The glycocalyx (Gcx) is a complex and poorly understood structure covering the luminal surface of endothelial cells. It is known to be a determinant of vascular rheology and permeability and may be a key control site for the vascular injuries caused by ischemia-reperfusion (I/R). We used intravital-microscopy to evaluate the effects of I/R injury on two properties of Gcx in mouse cremasteric microvessels: exclusion of macromolecules (anionic-dextrans) and intracapillary distribution of red blood cells (RBC). In this model, the Gcx is rapidly modified by I/R injury with an increase in 70-kDa anionic-dextran penetration without measurable effect on the penetration of 580-kDa anionic-dextran or on RBC exclusion. The effects of I/R injury appear to be mediated by the rapid production of reactive oxygen species (ROS) because they are ameliorated by the addition of exogenous superoxide dismutase-catalase. Intravenous application of allopurinol or heparin also inhibited the effects of I/R injury, and we interpret efficacy of allopurinol as evidence for a role for xanthine-oxidoreductase (XOR) in the response to I/R injury. Heparin, which is hypothesized to displace XOR from a heparin-binding domain in the Gcx, reduced the effects of I/R. The effects of I/R injury were also partially prevented or fully reversed by the intravascular infusion of exogenous hyaluronan. These data demonstrate: 1) the liability of Gcx during I/R injury; 2) the importance of locally produced ROS in the injury to Gcx; and 3) the potential importance of heparin-binding sites in modulating the ROS production. Our findings further highlight the relations between glycosaminoglycans and the pathophysiology of Gcx in vivo.

Background The age-related decline in mass, strength, and performance of skeletal muscle is associated with loss of independence, falls risk, disability, institutionalization, and death. Methods To determine whether a cocoa supplement enriched in flavonoids can improve plasma markers of oxidative stress and inflammation, physical performance and frailty in middle-aged and older subjects, we conducted a two-phase, randomized, double-blind, clinical trial. The initial study included 60 subjects (55- to 70-year-old) allocated into placebo (P), highly alkalinized (no-flavonoid; NF), or flavonoid-rich natural cocoa (F) beverage groups. The follow-up study included 74 older subjects (65- to 90-year-old) randomly distributed into NF or F groups. Subjects were instructed to consume the beverages once/day for up to 12-weeks. A comprehensive (aging relevant) set of end points were assessed, which included mean change in blood plasma metabolic and oxidative stress indicators, in physical performance tests and quality of life (QoL). Results In the initial study, the F group showed improved glycemia, triglyceridemia, High-density lipoprotein cholesterol, Low-density lipoprotein cholesterol, triglyceridemia/HDL index, and oxidative markers. Performance on the Up and Go test, skeletal muscle index, and quality of life also improved. In the follow-up study, F treatment was associated with significant improvements in metabolic, oxidative stress, and inflammatory endpoints and positive effects on physical performance, frailty indicators, and quality of life (F vs. NF group). Conclusions Regular flavonoids consumption positively affects blood oxidative stress and inflammation end points, cardiometabolic risk markers, physical performance, and quality of life. The sum of such effects may help to mitigate the extent of frailty development in the elderly people. Trial Registration NCT03585868

Postprandial hyperglycemia, in particular when accompanied by excessive hypertriglyceridemia, is associated with increased cardiovascular risk, mainly in overweight or obese subjects, as it favors oxidative stress, systemic inflammation and endothelial dysfunction. Thus, treatments that favorably modulate metabolism by reducing steep increases in postprandial serum glucose and triglycerides, are of considerable interest. Evidence suggests that (−)-epicatechin (EPI) is responsible for reductions in cardiometabolic risk associated with chocolate consumption these effects may be associated with favorable effects of EPI on postprandial metabolism. The aims of this study were to assess the effects of EPI on postprandial metabolism in normal-weight and overweight/obese subjects. Twenty adult volunteers (normal and overweight) underwent oral metabolic tolerance tests in the absence and presence of oral EPI (1 mg/kg). Metabolic responses were examined using indirect calorimetry and determining blood glucose and triglycerides at 0, 2 and 4 hours after metabolic load ingestion. Results show that EPI increased postprandial lipid catabolism, as evidenced by a significant decrease in the respiratory quotient, which implies an increase in fat oxidation. The effect was associated with significantly lower postprandial plasma glucose and triglycerides concentrations. The effects were more prominent in overweight subjects. In conclusion, EPI modulates postprandial metabolism by enhancing lipid oxidation accompanied by reductions in glycemia and triglyceridemia.

