The scratch wound assay as an alternative model of endothelial cell migration

We decided to assess the effect of platelets and podoplanin crosslinking in a different assay of cell migration and chose the scratch wound assay as our model. Initially, the experimental timecourse and effect of pre-treating the plates with gelatin were assessed. These

experiments found that pre-treatment with gelatin slowed migration, such that the wounds only healed by 20% in 24 hours (Figure 3.15A and Figure 3.16A). In comparison, wound closure after 24 hours was 50% on plates that had not been treated with gelatin (Figure 3.15B and Figure 3.16B). With these plates, the wound fully healed in 48 hours. Therefore, further experiments used plates that had not been pre-treated with gelatin. Addition of VEGF-C to these experiments did not accelerate wound healing (Figure 3.15 and Figure 3.16). Lack of effect was confirmed in further experiments that only compared basal and VEGF-C stimulated migration (Figure 3.17). Nevertheless, both unstimulated and VEGF-C stimulated cultures were used in subsequent experiments, as the transfilter assay had shown that the effect of platelets differed between these conditions.

Having characterised the assay, platelets were added to determine whether they would modulate LEC wound recovery. This variant of the assay was performed in two different ways: i) platelets were incubated with the monolayer for an hour before wounding; ii) the monolayer was scratched and washed before platelets were added. Both protocols were performed in the presence and absence of VEGF-C and images were taken from 0-40 hours. When the monolayer was scratched before platelets were added, subsequent addition of

125

platelets initially slowed wound healing, but by 40 hours healing was similar with or without platelets (Figure 3.18). This pattern was seen in either the presence (Figure 3.18A) or absence (Figure 3.18B) of VEGF-C. Conversely, if platelets were incubated with LEC before the monolayer was injured, the wound healed faster than in the absence of platelets (Figure 3.19). The effect of platelets was small, but significant, in the presence of VEGF-C (Figure 3.19A) and was more pronounced in the absence of VEGF-C (Figure 3.19B).

To help assess whether platelets might be physically obstructing wound healing rather than causing changes by binding podoplanin, antibody-mediated podoplanin crosslinking was used to assess the effect of podoplanin clustering on wound healing. In these experiments, the monolayer was wounded before podoplanin was crosslinked. To maintain consistency with previous measurements, these experiments were performed in culture medium with or without VEGF-C. We found that addition of the anti-human podoplanin alone had no effect on wound healing of either unstimulated or VEGF-C stimulated LEC (Figure 3.20). Crosslinking podoplanin had no effect in the presence of 300ng/ml VEGF-C (Figure 3.20A) and significantly promoted LEC wound recovery in the absence of VEGF-C (Figure 3.20B), compared to no antibodies but not compared to anti-podoplanin antibody alone.

126

Figure 3.15: Characterisation of the scratch wound assay. LEC were seeded onto plates that had been coated with 1% gelatin (A) or left untreated (B) and allowed to grow to confluence. The monolayers were wounded and phase contrast images were taken with a 4x objective to monitor wound closure. Images are representative of three independent experiments.

127

Figure 3.16: Effects of coating and VEGF-C on the time course of scratch wound recovery. Phase contrast images obtained during the scratch wound assay were analysed using ImageJ. The area of the wound in each image was calculated and expressed as a percentage of the original wound. (A)

Quantitation of scratch area on plates that had been pre-treated with 1% gelatin. ANOVA found no effect of time or VEGF-C (p>0.05). (B) Quantitation of wound area on plates that were not pre-treated with gelatin. ANOVA showed an effect of time on wound area (p<0.01). * = p<0.05 compared to wound area at time 0 hours by Dunnett’s test for both data sets. Data are mean ± SEM of three independent experiments.

128

Figure 3.17: Effect of VEGF-C treatment on LEC wound recovery. LEC were grown on uncoated plates until confluent. The monolayer was wounded, and then the cells were maintained in culture medium with or without 300ng/ml VEGF-C. Images were taken between 16 and 40 hours and used to determine percentage wound area. ANOVA showed that VEGF-C treatment did not affect wound recovery. Data are mean ± SEM of at least ten independent experiments.

129

Figure 3.18: Effect of adding platelets after wounding on LEC wound recovery. Monolayers of LEC were wounded and the detached cells washed away before 200µl Ca2+/Mg2+-free sterile PBS or 2x108 washed human platelets were added. Phase contrast images were used to quantify wound area. (A) Quantitation of wound area in the presence of 300ng/ml VEGF-C. ANOVA showed that the effects of time and platelets were significant (p<0.01 and p<0.05, respectively). (B) Quantitation of wound area in culture medium without VEGF-C. ANOVA showed that the effect of time was significant (p<0.01) but that the effect of platelets was not. Data are mean ± SEM of two independent experiments.

130

Figure 3.19: Effect of pre-incubation with platelets on LEC wound recovery 2x108 washed human platelets or 200µl Ca2+/Mg2+-free sterile PBS were incubated with LEC for an hour before the monolayer was wounded. Phase contrast images were taken using a 4x objective for up to 40 hours after wounding. From these images, percentage wound area was calculated. (A) Quantitation of wound healing in culture medium containing 300ng/ml VEGF-C. ANOVA showed that the effects of time and platelets were

significant (both p<0.01). (B) Quantitation of wound area in culture medium without VEGF-C. ANOVA showed that the effects of time and platelets were significant (both p<0.01). Data are mean ± SEM of at least four independent experiments.

131

Figure 3.20: Effect of podoplanin crosslinking on LEC wound recovery. Monolayers of LEC were wounded before the addition of 2µg/ml anti-human podoplanin, with or without subsequent crosslinking (1:15 anti-human

podoplanin and rat IgG2a). (A) Quantitation of wound area in culture medium containing 300ng/ml VEGF-C. ANOVA found no effect of treatment. (B) Quantitation of wound area in culture medium without VEGF-C. ANOVA showed an effect of treatment (p<0.05). Crosslinking was found to promote wound recovery compared to no antibodies, but not primary antibody alone, by Bonferroni test (p<0.05). Data are mean ± SEM of three independent experiments.

132