This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: To determine whether compounds that generate nitric oxide (NO) can relax carbachol contracted or resting ciliary muscle (CM) in vitro. This is an indicator of whether this class of compounds may be useful in enhancing uveoscleral outflow as an approach for lowering intraocular pressure (IOP) in glaucoma. To determine the effects of compounds on in vitro CM contraction/relaxation, and on IOP in vivo. To utilize the monkey anterior segment in organ culture to investigate the effects of gene therapy and other molecules on trabecular outflow which may also be important for glaucoma therapy. To determine the effects of in vivo gene therapy to the anterior segment. Overexpression of proteins that alter aqueous humor outflow may lead to intraocular pressure reduction that is important for glaucoma therapy. Alternatively, elevation of intraocular pressure may lead to a new glaucoma model. Progress: The CM relaxation response to nitric oxide donors was blocked by pretreatment with ODQ, an inhibitor of the guanylate cyclase pathway, prior to administering a nitric oxide donor. NO compounds have potential value in therapeutic areas where relaxation or contraction of the CM is desirable, such as in the treatment of glaucoma. The monkey organ-cultured anterior segment (MOCAS) system is being utilized to determine the effects of pharmacotherapy and gene therapy on trabecular outflow which may subsequently be utilized for glaucoma therapy to decrease intraocular pressure. Overexpression of the protein cochlin, which is elevated only in human glaucoma, increases intraocular pressure (IOP) and decreases trabecular outflow. The IOP elevation response in MOCAS following treatment with transforming growth beta-2 (TGF?2) is enhanced by including bovine serum albumin in the media. Studies to silence cochlin expression during TGF?2 treatment will determine if cochlin induction is necessary for the TGF?2-induced IOP elevation. Lentiviral vectors expressing genes that can potentially alter aqueous humor outflow have been injected into the anterior segment of living monkey eyes. Transduction of C3 transferase (capable of altering the actin cytoskeleton) in one eye did not alter the IOP. Ocular tissue is being examined to verify expression was present. Another gene, prostaglandin synthase (expected to enhance uveoscleral outflow), was delivered to the anterior segments of one eye of each of 5 monkeys. A transient reduction in IOP was detected in 3 of 5 eyes from 2 weeks to 5 months post-injection. Coexpression of green fluorescent protein was detectable in vivo for nearly 2 years. Tissues will be examined for PGFsynthase expression. Vitamin D applied topically to the eye lowers IOP. The mechanisms for this reduction are being investigated. This research used WNPRC Research Services. PUBLICATION: Lee E, Gabelt BT, Faralli JA, Peters DM, Brandt CR, Kaufman PL, Bhattacharya SK: COCH transgene expression by adenovirus in cultured human trabecular meshwork cells and its effect on outflow facility in monkey organ-cultured anterior segments. Invest Ophthalmol Vis Sci, Epub ahead of print, Nov 20, 2009.
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