Koralia E. Paschalaki

Endothelial von Willebrand factor regulates angiogenesis

The regulation of blood vessel formation is of fundamental importance to many physiological processes, and angiogenesis is a major area for novel therapeutic approaches to diseases from ischemia to cancer. A poorly understood clinical manifestation of pathological angiogenesis is angiodysplasia, vascular malformations that cause severe gastrointestinal bleeding. Angiodysplasia can be associated with von Willebrand disease (VWD), the most common bleeding disorder in man. VWD is caused by a defect or deficiency in von Willebrand factor (VWF), a glycoprotein essential for normal hemostasis that is involved in inflammation. We hypothesized that VWF regulates angiogenesis. Inhibition of VWF expression by short interfering RNA (siRNA) in endothelial cells (ECs) caused increased in vitro angiogenesis and increased vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2)-dependent proliferation and migration, coupled to decreased integrin αvβ3 levels and increased angiopoietin (Ang)-2 release. ECs expanded from blood-derived endothelial progenitor cells of VWD patients confirmed these results. Finally, 2 different approaches, in situ and in vivo, showed increased vascularization in VWF-deficient mice. We therefore identify a new function of VWF in ECs, which confirms VWF as a protein with multiple vascular roles and defines a novel link between hemostasis and angiogenesis. These results may have important consequences for the management of VWD, with potential therapeutic implications for vascular diseases.

Cryptogenic and Secondary Organizing Pneumonia: Clinical Presentation, Radiographic Findings, Treatment Response, and Prognosis

BACKGROUND Organizing pneumonia (OP) is a distinct clinical and pathologic entity. This condition can be cryptogenic (COP) or secondary to other known causes (secondary OP). In the present study, we reviewed the features associated with COP and secondary OP in patients from two teaching hospitals. METHODS The medical records of 61 patients with biopsy-proven OP were retrospectively reviewed. Forty patients were diagnosed with COP and 21 patients with secondary OP. The clinical presentation, radiographic studies, pulmonary function tests (PFTs), laboratory data, BAL findings, treatment, and outcome were analyzed. RESULTS The mean age at presentation was 60.46 ± 13.57 years. Malaise, cough, fever, dyspnea, bilateral alveolar infiltrates, and a restrictive pattern were the most common symptoms and findings. BAL lymphocytosis was observed in 43.8% of patients with OP. The relapse rate and mortality rate after 1 year of follow-up were 37.8% and 9.4%, respectively. The in-hospital mortality was 5.7%. The clinical presentation and radiographic findings did not differ significantly between patients with COP and secondary OP. A mixed PFT pattern (obstructive and restrictive physiology) and lower blood levels of serum sodium, serum potassium, platelets, albumin, protein, and pH were observed among patients with secondary OP. Higher blood levels of creatinine, bilirubin, Paco₂, and BAL lymphocytes were also more common among patients with secondary OP. There were no differences in the relapse rate or mortality between patients with COP and secondary OP. The 1-year mortality correlated with an elevated erythrocyte sedimentation rate, low albumin, and low hemoglobin levels. CONCLUSIONS The clinical and radiographic findings in patients with COP and secondary OP are similar and nonspecific. Although certain laboratory abnormalities are more common in secondary OP and can be associated with worse prognosis, they are likely due to the underlying disease. COP and secondary OP have similar treatment response, relapse rates, and mortality.

Cellular and molecular basis of von Willebrand disease: studies on blood outgrowth endothelial cells

Von Willebrand disease (VWD) is a heterogeneous bleeding disorder caused by decrease or dysfunction of von Willebrand factor (VWF). A wide range of mutations in the VWF gene have been characterized; however, their cellular consequences are still poorly understood. Here we have used a recently developed approach to study the molecular and cellular basis of VWD. We isolated blood outgrowth endothelial cells (BOECs) from peripheral blood of 4 type 1 VWD and 4 type 2 VWD patients and 9 healthy controls. We confirmed the endothelial lineage of BOECs, then measured VWF messenger RNA (mRNA) and protein levels (before and after stimulation) and VWF multimers. Decreased mRNA levels were predictive of plasma VWF levels in type 1 VWD, confirming a defect in VWF synthesis. However, BOECs from this group of patients also showed defects in processing, storage, and/or secretion of VWF. Levels of VWF mRNA and protein were normal in BOECs from 3 type 2 VWD patients, supporting the dysfunctional VWF model. However, 1 type 2M patient showed decreased VWF synthesis and storage, indicating a complex cellular defect. These results demonstrate for the first time that isolation of endothelial cells from VWD patients provides novel insight into cellular mechanisms of the disease.

