In humans, ageing and metabolic stress involving a sedentary lifestyle and excess caloric intake are significant risk factors for developing cardiovascular, cerebrovascular, and neurodegenerative diseases. The pathophysiology of these diseases involves hypertension and/or dysregulation of blood flow in critical organs. Small (resistance) arteries (<300 µm) regulate tissue blood flow and systemic vascular resistance. Endothelial dysfunction, hypercontractility, deficient autoregulation, structural remodeling, and arterial stiffening are hallmarks of vascular dysfunction. The flavonoid fisetin has known cardioprotective activities, but it is unknown whether it can alleviate vascular dysfunction in ageing and metabolic stress. The primary objectives of this study were to investigate small artery dysfunction in young vs. old mice with or without diet-induced metabolic stress, and to test whether Fisetin can reverse vascular dysfunction. A secondary objective was to test if Rho-kinase is involved in age- or diet-induced vascular dysfunction.
Animal treatments and procedures were approved by the relevant authorities. Wildtype C57BL/6NTac male mice were divided into 8 groups evenly distributed to intermittent Fisetin or Vehicle treatment (4 weeks; 20 mg/kg 2 days/week). Young mice (3.5 months at study end) were fed either normal diet (N=16) or a high-fat diet (60% saturated fat) with fructose (10%) in drinking water (N=16) for 10 weeks. Old mice (retired breeders; 17 months at study end) were fed either normal chow (N=15) or a high-fat/high-fructose diet (N=15) for 10 weeks. At study end, mice were humanely sacrificed, and 3rd order mesenteric arteries (150-200 µm) were quickly dissected free from fat and connective tissue. Using a pressure myograph system, arteries were cannulated under physiological pressure (60 mm Hg) and bathed in Krebs buffer (37°C; pH 7.40). We measured the following vascular responses: vasoconstriction to high-KCl (75 mM K+) and noradrenaline (NA, 1 µM); dilation to the endothelium-dependent vasodilator acetylcholine (ACh, 10 µM); myogenic tone (MT; pressure range 60-120 mm Hg); structural remodeling (passive lumen diameter, wall cross-sectional area, wall/lumen-ratio); and vascular stiffness (Young’s Modulus). We also tested the effects of Rho-kinase inhibition (Ripasudil, 400 nM) on MT. We performed factor analysis with age and diet as factors (plus age x diet interaction) in vehicle- vs. Fisetin-treated mice, using two- or three-way Anova with multiple comparisons. A P-value <0.05 was considered significant.
Ageing was associated with increased body weight (P<0.0001) and NA contractility (P<0.05), reduced endothelial function (P<0.05), and hypertrophic remodeling (P<0.05; P<0.01). Obesogenic diet was associated with increased body weight (P<0.0001), reduced endothelial function (P<0.05), reduced MT (P<0.001), and structural remodeling (P<0.05; P<0.01). An age x diet interaction was observed for MT (P<0.001) and vascular stiffness (P<0.001). Fisetin treatment was associated with a reduced age-dependent difference in NA contractility, reduced endothelial dysfunction, reduced MT in young mice, and reduced structural remodeling. Rho-kinase inhibition reduced MT in the young vehicle-treated mice (both diets) and reduced MT in all Fisetin-treated mice (young/old, both diets). This is consistent with a loss of Rho-kinase activity in ageing,1 which was partially restored by Fisetin treatment.
Overall, Fisetin treatment may have beneficial effects on aspects of the function of small arteries governing blood pressure and -flow.