9th Hatter Biannual Meeting: position document on ischaemia/reperfusion injury, conditioning and the ten commandments of cardioprotection.

In the 30 years since the original description of ischaemic preconditioning, understanding of the pathophysiology of ischaemia/reperfusion injury and concepts of cardioprotection have been revolutionised. In the same period of time, management of patients with coronary artery disease has also been transformed: coronary artery and valve surgery are now deemed routine with generally excellent outcomes, and the management of acute coronary syndromes has seen decade on decade reductions in cardiovascular mortality. Nonetheless, despite these improvements, cardiovascular disease and ischaemic heart disease in particular, remain the leading cause of death and a significant cause of long-term morbidity (with a concomitant increase in the incidence of heart failure) worldwide. The need for effective cardioprotective strategies has never been so pressing. However, despite unequivocal evidence of the existence of ischaemia/reperfusion in animal models providing a robust rationale for study in man, recent phase 3 clinical trials studying a variety of cardioprotective strategies in cardiac surgery and acute ST-elevation myocardial infarction have provided mixed results. The investigators meeting at the Hatter Cardiovascular Institute workshop describe the challenge of translating strong pre-clinical data into effective clinical intervention strategies in patients in whom effective medical therapy is already altering the pathophysiology of ischaemia/reperfusion injury-and lay out a clearly defined framework for future basic and clinical research to improve the chances of successful translation of strong pre-clinical interventions in man.

Comparative clinical- and cost-effectiveness of candesartan and losartan in the management of hypertension and heart failure: a systematic review, meta- and cost-utility analysis.

The UK National Health Service (NHS) currently spends in excess of £250 million per annum on angiotensin II receptor blockers (ARBs) for the treatment of hypertension and heart failure; with candesartan currently dominating the market. With the recent introduction of generic losartan, we set out to directly compare the branded market leader to its now cheaper alternative. The primary objectives were to compare the blood pressure (BP) lowering efficacy and cardiovascular outcomes of candesartan and losartan in the treatment of essential hypertension and chronic heart failure, respectively. The secondary objective was to model their comparative incremental cost-effectiveness in a UK NHS setting. The Cochrane Central Register of Controlled Trials (Cochrane Library 2009, issue 2), which contains the Hypertension and Heart Group's specialist register, Medline (1950-February 2010), and Embase (1980-February 2010) were included in the search strategy. Selection criteria were randomised studies of candesartan versus losartan in adults (> 18 years). The main outcome measures were as follows: Hypertension: mean change from baseline in trough (24 h postdose) systolic and diastolic BP. Heart failure: composite of cardiovascular death and hospital admission for management of heart failure. Two reviewers applied inclusion criteria, assessed trial quality, and extracted data. Eight (three of which met inclusion criteria) and zero trials compared candesartan directly with losartan in the treatment of hypertension and heart failure, respectively. A between-treatment difference of -1.96 mmHg [95% confidence interval (CI) -2.40 to -1.51] for trough diastolic BP and -3.00 mmHg (95% CI -3.79 to -2.22) for trough systolic BP in favour of candesartan was observed. Based on this differential, a 10-year Markov model estimates the cost per quality-adjusted life-year gained to exceed £40,000 for using candesartan in place of generic losartan. Candesartan reduces BP to a slightly greater extent when compared with losartan, however, such difference is unlikely to be cost-effective based on current acquisition costs, perceived NHS affordability thresholds and use of combination regimens. We could find no robust evidence supporting the superiority of candesartan over losartan in the treatment of heart failure. We therefore recommend using generic losartan as the ARB of choice which could save the UK NHS approximately £200 million per annum in drug costs.

Reciprocal regulation of human soluble and particulate guanylate cyclases in vivo.

