P-551: Blockage of the VEGF-C/VEGF receptor 3 pathway disrupts early pregnancy development in the rodent

Abstract

A major regulator of angiogenesis and/or lymphangiogenesis is VEGF-C acting through the VEGF receptor 3 (VEGFR-3). It is currently not known whether functionality of this pathway is important in the peri-implantation period. We tested whether a single dose of a blocking antibody against VEGFR-3 (mF4-31C1) on embryonic day (ED) 3.75 might interfere with normal embryonic development. Prospective animal laboratory. Adult pregnant female CD1 mice were randomized into 5 groups: baseline: sacrificed on ED 3.75; control: saline injection on ED 3.75; replacement control: Subcutaneous placement of three 10 mg/pellet 21-day release progesterone (P4) tabs (Innovative Research of America) on ED 3.5, and saline injection on ED 3.75; treatment: anti-VEGF-R3 antibody mF4-31C1 injection (160 mg/kg; ImClone Systems, New York) on ED 3.75; replacement plus treatment: Three P4 tabs ED 3.5, and anti-VEGF-R3 antibody ED 3.75. Animals were sacrificed on ED 6.5 or 13.5 (n=5 per group). Outcome parameters included: uterine and ovarian histology immunofluorescence (antibodies against the endothelial cell marker PECAM and against VEGFR-3), uterine weights, number of implantation sites, embryonic development, and maternal P4 levels. The Wilcoxon rank test was used for statistical analysis. PECAM staining mostly colocalizes with staining for VEGFR-3 when analyzed on ED 6.5 indicating that this receptor is primarily found on small vasculature in ovarian corpora lutea and uterus. Uterine weights increased in control and replacement control animals from 0.27±0.02g (ED 3.5) to 6.4±0.9g (ED 13.5) with the progression of pregnancy, whereas no weight increase was observed in the treatment and replacement plus treatment groups on ED 13.5 (p<0.01). Implantation sites were mostly absent on ED 13.5 in the treatment and the replacement plus treatment group. Histological evaluation of the uterus revealed the presence of minimal necrotic tissue with or without old blood on ED 13.5. In contrast in control and replacement control animals appropriate embryonic and placental structures were observed on ED 13.5. Endothelial cell staining with PECAM demonstrated that luteal vascular density (ovary) was similar in all groups on ED 6.5. In contrast uterine blood vessel density evaluated on ED 6.5 decreased in the two treatment groups by 25% when compared to controls (p<0.05). Progesterone (ED 13.5) levels fluctuated around 25 ng/ml in all groups on ED 13.5. Anti-VEGFR-3 antibody administered in the peri-implantation period interfered with in-vivo pregnancy development. The antibody disrupted formation and maintenance of peri-implantation blood vessels, possibly decreasing the blood supply to the developing embryo. In contrast the antibody did not affect ovarian P4 secretion or luteal endothelial cells. Interestingly, P4 replacement did not reverse the effects of the antibody, and disruption of pregnancy development occurred. Therefore, the VEGF-C/VEGFR-3 pathway seems to be important for early pregnancy development in the rodent. One mechanism of action seems to be the regulation of angiogenesis in the decidua during the peri-implantation period.