Using Metalloporphyrin Nanosensors for In Situ Monitoring and Measurement of Nitric Oxide and Peroxynitrite in a Single Human Neural Progenitor Cell.

Nitric oxide (NO) is an inorganic signaling molecule that plays a crucial role in the regulation of numerous physiological functions. An oxidation product of the cytoprotective NO is cytotoxic peroxynitrite (ONOO-). In biological systems, the concentrations of NO and ONOO- are typically transient, ranging from nanomolar to micromolar, and these increases are normally followed by a swift return to their basal levels due to their short life spans. To understand the vital physiological role of NO and ONOO- in vitro and in vivo, sensitive and selective methods are necessary for direct and continuous NO and ONOO- measurements in real time. Because electrochemical methods can be adjusted for selectivity, sensitivity, and biocompatibility in demanding biological environments, they are suitable for real-time monitoring of NO and ONOO- release. Metalloporphyrin nanosensors, described here, have been designed to measure the concentration of NO and ONOO- produced by a single human neural progenitor cell (hNPC) in real time. These nanosensors (200-300 nm in diameter) can be positioned accurately in the proximity of 4-5 ± 1 µm from an hNPC membrane. The response time of the sensors is better than a millisecond, while detection limits for NO and ONOO- are 1 × 10-9 and 3 × 10-9 mol/L, respectively, with a linear concentration response of up to about 1 µM. The application of these metalloporphyrin nanosensors for the efficient measurement of the concentrations of NO and ONOO- in hNPCs is demonstrated, providing an opportunity to observe in real time the molecular changes of the two signaling molecules in situ.

Eicosapentaenoic acid (EPA) reduces pulmonary endothelial dysfunction and inflammation due to changes in protein expression during exposure to particulate matter air pollution.

AIMS: Inhalation of air pollution small particle matter (PM) is a leading cause of cardiovascular (CV) disease. Exposure to PMs causes endothelial cell (EC) dysfunction as evidenced by nitric oxide (NO) synthase uncoupling, vasoconstriction and inflammation. Eicosapentaenoic acid (EPA) has been shown to mitigate PM-induced adverse cardiac changes in patients receiving omega-3 fatty acid supplementation. We set out to determine the pro-inflammatory effects of multiple PMs (urban and fine) on pulmonary EC NO bioavailability and protein expression, and whether EPA restores EC function under these conditions. METHODS AND RESULTS: We pretreated pulmonary ECs with EPA and then exposed them to urban or fine air pollution PMs. LC/MS-based proteomic analysis to assess relative expression levels. Expression of adhesion molecules was measured by immunochemistry. The ratio of NO to peroxynitrite (ONOO-) release, an indication of eNOS coupling, was measured using porphyrinic nanosensors following calcium stimulation. Urban/fine PMs also modulated 9/12 and 13/36 proteins, respectively, linked to platelet and neutrophil degranulation pathways and caused > 50% (p < 0.001) decrease in the stimulated NO/ONOO- release ratio. EPA treatment altered expression of proteins involved in these inflammatory pathways, including a decrease in peroxiredoxin-5 and an increase in superoxide dismutase-1. EPA also increased expression of heme oxygenase-1 (HMOX1), a cytoprotective protein, by 2.1-fold (p = 0.024). EPA reduced elevations in sICAM-1 levels by 22% (p < 0.01) and improved the NO/ONOO- release ratio by > 35% (p < 0.05). CONCLUSION: These cellular changes may contribute to anti-inflammatory, cytoprotective and lipid changes associated with EPA treatment during air pollution exposure.

Omega-3 and omega-6 fatty acids have distinct effects on endothelial fatty acid content and nitric oxide bioavailability.

