Adam M. Jacoby

Nebivolol Reduces Nitroxidative Stress and Restores Nitric Oxide Bioavailability in Endothelium of Black Americans

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 &bgr;1-selective agent with vasodilating and antioxidant properties. Methods and Results— The effects of nebivolol on endothelial nitric oxide (NO), superoxide (O2−), and peroxynitrite concentration (ONOO−) 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/O2−/ONOO− were measured simultaneously with nanosensors from single cells and shown to have significant interracial differences. The rate of NO release was ≈5 times slower in blacks than in whites (94 versus 505 nmol · L−1 · s−1), whereas the rates of release were faster by ≈2 times for O2− and ≈4 times for ONOO− (22.1 versus 9.4 nmol · L−1 · s−1 for O2− and 810 versus 209 nmol · L−1 · s−1 for ONOO−). Pretreatment with 1.0 to 5.0 &mgr;mol/L nebivolol restored NO bioavailability in endothelial cells from black donors with concurrent reductions in O2− and ONOO− release, similar to levels in the endothelium of whites. The effects of nebivolol were dose-dependent and not observed with atenolol; similar effects were observed with apocynin, an NAD(P)H oxidase inhibitor. Conclusions— Reduced endothelial NO bioavailability in American blacks is mainly due to excessive O2− and ONOO− generation by NAD(P)H and uncoupled endothelial NO synthase. Nebivolol decreased O2− and ONOO− concentrations and restored NO bioavailability in blacks to the level recorded in cells from whites, independently of &bgr;1-selective blockade.

Amorphous silica nanoparticles trigger nitric oxide/peroxynitrite imbalance in human endothelial cells: inflammatory and cytotoxic effects.

Background The purpose of this study was to investigate the mechanism of noxious effects of amorphous silica nanoparticles on human endothelial cells. Methods Nanoparticle uptake was examined by transmission electron microscopy. Electrochemical nanosensors were used to measure the nitric oxide (NO) and peroxynitrite (ONOO−) released by a single cell upon nanoparticle stimulation. The downstream inflammatory effects were measured by an enzyme-linked immunosorbent assay, real-time quantitative polymerase chain reaction, and flow cytometry, and cytotoxicity was measured by lactate dehydrogenase assay. Results We found that the silica nanoparticles penetrated the plasma membrane and rapidly stimulated release of cytoprotective NO and, to a greater extent, production of cytotoxic ONOO−. The low [NO]/[ONOO−] ratio indicated increased nitroxidative/oxidative stress and correlated closely with endothelial inflammation and necrosis. This imbalance was associated with nuclear factor κB activation, upregulation of key inflammatory factors, and cell death. These effects were observed in a nanoparticle size-dependent and concentration-dependent manner. Conclusion The [NO]/[ONOO−] imbalance induced by amorphous silica nanoparticles indicates a potentially deleterious effect of silica nanoparticles on vascular endothelium.

Amorphous silica nanoparticles aggregate human platelets: potential implications for vascular homeostasis

Background Amorphous silica nanoparticles (SiNP) can be used in medical technologies and other industries leading to human exposure. However, an increased number of studies indicate that this exposure may result in cardiovascular inflammation and damage. A high ratio of nitric oxide to peroxynitrite concentrations ([NO]/[ONOO−]) is crucial for cardiovascular homeostasis and platelet hemostasis. Therefore, we studied the influence of SiNP on the platelet [NO]/[ONOO−] balance and platelet aggregation. Methods Nanoparticle–platelet interaction was examined using transmission electron microscopy. Electrochemical nanosensors were used to measure the levels of NO and ONOO− released by platelets upon nanoparticle stimulation. Platelet aggregation was studied using light aggregometry, flow cytometry, and phase contrast microscopy. Results Amorphous SiNP induced NO release from platelets followed by a massive stimulation of ONOO− leading to an unfavorably low [NO]/[ONOO−] ratio. In addition, SiNP induced an upregulation of selectin P expression and glycoprotein IIb/IIIa activation on the platelet surface membrane, and led to platelet aggregation via adenosine diphosphate and matrix metalloproteinase 2-dependent mechanisms. Importantly, all the effects on platelet aggregation were inversely proportional to nanoparticle size. Conclusions The exposure of platelets to amorphous SiNP induces a critically low [NO]/[ONOO−] ratio leading to platelet aggregation. These findings provide new insights into the pharmacological profile of SiNP in platelets.

