Soluble guanylate cyclase stimulators in pulmonary hypertension.

Soluble guanylate cyclase (sGC) is a key enzyme in the nitric oxide (NO) signalling pathway. On binding of NO to its prosthetic haem group, sGC catalyses the synthesis of the second messenger cyclic guanosine monophosphate (cGMP), which promotes vasodilation and inhibits smooth muscle proliferation, leukocyte recruitment, platelet aggregation and vascular remodelling through a number of downstream mechanisms. The central role of the NO-sGC-cGMP pathway in regulating pulmonary vascular tone is demonstrated by the dysregulation of NO production, sGC activity and cGMP degradation in pulmonary hypertension (PH). The sGC stimulators are novel pharmacological agents that directly stimulate sGC, both independently of NO and in synergy with NO. Optimisation of the first sGC stimulator, YC-1, led to the development of the more potent and more specific sGC stimulators, BAY 41-2272, BAY 41-8543 and riociguat (BAY 63-2521). Other sGC stimulators include CFM-1571, BAY 60-4552, vericiguat (BAY 1021189), the acrylamide analogue A-350619 and the aminopyrimidine analogues. BAY 41-2272, BAY 41-8543 and riociguat induced marked dose-dependent reductions in mean pulmonary arterial pressure and vascular resistance with a concomitant increase in cardiac output, and they also reversed vascular remodelling and right heart hypertrophy in several experimental models of PH. Riociguat is the first sGC stimulator that has entered clinical development. Clinical trials have shown that it significantly improves pulmonary vascular haemodynamics and increases exercise ability in patients with pulmonary arterial hypertension (PAH), chronic thromboembolic PH and PH associated with interstitial lung disease. Furthermore, riociguat reduces mean pulmonary arterial pressure in patients with PH associated with chronic obstructive pulmonary disease and improves cardiac index and pulmonary vascular resistance in patients with PH associated with left ventricular systolic dysfunction. These promising results suggest that sGC stimulators may constitute a valuable new therapy for PH. Other trials of riociguat are in progress, including a study of the haemodynamic effects and safety of riociguat in patients with PH associated with left ventricular diastolic dysfunction, and long-term extensions of the phase 3 trials investigating the efficacy and safety of riociguat in patients with PAH and chronic thromboembolic PH. Finally, sGC stimulators may also have potential therapeutic applications in other diseases, including heart failure, lung fibrosis, scleroderma and sickle cell disease.

Definition and classification of pulmonary hypertension.

Pulmonary hypertension is defined as an increase of mean pulmonary arterial pressure ≥25 mmHg at rest as assessed by right heart catheterization. According to different combinations of values of pulmonary wedge pressure, pulmonary vascular resistance and cardiac output, a hemodynamic classification of pulmonary hypertension has been proposed. Of major importance is the pulmonary wedge pressure which allows to distinguish pre-capillary (pulmonary wedge pressure ≤15 mmHg) and post-capillary (pulmonary wedge pressure >15 mmHg) pulmonary hypertension. Pre-capillary pulmonary hypertension includes the clinical groups 1 (pulmonary arterial hypertension), 3 (pulmonary hypertension due to lung diseases and/or hypoxia), 4 (chronic thrombo-embolic pulmonary hypertension) and 5 (pulmonary hypertension with unclear and/or multifactorial mechanisms). Post-capillary pulmonary hypertension corresponds to the clinical group 2 (pulmonary hypertension due to left heart diseases).

Soluble guanylate cyclase agonists inhibit expression and procoagulant activity of tissue factor.

OBJECTIVE: Tissue factor (TF), a major initiator of blood coagulation, contributes to inflammation, atherosclerosis, angiogenesis, and vascular remodeling. Pharmacological agonists of soluble guanylate cyclase (sGC) attenuate systemic and pulmonary hypertension, vascular remodeling, and platelet aggregation. However, the influence of these novel pharmacophores on TF is unknown. METHODS AND RESULTS: We evaluated effects of BAY 41-2272 and BAY 58-2667 on expression and activity of TF in human monocytes and umbilical vein endothelial cells (HUVECs). Both compounds reduced expression of active TF protein in monocytes stimulated with lipopolysaccharide, as demonstrated by immunoblotting and a TF procoagulant activity assay. In-cell Western assay revealed that this effect was associated with a marked reduction of total and surface TF presentation. Furthermore, BAY 41-2272 and BAY 58-2667 decreased TF protein expression and the TF-dependent procoagulant activity in HUVECs stimulated with TNF-alpha. The sGC agonists also suppressed transcriptional activity of NF-kappaB. A siRNA-mediated knockdown of the alpha1-subunit of sGC in monocytes and HUVECs confirmed that the inhibitory effect of BAY 41-2272 and BAY 58-2667 on TF expression is mediated through the sGC-dependent mechanisms. CONCLUSIONS: Inhibition of TF expression and activity by sGC agonists might provide therapeutic benefits in cardiovascular diseases associated with enhanced procoagulant and inflammatory response.

