ROLE OF CAVEOLAE IN THE FORMATION OF THE PLACENTAL MICROVASCULATURE AND ITS INTERACTION WITH AQP1 AND VEGFR2

ESCUDERO, CARLOS A.; SIERRA, MATÍAS N.; MARTÍNEZ, NORA; REPPETTI, JULIETA; DAMIANO, ALICIA E.

BUENOS AIRES

Workshop; WORKSHOP IFIBIO; 2019

IFIBIO

ROLE OF CAVEOLAE IN THE FORMATION OF THE PLACENTAL MICROVASCULATURE AND ITS INTERACTION WITH AQP1 AND VEGFR2.Reppetti Julieta1, Sierra Matías N.2, Carlos A. Escudero3, Alicia E. Damiano1,2, Nora Martínez1.1 Laboratorio de Biología de la Reproducción. Instituto de Fisiología y Biofísica Bernardo Houssay (IFIBIO Houssay) UBA-CONICET. Buenos Aires, Argentina.2 Cátedra de Biología Celular y Molecular. Departamento de Ciencias Biológicas, Facultad de Farmacia y Bioquímica, UBA. Buenos Aires, Argentina.3 Laboratorio de Fisiología Vascular, Grupo de Investigación en Angiogénesis Tumoral (GIANT). Departamento de Ciencias Básicas, Universidad del Bío-Bío. Chillán, Chile.Introduction: A proper development of the placental vasculature is necessary to ensure a successful pregnancy. In the human placenta, there are two types of endothelial cells: the human placental microvascular endothelial cells (hPMECs) which are in the chorionic villi, and the macrovascular endothelial cells which are in the umbilical cord. These cells differ in morphology, gene expression and function. Caveolae are membrane domains that compartmentalize intracellular signaling pathways to orchestrate different cellular events, such as cell migration and invasion. Caveolae are a type of lipid rafts, coated with caveolin-1 (Cav-1) protein which can bind to different membrane receptor proteins and signaling molecules and concentrate these molecules in the caveolae. VEGFR2 is known to regulate endothelial survival, proliferation, migration and formation of the vascular tube. On the other hand, Aquaporin-1 (AQP1) is a transmembrane water channel which moves water in response to osmotic gradients. Recently, it was suggested that VEGFR2, AQPs and Cav-1 are also indispensable for efficient cell migration. Our hypothesis is that AQP1 and VEGFR2 located in the caveolae are essential for the proper formation of the human placental microvasculature.Objective: To evaluate the role of caveolae in the formation of the placental microvasculature and its interaction with AQP1 and VEGFR2.Materials and Methods: Primary culture of placental microvascular endothelial cells (hPMEC) was cultured in complete M199. This study was approved by the ethics committee of the Hospital Nacional Prof. Dr. A. Posadas. In Silico analysis of Cav-1 binding site in AQP1 and VEGFR2 was performed using BioEdit and PyMol programs. Gene expression of Cav-1 (sense 5?-TCTCTACACCGTTCCCATCC-3?; antisense 5?-CACAGACGGTGTGGACGTAG-3?), AQP1 (sense 5?-GAGTATGACCTGGATGCCGA-3?; antisense 5?-GGCCAGCTTGTCAGAGTGT-3?) and VEGFR2 (sense 5?-CTTCGAACGATCAGCATAAGAAACT-3?; antisense 5?-TGGTCATCAGCCCACTGGAT-3?) in microvascular and macrovascular endothelial cells was evaluated by RT-PCR. Protein expression and localization were studied by Western Blot and immunofluorescence. Co-localization of Cav-1 with AQP1 and VEGFR2 was assessed by immunoprecipitation assay. Cells were treated with 5 mM methyl-β-cyclodextrin (MβCD) to disrupt caveolae and with Tetraethylammonium chloride (TEA) to block AQP1. Cell viability was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. Macrovasculature cell line EA.hy926 (ATCC® CRL-2922 ?) derived from the fusion of human umbilical vein macrovascular endothelial cells (HUVEC) with the human cell line A549 were used as controls.Results: hPMEC cells express Cav-1, AQP1 and VEGFR2. All these proteins were localized in the cell membrane of hPMEC and AQP1 and VEGFR2 co-localize with Cav-1. MβCD significantly reduced cell migration in EA.hy926 cells (n=4; p