The tissue-engineered human cornea as a model to study expression of matrix metalloproteinases during corneal wound healing
ABSTRACT: Corneal injuries remain a major cause of consultation in the ophthalmology clinics worldwide. Repair of corneal wounds is a complex mechanism that involves cell death, migration, proliferation, differentiation, and extracellular matrix (ECM) remodeling. In the present study, we used a tissue-engineered, two-layers (epithelium and stroma) human cornea as a biomaterial to study both the cellular and molecular mechanisms of wound healing. Gene profiling on microarrays revealed important alterations in the pattern of genes expressed by tissue-engineered corneas in response to wound healing. Expression of many MMPs-encoding genes was shown by microarray and qPCR analyses to increase in the migrating epithelium of wounded corneas. Many of these enzymes were converted into their enzymatically active form as wound closure proceeded. In addition, expression of MMPs by human corneal epithelial cells (HCECs) was affected both by the stromal fibroblasts and the collagen-enriched ECM they produce. Most of all, results from mass spectrometry analyses provided evidence that a fully stratified epithelium is required for proper synthesis and organization of the ECM on which the epithelial cells adhere. In conclusion, and because of the many characteristics it shares with the native cornea, this human two layers corneal substitute may prove particularly useful to decipher the mechanistic details of corneal wound healing. Primary cultures of human corneal epithelial cells cultivated on BSA (number of replicates: 7), Collagen type I (number of replicates: 2), Collagen type IV (number of replicates: 2), Fibronectin (number of replicates: 2), Tenascin C (number of replicates: 2) and Laminin (number of replicates: 2) matrix. Central, internal and external ring of wounded Tissue-engineered human cornea.
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:To elucidate how the deficiency of a major corneal proteoglycan, lumican, affects corneal homeostasis, we used mass spectrometry to derive the proteome profile of the lumican-deficient and the heterozygous mouse corneas and compared these to the wild type corneal proteome. 2108 proteins were quantified in the mouse cornea. Selected proteins and transcripts were investigated by Western blot and quantitative RT-PCR, respectively. We observed major changes in the composition of the stromal extracellular matrix (ECM) proteins in the lumican-deficient mice. Lumican deficiency altered cellular proteins in the stroma and the corneal epithelium. The ECM changes included increases in fibril forming collagen type I, Collagen type VI, fibromodulin, perlecan, laminin ??, collagen type IV, nidogen/entactin and anchoring collagen type VII in the Lum?/? and the Lum?/? mouse corneas, while the stromal proteoglycans decorin, biglycan and keratocan were decreased in the Lum?/?( corneas. Cellular protein changes included increases in alcohol dehydrogenase, superoxide dismutase and decreases in epithelial cytokeratins 8 and 14. We also detected proteins that are novel to the cornea. The proteomes will provide an insight into the lumican-deficient corneal phenotype of stromal thinning and loss of transparency and a better understanding of pathogenic changes in corneal and ocular dystrophies.
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: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: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:The rabbit is commonly used to evaluate new corneal prosthetics and study corneal wound healing. Knowledge of the stiffness of the rabbit cornea would better inform the design and fabrication of keratoprosthetics and substrates with relevant mechanical properties for in vitro investigations of corneal cellular behavior. This study determined the elastic modulus of the rabbit corneal epithelium, anterior basement membrane (ABM), anterior and posterior stroma, Descemet's membrane (DM) and endothelium using atomic force microscopy (AFM). In addition, three-dimensional collagen fiber organization of the rabbit cornea was determined using nonlinear optical high-resolution macroscopy. The elastic modulus as determined by AFM for each corneal layer was: epithelium, 0.57 ± 0.29 kPa (mean ± SD); ABM, 4.5 ± 1.2 kPa, anterior stroma, 1.1 ± 0.6 kPa; posterior stroma, 0.38 ± 0.22 kPa; DM, 11.7 ± 7.4 kPa; and endothelium, 4.1 ± 1.7 kPa. The biophysical properties, including the elastic modulus, are unique for each layer of the rabbit cornea and are dramatically softer in comparison to the corresponding regions of the human cornea. Collagen fiber organization is also dramatically different between the two species, with markedly less intertwining observed in the rabbit vs. human cornea. Given that the substratum stiffness considerably alters the corneal cell behavior, keratoprosthetics that incorporate mechanical properties simulating the native human cornea may not elicit optimal cellular performance in rabbit corneas that have dramatically different elastic moduli. These data should allow for the design of substrates that better mimic the biomechanical properties of the corneal cellular environment.
