OBJECTIVE: To examine the in-vitro expression of matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in corneal stromal cells by distinguishing between fibroblasts and keratocytes of healthy and keratoconus (KC) corneas.
METHODS: Stromal cells were isolated from healthy and KC corneas (n = 8). A normal-glucose, serum-containing cell culture medium (NGSC-medium) was used for cultivation of healthy human corneal fibroblasts (HCFs) and KC human corneal fibroblasts (KC-HCFs). In order to obtain a keratocyte phenotype, the initial cultivation with NGSC-medium was changed to a low-glucose, serum-free cell culture medium for healthy (Keratocytes) and KC cells (KC-Keratocytes). Gene and protein expression of MMP-1, -2, -3, -7, -9 and TIMP-1, -2, -3 were measured by quantitative PCR and Enzyme-Linked Immunosorbent Assay (ELISA) from the cell culture supernatant.
RESULTS: KC-HCFs demonstrated a lower mRNA gene expression for MMP-2 compared to HCFs. In contrast to their respective fibroblast groups (either HCFs or KC-HCFs), Keratocytes showed a higher mRNA gene expression of TIMP-3, whereas TIMP-1 mRNA gene expression was lower in Keratocytes and KC-Keratocytes. Protein analysis of the cell culture supernatant revealed lower concentrations of MMP-1 in KC-HCFs compared to HCFs. Compared to Keratocytes, TIMP-1 concentrations was lower in the cell culture supernatant of KC-Keratocytes. In HCFs and KC-HCFs, protein levels of MMP-1 and TIMP-1 were higher and MMP-2 was lower compared to Keratocytes and KC-Keratocytes, respectively.
CONCLUSIONS: This study indicates an imbalance in MMP and TIMP expression between healthy and diseased cells. Furthermore, differences in the expression of MMPs and TIMPs exist between corneal fibroblasts and keratocytes, which could influence the specific proteolytic metabolism in-vivo and contribute to the progression of KC.

In the event of disease or injury, restoration of the native organization of cells and extracellular matrix is crucial for regaining tissue functionality. In the cornea, a highly organized collagenous tissue, keratocytes can align along the anisotropy of the physical microenvironment, providing a blueprint for guiding the organization of the collagenous matrix. Inspired by this physiological process, anisotropic contact guidance cues have been employed to steer the alignment of keratocytes as a first step to engineer in vitro cornea-like tissues. Despite promising results, two major hurdles must still be overcome to advance the field. First, there is an enormous design space to be explored in optimizing cellular contact guidance in three dimensions. Second, the role of contact guidance cues in directing the long-term deposition and organization of extracellular matrix proteins remains unknown. To address these challenges, here we combined two microengineering strategies-UV-based protein patterning (2D) and two-photon polymerization of topographies (2.5D)-to create a library of anisotropic contact guidance cues with systematically varying height (H, 0 µm ≤ H ≤ 20 µm) and width (W, 5 µm ≤ W ≤ 100 µm). With this unique approach, we found that, in the short term (24 h), the orientation and morphology of primary human fibroblastic keratocytes were critically determined not only by the pattern width, but also by the height of the contact guidance cues. Upon extended 7-day cultures, keratocytes were shown to produce a dense, fibrous collagen network along the direction of the contact guidance cues. Moreover, increasing the heights also increased the aligned fraction of deposited collagen and the contact guidance response of cells, all whilst the cells maintained the fibroblastic keratocyte phenotype. Our study thus reveals the importance of dimensionality of the physical microenvironment in steering both cellular organization and the formation of aligned, collagenous tissues.

Recent studies in rabbits and case reports in humans have demonstrated the efficacy of topical losartan in the treatment of corneal scarring fibrosis after a wide range of injuries, including chemical burns, infections, surgical complications, and some diseases. It is hypothesized that the effect of losartan on the fibrotic corneal stroma occurs through a two-phase process in which losartan first triggers the elimination of myofibroblasts by directing their apoptosis via inhibition of extracellular signal-regulated kinase (ERK)-mediated signal transduction, and possibly through signaling effects on the viability and development of corneal fibroblast and fibrocyte myofibroblast precursor cells. This first step likely occurs within a week or two in most corneas with fibrosis treated with topical losartan, but the medication must be continued for much longer until the epithelial basement membrane (EBM) is fully regenerated or new myofibroblasts will develop from precursor cells. Once the myofibroblasts are eliminated from the fibrotic stroma, corneal fibroblasts can migrate into the fibrotic tissue and reabsorb/reorganize the disordered extracellular matrix (ECM) previously produced by the myofibroblasts. This second stage is longer and more variable in different eyes of rabbits and humans, and accounts for most of the variability in the time it takes for the stromal opacity to be markedly reduced by topical losartan treatment. Eventually, keratocytes reemerge in the previously fibrotic stromal tissue to fine-tune the collagens and other ECM components and maintain the normal structure of the corneal stroma. The efficacy of losartan in the prevention and treatment of corneal fibrosis suggests that it acts as a surrogate for the EBM, by suppressing TGF beta-directed scarring of the wounded corneal stroma, until control over TGF beta action is re-established by a healed EBM, while also supporting regeneration of the EBM by allowing corneal fibroblasts to occupy the subepithelial stroma in the place of myofibroblasts.

