Corneal Tissue Engineering: An <em>In Vitro</em> 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 ...

University of Oklahoma Health Sciences Center

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.

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

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-β (TGF-β) 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-β3 treatment promotes assembly of a more normal stromal ECM and modulates the fibrotic phenotype in HKCs. Herein, we identify alterations in TGF-β signaling that contribute to the observed fibrotic phenotype in HKCs.

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

To determine the utility of human organotypic cornea cultures as a model to study herpes simplex virus type 1 (HSV-1)-induced inflammation and neovascularization.

The use of human cornea organotypic cultures to study herpes simplex virus type 1 (HSV-1)-induced inflammation.

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-β (TGF-β). We have previously identified alterations in the TGF-β 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-β isoforms. HKCs showed delayed contractility with decreased Collagen I:Collagen V ratios. TGF-β1 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.

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

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.

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

To establish an in vitro model that would mirror the in vivo corneal stromal environment in diabetes (DM) patients.

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

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)-β isoforms: TGF-β1 (T1), TGF-β2 (T2), and TGF-β3 (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-β1, 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-β pathway.

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

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.

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

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.

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

To delineate the role of Sphingolipids (SPLs) in the human cornea and their cross-talks with transforming growth factor beta (TGF-β) in order to develop novel, non-invasive therapies.

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

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 βIII 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.

Shrestha Priyadarsini

Department of Ophthalmology/Dean McGee Eye Institute

University of Oklahoma Health Sciences Center

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

Shrestha Priyadarsini

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Ophthalmology/Dean McGee Eye Institute

University of Oklahoma Health Sciences Center

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

Department of Ophthalmology/Dean McGee Eye Institute