Project description:To assess keratocyte backscattering, alignment, morphology, and connectivity in vivo following a full-thickness corneal injury using the Heidelberg Retina Tomograph Rostock Cornea Module (HRT-RCM), and to correlate these findings with en bloc three-dimensional (3-D) confocal fluorescence and second harmonic generation (SHG) imaging.Rabbit corneas were scanned in vivo both before and 3, 7, 14, and 28 days after transcorneal freeze injury (FI), which damages all corneal cell layers. Corneal tissue was also fixed and labeled for f-actin and nuclei en bloc, and imaged using 3-D confocal fluorescence microscopy and SHG imaging.Using the modified HRT-RCM, full-thickness scans of all cell layers were consistently obtained. Following FI, stromal cells repopulating the damaged tissue assumed an elongated fibroblastic morphology, and a significant increase in cellular light scattering was measured. This stromal haze gradually decreased as wound healing progressed. Parallel, interconnected streams of aligned corneal fibroblasts were observed both in vivo (from HRT-RCM reflection images) and ex vivo (from f-actin and nuclear labeling) during wound healing, particularly in the posterior cornea. Second harmonic generation imaging demonstrated that these cells were aligned parallel to the collagen lamellae.The modified HRT-RCM allows in vivo measurements of sublayer thickness, assessment of cell morphology, alignment and connectivity, and estimation of stromal backscatter during wound healing. In this study, these in vivo observations led to the novel finding that the pattern of corneal fibroblast alignment is highly correlated with lamellar organization, suggesting contact guidance of intrastromal migration that may facilitate more rapid wound repopulation.
Project description:Descemet's membrane (DM) helps maintain phenotype and function of corneal endothelial cells under physiological conditions, while little is known about the function of DM in corneal endothelial wound healing process. In the current study, we performed in vivo rabbit corneal endothelial cell (CEC) injury via CEC scraping, in which DM remained intact after CECs removal, or via DM stripping, in which DM was removed together with CECs. We found rabbit corneas in the CEC scraping group healed with transparency restoration, while there was posterior fibrosis tissue formation in the corneas after DM stripping on day 14. Following CEC scraping on day 3, cells that had migrated toward the central cornea underwent a transient fibrotic endothelial-mesenchymal transition (EMT) which was reversed back to an endothelial phenotype on day 14. However, in the corneas injured via DM stripping, most of the cells in the posterior fibrosis tissue did not originate from the corneal endothelium, and they maintained fibroblastic phenotype on day 14. We concluded that corneal endothelial wound healing in rabbits has different outcomes depending upon the presence or absence of Descemet's membrane. Descemet's membrane supports corneal endothelial cell regeneration in rabbits after endothelial injury.
Project description:Acetylcholine (ACh) has been reported to play various physiological roles, including wound healing in the cornea. Here, we study the role of ACh in the transition of corneal fibroblasts into myofibroblasts, and in consequence its role in the onset of fibrosis, in an in vitro human corneal fibrosis model. Primary human keratocytes were obtained from healthy corneas. Vitamin C (VitC) and transforming growth factor-?1 (TGF-?1) were used to induce fibrosis in corneal fibroblasts. qRT-PCR and ELISA analyses showed that gene expression and production of collagen I, collagen III, collagen V, lumican, fibronectin (FN) and alpha-smooth muscle actin (?-SMA) were reduced by ACh in quiescent keratocytes. ACh treatment furthermore decreased gene expression and production of collagen I, collagen III, collagen V, lumican, FN and ?-SMA during the transition of corneal fibroblasts into myofibroblasts, after induction of fibrotic process. ACh inhibited corneal fibroblasts from developing contractile activity during the process of fibrosis, as assessed with collagen gel contraction assay. Moreover, the effect of ACh was dependent on activation of muscarinic ACh receptors. These results show that ACh has an anti-fibrotic effect in an in vitro human corneal fibrosis model, as it negatively affects the transition of corneal fibroblasts into myofibroblasts. Therefore, ACh might play a role in the onset of fibrosis in the corneal stroma.