Response Of Microvascular Endothelial Cells Research Articles

Candidate Signature miRNAs from Secreted miRNAome of Human Lung Microvascular Endothelial Cells in Response to Different Oxygen Conditions: A Pilot Study.

Oxygen conditions in the lung determine downstream organ functionality by setting the partial pressure of oxygen, regulating the redox homeostasis and by activating mediators in the lung that can be propagated in the blood stream. Examples for such mediators are secreted soluble or vesicle-bound molecules (proteins and nucleic acids) that can be taken up by remote target cells impacting their metabolism and signaling pathways. MicroRNAs (miRNAs) have gained significant interest as intercellular communicators, biomarkers and therapeutic targets in this context. Due to their high stability in the blood stream, they have also been attributed a role as "memory molecules" that are able to modulate gene expression upon repeated (stress) exposures. In this study, we aimed to identify and quantify released miRNAs from lung microvascular endothelial cells in response to different oxygen conditions. We combined next-generation sequencing (NGS) of secreted miRNAs and cellular mRNA sequencing with bioinformatic analyses in order to delineate molecular events on the cellular and extracellular level and their putative interdependence. We show that the identified miRNA networks have the potential to co-mediate some of the molecular events, that have been observed in the context of hypoxia, hyperoxia, intermittent hypoxia and intermittent hypoxia/hyperoxia.

Calcium-activated Potassium Channels as Amplifiers of TRPV4-mediated Pulmonary Edema Formation in Male Mice.

As a mechanosensitive cation channel and key regulator of vascular barrier function, endothelial transient receptor potential vanilloid type 4 (TRPV4) contributes critically to ventilator-induced lung injury and edema formation. Ca2+ influx via TRPV4 can activate Ca2+-activated potassium (KCa) channels, categorized into small (SK1-3), intermediate (IK1), and big (BK) KCa, which may in turn amplify Ca2+ influx by increasing the electrochemical Ca2+ gradient and thus promote lung injury. The authors therefore hypothesized that endothelial KCa channels may contribute to the progression of TRPV4-mediated ventilator-induced lung injury. Male C57Bl/6J mice were ventilated for 2 h with low or high tidal volumes in the presence or absence of the nonselective KCa antagonists apamin and charybdotoxin or the selective IK1 antagonist TRAM34. Lung injury was similarly assessed in overventilated, endothelial-specific TRPV4-deficient mice or TRAM34-treated C57Bl/6J mice challenged with intratracheal acid installation. Changes in intracellular calcium Ca2+ concentration ([Ca2+]i) were monitored by real-time imaging in isolated-perfused lungs in response to airway pressure elevation or in human pulmonary microvascular endothelial cells in response to TRPV4 activation with or without inhibition of KCa channels. Analogously, changes in intracellular potassium concentration ([K+]i) and membrane potential were imaged in vitro. Endothelial TRPV4 deficiency or inhibition of KCa channels, and most prominently inhibition of IK1 by TRAM34, attenuated ventilator-induced lung injury as demonstrated by reduced lung edema, protein leak, and quantitative lung histology. All KCa antagonists reduced the [Ca2+]i response to mechanical stimulation or direct TRPV4 activation in isolated lungs. TRAM34 and charybdotoxin yet not apamin prevented TRPV4-induced potassium efflux and membrane hyperpolarization in human pulmonary microvascular endothelial cells. TRAM34 also attenuated the TRPV4 agonist-induced Ca2+ influx in vitro and reduced acid-induced lung injury in vivo. KCa channels, specifically IK1, act as amplifiers of TRPV4-mediated Ca2+ influx and establish a detrimental feedback that promotes barrier failure and drives the progression of ventilator-induced lung injury.

Organotypic heterogeneity in microvascular endothelial cell responses in sepsis-a molecular treasure trove and pharmacological Gordian knot.

