Jolyane Meloche

Signal Transducers and Activators of Transcription-3/Pim1 Axis Plays a Critical Role in the Pathogenesis of Human Pulmonary Arterial Hypertension

Background— Pulmonary artery hypertension (PAH) is a proliferative disorder associated with enhanced pulmonary artery smooth muscle cell proliferation and suppressed apoptosis. The sustainability of this phenotype required the activation of a prosurvival transcription factor like signal transducers and activators of transcription-3 (STAT3) and nuclear factor of activated T cell (NFAT). Because these factors are implicated in several physiological processes, their inhibition in PAH patients could be associated with detrimental effects. Therefore, a better understanding of the mechanism accounting for their expression/activation in PAH pulmonary artery smooth muscle cells is of great therapeutic interest. Methods and Results— Using multidisciplinary and translational approaches, we demonstrated that STAT3 activation in both human and experimental models of PAH accounts for the expression of both NFATc2 and the oncoprotein kinase Pim1, which trigger NFATc2 activation. Because Pim1 expression correlates with the severity of PAH in humans and is confined to the PAH pulmonary artery smooth muscle cell, Pim1 was identified as an attractive therapeutic target for PAH. Indeed, specific Pim1 inhibition in vitro decreases pulmonary artery smooth muscle cell proliferation and promotes apoptosis, all of which are sustained by NFATc2 inhibition. In vivo, tissue-specific inhibition of Pim1 by nebulized siRNA reverses monocrotaline-induced PAH in rats, whereas Pim1 knockout mice are resistant to PAH development. Conclusion— We demonstrated for the first time that inhibition of the inappropriate activation of STAT3/Pim1 axis is a novel, specific, and attractive therapeutic strategy to reverse PAH.

Role for DNA Damage Signaling in Pulmonary Arterial Hypertension

Background— Pulmonary arterial hypertension (PAH) is associated with sustained inflammation known to promote DNA damage. Despite these unfavorable environmental conditions, PAH pulmonary arterial smooth muscle cells (PASMCs) exhibit, in contrast to healthy PASMCs, a pro-proliferative and anti-apoptotic phenotype, sustained in time by the activation of miR-204, nuclear factor of activated T cells, and hypoxia-inducible factor 1-&agr;. We hypothesized that PAH-PASMCs have increased the activation of poly(ADP-ribose) polymerase-1 (PARP-1), a critical enzyme implicated in DNA repair, allowing proliferation despite the presence of DNA-damaging insults, eventually leading to PAH. Methods and Results— Human PAH distal pulmonary arteries and cultured PAH-PASMCs exhibit increased DNA damage markers (53BP1 and &ggr;-H2AX) and an overexpression of PARP-1 (immunoblot and activity assay), in comparison with healthy tissues/cells. Healthy PASMCs treated with a clinically relevant dose of tumor necrosis factor-&agr; harbored a similar phenotype, suggesting that inflammation induces DNA damage and PARP-1 activation in PAH. We also showed that PARP-1 activation accounts for miR-204 downregulation (quantitative reverse transcription polymerase chain reaction) and the subsequent activation of the transcription factors nuclear factor of activated T cells and hypoxia-inducible factor 1-&agr; in PAH-PASMCs, previously shown to be critical for PAH in several models. These effects resulted in PASMC proliferation (Ki67, proliferating cell nuclear antigen, and WST1 assays) and resistance to apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling and Annexin V assays). In vivo, the clinically available PARP inhibitor ABT-888 reversed PAH in 2 experimental rat models (Sugen/hypoxia and monocrotaline). Conclusions— These results show for the first time that the DNA damage/PARP-1 signaling pathway is important for PAH development and provide a new therapeutic target for this deadly disease with high translational potential.

