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Purpose: This study aimed to determine how different strain intensities-including normal, moderately increased, and high strain-influence protein expression profiles and related biological processes in human corneal stromal keratocytes.

Methods: A well-established in vitro model using the Flexcell FX-5000 Tension System, which replicates the natural corneal curvature and enables precise strain application to keratocytes, was used. Keratocytes were exposed to three strain levels: 3% (normal), 6% (moderately increased), and 12% (high). Following strain application, cells were collected for liquid chromatography-tandem mass spectrometry-based proteomic analysis to generate protein expression profiles. Differentially expressed proteins (DEPs) among the three groups were identified and subjected to biological pathway enrichment to reveal strain-dependent biological processes. Western blot analysis was performed to validate the expression of selected DEPs.

Results: Keratocytes exhibited strain intensity-dependent responses. Three percent strain maintained keratocytes in a quiescent phenotype, consistent with our previous findings. Six percent strain activated protective and adaptive programs to preserve tissue homeostasis under stress. In contrast, 12% strain suppressed immune-related processes and induced extracellular matrix (ECM) remodeling. Notably, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) and cathepsin L (CTSL)-two ECM remodeling-related proteins implicated in fibrotic responses-were significantly upregulated under 12% strain, highlighting a potential link between excessive mechanical stress and stromal fibrosis.

Conclusions: These findings demonstrate that corneal strain regulates keratocyte behavior in an intensity-dependent manner and suggest that high mechanical stress may drive pathologic stromal remodeling and fibrotic responses, offering mechanistic insights that may inspire future therapeutic strategies.

The purpose of this study was to examine the nature of the underlying molecular mechanisms of cell death in human keratocytes treated with nigericin, a known pyroptosis inducer. Human keratocytes were exposed to nigericin, and cell death was assessed through morphological analysis and detection of related molecular markers. Proteomic profiling was performed to identify cell death-related proteins, with key findings validated by western blot. Additionally, organelle disruptions were examined using immunostaining techniques. Pyroptosis-like cell death was observed morphologically in cultured keratocytes. Moreover, an elevated release of IL-1beta was detected, accompanied by a significant loss of mitochondrial membrane potential. However, nigericin treatment induced a form of non-inflammatory cell death characterized by extensive vacuolation, resembling paraptosis. This was accompanied by the absence of caspase-3 activation and endoplasmic reticulum (ER) stress markers, along with increased accumulation of the autophagic marker LC3-II. Proteomic analysis revealed the absence of key components of the canonical pyroptosis pathway, including proteins involved in inflammasome assembly and the gasdermin (GSDM) family. These results were further confirmed by western blot. Significant alterations were also observed in the Golgi apparatus, mitochondria, and lysosomes following nigericin treatment. These findings suggest that nigericin triggers a paraptosis-like cell death in human keratocytes, rather than pyroptosis, as keratocytes lack the canonical executors of pyroptosis. This highlights an alternative mechanism of cell death in the cornea, warranting further exploration to understand its role and potential therapeutic implications.

Purpose: To develop an in vitro model that mimics aspects of corneal healing in humans for uncovering key mechanisms involved in the mechanisms involved in the healing and scarring processes.

Methods: As part of the healing matrix, TGF-β1–induced and corneal-derived myofibroblasts were cultured in fibrin hydrogels with configurations that recapitulate the healthy (aligned) and wounded (random) microenvironment of the cornea.

Results: Evaluation of cellular alpha smooth muscle actin (α-SMA) and collagen hybridizing peptide (CHP) showed cell and matrix alignment, respectively. The aligned compared to the random constructs demonstrated an increased ability to synthesize total soluble proteins, including collagen type V, but collagen type I levels were reduced. This finding reveals a differential pattern for these proteins. Additionally, the collagen fibril diameters were larger in the aligned tissue constructs compared to the random constructs. Fibronectin and CHP colocalization patterns did not differ between groups; however, fibronectin and decorin were increased in the aligned group in contrast to tenascin C, which showed no difference.

Conclusions: These findings suggest that the alignment of the healing microenvironment plays a crucial role in modulating the structural properties of the extracellular matrix (ECM) and regulates the synthesis of key proteins that are closely involved in fibrillogenesis and are indicative of the quality of the deposited ECM.

Translational Relevance: We developed a three-dimensional in vitro model that closely mimics in vivo conditions to investigate the role of corneal myofibroblasts in healing and regeneration. Ultimately, this model can help develop targeted antifibrotic therapies to prevent corneal scarring.

