Role of Fibroblast Growth Factor Receptor 2 (FGFR2) in Corneal Stromal Thinning.

Purpose: To determine the role of fibroblast growth factor receptor 2 (FGFR2)-mediated signaling in keratocytes during corneal development, a keratocyte-specific FGFR2-knockout (named FGFR2cKO) mouse model was generated, and its phenotypic characteristics were determined. Methods: A FGFR2cKO mouse model was generated by the following method: FGFR2 flox mice were crossed with the inducible keratocyte specific-Cre mice (Kera-rtTA/tet-O-Cre). Both male and female FGFR2cKO- and control mice (1 to 3-months-old) were analyzed for changes in corneal topography and pachymetry maps using the optical coherence tomography (OCT) method. The comparative TUNEL assay and immunohistochemical analyses were performed using corneas of FGFR2cKO and control mice to determine apoptotic cells, and expression of collagen-1 and fibronectin. Transmission electron microscopic analysis was conducted to determine collagen structures and their diameters in corneas of FGFR2cKO and control mice. Results: OCT-analyses of corneas of FGFR2cKO mice (n = 24) showed localized central thinning and an increased corneal steepness compared to control mice (n = 23). FGFR2cKO mice further showed a decreased expression in collagen-1, decreased collagen diameters, acute corneal hydrops, an increased fibronectin expression, and an increased number of TUNEL-positive cells suggesting altered collagen structures and keratocytes' apoptosis in the corneas of FGFR2cKO mice compared to control mice. Conclusions: The FGFR2cKO mice showed several corneal phenotypes (as described above in the results) that are also exhibited by the human keratoconus corneas. The results suggested that the FGFR2cKO mouse model serves to elucidate not only the yet unknown role of FGFR2-mediated signaling in corneal physiology but also serves as a model to determine molecular mechanism of human keratoconus development.

Modeling Keratoconus Using Induced Pluripotent Stem Cells.

PURPOSE: To model keratoconus (KC) using induced pluripotent stem cells (iPSC) generated from fibroblasts of both KC and normal human corneal stroma by a viral method. METHODS: Both normal and KC corneal fibroblasts from four human donors were reprogramed directly by delivering reprogramming factors in a single virus using 2A "self-cleaving" peptides, using a single polycistronic lentiviral vector coexpressing four transcription factors (Oct 4, Sox2, Klf4, and Myc) to yield iPSC. These iPS cells were characterized by immunofluorescence detection using of stem cell markers (SSEA4, Oct4, and Sox2). The mRNA sequencing was performed and the datasets were analyzed using ingenuity pathways analysis (IPA) software. RESULTS: The generated stem cell-like clones expressed the pluripotency markers, SSEA4, Oct4, Sox2, Tra-1-60, and also expressed pax6. Our transcriptome analysis showed 4300 genes, which had 2-fold change and 870 genes with a q-value of <0.05 in keratoconus iPSC compared to normal iPSC. One of the genes that showed difference in KC iPSC was FGFR2 (down-regulated by 2.4 fold), an upstream target of Pi3-Kinase pathway, was further validated in keratoconus corneal sections and also KC iPSC-derived keratocytes (down regulated by 2.0-fold). Both normal and KC-derived keratocytes expressed keratocan, signature marker for keratocytes. KC iPSC-derived keratocytes showed adverse growth and proliferation and was further confirmed by using Ly2924002, a PI3k inhibitor, which severely affected the growth and differentiation in normal iPSC. CONCLUSIONS: Based on our result, we propose a model for KC in which inhibition FGFR2-Pi3-Kinase pathway affects the AKT phosphorylation, and thus affecting the keratocytes survival signals. This inhibition of the survival signals could be a potential mechanism for the KC-specific decreased cell survival and apoptosis of keratocytes.

Downregulation of ß-actin gene and human antigen R in human keratoconus.

