The Role of SLIT3-ROBO4 Signaling in Endoplasmic Reticulum Stress-Induced Delayed Corneal Epithelial and Nerve Regeneration.

Purpose: In the present study, we aim to elucidate the underlying molecular mechanism of endoplasmic reticulum (ER) stress induced delayed corneal epithelial wound healing and nerve regeneration. Methods: Human limbal epithelial cells (HLECs) were treated with thapsigargin to induce excessive ER stress and then RNA sequencing was performed. Immunofluorescence, qPCR, Western blot, and ELISA were used to detect the expression changes of SLIT3 and its receptors ROBO1-4. The role of recombinant SLIT3 protein in corneal epithelial proliferation and migration were assessed by CCK8 and cell scratch assay, respectively. Thapsigargin, exogenous SLIT3 protein, SLIT3-specific siRNA, and ROBO4-specific siRNA was injected subconjunctivally to evaluate the effects of different intervention on corneal epithelial and nerve regeneration. In addition, Ki67 staining was performed to evaluate the proliferation ability of epithelial cells. Results: Thapsigargin suppressed normal corneal epithelial and nerve regeneration significantly. RNA sequencing genes related to development and regeneration revealed that thapsigargin induced ER stress significantly upregulated the expression of SLIT3 and ROBO4 in corneal epithelial cells. Exogenous SLIT3 inhibited normal corneal epithelial injury repair and nerve regeneration, and significantly suppressed the proliferation and migration ability of cultured mouse corneal epithelial cells. SLIT3 siRNA inhibited ROBO4 expression and promoted epithelial wound healing under thapsigargin treatment. ROBO4 siRNA significantly attenuated the delayed corneal epithelial injury repair and nerve regeneration induced by SLIT3 treatment or thapsigargin treatment. Conclusions: ER stress inhibits corneal epithelial injury repair and nerve regeneration may be related with the upregulation of SLIT3-ROBO4 pathway.

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.

Corneal calcification of acellular porcine corneal stroma following lamellar keratoplasty.

PURPOSE: To describe the corneal calcification of acellular porcine corneal stroma (APCS) following lamellar keratoplasty (LKP) and identify risk factors. METHODS: Two cases of APCS calcification were evaluated by slit-lamp photography and anterior segment optical coherence tomography (AS-OCT). von Kossa staining and scanning electron microscope/energy-dispersive spectrometry (SEM/EDS) were performed on pathologic tissue. Associated graft and postoperative risk factors were analysed. Acellular porcine corneal stroma (APCS) cleanliness and element content after rinsing with sterilized water were observed by SEM/EDS and inductively coupled plasma mass spectrometry. Calcium metabolism-related proteins were analysed by protein mass spectrometry. Corneal epithelial defects and postoperative medications were reviewed. RESULTS: Two cases of APCS calcification occurred at 23 and 22 days postoperatively. Anterior segment optical coherence tomography (AS-OCT) and von Kossa staining demonstrated calcium deposition in the superficial stroma composed of calcium, phosphorus and oxygen conforming to the Ca/P ratio of hydroxyapatite. Phosphate crystals were present on the APCS surface and decreased with number of rinsing times. The phosphorus content of APCS was minimal after rinsing 10 times and avoiding excessive corneal swelling. Calcium metabolism-related proteins were downregulated in APCS. Patients with corneal calcification had 1-week postoperative corneal epithelial defects and were treated with three types of phosphorous eyedrops. CONCLUSIONS: Acellular porcine corneal stroma (APCS) calcification occurs in the superficial corneal stroma about 1 month after LKP. The application of AS-OCT, von Kossa staining and SEM/EDS provides a basis for the clinical and pathological diagnosis of corneal calcification. The associated risk factors were mainly high phosphorus content and downregulated calcium metabolism-related proteins in APCS. Postoperative epithelial defects, inflammation and use of phosphorous eyedrops may promote corneal calcification.

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.

Natural cross-linker-stabilized acellular porcine corneal stroma for lamellar keratoplasty.

