Macitentan slows down the dermal fibrotic process in systemic sclerosis: in vitro findings.

Systemic sclerosis (or scleroderma) is an autoimmune disease characterized by skin and internal organ fibrosis, caused by microvascular dysfunction. The microvascular damage seems to be a consequence of an endothelial autoimmune response, followed by activation of the inflammatory cascade and massive deposition of collagen. Endothelin-1 (ET-1) contributes to the inflammatory and fibrotic processes by increasing the concentration of pro-inflammatory and pro-fibrotic cytokines, and it is considered one of the most relevant mediators of vascular damage in scleroderma. It is indeed found in very high concentration in serum of sclerodermic patients. Moreover, in these pathological conditions there is an increased expression of ET-1 receptors (ETA and ETB), which mediate the detrimental action of ET-1, and often a change of ETA/ETB ratio. The aim of the present study is to evaluate the in vitro effect of macitentan, an orally active tissue-targeting dual endothelin receptor antagonist, and its major metabolite (ACT-132577) on alpha smooth muscle actin (alphaSMA) expression, evaluated on dermal fibroblasts from healthy subjects and on dermal fibroblasts from lesional and non-lesional skin from sclerodermic patients. The combination of macitentan and its major metabolite reduced the levels of αSMA after 48 h in sclerodermic fibroblasts from lesional skin. No relevant changes in αSMA levels were found in fibroblasts from non-lesional skin, whose behavior is similar to that of dermal fibroblasts from healthy patients.

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-ß1 (TGF-ß1), 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-ß1, 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.

Synergistic vascular protective effects of combined low doses of PPARalpha and PPARgamma activators in angiotensin II-induced hypertension in rats.

BACKGROUND AND PURPOSE: Protective cardiovascular effects of peroxisome proliferator activated receptor (PPAR)alpha and PPARgamma activators have been demonstrated. If used as vasoprotective agents in high risk vascular patients rather than for their metabolic benefits, these agents could be associated with unwanted side effects. As a proof of concept to support the use of combined low doses of PPARalpha and PPARgamma as vascular protective agents in high risk vascular patients, we tested the hypothesis that combined low doses of PPARalpha (fenofibrate) and PPARgamma (rosiglitazone) activators would provide vascular protective benefits similar to full individual doses of these PPAR agonists. EXPERIMENTAL APPROACH: Male Sprague-Dawley rats infused with Ang II (120 ng kg(-1) min(-1)) were treated with rosiglitazone (1 or 2 mg kg(-1) day(-1)) alone or concomitantly with fenofibrate (30 mg kg(-1) day(-1)) for 7 days. Thereafter, vessels was assessed on a pressurized myograph, while NAD(P)H oxidase activity was determined by lucigenin chemiluminescence. Inflammation was evaluated using ELISA for NFkappaB and Western blotting for adhesion molecules. KEY RESULTS: Ang II-induced blood pressure increase, impaired acetylcholine-induced vasorelaxation, altered vascular structure, and enhanced vascular NAD(P)H oxidase activity and inflammation were significantly reduced by low dose rosiglitazone+fenofibrate. CONCLUSIONS AND IMPLICATIONS: Combined low doses of PPARalpha and PPARgamma activators attenuated development of hypertension, corrected vascular structural abnormalities, improved endothelial function, oxidative stress, and vascular inflammation. These agents used in low-dose combination have synergistic vascular protective effects. The clinical effects of combined low-dose PPARalpha and PPARgamma activators as vascular protective therapy, potentially with reduced side-effects and drug interactions, should be assessed.

Chronic decrease in flow contributes to heart failure-induced endothelial dysfunction in rats.

