Eph-ephrin Signaling Affects Eye Lens Fiber Cell Intracellular Voltage and Membrane Conductance

The avascular eye lens generates its own microcirculation that is required for maintaining lifelong lens transparency. The microcirculation relies on sodium ion flux, an extensive network of gap junction (GJ) plaques between lens fiber cells and transmembrane water channels. Disruption of connexin proteins, the building blocks of GJs, or aquaporins, which make up water and adhesion channels, lead to lens opacification or cataracts. Recent studies have revealed that disruption of Eph-ephrin signaling, in particular the receptor EphA2 and the ligand ephrin-A5, in humans and mice lead to congenital and age-related cataracts. We investigated whether changes in lens transparency in EphA2 or ephrin-A5 knockout (–/–) mice is related to changes in GJ coupling and lens fluid and ion homeostasis. Immunostaining revealed changes in connexin 50 (Cx50) subcellular localization in EphA2–/– peripheral lens fibers and alteration in aquaporin 0 (Aqp0) staining patterns in ephrin-A5–/– and EphA2–/– inner mature fiber cells. Surprisingly, there was no obvious change in GJ coupling in knockout lenses. However, there were changes in fiber cell membrane conductance and intracellular voltage in knockout lenses from 3-month-old mice. These knockout lenses displayed decreased conductance of mature fiber membranes and were hyperpolarized compared to control lenses. This is the first demonstration that the membrane conductance of lens fibers can be regulated. Together these data suggest that EphA2 may be needed for normal Cx50 localization to the cell membrane and that conductance of lens fiber cells requires normal Eph-ephrin signaling and water channel localization.

Protein and Water Distribution Across Visual Axis in Mouse Lens: A Confocal Raman MicroSpectroscopic Study for Cold Cataract

Purpose: The aims of the study were to investigate cellular mechanisms of cold cataract in young lenses of wild-type C57BL/6J (B6WT) mice treated at different temperatures and to test a hypothesis that cold cataract formation is associated with the changes in lens protein and water distribution at different regions across lens fiber cells by Raman spectroscopy (RS).Methods: RS was utilized to scan the mouse lens at different regions with/without cold cataract. Three regions with various opacification along the equatorial axis in the anterior–posterior lens section were scanned. The intensity ratio of Raman bands at 2,935 and 3,390 cm−1 (Ip/Iw) were used to evaluate lens protein and water distribution. We further determined water molecular changes through Gaussian profiles of water Raman spectra.Results: Three specific regions 1, 2, and 3, located at 790–809, 515–534, and 415–434 μm away from the lens center, of postnatal day 14 B6WT lenses, were subjected to RS analysis. At 37°C, all three regions were transparent. At 25°C, only region 3 became opaque, while at 4°C, both regions 2 and 3 showed opacity. The sum of the difference between Ip/Iw and the value of linear fitting line from scattered-line at each scanning point was considered as fluctuation degree (FD) in each region. Among different temperatures, opaque regions showed relatively higher FD values (0.63 and 0.79 for regions 2 and 3, respectively, at 4°C, and 0.53 for region 3 at 25°C), while transparent regions provided lower FD values (less than 0.27). In addition, the decrease in Gaussian peak II and the rising of Gaussian peak III and IV from water Raman spectra indicated the instability of water molecule structure in the regions with cold cataract.Conclusion: Fluctuation degrees of RS data reveal new mechanistic information about cold cataract formation, which is associated with uneven distribution of lens proteins and water across lens fiber cells. It is possible that RS data partly reveals cold temperature-induced redistribution of lens proteins such as intermediate filaments in inner fiber cells. This lens protein redistribution might be related to unstable structure of water molecules according to Gaussian profiles of water RS.

