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Aryl hydrocarbon receptor (ahr): a therapeutic target for systemic hypertension associated with chronic intermittent hypoxia
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RESUMO: A síndrome da apneia obstrutiva do sono (SAOS) é uma perturbação respiratória
relacionada com o sono, que tem vindo a ser cada vez mais prevalente. Esta condição
clínica é responsável por uma multiplicidade de comorbilidades, com destaque para a
hipertensão arterial sistémica (HTA). A etiopatogenia da SAOS inclui quatro principais
características clínicas, nomeadamente a hipóxia crónica intermitente (HCI), a
hipercapnia, o aumento da pressão intratorácica e a fragmentação do sono. Destas, a HCI
assume-se como um factor chave no desenvolvimento de HTA.
A SAOS é uma das causas principais de HTA resistente, o que significa que ainda não existe
uma correta estratégia terapêutica para controlar este tipo particular de HTA. De facto, o
efeito anti-hipertensivo da pressão positiva contínua nas vias aérea (CPAP), a terapêutica
“gold-standard” para a SAOS, é moderado, e mesmo os fármacos anti-hipertensivos
tradicionais mostram alguma ineficácia no controlo da HTA induzida pela SAOS, impelindo
a procura de novas ferramentas terapêuticas. Assim, a investigação de novos alvos
terapêuticos tem-se tornado uma área bastante promissora. Modelos animais de SAOS,
reproduzindo uma das suas características fulcrais, a HCI, têm sido utilizados para
investigar mecanismos de HTA associados à HCI, de forma a procurar novas abordagens
terapêuticas, como é o caso desta dissertação.
O objetivo geral desta tese é, então, a descoberta de um novo alvo terapêutico para tratar
a HTA induzida pela HCI, com a utilização de um modelo animal.
A maioria das abordagens terapêuticas para a HTA da HCI consistem no bloqueio do
sistema simpático e/ou do sistema renina-angiotensina-aldosterona (SRAA), com alguns
resultados positivos, mas ainda com resultados clínicos pouco expressivos. Na verdade,
resultados anteriores do nosso grupo mostraram que o carvedilol (bloqueador α-e βadrenérgico) não teve nenhum efeito anti-hipertensivo num modelo animal de HTA
induzido por HIC.
Isso levou-nos a deduzir que outras vias de sinalização diretamente estimuladas pela HCI
ao nível celular deveriam ser investigadas e manipuladas farmacologicamente, de forma a inferirmos o seu efeito na pressão arterial. Tendo em conta o seu envolvimento na
regulação de processos adaptativos de longo-prazo, acreditamos que fatores de
transcrição poderão ser elementos de ligação plausíveis entre os ciclos de hipóxia
intermitente imediatos e os efeitos deletérios de longo prazo da SAOS-HCI, como a HTA.
Tendo isto em conta, assumimos que o fator de transcrição receptor de aril
hidrocarboneto (AHR) pudesse ser um putativo elemento mecanístico entre a HCI e a HTA
com base em quatro argumentos principais:
1) O factor indutível pela hipóxia 1α (HIF-1α), que faz parte duma via de sinalização
estimulada pela HCI, e o AHR partilham o mesmo parceiro de ligação nas suas vias de
activação canónicas, sugerindo uma eventual ligação entre o HIF-1α e o AHR;
2) O AHR tem um papel importante na regulação da pressão sanguínea;
3) Alguns dos mecanismos moleculares que originam a HTA são comuns para o AHR e
para a HCI (ativação do SRAA, stress oxidativo, disfunção endotelial, regulação de
áreas cardiovasculares centrais (por exemplo no tronco cerebral);
4) A HCI, como estado inflamatório crónico que é, pode levar à sobreprodução de certos
metabolitos que são ligados do AHR, como os metabolitos da quinurenina.
Desta forma, a hipótese de trabalho desta tese está alicerçada em duas premissas
principais: a HCI sobre-regula a via do AHR que, consequentemente, contribui para o
desenvolvimento da HTA.
Esta tese pretende investigar, pela primeira vez, o efeito da HCI na via do AHR e as
consequências na HTA induzida pela HCI da sua manipulação farmacológica.
De forma a concretizarmos este objetivo, ratos Wistar Han machos, com idades
compreendidas entre as 8 e as 12 semanas, foram expostos a um paradigma ligeiro de HCI
(5.6 ciclos/hora, 10.5 horas/dia, das 9.30h às 20h).
