2018 Shaffer Research Grants

Glaucoma is the leading cause of irreversible blindness in the world, which is projected to affect about 6.3 million Americans by 2050, and intraocular pressure (IOP) is a leading contributor to glaucoma. Our project addresses the major limitations of current glaucoma therapy by advancing a sustained-release IOP-lowering formulation that coordinates multiple targets to normalize IOP. Minimum performance metrics for the novel formulation will position us to move toward clinical trials and eventually to commercialization.

A healthy normally functioning eye is dependent upon a certain number of cells for good vision. The primary risk factor for glaucoma is elevated pressure inside the eye (intraocular pressure). The trabecular meshwork or TM is responsible for controlling this pressure, but with age and glaucoma the TM loses cells and sometimes can no longer perform its regulatory function. Glaucoma can be treated for some limited amount of time with a laser, which will burn holes in the TM, and restore the pressure to normal. Previously, we found that when human cadaver eyes are treated with a laser, there is increased cell division and migration to the areas burned. These laser-treated eyes were put into organ culture with growth medium, and when this medium was later collected, it now had molecules from the cells of the laser-treated eyes and is called “conditioned media”. When this conditioned medium was placed on non-laser treated human cadaver eyes, cell division and migration also increased in these untreated eyes, suggesting that the growth medium contained secreted substances from the laser treated TM cells. Laser-treated cells of the first eye produced a “laser factor” into the medium which increased TM cell division and migration. This project involves the isolation and identification of this factor. If we can identify what this substance is, it can be chemically synthesized and added to eye drops for the patient. Treatment with such eye drops could stimulate the stem cells of the TM in glaucomatous eyes to increase cell division and migrate to the areas needed to restore function to the eye. Our results indicate that a growth factor, PDGFbb, is a molecule important in cell division, and caused the largest amount of increase in cells. However, TNF-alpha and IL-1 seem promising as well, and there may be other factors that are important to this process. Further studies are ongoing on cell migration and on affirming our initial results with cell division.

Given that pressure-lowering eye drops are by far the most common treatment for glaucoma it is astounding that we know so little about how the effects of pressure are sensed and transduced in the eye. The project supported by GRF identified and studied new classes of ion channels that are specialized for pressure transduction. Our studies showed why and how pressure is such a potent regulator of ocular physiology and also identified pressure-sensing ion channels as targets for a novel generation of anti-glaucoma drugs. The take-home message is that trabecular meshwork cells, which play the central role in intraocular pressure regulation, exist in the state of “tensile balance” that is maintained by dynamic activation of at least three different pressure sensitive channels. Our results suggest that the loss of pressure regulation in glaucoma may occur in part due to the disturbed balance between these pressure sensors.

Glaucoma is an irreversible blinding disease in which the cells that comprise the optic nerve, the retinal ganglion cells (RGCs), are damaged and die. A major gap in taking care of glaucoma patients is that we do not have an objective test that measures how well the RGCs are functioning. There are actually over 30 types of RGCs, and our laboratory has recently identified specific types of RGCs that are more vulnerable in glaucoma. Taking advantage of this knowledge, we are developing novel methods to assess the function or health of RGCs that are more vulnerable versus more resistant to damage. A more sensitive and objective test of RGC function and health will greatly improve our ability to take care of glaucoma patients and their vision.

Critical barriers in the development of efficient IOP lowering therapies could be overcome if there were a better mechanistic understanding governing ECM homeostasis, TM stiffness and the pathobiological basis of altered ECM deposition in the aqueous humor outflow pathway leading to increased stiffness and outflow resistance. Our research shows that targeting CCN1 can be beneficial in increasing aqueous humor outflow. The data we find indicates that increasing CCN1 expression is able to attenuate the TGFβ2-mediated increase in TM stiffness by lowering pro-fibrogenic activity in the HTM cells caused by TGFβ2. We believe that since the CCN1 lowers the tissue stiffness and attenuates TGFβ2-mediated effects on HTM cells, CCN1 is a therapeutic target to lower IOP.

The mechanism by which elevated IOP levels induce degradation of the optical nerve head (ONH), the initial site of injury in glaucoma, is not yet understood but it is widely suspected that a main culprit is the lack of mechanical balance between IOP-induced strains and the stiffness of ONH and surrounding sclera. However, we do not have viable technology to test the stiffness of the back of the eye. We have developed a potential solution to this need, an all-optical approach to mechanical measurements using Brillouin light scattering. Our technology is already in human clinical trials for transparent ocular tissues (cornea, lens). In this grant we extended the reach of Brillouin microscopy to the back of the eye by using Adaptive Optics (AO). We have demonstrated that our new AO-Brillouin microscope enables imaging opaque tissues such as ONH and sclera, we have validated the instrument to show we can measure the expected mechanical differences at the back of the eye. Thus, this pilot grant provided proof-of-principle for the use of this new technology in glaucoma animal models to measure novel functional/mechanical parameters of the sclera and ONH.

Glaucoma is a group of optic neuropathies characterized by slow, progressive loss of retinal ganglion cells (RGCs), degeneration of the optic nerve and, consequently, loss of vision. Although the main risk factors associated with the development of the disease are elevated intraocular pressure and aging, genetic studies have described a number of loci in the genome that further increase the risk of glaucoma. Our previous work has indicated that upon intraocular pressure elevation RGCs become senescent and affect surrounding cells. In our project, we proposed to remove early senescent RGCs in the glaucomatous eye as a way to protect neighboring RGCs from death. Using mouse model of hypertension in the eye we were able to show that early removal of affected-senescent cells is beneficial for the eye in two ways: i) Fewer ganglion cells are lost in the retina; ii) Remaining cells in the retina are functional and overall visual functions are preserved. Therefore, our project provided a solid foundation for future studies on potential applications of senolytic drugs in glaucoma patients as a way to prevent the glaucoma progression.

Dr. Dorota Skowronska-Krawczyk was awarded the 2020 Shaffer Prize for Innovative Glaucoma Research. The Shaffer Prize, presented annually by Glaucoma Research Foundation, recognizes a researcher whose project best exemplifies the pursuit of innovative ideas in the quest to better understand glaucoma.

In this study, we have developed a new tool for understanding how the efforts of retinal neurons to repair themselves in glaucoma can ultimately contribute to their demise. Previous research had revealed that one potential means by which vision is lost is through a series of events that results in neuronal suicide. Retinal neurons essential for vision are challenged by disease-related changes, and their efforts to adapt to these challenges include cellular signaling that promotes repair but can also lead to cell death. We have established a tool that allows us to stimulate this signaling at various intensities and patterns to probe how and when it goes from beneficial to detrimental. Our findings so far suggest that modest stimulation may, when combined with other interventions, improve neuronal repair and survival.