EunDuck P. Kay

The ROCK Inhibitor Eye Drop Accelerates Corneal Endothelium Wound Healing

PURPOSE To evaluate the effect of Rho kinase (ROCK)-inhibitor eye drops on a corneal endothelial dysfunction primate model and human clinical case series of corneal endothelial dysfunction. METHODS As a corneal-endothelial partially injured model, the corneal endothelium of seven cynomolgus monkeys was damaged by transcorneal freezing; 10 mm of rock inhibitor Y-27632 was then applied topically 6 times daily. The phenotype of the reconstructed corneal endothelium was evaluated by immunohistochemical analysis and noncontact specular microscopy. For clinical study, the effect of Y-27632 eye drops after transcorneal freezing was evaluated in eight corneal endothelial dysfunction patients: four central corneal edema patients and four diffuse corneal edema patients. RESULTS Slit-lamp microscopy revealed that both Y-27632-treated and -nontreated corneas became hazy after transcorneal freezing, and then recovered their transparency within 4 weeks. ROCK inhibitor Y-27632 promoted recovery of corneal endothelial cell density and wound healing in terms of both morphology and function. The percentage of ZO-1 and Na(+)/K(+)-ATPase positive cells in the regenerated area in the Y-27632 group was significantly higher than in the controls. Noncontact specular microscopy revealed that corneal endothelial cell density was significantly higher in the Y-27632 group compared with the controls at 4 weeks; cell density reached approximately 3000 cells/mm(2), as opposed to 1500 cells/mm(2) in the control group. In addition to the animal study findings, the clinical study findings showed that Y-27632 eye drops effectively improved corneal edema of corneal endothelial dysfunction patients with central edema. CONCLUSIONS These findings show that rock inhibitor Y-27632 eye drops promote corneal endothelial wound healing in a primate animal model and suggest the possibility of Y-27632 as a novel therapeutic modality for certain forms of corneal endothelial dysfunction. (http://www.umin.ac.jp/ctr/ number, UMIN000003625.).

Epitheliomesenchymal Transdifferentiation of Cultured RPE Cells

Retinal pigment epithelium (RPE) cells of the proliferative vitreoretinopathy (PVR) membrane take on the shape of fibroblasts and participate in fibrosis, thus deviating from the character of epithelial cells. This study was undertaken to evaluate RPE cell transdifferentiation in vitro. During the culture of porcine RPE cells, primary and 10th-passaged RPE cells were investigated for cell growth in response to transforming growth factor (TGF) β2, change of phenotype and amount in collagen synthesis as well as expression of α-smooth-muscle actin (α-SMA). TGF-β2 inhibited the proliferation of the primary cultures of RPE cells in a dose-dependent manner, while the spindle-shaped 10th-passaged RPE cells were not inhibited by TGF-β2. The 10th-subcultured cells did not show much difference in the quality of collagen synthesis, other than type VIII collagen which was not produced. Collagen synthesis was dose-dependently stimulated by TGF-β2. The stimulation by TGF-β2 in the 10th-passaged RPE cells was much greater than in primary RPE cells. The 10th-subcultured RPE cells produced substantial α-SMA compared to α-SMA production by primary RPE cells. These results were also observed by confocal laser microscopy. These findings indicated that RPE metaplasia resulting in a change of biological cell behavior might be a necessary predisposing step in the development of PVR.

Corneal Endothelial Expansion Promoted by Human Bone Marrow Mesenchymal Stem Cell-Derived Conditioned Medium

Healthy corneal endothelium is essential for maintaining corneal clarity, as the damage of corneal endothelial cells and loss of cell count causes severe visual impairment. Corneal transplantation is currently the only therapy for severe corneal disorders. The greatly limited proliferative ability of human corneal endothelial cells (HCECs), even in vitro, has challenged researchers to establish efficient techniques for the cultivating HCECs, a pivotal issue for clinical applications. The aim of this study was to evaluate conditioned medium (CM) obtained from human bone marrow-derived mesenchymal stem cells (MSCs) (MSC-CM) for use as a consistent expansion protocol of HCECs. When HCECs were maintained in the presence of MSC-CM, cell morphology assumed a hexagonal shape similar to corneal endothelial cells in vivo, as opposed to the irregular cell shape observed in control cultures in the absence of MSC-CM. They also maintained the functional protein phenotypes; ZO-1 and Na+/K+-ATPase were localized at the intercellular adherent junctions and pump proteins of corneal endothelium were accordingly expressed. In comparison to the proliferative potential observed in the control cultures, HCECs maintained in MSC-CM were found to have more than twice as many Ki67-positive cells and a greatly increased incorporation of BrdU into DNA. MSC-CM further facilitated the cell migration of HCECs. Lastly, the mechanism of cell proliferation mediated by MSC-CM was investigated, and phosphorylation of Akt and ERK1/2 was observed in HCECs after exposure to MSC-CM. The inhibitor to PI 3-kinase maintained the level of p27Kip1 for up to 24 hours and greatly blocked the expression of cyclin D1 and D3 during the early G1 phase, leading to the reduction of cell density. These findings indicate that MSC-CM not only stimulates the proliferation of HCECs by regulating the G1 proteins of the cell cycle but also maintains the characteristic differentiated phenotypes necessary for the endothelial functions.

