Le niveau réduit d’AMPc dans la surexpression des protéines G(alpha)i et la prolifération accrue des cellules du muscle lisse vasculaire des rats spontanément hypertendus

Nous avons précédemment montré que les cellules musculaires lisses vasculaires(CMLV) des rats spontanément hypertendus (SHR) présentent une expression augmentée des protéines G inhibitrices (Gi) et une prolifération cellulaire accrue par rapport aux CMLV des rats Wystar-Kyoto (WKY). Le niveau d'AMPc s’est également avéré plus faible dans les CMLV de SHR. La présente étude a donc été entreprise afin d'examiner la contribution de la diminution du niveau intracellulaire d'AMPc à l’augmentation de l'expression des protéines Gi et à la prolifération accrue des CMLV de SHR et de continuer à explorer les mécanismes moléculaires sous-jacents responsables de cette réponse. Les CMLV de SHR ont montré par rapport aux CMLV des WKY une expression accrue de Giα-2 et Giα-3 qui a été diminué d'une manière dépendante de concentration par le dbcAMP, un analogue d'AMPc perméable à la membrane cellulaire. En outre, les fonctions augmentées des protéines Gi comme démontrées par l'amplification de l’inhibition de l'adénylate cyclase par les hormones inhibitrices et l'activité forskoline (FSK)-stimulée de l’adénylate cyclase par une faible concentration de GTPγS dans les CMLV de SHR ont également été restaurées aux niveaux de WKY par le dbcAMP. La prolifération accrue des CMLV de SHR a également été atténuée par le dbcAMP et la forskoline, un activateur de l'adénylate cyclase. De plus, dbcAMP a restauré la production augmentée d'anion superoxyde (O2-), l'activité de la NAD(P)H oxydase et l’expression accrue des protéines Nox 4 et p47phox observée dans les CMLV de SHR jusqu’au niveau contrôle. Par ailleurs, la phosphorylation accrue des PDGF-R, EGF-R, c-Src et ERK1/2 énoncée par les CMLV de SHR a également été diminuée par le dbcAMP d'une manière dépendante de concentration. Ces résultats suggèrent que le niveau réduit d'AMPc intracellulaire montré par les CMLV de SHR contribue à l'expression accrue des protéines Gi et à l’hyperprolifération cellulaire à travers l’augmentation du stress oxydatif, la transactivation des EGF-R, PDGF-R et la voie de signalisation des MAP kinases.

Mécanismes moléculaires de l’hypertrophie vasculaire dans le modèle animal d’hypertension essentielle (SHR)