Treatment with high dose icosapent ethyl (IPE), an ethyl ester of the omega-3 fatty acid eicosapentaenoic acid (EPA), significantly reduced ischemic events in patients with either cardiovascular disease (CV) or diabetes plus other risk factors (REDUCE-IT) but the mechanism is not well understood.  We compared the effects of EPA, docosahexaenoic acid (DHA), and the omega-6 fatty acid arachidonic acid (AA) on bioavailability of nitric oxide (NO) and fatty acid composition. Human umbilical vein endothelial cells (HUVECs) were pretreated with EPA, DHA, or AA (10 µM). Cells were stimulated with calcium ionophore and NO and peroxynitrite (ONOO-) were measured using porphyrinic nanosensors. Levels of EPA, DHA, AA and other fatty acids were measured by gas chromatography (GC). EPA treatment caused the greatest NO release (18%, p < 0.001) and reduction in ONOO- (13%, p < 0.05) compared to control; the [NO]/[ ONOO-] ratio increased by 35% (p < 0.001). DHA treatment increased NO levels by 12% (p < 0.01) but had no effect on ONOO- release. AA did not affect either NO or ONOO- release.  Fatty acid treatments increased their respective levels in endothelial cells.  EPA levels increased 10-fold to 4.59 mg/g protein (p < 0.001) with EPA treatment and the EPA/AA ratio increased by 10-fold (p < 0.001) compared to vehicle.  Only EPA increased docosapentaenoic acid (DPA, omega-3) levels by 2-fold (p < 0.001). AA alone decreased the EPA/AA ratio 4-fold (p<0.001). These findings support a preferential benefit of EPA on endothelial function and omega-3 fatty acid content.

Variable Effects Of LDL Subclasses Of Cholesterol On Endothelial Nitric Oxide/Peroxynitrite Balance - The Risks And Clinical Implications For Cardiovascular Disease.

BACKGROUND: Elevated levels of low density lipoprotein (LDL), "bad cholesterol", is not an accurate indicator of coronary disease. About 75% of patients with heart attacks have cholesterol levels that do not indicate a high risk for a cardiovascular event. LDL is comprised of three subclasses, with particles of different size and density. We used nanomedical systems to elucidate the noxious effects of LDL subclasses on endothelium. EXPERIMENTAL: Nanosensors were employed to measure the concentrations of nitric oxide (NO) and peroxynitrite (ONOO-) stimulated by LDL subclasses in HUVECs. N-LDL and ox-LDL (subclass A: 1.016-1.019 g/mL, subclass I: 1.024-1.029 g/mL, and subclass B: 1.034-1.053 g/mL) stimulated NO and ONOO- release. The concentrations ratio of (NO)/(ONOO-) was used to evaluate the noxious effects of the subclasses on endothelium. RESULTS: In HUVECs, the (NO)/(ONOO-) ratio for normal endothelium is about 5, but shifts to 2.7±0.4, 0.5±0.1, and 0.9±0.1 for subclasses A, B, and I, respectively. Ratios below 1.0 indicate an imbalance between NO and ONOO-, affecting endothelial function. LDL of 50% B and 50% I produced the most severe imbalance (0.45±0.04), whereas LDL of 60% A, 20% B, and 20% I had the most favorable balance of 5.66±0.69. Subclass B significantly elevated the adhesion of molecules and monocytes. The noxious effect was significantly higher for ox-LDL than n-LDL. CONCLUSION: Subclass B of "bad cholesterol" is the most damaging to endothelial function and can contribute to the development of atherosclerosis. Contrary to the current national guidelines, this study suggests that it's not the total LDL, rather it is the concentration of subclass B in relation to subclasses A and/or I, that should be used for diagnosis of atherosclerosis and the risk of heart attack. By utilizing specific pharmacological therapy to address the concentration of subclass B, there is a potential to significantly reduce the risk of heart attack and atherosclerosis.

A prospective study of contrast preservation using ultra-low contrast delivery technique versus standard automated contrast injector system in coronary procedures.

