Shaney Barratt

Detection of VEGF-Axxxb Isoforms in Human Tissues

Vascular Endothelial Growth Factor-A (VEGF-A) can be generated as multiple isoforms by alternative splicing. Two families of isoforms have been described in humans, pro-angiogenic isoforms typified by VEGF-A165a, and anti-angiogenic isoforms typified by VEGF-A165b. The practical determination of expression levels of alternative isoforms of the same gene may be complicated by experimental protocols that favour one isoform over another, and the use of specific positive and negative controls is essential for the interpretation of findings on expression of the isoforms. Here we address some of the difficulties in experimental design when investigating alternative splicing of VEGF isoforms, and discuss the use of appropriate control paradigms. We demonstrate why use of specific control experiments can prevent assumptions that VEGF-A165b is not present, when in fact it is. We reiterate, and confirm previously published experimental design protocols that demonstrate the importance of using positive controls. These include using known target sequences to show that the experimental conditions are suitable for PCR amplification of VEGF-A165b mRNA for both q-PCR and RT-PCR and to ensure that mispriming does not occur. We also provide evidence that demonstrates that detection of VEGF-A165b protein in mice needs to be tightly controlled to prevent detection of mouse IgG by a secondary antibody. We also show that human VEGF165b protein can be immunoprecipitated from cultured human cells and that immunoprecipitating VEGF-A results in protein that is detected by VEGF-A165b antibody. These findings support the conclusion that more information on the biology of VEGF-A165b isoforms is required, and confirm the importance of the experimental design in such investigations, including the use of specific positive and negative controls.

Vascular remodelling in the pathogenesis of idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible fibrosing interstitial pneumonia of unknown aetiology that usually leads to respiratory failure and death within 5 years of diagnosis. Alveolar epithelial cell injury, disruption of alveolar capillary membrane integrity and abnormal vascular repair and remodelling have all been proposed as possible pathogenic mechanisms. This review summarizes our current knowledge of the abnormalities in vascular remodelling observed in IPF and highlights several of the cytokines thought to play a pathogenic role, which may ultimately prove to be future therapeutic targets.

Differential Expression of VEGF-Axxx Isoforms Is Critical for Development of Pulmonary Fibrosis

Rationale: Fibrosis after lung injury is related to poor outcome, and idiopathic pulmonary fibrosis (IPF) can be regarded as an exemplar. Vascular endothelial growth factor (VEGF)‐A has been implicated in this context, but there are conflicting reports as to whether it is a contributory or protective factor. Differential splicing of the VEGF‐A gene produces multiple functional isoforms including VEGF‐A165a and VEGF‐A165b, a member of the inhibitory family. To date there is no clear information on the role of VEGF‐A in IPF. Objectives: To establish VEGF‐A isoform expression and functional effects in IPF. Methods: We used tissue sections, plasma, and lung fibroblasts from patients with IPF and control subjects. In a bleomycin‐induced lung fibrosis model we used wild‐type MMTV mice and a triple transgenic mouse SPC‐rtTA+/−TetoCre+/−LoxP‐VEGF‐A+/+ to conditionally induce VEGF‐A isoform deletion specifically in the alveolar type II (ATII) cells of adult mice. Measurements and Main Results: IPF and normal lung fibroblasts differentially expressed and responded to VEGF‐A165a and VEGF‐A165b in terms of proliferation and matrix expression. Increased VEGF‐A165b was detected in plasma of progressing patients with IPF. In a mouse model of pulmonary fibrosis, ATII‐specific deficiency of VEGF‐A or constitutive overexpression of VEGF‐A165b inhibited the development of pulmonary fibrosis, as did treatment with intraperitoneal delivery of VEGF‐A165b to wild‐type mice. Conclusions: These results indicate that changes in the bioavailability of VEGF‐A sourced from ATII cells, namely the ratio of VEGF‐Axxxa to VEGF‐Axxxb, are critical in development of pulmonary fibrosis and may be a paradigm for the regulation of tissue repair.

