Molecular Mechanisms Of Glaucoma

National Eye Institute

Investigators

Linked publications, trials & patents

Abstract

Several approaches for RGC neuroprotection have been suggested and tested in animal models. We previously demonstrated that intravitreal injection of human bone marrow-derived mesenchymal stem cells (BMSCs), or small extracellular vesicles secreted by BMSCs, provided statistically significant RGC neuroprotection when compared to control samples. Our data also revealed that miRNAs play an important role in the observed RGC neuroprotection. In our previous experiments, we have used relatively crude preparation of small extracellular vesicles isolated via differential centrifugation. This purification methodology has been used in multiple publications which utilize small extracellular vesicles to treat of pathological conditions in both cellular and animal experimental models. It is known that a heterogeneous population of small extracellular vesicles can be separated into individual fractions with distinct composition of proteins, mRNAs, and miRNAs. In our pilot experiments, we began fractionation of small extracellular vesicles by density using OptiPrep gradient. Our goal is to isolate fraction(s) of small extracellular vesicles (exosomes) possessing elevated neuroprotective properties compared with unfractionated vesicles to identify new miRNAs and proteins involved in observed neuroprotection. Using a described fractionation procedure, we have been able to isolate several fractions of exosomes having different density, which we subsequently characterized by the presence of different protein markers and by size distribution of small extracellular vesicles. These isolated fractions are currently being tested for their neuroprotective properties using in vitro retinal cultures and in vivo mouse models of optic neuropathies, specifically glaucoma. We will study the neuroprotective role of exosome fractions using existing mouse models of glaucoma as well as explore a novel mouse model of glaucoma in which elevation of intraocular pressure is induced by injection of silicon oil into the eye anterior chamber. We continue testing and confirming the neuroprotective properties of previously identified candidate miRNAs using in vitro and in vivo models. We also expanded a repertoire of cells that we use to isolate small extracellular vesicles for subsequent neuroprotection experiments. We began to test human induced pluripotent cells and human neuronal precursor cells. We adapted a protocol for exosome isolation from such cells. Exosomes from stem cells and neuronal precursor cells are currently being tested for their neuroprotective properties using in vivo and in vitro model systems. The composition of exosome possessing the highest neuroprotective activities will be further characterized for the identification of new, and confirmation of existing, candidate miRNAs, proteins, and other types of RNA. Subsequent testing of selected miRNAs in animal models and characterization of their targets in the retina are the next important steps in the search for new neuroprotective strategies.