BACKGROUND: We aimed to assess the decrease in contrast media volume (CMV) with ultra-low contrast delivery technique (ULCD) developed at our institution versus the usual automated contrast injector system (ACIS) contrast delivery in coronary procedures. METHODS: We analyzed the amount of contrast given in the consecutive 204 patients of the operators who use ULCD technique versus consecutive 200 patients of the other operators who use ACIS without ULCD technique for coronary angiograms and/or percutaneous coronary interventions (PCIs) from May 2017 to July 2018 at our center. We calculated the mean CMV between these groups. RESULTS: We observed a significant reduction in mean CMV with ULCD technique versus standard ACIS, respectively: angiogram 24.8 ± 15.8 mL (n = 194) vs 42.3 ± 25.1 mL (n = 200) (p < 0.0001); PCI 23.5 ± 19.7 mL (n = 52) vs 48.2 ± 30.8 mL (n = 16) (p < 0.0070); angiogram with ad hoc PCI 53.4 ± 32.1 mL (n = 23) vs 89.7 ± 35.6 mL (n = 16) (p < 0.0024); and overall angiogram and PCI 27.4 ± 20.5 mL (n = 204) vs 44.9 ± 28.0 mL (n = 181) (p < 0.0001). CONCLUSION: Our study showed a highly significant reduction in CMV using ULCD technique compared to standard ACIS contrast delivery in coronary invasive procedures. Even in the standard ACIS arm, CMV was significantly lower than values reported in literature, possibly due to operators' bias toward contrast preservation.

Eicosapentaenoic acid improves endothelial function and nitric oxide bioavailability in a manner that is enhanced in combination with a statin.

The endothelium exerts many vasoprotective effects that are largely mediated by release of nitric oxide (NO). Endothelial dysfunction represents an early but reversible step in atherosclerosis and is characterized by a reduction in the bioavailability of NO. Previous studies have shown that eicosapentaenoic acid (EPA), an omega-3 fatty acid (O3FA), and statins individually improve endothelial cell function, but their effects in combination have not been tested. Through a series of in vitro experiments, this study evaluated the effects of a combined treatment of EPA and the active metabolite of atorvastatin (ATM) on endothelial cell function under conditions of oxidative stress. Specifically, the comparative and time-dependent effects of these agents on endothelial dysfunction were examined by measuring the levels of NO and peroxynitrite (ONOO-) released from human umbilical vein endothelial cells (HUVECs). The data suggest that combined treatment with EPA and ATM is beneficial to endothelial function and was unique to EPA and ATM since similar improvements could not be recapitulated by substituting another O3FA docosahexaenoic acid (DHA) or other TG-lowering agents such as fenofibrate, niacin, or gemfibrozil. Comparable beneficial effects were observed when HUVECs were pretreated with EPA and ATM before exposure to oxidative stress. Interestingly, the kinetics of EPA-based protection of endothelial function in response to oxidation were found to be significantly different than those of DHA. Lastly, the beneficial effects on endothelial function generated by combined treatment of EPA and ATM were reproduced when this study was expanded to an ex vivo model utilizing rat glomerular endothelial cells. Taken together, these findings suggest that a combined treatment of EPA and ATM can inhibit endothelial dysfunction that occurs in response to conditions such as hyperglycemia, oxidative stress, and dyslipidemia.

Nanomedical studies of the restoration of nitric oxide/peroxynitrite balance in dysfunctional endothelium by 1,25-dihydroxy vitamin D3 - clinical implications for cardiovascular diseases.

BACKGROUND: Clinical studies indicate that vitamin D3 improves circulation and may have beneficial effects in hypertension. This study uses nanomedical systems to investigate the role of 1,25-dihydroxy vitamin D3 in the preservation/restoration of endothelial function in an angiotensin II (Ang II) cellular model of hypertension. METHODS: 1,25-dihydroxy vitamin D3-stimulated nitric oxide (NO) and peroxynitrite (ONOO-) concentrations were measured in situ with nanosensors (200-300 mm diameter with a detection limit of 1 nM) in human umbilical vein endothelial cells of African American (AA) and Caucasian American (CA) donors exposed to Ang II. The balance/imbalance between NO and ONOO- concentrations ([NO]/[ONOO-]) was simultaneously monitored and used as an indicator of endothelial nitric oxide synthase (eNOS) uncoupling and endothelial dysfunction. RESULTS: [NO]/[ONOO-] imbalance in Ang II-stimulated dysfunctional endothelium was 0.20±0.16 for CAs and 0.11±0.09 for AAs. Uncoupled eNOS and overexpression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase contributed to high production of ONOO-. Vitamin D3 treatment reversed [NO]/[ONOO-] to 3.0±0.1 in CAs and 2.1±0.1 in AAs - exceeding that observed in normal endothelium. Vitamin D3 restored uncoupled eNOS and endothelial function by increasing cytoprotective NO and decreasing the cytotoxic ONOO-. The beneficial effect of vitamin D3 is associated with a favorable rate of NO and ONOO- release, restoration of the [NO]/[ONOO-] and the overall decrease in the overexpression of eNOS, inducible nitric oxide synthase and NADPH oxidase. This effect of vitamin D3 may prove to be beneficial in the treatment of hypertension and other cardiovascular diseases, including heart failure, myocardial infarction, vasculopathy, stroke and diabetes.

