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Anticorpos Monoclonais Humanizados , Asma , Transcriptoma , Humanos , Asma/tratamento farmacológico , Asma/genética , Anticorpos Monoclonais Humanizados/uso terapêutico , Feminino , Masculino , Adulto , Antiasmáticos/uso terapêutico , Pessoa de Meia-Idade , Mucosa Nasal/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , EpigenomaRESUMO
An increase in intracellular calcium represents one of the early events during an ischaemic stroke. It triggers many downstream processes which promote the formation of brain oedema, the leading cause of death after an ischaemic stroke. As impairment of blood-brain barrier (BBB) accounts for much of oedema formation, the current study explored the impact of intracellular calcium on barrier integrity in relation to protein kinase C, caspase-3/7, plasminogen activators and the pro-oxidant enzyme NADPH oxidase. Human brain microvascular endothelial cells alone or in co-culture with human astrocytes were subjected to 4h of oxygen-glucose deprivation alone or followed by 20h of reperfusion (OGD±R) in the absence or presence of inhibitors for urokinase plasminogen activator (amiloride), NADPH oxidase (apocynin), intracellular calcium (BAPTA-AM) and protein kinase C-α (RO-32-0432). Endothelial cells with protein kinase C-α knockdown, achieved by siRNA, were also exposed to the above conditions. BBB permeability was measured by transendothelial electrical resistance and Evan's blue-albumin and sodium fluorescein flux. Intracellular calcium and total superoxide anion levels, caspase-3/7, NADPH oxidase, plasminogen activator and protein kinase C activities, stress fibre formation, the rate of apoptosis and BBB permeability were increased by OGD±R. Treatment with the specific inhibitors or knockdown of protein kinase C-α attenuated them. This study reveals successive increases in intracellular calcium levels and protein kinase C-α activity are key mechanisms in OGD±R-mediated impairment of BBB. Furthermore inhibition of protein kinase C-α may be therapeutic in restoring BBB function by reducing the rate of cytoskeletal reorganisation, oxidative stress and apoptosis.
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Apoptose , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Cálcio/metabolismo , Proteína Quinase C-alfa/metabolismo , Barreira Hematoencefálica/citologia , Permeabilidade Capilar , Linhagem Celular , Glucose/metabolismo , Humanos , NADPH Oxidases/metabolismo , Oxigênio/metabolismoRESUMO
Cerebral ischaemia, associated with neuroinflammation and oxidative stress, is known to perturb blood-brain barrier (BBB) integrity and promote brain oedema formation. Using an in vitro model of human BBB composed of brain microvascular endothelial cells and astrocytes, this study examined whether suppression of TNF-α, a potent pro-inflammatory cytokine, might attenuate ischaemia-mediated cerebral barrier damage. Radical decreases in transendothelial electrical resistance and concomitant increases in paracellular flux across co-cultures exposed to increasing periods of oxygen-glucose deprivation alone (0.5-20 h) or followed by 20 h of reperfusion (OGD ± R) confirmed the deleterious effects of ischaemic injury on cerebral barrier integrity and function which concurred with reductions in tight junction protein (claudin-5 and occludin) expressions. OGD ± R elevated TNF-α secretion, NADPH oxidase activity, O2(-) production, actin stress fibre formation, MMP-2/9 activities and apoptosis in both endothelial cells and astrocytes. Increases in MMP-2 activity were confined to its extracellular isoform and treatments with OGD+R in astrocytes where MMP-9 could not be detected at all. Co-exposure of individual cell lines or co-cultures to an anti-TNF-α antibody dramatically diminished the extent of OGD ± R-evoked oxidative stress, morphological changes, apoptosis, MMP-2/9 activities while improving the barrier function through upregulation of tight junction protein expressions. In conclusion, vitiation of the exaggerated release of TNF-α may be an important therapeutic strategy in preserving cerebral integrity and function during and following a cerebral ischaemic attack.
