The non-genomic effects of the PPARβγ agonist GW0742 on streptozotocin treated rat aorta

Copyright© Bentham Science Publishers; For any queries, please email at [email protected]: The ubiquitous nuclear receptor PPARβ/δ is increasingly being studied in regards to numerous diseases including diabetes following on the finding that PPARβ/δ agonist GW0742 controls Type 1 Diabetes in rats. Studies have shown that GW0742 has off target, non- PPARβ/δ effects in the cell although there are some key questions that remain to be addressed in respect to the significance of this control on vascular tone. Methods: Using isometric organ baths, rat aorta rings were exposed to ROCK inhibitors and the changes in contraction and dilation measured. Results: Our data shows that the PPARβ/δ agonist GW0742 (10 -7M) inhibits contractile responses to U46619 and phenylephrine, and that these responses are similar in normal and Streptozotocin (STZ) diabetic rat aorta. ROCK inhibitors Fasudil and Y27632 significantly reduced GW0742 mediated dilation of naïve rat aorta, but Fasudil had no effect on GW0742 dilation in STZ diabetic rat aorta. In contrast, STZ diabetic rat aorta pre-contracted with high [K +] Krebs lacked a dilatory response to GW0742, which taken together indicates that the mechanism of action of GW0742 mediated dilation changes in the diabetic state compared to non-diabetic state. Conclusion: This is the first direct evidence demonstrating the non- PPARβ/δ effect of GW0742 on contraction is irrespective to the diabetic state, and that GW0742 has the potential to induce vasodilation via multiple off-target mechanisms.Peer reviewedFinal Accepted Versio

Investigation of effect of hyperinsulinaemia on adipose tissue microvasculature.

Background: Obesity in Qatar is amongst the highest globally and constitutes a serious health risk. Obese individuals are at greater risk of vascular disease compared to the lean, especially in the insulin resistant state. The impact of obesity on endothelial vasomotor function is also adipose tissue depot-dependent, with the visceral environment being more pathogenic. It is, however, unclear how severe the impact of vascular dysfunction is in a relatively young and obese population. Further, it is becoming apparent that obesity-associated vascular dysfunction is heterogeneous. Recent research has focussed on different groups of obese subjects in order to elucidate the mediators of the differential cardiometabolic risk. In pathological obese (PO) subjects, sub-cutaneous adipocyte dysfunction and inflammation have been reported along with adipocyte hypertrophy. This led to the following hypotheses: 1. The enlarged adipocytes are more susceptible to hypoxia due to decreased capillary density of the depot and changes in vascular tone. Hypoxia leads to cell necrosis and the formation of immunological foci. 2. The more hypoxic and inflamed fat depot secretes less adiponectin, which then may determine the increased systemic insulin resistance and dyslipidaemia seen in the PO. Specifically differences between metabolically healthy but obese (MHO) and pathologically obese (PO) in relation to capillary density and vascular function was determined. To facilitate the overall objectives, in this study: 1. A cohort of MHO and PO subjects were identified. 2. Vascular differences in the adipose tissue of PO versus MHO by functional studies (myography) and histological assessment of vascular density were carried out, and, 3. Mechanisms that underlie these differences were investigated. Methods: Patients were recruited from the local hospital and blood and adipose tissue (Omental, OM; Subcutaneous, SC) samples collected. Fasting plasma glucose and insulin were assayed to determine insulin resistance status. Vascular function was assessed by wire myography. Cumulative concentration-response curves were generated for various vasoconstrictors (e.g. noradrenaline, potassium chloride), and vasodilators (e.g. acetylcholine, Sodium nitroprusside (SNP), and prostaglandin E2). Relaxation to acetylcholine was recorded in the absence or presence of Nω-Nitro-L¬arginine methyl ester (L-NAME), indomethacin, diclofenac, BaCl2, apamin+charybdotoxin, and arginine. mRNA expression of hypertension associated genes in stromal vascular fractions (SVFs) of both depots was assessed by real time RT-PCR. Paraffin-embedded tissues were used for histological studies. Results: OM arterioles were less sensitive to noradrenaline-mediated vasoconstriction compared with SC (log EC50 -5.9±0.2 vs. -6.5±0.1, p<0.05). Vasorelaxation to acetylcholine was attenuated in OM vessels compared with SC vessels (p<0.01). In contrast, relaxation to SNP was greater in OM compared with SC vessels (p<0.01). Acetylcholine curves for insulin-sensitive patients were less attenuated compared with insulin-resistant patients. L-NAME, apamin, charybdotoxin, and BaCl2 caused right-ward shifts of the acetylcholine curves, while indomethacin, diclofenac and arginine produced the reverse. In the whole group, COX2 mRNA, but not eNOS and COX1, were up regulated in OM compared with SC SVFs. However, when analyzed separately, in the OM compared to SC SVFs, of the MHO several genes were unregulated (AGT, ARG2, CLIC5, EPHX2, ITPR1 and PRKG1), while in the PO only two were significantly different between the depots (CLIC5 and PDE3B). Conclusions: Hyperinsulinaemia in adipose tissue microvessels was associated with і) vasocontractile insensitivity to noradrenaline and іі) to changes in NO-mediated vasodilation, at least partially mediated through components of the COX2 pathway

