Identification, Isolation, and Characterization of Melanocyte Precursor Cells in the Human Limbal Stroma.

Given their vital role in the homeostasis of the limbal stem cell niche, limbal melanocytes have emerged as promising candidates for tissue engineering applications. This study aimed to isolate and characterize a population of melanocyte precursors in the limbal stroma, compared with melanocytes originating from the limbal epithelium, using magnetic-activated cell sorting (MACS) with positive (CD117/c-Kit microbeads) or negative (CD326/EpCAM or anti-fibroblast microbeads) selection approaches. Both approaches enabled fast and easy isolation and cultivation of pure limbal epithelial and stromal melanocyte populations, which differed in phenotype and gene expression, but exhibited similar functional properties regarding proliferative potential, pigmentation, and support of clonal growth of limbal epithelial stem/progenitor cells (LEPCs). In both melanocyte populations, limbus-specific matrix (laminin 511-E8) and soluble factors (LEPC-derived conditioned medium) stimulated melanocyte adhesion, dendrite formation, melanogenesis, and expression of genes involved in UV protection and immune regulation. The findings provided not only a novel protocol for the enrichment of pure melanocyte populations from limbal tissue applying easy-to-use MACS technology, but also identified a population of stromal melanocyte precursors, which may serve as a reservoir for the replacement of damaged epithelial melanocytes and an alternative resource for tissue engineering applications.

Dysregulated Retinoic Acid Signaling in the Pathogenesis of Pseudoexfoliation Syndrome.

Pseudoexfoliation (PEX) syndrome, a stress-induced fibrotic matrix process, is the most common recognizable cause of open-angle glaucoma worldwide. The recent identification of PEX-associated gene variants uncovered the vitamin A metabolic pathway as a factor influencing the risk of disease. In this study, we analyzed the role of the retinoic acid (RA) signaling pathway in the PEX-associated matrix metabolism and evaluated its targeting as a potential candidate for an anti-fibrotic intervention. We provided evidence that decreased expression levels of RA pathway components and diminished RA signaling activity occur in an antagonistic crosstalk with TGF-ß1/Smad signaling in ocular tissues and cells from PEX patients when compared with age-matched controls. Genetic and pharmacologic modes of RA pathway inhibition induced the expression and production of PEX-associated matrix components by disease-relevant cell culture models in vitro. Conversely, RA signaling pathway activation by natural and synthetic retinoids was able to suppress PEX-associated matrix production and formation of microfibrillar networks via antagonization of Smad-dependent TGF-ß1 signaling. The findings indicate that deficient RA signaling in conjunction with hyperactivated TGF-ß1/Smad signaling is a driver of PEX-associated fibrosis, and that restoration of RA signaling may be a promising strategy for anti-fibrotic intervention in patients with PEX syndrome and glaucoma.

Long COVID: Association of Functional Autoantibodies against G-Protein-Coupled Receptors with an Impaired Retinal Microcirculation.

Long COVID (LC) describes the clinical phenotype of symptoms after infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnostic and therapeutic options are limited, as the pathomechanism of LC is elusive. As the number of acute SARS-CoV-2 infections was and is large, LC will be a challenge for the healthcare system. Previous studies revealed an impaired blood flow, the formation of microclots, and autoimmune mechanisms as potential factors in this complex interplay. Since functionally active autoantibodies against G-protein-coupled receptors (GPCR-AAbs) were observed in patients after SARS-CoV-2 infection, this study aimed to correlate the appearance of GPCR-AAbs with capillary microcirculation. The seropositivity of GPCR-AAbs was measured by an established cardiomyocyte bioassay in 42 patients with LC and 6 controls. Retinal microcirculation was measured by OCT-angiography and quantified as macula and peripapillary vessel density (VD) by the Erlangen-Angio Tool. A statistical analysis yielded impaired VD in patients with LC compared to the controls, which was accentuated in female persons. A significant decrease in macula and peripapillary VD for AAbs targeting adrenergic ß2-receptor, MAS-receptor angiotensin-II-type-1 receptor, and adrenergic α1-receptor were observed. The present study might suggest that a seropositivity of GPCR-AAbs can be linked to an impaired retinal capillary microcirculation, potentially mirroring the systemic microcirculation with consecutive clinical symptoms.

