Differentially Expressed Wound Healing-Related microRNAs in the Human Diabetic Cornea
ABSTRACT: MicroRNAs are powerful gene expression regulators, but their corneal repertoire and potential changes in corneal diseases remain unknown. Our purpose was to identify miRNAs altered in the human diabetic cornea by microarray analysis, and to examine their effects on wound healing in cultured telomerase-immortalized human corneal epithelial cells (HCEC) in vitro. Using microarrays, 29 miRNAs were identified as differentially expressed in diabetic samples. Two miRNA candidates showing the highest fold increased in expression in the diabetic cornea were confirmed by Q-PCR and further characterized. HCEC transfection with h-miR-146a or h-miR-424 significantly retarded wound closure, but their respective antagomirs significantly enhanced wound healing vs. controls. Cells treated with h-miR-146a or h-miR-424 had decreased p-p38 and p-EGFR staining, but these increased over control levels close to the wound edge upon antagomir treatment. In conclusion, several miRNAs with increased expression in human diabetic central corneas were found. Two such miRNAs inhibited cultured corneal epithelial cell wound healing. Dysregulation of miRNA expression in human diabetic cornea may be an important mediator of abnormal wound healing. Total RNA was extracted from age-matched human autopsy normal (n=6) and diabetic (n=6) central corneas, Flash Tag end-labeled, and hybridized to Affymetrix® GeneChip® miRNA Arrays. Select miRNAs associated with diabetic cornea were validated by quantitative RT-PCR (Q-PCR) and by in situ hybridization (ISH) in independent samples.
Project description:MicroRNAs are powerful gene expression regulators, but their corneal repertoire and potential changes in corneal diseases remain unknown. Our purpose was to identify miRNAs altered in the human diabetic cornea by microarray analysis, and to examine their effects on wound healing in cultured telomerase-immortalized human corneal epithelial cells (HCEC) in vitro. Total RNA was extracted from age-matched human autopsy normal (n=6) and diabetic (n=6) central corneas, Flash Tag end-labeled, and hybridized to Affymetrix® GeneChip® miRNA Arrays. Select miRNAs associated with diabetic cornea were validated by quantitative RT-PCR (Q-PCR) and by in situ hybridization (ISH) in independent samples. HCEC were transfected with human pre-miR™miRNA precursors (h-miR) or their inhibitors (antagomirs) using Lipofectamine 2000. Confluent transfected cultures were scratch-wounded with P200 pipette tip. Wound closure was monitored by digital photography. Expression of signaling proteins was detected by immunostaining and Western blot. Using microarrays, 29 miRNAs were identified as differentially expressed in diabetic samples. Two miRNA candidates showing the highest fold increased in expression in the diabetic cornea were confirmed by Q-PCR and further characterized. HCEC transfection with h-miR-146a or h-miR-424 significantly retarded wound closure, but their respective antagomirs significantly enhanced wound healing vs. controls. Cells treated with h-miR-146a or h-miR-424 had decreased p-p38 and p-EGFR staining, but these increased over control levels close to the wound edge upon antagomir treatment. In conclusion, several miRNAs with increased expression in human diabetic central corneas were found. Two such miRNAs inhibited cultured corneal epithelial cell wound healing. Dysregulation of miRNA expression in human diabetic cornea may be an important mediator of abnormal wound healing.
Project description:Wounds naturally produce electric signals which serve as powerful cues that stimulate and guide cell migration during wound healing. In diabetic patients, impaired wound healing is one of the most challenging complications in diabetes management. A fundamental gap in knowledge is whether diabetic wounds have abnormal electric signaling. Here we used a vibrating probe to demonstrate that diabetic corneas produced significantly weaker wound electric signals than the normal cornea. This was confirmed in three independent animal models of diabetes: db/db, streptozotocin-induced and mice fed a high-fat diet. Spatial measurements illustrated that diabetic cornea wound currents at the wound edge but not wound center were significantly weaker than normal. Time lapse measurements revealed that the electric currents at diabetic corneas lost the normal rising and plateau phases. The abnormal electric signals correlated significantly with impaired wound healing. Immunostaining suggested lower expression of chloride channel 2 and cystic fibrosis transmembrane regulator in diabetic corneal epithelium. Acute high glucose exposure significantly (albeit moderately) reduced electrotaxis of human corneal epithelial cells in vitro, but did not affect the electric currents at cornea wounds. These data suggest that weaker wound electric signals and impaired electrotaxis may contribute to the impaired wound healing in diabetes.
