Genotype-correlated expression of lysyl oxidase-like 1 in ocular tissues of patients with pseudoexfoliation syndrome/glaucoma and normal patients

Abstract

Pseudoexfoliation (PEX) syndrome is a generalized disease of the extracellular matrix and the most common identifiable cause of open-angle glaucoma. Two single nucleotide polymorphisms in the lysyl oxidase-like 1 (LOXL1) gene (rs1048661 and rs3825942) have been recently identified as strong genetic risk factors for both PEX syndrome and PEX glaucoma. Here we investigated the expression and localization of LOXL1, LOXL2, and lysyl oxidase (LOX) in tissues of PEX syndrome/glaucoma patients and controls in correlation with their individual single nucleotide polymorphism genotypes and stages of disease. LOXL1 ocular expression was reduced by approximately 20% per risk allele of rs1048661, whereas risk alleles of rs3825942, which were highly overrepresented in PEX cases, did not affect LOXL1 expression levels. Irrespective of the individual genotype, LOXL1 expression was significantly increased in early PEX stages but was decreased in advanced stages both with and without glaucoma compared with controls, whereas LOX and LOXL2 showed no differences between groups. LOXL1 was also found to be a major component of fibrillar PEX aggregates in both intra- and extraocular locations and to co-localize with various elastic fiber components. These findings provide evidence for LOXL1 involvement in the initial stages of abnormal fibrogenesis in PEX tissues. Alterations of LOXL1 activation, processing, and/or substrate specificity may contribute to the abnormal aggregation of elastic fiber components into characteristic PEX fibrils.

Figure 1

Quantitative determination of mRNA levels of LOX, LOXL1, and LOXL2 in ocular tissues of normal human donor eyes using real-time PCR technology. Data were normalized to GAPDH and are expressed as copies of gene of interest per copies of GAPDH. Values represent mean ± SD of three separate experiments.

Figure 2

Immunofluorescence labeling of LOXL1 in ocular tissues of normal human donor eyes. LOXL1 immunopositivity (green fluorescence, arrows) is observed in the corneal epithelium (A); intrascleral veins and fibrocytes (B); trabecular meshwork and Schlemm canal endothelium (C); vessels, dilator muscle, and stromal cells in the iris (D); ciliary epithelium and stromal vessels (E); and retinal ganglion cells (F). The inset in E shows weak LOXL1 immunopositivity at the tips of elastic fibers in the ciliary stroma. BV, blood vessel; CE, ciliary epithelium; DM, dilator muscle; EP, corneal epithelium; GCL, retinal ganglion cell layer; INL, inner nuclear layer; IPE, iris pigment epithelium; ONL, outer nuclear layer; PRL, photoreceptor layer; SC, Schlemm canal; SCL, sclera; ST, stroma; TM, trabecular meshwork. Original magnifications: ×100; ×250 (inset).

Figure 3

Association of LOXL1 genotypes and haplotypes with PEX syndrome/glaucoma. Percentage of SNP rs1048661 and rs3825942 genotypes and SNP haplotypes in PEX (n = 25) and control (n = 25) cases.

Figure 4

Genotype-correlated expression of LOXL1. A: LOXL1 mRNA expression in ciliary body tissue of both PEX (n = 25) and control (n = 25) eyes is significantly reduced per risk G allele of SNP rs1048661. B: LOXL1 protein expression (arrows) in the ciliary epithelium of control eyes with different genotypes is decreased per risk G allele of SNP rs1048661. CE, ciliary epithelium; ST, ciliary stroma. Original magnifications, ×100.

Figure 5

LOXL1 expression in ocular tissues of eyes with PEX syndrome/glaucoma at different stages of disease compared with control eyes. A: Quantitative determination of LOXL1 and LOXL2 mRNA expression in ciliary processes of control and PEX eyes without glaucoma at various stages of disease (PEX early, PEX late) and with glaucoma (PEXG) using real-time PCR technology. Data were normalized to GAPDH and are expressed as copies of gene of interest per copies of GAPDH. B: LOXL1 protein expression in ciliary processes of PEX eyes at early and late stages as compared with control eyes (rs1048661 genotype GG in all cases). Whereas early PEX stages disclose only minor focal PEX material deposits (arrows), advanced stages show prominent LOXL1-positive PEX material deposits on the surface of the ciliary epithelium (arrows). Cellular labeling for LOXL1 is increased in early stages of PEX (asterisk) and decreased in late stages as compared to controls. CE, ciliary epithelium; ST, ciliary stroma. Original magnifications, ×100.

Figure 6

Immunolocalization of LOXL1 in PEX material deposits in various intra- and extraocular tissues of PEX eyes. Immunopositive PEX material deposits (arrows) are present in the iris stroma, periphery of iris vessels, and on the surface of the iridal pigment epithelium (A); on the surface of the ciliary epithelium (B); in the periphery of episcleral veins (C); in myocardial tissue (D); in lung tissue, particularly in the periphery of pulmonary blood vessels (E); and in the periphery of hair follicles in the skin (F). Corresponding control tissues show weak staining for LOXL1 in vessel walls of myocardial (G) and lung tissue (H) as well as in epidermal cells and root sheaths of hair follicles in normal skin (I). J and K: Pre-adsorption of LOXL1 antibodies with the corresponding peptide completely abolished immunopositivity of PEX deposits (arrows) on the surface of the ciliary epithelium. L: PEX material deposits (arrows) in the iris were negative for LOX. BV, blood vessel; CE, ciliary epithelium; DM, dilator muscle; EP, epidermis; HF, hair follicle; IPE, iris pigment epithelium; ST, stroma. Original magnifications, ×100.

Figure 7

Double-labeling immunofluorescence of elastic fiber components (green fluorescence) and LOXL1 (red fluorescence) in ocular PEX material deposits; nuclear counterstaining with 4′,6′-diamino-2-phenylindole (blue fluorescence). A: Co-localization of tropoelastin and LOXL1 (arrows) in PEX material in the anterior border layer and periphery of vessels in the iris. B: Co-localization of fibrillin-1 and LOXL1 (arrows) in PEX material on the surface of the pigment epithelium and vessel walls of the iris. C: Lacking co-localization of fibulin-5 and LOXL1 in PEX aggregates in the wall of iridal vessels and on the surface of the iridal pigment epithelium. BV, blood vessel; IPE, iris pigment epithelium; ST, stroma. Original magnifications, ×100.

Figure 8

Immunogold localization of LOXL1 on ocular PEX material. A: Immunopositive PEX fibers emerging from the pre-equatorial lens epithelium (LE) into the lens capsule (LC). B: Gold marker for LOXL1 is evident within cytoplasmic vesicles (arrows) of the lens epithelium (LE) and along PEX fibers on its surface. C: Co-localization of LOXL1 (10-nm gold particles) and fibrillin-1 (18-nm gold particles) along developing PEX fibers in close association to the surface of the lens epithelium (LE). D: Co-localization of LOXL1 (10-nm gold particles) and fibrillin-1 (18-nm gold particles) along mature PEX fibers on the surface of the lens capsule. Scale bars = 0.5 μm.