Chronic heart failure (CHF) impairs endothelium-dependent, nitric oxide (NO)-mediated dilation. This decreased dilation may be partly secondary to the chronic decrease in blood flow, but this hypothesis has not yet been tested. Thus, we assessed whether a localized, chronic increase in blood flow in vivo reverses endothelial dysfunction of small arteries in rats with CHF. Two months after coronary artery ligation or sham surgery, second-order side branches of the superior mesenteric artery were ligated in order to obtain persistently elevated blood flow (HF) in the adjacent first-order side branch compared with normal vessels (NF). One month later, responses to acetylcholine and flow-mediated vasodilatation (FMD) were assessed in vitro in an arteriograph. Chronic heart failure induced a decrease in mesenteric blood flow (374 +/- 25 and 305 +/- 27 micro L/min for sham and CHF, respectively; P < 0.05). Neither CHF nor the chronic increase in flow affected the responses to acetylcholine. Chronic heart failure decreased FMD (maximal response in sham and control 34 +/- 6 and 13 +/- 4%, respectively; P < 0.05). Chronic increases in blood flow did not modify FMD in sham, but restored FMD in CHF rats (28 +/- 4%; P < 0.05 vs CHF NF). The restored response was abolished by an inhibitor of NO synthesis (N(G)-nitro-l-arginine). Chronic heart failure did not affect the abundance of mesenteric endothelial NO synthase (eNOS) mRNA. A chronic increase in flow significantly increased the abundance of eNOS mRNA in sham rats, but only moderately and non-significantly in CHF rats. Thus, endothelial dysfunction of small arteries in CHF appears to be largely the consequence of the chronic decrease in flow.

Chronic blockade of endothelin receptors improves ischemia-induced angiogenesis in rat hindlimbs through activation of vascular endothelial growth factor-no pathway.

This study investigated in vivo the putative angiogenic role of endothelin (ET)-1 in a model of ischemia-induced angiogenesis. Ischemia was produced by unilateral femoral artery occlusion in Wistar rats submitted to either chronic ET-1 infusion (2 nmol. kg(-1). min(-1)) or to a dual ET(A)/ET(B) receptor antagonist (bosentan, 100 mg. kg(-1). d(-1)) for 3 and 28 days. Arterial density was evaluated by microangiography and measurement of capillary and arteriolar density in hindlimb muscles. ET-1 infusion had no effect on ischemia-induced angiogenesis and was associated with a slight decrease in vascular endothelial growth factor (VEGF) content measured by Western blot analysis. Conversely, bosentan induced a marked increase in vessel density at 3 and 28 days (1.4-fold and 1.7-fold, respectively, compared with no treatment; P<0.05), which was associated with an increase in VEGF and endothelial NO synthase levels in ischemic legs (by 31+/-8% and 45+/-23%, respectively, at 3 days and by 65+/-13% and 55+/-15%, respectively, at 28 days; P<0.05 versus nontreated rats). At day 28, the proangiogenic effect of bosentan was abolished when NO synthesis inhibitor N(G)-nitro-L-arginine methyl ester (10 mg. kg(-1). d(-1)) or VEGF-neutralizing antibody (2.5 micro/kg twice a week) were coadministered with bosentan. Those results provide the first evidence of an early and sustained proangiogenic effect of endothelin antagonism associated with an upregulation of VEGF and endothelial NO synthase in vivo.

The deletion genotype of the angiotensin I-converting enzyme is associated with an increased vascular reactivity in vivo and in vitro.

OBJECTIVES: To define a link between the deletion genotype (DD) and vascular reactivity, we studied in vivo and in vitro phenylephrine (PE)-induced tone and the effect of angiotensin II (AII) at physiological (subthreshold) concentrations on PE-induced tone. BACKGROUND: The deletion allele (D) of the angiotensin I-converting enzyme (ACE) has been associated with a higher circulating and cellular ACE activity and possibly with some cardiovascular diseases. METHODS: During cardiac surgery PE-induced contraction was studied in patients with excessive hypotension. In parallel, excess material of internal mammary artery, isolated from patients operated for bypass surgery, was mounted in an organ chamber, in vitro, for isometric vascular wall force measurement. RESULTS: In patients under extracorporeal circulation, PE (25 to 150 microg) induced higher contractions in patients with the DD genotype (e.g., with PE 75 microg: 20.3 +/- 2.9 vs. 11.5 +/- 2.5 mm Hg/ml per min, DD vs. II/ID, n = 15 vs. 30, p < 0.03). In the mammary artery, in vitro, contractions to PE (0.1 to 100 micromol/liter) or AII (1 or 100 nmol/liter) were not affected by the genotype. Angiotensin II (10 pmol/liter) significantly potentiated PE (1 micromol/liter)-induced contraction in both groups. Potentiation of PE-induced tone by AII was significantly higher in the DD than in the II/ID group. CONCLUSIONS: The DD genotype was associated with an increased reactivity to PE in vivo and potentiating effect of exogenous AII in vitro. The higher response to PE in vivo might reflect a higher potentiation by endogenous AII. These data should be considered to understand possible link(s) between cardiovascular disorders and the ACE gene polymorphism.

Prolonged blockade of endothelin ET(A) receptors decreases vascular reactivity in the aorta of spontaneously hypertensive rats in vitro.