BACKGROUND & PURPOSE: We demonstrated previously that reciprocal regulation of soluble (sGC) and particulate (pGC) guanylate cyclases by NO and natriuretic peptides coordinates cyclic cGMP-mediated vasodilatation in vitro. Herein, we investigated whether such an interaction contributes to vascular homeostasis in mice and humans in vivo. EXPERIMENTAL APPROACH: Mean arterial blood pressure (MABP) changes in anaesthetized mice were monitored in response to i.v. administration of cGMP- and cAMP-dependent vasodilators in wild-type (WT), endothelial NO synthase (eNOS) and natriuretic peptide receptor (NPR)-A knockout mice. Forearm blood flow (FBF) in response to intra-brachial infusion of ANP (25, 50, 100, 200 pmol min(-1)) in the absence and presence of the NOS inhibitor NG-methyl-L-arginine (L-NMA; 4 micromol min(-1)) and the control constrictor noradrenaline (240 pmol min(-1)) was assessed in healthy volunteers. KEY RESULTS: Sodium nitroprusside (SNP; NO-donor) and atrial natriuretic peptide (ANP) produced dose-dependent reductions in MABP in WT animals that were significantly enhanced in eNOS KO mice. In NPR-A K mice, SNP produced a dose-dependent reduction in MABP that was significantly greater than that in WT mice. Responsiveness to the cAMP-dependent vasodilator epoprostenol was similar in WT, eNOS KO and NPR-A KO animals. ANP caused vasodilatation of the forearm resistance vasculature that was significantly greater in individuals lacking endothelium-derived NO (i.e. L-NMA treated). CONCLUSIONS & IMPLICATIONS: These data demonstrate that crosstalk occurs between the NO-sGC and ANP-pGC pathways to regulate cGMP-dependent vasodilatation in vivo in both mice and humans. These findings have implications for understanding the link between natriuretic peptide activity and cardiovascular risk.

Heterogenous nature of flow-mediated dilatation in human conduit arteries in vivo: relevance to endothelial dysfunction in hypercholesterolemia.

Flow-mediated dilatation (FMD) of conduit arteries is dependent on an intact endothelium, although the mechanisms are not fully understood. Using high-resolution ultrasound, we examined the role of endothelial mediators in radial artery dilatation in response to transient (short period of reactive hyperemia) and sustained (prolonged period of reactive hyperemia, hand warming, or an incremental infusion of acetylcholine into the distal radial artery) hyperemia. After short episodes of reactive hyperemia, FMD was abolished by local infusion of the nitric oxide synthesis inhibitor N:(G)monomethyl-L-arginine (5.3+/-1.2% versus 0.7+/-0.7%, P:<0.001). In contrast, basal vessel diameter and dilatation after prolonged episodes of reactive hyperemia, hand warming, and distal infusion of acetylcholine were not attenuated by nitric oxide synthesis inhibition. Inhibition of cyclooxygenase or local autonomic nervous system blockade also had no effect on FMD. Patients with hypercholesterolemia exhibited reduced FMD in response to transient hyperemia, but the response to sustained hyperemia was normal. These data suggest heterogeneity of endothelial responses to blood flow that are dependent on the characteristics of the flow stimulus. Dilatation after brief episodes of hyperemia is mediated by release of nitric oxide, whereas dilatation during sustained hyperemia is unaffected by NO synthesis inhibition. Hypercholesterolemia seems to differentially affect these pathways with impairment of the nitric oxide-dependent pathway and preservation of non nitric oxide-mediated dilatation to sustained flow stimuli.

KATP channel activation induces ischemic preconditioning of the endothelium in humans in vivo.