Corneal endothelial modulation : a factor released by leukocytes induces basic fibroblast growth factor that modulates cell shape and collagen

PURPOSE We have previously reported that corneal endothelial modulation takes place when rabbit corneal endothelial (CE) cells are exposed to corneal endothelium modulation factor (CEMF) released by polymorphonuclear leukocytes (PMN) (Kay, E. P., L. Rivela, and Y. G. He, 1990. Invest Ophthalmol Vis Sci. 31:313-322). The modulation was involved in phenotypic switches from polygonal cell shape to fibroblastic morphology and from basement membrane collagen (type IV-rich) synthesis to fibrillar collagen (type I-rich) synthesis. In the current study, we tested the effect of several growth-modulating factors on corneal endothelial modulation. METHODS The effect of basic fibroblast growth factor (bFGF) on cell proliferation was measured by [3H]thymidine incorporation into DNA and cell numbers. Collagen expression was determined by SDS-polyacrylamide gel electrophoresis and by Northern blot analysis. Transcription rate was determined by nuclear run-off assay. Basic fibroblast growth factor synthesis was analyzed by immunoblot assay and quantitated by ELISA assay. Immunofluorescent staining was used for in vivo localization of bFGF and its receptors. RESULTS Basic fibroblast growth factor (bFGF) supplemented with heparin is able to modulate the same phenotypes as observed in CEMF-induced modulation. Basic fibroblast growth factor has a marked stimulatory effect on cell proliferation, as shown by increased cell numbers and [3H]thymidine incorporation into DNA. It also has a strong effect on modulation of cell morphology and collagen phenotypes; the polygonal endothelial cells are induced to assume an elongated shape, and fibrillar collagen synthesis (types I and V) is turned on by bFGF, whereas type IV synthesis is markedly reduced. Such modulating effects of bFGF are augmented by CEMF. Furthermore, CEMF significantly increases production of bFGF in CE cells; the CEMF-treated CE cells synthesized bFGF seven times more than did the control cells. The induced bFGF has a major peptide band of 18.4 kD. Immunohistochemical analysis demonstrates that rabbit corneal endothelium in vivo stains for bFGF, while Descemets membrane requires prior digestion with proteinase K. In situ localization of bFGF receptors demonstrates that high affinity receptors for bFGF are present in corneal endothelium. However, neither transforming growth factor (TGF-beta), epidermal growth factor (EGF), nor retinoic acid (RA) alters qualitative collagen phenotypes; rabbit CE cells continue to synthesize type IV collagen as a predominant species under the influence of these factors. Unlike rabbit CE cells, bovine CE cells in culture produce predominantly fibrillar collagens (I, III, and V). Transforming growth factor enhances type III collagen synthesis and induces type I collagen, but none of these factors affects type IV collagen synthesis by bovine cells. Neither steady-state levels of collagen RNA nor relative transcription rates of the collagen genes are changed significantly by TGF-beta, EGF, or RA in either rabbit or bovine CE cells. CONCLUSIONS These findings suggest that bFGF is able to simultaneously modulate three phenotypic aspects of rabbit CE cells (cell proliferation, cell shape, and collagen expression). Furthermore, CEMF induces de novo synthesis of biologically active bFGF, indicating that bFGF, through the action of CEMF, is the key molecule during corneal endothelial modulation, which ultimately leads to corneal fibrosis (retrocorneal fibrous membrane).