Acellular porcine corneal stroma (APCS) is a promising alternative to human donor cornea for lamellar keratoplasty (LKP). However, the detergents, enzymes and physical forces used during decellularization unavoidably alter the cornea's extracellular matrix composition and disrupt its ultrastructure, making it less mechanically stable and liable to degradation both in vitro and in vivo. Herein, we systematically analyzed the low biomechanics and easy degradability of APCS in terms of structure and protein composition. Then, we introduced natural cross-linkers, namely proanthocyanidin (PA), epigallocatechin-3-gallate and genipin, to stabilize the APCS that exhibited color variations during crosslinking. Then, we developed a protective crosslinking system by combining cross-linkers with bovine serum albumin (BSA) to reduce color change, maintain transparency and improve the mechanical property of APCS. PA/BSA-crosslinked APCS (PA/BSA-APCS) shows favorable corneal transparency and swelling property; the improved overall and surface corneal biomechanics were comparable to those of human cornea, revealing strong resistance to enzymatic degradation and good biocompatibility. Results from LKP in the rabbit model showed complete re-epithelialization without graft melting, the stitches were scarcely loosened after the operation and more host keratocytes had migrated in PA/BSA-APCS at six months post-operation. Therefore, PA/BSA-APCS could be useful as a corneal substitute for tissue regeneration and the protective crosslinking system could be applicable in other bioengineering fields.

The proinflammatory cytokines IL-1ß and TNF-α modulate corneal epithelial wound healing through p16Ink4a suppressing STAT3 activity.

The proinflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) are involved in the corneal inflammatory response and wound healing following corneal injuries. However, the mechanism by which proinflammatory cytokines modulate corneal epithelial wound healing remains unclear. In this study, we found that IL-1ß or TNF-α was transiently elevated during corneal epithelial wound healing in mice. After corneal epithelial debridement, persistent treatment with IL-1ß or TNF-α restrained the level of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) and boosted the level of cell cycle inhibitor p16Ink4a , resulting in impaired corneal epithelial repair. When p16Ink4a was deleted, the p-STAT3 level in corneal epithelium was enhanced and corneal epithelial wound healing was clearly accelerated. In diabetic mice, IL-1ß, TNF-α, and p16Ink4a appeared a sustained and strong expression in the corneal epithelium, and p16Ink4a knockdown partially reverted the defective diabetic corneal epithelial repair. Furthermore, immunoprecipitation proved that p16Ink4a interacted with p-STAT3 and thus possibly suppressed the STAT3 activity. Our findings revealed a novel mechanism that the proinflammatory cytokines modulate corneal epithelial wound healing via the p16Ink4a -STAT3 signaling.

Ocular surface repair using decellularized porcine conjunctiva.

The primary functions of the conjunctiva embody ocular surface protection and the maintenance of the tear film equilibrium. Severe conjunctival defects such as symblepharon may impair the integrity of ocular surface and cause loss of visual functions. Here we report the use of a decellularized porcine conjunctiva (DPC) for conjunctival reconstruction in rabbit models and in clinic. Our results show that the major xenoantigens are efficiently removed, while abundant matrix components and integrated microstructures are well preserved in the DPC. These characteristics provide mechanical support and favorable histocompatibility for repairing damaged conjunctiva. The DPC application has demonstrated enhanced transplant stability and improved epithelial regeneration in severe ocular surface damage comparing to those of amniotic membrane (AM), the most frequently applied matrix for ocular surface reconstruction nowadays. In order to test the DPC performance in clinic, three patients with pterygium and one patient with symblepharon underwent transplant with DPC. The grafts in all cases were completely re-epithelized and no graft melt or fibroplasia were observed. These results suggest that the strategy we developed is feasible and effective for conjunctival reconstruction and ocular surface repair. STATEMENT OF SIGNIFICANCE: In this study, we adopted an innovative approach to prepare decellularized porcine conjunctiva (DPC). The intricate conjunctiva-specific structures and abundant matrix components were preserved in DPC, which offers favorable mechanical properties for graft. DPC has shown positive effects in ocular surface repair, which has been proven particularly in a rabbit model with severe symblepharon. Reconstructed conjunctiva by DPC exhibited epithelial heterogeneity, extremely resembling that of native conjunctiva. In addition, results from clinical studies were encouraging for pterygium and symblepharon and clinical application of DPC is promising.