The protective variant rs7173049 at LOXL1 locus impacts on retinoic acid signaling pathway in pseudoexfoliation syndrome.

LOXL1 (lysyl oxidase-like 1) has been identified as the major effect locus in pseudoexfoliation (PEX) syndrome, a fibrotic disorder of the extracellular matrix and frequent cause of chronic open-angle glaucoma. However, all known PEX-associated common variants show allele effect reversal in populations of different ancestry, casting doubt on their biological significance. Based on extensive LOXL1 deep sequencing, we report here the identification of a common non-coding sequence variant, rs7173049A>G, located downstream of LOXL1, consistently associated with a decrease in PEX risk (odds ratio, OR = 0.63; P = 6.33 × 10-31) in nine different ethnic populations. We provide experimental evidence for a functional enhancer-like regulatory activity of the genomic region surrounding rs7173049 influencing expression levels of ISLR2 (immunoglobulin superfamily containing leucine-rich repeat protein 2) and STRA6 [stimulated by retinoic acid (RA) receptor 6], apparently mediated by allele-specific binding of the transcription factor thyroid hormone receptor beta. We further show that the protective rs7173049-G allele correlates with increased tissue expression levels of ISLR2 and STRA6 and that both genes are significantly downregulated in tissues of PEX patients together with other key components of the STRA6 receptor-driven RA signaling pathway. siRNA-mediated downregulation of RA signaling induces upregulation of LOXL1 and PEX-associated matrix genes in PEX-relevant cell types. These data indicate that dysregulation of STRA6 and impaired retinoid metabolism are involved in the pathophysiology of PEX syndrome and that the variant rs7173049-G, which represents the first common variant at the broad LOXL1 locus without allele effect reversal, mediates a protective effect through upregulation of STRA6 in ocular tissues.

Association of Rare CYP39A1 Variants With Exfoliation Syndrome Involving the Anterior Chamber of the Eye.

Importance: Exfoliation syndrome is a systemic disorder characterized by progressive accumulation of abnormal fibrillar protein aggregates manifesting clinically in the anterior chamber of the eye. This disorder is the most commonly known cause of glaucoma and a major cause of irreversible blindness. Objective: To determine if exfoliation syndrome is associated with rare, protein-changing variants predicted to impair protein function. Design, Setting, and Participants: A 2-stage, case-control, whole-exome sequencing association study with a discovery cohort and 2 independently ascertained validation cohorts. Study participants from 14 countries were enrolled between February 1999 and December 2019. The date of last clinical follow-up was December 2019. Affected individuals had exfoliation material on anterior segment structures of at least 1 eye as visualized by slit lamp examination. Unaffected individuals had no signs of exfoliation syndrome. Exposures: Rare, coding-sequence genetic variants predicted to be damaging by bioinformatic algorithms trained to recognize alterations that impair protein function. Main Outcomes and Measures: The primary outcome was the presence of exfoliation syndrome. Exome-wide significance for detected variants was defined as P < 2.5 × 10-6. The secondary outcomes included biochemical enzymatic assays and gene expression analyses. Results: The discovery cohort included 4028 participants with exfoliation syndrome (median age, 78 years [interquartile range, 73-83 years]; 2377 [59.0%] women) and 5638 participants without exfoliation syndrome (median age, 72 years [interquartile range, 65-78 years]; 3159 [56.0%] women). In the discovery cohort, persons with exfoliation syndrome, compared with those without exfoliation syndrome, were significantly more likely to carry damaging CYP39A1 variants (1.3% vs 0.30%, respectively; odds ratio, 3.55 [95% CI, 2.07-6.10]; P = 6.1 × 10-7). This outcome was validated in 2 independent cohorts. The first validation cohort included 2337 individuals with exfoliation syndrome (median age, 74 years; 1132 women; n = 1934 with demographic data) and 2813 individuals without exfoliation syndrome (median age, 72 years; 1287 women; n = 2421 with demographic data). The second validation cohort included 1663 individuals with exfoliation syndrome (median age, 75 years; 587 women; n = 1064 with demographic data) and 3962 individuals without exfoliation syndrome (median age, 74 years; 951 women; n = 1555 with demographic data). Of the individuals from both validation cohorts, 5.2% with exfoliation syndrome carried CYP39A1 damaging alleles vs 3.1% without exfoliation syndrome (odds ratio, 1.82 [95% CI, 1.47-2.26]; P < .001). Biochemical assays classified 34 of 42 damaging CYP39A1 alleles as functionally deficient (median reduction in enzymatic activity compared with wild-type CYP39A1, 94.4% [interquartile range, 78.7%-98.2%] for the 34 deficient variants). CYP39A1 transcript expression was 47% lower (95% CI, 30%-64% lower; P < .001) in ciliary body tissues from individuals with exfoliation syndrome compared with individuals without exfoliation syndrome. Conclusions and Relevance: In this whole-exome sequencing case-control study, presence of exfoliation syndrome was significantly associated with carriage of functionally deficient CYP39A1 sequence variants. Further research is needed to understand the clinical implications of these findings.

