Quercetin improves lacrimal gland function through its anti-oxidant actions: Evidence from animal studies, and a pilot study in healthy human volunteers.

Anti-oxidant properties of polyphenols have been gaining medical attention as a preventive factor against aging and/or lifestyle diseases. In this study, we examined the anti-oxidant activity of quercetin improved tear function through its effects on the lacrimal gland in mice and humans. Six week-old diabetic mice, a model for decreased tear production, were fed for 12 weeks ad libitum with an experimental diet containing 0.5% quercetin. As a result, the tear volume was significantly improved compared to the control, despite no changes in body weight, food intake, lacrimal gland morphology or biochemical serum parameters. Moreover, significantly higher SOD-1 and SOD-2 protein levels were detected in the lacrimal glands of quercetin-treated mice by western blot. In addition, quercetin treatment of mouse corneal cell lines exposed to oxidative stress resulted in dose-dependent inhibition of ROS production and enhanced cell survival. Finally, we examined quercetin pharmacokinetics, specifically its presence in serum and tears subsequent to onion consumption in healthy volunteers, and found that the distribution of quercetin and its metabolite shifted from serum to tear following onion intake. An improvement in tear film stability also resulted following the intake by these healthy volunteers of a new, quercetin-rich onion cultivar ("Quergold") in powder form. These results suggested that quercetin improved tear function through its effects on the lacrimal gland in mice and humans.

Lost or fragmented bony septum of the optic canal facing the sphenoid sinus: a histological study using elderly donated cadavers.

PURPOSE: To histologically describe a direct contact (the so-called dehiscence) of the optic nerve (ON) and/or internal carotid artery (ICA) to the mucosa of posterior paranasal sinuses represented by the sphenoid sinus (SS). METHODS: Observations of histological sections of unilateral or bilateral skull bases (parasellar area and orbital apex) from 22 elderly cadavers were made. RESULTS: A bony septum was less than 300 µm between the SS and ICA and 200 µm between the SS and optic nerve. Parts of the septa were sometimes absent due to fragmentation and holes of the bony lamella (2/22 facing the ICA; 4 facing the ICA in combination with an absent bony septum facing the nerve). In these dehiscence sites, the SS submucosal tissue attached to a thick sheath (50-100 µm in thickness) enclosing the optic nerve and ophthalmic artery and/or the ICA adventitia (50-200 µm in thickness). The ICA sometimes contained a sclerotic plaque that attached to or even protruded into the SS. With or without dehiscence, the SS mucosa was always thin (50-100 µm in thickness) and accompanied no mononuclear cellular infiltration or tumor. CONCLUSIONS: A thin bony septum of the optic nerve or ICA had been notable as a danger point during surgery, but even a 0.05-mm-thick bone lamella might be an effective barrier against cellular infiltration or bacterial invasion from the SS. Fragmentation and holes of the bony lamella in 4 cadavers might allow cellular invasion to the optic nerve. Accordingly, unknown immunological cross talks might occur to cause demyelination.

Elevated Cytokine Levels in Aqueous Humor Are Associated with Peripheral Anterior Synechiae after Penetrating Keratoplasty.

Peripheral anterior synechiae (PAS) after corneal transplantation leads to refractory glaucoma and permanent loss of vision. However, the exact mechanism remains elusive. This study aimed to evaluate the association between cytokine levels in the aqueous humor (AqH) and the progression of PAS after penetrating keratoplasty (PKP). We measured 20 cytokine levels in AqH and assessed the correlation with PAS progression after PKP in 85 consecutive patients who underwent PKP. We also evaluated age-dependent alterations in PAS and cytokine levels in DBA2J mice. PAS developed in 38 (44.7%) of 85 eyes after PKP. The incidence of intraocular pressure increase after PKP was significantly greater in eyes with PAS (26.3%) than in those without PAS (2%, p = 0.0009). The PAS area at 12 months after PKP was significantly positively correlated with the preoperative levels of interleukin (IL)-6, interferon (IFN)-γ and monocyte chemotactic protein (MCP)-1 (p ≤ 0.049). In the DBA2J mice, an experimental glaucoma model that developed PAS at 50 weeks, the AqH levels of IL-2, IL-6, IL-10, IFN-γ, tumor necrosis factor-α, MCP-1 and granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly increased at 50 weeks compared to 8 weeks (p ≤ 0.021). In conclusion, inflammatory alterations in the AqH microenvironment, such as high preoperative specific cytokine levels, can lead to PAS formation and glaucoma.

