The consumption of cocoa products rich in (-)-epicatechin is associated with reduced cardiovascular risk and improved vascular function. However, little is known about (-)-epicatechin's effects on aged endothelium. In order to characterize the health restoring effects of (-)-epicatechin on aged endothelium and identify the underlying mechanisms, we utilized high passage number (i.e. aged) bovine coronary artery endothelial cells and aortas of 3 and 18 month old rats. We evaluated cell senescence (β-galactosidase), nitric oxide (NO) production through the endothelial nitric oxide synthase pathway, mitochondria related endpoints, citrate synthase activity and vascular relaxation. Cells were treated with water or (-)-epicatechin (1 μM) for 48 h and rats orally with either water or (-)-epicatechin (1 mg kg-1 day-1) for 15 days. Senescence associated β-galactosidase levels doubled in aged cells while those treated with (-)-epicatechin only evidenced an ∼40% increase. NO levels in cells decreased by ∼33% with aging and (-)-epicatechin normalized them. Endothelial nitric oxide synthase phosphorylation levels paralleled these results. Aging increased total protein and synthase acetylation levels and (-)-epicatechin partially restored them to those of young cells by stimulating sirtuin-1 binding to the synthase. Phosphorylated sirtuin-1, mitofilin, oxidative phosphorylation complexes and transcriptional factor for mitochondria were reduced by ∼40% with aging and were restored by (-)-epicatechin. (-)-Epicatechin enhanced acetylcholine induced aged aorta vasodilation and stimulated NO levels while reducing blood pressure. In conclusion, (-)-epicatechin reverses endothelial cell aging and restores key control elements of vascular function. These actions may partly explain the epidemiological evidence for the beneficial effects of cocoa consumption on the incidence of cardiac and vascular diseases.

Endothelial dysfunction (EnD) occurs with aging and endothelial nitric oxide (NO) production by NO synthase (NOS) can be impaired. Low NO levels have been linked to increased arginase (Ar) activity as Ar competes with NOS for L-arginine. The inhibition of Ar activity can reverse EnD and (−)-epicatechin (Epi) inhibits myocardial Ar activity. In this study, through in silico modeling we demonstrate that Epi interacts with Ar similarly to its inhibitor Norvaline (Norv). Using in vitro and in vivo models of aging, we examined Epi and Norv-inhibition of Ar activity and its endothelium-protective effects. Bovine coronary artery endothelial cells (BCAEC) were treated with Norv (10 μM), Epi (1 μM) or the combination (Epi + Norv) for 48 h. Ar activity increased in aged BCAEC, with decreased NO generation. Treatment decreased Ar activity to levels seen in young cells. Epi and Epi + Norv decreased nitrosylated Ar levels by ~25% in aged cells with lower oxidative stress (~25%) (dihydroethidium) levels. In aged cells, Epi and Epi + Norv restored the eNOS monomer/dimer ratio, protein expression levels and NO production to those of young cells. Furthermore, using 18 month old rats 15 days of treatment with either Epi (1 mg/kg), Norv (10 mg/kg) or combo, decreased hypertension and improved aorta vasorelaxation to acetylcholine, blood NO levels and tetra/dihydribiopterin ratios in cultured rat aortic endothelial cells. In conclusion, results provide evidence that inhibiting Ar with Epi reverses aged-related loss of eNOS function and improves vascular function through the modulation of Ar and eNOS protein levels and activity.

Evidence has implicated oxidative stress (OS) and inflammation as drivers of neurodegenerative pathologies. We previously reported on the beneficial effects of (-)-epicatechin (Epi) treatment, on aging-induced OS and its capacity...

Gulf War illness (GWI) afflicts military personnel who served during the Persian Gulf War and is notable for cognitive deficits, depression, muscle pain, weakness, intolerance to exercise, and fatigue. Suspect causal agents include the chemicals pyridostigmine (PB), permetrim (PM) and N,N-diethylm-toluamide (DEET) used as protectants against insects and nerve gases. No pre-clinical studies have explored the effects on skeletal muscle (SkM). Young male rats were provided PB, PM and DEET at equivalent human doses and physical restraint (to induce stress) for 3 weeks followed a 3-week recovery. GWI gastrocnemius weight was ~ 35% lower versus controls, which correlated with decreases in myofiber area, limb strength, and treadmill time/distance. In GWI rats, SkM fiber type relative abundance changed towards slow type I. Muscle wasting pathway proteins were upregulated while those that promote growth decreased as did mitochondrial endpoints and muscle ATP levels. Proteomic analysis of SkM also documented unique alterations in mitochondrial and metabolic pathways. Thus, exposure to GWI chemicals/stress adversely impacts key metabolic pathways leading to muscle atrophy and loss of function. These changes may account for GWI Veterans symptoms. Gulf War Illness (GWI) afflicts ~ 30% of the US military personnel who served in the 1990-1991 first Persian Gulf War. GWI comprises a gathering of symptoms that prominently affect the nervous and skeletal muscle (SkM) systems leading to cognitive deficits, muscle pain, weakness, exercise intolerance and fatigue 1,2. GWI affected Veterans continue to experience symptoms and altered function after 25+ years. However, the clinical presentation of GWI is unique to the 1990-1991 conflict, with no similar illness being reported in any other military campaign, indicating that the etiology cannot solely be attributed to combat-related stress 2. While the precise etiology is unknown, several hypotheses have been proposed most prominently, co-exposure to specific chemical agents and stress 2. Military personnel stationed in the battlefield are believed to have consumed the acetylcholinesterase inhibitor (AChEi) pyridostigmine bromide (PB) pills as a daily prophylactic treatment to protect against nerve gas 1,2. In addition, to reduce the risk of infections transmitted by vectors, personnel were also exposed to insecticides and insect repellants most commonly permethrin (PM) and N,Ndietyl-m-toluamide (DEET). PM is a widely used insecticide and intoxication leads to the opening of voltagegated sodium channels 3. DEET is also widely used and its target is unknown, but human poisoning can occur. AChEi, organophosphate toxicity and lethality have been related to the development of oxidative stress (OS) and mitochondrial dysfunction (MD) 2-4. Rodent models have been developed to examine the effects that GWI associated chemicals and stress have on physiological systems. A significant amount of pre-clinical work has focused on the nervous system as GWI