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Astrócitos/metabolismo , Barreira Hematoencefálica/metabolismo , Isquemia Encefálica/metabolismo , Estresse Oxidativo/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Apoptose/fisiologia , Barreira Hematoencefálica/efeitos dos fármacos , Isquemia Encefálica/patologia , Claudina-5/metabolismo , Técnicas de Cocultura/métodos , Células Endoteliais/citologia , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismoRESUMO
Ischaemic injury impairs the integrity of the blood-brain barrier (BBB). In this study, we investigated the molecular causes of this defect with regard to the putative correlations among NAD(P)H oxidase, plasminogen-plasmin system components, and matrix metalloproteinases. Hence, the activities of NAD(P)H oxidase, matrix metalloproteinase-2, urokinase-type plasminogen activator (uPA), and tissue-type plasminogen activator (tPA), and superoxide anion levels, were assessed in human brain microvascular endothelial cells (HBMECs) exposed to oxygen-glucose deprivation (OGD) alone or OGD followed by reperfusion (OGD + R). The integrity of an in vitro model of BBB comprising HBMECs and astrocytes was studied by measuring transendothelial electrical resistance and the paracellular flux of albumin. OGD with or without reperfusion (OGD ± R) radically perturbed barrier function while concurrently enhancing uPA, tPA and NAD(P)H oxidase activities and superoxide anion release in HBMECs. Pharmacological inactivation of NAD(P)H oxidase attenuated OGD ± R-mediated BBB damage through modulation of matrix metalloproteinase-2 and tPA, but not uPA activity. Overactivation of NAD(P)H oxidase in HBMECs via cDNA electroporation of its p22-phox subunit confirmed the involvement of tPA in oxidase-mediated BBB disruption. Interestingly, blockade of uPA or uPA receptor preserved normal BBB function by neutralizing both NAD(P)H oxidase and matrix metalloproteinase-2 activities. Hence, selective targeting of uPA after ischaemic strokes may protect cerebral barrier integrity and function by concomitantly attenuating basement membrane degradation and oxidative stress.
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Barreira Hematoencefálica/fisiopatologia , Encéfalo/fisiopatologia , Isquemia/fisiopatologia , Metaloproteinase 2 da Matriz/metabolismo , NADPH Oxidases/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/fisiologia , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/fisiologia , Células Cultivadas , Técnicas de Cocultura , Impedância Elétrica , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Glucose/deficiência , Humanos , Hipóxia , Metaloproteinase 9 da Matriz/metabolismo , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , Superóxidos/metabolismo , Ativador de Plasminogênio Tecidual/antagonistas & inibidores , Ativador de Plasminogênio Tecidual/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidoresRESUMO
Genes involved in cell death, inflammation and viral infection are common to both COPD and IPF. A link to rheumatic disease is unique to COPD, and IPF-specific analyses showed increases in gene expression of keratins, collagens, mucins and MMPs. https://bit.ly/3JoW73H.
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Endothelial progenitor cells (EPCs) are stem cells that can repair injured blood vessels through neovascularisation. This is achieved through secretion of growth factors and endothelial maturation. EPC numbers and function have been studied to determine their diagnostic, prognostic and therapeutic potential in many ischaemic diseases such as stroke. However their activation homing and migration is not definitively understood in stroke patients. In this study, we profiled the non-stroke control group recruited into the Dunhill Medical Trust Endothelial Progenitor Cell Study. Demographic, clinical and plasma levels of angiogenic regulators of participants were analysed to determine if there was any correlation with EPC numbers, subtypes and function. Participants with diabetes had significantly supressed EPC numbers (CD45-CD34 + CD133 + KDR+) and CD34 + KDR + and KDR + EPC subtypes. Male participants had significantly lower EPC numbers compared to female participants and the proliferative capacity of endothelial colony forming cells significantly decreased with increasing participant age. Pro-angiogenic proteins such as granulocyte colony-stimulating factor and stromal cell-derived factor were positively correlated with both undifferentiated and endothelial-committed EPC subtype numbers (CD133+, KDR+, CD34 + CD133+, CD34 + KDR+), whereas anti-angiogenic proteins such as thrombospondin-1 showed a negative correlation with undifferentiated EPC subtypes (CD133+, CD34 + CD133+) but a positive correlation with endothelial-committed EPC subtype numbers (KDR+, CD34 + KDR+). These results show that EPC numbers and subtypes are affected by many factors and larger studies which can analyse and deconvolute the interactions between comorbidities, plasma biomarker levels and EPC are needed.
