KLEIP Deficiency in Mice Causes Progressive Corneal Neovascular Dystrophy

Abstract

Purpose.: The BTB-kelch protein KLEIP/KLHL20 is an actin binding protein that regulates cell-cell contact formation and cell migration. The aim of our study was to characterize KLEIP's function in ocular health and disease in mice.

Methods.: KLEIP^−/− mice were generated, and corneas were examined histologically and stained for keratin-1, loricrin, keratin-12, keratin-14, CD31, LYVE-1, F4/80, E-cadherin, and Ki67. Corneal abrasions were performed after eyelid opening.

Results.: Corneas of KLEIP^+/+ and KLEIP^−/− mice were indistinguishable at birth. After eyelid opening corneal epithelial hyperplasia started to manifest in KLEIP^−/− mice, showing a progressive epithelial metaplasia leading to total corneal opacity. In KLEIP^−/− mice the initial stratified squamous corneal epithelium was altered to an epidermal histo-architecture showing several superficial keratinized cells, cell infiltrations into the stroma, and several apoptotic cells. Skin markers keratin 1 and loricrin were positive, and surface disease was accompanied by deep stromal vascularization. Expression analysis for E-cadherin in KLEIP^−/− corneas showed acellular areas in the squamous epithelium, indicating a progressive fragile corneal integrity. Removal of the virgin epithelium accelerated strongly development of the epithelial and stromal alterations, identifying mechanical injuries as the major trigger for corneal dystrophy formation and scarification in KLEIP^−/− mice.

Conclusions.: The data identify KLEIP as an important molecule regulating corneal epithelial integrity.

Introduction

Progredient opacity of the cornea due to stromal reorganization, keratinization of the corneal epithelium and neovascularization, is a major reason for blindness in man. ^1–3 In humans, only few genes have been identified so far that are linked to corneal dystrophy formation. For example, genomic mutations in the keratin-3 and keratin-12 genes are linked to an epithelial dystrophy phenotype, called Meesmann dystrophy, ⁴ and gene mutations in the transforming growth factor-β-induced (TGFBI) gene can cause corneal dystrophies, such as Reis-Bücklers dystrophy, Thiel-Behnke dystrophy, and granular type 1-, granular type 2-, and lattice type 1-dystrophy in humans. ⁵  

Genetic animal models to study onset, progression, and therapeutic intervention of corneal dystrophies currently are very limited. For example, keratin-12, a protein that forms intermediate filaments in epithelial cells, is expressed specifically in the corneal epithelium in mice. ⁶ Its gene silencing resulted in a fragile epithelium serving as a mouse model for Meesmann corneal dystrophy. ⁷ In addition to the keratins, genetic alterations in different proteoglycans can induce corneal malformations in mice. For example deletion of keratocan, a cornea-specific keratan sulphate proteoglycan, resulted in a thinner corneal stroma ⁸ and lumican, which gene inactivation in mice showed a cloudy and thin corneal stroma. ⁹ Another class of molecules that can cause corneal dystrophies in mice are transcription factors. Corneal-specific overexpression of the transcription factor Pax6 induced an abnormal cornea with altered epithelial cell morphology and neovascularization; ¹⁰ inactivation of zinc finger transcription factor Zeb1 in mice correlated with the posterior polymorphous corneal dystrophy, ¹¹ and tissue-specific deletion of transcription factor Pbx1 in the corneal epithelium resulted in a corneal dystrophy and clouding. ¹² Moreover, genetic inactivation of the transcriptions factors AP-2alpha, Klf4, Klf5, and Cited2 also induced corneal defects in mice. ^13–16 Together, these mouse models identified few genes that can cause corneal alterations in mice, but it is unclear mostly whether these genes can cause corneal dystrophies in patients as well. ¹⁷ Moreover, based on the diversity of corneal dystrophies in patients, several potential molecular regulators for corneal dystrophy formation still are not yet identified. 

