Marc Iglarz

Bosentan, Sildenafil, and Their Combination in the Monocrotaline Model of Pulmonary Hypertension in Rats:

The dual endothelin receptor antagonist, bosentan, and the phosphodiesterase inhibitor, sildenafil, are efficacious in experimental and clinical pulmonary hypertension (PHT). The effects of bosentan, sildenafil, and their combination were evaluated in rats with monocrotaline (MCT)-induced PHT. A first group consisted of control rats with no MCT injection. Four other groups of rats received MCT subcutaneously and were assigned to receive no treatment, 300 mg/kg/day bosentan as food admix, 100 mg/kg/day sildenafil in drinking water, or their combination for 4 weeks. The doses of bosentan and sildenafil were the maximally effective doses based on a dose-range–finding study. Mortality was 0%, 53%, 11%, 11%, and 0%, respectively, in the five different groups. Bosentan and sildenafil significantly attenuated the increase in mean pulmonary arterial pressure, and the combination had an additional effect. Similarly, bosentan, sildenafil, and, to a greater extent, their combination significantly reduced right ventricular (RV) hypertrophy. Bosentan, but not sildenafil, decreased norepinephrine and BNP plasma concentrations, reduced kidney weight, and normalized systemic hemodynamics. In conclusion, bosentan and sildenafil are efficacious in rats with chronic PHT, and their combination shows an additional effect for decreasing pulmonary arterial pressure, reducing plasma catecholamines, maintaining body weight, and reducing mortality.

At the heart of tissue: endothelin system and end-organ damage.

ET (endothelin)-1 was first described as a potent vasoconstrictor. Since then, many other deleterious properties mediated via its two receptors, ETA and ETB, have been described, such as inflammation, fibrosis and hyperplasia. These effects, combined with a wide tissue distribution of the ET system, its up-regulation in pathological situations and a local autocrine/paracrine activity due to a high tissue receptor binding, make the tissue ET system a key local player in end-organ damage. Furthermore, ET-1 interacts in tissues with other systems such as the RAAS (renin-angiotensin-aldosterone system) to exert its effects. In numerous genetically modified animal models, non-specific or organ-targeted ET-1 overexpression causes intense organ damage, especially hypertrophy and fibrosis, in the absence of haemodynamic changes, confirming a local activity of the ET system. ET receptor antagonists have been shown to prevent and sometimes reverse these tissue alterations in an organ-specific manner, leading to long-term benefits and an improvement in survival in different animal models. Potential for such benefits going beyond a pure haemodynamic effect have also been suggested by clinical trial results in which ET receptor antagonism decreased the occurrence of new digital ulcers in patients with systemic sclerosis and delayed the time to clinical worsening in patients with PAH (pulmonary arterial hypertension). The tissue ET system allows therapeutic interventions to provide organ selectivity and beneficial effects in diseases associated with tissue inflammation, hypertrophy or fibrosis.

Endothelin-2 induces oviductal contraction via endothelin receptor subtype A in rats

Proper function of the oviduct is critical to reproductive success with regulated contraction and relaxation facilitating transportation of the germ cells to the site of fertilization. Endothelin-2 (EDN2) is a potent vasoconstrictor produced by granulosa cells of the preovulatory follicle at the time of ovulation; however, whether this gonadotropin surge-induced peptide played a role in facilitating germ cell transportation by inducing oviductal contraction was unknown. The objectives of these experiments were (1) to determine whether the endothelin receptor system was present in the oviduct, (2) to test the hypothesis that EDN2 induces oviductal contraction via a specific endothelin receptor subtype, (3) to determine, as a possible alternate source of the ligand, whether mRNA for EDN2 was expressed in cumulus-oocyte complexes (COCs) within the oviduct, and (4) to determine whether EDN2 could overcome prostaglandin E(2) (PGE(2))-induced oviductal relaxation. Microarray and real-time PCR analysis indicated that mRNA for both the endothelin receptor subtypes (ET(A) and ET(B)) was present in the oviduct, whereas immunohistochemical examination revealed that ET(A) protein was the dominant isoform, present in the luminal epithelial cells of the oviduct. Real-time PCR analysis demonstrated that mRNA for EDN2 was expressed in COCs after ovulation. Isometric tension analysis indicated that EDN2 was a potent oviductal constrictor and that the contractile effect of EDN2 was mediated by the ET(A) and not the ET(B) receptor subtype. The oviductal contraction induced by EDN2 also reversed oviductal relaxation induced by PGE(2). In summary, ET(A) receptor-specific EDN2-induced contraction as a facilitator of oviductal function suggests a novel pathway involved in germ cell transport and hence mammalian fertility.