The purpose of this study was to evaluate transforming growth factor beta (TGFβ) isoform localization in rabbit corneas with spontaneous persistent epithelial defects (PEDs) after photorefractive keratectomy (PRK). Four cryofixed corneas from a previously reported series of PEDs in rabbits that had PRK were evaluated with triplex immunohistochemistry (IHC) for TGFβ3, myofibroblast marker alpha-smooth muscle actin (α-SMA) and mesenchymal marker vimentin. One cornea had sufficient remaining tissue for triplex IHC for TGFβ1, TGFβ2, or TGFβ3 (each with α-SMA and vimentin) using isoform-specific antibodies. All three TGFβ isoforms were detected in the subepithelial stroma at and surrounding the PED. Some of each TGFβ isoform co-localized with α-SMA of myofibroblasts, which could be TGFβ isoform autocrine production by myofibroblasts or TGFβ-1, -2, and -3 binding to these myofibroblasts.

The purpose of this study was to evaluate the localization of TGF beta-3 in situ in unwounded rabbit corneas and corneas that had epithelial-stromal injuries produced by photorefractive keratectomy (PRK) in rabbits and to evaluate the in vitro effects of TGF beta-3 compared to TGF beta-1 on alpha-smooth muscle actin (α-SMA) protein expression and myofibroblast development in corneal fibroblasts. Forty-eight New Zealand white rabbits underwent either -3 diopter (D) or -9D PRK and were studied from one to eight weeks (four corneas in each group at each time point) after surgery with immunohistochemistry for TGF beta-3, laminin alpha-5, and alpha-smooth muscle actin (α-SMA). Rabbit corneal fibroblasts were treated with activated TGF beta-1 and/or TGF beta-3 at different concentrations and duration of exposure and studied with immunocytochemistry for myofibroblast development and the expression of α-SMA using Jess automated Western blotting. TGF beta-3 was detected at high levels in the stroma of unwounded corneas and corneas at one to eight weeks after -3D or -9D PRK, as well as in the epithelium and epithelial basement membrane (EBM). No difference was noted between corneas that healed with and without myofibroblast-mediated fibrosis, although TGF beta-3 was commonly associated with myofibroblasts. TGF beta-3 effects on corneal fibroblasts in vitro were similar to TGF beta-1 in stimulating transition to α-SMA-positive myofibroblasts and promoting α-SMA protein expression. The corneal stromal localization pattern of TGF beta-3 protein in unwounded corneas and corneas after epithelial-stromal injury was found to be higher and different from TGF beta-1 and TGF beta-2 reported in previous studies. TGF beta-3 had similar effects to TGF beta-1 in driving myofibroblast development and α-SMA expression in corneal fibroblasts cultured in medium with 1% fetal bovine serum.

Fibroblasts isolated and expanded from ReLEx SMILE lenticules can be a source of human keratocytes. Since corneal keratocytes are quiescent cells, it is difficult to expand them in vitro in suitable numbers for clinical and experimental use. In the present study, this problem was solved by isolating and growing corneal fibroblasts (CFs) with a high proliferative potential and their reversion to keratocytes in a selective serum-free medium. Fibroblasts reversed into keratocytes (rCFs) had a dendritic morphology and ultrastructural signs of activation of protein synthesis and metabolism. The cultivation of CFs in a medium with 10% FCS and their reversion into keratocytes was not accompanied by the induction of myofibroblasts. After reversion, the cells spontaneously formed spheroids and expressed keratocan and lumican markers, but not mesenchymal ones. The rCFs had low proliferative and migratory activity, and their conditioned medium contained a low level of VEGF. CF reversion was not accompanied by a change with the levels of IGF-1, TNF-alpha, SDF-1a, and sICAM-1. In the present study, it has been demonstrated that fibroblasts from ReLEx SMILE lenticules reverse into keratocytes in serum-free KGM, maintaining the morphology and functional properties of primary keratocytes. These keratocytes have a potential for tissue engineering and cell therapy of various corneal pathologies.

