Search Results (79)

Background/Objective: A clinical need exists for more effective therapeutics and sustained drug delivery systems to promote ocular surface healing. This study tested the hypothesis that a novel biodegradable, thermoresponsive hydrogel loaded with the human recombinant (rh)MG53 protein, which we have demonstrated to promote corneal healing without fibrosis, would exhibit safety and biocompatibility in vitro and in vivo. Methods: Hydrogel optimization was performed based on varying concentrations of poloxamer 407, poloxamer 188, and hydroxypropyl methylcellulose. Hydrogels were characterized and potential toxicity was evaluated in vitro in cultured corneal epithelium, fibroblasts, and endothelium. In vivo safety and tolerability were assessed in mice and hydrogels were used to evaluate corneal healing following alkali injury. Results: The optimized hydrogel formulation did not result in any detrimental changes to the corneal cells and released functional rhMG53 protein for at least 24 h. In vivo rhMG53-loaded hydrogels improved re-epithelialization, reduced stromal opacification and vascularization, and promoted corneal nerve density. Mechanistically, rhMG53 reduced vascular endothelial cell migration and tube formation by inhibiting pSTAT3 signaling. Conclusions: Taken together, our poloxamer-based thermoresponsive hydrogel effectively released rhMG53 protein and enhanced multiple corneal healing outcomes. Full article

Corneal fibroblasts are central to normal and abnormal wound healing in the cornea. During the wound healing process, several biochemical and biophysical signals that are present in the extracellular matrix (ECM) play critical roles in regulating corneal fibroblast behavior. The translocation and activation of Yes-associated protein (YAP)—a main transcriptional factor in the Hippo signaling pathway—is one example of mechanotransduction involving these signals. However, how corneal fibroblasts integrate these simultaneous cues is unknown. In this study, we utilized well-defined micropatterns of aligned collagen fibrils and other ECM proteins to explore the effects of cell density, topography, geometric confinement, and ECM composition on the translocation of YAP in corneal fibroblasts. We observed that when human corneal fibroblasts (HTKs) were confined to narrow micropatterns (50 μm and 100 μm) of proteins, there was a high degree of cell alignment irrespective of cell seeding density. However, the location of YAP was dependent upon the cell seeding density, ECM composition, and topography. YAP was more nuclear-localized on substrates coated with aligned collagen fibrils or fibronectin as compared to substrates coated with monomeric collagen, random collagen fibrils, or poly-L-Lysine. In addition, we also observed that YAP nuclear localization was significantly reduced when HTKs were cultured on aligned collagen fibrils, monomeric collagen, or fibronectin in the presence of monoclonal blocking antibodies against α₅ or β₁ integrin subunits. Finally, we observed that HTK cells formed fibrillar fibronectin on both monomeric collagen and aligned collagen fibrils. These findings provide new insights into how simultaneous biochemical and biophysical cues affect YAP localization in corneal fibroblasts. Full article

Simple limbal epithelial transplantation (SLET) has emerged as an effective treatment option for limbal stem cell deficiency (LSCD). However, the precise molecular mechanisms underlying its success remain incompletely understood. This review delves into the proposed mechanisms involving the donor limbus, host microenvironment, and the amniotic membrane as a scaffold in SLET. The donor limbus contributes to SLET efficacy through various factors secreted by limbal epithelial stem cells, including hepatocyte growth factor (HGF), soluble Fms-like tyrosine kinase-1 (sFLT-1), and pigment epithelium-derived factor (PEDF), which support corneal healing and transparency. Additionally, the presence of melanocytes, immune cells, limbal fibroblasts, and adhesion molecules within the donor tissue helps preserve the integrity of the limbal niche. The host environment plays a critical role in supporting the transplanted stem cells, with mesenchymal stem cell-secreted factors promoting proliferation and differentiation. Although the amniotic membrane has traditionally been used as a scaffold, emerging evidence suggests that it may not always be necessary. Further studies are needed to validate this scaffold-free approach and to evaluate the vitality and functional contributions of individual components used in SLET. Understanding these complex interactions and molecular mechanisms sheds light on the importance of the donor tissue, host microenvironment, and scaffold in SLET, paving the way for the optimization of this technique for the effective treatment of LSCD. Full article