Transplantation of iPSC-derived corneal endothelial substitutes in a monkey corneal edema model.

OBJECTIVE: In order to provide regenerative therapy for millions of patients suffering from corneal blindness globally, we derived corneal endothelial cell substitute (CECSi) cells from induced pluripotent stem cells (iPSCs) to treat corneal edema due to endothelial dysfunction (bullous keratopathy). METHODS AND RESULTS: We developed an efficient xeno-free protocol to produce CECSi cells from both research grade (Ff-MH09s01 and Ff-I01s04) and clinical grade (QHJI01s04) iPSCs. CECSi cells formed a hexagonal confluent monolayer with Na, K-ATPase alpha 1 subunit expression (ATP1A1), tight junctions, N-cadherin adherence junction formation, and nuclear PITX2 expression, which are all characteristics of corneal endothelial cells. CECSi cells can be cryopreserved, and thawed CECSi cell suspensions also expressed N-cadherin and ATP1A1. Residual undifferentiated iPSCs in QHJI01s04-derived CECSi cells was below 0.01%. Frozen stocks of Ff-I01s04- and QHJI01s04-derived CECSi cells were transported, thawed and transplanted into a monkey corneal edema model. CECSi-transplanted eyes significantly reduced corneal edema compared to control group. CONCLUSION: Our results show a promising approach to provide bullous keratopathy patients with an iPS-cell-based cell therapy to recover useful vision.

Long-Term Outcome After Superficial Keratectomy of the Abnormal Epithelium for Partial Limbal Stem Cell Deficiency.

PURPOSE: To investigate the etiology and long-term surgical prognosis of the abnormal epithelium for partial limbal stem cell deficiency (LSCD), following superficial keratectomy DESIGN: Retrospective, interventional case series METHODS: This single-center, retrospective study was conducted to assess the prognosis of consecutive patients who underwent superficial keratectomy, with or without amniotic membrane transplantation (AMT), for the treatment of partial LSCD, from 2010 to 2019. We analyzed the etiologies of partial LSCD, surgical success rate, prognosis for recurrent cases, and the improvement in visual acuity. RESULTS: We included 40 patients (51 eyes) with partial LSCD. All eyes were in clinical stage II without dense fibrovascular tissue. Idiopathy was the most common etiology (39%), followed by multiple surgeries involving the corneoscleral limbus (19%). All eyes attained corneal reepithelialization and transparency. Furthermore, the visual acuity improved or remained unchanged postoperatively. We observed recurrence in 19 eyes (37%) with a mean follow-up period of 26.3 months. Despite no significant difference in the recurrence rates among different etiologies, postoperative delayed epithelialization and extensive limbal involvement were identified as risk factors for recurrence (P < .001 and P = .013, respectively). Repeat surgeries were performed in 16 eyes. The final success rate was 84%. CONCLUSIONS: Superficial keratectomy is useful for the treatment of partial LSCD of varied etiologies, with an expected improvement in visual acuity postoperatively. Although the procedure can be repeated and have a high success rate, there have been several cases of recurrence in the long-term postoperative course.

Long-term outcomes of cultivated cell sheet transplantation for treating total limbal stem cell deficiency.

PURPOSE: This study was conducted to determine the long-term outcomes of cultivated cell sheet transplantation (CCST), and to clarify risk factors that affected the outcomes. METHODS: We retrospectively analyzed the medical charts and photographs of 246 consecutive surgeries (162 eyes from 139 patients) that used CCST for treating total limbal stem cell deficiency. Deficiency types included Stevens-Johnson syndrome (SJS) or toxic epidermal necrolysis (SJS/TEN; n = 80), ocular surface burns (Burn; n = 75), ocular cicatricial pemphigoid (OCP) or pseudo-OCP (n = 58), and others (n = 33). The methods of CCST included 103 cultivated limbal epithelial transplantations (28 autologous, 62 allogeneic, and 13 living-related relatives), and 143 cultivated oral mucosal epithelial transplantations. We analyzed the rate of successful ocular surface reconstruction, clear corneas, and best-corrected visual acuity equal to or better than 20/200. RESULTS: With a mean observation period of 357 weeks, successful ocular surface reconstruction and clear corneas at last visit were achieved in 65.1% of the patients. Corrected visual acuity equal to or better than 20/200 was achieved in 19.0% of the patients. Eyes with SJS/TEN exhibited worse outcomes compared with the Burn group. Postoperative complications included corneal ulcerations or perforations (18.3%), glaucoma (13.8%), and infections (8.5%). Multivariate analyses using the logistic mixed-effects model indicated that the presence of preoperative corneal epithelial defects was a significant risk factor for postoperative failure. CONCLUSIONS: In this series, the success rate of CCST after a mean observation period of 357 weeks was 65.1%, and preoperative epithelial defect was the most relevant risk factor.