PURPOSE: The purpose of this study was to determine the expression levels and regulation of ß-actin in the stroma of keratoconus (KC) and normal corneas. METHODS: A total of 15 different human corneas from both KC and normal individuals were used for this study. Additionally, 3 Fuch's dystrophic corneas were also used. The ß-actin gene expression was analyzed at the transcriptional and translational levels in the epithelium and stroma of the KC and normal corneas. The human antigen R (HuR) gene expression was analyzed by real-time PCR in the stroma of five KC and five normal corneas. The keratocytes from three normal and three KC corneas were cultured in the presence of serum, and the expression levels of ß-actin and human antigen R (HuR) were analyzed by using confocal imaging in both normal and KC fibroblasts. RESULTS: The expression of the ß-actin gene was downregulated in the stroma of the six KC corneas but not in the stroma of six normal and Fuchs' dystrophic corneas. Immunofluorescence detection of ß-actin showed that it was absent in the KC fibroblast. The real-time PCR analysis of the HuR gene showed a relative 4.7-fold lower expression in KC corneas relative to the normal corneas, which was further confirmed by the immunofluorescence detection of HuR in fibroblasts of KC corneas. CONCLUSIONS: Although ubiquitous ß-actins are essential for cell survival during early embryogenesis, the effects on various stages of development are not well understood. Our results show that ß-actin is downregulated in the corneal stroma of patients with KC, which may be related to reduced levels of a stabilizing factor (HuR) for ß-actin mRNA. We propose that loss of ß-actin in the corneal stroma might be a triggering factor in the development of KC.

Stem cell markers in the human posterior limbus and corneal endothelium of unwounded and wounded corneas.

PURPOSE: The corneal endothelium is a monolayer of cells in the posterior cornea that is responsible for maintaining a clear cornea. Corneal endothelial cells may be induced to divide, but it has been held that they do not divide in the normal cornea of an adult human. Some studies have suggested that a stem cell population for the corneal endothelium exists. This population could give rise to mature corneal endothelial cells and may reside either in the peripheral corneal endothelium or in the adjacent posterior limbus. This study was initiated to demonstrate the presence of such stem cells in the region of the posterior limbus and to show the response of these cells to corneal wounding. METHODS: Unwounded and wounded corneas with their attached limbal sections were analyzed by immunofluorescence for the presence of nestin, telomerase, Oct-3/4, Pax-6, Wnt-1, and Sox-2. Alkaline phosphatase activity was observed with an enzyme-based reaction that produced a fluorescent product. RESULTS: In the unwounded cornea, stem cell markers nestin, alkaline phosphatase, and telomerase were found in the trabecular meshwork (TM) and in the transition zone between the TM and the corneal endothelial periphery (including Schwalbe's line). Telomerase was also present in the peripheral corneal endothelium. When wounded corneas and their attached limbii were tested, the same markers were found. However, after wounding, additional stem cell markers, Oct-3/4 (in the TM) and Wnt-1 (in both the TM and the transition zone), appeared. Moreover, the differentiation markers Pax-6 and Sox-2 were seen. Pax-6 and Sox-2 were also manifest in the peripheral endothelium post-wounding. CONCLUSIONS: Well documented specific stem cell markers were found in the TM and the transition zone of the human posterior limbus. Wounding of the corneas activated the production of two additional stem cell markers (Oct-3/4, Wnt-1) as well as two differentiation markers (Pax-6, Sox-2), the latter of which also appeared in the corneal endothelial periphery. It is suggested that stem cells reside in the posterior limbus and respond to corneal wounding to initiate an endothelial repair process. The stem cells may also contribute to a normal, slow replacement of corneal endothelial cells.

L-arginine-threonine-arginine (RTR) tetramer peptide inhibits ulceration in the alkali-injured rabbit cornea.

PURPOSE: Proline-glycine-proline (PGP) peptides have been identified as inflammatory mediators initiating neutrophil invasion into alkali-injured cornea. The complementary peptide, arginine-threonine-arginine (RTR), has been shown to bind to the PGP sequence and impede neutrophil infiltration. A prior study showed that L-RTR tetramer and D-RTR tetramer, used alternately (14 times a day), resulted in significantly reduced incidences of corneal ulceration and severity. The purpose of this experiment is to determine the effectiveness of both tetramers, used separately, compared with control. METHODS: Rabbit corneas were exposed to 1 N NaOH for 35 seconds. Sixteen animals were randomly assigned to each of 3 groups: 1) phosphate-buffered saline (PBS), 2) 1.5 mM L-RTR, or 3) 800 microM D-RTR. One drop of each was administered hourly (14 times a day) for 36 days. Additional studies were done to assess neutrophil infiltration into corneas with and without RTR treatment. RESULTS: The severity of corneal ulceration in both RTR groups was statistically significantly different from the 21st day of the experiment to the end. As a result of ulcers healing in the L-RTR group, there was a statistically significant reduction in the number of ulcers beginning on day 22 versus control. Although there was healing in the D-RTR group, the incidence of ulcers was not significantly different from control or L-RTR. Morphometric analysis revealed decreased neutrophil (PMN) invasion with RTR treatment compared with PBS control. CONCLUSIONS: Binding of the PGP molecules by RTR tetramer seems to deprive the cornea of this neutrophilic chemotactic stimulus, leading to a reduction in the severity and incidence of corneal ulceration.

