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Abstract
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.