Modulation of the vascular function by cannabinoids, calcitonin gene related peptide and connexin hemichannels

Blood vessel diameter and blood flow are tightly controlled by electrical signals and calcium ion (Ca2+) signals in smooth muscle cells (SMCs) and endothelial cells (ECs) in which selective and non-selective ion channels play a crucial role. In this doctoral thesis we characterized the targets of cannabinoid- and calcitonin gene related peptide (CGRP)-triggered vasodilation and the role of connexin channels in the modulation of blood vessel tone. Additionally, we investigated connexin channels as a putative target to protect blood vessels against cell death associated with stressful conditions like cryopreservation. Blood vessel tonus can be modulated by several factors, such as endothelial factors or factors released from perivascular nerves, thereby promoting vasodilation or vasoconstriction. Cannabinoids have been used for centuries for their psychoactive properties, but they have also a profound influence on the cardiovascular system, leading to vasodilation. Vasorelaxation by cannabinoids can be mediated by stimulation of cannabinoid receptors. Others pointed a role for vanilloid receptors on perivascular nerves and a subsequent release of the neuropeptide CGRP. This peptide is one of the most potent vasodilatory substances and can act via an endothelium-dependent or -independent process. In order to find out if a direct effect of the cannabinoid methanandamide was present in small mesenteric rat arteries, we performed experiments on acutely isolated SMCs. Our findings show that the cannabinoid methanandamide fails to increase the membrane potassium (K+) currents and fails to hyperpolarize the membrane potential. Moreover, the neuropeptide CGRP is acting specifically and directly on these cells by increasing the large-conductance calcium-dependent potassium (BKCa) channel activity in a receptor-, cyclic adenosine monophosphate- (cAMP), and protein kinase A- (PKA) dependent way and hyperpolarizes the membrane potential of these cells. This is consistent with earlier reports suggesting that methanandamide relaxes and hyperpolarizes intact rat mesenteric arteries by releasing the neuropeptide CGRP from perivascular nerves. Next to electrical changes, vessel diameter and blood flow are also controlled by Ca2+ signals and communicative pathways between vascular cells in which gap junctions (GJs) play a crucial role. GJs are dodecameric channels composed of connexin proteins that directly connect the cytoplasm of cells. These intercellular channels are composed of two hexameric 10 hemichannels (HCs) that may also be junctional as non-junctional HCs. Non-junctional (unapposed) HCs are present in the plasma membrane normally closed but may open by various messengers and conditions thereby forming a pore that allows passage of ions and messengers like ATP or other substances with a molecular weight below 1.5 kDa. HC opening allows Ca2+ entry into the cells but opening is also controlled by the intracellular (cytoplasmic) Ca2+ concentration ([Ca2+]i). Therefore, these channels are likely to contribute to Ca2+ oscillations which are repetitive [Ca2+]i changes. We investigated the role of HCs in Ca2+ oscillations by interfering with HC function via the application of peptides that are identical to connexin sequences on the connexin protein. We found that in small mesenteric artery fragments isolated from rat, norepinephrine-induced Ca2+ oscillations in SMCs were inhibited by the connexin mimetic peptide Gap27, by the TAT-L2 peptide and by the TAT-CT9 peptide. Gap27 inhibits HCs first and with some delay also GJs; it targets multiple connexins because it mimics well-conserved extracellular domains of the connexin protein. By contrast, TAT-L2 peptide has specificity at two levels: it only inhibits HCs but not GJs and it has specificity for Cx43 and not for other connexins. The latter is related to the fact that this peptide is identical to a part of the intracellularly located non-conserved domain. TAT-CT9 also has specificity for Cx43 but this peptide prevents the closure of HCs at high micromolar [Ca2+]i. In addition to allowing Ca2+ entry, open HCs can also allow the escape of ATP from the cell. We found that interfering with purinergic signaling, by promoting the degradation of ATP or by inhibiting purinergic receptors, norepinephrine-induced Ca2+ oscillations were also inhibited. Taken together, these findings suggest that ATP released by SMCs via connexin hemichannels (CxHCs) with subsequent activation of purinergic communication is involved in controlling vessel responses to norepinephrine. Tension measurements further confirmed the role of CxHCs in vascular smooth muscle tension since the contractility was also inhibited by Gap27, by TAT-L2 and by purinergic receptor antagonists. These data indicate that interfering with CxHCs offers a novel approach towards modulating blood vessel reactivity. GJs and CxHCs are important for vascular function, but they also play a role in cell death processes. GJs allow the passage of cell death messengers thereby contributing to the propagation of cell death known as bystander cell death. Unapposed CxHCs may promote cell death by uncontrolled opening and thereby contribute to the entrance or escape from 11 the cell of ions and small metabolites. Inhibiting connexin channels may thus lead to improved cell viability after exposure to stressful conditions like exposure to freezing conditions in the context of cryopreservation of cells and tissues. Cryopreserved blood vessels are being increasingly employed as grafts in vascular reconstruction procedures. However, the massive cell death provoked by the methods of cryopreservation used at most vascular banks is one of the main factors leading to the failure of grafting procedures performed using cryopreserved vascular allografts. We investigated whether blocking connexin channels could prevent cell death after cryopreservation. We report that Gap27 significantly reduced cell death in human femoral arteries and saphenous veins when present during cryopreservation/thawing. Veins had a better conserved intimal layer compared to arteries and had less endothelial cell death. This implies that veins are better suited for vessel replacement in patients with vascular diseases and a high risk of thrombosis because of the anti-thrombogenic effect of an intact endothelium. Most notably, blocking GJs and HCs with connexin mimetic peptides like Gap27 appears to be very efficient to prevent cell death in complex tissues like blood vessels undergoing cryopreservation. It is important to note here that blocking GJs and HCs is (in most cases) are not indicated in vivo because GJs are necessary for normal physiological function. However, inhibition of GJs and HCs is perfectly possible in tissues and organs isolated ex vivo for subsequent cold or cryo storage. Such procedure may well be indicated in the context of heart, lung and kidney transplantation and cryopreservation/vitrification of various cells and tissues. In conclusion, this work has improved our understanding of the vascular action of cannabinoids and CGRP, and has brought up connexins and their channels as interesting new targets to modulate blood vessel function and to protect vascular cell viability after cryopreservation

