αVβ8 integrin targeting to prevent posterior capsular opacification
Mahbubul H. Shihan, Samuel G. Novo, Yan Wang, Dean Sheppard, Amha Atakilit, Thomas D. Arnold, Nicole M. Rossi, Adam P. Faranda, Melinda K. Duncan
Mahbubul H. Shihan, Samuel G. Novo, Yan Wang, Dean Sheppard, Amha Atakilit, Thomas D. Arnold, Nicole M. Rossi, Adam P. Faranda, Melinda K. Duncan
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Research Article Cell biology Ophthalmology

αVβ8 integrin targeting to prevent posterior capsular opacification

Abstract

Fibrotic posterior capsular opacification (PCO), a major complication of cataract surgery, is driven by transforming growth factor–β (TGF-β). Previously, αV integrins were found to be critical for the onset of TGF-β–mediated PCO in vivo; however, the functional heterodimer was unknown. Here, β8 integrin–conditional knockout (β8ITG-cKO) lens epithelial cells (LCs) attenuated their fibrotic responses, while both β5 and β6 integrin–null LCs underwent fibrotic changes similar to WT at 5 days post cataract surgery (PCS). RNA-Seq revealed that β8ITG-cKO LCs attenuated their upregulation of integrins and their ligands, as well as known targets of TGF-β–induced signaling, at 24 hours PCS. Treatment of β8ITG-cKO eyes with active TGF-β1 at the time of surgery rescued the fibrotic response. Treatment of WT mice with an anti-αVβ8 integrin function blocking antibody at the time of surgery ameliorated both canonical TGF-β signaling and LC fibrotic response PCS, and treatment at 5 days PCS, after surgically induced fibrotic responses were established, largely reversed this fibrotic response. These data suggest that αVβ8 integrin is a major regulator of TGF-β activation by LCs PCS and that therapeutics targeting αVβ8 integrin could be effective for fibrotic PCO prevention and treatment.

Authors

Mahbubul H. Shihan, Samuel G. Novo, Yan Wang, Dean Sheppard, Amha Atakilit, Thomas D. Arnold, Nicole M. Rossi, Adam P. Faranda, Melinda K. Duncan

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Figure 4

The response of LCs lacking the β8 integrin gene to lens fiber cell removal.

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The response of LCs lacking the β8 integrin gene to lens fiber cell remo...
(A–F) While the unpregulation of α-SMA in WT LCs persisted until 5 days from 48 hours PCS in WT LCs (P = 0.028), β8ITG-cKO LCs attenuated α-SMA (A and B; P = 0.001) upregulation at 5 days PCS and tenascin C (A and C; P = 0.005 [48 hours]; P ≤ 0.001 [5 days]) and fibronectin (A and D) upregulation at 48 hours (P = 0.022) and 5 days (P = 0.005) PCS. In contrast, E cadherin (A and E) significantly downregulated in WT LCs by 48 hours PCS, an effect sustained at 5 days PCS (P ≤ 0.001), but this did not occur in β8ITG-cKO LCs (P = 0.390). Fiber cell regeneration measured by aquaporin 0 expression (A and F) occurs to a similar extent in β8ITG-cKO and WT LCs PCS. (G) Counting of cell nuclei associated with lens capsular bags PCS reveals that fewer cells were associated with β8ITG-cKO capsular bags compared with WT 5 days PCS (P ≤ 0.001). (H and I) WT LCs induce expression of the cell cycle marker Ki67 by 48 hours PCS (P = 0.002) while a significantly lower proportion of β8ITG-cKO LCs are in the cell cycle at this time (P = 0.021). Scale bar: 35 μm. Tenascin C, fibronectin, E-cadherin, aquaporin 0, and Ki67 (red); α-SMA (green); DNA (blue). n = 3 except for G, which had n = 6. Values are expressed as mean ± SEM presented for 1 representative experiment of 3 independent experiments, with similar results; asterisks indicate statistically significant MFI/nuclei per section between WT and β8ITG-cKO at an indicated time point PCS or between 2 PCS time points (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001); both Student’s 2-tailed t test (corrected for multiple comparisons using the Holm-Šídák method) and 1-way ANOVA with Tukey’s post hoc test (B–G) or Student’s 2-tailed t test (corrected for multiple comparisons using the Holm-Šídák method) (I). Graph colors: (B–G and I) red (WT), green (β8ITG-cKO). C, lens capsule; LC, lens cells; PCS, post cataract surgery; a, anterior; p, posterior.