Reversed Corneal Fibroblasts Therapy Restores Transparency of Mouse Cornea after Injury.

Cell-based therapies using corneal stromal stem cells (CSSC), corneal keratocytes, or a combination of both suppress corneal scarring. The number of quiescent keratocytes in the cornea is small; it is difficult to expand them in vitro in quantities suitable for transplantation. This study examined the therapeutic effect of corneal fibroblasts reversed into keratocytes (rCF) in a mouse model of mechanical corneal injury. The therapeutic effect of rCF was studied in vivo (slit lamp, optical coherence tomography) and ex vivo (transmission electron microscopy and immunofluorescence staining). Injection of rCF into the injured cornea was accompanied by recovery of corneal thickness, improvement of corneal transparency, reduction of type III collagen in the stroma, absence of myofibroblasts, and the improvement in the structural organization of collagen fibers. TEM results showed that 2 months after intrastromal injection of cells, there was a decrease in the fibril density and an increase in the fibril diameter and the average distance between collagen fibrils. The fibrils were well ordered and maintained the short-range order and the number of nearest-neighbor fibrils, although the averaged distance between them increased. Our results demonstrated that the cell therapy of rCF from ReLEx SMILe lenticules promotes the recovery of transparent corneal stroma after injury.

Impact of keratocyte differentiation on corneal opacity resolution and visual function recovery in male rats.

Intrastromal cell therapy utilizing quiescent corneal stromal keratocytes (qCSKs) from human donor corneas emerges as a promising treatment for corneal opacities, aiming to overcome limitations of traditional surgeries by reducing procedural complexity and donor dependency. This investigation demonstrates the therapeutic efficacy of qCSKs in a male rat model of corneal stromal opacity, underscoring the significance of cell-delivery quality and keratocyte differentiation in mediating corneal opacity resolution and visual function recovery. Quiescent CSKs-treated rats display improvements in escape latency and efficiency compared to wounded, non-treated rats in a Morris water maze, demonstrating improved visual acuity, while stromal fibroblasts-treated rats do not. Advanced imaging, including multiphoton microscopy, small-angle X-ray scattering, and transmission electron microscopy, revealed that qCSK therapy replicates the native cornea's collagen fibril morphometry, matrix order, and ultrastructural architecture. These findings, supported by the expression of keratan sulfate proteoglycans, validate qCSKs as a potential therapeutic solution for corneal opacities.

Rose Bengal Photodynamic Therapy (RB-PDT) Modulates the Inflammatory Response in LPS-Stimulated Human Corneal Fibroblasts By Influencing NF-κB and p38 MAPK Signaling Pathways.

PURPOSE: To investigate the effect of rose bengal photodynamic therapy on lipopolysaccharide-induced inflammation in human corneal fibroblasts. Furthermore, to analyze potential involvement of the mitogen-activated protein kinase and nuclear factor kappa B signaling pathways in this process. METHODS: Human corneal fibroblast cultures underwent 0-2.0 µg/mL lipopolysaccharide treatment, and 24 h later rose bengal photodynamic therapy (0.001% RB, 565 nm wavelength illumination, 0.17 J/cm2 fluence). Interleukin-6, interleukin-8, intercellular adhesion molecule-1, interferon regulatory factor-3, interferon α2, and interferon ß1 gene expressions were determined by quantitative PCR. Interleukin-6, interleukin-8, and C-C motif chemokine ligand-4 concentrations in the cell culture supernatant were measured by enzyme-linked immunosorbent assays and intercellular adhesion molecule-1 protein level in human corneal fibroblasts by western blot. In addition, the nuclear factor kappa B and mitogen-activated protein kinase signaling pathways were investigated by quantitative PCR and phosphorylation of nuclear factor kappa B p65 and p38 mitogen-activated protein kinase by western blot. RESULTS: Rose bengal photodynamic therapy in 2.0 µg/mL lipopolysaccharide-stimulated human corneal fibroblasts triggered interleukin-6 and interleukin-8 mRNA (p < .0001) and interleukin-6 protein increase (p < .0001), and downregulated intercellular adhesion molecule-1 expression (p < .001). C-C motif chemokine ligand-4, interferon regulatory factor-3, interferon α2, and interferon ß1 expressions remained unchanged (p ≥ .2). Rose bengal photodynamic therapy increased IκB kinase subunit beta, nuclear factor kappa B p65, extracellular signal-regulated kinases-2, c-Jun amino terminal kinase, and p38 transcription (p ≤ .01), and triggered nuclear factor kappa B p65 and p38 mitogen-activated protein kinase phosphorylation (p ≤ .04) in lipopolysaccharide treated human corneal fibroblasts. CONCLUSION: Rose bengal photodynamic therapy of lipopolysaccharide-stimulated human corneal fibroblasts can modify the inflammatory response by inducing interleukin-6 and interleukin-8 expression, and decreasing intercellular adhesion molecule-1 production. C-C motif chemokine ligand-4, interferon regulatory factor-3, and interferon α and ß expressions are not affected by rose bengal photodynamic therapy in these cells. The underlying mechanisms may be associated with nuclear factor kappa B and p38 mitogen-activated protein kinase pathway activation.

