Hyperglycemia Induces Tear Reduction and Dry Eye in Diabetic Mice through the Norepinephrine-α1 Adrenergic Receptor-Mitochondrial Impairment Axis of Lacrimal Gland.

Dry eye syndrome is a common complication in diabetic patients with a prevalence of up to 54.3%. However, the pathogenic mechanisms underlying hyperglycemia-induced tear reduction and dry eye remain less understood. The present study indicated that both norepinephrine (NE) and tyrosine hydroxylase levels were elevated in the lacrimal gland of diabetic mice, accompanied by increased Fos proto-oncogene (c-FOS)+ cells in the superior cervical ganglion. However, the elimination of NE accumulation by surgical and chemical sympathectomy significantly ameliorated the reduction in tear production, suppressed abnormal inflammation of the lacrimal gland, and improved the severity of dry eye symptoms in diabetic mice. Among various adrenergic receptors (ARs), the α1 subtype played a predominant role in the regulation of tear production, as treatments of α1AR antagonists improved tear secretion in diabetic mice compared with ßAR antagonist propranolol. Moreover, the α1AR antagonist alfuzosin treatment also alleviated functional impairments of the meibomian gland and goblet cells in diabetic mice. Mechanically, the α1AR antagonist rescued the mitochondrial bioenergetic deficit, increased the mitochondrial DNA copy numbers, and elevated the glutathione levels of the diabetic lacrimal gland. Overall, these results deciphered a previously unrecognized involvement of the NE-α1AR-mitochondrial bioenergetics axis in the regulation of tear production in the lacrimal gland, which may provide a potential strategy to counteract diabetic dry eye by interfering with the α1AR activity.

Involvement of aberrant acinar cell proliferation in scopolamine-induced dry eye mice.

Dry eye is a multifactorial disease that causes dryness, inflammation and damage of ocular surface. Subcutaneous injection of the muscarinic cholinergic antagonist scopolamine under desiccating stress reduces tear production and induces dry eye symptoms in mice. However, the expression profile and pathogenic changes of the lacrimal gland remain incompletely understood. In the present study, we performed comparative transcriptomic analysis of lacrimal glands from the control and scopolamine-treated mice. Primary analysis identified 677 upregulated genes and 269 downregulated genes in the lacrimal gland of mice with scopolamine treatment. Unexpectedly, KEGG pathway and hierarchical clustering analysis showed the enrichment of "DNA replication" and "cell cycle" categories in the upregulated genes. Subsequently, we confirmed that the acinar cells were the major proliferating cells of lacrimal gland, which exhibited significant increasing of the proliferating cell nuclear antigen (PCNA) expression after scopolamine treatment, accompanied with the upregulation of DNA damage marker γ-H2AX. More importantly, both prophylactic and therapeutic administration of the cyclin-dependent kinase (CDK) inhibitor AT7519 rescued the tear reduction and alleviated dry eye severity in the scopolamine-treated mice, including corneal epithelial barrier function, lacrimal and corneal inflammation, and conjunctival goblet cell density. Therefore, we conclude that aberrant acinar cell proliferation is involved in the scopolamine-induced tear reduction and dry eye onset, which can be improved by AT7519 treatment.

The senescence difference between the central and peripheral cornea induced by sutures.

INTRODUCTION: Cell senescence plays a regulatory role in tissue fibrosis. Corneal scarring is usually more severe in the central cornea based on clinical observation. In this study, we attempted to explore the senescence difference between the central and peripheral cornea in an in vivo mouse model with suture-induced senescence and in an in vitro model of senescence with hydrogen peroxide (H2O2)-induced rabbit corneal fibroblasts. METHODS: Male Balb/c mice (6-8 weeks) received sutures in the central, superior, inferior, nasal, and temporal cornea. The sutures were removed on the 14th day. Corneal neovascularization was observed under a slit lamp microscope with a digital camera. The fibroblasts isolated from the central and peripheral rabbit cornea were induced with H2O2 to establish the senescence model in vitro. Senescence was evaluated with SA-ß-gal staining and gene expression analysis of p21, p27, and p53. RESULTS: Senescent cells accumulated in the corneal stroma from the third day to the 14th day after the operation and peaked on the 14th day. More senescent keratocytes were observed in the peripheral cornea of the mouse model. In vitro, the peripheral corneal fibroblasts were more prone to senescence due to H2O2. The polymerase chain reaction results showed that the senescence-related genes p21, p27, and p53 were highly expressed in the peripheral corneal fibroblasts compared with the central corneal fibroblasts. CONCLUSIONS: Senescent fibroblasts can limit tissue fibrosis; hence, the senescence difference between the central and peripheral cornea may contribute to the difference in scarring.

