Imperial College London

ProfessorAnnaRandi

Faculty of MedicineNational Heart & Lung Institute

Head of Section for Vascular Science
 
 
 
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a.randi Website

 
 
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L-block, room 533Hammersmith HospitalHammersmith Campus

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Summary

 

Publications

Publication Type
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111 results found

Randi AM, Jones D, Peghaire C, Arachchillage DJet al., 2023, Mechanisms regulating heterogeneity of hemostatic gene expression in endothelial cells., J Thromb Haemost, Vol: 21, Pages: 3056-3066

The hemostatic system involves an array of circulating coagulation factors that work in concert with platelets and the vascular endothelium to promote clotting in a space- and time-defined manner. Despite equal systemic exposure to circulating factors, bleeding and thrombotic diseases tend to prefer specific sites, suggesting an important role for local factors. This may be provided by endothelial heterogeneity. Endothelial cells differ not only between arteries, veins, and capillaries but also between microvascular beds from different organs, which present unique organotypic morphology and functional and molecular profiles. Accordingly, regulators of hemostasis are not uniformly distributed in the vasculature. The establishment and maintenance of endothelial diversity are orchestrated at the transcriptional level. Recent transcriptomic and epigenomic studies have provided a global picture of endothelial cell heterogeneity. In this review, we discuss the organotypic differences in the hemostatic profile of endothelial cells; we focus on 2 major endothelial regulators of hemostasis, namely von Willebrand factor and thrombomodulin, to provide examples of transcriptional mechanisms that control heterogeneity; finally, we consider some of the methodological challenges and opportunities for future studies.

Journal article

Nagy D, Maude H, Birdsey GM, Randi AM, Cebola Iet al., 2023, Liver sinusoidal endothelial transcription factors in metabolic homeostasis and disease, Journal of Molecular Endocrinology, Vol: 71, Pages: 1-20, ISSN: 0952-5041

Liver sinusoidal endothelial cells (LSECs) are highly specialized endothelial cells that form the liver microvasculature. LSECs maintain liver homeostasis, scavenging bloodborne molecules, regulating immune response, and actively promoting hepatic stellate cell quiescence. These diverse functions are underpinned by a suite of unique phenotypical attributes distinct from other blood vessels. In recent years, studies have begun to reveal the specific contributions of LSECs to liver metabolic homeostasis and how LSEC dysfunction associates with disease aetiology. This has been particularly evident in the context of non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of metabolic syndrome, which is associated with loss of key LSEC phenotypical characteristics and molecular identity. Comparative transcriptome studies of LSECs and other endothelial cells, together with rodent knockout models, have revealed that loss of LSEC identity through disruption of core transcription factor activity leads to impaired metabolic homeostasis and to hallmarks of liver disease. This review explores the current knowledge of LSEC transcription factors, covering their roles in LSEC development and maintenance of key phenotypic features, which, when disturbed, lead to loss of liver metabolic homeostasis and promote features of chronic liver diseases, such as non-alcoholic liver disease.

Journal article

Mobayen G, Smith K, Ediriwickrema K, Starke RD, Solomonidis EG, Laffan MA, Randi AM, McKinnon TAJet al., 2023, von Willebrand factor binds to angiopoietin-2 within endothelial cells and after release from Weibel-Palade bodies, Journal of Thrombosis and Haemostasis, Vol: 21, Pages: 1802-1812, ISSN: 1538-7836

BACKGROUND: The von Willebrand factor (VWF) is a multimeric plasma glycoprotein essential for hemostasis, inflammation, and angiogenesis. The majority of VWF is synthesized by endothelial cells (ECs) and stored in Weibel-Palade bodies (WPB). Among the range of proteins shown to co-localize to WPB is angiopoietin-2 (Angpt-2), a ligand of the receptor tyrosine kinase Tie-2. We have previously shown that VWF itself regulates angiogenesis, raising the hypothesis that some of the angiogenic activity of VWF may be mediated by its interaction with Angpt-2. METHODS: Static-binding assays were used to probe the interaction between Angpt-2 and VWF. Binding in media from cultured human umbilical vein ECs s and in plasma was determined by immunoprecipitation experiments. Immunofluorescence was used to detect the presence of Angpt-2 on VWF strings, and flow assays were used to investigate the effect on VWF function. RESULTS: Static-binding assays revealed that Angpt-2 bound to VWF with high affinity (KD,app ∼3 nM) in a pH and calcium-dependent manner. The interaction was localized to the VWF A1 domain. Co-immunoprecipitation experiments demonstrated that the complex persisted following stimulated secretion from ECs and was present in plasma. Angpt-2 was also visible on VWF strings on stimulated ECs. The VWF-Angpt-2 complex did not inhibit the binding of Angpt-2 to Tie-2 and did not significantly interfere with VWF-platelet capture. CONCLUSIONS: Together, these data demonstrate a direct binding interaction between Angpt-2 and VWF that persists after secretion. VWF may act to localize Angpt-2; further work is required to establish the functional consequences of this interaction.