Identification of a Novel Role for Endothelins within the Oviduct

Endothelins were first identified as potent vasoactive peptides; however, diversity in the biological function of these hormones is now evident. We have identified a novel role for endothelins: a requirement for these peptides within the oviduct during fertilization and/or early embryo development. In vivo, treatment after ovulation with a dual endothelin receptor antagonist (tezosentan) decreased the number of two-cell embryos that could be collected from within the oviducts. In vitro fertilization experiments showed that gamete viability and their ability to fertilize were not affected by treatment with this antagonist, suggesting that the effect observed in vivo was mediated by the oviduct itself. Expression of mRNA for all three isoforms of the endothelins and both receptor subtypes was detectable within the oviduct. Expression of mRNA for endothelin-3 was regulated by gonadotropins in epithelial cells of the oviduct and increased specifically within the isthmus of this structure. Immunostaining revealed localization of both endothelin receptors A and B to the columnar epithelial cells within the oviduct, suggestive of a local role for endothelins in the regulation of epithelial function and ultimately oviductal secretions. A microarray analysis revealed three likely endothelin-regulated protein networks for future analysis: the TGFbeta, IL-10, and CCAAT/enhancer-binding protein superfamilies. Overall, these results suggest a novel and requisite role for endothelins within the oviduct during fertilization and/or early embryo development.

Renal, retinal and cardiac changes in type 2 diabetes are attenuated by macitentan, a dual endothelin receptor antagonist

AIMS Diabetes is known to cause alteration of the endothelin (ET) system. We have previously demonstrated that ETs regulate augmented production of extracellular matrix proteins causing structural alterations in type 1 diabetes. Here we investigated the effects of macitentan, an orally-active, tissue-targeting dual ET receptor antagonist on chronic complications in type 2 diabetes. MAIN METHODS db/db mice and their age- and sex-matched controls were examined after 2 and 4 months of diabetes. Groups of diabetic animals were treated with oral macitentan (25mg/kg/day). The animals were monitored with respect to body weight and blood glucose. Urine analyses were performed for albumin. Cardiac hemodynamic studies were carried out. Renal, cardiac and retinal tissues were analyzed for ET-1, transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), fibronectin (FN), extradomain B containing FN (EDB(+)FN) and collagen α-I (IV) mRNA. Cardiac atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were measured. Protein expressions were measured by ELISA and Western blot. Microscopic analyses were performed in the kidneys. KEY FINDINGS Diabetic animals showed hyperglycemia, increased urinary albumin and augmented serum creatinine levels. Diabetes caused increased renal, cardiac and retinal ET-1, TGF-β1, VEGF, FN, EDB(+)FN, collagen α-I(IV) mRNA expression along with increased FN and collagen protein and NF-κB activation. Diabetic mice also demonstrated mesangial expansion, cardiac dysfunction and increased expression of ANP and BNP. Treatment with macitentan attenuated such abnormalities. SIGNIFICANCE These experiments confirmed that ET system plays a significant role in the pathogenesis of chronic complications in type 2 diabetes. Such diabetes induced changes can be reduced macitentan therapy.

Comparison of pharmacological activity of macitentan and bosentan in preclinical models of systemic and pulmonary hypertension

AIMS The endothelin (ET) system is a tissular system, as the production of ET isoforms is mostly autocrine or paracrine. Macitentan is a novel dual ETA/ETB receptor antagonist with enhanced tissue distribution and sustained receptor binding properties designed to achieve a more efficacious ET receptor blockade. To determine if these features translate into improved efficacy in vivo, a study was designed in which rats with either systemic or pulmonary hypertension and equipped with telemetry were given macitentan on top of maximally effective doses of another dual ETA/ETB receptor antagonist, bosentan, which does not display sustained receptor occupancy and shows less tissue distribution. MAIN METHODS After establishing dose-response curves of both compounds in conscious, hypertensive Dahl salt-sensitive and pulmonary hypertensive bleomycin-treated rats, macitentan was administered on top of the maximal effective dose of bosentan. KEY FINDINGS In hypertensive rats, macitentan 30 mg/kg further decreased mean arterial blood pressure (MAP) by 19 mm Hg when given on top of bosentan 100 mg/kg (n=9, p<0.01 vs. vehicle). Conversely, bosentan given on top of macitentan failed to induce an additional MAP decrease. In pulmonary hypertensive rats, macitentan 30 mg/kg further decreased mean pulmonary artery pressure (MPAP) by 4 mm Hg on top of bosentan (n=8, p<0.01 vs. vehicle), whereas a maximal effective dose of bosentan given on top of macitentan did not cause any additional MPAP decrease. SIGNIFICANCE The add-on effect of macitentan on top of bosentan in two pathological models confirms that this novel compound can achieve a superior blockade of ET receptors and provides evidence for greater maximal efficacy.

