46 results found
Blandinières A, Randi AM, Paschalaki KE, et al., 2023, Results of an international survey about methods used to isolate human endothelial colony-forming cells: guidance from the SSC on Vascular Biology of the ISTH., J Thromb Haemost, Vol: 21, Pages: 2611-2619
BACKGROUND: Assessment of endothelial colony-forming cell (ECFC) number and vasculogenic properties is crucial for exploring vascular diseases and regeneration strategies. A previous survey of the Scientific and Standardization Committee on Vascular Biology of the International Society on Thrombosis and Haemostasis clarified key methodological points but highlighted a lack of standardization associated with ECFC culture. OBJECTIVES: The aim of this study was to provide expert consensus guidance on ECFC isolation and culture. METHODS: We surveyed 21 experts from 10 different countries using a questionnaire proposed during the 2019 International Society on Thrombosis and Haemostasis Congress in Melbourne (Australia) to attain a consensus on ECFC isolation and culture. RESULTS: We report here the consolidated results of the questionnaire. There was agreement on several general statements, mainly the technical aspects of ECFC isolation and cell culture. In contrast, on the points concerning the definition of a colony of ECFCs, the quantification of ECFCs, and the estimation of their age (in days or number of passages), the expert opinions were widely dispersed. CONCLUSION: Our survey clearly indicates an unmet need for rigorous standardization, multicenter comparison of results, and validation of ECFC isolation and culture procedures for clinical laboratory practice and robustness of results. To this end, we propose a standardized protocol for the isolation and expansion of ECFCs from umbilical cord and adult peripheral blood.
Kabir L, Maughan R, Paschalaki K, et al., 2022, Evidence for Mitochondrial Dysfunction in Blood-derived Endothelial Colony Forming Cells Isolated from Patients with Antiphospholipid Syndrome, Publisher: WILEY, Pages: 4515-4516, ISSN: 2326-5191
Havaki S, Evangelou K, Paschalaki K, et al., 2022, Reply: Identification of coronavirus particles by electron microscopy: a complementary tool for deciphering COVID-19, EUROPEAN RESPIRATORY JOURNAL, Vol: 60, ISSN: 0903-1936
Havaki S, Evangelou K, Paschalaki K, et al., 2022, Reply: Identification of coronavirus particles by electron microscopy: a complementary tool for deciphering COVID-19, EUROPEAN RESPIRATORY JOURNAL, Vol: 60, ISSN: 0903-1936
Evangelou K, Veroutis D, Paschalaki K, et al., 2022, Pulmonary infection by SARS-CoV-2 induces senescence accompanied by an inflammatory phenotype in severe COVID-19: possible implications for viral mutagenesis, EUROPEAN RESPIRATORY JOURNAL, Vol: 60, ISSN: 0903-1936
Khawaja A, Maughan R, Paschalaki K, et al., 2022, People living with HIV have higher frequencies of circulating endothelial colony-forming cells: steps towards patient-specific models to evaluate cardiovascular risk, Publisher: WILEY, Pages: 39-39, ISSN: 1464-2662
Paschalaki K, Rossios C, Pericleous C, et al., 2022, Inhaled corticosteroids reduce senescence in endothelial progenitor cells from COPD patients, Thorax, Vol: 77, ISSN: 0040-6376
Cellular senescence contributes to the pathophysiology of chronic obstructive pulmonarydisease (COPD) and cardiovascular disease. Using endothelial-colony-forming-cells (ECFC),we have demonstrated accelerated senescence in smokers and COPD patients compared tonon-smokers. Subgroup analysis suggests that ECFC from COPD patients on inhaledcorticosteroids (ICS) (n=14; 8 on ICS) exhibited significantly reduced senescence(Senescence-associated-beta galactosidase activity, p21CIP1), markers of DNA damageresponse (DDR) and IFN-γ-inducible-protein-10 compared to COPD patients not on ICS. Invitro studies using human-umbilical-vein-endothelial-cells showed a protective effect of ICSon the DDR, senescence and apoptosis caused by oxidative-stress, suggesting a protectivemolecular mechanism of action of corticosteroids on endothelium.