A potential link between desmoglein 3 and epidermal growth factor receptor in oral squamous cell carcinoma and its effect on cetuximab treatment efficacy.

Desmoglein (DSG) 3 is overexpressed in oral squamous cell carcinoma (OSCC). Epidermal growth factor receptor (EGFR) inhibitor cetuximab is widely used for OSCC treatment. Several evidences suggest a correlation between DSG3 and EGFR in epidermal keratinocytes. EGFR inhibition has been shown to enhance cell-cell adhesion and induce terminal differentiation in epidermal cells. Thus, here we investigated the DSG3-EGFR interaction in OSCC and its effect on cetuximab treatment. Cell lines established from the primary tumor and metastatic lymph nodes of four OSCC patients and three commercial OSCC cell lines were used for the experiments. Cells from metastatic lymph nodes of each patient expressed increased DSG3 and EGFR than cells from the primary tumor in the same patient. Cetuximab treatment increased DSG3 expression by up to 3.5-fold in seven of the 11 cell lines. A high calcium concentration increased the expression of DSG3 and EGFR in a dose-dependent manner. Strikingly, a high calcium-associated DSG3 induction enhanced cetuximab efficacy by up to 23% increase in cetuximab-low-sensitive cell lines. Our findings also suggest a correlation between DSG3 and EGFR in OSCC, and this affects cetuximab treatment efficacy.

Elevation of preoperative recipient aqueous cytokine levels in eyes with primary graft failure after corneal transplantation.

Purpose: Primary graft failure after corneal transplantation is caused by dysfunction of corneal endothelial cells. Recently, we demonstrated that preoperative recipients' aqueous cytokine levels are associated with rapid corneal endothelial cell loss after corneal transplantation. In the present study, we evaluated the preoperative inflammatory cytokine levels in the aqueous humor (AqH) of eyes with primary graft failure following corneal transplantation. Methods: Among the prospective consecutive case series (273 eyes), this study included patients who developed primary graft failure (eight eyes) and patients who underwent corneal transplantation for the treatment of bullous keratopathy (108 eyes) or cataract surgery (30 eyes). AqH samples were collected at the beginning of each surgery. The levels of the cytokines (interleukin [IL]-4, IL-6, IL-8, IL-10, IL-12p70, IL-17A, interferon [IFN]-γ, monocyte chemotactic protein [MCP]-1, E-selectin, P-selectin, and soluble intercellular adhesion molecule [sICAM]-1) in the AqH were measured with multiplex beads immunoassay. Results: In eyes with primary graft failure, the preoperative levels of aqueous protein (4.6-fold), interleukin (IL)-6 (179-fold), IL-17A (7.1-fold), MCP-1 (2.6-fold), IFN-γ (4.3-fold), E-selectin (2.3-fold), P-selectin (2.0-fold), and sICAM-1 (5.5-fold) were statistically significantly higher compared to the cataract controls (p<0.0021). There was no primary graft failure among the recipients who received corneal grafts of fellow eyes from the same donors. Conclusions: Preoperative levels of AqH cytokines, such as IL-6, IL-17A, MCP-1, IFN-γ, and sICAM-1, increased in eyes with primary graft failure after corneal transplantation. These cytokine levels could be prognostic biomarkers to predict primary graft failure after corneal transplantation.

Corneal Endothelial Regeneration Using Mesenchymal Stem Cells Derived from Human Umbilical Cord.

