Rafaela G. Feresin

Daily Blueberry Consumption Improves Blood Pressure and Arterial Stiffness in Postmenopausal Women with Pre- and Stage 1-Hypertension: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial

BACKGROUND Postmenopausal women have a high prevalence of hypertension and often develop arterial stiffness thereby increasing cardiovascular disease risk. Although antihypertensive drug therapies exist, increasing numbers of people prefer natural therapies. In vivo studies and a limited number of clinical studies have demonstrated the antihypertensive and vascular-protective effects of blueberries. OBJECTIVE To examine the effects of daily blueberry consumption for 8 weeks on blood pressure and arterial stiffness in postmenopausal women with pre- and stage 1-hypertension. DESIGN This was an 8-week, randomized, double-blind, placebo-controlled clinical trial. PARTICIPANTS/SETTING Forty-eight postmenopausal women with pre- and stage 1-hypertension recruited from the greater Tallahassee, FL, area participated. INTERVENTION Participants were randomly assigned to receive either 22 g freeze-dried blueberry powder or 22 g control powder. MAIN OUTCOME MEASURES Resting brachial systolic and diastolic blood pressures were evaluated and arterial stiffness was assessed using carotid-femoral pulse wave velocity and brachial-ankle pulse wave velocity. C-reactive protein, nitric oxide, and superoxide dismutase were measured at baseline, 4 weeks, and 8 weeks. STATISTICAL ANALYSES PERFORMED Statistical analysis was performed using a split plot model of repeated measures analysis of variance. RESULTS After 8 weeks, systolic blood pressure and diastolic blood pressure (131±17 mm Hg [P<0.05] and 75±9 mm Hg [P<0.01], respectively) and brachial-ankle pulse wave velocity (1,401±122 cm/second; P<0.01) were significantly lower than baseline levels (138±14 mm Hg, 80±7 mm Hg, and 1,498±179 cm/second, respectively), with significant (P<0.05) group×time interactions in the blueberry powder group, whereas there were no changes in the group receiving the control powder. Nitric oxide levels were greater (15.35±11.16 μmol/L; P<0.01) in the blueberry powder group at 8 weeks compared with baseline values (9.11±7.95 μmol/L), whereas there were no changes in the control group. CONCLUSIONS Daily blueberry consumption may reduce blood pressure and arterial stiffness, which may be due, in part, to increased nitric oxide production.

Differential Targeting of SLC30A10/ZnT10 Heterodimers to Endolysosomal Compartments Modulates EGF‐Induced MEK/ERK1/2 Activity

The solute carrier 30A (SLC30A) family of zinc exporters transports zinc into the lumen of intracellular organelles in order to prevent zinc toxicity. We reported that formation of tyrosine dimers is required for ZnT3 (zinc transporter 3) zinc transport activity and targeting to synaptic‐like microvesicles (SLMVs) in PC12 cells and the formation of ZnT3/ZnT10 heterodimers. Here, we focused on ZnT10 to determine the role of heterodimerization in the sorting of ZnTs in the endolysosomal pathway. Using cell fractionation, immunoprecipitation and immunofluorescence approaches, we found that ZnT10 resides in transferrin receptor and Rab5‐positive endosomes and forms covalent heterodimers and oligomers with ZnT2, ZnT3 and ZnT4. The interaction of ZnT10 with ZnT3, mediated by dityrosine bonds, was unable to target ZnT10 into SLMVs in vitro or into synaptic vesicles isolated from mouse brain in vivo. However, ZnT3/ZnT10 heterodimers regulate epidermal growth factor receptor (EGF‐R) signaling by increasing the phosphorylation of mitogen‐activated protein kinase kinase (MEK) and extracellular signal‐regulated kinase (ERK1/2), but not EGF‐R, C‐Raf or Akt phosphorylation in response to EGF. Further, mutation of tyrosine 4 in ZnT10 reduced ZnT3/ZnT10 dityrosine‐mediated heterodimerization and zinc transport, as well as MEK and ERK1/2 phosphorylation, which were also reduced by the zinc chelator TPEN. Phosphorylation of these kinases is likely to occur in the cytosol as no differences in phosphorylation were observed in membrane fractions of control and ZnT3/ZnT10‐expressing cells. We propose that ZnT10 plays a role in signal transduction, which is mediated by homo and heterodimerization with other ZnTs.

