ex-vivo-corneal-organ-culture-model-for-wound-healing-studies

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 ...

SUNY Upstate Medical University

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 β (TGFβ) 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 β1 and β5 in cell lysates, whereas recombinant USP10 removes ubiquitin from these integrin subunits. Correspondingly, the loss and gain of USP10 decreases and increases, respectively, αv/β1/β5 protein levels, without altering gene expression. Consequently, endogenous TGFβ is activated and the fibrotic markers alpha-smooth muscle actin (α-SMA) and cellular fibronectin (FN-EDA) are induced. Blocking either TGFβ signaling or cell-surface αv integrins after USP10 overexpression prevents or reduces fibrotic marker expression. Finally, silencing of USP10 in an  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.

The deubiquitylase USP10 regulates integrin β1 and β5 and fibrotic wound healing.

Exfoliation syndrome (XFS) is an age-related disease involving the deposition of aggregated fibrillar material (exfoliation material) at extracellular matrices in tissues that synthesize elastic fibers. Its main morbidity is in the eye, where exfoliation material accumulations form on the surface of the ciliary body, iris, and lens. Exfoliation glaucoma (XFG) occurs in a high proportion of persons with XFS and can be a rapidly progressing disease. Worldwide, XFG accounts for about 25% of open-angle glaucoma cases. XFS and XFG show a sharp age-dependence, similarly to the many age-related diseases classified as aggregopathies. Progress in understanding the cellular bases for XFS/XFG has been slowed by a lack of experimental models. Working with primary human tenon fibroblasts (TF) derived from trabeculectomies of XFG patients and age-matched primary open-glaucoma controls, we found that TF from XFG cells display many of the functional features observed in cells from other protein aggregate diseases, such as Parkinson, Alzheimer, Huntington, and age-related macular degeneration. We have documented defects in lysosomal positioning, microtubule organization, autophagy processing rate, and mitochondrial health. In regard to failure of lysosomal and autophagosome positioning in XFG cells, we have found that XFG TF are unable to establish the transnuclear microtubule organizing center that is required for efficient centripetal vesicular locomotion along microtubules. In regard to potential sources of the autophagy malfunction, we have directed our attention to a potential role of the lysyl oxidase-like 1 protein (LOXL1), the elastic fiber catalyst that displays variant-dependent association with risk for XFG. Our experiments show that (a) in XFG cells, a substantial fraction of LOXL1 is processed for degradation by the autophagic system; (b) most of the LOXL1 N-terminus domain exists in a highly disordered state, a condition known to greatly increase the frequency of polypeptide misfolding; (c) that maximum misfolding occurs at amino acid position 153, the location of the high risk variant G153D; and (d) that replacement of glycine (G) by aspartate (D) there results in a substantial decrease in disorder within the 20 amino acid surrounding domain. Finally, we show that clusterin, a protein that can be induced by the presence of intracellular, or extracellular aggregates, is uniformly overexpressed in XFG TF. The implications of our results for a theory relating XFG to cellular aggregopathy are discussed.

Article	Year
The deubiquitylase USP10 regulates integrin β1 and β5 and fibrotic wound healing. Journal of cell science\| PubMed ID: 28851806	2017
Exfoliation Syndrome: A Disease of Autophagy and LOXL1 Proteopathy. Journal of glaucoma\| PubMed ID: 29547474	2018

Audrey M. Bernstein

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