BACKGROUND: Endothelial dysfunction contributes to ischemia-reperfusion injury (IRI) and is reduced by ischemic preconditioning (IPC). IPC may involve activation of ATP-sensitive potassium channels (K(ATP)). We determined whether modulation of K(ATP) channels occurs in endothelial IPC in humans. METHODS AND RESULTS: IRI of the forearm was induced by inflating a blood pressure cuff to 200 mm Hg for 20 minutes in healthy volunteers. K(ATP) activation was modulated by intra-arterial glibenclamide (blocker) and diazoxide (opener). Endothelial function (response to intra-arterial acetylcholine) was assessed with forearm plethysmography before and after (1) 15-minute reperfusion, (2) IRI preceded by IPC (3 five-minute periods of ischemia), (3) IRI preceded by IPC with glibenclamide, (4) IPC followed by glibenclamide before IRI, (5) IRI preceded by diazoxide, and (6) IRI preceded by coinfusion of glibenclamide with diazoxide. IRI caused endothelial dysfunction (P=0.002), which IPC prevented (P=0.40). Glibenclamide abolished IPC when given contemporaneously with (P=0.003) or during IRI (P=0.0005). Diazoxide prevented endothelial dysfunction after IRI (P=0.68) but not when coinfused with glibenclamide. CONCLUSIONS: Glibenclamide abolishes and diazoxide mimics endothelial IPC in humans. The time course of the effect of glibenclamide suggests involvement of K(ATP) channels as effectors of endothelial IPC in vivo. These data may have implications for understanding the therapeutic role of agents that modulate K(ATP) channel function.

Transient limb ischemia induces remote ischemic preconditioning in vivo.

BACKGROUND: Ischemic preconditioning reduces local tissue injury caused by subsequent ischemia-reperfusion (IR), but may also have a salutary effect on IR injury of tissues remote from those undergoing preconditioning. We tested the hypothesis that limb ischemia induces remote preconditioning, reduces endothelial IR injury in humans, and reduces experimental myocardial infarct size. METHODS AND RESULTS: Endothelial IR injury of the human forearm was induced by 20 minutes of upper limb ischemia (inflation of a blood pressure cuff to 200 mm Hg) followed by reperfusion. Remote preconditioning was induced by three 5-minute cycles of ischemia of the contralateral limb. Venous occlusion plethysmography was used to assess forearm blood flow in response to acetylcholine at baseline and 15 minutes after reperfusion. Experimental myocardial infarction was achieved by 40 minutes of balloon occlusion of the left anterior descending artery in 15-kg pigs. Remote preconditioning was induced by four 5-minute cycles of lower limb ischemia. Triphenyltetrazolium staining was used to assess the extent of myocardial infarction. In the human study, the response to acetylcholine was significantly attenuated in the control group after 15 minutes' reperfusion, but remote preconditioning prevented this reduction. Limb ischemia caused a significant reduction in the extent of myocardial infarction relative to the area at risk compared with control (26+/-9% versus 53+/-8%, P<0.05). CONCLUSION: Remote ischemic preconditioning prevents IR-induced endothelial dysfunction in humans and reduces the extent of myocardial infarction in experimental animals. Transient limb ischemia is a simple preconditioning stimulus with important potential clinical applications.

Ischemic preconditioning prevents endothelial injury and systemic neutrophil activation during ischemia-reperfusion in humans in vivo.

BACKGROUND: Endothelial dysfunction leading to neutrophil infiltration of tissues has been implicated in tissue injury caused by ischemia-reperfusion (IR). Tissue injury during IR can be reduced by prior ischemic preconditioning (IPC). In humans, it is unclear whether endothelial dysfunction occurs during IR or whether IPC offers protection against endothelial dysfunction and inflammatory cell activation. We studied the effects of experimental IR on endothelial and neutrophil function in the human forearm in vivo and examined the protection afforded by IPC. METHOD AND RESULTS: The forearm was made ischemic for 20 minutes by inflating a blood pressure cuff to 200 mm Hg. We assessed endothelial function of conduit (radial artery flow-mediated dilation) and resistance vessels (blood flow responses to intra-arterial infusion of the endothelium-dependent dilator acetylcholine) in healthy volunteers before and after IR. IR reduced flow-mediated dilation of the radial artery at 15 minutes of reperfusion (7.7+/-1.5% to 3.5+/-0.9%) and the dilator response of resistance vessels to acetylcholine at 15, 30, and 60 minutes of reperfusion. IR did not reduce the dilator response of the radial artery to glyceryltrinitrate and only caused a small reduction of glyceryltrinitrate-induced dilation of resistance vessels at 60 minutes of reperfusion. IR caused an increase in neutrophil CD11b expression and platelet-neutrophil complexes in the circulating blood. IPC (three 5-minute episodes of ischemia) before IR prevented endothelial dysfunction and neutrophil activation. CONCLUSIONS: A clinically relevant period of ischemia-reperfusion causes profound and sustained endothelial dysfunction and systemic neutrophil activation. IPC attenuates both of these effects in humans.

