Nox4 and redox signaling mediate TGF-ß-induced endothelial cell apoptosis and phenotypic switch.

Transforming growth factor-ß (TGF-ß) triggers apoptosis in endothelial cells, while the mechanisms underlying this action are not entirely understood. Using genetic and pharmacological tools, we demonstrated that TGF-ß induced a moderate apoptotic response in human cultured endothelial cells, which was dependent upon upregulation of the Nox4 NADPH oxidase and production of reactive oxygen species (ROS). In contrast, we showed that ectopic expression of Nox4 via viral vectors (vNox4) produced an antiapoptotic effect. TGF-ß caused ROS-dependent p38 activation, whereas inhibition of p38 blunted TGF-ß-induced apoptosis. However, vNox4, but not TGF-ß, activated Akt, and inhibition of Akt attenuated the antiapoptotic effect of vNox4. Akt activation induced by vNox4 was accompanied by inactivation of the protein tyrosine phosphatase-1B (PTP1B) function and enhanced vascular endothelial growth factor receptor (VEGFR)-2 phosphorylation. Moreover, we showed that TGF-ß enhanced Notch signaling and increased expression of the arterial marker EphrinB2 in a redox-dependent manner. In summary, our results suggest that Nox4 and ROS have pivotal roles in mediating TGF-ß-induced endothelial apoptosis and phenotype specification. Redox mechanisms may influence endothelial cell functions by modulating p38, PTP1B/VEGFR/Akt and Notch signaling pathways.

Arginase II inhibition prevents nitrate tolerance.

BACKGROUND AND PURPOSE: Nitrate tolerance, the loss of vascular responsiveness with continued use of nitrates, remains incompletely understood and is a limitation of these therapeutic agents. Vascular superoxide, generated by uncoupled endothelial NOS (eNOS), may play a role. As arginase competes with eNOS for L-arginine and may exacerbate the production of reactive oxygen species (ROS), we hypothesized that arginase inhibition might reduce nitrate tolerance. EXPERIMENTAL APPROACH: Vasodilator responses were measured in aorta from C57Bl/6 and arginase II knockout (argII -/-) mice using myography. Uncoupling of eNOS, determined as eNOS monomer : dimer ratio, was assessed using low-temperature SDS-PAGE and ROS levels were measured using L-012 and lucigenin-enhanced chemiluminescence. KEY RESULTS: Repeated application of glyceryl trinitrate (GTN) on aorta isolated from C57Bl/6 mice produced a 32-fold rightward shift of the concentration-response curve. However this rightward shift (or resultant tolerance) was not observed in the presence of the arginase inhibitor (s)-(2-boronethyl)-L-cysteine HCl (BEC; 100 µM) nor in aorta isolated from argII -/- mice. Similar findings were obtained after inducing nitrate tolerance in vivo. Repeated administration of GTN in human umbilical vein endothelial cells induced uncoupling of eNOS from its dimeric state and increased ROS levels, which were reduced with arginase inhibition and exogenous L-arginine. Aortae from GTN tolerant C57Bl/6 mice exhibited increased arginase activity and ROS production, whereas vessels from argII -/- mice did not. CONCLUSION AND IMPLICATIONS: Arginase II removal prevents nitrate tolerance. This may be due to decreased uncoupling of eNOS and consequent ROS production.

3',4'-Dihydroxyflavonol down-regulates monocyte chemoattractant protein-1 in smooth muscle: role of focal adhesion kinase and PDGF receptor signalling.

