Endothelium-Derived Hyperpolarization and Coronary Vasodilation: Diverse and Integrated Roles of Epoxyeicosatrienoic Acids, Hydrogen Peroxide, and Gap Junctions.

Myocardial perfusion and coronary vascular resistance are regulated by signaling metabolites released from the local myocardium that act either directly on the VSMC or indirectly via stimulation of the endothelium. A prominent mechanism of vasodilation is EDH of the arteriolar smooth muscle, with EETs and H(2)O(2) playing important roles in EDH in the coronary microcirculation. In some cases, EETs and H(2)O(2) are released as transferable hyperpolarizing factors (EDHFs) that act directly on the VSMCs. By contrast, EETs and H(2)O(2) can also promote endothelial KCa activity secondary to the amplification of extracellular Ca(2+) influx and Ca(2+) mobilization from intracellular stores, respectively. The resulting endothelial hyperpolarization may subsequently conduct to the media via myoendothelial gap junctions or potentially lead to the release of a chemically distinct factor(s). Furthermore, in human isolated coronary arterioles dilator signaling involving EETs and H(2)O(2) may be integrated, being either complimentary or inhibitory depending on the stimulus. With an emphasis on the human coronary microcirculation, this review addresses the diverse and integrated mechanisms by which EETs and H(2)O(2) regulate vessel tone and also examines the hypothesis that myoendothelial microdomain signaling facilitates EDH activity in the human heart.

Wnt5a-induced Wnt1-inducible secreted protein-1 suppresses vascular smooth muscle cell apoptosis induced by oxidative stress.

OBJECTIVE: Apoptosis of vascular smooth muscle cells (VSMCs) contributes to thinning and rupture of the atherosclerotic plaque fibrous cap and is thereby associated with myocardial infarction. Wnt protein activation of ß-catenin regulates numerous genes that are associated with cell survival. We therefore investigated Wnt/ß-catenin survival signaling in VSMCs and assessed the presence of this pathway in human atherosclerotic plaques at various stages of the disease process. APPROACH AND RESULTS: Wnt5a induced ß-catenin/T-cell factor signaling and retarded oxidative stress (H2O2)-induced apoptosis in mouse aortic VSMCs. Quantification of mRNA levels revealed a >4-fold (P<0.05; n=9) increase in the expression of the Wnt/ß-catenin responsive gene, Wnt1-inducible secreted protein-1 (WISP-1), which was dependent on cAMP response element-binding protein and sustained in the presence of H2O2. Exogenous WISP-1 significantly reduced H2O2-induced apoptosis by 43% (P<0.05; n=3) and was shown using silencing small interfering RNA, to be important for Wnt5a-dependent survival responses to H2O2 (P<0.05; n=3). WISP-1 protein levels were significantly lower (≈50%) in unstable atherosclerosis compared with stable plaques (n=11 and n=14). CONCLUSIONS: These results indicate for the first time that Wnt5a induces ß-catenin survival signaling in VSMCs via WISP-1. The deficiency of the novel survival factor, WISP-1 in intimal VSMCs of unstable coronary plaques, suggests that there is altered Wnt/ß-catenin/ T-cell factor signaling with progressive atherosclerosis, and restoration of WISP-1 protein might be an effective stabilization factor for vulnerable atherosclerotic plaques.

Interactions between thromboxane A2, thromboxane/prostaglandin (TP) receptors, and endothelium-derived hyperpolarization.

Endothelium-dependent smooth muscle hyperpolarization (EDH) increasingly predominates over endothelium-derived nitric oxide (NO) as a participant in vasodilation as vessel size decreases. Its underlying nature is highly variable between vessel types, species, disease states, and exact experimental conditions, and is variably mediated by one or more transferable endothelium-derived hyperpolarizing factors and/or the electrotonic spread of endothelial hyperpolarization into the media via gap junctions. Although generally regarded (and studied) as a mechanism that is independent of NO and prostanoids, evidence has emerged that the endothelium-derived contracting factor and prostanoid thromboxane A2 can modulate several signalling components central to EDH, and therefore potentially curtail vasodilation through mechanisms that are distinct from those putatively involved in direct smooth muscle contraction. Notably, vascular production of thromboxane A2 is elevated in a number of cardiovascular disease states that promote endothelial dysfunction. This review will therefore discuss the mechanisms through which thromboxane A2 interacts with and modulates EDH, and will also consider the implications of such cross-talk in vasodilator control in health and disease.