Metformin treatment decreases nitroxidative stress, restores nitric oxide bioavailability and endothelial function beyond glucose control.

Reduction of nitric oxide (NO), a potent vasodilator, and an increase in cytotoxic peroxynitrite (ONOO-) may be associated with the uncoupling of NO synthase (eNOS) and endothelial cell (EC) dysfunction. In addition to its effect on glucose control, metformin, may also directly benefit in the restoration of the function of eNOS and EC. Obese Zucker rats were administered vehicle or 300 mg/kg/day metformin for 4 weeks. NO concentration [NO] and ONOO- concentration [ONOO-] were measured in aortic and glomerular endothelial cells from Zucker rats in vitro. Compared with controls, aortic and glomerular endothelial [NO] was reduced by 32% and 41%, while [ONOO-] release increased 79% and 69%, respectively. Metformin treatment increased aortic and glomerular endothelial [NO] by 37% and 57%, respectively, while decreasing [ONOO-] by 32% and 34%, compared with vehicle-treated animals. Treatment with metformin significantly restored the balance in the [NO]/[ONOO-] ratio with 101% and 138% increase for aortic and glomerular endothelial cells, respectively. Fasting glucose levels were not significantly changed. These findings indicate that metformin therapy has a direct and beneficial effect on arterial and renal EC function in obese rats, including enhanced NO release and reduced nitroxidative stress, beyond any effects on fasting glucose levels.

Development of novel FP-based probes for live-cell imaging of nitric oxide dynamics.

Nitric oxide () is a free radical with a wide range of biological effects, but practically impossible to visualize in single cells. Here we report the development of novel multicoloured fluorescent quenching-based probes by fusing a bacteria-derived -binding domain close to distinct fluorescent protein variants. These genetically encoded probes, referred to as geNOps, provide a selective, specific and real-time read-out of cellular dynamics and, hence, open a new era of bioimaging. The combination of geNOps with a Ca(2+) sensor allowed us to visualize and Ca(2+) signals simultaneously in single endothelial cells. Moreover, targeting of the probes was used to detect signals within mitochondria. The geNOps are useful new tools to further investigate and understand the complex patterns of signalling on the single (sub)cellular level.

The Abdominal Aortic Aneurysm and Intraluminal Thrombus: Current Concepts of Development and Treatment.

The pathogenesis of the abdominal aortic aneurysm (AAA) shows several hallmarks of atherosclerotic and atherothrombotic disease, but comprises an additional, predominant feature of proteolysis resulting in the degradation and destabilization of the aortic wall. This review aims to summarize the current knowledge on AAA development, involving the accumulation of neutrophils in the intraluminal thrombus and their central role in creating an oxidative and proteolytic environment. Particular focus is placed on the controversial role of heme oxygenase 1/carbon monoxide and nitric oxide synthase/peroxynitrite, which may exert both protective and damaging effects in the development of the aneurysm. Treatment indications as well as surgical and pharmacological options for AAA therapy are discussed in light of recent reports.

Using nanosensors for in situ monitoring and measurement of nitric oxide and peroxynitrite in a single cell.