Low Dose Iron Treatments Induce a DNA Damage Response in Human Endothelial Cells within Minutes.

Background Spontaneous reports from patients able to report vascular sequelae in real time, and recognition that serum non transferrin bound iron may reach or exceed 10μmol/L in the blood stream after iron tablets or infusions, led us to hypothesize that conventional iron treatments may provoke acute vascular injury. This prompted us to examine whether a phenotype could be observed in normal human endothelial cells treated with low dose iron. Methodology Confluent primary human endothelial cells (EC) were treated with filter-sterilized iron (II) citrate or fresh media for RNA sequencing and validation studies. RNA transcript profiles were evaluated 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 treatment types. Principal Findings Rapid changes in RNA transcript profiles were observed in endothelial cells treated with 10μmol/L iron (II) citrate, compared to media-treated cells. Clustering for Gene Ontology (GO) performed on all differentially expressed genes revealed significant differences in biological process terms between iron and media-treated EC, whereas 10 sets of an equivalent number of randomly selected genes from the respective EC gene datasets showed no significant differences in any GO terms. After 1 hour, differentially expressed genes clustered to 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 stimulus most significantly through DNA repair genes FANCG, BLM, and H2AFX. Comet assays demonstrated that 10μM iron treatment elicited DNA damage within 1 hour. This was accompanied by a brisk DNA damage response pulse, as ascertained by the development of DNA damage response (DDR) foci, and p53 stabilization. Significance These data suggest that low dose iron treatments are sufficient to modify the vascular endothelium, and induce a DNA damage response.

Pathogen Sensing Pathways in Human Embryonic Stem Cell Derived-Endothelial Cells: Role of NOD1 Receptors

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.

An autologous endothelial cell:peripheral blood mononuclear cell assay that detects cytokine storm responses to biologics

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

59 Endothelial colony forming cells (ECFC) are senescent and dysfunctional in COPD due to reduced sirtuin-1 levels

Introduction Cardiovascular disease (CVD) is a major cause of death in chronic obstructive pulmonary disease (COPD). Numerous studies describe clinical evidence of endothelial dysfunction in COPD but the molecular pathways which link COPD and CVD remain unclear. Endothelial colony forming cells (ECFC) could serve as a research tool to investigate endothelial defects in COPD patients. Aim and Objectives To examine whether ECFC from COPD patients exhibit dysfunctional characteristics, illustrating the underlying molecular process of endothelial dysfunction in COPD. Methods ECFC were isolated from peripheral blood samples received from 16 healthy non-smoking volunteers (age±SEM, 57±2.7 yr), 10 healthy smokers (57±2.6 yr) and 16 COPD patients (67±1.6 yr). The mononuclear fraction was placed in culture in the presence of endothelial growth factors. ECFC appeared between day 7 and 24, as characteristic cobblestone monolayers. The cells were grown to confluence in T-25 or T-75 flasks and used at passages 4 to 6 for all experiments. Endothelial senescence was measured by senescence-associated β-galactosidase (SA-β-Gal) activity and sirtuin (SIRT) 1 protein levels by Western blotting. Results ECFC from healthy smokers and COPD patients displayed significantly increased senescence and reduced SIRT1 protein levels compared to healthy non-smoking subjects. SIRT1 protein levels negatively correlated with endothelial senescence. Conclusions The results from our study demonstrate that ECFC from smokers and COPD patients display epigenetic molecular dysfunctions linked to increased senescence. These defects may contribute to endothelial dysfunction and cardiovascular events.

Downregulation of MicroRNA-126 Augments DNA Damage Response in Cigarette Smokers and COPD Patients

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T5 Circulating endothelial progenitor cells in smokers and patients with COPD are dysfunctional due to increased DNA damage and senescence

