Purpose. Human corneal endothelial cells (HCECs) are essential to visual function; however, since they have limited proliferative capacity in vivo, they are prone to corneal endothelial dysfunction. At present, the only treatment is a corneal transplantation from donor cadavers. Also, due to a global shortage of donor corneas, it is important to find alternative strategies. Recent studies highlight that stem cell–derived extracellular vesicles (EVs) play a relevant role in stem cell-induced regeneration by reprogramming injured cells and inducing proregenerative pathways. The aim of this work is to evaluate whether EVs derived from mesenchymal stem cells (MSC-EVs) are able to promote regeneration of damaged HCECs. Methods. We isolated HCECs from discarded corneas in patients undergoing corneal transplantation or enucleation ( N = 23 patients). Bone marrow mesenchymal stem cells (MSCs) were obtained from Lonza, cultured, and characterized. MSC-EVs were obtained from supernatants of MSCs. In order to establish a valid in vitro damage model to test the regenerative potential of EVs on HCECs, we evaluated the proliferation rate and the apoptosis after exposing the cells to serum-deprived medium at different concentrations for 24 hours. We then evaluated the HCEC migration through a wound healing assay. Results. In the selected serum deprivation damage conditions, the treatment with different doses of MSC-EVs resulted in a significantly higher proliferation rate of HCECs at all the tested concentrations of EVs ( 5 ‐ 20 × 10 3 MSC-EV/cell). MSC-EVs/cell induced a significant decrease in number of total apoptotic cells after 24 hours of serum deprivation. Finally, the wound healing assay showed a significantly faster repair of the wound after HCEC treatment with MSC-EVs. Conclusions. Results highlight the already well-known proregenerative potential of MSC-EVs in a totally new biological model, the endothelium of the cornea. MSC-EVs, indeed, induced proliferation and survival of HCECs, promoting the migration of HCECs in vitro.

Corneal endothelial dystrophy is a relevant cause of vision loss and corneal transplantation worldwide. In the present study, we analyzed the effect of mesenchymal stem cell (MSC)-derived extracellular vesicles (MSC-EVs) in an in vitro model of corneal dystrophy, characterized by endoplasmic reticulum stress. The effects of MSC-EVs were compared with those of serum-derived EVs, reported to display a pro-angiogenic activity. MSC-EVs were able to induce a significant down-regulation of the large majority of endoplasmic reticulum stress-related genes in human corneal endothelial cells after exposure to serum deprivation and tunicamycin. In parallel, they upregulated the Akt pathway and limited caspase-3 activation and apoptosis. At variance, the effect of the serum EVs was mainly limited to Akt phosphorylation, with minimal or absent effects on endoplasmic reticulum stress modulation and apoptosis prevention. The effects of MSC-EVs were correlated to the transfer of numerous endoplasmic reticulum (ER)-stress targeting miRNAs to corneal endothelial cells. These data suggest a potential therapeutic effect of MSC-EVs for corneal endothelial endoplasmic reticulum stress, a major player in corneal endothelial dystrophy.

Anti-angiogenic therapy is an important strategy to limit growth, development and expansion of solid tumors. However, resistance to VEGF-targeting agents may develop, due to activation of alternative pro-angiogenic pathways, indicating the need of multiple target strategy. Here we obtained tumor endothelial cells (TEC) either from total renal carcinomas or from renal cancer stem cells (CSC-TEC) and we tested the effect of a CD105 targeting monoclonal antibody, TRC105, alone or in association with anti-VEGF drugs. We demonstrated that TRC105 impaired the ability of TEC and CSC-TEC to organize in tubular structures, whereas it did not limit proliferation or survival. The combination of TRC105 with different anti-angiogenic drugs showed a synergistic effect of TRC105 only in combination with the tyrosine kinase inhibitor Sunitinib. In particular, TRC105 plus Sunitinib reduced tubulogenesis, proliferation and survival of CSC-TEC and tumor-derived TEC in a similar manner. At a molecular level, we showed that the combination of TRC105 and Sunitinib induced the phosphorylation of Smad 2/3 to promote endothelial cell death. Moreover, TRC105 enhanced the inhibitory effect of Sunitinib on VEGF signaling and reduced VEGFR2-Akt-Creb activation, suggesting a molecular cooperation between the two drugs. Our results highlight that the combined inhibition of VEGF and TGF-β pathway may have a potential use in renal cell carcinoma therapy.

Endothelial cells present in tumors show different origin, phenotype, and genotype with respect to the normal counterpart. Various mechanisms of intra-tumor vasculogenesis sustain the complexity of tumor vasculature, which can be further modified by signals deriving from the tumor microenvironment. As a result, resistance to anti-VEGF therapy and activation of compensatory pathways remain a challenge in the treatment of cancer patients, revealing the need to explore alternative strategies to the classical anti-angiogenic drugs. In this review, we will describe some alternative strategies to inhibit tumor vascularization, including targeting of antigens and signaling pathways overexpressed by tumor endothelial cells, the development of endothelial vaccinations, and the use of extracellular vesicles. In addition, anti-angiogenic drugs with normalizing effects on tumor vessels will be discussed. Finally, we will present the concept of endothelial demesenchymalization as an alternative approach to restore normal endothelial cell phenotype.

No abstract