Abstract/Summary

Background Interaction of podoplanin-lymphatic endothelial cells (LECs) with platelet-CLEC-2 plays several physiological roles including lymphatic vessel development and solid organ development. It is also involved in many pathological conditions including atherosclerosis, deep venous thrombosis, and metastasis. Podoplanin ligation by platelets or antibody-mediated crosslinking has been demonstrated to modulate VEGF-C-mediated LEC functions, however, the signalling mechanisms involved in this process are poorly understood. Aim Investigate LEC podoplanin signalling mechanisms and to determine how podoplanin ligation modulates VEGF-C-mediated LEC behaviour and signalling. Methods Trans-well migration and proliferation assays investigated the effect of platelets on LEC behaviour. The effect of podoplanin crosslinking by recombinant CLEC-2 or antibodies, in the presence and absence of VEGF-C, on ERM, ERK1/2 and AKT phosphorylation was assessed using Western blotting. Colocalisation of podoplanin with other membrane partner proteins was imaged using Total Internal Reflection Fluorescence microscopy (TIRF) combined with Direct Stochastic optical reconstruction microscopy (dSTORM). Results Co-culture of platelets with LECs induces clustering of podoplanin and inhibits VEGF-C-mediated cell migration. Mimicking the crosslinking of podoplanin using recombinant CLEC-2 or antibodies inhibits LEC migration and proliferation. Crosslinking of podoplanin does not change basal ERM phosphorylation or alter VEGF-C-mediated ERK1/2 or AKT phosphorylation. Podoplanin resides in CD9-containing domains which is altered following VEGF-C stimulation. Super-resolution dSTORM microscopy demonstrates that the association between podoplanin and CD9 is reduced following VEGF-C stimulation whereas the association between podoplanin and integrin β1 increases. Podoplanin and VEGF-3 weakly colocalise and this does not change following VEGF-C stimulation. CD9 and integrin β1 colocalise and VEGF-C increases their degree of colocalisation. Conclusion Crosstalk between podoplanin and VEGF-C mediated signalling is modulated by podoplanin ligation. Changes in CD9 and integrin β1 interactions with podoplanin may modulate podoplanin and VEGF-C-mediated signalling. This highlights a potential molecular mechanism through which podoplanin-dependent signalling may regulate VEGF-C-mediated signalling and cellular functions.