Fig 1: Inhibition of Notch4/Dll4 signaling decreased the expression of NF-?B, inhibited cell apoptosis and promoted cell proliferation in PMVECs. (A) Protein and (B) mRNA expression levels of NF-?B in PMVECs in the control, LPS and DAPT+LPS groups. (C) Cell viability in the control, LPS and DAPT+LPS groups was measured using an MTT assay. (D) Representative plots and quantitative flow cytometric analysis of the apoptotic rates of PMVECs in the control, LPS and DAPT+LPS groups. In the plots, quadrant a shows the proportion of dead/necrotic cells (Annexin V-FITC-/PI+), quadrant b shows the late apoptotic cells (Annexin V-FITC+/PI+), quadrant c shows the early apoptotic cells (Annexin V-FITC+/PI-) and quadrant d shows the live cells (Annexin V-FITC-/PI-). The sum of quadrants b and c was calculated as the apoptotic rate. Data are presented as the mean ± SD (n=6 per group). *P<0.05 with comparisons shown by lines. Dll4, delta-like canonical Notch ligand 4; PMVECs, pulmonary microvascular endothelial cells; LPS, lipopolysaccharide; PI, propidium iodide.
Fig 2: Extra-endothelial DLL4 signalling inhibits HemECs angiogenesis. (A) HemECs were co-cultured with normal human dermal fibroblasts and treated with conditioned media from CHO cells, untransduced (negative) or transduced with GFP (Ad.GFP) or sDLL4 adenovirus (ad.DLL4) and VEGF-A165a or VEGF-A165b; the cells were then stained for VE-cadherin. (B–D) Quantifications of (A): VEGF-A165a, not VEGF-A165b, induced endothelial tube extension, branching and coverage in the assay (n = 3; p < 0.001); conditioned media from CHO cells infected with ad.sDLL4 inhibited the in vitro angiogenic potential of HemECs, irrespective of the VEGF-A isoforms present (n = 3; p < 0.001, two-way ANOVA) compared with ad.GFP or untransduced conditioned medium. (E) Sections of IH were stained for NG2 and DLL4: typical stainings of proliferating and involuting IH are shown; relatively low NG2 staining was present in the proliferating phase; in the involuting phase, a relatively high proportion of DLL4 staining co-localized with NG2-positive cells in the more organized vasculature. (F) DLL4 protein expression was found in HemECs and IH pericytes, but VEGFR2 protein was undetectable in IH pericytes. Scale bars = 100 µm
Fig 3: VEGFR2 signalling, degradation and downstream targets are differentially regulated by VEGF-A isoforms. Cells were serum-starved prior to treatment with 2.5 nm VEGF-A165a, VEGF-A165b or both. (A, B) VEGF-A165a induced VEGFR2 signalling and downstream activation of ERK1/2 in both HemSCs and HemECs; VEGF-A165b weakly activated VEGFR2 and ERK1/2 in HemSCs and HemECs; VEGF-A165b inhibited VEGF-A165a-mediated VEGFR2 and ERK1/2 phosphorylation in both HemSCs and HemECs. (C) HemECs were treated with 2.5 nm VEGF-A165a and VEGF-A165b and protein extracted at the time points shown. (D) Quantification of (C): treatment with VEGF-A165b, but not VEGF-A165a, significantly reduced VEGFR2 levels; n = 3; p < 0.05, one-way ANOVA; *p < 0.05 compared with VEGF-A165b; # p < 0.05 compared with time 0. (E) HemECs were treated as above and RNA extracted and subjected to RT–qPCR for DLL4 expression; p < 0.001, ANOVA. (F) VEGF-A165a-induced DLL4 expression compared with untreated (n = 3; p < 0.05, one-way ANOVA); VEGF-A165b, alone or together with VEGF-A165a, did not induce DLL4 expression at the protein level; *p < 0.05, **p < 0.01, ***p < 0.01; NS, not significant
Fig 4: Inhibition of Notch4/Dll4 signaling decreases the expression of VEGF and its receptors in LPS-induced PMVECs. (A) The mRNA expression levels of Notch4 and Dll4 were determined by reverse transcription-quantitative PCR in PMVECs that were exposed to different concentrations of LPS. (B) Notch4, (C) Dll4, (D) VEGF, (E) Flt-1 and (F) Flk-1 mRNA levels were determined in PMVECs in the control, LPS and DAPT+LPS groups. (G) Western blot analysis of protein expression levels in the PMVEC treatment groups. Data are presented as the mean ± SD (n=6 per group). *P<0.05 with comparisons shown by lines. Dll4, delta-like canonical Notch ligand 4; PMVECs, pulmonary microvascular endothelial cells; LPS, lipopolysaccharide; Flt-1, FMS-like tyrosine kinase 1; Flk-1, fetal liver kinase 1.
Fig 5: Aberrant expression of Notch4, Dll4, NF-?B, VEGF, Flt-1 and Flk-1 in the rat lung after intrauterine infection. (A) The mRNA expression levels of Notch4, (B) Dll4, (C) NF-?B, (D) VEGF, (E) Flk-1 and (F) Flt-1 were evaluated in the two groups by reverse transcription-quantitative PCR. (G) The protein expression levels of Notch4, Dll4 and NF-?B, and (H) VEGF, Flt-1 and Flk-1 were determined in the two groups at P3 by western blotting. Data are presented as the mean ± SD (n=10 per group). *P<0.05. Dll4, delta-like canonical Notch ligand 4; Flt-1, FMS-like tyrosine kinase 1; Flk-1, fetal liver kinase 1; P, postnatal day.
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