Microvesicles derived from mesenchymal stem cells in diabetic-like conditions increase permeability in a retinal blood-barrier model

Background and aims: Loss of pericytes in the early phases of diabetic retinopathy may disrupt their stable association with endothelial cells (EC), leading to EC proliferation and, eventually, angiogenesis. Microvesicles (MV) are small membrane particles derived from different cells which contain biologically active proteins and RNA and are known to promote phenotypic changes in target cells. MV derived from injured cells may induce dedifferentiation of pericytes, allowing their detachment from vessels. We have previously shown that MV derived from mesenchymal stem cells (MSC), but not from EC, induce pericyte detachment and that diabetic-like conditions (high glucose and hypoxia) play a synergistic role in this destabilizing influence . This study aimed at evaluating whether MV produced by MSC in hypoxia and/or high glucose are able to influence the retinal blood-barrier permeability and to explore the possible role of matrix metalloproteases (MMP) in MV-induced pericyte detachment. Materials and methods: We used commercially available human microvascular EC (HMEC) and MSC from bone marrow, while human retinal pericytes (HRP) had been previously immortalized in our laboratory. A blood-barrier model was established by seeding HMEC on the porous membrane of transwell inserts, letting them adhere for 24 hrs and then adding HRP into the same insert. MV were extracted from the supernatant of MSC cultured in 1) physiological conditions (NG) 2) hypoxia (hypo) 3) high glucose (HG) 4) HG + hypo. These MV were subsequently added to the confluent HMEC/HRP co-cultures. After 2 hrs of MV exposure, FITC was added into the upper chamber and fluorescence measured in the lower chamber of the inserts after another 30’, 1, 2, 3, 4 and 24h. MMP expression in both MV and supernatants of HRP exposed to MV was evaluated by Zimography. Results were confirmed by pre-treatment of MV with batimastat, a MMP inhibitor, and subsequent exposure of HRP to them. Results: Permeability of EC-HRP co-cultures was increased by exposure to MSC-derived MV obtained in all the above conditions, the highest percentage increase occurring after 6h of total exposure to MV (2h pre-treatment + 4h FITC): NG-MV 134.42 ± 17.46% (p<0.05 vs control without MV), HG-MV 128.07 ± 3.81% (p=0.000), NG+hypo-MV 119.87 ± 15.56% (p<0.05), HG+hypo-MV 134.60 ± 15.21% (p<0.05). MSC-derived MV expressed MMP-2 and MMP-9. The same MMPs were expressed by HRP following 24h exposure to MV, MMP-2 to a much higher degree. HRP number decreased after 4h incubation with untreated MV (75.81 ± 9.22%, p<0.05 vs control), while pre-incubation of MV with batimastat completely reverted their effect on pericyte detachment. Conclusion: MSC-derived MV may play a role in vessel destabilization and increase of retinal blood-barrier permeability during the early stages of retinopathy. This effect may be mediated by increased MMP expression.