Cell sheet-based approach to study the diabetic corneal stroma.

Prolonged hyperglycemia during diabetes mellitus (DM) is associated with severe complications that may affect both the anterior and posterior ocular segments, leading to impaired vision or blindness. The cornea is a vital part of the eye that has a dual role as a protective transparent barrier and as a major refractive structure and is likewise negatively affected by hyperglycemia in DM. Understanding the cellular and molecular mechanisms underlying the phenotypic changes associated with DM is critical to developing targeted therapies to promote tissue integrity. In this proof-of-concept study, we applied a cell sheet-based approach to generate stacked constructs of physiological corneal thickness using primary human corneal fibroblasts isolated from cadaveric control (healthy), Type 1 DM and Type 2 DM corneal tissues. Self-assembled corneal stromal sheets were generated after 2 weeks in culture, isolated, and subsequently assembled to create stacked constructs, which were evaluated using transmission electron microscopy. Analysis of gene expression patterns revealed significant downregulation of fibrotic markers, α-smooth muscle actin, and collagen type 3, with stacking in Type 2 DM constructs when compared to controls. IGF1 expression was significantly upregulated in Type 2 DM constructs compared to controls with a significant reduction induced by stacking. This study describes the development of a thicker, self-assembled corneal stromal construct as a platform to evaluate phenotypic differences associated with DM-derived corneal fibroblasts and enable the development of targeted therapeutics to promote corneal integrity.

Sex Hormones, Growth Hormone, and the Cornea.

The growth and maintenance of nearly every tissue in the body is influenced by systemic hormones during embryonic development through puberty and into adulthood. Of the ~130 different hormones expressed in the human body, steroid hormones and peptide hormones are highly abundant in circulation and are known to regulate anabolic processes and wound healing in a tissue-dependent manner. Of interest, differential levels of sex hormones have been associated with ocular pathologies, including dry eye disease and keratoconus. In this review, we discuss key studies that have revealed a role for androgens and estrogens in the cornea with focus on ocular surface homeostasis, wound healing, and stromal thickness. We also review studies of human growth hormone and insulin growth factor-1 in influencing ocular growth and epithelial regeneration. While it is unclear if endogenous hormones contribute to differential corneal wound healing in common animal models, the abundance of evidence suggests that systemic hormone levels, as a function of age, should be considered as an experimental variable in studies of corneal health and disease.

Arginine Supplementation Promotes Extracellular Matrix and Metabolic Changes in Keratoconus.

Keratoconus (KC) is a common corneal ectatic disease that affects 1:500-1:2000 people worldwide and is associated with a progressive thinning of the corneal stroma that may lead to severe astigmatism and visual deficits. Riboflavin-mediated collagen crosslinking currently remains the only approved treatment to halt progressive corneal thinning associated with KC by improving the biomechanical properties of the stroma. Treatments designed to increase collagen deposition by resident corneal stromal keratocytes remain elusive. In this study, we evaluated the effects of arginine supplementation on steady-state levels of arginine and arginine-related metabolites (e.g., ornithine, proline, hydroxyproline, spermidine, and putrescine) and collagen protein expression by primary human corneal fibroblasts isolated from KC and non-KC (healthy) corneas and cultured in an established 3D in vitro model. We identified lower cytoplasmic arginine and spermidine levels in KC-derived constructs compared to healthy controls, which corresponded with overall higher gene expression of arginase. Arginine supplementation led to a robust increase in cytoplasmic arginine, ornithine, and spermidine levels in controls only and a significant increase in collagen type I secretion in KC-derived constructs. Further studies evaluating safety and efficacy of arginine supplementation are required to elucidate the potential therapeutic applications of modulating collagen deposition in the context of KC.

Mechanisms of Collagen Crosslinking in Diabetes and Keratoconus.

