Guohu Di

Substance P promotes diabetic corneal epithelial wound healing through molecular mechanisms mediated via the neurokinin-1 receptor

Substance P (SP) is a neuropeptide, predominantly released from sensory nerve fibers, with a potentially protective role in diabetic corneal epithelial wound healing. However, the molecular mechanism remains unclear. We investigated the protective mechanism of SP against hyperglycemia-induced corneal epithelial wound healing defects, using type 1 diabetic mice and high glucose–treated corneal epithelial cells. Hyperglycemia induced delayed corneal epithelial wound healing, accompanied by attenuated corneal sensation, mitochondrial dysfunction, and impairments of Akt, epidermal growth factor receptor (EGFR), and Sirt1 activation, as well as decreased reactive oxygen species (ROS) scavenging capacity. However, SP application promoted epithelial wound healing, recovery of corneal sensation, improvement of mitochondrial function, and reactivation of Akt, EGFR, and Sirt1, as well as increased ROS scavenging capacity, in both diabetic mouse corneal epithelium and high glucose–treated corneal epithelial cells. The promotion of SP on diabetic corneal epithelial healing was completely abolished by a neurokinin-1 (NK-1) receptor antagonist. Moreover, the subconjunctival injection of NK-1 receptor antagonist also caused diabetic corneal pathological changes in normal mice. In conclusion, the results suggest that SP-NK-1 receptor signaling plays a critical role in the maintenance of corneal epithelium homeostasis, and that SP signaling through the NK-1 receptor contributes to the promotion of diabetic corneal epithelial wound healing by rescued activation of Akt, EGFR, and Sirt1, improvement of mitochondrial function, and increased ROS scavenging capacity.

Ciliary Neurotrophic Factor Promotes the Activation of Corneal Epithelial Stem/Progenitor Cells and Accelerates Corneal Epithelial Wound Healing

Ciliary neurotrophic factor (CNTF), a well‐known neuroprotective cytokine, has been found to play an important role in neurogenesis and functional regulations of neural stem cells. As one of the most innervated tissue, however, the role of CNTF in cornea epithelium remains unclear. This study was to explore the roles and mechanisms of CNTF in the activation of corneal epithelial stem/progenitor cells and wound healing of both normal and diabetic mouse corneal epithelium. In mice subjecting to mechanical removal of corneal epithelium, the corneal epithelial stem/progenitor cell activation and wound healing were promoted by exogenous CNTF application, while delayed by CNTF neutralizing antibody. In cultured corneal epithelial stem/progenitor cells, CNTF enhanced the colony‐forming efficiency, stimulated the mitogenic proliferation, and upregulated the expression levels of corneal epithelial stem/progenitor cell‐associated transcription factors. Furthermore, the promotion of CNTF on the corneal epithelial stem/progenitor cell activation and wound healing was mediated by the activation of STAT3. Moreover, in diabetic mice, the content of CNTF in corneal epithelium decreased significantly when compared with that of normal mice, and the supplement of CNTF promoted the diabetic corneal epithelial wound healing, accompanied with the advanced activation of corneal epithelial stem/progenitor cells and the regeneration of corneal nerve fibers. Thus, the capability of expanding corneal epithelial stem/progenitor cells and promoting corneal epithelial wound healing and nerve regeneration indicates the potential application of CNTF in ameliorating limbal stem cell deficiency and treating diabetic keratopathy. Stem Cells 2015;33:1566–1576

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.

Corneal Epithelium-Derived Neurotrophic Factors Promote Nerve Regeneration.

Purpose To explore the neurotrophic factor expression in corneal epithelium and evaluate their effects on the trigeminal ganglion (TG) neurite outgrowth and corneal nerve regeneration in mice. Methods The expression of neurotrophic factors was compared among the intact, regenerating, and regenerated mouse corneal epithelium. Mouse primary TG neurons were treated with the conditioned medium of mouse corneal epithelial cells. Nerve growth factor (NGF) neutralizing antibody and glial cell-derived neurotrophic factor (GDNF) neutralizing antibody were used to evaluate their roles in mouse corneal nerve regeneration and TG neurite outgrowth. The promoting effects of NGF and GDNF for the corneal nerve regeneration were further evaluated in the diabetic mice. Results The expression of NGF and GDNF showed significant up-regulation in regenerating corneal epithelium and return to the preinjury levels in the regenerated epithelium, which was consistent with the progress of corneal subbasal nerve regeneration. The conditioned medium of corneal epithelial cells promoted the TG neurite outgrowth with extended branching and elongation. Furthermore, the blockage of either NGF or GDNF significantly impaired the promotion of the neurite outgrowth by the conditioned medium or the corneal nerve regeneration in normal mice. Moreover, the expression of NGF and GDNF was attenuated in the diabetic regenerating corneal epithelium as compared to that in normal mice, while exogenous NGF or GDNF supplement promoted the corneal epithelial and nerve regeneration in diabetic mice. Conclusions Corneal epithelium expresses multiple neurotrophic factors, among which NGF and GDNF may play an important role in the corneal nerve regeneration.

