Response Of Human Endothelial Cells Research Articles

The BCR::ABL1 tyrosine kinase inhibitors ponatinib and nilotinib differentially affect endothelial angiogenesis and signalling.

BCR::ABL1 inhibitors, the treatment of choice for the majority of patients with chronic myeloid leukaemia (CML), can cause vascular side effects that vary between agents. The exact underlying mechanisms are still poorly understood, but the vascular endothelium has been proposed as a site of origin. The present study investigates the effects of three BCR::ABL1 inhibitors, ponatinib, nilotinib and imatinib, on angiogenesis and signalling in human endothelial cells in response to vascular endothelial growth factor (VEGF). The experiments were performed in endothelial cells isolated from human umbilical veins. After exposure to imatinib, ponatinib and nilotinib, the angiogenic capacity of endothelial cells was assessed in spheroid assays. VEGF-induced signalling pathways were examined in Western blotting experiments using different specific antibodies. RNAi technology was used to downregulate proteins of interest. Intracellular cGMP levels were measured by ELISA. Imatinib had no effect on endothelial function. Ponatinib inhibited VEGF-induced sprouting, while nilotinib increased spontaneous and VEGF-stimulated angiogenesis. These effects did not involve wild-type ABL1 or ABL2, as siRNA-mediated knockdown of these kinases did not affect angiogenesis and VEGF signalling. Consistent with their effects on sprouting, ponatinib and nilotinib affected angiogenic pathways in opposite directions. While ponatinib inhibited VEGF-induced signalling and cGMP formation, nilotinib activated angiogenic signalling, in particular phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2). The latter occurred in an epidermal growth factor receptor (EGFR)-dependent manner possibly via suppressing Fyn-related kinase (FRK), a negative regulator of EGFR signalling. Both, pharmacological inhibition of Erk1/2 or EGFR suppressed nilotinib-induced angiogenic sprouting. These results support the notion that the vascular endothelium is a site of action of BCR::ABL1 inhibitors from which side effects may arise, and that the different vascular toxicity profiles of BCR::ABL1 inhibitors may be due to their different actions at the molecular level. In addition, the as yet unknown pro-angiogenic effect of nilotinib should be considered in the treatment of patients with comorbidities associated with pathological angiogenesis, such as ocular disease, arthritis or obesity.

Integrin α5β1 contributes to cell fusion and inflammation mediated by SARS-CoV-2 spike via RGD-independent interaction.

The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus infects host cells by engaging its spike (S) protein with human ACE2 receptor. Recent studies suggest the involvement of integrins in SARS-CoV-2 infection through interaction with the S protein, but the underlying mechanism is not well understood. This study investigated the role of integrin α5β1, which recognizes the Arg-Gly-Asp (RGD) motif in its physiological ligands, in S-mediated virus entry and cell-cell fusion. Our results showed that α5β1 does not directly contribute to S-mediated cell entry, but it enhances S-mediated cell-cell fusion in collaboration with ACE2. This effect cannot be inhibited by the putative α5β1 inhibitor ATN-161 or the high-affinity RGD-mimetic inhibitor MK-0429 but requires the participation of α5 cytoplasmic tail (CT). We detected a direct interaction between α5β1 and the S protein, but this interaction does not rely on the RGD-containing receptor binding domain of the S1 subunit of the S protein. Instead, it involves the S2 subunit of the S protein and α5β1 homo-oligomerization. Furthermore, we found that the S protein induces inflammatory responses in human endothelial cells, characterized by NF-κB activation, gasdermin D cleavage, and increased secretion of proinflammatory cytokines IL-6 and IL-1β. These effects can be attenuated by the loss of α5 expression or inhibition of the α5 CT binding protein phosphodiesterase-4D (PDE4D), suggesting the involvement of α5 CT and PDE4D pathway. These findings provide molecular insights into the pathogenesis of SARS-CoV-2 mediated by a nonclassical RGD-independent ligand-binding and signaling function of integrin α5β1 and suggest potential targets for antiviral treatment.

Role of human dural fibroblasts in the angiogenic responses of human endothelial cells: An in vitro dural model and the application of lab-on-a-chip for EDAS.

