Cellular senescence and new therapeutic approaches for pulmonary hypertension

L'hypertension artérielle pulmonaire (HTAP) idiopathique ou associée à une maladie sous-jacente est un trouble inexpliqué dont les formes graves, chez l'adulte comme le nouveau-né, restent mortels et pour lequel à ce jour, aucun traitement satisfaisant n'est disponible. Elle est caractérisée par une augmentation anormale de la pression artérielle pulmonaire (PAP) moyenne supérieur à 25 mmHg au repos et 30 mmHg à l'effort alors que chez un sujet sain elle est comprise entre 10 et 15 mmHg. L'hyperplasie des cellules musculaires lisses artérielle pulmonaire (CML-AP) est le principal déterminant du processus de remodelage vasculaire pulmonaire qui sous-tend l'hypertension artérielle pulmonaire.Il existe cependant des similitudes entre le cancer et l'hypertension artérielle pulmonaire. En effet, certains dysfonctionnements cellulaires et métaboliques sont communs aux deux maladies. En effet, des anomalies sont souvent retrouvées à la fois dans le cancer et dans les pathologies prolifératives, comme une mutation du gène codant pour le facteur de transcription p53, l'inactivation post-transcriptionnelle de p53 via l'inhibition de son interaction avec son régulateur négatif MDM2 (murine à double minute 2), l'augmentation de l'activité de la télomérase ainsi que la translocation de la sous-unité TERT vers la mitochondrie.Un moyen thérapeutique de choix dans le cancer ou autre pathologie proliférative est l'induction d'une sénescence cellulaire.Dans notre première étude, nous avons induit la sénescence des CML-AP en inhibant l'interaction p53-MDM2 en utilisant la Nutlin-3a, antagoniste spécifique et puissant de la liaison p53-MDM2, afin de limiter sa dégradation par le protéasome et ainsi augmenter son activité. Dans notre deuxième étude nous avons induit une sénescence des CML-AP en inhibant génétiquement ou pharmacologiquement la télomérase. Ces deux études ont montré que l'induction d'une sénescence cellulaire prévient et réverse partiellement l'HTAP dans différents modèles expérimentaux, en diminuant le nombre de CML-AP prolifératives et en augmentant le nombre de cellules sénescentes (p21-positives) sans induire davantage d'apoptose. Nous avons également confirmé l'implication de l'activité de la télomérase dans le développement de l'hypertension artérielle pulmonaire expérimentale ainsi que la translocation de la sous unité TERT dans la mitochondrie au cour de l'HTAP.La sénescence cellulaire semble donc être une cible thérapeutique de choix dans le traitement de l'hypertension artérielle pulmonaire.Pulmonary artery hypertension (PH) occurring as an idiopathic condition or associated with an underlying disease is an unexplained disorder whose sever forms in adults and neonates are fatal and for which no satisfactory treatment is available. PH is characterized by an abnormal increase in pulmonary artery pressure (PAP) greater than 25 mmHg at rest and 30 mmHg during exercise while in a healthy person is between 10 and 15 mmHg. Hyperplasia of pulmonary artery smooth muscle cells (PA-SMCs) is the primary determinant of the pulmonary vessel remodeling process that underlies PH.However, similarities exist between cancer and PH. Indeed, some cellular and metabolic dysfunctions are common to both diseases. Abnormalities are often found in both cancer and proliferative disorders, such as an inactivating mutation in the gene encoding the transcription factor p53, a post-transcriptional p53 inactivation via interaction of the p53 protein with its negative regulator MDM2 (mouse double minute 2), increasing the telomerase activity and translocation of the TERT subunit to mitochondria.A selecting means in therapeutic cancer or other proliferative diseases is the induction of cellular senescence.In our first study, we induced CML-AP senescence by inhibiting the p53-MDM2 interaction using Nutlin-3a, a specific and potent antagonist of the p53-MDM2 binding, to limit its degradation by the proteasome and thus increase its activity. In our second study we induced CML-AP senescence by genetical or pharmacological inhibition of telomerase. Both studies showed that the induction of cellular senescence in PA-SMCs prevents and partially reverses PH in different experimental models, by reducing the number of proliferative CML-AP and increasing the number of (p21-positive) senescent cells without inducing more apoptosis. We also confirmed the involvement of telomerase activity and the translocation of the TERT subunit in the mitochondria during PH development

Rapamycin reverses pulmonary artery smooth muscle cell proliferation in pulmonary hypertension.: mTOR Inhibition in Pulmonary Hypertension

