Un microarn au coeur de l'hypertension artérielle pulmonaire

L’hypertension artérielle pulmonaire (HTAP) est caractérisée par l’obstruction des artères pulmonaires, principalement due au phénotype pro-prolifératif/anti-apoptotique des cellules musculaires lisses de la paroi des artères pulmonaires (CMLAP). L’augmentation progressive des résistances vasculaires pulmonaires aboutit à une élévation de la pression pulmonaire qui va induire rapidement une insuffisance cardiaque droite et conduire au décès des patients à moyen terme. Plusieurs études ont démontré l’implication du facteur de transcription NFAT (nuclear factor of activated T cell) dans le maintien du phénotype pro-prolifératif/anti-apoptotique des CMLAP-HTAP. Cependant les voies de signalisation responsables de l’activation constitutive d’NFAT restent peu connues. Durant mon doctorat, j’ai étudié les mécanismes responsables de l’activation d’NFAT dans l’HTAP. Nous nous sommes intéressés au rôle des microARN et notamment à miR-204. Ainsi, les facteurs circulants augmentés dans HTAP, diminue l’expression de miR-204 via l’activation du facteur de transcription STAT3. Par un mécanisme de rétro-action positive, la diminution de miR-204 induit une suractivation de STAT3 aboutissant au phénotype pathologique. Ainsi, l’augmentation exogène de miR-204 permettrait de soigner l’HTAP in vitro et in vivo. Nous avons montré que miR-204 va également moduler l’expression de Runx2, facteur de transcription connu pour être impliqué dans la calcification. Dans les CMLAP-HTAP, la diminution de miR-204 est associée à une augmentation de l’expression de Runx2, connu comme un régulateur positif de l’activation du facteur de transcription HIF-1 impliqué dans l’HTAP. Ainsi la modulation de miR-204 affecte la prolifération et l’apoptose des CMLAP-HTAP par plusieurs axes de signalisation. Enfin, nous avons démontré l’implication du facteur de transcription Krüppel Like Factor 5 (KLF5) dans l’HTAP. La surexpression de KLF5 dans l’HTAP est secondaire à l’activation de STAT3, tandis que son inhibition diminue la prolifération et favorise l’apoptose des CMLAP-HTAP. In vivo, l’administration de siKLF5 renverse l’HTAP en diminuant les pressions pulmonaires, l’hypertrophie ventriculaire droite, la prolifération et augmentant l’apoptose des CMLAP des artères pulmonaires distales. Finalement, j’ai étudié différents aspects du développement de l’HTAP et notamment de l’activation de l’axe STAT3/NFAT. Nous avons pu mettre en évidence que cibler cette voie de signalisation par différents moyens (mimic miR-204, siRunx2, siSTAT3, siKLF5) semble une bonne stratégie pour traiter l’HTAP. Mots clés : l’hypertension artérielle pulmonaire, thérapeutique, prolifération, apoptose, microARN, facteur de transcription, réparation à l’ADN.Pulmonary arterial hypertension (PAH) is characterized by the obstruction of the pulmonary arteries, mainly due to the pro-proliferative and anti-apoptotic phenotype of the pulmonary artery smooth muscle cells (PASMC). The progressive increase of pulmonary vascular resistance first leads to an increase of pulmonary pressure and then leads to a right heart failure, which generates patient’s death within few years. Many studies demonstrated the implication of the transcription factor NFAT (nuclear factor of activated T cell), which maintains the pro-proliferative and anti-apoptotic phenotype in PAH-PASMC. However, pathways that lead to the constitutive NFAT activation remain unclear. During my doctorate, I studied mechanisms responsible for the activiation of NFAT in HTAP. We study the role of the microRNA and more exactly to miR-204. Thus, the circulating factors, which are increased in PAH and which decreased miR-204 expression in PAH, via the transcription factor STAT3 activation. Through a positive regulation loop mechanism, the decrease of miR-204 induces an overactivation sustain of STAT3 leading to the pathologique phenotype. Thus, the exogenous increase of miR-204 could treat PAH in vitro as well as in vivo. We demonstrated that miR-204 is able to modulate the expression of the transcription factor Runx2 known to be implicated in calcification. In PAH-PASMC, the decrease of miR-204 is associated to an increase of Runx2 expression, known as positive regulator of the HIF-1 activation implicated in PAH. Thus miR-204 modulations affected the proliferation and apoptosis of PAH-PASMC through many molecular axes. Finaly we reveal the implication of the transcription factor Kruppel Like Factor 5 (KLF5) in PAH. The KLF5 overexpressed in PAH is associated to the STAT3 activation, wherease its inhibition decreased the proliferation and promoted apoptosis in PAH-PASMC. In vivo, si KLF5 reversed PAH by decreasing pulmonary pressures, right ventricular hypertrophy, proliferation and increasing apoptosis in PASMC from distal PA. Finally, I studied many aspects implicated in PAH development and especially the STAT3/NFAT axis activation. We showed that targeting this pathway using many technics (mimic miR-204, siRunx2, siSTAT3, siKLF5) seem to be an interesting strategy to treat PAH. Key words: Pulmonary arterial hypertension, therapeutic, proliferation, apoptosis, microRNA, and transcription factor