In the last decades, it has become evident that endothelial cells (ECs) in the microvasculature play an important role in the pathophysiology of sepsis-associated multiple organ dysfunction syndrome (MODS). Studies on how ECs orchestrate leukocyte recruitment, control microvascular integrity and permeability, and regulate the haemostatic balance have provided a wealth of knowledge and potential molecular targets that could be considered for pharmacological intervention in sepsis. Yet, this information has not been translated into effective treatments. As MODS affects specific vascular beds, (organotypic) endothelial heterogeneity may be an important contributing factor to this lack of success. On the other hand, given the involvement of ECs in sepsis, this heterogeneity could also be leveraged for therapeutic gain to target specific sites of the vasculature given its full accessibility to drugs. In this review, we describe current knowledge that defines heterogeneity of organ-specific microvascular ECs at the molecular level and elaborate on studies that have reported EC responses across organ systems in sepsis patients and animal models of sepsis. We discuss hypothesis-driven, single-molecule studies that have formed the basis of our understanding of endothelial cell engagement in sepsis pathophysiology, and include recent studies employing high-throughput technologies. The latter deliver comprehensive data sets to describe molecular signatures for organotypic ECs that could lead to new hypotheses and form the foundation for rational pharmacological intervention and biomarker panel development. Particularly results from single cell RNA sequencing and spatial transcriptomics studies are eagerly awaited as they are expected to unveil the full spatiotemporal signature of EC responses to sepsis. With increasing awareness of the existence of distinct sepsis subphenotypes, and the need to develop new drug regimen and companion diagnostics, a better understanding of the molecular pathways exploited by ECs in sepsis pathophysiology will be a cornerstone to halt the detrimental processes that lead to MODS.

Altered gene expression in human brain microvascular endothelial cells in response to the infection of influenza H1N1 virus

Influenza viruses not only cause respiratory illness, but also have been reported to elicit neurological manifestations following acute viral infection. The central nervous system (CNS) has a specific defense mechanism against pathogens structured by cerebral microvasculature lined with brain endothelial cells to form the blood–brain barrier (BBB). To investigate the response of human brain microvascular endothelial cells (hBMECs) to the Influenza A virus (IAV), we inoculated the cells with the A/WSN/33 (H1N1) virus. We then conducted an RNAseq experiment to determine the changes in gene expression levels and the activated disease pathways following infection. The analysis revealed an effective activation of the innate immune defense by inducing the pattern recognition receptors (PRRs). Along with the production of proinflammatory cytokines, we detected an upregulation of interferons and interferon-stimulated genes, such as IFN-β/λ, ISG15, CXCL11, CXCL3 and IL-6, etc. Moreover, infected hBMECs exhibited a disruption in the cytoskeletal structure both on the transcriptomic and cytological levels. The RNAseq analysis showed different pathways and candidate genes associated with the neuroactive ligand-receptor interaction, neuroinflammation, and neurodegenerative diseases, together with a predicted activation of the neuroglia. Likewise, some genes linked with the mitochondrial structure and function displayed a significantly altered expression. En masse, this data supports that hBMECs could be infected by the IAV, which induces the innate and inflammatory immune response. The results suggest that the influenza virus infection could potentially induce a subsequent aggravation of neurological disorders.

Mechano-biological phenotype in post-infarction cardiac remodeling

Ischemic heart disease is one of the leading cause of death in developed countries. In 30% of patients with heart infarction, inadequate cardiac fibrosis leads to terminal failure. We believe that local mechanical forces (inherent to the pump function of the heart) after cardiac infarction play a key role in the outcome of cardiac remodeling, in particular by modifying the response of microvascular endothelial cells (MECs), main players accompanying myocardial healing processes. Dynamic data, accessible by ultrasound, are however not widely used in the clinic, and could help to understand why patients remain refractory to treatments for heart failure. We aimed at studying the mechano-biological coupling that exists between mechanical stress applied to ischemic lesions, and the local biological response of cardiac cells. Using a mouse model of myocardial infarction (coronary artery ligation), we analyzed heart deformation using dynamic ultrasound imaging 1 month post-infarction (speckle tracking, VevoLabs). Mice were then sacrificed, and the hearts were harvested and processed for immunohistochemical analysis. Multiparametric mechanical maps (strain, displacement and velocity) were obtained using a morphometry program developed in the laboratory, and compared to a spatial transcriptomic analysis in the corresponding section planes (Visium, 10X genomics), in 4 infarcted heart of various size and localization. The transcriptomic data were validated in situ by immunofluorescence, in contiguous mouse heart sections ( Fig. 1 ). Generated big data highlight a panel of transcripts which expression highly correlates with radial strain and displacement of the heart wall. Among these factors, neuregulin-1 (involved in cardiac remodeling), and TRPC6 or PECAM-1 (sensors of mechanical forces) were found highly expressed in the mouse heart sections (immunofluorescence). Neuregulin-1 and TRPC6 strongly co-localized with MECs and coronary artery SMCs bordering the infarcted area, while PECAM-1 activation (phosphorylation) in MECs was found highly correlated with radial strain. These results support a mechano-biological coupling in infarcted hearts, modulating the expression of factors involved in its repair. Understanding the biology associated with mechanical stress could improve the management of patients after a heart attack, on the sole basis of dynamic imaging.