BACKGROUND AND PURPOSE: We investigated the effects of a synthetic flavonol, 3',4'-dihydroxyflavonol (DiOHF) on the expression of monocyte chemoattractant protein-1 (MCP-1) in rat vascular smooth muscle cells. EXPERIMENTAL APPROACH: MCP-1 expression was assessed by quantitative real-time PCR and protein phosphorylation by immunoprecipitation and Western blots. KEY RESULTS: DiOHF (1-30 micromol x L(-1)) concentration-dependently reduced MCP-1 expression in both quiescent cells and cells stimulated with platelet-derived growth factor (PDGF) or interleukin 1-beta. The effect of DiOHF was associated with a suppression of focal adhesion kinase (FAK)-mediated signalling. In vitro kinase assays demonstrated that DiOHF is a potent inhibitor of FAK kinase activity (EC(50)= 2.4 micromol x L(-1)). Expression of FAK-related non-kinase reduced basal MCP-1 expression, but not that induced by PDGF or interleukin 1-beta. DiOHF also inhibited autophosphorylation of PDGF receptors. The PDGF receptor inhibitor AG-1296 potently suppressed basal and PDGF-induced MCP-1 expression. Inhibition of extracellular signal-regulated kinase activation by DiOHF, either directly or indirectly, may also be involved in its effects on MCP-1 expression. DiOHF had no inhibitory effect on either p38 or nuclear factor-kappaB activation. Moreover, DiOHF inhibited smooth muscle cell spreading (a FAK-mediated response) and proliferation. CONCLUSIONS AND IMPLICATIONS: This is the first report on a flavonoid compound (DiOHF) that is a potent FAK inhibitor. DiOHF also inhibits PDGF receptor autophosphorylation. These effects underlie the inhibitory action of DiOHF on MCP-1 expression in smooth muscle cells. Our results suggest that DiOHF might be a useful tool for dissection of the (patho)physiological roles of FAK signalling.

Neovascularization in an arterio-venous loop-containing tissue engineering chamber: role of NADPH oxidase.

Using an in vivo arterio-venous loop-containing tissue-engineering chamber, we have created a variety of vascularized tissue blocks, including functional myocardium. The viability of the transplanted cells is limited by the rate of neovascularization in the chamber. A Nox2-containing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is thought to have a critical role in ischaemic angiogenesis. In this study we investigated whether NADPH oxidase is involved in the neovascularization process in the tissue-engineering chamber. New blood vessels originating from the venous and the arterial ends of the loop could be identified after 3 days, and the vessel density (by lectin staining) peaked after 7 days and was maintained for at least 14 days. This was accompanied by granulation tissue formation and concomitant increase in the mRNA level of Nox4 NADPH oxidase. Although the total level of Nox2 mRNA in the chamber tissue decreased from day 3 to day 7, immunohistochemistry identified a strong expression of Nox2 in the endothelial cells of the new vessels. In human microvascular endothelial cells, the NADPH oxidase inhibitor apocynin reduced NADPH oxidase activity and inhibited the angiogenic responses in vitro. Local treatment with the NADPH oxidase inhibitors apocynin or gp91ds-tat peptide significantly suppressed the vessel growth in the chamber. In conclusion, NADPH oxidase-dependent redox signalling is important for neovascularization in this novel tissue-engineering chamber in vivo, and boosting this signalling might be a new approach to extending vascularization and tissue growth.

Failure of antioxidants to protect against angiotensin II-induced aortic rupture in aged apolipoprotein(E)-deficient mice.

BACKGROUND AND PURPOSE: Oxidative stress may be involved in the development of abdominal aortic aneurysms (AAAs). Previous studies indicate that antioxidants protect against AAA formation during chronic angiotensin (Ang) II infusion in apolipoprotein E-deficient (ApoE(0)) mice. We here examine if these protective effects also occurred in aged ApoE(0) mice. EXPERIMENTAL APPROACH: Male ApoE(0) mice (50-60 weeks) were randomly divided into 4 groups: saline, Ang II (1000 ng kg(-1) min(-1) for 4 weeks), Ang II plus antioxidants (0.1% vitamin E in food plus 0.1% vitamin C in drinking water), and Ang II plus losartan (30 mg kg(-1) day(-1)). KEY RESULTS: Exogenous Ang II increased systolic blood pressure by 40 mmHg and resulted in the formation of pseudoaneurysms (rupture and extramural haematoma) in the abdominal aorta in 50% of animals. True aneurysmal dilatation was rarely observed. Antioxidants decreased systemic oxidative stress (plasma malondialdehyde), but had only minor effects on aortic rupture, relative to the complete prevention by losartan. Immunohistochemistry revealed strong matrix metalloproteinase-9 (MMP-9) expression in atherosclerotic plaques and at the sites of rupture. Antioxidants did not affect tumour necrosis factor-alpha-stimulated MMP-9 release from U937 cells. In addition, antioxidants had little effects on Ang II-induced renal dysfunction. CONCLUSIONS AND IMPLICATIONS: In contrast to previous findings in younger mice, antioxidants had only minor effects on Ang II-induced aortic rupture in aged mice. Our results demonstrate that the pathological features of the aneurysmal remodelling induced by Ang II in old ApoE(0) mice are distinct from those of human AAA.