The BTB-kelch protein KLEIP (Kelch-like ECT2 interacting protein), also named KLHL20, has been identified first in MDCK cells where it co-localizes transiently with F-actin during the process of cell-cell contact induction. ¹⁸ Recruitment of KLEIP to cell adhesion sites depends on Rac1 activation and requires E-cadherin. ¹⁸ In addition, KLEIP is induced under hypoxic conditions ¹⁹ that can stimulate RhoA signaling during endothelial cell migration and in neurite outgrowth. ^20,21 Recent findings also identified KLEIP as a molecule acting together with the wnt/beta-catenin signaling pathway, which may regulate skin thickness. ²² In summary, the data highlight KLEIP as an important molecule in cell-cell contact formation, regulation of the cellular architecture, and cellular reorganization, and identified KLEIP as a factor regulating cell migration. Yet, its function in vivo still is unknown. 

To address KLEIP's function in vivo, we have generated KLEIP knockout mice, and identified KLEIP as an essential component for the maintenance of corneal integrity. KLEIP^−/− mice developed progressively a corneal dystrophy due to epithelial fragility, which was induced mainly by mechanical injuries of the cornea. Therefore, KLEIP-deficient mice represent a unique genetic model to study onset and progression of corneal damages in mice. 

Methods

Generation and Genotyping of KLEIP^−/− Mice

The 129SvEv embryonic stem cell clone XF202 (BayGenomics), carrying the β-geo (beta galactosidases and neomycin resistance) encoding vector pGT2Lxf in the KLEIP locus, was injected into C57/BL6 blastocytes, crossed, and maintained in C57/BL6 Ola mice. Currently, mice are in the F10 generation and named as B6.129-KLEIP ^{tm/ Mhm}. For genotyping, three primers were used, namely primer S1, which binds in intron 2 of genomic KLEIP; primer A1, which binds in the gene trap vector, and primer A2, which binds in exon 3 of KLEIP. Primer pair S1 and A2 generated the wild type band of 1349 base pair (bp) length, while primer pair S1 and A1 generated the transgenic signal of 591 bp length. Expression of lacZ was analyzed using primer pair L1 and L2 (Supplemental Fig. 1). For primer sequences see below. Mice were kept under specific pathogen-free conditions according to the animal facility regulations of the Medical Faculty Mannheim, Heidelberg University. All animal experiments were approved by the Regierungspräsidium Karlsruhe (protocol numbers 35-9185.83, 35-9185.81/G-82/11, and I-07/03), and are in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.  

Figure 1. 

 

KLEIP^−/− mice developed a corneal dystrophy. (A) Corneal dystrophy phenotype in a KLEIP^−/− mouse as indicated by a corneal opacity. A KLEIP^+/+ litter mate served as healthy control. (B) Incidence of corneal dystrophy in KLEIP^−/− mice. KLEIP^−/− mice started to manifest a corneal dystrophy three weeks after birth. At 16 weeks after birth, 90% of KLEIP^−/− mice had a severe corneal dystrophy. KLEIP^+/+ mice did not have a corneal dystrophy (n = 15 per group). (C) Top: Biopsies of KLEIP^+/+ and KLEIP^−/− corneal epithelial cells directly after abrasion showed expression of lacZ as an indicator for KLEIP expression. Bottom: RT-PCR analysis in mouse corneas (n = 9 pooled samples for each genotype) for KLEIP and lacZ expression. TBP served as a loading control. KLEIP^−/− _dys: KLEIP^−/− mice with corneal dystrophy. Scale bar: 50 μm.

Antibodies and Histologic Reagents

The following antibodies were used for the study: anti-mouse CD31, clone MEC 13.3 (BD Pharmingen, Heidelberg, Germany); anti-mouse Endomucin, clone V.1A7, and anti-mouse keratin-12 (Santa Cruz Biotechnology, Heidelberg, Germany); anti-mouse E-cadherin, clone ECCD-2 (Invitrogen, Darmstadt, Germany); anti-mouse LYVE-1 (RELIATech, Wolfenbüttel, Germany); anti-mouse F4/80, clone CI: A3-1, and donkey-anti-rabbit FITC (Dianova, Hamburg, Germany); anti-mouse keratin-1, anti-mouse keratin-14, and anti-mouse loricrin (Covance, München, Germany); anti-human Ki67 (Novocastra, Wetzlar, Germany); goat-anti-rat Alexa 546 and goat-anti-rabbit Alexa 488 (Molecular Probes, Darmstadt, Germany); HRP-conjugated antibodies and ABC-kit (VECTOR Laboratories, Dossenheim, Germany); and DAPI, HE staining solution, Sudan black B solution, Trichrome Stain (Masson) Kit, and anti-human SMA, clone 1A4 (Sigma-Aldrich, München, Germany). 

