Corneal Fibroblasts Research Articles

RHO-Associated Coiled-Coil-Containing Protein Kinase Inhibitors Significantly Modulate the Epithelial–Mesenchymal Transition Induced by TGF-β2 in the 2-D and 3-D Cultures of Human Corneal Stroma Fibroblasts

Background/Objectives: The objective of the present study was to examine the unidentified effects that RHO-associated coiled-coil-containing protein kinase 1 and 2 antagonists exert on the transforming growth factor beta2-induced epithelial–mesenchymal transition of the human corneal stroma. Methods: In the presence or absence of pan-RHO-associated coiled-coil-containing protein kinase inhibitors, ripasudil or Y27632 and RHO-associated coiled-coil-containing protein kinase 2 inhibitor, KD025, we analyzed the following: (1) planar proliferation caused by trans-endothelial electrical resistance and the cellular metabolic characteristics of the two-dimensional cultures of human corneal stroma fibroblasts; (2) the physical properties of a three-dimensional human corneal stroma fibroblasts spheroid; and (3) the gene expressions and their regulators in the extracellular matrix, along with the tissue inhibitors of metalloproteinases and matrix metalloproteinases and the endoplasmic reticulum stress-related factors of the two-dimensional and three-dimensional cultures in human corneal stroma fibroblasts. Results: Exposure to 5 nM of the transforming growth factor beta2 markedly increased the trans-endothelial electrical resistance values as well as the metabolic function in two-dimensional cultures of human corneal stroma fibroblasts. With an increase in stiffening, this exposure also reduced the size of three-dimensional human corneal stroma fibroblast spheroids, which are typical cellular phenotypes of the epithelial–mesenchymal transition. Both pan-RHO-associated coiled-coil-containing protein kinase inhibitors and RHO-associated coiled-coil-containing protein kinase 2 inhibitors substantially modulated these transforming growth factor beta2-induced effects, albeit in a different manner. Gene expression analysis supported such biological alterations via either with transforming growth factor beta2 alone or with the RHO-associated coiled-coil-containing protein kinase inhibitors variants with the noted exception being the transforming growth factor beta2-induced effects toward the three-dimensional human corneal stroma fibroblast spheroid. Conclusions: The findings presented herein suggest the following: (1) the epithelial–mesenchymal transition could be spontaneously evoked in the three-dimensional human corneal stroma fibroblast spheroid, and, therefore, the epithelial–mesenchymal transition induced by transforming growth factor beta2 could differ between two-dimensional and three-dimensional cultured HCSF cells; and (2) the inhibition of ROCK1 and 2 significantly modulates the transforming growth factor beta2-induced an epithelial–mesenchymal transition in both two-dimensionally and three-dimensionally cultured human corneal stroma fibroblasts, albeit in a different manner.

Anti-fibrotic effects of lisinopril (ACE inhibitor) and fasudil (ROCK inhibitor) in combination for canine corneal fibrosis invitro.

Corneal fibrosis is a leading cause of blindness in mammalian species and may result in compromised performance in sports and daily functions. This study evaluated the safety and anti-fibrotic effects of the FDA-approved drugs, angiotensin-converting enzyme inhibitor (ACE-I) lisinopril and rho-kinase inhibitor (ROCK-I) fasudil, alone and in combination, on the canine cornea using an established invitro model. To test the safety and efficacy of lisinopril and fasudil, primary canine corneal fibroblasts (CCFs) generated from donor corneas of healthy dogs (n = 20) were used. A series of dose-dependent and time-dependent assays with lisinopril (1-50 μM) and fasudil (1-10 nM) were performed. qRT-PCR, immunofluorescence (IF) staining, cell viability assay, cell proliferation assay, LIVE/DEAD viability/cytotoxicity assay, TUNEL assay, and total cell count were performed. A 25-μM lisinopril and 3-nM fasudil dose were safe, nontoxic, and optimal for therapeutic evaluations invitro. Treatments of lisinopril or fasudil, alone or in-combination, to CCFs grown in the presence of TGF-β1 (5 ng/mL) showed inhibition of myofibroblast formation based on phase-contrast microscopy. The qRT-PCR and IF studies showed a significant decrease in expression of profibrotic markers, including α-smooth muscle actin (α-SMA; p < .0001), fibronectin (FN; p = .0002), tenascin C (TNC; p < .0001), Collagen I (Col-I; p < .0001), Collagen IIIA1 (Co-IIIA1; p < .0001), and Collagen IV (Co-lV; p < .0001). An ophthalmic formulation consisting of lisinopril and fasudil may offer a safe and effective method to treat canine corneal fibrosis. Additional studies evaluating safety and efficacy of this formulation invivo are warranted.