A functional map of genomic HIF1α-DNA complexes in the eye lens revealed through multiomics analysis

Background During eye lens development the embryonic vasculature regresses leaving the lens without a direct oxygen source. Both embryonically and throughout adult life, the lens contains a decreasing oxygen gradient from the surface to the core that parallels the natural differentiation of immature surface epithelial cells into mature core transparent fiber cells. These properties of the lens suggest a potential role for hypoxia and the master regulator of the hypoxic response, hypoxia-inducible transcription factor 1 (HIF1), in the regulation of genes required for lens fiber cell differentiation, structure and transparency. Here, we employed a multiomics approach combining CUT&RUN, RNA-seq and ATACseq analysis to establish the genomic complement of lens HIF1α binding sites, genes activated or repressed by HIF1α and the chromatin states of HIF1α-regulated genes. Results CUT&RUN analysis revealed 8375 HIF1α-DNA binding complexes in the chick lens genome. One thousand one hundred ninety HIF1α-DNA binding complexes were significantly clustered within chromatin accessible regions (χ2 test p < 1 × 10− 55) identified by ATACseq. Formation of the identified HIF1α-DNA complexes paralleled the activation or repression of 526 genes, 116 of which contained HIF1α binding sites within 10kB of the transcription start sites. Some of the identified HIF1α genes have previously established lens functions while others have novel functions never before examined in the lens. GO and pathway analysis of these genes implicate HIF1α in the control of a wide-variety of cellular pathways potentially critical for lens fiber cell formation, structure and function including glycolysis, cell cycle regulation, chromatin remodeling, Notch and Wnt signaling, differentiation, development, and transparency. Conclusions These data establish the first functional map of genomic HIF1α-DNA complexes in the eye lens. They identify HIF1α as an important regulator of a wide-variety of genes previously shown to be critical for lens formation and function and they reveal a requirement for HIF1α in the regulation of a wide-variety of genes not yet examined for lens function. They support a requirement for HIF1α in lens fiber cell formation, structure and function and they provide a basis for understanding the potential roles and requirements for HIF1α in the development, structure and function of more complex tissues.

Exacerbation of Background Nuclear Cataracts in Sprague-Dawley Rats in Embryo-Fetal Development Studies With JNJ-42165279, a Fatty Acid Amide Hydrolase Inhibitor

Fetal examinations in embryo-fetal developmental (EFD) studies are based on macroscopic and dissecting microscopic evaluations, and histopathology is rarely performed other than to confirm macroscopic findings. Fetal lens examination is therefore generally limited to the presence, size, shape, and color of any abnormality. In a Sprague-Dawley rat EFD study with the fatty acid amide hydrolase (FAAH) inhibitor JNJ-42165279, an unusually high incidence of macroscopic granular foci was noted within the lens of gestation day 21 fetuses across all groups including controls, with higher incidence in the high-dose group. On histological evaluation of the lenses from fetuses with/without gross findings, primary lens fiber hypertrophy (swelling) and degeneration were observed across vehicle- and JNJ-42165279-exposed fetuses. In a follow-up study to investigate the progression or resolution of the fetal lens changes, animals exposed to suprapharmacological doses of JNJ-42165279 in utero had higher incidence of nuclear cataracts as detected via slit-lamp ophthalmic examinations on postnatal days 18 to 21 and 35 to 41. No histologic correlates for these cataracts were identified. We conclude that fetal primary lens fiber hypertrophy and nuclear cataracts at ophthalmology, are common background changes in this rat strain that are exacerbated by in utero exposure to the FAAH inhibitor JNJ-42165279.

Spatially-Resolved Proteomic Analysis of the Lens Extracellular Diffusion Barrier

