FAK Inhibition Attenuates Corneal Fibroblast Differentiation In Vitro.

Corneal fibrosis (or scarring) occurs in response to ocular trauma or infection, and by reducing corneal transparency, it can lead to visual impairment and blindness. Studies highlight important roles for transforming growth factor (TGF)-ß1 and -ß3 as modulators in corneal wound healing and fibrosis, leading to increased extracellular matrix (ECM) components and expression of α-smooth muscle actin (αSMA), a myofibroblast marker. In this study, human corneal fibroblasts (hCF) were cultured as a monolayer culture (2D) or on poly-transwell membranes to generate corneal stromal constructs (3D) that were treated with TGF-ß1, TGF-ß3, or TGF-ß1 + FAK inhibitor (FAKi). Results show that hCF 3D constructs treated with TGF-ß1 or TGF-ß3 impart distinct effects on genes involved in wound healing and fibrosis-ITGAV, ITGB1, SRC and ACTA2. Notably, in the 3D construct model, TGF-ß1 enhanced αSMA and focal adhesion kinase (FAK) protein expression, whereas TGF-ß3 did not. In addition, in both the hCF 2D cell and 3D construct models, we found that TGF-ß1 + FAKi attenuated TGF-ß1-mediated myofibroblast differentiation, as shown by abrogated αSMA expression. This study concludes that FAK signaling is important for the onset of TGF-ß1-mediated myofibroblast differentiation, and FAK inhibition may provide a novel beneficial therapeutic avenue to reduce corneal scarring.

Extracellular Vesicles in the Cornea: Insights from Other Tissues.

Extracellular vesicles (EVs) are phospholipid bilayer-bound particles secreted by cells that have been found to be important in mediating cell-cell communication, signal transduction, and extracellular matrix remodeling. Their role in both physiological and pathological processes has been established in different tissues throughout the human body. The human cornea functions as a transparent and refractive barrier that protects the intraocular elements from the external environment. Injury, infection, or disease may cause the loss of corneal clarity by altering extracellular matrix organization within the stroma that may lead to detrimental effects on visual acuity. Over the years, numerous studies have identified many of the growth factors (e.g., transforming growth factor-ß1, thrombospondin-1, and platelet-derived growth factor) important in corneal wound healing and scarring. However, the functional role of bound factors encapsulated in EVs in the context of corneal biology is less defined. In this review, we describe the discovery and characterization of EVs in the cornea. We focus on EV-matrix interactions, potential functions during corneal wound healing, and the bioactivity of mesenchymal stem cell-derived EVs. We also discuss the development of EVs as stable, drug-loaded therapeutics for ocular applications.

Modeling the cornea in 3-dimensions: Current and future perspectives.

The cornea is an avascular, transparent ocular tissue that serves as a refractive and protective structure for the eye. Over 90% of the cornea is composed of a collagenous-rich extracellular matrix within the stroma with the other 10% composed by the corneal epithelium and endothelium layers and their corresponding supporting collagen layers (e.g., Bowman's and Descemet's membranes) at the anterior and posterior cornea, respectively. Due to its prominent role in corneal structure, tissue engineering approaches to model the human cornea in vitro have focused heavily on the cellular and functional properties of the corneal stroma. In this review, we discuss model development in the context of culture dimensionality (e.g., 2-dimensional versus 3-dimensional) and expand on the optical, biomechanical, and cellular functions promoted by the culture microenvironment. We describe current methods to model the human cornea with focus on organotypic approaches, compressed collagen, bioprinting, and self-assembled stromal models. We also expand on co-culture applications with the inclusion of relevant corneal cell types, such as epithelial, stromal keratocyte or fibroblast, endothelial, and neuronal cells. Further advancements in corneal tissue model development will markedly improve our current understanding of corneal wound healing and regeneration.

Characterization of Tear Immunoglobulins in a Small-Cohort of Keratoconus Patients.