NO-independent stimulators and activators of soluble guanylate cyclase: discovery and therapeutic potential.

Soluble guanylate cyclase (sGC) is a key signal-transduction enzyme activated by nitric oxide (NO). Impaired bioavailability and/or responsiveness to endogenous NO has been implicated in the pathogenesis of cardiovascular and other diseases. Current therapies that involve the use of organic nitrates and other NO donors have limitations, including non-specific interactions of NO with various biomolecules, lack of response and the development of tolerance following prolonged administration. Compounds that activate sGC in an NO-independent manner might therefore provide considerable therapeutic advantages. Here we review the discovery, biochemistry, pharmacology and clinical potential of haem-dependent sGC stimulators (including YC-1, BAY 41-2272, BAY 41-8543, CFM-1571 and A-350619) and haem-independent sGC activators (including BAY 58-2667 and HMR-1766).

Inhaled agonists of soluble guanylate cyclase induce selective pulmonary vasodilation.

RATIONALE: Nitric oxide-independent agonists of soluble guanylate cyclase (sGC) have been developed. OBJECTIVES: We tested whether inhalation of novel dry-powder microparticle formulations containing sGC stimulators (BAY 41-2272, BAY 41-8543) or an sGC activator (BAY 58-2667) would produce selective pulmonary vasodilation in lambs with acute pulmonary hypertension. We also evaluated the combined administration of BAY 41-8543 microparticles and inhaled nitric oxide (iNO). Finally, we examined whether inhaling BAY 58-2667 microparticles would produce pulmonary vasodilation when the response to iNO is impaired. METHODS: In awake, spontaneously breathing lambs instrumented with vascular catheters and a tracheostomy tube, U-46619 was infused intravenously to increase mean pulmonary arterial pressure to 35 mm Hg. MEASUREMENTS AND MAIN RESULTS: Inhalation of microparticles composed of either BAY 41-2272, BAY 41-8543, or BAY 58-2667 and excipients (dipalmitoylphosphatidylcholine, albumin, lactose) produced dose-dependent pulmonary vasodilation and increased transpulmonary cGMP release without significant effect on mean arterial pressure. Inhalation of microparticles containing BAY 41-8543 or BAY 58-2667 increased systemic arterial oxygenation. The magnitude and duration of pulmonary vasodilation induced by iNO were augmented after inhaling BAY 41-8543 microparticles. Intravenous administration of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), which oxidizes the prosthetic heme group of sGC, markedly reduced the pulmonary vasodilator effect of iNO. In contrast, pulmonary vasodilation and transpulmonary cGMP release induced by inhaling BAY 58-2667 microparticles were greatly enhanced after treatment with ODQ. CONCLUSIONS: Inhalation of microparticles containing agonists of sGC may provide an effective novel treatment for patients with pulmonary hypertension, particularly when responsiveness to iNO is impaired by oxidation of sGC.

Soluble guanylate cyclase activator reverses acute pulmonary hypertension and augments the pulmonary vasodilator response to inhaled nitric oxide in awake lambs.