Corneal Endothelial Cell Fate Is Maintained by LGR5 Through the Regulation of Hedgehog and Wnt Pathway

Leucine‐rich repeat‐containing G protein‐coupled receptor 5 (LGR5), a target of Wnt signaling, is reportedly a marker of intestine, stomach, and hair follicle stem cells in mice. To gain a novel insight into the role of LGR5 in human corneal tissue, we performed gain‐ and loss‐of‐function studies. The findings of this study show for the first time that LGR5 is uniquely expressed in the peripheral region of human corneal endothelial cells (CECs) and that LGR5(+) cells have some stem/progenitor cell characteristics, and that in human corneal endothelium, LGR5 is the target molecule and negative feedback regulator of the Hedgehog (HH) signaling pathway. Interestingly, the findings of this study show that persistent LGR5 expression maintained endothelial cell phenotypes and inhibited mesenchymal transformation (MT) through the Wnt pathway. Moreover, R‐spondin‐1, an LGR5 ligand, dramatically accelerated CEC proliferation and also inhibited MT through the Wnt pathway. These findings provide new insights into the underlying homeostatic regulation of human corneal endothelial stem/progenitor cells by LGR5 through the HH and Wnt pathways. STEM Cells2013;31:1396–1407

Cryolathe Corneal Injury

Using a rabbit model of lamellar keratoplasty (LKP), we examined the effect of standardized Barraquer cryolathe freeze injury on corneal structure and keratocyte biochemistry. Cryolathe corneal freeze injury caused immediate disruption of the stromal keratocytes and initiated an inflammatory cell reaction peaking at 3 days following freeze injury. Frozen rabbit LKP corneas were hazy in the early postoperative period, an appearance analogous to the postoperative appearance of human keratomileusis and keratophakia cases. The clinical appearance correlated with keratocyte death and increased interfiber collagen distance. Keratocyte regeneration occurred within 21 days. The regenerating “keratoblasts” were characterized by active mitochondria and an increase in the rough endoplasmic reticulum. Keratocyte counts in the central cornea were found to correlate significantly with corneal clarity. Collagen synthesis by regenerating keratocytes in frozen LKP corneas was characterized by a decrease in the proportion of type V collagen relative to type I collagen when compared to that synthesized by fresh LKP corneas. Although the collagen phenotypic expression of type I and type V collagen was unaltered, there was a change in the solubility characteristics of newly-synthesized collagen in frozen LKP corneas, with a significant decrease in the salt-solubilized collagen. This finding suggests that the collagen synthesized by regenerating keratocytes was more quickly incorporated into mature collagen forms. These studies suggest that freeze injury produced by cryolathe refractive surgical procedures induces a significant early effect on corneal structure and keratocyte biochemistry.

Involvement of cyclin D and p27 in cell proliferation mediated by ROCK inhibitors Y-27632 and Y-39983 during corneal endothelium wound healing.

PURPOSE To investigate the molecular mechanism of Rho-associated kinase (ROCK) inhibitors Y-27632 and Y-39983 on corneal endothelial cell (CEC) proliferation and their wound-healing effect. METHODS The expression of G1 proteins of the cell cycle and expression of phosphorylated Akt in monkey CECs (MCECs) treated with Y-27632 were determined by Western blotting. The effect of Y-39983 on the proliferation of MCECs and human CECs (HCECs) was evaluated by both Ki67 staining and incorporation of BrdU. As an in vivo study, Y-39983 was topically instilled in a corneal-endothelial partially injured rabbit model, and CEC proliferation was then evaluated. RESULTS Investigation of the molecular mechanism of Y-27632 on CEC proliferation revealed that Y-27632 facilitated degradation of p27Kip1 (p27), and promoted the expression of cyclin D. When CECs were stimulated with Y-27632, a 1.7-fold increase in the activation of Akt was seen in comparison to the control after 1 hour. The presence of LY294002, the PI 3-kinase inhibitor, sustained the level of p27. When the efficacy of Y-39983 on cell proliferation was measured in a rabbit model, Y-39983 eye-drop instillation demonstrated rapid wound healing in a concentration range of 0.095 to 0.95 mM, whereas Y-27632 demonstrated rapid wound healing in a concentration range of 3 to 10 mM. CONCLUSIONS These findings show that ROCK inhibitors employ both cyclin D and p27 via PI 3-kinase signaling to promote CEC proliferation, and that Y-39983 may be a more potent agent than Y-27632 for facilitating corneal endothelium wound healing.

Endothelial mesenchymal transformation mediated by IL-1β-induced FGF-2 in corneal endothelial cells.

