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Keratocytes of the corneal stroma secrete a unique population of proteoglycan molecules considered essential for corneal transparency. In healing corneal wounds, keratocytes exhibit a myofibroblastic phenotype in response to transforming growth factor beta (TGF-beta), characterized by expression of alpha-smooth muscle actin. This study examined pro...
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... vivo keratocytes secrete keratan sulfate glycosaminoglycan chains modifying three proteins (lumican, keratocan, and mimecan) and dermatan sulfate chains primarily attached to decorin. Alteration of this proteoglycan secretion profile in response to TGF- β was examined by isolating keratan and dermatan sulfate-containing proteoglycans, metabolically labeled in the glycosaminoglycan moieties with [ 35 S]sulfate or in the protein component with [ 35 S] methionine. As shown in Fig. 3 A , incorporation of [ 35 S]sulfate into keratan sulfate decreased dramatically in response to TGF- β treatment, whereas incorporation into dermatan sulfate (Fig. 3 B ) was increased. Interestingly, labeling with [ 35 S]methionine documented a moderate decrease for both proteoglycan fractions in response to TGF- β . The ratio of sulfate (glycosaminoglycan) to methionine (protein) in the secreted proteoglycans consequently responded in an opposite fashion for the two proteoglycan fractions (Fig. 3 C ). Keratan sulfate/ protein ratios dropped to 10 –20% of the control value over a period of 2–3 days of TGF- β exposure, whereas dermatan sulfate proteoglycans increased by about 2-fold over the same time period. Qualitative SDS-PAGE gel analysis of the [ 35 S]sulfate-labeled proteoglycans (Fig. 4) was consistent with the results of the quantitative analysis of Fig. 3. Keratan sulfate proteoglycans (Fig. 4 A ) were markedly decreased in abundance and simultaneously underwent a decrease in molecular size in response to TGF- β . Sulfate-labeled dermatan sulfate proteoglycans (Fig. 4 B ), conversely, increased in abundance and in size during TGF- β treatment. A minor component of high molecular size was observed after 3 days of treatment. The primary dermatan sulfate-containing stromal proteoglycan is decorin (23), but biglycan, a similar proteoglycan that is not present in normal stroma, has been observed to accumulate in corneas with chronic pathological conditions (10). Biglycan protein contains two glycosaminoglycan attachment sites and in most tissues exhibits a higher molecular size than decorin (10,24). Immunoprecipitation of the sulfate-labeled dermatan sulfate-containing proteoglycans from TGF- β -treated cultures with monospecific antibodies to decorin and biglycan (Fig. 5) showed the high molecular weight dermatan sulfate proteoglycan accumulating in response to TGF- β to precipitate with the anti-biglycan antibody but not the antibody to decorin. Detection by immunoblotting of core proteins released from nonlabeled secreted proteoglycans (Fig. 6) supported the conclusions of Fig. 3, suggesting that KSPG proteins undergo a modest overall decrease after 5–6 days of exposure to TGF- β . Secreted decorin also decreased somewhat in abundance over the 6-day treatment period. Biglycan, on the other hand, was not detected in untreated cultures but appeared in the pool of secreted proteoglycan within 2 days of TGF- β treatment and increased over several days. Pools of mRNA for the KSPG proteins, detected by Northern blotting in Fig. 7, exhibited a pattern of alterations similar to that seen in the secreted proteoglycan proteins. Lumican transcripts underwent a moderate decrease, whereas those for mimecan exhibited a more marked reduction. Mimecan transcripts occur in several alternately spliced forms, and it appeared that TGF- β treatment shifted the ratio of these in addition to decreasing their overall abundance. Keratocan transcripts were present at the limit of detection and did not exhibit much apparent alteration during TGF- β treatment. RNA transcripts of decorin, similar to the protein levels, were reduced somewhat by TGF- β , and expression of biglycan mRNA, almost undetectable in untreated cultures, was rapidly up-regulated (Fig. 8). In most tissues, fibrosis is associated with marked accumulation of collagen. In cornea, type I and particularly type III collagens are more abundant in healing wounds and in fibrotic regions of chronic pathological corneas and mRNA pools for these proteins are elevated (25–31). We observed that [ 3 H]proline incorporation into pepsin-resistant protein was dramatically