Saima Muzaffar

Exogenous Hydrogen Sulfide Inhibits Superoxide Formation, NOX-1 Expression and Rac1 Activity in Human Vascular Smooth Muscle Cells

The activity of NADPH oxidase (NOX) is blocked by nitric oxide (NO). Hydrogen sulfide (H₂S) is also produced by blood vessels. It is reasonable to suggest that H₂S may have similar actions to NO on NOX. In order to test this hypothesis, the effect of sodium hydrosulfide (NaHS) on O₂^– formation, the expression of NOX-1 (a catalytic subunit of NOX) and Rac₁ activity (essential for full NOX activity) in isolated vascular smooth muscle cells (hVSMCs) was investigated. hVSMCs were incubated with the thromboxane A₂ analogue U46619 ± NaHS for 1 or 16 h, and O₂^– formation, NOX-1 expression and Rac₁ activity were assessed. The possible interaction between H₂S and NO was also studied by using an NO synthase inhibitor, L-NAME, and an NO donor, DETA-NONOate. The role of K_ATP channels was studied by using glibenclamide. NaHS inhibited O₂^– formation following incubation of 1 h (IC₅₀, 30 nM) and 16 h (IC₅₀, 20 nM), blocked NOX-1 expression and inhibited Rac₁ activity. These inhibitory effects of NaHS were mediated by the cAMP-protein-kinase-A axis. Exogenous H₂S prevents NOX-driven intravascular oxidative stress through an a priori inhibition of Rac₁ and downregulation of NOX-1 protein expression, an effect mediated by activation of the adenylylcyclase-cAMP-protein-kinase-G system by H₂S.

Nitroaspirins and Morpholinosydnonimine but Not Aspirin Inhibit the Formation of Superoxide and the Expression of gp91phox Induced by Endotoxin and Cytokines in Pig Pulmonary Artery Vascular Smooth Muscle Cells and Endothelial Cells

Background—Although nonsteroidal antiinflammatory drugs (NSAIDs) are ineffective in treating acute respiratory distress syndrome (ARDS), inhalational NO has proved to be useful. NO-donating NSAIDs may therefore be more effective in treating ARDS than NSAIDs alone. Because oxidant stress is central to the pathophysiology of ARDS, the effect of nitroaspirins (NCX 4016, NCX 4040, and NCX 4050) compared with morpholinosydnonimine (SIN-1; an NO donor) and aspirin (ASA) on superoxide (O2·−) formation and gp91phox (an active catalytic subunit of NADPH oxidase) expression in pig pulmonary artery vascular smooth muscle cells (PAVSMCs) and endothelial cells (PAECs) was investigated. Methods and Results—Cultured PAVSMCs and PAECs were incubated with lipopolysaccharide (LPS), tumor necrosis factor (TNF)-&agr;, and interleukin (IL)-1&agr; (with or without NO-ASA, SIN-1, or ASA) for 16 hours, and O2·− release was measured by use of the reduction of ferricytochrome c. The expression of gp91phox was assessed by use of Western blotting. LPS, TNF-&agr;, and IL-1&agr; all stimulated the formation of O2·− and expression of gp91phox in both PAVSMCs and PAECs, an effect inhibited by NADPH oxidase inhibitors, diphenyleneiodonium, and apocynin. SIN-1, NCX 4016, and NCX 4050 but not ASA alone inhibited the formation of O2·− and expression of gp91phox. Conclusions—LPS and cytokines promote the formation of O2·− in PAVSMCs and PAECs through an augmentation of NADPH oxidase activity, which in turn is prevented by NO. Thus, NO may play a protective role in preventing excess O2·− formation, but its negation by O2·− may augment the progress of ARDS. The inhibitory effect of nitroaspirins suggests that they may be therapeutically useful in treating ARDS through the suppression of NADPH oxidase upregulation and O2·− formation.