Histochemistry

Eyes from KLEIP^+/+ and KLEIP^−/− mice were enucleated, and corneas were isolated via cutting around the corneoscleral limbal ring. Eyelids were dissected using a scalpel. Tissues were fixed in 4% formaldehyde (PFA) or in Zn-fixative overnight at 4°C, or immediately frozen in liquid nitrogen, embedded in Tissue TEC (ornithine carbamoyltransferase [OCT]-medium) or dehydrated after PFA/Zn-fixation using ethanol-series (70%, 80%, 90%, and 100% ethanol, and 100% and 100% xylol, 5 minutes each), and embedded in paraffin. Both cryo and paraffin sections were cut to a size of 6-8 μm each. To analyze tissue structure of corneas and glands, mouse tissues were stained using Mayer's hematoxylin & eosin (HE), trichrome stain (keratin and collagen staining), and Sudan black (lipid staining). Apoptotic corneal cells were labeled using the TUNEL staining kit from Chemicon (ApopTag Red In Situ Apoptosis Detection Kit; Chemicon, Darmstadt, Germany).  

Isolation of mRNA, RT-PCR, and Primer Sequences

Eyes from KLEIP^+/+ and KLEIP^−/− mice were enucleated, and corneas were removed and mechanically homogenized. Total RNA was isolated using the RNeasy Mini Kit, (Qiagen, Hilden, Germany) followed by RT-PCR using the Superscript II RT Kit (Promega, Mannheim, Germany). PCRs were performed under the following conditions: 94°C for 3 minutes, 35× 94°C for 45 seconds, 58°C for 30 seconds, 72°C for 1:30 minutes, and a final extension at 72°C for 10 minutes. The following primers were used: 

• TBP :  
  forward 5′ GGA CCA GAA CAA CAG CCT TCC; reverse 5′ CAT GAT GAC TGC AGC AAA TCG
• lacZ :  
  forward (L1) 5′ TAT CGA TGA GCG TGG TGG TTA TGC C; reverse (L2) 5′ GCG CGT ACA TCG GGC AAA TAA TAT C
• KLEIP :  
  forward 5′ GTG ATG GCC TGG GTC AAA TAC; reverse 5′ GAG GAT CCA TCA TGG CCG CCT AC
• Primer  
  for genotyping: forward (S1) 5′ CAA GTG CGA TTG AAG CAT CC
• Primer  
  for genotyping: reverse (A1) 5′ ACC TGG CTC CTA TGG GAT AG
• Primer  
  for genotyping: reverse (A2) 5′ AAA CAT TGC TCG GAA GTA GG

lacZ Staining of Transgenic Mice

LacZ staining was performed as described. ²³ In brief, tissues were fixed in LacZ-fix solution containing 0.2% glutaraldehyde at 4°C overnight, washed 3 times in LacZ wash buffer, and incubated overnight at 37°C in lacZ stain containing 1 mg/mL X-Gal (BIOMOL prod.-No. 02249, Hamburg, Germany), 2.1 mg/mL K-ferrOcyanide, and 1.6 mg/mL K-ferricyanide. Then, 16 hours later tissues were washed 3 times in PBS, and postfixed in 2% PFA/0.1% glutaraldehyde/PBS overnight at 4°C. 

