Sulodexide Prevents Hyperglycemia-Induced Endothelial Dysfunction and Oxidative Stress in Porcine Retinal Arterioles

Abstract

Diabetes mellitus may cause severe damage to retinal blood vessels. The central aim of this study was to test the hypothesis that sulodexide, a mixture of glycosaminoglycans, has a protective effect against hyperglycemia-induced endothelial dysfunction in the retina. Functional studies were performed in isolated porcine retinal arterioles. Vessels were cannulated and incubated with highly concentrated glucose solution (HG, 25 mM D-glucose) +/- sulodexide (50/5/0.5 μg/mL) or normally concentrated glucose solution (NG, 5.5 mM D-glucose) +/- sulodexide for two hours. Endothelium-dependent and endothelium-independent vasodilatation were measured by videomicroscopy. Reactive oxygen species (ROS) were quantified by dihydroethidium (DHE) fluorescence. Using high-pressure liquid chromatography (HPLC), the intrinsic antioxidant properties of sulodexide were investigated. Quantitative PCR was used to determine mRNA expression of regulatory, inflammatory, and redox genes in retinal arterioles, some of which were subsequently quantified at the protein level by immunofluorescence microscopy. Incubation of retinal arterioles with HG caused significant impairment of endothelium-dependent vasodilation, whereas endothelium-independent responses were not affected. In the HG group, ROS formation was markedly increased in the vascular wall. Strikingly, sulodexide had a protective effect against hyperglycemia-induced ROS formation in the vascular wall and had a concentration-dependent protective effect against endothelial dysfunction. Although sulodexide itself had only negligible antioxidant properties, it prevented hyperglycemia-induced overexpression of the pro-oxidant redox enzymes, NOX4 and NOX5. The data of the present study provide evidence that sulodexide has a protective effect against hyperglycemia-induced oxidative stress and endothelial dysfunction in porcine retinal arterioles, possibly by modulation of redox enzyme expression.

Keywords: diabetic retinopathy; endothelial dysfunction; oxidative stress; sulodexide.

Figure 1

Concentration-dependent responses of porcine retinal arterioles after two hours of incubation with HG (highly concentrated glucose solution; 25 mM D-glucose), HG + Sulo 50 µg/mL (highly concentrated glucose solution + 50 µg/mL sulodexide), NG (normally concentrated glucose solution; 5.5 mM D-glucose), NG + Sulo 50 µg/mL (normally concentrated glucose solution + 50 µg/mL sulodexide) to the vasoconstrictor U46619 (A), the endothelium-independent vasodilator SNP (B), and to the endothelium-dependent vasodilator bradykinin (C). Data are presented as mean ± SE (n = 8 per concentration and group; * p < 0.05, HG versus all other groups).

Figure 2

Concentration-dependence studies for sulodexide. Vascular reactivity of retinal arterioles to the endothelium-independent vasodilator SNP (A) and to the endothelium-dependent vasodilator bradykinin (B) after incubation with highly concentrated glucose solution (HG) and different sulodexide concentrations for two hours: 50 µg/mL, 5 µg/mL, and 0.5 µg/mL. Vessels incubated with normally concentrated glucose solution (NG) served as controls. Data are expressed as mean ± SE (n = 8 per concentration and group; * p < 0.05, HG versus all other groups; # p < 0.05, HG + Sulo 0.5 µg/mL versus NG).

Figure 3

DHE staining of retinal arterioles in cryosections after two hours of incubation with HG (highly concentrated glucose solution; 25 mM D-glucose), HG + Sulo 50 µg/mL (highly concentrated glucose solution + 50 µg/mL sulodexide), NG (normally concentrated glucose solution; 5.5 mM D-glucose), and NG + Sulo 50 µg/mL (NG + 50 µg/mL sulodexide). The microphotographs show representative stainings of retinal arteriole cross-sections for the individual groups. Data are expressed as mean ± SE (n = 8 per group; * p < 0.05, *** p < 0.001). Scale bar = 50 µm.

Figure 4

Direct antioxidant properties of sulodexide tested on (A) superoxide formation by xanthine oxidase (20 mU/mL)/1 mM hypoxanthine with hydroethidine (50 µM), (B) nitration of phenol (5 mM) by peroxynitrite (250 µM), and (C) oxidation of ebselen (250 µM) by hydrogen peroxide (250 µM). Representative chromatograms are shown besides the quantification bar graphs. Data are expressed as mean ± SE (n = 3–5 per group; * p < 0.05; ** p < 0.01).

Figure 5

Messenger RNA expression of regulatory genes (SIRT1, FOXO1, (A)), hypoxic genes (HIF-1α, VEGF-A, (B)), inflammatory genes (NFκB, RANTES, IL-6, MCP-1, (C)), pro-oxidant redox genes (NOX2, NOX4, NOX5, (D)), and antioxidant redox genes (SOD1, SOD2, SOD3, CAT, GPX1, HO-1, (E)) after incubation for two hours with HG (highly concentrated glucose solution; 25 mM D-glucose), HG + Sulo 50 µg/mL (highly concentrated glucose solution + 50 µg/mL sulodexide), NG (normally concentrated glucose solution; 5.5 mM D-glucose), and NG + Sulo 50 µg/mL (NG + 50 µg/mL sulodexide). Data are expressed as mean ± SE (n = 8 per group; * p < 0.05, ** p < 0.01). In each case, mRNA expression levels were normalized to the NG group.

Figure 6

Immunofluorescence micrographs of retinal arteriole cross-sections (cryosections) stained with a primary antibody (Abcam, Waltham, MA, USA, dilution 1:200, incubation time: 2 h at RT) targeted against NOX2 (ab80508), NOX4 (ab154244), and NOX5 (ab191010) and a rhodamine Red-X-coupled secondary antibody (Dianova GmbH, Hamburg, Germany, 111-295-003, dilution 1:200, incubation time: 2 h at RT) (red color). Quantification of fluorescent intensities in the vascular wall of retinal arterioles normalized to NG. (A) Immunoreactivity to NOX2 was similar in all groups. (B) In contrast, immunoreactivity to NOX4 was elevated in the HG group. (C) Likewise, immunoreactivity to NOX5 was increased in the HG group. Data are expressed as mean ± SE (n = 8 per group; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Scale bar = 50 µm.