSEVs.

Novel Morphological Profiling Assay Connects ex Vivo Endothelial Cell Responses to Disease Severity in Liver Cirrhosis

Endothelial cell (EC) dysfunction in response to circulating plasma factors is a known causal factor in many systemic diseases. However, no appropriate assay is available to investigate this causality exvivo. In liver cirrhosis, systemic inflammation is identified as central mechanism in progression from compensated to decompensated cirrhosis (DC), but the role of ECs therein is unknown. We aimed to develop a novel exvivo assay for assessing EC responses to patient-derived plasma (PDP) and assess the potential of this assay in a cohort of liver cirrhosis patients. Image-based morphological profiling was utilized to assess the impact of PDP on cultured ECs. Endothelial cell (EC) monolayers were exposed to 25% stabilized PDP (20 compensated cirrhoses, 20 DCs, and 20 healthy controls (HCs). Single-cell morphological profiles were extracted by automated image-analysis following staining of multiple cellular components and high-content imaging. Patient profiles were created by dimension reduction and cell-to-patient data aggregation, followed by multivariate-analysis to stratify patients and identify discriminating features. Patient-derived plasma (PDP) exposure induced profound changes in EC morphology, displaying clear differences between controls and DC patients. Compensated cirrhosis patients showed overlap with healthy controls and DC patients. Supervised analysis showed Child-Pugh (CP) classcould be predicted from EC morphology. Most importantly, CP-C patients displayed distinct EC phenotypes, in whichmitochondrial changes were most discriminative. Morphological profiling presents a viable tool to assess the endothelium exvivo. We demonstrated that the EC phenotype corresponds with disease severity in liver cirrhosis. Moreover, our results suggest the presence of mitochondrial dysfunction in ECs of CP-C patient.

Comprehensive bioinformatics analysis of the solute carrier family and preliminary exploration of SLC25A29 in lung adenocarcinoma

According to the latest epidemiological investigation, lung adenocarcinoma (LUAD) is one of the most fatal cancer among both men and women. Despite continuous advancements in treatment approaches in recent years, the prognosis for LUAD remains relatively poor. Given the crucial role of the solute carrier (SLC) family in maintaining cellular energy metabolism stability, we conducted a comprehensive analysis of the association between SLC genes and LUAD prognosis. In the present study, we identified 71 genes among the SLC family members, of which 32 were downregulated and 39 were upregulated in LUAD samples. Based on these differentially expressed genes, a prognostic risk scoring model was established that was composed of five genes (SLC16A7, SLC16A4, SLC16A3, SLC12A8, and SLC25A15) and clinical characteristics; this model could effectively predict the survival and prognosis of patients in the cohort. Notably, SLC2A1, SLC25A29, and SLC27A4 were identified as key genes associated with survival and tumor stage. Further analysis revealed that SLC25A29 was underexpressed in LUAD tissue and regulated the phenotype of endothelial cells. Endothelial cell proliferation and migration increased and apoptosis decreased with a decrease in SLC25A29 expression. Investigation of the upstream regulatory mechanisms of SLC25A29 revealed that SLC25A29 expression gradually decreased as the lactate concentration increased. This phenomenon suggested that the expression of SLC25A29 may be related to lactylation modification. ChIP-qPCR experiments confirmed the critical regulatory role played by H3K14la and H3K18la modifications in the promoter region of SLC25A29. In conclusion, this study confirmed the role of SLC family genes in LUAD prognosis and revealed the role of SLC25A29 in regulating endothelial cell phenotypes. These study results provided important clues to further understand LUAD pathogenesis and develop appropriate therapeutic strategies.

3D bioprinted endothelial cell-microglia coculture for diabetic retinopathy modeling

Diabetic retinopathy (DR) is a common diabetes complication leading to vision impairment or blindness due to retinal vasculature alterations. Hyperglycemia induces structural alterations, inflammation, and angiogenic factor upregulation. Current treatments targeting vascular endothelial growth factor are insufficient for approximately 20% of DR patients, necessitating alternative approaches. Microglia (MG), essential for retinal homeostasis, remains underexplored in DR. This study used digital light processing bioprinting to construct a 3D coculture model of endothelial cells (ECs) and MG under varying glucose conditions, with a hydrogel stiffness of 4.6–7.1 kPa to mimic the extracellular matrix property of retina plexiform. Our results showed that high glucose levels influenced both EC and microglial phenotypes, gene expression, and angiogenic potential. Increasing glucose from 5 mM to 25 mM reduces drug efficacy by 17% for Aflibercept in EC monoculture, and 25% and 30% for Aflibercept and Conbercept in EC-MG coculture, respectively, suggesting that diabetic condition and MG presence could interfere with drug responses. In conclusion, our findings emphasize the importance of cellular interactions and microenvironmental factors in DR therapy, aiming to identify novel strategies and improve understanding of MG’s role in disease pathogenesis.