Keratoconus (KC) is classically considered a non-inflammatory condition caused by central corneal thinning that leads to astigmatism and reduced visual acuity. Previous studies have identified increased systemic levels of pro-inflammatory factors, including interleukin-6, tumor necrosis factor-α, and matrix metalloproteinase-9, suggesting that KC may have an inflammatory component in at least a subset of patients. In this study, we evaluated the levels of different immunoglobulins (light and heavy chains) based on Ig α, Ig λ, Ig κ, Ig µ, and Ig heavy chain subunits in non-KC tears (n = 7 control individuals) and KC tears (n = 7 KC patients) using tandem-liquid chromatography mass spectrometry. The most abundant Ig heavy chains detected in both control individuals and KC patients were Ig α-1 and Ig α-2 likely correlating to the higher IgA levels reported in human tears. We identified significant differences in immunoglobulin κ-chain V-II levels in KC patients compared to control individuals with no significant difference in Ig κ/Ig λ ratios or heavy chain levels. Our study supports previous findings suggesting that KC possesses a systemic component that may contribute to the KC pathology. Further studies are required to define causality and establish a role for systemic immune system-dependent factors and pro-inflammatory processes in KC development or progression.

Extracellular Vesicles Secreted by Corneal Epithelial Cells Promote Myofibroblast Differentiation.

The corneal epithelium mediates the initial response to injury of the ocular surface and secretes a number of profibrotic factors that promote corneal scar development within the stroma. Previous studies have shown that corneal epithelial cells also secrete small extracellular vesicles (EVs) in response to corneal wounding. In this paper, we hypothesized that EVs released from corneal epithelial cells in vitro contain protein cargo that promotes myofibroblast differentiation, the key cell responsible for scar development. We focused on the interplay between corneal epithelial-derived EVs and the stroma to determine if the corneal fibroblast phenotype, contraction, proliferation, or migration were promoted following vesicle uptake by corneal fibroblasts. Our results showed an increase in myofibroblast differentiation based on α-smooth muscle actin expression and elevated contractility following EV treatment compared to controls. Furthermore, we characterized the contents of epithelial cell-derived EVs using proteomic analysis and identified the presence of provisional matrix proteins, fibronectin and thrombospondin-1, as the dominant encapsulated protein cargo secreted by corneal epithelial cells in vitro. Proteins associated with the regulation of protein translation were also abundant in EVs. This paper reveals a novel role and function of EVs secreted by the corneal epithelium that may contribute to corneal scarring.

Extracellular Vesicles and Cell-Cell Communication in the Cornea.

One question that has intrigued cell biologists for many years is, "How do cells interact to influence one another's activity?" The discovery of extracellular vesicles (EVs) and the fact that they carry cargo, which directs cells to undergo changes in morphology and gene expression, has revolutionized this field of research. Little is known regarding the role of EVs in the cornea; however, we have demonstrated that EVs isolated from corneal epithelial cells direct corneal keratocytes to initiate fibrosis. Intriguingly, our data suggest that EVs do not penetrate epithelial basement membrane (BM), perhaps providing a mechanism explaining the importance of BM in the lack of scarring in scrape wounds. Since over 100-million people worldwide suffer from visual impairment as a result of corneal scarring, the role of EVs may be vital to understanding the mechanisms of wound repair. Therefore, we investigated EVs in ex vivo and in vivo-like three-dimensional cultures of human corneal cells using transmission electron microscopy. Some of the major findings were all three major cell types (epithelial, fibroblast, and endothelial cells) appear to release EVs, EVs can be identified using TEM, and EVs appeared to be involved in cell-cell communication. Interestingly, while our previous publication suggests that EVs do not penetrate the epithelial BM, it appears that EVs penetrate the much thicker endothelial BM (Descemet's membrane). These findings indicate the huge potential of EV research in the cornea and wound healing, and suggest that during homeostasis the endothelium and stromal cells are in communication. Anat Rec, 2019. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Biology of corneal fibrosis: soluble mediators, integrins, and extracellular vesicles.

Corneal fibrosis develops in response to injury, infection, postsurgical complications, or underlying systemic disease that disrupts the homeostasis of the tissue leading to irregular extracellular matrix deposition within the stroma. The mechanisms that regulate corneal scarring are focused heavily on the canonical transforming growth factor-ß pathway and relevant activators, and their role in promoting myofibroblast differentiation. In this paper, we discuss the biochemical pathways involved in corneal fibrosis in the context of different injury models-epithelial debridement, superficial keratectomy, and penetrating incision. We elaborate on the interplay of the major pro-fibrotic factors involved in corneal scar development (e.g., transforming growth factor-ß1, thrombospondin-1, and ανß6), and explore a novel role for extracellular vesicles secreted by the wounded epithelium and the importance of the basement membrane.

