Xiaowen Zhao

Overexpression of SIRT1 promotes high glucose-attenuated corneal epithelial wound healing via p53 regulation of the IGFBP3/IGF-1R/AKT pathway.

PURPOSE To investigate how Sirtuin (silent mating type information regulation 2 homolog) 1 (SIRT1) promotes high glucose-attenuated corneal epithelial wound healing. METHODS The effects of high glucose on SIRT1 expression were assessed in primary human corneal epithelial cells (CECs) in treatment of 5 mM d-glucose (normal glucose [NG]) and 25 mM D-glucose (high glucose [HG]) and corneas from Ins2(Akita/+) mice by Western blotting. The osmotic pressure of the NG medium was adjusted to that of the HG medium by adding 20 mM mannitol. Pifithrin-α (PFT-α) was used to inhibit the expression of p53 and an adenovirus was used for overexpression of SIRT1 in vivo and in vitro. How overexpression of SIRT1 promotes HG-attenuated corneal epithelial wound healing via p53 regulation of the IGFBP3 (insulin-like growth factor binding protein-3)/IGF-1 (insulin-like growth factor-1)/AKT pathway was investigated in CECs and Ins2(Akita/+) mice. RESULTS HG induced the downregulation of SIRT1 and the upregulation of p53 acetylation in primary human CECs and corneas from Ins2(Akita/+) mice. The results of cell migration assay and corneal wound healing from Ins2(Akita/+) mice demonstrated that SIRT1 overexpression strongly promoted wound healing in the presence of HG levels via the downregulation of the IGFBP3 protein. The levels of total p53 expression and acetylated p53 decreased dramatically in the presence of PFT-α, whereas the IGF-1R/AKT pathway was activated in CECs. The results of cell migration assay suggested this posttranslational modification of p53 was involved in the response to cell injury under HG conditions in CECs. CONCLUSIONS The molecular mechanism by which SIRT1 promotes corneal epithelial wound healing was involved in an enhancement of the IGFBP3/IGF-1/AKT pathway through the deacetylation of p53. This study also suggests that SIRT1 has a protective role in the pathogenesis of diabetic keratopathy.

MicroRNA-182 mediates Sirt1-induced diabetic corneal nerve regeneration

Sensory neurons are particularly susceptible to neuronal damage in diabetes, and silent mating type information regulation 2 homolog 1 (Sirt1) has been recently identified as a key gene in neuroprotection and wound healing. We found that the expression of Sirt1 was downregulated in trigeminal sensory neurons of diabetic mice. A microRNA microarray analysis identified microRNA-182 (miR-182) as a Sirt1 downstream effector, and the expression level of miR-182 was increased by Sirt1 overexpression in trigeminal neurons; Sirt1 bound to the promoter of miR-182 and regulated its transcription. We also revealed that miR-182 enhanced neurite outgrowth in isolated trigeminal sensory neurons and overcame the detrimental effects of hyperglycemia by stimulating corneal nerve regeneration by decreasing the expression of one of its target genes, NOX4. Furthermore, the effects of miR-182 on corneal nerve regeneration are associated with a functional recovery of corneal sensation in hyperglycemic conditions. These data demonstrate that miR-182 is a key regulator in diabetic corneal nerve regeneration through targeting NOX4, suggesting that miR-182 might be a potential target for the treatment of diabetic sensory nerve regeneration and diabetic keratopathy.

MicroRNA-204-5p–Mediated Regulation of SIRT1 Contributes to the Delay of Epithelial Cell Cycle Traversal in Diabetic Corneas