Collagen crosslinking provides the mechanical strength required for physiological maintenance of the extracellular matrix in most tissues in the human body, including the cornea. Aging and diabetes mellitus (DM) are processes that are both associated with increased collagen crosslinking that leads to increased corneal rigidity. By contrast, keratoconus (KC) is a corneal thinning disease associated with decreased mechanical stiffness leading to ectasia of the central cornea. Studies have suggested that crosslinking mediated by reactive advanced glycation end products during DM may protect the cornea from KC development. Parallel to this hypothesis, riboflavin-mediated photoreactive corneal crosslinking has been proposed as a therapeutic option to halt the progression of corneal thinning by inducing intra- and intermolecular crosslink formation within the collagen fibrils of the stroma, leading to stabilization of the disease. Here, we review the pathobiology of DM and KC in the context of corneal structure, the epidemiology behind the inverse correlation of DM and KC development, and the chemical mechanisms of lysyl oxidase-mediated crosslinking, advanced glycation end product-mediated crosslinking, and photoreactive riboflavin-mediated corneal crosslinking. The goal of this review is to define the biological and chemical pathways important in physiological and pathological processes related to collagen crosslinking in DM and KC.

Association between Diabetes and Keratoconus: A Retrospective Analysis.

Keratoconus (KC) and chronic diabetes mellitus (DM) are both associated with significant defects in the human corneal structure. Studies have long suggested that DM is linked to KC, mainly via the crosslinking mechanism, but scientific evidences are lacking. The role of altered systemic metabolism is well-established in both DM and KC with studies suggesting localized altered cellular metabolism leading to the development of corneal pathologies. We have previously characterized the metabolic defects associated with both conditions using targeted metabolomics. To compare metabolic differences between KC and DM-derived corneal fibroblasts, we performed a respective study of two cohorts of the KC and DM populations using a retrospective analysis of targeted metabolomics data. The goal of this study was to identify the group of differentially regulated metabolites, in KC versus DM, so that we may unravel the link between the two devastating corneal pathologies.

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea.

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide suffering from corneal vision loss1. To address the demand for viable human corneas, significant progress in three-dimensional (3D) tissue engineering has been made2,3,4. These cornea models range from simple monolayer systems to multilayered models, leading to 3D full-thickness corneal equivalents2. However, the use of a 3D tissue-engineered cornea in the context of in vitro disease models studied to date lacks resemblance to the multilayered 3D corneal tissue structure, function, and the networking of different cell types (i.e., nerve, epithelium, stroma, and endothelium)2,3. In addition, the demand for in vitro cornea tissue models has increased in an attempt to reduce animal testing for pharmaceutical products. Thus, more sophisticated models are required to better match systems to human physiological requirements, and the development of a model that is more relevant to the patient population is absolutely necessary. Given that multiple cell types in the cornea are affected by diseases and dystrophies, such as Keratoconus, Diabetic Keratopathy, and Fuchs, this model includes a 3D co-culture model of primary human corneal fibroblasts (HCFs) from healthy donors and neurons from the SH-SY5Y cell line. This allows us for the first time to investigate the interactions between the two cell types within the human corneal tissue. We believe that this model could potentially dissect the underlying mechanisms associated with the stromal-nerve interactions of corneal diseases that exhibit nerve damages. This 3D model mirrors the basic anatomical and physiological nature of the corneal tissue in vivo and can be used in the future as a tool for investigating corneal defects as well as screening the efficacy of various agents before animal testing.

3D Stacked Construct: A Novel Substitute for Corneal Tissue Engineering.

Corneal trauma/injury often results in serious complications including permanent vision loss or loss of visual acuity which demands corneal transplantations or treatment with allogenic graft tissues. There is currently a huge shortage of donor tissue worldwide and the need for human corneal equivalents increases annually. In order to meet such demand the current clinical approach of treating corneal injuries is limited and involves synthetic and allogenic materials which have various shortcomings when it comes to actual transplantations. In this study we introduce the newly developed, next generation of our previously established 3D self-assembled constructs, where multiple constructs are grown and stacked on top of each other without any other artificial product. This new technology brings our 3D in vitro model closer to what is seen in vivo and provides a solid foundation for future studies on corneal biology.Lipids are known for playing a vital role during metabolism and diseased state of various tissues and Sphingolipids are one such class of lipids which are involved in various cellular mechanisms and signaling processes. The impacts of Sphingolipids that have been documented in several human diseases often involve inflammation, neovascularization, tumorigenesis, and diabetes, but these conditions are not yet thoroughly studied. There is very little information about the exact role of Sphingolipids in the human cornea and future studies aiming at dissecting the mechanisms and pathways involved in order to develop novel therapies. We believe that our novel 3D stacked model can be used to delineate the role of Sphingolipids in the human cornea and provide new insights for understanding and treating various human corneal diseases.

