Effects of Mitochondrial-Targeted Antioxidants on Real-Time Blood Nitric Oxide and Hydrogen Peroxide Release in Acute Hyperglycemia Rats

Acute hyperglycemia in non-diabetic subjects can impair vascularendothelial function, causing decreased endothelium-derived nitric oxide (NO) release and increased reactive oxygen species (ROS), such assuperoxide and hydrogen peroxide (H2O2). Hyperglycemia may induce mitochondrial dysfunction leading to ROS production and exacerbation of vascular endothelial dysfunction. We investigated whether mitochondrial-targeted antioxidants mitigate acute hyperglycemia-induced oxidative stress and reduced blood NO. To test this hypothesis, blood NO or H2O2 levels were measured simultaneously using NO or H2O2 microsensors (100 µm) which were placed into the femoral veins of anesthesized male Sprague-Dawley rats. Acute hyperglycemia was induced by infusion 20% D-glucose intravenously with or without mitochondria-targeted antioxidants (mitoquinone: mitoQ, MW=1714 g/mol, 2.3 mg/Kg; SS-31: (D-Arg)-Dmt-Lys-Phe-Amide, MW=640g/mol, 2.7 mg/Kg) for 3 hours. We found that acute hyperglycemia (200 mg/dL) significantly increased blood H2O2 by 3.0±0.5 M (n=7) and reduced blood NO by 68.0±13.5 nM (n=9) compared to the saline group at end of infusion (both p\u3c0.05). MitoQ significantly attenuated hyperglycemia– induced H2O2 levels by 2.5±0.2 M (n=7) and increased blood NO levels by 59.3±9.7 nM (n=5) (both p\u3c0.05 compared to hyperglycemia). Similarly, SS-31 significantly reduced hyperglycemia-induced blood H2O2 level by 4.0±0.6 M (n=5) and enhanced blood NO levels by 52.8±7.7 nM (n=6) at end of infusion (both p\u3c0.05 compared to hyperglycemia). In summary, acute hyperglycemia induces mitochondria-derived ROS which in turn contribute to vascular endothelial dysfunction. Therefore, mitochondria-targeted antioxidants are useful to attenuate acute hyperglycemia-induced vascular endothelial dysfunction and oxidative stress

The role of endothelial nitric oxide synthase (eNOS) uncoupling in acute hyperglycemia – induced oxidative stress and vascular endothelial dysfunction by measuring blood nitric oxide and hydrogen peroxide in real-time

Acute hyperglycemia can impair vascular endothelial function in non-diabetic subjects in addition to diabetic patients. Decreased eNOS derived nitric oxide (NO) bioavailability and increased reactive oxygen species (ROS), such as superoxide (SO) and hydrogen peroxide (H2O2), are the major characteristics of vascular endothelial dysfunction. Furthermore, eNOS can change from coupled to an uncoupled status resulting in SO production instead of NO production. The role of eNOS uncoupling in acute hyperglycemia induced vascular dysfunction is unclear in vivo. In this study we hypothesized that acute hyperglycemia (200 mg/dL) would increase H2O2 and decrease NO release in blood relative to saline control. By contrast, 5,6,7,8-tetrahydrobiopterin (BH4, an essential cofactor of coupled eNOS) (MW=241.247 g/mol, 6.5 mg/kg) or L-arginine (the substrate of coupled eNOS) (MW=210.66 g/mol, 600 mg/kg) would attenuate acute hyperglycemia-induced blood NO/H2O2 change. However, 7,8-dihydrobiopterin (BH2, an oxidized form of BH4 and serves as a cofactor for uncoupled eNOS) (MW=239.231 g/mol, 4 mg/kg) will exacerbate acute hyperglycemia-induced blood NO/H2O2 change. Blood NO or H2O2 levels were measured simultaneously using calibrated NO or H2O2 microsensors (100 µm WPI Inc.) by placing them into the femoral veins of male Sprague-Dawley rats. The electrical traces were recorded at baseline and throughout 3 hours of infusion with saline or 20% D-glucose with or without a drug and converted into concentration based on the calibration curve. We found that acute hyperglycemia (200 mg/dL) significantly increased H2O2 (n=6) and reduced NO (n=6) blood levels compared to the saline group (n=7, p2 exacerbated hyperglycemia– induced increased H2O2 levels (n=7) and decreased NO levels (n=4) (p4 (n=6), significantly attenuated hyperglycemia– induced increased H2O2 levels and decreased NO levels (p2O2 (n=5) and NO (n=6) blood levels as BH4, showing significant reduction of blood H2O2 and enhancement of blood NO (p2O2 and reduced NO blood levels. Uncoupled eNOS serves as a significant source mediating acute hyperglycemia-induced vascular dysfunction. Therefore, promotion of eNOS coupling may be effective in protecting vascular endothelial function from hyperglycemic insult

