Characterization of Retinal Microvascular Complications and the Effects of Endoplasmic Reticulum Stress in Mouse Models of Diabetic Atherosclerosis

Abstract

Purpose: Recent evidence suggests that there is a correlation between the micro- and macrovascular complications of diabetes mellitus. The aim of this study is to investigate the molecular mechanisms by which diabetes promotes the development of microvascular disease (diabetic retinopathy [DR]) through characterization of the effects of hyperglycemia in the retina of mouse models of diabetic atherosclerosis.

Methods: Hyperglycemia was induced in apolipoprotein E-deficient (ApoE-/-) mice, a model of accelerated atherosclerosis, either through streptozotocin (STZ) injection or introduction of the Ins2Akita mutation (ApoE-/-Ins2+/Akita). Another subset of ApoE-/- mice was supplemented with glucosamine (GlcN). To attenuate atherosclerosis, subsets of mice from each experimental group were treated with the chemical chaperone, 4-phenylbutyric acid (4PBA). Eyes from 15-week-old mice were either trypsin digested and stained with periodic acid-Schiff (PAS) or frozen for cryostat sectioning and immunostained for endoplasmic reticulum (ER) stress markers, including C/EBP homologous protein (CHOP) and 78-kDa glucose-regulated protein (GRP78). PAS-stained retinal flatmounts were analyzed for microvessel density, acellular capillaries, and pericyte ghosts.

Results: Features of DR, including pericyte ghosts and reduced microvessel density, were observed in hyperglycemic and GlcN-supplemented mice. Treatment with 4PBA reduced ER stress in the retinal periphery and attenuated DR in the experimental groups.

Conclusions: Mouse models of diabetic atherosclerosis show characteristic pathologies of DR that correlate with atherosclerosis. The increased magnitude of these changes and responses to 4PBA in the peripheral retina suggest that future studies should be aimed at assessing regional differences in mechanisms of ER stress-related pathways in these mouse models.

Figure 1.

Retinal microvascular density. (A) Representative image of a retinal flatmount prepared by trypsin digestion and PAS staining. The central retinal region is defined as the circular region closest to the optic disc, while the periphery is the outer ring. (B) Microvessel density in the midretina showing the effects of 4PBA. n = 5–8. (C) Microvessel density was quantified in each treatment group in the central (C), mid (M), and peripheral (P) retina. *P < 0.05, **P < 0.005, and ***P < 0.0005 as compared to the same region in control mice. ^≠P < 0.05, ^≠≠P < 0.005, ^≠≠≠P < 0.0005, and ^≠≠≠≠P < 0.00005 as compared to a different region within the same group. ^γP < 0.05 and ^γγP < 0.005 as compared to non-4PBA-treated mice of the same group. Refer to legend below graph. (D) Representative images of 4PBA and non-4PBA-treated groups showing average microvessel density. Scale bar: 50 µm.

Figure 2.

Acellular capillaries. (A) Representative images of acellular capillaries in 4PBA and non-4PBA-treated sections from each group. Examples of acellular capillaries are indicated by black arrows. Scale bar: 10 µm. (B) Acellular capillaries were quantified in the midregion of the retina. n = 5–7. ^≠P < 0.05 and ^≠≠P < 0.005 as compared to non-4PBA-treated mice of the same group.

Figure 3.

Pericyte ghosts. (A) Representative images of pericyte ghosts in 4PBA and non-4PBA-treated sections from each group. Examples of pericyte ghosts are indicated by black arrowheads. Scale bar: 10 µm. (B) Pericyte ghosts were quantified in the central, mid-, and peripheral retina. Data are presented as the average number of pericyte ghosts per quantification box. n = 3–4. *P < 0.05 and **P < 0.005 as compared to the same region in control mice. ^≠P < 0.05, ^≠≠P < 0.005, ^≠≠≠P < 0.0005, and ^≠≠≠≠P < 0.00005 as compared to the same region in non-4PBA-treated mice of the same group.

Figure 4.

GRP78 and CHOP expression in retinas. Representative images of cryostat sectioned retinas from 15-week-old mice immunostained for GRP78 (A) and CHOP (C). Scale bar: 50 µm. Fluorescence intensity of GRP78 (B) and CHOP (D) was measured using ImageJ software and normalized to account for changes in area. n = 3–6. *P < 0.05 and **P < 0.005 as compared to the same region in control mice. ^≠P < 0.05 and ^≠≠P < 0.005 as compared to the same region in non-4PBA-treated mice of the same group.