Macromolecular crowding agent dependent extracellular matrix deposition and growth factor retention in human corneal fibroblast cultures

The major obstacle in the commercialisation and clinical translation of tissue engineered medicines is the required for the development of implantable tissue surrogates prolonged in vitro culture. Macromolecular crowding (MMC) enhances and accelerates extracellular matrix (ECM) deposition, thus offering an opportunity to bridge the gap between research and development in tissue engineered substitutes. However, the optimal MMC agent is still elusive. Herein, we first assessed the biophysical properties of the most widely used MMC agents [κλ carrageenan (κλ CR), λ carrageenan (λ CR) and Ficoll™ cocktail (FC)] and then assessed their effect in basic cell function, ECM deposition and growth factor retention in human corneal fibroblast (hCF) cultures. Dynamic light scattering analysis revealed that both CR macromolecules had significantly lower and higher zeta potential and hydrodynamic radius, respectively, than the FC. None of the MMC agents affected hCF morphology and all induced similar hCF viability, proliferation and metabolic activity. Electrophoresis and immunofluorescence analyses made apparent that at day 10 (longest time point assessed), the FC brought about the highest fibronectin and collagen types I, III, IV, V and VI deposition. Deposited ECM pattern analysis showed that at day 10, the FC induced the lowest lacunarity and normalised end points and the highest fractal dimension and % high density matrix. Further immunofluorescence analysis revealed no significant differences between the groups in vimentin, aldehyde dehydrogenase 3 family member A1, keratocan, paired box protein 6 and α-smooth muscle actin. Importantly, at day 10, the FC resulted in the highest growth factor retention (20 molecules). Our data clearly illustrate a MMC agent dependent cell response, with the FC having the highest positive effect in hCF cultures.

TGF-β1-mediated upregulation of LMCD1 drives corneal myofibroblast differentiation and corneal fibrosis

Transforming growth factor β1 (TGF-β1) drives corneal fibroblasts to differentiate into corneal myofibroblasts and plays a key role in corneal fibrosis. However, the role of LIM and cysteine-rich domains-1 (LMCD1) in TGF-β1-induced corneal myofibroblast differentiation and corneal fibrosis remains elusive. Thus, this study aimed to investigate the expression, regulatory mechanism, and role of LMCD1 in TGF-β1-induced corneal myofibroblast differentiation and corneal fibrosis. The expression of LMCD1 in TGF-β1-stimulated corneal fibroblasts was found to be upregulated through mRNA sequencing, quantitative PCR (qPCR), and Western blotting. Moreover, LMCD1 was identified to be upregulated in a mouse model of corneal fibrosis via qPCR and Western blotting. Additionally, our results demonstrated that the increase in LMCD1 expression induced by TGF-β1 in corneal fibroblasts was primarily regulated by the SMAD3 signaling pathway. Furthermore, LMCD1 knockdown significantly inhibited TGF-β1-induced corneal fibroblast-to-myofibroblast differentiation and simultaneously activated SMAD3, JNK, and p38 by promoting TGF-β1 transcription. These findings collectively suggest that LMCD1 could upregulate alpha-smooth muscle actin (α-SMA) expression and downregulate TGF-β1 expression in corneal myofibroblast differentiation. Consequently, upregulation of LMCD1 expression could potentially serve as a strategy to mediate the TGF-β1 signaling pathway in corneal myofibroblast differentiation and corneal fibrosis, laying a theoretical reference for corneal fibrosis and contributing to the development of effective therapeutic strategies for corneal fibrosis.