Corneal blindness is the third leading cause of blindness in the world, and one of the main etiologies is dysfunction of the corneal endothelium. Current treatment of corneal endothelial disease is allogenic corneal transplantation, which is limited by the global shortage of donor corneas and immunological rejection. The corneal endothelium consists of a monolayer of cells derived from the neural crest and mesoderm. Its main function is to prevent corneal edema by tight junctions formed by zonular occludens-1 (ZO-1) and Na, K-ATPase pump function. The human umbilical cord (UC) is a rich source of mesenchymal stem cells (MSCs). UC-MSCs that have multi-lineage potential may be an accessible allogenic source. After inducing differentiation with medium containing glycogen synthase kinase (GSK) 3-ß inhibitor, UC-MSCs formed polygonal corneal endothelial-like cells that functioned as tissue-engineered corneal endothelium (UTECE). Expressions of major corneal endothelial markers were confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative RT-PCR (qRT-PCR). Western blotting confirmed the expression of Na,K-ATPase and PITX2, the functional and developmental markers of corneal endothelial cells. Immunohistochemistry revealed the localization of Na,K-ATPase and ZO-1 in cell-cell junctions, suggesting the presence of tight junctions. In vitro functional analysis revealed that UTECE had significantly high pump function compared with UC-MSCs. Moreover, UTECE transplanted into a rabbit model of bullous keratopathy successfully maintained corneal thickness and transparency. Our findings suggest that UTECE may be used as a source of allogenic cells for the treatment of corneal endothelial disease.

The effects of 3% diquafosol sodium eye drop application on meibomian gland and ocular surface alterations in the Cu, Zn-superoxide dismutase-1 (Sod1) knockout mice.

PURPOSE: The purpose of the study is to investigate the effect of 3% diquafosol sodium eye drops on meibomian gland and ocular surface alterations in the superoxide dismutase-1 (Sod1 -/- ) mice in comparison to the wild-type mouse. METHODS: Three percent diquafosol sodium eye drop was instilled to 20 eyes of 10 50-week-old male Sod1 -/- mice and 22 eyes of 11 C57BL/6 strain 50-week-old wild-type (WT) male mice six times a day for 2 weeks. Aqueous tear secretion quantity was measured with phenol red-impregnated cotton threads without anesthesia. Tear film stability and corneal epithelial damage were assessed by fluorescein and lissamine green staining. We also performed oil red O (ORO) lipid staining to evaluate the lipid changes in the meibomian glands. Meibomian gland specimens underwent hematoxylin and eosin staining to examine histopathological changes and meibomian gland acinar unit density after sacrifice. Immunohistochemistry staining was performed using cytokeratin 4, cytokeratin 13, and transglutaminase-1 antibodies. Quantitative real-time polymerase chain reaction for cytokeratin 4, cytokeratin 13, and transglutaminase-1 mRNA expression was also performed. RESULTS: The aqueous tear quantity, the mean tear film breakup time, and the number of lipid droplets significantly improved in the Sod1 -/- mice with treatment. The mean meibomian acinar unit density did not change in the Sod1 -/- mice and WT mice after treatment. Application of 3% diquafosol sodium eye drop significantly decreased the corneal fluorescein and lissamine green staining scores in the Sod1 -/- mice after 2 weeks. We showed a notable increase in cytokeratin 4, cytokeratin 13 immunohistochemistry staining, and cytokeratin 4, cytokeratin 13 mRNA expressions with a marked decrease in immunohistochemistry staining and significant decline in mRNA expression of transglutaminase-1 after 3% diquafosol sodium treatment. CONCLUSION: Topical application of 3% diquafosol sodium eye drop improved the number of lipid droplets, tear stability, and tear production which in turn appeared to have a favorable effect on the ocular surface epithelium. Three percent diquafosol sodium eye drop may be a potential treatment for age-related meibomian gland and dry eye disease based on the observations of the current study.

Preoperative Aqueous Cytokine Levels are Associated With Endothelial Cell Loss After Descemet's Stripping Automated Endothelial Keratoplasty.