Topical calcitriol application promotes diabetic corneal wound healing and reinnervation through inhibiting NLRP3 inflammasome activation.

Vitamin D (VD) deficiency delays corneal wound healing in those with diabetes, which cannot be rescued with supplemental diet. Here, we employed topical calcitriol application to evaluate its efficiency in corneal wound healing and reinnervation in diabetic mice. Type 1 diabetic mice were topically administrated calcitriol, or subconjunctivally injected with NLRP3 antagonist MCC950 or IL-1ß blocking antibody after epithelial debridement. Serum VD levels, corneal epithelial defect, corneal sensation and nerve density, NLRP3 inflammasome activation, neutrophil infiltration, macrophage phenotypes, and gene expressions were examined. Compared with those of normal mice, diabetic mice showed reduced serum VD levels. Topical calcitriol application promoted corneal wound healing and nerve regeneration, as well as sensation recovery in diabetic mice. Moreover, calcitriol ameliorated neutrophil infiltration and promoted the M1-to-M2 macrophage transition, accompanied by suppressed overactivation of the NLRP3 inflammasome. Treatment with NLRP3 antagonist or IL-1ß blockage demonstrated similar improvements as those of topical calcitriol application. Additionally, calcitriol administration upregulated desmosomal and hemidesmosomal gene expression in the diabetic cornea. In conclusion, topical calcitriol application promotes corneal wound healing and reinnervation during diabetes, which may be related to the suppression of the overactivation of NLRP3 inflammasome.

Autocrine IL-1ß mediates the promotion of corneal neovascularization by senescent fibroblasts.

Our previous study has confirmed that senescent fibroblasts promote corneal neovascularization (CNV) partially via the enhanced secretion of matrix metalloproteases (MMPs). However, the regulation of MMP expression in senescent fibroblasts remained unclear. In this study, we identified that the expression and secretion levels of interleukin-1ß (IL-1ß) were significantly upregulated in senescent human corneal fibroblasts than that in normal fibroblasts. Moreover, compared with vehicle-pretreated senescent fibroblasts, IL-1ß pretreatment enhanced the expression of angiogenic factors but reduced the expression of angiostatic factors in senescent fibroblasts. When cocultured with human umbilical vein endothelial cells, IL-1ß-pretreated senescent fibroblasts more strongly promoted their proliferation, migration, and tube-formation capacities than the vehicle-controlled senescent fibroblasts. In addition, either interleukin-1 receptor antagonist or anti-IL-1ß neutralization completely inhibited the promotion of senescent fibroblasts in vascular tube formation in vitro and CNV in vivo. Therefore, we concluded that autocrine IL-1ß mediated the promotion of senescent fibroblasts on corneal neovascularization.

Epithelium-derived miR-204 inhibits corneal neovascularization.

