Hevin Plays a Pivotal Role in Corneal Wound Healing

<div><p>Background</p><p>Hevin is a matricellular protein involved in tissue repair and remodeling via interaction with the surrounding extracellular matrix (ECM) proteins. In this study, we examined the functional role of hevin using a corneal stromal wound healing model achieved by an excimer laser-induced irregular phototherapeutic keratectomy (IrrPTK) in hevin-null (hevin^-/-) mice. We also investigated the effects of exogenous supplementation of recombinant human hevin (rhHevin) to rescue the stromal cellular components damaged by the excimer laser.</p> <p>Methodology/Principal Findings</p><p>Wild type (WT) and <i>hevin</i>^<i>-/-</i> mice were divided into three groups at 4 time points- 1, 2, 3 and 4 weeks. Group I served as naïve without any treatment. Group II received epithelial debridement and underwent IrrPTK using excimer laser. Group III received topical application of rhHevin after IrrPTK surgery for 3 days. Eyes were analyzed for corneal haze and matrix remodeling components using slit lamp biomicroscopy, in vivo confocal microscopy, light microscopy (LM), transmission electron microscopy (TEM), immunohistochemistry (IHC) and western blotting (WB). IHC showed upregulation of hevin in IrrPTK-injured WT mice. <i>Hevin</i>^<i>-/-</i> mice developed corneal haze as early as 1-2 weeks post IrrPTK-treatment compared to the WT group, which peaked at 3-4 weeks. They also exhibited accumulation of inflammatory cells, fibrotic components of ECM proteins and vascularized corneas as seen by IHC and WB. LM and TEM showed activated keratocytes (myofibroblasts), inflammatory debris and vascular tissues in the stroma. Exogenous application of rhHevin for 3 days reinstated inflammatory index of the corneal stroma similar to WT mice. </p> <p>Conclusions/Significance</p><p>Hevin is transiently expressed in the IrrPTK-injured corneas and loss of hevin predisposes them to aberrant wound healing. <i>Hevin</i>^<i>-/-</i> mice develop early corneal haze characterized by severe chronic inflammation and stromal fibrosis that can be rescued with exogenous administration of rhHevin. Thus, hevin plays a pivotal role in the corneal wound healing.</p> </div

Light and Transmission electron microscopy in wild type and IrrPTK-treated corneas.

<p><i>Hevin^-/-</i> mice (Figure 11F) showed distinct cellular activity and epithelial cellular disorganization after IrrPTK surgery compared to the WT corneas (Figure 11B). Large vacuolated keratocytes observed in transmission electron micrographs of <i>Hevin^-/-</i> mice (Figure 11G-H) compared to the WT corneas (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081544#pone-0081544-g011" target="_blank">Figure 11C-D</a>). </p

Mean corneal curvature measured by keratometer and mean spherical error measured by refractometer (n = 7).

a<p>PR = post-ReLEx.</p>b<p>PLR = post-lenticule re-implantation.</p>c<p>D = diopter.</p>d<p><i>p</i> values relative to the keratometry before ReLEx.</p>e<p><i>p</i> values relative to the spherical error before ReLEx.</p

<i>Hevin^-/-</i> mice prolonged the inflammatory state of the corneal fibroblasts.

<p>WT mouse cornea showed fewer CD11b in weeks 1 and 2 following IrrPTK (B,C), which reduced progressively in week 3 and 4 (D,E) during wound healing. <i>Hevin^-/-</i> mice showed significantly large number of CD11b+ cells in the naïve state (F), with persistent increase over 4-weeks post-op (G-J). Application of rhHevin (K-N) reduced CD11b+ve inflammatory in <i>Hevin^-/-</i> mice by week 4 (N). Western blot analysis showed ~2-fold increase in CD11b expression in <i>Hevin^-/-</i> mice compared to the naïve WT corneas and further increase at 4 weeks after IrrPTK surgery, which can be reduced to near normal levels after rhHevin treatment (O). ***, P<0.001. Scale bar = 25μm.</p

<i>Hevin^-/-</i> mice exhibit excessive cell death in the corneal stroma after IrrPTK surgery.

<p>WT naïve mouse showed no TUNEL+ve cells in the corneal stroma (A), whereas few apoptotic cells (^) were seen in naïve state of <i>Hevin^-/-</i> mice (B,C). IrrPTK-treatment significantly increased cell death in <i>Hevin^-/-</i> corneal stroma (E) compared to the fewer TUNEL+ve cells observed in WT stroma (D). rhHevin prevented these cells from undergoing cell death after IrrPTK (F). Scale bar = 25μm.</p

Hevin deletion leads to early stage fibrosis in mouse cornea.