Journal article

Lang M, Leung K-Y, Greene N, Malone K, Saginc Giannoustas G, Randi A, Kiprianos A, Maughan R, Pericleous C, Mason Jet al., 2023, The actions of methotrexate on endothelial cells are dependent on the shear stress-induced regulation of one carbon metabolism, Frontiers in Immunology, Vol: 14, Pages: 1-16, ISSN: 1664-3224

Objectives: The disease-modifying anti-rheumatic drug methotrexate (MTX) is recognized to reduce cardiovascular risk in patients with systemic inflammatory diseases. However, the molecular basis for these cardioprotective effects remains incompletely understood. This study evaluated the actions of low-dose MTX on the vascular endothelium.Methods: Human endothelial cells (EC) were studied under in vitro conditions relevant to inflammatory arthritis. These included culture in a pro-inflammatory microenvironment and exposure to fluid shear stress (FSS) using a parallel plate model. Respectively treated cells were analyzed by RNA sequencing and quantitative real-time PCR for gene expression, by immunoblotting for protein expression, by phosphokinase activity arrays, by flow cytometry for cell cycle analyses and by mass spectrometry to assess folate metabolite levels.Results: In static conditions, MTX was efficiently taken up by EC and caused cell cycle arrest concurrent with modulation of cell signaling pathways. These responses were reversed by folinic acid (FA), suggesting that OCM is a predominant target of MTX. Under FSS, MTX did not affect cell proliferation or pro-inflammatory gene expression. Exposure to FSS downregulated endothelial one carbon metabolism (OCM) as evidenced by decreased expression of key OCM genes and metabolites.Conclusion: We found that FSS significantly downregulated OCM and thereby rendered EC less susceptible to the effects of MTX treatment. The impact of shear stress on OCM suggested that MTX does not directly modulate endothelial function. The cardioprotective actions of MTX likely reflect direct actions on inflammatory cells and indirect benefit on the vascular endothelium.

Journal article

Bosseboeuf E, Chikh A, Chaker AB, Mitchell TP, Vignaraja D, Rajendrakumar R, Khambata RS, Nightingale TD, Mason JC, Randi AM, Ahluwalia A, Raimondi Cet al., 2023, Neuropilin-1 interacts with VE-cadherin and TGFBR2 to stabilize adherens junctions and prevent activation of endothelium under flow, SCIENCE SIGNALING, Vol: 16, ISSN: 1945-0877

Journal article

Sidonio RF, Bryant PC, Di Paola J, Hale S, Heiman M, Horowitz GS, Humphrey C, Jaffray J, Joyner LC, Kasthuri R, Konkle BA, Kouides PA, Montgomery R, Neeves K, Randi AM, Scappe N, Tarango C, Tickle K, Trapane P, Wang M, Waters B, Flood VHet al., 2023, Building the foundation for a community-generated national research blueprint for inherited bleeding disorders: research priorities for mucocutaneous bleeding disorders, Expert Review of Hematology, Vol: 16, Pages: 39-54, ISSN: 1747-4086

BACKGROUND: Excessive or abnormal mucocutaneous bleeding (MCB) may impact all aspects of the physical and psychosocial wellbeing of those who live with it (PWMCB). The evidence base for the optimal diagnosis and management of disorders such as inherited platelet disorders, hereditary hemorrhagic telangiectasia (HHT), hypermobility spectrum disorders (HSD), Ehlers-Danlos syndromes (EDS), and von Willebrand disease (VWD) remains thin with enormous potential for targeted research. RESEARCH DESIGN AND METHODS: National Hemophilia Foundation and American Thrombosis and Hemostasis Network initiated the development of a National Research Blueprint for Inherited Bleeding Disorders with extensive all-stakeholder consultations to identify the priorities of people with inherited bleeding disorders and those who care for them. They recruited multidisciplinary expert working groups (WG) to distill community-identified priorities into concrete research questions and score their feasibility, impact, and risk. RESULTS: WG2 detailed 38 high priority research questions concerning the biology of MCB, VWD, inherited qualitative platelet function defects, HDS/EDS, HHT, bleeding disorder of unknown cause, novel therapeutics, and aging. CONCLUSIONS: Improving our understanding of the basic biology of MCB, large cohort longitudinal natural history studies, collaboration, and creative approaches to novel therapeutics will be important in maximizing the benefit of future research for the entire MCB community.

