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However, the effect of changes in the maternal microenvironment on the mechanisms associated with the secretion of EVs from placental cells remains to be fully established. Here, we evaluated the effect of high glucose on proteins associated with the trafficking and release of different populations of EVs from placental cells. BeWo and HTR8\/SVneo cells were used as placental models and cultured under 5\u2010mM D\u2010glucose (i.e. control) or 25\u2010mM D\u2010glucose (high glucose). Cell\u2010conditioned media (CCM) and cell lysate were collected after 48\u00a0h. Different populations of EVs were isolated from CCM by ultracentrifugation (i.e. pellet 2K\u2010g, pellet 10K\u2010g, and pellet 100K\u2010g) and characterised by Nanoparticle Tracking Analysis. Quantitative proteomic analysis (IDA\/SWATH) and multiple reaction monitoring protocols at high resolution (MRM\n                    <jats:sup>HR<\/jats:sup>\n                    ) were developed to quantify 37 proteins related to biogenesis, trafficking\/release and recognition\/uptake of EVs. High glucose increased the secretion of total EVs across the pellets from BeWo cells, an effect driven mainly by changes in the small EVs concentration in the CCM. Interestingly, no effect of high glucose on HTR8\/SVneo cells EVs secretion was observed. High glucose induces changes in proteins associated with vesicle trafficking in BeWo cells, including Heat Shock Protein Family A (Hsp70) Member 9 (HSPA9) and Member 8 (HSPA8). For HTR8\/SVneo, altered proteins including prostaglandin F2\u03b1 receptor regulatory protein (FPRP), RAB5A, RAB35, RAB5B, and RB11B, STAM1 and TSG101. These proteins are associated with the secretion and trafficking of EVs, which could explain in part, changes in the levels of circulating EVs in diabetic pregnancies. Further, we identified that proteins RAB11B, PDCD6IP, STAM, HSPA9, HSPA8, SDCBP, RAB5B, RAB5A, RAB7A and ERAP1 regulate EV release in response to high and low glucose when overexpressed in cells. Interestingly, immunohistochemistry analysis of RAB7A revealed distinct changes in placental tissues obtained from women with normal glucose tolerance (NGT,\n                    <jats:italic>n<\/jats:italic>\n                    \u00a0=\u00a06) and those with GDM (\n                    <jats:italic>n<\/jats:italic>\n                    \u00a0=\u00a06), influenced by diet or insulin treatment. High glucose regulation of proteins involved in intercellular dynamics and the trafficking of multivesicular bodies to the plasma membrane in placental cells is relevant in the context of GDM pregnancies.\n                  <\/jats:p>","DOI":"10.1002\/jex2.135","type":"journal-article","created":{"date-parts":[[2024,1,16]],"date-time":"2024-01-16T00:20:43Z","timestamp":1705364443000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Differential response of placental cells to high D\u2010glucose and its impact on extracellular vesicle biogenesis and trafficking via small GTPase Ras\u2010related protein RAB\u20107A"],"prefix":"10.1002","volume":"3","author":[{"given":"Carlos","family":"Palma","sequence":"first","affiliation":[{"name":"Translational Extracellular Vesicles in Obstetrics and Gynae\u2010Oncology Group, Faculty of Medicine, University of Queensland Centre for Clinical Research, Royal Brisbane and Women's Hospital The University of Queensland  Brisbane Queensland Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew","family":"Lai","sequence":"additional","affiliation":[{"name":"Translational Extracellular Vesicles in Obstetrics and Gynae\u2010Oncology Group, Faculty of Medicine, University of Queensland Centre for Clinical Research, Royal Brisbane and Women's Hospital The University of Queensland  Brisbane Queensland Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katherin","family":"Scholz\u2010Romero","sequence":"additional","affiliation":[{"name":"Translational Extracellular Vesicles in Obstetrics and Gynae\u2010Oncology Group, Faculty of Medicine, University of Queensland Centre for Clinical Research, Royal Brisbane and Women's Hospital The University of Queensland  Brisbane Queensland Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haarika","family":"Chittoory","sequence":"additional","affiliation":[{"name":"UQ Centre for Clinical Research, Faculty of Medicine The University of Queensland  Brisbane Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7011-9889","authenticated-orcid":false,"given":"Benjamin","family":"Van Haeringen","sequence":"additional","affiliation":[{"name":"UQ Centre for Clinical Research, Faculty of Medicine The University of Queensland  Brisbane Australia"},{"name":"Pathology Queensland The Royal Brisbane and Women's Hospital  Brisbane Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Flavio","family":"Carrion","sequence":"additional","affiliation":[{"name":"Departamento de Investigaci\u00f3n, Postgrado y Educaci\u00f3n Continua (DIPEC), Facultad de Ciencias de la Salud Universidad del Alba  Santiago Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aase","family":"Handberg","sequence":"additional","affiliation":[{"name":"Department of Clinical Biochemistry Aalborg University Hospital  Aalborg Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Martha","family":"Lappas","sequence":"additional","affiliation":[{"name":"Obstetrics, Nutrition and Endocrinology Group, Department of Obstetrics and Gynaecology University of Melbourne  Victoria Australia"},{"name":"Mercy Perinatal Research Centre Mercy Hospital for Women  Victoria Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sunil R","family":"Lakhani","sequence":"additional","affiliation":[{"name":"UQ Centre for Clinical Research, Faculty of Medicine The University of Queensland  Brisbane Australia"},{"name":"Pathology Queensland The Royal Brisbane and Women's Hospital  Brisbane Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amy E","family":"McCart Reed","sequence":"additional","affiliation":[{"name":"UQ Centre for Clinical Research, Faculty of Medicine The University of Queensland  Brisbane Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"H. 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