Toll-like receptor 4 in bone marrow-derived cells contributes to the progression of diabetic retinopathy

Abstract

Diabetic retinopathy (DR) is a major microvascular complication in diabetics, and its mechanism is not fully understood. Toll-like receptor 4 (TLR4) plays a pivotal role in the maintenance of the inflammatory state during DR, and the deletion of TLR4 eventually alleviates the diabetic inflammatory state. To further elucidate the mechanism of DR, we used bone marrow transplantation to establish reciprocal chimeric animals of TLR4 mutant mice and TLR4 WT mice combined with diabetes mellitus (DM) induction by streptozotocin (STZ) treatment to identify the role of TLR4 in different cell types in the development of the proinflammatory state during DR. TLR4 mutation did not block the occurrence of high blood glucose after STZ injection compared with WT mice but did alleviate the progression of DR and alter the expression of the small vessel proliferation-related genes, vascular endothelial growth factor (VEGF), and hypoxia inducible factor-1α (HIF-1α). Grafting bone marrow-derived cells from TLR4 WT mice into TLR4 mutant mice increased the levels of TNF-α, IL-1β, and MIP-2 and increased the damage to the retina. Similarly, VEGF and HIF-1α expression were restored by the bone marrow transplantation. These findings identify an essential role for TLR4 in bone marrow-derived cells contributing to the progression of DR.

Figure 1

The study flow chart of the whole experiment.

Figure 2

Ultrastructural signs of the blood retina barrier. (a to d) Representative electron micrographs of the retinal capillary ultrastructure from the inner layer are shown. (a) Capillary from the WT/WT (donor/recipient) group, showing a thick BM (0.18 μm, arrowhead) with pericyte edema (∗); inset, monocyte infiltration in the retinal tissue (★). (b) Capillary from the WT/Mut group. Protrusion of some villi into the lumen in endothelial cells (arrow). Reduced thickening of the basal membrane (0.15 μm, arrowhead). (c) Capillary from the Mut/WT group, showing a relatively thin BM (0.12 μm, arrowhead). Part of the crest of the mitochondria is shortened (arrow); inset, the obvious swelling of the mitochondrial crest. (d) Capillary from the Mut/Mut group, showing a thin BM (0.10 μm, arrowhead). The structure of the mitochondria is intact (arrow, magnification is indicated by the bar in the figure, n = 3 mice/group). (e) Vascular basement membrane thickness was measured in 12 retinal vessels from each group (from three mice). Basement membrane thickness was measured at 5 locations around the perimeter of the vessel and averaged to obtain a value for each vessel. *P < 0.01, WT/WT group compared with the Mut/WT group, ^# P < 0.01, WT/WT group compared with the Mut/Mut group, and ^Δ P < 0.01, WT/WT group compared with the WT/Mut group.

Figure 3

Levels of the inflammatory mediators TNF-α, IL-1β, and MIP-2 in whole retinas from each group as detected by ELISA. The data are represented as the means ± SE (4–6 mice per group). ^◆ P < 0.05, compared between WT/WT and Mut/Mut groups in the same time point; ^▲ P < 0.05, compared between WT/Mut and Mut/Mut groups in the same time point; ^★ P < 0.05, compared between WT/Mut and Mut/WT groups in the same time point; ^# P > 0.05, compared between Mut/WT and Mut/Mut groups in the same time point; *P > 0.05, ANOVA, compared among the four groups.

Figure 4

VEGF and HIF-1α mRNA expression in whole retinal tissue as detected by qt-PCR. The data are represented as the means ± SD (4–6 mice in each group), ^◆ P < 0.05, compared between WT/WT and Mut/Mut groups; ^▲ P < 0.05, compared between WT/Mut and Mut/Mut groups; ^★ P > 0.05, compared between Mut/WT and Mut/Mut groups in the same time point.