Journal article

Schafer CM, Martin-Almedina S, Kurylowicz K, Dufton N, Osuna-Almagro L, Wu M-L, Johnson CF, Shah AV, Haskard DO, Buxton A, Willis E, Wheeler K, Turner S, Chlebicz M, Scott RP, Kovats S, Cleuren A, Birdsey GM, Randi AM, Griffin CTet al., 2023, Cytokine-Mediated Degradation of the Transcription Factor ERG Impacts the Pulmonary Vascular Response to Systemic Inflammatory Challenge., bioRxiv

BACKGROUND: During infectious diseases, pro-inflammatory cytokines transiently destabilize interactions between adjacent vascular endothelial cells (ECs) to facilitate the passage of immune molecules and cells into tissues. However, in the lung the resulting vascular hyperpermeability can lead to organ dysfunction. Previous work identified the transcription factor ERG as a master regulator of endothelial homeostasis. Here we investigate whether the sensitivity of pulmonary blood vessels to cytokine-induced destabilization is due to organotypic mechanisms affecting the ability of endothelial ERG to protect lung ECs from inflammatory injury. METHODS: Cytokine-dependent ubiquitination and proteasomal degradation of ERG was analyzed in cultured Human Umbilical Vein ECs (HUVECs). Systemic administration of TNFα or the bacterial cell wall component lipopolysaccharide (LPS) was used to cause a widespread inflammatory challenge in mice; ERG protein levels were assessed by immunoprecipitation, immunoblot, and immunofluorescence. Murine Erg deletion was genetically induced in ECs ( Erg fl/fl ;Cdh5(PAC)Cre ERT2 ), and multiple organs were analyzed by histology, immunostaining, and electron microscopy. RESULTS: In vitro, TNFα promoted the ubiquitination and degradation of ERG in HUVECs, which was blocked by the proteasomal inhibitor MG132. In vivo, systemic administration of TNFα or LPS resulted in a rapid and substantial degradation of ERG within lung ECs, but not ECs of the retina, heart, liver, or kidney. Pulmonary ERG was also downregulated in a murine model of influenza infection. Erg fl/fl ;Cdh5(PAC)-Cre ERT2 mice spontaneously recapitulated aspects of inflammatory challenges, including lung-predominant vascular hyperpermeability, immune cell recruitment, and fibrosis. These phenotypes were associated with a lung-specific decrease in the expression of Tek , a gene target of ERG previously implicated in maintaining pulmonary vascular stability during infl

Journal article

Kharwadkar R, Ulrich BJ, Chu M, Koh B, Hufford MM, Fu Y, Birdsey GM, Porse BT, Randi AM, Kaplan MHet al., 2023, ERG functionally overlaps with other Ets proteins in promoting TH9 cell expression of Il9 during allergic lung inflammation., Journal of Immunology, ISSN: 0022-1767

CD4+ TH cells develop into subsets that are specialized in the secretion of particular cytokines to mediate restricted types of inflammation and immune responses. Among the subsets that promote development of allergic inflammatory responses, IL-9-producing TH9 cells are regulated by a number of transcription factors. We have previously shown that the E26 transformation-specific (Ets) family members PU.1 and Ets translocation variant 5 (ETV5) function in parallel to regulate IL-9. In this study we identified a third member of the Ets family of transcription factors, Ets-related gene (ERG), that mediates IL-9 production in TH9 cells in the absence of PU.1 and ETV5. Chromatin immunoprecipitation assays revealed that ERG interaction at the Il9 promoter region is restricted to the TH9 lineage and is sustained during murine TH9 polarization. Knockdown or knockout of ERG during murine or human TH9 polarization in vitro led to a decrease in IL-9 production in TH9 cells. Deletion of ERG in vivo had modest effects on IL-9 production in vitro or in vivo. However, in the absence of PU.1 and ETV5, ERG was required for residual IL-9 production in vitro and for IL-9 production by lung-derived CD4 T cells in a mouse model of chronic allergic airway disease. Thus, ERG contributes to IL-9 regulation in TH9 cells.