Rapid porcine corneal decellularization through the use of sodium N-lauroyl glutamate and supernuclease.

Corneal decellularization represents a promising alternative source of human donor with global shortage. Multiple methods have been developed for the preparation of decellularized porcine corneal stroma. However, most strategies relied on long-time treatment to facilitate the entry of detergents or nucleases, which may cause irreversible ultrastructural damage. Here, we developed a rapid decellularization method for porcine corneal stroma through the combined mild detergent sodium N-lauroyl glutamate (SLG) and supernuclease. Compared with traditional methods, the novel decellularization method allowed the efficient removal of xenoantigen DNA within 3 h, while retaining the ultrastructure, transparency, and mechanical properties of porcine corneas. When transplanted in rabbit model for 1 month, the decellularized porcine corneal grafts presented favorable transparency and biocompatibility without immune rejection. Therefore, the combined use of detergent SLG and supernuclease may serve as a promising method for the clinical use of decellularized porcine cornea.

Different Effects of Pro-Inflammatory Factors and Hyperosmotic Stress on Corneal Epithelial Stem/Progenitor Cells and Wound Healing in Mice.

Chronic inflammation and severe dry eye are two important adverse factors for the successful transplant of cultured limbal stem cells. The aim of this study was to investigate the effects of inflammation and hyperosmotic stress (a key pathological factor in dry eye) on corneal epithelial stem cells (CESCs) and corneal epithelial wound healing. We observed that the CESCs exhibited significant morphological changes when treated with interleukin-1 beta (IL-1ß), tumor necrosis factor alpha (TNF-α), or hyperosmotic stress. Colony-forming efficiency or colony-forming size was decreased with the increasing concentrations of IL-1ß, TNF-α, or hyperosmotic stress, which was exacerbated when treated simultaneously with pro-inflammatory factors and hyperosmotic stress. However, the colony-forming capacity of CESCs recovered more easily from pro-inflammatory factor treatment than from hyperosmotic stress treatment. Moreover, when compared with pro-inflammatory factors treatment, hyperosmotic stress treatment caused a more significant increase of apoptotic and necrotic cell numbers and cell cycle arrest in the G2/M phase. Furthermore, the normal ability of corneal epithelial wound healing in the mice model was suppressed by both pro-inflammatory factors and hyperosmotic stress treatment, and especially severely by hyperosmotic stress treatment. In addition, inflammation combined with hyperosmotic stress treatment induced more serious epithelial repair delays and apoptosis in corneal epithelium. Elevated levels of inflammatory factors were found in hyperosmotic stress-treated cells and mice corneas, which persisted even during the recovery period. The results suggested that pro-inflammatory factors cause transient inhibition, while hyperosmotic stress causes severe apoptosis and necrosis, persistent cell cycle arrest of CESCs, and severe corneal wound healing delay. Stem Cells Translational Medicine 2019;8:46-57.

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.

BRD4 inhibitor JQ1 inhibits and reverses mechanical injury-induced corneal scarring.

Corneal scarring is characterized by the improper deposition of extracellular matrix components and myofibroblast differentiation from keratocytes. The bromodomain-containing protein 4 (BRD4) inhibitor JQ1 has been shown to attenuate pathological fibrosis. The present study aimed to explore the potential therapeutic effect of JQ1 on mechanical injury-induced mouse corneal scarring and TGFß-induced human corneal myofibroblast differentiation and the related mechanism. The corneal scarring and myofibroblast differentiation were evaluated with clinical observation and fibrosis-related gene expression analysis. In mice, subconjunctivally injected JQ1 suppressed the initial development and reversed the established progression of corneal scarring, while having no impairment on the epithelial regenerative capacity. BRD4 inhibition with either JQ1 or small-interfering RNA inhibited the differentiation and promoted the dedifferentiation of human corneal myofibroblasts. Moreover, JQ1 attenuated the accumulation of intracellular reactive oxygen species induced by TGFß treatment, induced Nrf2 nuclear translocation and activated the expression of Nrf2-ARE downstream antioxidant genes. In conclusion, this study implicates that JQ1 suppresses and reverses corneal scarring through the regulation of BRD4 inhibition and Nrf2-dependant antioxidant induction.

