Exfoliation glaucoma (XFG) is a sequel to exfoliation syndrome (XFS), which is characterized by the production and accumulation of extracellular fibrillary material (XFM) throughout the body.1,2 Clinically, XFG is an aggressive form of glaucoma which is relatively refractory to current medical therapies. Within the eye, XFM presents as whitish deposits on the anterior capsule of the crystalline lens, and can be found throughout the anterior portion of the eye including the lens, trabecular meshwork, and the Schlemm canal. It is identified electron microscopically as having a classical “beads on a string” morphology (Fig. 1). XFM impairs aqueous humor outflow facility through the trabecular meshwork and the Schlemm canal, leading to increased intraocular pressure. If untreated, retinal ganglion cell death and irreversible blindness can occur.1,2 XFM is a highly cross-linked and glycosylated mass built of a protein core containing glycoproteins, basement membrane proteins, proteases, and several other proteins linked to neurodegenerative and ocular diseases.3
Early work identifying nonprotein components of XFM relied on light and electron microscopy, immunostaining/immunocytochemical techniques and basic amino acid analysis, as the insolubility of the XFM created analytical chemistry challenges. In 1973, a study of the amino acid composition of XFM (after 24 h hydrolysis with 6N HCL at 110°C) found amyloid, basement membrane, and elastic microfibril components (all of which have been since confirmed using additional methods, see below). Of note, collagen components were not found4 but later studies identified the presence of collagen in XFM.5 Work in the late 1970s and early 1980s determined that the XFM reacted with multiple histochemical stains indicating the presence of glycosaminoglycans. Additional immunohistochemical staining over the next 2 decades revealed the presence of specific glycosaminoglycans including heparin sulfate, chondroitin sulfate, and hyaluronan (reviewed in Ritch and Schlötzer-Schrehardt2). This work was followed up with staining of HNK-1 (a carbohydrate epitope that may stabilize the XFM) along with many studies indicating a complex mixture of glycoconjugates.2,6 These early studies laid the groundwork for the nonprotein composition of XFM. Taken together, abnormal expression and degradation of macromolecules including glycosaminoglycans appears to play an important role in XFM development and XFG pathogenesis. Although the fundamental biochemical analyses have identified the outer components, the molecular mechanisms that are dysregulated to allow such aggregation still needs to be elucidated.
Extensive work using immunohistochemical approaches laid the foundation for the fundamental understanding of the protein components that make up XFM. Specifically, XFM was found to be composed of a variety of proteins, including basement membrane proteins (including laminin, fibronectin),5,7 elastic fiber proteins (including fibrillin-1, and vitronectin),8,9 latent TGFβ binding proteins and latent TGFβ1,10 and the chaperone protein clusterin.11 Notably, other studies have identified increased TGFβ1 in the aqueous humor of XFG patients.12 Analysis of this work suggests that the homeostatic mechanisms that regulate the generation and degradation of the basement membrane and fiber proteins is involved in the pathogenesis of this disorder.
A major obstacle in identifying the protein components of XFM was breaking down the fibrous material to enable mass spectrometry-based approaches. A new strategy to dissolve XFM was found in 2007, leading to the identification of several novel protein components. Using a combination of cyanogen bromide and formic acid digestions on the lens capsules of XFG and control patients along with quadrupole time-of-flight mass spectrometry and liquid chromatography coupled to tandem mass spectrometry analyses allowed a more complete XFM analysis.13 These analyses found known XFM proteins, including fibrillin-1, fibronectin, vitronectin, TIMP-3, and clusterin, among others. Notably, this novel solubilization technique led to the identification of fibulin-2, desmocollin-2, syndecan-3, versican, members of the ADAM family of metalloproteinases and the complement protein C1q. Importantly, the presence of the proteins was confirmed by immunostaining of XFM, along with classically known components including fibrilin-1 and fibronectin.13 In addition to the complement protein C1q localizing to XFM, the authors also found complement proteins C3c and C4c within XFM.13 Interestingly, other XFM components clusterin and vitronectin are regulators of the complement activity, suggesting that the complement cascade may also be involved in the formation of XFM. This work has recently been supported by a report demonstrating significantly elevated levels of complement components C3a and C5b-9 in the aqueous humor of XFG patients compared with controls.14 The repeated finding of TIMP-3, the novel identification of the ADAM family of proteinases, along with the data demonstrating dysregulated levels of matrix metalloproteinases in the aqueous humor of XFG patients support the hypothesis that dysregulation of protein degradation is one possible mechanism of XFM generation and stabilization.
Using an adapted solubilization technique based on the work mentioned above, linear ion trap mass spectrometry identified several proteins including latent-TGFβ binding protein-2, C3, clusterin, LOXL1 and apolipoprotein E (ApoE).15 The presence of both LOXL1 and ApoE were verified using immunohistochemistry on XFS and normal lens capsules. LOXL1 is a copper dependent secreted cross-linking oxidase. Importantly, single nucleotide polymorphisms associated with glaucoma have been found throughout the LOXL1 gene and represent the strongest genetic association with XFG to date. Levels of LOXL1 protein are altered in XFG patients, and LOXL1 increases in response to TGFβ in cultured human trabecular meshwork cells.16–18 Interestingly, the other novel component of XFM was ApoE, which is also linked to both age related macular degeneration and Alzheimer disease, both of which feature protein-rich deposits, drusen and plaques, respectively.19,20 Although reports on ApoE genotypes have been variable in their association with XFG,21,22 the presence of protein within XFM suggests unknown disease relevance. The advances in solubilization of XFM has allowed for the determination of novel and intriguing protein components within XFM including ApoE and LOXL1. Although it is unclear what the function of these components are in XFM, the genetic linkage to XFG (in the case of LOXL1) and the association with diseases that have protein aggregates (ApoE) suggest some role in pathogenesis and warrants further investigation. Notable proteins found in XFM (Table 1).
XFG is a complex, multifaceted disease characterized by the proteinaceous aggregates known as XFM. While many components of XFM have been identified including fibrous proteins (fibrilin-1, elastin, fibulin, etc), inhibitors of proteinases (TIMP-3), complement factors (C1q and C3), ApoE which is associated with the protein aggregate generation in Alzheimer and the protein most highly associated with XFG through genetic studies LOXL1; there are still many questions that need to be answered. These questions include:
- Are there other proteins found with XFM?
- What are the dysregulated molecular mechanisms that allow for the formation of XFM?
- Is the complement system involved in XFG?
- Can we identify a therapeutic intervention to either halt the production of XFM or breakdown the current XFM in patients effected by XFG?
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