Differential upregulation of Nox homologues of NADPH oxidase by tumor necrosis factor-alpha in human aortic smooth muscle and embryonic kidney cells.

NADPH oxidases are important sources of vascular superoxide, which has been linked to the pathogenesis of atherosclerosis. Previously we demonstrated that the Nox4 subunit of NADPH oxidase is a critical catalytic component for superoxide production in quiescent vascular smooth muscle cells. In this study we sought to determine the role of Nox4 in superoxide production in human aortic smooth muscle cells (AoSMC) and embryonic kidney (HEK293) cells under proinflammatory conditions. Incubation with tumor necrosis factor-alpha (TNF-alpha, 10 ng/ml) for 12 h increased superoxide production in both cell types, whereas angiotensin II, platelet-derived growth factor or interleukin-1beta had little effects. Superoxide production was completely abolished by the NADPH oxidase inhibitors diphenyline iodonium and apocynin, but not by inhibitors of xanthine oxidase, nitric oxide synthase or mitochondrial electron transport. TNF-alpha upregulated the expression of Nox4 in AoSMC at both message and protein levels, while Nox1 and Nox2 were unchanged. In contrast, upregulation of Nox2 appeared to mediate the enhanced superoxide production by TNF-alpha in HEK293 cells. We suggest that Nox4 may be involved in increased superoxide generation in vascular smooth muscle cells under proinflammatory conditions.

Activation of IP prostanoid receptors prevents cardiomyocyte hypertrophy via cAMP-dependent signaling.

The antihypertrophic action of angiotensin-converting enzyme inhibitors in the heart results partly from local potentiation of bradykinin. We have demonstrated that the antihypertrophic action of bradykinin is mediated by the release of nitric oxide from endothelium and elevation of cardiomyocyte cGMP. Whether other paracrine factors derived from the coronary endothelium, such as prostacyclin (PGI2), may act to prevent hypertrophy has not been explored. In the vasculature, activation by PGI2 of IP and EP1 prostanoid receptors elicits vasodilatation (via cAMP-dependent signaling) and vasoconstriction, respectively. The present objective was to determine whether IP prostanoid receptor activation has antihypertrophic actions in adult rat cardiomyocytes (ARCM). The selective IP agonist cicaprost (1 microM) virtually abolished the increase in [3H]phenylalanine incorporation (a marker of hypertrophy) induced either by endothelin-1 (ET-1; 60 nM, n = 10, P < 0.005) or by angiotensin II (1 microM, n = 6, P < 0.005). Cicaprost also inhibited ET-1 induction of c-fos mRNA expression, an additional marker of hypertrophy in ARCM (n = 5, P < 0.005). In the absence of hypertrophic stimuli, cicaprost alone did not significantly influence either marker. The antihypertrophic actions of cicaprost were mimicked by the dual IP/EP1 agonist iloprost (1 microM) in the presence of the EP1 antagonist AH-6809 (3 microM). Furthermore, cicaprost modestly but significantly increased cardiomyocyte cAMP content by 13 +/- 6% (P < 0.05, n = 4), and the antihypertrophic effect of cicaprost was lost in the presence of the cAMP-dependent protein kinase inhibitor H-89 (1 microM, n = 5, P < 0.05). However, ET-1 also induced increases in the activity of the intracellular growth signals ERK1 (by 3-fold) and ERK2 (by 5-fold) in ARCM, and these were not inhibited by cicaprost (P < 0.01, n = 5). Activation of IP receptors thus represents a novel approach to prevention of hypertrophy, and this effect is linked to cAMP-dependent signaling.

Endothelial dysfunction induced by oxidized low-density lipoproteins in isolated mouse aorta: a comparison with apolipoprotein-E deficient mice.

