The Role of Heparan Sulfate and Neuropilin 2 in VEGFA Signaling in Human Endothelial Tip Cells and Non-Tip Cells during Angiogenesis In Vitro.

Marchien G Dallinga,Yasmin I Habani,Ingeborg Klaassen,Geesje M Dallinga-Thie,Reinier O Schlingemann,Alinda W M Schimmel,Cornelis J F Van Noorden

doi:10.3390/cells10040926

Abstract

During angiogenesis, vascular endothelial growth factor A (VEGFA) regulates endothelial cell (EC) survival, tip cell formation, and stalk cell proliferation via VEGF receptor 2 (VEGFR2). VEGFR2 can interact with VEGFR2 co-receptors such as heparan sulfate proteoglycans (HSPGs) and neuropilin 2 (NRP2), but the exact roles of these co-receptors, or of sulfatase 2 (SULF2), an enzyme that removes sulfate groups from HSPGs and inhibits HSPG-mediated uptake of very low density lipoprotein (VLDL), in angiogenesis and tip cell biology are unknown. In the present study, we investigated whether the modulation of binding of VEGFA to VEGFR2 by knockdown of SULF2 or NRP2 affects sprouting angiogenesis, tip cell formation, proliferation of non-tip cells, and EC survival, or uptake of VLDL. To this end, we employed VEGFA splice variant 121, which lacks an HSPG binding domain, and VEGFA splice variant 165, which does have this domain, in in vitro models of angiogenic tip cells and vascular sprouting. We conclude that VEGFA165 and VEGFA121 have similar inducing effects on tip cells and sprouting in vitro, and that the binding of VEGFA165 to HSPGs in the extracellular matrix does not seem to play a role, as knockdown of SULF2 did not alter these effects. Co-binding of NRP2 appears to regulate VEGFA–VEGFR2-induced sprout initiation, but not tip cell formation. Finally, as the addition of VLDL increased sprout formation but not tip cell formation, and as VLDL uptake was limited to non-tip cells, our findings suggest that VLDL plays a role in sprout formation by providing biomass for stalk cell proliferation.

Highlights

Differential binding of vascular endothelial growth factor A (VEGFA) splice variants to heparan sulfate proteoglycans (HSPGs) on the endothelial cells or in the extracellular matrix (ECM) may have an effect on downstream VEGF signaling, as VEGFA165 has an heparan sulfate (HS)
We investigated whether the differential HSPG-binding capacity of VEGFA splice variants or the presence of VEGF receptor 2 (VEGFR2) co-receptors Neuropilin 1 (NRP1) and neuropilin 2 (NRP2) regulates of VEGFA splice variants or the presence of VEGFR2 co-receptors NRP1 and NRP2 regutip cell formation and sprouting angiogenesis
Since we have shown that expression of sulfatase 2 (SULF2) was stronger in CD34+ tip cells compared to CD34- cells, a finding recently reported by others [56], sulfation of HSPGs in the ECM may well play a regulatory role in vessel sprouting and maturation during angiogenesis in vivo

Summary

Introduction

Sprouting angiogenesis is the complex process of blood vessel growth from the existing vasculature. It is characterized by differentiation of ECs into subtypes with distinct functions: tip cells are non-proliferating and highly migratory and lead the growing vessel sprout towards a source of growth factors. Tip cells suppress the tip cell phenotype in adjacent stalk cells that are proliferating and start forming a lumen, and further down the sprout are phalanx cells that initiate vessel maturation [1,2]. The main receptor for angiogenic signaling by VEGFA is VEGFR2, and its stimulation induces tip cell migration, expression of tip cell genes, proliferation of stalk cells, and EC survival [2,3]. Binding of VEGFA to VEGFR2 is affected by a number of factors, which include binding to extracellular matrix (ECM) components such as HSPGs, or simultaneous binding of VEGFA to one of its NRP co-receptors (NRP1 or NRP2) [4,5,6]

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Conclusion