Journal article

Gomez-Salinero J, Itkin T, Houghton S, Badwe C, Lin Y, Kalna V, Dufton N, Peghaire C, Yokoyama M, Wingo M, Lu T, Li G, Xiang JZ, Hsu Y-MS, Redmond D, Schreiner R, Birdsey G, Randi A, Rafii Set al., 2022, Cooperative ETS transcription factors enforce adult endothelial cell fate and cardiovascular homeostasis., Nature Cardiovascular Research, Vol: 1, Pages: 882-899, ISSN: 2731-0590

Current dogma dictates that, during adulthood, endothelial cells (ECs) are locked in an immutable stable homeostatic state. By contrast, herein we show that maintenance of EC fate and function are linked and active processes, which depend on the constitutive cooperativity of only two ETS transcription factors (TFs), ERG and Fli1. Although deletion of either ERG or Fli1 manifests subtle vascular dysfunction, their combined genetic deletion in adult ECs results in acute vasculopathy and multi-organ failure, due to loss of EC fate and integrity, hyperinflammation and spontaneous thrombosis, leading to death. ERG and Fli1 co-deficiency causes rapid transcriptional silencing of pan and organotypic vascular core genes, with dysregulation of inflammation and coagulation pathways. Vascular hyperinflammation leads to impaired hematopoiesis with myeloid skewing. Accordingly, enforced ERG and FLI1 expression in adult human mesenchymal stromal cells activates vascular programs and functionality, enabling in vivo engraftment of a perfusable vascular network. Genome-wide association study analysis identified vascular diseases that are associated with FLI1/ERG mutations. Constitutive expression of ERG and Fli1 upholds EC fate, physiological function and resilience in adult vasculature, whereas their functional loss can contribute to systemic human diseases.

Journal article

King O, Cruz-Moreira D, Sayed A, Kermani F, Kit-Anan W, Sunyovszki I, Wang BX, Downing B, Fourre J, Hachim D, Randi AM, Stevens MM, Rasponi M, Terracciano CMet al., 2022, Functional microvascularization of human myocardium in vitro, Cell Reports: Methods, Vol: 2, Pages: 1-16, ISSN: 2667-2375

In this study, we report static and perfused models of human myocardial-microvascular interaction. In static culture, we observe distinct regulation of electrophysiology of human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CMs) in co-culture with human cardiac microvascular endothelial cells (hCMVECs) and human left ventricular fibroblasts (hLVFBs), including modification of beating rate, action potential, calcium handling, and pro-arrhythmic substrate. Within a heart-on-a-chip model, we subject this three-dimensional (3D) co-culture to microfluidic perfusion and vasculogenic growth factors to induce spontaneous assembly of perfusable myocardial microvasculature. Live imaging of red blood cells within myocardial microvasculature reveals pulsatile flow generated by beating hiPSC-CMs. This study therefore demonstrates a functionally vascularized in vitro model of human myocardium with widespread potential applications in basic and translational research.

Journal article

D'Amico G, Fernandez I, Gómez-Escudero J, Kim H, Maniati E, Azman MS, Mardakheh FK, Serrels B, Serrels A, Parsons M, Squire A, Birdsey GM, Randi AM, Bolado-Carrancio A, Gangeswaran R, Reynolds LE, Bodrug N, Wang Y, Wang J, Meier P, Hodivala-Dilke KMet al., 2022, ERG activity is regulated by endothelial FAK coupling with TRIM25/USP9x in vascular patterning, Development, Vol: 149, ISSN: 0950-1991

Precise vascular patterning is critical for normal growth and development. The ERG transcription factor drives Delta like ligand 4 (DLL4)/Notch signalling and is thought to act as pivotal regulators of endothelial cell (EC) dynamics and developmental angiogenesis. However, molecular regulation of ERG activity remains obscure. Using a series of EC specific Focal Adhesion Kinase (FAK)-knockout (KO) and point-mutant FAK-knockin mice, we show that loss of ECFAK, its kinase activity or phosphorylation at FAK-Y397, but not FAK-Y861, reduces ERG and DLL4 expression levels together with concomitant aberrations in vascular patterning. Rapid Immunoprecipitation Mass Spectrometry of Endogenous Proteins identified that endothelial nuclear-FAK interacts with the de-ubiquitinase USP9x and the ubiquitin ligase TRIM25 enzymes. Further in silico analysis corroborates that ERG interacts with USP9x and TRIM25. Moreover, ERG levels are reduced in FAKKO ECs via a ubiquitin-mediated post-translational modification programme involving USP9x and TRIM25. Re-expression of ERG in vivo and in vitro rescues the aberrant vessel sprouting defects observed in the absence of ECFAK. Our findings identify ECFAK as a regulator of retinal vascular patterning by controlling ERG protein degradation via TRIM25/USP9x.

Journal article

Paschalaki K, Rossios C, Pericleous C, MacLeod M, Rothery S, Donaldson G, Wedzicha J, Gorgoulis V, Randi A, Barnes Pet 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.

