Ursodeoxycholic Acid Attenuates Endoplasmic Reticulum Stress-Related Retinal Pericyte Loss in Streptozotocin-Induced Diabetic Mice

Abstract

Loss of pericytes, an early hallmark of diabetic retinopathy (DR), results in breakdown of the blood-retinal barrier. Endoplasmic reticulum (ER) stress may be involved in this process. The purpose of this study was to examine the effects of ursodeoxycholic acid (UDCA), a known ameliorator of ER stress, on pericyte loss in DR of streptozotocin- (STZ-) induced diabetic mice. To assess the extent of DR, the integrity of retinal vessels and density of retinal capillaries in STZ-induced diabetic mice were evaluated. Additionally, induction of ER stress and the unfolded protein response (UPR) were assessed in diabetic mice and human retinal pericytes exposed to advanced glycation end products (AGE) or modified low-density lipoprotein (mLDL). Fluorescein dye leakage during angiography and retinal capillary density were improved in UDCA-treated diabetic mice, compared to the nontreated diabetic group. Among the UPR markers, those involved in the protein kinase-like ER kinase (PERK) pathway were increased, while UDCA attenuated UPR in STZ-induced diabetic mice as well as AGE- or mLDL-exposed retinal pericytes in culture. Consequently, vascular integrity was improved and pericyte loss reduced in the retina of STZ-induced diabetic mice. Our findings suggest that UDCA might be effective in protecting against DR.

Figure 1

Effect of UDCA on vascular integrity in STZ-induced diabetic mice. (a) Representative fundus images and fluorescein angiography (FA) of early- and late-phase DR in control nontreated and UDCA-treated diabetic mice. (b) Fluorescein leakage was increased in diabetic mice (n = 8) at the late phase of fluorescein angiography, compared to control mice (n = 8), and decreased in diabetic mice treated with 100 mg/kg UDCA (n = 11). Fluorescein angiographs of the early phase were obtained 3 min after fluorescein injection (early FA), while those of the late phase (late FA) were taken at 15 min. ^##P < 0.001 versus control mice, ^∗∗P < 0.01 versus nontreated diabetic mice.

Figure 2

Effect of UDCA on pericyte loss in STZ-induced diabetic mice. (a) Representative images of FITC staining in control, nontreated diabetic, and UDCA-treated diabetic mice. Original magnification: ×200; scale bar: 500 μm. (b) Capillary density was lower in nontreated diabetic mice, while no differences were observed between UDCA-treated diabetic and control mice. Fifteen measures of capillary density were averaged for each retina from control (n = 5), nontreated diabetic mice (n = 5), and UDCA-treated diabetic mice (n = 5). ^##P < 0.001 versus control mice, ^#P < 0.005 versus nontreated diabetic mice. (c) Representative images of PDGFR-β staining for retinal pericytes in control, nontreated diabetic and UDCA-treated diabetic mice. Original magnification: ×200; scale bar: 200 μm. (d) The number of pericytes was lower in nontreated diabetic mice while no differences between UDCA-treated diabetic and control mice were evident. Measurements of pericyte number were averaged for each retina from control (n = 5), nontreated diabetic mice (n = 5), and UDCA-treated diabetic mice (n = 6). CTL: control mice, DM: nontreated diabetic mice, and +UDCA: diabetic mice treated with UDCA, ^##P < 0.001 versus control mice.

Figure 3

Induction of UPR and inflammatory cytokines and UDCA-mediated attenuation in diabetic mice. Levels of UPR markers and inflammatory cytokines were assessed via western blot analysis of eye tissue from control (n = 5), nontreated DM (n = 5), and UDCA-treated DM (n = 5) groups. (a) Representative results of western blot analyses are shown. (b) Increased levels of GRP78, pPERK, peIF2α, MCP-1, and TNF α in diabetic mice, compared to the control group, were attenuated by UDCA. ATF6 and CHOP expression was not significantly different among groups. CTL: control mice, DM: nontreated diabetic mice, and +UDCA: diabetic mice treated with UDCA, ^∗P < 0.05, ^∗∗P < 0.01, ^#P < 0.005.

Figure 4

Body weights and blood glucose levels throughout the experimental period. No significant differences were evident in body weight (a) or blood glucose levels (b) between control and diabetic mouse groups with or without UDCA treatment. CTL: control mice, DM: nontreated diabetic mice, and +UDCA: diabetic mice treated with UDCA.

Figure 5

AGE-induced UPR, inflammatory cytokine expression, and cell death are attenuated by UDCA in retinal pericytes. (a) Typical results of five independent western blots are shown. (b) Increase in pPERK, peIF2α, CHOP, and MCP-1 was attenuated upon cotreatment with UDCA. GRP78, ATF6, and TNF-α did not display significant changes in expression among groups. (c, d) PI staining demonstrating a protective effect of UDCA against cell death in AGE-exposed retinal pericytes. Original magnification: ×100; scale bar: 100 μm. CTL: control pericytes, AGE: AGE-exposed pericytes, and +UDCA: AGE-exposed pericytes cotreated with UDCA, ^∗P < 0.05, ^∗∗P < 0.01, ^##P < 0.001.

Figure 6

mLDL-induced UPR, inflammatory cytokine expression, and cell death are attenuated by UDCA in retinal pericytes. (a) Typical results of four independent western analyses are shown. (b) Levels of pPERK, peIF2α, CHOP, and MCP-1 were increased and attenuated upon cotreatment with UDCA. GRP78, ATF6, and TNF-α did not display significant changes in expression. (c, d) PI staining demonstrating the protective effect of UDCA against cell death in mLDL-exposed retinal pericytes. Original magnification: ×100; scale bar: 100 μm. CTL: control pericytes, mLDL: mLDL-exposed pericytes, and +UDCA: mLDL-exposed pericytes cotreated with UDCA, ^∗P < 0.05, ^##P < 0.001.