Dehydroepiandrosterone inhibits the Src/STAT3 constitutive activation in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is an obstructive vasculopathy characterized by enhanced pulmonary artery smooth muscle cell (PASMC) proliferation and suppressed apoptosis. This phenotype is sustained by the activation of the Src/signal transducer and activator of transcription 3 (STAT3) axis, maintained by a positive feedback loop involving miR-204 and followed by an aberrant expression/activation of its downstream targets such as Pim1 and nuclear factor of activated T-cells (NFATc2). Dehydroepiandrosterone (DHEA) is a steroid hormone shown to reverse vascular remodeling in systemic vessels. Since STAT3 has been described as modulated by DHEA, we hypothesized that DHEA reverses human pulmonary hypertension by inhibiting Src/STAT3 constitutive activation. Using PASMCs isolated from patients with PAH (n = 3), we demonstrated that DHEA decreases both Src and STAT3 activation (Western blot and nuclear translocation assay), resulting in a significant reduction of Pim1, NFATc2 expression/activation (quantitative RT-PCR and Western blot), as well as Survivin and upregulation of bone morphogenetic protein receptor 2 (BMPR2) and miR-204. Src/STAT3 axis inhibition by DHEA is associated with 1) mitochondrial membrane potential (tetramethylrhodamine methyl-ester perchlorate; n = 150; P < 0.05) depolarization increasing apoptosis by 25% (terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling; n = 150; P < 0.05); and 2) decreased intracellular Ca(2+) concentration (fluo-3 AM; n = 150; P < 0.05) and proliferation by 30% (PCNA). Finally, in vivo similarly to STAT3 inhibition DHEA improves experimental PAH (monocrotaline rats) by decreasing mean PA pressure and right ventricle hypertrophy. These effects were associated with the inhibition of Src, STAT3, Pim1, NFATc2, and Survivin and the upregulation of BMPR2 and miR-204. We demonstrated that DHEA reverses pulmonary hypertension in part by inhibiting the Src/STAT3.

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

BackgroundPulmonary 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 resultsWe 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.ConclusionWe 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.

MiR-223 Reverses Experimental Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a devastating disease affecting lung vasculature. The pulmonary arteries become occluded due to increased proliferation and suppressed apoptosis of the pulmonary artery smooth muscle cells (PASMCs) within the vascular wall. It was recently shown that DNA damage could trigger this phenotype by upregulating poly(ADP-ribose)polymerase 1 (PARP-1) expression, although the exact mechanism remains unclear. In silico analyses and studies in cancer demonstrated that microRNA miR-223 targets PARP-1. We thus hypothesized that miR-223 downregulation triggers PARP-1 overexpression, as well as the proliferation/apoptosis imbalance observed in PAH. We provide evidence that miR-223 is downregulated in human PAH lungs, distal PAs, and isolated PASMCs. Furthermore, using a gain and loss of function approach, we showed that increased hypoxia-inducible factor 1α, which is observed in PAH, triggers this decrease in miR-223 expression and subsequent overexpression of PARP-1 allowing PAH-PASMC proliferation and resistance to apoptosis. Finally, we demonstrated that restoring the expression of miR-223 in lungs of rats with monocrotaline-induced PAH reversed established PAH and provided beneficial effects on vascular remodeling, pulmonary resistance, right ventricle hypertrophy, and survival. We provide evidence that miR-223 downregulation in PAH plays an important role in numerous pathways implicated in the disease and restoring its expression is able to reverse PAH.

STAT3 signaling in pulmonary arterial hypertension.

Pulmonary artery hypertension (PAH) is a proliferative disorder associated with enhanced pulmonary artery smooth muscle cell proliferation and suppressed apoptosis. The sustainability of this phenotype requires the activation of pro-survival transcription factor like the signal transducers and activators of transcription-3 (STAT3). Using multidisciplinary and translational approaches, we and others have demonstrated that STAT3 activation in both human and experimental models of PAH accounts for the modulation of the expression of several proteins already known as implicated in PAH pathogenesis, as well as for signal transduction to other transcription factors. Furthermore, recent data demonstrated that STAT3 could be therapeutically targeted in different animal models and some molecules are actually in clinical trials for cancer or PAH treatment.

RAGE-Dependent Activation of the Oncoprotein Pim1 Plays a Critical Role in Systemic Vascular Remodeling Processes