Unravelling the interplay of sphingolipids and TGF-ß signaling in the human corneal stroma.

PURPOSE: To delineate the role of Sphingolipids (SPLs) in the human cornea and their cross-talks with transforming growth factor beta (TGF-ß) in order to develop novel, non-invasive therapies. METHODS: Human corneal fibroblasts (HCFs) were harvested from healthy donors, stimulated with Vitamin C to promote extracellular matrix assembly, treated with exogenous sphingosine-1-phosphate (S1P) or sphingosine kinase inhibitor 2 (SPHK I2) and isolated after 4 weeks for further analysis. RESULTS: Data showed that S1P led to a significant decrease in cellular migration where SPHK I2 just delayed it for 24h. Significant modulation of the sphingolipid pathway was also noted. Sphingosine kinase-1 (SphK1) was significantly downregulated upon exogenous stimulation with S1P at a concentration of 5µM and Sphingosine kinase-2 (SphK2) was also significantly downregulated at concentrations of 0.01µM, 0.1µM, and 5µM; whereas no effects were observed upon stimulation with SPHK I2. S1PR3 was significantly downregulated by 0.1µM and 5µM S1P and upregulated by 5µM and 10µM SPHK I2. Furthermore, both S1P and SPHK I2 regulated corneal fibrosis markers such as alpha-smooth muscle actin, collagen I, III, and V. We also investigated the interplay between two TGF-ß isoforms and S1P/SPHK I2 treatments and found that TGF-ß1 and TGF-ß3 were both significantly upregulated with the 0.1µM S1P but were significantly downregulated with the 5µM S1P concentration. When TGF-ß1 was compared directly to TGF-ß3 expression, we observed that TGF-ß3 was significantly downregulated compared to TGF-ß1 in the 5µM concentration of S1P. No changes were observed upon SPHK I2 treatment. CONCLUSION: Our study delineates the role of sphingolipids in the human cornea and highlights their different activities based on the cell/tissue type.

Unravelling the stromal-nerve interactions in the human diabetic cornea.

Corneal defects due to diabetes mellitus (DM) may cause severe vision impairments. Current studies focus on the corneal epithelium and nerve defects neglecting the corneal stroma. The aim of this study was to develop a 3D in vitro model to examine the interactions between corneal stroma and nerves in the context of DM. Primary human corneal stromal fibroblasts isolated from healthy (HCFs), Type 1 (T1DM) and Type 2 (T2DM) patients were stimulated with stable ascorbic acid to secrete and assemble an extracellular matrix (ECM). Human neuronal cells were then seeded on top and differentiated to create the 3D co-cultures. Our data revealed successful co-culture of stromal fibroblasts and neuronal cells with large elongated neuron extensions. T2DM showed significant upregulation of Collagen III and IGF1 when compared to T1DM. Interestingly, upon nerve addition, those markers returned to HCF levels. Neuronal markers were also differentially modulated with T2DM co-cultures expressing high levels of ßIII tubulin where T1DM co-cultures expressed Substance P. . Overall, our unique 3D co-culture model provides us with a tool that can be utilized for both molecular and therapeutic studies for diabetic keratopathy.

Acute hypoxia influences collagen and matrix metalloproteinase expression by human keratoconus cells in vitro.

