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The role of peroxisome proliferator-activated receptors in the rostral ventrolateral medulla in blood pressure lowering effect of rosiglitazone in spontaneously hypertensive ratKung, Sui-sum 28 August 2008 (has links)
Background: The rostral ventrolateral medulla (RVLM), location of the
sympathetic premotor neurons, plays a pivotal role in central cardiovascular
regulation. The peroxisome proliferator-activated receptors-£^ (PPAR£^) agonist
is commonly prescribed for the treatment of type II diabetes mellitus by its
insulin sensitizing ability. Intriguingly, both animal and human studies
revealed that PPAR£^ agonist also possesses blood pressure lowering effect
although the underlying mechanism is unknown. We designed a study to
evaluate the hypothesis that activation of PPAR£^ in the RVLM mediates the
blood pressure lowering effect of PPAR£^ agonist, rosiglitazone.
Materials and Methods: The 12-week spontaneously hypertensive rats (SHR)
and the age-matched normotensive Wistar Kyoto (WKY) rats were used in
this study. Basal systemic arterial pressure (SAP) and heart rate (HR) were
measured for one week, followed by oral administration of a synthetic PPAR£^
agonist, rosiglitazone (80 mg/kg/day), or saline for 7 days. The hemodynamic
profile was recorded for 4 weeks post treatment. The role of PPAR£^ in the
RVLM on blood pressure lowering effect of rosiglitazone was examined by
microinjection bilaterally into the RVLM of the PPAR£^ antagonist, GW9662
(5 nmol). In a separated series of experiments, the RVLM of SHR or WKY
rats was removed at the end of rosiglitazone or saline treatment. Protein
expression of PPAR£\, PPAR£]/£_ or PPAR£^ in the RVLM was analyzed by
Western blotting. To ascertain that changes in protein expression are not
secondary to perturbation of SAP, expression of PPARs was also examined inSHR that received oral administration of a calcium channel inhibitor,
amlodipine (16 mg/kg/day), for 7 days.
Results: Compared to saline intake, rosiglitazone significantly lowered the
mean SBP (MSBP, 159.2¡Ó9.9 mmHg vs. 139.8¡Ó12.6 mmHg) in SHR, but not
WKY rats. This blood pressure lowering effect of rosiglitazone in SHR lasted
for at least 10 days post treatment. Rosiglitazone treatment, on the other hand,
had no significant effect on HR in SHR or WKY rats. At the end of 7-day
treatment, microinjection bilaterally into the RVLM of PPAR£^ antagonist,
GW9662 (5 nmol), significantly reversed the blood pressure lowering effect of
rosiglitazone in SHR. In addition, protein expression of PPAR£\ or PPAR£^
was significantly upregulated in the RVLM of the SHR but not WKY rats that
received rosiglitazone treatment. Oral intake of amlodipine (16 mg/kg/day) for
7 days in SHR significantly lowered MSBP (164.8¡Ó7.7 mmHg to 131.8¡Ó7.8
mmHg), but did not affect protein expression of PPAR£\, PPAR£]/£_ or PPAR£^
in the RVLM of SHR.
Conclusion: These results suggest that oral administration of rosiglitazone
exerts blood pressure lowering effect via activation of PPARs in the RVLM of
SHR. Moreover, upregulation of PPAR£\ or PPAR£^ in the RVLM may
underlie the antihypertensive effect of rosiglitazone.
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Molecular Mechanism of PPAR in the Regulation of Age-Related InflammationChung, Jae, Seo, Arnold Y., Chung, Sang Woon, Kim, Mi Kyung, Leeuwenburgh, Christiaan, Yu, Byung Pal, Chung, Hae Young 01 April 2008 (has links)
Evidence from many recent studies has linked uncontrolled inflammatory processes to aging and aging-related diseases. Decreased a nuclear receptor subfamily of transcription factors, peroxisome proliferator-activated receptors (PPARs) activity is closely associated with increased levels of inflammatory mediators during the aging process. The anti-inflammatory action of PPARs is substantiated by both in vitro and in vivo studies that signify the importance of PPARs as major players in the pathogenesis of many inflammatory diseases. In this review, we highlight the molecular mechanisms and roles of PPARα, γ in regulation of age-related inflammation. By understanding these current findings of PPARs, we open up the possibility of developing new therapeutic agents that modulate these nuclear receptors to control various inflammatory diseases such as atherosclerosis, vascular diseases, Alzheimer's disease, and cancer.
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ALTERATIONS OF MITOCHONDRIAL BIOGENESIS AND ALTERATIONS OF MITOCHONDRIAL ANTIOXIDANT DEFENSE IN FRIEDREICH’S ATAXIAMarmolino, Daniele 25 January 2011 (has links)
Friedreich’s ataxia (FRDA) is an autosomal recessive inherited disorder affecting approximately 1 every 40,000 individuals in Western Europe, is characterized by progressive gait and limb ataxia, dysarthria, areflexia, loss of vibratory and position sense, and a progressive weakness of central origin. Additional features particularly include an hypertrophic cardiomyopathy that can cause premature death. A large GAA repeat expansion in the first intron of the FXN gene is the most common mutation underlying FRDA. Patients show severely reduced levels of the FXN-encoded mitochondrial protein frataxin.
Frataxin function is not yet completely elucidated. In frataxin deficiency conditions abnormalities of iron metabolism occur: decreased activities of iron-sulfur cluster (ISC) containing proteins, accumulation of iron in mitochondria and depletion in the cytosol, enhanced cellular iron uptake, and, in some models, reduced heme synthesis.
Evidence of oxidative stress has also been found in most though not all models of frataxin deficiency. Accordingly, yfh1-deficient yeast and cells from FRDA patients are highly sensitive to oxidants. Respiratory chain dysfunction further aggravate oxidative stress by increasing leakage of electrons and the formation of superoxide. Frataxin deficient cells not only generate more free radicals, but, they also show a reduced ability to mobilize antioxidant defenses, in particular to induce superoxide dismutase 2 (SOD2).