Encephaloduroarteriosynangiosis (EDAS), an indirect anastomosis procedure, is widely accepted as a primary treatment for moyamoya disease (MMD) to improve collateral blood flow. During surgical intervention, dural fibroblasts (DuF) are thought to produce various proteins that create an angiogenic microenvironment. However, the biophysiological evidence supporting the angiogenic properties of this surgical technique has not been thoroughly elucidated. The purpose of these studies was to determine whether DuF releases pro-angiogenic factors and chemokines and promotes angiogenic properties in human endothelial cells (ECs) under IL-1β-mediated wound conditions, which are expected to occur during the process of neo-vascularization within the dura mater. Furthermore, a microfluidic chemotaxis platform was implemented to investigate the angiogenic activity of ECs in response to a reconstituted dura model. Transcriptome sequencing revealed that IL-1β stimulation on DuF induced a significant upregulation of various pro-angiogenic genes, including IL-6, IL-8, CCL-2, CCL-5, SMOC-1, and SCG-2 (p < 0.05). Moreover, compared to ECs cultured in naïve media or naïve DuF media, those exposed to IL-1β-DuF conditioned media expressed higher mRNA and protein levels of these pro-angiogenic factors (p < 0.001). ECs co-cultured with IL-1β-DuF also exhibited considerable migration on the microfluidic chemotaxis platform. Furthermore, the chemotactic effects on the ECs were reduced upon neutralization of IL-8 or inhibition of NF-κB signaling. Our findings demonstrate that IL-1β-DuFs release factors that activate and enhance the angiogenic properties of ECs. These results suggest a potential interaction between DuF and ECs following EDAS for MMD, and these components could be targeted for the development of therapeutic biomarkers.

Sex-dependent PD-L1/sPD-L1 trafficking in human endothelial cells in response to inflammatory cytokines and VEGF

Programmed cell death 1 ligand 1 (PD-L1) expressed in non-immune cells is involved in immune-mediated tissue damage in the context of inflammatory conditions and tumor immune escape. Emerging evidence suggests soluble (s)PD-L1 as a marker of inflammation. Based on well-established sex-specific differences in immunity, we tested the novel hypotheses that (i) endothelial cell PD-L1 is modulated by inflammatory cytokines and vascular endothelial growth factor (VEGF) in a sex-specific fashion, and (ii) the endothelium is a source of sPD-L1. After exposure of human umbilical vein endothelial cells (HUVECs) to lipopolysaccharide, interleukin (IL)1β or VEGF for 24 h, total PD-L1 levels were upregulated solely in cells from female donors, while being unchanged in those from male donors. Accordingly, exposure to synovial fluids from patients with inflammatory arthritis upregulated PD-L1 levels in HUVECs from female donors only. Membrane PD-L1 expression as measured by flow cytometry was unchanged in response to inflammatory stimuli. However, exposure to 2 ng/mL IL-1β or 50 ng/mL VEGF time-dependently increased sPD-L1 release by HUVECs from female donors. Treatment with the metalloproteinase (MMP) inhibitor GM6001 (10 μM) prevented IL-1β-induced sPD-L1 release and enhanced membrane PD-L1 levels. The anti-VEGF agents bevacizumab and sunitinib reduced both VEGF-induced PD-L1 accumulation and sPD-L1 secretion. Thus, inflammatory agents and VEGF rapidly increased endothelial PD-L1 levels in a sex-specific fashion. Furthermore, the vascular endothelium may be a sPD-L1 source, whose production is MMP-dependent and modulated by anti-VEGF agents. These findings may have implications for sex-specific immunity, vascular inflammation and response to anti-angiogenic therapy.

Angiotensin II Increases Oxidative Stress and Inflammation in Female, But Not Male, Endothelial Cells.

Women are at elevated risk for certain cardiovascular diseases, including pulmonary arterial hypertension, Alzheimer's disease, and vascular complications of diabetes. Angiotensin II (AngII), a circulating stress hormone, is elevated in cardiovascular disease; however, our knowledge of sex differences in the vascular effects of AngII are limited. We therefore analyzed sex differences in human endothelial cell response to AngII treatment. Male and female endothelial cells were treated with AngII for 24h and analyzed by RNA sequencing. We then used endothelial and mesenchymal markers, inflammation assays, and oxidative stress indicators to measure female and male endothelial cell functional changes in response to AngII. Our data show that female and male endothelial cells are transcriptomically distinct. Female endothelial cells treated with AngII had widespread gene expression changes related to inflammatory and oxidative stress pathways, while male endothelial cells had few gene expression changes. While both female and male endothelial cells maintained their endothelial phenotype with AngII treatment, female endothelial cells showed increased release of the inflammatory cytokine interleukin-6 and increased white blood cell adhesion following AngII treatment concurrent with a second inflammatory cytokine. Additionally, female endothelial cells had elevated reactive oxygen species production compared to male endothelial cells after AngII treatment, which may be partially due to nicotinamide adenine dinucleotide phosphate oxidase-2 (NOX2) escape from X-chromosome inactivation. These data suggest that endothelial cells have sexually dimorphic responses to AngII, which could contribute to increased prevalence of some cardiovascular diseases in women. The online version contains supplementary material available at 10.1007/s12195-023-00762-2.