Acute systemic inflammation impairs endothelium-dependent dilatation in humans.

BACKGROUND: We tested the hypothesis that endothelial dysfunction underlies the association between an acute inflammatory episode and the transiently increased risk of a cardiovascular event by examining the effects of an experimental inflammatory stimulus on endothelium-dependent vasodilation. METHODS AND RESULTS: Salmonella typhi vaccine was used to generate a systemic inflammatory response in healthy volunteers. In 12 subjects, dilatation of the brachial artery to flow and to sublingual nitroglycerin (NTG) was recorded (conduit vessel response), and in 6 subjects, venous occlusion plethysmography was used to measure forearm blood flow during intrabrachial infusion of the endothelium-dependent dilators acetylcholine (ACh) and bradykinin (BK) and the endothelium-independent dilators NTG and verapamil (resistance vessel response). Responses were assessed 16 hours before and 8 and 32 hours after vaccination. Vaccination resulted in elevations in white cell count and serum levels of interleukin-6 and interleukin-1 receptor antagonist. Eight hours after vaccination, resistance vessel responses to BK (P:=0.0099) and ACh (P:=0.0414) were markedly attenuated, and brachial artery flow-mediated dilatation was depressed. Resistance vessel responses to verapamil and NTG were unchanged, as was the conduit vessel response to NTG. Thirty-two hours after vaccination, resistance vessel responses to BK and ACh had returned to normal. CONCLUSIONS: S typhi vaccine generates a mild inflammatory reaction associated with temporary but profound dysfunction of the arterial endothelium in both resistance and conduit vessels to both physical and pharmacological dilator stimuli. This finding might explain the association between infection and inflammation and the enhanced risk of an acute cardiovascular event.

Protection against lipopolysaccharide-induced endothelial dysfunction in resistance and conduit vasculature of iNOS knockout mice.

Endothelial dysfunction is a characteristic of, and may be pathogenic in, inflammatory cardiovascular diseases, including sepsis. The mechanism underlying inflammation-induced endothelial dysfunction may be related to the expression and activity of inducible nitric oxide synthase (iNOS). This possibility was investigated in isolated resistance (mesenteric) and conduit (aorta) arteries taken from lipopolysaccharide (LPS)-treated (12.5 mg/kg i.v.) or saline-treated iNOS knockout (KO) and wild-type (WT) mice. LPS pretreatment (for 15 h, but not 4 h) profoundly suppressed responses to acetylcholine (ACh) and significantly reduced sensitivity to the NO donor spermine-NONOate (SPER-NO) in aorta and mesenteric arteries of WT mice. This effect was temporally associated with iNOS protein expression in both conduit and resistance arteries and with a 10-fold increase in plasma NOx levels. In contrast, no elevation of plasma NOx was observed in LPS-treated iNOS KO animals, and arteries dissected from these animals did not express iNOS or display hyporeactivity to ACh or SPER-NO. The mechanism underlying this phenomenon may be suppression of eNOS expression, as observed in arteries of WT animals, that was absent in arteries of iNOS KO animals. These results clearly demonstrate that iNOS induction plays an integral role in mediation of the endothelial dysfunction associated with sepsis in both resistance and conduit arteries.

Effects of S-nitroso-glutathione in the human forearm circulation: evidence for selective inhibition of platelet activation.