Introduction Cardiovascular disease (CVD) is a major cause of death in smokers, particularly in patients with chronic obstructive pulmonary disease (COPD). Circulating endothelial progenitor cells (EPC) are required for endothelial homeostasis, and their dysfunction contributes to CVD. DNA damage has been recognised as an important contributor to CVD. Our aim was to investigate whether EPC from smokers and COPD patients are dysfunctional, and the role of DNA damage pathways in mediating endothelial dysfunction in these patients. Methods To investigate EPC dysfunction in smokers, we isolated and expanded blood outgrowth endothelial cells (BOEC) from peripheral blood samples of healthy non-smokers, healthy smokers and COPD patients. Endothelial senescence was measured by senescence-associated β-galactosidase (SA-β-Gal) activity. Expression of sirtuin (SIRT)-1, p16, p21, γ-H2AX and 53BP1 were measured by Western blotting and/or immunofluorescence confocal microscopy. SIRT1 activity was measured using a SIRT1 fluorescent activity assay kit. To investigate angiogenesis in vivo, BOEC were labelled with Vybrant DiI Cell-Labelling Solution, mixed with Matrigel and injected subcutaneously into the back of NOD. CB17-Prkdcscid/NcrCrl mice. Seven days later, the mice were sacrificed and the plugs were cryosectioned. Results BOEC from smokers and COPD patients showed increased DNA double-strand breaks (measured by γ-H2AX, 53BP1) and senescence (senescence associated-β-galactosidase activity, p16 and p21 levels) compared to non-smokers. Senescence negatively correlated with sirtuin-1 (SIRT1) expression and activity, a protein deacetylase that inhibits DNA damage and cellular senescence. Inhibition of DNA damage response by silencing of ataxia telangiectasia-mutated (ATM) kinase resulted in up-regulation of SIRT1 expression and decreased senescence. Interestingly, treatment of BOEC from COPD patients with the SIRT1 activator resveratrol or a selective ATM inhibitor rescued the senescent phenotype. Using the in vivo Matrigel plug assay, BOEC from COPD patients displayed reduced angiogenesis (capillary-like structures) and increased DNA damage, senescence and apoptosis (measured by 53BP1, p16, TUNEL and cleaved-caspase 3 staining) compared to non-smokers. Conclusions BOEC from smokers and COPD patients show reduced angiogenesis in vivo and display increased DNA damage and senescence, associated with reduced SIRT1 expression. These defects may contribute to endothelial dysfunction and cardiovascular events in smokers and COPD patients and could potentially constitute therapeutic targets for intervention.

Endothelial progenitor cells in smokers are dysfunctional because of increased DNA damage and senescence

Abstract Background Cardiovascular disease (CVD) is a major cause of death in smokers, especially in patients with chronic obstructive pulmonary disease (COPD). The molecular pathways that lead to endothelial dysfunction and CVD due to cigarette smoke remain unclear. DNA damage has been recognised as an important contributor in ageing disorders, including CVD. Circulating endothelial progenitor cells (EPC) are required for endothelial homoeostasis, and their dysfunction contributes to CVD. This study aimed to examine whether circulating EPC (also called blood outgrowth endothelial cells [BOEC]) from smokers and COPD patients are dysfunctional, and to investigate the role of DNA damage pathways in mediating endothelial dysfunction in these patients. Methods BOEC were isolated from peripheral blood samples received from 16 healthy non-smokers (five men, 11 women; mean age 57 years [SE 2·7]), ten healthy smokers (five men, five women; 57 years [2·6]), and 16 COPD patients (11 men, five women; 67 years [1·6]). Endothelial senescence was measured by senescence-associated β-galactosidase (SA-β-gal) activity. Protein levels of sirtuin 1 (SIRT1) were measured by western blotting, expression of p16, γ-H2AX, and 53BP1 by immunofluorescence, and p21 by western blotting and immunofluorescence. SIRT1 activity was measured with a SIRT1 fluorescent activity assay kit. Findings BOEC from smokers and COPD patients showed evidence of increased DNA double-strand breaks (increased γ-H2AX, 53BP1) compared with non-smokers. BOEC from healthy smokers and COPD patients displayed increased senescence (measured by SA-β-gal activity, p16, and p21) and decreased SIRT1 expression and activity compared with controls. SIRT1 protein levels and activity negatively correlated with senescence, indicating a regulatory role of SIRT1 on senescence. Interestingly, treatment of BOEC from COPD patients with a SIRT1 activator (resveratrol) rescued the senescent phenotype. Interpretation The results from our study demonstrate that BOEC from smokers and COPD patients display increased DNA damage and senescence, associated with reduced SIRT1 expression and activity. These defects may contribute to endothelial dysfunction and cardiovascular events in people who smoke and could potentially constitute therapeutic targets for intervention. Funding Imperial College London.