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1 Endothelial cells, under given circumstances, can initiate contraction (constriction) of the vascular smooth muscle cells that surround them. Such endothelium‐dependent, acute increases in contractile tone can be due to the withdrawal of the production of nitric oxide, to the production of vasoconstrictor peptides (angiotensin II, endothelin‐1), to the formation of oxygen‐derived free radicals (superoxide anions) and/or the release of vasoconstrictor metabolites of arachidonic acid. The latter have been termed endothelium‐derived contracting factor (EDCF) as they can contribute to moment‐to‐moment changes in contractile activity of the underlying vascular smooth muscle cells. 2 To judge from animal experiments, EDCF‐mediated responses are exacerbated by aging, spontaneous hypertension and diabetes. 3 To judge from human studies, they contribute to the blunting of endothelium‐dependent vasodilatations in aged subjects and essential hypertensive patients. 4 Since EDCF causes vasoconstriction by activation of the TP‐receptors on the vascular smooth muscle cells, selective antagonists at these receptors prevent endothelium‐dependent contractions, and curtail the endothelial dysfunction in hypertension and diabetes. British Journal of Pharmacology (2005) 144, 449–458. doi:10.1038/sj.bjp.0706042

Endothelial cells regulate vascular tone by releasing various contracting and relaxing factors including nitric oxide (NO), arachidonic acid metabolites (derived from cyclooxygenases, lipoxygenases, and cytochrome P450 monooxygenases), reactive oxygen species, and vasoactive peptides. Additionally, another pathway associated with the hyperpolarization of the underlying smooth muscle cells plays a predominant role in resistance arteries. Endothelial dysfunction is a multifaceted disorder, which has been associated with hypertension of diverse etiologies, involving not only alterations of the L-arginine NO-synthase–soluble guanylyl cyclase pathway but also reduced endothelium-dependent hyperpolarizations and enhanced production of contracting factors, particularly vasoconstrictor prostanoids. This brief review highlights these different endothelial pathways as potential drug targets for novel treatments in hypertension and the associated endothelial dysfunction and end-organ damage.

Endothelial dysfunction is a common feature of hypertension, and it results from the imbalanced release of endothelium-derived relaxing factors (EDRFs; in particular, nitric oxide) and endothelium-derived contracting factors (EDCFs; angiotensin II, endothelins, uridine adenosine tetraphosphate, and cyclooxygenase-derived EDCFs). Thus, drugs that increase EDRFs (using direct nitric oxide releasing compounds, tetrahydrobiopterin, or l -arginine supplementation) or decrease EDCF release or actions (using cyclooxygenase inhibitor or thromboxane A2/prostanoid receptor antagonists) would prevent the dysfunction. Many conventional antihypertensive drugs, including angiotensin-converting enzyme inhibitors, calcium channel blockers, and third-generation β-blockers, possess the ability to reverse endothelial dysfunction. Their use is attractive, as they can address arterial blood pressure and vascular tone simultaneously. The severity of endothelial dysfunction correlates with the development of coronary artery disease and predicts future cardiovascular events. Thus, endothelial dysfunction needs to be considered as a strategic target in the treatment of hypertension.

Sodium nitrite (NaNO 2 ) induces relaxation in isolated arteries partly through an endothelium-dependent mechanism involving NO-eNOS-sGC-cGMP pathway. The present study was designed to investigate the effect of chronic NaNO 2 administration on arterial systolic blood pressure (SBP) and vascular function in hypertensive rats. NaNO 2 (150 mg L−1) was given in drinking water for four weeks to spontaneously (SHR) and Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) treated hypertensive SD rats. Arterial SBP and vascular function in isolated aortae were studied. Total plasma nitrate/nitrite and vascular cyclic guanosine monophosphate (cGMP) levels were measured using commercially available assay kits. Vascular nitric oxide (NO) levels were evaluated by DAF-FM fluorescence while the proteins involved in endothelial nitric oxide synthase (eNOS) activation was determined by Western blotting. NaNO 2 treatment reduced SBP, improved the impaired endothelium-dependent relaxation, increased plasma total nitrate/nitrite level and vascular tissue NO and cGMP levels in SHR. Furthermore, increased presence of phosphorylated eNOS and Hsp-90 was observed in NaNO 2 -treated SHR. The beneficial effect of nitrite treatment was not observed in L-NAME treated hypertensive SD rats. The present study provides evidence that chronic treatment of genetically hypertensive rats with NaNO 2 improves endothelium-dependent relaxation in addition to its antihypertensive effect, partly through mechanisms involving activation of eNOS.