Molecular genetic studies in pregnancies affected by preeclampsia and intrauterine growth restriction

Preeclampsia and fetal growth restriction (FGR) are common and costly obstetric complications. Both conditions are associated with immediate and remote mortality and morbidity for the mother and the offspring. Impaired placentation and aberrant maternal systemic responses are implicated as pathophysiological mechanisms in preeclampsia and FGR. Both preeclampsia and FGR are known to have a clear genetic basis. This study has investigated the roles of several candidate genes including those previously associated with diabetes (TCF7L2, FTO, PPAR-g, CDKN2B-AS1 and KCNJ11), and epidermal growth factor (EGF). Functional consequences of variants within the EGF gene were also investigated. A bidirectional association between type 2 diabetes (T2D) and preeclampsia is consistently reported, whereby each condition is associated with an increased risk of the other. Furthermore, fetal growth restriction, which complicates 30% of preeclamptic pregnancies, predisposes the offspring to an increased risk of type 2 diabetes and coronary artery disease (CAD) later in life. 11 single nucleotide polymorphisms (SNPs) reproducibly associated with T2D in the TCF7L2, FTO, PPAR-y, CDKN2B-AS1 and KCNJ11 genes were investigated as susceptibility loci for preeclampsia and fetal growth restriction in a maternal case control study. The study group consisted of 448 white western European women with preeclampsia, 673 controls with no evidence of preeclampsia, 243 women with pregnancies complicated by FGR, and 570 controls with no evidence of growth restriction. A maternal haplotype on the T2D region of the CDKN2BAS1 gene on chromosome 9p21 was found to be a risk variant for fetal growth restriction (P=O.005).The other 9 investigated SNPs in TCF7L2, FTO, PPAR-y, and KCNJ11 showed no association with growth restricted pregnancies. None of the SNPs investigated showed an association with preeclampsia. These findings suggest that some maternal diabetogenic risk variants are associated with an altered risk of FGR pregnancy but not preeclampsia. The results require replication in a larger sample and fetal-maternal gene interactions merit investigation. Epidermal growth factor (EGF) is described as a major regulator of the placentation process. It also helps to maintain an adequate blood supply to the growing fetus through its effects on umbilical vessel tone. Investigating the role of two genetic variants of the EGF gene in susceptibility to preeclampsia and FGR showed that the maternal variants, rs4444903 in the 5'UTR and rs2237051 in exon 14 of the EGF gene has no effect on the risk of preeclampsia or FGR pregnancy. The G allele of the SNP rs4444903 was associated with higher systolic blood pressure measures in the control group. The G allele of the rs4444903 and the A allele of rs2237051 have been associated with increased risk for FGR and lower birth weight in a previous study from our laboratory. This led to investigations to characterize the functional consequences of the two SNPs in the EGF gene on transcription, translation and ribonucleic acid (RNA) splicing using a variety of methods. These experiments have shown that the G allele of rs4444903 was transcriptionally more active than the A allele in hepatocellular carcinoma (HepG2) and more active than EGFP on its own in choriocarcinoma (Jeg-3) cell lines using a luciferase reporter gene assay. There was no effect of this variant on translational efficiency in the cell lines investigated using reporter gene assays, or in a cell free environment using an in vitro translation assay. DNA-protein interaction was investigated using nuclear extract from HepG2 cells to further define the mechanism by which the G allele exerts its higher transcriptional activity. Initial experiments suggest that the Sp1 transcription factor interacts with and represses the A allele of the rs4444903 SNP. The study also demonstrated no evidence of higher activity of the G allele on EGF expression in vivo using term placental tissues. It was expected that higher EGF expression as a function of genotype at rs4444903 SNP may lead to down regulation of the EGFR in the placenta, which was not confirmed in this study. SNP rs2237051 in exon 14 of the EGF gene is in strong linkage disequilibrium with rs4444903, and disrupts a predicted exon splicing enhancer region. This polymorphism was investigated using a minigene assay, but there was no evidence that it affected splicing of exon 14. Taken together, these findings provide no evidence that EGF genetic variants alter the risk of preeclampsia or FGR though functioning