Single-Cell RNA sequencing investigation of female-male differences under PAD conditions.

Peripheral arterial disease (PAD) is an age-related medical condition affecting mostly muscular arteries of the limb. It is the 3rd leading cause of atherosclerotic morbidity. The mechanical environment of endothelial cells (ECs) in PAD is characterized by disturbed blood flow (d-flow) and stiff extracellular matrices. In PAD, the stiffness of arteries is due to decreased elastin function and increased collagen content. These flow and stiffness parameters are largely missing from current models of PAD. It has been previously proven that ECs exposed to d-flow or stiff substrates lead to proatherogenic pathways, but the effect of both, d-flow and stiffness, on EC phenotype has not been fully investigated. In this study, we sought to explore the effect of sex on proatherogenic pathways that could result from exposing endothelial cells to a d-flow and stiff environment. We utilized the scRNA-seq tool to analyze the gene expression of ECs exposed to the different mechanical conditions both in vitro and in vivo. We found that male ECs exposed to different mechanical stimuli presented higher expression of genes related to fibrosis and d-flow in vitro. We validated our findings in vivo by exposing murine carotid arteries to d-flow via partial carotid artery ligation. Since women have delayed onset of arterial stiffening and subsequent PAD, this work may provide a framework for some of the pathways in which biological sex interacts with sex-based differences in PAD.

Using extracellular matrix derived from sugen-chronic hypoxia lung tissue to study pulmonary arterial hypertension.

Pulmonary arterial hypertension has characteristic changes to the mechanical environment, extracellular matrix, and cellular proliferation. In order to develop a culture system to investigate extracellular matrix (ECM) compositional-dependent changes in pulmonary arterial hypertension, we decellularized and characterized protein and lipid profiles from healthy and Sugen-Chronic Hypoxia rat lungs. Significant changes in lipid profiles were observed in intact Sugen-Hypoxia lungs compared with healthy controls. Decellularized lung matrix retained lipids in measurable quantities in both healthy and Sugen-Chronic Hypoxia samples. Proteomics revealed significantly changed proteins associated with pulmonary arterial hypertension in the decellularized Sugen-Chronic Hypoxia lung ECM. We then investigated the potential role of healthy vs. Sugen-Chronic Hypoxia ECM with controlled substrate stiffness to determine if the ECM composition regulated endothelial cell morphology and phenotype. CD117+ rat lung endothelial cell clones were plated on the variable stiffness gels and cellular proliferation, morphology, and gene expression were quantified. Sugen-Chronic Hypoxia ECM on healthy stiffness gels produced significant changes in cellular gene expression levels of Bmp2, Col1α1, Col3α1 and Fn1. The signaling and cell morphology observed at low substrate stiffness suggests early changes to the ECM composition can initiate processes associated with disease progression. These data suggest that Sugen-Chronic Hypoxia ECM can be used to investigate cell-ECM interactions relevant to pulmonary arterial hypertension.

RNA-Binding Proteins Regulate Post-Transcriptional Responses to TGF-β to Coordinate Function and Mesenchymal Activation of Murine Endothelial Cells.