Ultraviolet B Light‐induced Nitric Oxide/Peroxynitrite Imbalance in Keratinocytes—Implications for Apoptosis and Necrosis

Elevation of nitric oxide (NO˙) can either promote or inhibit ultraviolet B light (UVB)‐induced apoptosis. In this study, we determined real‐time concentration of NO˙ and peroxynitrite (ONOO−) and their role in regulation of membrane integrity and apoptosis. Nanosensors (diameter 300–500 nm) were used for direct in situ simultaneous measurements of NO˙ and ONOO− generated by UVB in cultured keratinocytes and mice epidermis. An exposure of keratinocytes to UVB immediately generated ONOO− at maximal concentration of 190 nm followed by NO˙ release with a maximal concentration of 91 nm. The kinetics of UVB‐induced NO˙/ONOO− was in contrast to cNOS agonist stimulated NO˙/ONOO− from keratinocytes. After stimulating cNOS by calcium ionophore (CaI), NO˙ release from keratinocytes was followed by ONOO− production. The [NO˙] to [ONOO−] ratio generated by UVB decreased below 0.5 indicating a serious imbalance between cytoprotective NO˙ and cytotoxic ONOO−—a main component of nitroxidative stress. The NO˙/ONOO− imbalance increased membrane damage and cell apoptosis was partially reversed in the presence of free radical scavenger. The results suggest that UVB‐induced and cNOS‐produced NO˙ is rapidly scavenged by photolytically and enzymatically generated superoxide (O2˙−) to produce high levels of ONOO−, which enhances oxidative injury and apoptosis of the irradiated cells.

Effects of angiotensin receptor blockers on endothelial nitric oxide release: the role of eNOS variants

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • Angiotensin II receptor blockers improve endothelial cell-dependent vasodilation in patients with hypertension through suppression of angiotensin II type 1 receptors but may have additional and differential effects on endothelial nitric oxide synthase (eNOS) function. WHAT THIS STUDY ADDS • The key finding from this study is that angiotensin II receptor blockers (ARBs) differentially enhanced nitric oxide (NO) release in a manner influenced by certain genetic variants of eNOS. This finding provides new insights into the effects of ARBs on endothelial cell-dependent vasodilation and eNOS function that are of high importance in vascular medicine and clinical pharmacology. AIM Angiotensin II receptor blockers (ARBs) improve endothelial cell (EC)-dependent vasodilation in patients with hypertension through suppression of angiotensin II type 1 receptors but may have additional and differential effects on endothelial nitric oxide (NO) synthase (eNOS) function. To investigate this question, we tested the effects of various ARBs on NO release in ECs from multiple donors, including those with eNOS genetic variants linked to higher cardiovascular risk. METHODS The effects of ARBs (losartan, olmesartan, telmisartan, valsartan), at 1 µm, on NO release were measured with nanosensors in human umbilical vein ECs obtained from 18 donors. NO release was stimulated with calcium ionophore (1 µm) and its maximal concentration was correlated with eNOS variants. The eNOS variants were determined by a single nucleotide polymorphism in the promoter region (T-786C) and in the exon 7 (G894T), linked to changes in NO metabolism. RESULTS All of the ARBs caused an increase in NO release as compared with untreated samples (P < 0.01, n= 4-5 in all eNOS variants). However, maximal NO production was differentially influenced by eNOS genotype. Olmesartan increased maximal NO release by 30%, which was significantly greater (P < 0.01, n= 4-5 in all eNOS variants) than increases observed with other ARBs. CONCLUSIONS The ARBs differentially enhanced NO release in ECs in a manner influenced by eNOS single nucleotide polymorphisms. These findings provide new insights into the effects of ARBs on EC-dependent vasodilation and eNOS function.