This is a competitive revision in response to notice NOT-OD-09-058 entitled """"""""NIH announces the availability of recovery act funds for competitive revision applications"""""""" The long-term objective of this grant is to identify cell-matrix signaling mechanisms that can be used as therapeutic targets to control steroid induced glaucoma. The glaucomas, which lead to irreversible loss of retinal ganglion cells, affect approximately 67 million people worldwide. They are commonly associated with elevated levels of intraocular pressure (IOP) due to a reduction in aqueous humor outflow from the trabecular meshwork (TM). Although a number of physiological factors are known to regulate outflow facility, one of the key factors that have emerged as an important regulatory mechanism for outflow facility is the contractile properties of the TM. At the present, it is unclear what molecular events regulate contractility in the TM. It is well established that the signaling properties of extracellular matrix (ECM) proteins and their receptors (integrins) play a critical role in regulating cell contractility and we proposed that activation of one or more of the components in these signaling pathways may be responsible for the formation of cross-linked actin networks (CLANs) observed in steroid induced glaucoma. In this competitive revision we plan to expand the scope of the original grant by utilizing new methodology to examine the relationship between integrin and G-protein signaling and steroid induced changes in the actin cytoskeleton of cultured TM cells. The prior aims focused on integrin and syndecan signaling pathways in order to determine which integrins and syndecans were involved in CLAN formation and demonstrating that activation of these pathways influence outflow facility in cultured anterior segments. These studies indicated that both 23 integrins and CD47, a member of both the IgG superfamily and the G-protein- coupled receptor family, may be part of the signaling mechanism that alters the organization of the actin network into cross- linked actin networks (CLANs) in steroid induced glaucoma. To aid in the identification of components in this 23 integrin/CD47 signaling pathway that may be upregulated by steroids we plan to use gene microarrays to identify components of the 23 integrin and CD47 signaling pathways that are unregulated by steroid treatment and activated by both 23 integrin and CD47 in TM cells in culture. Quantitative phosphoproteomics will be used to determine if changes in the phosphorylation state and hence activity of proteins in the 23 integrin/CD47 signaling pathway(s) are upregulated by steroid treatment and correlate with the activation of CLAN formation by 23 integrin/CD47 in TM cells in culture.
Glaucoma is the second most common cause of blindness in the U.S. and the most common cause of blindness among African-Americans. Attempts to understand the cause(s) of this disease have been hampered by the lack of well defined cell culture systems. The goal of this project is to identify the signaling pathway(s) activated during steroid induced glaucoma (SIG) so we identify potential therapeutic targets to reduce SIG.
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