Molecular changes in selected epithelial proteins in human keratoconus corneas compared to normal corneas.

PURPOSE: The purpose of the study was to determine molecular changes in selected epithelial proteins in human keratoconus (KC) corneas compared to normal corneas. METHODS: Two-dimensional (2-D) gel electrophoretic profiles of epithelial cell proteins from normal and keratoconus corneas were compared, and the selected protein spots that showed either up- or downregulation were identified. The desired spots were identified after trypsin digestion and mass spectrometric analysis. Based on the results, two proteins, alpha-enolase and beta-actin, were further analyzed by immunohistochemical and western blot methods, using respective antibodies. To determine the presence of mRNA of the two proteins in the epithelial cells, RT-PCR studies were performed. RESULTS: On comparison of the 2-D gel electrophoretic protein profiles, two protein spots were identified in normal corneas that were either absent or present at lower levels in keratoconus corneas. The two spots were determined to be alpha-enolase (48 kDa) and beta-actin (42 kDa) by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF), and ES-MS/MS mass spectrometric methods. Immunohistochemical analysis revealed that alpha-enolase and beta-actin were present at extremely low levels in the epithelial superficial and wing cells of the keratoconus corneas compared to these cells of normal corneas. 2-D gel electrophoresis followed by western blot analysis revealed relatively greater degradation of the two proteins in the keratoconus corneas compared to normal corneas. RT-PCR analysis showed the mRNA expression of the two proteins in the epithelial cells of both normal and keratoconus corneas. CONCLUSIONS: The results showed relatively low or negligible levels of alpha-enolase and beta-actin in the wing and superficial epithelial cells of keratoconus corneas compared to normal corneas. This was attributed to relatively greater degradation of the two proteins in keratoconus corneas compared to normal corneas.

Fibrin sealant in corneal stem cell transplantation.

PURPOSE: To determine if transplanted corneal epithelial stem cells are safely and efficiently attached to the deficient limbal niche with use of fibrin sealant. The primary outcome is measured with respect to the stability of the transplant, with secondary qualitative evaluations of inflammation, patient comfort, speed of operation, and incidence of complications. METHODS: This retrospective case study examined a total of 114 corneal stem cell reconstructions performed in 95 patients from 1996 to 2004 using corneal stem cells primarily, with a minority of amnion alone, or both. Fibrin sealant was used as the only technique of stem cell adhesion for limbal reconstruction for primary or recurrent pterygia and various stem cell-deficient diseases from 2000 to 2004. RESULTS: The fibrin sealant group showed 1 small recurrence of pterygium but no complications. With sutures, there were 3 recurrences in the pterygia group. After completion of all surgical procedures, all patients were free of pterygia. Miscellaneous stem cell deficiencies were included to demonstrate that corneal stem cell transplants can be used in other corneal procedures in addition to pterygia. CONCLUSIONS: Fibrin sealant alone effectively and safely attached corneal stem cell transplants to the limbal niche. The additional qualitative observations of a reduction in operation time, postoperative pain, and inflammation augurs for more extensive use of fibrin sealants in ophthalmology.

Permanent corneal edema resulting from the treatment of PTK corneal haze with mitomycin: a case report.

A 39-year-old man underwent phototherapeutic keratectomy via excimer laser for recurrent corneal erosions secondary to basement membrane dystrophy with the subsequent development of irregular astigmatism and central stromal opacity. The cornea was scraped and treated with 0.02% mitomycin C using a total of 14 drops over a period of 6 days. Corneal edema developed as a consequence of low endothelial cell count with dysfunctional cells. A corneal transplant restored acuity of 20/20 with binocular vision. It is believed that the underlying endothelium was exposed to toxic doses of mitomycin C sufficient to damage and destroy vital cells. The author reports this case not to criticize the use of mitomycin C in visually disabling post-phototherapeutic keratectomy or photorefractive keratectomy haze but to apprize colleagues of a potential pitfall. The author believes that the use of mitomycin C as a 1-time application at the end of surgery is a safe and valuable adjunct to recover vision when no other is known. However, continued topical application of mitomycin C to the central cornea, in the face of an epithelial defect or an epithelium with inadequate barrier function, increases the risk of endothelial damage.