Keratocytes Research Articles

Characteristics of Toxic Keratopathy, an In Vivo Confocal Microscopy Study.

PurposeToxic keratopathy (TK) involves complex clinical manifestations and is difficult to differentiate from other ocular surface diseases by conventional slit-lamp examination. The challenge faced by clinicians in confidently diagnosing TK cannot be underestimated. This study aimed to explore the microstructural characteristics and diagnostic parameters by in vivo confocal microscopy (IVCM) in TK.MethodsIn this prospective, cross-sectional, comparative study, slit-lamp and IVCM examinations were performed on 20 normal eyes and 54 eyes with TK. Based on slit-lamp imaging, TK subjects were divided into four groups: superficial punctate keratitis (n = 10 eyes), pseudodendritic keratitis (n = 14 eyes), ulcerative keratitis (UK; n = 16 eyes), and ring keratitis (RK; n = 14 eyes). The microstructural characteristics of TK were described according to the following IVCM parameters: basal cell (BC) density, dendritiform cell (DC) density, DC size, corneal nerve fiber (CNF) length, nerve tortuosity, and keratocyte reflectivity. A receiver operating characteristic (ROC) curve model was also formulated to compare the predictive power of BC density, DC density, and CNF length.ResultsTK eyes showed significantly higher values for DC density (45.8 cells/mm2; range, 25.0–100.0) compared with normal eyes (24.0 cells/mm2; range, 20.8–32.3; P = 0.013; DC size (111.0 µm2; range, 92.0–137.8) compared with normal eyes (63.7 µm2; range, 47.7–70.3; P < 0.001); nerve tortuosity (0.08; range, 0.05–0.09) compared with normal eyes (0.04; range, 0.02–0.04; P < 0.001); and keratocyte reflectivity. BC density and CNF length values were found to be significantly less than those for normal controls (both P < 0.001). In all subgroups, CNF length appeared to be significantly lower than that of controls (all P < 0.001), and DC density was only statistically significantly higher in the UK (P = 0.003) and RK (P < 0.001) groups. Corneal fluorescein staining had no correlation with the analyzed IVCM parameters (all P ˃ 0.05). However, the increase in DC density and DC size showed negative correlations to CNF length (density: r = −0.325, P < 0.005; size: r = −0.493, P < 0.005), as well as positive correlations to duration and frequency of topical eye drops and DC size (density: r = 0.361, P < 0.05; size: r = 0.581, P < 0.05). A ROC curve showed that CNF length had the strongest predictive power, with the estimated area under the curve being 0.992 ± 0.008.ConclusionsLower BC density and CNF length, greater DC density and DC size, and greater keratocyte reflectivity were the microstructural characteristics of TK. The role of subbasal nerve, inflammatory response, and limbal stem cells in the progression of TK and the appropriate treatment of different TK stages are future research directions.Translational RelevanceThe evaluation of basal cells, subbasal nerve, and dendritiform cells is helpful to our understanding of the pathological process of TK.

Wedelolactone ameliorates Pseudomonas aeruginosa-induced inflammation and corneal injury by suppressing caspase-4/5/11/GSDMD-mediated non-canonical pyroptosis

Pseudomonas aeruginosa (P. aeruginosa) keratitis, a worldwide leading cause of corneal perforation and blindness, which is associated with contact lens usage. Increasing evidence has indicated that pyroptosis, a novel proinflammatory programmed cell death, is linked with ocular diseases, little is known about the role of noncanonical pyroptosis in microbial keratitis. Here, we first indicated the involvement of noncanonical pyroptosis in P. aeruginosa keratitis and investigated whether wedelolactone (WDL), a major active component of Eclipta prostrate known to target caspase-11, could alleviate P. aeruginosa keratitis development. We found the expression of caspase-4/5/11 and cleaved GSDMD in corneas of P. aeruginosa keratitis patients, animal models and lipopolysaccharide (LPS)-induced primary cultured human corneal keratocytes (piHCKs) were increased. Combining ciprofloxacin with WDL significantly ameliorated the severity of P. aeruginosa keratitis, as manifested by decreased inflammatory responses and reduced corneal epithelial defects. Consistent with these findings, WDL also dose-dependently alleviated LPS-induced noncanonical pyroptosis by reversing the increased expression of caspase-4/5 and GSDMD in piHCKs. In summary, our results demonstrated that by targeting the activation of caspase-4/5/11, wedelolactone inhibited the development of P. aeruginosa keratitis and suppressed the release of proinflammatory cytokines. Wedelolactone may be a promising anti-inflammatory candidate to combat P. aeruginosa keratitis.

