Pascal Bousquet’s research while affiliated with University of Strasbourg and other places

Imidazoline receptor antisera selected/Nischarin was proposed several years ago as the functional entity for the I1 medullary receptors (I1Rs) targeted, together with α2-adrenoceptors, by the centrally acting antihypertensive drugs, such as clonidine. The objective of this study was to test this assumption using a pyrroline analog of clonidine, LNP599, which, unlike clonidine and related compounds, displays high selectivity toward I1Rs. Cardiovascular effects of LNP599 (3 mg/kg intravenous) were evaluated in anesthetized, artificially ventilated nischarin mutant rats expressing a truncated form of nischarin lacking the putative imidazoline binding site. LNP599 induced a rapid and pronounced fall in arterial blood pressure in wild-type animals (-42.7% ± 11.0% after 15 minutes), associated with a ≈30% heart rate reduction. Similar effects were obtained in homozygous and heterozygous nischarin mutant rats. The observation that the hypotensive response to I1R activation is not affected by the absence of the putative imidazoline binding site on nischarin strongly suggests that nischarin cannot be regarded as the functional I1R. Carbohydrate regulation was improved in nischarin mutant rats, further supporting the conclusion that nischarin and I1R are 2 distinct molecular entities.

Background/Objectives Overweight and obesity are undoubtable risk factors for type 2 diabetes and cardiovascular diseases and significantly contribute to the global morbi-mortality. We previoulsy reported that LNP599, a pharmacological imidazoline-like activator of hepatic AMPK/adiponectin signaling, protects against the development of adiposity and obesity and the associated cardio-metabolic disorders, suggesting that it may be a suitable drug candidate for a therapeutic approach targeting the development of obesity at very early stages. The objective of the present study was to evaluate the metabolic effects of LNP599 in a model of diet-induced overweight and metabolic disorders in a nonhuman primate, the common marmoset (Callithrix jacchus), and more particularly to establish the impact of the compound on cholesterol homeostasis, i.e., HDL and LDL/VLDL lipoproteins. Methods Marmosets were fed normal (NC) or hypercaloric (HC) chow during 16 weeks. Diet-induced changes in body weight and metabolism were assessed. Effects of LNP599 were evaluated in a subset of HC animals (HC-LNP) receiving the compound at a daily dose of 10 mg/kg over the 16 weeks. Results HC-feeding induced significant overweight associated with a marked dyslipidemia (hypertriglyceridemia, hypercholesterolemia, and reduced HDL over LDL/VLDL cholesterol ratio). LNP599 blunted the diet-induced body weight gain and largely protected against the development of hypertriglyceridemia. Total cholesterol was unchanged but the ratio of HDL over LDL/VLDL cholesterol was more than doubled. Conclusions The profile of metabolic troubles obtained upon enriched diet mimicked the disorders associated with spontaneous obesity in marmosets. HC marmosets represent an experimental model of high clinical relevance to study the pathophysiology of obesity and related dyslipidemia and to evaluate the effects of emerging therapies targeting these disorders. Our data confirm the preventing effects of LNP599 in a nonhuman primate model and demonstrate for the first time the high potency of this drug in promoting HDL-cholesterol.

The sympathetic nervous system (SNS) plays a fundamental role in the pathophysiology of cardiovascular diseases, including primary arterial hypertension. In this study, we aimed to investigate whether the expression of the rate-limiting enzyme in catecholamine synthesis, tyrosine hydroxylase (TH), and the β2-adrenergic receptor (β2-AR) in immune cells from peripheral blood, reflect central SNS activity in spontaneously hypertensive rats (SHR). TH expression in the lower brainstem and adrenal glands and β2-AR expression in the lower brainstem were analyzed by western blot analyses. In the leukocytes, TH and β2-AR expression was evaluated by flow cytometry before and after chronic treatment with the centrally-acting sympathoinhibitory drug clonidine. Western blot analyses showed increased TH and β2-AR expression in the lower brainstem and increased TH in adrenal glands from SHR compared to normotensive Wistar Kyoto rats (WKY). Lower brainstem from SHR treated with clonidine presented reduced TH and β2-AR levels, and adrenal glands had decreased TH expression compared to SHR treated with vehicle. Flow cytometry showed that the percentage of leukocytes that express β2-AR is higher in SHR than in WKY. However, the percentage of leukocytes that expressed TH was higher in WKY than in SHR. Moreover, chronic treatment with clonidine normalized the levels of TH and β2-AR in leukocytes from SHR to similar levels of those of WKY. Our study demonstrated that the percentage of leukocytes expressing TH and β2-AR was altered in arterial hypertension and can be modulated by central sympathetic inhibition with clonidine treatment.