VEGF isoforms have differential effects on permeability of human pulmonary microvascular endothelial cells

BackgroundAlternative splicing of Vascular endothelial growth factor-A mRNA transcripts (commonly referred as VEGF) leads to the generation of functionally differing isoforms, the relative amounts of which have potentially significant physiological outcomes in conditions such as acute respiratory distress syndrome (ARDS). The effect of such isoforms on pulmonary vascular permeability is unknown. We hypothesised that VEGF165a and VEGF165b isoforms would have differing effects on pulmonary vascular permeability caused by differential activation of intercellular signal transduction pathways.MethodTo test this hypothesis we investigated the physiological effect of VEGF165a and VEGF165b on Human Pulmonary Microvascular Endothelial Cell (HPMEC) permeability using three different methods: trans-endothelial electrical resistance (TEER), Electric cell-substrate impedance sensing (ECIS) and FITC-BSA passage. In addition, potential downstream signalling pathways of the VEGF isoforms were investigated by Western blotting and the use of specific signalling inhibitors.ResultsVEGF165a increased HPMEC permeability using all three methods (paracellular and transcellular) and led to associated VE-cadherin and actin stress fibre changes. In contrast, VEGF165b decreased paracellular permeability and did not induce changes in VE-cadherin cell distribution. Furthermore, VEGF165a and VEGF165b had differing effects on both the phosphorylation of VEGF receptors and downstream signalling proteins pMEK, p42/44MAPK, p38 MAPK, pAKT and peNOS. Interestingly specific inhibition of the pMEK, p38 MAPK, PI3 kinase and eNOS pathways blocked the effects of both VEGF165a and VEGF165b on paracellular permeability and the effect of VEGF165a on proliferation/migration, suggesting that this difference in cellular response is mediated by an as yet unidentified signalling pathway(s).ConclusionThis study demonstrates that the novel isoform VEGF165a and VEGF165b induce differing effects on permeability in pulmonary microvascular endothelial cells.

S137 Vascular Endothelial Growth Factor (vegf) Expression In The Ipf Lung – A Role For Anti-angiogenic Isoforms?

Introduction VEGF has been implicated in the pathogenesis of IPF. Differential splicing of the VEGF gene produces an alternative family of isoforms (VEGFxxxb) that have anti-angiogenic properties, in contrast to conventional isoforms (VEGFxxx). Currently available literature on the role of VEGF in IPF has not differentiated between these families of isoforms and thus a degree of literature re-appraisal is required. Hypotheses The balance of VEGFxxx:VEGFxxxb isoforms may be important in IPF pathogenesis VEGFxxxb isoforms may abrogate the development of IPF Methods Human lung sections and BALF were used to quantify isoform expression in the IPF lung and were compared to controls (ELISA and IHC). Explanted ‘normal’ (NF) and ‘fibrotic’ (FF) fibroblasts were grown in culture with subsequent total RNA and cell lysate extraction (qPCR and WB). Wild-type mice were administered bleomycin (BLM) then received bi-weekly therapeutic intraperitoneal (IP) injections of rhVEGF165b (from day 10). Fibrosis was assessed histologically (Masson’s Trichrome and Lung fibrosis score). Results In the IPF lung, the alveolar epithelium was the most prominent site for total VEGF (PanVEGF isoforms) but also for VEGF165b (n = 10). Addiitonal staining was noted in fibroblasts and lung inflammatory cells. Alveolar and fibrotic cells in the least fibrotic areas of the IPF lung expressed significantly less VEGF165b than severely fibrotic areas (p < 0.001, n = 10). Examination of IPF BALF by ELISA revealed that total VEGF expression was significantly lower compared to control (IPF: 18.04 pg/ml +/- 6.13 n = 15, CTRL 85.72 pg/ml +/- 17.08 n = 13), whilst VEGF165b could not be detected in identical samples. Explanted NF and FF express comparable quantities of VEGFxxx and VEGFxxxb isoforms at the mRNA and protein level. Rh VEGF165 increases the mRNA expression of fibronectin (p < 0.001, n = 4) an effect not seen following the administration of rhVEGF165b. Administration of rhVEGF165b to mice attenuated the development of BLM-induced pulmonary fibrosis (Masson’s Trichrome (Figure 1) and lung fibrosis score (mean score: BLM alone 41.20 vs VEGF165b 30.67, p < 0.01, n = 6 per group)). Conclusion Differential expression of VEGFxxx and VEGFxxxb isoforms occurs in the IPF lung. In vitro, recombinant proteins appear to have differential effects on ECM synthesis and in vivo attenuate the formation of pulmonary fibrosis. A mouse overexpressing VEGF165b in the lung has been developed to study this concept in greater detail. Abstract S137 Figure 1 The effect of VEGF165b on the development of murine BLM-induced pulmonary fibrosis Bleomycin was given to WT type mice (n = 6 per group) by oro-pharyngeal aspiration (Day 0). rhVEGF165b was administered by IP injection (1µg per mouse, bi-weekly) from days 10 to 21. Fibrosis was scored and examined histologically by Masson’s trichrome staining. The development of BLM-induced fibrosis was attenuated in mice receiving rhVEGF165b