increased in response to TGF- β (Fig. 9 A ). This pepsin-resistant protein band showed reactivity with both collagen types I and type III antibodies in Western blotting. Increased pools of collagen α 2(I) and α 1(III) mRNA were observed in the cells by Northern blotting (Fig. 9 B ). This up-regulation of fibrotic collagen expression in response to TGF- β correlates with biglycan expression implicating myofibroblasts in secretion of fibrotic tissue. In this study, we found that bovine keratocytes respond to TGF- by the development of extensive F-actin stress fibers terminating at paxillin-containing focal adhesions. The formation of new cytoskeletal elements correlates with accumulation of cell-associated fibronectin and its receptor, α 5 integrin. Such a fibronectin matrix and associated cytoskeleton is not observed in keratocytes in quiescent cultures (Fig. 1 A ), nor is it characteristic of keratocytes in vivo (13). Expression of and interaction with a fibronectin matrix, however, represents a classic marker of the fibroblastic phenotype and is exhibited by most adherent cells in culture as well as by keratocytes that populate healing corneal wounds (13,15,25). Stromal fibronectin accumulation is also observed in human corneas from several chronic pathological conditions as well as in acute healing wounds (29,32). -Smooth muscle actin is a protein originally thought to be restricted to muscle cells, and its appearance in fibroblastic cells has given rise to the term “myofibroblast.” Corneal myofibroblasts are contractile and have been suggested to play a role in wound closure (14). Accumulation of α -smooth muscle actin in rabbit keratocytes has been shown to depend on interaction of the fibronectin receptor with its ligand (the amino acid sequence RGD) (33). Consequently, it is thought that the corneal myofibroblast represents a phenotype derived from fibroblasts, not directly from the differentiated keratocytes, which lack cell-associated fibronectin (34). Myofibroblastic cells are widely observed in normal and pathological tissue environments, and in lung and liver there is an association of myofibroblastic cell population with tissue fibrosis (35–37). In healing corneal wounds, increased collagen deposition is associated with the presence of keratocytes containing α -smooth actin, suggesting that the myofibroblastic phenotype identifies cells responsible for deposition of fibrotic tissue (38). Only low level collagen expression is observed in normal adult cornea, but in the late phase of healing wounds, a marked increase in collagen biosynthesis and mRNA pools has been demonstrated (39). Type III collagen, not a component of normal stroma, is detected in stromal fibrotic tissue (26,27,29, 30) and as such represents a good marker for fibrosis. Our demonstration that up-regulation of type I and III collagen expression (Fig. 9) correlates with α -smooth actin accumulation in vitro strengthens the hypothesized link between myofibroblastic cells and fibrotic tissue deposition in the cornea. Numerous earlier reports have documented lack of keratan sulfate biosynthesis by fibroblastic cultures of keratocytes maintained in fetal bovine serum and subcultured by trypsinization (40). Our more recent studies found that keratocytes in serum-free or reduced serum media secrete keratan sulfate for extended periods (18,19). Cell proliferation induced by basic fibroblast growth factor does not down-regulate keratan sulfate biosynthesis, suggesting that cell division per se is not involved. Our current data show a dramatic and rapid effect of TGF- β on keratan sulfate biosynthesis. Stromal fibroblast cultures both produce and respond to TGF- in an auto-crine fashion (16,41). TGF- is also present in wounded and pathological corneas (42). Considered together, these results suggest that TGF- β may be the primary agent responsible for keratan sulfate loss in fibroblastic cultures and in the cornea during wound healing and inflammation. The loss of keratan sulfate must precede or be independent of myofibroblast formation, because although not all cultures of stromal fibroblasts exhibit α smooth actin expression, none have been found to express keratan sulfate (16). In the current experiments, exposure to TGF- caused a moderate decrease in mRNA pools for keratan sulfate-linked proteins and a similar decrease in KSPG core proteins labeled with methionine or detected by immunoblotting. Incorporation of sulfate into keratan sulfate on these proteoglycans, on the other hand, was rapidly and dramatically reduced by TGF- β , resulting in a change in ...
