Background-Nebivolol and carvedilol are third-generation ␤-adrenoreceptor antagonists, which unlike classic ␤-blockers, have additional endothelium-dependent vasodilating properties specifically related to microcirculation by a molecular mechanism that still remains unclear. We hypothesized that nebivolol and carvedilol stimulate NO release from microvascular endothelial cells by extracellular ATP, which is a well-established potent autocrine and paracrine signaling factor modulating a variety of cellular functions through the activation of P2-purinoceptors. Methods and Results-Contraction and relaxation of renal glomerular vasculature were measured by determination of intracapillary volume with [ 3 H]-inulin. Biologically active NO was measured with highly sensitive porphyrinic NO microsensors in a single glomerular endothelial cell (GEC). Extracellular ATP was measured by a luciferin-luciferase assay. Enzymatic degradation of extracellular ATP by apyrase and blockade of P2Y-purinoceptors by suramin or reactive blue 2 inhibited both ␤-blocker-induced glomerular vasorelaxations and ␤-blocker-stimulated NO release from GECs. Both ␤-blocker-induced vasorelaxations were in the micromolar concentration range identical to that required for the ␤-blocker stimulation of ATP and NO release from GECs. The maximum of NO release for nebivolol and carvedilol was very similar (188Ϯ14 and 226Ϯ17, respectively). Blockade of ATP release by a mechanosensitive ion channel blocker, Gd 3ϩ , inhibited the ␤-blocker-dependent release of ATP and NO from GECs. Conclusions-These results demonstrate for the first time that nebivolol and carvedilol induce relaxation of renal glomerular microvasculature through ATP efflux with consequent stimulation of P2Y-purinoceptor-mediated NO

Background-Alterations in endothelial function may contribute to increased susceptibility of black Americans to cardiovascular disease. The ability to pharmacologically reverse endothelial dysfunction in blacks was tested with nebivolol, a ␤ 1 -selective agent with vasodilating and antioxidant properties. Methods and Results-The effects of nebivolol on endothelial nitric oxide (NO), superoxide (O 2 Ϫ ), and peroxynitrite concentration (ONOO Ϫ ) release were studied in human umbilical vein endothelial cells and iliac artery endothelial cells isolated from age-matched black and white donors. Kinetics and concentrations of NO/O 2 Ϫ /ONOO Ϫ were measured simultaneously with nanosensors from single cells and shown to have significant interracial differences. The rate of NO release was Ϸ5 times slower in blacks than in whites (94 versus 505 nmol · L Ϫ1 · s

Background-The prevalence of the endothelium-impaired function disorders, such as hypertension and diabetes mellitus, and the severity of their complications are considerably greater in blacks than whites. Evidence has accumulated that superoxide (O 2 Ϫ ) production and its interaction with nitric oxide (NO), yielding the strong oxidant peroxynitrite (ONOO Ϫ ), play central roles in vascular pathophysiology. We hypothesized that the differences in endothelial NO/O 2 Ϫ /ONOO Ϫ metabolism may highlight the potential predisposition to endothelial dysfunction and cardiovascular complications prevalent in blacks. Methods and Results-Highly sensitive tandem electrochemical NO/O 2 Ϫ /ONOO Ϫ nanosensors were positioned in single human umbilical vein endothelial cells (HUVECs) isolated from blacks and whites, and the kinetics of NO/O 2 Ϫ /ONOO

Abstract-This study investigated the process of nitric oxide (NO) release from platelets after stimulation with different angiotensin II type 1 (AT 1 )-receptor antagonists and its effect on platelet adhesion and aggregation. Angiotensin II AT 1 -receptor antagonist-stimulated NO release in platelets was compared with that in human umbilical vein endothelial cells by using a highly sensitive porphyrinic microsensor. In vitro and ex vivo effects of angiotensin II AT 1 -receptor antagonists on platelet adhesion to collagen and thromboxane A 2 analog U46619-induced aggregation were evaluated. Losartan, EXP3174, and valsartan alone caused NO release from platelets and endothelial cells in a dose-dependent manner in the range of 0.01 to 100 mol/L, which was attenuated by NO synthase inhibitor N G -nitro-L-arginine methyl ester. The angiotensin II AT 1 -receptor antagonists had more than 70% greater potency in NO release in platelets than in endothelial cells. The degree of inhibition of platelet adhesion (collagen-stimulated) and aggregation (U46619-stimulated) elicited by losartan, EXP3174, and valsartan, either in vitro or ex vivo, closely correlated with the NO levels produced by each of these drugs alone. The inhibiting effects of angiotensin II AT 1 -receptor antagonists on collagen-stimulated adhesion and U46619-stimulated aggregation of platelets were significantly reduced by pretreatment with N G -nitro-L-arginine methyl ester. Neither the AT 2 receptor antagonist PD123319, the cyclooxygenase synthase inhibitor indomethacin, nor the selective thromboxane A 2 /prostaglandin H 2 receptor antagonist SQ29,548 had any effect on angiotensin II AT 1 -receptor antagonist-stimulated NO release in platelets and endothelial cells. Key Words: platelets Ⅲ nitric oxide Ⅲ endothelium Ⅲ angiotensin II Ⅲ angiotensin antagonist P latelets play an important role in arterial thrombosis and the onset of acute myocardial infarction after atherosclerotic plaque rupture. Inhibition of platelet aggregation has become a critical step in preventing thrombotic events that are associated with stroke, heart attack, and peripheral arterial thrombosis. 1 Thrombosis is a multicellular event in which other cells, such as endothelial cells, are involved in the regulation of platelet reactivity. During the past several years, clear evidence has emerged that a concerted action of nitric oxide (NO) generated by either endothelial or platelet NO synthases regulates platelet activation, causing inhibition of adhesion and aggregation. 2,3 Recently developed nonpeptide angiotensin II (Ang II) AT 1 -receptor antagonists (AT 1 -As) make up a new generation of antihypertensive agents that also modulate hemostasis, 4,5 and apparently this effect is not solely a result of Ang II-receptor blockage. Ang II induces an early phase of platelet activation 6 and increases secretion of plasminogen activator inhibitor type I from vascular endothelial cells, 7 whereas AT 1 -As inhibit the vasoconstrictor and platelet aggregation effects induced by tromboxane A 2 (T...

