SMAD4-Dependent Signaling Pathway Involves in the Pathogenesis of TGFBR2-Related CE-like Phenotype.

(1) Background: Our previous data indicated that disturbance of the Transforming Growth Factor beta (TGFB) signaling pathway via its Type-2 Receptor (TGFBR2) can cause a Corneal Ectasia (CE)-like phenotype. The purpose of this study is to elucidate whether the SMAD4-dependent signaling pathway is involved in the TGFBR2-related CE-like pathogenesis. (2) Methods: Smad4 was designed to be conditionally knocked out from keratocytes. Novel triple transgenic mice, KerartTA; Tet-O-Cre; Smad4flox/flox (Smad4kera-cko), were administered with doxycycline (Dox). Optical Coherence Tomography (OCT) was performed to examine Central Corneal Thickness (CCT), Corneal Radius, Anterior Chamber and CE-like phenotype and compared to the littermate Control group (Smad4Ctrl). (3) Results: The OCT revealed normal cornea in the Smad4Ctrl and a CE-like phenotype in the Smad4kera-cko cornea, in which the overall CCT in Smad4kera-cko was thinner than that of Smad4Ctrl at P42 (n = 6, p < 0.0001) and showed no significant difference when compared to that in Tgfbr2kera-cko. Furthermore, the measurements of the Anterior Chamber and Corneal Radius indicated a substantial ectatic cornea in the Smad4kera-cko compared to Smad4Ctrl. The H&E staining of Smad4kera-cko mimics the finding in the Tgfbr2kera-cko. The positive immunostaining of cornea-specific marker K12 indicating the cell fate of cornea epithelium remained unchanged in Smad4kera-cko and the Proliferating Cell Nuclear Antigen (PCNA) immunostaining further indicated an enhanced proliferation in the Smad4kera-cko. Both immunostainings recapitulated the finding in Tgfbr2kera-cko. The Masson's Trichrome staining revealed decreased collagen formation in the corneal stroma from both Smad4kera-cko and Tgfbr2kera-cko. The collagen type 1 (Col1a1) immunostaining further confirmed the reduction in collagen type 1 formation in Smad4kera-cko. (4) Conclusions: The aforementioned phenotypes in the Smad4kera-cko strain indicated that the SMAD4-dependent signaling pathway is involved in the pathogenesis of the CE-like phenotype observed in Tgfbr2kera-cko.

Transforming growth factor beta receptor 2 (Tgfbr2) deficiency in keratocytes results in corneal ectasia.

PURPOSE: We hypothesized that Transforming growth factor beta receptor 2 (Tgfbr2) deletion in keratocyte (Tgfbr2kera-cko), the corneal stroma cell, can result in corneal thinning and generate a potential model for Cornea Ectasia (CE). METHODS: Corneal thickness of Tgfbr2kera-cko and Tgfbr2Ctrl was examined with Optical Coherence Tomography (OCT) at post-natal (P) days 42 and 70, respectively. Histological H&E staining, transmission electron micrograph (TEM), and immunofluorescence staining (IFS) were harnessed to examine corneal cell morphology, proliferation, differentiation, and collagen fibrils. RESULTS: Slit-Lamp revealed that corneas were transparent in both Tgfbr2kera-cko and Tgfbr2Ctrl, however, Tgfbr2kera-cko cornea was 33.5% and 42.9% thinner as compared with those of Tgfbr2Ctrl at P42 and P70, respectively. H&E and semithin section staining with toluidine blue-O confirmed that Tgfbr2kera-cko cornea has a thinner stroma. In contrast, the epithelium in Tgfbr2kera-cko was substantially thicker. The cell proliferation marker Ki67 expression level increased ∼9% in Tgfbr2kera-cko corneal epithelium as compared with that in Tgfbr2Ctrl, however, the Krt14 and Krt12 expression pattern was not obviously changed in Tgfbr2kera-cko corneal epithelium. It was noticed that Col1a1 expression was substantially reduced in Tgfbr2kera-cko as compared with that in Tgfbr2Ctrl. TEM showed that keratocytes were unhealthy and stromal collagen fibril density was significantly reduced in Tgfbr2kera-cko as compared with that in Tgfbr2Ctrl cornea. Moreover, mechanical eye-rubbing on Tgfbr2kera-cko resulted in corneal hydrops and edema. CONCLUSION: Tgfbr2 in keratocytes is indispensable for the corneal stroma at postnatal homeostasis. The cornea phenotype manifested in these Tgfbr2kera-cko mice resembles corneal ectasia disease in humans.

Inducible Slc4a11 Knockout Triggers Corneal Edema Through Perturbation of Corneal Endothelial Pump.