Comparative analysis of the biocompatibility of endothelial cells on surfaces treated by thermal plasma and cold atmospheric plasma.

In recent years, cold atmospheric plasma (CAP) is used for surface disinfection. However, little is known about its ability to improve biocompatibility of metallic surfaces when compared to thermal plasma methods. In this context, the study aimed to evaluate the response of human endothelial cells (Ea.hy926) on titanium surfaces treated by non-thermal plasma method and thermal plasma method under nitriding atmosphere. The wettability was characterized by the sessile drop method, the topography and roughness were evaluated by atomic force microscopy (AFM), and the microstructure by grazing angle X-ray diffraction (GIXRD). Endothelial cells were cultured and evaluated for morphology by scanning electron microscopy and viability by an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. CAP treatment reduced the contact angle of the Ti surface (13.43° ± 1.48; p<0.05), increasing hydrophilicity. Rz roughness was higher on the nitrided surface (220.44±20.30; p< 0.001) compared to the CAP treated surfaces (83.29 ± 11.61; p< 0.001) and polished (75.98 ±34.21a); p<0.001). The different applied plasma treatments created different titanium surfaces improving the biocompatibility of endothelial cells, however CAP results demonstrate its potential for biomedical applications, considering the low cost and ease of use of the technique, allowing surface treatments before clinical procedures.

Jujuboside B Inhibited High Mobility Group Box Protein 1-Mediated Severe Inflammatory Responses in Human Endothelial Cells and Mice.

High mobility group box protein 1 (HMGB1) is a biomolecule that acts as an alerting signal of late sepsis by accelerating the production of proinflammatory cytokines, and eventually leads to various inflammation-related symptoms. When released into plasma at high concentration, it disrupts precise diagnosis and prognosis and worsens the survival of patients with systemic inflammatory conditions. Jujuboside B (JB) is a natural compound pressed from the seed of Zizyphi Spinosi Semen, which is known for its medical efficacies in treating various conditions such as hyperlipidemia, hypoxia, and platelet aggregation. Nevertheless, the medicinal activity of JB on HMGB1-involved inflammatory response in vascular cells in the human body is still ambiguous. Therefore, we hypothesized that JB could regulate the lipopolysaccharide (LPS)-induced dynamics of HMGB1 and its mediated cascade in inflammatory responses in human umbilical vein endothelial cells (HUVECs). In this experiment, JB and HMGB1 were administered in that order. In vitro and in vivo permeability, and cell viability, adhesion, and excavation of leukocytes, development of cell adhesion molecules, and lastly production of proinflammatory substances were investigated on human endothelial cells and mouse disease models to investigate the efficacy of JB in inflammatory condition. JB substantially blocked the translocation of HMGB1 from HUVECs and controlled HMGB1-induced adhesion and extravasation of the neutrophils through LPS-treated HUVECs. Moreover, JB decreased the formation of HMGB1 receptors and continually prevented HMGB1-induced proinflammatory mechanisms by blocking transcription of nuclear factor-κB and synthesis of tumor necrosis factor-α. In conclusion, JB demonstrated preventive effects against inflammatory pathologies and showed the potential to be a candidate substance for various inflammatory diseases by regulating HMGB1-mediated cellular signaling.

Epigenetic BET reader inhibitor apabetalone (RVX-208) counters proinflammatory aortic gene expression in a diet induced obesity mouse model and in human endothelial cells