Abrasions

Abrasions were performed directly after eyelid opening to monitor epithelial wound healing capacity in KLEIP^+/+ and KLEIP^−/− mice. Mice were anesthetized using isoflurane (Baxter, Unterschleißheim, Germany) and, for postoperative analgesia, treated mice received 200 mg/kg body weight Metamizol (Ratiopharm, Ulm, Germany). For wounding, the corneal epithelium was moistened via short-time incubation using a 2.5% ethanol/water solution. Afterwards, approximately 40% of the central corneal epithelium was removed via a hockey spatula. Cornea epithelial wounding was monitored using Thilorbin (Alcon, Freiburg, Germany), and dystrophy development was assessed after 1, 6, and 12 hours, and 1, 3, 7, and 14 days after abrasion. 

Results

Generation of KLEIP-Deficient Mice

To dissect the function of the BTB-kelch protein KLEIP/KLHL20 during vertebrate development, the KLEIP gene was disrupted in mice. The genetic modified embryonic stem cell line XF202, targeting the KLEIP gene, was used that was generated by the gene trap technology (Supplemental Fig. 1A, B, C, D). XF202 cells were injected into blastocysts and transferred into pseudo-pregnant foster mothers. Born chimeras were crossed into C57/Bl6 Ola mice, and a genotyping protocol was established (Supplemental Fig. 1E). KLEIP-deficient embryos and mice initially were analyzed for KLEIP expression by RT-PCR analysis, which showed the expected loss of KLEIP expression (Supplemental Fig. 1F). To analyze the effect of KLEIP knock down on viability and fertility in mice, the ratio of KLEIP^+/+, KLEIP^+/−, and KLEIP^−/− mice was determined. Interestingly, we observed a reduced number for KLEIP^−/− mice at P28 (wild-type mice versus heterozygous mice versus homozygous mice: 33% versus 54% versus 13%), showing that approximately 50% of KLEIP^−/− mice die until day 28 of postnatal development. Yet, reasons for this reduced viability of KLEIP^−/− mice presently are unclear. KLEIP^−/− mice that survived beyond P28 were fertile.  

KLEIP Deficiency in Mice Progressively Resulted in a Corneal Dystrophy, Which Is Associated with an Epithelial Hyperplasia and an Altered Corneal Epithelial Cell Differentiation

KLEIP^−/− mice displayed, starting earliest in the third postnatal week, a whitish corneal opacification on both eyes that was macroscopically visible (Fig. 1A). The onset of this pathology varied between weeks 3 and 16, when 90% of the surviving animals had a corneal opacification (Fig. 1B). Expression analysis in KLEIP^+/+ and KLEIP^−/− eyes confirmed expression of KLEIP or lacZ as a marker for KLEIP within the corneal epithelium, suggesting a direct function of KLEIP in the mouse cornea (Fig. 1C). In addition, weak expression of lacZ also was observed in the retina and in the ciliary marginal zone (not shown). 

To characterize in detail onset and progression of corneal dystrophy in KLEIP^−/− mice, corneas of KLEIP^+/+ and KLEIP^−/− mice were analyzed histologically at different time points. While KLEIP^+/+ mice showed a normal corneal anatomical structure consisting of the epithelium, stroma, Descemet's membrane, and the endothelium, all KLEIP^−/− mice had a progressive epithelial metaplasia leading to a corneal opacity (Fig. 2). Histologic analysis of the altered corneas in KLEIP^−/− mice performed at different time points revealed a reproducible sequence. Initially, we observed epithelial hyperplasia. This was followed by metaplasia with a reorganization of the original non-keratinized stratified squamous epithelium towards a keratinized cell population on the surface (Figs. 2, 3A–C). This was accompanied by focal epithelial indentations into the corneal stroma, and a massive thickening of the epithelial layer (Fig. 2). The final stage of the disease resulted in a situation in which the tissue displayed an epidermal organization pattern with a keratinized superficial layer (Figs. 2, 3A–C). Immunostaining of the corneal epithelium in KLEIP^−/− mice revealed Ki67-positive, proliferating cells within the epithelium and stroma (Fig. 3D), and altered expression of skin- and cornea-specific keratins. ²⁴ While KLEIP^+/+ corneas expressed only keratin-12 and keratin-14, KLEIP-deficient corneas expressed strongly skin-specific keratin-1, loricrin, and keratin-14, but only weakly keratin-12, indicating an altered differentiation in KLEIP-deficient corneas from a cornea-like into a skin-like phenotype (Figs. 3B, C). 

