Arginase inhibition by (-)-Epicatechin reverses endothelial cell aging.

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.

(-)-Epicatechin induced reversal of endothelial cell aging and improved vascular function: underlying mechanisms.

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 (ß-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 ß-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.

Effects of indomethacin on prostanoid synthesis and vasomotor responsiveness in endothelium-denuded abdominal and thoracic aortas of Wistar rats.

In endothelium-denuded abdominal (but not thoracic) aortas of rats, the nonselective cyclooxygenase (COX) inhibitor, indomethacin, suppressed contractions evoked by α-adrenergic agonists hypothetically mediated by prostanoids. We aimed to identify these non-endothelial-derived contractile prostanoids released by α-adrenergic receptors activation. Endothelium-denuded abdominal and thoracic aortas of Wistar rats were used for biochemical and functional analyses. Western blot analysis showed that COX-1 and COX-2 protein levels were respectively equivalent in endothelium-denuded abdominal and thoracic aortas. Enzyme immunoassay data supported direct evidence of phenylephrine-stimulated release of prostanoids (PGI2, PGE2, and PGF2α) by thoracic and abdominal aortas without endothelium, and their almost complete inhibition by 1 µM indomethacin. Isometric force measurements established that 10 µM indomethacin-but no lower concentrations-inhibited the contractions evoked by phenylephrine in endothelium-denuded abdominal aorta. In this preparation, 10 µM indomethacin also depressed the contractions provoked by angiotensin II and high K+ (80 mM). In fact, indomethacin (up to 1 mM) caused concentration-dependent reductions in all abovementioned contractile responses. In endothelium-denuded thoracic aortas, however, only 1 mM indomethacin significantly depressed the contractile activity stimulated by either phenylephrine, angiotensin II, or high K+. Hence, there was a clear quantitative difference in response to indomethacin between abdominal and thoracic aortas without endothelium. Altogether, the results indicate that prostanoids induced by phenylephrine in abdominal and thoracic aortas were derived from non-endothelial COX-mediated metabolism; notably, the decrease in prostanoid synthesis could not account for the inhibition of vasoconstrictor responses by indomethacin: Through COX-independent actions, indomethacin inhibited aortic smooth muscle contractility.

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.

Activation of upregulated angiotensin II type 2 receptors decreases carotid pulse pressure in rats with suprarenal abdominal aortic coarctation.

Our aim was to determine whether angiotensin type 2 receptors (AT2R) are involved in the depression of carotid pulse pressure (PP) in rats with suprarenal aortic coarctation (SrC). We tested the effects of losartan, PD123319, and CGP42112 on PP in SrC and Sham-operated anesthetized rats. PP increased in SrC rats. Neither losartan nor PD123319 affected PP in SrC and Sham-operated rats. CGP42112 reduced PP, in SrC rats, combined with losartan. Moreover, PD123319 blocked this effect. AT2R protein increased in the thoracic aortas of SrC rats. Thus, upregulated AT2R stimulation by CGP42112 mediates depression of PP in rats under pressure overloading.

Leptin mediated ObRb receptor increases expression of adhesion intercellular molecules and cyclooxygenase 2 on murine aorta tissue inducing endothelial dysfunction.

There are several reports showing that hyperleptinemia positively correlates with atherogenic process including promotion of platelet aggregation, thrombosis, and production of inflammatory cytokines. Thereby endothelial dysfunction takes place and underlies metabolic and vascular alterations that contribute to the development of both cardiovascular disease and type 2 diabetes. It has been also proposed that endothelial dysfunction may antecede the development of insulin resistance and diabetes. Endothelial cells participate in vasoregulation by the modulation of nitric oxide production and prostaglandin; in addition these cells are major vector in angiogenesis and recruitment of leukocytes and adhesion molecules. Nevertheless mechanisms underlying vascular dysfunction remain to be fully elucidated. This experiment in vitro revealed that the addition of leptin to cultivated endothelial cells elicited a significant molecular expression of both COX 2 and ICAM-1: in addition, the response showed a positive relationship with leptin concentration and the time of incubation. Thus, it may be suggested that leptin acts directly on the endothelium by activating its specific receptor which in turn initiates the molecular response related with the production of factors involved in the inflammatory response. Alterations on prostaglandins and recruiting molecules of adhesion are relevant stages of the endothelial damage.

Paragonimiasis pulmonar en el Río Canande, Provincia de Esmeraldas / Pulmonary Paragonimiasis in the Canande River, Esmerald Province

Se estudiaron 137 muestras de esputo de adultos que habitan en un área endémica para paragonimiasis en laprovincia de Esmeraldas. En 10 individuos (7.11 por ciento) se observaron huevos de Paragonimus mexicanus en el esputo...