La voie de signalisation des phosphoinositides joue un rôle clé dans la régulation du tonus vasculaire. Plusieurs études rapportent une production endogène de l’angiotensin II (Ang II) et de l’endothéline-1 (ET-1) par les cellules musculaires lisses vasculaires (CMLVs) de rats spontanément hypertendus (spontaneously hypertensive rats : SHR). De plus, l’Ang II exogène induit son effet prohypertrophique sur les CMLVs selon un mécanisme dépendant de la protéine Gqα et de la PKCẟ. Cependant, le rôle de l’axe Gqα/PLCβ/PKCẟ dans l’hypertrophie des CMLVs provenant d’un modèle animal de l’hypertension artérielle n’est pas encore étudié. L’objectif principal de cette thèse est d’examiner le rôle de l’axe Gqα/PLCβ1 dans les mécanismes moléculaires de l’hypertrophie des CMLVs provenant d’un modèle animal d’hypertension artérielle essentielle (spontaneously hypertensive rats : SHR). Nos premiers résultats indiquent que contrairement aux CMLVs de SHR âgés de 12 semaines (absence d’hypertrophie cardiaque), les CMLVs de SHR âgés de 16 semaines (présence d’hypertrophie cardiaque) présentent une surexpression protéique endogène de Gqα et de PLCβ1 par rapport aux CMLVs de rats WKY appariés pour l’âge. L’inhibition du taux d’expression protéique de Gqα et de PLCβ1 par des siRNAs spécifiques diminue significativement le taux de synthèse protéique élevé dans les CMLVs de SHR. De plus, la surexpression endogène des Gqα et PLCβ1, l’hyperphosphorylation de la molécule ERK1/2 et le taux de synthèse protéique élevé dans les CMLVs de SHR de 16 semaines ont été atténués significativement par des antagonistes des récepteurs AT1 (losartan) et ETA (BQ123), mais pas par l’antagoniste du récepteur ETB (BQ788). L’inhibition pharmacologique des MAPKs par PD98059 diminue significativement la surexpression endogène de Gqα/PLCβ1 et le taux de synthèse protéique élevé dans les CMLVs de SHR. D’un côté, l’inhibition du stress oxydatif (par DPI, inhibiteur de la NAD(P)H oxidase, et NAC , molécule anti-oxydante), de la molécule c-Src (PP2) et des récepteurs de facteurs de croissance (AG1024 (inhibiteur de l’IGF1-R), AG1478 (inhibiteur de l’EGFR) et AG1295 (inhibiteur du PDGFR)) a permis d’atténuer significativement la surexpression endogène élevée de Gqα/PLCβ1 et l’hypertrophie des CMLVs de SHR. D’un autre côté, DPI, NAC et PP2 atténuent significativement l’hyperphosphorylation de la molécule c-Src, des RTKs (récepteurs à activité tyrosine kinase) et de la molécule ERK1/2. Dans une autre étude, nous avons aussi démontré que la PKCẟ montre une hyperphosphorylation en Tyr311 dans les CMLVs de SHR comparées aux CMLVs de WKY. La rottlerin, utilisée comme inhibiteur spécifique de la PKCẟ, inhibe significativement cette hyperphosphorylation en Tyr311 dépendamment de la concentration. L’inhibition de l’activité de la PKCẟ par la rottlerin a été aussi associée à une atténuation significative de la surexpression protéique endogène de Gqα/PLCβ1 et l’hypertrophie des CMLVs de SHR. De plus, l’inhibition pharmacologique de l’activité de la PKCẟ, en amont du stress oxydatif, a permis d’inhiber significativement l’activité de la NADPH, le taux de production élevée de l’ion superoxyde ainsi que l’hyperphosphorylation de la molécule ERK1/2, de la molécule c-Src et des RTKs. À notre surprise, nous avons aussi remarqué une surexpression protéique de l’EGFR et de l’IGF-1R dans les CMLVs de SHR à l’âge de 16 semaines. L’inhibition pharmacologique de l’activité de la PKCẟ, de la molécule c-Src et du stress oxydatif a permis d’inhiber significativement la surexpression protéique endogène de ces RTKs. De plus, l’inhibition de l’expression protéique de l’EGFR et de la molécule c-Src par des siRNA spécifiques atténue significativement le taux d’expression protéique élevé de Gqα et de PLCβ1 ainsi que le taux de synthèse protéique élevé dans les CMLVs de SHR. Des siRNAs spécifiques à la PKCẟ ont permis d’atténuer significativement le taux de synthèse protéique élevé dans les CMLVs de SHR et confirment le rôle important de la PKCẟ dans les mécanismes moléculaires de l’hypertrophie des CMLVs selon une voie dépendante du stress oxydatif. En conclusion, ces résultats suggèrent un rôle important de l’activation endogène de l’axe Gqα-PLCβ-PKCẟ dans le processus d’hypertrophie vasculaire selon un mécanisme impliquant une activation endogène des récepteurs AT1/ETa, de la molécule c-Src, du stress oxidatif, des RTKs et des MAPKs.

Baroreflex sensitivity and oxidative stress in the LDL receptor knockout mice

This study alms at observing the effect of low-density lipoprotein (LDL) receptor deficiency in cholesterol blood levels, baroreflex sensitivity (BRS), nitric oxide (NO) bioavailability, and oxidative stress. The lack of LDL receptors in mice significantly increased the cholesterol blood levels (179 +/- 35 vs. 109 +/- 13 mg/dL) in the knockout (KO) mice compared to control. There was no difference in basal mean arterial pressure and heart rate between the groups. However, in KO mice the BRS was significantly attenuated and the antioxidant enzyme activities, measured in erythrocytes and heart, were significantly decreased. On the other hand, the oxidative damage measured by chemiluminescence and carbonyls was increased, while total plasma nitrate levels were lower in KO mice, indicating a decrease in NO availability. In conclusion, these results indicate that the lack of LDL receptor increased cholesterol blood levels, induced oxidative stress and decreased BRS. (C) 2008 Elsevier GmbH. All rights reserved.

