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.

Dimensionality Matters: Exploiting UV-Photopatterned 2D and Two-Photon-Printed 2.5D Contact Guidance Cues to Control Corneal Fibroblast Behavior and Collagen Deposition.

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.

Two-phase mechanism in the treatment of corneal stromal fibrosis with topical losartan.

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.

Corneal stromal localization of TGF beta isoforms in spontaneous persistent epithelial defects after PRK in rabbits.

The purpose of this study was to evaluate transforming growth factor beta (TGFß) 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 TGFß3, myofibroblast marker alpha-smooth muscle actin (α-SMA) and mesenchymal marker vimentin. One cornea had sufficient remaining tissue for triplex IHC for TGFß1, TGFß2, or TGFß3 (each with α-SMA and vimentin) using isoform-specific antibodies. All three TGFß isoforms were detected in the subepithelial stroma at and surrounding the PED. Some of each TGFß isoform co-localized with α-SMA of myofibroblasts, which could be TGFß isoform autocrine production by myofibroblasts or TGFß-1, -2, and -3 binding to these myofibroblasts.

Short-Term Effect of Rose Bengal Photodynamic Therapy (RB-PDT) on Collagen I, Collagen V, NF-κB, LOX, TGF-ß and IL-6 Expression of Human Corneal Fibroblasts, In Vitro.

PURPOSE: To investigate collagen I, collagen V, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), lysyl oxidase (LOX), transforming growth factor ß1 (TGF-ß1) and interleukin-6 (IL-6) expression in healthy and keratoconus human corneal fibroblasts (HCFs and KC-HCFs), 24 h after Rose Bengal photodynamic therapy (RB-PDT). METHODS: HCFs were isolated from healthy human corneal donors (n = 5) and KC-HCFs from elective penetrating keratoplasties (n = 5). Both cell cultures underwent RB-PDT (0.001% RB concentration, 0.17 J/cm2 fluence) and 24 h later collagen I, collagen V, NF-κB, LOX, TGF-ß1 and IL-6 mRNA and protein expression have been determined using qPCR and Western blot, IL-6 concentration in the cell culture supernatant by ELISA. RESULTS: TGF-ß1 mRNA expression was significantly lower (p = 0.02) and IL-6 mRNA expression was significantly higher in RB-PDT treated HCFs (p = 0.01), than in HCF controls. COL1A1, COL5A1 and TGF-ß1 mRNA expression was significantly lower (p = 0.04; p = 0.02 and p = 0.003) and IL-6 mRNA expression was significantly higher (p = 0.02) in treated KC-HCFs, than in KC-HCF controls. TGF-ß1 protein expression in treated HCFs was significantly higher than in HCF controls (p = 0.04). IL-6 protein concentration in the HCF and KC-HCF culture supernatant after RB-PDT was significantly higher than in controls (p = 0.02; p = 0.01). No other analyzed mRNA and protein expression differed significantly between the RB-PDT treated and untreated groups. CONCLUSIONS: Our study demonstrates that RB-PDT reduces collagen I, collagen V and TGF-ß1 mRNA expression, while increasing IL-6 mRNA and protein expression in KC-HCFs. In HCFs, RB-PDT increases TGF-ß1 and IL-6 protein level after 24 h.

Transforming growth factor beta-3 localization in the corneal response to epithelial-stromal injury and effects on corneal fibroblast transition to 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.

3D bioprinting of corneal decellularized extracellular matrix: GelMA composite hydrogel for corneal stroma engineering.

Millions of individuals across the world suffer from corneal stromal diseases that impair vision. Fortunately, three-dimensional (3D) bioprinting technology which has revolutionized the field of regenerative tissue engineering makes it feasible to create personalized corneas. In this study, an artificial cornea with a high degree of precision, smoothness, and programmable curvature was prepared by using digital light processing (DLP) 3D bioprinting in one piece with no support structure, and the construct was then confirmed by optical coherence tomography (OCT). On the basis of this approach, we developed a novel corneal decellularized extracellular matrix/gelatin methacryloyl (CECM-GelMA) bioink that can produce complex microenvironments with highly tunable mechanical properties while retaining high optical transmittance. Furthermore, the composite hydrogel was loaded with human corneal fibroblasts (hCFs), and in vitro experiments showed that the hydrogel maintained high cell viability and expressed core proteins. In vivo tests revealed that the hydrogel might promote epithelial regeneration, keep the matrix aligned, and restore clarity. This demonstrates how crucial a role CECM plays in establishing a favorable environment that encourages the transformation of cell function. Therefore, artificial corneas that can be rapidly customized have a huge potential in the development of in vitro corneal matrix analogs.