Lodge K, Vassallo A, Liu B, et al., 2021, Hypoxia increases the potential for neutrophil-mediated endothelial damage in COPD, American Journal of Respiratory and Critical Care Medicine, Vol: 205, ISSN: 1073-449X
Rationale: Chronic obstructive pulmonary disease (COPD) patients experience excess cardiovascular morbidity and mortality, and exacerbations further increase the risk of such events. COPD is associated with persistent blood and airway neutrophilia, and systemic and tissue hypoxia. Hypoxia augments neutrophil elastase release, enhancing capacity for tissue injury.Objective: To determine whether hypoxia-driven neutrophil protein secretion contributes to endothelial damage in COPD.Methods: The healthy human neutrophil secretome generated under normoxic or hypoxic conditions was characterised by quantitative mass spectrometry, and the capacity for neutrophil-mediated endothelial damage assessed. Histotoxic protein levels were measured in normoxic versus hypoxic neutrophil supernatants and plasma from exacerbating COPD patients and healthy controls.Main results: Hypoxia promoted PI3Kγ-dependent neutrophil elastase secretion, with greater release seen in neutrophils from COPD patients. Supernatants from neutrophils incubated under hypoxia caused pulmonary endothelial cell damage and identical supernatants from COPD neutrophils increased neutrophil adherence to endothelial cells. Proteomics revealed differential neutrophil protein secretion under hypoxia and normoxia; hypoxia augmented secretion of a subset of histotoxic granule and cytosolic proteins, with significantly greater release seen in COPD neutrophils. The plasma of COPD patients had higher content of hypoxia-upregulated neutrophil-derived proteins and protease activity, and vascular injury markers.Conclusions: Hypoxia drives a destructive ‘hyper-secretory’ neutrophil phenotype conferring enhanced capacity for endothelial injury, with a corresponding signature of neutrophil degranulation and vascular injury identified in COPD patient plasma. Thus, hypoxic enhancement of neutrophil degranulation may contribute to increased cardiovascular risk in COPD. These insights may identify new therapeutic o
Paschalaki K, Evangelou K, Veroutis D, et al., 2021, Alveolar type II cells harbouring SARS-CoV-2 show senescence with a proinflammatory phenotype, Publisher: EUROPEAN RESPIRATORY SOC JOURNALS LTD, ISSN: 0903-1936
Paschalaki K, Rossios C, Gorgoulis V, et al., 2021, Inhaled corticosteroids reduce senescence in endothelial progenitor cells from COPD patients, Publisher: EUROPEAN RESPIRATORY SOC JOURNALS LTD, ISSN: 0903-1936
Khawaja AA, Maughan RT, Paschalaki KE, et al., 2020, Modelling endothelial function in vitro and via blood sampling to evaluate cardiovascular risk in people living with HIV, Publisher: WILEY, Pages: 39-39, ISSN: 1464-2662
Khawaja AA, Maughan RT, Paschalaki KE, et al., 2020, Modelling endothelial function in vitro and via blood sampling to assess cardiovascular risk in people living with HIV, Publisher: JOHN WILEY & SONS LTD, Pages: 105-105
Guy A, Danaee A, Paschalaki K, et al., 2020, Absence of JAK2V617F mutated endothelial colony-forming cells in patients with JAK2V617F myeloproliferative neoplasms and splanchnic vein thrombosis, HemaSphere, Vol: 4, Pages: 1-4, ISSN: 2572-9241
Philadelphia (Ph)-negative myeloproliferative neoplasms (MPN) are acquired hematologic diseases with increased production of mature blood cells. They include polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF). The most frequent molecular abnormality found in Ph negative MPN is JAK2V617F, an activating mutation of JAK2 which is responsible for constitutive signaling of various cytokine receptors. Arterial and venous thromboses are the main complications of these diseases and are responsible for high rates of morbidity and mortality. Of note there is a disproportionate incidence of thrombosis at unusual sites including splanchnic vein thrombosis.1 Splanchnic vein thromboses (SVT) involve one or more abdominal veins, the two most frequent are Portal Vein Thrombosis (PVT) and Budd Chiari Syndrome (BCS). Pathophysiology of thrombosis in MPN is complex and involves abnormalities in blood cells, plasma factors, and endothelial cells (ECs). Several