Blueberry Metabolites Restore Composition of Glycosaminoglycan Structures in Diabetic Human Vascular Endothelial Cells
Authors: Cutler Brett , Chua Jie Shi , Kuberan Balagurunathan , Velayutham Anandh Babu Pon 
Published: November 2023

BackgroundHigh glucose induced vascular inflammation plays a major role in the development of vascular complications in diabetes. The vascular endothelium is covered with the glycocalyx that acts as an orchestrator of vascular homeostasis. It is comprised of a mesh of glycosaminoglycans (GAG), proteoglycans including heparan sulfate (HS) proteoglycans, and plasma proteins. The structure and function of the glycocalyx is severely perturbed in the vessels of diabetic patients leading to enhanced sensitivity of the vasculature towards atherogenic stimuli. Hence, restoration of the glycocalyx structure is predicted to ameliorate vascular complications in diabetes. The vascular beneficial effects of blueberry polyphenols may be mediated by their circulating metabolites but their activities are unknown as many of these metabolites are not commercially available.HypothesisBlueberry metabolites (BBM) restore/reestablish the glycocalyx in diabetic human aortic endothelial cells.MethodsWe synthesized blueberry metabolites (BBM) such as vanillic acid‐4‐sulfate (V4S), isovanillic acid‐3‐sulfate (IV3S) and benzoic acid‐4‐sulfate (B4S). The BBM mixture used in the present study contained V45, IV3S, B4S, hydroxyhippuric acid, and hippuric acid at serum concentrations reported following consumption of 240 g of blueberries in humans. Human aortic endothelial cells isolated from healthy individuals (HAEC) and diabetic patients (diabetic‐HAEC) (Lonza) were grown to 70% confluency in EGM‐2 complete medium. Medium was replaced with sulfate‐free DMEM/F12 medium supplemented with [35S]Na2SO4 ± BBM mixture and incubated over physiological shear for 3 days. Following the incubation, the medium, which contains the shedded GAG, and the cell components were collected separately. GAG were isolated using DEAE‐Sepharose columns, the elution was concentrated using centrifugal filter, and the radioactivity was analyzed with a liquid scintillation counter. The GAG amounts isolated were normalized for protein concentration. The samples were analyzed with high‐pressure liquid chromatography (HPLC) coupled to an inline flow scintillation analyzer. The DEAE‐3SW column was used to analyze the relative amount of heparan sulfate (HS) and chondroitin sulfate (CS) in the samples, and a Dionex CarboPac PA1 column was used to analyze the disaccharide composition of the HS after digestion with heparitinases.ResultsMetabolic radiolabelling showed a significant decrease in [35S] sulfate incorporation into the cell surface GAG (HS and CS together) produced by diabetic‐HAEC as compared to HAEC. This indicates a reduced synthesis of sulfated GAG in the glycocalyx of the diabetic‐HAEC. However, BBM treatment significantly increased (p<0.05) the sulfated GAG production as shown by increased [35S] sulfate incorporation in cell surface GAG. The GAG sheds into the culture medium as well as the disaccharide composition of HS were similar among groups.ConclusionMetabolites of blueberry polyphenols restored GAG composition in diabetic endothelial cells. Blueberry might complement conventional therapies to improve diabetic vascular complications.Support or Funding InformationSupported by University of Utah Seed Grant and College of Health Research Pilot Grant.