Folic acid administration reduces neointimal thickening, augments neo-vasa vasorum formation and reduces oxidative stress in saphenous vein grafts from pigs used as a model of diabetes.

AIMS/HYPOTHESIS: There is evidence that plasma homocysteine augments vein graft failure and that it augments both micro- and macro-angiopathy in patients with diabetes mellitus. It is therefore suggested that homocysteine may augment vein graft thickening, a major cause of vein graft failure, in diabetic patients, as well as impairing adaptive growth of a new vasa vasorum, possibly through overproduction of superoxide. In order to test these proposals, the effect of folic acid administration, which lowers plasma homocysteine, on vein graft thickening and microvessel density was studied in pigs used as a model of diabetes. METHODS: Non-ketotic hyperglycaemia was induced in Landrace pigs by intravenous injection of streptozotocin, and folic acid was fed daily for 1 month. Vein grafts were excised and the thickness of the neointima and media and microvessel density were assessed by planimetry and superoxide formation. RESULTS: Plasma total homocysteine was significantly reduced by folic acid in both control and diabetic pigs, whereas glucose was unchanged. Compared with controls, diabetic pigs showed increased neointimal thickness and superoxide formation and decreased adventitial microvessel density. Folic acid reduced neointimal thickness and superoxide formation and augmented microvessel density in diabetic but not in control pigs. CONCLUSIONS: Folic acid administration reduces neointimal thickening, augments vasa vasorum neoformation and reduces oxidative stress in saphenous vein grafts from diabetic pigs. Folic acid may therefore be particularly effective in reducing vein graft failure in diabetic patients.

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

BACKGROUND AND PURPOSE: Superoxide (O(2)(*-)), 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 (H(2)S) is formed by blood vessels, the effect of sodium hydrosulphide (NaHS) and the H(2)S-donating derivative of sildenafil, ACS6, on O(2)(*-) formation and the expression of gp91(phox) (a catalytic subunit of NADPH oxidase) in porcine pulmonary arterial endothelial cells (PAECs) was investigated. EXPERIMENTAL APPROACH: PAECs were incubated with 10 ng mL(-1) tumour necrosis factor-alpha (TNFalpha) (+/-NaHS or ACS6), both of which released H(2)S, for 2 h or 16 h. O(2)(*-) was measured. Expression of gp91(phox) was measured by western blotting and the role of cyclic AMP (cAMP) and/or cyclic GMP was assessed using protein kinase inhibitors. KEY RESULTS: After either 2- or 16-h incubations, O(2)(*-) formation by PAECs was inhibited by NaHS or ACS6, with IC(50) values of about 10 nM and less than 1 nM, respectively. Both 100 nM NaHS and 1 nM ACS6 completely inhibited gp91(phox) expression induced by TNFalpha. The effects of NaHS were blocked by the inhibition of protein kinase A (PKA), but not PKG, and not by the inhibition of guanylyl cyclase. Effects of ACS6 were blocked by inhibition of both PKA and PKG. Both NaHS and ACS6 augmented cAMP formation. CONCLUSION AND IMPLICATIONS: H(2)S inhibited O(2)(*-) formation and upregulation of NADPH oxidase in PAECs through the adenylyl cyclase-PKA pathway. ACS6 may be effective in treating ARDS through both elevation of cAMP and inhibition of phosphodiesterase type 5 activity.

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

BACKGROUND AND PURPOSE: To determine whether there is an association between vascular NADPH oxidase (NOX), superoxide, the small GTPase Rac(1) and PDE type 5 (PDE5) in human vascular smooth muscle cell (hVSMCs). EXPERIMENTAL APPROACH: hVSMCs were incubated with xanthine-xanthine oxidase (X-XO; a superoxide generating system) or the thromboxane A(2) analogue, U46619 (+/-superoxide dismutase (SOD) or apocynin) for 16 h. The expression of PDE5 and NOX-1 was assessed using Western blotting and superoxide measured. The role of Rac(1) in superoxide generation was assessed by overexpressing either the dominant-negative or constitutively active Rac isoforms. The effects of iloprost, DETA-NONOate and the Rho-kinase inhibitor, Y27632, on PDE5 and NOX-1 expression were also studied. KEY RESULTS: Following 16 h incubation, U46619 and X-XO promoted the expression of PDE5 and NOX-1, an effect blocked by SOD or apocynin when co-incubated over the same time course. X-XO and U46619 both promoted the formation of superoxide. Overexpression of dominant-negative Rac(1) or addition of iloprost, DETA-NONOate or Y27632 completely blocked both superoxide release and PDE5 protein expression and activity. CONCLUSIONS AND IMPLICATIONS: These data demonstrate that superoxide derived from NOX upregulates the expression of PDE5 in human VSMCs. As PDE5 hydrolyses cyclic GMP, this effect may blunt the vasculoprotective actions of NO.