Neodymium-Facilitated Visualization of Extreme Phosphate Accumulation in Fibroblast Filopodia: Implications for Intercellular and Cell–Matrix Interactions

A comprehensive understanding of intercellular and cell–matrix interactions is essential for advancing our knowledge of cell biology. Existing techniques, such as fluorescence microscopy and electron microscopy, face limitations in resolution and sample preparation. Supravital lanthanoid staining provides new opportunities for detailed visualization of cellular metabolism and intercellular interactions. This study aims to describe the structure, elemental chemical, and probable origin of zones of extreme lanthanoid (neodymium) accumulation that form during preparation for scanning electron microscopy (SEM) analysis in corneal fibroblasts filopodia. The results identified three morphological patterns of neodymium staining in fibroblast filopodia, each exhibiting asymmetric staining within a thin, sharp, and extremely bright barrier zone, located perpendicular to the filopodia axis. Semi-quantitative chemical analyses showed neodymium-labeled non-linear phosphorus distribution within filopodia, potentially indicating varying phosphate anion concentrations and extreme phosphate accumulation at a physical or physicochemical barrier. Phosphorus zones labeled with neodymium did not correspond to mitochondrial clusters. During apoptosis, the number of filopodia with extreme and asymmetric phosphorus accumulation increases. Supravital lanthanoid staining coupled with SEM allows detailed visualization of intercellular and cell–matrix interactions with high contrast and resolution. These results enhance our understanding of phosphate anion accumulation and transfer mechanisms in cells under normal conditions and during apoptosis.

Decreased Circulating Gonadotropin-Releasing Hormone Associated with Keratoconus.

Keratoconus (KC) is a corneal thinning dystrophy that leads to visual impairment. While the cause of KC remains poorly understood, changes in sex hormone levels have been correlated with KC development. This study investigated circulating gonadotropin-releasing hormone (GnRH) in control and KC subjects to determine if this master hormone regulator is linked to the KC pathology. Plasma and saliva were collected from KC subjects (n = 227 and n = 274, respectively) and non-KC controls (n = 58 and n = 101, respectively), in concert with patient demographics and clinical features. GnRH levels in both plasma and saliva were significantly lower in KC subjects compared to controls. This finding was retained in plasma when subjects were stratified based on age, sex, and KC severity. Control and KC corneal fibroblasts (HKCs) stimulated with recombinant GnRH protein in vitro revealed significantly increased luteinizing hormone receptor by HKCs and reduced expression of α-smooth muscle actin with treatment suggesting that GnRH may modulate hormonal and fibrotic responses in the KC corneal stroma. Further studies are needed to reveal the role of the hypothalamic-pituitary-gonadal axis in the onset and progression of KC and to explore this pathway as a novel therapeutic target.

Coenzyme Q10 and Vitamin E Regulate the Bioactivity of Human Corneal Fibroblast Cells.

Purpose: Corneal fibroblasts are involved in the wound healing of the cornea with proliferation, migration, and differentiation processes. Coenzyme Q10 (CoQ10) and vitamin E can enhance corneal wound healing when applied after a corneal lesion as an eye drop. Thus, this study was performed to determine the potential efficiency of a CoQ10 ophthalmical solution containing a CoQ10 and vitamin E D-α-tocopherol polyethylene glycol 1000 succinate (TPGS)-derived formulation in human corneal fibroblasts (HCFs) in vitro. Methods: Primary HCFs were obtained from cadaveric corneal tissue, and cell viability was determined using MTT assay at 24 and 72 h. Cell migration was evaluated using an in vitro wound healing assay, and mRNA expressions of collagen type I (COL-I), collagen type III (COL-III), lumican, hyaluronan, matrix metalloproteinase (MMP)-1, MMP-2, MMP-9, tissue inhibitors of MMP (TIMP)-1, TIMP-2, interleukin (IL)-1β, IL-6, IL-8, and IL-10 were assessed using reverse transcription polymerase chain reaction at 24 and 72 h. Results: At various concentrations of CoQ10 ophthalmical solution (CoQ10-os), cell viability and wound healing rates of HCFs increased compared with the control group. The expressions of COL-I, COL-III, lumican, and hyaluronan were increased by CoQ10-os, whereas those of MMP-1, MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 were not affected by CoQ10-os at 24 and 72 h. In treating HCFs with a CoQ10-os medium, IL-1β, IL-6, and IL-8 decreased, whereas IL-10 was significantly increased in a time- and dose-dependent manner. Conclusions: The findings indicate that CoQ10 and vitamin E-TPGS are potent regulators of the bioactivity of HCFs, thus supporting their potential application as ophthalmical solutions in therapies aimed at the fast regeneration of damaged cornea tissues.

