Ronald Van Beneden

Bosentan for the prevention of overcirculation-induced experimental pulmonary arterial hypertension.

Background—The dual endothelin-receptor antagonist bosentan has been reported to improve pulmonary arterial hypertension, but the role of endothelins in the pathogenesis of the condition remains uncertain. We investigated the roles of endothelin-1 (ET-1), nitric oxide (NO), vascular endothelial growth factor (VEGF), and tenascin in overcirculation-induced pulmonary hypertension in piglets, as a model of early pulmonary arterial hypertension, with or without bosentan therapy. Methods and Results—Thirty 3-week-old piglets were randomized to placebo or to bosentan 15 mg/kg BID after the anastomosis of the left subclavian artery to the pulmonary arterial trunk or after a sham operation. Three months later, the animals underwent a hemodynamic evaluation followed by cardiac and pulmonary tissue sampling for morphometry, immunohistochemistry, and real-time quantitative PCR. Chronic systemic-to-pulmonary shunting increased circulating plasma ET-1, pulmonary mRNA for ET-1, ETB receptor, inducible NO synthase, VEGF, and pulmonary ET-1 and VEGF proteins. There were increases in myocardial mRNA for ETA receptor and VEGF and in myocardial VEGF protein. Pulmonary and myocardial tissue mRNA for tenascin did not change. Normalized-flow pulmonary artery pressure increased from 20 (2) to 33 (1) mm Hg [mean (SEM)], arteriolar medial thickness increased on average by 83%, and these changes were completely prevented by bosentan therapy. Right ventricular end-systolic elastance increased in proportion to pulmonary arterial elastance with or without bosentan. Conclusions—Experimental overcirculation-induced pulmonary arterial hypertension appears to be causally related to an activation of the pulmonary ET-1 system and as such is completely prevented by the dual endothelin receptor antagonist bosentan.

Induction of MafBx and Murf ubiquitin ligase mRNAs in rat skeletal muscle after LPS injection.

MafBx and Murf are two new rat E3 ubiquitin ligases induced in muscle atrophy. Our goal was to investigate whether lipopolysaccharide (LPS) injection, a model of muscle catabolism, is associated with increased expression of MafBx and Murf. LPS (750 μg/100 g body weight) induces MafBx and Murf mRNA (respectively, 23‐fold and 33‐fold after 12 h; P<0.001). A transient induction of tumor necrosis factor‐α mRNA (21‐fold; P<0.001 at 3 h) and a decrease of insulin like growth factor‐I mRNA (50%; P<0.001 at 6 h), two potential regulators of the ubiquitin–proteasome system were also demonstrated. In summary, MafBx and Murf mRNA are up‐regulated in response to LPS and might play a role in the muscle proteolysis observed.

Signaling molecules in overcirculation-induced pulmonary hypertension in piglets: effects of sildenafil therapy.

Background—The phosphodiesterase type-5 (PDE-5) inhibitor sildenafil has been reported to improve pulmonary arterial hypertension (PAH), but the mechanisms that account for this effect are incompletely understood. Severe pulmonary hypertension has been characterized by defects in a signaling pathway involving angiopoietin-1 and the bone morphogenetic receptor-2 (BMPR-2). We investigated the effects of sildenafil on hemodynamics and signaling molecules in a piglet overcirculation-induced model of early PAH. Methods and Results—Thirty 3-week-old piglets were randomized to placebo or sildenafil therapy 0.75 mg/kg TID after anastomosis of the left subclavian artery to the pulmonary arterial trunk or after a sham operation. Three months later, the animals underwent a hemodynamic evaluation followed by pulmonary tissue sampling for morphometry, immunohistochemistry or radioimmunoassay, and real-time quantitative-polymerase chain reaction. Chronic systemic-to-pulmonary shunting increased pulmonary mRNA for angiopoietin-1, endothelin-1 (ET-1), angiotensin II, inducible nitric oxide synthase, vascular endothelial growth factor, and PDE-5. Pulmonary messenger RNA for BMPR-1A and BMPR-2 decreased. Pulmonary angiotensin II, ET-1, and vascular endothelial growth factor proteins increased. Pulmonary artery pressure increased from 20±2 to 33±1 mm Hg, and arteriolar medial thickness increased by 91%. The expressions of angiopoietin-1, ET-1, and angiotensin II were tightly correlated to pulmonary hypertension. Sildenafil prevented the increase in pulmonary artery pressure, limited the increase in medial thickness to 41%, and corrected associated biological perturbations except for the angiopoietin-1/BMPR-2 pathway, PDE-5, and angiotensin II. Conclusions—Sildenafil partially prevents overcirculation-induced PAH and associated changes in signaling molecules. Angiotensin II, PDE-5, and angiopoietin-1/BMPR-2 signaling may play a dominant role in the early stages of the disease.