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.

The Cornea: No Difference in the Wound Healing Response to Injury Related to Whether, or Not, There's a Bowman's Layer.

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.

Standardization of corneal alkali burn methodology in rabbits.

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.

Reduced OPA1, Mitochondrial Fragmentation and Increased Susceptibility to Apoptosis in Granular Corneal Dystrophy Type 2 Corneal Fibroblasts.

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.

In Vitro Expression Analysis of Cytokines and ROS-Related Genes in Human Corneal Fibroblasts and Keratocytes of Healthy and Keratoconus Corneas.

PURPOSE: To investigate expression of cytokines and ROS-related genes in stromal cells of healthy and keratoconus (KC) corneas. METHODS: Expression analysis was performed for cytokines including several interleukins (IL), Tumor necrosis factor-α (TNF-α), Transforming growth factor-ß1 (TGF-ß1), 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. RESULTS: 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. CONCLUSION: Increased expression of several inflammatory markers described as altered in KC was not evident in KC cells in vitro.

Hydroxycamptothecin and Substratum Stiffness Synergistically Regulate Fibrosis of Human Corneal Fibroblasts.

Corneal fibrosis is a common outcome of inappropriate repair associated with trauma or ocular infection. Altered biomechanical properties with increased corneal stiffness is a feature of fibrosis that cause corneal opacities, resulting in severe visual impairment and even blindness. The present study aims to determine the effect of hydroxycamptothecin (HCPT) and matrix stiffness on transforming growth factor-ß1 (TGF-ß1)-induced fibrotic processes in human corneal fibroblasts (HTK cells). HTK cells were cultured on substrates with different stiffnesses ("soft", ∼261 kPa; "stiff", ∼2.5 × 103 kPa) and on tissue culture plastic (TCP, ∼106 kPa) and simultaneously treated with or without 1 µg/mL HCPT and 10 ng/mL TGF-ß1. We found that HCPT induced decreased cell viability and antiproliferative effects on HTK cells. TGF-ß1-induced expression of fibrosis-related genes (FN1, ACTA2) was reduced if the cells were simultaneously treated with HCPT. Substrate stiffness did not affect the expression of fibrosis-related genes. The TGF-ß1 induced expression of FN1 on both soft and stiff substrates was reduced if cells were simultaneously treated with HCPT. However, this trend was not seen for ACTA2, i.e., the TGF-ß1 induced expression of ACTA2 was not reduced by simultaneous treatment of HCPT in either soft or stiff substrate. Instead, HCPT treatment in the presence of TGF-ß1 resulted in increased gene expression of keratocyte phenotype makers (LUM, KERA, AQP1, CHTS6) on both substrate stiffnesses. In addition, the protein expression of keratocyte phenotype makers LUM and ALDH3 was increased in HTK cells simultaneously treated with TGF-ß1 and HCPT on stiff substrate as compared to control, i.e., without HCPT. In conclusion, we found that HCPT can reduce TGF-ß1-induced fibrosis and promote the keratocyte phenotype in a substrate stiffness dependent manner. Thus, HCPT stimulation might be an approach to stimulate keratocytes in the appropriate healing stage to avoid or reverse fibrosis and achieve more optimal corneal wound healing.

Effect of Conditioned Media of Limbal Epithelial Cells and Corneal Stromal Cells on Functional Activity of Limb Mesenchymal Stem Cells.

We studied the effect of conditioned media from limbal epithelial stem cells, fibroblasts, and corneal keratocytes on the functional activity of human limbal mesenchymal stem cells. It was shown that the conditioned media from limbal epithelial stem cells reduced proliferative activity and inhibited migration of limbal mesenchymal stem cells. In the conditioned media of limbal epithelial stem cells, increased concentrations of VEGF and TNFα and reduced concentration of BDNF, vimentin, and fibronectin were found. The conditioned medium from corneal stromal cells did not affect functional activity of mesenchymal stem cells in the limbus. These data contribute to the understanding of the interaction of cells in the limbal niche and with corneal cells essential for the maintenance of the cellular homeostasis in the cornea.

Hypoxia-induced mesenchymal stem cells inhibit corneal fibroblast proliferation by regulating the WWP2/Notch1 axis.