The role of lysyl oxidase-like 1 (LOXL1) in exfoliation syndrome and glaucoma.

Exfoliation syndrome (XFS) is an age-related systemic disease that affects the extracellular matrix. It increases the risk of glaucoma (exfoliation glaucoma, XFG) and susceptibility to diseases of elastin-rich connective tissues. LOXL1 (lysyl oxidase-like 1) is still recognized as the major genetic effect locus in XFS and XFG in all populations worldwide, although its genetic architecture is incompletely understood. LOXL1 is a key cross-linking enzyme in elastic fiber formation and remodeling, which is compatible with the pathogenetic concept of XFS as a specific type of elastosis. This review provides an overview on the current knowledge about the role of LOXL1 in the etiology and pathophysiology of XFS and XFG. It covers the known genetic associations at the LOXL1 locus, potential mechanisms of gene regulation, implications of LOXL1 in XFS-associated fibrosis and connective tissue homeostasis, its role in the development of glaucoma and associated systemic diseases, and the currently available LOXL1-based in vivo and in vitro models. Finally, it also identifies gaps in knowledge and suggests potential areas for future research.

VIP changes during daytime in chicken intrinsic choroidal neurons.

PURPOSE: Ocular autonomic control is mediated by sympathetic and parasympathetic nerve fibres. Their interactions are complemented by primary afferent nerve fibers of and intrinsic choroidal neurons (ICN). As the vasodilatative neuropeptide, vasoactive intestinal peptide (VIP), is expressed in extrinsic and intrinsic ocular neurons, it is of special interest in ophthalmic research. Since circadian changes of ocular blood flow are known in humans and birds, this study aimed at investigating VIP expression at different daytimes in chicken choroid, the preferred model species in ICN research. METHODS: 12 eyes of 12 chickens were retrieved, slaughtered at 8.00-9.30 a.m. (n = 6) and 8.00 p.m. (n = 6), respectively, and choroidal wholemounts were prepared for immunofluorescence of VIP. VIP-positive ICN of both groups were quantified and density of VIP-positive axons assessed semi-quantitatively. In 28 additional eyes retrieved in the morning (n = 14) and evening (n = 14), choroidal VIP content was determined by ELISA. Morning and evening data were analyzed statistically. NADPH-diaphorase (NADPH-d, ICN cell marker) was done at additional 12 whole mount choroids of 12 chicken, retrieved in the morning (n = 6) and evening (n = 6). RESULTS: (1) Numbers of VIP positive neurons differed significantly between morning: (239.17 ±â€¯113.9) and evening: (550.83 ±â€¯245.7; p = 0.018). (2) Numbers of VIP-positive perikarya were significantly more accumulated in the temporal part of the choroid in the evening than in the morning (p = 0.026). (3) VIP positive axon density was found to be similar throughout the choroid in the morning and evening. (4) Number of NADPH-d positive neurons was not significantly different between morning (848.8 ±â€¯399.5) and evening (945.8 ±â€¯622.1, p > 0.05). (5) ELISA demonstrated a significant difference of VIP content (p = 0.012) in tissues harvested in the morning (145.41 ±â€¯43.3 pg/ml) compared to evening (221.44 ±â€¯106.3 pg/ml). CONCLUSIONS: As VIP positive axon density was similar in the morning and the evening throughout the choroid, PPG and ICN seemed to contribute equally to the axon network. Yet, changes in the total choroidal VIP content, the numbers of VIP positive perikarya, reflecting the intracellular VIP content, and their topographical distribution at two different days-times argue for a different status of activation of both neuronal sources in contrast to the equal amount of NADPHD-d positive neurons. The higher VIP content in the evening, compared to the morning, correlates with a known circadian rhythm of a lower IOP and a higher choroidal thickness at night. Thus, these changes may argue for a potential role of ICN in the regulation of ocular homeostasis and integrity.