Figure 2. 

 

Progression of corneal dystrophy formation in KLEIP^−/− mice. Progression of corneal dystrophy formation was monitored during the first four months of life in five KLEIP^+/+ and five KLEIP^−/− mice. Macroscopic (top) and histologic (bottom) disease progression is shown in one eye of a KLEIP^−/− mouse (right) in comparison to a KLEIP^+/+ mouse (left) for each time point. During the whole observation period eyes in KLEIP^+/+ mice remained healthy (left). KLEIP^−/− deficient mice were born with normal functional eyelids and corneas, as can be seen after eyelid opening (week 3). During the following weeks KLEIP^−/− mice had a corneal plaque (as of week 8), which was vascularized at weeks 10 to 12 (top). HE stainings of corneal sections from KLEIP^−/− mice showed morphologic alterations during disease progression (bottom), cell infiltrations into the stroma (weeks 8 to 10), diffuse progressive epithelial metaplasia, and indentations into the corneal stroma (weeks 10 to 12) and corneal neovascularization (weeks 10 to 12). Highlighted areas within section “12 weeks” correspond to the higher magnification (right) showing an epithelial hyperplasia (asterisk), cell infiltrations (point), and neovascularization (arrows). Scale bar: 100 μm.

Figure 3. 

 

Corneas of KLEIP^−/− mice contained several keratinized and proliferating cells, and expressed skin-specific keratins. (A) Masson trichrome staining in corneas of KLEIP^−/− and KLEIP^+/+ mice. In comparison to corneas of KLEIP^+/+ mice, corneas of KLEIP^−/− mice showed a thickened stroma (collagen green), a thickened and keratinized epithelium (red) and cell infiltrations into the stroma (nuclei dark brown/black). (B) Corneas in KLEIP^−/− mice expressed skin-specific markers keratin-1, loricrin, and keratin-14 (brown), whereas corneas of KLEIP^+/+ mice expressed keratin-14 only. (C) Expression of cornea-specific keratin-12 (green) in KLEIP^−/− corneas was reduced strongly as compared to KLEIP^+/+ corneas. (D) In contrast to KLEIP^+/+ mice, corneas of KLEIP^−/− mice contained proliferating cells within the epithelium and stroma (Ki67 brown). Tissue sections were counterstained with HE. Scale bars: 100 μm.

Periocular Lacrimal Glands and Embryonic Development of the Eye Remained Unaltered in KLEIP^−/− Mice

Next, we analyzed histologically the meibomian glands, goblet cells, and lacrimal glands in KLEIP^−/− mice, because dysfunction of these glands finally can lead to corneal damages ²⁴ as observed in KLEIP^−/− mice. However, examination of the meibomian glands, goblet cells, and lacrimal glands did not reveal major structural differences in KLEIP^+/+ and KLEIP^−/− mice (Supplemental Fig. 2) suggesting functional tear layer-producing tissues. Furthermore, we addressed the question whether KLEIP deletion in mice may affect embryonic eye development and whether other structures in the eye in KLEIP^−/− mice are altered by the KLEIP deletion as well. To this end we assessed macroscopically and histologically KLEIP^−/− embryos and newborns ranging from age E11, E14, E18.5, and P0 for eye development. However, we could not observe obvious histologic and anatomical differences between KLEIP^−/− and KLEIP^+/+ eyes (Supplemental Fig. 3). In addition, besides corneal opacity in juvenile and adult KLEIP^−/− mice, eyes in KLEIP^−/− did not have obvious pathologic alterations in other structures of the eye: retina, choroid, sclera, iris, and lens were not affected by the KLEIP deletion (data not shown). Together, the data indicate that KLEIP deletion in mice leads to a progressive corneal dystrophy. 

