Abstract-Recent reports indicate that (Ϫ)-epicatechin can exert cardioprotective actions, which may involve endothelial nitric oxide synthase (eNOS)-mediated nitric oxide production in endothelial cells. However, the mechanism by which (Ϫ)-epicatechin activates eNOS remains unclear. In this study, we proposed to identify the intracellular pathways involved in (Ϫ)-epicatechin-induced effects on eNOS, using human coronary artery endothelial cells in culture. Key Words: flavonoids Ⅲ ischemia Ⅲ polyphenols Ⅲ endothelial cells Ⅲ endothelial nitric oxide synthase C ardiovascular diseases (CVD) are is a leading cause of morbidity and mortality, affecting Western industrialized countries as well as developing countries. 1,2 CVD imposes major direct and indirect costs on health care systems, ranging from hospitalizations, drugs, and rehabilitation services to losses of productivity due to premature mortality and short-and long-term disability. 1 The incidence of CVD continues to increase; thus, it is important to identify potential therapeutic agents to prevent and treat CVD.CVD is importantly linked to endothelial cell dysfunction. Endothelial cell dysfunction is associated with conditions, such as obesity, smoking, diabetes, and hormonal changes. The vascular endothelium mediates many important physiological functions, such as responses to shear stress, angiogenesis, vascular remodeling, inflammation, and coagulation. It also participates in metabolic and synthetic processes. 3 A key modulator of endothelial cell activity is nitric oxide (NO), which under physiological conditions is mainly produced by the endothelial nitric oxide synthase (eNOS) isoform. NO regulates vascular tone, proliferation of vascular smooth muscle cells, and hemostasis, among other important functions. Disruptions in the physiological production of NO triggers endothelial cell dysfunction, resulting in an increased susceptibility to CVD. 4 Therefore, strategies aimed at "physiologically" increasing NO bioavailability are promising for the prevention and therapy of CVD.The induction of NO synthesis by flavonoid-containing compounds has received widespread attention, as their effects appear to positively impact CVD. Epidemiological studies indicate that the regular dietary intake of plant-derived foods and beverages high in flavonoids is inversely associated with the incidence of CVD. 5 Although these compounds are pleiotropic in nature, many of their effects may be explained by improving endothelial function. The regular consumption of cacao products high in flavonoid content has also been demonstrated to provide beneficial cardiovascular effects. 5 Natural cacao products are rich in monomeric and polymeric forms of the flavonoids (Ϫ)-epicatechin (EPI) and catechin (60/40 ratio) and can contain up to 10% flavonoids by weight. 6 The consumption of cacao products can ameliorate

In diabetes mellitus, alterations in cardiac structure/function in the absence of ischemic heart disease, hypertension or other cardiac pathologies is termed diabetic cardiomyopathy. In the United States, the prevalence of diabetes mellitus continues to rise and the disease currently affects about 8% of the general population. Hence, it is imperative the use of appropriate diagnostic strategies for diabetic cardiomyopathy, which may help correctly identify the disease at early stages and implement suitable corrective therapies. Currently, there is no single diagnostic method for the identification of diabetic cardiomyopathy. Diabetic cardiomyopathy is known to induce changes in cardiac structure such as, myocardial hypertrophy, fibrosis and fat droplet deposition. Early changes in cardiac function are typically manifested as abnormal diastolic function that with time leads to loss of contractile function. Echocardiography based methods currently stands as the preferred diagnostic approach for diabetic cardiomyopathy, due to its wide availability and economical use. In addition to conventional techniques, magnetic resonance imaging and spectroscopy along with contrast agents are now leading new approaches in the diagnosis of myocardial fibrosis, and cardiac and hepatic metabolic changes. These strategies can be complemented with serum biomarkers so they can offer a clear picture as to diabetes-induced changes in cardiac structure/function even at very early stages of the disease. This review article intends to provide a summary of experimental and routine tools currently available to diagnose diabetic cardiomyopathy induced changes in cardiac structure/function. These tools can be reliably used in either experimental models of diabetes or for clinical applications.

The consumption of cacao-derived (i.e., cocoa) products provides beneficial cardiovascular effects in healthy subjects as well as individuals with endothelial dysfunction such as smokers, diabetics, and postmenopausal women. The vascular actions of cocoa are related to enhanced nitric oxide (NO) production. These actions can be reproduced by the administration of the cacao flavanol (Ϫ)-epicatechin (EPI). To further understand the mechanisms behind the vascular action of EPI, we investigated the effects of Ca 2ϩ depletion on endothelial nitric oxide (NO) synthase (eNOS) activation/phosphorylation and translocation. Human coronary artery endothelial cells were treated with EPI or with bradykinin (BK), a well-known Ca 2ϩ -dependent eNOS activator. Results demonstrate that both EPI and BK induce increases in intracellular calcium and NO levels. However, under Ca 2ϩ -free conditions, EPI (but not BK) is still capable of inducing NO production through eNOS phosphorylation at serine 615, 633, and 1177. Interestingly, EPI-induced translocation of eNOS from the plasmalemma was abolished upon Ca 2ϩ depletion. Thus, under Ca 2ϩ -free conditions, EPI can stimulate NO synthesis independent of calmodulin binding to eNOS and of its translocation into the cytoplasm. We also examined the effect of EPI on the NO/cGMP/vasodilator-stimulated phosphoprotein (VASP) pathway activation in isolated Ca 2ϩ -deprived canine mesenteric arteries. Results demonstrate that under these conditions, EPI induces the activation of this vasorelaxation-related pathway and that this effect is inhibited by pretreatment with nitro-L-arginine methyl ester, suggesting a functional relevance for this phenomenon.

Polyphenolic compounds of the flavanoid family are abundantly present in cacao seed and its cocoa products. Results from studies using cocoa products indicate beneficial effects of flavanols on cardiovascular endpoints. Evidence indicates that (-)-epicatechin is the main cacao flavanol associated with cardiovascular effects, so the accurate quantification of its content in cacao seeds or cocoa products is important. Common methods for the quantification of phenolic content in cocoa products are based on the reaction of phenols with colorimetric reagents such as the FolinCiocalteu (FC) In this study, we compared the FC method of phenolic determinations using 2 different standards (gallic acid and (-)-epicatechin) to construct calibration curves. We compare these results with those obtained from a simple fluorometric method (Ex 280 /Em 320 nm) used to determine catechin/(-)-epicatechin content in samples of cacao seeds and cocoa products. Values obtained from the FC method determination of polyphenols yield an overestimation of phenol (flavonoid) content when gallic acid is used as standard. Moreover, the epicatechin is a more reliable standard because of its abundance in cacao seeds and cocoa products. The use of fluorometric spectra yields a simple and highly quantitative means for a more precise and rapid quantification of cacao catechins. Fluorometric values are essentially in agreement with those reported using more cumbersome methods. In conclusion, the use of fluorescence emission spectra is a quick, practical and suitable means to quantifying catechins in cacao seeds and cocoa products.

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