We characterized the acute effects of oxidized low-density lipoproteins (oxidized-LDL) on vascular reactivity in isolated aorta from wild-type C57BL/6J mice, and compared these with the chronic alterations in vascular function observed in apolipoprotein-E gene knockout [ApoE(-/-)] mice fed a high-fat diet, which results in hyperlipidemia and atherosclerosis. In the abdominal (but not thoracic) aorta, oxidized-LDL (100 microg/ml) reduced relaxations induced by acetylcholine (10(-9) M-10(-5) M), which are mediated entirely by nitric oxide (NO). The relaxations induced by the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 10(-8) M-10(-4) M), the cyclic GMP analogue 8-bromo cyclic GMP (100 microM) and the nonspecific vasodilator papaverine (100 microM) were not changed by oxidized-LDL. Native LDL had no effect on vasorelaxations. The attenuation of endothelium-dependent relaxations caused by oxidized-LDL mimicked the endothelial dysfunction found in ApoE(-/-) mice. These results are consistent with the suggestion that oxidized-LDL has an important role in the pathogenesis of endothelial NO dysfunction associated with hyperlipidemia and atherosclerosis in these mice.

Endothelium-dependent vasorelaxation independent of nitric oxide and K(+) release in isolated renal arteries of rats.

1. We investigated whether K(+) can act as an endothelium-derived hyperpolarizing factor (EDHF) in isolated small renal arteries of Wistar-Kyoto rats. 2. Acetylcholine (0.001 - 3 microM) caused relaxations that were abolished by removal of the endothelium. However, acetylcholine-induced relaxations were not affected by the nitric oxide (NO) synthase inhibitor N:(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM), by L-NAME plus the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 1 microM) or by L-NAME plus the cyclo-oxygenase inhibitor indomethacin (10 microM). In rings precontracted with high-K(+)(60 mM) physiological salt solution in the presence of L-NAME, acetylcholine-induced relaxations were abolished. 3. L-NAME-resistant relaxations were abolished by the large-conductance Ca(2+)-activated K(+) channel inhibitor charybdotoxin plus the small-conductance Ca(2+)-activated K(+) channel inhibitor apamin, while the inward rectifier K(+) channel inhibitor Ba(2+) or the gap junction inhibitor 18alpha-glycyrrhetinic acid had no effect. Acetylcholine-induced relaxation was unchanged by ouabain (10 microM) but was partially inhibited by a higher concentration (100 microM). 4. In half of the tissues tested, K(+)(10 mM) itself produced L-NAME-resistant relaxations that were blocked by ouabain (10 microM) and partially reduced by charybdotoxin plus apamin, but not affected by 18alpha-glycyrrhetinic acid or Ba(2+). However, K(+) did not induce relaxations in endothelium-denuded tissues. 5. In conclusion, acetylcholine-induced relaxations in this tissue are largely dependent upon hyperpolarization mechanisms that are initiated in the endothelium but do not depend upon NO release. K(+) release cannot account for endothelium-dependent relaxation and cannot be an EDHF in this artery. However, K(+) itself can initiate endothelium-dependent relaxations via a different pathway from acetylcholine, but the mechanisms of K(+)-induced relaxations remain to be clarified.

Endothelial dysfunction limits the antihypertrophic action of bradykinin in rat cardiomyocytes.

We have previously demonstrated that bradykinin blocks hypertrophy of isolated cardiomyocytes: this is dependent on the release of nitric oxide from endothelial cells. In the present study, we investigated the influence of endothelial dysfunction on the antihypertrophic action of bradykinin. Angiotensin II (1 microM) induced a 34 +/- 2% increase in [3H]phenylalanine incorporation (P<0.001), an in vitro marker of hypertrophy, in adult rat cardiomyocytes co-cultured with bovine aortic endothelial cells. This response was blocked by bradykinin (10 microM), but restored by the nitric oxide synthase inhibitor. N(omega)-monomethyl-L-arginine (100 microM). However, the antihypertrophic effect of bradykinin in co-culture was abolished by 24 h pretreatment of endothelial cells with high glucose (25 mM, to mimic hyperglycemia) and attenuated by hydrogen peroxide (100 microM, to mimic oxidative stress). Pretreatment with oxidized low-density lipoprotein (100 microg/ml for 24 h, to mimic hyperlipidemia) was without effect. The hypertrophic response to angiotensin II was not modified by endothelial cell pretreatment. Furthermore, the ability of bradykinin to elevate cGMP (a marker for nitric oxide) in cardiomyocytes co-cultured with endothelial cells was attenuated by pretreatment with either high glucose or hydrogen peroxide. In conclusion, loss of the cardioprotective action of bradykinin against angiotensin II-induced hypertrophy was associated with impaired nitric oxide release from dysfunctional endothelial cells.