Effect of Thyroxine on Transforming Growth Factor β1, Collagen I, and V Expression in Keratoconus Corneal Fibroblasts and Keratocytes, in Vitro

ABSTRACT Background Keratoconus (KC) is a corneal disorder, associated with oxidative stress, hypoxia and as several times discussed, potentially with thyroid gland dysfunction. We aimed to investigate the effect of thyroxine on transforming growth factor β1 (TGF-β1), collagen I and V (Col I and V) expression in human corneal fibroblasts (HCFs) and human keratocytes of KC corneas, in vitro. Methods Primary human KC-keratocytes and normal keratocytes were isolated and cultured as corneal fibroblasts or keratocytes. The effect of 0.1 µg/ml and 1.0 µg/ml thyroxine on TGF-β1, Col I and Col V expression was investigated by qPCR, Western blot, and ELISA. Proliferation assay was performed using BrdU ELISA to observe the 24h effect of 1.0 µg/ml thyroxine on keratocytes, in vitro. Results TGFB1 mRNA expression of normal keratocytes increased following 1.0 µg/ml thyroxine stimulation for 24 h (p = .036), without changes in protein expression. Col I protein expression of KC-HCFs increased following 1.0 µg/ml thyroxine stimulation for 24 h (p = .0003). Proliferation of normal and KC keratocytes increased following a 7-day growth period and 24 hours thyroxine administration (p = .018; p = .024). Conclusions Thyroxine may affect the Col I protein expression in KC-HCFs, but not in KC keratocytes, in vitro. Thyroxine administration has no effect on TGF-β1, collagen I and V expression of keratoconus keratocytes. Therefore, an increased thyroxine concentration alone seems not to be causally related to the development of keratoconus.

L-carnitine suppresses transient receptor potential vanilloid type 1 activity and myofibroblast transdifferentiation in human corneal keratocytes

Corneal stromal wound healing is a well-balanced process promoted by overlapping phases including keratocyte proliferation, inflammatory-related events, and tissue remodeling. L-carnitine as a natural antioxidant has shown potential to reduce stromal fibrosis, yet the underlying pathway is still unknown. Since transient receptor potential vanilloid 1 (TRPV1) is a potential drug target for improving the outcome of inflammatory/fibrogenic wound healing, we investigated if L-carnitine can mediate inhibition of the fibrotic response through suppression of TRPV1 activation in human corneal keratocytes (HCK). We determined TRPV1-induced intracellular calcium transients using fluorescence calcium imaging, channel currents by planar patch-clamping, and cell migration by scratch assay for wound healing. The potential L-carnitine effect on TRPV1-induced myofibroblast transdifferentiation was evaluated by immunocytochemical detection of alpha smooth muscle actin. RT-PCR analysis confirmed TRPV1 mRNA expression in HCK. L-carnitine (1 mmol/l) inhibited either capsaicin (CAP) (10 µmol/l), hypertonic stress (450 mOsmol/l), or thermal increase (>43 °C) induced Ca2+ transients and corresponding increases in TRPV1-induced inward and outward whole-cell currents. This was accompanied by suppression of injury-induced increases in myofibroblast transdifferentiation and cell migration. In conclusion, L-carnitine contributes to inhibit stromal scarring through suppressing an injury-induced intrinsic TRPV1 activity that is linked with induction of myofibroblast transdifferentiation in HCK cells.

