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Maria Gomez-Caraballo, Gaurang Patel, Wen Fury, Hua Yang, Ming Yuan, Ying Hu, Carl Romano, Daniel Stamer; Immuno-labeling of human anterior segments localizes trabecular meshwork-specific proteins identified by scRNA-seq. Invest. Ophthalmol. Vis. Sci. 2020;61(7):4625.
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Dysfunction of the trabecular meshwork (TM) is responsible for ocular hypertension in primary open-angle glaucoma (POAG); however, TM-specific proteins are still unknown. We performed single-cell RNA sequencing (scRNA-seq) of TM and surrounding cells from human donor eyes. Candidate genes were mapped in human TM tissues via RNAscope®. The purpose of this study was validate RNA signatures and localize candidate gene products using immuno-labeling of iridocorneal angle sections.
Anterior segments from two human donor eyes were paraffin-embedded and sectioned for immuno-labeling of control proteins: CD31 (Schlemm’s canal, SC) and myocilin (TM), and candidate gene products: R-Spondin 2 (RSPO2), R-Spondin 4 (RSPO4), Chromogranin B (CHGB), Trefoil Factor 3, Transmembrane Protein 88, DNAX-Activation Protein 1, Proteolipid Protein 1, and Fms-Like Tyrosine Kinase 4. Sections underwent deparaffinization, antigen retrieval in citric acid buffer, and blocking in 5% normal serum at room temperature (RT) for 1 hour. Primary antibodies were incubated overnight at 4°C. Secondary antibodies were incubated with/without primary antibodies at RT for 1 hour. Experimental and control sections were imaged using a Nikon confocal microscope at identical settings. Z-stacks were merged and images were scored 0 (no labeling), 1+ (modest labeling) and 2+ (intense labeling).
After optimization of antibodies, we visualized positive labeling from RSPO2, RSPO4, and CHGB proteins. RSPO2 and CHGB scored 2+ in the uveal, corneoscleral, and juxtacanalicular (JCT) TM, and was not present in SC cells. This distribution was consistent with the localization of these genes by RNAscope®. RSPO4 expression scored 2+ in the corneoscleral and 1+ in the JCT and uveal TM. Interestingly, we observed 1+ labeling of R-spondin-4 at SC; similar, albeit weaker, to that of the CD31 (2+). Perinuclear Myocilin staining was detected in the uveal, corneoscleral, and JCT TM with 2+ intensity. Other candidate proteins were undetectable using commercial antibodies
We have confirmed TM localization of the gene product candidates RSPO2, RSPO4, and CHGB, which are all secreted proteins. Interestingly, R-spondin proteins enhance wnt signaling, a known regulatory pathway of outflow resistance and intraocular pressure. These genes represent potential TM biomarkers in the study of the TM’s role in POAG pathology.
This is a 2020 ARVO Annual Meeting abstract.
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