Endothelial cells (ECs) are primed to respond to various signaling cues. For example, TGF (transforming growth factor)-β has major effects on EC function and phenotype by driving ECs towards a more mesenchymal state (ie, triggering endothelial to mesenchymal activation), a dynamic process associated with cardiovascular diseases. Although transcriptional regulation triggered by TGF-β in ECs is well characterized, post-transcriptional regulatory mechanisms induced by TGF-β remain largely unknown. Using RNA interactome capture, we identified global TGF-β driven changes in RNA-binding proteins in ECs. We investigated specific changes in the RNA-binding patterns of hnRNP H1 (heterogeneous nuclear ribonucleoprotein H1) and Csde1 (cold shock domain containing E1) using RNA immunoprecipitation and overlapped this with RNA-sequencing data after knockdown of either protein for functional insight. Using a modified proximity ligation assay, we visualized the specific interactions between hnRNP H1 and Csde1 and target RNAs in situ both in vitro and in mouse heart sections. Characterization of TGF-β-regulated RBPs (RNA-binding proteins) revealed hnRNP H1 and Csde1 as key regulators of the cellular response to TGF-β at the post-transcriptional level, with loss of either protein-promoting mesenchymal activation in ECs. We found that TGF-β drives an increase in binding of hnRNP H1 to its target RNAs, offsetting mesenchymal activation, but a decrease in Csde1 RNA-binding, facilitating this process. Both, hnRNP H1 and Csde1, dynamically bind and regulate specific subsets of mRNAs related to mesenchymal activation and endothelial function. Together, we show that RBPs play a key role in the endothelial response to TGF-β stimulation at the post-transcriptional level and that the RBPs hnRNP H1 and Csde1 serve to maintain EC function and counteract mesenchymal activation. We propose that TGF-β profoundly modifies RNA-protein interaction entailing feedback and feed-forward control at the post-transcriptional level, to fine-tune mesenchymal activation in ECs.

Endothelial CD34 expression and regulation of immune cell response in-vitro

Endothelial cells cover the lining of different blood vessels and lymph nodes, and have major functions including the transport of blood, vessel homeostasis, inflammatory responses, control of transendothelial migration of circulating cells into the tissues, and formation of new blood vessels. Therefore, understanding these cells is of major interest. The morphological features, phenotype and function of endothelial cells varies according to the vascular bed examined. The sialomucin, CD34, is widely used as an endothelial marker. However, CD34 is differentially expressed on endothelial cells in different organs and in pathological conditions. Little is known about regulation of endothelial CD34 expression or function. Expression of CD34 is also strongly regulated in-vitro in endothelial cell models, including human umbilical vein endothelial cells (HUVEC) and endothelial colony forming cells (ECFC). We have therefore analysed the expression and function of CD34 by comparing CD34high and CD34low endothelial cell subpopulations. Transcriptomic analysis showed that CD34 gene and protein expressions are highly correlated, that CD34high cells proliferate less but express higher levels of IL-33 and Angiopoietin 2, compared with CD34low cells. Higher secretion levels of IL-33 and Angiopoietin 2 by CD34high HUVECs was confirmed by ELISA. Finally, when endothelial cells were allowed to interact with peripheral blood mononuclear cells, CD34high endothelial cells activated stronger proliferation of regulatory T lymphocytes (Tregs) compared to CD34low cells whereas expansion of other CD4+-T cell subsets was equivalent. These results suggest that CD34 expression by endothelial cells in-vitro associates with their ability to proliferate and with an immunogenic ability that favours the tolerogenic response.

Reactive astrocytes associated with prion disease impair the blood brain barrier

BackgroundImpairment of the blood-brain barrier (BBB) is considered to be a common feature among neurodegenerative diseases, including Alzheimer's, Parkinson's and prion diseases. In prion disease, increased BBB permeability was reported 40 years ago, yet the mechanisms behind the loss of BBB integrity have never been explored. Recently, we showed that reactive astrocytes associated with prion diseases are neurotoxic. The current work examines the potential link between astrocyte reactivity and BBB breakdown. ResultsIn prion-infected mice, the loss of BBB integrity and aberrant localization of aquaporin 4 (AQP4), a sign of retraction of astrocytic endfeet from blood vessels, were noticeable prior to disease onset. Gaps in cell-to-cell junctions along blood vessels, together with downregulation of Occludin, Claudin-5 and VE-cadherin, which constitute tight and adherens junctions, suggested that loss of BBB integrity is linked with degeneration of vascular endothelial cells. In contrast to cells isolated from non-infected adult mice, endothelial cells originating from prion-infected mice displayed disease-associated changes, including lower levels of Occludin, Claudin-5 and VE-cadherin expression, impaired tight and adherens junctions, and reduced trans-endothelial electrical resistance (TEER). Endothelial cells isolated from non-infected mice, when co-cultured with reactive astrocytes isolated from prion-infected animals or treated with media conditioned by the reactive astrocytes, developed the disease-associated phenotype observed in the endothelial cells from prion-infected mice. Reactive astrocytes were found to produce high levels of secreted IL-6, and treatment of endothelial monolayers originating from non-infected animals with recombinant IL-6 alone reduced their TEER. Remarkably, treatment with extracellular vesicles produced by normal astrocytes partially reversed the disease phenotype of endothelial cells isolated from prion-infected animals. ConclusionsTo our knowledge, the current work is the first to illustrate early BBB breakdown in prion disease and to document that reactive astrocytes associated with prion disease are detrimental to BBB integrity. Moreover, our findings suggest that the harmful effects are linked to proinflammatory factors secreted by reactive astrocytes.