Creatine increases IGF-I and myogenic regulatory factor mRNA in C2C12 cells

Addition of creatine to the differentiation medium of C2C12 cells leads to hypertrophy of the myotubes. To investigate the implication of insulin‐like growth factor I (IGF‐I) and myogenic regulatory factors (MRFs) in this hypertrophy, their mRNA levels were assessed during the first 72 h of differentiation. Creatine significantly increased the IGF‐I mRNA level over the whole investigated period of time, whereas the MRF mRNA levels were only augmented at precise moments, suggesting a general activation mechanism for IGF‐I and a specifically regulated mechanism for MRF transcription. Our results suggest therefore that creatine‐induced hypertrophy of C2C12 cells is at least partially mediated by overexpression of IGF‐I and MRFs.

Expression of the serotonin 1b receptor in experimental pulmonary hypertension

The pathogenesis of pulmonary arterial hypertension (PAH) remains uncertain. Both the serotonin and endothelin (ET) systems are believed to be involved. Recent studies pointed to the importance of the serotonin 2B receptor as a limiting step. The current authors investigated the lung tissue expression of serotonin receptors and of the serotonin transporter (5‐HTT) by real-time-quantitative polymerase chain reaction in chronic overcirculation-induced PAH in growing piglets, with and without treatment with the dual ET receptor blocker bosentan. Pulmonary haemodynamic changes were described by pulmonary arterial impedance spectra. Three months after the surgical anastomosis of the left subclavian artery to the pulmonary arterial trunk, there was a shift of the impedance spectra to higher ratios of pressure and flow moduli, with increases in both 0 Hz impedance and characteristic impedance, and these changes were completely prevented by bosentan therapy. There was an increase in the expression of the serotonin 1B receptor. There was no change in the expression of the 5‐HTT, and of the serotonin 2B, 1D, and 4 receptors. The overexpression of the serotonin 1B receptor was partially prevented by bosentan therapy. The present authors conclude that this early pulmonary arterial hypertension model is characterised by an endothelin receptor-dependent increased expression of the serotonin 1B receptor.

Increased expression of endothelin-1 and its mitogenic receptor ETA in human papillary thyroid carcinoma.

objective Since the isolation of endothelin‐1 (ET‐1) in 1988, there has been tremendous interest in the pathophysiological roles of ET‐1 as a vasoconstrictive and mitogenic peptide. Whereas ET‐1 is mainly released by vascular endothelial cells, it also proved to be produced by various tissues including the thyroid. Because of its mitogenic properties in malignancy and its role as an inflammatory modulator, ET‐1 could be involved in thyroid carcinogenesis and thyroiditis.

Interrelated overexpression of endothelial and inducible nitric oxide synthases, endothelin-1 and angiogenic factors in human papillary thyroid carcinoma

Objective  Nitric oxide (NO) and endothelin‐1 (ET‐1) are involved in carcinogenesis. Overexpression of the ET‐1 axis has been demonstrated in papillary thyroid carcinoma (PTC). This study investigated the expression of NO synthases (NOS) and their relationship with expression of ET‐1 and angiogenic markers in PTC.

