Eicosapentaenoic Acid Improves Endothelial Nitric Oxide Bioavailability Via Changes in Protein Expression During Inflammation.

BACKGROUND: Endothelial cell (EC) dysfunction involves reduced nitric oxide (NO) bioavailability due to NO synthase uncoupling linked to increased oxidation and reduced cofactor availability. Loss of endothelial function and NO bioavailability are associated with inflammation, including leukocyte activation. Eicosapentaenoic acid (EPA) administered as icosapent ethyl reduced cardiovascular events in REDUCE-IT (Reduction of Cardiovascular Events With Icosapent Ethyl-Intervention Trial) in relation to on-treatment EPA blood levels. The mechanisms of cardiovascular protection for EPA remain incompletely elucidated but likely involve direct effects on the endothelium. METHODS AND RESULTS: In this study, human ECs were treated with EPA and challenged with the cytokine IL-6 (interleukin-6). Proinflammatory responses in the ECs were confirmed by ELISA capture of sICAM-1 (soluble intercellular adhesion molecule-1) and TNF-α (tumor necrosis factor-α). Global protein expression was determined using liquid chromatography-mass spectrometry tandem mass tag. Release kinetics of NO and peroxynitrite were monitored using porphyrinic nanosensors. IL-6 challenge induced proinflammatory responses from the ECs as evidenced by increased release of sICAM-1 and TNF-α, which correlated with a loss of NO bioavailability. ECs pretreated with EPA modulated expression of 327 proteins by >1-fold (P<0.05), compared with IL-6 alone. EPA augmented expression of proteins involved in NO production, including heme oxygenase-1 and dimethylarginine dimethylaminohydrolase-1, and 34 proteins annotated as associated with neutrophil degranulation. EPA reversed the endothelial NO synthase uncoupling induced by IL-6 as evidenced by an increased [NO]/[peroxynitrite] release ratio (P<0.05). CONCLUSIONS: These direct actions of EPA on EC functions during inflammation may contribute to its distinct cardiovascular benefits.

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.

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.