Mesenchymal stem cell-derived microvesicles induce pericyte destabilization in diabetic-like conditions

Background and aims: During angiogenesis, the stable association between pericytes and endothelial cells (EC) in pre-existing vasculature is disrupted, leading to EC proliferation. Recent findings suggest the existence of a novel way of intercellular communication, where the ‘units’ of information are microvesicles (MV), or exosomes, derived from different cells and containing biologically active proteins and RNA, that may promote phenotypic changes in other target cells. MV derived from injured cells may induce dedifferentiation of pericytes allowing their detachment from vessels. The literature on the molecular interactions between EC-derived MV and pericytes for the angiogenic switch is growing, but little is known about mesenchymal stem cells (MSC), a self-renewing cells that can be found in most tissues, to activate pericytes. Our study aimed at evaluating if MV produced by MSC in hypoxia and/or hyperglycaemic-like conditions are able to induce pericyte detachment and influence their survival. Materials and methods: MSC from bone marrow and human microvascular EC were of commercial origin, while human retinal pericytes (HRP) were immortalized in our laboratory. MV were extracted from the supernatant of MSC or EC cultured in hypoxic and/or high glucose (HG) conditions and added to HRP cultured in physiological conditions both on plastic and EC-produced extracellular matrix (ECM). We evaluated HRP detachment after 2, 4, 6 and 24h exposure to MV by cell counts, as well as viability, cytotoxicity and apoptosis by an ELISA/fluorimetric assay. Motility and cell modifications were analyzed by a MicroImage analysis system. Results: The number of attached HRP decreased in a time-dependent manner after addition to the culture medium of MSC-derived MV obtained in all the above-described conditions, the higher decrease occurring after 4h of MV exposure. HRP number on plastic after 4h: with MV obtained in physiological conditions (NG) -35.8% vs control (HRP not exposed to MV, p<0.05, n=5); MV in HG -27.1% (p<0.05), MV in NG+hypoxia -27.2% (p<0.05), MV in HG+hypoxia -37.0% (p<0.005). HRP number on EC-produced ECM after 4h: MV in NG -30.1% vs control (p<0.05); MV in HG -52.3% (p<0.005), MV in NG+hypoxia -40.3% (p<0.005), MV in HG+hypoxia -41.0% (p<0.005). Apoptosis and cytotoxicity did not change significantly. Trypan blue staining and TUNEL assay showed that detached cells were alive. Increased cell motility was observed in the presence of MV, reaching its maximum after 1h exposition of HRP to MV (+56.0% vs control, p<0.05), while microscopy observation showed evidence of cell contraction. Interestingly, EC-derived MV had no effects on pericyte viability/destabilization. Conclusion: We conclude that MSC-derived MV induce HRP detachment, a possible indicator of destabilization. Diabetic-like conditions (hyperglycaemia and hypoxia) may play a synergistic role in influencing vessel destabilization during the stages of retinopathy that precede angiogenesis. Since this role seems to be MSC-specific, EC-derived MV having no effect, further studies are needed to better characterise MSC-HRP interactions.