Opposing effect of chlorogenic acid on induction of beige adipocyte phenotype in 3T3-L1 adipocytes and angiogenic phenotype in endothelial cells

Opposing effect of chlorogenic acid on induction of beige adipocyte phenotype in 3T3-L1 adipocytes and angiogenic phenotype in endothelial cells

Differential Expression of the MiT/TFE Transcription Factor Family in Vascular Endothelial Cells under Low Oscillatory Shear Stress

Low Oscillating Shear Stress (LOSS) is known to promote inflammation and atherosclerosis, impacting the phenotype and function of Vascular Endothelial Cells (VECs). The microphthalmia/transcription factor E (MiT/TFE) family of transcription factors regulates autophagy and lysosomal biogenesis in response to internal and external stressors. However, whether LOSS affects the expression of MiT/TFE family members in Human Umbilical Vein Endothelial Cells (HUVECs) has remained unclear. In this study, we utilized qPCR and WB to examine how LOSS influenced the mRNA and protein expression of MiT/TFE family members (MITF, TFEB, TFEC, and TFE3) in HUVECs in vitro. The in vitro findings were further confirmed using immunohistochemistry in a mouse model with a partially ligated carotid artery. Our results showed that LOSS down-regulated the expressions of TFEC and TFE3, while having no impact on MITF and TFEB expressions in HUVECs, the down-regulated expression of TFE3 were validated in the animal model. In conclusion, this study systematically explored the expression patterns of the MiT/TFE family in VECs under LOSS, providing new insights into the molecular mechanisms of abnormal hemodynamics-induced vascular endothelial inflammation-related diseases like atherosclerosis.

МЕХАНИЗМЫ ТРОМБО-ВОСПАЛЕНИЯ У ПАЦИЕНТОВ С СИСТЕМНОЙ КРАСНОЙ ВОЛЧАНКОЙ

Introduction. Systemic lupus erythematosus (SLE) is a systemic autoimmune inflammatory disease. According to different authors, the incidence of thrombotic complications in this disease is higher than the average in the population and can be up to 40%. Aim. The aim of the present review is to provide an overview of mechanisms of thrombosis in patients with systemic lupus erythematosus. Material and methods. Analytical review of foreign and native publications in PubMed and eLibrary.ru between 1985-2020. Results and discussion. SLE represents a model of inflammation-induced thrombosis. The hypercoagulability state which characterizes SLE seems to be associated with inflammation-driven alterations of the thrombotic balance. Tissue-nonspecific autoantibodies, as well as cytokines and acute phase proteins induce a proinflammatory endothelial cell phenotype. Platelets are activated following endothelial dysfunction; they participate in inflammatory and thrombotic events occurring in an injured area. One of the possible initiators of the thrombosis process is considered to be an interaction of antiphospholipid antibodies with phospholipids of platelet membranes, endothelium and phospholipid-linked plasma proteins. Local unbalanced thrombin generation leading to hypercoagulability is another source central for thrombosis. Patients with systemic lupus erythematosus show multidirectional disorders of the coagulation system, with predominance of hypercoagulation especially in patients with II-III degree of disease activity. Conclusion. The mechanism of thrombo-inflammation in systemic lupus erythematosus is multifactorial, involving all components of the hemostatic system. Chronic endothelial dysfunction, platelet activation, hypercoagulation, suppression of fibrinolysis reflects an interplay between thrombosis and inflammation. Persisting autoimmune inflammation plays a key role in these abnormalities.