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.

BACKGROUND: The 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. METHODS: hVSMCs 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. RESULTS: NONOate 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. CONCLUSIONS: NO 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.

Reactive oxygen species and erectile dysfunction: possible role of NADPH oxidase.

Erectile dysfunction (ED) is a widespread condition, the incidence of which is increasing globally. ED is also indicative of underlying vasculopathy and represents a predictor of more serious cardiovascular disorders. Understanding the aetiology of ED may therefore provide invaluable pointers to the pathobiology of other cardiovascular diseases (CVDs) and syndromes. It follows, too, that therapeutic interventions that are successful in treating ED may, ipso facto, be effective in treating the early stages of conditions that include atherosclerosis, angina, plaque rupture and diabetic angiopathy. One common pathological denominator in both CVD and ED is oxidative stress, that is, the overproduction of reactive oxygen species (ROS), in particular, superoxide (O(2)(*-)) and hydrogen peroxide (H(2)O(2)). In this review, therefore, we consider the aetiology and pathobiology of O(2)(*-) in promoting ED and focus on NADPH oxidase as an inducible source of O(2)(*-) and H(2)O(2). Therapeutic strategies aimed at reducing oxidative stress to improve erectile function are also discussed.

Acute hypoxia simultaneously induces the expression of gp91phox and endothelial nitric oxide synthase in the porcine pulmonary artery.

BACKGROUND: The effect of hypoxia on the formation of superoxide (O2-), the expression of gp91phox and endothelial NO synthase (eNOS) were studied in pig intact pulmonary artery (PA) segments and PA vascular smooth muscle cells (PAVSMCs) and PA endothelial cells (PAECs). METHODS: Segments and cells were incubated under hypoxic conditions for 2 hours (with or without enzyme inhibitors) and the formation of O2- measured spectrophotometrically. Protein expression was assessed using Western blotting and immunocytochemistry. RESULTS: Hypoxia promoted the formation of O2- in PA segments, PAVSMCs and PAECs, an effect inhibited by diphenylene iodonium and apocynin (NAD[P]H oxidase inhibitors). Hypoxia induced O2- formation was enhanced by inhibition of eNOS and augmented by endotoxin and cytokines and re-oxygenation. Hypoxia also promoted the expression of gp91phox and eNOS. In intact PA segments hypoxia induced the expression of nitrotyrosine and eNOS in the endothelium. CONCLUSIONS: The simultaneous upregulation of NAD[P]H oxidase and eNOS in response to hypoxia in the PA results in the simultaneous formation of O2-, NO, and ONOO-. This may represent either a protective mechanism designed to counter the pro-oxidant effect of hypoxia or a novel pathological mechanism underlying the progression of acute respiratory distress syndrome (ARDS).

Low micromolar concentrations of copper augment the impairment of endothelium-dependent relaxation of aortae from diabetic rabbits.