Effects of Mechanical Compression on Cell Morphology and Function in Human Corneal Fibroblasts

ABSTRACT Purpose: To explore the effect of mechanical compression (similar to that induced by eye rubbing) on cell morphology, proliferation, apoptosis, and extracellular matrix synthesis and degradation in human corneal fibroblasts. Materials and methods: Human corneal fibroblasts were isolated from corneal lenticule tissue of 10 patients after small incision lenticule extraction surgery. A system was established to mechanically compress corneal fibroblasts with pressure ranging from 0 to 524 Pa. Morphological changes, cell proliferation, apoptosis, and corneal matrix synthesis and degradation were examined using microscopy imaging, bromodeoxyuridine staining, flow cytometry, and qPCR analysis in human corneal fibroblasts after mechanical compression. Results: Human corneal fibroblasts showed short and thick cytoplasmic extensions, as well as a relatively low aspect ratio, suggesting significant morphological alterations caused by high levels of compressive stress. Mechanical compression inhibited cell proliferation and promoted cell apoptosis. Furthermore, compressive stress led to significant elevation in the expression of genes related to extracellular matrix degradation (matrix metalloproteinases MMP1 and MMP9) as early as 6 h after compression and moderate changes in the expression of tissue inhibitors of metalloproteinases. In addition, the mRNA expression levels of COL1A1, lumican, and vimentin were reduced 24 and 48 h after mechanical compression. Conclusions: Mechanical compression alters cell morphology, inhibits proliferation, induces apoptosis, upregulates genes related to extracellular matrix degradation, and downregulates corneal structural genes in human corneal fibroblasts. This study provides evidence that compressive stress significantly influences corneal keratocytes. Our findings suggest that this mechanical effect may be related to keratoconus associated with chronic eye rubbing.

Tailoring therapeutic properties of silver nanoparticles for effective bacterial keratitis treatment

The formulation of nanoparticles with intrinsically therapeutic properties in a tailorable and appropriate manner is critical in nanomedicine for effective treatments of infectious diseases. Here, we present a biomedical strategy to formulate silver nanoparticles (AgNPs) as intrinsically therapeutic agents for the treatment of Staphylococcus aureus (S. aureus) keratitis. Specifically, AgNPs are controllably obtained as spheres, wrapped with a biopolymer, and varied in sizes. in vitro and in vivo studies indicate that biological interactions between the AgNPs and corneal keratocytes, S. aureus bacteria, and blood vessels are strongly determined by the particle sizes. As the size increased from 3.3 ± 0.7 to 37.2 ± 5.3 nm, the AgNPs exhibit better ocular biocompatibility and stronger antiangiogenic activity, but poorer bactericidal performance. In a rabbit model of S. Aureus-induced keratitis, intrastromal injection of AgNP formulations (single dose) show substantial influences of particle size on the treatment efficacy. As the trade-off, AgNPs with medium size of 15.0 ± 3.6 nm reveal as the best therapeutic agent that could offer ∼5.6 and ∼9.1-fold greater corneal thickness recovery respectively compared to those with smaller and larger sizes at 3 days post-administration. These findings suggest an important advance in structural design for formulating intrinsically therapeutic nano-agents toward the efficient management of infectious diseases.

Corneal confocal microscopy identifies a reduction in corneal keratocyte density and sub-basal nerves in children with type 1 diabetes mellitus

PurposeTo assess whether alterations in stromal keratocyte density are related to loss of corneal nerve fibres in children with type 1 diabetes mellitus (T1DM).MethodsTwenty participants with T1DM and 20 age-matched...

Epigenetic Landscape Analysis of the Long Non-Coding RNA and Messenger RNA in a Mouse Model of Corneal Alkali Burns.