Purpose: The conventional Slc4a11 knockout (KO) shows significant corneal edema at eye opening, a fact that complicates the study of the initial events leading to edema. An inducible KO would provide opportunities to examine early events following loss of Slc4a11 activity. Methods: Slc4a11 Flox (SF) mice were crossed with mice expressing the estrogen receptor Cre Recombinase fusion protein and fed tamoxifen (Tm) for two weeks. Corneal thickness (CT) was measured by OCT. At eight weeks endpoint, oxidative damage, tight junction integrity, stromal lactate concentration, endothelial permeability, differentially expressed transporters, and junction proteins were determined. Separately, a keratocyte only inducible Slc4a11 KO was also examined. Results: At four weeks post-Tm induction Slc4a11 transcript levels were 2% of control. Corneal thickness increased gradually and was 50% greater than Wild Type (WT) after eight weeks with significantly altered endothelial morphology, increased nitrotyrosine staining, significantly higher stromal lactate, decreased expression of lactate transporters and Na-K ATPase activity, higher ATP, altered expression of tight and adherens junctions, and increased fluorescein permeability. No significant differences in CT were found between WT and keratocyte only Slc4a11 KO. Conclusions: The Slc4a11 inducible KO shows development of a similar phenotype as the conventional KO, thereby validating the model and providing a tool for further use in examining the sequence of cellular events by use of noninvasive in vivo physiological probes.

Shp2-mediated MAPK pathway regulates ΔNp63 in epithelium to promote corneal innervation and homeostasis.

Corneal nerve fibers serving sensory, reflex, and neurotrophic functions sustain corneal homeostasis and transparency to promote normal visual function. It is not known whether corneal epithelium is also important for the corneal innervation. Herein, we generated a compound transgenic mouse strain, K14rtTA;tetO-Cre (TC);Shp2flox/flox, in which Shp2 was conditionally knocked out from K14-positive cells including corneal epithelium (Shp2K14ce-cko) upon doxycycline (dox) administration. Our data reveal that Shp2K14ce-cko caused corneal denervation. More specifically, corneal epithelium thickness and corneal sensitivity reduced dramatically in Shp2K14ce-cko mice. In addition, corneal epithelial wound healing after debridement was delayed substantially in the mutant mice. These defects manifested in Shp2K14ce-cko mice resemble the symptoms of human neurotrophic keratopathy. Our in vitro study shows that neurite outgrowth of the mouse primary trigeminal ganglion cells (TGCs) was inhibited when as cocultured with mouse corneal epithelial cells (TKE2) transfected by Shp2-, Mek1/2-, or ∆Np63-targeted siRNA but not by Akt1/2-targeted siRNA. Furthermore, ∆Np63 RNA interference downregulated Ngf expression in TKE2 cells. Cotransfection experiments reveal that Shp2 tightly monitored ΔNp63 protein levels in HEK293 and TKE2 cells. Taken together, our data suggest that the Shp2-mediated MAPK pathway regulated ΔNp63, which in turn positively regulated Ngf in epithelium to promote corneal innervation and epithelial homeostasis.

Aberrant expression of a stabilized ß-catenin mutant in keratocytes inhibits mouse corneal epithelial stratification.

We previously reported that genetic deletion of ß-catenin in mouse corneal keratocytes resulted in precocious corneal epithelial stratification. In this study, to strengthen the notion that corneal keratocyte-derived Wnt/ß-catenin signaling regulates corneal epithelial stratification during mouse development, we examined the consequence of conditional overexpression of a stabilized ß-catenin mutant (Ctnnb1ΔE3) in corneal keratocytes via a doxycycline (Dox)-inducible compound transgenic mouse strain. Histological analysis showed that conditional overexpression of Ctnnb1ΔE3 in keratocytes inhibited corneal epithelial stratification during postnatal development. Unlike the corneal epithelium of the littermate controls, which consisted of 5-6 cell layers at postnatal day 21 (P21), the mutant corneal epithelium contained 1-2 or 2-3 cell layers after Dox induction from embryonic day 0 (E0) to P21 and from E9 to P21, respectively. X-gal staining revealed that Wnt/ß-catenin signaling activity was significantly elevated in the corneal keratocytes of the Dox-induced mutant mice, compared to the littermate controls. Furthermore, RT-qPCR and immunostaining data indicated that the expression of Bmp4 and ΔNp63 was downregulated in the mutant corneas, which was associated with reduced corneal epithelial proliferation in mutant epithelium, as revealed by immunofluorescent staining. However, the expression of Krt12, Krt14 and Pax6 in the mutant corneas was not altered after overexpression of Ctnnb1ΔE3 mutant protein in corneal keratocytes. Overall, mutant ß-catenin accumulation in the corneal keratocytes inhibited corneal epithelial stratification probably through downregulation of Bmp4 and ΔNp63 in the corneal epithelium.

