Fig 1: Downregulation of NRP2 suppresses PNET angiogenesis and tumor growth in vivo and correlates with increased survival in patients. a The xenograft experiments in vivo with the mouse model with BON cells. After mice were injected with anti-NRP2 antibody or PBS intraperitoneally, the xenografts were dissected. b H&E staining was performed to determine the number of vessels in the xenograft tumors. The vascular number was calculated as the mean counts of vessels in 5 fields under 20 × magnification. ***P ≤ 0.001 by Student’s t test. c Tumor sizes were measured every other day after injection with PBS or NRP2 antibody. d Survival curve of PNET patients in the high and low NRP2 expression groups
Fig 2: Gene and protein expression of vascular endothelial growth factor receptor-3 (VEGFR-3)/Flt-4 and neuropilin-2 (NRP-2) in dermal lymphatic microvascular endothelial cells (LMVECs) is significantly decreased by conditioning with systemic sclerosis (SSc) serum. LMVECs were treated for 48 h with sera from healthy controls (n = 8) or from patients with early diffuse cutaneous SSc (n = 8). Quantification of VEGFR3/FLT4 and NRP2 gene expression by real-time reverse transcription PCR is reported in the histograms. Gene expression levels in LMVECs treated with healthy serum were set to 1; the other results are normalized to this value. The reference gene used was 18S ribosomal RNA. Bars represent the mean ± SEM values of triplicate determinations from each one of the three LMVEC lines. * p < 0.001 vs. healthy serum (unpaired Student’s t-test). Representative immunoblots of VEGFR-3/Flt-4 and NRP-2 proteins are shown at the bottom; α-tubulin was measured as a loading control.
Fig 3: Expressions analysis of miR-331-3p and NRP2 in CRC tissue samples and cells. (A and B) The relative expression of miR-331-3p in CRC tissue samples (n=54) and cell lines were measured by RT-qPCR. (C and D) The relative expression of NRP2 in CRC tissues and cell lines were detected by RT-qPCR. (E) Expression of NRP2 in CRC tissues was measured by IHC. (F) Correlation between expression of miR-331-3p and NRP2. *P<0.05, **P<0.01, ***P<0.001. The data are from at least 3 independent experiments. CRC, colorectal carcinoma; NRP2, neuropilin-2; IHC, immunohistochemistry.
Fig 4: Proposed model of vascular NRP2 triggers PNET angiogenesis via activating SSH1-cofilin pathway
Fig 5: NRP2 modulates angiogenesis by promoting HUVEC migration via a VEGF/VEGFR2-independent pathway. a HUVECs were cultured in the presence or absence of conditioned medium from BON cells (treatment and control, respectively) and then transfected with a vector control or NRP2 overexpression plasmid before they were seeded for the capillary tube formation assay. Representative images at 4, 12 and 24 h after plating are shown. b Quantification of the number of complete and broken tubes at 6 h from a representative experiment. Data are shown as the mean ± SD of three independent experiments. *P ≤ 0.05 by Student’s t test. c HUVECs were treated with conditioned medium from BON cells for 24 h and then transfected with a vector or NRP2 overexpression plasmid before they were subjected to a CCK8 assay. d After HUVECs were cultured in the presence or absence of conditioned medium from BON cells (treatment and control, respectively) and transduced with the NRP2-overexpressing plasmid, flow cytometry was performed to assess apoptosis. e Representative images for the wound-healing assay at 0, 24 and 48 h after scratching for the 4 different cell groups (HUVECs with or without NRP2 overexpression cultured in the presence or absence of conditioned medium from BON cells). f Quantification of the healing rate at 48 h after wound-healing assays in HUVECs cultured in the presence or absence of conditioned medium from BON cells followed by transfection with empty vector or NRP2 plasmid. The data are shown as the means ± SD of three independent experiments. ***P ≤ 0.001 by Student’s t test. g HUVECs were transfected with empty vector or an NRP2 overexpression plasmid and then treated with the VEGFR2-specific inhibitor KI8751. Western blotting assays were performed to determine the levels of VEGFR2 phosphorylation at Tyr951 as well as the total protein levels of VEGFR2, CD31, CD34 and GAPDH. h Control and NRP2-overexpressing HUVECs were treated with KI8751 and PBS and evaluated for tube formation. i After HUVECs were cultured in the presence or absence of conditioned medium from BON cells for 24 h, they were transfected with a vector or NRP2 overexpression plasmid. These cells were subsequently treated with PBS (control) or KI8751, and a wound-healing assay was performed. Representative image of three independent experiments is shown. j Qualification of the wound-healing rate at 48 h in HUVEC-vector or HUVEC-NRP2 cells treated with PBS or KI8751. The data are shown as the mean ± SD of three independent experiments. **P ≤ 0.01 by Student’s t test. k After HUVECs were transfected with empty vector or an NRP2 overexpression plasmid, they were treated with PBS or KI8751 and subjected to the CCK8 assay at 24, 48 and 72 h after treatment. l Flow cytometry assay was performed in the 4 cell groups described in Fig. 2k
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