Low nanomolar thapsigargin inhibits the replication of vascular smooth muscle cells through reversible endoplasmic reticular stress.

Thapsigargin (TG), an inhibitor of Ca(2+) ATPase pumps in the endoplasmic reticulum (ER), inhibits replication of human vascular smooth muscle cell (hVSMC) at low nM concentrations. TG blocks replication of other cell types through promotion of ER stress (ERS). In order to determine whether ERS may mediate the cytostatic effect of TG in hVSMCs, the effect of TG on ERS in hVSMCs was studied by assessing markers of ERS: Immunoglobulin Heavy Chain Binding Protein (BiP), growth inhibitory transcription factor, GADD153, phosphorlylated eukaryotic initiation factor 2α (p-eIF2α) and phosphorlylated protein kinase R (p-PKR). hVSMCs derived from saphenous veins were rendered quiescent with serum-free medium for 96 h incubated with 10 nM TG at 37 °C for 24 h, then washed free of TG and incubated with 10% foetal calf serum (FCS) for a further 24 h. At selected times, BiP, GADD153, p-eIF2α, p-PKR and cyclin D1 expression was assessed. TG promoted a marked increase in BiP and GADD153, but suppressed cyclin D1 mRNA and protein expression. Under serum-free conditions p-eIF2α and p-PKR expression was not enhanced by TG. 15-24 h After removal of TG all these factors returned to levels seen in control cells. These data demonstrate that the inhibitory effect of 10nM TG on hVSMC replication is mediated through induction of ERS and associated factors that cessate replication and is reversible. These observations have implications in the aetiology and treatment of diseases that include atherogenesis, vein graft failure and restenosis.

Oxidative stress and vein graft failure: a focus on NADH oxidase, nitric oxide and eicosanoids.

Recent interest has focused on superoxide and the upregulation of NADPH oxidase expression in the aetiology of vein graft failure. Implantation of saphenous vein grafts promotes upregulation of NADPH oxidase through a number of distinct interrelated mechanisms: (a) endothelial denudation, (b) factors released by adherent platelets, monocytes and neutrophils, (c) hypoxia and (d) altered prostacyclin (PGI(2)) and enhanced isoprostane formation. These, in turn, impact on neointima (NI) formation (vascular smooth muscle cell [VSMC] replication and migration) and metalloproteinase (MMP) expression, key events in vein graft thickening. NADPH oxidase in the aetiology of vein graft failure will be discussed in this review with particular reference to nitric oxide and eicosanoids and related drugs that inhibit its activity and expression.

Pathophysiology of saphenous vein graft failure: a brief overview of interventions.

Coronary artery bypass graft surgery (CABG) is widely used for the treatment of atheromatous stenosis of coronary arteries. However, as many as 50% of grafts fail within 10 years after CABG due to neointima (NI) formation, a process involving the proliferation of vascular smooth muscle cells (VSMCs) and superimposed atherogenesis. To date no therapeutic intervention has proved successful in treating late vein graft failure. However, several diverse approaches aimed at preventing neointimal formation have been devised which have yielded promising results. In this review, therefore, we will summarise the pathophysiology of vein graft disease and then briefly consider interventional approaches to prevent late vein graft failure which include surgical technique, conventional pharmacology, external sheaths, cytostatic drugs and gene transfer.

Role of osteopontin in the development of neointimal hyperplasia in vein grafts.

OBJECTIVES: Neointimal hyperplasia and superimposed atherosclerosis are central to late vein graft failure following coronary artery bypass grafting. Recent studies on post-injury arterial vessels have suggested a role of osteopontin (OPN) in the process of vascular remodelling. This study was designed to assess the in vivo performance of OPN following vein grafting. METHODS: Bilateral saphenous vein-carotid artery interposition grafting was performed in 16 Large White pigs (35-45 kg). All patent vein grafts were removed and fixed at 1, 2, 4 (n = 8 grafts in each group) and 12 weeks (n = 6 grafts) following surgery. Multiple histological sections from each graft were prepared. The expression of OPN in the vein grafts was determined by immunostaining and western blot assay. Proliferating cell nuclear antigen (PCNA) was detected by immunocytochemistry. Vein graft morphology was assessed using computer-aided planimetry. RESULTS: The expression of OPN remarkably increased in the intima of the vein grafts at the first week postoperatively and then gradually declined from the second postoperative week, although OPN expression remained significantly higher than the baseline level at the end of the 3-month study period. More importantly, the number of PCNA-positive cells and matrix metalloproteinases (MMPs) expression correlated well with the OPN expression. CONCLUSIONS: Early induction of OPN in vein grafts may contribute to the subsequent increase in MMPs activities as well as vascular smooth muscle cell proliferation. Therefore, OPN could play an important role in the development of neointimal hyperplasia in venous conduits after coronary artery bypass grafting.