Mitoquinone (mitoQ) Exerts Antioxidant Effects Independent of Mitochondrial Targeted Effects in Phorbol-12-myristate-13-acetate (PMA) or N-formyl-L-methiony-L-leucyl-L-phenylalanine (fMLP) Stimulated Polymorphonuclear Leukocyte (PMN) Superoxide (SO) Release

MitoQ is a mitochondrial-targeted coenzyme Q antioxidant analog that dose-dependently restored cardiac function and reduced infarct size in isolated perfused rat hearts subjected to ischemia reperfusion (I/R). Moreover, mitoQ also dose-dependently attenuated PMA stimulated PMN superoxide (SO) release at the same concentration (10uM) as the cardioprotective dose. NADPH oxidase is the principle source of PMN SO release. We speculate that mitoQ may exert antioxidant effects independent of the mitochondria. Therefore, we hypothesized that inhibition of mitoQ on PMN-SO release will be similar as other coenzyme Q analogs: coenzyme Q1 and decylubiquinone without affecting cell viability. SO release was measured spectrophotometrically from isolated rat PMNs measured by the reduction of ferricytochrome c and were stimulated with 100nM PMA. The absorbance was measured at 550 nm up to 360sec. Positive control samples were given SO dismutase (SOD; 10ug/ml) which inhibited PMA induced SO release by \u3e90%. MitoQ significantly inhibited SO release by 56 + 3% (10uM, n=10 ,

The Effects of Protein Kinase C Beta II Peptide Modulation on Superoxide Release in Rat Polymorphonuclear Leukocytes

Phorbol 12-myristate 13-acetate (PMA; a diacylglycerol mimetic) is known to augment polymorphonuclear leukocyte (PMN) superoxide (SO) release via protein kinase C (PKC) activation. However, the role of PKC beta II (βII) mediating this response is not known. It’s known that myristic acid (myr-) conjugation facilitates intracellular delivery of the cargo sequence, and that putative PKCβII activator and inhibitor peptides work by augmenting or attenuating PKCβII translocation to cell membrane substrates (e.g. NOX-2). Therefore, we hypothesize that myr- conjugated PKCβII peptide-activator (N-myr-SVEIWD; myr-PKCβ+) would increase PMA-induced rat PMN SO release, whereas, myr-PKCβII peptide-inhibitor (N-myr-SLNPEWNET; myr-PKCβ-) would attenuate this response compared to non-drug treated controls. Rat PMNs (5x106) were incubated for 15min at 370C in the presence/absence of myr-PKCβ+/- (20 μM) or SO dismutase (SOD;10μg/mL; n=8) as positive control. PMA (100nM) induced PMN SO release was measured spectrophotometrically at 550nm via reduction of ferricytochrome c for 390 sec. PMN SO release increased absorbance to 0.39±0.04 in non-drug treated controls (n=28), and 0.49±0.05 in myr-PKCβ+(n=16). This response was significantly increased from 180 seconds to 240 seconds (p\u3c0.05). By contrast, myr-PKCβ- (0.26±0.03; n=14) significantly attenuated PMA-induced SO release compared to non-drug controls and myr-PKCβ+ (p\u3c0.05). SOD-treated samples showed \u3e90% reduction of PMA-induced SO release and was significantly different from all groups (p\u3c0.01). Cell viability ranged between 94± to 98±2% in all groups as determined by 0.2% trypan blue exclusion. Preliminary results suggest that myr-PKCβ- significantly attenuates PMA-induced SO release, whereas myr-PKCβ+ significantly augments PMA-induced SO release, albeit transiently. Additional dose response and western blot experiments are planned with myr-PKCβ+/- in PMA-induced PMN SO release assays. This research was supported by the Department of Bio-Medical Sciences and the Division of Research at PCOM and by Young Therapeutics, LLC

Protein Kinase C Beta II Peptide Inhibitor Elicits Robust Effects on Attenuating Myocardial Ischemia/Reperfusion Injury

Reperfusion injury contributes to myocardial tissue damage following a heart attack partly due to the generation of reactive oxygen species (ROS) upon cardio-angioplasty. Protein kinase C beta II (PKCβII) inhibition during reperfusion with peptide inhibitor (N-myr-SLNPEWNET; PKCβII-) decreases ROS release and leukocyte infiltration in rat hind-limb and myocardial ischemia/reperfusion (I/R) studies, respectively. However, the role of activating PKCβII during reperfusion has not been previously determined. In this study, we hypothesize that myristoylated (myr)-PKCβII- will decrease infarct size and improve post-reperfused cardiac function compared to untreated controls, whereas PKCβII peptide activator (N-myr-SVEIWD; myr-PKCβII+) will show no improvement compared to control. Myristoylation of PKCβII peptides facilitate their entry into the cell in order to affect PKCβII activity by either augmenting or attenuating its translocation to cell membrane proteins, such as NOX-2. Isolated perfused rat hearts were subjected to global I(30min)/R(50min) and infused with myr-PKCβII+ (20μM; n=9), myr-PKCβII- (20µM; n=8), or plasma (control; n=9) at reperfusion. Hearts were frozen (-20oC), sectioned and stained using 1% triphenyltetrazolium chloride to differentiate necrotic tissue. The measurement of Left ventricular (LV) cardiac function was determined using a pressure transducer and infarct size was calculated as percent dead tissue vs. total heart tissue weight. Myr-PKCβII- significantly improved LV end-diastolic pressure 37±7 mmHg compared to control (58±5; p\u3c0.01) and myr-PKCβII+ (58±4; p\u3c0.01). Myr-PKCβII- significantly reduced infarct size to 14±3% compared to control (26±5%; p\u3c0.01), while myr-PKCβII+ (25±3%) showed no difference. The data indicate that myr-PKCβII- may be a putative treatment to reduce myocardial reperfusion injury when given to heart attack patients during cardio-angioplasty. Future studies are planned to determine infarct size by Image J analysis

