Thursday, May 2, 2024
5/2/2024
Project Summary The proposed research is relevant to public health because corneal blindness is the 4th leading cause of blindness globally and is often preventable with proper healthcare. Blindness arises due to damaged integrity of the corneal epithelium as the result of physical, chemical, or thermal trauma and/or as a consequence of diseases, surgery, or anti-cancer drugs. Regardless of the cause, disruption of the epithelial layer is extremely painful due to the dense sensory nerve innervation, makes the eye vulnerable to infection, and can result in blindness. Despite the widespread incidence and the array of medical problems associated with epithelial damage, there are no FDA-approved drugs that promote corneal wound healing or epithelial homeostasis. It is well-established that proteins of the receptor tyrosine kinase (RTK) family can induce cell proliferation, migration, survival, and differentiation of epithelial cells. We are investigating the signaling axis of one of these proteins, c- Met, and its cognate ligand hepatocyte growth factor (HGF). c-Met activity can not only promote the wound healing/re-epithelialization phenotypes stated previously but can also prevent transformation of keratocytes to fibroblasts and promote neuronal growth. However, c-Met is limited for therapeutic reasons in that RTKs have transient activation and are desensitized following ligand stimulation. We hypothesize that extending c-Met receptor activity by disrupting its downregulation will help restore corneal homeostasis following wounding. This hypothesis will be tested through the following aims: Aim 1: Determine if ubiquitylation of the c- Met receptor limits its signaling in corneal epithelial cells in vitro. We hypothesize that inhibiting negative regulation of c-Met signaling will prolong receptor activity and accelerate healing. We will test this by blocking c- Met ubiquitylation in vitro by genetic knockout of CBL genes from immortalized human corneal epithelial cells and measuring receptor phosphorylation, trafficking, in vitro wound healing, and cell migration and proliferation. Aim 2: Determine effect of CBL knockout and c-Met activation in corneal wound healing murine models in vivo. We hypothesize that HGF-induced c-Met signaling will not only aid in re-epithelialization, but also will decrease fibrotic markers in the stromal layer and promote axonal outgrowth of corneal nerves. We will test this by using corneal epithelial-specific, inducible CBL knockout murine models. We will wound the corneas and monitor gross corneal morphology and homeostasis, re-epithelialization, fibrosis and inflammatory markers, and nerve restoration. These studies are innovative because we will manipulate c-Met signaling regulation to extend signaling and promote healing of all layers of the cornea. The proposed research is relevant to the National Eye Institute’s mission of eliminating vision loss and improving quality of life through research.
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