Corneas in KLEIP^−/− Mice Were Highly Vascularized

Healthy corneas are optically transparent and avascular due to soluble VEGF receptor sFlt-1. ²⁵ In contrast to KLEIP^+/+ mice, which did not show any blood vessels in the corneas, we observed in KLEIP^−/− whole mount cornea samples macroscopically a substantial formation of blood vessels that originated typically in the limbal circumference, and could be visualized further by whole mount endothelial specific endomucin staining (Figs. 2, 4A). Cross-sections of KLEIP^−/− corneas identified location of blood (CD31 staining) and lymphatic vessels (LYVE-1 staining) mostly in the corneal stroma (Fig. 4B). These blood vessels were covered by pericytes as indicated by alpha SMA staining (Fig. 4C). In addition to newly formed blood vessels in the corneal stroma of KLEIP^−/− mice, we also observed several F4/80-positive cells, indicating infiltrating macrophages (Fig. 4C). The control group of KLEIP^+/+ mice, however, was completely negative for F4/80-positive cells. In conclusion, the data showed a strong induction of angiogenesis and lymphangiogenesis, and infiltrations of macrophages in KLEIP^−/− corneas. 

Figure 4. 

 

KLEIP deficiency in mice induced corneal neovascularization. (A) Brightfield image (top) and whole mount endomucin staining (middle and bottom) of KLEIP^−/− corneas showed newly formed blood vessels. Corneas of KLEIP^+/+ mice remained avascular. Scale bars: 500 μm. (B) Corneal sections of KLEIP^−/− mice identified blood vessels and lymphatic vessels (CD31 red, LYVE-1 green, DAPI blue) predominantly in the corneal stroma. Corneas of KLEIP^+/+ mice were negative for both markers. (C) Top: Blood vessel maturation in KLEIP^−/− corneas as indicated by CD31 staining (green) and alpha SMA (red) staining. Bottom: KLEIP deficiency resulted in corneal infiltrations of macrophages (F4/80 red), while macrophages were not detectable in KLEIP^+/+ corneas. Nuclei were stained with DAPI (blue). Scale bars: 100 μm.

KLEIP Regulated Corneal Epithelial Integrity and Development of Corneal Diseases in KLEIP^−/− Mice Was Intensified through Experimental Mechanical Injury

KLEIP co-localizes transiently with E-cadherin in cultured MDCK cells, suggesting that it regulates formation of cell-cell contacts. ¹⁸ Therefore, we hypothesized that E-cadherin localization could be altered in the epithelium of KLEIP-deficient corneas, and expression of E-cadherin in KLEIP^+/+ and KLEIP^−/− mice was examined. In KLEIP^+/+ corneas, both epithelial cell layers, namely the squamous cell layer and the basal cell layer, were positive for E-cadherin, and both layers displayed the physiologic architecture (Fig. 5A, left). In contrast, squamous and basal cell layers in KLEIP^−/− mice largely were disorganized and characterized by acellular areas in the squamous cell layer, suggesting loosened cell-cell contacts and an incomplete regeneration of the corneal epithelium in KLEIP^−/− corneas (Fig. 5A, right). Furthermore, corneas of KLEIP^−/− mice displayed several apoptotic cells, which were absent in KLEIP^+/+ corneas (Fig. 5B). This observation suggested that high proliferative activity and massive corneal epithelium formation in KLEIP^−/− mice (Figs. 2, 3) is a compensatory mechanism due to corneal epithelial fragility, apoptosis, and damage. Next, we hypothesized that the corneal epithelium in KLEIP^−/− mice makes corneas more prone to mechanical injury, and an experimental mechanical injury would induce rapidly a strong corneal dystrophy in KLEIP^−/− mice. To test this hypothesis, abrasions were performed by removing mechanically 40% of the cornea in 18-day-old KLEIP^+/+ and KLEIP^−/− mice, and corneal wound closure was analyzed (Fig. 6). In KLEIP^+/+ mice a rapid corneal regeneration occurred, and after three days injured corneas were indistinguishable from untreated eyes (Fig. 6A top, B). In contrast, corneal abrasions in KLEIP^−/− mice led rapidly to corneal opacity within seven days in all treated animals (Fig. 6A bottom, B). Injured KLEIP^−/− corneas showed similar histologic alterations, such as epithelial hyperplasia, stromal infiltrations, and superficial keratinized cells (Fig. 6C right) as in 15-week-old non-injured KLEIP^−/− mice (Fig. 2). Likewise, analysis for E-cadherin expression in KLEIP^−/− corneas 12 hours after abrasion showed disorganized and acellular areas in the squamous epithelial cell layer (Fig. 6D), which were similar to the non-injured 15-week-old KLEIP^−/− mice (Fig. 5). Together, the data indicate that KLEIP regulates corneal epithelial integrity, and loss of KLEIP expression makes corneas more fragile and sensitive to mechanical injury. 