Peroxisome proliferator-activated receptor (PPAR) isoform-gamma play a key role in numerous cellular functions and is a key regulator of mitochondrial biogenesis and of the ROS metabolism. Recruitment of the PPAR coactivator-1a (PGC-1a) mediates many effects of the PPAR-γ activation.
In a first work we assessed the potential beneficial effects of a potent PPAR-gamma agonist on frataxin expression in primary fibroblasts from healthy controls and FRDA patients, and Neuroblastoma cells. We used the APAF molecule (1-0-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocoline; C33H66NO9P). Our results show that this compound is able to increase frataxin amount both at transcriptional and post-transcriptional level. At a dose of 20µM frataxin mRNA significantly increases in both controls (p=0.03) and FRDA patients (p=0.002) fibroblasts (1). The finding was confirmed in Neuroblastoma cells (p=0.042). According to previous publications APAF, as others PPAR-gamma agonists is able to up-regulate PGC-1a transcription.
In a second part of the study we investigate the role of the PPAR-gamma/PGC-1a pathway in the pathogenesis of FRDA. We performed a microarray analysis of heart and skeletal muscle in a mouse model of frataxin deficiency and we found molecular evidence of increased lipogenesis in skeletal muscle and alteration of fiber-type composition in heart, consistent with insulin resistance and cardiomyopathy, respectively. Since the PPAR-gamma pathway is known to regulate both processes, we hypothesized that dysregulation of this pathway could play a key role in frataxin deficiency. We confirmed this by showing a coordinate dysregulation of Pgc1a and the transcription factor Srebp1 in cellular and animal models of frataxin deficiency, and in cells from FRDA patients, who have marked insulin resistance. Particularly, PGC-1a was found significantly reduced (2) in primary fibroblasts and lymphocytes from FRDA patients (p<0.05). Furthermore, PGC-1a mRNA levels strongly correlate with frataxin relative mRNA levels (r2=0.9, p<0.001). According to this observation, in C2C12 myoblasts, PGC-1a and a reporter gene under the control of the PGC-1a promoter are rapidly down-regulated (p<0.05) when frataxin expression is inhibited by an shRNA in vitro. To further investigate this relation, we then generate PGC-1a deficient fibroblasts cells using a specific siRNA; at 72 hours of transfection frataxin was found down-regulate (p<0.05) in control cells.
Taken together those data indicate that some mechanism directly links an early effect of frataxin deficiency with reduced PGC-1a transcription in this cell type, and presumably in other cells that also down-regulate PGC-1α when frataxin levels are low.
Finally, since PGC-1a has also emerged as a key factor in the induction of many antioxidant programs in response to oxidative stress, both in vivo and in vitro, in particular in neurons, we tested whether the PGC-1a down-regulation occurring in FRDA cells could be in part responsible for the blunted antioxidant response observed in frataxin deficiency.
Using primary fibroblasts from FRDA patients we found reduced SOD2 levels (p<0.05), according to PGC1 and frataxin reduced levels. Our finding confirm previous publications showing that PGC-1a directly regulate SOD2 levels in vitro and in vivo. We then tested the response to oxidative stress induced by the addition of hydrogen peroxide (H2O2) at different time and doses. Our data show that H2O2 directly increase PGC-1a and SOD2 levels (p<0.01 and p<0.05) in control cells; no effect was observed in FRDA cells, suggesting a lack in the activation of this response. Moreover, PGC-1α direct silencing, using a specific siRNA, in control fibroblasts led to a similar loss of SOD2 response (p<0.001) to oxidative stress as observed in FRDA fibroblasts, confirming its crucial role in this response (3). We then measured the same parameters after pharmacological manipulations of PGC-1a. PGC-1a activation with the PPAR agonist (Pioglitazone) or with a cAMP-dependent protein kinase (AMPK) agonist (AICAR) restored normal SOD2 induction (4) in FRDA cells (p<0.01). In vivo treatment of the KIKO mice (35-40% of wiled-type frataxin) with Pioglitazone significantly up-regulate SOD2 (5) in cerebellum (p<0.01) and spinal cord (p<0.05), two primary affected tissues in patients.
The search for experimental drugs increasing the amount of frataxin is a very active and timely area of investigation. In cellular and in animal model systems, the replacement of frataxin function seems to alleviate the symptoms or completely reverts the phenotype. Therefore, drugs that are able to increase directly the amount of frataxin, at least up to the level of an asymptomatic carrier, are attractive candidates for new approaches to the therapy of FRDA. Our findings show (1) that a potent PPAR-gamma agonists can increase frataxin expression. We do also show a regulatory loop between frataxin and PGC-1a. Thus, we suggest that this loop could play a critical role in the pathogenesis of the disease and breaking this loop could help to slow down the pathological phenotype observed in FRDA patients. Particularly, PGC-1α down-regulation (3) is likely to contribute to the blunted antioxidant response observed in cells from FRDA patients. This response can be restored by AMPK and PPAR agonists in vitro (4) and in vivo, as shown by Pioglitazone treatment (5) in a mouse model for the disease. To conclude, our study provide evidences that PPAR-gamma agonists are a potential treatment for Friedreich’s ataxia, consisting with their action on both mitochondrial biogenesis and oxidative stress defenses.