Type-C natriuretic peptide prevents development of experimental atherosclerosis in rabbits.

1. We investigated the effect of local administration of type-C natriuretic peptide (CNP) on the endothelial dysfunction and development of an atheroma-like neointima induced by a peri-arterial collar in rabbits. 2. Peri-arterial collars were placed on both common carotid arteries allowing local treatment of the collared region with either CNP (10 micromol/L) or saline. After 7 days, uncollared (control) and collared sections were taken from both arteries for pharmacological and morphological analysis. 3. Application of the collar markedly attenuated (P < 0.05) endothelium-dependent vasorelaxation induced by acetylcholine (ACh); inhibition of 5-hydroxytryptamine contraction was 80+/-5% in control sections compared with 44+/-4% in collared sections from the same arteries. Local infusion of CNP (10 micromol/L) into the collar restored ACh-induced vasorelaxation (74+/-3% from collared arteries + CNP vs 77+/-2% from control sections from the same arteries). 4. Type-C natriuretic peptide treatment also reduced (P < 0.05) intimal thickening compared with contralateral collared arteries (intima/media ratio 0.06+/-0.01 vs 0.16+/-0.01). 5. These results provide evidence that locally administered CNP is effective in preventing the endothelial dysfunction and development of a neointima in this model.

Cyclosporin A and tacrolimus (FK506) suppress expression of inducible nitric oxide synthase in vitro by different mechanisms.

1 The effects of the immunosuppressant drugs cyclosporin A and tacrolimus (FK506) on nitric oxide synthesis were examined in a murine macrophage cell line (J774) and rat vascular smooth muscle cells (VSMC) in culture for 24 and 48 h, respectively. 2 Cyclosporin A (0.01-10 microM) inhibited by up to 90% accumulation of nitrite induced by lipopolysaccharide (LPS) in both cell lines, but FK506 (0.01-10 microM) had a weaker effect on nitrite accumulation in these cells. Cyclosporin A and FK506 (at 1 microM) also significantly inhibited nitrite production induced by recombinant murine interferon-gamma (rIFNgamma) and recombinant murine interleukin-1beta (rIL-1beta) in J774 and VSMC, respectively. 3 In J774 cells, cyclosporin A (but not FK506) at 1 microM was inhibitory when co-incubated with the inducing agents but not when the cells were treated with the immunosuppressant before or after the inducer. In VSMC, nitrite production was inhibited by co-incubation of cyclosporin A or FK506 with the inducer, or when the immunosuppressants were pre-incubated with cells. In contrast, N-monomethyl L-arginine (NMMA) abolished nitrite production when incubated with either cell type during or after addition of inducing agent, but not if cells were preincubated with NMMA. 4 RNA extracted from treated J774 and VSMC was subjected to reverse transcription-polymerase chain reaction (RT-PCR). Cyclosporin A, but not FK506, suppressed expression of mRNA for NOS2 in a concentration-dependent manner when co-incubated with LPS. 5 The fact that the potency difference between cyclosporin A and FK506 for NO suppression is the opposite to that for inhibition of interleukin-2 generation suggests that the immunosuppressants act in J774 macrophages and VSMC through intracellular mechanisms that differ from those elucidated in T-cells. Cyclosporin A suppresses NOS2 gene transcription, but FK506 acts post-transcriptionally to suppress NO generation in VSMC. 6 Taken together the present data suggest that therapeutic concentrations of cyclosporin A, but not FK506, might well suppress NO production, but FK506 would not have this effect. Suppression of NO might contribute to the side effects of hypertension and nephrotoxicity associated with long-term use of cyclosporin A to prevent transplant rejection.

