Weiyun Shi

Macrophage Depletion Impairs Corneal Wound Healing after Autologous Transplantation in Mice

Purpose Macrophages have been shown to play a critical role in the wound healing process. In the present study, the role of macrophages in wound healing after autologous corneal transplantation was investigated by depleting local infiltrated macrophages. Methods Autologous corneal transplantation model was used to induce wound repair in Balb/c mice. Macrophages were depleted by sub-conjunctival injections of clodronate-containing liposomes (Cl2MDP-LIP). The presence of CD11b+ F4/80+ macrophages, α-smooth muscle actin+ (α-SMA+) myofibroblasts, CD31+ vascular endothelial cells and NG2 + pericytes was examined by immunohistochemical and corneal whole-mount staining 14 days after penetrating keratoplasty. Peritoneal macrophages were isolated from Balb/c mice and transfused into conjunctiva to examine the recovery role of macrophages depletion on wound healing after autologous corneal transplantation. Results Sub-conjunctival Cl2MDP-LIP injection significantly depleted the corneal resident phagocytes and infiltrated macrophages into corneal stroma. Compared with the mice injected with PBS-liposome, the Cl2MDP-LIP-injected mice showed few inflammatory cells, irregularly distributed extracellular matrix, ingrowth of corneal epithelium into stroma, and even the detachment of donor cornea from recipient. Moreover, the number of macrophages, myofibroblasts, endothelial cells and pericytes was also decreased in the junction area between the donor and recipient cornea in macrophage-depleted mice. Peritoneal macrophages transfusion recovered the defect of corneal wound healing caused by macrophages depletion. Conclusions Macrophage depletion significantly impairs wound healing after autologous corneal transplantation through at least partially impacting on angiogenesis and wound closure.

Trichostatin A inhibits transforming growth factor-β-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-β (TGF-β)-induced myofibroblast differentiation of corneal fibroblasts in vitro. Human immortalized corneal fibroblasts were treated with TGF-β 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, H2O2, 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-β–induced myofibroblast differentiation as compared with treatment with DPI or NAC. Furthermore, TSA also decreased cellular ROS and H2O2 accumulation induced by TGF-β, 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-β–induced ROS accumulation and myofibroblast differentiation via enhanced Nrf2-ARE signaling.

Xenogeneic acellular conjunctiva matrix as a scaffold of tissue-engineered corneal epithelium.

Amniotic membrane-based tissue-engineered corneal epithelium has been widely used in the reconstruction of the ocular surface. However, it often degrades too early to ensure the success of the transplanted corneal epithelium when treating patients with severe ocular surface disorders. In the present study, we investigated the preparation of xenogeneic acellular conjunctiva matrix (aCM) and evaluated its efficacy and safety as a scaffold of tissue-engineered corneal epithelium. Native porcine conjunctiva was decellularized with 0.1% sodium dodecyl sulfate (SDS) for 12 h at 37°C and sterilized via γ-irradiation. Compared with native conjunctiva, more than 92% of the DNA was removed, and more than 90% of the extracellular matrix components (glycosaminoglycan and collagen) remained after the decellularization treatment. Compared with denuded amniotic membrane (dAM), the aCM possessed favorable optical transmittance, tensile strength, stability and biocompatibility as well as stronger resistance to degradation both in vitro and in vivo. The corneal epithelial cells seeded on aCM formed a multilayered epithelial structure and endured longer than did those on dAM. The aCM-based tissue-engineered corneal epithelium was more effective in the reconstruction of the ocular surface in rabbits with limbal stem cell deficiency. These findings support the application of xenogeneic acellular conjunctiva matrix as a scaffold for reconstructing the ocular surface.

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. J. Cell. Physiol. 227: 1148–1156, 2012.

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-β3-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.

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.

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 β-galactosidase (SA-β-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-β-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.

VEGF-B promotes recovery of corneal innervations and trophic functions in diabetic mice.

Vascular endothelial growth factor (VEGF)-B possesses the capacity of promoting injured peripheral nerve regeneration and restore their sensory and trophic functions. However, the contribution and mechanism of VEGF-B in diabetic peripheral neuropathy remains unclear. In the present study, we investigated the expression and role of VEGF-B in diabetic corneal neuropathy by using type 1 diabetic mice and cultured trigeminal ganglion (TG) neurons. Hyperglycemia attenuated the endogenous expression of VEGF-B in regenerated diabetic corneal epithelium, but not that of VEGF receptors in diabetic TG neurons and axons. Exogenous VEGF-B promoted diabetic corneal nerve fiber regeneration through the reactivation of PI-3K/Akt-GSK3β-mTOR signaling and the attenuation of neuronal mitochondria dysfunction via the VEGF receptor-1 and neuropilin-1. Moreover, VEGF-B improved corneal sensation and epithelial regeneration in both normal and diabetic mice, accompanied with the elevated corneal content of pigment epithelial-derived factor (PEDF). PEDF blockade partially abolished trophic function of VEGF-B in diabetic corneal re-innervation. In conclusion, hyperglycemia suppressed endogenous VEGF-B expression in regenerated corneal epithelium of diabetic mice, while exogenous VEGF-B promoted recovery of corneal innervations and trophic functions through reactivating PI-3K/Akt-GSK-3β-mTOR signaling, attenuating neuronal oxidative stress and elevating PEDF expression.

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‐1β), tumor necrosis factor alpha (TNF‐α), or hyperosmotic stress. Colony‐forming efficiency or colony‐forming size was decreased with the increasing concentrations of IL‐1β, 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

Cyclosporine a drug-delivery system for high-risk penetrating keratoplasty: Stabilizing the intraocular immune microenvironment

Cyclosporine A (CsA) is an essential medication used to prevent corneal allograft rejection. Our preliminary studies revealed that CsA drug-delivery system (DDS) was more effective in preventing high-risk corneal allograft rejection than topical CsA application. However, the impacts of CsA DDS on the intraocular immune microenvironment were not fully elucidated. In the present study, we investigated the effect of CsA DDS on the cornea allograft, aqueous humor, and iris-ciliary body using a rabbit model of high-risk penetrating keratoplasty. New Zealand white rabbits were divided into four groups: a normal control group, an untreated group, a CsA eye drop group and a CsA DDS group. Graft survival was monitored for 12 weeks, and the therapeutic effects of CsA DDS were evaluated at 3 and 12 weeks after high-risk keratoplasty. In the CsA DDS group, the mean graft survival time was significantly prolonged when compared with the untreated and CsA eye drop groups. At all time-points, Langerhans cell density, inflammatory cell density, and central corneal thickness in the CsA DDS group were much lower(all p < 0.01) than the untreated and CsA eye drop groups, in which their parameters were significantly higher than the normal control group (all p < 0.01). Compared with the untreated and CsA eye drop groups, an implanted CsA DDS markedly decreased the CD11b+ and CD8+ T cell infiltration in the corneal grafts. CsA DDS treatment also greatly reduced the CD4+ T cell density and the expression of interferon-gamma, interleukin-2 (IL-2), IL-6, CD80, and CD86 mRNA both in the corneal graft and iris-ciliary body (all p < 0.01). Moreover, CsA DDS significantly reduced the IL-2 level in aqueous humor (p < 0.01). Taken together, our results suggest that CsA DDS implanted into the anterior chamber create a relative immunosuppressive microenvironment in the corneal graft, iris-ciliary body, and aqueous humor. Stabilizing the intraocular immune microenvironment could partially elucidate the mechanism of CsA DDS in suppressing corneal graft rejection.