Renal microvascular endothelial cell responses in sepsis-induced acute kidney injury.

Microvascular endothelial cells in the kidney have been a neglected cell type in sepsis-induced acute kidney injury (sepsis-AKI) research; yet, they offer tremendous potential as pharmacological targets. As endothelial cells in distinct cortical microvascular segments are highly heterogeneous, this Review focuses on endothelial cells in their anatomical niche. In animal models of sepsis-AKI, reduced glomerular blood flow has been attributed to inhibition of endothelial nitric oxide synthase activation in arterioles and glomeruli, whereas decreased cortex peritubular capillary perfusion is associated with epithelial redox stress. Elevated systemic levels of vascular endothelial growth factor, reduced levels of circulating sphingosine 1-phosphate and loss of components of the glycocalyx from glomerular endothelial cells lead to increased microvascular permeability. Although coagulation disbalance occurs in all microvascular segments, the molecules involved differ between segments. Induction of the expression of adhesion molecules and leukocyte recruitment also occurs in a heterogeneous manner. Evidence of similar endothelial cell responses has been found in kidney and blood samples from patients with sepsis. Comprehensive studies are needed to investigate the relationships between segment-specific changes in the microvasculature and kidney function loss in sepsis-AKI. The application of omics technologies to kidney tissues from animals and patients will be key in identifying these relationships and in developing novel therapeutics for sepsis.

Endothelial Dysfunction and Impaired Neurovascular Coupling Responses Precede Cognitive Impairment in a Mouse Model of Geriatric Sepsis.

Sepsis is a life-threatening condition, the incidence of which is significantly increased in elderly patients. One of the long-lasting effects of sepsis is cognitive impairment defined as a new deficit or exacerbation of preexisting deficits in global cognition or executive function. Normal brain function is dependent on moment-to-moment adjustment of cerebral blood flow to match the increased demands of active brain regions. This homeostatic mechanism, termed neurovascular coupling (NVC, also known as functional hyperemia), is critically dependent on the production of vasodilator NO by microvascular endothelial cells in response to mediators released from activated astrocytes. The goal of this study was to test the hypothesis that sepsis in aging leads to impairment of NVC responses early after treatment and that this neurovascular dysfunction associates with impairments in cognitive performance and vascular endothelial dysfunction. To test this hypothesis, we used a commonly studied bacterial pathogen, Listeria monocytogenes, to induce sepsis in experimental animals (males, 24 months of age) and subjected experimental animals to a standard clinical protocol of 3 doses of ampicillin i.p. and 14 days of amoxicillin added to the drinking water. NVC responses, endothelial function and cognitive performance were measured in septic and age-matched control groups within 14 days after the final antibiotic treatment. Our data demonstrate that sepsis in aging significantly impairs NVC responses measured in somatosensory cortex during whisker stimulation, significantly impairs endothelial function in isolated and pressure cannulated aorta rings in response to acetylcholine stimulation. No significant impairment of cognitive function in post-sepsis aged animals has been observed when measured using the PhenoTyper homecage based system. Our findings suggest that sepsis-associated endothelial dysfunction and impairment of NVC responses may contribute to long-term cognitive deficits in older sepsis survivors.

Transient exposure to elevated glucose levels causes persistent changes in dermal microvascular endothelial cell responses to injury.