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... Corneal fibrosis is a leading cause of blindness worldwide, affecting over 10 million people [1]. Injury or trauma to the cornea can initiate resident keratocyte differentiation into myofibroblasts, causing high expression levels of α smooth muscle actin (αSMA) and Collagen III, which can cause irregular deposition of extracellular matrix (ECM) components [2,3]. This disruption of the homeostatic corneal environment can cause cornea scarring, which can result in vision loss. ...
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.
... Their differentiation into fibroblasts and subsequently into myofibroblasts leads to important changes in corneal morphology, transparency, and vision [72][73][74][75]. Thus, the genetic markers of the three cell types were studied: LUMI, the major proteoglycan expressed by keratocytes that plays an important role in ECM regulation [76][77][78]; THY-1, a corneal fibroblast surface antigen [79] and α-SMA, a marker of myofibroblast differentiation [76]. In addition, changes in the expression of important components of the extracellular matrix were studied, including COL1A1, the most abundant component of the ECM [80] and the most abundant collagen type in the corneal stroma [81,82]; COL3A1, which is controversial because there is still no consensus regarding its presence in healthy corneas. ...
... Their differentiation into fibroblasts and subsequently into myofibroblasts leads to important changes in corneal morphology, transparency, and vision [72][73][74][75]. Thus, the genetic markers of the three cell types were studied: LUMI, the major proteoglycan expressed by keratocytes that plays an important role in ECM regulation [76][77][78]; THY-1, a corneal fibroblast surface antigen [79] and α-SMA, a marker of myofibroblast differentiation [76]. In addition, changes in the expression of important components of the extracellular matrix were studied, including COL1A1, the most abundant component of the ECM [80] and the most abundant collagen type in the corneal stroma [81,82]; COL3A1, which is controversial because there is still no consensus regarding its presence in healthy corneas. ...
The Boston keratoprosthesis (BKPro) is a medical device used to restore vision in complicated cases of corneal
blindness. This device is composed by a front plate of polymethylmethacrylate (PMMA) and a backplate usually
made of titanium (Ti). Ti is an excellent biomaterial with numerous applications, although there are not many
studies that address its interaction with ocular cells. In this regard, despite the good retention rates of the BKPro,
two main complications compromise patients’ vision and the viability of the prosthesis: imperfect adhesion of the
corneal tissue to the upside of the backplate and infections. Thus, in this work, two topographies (smooth and
rough) were generated on Ti samples and tested with or without functionalization with a dual peptide platform.
This molecule consists of a branched structure that links two peptide moieties to address the main complications
associated with BKPro: the well-known RGD peptide in its cyclic version (cRGD) as cell pro-adherent motif and
the first 11 residues of lactoferrin (LF1–11) as antibacterial motif. Samples were physicochemically characterized,
and their biological response was evaluated in vitro with human corneal keratocytes (HCKs) and against the
gram-negative bacterial strain Pseudomonas aeruginosa. The physicochemical characterization allowed to verify
the functionalization in a qualitative and quantitative manner. A higher amount of peptide was anchored to the
rough surfaces. The studies performed using HCKs showed increased long-term proliferation on the functionalized
samples. Gene expression was affected by topography and peptide functionalization. Roughness promoted
α-smooth muscle actin (α-SMA) overexpression, and the coating notably increased the expression of extracellular
matrix components (ECM). Such changes may favour the development of unwanted fibrosis, and thus, corneal
haze. In contrast, the combination of the coating with a rough topography decreased the expression of α-SMA and
ECM components, which would be desirable for the long-term success of the prosthesis. Regarding the antibacterial
activity, the functionalized smooth and rough surfaces promoted the death of bacteria, as well as a
perturbation in their wall definition and cellular morphology. Bacterial killing values were 58 % for smooth
functionalised and 68 % for rough functionalised samples. In summary, this study suggests that the use of the
dual peptide platform with cRGD and LF1-11 could be a good strategy to improve the in vitro and in vivo performance
of the rough topography used in the commercial BKPro.
... TGF-β has been shown to drive corneal wound healing, where TGF-β isoforms regulate myofibroblast differentiation [13]. In vivo studies have shown the TGF-β superfamily is structurally related [14,15] where these multifunctional growth factors play prominent roles in fibrosis via myofibroblast differentiation [4,5,[16][17][18], embryonic development, granulation [14], cell contraction and loss of transparency, specifically in corneal tissue. TGF-β can maintain its production in an autocrine manner in myofibroblasts, necessitating additional mechanisms to halt the stromal repair processes once the wound is closed [9]. ...