PurposeCorneal alkali burns (CABs) are a common clinical ocular disease, presenting a poor prognosis. Although some long noncoding RNAs (lncRNAs) reportedly play a key role in epigenetic regulation associated with CABs, studies regarding the lncRNA signature in CABs remain rare and elusive.MethodsA CAB model was established in C57BL/6J mice and profiling of lncRNA expressions was performed by RNA-Seq. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to predicate the related pathological pathways and candidate genes. RT-qPCR was used to verify the expression pattern of lncRNAs and related mRNAs, both in vitro and in vivo. Data were statistically analyzed by GraphPad Prism version 6.0.ResultsIn all, 4436 aberrantly expressed lncRNAs were identified in CAB mice when compared with control mice. In the top 13 aberrantly expressed lncRNAs, Bc037156 and 4930511E03Rik were confirmed as the most significantly altered lncRNAs. Pathway analysis revealed that mitogen-activated protein kinase (MAPK) signaling pathway was most enriched. Following 4930511E03Rik siRNA treated, Srgn, IL-1β and Cxcr2 were significant upregulated in corneal epithelial cells, corneal keratocytes, and bone marrow dendritic cells, with NaOH treatment. Moreover, after Bc037156 siRNA treated, expression levels of IL-1β and Srgn were significantly downregulated in the three cell lines.ConclusionsOur study suggests that Bc037156 and 4930511E03Rik may be involved in inflammation, immune response, and neovascularization by regulating Srgn, IL-1β, and Cxcr2 expression after CAB. These candidate lncRNAs and mRNAs may be the potential targets for the treatment strategy of the alkali injured cornea.

Plasma membrane disruption (PMD) formation and repair in mechanosensitive tissues.

Mammalian cells employ an array of biological mechanisms to detect and respond to mechanical loading in their environment. One such mechanism is the formation of plasma membrane disruptions (PMD), which foster a molecular flux across cell membranes that promotes tissue adaptation. Repair of PMD through an orchestrated activity of molecular machinery is critical for cell survival, and the rate of PMD repair can affect downstream cellular signaling. PMD have been observed to influence the mechanical behavior of skin, alveolar, and gut epithelial cells, aortic endothelial cells, corneal keratocytes and epithelial cells, cardiac and skeletal muscle myocytes, neurons, and most recently, bone cells including osteoblasts, periodontal ligament cells, and osteocytes. PMD are therefore positioned to affect the physiological behavior of a wide range of vertebrate organ systems including skeletal and cardiac muscle, skin, eyes, the gastrointestinal tract, the vasculature, the respiratory system, and the skeleton. The purpose of this review is to describe the processes of PMD formation and repair across these mechanosensitive tissues, with a particular emphasis on comparing and contrasting repair mechanisms and downstream signaling to better understand the role of PMD in skeletal mechanobiology. The implications of PMD-related mechanisms for disease and potential therapeutic applications are also explored.

Transplantation of human induced pluripotent stem cell-derived neural crest cells for corneal endothelial regeneration

BackgroundThe corneal endothelium maintains corneal hydration through the barrier and pump function, while its dysfunction may cause corneal edema and vision reduction. Considering its development from neural crest cells (NCCs), here we investigated the efficacy of the human induced pluripotent stem cell (hiPSC)-derived NCCs for corneal endothelial regeneration in rabbits.MethodsDirected differentiation of hiPSC-derived NCCs was achieved using the chemically defined medium containing GSK-3 inhibitor and TGF-β inhibitor. The differentiated cells were characterized by immunofluorescence staining, FACS analysis, and in vitro multi-lineage differentiation capacity. For in vivo functional evaluation, 1.0 × 106 hiPSC-derived NCCs or NIH-3 T3 fibroblasts (as control) combined with 100 μM Y-27632 were intracamerally injected into the anterior chamber of rabbits following removal of corneal endothelium. Rabbit corneal thickness and phenotype changes of the transplanted cells were examined at 7 and 14 days with handy pachymeter, dual-immunofluorescence staining, and quantitative RT-PCR.ResultsThe hiPSC-derived NCCs were differentiated homogenously through 7 days of induction and exhibited multi-lineage differentiation capacity into peripheral neurons, mesenchymal stem cells, and corneal keratocytes. After 7 days of intracameral injection in rabbit, the hiPSC-derived NCCs led to a gradual recovery of normal corneal thickness and clarity, when comparing to control rabbit with fibroblasts injection. However, the recovery efficacy after 14 days deteriorated and caused the reappearance of corneal edema. Mechanistically, the transplanted cells exhibited the impaired maturation, cellular senescence, and endothelial-mesenchymal transition (EnMT) after the early stage of the in vivo directional differentiation.ConclusionsTransplantation of the hiPSC-derived NCCs rapidly restored rabbit corneal thickness and clarity. However, the long-term recovery efficacy was impaired by the improper maturation, senescence, and EnMT of the transplanted cells.