SREBP2 regulates the endothelial response to cytokines via direct transcriptional activation of KLF6

The transcription factor SREBP2 is the main regulator of cholesterol homeostasis and is central to the mechanism of action of lipid-lowering drugs, such as statins, which are responsible for the largest overall reduction in cardiovascular risk and mortality in humans with atherosclerotic disease. Recently, SREBP2 has been implicated in leukocyte innate and adaptive immune responses by upregulation of cholesterol flux or direct transcriptional activation of pro-inflammatory genes. Here, we investigate the role of SREBP2 in endothelial cells (ECs), since ECs are at the interface of circulating lipids with tissues and crucial to the pathogenesis of cardiovascular disease. Loss of SREBF2 inhibits the production of pro-inflammatory chemokines but amplifies type I interferon response genes in response to inflammatory stimulus. Furthermore, SREBP2 regulates chemokine expression not through enhancement of endogenous cholesterol synthesis or lipoprotein uptake but partially through direct transcriptional activation. Chromatin immunoprecipitation sequencing of endogenous SREBP2 reveals that SREBP2 bound to the promoter regions of two nonclassical sterol responsive genes involved in immune modulation, BHLHE40 and KLF6. SREBP2 upregulation of KLF6 was responsible for the downstream amplification of chemokine expression, highlighting a novel relationship between cholesterol homeostasis and inflammatory phenotypes in ECs.

Vascular Inflammatory Diseases and Endothelial Phenotypes

The physiological functions of endothelial cells control vascular tone, permeability, inflammation, and angiogenesis, which significantly help to maintain a healthy vascular system. Several cardiovascular diseases are characterized by endothelial cell activation or dysfunction triggered by external stimuli such as disturbed flow, hypoxia, growth factors, and cytokines in response to high levels of low-density lipoprotein and cholesterol, hypertension, diabetes, aging, drugs, and smoking. Increasing evidence suggests that uncontrolled proinflammatory signaling and further alteration in endothelial cell phenotypes such as barrier disruption, increased permeability, endothelial to mesenchymal transition (EndMT), and metabolic reprogramming further induce vascular diseases, and multiple studies are focusing on finding the pathways and mechanisms involved in it. This review highlights the main proinflammatory stimuli and their effects on endothelial cell function. In order to provide a rational direction for future research, we also compiled the most recent data regarding the impact of endothelial cell dysfunction on vascular diseases and potential targets that impede the pathogenic process.

#4451 MONOCYTE MIGRATION AND ACTIVATION IN ANCA-ASSOCIATED GLOMERULONEPHRITIS

Abstract Background and Aims Glomerular injury in Anti-Neutrophil Cytoplasmic Antibodies (ANCA)-associated glomerulonephritis (AGN) is associated with macrophage infiltration. To date, their role and origin remain to be fully elucidated. Activated blood monocytes likely transmigrate and differentiate into kidney macrophages depending on the local microenvironment. Even in remission, some studies indicate sustained monocyte activation and upregulation of adhesion markers. The aim of this study was to research the intrinsic migration capacity of monocytes in AGN. Targeting monocyte migration might open new therapeutic avenues in the treatment of AGN. Method Transendothelial migration of monocytes was tested in AGN patients with active disease (n = 2), in remission (n = 8) and healthy controls (n = 6). To assess their adhesive and migratory capacity, freshly isolated CD14 positive monocytes were added to confluent TNF-α or IL1β overnight-stimulated Human Aortic Endothelial Cell (HAEC) for 30 minutes and subsequently fixed. Monocyte adhesion and transmigration was visualized by phase-contrast microscopy and quantified using ImageJ. To unravel the potential mechanisms driving these differences in monocyte migration, we performed bulk RNA-sequencing of monocytes from AGN patients with active (n = 4) and stable (n = 10) disease, and healthy controls (HC) (n = 6). Results Monocyte adhesion, but not migration, was significantly increased during active disease, independently of the stimuli used to mimic the pro-inflammatory phenotype of endothelial cells (EC) (Figure 1). During remission, decreased adhesion was found on the IL1β-stimulated ECs. While CD11b mRNA expression was upregulated, CD11a expression was downregulated in monocytes from active AGN patients compared to HC. Two genes involved in paracellular monocyte transmigration (JAML, PECAM-1) were significantly decreased. Most AGN patients were treated with corticosteroids at time of experiments. Conclusion These results suggest a remarkable increase in monocyte adhesion, but lower intrinsic migration capacity, in a 30 minute time-frame during active AGN. Our findings on migration are surprising in the light of theorized enhanced monocyte extravasation towards diseased AGN kidneys. Therefore, it could be speculated that after a longer period of time, a subset of the adherent monocytes would ultimately migrate and differentiate into kidney macrophages. In remission, long-term immunosuppressive treatment or chronic inflammation might decrease monocyte adhesion. This was the first ex-vivo study to research monocyte migration in AGN, further research is required to validate findings and to develop new therapies targeting monocyte migration.

