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Xinbo Li, Ted S Acott, Mary J Kelley; Gap and tight intercellular junctions in trabecular meshwork and Schlemm’s canal cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4678. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Primary open-angle glaucoma (POAG) is one of the leading causes of blindness worldwide. Trabecular meshwork (TM) and Schlemm’s canal (SC) are important components of the aqueous humor drainage pathways and relate intraocular pressure (IOP). Electron microscopy previously showed gap and tight junctions in this region. Specific junctional protein involvement was not determined. Here, we investigated gap junction protein Cx43, tight junction protein claudin-1, PDZ domain containing protein ZO-1, AF-6 and MUPP1, and pore forming protein plasmalemma vesicle associated protein (PLVAP) expression and their relationships in TM and SC cells or tissues.
Porcine SC cells were isolated utilizing anti-CD31 antibody and magnetic beads; human TM and SC cells were separated using laser capture microdissection (LCM) from 8 µm frozen sections perfusion-labeled with fluorescent microspheres. Real time PCR (RT-PCR), Western blot, and single or double immunofluorescence (IF) labeling were used for assessing mRNA and protein expression. Co-immunoprecipitation (IP) and GST pull-down assay were used to evaluate protein-protein interactions.
Claudin-1 mRNA is highly expressed by isolated porcine SC cells and LCM-separated SC cells, while Cx43, ChI3L1, MGP, AQP-1 are found in both TM and SC cells. Double IF showed partial co-localization of claudin-1 with PLVAP and ZO-1, as well as ZO-1 with PLVAP, and AF-6 and MUPP1 with claudin-1 in SC cells. ZO-1 partially co-localized with Cx43 in both TM and SC cells. Co-IP showed Cx43 and ZO-1 both immuno-precipitated with claudin-1 using lysates from SC cells. GST pull down showed Cx43 binds the PDZ2 domain of ZO-1, while claudin-1 binds the PDZ1 domain of ZO-1. Carbenoxolone (CBX, 10µM), a gap junction blocker, significantly increases outflow facility (n=6), as determined by one-way ANOVA, with Dunnett’s Multiple Comparison Test.
Claudin-1 and PLVAP are highly expressed in cultured SC cells and tissues, while Cx43 and ZO-1 are present in both TM and SC cells and tissues. Gap junction and tight junction intercellular communication may be involved in outflow facility regulation. Claudin-1, Cx43 and ZO-1 may associate with pore forming protein PLVAP in human SC, potentially affecting IOP homeostasis.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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