Molecular genetic studies in pregnancies affected by preeclampsia and intrauterine growth restriction

Preeclampsia and fetal growth restriction (FGR) are common and costly obstetric complications. Both conditions are associated with immediate and remote mortality and morbidity for the mother and the offspring. Impaired placentation and aberrant maternal systemic responses are implicated as pathophysiological mechanisms in preeclampsia and FGR. Both preeclampsia and FGR are known to have a clear genetic basis. This study has investigated the roles of several candidate genes including those previously associated with diabetes (TCF7L2, FTO, PPAR-g, CDKN2B-AS1 and KCNJ11), and epidermal growth factor (EGF). Functional consequences of variants within the EGF gene were also investigated. A bidirectional association between type 2 diabetes (T2D) and preeclampsia is consistently reported, whereby each condition is associated with an increased risk of the other. Furthermore, fetal growth restriction, which complicates 30% of preeclamptic pregnancies, predisposes the offspring to an increased risk of type 2 diabetes and coronary artery disease (CAD) later in life. 11 single nucleotide polymorphisms (SNPs) reproducibly associated with T2D in the TCF7L2, FTO, PPAR-y, CDKN2B-AS1 and KCNJ11 genes were investigated as susceptibility loci for preeclampsia and fetal growth restriction in a maternal case control study. The study group consisted of 448 white western European women with preeclampsia, 673 controls with no evidence of preeclampsia, 243 women with pregnancies complicated by FGR, and 570 controls with no evidence of growth restriction. A maternal haplotype on the T2D region of the CDKN2BAS1 gene on chromosome 9p21 was found to be a risk variant for fetal growth restriction (P=O.005).The other 9 investigated SNPs in TCF7L2, FTO, PPAR-y, and KCNJ11 showed no association with growth restricted pregnancies. None of the SNPs investigated showed an association with preeclampsia. These findings suggest that some maternal diabetogenic risk variants are associated with an altered risk of FGR pregnancy but not preeclampsia. The results require replication in a larger sample and fetal-maternal gene interactions merit investigation. Epidermal growth factor (EGF) is described as a major regulator of the placentation process. It also helps to maintain an adequate blood supply to the growing fetus through its effects on umbilical vessel tone. Investigating the role of two genetic variants of the EGF gene in susceptibility to preeclampsia and FGR showed that the maternal variants, rs4444903 in the 5'UTR and rs2237051 in exon 14 of the EGF gene has no effect on the risk of preeclampsia or FGR pregnancy. The G allele of the SNP rs4444903 was associated with higher systolic blood pressure measures in the control group. The G allele of the rs4444903 and the A allele of rs2237051 have been associated with increased risk for FGR and lower birth weight in a previous study from our laboratory. This led to investigations to characterize the functional consequences of the two SNPs in the EGF gene on transcription, translation and ribonucleic acid (RNA) splicing using a variety of methods. These experiments have shown that the G allele of rs4444903 was transcriptionally more active than the A allele in hepatocellular carcinoma (HepG2) and more active than EGFP on its own in choriocarcinoma (Jeg-3) cell lines using a luciferase reporter gene assay. There was no effect of this variant on translational efficiency in the cell lines investigated using reporter gene assays, or in a cell free environment using an in vitro translation assay. DNA-protein interaction was investigated using nuclear extract from HepG2 cells to further define the mechanism by which the G allele exerts its higher transcriptional activity. Initial experiments suggest that the Sp1 transcription factor interacts with and represses the A allele of the rs4444903 SNP. The study also demonstrated no evidence of higher activity of the G allele on EGF expression in vivo using term placental tissues. It was expected that higher EGF expression as a function of genotype at rs4444903 SNP may lead to down regulation of the EGFR in the placenta, which was not confirmed in this study. SNP rs2237051 in exon 14 of the EGF gene is in strong linkage disequilibrium with rs4444903, and disrupts a predicted exon splicing enhancer region. This polymorphism was investigated using a minigene assay, but there was no evidence that it affected splicing of exon 14. Taken together, these findings provide no evidence that EGF genetic variants alter the risk of preeclampsia or FGR though functioning

