Introduction Fibrosis is a constituent of various respiratory diseases including asthma, COPD, idiopathic pulmonary fibrosis (IPF) and pulmonary hypertension. Research shows that Wnt genes, critical for lung development, are reactivated post cellular injury and that the canonical Wnt/β-catenin pathway is mis-expressed in these diseases. The non-canonical Wnt/PCP pathway can inhibit canonical signalling and also cause co-ordinated, directional cell migration. Moreover components of this pathway are suppressed in many respiratory fibro-proliferative disorders. We investigated the role of non-canonical Wnts in a tractable model of lung repair, by comparing the rate of wound healing using either canonical Wnt4 or non-canonical Wnt5a ligands. We hypothesised that Wnt5a would exhibit a higher rate of wound healing than Wnt4, coupled with increased cytoskeleton dynamics, favouring co-ordinated and directional cell migration.
Methods Confluent A549 cells were scratched with a 10µl pipette tip to induce a ‘wound’ and treated with either recombinant canonical Wnt4 or non-canonical Wnt5a, or cultured in control serum free medium. Wound area was measured at t=0, t-6 and t=24h post scratch to determine the rate of wound healing using time-lapse imaging. Phalloidin labelling and imaging was used to determine levels and distribution of F-actin at those times.
Results The addition of Wnt5a healed 4 times the area either Wnt4 or Control did using concentrations that induce maximal activity at t=6 (5.49, 1.23 and 1.01×105µm2 respectively) and t=24 (8.62, 2.42 and 2.39×105µm2 respectively) hours post scratch (p<1x10–7). At 0.5µg/ml Wnt5a healed 2 times the area Wnt4 did at it’s ED50 dose with Wnt4 at 5xED50, at t=6 (1.99 and 1.01 x105µm2 respectively) and t=24 (4.29 and 1.80x105µm2 respectively) hours post scratch (p<0.01). Wnt5a treatment increased the frequency of focal F-actin enrichment towards the wound edge versus Wnt4 or control.
Conclusion Wnt5a is more efficient at wound healing in A549 cells than Wnt4. Wnt5a associated increase in focal F-actin enrichment amplified wound healing is associated with increased cytoskeletal dynamics and directional movement. In the future, novel therapies based around Wnt5a have the potential to be used to enhance repair in fibrotic respiratory diseases.
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