Keratoconus (KC) is a progressive corneal ectasia linked to thinning of the central cornea. Hard contact lenses, rigid gas permeable lenses, and scleral lenses are the primary treatment modalities for early to mid- stages of KC to correct refractive error and astigmatism that develops as a result of an irregular corneal structure. These treatments are associated with significant drawbacks, including reduced availability of the tear film and oxygen to the corneal epithelium and stroma. However, it remains unknown whether hypoxia affects corneal integrity in the KC pathobiology. A number of studies have associated elevated oxidative stress with KC both in vitro and ex vivo. We hypothesized that KC-derived corneal fibroblasts are more susceptible to hypoxia-induced oxidative stress compared to healthy controls leading to exacerbation of corneal thinning in KC. This study investigated the effects of hypoxia on ECM secretion, assembly, and matrix metalloproteinase (MMP) expression in human corneal fibroblasts from healthy controls (HCFs) and KC patients (HKCs) in vitro. HCFs and HKCs were cultured in 3D constructs for 3 weeks and maintained or transferred to normoxic (21% O2) or hypoxic (2% O2) conditions, respectively, for 1 additional week. At the 4 week time-point, constructs were isolated and probed for Collagen I, III, and V, keratocan and MMP-1, -2, -3, -9, and -13, as well as hypoxia markers, hypoxia inducible factor-1α and lactoferrin. Conditioned media was also collected and probed for Collagen I, III, and V by Western blot. Thickness of the ECM assembled by HCFs and HKCs was measured using immunofluorescence microscopy. Results showed that hypoxia significantly reduced Collagen I secretion in HKCs, as well as upregulated the expression of MMP-1 and -2 with no significant effects on MMP-3, -9, or -13. ECM thickness was reduced in both cell types following 1 week in a low oxygen environment. Our study shows that hypoxia influences collagen and MMP expression by HKCs, which may have consequential effects on ECM structure in the context of KC.

Analysis of sphingolipids in human corneal fibroblasts from normal and keratoconus patients.

The pathophysiology of human keratoconus (KC), a bilateral progressive corneal disease leading to protrusion of the cornea, stromal thinning, and scarring, is not well-understood. In this study, we investigated a novel sphingolipid (SPL) signaling pathway through which KC may be regulated. Using human corneal fibroblasts (HCFs) and human KC cells (HKCs), we examined the SPL pathway modulation. Both cell types were stimulated by the three transforming growth factor (TGF)-ß isoforms: TGF-ß1 (T1), TGF-ß2 (T2), and TGF-ß3 (T3). All samples were analyzed using lipidomics and real-time PCR. Our data showed that HKCs have increased levels of signaling SPLs, ceramide (Cer), and sphingosine 1-phosphate (S1P). Treatment with T1 reversed the increase in Cer in HKCs and treatment with T3 reversed the increase in S1P. S1P3 receptor mRNA levels were also significantly upregulated in HKCs, but were reduced to normal levels following T3 treatment. Furthermore, stimulation with Cer and S1P led to significant upregulation of fibrotic markers in HCFs, but not in HKCs. Additionally, stimulation with a Cer synthesis inhibitor (FTY720) led to significant downregulation of specific fibrotic markers in HKCs (TGF-ß1, collagen type III, and α smooth muscle actin) without an effect on healthy HCFs, suggesting a causative role of Cer and S1P in fibrogenesis. Overall, this study suggests an association of the SPL signaling pathway in KC disease and its relation with the TGF-ß pathway.

Establishment of a 3D In Vitro Model to Accelerate the Development of Human Therapies against Corneal Diabetes.

PURPOSE: To establish an in vitro model that would mirror the in vivo corneal stromal environment in diabetes (DM) patients. METHODS: Human corneal fibroblasts from Healthy (HCFs), Type 1DM (T1DM) and Type 2DM (T2DM) donors were isolated and cultured for 4 weeks with Vitamin C stimulation in order to allow for extracellular matrix (ECM) secretion and assembly. RESULTS: Our data indicated altered cellular morphology, increased cellular migration, increased ECM assembly, and severe mitochondrial damage in both T1DM and T2DMs when compared to HCFs. Furthermore, we found significant downregulation of Collagen I and Collagen V expression in both T1DM and T2DMs. Furthermore, a significant up regulation of fibrotic markers was seen, including α-smooth muscle actin in T2DM and Collagen III in both T1DM and T2DMs. Metabolic analysis suggested impaired Glycolysis and Tricarboxylic acid cycle (TCA) pathway. CONCLUSION: DM has significant effects on physiological and clinical aspects of the human cornea. The benefits in developing and fully characterizing our 3D in vitro model are enormous and might provide clues for the development of novel therapeutics.