Purpose: To evaluate the association between endothelial cell density (ECD) after Descemet's stripping automated endothelial keratoplasty (DSAEK) and preoperative cytokine levels in the aqueous humor (AqH). Methods: This prospective consecutive case series included 97 consecutive patients who underwent DSAEK (64 eyes) or cataract surgery (33 eyes). AqH samples were collected at the beginning of each surgery. The levels of cytokines (IL-1α, IL-1ß, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IFN-α, IFN-γ, monocyte chemotactic protein [MCP]-1, E-selectin, P-selectin, and soluble intercellular adhesion molecule [sICAM]-1) in the AqH were measured by multiplex beads immunoassay. The correlations between preoperative aqueous cytokine levels and the ECD at 12 months after DSAEK were analyzed. Results: The ECD decreased from 2747 ± 259 cells/mm2 in the donor graft to 1235 ± 607 cells/mm2 at 12 months after DSAEK. In all subjects undergoing DSAEK, the postoperative ECD at 12 months was significantly correlated with the preoperative levels of MCP-1 (r = -0.467, 95% confidence interval [CI]: -0.650 to -0.222, P = 0.0003). In an analysis excluding Fuchs endothelial corneal dystrophy (11 eyes), the ECD at 12 months after DSAEK was significantly correlated with preoperative levels of IL-17A (r = -0.635, 95% CI: -0.819 to -0.319, P = 0.0004), MCP-1 (r = -0.605, 95% CI: -0.779 to -0.345, P < 0.0001), IFN-γ (r = -0.633, 95% CI: -0.796 to -0.385, P < 0.0001), E-selectin (r = -0.516, 95% CI: -0.756 to -0.276, P = 0.0004), and sICAM-1 (r = -0.537, 95% CI: -0.735 to -0.253, P = 0.0005). Conclusions: Higher preoperative levels of IL-17A, MCP-1, IFN-γ, E-selectin, and sICAM-1 in the AqH were associated with lower ECD after DSAEK for bullous keratopathy.

Elevated Aqueous Cytokine Levels in Eyes With Ocular Surface Diseases.

PURPOSE: To evaluate cytokine and protein levels in the aqueous humor (AqH) of eyes with ocular surface diseases. DESIGN: Prospective consecutive case series. METHODS: This study includes 14 patients (aged 62.4 ± 13.7 years) with chronic-phase ocular surface diseases (4 with ocular cicatricial pemphigoid, 5 with chemical burns, 2 with a thermal burn, 2 with Stevens-Johnson syndrome, and 1 with exposure keratitis), 14 matched patients without ocular surface disease (controls with corneal scar), and 30 patients who underwent cataract surgery (healthy controls). AqH samples were collected at the beginning of surgery. AqH levels of cytokines (interleukin [IL]-1α, IL-1ß, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, monocyte chemotactic protein [MCP]-1, interferon [IFN]-α, IFN-γ, macrophage inflammatory protein [MIP]-1α, MIP-1ß, P-selectin, E-selectin, soluble-intercellular adhesion molecule [s-ICAM]-1, tumor necrosis factor [TNF]-α, granulocyte-macrophage colony-stimulating factor [GM-CSF], IFN-γ-induced protein [IP]-10) were measured using multiplex beads immunoassays. RESULTS: The levels of IL-6, IL-10, IL-17A, GM-CSF, E-selectin, P-selectin, and s-ICAM in AqH were significantly elevated in eyes with ocular surface diseases (in pg/mL: 1696 ± 804, 4.0 ± 1.0, 24.3 ± 9.8, 26.0 ± 18.3, 5150 ± 1232, 13122 ± 7219, and 7914 ± 2813, respectively), compared to healthy controls (IL-6: 6.36 ± 0.94, P = .001; IL-10: 1.68 ± 0.04, P = .0006; IL-17A: 3.7 ± 0.2, P = .008; GM-CSF: 2.7 ± 0.3, P = .007; E-selectin: 2093 ± 37, P = .0001; P-selectin: 3658 ± 137, P = .0001; sICAM-1: 1397 ± 119, P = .008). The levels of IL-6, IL-17A, E-selectin, and P-selectin in AqH were significantly higher in eyes with ocular surface diseases compared to those with corneal scar (IL-6: 44.1 ± 15.0, P = .0077; IL-17A: 4.1 ± 0.7, P = .034; E-selectin: 2439 ± 302, P = .039; and P-selectin: 5673 ± 1553, P = .017). CONCLUSIONS: Multiple AqH cytokine levels were elevated in chronic ocular surface diseases.

Preoperative Aqueous Cytokine Levels Are Associated With a Rapid Reduction in Endothelial Cells After Penetrating Keratoplasty.