The endothelium mediates relaxations (dilatations) of the underlying vascular smooth muscle cells. The endothelium-dependent relaxations are due to the release of non-prostanoid vasodilator substances. The best characterized endothelium-derived relaxing factor (EDRF) is nitric oxide (NO). The endothelial cells also release substances (endothelium-derived hyperpolarizing factor, EDHF) that cause hyperpolarization of the cell membrane of the underlying vascular smooth muscle. The release of EDRF from the endothelium can be mediated by both pertussis toxin-sensitive G i (alpha 2 -adrenergic activation, serotonin, thrombin) and insensitive G q (adenosine diphosphate, bradykinin) coupling proteins. The ability of the endothelial cell to release relaxing factors can be upregulated by impregnation with estrogens, exercise and antioxidants, and down-regulated by oxidative stress and increased presence of oxidized LDL. Following injury or apoptotic death, the endothelium regenerates. However, in regenerated endothelial cells, there is an early selective loss of the pertussis-toxin sensitive mechanisms of EDRF-release. Functional studies suggest that abnormal handling of LDL because of increased oxidative stress play a key role in this selective loss. Genomic analysis demonstrates the emergence of fatty acid binding protein-A (A-FBP) and metalloproteinase-7 (MMP7) in regenerated endothelial cells. The reduced release of NO resulting from the endothelial dysfunction in regenerated areas creates a locus minoris resistentiae which favors the occurrence of vasospasm and thrombosis as well as the initiation of atherosclerosis.

To determine whether or not the antioxidants N-acetylcysteine (NAC) and allopurinol (ALP) confer synergistic cardioprotection against myocardial ischemia/reperfusion (MI/R) injury by stabilizing hypoxia inducible factor 1α (HIF-1α)/heme oxygenase 1 (HO-1) signaling in diabetic myocardium. Control or diabetic [streptozotocin (STZ)-induced] Sprague Dawley rats received vehicle or NAC, ALP or their combination for four weeks starting one week after STZ injection. The animals were then subjected to thirty minutes of coronary artery occlusion followed by two hours reperfusion in the absence or presence of the selective HO-1 inhibitor, tin protoporphyrin-IX (SnPP-IX) or the HIF-1α inhibitor 2-Methoxyestradiol (2ME2). Cardiomyocytes exposed to high glucose were subjected to hypoxia/re-oxygenation in the presence or absence of HIF-1α and HO-1 achieved by gene knock-down with related siRNAs. Myocardial and plasma levels of 15-F2t-isoprostane, an index of oxidative stress, were significantly increased in diabetic rats while cardiac HO-1 protein and activity were reduced; this was accompanied with reduced cardiac protein levels of HIF-1α, and increased post-ischemic myocardial infarct size and cellular injury. NAC and ALP given alone and in particular their combination normalized cardiac levels of HO-1 and HIF-1α protein expression and prevented the increase in 15-F2t-isoprostane, resulting in significantly attenuated post-ischemic myocardial infarction. NAC and ALP also attenuated high glucose-induced post-hypoxic cardiomyocyte death in vitro. However, all the above protective effects of NAC and ALP were cancelled either by inhibition of HO-1 or HIF-1α with SnPP-IX and 2ME2 in vivo or by HO-1 or HIF-1α gene knock-down in vitro. NAC and ALP confer synergistic cardioprotection in diabetes via restoration of cardiac HIF-1α and HO-1 signaling.

Rapid non-genomic effects of 17β-estradiol are elicited by the activation of different estrogen receptor-α isoforms. Presence of surface binding sites for estrogen have been identified in cells transfected with full-length estrogen receptor-α66 (ER66) and the truncated isoforms, estrogen receptor-α46 (ER46) and estrogen receptor-α36 (ER36). However, the binding affinities of the membrane estrogen receptors (mERs) remain unknown due to the difficulty of developing of stable mER-transfected cell lines with sufficient mER density, which has largely hampered biochemical binding studies. The present study utilized cell-free expression systems to determine the binding affinities of 17β-estradiol to mERs, and the relationship among palmitoylation, membrane insertion and binding affinities. Saturation binding assays of human mERs revealed that [³H]-17β-estradiol bound ER66 and ER46 with Kd values of 68.81 and 60.72 pM, respectively, whereas ER36 displayed no specific binding within the tested concentration range. Inhibition of palmitoylation or removal of the nanolipoprotein particles, used as membrane substitute, reduced the binding affinities of ER66 and ER46 to 17β-estradiol. Moreover, ER66 and ER46 bound differentially with some estrogen receptor agonists and antagonists, and phytoestrogens. In particular, the classical estrogen receptor antagonist, ICI 182,780, had a higher affinity for ER66 than ER46. In summary, the present study defines the binding affinities for human estrogen receptor-α isoforms, and demonstrates that ER66 and ER46 show characteristics of mERs. The present data also indicates that palmitoylation and membrane insertion of mERs are important for proper receptor conformation allowing 17β-estradiol binding. The differential binding of ER66 and ER46 with certain compounds substantiates the prospect of developing mER-selective drugs.