Complete metabolome and lipidome analysis reveals novel biomarkers in the human diabetic corneal stroma.

Prolonged hyperglycemia during diabetes mellitus can cause severe ophthalmic complications affecting both the anterior and posterior ocular segments leading to impaired vision or blindness. Diabetes-induced corneal pathologies are associated with decreased wound healing capacity, corneal edema, and altered epithelial basement membrane. The mechanism by which diabetes modulates structure and function within the corneal stroma are unknown. In our study, we characterized the effects of diabetes on extracellular matrix, lipid transport, and cellular metabolism by defining the entire metabolome and lipidome of Type 1 and Type 2 human diabetic corneal stroma. Significant increases in Collagen I and III were found in diabetic corneas suggesting that diabetes promotes defects in matrix structure leading to scarring. Furthermore, increased lipid content, including sphingosine-1-phosphate and dihydrosphingosine, in diabetic corneas compared to healthy controls were measured suggesting altered lipid retention. Metabolomics analysis identified elevated tryptophan metabolites, independent of glucose metabolism, which correlated with upregulation of the Kynurenine pathway in diabetic corneas. We also found significant upregulation of novel biomarkers aminoadipic acid, D,L-pipecolic acid, and dihydroorotate. Our study links aberrant tryptophan metabolism to end-stage pathologies associated with diabetes indicating the potential of the Kynurenine pathway as a therapeutic target for inhibiting diabetes-associated defects in the eye.

Human Keratoconus Cell Contractility is Mediated by Transforming Growth Factor-Beta Isoforms.

Keratoconus (KC) is a progressive disease linked to defects in the structural components of the corneal stroma. The extracellular matrix (ECM) is secreted and assembled by corneal keratocytes and regulated by transforming growth factor-ß (TGF-ß). We have previously identified alterations in the TGF-ß pathway in human keratoconus cells (HKCs) compared to normal corneal fibroblasts (HCFs). In our current study, we seeded HKCs and HCFs in 3D-collagen gels to identify variations in contractility, and expression of matrix metalloproteases (MMPs) by HKCs in response the TGF-ß isoforms. HKCs showed delayed contractility with decreased Collagen I:Collagen V ratios. TGF-ß1 significantly increased ECM contraction, Collagen I, and Collagen V expression by HKCs. We also found that HKCs have significantly decreased Collagen I:Collagen III ratios suggesting a potential link to altered collagen isoform expression in KC. Our findings show that HKCs have significant variations in collagen secretion in a 3D collagen gel and have delayed contraction of the matrix compared to HCFs. For the first time, we utilize a collagen gel model to characterize the contractility and MMP expression by HKCs that may contribute to the pathobiology of KC.

Keratoconus in vitro and the key players of the TGF-ß pathway.

PURPOSE: Keratoconus (KC) is a corneal thinning disease of unknown etiology whose pathophysiology is correlated with the presence of a thin corneal stroma and altered extracellular matrix (ECM). Transforming growth factor-ß (TGF-ß) signaling is a key regulator of ECM secretion and assembly in multiple tissues, including the anterior segment of the eye, and it has been linked to KC. We have previously shown that human keratoconus cells (HKCs) have a myofibroblast phenotype and altered ECM assembly compared to normal human corneal fibroblasts (HCFs). Moreover, TGF-ß3 treatment promotes assembly of a more normal stromal ECM and modulates the fibrotic phenotype in HKCs. Herein, we identify alterations in TGF-ß signaling that contribute to the observed fibrotic phenotype in HKCs. METHODS: HCFs and HKCs were stimulated with TGF-ß1, TGF-ß2, or TGF-ß3 isoforms (0.1 ng/mL) in the presence of a stable vitamin C derivative (0.5 mM) for 4 weeks. All samples were examined using RT-PCR and western blotting to quantify changes in the expressions of key TGF-ß signaling molecules between HCFs and HKCs. RESULTS: We found a significant downregulation in the SMAD6 and SMAD7 expressions by HKCs when compared to HCFs (p≤0.05). Moreover, stimulation of HKCs with any of the three TGF-ß isoforms did not significantly alter the expressions of SMAD6 or SMAD7. HCFs also showed an upregulation in TGF-ßRI, TGF-ßRII, and TGF-ßRIII following TGF-ß3 treatment, whereas HKCs showed a significant two-fold downregulation. CONCLUSIONS: Overall, our data shows the decreased expressions of the regulatory SMADs SMAD6 and SMAD7 by HKCs contribute to the pathological ECM structure observed in KC, and TGF-ß3 may attenuate this mechanism by downregulating the expression of the key profibrotic receptor, TGF-ßRII. Our study suggests a significant role of altered regulation of TGF-ß signaling in KC progression and that it may enable novel therapeutic developments targeting TGF-ß receptor regulation.