groups, using different techniques, have shown JAK2V617F expression in endothelial cells (Supplemental Fig. 1, http://links.lww.com/HS/A79). Using laser capture microdissection, JAK2V617F was demonstrated in ECs from hepatic venules in 2 of 3 patients with PV and BCS.2JAK2V617F endothelial cells were demonstrated in microdissected splenic capillaries and in ECs cultured from splenic vein in patients with myelofibrosis but without SVT.3 Although these teams performed experiments to ensure that the DNA they obtained originated from ECs, it is difficult to completely rule out a possible contamination by blood cells. Analysis of endothelial progenitor cells, specifically endothelial colony forming cells (ECFCs), is an alternative way to look for JAK2V617F ECs. Indeed, ECFCs are reported to be the only “true” endothelial progenitor cells, as they are the only ones able to generate blood vessels in vivo: they display clonogenic potential, endothelial but not myeloid cell surface markers, and prono
Paschalaki KE, Randi AM, 2018, Recent advances in endothelial colony forming cells toward their use in clinical translation, Frontiers in Medicine, Vol: 5, ISSN: 2296-858X
The term “Endothelial progenitor cell” (EPC) has been used to describe multiple cell populations that express endothelial surface makers and promote vascularisation. However, the only population that has all the characteristics of a real “EPC” is the Endothelial Colony Forming Cells (ECFC). ECFC possess clonal proliferative potential, display endothelial and not myeloid cell surface markers, and exhibit pronounced postnatal vascularisation ability in vivo. ECFC have been used to investigate endothelial molecular dysfunction in several diseases, as they give access to endothelial cells from patients in a non-invasive way. ECFC also represent a promising tool for revascularization of damaged tissue. Here we review the translational applications of ECFC research. We discuss studies which have used ECFC to investigate molecular endothelial abnormalities in several diseases and review the evidence supporting the use of ECFC for autologous cell therapy, gene therapy and tissue regeneration. Finally, we discuss ways to improve the therapeutic efficacy of ECFC in clinical applications, as well as the challenges that must be overcome to use ECFC in clinical trials for regenerative approaches.
Paschalaki KE, Zampetaki A, Baker JR, et al., 2017, Downregulation of MicroRNA-126 Augments DNA Damage Response in Cigarette Smokers and COPD Patients., Am J Respir Crit Care Med
Mollet IG, Patel D, Govani FS, et al., 2016, Low Dose Iron Treatments Induce a DNA Damage Response in Human Endothelial Cells within Minutes, PLOS One, Vol: 11, ISSN: 1932-6203
BackgroundSpontaneous reports from patients able to report vascular sequelae in real time, and recognitionthat serum non transferrin bound iron may reach or exceed 10μmol/L in the bloodstream after iron tablets or infusions, led us to hypothesize that conventional iron treatmentsmay provoke acute vascular injury. This prompted us to examine whether a phenotypecould be observed in normal human endothelial cells treated with low dose iron.MethodologyConfluent primary human endothelial cells (EC) were treated with filter-sterilized iron (II) citrateor fresh media for RNA sequencing and validation studies. RNA transcript profiles wereevaluated using directional RNA sequencing with no pre-specification of target sequences.Alignments were counted for exons and junctions of the gene strand only, blinded to treatmenttypes.Principal FindingsRapid changes in RNA transcript profiles were observed in endothelial cells treated with10μmol/L iron (II) citrate, compared to media-treated cells. Clustering for Gene Ontology(GO) performed on all differentially expressed genes revealed significant differences in biologicalprocess terms between iron and media-treated EC, whereas 10 sets of an equivalentnumber of randomly selected genes from the respective EC gene datasets showed no significantdifferences in any GO terms. After 1 hour, differentially expressed genes clusteredto vesicle mediated transport, protein catabolism, and cell cycle (Benjamini p = 0.0016,0.0024 and 0.0032 respectively), and by 6 hours, to cellular response to DNA damage stimulusmost significantly through DNA repair genes FANCG, BLM, and H2AFX. Comet assays demonstrated that 10μM iron treatment elicited DNA damage within 1 hour. This wasaccompanied by a brisk DNA damage response pulse, as ascertained by the developmentof DNA damage response (DDR) foci, and p53 stabilization.SignificanceThese data suggest that low dose iron treatments are sufficient to modify the vascular endothelium,and induce a DNA damage