The endothelium can evoke relaxations (dilatations) of the underlying vascular smooth muscle, by releasing vasodilator substances. The best characterized endothelium-derived relaxing factor is nitric oxide (NO), which is synthesized by the endothelial isoform of nitric oxide synthase (eNOS). Endothelium-dependent relaxations involve both pertussis-toxin-sensitive G i (e.g., responses to serotonin, sphingosine 1-phosphate, alpha 2 -adrenergic agonists, and thrombin) and pertussis-toxin-insensitive G q (e.g., adenosine diphosphate and bradykinin) coupling proteins. eNOS undergoes a complex pattern of intracellular regulation, including post-translational modifications involving enzyme acylation and phosphorylation. eNOS is reversibly targeted to signal-transducing plasmalemmal caveolae where the enzyme interacts with a number of regulatory proteins, many of which are modified in cardiovascular disease states. The release of nitric oxide by the endothelial cell can be up- (e.g., by estrogens, exercise, and dietary factors) and down-regulated (e.g. oxidative stress, smoking, and oxidized low-density lipoproteins). It is reduced in the course of vascular disease (e.g., diabetes and hypertension). Arteries covered with regenerated endothelium (e.g. following angioplasty) selectively lose the pertussis-toxin-sensitive pathway for NO release which favors vasospasm, thrombosis, penetration of macrophages, cellular growth, and the inflammatory reaction leading to atherosclerosis. The unraveling of the complex interaction of the pathways regulating the presence and the activity of eNOS will enhance the understanding of the perturbations in endothelium-dependent signaling that are seen in cardiovascular disease states, and may lead to the identification of novel targets for therapeutic intervention.

Background: Aging, obesity, and diabetes favor vascular dysfunction. Endothelial activation of adenosine monophosphate-activated protein kinase (AMPK) has protective effects in diabetes. Methods: Mice with constitutive endothelial activation of AMPK (CA-AMPK) were given a high fat diet to induce obesity or kept on standard chow as lean controls for up to 2 years. A subset of obese animals was changed to standard chow after 30 weeks of high fat feeding. En­dothelium-dependent and endothelium-independent responses were examined by isometric tension recording. Results and Conclusion: Endothelium-dependent nitric oxide (NO)- and apamin plus charybdotoxin-sensitive relaxations were preserved and similar between aortic or renal arterial preparations of lean and obese CA-AMPK mice and their wild-type littermates. Despite comparable release of vasoconstrictor prostanoids, cyclooxygenase-dependent contractions were enhanced during NO synthase inhibition in carotid arterial rings of obese CA-AMPK mice. Contractions to the α 1 -adrenoceptor agonist phenylephrine were augmented in renal arteries of obese animals, a genotype-independent phenomenon reversible by weight loss. These data indicate a higher α 1 -adrenergic reactivity in renal arteries of aged mice with obesity. The current results highlight the potential of weight loss to alleviate vascular dysfunction. However, endothelial activation of the AMPK pathway in obesity may not be sufficient to prevent vascular dysfunction without lifestyle changes.

In the spontaneously hypertensive rat (SHR) and aging Wistar–Kyoto rats (WKY), acetylcholine releases an endothelium‐derived contracting factor (EDCF) produced by endothelial cyclooxygenase‐1, which stimulates thromboxane A2 receptors (TP receptors) on vascular smooth muscle. The purpose of the present study was to identify this EDCF by measuring changes in isometric tension and the release of various prostaglandins by acetylcholine. In isolated aortic rings of SHR, U 46619, prostaglandin (PG) H2, PGF2α, PGE2, PGD2, prostacyclin (PGI2) and 8‐isoprostane, all activate TP receptors of the vascular smooth muscle to produce a contraction (U 46619≫8‐isoprostane=PGF2α=PGH2>PGE2=PGD2>PGI2). The contractions produced by PGH2 and PGI2 were fast and transient, mimicking endothelium‐dependent contractions. PGI2 did not relax isolated aortic rings of WKY and SHR. Acetylcholine evoked the endothelium‐dependent release of thromboxane A2, PGF2α, PGE2, PGI2 and most likely PGH2 (PGI2≫PGF2αPGE2>TXA2>8‐isoprostane, PGD2). Dazoxiben abolished the production of thromboxane A2, but did not influence the endothelium‐dependent contractions to acetylcholine. The release of PGI2 was significantly larger in the aorta of SHR than in WKY, and the former was more sensitive to the contractile effect of PGI2 than the latter. The inhibition of PGI‐synthase was associated with an increase in PGH2 spillover and the enhancement of acetylcholine‐induced endothelium‐dependent contractions. Thus, in the aorta of SHR and aging WKY, the endothelium‐dependent contractions elicited by acetylcholine most likely involve the release of PGI2 with a concomitant contribution of PGH2. British Journal of Pharmacology (2005) 146, 834–845. doi:10.1038/sj.bjp.0706390