The differentiation of keratocytes to fibroblasts and myofibroblasts is an essential requisite during corneal wound closure. The aim of this study is to uncover factors involved in differentiation-dependent alteration in the protein profile of human corneal stromal cells using quantitative proteomics. Human corneal fibroblasts were cultured and differentiated into keratocytes in serum-free media and myofibroblasts through treatment with TGF-β. The protein cell lysates from the donors were tryptic and were digested and labeled using a 3-plex iTRAQ kit. The labeled peptides were subjected to LCMS analysis. Biological functional analysis revealed a set of crucial proteins involved in the differentiation of human corneal stromal cells which were found to be significantly enriched. The selected proteins were further validated by immunohistochemistry. Quantitative proteomics identified key differentially expressed proteins which are involved in cellular signaling pathways. Proteins involved in integrin signaling (Ras-RAP1b, TLN and FN) and SLIT-ROBO pathways (PFN1, CAPR1, PSMA5) as well as extracellular matrix proteins (SERPINH1, SPARC, ITGβ1, CRTAP) showed enhanced expression in corneal fibroblasts and myofibroblasts compared to keratocytes, indicating their possible role in wound healing. Corneal stromal cell differentiation is associated with the activation of diverse molecular pathways critical for the repair of fibroblasts and myofibroblasts. Identified proteins such as profilin 1 and talin could play a tentative role in corneal healing and serve as a potential target to treat corneal fibrosis.
... In vivo confocal microscopy studies revealed that immune response to corneal intraepithelial adenoviral infection includes the recruitment of dendritic cells initially as innate immunity followed by an anterior stromal reaction at the second week compatible with SEIs (18,19) . The stromal reaction was thought to include keratocytes, highly active myofibroblasts, and infiltrated inflammatory cells (18,20) . Besides, midstromal infiltrates in form of hyperreflective plaques compatible with the healing process were observed in as late as 24 weeks. ...
Purpose:
To examine the effect of subepithelial corneal infiltrates on corneal biomechanical properties after epidemic keratoconjunctivitis compared to that in healthy controls.
Methods:
The cross-sectional study included consecutive patients with bilateral subepithelial corneal infiltrates after epidemic keratoconjunctivitis and healthy controls. Best corrected visual acuity corneal subepithelial infiltrate scoring Fantes grading scale, and central corneal thickness were measured. Corneal hysteresis corneal resistance factor Goldmann correlated intraocular pressure and corneal compensated intraocular pressure were assessed using an ocular response analyzer.
Results:
This study included 66 eyes of 33 patients with subepithelial corneal infiltrates following epidemic keratoconjunctivitis and randomly selected 37 eyes of 37 healthy volunteers. The mean Fantes and CSIS scores were 1.8 ± 0.8 and 2.9 ± 1.3, respectively, in the first involved eyes and 1.3 ± 1.1 and 1.9 ± 1.7, respectively, in the fellow eyes (p=0.009 and p=0.002, respectively). The first (526.1 ± 28.1 µm; p=0.005) and second involved eyes (523.4 ± 38.1 µm; p=0.044) had significantly thinner corneas compared to that in healthy controls (557.0 ± 38.1 µm). While best-corrected visual acuity showed a positive correlation with corneal resistance factor (r=0.363, p=0.045) and corneal hysteresis (r=0.414, p=0.021), corneal subepithelial infiltrate scoring showed a negative correlation with Goldmann correlated intraocular pressure (r=-0.479, p=0.006) and corneal compensated intraocular pressure (r=-0.413, p=0.021).
Conclusion:
Eyes with subepithelial corneal infiltrates had significantly thinner corneas compared to that in healthy controls. A positive correlation of the corneal resistance factor and corneal hysteresis with best-corrected visual acuity and a negative correlation of the Goldmann correlated intraocular pressure and corneal compensated intraocular pressure with corneal subepithelial infiltrate scoring should be taken into account when measuring intraocular pressure values in patients with subepithelial corneal infiltrates.