Both diabetes mellitus (DM) and elevated plasma copper concentrations are risk factors for cardiovascular disease (CVD). DM is associated with impaired endothelial nitric oxide (NO) and with excess superoxide (O2*-) formation. Copper is also elevated in DM and is also associated with the generation of O2*-. To explore possible interactions between DM and copper, the effect of exogenous copper (CuCl2) on endothelium-dependent relaxation and cyclic guanosine monophosphate (GMP) formation was investigated in aortae from diabetic rabbits. Rabbits were rendered diabetic by intravenous injection of alloxan. Six months after induction of DM, the aortae were excised, cut into rings, and mounted in an organ bath for isometric measurement of acetylcholine (Ach)-evoked relaxation in rings precontracted with phenylephrine (PE). In parallel studies, cyclic (c)GMP formation by aortic rings following stimulation with Ach, calcium ionophore A23187 (A23187) and sodium nitroprusside (SNP) was assessed using radioimmunoassay. The effect of copper on these parameters was then studied using the same methods. Ach-evoked relaxation and Ach- and A23187-evoked cGMP formation were significantly impaired in aortae from diabetic rabbits compared to controls, effects that were reversed with superoxide dismutase (SOD) and catalase (CAT). In contrast, there were no significant differences in SNP-stimulated relaxation or cGMP formation in aortae from diabetic rabbits compared to controls. Copper (1 to 10 micromol/L) promoted a further significant inhibition of Ach-stimulated relaxation in aortae from diabetic but not control rabbits. This reduction by copper was again reversed by SOD and CAT. We conclude that copper augments the reduction of NO bioavailability, which is already impaired in aortae from diabetic rabbits due to excess production of O2*- and H2O2. These results indicate that patients with DM may be susceptible to copper-mediated vasculopathy at much lower concentrations than those that promote vasculopathy in nondiabetic patients.

Role of the endothelium and nitric oxide synthases in modulating superoxide formation induced by endotoxin and cytokines in porcine pulmonary arteries.

BACKGROUND: The interactive roles of cytokines, endotoxins, superoxide (O(2)(*-) ) and nitric oxide (NO) in the pathogenesis of adult respiratory distress syndrome (ARDS) have not been fully elucidated. The effects of tumour necrosis factor-alpha (TNF-alpha), interleukin 1alpha (IL-1alpha), and lipopolysaccharide (LPS) and the role of NO and the endothelium in mediating O(2)(*-) formation were therefore investigated in intact porcine pulmonary arteries in vitro. METHODS: Intrapulmonary artery (PA) segments were obtained from White Landrace pigs (25-35 kg) and incubated with LPS, IL-1alpha, and TNF-alpha and O(2)(*-) release was measured by the superoxide dismutase (SOD) inhibitable reduction of ferricytochrome c. The source of O(2)(*-) formation was determined using a number of enzyme inhibitors. The role of NO was explored using NO synthase (NOS) inhibitors and the distribution of NOS isoforms and peroxynitrite (ONOO(-), an index of NO-O(2)(*-) interactions) assessed by immunocytochemistry. RESULTS: LPS, IL-1alpha, and TNF-alpha promoted the formation of O(2)(*-) from PA compared with untreated controls in a time and dose dependent manner, an effect markedly enhanced by removal of the endothelium but completely inhibited by the NADPH oxidase inhibitor diphenylene iodonium chloride (DPI). L-NAME and the eNOS inhibitor N(5)-(1-iminoethyl)-ornithine (L-NIO) enhanced O(2)(*-) formation from PA (with endothelium) in response to IL-1alpha and TNF-alpha but had no effect on LPS mediated O(2)(*-) formation, whereas L-NAME and the iNOS inhibitor L-N(6)-(1-iminoethyl)-lysine-HCl (L-NIL) enhanced O(2)(*-) formation only in response to LPS. CONCLUSIONS: LPS, IL-1alpha, and TNF-alpha promote O(2)(*-) formation through an upregulation of NADPH oxidase activity which is augmented by removal of the endothelium, as well as the inhibition of eNOS (in the case of cytokines) and iNOS (in the case of LPS). The concomitant expression of NOS isoforms (and NO formation) with that of NADPH oxidase may therefore constitute a protective system designed to remove O(2)(*-) through the formation of ONOO(-). If this is so, the integrity of the endothelium may be axiomatic in the progression and severity of ARDS.

Homocysteine enhances impairment of endothelium-dependent relaxation and guanosine cyclic monophosphate formation in aortae from diabetic rabbits.