MicroRNA-204 (miR-204) is highly expressed in cornea, here we explored the role and mechanism of miR-204 in corneal neovascularization (CNV). Mouse CNV was induced by intrastromal placement of suture in BALB/c mice with the subconjunctival injection of miR-204 agomir or negative control. Human primary limbal epithelial cells (LECs) and immortalized microvascular endothelial cells (HMECs) were used to evaluate the expression changes and anti-angiogenic effects of miR-204 under biomechanical stress (BS). The expression and localization of miR-204, vascular endothelial growth factor (VEGF) and their receptors were detected by quantitative real-time PCR, in situ hybridization, immunohistochemistry and Western blot. The results showed that miR-204 expression was mainly localized in epithelium and down-expressed in vascularized cornea. Subconjunctival injection of miR-204 agomir inhibited CNV and reduced the expression of VEGF and VEGF receptor 2. Similarly, miR-204 overexpression attenuated the increased expression of VEGF by biomechanical stress in LECs, and suppressed the proliferation, migration, and tube formation of HMECs. These novel findings indicate that epithelium-derived miR-204 inhibits suture-induced CNV through regulating VEGF and VEGF receptor 2.

Protective efficacy of a peptide derived from a potential adhesin of Pseudomonas aeruginosa against corneal infection.

Dissecting the interactions between Pseudomonas aeruginosa and corneal cells is important to identify a novel target for prevention and treatment of Pseudomonas keratitis. The current study began with a peptide identified by phage display, and was to investigate the protective efficacy against P. aeruginosa infection in cornea. The original peptide Pc-E, with high homology to a hypothetical membrane protein (HmpA) in P. aeruginosa, and the derived peptide Pc-EP, with the same sequence as a region in HmpA, were synthesized. Peptide Pc-EP could directly bind to HCEC, stronger than Pc-E, and specifically activate toll-like receptor 5, and thereby significantly induce the production of pro-inflammatory factors, such as IL-1ß, IL-6, IFN-γ and IL-17. Moreover, Pc-EP could act as an antagonist to inhibit the adhesion of wild-type P. aeruginosa to HCEC and mouse corneas. No inhibitory effect was observed on the adhesion of the strain loss of HmpA. When compared to the wild-type strain, the adhesion of the hmpA mutant to corneal cells was significantly decreased. Treatment of infected mouse corneas with Pc-EP before infection significantly decreased the bacterial load in the cornea and attenuated the corneal pathology. These results indicate that Pc-EP can be a useful prophylactic agent for P. aeruginosa keratitis.

Inductive differentiation of conjunctival goblet cells by γ-secretase inhibitor and construction of recombinant conjunctival epithelium.

γ-secretase inhibitor has been shown to promote intestinal goblet cell differentiation. We now demonstrated that the in vitro addition of γ-secretase inhibitor in the culture of human conjunctival epithelial cells significantly promoted the differentiation of conjunctival goblet cells with typical droplet-like phenotype, positive periodic acid-Schiff and goblet cell-specific Muc5Ac, cytokeratin 7 and Helix pomatia agglutinin lectin staining. Moreover, topical application of γ-secretase inhibitor promoted the differentiation of mouse conjunctival goblet cells in vivo. Furthermore, the expression of Notch target gene HES-1 was down-regulated during the differentiation of conjunctival goblet cells. In addition, we found that the recombinant conjunctival epithelium on amniotic membrane showed less goblet cell density and abnormal location when compared with normal conjunctival epithelium, which were improved by the addition of γ-secretase inhibitor in the final induction.

Trichostatin A inhibits transforming growth factor-ß-induced reactive oxygen species accumulation and myofibroblast differentiation via enhanced NF-E2-related factor 2-antioxidant response element signaling.

