Matthias Zenkel

Corneal Limbal Microenvironment Can Induce Transdifferentiation of Hair Follicle Stem Cells into Corneal Epithelial‐like Cells

The aim of this study was to investigate the transdifferentiation potential of murine vibrissa hair follicle (HF) stem cells into corneal epithelial‐like cells through modulation by corneal‐ or limbus‐specific microenvironmental factors. Adult epithelial stem cells were isolated from the HF bulge region by mechanical dissection or fluorescence‐activated cell sorting using antibodies to α6 integrin, enriched by clonal expansion, and subcultivated on various extracellular matrices (type IV collagen, laminin‐1, laminin‐5, fibronectin) and in different conditioned media derived from central and peripheral corneal fibroblasts, limbal stromal fibroblasts, and 3T3 fibroblasts. Cellular phenotype and differentiation were evaluated by light and electron microscopy, real‐time reverse transcription‐polymerase chain reaction, immunocytochemistry, and Western blotting, using antibodies against putative stem cell markers (K15, α6 integrin) and differentiation markers characteristic for corneal epithelium (K12, Pax6) or epidermis (K10). Using laminin‐5, a major component of the corneo‐limbal basement membrane zone, and conditioned medium from limbal stromal fibroblasts, clonally enriched HF stem and progenitor cells adhered rapidly and formed regularly arranged stratified cell sheets. Conditioned medium derived from limbal fibroblasts markedly upregulated expression of cornea‐specific K12 and Pax6 on the mRNA and protein level, whereas expression of the epidermal keratinocyte marker K10 was strongly downregulated. These findings suggest that adult HF epithelial stem cells are capable of differentiating into corneal epithelial‐like cells in vitro when exposed to a limbus‐specific microenvironment. Therefore, the HF may be an easily accessible alternative therapeutic source of autologous adult stem cells for replacement of the corneal epithelium and restoration of visual function in patients with ocular surface disorders. STEM CELLS 2009;27:642–652

Common Genetic Determinants of Intraocular Pressure and Primary Open-Angle Glaucoma

Intraocular pressure (IOP) is a highly heritable risk factor for primary open-angle glaucoma and is the only target for current glaucoma therapy. The genetic factors which determine IOP are largely unknown. We performed a genome-wide association study for IOP in 11,972 participants from 4 independent population-based studies in The Netherlands. We replicated our findings in 7,482 participants from 4 additional cohorts from the UK, Australia, Canada, and the Wellcome Trust Case-Control Consortium 2/Blue Mountains Eye Study. IOP was significantly associated with rs11656696, located in GAS7 at 17p13.1 (p = 1.4×10−8), and with rs7555523, located in TMCO1 at 1q24.1 (p = 1.6×10−8). In a meta-analysis of 4 case-control studies (total N = 1,432 glaucoma cases), both variants also showed evidence for association with glaucoma (p = 2.4×10−2 for rs11656696 and p = 9.1×10−4 for rs7555523). GAS7 and TMCO1 are highly expressed in the ciliary body and trabecular meshwork as well as in the lamina cribrosa, optic nerve, and retina. Both genes functionally interact with known glaucoma disease genes. These data suggest that we have identified two clinically relevant genes involved in IOP regulation.

Genotype-Correlated Expression of Lysyl Oxidase-Like 1 in Ocular Tissues of Patients with Pseudoexfoliation Syndrome/Glaucoma and Normal Patients

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.

