Excess vascular endothelial growth factor-A disrupts pericyte recruitment during blood vessel formation

Abstract

Pericyte investment into new blood vessels is essential for vascular development such that misregulation within this phase of vessel formation can contribute to numerous pathologies including arteriovenous and cerebrovascular malformations. It is critical therefore to illuminate how angiogenic signaling pathways intersect to regulate pericyte migration and investment. Here, we disrupted vascular endothelial growth factor-A (VEGF-A) signaling in ex vivo and in vitro models of sprouting angiogenesis, and found pericyte coverage to be compromised during VEGF-A perturbations. Pericytes had little to no expression of VEGF receptors, suggesting VEGF-A signaling defects affect endothelial cells directly but pericyte indirectly. Live imaging of ex vivo angiogenesis in mouse embryonic skin revealed limited pericyte migration during exposure to exogenous VEGF-A. During VEGF-A gain-of-function conditions, pericytes and endothelial cells displayed abnormal transcriptional changes within the platelet-derived growth factor-B (PDGF-B) and Notch pathways. To further test potential crosstalk between these pathways in pericytes, we stimulated embryonic pericytes with Notch ligands Delta-like 4 (Dll4) and Jagged-1 (Jag1) and found induction of Notch pathway activity but no changes in PDGF Receptor-β (Pdgfrβ) expression. In contrast, PDGFRβ protein levels decreased with mis-regulated VEGF-A activity, observed in the effects on full-length PDGFRβ and a truncated PDGFRβ isoform generated by proteolytic cleavage or potentially by mRNA splicing. Overall, these observations support a model in which, during the initial stages of vascular development, pericyte distribution and coverage are indirectly affected by endothelial cell VEGF-A signaling and the downstream regulation of PDGF-B-PDGFRβ dynamics, without substantial involvement of pericyte Notch signaling during these early stages.

The pericyte microenvironment during vascular development

“…Interestingly, this elegant study found that Notch pathway signals, known to be important for vSMC differentiation and development, were not required for the differentiation, recruitment, or retention of microvascular pericytes. These observations are consistent with CNS‐focused and developmental studies . However, these results may be context‐dependent and could be confounded by the fact that, although platelet‐derived growth factor receptor‐β (PDGFRβ) and neural‐glial antigen‐2 (NG2, gene name: Cspg4 ) are useful markers for pericytes, they are also expressed by other cell types such as vSMCs and brain glia, and oligodendrocyte precursor cells, respectively.…”

“…Previous studies have suggested that the endothelium acquires pericyte associations primarily, if not exclusively, after endothelial sprouting events begin to establish basic vascular networks, perhaps allowing greater plasticity in endothelial remodeling . We, and others, have found that mouse embryonic stem cells give rise not only to primitive endothelial cell networks, but also to presumptive pericytes (or pericyte precursors) during the earliest stages of cardiovascular development . Pericytes seem to emerge at approximately the same time as, or even prior to, endothelial cell differentiation, homing to endothelium engaged in both vasculogenic (Payne, L.B.…”

“…The tip cell would eventually resume its outward migration, and the pericyte would continue to keep pace with vessel sprouting (Figure and Movie ). Considering these anecdotal observations alongside current published data suggests a model in which endothelial‐secreted cues such as PDGF‐BB are critical for synchronizing pericyte migration along growing sprouts. As endothelial cells extend and retract in a dynamic competition for the tip cell position, pericytes likely engage recruitment ligands via receptor binding and dynamically reduce or deplete local concentrations of these chemo‐attractants at the leading front.…”

“…We recently visualized NG2‐DsRed‐positive pericytes migrating along sprouting endothelial cells in an ex vivo model of mouse embryonic (E14.5) back skin (Figure and Movie ) . Throughout remodeling vascular networks, pericyte migration appeared to be tightly entrained to the endothelial sprout.…”

“…Pericytes seem to emerge at approximately the same time as, or even prior to, endothelial cell differentiation, homing to endothelium engaged in both vasculogenic (Payne, L.B. and Chappell, J.C., Unpublished observations) and angiogenic processes . Nevertheless, whether pericytes, or their precursors, directly engage with the developing endothelium to actively coordinate the formation of primitive blood vessels remains an ongoing area of investigation.…”