Trichostatin A (TSA) has been shown to prevent fibrosis in vitro and in vivo. The present study aimed at investigating the role of reactive oxygen species (ROS) scavenging by TSA on transforming growth factor-ß (TGF-ß)-induced myofibroblast differentiation of corneal fibroblasts in vitro. Human immortalized corneal fibroblasts were treated with TGF-ß in the presence of TSA, the NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI), the antioxidant N-acetyl-cysteine (NAC), the NF-E2-related factor 2-antioxidant response element (Nrf2-ARE) activator sulforaphane, or small interfering RNA. Myofibroblast differentiation was assessed by α-smooth muscle actin (α-SMA) expression, F-actin bundle formation, and collagen gel contraction. ROS, H(2)O(2), intracellular glutathione (GSH) level, cellular total antioxidant capacity, and the activation of Nrf2-ARE signaling were determined with various assays. Treatment with TSA and the Nrf2-ARE activator resulted in increased inhibition of the TGF-ß-induced myofibroblast differentiation as compared with treatment with DPI or NAC. Furthermore, TSA also decreased cellular ROS and H(2)O(2) accumulation induced by TGF-ß, whereas it elevated intracellular GSH level and cellular total antioxidant capacity. In addition, TSA induced Nrf2 nuclear translocation and up-regulated the expression of Nrf2-ARE downstream antioxidant genes, whereas Nrf2 knockdown by RNA interference blocked the inhibition of TSA on myofibroblast differentiation. In conclusion, this study provides the first evidence implicating that TSA inhibits TGF-ß-induced ROS accumulation and myofibroblast differentiation via enhanced Nrf2-ARE signaling.

Role of senescent fibroblasts on alkali-induced corneal neovascularization.

Cellular senescence acts as a potent regulator of tumor suppression and fibrosis limitation; however, its contribution and crosstalk with neovascularization during normal wound healing has not been examined. Here, we explored the role of senescent fibroblasts on neovascularization with a mouse model of alkali-induced corneal wound healing. Senescent cells accumulated in corneal stroma from day 7 to 27 after alkali burn and peaked on day 14, which was consistent with the development of corneal neovascularization (CNV). In vitro and in vivo assays confirmed that the senescent cells were derived primarily from activated corneal fibroblasts. Furthermore, senescent corneal fibroblasts exhibited enhanced synthesis and secretion of extracellular matrix-degrading enzymes (matrix metalloproteinases 2, 3, and 14 and tissue- and urokinase-type plasminogen activators) and angiogenic factors (vascular endothelial growth factor) and decreased expression of anti-angiogenic factors (pigment epithelium-derived factor and thrombospondins), which supported the proliferation, migration, and promotion of tube formation of vascular endothelial cells. Intrastromal injection of premature senescent fibroblasts induced CNV earlier than that of normal fibroblasts, while matrix metalloproteinase inhibitors blocked the early onset of senescent cell-induced CNV. Therefore, senescent fibroblasts promoted the alkali-induced CNV partially via the enhanced secretion of matrix metalloproteases.

Pluripotin enhances the expansion of rabbit limbal epithelial stem/progenitor cells in vitro.

This study was designed to demonstrate the effects of pluripotin on the proliferation, senescence and colony formation efficiency of rabbit limbal epithelial cells (RLECs) in vitro. Rabbit primary limbal epithelial cells were harvested and cultured in the presence of pluripotin. The cell proliferation was measured using the 3-(4,5)-dimethylthiahiazo(-z-y1)-3 5-di-phenytetrazoliumromide (MTT) assay and was also observed by confocal microscopy with Ki67 staining, whereas cell senescence was detected by senescence-associated ß-galactosidase (SA-ß-gal) staining. The colony morphology, colony-forming efficiency and colony size were observed and compared. The characteristics of the proliferating cells were examined by immunofluorescent staining using antibodies against deltaNP63, ABCG2 and Keratin 3/12. The results showed that pluripotin significantly promoted the proliferation of RLECs and increased the dividing cells with positive Ki67 staining at the concentrations lower than 400 nM. The colony-forming efficiency increased from 13.5% in the control cells to 26.4% in the 200 or 400 nM pluripotin-treated cells. The number of colonies of moderate size (600-900 µm) increased significantly in the presence of pluripotin (above 60.0% at 200 nM or 400 nM) compared with the untreated normal cells (18.6%), whereas the number of small-sized colonies (<600 µm) decreased from 79.5% for the control cells to lower than 35.0% at 200 nM or 400 nM pluripotin. Moreover, the cells treated with pluripotin stained negative with SA-ß-gal, while the untreated cells showed visible positive staining. Immunofluorescent staining suggested that the pluripotin treatment resulted in higher positive staining for the limbal stem cell markers (deltaNP63 and ABCG2) and down-regulated of differentiated corneal epithelial cell marker (Keratin 3/12). This study confirmed that the small molecular compound pluripotin promoted the proliferation of rabbit limbal epithelial cells by improving the expansion of limbal stem/progenitor cells in vitro.

