We evaluated whether physiological and pre-eclamptic (PE) placentae, characterized by exacerbated inflammation, presented alterations in pro-inflammatory High Mobility Group Box 1 (HMGB1) and its Receptor of Advanced Glycation End products (RAGE) expression. Moreover, we investigated, in physiological placental tissue, the ability of Low Molecular Weight Heparin (LMWH) to modify HMGB1 structural conformation thus inhibiting RAGE binding and HMGB1/RAGE axis inflammatory activity. HMGB1, RAGE, IL-6 and TNFα (HMGB1/RAGE targets) mRNA expression were assessed by Real Time PCR. HMGB1, RAGE protein levels were assessed by western blot assay. Physiological term placental explants were treated by 0.5 U LMWH for 24 or 48 h. HMGB1 and RAGE expression and association were evaluated in LMWH explants by RAGE immunoprecipitation followed by HMGB1 immunoblot. HMGB1 spatial localization was evaluated by immuofluorescent staining (IF). HMGB1 expression was increased in PE relative to physiological placentae while RAGE was unvaried. 24 h LMWH treatment significantly up-regulated HMGB1 expression but inhibited HMGB1/RAGE complex formation in physiological explants. RAGE expression decreased in treated relative to untreated explants at 48 h. IF showed HMGB1 localization in both cytoplasm and nucleus of mesenchymal and endothelial cells but not in the trophoblast. IL-6 and TNFα gene expression were significantly increased at 24 h relative to controls, while they were significantly down-regulated in 48 h vs. 24 h LMWH explants. Our data depicted a new molecular mechanism through which LMWH exerts its anti-inflammatory effect on PE placentae, underlying the importance of HMGB1/RAGE axis in PE inflammatory response.
Ovarian cancer (OC) has the highest mortality rate of all gynecological cancers. We previously demonstrated that PDMSCs produce soluble factors able to inhibit OC cell cycle and growth through a pathway that still remains to be determined. Hypoxia Inducible Factor-1 alpha (HIF-1a), main player in cellular response to hypoxia, promotes the expression of VEGF, responsible for neo-angiogenesis. HIF-1a overexpression has been described in most human malignancies and it is linked to bad cancer prognosis. Activating Protein-1 (AP-1) molecules are pivotal regulators of OC cell cycle progression and metastatization. Herein, we investigated HIF-1a, VEGF and JunB expression in OC ES-2 and SKOV-3 cells treated by PDMSCs conditioned medium (CM) in order to verify our hypothesis that PDMSCs could act through the modulation of HIF-1a and anti-proliferative AP-1 molecules.Methods: PDMSCs were isolated from physiological human term placentae (n=12). CM will be produced by incubating PDMSCs for 24-48 hours in DMEM medium. CM will be harvested and filtered to remove cellular debris. ES-2 and SKOV-3 cells were treated by 24/48h CMs for 24h or 48h and mRNA was isolated. Expression of HIF-1a, VEGF and JunB was assessed by Real Time PCR.
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