Substance P promotes transforming growth factor-ß-induced collagen synthesis in human corneal fibroblasts.

Corneal fibroblasts maintain homeostasis of the corneal stroma by mediating the synthesis and degradation of extracellular collagen, and these actions are promoted by transforming growth factor-ß (TGF-ß) and interleukin-1ß (IL-1ß), respectively. The cornea is densely innervated with sensory nerve fibers that are not only responsible for sensation but also required for physiological processes such as tear secretion and wound healing. Loss or dysfunction of corneal nerves thus impairs corneal epithelial wound healing and can lead to neurotrophic keratopathy. The sensory neurotransmitter substance P (SP) promotes corneal epithelial wound healing by enhancing the stimulatory effects of growth factors and fibronectin. We have now investigated the role of SP in collagen metabolism mediated by human corneal fibroblasts in culture. Although SP alone had no effect on collagen synthesis or degradation by these cells, it promoted the stimulatory effect of TGF-ß on collagen type I synthesis without affecting that of IL-1ß on the expression of matrix metalloproteinase-1. This effect of SP on TGF-ß-induced collagen synthesis was accompanied by activation of p38 mitogen-activated protein kinase (MAPK) signaling and was attenuated by pharmacological inhibition of p38 or of the neurokinin-1 receptor. Our results thus implicate SP as a modulator of TGF-ß-induced collagen type I synthesis by human corneal fibroblasts, and they suggest that loss of this function may contribute to the development of neurotrophic keratopathy.NEW & NOTEWORTHY This study investigates the role of substance P (SP) in collagen metabolism mediated by human corneal fibroblasts in culture. We found that, although SP alone had no effect on collagen synthesis or degradation by corneal fibroblasts, it promoted the stimulatory effect of transforming growth factor-ß on collagen type I synthesis without affecting that of interleukin-1ß on the expression of matrix metalloproteinase-1.

Pharmacological Stimulation of Soluble Guanylate Cyclase Counteracts the Profibrotic Activation of Human Conjunctival Fibroblasts.