PurposeThe presence of a physical barrier to molecular diffusion through lenticular extracellular space has been repeatedly detected in multiple species. This extracellular diffusion barrier has been proposed to restrict the movement of solutes into the lens and to direct nutrients into the lens core via the sutures at both poles. The purpose of this study is to characterize the molecular components that could contribute to the formation of this barrier.MethodsThree distinct regions in the bovine lens cortex were captured by laser capture microdissection guided by dye penetration. Proteins were digested by endoproteinase Lys C and trypsin. Mass spectrometry-based quantitative proteomic analysis followed by gene ontology (GO) and protein-protein interaction network analysis was performed.ResultsDye penetration showed that lens fiber cells first shrink the extracellular spaces of the broad sides of fiber cells followed by closure of the extracellular space between narrow sides at normalized lens distance (r/a) of 0.9. Accompanying the closure of extracellular space of the broad sides, dramatic proteomic changes were detected including up-regulation of several cell junctional proteins. AQP0 and its interacting partners ERM proteins were among a few proteins that were upregulated accompanying the closure of extracellular space of the narrow sides suggesting a particularly important role for the major lens membrane protein AQP0 in controlling the narrowing of the extracellular spaces between lens fiber cells. The results also provided important information related to biological processes that occur during fiber cell differentiation such as organelle degradation, cytoskeletal remodeling and GSH synthesis.ConclusionsThe formation of lens extracellular diffusion barrier is accompanied by significant membrane and cytoskeletal protein remodeling.

Time Course of Lens Epithelial Cell Behavior in Rabbit Eyes following Lens Extraction and Implantation of Intraocular Lens

Background. After cataract surgery, some lens epithelial cells (LECs) transdifferentiate into myofibroblast-like cells, which causes fibric posterior capsule opacification (PCO). Residual LECs differentiate into lens fiber cells, forming Elschnig pearls with PCO. This study was carried out to identify the time course of both types of LEC behavior in rabbit eyes following lens extraction and implantation of an intraocular lens (IOL). Methods. Phacoemulsification and implantation of posterior chamber IOLs were performed in rabbit eyes. Following enucleation, immunohistochemical methods were used to detect α-smooth muscle actin (α-SMA), a marker for myofibroblast-like cells, in the pseudophakic rabbit eyes. A mouse monoclonal antibody against α-SMA was used. Results. Soon after the operation, the LECs migrated and covered the lens capsule. Thereafter, the LECs around the anterior capsular margin were always positive for α-SMA. However, the distributions of these cells were not consistent. In some specimens, α-SMA-positive LECs were present around the IOL optic early after surgery, but most of them had disappeared several weeks after the surgery. The residual cells induced fibrotic PCO. In the other specimens, most LECs around the IOL optic except the anterior capsular margin were negative for α-SMA. In the peripheral region covered by the peripheral anterior and posterior capsules, LECs on the posterior capsule always differentiated into lens fiber cells and formed a Soemmering ring. Thereafter, migration of lens fiber cells from the Soemmering ring and differentiation of LECs in situ on the central posterior capsule consisted of Elschnig pearls type of PCO. Conclusions. Although postoperative LEC behavior is not consistent, residual α-SMA-positive LECs induced fibrotic PCO. The lens fiber cells that migrated from the peripheral capsular bag or that were differentiated in situ covered the central posterior capsule, forming Elschnig pearls with PCO.

Mechanosensitive collaboration between integrins and connexins allows nutrient and antioxidant transport into the lens

The delivery of glucose and antioxidants is vital to maintain homeostasis and lens transparency. Here, we report a new mechanism whereby mechanically activated connexin (Cx) hemichannels serve as a transport portal for delivering glucose and glutathione (GSH). Integrin α6β1 in outer cortical lens fiber activated by fluid flow shear stress (FFSS) induced opening of hemichannels. Inhibition of α6 activation prevented hemichannel opening as well as glucose and GSH uptake. The activation of integrin β1, a heterodimeric partner of α6 in the absence of FFSS, increased Cx50 hemichannel opening. Hemichannel activation by FFSS depended on the interaction of integrin α6 and Cx50 C-terminal domain. Moreover, hemichannels in nuclear fiber were unresponsive owing to Cx50 truncation. Taken together, these results show that mechanically activated α6β1 integrin in outer cortical lens fibers leads to opening of hemichannels, which transport glucose and GSH into cortical lens fibers. This study unveils a new transport mechanism that maintains metabolic and antioxidative function of the lens.