Figure 5. 

 

Corneal epithelium of KLEIP^−/− mice showed disorganized, acellular and apoptotic areas. (A) Corneas of KLEIP^+/+ mice showed intact squamous and basal cell layers as indicated by E-cadherin staining. Squamous and basal epithelial cell layers in KLEIP^−/− mice largely were disorganized, and the squamous cell layer showed several acellular areas. Scale bar: 100 μm. (B) Non-dystrophic KLEIP^−/− corneas of 3-week-old mice showed several apoptotic cells (top, scale bars: 500 μm). Apoptotic cells (red) were detected in the squamous epithelial cell layer (S) but not in the basal layer (B) as indicated by TUNEL staining (bottom, scale bars: 100 μm).

Figure 6. 

 

Corneal epithelial abrasion strongly accelerated corneal dystrophy development in KLEIP^−/− mice. (A) Left: Brightfield images of mouse eyes directly after abrasions. A 40% removal of the corneal epithelium was visualized using Thilorbin. Middle: Corresponding untreated eyes served as controls for dystrophy development. Pictures were taken 14 days later. Right: Brightfield images of mechanical treated eyes 14 days after corneal abrasion. Injured eyes developed a severe corneal dystrophy in KLEIP^−/− mice, but not in KLEIP^+/+ mice. Scale bar: 500 μm. (B) Quantification of data shown in (A), n = 7 per group. All KLEIP^−/− mice had a corneal dystrophy on the treated eye within 7 days after abrasion. Of those mice 14% also had a corneal dystrophy on the control eye, whereas KLEIP^+/+ mice had no dystrophy either in the untreated or in the treated eye. (C) HE (top) and Masson trichrome (bottom) staining of corneal sections after corneal abrasion. Thickening of the epithelium and stroma, cell infiltrations in the stroma, and a keratinized epithelium were induced strongly in corneas of KLEIP^−/− mice, but not in corneas of KLEIP^+/+ mice. Scale bar: 100 μm. (D) E-cadherin expression in the squamous (S) and basal (B) corneal epithelial cell layers 12 hours after abrasions in 18-day-old KLEIP^−/− and KLEIP^+/+ mice. KLEIP^−/− corneas showed several acellular areas in the squamous epithelial cell layer 12 hours after abrasions, which were not present in KLEIP^+/+ corneas. Scale bar: 100 μm.

Discussion

Our observations and data reveal that KLEIP^−/− mice have a distinct phenotype of corneal opacification, epidermal metaplasia of the epithelium, and stromal vascularization. This is accompanied by lymphangiogenesis and inflammatory changes. This exclusive observation of the described changes in KLEIP^−/− mice would make the phenotype eligible to be termed a corneal dystrophy. 

