Background: Recently, neuropilins (NRPs) were identified as new VEGF receptors (VEGFRs) and VEGFR-3 was revealed to be implicated in angiogenesis besides in lymphangiogenesis. However, quantitative expression and regulation of NRPs and VEGFRs remain unclear. The objective of this study was to compare the expression of NRPs and VEGFRs and investigate their regulation by tumor necrosis factor (TNF-alpha) in human endothelial cells (ECs) as well as their roles in VEGF(165)-induced EC proliferation.
Materials and methods: Human umbilical vein ECs (HUVECs) were treated with TNF-alpha (2 ng/ml) or PBS for 24 h. The mRNA and protein levels of NRP-1, NRP-2, VEGFR-1, VEGFR-2, and VEGFR-3 were semiquantitatively determined by real-time PCR and Western blot, respectively. Real-time PCR data were presented as the difference of reaction cycle thresholds (Ct) between beta-actin and each of the genes of interest (2(-DeltaCt)). EC proliferation in response to VEGF(165) (10 ng/ml) with or without anti-VEGFR-2 neutralization antibody pretreatment was analyzed by [(3)H]thymidine incorporation.
Results: In PBS-treated HUVECs, mRNA levels of NRPs and VEGFRs were NRP-1 (0.013), NRP-2 (0.007), VEGFR-2 (0.006), VEGFR-1 (0.0024), and VEGFR-3 (0.0009). After TNF-alpha treatment, mRNA levels of VEGFR-2, VEGFR-3, and NRP-1 were significantly reduced by 72, 65, and 53%, respectively (P < 0.05). The protein expression of all NRPs and VEGFRs were also detected by Western blot. TNF-alpha significantly reduced protein levels of VEGFR-2, VEGFR-3, and NRP-1 by 59, 35, and 22%, respectively. However, both mRNA and protein levels of VEGFR-1 and NRP-2 were not affected by TNF-alpha. Furthermore, TNF-alpha treatment significantly reduced EC proliferation in response to VEGF(165) by 67%. After blocking VEGFR-2 with neutralization antibody, TNF-alpha treatment elicited a 30% reduction of EC proliferation in response to VEGF(165).
Conclusions: These data demonstrate that HUVECs express higher mRNA levels of NRP-1 and NRP-2 than those of VEGFRs, and TNF-alpha treatment significantly decreases the expression of VEGFR-2, VEGFR-3, and NRP-1, which may be responsible for TNF-alpha-induced reduction of EC proliferation in response to VEGF(165).