EuroSciCon CONFERENCE ON NANOTECHNOLOGY 2019, June 8-10, Prague, Czech Republic
Daniela Rebleanu1, Geanina Voicu1, Cristina Ana Constantinescu1, Letitia Ciortan1, Razvan Daniel Macarie1, Mihaela Avadanei1, Agneta Simionescu2, Ileana Manduteanu1, Manuela Calin1
1Institute of Cellular Biology and Pathology “Nicolae Simionescu” of Romanian Academy, Bucharest, Romania; 2Department of Bioengineering, Clemson University, United States of America
Introduction. Accumulating data indicate that diabetes accelerates vascular aortic disease. In the early phase, inflammation may induce in a subset of valvular endothelial cells (VEC) an endothelial to mesenchymal transformation (EndMT) and this process is governed by specific regulatory proteins.
Purpose. We hypothesize that, the silencing of the molecules critically involved in the EndMT, such as SMAD3 may be a therapeutic strategy in the early stages of aortic valve disease in diabetes.
Methods. VEC were isolated from human aortic valves of patients who have undergone valve replacement and separated from valvular interstitial cells using anti-CD31 magnetic beads. The cells were exposed for 5 days to HGMO (25mM glucose and osteogenic factors: 50 µg/ml ascorbic acid, 10 mM β-glycerophosphate, 10 nM Dexamethasone) in order to induce EndMT. Then, the cells were treated with polyplexes made from C60-PEI and various sequences of shRNA-SMAD3 specifically designed for SMAD3 silencing and analyzed after 48 hours for gene expression (RealTime PCR) and after72 hours for protein expression (Western Blot).
Results. The SMAD3 mRNA and protein level was significantly reduced by ~90% and ~ 65%, respectively in VEC exposed to HGMO medium and transfected with polyplexes C60-PEI/shRNA-SMAD3 as compared with non-transfected cells. The down-regulation of SMAD3 determines an increase in the expression of the endothelial marker CD31 and a decrease in the expression of mesenchymal marker aSMA.
Conclusions. This study indicates that the silencing of SMAD3 determines a reversal of the EndMT process induced by high glucose and osteogenic factors in VEC.
Acknowledgements. Work supported by the Competitiveness Operational Program 2014-2020, Priority Axis1/Action 1.1.4/, THERAVALDIS Project, contract no.115/13.09.2016/ MySMIS:104362.
Keywords: endothelial to mesenchymal transformation, high glucose, SMAD3, valvular endothelial cells.