Corneal Epithelial-Stromal Fibroblast Constructs to Study Cell-Cell Communication in Vitro.

Cell-cell communication plays a fundamental role in mediating corneal wound healing following injury or infection. Depending on the severity of the wound, regeneration of the cornea and the propensity for scar development are influenced by the acute resolution of the pro-fibrotic response mediated by closure of the wound via cellular and tissue contraction. Damage of the corneal epithelium, basement membrane, and anterior stroma following a superficial keratectomy is known to lead to significant provisional matrix deposition, including secretion of fibronectin and thrombospondin-1, as well as development of a corneal scar. In addition, corneal wounding has previously been shown to promote release of extracellular vesicles from the corneal epithelium, which, in addition to soluble factors, may play a role in promoting tissue regeneration. In this study, we report the development and characterization of a co-culture system of human corneal epithelial cells and corneal stromal fibroblasts cultured for 4 weeks to allow extracellular matrix deposition and tissue maturation. The secretion of provisional matrix components, as well as small and large extracellular vesicles, was apparent within the constructs, suggesting cell-cell communication between epithelial and stromal cell populations. Laminin-1ß was highly expressed by the corneal epithelial layer with the presence of notable patches of basement membrane identified by transmission electron microscopy. Interestingly, we identified expression of collagen type III, fibronectin, and thrombospondin-1 along the epithelial-stromal interface similar to observations seen in vivo following a keratectomy, as well as expression of the myofibroblast marker, α-smooth muscle actin, within the stroma. Our results suggest that this corneal epithelial-stromal model may be useful in the study of the biochemical phenomena that occur during corneal wound healing.

Transient Mitomycin C-treatment of human corneal epithelial cells and fibroblasts alters cell migration, cytokine secretion, and matrix accumulation.

A single application of Mitomycin C (MMC) is used clinically in ophthalmology to reduce scarring and enhance wound resolution after surgery. Here we show in vitro that a 3-hour MMC treatment of primary and telomerase immortalized human corneal limbal epithelial (HCLE) cells impacts their migration and adhesion. Transient MMC treatment induces HCLE expression of senescence associated secretory factors, cytokine secretion, and deposition of laminin 332 for several days. Transient MMC treatment also reduces migration and deposition of transforming growth factor-ß1 (TGFß1)-stimulated collagen by corneal fibroblasts. Using conditioned media from control and MMC treated cells, we demonstrate that factors secreted by MMC-treated corneal epithelial cells attenuate collagen deposition by HCFs whereas those secreted by MMC-treated HCFs do not. These studies are the first to probe the roles played by corneal epithelial cells in reducing collagen deposition by corneal fibroblasts in response to MMC.

Epidermal Growth Factor Stimulates Transforming Growth Factor-Beta Receptor Type II Expression In Corneal Epithelial Cells.

We previously demonstrated that inhibition of epidermal growth factor receptor (EGFR) slowed corneal epithelial migration. Here we examine the effect of EGF on transforming growth factor-beta receptor II (TGF-ßRII) in a corneal wound-healing model and primary human corneal epithelial cells (pHCE). Corneal debridement wounds were made and allowed to heal ± Tyrphostin AG1478 (EGFR inhibitor), and assayed for EGFR activation and EGFR and TGF-ßRII localization. Primary HCE were treated with EGF ± U0126 (MEK inhibitor) and assayed for TGF-ßRII expression. EGFR activation was maximal 15 minutes after wounding and localized in the migrating epithelial cells. TGF-ßRII localization was also observed in the migrating epithelium and was reduced when EGFR was blocked. When pHCE were treated with EGF for 6 hours, the cells produced enhanced levels of TGF-ßRII, which was blocked by U0126. Downstream signaling pathways of MEK (p38MAPK and ERK1/2MAPK) were then examined, and TGF-ß1 and EGF were found to have differential effects on the phosphorylation of p38 and ERK1/2, with TGF-ß1 upregulating p-p38 but not pERK1/2 and EGF upregulating pERK1/2 but not p-p38. Taken together, these data indicate that EGF stimulates TGF-ßRII through ERK1/2 and EGFR signaling, suggesting interplay between EGF- and TGF-ß-signaling pathways during corneal wound repair.