PURPOSE: To evaluate the influence of preoperative inflammatory cytokine levels in the aqueous humor (AqH) on the endothelial cell density (ECD) after penetrating keratoplasty (PKP). DESIGN: Prospective, interventional, consecutive case series. METHODS: This study includes 70 consecutive patients (mean age 73.7 ± 10.6 years) who underwent PKP (37 eyes) or cataract surgery (controls, 33 eyes). A total of 70 AqH samples were collected at the beginning of each surgery. The levels of cytokines (interleukin [IL]-1α, IL-1ß, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, interferon [IFN]-α, IFN-γ, monocyte chemotactic protein [MCP]-1, E-selectin and P-selectin) in AqH were measured by multiplex bead immunoassay. The subjects who underwent PKP were classified into 2 groups: ECD ≥ 1200 cells/mm2 at 6 months (24 eyes), and ECD < 1200 cells/mm2 at 6 months (13 eyes). RESULTS: The ECD at 3 months significantly correlated with the preoperative levels of IL-10 (r = -0.428, P = .02) and IFN-γ (r = -0.412, P = .029). The ECD at 6 months significantly correlated with the preoperative levels of IL-10 (r = -0.399, P = .024), MCP-1 (r = -0.444, P = .011), and IFN-γ (r = -0.474, P = .006). The preoperative levels of IL-6, IL-10, MCP-1, IFN-γ, and P-selectin in AqH were significantly higher in eyes with ECD < 1200 cells/mm2 compared with those with ECD ≥ 1200 cells/mm2 at 6 months (P < .05). CONCLUSIONS: Higher preoperative levels of IL-10, MCP-1, and IFN-γ in the AqH were associated with low ECD after PKP.

Iris Damage Is Associated With Elevated Cytokine Levels in Aqueous Humor.

Purpose: To evaluate the association between iris damage and cytokine levels in the aqueous humor (AqH). Methods: A total of 201 AqH samples from 201 consecutive patients (mean age 73.7 ± 10.6) were collected at the beginning of corneal transplantation or cataract surgery. Iris damage of each case was assessed from preoperative slit-lamp findings based on its severity. The subjects were classified into three groups: eyes without iris damage (126 eyes), eyes with mild iris damage (51 eyes), and eyes with severe iris damage (24 eyes). The levels of cytokines (IL-1α, IL-1ß, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17a, interferon gamma-induced protein [IP]-10, monocyte chemotactic protein [MCP]-1, IFN-α, IFN-γ, macrophage inflammatory protein [MIP]-1α, MIP-1ß, P-selectin, E-selectin, soluble intercellular adhesion molecule [sICAM]-1, TNF-α, and granulocyte-macrophage colony-stimulating factor [GM-CSF]) in AqH were measured by multiplex beads immunoassay. Results: The levels of aqueous protein, IL-1α, IL-1ß, IL-4, IL-6, IL-8, IL-10, IL-17A, MCP-1, TNF-α, E-selectin, P-selectin, and sICAM-1 in eyes with mild and severe iris damage were higher than in those without iris damage (P < 0.033). Multivariate analyses of clinical factors revealed that iris damage was associated with the history of complicated glaucoma, and the number of previous intraocular surgeries. The levels of AqH IL-6, IL-8, IL-13, MIP-1α, TNF-α, and sICAM-1 were significantly elevated in eyes with mild and severe iris damage in phakic eyes, and the levels of AqH IL-8 and sICAM-1 were significantly elevated in eyes with severe iris damage in pseudophakic eyes, compared with the eyes without iris damage (P < 0.045). Conclusions: Iris damage was associated with the elevation in the levels of aqueous protein and cytokines.

Skin-Derived Precursors as a Source of Progenitors for Corneal Endothelial Regeneration.

