Development of a Glycosaminoglycan Derived, Selectin Targeting Anti-Adhesive Coating to Treat Endothelial Cell Dysfunction

Figures

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  Figure 1. Selectin expression on SMCs and ECs under various conditions. Expression was quantified using primary anti-E-selectin and secondary horseradish peroxidase (HRP)-conjugated antibodies. All cultures exhibited significant selectin expression; however, selectin levels did not change when stimulated with TNF-α. n = 3; p < 0.05.

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  Figure 2. EC-SEAL variant binding to No Cells, SMCs and ECs (with and without 5 ng/mL TNF-α). (A) No Cells; (B) SMCs; and (C) ECs with and without 5 ng/mL TNF-α were treated with 3 μM of each EC-SEAL variant. Absorbance was measured using a streptavidin-HRP assay to quantify biotinylated peptides on each molecule bound to the surface. None of the EC-SEAL variants displayed a significant difference in binding to unstimulated ECs and ECs stimulated with TNF-α. Variants containing the peptide IEL showed the highest binding affinity to ECs. n = 3; p < 0.05.

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  Figure 3. IEL variant binding to No Cells, SMCs and ECs (with and without 5 ng/mL TNF-α). Concentration of IEL variants = 3 μM. Absorbance was measured using a streptavidin-HRP assay to quantify biotinylated peptides on each molecule remaining bound to the surface. Each letter represents groups that exhibited statistically equivalent binding to ECs. Number of molecules bound to EC surface: A > B > C > D. n = 2; p < 0.05.

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  Figure 4. Impact of DS-IEL10 and DS-IEL30 on platelet activation markers NAP-2 and PF-4. ECs were stimulated with 5 ng/mL TNF-α and treated with 3–30 µM EC-SEAL variants or control (No Treatment). PRP was added and NAP-2 and PF-4 levels in the collected PRP were recorded using sandwich ELISAs. DS-IEL30 showed the greatest reduction in platelet activation. * represents a significant difference from the control (No Treatment); # represents a significant difference when compared to all other treatment groups. n = 3; p < 0.05.

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  Figure 5. Dose-dependent response of DS-IEL30 on platelet activation. ECs were stimulated with 5 ng/mL TNF-α and treated with 3–30 µM DS-IEL30 or control (No Treatment). PRP was added and NAP-2 and PF-4 levels in the collected PRP were recorded using sandwich ELISAs. DS-IEL30 at 30 µM proved to be the most effective dose to reduce platelet activation. * represents a significant difference from the control (No Treatment); # represents a significant difference when compared to all other treatment groups. n = 2; p < 0.05.

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  Figure 6. Platelets binding to ECs in the presence of EC-SEAL. ECs (green) were stimulated with 5 ng/mL TNF-α and treated with: control media (A); or 30 µM DS-IEL30 (B). Platelets (red) were isolated from human whole blood and incubated on ECs. Cultures treated with DS-IEL30 showed less platelet binding, particularly on EC surfaces. The 10× images were acquired using a fluorescent microscope. Scale bar = 250 µm. n = 3.

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  Figure 7. EC-SEAL binding to recombinant human E-selectin. Absorbance was measured using a streptavidin-HRP assay to quantify biotinylated peptides on DS-IEL30 bound to the surface. DS-IEL30 exhibits preferential binding to selectin over BSA and is dependent upon the concentration of selectin protein present. n = 3; p < 0.05.

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  Figure 8. Antibody binding to recombinant human E-selectin. Expression was quantified using primary anti-E-selectin and secondary HRP-conjugated antibodies. Antibodies display preferential binding to selectin over BSA and binding is dependent upon the concentration of selectin protein present. n = 2; p < 0.05.

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  Figure 9. B-Mode ultrasound images of the IVC before and after partial ligation surgery. Representative images from: (A) Baseline (pre-surgery); (B) Saline; (C) Heparin; and (D) EC-SEAL (DS-IEL30) at 6 h after partial ligation are shown. IVC is outlined in red and thrombus is outlined in yellow. White arrows represent the location of the 6-0 silk suture creating the partial ligation.

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  Figure 10. Thrombus percentage in DVT mouse model. Ultrasound images were utilized to calculate thrombus percentage by dividing thrombus volume by total IVC volume between the partial ligation suture and iliac bifurcation. Both heparin (n = 6) and EC-SEAL (n = 4) decreased thrombus percentage compared to saline controls (n = 6). * represents a significant difference from the control group (Saline). p < 0.05.

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  Figure 11. Whole blood clotting time (aPTT) in the presence of heparin and EC-SEAL. aPTT measurements were recorded with varying concentrations of heparin and EC-SEAL. Heparin exhibited a much greater effect on aPTT than EC-SEAL, likely indicating different mechanisms of reducing thrombus formation in vivo. n = 2; p < 0.05.

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  Scheme 1. GAG-derived, selectin targeting anti-adhesive coating (EC-SEAL) synthesis.