Journal article

Smadja DM, Mentzer SJ, Fontenay M, Laffan MA, Ackermann M, Helms J, Jonigk D, Chocron R, Pier GB, Gendron N, Pons S, Diehl J-L, Margadant C, Guerin C, Huijbers EJM, Philippe A, Chapuis N, Nowak-Sliwinska P, Karagiannidis C, Sanchez O, Kuempers P, Skurnik D, Randi AM, Griffioen AWet al., 2021, COVID-19 is a systemic vascular hemopathy: insight for mechanistic and clinical aspects, ANGIOGENESIS, Vol: 24, Pages: 755-788, ISSN: 0969-6970

Journal article

McCracken IR, Saginc G, He L, Huseynov A, Daniels A, Fletcher S, Peghaire C, Kalna V, Andaloussi-Mäe M, Muhl L, Craig NM, Griffiths SJ, Haas JG, Tait-Burkard C, Lendahl U, Birdsey GM, Betsholtz C, Noseda M, Baker AH, Randi AMet al., 2021, Lack of evidence of ACE2 expression and replicative infection by SARSCoV-2 in human endothelial cells, Circulation, Vol: 143, Pages: 865-868, ISSN: 0009-7322

Journal article

Dufton NP, Peghaire CR, Osuna-Almagro L, Raimondi C, Kalna V, Chauhan A, Webb G, Yang Y, Birdsey GM, Lalor P, Mason JC, Adams DH, Randi AMet al., 2020, Dynamic regulation of canonical TGF beta signalling by endothelial transcription factor ERG protects from liver fibrogenesis (vol 31, pg 450, 2017), Nature Communications, Vol: 11, Pages: 1-1, ISSN: 2041-1723

Journal article

Peghaire C, Dufton N, Lang M, Salles I, Ahnstroem J, Kalna V, Raimondi C, Pericleous C, Inuabasi L, Kiseleva R, Muzykantov V, Mason J, Birdsey G, Randi Aet al., 2019, The transcription factor ERG regulates a low shear stress-induced anti-thrombotic pathway in the microvasculature, Nature Communications, Vol: 10, Pages: 1-17, ISSN: 2041-1723

Endothelial cells actively maintain an anti-thrombotic environment; loss of this protective function may lead to thrombosis and systemic coagulopathy. The transcription factor ERG is essential to maintain endothelial homeostasis. Here we show that inducible endothelial ERG deletion (ErgiEC-KO) in mice is associated with spontaneous thrombosis, hemorrhages and systemic coagulopathy. We find that ERG drives transcription of the anti-coagulant thrombomodulin (TM), as shown by reporter assays and chromatin immunoprecipitation. TM expression is regulated by shear stress (SS) via Krüppel-like factor 2 (KLF2). In vitro, ERG regulates TM expression under low SS conditions, by facilitating KLF2 binding to the TM promoter. However, ERG is dispensable for TM expression in high SS conditions. In ErgiEC-KO mice, TM expression is decreased in liver and lung microvasculature exposed to low SS but not in blood vessels exposed to high SS. Our study identifies an endogenous, vascular bed- specific anti-coagulant pathway in microvasculature exposed to low SS.

Journal article

Lasch M, Kleinert EC, Meister S, Kumaraswami K, Buchheim J-I, Grantzow T, Lautz T, Salpisti S, Fischer S, Troidl K, Fleming I, Randi AM, Sperandio M, Preissner KT, Deindl Eet al., 2019, Extracellular RNA released due to shear stress controls natural bypass growth by mediating mechanotransduction in mice, BLOOD, Vol: 134, Pages: 1469-1479, ISSN: 0006-4971

Journal article

Smadja DM, Melero-Martin JM, Eikenboom J, Bowman M, Sabatier F, Randi AMet al., 2019, Standardization of methods to quantify and culture endothelial colony-forming cells derived from peripheral blood Position paper from the International Society on Thrombosis and Haemostasis SSC, JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Vol: 17, Pages: 1190-1194, ISSN: 1538-7933

Journal article

Ishihara J, Ishihara A, Starke RD, Peghaire CR, Smith KE, McKinnon TAJ, Tabata Y, Sasaki K, White MJV, Fukunaga K, Laffan MA, Lutolf MP, Randi AM, Hubbell JAet al., 2019, The heparin binding domain of von Willebrand factor binds to growth factors and promotes angiogenesis in wound healing, Blood, Vol: 133, Pages: 2559-2569, ISSN: 0006-4971

