Dynamic phosphorylation of VE-cadherin Y685 throughout mouse estrous cycle in ovary and uterus.

We previously reported that vascular endothelial growth factor induced vascular endothelial (VE)-cadherin tyrosine phosphorylation at Y685 in a Src-dependent manner in vitro. Here, we studied the occurrence of Y685 phosphorylation in vivo in the female reproductive tract because it is a unique model of physiological vascular remodeling dependent on vascular endothelial growth factor. We first developed and characterized an anti-phospho-specific antibody against the site Y685 of VE-cadherin to monitor VE-cadherin phosphorylation along the four phases of mouse estrous cycle, termed proestrus, estrus, metestrus, and diestrus. A dynamic profile of tyrosine phosphorylated proteins was observed in both uterus and ovary throughout mouse estrous cycle, including kinase Src, which was found highly active at the estrus phase. The extent of tyrosine phosphorylated VE-cadherin was low at proestrus but strongly increased at estrus and returned to baseline at metestrus and diestrus, suggesting a potent hormonal regulation of this specific process. Indeed, C57Bl/6 female mice treatment with pregnant mare serum gonadotropin and human chorionic gonadotropin confirmed a significant increase in phosphoY685-VE-cadherin compared with that in untreated mice. These results demonstrate that VE-cadherin tyrosine phosphorylation at Y685 is a physiological and hormonally regulated process in female reproductive organs. In addition, this process was concomitant with the early steps of vascular remodeling taking place at estrus stage, suggesting that phosphoY685-VE-cadherin is a biomarker of endothelial cell activation in vivo.

VE-cadherin Y685F knock-in mouse is sensitive to vascular permeability in recurrent angiogenic organs.

Covalent modifications such as tyrosine phosphorylation are associated with the breakdown of endothelial cell junctions and increased vascular permeability. We previously showed that vascular endothelial (VE)-cadherin was tyrosine phosphorylated in vivo in the mouse reproductive tract and that Y685 was a target site for Src in response to vascular endothelial growth factor in vitro. In the present study, we aimed to understand the implication of VE-cadherin phosphorylation at site Y685 in cyclic angiogenic organs. To achieve this aim, we generated a knock-in mouse carrying a tyrosine-to-phenylalanine point mutation of VE-cadherin Y685 (VE-Y685F). Although homozygous VE-Y685F mice were viable and fertile, the nulliparous knock-in female mice exhibited enlarged uteri with edema. This phenotype was observed in 30% of females between 4 to 14 mo old. Histological examination of longitudinal sections of the VE-Y685F uterus showed an extensive disorganization of myometrium and endometrium with highly edematous uterine glands, numerous areas with sparse cells, and increased accumulation of collagen fibers around blood vessels, indicating a fibrotic state. Analysis of cross section of ovaries showed the appearance of spontaneous cysts, which suggested increased vascular hyperpermeability. Electron microscopy analysis of capillaries in the ovary showed a slight but significant increase in the gap size between two adjacent endothelial cell membranes in the junctions of VE-Y685F mice (wild-type, 11.5 ± 0.3, n = 78; and VE-Y685F, 12.48 ± 0.3, n = 65; P = 0.045), as well as collagen fiber accumulation around capillaries. Miles assay revealed that either basal or vascular endothelial growth factor-stimulated permeability in the skin was increased in VE-Y685F mice. Since edema and fibrotic appearance have been identified as hallmarks of initial increased vascular permeability, we conclude that the site Y685 in VE-cadherin is involved in the physiological regulation of capillary permeability. Furthermore, this knock-in mouse model is of potential interest for further studies of diseases that are associated with abnormal vascular permeability.