Background/objectives: We previously observed that selective agonists of the sympatho-inhibitory I1 imidazoline receptors (LNP ligands) have favorable effects on several cardiovascular and metabolic disorders defining the metabolic syndrome, including body weight. The objectives of this study were to explore the effects of LNPs on adiposity and the mechanisms involved, and to evaluate their impact on metabolic homeostasis. Methods: Young Zucker fa/fa rats were treated with LNP599 (10 mg/kg/day) for 12 weeks. Effects on body weight, adiposity (regional re-distribution, morphology, and function of adipose tissues), cardiovascular and metabolic homeostasis, and liver function were evaluated. Direct effects on insulin and AMP-activated protein kinase (AMPK) signaling were studied in human hepatoma HepG2 cells. Results: LNP599 treatment limited the age-dependent remodeling and inflammation of subcutaneous, epididymal, and visceral adipose tissues, and prevented total fat deposits and the development of obesity. Body-weight stabilization was not related to reduced food intake but rather to enhanced energy expenditure and thermogenesis. Cardiovascular and metabolic parameters were also improved and were significantly correlated with body weight but not with plasma norepinephrine. Insulin and AMPK signaling were enhanced in hepatic tissues of treated animals, whereas blood markers of hepatic disease and pro-inflammatory cytokine levels were reduced. In cultured HepG2 cells, LNP ligands phosphorylated AMPK and the downstream acetyl-CoA carboxylase and prevented oleic acid-induced intracellular lipid accumulation. They also significantly potentiated insulin-mediated AKT activation and this was independent from AMPK. Conclusions: Selective I1 imidazoline receptor agonists protect against the development of adiposity and obesity, and the associated cardio-metabolic disorders. Activation of I1 receptors in the liver, leading to stimulation of the cellular energy sensor AMPK and insulin sensitization, and in adipose tissues, leading to improvement of morphology and function, are identified as peripheral mechanisms involved in the beneficial actions of these ligands.

Aims Pathophysiology of reflex syncope is not fully understood but a vagal overactivity might be involved in this syncope. Previously, overexpression of muscarinic M2 receptors and acetylcholinesterase was found in particular in the heart and in lymphocytes of rabbits with vagal overactivity as well as in hearts of Sudden Infant Death Syndromes. The aim of this present study was to look at M2 receptor expression in blood of patients with reflex syncope. The second objective was to measure acetylcholinesterase expression in these patients. Methods and results 136 subjects were enrolled. This monocenter study pooled 45 adults exhibiting recurrent reflex syncope compared with 32 healthy adult volunteers (18–50 years) and 38 children exhibiting reflex syncope requiring hospitalization compared with 21 controls (1–17 years). One blood sample was taken from each subject and blood mRNA expression of M2 receptors was assessed by qRT-PCR. Taking into account the non-symmetric distributions of values in both groups, statistical interferences were assessed using bayesian techniques. A M2 receptor overexpression was observed in adult and pediatric patients compared to controls. The medians [q1;q3] were 0.9 [0.3;1.9] in patients versus 0.2 [0.1;1.0] in controls; the probability that M2 receptor expression was higher in patients than in controls (Pr[patients>controls]) was estimated at 0.99. Acetylcholinesterase expression was also increased 0.7 [0.4;1.6] in patients versus 0.4 [0.2;1.1] in controls; the probability that acetylcholinesterase expression was higher in patients than in controls (Pr[patients>controls]) was estimated at 0.97. Both in adults and children, the expression ratio of M2 receptors over acetylcholinesterase was greater in the patient group compared with the control group. Conclusion M2 receptor overexpression has been detected in the blood of both, adults and children, exhibiting reflex syncope. As in our experimental model, i.e. rabbits with vagal overactivity, acetylcholinesterase overexpression was associated with M2 receptor overexpression. For the first time, biological abnormalities are identified in vagal syncope in which only clinical signs are, so far, taken into account for differential diagnosis and therapeutic management. Further work will be needed to validate potential biomarkers of risk or severity associated with the cholinergic system.