During wound healing, the distribution, availability, and signaling of growth factors (GFs) are orchestrated by their binding to extracellular matrix components in the wound microenvironment. Extracellular matrix proteins have been shown to modulate angiogenesis and promote wound healing through GF binding. The hemostatic protein von Willebrand factor (VWF) released by endothelial cells (ECs) in plasma and in the subendothelial matrix has been shown to regulate angiogenesis; this function is relevant to patients in whom VWF deficiency or dysfunction is associated with vascular malformations. Here, we show that VWF deficiency in mice causes delayed wound healing accompanied by decreased angiogenesis and decreased amounts of angiogenic GFs in the wound. We show that in vitro VWF binds to several GFs, including vascular endothelial growth factor-A (VEGF-A) isoforms and platelet-derived growth factor-BB (PDGF-BB), mainly through the heparin-binding domain (HBD) within the VWF A1 domain. VWF also binds to VEGF-A and fibroblast growth factor-2 (FGF-2) in human plasma and colocalizes with VEGF-A in ECs. Incorporation of the VWF A1 HBD into fibrin matrices enables sequestration and slow release of incorporated GFs. In vivo, VWF A1 HBD-functionalized fibrin matrices increased angiogenesis and GF retention in VWF-deficient mice. Treatment of chronic skin wounds in diabetic mice with VEGF-A165 and PDGF-BB incorporated within VWF A1 HBD-functionalized fibrin matrices accelerated wound healing, with increased angiogenesis and smooth muscle cell proliferation. Therefore, the VWF A1 HBD can function as a GF reservoir, leading to effective angiogenesis and tissue regeneration.

Journal article

Kalna V, Yang Y, Peghaire C, Frudd K, hannah R, Shah A, Osuna Almagro L, Boyle J, gottgens B, Ferrer J, Randi A, Birdsey Get al., 2019, The transcription factor ERG regulates super-enhancers associated with an endothelial-specific gene expression program, Circulation Research, Vol: 124, Pages: 1337-1349, ISSN: 0009-7330

Rationale:The ETS (E-26 transformation-specific) transcription factor ERG (ETS-related gene) is essential for endothelial homeostasis, driving expression of lineage genes and repressing proinflammatory genes. Loss of ERG expression is associated with diseases including atherosclerosis. ERG’s homeostatic function is lineage-specific, because aberrant ERG expression in cancer is oncogenic. The molecular basis for ERG lineage-specific activity is unknown. Transcriptional regulation of lineage specificity is linked to enhancer clusters (super-enhancers).Objective:To investigate whether ERG regulates endothelial-specific gene expression via super-enhancers.Methods and Results:Chromatin immunoprecipitation with high-throughput sequencing in human umbilical vein endothelial cells showed that ERG binds 93% of super-enhancers ranked according to H3K27ac, a mark of active chromatin. These were associated with endothelial genes such as DLL4 (Delta-like protein 4), CLDN5 (claudin-5), VWF (von Willebrand factor), and CDH5 (VE-cadherin). Comparison between human umbilical vein endothelial cell and prostate cancer TMPRSS2 (transmembrane protease, serine-2):ERG fusion-positive human prostate epithelial cancer cell line (VCaP) cells revealed distinctive lineage-specific transcriptome and super-enhancer profiles. At a subset of endothelial super-enhancers (including DLL4 and CLDN5), loss of ERG results in significant reduction in gene expression which correlates with decreased enrichment of H3K27ac and MED (Mediator complex subunit)-1, and reduced recruitment of acetyltransferase p300. At these super-enhancers, co-occupancy of GATA2 (GATA-binding protein 2) and AP-1 (activator protein 1) is significantly lower compared with super-enhancers that remained constant following ERG inhibition. These data suggest distinct mechanisms of super-enhancer regulation in endothelial cells and highlight the unique role of ERG in controlling a core subset of super-enhancers. Most disease-assoc

Journal article

Issitt T, Bosseboeuf E, De Winter N, Dufton N, Gestri G, Senatore V, Chikh A, Randi AM, Raimondi Cet al., 2019, Neuropilin-1 controls endothelial homeostasis by regulating mitochondrial function and iron-dependent oxidative stress via ABCB8, iScience, Vol: 11, Pages: 205-223, ISSN: 2589-0042

The transmembrane protein Neuropilin-1 (NRP1) promotes vascular endothelial growth factor (VEGF) and extracellular matrix signalling in endothelial cells (ECs). Although it is established that NRP1 is essential for angiogenesis, little is known about its role in EC homeostasis. Here, we report that NRP1 promotes mitochondrial function in ECs by preventing iron accumulation and iron-induced oxidative stress through a VEGF-independent mechanism in non-angiogenic ECs. Furthermore, NRP1-deficient ECs have reduced growth and show the hallmarks of cellular senescence. We show that a subcellular pool of NRP1 localises in mitochondria and interacts with the mitochondrial transporter ATP-binding-cassette-B8 (ABCB8). NRP1 loss reduces ABCB8 levels, resulting in iron accumulation, iron-induced mitochondrial superoxide production and iron-dependent EC senescence. Treatment of NRP1-deficient ECs with the mitochondria-targeted antioxidant compound mitoTEMPO or with the iron chelator deferoxamine restores mitochondrial activity, inhibits superoxide production and protects from cellular senescence. This finding identifies an unexpected role of NRP1 in EC homeostasis.

