Aldo Moreno-Ulloa

Cell membrane mediated (-)-epicatechin effects on upstream endothelial cell signaling: evidence for a surface receptor.

The consumption of cacao-derived products, particularly in the form of dark chocolate is known to provide beneficial cardiovascular effects in normal individuals and in those with vascular dysfunction (reduced nitric oxide [NO] bioavailability and/or synthesis). Upstream mechanisms by which flavonoids exert these effects are poorly understood and may involve the participation of cell membrane receptors. We previously demonstrated that the flavanol (-)-epicatechin (EPI) stimulates NO production via Ca(+2)-independent eNOS activation/phosphorylation. We wished to investigate the plausible participation of a cell surface receptor using a novel cell-membrane impermeable EPI-Dextran conjugate (EPI-Dx). Under Ca(2+)-free conditions, human coronary artery endothelial cells (HCAEC) were treated for 10min with EPI or EPI-Dx at equimolar concentrations (100nM). Results demonstrate that both EPI and EPI-Dx induced the phosphorylation/activation of PI3K, PDK-1, AKT and eNOS. Interestingly, EPI-Dx effects were significantly higher in magnitude than those of EPI alone. The capacity of EPI-Dx to stimulate cell responses supports the existence of an EPI cell membrane receptor mediating eNOS activation.

Recovery of Indicators of Mitochondrial Biogenesis, Oxidative Stress, and Aging With (−)-Epicatechin in Senile Mice

There is evidence implicating oxidative stress (OS) as the cause of the deleterious effects of aging. In this study, we evaluated the capacity of the flavanol (-)-epicatechin (Epi) to reduce aging-induced OS and restore mitochondrial biogenesis, as well as, structural and functional endpoints in aged mice. Senile (S; 26-month-old) C57BL/6 male mice were randomly assigned to receive either water (vehicle) or 1mg/kg of Epi via oral gavage (twice daily) for 15 days. Young (Y; 6-month-old) mice were used as controls. In S brain, kidney, heart, and skeletal muscle (compared with Y animals) an increase in OS was observed as evidenced by increased protein-free carbonyls and decreased reduced glutathione levels as well as sirtuin 3, superoxide dismutase 2, catalase, thioredoxin and glutathione peroxidase protein levels. Well-recognized factors (eg, sirtuin 1) that regulate mitochondrial biogenesis and mitochondrial structure- and/or function-related endpoints (eg, mitofilin and citrate synthase) protein levels were also reduced in S organs. In contrast, the aging biomarker senescence-associated β-galactosidase was increased in S compared with Y animals, and Epi administration reduced levels towards those observed in Y animals. Altogether, these data suggest that Epi is capable of shifting the biology of S mice towards that of Y animals.

The effects of (−)-epicatechin on endothelial cells involve the G protein-coupled estrogen receptor (GPER)

We have provided evidence that the stimulatory effects of (-)-epicatechin ((-)-EPI) on endothelial cell nitric oxide (NO) production may involve the participation of a cell-surface receptor. Thus far, such entity(ies) has not been fully elucidated. The G protein-coupled estrogen receptor (GPER) is a cell-surface receptor that has been linked to protective effects on the cardiovascular system and activation of intracellular signaling pathways (including NO production) similar to those reported with (-)-EPI. In bovine coronary artery endothelial cells (BCAEC) by the use of confocal imaging, we evidence the presence of GPER at the cell-surface and on F-actin filaments. Using in silico studies we document the favorable binding mode between (-)-EPI and GPER. Such binding is comparable to that of the GPER agonist, G1. By the use of selective blockers, we demonstrate that the activation of ERK 1/2 and CaMKII by (-)-EPI is dependent on the GPER/c-SRC/EGFR axis mimicking those effects noted with G1. We also evidence by the use of siRNA the role that GPER has on mediating ERK1/2 activation by (-)-EPI. GPER appears to be coupled to a non Gαi/o or Gαs, protein subtype. To extrapolate our findings to an ex vivo model, we employed phenylephrine pre-contracted aortic rings evidencing that (-)-EPI can mediate vasodilation through GPER activation. In conclusion, we provide evidence that suggests the GPER as a potential mediator of (-)-EPI effects and highlights the important role that GPER may have on cardiovascular system protection.

