AMPK stimulation inhibits YAP/TAZ signaling to ameliorate hepatic fibrosis.

Hepatic fibrosis is driven by the activation of hepatic stellate cells (HSCs). The Hippo pathway and its effectors, YAP and TAZ, are key regulators of HSC activation and fibrosis. However, there is a lack of mechanistic understanding of YAP/TAZ regulation in HSCs. Here we show that AMPK activation leads to YAP/TAZ inhibition and HSC inactivation in vitro, while the expression of a kinase-inactive mutant reversed these effects compared to wild type AMPKɑ1. Notably, the depletion of LATS1/2, an upstream kinase of YAP/TAZ signaling, rescues YAP/TAZ activation, suggesting that AMPK may be mediating YAP/TAZ inhibition via LATS1/2. In the carbon tetrachloride mouse model of fibrosis, pharmacologic activation of AMPK in HSCs inhibits YAP/TAZ signaling and reduces fibrosis. The findings implicate AMPK as a critical regulator of YAP/TAZ signaling and HSC inactivation and highlight AMPK activation as a therapeutic target for the treatment of hepatic fibrosis.

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.

Targeting acid ceramidase ameliorates fibrosis in mouse models of non-alcoholic steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD) is a common cause of liver disease worldwide, and is characterized by the accumulation of fat in the liver. Non-alcoholic steatohepatitis (NASH), an advanced form of NAFLD, is a leading cause of liver transplantation. Fibrosis is the histologic feature most associated with liver-related morbidity and mortality in patients with NASH, and treatment options remain limited. In previous studies, we discovered that acid ceramidase (aCDase) is a potent antifibrotic target using human hepatic stellate cells (HSCs) and models of hepatic fibrogenesis. Using two dietary mouse models, we demonstrate that depletion of aCDase in HSC reduces fibrosis without worsening metabolic features of NASH, including steatosis, inflammation, and insulin resistance. Consistently, pharmacologic inhibition of aCDase ameliorates fibrosis but does not alter metabolic parameters. The findings suggest that targeting aCDase is a viable therapeutic option to reduce fibrosis in patients with NASH.

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.

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. RNAseq based transcriptomic profiling of young adult C57BL/6J mouse lens epithelial and fiber cells revealed that few genes are differentially expressed between the sexes. In contrast, lens cells from aged (24 month old) male and female C57BL/6J mice differentially expressed many genes, including several whose expression is lens preferred. Like cataracts, posterior capsular opacification (PCO), a major sequela of cataract surgery, may also be more prevalent in women. Lens epithelial cells isolated from mouse eyes 24 h after lens fiber cell removal exhibited numerous transcriptomic differences between the sexes, including genes implicated in complement cascades and extracellular matrix regulation, and these differences are much more pronounced in aged mice than in young mice. These results provide an unbiased basis for future studies on how sex affects the lens response to aging, cataract development, and cataract surgery.

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. EdgeR estimated differential gene expression in pairwise contrasts, and Advaita's Ipathway guide and custom R scripts were used to evaluate the potential biological significance of differentially expressed genes (DEGs). This analysis revealed age-dependent decreases in lens differentiation marker expression in both LECs and LFCs, with gamma crystallin transcripts downregulating nearly 50 fold in aged LFCs. The expression of the transcription factors Hsf4 and Maf, which are known to activate lens fiber cell preferred genes, are downregulated, while FoxE3, which represses gamma crystallin expression, is upregulated in aged fibers. Aged LECs upregulate genes controlling the immune response, complement pathways, and cellular stress responses, including glutathione peroxidase 3 (Gpx3). Aged LFCs exhibit broad changes in the expression of genes regulating cell communication, and upregulate genes involved in antigen processing/presentation and cholesterol metabolism, while changes in the expression of mitochondrial respiratory chain genes are consistent with mitochondrial stress, including upregulation of NDufa4l2, which encodes an alternate electron transport chain protein. However, age did not profoundly affect the response of LECs to injury as both young and aged LECs upregulate inflammatory gene signatures at 24 h post injury to similar extents. These RNAseq profiles provide a rich data set that can be mined to understand the genetic regulation of lens aging and how this impinges on the pathophysiology of age related cataract.

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.

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. While animals from this line have normal lenses, upon lens fiber cell removal which models cataract surgery, FNcKO LCs exhibit a greatly attenuated fibrotic response from 3 days PCS onward as assessed by a reduction in surgery-induced cell proliferation, and fibrotic extracellular matrix (ECM) production and deposition. This is correlated with less upregulation of Transforming Growth Factor ß (TGFß) and integrin signaling in FNcKO LCs PCS concomitant with sustained Bone Morphogenetic Protein (BMP) signaling and elevation of the epithelial cell marker E cadherin. Although the initial fibrotic response of FNcKO LCs was qualitatively normal at 48 h PCS as measured by the upregulation of fibrotic marker protein αSMA, RNA sequencing revealed that the fibrotic response was already quantitatively attenuated at this time, as measured by the upregulation of mRNAs encoding molecules that control, and are controlled by, TGFß signaling, including many known markers of fibrosis. Most notably, gremlin-1, a known regulator of TGFß superfamily signaling, was upregulated sharply in WT LCs PCS, while this response was attenuated in FNcKO LCs. As exogenous administration of either active TGFß1 or gremlin-1 to FNcKO lens capsular bags rescued the attenuated fibrotic response of fibronectin null LCs PCS including the loss of SMAD2/3 phosphorylation, this suggests that fibronectin plays multifunctional roles in fibrotic PCO development.

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.

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 (TGFß) signaling is important, the mechanisms by which the TGFß 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. Bioinformatic analysis was performed with iPathwayGuide. Expression dynamics were determined by immunofluorescence. Results: The LEC transcriptome is massively altered by 24 hours PCS. The differentially expressed genes included those important for lens biology, and fibrotic markers. However, the most dramatic changes were in the expression of genes regulating the innate immune response, with the top three altered genes exhibiting greater than 1000-fold upregulation. Immunolocalization revealed that CXCL1, S100a9, CSF3, COX-2, CCL2, LCN2, and HMOX1 protein levels upregulate in LECs between 1 hour and 6 hours PCS and peak at 24 hours PCS, while their levels sharply attenuate by 3 days PCS. This massive upregulation of known inflammatory mediators precedes the infiltration of neutrophils into the eye at 18 hours PCS, the upregulation of canonical TGFß signaling at 48 hours PCS, and the infiltration of macrophages at 3 days PCS. Conclusions: These data demonstrate that LECs produce proinflammatory cytokines immediately following lens injury that could drive postsurgical flare, and suggest that inflammation may be a major player in the onset of lens-associated fibrotic disease PCS.

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).