Aldo Moreno Ulloa | Centro de Investigación Científica y de Educación Superior de Ensenada, BC. (original) (raw)

Research articles by Aldo Moreno Ulloa

In heart failure patients the consumption of (-)-epicatechin ((-)-Epi)-rich cocoa can restore ske... more In heart failure patients the consumption of (-)-epicatechin ((-)-Epi)-rich cocoa can restore skeletal muscle (SkM) mitochondrial structure and decrease biomarkers of oxidative stress. However, nothing is known about its effects on exercise capacity and underlying mechanisms in normal, sedentary subjects. Twenty normal, sedentary subjects (~50 years old) were randomized to placebo or dark chocolate (DC) groups and consumed 20 g of the products for 3 months. Subjects underwent before and after treatment, bicycle ergometry to assess VO2 max and work, SkM biopsy to assess changes in mitochondrial density, function and oxidative stress and blood sampling to assess metabolic endpoints. Seventeen subjects completed the trial. In the DC group (n=9), VO2 max increased (17% increase, p=0.056) as well as maximum work (watts) achieved (p=0.026) with no changes with placebo (n=8). The DC group evidenced increases in HDL levels (p=0.005) and decreased triglycerides (p=0.07). With DC, SkM evidenced significant increases in protein levels for LKB1, AMPK and PGC1α and in their active forms (phosphorylated AMPK and LKB1) as well as in citrate synthase activity while no changes were observed in mitochondrial density. With DC, significant increases in SkM reduced glutathione levels and decreases in protein carbonylation were observed. Improvements in maximum work achieved and VO2 max may be due to DC activation of upstream control systems and enhancement of SkM mitochondria efficiency. Larger clinical studies are warranted to confirm these observations.

Bioorganic & Medicinal Chemistry Letters, 2013

Impaired mitochondrial function represents an early manifestation of endothelial dysfunction and ... more 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.

Diabetes and Vascular Disease Research, 2016

(-)-Epicatechin increases indicators associated with mitochondrial biogenesis in endothelial cell... more (-)-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.

Diabetes has become a worldwide epidemic, and is growing at a rapid rate with drastic projections... more 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

(−)-Epicatechin ((−)-EPI), a naturally occurring flavanol, has emerged as a likely candidate for ... more (−)-Epicatechin ((−)-EPI), a naturally occurring flavanol, has emerged as a likely candidate for cocoa-based product reported reductions in cardiometabolic risk. The present study aimed to determine the safety, tolerability, pharmacokinetics and pharmacodynamics of purified (−)-EPI administered to healthy volunteers. In this phase I, open-label, two-part single-and multiple-dose study, subjects received either a single dose (n = 9) of 50, 100 or 200 mg or multiple doses (n = 8) of 50 mg daily (q.d.) or twice daily (b.i.d) for 5 days. Blood was collected at 0, 0.5, 1, 2, 4 and 6 h after (−)-EPI administration in the single and multiple dose groups (blood collection repeated in day 5). Samples were analyzed by HPLC-HR-ESI-MS for EPI and metabolite quantification. In the q.d. and b.i.d. groups, blood samples were analyzed for NO surrogates and follistatin levels as well as, platelet mitochondrial complexes I, V and citrate synthase activity levels. (−)-EPI was well tolerated and readily absorbed with further phase 2 metabolism. On day 5, in the q.d. and b.i.d. groups, there were significant increases in plasma nitrite of 30% and 17%, respectively. In the q.d. group on day 5 vs. day 1, platelet mitochondrial complexes I, IV and citrate synthase activities demonstrated a significant increase of ∼92, 62 and 8%, respectively. Average day 5 follistatin AUC levels were ∼2.5 fold higher vs. day 1 AUC levels in the b.i.d. group. (−)-EPI was safe to use, with no observed adverse effects, and our findings suggest that increases in NO metabolites, mitochondrial enzyme function and plasma follistatin levels may underlie some of the beneficial effects of cocoa products or (−)-EPI as reported in other studies.

We have provided evidence that the stimulatory effects of (-)-epicatechin ((-)-EPI) on endothelia... more 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

Titanium (Ti) and its alloys are amongst the most commonly-used biomaterials in orthopedic and de... more 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. 868 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.

The purpose of the present study was to synthetize 80 nm diameter TiO2 nanotubes (NTs) on Ti6Al4V... more 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 emission-scanning 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.

Objective. To evaluate the effects of ezetimibe/simvastatin (EZE/SIMV) and rosuvastatin (ROSUV) o... more 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 (í µí± < 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.

There is evidence implicating oxidative stress (OS) as the cause of the deleterious effects of ag... more 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 consumption of cacao-derived products, particularly in the form of dark chocolate is known to... more 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

The consumption of moderate amounts of cocoa products has been associated with reductions in the ... more The consumption of moderate amounts of cocoa products has been associated with reductions in the incidence of cardiovascular diseases. In animal studies, the flavanol (-)-epicatechin (Epi) yields cardioprotection. The effects may be partly due to its capacity to stimulate endothelial nitric oxide synthase (eNOS). The sustained activation of eNOS, as observed with exercise, can serve as a trigger of muscle angiogenesis via the activation of vascular endothelial growth factor (VEGF)–related events. Experiments were pursued to examine the potential of Epi to stimulate myocardial angiogenesis and determine the effects that its combined use with exercise (Ex) may trigger. Hearts obtained from a previous study were used for this purpose. Animals received 1 mg/kg of Epi or water (vehicle) via oral gavage (twice daily). Epi and/or Ex (by treadmill) was provided for 15 days. Results indicate that Ex or Epi significantly stimulate myocardial angiogenesis by ;30% above control levels. The use of Epi-Ex lead to further significant increases (to ;50%). Effects were associated with increases in protein levels and/or activation of canonical angio-genesis pathway associated events (HIF1a, VEGF, VEGFR2, PI3K, PDK, AKT, eNOS, NO, cGMP, MMP-2/-9, Src-1, and CD31). Thus, the use of Epi may represent a safe and novel means to stimulate myocardial angiogenesis.

Conference Presentations by Aldo Moreno Ulloa

background: We have previously shown in patients with heart failure and type II diabetes that tr... more background: We have previously shown in patients with heart failure and type II diabetes that treatment with DC improves mitochondrial structure, indicators of mitochondrial biogenesis and oxidative stress in skeletal muscle (SkM).Methods: Seventeen sedentary subjects (mean age of 50) were randomized to placebo (n=8) or DC groups (n=9) and consumed 2 squares of Hershey’s Extra Dark chocolate or placebo (20 g) for 3 months. Epicatechin levels were measured in a subset of patients to optimize dosing (~100 nM based on prior studies). Subjects underwent bicycle ergometry to assess VO2 max and power, lipid profile and SkM biopsy to investigate underlying biochemical mechanisms.Results: In the DC group, there was a trend for an increase in VO2 max (p=0.056 before vs. after) with no changes in the placebo group. In the DC group post treatment (Figure 1) there was an increase in maximum work (watts) achieved (p=0.026). The DC group showed significant increases in HDL levels (p=0.005). SkM biochemistry (Western blots) showed significant increases in protein levels for metabolic and/or mitochondrial biogenesis control enzymes (PGC1α, AMPK and LKB1) and in their phosphorylation levels.conclusion: Treatment of sedentary, older subjects with DC improves HDL cholesterol in association with upstream regulators of SkM metabolic control. There were improvements in maximum work without an apparent concomitant increase in VO2 max which may suggest that DC enhances efficiency of SkM mitochondria.

Bioorganic & Medicinal Chemistry Letters, 2013

Diabetes and Vascular Disease Research, 2016