We investigated the effect of prolonged endothelin-1 type A (ET(A)) receptors blockade on the constrictor response to phenylephrine and the dilator response to acetylcholine (ACh) in isolated aortic rings from normotensive [Wistar-Kyoto (WKY)] rats and spontaneously hypertensive rats (SHRs). Animals were treated for 2 weeks with the ET(A)-receptor blocker LU135252 (50 mg/kg/day; n = 8). LU135252 treatment did not affect blood pressure in both strains. In isolated aortic segments, dilation to ACh and contractions to phenylephrine were decreased only in SHRs. Nitric oxide (NO) synthesis blockade (L-NAME, 0.1 mM) inhibited 90+/-11% (WKY rats) and 76+/-8% (SHRs) of ACh-induced dilation. Cyclooxygenases blockade (indomethacin, 10 microM) had no effect in both strains. Endothelium-derived hyperpolarizing factor(s) (EDHF) blockade (KCl, 20 mM) suppressed the remaining ACh-induced dilation in both strains. Treatment with LU135252 significantly decreased NO-dependent dilation, as compared with controls [70+/-8% vs. 90+/-11% (WKY rats) and 54+/-6% vs. 76+/-8% (SHRs) of total dilation; p<0.05]. On the other hand, EDHF-dependent dilation was significantly higher in the LU135252 groups [29+/-5% vs. 10+/-3% (WKY rats) and 44+/-7% vs. 19+/-4% (SHRs) of total dilation; p<0.05]. Thus prolonged ET(A)-receptor blockade decreased the responsiveness to phenylephrine and ACh in SHR aortas and changed the proportion of dilator agents in ACh-induced dilation.

Chronic endothelin-1 improves nitric oxide-dependent flow-induced dilation in resistance arteries from normotensive and hypertensive rats.

Endothelin-1 (ET-1) is released on stimulation by shear stress of the vascular wall. In several pathological situations, an involvement of ET-1 is suspected. Nevertheless, the effect of a chronic increase in circulating ET-1 on vascular tone in resistance arteries is not yet fully understood. We investigated the response to tensile stress (pressure-induced myogenic tone) and shear stress (flow-induced dilation, FD) of rat mesenteric resistance arteries cannulated in an arteriograph. Intraluminal diameter was measured continuously. Rats (normotensive Wistar-Kyoto rats [WKYs] and spontaneously hypertensive rats [SHRs]) were treated for 2 weeks with ET-1 (5 pmol. kg(-1). min(-1) SC; n=8 to 16 per group). Systolic arterial blood pressure increased significantly in ET-1-treated rats (171+/-7 versus 196+/-6 mm Hg in WKYs and 216+/-8 versus 245+/-6 mm Hg in SHRs, P<0.05). Passive arterial diameter in isolated resistance arteries ranged from 78+/-9 to 169+/-4 microm in WKYs and from 62+/-6 to 149+/-7 microm in SHRs (pressure from 10 to 150 mm Hg). Myogenic tone was not significantly affected by chronic ET-1. Flow (9 to 150 microL/min) significantly increased the arterial diameter by 2+/-0.5 to 22+/-2 microm in WKYs and by 1.3+/-0. 7 to 8.3+/-0.8 microm in SHRs (P<0.001 versus WKYs). The NO synthesis blocker N(G)-nitro-L-arginine methyl ester (L-NAME; 100 micromol/L) attenuated FD in WKYs (eg, 22+/-2 versus 15+/-3 microm after L-NAME, flow=150 microL/min) and, to a lesser extent, in SHRs (P<0.001 versus WKYs). The cyclooxygenase inhibitor indomethacin (3 micromol/L) attenuated the remaining FD in WKYs (eg, 15+/-3 versus 8+/-3 microm, flow=150 microL/min) and in SHRs (eg, 7.5+/-0.5 versus 5.0+/-0.6 microm). Chronic ET-1 significantly increased FD in SHRs but not in WKYs. In both strains, NO-dependent FD was significantly increased by chronic ET-1. Furthermore, indomethacin-sensitive FD was increased by chronic ET-1 in SHRs only. Thus, chronic ET-1 increased NO-dependent FD in resistance mesenteric arteries from both WKYs and SHRs and increased indomethacin-sensitive FD in SHRs only.

Chronic blockade of endothelin ETA receptors improves flow dependent dilation in resistance arteries of hypertensive rats.

