Figure 3. sFLT1 alters the vascular endothelium by decreasing eNOS function.

(A) VEGF induces dose-dependent activation of eNOS in HUVECs, as measured by eNOS phosphorylation at Ser1177 (phospho-eNOS) relative to total eNOS by Western blotting. This is inhibited by excess sFLT1 (1 μg/ml). Representative of 4 individual experiments. (B) Densitometry of phospho-eNOS/total eNOS bands plotted as a percentage of control. VEGF-induced eNOS phosphorylation in HUVECs was approximately 2.5-fold above control at 10 ng/ml of VEGF (P < 0.001 vs. all other groups). The increase in VEGF-induced phosphorylation was blocked when cells were preincubated with excess of sFLT1; *P < 0.001. Analysis by 2-way repeated-measures ANOVA, n = 4 individual experiments. (C) IHC for phospho-eNOS expression in mesenteric resistance vessels from gd17 pregnant mice given CMV-null (left) or sFlt1 (right). Images are representative of 4–5 mice per group. Scale bar: 100 μm. (D) Administration of l-NAME in drinking water during pregnancy phenocopies sFlt1-induced Ang II hypersensitivity. Pregnant mice were given 1 g/l l-NAME in drinking water at gd8 and then implanted with s.c. osmotic minipumps infusing Ang II or saline control at gd12. Blood pressure became significantly elevated only in mice given l-NAME and Ang II (n = 4 per group; daily mean ± SEM). Two-way repeated-measures ANOVA, *P < 0.01. (E) Comparison of MAP and plasma sFLT1 between l-NAME + saline and l-NAME + Ang II pregnant mice (n = 4 per group). ΔMAP was obtained by subtraction of individual pressure at gd17 from that at gd7. Gd7 was selected as the latest time point when all animals had no pressure differences (prior to intervention, permitting comparison). *P < 0.01. Analysis by 1-way ANOVA with Bonferroni’s post-hoc test. (F) Representative images of aorta isolated from CMV-null (left), sFlt1 (middle), and l-NAME (right) mice at gd17 staining for MitoSOX Red (top; scale bar: 50 μm) and nitrotyrosine (bottom; scale bar: 20 μm). All data represent the mean ± SEM.