The role of mitochondria and KATP channels in the vasodilatation response to simvastatin: comparison with the effects of simvastatin in pancreatic β-cells

Clinical trials have established the efficacy and safety of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) in lowering cardiovascular morbidity and mortality in patients with and without coronary artery diseases. Traditionally, the beneficial effects of statins have been ascribed entirely to their ability to lower serum cholesterol. However, evidence indicates that statins may exert cholesterol-independent or pleiotropic effects. As well as reducing plasma cholesterol levels, statins induce acute vasorelaxation which may contribute to the overall benefits of statins in the treatment of cardiovascular disease. The mechanism underlying this relaxation is unknown. Statins have been shown to alter mitochondrial function. Therefore, the aim of this study was to determine the role of the mitochondria in the relaxation to statins. Changes in rhodamine 123 fluorescence showed that simvastatin, but not pravastatin, depolarized the membrane potential of mitochondria in both isolated smooth muscle cells and intact blood vessels. As simvastatin, but not pravastatin, causes relaxation of the porcine coronary artery, this could be due to this effect on mitochondria. Mitochondria are known as the energy generating centre of the cells. However, there is growing consensus that mitochondria actively participate in intracellular signalling, such as production of reactive oxygen species (ROS) and regulation of the intracellular Ca2+ concentration. Moreover, ROS could play an important supportive role in a variety of vascular cell signalling processes, including activation of nitric oxide synthase (NOS), modulation of intracellular Ca2+, and AMP kinase activation. Therefore, this study investigated whether the relaxation to the lipophilic statin simvastatin is due to an effect on the mitochondria. Relaxation of porcine coronary artery segments by statins was measured using isolated tissue baths. Simvastatin, but not pravastatin, produced a slow relaxation of the coronary artery, which was independent of K+ channel activation, nitric oxide, cyclo-oxygenase, or the endothelium. The relaxation was attenuated by the mitochondrial complex I inhibitor rotenone and the complex III inhibitor myxothiazol, or a combination of the two. Simvastatin inhibited calcium-induced contractile responses, and this inhibition was partially reversed by incubation with the complex I inhibitor rotenone suggesting that mitochondrial function is required for the effect of simvastatin on calcium influx. The effect of mitochondrial complex III inhibitor, antimycin A, was examined as a comparison with simvastatin. Antimycin A induced porcine coronary relaxation and inhibited Ca2+ influx in isolated porcine coronary smooth muscle cells. Evidence from a number of clinical trials highlights a potential association between treatment with lipophilic statins and increased risk of development of diabetes. The close connection between energy metabolism and insulin secretion in pancreatic β-cells suggests that the glycaemic effects of simvastatin may also result from a direct mitochondrial action with reduction in insulin secretion and, hence, result in a reduced control of plasma glucose levels. Although simvastatin depolarized mitochondria in pancreatic β-cells, it also directly inhibited KATP channels. Pravastatin, on the other hand, had no effect on either measurement, suggesting that these phenomena relate to the lipophilicity of the compounds. The inhibition of KATP channels by simvastatin is likely to underlie the increase in insulin secretion observed within days of simvastatin treatment. On the other hand, the effects on mitochondrial membrane potential may be detrimental, particularly with chronic treatment, although further studies are required in order to determine whether this plays a role in the increased risk of diabetes observed with lipophilic statins. Overall, our results demonstrated that simvastatin alters mitochondrial membrane potential in vascular smooth muscle cells and pancreatic β-cells. The relaxation to simvastatin in the porcine coronary artery is dependent, in part, upon mitochondrial activity. Alteration of mitochondrial membrane potential by simvastatin may lead to inhibition of calcium influx, hence stimulation of relaxation. On the other hand, the effects on mitochondrial membrane potential in pancreatic β-cell may be detrimental, particularly with chronic treatment due to the increased risk of diabetes observed with lipophilic statins