Aim: This study aimed to explore the role of hypoxic mesenchymal stem cells (MSCs) in corneal alkali burns and the underlying mechanism. Materials & methods: Rat corneal fibroblasts were incubated with IL-6, followed by treatment with hypoxic MSC supernatant. A rat corneal alkali burn model was implemented and processed with hypoxic MSCs. The associated factors were detected by corresponding methods. Results: Hypoxic MSCs reduced the Notch1 level and the proliferation of rat corneal fibroblasts. Hypoxic MSCs or WWP2 overexpression in MSCs enhanced ubiquitination of Notch1. WWP2 interacted with Notch1, and WWP2 silencing reversed the effects of the hypoxic MSCs. Hypoxic MSC treatment in vivo decreased the corneal neovascularization scores and opacity scores. Conclusion: Hypoxic MSCs inhibited inflammation and alleviated corneal injury in alkali burns via the WWP2/Notch1 axis.

Acute ocular chemical burns are ophthalmic emergencies which require immediate diagnosis and treatment. Quiescent corneal cells differentiate into active fibroblast and myofibroblast phenotypes after corneal injury. Mesenchymal stem cells (MSCs) under hypoxia treatment are applied for the treatment of acute ocular chemical burns. We aimed to explore the role of hypoxic MSCs in corneal alkali burns and the underlying mechanism. The result showed that hypoxic MSCs reduced the proliferation of rat corneal fibroblasts, implying an anti-inflammatory effect. In vivo, treatment with hypoxic MSCs decreased the corneal neovascularization scores and opacity scores, indicating a protective effect on corneal alkali burns. We concluded that hypoxic MSCs could alleviate corneal injury in alkali burns and may be a promising therapeutic option for corneal alkali burns.

Corneal fibroblast collagen type IV negative feedback modulation of TGF beta: A fibrosis modulating system likely active in other organs.

Collagen type IV (COL IV) is a major component of basement membranes (BM) in all organs. It serves functions related to BM organization and modulates the passage of growth factors from one tissue compartment to another. COL IV binds transforming growth factor (TGF) beta-1 and TGF beta-2 and, therefore, is a major modulator of TGF beta pro-fibrotic functions. After fibrotic corneal injury, TGF beta enters into the stroma from the tears, epithelium, endothelium and/or aqueous humor and markedly upregulates COL IV production in corneal fibroblasts in the adjacent stroma far removed from BMs. It is hypothesized this non-BM stromal COL IV binds TGF beta-1 (and likely TGF beta-2) in competition with the binding of the growth factors to TGF beta cognate receptors and serves as a negative feedback regulatory pathway to mitigate the effects of TGF beta on stromal cells, including reducing the developmental transition of corneal fibroblasts and fibrocytes into myofibroblasts. Losartan, a known TGF beta signaling inhibitor, when applied topically to the cornea after fibrotic injury, alters this COL IV-TGF beta pathway by down-regulating COL IV production by corneal fibroblasts. Non-BM COL IV produced in response to injuries in other organs, including the lung, skin, liver, kidney, and gut, may participate in similar COL IV-TGF beta pathways and have an important role in controlling TGF beta pro-fibrotic effects in these organs.

Role of Damage-Associated Molecular Patterns (DAMPs/Alarmins) in Severe Ocular Allergic Diseases.

Severe ocular allergic diseases, such as atopic keratoconjunctivitis and vernal keratoconjunctivitis, cause severe allergic inflammation in the conjunctiva and corneal epithelial damage, resulting in visual disturbances. The involvement of damage (danger)-associated molecular patterns (DAMPs/alarmins) in the pathogenesis of these diseases has been recognized. Alarmins released from damaged corneal epithelial cells or eosinophils play a critical role in the induction of corneal lesions, vicious loop of corneal injury, and exacerbation of conjunctival allergic inflammation. Alarmins in the conjunctiva also play an essential role in the development of both allergic inflammation, based on the acquired immune system, and type 2 inflammation by innate immune responses in the ocular surface. Therefore, alarmins may be a potentially important therapeutic target in severe refractory ocular allergic diseases.

Fibrosis Is a Basement Membrane-Related Disease in the Cornea: Injury and Defective Regeneration of Basement Membranes May Underlie Fibrosis in Other Organs.

Every organ develops fibrosis that compromises functions in response to infections, injuries, or diseases. The cornea is a relatively simple, avascular organ that offers an exceptional model to better understand the pathophysiology of the fibrosis response. Injury and defective regeneration of the epithelial basement membrane (EBM) or the endothelial Descemet's basement membrane (DBM) triggers the development of myofibroblasts from resident corneal fibroblasts and bone marrow-derived blood borne fibrocytes due to the increased entry of TGF beta-1/-2 into the stroma from the epithelium and tears or residual corneal endothelium and aqueous humor. The myofibroblasts, and disordered extracellular matrix these cells produce, persist until the source of injury is removed, the EBM and/or DBM are regenerated, or replaced surgically, resulting in decreased stromal TGF beta requisite for myofibroblast survival. A similar BM injury-related pathophysiology can underly the development of fibrosis in other organs such as skin and lung. The normal liver does not contain traditional BMs but develops sinusoidal endothelial BMs in many fibrotic diseases and models. However, normal hepatic stellate cells produce collagen type IV and perlecan that can modulate TGF beta localization and cognate receptor binding in the space of Dissé. BM-related fibrosis is deserving of more investigation in all organs.