BACKGROUND: Inhaled nitric oxide (NO) is a potent and selective pulmonary vasodilator, which induces cGMP synthesis by activating soluble guanylate cyclase (sGC) in ventilated lung regions. Carbon monoxide (CO) has also been proposed to influence smooth muscle tone via activation of sGC. We examined whether direct stimulation of sGC by BAY 41-2272 would produce pulmonary vasodilation and augment the pulmonary responses to inhaled NO or CO. METHODS AND RESULTS: In awake, instrumented lambs, the thromboxane analogue U-46619 was intravenously administered to increase mean pulmonary arterial pressure to 35 mm Hg. Intravenous infusion of BAY 41-2272 (0.03, 0.1, and 0.3 mg x kg(-1) x h(-1)) reduced mean pulmonary arterial pressure and pulmonary vascular resistance and increased transpulmonary cGMP release in a dose-dependent manner. Larger doses of BAY 41-2272 also produced systemic vasodilation and elevated the cardiac index. N(omega)-nitro-l-arginine methyl ester abolished the systemic but not the pulmonary vasodilator effects of BAY 41-2272. Furthermore, infusing BAY 41-2272 at 0.1 mg x kg(-1) x h(-1) potentiated and prolonged the pulmonary vasodilation induced by inhaled NO (2, 10, and 20 ppm). In contrast, inhaled CO (50, 250, and 500 ppm) had no effect on U-46619-induced pulmonary vasoconstriction before or during administration of BAY 41-2272. CONCLUSIONS: In lambs with acute pulmonary hypertension, BAY 41-2272 is a potent pulmonary vasodilator that augments and prolongs the pulmonary vasodilator response to inhaled NO. Direct pharmacological stimulation of sGC, either alone or in combination with inhaled NO, may provide a novel approach for the treatment of pulmonary hypertension.

Role of nitrosative stress and activation of poly(ADP-ribose) polymerase-1 in cardiovascular failure associated with septic and hemorrhagic shock.

Reactive oxygen and nitrogen species, particularly peroxynitrite, are potent inducers of tissue damage during systemic inflammatory response and circulatory shock. Recent evidence indicates that the toxicity of these species largely depends on their ability to trigger activation of the nuclear enzyme poly(adenosine 5'-diphosphate ribose) polymerase-1 (PARP-1). Following excessive activation, PARP-1 depletes the intracellular stores of its substrate, nicotinamide adenine dinucleotide, thus slowing glycolysis, generation of high energy phosphates, and mitochondrial electron transport. Consequently, the severe metabolic crisis induced by PARP-1 activation results in acute cell dysfunction and necrotic cell death. In addition, activation of PARP-1 plays an important role in the upregulation of inflammatory cascades via a functional association with mitogen-activated protein kinases and several transcription factors, such as nuclear factor kappa B, resulting in augmented expression of pro-inflammatory cytokines, chemokines, adhesion molecules, and enzymes. In severe sepsis and hemorrhage, PARP-1 activation has emerged as one of the central mechanisms of systemic inflammation, endothelial dysfunction, peripheral vascular failure, and reduction of cardiac contractility. Innovative therapeutic strategies based on the pharmacological inhibition of PARP-1 catalytic activity might provide benefits by preventing tissue injury, organ dysfunction, and lethality associated with these conditions.

NOS3 deficiency augments hypoxic pulmonary vasoconstriction and enhances systemic oxygenation during one-lung ventilation in mice.

Nitric oxide (NO), synthesized by NO synthases (NOS), plays a pivotal role in regulation of pulmonary vascular tone. To examine the role of endothelial NOS (NOS3) in hypoxic pulmonary vasoconstriction (HPV), we measured left lung pulmonary vascular resistance (LPVR), intrapulmonary shunting, and arterial PO2 (PaO2) before and during left mainstem bronchus occlusion (LMBO) in mice with and without a deletion of the gene encoding NOS3. The increase of LPVR induced by LMBO was greater in NOS3-deficient mice than in wild-type mice (151 +/- 39% vs. 109 +/- 36%, mean +/- SD; P < 0.05). NOS3-deficient mice had a lower intrapulmonary shunt fraction than wild-type mice (17.1 +/- 3.6% vs. 21.7 +/- 2.4%, P < 0.05) during LMBO. Both real-time PaO2 monitoring with an intra-arterial probe and arterial blood-gas analysis during LMBO showed higher PaO2 in NOS3-deficient mice than in wild-type mice (P < 0.05). Inhibition of all three NOS isoforms with Nomega-nitro-L-arginine methyl ester (L-NAME) augmented the increase of LPVR induced by LMBO in wild-type mice (183 +/- 67% in L-NAME treated vs. 109 +/- 36% in saline treated, P < 0.01) but not in NOS3-deficient mice. Similarly, systemic oxygenation during one-lung ventilation was augmented by L-NAME in wild-type mice but not in NOS3-deficient mice. These findings indicate that NO derived from NOS3 modulates HPV in vivo and that inhibition of NOS3 improves systemic oxygenation during acute unilateral lung hypoxia.