Angiotensin II modulates CD40 expression in vascular smooth muscle cells

The signalling pathway CD40/CD40L (CD40 ligand) plays an important role in atherosclerotic plaque formation and rupture. AngII (angiotensin II), which induces oxidative stress and inflammation, is also implicated in the progression of atherosclerosis. In the present study, we tested the hypothesis that AngII increases CD40/CD40L activity in vascular cells and that ROS (reactive oxygen species) are part of the signalling cascade that controls CD40/CD40L expression. Human CASMCs (coronary artery smooth muscle cells) in culture exposed to IL (interleukin)-1 beta or TNF-alpha (tumour necrosis factor-a) had increased superoxide generation and enhanced CD40 expression, detected by EPR (electron paramagnetic resonance) and immunoblotting respectively. Both phenomena were abolished by previous incubation with membrane-permeant antioxidants or cell transfection with P22(phox) antisense. AngII (50-200 nmol/l) induced an early and sustained increase in CD40 mRNA and protein expression in CASMCs, which was blocked by treatment with antioxidants. Increased CD40 expression led to enhanced activity of the pathway, as AngII-treated cells stimulated with recombinant CD40L released higher amounts of IL-8 and had increased COX-2 (cyclo-oxygenase-2) expression. We conclude that AngII stimulation of vascular cells leads to a ROS-dependent increase in CD40/CD40L signalling pathway activity. This phenomenon may be an important mechanism modulating the arterial injury observed in atherosclerosis-related vasculopathy.

Efeito da apocinina na hipertensão, disfunção endotelial e hipertrofia ventricular esquerda em Ratos Espontaneamente Hipertensos (SHR)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Papel das espécies reativas de oxigênio na hiporreatividade de aortas à fenilefrina observada no final da prenhez de Ratas Espontaneamente Hipertensas (SHR)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

CYBA C242T polymorphism is associated with obesity and diabetes mellitus in Brazilian hypertensive patients

Diabet. Med. 29, e55e61 (2012) Abstract Aims The CYBA C242T polymorphism has been associated with cardiovascular phenotypes such as hypertension and atherosclerosis, but available data are conflicting. This report investigated the impact of this variant on hypertension and metabolic determinants of cardiovascular risk in a large Brazilian sample. Methods We cross-sectionally evaluated 1856 subjects (826 normotensive subjects and 1030 hypertensive patients) by clinical history, anthropometry, laboratory analysis and genotyping of the CYBA C242T polymorphism. Results Genotype frequencies in the whole population were consistent with the HardyWeinberg equilibrium and genotype distributions were not different between hypertensive and normotensive subjects. Hypertensive patients with the CC genotype presented lower fasting plasma glucose levels (5.9 +/- 0.1 vs. 6.2 +/- 0.1 mmol/l, P = 0.020) and waist circumference (94.5 +/- 0.6 vs. 96.3 +/- 0.6 cm, P = 0.028) than CT + TT ones. Similarly, the prevalence of diabetes mellitus and obesity was also lower in hypertensive patients carrying the CC genotype (16% vs. 21%, P = 0.041; 36% vs. 43%, P = 0.029, respectively). In addition, multiple and logistic regression analysis demonstrated that the CYBA C242T polymorphism was associated with glucose levels, waist circumference, obesity and diabetes mellitus in hypertensive patients independently of potential confounders. Conversely, in normotensive subjects, no significant difference in studied variables was detected between the genotype groups. Conclusions These data suggest that the T allele of the CYBA C242T polymorphism may be used as a marker for adverse metabolic features in Brazilian subjects with systemic hypertension.