Role of Dendritic Cells and Inflammatory Cells in Herpetic Endotheliitis: Analysis Using In Vivo Confocal Microscopy.

PURPOSE: To observe the pathological changes in dendritic cells (DCs) and inflammatory cells in the corneal epithelium and endothelium using in vivo confocal microscopy during the management of herpetic endotheliitis. METHODS: A total of 110 eyes with herpetic endotheliitis were included. All patients were treated with antiviral agents combined with glucocorticoids. Changes in corneal edema were observed using slit-lamp microscopy and anterior segment optical coherence tomography. DCs and inflammatory cells in the epithelium and endothelium were detected using in vivo confocal microscopy before treatment and at 1 to 2 weeks and 1 and 3 months after treatment. Recurrence was monitored for 2 years. The contralateral normal eyes were evaluated as controls. RESULTS: Mean density of DCs decreased at 1 month after treatment (100 ± 14 cells/mm) compared with before treatment (148 ± 26 cells/mm, P < 0.001). At 3 months, DCs returned to small and dendritiform reflective corpuscular cells at a density of 44 ± 11 cells/mm (P < 0.001), and the mean density of endothelial cells (2011 ± 173 cells/mm) was significantly lower than in controls (2472 ± 233 cells/mm, P = 0.002). Inflammatory cells residing in the epithelium were significantly reduced in number and disappeared at 1 to 2 weeks, and those at the endothelial surface almost disappeared at 1 month. There was no relapse during the follow-up evaluation. CONCLUSIONS: DCs and inflammatory cells in the epithelial and endothelial cell layers of the cornea changed constantly in density, morphology, and distribution during the therapeutic process of herpetic endotheliitis. Adequate understanding of these alterations may help to guide the management of this disease.

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.

Subconjunctival Bevacizumab Injection Impairs Corneal Innervations and Epithelial Wound Healing in Mice.

Purpose: To investigate the effects of subconjunctival bevacizumab injection on the corneal nerve, sensitivity, and epithelial wound healing in mice. Methods: Adult C57BL/6 mice were treated with subconjunctival injection of 1, 2, 5, or 25 mg/mL bevacizumab. The corneal nerve was observed with whole-mount anti-ß3-tubulin fluorescence staining. Corneal sensitivity was measured with a Cochet-Bonnet esthesiometer. The protein levels of pigment epithelium-derived factor (PEDF), nerve growth factor (NGF), glial-derived neurotrophic factor (GDNF), and ciliary neurotrophic factor (CNTF) were measured by ELISA. The corneal epithelial wound-healing rate was evaluated by fluorescein staining. The recovery of impaired mouse corneal innervations and epithelial wound-healing rate following bevacizumab injection was evaluated with the co-injection of PEDF, NGF, or CNTF. Results: Subconjunctival bevacizumab injection caused apparent corneal nerve degeneration, attenuated corneal sensitivity, and delayed corneal epithelial wound healing and nerve regeneration in normal mice, which was more significant with increased concentration and times of the bevacizumab injection. However, the corneal nerve and sensitivity gradually improved and recovered in mice with a single injection of 1 to 5 mg/mL bevacizumab. Moreover, the bevacizumab injection significantly decreased the corneal PEDF, NGF, and CNTF content, whereas exogenous PEDF, NGF, or CNTF supplement attenuated impairment of the corneal nerve, sensitivity, and epithelial wound healing after subconjunctival bevacizumab injection. Conclusions: Subconjunctival bevacizumab injection impairs corneal innervations, epithelial wound healing, and nerve regeneration in normal mice, which may be caused by the reduction of neurotrophic factor content in the cornea.

