Expression and localization of vascular endothelial growth factor and its receptors in the pig uterus during peri-implantation and determination of the in vitro effect of the angiogenesis inhibitor SU5416 on VEGF system expression

Implantation of the embryo and development of a network of connections towards maternal circulation are evolutionary advantages of eutherian mammals [1]. Sows develop a diffuse placenta, mutually folded and epitheliochorial, where the fetal tissue does not invade the maternal endometrium [2]. During peri-implantation, the uterine luminal epithelium and the trophectoderm of the conceptus develop adhesion competence simultaneously to initiate signaling towards implantation within a restricted period of the uterine cycle called the “window of receptivity” [3], which is key to survival and development [4].

During gestation, a prenatal mortality rate of approximately 40% is reported [5,6]. These spontaneous losses occur during peri-implantation (10–30 gestation days) and mid-gestation (50–70 days) [5,7]. While the potential mechanisms involved in embryonic survival are still being investigated, angiogenesis, a process that begins around day 15 post-insemination [8], seems to be crucial in the successful development of conceptus during gestation [9]. In other words, a deficient supply of uterine blood caused by an undeveloped maternal-embryonic interface due to angiogenesis inhibition could be associated with the loss of embryos during peri-implantation.

Angiogenesis is a complex process that leads to the formation of new blood vessels from pre-existing vessels and depends on a strict regulation of promoting and inhibitory factors [10], such as vascular endothelial growth factor (VEGF) and its membrane receptors, that are among the most important ligand-receptor systems involved in the initiation of the angiogenesis signal transduction cascade [11]. The biological functions of VEGF-A are mediated by its specific binding to two receptor tyrosine kinases (RTKs), VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1) [12]. VEGF-A stimulates the migration, proliferation, and vascular permeability of endothelial cells (ECs) under physiological and pathological conditions [13]. During implantation in mammals such as human [14], mouse [15], pig [16], and cattle [17], VEGF appears to be one of several crucial angiogenic elements. Since the endometrium exhibits rapid cyclical growth and regression throughout the reproductive life of the sow, an increase in VEGF expression during gestation has been reported [8,18].

VEGFR2 appears to be the main signaling receptor in ECs in blood vessels, whose activation promotes intracellular signal transduction in pathways involved in the proliferation and migration of ECs and in greater survival and vascular permeability [19]. In addition, an autophosphorylation capability at least 10 times greater than that of VEGFR1 has been reported [20]. Moreover, chemotactic and differentiation effects in ECs have been attributed to VEGFR1; however, due to its limited signaling capacity compared to VEGFR2, it seems to be acting as a “decoy” receptor [18]. With the use of genetic approaches [21], neutralizing antibodies [22], and synthetic inhibitors of tyrosine kinase activity [23] it has been demonstrated that blocking VEGF signaling via VEGFR2 results in the inhibition of mitogenesis in ECs. Therefore, VEGFR2-mediated inhibition of the RTKs pathway could result in disruption of angiogenesis. Inhibitor SU5416 is a highly bound, small molecule, synthetic RTKs inhibitor of VEGFR2 [24]. It acts selectively as a competitive inhibitor, binding to the adenosine triphosphate (ATP) catalytic site, within the kinase domain of the receptor, which induces a blockade of VEGFR2 signaling, inhibiting the VEGF-dependent proliferation of ECs in vitro and animal models [25].

Although the endometrial neovascularization process involved in the establishment of pregnancy has been the subject of several studies in recent years, much remains to be clarified regarding this phenomenon [26]. Given the limited information available on the processes that lead to high levels of mortality during pregnancy, or on the expression and localization of molecules associated with uterine angiogenesis in the early stages of pregnancy in the sow, the objective of the present study was to identify and locate the proteins and mRNA expression of VEGF-A, VEGFR1, and VEGFR2 in the endometrial implantation sites, at different stages of peri-implantation and in uterine tissue during the estrous cycle (EC), in order to know their expression pattern and localization. Additionally, a primary culture of sow endometrial epithelial cells was established to define the potential of the selective inhibition of VEGFR2 after treatment with inhibitor SU5416 and determine its effects on the expression pattern of the VEGF system.

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