3D in vitro model for human corneal endothelial cell maturation.

Corneal endothelium is a cellular monolayer positioned on the Descemet's membrane at the anterior cornea, and it plays a critical role in maintaining corneal clarity. Our present study examines the feasibility of utilizing our 3-dimensional (3D) corneal stromal construct, which consists of human corneal fibroblasts (HCF) and their self-assembled matrix, to observe the development and maturation of human corneal endothelial cells (HCEndoCs) in a co-culture model. Three-dimensional HCF constructs were created by growing the HCFs on Transwell membranes in Eagles' minimum essential medium (EMEM) + 10% FBS + 0.5 mM Vitamin C (VitC) for about 4 weeks. HCEndoCs, either primary (pHCEndoC) or cell line (HCEndoCL), were either seeded in chamber slides, directly on the Transwell membranes, or on the 3D HCF constructs and cultivated for 5 days or 2 weeks. The HCEndoCs that were seeded directly on the Transwell membranes were exposed indirectly to HCF by culturing the HCF on the plate beneath the membrane. Cultures were examined for morphology and ultrastructure using light and transmission electron microscopy (TEM). In addition, indirect-immunofluorescence microscopy (IF) was used to examine tight junction formation (ZO-1), maturation (ALDH1A1), basement membrane formation (Laminin), cell proliferation (Ki67), cell death (caspase-3), and fibrotic response (CTGF). As expected, both pHCEndoCs and HCEndoCLs formed monolayers on the constructs; however, the morphology of the HCEndoCLs appeared to be similar to that seen in vivo, uniform and closely packed, whereas the pHCEndoCs remained elongated. The IF data showed that laminin localization was present in the HCEndoCs' cytoplasm as cell-cell contact increased, and when they were grown in the 3D co-culture, the beginnings of what appears to be a continuous DM-like structure was observed. In addition, in co-cultures, ALDH1A1-positive HCEndoCs were present, ZO-1 expression localized within the tight junctions, minimal numbers of HCEndoCs were Ki67-or Caspase-3-positive, and CTGF was positive in both the HCEndoCs cytoplasm and the matrix of the co-culture. Also, laminin localization was stimulated in HCEndoCs upon indirect stimuli secreted by HCF. The present data suggests our 3D co-culture model is useful for studying corneal endothelium maturation in vitro since the co-culture promotes new DM-like formation, HCEndoCs develop in vivo-like characteristics, and the fibrotic response is activated. Our current findings are applicable to understanding the implications of corneal endothelial injection therapy, such as if the abnormal DM has to be removed from the patient, the newly injected endothelial cells will seed onto the wound area and deposit a new DM-like membrane. However, caution should be observed and as much of the normal DM should be left intact since removal of the DM can cause a posterior stromal fibrotic response.

Initiation of fibrosis in the integrin Αvß6 knockout mice.

We previously demonstrated that ß6 knockout mice showed impaired wound repair in corneal debridement and keratectomy wounds. In the current investigation, we continued our examination of integrin αvß6 in order to determine if it was required for the initiation of wound healing in a corneal wound model that normally heals in a fibrotic manner. A full-thickness corneal incision was made in C57BL/6 J wild type (WT) and C57BL/6-Itgb6 KO (ß6-/-) mice. The mice were observed at 3, 7, 14, and 28 days post-incision. The morphology of corneal restoration was observed in tissue sections stained with hemotoxilin and eosin (H&E). In addition, indirect-immunofluorescence (IF) was performed on sections and/or whole mounts to evaluate the immunolocalization of α-smooth muscle actin (SMA) and thrombospondin-1 (TSP-1). H&E staining revealed that the corneas in ß6-/- mice healed slower than those in WT mice, with an obvious delay in the restoration of the stromal matrix and epithelium. In sections at 3 and 7 days, SMA and TSP-1 were greatly reduced in the ß6-/- mice as compared to WT, but peaked at 28 days after incision. Whole mount SMA IF results were consistent with those from sections. Therefore, the initiation of fibrosis was inhibited by the lack of αvß6; however, there appeared to be an alternate mechanism that initiated fibrosis 7-14 days later. Localization of TSP-1 correlated with expression of SMA whether wound healing was delayed or initiated immediately after wounding.