Effects of moderate electrical stimulation on reactive species production by primary rat skeletal muscle cells: Cross talk between superoxide and nitric oxide production

The effects of a moderate electrical stimulation on superoxide and nitric oxide production by primary cultured skeletal muscle cells were evaluated. The involvement of the main sites of these reactive species production and the relationship between superoxide and nitric oxide production were also examined. Production of superoxide was evaluated by cytochrome c reduction and dihydroethidium oxidation assays. Electrical stimulation increased superoxide production after 1?h incubation. A xanthine oxidase inhibitor caused a partial decrease of superoxide generation and a significant amount of mitochondria-derived superoxide was also observed. Nitric oxide production was assessed by nitrite measurement and by using 4,5-diaminofluorescein diacetate (DAF-2-DA) assay. Using both methods an increased production of nitric oxide was obtained after electrical stimulation, which was also able to induce an increase of iNOS content and NF-?B activation. The participation of superoxide in nitric oxide production was investigated by incubating cells with DAF-2-DA in the presence or absence of electrical stimulation, a superoxide generator system (xanthinexanthine oxidase), a mixture of NOS inhibitors and SOD-PEG. Our data show that the induction of muscle contraction by a moderate electrical stimulation protocol led to an increased nitric oxide production that can be controlled by superoxide generation. The cross talk between these reactive species likely plays a role in exercise-induced maintenance and adaptation by regulating muscular glucose metabolism, force of contraction, fatigue, and antioxidant systems activities. J. Cell. Physiol. 227: 25112518, 2012. (c) 2011 Wiley Periodicals, Inc.

Endothelial dysfunction in the pulmonary artery induced by concentrated fine particulate matter exposure is associated with local but not systemic inflammation

Clinical evidence has identified the pulmonary circulation as an important target of air pollution. It was previously demonstrated that in vitro exposure to fine particulate matter (aerodynamic diameter <= 2.5 mu m, PM2.5) induces endothelial dysfunction in isolated pulmonary arteries. We aimed to investigate the effects of in vivo exposure to urban concentrated PM2.5 on rat pulmonary artery reactivity and the mechanisms involved. For this, adult Wistar rats were exposed to 2 weeks of concentrated Sao Paulo city air PM2.5 at an accumulated daily dose of approximately 600 mu g/m(3). Pulmonary arteries isolated from PM2.5-exposed animals exhibited impaired endothelium-dependent relaxation to acetylcholine without significant changes in nitric oxide donor response compared to control rats. PM2.5 caused vascular oxidative stress and enhanced protein expression of Cu/Zn- and Mn-superoxide dismutase in the pulmonary artery. Protein expression of endothelial nitric oxide synthase (eNOS) was reduced, while tumor necrosis factor (TNF)-alpha was enhanced by PM2.5 inhalation in pulmonary artery. There was a significant positive correlation between eNOS expression and maximal relaxation response (E-max) to acetylcholine. A negative correlation was found between vascular TNF-alpha expression and E-max to acetylcholine. Plasma cytokine levels, blood cells count and coagulation parameters were similar between control and PM2.5-exposed rats. The present findings showed that in vivo daily exposure to concentrated urban PM2.5 could decrease endothelium-dependent relaxation and eNOS expression on pulmonary arteries associated with local high TNF-alpha level but not systemic pro-inflammatory factors. Taken together, the present results elucidate the mechanisms underlying the trigger of cardiopulmonary diseases induced by urban ambient levels of PM2.5. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: A role for angiotensin type 1 receptor

Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothesized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through renin-angiotensin system (RAS) activation. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected by the previous administration of losartan (10 mg/kg; p.o. gavage), a selective ATI receptor antagonist. Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxidative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P) H oxidase-mediated vascular generation of superoxide anion and p47phox translocation (cytosol to membrane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed increased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased phenylephrine-induced contraction in endothelium-intact aortas. Ethanol significantly decreased plasma and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates RAS activity and induces vascular oxidative stress and redox-signaling activation through AT(1)-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage. (c) 2012 Elsevier Inc. All rights reserved.