Objectives: The objective of this study was to investigate the effects of a new I1-imidazoline receptor-selective pyrroline compound on the hemodynamic, metabolic and microvascular alterations in a high-fat diet (HFD)-induced model of metabolic syndrome in rats. Methods: In total, twenty adult male Wistar rats were fed a high-fat diet (HFD, n = 20) for 20 weeks. Thereafter, the rats received a new pyrroline compound selective for I1-imidazoline receptors (LNP599; 10 mg/kg/day) or vehicle (n = 10/group) orally by gavage for 4 weeks. Functional microcirculation was assessed using intravital video microscopy, and structural microcirculation was evaluated using histochemical analysis. Results: LNP599 induced concomitant reductions in the SBP, HR and plasma catecholamine levels. The animals treated with this new antihypertensive compound also presented an improvement in body weight and the metabolic parameters related to metabolic syndrome, such as the glucose and lipid profiles. These effects were accompanied by a reversal of the functional and structural capillary rarefaction in the skeletal muscle. Conclusions: The modulation of the sympathetic nervous system by a selective agonist for I1-imidazoline receptors improves the hemodynamic and metabolic parameters in an experimental model of metabolic syndrome. LNP599 can also contribute to the restoration of microcirculatory parameters.

Background: We aimed to investigate the effects of chronic oral treatment with centrally acting antihypertensive drugs, such as clonidine (CLO), an α2-adrenoceptor agonist, or LNP599, a selective I1 imidazoline receptor agonist, on brain microvascular function in rats with high-fat diet (HFD)-induced metabolic syndrome. Methods: Male Wistar Kyoto rats were maintained on a normal diet (CON) or a HFD for 20 weeks. After this period, the HFD group received oral CLO (0.1 mg/kg), LNP599 (20 mg/kg), or vehicle daily for 4 weeks. Systolic blood pressure and heart rate (HR) were evaluated by photoplethysmography. Functional capillary density, endothelial function, and endothelial-leukocyte interactions in the brain were investigated by intravital video microscopy. Cerebral microcirculatory flow was evaluated by laser speckle contrast imaging. Brain tissue endothelial nitric oxide synthase, oxidative enzyme, and inflammatory marker expression levels were analyzed. Results: Metabolic syndrome decreased brain functional capillary density and microvascular blood perfusion, changes accompanied by deficient brain microcirculation vasodilatory responses to acetylcholine. Significant numbers of rolling and adherent leukocytes were also observed in the brain venules. Chronic sympathetic inhibition with clonidine and LNP599 reduced blood pressure and HR. These effects were accompanied by reversals of cerebral capillary rarefaction, improvements in cerebral microvascular blood flow and endothelial function, and decreases in endothelial-leukocyte interactions in the cerebral venules. Conclusions: Our results suggest that central sympathetic inhibition exerts beneficial effects by increasing perfusion and reducing inflammatory marker expression and oxidative stress in the brains of rats with metabolic syndrome. Centrally acting antihypertensive drugs may be helpful in regulating cerebral microcirculatory function and vascular inflammation in metabolic syndrome.

This study investigates the expression of M2 muscarinic receptors’ in the blood samples of infants who experienced apparent life-threatening events.About half of the apparent life-threatening events (ALTEs) that occur among infants remain unexplained and are called idiopathic.1 Yet there is no biomarker associated with idiopathic ALTEs. Although the connection between ALTEs and sudden infant death syndrome (SIDS) remains controversial, the cholinergic system has been investigated in the pathogenesis of both.2- 4 We investigate the M2 muscarinic receptors’ expression in the blood samples of infants who experienced severe ALTEs.