OBJECTIVE: Nitric oxide (NO) is a vasodilator and inhibitor of platelet function. The clinical use of NO donors as inhibitors of platelet activation is limited by their concomitant hypotensive effect. S-nitroso-glutathione (GSNO) has a significant antiplatelet effect at doses that cause only a small decrease in blood pressure in rats. The aim of this study was to examine the antiplatelet and vasodilator properties of this nitrosothiol in the human forearm. METHODS: Forearm blood flow was measured by forearm occlusion plethysmography in five healthy males. Ex vivo platelet aggregation to ADP was performed in a platelet ionised calcium lumi-aggregometer. RESULTS: Intra-arterial infusion of GSNO (0.2, 1, and 5 nmol.min-1) resulted in inhibition of ADP (1-10 microM) induced platelet aggregation. This inhibition was submaximal for 0.2 and maximal for 1 and 5 nmol.min-1. However, the antiaggregatory effect observed at the lowest dose of GSNO was accompanied only by a threshold increase in forearm blood flow. CONCLUSIONS: These results show that GSNO is more effective as an inhibitor of platelet activation than as a vasodilator, suggesting that it is possible to achieve selective antiplatelet and potentially antithrombotic effects with NO donors.

Intravenous Furosemide for Acute Decompensated Congestive Heart Failure: What Is the Evidence?

Use of intravenous furosemide rather than oral administration in acute decompensated congestive cardiac failure is universally recommended in international guidelines. We argue that this recommendation is not supported by the existing evidence, and suggest that trials should be performed to determine whether larger doses of oral furosemide should be prescribed prior to an IV switch. This could reduce length of hospital admissions and allow for more patients to be managed in the primary care setting.

Changes in endothelium-dependent vasodilatation and alpha-adrenergic responses in resistance vessels during the menstrual cycle in healthy women.

During the menstrual cycle, changes in endothelium-dependent vasodilatation have been demonstrated in conduit vessels in vivo, but responses in resistance vessels have not been studied. The aim of this study was to examine endothelium-dependent vasodilatation, the effects of local nitric oxide synthesis, and alpha-adrenergic constriction in resistance vessels during the menstrual cycle in 15 healthy female volunteers (mean age, 28.07 +/- 2.1 yr). Forearm blood flow in response to intrabrachial infusion of bradykinin (10, 30, and 100 pmol/min; endothelium-dependent vasodilator), glyceryl trinitrate (4, 8, and 16 nmol/min; endothelium-independent vasodilator), noradrenaline (60, 120, and 240 pmol/min; alpha-adrenergic receptor agonist), and N(G)-monomethyl-L-arginine (1, 2, and 4 micromol/min; nitric oxide synthase inhibitor) was assessed by venous occlusion plethysmography. All subjects were studied in early menstrual phase (days 1--4) and midcycle (days 10-13). Vasodilator response to bradykinin, expressed as the within-subject mean difference in the area under the dose-response curve between phases, was significantly increased at midcycle compared with that in the early menstrual phase (486.5 +/- 165.0; P = 0.01), whereas there was no significant difference in response to glyceryl trinitrate (185.8 +/- 239.0; P = 0.45). The vasoconstrictor response to noradrenaline was significantly greater at midcycle (97.1 +/- 39.4; P = 0.027), but the response to N(G)-monomethyl-L-arginine was not significantly different (17.5 +/- 35.2; P = 0.63). Serum estradiol was approximately 3-fold higher at midcycle, with a mean difference of 252.3 +/- 56.0 pmol/L (P = 0.0005). Progesterone concentrations were not significantly different (-0.11 +/- 0.1 nmol/L; P = 0.28). Differences in endogenous estrogen levels between menstrual phases may underlie changes in bradykinin and noradrenaline responses. If exogenous estrogens have similar effects, the balance of these two opposing actions may determine whether estrogen replacement in postmenopausal women has beneficial or harmful effects on the vasculature.

Effects of sodium intake on the pressor and renal responses to nitric oxide synthesis inhibition in normotensive individuals with different sodium sensitivity.