Objective— Vascular remodeling diseases (VRD) are mainly characterized by inflammation and a vascular smooth muscle cells (VSMCs) proproliferative and anti-apoptotic phenotype. Recently, the activation of the advanced glycation endproducts receptor (RAGE) has been shown to promote VSMC proliferation and resistance to apoptosis in VRD in a signal transducer and activator of transcription (STAT)3-dependant manner. Interestingly, we previously described in both cancer and VRD that the sustainability of this proproliferative and antiapoptotic phenotype requires activation of the transcription factor NFAT (nuclear factor of activated T-cells). In cancer, NFAT activation is dependent of the oncoprotein provirus integration site for Moloney murine leukemia virus (Pim1), which is regulated by STAT3 and activated in VRD. Therefore, we hypothesized that RAGE/STAT3 activation in VSMC activates Pim1, promoting NFAT and thus VSMC proliferation and resistance to apoptosis. Methods/Results— In vitro, freshly isolated human carotid VSMCs exposed to RAGE activator N&egr;-(carboxymethyl)lysine (CML) for 48 hours had (1) activated STAT3 (increased P-STAT3/STAT3 ratio and P-STAT3 nuclear translocation); (2) increased STAT3-dependent Pim1 expression resulting in NFATc1 activation; and (3) increased Pim1/NFAT-dependent VSMC proliferation (PCNA, Ki67) and resistance to mitochondrial-dependent apoptosis (TMRM, Annexin V, TUNEL). Similarly to RAGE inhibition (small interfering RNA [siRNA]), Pim1, STAT3 and NFATc1 inhibition (siRNA) reversed these abnormalities in human carotid VSMC. Moreover, carotid artery VSMCs isolated from Pim1 knockout mice were resistant to CML-induced VSMC proliferation and resistance to apoptosis. In vivo, RAGE inhibition decreases STAT3/Pim1/NFAT activation, reversing vascular remodeling in the rat carotid artery-injured model. Conclusion— RAGE activation accounts for many features of VRD including VSMC proliferation and resistance to apoptosis by the activation of STAT3/Pim1/NFAT axis. Molecules aimed to inhibit RAGE could be of a great therapeutic interest for the treatment of VRD.

Adaptation and Remodelling of the Pulmonary Circulation in Pulmonary Hypertension

Pulmonary arterial hypertension (PAH) is characterized by remodelling of pulmonary arteries caused by a proliferation/apoptosis imbalance within the vascular wall. This pathological phenotype seems to be triggered by different environmental stress and injury events such as increased inflammation, DNA damage, and epigenetic deregulation. It appears that one of the first hit to occur is endothelial cells (ECs) injury and apoptosis, which leads to paracrine signalling to other ECs, pulmonary artery smooth muscle cells (PASMCs), and fibroblasts. These signals promote a phenotypic change of surviving ECs by disturbing different signalling pathways leading to sustained vasoconstriction, proproliferative and antiapoptotic phenotype, deregulated angiogenesis, and formation of plexiform lesions. EC signalling also recruits proinflammatory cells, leading to pulmonary infiltration of lymphocytes, macrophages, and dendritic cells, sustaining the inflammatory environment and autoimmune response. Finally, EC signalling promotes proliferative and antiapoptotic PAH-PASMC phenotypes, which acquire migratory capacities, resulting in increased vascular wall thickness and muscularization of small pulmonary arterioles. Adaptation and remodelling of pulmonary circulation also involves epigenetic components, such as microRNA deregulation, DNA methylation, and histone modification. This review will focus on the different cellular and epigenetic aspects including EC stress response, molecular mechanisms contributing to PAH-PASMC and PAEC proliferation and resistance to apoptosis, as well as epigenetic control involved in adaptation and remodelling of the pulmonary circulation in PAH.

Potassium-Channel Subfamily K-Member 3 (KCNK3) Contributes to the Development of Pulmonary Arterial Hypertension

Background— Mutations in the KCNK3 gene have been identified in some patients suffering from heritable pulmonary arterial hypertension (PAH). KCNK3 encodes an outward rectifier K+ channel, and each identified mutation leads to a loss of function. However, the pathophysiological role of potassium channel subfamily K member 3 (KCNK3) in PAH is unclear. We hypothesized that loss of function of KCNK3 is a hallmark of idiopathic and heritable PAH and contributes to dysfunction of pulmonary artery smooth muscle cells and pulmonary artery endothelial cells, leading to pulmonary artery remodeling: consequently, restoring KCNK3 function could alleviate experimental pulmonary hypertension (PH). Methods and Results— We demonstrated that KCNK3 expression and function were reduced in human PAH and in monocrotaline-induced PH in rats. Using a patch-clamp technique in freshly isolated (not cultured) pulmonary artery smooth muscle cells and pulmonary artery endothelial cells, we found that KCNK3 current decreased progressively during the development of monocrotaline-induced PH and correlated with plasma-membrane depolarization. We demonstrated that KCNK3 modulated pulmonary arterial tone. Long-term inhibition of KCNK3 in rats induced distal neomuscularization and early hemodynamic signs of PH, which were related to exaggerated proliferation of pulmonary artery endothelial cells, pulmonary artery smooth muscle cell, adventitial fibroblasts, and pulmonary and systemic inflammation. Lastly, in vivo pharmacological activation of KCNK3 significantly reversed monocrotaline-induced PH in rats. Conclusions— In PAH and experimental PH, KCNK3 expression and activity are strongly reduced in pulmonary artery smooth muscle cells and endothelial cells. KCNK3 inhibition promoted increased proliferation, vasoconstriction, and inflammation. In vivo pharmacological activation of KCNK3 alleviated monocrotaline-induced PH, thus demonstrating that loss of KCNK3 is a key event in PAH pathogenesis and thus could be therapeutically targeted.