Flagellin from gram-negative bacteria is a potent mediator of acute pulmonary inflammation in sepsis.

Flagellin is a recently identified bacterial product that elicits immune response via toll-like receptor 5. Here, we demonstrate that flagellin is an extraordinarily potent proinflammatory stimulus in the lung during sepsis. In vitro, flagellin triggers the production of interleukin (IL)-8 by human lung epithelial (A549) cells, with 50% of the maximal response obtained at a concentration of 2 x 10(-14) M. Flagellin also induces the expression of ICAM-1 in vitro. Intravenous administration of flagellin to mice elicited a severe acute lung inflammation that was significantly more pronounced than following lipopolysaccharide (LPS) administration. Flagellin induced a local release of proinflammatory cytokines, the accumulation of inflammatory cells, and the development of pulmonary hyperpermeability. These effects were associated with the nuclear translocation of the transcription NF-kappaB in the lung. Flagellin remained active in inducing pulmonary inflammation at doses as low as 10 ng/mouse. In the plasma of patients with sepsis, flagellin levels amounted to 7.1 +/- 0.1 ng/mL. Plasma flagellin levels showed a significant positive correlation with the lung injury score, with the alveolar-arterial oxygen difference as well as with the duration of the sepsis. Flagellin emerges as a potent trigger of acute respiratory complications in gram-negative bacterial sepsis.

Soluble guanylate cyclase stimulation prevents fibrotic tissue remodeling and improves survival in salt-sensitive Dahl rats.

BACKGROUND: A direct pharmacological stimulation of soluble guanylate cyclase (sGC) is an emerging therapeutic approach to the management of various cardiovascular disorders associated with endothelial dysfunction. Novel sGC stimulators, including riociguat (BAY 63-2521), have a dual mode of action: They sensitize sGC to endogenously produced nitric oxide (NO) and also directly stimulate sGC independently of NO. Little is known about their effects on tissue remodeling and degeneration and survival in experimental malignant hypertension. METHODS AND RESULTS: Mortality, hemodynamics and biomarkers of tissue remodeling and degeneration were assessed in Dahl salt-sensitive rats maintained on a high salt diet and treated with riociguat (3 or 10 mg/kg/d) for 14 weeks. Riociguat markedly attenuated systemic hypertension, improved systolic heart function and increased survival from 33% to 85%. Histological examination of the heart and kidneys revealed that riociguat significantly ameliorated fibrotic tissue remodeling and degeneration. Correspondingly, mRNA expression of the pro-fibrotic biomarkers osteopontin (OPN), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and plasminogen activator inhibitor-1 (PAI-1) in the myocardium and the renal cortex was attenuated by riociguat. In addition, riociguat reduced plasma and urinary levels of OPN, TIMP-1, and PAI-1. CONCLUSIONS: Stimulation of sGC by riociguat markedly improves survival and attenuates systemic hypertension and systolic dysfunction, as well as fibrotic tissue remodeling in the myocardium and the renal cortex in a rodent model of pressure and volume overload. These findings suggest a therapeutic potential of sGC stimulators in diseases associated with impaired cardiovascular and renal functions.

CB1 cannabinoid receptors promote oxidative stress and cell death in murine models of doxorubicin-induced cardiomyopathy and in human cardiomyocytes.

