Compositional Analysis of Extracellular Aggregates in the Eyes of Patients With Exfoliation Syndrome and Exfoliation Glaucoma.

Purpose: Exfoliation syndrome (XFS) is a condition characterized by the production of insoluble fibrillar aggregates (exfoliation material; XFM) in the eye and elsewhere. Many patients with XFS progress to exfoliation glaucoma (XFG), a significant cause of global blindness. We used quantitative mass spectrometry to analyze the composition of XFM in lens capsule specimens and in aqueous humor (AH) samples from patients with XFS, patients with XFG and unaffected individuals. Methods: Pieces of lens capsule and samples of AH were obtained with consent from patients undergoing cataract surgery. Tryptic digests of capsule or AH were analyzed by high-performance liquid chromatography-mass spectrometry and relative differences between samples were quantified using the tandem mass tag technique. The distribution of XFM on the capsular surface was visualized by SEM and super-resolution light microscopy. Results: A small set of proteins was consistently upregulated in capsule samples from patients with XFS and patients with XFG, including microfibril components fibrillin-1, latent transforming growth factor-ß-binding protein-2 and latent transforming growth factor-ß-binding protein-3. Lysyl oxidase-like 1, a cross-linking enzyme associated with XFS in genetic studies, was an abundant XFM constituent. Ligands of the transforming growth factor-ß superfamily were prominent, including LEFTY2, a protein best known for its role in establishing the embryonic body axis. Elevated levels of LEFTY2 were also detected in AH from patients with XFG, a finding confirmed subsequently by ELISA. Conclusions: This analysis verified the presence of suspected XFM proteins and identified novel components. Quantitative comparisons between patient samples revealed a consistent XFM proteome characterized by strong expression of fibrillin-1, lysyl oxidase-like-1, and LEFTY2. Elevated levels of LEFTY2 in the AH of patients with XFG may serve as a biomarker for the disease.

Increased Aqueous Humor GDF15 Is Associated with Worse Visual Field Loss in Pseudoexfoliative Glaucoma Patients.

Purpose: To determine whether increased growth differentiation factor 15 (GDF15) in aqueous humor (AH) is associated with worse visual field loss in patients with pseudoexfoliative glaucoma (PXG). Methods: We recruited 12 patients (6 males, 6 females) with primary open-angle glaucoma (POAG) or PXG who were scheduled to undergo glaucoma surgery. AH was obtained from the initial peripheral paracentesis for the planned glaucoma surgery, and GDF15 levels were quantified with enzyme-linked immunosorbent assay by an investigator masked to clinical information. Humphrey visual field testing was performed as a part of routine care; results were obtained by reviewing the medical record. Results: AH GDF15 was detectable in patients with POAG and PXG. Increased AH GDF15 was significantly associated with worse mean deviation in patients with POAG (r = -0.94; 95% confidence interval [CI], -0.99 to -0.33; P = 0.02) and PXG (r = -0.92; 95% CI, -0.99 to -0.41; P = 0.01). Conclusions: AH GDF15 is detectable in patients with PXG and POAG. Elevated AH GDF15 is strongly associated with worse mean deviation in both subgroups. These findings suggest that GDF15 may be a molecular marker of glaucoma severity that is generalizable to multiple types of glaucoma regardless of the underlying etiology. Translational Relevance: This study provides proof of concept that GDF15, a molecular marker of retinal ganglion stress that was initially identified in rodent models, may have clinical utility as a measure of glaucoma severity not only in POAG but also in PXG.

Birc7: A Late Fiber Gene of the Crystalline Lens.