Inhibition of Human Corneal Myofibroblast Formation.

Purpose: Transforming growth factor-beta (TGF-ß) isoform 1 (T1) is involved in corneal fibrotic wound healing by stimulating myofibroblast transformation and altering fibrotic gene expression. In this study, two specific inhibitors were used to dissect the relationship between myofibroblast generation and the TGF-ß/Smad- or TGF-ß/p38-signaling pathway in human corneal fibroblasts (HCF). Methods: In HCF, Trx-SARA (Smad-pathway inhibitor) was used to block the TGF-ß/Smad-signaling pathway, and the p38 inhibitor (p38inh, SB202190) was used to inhibit p38MAPK, thus blocking the TGF-ß/p38-signaling pathway. HCF ± Trx-SARA or Trx-GA (SARA control) were serum starved overnight in Eagle's minimum essential medium (EMEM) ± p38inh, grown in EMEM ± T1 ± p38inh for 24 hours, and then processed for indirect-immunofluorescence, Western blot, or quantitative real-time polymerase chain reaction to examine α-smooth muscle actin (αSMA) and other fibrotic genes, such as fibronectin, thrombospondin1, and type III collagen. In addition, the morphology and the effect of p38inh on myofibroblast phenotype after myofibroblast formation were examined. Results: We observed that Trx-SARA had little effect on αSMA expression, indicating that blocking the Smad pathway did not significantly inhibit myofibroblast formation. However, p38inh did significantly inhibit αSMA and other fibrotic genes, thus efficiently preventing the transition of HCFs to myofibroblasts. In addition, morphology changed and αSMA decreased in myofibroblasts exposed to p38inh medium, as compared with controls. Conclusions: HCF transition to myofibroblasts was mainly through the p38 pathway. Therefore, blocking the p38 pathway may be a potential therapeutic tool for human corneal fibrosis prevention/treatment, because it controls myofibroblast formation in human corneal cells, while leaving other functions of T1 unaffected.

Hypoxia modulates the development of a corneal stromal matrix model.

Deposition of matrix proteins during development and repair is critical to the transparency of the cornea. While many cells respond to a hypoxic state that can occur in a tumor, the cornea is exposed to hypoxia during development prior to eyelid opening and during the diurnal sleep cycle where oxygen levels can drop from 21% to 8%. In this study, we used 2 three-dimensional (3-D) models to examine how stromal cells respond to periods of acute hypoxic states. The first model, a stromal construct model, is a 3-D stroma-like construct that consists of human corneal fibroblasts (HCFs) stimulated by a stable form of ascorbate for 1, 2, and 4 weeks to self-assemble their own extracellular matrix. The second model, a corneal organ culture model, is a corneal wound-healing model, which consists of wounded adult rat corneas that were removed and placed in culture to heal. Both models were exposed to either normoxic or hypoxic conditions for varying time periods, and the expression and/or localization of matrix proteins was assessed. No significant changes were detected in Type V collagen, which is associated with Type I collagen fibrils; however, significant changes were detected in the expression of both the small leucine-rich repeating proteoglycans and the larger heparan sulfate proteoglycan, perlecan. Also, hypoxia decreased both the number of Cuprolinic blue-positive glycosaminoglycan chains along collagen fibrils and Sulfatase 1, which modulates the effect of heparan sulfate by removing the 6-O-sulfate groups. In the stromal construct model, alterations were seen in fibronectin, similar to those that occur in development and after injury. These changes in fibronectin after injury were accompanied by changes in proteoglycans. Together these findings indicate that acute hypoxic changes alter the physiology of the cornea, and these models will allow us to manipulate the conditions in the extracellular environment in order to study corneal development and trauma.

