Investigating the role of Plasminogen Activator Inhibitor-1 (PAI-1) in lamina cribrosa fibrosis using single cell RNA sequencing.

Abstract

Purpose : PAI-1, through its inhibition of urokinase-type and tissue-type plasminogen activators (uPA, tPA), plays a key role in fibrosis, inhibiting cellular degradation of extracellular matrix proteins. Elevated levels of PAI-1 have previously been found in studies of liver, kidney, heart and lung fibrosis. The aim of this study was to analyse the difference in gene expression between normal and glaucoma lamina cribrosa (LC) cells, in particular focusing on genes involved in fibrosis.

Methods : We obtained LC cells from 2 normal and 2 confirmed glaucoma donors. Cells were cultured and dissociated using papain dissociation method. The suspension viability was >90%. Cells from all 4 individuals were pooled and the suspension was processed on the 10x Genomics platform at Queens University Belfast (QUB) Genomics Core Technology Unit to capture a total of ~5000 cells. Sequencing was performed on an Illumina Novaseq which generated ~800 million reads. Bioinformatic analysis was performed in QUB, with SNP data used to demultiplex the individuals using the Demuxlet package.

Results : We found that the expression levels of PAI-1 and related proteins such as Vitronectin (VTN), Thrombospondin 1 (THBS1) and Somatomedin B and Thrombospondin Type 1 Domain Containing (SBSPON) are significantly enhanced in glaucomatous LC cells. tPA was overexpressed in normal LC cells compared to glaucoma LC cells.

Conclusions : Single cell RNA sequencing has revealed glaucomatous LC cells show a differential expression of pro-fibrotic genes. Increased PAI-1 leads to excess collagen accumulation and reduced extracellular matrix proteolysis, thus contributing to LC fibrosis and optic disc cupping. The overexpression of tPA in normal LC cells and its relevance to the fibrinolysis pathway may represent a novel therapeutic path.

This is a 2021 ARVO Annual Meeting abstract.