Project description:The diabetic cornea exhibits pathological alterations, such as delayed epithelial wound healing and nerve regeneration. We investigated the role of semaphorin (SEMA) 3C in corneal wound healing and reinnervation in normal and diabetic B6 mice. Wounding induced the expression of SEMA3A, SEMA3C, and their receptor neuropilin-2 (NRP2), but not NRP1, in normal corneal epithelial cells; this upregulation was suppressed for SEMA3C and NRP2 in diabetic corneas. Injections of Sema3C-specific small interfering RNA and NRP2-neutralizing antibodies in wounded mice resulted in a decrease in the rate of wound healing and regenerating nerve fibers, whereas exogenous SEMA3C had opposing effects in diabetic corneas. NRP1 neutralization, on the other hand, decreased epithelial wound closure but increased sensory nerve regeneration in diabetic corneas, suggesting a detrimental role in nerve regeneration. Taken together, epithelium-expressed SEMA3C plays a role in corneal epithelial wound closure and sensory nerve regeneration. The hyperglycemia-suppressed SEMA3C/NRP2 signaling may contribute to the pathogenesis of diabetic neurotrophic keratopathy, and SEMA3C might be used as an adjunctive therapeutic for treating the disease.
Project description:Small non-coding RNAs, in particular microRNAs (miRNAs), regulate fine-tuning of gene expression and can impact a wide range of biological processes. However, their roles in normal and diseased limbal epithelial stem cells (LESC) remain unknown. Using deep sequencing analysis, we investigated miRNA expression profiles in central and limbal regions of normal and diabetic human corneas. We identified differentially expressed miRNAs in limbus vs. central cornea in normal and diabetic (DM) corneas including both type 1 (T1DM/IDDM) and type 2 (T2DM/NIDDM) diabetes. Some miRNAs such as miR-10b that was upregulated in limbus vs. central cornea and in diabetic vs. normal limbus also showed significant increase in T1DM vs. T2DM limbus. Overexpression of miR-10b increased Ki-67 staining in human organ-cultured corneas and proliferation rate in cultured corneal epithelial cells. MiR-10b transfected human organ-cultured corneas showed downregulation of PAX6 and DKK1 and upregulation of keratin 17 protein expression levels. In summary, we report for the first time differential miRNA signatures of T1DM and T2DM corneal limbus harboring LESC and show that miR-10b could be involved in the LESC maintenance and/or their early differentiation. Furthermore, miR-10b upregulation may be an important mechanism of corneal diabetic alterations especially in the T1DM patients.
Project description:Purpose. Diabetic corneas display altered basement membrane and integrin markers, increased expression of proteinases, decreased hepatocyte growth factor (HGF) receptor, c-met proto-oncogene, and impaired wound healing. Recombinant adenovirus (rAV)-driven c-met overexpression in human organ-cultured corneas was tested for correction of diabetic abnormalities. Methods. Forty-six human corneas obtained postmortem from 23 donors with long-term diabetes (5 with diabetic retinopathy) were organ cultured and transduced with rAV-expressing c-met gene (rAV-cmet) under the cytomegalovirus promoter at approximately 10(8) plaque-forming units per cornea for 48 hours. Each control fellow cornea received control rAV (rAV expressing the beta-galactosidase gene or vector alone). After an additional 4 to 5 days of incubation, 5-mm epithelial wounds were created with n-heptanol, and healing was monitored. The corneas were analyzed afterward by immunohistochemistry and Western blot analysis. Signaling molecule expression and role was examined by immunostaining, phosphokinase antibody arrays, Western blot analysis, and inhibitor analysis. Results. rAV-cmet transduction led to increased epithelial staining for c-met (total, extracellular, and phosphorylated) and normalization of the patterns of select diabetic markers compared with rAV-vector-transduced control fellow corneas. Epithelial wound healing time in c-met-transduced diabetic corneas decreased twofold compared with rAV-vector-transduced corneas and became similar to normal. c-Met action apparently involved increased activation of p38 mitogen-activated protein kinase. c-Met transduction did not change tight junction protein patterns, suggesting unaltered epithelial barrier function. Conclusions. rAV-driven c-met transduction into diabetic corneas appears to restore HGF signaling, normalize diabetic marker patterns, and accelerate wound healing. c-Met gene therapy could be useful for correcting human diabetic corneal abnormalities.