BackgroundThe purpose of this study was to determine whether elevated glucose can induce a dermal microvascular endothelial cell metabolic memory, thus affecting angiogenesis in the repair process of mammalian cutaneous wound. We hypothesized that transient elevated glucose levels cause sustained alteration of endothelial cell responses to injury and persistent epigenetic changes in gene expression.MethodsHuman dermal microvascular endothelial cells were exposed to experimental conditions with or without 30 mM D-glucose. The control group was maintained at 5 mM D-glucose; while in the transient glucose group, after being exposed to 30 mM D-glucose for two days, then being put under the control conditions during the experiment. Besides, in the whole process of the experiment, the chronic glucose group was kept in the condition with 30 mM D-glucose. Proliferation, migration, tube formation, gene expression and histone methylation were assessed for individual conditions.ResultsTransient elevated glucose caused sustained effects on endothelial cell migration, tube formation and TIMP3 gene expression. The effects on TIMP3 expression were associated with persistent changes in histone modification at the 5' end of the TIMP3 gene, suggesting an epigenetic effect.ConclusionsHyperglycemia induced metabolic memory could promote the regulation of TIMP3, and it can be used as a possible innovative molecular target for therapeutic intervention in the treatment of chronic non-healing diabetic wounds.

ZNF667 attenuates leukocyte-endothelial adhesion via downregulation of P-selectin in skin flap following remote limb ischemic preconditioning.

We assessed the effects and potential mechanism of romote ischemic preconditioning (RIPC) on leukocytes-endothelium cell adhesion in the flap microvessel after ischemia-reperfusion (I/R) injury. Eight hours after reperfusion, edema and intravascular leukocyte aggregation were reduced and microvessels were more obvious in the group with superficial inferior epigastric artery (SIEA) perforator flap (SIEA-flap) subjected to RIPC than in the I/R group. Zinc finger protein 667 (ZNF667) was significantly increased but P-selectin was decreased in the flaps subjected to RIPC, compared to those in the I/R group. The low expression of P-selectin was associated with ZNF667 expression and activation in human dermal microvascular endothelial cells in response to hypoxic preconditioning. ZNF667 bound to the P-selectin promoter region, suppressing its transcription through a special core sequence. The ablation of P-selectin by small interfering RNA effectively prevented the leukocytes-endothelium cell adhesion effect of ZNF667-knockdown. ZNF667 upregulation attenuates leukocyte-endothelial cell adhesion by negatively regulating the expression of P-selectin in SIEA-flap subjected to RIPC.

Shear Stress Rescued the Neuronal Impairment Induced by Global Cerebral Ischemia Reperfusion via Activating PECAM-1-eNOS-NO Pathway.

Microvessel hypoperfusion following ischemic stress resulted in a decreased shear stress of brain microvascular endothelial cells (BMECs) and contributed to abnormal expression of PECAM-1 after global cerebral ischemia/reperfusion (I/R) injury. Here, we identified novel pathophysiologic and rehabilitative procedures specific to shear stress in microvascular endothelial cells in response to global cerebral I/R injury. We found that the decrease in cerebral blood flow of gerbils after global cerebral I/R injury reduces shear stress, and the abnormal change in shear stress leads to microvascular endothelial cell and neuron damage. Nevertheless, suitable high levels of shear stress contribute to rescuing the dysfunction and malformation of BMECs via regulating the PECAM-1-eNOS-NO pathway to enhance nitric oxide release, decrease the expression of caspase-3 to reduce apoptosis, and improve the shear-adaptability of endothelial cells, thereby playing a protective role in the gerbil brain.