... Type III collagen fibres have a larger diameter than type I collagen fibres, which contributes to the light scattering disorder. Unlike quiescent keratocytes which mainly secrete keratan sulphates, myofibroblasts produce abnormal proteoglycans and mainly dermatan sulphates [18] which do not contribute to the regular spacing of collagen fibrils, and to the uniformity of their diameters. These structural modifications of the extracellular matrix, the density of which is increased, and the presence of the myofibroblasts that induce them, are at the origin of the loss of corneal transparency and therefore the corneal haze. ...
Corneal haze represents subepithelial corneal fibrosis, a manifestation of a pathological healing process. It occurs as a result of an epithelial-stromal lesion involving a break in the epithelial barrier. It is an inflammatory response that involves the migration, multiplication and differentiation of keratocytes into mature myofibroblasts, causing loss of corneal transparency. Although it is a transient phenomenon, this complication is feared following refractive photokeratectomy (PRK), because it can cause alterations in the quality of vision, refractive regression and decreased visual acuity. The severity of these symptoms is correlated with the severity of the corneal haze, which can be assessed clinically or by objective means such as corneal densitometry measurement. The frequency and severity of corneal haze increase with the depth of photoablation in PRK and are therefore increased during the treatment of severe ametropia. Considering that no consensus exists, the application of mitomycin C (MMC) intraoperatively and topical corticosteroids postoperatively are conventionally used to inhibit collagen synthesis, sometimes in combination with various protocols depending on the center or surgeon. This review of the literature reports the current knowledge on corneal haze, in order to better understand it and optimise its prevention in the context of a decreased MMC supply, which has occurred in the past and could recur in the future.
... Corneal stroma keratocytes produce a specific and balanced population of proteoglycans (lumican, keratocan, mimecan, decorin), the arrangement of which is essential for corneal transparency [15] . Among these, lumican seems to be the main orchestrator in order to produce and maintain a regular structure of corneal stroma, which is an essential requisite for transparency. ...
Inflammatory events in the corneal stroma may activate keratocytes and trigger their transition towards myofibroblasts, which now produce different extracellular matrix (ECM) proteins thus causing corneal opacification.Corneal haze is a frequent side effect after photorefractive keratectomy (PRK) to correct high myopia.Currently, a preventive treatment with mitomycin-c can be used to limit the occurrence of this phenomenon. However, mitomycin-c is a toxic drug, not devoid of side effects, which may occasionally involve the corneal endothelium. Therefore, we have searched for a less risky, natural way, to prevent keratocytes transition. To this purpose, we have used as markers of the phenotype switch the proteins lumican (highly expressed by keratocytes and much less by myofibroblasts) and smooth muscle actin (αSMA) (highly expressed by myofibroblasts and poorly found in keratocytes), beside Fibronectin (Fn), the expression of which is also increased by transforming growth factor-beta (TGFβ treatment. Treatment of human keratocytes with TGFβ was used to induce the protein shift. Among different possible candidates, we have found that vitamins A and E, hyaluronic and lactobionic acids may prevent, either alone, or much better in association, the shift in the ratio between lumican and αSMA and the increased Fn expression. In conclusion, it could be speculated that topic treatment of the ocular surface with an association of these four compounds could be able to prevent or at least limit the occurrence of post-PRK corneal haze, with the additional advantage of lubrication, hydration and antioxidant defense exerted by these molecules.
... The transition of quiescent stromal cells to myofibroblasts is presumably mediated by TGF (3,7). The overproduction of matrix by myo fibroblasts results in an unorganized deposition of ECM proteins such as type III collagen that leads to fibrosis and, eventually, scar formation (8). Crosstalk between immune cells recruited to the wounded tissue, activated resident immune cells, and stromal cells controls the delicate balance between regenerative repair that main tains corneal clarity and scar formation that causes loss of vision. ...