Background and aimsObese patients are at risk for type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD). A lipid-rich diet promotes arterial changes by inducing hypertension, oxidative stress, and inflammation. Bromodomain and extraterminal (BET) proteins contribute to endothelial and immune cell activation in vitro and in atherosclerosis mouse models. We aim to determine if BET inhibition can reduce lipid-rich diet-induced vascular inflammation in mice. MethodsBody weight, serum glucose and lipid levels were measured in mice fed a high-fat diet (HFD) or low-fat diet (LFD) for 6 weeks and at study termination. BET inhibitors apabetalone and JQ1 were co-administered with the HFD for additional 16 weeks. Aortic gene expression was analyzed post necropsy by PCR, Nanostring nCounter® Inflammation Panel and bioinformatics pathway analysis. Transcription changes and BRD4 chromatin occupancy were analyzed in primary human endothelial cells in response to TNFα and apabetalone. ResultsHFD induced weight gain, visceral obesity, high fasting blood glucose, glucose intolerance and insulin resistance compared to LFD controls. HFD upregulated the aortic expression of 47 genes involved in inflammation, innate immunity, cytoskeleton and complement pathways. Apabetalone and JQ1 treatment reduced HFD-induced aortic expression of proinflammatory genes. Congruently, bioinformatics predicted enhanced signaling by TNFα in the HFD versus LFD aorta, which was countered by BETi treatment. TNFα-stimulated human endothelial cells had increased expression of HFD-sensitive genes and higher BRD4 chromatin occupancy, which was countered by apabetalone treatment. ConclusionsHFD induces vascular inflammation in mice through TNFα signaling. Apabetalone treatment reduces this proinflammatory phenotype, providing mechanistic insight into how BET inhibitors may reduce CVD risk in obese patients.

Abstract 12127: Protein Disulfide Isomerase A1 as Novel Redox Sensor in VEGFR2 Signaling and Angiogenesis

Background: VEGFR2 signaling in endothelial cells (ECs) is regulated by reactive oxygen species (ROS) derived from NADPH oxidases (NOXs) and mitochondria, which plays an important role in postnatal angiogenesis. However, it remains unclear how highly diffusible ROS signal enhances VEGFR2 signaling and reparative angiogenesis. Protein disulfide isomerase A1 (PDIA1) functions as an oxidoreductase depending on the redox environment. However, role of PDIA1 in ROS-dependent VEGFR2 signaling and angiogenesis is entirely unknown. Results: Here we showed that PDIA1 co-immunoprecipitated with VEGFR2 or colocalized with either VEGFR2 or early endosome marker Rab5, but not late endosome marker Rab7, at the perinuclear region in human ECs in response to VEGF. PDIA1 silencing significantly reduced VEGF-induced EC migration (43.3%), proliferation (52.8%) and spheroid sprouting via inhibiting VEGFR2 signaling. Mechanistically, VEGF stimulation rapidly increased Cys-OH formation of PDIA1 via the NOX4-mitochondrial ROS axis. Experiments using “redox-dead” mutant PDIA1 with replacement of the active four Cys residues with Ser revealed that VEGF-induced PDIA1 CysOH formation promoted angiogenic responses by increasing phosphorylation of VEGFR2 at Y1175 via oxidative inactivation of PTP1B. In vivo, Pdia1+/- mice showed impaired angiogenesis in developmental retina and Matrigel plug models as well as ex vivo aortic ring sprouting model. Hindlimb ischemia model revealed that PDIA1 expression was markedly increased in angiogenic CD31 positive ECs of ischemic muscles, and that limb perfusion recovery and neovascularization (48%) in response to ischemic injury were significantly impaired in EC-specific Pdia1 conditional knockout mice. Conclusion: PDIA1 can sense VEGF-induced H2O2 signal via CysOH formation to promote VEGFR2 signaling and angiogenesis in ECs via oxidative inactivation of PTP1B, thereby enhancing postnatal angiogenesis. The oxidized PDIA1 is a potential therapeutic target for treatment of ischemic vascular diseases

Complement Regulation in Immortalized Fibroblast-like Synoviocytes and Primary Human Endothelial Cells in Response to SARS-CoV-2 Nucleocapsid Protein and Pro-Inflammatory Cytokine TNFα.

Background: Case reports are available showing that patients develop symptoms of acute arthritis during or after recovery from SARS-CoV-2 infection. Since the interrelation is still unknown, our aim was to study the impact of the SARS-CoV-2 nucleocapsid protein (NP) on human fibroblast-like synoviocytes and human endothelial cells (hEC) in terms of complement and cytokine regulation. Methods: Non-arthritic (K4IM) synoviocyte, arthritic (HSE) synoviocyte cell lines and primary hEC were stimulated with recombinant NP and/or TNFα. Analyses of cell viability, proliferation, gene and protein expression of cytokines and complement factors were performed. Results: NP suppressed significantly the vitality of hEC and proliferation of HSE. NP alone did not induce any significant changes in the examined gene expressions. However, NP combined with TNFα induced significantly higher TNFα in HSE and K4IM as well as higher IL-6 and CD55 gene expression in HSE and suppressed C3aR1 gene expression in hEC. HSE proliferated twice as fast as K4IM, but showed significantly lesser gene expressions of CD46, CD55, CD59 and TNFα with significantly higher IL-6 gene expression. CD35 gene expression was undetectable in K4IM, HSE and hEC. Conclusions: NP might contribute in combination with other inflammatory factors to complement regulation in arthritis.