Notch signaling modulates proliferative vitreoretinopathy via regulating retinal pigment epithelial-to-mesenchymal transition.

Elevated Notch signaling has been verified in a large range of fibrotic diseases developed in the kidney, liver, and lung, inducing the development of the epithelial-mesenchymal transition (EMT). The aim of this study was to observe the involvement of Notch signaling in the EMT of retinal pigment epithelial (RPE) cells and the pathogenesis of proliferative vitreoretinopathy (PVR). In vitro cultivated human RPE cells (ARPE-19) were treated with 10 ng/mL transforming growth factor (TGF)-ß1 for 24, 48, and 72 h. The expression levels of ZO-1, α-SMA, vimentin, Notch1 intracellular domain (NICD1), and Hes-1 were evaluated with quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence staining or Western blot. TGF-ß1 induced EMT and the activation of Notch signaling in ARPE-19 cells. To examine the effect of Notch inhibition on TGF-ß1-induced EMT and PVR formation, ARPE-19 cells were preincubated with γ-secretase inhibitor LY411575 before TGF-ß1 treatment. Mouse PVR model was used for in vivo study. ARPE-19 cells were injected intravitreously with or without the LY411575 to examine the effect of Notch inhibition on PVR formation. LY411575 significantly attenuated EMT by inhibiting the Notch signaling activation in vitro. PVR was induced by intravitreal injections of ARPE-19 cells, while LY411575 inhibited mouse PVR formation in vivo. Notch signaling plays a critical role in TGF-ß1-induced EMT in vitro and mice PVR model, which provides a novel insight into the pathogenesis of PVR. The specific inhibition of Notch signaling by γ-secretase inhibitor may provide a new approach for the prevention of PVR.

Immunological Properties of Corneal Epithelial-Like Cells Derived from Human Embryonic Stem Cells.

Transplantation of ex vivo expanded corneal limbal stem cells (LSCs) has been the main treatment for limbal stem cell deficiency, although the shortage of donor corneal tissues remains a major concern for its wide application. Due to the development of tissue engineering, embryonic stem cells (ESCs)-derived corneal epithelial-like cells (ESC-CECs) become a new direction for this issue. However, the immunogenicity of ESC-CECs is a critical matter to be solved. In the present study, we explored the immunological properties of ESC-CECs, which were differentiated from ESCs. The results showed that ESC-CECs had a similar character and function with LSCs both in vitro and in vivo. In ESC-CECs, a large number of genes related with immune response were down-regulated. The expressions of MHC-I, MHC-II, and co-stimulatory molecules were low, but the expression of HLA-G was high. The ESC-CECs were less responsible for T cell proliferation and NK cell lysis in vitro, and there was less immune cell infiltration after transplantation in vivo compared with LSCs. Moreover, the immunological properties were not affected by interferon-γ. All these results indicated a low immunogenicity of ESC-CECs, and they can be promising in clinical use.

Notch signaling regulates M2 type macrophage polarization during the development of proliferative vitreoretinopathy.

Macrophages play an important role in the pathogenesis of proliferative vitreoretinopathy (PVR). M2 macrophages can promote tissue remodeling and repair. In this study, CD206 positive M2 type macrophages were found in preretinal fibrous membranes of the mouse model of PVR induced by the intravitreal injection of retinal pigment epithelial (RPE) cells. Notch signaling determines M2 macrophage polarization. The specific inhibition of Notch signaling pathway by the intravitreal injection of γ-secretase inhibitor DAPT attenuated RPE cells-induced PVR formation as demonstrated by the decreased expression of α-SMA, and inhibited M2 type macrophage infiltation as demonstrated by the decreased expression of Arg-1. Notch signaling may modulate PVR formation by regulating M2 type macrophage polarization.