Advanced glycation end-products disrupt brain microvascular endothelial cell barrier: The role of mitochondria and oxidative stress

During diabetes mellitus, advanced glycation end-products (AGEs) are major contributors to the development of alterations in cerebral capillaries, leading to the disruption of the blood-brain barrier (BBB). Consequently, this is often associated with an amplified oxidative stress response in microvascular endothelial cells. As a model to mimic brain microvasculature, the bEnd.3 endothelial cell line was used to investigate cell barrier function. Cells were exposed to native bovine serum albumin (BSA) or modified BSA (BSA-AGEs). In the presence or absence of the antioxidant compound, N-acetyl-cysteine, cell permeability was assessed by FITC-dextran exclusion, intracellular free radical formation was monitored with H2DCF-DA probe, and mitochondrial respiratory and redox parameters were analyzed. We report that, in the absence of alterations in cell viability, BSA-AGEs contribute to an increase in endothelial cell barrier permeability and a marked and prolonged oxidative stress response. Decreased mitochondrial oxygen consumption was associated with these alterations and may contribute to reactive oxygen species production. These results suggest the need for further research to explore therapeutic interventions to restore mitochondrial functionality in microvascular endothelial cells to improve brain homeostasis in pathological complications associated with glycation.

Metabolic pathway alterations in microvascular endothelial cells in response to hypoxia.

The vasculature within a tumor is highly disordered both structurally and functionally. Endothelial cells that comprise the vasculature are poorly connected causing vessel leakage and exposing the endothelium to a hypoxic microenvironment. Therefore, most anti-angiogenic therapies are generally inefficient and result in acquired resistance to increased hypoxia due to elimination of the vasculature. Recent studies have explored the efficacy of targeting metabolic pathways in tumor cells in combination with anti-angiogenic therapy. However, the metabolic alterations of endothelial cells in response to hypoxia have been relatively unexplored. Here, we measured polar metabolite levels in microvascular endothelial cells exposed to short- and long-term hypoxia with the goal of identifying metabolic vulnerabilities that can be targeted to normalize tumor vasculature and improve drug delivery. We found that many amino acid-related metabolites were altered by hypoxia exposure, especially within alanine-aspartate-glutamate, serine-threonine, and cysteine-methionine metabolism. Additionally, there were significant changes in de novo pyrimidine synthesis as well as glutathione and taurine metabolism. These results provide key insights into the metabolic alterations that occur in endothelial cells in response to hypoxia, which serve as a foundation for future studies to develop therapies that lead to vessel normalization and more efficient drug delivery.

EphA2 contributes to disruption of the blood-brain barrier in cerebral malaria.

Disruption of blood-brain barrier (BBB) function is a key feature of cerebral malaria. Increased barrier permeability occurs due to disassembly of tight and adherens junctions between endothelial cells, yet the mechanisms governing junction disassembly and vascular permeability during cerebral malaria remain poorly characterized. We found that EphA2 is a principal receptor tyrosine kinase mediating BBB breakdown during Plasmodium infection. Upregulated on brain microvascular endothelial cells in response to inflammatory cytokines, EphA2 is required for the loss of junction proteins on mouse and human brain microvascular endothelial cells. Furthermore, EphA2 is necessary for CD8+ T cell brain infiltration and subsequent BBB breakdown in a mouse model of cerebral malaria. Blocking EphA2 protects against BBB breakdown highlighting EphA2 as a potential therapeutic target for cerebral malaria.

Circulating neuregulin1-β in heart failure with preserved and reduced left ventricular ejection fraction.

AimsNeuregulin1‐β (NRG1‐β) is released from microvascular endothelial cells in response to inflammation with compensatory cardioprotective effects. Circulating NRG1‐β is elevated in heart failure (HF) with reduced ejection fraction (HFrEF) but not studied in HF with preserved EF (HFpEF).Methods and resultsCirculating NRG1‐β was quantified in 86 stable patients with HFpEF (EF ≥45% and N‐terminal pro‐brain natriuretic peptide >300 ng/L), in 86 patients with HFrEF prior to and after left ventricular assist device (LVAD) and/or heart transplantation (HTx) and in 21 healthy controls. Association between NRG1‐β and the composite outcome of all‐cause mortality/HF hospitalization in HFpEF and all‐cause mortality/HTx/LVAD implantation in HFrEF with and without ischaemia assessed as macrovascular coronary artery disease was assessed. In HFpEF, median (25th–75th percentile) NRG1‐β was 6.5 (2.1–11.3) ng/mL; in HFrEF, 3.6 (2.1–7.6) ng/mL (P = 0.035); after LVAD, 1.7 (0.9–3.6) ng/mL; after HTx 2.1 (1.4–3.6) ng/mL (overall P < 0.001); and in controls, 29.0 (23.1–34.3) ng/mL (P = 0.001). In HFrEF, higher NRG1‐β was associated with worse outcomes (hazard ratio per log increase 1.45, 95% confidence interval 1.04–2.03, P = 0.029), regardless of ischaemia. In HFpEF, the association of NRG1‐β with outcomes was modified by ischaemia (log‐rank P = 0.020; P interaction = 0.553) such that only in ischaemic patients, higher NRG1‐β was related to worse outcomes. In contrast, in patients without ischaemia, higher NRG1‐β trended towards better outcomes (hazard ratio 0.71, 95% confidence interval 0.48–1.05, P = 0.085).ConclusionsNeuregulin1‐β was reduced in HFpEF and further reduced in HFrEF. The opposing relationships of NRG1‐β with outcomes in non‐ischaemic HFpEF compared with HFrEF and ischaemic HFpEF may indicate compensatory increases of cardioprotective NRG1‐β from microvascular endothelial dysfunction in the former (non‐ischaemic HFpEF), but this compensatory mechanism is overwhelmed by the presence of ischaemia in the latter (HFrEF and ischaemic HFpEF).