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Propriedades antiaterogênicas de novas tiazolidino-2,4-dionas / Antiatherogenic properties of new thiazolidin-2,4-dionesCésar, Fernanda Andrade de 20 March 2013 (has links)
Tiazolidinadionas (TZDs) são agentes sensibilizadores de insulina que agem por ligação ao receptor gama ativado por proliferador de peroxissomos (PPARγ). Elas têm apresentado efeitos cardioprotetores em humanos e propriedades anti-aterogênicas em modelos animais. Estudos in vitro têm sugerido que esses efeitos anti-aterogênicos da ativação de PPARγ ocorrem por inibição da expressão de genes pro-inflamatórios e por aumentar o efluxo de colesterol via ativação dos receptores LXR-ABCA1. Entretanto, vários efeitos colaterais são associados ao tratamento com as TZDs, tornando necessária a pesquisa por novos compostos desta classe. Neste estudo, 14 novas tiazolidina-2,4- dionas, que são TZDs modificadas por bioisosterismo, foram avaliadas quanto à expressão de fatores aterogênicos e inflamatórios em linhagens de macrófagos J774 e RAW 264.7 e em camundongos com deleção genética para o receptor de LDL (LDLr-/-). Após a avaliação da citotoxicidade em macrófagos, foram eleitas cinco TZDs, denominadas de GQ-11, GQ-97, GQ-177, GQ-145 e LYSO-7. Três destas TZDs (GQ- 145, GQ-177 e LYSO-7) aumentaram significativamente a expressão de RNAm dos fatores de transcrição PPARγ1, PPARγ2 e do receptor CD36, assim como também aumentaram a expressão gênica de ABCA1 em 2.9, 3.5 e 6.7 vezes, respectivamente. Em adição, estas TZDs diminuíram a expressão gênica de iNOS, COX2, VCAM e IL-6 associado a redução na produção de nitritos, mas apenas a LYSO-7 reduziu significativamente a expressão desses genes quando comparada à rosiglitazona (RSG), além de diminuir a expressão da proteína-1 quimiotática para monócitos (MCP-1). No estudo experimental, os camundongos LDLr-/- machos foram alimentados com dieta hipercolesterolêmica por 16 semanas e quatro semanas antes da eutanásia receberam os derivados tiazolidínicos (20 mg/kg/dia) por gavagem. GQ-177 inibiu a progressão da placa aterosclerótica associada à aumento nas concentrações plasmáticas de HDL-C, com elevação na expressão de ABCA1, e redução da via inflamatória CD40-CD40L. LYSO-7 também mostrou inibição da aterogênese associada à redução das concentrações plasmáticas de colesterol total e triacilgliceróis, com diminuição na interação entre CD40-CD40L e expressão de citocinas inflamatórias. A GQ-145 não alterou os níveis plasmáticos dos lipídeos, mas aumentou a expressão de todos os genes pró-aterogênicos e pró-inflamatórios. Adicionalmente, as vias de ativação destas novas TZDs também foram estudadas por ensaio de luciferase, como gene repórter. A GQ-177 induziu ativação de PPARγ e ligação ao seu domínio, enquanto a LYSO-7 estimulou ativação de PPARα e PPARδ. Portanto, conclui-se que as novas TZDs, especialmente a GQ-177 e a LYSO-7, podem apresentar propriedades ateroprotetoras associadas ao transporte reverso de colesterol e aos efeitos antiinflamatórios, e poderiam ser uma alternativa promissora para o tratamento da aterosclerose. Porém, estudos complementares são requeridos para caracterizar as vias de sinalização intracelular, visto que as duas demonstraram ativar diferentes isotipos do fator de transcrição PPAR. / Thiazolidinediones (TZDs) are insulin-sensitizing agents that act by binding to peroxisome proliferator-activated receptor-γ (PPARγ). They have been demonstrated to possess cardioprotective effects in humans and antiatherogenic properties in animal models. In vitro studies have also suggested that these antiatherogenic effects of PPARγ activation occur by inhibiting the inflammatory gene expression and by increasing cholesterol efflux via LXR-ABCA1 activation. However, several side effects are associated with TZDs treatment making necessary the search for new compounds. In this study, 14 new thiazolidine-2,4-diones, modified TZDs by bioisosterism, were tested for aterogenic and inflammtary factors in RAW 264.7 macrophages and in low-density lipoprotein receptor-deficient mice. After the citotoxicity evaluation in RAW 264.7 macrophages the TZDs named GQ-11, GQ-97, GQ-177, GQ-145 e LYSO-7 were selected for this study. Three of these TZDs (GQ-177, GQ-145 and LYSO-7) significantly increased the expression of PPARγ1, PPARγ2 and CD36 mRNA, and enhanced the expression of ABCA1 mRNA in 2.9, 3.5 and 6.7 fold, respectively. Moreover, they also significantly decreased the expression of iNOS, COX2, VCAM and IL-6 mRNA in relation to control, and these results are associated to reduction on nitrits concentration. In addition, LYSO-7 significantly reduced the expression of these genes when compared to rosiglitazone, and decreased expression of MCP1 mRNA. In the experimental study, male LDLr-/- mice were fed an atherogenic diet containing 0.5% cholesterol for 16 weeks, and 4 weeks before euthanasia they received TZDs (20mg/kg/ per day) by gavage. GQ-177 treatment inhibited progression of atherosclerotic plaque associated to increased plasma concentrations of HDL-C, with enhance of ABCA1 expression and reduction on CD40-CD40L interaction. LYSO-7 treatment also showed inhibition of the atherogenesis associated to decreased plasma concentrations of total cholesterol and TAG, with reduction on CD40-CD40L pathway and inflammatory cytokines expression.GQ-145 did not alter the lipid plasma levels and increased the expression of all pro-atherogenic and pro-inflammatory genes. Furthermore, the activation of PPARs has also been studied, by luciferase assay as reporter gene. GQ-177 induced activation of PPARγ, whereas LYSO-7 stimulated activation of PPARα and PPARβ/δ. Altogether, our data suggest that the new TZDs derivatives, specially GQ- 177 and LYSO-7, may have atheroprotective properties associated with the reverse cholesterol transport and anti-inflammatory effects, and could be a promising alternative for the treatment of atherosclerosis. However, further studies are warranted in order to characterize the pathways of intracellular signaling since both have demonstrated to activate different isotypes of PPAR.