The role of mitochondria and KATP channels in the vasodilatation response to simvastatin: comparison with the effects of simvastatin in pancreatic β-cells

Clinical trials have established the efficacy and safety of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) in lowering cardiovascular morbidity and mortality in patients with and without coronary artery diseases. Traditionally, the beneficial effects of statins have been ascribed entirely to their ability to lower serum cholesterol. However, evidence indicates that statins may exert cholesterol-independent or pleiotropic effects. As well as reducing plasma cholesterol levels, statins induce acute vasorelaxation which may contribute to the overall benefits of statins in the treatment of cardiovascular disease. The mechanism underlying this relaxation is unknown. Statins have been shown to alter mitochondrial function. Therefore, the aim of this study was to determine the role of the mitochondria in the relaxation to statins. Changes in rhodamine 123 fluorescence showed that simvastatin, but not pravastatin, depolarized the membrane potential of mitochondria in both isolated smooth muscle cells and intact blood vessels. As simvastatin, but not pravastatin, causes relaxation of the porcine coronary artery, this could be due to this effect on mitochondria. Mitochondria are known as the energy generating centre of the cells. However, there is growing consensus that mitochondria actively participate in intracellular signalling, such as production of reactive oxygen species (ROS) and regulation of the intracellular Ca2+ concentration. Moreover, ROS could play an important supportive role in a variety of vascular cell signalling processes, including activation of nitric oxide synthase (NOS), modulation of intracellular Ca2+, and AMP kinase activation. Therefore, this study investigated whether the relaxation to the lipophilic statin simvastatin is due to an effect on the mitochondria. Relaxation of porcine coronary artery segments by statins was measured using isolated tissue baths. Simvastatin, but not pravastatin, produced a slow relaxation of the coronary artery, which was independent of K+ channel activation, nitric oxide, cyclo-oxygenase, or the endothelium. The relaxation was attenuated by the mitochondrial complex I inhibitor rotenone and the complex III inhibitor myxothiazol, or a combination of the two. Simvastatin inhibited calcium-induced contractile responses, and this inhibition was partially reversed by incubation with the complex I inhibitor rotenone suggesting that mitochondrial function is required for the effect of simvastatin on calcium influx. The effect of mitochondrial complex III inhibitor, antimycin A, was examined as a comparison with simvastatin. Antimycin A induced porcine coronary relaxation and inhibited Ca2+ influx in isolated porcine coronary smooth muscle cells. Evidence from a number of clinical trials highlights a potential association between treatment with lipophilic statins and increased risk of development of diabetes. The close connection between energy metabolism and insulin secretion in pancreatic β-cells suggests that the glycaemic effects of simvastatin may also result from a direct mitochondrial action with reduction in insulin secretion and, hence, result in a reduced control of plasma glucose levels. Although simvastatin depolarized mitochondria in pancreatic β-cells, it also directly inhibited KATP channels. Pravastatin, on the other hand, had no effect on either measurement, suggesting that these phenomena relate to the lipophilicity of the compounds. The inhibition of KATP channels by simvastatin is likely to underlie the increase in insulin secretion observed within days of simvastatin treatment. On the other hand, the effects on mitochondrial membrane potential may be detrimental, particularly with chronic treatment, although further studies are required in order to determine whether this plays a role in the increased risk of diabetes observed with lipophilic statins. Overall, our results demonstrated that simvastatin alters mitochondrial membrane potential in vascular smooth muscle cells and pancreatic β-cells. The relaxation to simvastatin in the porcine coronary artery is dependent, in part, upon mitochondrial activity. Alteration of mitochondrial membrane potential by simvastatin may lead to inhibition of calcium influx, hence stimulation of relaxation. On the other hand, the effects on mitochondrial membrane potential in pancreatic β-cell may be detrimental, particularly with chronic treatment due to the increased risk of diabetes observed with lipophilic statins