Scleral ultrastructure and biomechanical changes in rabbits after negative lens application.

AIM: To address the microstructure and biomechanical changes of the sclera of rabbits after negative lens application by spectacle frame apparatus. METHODS: Five New Zealand rabbits of seven weeks post-natal were treated with -8 D lens monocularly over the course of two weeks. Refractive errors and axial length (AXL) were measured at the 1st, 7th and 14th days of the induction period. Ultrastructure of sclera was determined with electron microscopy. Biomechanical properties were tested by an Instron 5565 universal testing machine. RESULTS: Lens-induced (LI) eyes elongated more rapidly compared with fellow eyes with AXL values of 15.56±0.14 and 15.21±0.14 mm (P<0.01). Fibril diameter was significantly smaller in the LI eyes compared with control ones in the inner, middle, and outer layers (inner layer, 63.533 vs 76.467 nm; middle layer, 92.647 vs 123.984 nm; outer layer, 86.999 vs 134.257 nm, P<0.01, respectively). In comparison with control eyes, macrophage-like cells that engulfed fibroblasts, dilated endoplasmic reticulum, and vacuoles in fibroblasts were observed in the inner and middle stroma in the LI eyes. Ultimate stress and Young's modulus were lower in the LI eyes compared with those in the control eyes. CONCLUSION: Negative lens application alters eye growth, and results in axial elongation with changes in scleral ultrastructural and mechanical properties.

Generation and Characterization of a Novel Mouse Line, Keratocan-rtTA (KeraRT), for Corneal Stroma and Tendon Research.

Purpose: We created a novel inducible mouse line Keratocan-rtTA (KeraRT) that allows specific genetic modification in corneal keratocytes and tenocytes during development and in adults. Methods: A gene-targeting vector (Kera- IRES2-rtTA3) was constructed and inserted right after the termination codon of the mouse Kera allele via gene targeting techniques. The resulting KeraRT mouse was crossed to tet-O-Hist1H2B-EGFP (TH2B-EGFP) to obtain KeraRT/TH2B-EGFP compound transgenic mice, in which cells expressing Kera are labeled with green fluorescence protein (GFP) by doxycycline (Dox) induction. The expression patterns of GFP and endogenous Kera were examined in KeraRT/TH2B-EGFP. Moreover, KeraRT was bred with tet-O-TGF-α to generate a double transgenic mouse, KeraRT/tet-O-TGF-α, to overexpress TGF-α in corneal keratocytes upon Dox induction. Results: Strong GFP-labeled cells were detected in corneal stroma, limbs, and tail when KeraRT/TH2B-EGFP mice were fed Dox chow. There was no GFP in any single transgenic KeraRT or TH2B-EGFP mouse. Histological analysis showed that GFP in the cornea was limited to stromal keratocytes of KeraRT/TH2B-EGFP, which is consistent with Kera expression. Induction of GFP occurred in 24 hours and reached a plateau by 7 days after Dox induction. GFP could be detected 3-months after induction of KeraRT/TH2B-EGFP. Ectopic expression of TGF-α in corneal keratocytes caused hyperplasia in the corneal epithelium and stroma. Conclusions: The novel Dox inducible KeraRT driver mouse line is a useful genetic tool for gene manipulation and elucidating gene functions in corneal stroma and tendons of limbs and tail during embryonic development, homeostasis and pathogenesis.

Meibomian Gland Absence Related Dry Eye in Ectodysplasin A Mutant Mice.

Meibomian gland dysfunction is the most frequent cause of evaporative dry eye, yet its underlying pathophysiology is unknown. To gain insight into this pathophysiology, we characterized the time-dependent tear film and ocular surface changes occurring in X-linked anhidrotic-hypohidrotic ectodermal dysplasia mice (Tabby), which lack the meibomian gland. These mice sequentially developed corneal epithelial defects, central corneal stromal edema, neovascularization, and pannus 8 to 16 weeks after birth. Aqueous tear secretion was normal, whereas tear break-up time and ex vivo tear evaporation times were all shortened. Corneal epithelial microvilli were less numerous, conjunctival goblet cell density was unaffected, and MUC5AC and MUC5B gene expression was increased. Markers of squamous metaplasia (cytokeratin 10 and small proline-rich protein 1B) were noticed in the corneal epithelium of Tabby mice as early as the fourth week. Taken together, the Tabby mouse is a relevant meibomian gland dysfunction-related dry eye model that may lead to a better understanding of how meibomian glands are related to ocular surface health. This model may also help with discovering novel drug options for treating evaporative dry eye.

Mouse Corneal Stroma Fibroblast Primary Cell Culture.

This protocol is developed for primary cell culture of cornea stromal keratocytes isolated from neonatal mouse eyeballs. It provides an optimal condition to isolate stromal keratocytes which maintain high viability for cell culture.