Fig 1.
Schematic of the workflow for the study.
(A) Schematic of organ culture system. Fluorescent beads (red dots) are perfused through a human anterior segment in organ culture. After 1 hour the anterior segment is divided into low, medium and high outflow regions based on the intensity of the fluorescent beads (red regions). (B) Images show steps in the DSP process. Tissue sections on a slide were labeled with DSP probes and ROIs were selected based on tissue morphology and fibronectin immunostaining. Barcodes in ROIs were cleaved using UV light and collected into a 96-well plate for sequencing. (C) Actual scan of fibronectin labeled tissue from a high outflow region with the ROI drawn. (D-E) Images of fibronectin (red) labeled tissue sections. Dashed line in panel E shows a typical size and location of the ROI used for the study. Nuclei (blue) were labeled with Hoechst 33342.
Fig 2.
Volcano plot of differentially expressed genes.
Plot shows differential expression of genes in high and low outflow areas. Each dot represents a specific gene. The solid horizontal red line shows the cutoff for genes with an adjusted p value <0.05. Only statistically significant genes are labeled.
Fig 3.
Biological categories of genes exhibiting differential expression in high outflow and low outflow regions of the TM.
Biological processes were identified through gene ontology enrichment analysis using the following databases: DAVID Bioinformatics Resources, Gene Cards and UniProt.
Table 1.
ECM/cell adhesion genes differentially expressed compared to the high outflow regions.
Unless otherwise noted with a citation, the function of genes was determined using GeneCards®, the human gene database or UniProtKB/Swiss site.
Fig 4.
Box plots showing distribution of ADAM15, LDB3, CRKL and BGN gene expression in high and low outflow regions.
Each dot represents the log2FC of the gene in one high or low outflow region. The horizontal bar represents the median of the log2FC for that gene within the interquartile range (IQR). The whiskers show the minimum and maximum log2FC change of that gene and its variability in comparison to the IQR as well as outliers. Points seen outside the box, but within the whiskers, represent data points that fall within the min (Q1-1.5* IQR) and max (Q3 + 1.5* IQR). Only data points outside the whiskers are considered outliers.
Fig 5.
Localization of ADAM15 in high and low outflow segments of TM.
(A) ADAM15 labeling (red) in high outflow regions and low outflow regions. AC, anterior chamber, SC, Schlemm’s canal, Bar = 50μm. Arrowheads show ADAM15 in the beams and along Schlemm’s canal. Sections were labeled with a rabbit monoclonal anti-ADAM15 antibody which was detected using Alexa 546-conjugated goat anti-rabbit IgG. Nuclei were labeled with Hoechst 33342 (blue). (B) Quantification of labeling along beams versus JCT/IW, OW and beams/JCT/IW in low and high outflow regions. * p<0.05 C) Box plots showing distribution of ADAM15 labeling in high and low outflow regions. Each dot represents the integrated density in one high or low outflow region. The horizontal bar represents the median of the integrated density. (D) Schematic of the trabecular meshwork/Schlemm’s canal outflow pathway. Aqueous humor (green arrows) flows through the trabecular beams and the JCT. It then crosses the IW of Schlemm’s Canal to exit either paracellularly or transcellularly into the lumen of Schlemm’s Canal and into the OW of Schlemm’s Canal. The beams consist of a collagenous/elastic matrix surrounded by endothelial-like cells. The beams are connected to each other by cytoplasmic extensions between the cells surrounding the beams. IW, inner wall; OW outer wall, JCT, Juxtacanalicular region.
Table 2.
Genes involved in signal transduction events.
Unless otherwise noted with a citation, the function of genes was determined using GeneCards®, the human gene database or UniProtKB/Swiss site.
Table 3.
Genes involved in transcription.
Unless otherwise noted with a citation, the function of genes was determined using GeneCards®, the human gene database or UniProtKB/Swiss site.
Fig 6.
Box plots showing distribution of various ECM genes and TGFβ2 in high and low outflow regions.
Each dot represents the log2FC of the gene in one high or low outflow region. DCN was the only gene which showed a preferential distribution to the high outflow region of the TM. The horizontal bar represents the median of the log2FC for that gene within the IQR. The whiskers show the minimum and maximum log2FC change of that gene and its variability in comparison to the IQR as well as outliers. Points seen outside the box but within the whiskers represent data points that fall within the min (Q1-1.5* IQR) and max (Q3 + 1.5* IQR). Only data points outside the whiskers are considered outliers. FN1, fibronectin; VCAN, versican, DCN, decorin; THBS2, thrombospondin 2; MMP2, matrix metalloproteinase 2; TGFB2, transforming growth factor 2; TIMP1, TIMP metallopeptidase inhibitor 1.
Fig 7.
Box plots showing distribution of various integrin genes in high and low outflow regions.
Each dot represents the log2FC of the gene in one high or low outflow region. Of all these integrins, only the ITGA8 showed enrichment in the high outflow regions of the TM. The horizontal bar represents the median of the log2FC for that gene within the IQR. The whiskers show the minimum and maximum log2 FC change of that gene and its variability in comparison to the IQR as well as outliers. Points seen outside the box but within the whiskers represent data points that fall within the min (Q1-1.5* IQR) and max (Q3 + 1.5* IQR). Only data points outside the whiskers are considered outliers. ITGA2, α2 integrin; ITGA4, α4 integrin; ITGA5, α5 integrin; ITGA7, α7 integrin; ITGA8, α8 integrin; ITGA9, α9 integrin; ITGB1, β1 integrin; ITGB3, β3 integrin.