Effects of Vitamin E on Bone Biomechanical and Histomorphometric Parameters in Ovariectomized Rats

The present study examined the dose-dependent effect of vitamin E in reversing bone loss in ovariectomized (Ovx) rats. Sprague-Dawley rats were either Sham-operated (Sham) or Ovx and fed control diet for 120 days to lose bone. Subsequently, rats were divided into 5 groups (n = 12/group): Sham, Ovx-control, low dose (Ovx + 300 mg/kg diet; LD), medium dose (Ovx + 525 mg/kg diet; MD), and high dose (Ovx + 750 mg/kg diet; HD) of vitamin E and sacrificed after 100 days. Animals receiving MD and HD of vitamin E had increased serum alkaline phosphatase compared to the Ovx-control group. Bone histomorphometry analysis indicated a decrease in bone resorption as well as increased bone formation and mineralization in the Ovx groups supplemented with MD and HD of vitamin E. Microcomputed tomography findings indicated no effects of vitamin E on trabecular bone of fifth lumbar vertebrae. Animals receiving HD of vitamin E had enhanced fourth lumbar vertebra quality as evidenced by improved ultimate and yield load and stress when compared to Ovx-control group. These findings demonstrate that vitamin E improves bone quality, attenuates bone resorption, and enhances the rate of bone formation while being unable to restore bone density and trabecular bone structure.

Zinc regulates Nox1 expression through a NF-κB and mitochondrial ROS dependent mechanism to induce senescence of vascular smooth muscle cells

Aims The role of oxidative stress and inflammation in the development and progression of cardiovascular diseases (CVD) is well established. Increases in oxidative stress can further exacerbate the inflammatory response and lead to cellular senescence. We previously reported that angiotensin II (Ang II) and zinc increase reactive oxygen species (ROS) and cause senescence of vascular smooth muscle cells (VSMCs) and that senescence induced by Ang II is a zinc‐dependent process. Zinc stimulated NADPH oxidase (Nox) activity; however, the role of Nox isoforms in zinc effects was not determined. Results Here, we show that downregulation of Nox1, but not Nox4, by siRNA prevented both Ang II‐ and zinc‐induced senescence in VSMCs. On the other hand, overexpression of Nox1 induced senescence, which was associated with reduced proliferation, reduced expression of telomerase and increased DNA damage. Zinc increased Nox1 protein expression, which was inhibited by chelation of zinc with TPEN and by overexpression of the zinc exporters ZnT3 and ZnT10. These transporters work to reduce cytosolic zinc, suggesting that increased cytosolic zinc mediates Nox1 upregulation. Other metals including copper, iron, cobalt and manganese failed to upregulate Nox1, suggesting that this pathway is zinc specific. Nox1 upregulation was inhibited by actinomycin D (ACD), an inhibitor of transcription, by inhibition of NF‐&kgr;B, a known Nox1 transcriptional regulator and by N‐acetyl cysteine (NAC) and MitoTEMPO, suggesting that NF‐&kgr;B and mitochondrial ROS mediate zinc effects. Supporting this idea, we found that zinc increased NF‐&kgr;B activation in the cytosol, stimulated the translocation of the p65 subunit to the nucleus, and that zinc accumulated in mitochondria increasing mitochondrial ROS, measured using MitoSox. Further, zinc‐induced senescence was reduced by inhibition of NF‐&kgr;B or reduction of mitochondrial ROS with MitoTEMPO. NF‐&kgr;B activity was also reduced by MitoTEMPO, suggesting that mitochondrial ROS is upstream of NF‐&kgr;B. Innovation and conclusion Our data demonstrate that altered zinc distribution leading to accumulation of zinc in the mitochondria increases mitochondrial ROS production causing NF‐&kgr;B activation which in turn upregulates Nox1 expression inducing senescence of VSMCs. Graphical abstract Figure. No Caption available. HighlightsIncreased Nox1 expression by zinc mediates senescence of VSMCs by telomere‐dependent and ‐independent mechanisms.Overexpression of ZnT3 or ZnT10 reduces Nox1 expression, while ZnT3 gene deficiency increases Nox1.Zinc accumulates in mitochondria increasing mitochondrial ROS.Zinc stimulates NF‐&kgr;B activation in VSMCs.Inhibition of NF‐&kgr;B activation and reduction of mitochondrial ROS reduces Nox1 expression and prevents zinc‐induced senescence.

