Crosstalk between SHANK3 and VEGF signalling in Endothelial cells
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SHANK3 (SH3 and multiple ankyrin repeat domains) is a well- known postsynaptic density (PSD) scaffolding protein, particularly important for its roles in synapse formation, signal transduction, and structural organization in the nervous system. However, its role in endothelial cells remains largely unknown. Recent findings from our laboratory suggest that SHANK3 contributes to endothelial junctional morphology, cell motility, and barrier integrity- processes mainly influenced and regulated by vascular endothelial growth factor (VEGF) in endothelial cells. This thesis, therefore, investigated the potential crosstalk between SHANK3 and VEGF signalling and its downstream proteins ERK and AKT and whether these pathways are linked or are completely independent of each other.
To achieve this, SHANK3 expression was reduced using small interfering RNA (siRNA)-mediated gene silencing in human umbilical vein endothelial cells (HUVECs) combined with VEGF treatment. Immunofluorescence staining, confocal microscopy, and Western blot analysis were used to assess SHANK3 expression, knockdown efficiency, and its potential involvement with VEGF signalling through the downstream signalling proteins extracellular signal-regulated kinase (ERK) and protein kinase B (AKT/PKB).
The results showed that SHANK3 localizes to the cell periphery and throughout the cytoplasm in a confluency-dependent manner. Confocal imaging further suggested the presence of SHANK3 near the endothelial cell junctions. However, the effects of SHANK3 depletion on VEGF-mediated signalling and on downstream signalling proteins could not be determined, as no consistent changes in VEGFR2, ERK or AKT activation were observed, possibly due to variable/incomplete siRNA knockdown efficiency. While these findings suggest that SHANK3 is associated with endothelial cell structures and junctional regions, its role in VEGF-mediated signalling remains unclear, highlighting the need for more effective gene-silencing approaches in future studies.