Effects of estrogen-related receptor alpha on inflammatory response in pulmonary microvascular endothelial cells

Objective To investigate the effect of estrogen-related receptor alpha(ERRα)on lipopolysaccharide-induced inflammatory response in rat pulmonary microvascular endothelial cells (PMVECs) and its mechanism. Methods PMVECs were cultured in vitro. When the cells were in the logarithmic growth phase, the cell were ransfected with lentivirus, and a stable low-expression ERRα cell line was constructed. The cells were divided into four groups: Ctr group (normal control group), Ctr+LPS group (normal cell+LPS treatment group), shERRα1 (shERRα1 gene knockdown group), and shERRα1+LPS group (shERRα1 gene knockdown +LPS treatment group). After 20 μg/mL LPS stimulated cells in the control group and shERRα1 group for 6, 12 and 24 h, cell counting kit-8 (cck-8) was used to detect the cell proliferation ability of each group, and enzyme-linked immunosorbent assay (ELISA) was used to detect the concentrations of tumor necrosis factor alpha (TNF-α) and Interleukin-1β (IL-1β) in cell culture fluid. After 12 h LPS stimulation, the expression levels of ERRα and NF-κB related proteins (p-p65, p65, P-IKBα, IKBα) were measured by Western blot. Pairwise comparisons were performed with SNK-q test (two-tailed), and multiple-group comparisons were performed with one-way ANOVA. The non-parametric test of rank transformation was used when homogeneity of variance were not met. P value<0.05 was considered significantly different. Results Compared with the control group, ERRα expression in the shERRα group was significantly decreased (0.09±0.01 vs 0.15±0.01). At 6, 12 and 24 h after LPS stimulation, compared with the control group, the cell proliferation ability (%) of the shERRα1+LPS group was significantly reduced (99.68±4.53 vs 48.62±1.60) and the concentration of TNF-α (ng/mL) (15.76±3.38 vs 5 498.91±367.95) and IL-1β (ng/mL) (14.41±3.86 vs 6 014.92±277.33) in the cell culture supernatant were significantly increased. The change was most obvious after 12 h stimulation. Meanwhile the expression of p-p65 (0.30±0.50 vs 1.05±0.07) and p-IKBα (0.27±0.04 vs 0.77±0.06) were increased significantly, while the expression of IKBα (0.96±0.07 vs 0.14±0.04) was decreased significantly in the shERRα1+LPS group (all P<0.05). Conclusion ERRα gene attenuates LPS-induced inflammatory response in rat pulmonary microvascular endothelial cells by inhibiting NF-κB signaling pathway activation. Key words: Sepsis; Estrogen-related receptor alpha; NF-κB pathway; Gene knockdown

Basic Fibroblast Growth Factor Reduces Permeability and Apoptosis of Human Brain Microvascular Endothelial Cells in Response to Oxygen and Glucose Deprivation Followed by Reoxygenation via the Fibroblast Growth Factor Receptor 1 (FGFR1)/ERK Pathway.