Antiobesity and antidiabetic activity of P. balsamifera, its active Salicortin, and L. laricina, medicinal plants from the traditional pharmacopoeia of the James Bay Cree

La prévalence de l’obésité, du diabète de type 2, et du syndrome métabolique, sont à la hausse chez les Cris d’Eeyou Istchee (CEI-Nord du Québec). Ces problèmes sont aggravés par leur diète non traditionnelle, leur sédentarité, ainsi que par une résistance culturelle aux produits pharmaceutiques. Afin de développer des traitements antidiabétiques culturellement adaptés, notre équipe a effectué une enquête ethnobotanique qui a identifié 17 plantes provenant de la pharmacopée traditionnelle des CEI. À partir des études de criblage effectuées in vitro, deux plantes parmi les 17 ont attiré notre attention. Populus balsamifera L. (Salicaceae) pour ses propriétés anti-obésité et Larix laricina K. Koch (Pinaceae) pour ses propriétés antidiabétiques. P. balsamifera et son composé actif salicortin ont inhibé l’accumulation de triglycérides durant l’adipogénèse dans les adipocytes 3T3-L1. L. laricina a augmenté le transport de glucose et l’activation de l’AMPK dans les cellules musculaires C2C12, l’adipogénèse dans les 3T3-L1 et a démontré un fort potentiel découpleur (propriété anti-obésité). Les objectifs de cette thèse sont d'évaluer les potentiels anti-obésité et antidiabétique et d’élucider les mécanismes d'action de P. balsamifera, salicortin, et L. laricina chez la souris C57BL/6 rendue obèse par une diète riche en gras (HFD). Les souris ont été soumises pendant huit (étude préventive) ou seize semaines (étude traitement) à une HFD, ou à une HFD dans laquelle P. balsamifera, salicortin, ou L. laricina a été incorporé soit dès le départ (prévention), ou dans les 8 dernières des 16 semaines d'administration de HFD (traitement). iv Les résultats démontrent que P. balsamifera (dans les deux études) et salicortin (évalué dans l’étude traitement) diminuent: le poids corporel, le gras rétropéritonéal, la sévérité de la stéatose et l’accumulation de triglycérides hépatique (ERK impliqué), les niveaux de glycémie et d'insuline, et le ratio leptine/adiponectine. Dans les deux études, P. balsamifera a significativement réduit la consommation de nourriture mais cet effet coupe-faim nécessite d’être approfondi. Dans l'étude préventive, P. balsamifera a augmenté la dépense énergétique (hausse de la température à la surface de la peau et de l’activation de la protéine découplante-1; UCP-1). Les voies de signalisation activées par P. balsamifera et par salicortin (de façon plus modeste) sont impliquées dans: la production de glucose hépatique (Akt), l’expression de Glut4 dans le muscle squelettique, la captation du glucose et du métabolisme des lipides (Akt dans le tissu adipeux), la différenciation des adipocytes (ERK et PPARg), l’inflammation dans le foie (IKKαβ), et l'oxydation des acides gras dans le muscle, le foie, ou le tissu adipeux (PPARa et CPT-1). D’autre part, L. laricina a également diminué les niveaux de glycémie et d’insuline, le ratio leptine/adiponectine, le gras rétropéritonéal et le poids corporel. Ces effets ont été observés en conjonction avec une augmentation de la dépense énergétique: hausse de température à la surface de la peau (prévention) et amélioration de la fonction mitochondriale et de la synthèse d'ATP (traitement). En conclusion, l’utilisation de P. balsamifera, salicortin et L. laricina comme des traitements alternatifs et culturellement adaptés aux CEI représente une contribution importante dans la prévention et le traitement de l’obésité et du diabète.The prevalence of obesity, insulin resistance, and the metabolic syndrome is increasing among the Cree of Eeyou Istchee (CEI - Northern Quebec). Non-traditional diet and sedentary lifestyle along with cultural disconnect of modern type 2 diabetes (T2D) therapies are involved. In order to establish culturally adapted antidiabetic treatments, our research team conducted an ethnobotanical survey, where 17 plants were identified from the CEI traditional pharmacopoeia. Based on data obtained from in vitro screening studies, two plant species out of 17 were of particular interest for their properties as antiobesity, namely Populus balsamifera L. (Salicaceae), and antidiabetic agents, namely Larix laricina K. Koch (Pinaceae). P. balsamifera and its active salicortin inhibited triglyceride accumulation during adipogenesis in 3T3-L1 adipocytes. L. laricina increased glucose uptake and AMPK activation in C2C12 myotubes, adipogenesis in the 3T3-L1 adipocyte cell line, and was observed as one of the strongest uncouplers, severely disrupting mitochondrial function (increasing fuel consumption/metabolic rate; antiobesity property). The purpose of this PhD thesis is to evaluate the antiobesity and antidiabetic potential of P. balsamifera, salicortin, and L. laricina, in an in vivo model of diet-induced obese (DIO) C57BL/6 mice, as well as to investigate their possible mechanisms of action. Mice were subjected for eight (prevention study) or sixteen weeks (treatment study) to a high fat diet (HFD), or HFD to which P. balsamifera, salicortin, or L. laricina were incorporated either at onset (prevention), or in the last 8 of the 16 weeks of administration of the HFD (treatment). The results showed that P. balsamifera (in either study) and salicortin (incorporated in HFD only in treatment study) decreased the weight of whole vii body, retroperitoneal fat pad, reduced the severity of hepatic macrovesicular steatosis and triglyceride accumulation (ERK pathway implicated). They also decreased glycemia and improved insulin sensitivity by diminishing insulin levels, and altering adipokine secretion whereby reducing the leptin/adiponectin ratio. In both studies, P. balsamifera significantly reduced food intake. This appetite-reducing effect needs to be investigated further. In the prevention study this was accompanied by an increase in energy expenditure (increase in skin temperature and tends to increase expression of uncoupling protein-1; UCP-1). The signaling pathways activated by P. balsamifera and slightly by salicortin are implicated in either controlling hepatic glucose output (Akt), skeletal muscle Glut4 expression, glucose uptake and lipid metabolism in adipose tissue (Akt), adipocyte differentiation (ERK pathway and PPARg), decreasing the hepatic inflammatory state (IKKab), and increasing muscular, hepatic, or adipose tissue fatty acid oxidation (PPARa, CPT-1). As for L. laricina, it effectively decreased glycemia levels, insulin levels and the leptin/adiponectin ratio, improved insulin sensitivity and slightly decreased abdominal fat pad and body weights. This occurred in conjunction with increased energy expenditure as demonstrated by elevated skin temperature in the prevention study, and tendency to improve mitochondrial function and ATP synthesis in the treatment protocol. In conclusion, these results represent a major contribution, identifying P. balsamifera, salicortin, and L. laricina, as promising alternative, and culturally adapted therapies for the prevention and treatment care of obesity and diabetes among the CEI