Proinflammatory Cytokines Are Involved in the Initiation of the Abnormal Matrix Process in Pseudoexfoliation Syndrome/Glaucoma

Pseudoexfoliation (PEX) syndrome, which is an age-related, generalized elastotic matrix process, currently represents the most common identifiable risk factor for open-angle glaucoma. Dysregulated expression of proinflammatory cytokines has been implicated in the initiation of various fibrotic disorders and in the pathophysiology of glaucoma. Here we investigated the presence, expression, regulation, and functional significance of proinflammatory cytokines in eyes with early and late stages of PEX syndrome/glaucoma in comparison with normal and glaucomatous control eyes using multiplex bead analysis, immunoassays, real-time PCR, Western blotting, immunohistochemistry, and cell culture models. Early stages of PEX syndrome were characterized by approximately threefold (P < 0.005) elevated interleukin (IL)-6 and IL-8 levels in the aqueous humor and a concomitant approximately twofold (P < 0.001) increase in mRNA expression levels in anterior segment tissues as compared with controls. In contrast, late stages of PEX syndrome/glaucoma did not differ significantly from controls. IL-6, IL-6 receptor, and phospho-signal transducer and activator of transcription 3 could be mainly localized to walls of iris vessels and to the nonpigmented epithelium of ciliary processes. IL-6 and IL-8 were significantly up-regulated by ciliary epithelial cells in response to hypoxia or oxidative stress in vitro, whereas IL-6, but not IL-8, induced the expression of transforming growth factor-beta1 and elastic fiber proteins. These findings support a role for a stress-induced, spatially, and temporally restricted subclinical inflammation in the onset of the fibrotic matrix process characteristic of PEX syndrome/glaucoma.

Variants in ASB10 are associated with open-angle glaucoma

The molecular events responsible for obstruction of aqueous humor outflow and the loss of retinal ganglion cells in glaucoma, one of the main causes of blindness worldwide, remain poorly understood. We identified a synonymous variant, c.765C>T (Thr255Thr), in ankyrin repeats and suppressor of cytokine signaling box-containing protein 10 (ASB10) in a large family with primary open angle glaucoma (POAG) mapping to the GLC1F locus. This variant affects an exon splice enhancer site and alters mRNA splicing in lymphoblasts of affected family members. Systematic sequence analysis in two POAG patient groups (195 US and 977 German) and their respective controls (85 and 376) lead to the identification of 26 amino acid changes in 70 patients (70 of 1172; 6.0%) compared with 9 in 13 controls (13 of 461; 2.8%; P = 0.008). Molecular modeling suggests that these missense variants change ASB10 net charge or destabilize ankyrin repeats. ASB10 mRNA and protein were found to be strongly expressed in trabecular meshwork, retinal ganglion cells and ciliary body. Silencing of ASB10 transcripts in perfused anterior segment organ culture reduced outflow facility by ∼50% compared with control-infected anterior segments (P = 0.02). In conclusion, genetic and molecular analyses provide evidence for ASB10 as a glaucoma-causing gene.

LOXL1 Deficiency in the Lamina Cribrosa as Candidate Susceptibility Factor for a Pseudoexfoliation-Specific Risk of Glaucoma

PURPOSE To test the hypothesis that a primary disturbance in lysyl oxidase-like 1 (LOXL1) and elastin metabolism in the lamina cribrosa of eyes with pseudoexfoliation syndrome constitutes an independent risk factor for glaucoma development and progression. DESIGN Observational, consecutive case series. PARTICIPANTS Posterior segment tissues obtained from 37 donors with early and late stages of pseudoexfoliation syndrome without glaucoma, 37 normal age-matched control subjects, 5 eyes with pseudoexfoliation-associated open-angle glaucoma, and 5 eyes with primary open-angle glaucoma (POAG). METHODS Protein and mRNA expression of major elastic fiber components (elastin, fibrillin-1, fibulin-4), collagens (types I, III, and IV), and lysyl oxidase crosslinking enzymes (LOX, LOXL1, LOXL2) were assessed in situ by quantitative real-time polymerase chain reaction, (immuno)histochemistry, and light and electron microscopy. Lysyl oxidase-dependent elastin fiber assembly was assessed by primary optic nerve head astrocytes in vitro. MAIN OUTCOME MEASURES Expression levels of elastic proteins, collagens, and lysyl oxidases in the lamina cribrosa. RESULTS Lysyl oxidase-like 1 proved to be the major lysyl oxidase isoform in the normal lamina cribrosa in association with a complex elastic fiber network. Compared with normal and POAG specimens, lamina cribrosa tissues obtained from early and late stages of pseudoexfoliation syndrome without and with glaucoma consistently revealed a significant coordinated downregulation of LOXL1 and elastic fiber constituents on mRNA and protein level. In contrast, expression levels of collagens and other lysyl oxidase isoforms were not affected. Dysregulated expression of LOXL1 and elastic proteins was associated with pronounced (ultra)structural alterations of the elastic fiber network in the laminar beams of pseudoexfoliation syndrome eyes. Inhibition of LOXL1 interfered with elastic fiber assembly by optic nerve head astrocytes in vitro. CONCLUSIONS The findings provide evidence for a pseudoexfoliation-specific elastinopathy of the lamina cribrosa resulting from a primary disturbance in LOXL1 regulation and elastic fiber homeostasis, possibly rendering pseudoexfoliation syndrome eyes more vulnerable to pressure-induced optic nerve damage and glaucoma development and progression.

