ABSTRACT
Corneal scarring following moderate to severe injury is inevitable. Despite significant advancements in the field, current treatments following these types of injuries are limited, and often, the visual recovery is poor. One of the problems and limitations is that corneal wound healing is a complex process, involving corneal cells, extracellular matrix components and growth factors. Therefore, further understanding is required, along with new treatments and techniques to reduce or prevent corneal scarring following injury. Two isoforms of transforming growth factor-beta (TGF-ß), TGF-ß1 and -ß3 (T1 and T3, respectively), are associated with corneal wound healing. T1 has been shown to drive the corneal keratocytes to differentiate into myofibroblasts; whereas, T3 has been found to inhibit fibrotic markers. In the current study, we examined whether the fibrotic characteristics expressed by human corneal fibroblasts (HCF) in our 3-dimensional (3D) construct following T1 stimulation could be reversed by introducing T3 to the in vitro system. To do this, HCF were isolated and cultured in 10% serum, and when they reached confluence, the cells were stimulated with a stable Vitamin C (VitC) derivative for 4 weeks, which allowed them to secrete a self-assembled matrix. Three conditions were tested: (1) CONTROL: 10% serum (S) only, (2) T1: 10%S + T1, or (3) Rescue: 10%S + T1 for two weeks and then switched to 10%S + T3 for another two weeks. At the end of 4 weeks, the constructs were processed for analysis by indirect-immunofluorescence (IF) and transmission electron microscopy (TEM). Different collagens that are normally present in healthy corneas in vivo, such as Type I and V, as well as Type III, which is a fibrotic indicator, were examined. In addition, we examined smooth muscle actin (SMA), a marker of myofibroblasts, and thrombospondin-1 (TSP-1), a multifunctional matrix protein known to activate the latent complex of TGF-ß and appear upon wounding in vivo. Our data showed high expression of collagens type I and V under all conditions throughout the 3D constructs; however, type III and SMA expression were higher in the constructs that were stimulated with T1 and reduced to almost nothing in the Rescue samples. A similar pattern was seen with TSP-1, where TSP-1 expression following "rescue" was decreased considerably. Overall, this data is in agreement with our previous observations that T3 has a significant non-fibrotic effect on HCFs, and presents a novel model for the "rescue" of both cellular and matrix fibrotic components with a single growth factor.
Subject(s)
Corneal Diseases/pathology , Corneal Keratocytes/ultrastructure , Transforming Growth Factor beta3/metabolism , Actins/immunology , Actins/metabolism , Antibodies/analysis , Cells, Cultured , Corneal Diseases/immunology , Corneal Diseases/metabolism , Corneal Keratocytes/metabolism , Extracellular Matrix/metabolism , Extracellular Matrix/ultrastructure , Fibrosis/metabolism , Fibrosis/pathology , Fluorescent Antibody Technique, Indirect , Humans , Microscopy, Electron, TransmissionABSTRACT
Keratoconus (KC) affects 1:2000 people and is a disorder where cornea thins and assumes a conical shape. Advanced KC requires surgery to maintain vision. The role of oxidative stress in KC remains unclear. We aimed to identify oxidative stress levels between human corneal keratocytes (HCKs), fibroblasts (HCFs) and keratoconus cells (HKCs). Cells were cultured in 2D and 3D systems. Vitamin C (VitC) and TGF-ß3 (T3) were used for 4 weeks to stimulate self-assembled extracellular matrix (ECM). No T3 used as controls. Samples were analyzed using qRT-PCR and metabolomics. qRT-PCR data showed low levels of collagen I and V, as well as keratocan for HKCs, indicating differentiation to a myofibroblast phenotype. Collagen type III, a marker for fibrosis, was up regulated in HKCs. We robustly detected more than 150 metabolites of the targeted 250 by LC-MS/MS per condition and among those metabolites several were related to oxidative stress. Lactate levels, lactate/malate and lactate/pyruvate ratios were elevated in HKCs, while arginine and glutathione/oxidized glutathione ratio were reduced. Similar patterns found in both 2D and 3D. Our data shows that fibroblasts exhibit enhanced oxidative stress compared to keratocytes. Furthermore the HKC cells exhibit the greatest level suggesting they may have a myofibroblast phenotype.