PURPOSE: A distinct subset of genes, so-called "late fiber genes," is expressed in cells bordering the central, organelle-free zone (OFZ) of the lens. The purpose of this study was to identify additional members of this group. METHODS: Fiber cells were harvested from various layers of the lens by laser micro-dissection and subjected to microarray, in situ hybridization, and Western blot analysis. RESULTS: Expression of Livin, a member of the inhibitor of apoptosis protein (IAP) family encoded by Birc7, was strongly upregulated in deep cortical fiber cells. The depth-dependent distribution of Livin mRNA was confirmed by quantitative PCR and in situ hybridization. The onset of Livin expression coincided with loss of organelles from primary fiber cells. Livin expression peaked at 1 month but was sustained even in aged lenses. Antibodies raised against mouse Livin labeled multiple bands on immunoblots, reflecting progressive proteolysis of the parent molecule during differentiation. Mice harboring a floxed Birc7 allele were generated and used to conditionally delete Birc7 in lens. Lenses from knockout mice grew normally and retained their transparency, suggesting that Livin does not have an indispensable role in fiber cell differentiation. CONCLUSIONS: Birc7 is a late fiber gene of the mouse lens. In tumor cells, Livin acts as an antiapoptotic protein, but its function in the lens is enigmatic. Livin is a RING domain protein with putative E3 ubiquitin ligase activity. Its expression in cells bordering the OFZ is consistent with a role in organelle degradation, a process in which the ubiquitin proteasome pathway has been implicated previously.

The penny pusher: a cellular model of lens growth.

PURPOSE: The mechanisms that regulate the number of cells in the lens and, therefore, its size and shape are unknown. We examined the dynamic relationship between proliferative behavior in the epithelial layer and macroscopic lens growth. METHODS: The distribution of S-phase cells across the epithelium was visualized by confocal microscopy and cell populations were determined from orthographic projections of the lens surface. RESULTS: The number of S-phase cells in the mouse lens epithelium fell exponentially, to an asymptotic value of approximately 200 cells by 6 months. Mitosis became increasingly restricted to a 300-µm-wide swath of equatorial epithelium, the germinative zone (GZ), within which two peaks in labeling index were detected. Postnatally, the cell population increased to approximately 50,000 cells at 4 weeks of age. Thereafter, the number of cells declined, despite continued growth in lens dimensions. This apparently paradoxical observation was explained by a time-dependent increase in the surface area of cells at all locations. The cell biological measurements were incorporated into a physical model, the Penny Pusher. In this simple model, cells were considered to be of a single type, the proliferative behavior of which depended solely on latitude. Simulations using the Penny Pusher predicted the emergence of cell clones and were in good agreement with data obtained from earlier lineage-tracing studies. CONCLUSIONS: The Penny Pusher, a simple stochastic model, offers a useful conceptual framework for the investigation of lens growth mechanisms and provides a plausible alternative to growth models that postulate the existence of lens stem cells.

Cadm1 expression and function in the mouse lens.

PURPOSE: The immunoglobulin superfamily member Cadm1 is a single-pass, type 1 membrane protein that mediates calcium-independent, cell-cell adhesion. Cadm1 has been implicated in tumor formation and synaptogenesis. A recent analysis of mouse lens cell membranes identified Cadm1 as a major constituent of the fiber cell membrane proteome. Here the authors examined the expression and function of Cadm1 in the mouse lens. METHODS: Cadm1 expression was analyzed by Western blotting and immunofluorescence. The morphology of individual wild-type and Cadm1-null lens cells was visualized by confocal microscopy. RESULTS: Cadm1 was present in epithelial and superficial fiber cells as a heavily glycosylated protein with an apparent molecular mass of ≈80 kDa. Analysis of proteins extracted from various strata of the lens indicated that Cadm1 was degraded during fiber cell differentiation, at approximately the same time as the lens organelles, an observation confirmed by confocal microscopy. In epithelial cells, Cadm1 was enriched in basolateral membranes, whereas, in fiber cells, expression was restricted to the lateral membranes. Lenses from Cadm1-null mice were of normal size and transparency. The three-dimensional morphology of the cells in the epithelial layer was unaltered in the absence of Cadm1. However, in contrast to wild-type lens fiber cells, Cadm1-null fiber cells had an irregular, highly undulating morphology. CONCLUSIONS: Cadm1 is an abundant component of the lens fiber cell membrane. Although not essential for lens transparency, Cadm1 has an indispensable role in establishing and maintaining the characteristic three-dimensional architecture of the lens fiber cell mass.