Journal article

Randi AM, 2018, Von Willebrand Factor in Angiogenesis and Angiodysplasia, 60th Annual Meeting of the American-Society-of-Hematology (ASH), Publisher: AMER SOC HEMATOLOGY, ISSN: 0006-4971

Conference paper

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.

Journal article

Nowak-Sliwinska P, Alitalo K, Allen E, Anisimov A, Aplin AC, Auerbach R, Augustin HG, Bates DO, van Beijnum JR, Bender RHF, Bergers G, Bikfalvi A, Bischoff J, Böck BC, Brooks PC, Bussolino F, Cakir B, Carmeliet P, Castranova D, Cimpean AM, Cleaver O, Coukos G, Davis GE, De Palma M, Dimberg A, Dings RPM, Djonov V, Dudley AC, Dufton NP, Fendt S-M, Ferrara N, Fruttiger M, Fukumura D, Ghesquière B, Gong Y, Griffin RJ, Harris AL, Hughes CCW, Hultgren NW, Iruela-Arispe ML, Irving M, Jain RK, Kalluri R, Kalucka J, Kerbel RS, Kitajewski J, Klaassen I, Kleinmann HK, Koolwijk P, Kuczynski E, Kwak BR, Marien K, Melero-Martin JM, Munn LL, Nicosia RF, Noel A, Nurro J, Olsson A-K, Petrova TV, Pietras K, Pili R, Pollard JW, Post MJ, Quax PHA, Rabinovich GA, Raica M, Randi AM, Ribatti D, Ruegg C, Schlingemann RO, Schulte-Merker S, Smith LEH, Song JW, Stacker SA, Stalin J, Stratman AN, Van de Velde M, van Hinsbergh VWM, Vermeulen PB, Waltenberger J, Weinstein BM, Xin H, Yetkin-Arik B, Yla-Herttuala S, Yoder MC, Griffioen AWet al., 2018, Consensus guidelines for the use and interpretation of angiogenesis assays, Angiogenesis, Vol: 21, Pages: 425-532, ISSN: 0969-6970

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.

Journal article

Randi AM, Smith KE, Castaman G, 2018, von Willebrand factor regulation of blood vessel formation, Blood, Vol: 132, Pages: 132-140, ISSN: 1528-0020

Several important physiological processes, from permeability to inflammation to haemostasis, take place at the vessel wall and are regulated by endothelial cells (EC). Thus, proteins that have been identified as regulators of one process are increasingly found to be involved in other vascular functions. Such is the case for Von Willebrand Factor (VWF), a large glycoprotein best known for its critical role in haemostasis. In vitro and in vivo studies have shown that lack of VWF causes enhanced vascularisation, both constitutively and following ischemia. This evidence is supported by studies on blood outgrowth endothelial cells (BOEC) from patients with lack of VWF synthesis (type 3 von Willebrand disease [VWD]). The molecular pathways are likely to involve VWF binding partners, such as integrin αvβ3, and components of Weibel Palade bodies (WPB), such as Angiopoietin-2 and Galectin-3, whose storage is regulated by VWF; these converge on the master regulator of angiogenesis and endothelial homeostasis, vascular endothelial growth factor (VEGF) signalling. Recent studies suggest that the roles of VWF may be tissue-specific. The ability of VWF to regulate angiogenesis has clinical implications for a subset of VWD patients with severe, intractable gastrointestinal bleeding due to vascular malformations. In this article, we review the evidence showing that VWF is involved in blood vessel formation, discuss the role of VWF high molecular weight multimers in regulating angiogenesis, and the value of studies on BOEC in developing a precision medicine approach to validate novel treatments for angiodysplasia in congenital VWD and acquired von Willebrand syndrome.

