Nilima Shukla

Sildenafil inhibits the formation of superoxide and the expression of gp47phox NAD[P]H oxidase induced by the thromboxane A2 mimetic, U46619, in corpus cavernosal smooth muscle cells

To assess the effect of sildenafil on superoxide formation and p47phox (the active subunit of NADPH oxidase) expression in cultured corpus cavernosal smooth muscle cells (CVSMCs).

H2S-donating sildenafil (ACS6) inhibits superoxide formation and gp91phox expression in arterial endothelial cells: role of protein kinases A and G

Superoxide (O2•−), derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, is associated with acute respiratory distress syndrome (ARDS). NADPH oxidase activity and expression are blocked by nitric oxide (NO) and sildenafil. As another gas, hydrogen sulphide (H2S) is formed by blood vessels, the effect of sodium hydrosulphide (NaHS) and the H2S‐donating derivative of sildenafil, ACS6, on O2•− formation and the expression of gp91phox (a catalytic subunit of NADPH oxidase) in porcine pulmonary arterial endothelial cells (PAECs) was investigated.

Iloprost inhibits superoxide formation and gp91phox expression induced by the thromboxane A2 analogue U46619, 8‐isoprostane F2α, prostaglandin F2α, cytokines and endotoxin in the pig pulmonary artery

Since the roles of thromboxane A2 (TXA2), prostacyclin (PGI2) and 8‐isoprostane F2α in mediating vascular O2•− formation and its relation to adult respiratory distress syndrome (ARDS) is unknown, the effects of these eicosanoids on the expression of gp91phox (catalytic subunit of NADPH oxidase) and O2•− release from cultured pig pulmonary artery (PA) segments, PA vascular smooth muscle cells (PAVSMCs) and PA endothelial cells (PAECs) were investigated. PA segments, PAVSMCs and PAECs were incubated with the TXA2 analogue, U46619, (±LPS, tumour necrosing factor‐alpha (TNF‐α) or IL‐1α), 8‐isoprostane F2α and±iloprost (a stable PGI2 analogue) for 16 h. The formation of superoxide dismutase‐inhibitable O2•− was then measured spectrophotometrically and gp91phox expression assessed using Western blotting. In parallel experiments, whole PA segments were treated with LPS, TNF‐α and IL‐α after which time TXA2, PGI2, PGF2α and 8‐isoprostane F2α formation was measured using enzyme‐linked immunoassays. U46619, PGF2α and 8‐isoprostane F2α promoted the formation of O2•− in PA segments, PAVSMCs and PAECs, an effect inhibited by diphenyleneiodonium and apocynin (both NADPH oxidase inhibitors) and upregulated the expression of gp91phox in PAECs and PAVSMCs. These effects were augmented by LPS, TNF‐α and IL‐1α but inhibited by iloprost. Under identical incubation conditions, IL‐1α, LPS and TNF‐α all induced an increase in the formation of TXA2, PGF2α and 8‐isoprostane F2α but reduced the concomitant formation of PGI2. These data demonstrate that LPS and cytokines influence the relative balance of TXA2, PGI2, PGF2α and 8‐isoprostane F2α in pig PA, which in turn alter NADPH oxidase expression and O2•− formation. These novel findings have implications in devising effective strategies for treating ARDS.

Sildenafil citrate and sildenafil nitrate (NCX 911) are potent inhibitors of superoxide formation and gp91phox expression in porcine pulmonary artery endothelial cells