The cytotoxic peroxynitrite (ONOO(-)) is an oxidation product of the cytoprotective nitric oxide (NO). Our studies support the hypothesis that the concentration ratio of NO and ONOO(-), [NO]/[ONOO(-)] can be a marker of nitroxidative imbalance, which subsequently correlates well with endothelial dysfunction and dysfunction of the cardiovascular system. Nanosensors, described here, have been used for simultaneous monitoring and measurement of NO and ONOO(-) release from a single endothelial cell. These nanosensors, with a diameter of 200-300 nm, can be positioned accurately in close proximity of 5-10 µm from the endothelial cell membrane. The response time of the sensors is better than a millisecond and the detection limit is 10(-9) M, with a linear concentration response of up to about 2 µM. The application of these sensors for the measurement of the balance and imbalance of [NO]/[ONOO(-)] in normal and dysfunctional endothelium is demonstrated.

Amlodipine increased endothelial nitric oxide and decreased nitroxidative stress disproportionately to blood pressure changes.

BACKGROUND: Clinical trials have shown that amlodipine reduces cardiovascular events at a rate that is not predicted by changes in brachial arterial pressure alone. These findings may be explained, in part, by the pleiotropic effects of amlodipine on endothelial cell (EC) function. In this study, we elucidated the effect of amlodipine on nitric oxide (NO) bioavailability and cytotoxic peroxynitrite (ONOO(-)) and blood pressure (BP). METHODS: Spontaneously hypertensive rats (SHRs) were treated with vehicle or amlodipine (5 mg/kg/day) for 8 weeks and compared with untreated, baseline rats. NO and ONOO(-) release from aortic and glomerular ECs were measured ex vivo using amperometric nanosensors following maximal stimulation with calcium ionophore. BP was measured using the tail-cuff method. RESULTS: As compared with baseline, vehicle treatment had reduced aortic endothelial NO release from 157 ± 11 nM to 55 ± 6 nM and increased ONOO(-) from 69 ± 7 nM to 156 ± 19 nM. The NO/ONOO(-) ratio, a comprehensive measurement of eNOS function, decreased from 2.3 ± 0.3 to 0.3 ± 0.1. Compared with vehicle, amlodipine treatment restored NO to 101 ± 3 nM, decreased ONOO(-) to 50 ± 4 nM, and increased the NO/ONOO(-) ratio to 2.0 ± 0.2, a level similar to baseline. Similar changes were observed for glomerular ECs. Mean arterial blood pressure increased from 149 ± 3 mm Hg (baseline) to 174 ± 1 mm Hg (vehicle). Amlodipine slightly, but significantly, decreased mean arterial blood pressure to 167 ± 3 mm Hg vs. vehicle treatment. CONCLUSIONS: Amlodipine increased NO bioavailability and decreased nitroxidative stress in SHRs with EC dysfunction disproportionately to BP changes. These direct, vascular effects of amlodipine on EC function may contribute to reduced risk for atherothrombotic events as observed in clinical trials.

Effect of glyceryl trinitrate on staphylococcus aureus growth and leukocyte activation during simulated extracorporeal circulation.

BACKGROUND: Previously, nitric oxide has been shown to possess antimicrobial effects. In this study, we aim to test the effect of glyceryl trinitrate (GTN) on Staphylococcus aureus growth during simulated extracorporeal circulation (SECC) and also to examine the effect of S. aureus, alone and in combination with GTN, on activation markers of the innate immune system during SECC. METHODS: In an in vitro system of SECC, we measured GTN-induced changes in markers of leukocyte activation in whole blood caused by S. aureus infestation, as well as the effect of GTN on S. aureus growth. RESULTS: GTN had no effect on S. aureus growth after 240 minutes SECC. Staphylococcus aureus reduced the expression of granulocyte Fcγ-receptor CD32 but stimulated the expression of monocyte CD32. Staphylococcus aureus stimulated expression of some leukocyte adhesion key proteins, activation marker CD66b, lipopolysaccharide-receptor CD14, and C3b-receptor CD35. Staphylococcus aureus and GTN addition induced significant increases in monocyte CD63 (lysosomal granule protein) levels. CONCLUSION: GTN does not affect S. aureus growth during SECC and has no effect on SECC-induced leukocyte activation.

