Exfoliation syndrome (XFS) is an age-related, genetically predisposed, generalized disorder of the extracellular matrix, which is characterized by the excessive production and accumulation of an abnormal, extracellular, fibrillar material (XFM) in many intraocular and extraocular tissues.1 The structure and composition of XFM as well as the cells involved in the XFS process have been extensively characterized.2 However, the exact biochemical composition of XFM as well as the molecular pathology of XFS still remain elusive.
In the eye, XFM appears macroscopically as whitish, fluffy deposits on the surfaces of the anterior segment. By light microscopy, XFM displays a bush-like, nodular or feathery structure. Transmission electron microscopy shows that XFM is composed of randomly arranged, electron dense, fuzzy fibrils with a periodic cross-banding pattern of about 50 nm, likely to originate from the lateral aggregation of newly synthesized or preexisting microfibrillar subunits into mature exfoliation fibrils (Fig. 1).
XFM appears to be actively and multifocally produced by various cell types, dispersed through the aqueous humor and deposited on all ocular structures of the anterior eye segment. Cells mainly involved in the exfoliation process are corneal endothelial cells, preequatorial lens epithelial cells, all cells of the iris, nonpigmented ciliary epithelial cells, and trabecular endothelial cells. These cells display ultrastructural evidence of an active fibrillogenesis and signs of a metabolically activated state, such as a prominent rough endoplasmic reticulum, increased vesicular transport to the cell surface, and membrane concavities containing microfibrils that are ejected into the extracellular space.
On the basis of immunohistochemical and biochemical approaches, XFM is considered a highly cross-linked and glycosylated complex of glycoproteins and proteoglycans. By immunohistochemistry, exfoliation fibrils predominantly contain fibrillin-1 and other epitopes of the elastic fiber system, such as latent transforming growth factor binding proteins (LTBP-1, -2, -3, -4), microfibril-associated glycoprotein (MAGP-1), fibulins (FBLN-2, -4, -6), emilin, vitronectin, amyloid P, tropoelastin, and elastin. Therefore, it is likely that the accumulation of XFM results from the excessive production and abnormal aggregation of elastic microfibrillar components. This theory is further corroborated by immunoelectron microscopy displaying a prominent staining for fibrillin-1 to exfoliation fibrils close to the cell membrane of various intraocular and extraocular cells from which newly synthesized fibrils appear to emerge (Fig. 2). In addition, as the banding pattern of XFM resembles the “bead on a string” appearance of isolated fibrillin microfibrils, fibrillin-1 seems to be its integral constituent, forming the core of the abnormal deposits. As epitopes of the basement membrane system, such as laminin, nidogen/entactin, and fibronectin, have been localized to XFM, its production has also been suggested to result from an abnormal basement membrane metabolism. Further, enzymatically active components, such as lysyl oxidase-like 1 (LOXL1), a key enzyme of elastogenesis and elastic fiber homeostasis, and the extracellular chaperone clusterin, have been found to be major components of exfoliation deposits.3–5 These findings suggest the involvement of abnormal cross-linking and protein misfolding processes, both leading to the aggregation and stable accumulation of exfoliation deposits, as part of the pathology of XFS.
Biochemical approaches have confirmed most of these components and revealed some additional components of XFM, such as apolipoprotein E, complement factors (C1q and C3), proteoglycans (syndecan-3 and versican), proteases from the ADAM (A Disintegrin And Metalloprotease) family of proteases (ADAM19 and ADAM21), and tissue inhibitor of matrix metalloproteinases (TIMP)-3, suggesting the involvement of subtle inflammatory processes and abnormal protein degradation.4,6 Further, histochemical, immunohistochemical, and lectin-binding studies have identified numerous carbohydrate components, such as heparan-sulfate, chondroitin-sulfate, and dermatan-sulfate proteoglycan, and the HNK-1 epitope, a carbohydrate moiety of many cell adhesion–related glycoproteins, suggesting the involvement of abnormal glycosylation processes. Interestingly, the HNK-1 epitope appears to be absent from extraocular deposits. Generally, on comparing intraocular and extraocular XFM, both seem to share the same protein components and differ in their carbohydrate composition only, arguing for a common, systemic process underlying the formation of abnormal deposits.2
At present, it is unknown which of the various components represent primary products of a disturbed cellular metabolism or which become secondarily incorporated into exfoliation deposits. Therefore, further studies are required to elucidate the primary products and processes leading to the production, aggregation, and stable accumulation of XFM.