Acute respiratory distress syndrome (ARDS) is associated with increased superoxide (O2•−) formation in the pulmonary vasculature and negation of the bioavailability of nitric oxide (NO). Since NO inhibits NADPH oxidase expression through a cyclic GMP‐mediated mechanism, sildenafil, a type V phosphodiesterase inhibitor, may be therapeutically effective in ARDS through an augmentation of NO‐mediated inhibition of NADPH oxidase. Therefore, the effect of sildenafil citrate and NO‐donating sildenafil (NCX 911) on O2•− formation and gp91phox (active catalytic subunit of NADPH oxidase) expression was investigated in cultured porcine pulmonary artery endothelial cells (PAECs). PAECs were incubated with 10u2003nM TXA2 analogue, 9,11‐dideoxy‐9α,11α‐methanoepoxy‐prostaglandin F2α (U46619) (±sildenafil or NCX 911), for 16u2003h and O2•− formation measured spectrophometrically and gp91phox using Western blotting. The role of the NO‐cGMP axis was studied using morpholinosydnonimine hydrochloride (SIN‐1), the diethylamine/NO complex (DETA‐NONOate), the guanylyl cyclase inhibitor, 1H‐{1,2,4}oxadiazolo{4,3‐a}quinoxalin‐1‐one (ODQ), and the protein kinase G inhibitor, 8‐bromoguanosine‐3′,5′‐cyclic monophosphorothioate, Rp‐isomer (Rp‐8‐Br‐cGMPS). NO release was studied using a fluorescence assay and O2•−–NO interactions by measuring nitrites. After a 16‐h incubation with 10u2003nM U46619, both NCX 911 and sildenafil elicited a concentration‐dependent inhibition of O2•− formation and gp91phox expression, NCX 911 being more potent (IC50; 0.26u2003nM) than sildenafil citrate (IC50; 1.85u2003nM). These inhibitory effects were reversed by 1u2003μM ODQ and 10u2003μM Rp‐8‐Br‐cGMPS. NCX 911 stimulated the formation of cGMP in PAECs and generated NO in a cell‐free system to a greater degree than sildenafil citrate. The inhibitory effect of sildenafil was augmented by 1u2003μM SIN‐1 and blocked partially by the eNOS inhibitor 10u2003μM N5‐(1‐iminoethyl)‐ornithine (L‐NIO). Acutely, sildenafil and NCX 911 also inhibited O2•− formation, again blocked by 1u2003μM ODQ. NCX 911 reacted with O2•− generated by xanthine oxidase, an effect that was inhibited by superoxide dismutase (500u2003Uu2003ml−1). Since O2•− formation plays contributory role in ARDS, both sildenafil citrate and NCX 911 may be indicated for treating ARDS through suppression of NADPH oxidase expression and therefore of O2•− formation and preservation of NO bioavailability.

Sildenafil inhibits the up-regulation of phosphodiesterase type 5 elicited with nicotine and tumour necrosis factor-α in cavernosal vascular smooth muscle cells: mediation by superoxide

To determine whether there is an association between vascular phosphodiesterase type 5 (PDE‐5) and NADPH oxidase (NOX) in cavernosal vascular smooth muscle cells (CVSMCs), and to study the actions of the PDE‐5 inhibitor sildenafil; the pro‐erectile actions of nitric oxide (NO) are reduced by PDE‐5 which hydrolyses cGMP to inactive GMP, thus an up‐regulation of PDE‐5 and over‐production of O2‐ derived from NOX might promote erectile dysfunction (ED).

Acute inhibition of superoxide formation and Rac1 activation by nitric oxide and iloprost in human vascular smooth muscle cells in response to the thromboxane A2 analogue, U46619

BACKGROUNDnThe over-production of superoxide (O(2)(-)) derived from NADPH oxidase (NOX) plays a central role in cardiovascular diseases. By contrast, nitric oxide (NO) and prostacyclin (PGI(2)) are vasculoprotective. The effect of the NO donor, NONOate and iloprost on O(2)(-) formation, p47(phox) and Rac(1) activation in human vascular smooth muscle cells (hVSMCs) was investigated.nnnMETHODSnhVSMCs were incubated with 10nM thromboxane A(2) analogue, U46619 for 16h, and then with apocynin (a NOX inhibitor), NONOate or iloprost for 1h and O(2)(-) measured spectrophometrically. The role of cyclic AMP and cyclic GMP was examined by co-incubation of drugs with protein kinase (PK) A and G inhibitors listed above. Rac(1) was studied using pull-down assays.nnnRESULTSnNONOate and iloprost inhibited O(2)(-) formation, acutely, effects blocked by inhibition of PKG and PKA, respectively. Rac(1) and p47(phox) activation and translocation to the plasma membrane was completely inhibited by NONOate and iloprost, effects again reversed by co-incubation with PKG or PKA inhibitors.nnnCONCLUSIONSnNO and PGI(2) block the acute activity of NOX in hVSMCs via the cGMP-PKG axis (for NO) and by the cAMP-PKA axis (for iloprost) through inhibition of Rac(1) and p47(phox) translocation. These findings have implications in the pathophysiology and treatment of CVD.

Nitric oxide donating aspirins: novel drugs for the treatment of saphenous vein graft failure