Pathogenic Role of Endoplasmic Reticulum Stress in Diabetic Corneal Endothelial Dysfunction.

PURPOSE: Progressive corneal edema and endothelial cell loss represent the major corneal complications observed in diabetic patients after intraocular surgery. However, the underlying pathogenesis and potential treatment remain incompletely understood. METHODS: We used streptozotocin-induced type 1 diabetic mice and db/db type 2 diabetic mice as diabetic animal models. These mice were treated with the endoplasmic reticulum (ER) stress agonist thapsigargin; 60-mmHg intraocular pressure (IOP) with the ER stress antagonist 4-phenylbutyric acid (4-PBA); mitochondria-targeted antioxidant SkQ1; or reactive oxygen species scavenger N-acetyl-l-cysteine (NAC). Corneal thickness and endothelial cell density were measured before and after treatment. Human corneal endothelial cells were treated with high glucose with or without 4-PBA. The expression of corneal endothelial- and ER stress-related genes was detected by western blot and immunofluorescence staining. Mitochondrial bioenergetics were measured with an Agilent Seahorse XFp Analyzer. RESULTS: In diabetic mice, the appearance of ER stress preceded morphological changes in the corneal endothelium. The persistent ER stress directly caused corneal edema and endothelial cell loss in normal mice. Pharmacological inhibition of ER stress was sufficient to mitigate corneal edema and endothelial cell loss in both diabetic mice after high IOP treatment. Mechanistically, inhibiting ER stress ameliorated the hyperglycemia-induced mitochondrial bioenergetic deficits and improved the barrier and pump functional recovery of the corneal endothelium. When compared with NAC, 4-PBA and SkQ1 exhibited better improvement of corneal edema and endothelial cell loss in diabetic mice. CONCLUSIONS: Hyperglycemia-induced ER stress contributes to the dysfunction of diabetic corneal endothelium, and inhibiting ER stress may offer therapeutic potential by improving mitochondrial bioenergetics.

Different cellular effects of four anti-inflammatory eye drops on human corneal epithelial cells: independent in active components.

PURPOSE: To evaluate and compare the cellular effects of four commercially available anti-inflammatory eye drops and their active components on human corneal epithelial cells (HCECs) in vitro. METHODS: The cellular effects of four eye drops (Bromfenac Sodium Hydrate Eye Drops, Pranoprofen Eye Drops, Diclofenac Sodium Eye Drops, and Tobramycin & Dex Eye Drops) and their corresponding active components were evaluated in an HCEC line with five in vitro assays. Cell proliferation and migration were measured using 3-(4,5)-dimethylthiahiazo (-z-y1)-3 5-di-phenytetrazoliumromide (MTT) assay and transwell migration assay. Cell damage was determined with the lactate dehydrogenase (LDH) assay. Cell viability and median lethal time (LT50) were measured by 7-amino-actinomycin D (7-AAD) staining and flow cytometry analysis. RESULTS: Cellular effects after exposure of HCECs to the four anti-inflammatory eye drops were concentration dependent. The differences of cellular toxicity on cell proliferation became significant at lower concentrations (<0.002%). Diclofenac Sodium Eye Drops showed significant increasing effects on cell damage and viability when compared with the other three solutions. Tobramycin & Dex Eye Drops inhibited the migration of HCECs significantly. Tobramycin & Dex Eye Drops showed the quickest effect on cell viability: the LT50 was 3.28, 9.23, 10.38, and 23.80 min for Tobramycin & Dex Eye Drops, Diclofenac Sodium Eye Drops, Pranoprofen Eye Drops, and Bromfenac Sodium Hydrate Eye Drops, respectively. However, the comparisons of cellular toxicity revealed significant differences between the eye drops and their active components under the same concentration. The corneal epithelial toxicity differences among the active components of the four eye drops became significant as higher concentration (>0.020%). CONCLUSIONS: The four anti-inflammatory eye drops showed different cellular effects on HCECs, and the toxicity was not related with their active components, which provides new reference for the clinical application and drug research and development.