Human corneal dystrophies can be divided into four groups according to the affected anatomical structure in the cornea, namely into epithelial, Bowman Layer, stromal, and endothelial dystrophies. ⁵ Corneal epithelial dystrophies, in particular Meesmann corneal dystrophy, are caused partially in human by mutations in the keratin-3 and keratin-12 genes. ⁵ In mice, homozygous deletion of the keratin-12 gene led to a fragile and thinner corneal epithelium. ⁷ Keratin-12 is a protein belonging to the intermediate filaments and is expressed specifically in the corneal epithelium. ^6,26 In our study, mice that harbored a homozygous deletion for the BTB-kelch KLEIP gene also showed a corneal dystrophy in the epithelium, but corneal alterations were different from the keratin-12^−/− mice. While corneas in keratin-12^−/− mice are thinner, ⁷ corneas in KLEIP^−/− mice experienced an epithelial hyperplasia, including keratinized cells, an altered expression of keratins, and they showed several stromal infiltrations. This phenotype suggests a different function for KLEIP in maintenance of the corneal integrity as compared to keratin-12. 

KLEIP has been identified originally as a molecule regulating transiently cell-cell contact sites via E-cadherin localization in MDCK cells, ¹⁸ which implied that KLEIP also may regulate corneal integrity in epithelial cells via cell-cell contact formation. Although corneas in newborn KLEIP^−/− mice initially are normal, after eyelid opening an epithelial hyperplasia developed progressively, leading to a corneal opacity. Experimental mechanical injury of the cornea strongly and rapidly induced formation of a corneal dystrophy within few days, which clearly showed that mechanical injury is the major trigger for corneal dystrophy development in KLEIP^−/− mice. This is supported strongly by the observation that the corneal epithelium in 4-month-old KLEIP^−/− mice showed a similar epithelial metaplasia and acellular epithelial areas as compared to KLEIP^−/− corneas 12 hours after abrasions. Therefore, the data show that loss of KLEIP expression makes corneas more fragile and sensitive to mechanical injury. As a compensatory mechanism to the injuries, corneal epithelial cells start to proliferate. Insufficient wound healing based on loosened cell-cell contacts promotes a continuous epithelial cell proliferation and epithelial metaplasia, and finally a total corneal opacity in KLEIP^−/− mice. Interestingly, this observation resembles closely a spontaneous scarification with neovascularization as seen in connection with external triggers in patients. For instance, chemical and thermal injury ²⁷ to a healthy cornea leads to a similar loss of epithelial and stromal clarity, and eventually neovascularization. 

Healthy corneas usually are avascular as a major prerequisite for corneal transparency, which is regulated mainly by the soluble VEGF receptor sFlt-1. ²⁵ sFlt-1 binds VEGF with high affinity, acts as a trap for VEGF and, therefore, prevents formation of new blood vessels driven by VEGF. ²⁸ A recent study also suggested an additional mechanism showing that transcription factor FoxC1 regulates bioavailability of VEGF by modifying expression of metalloproteinases. ²⁹ In this study KLEIP deficiency in mice resulted in a strong formation of new blood and lymphatic vessels in the corneal stroma. Although KLEIP is known to be involved in endothelial cell function and angiogenesis²⁰, the data suggest that vascularization of corneas in KLEIP^−/− mice is a secondary phenomenon. It could be due to high proliferative activity and scarification of the corneal epithelium. 

In summary, our data suggest a new important function for KLEIP as an essential regulator for corneal integrity in mice. The data establish KLEIP^−/− mice as a unique mouse model to study corneal dystrophy formation and scarification, and it is tempting to speculate that patients suffering from corneal epithelial dystrophies may show genetic alterations in the human KLEIP gene. Further research will be directed now at elucidating the role of KLEIP in physiologic regeneration of the corneal epithelium, and in the formation of cell-cell contacts. Also, pharmacologic ways to support the apparently fragile equilibrium of the cornea in KLEIP-deficiency merits deeper consideration. Clinically, it would be desirable if the spontaneous pathology in KLEIP knockout animals could be used to study mechanisms of progressive corneal scarring. 

Supplementary Materials

pdf S1 

Acknowledgments

The authors would like to thank the Nikon Imaging Center of Heidelberg University. 

Footnotes

 Supported by the Deutsche Forschungsgemeinschaft (KR1887/4-3, KR1887/5-1, and INST 91027/10-1 FUGG).

Footnotes

 Disclosure: N. Hahn, None; C.T. Dietz, None; S. Kühl, None; U. Vossmerbaeumer, None; J. Kroll, None