The hallmark of exfoliation syndrome is the pathologic production and stable accumulation of an abnormal, fibrillar, extracellular material (XFM). Although the exact biochemical composition of the pathologic matrix product is still not known, immunohistochemical and biochemical approaches have shown XFM to represent a highly glycosylated, cross-linked, and enzymatically resistant glycoprotein/proteoglycan complex, composed of a protein core surrounded by glycoconjugates. The protein core includes basement membrane components, such as laminin, nidogen, and fibronectin, and components of the elastic fiber system, such as fibrillin-1, elastin, and latent transforming growth factor binding proteins, as well as enzymatically active components, such as metalloproteinases, the extracellular chaperone clusterin, and the cross-linking enzyme lysyl oxidase-like 1 (LOXL1). Ultrastructural evidence suggests that XFM is multifocally produced by intraocular cells, such as ciliary epithelial cells, preequatorial epithelial cells of the lens, trabecular and corneal endothelial cells, all cell types of the iris, as well as by extraocular cells, such as fibrocytes, vascular cells, and muscle cells. All cells involved in the exfoliation syndrome process disclosed common ultrastructural signs of active fibrillogenesis and metabolic activation, such as increased vesicular transport to the cell surface, XFM formation within infoldings of cellular surfaces, and a prominent rough endoplasmic reticulum. Finally, cells involved in the production of XFM displayed a gene expression pattern characterized by the upregulation of elastic components, the transient upregulation of LOXL1, and the dysregulated expression of cytoprotective gene products, matrix metalloproteinases, and their inhibitors, possibly leading to the accumulation and stable deposition of XFM.
Purpose The cross‐linking enzyme lysyl oxidase‐like 1 ( LOXL 1) and profibrotic transforming growth factor ( TGF )‐ß1 play key roles in pathophysiology of pseudoexfoliation ( PEX ) syndrome/glaucoma. The purpose of this study was to investigate the interaction between LOXL 1 and TGF ‐ß1 with respect to the PEX ‐specific disordered matrix metabolism. Methods Primary human Tenon's capsule fibroblasts ( hTCF ) obtained from patients were treated with TGF ‐ß1 (0‐10 ng/ml) for 12‐72 hours without or with preincubation with inhibitors of TGF ‐ β signalling pathways. Expression of LOXL 1 and PEX ‐specific extracellular matrix components was examined by using quantitative RT ‐ PCR and Western immunoblot analysis. Direct binding of LOXL 1 to TGF ‐ß1 was analyzed by blot overlay assay and solid phase ELISA using purified LOXL 1, recombinant human TGF ‐ß1, TGF ß1‐ LAP . The effect of LOXL 1 on TGF ‐ß1 signaling was analyzed using TGF ‐ß receptor signaling real time PCR assays (BioRad) after transient transfection of hTCF with a full‐length pCMV 6‐ LOXL 1 vector construct/with empty vector . Results TGF ß1 significantly increased LOXL 1 expression, secretion and enzymatic activity and correlated with enhanced expression of BMP ‐1, elastin, fibrillin‐1, fibulin‐4 and fibulin‐5 with peak effects at 10 ng/ml for 48 hours. This induction was blocked by TGF ‐ β receptor inhibitors and inhibitors of the canonical Smad and non‐canonical signaling pathways. Direct binding between LOXL 1 and TGF ß1‐ LAP was demonstrated by Blot overlay assays and ELISA . LOXL 1 overexpression temporarily upregulates different transcriptional regulators and some protein kinases of p38‐ MAPK signalling pathway after 12 to 24 hours post‐transfection. Conclusions The results of this study indicate that the interaction of LOXL 1 and TGF ‐ß1 plays an important role in the PEX ‐associated abnormal matrix metabolism and fibrosis.
Evidence suggests that chronically increased stress conditions in the anterior eye segment constitute major mechanisms involved in the pathobiology of pseudoexfoliation (PEX) syndrome. The expression of stress-related genes in eyes from patients with and without PEX syndrome/glaucoma was investigated to determine whether PEX syndrome is associated with an altered cellular stress response.cDNA array hybridization, quantitative real-time PCR, Western blot analysis, and immunohistochemistry were applied to analyze the mRNA and protein expression of stress-related genes in anterior segment tissues of PEX eyes, with and without glaucoma, and to compare them with normal and glaucomatous control eyes.Hybridization of cDNA arrays identified a set of stress-related candidate genes for differential expression in PEX syndrome/glaucoma, of which 10 were confirmed by real-time PCR in ciliary processes and iris tissue. The expression of MAPKp38, heat shock proteins (HSP40, HSP60), and superoxide dismutase (SOD2) was increased up to threefold in PEX specimens. In contrast, a large set of cytoprotective gene products, including antioxidant defense enzymes (glutathione S-transferases mGST1 and GSTT1), ubiquitin-conjugating enzymes (UBE2A, UBE2B), the DNA repair protein MLH1, and the stress-inducible transcription factor GADD153, were found to be consistently downregulated up to threefold in PEX specimens on both the mRNA and protein levels.The present findings provide evidence of alterations in cytoprotective mechanisms including antioxidant defense, proteasome function, endoplasmic reticulum-related stress response, and DNA repair in anterior segment tissues of PEX eyes. The resultant enhanced sensitivity and vulnerability to cellular stress conditions may therefore be one contributing factor in the pathobiology of PEX syndrome.
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