Modeling Cataract Surgery in Mice.

Cataract surgery (CS) is an effective treatment for cataracts, a major cause of visual disability worldwide. However, CS leads to ocular inflammation, and in the long term, it can result in posterior capsular opacification (PCO) and/or lens dislocation driven by the post-surgical overgrowth of lens epithelial cells (LECs) and their conversion to myofibroblasts and/or aberrant fiber cells. However, the molecular mechanisms by which CS results in inflammation and PCO are still obscure because most in vitro models do not recapitulate the wound healing response of LECs seen in vivo, while traditional animal models of cataract surgery, such as rabbits, do not allow the genetic manipulation of gene expression to test mechanisms.

Reduction of lens size in PAX6-related aniridia.

Heterozygous mutation of PAX6 in humans leads to congenital aniridia (OMIM 106210) which is typified by congenital iris and foveal defects, and later onset glaucoma, aniridic keratopathy, and cataract. Mice heterozygous for Pax6 mutations phenocopy many aspects of aniridia including the iris defects, keratopathy and cataract, although Pax6 mutant mice have small lenses, a phenotype which is not typically reported in human aniridia, perhaps due to difficulties in measuring lens diameter during typical ophthalmic examinations as the lens periphery is shielded by the iris. In order to overcome this, records of patients diagnosed with congenital aniridia between April 2015 and May 2021 at the Necker-Enfants Malades Hospital, and genetically confirmed with a disease-causing PAX6 variant, were retrospectively reviewed for those with normal axial length whose iris defects allowed visualization of the lens margins and corneal diameter to allow calculation of a lens/corneal diameter ratio.

ATF4 May Be Essential for Adaption of the Ocular Lens to Its Avascular Environment.

The late embryonic mouse lens requires the transcription factor ATF4 for its survival although the underlying mechanisms were unknown. Here, RNAseq analysis revealed that E16.5 null mouse lenses downregulate the mRNA levels of lens epithelial markers as well as known markers of late lens fiber cell differentiation.

The Immediate Early Response of Lens Epithelial Cells to Lens Injury.

Cataracts are treated by lens fiber cell removal followed by intraocular lens (IOL) implantation into the lens capsule. While effective, this procedure leaves behind numerous lens epithelial cells (LECs) which undergo a wound healing response that frequently leads to posterior capsular opacification (PCO). In order to elucidate the acute response of LECs to lens fiber cell removal which models cataract surgery (post cataract surgery, PCS), RNA-seq was conducted on LECs derived from wild type mice at 0 and 6 h PCS.

Congenital aniridia beyond black eyes: From phenotype and novel genetic mechanisms to innovative therapeutic approaches.

Congenital PAX6-aniridia, initially characterized by the absence of the iris, has progressively been shown to be associated with other developmental ocular abnormalities and systemic features making congenital aniridia a complex syndromic disorder rather than a simple isolated disease of the iris. Moreover, foveal hypoplasia is now recognized as a more frequent feature than complete iris hypoplasia and a major visual prognosis determinant, reversing the classical clinical picture of this disease. Conversely, iris malformation is also a feature of various anterior segment dysgenesis disorders caused by PAX6-related developmental genes, adding a level of genetic complexity for accurate molecular diagnosis of aniridia.

αVβ8 integrin targeting to prevent posterior capsular opacification.

Fibrotic posterior capsular opacification (PCO), a major complication of cataract surgery, is driven by transforming growth factor-β (TGF-β). Previously, αV integrins were found to be critical for the onset of TGF-β-mediated PCO in vivo; however, the functional heterodimer was unknown. Here, β8 integrin-conditional knockout (β8ITG-cKO) lens epithelial cells (LCs) attenuated their fibrotic responses, while both β5 and β6 integrin-null LCs underwent fibrotic changes similar to WT at 5 days post cataract surgery (PCS).

The effect of sex on the mouse lens transcriptome.

The transcriptome of mammalian tissues differs between males and females, and these differences can change across the lifespan, likely regulating known sexual dimorphisms in disease prevalence and severity. Cataract, the most prevalent disease of the ocular lens, occurs at similar rates in young individuals, but its incidence is elevated in older women compared to men of the same age. However, the influence of sex on the lens transcriptome was unknown.

The aging mouse lens transcriptome.