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Propriedades antiaterogênicas de novas tiazolidino-2,4-dionas / Antiatherogenic properties of new thiazolidin-2,4-dionesFernanda Andrade de César 20 March 2013 (has links)
Tiazolidinadionas (TZDs) são agentes sensibilizadores de insulina que agem por ligação ao receptor gama ativado por proliferador de peroxissomos (PPARγ). Elas têm apresentado efeitos cardioprotetores em humanos e propriedades anti-aterogênicas em modelos animais. Estudos in vitro têm sugerido que esses efeitos anti-aterogênicos da ativação de PPARγ ocorrem por inibição da expressão de genes pro-inflamatórios e por aumentar o efluxo de colesterol via ativação dos receptores LXR-ABCA1. Entretanto, vários efeitos colaterais são associados ao tratamento com as TZDs, tornando necessária a pesquisa por novos compostos desta classe. Neste estudo, 14 novas tiazolidina-2,4- dionas, que são TZDs modificadas por bioisosterismo, foram avaliadas quanto à expressão de fatores aterogênicos e inflamatórios em linhagens de macrófagos J774 e RAW 264.7 e em camundongos com deleção genética para o receptor de LDL (LDLr-/-). Após a avaliação da citotoxicidade em macrófagos, foram eleitas cinco TZDs, denominadas de GQ-11, GQ-97, GQ-177, GQ-145 e LYSO-7. Três destas TZDs (GQ- 145, GQ-177 e LYSO-7) aumentaram significativamente a expressão de RNAm dos fatores de transcrição PPARγ1, PPARγ2 e do receptor CD36, assim como também aumentaram a expressão gênica de ABCA1 em 2.9, 3.5 e 6.7 vezes, respectivamente. Em adição, estas TZDs diminuíram a expressão gênica de iNOS, COX2, VCAM e IL-6 associado a redução na produção de nitritos, mas apenas a LYSO-7 reduziu significativamente a expressão desses genes quando comparada à rosiglitazona (RSG), além de diminuir a expressão da proteína-1 quimiotática para monócitos (MCP-1). No estudo experimental, os camundongos LDLr-/- machos foram alimentados com dieta hipercolesterolêmica por 16 semanas e quatro semanas antes da eutanásia receberam os derivados tiazolidínicos (20 mg/kg/dia) por gavagem. GQ-177 inibiu a progressão da placa aterosclerótica associada à aumento nas concentrações plasmáticas de HDL-C, com elevação na expressão de ABCA1, e redução da via inflamatória CD40-CD40L. LYSO-7 também mostrou inibição da aterogênese associada à redução das concentrações plasmáticas de colesterol total e triacilgliceróis, com diminuição na interação entre CD40-CD40L e expressão de citocinas inflamatórias. A GQ-145 não alterou os níveis plasmáticos dos lipídeos, mas aumentou a expressão de todos os genes pró-aterogênicos e pró-inflamatórios. Adicionalmente, as vias de ativação destas novas TZDs também foram estudadas por ensaio de luciferase, como gene repórter. A GQ-177 induziu ativação de PPARγ e ligação ao seu domínio, enquanto a LYSO-7 estimulou ativação de PPARα e PPARδ. Portanto, conclui-se que as novas TZDs, especialmente a GQ-177 e a LYSO-7, podem apresentar propriedades ateroprotetoras associadas ao transporte reverso de colesterol e aos efeitos antiinflamatórios, e poderiam ser uma alternativa promissora para o tratamento da aterosclerose. Porém, estudos complementares são requeridos para caracterizar as vias de sinalização intracelular, visto que as duas demonstraram ativar diferentes isotipos do fator de transcrição PPAR. / Thiazolidinediones (TZDs) are insulin-sensitizing agents that act by binding to peroxisome proliferator-activated receptor-γ (PPARγ). They have been demonstrated to possess cardioprotective effects in humans and antiatherogenic properties in animal models. In vitro studies have also suggested that these antiatherogenic effects of PPARγ activation occur by inhibiting the inflammatory gene expression and by increasing cholesterol efflux via LXR-ABCA1 activation. However, several side effects are associated with TZDs treatment making necessary the search for new compounds. In this study, 14 new thiazolidine-2,4-diones, modified TZDs by bioisosterism, were tested for aterogenic and inflammtary factors in RAW 264.7 macrophages and in low-density lipoprotein receptor-deficient mice. After the citotoxicity evaluation in RAW 264.7 macrophages the TZDs named GQ-11, GQ-97, GQ-177, GQ-145 e LYSO-7 were selected for this study. Three of these TZDs (GQ-177, GQ-145 and LYSO-7) significantly increased the expression of PPARγ1, PPARγ2 and CD36 mRNA, and enhanced the expression of ABCA1 mRNA in 2.9, 3.5 and 6.7 fold, respectively. Moreover, they also significantly decreased the expression of iNOS, COX2, VCAM and IL-6 mRNA in relation to control, and these results are associated to reduction on nitrits concentration. In addition, LYSO-7 significantly reduced the expression of these genes when compared to rosiglitazone, and decreased expression of MCP1 mRNA. In the experimental study, male LDLr-/- mice were fed an atherogenic diet containing 0.5% cholesterol for 16 weeks, and 4 weeks before euthanasia they received TZDs (20mg/kg/ per day) by gavage. GQ-177 treatment inhibited progression of atherosclerotic plaque associated to increased plasma concentrations of HDL-C, with enhance of ABCA1 expression and reduction on CD40-CD40L interaction. LYSO-7 treatment also showed inhibition of the atherogenesis associated to decreased plasma concentrations of total cholesterol and TAG, with reduction on CD40-CD40L pathway and inflammatory cytokines expression.GQ-145 did not alter the lipid plasma levels and increased the expression of all pro-atherogenic and pro-inflammatory genes. Furthermore, the activation of PPARs has also been studied, by luciferase assay as reporter gene. GQ-177 induced activation of PPARγ, whereas LYSO-7 stimulated activation of PPARα and PPARβ/δ. Altogether, our data suggest that the new TZDs derivatives, specially GQ- 177 and LYSO-7, may have atheroprotective properties associated with the reverse cholesterol transport and anti-inflammatory effects, and could be a promising alternative for the treatment of atherosclerosis. However, further studies are warranted in order to characterize the pathways of intracellular signaling since both have demonstrated to activate different isotypes of PPAR.
