Cellular Contractility Profiles of Human Diabetic Corneal Stromal Cells

Abstract

Diabetic keratopathy is a corneal complication of diabetes mellitus (DM). Patients with diabetic keratopathy are prone to developing corneal haze, scarring, recurrent erosions, and significant wound healing defects/delays. The purpose of this study was to determine the contractility profiles in the diabetic human corneal stromal cells and characterize their molecular signatures. Primary human corneal fibroblasts from healthy, Type 1 DM (T1DM), and Type 2 DM (T2DM) donors were cultured using an established 3D collagen gel model. We tracked, measured, and quantified the contractile footprint over 9 days and quantified the modulation of specific corneal/diabetes markers in the conditional media and cell lysates using western blot analysis. Human corneal fibroblasts (HCFs) exhibited delayed and decreased contractility compared to that from T1DMs and T2DMs. Compared to HCFs, T2DMs demonstrated an initial downregulation of collagen I (day 3), followed by a significant upregulation by day 9. Collagen V was significantly upregulated in both T1DMs and T2DMs based on basal secretion, when compared to HCFs. Cell lysates were upregulated in the myofibroblast-associated marker, α-smooth muscle actin, in T2DMs on day 9, corresponding to the significant increase in contractility rate observed at the same time point. Furthermore, our data demonstrated a significant upregulation in IGF-1 expression in T2DMs, when compared to HCFs and T1DMs, at day 9. T1DMs demonstrated significant downregulation of IGF-1 expression, when compared to HCFs. Overall, both T1DMs and T2DMs exhibited increased contractility associated with fibrotic phenotypes. These findings, and future studies, may contribute to better understanding of the pathobiology of diabetic keratopathy and ultimately the development of new therapeutic approaches.

Figure 1

Quantification of contraction collagen matrix in HCFs, T1DMs, and T2DMs from days 0 to 9. A significant reduction is observed in the area of the collagen matrix between the three cell types correlating with increased contractility. Two-way ANOVA (n = 12) was used to analyze the results. Error bars represent standard deviation (∗∗∗∗ denotes p < 0.0001).

Figure 2

(a) Collagen I (Col I), (b) collagen III (Col III), (c) collagen V (Col V), and (d) α-SMA (SMA) protein levels in HCF, T1DM, and T2DM on days 3 and 9. Col I was significantly increased on day 9 in the T1DM and T2DM compared to the HCFs and between days 3 and 9. Significant increases in Col V and SMA were seen at day 9 for the T2DM when compared to HCFs at day 9. Welch's unpaired t-test and/or ordinary one-way ANOVA (n = 4) was used to analyze the results. Error bars represent standard deviation (∗∗ denotes p < 0.01 and ∗ denotes p < 0.05).

Figure 3

Lysate ratios of (a) Col I/Col III and (b) Col I/Col V. Welch's unpaired t-test and/or ordinary one-way ANOVA (n = 4) was used to analyze the results. Error bars represent standard deviation.

Figure 4

(a) Collagen I (Col I), (b) collagen III (Col III), and (c) collagen V (Col V) protein levels in conditioned media of HCF, T1DM, and T2DM on days 3 and 9. Col I expression significantly decreased on day 3 but not at day 9 compared to the HCFs. T1DM and T2DM show significant increases in Col V expression, followed by significant decreases of secretion at day 9. Welch's unpaired t-test and/or ordinary one-way ANOVA (n = 4) was used to analyze the results. Error bars represent standard deviation (∗∗∗∗ denotes p < 0.0001, ∗∗∗ denotes p < 0.001, ∗∗ denotes p < 0.01, and ∗ denotes p < 0.05).

Figure 5

Collagen secretion ratio of (a) Col I/Col III and (b) Col I/Col V. T1DMs had a significant difference between its day 3 and 9 Col I/Col III ratio. For the Col I/Col V ratio, both days of the T1DM and T2DM showed significant decreases of secretion, as well as the HCFs at day 9. Welch's unpaired t-test and/or ordinary one-way ANOVA (n = 4) was used to analyze the results. Error bars represent standard deviation (∗∗∗∗ denotes p < 0.0001 and ∗∗ denotes p < 0.01).

Figure 6

(a) Insulin-like growth factor 1 (IGF-1) and (b) insulin-like growth factor 1 receptor (IGF-1R) protein expression in HCF, T1DM, and T2DM on days 3 and 9. Welch's unpaired t-test (n = 4) was used to analyze the results. Error bars represent the standard deviation (∗∗∗∗ denotes p < 0.0001, ∗∗∗ denotes p < 0.001, and ∗∗ denotes p < 0.01).