OBJECTIVE: The present study evaluated the role of nitric oxide (NO) in the systemic vascular and renal adaptation to changes in dietary sodium intake. DESIGN AND METHODS: Seven healthy normotensive male subjects were randomized to high or low sodium diets in a double blind crossover design (7 days on each diet). The NO synthesis inhibitor, NGmonomethyl-L-arginine (L-NMMA) was infused systemically (1.8 mg/kg over 30 min) at the end of each dietary period and its effects on blood pressure, renal plasma flow, glomerular filtration rate, urinary flow rate and sodium excretion were measured. RESULTS: Blood pressure increased in response to L-NMMA on a high sodium diet only (area under time curve percentage change in mean blood pressure, low sodium = -94.5 +/- 164.3; high sodium = 391.1 +/- 228.6; P < 0.05 low versus high). The increase in blood pressure was directly and significantly associated with the individual salt sensitivity, defined by the difference in systemic mean blood pressure between high and low sodium diets (r = 0.756; P < 0.05). L-NMMA also reduced renal plasma flow and urinary flow rate in subjects on high sodium diet. CONCLUSIONS: The data support a significant influence of endogenous NO in the systemic and renal vascular adaptation to a high sodium diet in normotensive men. In addition, the direct association between the individual sodium-sensitivity and the pressor response to L-NMMA suggests that there is increased dependence of vascular tone on NO in normotensive subjects whose blood pressure is more sodium sensitive.

Metabolism of methylarginines by human vasculature; implications for the regulation of nitric oxide synthesis.

1. The metabolism of methylarginines by human cultured endothelial cells and human saphenous vein was studied in vitro. The human endothelial cell line (SGHEC-7), primary cultures of human umbilical vein endothelial cells (HUVEC) and human saphenous vein were incubated with [14C]-monomethyl-L-arginine ([14C]-L-NMMA) and the cytosolic extract analysed by high performance liquid chromatography (h.p.l.c.) with on-line radioisotope detection. 2. SGHEC-7, HUVEC and human saphenous vein metabolized [14C]-L-NMMA to a compound which co-eluted with [14C]-citrulline. A second metabolite which co-eluted with [14C]-arginine was evident on the radiochromatograms of HUVEC cytosol and saphenous vein extracts. 3. The intracellular levels of [14C]-L-NMMA and [14C]-citrulline in SGHEC-7 cells incubated with [14C]-L-NMMA (0.5 microCi ml-1: 8.9 microM) for 1 h were 113 +/- 22 and 67.6 +/- 6.2 pmol mg-1 cell protein respectively (n = 7). Co-incubation with NGNGdimethyl-L-arginine (ADMA; 100 microM) but not NGNGdimethyl-L-arginine (SDMA; 100 microM) reduced the intracellular level of [14C]-citrulline to 26.3 +/- 3.7 pmol mg-1 cell protein (P < 0.01; n = 3) without reducing the intracellular level of [14C]-L-NMMA. 4. The intracellular levels of [14C]-citrulline in SGHEC-7 cells incubated with [14C]-L-NMMA for 1 h were reduced following co-incubation with NGnitro-L-arginine methylester (L-NAME; 1 mM), NGnitro-L-arginine (L-NOARG; 1 mM) and L-canavanine (1 mM) to 47.1 +/- 6.2, 24.7 +/- 3.6 and 12.5 +/- 2.8% of control levels (P < 0.001; n = 9). ADMA (1 mM; n = 3) reduced intracellular [14C]-citrulline levels to4 +/- 4% of control (P<0.01) but SDMA (1 mM; n = 3) had no effect.5. The accumulation of endogenously synthesized ADMA in the culture supernatant of SGHEC-7 cells was increased by co-incubation with L-NMMA (1 mM) from 1.98 +/- 0.08 to 2.74 +/- 0.36 nmol mg- cell protein, an increase of 40%.6. These results demonstrate that human vasculature possesses an enzyme which has similar properties to dimethylarginase; human endothelial cells and human saphenous vein metabolize L-NMMA to citrulline via a process inhibited by ADMA but not SDMA. The increase in endothelium-derivedADMA following co-incubation with L-NMMA is consistent with competition between ADMA and L-NMMA for dimethylarginase. Inhibition of this enzyme might increase the intracellular concentration of ADMA, an endogenously produced compound that inhibits nitric oxide synthesis.