Paschalaki KE, Jacob J, Wells AU, 2016, Monitoring of lung involvement in rheumatologic disease, Respiration, Vol: 91, Pages: 89-98, ISSN: 1423-0356
The monitoring of lung involvement in patients with connective tissue diseases is central to optimal long-term management and is directed towards: (a) the detection of supervening lung involvement not present at presentation and (b) the identification of disease progression in established lung disease. For both goals, accurate surveillance requires multi-disciplinary evaluation with the integration of symptomatic change, serial pulmonary function trends and imaging data. Evaluated in isolation, each of these monitoring domains has significant limitations. Symptomatic change may be confounded by a wide variety of systemic factors. Pulmonary function tests provide the most reliable data, but are limited by measurement variability, the heterogeneity of functional patterns and the confounding effects of non-pulmonary factors. Chest radiography is insensitive to change but may provide rapid confirmation of major disease progression or alert the clinician to respiratory co-morbidities. Although high-resolution computed tomography has a central role in assessing disease severity, it should be used very selectively as a monitoring tool due to the associated radiation burden. Ancillary tests include echocardiography and exercise testing to proactively identify cases of pulmonary hypertension and worsening of oxygenation. In summary, a multi-disciplinary approach is essential for the identification of disease progression and prompt treatment of comorbidities that severely impact on the morbidity and mortality of disease.
Reed DM, Paschalaki KE, Starke RD, et al., 2015, An autologous endothelial cell: peripheral blood mononuclear cell assay that detects cytokine storm responses to biologics, The FASEB Journal, Vol: 29, Pages: 2595-2602, ISSN: 0892-6638
There is an urgent unmet need for human tissue bioassays to predict cytokine storm responses to biologics. Current bioassays that detect cytokine storm responses in vitro rely on endothelial cells, usually from umbilical veins or cell lines, cocultured with freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy adult volunteers. These assays therefore comprise cells from 2 separate donors and carry the disadvantage of mismatched tissues and lack the advantage of personalized medicine. Current assays also do not fully delineate mild (such as Campath) and severe (such as TGN1412) cytokine storm‐inducing drugs. Here, we report a novel bioassay where endothelial cells grown from stem cells in the peripheral blood (blood outgrowth endothelial cells) and PBMCs from the same donor can be used to create an autologous coculture bioassay that responds by releasing a plethora of cytokines to authentic TGN1412 but only modestly to Campath and not to control antibodies such as Herceptin, Avastin, and Arzerra. This assay performed better than the traditional mixed donor assay in terms of cytokine release to TGN1412 and, thus, we suggest provides significant advancement and a definitive system by which biologics can be tested and paves the way for personalized medicine.—Reed, D. M., Paschalaki, K. E., Starke, R. D., Mohamed, N. A., Sharp, G., Fox, B., Eastwood, D., Bristow, A., Ball, C., Vessillier, S., Hansel, T. T., Thorpe, S. J., Randi, A. M., Stebbings, R., Mitchell, J. A. An autologous endothelial cell:peripheral blood mononuclear cell assay that detects cytokine storm responses to biologics. FASEB J. 29, 2595‐2602 (2015). www.fasebj.org
Reed D, Kirkby N, Mohamed N, et al., 2014, PRINCIPAL COMPONENT ANALYSIS OF 17 CYTOKINES AND CHEMOKINES SUGGESTS THAT AUTOLOGOUS ENDOTHELIAL CELL: PBMC CO-CULTURES DELINEATE SEVERE AND MILD CYTOKINE STORM CAUSING BIOLOGICS: A NEW ASSAY FOR CYTOKINE STORM DETECTION AND RESEARCH, Publisher: WILEY-BLACKWELL, Pages: 142-143, ISSN: 1742-7835