BackgroundDisruption of the blood–brain barrier (BBB) is a mechanism in the pathogenesis of traumatic brain injury. Basic fibroblast growth factor (bFGF) is expressed in angiogenesis, neurogenesis, and neuronal survival. This study aimed to investigate the role of bFGF in vitro in human brain microvascular endothelial cells (HBMECs) challenged by oxygen-glucose deprivation/reperfusion (OGD/R).Material/MethodsHBMECs were cultured in glucose-free medium and an environment with <0.5% oxygen in an anaerobic chamber. Immunocytochemistry, Western blot, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were used to measure the protein and mRNA expression levels of bFGF, tight junction, adherens junction, apoptotic proteins, and matrix metalloproteinases (MMPs). The effects of bFGF on the viability of HBMECs was evaluated using the cell counting kit-8 (CCK-8) assay. Cell apoptosis was evaluated using the TUNEL assay, and endothelial permeability was quantified using a transwell migration assay with fluorescein isothiocyanate (FITC) conjugated with dextran. The effects of bFGF were evaluated following inhibition of fibroblast growth factor receptor 1 (FGFR1) with PD173074 and inhibition of ERK with PD98059.ResultsFollowing OGD/R of HBMECs, bFGF significantly reduced cell permeability and apoptosis and significantly inhibited the down-regulation of the expressions of proteins associated with tight junctions, adherens junctions, apoptosis and matrix metalloproteinases (MMPs). The effects of bFGF were mediated by the activation of FGFR1 and ERK, as they were blocked by FGFR1 and ERK inhibitors.ConclusionsPermeability and apoptosis of HBMECs challenged by OGD/R were reduced by bFGF by activation of the FGFR1 and the ERK pathway.

Circular RNA Transcriptomic Analysis of Primary Human Brain Microvascular Endothelial Cells Infected with Meningitic Escherichia coli

With their essential regulatory roles in gene expression and high abundance in the brain, circular RNAs (circRNAs) have recently attracted considerable attention. Many studies have shown that circRNAs play important roles in the pathology of CNS diseases, but whether circRNAs participate in E. coli-induced bacterial meningitis is unclear. We used high-throughput sequencing to analyze the transcriptional profiles of host circRNAs in primary brain microvascular endothelial cells in response to meningitic E. coli. A total of 308 circRNAs were significantly altered, including 140 upregulated and 168 downregulated ones (p < 0.05). Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology enrichment of the parental genes of these altered circRNAs indicated that they are likely to be involved in diverse biological processes via influencing the expression of their parental genes. Coupled with our previous mRNA and microRNA sequencing data, a competing endogenous RNA analysis was performed, and the potential regulatory network was preliminarily constructed and validated. By revealing the transcriptional profiles of the host circRNAs involved in E. coli meningitis, it is envisaged that the novel insight gained into the regulatory mechanisms of circRNAs in the development of bacterial meningitis will lead to better understanding of how to prevent and treat bacterial CNS infections.

Phenotypic and Sex Differences in Microvesicles Released from Cultured Human Brain Microvascular Endothelial Cells in Response to Proinflammatory and Prothrombotic Stimuli