Fabrication and characterization of an electrospun silk fibroin/gelatin transparent graft material for corneal epithelial regeneration

The damaged corneal epithelial causing loss of vision can be restored by epithelial tissue regeneration through tissue engineering approach that provides an engineered corneal substitute with appropriate properties and nanofibrous architecture. This works reports a polymeric matrix that was derived from silk fibroin (SF) and gelatin (GE) natural polymer blend with optimized composition resembling the structure and function of the native epithelial tissue. SF/GE blend spinning solutions prepared with different volume ratios of SF/GE: 70/30, 50/50, and 30/70 were used to fabricate electrospun mats. The fabricated two-dimensional mats had nanofibrous porous architecture having interconnected pores with pore size range of 175–267 nm suitable for corneal epithelial regeneration and 891.2–558 nm fiber diameter range as revealed by scanning electron microscopic image analysis. The performed Fourier transform infrared spectra confirmed the presence of individual polymers in the scaffolds and the scaffolds are hydrophilic in nature. Among the scaffolds, SF/GE 70:30 and SF/GE 50:50 composition possess desired porosity range of 87–88%. The tensile strength was slightly decreased with increase in GE content and the strength obtained was in the range 2.74–2.26 Mpa. An enhanced transparency was obtained with increased GE concentration, the transparency range of 73–82% observed with SF/GE:50/50 matches with the transparency of natural corneal epithelium. The biocompatibility of the scaffolds was confirmed by cell attachment, cytotoxicity and ROS evaluation by culturing rabbit corneal fibroblast cells (SIRC) on the scaffolds. Among the scaffolds, SF/GE: 70/30 exhibited the highest cell viability, but with less transparency ranging 55–71%. Therefore, considering all the properties, SF/GE with 50:50 ratio scaffold may be a suitable substrate in the field of corneal tissue engineering.

Generating a 3D Co-Culture Model of Human Corneal Fibroblasts and Neurons

Generating a 3D Co-Culture Model of Human Corneal Fibroblasts and Neurons

Proteomic Characterization of Corneal Epithelial and Stromal Cell-Derived Extracellular Vesicles.

Communication between the different layers of the cornea (epithelium and stroma) is a complex, yet crucial element in the corneal healing process. Upon corneal injury, it has been reported that the bi-directional cross talk between the epithelium and stroma via the vesicular secretome, namely, extracellular vesicles (EVs), can lead to accelerated wound closure upon injury. However, the distinct protein markers of EVs derived from human corneal epithelial (HCE) cells, keratocytes (HCKs), fibroblasts (HCFs), and myofibroblasts (HCMs) remain poorly understood. All EVs were enriched for CD81 and showed increased expression levels of ITGAV and FN1 in HCM-EVs compared to HCE- and HCF-EVs. All EVs were negative for GM130 and showed minimal differences in biophysical properties (particle concentration, median particle size, and zeta potential). At the proteomic level, we show that HCM-EVs are enriched with proteins associated with fibrosis pathways, such as COL6A1, COL6A2, MMP1, MMP2, TIMP1, and TIMP2, compared to HCE-, HCK-, and HCF-EVs. Interestingly, HCE-EVs express proteins involved with the EIF-2 signaling pathway (stress-induced signals to regulate mRNA translation), such as RPS21, RALB, EIF3H, RALA, and others, compared to HCK-, HCF-, and HCM-EVs. In this study, we isolated EVs from cell-conditioned media from HCE, HCKs, HCFs, and HCMs and characterized their biophysical and protein composition by Western blot, nanoparticle tracking analysis, and proteomics. This study supports the view that EVs from the corneal epithelium and stroma have a distinct molecular composition and may provide novel protein markers to distinguish the difference between HCE-, HCK-, HCF-, and HCM-EVs.