Iniciámos as nossas experiências expondo grupos de ratos a vários períodos de HCI (14, 21
e 60 dias), e para cada período foi também utilizado um grupo controlo de animais
expostos a condições de normóxia (Nx) durante o mesmo intervalo de tempo (n=5-8
animais por grupo). No final da exposição, os animais foram occisados e recolheu-se sangue e alguns órgãos (córtex e medula renais, fígado, tecido adiposo visceral, baço e
hipocampo).
Desses órgãos, procedemos à extração do ácido ribonucleico (ARN) total para se proceder
à análise de reação em cadeia da polimerase (RCP) para alguns genes, nomeadamente
relacionados com a via do AHR (Ahr, Cyp1a1, Cyp1b1, Cyp1a2, Arnt), da família do HIF
(Hif1a, Epas1, Hif3a, Vegfa), da via do NF-kB pathway (Rela, Nfkb2, Il6, Il1b), do SRAA
(Ren, Ace, Agtr1) e marcadores de transição epitelio-mesenquimal e de fibrose (Fn1, Vim,
Cdh1, Col1a1, Acta2).
Além disso, considerando que a HCI é uma condição caracterizada pela presença de stress
oxidativo, analisou-se, por cromatografia líquida de alta performance, a dinâmica da
cisteína, um tiol antioxidante, em rins de ratos expostos aos vários períodos de HCI.
Também se quantificou, através de cromatografia liquida acoplada a espetrometria de
massa, vários metabolitos do triptofano no plasma e urina em vários períodos de HCI (1, 7,
14, 21 e 60 dias) e em rins de animais expostos a 21 dias de HCI.
No seguimento dessas experiências, modulou-se farmacologicamente a via do AHR com
um antagonista, CH-223191 (5 mg/kg, uma vez por dia, por gavagem, em 1 m de óleo
vegetal). Este antagonista foi utilizado em dois conjuntos principais de experiências. Um
conjunto foi desenhado para verificar o potencial efeito preventivo deste composto na
HTA induzida pela HCI, enquanto o outro conjunto foi desenhado mais especificamente
para aferir o potencial efeito reversor deste composto.
A pressão arterial e a frequência cardíaca foram medidas por radiotelemetria às 8h
(período ativo, luzes apagadas) e às 18h (período inativo, luzes acesas).
Após 21 dias de HCI, genes do SRAA estavam sobre-expressos no rim, bem como alguns
genes da família dos HIFs (Hif1a, Epas1 and Hif3a). Rela estava sobre-regulado após 21
dias de HCI e o Nfkb2 após 60 dias, ambos no córtex renal. As expressões de mRNA do
Nfkb2 e do Il6 estavam aumentadas, reflectindo os efeitos inflamatórios da HCI sobre o
tecido adiposo. Durante o mesmo período de tempo (21 dias), um processo de transição
epitelio-mesenquimal parecia estar a ocorrer no rim, com sobre-expressões dos genes de
Fn1 e Vim.A dinâmica renal da cisteína também se alterou com a hipóxia. Ao longo do tempo, o
conteúdo total de cisteína diminuiu, bem como a fração ligada às proteínas. Além disso,
uma variação em forma de U no par redox (cisteína reduzida:oxidada) foi também
observada, sugerindo que o rim é susceptível à lesão oxidativa desencadeada pela HCI.
Adicionalmente, observou-se, no rim, um elevado rácio quinurenina:triptofano, revelando
um estado pró-quinurenina, um conhecido ligando do AHR, que poderá promover,
potencialmente, a activação do AHR. Em contraste, no plasma, encontraram-se vários
metabolitos relacionados com a via da serotonina aumentados.
O antagonista do AHR, CH-223191, foi capaz de prevenir e reverter parcialmente a HTA
durante a fase ativa dos ratos (ou seja, no período em que as luzes estão desligadas).
Contudo, durante o período inativo (fase em que as luzes estão ligadas e que ocorre
simultaneamente com os ciclos de HCI), o efeito anti-hipertensivo do antagonista foi
inexistente.
Adicionalmente, o composto não recuperou a perda do perfil “dipping” da pressão arterial
e da frequência cardíaca causado pela HCI.
Em conclusão, a via do AHR parece estar sobre-activada no rim, após a exposição à HCI, e
a sua modulação farmacológica interferiu no aumento da pressão arterial induzida pela
HCI, tendo um significante efeito anti-hipertensivo durante o período ativo dos animais.
Estes resultados estimulam estudos futuros de forma a clarificar o pleno potencial desta
via como novo alvo terapêutico para tratar a HTN induzida pela HCI e SAOS, bem como o
seu mecanismo de ação.