Cell Adhesion Molecules and Stem Cell-Niche-Interactions in the Limbal Stem Cell Niche.

Interactions between stem cells and their microenvironment are critical for regulation and maintenance of stem cell function. To elucidate the molecular interactions within the human limbal epithelial stem/progenitor cell (LEPC) niche, which is essential for maintaining corneal transparency and vision, we performed a comprehensive expression analysis of cell adhesion molecules (CAMs) using custom-made quantitative real-time polymerase chain reaction (qRT-PCR) arrays and laser capture-microdissected LEPC clusters, comprising LEPCs, melanocytes, mesenchymal cells, and transmigrating immune cells. We show that LEPCs are anchored to their supporting basement membrane by the laminin receptors α3ß1 and α6ß4 integrin and the dystroglycan complex, while intercellular contacts between LEPCs and melanocytes are mediated by N-, P-, and E-cadherin together with L1-CAM, a member of the immunoglobulin superfamily (Ig)CAMs. In addition to the LEPC-associated heparan sulfate proteoglycans syndecan-2, glypican-3, and glypican-4, the IgCAM members ICAM-1 and VCAM-1 were found to be variably expressed on LEPCs and associated niche cells and to be dynamically regulated in response to chemokines such as interferon-γ to enhance interactions with immune cells. Moreover, junctional adhesion molecule JAM-C accumulating in the subepithelial limbal matrix, appeared to be involved in recruitment of immune cells, while mesenchymal stromal cells appeared to use the nephronectin receptor integrin α8 for approaching the limbal basement membrane. In summary, we identified a novel combination of cell surface receptors that may regulate both stable and dynamic cell-matrix and cell-cell interactions within the limbal niche. The findings provide a solid foundation for further functional studies and for advancement of our current therapeutic strategies for ocular surface reconstruction.

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.

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.

Reproducibility of graft preparations in Descemet's membrane endothelial keratoplasty.

PURPOSE: To assess the reproducibility of manual graft preparation and evaluate the incidence rate and nature of structural anomalies of Descemet's membrane (DM) preventing successful graft preparation in DM endothelial keratoplasty (DMEK). DESIGN: Prospective, single-center, nonrandomized, consecutive case series. PARTICIPANTS: We analyzed 350 corneoscleral buttons from donors aged 18-95 years stored in Optisol-GS or Dulbecco's modified Eagle's medium and used for DMEK surgery in 343 consecutive patients with Fuchs' endothelial dystrophy or pseudophakic bullous keratopathy. METHODS: Residual endothelial cell-DM complexes obtained after successful DM stripping for DMEK and whole donor corneas obtained after unsuccessful DM stripping were examined by transmission electron microscopy and immunohistochemistry. MAIN OUTCOME MEASURES: Accuracy of the cleavage plane between DM and corneal stroma and structural abnormalities of the DM-stroma interface. RESULTS: Uneventful manual separation without any disruption of DM was achieved in 335 of 350 donor corneas (95.7%) by use of a previously established bimanual submerged preparation technique. Correspondingly, the peeled DM specimens revealed a regular and smooth cleavage plane exposing the amorphous interfacial matrix on their anterior surface. Although 8 of 350 donor corneas (2.3%) showed focal adhesions of DM to the corneal stroma and developed isolated tears during stripping, preparation of the graft could be successfully completed. However, 7 of the 350 donor corneas (2.0%) showed extremely strong adhesion and multiple tears of DM, preventing successful preparation of the graft. These specimens revealed either ultrastructural (peg-like interlockings) or biochemical abnormalities (increased staining intensities for adhesive glycoproteins) along the DM-stroma interface. CONCLUSIONS: Using an appropriate technique, manual preparation of grafts for DMEK with reproducible tissue qualities is possible in the vast majority (98%) of donor corneas. Although a relatively rare phenomenon, interindividual variations in DM structure and composition may be responsible for failure of graft preparation in about 2% of donor corneas. FINANCIAL DISCLOSURE(S): The authors have no proprietary or commercial interest in any of the materials discussed in this article.