Topical losartan inhibits corneal scarring fibrosis and collagen type IV deposition after Descemet's membrane-endothelial excision in rabbits.

The purpose of this study was to examine the effect of topical and/or oral angiotensin converting enzyme II inhibitor and TGF-beta signaling blocker losartan on corneal stromal fibrosis that developed in rabbit corneas after Descemetorhexis removal of central Descemet's membrane and corneal endothelium. Twenty-eight New Zealand white rabbits were included and either had 8 mm central Descemetorhexis or sham control surgery without Descemetorhexis in one eye. Groups of 4 eyes without Descemetorhexis were treated for one month with no medications, topical losartan or oral losartan. Groups of 4 eyes with Descemetorhexis were treated with topical and oral vehicle, topical losartan, oral losartan, or both topical losartan and oral losartan for one month. Standardized slit lamp photos were obtained with central opacity intensity measured with ImageJ. The posterior fibrotic zone of corneas was measured on immunohistochemistry for alpha-smooth muscle actin (SMA) and keratocan using QuPath analysis. Collagen type IV expression in the posterior cornea was quantitated with ImageJ and duplex immunohistochemistry for collagen type IV and TGF beta-1. After Descemetorhexis, topical, but not oral, losartan decreased the intensity of central stromal opacity, reduced peripheral corneal scarring, and decreased alpha-smooth muscle actin myofibroblast fibrosis area compared to corneas that had Descemetorhexis and treatment with vehicles alone. Topical losartan decreased posterior stromal cellular, non-Descemet's membrane, collagen type IV production, that is likely stimulated by TGF beta as part of a negative regulatory feedback mechanism, compared to vehicle treatment at one month after Descemetorhexis. Topical losartan is likely to be effective in reducing corneal scarring fibrosis produced by traumatic injury, microbial infection, and some corneal diseases and surgeries.

Sulforaphane inhibits cytokine-stimulated chemokine and adhesion molecule expressions in human corneal fibroblasts: Involvement of the MAPK, STAT, and NF-κB signaling pathways.

Chemokines and adhesion molecules are major inflammatory mediators of chronic and recurrent vernal keratoconjunctivitis (VKC). Sulforaphane (SFN) is a natural plant extract that is known to have anti-inflammatory and antioxidant properties. SFN is demonstrated to be effective against a variety of human diseases. The current investigation examines the effects and the molecular mechanisms of SFN on cytokine-induced human corneal fibroblasts (HCFs) expression of adhesion molecules and chemokines. HCFs were exposed to both interleukin (IL)-4 and tumor necrosis factor (TNF)-α in the absence or presence of SFN treatment. The levels of thymus- and activation-regulated chemokine (TARC) and eotaxin-1 in culture supernatants were evaluated using enzyme-linked immunosorbent assay (ELISA). Reverse transcription-polymerase chain reaction analysis (RT-PCR) enabled quantification of mRNA levels of vascular cell adhesion molecule (VCAM)-1, eotaxin-1, and TARC along with cytokine receptors. An immunoblotting assay was used to evaluate the activities of VCAM-1, nuclear factor-kappa B (NF-κB), mitogen-activated protein kinases (MAPKs), signal transducer and activator of transcription factor (STAT)6 pathways, along with the expression of the cytokine receptors including IL-4 receptor (R)α, IL-13Rα1, TNFRI, as well as TNFRII. SFN inhibited TARC and eotaxin-1 release in HCFs stimulated by TNF-α and IL-4 in a manner dependent on dose and time. SFN suppressed transcriptions of TARC, eotaxin-1, and VCAM-1. Furthermore, the mRNA and protein expression levels of IL-4Rα, TNFRI, and TNFRII were also attenuated by SFN exposure, however, those of IL-13Rα1 remained unaffected. In addition, SFN downregulated the expression of VCAM-1 and the phosphorylation of MAPKs, IκBα, and STAT6. These results suggest that SFN inhibited cytokine-stimulated TARC, eotaxin-1 secretion as well as VCAM-1 expression in HCFs, with these effects likely occurring as a result of cytokine receptor inhibition and attenuation of MAPK, NF-κB, and STAT6 signaling. SFN may therefore have therapeutic potential in VKC treatment.