Association between the C242T polymorphism in the p22phox gene with arterial stiffness in the Brazilian population

de Oliveira Alvim R, Lima Santos PCJ, Goncalves Dias R, Rodrigues MV, de Sa Cunha R, Mill JG, Junior WN, Krieger JE, Pereira AC. Association between the C242T polymorphism in the p22phox gene with arterial stiffness in the Brazilian population. Physiol Genomics 44: 587-592, 2012. First published April 10, 2012; doi:10.1152/physiolgenomics.00122.2011.-NADPH oxidase p22phox subunit is responsible for the production of reactive oxygen species in the vascular tissue. The C242T polymorphism in the p22phox gene has been associated with diverse coronary artery disease phenotypes, but the findings about the protective or harmful effects of the T allele are still controversial. Our main aim was to assess the effect of p22phox C242T genotypes on arterial stiffness, a predictor of late morbidity and mortality, in individuals from the general population. We randomly selected 1,178 individuals from the general population of Vitoria City, Brazil. Genotypes for the C242T polymorphism were detected by PCR-RFLP, and pulse wave velocity (PWV) values were measured with a noninvasive automatic device Complior. p22phox and TNF-alpha gene expression were quantified by real-time PCR in human arterial mammary smooth muscle cells. In both the entire and nonhypertensive groups: individuals carrying the TT genotype had higher PWV values and higher risk for increased arterial stiffness [odds ratio (OR) 1.93, 95% confidence interval (CI) 1.27-2.92 and OR 1.78, 95% CI 1.07-2.95, respectively] compared with individuals carrying CC + CT genotypes, even after adjustment for covariates. No difference in the p22phox gene expression according C242T genotypes was observed. However, TNF-alpha gene expression was higher in cells from individual carrying the T allele, suggesting that this genetic marker is associated with functional phenotypes at the gene expression level. In conclusion, we suggest that p22phox C242T polymorphism is associated with arterial stiffness evaluated by PWV in the general population. This genetic association shed light on the understanding of the genetic modulation on vascular dysfunction mediated by NADPH oxidase.

Insulin alone or with captopril: effects on signaling pathways (AKT and AMPK) and oxidative balance after ischemia-reperfusion in isolated hearts

Insulin and the inhibition of the reninangiotensin system have independent benefits for ischemiareperfusion injury, but their combination has not been tested. Our aim was to evaluate the effects of insulin+captopril on insulin/angiotensin signaling pathways and cardiac function in the isolated heart subjected to ischemiareperfusion. Isolated hearts were perfused (Langendorff technique) with KrebsHenseleit (KH) buffer for 25 min. Global ischemia was induced (20 min), followed by reperfusion (30 min) with KH (group KH), KH+angiotensin-I (group A), KH+angiotensin-I+captopril (group AC), KH+insulin (group I), KH+insulin+angiotensin-I (group IA), or KH+insulin+angiotensin-I+captopril (group IAC). Group A had a 24% reduction in developed pressure and an increase in end-diastolic pressure vs. baseline, effects that were reverted in groups AC, IA, and IAC. The phosphorylation of protein kinase B (AKT) was higher in groups I and IA vs. groups KH and A. The phosphorylation of AMP-activated protein kinase (AMPK) was similar to 31% higher in groups I, IA, and IAC vs. groups KH, A, and AC. The tert-butyl hydroperoxide (tBOOH)-induced chemiluminescence was lower (similar to 2.2 times) in all groups vs. group KH and was similar to 35% lower in group IA vs. group A. Superoxide dismutase content was lower in groups A, AC, and IAC vs. group KH. Catalase activity was similar to 28% lower in all groups (except group IA) vs. group KH. During reperfusion of the ischemic heart, insulin activates the AKT and AMPK pathways and inhibits the deleterious effects of angiotensin-I perfusion on SOD expression and cardiac function. The addition of captopril does not potentiate these effects.

The role of reactive oxygen species in the modulation of the contraction induced by angiotensin II in carotid artery from diabetic rat