Pressure-Dependent Elevation of Vasoactive Intestinal Peptide Level in Chicken Choroid.

PURPOSE: Autonomic control is important in maintaining ocular integrity. As recent data suggested that intrinsic choroidal neurons (ICN), an intrinsic choroidal autonomic control, may regulate choroidal thickening via release of the vasodilative vasoactive intestinal peptide (VIP), it was the aim of the study to investigate the level of choroidal VIP (VIPchor) in the presence of an increased atmospheric pressure in a chicken model. METHODS: Chicken choroidal whole mounts were exposed to ambient pressure (n = 20) and 40 mm Hg (n = 20) in a PC-controlled, open chamber system for 24 and 72 h, respectively. The VIP concentration was analyzed by ELISA, and the total protein concentration was measured by the BCA assay. Statistical analysis was done using an unpaired two-tailed t-test. RESULTS: The pressurization systems enabled choroidal whole mount pressurization (40 mm Hg) with humidifying, pressure, temperature, and gas exchange. Overall, the VIPchor level concentration was significantly increased at 40 mmHg compared to the ambient pressure (30.09 ± 7.18 pg vs. 20.69 ± 3.24 pg; p < 0.0001). Subgroup analysis yielded a significantly increased VIPchor level at 40 mmHg compared to the ambient pressure after 24 h (28.42 ± 6.03 pg vs. 20.76 ± 4.06 pg; p = 0.005) and 72 h (31.77 ± 7.82 pg vs. 20.61 ± 2.12 pg; p = 0.002), respectively. The VIPchor elevation at 40 mm Hg ranged between 1.37- (24 h) and 1.54-fold (72 h) compared to the ambient pressure. No difference was observed between the VIPchor level at 24 h and 72 h (p > 0.05). CONCLUSIONS: The increase of the total choroidal VIP level, representing the intracellular VIP content, in the presence of an increased ambient pressure argues for a retention of VIP within the neurons, decreasing both vasodilatation and, consequently, choroid thickness. This finding might be a passive or even active function of ICN in the regulation of choroidal thickness, ocular integrity and IOP.

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.

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.

Melanocytes as emerging key players in niche regulation of limbal epithelial stem cells.

PURPOSE: Limbal melanocytes (LMel) represent essential components of the corneal epithelial stem cell niche and are known to protect limbal epithelial stem/progenitor cells (LEPCs) from UV damage by transfer of melanosomes. Here, we explored additional functional roles for LMel in niche homeostasis, immune regulation and angiostasis. METHODS: Human corneoscleral tissues were morphologically analyzed in normal, inflammatory and wound healing conditions. The effects of LMel on LEPCs were analyzed in direct and indirect co-culture models using electron microscopy, immunocytochemistry, qRT-PCR, Western blotting and functional assays; limbal mesenchymal stromal cells and murine embryonic 3T3 fibroblasts served as controls. The immunophenotype of LMel was assessed by flow cytometry before and after interferon-γ stimulation, and their immunomodulatory properties were analyzed by mixed lymphocytes reaction, monocyte adhesion assays and cytometric bead arrays. Their angiostatic effects on human umbilical cord endothelial cells (HUVECs) were evaluated by proliferation, migration, and tube formation assays. RESULTS: LMel and LEPCs formed structural units in the human limbal stem cell niche in situ, which could be functionally replicated, including melanosome transfer, by co-cultivation in vitro. LMel supported LEPCs during clonal expansion and during epithelial wound healing by stimulating proliferation and migration, and suppressed their differentiation through direct contact and paracrine effects. Under inflammatory conditions, LMel were increased in numbers and upregulated expression of ICAM-1 and MHC II molecules (HLA-DR), but lacked expression of HLA-G, -DP, -DQ and costimulatory molecules CD80 and CD86. They were also found to be potent suppressors of alloreactive T- cell proliferation and cytokine secretion, which largely depended on direct cell-cell interaction. Moreover, the LMel secretome exerted angiostatic activity by inhibiting vascular endothelial cell proliferation and capillary network formation. CONCLUSION: These findings suggest that LMel are not only professional melanin-producing cells, but exert various non-canonical functions in limbal niche homeostasis by regulating LEPC maintenance, immune responses, and angiostasis. Their potent regulatory, immunomodulatory and anti-angiogenic properties may have important implications for future regenerative cell therapies.

