VEGF Affects Postnatal Thymic Development Through Distinct Receptor Pathways

Neonatal thymic growth is dependent on high levels of vascular endothelial growth factor (VEGF); Inhibiting VEGF through a decoy fusion receptor (Aflibercept) resulted in a dramatic decrease in endothelial cells (ECs) and thymocytes, accompanied by significant changes affecting both thymic epithelial cells (TECs) and thymic mesenchymal cells (TMCs). Indeed, cTEC:mTEC ratio was decreased, reflecting a relative loss of cTECs. We have identified a subpopulation of thymic mesenchymal cells (TMCs) characterized by the expression of CD146 and displaying pericyte features, distinct from parenchymal CD140a+ TMCs. We found that Aflibercept decreased CD146+ numbers but not CD140a+ TMCs. Of note, the effects in both the TEC and TMC compartments upon VEGF inhibition in the neonatal thymus recapitulated the thymic stroma maturation occurring from neonatal to adult stages of development. While high expression of Flk1 (encoding VEGFR2) characterized thymic endothelium its co-receptor Neuropilin-1 (Nrp1) was expressed on endothelium as well as both CD140a+ and CD146+ TMCs. Specific blockade of VEGFR2 reduced ECs and CD146+ TMCs, but thymocyte numbers and TECs remained unchanged. In contrast, NRP1 blockade rapidly decreased the cTEC:mTEC ratio but did not affect endothelium. However, only combined blockade of VEGFR2 and NRP1 recapitulated all the stromal changes observed early after VEGF-blockade. Moreover, only the combination receptor blockade induced a significant decrease in thymocytes, a result of reduced proliferation and increased apoptosis of early thymocytes. In order to investigate the pathways affected by VEGF blockade in the neonatal thymus, we performed RNA-Seq analysis of all thymic stromal populations from each experimental group, focusing on which receptor inhibition recapitulated the transcriptional changes observed after Aflibercept. VEGFR2 blockade showed the highest transcriptional similarity with Aflibercept in ECs and CD146+ TMCs, altering the expression of genes involved in cell cycle, metabolism and chemokine regulation. However, transcriptional changes after NRP1-blockade were most similar to those seen in TECs after Aflibercept, with downstream targets involved in cell migration, among others. Of note, the cell type-specificity of these transcriptional events were consistent with the phenotypic and functional effects seen with the receptor blockade. Given the pattern of Flk1 and Nrp1 expression in the thymic stroma, our data suggest that during development, VEGF affects TECs independent of VEGFR2 driven angiogenesis, through an indirect effect induced via NRP1 signaling on TMCs and/or ECs, uncovering a new mechanism by which VEGF regulates the neonatal thymic microenvironment.