Smooth muscle cell proliferation and endothelin pathway in pulmonary arterial hypertension: Comparative effects of current and putative therapeutic agents.

Pulmonary arterial hypertension (PAH) is characterised by a progressive increase in pulmonary vascular resistance (PVR) leading to right ventricular failure or a premature death. Its prevalence varies from 15-50 patients per million populations with an average age of 45 years. Prostacyclin (PGI2) analogues are used to treat PAH, but do not cure the condition. PGI2 binds to prostacyclin receptors (IP) in the cell membrane stimulating adenylate cyclase to increase intracellular cyclic 3’5’-adenosine monophosphate (cAMP). Once elevated, cAMP is rapidly broken down by phosphodiesterases (PDEs), specifically 1, 3, 4 which appear responsible for regulating levels in the lungs. One of the reasons for the ineffectiveness of PGI2 in PAH could be high activity of these PDE isoforms that specifically break down cAMP. So in chapter 3, I sought to evaluate the effect of prostacyclin analogues (e.g. treprostinil) on cAMP and cell proliferation in PAH. I was also interested to check whether phosphodiesterase inhibitors could potentiate the effects of PGI2 analogues. Elevated levels of endothelial ET-1 in idiopathic PAH have been shown to be correlated with increased right atrial pressure, pulmonary artery oxygen saturation and pulmonary vascular resistance. This has led to the use of ET-1 antagonists to treat PAH alone or in combination with other classical drugs. In chapter 4, I investigated the interaction between treprostinil, and endothelin antagonists on the ET-1 levels and growth characteristics of pulmonary smooth muscle cell cells (PASMCs) derived from IPAH patients. Vascular remodelling has been considered a pseudo-malignant disorder and mediators from cancer research have been described as targets for therapeutic interventions for PAH (Paulin et al., 2011). In chapter 5 I evaluated the antiproliferative effects of an already established anti-cancer drug ispinesib (Purcell et al., 2010) and thus established a rationale for investigating this agent in PAH settings