ABSTRACT: Obstructive sleep apnea (OSA) is a sleep-related breathing disorder that has becoming a more prevalent clinical condition in recent years. This clinical condition is responsible for a wide range of comorbidities, in particular systemic hypertension (HTN). OSA’s etiopathogenesis includes four clinical features, namely, chronic intermittent hypoxia (CIH), hypercapnia, increased intrathoracic pressure and sleep fragmentation. Among them, CIH is a well-established factor in the development of HTN. OSA is a major cause of resistant HTN, which means that the correct therapeutic strategy to control this particular type of HTN is still undefined. In fact, the antihypertensive effect of continuous positive airway pressure - CPAP (OSA’s gold standard therapeutic procedure) is moderate, and even traditional antihypertensive drugs show some lack of efficacy to control OSA-induced HTN, urging to find new suitable antihypertensive drugs. Therefore, the investigation for novel therapeutic targets is a hot topic in the field of OSA. Animal models of OSA, reproducing its major feature, CIH, have been used to investigate the mechanisms of HTN associated to CIH, in order to seek new therapeutic approaches, as this is the case of the present dissertation. The general goal of this thesis is to find a novel therapeutic target to HTN induced by CIH, with the use of an animal model. Most of the pharmacological approaches for HTN in CIH consisted in the abrogation of the sympathetic and/or the renin-angiotensin-aldosterone systems (RAAS), with some positive results, but still with weak outcomes. Indeed, previous results from our group showed that carvedilol (an α-and β -adrenergic blocker) had no antihypertensive effect in an animal model of HTN induced by CIH. These facts led us to deduce that other signaling pathways directly stimulated by CIH at cellular level should be investigated and pharmacologically manipulated to infer its effect over BP. We believe that transcription factors, as they are involved in long-term adaptive regulations, are plausible mechanistic bridges between the immediate cyclical intermittent hypoxia cycles and the long-term deleterious effects of CIH-OSA, as HTN. With this in mind, four major arguments converge to sustain the assumption that the transcription factor aryl hydrocarbon receptor (AHR) might be a putative mechanistic link between CIH and HTN: 1) Hypoxia inducible factor 1α (HIF-1α), a hypoxic signaling pathway known to be triggered by CIH, and AHR share the same binding partner to assist their canonical activation routes, suggesting a potential crosstalk/interplay between HIF-1 and AHR; 2) AHR has an important role in the regulation of blood pressure; 3) Some of the molecular mechanisms leading to HTN are common for AHR and CIH (RAAS activation, oxidative stress, endothelial dysfunction, regulation of central cardiovascular areas (for instance, in brainstem); 4) CIH, as a chronic inflammatory state, can lead to the overproduction of certain metabolites known to be AHR ligands (e.g. kynurenine metabolites). Thus, the working hypothesis of this thesis is the following: CIH upregulates the AHR circuitry, and consequently contributes to the development of systemic HTN. This thesis aims to investigate, for the first time, the effect of CIH on the AHR circuitry and the consequences of its pharmacological manipulation on HTN induced by CIH. In order to pursue this objective, male Wistar Han rats, aged 8-12 weeks, were exposed to a mild CIH paradigm (5.6 cycles/hour, 10.5 hours/day, from 9.30am to 8pm). We initiated our experiments by exposing groups of rats to a time-course of CIH. Groups of rats were exposed to 14, 21 and 60 days of CIH, and for each time-point, a corresponding control/normoxic (Nx) group was also used (n=5-8/group). At the end of the exposure, the animals were sacrificed and blood and several organs (renal cortex, renal medulla, liver, visceral adipose tissue, spleen and hippocampus) were collected. From those organs, we extracted total ribonucleic acids (RNA) to perform quantitative polymerase chain reaction analysis (qPCR) for several genes, namely genes involved in the AHR pathway (Ahr, Cyp1a1, Cyp1b1, Cyp1a2, Arnt), hypoxia inducible factor (HIF) family (Hif1a, Epas1, Hif3a, Vegfa), NF-kB pathway (Rela, Nfkb2, Il6, Il1b), RAAS members (Ren,Ace, Agtr1) and epithelial-mesenchymal transition and fibrosis markers (Fn1, Vim, Cdh1, Col1a1, Acta2). Moreover, considering that CIH is an oxidative stress disorder, we analyzed, by HighPerformance Liquid Chromatography with Fluorescence Detection (HPLC-FD), the dynamic of cysteine (Cys), an important antioxidant thiol, in the renal tissues of rats exposed to the CIH time-course. We also quantified several tryptophan metabolites by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in plasma and urine of several time-points (1, 7, 14, 21 and 60 days) and in kidney of CIH-exposed rats (21 days). Following these