VEGF (Vascular Endothelial Growth Factor) and Fibrotic Lung Disease

Interstitial lung disease (ILD) encompasses a group of heterogeneous diseases characterised by varying degrees of aberrant inflammation and fibrosis of the lung parenchyma. This may occur in isolation, such as in idiopathic pulmonary fibrosis (IPF) or as part of a wider disease process affecting multiple organs, such as in systemic sclerosis. Anti-Vascular Endothelial Growth Factor (anti-VEGF) therapy is one component of an existing broad-spectrum therapeutic option in IPF (nintedanib) and may become part of the emerging therapeutic strategy for other ILDs in the future. This article describes our current understanding of VEGF biology in normal lung homeostasis and how changes in its bioavailability may contribute the pathogenesis of ILD. The complexity of VEGF biology is particularly highlighted with an emphasis on the potential non-vascular, non-angiogenic roles for VEGF in the lung, in both health and disease.

T2 VEGF signalling:differences in isoforms?

Vascular endothelial growth factor (VEGF) undergoes alternate splicing producing isoforms with differing functional effects. The most biologically active and extensively studied isoform is VEGF165. An isoform that causes inhibition of endothelial proliferation, migration and permeability, VEGF165b has also been identified (Bates et al. 2002). We have previously investigated the downstream signalling mechanisms in response to VEGF165 in pulmonary and systemic endothelial cells and have now compared these to the effects of VEGF165b. Abstract T2 Figure 1. HMVEC-L and HUVEC cells stimulated with 20 ng/ml of VEGF165a, VEGF165b, VEGF165 a+b or without any stimulation (control) A, immunoblotting of primary HUMVECL treated for 5 to 10min and immunoblotted for the phosphorylation of p-VEGFR-2, p-MEK, p-MAPK and p-eNOS using phosphospecific antibodies (comparable results for HUVEC cells, data not shown). B C, Measurement of TEER by Endohm (inserts 0.4mm pore size). VEGF165a reduces resistance (increased permeability) ***p < 0.001 (45min onwards) and VEGF165b increases resistance (decrease permeability) **p < 0.01 (15 to 45min) compared with control. In combination VEGF165b ameliorates VEGF165a effect in HMVEC-L. D E, Electrical Cell-Substrate Impedance Sensor (ECIS) measurement (B well assay 8W10E+); VEGF165a reduces the resistance (increased permeability) VEGF165b increases resistance (decreases permeability) in comparison to control. Data were analysed using one-way ANOVA and Bonferroni post test analysis. HUVEC and Human Lung Microvascular Endothelial Cells (HMVEC-L) were treated with both VEGF isoforms. Phosphorylation of VEGFR-2 (tyr1175 and tyr1214) was measured along with phosphorylation/activation of pMEK1/2, p44/42MAPK (regulating cell proliferation) and eNOS (involved in cell permeability). We have previously shown the functional effects of VEGF165/VEGF165b on HMVEC-L proliferation (Varet et al. 2010). We have now explored the functional effects of VEGF165/ VEGF165b on cell permeability parameters by Endohm and Electrical Cell-Substrate Impedance Sensor (ECIS) measurements and modification in VE-cadherin cell distribution. The effects of the eNOS inhibitor L-NIO were also investigated. VEGF165 induced maximal phosphorylation of VEGFR-2 at tyr1175 and tyr1214 between 5 and 10min (>10 fold increase), VEGF165b induced less than 5 fold increase compared to control. Comparable results for both isoforms were seen for activation of pMEK1/2, p42/44MAPK and eNOS. The two permeability assessments showed an increase in cells permeability due to VEGF165 (HUVEC p < 0.001); (HMVEC-l p < 0.01) in contrast to VEGF165b. This may be reflected by the differential changes in the cellular distribution of VE-cadherin induced in both cell types by VEGF isofroms. Treatment with L-NIO inhibited the effect of VEGF165b suggesting a potential regulatory mechanism.. VEGF165b induces differential responses to VEGF165 in HUVEC and HMVEC-L. These observations suggest separate pathways for the regulation of mitogenesis and permeability which may be targeted.