Age is a major risk factor for cataract (ARC). However, the influence of aging on the lens transcriptome is under studied. Lens epithelial (LEC) and fiber cells (LFC) were isolated from young (3 month old) and aged (24 month old) C57BL/6J mice, and the transcriptome elucidated via RNAseq.

A simple method for quantitating confocal fluorescent images.

Western blotting (WB), enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FC) have long been used to assess and quantitate relative protein expression in cultured cells and tissue samples. However, WB and ELISA have limited ability to meaningfully quantitate relative protein levels in tissues with complex cell composition, while tissue dissociation followed by FC is not feasible when tissue is limiting and/or cells difficult to isolate. While protein detection in tissue using immunofluorescent (IF) probes has traditionally been considered a qualitative technique, advances in probe stability and confocal imaging allow IF data to be easily quantitated, although reproducible quantitation of relative protein expression requires careful attention to appropriate controls, experiment design, and data collection.

Morphometric analysis of the lens in human aniridia and mouse Small eye.

Congenital aniridia is caused by heterozygous mutations in the PAX6 gene. In this disease, congenital iris and foveal hypoplasia is associated with juvenile onset cataract, glaucoma, and corneal keratopathy. In rodents, Pax6 mutations result in a congenital reduction in ocular size that is not typically described in human aniridia.

Fibronectin has multifunctional roles in posterior capsular opacification (PCO).

Fibrotic posterior capsular opacification (PCO), one of the major complications of cataract surgery, occurs when lens epithelial cells (LCs) left behind post cataract surgery (PCS) undergo epithelial to mesenchymal transition, migrate into the optical axis and produce opaque scar tissue. LCs left behind PCS robustly produce fibronectin, although its roles in fibrotic PCO are not known. In order to determine the function of fibronectin in PCO pathogenesis, we created mice lacking the fibronectin gene (FN conditional knock out -FNcKO) from the lens.

A new transgenic reporter line reveals Wnt-dependent Snai2 re-expression and cranial neural crest differentiation in Xenopus.

During vertebrate embryogenesis, the cranial neural crest (CNC) forms at the neural plate border and subsequently migrates and differentiates into many types of cells. The transcription factor Snai2, which is induced by canonical Wnt signaling to be expressed in the early CNC, is pivotal for CNC induction and migration in Xenopus. However, snai2 expression is silenced during CNC migration, and its roles at later developmental stages remain unclear.

Cataract surgeon viewpoints on the need for novel preventative anti-inflammatory and anti-posterior capsular opacification therapies.

To determine cataract surgeon viewpoints on the efficacy of available therapies/preventatives for two common sequelae of cataract surgery: inflammation and posterior capsular opacification (PCO). Cataract surgeons practicing worldwide specializing in adult, pediatric and veterinary patients were interviewed between March and August 2018. Ocular inflammation following cataract surgery is treated by either corticosteroids and/or nonsteroidal anti-inflammatories (NSAIDs).

Lens Epithelial Cells Initiate an Inflammatory Response Following Cataract Surgery.

Purpose: Lens epithelial cell (LEC) conversion to myofibroblast is responsible for fibrotic cataract surgery complications including posterior capsular opacification. While transforming growth factor beta (TGFβ) signaling is important, the mechanisms by which the TGFβ pathway is activated post cataract surgery (PCS) are not well understood.

Methods: RNA-seq was performed on LECs obtained from a mouse cataract surgery model at the time of surgery and 24 hours later.

Spatiotemporal dynamics of canonical Wnt signaling during embryonic eye development and posterior capsular opacification (PCO).

The appropriate spatial and temporal regulation of canonical Wnt signaling is vital for eye development. However, the literature often conflicts on the distribution of canonical Wnt signaling in the eye. Here, using a sensitive mouse transgenic reporter line, we report a detailed re-evaluation of the spatiotemporal dynamics of canonical Wnt signaling in the developing eye.

β1-Integrin Deletion From the Lens Activates Cellular Stress Responses Leading to Apoptosis and Fibrosis.

Purpose: Previous research showed that the absence of β1-integrin from the mouse lens after embryonic day (E) 13.5 (β1MLR10) leads to the perinatal apoptosis of lens epithelial cells (LECs) resulting in severe microphthalmia. This study focuses on elucidating the molecular connections between β1-integrin deletion and this phenotype.

Intravitreal methotrexate infusion for proliferative vitreoretinopathy.

Purpose: The purpose of this study was to evaluate intravitreal methotrexate infusion (IMI) during pars plana vitrectomy (PPV) for retinal detachment in patients with high risk for the development of proliferative vitreoretinopathy (PVR).