Aminoguanidine selectively inhibits inducible nitric oxide synthase.

1. Endotoxin induces nitric oxide synthase in vascular tissue, including rat main pulmonary artery. Currently available agents that cause inhibition of nitric oxide synthase are relatively non-selective between the constitutive and inducible forms of the enzyme. 2. Aminoguanidine caused a dose-dependent increase in phenylephrine-induced tension in intact and endothelium-denuded pulmonary artery rings from endotoxin-treated rats, but had no effect on sham-treated controls. 3. Contraction caused by aminoguanidine in endothelium-denuded vessels from endotoxin-treated rats was unaffected by indomethacin (10 microM), and by cimetidine and mepyramine (both 10 microM), excluding an effect of aminoguanidine mediated by arachidonic acid metabolites or histamine. 4. Contraction caused by aminoguanidine in endothelium-denuded vessels from endotoxin-treated rats was abolished by L-arginine (2 mM) and L-NG-monomethyl arginine (300 microM), but unaffected by D-arginine and D-NG-monomethyl arginine, suggesting that its action is mediated by the L-arginine/nitric oxide pathway. 5. Aminoguanidine had no effect on acetylcholine-induced relaxation of intact vessels from shamtreated rats. However, relaxation of artery rings from endotoxin-treated rats by L-arginine was competitively inhibited by aminoguanidine.6. These results in isolated main pulmonary arteries of the rat confirm previous reports that aminoguanidine is a selective inhibitor of inducible nitric oxide synthase.

Autoregulation of nitric oxide-soluble guanylate cyclase-cyclic GMP signalling in mouse thoracic aorta.

1. The sensitivity of the soluble guanylate cyclase (sGC)-cyclic guanosine-3',5'-monophosphate (cyclic GMP) system to nitric oxide (NO) was investigated in mouse aorta from wild type (WT) and NO synthase (NOS) knockout (KO) animals. 2. The NO donor, spermine-NONOate (SPER-NO) was more potent in aortas from eNOS KO mice compared to WT (pEC50 7.30+/-0.06 and 6.56+/-0.04, respectively; n=6; P<0.05). In contrast, the non-NO based sGC activator, YC-1 was equipotent in vessels from eNOS WT and KO mice. The sensitivity of aortas from nNOS and iNOS KO animals to SPER-NO was unchanged. Forskolin (an adenylate cyclase activator), was equipotent in vessels from eNOS WT and KO animals. 3. The cyclic GMP analogue, 8-Br-cGMP was equipotent in eNOS WT and KO mice (pEC50 4. 38+/-0.04 and 4.40+/-0.05, respectively; n=5; P>0.05). Zaprinast (10-5 M) a phosphodiesterase type V (PDE V) inhibitor, had no effect on the response to SPER-NO in vessels from eNOS WT or KO mice. 4. The NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 3x10-4 M) increased the potency of SPER-NO in aortas from WT mice (pEC50 6. 64+/-0.02 and 7.37+/-0.02 in the absence and presence of L-NAME, respectively; n=4; P<0.05). 5. In summary, there is increased sensitivity of vessels from eNOS KO animals to NO. Cyclic AMP-mediated dilatation is unchanged, consistent with a specific up-regulation of sGC - cyclic GMP signalling. The functional activity of cyclic GMP-dependent protein kinase (G-kinase) and PDE V was also unchanged, suggesting that sGC is the site of up-regulation. These alterations in the sensitivity of the sGC - cyclic GMP pathway might represent a mechanism for the dynamic regulation of NO bioactivity.

