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. 2022 Jan 12;9(1):29.
doi: 10.3390/bioengineering9010029.

Radiation-Induced Alterations in Proliferation, Migration, and Adhesion in Lens Epithelial Cells and Implications for Cataract Development

Graysen Vigneux  1 , Jake Pirkkanen  2   3 , Taylor Laframboise  2   3 , Hallie Prescott  2 , Sujeenthar Tharmalingam  1   2   3   4 , Christopher Thome  1   2   3   4
Affiliations

Radiation-Induced Alterations in Proliferation, Migration, and Adhesion in Lens Epithelial Cells and Implications for Cataract Development

Graysen Vigneux et al. Bioengineering (Basel). .

Abstract

The lens of the eye is one of the most radiosensitive tissues. Although the exact mechanism of radiation-induced cataract development remains unknown, altered proliferation, migration, and adhesion have been proposed as factors. Lens epithelial cells were exposed to X-rays (0.1-2 Gy) and radiation effects were examined after 12 h and 7 day. Proliferation was quantified using an MTT assay, migration was measured using a Boyden chamber and wound-healing assay, and adhesion was assessed on three extracellular matrices. Transcriptional changes were also examined using RT-qPCR for a panel of genes related to these processes. In general, a nonlinear radiation response was observed, with the greatest effects occurring at a dose of 0.25 Gy. At this dose, a reduction in proliferation occurred 12 h post irradiation (82.06 ± 2.66%), followed by an increase at 7 day (116.16 ± 3.64%). Cell migration was increased at 0.25 Gy, with rates 121.66 ± 6.49% and 232.78 ± 22.22% greater than controls at 12 h and 7 day respectively. Cell adhesion was consistently reduced above doses of 0.25 Gy. Transcriptional alterations were identified at these same doses in multiple genes related to proliferation, migration, and adhesion. Overall, this research began to elucidate the functional changes that occur in lens cells following radiation exposure, thereby providing a better mechanistic understanding of radiation-induced cataract development.

Keywords: adhesion; cataract; ionizing radiation; lens epithelial cell; migration; proliferation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Cell proliferation following ionizing radiation exposure in HLE-B3 cells. Proliferation was quantified using the MTT assay at 12 h (A) and 7 days (B) post exposure. Data are presented as a percentage of unirradiated control cell proliferation. Bars represent the average of three independent experimental replicates ± SEM. Data were analyzed using a one-way ANOVA followed by Dunnett’s multiple comparisons test (* p < 0.05, ** p < 0.01).
Figure 2
Figure 2
Cell migration following ionizing radiation exposure in HLE-B3 cells. Migration was quantified using the Boyden chamber assay at 12 h (A) and 7 days (B) post exposure. Data are presented as a percentage of unirradiated control cell migration. Bars represent the average of three independent experimental replicates ± SEM. Data were analyzed using a one-way ANOVA followed by Dunnett’s multiple comparisons test (** p < 0.01).
Figure 3
Figure 3
Cell migration following ionizing radiation exposure in HLE-B3 cells. Migration was quantified using the wound-healing assay at 12 h (A,B) and 7 days (C,D) post exposure. The percent of wound closure was measured every 12 h up to 48 h post scratch (A,C). The average rate of wound closure across the first 24 h was calculated and was plotted as a percentage of unirradiated controls (B,D). Data points represent the average of three independent experimental replicates ± SEM. Data were analyzed using a one-way ANOVA followed by Dunnett’s multiple comparisons test (*** p < 0.001). Note: data points overlap one another at 36 and 48 h once wounds reach full closure (A,C).
Figure 4
Figure 4
Cell adhesion following ionizing radiation exposure in HLE-B3 cells. Adhesion was quantified on three different extracellular matrices (fibronectin, laminin and collagen IV) at 12 h (A–C) and 7 days (D–F) post exposure. Data are presented as a percentage of unirradiated control cell adhesion. Bars represent the average of three independent experimental replicates ± SEM. Data were analyzed using a one-way ANOVA followed by Dunnett’s multiple comparisons test (* p < 0.05, ** p < 0.01, *** p < 0.001).
Figure 5
Figure 5
Relative transcript expression of genes related to cell proliferation in HLE-B3 cells following ionizing radiation exposure. Grey squares represent 12 h post irradiation data, while 7 day post irradiation data are represented by black circles. Data are presented as a relative expression compared to unirradiated control cells. Data points represent the average of three independent experimental replicates ± SEM. Data were analyzed using a one-way ANOVA followed by Dunnett’s multiple comparisons test (* p < 0.05, ** p < 0.01). Abbreviations: (A) Fibroblast Growth Factor 2 (FGF2); (B) Platelet Derived Growth Factor D (PDGFD); (C) Mitogen-Activated Protein Kinase 1 (MAPK1); (D) Insulin-Like Growth Factor 1 (IGF1); (E) Transforming Growth Factor Beta 2 (TGFB2); (F) Epidermal Growth Factor (EGF).
Figure 6
Figure 6
Relative transcript expression of genes related to cell migration in HLE-B3 cells following ionizing radiation exposure. Grey squares represent 12 h post irradiation data, while 7 day post irradiation data are represented by black circles. Data are presented as a relative expression compared to unirradiated control cells. Data points represent the average of three independent experimental replicates ± SE. Data were analyzed using a one-way ANOVA followed by Dunnett’s multiple comparisons test (* p < 0.05, ** p < 0.01). Abbreviations: (A) Matrix Metallopeptidase 9 (MMP9); and (B) Protein Tyrosine Kinase 2 (PTK2).
Figure 7
Figure 7
Relative transcript expression of genes related to cell adhesion in HLE-B3 cells following ionizing radiation exposure. Grey squares represent 12 h post irradiation data, while 7 day post irradiation data are represented by black circles. Data are presented as a relative expression compared to unirradiated control cells. Data points represent the average of three independent experimental replicates ± SE. Data were analyzed using a one-way ANOVA followed by Dunnett’s multiple comparisons test (* p < 0.05, ** p < 0.01, *** p < 0.001). Abbreviations: (A) Integrin Subunit Alpha 5 (ITGA5); (B) Intercellular Adhesion Molecule 1 (ICAM1); (C) Cadherin 2 (CDH2); (D) Integrin Subunit Beta 1 (ITGB1).
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