Dysfunction of the limbal epithelial stem cell niche in aniridia-associated keratopathy.

PURPOSE: Abnormalities in the limbal niche microenvironment have been suggested to be causally involved in aniridia-associated keratopathy (AAK), but histological analyses on the limbal structure and composition in AAK are lacking. Here, we investigated morphologic and molecular alterations of the limbal epithelial stem cell niche in human congenital aniridia. METHODS: The blind, buphthalmic and painful left eye of a 16-year old girl with congenital aniridia and juvenile glaucoma had to be enucleated because of uncontrolled intraocular pressure. The diagnosis of AAK was based on classical clinical features and partial limbal stem cell deficiency in the superior half. Genetic analysis identified a large heterozygous PAX6 gene deletion encompassing exons 11-15 as well as exon 9 of the neighboring ELP4 gene. Three limbal biopsies were taken from the superior, nasal and temporal regions to isolate and cultivate limbal epithelial progenitor cells and subject them to mRNA expression analyses. The globe was vertically bisected and processed for light and transmission electron microscopy and immunohistochemistry. RESULTS: Comparative analysis of the superior and inferior limbal zones showed a gradual degradation of palisade structures associated with the transition from a hyperplastic to an attenuated corneal epithelium, inflammatory cell infiltrations and basement membrane irregularities. The clinically unaffected inferior part revealed no distinct stem cell clusters in the preserved palisade region, but a uniform population of hyperproliferative, undifferentiated progenitor cells in the basal/suprabasal layers of limbal and corneal epithelia, which gave rise to maldifferentiated epithelial cells exhibiting a conjunctival/epidermal phenotype and nuclear-to-cytoplasmic translocation of Pax6. The structure of the limbal niche was fundamentally perturbed, showing marked alterations in extracellular matrix composition, dislocation of atypical melanocytes lacking melanosomes and melanin, aberrant Wnt/ß-catenin and retinoic acid signaling, and massive immune cell infiltration. CONCLUSIONS: Considering the limitations of a single Case study, the findings suggest that ocular surface alterations in AAK are caused by a primary dysfunction and gradual breakdown of the limbal stem cell niche through Pax6-related effects on both melanogenesis and epithelial differentiation.

Potential Functional Restoration of Corneal Endothelial Cells in Fuchs Endothelial Corneal Dystrophy by ROCK Inhibitor (Ripasudil).

PURPOSE: Rho-associated kinase (ROCK) inhibitors have been successfully used as a rescue strategy in eyes that failed to clear after descemetorhexis without endothelial graft for treatment of Fuchs endothelial corneal dystrophy (FECD). The functional mechanisms by which ROCK inhibitors modulate corneal endothelial cell regeneration in FECD patients have, however, not been clarified. Here, we analyzed the effect of the ROCK inhibitor ripasudil on corneal endothelial cells of FECD patients and normal donors using ex vivo tissue and in vitro cellular models. DESIGN: Experimental study: laboratory investigation. METHODS: This institutional study used endothelial cell-Descemet membrane lamellae from FECD patients (n = 450) undergoing Descemet membrane endothelial keratoplasty (FECD ex vivo model), normal research-grade donor corneas (n = 30) after scraping off central endothelial cells (ex vivo wound healing model), normal donor corneas (n = 20) without endothelial injury, and immortalized cell lines (n = 3) generated from FECD patients (FECD in vitro model). Descemet membrane lamellae were dissected into halves and incubated for 24-72 hours in storage medium with or without a single dose of 30 µM ripasudil. The effects of ripasudil on expression of genes and proteins related to endothelial cell proliferation, migration, functionality, and endothelial-to-mesenchymal transition were analyzed and complemented by functional assays on FECD cell lines. RESULTS: A single dose of ripasudil induced significant upregulation of genes and proteins related to cell cycle progression, cell-matrix adhesion and migration, as well as endothelial barrier and pump function up to 72 hours, whereas classical markers of endothelial-to-mesenchymal transition were downregulated in both FECD and normal specimens compared to unstimulated controls ex vivo. In addition to stimulation of proliferation and migration, ripasudil-induced changes in expression of functional signature genes could be also verified in FECD cell lines in vitro. CONCLUSIONS: These data support the concept that inhibition of ROCK signaling represents a potent tool in regenerative therapies in FECD patients through reactivation of cell proliferation and migration as well as restoration of endothelial pump and barrier function without inducing adverse phenotypic changes.

