The deubiquitylase USP10 regulates integrin ß1 and ß5 and fibrotic wound healing.

Scarring and fibrotic disease result from the persistence of myofibroblasts characterized by high surface expression of αv integrins and subsequent activation of the transforming growth factor ß (TGFß) proteins; however, the mechanism controlling their surface abundance is unknown. Genetic screening revealed that human primary stromal corneal myofibroblasts overexpress a subset of deubiquitylating enzymes (DUBs), which remove ubiquitin from proteins, preventing degradation. Silencing of the DUB USP10 induces a buildup of ubiquitin on integrins ß1 and ß5 in cell lysates, whereas recombinant USP10 removes ubiquitin from these integrin subunits. Correspondingly, the loss and gain of USP10 decreases and increases, respectively, αv/ß1/ß5 protein levels, without altering gene expression. Consequently, endogenous TGFß is activated and the fibrotic markers alpha-smooth muscle actin (α-SMA) and cellular fibronectin (FN-EDA) are induced. Blocking either TGFß signaling or cell-surface αv integrins after USP10 overexpression prevents or reduces fibrotic marker expression. Finally, silencing of USP10 in an ex vivo cornea organ culture model prevents the induction of fibrotic markers and promotes regenerative healing. This novel mechanism puts DUB expression at the head of a cascade regulating integrin abundance and suggests USP10 as a novel antifibrotic target.

Isolation and Characterization of Cutaneous Epithelial Stem Cells.

The outer layer of mammalian skin is a multilayered epithelium that perpetually renews multiple differentiated lineages. During homeostasis, the maintenance of skin epithelial turnover is ensured by regionalized populations of stem cells that largely remain dedicated to distinct epithelial lineages including squamous, follicular, sebaceous, Merkel, and sweat glands. Cutting edge developments in this field have focused on: (1) stem cell activation cues derived from a number of extrinsic sources including neurons, dermal fibroblasts and adipocyte, and immune cells; and (2) characterization of epithelial stem cell homeostasis via hierarchical versus stochastic paradigms. The techniques outlined in this chapter are designed to facilitate such studies and describe basic procedures for cutaneous stem cell isolation and purification, which are based on leveraging their unique expression of surface proteins for simultaneous targeting and purifying of multiple subpopulations in adult skin. In addition, protocols for assessment of in vitro and ex vivo progenitor capacity as well as techniques to visualize progenitor populations in whole skin are discussed.

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies.

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

Autophagy and Mitochondrial Dysfunction in Tenon Fibroblasts from Exfoliation Glaucoma Patients.

PURPOSE: To test the hypothesis that autophagy dysfunction is involved in exfoliation syndrome (XFS), a systemic disorder of extracellular elastic matrices that causes a distinct form of human glaucoma. METHODS: Fibroblasts derived from tenon tissue discards (TFs) from filtration surgery to relieve intraocular pressure in XFS patients were compared against age-matched TFs derived from surgery in primary open-angle glaucoma (POAG) patients or from strabismus surgery. Differential interference contrast light, and electron microscopy were used to examine structural cell features. Immunocytochemistry was used to visualize LOXL1 and Fibulin-5, lysosomes, endosomes, Golgi, and microtubules. Light scatter, Cyto-IDTM and JC1 flow cytometry were used to measure relative cell size, autophagic flux rate and mitochondrial membrane potential (MMPT), respectively. Enhanced autophagy was induced by serum withdrawal. RESULTS: In culture, XFS-TFs were 1.38-fold larger (by light scatter ratio, p = 0.05), proliferated 42% slower (p = 0.026), and were morphologically distinct in 2D and 3D culture compared to their POAG counterparts. In extended 3D cultures, XFS-TFs accumulated 8-10 times more Fibulin-5 than the POAG-TFs, and upon serum withdrawal, there were marked deficiencies in relocation of endosomes and lysosomes to the perinuclear area. Correspondingly, the XFS-TFs displayed significant accumulation of the autophagasome marker LC3 II (3.9 fold increase compared to POAG levels, p = 0.0001) and autophagic flux rate as measured by Cyto-ID dye was 53% lower in XFS-TFs than in POAG-TFs (p = 0.01), indicating reduced clearance of autophagasomes. Finally the percent of cells with diminished MMPT was 3-8 times larger in the XFS-TFs than in POAG-TFs (p = 0.02). CONCLUSIONS: Our results provide for the first time a link between XFS pathology to autophagy dysfunction, a major contributor to multiple age related diseases systemically throughout the body, in the brain and in the retina. A diminished capacity for degradation of denatured protein and aging cellular organelles may underpin the development of extracellular protein aggregates in XFS.

Sexual selection is influenced by both developmental and adult environments.

Sexual selection is often assumed to be strong and consistent, yet increasing research shows it can fluctuate over space and time. Few experimental studies have examined changes in sexual selection in response to natural environmental variation. Here, we use a difference in resource quality to test for the influence of past environmental conditions and current environmental conditions on male and female mate choice and resulting selection gradients for leaf-footed cactus bugs, Narnia femorata. We raised juveniles on natural high- and low-quality diets, cactus pads with and without ripe cactus fruits. New adults were again assigned a cactus pad with or without fruit, paired with a potential mate, and observed for mating behaviors. We found developmental and adult encounter environments affected mating decisions and the resulting patterns of sexual selection for both males and females. Males were not choosy in the low-quality encounter environment, cactus without fruit, but they avoided mating with small females in the high-quality encounter environment. Females were choosy in both encounter environments, avoiding mating with small males. However, they were the choosiest when they were in the low-quality encounter environment. Female mate choice was also context dependent by male developmental environment. Females were more likely to mate with males that had developed on cactus with fruit when they were currently in the cactus with fruit environment. This pattern disappeared when females were in the cactus without fruit environment. Altogether, these results experimentally demonstrate context-dependent mate choice by both males and females. Furthermore, we demonstrate that simple, seasonal changes in resources can lead to fluctuations in sexual selection.