Conjunctival fibrosis is a serious clinical concern implicated in a wide spectrum of eye diseases, including outcomes of surgery for pterygium and glaucoma. It is mainly driven by chronic inflammation that stimulates conjunctival fibroblasts to differentiate into myofibroblasts over time, leading to abnormal wound healing and scar formation. Soluble guanylate cyclase (sGC) stimulation was found to suppress transforming growth factor ß (TGFß)-induced myofibroblastic differentiation in various stromal cells such as skin and pulmonary fibroblasts, as well as corneal keratocytes. Here, we evaluated the in vitro effects of stimulation of the sGC enzyme with the cell-permeable pyrazolopyridinylpyrimidine compound BAY 41-2272 in modulating the TGFß1-mediated profibrotic activation of human conjunctival fibroblasts. Cells were pretreated with the sGC stimulator before challenging with recombinant human TGFß1, and subsequently assayed for viability, proliferation, migration, invasiveness, myofibroblast marker expression, and contractile properties. Stimulation of sGC significantly counteracted TGFß1-induced cell proliferation, migration, invasiveness, and acquisition of a myofibroblast-like phenotype, as shown by a significant downregulation of FAP, ACTA2, COL1A1, COL1A2, FN1, MMP2, TIMP1, and TIMP2 mRNA levels, as well as by a significant reduction in α-smooth muscle actin, N-cadherin, COL1A1, and FN-EDA protein expression. In addition, pretreatment with the sGC stimulator was capable of significantly dampening TGFß1-induced acquisition of a contractile phenotype by conjunctival fibroblasts, as well as phosphorylation of Smad3 and release of the proinflammatory cytokines IL-1ß and IL-6. Taken together, our findings are the first to demonstrate the effectiveness of pharmacological sGC stimulation in counteracting conjunctival fibroblast-to-myofibroblast transition, thus providing a promising scientific background to further explore the feasibility of sGC stimulators as potential new adjuvant therapeutic compounds to treat conjunctival fibrotic conditions.

Mesenchymal Stem Cell-Laden In Situ-Forming Hydrogel for Preventing Corneal Stromal Opacity.

PURPOSE: The aims of this study were to construct a mesenchymal stem cell (MSC)-laden in situ-forming hydrogel and study its effects on preventing corneal stromal opacity. METHODS: The native gellan gum was modified by high temperature and pressure, and the rabbit bone marrow MSCs were encapsulated before adding Ca 2+ to initiate cross-linking. The effects of the hydrogel on 3D culture and gene expression of the rabbit bone marrow MSCs were observed in vitro. Then, the MSC-hydrogel was used to repair corneal stromal injury in New Zealand white rabbits within 28 days postoperation. RESULTS: The short-chain gellan gum solution has a very low viscosity (<0.1 Pa·s) that is ideal for encapsulating cells. Moreover, mRNA expressions of 3D-cultured MSCs coding for corneal stromal components (decorin, lumican, and keratocan) were upregulated (by 127.8, 165.5, and 25.4 times, respectively) ( P < 0.05) on day 21 in vitro and were verified by Western blotting results. For the in vivo study, the corneal densitometry of the experimental group was (20.73 ± 1.85) grayscale units which was lower than the other groups ( P < 0.05). The MSC-hydrogel downregulated mRNA expression coding for fibrosis markers (α-smooth muscle actin, vimentin, collagen type 5-α1, and collagen type 1-α1) in the rabbit corneal stroma. Furthermore, some of the 5-ethynyl-2'-deoxyuridine (EdU)-labeled MSCs integrated into the upper corneal stroma and expressed keratocyte-specific antigens on day 28 postoperation. CONCLUSIONS: The short-chain gellan gum allows MSCs to slowly release to the corneal stromal defect and prevent corneal stromal opacity. Some of the implanted MSCs can integrate into the corneal stroma and differentiate into keratocytes.

Benzalkonium chloride greatly deteriorates the biological activities of human corneal stroma fibroblasts in a concentration-dependent manner.

BACKGROUND: Corneal tissues indirectly obtain nutritional needs and oxygen to maintain their homeostasis, and therefore, benzalkonium chloride (BAC) containing ocular instillations for medical therapy may, in turn, induce toxic effects more than expected in corneal tissues, especially the inside stroma layer. METHODS: To evaluate the effects of very low concentrations (10-8%, 10-6%, or 10-4%) of BAC on human corneal stroma, we used two-dimensional (2D) cultures of human corneal stromal fibroblast (HCSF) cells and carried out the following analyses: (1) cell viability measurements, (2) Seahorse cellular bio-metabolism analysis, and (3) the expression of ECM molecules and endoplasmic reticulum (ER) stress-related molecules. RESULTS: In the absence and presence of 10-8%, 10-6%, or 10-4% concentrations of BAC, cell viability deteriorated and this deterioration was dose-dependent. The results showed that maximal mitochondrial respiration was decreased, the mRNA expression of most of ECM proteins was decreased, and ER stress-related molecules were substantially and dose-dependently down-regulated in HCSFs by the BAC treatment. CONCLUSIONS: The findings reported herein indicate that the presence of BAC, even at such low concentrations, is capable of causing the deterioration of cellular metabolic functions and negatively affecting the response to ER stress in HCSF cells resulting in a substantially decreased cellular viability.