Corneal blindness is the fourth leading cause of blindness in the world. Current treatment is allogenic corneal transplantation, which is limited by shortage of donors and immunological rejection. Skin-derived precursors (SKPs) are postnatal stem cells, which are self-renewing, multipotent precursors that can be isolated and expanded from the dermis. Facial skin may therefore be an accessible autologous source of neural crest derived cells. SKPs were isolated from facial skin of Wnt1-Cre/Floxed EGFP mouse. After inducing differentiation with medium containing retinoic acid and GSK 3-ß inhibitor, SKPs formed polygonal corneal endothelial-like cells (sTECE). Expression of major corneal endothelial markers were confirmed by Reverse transcription polymerase chain reaction (RT-PCR) and quantitative Real time polymerase chain reaction (qRT-PCR). Western blots confirmed the expression of Na, K-ATPase protein, the major functional marker of corneal endothelial cells. Immunohistochemistry revealed the expression of zonular occludens-1 and Na, K-ATPase in cell-cell junctions. In vitro functional analysis of Na, K-ATPase pump activity revealed that sTECE had significantly high pump function compared to SKPs or control 3T3 cells. Moreover, sTECE transplanted into a rabbit model of bullous keratopathy successfully maintained corneal thickness and transparency. Furthermore, we successfully induced corneal endothelial-like cells from human SKPs, and showed that transplanted corneas also maintained corneal transparency and thickness. Our findings suggest that SKPs may be used as a source of autologous cells for the treatment of corneal endothelial disease. Stem Cells Translational Medicine 2017;6:788-798.

Accelerated Epithelial Proliferation in a Rabbit Model of Bullous Keratopathy.

Purpose: Long standing bullous keratopathy (BK) is associated with peripheral conjunctivalization and limbal deficiency. We hypothesized that cases of limbal deficiency may be induced due to accelerated proliferation of corneal epithelial cells. To investigate this hypothesis, we examined the influence of BK on the corneal epithelium in a rabbit model. Methods: Continuous curvilinear descemetorhexis was performed to remove a circular section of the corneal endothelium and Descemet's membrane (DM) using inverted Shinskey hook. Corneal tissue sections of BK eyes were observed by histochemical analysis using BrdU pulse chase methods for evaluation of corneal epithelial cell proliferation. Results: Rabbit corneas immediately became stromal edematous after DM stripping surgery, and a week later their thickness was five times that of control cornea. Vascularization of the peripheral cornea was observed in BK eyes, however, conjunctivalization was rarely observed at 6 weeks. The number of BrdU positive cells tended to be lower in the BK cornea epithelium compared to the control cornea epithelium. The number of Ki67 positive cells also showed a tendency to increase in the BK corneal epithelium. Telomere intensity in BK was similar to control corneal epithelium. Conclusion: BK may accelerate the proliferation of corneal epithelial cells in a rabbit model.

Elevated Cytokine Levels in the Aqueous Humor of Eyes With Bullous Keratopathy and Low Endothelial Cell Density.

PURPOSE: To evaluate cytokine levels in the aqueous humor (AqH) of eyes with bullous keratopathy (BK) and low endothelial cell density (ECD). METHODS: A total of 145 AqH samples (60 BK, 16 low ECD, 35 corneal diseases with normal ECD, and 34 normal controls) were collected from consecutive patients who underwent corneal transplantation or cataract surgery. None of the patients had any clinically apparent inflammation at the time of AqH collection. The AqH levels of cytokines (IL-1α, IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IFN-α, IFN-γ, monocyte chemotactic protein [MCP]-1, TNF-α, E-selectin, P-selectin, soluble intercellular adhesion molecule [sICAM]-1, granulocyte-macrophage colony-stimulating factor [GM-CSF], macrophage inflammatory protein [MIP] -1α and MIP-1ß) were compared among the groups. RESULTS: The levels of IL-1α, IL-8, IL-17A, TNF-α, GM-CSF, MIP-1α, IFN-γ, and E-selectin in the AqH were significantly elevated in BK and low ECD eyes, compared with healthy controls (all P < 0.03). The levels of IL-4, IL-6, IL-10, IL-12p70, IL-13, MCP-1, MIP-1ß, P-selectin, and sICAM-1 were significantly elevated only in BK eyes, compared with healthy control (all P < 0.001). There were no significant differences in AqH cytokine levels between corneal diseases with normal ECD and normal control eyes. Among BK eyes, the IL-6 and IFN-γ levels were elevated in eyes with pseudophakic BK (PBK), Fuchs' endothelial corneal dystrophy (FECD), postkeratoplasty, posttrabeculectomy, and postlaser iridotomy (LI) (all, P < 0.04), whereas IL-1α, IL-10, IL-17A, MIP-1ß, and sICAM-1 levels were elevated only in PBK, postkeratoplasty, posttrabeculectomy, and post-LI eyes (all, P < 0.05). CONCLUSIONS: Subclinical elevation of AqH cytokine levels may cause endothelial cell loss.

