Transforming growth factor-β3 regulates assembly of a non-fibrotic matrix in a 3D corneal model

Abstract

Purpose Corneal tissue engineering has attracted the attention of many researchers over the years in part due to the cornea’s avascularity and relatively, straight forward structure. However, the highly organized and structured nature of this optically clear tissue has presented a great challenge. We have previously developed a model where human corneal fibroblasts (HCFs) are stimulated by a stable Vitamin C (VitC) derivative to self-assemble an extracellular matrix (ECM). Addition of TGF-β1 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 TGF-β isoforms (-β1, -β2 and -β3) on ECM production, as well as, expression of fibrotic markers. Methods HCFs were grown in four media conditions for 4 weeks: (Control) VitC only; (T1) VitC+TGF-β1; (T2) VitC+TGF-β2; or (T3) VitC+TGF-β3. Cultures were analyzed with Western Blots, TEM and indirect-immunofluorescence (IF). Results Compared to controls, all TGF-β isoforms stimulated matrix production by ~3 times. 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. Conclusions HCFs stimulated by VitC and TGF-β3 appear to generate a matrix that mimics the normal adult or developing human cornea; whereas, TGF-β1 and -β2 are driving the constructs toward a more fibrotic path.

Human Corneal Fibroblast Pattern Evolution and Matrix Synthesis on Mechanically Biased Substrates

“…At the end of the experimental period, the constructs were examined for the expression of intracellular and extracellular components, which are indicators of a normal or fibrotic corneal fibroblast phenotype. 25,43,44 We found that PHCFs on both substrates produced markers consistent with a normal, nonfibrotic culture system (Fig. 6).…”

Corneal Stromal Cell Plasticity: In Vitro Regulation of Cell Phenotype Through Cell-Cell Interactions in a Three-Dimensional Model

“…6,24 TGF-b1 induces fibroblastic and myofibroblastic differentiation by stimulating cell stratification and matrix component production, while increasing fibrotic protein marker expression 36 such as a-SMA, which was confirmed by immunohistochemical data. The action of TGF-b1 was very potent in that it blocked epithelial cell proliferation (data not shown) while stimulating the proliferation and re-activation of the stromal cells.…”

Bioengineering Organized, Multilamellar Human Corneal Stromal Tissue by Growth Factor Supplementation on Highly Aligned Synthetic Substrates

“…The TGF-b3 was most effective to induce the stromal matrix without expression of fibrotic markers. 16 These findings suggest an effective way to reproduce native corneal stromal tissue with growth factor manipulation. In this report, the effects of FGF-2 and TGF-b3, individually and together, are combined with a highly aligned fibrous substrate in an effort to generate corneal stromal tissue constructs that reflect the structural organization and composition of native tissue, and that are of dimensions that might be considered for further implementation in animal models.…”

“…36 The TGF-b3 isoform is responsible for scarless healing of fetal wounds in which the collagen organization is maintained, suggesting it has an in vivo biological role distinct from the other two isoforms. 37 Recently, Karamichos et al 16 systematically compared the influence of three isoforms of TGF-b on the expression of fibrotic markers and ECM production by human corneal fibroblasts in an in vitro model. Unlike TGF-b1 and TGF-b2, low concentrations of TGF-b3 (0.1 ng/mL) suppressed the expression of fibrotic markers type III collagen and a-SMA.…”

“…Zieske and collaborators have shown TGF-b to induce stromal tissue secretion by primary human corneal fibroblasts. [13][14][15][16] These constructs were up to 60 mm thick and multilayered. The TGF-b3 was most effective to induce the stromal matrix without expression of fibrotic markers.…”