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 mitochondria 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.

Endothelial luminal glycocalyx (ELG) is a multifunctional complex structure made off of a diversity of glycosilated proteins, and glycosaminoglycans (GAG). Coronary ELG may participate as a sensor of coronary flow (CF) to induce inotropic and dromotropic effects. In isolated perfused guinea pig heart we tested the role of glycosidic groups of glycans bound to proteins and GAG of the ELG on CF-induced inotropic and dromotropic effects. To study the role of saccharide related groups of certain glycans, they were removed by selective enzyme hydrolysis or bound to a selective plant lectin. CF-induced positive inotropic and positive dromotropic control curves were obtained and the effects of intracoronary infusion of enzyme or lectin determined. The analyzed groups were as follow: 1) Fucosidase enzyme and Ulex europeasus lectin; hydrolysis and binding respectively (H&Br) to alpha-linked fucosyl related groups. 2). Endoglycanase-H and Lycopersicon esculentum (H&Br to N-linked beta-1,3GlcNAc related groups). 3) O-glycanase and Arachis hypogea (H&Br to O-linked beta-Gal1, 3GalNac related groups). 4) Sialidase and Maackia amurensis (H&Br to neuraminic acid related groups). In treatments 1-3 both. lectin and corresponding enzyme, equally depressed CF-positive dromotropic effects without affecting positive inotropic effects. In treatment 4 both lectin and enzyme equally depressed CF-positive inotropic effects without dromotropic effects. The differential role of GAG hyaluran or heparan groups on CF-positive inotropism and positive dromotropism respectively was shown. Infusing hyaluranidase removed hyaluran that solely inhibited CF- inotropism while removal of heparan with heparinase solely inhibited CF-dromotropism. Only the effects of hyaluronidase were reversed infusing hyaluronidate. Our results indicate glycans of ELG are elements of complex multimolecular sensors of coronary flow.

17Beta-estradiol (E2) plays an important role in Ca2+ fluxes in several cell types. It has been proposed that some of its effects are of nongenomic origin E2 at vascular smooth muscle level can block calcium entry through L-type calcium channels, this mechanism cannot include vascular endothelial cells (VECs), in which increases in the intracellular calcium concentration ([Ca2+]i) are necessary to NO synthesis. We used male rat aorta ECs in culture loaded with fura-2 and a fluorescence imaging system to evaluate the short-term effects of E2 on [Ca2+]i kinetics. We explored the participation of the intracellular steroid receptor on the effects induced by E2, using tamoxifen (1 microM) and ICI 182,780 (10 microM). Our results showed that E2 (like bradykinin) induced an increase in [Ca2+]i. Such agonist-like effects showed a biphasic curve behavior. The 17beta-estradiol effects were not modified by the presence of the intracellular estradiol-receptor antagonist tamoxifen, but it is blocked in the presence of the ICI 182,780. The 17beta-estradiol effects were obtained even with restriction of steroid-free diffusion into cells (17beta-estradiol-bovine serum albumin). Phospholipase Cbeta activity is involved in these effects, because U-73122, a PLCbeta inhibitor, blocked E2 effects. All E2 effects were of rapid onset (milliseconds), exerted at the membrane level, and of rapid offset. We conclude that estradiol can influence the endothelium physiologic responses through effects of nongenomic origin.

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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.