PURPOSE We investigated how the microRNA (miRNA) modifies the expression of silent mating type information regulation 2 homolog 1 (SIRT1) in diabetic corneas. METHODS The bioinformatic assay was used to predict which miRNAs might regulate the expression of SIRT1. A lipid transfection protocol was used to upregulate or knockdown the miRNA expression in TKE2 cells. Adenovirus-expressing short interfering RNA was used to knockdown the expression of SIRT1 in TKE2 cells and Ins2(Akita/+) mice were used to evaluate how miRNA promotes diabetic corneal epithelial wound healing. Cell cycle status was determined by flow cytometry assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to analyze the cell viability. RESULTS Nine miRNAs were selected for quantitative PCR (qPCR) detection after bioinformatics analysis. The miR-204-5p merited further investigation, because it was increased almost 5-fold in diabetic corneal epithelia compared to nondiabetic control corneal epithelia. Using luciferase activity assay, we identified SIRT1 was a direct target of miR-204-5p. The results of flow cytometry and MTT assay demonstrated that downregulation of miR-204-5p increased TKE2 cell growth and restored cell cycle progression in high glucose (HG) conditions by the regulation of Cyclin D1 and p16. Furthermore, we showed downregulation of miR-204-5p promoted HG attenuation of corneal epithelial wound healing via upregulation of SIRT1 in Ins2(Akita/+) mice. CONCLUSIONS Our data provide firm evidence of a role for miR-204-5p in the direct regulation of SIRT1 in diabetic corneas and identified the miR-204-5p-mediated regulation of SIRT1 contributes to the delay of epithelial cell cycle traversal in diabetic keratopathy. : Chinese Abstract.

Mutations in the ABCA3 Gene Are Associated With Cataract-Microcornea Syndrome

PURPOSE Cataract-microcornea syndrome (CCMC) is an autosomal dominant inherited disease characterized by the association of congenital cataract and microcornea without any other systemic anomaly or dysmorphism. Although mutations of several genes have been shown to cause dominant CCMC, in many patients the causative gene has not yet been identified. Our aim was to identify the disease-associated gene in Chinese patients with CCMC. METHODS The CCMC patients from two unrelated Chinese families and 26 sporadic patients were enrolled. All the patients were screened by Sanger sequencing with no identified mutations. Genetic variations were screened by whole-exome sequencing and then validated using Sanger sequencing. RESULTS By sequencing the whole exome of three patients in a Chinese four-generation dominant CCMC family (Family A), three heterozygous missense mutation (c.115C>G, c.277G>A, and c.4393G>A) were identified in ATP-binding cassette protein A3 (ABCA3). At highly conserved positions, changes (c.115C>G and c.4393G>A) were predicted to have functional impacts and completely cosegregated with the phenotype. We further confirmed our finding by identifying another heterozygous missense mutation, c.2408C>T, in ABCA3 in an additional dominant CCMC family (Family B), which also cosegregated with the phenotype. Moreover, four heterozygous mutations, two missense mutations (c.4253A>T, c.2069A>T) and two splice site mutations (c.4053+2T>C, c.2765-1G>T) were identified from the sporadic patients. The ABCA3 protein was expressed in human lens capsule, choroid-retinal pigment epithelium and retinal pigment epithelial cells. CONCLUSIONS Mutations in the human ABCA3 gene were associated with lethal respiratory distress. Our study showed, for the first time to our knowledge, that mutations in ABCA3 were associated with CCMC, warranting further investigations on the pathogenesis of this disorder.

Characterization of the corneal surface in limbal stem cell deficiency and after transplantation of cultured allogeneic limbal epithelial cells

PurposeThe objective of this study was to characterize the changes that occur in the cornea during Limbal Stem Cell Deficiency (LSCD) and on the corneal surface after transplantation of ex vivo cultured allogeneic limbal epithelial transplantation (CALET).MethodsForty-one pannus were analyzed to characterize the changes found in the cornea in LSCD. Nineteen impression cytology samples, including 14 pannus and five corneal buttons, obtained during subsequent procedures from patients who had undergone CALET were examined to assess the effect of CALET and to determine the long-term fate of donor cells. The presence of donor and recipient epithelial cells in each sample was determined by short tandem repeat (STR) amplification and fluorescent-multiplex polymerase chain reaction (PCR). Phenotypic analysis of the epithelium was performed by immunohistochemistry and real-time PCR.ResultsThe expression of lineage markers was similar between pannus and conjunctivae, but not to corneas. Objective long-term benefits from the transplantation were recorded in most cases. After CALET, the lineage markers in the excised corneal buttons and pannus showed a limbus phenotype. DNA analysis of the 19 cases showed no donor cells present on the ocular surface beyond three months after CALET.ConclusionsLSCD was characterized by ingrowth of abnormal, inflamed tissue with a conjunctival phenotype. CALET was a useful technique for restoring the ocular surface in LSCD. However, such benefits did not necessarily correlate with survival of measurable numbers of donor cells on the ocular surface. The absence of donor DNA beyond three months raises questions regarding the period of ongoing immunosuppression and the origin of the regenerated corneal epithelium.