AIMS: Here we investigated the mechanisms by which cardiovascular CB1 cannabinoid receptors may modulate the cardiac dysfunction, oxidative stress, and interrelated cell death pathways associated with acute/chronic cardiomyopathy induced by the widely used anti-tumour compound doxorubicin (DOX). METHODS AND RESULTS: Both load-dependent and -independent indices of left-ventricular function were measured by the Millar pressure-volume conductance system. Mitogen-activated protein kinase (MAPK) activation, cell-death markers, and oxidative/nitrosative stress were measured by molecular biology/biochemical methods and flow cytometry. DOX induced left-ventricular dysfunction, oxidative/nitrosative stress coupled with impaired antioxidant defense, activation of MAPK (p38 and JNK), and cell death and/or fibrosis in hearts of wide-type mice (CB1(+/+)), and these effects were markedly attenuated in CB1 knockouts (CB1(-/-)). In human primary cardiomyocytes expressing CB1 receptors (demonstrated by RT-PCR, western immunoblot, and flow cytometry) DOX, likewise the CB1 receptor agonist HU210 and the endocannabinoid anandamide (AEA), induced MAPK activation and cell death. The DOX-induced MAPK activation and cell death were significantly enhanced when DOX was co-administered with CB1 agonists AEA or HU210. Remarkably, cell death and MAPK activation induced by AEA, HU210, and DOX +/- AEA/HU210 were largely attenuated by either CB1 antagonists (rimonabant and AM281) or by inhibitors of p38 and JNK MAPKs. Furthermore, AEA or HU210 in primary human cardiomyocytes triggered increased reactive oxygen species generation. CONCLUSION: CB1 activation in cardiomyocytes may amplify the reactive oxygen/nitrogen species-MAPK activation-cell death pathway in pathological conditions when the endocannabinoid synthetic or metabolic pathways are dysregulated by excessive inflammation and/or oxidative/nitrosative stress, which may contribute to the pathophysiology of various cardiovascular diseases.

Microparticles for inhalational delivery of antipseudomonal antibiotics.

Chronic pseudomonal bronchopulmonary infections in cystic fibrosis patients are frequently controlled with inhaled antibiotics. Dry-powder inhalable antibiotics are an attractive alternative to nebulized medications. We produced and evaluated microparticles composed of dipalmitoylphosphatidylcholine, albumin, and lactose as a model system for intrapulmonary delivery of ceftazidime, ciprofloxacin, and several combinations of the two, none of which is presently available for inhalation. Microparticles containing one or both antibiotics were prepared by spray-drying. Their Anderson cascade impactor deposition profiles showed 10-30% fine particle fractions of the nominal dose. Microparticles containing varying amounts of each antibiotic showed statistically different deposition profiles. Aerodynamics and deposition of microparticles co-encapsulating both antibiotics were similar to those of single-drug microparticles with the same proportion of ciprofloxacin alone. The antipseudomonal activities of microparticles co-encapsulating half of the 50% effective concentration (EC(50)) of both ceftazidime and ciprofloxacin (5 mg of particles containing 5% ceftazidime and 10% ciprofloxacin) were at least additive compared to particles containing the EC(50) of each antibiotic separately (5 mg of particles containing 10% ceftazidime or 5 mg of particles containing 20% ciprofloxacin). Co-encapsulation of the antibiotics in microparticles ensures co-deposition at desired ratios, improves the particles' aerodynamics and fine particle fraction, as compared to microparticles with equivalent amounts of ceftazidime alone, and achieves additive antipseudomonal activity.

Congenital NOS2 deficiency prevents impairment of hypoxic pulmonary vasoconstriction in murine ventilator-induced lung injury.

Hypoxic pulmonary vasoconstriction (HPV) preserves systemic arterial oxygenation during lung injury by diverting blood flow away from poorly ventilated lung regions. Ventilator-induced lung injury (VILI) is characterized by pulmonary inflammation, lung edema, and impaired HPV leading to systemic hypoxemia. Studying mice congenitally deficient in inducible nitric oxide synthase (NOS2) and wild-type mice treated with a selective NOS2 inhibitor, L-N(6)-(1-iminoethyl)lysine (L-NIL), we investigated the contribution of NOS2 to the impairment of HPV in anesthetized mice subjected to 6 h of either high tidal volume (HV(T)) or low tidal volume (LV(T)) ventilation. HPV was estimated by measuring the changes of left lung pulmonary vascular resistance (LPVR) in response to left mainstem bronchus occlusion (LMBO). LMBO increased the LPVR similarly in wild-type, NOS2(-/-), and wild-type mice treated with L-NIL 30 min before commencing 6 h of LV(T) ventilation (96% +/- 30%, 103% +/- 33%, and 80% +/- 16%, respectively, means +/- SD). HPV was impaired in wild-type mice subjected to 6 h of HV(T) ventilation (23% +/- 16%). In contrast, HPV was preserved after 6 h of HV(T) ventilation in NOS2(-/-) and wild-type mice treated with L-NIL either 30 min before or 6 h after commencing HV(T) ventilation (66% +/- 22%, 82% +/- 29%, and 85% +/- 16%, respectively). After 6 h of HV(T) ventilation and LMBO, systemic arterial oxygen tension was higher in NOS2(-/-) than in wild-type mice (192 +/- 11 vs. 171 +/- 17 mmHg; P < 0.05). We conclude that either congenital NOS2 deficiency or selective inhibition of NOS2 protects mice from the impairment of HPV occurring after 6 h of HV(T) ventilation.