In Vivo Confocal Microscopic Evaluation of the Limbus and Cornea in Vogt Koyanagi Haradas Syndrome

ABSTRACT Aim To elucidate the microarchitecture of limbus and cornea in subjects with Vogt Koyanagi Haradas syndrome (VKH) Methods From January 2019 to December 2019 a total of 17 VKH subjects, 5 acute and 12 chronic, 18 non VKH uveitis controls, and 6 healthy controls were recruited for this study . In vivo confocal microscopic (IVCM) analysis of the limbal basal epithelium, scleral side of the limbus, limbal niche, corneal keratocytes, and corneal nerves was carried out . Results Absence of pigmented cells in the limbal basal epithelium, presence of inflammatory cells on the scleral side of the limbus, fibrotic niche, degenerated keratocytes, thinning, and beading of corneal nerves were noted in VKH eyes as compared to controls. Conclusion Presence of inflammatory cells and depigmentation of limbus in chronic VHK points to disease progression.Keratocyte and corneal nerve changes in Vogt Koyanagi Haradas syndrom are novel findings.

Safety profiles of terahertz scanning in ophthalmology

Terahertz (THz) technology has emerged recently as a potential novel imaging modality in biomedical fields, including ophthalmology. However, the ocular biological responses after THz electromagnetic exposure have not been investigated. We conducted a rabbit study to evaluate the safety profiles of THz scanning on eyes, at a tissue, cellular, structural and functional level. Eight animals (16 eyes) were analysed after excessive THz exposure (control, 1 h, 4 h, and 1 week after continuous 4-h exposure; THz frequency = 0.3 THz with continuous pulse generated at 40 µW). We found that at all the time points, the corneas and lens remained clear with no corneal haze or lens opacity formation clinically and histopathologically. No thermal effect, assessed by thermographer, was observed. The rod and cone cell-mediated electroretinography responses were not significantly altered, and the corneal keratocytes activity as well as endothelial viability, assessed by in-vivo confocal microscopy, was not affected. Post-exposed corneas, lens and retinas exhibited no significant changes in the mRNA expression of heat shock protein (HSP)90AB1), DNA damage inducible transcript 3 (DDIT3), and early growth response (EGR)1. These tissues were also negative for the inflammatory (CD11b), fibrotic (fibronectin and α-smooth muscle actin), stress (HSP-47) and apoptotic (TUNEL assay) responses on the immunohistochemical analyses. The optical transmittance of corneas did not change significantly, and the inter-fibrillar distances of the corneal stroma evaluated with transmission electron microscopy were not significantly altered after THz exposure. These results provide the basis for future research work on the development of THz imaging system for its application in ophthalmology.

Corneal Keratocyte Density and Corneal Nerves Are Reduced in Patients With Severe Obesity and Improve After Bariatric Surgery.