Keratocytes are the primary resident cells in the corneal stroma. They play an essential role in maintaining corneal physiological function. Studying the factors that affect the phenotype and behavior of keratocytes offers meaningful perspectives for improving the understanding and treatment of corneal injuries. In this study, 3% strain was applied to human keratocytes using the Flexcell® Tension Systems. Real-time quantitative PCR (RT-qPCR) and western blot were used to investigate the influence of strain on the expression of intracellular aldehyde dehydrogenase 3A1 (ALDH3A1). ALDH3A1 knockdown was achieved using double-stranded RNA-mediated interference (RNAi). Immunofluorescence (IF) staining was employed to observe the impact of changes in ALDH3A1 expression on nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) nuclear translocation. Keratocyte proliferation and migration were assessed by bromodeoxyuridine (BrdU) assay and scratch wound healing assay, respectively. Mouse injury models and single-cell RNA sequencing of keratocytes from keratoconus patients were used to assess how strain influenced ALDH3A1 in vivo. Our results demonstrate that 3% strain suppresses keratocyte proliferation and increases ALDH3A1. Increased ALDH3A1 inhibits NF-κB nuclear translocation, a key step in the activation of the NF-κB signaling pathway. Conversely, ALDH3A1 knockdown promotes NF-κB nuclear translocation, ultimately enhancing keratocyte proliferation and migration. Elevated ALDH3A1 levels were also observed in mouse injury models with increased corneal strain and keratoconus patients. These findings provide valuable insights for further research into the role of corneal strain and its connection to corneal injury repair.

Interleukin-1 (IL-1) and transforming growth factor-beta (TGFβ) are important cytokines involved in corneal wound healing. Here, we studied the effect of these cytokines on corneal stromal cell (keratocyte) differentiation. IL-1β treatment resulted in reduced keratocyte phenotype, as evident by morphological changes and decreased expression of keratocyte markers, including keratocan, lumican, ALDH3A1, and CD34. TGFβ1 treatment induced keratocyte differentiation towards the myofibroblast phenotype. This was inhibited by simultaneous treatment with IL-1β, as seen by inhibition of α-SMA expression, morphological changes, and reduced contractibility. We found that the mechanism of crosstalk between IL-1β and TGFβ1 occurred via regulation of the NF-κB signaling pathway, since the IL-1β induced inhibition of TGFβ1 stimulated keratocyte-myofibroblast differentiation was abolished by a specific NF-κB inhibitor, TPCA-1. We further found that Smad7 participated in the downstream signaling. Smad7 expression level was negatively regulated by IL-1β and positively regulated by TGFβ1. TPCA-1 treatment led to an overall upregulation of Smad7 at mRNA and protein level, suggesting that NF-κB signaling downregulates Smad7 expression levels in keratocytes. All in all, we propose that regulation of cell differentiation from keratocyte to fibroblast, and eventually myofibroblast, is closely related to the opposing effects of IL-1β and TGFβ1, and that the mechanism of this is governed by the crosstalk of NF-κB signaling.

Purpose/aim: Corneal injury is a major cause of impaired vision around the globe. The fine structure of the corneal stroma plays a pivotal role in the phenotype and behavior of the embedded cells during homeostasis and healing after trauma or infection. In order to study healing processes in the cornea, it is important to create culture systems that functionally mimic the natural environment.

Materials and methods: Collagen solution was vitrified on top of a grated film to achieve thin collagen films with parallel microgrooves. Keratocytes (corneal stromal cells) were cultured on the films either as a single layer or as stacked layers of films and cells. SEM and F-actin staining were used to analyze the pattern transference onto the collagen and the cell orientation on the films. Cell viability was analyzed with MTS and live/dead staining. Keratocytes, fibroblasts, and myofibroblasts were cultured to study the pattern’s effect on phenotype.

Results: A microstructured collagen film-based culture system that guides keratocytes (stromal cells) to their native, layerwise perpendicular orientation in 3D and that can support fibroblasts and myofibroblasts was created. The films are thin and transparent enough to observe cells at least three layers deep. The cells maintain viability in 2D and 3D cultures and the films can support fibroblast and myofibroblast phenotypes.

Conclusions: The films provide an easily reproducible stroma model that maintains high cell viability and improves the preservation of the keratocyte phenotype in keratocytes that are differentiated to fibroblasts.

Scar formation as a result of corneal wound healing is a leading cause of blindness. It is a challenge to understand why scar formation is more likely to occur in the central part of the cornea as compared to the peripheral part. The purpose of this study was to unravel the underlying mechanisms. We applied RNA-seq to uncover the differences of expression profile in keratocytes in the central/peripheral part of the cornea. The relative quantity of mitochondrial RNA was measured by multiplex qPCR. The characterization of mitochondrial RNA in the cytoplasm was confirmed by immunofluoresence microscope and biochemical approach. Gene expression was analyzed by western blot and RT qPCR. We demonstrate that the occurrence of mitochondrial DNA common deletion is greater in keratocytes from the central cornea as compared to those of the peripheral part. The keratocytes with CD have elevated oxidative stress levels, which leads to the leakage of mitochondrial double-stranded RNA into the cytoplasm. The cytoplasmic mitochondrial double-stranded RNA is sensed by MDA5, which induces NF-κB activation. The NF-κB activation thereafter induces fibrosis-like extracellular matrix expressions and IL-8 mRNA transcription. These results provide a novel explanation of the different clinical outcome in different regions of the cornea during wound healing.