Corneal epithelial-stromal constructs to study differences associated with diabetes mellitus

Diabetes mellitus (DM) is a common metabolic disease associated with severe macrovascular and microvascular complications that influence nearly every tissue in the body, including the anterior and posterior segments of the eye. In the cornea, DM is associated with recurrent epithelial erosion and reduced wound-healing capacity, which increases the risk of corneal scarring. We previously developed a co-culture model of the cornea consisting of immortalized human corneal epithelial cells (hCE-TJ) overlaying a self-assembled stromal layer generated by human corneal fibroblasts (hCFs) over a 4-week period. In this study, we investigated epithelial-stromal constructs generated from hCFs derived from subjects with Type 1 (T1DM) or 2 diabetes (T2DM) compared to controls. We found that T2DM constructs exhibited a disrupted epithelium and a thicker, stratified stromal layer compared to controls or T1DM. Both T1DM and T2DM stromal constructs expressed lower expression of thrombospondin-1 in isolated extracellular vesicles (EVs) compared to controls with no significant difference observed in the presence of epithelial cells, suggesting that reduced provisional matrix secretion in the corneal stroma may be a factor that promotes delayed corneal wound healing in diabetes. The tetraspanins are established extracellular vesicle (EV) markers and include CD63, CD81, and CD9, and were highly expressed by EVs in all three cell types. Control corneal stromal fibroblasts produced more and larger EVs when compared to T1DM and T2DM hCF-derived EVs, supporting a role for altered cell-cell communication in the context of DM. Further characterization of EVs and their cargo is expected to aid in the development of targeted treatments to improve corneal wound healing.

Sodium butyrate alleviates lipopolysaccharide-induced inflammation through JAK/STAT signalling in primary human corneal fibroblasts

BackgroundBacterial keratitis is a common cause of blindness. Antibiotic treatment leads to the rapid release of lipopolysaccharide (LPS), which can activate corneal fibroblasts and cause persistent and excessive inflammatory responses. The anti-inflammatory drugs currently used to treat keratitis have serious side effects. Therefore, the ability of sodium butyrate (NaB), which can suppress the production of proinflammatory cytokines and promote the production of anti-inflammatory cytokines, to ameliorate keratitis was assessed in the present study. MethodsThe effect of NaB on the viability of primary human corneal fibroblasts was assayed with a CCK-8 kit. Cell migration was assessed by an in vitro scratch assay. Cell phenotypes were assessed by Western blotting and immunofluorescence staining. An antibody array was used to measure the production of proinflammatory cytokines and chemokines. ResultsAt 0–1 mM, NaB had no significant effect on cell viability, promoted the expression of the keratocyte marker keratocan and inhibited the fibroblast marker vimentin. Inhibition of cell migration was observed in the wound healing assay. By targeting the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling pathway, NaB decreased the levels of inflammation-related cytokines and chemokines whose expression was induced by LPS. ConclusionsNaB maintained the keratocyte phenotype, inhibited cell migration, and relieved LPS-induced inflammatory responses through the JAK/STAT signalling pathway.

Expression of matrix metalloproteinases and their inhibitors in corneal stromal fibroblasts and keratocytes from healthy and keratoconus corneas.

