Custom RT-qPCR-array for glaucoma filtering surgery prognosis

Abstract

Excessive subconjunctival scarring is the main reason of failure of glaucoma filtration surgery. We analyzed conjunctival and systemic gene expression patterns after non penetrating deep sclerectomy (NPDS). To find expression patterns related to surgical failure and their correlation with the clinical outcomes. This study consisted of two consecutive stages. The first was a prospective analysis of wound-healing gene expression profile of six patients after NPDS. Conjunctival samples and peripheral blood samples were collected before and 15, 90,180, and 360 days after surgery. In the second stage, we conducted a retrospective analysis correlating the late conjunctival gene expression and the outcome of the NPDS for 11 patients. We developed a RT-qPCR Array for 88 key genes associated to wound healing. RT-qPCR Array analysis of conjunctiva samples showed statistically significant differences in 29/88 genes in the early stages after surgery, 20/88 genes between 90 and 180 days after surgery, and only 2/88 genes one year after surgery. In the blood samples, the most important changes occurred in 12/88 genes in the first 15 days after surgery. Correspondence analyses (COA) revealed significant differences between the expression of 20/88 genes in patients with surgical success and failure one year after surgery. Different expression patterns of mediators of the bleb wound healing were identified. Examination of such patterns might be used in surgery prognosis. RT-qPCR Array provides a powerful tool for investigation of differential gene expression wound healing after glaucoma surgery.

Genetic and Protein Expression Profile of Conjunctival Bleb Hyperfibrosis in Early Failure of Glaucoma Surgery

“…IL-8 also increases proliferation and migration of endothelial cells as well as wound angiogenesis [33,34]. Our results, in the same line as those of others [6], show that overexpression of VEGF-A and IL-8 could be used as an indicator of conjunctival fibrosis following filtering surgery.…”

“…One year after surgery, however, only the expression of TGF-β1 and ITG-β3 was reduced. And, in peripheral blood, the most evident changes occurred also during the first 15 days after surgery, when 12 genes (IL-1β, CEBPD, HIF-1α, ITG-β2, TNFRSF1A and CASP1, among others) were induced [6]. These results suggested that conjunctival fibrosis developed after filtration surgery could have a significant genetic component driven by an altered expression of specific genes.…”

“…Likewise, a previous correspondence analysis revealed VEGF-A as the only tested gene differentially expressed in the patients with surgical failure versus patients with successful surgery [6]. VEGF-A is a glycoprotein highly expressed in acute wounds [23] that has been involved in many processes linked to wound healing repair.…”

“…Both genes, involved in the cell cycle control, were found significantly overexpressed. CDKN1A was previously reported to be upregulated in the conjunctiva during the postoperative period of filtering surgery, although the expression of C-MYC was reduced [6]. It is known that the C-MYC oncogene promotes cell growth by counteracting the action of several negative regulators of the cell cycle [35,36] such as CDKN1A in a TGF-β-dependent manner [37].…”

“…Therefore, apart from the acquired profibrotic factors, it is suspected that the individual susceptibility to early fibrosis of the filtration bleb could be driven by changes in the conjunctival gene expression patterns. A prospective study comparing conjunctival gene expression at different time points after surgery revealed that IL-8, VEGF-A, CDKN1A, CDKN2A, and TGF-α resulted upregulated while others such as MMP2, TGF-β2, C-MYC, CTGF, and IL-18 were downregulated [6]. Upregulation of the profibrotic VEGF-A and downregulation of antifibrotic genes, including IFN-b1, IL-6, F2, and COL3A1 have been found in glaucoma patients with surgical failure in comparison with those with surgical glaucoma surgery and patients with no previous surgery, respectively [7].…”