Subject(s)
Corneal Keratocytes/pathology , Keratoconus/pathology , Oxidative Stress , Arginine/metabolism , Ascorbic Acid/pharmacology , Cell Differentiation , Cells, Cultured , Collagen Type I/biosynthesis , Collagen Type III/biosynthesis , Collagen Type IV/biosynthesis , Cornea/cytology , Cornea/pathology , Corneal Keratocytes/cytology , Extracellular Matrix , Fibroblasts/cytology , Glutathione/metabolism , Humans , Lactic Acid/metabolism , Malates/metabolism , Metabolomics , Myofibroblasts/cytology , Proteoglycans/biosynthesis , Pyruvic Acid/metabolism , Transforming Growth Factor beta3/pharmacologyABSTRACT
Corneal tissue engineering has attracted the attention of many researchers over the years, in part due to the cornea's avascularity and relatively straightforward structure. However, the highly organized and structured nature of this optically clear tissue has presented a great challenge. We have previously developed a model in which human corneal fibroblasts (HCFs) are stimulated by a stable vitamin C (VitC) derivative to self-assemble an extracellular matrix (ECM). Addition of TGFß1 enhanced the assembly of ECM; however, it was accompanied by the upregulation of specific fibrotic markers. In this study, we tested the effects of all three TGFß isoforms (-ß1, -ß2 and -ß3) on ECM production, as well as expression of fibrotic markers. HCFs were grown in four media conditions for 4 weeks: control, VitC only; T1, VitC + TGFß1; T2, VitC + TGFß2; and T3, VitC + TGFß3. The cultures were analysed with western blots, TEM and indirect immunofluorescence (IF). Compared to controls, all TGFß isoforms stimulated matrix production by about three-fold. IF showed the presence of type III collagen and smooth muscle actin (SMA) in T1 and T2; however, T3 showed little to no expression. In western blots, T3 stimulated a lower type III:type I collagen ratio when compared to the other conditions. In addition, TEM indicated that T3 stimulated a higher level of matrix alignment and organization. HCFs stimulated by VitC and TGFß3 appear to generate a matrix that mimics the normal adult or developing human cornea, whereas TGF-ß1 and -ß2 drive the constructs towards a more fibrotic path.
Subject(s)
Cornea/drug effects , Cornea/pathology , Extracellular Matrix/metabolism , Models, Biological , Transforming Growth Factor beta3/pharmacology , Actins/metabolism , Cell Count , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Collagen Type I/metabolism , Collagen Type III/metabolism , Extracellular Matrix/drug effects , Extracellular Matrix/ultrastructure , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibrosis , Fluorescent Antibody Technique , Humans , Protein Isoforms/pharmacologyABSTRACT
Our goal was to develop a 3-D multi-cellular construct using primary human corneal fibroblasts cultured on a disorganized collagen substrate in a scaffold-free environment and to use it to determine the regulation of proteoglycans over an extended period of time (11 weeks). Electron micrographs revealed multi-layered constructs with cells present in between alternating parallel and perpendicular arrays of fibrils. Type I collagen increased 2-4-fold. Stromal proteoglycans including lumican, syndecan4, decorin, biglycan, mimecan, and perlecan were expressed. The presence of glycosaminoglycan chains was demonstrated for a subset of the core proteins (lumican, biglycan, and decorin) using lyase digestion. Cuprolinic blue-stained cultures showed that sulfated proteoglycans were present throughout the construct and most prominent in its mid-region. The size of the Cuprolinic-positive filaments resembled those previously reported in a human corneal stroma. Under the current culture conditions, the cells mimic a development or nonfibrotic repair phenotype.