Neuropeptide FF Promotes Recovery of Corneal Nerve Injury Associated With Hyperglycemia

PURPOSE To investigate how the neuropeptide FF (NPFF) promotes the recovery of corneal nerve injury associated with hyperglycemia. METHODS Gene expression was analyzed using neurotrophin and receptor RT2 profiler polymerase chain reaction arrays in trigeminal (TG) sensory neurons. The role of NPFF in the regeneration of diabetic TG nerves was investigated in vitro by using cultured TG neurons from diabetic BKS.Cg-m+/+Leprdb/J (db/db) mice and in vivo by following corneal injury healing responses. RF9, a selective NPFF receptor (NPFF2R) antagonist, was used to prevent the interactions between NPFF and NPFF2R. RESULTS Using a mRNA real-time PCR array, NPFF was found to be significantly lower in diabetic TG sensory neurons. Hyperglycemia induced the deficiency of ocular properties in db/db mice. The application of NPFF enhanced neurite elongation in diabetic TG neurons. Through subconjunctival injection, NPFF promoted corneal nerve injury recovery and epithelial wound healing in db/db mice. Furthermore, the application of NPFF rescued the activation of SIRT1 and PPAR-gamma, and downregulated the expression of PTEN and Rb in diabetic TG neurons. The promotion of NPFF on diabetic corneal epithelial healing and corneal innervations was completely abolished by RF9. Moreover, subconjunctivally injected NPFF accelerated the reinnervation of corneal nerves via the ERK1/2 pathway. CONCLUSIONS These results indicate that NPFF signaling through NPFFR2 contributes to diabetic corneal nerve injury recovery and epithelial wound healing. Neuropeptide FF is a potential neuroregenerative factor for diabetic sensory nerve injury. Chinese Abstract.

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.

Decreased Integrity, Content, and Increased Transcript Level of Mitochondrial DNA Are Associated with Keratoconus.

Oxidative stress may play an important role in the pathogenesis of keratoconus (KC). Mitochondrial DNA (mtDNA) is involved in mitochondrial function, and the mtDNA content, integrity, and transcript level may affect the generation of reactive oxygen species (ROS) and be involved in the pathogenesis of KC. We designed a case-control study to research the relationship between KC and mtDNA integrity, content and transcription. One-hundred ninety-eight KC corneas and 106 normal corneas from Chinese patients were studied. Quantitative real-time PCR was used to measure the relative mtDNA content, transcript levels of mtDNA and related genes. Long-extension PCR was used to detect mtDNA damage. ROS, mitochondrial membrane potential and ATP were measured by respective assay kit, and Mito-Tracker Green was used to label the mitochondria. The relative mtDNA content of KC corneas was significantly lower than that of normal corneas (P = 9.19×10−24), possibly due to decreased expression of the mitochondrial transcription factor A (TFAM) gene (P = 3.26×10−3). In contrast, the transcript levels of mtDNA genes were significantly increased in KC corneas compared with normal corneas (NADH dehydrogenase subunit 1 [ND1]: P = 1.79×10−3; cytochrome c oxidase subunit 1 [COX1]: P = 1.54×10−3; NADH dehydrogenase subunit 1, [ND6]: P = 4.62×10−3). The latter may be the result of increased expression levels of mtDNA transcription-related genes mitochondrial RNA polymerase (POLRMT) (P = 2.55×10−4) and transcription factor B2 mitochondrial (TFB2M) (P = 7.88×10−5). KC corneas also had increased mtDNA damage (P = 3.63×10−10), higher ROS levels, and lower mitochondrial membrane potential and ATP levels compared with normal corneas. Decreased integrity, content and increased transcript level of mtDNA are associated with KC. These changes may affect the generation of ROS and play a role in the pathogenesis of KC.