Differential Gene and Protein Expression of Conjunctival Bleb Hyperfibrosis in Early Failure of Glaucoma Surgery

Abstract

The early failure of glaucoma surgery is mainly caused by over-fibrosis at the subconjunctival space, causing obliteration of the filtration bleb. Because fibrosis has a suspected basis of genetic predisposition, we have undertaken a prospective study to identify upregulated profibrotic genes in a population of glaucoma patients with signs of conjunctival fibrosis and early postoperative surgical failure. Clinical data of re-operated fibrosis patients, hyperfibrosis patients who re-operated more than once in a short time, and control patients with no fibrosis were recorded and analyzed at each follow-up visit. Conjunctival-Tenon surgical specimens were obtained intraoperatively to evaluate the local expression of a panel of genes potentially associated with fibrosis. In order to correlate gene expression signatures with protein levels, we quantified secreted proteins in primary cultures of fibroblasts from patients. Expression of VEGFA, CXCL8, MYC, and CDKN1A was induced in the conjunctiva of hyperfibrosis patients. VEGFA and IL8 protein levels were also increased in fibroblast supernatants. We propose that an increase in these proteins could be useful in detecting conjunctival fibrosis in glaucoma patients undergoing filtering surgery. Molecular markers could be crucial for early detection of patients at high risk of failure of filtration surgery, leading to more optimal and personalized treatments.

Keywords: conjunctival fibrosis; conjunctival filtration bleb; gene expression; glaucoma surgery failure; profibrotic genes.

Figure 1

Expression changes of conjunctival genes in fibrosis patients in comparison with non-fibrosis patients. Expression levels of cDNA were measured by relative-quantitative real-time PCR in the conjunctiva of glaucoma patients with signs of fibrosis (fibrosis, hyperfibrosis) and with no fibrosis (control). The expression of all the genes of interest (VEGFA, CXCL8, IL18, TFGA, TFGB1, MYC, THBS1, CDKN1A, CDKN2A) were normalized to 18S levels to determine changes between conjunctival tissue from (a) fibrosis patients that had to be re-operated once (n = 41) and (b) hyperfibrosis patients re-operated more than once in a short time (n = 6) versus control patients (n = 32). Data are presented as mean ± SEM of ΔCts of all experiments performed by triplicate (* p < 0.05, ** p < 0.01, and *** p < 0.001 as indicated, Student’s test). Note that the decrease in ΔCt values represents an increase in gene expression.

Figure 2

Levels of protein secreted by cultured fibroblasts from fibrosis patients compared with those from non-fibrosis patients. Protein expression levels were quantified in supernatants of primary cultures of conjunctival fibroblasts from patients with no fibrosis (control) and patients with early signs of fibrosis (hyperfibrosis). ELISA was used to measure the levels of VEGFA, IL8, TGFB1, and TSP1 in cell supernatants. (a) Protein levels are plotted by a vertical bar for each patient analyzed to show individual differences (1–5 control, 6–11 hyperfibrosis). (b) Results are presented as the mean ± SEM in hyperfibrosis patients and non-fibrosis patients of two independent experiments performed by duplicate (ns: non-significant, *** p < 0.001, **** p < 0.0001 as indicated, Student’s test).