Bromodomain Containing Protein-4: The Epigenetic Origin of Pulmonary Arterial Hypertension

Rationale: Pulmonary arterial hypertension (PAH) is a vasculopathy characterized by enhanced pulmonary artery (PA) smooth muscle cell (PASMC) proliferation and suppressed apoptosis. Decreased expression of microRNA-204 has been associated to this phenotype. By a still elusive mechanism, microRNA-204 downregulation promotes the expression of oncogenes, including nuclear factor of activated T cells, B-cell lymphoma 2, and Survivin. In cancer, increased expression of the epigenetic reader bromodomain-containing protein 4 (BRD4) sustains cell survival and proliferation. Interestingly, BRD4 is a predicted target of microRNA-204 and has binding sites on the nuclear factor of activated T cells promoter region. Objective: To investigate the role of BRD4 in PAH pathogenesis. Methods and Results: BRD4 is upregulated in lungs, distal PAs, and PASMCs of patients with PAH compared with controls. With mechanistic in vitro experiments, we demonstrated that BRD4 expression in PAH is microRNA-204 dependent. We further studied the molecular downstream targets of BRD4 by inhibiting its activity in PAH–PASMCs using a clinically available inhibitor JQ1. JQ1 treatment in PAH–PASMCs increased p21 expression, thus triggering cell cycle arrest. Furthermore, BRD4 inhibition, by JQ1 or siBRD4, decreased the expression of 3 major oncogenes, which are overexpressed in PAH: nuclear factor of activated T cells, B-cell lymphoma 2, and Survivin. Blocking this oncogenic signature led to decreased PAH-PASMC proliferation and increased apoptosis in a BRD4-dependent manner. Indeed, pharmacological JQ1 or molecular (siRNA) inhibition of BRD4 reversed this pathological phenotype in addition to restoring mitochondrial membrane potential and to increasing cells spare respiratory capacity. Moreover, BRD4 inhibition in vivo reversed established PAH in the Sugen/hypoxia rat model. Conclusions: BRD4 plays a key role in the pathological phenotype in PAH, which could offer new therapeutic perspectives for patients with PAH. # Novelty and Significance {#article-title-53}RATIONALE Pulmonary arterial hypertension (PAH) is a vasculopathy characterized by enhanced pulmonary artery (PA) smooth muscle cell (PASMC) proliferation and suppressed apoptosis. Decreased expression of microRNA-204 has been associated to this phenotype. By a still elusive mechanism, microRNA-204 downregulation promotes the expression of oncogenes, including nuclear factor of activated T cells, B-cell lymphoma 2, and Survivin. In cancer, increased expression of the epigenetic reader bromodomain-containing protein 4 (BRD4) sustains cell survival and proliferation. Interestingly, BRD4 is a predicted target of microRNA-204 and has binding sites on the nuclear factor of activated T cells promoter region. OBJECTIVE To investigate the role of BRD4 in PAH pathogenesis. METHODS AND RESULTS BRD4 is upregulated in lungs, distal PAs, and PASMCs of patients with PAH compared with controls. With mechanistic in vitro experiments, we demonstrated that BRD4 expression in PAH is microRNA-204 dependent. We further studied the molecular downstream targets of BRD4 by inhibiting its activity in PAH-PASMCs using a clinically available inhibitor JQ1. JQ1 treatment in PAH-PASMCs increased p21 expression, thus triggering cell cycle arrest. Furthermore, BRD4 inhibition, by JQ1 or siBRD4, decreased the expression of 3 major oncogenes, which are overexpressed in PAH: nuclear factor of activated T cells, B-cell lymphoma 2, and Survivin. Blocking this oncogenic signature led to decreased PAH-PASMC proliferation and increased apoptosis in a BRD4-dependent manner. Indeed, pharmacological JQ1 or molecular (siRNA) inhibition of BRD4 reversed this pathological phenotype in addition to restoring mitochondrial membrane potential and to increasing cells spare respiratory capacity. Moreover, BRD4 inhibition in vivo reversed established PAH in the Sugen/hypoxia rat model. CONCLUSIONS BRD4 plays a key role in the pathological phenotype in PAH, which could offer new therapeutic perspectives for patients with PAH.