Acetylcholine (ACh) has been reported to play various physiological roles, including wound healing in the cornea. Here, we study the role of ACh in the transition of corneal fibroblasts into myofibroblasts, and in consequence its role in the onset of fibrosis, in an in vitro human corneal fibrosis model. Primary human keratocytes were obtained from healthy corneas. Vitamin C (VitC) and transforming growth factor-β1 (TGF-β1) were used to induce fibrosis in corneal fibroblasts. qRT-PCR and ELISA analyses showed that gene expression and production of collagen I, collagen III, collagen V, lumican, fibronectin (FN) and alpha-smooth muscle actin (α-SMA) were reduced by ACh in quiescent keratocytes. ACh treatment furthermore decreased gene expression and production of collagen I, collagen III, collagen V, lumican, FN and α-SMA during the transition of corneal fibroblasts into myofibroblasts, after induction of fibrotic process. ACh inhibited corneal fibroblasts from developing contractile activity during the process of fibrosis, as assessed with collagen gel contraction assay. Moreover, the effect of ACh was dependent on activation of muscarinic ACh receptors. These results show that ACh has an anti-fibrotic effect in an in vitro human corneal fibrosis model, as it negatively affects the transition of corneal fibroblasts into myofibroblasts. Therefore, ACh might play a role in the onset of fibrosis in the corneal stroma.

Corneal tissue engineering is an alternative way to solve the problem of lack of corneal donor tissue in corneal transplantation. Keratocytes with a normal phenotype and function in tissue-engineered cornea would be critical for corneal regeneration. Although the role of extracellular/substrate material stiffness is well-known for the regulation of the cell phenotype and cell behavior in many different cell types, its effects in keratocyte culture have not yet been thoroughly studied. This project studied the effect of substrate stiffness on the keratocyte phenotype marker expression and typical cell behavior (cell adhesion, proliferation, and migration), and the possible mechanisms involved. Human primary keratocytes were cultured on tissue culture plastic (TCP, similar to 10(6) kPa) or on plates with the stiffness equivalent of physiological human corneal stroma (25 kPa) or vitreous body (1 kPa). The expression of keratocyte phenotype markers, cell adhesion, proliferation, and migration were compared. The results showed that the stiffness of the substrate material regulates the phenotype marker expression and cell behavior of cultured keratocytes. Physiological corneal stiffness (25 kPa) superiorly preserved the cell phenotype when compared to the TCP and 1 kPa group. Keratocytes had a larger cell area when cultured on 25 kPa plates as compared to on TCP. Treatment of cells with NSC 23766 (Rac1 inhibitor) mimicked the response in the cell phenotype and behavior seen in the transition from soft materials to stiff materials, including the cytoskeletal structure, expression of keratocyte phenotype markers, and cell behavior. In conclusion, this study shows that substrate stiffness regulates the cell phenotype marker expression and cell behavior of keratocytes by Rac1-mediated cytoskeletal reorganization. This knowledge contributes to the development of corneal tissue engineering.

Corneal injury due to ocular trauma or infection is one of the most challenging vision impairing pathologies that exists. Many studies focus on the pro-inflammatory and pro-angiogenic effects of interleukin-1 beta(IL-1 beta) on corneal wound healing. However, the effect of IL-1 beta on keratocyte phenotype and corneal repair, as well as the underlying mechanisms, is not clear. This study reports, for the first time, that IL-1 beta induces phenotype changes of keratocytes in vitro, by significantly down-regulating the gene and protein expression levels of keratocyte markers (Keratocan, Lumican, Aldh3a1 and CD34). Furthermore, it is found that the NF-kappa B pathway is involved in the IL-1 beta-induced changes of keratocyte phenotype, and that the selective IKK beta inhibitor TPCA-1, which inhibits NF-kappa B, can preserve keratocyte phenotype under IL-1 beta simulated pathological conditions in vitro. By using a murine model of corneal injury, it is shown that sustained release of TPCA-1 from degradable silk fibroin hydrogels accelerates corneal wound healing, improves corneal transparency, enhances the expression of keratocyte markers, and supports the regeneration of well-organized epithelium and stroma. These findings provide insights not only into the pathophysiological mechanisms of corneal wound healing, but also into the potential development of new treatments for patients with corneal injuries.