In vitro culture of human fetal corneal endothelial cells.

PURPOSE: To explore and optimize a proper culture system for human fetal corneal endothelial cells (hFCECs), including the methods of primary culture, passage and cryopreservation. METHODS: Fresh fetal corneas were explanted to propagate primary corneal endothelial cells. The cells were cultured in DMEM/F-12 supplemented with 10% fetal bovine serum (FBS) in the absence or presence of the extracts from bovine corneal endothelium cells (bCECs), and the cells were identified with immunocytochemical staining. The passage and cryopreservation of hFCECs were optimized according to previous reports on adult corneal endothelial cells. RESULTS: Using the explant culture method, hFCECs migrated successfully within 3 days and assumed polygonal-shaped corneal endothelial morphology. The optimizing methods were 0.125% trypsin + 0.02% EDTA for passage and 10% DMSO + 90% FBS for cryopreservation. Recovered hFCECs from cryopreservation remained typical morphology and immunological markers of corneal endothelial cells, including positive staining of NSE, Nestin, Ki67 and ZO-1, and negative staining of CK3/12, which demonstrated that they retained the characterizations of corneal endothelial cells and proliferative capacity. Moreover, the extracts from bCECs can promote the proliferative capacity of hFCECs significantly, while maintaining their typical endothelial morphology. CONCLUSIONS: The culture conditions of human fetal corneal endothelial cells were firstly optimized, including the primary culture, passage and cryopreservation. Meanwhile, we confirmed that the extracts from bovine corneal endothelium promoted the proliferative capacity while maintaining the morphology of hFCECs in vitro.

Development of a novel ex vivo model of corneal fungal adherence.

PURPOSE: To construct a suitable ex vivo model for the research of molecular mechanisms and the pharmacological screening of fungal adherence on the corneal surface. MATERIALS AND METHODS: Mouse eyes were divided into three groups as follows: a control group with normal corneal epithelium, a group with corneal epithelium that was needle-scarified, and a group with corneal epithelium that was completely debrided. All 96 corneas were placed in organ culture and inoculated with 5 µl spore suspensions of Candida albicans at 109, 108, or 107 colony-forming units (CFU)/ml and incubated for 0, 30, 60, or 120 min. The corneas were homogenated and diluted for quantification by counting the CFU. The effects of amphotericin B or chondroitin sulfate on the adherence of the fungal spores were evaluated with the ex vivo organ culture model and were also compared with the human corneal epithelium monolayer model in vitro. RESULTS: Compared with the normal corneas with intact epithelium, the corneas with scarified and debrided epithelium adhered more spores for above two and four folds. The spore adhesion on the corneal surface was in an inoculation concentration- and incubation time-dependent manner. Moreover, both amphotericin B and chondroitin sulfate inhibited the adhesion of C. albicans spores on the corneal surface, but the inhibitory rates were different between the ex vivo corneal organ culture model and the in vitro corneal epithelium monolayer model. CONCLUSIONS: The corneal organ culture was a suitable ex vivo model for the research of fungal adhesion mechanisms and drug screening.

Hyperglycemia-induced severe mitochondrial bioenergetic deficit of lacrimal gland contributes to the early onset of dry eye in diabetic mice.