To examine the in-vitro expression of matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in corneal stromal cells by distinguishing between fibroblasts and keratocytes of healthy and keratoconus (KC) corneas. Stromal cells were isolated from healthy and KC corneas (n = 8). A normal-glucose, serum-containing cell culture medium (NGSC-medium) was used for cultivation of healthy human corneal fibroblasts (HCFs) and KC human corneal fibroblasts (KC-HCFs). In order to obtain a keratocyte phenotype, the initial cultivation with NGSC-medium was changed to a low-glucose, serum-free cell culture medium for healthy (Keratocytes) and KC cells (KC-Keratocytes). Gene and protein expression of MMP-1, -2, -3, -7, -9 and TIMP-1, -2, -3 were measured by quantitative PCR and Enzyme-Linked Immunosorbent Assay (ELISA) from the cell culture supernatant. KC-HCFs demonstrated a lower mRNA gene expression for MMP-2 compared to HCFs. In contrast to their respective fibroblast groups (either HCFs or KC-HCFs), Keratocytes showed a higher mRNA gene expression of TIMP-3, whereas TIMP-1 mRNA gene expression was lower in Keratocytes and KC-Keratocytes. Protein analysis of the cell culture supernatant revealed lower concentrations of MMP-1 in KC-HCFs compared to HCFs. Compared to Keratocytes, TIMP-1 concentrations was lower in the cell culture supernatant of KC-Keratocytes. In HCFs and KC-HCFs, protein levels of MMP-1 and TIMP-1 were higher and MMP-2 was lower compared to Keratocytes and KC-Keratocytes, respectively. This study indicates an imbalance in MMP and TIMP expression between healthy and diseased cells. Furthermore, differences in the expression of MMPs and TIMPs exist between corneal fibroblasts and keratocytes, which could influence the specific proteolytic metabolism in-vivo and contribute to the progression of KC.

A novel intralamellar semi‐bioresorbable keratoprosthesis—Part B: Surface functionalization and physico‐chemical characterization toward site‐specific cellular activity

AbstractIn this article, site‐specific functionalized keratoprosthesis (KPro) was accomplished to meet epithelial on‐growth on the anterior optical surface, inhibit cell adhesion on the posterior optic surface, and promote cell ingrowth at the peripheral tissue‐haptic‐flange interface toward tissue integration. Gelatin immobilization on PC4 hydrogel surfaces was achieved through acid hydrolysis followed by EDC/NHS chemistry, and polyethylene glycol‐diacrylate (PEGDA)‐modified inter‐penetrating network (IPN) surfaces were prepared using photo‐polymerization of ethylene glycol‐diacrylate (EGDA) for enhancing and inhibiting cellular growth, respectively. The surface chemical structures of modified hydrogels were characterized using attenuated total reflectance‐infrared (ATR‐IR) spectroscopy and a bicinchoninic acid (BCA) protein adsorption assay. Further, suitable differentially functionalized surfaces were prepared and characterized using the universal testing machine (UTM), refractometer, atomic force microscopy (AFM), and contact angle measurements to understand mechanical, optical, surface morphological, and wettability properties, respectively. Finally, in vitro site‐specific cell adhesion, cell inhibition, and cell ingrowth functionalities were tested using isolated goat corneal limbal epithelial stem cells and corneal stromal fibroblast cells, respectively. Overall, the two connective articles represent a successful endeavor to develop a more esthetically pleasing, mechanically stable, tissue‐free hydrogel KPro with site‐specific surface functionality nearly mimicking the cornea's functionality. A further pre‐clinical investigation is necessary to advance this functional novel KPro to reality, especially the tissue integration aspect.

Toxicity and efficacy of type I interferons on the ocular surface: in vitro, animal, and clinical studies

PurposeTo investigate the toxicity of type I interferons (IFNs) on the ocular surface and assess their efficacy in ocular surface tumors. MethodsWe examined the effects of IFN-α2a, IFN-α2b and IFN-β on corneal epithelial cells and stromal fibroblasts in vitro as well as the impact of IFN-α2a on the ocular surface in mice. Additionally, we analyzed the therapeutic and adverse effects of topically administered IFN-α2a and IFN-α2b in patients with ocular surface tumors. Risk factors contributing to side effects were explored. ResultsIFN-α2a, IFN-α2b or IFN-β reduced cell viability and induced pro-inflammatory cytokines in corneal epithelial cells and stromal fibroblasts. Furthermore, IFNs enhanced the expression of major histocompatibility complex class II and CD40 in corneal epithelial cells. In mice, subconjunctival IFN-α2a injection did not induce corneal epithelial defects or opacity, nor did it reduce aqueous tears or conjunctival goblet cells. In patients, topical IFN-α2a or IFN-α2b administration decreased tumor size and prevented recurrence; however, it was associated with mild side effects, including corneal epitheliopathy and conjunctival hyperemia. These complications were associated with longer IFN use, the presence of underlying ocular surface disease and concurrent use of mitomycin C or anti-glaucoma eye drops. ConclusionAlthough type I IFNs cause direct toxicity on corneal cells, they do not induce significant side effects on the healthy ocular surface. Considering its therapeutic and preventive effects, topical type I IFN is safe and effective for treating ocular surface tumors. The potential for ocular side effects should be considered in eyes with identified risk factors.