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.

Mesenchymal Stem Cells Promote Diabetic Corneal Epithelial Wound Healing Through TSG-6–Dependent Stem Cell Activation and Macrophage Switch

Purpose To explore the role and mechanism of bone marrow-derived mesenchymal stem cells (BM-MSCs) in corneal epithelial wound healing in type 1 diabetic mice. Methods Diabetic mice were treated with subconjunctival injections of BM-MSCs or recombinant tumor necrosis factor-α-stimulated gene/protein-6 (TSG-6). The corneal epithelial wound healing rate was examined by fluorescein staining. The mRNA and protein expression levels of TSG-6 were measured by quantitative RT-PCR and Western blot. The infiltrations of leukocytes and macrophages were analyzed by flow cytometry and immunofluoresence staining. The effect of TSG-6 was further evaluated in cultured limbal epithelial stem/progenitor cells, macrophages, and diabetic mice by short hairpin RNA (shRNA) knockdown. Results Local MSC transplantation significantly promoted diabetic corneal epithelial wound healing, accompanied by elevated corneal TSG-6 expression, increased corneal epithelial cell proliferation, and attenuated inflammatory response. Moreover, in cultured human limbal epithelial stem/progenitor cells, TSG-6 enhanced the colony-forming efficiency, stimulated mitogenic proliferation, and upregulated the expression level of ΔNp63. Furthermore, in diabetic mouse cornea and in vitro macrophage culture, TSG-6 alleviated leukocyte infiltration and promoted the polarization of recruited macrophages to anti-inflammatory M2 phenotypes with increased phagocytotic capacity. In addition, the promotion of epithelial stem/progenitor cell activation and macrophage polarization by MSC transplantation was largely abrogated by shRNA knockdown of TSG-6. Conclusions This study provided the first evidence of TSG-6 secreted by MSCs promoting corneal epithelial wound healing in diabetic mice through activating corneal epithelial stem/progenitor cells and accelerating M2 macrophage polarization.

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.

Netrin-1 promotes diabetic corneal wound healing through molecular mechanisms mediated via the adenosine 2B receptor

Netrins are secreted chemoattractants with the roles in axon guidance, cell migration and epithelial plasticity. In the present study, we investigated the roles of netrin-1 in the regulation of corneal epithelial wound healing, inflammation response and nerve fiber regeneration in diabetic mice and cultured corneal epithelial cells. In diabetic mice, the expression of netrin-1 was decreased when compared with that of normal mice. Furthermore, high glucose blocked the wounding-induced up-regulation of netrin-1 expression in corneal epithelial cells. Exogenous netrin-1 promoted the corneal epithelial wound healing in diabetic mice, and facilitated the proliferation and migration by reactivating the phosphorylation of ERK and EGFR in high-glucose treated corneal epithelial cells. Moreover, netrin-1 decreased the neutrophil infiltration and promoted M2 macrophage transition, accompanied with the attenuated expression of pro-inflammatory factors in diabetic mouse corneal epithelium. The promotions of netrin-1 on corneal epithelial wound healing and inflammation resolution were mediated at least through the adenosine 2B receptor. In addition, netrin-1 promoted the regeneration of corneal nerve fibers that was impaired in diabetic mice. Taken together, netrin-1 regulates corneal epithelial wound healing, inflammation response and nerve fiber regeneration in diabetic mice, indicating the potential application for the therapy of diabetic keratopathy.

Epithelium-derived miR-204 inhibits corneal neovascularization

Abstract 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. HighlightsMiR‐204 was down‐expressed in corneal epithelium with neovascularization.Exogenous miR‐204 suppressed corneal neovascularization.MiR‐204 suppressed VEGF‐A and VEGFR2 expression.MiR‐204 attenuated biomechanical stress‐induced VEGF‐A expression in HMECs.