At the molecular level, cells involved in the abnormal matrix process are characterized by a differential mRNA and protein expression pattern as compared with normal cells.7–11 A hallmark of the disease is the increased expression of elastic fiber components, such as elastin, fibrillin-1, LTBP-1, -2, and fibulin-2, -4, -6, all of them being present in XFM, whereas collagens (Col I, Col II, Col III, Col IV) display no differential expression and are absent from exfoliation deposits.
In early stages of the disease, the upregulation of elastic fiber components is paralleled by the upregulation of the extracellular matrix cross-linking enzyme LOXL1, the major genetic risk factor for XFS and exfoliation glaucoma (XFG). In contrast to LOXL1, other LOX isoenzymes display no differential expression and are absent from XFM. Therefore, it appears that LOXL1 is selectively upregulated and activated in the initial phase of the fibrotic process and participates in the cross-linking of the newly synthesized extracellular matrix and finally in the accumulation of XFM.
In advanced stages of the disease, LOXL1 expression is downregulated, possibly by compensatory mechanisms, as the protein accumulates in the extracellular space. The resulting deficiency of LOXL1 expression may predispose to an insufficient cross-linking of elastic fibers and elastotic matrix alterations, characteristic of eyes with late-stage XFS and XFG.
Several genetic variants of the LOXL1 gene have been identified as a strong risk factor for the development of XFS and XFG. However, the mechanisms by which these variants confer risk for the disease still remain unknown and may include abnormal mRNA and protein expression levels, or an altered substrate specificity of the LOXL1 protein. In our Caucasian study population, the risk alleles of 2 nonsynonymous single-nucleotide polymorphisms (SNPs) in exon 1 (rs1048661 and rs3825942) and 3 SNPs (rs2165241, rs2028387, and rs1992314) in intron 1 of the LOXL1 gene are correlated with a reduced expression of LOXL1 mRNA in ocular tissues and form the risk haplotype GGTCA. However, it still remains to be determined whether 1 of these SNPs or their haplotype has direct functional consequences on the expression of LOXL1 or tag another yet unknown genetic risk variant predisposing for the disease.
Cells involved in the production of XFM also displayed an increased expression of matrix metalloproteinase (MMP)-2 and tissue inhibitors of matrix metalloproteinase (TIMP)-1 and TIMP-2, whereas members of the ADAM family of proteases (ADAM19 and ADAM21) showed no differential expression. Importantly, the upregulation of TIMP-1 and TIMP-2 is more pronounced than the upregulation of MMP-2, possibly leading to a proteolytic imbalance and the inhibition of matrix degradation and the accumulation of extracellular deposits in eyes with XFS. Further, inflammatory mediators, such as interleukin (IL)-6 and IL-8, displayed a transiently increased expression in the iris and the ciliary processes of eyes with XFS leading to transiently increased aqueous levels of these cytokines. The activation of IL-6 signaling pathways, reflected by the phosphorylation of signal transducer and activator of transcription (STAT)-3, may then lead to the stimulation of extracellular matrix synthesis and induction of the fibrogenic growth factor TGF-β1, and to increased aqueous TGF-β1 levels through the enhancement of vascular permeability in the iris. Moreover, cells involved in the disease displayed a downregulation of cytoprotective gene products, such as ubiquitin conjugating enzymes (UBE2A and UBE2B), antioxidative enzymes (glutathione-S-transferase T1 and microsomal glutathione-S-transferase), DNA repair proteins (excision repair cross-complementation 1, ERCC1 and MutL homolog 1, MLH1), a stress-inducible transcription factor (growth arrest and DNA damage-inducible protein, GADD153), and the extracellular chaperone clusterin, indicating impaired cytoprotective mechanisms in eyes with XFS.
XFS is a complex genetic disease; therefore, in addition to genetic variants in the LOXL1 gene, further genetic and/or nongenetic factors seem necessary for the initiation of the abnormal matrix process. Chronic stress conditions, such as increased oxidative stress and a pronounced anterior chamber hypoxia are well-known characteristics of eyes with XFS and may represent potential triggering factors. Chronically increased cellular stress may be intensified by impaired cytoprotective mechanisms and lead to the upregulation of proinflammatory cytokines, such as IL-6 and IL-8, in certain ocular tissues. Once activated, IL-6 and IL-8 may lead, either by increasing vascular permeability or by direct upregulation, to increased TGF-β1 levels in the aqueous humor of eyes with XFS. The profibrotic growth factor TGF-β1, in turn, may upregulate LOXL1 and elastic components, leading to the production, aggregation, and stable accumulation of XFM.
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