Expression of insulin-like growth factor 2 receptor in corneal keratocytes during differentiation and in response to wound healing.

PURPOSE: Insulin-like growth factor 2 receptor (IGF2R) associates with ligands that influence wound healing outcomes. However, the expression pattern of IGF2R and its role in the cornea is unknown. METHODS: Human keratocytes were isolated from donor corneas. Fibroblasts (fibroblast growth factor 2 [FGF2]-treated) or myofibroblasts (TGF-ß1-treated) were analyzed for IGF2R and α-smooth muscle actin (α-SMA) expression by Western blotting and immunolocalization. Mouse corneas were wounded in vivo and porcine corneas ex vivo. The IGF2R and α-SMA protein expression were visualized and quantified by immunohistochemistry. The IGF2R gene expression in human corneal fibroblasts was knocked-down with targeted lentiviral shRNA. RESULTS: The IGF2R is expressed in epithelial and stromal cells of normal human, mouse, and porcine corneas. The IGF2R increases (11.2 ± 0.4-fold) in the epithelial and (11.7 ± 0.9-fold) stromal layers of in vivo wounded mouse corneas. Double-staining with α-SMA- and IGF2R-specific antibodies reveals that IGF2R protein expression is increased in stromal myofibroblasts in the wounded cornea relative to keratocytes in the normal cornea (11.2 ± 0.8-fold). Human primary stromal keratocytes incubated with FGF2 or TGF-ß1 in vitro demonstrate increased expression (2.0 ± 0.4-fold) of IGF2R in myofibroblasts relative to fibroblasts. Conversion of IGF2R shRNA-lentiviral particle transduced corneal fibroblasts to myofibroblasts reveals a dependence on IGF2R expression, as only 40% ± 10% of cells transduced converted to myofibroblasts compared to 86% ± 3% in control cells. CONCLUSIONS: The IGF2R protein expression is increased during corneal wound healing and IGF2R regulates human corneal fibroblast to myofibroblast differentiation.

Conspecific and heterospecific cues override resource quality to influence offspring production.

Animals live in an uncertain world. To reduce uncertainty, animals use cues that can encode diverse information regarding habitat quality, including both non-social and social cues. While it is increasingly appreciated that the sources of potential information are vast, our understanding of how individuals integrate different types of cues to guide decision-making remains limited. We experimentally manipulated both resource quality (presence/absence of cactus fruit) and social cues (conspecific juveniles, heterospecific juveniles, no juveniles) for a cactus-feeding insect, Narniafemorata (Hemiptera: Coreidae), to ask how individuals responded to resource quality in the presence or absence of social cues. Cactus with fruit is a high-quality environment for juvenile development, and indeed we found that females laid 56% more eggs when cactus fruit was present versus when it was absent. However, when conspecific or heterospecific juveniles were present, the effects of resource quality on egg numbers vanished. Overall, N. femorata laid approximately twice as many eggs in the presence of heterospecifics than alone or in the presence of conspecifics. Our results suggest that the presence of both conspecific and heterospecific social cues can disrupt responses of individuals to environmental gradients in resource quality.

TRPV1 potentiates TGFß-induction of corneal myofibroblast development through an oxidative stress-mediated p38-SMAD2 signaling loop.

Injuring mouse corneas with alkali causes myofibroblast expression leading to tissue opacification. However, in transient receptor potential vanilloid 1 channel (TRPV1-/-) knockout mice healing results in transparency restoration. Since TGFß is the primary inducer of the myofibroblast phenotype, we examined the mechanism by which TRPV1 affects TGFß-induced myofibroblast development. Experiments were performed in pig corneas and human corneal fibroblasts (HCFs). Immunohistochemical staining of α-smooth muscle actin (α-SMA) stress fibers was used to visualize myofibroblasts. Protein and phosphoprotein were determined by Western blotting. siRNA transfection silenced TRPV1 gene expression. Flow cytometry with a reactive oxygen species (ROS) reporting dye analyzed intracellular ROS. [Ca2+]I was measured by loading HCF with fura2. In organ cultured corneas, the TRPV1 antagonist capsazepine drastically reduced by 75% wound-induced myofibroblast development. In HCF cell culture, TGF-ß1 elicited rapid increases in Ca2+ influx, phosphorylation of SMAD2 and MAPKs (ERK1/2, JNK1/2 and p38), ROS generation and, after 72 hrs myofibroblast development. SMAD2 and p38 activation continued for more than 16 h, whereas p-ERK1/2 and p-JNK1/2 waned within 90 min. The long-lived SMAD2 activation was dependent on activated p38 and vice versa, and it was essential to generate a > 13-fold increase in α-SMA protein and a fully developed myofibroblast phenotype. These later changes were markedly reduced by inhibition of TRPV1 or reduction of the ROS generation rate. Taken together our results indicate that in corneal derived fibroblasts, TGFß- induced myofibroblast development is highly dependent on a positive feedback loop where p-SMAD2-induced ROS activates TRPV1, TRPV1 causes activation of p38, the latter in turn further enhances the activation of SMAD2 to establish a recurrent loop that greatly extends the residency of the activated state of SMAD2 that drives myofibroblast development.