We examined in a rat model of Gulf War illness (GWI), the potential of (−)-epicatechin (Epi) to reverse skeletal muscle (SkM) atrophy and dysfunction, decrease mediators of inflammation and normalize metabolic perturbations. Male Wistar rats (n = 15) were provided orally with pyridostigmine bromide (PB) 1.3 mg/kg/day, permethrin (PM) 0.13 mg/kg/day (skin), DEET 40 mg/kg/day (skin) and were physically restrained for 5 min/day for 3 weeks. A one-week period ensued to fully develop the GWI-like profile followed by 2 weeks of either Epi treatment at 1 mg/kg/day by gavage (n = 8) or water (n = 7) for controls. A normal, control group (n = 15) was given vehicle and not restrained. At 6 weeks, animals were subjected to treadmill and limb strength testing followed by euthanasia. SkM and blood sampling was used for histological, biochemical and plasma pro-inflammatory cytokine and metabolomics assessments. GWI animals developed an intoxication profile characterized SkM atrophy and loss of function accompanied by increases in modulators of muscle atrophy, degradation markers and plasma pro-inflammatory cytokine levels. Treatment of GWI animals with Epi yielded either a significant partial or full normalization of the above stated indicators relative to normal controls. Plasma metabolomics revealed that metabolites linked to inflammation and SkM waste pathways were dysregulated in the GWI group whereas Epi, attenuated such changes. In conclusion, in a rat model of GWI, Epi partially reverses detrimental changes in SkM structure including modulators of atrophy, inflammation and select plasma metabolites yielding improved function.

(−)-Epicatechin and (+)-epicatechin, upregulate neurogenesis markers likely through stimulation of capillary formation and nitric oxide triggering, improvements in memory.

No abstract

Early menopause occurs in women under age 45 and is seen in ~5% of women. Women who experience estrogen deficiency at an early age (before menopause) are known to be at increased overall risk for premature morbidity and mortality. Also, higher rates of physical limitation are known to occur in surgically menopausal vs. naturally menopausal women. To the extent that muscle mass and strength decreases, it can foster lesser levels of overall physical activity which, can promote a further deterioration of the musculoskeletal system including bone density. We previously documented the capacity of the cacao flavanol (‐)‐epicatechin (Epi) to restore skeletal muscle (SkM) mass and strength in various models of disease. Our research group implemented a female Wistar rat model whereby ovariectomy (OVX) was used to assess its impact on SkM structure/function as well as on modulators of muscle mass (atrophy). Using this model, we tested the hypothesis that Epi can restore SkM structure and function and recognized modulators of muscle mass. For these experiments, female rats at the age of 6 months (n=9‐10/subgroups) were subjected to OVX and allowed to evolve for a period of 12 weeks. A subgroup was treated with Epi at the dose of 1 mg/kg/day (by gavage in water) 8 weeks after OVX. Intact animals subjected to a sham OVX and vehicle (water) were used as controls. Front limb strength (in Newtons =N) was assessed weekly resulting in a significant progressive drop in force with OVX that was fully restored with 4 weeks of Epi treatment (figure 1 top graphs). Overall exercise capacity was assessed by treadmill (bottom panels) demonstrating a decrease with OVX and an improvement in total treadmill time (in minutes) with Epi that was greater vs. controls. At the time of sacrifice, muscle weight of gastrocnemius was recorded and values normalized to tibial length demonstrating the development of significant atrophy with OVX and the restoring effects of Epi treatment. An assessment of factors recognized to induce a loss of muscle mass was pursued. Myostatin levels were measured in blood by ELISA while the relative protein levels for the recognized inducers of muscle atrophy Murf‐1 and atrogin‐1 by Western blots of gastrocnemius lysates. Relative changes were normalized for loading differences using GAPDH. Blood levels of the muscle atrophy inducer factor myostatin were significantly increased with OVX and were fully normalized by Epi. OVX led to the significant upregulation of Murf‐1 and atrogin‐1 and the normalization of such effects with Epi. OXV led to significant increases in pro‐inflammatory cytokines TNF‐α, IL‐1β, IL‐2 and IFN‐γ andf plasma oxidative stress leveles (protein carbonylation) that were completely normalized with Epi. Altogether, results demonstrate that OVX can significantly compromise SkM structure and function which can be fully reversed by the use of Epi. These effects are likely the results of the effects on recognized modulators of inflammation, oxidative stress and atrophy. Clinical trials are warranted as per ...