EFFECTS OF (−)-EPICATECHIN AND DERIVATIVES ON NITRIC OXIDE MEDIATED INDUCTION OF MITOCHONDRIAL PROTEINS

Impaired mitochondrial function represents an early manifestation of endothelial dysfunction and likely contributes to the development of cardiovascular diseases (CVD). The stimulation of mitochondrial function and/or biogenesis is seen as a means to improve the bioenergetic and metabolic status of cells and thus, reduce CVD. In this study we examined the capacity of the flavanol (-)-epicatechin and two novel derivatives to enhance mitochondrial function and protein levels in cultured bovine coronary artery endothelial cells. As nitric oxide production by endothelial cells is suspected in mediating mitochondria effects (including biogenesis), we also examined the dependence of responses on this molecule using an inhibitor of nitric oxide synthase. Results indicate that the flavanol (-)-epicatechin and derivatives are capable of stimulating mitochondrial function as assessed by citrate synthase activity as well as induction of structural (porin, mitofilin) and oxidative phosporylation protein levels (complex I and II). Effects were blocked by the use of the chemical inhibitor of the synthase thus, evidencing a role for nitric oxide in mediating these effects. The results observed indicate that the three agents are effective in enhancing mitochondria function and protein content. The effects noted for (-)-epicatechin may serve to explain the healthy effects on cardiometabolic risk ascribed to the consumption of cocoa products.

(-)-Epicatechin-induced recovery of mitochondria from simulated diabetes: Potential role of endothelial nitric oxide synthase

(-)-Epicatechin increases indicators associated with mitochondrial biogenesis in endothelial cells and myocardium. We investigated endothelial nitric oxide synthase involvement on (-)-epicatechin-induced increases in indicators associated with mitochondrial biogenesis in human coronary artery endothelial cells cultured in normal-glucose and high-glucose media, as well as to restore indicators of cardiac mitochondria from the effects of simulated diabetes. Here, we demonstrate the role of endothelial nitric oxide synthase on (-)-epicatechin-induced increases in mitochondrial proteins, transcription factors and sirtuin 1 under normal-glucose conditions. In simulated diabetes endothelial nitric oxide synthase function, mitochondrial function–associated and biogenesis-associated indicators were adversely impacted by high glucose, effects that were reverted by (-)-epicatechin. As an animal model of type 2 diabetes, 2-month old C57BL/6 mice were fed a high-fat diet for 16 weeks. Fasting and fed blood glucose levels were increased and NO plasma levels decreased. High-fat-diet-fed mice myocardium revealed endothelial nitric oxide synthase dysfunction, reduced mitochondrial activity and markers of mitochondrial biogenesis. The administration of 1 mg/kg (-)-epicatechin for 15 days by oral gavage shifted these endpoints towards control mice values. Results suggest that endothelial nitric oxide synthase mediates (-)-epicatechin-induced increases of indicators associated with mitochondrial biogenesis in endothelial cells. (-)-Epicatechin also counteracts the negative effects that high glucose or simulated type 2 diabetes has on endothelial nitric oxide synthase function.

Improved Osteoblast and Chondrocyte Adhesion and Viability by Surface-Modified Ti6Al4V Alloy with Anodized TiO2 Nanotubes Using a Super-Oxidative Solution

