Dietary Blueberry Ameliorates Vascular Complications in Diabetic Mice Possibly through NOX4 and Modulates Composition and Functional Diversity of Gut Microbes.

SCOPE: In diabetes, endothelial inflammation and dysfunction play a pivotal role in the development of vascular disease. This study investigates the effect of dietary blueberries on vascular complications and gut microbiome in diabetic mice. METHODS AND RESULTS: Seven-week-old diabetic db/db mice consume a standard diet (db/db) or a diet supplemented with 3.8% freeze-dried blueberry (db/db+BB) for 10 weeks. Control db/+ mice are fed a standard diet (db/+). Vascular inflammation is assessed by measuring monocyte binding to vasculature and inflammatory markers. Isometric tension procedures are used to assess mesenteric artery function. db/db mice exhibit enhanced vascular inflammation and reduced endothelial-dependent vasorelaxation as compared to db/+ mice, but these are improved in db/db+BB mice. Blueberry supplementation reduces the expression of NOX4 and IκKß in the aortic vessel and vascular endothelial cells (ECs) isolated from db/db+BB compared to db/db mice. The blueberry metabolites serum reduces glucose and palmitate induced endothelial inflammation in mouse aortic ECs. Further, blueberry supplementation increases commensal microbes and modulates the functional potential of gut microbes in diabetic mice. CONCLUSION: Dietary blueberry suppresses vascular inflammation, attenuates arterial endothelial dysfunction, and supports the growth of commensal microbes in diabetic mice. The endothelial-specific vascular benefits of blueberries are mediated through NOX4 signaling.

Methods to Analyze the Effect of Diet-Derived Metabolites on Endothelial Inflammation and Cell Surface Glycosaminoglycans.

The glycocalyx is a biologically active barrier that covers the luminal side of the vascular endothelium and it is comprised of proteoglycans [core proteins with glycosaminoglycans (GAG) side chains], glycoproteins, and plasma proteins. Evidence shows that the disruption in the structure and function of the endothelial glycocalyx exacerbates vascular inflammation and atherosclerosis. The GAG components of the glycocalyx undergo remodeling in the setting of diabetes and these alterations in endothelial GAGs negatively impact the vascular function. Hence, the preservation and restoration of GAGs in altered vasculature may be a novel strategy to ameliorate vascular complications in diabetes and metabolic syndrome. Human studies support the beneficial vascular effects of flavonoids which are widely found in fruits and vegetables. Flavonoids are extensively metabolized by the intestinal microbiota and digestive enzymes in humans, suggesting that their biological activities may be mediated by their circulating metabolites. Studies indicate that counteracting the damage to GAGs using dietary compounds improve vascular complications. In this article, we describe the methods to analyze the effect of diet-derived metabolites such as metabolites of flavonoids on endothelial inflammation and cell surface glycosaminoglycans.

Circulating metabolites of strawberry mediate reductions in vascular inflammation and endothelial dysfunction in db/db mice.

BACKGROUND: Cardiovascular disease is 2-4-fold more prevalent in patients with diabetes. Human studies support the cardiovascular benefits of strawberry consumption but the effects of strawberry on diabetic vasculature are unknown. We tested the hypothesis that dietary strawberry supplementation attenuates vascular inflammation and dysfunction in diabetic mice. METHODS: Seven-week-old diabetic db/db mice that consumed standard diet (db/db) or diet supplemented with 2.35% freeze-dried strawberry (db/db + SB) for ten weeks were compared to non-diabetic control mice (db/+). Indices of vascular inflammation and dysfunction were measured. Endothelial cells (ECs) were isolated from the vasculature to determine the influence of strawberry on them. The effect of metabolites of strawberry on endothelial inflammation was determined by incubating mouse aortic ECs (MAECs) with ±5% serum, obtained from strawberry fed mice (metabolites serum) or standard diet fed mice (control serum) ±â€¯25 mM glucose and 100 µM palmitate. RESULTS: db/db mice exhibited an increased monocyte binding to vessel, elevated blood pressure, and reduced endothelial-dependent vasorelaxation compared with db/+ mice but each defect was attenuated in db/db + SB mice. The elevation of inflammatory molecules, NOX2 and inhibitor-κB kinase observed in ECs from db/db vs. db/+ mice was suppressed in db/db + SB mice. Glucose and palmitate increased endothelial inflammation in MAECs but were normalized by co-incubation with metabolites serum. CONCLUSIONS: Dietary supplementation of strawberry attenuates indices of vascular inflammation and dysfunction in diabetic db/db mice. The effect of strawberry on vasculature is endothelial-dependent and possibly mediated through their circulating metabolites. Strawberry might complement conventional therapies to improve vascular complications in diabetics.

Blueberry metabolites restore cell surface glycosaminoglycans and attenuate endothelial inflammation in diabetic human aortic endothelial cells.