Project description:MicroRNAs (miRNAs) are small, stable non-coding RNA molecules with regulatory function and marked tissue specificity that post-transcriptionally regulate gene expression, however their role in fungal keratitis remain unknown. Our purpose was to identify the miRNAs in human cornea from fungal keratitis patients and understand their key role in regulation of pathogenesis. Corneal samples from normal cadaver (n=3) and fungal keratitis (n=5) patients were pooled separately and total RNA was extracted. Deep sequencing was done using Illumina HiSeq1000 platform to identify miRNA profile. We identified seventy five differentially expressed miRNAs in fungal keratitis corneas. Select miRNAs were validated by real-time RT-PCR (Q-PCR). We predicted their role in regulating target genes in several pathways by combining miRNA target genes and pathway analysis, and mRNA expression of select target genes were further analysed by Q-PCR. MiR-21-5p, miR-223-3p, miR-146b-5p, miR-155-5p, miR-511-5p were found to be involved in inflammatory and immune responses, regulating Toll like receptor signaling pathways, which is of particular interest. MiR-451a with an increased expression in keratitis may have a role in wound healing by targeting Macrophage Migration Inhibitory Factor (MIF). Further, we highlighted that Neurotrophin signaling pathway may play a role in wound healing process. One novel miRNA was also detected in cornea. In conclusion, several miRNAs with high expression in fungal keratitis corneas point towards their role in regulation of pathogenesis. Further insights in understanding miRNAs role in wound healing and inflammation may help design new therapeutic strategies. Control and fungal keratitis, human corneal miRNA profiles were generated using IlluminaHiseq1000 platform
Project description:Wound healing is characterized by cell and extracellular matrix changes mediating cell migration, fibrosis, remodeling and regeneration. We previously demonstrated that chick fetal wound healing shows a regenerative phenotype regarding the cellular and molecular organization of the cornea. However, the chick corneal stromal structure is remarkably complex in the collagen fiber/lamellar organization, involving branching and anastomosing of collagen bundles. It is unknown whether the chick fetal wound healing is capable of recapitulating this developmentally regulated organization pattern. The purpose of this study was to examine the three-dimensional collagen architecture of wounded embryonic corneas, whilst identifying temporal and spatial changes in collagen organization during wound healing. Linear corneal wounds that traversed the epithelial layer, Bowman´s layer, and anterior stroma were generated in chick corneas on embryonic day 7. Irregular thin collagen fibers are present in the wounded cornea during the early phases of wound healing. As wound healing progresses, the collagen organization dramatically changes, acquiring an orthogonal arrangement. Fourier transform analysis affirmed this observation and revealed that adjacent collagen lamellae display an angular displacement progressing from the epithelium layer towards the endothelium. These data indicate that the collagen organization of the wounded embryonic cornea recapitulate the native macrostructure.
Project description:The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.
Project description:Patients with diabetes mellitus (DM) may develop corneal complications and delayed wound healing. The aims of this study are to characterize the molecular signatures and biological pathways leading to delayed epithelial wound healing and to delineate the involvement of TGF?3 therein. Genome-wide cDNA microarray analysis revealed 1,888 differentially expressed genes in the healing epithelia of normal (NL) versus type 1 DM rat corneas. Gene ontology and enrichment analyses indicated TGF? signaling as a major altered pathway. Among three TGF? isoforms, TGF-?1 and ?3 were upregulated in response to wounding in NL corneal epithelial cells (CECs), whereas the latter was greatly suppressed by hyperglycemia in rat type 1 and 2 and mouse type 1 DM models. Functional analysis indicated that TGF-?3 contributed to wound healing in NL corneas. Moreover, exogenously added TGF-?3 accelerated epithelial wound closure in type 2 rat and type 1 mouse DM corneas via Smad and PI3K-AKT signaling pathways, autoregulation, and/or upregulation of Serpine1, a well-known TGF? target gene. Taken together, our study for the first time provides a comprehensive list of genes differentially expressed in the healing CECs of NL versus diabetic corneas and suggests the therapeutic potential of TGF-?3 for treating corneal and skin wounds in diabetic patients.
Project description:Diabetic corneas overexpress proteinases including matrix metalloproteinase-10 (M10) and cathepsin F (CF). Our purpose was to assess if silencing M10 and CF in organ-cultured diabetic corneas using recombinant adenovirus (rAV)-driven small hairpin RNA (rAV-sh) would normalize slow wound healing, and diabetic and stem cell marker expression.Sixteen pairs of organ-cultured autopsy human diabetic corneas (four per group) were treated with rAV-sh. Proteinase genes were silenced either separately, together, or both, in combination (Combo) with rAV-driven c-met gene overexpression. Fellow control corneas received rAV-EGFP. Quantitative RT-PCR confirmed small hairpin RNA (shRNA) silencing effect. Ten days after transfection, 5-mm epithelial wounds were made with n-heptanol and healing time recorded. Diabetic, signaling, and putative stem cell markers were studied by immunofluorescence of corneal cryostat sections.Proteinase silencing reduced epithelial wound healing time versus rAV-enhanced green fluorescent protein (EGFP) control (23% for rAV-shM10, 31% for rAV-shCF, and 36% for rAV-shM10 + rAV-shCF). Combo treatment was even more efficient (55% reduction). Staining patterns of diabetic markers (α₃β₁ integrin and nidogen-1), and of activated epidermal growth factor receptor and its signaling target activated Akt were normalized upon rAV-sh treatment. Combo treatment also restored normal staining for activated p38. All treatments, especially the combined ones, increased diabetes-altered staining for putative limbal stem cell markers, ΔNp63α, ABCG2, keratins 15 and 17, and laminin γ3 chain.Small hairpin RNA silencing of proteinases overexpressed in diabetic corneas enhanced corneal epithelial and stem cell marker staining and accelerated wound healing. Combined therapy with c-met overexpression was even more efficient. Specific corneal gene therapy has a potential for treating diabetic keratopathy.