Inhibition of phosphodiesterase 1 augments the pulmonary vasodilator response to inhaled nitric oxide in awake lambs with acute pulmonary hypertension.

Phosphodiesterase 1 (PDE1) modulates vascular tone and the development of tolerance to nitric oxide (NO)-releasing drugs in the systemic circulation. Any role of PDE1 in the pulmonary circulation remains largely uncertain. We measured the expression of genes encoding PDE1 isozymes in the pulmonary vasculature and examined whether or not selective inhibition of PDE1 by vinpocetine attenuates pulmonary hypertension and augments the pulmonary vasodilator response to inhaled NO in lambs. Using RT-PCR, we detected PDE1A, PDE1B, and PDE1C mRNAs in pulmonary arteries and veins isolated from healthy lambs. In 13 lambs, the thromboxane A(2) analog U-46619 was infused intravenously to increase mean pulmonary arterial pressure to 35 mmHg. Four animals received an intravenous infusion of vinpocetine at incremental doses of 0.3, 1, and 3 mg.kg(-1).h(-1). In nine lambs, inhaled NO was administered in a random order at 2, 5, 10, and 20 ppm before and after an intravenous infusion of 1 mg.kg(-1).h(-1) vinpocetine. Administration of vinpocetine did not alter pulmonary and systemic hemodynamics or transpulmonary cGMP or cAMP release. Inhaled NO selectively reduced mean pulmonary arterial pressure, pulmonary capillary pressure, and pulmonary vascular resistance index, while increasing transpulmonary cGMP release. The addition of vinpocetine enhanced pulmonary vasodilation and transpulmonary cGMP release induced by NO breathing without causing systemic vasodilation but did not prolong the duration of pulmonary vasodilation after NO inhalation was discontinued. Our findings demonstrate that selective inhibition of PDE1 augments the therapeutic efficacy of inhaled NO in an ovine model of acute chemically induced pulmonary hypertension.

Combination of intravenously infused methylene blue and inhaled nitric oxide ameliorates endotoxin-induced lung injury in awake sheep.

OBJECTIVE: To evaluate the effects of a combination of methylene blue, an inhibitor of the nitric oxide pathway, and inhaled nitric oxide on endotoxin-induced acute lung injury in awake sheep. DESIGN: Prospective, randomized, controlled experimental study. SETTING: University animal laboratory. SUBJECTS: Twenty-four yearling, awake sheep. INTERVENTIONS: The sheep were anesthetized and instrumented with vascular catheters. After 1 wk of recovery, the animals underwent tracheotomy and were subjected to intravenous infusions of endotoxin 10 ng x kg-1 x min-1 and isotonic saline 3 mL x kg-1 x hr-1 for 8 hrs. The sheep were randomly assigned to three groups of eight animals each: a) the control group received endotoxin and saline; b) the INO group received endotoxin, saline, and inhaled nitric oxide 40 ppm for 5 hrs; and c) the MB/INO group received endotoxin, saline, and methylene blue 3 mg/kg as an intravenous bolus injection followed by a continuous infusion of 3 mg x kg-1 x min-1 for 6 hrs in combination with inhaled nitric oxide 40 ppm for 5 hrs. MEASUREMENTS AND MAIN RESULTS: Hemodynamic variables and blood gases were determined hourly. In the early phase of endotoxemia (0-2 hrs), methylene blue/inhaled nitric oxide reduced the increments in pulmonary arterial pressure, pulmonary microvascular pressure, and pulmonary vascular resistance index by 60% compared with the controls and to a greater extent than did inhaled nitric oxide alone. During the late phase, all the preceding variables returned closely to baseline following inhaled nitric oxide or methylene blue/inhaled nitric oxide but remained remarkably elevated in the control group. Inhaled nitric oxide and methylene blue/inhaled nitric oxide reduced the increase in extravascular lung water by 40% and 80%, respectively. Inhaled nitric oxide transiently attenuated the increase in venous admixture and did not prevent a decrease in arterial oxygenation. In the methylene blue/inhaled nitric oxide group, blood gases remained unchanged from baseline. CONCLUSIONS: In sheep, methylene blue/inhaled nitric oxide protects more efficiently against acute lung injury than inhaled nitric oxide alone, as indicated by a milder pulmonary hypertension, less extravascular lung water accumulation, and maintained gas exchange.

