Thomas Blythe

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

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.

S64 Alveolar epithelial type II cell expression of VEGF-Axxxa is critical for development of Idiopathic Pulmonary Fibrosis (IPF): an anti-fibrotic role for VEGF-Axxxb anti-angiogenic isoforms?

Introduction VEGF has been implicated in the development of IPF. Alternative splicing of the VEGF-A gene generates numerous isoforms. The differential effects of these isoforms, in particular the VEGF-Axxxb family, thought to have several opposing functions to the conventional family of isoforms (VEGF-Axxxa), have not been considered. Hypothesis The balance of VEGF-Axxxa:VEGF-Axxxb isoform expression is important in the pathogenesis of IPF. VEGF-Axxxb isoforms may be protective against the formation of pulmonary fibrosis (PF). Methods Normal and IPF lung lysates (n = 5) were analysed by western blotting (WB), and ELISA using an antibodies specific for PanVEGF-A and VEGF-Axxxb isoforms. The Bleomycin (BLM)-induced model of PF was used in conjunction with two transgenic (TG) mouse models, developed to explore the role of ATII-derived VEGF in the development of PF: 1) a conditionally inducible, ATII-specific, VEGF knock-out mouse (STCLL mice) and 2) a TG mouse over-expressing VEGF-Axxxb in ATII cells (MMTV-VEGF165b). To explore the therapeutic potential of VEGF-Axxxb in PF, wild-type mice were administered intraperitoneal (IP) injections of VEGF-A165b, commencing 10 days after BLM challenge. In all experiments fibrosis was assessed histologically using Masson’s Trichrome, with blinded scoring of tissue sections. Results By WB (n = 3) and ELISA (n = 5) there was no significant difference in PanVEGF-A expression between normal and IPF lung homogenates (t-test, p > 0.05). In contrast, VEGF-Axxxb expression was significantly increased in these same IPF samples compared to control, by ELISA (t-test, ****p < 0.0001) and WB (Densitometry: t-test, *p < 0.05). Specific deletion of VEGF-A from ATII cells of mice ameliorated the development of BLM-induced pulmonary fibrosis (n = 5, Lung fibrosis score: ANOVA with Holm’s Sidak **p < 0.01). Over-expression of VEGF-Axxxb in ATII cells also ameliorated the development of pulmonary fibrosis (n = 6, Lung fibrosis score: ANOVA with Holm’s Sidak ***p < 0.001). Furthermore, delivery of VEGF-A165b, specifically during the fibrotic phase of the BLM model, also attenuated lung fibrosis development (n = 6, Lung fibrosis score: ANOVA with Holm’s Sidak *p < 0.05). Conclusion Changes in the bioavailability of ATII cell-derived VEGF-A, namely the ratio of VEGF-Axxxa:VEGF-Axxxb, appear critical to the development of pulmonary fibrosis. This data suggests that more a targeted approach to anti-VEGF-A therapy in IPF should be explored.

P141 Differential expression of conventional and inhibitory VEGFA isoforms in normal and fibrotic fibroblasts–a potential role in IPF pathogenesis?