Role for miR-204 in human pulmonary arterial hypertension

Reduced miR-204 expression facilitates the excessive proliferation and apoptosis resistance of pulmonary artery smooth muscle cells characteristic of human pulmonary arterial hypertension

Increasing pulmonary artery pulsatile flow improves hypoxic pulmonary hypertension in piglets

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular failure. Current treatments are limited. Physiologically, pulsatile blood flow is detrimental to the vasculature. In response to sustained pulsatile stress, vessels release nitric oxide (NO) to induce vasodilation for self-protection. Based on this observation, this study developed a protocol to assess whether an artificial pulmonary pulsatile blood flow could induce an NO-dependent decrease in pulmonary artery pressure. One group of piglets was exposed to chronic hypoxia for 3 weeks and compared to a control group of piglets. Once a week, the piglets underwent echocardiography to assess PAH severity. At the end of hypoxia exposure, the piglets were subjected to a pulsatile protocol using a pulsatile catheter. After being anesthetized and prepared for surgery, the jugular vein of the piglet was isolated and the catheter was introduced through the right atrium, the right ventricle and the pulmonary artery, under radioscopic control. Pulmonary artery pressure (PAP) was measured before (T0), immediately after (T1) and 30 min after (T2) the pulsatile protocol. It was demonstrated that this pulsatile protocol is a safe and efficient method of inducing a significant reduction in mean PAP via an NO-dependent mechanism. These data open up new avenues for the clinical management of PAH

Olfactory receptors in pulmonary arterial hypertension: A novel pathway of vascular remodeling?

Pulmonary arterial hypertension (PAH) is due to progressive obstruction of pulmonary arteries, thus leading to right heart failure and death. Breath volatile organic compounds (VOCs) can discriminate PAH and controls. Thus, a unique breath-print of PAH is detected using an artificial nose. VOCs target olfactory receptors (ORs) in olfaction. Interestingly, ORs are detected in peripheral tissues not related to olfaction and their deregulation is associated to cancer development. PSGR, encoded by the OR51E2 gene, is one of the ORs. Because vascular cells in PAH exhibit properties of cancer cells, we propose the ground-breaking hypothesis that ORs participate to vascular remodeling leading to PAH. Thus we aim to determine whether a deregulated expression and function of the pulmonary vascular PSGR could participate to the pathological phenotype of vascular cells, and its potential use as a novel therapeutic target in PAH. PSGR gene and protein expression were assessed in total lung, distal pulmonary arteries and PASMCs from PAH patients compared to controls using qRT-PCR and western blot. We evaluated proliferation (Ki67) and apoptosis (TMRM) after siRNA-directed silencing of PSGR expression in PASMCs. We demonstrate that PSGR expression is significantly increased (50%) in PASMC, in total lung and isolated pulmonary arteries from PAH patients compared to controls. PSGR silencing in PAH-PASMCs decreased both cell proliferation (20%) and resistance to apoptosis (25%). This deregulated OR expression in PAH PASMCs opens a new avenue in PAH pathophysiology. The whole spectrum of ORs is currently investigated using microarrays and deregulated ORs will be evaluated both in vitro and in vivo