Reed DM, Foldes G, Gatheral T, et al., 2014, Pathogen Sensing Pathways in Human Embryonic Stem Cell Derived-Endothelial Cells: Role of NOD1 Receptors, PLOS One, Vol: 9, ISSN: 1932-6203
Human embryonic stem cell-derived endothelial cells (hESC-EC), as well as other stem cell derived endothelial cells, have a range of applications in cardiovascular research and disease treatment. Endothelial cells sense Gram-negative bacteria via the pattern recognition receptors (PRR) Toll-like receptor (TLR)-4 and nucleotide-binding oligomerisation domain-containing protein (NOD)-1. These pathways are important in terms of sensing infection, but TLR4 is also associated with vascular inflammation and atherosclerosis. Here, we have compared TLR4 and NOD1 responses in hESC-EC with those of endothelial cells derived from other stem cells and with human umbilical vein endothelial cells (HUVEC). HUVEC, endothelial cells derived from blood progenitors (blood outgrowth endothelial cells; BOEC), and from induced pluripotent stem cells all displayed both a TLR4 and NOD1 response. However, hESC-EC had no TLR4 function, but did have functional NOD1 receptors. In vivo conditioning in nude rats did not confer TLR4 expression in hESC-EC. Despite having no TLR4 function, hESC-EC sensed Gram-negative bacteria, a response that was found to be mediated by NOD1 and the associated RIP2 signalling pathways. Thus, hESC-EC are TLR4 deficient but respond to bacteria via NOD1. This data suggests that hESC-EC may be protected from unwanted TLR4-mediated vascular inflammation, thus offering a potential therapeutic advantage.
Starke RD, Paschalaki K, Millar CM, et al., 2014, Von Willebrand factor (VWF) regulates angiogenesis through angiopoietin-2 (Ang-2)-dependent and independent pathways, ANGIOGENESIS, Vol: 17, Pages: 290-290, ISSN: 0969-6970
Wright WR, Paschalaki KE, Gashaw HH, et al., 2013, Characterisation of the phospholipase A2 isoforms supporting prostacyclin production synthesis by endothelial cells, Pharmacology 2013
Paschalaki K, Starke RD, Hu Y, et al., 2013, CIRCULATING ENDOTHELIAL PROGENITOR CELLS IN SMOKERS AND PATIENTS WITH COPD ARE DYSFUNCTIONAL DUE TO INCREASED DNA DAMAGE AND SENESCENCE, Winter Meeting of the British-Thoracic-Society, Publisher: BMJ PUBLISHING GROUP, Pages: A2-A3, ISSN: 0040-6376
Paschalaki KE, Starke RD, Hu Y, et al., 2013, Dysfunction of endothelial progenitor cells from smokers and chronic obstructive pulmonary disease patients due to increased DNA damage and senescence, Stem Cells, Vol: 31, Pages: 2813-2826, ISSN: 1066-5099
Cardiovascular disease (CVD) is a major cause of death in smokers, particularly in those with chronic obstructive pulmonary disease (COPD). Circulating endothelial progenitor cells (EPC) are required for endothelial homeostasis, and their dysfunction contributes to CVD. To investigate EPC dysfunction in smokers, we isolated and expanded blood outgrowth endothelial cells (BOEC) from peripheral blood samples from healthy nonsmokers, healthy smokers, and COPD patients. BOEC from smokers and COPD patients showed increased DNA double‐strand breaks and senescence compared to nonsmokers. Senescence negatively correlated with the expression and activity of sirtuin‐1 (SIRT1), a protein deacetylase that protects against DNA damage and cellular senescence. Inhibition of DNA damage response by silencing of ataxia telangiectasia mutated (ATM) kinase resulted in upregulation of SIRT1 expression and decreased senescence. Treatment of BOEC from COPD patients with the SIRT1 activator resveratrol or an ATM inhibitor (KU‐55933) also rescued the senescent phenotype. Using an in vivo mouse model of angiogenesis, we demonstrated that senescent BOEC from COPD patients are dysfunctional, displaying impaired angiogenic ability and increased apoptosis compared to cells from healthy nonsmokers. Therefore, this study identifies epigenetic regulation of DNA damage and senescence as pathogenetic mechanisms linked to endothelial progenitors' dysfunction in smokers and COPD patients. These defects may contribute to vascular disease and cardiovascular events in smokers and could therefore constitute therapeutic targets for intervention.