experiments, we modulated pharmacologically the AHR pathway, with an AHR antagonist, CH-223191 (5 mg/kg, once a day, by oral gavage in 1 ml of vegetable oil). We used the antagonist in two major sets of experiments. One set was designed to investigate particularly the preventive role of this compound on CIH-induced HTN, while the other aimed to determine its capacity to revert this outcome. BP and HR measurements were determined by radiotelemetry, at 8am (active period, lights-off) and 6pm (inactive period, lights-on). Following 21 days of CIH exposure, RAAS genes were overexpressed in the kidney, as well as some HIF family genes (Hif1a, Epas1 and Hif3a). Rela mRNA expression was upregulated after 21 days and Nkfb2 after 60 days of CIH, in the renal cortex. Visceral adipose tissue mRNA expressions of Nfkb2 and Il6 were increased, reflecting the inflammatory effects of CIH on the adipose tissue. In the same time-point (21 days), expression of epithelial-mesenchymal transition genes was increased (Fn1 and Vim overexpressions) in the kidney. Kidney cysteine dynamic was altered with hypoxia. Over time, we found an overall decrease in total cysteine content and in the protein bound fraction, and a U-shaped variation in their redox couple (reduced/oxidized), suggesting that kidney is susceptible to the oxidative damage triggered by CIH.In addition, we observed, in the kidney, an elevated kynurenine:tryptophan ratio (KTR), revealing a pro-kynurenine state, which may lead to AHR activation. In contrast, we found increased plasma levels of several serotonin-related metabolites. The AHR antagonist, CH-223191, was able to prevent and revert partially the HTN during the active phase of the animals (lights-off period). However, during the inactive phase (lights-on, simultaneous to the hypoxic cycles), the antihypertensive effect of the AHR antagonist was absent. Also, the compound did not recover the loss of the dipping profile of BP and HR originated by CIH. In conclusion, the AHR pathway seems to be overactivated in the kidney, upon CIH exposure, and its pharmacological modulation was able to counteract the increased BP, showing a significant antihypertensive during the active period. These results stimulate further studies to clarify the full potential of this pathway as a novel therapeutic target to HTN induced by CIH/OSA, as well as their mechanisms of action.
ABSTRACT: Obstructive sleep apnea (OSA) is a sleep-related breathing disorder that has becoming a more prevalent clinical condition in recent years. This clinical condition is responsible for a wide range of comorbidities, in particular systemic hypertension (HTN). OSA’s etiopathogenesis includes four clinical features, namely, chronic intermittent hypoxia (CIH), hypercapnia, increased intrathoracic pressure and sleep fragmentation. Among them, CIH is a well-established factor in the development of HTN. OSA is a major cause of resistant HTN, which means that the correct therapeutic strategy to control this particular type of HTN is still undefined. In fact, the antihypertensive effect of continuous positive airway pressure - CPAP (OSA’s gold standard therapeutic procedure) is moderate, and even traditional antihypertensive drugs show some lack of efficacy to control OSA-induced HTN, urging to find new suitable antihypertensive drugs. Therefore, the investigation for novel therapeutic targets is a hot topic in the field of OSA. Animal models of OSA, reproducing its major feature, CIH, have been used to investigate the mechanisms of HTN associated to CIH, in order to seek new therapeutic approaches, as this is the case of the present dissertation. The general goal of this thesis is to find a novel therapeutic target to HTN induced by CIH, with the use of an animal model. Most of the pharmacological approaches for HTN in CIH consisted in the abrogation of the sympathetic and/or the renin-angiotensin-aldosterone systems (RAAS), with some positive results, but still with weak outcomes. Indeed, previous results from our group showed that carvedilol (an α-and β -adrenergic blocker) had no antihypertensive effect in an animal model of HTN induced by CIH. These facts led us to deduce that other signaling pathways directly stimulated by CIH at cellular level should be investigated and pharmacologically manipulated to infer its effect over BP. We believe that transcription factors, as they are involved in long-term adaptive regulations, are plausible mechanistic bridges between the immediate cyclical intermittent hypoxia cycles and the long-term deleterious effects of CIH-OSA, as HTN. With this in mind, four major arguments converge to sustain the assumption that the transcription factor aryl hydrocarbon receptor (AHR) might be a putative mechanistic link between CIH and HTN: 1) Hypoxia inducible factor 1α (HIF-1α), a hypoxic signaling pathway known to be triggered by CIH, and AHR share the same binding partner to assist their canonical activation routes, suggesting