Dry eye and diabetic keratopathy represent the major diabetic complications in ocular surface. Here we found that diabetic mice exhibited the early onset of reduced tear secretion and lacrimal gland weight compared to the symptoms of diabetic keratopathy. Considering to the high bioenergetic needs in lacrimal gland and cornea, we hypothesized that hyperglycemia may cause different severity of mitochondrial bioenergetic deficit between them. Through the measurement of oxygen consumption rate (OCR) and basal extracellular acidification rate (ECAR), we found the apparent alterations of mitochondrial bioenergetic profiles in diabetic lacrimal gland and cornea, accompanied with the mtDNA damage and copy number reduction, as well as the reduced glutathione content. Comparative analysis revealed that mouse lacrimal gland cells exhibited 2-3 folds higher of basal, ATP production, maximal OCR and basal ECAR than corneal epithelial cells in normoglycemia. However, the differences were slightly significant or even not detected in hyperglycemia. Accordingly, the mitochondrial bioenergetic metabolism of lacrimal gland was more compromised than that of corneal epithelium in diabetic mice. Through the administration of mitochondrial-targeted antioxidant SkQ1, the severity of dry eye and diabetic keratopathy was significantly attenuated with the improved mitochondrial function. These results indicate that the susceptibility of mitochondrial bioenergetic deficit in diabetic lacrimal gland may contribute to the early onset of dry eye, while mitochondria-targeted antioxidant possesses therapeutic potential for diabetic dry eye and keratopathy.

TGFbeta mediated transition of corneal fibroblasts from a proinflammatory state to a profibrotic state through modulation of histone acetylation.

Corneal fibroblasts exhibit different phenotypes in different phases of corneal wound healing. In the inflammatory phase, the cells assume a proinflammatory phenotype and produce large amounts of cytokines and chemokines, but in the proliferative and remodeling phases, they adapt a profibrotic state, differentiate into myofibroblasts and increase extracellular matrix protein synthesis, secretion, and deposition. In the present study, the molecular mechanisms regulating the transition of corneal fibroblasts from the proinflammatory state to the profibrotic state were investigated. Corneal fibroblasts were treated with TGFbeta, a known profibrotic and anti-inflammatory factor in wound healing, in the absence or presence of trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor. The results revealed that TGFbeta induced the profibrotic transition of corneal fibroblasts, including increased extracellular matrix synthesis, morphological changes, and assembly of actin filaments. Meanwhile, proinflammatory gene expressions of corneal fibroblasts were down-regulated with the treatment of TGFbeta, as confirmed by cDNA microarray, real time PCR and ELISA. Moreover, TSA reversed the TGFbeta-mediated transition of corneal fibroblasts from the proinflammatory state to the profibrotic state, as accompanied by histone hyperacetylations. In conclusion, TGFbeta suppressed the production of proinflammatory factors and enhanced the expression of matrix remodeling genes of corneal fibroblasts in the transition from the proinflammatory state to the profibrotic state, and the dual roles of TGFbeta on the phenotype regulations of corneal fibroblasts were mediated by altered histone acetylation.

Sustained Release of TPCA-1 from Silk Fibroin Hydrogels Preserves Keratocyte Phenotype and Promotes Corneal Regeneration by Inhibiting Interleukin-1ß Signaling.

Corneal injury due to ocular trauma or infection is one of the most challenging vision impairing pathologies that exists. Many studies focus on the pro-inflammatory and pro-angiogenic effects of interleukin-1ß (IL-1ß) on corneal wound healing. However, the effect of IL-1ß on keratocyte phenotype and corneal repair, as well as the underlying mechanisms, is not clear. This study reports, for the first time, that IL-1ß induces phenotype changes of keratocytes in vitro, by significantly down-regulating the gene and protein expression levels of keratocyte markers (Keratocan, Lumican, Aldh3a1 and CD34). Furthermore, it is found that the NF-κB pathway is involved in the IL-1ß-induced changes of keratocyte phenotype, and that the selective IKKß inhibitor TPCA-1, which inhibits NF-κB, can preserve keratocyte phenotype under IL-1ß simulated pathological conditions in vitro. By using a murine model of corneal injury, it is shown that sustained release of TPCA-1 from degradable silk fibroin hydrogels accelerates corneal wound healing, improves corneal transparency, enhances the expression of keratocyte markers, and supports the regeneration of well-organized epithelium and stroma. These findings provide insights not only into the pathophysiological mechanisms of corneal wound healing, but also into the potential development of new treatments for patients with corneal injuries.