Corneal keratocytes, fibroblasts, and myofibroblasts exhibit distinct transcriptional profiles in vitro.

After stromal injury to the cornea, the release of growth factors and pro-inflammatory cytokines promotes the activation of quiescent keratocytes into a migratory fibroblast and/or fibrotic myofibroblast phenotype. Persistence of the myofibroblast phenotype can lead to corneal fibrosis and scarring, which are leading causes of blindness worldwide. This study aims to establish comprehensive transcriptional profiles for cultured corneal keratocytes, fibroblasts, and myofibroblasts to gain insights into the mechanisms through which these phenotypic changes occur. Primary rabbit corneal keratocytes were cultured in either defined serum-free media (SF), fetal bovine serum (FBS) containing media, or in the presence of TGF-β1 to induce keratocyte, fibroblast, or myofibroblast phenotypes, respectively. Bulk RNA sequencing followed by bioinformatic analyses was performed to identify significant differentially expressed genes (DEGs) and enriched biological pathways for each phenotype. Genes commonly associated with keratocytes, fibroblasts, or myofibroblasts showed high relative expression in SF, FBS, or TGF-β1 culture conditions, respectively. Differential expression and functional analyses revealed novel DEGs for each cell type, as well as enriched pathways indicative of differences in proliferation, apoptosis, extracellular matrix (ECM) synthesis, cell-ECM interactions, cytokine signaling, and cell mechanics. Overall, these data demonstrate distinct transcriptional differences among cultured corneal keratocytes, fibroblasts, and myofibroblasts. We have identified genes and signaling pathways that may play important roles in keratocyte differentiation, including many related to mechanotransduction and ECM biology. Our findings have revealed novel molecular markers for each cell type, as well as possible targets for modulating cell behavior and promoting physiological corneal wound healing.

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

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

The Role of Vimentin in Human Corneal Fibroblast Spreading and Myofibroblast Transformation.

Vimentin has been reported to play diverse roles in cell processes such as spreading, migration, cell-matrix adhesion, and fibrotic transformation. Here, we assess how vimentin impacts cell spreading, morphology, and myofibroblast transformation of human corneal fibroblasts. Overall, although knockout (KO) of vimentin did not dramatically impact corneal fibroblast spreading and mechanical activity (traction force), cell elongation in response to PDGF was reduced in vimentin KO cells as compared to controls. Blocking vimentin polymerization using Withaferin had even more pronounced effects on cell spreading and also inhibited cell-induced matrix contraction. Furthermore, although absence of vimentin did not completely block TGFβ-induced myofibroblast transformation, the degree of transformation and amount of αSMA protein expression was reduced. Proteomics showed that vimentin KO cells cultured in TGFβ had a similar pattern of protein expression as controls. One exception included periostin, an ECM protein associated with wound healing and fibrosis in other cell types, which was highly expressed only in Vim KO cells. We also demonstrate for the first time that LRRC15, a protein previously associated with myofibroblast transformation of cancer-associated fibroblasts, is also expressed by corneal myofibroblasts. Interestingly, proteins associated with LRRC15 in other cell types, such as collagen, fibronectin, β1 integrin and α11 integrin, were also upregulated. Overall, our data show that vimentin impacts both corneal fibroblast spreading and myofibroblast transformation. We also identified novel proteins that may regulate corneal myofibroblast transformation in the presence and/or absence of vimentin.