Case Report: Neutralization of Autoantibodies Targeting G-Protein-Coupled Receptors Improves Capillary Impairment and Fatigue Symptoms After COVID-19 Infection.

Clinical features of Coronavirus disease 2019 (COVID-19) are caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Acute infection management is a substantial healthcare issue, and the development of long-Covid syndrome (LCS) is extremely challenging for patients and physicians. It is associated with a variety of characteristics as impaired capillary microcirculation, chronic fatigue syndrome (CFS), proinflammatory cytokines, and functional autoantibodies targeting G-protein-coupled receptors (GPCR-AAbs). Here, we present a case report of successful healing of LCS with BC 007 (Berlin Cures, Berlin, Germany), a DNA aptamer drug with a high affinity to GPCR-AAbs that neutralizes these AAbs. A patient with a documented history of glaucoma, recovered from mild COVID-19, but still suffered from CFS, loss of taste, and impaired capillary microcirculation in the macula and peripapillary region. He was positively tested for various targeting GPCR-AAbs. Within 48 h after a single BC 007 treatment, GPCR-AAbs were functionally inactivated and remained inactive during the observation period of 4 weeks. This observation was accompanied by constant improvement of the fatigue symptoms of the patient, taste, and retinal capillary microcirculation. Therefore, the removal of GPCR-AAb might ameliorate the characteristics of the LCD, such as capillary impairment, loss of taste, and CFS.

Retinal Microcirculation as a Correlate of a Systemic Capillary Impairment After Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), affects the pulmonary systems via angiotensin-converting enzyme-2 (ACE-2) receptor, being an entry to systemic infection. As COVID-19 disease features ACE-2 deficiency, a link to microcirculation is proposed. Optical coherence tomography angiography (OCT-A) enables non-invasive analysis of retinal microvasculature. Thus, an impaired systemic microcirculation might be mapped on retinal capillary system. As recent OCT-A studies, analyzing microcirculation in two subdivided layers, yielded contrary results, an increased subdivision of retinal microvasculature might offer an even more fine analysis. The aim of the study was to investigate retinal microcirculation by OCT-A after COVID-19 infection in three subdivided layers (I). In addition, short-term retinal affections were monitored during COVID-19 disease (II). Considering (I), a prospective study (33 patientspost-COVID and 28 controls) was done. Macula and peripapillary vessel density (VD) were scanned with the Spectralis II. Macula VD was measured in three layers: superficial vascular plexus (SVP), intermediate capillary plexus (ICP), and deep capillary plexus (DCP). Analysis was done by the EA-Tool, including an Anatomical Positioning System and an analysis of peripapillary VD by implementing Bruch's membrane opening (BMO) landmarks. Overall, circular (c1, c2, and c3) and sectorial VD (s1-s12) was analyzed. Considering (II), in a retrospective study, 29 patients with severe complications of COVID-19 infection, hospitalized at the intensive care unit, were monitored for retinal findings at bedside during hospitalization. (I) Overall (p = 0.0133) and circular (c1, p = 0.00257; c2, p = 0.0067; and c3, p = 0.0345). VD of the ICP was significantly reduced between patientspost-COVID and controls, respectively. Overall (p = 0.0179) and circular (c1, p = 0.0189) peripapillary VD was significantly reduced between both groups. Subgroup analysis of hospitalized vs. non-hospitalized patientspost-COVID yielded a significantly reduced VD of adjacent layers (DCP and SVP) with increased severity of COVID-19 disease. Clinical severity parameters showed a negative correlation with VD (ICP) and peripapillary VD. (II) Funduscopy yielded retinal hemorrhages and cotton wool spots in 17% of patients during SARS-CoV-2 infection. As VD of the ICP and peripapillary regions was significantly reduced after COVID-19 disease and showed a link to clinical severity markers, we assume that the severity of capillary impairment after COVID-19 infection is mapped on retinal microcirculation, visualized by non-invasive OCT-A.

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 alpha6 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, alpha6 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.