Paschalaki KE, Starke RD, Hu Y, et al., 2013, Endothelial progenitor cells are dysfunctional in smokers and COPD patients due to increased DNA damage and senescence, EUROPEAN RESPIRATORY JOURNAL, Vol: 42, ISSN: 0903-1936
Reed D, Gashaw H, Bailey L, et al., 2013, Use of 'same donor' endothelial cells and PBMC in co-culture to detect cytoldne storm reactions to a TGN1412-like anti-CD28 antibody: A novel assay for biologic drug safety screening, 49th Congress of the European-Societies-of-Toxicology (EUROTOX), Publisher: ELSEVIER IRELAND LTD, Pages: S164-S164, ISSN: 0378-4274
Bikov A, Paschalaki K, Logan-Sinclair R, et al., 2013, Standardised exhaled breath collection for the measurement of exhaled volatile organic compounds by proton transfer reaction mass spectrometry, BMC Pulmonary Medicine, Vol: 13, Pages: 1-7, ISSN: 1471-2466
BackgroundExhaled breath volatile organic compound (VOC) analysis for airway disease monitoring is promising. However, contrary to nitric oxide the method for exhaled breath collection has not yet been standardized and the effects of expiratory flow and breath-hold have not been sufficiently studied. These manoeuvres may also reveal the origin of exhaled compounds.Methods15 healthy volunteers (34 ± 7 years) participated in the study. Subjects inhaled through their nose and exhaled immediately at two different flows (5 L/min and 10 L/min) into methylated polyethylene bags. In addition, the effect of a 20 s breath-hold following inhalation to total lung capacity was studied. The samples were analyzed for ethanol and acetone levels immediately using proton-transfer-reaction mass-spectrometer (PTR-MS, Logan Research, UK).ResultsEthanol levels were negatively affected by expiratory flow rate (232.70 ± 33.50 ppb vs. 202.30 ± 27.28 ppb at 5 L/min and 10 L/min, respectively, p < 0.05), but remained unchanged following the breath hold (242.50 ± 34.53 vs. 237.90 ± 35.86 ppb, without and with breath hold, respectively, p = 0.11). On the contrary, acetone levels were increased following breath hold (1.50 ± 0.18 ppm) compared to the baseline levels (1.38 ± 0.15 ppm), but were not affected by expiratory flow (1.40 ± 0.14 ppm vs. 1.49 ± 0.14 ppm, 5 L/min vs. 10 L/min, respectively, p = 0.14). The diet had no significant effects on the gasses levels which showed good inter and intra session reproducibility.ConclusionsExhalation parameters such as expiratory flow and breath-hold may affect VOC levels significantly; therefore standardisation of exhaled VOC measurements is mandatory. Our preliminary results suggest a different origin in the respiratory tr
Mangles SE, Paschalaki KE, Starke RD, et al., 2013, Increased fibrinolysis on blood outgrowth endothelial cells (BOEC) from chronic thromboembolic pulmonary hypertension (CTEPH) patients, JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Vol: 11, Pages: 920-921, ISSN: 1538-7933
Starke RD, Paschalaki KE, Dyer CEF, et al., 2013, Defective angiopoietin-2 release from von Willebrand disease patients' blood outgrowth endothelial cells, JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Vol: 11, Pages: 175-175, ISSN: 1538-7933
Starke R, Paschalaki K, Dyer C, et al., 2013, DEFECTIVE VON WILLEBRAND FACTOR AND ANGIOPOIETIN-2 RELEASE FROM VON WILLEBRAND DISEASE PATIENTS' BLOOD OUTGROWTH ENDOTHELIAL CELLS, Annual Conference of the British-Cardiovascular-Society (BCS), Publisher: BMJ PUBLISHING GROUP, Pages: A106-A106, ISSN: 1355-6037
This data is extracted from the Web of Science and reproduced under a licence from Thomson Reuters. You may not copy or re-distribute this data in whole or in part without the written consent of the Science business of Thomson Reuters.