Study of the differentiation of pheripheric NK cells into non cytotoxic, pro-angiogenic decidual NK cells and their association in the pathophysiology of preeclampsia

• Grant number: 12/16025-0
• Support Opportunities: Scholarships abroad - Research
• Start date: March 18, 2013
• End date: March 04, 2014
• Field of knowledge: Health Sciences - Medicine - Maternal and Child Health
• Principal Investigator: Ricardo de Carvalho Cavalli
• Grantee: Ricardo de Carvalho Cavalli
• Host Investigator: S. Ananth Karumanchi
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Institution abroad: Harvard University, Cambridge, United States

Abstract

Background: recent data have suggested that natural killer (NK) cells in the maternal-fetal interface are important regulators of the remodeling of maternal spiral arteries through its ability to produce angiogenic factors. In humans, some combinations of maternal NK KIR cell haplotypes (KIR AA) and paternal HLA-C alleles (HLA-C2) have been associated with pre-eclampsia, intrauterine growth restriction and recurrent abortion, suggesting that tissue NK (dNK) have a role in the pathogenesis of these disorders. Objective: to differentiate peripheral blood NK cells into tissue NK cells. To characterize pro-angiogenic dNK-like cells generated in vitro by exposure of pNK cells to hypoxia and to TGF²; to characterize the secretory profile of the angiogenic molecule of dNK-like cells and to establish whether dNK-like are terminally differentiated. Materials and methods: pNK cells will be converted into dNK-like cells by exposure to hypoxia and TGF² for one week or culture under normoxia conditions without TGF² as a control. The supernatants of the cell cultures will be tested for Ang1, Ang2, VEGF-C, PLGF and IL-8 content by ELISA. dNK-like cells induced by TGF² and hypoxia will be returned to normoxia conditions in the absence of TGF². The supernatants of the cell culture will be collected at different time points during the study (3, 5 and 7 days) for the determination of VEGF-A content by ELISA. The cells will be labeled with monoclonal antibodies to evaluate the expression of the CD9 marker of dNK cells by NK CD56Bright CD16- cells by flow cytometry analysis. Its cytotoxic activity against the K562 target cells will be evaluated by 51Cr release assays. In all experiments, dNK-like cells maintained under hypoxia in the presence of TGF² will be used as control. Samples from at least 5 independent donors will be analyzed. In a second approach, we will generate clones of pNK cells treated under hypoxia and in the presence of TGF² and determine whether they maintain the properties of dNK-like cells under condition of normoxia in the absence of TGF². The Student t-test will be used to compare the experimental and control data. (AU)