The favorable kinetics and balance of nebivolol-stimulated nitric oxide and peroxynitrite release in human endothelial cells.

BACKGROUND: Nebivolol is a third-generation beta-blocker used to treat hypertension. The vasodilation properties of nebivolol have been attributed to nitric oxide (NO) release. However, the kinetics and mechanism of nebivolol-stimulated bioavailable NO are not fully understood. METHODS: Using amperometric NO and peroxynitrite (ONOO⁻) nanosensors, ß3-receptor (agonist: L-755,507; antagonists: SR59230A and L-748,337), ATP efflux (the mechanosensitive ATP channel blocker, gadolinium) and P2Y-receptor (agonists: ATP and 2-MeSATP; antagonist: suramin) modulators, superoxide dismutase and a NADPH oxidase inhibitor (VAS2870), we evaluated the kinetics and balance of NO and ONOO⁻ stimulated by nebivolol in human umbilical vein endothelial cells (HUVECs). NO and ONOO⁻ were measured with nanosensors (diameter ~ 300 nm) placed 5 ± 2 µm from the cell membrane and ATP levels were determined with a bioluminescent method. The kinetics and balance of nebivolol-stimulated NO and ONOO⁻ were compared with those of ATP, 2-MeSATP, and L-755,507. RESULTS: Nebivolol stimulates endothelial NO release through ß3-receptor and ATP-dependent, P2Y-receptor activation with relatively slow kinetics (75 ± 5 nM/s) as compared to the kinetics of ATP (194 ± 10 nM/s), L-755,507 (108 ± 6 nM/s), and 2-MeSATP (105 ± 5 nM/s). The balance between cytoprotective NO and cytotoxic ONOO- was expressed as the ratio of [NO]/[ONOO⁻] concentrations. This ratio for nebivolol was 1.80 ± 0.10 and significantly higher than that for ATP (0.80 ± 0.08), L-755,507 (1.08 ± 0.08), and 2-MeSATP (1.09 ± 0.09). Nebivolol induced ATP release in a concentration-dependent manner. CONCLUSION: The two major pathways (ATP efflux/P2Y receptors and ß3 receptors) and several steps of nebivolol-induced NO and ONOO⁻ stimulation are mainly responsible for the slow kinetics of NO release and low ONOO⁻. The net effect of this slow kinetics of NO is reflected by a favorable high ratio of [NO]/[ONOO⁻] which may explain the beneficial effects of nebivolol in the treatment of endothelial dysfunction, hypertension, heart failure, and angiogenesis.

Deletion of the activated protein-1 transcription factor JunD induces oxidative stress and accelerates age-related endothelial dysfunction.

BACKGROUND: Reactive oxygen species are major determinants of vascular aging. JunD, a member of the activated protein-1 family of transcription factors, is emerging as a major gatekeeper against oxidative stress. However, its contribution to reactive oxygen species homeostasis in the vasculature remains unknown. METHODS AND RESULTS: Endothelium-dependent vasorelaxation was impaired in young and old JunD(-/-) mice (6 and 22 months old) compared with age-matched wild-type mice. JunD(-/-) mice displayed an age-independent decline in endothelial nitric oxide release and endothelial nitric oxide synthase activity and increased mitochondrial superoxide formation and peroxynitrite levels. Furthermore, vascular expression and activity of the free radical scavengers manganese and extracellular superoxide dismutase and aldehyde dehydrogenase 2 were reduced, whereas the NADPH oxidase subunits p47phox, Nox2, and Nox4 were upregulated. These redox changes were associated with premature vascular aging, as shown by reduced telomerase activity, increased ß-galactosidase-positive cells, upregulation of the senescence markers p16(INK4a) and p53, and mitochondrial disruption. Interestingly, old wild-type mice showed a reduction in JunD expression and transcriptional activity resulting from promoter hypermethylation and binding with tumor suppressor menin, respectively. In contrast, JunD overexpression blunted age-induced endothelial dysfunction. In human endothelial cells, JunD knockdown exerted a similar impairment of the O2(-)/nitric oxide balance that was prevented by concomitant NADPH inhibition. In parallel, JunD expression was reduced in monocytes from old versus young healthy subjects and correlated with mRNA levels of scavenging and oxidant enzymes. CONCLUSIONS: JunD provides protection in aging-induced endothelial dysfunction and may represent a novel target to prevent reactive oxygen species-driven vascular aging.