<p>Activated keratocytes produced during the corneal wound healing results in the corneal fibrosis, which can be detected using immunostaining with αSMA protein. WT and <i>Hevin^-/-</i> naive corneas showed negative expression of αSMA protein (A,F). IrrPTK-surgery results in early development of αSMA-positive cells (^) as early as Week 1 and 2 in <i>Hevin^-/-</i> mouse (G, H), with further increase in week 3 and 4 (I, J). On the other hand, WT mouse cornea showed nominal increase in the early stages of wound healing (B,C) but peaked in week 3 and 4 (D, E). Exogenous treatment of rhHevin to the IrrPTK tissues at week 4 significantly reduced αSMA-positive cells in both WT and <i>Hevin^-/-</i> mice as observed by immunohistochemistry (L,N) and western blotting (O). *, P<0.001. Scale bar = 25μm.</p

Expression ofα-smooth muscle actin (α-SMA) in the post-operative central corneas and peripheral flaps.

<p>(A–D) α-SMA (green), a marker of myofibroblasts, was not present in the central corneas on week 8 and 16 after both ReLEx and refractive lenticule re-implantation. (E–H) α-SMA (green) was expressed at the flap periphery and co-localized with F-actin (red) subepithelially on week 8 post-ReLEx and lenticule re-implantation, but was absent 16 weeks after both surgical procedures. In pane A–H, α-SMA (green) was double immunostained with F-actin marker (red), phalloidin. Nuclei were counterstained using DAPI (blue). Arrowheads indicate the location of the laser incision site or lenticular interface. PR: post-ReLEx, PLR: post-lenticule re-implantation. Scale bar: 50 µm.</p

Loss of hevin results in excessive accumulation of Collagen I and Collagen IV in corneal stroma.

<p>Immunostaining of Col I showed even distribution of the ECM protein across the corneal stroma in both WT (A-E) and <i>Hevin^-/-</i> (F-J) mouse. Marginal increase in Col I expression (^) observed in anterior stroma of IrrPTK samples (C, H, I). rhHevin treatment did not show any adverse effects on Col I staining (K-N). Western blot (O) confirm the increase of Col I expression in IrrPTK group. Col IV initially absent in naïve WT (A’) and <i>Hevin^-/-</i> (F’) mouse showed increased and persistence expression (^) post-IrrPTK (B’-E’, G’-J’). rhHevin treatment reduced Col IV production in the WT (L’) but not in the <i>Hevin^-/-</i> mice (N’). Similar observations were made in Western Blot analysis (O’). *, P<0.001. Scale bar = 25μm.</p

In vivo slit lamp biomicroscopy in WT and <i>Hevin^-/-</i> mouse.

<p>Naïve WT (A-D) and <i>Hevin^-/-</i> (A’-D’) mice corneas served as control without any treatment. IrrPTK-surgery in WT mouse developed corneal haze from Week 2 (F) but more prominently seen in weeks 3-4 (G-H). In <i>Hevin^-/-</i> mouse, IrrPTK-treatment began to develop haze as early as Week 1 (E’), with increasing severity from Weeks 2-4 (F’-H’). In both WT and <i>hevin</i>^<i>-/-</i>, addition of exogenous rhHevin significantly reduced corneal haze and neovascularization in the zone of laser ablation (I-L; I’-L’).</p

Flow diagram describing the experimental plan and data collection for the study.

<p>Eight-10 weeks old WT and <i>hevin^-/-</i> mice were divided into 3 groups: (I) Naïve, (II) Irregular Phototherapeutic Keratectomy (IrrPTK), (III) IrrPTK + rhHevin. Group I without any treatment served as naïve control. Groups II and III underwent IrrPTK after epithelial debridement. rhHevin (10mg/Kg) was topically administered to Group III animals every 24 hours for 3 days until epithelial closure. Each group consists of 4 time points at 1, 2, 3, 4 weeks with 6-10 eyes at each time point. Slit lamp and confocal biomicroscopy were used for clinical follow-up every week for 4 weeks. At 4 weeks, mice were euthanized and eyes were collected at each time point for biochemical analysis using TUNEL assay, immunohistochemistry, western blot and transmission electron microscopy. </p