Induction of Fibroblast Senescence During Mouse Corneal Wound Healing.

Purpose: To investigate the presence and role of fibroblast senescence in the dynamic process of corneal wound healing involving stromal cell apoptosis, proliferation, and differentiation. Methods: An in vivo corneal wound healing model was performed using epithelial debridement in C57BL/6 mice. The corneas were stained using TUNEL, Ki67, and α-smooth muscle actin (α-SMA) as markers of apoptosis, proliferation, and myofibroblastic differentiation, respectively. Cellular senescence was confirmed by senescence-associated ß-galactosidase (SA-ß-gal) staining and P16Ink4a expression. Mitogenic response and gene expression were compared among normal fibroblasts, H2O2-induced senescent fibroblasts, and TGF-ß-induced myofibroblasts in vitro. The senescence was further detected in mouse models of corneal scarring, alkali burn, and penetrating keratoplasty (PKP). Results: The apoptosis and proliferation of corneal stromal cells were found to peak at 4 and 24 hours after epithelial debridement. Positive staining of SA-ß-gal was observed clearly in the anterior stromal cells at 3 to 5 days. The senescent cells displayed P16Ink4a+ vimentin+ α-SMA+, representing the major origin of activated corneal resident fibroblasts. Compared with normal fibroblasts and TGF-ß-induced myofibroblasts, H2O2-induced senescent fibroblasts showed a nonfibrogenic phenotype, including a reduced response to growth factor basic fibroblast growth factor (bFGF) or platelet-derived growth factor-BB (PDGF-BB), increased matrix metalloproteinase (MMP)1/3/13 expression, and decreased fibronectin and collagen I expression. Moreover, cellular senescence was commonly found in the mouse corneal scarring, alkali burn, and PKP models. Conclusions: Corneal epithelial debridement induced the senescence of corneal fibroblasts after apoptosis and proliferation. The senescent cells displayed a nonfibrogenic phenotype and may be involved in the self-limitation of corneal fibrosis.

Therapeutic Effects of STAT3 Inhibition on Experimental Murine Dry Eye.

Purpose: To investigate the therapeutic effects of targeting signal transducer and activator of transcription-3 (STAT3) activation on the ocular surface damage of dry eye in mice. Methods: Adult Balb/C and C57BL/6 mice with benzalkonium chloride (BAC) treatment, lacrimal gland excision, and meibomian gland dysfunction were used as dry eye models. The levels of phosphorylated STAT3 (p-STAT3) were detected with immunofluorescence staining and Western blotting. STAT3 inhibition was performed by topical application of STAT3 inhibitor S3I-201. Corneal epithelial barrier function, tear production, and conjunctival goblet cell density were quantified with fluorescein sodium staining, phenol red cotton test, and histochemical staining. The expressions of matrix metalloproteinase (MMP)-3/9, TUNEL, and inflammation cytokines were assessed with immunofluorescence staining, qPCR, and ELISA assays. The therapeutic effect of S3I-201 was further compared with the Janus kinase inhibitor tofacitinib and ruxolitinib. Results: Elevated levels of nuclear p-STAT3 were detected in the corneal and conjunctival epithelium of three dry eye models. Topical application of S3I-201 improved corneal epithelial barrier function, increased tear production and conjunctival goblet cell density in BAC-induced dry eye mice. Moreover, S3I-201 decreased the expression of MMP-3/9, suppressed the apoptosis of corneal and conjunctival epithelial cells, and reduced the levels of IL-1ß, IL-6, IL-17A, and IFN-γ. Compared with tofacitinib and ruxolitinib, the STAT3 inhibitor S3I-201 showed superior improvement of tear production and inflammatory cytokine expression in lacrimal gland. Conclusions: Elevated STAT3 activation is involved in the pathogenesis of dry eye, while targeting STAT3 effectively alleviates BAC-induced ocular surface damage.