The stratified syncytium of the vertebrate lens.

The fusion of cells to generate syncytial tissues is a crucial event in the development of many organisms. In the lens of the vertebrate eye, proteins and other macromolecules diffuse from cell to cell via the large molecule diffusion pathway (LMDP). We used the tamoxifen-induced expression of GFP to investigate the nature and role of the LMDP in living, intact lenses. Our data indicate that the LMPD preferentially connects cells lying within a stratum of the lens cortex and that formation of the LMPD depends on the expression of Lim2, a claudin-like molecule. The conduits for intercellular protein exchange are most likely regions of partial cellular fusion, which are commonly observed in wild-type lenses but rare or absent in Lim2-deficient lenses. The observation that lens tissue constitutes a stratified syncytium has implications for the transparency, refractive function and pathophysiology of the tissue.

DNase I and fragmented chromatin during nuclear degradation in adult bovine lens fibers.

PURPOSE: Nuclear loss is a most remarkable organelle disappearance during terminal differentiation of lens fiber cells given that it implicates the full degradation of a major molecular component, DNA. Consequently, to gain insight into the progression of DNA cleavage we analyzed the appearance of single strand breaks in relationship with chromatin condensation. To assess a possible involvement of DNase I in DNA fragmentation we explored its localization in lens fibers having different degrees of nuclear breakdown, evaluated by the state of chromatin, nuclear envelope, and DNA. METHODS: Whole mounts of adult bovine lens epithelium as well as lens cryosections were utilized to examine, using antibodies or specific molecular probes, the localization of DNase I, nuclear membrane, lamins, and DNA 3'-OH-free termini. Nuclease activity gel and western blot assays were used to characterize DNase I in different lens fiber extracts. RESULTS: Nuclear morphology was found to undergo significant changes from the onset of fiber differentiation. Initial spherical nuclei present at early fibergenesis stages evolve to elongated ones in mature fibers. Chromatin did not present signs of condensation in these nuclei. However, nuclei from fibers located deeper in lens volume exhibited some chromatin condensation and fragmentation while the nuclear lamina appeared undamaged. At more advanced stages, different patterns of nuclear envelope integrity and chromatin condensation and cleavage were observed. DNase I was found in the cytoplasm in the very initial fibers and then in the nuclear territory. DNase I appeared closely associated with fully condensed and fragmented chromatin at the final phases of nuclear breakdown. CONCLUSIONS: DNase I is a nuclease present in bovine lens fibers and can be considered as an enzyme producing final DNA cleavage since it is closely associated with highly fragmented DNA in disintegrating nuclei.

Bovine DNase I: gene organization, mRNA expression, and changes in the topological distribution of the protein during apoptosis in lens epithelial cells.

Genomic DNA sequencing and alignment with the known DNase I mRNA showed that the bovine gene consists of 9 exons and that only the last 8 encode the protein, since initial ATG was found at exon II. RT-PCR was used to identify DNase I mRNA in lens epithelium in vivo and in cultured epithelial cells. We found DNase I transcripts having the same nucleotide sequence as the pancreas form and others lacking almost all exon V. The lens protein presented a slightly higher relative molecular weight than the pancreatic enzyme. Lens DNase I was located in secretory pathway organelles and excluded from the nucleus. Nevertheless, in apoptotic lens epithelial cells in vitro, DNase I translocated to the nucleus and co-localized with TUNEL positive chromatin aggregates. These results indicate that cells in the lens epithelium constitutively express DNase I, and suggest a direct involvement of this nuclease in the final phases of chromatin degradation.