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Die Bedeutung von Angiotensin II und "Peroxisome Proliferator-Activated Receptors" in vaskulären Zellen bei der Entstehung der AtheroskleoseKintscher, Ulrich 26 March 2004 (has links)
Die Entstehung atherosklerotischer Gefässwandveränderungen ist ein chronischer Prozess, welcher durch vaskuläre pro-atherosklerotische Wachstumsfaktoren und anti-atherosklerotisch wirkende Modulatoren in der Gefässwand reguliert wird. Angiotensin II ist sowohl in in-vitro als auch in in-vivo Studien als ein zentraler pro-atherosklerotischer Wachstumsfaktor identifiziert worden. Pro-atherosklerotische in-vitro Effekte von Angiotensin II resultieren in einer massiven Zunahme atherosklerotischer Läsionen durch Angiotensin II im Tiermodell. Die pro-atherosklerotische Funktion von Angiotensin II wird parallel durch klinische Daten unterstützt, in denen gezeigt wird, dass die Angiotensin II Blockade durch ACE-Inhibitoren oder AT1-R Antagonisten zu einer Reduktion atherosklerotischer Gefässwandveränderungen führt, was letztendlich in einer Senkung der kardiovaskulären Mortalität resultiert. Demgegenüber stehen multiple atherosklerose-hemmende Faktoren in der Gefässwand. In der Gruppe dieser Faktoren spielen die nukleären Hormonrezeptoren der Peroxisome Proliferator-Activated Receptors (PPARs) eine wichtige pathophysiologische und klinische Rolle. Aktiviert durch endogene Liganden supprimieren PPARs pro-atherosklerotische Abläufe in der Gefässwand. Eine zusätzliche Aktivierung wird durch exogene, synthetische Liganden erreicht, welche in der Therapie metabolischer Erkrankungen bereits klinisch eingesetzt werden. Zwei PPAR Isoformen sind in der Gefässwand überwiegend exprimiert, PPARa und PPARg. Beide Isoformen vermitteln anti-atherosklerotische Effekte durch die transkriptionelle Regulation unterschiedlicher Zielgene. Wir untersuchten die PPAR-vermittelte Regulation dieser Zielgene und konnten neue molekulare Wirkmechanismen, welche den anti-atherosklerotischen Wirkungen von PPARa- und PPARg-Liganden zugrunde liegen identifizieren. In weiterführenden Arbeiten konnten wir zeigen, dass multiple bidirektionale Interaktionen zwischen Angiotensin II und PPARs bestehen, welche einen wesentlichen Einfluss auf die Entstehung und Progression atherosklerotischer Gefässveränderungen haben. / The development of atherosclerosis is a chronic process, which is tightly regulated by pro-atherosclerotic vascular growth factors and anti-atherosclerotic modulators in the vessel wall. Angiotensin II has been identified in in-vitro and in-vivo studies as a central pro-atherosclerotic growth factor. Angiotensin II-infusion in mice results in a profound induction of atherosclerotic lesions in the aorta. In parallel, clinical studies have demonstrated that blockade of the renin-angiotensin-system by AT1R-antagonists or ACE-inhibitors significantly lowers the incidence of atherosclerotic diseases. The pro-atherosclerotic actions of angiotensin II are counterbalanced by multiple anti-atherosclerotic modulators present in the vessel wall. The peroxisome proliferators-activated receptors (PPARs), including the isoforms alpha and gamma, are ligand-activated nuclear hormone receptors, present in the vascular wall, and exerting major anti-atherosclerotic actions. They function as transcriptional regulators of target genes involved in atherogenesis. We investigated PPAR-mediated target gene expression in vascular and inflammatory cells, and identified new anti-atherosclerotic mechanisms of these receptors. Finally we characterized bidirectional interactions between angiotensin II and PPARs, which might play a major role in the development of atherosclerosis in humans.
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Caracterização estrutural dos complexos entre os receptores ativadores da proliferação de peroxissomos (PPARs) dos tipos alfa e gama e seus agonistas / Structural characterization of the peroxisome proliferator-activated receptors (PPARs) types alpha and gamma complexes and its agonistsSantos, Jademilson Celestino dos 25 April 2014 (has links)
Os receptores ativadores da proliferação de peroxissomos (PPARs) são fatores de transcrição dependentes da ligação de ligantes e possuem um papel chave no controle do metabolismo dos lipídios e da glicose. Existem três isotipos desse receptor: PPARα, PPARβ e PPARγ. O PPARγ é alvo molecular para os compostos TZDs, os quais são fármacos usados clinicamente no controle da diabetes do tipo 2, aumentando a sensibilidade à insulina. Enquanto que os fibratos são os fármacos que atuam no PPARα e são utilizados para diminuir os níveis de triglicerídeos. A maioria dos pacientes que sofrem com a diabetes do tipo 2 apresentam desordens no metabolismo de lipídios. Mesmo com a existência de fármacos capazes de controlar estas desordens metabólicas, a busca de um agonista dual para os PPARα e PPARγ é um grande desafio no controle da síndrome metabólica, uma vez que este composto pode combinar os dois efeitos terapêuticos em uma única molécula. O GL479 é um agonista dual que foi sintetizado com dois grupos farmacóforos, ligando-se tanto ao PPARα quanto ao PPARγ. Dentro desse contexto, este estudo apresenta as bases estruturais de interação do agonista dual GL479 aos PPARs por meio da determinação estrutural dos complexos PPARα-LBD:GL479 e PPARγ-LBD:GL479. A análise detalhada desses complexos revelou diferentes modos de interação do ligante em cada receptor, porém em ambos os casos o GL479 interage com a Tyr da H12. Na estrutura do PPARα-LBD, o ligante adquiriu a característica de um agonista total e no caso do PPARγ-LBD, o GL479 adotou características de um agonista parcial dependente da interação com a H12. Além das analises do agonista dual, 16 compostos foram identificados por docking como ligantes do PPARγ. Três desses ligantes (8, 10 e 15) foram caracterizados por ThermoFluor e fluorescência de polarização com valores de IC50 menor que 10 µM. Adicionalmente, um dos compostos identificados no docking (16) foi cocristalizado com PPARγ-LBD. A conformação adotada pelo ligante não permitiu que ele interagisse diretamente com a H12, sugerindo que este composto possa atuar como um agonista parcial independente da H12. Todas estas descobertas podem ser exploradas no desenho de novos moduladores dos PPARs com menores efeitos adversos ou até mesmo na busca de agonistas duais PPARα ⁄γ, que combine os efeitos terapêuticos no tratamento da diabetes do tipo 2 e da dislipidemia. / Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors that control various functions in human organism and they play key roles in the control of glucose and lipid metabolism. There are three different PPAR isotypes: PPARα, PPARβ e PPARγ. PPARγ is a molecular target of TZD agonists, which are clinically used drugs in the control of type 2 diabetes by increasing insulin sensitivity. Whereas fibrates are drugs that act on PPARα