The modulation played by reactive oxygen species on the angiotensin II-induced contraction in type I-diabetic rat carotid was investigated. Concentration-response curves for angiotensin II were obtained in endothelium-intact or endothelium-denuded carotid from control or streptozotocin-induced diabetic rats, pre-treated with tiron (superoxide scavenger), PEG-catalase (hydrogen peroxide scavenger), dimethylthiourea (hydroxyl scavenger), apocynin [NAD(P) H oxidase inhibitor], SC560 (cyclooxygenase-1 inhibitor), SC236 (cyclooxygenase-2 inhibitor) or Y-27632 (Rho-kinase inhibitor). Reactive oxygen species were measured by flow cytometry in dihydroethidium (DHE)-loaded endothelial cells. Cyclooxygenase and AT1-receptor expression was assessed by immunohistochemistry. Diabetes increased the angiotensin II-induced contraction but reduced the agonist potency in rat carotid. Endothelium removal, tiron or apocynin restored the angiotensin II-induced contraction in diabetic rat carotid to control levels. PEG-catalase, DMTU or SC560 reduced the angiotensin II-induced contraction in diabetic rat carotid at the same extent. SC236 restored the angiotensin II potency in diabetic rat carotid. Y-27632 reduced the angiotensin II-induced contraction in endothelium-intact or -denuded diabetic rat carotid. Diabetes increased the DHE-fluorescence of carotid endothelial cells. Apocynin reduced the DHE-fluorescence of endothelial cells from diabetic rat carotid to control levels. Diabetes increased the muscular cyclooxygenase-2 expression but reduced the muscular AT1-receptor expression in rat carotid. In summary, hydroxyl radical, hydrogen peroxide and superoxide anion-derived from endothelial NAD(P) H oxidase mediate the hyperreactivity to angiotensin II in type I-diabetic rat carotid, involving the participation of cyclooxygenase-1 and Rho-kinase. Moreover, increased muscular cyclooxygenase-2 expression in type I-diabetic rat carotid seems to be related to the local reduced AT1-receptor expression and the reduced angiotensin II potency. (C) 2011 Elsevier B. V. All rights reserved.

Ruolo delle specie reattive dell'ossigeno e di caveole/raft lipidici nella biosegnalazione in linee cellulari umane

Membrane lipid rafts are detergent-resistant microdomains containing glycosphingolipids, cholesterol and glycosylphosphatidylinositol-linked proteins; they seem to be actively involved in many cellular processes including signal transduction, apoptosis, cell adhesion and migration. Lipid rafts may represent important functional platforms where redox signals are produced and transmitted in response to various agonists or stimuli. In addition, a new concept is emerging that could be used to define the interactions or amplification of both redox signalling and lipid raft-associated signalling. This concept is characterized by redox-mediated feed forward amplification in lipid platforms. It is proposed that lipid rafts are formed in response to various stimuli; for instance, NAD(P)H oxidase (Nox) subunits are aggregated or recruited in these platforms, increasing Nox activity. Superoxide and hydrogen peroxide generation could induce various regulatory activities, such as the induction of glucose transport activity and proliferation in leukaemia cells. The aim of our study is to probe: i) the involvement of lipid rafts in the modulation of the glucose transporter Glut1 in human acute leukemia cells; ii) the involvement of plasma membrane caveolae/lipid rafts in VEGF-mediated redox signaling via Nox activation in human leukemic cells; iii) the role of p66shc, an adaptor protein, in VEGF signaling and ROS production in endothelial cells (ECs); iv) the role of Sindecan-2, a transmembrane heparan sulphate proteoglycan, in VEGF signaling and physiological response in ECs and v) the antioxidant and pro-apoptotic activities of simple dietary phenolic acids, i. e. caffeic, syringic and protocatechuic acids in leukemia cells, characterized by a very high ROS content. Our results suggest that the role played by NAD(P)H oxidase-derived ROS in the regulation of glucose uptake, proliferation and migration of leukaemia and endothelial cells could likely occur through the control of lipid raft-associated signalling.

Ursachen der Anthrazyklin-induzierten Kardiotoxizität : Einfluss der individuellen genetischen Ausstattung, des oxidativen Stresses und der Metabolisierung