BACKGROUNDnA new class of nitric oxide donating aspirin (NO-ASA) drugs may increase the therapeutic impact of aspirin in saphenous vein coronary artery bypass grafting (CABG) not only through the inhibition of thrombosis but also through a reduction of vasospasm and inhibition of vascular smooth muscle cell (VSMC) proliferation (effects that are inhibited by NO but not ASA). In order to test this proposal the effect of three NO-ASA drugs (NCX4040, NCX4050, and NCX4060) on in vitro relaxation and cyclic guanosine monophosphate (cGMP) formation in the human isolated saphenous vein and the proliferation of human VSMCs was investigated.nnnMETHODSnSaphenous vein segments were obtained from 30 patients undergoing CABG (median age, 59 years; range, 49 to 68). The effect of the NO-ASA adducts, ASA alone, and sodium nitroprusside (NO donor) were investigated on (1) relaxation of phenylephrine-stimulated contraction using an organ bath, (2) cyclic guanosine monophosphate (cGMP) formation using an enzyme-linked immunosorbent assay, and (3) the proliferation of VSMCs derived from saphenous vein using bromo-deoxyuridine (BRDU) incorporation.nnnRESULTSnAll three NO-ASA adducts (at concentrations that inhibited responses by 50% [IC50s] between 1 micromol/L and 100 micromol/L) and nitroprusside (at IC50s between 0.5 and 10 micromol/L) elicited relaxation of isolated human saphenous vein, promoted cGMP formation, and inhibited VSMC proliferation whereas ASA alone (up to 100 micromol/L) had no effect on any variable.nnnCONCLUSIONSnThese data indicate that the NO-ASA adducts by virtue of their capacity to release NO and stimulate guanylyl cyclase may be useful not only in the prevention of thrombosis following CABG but also the reduction of saphenous vein graft spasm and neointima formation.

The administration of folic acid improves erectile function and reduces intracavernosal oxidative stress in the diabetic rabbit.

To test the possibility that folic acid (FA) may be a means of treating erectile dysfunction (ED) in diabetes mellitus (DM), by studying the effect of FA administration to DM rabbits on cavernosal function and intrapenile oxidative stress.

The administration of folic acid reduces intravascular oxidative stress in diabetic rabbits

There is evidence that plasma homocysteine augments angiopathy in patients with diabetes mellitus. Although lowering homocysteine with folic acid improves endothelial function, the precise mechanisms underlying this effect are unknown. To study this area further, the effect of administration of folic acid to diabetic rabbits on intraaortic oxidative stress was studied by assessing the formation of superoxide (O(2)(-)), 8-isoprostane F(2alpha) (8-IPF(2alpha)), and prostacyclin (as 6-keto-PGF(1alpha)) as well as acetylcholine-stimulated relaxation and gp47(phox) content. Nonketotic diabetes mellitus was induced in New Zealand rabbits with alloxan, and low- and high-dose folic acid was administered daily for 1 month. Rabbits were killed, aortae were excised, and rings were prepared. Rings were mounted in an organ bath, and relaxation was elicited with acetylcholine. The O(2)(-) release was measured spectrophotometrically; the gp47(phox) expression, by Western blotting; and the 8-IPF(2alpha) and 6-keto-PGF(1alpha) formation, by enzyme-linked immunosorbent assay. Blood was collected for measurement of homocysteine, red blood cell folate, and glucose. In aortae from the diabetic rabbits, acetylcholine-induced relaxation was significantly impaired compared with that in untreated controls. The O(2)(-) release, p47(phox) expression, and 8-IPF(2alpha) formation were all enhanced and 6-keto-PGF(1alpha) formation was reduced compared with controls. All these effects were reversed by both low- and high-dose folic acid. Plasma total homocysteine was reduced by high-dose, but not low-dose, folic acid. Red blood cell folate was elevated in both groups. The improvement of endothelial function in patients receiving folic acid may be due to inhibition of nicotinamide adenine nucleotide phosphate oxidase (NADPH) oxidase expression and therefore conservation of nitric oxide and prostacyclin bioavailability, 2 vasculoprotective factors.

Thapsigargin inhibits angiogenesis in the rat isolated aorta: studies on the role of intracellular calcium pools

OBJECTIVEnSince the role of Ca2+ in angiogenesis is not fully understood, we investigated the effect of thapsigargin (TG: depletes intracellular Ca2+ pools) and other Ca2+ modulators [ionomycin, calcium ionophore A23187 and dibutyrylhydroquinone (DBHQ)] on in vitro angiogenesis by rat aortic rings.nnnMETHODSnAortae from Sprague-Dawley rats were cut into 2-mm rings, embedded in a fibrin clot and cultured for 15 days in serum-free medium containing drugs and the microvessels counted. Rings were also pre-treated with TG and Ca2+ modulators for 1 h prior to embedding and culture. Viability was examined by the measurement of lactic acid dehydrogenase release. Rings were also treated with hydrocortisone and lavendustin A (a tyrosine kinase inhibitor), as positive controls. The effect of TG on the proliferation and migration of human umbilical artery endothelial cells (HUVECs) was studied in parallel.nnnRESULTSnTG significantly inhibited microvessel formation and HUVEC proliferation and migration in a dose-dependent manner, all at <10 nmol/l, without affecting viability. In contrast, ionomycin, A23187 and DBHQ were cytotoxic at inhibitory concentrations. Continual exposure to hydrocortisone and lavendusin A also inhibited angiogenesis without affecting viability.nnnCONCLUSIONnSince low concentrations of TG deplete intracellular Ca2+ stores, it is concluded that these pools play a central role in mediating angiogenesis.