Rbfox2 emerges as a post-transcriptional regulator of VEGF-A signalling and angiogenesis in cardiac endothelial cells

Cardiac endothelial cells (ECs) are highly heterogeneous, ensuring sufficient oxygen and nutrient supply and serving as a primary response to patho/physiological changes in conditions. ECs are primed to respond to cues such as growth factor signalling. For example, growth factor stimulation by VEGF-A (vascular endothelial growth factor-A) prompts ECs to undergo a range of phenotypic alterations to facilitate the formation of new blood vessels from pre-existing ones (angiogenesis), essential to ensuring that all tissue remains adequately perfused with oxygen and nutrients. In the cardiac context, the possibility to selectively stimulate angiogenesis following pathological heart damage, for example myocardial infarction, has been suggested as a promising therapeutic target to improve blood flow to the damaged area and improve repair. RNA binding proteins (RBPs) interact with their RNAs to regulate all aspects of the RNA life cycle, for example, regulating splicing, RNA export and localisation, translational efficiency and RNA stability. Given the dynamic nature of RNA-protein interactions, RBPs offer a primary, post-transcriptional response to changes in cellular conditions and likely play a key role determining the post-transcriptional EC response to VEGF-A stimulation.

Using RNA interactome capture, we identified global acute (15 mins) and prolonged (24 h) changes in the mRNA bound proteome of cardiac ECs following VEGF-A stimulation. Proteomic analysis revealed that VEGF-A results in distinct, dynamic changes in RNA binding patterns. Visualisation of differentially bound RBPs revealed the RBP Rbfox2 was the most significantly downregulated RBP after 15 minutes of VEGF-A stimulation. Subsequent immunofluorescence staining and nuclear cytoplasmic fractionation determined that VEGF-A stimulation results in an acute decrease in nuclear Rbfox2, which closely correlates with the decreased RNA binding activity. Interestingly, we found that Proteinkinase B/ Akt drives differential isoform expression of Rbfox2 downstream of VEGF-A..

Loss of Rbfox2 promoted increased tubule formation, sprouting and proliferation in HUVECs and mouse cardiac ECs, suggesting that the decreased RNA binding activity induced in Rbfox2 upon VEGF-A stimulation may act to promote angiogenesis. Using RNA immunoprecipitation and RNA sequencing (RIP-seq), we identified the target RNAs to which Rbfox2 differentially binds in a VEGF-A dependent manner. This uncovered an enrichment of differential binding to genes with central roles in angiogenic processes. RNA sequencing revealed differential expression and splicing of these genes upon Rbfox2 knockdown in cardiac ECs.

Together, our data suggests that Rbfox2 plays a key role in determining the angiogenic response of ECs to VEGF-A stimulation, with the VEGF-A driven decrease in Rbfox2 promoting angiogenesis. Therefore, selectively modulating the interaction of Rbfox2 with angiogenic target RNAs may be a potential therapeutic avenue to promote new vessel formation post-infarct.