NRP2 as an emerging angiogenic player; promoting endothelial cell adhesion and migration by regulating recycling of α5 integrin

Abstract

Angiogenesis relies on the ability of endothelial cells (ECs) to migrate over the extracellular matrix via integrin receptors to respond to an angiogenic stimulus. Of the two neuropilin (NRP) orthologs to be identified, both have been reported to be expressed on normal blood and lymphatic ECs, and to play roles in the formation of blood and lymphatic vascular networks during angiogenesis. Whilst the role of NRP1 and its interactions with integrins during angiogenesis has been widely studied, the role of NRP2 in ECs is poorly understood.Here we demonstrate that NRP2 promotes EC adhesion and migration over fibronectin (FN) matrices in a mechanistically distinct fashion to NRP1, showing no dependence on β3 integrin (ITGB3) expression, or VEGF stimulation. Furthermore, we highlight evidence of a regulatory crosstalk between NRP2 and α5 integrin (ITGA5) in ECs, with NRP2 depletion eliciting an upregulation of ITGA5 expression and disruptions in ITGA5 cellular organisation. Finally, we propose a mechanism whereby NRP2 regulates ITGA5 trafficking in ECs by promoting Rab11-dependent recycling of ITGA5. NRP2 depleted ECs were found to exhibit reduced levels of total ITGA5 subunit recycling compared to wild-type (WT) ECs, where we demonstrate a strong co-localisation between NRP2, ITGA5 and Rab11. Our findings expose NRP2 as a novel angiogenic player by promoting ITGA5-mediated EC adhesion and migration on FN.

Endothelial neuropilin‐2 influences angiogenesis by regulating actin pattern development and α5‐integrin‐ p ‐FAK complex recruitment to assembling adhesion sites

“…We have previously shown that endothelial NRP2 drives a VEGF‐independent, Rac1‐mediated mechanism of cell adhesion and migration over fibronectin (FN) matrices and regulates FA turnover by promoting basal α5‐integrin recycling 24 . Here we show that NRP2 regulates VASP‐mediated control of actin pattern development to support the protrusion of dynamic lamellipodia at the leading edge of migrating cells.…”

“…We have previously used multiple specific NRP2 siRNAs in both immortalized and primary mouse‐lung microvascular ECs (mLMECs) to identify endothelial NRP2 as a key regulator of EC adhesion and migration by promoting FA turnover and Rac1 activation, independent of VEGF‐stimulation. Label‐free quantitative mass spectrometry was additionally utilized to elucidate candidate NRP2 interactions with several endocytic and recycling‐associated trafficking proteins 24 …”

“…To further elucidate the mechanisms by which NRP2 controls the remodeling of the actin cytoskeleton, we re‐examined our label‐free mass spectrometry data, originally performed to take an unbiased approach to identifying candidate proteins associating with NRP2 in mLMECs 24 . This re‐analysis revealed a large number of proteins known to be involved in regulating the actin cytoskeleton.…”

“…Neuropilin‐2 (NRP2) is a single pass transmembrane receptor whose upregulation is consistent with an accelerated cancer progression in a number of cell types (eg, neuroblastomas, 17 non‐small‐cell lung carcinoma [NSCLC], 18 human prostate carcinoma, melanoma, 19 lung cancer, 20 myeloid leukemia, 21 breast cancer, 22 and pancreatic cancer 23 ). As a selective co‐receptor for members of the VEGF family of growth factors, the function of NRP2 has also been implicated in influencing EC adhesion, migration and permeability during angiogenesis, under both physiological and pathological conditions 24‐26 . NRP2 has since emerged as a promising candidate for targeted therapy against tumorigenesis, however the mechanisms by which it integrates and disseminates ECM and growth factor signals to coordinate EC responses during angiogenesis is as yet unclear.…”

NRP2 as an Emerging Angiogenic Player; Promoting Endothelial Cell Adhesion and Migration by Regulating Recycling of α5 Integrin

“…Robinson is also partially funded by the BBSRC Institute Strategic Programme Gut Microbes and Health BB/R012490/1 and its constituent project (BBS/E/F/000PR10355). This manuscript has been released as a pre-print at BioRxiv (Alghamdi et al, 2020).…”