Boysenberry polyphenol inhibits endothelial dysfunction and improves vascular health

Abstract

Endothelial cells have an important role in maintaining vascular homeostasis. Age-related disorders (including obesity, diabetes, and hypertension) or aging per se induce endothelial dysfunction that predisposes to the development of atherosclerosis. Polyphenols have been reported to suppress age-related endothelial cell disorders, but their role in vascular function is yet to be determined. We investigated the influence of boysenberry polyphenol on vascular health under metabolic stress in a murine model of dietary obesity. We found that administration of boysenberry polyphenol suppressed production of reactive oxygen species (ROS) and increased production of nitric oxide (NO) in the aorta. It has been reported that p53 induces cellular senescence and has a crucial role in age-related disorders, including heart failure and diabetes. Administration of boysenberry polyphenol significantly reduced the endothelial p53 level in the aorta and ameliorated endothelial cell dysfunction in iliac arteries under metabolic stress. Boysenberry polyphenol also reduced ROS and p53 levels in cultured human umbilical vein endothelial cells (HUVECs), while increasing NO production. Uncoupled endothelial nitric oxide synthase (eNOS monomer) is known to promote ROS production. We found that boysenberry polyphenol reduced eNOS monomer levels both in vivo and in vitro, along with an increase of eNOS dimerization. To investigate the components of boysenberry polyphenol mediating these favorable biological effects, we extracted the anthocyanin fractions. We found that anthocyanins contributed to suppression of ROS and p53, in association with increased NO production and eNOS dimerization. In an ex vivo study, anthocyanins promoted relaxation of iliac arteries from mice with dietary obesity. These findings indicate that boysenberry polyphenol and anthocyanins, a major component of this polyphenol, inhibit endothelial dysfunction and contribute to maintenance of vascular homeostasis.

Fig 1. Boysenberry polyphenol inhibits endothelial dysfunction and improves vascular function in obese mice.

Wild-type mice were fed normal chow (NC) or a high fat diet (HFD). In some groups, boysenberry polyphenol (BP; 0.1% in the drinking water) was administrated in addition to the HFD (HFD+BP). A. DHE staining of aortas from NC, HFD, and HFD+BP mice (Scale bar = 100 μm). The right graph shows the relative fluorescence intensity of DHE (n = 8, 9, and 9). B. Immunofluorescence staining and quantification of relative p53 expression by EC in the aorta (n = 6, 6, and 6). C. DAR-4M staining of the aorta to detect nitric oxide (Scale bar = 100 μm). The right graph shows the relative fluorescence intensity (n = 9, 9, and 10). D. Western blot analysis of eNOS dimer, eNOS monomer, and α-tubulin in the aorta. The right panel shows quantification of the eNOS dimer/monomer ratio adjusted for α-tubulin (n = 8, 7, and 8). E. The left panel indicates endothelium-dependent relaxation of iliac arteries in response to escalating doses of acetylcholine (Ach). The right panel indicates endothelium-independent relaxation of iliac arteries in response to escalating doses of sodium nitroprusside (SNP) (n = 6, 8, and 8). Data were analyzed by the 2-tailed Student’s t-test (C), 2-way ANOVA (A, B and D), followed by Tukey’s multiple comparison test, or repeated measures followed by Tukey’s multiple comparison test (E). *P < 0.05; **P < 0.01. Values represent the mean ± SEM.

Fig 2. Boysenberry polyphenol inhibits metabolic stress-induced endothelial dysfunction.

Human umbilical vein endothelial cells (HUVECs) were treated with BSA (Con group), palmitic acid (500 μM) (PA group(6hr)), or PA (500 μM) + BP (10 μg/ml) (PA+BP group). A. DHE staining of HUVECs in Con, PA, and PA+BP groups (Scale bar = 100 μm). The right graph shows the relative fluorescence intensity of DHE (n = 4, 4, and 4). B. Western blot analysis of p53 expression in HUVECs. The right panel displays quantification of p53 relative to the β-actin loading control (n = 6, 6, and 6). C. DAR-4M staining of HUVECs to detect nitric oxide (Scale bar = 100 μm). The right graph shows the relative fluorescence intensity (n = 4, 4, and 4). D. Western blot analysis of eNOS dimer, eNOS monomer, and α-tubulin in HUVECs. The right panel shows quantification of the eNOS dimer/ monomer ratio adjusted for α-tubulin (n = 3, 3, and 3). Data were analyzed by 2-way ANOVA, followed by Tukey’s multiple comparison test. *P < 0.05; **P < 0.01. Values represent the mean ± SEM.

Fig 3. Anthocyanins purified from boysenberry polyphenol protect endothelial cells.

Human umbilical vein endothelial cells (HUVECs) were treated with BSA (Con group), palmitic acid (500 μM) (PA group), or PA (500 μM) + anthocyanins (AC) (10 μg/ml) (PA+AC group). A. DHE staining of HUVECs in Con, PA, and PA+AC groups (Scale bar = 100 μm). The right graph shows the relative fluorescence intensity of DHE (n = 4, 4, and 4). B. Western blot analysis of p53 expression in HUVECs. The right panel displays quantification of p53 relative to the β-actin loading control (n = 3, 3, and 3). C. DAR-4M staining of HUVECs for nitric oxide (Scale bar = 100 μm). The right graph shows the relative fluorescence intensity (n = 4, 4, and 4). D. Western blot analysis of eNOS dimer, eNOS monomer, and α-tubulin expression in HUVECs. The right panel displays quantification of the eNOS dimer/ monomer ratio adjusted for α-tubulin (n = 3, 3, and 3). Data were analyzed by 2-way ANOVA, followed by Tukey’s multiple comparison test. *P < 0.05; **P < 0.01. Values represent the mean ± SEM.

Fig 4. Anthocyanins purified from boysenberry polyphenol improve vascular function.

Iliac arteries or aortas extracted from mice with dietary obesity were incubated ex vivo with PBS (Con group) or anthocyanins (AC group) for 6 hours, and the following studies were performed. A. The left panel shows endothelium-dependent relaxation of iliac arteries in response to escalating doses of acetylcholine (Ach). The right panel indicates endothelium-independent relaxation of iliac arteries in response to escalating doses of sodium nitroprusside (SNP) (n = 4 and 4). B. DHE staining of ex vivo-incubated aortas in Con and AC groups (Scale bar = 100 μm). The right graph shows the relative fluorescence intensity of DHE (n = 8 and 8). C. Immunofluorescence staining for quantification of relative p53 expression in EC in the aorta (n = 5 and 5). D. DAR-4M staining of ex vivo-incubated aortas to detect nitric oxide (Scale bar = 100 μm). The right graph shows the relative fluorescence intensity (n = 6 and 6). E. Western blot analysis of eNOS dimer, eNOS monomer and α-tubulin in ex vivo incubated aortas. The right panel displays quantification of the eNOS dimer/ monomer ratio adjusted for α-tubulin (n = 5 and 6). Data were analyzed by the 2-tailed Student’s t-test (B-E) or repeated measures (A). *P < 0.05; **P < 0.01. Values represent the mean ± SEM.