Superoxide from NADPH oxidase upregulates type 5 phosphodiesterase in human vascular smooth muscle cells: inhibition with iloprost and NONOate

To determine whether there is an association between vascular NADPH oxidase (NOX), superoxide, the small GTPase Rac1 and PDE type 5 (PDE5) in human vascular smooth muscle cell (hVSMCs).

NADPH oxidase 4 mediates upregulation of type 4 phosphodiesterases in human endothelial cells

The protective actions of prostacyclin (PGI2) are mediated by cyclic AMP (cAMP) which is reduced by type 4 phosphodiesterases (PDE4) which hydrolyze cAMP. Superoxide

NADPH oxidase 1 mediates upregulation of thromboxane A2 synthase in human vascular smooth muscle cells: inhibition with iloprost.

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Contents Vol. 45, 2008

from NADPH oxidase (Nox) is associated with impaired PGI2 bioactivity. The objective of this study, therefore, was to study the relationship between Nox and PDE4 expression in human umbilical vein endothelial cells (HUVECs). HUVECs were incubated with the thromboxane A2 analog, U46619, 8‐isoprostane F2α (8IP), or tumor necrosing factor alpha (TNFα) [±iloprost (a PGI2 analog)] and the expression of PDE4A, B, C, and D and splice variants thereof assessed using Western blotting and qPCR and mRNA silencing of Nox4 and Nox5. Effects on cell replication and angiogenesis were also studied. U46619, 8IP, and TNFα increased the expression of Nox 4 and Nox 5 and all PDE4 isoforms as well as cell replication and tubule formation (index of angiogenesis), effects inhibited by mRNA silencing of Nox4 (but not Nox5) and iloprost and rolipram. These data demonstrate that upregulation of Nox4 leads to an upregulation of PDE4A, B, and D and increased hydrolysis of cAMP which in turn augments cell replication and angiogenesis. This mechanism may be central to vasculopathies associated with endothelial dysfunction since the PGI2–cAMP signaling axis plays a key role in mediating functions that include hemostasis and angiogenesis. J. Cell. Physiol. 227: 1941–1950, 2012.

Inhaled prostacyclin is safe, effective, and affordable in patients with pulmonary hypertension, right-heart dysfunction, and refractory hypoxemia after cardiothoracic surgery

Thromboxane A(2) (TXA(2)) upregulates and activates NADPH oxidase (Nox) both of which are associated with cardiovascular disease. The aim of this study, therefore, was to investigate the relationship between thromboxane A(2) synthase (TXAS) status and Nox in human vascular smooth muscle cells (hVSMCs), in particular, whether superoxide (O(2)(▪-)) derived from Nox influences TXAS expression and activity. hVSMCs were incubated with TNFα: (10 ng/ml), TXA(2) mimetic U46619 (100 nM), 8-isoprostane F(2α) (8-IP; 100 nM) and hypoxia. Expression of TXAS was assessed using western blotting and quantitative PCR. The role of Nox1 and Nox4 was studied using apocynin and mRNA silencing. The effect of the thromboxane receptor antagonist picotamide and of iloprost, a prostacyclin (PGI(2)) analogue was also studied. TNF-α, U46619 and 8-IP and hypoxia all augmented TXAS expression as well as TXA(2) formation, effects inhibited by apocynin. Nox-1 (but not Nox4) gene silencing inhibited the increase in TXAS expression and activity. Both picotamide and iloprost inhibited the upregulation of TXAS as well as TXA(2) formation induced by TNF-α, U46619 and 8-isoprostane F(2α) and hypoxia. It is concluded that upregulation of TXA(2) synthase expression and activity in human VSMCs is mediated by an a priori upregulation of Nox1 and represents a self amplifying cascade. The inhibition of this effect with iloprost consolidates that PGI(2) plays a protective anti-oxidative role in the vasculature and that picotamide and like drugs may be effective in reducing the incidence of cardiovascular disease associated with an oxidative aetiology.

Superoxide auto-augments superoxide formation and upregulates gp91phox expression in porcine pulmonary artery endothelial cells: Inhibition by iloprost

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