Background-Although statins preserve endothelial function by reducing serum cholesterol levels, it has been suggested they may also stimulate nitric oxide (NO) synthase in endothelium with concurrent increase in superoxide (O 2 Ϫ ) generation, leading to impairment of NO activity. Therefore, measurements of biologically active NO and O 2 Ϫ in endothelium after exposure to the HMG-CoA reductase inhibitor cerivastatin were undertaken to evaluate its potential effect on NO biological activity. Methods and Results-Highly sensitive electrochemical NO and O 2Ϫ microsensors were placed near the surface of a single human umbilical vein endothelial cell, and the kinetics of NO and O 2 Ϫ release were recorded in vitro. Cerivastatin demonstrated a time-dependent effect on NO release in endothelial cells. The initial release (approximately the first 3 minutes) was concentration-dependent (0.01 to 10 mol/L) and was similar to that observed for typical NO synthase agonists calcium ionophore or acetylcholine. Cerivastatin stimulated NO release at a favorable rate and scavenged O 2 Ϫ , which led to the preservation of the active concentration of NO. The sustained effect (after Ϸ6 hours) of cerivastatin on endothelium was associated with an Ϸ35% increase in NO release as compared with the initial effect. In contrast to the initial effect, the sustained effect of cerivastatin was shown at concentrations Ϸ100-fold lower and was dependent on inhibition of endothelial HMG-CoA reductase. Conclusions-These data provide direct evidence to prove that in the presence of cerivastatin, the NOS system in endothelium operates with high efficiency toward increasing NO

Cyclic peptides containing the Arg-Gly-Asp (RGD) sequence have been shown to specifically bind the angiogenesis biomarker α V β 3 integrin. We report the synthesis, chemical characterization, and biological evaluation of two novel dimeric cyclic RGD-based molecular probes for the targeted imaging of α V β 3 activity (a radiolabeled version, 64Cu-NOTA-PEG4-cRGD2, for PET imaging, and a fluorescent version, FITC-PEG4-cRGD2, for in vitro work). We investigated the performance of this probe at the receptor, cell, organ, and whole-body levels, including its use to detect diabetes associated impairment of ischemia-induced myocardial angiogenesis. Both versions of the probe were found to be stable, demonstrated fast receptor association constants, and showed high specificity for α V β 3 in HUVECs (K d ~ 35 nM). Dynamic PET-CT imaging indicated rapid blood clearance via kidney filtration, and accumulation within α V β 3-positive infarcted myocardium. 64Cu-NOTA-PEG4-cRGD2 demonstrated a favorable biodistribution, slow washout, and excellent performance with respect to the quality of the PET-CT images obtained. Importantly, the ratio of probe uptake in infarcted heart tissue compared to normal tissue was significantly higher in non-diabetic rats than in diabetic ones. Overall, our probes are promising agents for non-invasive quantitative imaging of α V β 3 expression, both in vitro and in vivo.

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Atopic dermatitis is a heterogeneous disease, in which the pathogenesis is associated with mutations in genes encoding epidermal structural proteins, barrier enzymes, and their inhibitors; the role of genes regulating innate and adaptive immune responses and environmental factors inducing the disease is also noted. Recent studies point to the key role of epigenetic changes in the development of the disease. Epigenetic modifications are mainly mediated by DNA methylation, histone acetylation, and the action of specific non-coding RNAs. It has been documented that the profile of epigenetic changes in patients with atopic dermatitis (AD) differs from that observed in healthy people. This applies to the genes affecting the regulation of immune response and inflammatory processes, e.g., both affecting Th1 bias and promoting Th2 responses and the genes of innate immunity, as well as those encoding the structural proteins of the epidermis. Understanding of the epigenetic alterations is therefore pivotal to both create new molecular classifications of atopic dermatitis and to enable the development of personalized treatment strategies.