High-glucose-induced endothelial cell injury is inhibited by a Peptide derived from human apolipoprotein E

Abstract

Although the importance of human apolipoprotein E (apoE) in vascular diseases has clearly been established, most of the research on apoE has focused on its role in cholesterol metabolism. In view of the observation that apoE and its functional domains impact extracellular matrix (ECM) remodeling, we hypothesized that apoE could also confer protection against ECM degradation by mechanisms independent of its role in cholesterol and lipoprotein transport. The ECM degrading enzyme, heparanase, is secreted by cells as pro-heparanase that is internalized through low-density lipoprotein (LDL) receptor-related protein-1 (LRP-1) to become enzymatically active. Both apoE and pro-heparanase bind the LRP-1. We further hypothesized that an apoE mimetic peptide (apoEdp) would inhibit the production of active heparanase by blocking LRP-1-mediated uptake of pro-heparanase and thereby decrease degradation of the ECM. To test this hypothesis, we induced the expression of heparanase by incubating human retinal endothelial cells (hRECs) with high glucose (30 mM) for 72 hours. We found that elevated expression of heparanase by high glucose was associated with increased shedding of heparan sulfate (ΔHS) and the tight junction protein occludin. Treatment of hRECs with 100 µM apoEdp in the presence of high glucose significantly reduced the expression of heparanase, shedding of ΔHS, and loss of occludin as detected by Western blot analysis. Either eye drop treatment of 1% apoEdp topically 4 times a day for 14 consecutive days or intraperitoneal injection (40 mg/kg) of apoEdp daily for 14 consecutive days in an in vivo mouse model of streptozotocin-induced diabetes inhibited the loss of tight junction proteins occludin and zona occludin- 1 (ZO-1). These findings imply a functional relationship between apoE and endothelial cell matrix because the deregulation of these molecules can be inhibited by a short peptide derived from the receptor-binding region of apoE. Thus, strategies targeting ECM-degrading enzymes could be therapeutically beneficial for treating diabetic retinopathy.

Figure 1. The protective effect of apoEdp on hRECs injured by high glucose in the cell medium.

(A). The hRECs were treated with varying doses of 25, 50, and 100 µM of apoEdp in the presence of 30 mM D-glucose or L-glucose (osmotic control). Western blot analysis of proteins extracted from high sugar-injured cells following 72 hours of apoEdp treatment revealed dose-dependent inhibition of heparanase expression. The 100 µM apoEdp treatment inhibited (*P≤0.05) heparanase expression to the lowest level of the 3 treatment groups assessed. (B). To detect the effect of hyperglycemia-induced HS loss (Δ-HS) in hRECs treated with 100 µM of apoEdp, a commercial monoclonal antibody (3G10) specific to neoepitope generated by heparanase digestion of HSPG was used. A 100 µM apoEdp treatment significantly inhibited (*P≤0.05) the loss of Δ-HS in hRECs after 72 hours of high glucose treatment compared to mock treatment (high glucose but no apoEdp treatment). (C). ApoEdp (100 µM) treatment prevented the loss of tight junction protein occludin in hRECs under hyperglycemic conditions. The hRECs were incubated for 72 hours with or without apoEdp (100 µM) in the presence of hyperglycemia and assayed for the expression of occludin. Each value represents the mean ± SE of results in three independent experiments (*P≤0.05). β-actin served as the loading control.

Figure 2. ApoEdp treatment prevents retinal tight junction protein loss diabetic mice.

(A). RNA samples isolated from different experimental groups of mice retinas were assessed for occludin-specific mRNA using one-step real-time RT-PCR. ApoEdp treatment increased retinal occludin specific mRNA>2-fold in drop-treated eyes and >2.5 fold in animals given intraperitoneal injection. (B). ApoEdp treatment inhibits the loss of retinal occludin-specific protein expression in streptozotocin-induced diabetic mice as determined by Western blot analysis. Significantly upregulated expression of occludin was detected in both routes (topical and intraperitoneal) in the retinas of diabetic mice.

Figure 3. ApoEdp treatment restores retinal ZO-1 protein in diabetic mice as determined by immunohistochemistry.

(A). Immunohistochemical analysis of formalin-fixed sections of mouse retinas were stained with zona occludin-1(ZO-1) specific antibody. Mean ±SEM of ZO-1 positive cells in five high power (×400) fields were counted. The arrows in the four photos indicate a ZO-1 positive cell. (B): The immunohistochemical quantitative analysis of ZO-1 protein in mouse retinas. Four treatment groups were used. The number of positive cells per high-power field is determined and the (mean ±SEM) of the ZO-1 positive cells are provided. The significant difference was detected in the ZO-1 cells between the diabetic, untreated eye, and the two groups of apoEdp treated mice retinas. Treatment with either topical 1% apoEdp drops or intraperitoneal injections of apoEdp for 14 consecutive days significantly improved the relationship of the number of increased positive cells compared to the untreated, diabetic retina.

Figure 4. ApoEdp inhibits VEGF in the retinas of diabetic mice.

Total protein extracts from the mouse retinas of diabetic mice were analyzed by Western blot to detect the VEGF expression. ApoEdp treatment significantly inhibited the expression of VEGF in diabetic mice retinas.

Figure 5. Possible mechanisms of apoEdp mediated heparanase inhibition.

(A). Heparanase is produced as a larger precursor protein (pro-heparanase). LRP-1 on ECs known to rapidly bind the secreted pro-heparanase and transfer the internalized pro-heparanase to late endosomes/lysosomes. Pro-heparanase is proteolytically cleaved into enzymatically active form in intracellular lysosomal/endosomal compartments and remains localized. (B). Human apoE protein binds to the same receptor LRP present on the cell surface ECM. (C) The apoEdp competitively inhibits hREC uptake and internalization through LRP-1 arresting the production of active heparanase.

Figure 6. ApoEdp inhibits diabetic endothelial cell injury:

Working model of apoEdp-mediated inhibition of hyperglycemia-induced expression of active heparanase by hRECs and related degradation of ECM.