and are used to lower serum triglyceride levels. The most patients who have type 2 diabetes also display lipid metabolism disorders. Even with the existence of drugs that can control these metabolic disorders, the search of dual agonist for PPARα and PPAR γ is a major challenge in the control of metabolic syndrome, because this compound could combine both therapeutic effects in a single molecule. GL479 is a dual agonist that was synthesized based on a combination of two key pharmacophores, with the ability to bind in the both PPARs, α, and γ. Thus, this study reveals the structural basis for this dual agonist GL479 by structural determination of the complexes PPARα-LBD:GL479 and PPARγ-LBD:GL479. The detailed analysis of these complexes showed different ligand binding modes for each receptor, however, in the both cases the GL479 interacted with the Tyr of H12. In the PPARα-LBD structure the ligand acquired the features of full agonist and in the case of PPARγ-LBD, GL479 adopted features of a partial agonist dependent of H12 interaction. In addition to the dual agonist analysis, sixteen compounds were identified as PPARγ ligand by docking. Three of these ligands were characterized by ThermoFluor and fluorescence polarization, which resulted in IC50 values smaller than 10 µM. Additionally, one of the compounds, identified by docking, was co-crystallized with PPARγ. The ligand conformation adopted would not allow it a direct interaction with the H12. These contacts were mediated by one water molecule, suggesting this compound might also act as a partial agonist, independent of H12 interaction. All these findings may be explored for the design of PPARs novel modulators with lower side effects, as well, in the exploration of dual agonists PPARα ⁄ γ that combines the therapeutic effects in the treatment of type 2 diabetes and dyslipidemia.
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Caracterização estrutural dos complexos entre os receptores ativadores da proliferação de peroxissomos (PPARs) dos tipos alfa e gama e seus agonistas / Structural characterization of the peroxisome proliferator-activated receptors (PPARs) types alpha and gamma complexes and its agonistsJademilson Celestino dos Santos 25 April 2014 (has links)
Os receptores ativadores da proliferação de peroxissomos (PPARs) são fatores de transcrição dependentes da ligação de ligantes e possuem um papel chave no controle do metabolismo dos lipídios e da glicose. Existem três isotipos desse receptor: PPARα, PPARβ e PPARγ. O PPARγ é alvo molecular para os compostos TZDs, os quais são fármacos usados clinicamente no controle da diabetes do tipo 2, aumentando a sensibilidade à insulina. Enquanto que os fibratos são os fármacos que atuam no PPARα e são utilizados para diminuir os níveis de triglicerídeos. A maioria dos pacientes que sofrem com a diabetes do tipo 2 apresentam desordens no metabolismo de lipídios. Mesmo com a existência de fármacos capazes de controlar estas desordens metabólicas, a busca de um agonista dual para os PPARα e PPARγ é um grande desafio no controle da síndrome metabólica, uma vez que este composto pode combinar os dois efeitos terapêuticos em uma única molécula. O GL479 é um agonista dual que foi sintetizado com dois grupos farmacóforos, ligando-se tanto ao PPARα quanto ao PPARγ. Dentro desse contexto, este estudo apresenta as bases estruturais de interação do agonista dual GL479 aos PPARs por meio da determinação estrutural dos complexos PPARα-LBD:GL479 e PPARγ-LBD:GL479. A análise detalhada desses complexos revelou diferentes modos de interação do ligante em cada receptor, porém em ambos os casos o GL479 interage com a Tyr da H12. Na estrutura do PPARα-LBD, o ligante adquiriu a característica de um agonista total e no caso do PPARγ-LBD, o GL479 adotou características de um agonista parcial dependente da interação com a H12. Além das analises do agonista dual, 16 compostos foram identificados por docking como ligantes do PPARγ. Três desses ligantes (8, 10 e 15) foram caracterizados por ThermoFluor e fluorescência de polarização com valores de IC50 menor que 10 µM. Adicionalmente, um dos compostos identificados no docking (16) foi cocristalizado com PPARγ-LBD. A conformação adotada pelo ligante não permitiu que ele interagisse diretamente com a H12, sugerindo que este composto possa atuar como um agonista parcial independente da H12. Todas estas descobertas podem ser exploradas no desenho de novos moduladores dos PPARs com menores efeitos adversos ou até mesmo na busca de agonistas duais PPARα ⁄γ, que combine os efeitos terapêuticos no tratamento da diabetes do tipo 2 e da dislipidemia. / Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors that control various functions in human organism and they play key roles in the control of glucose and lipid metabolism. There are three different PPAR isotypes: PPARα, PPARβ e PPARγ. PPARγ is a molecular target of TZD agonists, which are clinically used drugs in the control of type 2 diabetes by increasing insulin sensitivity. Whereas fibrates are drugs that act on PPARα and are used to lower serum triglyceride levels. The most patients who have type 2 diabetes also display lipid metabolism disorders. Even with the existence of drugs that can control these metabolic disorders, the search of dual agonist for PPARα and PPAR γ is a major challenge in the control of metabolic syndrome, because this compound could combine both therapeutic effects in a single molecule. GL479 is a dual agonist that was synthesized based on a combination of two key pharmacophores, with the ability to bind in the both PPARs, α, and γ. Thus, this study reveals the structural basis for this dual agonist GL479 by structural determination of the complexes PPARα-LBD:GL479 and PPARγ-LBD:GL479. The detailed analysis of these complexes showed different ligand binding modes for each receptor, however, in the both cases the GL479 interacted with the Tyr of H12. In the PPARα-LBD structure the ligand acquired the features of full agonist and in the case of PPARγ-LBD, GL479 adopted features of a partial agonist dependent of H12 interaction. In addition to the dual agonist analysis, sixteen compounds were identified as PPARγ ligand by docking. Three of these ligands were characterized by ThermoFluor and fluorescence polarization, which resulted in IC50 values smaller than 10 µM. Additionally, one of the compounds, identified by docking, was co-crystallized with PPARγ. The ligand conformation adopted would not allow it a direct interaction with the H12. These contacts were mediated by one water molecule, suggesting this compound might also act as a partial agonist, independent of H12 interaction. All these findings may be explored for the design of PPARs novel modulators with lower side effects, as well, in the exploration of dual agonists PPARα ⁄ γ that combines the therapeutic effects in the treatment of type 2 diabetes and dyslipidemia.