Left ventricular pressure-volume relationship in a rat model of advanced aging-associated heart failure.

Aging is associated with profound changes in the structure and function of the heart. A fundamental understanding of these processes, using relevant animal models, is required for effective prevention and treatment of cardiovascular disease in the elderly. Here, we studied cardiac performance in 4- to 5-mo-old (young) and 24- to 26-mo-old (old) Fischer 344 male rats using the Millar pressure-volume (P-V) conductance catheter system. We evaluated systolic and diastolic function in vivo at different preloads, including preload recruitable stroke work (PRSW), maximal slope of the systolic pressure increment (+dP/dt), and its relation to end-diastolic volume (+dP/dt-EDV) as well as the time constant of left ventricular pressure decay, as an index of relaxation. The slope of the end-diastolic P-V relation (EDPVR), an index of left ventricular stiffness, was also calculated. Aging was associated with decrease in left ventricular systolic pressure, +dP/dt, maximal slope of the diastolic pressure decrement, +dP/dt-EDV, PRSW, ejection fraction, stroke volume, cardiac and stroke work indexes, and efficiency. In contrast, total peripheral resistance, left ventricular end-diastolic volume, left ventricular end-diastolic pressure, and EDPVR were greater in aging than in young animals. Taken together, these data suggest that advanced aging is characterized by decreased systolic performance accompanied by delayed relaxation and increased diastolic stiffness of the heart in male Fischer 344 rats. P-V analysis is a sensitive method to determine cardiac function in rats.

Novel endothelin receptor antagonist attenuates endotoxin-induced lung injury in sheep.

OBJECTIVE: To evaluate the cardiopulmonary effects of the novel endothelin receptor antagonist tezosentan in endotoxin-induced lung injury in sheep and to assess the dose response to tezosentan and endothelin-1 in healthy sheep. DESIGN: Prospective, randomized, controlled experimental study. SETTING: University animal laboratory. SUBJECTS: Twenty-one yearling sheep. INTERVENTIONS: Seventeen awake, chronically instrumented sheep were subjected to intravenous infusion of Ringer's lactate for 24 hrs. The animals were randomly assigned to a sham-operated group (n = 3), a lipopolysaccharide group (n = 7) receiving an intravenous infusion of Escherichia coli lipopolysaccharide 15 ng x kg x min, and a tezosentan group (n = 7) subjected to lipopolysaccharide and, from 4 hrs, an intravenous injection of tezosentan 3 mg/kg followed by infusion of 1 mg x kg x hr. In addition, four healthy sheep, exposed to an intravenous infusion of endothelin-1 at 20 ng x kg x min, after 1 hr received tezosentan in stepwise increasing doses of 0.5, 1, and 2 mg x kg x hr that were maintained for 1 hr each. After a 4-hr recovery, the sheep received infusions of tezosentan at the same dose rates as a pretreatment to endothelin-1. MEASUREMENTS AND MAIN RESULTS: In the sham-operated sheep, all cardiopulmonary variables remained unchanged. Lipopolysaccharide caused pulmonary hypertension, increased extravascular lung water index, and induced arterial hypoxemia. Tezosentan decreased the increments in pulmonary vascular resistance and extravascular lung water index by as much as 60% and 70%, respectively. In parallel, tezosentan ameliorated arterial hypoxemia, increased cardiac index, attenuated the decrease in stroke volume index, and reduced systemic vascular resistance. Compared with the lipopolysaccharide group, tezosentan further increased plasma concentrations of endothelin-1. In healthy animals, the administration of endothelin-1 induced systemic and pulmonary hypertension, increased extravascular lung water index, and evoked bradycardia and a decrease in cardiac index. These changes were attenuated by tezosentan infused at 1 and 2 mg x kg x hr. CONCLUSIONS: In an ovine model of endotoxin-induced lung injury, tezosentan ameliorates pulmonary hypertension, lung edema, cardiac dysfunction, and arterial hypoxemia. Tezosentan counteracts the hemodynamic effects of endothelin-1 in a dose-dependent manner.