Genome-wide association study with DNA pooling identifies variants at CNTNAP2 associated with pseudoexfoliation syndrome

Genetic and nongenetic factors contribute to development of pseudoexfoliation (PEX) syndrome, a complex, age-related, generalized matrix process frequently associated with glaucoma. To identify specific genetic variants underlying its etiology, we performed a genome-wide association study (GWAS) using a DNA-pooling approach. Therefore, equimolar amounts of DNA samples of 80 subjects with PEX syndrome, 80 with PEX glaucoma (PEXG) and 80 controls were combined into separate pools and hybridized to 500K SNP arrays (Affymetrix). Array probe intensity data were analyzed and visualized with expressly developed software tools GPFrontend and GPGraphics in combination with GenePool software. For replication, independent German cohorts of 610 unrelated patients with PEX/PEXG and 364 controls as well as Italian cohorts of 249 patients and 190 controls were used. Of 19, 17 SNPs showing significant allele frequency difference in DNA pools were confirmed by individual genotyping. Further single genotyping at CNTNAP2 locus revealed association between PEX/PEXG for two SNPs, which was confirmed in an independent German but not the Italian cohort. Both SNPs remained significant in the combined German cohorts even after Bonferroni correction (rs2107856: Pc=0.0108, rs2141388: Pc=0.0072). CNTNAP2 was found to be ubiquitously expressed in all human ocular tissues, particularly in retina, and localized to cell membranes of epithelial, endothelial, smooth muscle, glial and neuronal cells. Confirming efficiency of GWAS with DNA-pooling approach by detection of the known LOXL1 locus, our study data show evidence for association of CNTNAP2 with PEX syndrome and PEXG in German patients.

Regulation of lysyl oxidase-like 1 (LOXL1) and elastin-related genes by pathogenic factors associated with pseudoexfoliation syndrome.

PURPOSE Pseudoexfoliation (PEX) syndrome/glaucoma is a complex, late-onset disorder of the elastic fiber system. Strong genetic risk is conferred by the lysyl oxidase-like 1 (LOXL1) gene, but additional comodulating factors are necessary for the manifestation of the disease. The aim of this study was to analyze the effect of various PEX-associated pathogenic factors on the genotype-correlated expression of LOXL1 and elastin-related genes. METHODS Cultured human Tenons capsule fibroblasts with high- and low-risk LOXL1 haplotypes were exposed to transforming growth factor (TGF)-β1, interleukin (IL)-6, homocysteine, oxidative stress, hypoxia, or ultraviolet (UV) radiation. Changes in the expression of LOXL1 and elastic constituents of PEX material and TGF-β1 were assessed by quantitative real-time PCR, Western blotting, immunohistochemistry, and electron microscopy. RESULTS Treatment of fibroblasts with TGF-β1, oxidative stress, UV light, and hypoxia induced a significant increase in expression levels of LOXL1 and elastic proteins, whereas the effect of IL-6 was limited to induction of elastic constituents. Immunohistochemistry and electron microscopy confirmed an upregulation of LOXL1 and elastic fiber proteins and their assembly into extracellular microfibrillar networks with focal aggregation of microfibrils into PEX-like fibrils on stimulation with TGF-β1 and oxidative stress. Basal and stimulated expression of LOXL1 mRNA and protein was slightly decreased in cells carrying the high-risk compared with the low-risk haplotype of LOXL1, but the differences between groups were statistically not significant. CONCLUSIONS The findings support the notion that both genetic and nongenetic fibrogenic factors, particularly TGF-β1 and oxidative stress, may cooperate in the stable accumulation of PEX aggregates.