Mouse Models of Corneal Scarring.

Corneal scarring is an obligatory consequence of stroma corneal injury and is a major cause of decreased visual quality and vision loss worldwide. There are currently no satisfactory intervention therapies for corneal fibrosis. In this chapter, we describe well-established in vivo corneal wound models to allow researchers to investigate epithelial and stromal responses to corneal injury.

Development of wound healing models to study TGFß3's effect on SMA.

The goal of this study was to test the efficacy of transforming growth factor beta 3 (TGFß3) in reducing α-smooth muscle actin (SMA) expression in two models-an ex vivo organ culture and an in vitro 3D cell construct-both of which closely mimic an in vivo environment. For the ex vivo organ culture system, a central 6.0 mm corneal keratectomy was performed on freshly excised rabbit globes The corneas were then excised, segregated into groups treated with 1.0 ng/ml TGFß1 or ß3 (T1 or T3, respectively), and cultured for 2 weeks. The corneas were assessed for levels of haze and analyzed for SMA mRNA levels. For the 3D in vitro model, rabbit corneal fibroblasts (RbCFs) were cultured for 4 weeks on poly-transwell membranes in Eagle's minimum essential media (EMEM) + 10% FBS + 0.5 mM vitamin C ± 0.1 ng/ml T1 or T3. At the end of 4 weeks, the constructs were processed for analysis by indirect-immunofluorescence (IF) and RT-qPCR. The RT-qPCR data showed that SMA mRNA expression in T3 samples for both models was significantly lower (p < 0.05) than T1 treatment (around 3-fold in ex vivo and 2-fold in constructs). T3 also reduced the amount of scarring in ex vivo corneas as compared with the T1 samples. IF data from RbCF constructs confirmed that T3-treated samples had up to 4-fold (p < 0.05) lower levels of SMA protein expression than samples treated with T1. These results show that T3 when compared to T1 decreases the expression of SMA in both ex vivo organ culture and in vitro 3D cell construct models. Understanding the mechanism of T3's action in these systems and how they differ from simple cell culture models, may potentially help in developing T3 as an anti-scarring therapy.

TGF-ß-target genes are differentially regulated in corneal epithelial cells and fibroblasts.

PURPOSE: Transforming growth factor-beta (TGF-ß) activates the canonical Smad pathway, which includes the Smad family of proteins and SARA (Smad Anchor for Receptor Activation) and other less understood pathways, including one involving p38MAPK. The goal of the current research was to determine if corneal epithelial cells and fibroblasts used the classical or alternative TGF-ß-signaling pathways. To examine this question, we made use of Trx-SARA, which inhibits native SARA, thus blocking the Smad pathway. METHODS: A human corneal epithelial cell line (HCE-TJ), and stromal fibroblasts (HCF) were infected with retroviruses (RTV) containing either Trx-SARA or Trx-GA (a control plasmid). The effect of Trx-SARA on thrombospondin-1 (TSP-1) expression in both cell types, p15ink4b expression in HCE-TJ, and cellular fibronectin (cFN) expression in HCF was determined. In addition, the effect of p38MAPK inhibitor on TSP-1 and p15ink4b were examined. RESULTS: In HCE-TJ with TGF-ß1, TSP-1-protein levels increased and peaked at 24 hours. Trx-SARA reduced TSP-1 expression in HCE-TJ, but had no effect on p15ink4b. With HCF, Trx-SARA failed to reduce TSP-1 expression; however, cFN expression decreased and proliferation was inhibited. By blocking the p38MAPK pathway, TSP-1 expression was reduced in HCF and p15ink4b expression was decreased in HCE-TJ. CONCLUSIONS: Surprisingly, TSP-1 was regulated through the Smad pathway in HCE-TJ and the p38MAPK pathway in HCF. The p38MAPK pathway also induced p15ink4b in HCE-TJ. Our results indicate that not all TGF-ß-target proteins require the Smad pathway, and it may be possible to block certain TGF-ß-target proteins without blocking the expression of all the TGF-ß-target proteins.

PDGFRα Is a Key Regulator of T1 and T3's Differential Effect on SMA Expression in Human Corneal Fibroblasts.