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Ageing impairs endothelial function and predisposes the person to ischaemic stroke (IS). Endothelial progenitor cells (EPCs) repair endothelial damage and induce post-ischaemic neovascularisation. Given the prevalence of IS in older population, this study explored whether changes in EPC number and function may reliably predict the type or outcome of stroke in patients ≥ 65 years of age. For this, blood samples were collected once from healthy volunteers (HVs, n = 40) and four times (admission and days 7, 30 and 90 post-stroke) from participants with lacunar (n = 38) or cortical (n = 43) stroke. EPCs were counted with flow cytometry and defined as non-haematopoietic cells (CD45-) expressing markers for stemness (CD34 +), immaturity (CD133 +) and endothelial maturity (KDR +). Clonogenesis, tubulogenesis, migration and proliferation assays were performed as measures of EPC functionality. Biochemical profile of plasma inflammatory and angiogenic agents were studied using specific ELISAs. Primary outcome was disability or dependence on day 90 post-stroke, assessed by the modified Rankin Scale (mRS). Compared to HVs, EPC numbers were higher in stroke patients at all time points studied, reaching significance at baseline and day 30. No differences in EPC counts and functionality were observed between lacunar and cortical stroke groups at any time. Plasma endostatin, PDGF-BB, TNF-α and VEGF levels were higher in stroke patients vs HVs. Patient outcome, evaluated by mRS on day 90 post-stroke, did not correlate with EPC count or functionality. Baseline EPC counts may serve as a diagnostic marker for stroke but fail to distinguish between different stroke subtypes and predict post-stroke outcome.
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Isquemia Encefálica , Células Progenitoras Endoteliais , AVC Isquêmico , Acidente Vascular Cerebral , Humanos , Idoso , Células Progenitoras Endoteliais/metabolismo , Acidente Vascular Cerebral/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Biomarcadores/metabolismo , AVC Isquêmico/metabolismoRESUMO
Endothelial progenitor cells (EPCs), expressing markers for stemness (CD34), immaturity (CD133) and endothelial maturity (KDR), may determine the extent of post-stroke vascular repair. Given the prevalence of stroke in elderly, this study explored whether variations in plasmatic availability of certain EPC subtypes could predict the severity and outcome of disease in older patients. Blood samples were collected from eighty-one consented patients (≥ 65 years) at admission and days 7, 30 and 90 post-stroke. EPCs were counted with flow cytometry. Stroke severity and outcome were assessed using the National Institutes of Health Stroke Scale, Barthel Index and modified Rankin Scale. The levels of key elements known to affect EPC characteristics were measured by ELISA. Diminished total antioxidant capacity and CD34 + KDR + and CD133 + KDR + counts in early phases of stroke were associated with disease severity and worse functional outcome at day 90 post-stroke. Baseline levels of angiogenic agent PDGF-BB, but not VEGF, positively correlated with CD34 + KDR + numbers at day 90. Baseline LDL-cholesterol levels were inversely correlated with CD34 + KDR+, CD133 + KDR + and CD34 + CD133 + KDR + numbers at day 90. Close correlation between baseline CD34 + KDR + and CD133 + KDR + counts and the outcome of stroke proposes these particular EPC subtypes as potential prognostic markers for ischaemic stroke.
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Isquemia Encefálica , Células Progenitoras Endoteliais , AVC Isquêmico , Acidente Vascular Cerebral , Humanos , Idoso , Biomarcadores , Antígenos CD34 , Citometria de FluxoRESUMO
Improved understanding of the key mechanisms underlying cerebral ischemic injury is essential for the discovery of efficacious novel therapeutics for stroke. Through detailed analysis of plasma samples obtained from a large number of healthy volunteers (n = 90) and ischemic stroke patients (n = 81), the current study found significant elevations in the levels of TNF-α at baseline (within the first 48 h of stroke) and on days 7, 30, 90 after ischaemic stroke. It then assessed the impact of this inflammatory cytokine on an in vitro model of human blood-brain barrier (BBB) and revealed dramatic impairments in both barrier integrity and function, the main cause of early death after an ischemic stroke. Co-treatment of BBB models in similar experiments with outgrowth endothelial cell-derived conditioned media (OEC-CM) negated the deleterious effects of TNF-α on BBB. Effective suppression of anti-angiogenic factor endostatin, stress fiber formation, oxidative stress, and apoptosis along with concomitant improvements in extracellular matrix adhesive and tubulogenic properties of brain microvascular endothelial cells and OECs played an important role in OEC-CM-mediated benefits. Significant increases in pro-angiogenic endothelin-1 and monocyte chemoattractant protein-1 in OEC-CM compared to the secretomes of OEC and HBMEC, detected by proteome profiling assay, accentuate the beneficial effects of OEC-CM. In conclusion, this reverse translational study identifies TNF-α as an important mediator of post-ischemic cerebral barrier damage and proposes OEC-CM as a potential vasculoprotective therapeutic strategy by demonstrating its ability to regulate a wide range of mechanisms associated with BBB function. Clinical trial registration NCT02980354.