Introduction Vascular endothelial growth factor (VEGFA) has been implicated in the pathogenesis of Idiopathic Pulmonary Fibrosis (IPF). Two families of endogenous isoforms exist formed by alternative splicing of mRNA transcripts: the conventional potent angiogenic and mitogenic isoforms (VEGFxxxa family) and the VEGFxxxb family that is thought to have contrasting inhibitory functions. Hypothesis We hypothesise that differential expression of VEGFxxxa and VEGFxxxb isoforms by fibroblasts may influence the development of IPF. Methods Normal (NF) and fibrotic fibroblasts (FF) (from patients with proven UIP) were extracted from lung samples using the explant method. The expression of VEGFxxxa and VEGFxxxb by NF and FF was analysed at the mRNA level by RT-PCR and quantified by qPCR. Protein expression was determined by western blotting (WB) and ELISA. We sought to establish a potential functional effect of recombinant VEGF165a and VEGF165b proteins on fibroblasts by assessing the expression of a) the extracellular matrix (ECM) protein fibronectin and b) α-SMA, a marker of myofibroblast differentiation. Results Both NF and FF expressed VEGFxxxa and VEGFxxxb isoforms at the mRNA level as determined by RT-PCR with confirmation by direct sequencing. There was no statistical difference in total VEGF mRNA expression between the two cell types by qPCR (p = 0.9307, NF n = 5, FF n = 6), but FF expressed significantly more VEGF165b mRNA than NF (p = 0.05, NF n = 5, FF n = 6). Total VEGF protein expression was significantly increased in FF (mean expression NF = 180.5pg/ml vs FF 332.0pg/ml, p = 0.0012) by ELISA and confirmed by WB. Furthermore, increased VEGF165b protein expression was also observed in FF by WB. Recombinant VEGF165b had no effect on fibronectin or α-SMA expression in NF, but VEGF165a (10ng/µl) significantly increased expression of fibronectin (p < 0.05). Interestingly, co-administration of VEGF165a with VEGF165b inhibited both α-SMA and fibronectin expression in these cells (Figure 1). Conclusion Differential expression of VEGF isoforms between NF and FF suggests a potential role in the development of IPF. Furthermore, results suggest that factors altering the balance of splice variants may influence the surrounding fibrotic milieu. Abstract P141 Figure 1.

S56 Unravelling VEGF165 Signalling in the Lung

Introduction Vascular endothelial growth factor (VEGF) is a potent mitogenic, angiogenic and permeability factor that has been implicated in the development of lung injury and repair in a number of respiratory diseases such as ARDS and IPF VEGF165a functions via VEGF receptors in particular VEGFR-2, leading to a diverse and complex network of signalling pathways including activation of both the MAPK pathway and eNOS This results in changes to cell permeability, migration and proliferation. We have investigated the downstream signalling mechanisms regulated by VEGF165a in pulmonary and systemic endothelial cells. Understanding the signalling pathway used by VEGF165a to regulate lung biology is critical to preferentially induce specific beneficial effects. Methods Human Umbilical Vein Endothelial Cells (HUVEC) and Human Lung Microvascular Endothelial Cells (HUMVEC-L) were treated with 20ng/ml of VEGF165a lysed and studied using phosphospecific antibodies which measure the phosphorylation/activation of key signalling molecules. Phosphorylation of VEGFR-2 was measured using phosphotyrosine-specific antibody to tyr1175 and tyr1214. Phosphorylation and hence activation of MEK, MAPK and eNOS were also measured. The effects of VEGF isoforms on cell permeability in a time and dose dependent manner were measured by using a transwell system and “Electrical Cell-Substrate Impedance Sensor” (ECIS). Changes in the cellular distribution of VE-cadherin a protein known to be involved in the regulation of cell permeability was assessed by immunofluorescent labelling and confocal microscopy. Results Phosphorylation of VEGFR-2 at tyr1175 and tyr1214was induced between 5 and 10min (n=4; >5 fold increase). Activation of MEK and p44/42 MAPK (members of the MAPK pathway which regulates cell proliferation) were seen over a similar time course to that of VEGFR-2 (n=4; >5 fold increase) (Figs 1A, B). Phosphorylation of eNOS which regulates cell permeability was also observed (n=3; >2 fold) and indeed VEGF165a increased permeability in both HUVEC and HUMVEC-L (Huvec p<0.001); (Humvec-l p<0.01) (Fig 1). Finally we showed that in both cell types VEGF induced changes in the cellular distribution of VE-cadherin. Abstract S56 Figure 1 Conclusion These results demonstrate that signalling pathways, previously suggested to induce mitogenesis or permeability are activated by VEGF 165a in HUVEC and HMVEC-l cells, identifying potential future therapeutic targets.