Fibroblast growth factor-2 did not restore plasminogen system activity in endothelial cells on glycated collagen

•Glycated collagen decreased endothelial plasminogen activity.•FGF-2 increased plasminogen activity in cells on native but not glycated collagen.•PAI-1 bound to glycated but not native collagen.•FGF-2 decreased total PAI-1 but not PAI-1 bound to glycated collagen.•PAI-1 binding to glycated collagen may be more important than total PAI-1.

People with diabetes experience morbidity and mortality from unregulated microvascular remodeling, which may be linked to hyperglycemia. Elevated glucose leads to extracellular matrix collagen glycation, which delays endothelial capillary-like tube formation in vitro. Glucose also increases endothelial cell fibroblast growth factor-2 (FGF-2) release and extracellular matrix storage, which should increase tube formation. In this study, we determined if FGF-2 could restore plasminogen system activity and angiogenic function in endothelial cells on glycated collagen. Human umbilical vein endothelial cells cultured on native or glycated collagen substrates were stimulated with FGF-2. Plasminogen system activity, cell migration, and capillary-like tube formation were measured, along with plasminogen system protein and mRNA levels. Glycated collagen decreased endothelial cell plasminogen system activity, cell migration, and tube length. FGF-2 did not restore plasminogen system activity or tube formation in cells on glycated collagen, despite decreasing plasminogen activator inhibitor-1 (PAI-1) protein level. We now show that PAI-1 binds to glycated collagen, which may localize PAI-1 to the extracellular matrix. These data suggest that FGF-2 may not restore angiogenic functions in endothelial cells on glycated collagen due to PAI-1 bound to glycated collagen.

Graphical abstractFigure 1. Glycated collagen decreased endothelial cell uPA activity, 3D migration, and tube length compared to native collagen. FGF-2 only partially abrogated this effect. A) uPA activity was measured using Chromozym PL. HUVEC seeded on native and glycated collagen (50 µg/ml) coated substrates for 48 h were stimulated with 50 ng/ml FGF-2 for 24 h. B) 3D cell migration was measured using a Boyden chamber. HUVEC ± 50 ng/ml FGF-2 were added to a Transwell insert coated with 100 µg/ml native or glycated collagen. After 24 h, cells that migrated to the chamber bottom were labeled with Hoechst, imaged by fluorescent microscopy, and quantified with ImageJ. Samples were normalized to native collagen without FGF-2. C) For tube formation, HUVEC were added to native and glycated collagen gels (4 mg/ml) ± FGF-2 (50 ng/ml). After 18 h, samples were imaged by phase contrast microscopy and tube length was analyzed. D) Sample tube formation images, with tubes indicated by black arrows. *p<0.01; **p<0.01 glycated vs. native collagen.Figure optionsDownload full-size imageDownload as PowerPoint slide