Engineered three-dimensional rabbit oral epithelial-mesenchymal-muscular hybrid sheets.

Regenerative muscles are required for swallowing and mastication, and are important for functional recovery from diseases involving oral muscular defects. Therefore, we generated three-layer hybrid sheets, similar to oral mucosal structures containing submucosal muscles, using rabbit oral mucosa epithelial, mesenchymal, and myoblastic progenitor cells, and examined the structural proteins. Each cell type was obtained from rabbit oral mucosa using enzymatic digestion. Isolated mesenchymal and myoblastic cells were multi-differentiated into osteoblasts, adipocytes, and chondrocytes or myotubes. Isolated epithelial cells were cultured on collagen gels containing isolated mesenchymal cells for 2 weeks, and these epithelial-mesenchymal cell sheets were laminated onto myoblastic cell sheets. The engineered hybrid sheets were multi-stratified in the epithelial and myoblastic layers in a time-dependent manner, expressing intermediate cytoskeletal filament proteins of epithelium and muscle. Hybrid sheets also expressed extracellular matrix basement membrane proteins. Immature cell markers for epithelial and myoblastic cells were observed continuously in hybrid sheet cultures. We established engineered three-dimensional rabbit oral mucosa hybrid sheets containing each immature cell type in vitro.

Ocular surface reconstruction by cultivated epithelial sheet transplantation.

Recent advances in ocular surface reconstruction for patients with severe ocular surface diseases have significantly improved the prognosis of patients with vision-impairing corneal abnormalities. The history of cultivated epithelial sheet transplantation is short, and debate on the current approaches for these procedures is continuing. Limbal stem cell transplantation, including conjunctivolimbal autograft and keratolimbal allograft, has brought opportunities for vision improvement. In addition, the use of cultivated limbal epithelial transplantation from both allogeneic and autologous sources has provided further options for immediate postoperative epithelialization of the corneal surface. Finally, cultivated oral mucosal epithelial transplantation, which allows autologous transplantation for patients with bilateral limbal stem cell deficiency, has provided the best overall midterm and long-term results. Its biggest advantages are the absence of rejection reactions and the reduction of postoperative complications associated with steroid therapy. However, a solitary surgical approach is not sufficient for obtaining a good clinical outcome. To maximize the possibility of success using these procedures, it is important to preoperatively enhance aggressive treatment of the ocular surface, especially with factors that facilitate moisture retention. In this review article, we also discuss our clinical experience in relation to these surgical procedures.

The effects of 3% diquafosol sodium application on the tear functions and ocular surface of the Cu,Zn-superoxide dismutase-1 (Sod1)-knockout mice.

PURPOSE: To investigate the role of a water and mucin secretagogue (3% diquafosol sodium eye drops) on the tear function and conjunctival ocular surface changes in Sod1(-/-) in comparison to the wild-type (WT) mice. METHODS: Fourteen eyes of 7 Sod1(-/-) male mice with C57BL/background and 14 eyes of 7 C57BL6 strain wild-type male mice were examined at 40 weeks in this study. All mice had application of 3% diquafosol ophthalmic solution six times a day for 2 weeks. Tear film stability and corneal epithelial damage was evaluated by fluorescein and Rose Bengal stainings. Anterior segment photography was performed before and after eye drop instillations. Aqueous tear quantity was measured with phenol red-impregnated cotton threads without anesthesia. Animals were sacrificed at 42 weeks after diquafosol treatment and the whole globe specimens were subjected to periodic acid Schiff staining. Goblet cell density was quantified by J Image software. Quantitative real-time PCR for conjunctival muc 5AC messenger RNA expression was also performed. RESULTS: Sod1(-/-) mice had significantly higher fluorescein staining scores compared to the WT mice before eye drop instillation. The mean tear film breakup time, Rose Bengal staining scores, and muc5 messenger RNA expression improved significantly with diquafosol treatment in both the WT and the knockout mice. The mean fluorescein staining score and aqueous tear quantity significantly improved in the Sod1(-/-) mice with treatment. A notable and consistent increase in goblet cells and decrease in inflammatory cell infiltrates could be confirmed in all specimens after 2 weeks of diquafosol eye drop application. CONCLUSIONS: Three percent diquafosol ophthalmic solution appears to be effective in the treatment of ocular surface disease in this age-related dry eye disease mouse model.