The avascular nature of cornea tissue limits its regenerative potential, which may lead to incomplete healing and formation of scars when damaged. Here, we applied micro- and ultrafine porcine urinary bladder matrix (UBM) particulate to promote type 2 immune responses in cornea wounds. Results demonstrated that UBM particulate substantially reduced corneal haze formation as compared to the saline-treated group. Flow cytometry and gene expression analysis showed that UBM particulate suppressed the differentiation of corneal stromal cells into α-smooth muscle actin–positive (αSMA+) myofibroblasts. UBM treatments up-regulated interleukin-4 (IL-4) produced primarily by eosinophils in the wounded corneas and CD4+ T cells in draining lymph nodes, suggesting a cross-talk between local and peripheral immunity. Gata1−/− mice lacking eosinophils did not respond to UBM treatment and had impaired wound healing. In summary, stimulating type 2 immune responses in the wounded cornea can promote proregenerative environments that lead to improved wound healing for vision restoration.
... Congenital or acquired LSCD impairs corneal epithelium renewal, resulting in progressive opacification, chronic ulceration, conjunctivalization, and neovascularization with blindness, disfigurement, and occasionally discomfort as a result. If the damage extends to the anterior corneal stroma, fibroblastic cells derived from stromal keratocytes may produce long-lasting opaque scar tissue [283,284]. In many cases, stromal opacities and neovascularization improve once the corneal epithelial phenotype is restored by limbal stem cells and corneal epithelium regeneration. ...
Each day, about 2000 U.S. workers have a job-related eye injury requiring medical treatment. Corneal diseases are the fifth cause of blindness worldwide. Most of these diseases can be cured using one form or another of corneal transplantation, which is the most successful transplantation in humans. In 2012, it was estimated that 12.7 million people were waiting for a corneal transplantation worldwide. Unfortunately, only 1 in 70 patients received a corneal graft that same year. In order to provide alternatives to the shortage of graftable corneas, considerable progress has been achieved in the development of living corneal substitutes produced by tissue engineering and designed to mimic their in vivo counterpart in terms of cell phenotype and tissue architecture. Most of these substitutes use synthetic biomaterials combined with immortalized cells, which makes them dissimilar from the native cornea. However, studies have emerged that describe the production of tridimensional (3D) tissue-engineered corneas using untransformed human corneal epithelial cells grown on a totally natural stroma synthesized by living corneal fibroblasts, that also show appropriate histology and expression of both extracellular matrix (ECM) components and integrins. This review highlights contributions from laboratories working on the production of human tissue-engineered corneas (hTECs) as future substitutes for grafting purposes. It overviews alternative models to the grafting of cadaveric corneas where cell organization is provided by the substrate, and then focuses on their 3D counterparts that are closer to the native human corneal architecture because of their tissue development and cell arrangement properties. These completely biological hTECs are therefore very promising as models that may help understand many aspects of the molecular and cellular mechanistic response of the cornea toward different types of diseases or wounds, as well as assist in the development of novel drugs that might be promising for therapeutic purposes.
... To further confirm its ability in maintaining the keratocyte function, a set of genes involved in the synthesis of a functional product by keratocytes was assessed by qRT-PCR analysis. Keratocytes typically express a higher amount of keratan sulfate and a lesser amount of collagen type I. 28 Our results showed an increased expression of keratan sulfate and reduction of collagen type 1 (COL-1) expression. It is also worth noting that little or no expression of α-SMA was observed, which is typically a hallmark of myofibroblast cells. ...
... It is also worth noting that little or no expression of α-SMA was observed, which is typically a hallmark of myofibroblast cells. 28 An expression pattern of a higher keratan sulfate and a lower pattern of COL-I was observed in the cells encapsulated and cultured for 14 days in dCMH ( Figure 5B). The above data suggest that our The myofibroblast cells are typically contractile and they play a major role in extensive matrix contraction when they are cultured on a substrate. ...
... A major challenge of the in vitro culture of keratocytes is their transdifferentiation toward myofibroblasts, which are associated with the production of higher amounts of collagen type I and a lower amount of keratan sulfate. 28 We evaluated the maintenance of keratocyte function through immunostaining of the cells encapsulated and cultured in dCMH. It was found that in the presence of hydrogel, cells synthesized more keratan sulfate and less COL-I, which is a characteristic feature of keratocytes ( Figure 6A). ...