Increased expression of endothelin-1 converting enzyme in human thyroid carcinoma

Endothelin-l (ET-l), a 21amino-acid peptide possessing vasoconstrictive and mitogenic properties, is principally released by the vascular endothelium (Yanagisawa et al., 1988; Donckier et al., 1991) but can also be produced by thyroid follicular cells (Lenziardi et al. 1995). In the September 2003 issue of Clinical Endocrinology, we demonstrated that both ET-1 mRNA and ET-l peptide as well as the effector receptor ETAR mRNA levels were increased in thyroid papillary carcinoma and Hashimotos thyroiditis (Donckier et al., 2003). ET-1 peptide is synthesized through successive proteolytic cleavage of a 203-amino-acid precursor to obtain Big-endothelin-1 (1-38Big-ET-l). During secretion, 1-38Big-ET-l is cleaved by a specific membrane-bound metalloproteinase, the ET-l converting enzyme (ECE-1) that leads to the equimolar secretion of the ET-1 active form (1-21ET-1) and the C-terminal fragment (22-38CTF or 22-38Big-ET-1) (Kido et al., 1997). The physiological importance of this cleavage is indicated by the reported 140-fold increase in vasoconstrictor potency upon cleavage to 1-21ET-l (Kimura et al., 1989). Because the amounts of cross-reactivity from our anti-ET-l antibody were 100%, 87% and 0%, respectively, for 1-21ET-1, 1-38Big-ET-l and 22-38CTF, our study suggested that the increased ET-l immunostaining, as evaluated by counting the number of positive cells among 1000 follicular cells, may result from mixed labelling of 1-21ET-1 and 1-38Big-ET-l. In this respect, we considered it of importance to demonstrate that increased expression of the ET-1 peptide could be related to an increased expression of the ECE-l.

Characterization of xenoreactive natural secreting cells in rats

Abstract: IgM xenoreactive natural antibodies (XNA) and the activation of complement are thought to be the main components involved in the hyperacute rejection (HAR) of discordant xenografts. Nevertheless few studies have focused on the B cell population responsible for the production of XNA. Using the guinea pig to rat discordant xenograft model we have developed a xenoreactive ELISA‐spot technique allowing the specific detection of XNA secreting cells in rats. In order to detect rat XNA‐secreting cells, guinea pig platelet membrane extracts, which have been shown to share major xenoantigens as recognized by XNA, with endothelial cells, were used as targets for rat XNA. Adult LOU/C rats lymphocytes from spleen, mesenteric lymph nodes, Peyers patches, bone marrow, and the peritoneal cavity were used as source of XNA secreting cells. The number of IgM, IgG2a, Ig k light chain XNA, and IgM XNA secreting cells was measured using standard and xenoreactive ELISA‐spot techniques respectively. Data presented here show that IgM XNA secreting cells are almost exclusively located in the spleen. Moreover, IgM XNA secreting cells represent around 0.0001% of spleen lymphocytes corresponding to 0.1% of the IgM secreting cells in the spleen. Although circulating IgG2a XNA are detectable, by cellular ELISA, in normal LOU/C rats, IgG2a secreting cells were not found in any of the cell suspensions analyzed. In contrast, Ig k light chain XNA were found in the spleen, mesenteric lymph nodes, and the bone marrow but not in the peritoneal cavity. Taken together these results suggest that extremely low numbers of secreting cells producing isotypes other than IgM and IgG2a might exist in different rat organs but are not detectable using the xenoreactive ELISA‐spot technique. In conclusion, our data show that only 0.0001% of spleen lymphocytes were found to be IgM XNA secreting cells. Moreover this IgM XNA secreting cells population is located almost exclusively in the spleen further illustrating the role of the spleen in the production of XNA.

Superiority of big endothelin-1 and endothelin-1 over natriuretic peptides to predict survival in severe congestive heart failure

Superiority of Big Endothelin-1 and Endothelin-1 over Natriuretic Peptides To Predict Survival in Severe Congestive Heart Failure Michel F. Rousseau,RonaldVanBeneden,OlivierGurne, PhilippeL. Selvais, Annie R. Robert, Sylvie A. Ahn, Hubert G. Pouleur, Jean Marie Ketelslegers2—Division of Cardiology, University of Louvain, Brussels, Belgium; Diabetes and Nutrition Unit, University of Louvain, Brussels, Belgium; School of Public Health, University of Louvain, Brussels, Belgium