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-κB, VEGF, Flt-1 and Flk-1 in the rat lung after intrauterine infection. (A) The mRNA expression levels of Notch4, (B) Dll4, (C) NF-κB, (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-κB, 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.