Description of the sphingolipid content and subspecies in the diabetic cornea.

PURPOSE: Diabetes mellitus (DM) is characterized by high blood sugar levels over a prolonged period. Long term complications include but not limited heart disease, stroke, kidney failure, and ocular damage. An estimated 382 million people are diagnosed with Type 2 DM accounting for 90% of the cases. Common corneal dysfunctions associated with DM result in impaired vision due to decreased wound healing, corneal edema, and altered epithelial basement membrane. Lipids play a fundamental role in tissue metabolism and disease states. We attempt to determine the role of sphingolipids (SPL) in human Type I and Type II diabetic corneas. MATERIALS AND METHODS: Cadaver corneas from healthy (non-diabetic/no ocular trauma), Type I (T1DM), and Type II diabetic (T2DM) donors were obtained and processed for lipidomics using LC-MS/MS. RESULTS: Our data show significant differences in the SPL composition between control, T1DM and T2DM corneas. Both T1DM and T2DM showed a 10-folddownregulation of sphingomyelin(SM), 5-fold up regulation of Ceramides (Cer) and 2-fold upregulation of monohexosylceramides (MHC). Differences were also seen in total amounts of SPL where Cer was increased by approximately 3 fold in both T1DM and T2DM where SM decreased by 50% in both T1DM and T2DM when compared to healthy controls. No differences were seen in MHC amounts. CONCLUSIONS: Overall, our data indicate major differences in SPL distribution in human diabetic corneas. Information on the sphingolipids role in cornea, corneal cell physiology, and diseases are very limitedwhich highlights the importance of these findings.

Gross cystic disease fluid protein-15/prolactin-inducible protein as a biomarker for keratoconus disease.

Keratoconus (KC) is a bilateral degenerative disease of the cornea characterized by corneal bulging, stromal thinning, and scarring. The etiology of the disease is unknown. In this study, we identified a new biomarker for KC that is present in vivo and in vitro. In vivo, tear samples were collected from age-matched controls with no eye disease (n = 36) and KC diagnosed subjects (n = 17). Samples were processed for proteomics using LC-MS/MS. In vitro, cells were isolated from controls (Human Corneal Fibroblasts-HCF) and KC subjects (Human Keratoconus Cells-HKC) and stimulated with a Vitamin C (VitC) derivative for 4 weeks, and with one of the three transforming growth factor-beta (TGF-ß) isoforms. Samples were analyzed using real-time PCR and Western Blots. By using proteomics analysis, the Gross cystic disease fluid protein-15 (GCDFP-15) or prolactin-inducible protein (PIP) was found to be the best independent biomarker able to discriminate between KC and controls. The intensity of GCDFP-15/PIP was significantly higher in healthy subjects compared to KC-diagnosed. Similar findings were seen in vitro, using a 3D culture model. All three TGF-ß isoforms significantly down-regulated the expression of GCDFP-15/PIP. Zinc-alpha-2-glycoprotein (AZGP1), a protein that binds to PIP, was identified by proteomics and cell culture to be highly regulated. In this study by different complementary techniques we confirmed the potential role of GCDFP-15/PIP as a novel biomarker for KC disease. It is likely that exploring the GCDFP-15/PIP-AZGP1 interactions will help better understand the mechanism of KC disease.