Keratocyte-Derived Myofibroblasts: Functional Differences With Their Fibroblast Precursors.

Purpose: In this study, we aim to elucidate functional differences between fibroblasts and myofibroblasts derived from a keratocyte lineage to better understand corneal scarring. Methods: Corneal fibroblasts, derived from a novel triple transgenic conditional KeraRT/tetO-Cre/mTmG mouse strain that allows isolation and tracking of keratocyte lineage, were expanded, and transformed by exposure to transforming growth factor (TGF)-ß1 to myofibroblasts. The composition and organization of a fibroblast-built matrix, deposited by fibroblasts in vitro, was analyzed and compared to the composition of an in vitro matrix built by myofibroblasts. Second harmonic generation microscopy (SHG) was used to study collagen organization in deposited matrix. Different extracellular matrix proteins, expressed by fibroblasts or myofibroblasts, were analyzed and quantified. Functional assays compared latent (TGF-ß) activation, in vitro wound healing, chemotaxis, and proliferation between fibroblasts and myofibroblasts. Results: We found significant differences in cell morphology between fibroblasts and myofibroblasts. Fibroblasts expressed and deposited significantly higher quantities of fibril forming corneal collagens I and V. In contrast, myofibroblasts expressed and deposited higher quantities of fibronectin and other non-collagenous matrix components. A significant difference in the activation of latent TGF-ß activation exists between fibroblasts and myofibroblasts when measured with a functional luciferase assay. Fibroblasts and myofibroblasts differ in their morphology, extracellular matrix synthesis, and deposition, activation of latent TGF-ß, and chemotaxis. Conclusions: The differences in the expression and deposition of extracellular matrix components by fibroblasts and myofibroblasts are likely related to critical roles they play during different stages of corneal wound healing.

Effect of adlay seed extract on inflammation and fibrogenesis in human corneal activated keratocytes at transcriptional level.

Corneal activated keratocytes (CAKs) -representing the injured phenotype of corneal stromal cells- are associated with several corneal diseases. Inflammatory cytokines are the key drivers of CAK formation subsequently leading to fibrogenesis. This study aimed to investigate the effect of adlay seed extract on the expression of genes involved in inflammation (IL-6, IL-1b, LIF) and fibrogenesis (TGF-ß) in CAK cells. CAKs (106 cells/10 cm2) were exposed to methanolic (MeOH) and residual (Res) extract of adlay seed (1 mg/ml, 24 h). The control group received the vehicle solution without extract at the same time and condition. Then, RNA extraction, cDNA synthesis, and real-time PCR were performed to quantify the relative expression of IL-6, IL-1b, LIF, and TGF-ß in the treated vs. control cells. This study showed that the MeOH extract of adlay seed could significantly downregulate the expression of IL-6 and IL-1b in the CAKs, while the Res extract led to a significant decrease in TGF-ß gene expression. We showed that CAK treatment with adlay seed extract could decrease the expression of genes related to inflammation and fibrogenesis. However, the genes to be targeted depended on the method of extraction. This proof-of-concept study could provide groundwork for the treatment of corneal stromal diseases and ocular regenerative medicine in the future.

An inspired microenvironment of cell replicas to induce stem cells into keratocyte-like dendritic cells for corneal regeneration.