PurposeObesity is associated with peripheral neuropathy, which bariatric surgery may ameliorate. The aim of this study was to assess whether corneal confocal microscopy can show a change in corneal nerve morphology and keratocyte density in subjects with severe obesity after bariatric surgery.MethodsTwenty obese patients with diabetes (n = 13) and without diabetes (n = 7) underwent assessment of hemoglobin A1c (HbA1c), lipids, IL-6, highly sensitive C-reactive protein (hsCRP), and corneal confocal microscopy before and 12 months after bariatric surgery. Corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), corneal nerve fiber length (CNFL), and keratocyte density (KD) from the anterior, middle, and posterior stroma were quantified. Twenty-two controls underwent assessment at baseline only.ResultsCNFL (P < 0.001), CNBD (P < 0.05), and anterior (P < 0.001), middle (P < 0.001), and posterior (P < 0.001) keratocyte densities were significantly lower in obese patients compared to controls, and anterior keratocyte density (AKD) correlated with CNFL. Twelve months after bariatric surgery, there were significant improvements in body mass index (BMI; P < 0.001), HDL cholesterol (P < 0.05), hsCRP (P < 0.001), and IL-6 (P < 0.01). There were significant increases in CNFD (P < 0.05), CNBD (P < 0.05), CNFL (P < 0.05), and anterior (P < 0.05) and middle (P < 0.001) keratocyte densities. The increase in AKD correlated with a decrease in BMI (r = –0.55, P < 0.05) and triglycerides (r = –0.85, P < 0.001). There were no significant correlations between the change in keratocyte densities and corneal nerve fiber or other neuropathy measures.ConclusionsCorneal confocal microscopy demonstrates early small fiber damage and reduced keratocyte density in obese patients. Bariatric surgery leads to weight reduction and improvement in lipids and inflammation and an improvement in keratocyte density and corneal nerve regeneration.

Quantitative measurement of mechanical properties in wound healing processes in a corneal stroma model by using vibrational optical coherence elastography (OCE)

Corneal wound healing, caused by frequent traumatic injury to the cornea and increasing numbers of refractive surgeries, has become a vital clinical problem. In the cornea, wound healing is an extremely complicated process. However, little is known about how the biomechanical changes in wound healing response of the cornea. Collagen-based hydrogels incorporating corneal cells are suitable for replicating a three-dimensional (3D) equivalent of the cornea in-vitro. In this study, the mechanical properties of corneal stroma models were quantitatively monitored by a vibrational optical coherence elastography (OCE) system during continuous culture periods. Specifically, human corneal keratocytes were seeded at 5 × 105 cells/mL in the hydrogels with a collagen concentration of 3.0 mg/mL. The elastic modulus of the unwounded constructs increased from 2.950 ± 0.2 kPa to 11.0 ± 1.4 kPa, and the maximum thickness decreased from 1.034 ± 0.1 mm to 0.464 ± 0.09 mm during a 15-day culture period. Furthermore, a traumatic wound in the construct was introduced with a size of 500 µm. The elastic modulus of the neo-tissue in the wound area increased from 1.488 ± 0.4 kPa to 6.639 ± 0.3 kPa over 13 days. This study demonstrates that the vibrational OCE system is capable of quantitative monitoring the changes in mechanical properties of a corneal stroma wound model during continuous culture periods and improves our understanding on corneal wound healing processes.

Lycium barbarum Polysaccharide Suppresses Expression of Fibrotic Proteins in Primary Human Corneal Fibroblasts.

(1) Objective: To study the anti-fibrotic effects of Lycium barbarum polysaccharides (LBP) on corneal stromal fibroblasts and assess LBP’s effect on cell viability. (2) Methods: Primary human corneal keratocytes of passage 3 to 6 were used for all experiments. Cells are pretreated with LBP solution for 24 h and then transforming growth factor beta 1 (TGFβ1) for 48 h and collected for experiments. Fibrotic protein analysis was performed using immunofluorescence and Western blot. The effect of LBP on cell viability was assessed using the MTS assay. (3) Results: LBP significantly reduced the expression of fibrotic proteins, including α-SMA and extracellular matrix proteins (collagen type I and III). LBP significantly decreased the viability of myofibroblasts but not the fibroblasts. Conclusions: In this study, LBP was effective in the prevention of fibrosis gene expression. Further studies to assess the underlying mechanism and pharmacological properties will facilitate the formation of a topical LBP solution for in vivo studies.

Methods for Investigating Corneal Cell Interactions and Extracellular Vesicles In Vitro.

