Abstract
Purpose :
Glaucoma trabecular meshwork (GTM) tissue is characterized by excess, disorganized extracellular matrix (ECM). Recently, we recently identified a single gene variant, rs2228262 N700S, in thrombospondin-1 (THBS1; TSP-1) to be significantly associated with glaucoma. Extracellularly, TSP-1 binds and organizes many ECM molecules and cell surface receptors. In this study, we investigated which genes and proteins were altered in glaucoma TM cells with and without the TSP-1 variant.
Methods :
Primary GTM cells, derived from age-matched human cadaver eyes, were cultured at identical passage number in the presence of 0.1 mM ascorbate. DNA genotyping of rs2228262 was performed. RNA was isolated, hybridized with unique fluorescent barcodes, and immobilized on human fibrosis v2 cartridges (Nanostring) containing 770 gene targets. The cartridges were scanned and fluorescently-labeled barcodes were digitally counted. Rosalind® software identified differentially expressed genes in wild-type and heterozygous THBS1 cells. Taqman qPCR was used to validate results. TM cellular proteins were homogenized by bead beating, labeled with tandem mass tags (TMT), and loaded onto an Orbitrap Fusion mass spectrometer.
Results :
GTM wild-type (n=4) versus GTM heterozygous (n=4) cells were analyzed. Twenty-seven genes were up-regulated in heterozygous THBS1 glaucoma cells, while 4 were down-regulated. Nine genes were related to ECM organization (COL6A3, COL14A1, CSTL, ELN, MMP2, MMP16, PRKCA, TIMP1, VCAM1). Gene Ontology analyses identified extracellular structure and organization being significantly affected in glaucoma TM cells with the THBS1 SNP. Taqman analysis validated gene changes in additional GTM cells with and without the gene variant. Proteomics analysis showed significantly increased protein levels of CSTL and VCAM1 in THBS1 heterozygous glaucoma cells, and while COL4A1 levels were reduced.
Conclusions :
Nanostring, qPCR, and proteomic analyses identified CTSL and VCAM1 to be significantly increased in THBS1 heterozygous GTM cells. CTSL is a lysosomal cysteine proteinase important for protein degradation, while VCAM1 mediates cell adhesion. These results suggest that a single amino acid change in TSP-1 may alter the structure and organization of ECM, changing cell surface receptors, affecting GTM cellular functions.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.