ObjectiveActivation of brain microvascular endothelium by inflammatory and procoagulant stimuli increases expression of surface cell‐adhesion molecules (CAMs), releases endothelium‐derived microvesicles (MV) into the circulation, and increases permeability of the blood‐brain barrier (BBB). This study evaluated characteristics of MV released from human brain microvascular endothelial cells (HBMEC) following stimulation with proinflammatory or procoagulant stimuli and examined their effect on monolayer permeability.MethodsCultured HBMECs from age‐matched male and female donors were treated for 24 hours with medium supplemented with tumor necrosis factor alpha (TNFα, 20 ng/mL), thrombin (THR, 2 U/mL), or vehicle (control). MVs released into the medium were isolated by differential centrifugation, labeled with annexin V together with antibodies against PECAM‐1, ICAM‐1, E‐selectin, integrin aV, or MCAM, and then analyzed by digital flow cytometry. Permeability (mg/mL FITC‐dextran) of the HBMEC monolayer in response to MV treatment was quantified by fluorescence microplate assay of passage of FITC‐dextran across monolayer cells cultured on microporous supports.ResultsFewer specific antigen‐positive MV were released from unstimulated male compared to female HBMECs. TNFa, but not THR induced early‐stage apoptosis; determined by fluorogenic Caspase‐3 assay in the HBMEC monolayers. TNFa induced release of all MV subtypes from male cells, whereas only integrin aV, ICAM‐1 and E‐selectin positive MVs were released from female cells. THR increased release of PECAM‐1, ICAM‐1, and MCAM positive MVs from male cells, but did not increase release of MV in female cells. After 24 hours exposure to MV (2,500 MV/mL) derived from TNFa‐treated female cells, permeability increased by 17%. Permeabiity was not increased when female cells were exposed to THR‐generated MV.ConclusionsThese results indicate that: 1) inflammatory and procoagulant stimuli release a different array of MV subtypes from male and female HBMECs; 2) specific stimuli‐derived MVs can modify HMBMEC permeability.Support or Funding Information(Supported by NIH P50 AG44170 and the Mayo Clinic Fnd.).

Metformin represses glucose starvation induced autophagic response in microvascular endothelial cells and promotes cell death

Metformin, the most frequently administered drug for the treatment of type 2 diabetes, is being investigated for its potential in the treatment of various types of cancer; however, the cellular basis for this putative anti-cancer action remains controversial. In the current study we examined the effect of metformin on endoplasmic reticulum (ER) stress and autophagy in glucose-starved micro-vascular endothelial cells (MECs). The rationale for our experimental protocol is that in a growing tumor MECs are subjected to hypoxia and nutrient/glucose starvation that results from the reduced supply and relatively high consumption of glucose. Mouse MECs (MMECs) were glucose-starved for up to 48h in the absence or presence of metformin (50μM and 2mM) and the status of ER stress, autophagic, cell survival and apoptotic markers were assessed. Activation of ER stress and autophagy was observed in glucose starved MECs as evidenced by the significant increase in the levels of ER stress and autophagic markers while accumulation of LC3B stained punctae in the MECs confirmed autophagic activation. Treatment with 2mM metformin, independent of AMPK, significantly reversed glucose starvation induced ER stress and autophagy in MECs, but, at 24h, did not decrease cell viability; however, at 48h, persistent ER stress and metformin associated inhibition of autophagy decreased cell viability, caused cell cycle arrest in G2/M and increased the number of cells in the sub-G0/G1 phase of cell cycle. Treatment with metformin reduced phosphorylation of Akt and mTOR and inhibited downstream targets of mTOR. Our findings support the argument that treatment with metformin when used in combination with glycolytic inhibitors will inhibit pro-survival autophagy and promote cell death and potentially prove to be the basis for an effective anti-cancer strategy.

Expression Profiling of Long Noncoding RNA Splice Variants in Human Microvascular Endothelial Cells: Lipopolysaccharide Effects In Vitro.

Endothelial cell interactions with lipopolysaccharide (LPS) involve both activating and repressing signals resulting in pronounced alterations in their transcriptome and proteome. Noncoding RNAs are now appreciated as posttranscriptional and translational regulators of cellular signaling and responses, but their expression status and roles during endothelial interactions with LPS are not well understood. We report on the expression profile of long noncoding (lnc) RNAs of human microvascular endothelial cells in response to LPS. We have identified a total of 10,781 and 8310 lncRNA transcripts displaying either positive or negative regulation of expression, respectively, at 3 and 24 h posttreatment. A majority of LPS-induced lncRNAs are multiexonic and distributed across the genome as evidenced by their presence on all chromosomes. Present among these are a total of 44 lncRNAs with known regulatory functions, of which 41 multiexonic lncRNAs have multiple splice variants. We have further validated splice variant-specific expression of EGO (NONHSAT087634) and HOTAIRM1 (NONHSAT119666) at 3 h and significant upregulation of lnc-IL7R at 24 h. This study illustrates the genome-wide regulation of endothelial lncRNA splice variants in response to LPS and provides a foundation for further investigations of differentially expressed lncRNA transcripts in endothelial responses to LPS and pathophysiology of sepsis/septic shock.