AIMS/HYPOTHESIS: Both diabetes mellitus and hyperhomocysteinaemia are risk factors for cardiovascular disease and are associated with impaired endothelial nitric oxide and with excess superoxide formation. To explore potential vasculopathic interactions between these risk factors, the effect of homocysteine on endothelium-dependent relaxation and cyclic GMP formation was investigated in aortae from diabetic rabbits. METHODS: Rabbits were rendered diabetic by intravenous injection of alloxan. Six months later, the aortae were excised, cut into rings and mounted in an organ bath for isometric measurement of acetylcholine-evoked relaxation in rings pre-contracted with phenylephrine. Cyclic GMP formation by aortic rings after stimulation with acetylcholine, calcium ionophore A23187 and sodium nitroprusside was assessed using radioimmunoassay. The effect of homocysteine on these parameters was then studied. RESULTS: Ach-evoked relaxation and cyclic GMP formation induced with acetylcholine and calcium ionophore A23187 were impaired in aortae from diabetic rabbits compared with the control rabbits, effects that were reversed with superoxide dimutase (SOD) and augmented by 10-100 micro mol/l homocysteine, an effect again reversed by SOD. CONCLUSION/INTERPRETATION: These data show that the bioavailability of nitric oxide is reduced in aortae from diabetic rabbits due to excess production of superoxide, an effect augmented by homocysteine. These results indicate that patients with diabetes mellitus could be susceptible to homocysteine-mediated angiopathy at lower concentrations than those that promote vasculopathy in non-diabetic patients.

External supports and the prevention of neointima formation in vein grafts.

AIMS AND METHODOLOGY: the aim of this review is to provide an overview of the aetiology of neointima formation in vein grafts and to highlight the use of an external support to modulate this phenomenon. A systematic literature review was performed via computerised search on MEDLINE, OVID and the Cochrane Library. The search terms initially employed were broad-based; "vein graft", "neointima" and "external stent". Subsequently, more specific search terms were utilised; "perivenous mesh", "external prosthesis" and "varicose vein". Articles from indexed journals relevant to the objective, external venous supports, from the earliest reports in the 1960's to the latest in 2001 were included to obtain an exhaustive list. Reviews, abstracts and proceedings of scientific meetings, case reports and the results of both animal model investigations and human clinical trials in all languages were included. Articles describing an external support employed in both peripheral and aortocoronary bypass investigations were included.

Oxygen free radicals and the penis.

Penile erection is dependent upon vascular smooth muscle relaxation in erectile tissue and penile arteries, the principal mediator of relaxation being nitric oxide (NO). Evidence from basic scientific studies indicates that oxidative stress mediated through the superoxide radical (superoxide) and other reactive oxygen species (ROS) may be central to impaired cavernosal function in erectile dysfunction (ED). Increased inactivation of NO by superoxide results in impaired penile NO transmission and smooth muscle relaxation. Furthermore, propagation of endothelial dysfunction by ROS may result in chronic impairment of penile vascular function, a process analogous to early atherogenesis. Indeed, ED and atherosclerosis are closely linked through shared risk factors. Given our current understanding of ED pathophysiology, antioxidants may be of benefit in both the short- and long-term. Evidence supporting the paradigm of antioxidant therapy for the prevention or treatment of ED is presented herein.

Does external stenting reduce porcine vein-graft occlusion via an action on vascular nerves?

BACKGROUND: Neural reorganization occurs in porcine vein grafts and placement of an external stent reduces graft occlusion. AIM OF THE STUDY: To determine the effect of external stenting on the innervation of porcine vein grafts. METHODS: Saphenous vein into carotid artery grafting (with and without external stents) was performed in 16 pigs. After one and six months, grafts were removed, nerves were counted, and neointima was assessed. RESULTS: In vein graft compared to ungrafted vein, there was a significant (p < 0.05) decrease in medial perivascular nerves, but a dramatic increase in paravascular nerves in the adventitia (p < 0.05). In stented vein grafts there was also a reduction of perivascular nerves and the paravascular nerve proliferation observed in vein grafts at one month was inhibited (p < 0.05). Neointima formation and the appearance of large paravascular nerve bundles in the adventitia of vein grafts were abolished by external stenting. CONCLUSIONS: Neural reorganization plays a role in vein-graft failure, possibly through the local release of mitogens; the prevention of this reorganization contributes to the inhibitory effect of the external stent on neointima formation.

The effect of sildenafil on corpus cavernosal smooth muscle relaxation and cyclic GMP formation in the diabetic rabbit.