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Régulation de l'expression de PPARγ dans l'arthroseNebbaki, Salwa Sarah 06 1900 (has links)
L’arthrose (OA) est une maladie dégénérative très répondue touchant les articulations. Elle est caractérisée par la destruction progressive du cartilage articulaire, l’inflammation de la membrane synoviale et le remodelage de l’os sous chondral. L’étiologie de cette maladie n’est pas encore bien définie. Plusieurs études ont été menées pour élucider les mécanismes moléculaires et cellulaires impliqués dans le développement de l’OA.
Les effets protecteurs du récepteur activé par les proliférateurs de peroxysomes gamma (PPARγ) dans l'OA sont bien documentés. Il a été démontré que PPARγ possède des propriétés anti-inflammatoires et anti-cataboliques. Aussi, plusieurs stimuli ont été impliqués dans la régulation de l’expression de PPARγ dans différents types cellulaires. Cependant, les mécanismes exacts responsables de cette régulation ainsi que le profil de l’expression de ce récepteur au cours de la progression de l’OA ne sont pas bien connus.
Dans la première partie de nos travaux, nous avons essayé d’élucider les mécanismes impliqués dans l’altération de l’expression de PPARγ dans cette maladie. Nos résultats ont confirmé l’implication de l’interleukine-1β (IL-1β), une cytokine pro-inflammatoire, dans la réduction de l’expression de PPARγ au niveau des chondrocytes du cartilage articulaire. Cet effet coïncide avec l'induction de l’expression du facteur de transcription à réponse précoce de type 1 (Egr-1). En plus, la diminution de l'expression de PPARγ a été associée au recrutement d'Egr-1 et la réduction concomitante de la liaison de Sp1 au niveau du promoteur de PPARγ.
Dans la deuxième partie de nos travaux, nous avons évalué le profil d’expression de ce récepteur dans le cartilage au cours de la progression de cette maladie. Le cochon d’inde avec OA spontanée et le chien avec OA induite par rupture du ligament croisé antérieur (ACLT) deux modèles animaux d’OA ont été utilisés pour suivre l’expression des trois isoformes de PPARs : PPAR alpha (α), PPAR béta (β) et PPAR gamma (γ) ainsi que la prostaglandine D synthase hématopoïétique (H-PGDS) et la prostaglandine D synthase de type lipocaline (L-PGDS) deux enzymes impliquées dans la production de l’agoniste naturel de PPARγ, la 15-Deoxy-delta(12,14)-prostaglandine J(2) (15d-PGJ2).
Nos résultats ont démontré des changements dans l’expression de PPARγ et la L-PGDS. En revanche, l’expression de PPARα, PPARβ et H-PGDS est restée stable au fil du temps. La diminution de l’expression de PPARγ dans le cartilage articulaire semble contribuer au développement de l’OA dans les deux modèles animaux. En effet, le traitement des chondrocytes par de siRNA dirigé contre PPARγ a favorisé la production des médiateurs arthrosiques tels que l'oxyde nitrique (NO) et la métalloprotéase matricielle de type 13 (MMP-13), confirmant ainsi le rôle anti-arthrosique de ce récepteur. Contrairement à ce dernier, le niveau d'expression de la L-PGDS a augmenté au cours de la progression de cette maladie. La surexpression de la L-PGDS au niveau des chondrocytes humains a été associée à la diminution de la production de ces médiateurs arthrosiques, suggérant son implication dans un processus de tentative de réparation.
En conclusion, l’ensemble de nos résultats suggèrent que la modulation du niveau d’expression de PPARγ, de la L-PGDS et d’Egr-1 au niveau du cartilage articulaire pourrait constituer une voie thérapeutique potentielle dans le traitement de l’OA et probablement d’autres formes d'arthrite. / Osteoarthritis (OA) is the most common degenerative joint disease. It is characterised by progressive destruction of articular cartilage, synovial inflammation and subchondral bone remodelling. The complete etiology of OA is still not well defined. Several studies have been carried out to elucidate the molecular and cellular mechanisms involved in OA development.
The protective effects of Peroxisome proliferator-activated receptor gamma (PPARγ) in OA have been well documented. It has been demonstrated that PPARγ exhibit anti-inflammatory and anti-catabolic properties. Although many stimuli have been reported to regulate the expression of PPARγ in several cell types. However, little information is available on the exact mechanisms that govern its regulation as well as the expression profile of this recepteur during the course of the disease.
In the first part of this work, we tried to elucidate the mechanisms involved in the alteration of PPARγ expression in OA. Our findings confirm that interleukin-1 beta (IL-1β), a proinflammatory cytokine, down regulate the expression of PPARγ in articular chondrocytes. This effect coincided with the induction of early growth response protein-1 (Egr-1) expression. In addition, down regulation of PPARγ expression was associated with Egr-1 recruitment to and concomitant reduction in Sp1 occupancy at PPARγ promoter.