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

The protective actions of prostacyclin (PGI(2) ) are mediated by cyclic AMP (cAMP) which is reduced by type 4 phosphodiesterases (PDE4) which hydrolyze cAMP. Superoxide (O2(-)) from NADPH oxidase (Nox) is associated with impaired PGI(2) 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 A(2) analog, U46619, 8-isoprostane F(2α) (8IP), or tumor necrosing factor alpha (TNFα) [±iloprost (a PGI(2) 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 PGI(2)-cAMP signaling axis plays a key role in mediating functions that include hemostasis and angiogenesis.

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

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.

Characterization of the differential response of endothelial cells exposed to normal and elevated laminar shear stress.

Most acute coronary events occur in the upstream region of stenotic atherosclerotic plaques that experience laminar shear stress (LSS) elevated above normal physiological levels. Many studies have described the atheroprotective effect on endothelial behavior of normal physiological LSS (approximately 15 dynes/cm(2)) compared to static or oscillatory shear stress (OSS), but it is unknown whether the levels of elevated shear stress imposed by a stenotic plaque would preserve, enhance or reverse this effect. Therefore we used transcriptomics and related functional analyses to compare human endothelial cells exposed to laminar shear stress of 15 (LSS15-normal) or 75 dynes/cm(2) (LSS75-elevated). LSS75 upregulated expression of 145 and downregulated expression of 158 genes more than twofold relative to LSS15. Modulation of the metallothioneins (MT1-G, -M, -X) and NADPH oxidase subunits (NOX2, NOX4, NOX5, and p67phox) accompanied suppression of reactive oxygen species production at LSS75. Shear induced changes in dual specificity phosphatases (DUSPs 1, 5, 8, and 16 increasing and DUSPs 6 and 23 decreasing) were observed as well as reduced ERK1/2 but increased p38 MAP kinase phosphorylation. Amongst vasoactive substances, endothelin-1 expression decreased whereas vasoactive intestinal peptide (VIP) and prostacyclin expression increased, despite which intracellular cAMP levels were reduced. Promoter analysis by rVISTA identified a significant over representation of ATF and Nrf2 transcription factor binding sites in genes upregulated by LSS75 compared to LSS15. In summary, LSS75 induced a specific change in behavior, modifying gene expression, reducing ROS levels, altering MAP kinase signaling and reducing cAMP levels, opening multiple avenues for future study.

Endothelin-1 (ET-1) and vein graft failure and the therapeutic potential of ET-1 receptor antagonists.

Despite the exploration of a large number of disparate drugs in animal models and clinical trials, no pharmacological intervention, with the exception of aggressive lipid lowering therapy has reduced late vein graft failure in man. The importance of devising more effective strategies is exemplified by the enormous economic consequences of vein graft failure. Worldwide, there are currently more than 1,000,000 coronary artery bypass graft surgery (CABG) operations a year, the same number of patients undergoing infrainguinal bypass for vascular diseases of the lower limb. The pathophysiology of vein graft failure is complex, involving disparate factors that include adhesion of platelets and leukocytes, rheological forces, metalloproteinase expression, proliferation and migration of vascular smooth muscle cells, neointima formation, oxidative stress, hypoxia and neural re-organisation. Although this diverse etiology may seem to preclude any single drug type as being effective in mediating vein graft failure: one factor that is involved in every facet of vein graft pathobiology is endothelin-1 (ET-1). As such a single drug type (ET(A) antagonist) may prove to be the magic bullet in this scenario. Thus, in this review, we will consider the etiology of vein graft disease in relation to ET-1 and will then present an argument (with evidence) that specific ET(A) receptor antagonists constitute a potentially effective means of preventing vein graft failure.

8-isoprostane F2α up-regulates the expression of type 5 phosphodiesterase in cavernosal vascular smooth muscle cells: inhibition with sildenafil, iloprost, nitric oxide and picotamide.