Pharmacologic modulation of intracellular Na+ concentration with ranolazine impacts inflammatory response in humans and mice

Changes in Ca2+ influx during proinflammatory stimulation modulates cellular responses, including the subsequent activation of inflammation. Whereas the involvement of Ca2+ has been widely acknowledged, little is known about the role of Na+. Ranolazine, a piperazine derivative and established antianginal drug, is known to reduce intracellular Na+ as well as Ca2+ levels. In stable coronary artery disease patients (n = 51) we observed reduced levels of high-sensitive C-reactive protein (CRP) 3 mo after the start of ranolazine treatment (n = 25) as compared to the control group. Furthermore, we found that in 3,808 acute coronary syndrome patients of the MERLIN-TIMI 36 trial, individuals treated with ranolazine (1,934 patients) showed reduced CRP values compared to placebo-treated patients. The antiinflammatory effects of sodium modulation were further confirmed in an atherosclerotic mouse model. LDL-/- mice on a high-fat diet were treated with ranolazine, resulting in a reduced atherosclerotic plaque burden, increased plaque stability, and reduced activation of the immune system. Pharmacological Na+ inhibition by ranolazine led to reduced express of adhesion molecules and proinflammatory cytokines and reduced adhesion of leukocytes to activated endothelium both invitro and invivo. We demonstrate that functional Na+ shuttling is required for a full cellular response to inflammation and that inhibition of Na+ influx results in an attenuated inflammatory reaction. In conclusion, we demonstrate that inhibition of Na+-Ca2+ exchange during inflammation reduces the inflammatory response in human endothelial cells invitro, in a mouse atherosclerotic disease model, and in human patients.

Exercise improves angiogenic function of circulating exosomes in type 2 diabetes: Role of exosomal SOD3.

Exosomes, key mediators of cell-cell communication, derived from type 2 diabetes mellitus (T2DM) exhibit detrimental effects. Exercise improves endothelial function in part via the secretion of exosomes into circulation. Extracellular superoxide dismutase (SOD3) is a major secretory copper (Cu) antioxidant enzyme that catalyzes the dismutation of O2•- to H2 O2 whose activity requires the Cu transporter ATP7A. However, the role of SOD3 in exercise-induced angiogenic effects of circulating plasma exosomes on endothelial cells (ECs) in T2DM remains unknown. Here, we show that both SOD3 and ATP7A proteins were present in plasma exosomes in mice, which was significantly increased after two weeks of volunteer wheel exercise. A single bout of exercise in humans also showed a significant increase in SOD3 and ATP7A protein expression in plasma exosomes. Plasma exosomes from T2DM mice significantly reduced angiogenic responses in human ECs or mouse skin wound healing models, which was associated with a decrease in ATP7A, but not SOD3 expression in exosomes. Exercise training in T2DM mice restored the angiogenic effects of T2DM exosomes in ECs by increasing ATP7A in exosomes, which was not observed in exercised T2DM/SOD3-/- mice. Furthermore, exosomes overexpressing SOD3significantly enhanced angiogenesis in ECs by increasing local H2 O2 levels in a heparin-binding domain-dependent manner as well as restored defective wound healing and angiogenesis in T2DM or SOD3-/- mice. In conclusion, exercise improves the angiogenic potential of circulating exosomes in T2DM in a SOD3-dependent manner. Exosomal SOD3may provide an exercise mimetic therapy that supports neovascularization and wound repair in cardiometabolic disease.