Identification and validation of G protein-coupled receptors modulating flow-dependent signaling pathways in vascular endothelial cells

Vascular endothelial cells are exposed to mechanical forces due to their presence at the interface between the vessel wall and flowing blood. The patterns of these mechanical forces (laminar vs. turbulent) regulate endothelial cell function and play an important role in determining endothelial phenotype and ultimately cardiovascular health. One of the key transcriptional mediators of the positive effects of laminar flow patterns on endothelial cell phenotype is the zinc-finger transcription factor, krüppel-like factor 2 (KLF2). Given its importance in maintaining a healthy endothelium, we sought to identify endothelial regulators of the KLF2 transcriptional program as potential new therapeutic approaches to treating cardiovascular disease. Using an approach that utilized both bioinformatics and targeted gene knockdown, we identified endothelial GPCRs capable of modulating KLF2 expression. Genetic screening using siRNAs directed to these GPCRs identified 12 potential GPCR targets that could modulate the KLF2 program, including a subset capable of regulating flow-induced KLF2 expression in primary endothelial cells. Among these targets, we describe the ability of several GPCRs (GPR116, SSTR3, GPR101, LGR4) to affect KLF2 transcriptional activation. We also identify these targets as potential validated targets for the development of novel treatments targeting the endothelium. Finally, we highlight the initiation of drug discovery efforts for LGR4 and report the identification of the first known synthetic ligands to this receptor as a proof-of-concept for pathway-directed phenotypic screening to identify novel drug targets.

Microenvironment commits breast tumor ECs to dedifferentiation by micro-RNA-200-b-3p regulation and extracellular matrix remodeling.

Introduction: Hypoxia shapes the tumor microenvironment, modulates distinct cell population activities, and activates pathological angiogenesis in cancer, where endothelial cells (ECs) are the most important players. This study aimed to evidence the influences of the tumor microenvironment on the global gene expression pattern characteristic for ECs and the distinct responses displayed by tumor-derived ECs in comparison to the healthy endothelium during endothelial to mesenchymal transition (EndMT) and its regulation by miR-200-b-3p. Methodology: Immortalized lines of ECs from the same patient with breast cancer, healthy breast tissue (HBH.MEC), and primary tumor (HBCa.MEC) were used. The experiments were performed in normoxia and hypoxia for 48h. By using the wound healing test, we investigated the migration abilities of ECs. Global gene expression analysis with NGS was carried out to detect new pathways altered in pathological ECs and find the most changed miRNAs. The validation of NGS data from RNA and miRNA was estimated by qPCRs. Mimic miR-200b-3p was used in HBH.MEC, and the targets VEGF, Bcl2, ROCK2, and SP1 were checked. Results: Hypoxia influences EC migration properties in wound healing assays. In hypoxia, healthy ECs migrate slower than they do in normoxia, as opposed to HBCa.MEC, where no decreased migration ability is induced by hypoxia due to EndMT features. NGS data identified this process to be altered in cancer ECs through extracellular matrix (ECM) organization. The deregulated genes, validated by qPCR, included SPP1, ITGB6, COL4A4, ADAMST2, LAMA1, GAS6, PECAM1, ELN, FBLN2, COL6A3, and COL9A3. NGS also identified collagens, laminins, fibronectins, and integrins, as being deregulated in tumor-derived ECs. Moreover, the analysis of the 10 most intensively modified miRNAs, when breast tumor-derived ECs were compared to healthy ECs, shed light on miR-200b-3p, which is strongly upregulated in HBCa.MECs when compared to HBH.MECs. Discussion and conclusion: The pathological ECs differed significantly, both phenotypically and functionally, from the normal corresponding tissue, thus influencing their microenvironment cross-talk. The gene expression profile confirms the EndMT phenotype of tumor-derived ECs and migratory properties acquisition. Moreover, it indicates the role of miR-200b-3p, that is, regulating EndMT in pathological ECs and silencing several angiogenic growth factors and their receptors by directly targeting their mRNA transcripts.