Expression of Otx Genes in Müller Cells Using an In Vitro Experimental Model of Retinal Hypoxia

Abstract

Introduction: Müller glial cells typically activate to react to hypoxic tissue damage in several retinal diseases. We evaluated the in vitro response to a hypoxia-mimicking stimulus on the expression of a set of genes, known to contribute to eye morphogenesis and cell differentiation.

Materials and methods: A MIO-M1 Müller cell line was cultured in a hypoxia-mimicking environment by the addition of cobalt chloride to the culture medium, followed by a recovery time in which we mimic restoration from the hypoxic insult. The HIF-1α protein and VEGF-A gene expression were quantified to verify the induction of a hypoxia-like state.

Results: Among the genes under study, we did not observe any difference in the expression levels of Otx1 and Otx2 during treatment; conversely, Otx1 was overexpressed during recovery steps. The VEGF-A gene was strongly upregulated at both the CoCl₂ and recovery time points. The transactivated isoform (TA) of the TP73 gene showed an overexpression in long-term exposure to the hypoxic stimulus with a further increase after recovery. Discussion. Our molecular analysis is able to describe the activation of a set of genes, never before described, that can drive the response to a hypoxia-like status. The improved comprehension of these cellular events will be useful for designing new therapeutical approaches for retinal pathologies.

Figure 1

Schematic representation of two CoCl₂ treatments. (1) Exposure of MIO-M1 cells to 100 µM CoCl₂ for 48 h followed by 24 h of recovery in a complete medium (DMEM + 10% FBS + 1% L-Glut + 1% Pen/Strep). (2) 24 h exposure to 100 µM CoCl₂ followed by 24 h of recovery. CTR: control sample. White arrow: medium added with CoCl₂. Black arrow: medium without CoCl₂. Black vertical lines indicate timepoints of cell collection.

Figure 2

Morphological analysis of MIO-M1 Müller cells in different experimental conditions. (a) CTR 24 h; (b) 24 h CoCl2; (c) 24 h CoCl₂ + 24 h REC; (d) CTR 48 h; (e) 48 h CoCl2; and (f) 48 h CoCl2 + 24 h REC. Black bar: 100 μM.

Figure 3

Analysis of HIF-1α and β-actin protein expression levels by western blot evaluated in MIO-M1 cells at different time points of treatment. (a) Representative immunoreactive bands for HIF-1α (top; 120 kDa) and β-actin (bottom; 43 kDa). For each sample, 200 μg of protein was loaded on SDS-8% polyacrylamide gel. (b) Relative expression of HIF-1α protein, normalized to the respective β-actin, is represented as mean ± SD of three repeated experiments. ^∗∗∗p < 0.001 (24 h CoCl₂ vs. 24 h CTR); ^∗∗∗∗p < 0.0001 (48 h CoCl₂ vs. 24 h CTR); and p < 0.0001 (48 h CoCl₂ vs. 48 h CTR) by one-way ANOVA and Tukey's post hoc test.

Figure 4

Molecular characterization of gene expression in untreated MIO-M1 cells. Each gene was normalized to β-actin expression level, to obtain a value representing the fold change between the two analyzed genes. On the Y-axis, the fold change value is reported. Each value is indicated as median ± SD.

Figure 5

qRT-PCR on CTR- and CoCl₂-treated MIO-M1 Müller cells. Each graph represents the expression levels of a single studied gene in different experimental conditions. All values are indicated as median ± SD. On the Y-axis, the fold change value is reported. Statistical analysis was performed by one-way ANOVA and Tukey's post hoc test. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; and ^∗∗∗∗p < 0.0001.