Amelioration of Fibrosis via S1P Inhibition Is Regulated by Inactivation of TGF-β and SPL Pathways in the Human Cornea.

Human corneal fibrosis can lead to opacity and ultimately partial or complete vision loss. Currently, corneal transplantation is the only treatment for severe corneal fibrosis and comes with the risk of rejection and donor shortages. Sphingolipids (SPLs) are known to modulate fibrosis in various tissues and organs, including the cornea. We previously reported that SPLs are tightly related to both, transforming growth factor beta (TGF-β) signaling and corneal fibrogenesis. The aim of this study was to investigate the effects of sphingosine-1-phosphate (S1P) and S1P inhibition on specific TGF-β and SPL family members in corneal fibrosis. Healthy human corneal fibroblasts (HCFs) were isolated and cultured in EMEM + FBS + VitC (construct medium) on 3D transwells for 4 weeks. The following treatments were prepared in a construct medium: 0.1 ng/mL TGF-β1 (β1), 1 μM sphingosine-1-phosphate (S1P), and 5 μM Sphingosine kinase inhibitor 2 (I2). Five groups were tested: (1) control (no treatment); rescue groups; (2) β1/S1P; (3) β1/I2; prevention groups; (4) S1P/β1; and (5) I2/β1. Each treatment was administered for 2 weeks with one treatment and switched to another for 2 weeks. Using Western blot analysis, the 3D constructs were examined for the expression of fibrotic markers, SPL, and TGF-β signaling pathway members. Scratch assays from 2D cultures were also utilized to evaluate cell migration We observed reduced fibrotic expression and inactivation of latent TGF-β binding proteins (LTBPs), TGF-β receptors, Suppressor of Mothers Against Decapentaplegic homologs (SMADs), and SPL signaling following treatment with I2 prevention and rescue compared to S1P prevention and rescue, respectively. Furthermore, we observed increased cell migration following stimulation with I2 prevention and rescue groups, with decreased cell migration following stimulation with S1P prevention and rescue groups after 12 h and 18 h post-scratch. We have demonstrated that I2 treatment reduced fibrosis and modulated the inactivation of LTBPs, TGF-β receptors, SPLs, and the canonical downstream SMAD pathway. Further investigations are warranted in order to fully uncover the potential of utilizing SphK I2 as a novel therapy for corneal fibrosis.

Manipulating mitochondrial pyruvate carrier function causes metabolic remodeling in corneal myofibroblasts that ameliorates fibrosis

Myofibroblasts are key cellular effectors of corneal wound healing from trauma, surgery, or infection. However, their persistent deposition of disorganized extracellular matrix can also cause corneal fibrosis and visual impairment. Recent work showed that the PPARγ agonist Troglitazone can mitigate established corneal fibrosis, and parallel in vitro data suggested this occurred through inhibition of the mitochondrial pyruvate carrier (MPC) rather than PPARγ. In addition to oxidative phosphorylation (Ox-Phos), pyruvate and other mitochondrial metabolites provide carbon for the synthesis of biological macromolecules. However, it is currently unclear how these roles selectively impact fibrosis. Here, we performed bioenergetic, metabolomic, and epigenetic analyses of corneal fibroblasts treated with TGF-β1 to stimulate myofibroblast trans-differentiation, with further addition of Troglitazone or the MPC inhibitor UK5099, to identify MPC-dependencies that may facilitate remodeling and loss of the myofibroblast phenotype. Our results show that a shift in energy metabolism is associated with, but not sufficient to drive cellular remodeling. Metabolites whose abundances were sensitive to MPC inhibition suggest that sustained carbon influx into the Krebs’ cycle is prioritized over proline synthesis to fuel collagen deposition. Furthermore, increased abundance of acetyl-CoA and increased histone H3 acetylation suggest that epigenetic mechanisms downstream of metabolic remodeling may reinforce cellular phenotypes. Overall, our results highlight a novel molecular target and metabolic vulnerability that affects myofibroblast persistence in the context of corneal wounding.