Sildenafil, a type V phosphodiesterase inhibitor, enhances smooth muscle relaxation in normal human and rabbit corpus cavernosum. We investigated the in vitro effects of sildenafil on non-adrenergic, non-cholinergic and nitric oxide (NO)-mediated cavernosal smooth muscle relaxation in diabetic rabbits, since alterations in this pathway are recognised in diabetic erectile dysfunction. Diabetes mellitus was induced in male New Zealand White rabbits with alloxan. Cavernosal strips from age-matched control, 3- and 6-month diabetic animals were mounted in organ baths. Relaxation responses to electrical field stimulation (1-20 Hz) or sodium nitroprusside (10(-8)-10(-4) M) were assessed in the absence and presence of sildenafil (10(-8) and 10(-7) M). The effect of sildenafil on cGMP formation by the corpus cavernosum was also assessed following stimulation with sodium nitroprusside, A23187 and acetylcholine. Sodium nitroprusside-stimulated relaxations were significantly (P<0.03) impaired in the corpus cavernosum from both diabetic groups, (IC(50)=4.6 x 10(-6) M following 3 months of diabetes mellitus and 4.0 x 10(-6) M following 6 months of diabetes mellitus; compared to 7.5 x 10(-7) M for pooled age-matched controls). Sildenafil (10(-7) M) significantly enhanced sodium nitroprusside-stimulated relaxation in control (P<0.05) and diabetic groups (P<0.03). Electrical field stimulation-mediated relaxations of the corpus cavernosum were significantly impaired after 6-month diabetes mellitus and enhanced by sildenafil (10(-8) M). cGMP formation by the diabetic corpus cavernosum was impaired significantly, but restored towards normal by sildenafil. We suggest that the impairment of NO-mediated relaxation of the corpus cavernosum reflect, at least in part, a defect in guanylyl cyclase activity. These findings support the use of sildenafil as an effective, orally administered, treatment for diabetic erectile dysfunction.

The systemic effects of intermittent claudication are reversed by angioplasty.

INTRODUCTION: exercise in patients with intermittent claudication causes systemic effects, the consequences of which are unknown. This study investigates whether successful PTA reverses the systemic effects. PATIENTS AND METHODS: ten patients with IC were recruited prior to PTA. Having emptied their bladders and rested for 1 h, pre-exercise blood and urine samples were collected. Patients underwent treadmill exercise to maximum walking time and blood samples were collected at 10, 20 and 30 min. A second urine sample was collected at 60 min. Total antioxidant capacity (TAC) and von Willebrands Factor (vWF) were measured in blood and albumin/creatinine ratio (ACR) and retinol binding protein/creatinine ratio (RBP/Cr) in urine. Patients were recalled 2 weeks after successful angioplasty and the protocol repeated. Following PTA patients walked for a maximum of 5 min. RESULTS: there was no significant change in vWF. Exercise in claudicants induced a significant increase in median ACR (pre/post exercise=0.85 p =0.03) and in median RBP/Cr (pre/post exercise=1.8 p =0.04). These changes were no longer evident after successful PTA. TAC was significantly different before and after angioplasty at all time intervals. CONCLUSION: successful PTA reverses glomerular effects of exercise in claudicants. Future work should investigate the use of PTA in conjunction with exercise in the treatment of peripheral vascular disease.

Clinical and angiographic outcome of different surgical strategies of bilateral internal mammary artery grafting.

Long-term survival, relief of angina, and prevention of myocardial infarction after coronary revascularization are related to the preoperative status of the patient, progression of coronary artery atherosclerosis, and the patency of the conduits used. The increased use of the internal mammary artery for coronary grafting depends upon the accumulation of data on superior late patency compared with venous conduits. These data have supported the simultaneous use of both left and right internal mammary arteries with reported improved late survival. However, controversy still surrounds the clinical and angiographic outcomes of some of the surgical strategies of bilateral internal mammary artery grafting. This review examines a range of surgical strategies of bilateral internal mammary artery grafting and their mid- and long-term clinical and angiographic outcomes. From the available data, careful preoperative selection of patients is paramount. Clinical and angiographic outcome of bilateral internal mammary grafting is superior to single internal mammary grafting with supplemental vein grafts when pedicled, sequential, or free aorto-coronary internal mammary artery is used. Further studies are needed to evaluate the midterm and long-term clinical and angiographic outcomes of complex strategies such as Y or T procedures.