Response of human endothelial cells to the vortex flow in an immediately expanding flow chamber

Response of human endothelial cells to the vortex flow in an immediately expanding flow chamber

Porphyromonas gingivalis Gingipains-Mediated Degradation of Plasminogen Activator Inhibitor-1 Leads to Delayed Wound Healing Responses in Human Endothelial Cells

Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, is constitutively produced by endothelial cells and plays a vital role in maintaining vascular homeostasis. Chronic periodontitis is an inflammatory disease characterized by bleeding of periodontal tissues that support the tooth. In this study, we aimed to determine the role of PAI-1 produced by endothelial cells in response to infections caused by the primary periodontal pathogen Porphyromonas gingivalis. We demonstrated that P. gingivalis infection resulted in significantly reduced PAI-1 levels in human endothelial cells. This reduction in PAI-1 levels could be attributed to the proteolysis of PAI-1 by P. gingivalis proteinases, especially lysine-specific gingipain-K (Kgp). We demonstrated the roles of these degradative enzymes in the endothelial cells using a Kgp-specific inhibitor and P. gingivalis gingipain-null mutants, in which the lack of the proteinases resulted in the absence of PAI-1 degradation. The degradation of PAI-1 by P. gingivalis induced a delayed wound healing response in endothelial cell layers via the low-density lipoprotein receptor-related protein. Our results collectively suggested that the proteolysis of PAI-1 in endothelial cells by gingipains of P. gingivalis might lead to the deregulation of endothelial homeostasis, thereby contributing to the permeabilization and dysfunction of the vascular endothelial barrier.

Effect of cellular communication network factor 2/connective tissue growth factor on tube formation by endothelial cells derived from human periodontal ligaments

ObjectivesTo clarify the role of cellular communication network factor 2/connective tissue growth factor (CCN2/CTGF) in periodontal tissue regeneration by investigating, the proliferative and tubulogenic responses of human endothelial cells obtained from the periodontal ligament to CCN2/CTGF. DesignEndothelial cells were seeded on agar gel medium with or without 50 ng/mL recombinant CCN2/CTGF (rCCN2/CTGF) and cultured for 6 h. Cells were morphologically and phenotypically analyzed by immunofluorescent microscopy. A colorimetric assay was used to evaluate cell proliferation, and transmission electron microscopy (TEM) was used for ultrastructural analysis. ResultsThe proliferation of endothelial cells was best promoted by rCCN2/CTGF at 50 ng/mL. In the control group, tube formation was not observed within 6 h. In contrast, endothelial cells seeded on the agar with 50 ng/mL rCCN2/CTGF clearly showed proliferation with network formation. Under a two-dimensional culture condition, a dense network of endothelial cells was not constructed on the plastic bottom. However, drastic morphological change was observed in the endothelial cells on the agar containing rCCN2/CTGF. The endothelial cells in the dense network were interconnected with each other and showed a tube-like structure. Tight junctions or adherens junctions were observed between the adjoining endothelial cells in the dense network. ConclusionsCCN2/CTGF was found to promote the proliferation and tubulogenesis of endothelial cells from the periodontal ligament. These results suggest that CCN2/CTGF may contribute to the regeneration of damaged periodontal tissue by activating the remaining endothelial cells.

Spike protein 1 of SARS‐CoV‐2 induces an immune and inflammatory response and increases expression of ACE2 in human endothelial cells