Dipeptidyl peptidase-4 inhibition with saxagliptin enhanced nitric oxide release and reduced blood pressure and sICAM-1 levels in hypertensive rats.

Most patients with diabetes also have hypertension, a risk factor associated with atherothrombotic disease and characterized by endothelial cell (EC) dysfunction and loss of nitric oxide (NO) bioavailability. Recent studies suggest a possible antihypertensive effect with dipeptidyl peptidase-4 (DPP4) inhibition; however, the underlying mechanism is not understood. In this study, we tested the effects of the DPP4 inhibitor, saxagliptin, on EC function, blood pressure, and soluble intercellular adhesion molecule 1 (sICAM-1) levels in hypertensive rats. Spontaneously hypertensive rats were treated with vehicle or saxagliptin (10 mg·kg(-1)·day(-1)) for 8 weeks. NO and peroxynitrite (ONOO(-)) release from aortic and glomerular ECs was stimulated with calcium ionophore and measured using electrochemical nanosensor technology. Changes in EC function were correlated with fasting glucose levels. Saxagliptin treatment was observed to increase aortic and glomerular NO release by 22% (P < 0.001) and 23% (P < 0.001), respectively, with comparable reductions in ONOO(-) levels; the NO/ONOO(-) ratio increased by >50% in both EC types (P < 0.001) as compared with vehicle. Saxagliptin also reduced mean arterial pressure from 170 ± 10 to 158 ± 10 mm Hg (P < 0.001) and decreased sICAM-1 levels by 37% (P < 0.01). The results of this study suggest that DPP4 inhibition reduces blood pressure and inflammation in hypertensive rats while increasing NO bioavailability.

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.

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.

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.

Poly(ADP-ribose) polymerase-1 protects from oxidative stress induced endothelial dysfunction.

BACKGROUND: Generation of reactive oxygen species (ROS) is a key feature of vascular disease. Activation of the nuclear enzyme poly (adenosine diphosphate [ADP]-ribose) polymerase-1 (PARP-1) is a downstream effector of oxidative stress. METHODS: PARP-1(-/-) and PARP-1(+/+) mice were injected with paraquat (PQ; 10 mg/kg i.p.) to induce intracellular oxidative stress. Aortic rings were suspended in organ chambers for isometric tension recording to analyze vascular function. RESULTS: PQ treatment markedly impaired endothelium-dependent relaxations to acetylcholine in PARP-1(-/-), but not PARP-1(+/+) mice (p<0.0001). Maximal relaxation was 45% in PQ treated PARP-1(-/-) mice compared to 79% in PARP-1(+/+) mice. In contrast, endothelium-independent relaxations to sodium nitroprusside (SNP) were not altered. After PQ treatment, l-NAME enhanced contractions to norepinephrine by 2.0-fold in PARP-1(-/-) mice, and those to acetylcholine by 3.3-fold, respectively, as compared to PARP-1(+/+) mice. PEG-superoxide dismutase (SOD) and PEG-catalase prevented the effect of PQ on endothelium-dependent relaxations to acetylcholine in PARP-1(-/-) mice (p<0.001 vs. PQ treated PARP-1(+/+) mice. Indomethacin restored endothelium-dependent relaxations to acetylcholine in PQ treated PARP-1(-/-) mice (p<0.05 vs. PQ treated PARP-1(+/+). CONCLUSION: PARP-1 protects from acute intracellular oxidative stress induced endothelial dysfunction by inhibiting ROS induced production of vasoconstrictor prostanoids.