Faculty Bibliography

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 (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.

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/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.

AIMS/OBJECTIVE: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/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/CONCLUSIONS: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.

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/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.

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.