Methods: Patients presenting with severe recurrent PVR with tractional retinal detachment and/or a history of severe ocular inflammation were treated with IMI. Clinical outcomes were determined from a retrospective medical chart review.

β1-integrin controls cell fate specification in early lens development.

Integrins are heterodimeric cell surface molecules that mediate cell-extracellular matrix (ECM) adhesion, ECM assembly, and regulation of both ECM and growth factor induced signaling. However, the developmental context of these diverse functions is not clear. Loss of β1-integrin from the lens vesicle (mouse E10.

The molecular mechanisms underlying lens fiber elongation.

Lens fiber cells are highly elongated cells with complex membrane morphologies that are critical for the transparency of the ocular lens. Investigations into the molecular mechanisms underlying lens fiber cell elongation were first reported in the 1960s, however, our understanding of the process is still poor nearly 50 years later. This review summarizes what is currently hypothesized about the regulation of lens fiber cell elongation along with the available experimental evidence, and how this information relates to what is known about the regulation of cell shape/elongation in other cell types, particularly neurons.

Prox1 and fibroblast growth factor receptors form a novel regulatory loop controlling lens fiber differentiation and gene expression.

Lens epithelial cells differentiate into lens fibers (LFs) in response to a fibroblast growth factor (FGF) gradient. This cell fate decision requires the transcription factor Prox1, which has been hypothesized to promote cell cycle exit in differentiating LF cells. However, we find that conditional deletion of Prox1 from mouse lenses results in a failure in LF differentiation despite maintenance of normal cell cycle exit.

Unfolded-protein response-associated stabilization of p27(Cdkn1b) interferes with lens fiber cell denucleation, leading to cataract.

Failure of lens fiber cell denucleation (LFCD) is associated with congenital cataracts, but the pathobiology awaits elucidation. Recent work has suggested that mechanisms that direct the unidirectional process of LFCD are analogous to the cyclic processes associated with mitosis. We found that lens-specific mutations that elicit an unfolded-protein response (UPR) in vivo accumulate p27(Cdkn1b), show cyclin-dependent kinase (Cdk)-1 inhibition, retain their LFC nuclei, and are cataractous.

Development of novel filtering criteria to analyze RNA-sequencing data obtained from the murine ocular lens during embryogenesis.

Next-generation sequencing of the transcriptome (RNA-Seq) is a powerful method that allows for the quantitative determination of absolute gene expression, and can be used to investigate how these levels change in response to an experimental manipulation or disease condition. The sensitivity of this method allows one to analyze transcript levels of all expressed genes, including low abundance transcripts that encode important regulatory molecules, providing valuable insights into the global effects of experimental manipulations. However, this increased sensitivity can also make it challenging to ascertain which expression changes are biologically significant.

The Zeb proteins δEF1 and Sip1 may have distinct functions in lens cells following cataract surgery.

Purpose: Posterior capsular opacification (PCO), the most prevalent side effect of cataract surgery, occurs when residual lens epithelial cells (LECs) undergo fiber cell differentiation or epithelial-to-mesenchymal transition (EMT). Here, we used a murine cataract surgery model to investigate the role of the Zeb proteins, Smad interacting protein 1 (Sip1) and δ-crystallin enhancer-binding factor 1 (δEF1), during PCO.

Methods: Extracapsular extraction of lens fiber cells was performed on wild-type and Sip1 knockout mice.

Beta-1 integrin is important for the structural maintenance and homeostasis of differentiating fiber cells.

β1-Integrin is a heterodimeric transmembrane protein that has roles in both cell-extra-cellular matrix and cell-cell interactions. Conditional deletion of β1-integrin from all lens cells during embryonic development results in profound lens defects, however, it is less clear whether this reflects functions in the lens epithelium alone or whether this protein plays a role in lens fibers. Thus, a conditional approach was used to delete β1-integrin solely from the lens fiber cells.

Lens extrusion from Laminin alpha 1 mutant zebrafish.

We report analysis of the ocular lens phenotype of the recessive, larval lethal zebrafish mutant, lama1 (a69/a69). Previous work revealed that this mutant has a shortened body axis and eye defects including a defective hyaloid vasculature, focal corneal dysplasia, and loss of the crystalline lens. While these studies highlight the importance of laminin α1 in lens development, a detailed analysis of the lens defects seen in these mutants was not reported.