Hyperglycaemia abolishes the antihypertrophic efficacy of bradykinin in rat ventricular myocytes.

1. Bradykinin inhibits hypertrophy of rat ventricular myocytes, but only in the presence of endothelial cells. 2. The influence of hyperglycaemia on the ability of bradykinin to prevent hypertrophy was investigated in adult rat ventricular myocytes cocultured with bovine aortic endothelial cells (BAEC). 3. In myocytes cocultured with normal BAEC, angiotensin II (AngII; 1 mumol/L) significantly increased [3H]-phenylalanine incorporation (an in vitro marker of hypertrophy) by 32 +/- 2%. This was abolished by bradykinin (10 mumol/L). 4. Pretreatment of BAEC with high glucose (25 mmol/L for 24 h) prior to coculture with myocytes reduced the antihypertrophic effect of bradykinin, but did not modulate the hypertrophic effect of AngII. 5. Pretreatment of BAEC with hyperglycaemia abolishes the antihypertrophic efficacy of bradykinin in rat ventricular myocytes cocultured with BAEC. This has implications for the action of angiotensin-converting enzyme inhibitors.

Lipocortin-1 preserves myocardial responsiveness to beta-adrenergic stimulation in rat papillary muscle.

1. During septic shock, myocardial contractile dysfunction is accompanied by the release of cytokines and enhanced production of nitric oxide, and the contractile dysfunction is prevented by glucocorticoids. 2. Myocardial dysfunction was induced in vitro by incubation of rat papillary muscle for 15 h with endotoxin (lipopolysaccharide, LPS) and interferon-gamma (IFN-gamma). 3. Both baseline contractile function and inotropic responsiveness to isoprenaline were markedly reduced by the combination of LPS plus IFN-gamma. 4. Lipocortin-1 (LC-1) is induced by glucocorticoids, and LC-1(2-26), its N-terminal fragment, protected the papillary muscle inotropic responsiveness to isoprenaline, but did not affect the decline in baseline contractile function induced by LPS plus IFN-gamma. 5. The mechanisms of this protective action need to be explored further, but LC-1 may prove to be a novel cardioprotective agent for the management of septic shock.

Dexamethasone inhibits endotoxin-induced changes in calcium and contractility in rat isolated papillary muscle.

This study investigates whether endotoxin-induced contractile dysfunction is associated with a defect in the modulation of calcium homeostasis and the potential mechanisms involved. Treatment of rats in vivo with endotoxin significantly decreased the magnitude of contractile transients in electrically stimulated left ventricular papillary muscle isolated after an equilibration period of 6 hours. Although no significant difference was found in the peak intracellular calcium concentration ([Ca2+]i) between the endotoxin-treated and control groups, resting [Ca2+]i) was significantly elevated in the endotoxin-treated group, producing a smaller Ca2+ transient (basal-peak difference) in this group. Pretreatment of rats with dexamethasone prevented the endotoxin-induced decrease in peak tension and inhibited the elevation in resting [Ca2+]i, with a resultant maintenance of Ca2+ transient magnitude. Similar observations were made during stimulation of the muscles by the beta-adrenoceptor agonist, isoprenaline. These results show that endotoxin-induced reduction of cardiac contractile performance is mediated, at least in part, by elevating resting [Ca2+]i, and a glucocorticoid protected from these negative effects. While endotoxin reduces the magnitude of the Ca2+ transient it does not alter peak [Ca2+]i availability. Further investigation is required to determine whether endotoxin decreases contractile performance by reducing the sensitivity of cardiac myofilaments to calcium.

Nitric oxide in atherosclerosis: vascular protector or villain?