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Lesões Encefálicas , Isquemia Encefálica , AVC Isquêmico , Acidente Vascular Cerebral , Humanos , Células Endoteliais/metabolismo , Meios de Cultivo Condicionados/farmacologia , Meios de Cultivo Condicionados/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Acidente Vascular Cerebral/metabolismo , Pesquisa Translacional Biomédica , AVC Isquêmico/metabolismoRESUMO
Gene changes observed in asthma bronchial epithelial cells are maintained following repeated culture, presenting with an exaggerated response to viral infection and immune responses as well as having differences in the rate of cell division and replication https://bit.ly/3Cq2xKf.
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Ageing is characterised by a progressive loss of vascular endothelial function and integrity. Endothelial progenitor cells (EPCs) play an integral role in endothelial regeneration but are prone to age-dependent changes which may accelerate their senescence and diminish their availability and functionality. Considering these, we firstly investigated the quantity of circulating EPCs in older (73.3 ± 7.2 years) and younger (40.2 ± 14.3 years) healthy volunteers and showed sharp declines in the number of EPCs expressing stemness markers (CD34 + and/or CD133 + ) in older people. These coincided with the decreases in total anti-oxidant capacity (TAC) and concomitant increases in plasma levels of pro-inflammatory cytokine, TNF-α and anti-angiogenic factor, endostatin and thrombospondin-1. The subsequent experimental studies to scrutinise the effect of ageing on molecular and functional properties of outgrowth endothelial cells (OECs), the functional subtype of EPCs, showed that chronological ageing, mimicked by replicative senescence, profoundly impaired proliferation, migration, tubulogenesis, and blood-brain barrier (BBB)-forming capacity of these cells. Similar to those seen in the clinical observational studies, senescent OECs also manifested decreased TAC and increased pro-oxidant NADPH oxidase activity and endostatin level. Suppressing oxidative stress level using structurally and functionally distinct anti-oxidants, namely vitamin C or VAS2870, an NADPH oxidase inhibitor, delayed OEC senescence and restored their tubulogenic and BBB-forming capacities. In conclusion, the enhanced oxidative stress level that develops during physiological ageing may promote EPC senescence and evoke endothelial dysfunction. Effective control of oxidative stress using either compound somewhat delays both phenomena and augments EPC functionality.
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Células Progenitoras Endoteliais , Idoso , Antioxidantes/farmacologia , Células Cultivadas , Senescência Celular/fisiologia , Endostatinas/farmacologia , Humanos , NADPH Oxidases , Estresse OxidativoRESUMO
Asthma affects more than 300 million people globally and is both under diagnosed and under treated. The most recent and largest genome-wide association study investigating moderate to severe asthma to date was carried out in 2019 and identified 25 independent signals. However, as new and in-depth downstream databases become available, the translational analysis of these signals into target genes and pathways is timely. In this study, unique (U-BIOPRED) and publicly available datasets (HaploReg, Open Target Genetics and GTEx) were investigated for the 25 GWAS signals to identify 37 candidate causal genes. Additional traits associated with these signals were identified through PheWAS using the UK Biobank resource, with asthma and eosinophilic traits amongst the strongest associated. Gene expression omnibus dataset examination identified 13 candidate genes with altered expression profiles in the airways and blood of asthmatic subjects, including MUC5AC and STAT6. Gene expression analysis through publicly available datasets highlighted lung tissue cell specific expression, with both MUC5AC and SLC22A4 genes showing enriched expression in ciliated cells. Gene enrichment pathway and interaction analysis highlighted the dominance of the HLA-DQA1/A2/B1/B2 gene cluster across many immunological diseases including asthma, type I diabetes, and rheumatoid arthritis. Interaction and prediction analyses found IL33 and IL18R1 to be key co-localization partners for other genes, predicted that CD274 forms co-expression relationships with 13 other genes, including the HLA-DQA1/A2/B1/B2 gene cluster and that MUC5AC and IL37 are co-expressed. Drug interaction analysis revealed that 11 of the candidate genes have an interaction with available therapeutics. This study provides significant insight into these GWAS signals in the context of cell expression, function, and disease relationship with the view of informing future research and drug development efforts for moderate-severe asthma.
