Von Willebrand factor, angiodysplasia and angiogenesis

The large multimeric glycoprotein Von Willebrand factor (VWF) is best known for its role in haemostasis; however in recent years other functions of VWF have been identified, indicating that this protein is involved in multiple vascular processes. We recently described a new role for VWF in controlling angiogenesis, which may have significant clinical implications for patients with Von Willebrand disease (VWD), a genetic or acquired condition caused by the deficiency or dysfunction of VWF. VWD can be associated with angiodysplasia, a condition of degenerative blood vessels often present in the gastrointestinal tract, linked to dysregulated angiogenesis. Angiodysplasia can cause severe intractable bleeding, often refractory to conventional VWD treatments. In this review we summarise the evidence showing that VWF controls angiogenesis, and review the angiogenic pathways which have been implicated in this process. We discuss the possible mechanisms though which VWF regulates angiopoietin-2 (Ang-2) and integrin αvβ3, leading to signalling through vascular endothelial growth factor receptor-2 (VEGFR2), one of the most potent activators of angiogenesis. We also review the evidence that links VWF with angiodysplasia, and how the newly identified function of VWF in controlling angiogenesis may pave the way for the development of novel therapies for the treatment of angiodysplasia in congenital VWD and in acquired conditions such as Heyde syndrome

Neuropilin-1 Controls Endothelial Homeostasis by Regulating Mitochondrial Function and Iron-Dependent Oxidative Stress.

The transmembrane protein neuropilin-1 (NRP1) promotes vascular endothelial growth factor (VEGF) and extracellular matrix signaling 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 localizes 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

Liver sinusoidal endothelial transcription factors in metabolic homeostasis and disease.

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

Regulation of endothelial homeostasis, vascular development and angiogenesis by the transcription factor ERG

Over the last few years, the ETS transcription factor ERG has emerged as a major regulator of endothelial function. Multiple studies have shown that ERG plays a crucial role in promoting angiogenesis and vascular stability during development and after birth. In the mature vasculature ERG also functions to maintain endothelial homeostasis, by transactivating genes involved in key endothelial functions, while repressing expression of pro-inflammatory genes. Its homeostatic role is lineage-specific, since ectopic expression of ERG in non-endothelial tissues such as prostate is detrimental and contributes to oncogenesis. This review summarises the main roles and pathways controlled by ERG in the vascular endothelium, its transcriptional targets and its functional partners and the emerging evidence on the pathways regulating ERG's activity and expression

von Willebrand factor binds to angiopoietin-2 within endothelial cells and after release from Weibel-Palade bodies

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

The actions of methotrexate on endothelial cells are dependent on the shear stress-induced regulation of one carbon metabolism

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

Author Correction: Dynamic regulation of canonical TGFβ signalling by endothelial transcription factor ERG protects from liver fibrogenesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Cytokine-mediated degradation of the transcription factor ERG impacts the pulmonary vascular response to systemic inflammatory challenge

BACKGROUND: During infectious diseases, proinflammatory 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 (erythroblast transformation-specific-related gene) 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 were analyzed in cultured HUVECs (human umbilical vein ECs). Systemic administration of TNFα (tumor necrosis factor alpha) or the bacterial cell wall component lipopolysaccharide 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 (Ergfl/fl;Cdh5[PAC]-CreERT2), 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 lipopolysaccharide 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. Ergfl/fl;Cdh5(PAC)-CreERT2 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 inflammation. CONCLUSIONS: Collectively, our data highlight a unique role for ERG in pulmonary vascular function. We propose that cytokine-induced ERG degradation and subsequent transcriptional changes in lung ECs play critical roles in the destabilization of pulmonary blood vessels during infectious diseases

An Anti-Human ICAM-1 Antibody Inhibits Rhinovirus-Induced Exacerbations of Lung Inflammation

Human rhinoviruses (HRV) cause the majority of common colds and acute exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Effective therapies are urgently needed, but no licensed treatments or vaccines currently exist. Of the 100 identified serotypes, ∼90% bind domain 1 of human intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor, making this an attractive target for development of therapies; however, ICAM-1 domain 1 is also required for host defence and regulation of cell trafficking, principally via its major ligand LFA-1. Using a mouse anti-human ICAM-1 antibody (14C11) that specifically binds domain 1 of human ICAM-1, we show that 14C11 administered topically or systemically prevented entry of two major groups of rhinoviruses, HRV16 and HRV14, and reduced cellular inflammation, pro-inflammatory cytokine induction and virus load in vivo. 14C11 also reduced cellular inflammation and Th2 cytokine/chemokine production in a model of major group HRV-induced asthma exacerbation. Interestingly, 14C11 did not prevent cell adhesion via human ICAM-1/LFA-1 interactions in vitro, suggesting the epitope targeted by 14C11 was specific for viral entry. Thus a human ICAM-1 domain-1-specific antibody can prevent major group HRV entry and induction of airway inflammation in vivo