1. Nitric oxide (NO) has important roles in physiological vasodilatation, cytotoxicity and vascular disease. Nitric oxide and prostacyclin (PGI2), both released from the endothelium, act synergistically to inhibit platelet aggregation and adhesion. These autacoids also inhibit the adhesion and migration of leucocytes and, in some arteries, they synergize in terms of vasodilation. 2. The development of atherosclerosis and hyperlipaemia per se is accompanied by impairment of endothelium-dependent vasodilation. 3. Atherosclerosis is associated with marked changes in the activity of isoforms of NO synthase (NOS) in the artery wall, including increased expression of the NOS2 (inducible) isoform in complex human lesions as well as in the neointima of experimental animal models. 4. Failure of NO release from the endothelium with normal physiological stimuli, which has been attributed to a defect in the operation of the endothelial NOS (NOS3), provides conditions propitious for leucocyte adhesion, vasospasm, thrombosis and, in addition, may promote increased proliferation of intimal cells. 5. Nitric oxide and superoxide anions generated by inflammatory cells in atherosclerosis react to form cytodestructive peroxynitrite radicals, potentially causing injury to the endothelium and myocytes, and this may be a factor in apoptosis of cells leading to plaque rupture. 6. We have been able to reverse these NO defects with therapeutic agents, including angiotensin-converting enzyme inhibitors, antagonists of platelet-activating factor and NO donor compounds, all offering promise in protecting against some manifestations of vascular disease.

A platelet-activating factor antagonist (WEB 2170) preserves endothelium-dependent vasodilatation and prevents development of a neo-intima induced by a periarterial collar in rabbit carotid arteries.

Platelet-activating factor (PAF) may be involved in adhesion of leucocytes and migration of cells during vascular remodelling for it is expressed in leucocytes after cytokine priming and is required for cell adhesion. We studied the effects of WEB 2170, a potent PAF antagonist, on the development of an atheroma-like neo-intima induced by a peri-arterial collar in rabbits. Either WEB 2170 (3 mg/kg/day) or vehicle was given by subcutaneous injection once a day for 4 or 9 days, and on day 3 peri-arterial collars were applied to both carotid arteries in all animals. Two or 7 days after implanting the collars vasodilator responses to the endothelium-dependent vasodilator, acetylcholine and the endothelium-independent vasodilator, sodium nitroprusside were studied in isolated artery rings from both groups of rabbits. Neo-intima formation after 7 days (day 10 of treatment) was measured by light microscopy as the ratio of cross-sectional areas of intima and media, and expression of inducible nitric oxide synthase (iNOS) was studied by immunohistochemistry. PAF-induced platelet aggregation ex vivo was inhibited specifically in WEB 2170-treated rabbits. At day 5, acetylcholine-induced vasorelaxation in collared artery rings was markedly impaired as compared to control sections from both vehicle- and WEB 2170-treated rabbits. At day 10, acetylcholine-induced vasorelaxation in collared artery rings from vehicle rabbits was markedly less than in controls, but in WEB 2170-treated rabbits, the acetylcholine response in collared arteries was similar to control sections. Intimal thickening was much reduced in WEB 2170-treated rabbits, ratios of intima/media areas being vehicle: 0.21 +/- 0.02 (n = 5) and WEB 2170: 0.07 +/- 0.01 (n = 7; p < 0.01). Immunofluorescence showed expression of iNOS only in the neo-intima of vehicle-treated, collared arteries, but not in the residual neo-intima of WEB 2170-treated, collared arteries. These results suggest that WEB 2170 is effective in preserving endothelial function, prevents the development of neo-intima and blocks iNOS expression in the neo-intima in this model.

Cardiodepressant effects of interferon-gamma and endotoxin reversed by inhibition of NO synthase 2 in rat myocardium.