Purpose: The goal of this study was to examine the mechanism behind the unique differential action of transforming growth factor ß3 (TGF-ß3) and TGF-ß1 on SMA expression. It was our hypothesis that platelet-derived growth factor receptor α (PDGFRα) played a key role in determining TGF-ß3's response to wounding. Methods: A stable cell line, human corneal fibroblast (HCF)-P, was created from HCFs by knocking down PDGFRα expression using a lentivirus-delivered shRNA sequence. A three-dimensional (3D) in vitro model was constructed by culturing HCF or HCF-P on poly-transwell membranes for 4 weeks in the presence and absence of 0.1 ng/mL TGF-ß1 or -ß3. At the end of 4 weeks, the constructs were processed for immunofluorescence and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In addition, HCF and HCF-P cell migration was evaluated. Results: In HCF, TGF-ß3 treatment resulted in significantly lower α-smooth muscle actin (SMA) mRNA expression and immunolocalization when compared to TGF-ß1, while in HCF-P, both TGF-ß1 and -ß3 treatment increased the SMA mRNA expression and immunolocalization compared to both the untreated HCF-P control and TGF-ß3-treated HCF. Human corneal fibroblast-P also had a lower migration rate and construct thickness when compared to HCF. Conclusions: These results show that TGF-ß3 decreases SMA in HCF, while remarkably increasing SMA in HCF-P, thus indicating that the presence or absence of PDGFRα elicits contrasting responses to the same TGF-ß3 treatment. Understanding the role of PDGFRα in TGF-ß3's ability to stimulate SMA may potentially help in understanding the differential functions of TGF-ß1 and TGF-ß3 in corneal wound healing.

Potential role of corneal epithelial cell-derived exosomes in corneal wound healing and neovascularization.

Specific factors from the corneal epithelium underlying the stimulation of stromal fibrosis and myofibroblast formation in corneal wound healing have not been fully elucidated. Given that exosomes are known to transfer bioactive molecules among cells and play crucial roles in wound healing, angiogenesis, and cancer, we hypothesized that corneal epithelial cell-derived exosomes may gain access to the underlying stromal fibroblasts upon disruption of the epithelial basement membrane and that they induce signaling events essential for corneal wound healing. In the present study, exosome-like vesicles were observed between corneal epithelial cells and the stroma during wound healing after corneal epithelial debridement. These vesicles were also found in the stroma following anterior stromal keratectomy, in which surgical removal of the epithelium, basement membrane, and anterior stroma was performed. Exosomes secreted by mouse corneal epithelial cells were found to fuse to keratocytes in vitro and to induce myofibroblast transformation. In addition, epithelial cell-derived exosomes induced endothelial cell proliferation and ex vivo aortic ring sprouting. Our results indicate that epithelial cell-derived exosomes mediate communication between corneal epithelial cells and corneal keratocytes as well as vascular endothelial cells. These findings demonstrate that epithelial-derived exosomes may be involved in corneal wound healing and neovascularization, and thus, may serve as targets for potential therapeutic interventions.

Human Corneal Fibroblast Pattern Evolution and Matrix Synthesis on Mechanically Biased Substrates.

In a fibroblast colony model of corneal stromal development, we asked how physiological tension influences the patterning dynamics of fibroblasts and the orientation of deposited extracellular matrix (ECM). Using long-term live-cell microscopy, enabled by an optically accessible mechanobioreactor, a primary human corneal fibroblast colony was cultured on three types of substrates: a mechanically biased, loaded, dense, disorganized collagen substrate (LDDCS), a glass coverslip, and an unloaded, dense, disorganized collagen substrate (UDDCS). On LDDCS, fibroblast orientation and migration along a preferred angle developed early, cell orientation was correlated over long distances, and the colony pattern was stable. On glass, fibroblast orientation was poorly correlated, developed more slowly, and colony patterns were metastable. On UDDCS, cell orientation was correlated over shorter distances compared with LDDCS specimens. On all substrates, the ECM pattern reflected the cell pattern. In summary, mechanically biasing the collagen substrate altered the early migration behavior of individual cells, leading to stable emergent cell patterning, which set the template for newly synthesized ECM.