Effects of Obesity on Bone Mass and Quality in Ovariectomized Female Zucker Rats

Obesity and osteoporosis are two chronic conditions that have been increasing in prevalence. Despite prior data supporting the positive relationship between body weight and bone mineral density (BMD), recent findings show excess body weight to be detrimental to bone mass, strength, and quality. To evaluate whether obesity would further exacerbate the effects of ovariectomy on bone, we examined the tibiae and fourth lumbar (L4) vertebrae from leptin receptor-deficient female (Lepr fa/fa) Zucker rats and their heterozygous lean controls (Lepr fa/+) that were either sham-operated or ovariectomized (Ovx). BMD of L4 vertebra was measured using dual-energy X-ray absorptiometry, and microcomputed tomography was used to assess the microstructural properties of the tibiae. Ovariectomy significantly (P < 0.001) decreased the BMD of L4 vertebrae in lean and obese Zucker rats. Lower trabecular number and greater trabecular separation (P < 0.001) were also observed in the tibiae of lean- and obese-Ovx rats when compared to sham rats. However, only the obese-Ovx rats had lower trabecular thickness (Tb.Th) (P < 0.005) than the other groups. These findings demonstrated that ovarian hormone deficiency adversely affected bone mass and quality in lean and obese rats while obesity only affected Tb.Th in Ovx-female Zucker rats.

A Calcium-Collagen Chelate Dietary Supplement Attenuates Bone Loss in Postmenopausal Women with Osteopenia: A Randomized Controlled Trial

Menopause leads to an increased risk for osteoporosis in women. Although drug therapies exist, increasing numbers of people prefer alternative therapies such as dietary supplements, for example, calcium, vitamin D, and collagen hydrolysates for the prevention and treatment of osteoporosis. We have previously shown that a 3-month intervention using a calcium-collagen chelate (CC) dietary supplement was efficacious in improving bone mineral density (BMD) and blood biomarkers of bone turnover in osteopenic postmenopausal women. This study reports the long-term efficacy of CC in reducing bone loss in postmenopausal women with osteopenia. Thirty-nine women were randomly assigned to one of two groups: 5 g of CC containing 500 mg of elemental calcium and 200 IU vitamin D (1,25-dihydroxyvitamin D3) or control (500 mg of calcium and 200 IU vitamin D) daily for 12 months. Total body, lumbar, and hip BMD were evaluated at baseline, 6 and 12 months using dual-energy X-ray absorptiometry. Blood was collected at baseline, 6 and 12 months to assess levels of blood biomarkers of bone turnover. Intent-to-treat (ITT) analysis was performed using repeated measures analysis of variance pairwise comparisons and multivariate analysis to assess time and group interactions. The loss of whole body BMD in women taking CC was substantially lower than that of the control group at 12 months in those who completed the study and the ITT analysis, respectively (CC: -1.33% and -0.33% vs. control: -3.75% and -2.17%; P=.026, P=.035). The CC group had significantly reduced levels of sclerostin and tartrate-resistant acid phosphatase isoform 5b (TRAP5b) (P<.05), and higher bone-specific alkaline phosphatase/TRAP5b ratio (P<.05) than control at 6 months. These results support the use of CC in reducing bone loss in osteopenic postmenopausal women.

Blackberry, raspberry and black raspberry polyphenol extracts attenuate angiotensin II-induced senescence in vascular smooth muscle cells

Activation of angiotensin II (Ang II) signaling during aging increases reactive oxygen species (ROS) leading to vascular senescence, a process linked to the onset and progression of cardiovascular diseases (CVD). Consumption of fruits and vegetables, particularly berries, is associated with decreased incidence of CVD, which has mainly been attributed to the polyphenol content of these foods. Thus, the objective of this study was to investigate the role of blackberry (BL), raspberry (RB), and black raspberry (BRB) polyphenol extracts in attenuating Ang II-induced senescence in vascular smooth muscle cells (VSMCs) and to determine the molecular mechanisms involved. BL, RB and BRB polyphenol extracts (200 μg ml-1) attenuated Ang II-induced senescence, denoted by decreased number of cells positive for senescence associated β-galactosidase (SA-β-gal) and down-regulation of p21 and p53 expression, which were associated with decreased ROS levels and Ang II signaling. BL polyphenol extract increased superoxide dismutase (SOD) 1 expression, attenuated the up-regulation of Nox1 expression and the phosphorylation of Akt, p38MAPK and ERK1/2 induced by Ang II, and reduced senescence in response to Nox1 overexpression. In contrast, RB and BRB polyphenol extracts up-regulated the expression of SOD1, SOD2, and glutathione peroxidase 1 (GPx1), but exerted no effect on Nox1 expression nor on senescence induced by Nox1 overexpression. BRB reduced signaling similar to BL, while RB was unable to reduce Akt phosphorylation. Furthermore, we demonstrated that inhibition of Akt, p38MAPK and ERK1/2 as well as down-regulation of Nox1 by siRNA prevented senescence induced by Ang II. Our findings indicate that Ang II-induced senescence is attenuated by BL polyphenols through a Nox1-dependent mechanism and by RB and BRB polyphenols in a Nox1-independent manner, likely by increasing the cellular antioxidant capacity.