Parenteral administration of glipizide sodium salt, an inhibitor of adenosine triphosphate-sensitive potassium channels, prolongs short-term survival after severe controlled hemorrhage in rats.

OBJECTIVE: Recent experimental evidence suggests that activation of adenosine triphosphate (ATP)-sensitive potassium channels contributes to vascular failure and early mortality after hemorrhagic shock. The present investigation evaluated the effects of the water-soluble sodium salt of glipizide, an inhibitor of ATP-sensitive potassium channels, in anesthetized and awake rats subjected to severe controlled hemorrhage. DESIGN: Prospective, randomized, controlled study. SETTING: Animal research laboratory. SUBJECTS: Male Wistar rats. INTERVENTIONS: Anesthetized rats were subjected to bleeding to reduce mean arterial pressure to either 40 or 35 mm Hg, which was maintained constant for 60 mins. In addition, awake rats underwent blood withdrawal of 4.25 mL/100 g over 20 mins. At the end of the hemorrhage period and 30 mins later, the animals received intravenous (5 and 20 mg/kg) or intramuscular (10 and 40 mg/kg) injections of glipizide sodium salt or vehicle. MEASUREMENTS AND MAIN RESULTS: In anesthetized rats subjected to pressure-controlled hemorrhage, glipizide sodium salt improved mean arterial pressure in a dose-dependent manner. Compared with the vehicle-treated animals, mean arterial pressure increased from 41.6 +/- 4.6 to 63.1 +/- 3.1 mm Hg in the 20 mg/kg intravenous group and from 33.2 +/- 4.9 to 54.0 +/- 4.7 mm Hg in the 40 mg/kg intramuscular group 60 mins after a 40-mm Hg shock. Furthermore, the drug did not affect the hemorrhage-induced changes in blood glucose concentrations. However, the higher doses of glipizide sodium salt attenuated the increments in plasma concentrations of lactate, alanine aminotransferase, creatinine, and amylase. Moreover, the higher doses markedly improved short-term survival after pressure- and volume-controlled bleeding. Overall, the intramuscular injections of the drug exerted salutary effects that were comparable to the intravenous administration. CONCLUSIONS: In rats, parenteral administration of the water-soluble glipizide sodium salt attenuates vascular and end-organ dysfunction associated with severe hemorrhagic shock and prolongs short-term survival. The intramuscular administration provides comparable benefits as obtained by the intravenous injection.

Aerosolized linear polyethylenimine-nitric oxide/nucleophile adduct attenuates endotoxin-induced lung injury in sheep.

Pulmonary hypertension and edema are mainstays of acute lung injury (ALI). We synthesized linear polyethylenimine-nitric oxide/nucleophile adduct (DS-1), a water-soluble nitric oxide donor, and demonstrated that it is a potent relaxant of precontracted rat aortic rings without inducing desensitization. Moreover, DS-1 does not suppress the viability of human pulmonary epithelial cells in vitro. We also tested whether DS-1 counteracts ALI in endotoxemic sheep. Animals were instrumented for a chronic study. In 16 awake, spontaneously breathing sheep, Escherichia coli endotoxin (10 ng/kg/minute) was infused for 8 hours. From 2 hours of endotoxemia, sheep received either nebulized DS-1 (1 mg/kg/hour) or isotonic saline. DS-1 reduced endotoxin-induced rises in pulmonary arterial and microwedge pressures and vascular resistance index by 40-70%. In parallel, DS-1 decreased the accumulation of extravascular lung water by 60-70% and reduced the increment in right ventricle stroke work index and the falls in right ventricle ejection fraction, stroke volume, and left ventricle stroke work indices. Furthermore, DS-1 reduced venous admixture and improved arterial oxygen saturation. In four healthy animals, DS-1 alone slightly increased arterial oxygenation but had no other effects. Thus, aerosolized DS-1 attenuates endotoxin-induced ALI in sheep by reducing pulmonary hypertension and edema and improving myocardial function and gas exchange.