BACKGROUND: Glycosaminoglycan (GAG), a major component of the endothelial glycocalyx, is severely perturbed in diabetic vasculature leading to endothelial inflammation and vascular disease in diabetes. We tested the hypothesis that blueberry metabolites (BBM) ameliorate endothelial inflammation in diabetic endothelial cells (ECs) by restoring cell surface GAGs. METHODS: ECs isolated from healthy individuals [human aortic ECs (HAECs)] and diabetic patients (diabetic HAECs) were treated with ±BBM (benzoic acid-4-sulfate, hippuric acid, hydroxyhippuric acid, isovanillic acid-3-sulfate, and vanillic acid-4-sulfate at concentrations known to circulate in human plasma following blueberry consumption) for 3 days, and indices for endothelial inflammation were measured. To analyze GAGs, ECs were incubated with sulfate-free medium supplemented with [35S] Na2SO4 ±â€¯BBM. Total GAGs in ECs and medium were purified using DEAE-Sepharose column and were analyzed with high-pressure liquid chromatography coupled to an inline flow scintillation analyzer. Heparan sulfate/chondroitin sulfate ratio and disaccharide composition of GAGs from the medium were analyzed using DEAE-3SW column and Dionex CarboPac PA1 column, respectively. RESULTS: BBM suppressed diabetes-induced monocyte binding to ECs, and reduced the expression of inflammatory markers in diabetic HAECs. Diabetic HAECs displayed a decrease in [35S] sulfate incorporation into the cell surface GAGs indicating the dysregulation of sulfated GAGs. However, treatment with BBM restored the levels of GAGs in diabetic HAECs. The composition, heparan sulfate/chondroitin sulfate ratio, and disaccharide composition of GAGs from medium were similar among groups. CONCLUSIONS: BBM restored cell surface GAGs and attenuated endothelial inflammation in diabetic HAECs. Blueberry might complement conventional therapies to improve vascular complications in diabetes.

Blueberry Metabolites Attenuate Lipotoxicity-Induced Endothelial Dysfunction.

SCOPE: Lipotoxicity-induced endothelial dysfunction is an important vascular complication associated with diabetes. Clinical studies support the vascular benefits of blueberry anthocyanins, but the underlying mechanism is unclear. The hypothesis that metabolites of blueberry anthocyanins attenuate lipotoxicity-induced endothelial dysfunction was tested. METHODS AND RESULTS: Human aortic endothelial cells (HAECs) were treated for 6 h with either: (i) the parent anthocyanins (malvidin-3-glucoside and cyanidin-3-glucoside); or (ii) the blueberry metabolites (hydroxyhippuric acid, hippuric acid, benzoic acid-4-sulfate, isovanillic acid-3-sulfate, and vanillic acid-4-sulfate), at concentrations known to circulate in humans following blueberry consumption. For the last 5 h HAECs were treated with palmitate or vehicle. HAECs treated with palmitate displayed elevated reactive oxygen species generation, increased mRNA expression of NOX4, chemokines, adhesion molecules, and IκBα, exaggerated monocyte binding, and suppressed nitric oxide production. Of note, the damaging effects of palmitate were ameliorated in HAECs treated with blueberry metabolites but not parent anthocyanins. Further, important translational relevance of these results was provided by our observation that palmitate-induced endothelial dysfunction was lessened in arterial segments that incubated concurrently with blueberry metabolites. CONCLUSION: The presented findings indicate that the vascular benefits of blueberry anthocyanins are mediated by their metabolites. Blueberries might complement existing therapies to lessen vascular complications.

Mechanistic insights into the vascular effects of blueberries: Evidence from recent studies.

Cardiovascular disease is the leading cause of death in the United States. Dietary habits influence a variety of cardiovascular complications such as peripheral artery disease, heart failure, and kidney disease. We along with others have previously reported the cardiovascular beneficial effects of dietary flavonoids. Anthocyanins, one class of flavonoids widely available in berries, have recently drawn wide scientific attention because of their diverse health benefits. Epidemiological, clinical, and animal studies indicate that blueberry anthocyanins exert protection against cardiovascular complications by acting on multiple targets in the vascular system. These include activating endothelial nitric oxide synthase signaling, reducing oxidative stress, improving inflammatory pathways, and ameliorating dyslipidemia. Anthocyanins are extensively metabolized in humans suggesting that their vascular benefits are likely mediated by their circulating metabolites. However, the bioactivities of blueberry metabolites are unknown. Evaluating the bioactivities of metabolites, analyzing their structure-activity relationship, and well-designed human trials are needed to understand the potential vascular effects of blueberries and their metabolites. Understanding the vascular effects will provide a solid scientific foundation to recommend blueberries to improve vascular health. This review highlights the recent developments in the understanding of the vascular effects of blueberries with special emphasis on the molecular mechanisms involved.