Corneal scarring is one of the major causes of blindness, affecting millions worldwide. Despite recent advancements in surgical strategies, there is an unmet need for a clinically feasible material and methods to prevent scarring following corneal injury. In this study, we report the potential utility of a hydrogel derived from cadaveric animal corneas, using a decellularized corneal matrix hydrogel (abbreviated as dCMH), which is prepared by a simple method. This hydrogel is easily injectable, biocompatible, and has the ability to maintain good shape-retention properties at 37 °C, which make it suitable for in vivo applications. Furthermore, our gene expression studies and immunofluorescence studies indicate that dCMH maintains the morphology and function of keratocytes in vitro and prevents their transdifferentiation to myofibroblasts. From the above results, it is evident that dCMH maintains the keratocytes with the ability to regenerate the corneal defect without scar. We thus suggest a simple yet effective approach for corneal tissue decellularization and that dCMH can be a promising material for prophylaxis against blinding scar formation in an injured cornea.
... Wrighton et al. (2008) identified TGFβRI and TGFβRII as Hsp90-interacting proteins and demonstrated that Hsp90 inhibition with 17AAG increased ubiquitination and degradation of TGFβ receptors thus blocking the TGFβ signaling pathway. Since the TGFβ pathway is critically implicated in corneal wound healing response and KFM transformation (Fini, 1999;Folger et al., 2001;Funderburgh et al., 2001;Jester et al., 1999;Kaur et al., 2009;Sakamoto et al., 2000;Tandon et al., 2010), we evaluated the effect of disrupting the TGF signaling pathway via Hsp90 inhibition in rabbit stromal cells in vitro and in vivo. ...
The transformation of quiescent keratocytes to activated fibroblasts and myofibroblasts (KFM transformation) largely depends on transforming growth factor beta (TGFβ) signaling. Initiation of the TGFβ signaling cascade results from binding of TGFβ to the labile type I TGFβ receptor (TGFβRI), which is stabilized by the 90 kDa heat shock protein (Hsp90). Since myofibroblast persistence within the corneal stroma can result in stromal haze and corneal fibrosis in patients undergoing keratorefractive therapy, modulation of TGFβ signaling through Hsp90 inhibition would represent a novel approach to prevent myofibroblast persistence.
In vitro, rabbit corneal fibroblasts (RCFs) or stratified immortalized human corneal epithelial cells (hTCEpi) were treated with a Hsp90 inhibitor (17AAG) in the presence/absence of TGFβ1. RCFs were cultured either on tissue culture plastic, anisotropically patterned substrates, and hydrogels of varying stiffness. Cellular responses to both cytoactive and variable substrates were assessed by morphologic changes to the cells, and alterations in expression patterns of key keratocyte and myofibroblast proteins using PCR, Western blotting and immunocytochemistry. Transepithelial electrical resistance (TEER) measurements were performed to establish epithelial barrier integrity. In vivo, the corneas of New Zealand White rabbits were wounded by phototherapeutic keratectomy (PTK) and treated with 17AAG (3× or 6× daily) either immediately or 7 days after wounding for 28 days. Rabbits underwent clinical ophthalmic examinations, SPOTS scoring and advanced imaging on days 0, 1, 3, 7, 10, 14, 21 and 28. On day 28, rabbits were euthanized and histopathology/immunohistochemistry was performed.
In vitro data demonstrated that 17AAG inhibited KFM transformation with the de-differentiation of spindle shaped myofibroblasts to dendritic keratocyte-like cells accompanied by significant upregulation of corneal crystallins and suppression of myofibroblast markers regardless of TGFβ1 treatment. RCFs cultured on soft hydrogels or patterned substrates exhibited elevated expression of α-smooth muscle actin (αSMA) in the presence of 17AAG. Treatment of hTCEpi cells disrupted zonula occludens 1 (ZO-1) adherens junction formation. In vivo, there were no differences detected in nearly all clinical parameters assessed between treatment groups. However, rabbits treated with 17AAG developed greater stromal haze formation compared with controls, irrespective of frequency of administration. Lastly, there was increased αSMA positive myofibroblasts in the stroma of 17AAG treated animals when compared with controls.
Hsp90 inhibition promoted reversion of the myofibroblast to keratocyte phenotype, although this only occurred on rigid substrates. By contrast, in vivo Hsp90 inhibition was detrimental to corneal wound healing likely due to impairment in corneal epithelial closure and barrier function restoration. Collectively, our data demonstrated a strong interplay in vitro between biophysical cues and soluble signaling molecules in determining corneal stromal cell phenotype.