Corneal stromal disorders due to the loss of keratocytes can affect visual impairment and blindness. Corneal cell therapy is a promising therapeutic strategy for healing corneal tissue or even enhancing corneal function upon advanced disorders, however, the sources of corneal keratocytes are limited for clinical applications. Here, the capacity of cell-imprinted substrates fabricated by molding human keratocyte templates to induce differentiation of human adipose-derived stem cells (hADSCs) into keratocytes, is presented. Keratocytes are isolated from human corneal stroma and grown to transmit their ECM architecture and cell-like topographies to a PDMS substrate. The hADSCs are then seeded on cell-imprinted substrates and their differentiation to keratocytes in DMEM/F12 (with and without chemical factors) are evaluated by real-time PCR and immunocytochemistry. The mesenchymal stem cells grown on patterned substrates present gene and protein expression profiles similar to corneal keratocytes. In contrast, a negligible expression of myofibroblast marker in the hADSCs cultivated on the imprinted substrates, is observed. Microscopic analysis reveals dendritic morphology and ellipsoid nuclei similar to primary keratocytes. Overall, it is demonstrated that biomimetic imprinted substrates would be a sufficient driver to solely direct the stem cell fate toward target cells which is a significant achievement toward corneal regeneration.

P05-A152†Human platelet lysate for cornea organ culture.

PURPOSE: Comprehensible concerns have been raised regarding the safety of FBS-based culture media. In this talk we discuss the benefits of using human platelet lysate (HPL) for the xeno-free culture of human donor corneas, isolated corneal stromal keratocytes (CSK) and stromal fibroblasts (SF). METHODS: 32 human corneas unsuitable for transplantation from 16 human donors were cultured for 25-days in either 2%FBS or 2%HPL medium and compared by phase contrast microscopy (ECD and morphology), and next generation sequencing (NGS). Effects of 0.5%FBS, 5%FBS, 0.5%HPL, 2%HPL and 10%hPL on cultured human CSK and SF were evaluated. RESULTS: Differential cornea culture showed lower endothelial cell loss in the 2%HPL vs. 2%FBS group (ECL hPL=-0.7% vs. FBS=-3.8%; p=0.01). 2%HPL led to the upregulation of cytoprotective, anti-inflammatory and anti-fibrotic genes (e.g. HMOX1, SERPINE1, ANGPTL4, LEFTY2) and the downregulation of pro-inflammatory/apoptotic genes (e.g. CXCL14, SIK1B, PLK5, PPP2R3B). CSK/SF cell viability remained high in all groups (98-100%). Cell numbers and proliferation rates increased (p=0.024-0.001), CSK marker expression decreased with higher fractions of HPL and FBS (p<0.001). SMA1 increased with higher amounts of FBS (p=0.003) but decreased with incremental HPL substitution in both cell types (p=0.014). HPL contained more TGF-ß1 (100%hPL 1.861±0.231ng/ml vs. 100%FBS 0.015±0.010ng/ml, p<0.001). bFGF and HGF were only detectable in 100% hPL (bFGF 0.067±0.017ng/ml, HGF 1.074±0.050ng/ml). CONCLUSION: 2%HPL is a suitable xeno-free substitution for 2%FBS in human cornea organ culture, inducing less ECL and potentially beneficial alterations in gene expression. CSK and SF can be cultured with xeno-free hPL. To maintain CSK characteristics substitution must remain minimal (0.5% hPL/FBS). hPL contains the antifibrotic HGF und bFGF, suppressing myofibroblast conversion.

Growth factors and mechano-regulated reciprocal crosstalk with extracellular matrix tune the keratocyte-fibroblast/myofibroblast transition.

Improper healing of the cornea after injury, infections or surgery can lead to corneal scar formation, which is associated with the transition of resident corneal keratocytes into activated fibroblasts and myofibroblasts (K-F/M). Myofibroblasts can create an extracellular matrix (ECM) niche in which fibrosis is promoted and perpetuated, resulting in progressive tissue opacification and vision loss. As a reversion back to quiescent keratocytes is essential to restore corneal transparency after injury, we characterized how growth factors with demonstrated profibrotic effects (PDGF, FGF, FBS, TGFß1) induce the K-F/M transition, and whether their withdrawal can revert it. Indeed, the upregulated expression of αSMA and the associated changes in cytoskeletal architecture correlated with increases in cell contractility, fibronectin (Fn) and collagen matrix density and Fn fiber strain, as revealed by 2D cell culture, nanopillar cellular force mapping and a FRET-labeled Fn tension probe. Substrate mechanosensing drove a more complete K-F/M transition reversal following growth factor withdrawal on nanopillar arrays than on planar glass substrates. Using decellularized ECM scaffolds, we demonstrated that the K-F/M transition was inhibited in keratocytes reseeded onto myofibroblast-assembled, and/or collagen-1-rich ECM. This supports the presence of a myofibroblast-derived ECM niche that contains cues favoring tissue homeostasis rather than fibrosis.