a potential crosstalk/interplay between HIF-1 and AHR; 2) AHR has an important role in the regulation of blood pressure; 3) Some of the molecular mechanisms leading to HTN are common for AHR and CIH (RAAS activation, oxidative stress, endothelial dysfunction, regulation of central cardiovascular areas (for instance, in brainstem); 4) CIH, as a chronic inflammatory state, can lead to the overproduction of certain metabolites known to be AHR ligands (e.g. kynurenine metabolites). Thus, the working hypothesis of this thesis is the following: CIH upregulates the AHR circuitry, and consequently contributes to the development of systemic HTN. This thesis aims to investigate, for the first time, the effect of CIH on the AHR circuitry and the consequences of its pharmacological manipulation on HTN induced by CIH. In order to pursue this objective, male Wistar Han rats, aged 8-12 weeks, were exposed to a mild CIH paradigm (5.6 cycles/hour, 10.5 hours/day, from 9.30am to 8pm). We initiated our experiments by exposing groups of rats to a time-course of CIH. Groups of rats were exposed to 14, 21 and 60 days of CIH, and for each time-point, a corresponding control/normoxic (Nx) group was also used (n=5-8/group). At the end of the exposure, the animals were sacrificed and blood and several organs (renal cortex, renal medulla, liver, visceral adipose tissue, spleen and hippocampus) were collected. From those organs, we extracted total ribonucleic acids (RNA) to perform quantitative polymerase chain reaction analysis (qPCR) for several genes, namely genes involved in the AHR pathway (Ahr, Cyp1a1, Cyp1b1, Cyp1a2, Arnt), hypoxia inducible factor (HIF) family (Hif1a, Epas1, Hif3a, Vegfa), NF-kB pathway (Rela, Nfkb2, Il6, Il1b), RAAS members (Ren,Ace, Agtr1) and epithelial-mesenchymal transition and fibrosis markers (Fn1, Vim, Cdh1, Col1a1, Acta2). Moreover, considering that CIH is an oxidative stress disorder, we analyzed, by HighPerformance Liquid Chromatography with Fluorescence Detection (HPLC-FD), the dynamic of cysteine (Cys), an important antioxidant thiol, in the renal tissues of rats exposed to the CIH time-course. We also quantified several tryptophan metabolites by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in plasma and urine of several time-points (1, 7, 14, 21 and 60 days) and in kidney of CIH-exposed rats (21 days). Following these experiments, we modulated pharmacologically the AHR pathway, with an AHR antagonist, CH-223191 (5 mg/kg, once a day, by oral gavage in 1 ml of vegetable oil). We used the antagonist in two major sets of experiments. One set was designed to investigate particularly the preventive role of this compound on CIH-induced HTN, while the other aimed to determine its capacity to revert this outcome. BP and HR measurements were determined by radiotelemetry, at 8am (active period, lights-off) and 6pm (inactive period, lights-on). Following 21 days of CIH exposure, RAAS genes were overexpressed in the kidney, as well as some HIF family genes (Hif1a, Epas1 and Hif3a). Rela mRNA expression was upregulated after 21 days and Nkfb2 after 60 days of CIH, in the renal cortex. Visceral adipose tissue mRNA expressions of Nfkb2 and Il6 were increased, reflecting the inflammatory effects of CIH on the adipose tissue. In the same time-point (21 days), expression of epithelial-mesenchymal transition genes was increased (Fn1 and Vim overexpressions) in the kidney. Kidney cysteine dynamic was altered with hypoxia. Over time, we found an overall decrease in total cysteine content and in the protein bound fraction, and a U-shaped variation in their redox couple (reduced/oxidized), suggesting that kidney is susceptible to the oxidative damage triggered by CIH.In addition, we observed, in the kidney, an elevated kynurenine:tryptophan ratio (KTR), revealing a pro-kynurenine state, which may lead to AHR activation. In contrast, we found increased plasma levels of several serotonin-related metabolites. The AHR antagonist, CH-223191, was able to prevent and revert partially the HTN during the active phase of the animals (lights-off period). However, during the inactive phase (lights-on, simultaneous to the hypoxic cycles), the antihypertensive effect of the AHR antagonist was absent. Also, the compound did not recover the loss of the dipping profile of BP and HR originated by CIH. In conclusion, the AHR pathway seems to be overactivated in the kidney, upon CIH exposure, and its pharmacological modulation was able to counteract the increased BP, showing a significant antihypertensive during the active period. These results stimulate further studies to clarify the full potential of this pathway as a novel therapeutic target to HTN induced by CIH/OSA, as well as their mechanisms of action.
Descrição
Palavras-chave
Obstructive sleep apnea Chronic intermittent hypoxia Systemic hypertension Blood pressure Rat model Cysteine Oxidative stress Kidney Aryl hydrocarbon receptor Kynurenine CH-223191