OBJECTIVES: To explore the possible role of of 8-isoprostane F(2α) (8-IPF(2α) ) in the aetiology of erectile dysfunction (ED), as the over-production of superoxide (O(2)(-)) derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase results in the formation of 8-IPF(2α) in vascular tissue, which has similar properties to thromboxane A(2) (TXA(2) ). TXA(2) is vasoconstrictor and up-regulates the expression of NADPH oxidase and phosphodiesterase type 5 (PDE5). MATERIALS AND METHODS: Cavernosal vascular smooth muscle cells (CVSMCs) were incubated with 8-IPF(2α) or the TXA(2) analogue, U46619, ±sildenafil, iloprost (a stable prostacyclin [PGI(2) ] analogue) or the nitric oxide (NO) donor NONOate for 16 h. The formation of O(2)(-) was then measured, PDE5 expression assessed using Western blotting and PGI(2) and 8-IPF(2α) formation measured using enzyme-linked immunoassays. RESULTS: 8-IPF(2α) promoted the formation of O(2)(-) , an effect inhibited by apocynin (an NADPH oxidase inhibitor) and up-regulated the expression of PDE5. Under identical incubation conditions, 8-IPF(2α) induced an increase in the formation of 8-IPF(2α) but reduced the formation of PGI(2) . All, these effects were reversed by sildenafil, iloprost, NONOate and picotamide. CONCLUSIONS: These data show that O(2) (-) derived from NADPH oxidase influences the relative balance of PGI(2) and 8-IPF(2α) in CVSMCs, which in turn alters the degree of PDE5 expression. This is a novel pathogenic mechanism underlying ED and a novel mechanism of action of sildenafil.

Animal models for studying neointima formation.

Neointima (NI) formation following arterial bypass graft surgery or balloon angioplasty is considered central to subsequent failure after these procedures. The NI promotes accelerated atherogenesis, re-occlusion and thrombosis resulting in a failure rate as high as 50% within 1-10 years. Furthermore, despite the relative success of statins and drug eluting stents, drugs that reduce the failure rate have as yet not been implemented. In turn, animal models are a crucial means of testing potential interventions, in particular, drugs. The objective of this review therefore is to provide a survey of all the possible models that can be used to explore the effects of drugs on NI formation. The review will focus on the most commonly used of species, namely the rat, rabbit, mouse, pig and dog.

Effect of hydrogen sulphide-donating sildenafil (ACS6) on erectile function and oxidative stress in rabbit isolated corpus cavernosum and in hypertensive rats.

OBJECTIVE To study the effect of the H(2)S-donating derivative of sildenafil (ACS6) compared to sildenafil citrate and sodium hydrosulphide (NaHS) on relaxation, superoxide formation and NADPH oxidase and type 5 phosphodiesterase (PDE5) expression in isolated rabbit cavernosal tissue and smooth muscle cells (CSMCs), and in vivo on indices of oxidative stress induced with buthionine sulphoximine (BSO). MATERIALS AND METHODS Relaxation was studied in an organ bath in response to carbachol and after incubation with interleukin-1beta for 12 h. CSMCs were incubated with tumour-necrosis factor-alpha or the thromboxane A(2) (TXA(2)) analogue, U46619, with or with no sildenafil citrate, ACS6 or NaHS for 16 h. Superoxide formation and the expression of p47(phox) (an active subunit of the NADPH oxidase complex) and PDE5 protein was then assessed using Western blotting. Rats were also treated with BSO (with or with no sildenafil citrate or ACS6) for 7 days; cavernosal cGMP, cAMP, glutathionine and plasma TXA(2) and 8-isoprostane F(2alpha) was measured by enzyme-linked immunosorbent assay. RESULTS ACS6 and sildenafil citrate relaxed cavernosal smooth muscle equipotently; NaHS alone had little effect at up to 100 microm. The formation of superoxide and expression of p47(phox) and PDE5 was reduced by ACS6, sildenafil citrate and NaHS (order of potency: ACS6 > sildenafil citrate > NaHS). The effects of ACS6 were blocked by inhibitors of protein kinase A (PKA) and PKG. In rats treated with BSO, both ASC6 and sildenafil citrate reduced the increased plasma levels of TXA(2) and 8-isoprostane F(2alpha) but increased cGMP, cAMP and glutathionine levels in corpus cavernosum. CONCLUSIONS By virtue of a dual action on PKA and PKG activation, ACS6 not only promotes erection, acutely, but might also have a long-term beneficial effect through inhibition of oxidative stress and downregulation of PDE5.