Science and medicine have become increasingly "human-centric" over the years. A growing shift away from the use of animals in basic research has led to the development of sophisticated in vitro models of various tissues utilizing human-derived cells to study physiology and disease. The human cornea has likewise been modeled in vitro using primary cells derived from corneas obtained from cadavers or post-transplantation. By utilizing a cell's intrinsic ability to maintain its tissue phenotype in a pre-designed microenvironment containing the required growth factors, physiological temperature, and humidity, tissue-engineered corneas can be grown and maintained in culture for relatively long periods of time on the scale of weeks to months. Due to its transparency and avascularity, the cornea is an optimal tissue for studies of extracellular matrix and cell-cell interactions, toxicology and permeability of drugs, and underlying mechanisms of scarring and tissue regeneration. This paper describes methods for the cultivation of corneal keratocytes, fibroblasts, epithelial, and endothelial cells for in vitro applications. We also provide detailed, step-by-step protocols for assembling and culturing 3D constructs of the corneal stroma, epithelial- and endothelial-stromal co-cultures and isolation of extracellular vesicles. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Isolating and culturing human corneal keratocytes and fibroblasts Basic Protocol 2: Isolating and culturing human corneal epithelial cells Basic Protocol 3: Isolating and culturing human corneal endothelial cells Basic Protocol 4: 3D corneal stromal construct assembly Basic Protocol 5: 3D corneal epithelial-stromal construct assembly Basic Protocol 6: 3D corneal endothelial-stromal construct assembly Basic Protocol 7: Isolating extracellular vesicles from corneal cell conditioned medium Support Protocol: Cryopreserving human corneal fibroblasts, corneal epithelial cells, and corneal endothelial cells.

ECM Stiffness Controls the Activation and Contractility of Corneal Keratocytes in Response to TGF-β1

After surgery or traumatic injury, corneal wound healing can cause a scarring response that stiffens the tissue and impairs ocular function. This fibrosis is caused in part by the activation of corneal keratocytes from a native mechanically quiescent state to an activated myofibroblastic state. This transformation is tied to signaling downstream of transforming growth factor-β1 (TGF-β1). Here, to better understand how biochemical and biophysical cues interact to regulate keratocyte activation and contractility, we cultured primary rabbit corneal keratocytes on flexible substrata of varying stiffness in the presence (or absence) of TGF-β1. Time-lapse fluorescence microscopy was used to assess changes in keratocyte morphology, as well as to quantify the dynamic traction stresses exerted by cells under different experimental conditions. In other experiments, keratocytes were fixed after 5 days of culture and stained for markers of both contractility and myofibroblastic activation. Treatment with TGF-β1 elicited distinct phenotypes on substrata of different stiffnesses. Cells on soft (1 kPa) gels formed fewer stress fibers and retained a more dendritic morphology, indicative of a quiescent keratocyte phenotype. Keratocytes cultured on stiff (10 kPa) gels or collagen-coated glass coverslips, however, had broad morphologies, formed abundant stress fibers, exhibited greater levels of α-smooth muscle actin (α-SMA) expression, and exerted larger traction forces. Confocal images of phospho-myosin light chain (pMLC) immunofluorescence, moreover, revealed stiffness-dependent differences in the subcellular distribution of actomyosin contractility, with pMLC localized at the tips of thin cellular processes in mechanically quiescent cells. Importantly, keratocytes cultured in the absence of TGF-β1 showed no stiffness-dependent differences in α-SMA immunofluorescence, suggesting that a stiff microenvironment alone is insufficient to induce myofibroblastic activation. Taken together, these data suggest that changes in ECM stiffness can modulate the morphology, cytoskeletal organization, and subcellular pattern of force generation in corneal keratocytes treated with TGF-β1.