Untreated hypertension decreases heritability of cognition in late middle age

Abstract Hypertension is a risk factor for cognitive decline, but the mechanisms underlying the effects of hypertension on cognition, particularly in midlife, are unclear. We examined whether hypertension modifies genetic influences on individual differences in cognition. Nine cognitive domains and general cognitive ability were assessed in a sample of 1,237 male twins aged 51-60 who were divided into three blood pressure groups: nonhypertensive; medicated hypertensive; and unmedicated hypertensive. Heritability was significantly lower among unmedicated hypertensives compared to medicated hypertensives and non-hypertensives for visual-spatial ability (p = 0.013) and episodic memory (p = 0.004). There were no heritability differences between non-hypertensives and medicated hypertensives. In addition, there were no significant differences in mean level cognition across the three blood pressure groups. These results suggest that in middle-aged men, untreated hypertension suppresses normal genetic influences on individual differences in certain domains of cognition prior to the emergence of hypertension-related effects on cognitive performance. These results further suggest that antihypertensive medication may protect against or reverse this effect

Adult Romantic Attachment, Negative Emotionality, and Depressive Symptoms in Middle Aged Men: A Multivariate Genetic Analysis

Adult romantic attachment styles reflect ways of relating in close relationships and are associated with depression and negative emotionality. We estimated the extent to which dimensions of romantic attachment and negative emotionality share genetic or environmental risk factors in 1,237 middle-aged men in the Vietnam Era Twin Study of Aging (VETSA). A common genetic factor largely explained the covariance between attachment-related anxiety, attachment-related avoidance, depressive symptoms, and two measures of negative emotionality: Stress-Reaction (anxiety), and Alienation. Multivariate results supported genetic and environmental differences in attachment. Attachment-related anxiety and attachment-related avoidance were each influenced by additional genetic factors not shared with other measures; the genetic correlation between the attachment measure-specific genetic factors was 0.41, indicating some, but not complete overlap of genetic factors. Genetically informative longitudinal studies on attachment relationship dimensions can help to illuminate the role of relationship-based risk factors in healthy aging

Structure-based design of a bromodomain and extraterminal domain (BET) inhibitor selective for the N-terminal bromodomains that retains an anti-inflammatory and antiproliferative phenotype

The bromodomain and extraterminal domain (BET) family of epigenetic regulators comprises four proteins (BRD2, BRD3, BRD4, BRDT), each containing tandem bromodomains. To date, small molecule inhibitors of these proteins typically bind all eight bromodomains of the family with similar affinity, resulting in a diverse range of biological effects. To enable further understanding of the broad phenotype characteristic of pan-BET inhibition, the development of inhibitors selective for individual, or sets of, bromodomains within the family is required. In this regard, we report the discovery of a potent probe molecule possessing up to 150-fold selectivity for the N-terminal bromodomains (BD1s) over the C-terminal bromodomains (BD2s) of the BETs. Guided by structural information, a specific amino acid difference between BD1 and BD2 domains was targeted for selective interaction with chemical functionality appended to the previously developed I-BET151 scaffold. Data presented herein demonstrate that selective inhibition of BD1 domains is sufficient to drive anti-inflammatory and antiproliferative effects

The discovery of I-BRD9, a selective cell active chemical probe for bromodomain containing protein 9 inhibition

Acetylation of histone lysine residues is one of the most well-studied post-translational modifications of chromatin, selectively recognized by bromodomain “reader” modules. Inhibitors of the bromodomain and extra terminal domain (BET) family of bromodomains have shown profound anticancer and anti-inflammatory properties, generating much interest in targeting other bromodomain-containing proteins for disease treatment. Herein, we report the discovery of I-BRD9, the first selective cellular chemical probe for bromodomain-containing protein 9 (BRD9). I-BRD9 was identified through structure-based design, leading to greater than 700-fold selectivity over the BET family and 200-fold over the highly homologous bromodomain-containing protein 7 (BRD7). I-BRD9 was used to identify genes regulated by BRD9 in Kasumi-1 cells involved in oncology and immune response pathways and to the best of our knowledge, represents the first selective tool compound available to elucidate the cellular phenotype of BRD9 bromodomain inhibition