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

OBJECTIVE: 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. MATERIALS AND METHODS: To investigate the effect of administering FA to DM rabbits on erectile function and oxidative stress the formation of superoxide (O(2)(-)), 8-isoprostane F(2 alpha) (8-IPF(2 alpha)) and prostacyclin (as 6-keto-PGF(1 alpha)) were assessed, as well as carbachol- and electrical field stimulated (EFS) relaxation and p47(phox) content (active component of NADPH oxidase complex). Non-ketotic DM was induced in New Zealand rabbits with alloxan and FA administered orally daily for 1 month. Rabbits were killed, penises excised and segments prepared. These were mounted in an organ bath and relaxation elicited with carbachol or EFS. O(2)(-) release was measured spectrophotometrically, p47(phox) expression by Western blotting and 8-IPF(2 alpha) and 6-keto-PGF(1 alpha) formation by enzyme-linked immunosorbant assay. Blood was collected for measurement of homocysteine, red blood cell (RBC) folate and glucose. RESULTS: In cavernosal tissue from DM rabbits, carbachol-and EFS-induced relaxation was significantly impaired compared with the untreated controls. O(2)(-) release, p47(phox) expression and 8-IPF(2 alpha) formation were all enhanced and 6-keto-PGF(1 alpha) formation reduced compared with the controls. All these effects were reversed by FA. Plasma total homocysteine was reduced and RBC folate elevated. CONCLUSIONS: The administration of FA may constitute a strategy for reducing ED in patients with DM.

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 (H(2)S) is also produced by blood vessels. It is reasonable to suggest that H(2)S may have similar actions to NO on NOX. In order to test this hypothesis, the effect of sodium hydrosulfide (NaHS) on O(2)(-) formation, the expression of NOX-1 (a catalytic subunit of NOX) and Rac(1) activity (essential for full NOX activity) in isolated vascular smooth muscle cells (hVSMCs) was investigated. hVSMCs were incubated with the thromboxane A(2) analogue U46619 +/- NaHS for 1 or 16 h, and O(2)(-) formation, NOX-1 expression and Rac(1) activity were assessed. The possible interaction between H(2)S 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 O(2)(-) formation following incubation of 1 h (IC(50), 30 nM) and 16 h (IC(50), 20 nM), blocked NOX-1 expression and inhibited Rac(1) activity. These inhibitory effects of NaHS were mediated by the cAMP-protein-kinase-A axis. Exogenous H(2)S prevents NOX-driven intravascular oxidative stress through an a priori inhibition of Rac(1) and downregulation of NOX-1 protein expression, an effect mediated by activation of the adenylylcyclase-cAMP-protein-kinase-G system by H(2)S.

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.

Homocysteine and copper interact to promote type 5 phosphodiesterase expression in rabbit cavernosal smooth muscle cells.

AIM: To study the effects of homocysteine and copper on type 5 phosphodiesterase (PDE5) expression in cavernosal vascular smooth muscle cells (CVSMCs) and to investigate superoxide (O(2)(.-)) derived from nicotinamide adenine dinucleotide phosphate oxidase as homocysteine and copper generate O(2)(.-), and O(2)(.-) upregulates PDE5 expression. METHODS: CVSMCs derived from rabbit penis were incubated with homocysteine or copper chloride with or without superoxide dismutase (SOD), catalase, sildenafil citrate, or apocynin (nicotinamide adenine dinucleotide phosphate inhibitor) for 16 h. The expression of PDE5 and of glyceraldehyde-3-phosphate dehydrogenase (internal standard) was assessed using Western blot analysis. In parallel, O(2)(.-) was measured spectrophotometrically. RESULTS: CuCl(2) alone (up to 10 micromol/L) and homocysteine alone (up to 100 micromol/L) had no effect on O(2)(.-) formation in CVSMCs compared to controls. In combination, however, homocysteine and CuCl(2) markedly increased O(2)(.-) formation, an effect blocked by SOD, catalase, apocynin, and sildenafil (1 micromol/L) when co-incubated over the same time course. PDE5 expression was also significantly increased in CVSMCs incubated with homocysteine and CuCl(2), compared to controls. This effect was also negated by 16-h co-incubation with SOD, catalase, apocynin and sildenafil. CONCLUSION: This represents a novel pathogenic mechanism underlying ED, and indicates that the therapeutic actions of prolonged sildenafil use are mediated in part through inhibition of this pathway.