It is now well established that a genetic component may contribute to glaucoma, and several glaucoma-associated genes have been identified. The first identified and the most studied gene is MYOCILIN, which is heavily expressed in and secreted by the trabecular meshwork, one of the key components of the eye aqueous humor outflow system. The functions of the encoded protein, myocilin, are still not fully understood. To study mechanisms of human myocilin action, we produced stably transfected HEK293 cell lines expressing wild-type or mutant (Tyr437His and Ile477Asn) myocilins under an inducible promoter. Expression of the two mutant myocilins led to increased endoplasmic reticulum stress and induced apoptosis in cells after oxidative challenge. The Y437H mutant myocilin cell line showed the highest sensitivity to oxidative stress. Several antioxidant genes were down-regulated in the Y437H mutant myocilin cell line, but not in other cell lines. The Y437H mutant myocilin cell line also produced more reactive oxygen species than other cell lines examined. Consistent with the data obtained in cultured cells, the endoplasmic reticulum stress marker, 78 kDa glucose-regulated protein was up-regulated, while antioxidant proteins, paraoxonase 2 and glutathione peroxidase 3, were down-regulated in the eye angle tissue of transgenic mice expressing the Y437H myocilin mutant. Our results suggest that expression of mutated myocilins may have a sensitization effect which can lead to a severe phenotype in combination with oxidative stress. Mutant myocilins may confer different sensitivity to oxidative stress depending upon the mutation. The synergystic effect of mutant myocilin and oxidative stress may lead to the loss of cells in the trabecular meshwork which is regarded as one of potential mechanisms for glaucoma pathogenesis. Myocilin may induce formation of stress fibers in primary cultures of human trabecular meshwork or NIH3T3 cells. In continuation of this research, we demonstrated that myocilin and its proteolytic fragments are not only able to induce the formation of stress fibers but also stimulate cell migration. The stimulation of cell migration occurs through activation of the -catenin-focal adhesion kinase (FAK)-serine/treonine kinase (AKT) signaling pathway. Myocilin treatment also induced an elevation of the NFATc1 transcription factor level but its dephosphorylation was not required for myocilin-induced cell migration. The myocilin effects were demonstrated both in cell culture and in the eyes of transgenic mice expressing elevated levels of myocilin in the drainage structures. Besides studying the pathophysiology of glaucoma, we are also interested in potential treatments for the disease. Glaucoma is associated with impairment in retrograde transport of neurotrophic factors to retinal ganglion cell bodies. Mesenchymal stem cell (MSC) transplantation appears to be protective in a variety of neurodegenerative disorders of the brain and spinal cord, in part by neurotrophic factor secretion. Thus, we have been investigating a potential role of MSC transplantation as a therapy for glaucoma. We demonstrated in vitro that transplantation of MSCs is neuroprotective to retinal ganglion cells in a retinal explant model. This effect is observed in retinal explants derived from healthy tissue as well as explants derived from eyes in which the optic nerve had been crushed one week prior to isolation. In vivo, MSCs transplantation into the vitreous cavity of rats with laser-induced ocular hypertension slows retinal ganglion cell loss. However, systemic administration of MSCs was not beneficial in this model. Current research is exploring the mechanism of MSC-mediated neuroprotection, which might involve the production and secretion of neurotrophic factors.
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