Keratocyte mechanobiology

In vivo, corneal keratocytes reside within a complex 3D extracellular matrix (ECM) consisting of highly aligned collagen lamellae, growth factors, and other extracellular matrix components, and are subjected to various mechanical stimuli during developmental morphogenesis, fluctuations in intraocular pressure, and wound healing. The process by which keratocytes convert changes in mechanical stimuli (e.g. local topography, applied force, ECM stiffness) into biochemical signaling is known as mechanotransduction. Activation of the various mechanotransductive pathways can produce changes in cell migration, proliferation, and differentiation. Here we review how corneal keratocytes respond to and integrate different biochemical and biophysical factors. We first highlight how growth factors and other cytokines regulate the activity of Rho GTPases, cytoskeletal remodeling, and ultimately the mechanical phenotype of keratocytes. We then discuss how changes in the mechanical properties of the ECM have been shown to regulate keratocyte behavior in sophisticated 2D and 3D experimental models of the corneal microenvironment. Finally, we discuss how ECM topography and protein composition can modulate cell phenotypes, and review the different methods of fabricating in vitro mimics of corneal ECM topography, novel approaches for examining topographical effects in vivo, and the impact of different ECM glycoproteins and proteoglycans on keratocyte behavior.

Corneal fibroblasts: Function and markers

Corneal stromal keratocytes contribute to the maintenance of corneal transparency and shape by synthesizing and degrading extracellular matrix. They are quiescent in the healthy cornea, but they become activated in response to insults from the external environment that breach the corneal epithelium, with such activation being associated with phenotypic transformation into fibroblasts. Corneal fibroblasts (activated keratocytes) act as sentinel cells to sense various external stimuli—including damage-associated molecular patterns derived from injured cells, pathogen-associated molecular patterns of infectious microorganisms, and inflammatory mediators such as cytokines—under pathological conditions such as trauma, infection, and allergy. The expression of various chemokines and adhesion molecules by corneal fibroblasts determines the selective recruitment and activation of inflammatory cells in a manner dependent on the type of insult. In infectious keratitis, the interaction of corneal fibroblasts with various components of microbes and with cytokines derived from infiltrated inflammatory cells results in excessive degradation of stromal collagen and consequent corneal ulceration. Corneal fibroblasts distinguish between type 1 and type 2 inflammation through recognition of corresponding cytokines, with their activation by type 2 cytokines contributing to the pathogenesis of corneal lesions in severe ocular allergic diseases. Pharmacological targeting of corneal fibroblast function is thus a potential novel therapeutic approach to prevention of excessive corneal stromal inflammation, damage, and scarring.

Toward understanding the purely geometric effects of silver nanoparticles on potential application as ocular therapeutics via treatment of bacterial keratitis

Understanding a complex interaction between therapeutic nanoparticles and biological entities is crucially important for the development of effective disease treatments in the modern nanopharmaceuticals and nanomedicines. Herein, we present a strategy to thoroughly assess geometrical impacts of silver nanoparticles (AgNPs, one of the most promising nanotherapeutic agents) on their biological activities toward treatment of Staphylococcus aureus (S. aureus)-induced keratitis. Specifically, three types of differently shaped AgNPs including silver nanorods (R-Ag), silver nanotriangles (T-Ag), and silver nanospheres (SAg) are employed and interferences of particle surface area and functionality are eliminated to reflect purely geometric effects. Ocular biocompatibility studies on rabbit corneal keratocytes reveal that SAg is the least cytotoxic agent while R-Ag, because of its strongest cellular uptake, induces highest cytotoxic levels. Moreover, SAg is demonstrated to outperform R-Ag and T-Ag in killing S. aureus, possibly due to a predominance of specific particle density and high-atom-density {111} facets of the SAg when interacting with the bacteria. In contrast, owing to its predominance of sharp-tip effects on vascular endothelial cells, R-Ag can suppress blood vessel development in cornea at a greatest extent. In a rabbit model of S. aureus-induced keratitis, intrastromal administration of the differently shaped AgNPs exhibits critical roles of the particle geometry at comparable conditions (i.e., total surface area and functionality) in attenuating progression of S. aureus-induced keratitis. As a compromise among ocular biocompatibility, anti-bacterial activity, and anti-angiogenic capability, SAg shows as the most effective agent that could repair infectious corneal tissues 1.2 and 4-fold greater than the anisotropic counterparts (R-Ag and T-Ag). These findings therefore suggest a promising strategy for a clear-cut evaluation on geometric effects of therapeutic nanoparticles toward preclinical treatment of eye-related microbial infections.