OBJECTIVE: Flow (shear stress)-induced dilation (FD) is attenuated in hypertension. Flow triggers the release by endothelial cells of dilators, such as NO or cyclo-oxygenase (COX) derivatives and constrictor factors such as endothelin-1 (ET-1) which might be involved in several cardiovascular diseases. We hypothesized that ET-1 might play a functional role in FD and participate in the endothelial dysfunction in hypertension. METHODS: We investigated the effect of chronic treatment with the ETA receptor blocker LU135252 (50 mg/kg/day) for 2 weeks on the dilator response to flow in normotensive (Wistar-Kyoto; WKY) or hypertensive (SHR, n = 7 or 8 per group) rats. RESULTS: Systolic arterial pressure was not significantly affected by chronic ETA receptor blockade in both strains. In mesenteric resistance arteries (diameter: approximately 100 microns), isolated in vitro, FD was lower and myogenic tone higher in SHR than in WKY rats. Chronic ETA receptor blockade increased FD by 73% (7.5 +/- 1.5 to 13.0 +/- 2.7 microns dilation with a flow-rate of 150 microliters/min) in SHR (no effect in WKY). The participation of NO to FD was increased in SHR and the participation of dilator COX product(s) (blocked by indomethacin 10 mumol/l) to FD was significantly increased in SHR and in WKY. In control rats FD was improved by acute ETA receptor blockade in WKY rats (18.5 +/- 2.0 to 23.2 +/- 1.8 microns dilation to flow-rate of 150 microliters/min) and significantly more in SHR (6.0 +/- 1.8 to 15.1 +/- 1.6 microns). Acetylcholine-induced dilation was also improved by chronic ETA receptor blockade (no effect of an acute blockade). Myogenic and phenylephrine-induced tone were not affected by chronic or acute ETA receptor blockade. The improvement of endothelium-dependent dilation was not related to a change in blood pressure. CONCLUSION: Chronic ETA receptor blockade increased flow-induced dilation in SHR possibly by suppressing flow-induced ETA stimulation and by improving the release of dilator products by the endothelium.

Chronic endothelin-1-induced changes in vascular reactivity in rat resistance arteries and aorta.

The role of endothelin-1 in vascular homeostasis is not yet clearly established. We investigated the responses to phenylephrine and acetylcholine in rat mesenteric resistance artery and aorta mounted in vitro in myographs after a 2-week treatment with endothelin-1 (5 pmol kg(-1) min(-1), n = 8). Systolic arterial blood pressure increased in endothelin-1-treated rats (171 +/- 7 mmHg vs. 196 +/- 6 mmHg, P < 0.05). In the aorta, chronic endothelin-1 significantly increased the dilator response to acetylcholine (maximal dilatation: 76 +/- 3 vs. 86 +/- 3% in control, P < 0.05). Acetylcholine-induced dilatation was decreased by nitric oxide (NO) synthase inhibition with NG-nitro-L-arginine methyl ester (L-NAME 100 micromol/l) and partly restored by cyclooxygenases inhibition (indomethacin, 10 micromol/l). In endothelin-1-treated rats, L-NAME-sensitive acetylcholine dilatation was lower than in the control, but dilator cyclooxygenase product(s) were found instead of constrictor cyclooxygenase product(s). In mesenteric resistance arteries chronic endothelin-1 increased the participation of cyclooxygenase products in acetylcholine-induced dilatation from 10 +/- 2 to 19 +/- 3%. In both types of arteries, phenylephrine-induced contraction was not affected by chronic endothelin-1. Thus chronic endothelin-1 increased the participation of dilator cyclooxygenase product(s) in acetylcholine-induced dilatation in the aorta and the mesenteric resistance arteries.

Determination of 2-n-propylquinoline in mouse plasma and liver by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed for the specific determination of 2-n-propylquinoline, a new anti-leishmaniasis drug, in plasma and liver homogenates of mice. 2-n-Propylquinoline was extracted with methyl-tert.-butyl ether with quinoline as internal standard. Separation was carried out using a Nucleosil C18 column. The mobile phase consisted of methanol-0.005 M ammonium acetate buffer (60:40) at pH 5.5 and 8 for plasma and liver homogenates, respectively. Detection was monitored at 233 nm. The method was validated and shown to be accurate and precise for plasma and liver homogenates. Extraction yield was 96% in plasma and 81% in liver homogenates. This method was used to determine the pharmacokinetic profile of 2-n-propylquinoline following oral administration to mice.