Bowman\'s layer is an acellular layer in the anterior stroma found in the corneas of humans, most other primates, chickens, and some other species. Many other species, however, including the rabbit, dog, wolf, cat, tiger, and lion, do not have a Bowman\'s layer. Millions of humans who have had photorefractive keratectomy over the past thirty plus years have had Bowman\'s layer removed by excimer laser ablation over their central cornea without apparent sequelae. A prior study showed that Bowman\'s layer does not contribute significantly to mechanical stability within the cornea. Bowman\'s layer does not have a barrier function, as many cytokines and growth factors, as well as other molecules, such as EBM component perlecan, pass bidirectionally through Bowman\'s layer in normal corneal functions, and during the response to epithelial scrape injury. We hypothesized that Bowman\'s layer represents a visible indicator of ongoing cytokine and growth factor-mediated interactions that occur between corneal epithelial cells (and corneal endothelial cells) and stromal keratocytes that maintain the normal corneal tissue organization via negative chemotactic and apoptotic effects of modulators produced by the epithelium on stromal keratocytes. Interleukin-1 alpha, produced constitutively by corneal epithelial cells and endothelial cells, is thought to be one of these cytokines. Bowman\'s layer is destroyed in corneas with advanced Fuchs\' dystrophy or pseudophakic bullous keratopathy when the epithelium becomes edematous and dysfunctional, and fibrovascular tissue commonly develops beneath and/or within the epithelium in these corneas. Bowman\'s-like layers have been noted to develop surrounding epithelial plugs within the stromal incisions years after radial keratotomy. Although there are species-related differences in corneal wound healing, and even between strains within a species, these differences are not related to the presence or absence of Bowman\'s layer.

The progressive degeneration of granular corneal dystrophy type 2 (GCD2) corneal fibroblasts is associated with altered mitochondrial function, but the underlying mechanisms are incompletely understood. We investigated whether an imbalance of mitochondrial dynamics contributes to mitochondrial dysfunction of GCD2 corneal fibroblasts. Transmission electron microscopy revealed several small, structurally abnormal mitochondria with altered cristae morphology in GCD2 corneal fibroblasts. Confocal microscopy showed enhanced mitochondrial fission and fragmented mitochondrial tubular networks. Western blotting revealed higher levels of MFN1, MFN2, and pDRP1 and decreased levels of OPA1 and FIS1 in GCD2. OPA1 reduction by short hairpin RNA (shRNA) resulted in fragmented mitochondrial tubular networks and increased susceptibility to mitochondrial stress-induced apoptosis. A decrease in the mitochondrial biogenesis-related transcription factors NRF1 and PGC1α was observed, while there was an increase in the mitochondrial membrane proteins TOM20 and TIM23. Additionally, reduced levels of mitochondrial DNA (mtDNA) were exhibited in GCD2 corneal fibroblasts. These observations suggest that altered mitochondrial fission/fusion and biogenesis are the critical molecular mechanisms that cause mitochondrial dysfunction contributing to the degeneration of GCD2 corneal fibroblasts.

Alkali burns are one of the most common injuries used in corneal wound healing studies. Investigators have used different conditions to produce corneal alkali injuries that have varied in sodium hydroxide concentration, application methods, and duration of exposure. A critical factor in the subsequent corneal healing responses, including myofibroblast generation and fibrosis localization, is whether, or not, Descemet\'s membrane and the endothelium are injured during the initial exposure. After exposures that produce injuries confined to the epithelium and stroma, anterior stromal myofibroblasts and fibrosis are typical, with sparing of the posterior stroma. However, if there is also injury to Descemet\'s membrane and the endothelium, then myofibroblast generation and fibrosis is noted full corneal thickness, with predilection to the most anterior and most posterior stroma and a tendency for relative sparring of the central stroma that is likely related to the availability of TGF beta from the tears, epithelium, and the aqueous humor. A method is described where a 5 mm diameter circle of Whatman #1 filter paper wetted with only 30 μL of alkali solution is applied for 15 s prior to profuse irrigation in rabbit corneas. When 0.6N, or lower, NaOH is used, then the injury, myofibroblasts, and fibrosis generation are limited to the epithelium and stroma. Use of 0.75N NaOH triggers injury to Descemet\'s membrane and the corneal endothelium with fibrosis throughout the stroma, but rare corneal neovascularization (CNV) and persistent epithelial defects (PED). Use of 1N NaOH with this method produces greater stromal fibrosis and increased likelihood that CNV and PED will occur in individual corneas.

UNASSIGNED: To investigate expression of cytokines and ROS-related genes in stromal cells of healthy and keratoconus (KC) corneas.
UNASSIGNED: Expression analysis was performed for cytokines including several interleukins (IL), Tumor necrosis factor-α (TNF-α), Transforming growth factor-β1 (TGF-β1), Interferon-γ (IFN-γ) and ROS-related genes such as Catalase, Glutathione peroxidase 1, NADPH oxidase 1, superoxide dismutase 1 in corneal fibroblasts (HCFs/KC-HCFs) or keratocytes (Keratocytes/KC-Keratocytes) by qPCR and ELISA.
UNASSIGNED: Gene and protein expression of most inflammatory markers was decreased in keratocytes compared to fibroblasts, whereas no differences were found between healthy and keratoconus cells for the majority of cytokines measured. TNF-α expression was increased at gene (KC keratocytes) and protein levels (supernatant of Keratocytes/KC-Keratocytes) compared to corneal fibroblasts. No differential expression of ROS-related genes was detected between healthy and diseased cells in both fibroblasts and keratocytes.
UNASSIGNED: Increased expression of several inflammatory markers described as altered in KC was not evident in KC cells in vitro.