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 Atf4 null mouse lenses downregulate the mRNA levels of lens epithelial markers as well as known markers of late lens fiber cell differentiation. However, a comparison of this list of differentially expressed genes (DEGs) with other known transcriptional regulators of lens development indicated that ATF4 expression is not directly controlled by the previously described lens gene regulatory network. Pathway analysis revealed that the Atf4 DEG list was enriched in numerous genes involved in nutrient transport, amino acid biosynthesis, and tRNA charging. These changes in gene expression likely result in the observed reductions in lens free amino acid and glutathione levels, which would result in the observed low levels of extractable lens protein, finally leading to perinatal lens disintegration. These data demonstrate that ATF4, via its function in the integrated stress response, is likely to play a crucial role in mediating the adaption of the lens to the avascularity needed to maintain lens transparency.

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. This analysis found that LECs upregulate the expression of numerous proinflammatory cytokines and profibrotic regulators by 6 h PCS suggesting rapid priming of pathways leading to inflammation and fibrosis PCS. LECs also highly upregulate the expression of numerous immediate early transcription factors (IETFs) by 6 h PCS and immunolocalization found elevated levels of these proteins by 3 h PCS, and this was preceded by the phosphorylation of ERK1/2 in injured LECs. Egr1 and FosB were among the highest expressed of these factors and qRT-PCR revealed that they also upregulate in explanted mouse lens epithelia suggesting potential roles in the LEC injury response. Analysis of lenses lacking either Egr1 or FosB revealed that both genes may regulate a portion of the acute LEC injury response, although neither gene was essential for expression of either proinflammatory or fibrotic markers at later times PCS suggesting that IETFs may work in concert to mediate the LEC injury response following cataract surgery.

αVß8 integrin targeting to prevent posterior capsular opacification.

Fibrotic posterior capsular opacification (PCO), a major complication of cataract surgery, is driven by transforming growth factor-ß (TGF-ß). Previously, αV integrins were found to be critical for the onset of TGF-ß-mediated PCO in vivo; however, the functional heterodimer was unknown. Here, ß8 integrin-conditional knockout (ß8ITG-cKO) lens epithelial cells (LCs) attenuated their fibrotic responses, while both ß5 and ß6 integrin-null LCs underwent fibrotic changes similar to WT at 5 days post cataract surgery (PCS). RNA-Seq revealed that ß8ITG-cKO LCs attenuated their upregulation of integrins and their ligands, as well as known targets of TGF-ß-induced signaling, at 24 hours PCS. Treatment of ß8ITG-cKO eyes with active TGF-ß1 at the time of surgery rescued the fibrotic response. Treatment of WT mice with an anti-αVß8 integrin function blocking antibody at the time of surgery ameliorated both canonical TGF-ß signaling and LC fibrotic response PCS, and treatment at 5 days PCS, after surgically induced fibrotic responses were established, largely reversed this fibrotic response. These data suggest that αVß8 integrin is a major regulator of TGF-ß activation by LCs PCS and that therapeutics targeting αVß8 integrin could be effective for fibrotic PCO prevention and treatment.

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. Here we describe the methods used to quantify the data presented in Shihan et al. Matrix Biology, 2020 which lays out a workflow where IF data collected on a confocal microscope can be used to quantitate the relative levels of a molecule of interest by measuring mean fluorescent intensity across a region of interest, cell number, and the percentage of cells in a sample "positive" for staining with the fluorescent probe of interest. Overall, this manuscript discusses considerations for collecting quantifiable fluorescent images on a confocal microscope and provides explicit methods for quantitating IF data using FIJI-ImageJ.

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

Purpose: 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). Methods: Cataract surgeons practicing worldwide specializing in adult, pediatric and veterinary patients were interviewed between March and August 2018. Results: Ocular inflammation following cataract surgery is treated by either corticosteroids and/or nonsteroidal anti-inflammatories (NSAIDs). Adult and pediatric cataract surgeons are satisfied with current treatments whereas this inflammation is still considered a problem by some in veterinary practice due to its slow resolution. Yttrium-aluminum-garnet (YAG) laser therapy is the PCO treatment of choice for adult cataract surgeons and they are generally pleased with its outcome. However, pediatric cataract surgeons find YAG problematic, especially in patients under 6 years of age, and invasive surgery is often needed to correct PCO/visual axis opacification (VAO). Veterinary ophthalmologists report that YAG is not effective for PCO in animals, especially dogs, due to the density of the fibrotic plaques; 86% of adult and 100% of veterinary and pediatric cataract surgeons surveyed agree that effective anti-PCO therapeutics would improve clinical care. Conclusions: Surgeons treating human patients are pleased with the available treatments for ocular inflammation following cataract surgery, although some veterinary ophthalmologists disagree. The surgeons surveyed agree that PCO/VAO remains an unsolved problem in pediatric and veterinary cataract surgery while the long-term outcome of adult cataract surgery could be improved by additional attention to this issue.

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. Canonical Wnt activity was dynamic in the optic vesicle and later in the retina, while it was absent from the ectodermal precursors of the lens and corneal epithelium. However, later in corneal development, canonical Wnt reporter activity was detected in corneal stroma and endothelium precursors as they form from the neural crest, although this was lost around birth. Interestingly, while no canonical Wnt signaling was detected in the corneal limbus or basal cells at any developmental stage, it was robust in adult corneal wing and squamous epithelial cells. While canonical Wnt reporter activity was also absent from the postnatal lens, upon lens injury intended to model cataract surgery, it upregulated within 12 h in remnant lens epithelial cells, and co-localized with alpha smooth muscle actin in fibrotic lens epithelial cells from 48 h post-surgery onward. This pattern correlated with downregulation of the inhibitor of canonical Wnt signaling, Dkk3. These data demonstrate that canonical Wnt signaling is dynamic within the developing eye and upregulates in lens epithelial cells in response to lens injury. As canonical Wnt signaling can collaborate with TGFß to drive fibrosis in other systems, these data offer the first evidence in a lens-injury model that canonical Wnt may synergize with TGFß signaling to drive fibrotic posterior capsular opacification (PCO).