Endogenous nitric oxide (NO) signalling pathways within the myocardium depress myocardial contractile function in septic shock and some cardiomyopathies. We have explored the role of NO synthases (NOSs) in mediating the cardiodepressant actions of interferon-gamma (IFN-gamma) and lipopolysaccaride (LPS) in rat papillary muscle. Muscles from the right ventricle were electrically stimulated (0.2 Hz) at 30 degrees C and isometric contraction monitored. Exposure to IFN-gamma and LPS for 15 h in vitro significantly decreased the peak tension (PT for IFN-gamma + LPS, from 0.13 +/- 0.03 to 0.07 +/- 0.02 g) and rate of tension development (dT/dt for IFN-gamma + LPS, from 1.78 +/- 0.36 to 1.17 +/- 0.28 g/s) compared to untreated controls, and this was prevented by dexamethasone (1 microM) and partly reversed by a non-specific NOS inhibitor, NG-nitro-L-arginine (NOLA, 30 microM). Likewise, the maximum inotropic response of the papillary muscles to isoprenaline (0.001-10 microM) decreased significantly after 15 h treatment with IFN-gamma and LPS (PT from 83 +/- 18 to 28 +/- 6%; +dT/dt from 83 +/- 12 to 31 +/- 7%; -dT/dt from 83 +/- 12 to 38 +/- 6%). Again, the depressant effects of IFN-gamma and LPS on inotropic responsiveness to isoprenaline were completely prevented by pretreatment with dexamethasone (1 microM), by a specific inhibitor of NOS2, mercaptoethylguanidine (MEG, 30 microM) and by NOLA. Whereas dexamethasone and NOLA protected against the attenuation of baseline contractions induced by LPS and IFN-gamma, MEG did not. Western blot analysis of cardiac myocytes showed that there was no constitutive expression of NOS2, but IFN-gamma and LPS induced expression of NOS2, and this was prevented by dexamethasone. Thus IFN-gamma, in the presence of LPS, reduced papillary muscle contraction and decreased responsiveness to beta-adrenoceptor stimulation through induction of NOS2 in the muscle. Increased NO production may contribute to the cardiac depression during septic shock and anti-cancer therapy with cytokines, and perhaps in heart failure.

CGRP and nitric oxide of neuronal origin and their involvement in neurogenic vasodilatation in rat skin microvasculature.

1. Sensory nerves are important for the initiation of neurogenic inflammation and tissue repair. Both calcitonin gene-related peptide (CGRP) and nitric oxide (NO) have been implicated in neurogenic vasodilatation and inflammatory responses. 2. A blister model in the rat hind footpad was used as a site to induce neurogenic vasodilatation in response to antidromic electrical stimulation of the sciatic nerve. Blood flux was monitored with a laser Doppler flow monitor. 3. The quantitative contributions of CGRP and NO to vasodilatation were examined by use of the CGRP receptor antagonist CGRP8-37 and NO synthase inhibitors 7-nitroindazole (7-NI), 3-bromo 7-NI and N(G)-nitro L-arginine methyl ester (L-NAME). The potential modulatory role of endothelin was examined by use of the ET(A) receptor antagonist BQ-123. 4. CGRP8-37 (10 microM) was perfused over the blister base before nerve stimulation and continuously throughout the post-stimulation period, resulting in a significant reduction (41%) in the blood flux vascular response. 5. Pretreatment with the specific neuronal NO synthase inhibitors, 7-NI and 3-bromo 7-NI (10 mg kg(-1), i.v.), and of the non-specific L-NAME (100 microM), resulted in significant inhibition of the blood flux response (36%, 72% and 57% decrease, respectively). In contrast, 7-NI treatment in young rats pretreated with capsaicin had no further effect on the vascular response, suggesting that the source of NO is the sensory nerves. 6. BQ-123 (10 microM) significantly enhanced the stimulation-induced blood flux response (61% increase). When 7-NI was co-administered with either CGRP8-37 or BQ-123, the drug actions were additive, suggesting that there was no interaction between NO and CGRP or endothelin. 7. These data suggest that both NO and CGRP participate in neurogenic vasodilatation in rat skin microvasculature and that this response is modulated by endogenous endothelin.

A nitric oxide donor (spermine-NONOate) prevents the formation of neointima in rabbit carotid artery.

1. In the present study we investigated the effect of spermine diazeniumdiolate (spermine-NONOate), a nitric oxide donor, on the early development of atheroma-like lesions induced by a peri-arterial collar in rabbits. 2. Spermine-NONOate was given locally by incorporating the compound (1 mg/mL) into a silastic collar, which was applied on one common carotid artery of rabbit while the other carotid artery had a placebo collar (without compound) applied. 3. Fourteen days postimplantation, both carotid arteries were dissected free for histological study (n = 6). 4. After 14 days with collars, treatment with spermine-NONOate had significantly reduced (by 74%) the thickness of the neointima in comparison with the contralateral collared artery without compound. Blood pressure did not change during treatment. Nitric oxide, detected as nitrite, was still released from spermine-NONOate silastic collars after 14 days implantation. 5. These results suggest that locally administered spermine-NONOate is effective in slowing the development of neointima in this model.