In the second part of this work, we evaluated the expression profile of this receptor in cartilage during the progression of OA. Spontaneous Hartley guinea pig model and anterior cruciate ligament transection (ACLT) dog model were used to follow the expression of three isoforms of PPARs: PPAR alpha (α), PPAR beta (β) and PPAR gamma (γ) as well as hematopoietic prostaglandin D synthase (H-PGDS) and lipocalin-type prostaglandin D synthase (L-PGDS) two enzymes involved in the production of the natural agonist PAARγ, 15-Deoxy-delta(12,14)-prostaglandin J(2) (15d PGJ2).
Our reultats showed changes in the expression of PPARγ and L-PGDS. In contrast, the level of PPARα, PPARβ and H-PGDS was constant over time. The decrease in PPARγ levels in articular chondrocytes suggest that it may be a contributing factor in OA development in both animal models used in this study. Furthermore, siRNA silencing of PPARγ resulted in an enhanced production of osteoarthric mediators such as matrix metalloproteinase-13 (MMP-13) and nitric oxide (NO). Thus, confirming the anti-arthritic role of this receptor. In contrast, unlike the later, there was an increase in the expression level of L-PGDS during disease progression. The overexpression of L-PGDS in human chondrocytes was associated with reduced production of these osteoarthric mediators, suggesting its involvement in repair process.
In summary, our data suggest that the modulation of PPARγ, L-PGDS and Egr-1 expression levels in articular cartilage may be a potential therapeutic approach in the treatment of OA and probably other forms of arthritis.
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Alterations of mitochondrial biogenesis and alterations of mitochondrial antioxidant defense in Friedreich's ataxiaMarmolino, Daniele 25 January 2011 (has links)
Friedreich’s ataxia (FRDA) is an autosomal recessive inherited disorder affecting approximately 1 every 40,000 individuals in Western Europe, is characterized by progressive gait and limb ataxia, dysarthria, areflexia, loss of vibratory and position sense, and a progressive weakness of central origin. Additional features particularly include an hypertrophic cardiomyopathy that can cause premature death. A large GAA repeat expansion in the first intron of the FXN gene is the most common mutation underlying FRDA. Patients show severely reduced levels of the FXN-encoded mitochondrial protein frataxin.<p>Frataxin function is not yet completely elucidated. In frataxin deficiency conditions abnormalities of iron metabolism occur: decreased activities of iron-sulfur cluster (ISC) containing proteins, accumulation of iron in mitochondria and depletion in the cytosol, enhanced cellular iron uptake, and, in some models, reduced heme synthesis. <p>Evidence of oxidative stress has also been found in most though not all models of frataxin deficiency. Accordingly, yfh1-deficient yeast and cells from FRDA patients are highly sensitive to oxidants. Respiratory chain dysfunction further aggravate oxidative stress by increasing leakage of electrons and the formation of superoxide. Frataxin deficient cells not only generate more free radicals, but, they also show a reduced ability to mobilize antioxidant defenses, in particular to induce superoxide dismutase 2 (SOD2).<p>Peroxisome proliferator-activated receptor (PPAR) isoform-gamma play a key role in numerous cellular functions and is a key regulator of mitochondrial biogenesis and of the ROS metabolism. Recruitment of the PPAR coactivator-1a (PGC-1a) mediates many effects of the PPAR-γ activation.<p>In a first work we assessed the potential beneficial effects of a potent PPAR-gamma agonist on frataxin expression in primary fibroblasts from healthy controls and FRDA patients, and Neuroblastoma cells. We used the APAF molecule (1-0-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocoline; C33H66NO9P). Our results show that this compound is able to increase frataxin amount both at transcriptional and post-transcriptional level. At a dose of 20µM frataxin mRNA significantly increases in both controls (p=0.03) and FRDA patients (p=0.002) fibroblasts (1). The finding was confirmed in Neuroblastoma cells (p=0.042). According to previous publications APAF, as others PPAR-gamma agonists is able to up-regulate PGC-1a transcription.<p>In a second part of the study we investigate the role of the PPAR-gamma/PGC-1a pathway in the pathogenesis of FRDA. We performed a microarray analysis of heart and skeletal muscle in a mouse model of frataxin deficiency and we found molecular evidence of increased lipogenesis in skeletal muscle and alteration of fiber-type composition in heart, consistent with insulin resistance and cardiomyopathy, respectively. Since the PPAR-gamma pathway is known to regulate both processes, we hypothesized that dysregulation of this pathway could play a key role in frataxin deficiency. We confirmed this by showing a coordinate dysregulation of Pgc1a and the transcription factor Srebp1 in cellular and animal models of frataxin deficiency, and in cells from FRDA patients, who have marked insulin resistance. Particularly, PGC-1a was found significantly reduced (2) in primary fibroblasts and lymphocytes from FRDA patients (p<0.05). Furthermore, PGC-1a mRNA levels strongly correlate with frataxin relative mRNA levels (r2=0.9, p<0.001). According to this observation, in C2C12 myoblasts, PGC-1a and a reporter gene under the control of the PGC-1a promoter are rapidly down-regulated (p<0.05) when frataxin expression is inhibited by an shRNA in vitro. To further investigate this relation, we then generate PGC-1a deficient fibroblasts cells using a specific siRNA; at 72 hours of transfection frataxin was found down-regulate (p<0.05) in control cells. <p>Taken together those data indicate that some mechanism directly links an early effect of frataxin deficiency with reduced PGC-1a transcription in this cell type, and presumably in other cells that also down-regulate PGC-1α when frataxin levels are low.<p>Finally, since PGC-1a has also emerged as a key factor in the induction of many antioxidant programs in response to oxidative stress, both in vivo and in vitro, in particular in neurons, we tested whether the PGC-1a down-regulation occurring in FRDA cells could be in part responsible for the blunted antioxidant response observed in frataxin deficiency.<p>Using primary fibroblasts from FRDA patients we found reduced SOD2 levels (p<0.05), according to PGC1& / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished
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