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Ischemic stroke is a devastating, life altering event which can severely reduce patient quality of life. Despite years of research there have been minimal therapeutic advances. Endothelial progenitor cells (EPCs), stem cells involved in both vasculogenesis and angiogenesis, may be a potential therapeutic target. After a stroke, EPCs migrate to the site of ischemic injury to repair cerebrovascular damage, and their numbers and functional capacity may determine patients' outcome. This study aims to determine whether the number of circulating EPCs and their functional aspects may be used as biomarkers to identify the type (cortical or lacunar) and/or severity of ischemic stroke. The study will also investigate if there are any differences in these characteristics between healthy volunteers over and under 65 years of age. 100 stroke patients (50 lacunar and 50 cortical strokes) will be recruited in this prospective, observational case-controlled study. Blood samples will be taken from stroke patients at baseline (within 48 hours of stroke) and days 7, 30 and 90. EPCs will be counted with flow cytometry. The plasma levels of pro- and anti-angiogenic factors and inflammatory cytokines will also be determined. Outgrowth endothelial cells will be cultured to be used in tube formation, migration and proliferation functional assays. Primary outcome is disability or dependence on day 90 after stroke, assessed by the modified Rankin Scale. Secondary outcomes are changes in circulating EPC numbers and/or functional capacity between patient and healthy volunteers, between patient subgroups and between elderly and young healthy volunteers. Recruitment started in February 2017, 167 participants have been recruited. Recruitment will end in November 2019. West Midlands - Coventry & Warwickshire Research Ethics Committee approved this study (REC number: 16/WM/0304) on September 8, 2016. Protocol version: 2.0. The Bayraktutan Dunhill Medical Trust EPC Study was registered in ClinicalTrials.gov (NCT02980354) on November 15, 2016. This study will determine whether the number of EPCs can be used as a prognostic or diagnostic marker for ischemic strokes and is a step towards discovering if transplantation of EPCs may aid patient recovery.
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Breakdown of blood-brain barrier, formed mainly by brain microvascular endothelial cells (BMECs), represents the major cause of mortality during early phases of ischemic strokes. Hence, discovery of novel agents that can effectively replace dead or dying endothelial cells to restore blood-brain barrier integrity is of paramount importance in stroke medicine. Although endothelial progenitor cells (EPCs) represent one such agents, their rarity in peripheral blood severely limits their adequate isolation and therapeutic use for acute ischemic stroke which necessitate their ex vivo expansion and generate early EPCs and outgrowth endothelial cells (OECs) as a result. Functional analyses of these cells, in the present study, demonstrated that only OECs endocytosed DiI-labelled acetylated low-density lipoprotein and formed tubules on matrigel, prominent endothelial cell and angiogenesis markers, respectively. Further analyses by flow cytometry demonstrated that OECs expressed specific markers for stemness (CD34), immaturity (CD133) and endothelial cells (CD31) but not for hematopoietic cells (CD45). Like BMECs, OECs established an equally tight in vitro model of human BBB with astrocytes and pericytes, suggesting their capacity to form tight junctions. Ischemic injury mimicked by concurrent deprivation of oxygen and glucose (4 hours) or deprivation of oxygen and glucose followed by reperfusion (20 hours) affected both barrier integrity and function in a similar fashion as evidenced by decreases in transendothelial electrical resistance and increases in paracellular flux, respectively. Wound scratch assays comparing the vasculoreparative capacity of cells revealed that, compared to BMECs, OECs possessed a greater proliferative and directional migratory capacity. In a triple culture model of BBB established with astrocytes, pericytes and BMEC, exogenous addition of OECs effectively repaired the damage induced on endothelial layer in serum-free conditions. Taken together, these data demonstrate that OECs may effectively home to the site of vascular injury and repair the damage to maintain (neuro)vascular homeostasis during or after a cerebral ischemic injury.