This review describes the molecular mechanism of endothelial mesenchymal transformation (EMT) mediated by fibroblast growth factor-2 (FGF-2) in corneal endothelial cells (CECs). Corneal fibrosis is not frequently observed in corneal endothelium/Descemets membrane complex; but when this pathologic tissue is produced, it causes a loss of vision by physically blocking light transmittance. Herein, we will address the cellular activities of FGF-2 and its signaling pathways during the EMT process. Furthermore, we will discuss the role of inflammation on FGF-2-mediated EMT. Interleukin-1β (IL-1β) greatly upregulates FGF-2 production in CECs, thus leading to FGF-2-mediated EMT; the whole spectrum of the injury-mediated inflammation (IL-1β pathway) and the subsequent EMT process (FGF-2 pathway) will be briefly discussed. Intervention in the two pathways will provide the means to block EMT before inflammation causes an irreversible change, such as the production of retrocorneal fibrous membrane observed in human eyes.

NF-κB Is the Transcription Factor for FGF-2 That Causes Endothelial Mesenchymal Transformation in Cornea

PURPOSE To determine the role of nuclear factor-κB (NF-κB) during FGF-2-mediated endothelial mesenchymal transformation (EMT) in response to interleukin (IL)-1β stimulation in corneal endothelial cells (CECs). METHODS Expression and/or activation of IL-1 receptor-associated protein kinase (IRAK), TNF receptor-associated factor 6 (TRAF6), phosphatidylinositol 3-kinase (PI 3-kinase), IκB kinase (IKK), IκB, NF-κB, and FGF-2 were analyzed by immunoblot analysis. Cell proliferation was measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. NF-κB activity was measured by NF-κB ELISA kit, while binding of NF-κB to the promoter region of FGF-2 gene was determined by chromatin immunoprecipitation. RESULTS Brief stimulation of CECs with IL-1β upregulated expression of IRAK and TRAF6 and activated PI 3-kinase; expression of IRAK and TRAF6 reached maximum within 60 minutes, after which the expression disappeared, while PI 3-kinase activity was observed up to 4 hours after IL-1β stimulation. Use of specific inhibitor to PI 3-kinase or IRAK demonstrated that IRAK activates PI 3-kinase, the signaling of which phosphorylates IKKα/β and degrades IκB, subsequently leading to activation of NF-κB. The induction of FGF-2 by IL-1β was completely blocked by inhibitors to NF-κB activation (sulfasalazine) or PI 3-kinase (LY294002), and both inhibitors greatly blocked cell proliferation of CECs. Chromatin immunoprecipitation further demonstrated that NF-κB is the transcription factor of FGF-2 as NF-κB binds the putative NF-κB binding site of the FGF-2 promoter. CONCLUSIONS These data suggest that IL-1β signaling combines the canonical pathway and the PI 3-kinase signaling to upregulate FGF-2 production through NF-κB, which plays a key role as a transcription factor of FGF-2 gene.

Two Populations of p27 Use Differential Kinetics to Phosphorylate Ser-10 and Thr-187 via Phosphatidylinositol 3-Kinase in Response to Fibroblast Growth Factor-2 Stimulation

The cyclin-dependent kinase inhibitor p27 regulates cell cycle progression. We investigated whether FGF-2 uses PI 3-kinase to facilitate phosphorylation of p27 on serine 10 (Ser-10) and threonine 187 (Thr-187) and whether the two phosphorylation sites were differentially regulated. FGF-2 stimulation dramatically increased p27 phosphorylation at Ser-10 and Thr-187 using differential kinetics, and the FGF-2-induced p27 phosphorylation was completely blocked at both sites by LY294002. We determined the physical and biochemical interaction of p27 with the Cdk2-cyclin E complex in response to FGF-2 stimulation. Maximal p27 binding to Cdk2-cyclin E occurred at 12 h; the maximal level of p27 phosphorylation at Thr-187 in the ternary complex was observed at 16 h; ubiquitination of the Thr-187-phosphorylated p27 (pp27Thr-187) was observed starting at 12 h and continuing up to 24 h. However, maximum p27 phosphorylation at Ser-10 occurred in the nucleus 6 h after FGF-2 stimulation; maximal export of Ser-10-phosphorylated p27 (pp27Ser-10) occurred 8 h after FGF-2 treatment, and pp27Ser-10 was simultaneously ubiquitinated. We further investigated which of the two phosphorylated p27 was involved in G1/S progression. LY294002 blocked 64% of the cell proliferation stimulated by FGF-2. Use of leptomycin B to block nuclear export of pp27Ser-10 greatly decreased the FGF-2-stimulated cell proliferation (44%), suggesting that phosphorylation of p27 at Ser-10 is the major mechanism for G1/S transition. Our results suggest that differential kinetics are observed in p27 phosphorylation at Ser-10 and Thr-187 and that pp27Thr-187 and pp27Ser-10 may represent two populations of p27 observed in the G1 phase of the cell cycle.