Doxorubizin (Dox) gehört zur Gruppe der Anthrazykline, welche seit mehreren Jahrzehnten erfolgreich gegen ein breites Spektrum an Tumoren eingesetzt wird. Neben der guten Wirksamkeit besitzt Dox jedoch auch ein sehr hohes Nebenwirkungspotential. Die wohl folgenschwerste Nebenwirkung stellt die irreversible Schädigung des Herzens dar. Zahlreiche Faktoren, wie zum Beispiel die kumulative Dox-Dosis konnten bereits mit einer erhöhten Inzidenz an kardialen Schäden in Verbindung gebracht werden. Bislang ungeklärt war jedoch die Frage, warum Patienten unterschiedlich sensibel auf die Verabreichung von Dox reagierten. rnAn dem Patientenkollektiv der Ricover60-Studie wurde der Einfluss der individuellen genetischen Ausstattung auf die Entstehung der Anthrazyklin-induzierten Herzschädigung untersucht. Alle Patienten mit Dox-induzierten Herzschäden wurden identifiziert und auf das Vorhandensein von genetischen Polymorphismen der NAD(P)H-Oxidase (CYBA, RAC2 und NCF4) und der Anthrazyklin-Transporter (MRP1 und MRP2) untersucht. Sowohl für CYBA als auch für RAC2 konnte eine Anreicherung bestimmter Genotypen (CYBA: CT/TT; RAC2: TA/AA) in der Gruppe der herzgeschädigten Patienten nachgewiesen werden. In der Multivariaten Analyse von RAC2 erreichte diese Anreicherung ein signifikantes Niveau (p=0.028). Damit konnte für diesen Polymorphismus die klinische Relevanz bestätigt werden.rnDie Ursachen der Dox-induzierten Toxizität wurden außerdem an verschiedenen Mäusestämmen und Zelllinien untersucht. Balb/c- und C57BL/6-Mäuse, die bekanntermassen unterschiedlich sensibel auf Dox reagierten, wurden mit Dox behandelt. Anschliessend wurden die Organe Herz, Leber und Blut via HPLC untersucht. Es konnte gezeigt werden, dass sich 1. die Hauptanreicherungsorte für Dox und Doxol (Balb/c: Herz und Blut versus C57BL/6: Leber), 2. die nachgewiesenen Gesamtmengen an Dox+Doxol+Doxon in den drei Organen (MengeC57BL/6 > MengeBalb/c) sowie 3. die An- und Abflutungsgeschwindigkeiten von Dox zwischen den beiden Mäusestämmen unterscheiden. Schlussendlich konnte im Vergleich zu den Balb/c-Mäusen, bei den C57BL/6-Mäusen eine stärkere kardiale Anreicherung von Dox nach der mehrmaligen Dox-Injektion nachgewiesen werden. Somit scheinen der deutlich höhere Dox-Gehalt und die längere Verweilzeit in den Herzen für die stärkere kardiale Schädigung der C57BL/6-Mäuse verantwortlich zu sein. Hingegen verlief die Art der Dox-Metabolisierung in beiden Mäusestämmen ähnlich. rnBei der Betrachtung des oxidativen Stresses konnte gezeigt werden, dass in den Herzen der C57BL/6-Mäusen ein gröβerer oxidativer Stress vorlag, als bei den Balb/c-Mäusen. Ähnlich wie bei der Ricover60-Studie ließ sich auch bei den Mäusen eine Beteiligung der NAD(P)H-Oxidase am Dox-induzierten oxidativen Stress nachweisen. rnMit der HTETOP-Zelllinie konnte gezeigt werden, dass Dox unter physiologischen Bedingungen oxidativen Stress auslösen kann. Die Art und die Konzentration der gebildeten ROS waren abhängig von der Dox-Konzentration, der Einwirkzeit und der Kompensationsfähigkeit der Zellen. Durch die Gabe von Dex ließ sich das Ausmaß des oxidativen Stresses lediglich in den Mäuseherzen reduzieren. In den HTETOP-Zellen zeigte Dex selbst stressauslösende Eigenschaften. Durch die Behandlung mit Dex / DOXY konnte gezeigt werden, dass die Hemmung der Topo IIα selbst oxidativen Stress in den HTETOP-Zellen auslöst. Jedoch scheint weder die Topo IIalpha-Hemmung, noch der Dox-induzierte oxidative Stress bei physiologischen Dox-Konzentrationen (< 1 µM) eine entscheidende Rolle für die Toxizität zu spielen. rnIn der Mikroarray-Analyse der HTETOP-Zellen konnten verschiedene Gene identifiziert werden, die in den oxidativen Stress involviert sind und die durch die Gabe von Dox differentiell reguliert werden. Durch die Komedikation mit Dex / DOXY ließen sich diese Veränderungen teilweise modulieren. rn