International audiencePulmonary artery (PA) smooth muscle cell (SMC) proliferation in pulmonary hypertension (PH) may be linked to dysregulated mammalian target of rapamycin (mTOR) signaling. The mTOR pathway involves two independent complexes, mTORC1 and mTORC2, which phosphorylate S6 kinase (S6K) and serine/threonine kinase (Akt), respectively, and differ in their sensitivity to rapamycin. Here, we evaluated rapamycin-sensitive mTOR substrates and PA-SMC proliferation in rats with monocrotaline (MCT)-induced PH (MCT-PH). Compared with cells from control rats, cultured PA-SMCs from MCT-PH rats exhibited increased growth responses to platelet-derived growth factor, serotonin (5-hydroxytryptophan), IL-1β, insulin-like growth factor-1, or fetal calf serum (FCS), with increases in phosphorylated (Ser-473)Akt, (Thr-308)Akt, glycogen synthase kinase (GSK)3, and S6K reflecting activated mTORC1 and mTORC2 signaling. Treatment with rapamycin (0.5 μM) or the Akt inhibitor, A-443654 (0.5 μM), reduced FCS-stimulated growth of PA-SMCs from MCT-PH rats to the level in control rats while inhibiting Akt, GSK3, and S6K activation. Neither the tyrosine kinase inhibitor, imatinib (0.1 μM), nor the 5-hydroxytryptophan transporter inhibitor, fluoxetine (5 μM), normalized the increased PA-SMC growth response to FCS. Rapamycin treatment (5 mg/kg/d) of MCT-PH rats from Day 21 to Day 28 markedly reduced phoshop (p)-Aky, p-GSK3, and p-S6K in PAs, and normalized growth of derived PA-SMCs. This effect was not observed after 1 week of imatinib (100 mg/kg/d) or fluoxetine (20 mg/kg/d). Rapamycin given preventively (Days 1-21) or curatively (Days 21-42) inhibited MCT-PH to a greater extent than did imatinib or fluoxetine. Experimental PH in rats is associated with a sustained proliferative PA-SMC phenotype linked to activation of both mTORC1 and mTORC2 signaling and is suppressed by rapamycin treatment

Activation of Lung p53 by Nutlin-3a Prevents and Reverses Experimental Pulmonary Hypertension.

International audienceBACKGROUND: Induction of cellular senescence through activation of the p53 tumor suppressor protein is a new option for treating proliferative disorders. Nutlins prevent the ubiquitin ligase MDM2 (murine double minute 2), a negative p53 regulator, from interacting with p53. We hypothesized that cell senescence induced by Nutlin-3a exerted therapeutic effects in pulmonary hypertension (PH) by limiting the proliferation of pulmonary artery smooth muscle cells (PA-SMCs). METHODS AND RESULTS: Nutlin-3a treatment of cultured human PA-SMCs resulted in cell growth arrest with the induction of senescence but not apoptosis; increased phosphorylated p53 protein levels; and expression of p53 target genes including p21, Bax, BTG2, and MDM2. Daily intraperitoneal Nutlin-3a treatment for 3 weeks dose-dependently reduced PH, right ventricular hypertrophy, and distal pulmonary artery muscularization in mice exposed to chronic hypoxia or SU5416/hypoxia. Nutlin-3a treatment also partially reversed PH in chronically hypoxic or transgenic mice overexpressing the serotonin-transporter in SMCs (SM22-5HTT+ mice). In these mouse models of PH, Nutlin-3a markedly increased senescent p21-stained PA-SMCs; lung p53, p21, and MDM2 protein levels; and p21, Bax, PUMA, BTG2, and MDM2 mRNA levels; but induced only minor changes in control mice without PH. Marked MDM2 immunostaining was seen in both mouse and human remodeled pulmonary vessels, supporting the use of Nutlins as a PH-targeted therapy. PH prevention or reversal by Nutlin-3a required lung p53 stabilization and increased p21 expression, as indicated by the absence of Nutlin-3a effects in hypoxia-exposed p53(-/-) and p21(-/-) mice. CONCLUSIONS: Nutlin-3a may hold promise as a prosenescence treatment targeting PA-SMCs in PH

Inhibition of gut- and lung-derived serotonin attenuates pulmonary hypertension in mice.

International audienceDecreasing the bioavailability of serotonin (5-HT) by inhibiting its biosynthesis may represent a useful adjunctive treatment of pulmonary hypertension (PH). We assessed this hypothesis using LP533401, which inhibits the rate-limiting enzyme tryptophan hydroxylase 1 (Tph1) expressed in the gut and lung, without inhibiting Tph2 expressed in neurons. Mice treated repeatedly with LP533401 (30-250 mg/kg per day) exhibited marked 5-HT content reductions in the gut, lungs, and blood, but not in the brain. After a single LP533401 dose (250 mg/kg), lung and gut 5-HT contents decreased by 50%, whereas blood 5-HT levels remained unchanged, suggesting gut and lung 5-HT synthesis. Treatment with the 5-HT transporter (5-HTT) inhibitor citalopram decreased 5-HT contents in the blood and lungs but not in the gut. In transgenic SM22-5-HTT+ mice, which overexpress 5-HTT in smooth muscle cells and spontaneously develop PH, 250 mg/kg per day LP533401 or 10 mg/kg per day citalopram for 21 days markedly reduced lung and blood 5-HT levels, right ventricular (RV) systolic pressure, RV hypertrophy, distal pulmonary artery muscularization, and vascular Ki67-positive cells (P < 0.001). Combined treatment with both drugs was more effective in improving PH-related hemodynamic parameters than either drug alone. LP533401 or citalopram treatment partially prevented PH development in wild-type mice exposed to chronic hypoxia. Lung and blood 5-HT levels were lower in hypoxic than in normoxic mice and decreased further after LP533401 or citalopram treatment. These results provide proof of concept that inhibiting Tph1 may represent a new therapeutic strategy for human PH

Role for Telomerase in Pulmonary Hypertension

-Cells exhibiting dysregulated growth may express telomerase reverse transcriptase (TERT), whose dual function consists in maintaining telomere length, in association with the RNA template molecule TERC and controlling cell growth. Here, we investigated lung TERT in human and experimental pulmonary hypertension (PH) and its role in controlling pulmonary artery-smooth muscle cell (PA-SMC) proliferation.status: publishe