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. Full article

Background/Objectives: Pterygium is a nonneoplastic elastotic degeneration characterized by subepithelial growth. It manifests as an ocular lesion originating from the bulbar conjunctiva, extending to the corneal surface, and reaching the visual axis in some cases. Although the exact cause is unknown, prolonged exposure to ultraviolet radiation is considered the most significant contributing factor. Chronic irritation and actinic damage are likely responsible for the typical fibrovascular reactions observed in pterygium. Additionally, growth factors, cytokines, and matrix metalloproteinases play roles in the pathogenesis of pterygium. This study compared recurrent and primary pterygium cases at the molecular level to gain new insights into the etiology of pterygium. Methods: Total protein was extracted from surgical samples of patients with primary and recurrent pterygium, and the levels of transforming growth factor beta 1 (TGF-β1), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), IL-8, and IL-10 were analyzed using the enzyme-linked immunosorbent assay technique. Target gene expression levels were analyzed using the ΔΔCt method after cDNA synthesis from isolated RNA, with normalization to GAPDH and quantification performed with SYBR Green PCR Master Mix. Results: Among the studied cytokines, IL-10 levels were higher in primary pterygium than in recurrent pterygium (722.0 ± 600.9/421.4 ± 266.8) (p = 0.0054). Other cytokines (IL-6, IL-8, IL-1β, and TGF-β1) were detected at similar levels in both primary and recurrent pterygium (p = 0.2986). Additionally, the TGF-β1 gene expression was found to be significantly upregulated in recurrent pterygium tissue compared to primary pterygium tissue (p = 0.034). Conclusions: This increase suggests that TGF-β1 may contribute to the recurrence mechanisms of pterygium through processes such as fibroblast activation and tissue remodeling. The higher levels of IL-10 in primary pterygium compared to recurrent pterygium indicate an enhanced early protective response aimed at limiting pterygium progression and controlling the inflammatory process. Full article

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. Full article

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. Full article

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. Full article

Limbal mesenchymal stromal cells (LMSCs) reside in the limbal niche, supporting corneal integrity and facilitating regeneration. While mesenchymal stem/stromal cells (MSCs) are used in regenerative therapies, there is limited knowledge about LMSC subpopulations and their characteristics. This study characterized human LMSC subpopulations through the flow cytometric assessment of fifteen cell surface markers, including MSC, wound healing, immune regulation, ASC, endothelial, and differentiation markers. Primary LMSCs were established from remnant human corneal transplant specimens and passaged eight times to observe changes during subculture. The results showed the consistent expression of typical MSC markers and distinct subpopulations with the passage-dependent expression of wound healing, immune regulation, and differentiation markers. High CD166 and CD248 expressions indicated a crucial role in ocular surface repair. CD29 expression suggested an immunoregulatory role. Comparable pigment-epithelial-derived factor (PEDF) expression supported anti-inflammatory and anti-angiogenic roles. Sustained CD201 expression indicated maintained differentiation capability, while VEGFR2 expression suggested potential endothelial differentiation. LMSCs showed higher VEGF expression than fibroblasts and endothelial cells, suggesting a potential contribution to ocular surface regeneration through the modulation of angiogenesis and inflammation. These findings highlight the heterogeneity and multipotent potential of LMSC subpopulations during in vitro expansion, informing the development of standardized protocols for regenerative therapies and improving treatments for ocular surface disorders. Full article

The functioning of the human cornea heavily relies on the maintenance of its extracellular matrix (ECM) mechanical properties. Within this context, corneal stromal fibroblasts (CSFs) are essential, as they are responsible for remodeling the corneal ECM. In this study, we used a decellularized human amniotic membrane (dHAM) and a custom fibrillar collagen film (FCF) to explore the effects of fibrillar materials on human CSFs. Our findings indicate that substrates like FCF can enhance the early development of focal adhesions (FAs), leading to the activation and propagation of mechanotransduction signals. This is primarily achieved through FAK autophosphorylation and YAP1 nuclear translocation pathways. Remarkably, inhibiting FAK autophosphorylation negated the observed changes. Proteome analysis further confirmed the central role of FAs in mechanotransduction propagation in CSFs cultured on FCF. This analysis also highlighted complex signaling pathways, including chromatin epigenetic modifications, in response to fibrillar substrates. Overall, our research highlights the potential pathways through which CSFs undergo behavioral changes when exposed to fibrillar substrates, identifying FAs as essential mechanotransducers. Full article

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. Full article

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. Full article

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. Full article

Backgrond: Intracorneal ring segments (ICRSs) are utilized to correct refractive changes impacting visual acuity, commonly implanted via femtosecond laser but can also inserted manually. Corneal deposits alongside the ICRS channels are seen commonly. Methods: This study explores the histological characteristics of corneal deposits following manual ICRS implantation, comparing them to previously published articles describing femtosecond laser-assisted cases. Results: This is a retrospective analysis of three cases involving manual ICRS implantation, accumulation of whitish deposits and later explanation of the corneas due to penetrating keratoplasty (PKP). Patient demographics, ocular history, and surgical details were collected. Histological analysis employed Hematoxylin and Eosin (H&E) and Masson’s trichrome staining. Whitish deposits along ICRS tracts were observed in all cases, with minimal fibroblastic transformation of keratocytes adjacent to the segments. Comparing these cases of manual to femtosecond laser-assisted ICRS implantation, in most cases, similar deposits were identified, indicating the deposits’ association with the stromal tissue reaction to the ring segment and not to the surgical technique. Conclusions: This study contributes insights into the histopathology of manually implanted ICRS, emphasizing the shared nature of deposits in both insertion methods. The findings highlight the link between deposits and the stromal tissue reaction to the ring segment, irrespective of the insertion technique. Full article