Regulation of nitric oxide synthesis by dimethylarginine dimethylaminohydrolase.

1. Dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that metabolizes the endogenous nitric oxide synthase inhibitors NG-monomethyl-arginine and NG,NG-dimethy-L-arginine to citrulline, was identified by Western blotting in rat and human tissue homogenates. 2. S-2-amino-4(3-methylguanidino)butanoic acid (4124W) inhibited the metabolism of [14C]-NG-monomethyl-L-arginine to [14C]-citrulline by rat liver homogenates (IC50 416 +/- 66 microM; n = 9), human cultured endothelial cells (IC50 250 +/- 34 microM; n = 9) and isolated purified dimethylarginine dimethylaminohydrolase. 3. Addition of 4124W to culture medium increased the accumulation of endogenously-generated NG,NG-dimethy-L-arginine in the supernatant of human cultured endothelial cells from 3.1 +/- 0.3 to 5 +/- 0.7 microM (n = 15; P < 0.005). 4. 4124W (1 microM - 1 mM) had no direct effect on endothelial nitric oxide synthase activity but caused endothelium-dependent contraction of rat aortic rings (1 mM 4124W increased tone by 81.5 +/- 9.6% of that caused by phenylephrine 100 nM). This effect was reversed by L-arginine (100 microM). 4124W reversed endothelium-dependent relaxation of human saphenous vein (19.2 +/- 6.7% reversal of bradykinin-induced relaxation at 1 mM 4124W). 5. These data suggest that inhibition of dimethylarginine dimethylaminohydrolase increases the intracellular contraction of NG,NG-dimethyl-L-arginine sufficiently to inhibit nitric oxide synthesis. Inhibiting the activity of DDAH may provide an alternative mechanism for inhibition of nitric oxide synthases and changes in the activity of DDAH could contribute to pathophysiological alterations in NO generation.

Intrinsic defence capacity and therapeutic potential of natriuretic peptides in pulmonary hypertension associated with lung fibrosis.

BACKGROUND AND PURPOSE: Idiopathic pulmonary fibrosis (IPF) is a progressive fibro-proliferative disorder refractory to current therapy commonly complicated by the development of pulmonary hypertension (PH); the associated morbidity and mortality are substantial. Natriuretic peptides possess vasodilator and anti-fibrotic actions, and pharmacological augmentation of their bioactivity ameliorates renal and myocardial fibrosis. Here, we investigated whether natriuretic peptides possess an intrinsic cytoprotective function preventing the development of pulmonary fibrosis and associated PH, and whether therapeutics targeting natriuretic peptide signalling demonstrate efficacy in this life-threatening disorder. EXPERIMENTAL APPROACH: Pulmonary haemodynamics, right ventricular function and markers of lung fibrosis were determined in wild-type (WT) and natriuretic peptide receptor (NPR)-A knockout (KO) mice exposed to bleomycin (1 mg·kg(-1) ). Human myofibroblast differentiation was studied in vitro. KEY RESULTS: Exacerbated cardiac, vascular and fibrotic pathology was observed in NPR-A KO animals, compared with WT mice, exposed to bleomycin. Treatment with a drug combination that raised circulating natriuretic peptide levels (ecadotril) and potentiated natriuretic peptide-dependent signalling (sildenafil) reduced indices of disease progression, whether administered prophylactically or to animals with established lung disease. This positive pharmacodynamic effect was diminished in NPR-A KO mice. Atrial natriuretic peptide and sildenafil synergistically reduced TGFß-induced human myofibroblast differentiation, a key driver of remodelling in IPF patients. CONCLUSIONS AND IMPLICATIONS: These data highlight an endogenous host-defence capacity of natriuretic peptides in lung fibrosis and PH. A combination of ecadotril and sildenafil reversed the pulmonary haemodynamic aberrations and remodelling that characterize the disease, advocating therapeutic manipulation of natriuretic peptide bioactivity in patients with IPF.