Chymase: a multifunctional player in pulmonary hypertension associated with lung fibrosis

Limited literature sources implicate mast-cell mediator chymase in the pathologies of pulmonary hypertension and pulmonary fibrosis. However, there is no evidence on the contribution of chymase to the development of pulmonary hypertension associated with lung fibrosis, which is an important medical condition linked with increased mortality of patients who already suffer from a life-threatening interstitial lung disease. The aim of this study was to investigate the role of chymase in this particular pulmonary hypertension form, by using a bleomycin-induced pulmonary hypertension model. Chymase inhibition resulted in attenuation of pulmonary hypertension and pulmonary fibrosis, as evident from improved haemodynamics, decreased right ventricular remodelling/hypertrophy, pulmonary vascular remodelling and lung fibrosis. These beneficial effects were associated with a strong tendency of reduction in mast cell number and activity, and significantly diminished chymase expression levels. Mechanistically, chymase inhibition led to attenuation of transforming growth factor β1 and matrix-metalloproteinase-2 contents in the lungs. Furthermore, chymase inhibition prevented big endothelin-1-induced vasoconstriction of the pulmonary arteries. Therefore, chymase plays a role in the pathogenesis of pulmonary hypertension associated with pulmonary fibrosis and may represent a promising therapeutic target. In addition, this study may provide valuable insights on the contribution of chymase in the pulmonary hypertension context, in general, regardless of the pulmonary hypertension form. Chymase plays an important role in pathology of pulmonary hypertension associated with lung fibrosis http://ow.ly/MVYW6

Endothelin receptor antagonist macitentan or deletion of mouse mast cell protease 4 delays lesion development in atherosclerotic mice

AIMS To determine the impact of mixed endothelin receptor antagonist and mouse mast cell protease-4 (mMCP-4) in the development of atherosclerosis in the mouse model. MATERIALS AND METHODS Apolipoprotein E (ApoE) KO mice were crossed with mMCP-4 KO mice to generate ApoE/mMCP-4 double KO mice. Atherosclerosis was induced with a normal- or high-fat diet for 12, 27 or 52weeks. Macitentan (30mg/kg/day), a dual ETA/ETB receptor antagonist, was given orally for 6weeks (27week protocol). At sacrifice, aortas and brachiocephalic arteries (BCAs) were collected. En face Sudan IV staining was performed on aortas and BCA sections were subjected to Massons trichrome stain and α-smooth muscle actin labeling. KEY FINDINGS Under normal diet, both macitentan treatment and the absence of mMCP-4 reduced the development of aortic atherosclerotic lesions in 27-week old ApoE KO mice, but mMCP-4 deletion failed to maintain this effect on 52-week old mice. Under high-fat diet (WD), macitentan, but not the absence of mMCP-4, reduced aortic lesion development in ApoE KO mice. On BCA lesions of 27-week old WD mice, macitentan treatment had a small impact while mMCP-4 deletion showed improved features of plaque stability. SIGNIFICANCE These results suggest that the inhibition of mMCP-4 reduces lesion spreading in the earlier phases of atherosclerosis development and can help stabilise the more advanced plaque. Macitentan treatment was more effective to prevent lesion spreading but did not improve plaque features to the same extent.

Discovery of novel bridged tetrahydronaphthalene derivatives as potent T/L-type calcium channel blockers.

Chemical evolution of mibefradil resulted in the identification of novel bridged tetrahydronaphthalene derivatives as potent T/L-type calcium channel blockers. A SAR study, in vitro and in vivo DMPK properties as well as the in vivo antihypertensive effect in rats are presented.

Dual ETA/ETB blockade with macitentan improves both vascular remodeling and angiogenesis in pulmonary arterial hypertension:

Dysregulated metabolism and rarefaction of the capillary network play a critical role in pulmonary arterial hypertension (PAH) etiology. They are associated with a decrease in perfusion of the lungs, skeletal muscles, and right ventricle (RV). Previous studies suggested that endothelin-1 (ET-1) modulates both metabolism and angiogenesis. We hypothesized that dual ETA/ETB receptors blockade improves PAH by improving cell metabolism and promoting angiogenesis. Five weeks after disease induction, Sugen/hypoxic rats presented severe PAH with pulmonary artery (PA) remodeling, RV hypertrophy and capillary rarefaction in the lungs, RV, and skeletal muscles (microCT angiogram, lectin perfusion, CD31 staining). Two-week treatment with dual ETA/ETB receptors antagonist macitentan (30 mg/kg/d) significantly improved pulmonary hemodynamics, PA vascular remodeling, and RV function and hypertrophy compared to vehicle-treated animals (all P = 0.05). Moreover, macitentan markedly increased lung, RV and quadriceps perfusion, and microvascular density (all P = 0.05). In vitro, these effects were associated with increases in oxidative phosphorylation (oxPhox) and markedly reduced cell proliferation of PAH-PA smooth muscle cells (PASMCs) treated with macitentan without affecting apoptosis. While macitentan did not affect oxPhox, proliferation, and apoptosis of PAH–PA endothelial cells (PAECs), it significantly improved their angiogenic capacity (tube formation assay). Exposure of control PASMC and PAEC to ET-1 fully mimicked the PAH cells phenotype, thus confirming that ET-1 is implicated in both metabolism and angiogenesis abnormalities in PAH. Dual ETA/ETB receptor blockade improved the metabolic changes involved in PAH-PASMCs’ proliferation and the angiogenic capacity of PAH-PAEC leading to an increased capillary density in lungs, RV, and skeletal muscles.