Titanium (Ti) and its alloys are amongst the most commonly-used biomaterials in orthopedic and dental applications. The Ti-aluminum-vanadium alloy (Ti6Al4V) is widely used as a biomaterial for these applications by virtue of its favorable properties, such as high tensile strength, good biocompatibility and excellent corrosion resistance. TiO2 nanotube (NTs) layers formed by anodization on Ti6Al4V alloy have been shown to improve osteoblast adhesion and function when compared to non-anodized material. In his study, NTs were grown on a Ti6Al4V alloy by anodic oxidation for 5 min using a super-oxidative aqueous solution, and their in vitro biocompatibility was investigated in pig periosteal osteoblasts and cartilage chondrocytes. Scanning electron microscopy (SEM), energy dispersion X-ray analysis (EDX) and atomic force microscopy (AFM) were used to characterize the materials. Cell morphology was analyzed by SEM and AFM. Cell viability was examined by fluorescence microscopy. Cell adhesion was evaluated by nuclei staining and cell number quantification by fluorescence microscopy. The average diameter of the NTs was 80 nm. The results demonstrate improved cell adhesion and viability at Day 1 and Day 3 of cell growth on the nanostructured material as compared to the non-anodized alloy. In conclusion, this study evidences the suitability of NTs grown on Ti6Al4V alloy using a super-oxidative water and a short anodization process to enhance the adhesion and viability of osteoblasts and chondrocytes. The results warrant further investigation for its use as medical implant materials.

Effects of Ezetimibe/Simvastatin and Rosuvastatin on Oxidative Stress in Diabetic Neuropathy: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial

Objective. To evaluate the effects of ezetimibe/simvastatin (EZE/SIMV) and rosuvastatin (ROSUV) on oxidative stress (OS) markers in patients with diabetic polyneuropathy (DPN). Methods. We performed a randomized, double-blind, placebo-controlled phase III clinical trial in adult patients with Type 2 Diabetes Mellitus (T2DM) and DPN, as evaluated by composite scores and nerve conduction studies (NCS). Seventy-four subjects with T2DM were allocated 1 : 1 : 1 to placebo, EZE/SIMV 10/20 mg, or ROSUV 20 mg for 16 weeks. All patients were assessed before and after treatment: primary outcomes were lipid peroxidation (LPO), and nitric oxide (NO) surrogate levels in plasma; secondary outcomes included NCS, neuropathic symptom scores, and metabolic parameters. Data were expressed as mean ± SD or SEM, frequencies, and percentages; we used nonparametric analysis. Results. LPO levels were reduced in both statin arms after 16 weeks of treatment (p < 0.05 versus baseline), without changes in the placebo group. NO levels were not significantly affected by statin treatment, although a trend towards significance concerning increased NO levels was noted in both statin arms. No significant changes were observed for the NCS or composite scores. Discussion. EZE/SIMV and ROSUV are superior to placebo in reducing LPO in subjects with T2DM suffering from polyneuropathy. This trial is registered with NCT02129231.

Mortality reduction among persons with type 2 diabetes: (−)-Epicatechin as add-on therapy to metformin?

Diabetes has become a worldwide epidemic, and is growing at a rapid rate with drastic projections for developing countries. Mexico occupies the ninth place worldwide for type 2 diabetes prevalence, and in the foreseeable future, it is expected rise to the seventh place. Myocardial infarction is the most common cause of death in these patients. Although several drugs are approved for the treatment of type 2 diabetes that reduce factors associated with myocardial infarction, an excess risk of death is still present. In this regard, the American Diabetes Association recommends metformin (oral glucose lowering drug) as the first-line therapy in type 2 diabetic subjects, based on its amply confirmed positive metabolic effects; however, its capacity to reduce cardiovascular mortality in type 2 diabetic subjects is inconclusive. Thus, mortality reduction in these patients has been an elusive goal, and is therefore, imperative to evaluate new pharmacological interventions that may favorably impact mortality in these individuals. On the other hand, epidemiological studies have suggested that moderate consumption of cacao-derived products (i.e., chocolate and cocoa) may reduce the risk of diabetes, myocardial infarction, and cardiovascular disease-associated mortality. Moreover, interventional studies have also suggested that dark chocolate and cocoa consumption is vasculoprotective in normal and type 2 diabetic individuals. (-)-Epicatechin ((-)-EPI) is the main flavanol present in cacao, and suggested to be responsible for the beneficial effects observed after dark chocolate/cocoa consumption. Interestingly, in vivo studies have evidenced the capacity of (-)-EPI to reduce infarct size, and preserve cardiac mechanics in rodent models of ischaemia-reperfusion injury. Nonetheless, long-term studies using (-)-EPI and evaluating its effects on mortality are lacking. Thus, based on their particular properties, it is valid to speculate that (-)-EPI and metformin in conjunction may favorably impact mortality in type 2 diabetic individuals. Here, we provide the evidence that allow us to propose our hypothesis, and further suggest a reasonable way to perform the study needed for such investigation.