Pleuroparenchymal sarcoidosis - A recognised but rare manifestation of disease

Pleural involvement is rare in sarcoidosis. The presence of a large symptomatic effusion in a patient with sarcoidosis should therefore prompt further investigation for an alternate aetiology. Here we present a case of confirmed pleuro-parenchymal sarcoidosis. We discuss the important differential diagnoses and review the current literature.

Physiological predictors of Hypoxic Challenge Testing (HCT) outcomes in Interstitial Lung Disease (ILD)

BACKGROUND Pre-flight risk assessments are currently recommended for all Interstitial Lung Disease (ILD) patients. Hypoxic challenge testing (HCT) can inform regarding the need for supplemental in-flight oxygen but variables which might predict the outcome of HCT and thus guide referral for assessment, are unknown. METHODS A retrospective analysis of ILD patients attending for HCT at three tertiary care ILD referral centres was undertaken to investigate the concordance between HCT and existing predictive equations for prediction of in-flight hypoxia. Physiological variables that might predict a hypoxaemic response to HCT were also explored with the aim of developing a practical pre-flight assessment algorithm for ILD patients. RESULTS A total of 106 ILD patients (69 of whom (65%) had Idiopathic Pulmonary Fibrosis (IPF)) underwent HCT. Of these, 54 (51%) patients (of whom 37 (69%) had IPF) failed HCT and were recommended supplemental in-flight oxygen. Existing predictive equations were unable to accurately predict the outcome of HCT. ILD patients who failed HCT had significantly lower resting SpO2, baseline PaO2, reduced walking distance, FEV1, FVC and TLCO, but higher GAP index than those who passed HCT. CONCLUSIONS TLCO >50% predicted and PaO2 >9.42 kPa were independent predictors for passing HCT. Using these discriminators, a novel, practical pre-flight algorithm for evaluation of ILD patients is proposed.

Idiopathic Pulmonary Fibrosis (IPF): An Overview

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterised by chronic, progressive scarring of the lungs and the pathological hallmark of usual interstitial pneumonia. Current paradigms suggest alveolar epithelial cell damage is a key initiating factor. Globally, incidence of the disease is rising, with associated high morbidity, mortality, and economic healthcare burden. Diagnosis relies on a multidisciplinary team approach with exclusion of other causes of interstitial lung disease. Over recent years, two novel antifibrotic therapies, pirfenidone and nintedanib, have been developed, providing treatment options for many patients with IPF, with several other agents in early clinical trials. Current efforts are directed at identifying key biomarkers that may direct more customized patient-centred healthcare to improve outcomes for these patients in the future.