Combination of natural scaffolds and conditional medium to induce the differentiation of adipose-derived mesenchymal stem cells into keratocyte-like cells and its safety evaluation in the animal cornea.

Keratocytes are the main cellular components of the corneal stroma. This cell is quiescent and cannot be cultured easily. The aim of this study was to investigate differentiate human adipose mesenchymal stem cells (hADSCs) into corneal keratocyte cells by combining natural scaffolds and conditioned medium (CM) and evaluating their safety in the rabbit's cornea. Keratocytes were cultured in an optimal culture medium and this medium was collected and kept as a CM. hADSCs were cultured on the decellularized human small incision lenticule extraction (SMILE) lenticule (SL), amniotic membrane (AM), and collagen-coated plates, and were exposed to keratocyte-CM (KCM) for 7, 14, and 21 days. Differentiation was evaluated using Real-time PCR and immunocytochemistry (ICC). hADSCs were cultured on the SL scaffolds and implanted in the corneal stroma of 8 New Zealand male rabbits. Rabbits were followed for 3 months and the safety was evaluated by clinical and histological variables. Real-time PCR results showed a significant increase in the expression of keratocyte-specific markers on the 21 day of differentiation compared to the control group. ICC also confirmed the induction of differentiation. Implantation of SLs containing differentiated cells in the cornea of animals showed no serious complications including neovascularization, corneal opacity, inflammation, or signs of tissue rejection. Furthermore, the evaluation of the presence of keratocyte-like cells after three months in the rabbit stroma was confirmed by Real-time PCR and immunohistochemistry (IHC) analysis. Our results showed that combination of combination of corneal extracellular matrix and KCM can induced keratocytes differentiation of hADSC and can be introduced as a alternative method to supply the required keratocytes in corneal tissue engineering.

Derivation of Human Corneal Keratocytes from ReLEx SMILE Lenticules for Cell Therapy and Tissue Engineering.

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.

Evidence of Disruption in Neural Regeneration in Dry Eye Secondary to Rheumatoid Arthritis.

The purpose of our study was to analyze abnormal neural regeneration activity in the cornea through means of confocal microscopy in rheumatoid arthritis patients with concomitant dry eye disease. We examined 40 rheumatoid arthritis patients with variable severity and 44 volunteer age- and gender-matched healthy control subjects. We found that all examined parameters were significantly lower (p < 0.05) in rheumatoid arthritis patients as opposed to the control samples: namely, the number of fibers, the total length of the nerves, the number of branch points on the main fibers and the total nerve-fiber area. We examined further variables, such as age, sex and the duration of rheumatoid arthritis. Interestingly, we could not find a correlation between the above variables and abnormal neural structural changes in the cornea. We interpreted these findings via implementing our hypotheses. Correspondingly, one neuroimmunological link between dry eye and rheumatoid arthritis could be through the chronic Piezo2 channelopathy-induced K2P-TASK1 signaling axis. This could accelerate neuroimmune-induced sensitization on the spinal level in this autoimmune disease, with Langerhans-cell activation in the cornea and theorized downregulated Piezo1 channels in these cells. Even more importantly, suggested principal primary-damage-associated corneal keratocyte activation could be accompanied by upregulation of Piezo1. Both activation processes on the periphery would skew the plasticity of the Th17/Treg ratio, resulting in Th17/Treg imbalance in dry eye, secondary to rheumatoid arthritis. Hence, chronic somatosensory-terminal Piezo2 channelopathy-induced impaired Piezo2-Piezo1 crosstalk could result in a mixed picture of disrupted functional regeneration but upregulated morphological regeneration activity of these somatosensory axons in the cornea, providing the demonstrated abnormal neural corneal morphology.