The promotion of antibacterial effects of ti6al4v alloy modified with TiO 2 nanotubes using a superoxidized solution

The purpose of the present study was to synthetize 80 nm diameter TiO2 nanotubes (NTs) on Ti6Al4V alloy using a commercially superoxidized water (SOW) enriched with fluoride to reduce anodization time and promote the antibacterial efficacy against Staphylococcus aureus (S. aureus). The alloy discs were anodized for 5 min and as a result, NTs of approximately 80 nm diameters were obtained with similar morphology as reported in previous studies using longer anodization times (1-2 h). Filed emissionscanning electron microscopy (FE-SEM) and energy dispersive X-ray spectroscopy (EDX) were used to characterize the materials surfaces. The NTs showed significantly decreased S. aureus viability after 1, 3, and 5 days of culture in comparison to nonanodized alloy. Likewise, SEM analysis also suggested lower bacterial adhesion on the NTs surface. No differences in bacterial morphology and topography were observed on both materials, as analyzed by SEM and atomic force microscopy (AFM). In conclusion, 80 nm diameter NTs were grown on Ti6Al4V alloy in 5 min by using a SOW solution enriched with fluoride, which resulted in a material with promoted antibacterial efficacy against S. aureus for up to 5 days of in vitro culture when compared to nonanodized alloy.

(-)-Epicatechin stimulates mitochondrial biogenesis and cell growth in C2C12 myotubes via the G-protein coupled estrogen receptor

Abstract We have reported on the capacity of (‐)‐epicatechin ((‐)‐EPI) to stimulate mitochondrial biogenesis (MiB) in mouse skeletal muscle (SkM). However, the mechanisms mediating the effects of (‐)‐EPI are not fully understood. We previously identified a role of the G‐protein coupled estrogen receptor (GPER) in modulating the vascular effects of (‐)‐EPI. We therefore tested the hypothesis that GPER mediates (at least in part) the stimulatory effects of (‐)‐EPI on MiB in SkM cells. As an in vitro model, we employed mouse SkM‐derived C2C12 myoblasts differentiated into myotubes. Using confocal microscopy, we detected GPER at the cell surface and cytoplasm in C2C12 myotubes. Treatment with (‐)‐EPI (3 and 10 &mgr;M) resulted in the stimulation of MiB as per increases in mitochondrial inner (MitoTracker Red FM fluorescence staining) and outer membrane (porin protein levels) markers, transcription factors involved in MiB stimulation (i.e., nuclear respiratory factor‐2 [NRF‐2] and mitochondrial transcription factor A [TFAM] protein levels) and citrate synthase (CS) activity levels. (‐)‐EPI‐treated myotubes were longer and wider compared to vehicle‐treated myotubes. The effects of (‐)‐EPI on myotube mitochondria and cell size were larger in magnitude to those observed with the GPER agonist G‐1. The chemical blockade and down‐regulation (siRNA) of GPER evidenced a partial and complete blockade of measured endpoints following (‐)‐EPI‐ or G‐1‐treatment, respectively. Altogether, results indicate that GPER is expressed in muscle cells and appears to mediate to a significant extent, the stimulatory effects of (‐)‐EPI on MiB. Thus, GPER activation may account for the stimulatory effects of (‐)‐EPI on SkM structure/function. Graphical abstract Figure. No Caption available.