Introduction Interferon (IFN) alpha (IFNα) and lambda3 (IFNλ3) constitute the first line of immunity against viral infection by increasing expression of interferon-stimulated genes (ISGs). Both are produced in SARS-CoV-2 infection and have been considered therapeutically in COVID-19. In epithelial cells IFNs have been shown to influence the expression of angiotensin-converting enzyme 2 (ACE2), the receptor for S-protein (S1P) of SARS-CoV-2. ACE2 is part of the vascular renin angiotensin system and may be important in COVID-19-associated endotheliitis. Whether SP1 influences immune responses in endothelial cells unknown. We investigated effects of SP1 on ACE2 expression and immune responses in human endothelial cells. Methods Culturedhuman Microvascular, Lymphatic, Aortic and Pulmonary Endothelial Cells (MEC, LEC, AEC, and PEC respectively) were stimulated with SP1 of SARS-CoV-2 (1µg/106 cells), IFNα (100ng/mL) or IFNλ3 (100IU/mL). Because ACE2, metalloproteinase domain 17 (ADAM17) and type II transmembrane serine protease (TMPRSS2) are important for SARS-CoV-2 infection, cells were treated with inhibitors of ADAM17 (marimastat, 3.8nM and TAPI-1, 100nM), ACE2 (MLN4760, 440pM), and TMPRSS2 (camostat, 50µM). Expression of ISGs (ISG15, IFIT1, and MX1) was investigated by real-time PCR (5h), cytokine by ELISA and protein expression by immunoblotting (24h). Results AEC, LEC, MEC, and PEC stimulated with SP1 exhibited increased expression of ISGs: ISG15 (1.9-2.3-fold), IFIT1 (2.0-7.9-fold), MX1 (3.1-5.8-fold), indicating activation of the IFN pathway. MEC and LEC exhibited higher responses to IFNα activation. MEC (ISG15: 16-fold, IFIT1: 21-fold, MX1: 31-fold) and LEC (ISG15: 48-fold, IFIT1: 105-fold, MX1: 134-fold). Only MEC exhibited ISG expression induced by IFNλ3 (ISG15: 1.7-fold, IFIT1: 1.9-fold, MX1: 1.7-fold) (P<0.05). IL-6 production was increased in MEC stimulated with IFNα (1230pg/mL) and IFNλ3 (1124pg/mL) vs control (591pg/mL). Marimastat, but not camostat or MLN4760 inhibited SP1 effects. IFNα induced IL-6 production in AEC and PEC (178pg/mL vs control 109pg/mL). No changes in IL-6 were observed in LEC. IFNα increased expression of the short form of ACE2 (75kDa) (63%), ADAM17 (36%), and TMPRSS2 (65%) in MECs. This was associated with increased activation of pro-inflammatory pathways with increased phosphorylation of Stat1 (134%), Stat2 (102%), ERK1/2 (42%). IFNλ3 also increased phosphorylation of Stat2 (60%) and ERK1/2 (75%). In MEC, phosphorylation of the eNOS activation motif Ser1177 was reduced by IFNLα and (40%) and IFNλ3 (40%). Conclusions In human endothelial cells from multiple vascular beds, SP1, IFNα and IFNλ3 induced an immune response characterised by increased ISG expression and IL-6 production, processes that involve ADAM17. These effects were associated with increased expression of the short form of ACE2 and activation of pro-inflammatory pathways. Our novel findings demonstrate that SP-1 induces an endothelial immune and inflammatory response that may be important in endotheliitis associated with COVID-19.

Longitudinal shear stress response in human endothelial cells to atheroprone and atheroprotective conditions

The two main blood flow patterns, namely, pulsatile shear (PS) prevalent in straight segments of arteries and oscillatory shear (OS) observed at branch points, are associated with atheroprotective (healthy) and atheroprone (unhealthy) vascular phenotypes, respectively. The effects of blood flow-induced shear stress on endothelial cells (ECs) and vascular health have generally been studied using human umbilical vein endothelial cells (HUVECs). While there are a few studies comparing the differential roles of PS and OS across different types of ECs at a single time point, there is a paucity of studies comparing the temporal responses between different EC types. In the current study, we measured OS and PS transcriptomic responses in human aortic endothelial cells (HAECs) over 24 h and compared these temporal responses of HAECs with our previous findings on HUVECs. The measurements were made at 1, 4, and 24 h in order to capture the responses at early, mid, and late time points after shearing. The results indicate that the responses of HAECs and HUVECs are qualitatively similar for endothelial function-relevant genes and several important pathways with a few exceptions, thus demonstrating that HUVECs can be used as a model to investigate the effects of shear on arterial ECs, with consideration of the differences. Our findings show that HAECs exhibit an earlier response or faster kinetics as compared to HUVECs. The comparative analysis of HAECs and HUVECs presented here offers insights into the mechanisms of common and disparate shear stress responses across these two major endothelial cell types.

Aggregatibacter actinomycetemcomitans induces a proatherosclerotic response in human endothelial cells in a three‐dimensional collagen scaffold model