The corneal fibroblast: The Dr. Jekyll underappreciated overseer of the responses to stromal injury.

PURPOSE: To review the functions of corneal fibroblasts in wound healing. METHODS: Literature review. RESULTS: Corneal fibroblasts arise in the corneal stroma after anterior, posterior or limbal injuries and are derived from keratocytes. Transforming growth factor (TGF) ß1 and TGFß2, along with platelet-derived growth factor (PDGF), are the major modulators of the keratocyte to corneal fibroblast transition, while fibroblast growth factor (FGF)-2, TGFß3, and retinoic acid are thought to regulate the transition of corneal fibroblasts back to keratocytes. Adequate and sustained levels of TGFß1 and/or TGFß2, primarily from epithelium, tears, aqueous humor, and corneal endothelium, drive the development of corneal fibroblasts into myofibroblasts. Myofibroblasts have been shown in vitro to transition back to corneal fibroblasts, although apoptosis of myofibroblasts has been documented as a major contributor to the resolution of fibrosis in several in situ corneal injury models. Corneal fibroblasts, aside from their role as a major progenitor to myofibroblasts, also perform many critical functions in the injured cornea, including the production of critical basement membrane (BM) components during regeneration of the epithelial BM and Descemet's membrane, production of non-basement membrane-associated stromal collagen type IV to control and downregulate TGFß effects on stromal cells, release of chemotactic chemokines that attract bone marrow-derived cells to the injured stroma, production of growth factors that modulate regeneration and maturation of the overlying epithelium, and production of collagens and other ECM components that contribute to stromal integrity after injury. CONCLUSIONS: Corneal fibroblasts are major contributors to and overseers of the corneal response to injuries.

Stiffness-dependent dynamic effect of inflammation on keratocyte phenotype and differentiation.

Although extensive studies have evaluated the regulation effect of microenvironment on cell phenotype and cell differentiation, further investigations in the field of the cornea are needed to gain sufficient knowledge for possible clinical translation. This study aims to evaluate the regulation effects of substrate stiffness and inflammation on keratocyte phenotype of corneal fibroblasts, as well as the differentiation from stem cells towards keratocytes. Soft and stiff substrates were prepared based on polydimethylsiloxane. HTK and stem cells were cultured on these substrates to evaluate the effects of stiffness. The possible synergistic effects between substrate stiffness and inflammatory factor IL-1ßwere examined by qPCR and immunofluorescence staining. In addition, macrophages were cultured on soft and stiff substrates to evaluate the effect of substrate stiffness on the synthesis of inflammatory factors. The conditioned medium of macrophages (Soft-CM and Stiff-CM) was collected to examine the effects on HTK and stem cells. It was found that inflammatory factor IL-1ßpromoted keratocyte phenotype and differentiation when cells were cultured on soft substrate (∼130 kPa), which were different from cells cultured on stiff substrate (∼2 × 103kPa) and TCP (∼106kPa). Besides, macrophages cultured on stiff substrates had significantly higher expression ofIL-1ßandTnf-αas compared to the cells cultured on soft substrates. And Stiff-CM decreased the expression of keratocyte phenotype markers as compared to Soft-CM. The results of our study indicate a stiffness-dependent dynamic effect of inflammation on keratocyte phenotype and differentiation, which is of significance not only in gaining a deeper knowledge of corneal pathology and repair, but also in being instructive for scaffold design in corneal tissue engineering and ultimate regeneration.

The Soluble Guanylate Cyclase Stimulator BAY 41-2272 Attenuates Transforming Growth Factor ß1-Induced Myofibroblast Differentiation of Human Corneal Keratocytes.

