Fig 1: Fasudil decreases blood pressure induced by sFlt infusion in pregnant mice(A) Non-invasive rat tail blood pressure detecting method was used to measure blood pressure on day 13 of gestation. Student t test was used for statistical analyses. (B) Non-invasive rat tail blood pressure detecting method was used to measure systolic blood pressure on day 19 of gestation after 6 days Fasudil treatment or ROCK knockdown by siRNA. (C) ELISA was used to detect the concentration of Angiotensin converting enzyme (ACE) in peripheral blood. (D) The proteinuria was measured on day 19 of gestation after 6 days Fasudil treatment or ROCK knockdown by siRNA. The PE mice treated with saline were used as control. ELISA was used to detect the concentration of s-Flt in peripheral blood (E) and cord blood (F). Number of animal for each group=8. Data were expressed as mean ± standard error. ANOVA with post hoc Tukey’s test was used for statistical analyses. *p<0.05, **p<0.01, ***p<0.001.
Fig 2: NK cell HIF-1α deficiency increases VEGF bioavailability and endothelial cell migration. a Determination of levels of VEGF and sVEGFR1 protein in MC38 isografts implanted in WT and HIF-1α KO mice by ELISA at endpoint, day 14 (n = 8). b Immunoblotting for VEGFR2 and phosphotyrosine (p-Tyr) after immunoprecipitation of VEGFR2 from MC38 tumour lysates and ratio of p-Tyr and VEGFR2 signal intensities as measured by photon emission at endpoint, day 14 (n = 8). c Gene expression analysis for soluble variant of FLT1 and total form of FLT1 on sorted NK cells and endothelial cells from naïve spleens from WT and HIF-1α KO mice (n = 6). d Gene expression analysis for soluble variant of FLT1 and total form of FLT1 on sorted intratumoural NK cells and endothelial cells from MC38 tumours injected subcutaneously in WT and HIF-1α KO mice at endpoint, day 10 (n = 10). Statistical significance was determined by an unpaired Student’s t-test or one-way analysis of variance, where appropriate. Bars represent mean values; error bars indicate the s.e.m. Statistical significance is indicated as *P < 0.05, **P < 0.01, and ***P < 0.001
Fig 3: Reconstitution of sVEGFR1 rescues the HIF-1α KO phenotype. a Tumour volume analysis of MC38 isografts implanted in WT and HIF-1α KO mice after intratumoural injection with recombinant sVEGFR1 protein or sVEGFR1 vector at day 4, 6, 8, and 12 at endpoint, day 14. Control mice received intratumoural injections of 100 μl PBS or ctrl vector. b Quantitative analysis of CD31-positive endothelial cells and pericyte coverage as assessed by α-SMA/CD31 co-localisation at endpoint, day 14. c Quantitative analysis of hypoxic tumour areas with the specific marker GLUT1 at endpoint, day 14. d Quantitative analysis of caspase-3-positive areas at endpoint, day 14 (n = 10 for ctrl group; n = 5 for sVEGFR1 protein injection group; n = 3 for sFLT1 vector injection group). Statistical significance was determined by an unpaired Student’s t-test or one-way analysis of variance, where appropriate. Bars represent mean values; error bars indicate the s.e.m. Statistical significance is indicated as *P < 0.05, **P < 0.01, and ***P < 0.001
Fig 4: VEGFR1 and VEGFR3 are Predominant in Trophoblasts.Relative mRNA expression of VEGFR1, VEGFR2, VEGFR3 in both HUVECs and BeWo cells (A); VEGFR gene expression in BeWo cells (B); and HUVECs (C). BeWo cells treated with either a vehicle or sFlt-1 showing the protein levels of pVEGFR1 (D); and pVEGFR3 (E); One Way ANOVA, Tukey’s post hoc test for A-C. Unpaired t test in D,E. *p<0.05,***p<0.001, ****p<0.0001.
Fig 5: VEGFR1 and VEGFR3 induce leptin production in trophoblasts.Western blot showing the effect of VEGFR1 siRNA (A) and VEGFR3 siRNA (B) on their respective receptor protein expression. Leptin secretion level measured in the media of BeWo cells treated with sFlt-1, siVEGFR1, siVEGFR3, siVEGFR1+ siVEGFR3, or a vehicle (C); Leptin secretion level measured in the media of BeWo cells treated with sFlt-1, VEGF165b, VEGFc, vehicle or a combination (D). Unpaired t-test in figure 3A–D. One Way ANOVA, Fisher’s LSD post hoc test for 3E-F. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Supplier Page from R&D Systems, a Bio-Techne Brand for Recombinant Mouse VEGFR1/Flt-1 Fc Chimera Protein, CF