Krüppel-like Factor 5 contributes to pulmonary artery smooth muscle proliferation and resistance to apoptosis in human pulmonary arterial hypertension

Background Pulmonary arterial hypertension (PAH) is a vascular remodeling disease characterized by enhanced proliferation of pulmonary artery smooth muscle cell (PASMC) and suppressed apoptosis. This phenotype has been associated with the upregulation of the oncoprotein survivin promoting mitochondrial membrane potential hyperpolarization (decreasing apoptosis) and the upregulation of growth factor and cytokines like PDGF, IL-6 and vasoactive agent like endothelin-1 (ET-1) promoting PASMC proliferation. Krüppel-like factor 5 (KLF5), is a zinc-finger-type transcription factor implicated in the regulation of cell differentiation, proliferation, migration and apoptosis. Recent studies have demonstrated the implication of KLF5 in tissue remodeling in cardiovascular diseases, such as atherosclerosis, restenosis, and cardiac hypertrophy. Nonetheless, the implication of KLF5 in pulmonary arterial hypertension (PAH) remains unknown. We hypothesized that KLF5 up-regulation in PAH triggers PASMC proliferation and resistance to apoptosis. Methods and results We showed that KFL5 is upregulated in both human lung biopsies and cultured human PASMC isolated from distal pulmonary arteries from PAH patients compared to controls. Using stimulation experiments, we demonstrated that PDGF, ET-1 and IL-6 trigger KLF-5 activation in control PASMC to a level similar to the one seen in PAH-PASMC. Inhibition of the STAT3 pathway abrogates KLF5 activation in PAH-PASMC. Once activated, KLF5 promotes cyclin B1 upregulation and promotes PASMC proliferation and triggers survivin expression hyperpolarizing mitochondria membrane potential decreasing PASMC ability to undergo apoptosis. Conclusion We demonstrated for the first time that KLF5 is activated in human PAH and implicated in the pro-proliferative and anti-apoptotic phenotype that characterize PAH-PASMC. We believe that our findings will open new avenues of investigation on the role of KLF5 in PAH and might lead to the identification of new therapeutic targets.</p

PSGR olfactory receptor: A new potential target in pulmonary arterial hypertension

International audiencePulmonary arterial hypertension {PAH) is a rare progressive disease due to dista lvascular remodeling, leading to right heart failure and death. PSGR is an olfactory receptor {OR) that has been recently detected in peripheral tissues. Moreover, PSGR overexpression is associated with pro-proliferative phenotype in prostate cancer. Since PAH vascular cells are characterized by cancer-like over-proliferation, we hypothesized that PSGR might participate in the vascular remodeling leading to PAH. Here we aimed to determine whether upregulation of PSGR is implicated in PAH pathological phenotype, and to explore PSGR as a novel therapeutic target in PAH. PSGR gene and protein expressions were assessed in total lung, distal pulmonary arteries and Pulmonary Artery Smooth Muscle Cells {PASMC) and Endothelial Cells from PAH patients and controls using qRT-PCR and western blot. We evaluated proliferation and apoptosis using Ki67 and TMRM. siRNAdirected silencing of PSGR and STAT3, was used to inhibit the specific expression in PASMCs, whereas PP2 was used to inhibit Src activation. We demonstrate that PSGR expression is significantly increased in PASMCs and isolated pulmonary arteries of PAH patients. We also show a trend to decreased Src activation and restored BMPR2 expression as a function of PSGR inhibition in PAH-PASMC. Moreover, inhibition of STAT3 and/or Src partially decreased PSGR mRNA expression. PSGR silencing reversed the PAH pro-proliferative phenotype in human PASMC. To conclude, overexpression of PSGR leads to a pro-proliferative phenotype of PASMCs in PAH, which could be decreased by PSGR inhibition. Src-STAT3 pathway activation is potentially the bridge linking PSGR and the pathophysiology

Functional interaction between PDGFβ and GluN2B-containing NMDA receptors in smooth muscle cell proliferation and migration in pulmonary arterial hypertension

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