Impact of age on aortic wave reflection responses to metaboreflex activation and its relationship with leg lean mass in post-menopausal women

Wave reflection (augmentation pressure [AP] and index [AIx]) is greater in older women than men. Resting AP is a better wave reflection index than AIx in older adults. The negative relationship between wave reflection and lean mass (LM) has been inconsistent. We investigated the impact of age and LM on aortic hemodynamic responses to metaboreflex activation in post-menopausal women. Post-menopausal women, younger and older (n=20 per group) than 60 years, performed 2-min isometric handgrip at 30% of maximal force followed by 3-min post-exercise muscle ischemia (PEMI). We measured carotid-femoral pulse wave velocity (cfPWV) and femoral-ankle PWV (faPWV) at rest, and aortic systolic blood pressure (aSBP), pulse pressure (aPP), AP, AIx, and AIx-adjusted for heart rate (AIx@75) at rest and during PEMI using tonometry. Arm and leg LM were measured by DEXA. Resting cfPWV, aSBP, and aPP were higher, while AIx@75 and leg LM were lower in older than younger women. aSBP and aPP increased similarly during PEMI in both groups. Increases in AP (P<0.05), AIx (P<0.05), and AIx@75 (P<0.01) during PEMI were greater in older than younger women. From these responses, only AP during PEMI was correlated (P<0.05) positively with aSBP and aPP responses, and negatively with leg LM. Resting faPWV, but not cfPWV, was correlated (P<0.01) with AP, aSBP, and aPP during PEMI. Therefore, PEMI induces greater wave reflection responses in older than younger post-menopausal women. Our findings suggest that the increased AP response to PEMI is related to leg arterial stiffness and muscle loss in older women.

Dietary phosphorus exacerbates bone loss induced by cadmium in ovariectomized rats

ObjectivePostmenopausal bone loss can be exacerbated by environmental contaminants, including the heavy metal cadmium (Cd). We hypothesized that incorporating phosphorus (P) into the diet would lead to the chelation of Cd into P, preventing its absorption and subsequent bone loss. MethodsTo test this hypothesis, we used ovariectomized rats as a model of postmenopausal osteoporosis to examine the deleterious effects of Cd on bone with and without added P. Fifty 3-month-old ovariectomized Sprague-Dawley rats were assigned to five treatment groups (n = 10 per group) for 3 months as follows: (1) control; (2) 50 ppm Cd; (3) 50 ppm Cd plus 1.2% P; (4) 200 ppm Cd; and (5) 200 ppm Cd plus 1.2% P. ResultsCd plus P caused a significant loss of whole body (P = 0.0001 and P < 0.001) and femoral (P = 0.0005 and P < 0.001) bone mineral density (BMD) and bone mineral content, respectively, and a loss of fourth lumbar vertebra BMD and bone mineral content (P < 0.0001 and P < 0.001, respectively). Nonetheless, 200 ppm Cd plus 1.2% P had the most deleterious effects on whole body and femoral BMD. For femoral neck microstructural properties, 50 ppm Cd plus 1.2% P caused an increase in trabecular separation, whereas 200 ppm Cd plus 1.2% P caused a decrease in bone volume–to–total volume ratio, a decrease in trabecular number, and an increase in trabecular separation and structural model index. ConclusionsOur findings indicate that Cd exposure, along with high intake of P, may be a public health hazard with respect to bone health.

Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro

Epidemiological studies indicate that increased flavonoid intake correlates with decreased mortality due to cardiovascular diseases (CVD) in the United States (US) and Europe. Berries are widely consumed in the US and have a high polyphenolic content. Polyphenols have been shown to interact with many molecular targets and to exert numerous positive biological functions, including antioxidant, anti-inflammatory, and cardioprotective effects. Polyphenols isolated from blackberry (BL), raspberry (RB), and black raspberry (BRB) reduce oxidative stress and cellular senescence in response to angiotensin II (Ang II). This work provides a detailed description of the protocol used to prepare the polyphenol extracts from freeze-dried berries. Polyphenol extractions from freeze-dried berry powder were performed using 80% aqueous ethanol and an ultrasonic-assisted extraction method. The crude extract was further purified and fractionated using chloroform and ethyl acetate, respectively. The effects of both crude and purified extracts were tested on Vascular Smooth Muscle Cells (VSMCs) in culture.