Corneal transparency, necessary for vision and depending on the high organization of stromal extracellular matrix, is maintained by keratocytes. Severe or continuous corneal injuries determine exaggerated healing responses resulting in the formation of irreversible fibrotic scars and vision impairment. Soluble guanylate cyclase (sGC) stimulation demonstrated antifibrotic effects in both experimental fibrosis and human lung and skin fibroblasts. Here, we assessed whether sGC stimulation with BAY 41-2272 could attenuate transforming growth factor ß1 (TGFß1)-induced myofibroblast differentiation of human corneal keratocytes. Cells were challenged with TGFß1, with/without BAY 41-2272 preincubation, and subsequently assessed for viability, proliferation, migration, chemoinvasion, as well for the expression of myofibroblast/fibroblast activation markers and contractile abilities. Treatment with BAY 41-2272 did not affect keratocyte viability, while preincubation of cells with the sGC stimulator was able to inhibit TGFß1-induced proliferation, wound healing capacity, and invasiveness. BAY 41-2272 was also able to attenuate TGFß1-induced myofibroblast-like profibrotic phenotype of keratocytes, as demonstrated by the significant decrease in ACTA2, COL1A1, COL1A2, FN1 and PDPN gene expression, as well as in α-smooth muscle actin, α-1 chain of type I collagen, podoplanin, vimentin and N-cadherin protein expression. Finally, BAY 41-2272 significantly counteracted the TGFß1-induced myofibroblast-like ability of keratocytes to contract collagen gels, reduced phosphorylated Smad3 protein levels, and attenuated gene expression of proinflammatory cytokines. Collectively, our data show for the first time that BAY 41-2272 is effective in counteracting keratocyte-to-myofibroblast transition, thus providing the rationale for the development of sGC stimulators as novel promising modulators of corneal scarring and fibrosis.

Morphological analysis of cell distribution and network structure via gap junctions in swine corneal stroma.

Because of corneal transplantation limitations, there is a need for cornea-specific regenerative medicine. The development of such regenerative medicine has been delayed because of the complex and unique structure of the corneal stroma. Few studies have explored the corneal stroma cell distribution and cell types in vivo. This study investigated regional differences in morphological characteristics and distributions of corneal keratocytes and immunocompetent cells in the corneal stroma to clarify their functions and structural characteristics. The porcine eyeballs were subjected to light microscopy, transmission electron microscopy, scanning electron microscopy, and immunofluorescence staining analyses. Corneal cells were primarily located in the limbus, rather than the center of the cornea; the long keratocyte diameter was largest on the epithelial side of the corneal limbus, while the short diameter was largest on the endothelial side of the central cornea. Moreover, there were significantly more corneal cells on the epithelial side than on the endothelial side in both the central and limbus areas. Gap junctions between cells in the corneal stroma were present on the surfaces of cytoplasmic processes. Many cytoplasmic processes were scattered throughout the corneal stroma; they were connected both vertically and horizontally, forming an intercellular network. Additionally, immunocompetent cells on the epithelial side suggested to participate in this network via gap junctions. The morphology of keratocytes and immunocompetent cells on the epithelial side suggests that they play important roles in corneal homeostasis.

Notch1 signaling in keratocytes maintains corneal transparency by suppressing VEGF expression.

The cornea fends off chemicals, dirt, and infectious particles and provides most of the eye's focusing power. Corneal transparency is of paramount importance to normal vision, yet how it is established and maintained remains unclear. Here, we ablated Notch1 in keratocytes using Twist2-Cre mice and found that Twist2-Cre; Notch1f/f mice developed stroma expansion and neovascularization, followed by hyperproliferation and metaplasia of corneal epithelial progenitor cells and plaque formation at central cornea, leading to loss of transparency. Development of these phenotypes does not involve bacteria-caused inflammation; instead, Notch1 deletion upregulates Vegfa and Vegfc via Hif1α in keratocytes. Vascular endothelial growth factor (VEGF) receptor inhibitor axitinib prevented development of these anomalies in Twist2-Cre; Notch1f/f mice, suggesting that VEGFs secreted by keratocytes promote not only neovascularization but also proliferation and metaplasia of epithelial progenitor cells at central cornea. This study uncovers a Notch1-Hif1α-VEGF pathway in keratocytes that maintains corneal transparency and represents a potential target for treatment of related corneal disorders.