AbstractThe role of periodontopathogens in inflammatory endothelial dysfunction is not known. This study characterizes a three‐dimensional model with human coronary artery endothelial cells on three‐dimensional (HCAEC‐3D) type I collagen scaffolds to evaluate whether infection with Aggregatibacter actinomycetemcomitans induces a proinflammatory response associated with atherosclerosis. The HCAEC‐3D culture was physicochemically characterized with regard to biocompatibility and barrier function. Then, the culture was infected with A. actinomycetemcomitans strain ATCC 29522 at multiplicities of infection (MOIs) of 1:1, 1:10, and 1:100. Cultures without infection and stimulated with A. actinomycetemcomitans lipopolysaccharide were used as controls. The secretion of soluble factors (IL‐6, IL‐1β, MCP‐1, RANTES, MIP‐1, IL‐8, IL‐1α, and TNF‐α) was evaluated via flow cytometry; TGF‐β1 was evaluated via enzyme‐linked immunosorbent assay (ELISA). The adhesion and migration of fluorescent human THP‐1 monocytes was evaluated. IL‐8, MCP‐1, and IL‐6 secretion increased in a dose‐dependent manner with A. actinomycetemcomitans infection and was significantly greater than that under control treatment. The concentration of TGF‐β1 was significantly higher at MOI 1:100 than in controls. Treatment of the 3D cultures with A. actinomycetemcomitans at different MOIs induced significant differences in the adhesion of monocytes to the endothelium compared to the control without infection. Lastly, conditioned media from 3D cultures treated with A. actinomycetemcomitans induced monocyte migration. The effects of IL‐8, MCP‐1, IL‐6, and TGF‐β1 on the endothelium indicate the ability of A. actinomycetemcomitans to induce an inflammatory response through a mechanism of monocyte adhesion and migration and endothelial dysfunction.

Sex Differences in the Pro-Angiogenic Response of Human Endothelial Cells: Focus on PFKFB3 and FAK Activation.

Female hormones and sex-specific factors are established determinants of endothelial function, yet their relative contribution to human endothelium phenotypes has not been defined. Using human umbilical vein endothelial cells (HUVECs) genotyped by donor's sex, we investigated the influence of sex and estrogenic agents on the main steps of the angiogenic process and on key proteins governing HUVEC metabolism and migratory properties. HUVECs from female donors (fHUVECs) showed increased viability (p < 0.01) and growth rate (p < 0.01) compared with those from males (mHUVECs). Despite higher levels of G-protein coupled estrogen receptor (GPER) in fHUVECs (p < 0.001), treatment with 17β-estradiol (E2) and the selective GPER agonist G1 (both 1–100 nM) did not affect HUVEC viability. Migration and tubularization in vitro under physiological conditions were higher in fHUVECs than in mHUVECs (p < 0.05). E2 treatment (1–100 nM) upregulated the glycolytic activator PFKFB3 with higher potency in fHUVECs than in mHUVECs, despite comparable baseline levels. Moreover, Y576/577 phosphorylation of focal adhesion kinase (FAK) was markedly enhanced in fHUVECs (p < 0.001), despite comparable Src activation levels. While the PI3K inhibitor LY294002 (25 µM) inhibited HUVEC migration (p < 0.05), Akt phosphorylation levels in fHUVECs and mHUVECs were comparable. Finally, digitoxin treatment, which inhibits Y576/577 FAK phosphorylation, abolished sexual dimorphism in HUVEC migration. These findings unravel complementary modulation of HUVEC functional phenotypes and signaling molecules involved in angiogenesis by hormone microenvironment and sex-specific factors, and highlight the need for sex-oriented pharmacological targeting of endothelial function.

Vascular endothelial cells evade complement‐mediated membrane injury via Weibel‐Palade body mobilization

BackgroundDefective complement inhibition can lead to the formation of membrane attack complexes (MAC; C5b‐9) on the plasma membranes of vascular endothelial cells, resulting in injury that drives the progression of thrombotic microangiopathy (TMA), a key pathology in kidney disease. Objective/MethodsWe examined the response of human endothelial cells to complement‐mediated damage using blood outgrowth endothelial cells (BOECs) derived from healthy donors. BOECs were sensitized to complement factors present in normal human serum to induce the formation of C5b‐9 on their plasma membranes. ResultsThis triggered an expected abrupt rise in intracellular Ca2+ reflecting membrane leakage. Remarkably, while intracellular Ca2+ remained elevated, membrane leakage ceased within 30 minutes, and cells did not show significant death. Extensive mobilization of Weibel‐Palade bodies (WPBs) was observed along with secretion of von Willebrand factor (VWF). The potential role of WPBs and VWF in mitigating complement‐mediated damage was examined by comparing the effects of C5b‐9 on BOECs derived from von Willebrand disease (VWD) patients expressing reduced amounts of VWF, lacking expression of functional VWF, or lacking both VWF and WPBs. BOECs lacking WPBs were not resistant to complement‐mediated damage, but became resistant when transfected to express VWF (and thus WPBs). ConclusionWe conclude that BOECs exposed to C5b‐9 attack respond by mobilizing WPBs, which mitigate and repair damage by fusing with the plasma membrane. We propose that a similar cell‐specific response may protect the vascular endothelium from complement‐mediated damage in vivo.