Journal article

Schmid CD, Schledzewski K, Mogler C, Waldburger N, Kalna V, Marx A, Randi AM, Geraud C, Goerdt S, Koch P-Set al., 2018, GPR182 is a novel marker for sinusoidal endothelial differentiation with distinct GPCR signaling activity in vitro, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Vol: 497, Pages: 32-38, ISSN: 0006-291X

Journal article

Dufton NP, peghaire CR, Osuna-Almagro L, Raimondi C, Kalna V, Chuahan A, Webb G, Yang Y, Birdsey GM, Lalor P, Mason JC, Adams D, Randi AMet al., 2017, Dynamic regulation of canonical TGFβ signaling by endothelial transcription factor ERG protects from liver fibrogenesis, Nature Communications, Vol: 8, Pages: 1-14, ISSN: 2041-1723

The role of the endothelium in protecting from chronic liver disease and TGFβ-mediated fibrosis remains unclear. Here we describe how the endothelial transcription factor ETS-related gene (ERG) promotes liver homoeostasis by controlling canonical TGFβ-SMAD signalling, driving the SMAD1 pathway while repressing SMAD3 activity. Molecular analysis shows that ERG binds to SMAD3, restricting its access to DNA. Ablation of ERG expression results in endothelial-to-mesenchymal transition (EndMT) and spontaneous liver fibrogenesis in EC-specific constitutive hemi-deficient (ErgcEC-Het) and inducible homozygous deficient mice (ErgiEC-KO), in a SMAD3-dependent manner. Acute administration of the TNF-α inhibitor etanercept inhibits carbon tetrachloride (CCL4)-induced fibrogenesis in an ERG-dependent manner in mice. Decreased ERG expression also correlates with EndMT in tissues from patients with end-stage liver fibrosis. These studies identify a pathogenic mechanism where loss of ERG causes endothelial-dependent liver fibrogenesis via regulation of SMAD2/3. Moreover, ERG represents a promising candidate biomarker for assessing EndMT in liver disease.

Journal article

Perbellini F, Watson SA, Scigliano M, Alayoubi S, Tkach S, Bardi I, Quaife N, Kane C, Dufton NP, Simon A, Sikkel MB, Faggian G, Randi AM, Gorelik J, Harding S, Terracciano CMNet al., 2017, Investigation of cardiac fibroblasts using myocardial slices, Cardiovascular Research, Vol: 114, Pages: 77-89, ISSN: 1755-3245

AimsCardiac fibroblasts (CFs) are considered the principal regulators of cardiac fibrosis. Factors that influence CF activity are difficult to determine. When isolated and cultured in vitro, CFs undergo rapid phenotypic changes including increased expression of α-SMA. Here we describe a new model to study CFs and their response to pharmacological and mechanical stimuli using in vitro cultured mouse, dog and human myocardial slices.Methods and resultsUnloading of myocardial slices induced CF proliferation without α-SMA expression up to 7 days in culture. CFs migrating onto the culture plastic support or cultured on glass expressed αSMA within 3 days. The cells on the slice remained αSMA(−) despite transforming growth factor-β (20 ng/ml) or angiotensin II (200 µM) stimulation. When diastolic load was applied to myocardial slices using A-shaped stretchers, CF proliferation was significantly prevented at Days 3 and 7 (P < 0.001).ConclusionsMyocardial slices allow the study of CFs in a multicellular environment and may be used to effectively study mechanisms of cardiac fibrosis and potential targets.

Journal article

Paschalaki KE, Zampetaki A, Baker JR, Birrell MA, Starke RD, Belvisi MG, Donnelly LE, Mayr M, Randi AM, Barnes PJet al., 2017, Downregulation of MicroRNA-126 Augments DNA Damage Response in Cigarette Smokers and COPD Patients., Am J Respir Crit Care Med

Journal article

Shah AV, Birdsey GM, Peghaire C, Pitulescu ME, Dufton NP, Yang Y, Weinberg I, Osuna Almagro L, Payne L, Mason JC, Gerhardt H, Adams RH, Randi AMet al., 2017, The endothelial transcription factor ERG mediates Angiopoietin-1-dependent control of Notch signalling and vascular stability, Nature Communications, Vol: 8, Pages: 1-16, ISSN: 2041-1723

Notch and Angiopoietin-1 (Ang1)/Tie2 pathways are crucial for vascular maturation and stability. Here we identify the transcription factor ERG as a key regulator of endothelial Notch signalling. We show that ERG controls the balance between Notch ligands by driving Delta-like ligand 4 (Dll4) while repressing Jagged1 (Jag1) expression. In vivo, this regulation occurs selectively in the maturing plexus of the mouse developing retina, where Ang1/Tie2 signalling is active. We find that ERG mediates Ang1-dependent regulation of Notch ligands and is required for the stabilizing effects of Ang1 in vivo. We show that Ang1 induces ERG phosphorylation in a phosphoinositide 3-kinase (PI3K)/Akt-dependent manner, resulting in ERG enrichment at Dll4 promoter and multiple enhancers. Finally, we demonstrate that ERG directly interacts with Notch intracellular domain (NICD) and β-catenin and is required for Ang1-dependent β-catenin recruitment at the Dll4 locus. We propose that ERG coordinates Ang1, β-catenin and Notch signalling to promote vascular stability.

Journal article

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