Exploring functional candidate genes for genetic association in German patients with pseudoexfoliation syndrome and pseudoexfoliation glaucoma.

PURPOSE Pseudoexfoliation (PEX) syndrome is a generalized elastic microfibrillopathy characterized by fibrillar deposits in intra- and extraocular tissues. Genetic and nongenetic factors are known to be involved in its etiopathogenesis. This study was focused on six functional candidate genes involved in PEX material deposition and the analysis of their potential association with PEX syndrome and PEX glaucoma (PEXG). METHODS Fifty single-nucleotide polymorphisms (SNPs) capturing >95% of overall genetic variance observed in Europeans at loci for FBN1, LTBP2, MFAP2, TGM2, TGF-b1, and CLU were genotyped in 333 unrelated PEX-affected and 342 healthy individuals of German origin, and a genetic association study was performed. To replicate the findings, two SNPs of the CLU gene were genotyped in a further 328 unrelated German patients with PEX as well as in 209 Italian patients with PEX and 190 Italian control subjects. RESULTS Association with PEX was observed only for the SNP rs2279590 in intron 8 of the CLU gene coding for clusterin (corrected P = 0.0347, OR = 1.34) in our first German cohort. Likewise, a frequent haplotype encompassing the associated risk allele showed nominally significant association. None of remaining SNPs or SNP haplotypes were associated with PEX. The association found was confirmed in a second German cohort (P = 0.0244) but not in the Italian cohort (P = 0.7173). In addition, the association with CLU SNP rs2279590 was more significant in German patients with PEX syndrome than in those with PEXG. CONCLUSIONS Genetic variants in the gene encoding clusterin may represent a risk factor for PEX in German patients but not in Italian patients. Variants in FBN1, LTBP2, MFAP2, TGF-b1, and TGM2 do not play a major role in the etiology of PEX syndrome, at least in German patients.

Four Subunits (αβγδ) of the Epithelial Sodium Channel (ENaC) Are Expressed in the Human Eye in Various Locations

PURPOSE The epithelial sodium channel (ENaC) is typically expressed in sodium-absorbing epithelia. Several reports suggest that ENaC is also expressed in ocular tissues and may play a role in aqueous humor secretion and glaucoma. However, the precise localization of ENaC in the human eye is still unclear. Here, the authors studied ENaC expression in 12 normal human donor eyes and in six eyes of patients with glaucoma. METHODS Quantitative real-time PCR was used to investigate the expression of α-, β-, γ-, and δ-ENaC transcripts in ocular tissues. In addition, the authors performed immunohistochemical studies using recently generated antibodies against human β- and γ-ENaC. RESULTS At the mRNA level, all four ENaC subunits were found to be expressed in a wide range of ocular tissues from normal and glaucomatous human eyes, with the cornea, ciliary body, iris, and retina showing the highest expression levels. At the protein level, β- and γ-ENaC subunits showed distinct distribution patterns and could be immunolocalized primarily to the cell membranes of epithelial cells of the cornea and to the conjunctiva, iris, ciliary body, lens, and retinal pigment epithelium but also to vascular endothelial cells, smooth muscle cells, stromal cells, and retinal neurons. The authors found no altered mRNA level of any subunit in glaucomatous eyes. CONCLUSIONS All four ENaC subunits (αβγδ) are expressed in the normal human eye, with distinct localization of subunits possibly reflecting different functional states of the channel. The (patho-)physiological roles of ENaC in the various localizations in the eye remain to be determined.