Loss of cystatin C regulates permeability and inflammatory pathways in retina

Abstract

Cystatin C has been linked to inflammation in other diseases, such as epilepsy and Alzheimer's disease. These studies were designed to investigate whether Cystatin C regulates retinal inflammation and permeability. To address this question, we used Cystatin C knockout mice in a retinal ischemia/reperfusion model to determine whether Cystatin C regulated retinal damage, as well as inflammatory mediators and retinal permeability. To support the mouse work, we also used primary retinal endothelial cells cultured in normal and high glucose. Ischemia/reperfusion in Cystatin C knockout mice caused increased formation of degenerate capillaries. Loss of Cystatin C increased fluorescein leakage in the retina, which was accompanied by reduced levels of zonula occludin 1 (ZO-1) and occludin proteins. When REC were grown in high glucose, recombinant Cystatin C decreased retinal permeability, while Cystatin C siRNA increased dextran flux compared to high glucose alone. Recombinant Cystatin C decreased levels of interleukin-1-beta (IL-1β) and high mobility group box 1 (HMGB1) levels. In conclusion, loss of Cystatin C increased vascular damage in response to ischemia/reperfusion. Cystatin C regulated permeability and inflammatory mediators in the retina in response to stressors. Cystatin C offers a new target for retinal disease therapeutic development.

Keywords: Cystatin C; Inflammation; Ischemia/reperfusion; Permeability.

Figure 1.

Validation of the Cystatin C KO mice. Panel A is genotyping, Panel B is immunostaining and Panel C is Western blot data confirming the lack of Cystatin C in the knockout mice compared to control. In Panel A, Ctrl 1-3 are samples from C57BL/6 mice and Cst 1-5 are samples from Cystatin C KO mice. The ladder is 1kB. 270bp is the expected size of knockout cystatin levels, while 192bp is the expected size for normal cystatin C. Panel B is Western blotting for Cystatin C in whole retinal lysates. *P<0.05 vs. C57BL/6.

Figure 2.

Ischemia/reperfusion in the Cystatin C mice exacerbates retinal damage. Data is from control mice, mice exposed to ischemia/reperfusion (I/R), and cystatin C knockout mice exposed to nothing or I/R. Data show vascular changes (degenerate capillaries). *P<0.05 vs. C57BL/6, #P<0.05 vs. I/R. N=5.

Figure 3.

Cystatin C regulates inflammatory mediators in mouse retina. Western blot data from whole retinal lysates from Cystatin C knockout (Cys C KO) and C57BL/6 (Ctrl) mice for HMGB1 (A), IL-1B (B) and TNFα (C). *P<0.05 vs. C57BL/6. N=5. Data are mean ± SEM.

Figure 4.

Cystatin C regulates permeability in the mouse retina. Panel A shows fluorescein angiography in Cystatin C knockout (Cys C KO) vs. C57BL/6 (Ctrl) mice. Panels B-C show Western blot data from whole retinal lysates for occludin (B) and ZO-1 (C). *P<0.05 vs. C57BL/6. N=7 for fluorescein angiography, N=5 for western blot samples. Data are mean ± SEM.

Figure 5.

Cystatin C decreased inflammatory mediators in retinal endothelial cells. Retinal endothelial cells (REC) grown in normal or high glucose were treated with recombinant Cystatin C. The data show that IL-1β (A) and HMGB1 (B) were decreased compared to high glucose alone. *P<0.05 vs. normal glucose, #P<0.05 vs. high glucose. N=5. Data are mean ± SEM.

Figure 6.

Loss of Cystatin C decreased permeability, as well as ZO-1 and occludin protein levels. Retinal endothelial cells (REC) were grown in normal or high glucose and treated with Cystatin C siRNA, recombinant cystatin C, or scrambled siRNA. Panel A is a permeability assay for cells treated with either cystatin C siRNA, recombinant Cystatin C or scrambled siRNA. Panel B-D shows protein levels in cells treated with cystatin C siRNA. Panel B is a control to show successful knockdown of Cystatin C with siRNA. Panel C (occludin) and Panel D (ZO-1) show that protein levels are decreased compared to high glucose alone. *P<0.05 vs. normal glucose, #P<0.05 vs. high glucose. N=5. Data are mean ± SEM.