Abstract 17072: Acetylcholine Signaling and Endothelial to Mesenchymal Transition in Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is characterized by severe pulmonary vascular remodeling. Transition of endothelial cells (EC) to mesenchymal cells (EndMT) contributes to vascular remodeling; however, the role of EndMT and underlying mechanisms in PAH remain unclear. While nicotinic acetylcholine (Ach) receptor (nAChR)-mediated pathway regulates epithelial to mesenchymal transition and promotes mesenchymal cell proliferation, its role in EndMT is unknown. In this study, we investigate EndMT in PAH and delineate the mechanisms. Methods: PAH was induced in Sprague Dawley rats by SU5416 (20 mg/kg; s.c.), followed by 3 wks of hypoxia (3 wk PAH) or 3wks of hypoxia with additional 4 wks of normoxia (7 wk PAH). Rats without SU5416 and kept at normoxia served as controls. At the end of experiments, hemodynamic measurements were performed. Lung EC were then isolated and purified using CD31 antibody conjugated beads and passage 3-4 were used. EndMT was assessed by dual staining of EC markers (von Willebrand factor and Griffonia simplicifolia) and α-smooth muscle actin (α-SMA) and by the mRNA expression of EC and mesenchymal genes. Lung ACh was measured by ELISA. Results: PAH rats had elevated PA pressures at both 3 wk (PAH vs. CON in mm Hg: 77.3 vs. 24.8, p<0.05, n=4-5) and 7 wk (65.5 vs. 28.6, p<0.05, n=10-12). EndMT was evidenced in PAH (% of cells positive with both EC markers and α- SMA: 3 wks: PAH 4.2% vs. CON 1.6%, p<0.05; 7 wks: PAH 38.5% vs. CON 4.5%, p<0.05), which was associated with significantly increased expression of mesenchymal gene (CD44, PAI1, and α-SMA) and decreased expression of vascular endothelial cadherin at 7 wk. The lungs of PAH rats had higher levels of ACh (PAH vs. CON in pg/mL, 3wk: 78.7 vs. 47.3, p<0.05, n=7; 7wk: 85.1 vs. 45.6, p<0.05, n=7). A significant increase in α7 nAChR expression was found in 7wk PAH EC. In vitro , treatment with ACh markedly induced EndMT in lung EC from normal rats, which was assessed by increased dual staining of EC markers and α-SMA and increased expression of mesenchymal genes (α-SMA, PAI1, and pro-collagen 1, p<0.05 for all). Conclusions: EndMT in PAH is associated with increased lung ACh levels and α7nAChR expression in lung ECs. ACh/nAChR-mediated EndMT may contribute to the vascular remodeling in PAH.

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Abstract 18446: Identification of a Novel Cellular Actor Participating in Vascular Remodeling During Pulmonary Arterial Hypertension : the PW1+ Progenitor Cells

Circulation ◽

10.1161/circ.130.suppl_2.18446 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

France Dierick

Keyword(s):

Bone Marrow ◽

Pulmonary Arterial Hypertension ◽

Arterial Hypertension ◽

Vascular Remodeling ◽

Bone Marrow Cells ◽

Mouse Lung ◽

Pulmonary Vessels ◽

Pulmonary Arterial ◽

Lung Vessels

AIM: PW1+ progenitors were identified in various adult tissues and can differentiate in smooth muscle cells (SMC) in vitro. Our hypothesis is that PW1+ progenitors are recruited to participate in the vascular remodeling during pulmonary arterial hypertension (PAH). METHODS: PW1IRESnLacZ+/- mice express the β-galactosidase as a reporter gene for PW1 expression allowing to follow the lineage of PW1+ cells during a few days. These mice were exposed to chronic hypoxia (CH) to induce PAH, lung vessels neomuscularisation and SMC proliferation. PW1+ and β-Gal+ cells were studied by FACS and by immunofluorescence. RESULTS: PW1+ cells are localized in the lung parenchyma and in the perivascular zone in rodent and human lung. Two PW1+ populations were identified by flow cytometry in the mouse lung 1/ a Sca-1high/CD34high/PDGFR-α+ population which differentiates into calponin+ or α-SMA+ SMC and into vWF+ endothelial cell and 2/ a CD34-/CD146+ population expressing pericyte markers. After 2-4 days of CH, the number of lung PW1+ cells is increased (x3.5, p<0.01) and, in small pulmonary vessels media, the proportion of β-Gal+ SMC derived from PW1+ cells is increased (64±6% vs 35±3%, p<0.05) suggesting a recruitment and differentiation of PW1+ cells into lung vascular SMC. Moreover WT mice irradiated and engrafted with GFP+/β-Gal+ bone marrow cells do not show any increase in GFP+ SMC in lung vessels and do not show any β-Gal+ cells in the lung indicating that the lung PW1+ progenitors are not derived from bone marrow . Moreover, in the human PAH lung, PW1+ cells were observed in remodeled vascular structures: in the media of remodeled vessel and in plexiform lesions. CONCLUSION: These results suggest that lung resident PW1+ progenitors are recruited to participate in the vascular remodeling of small pulmonary vessels in experimental and human PAH. These progenitors show characteristics of pericytes and of vascular progenitors.

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LncRNA-SMILR modulates RhoA/ROCK signaling by targeting miR-141 to regulate vascular remodeling in pulmonary arterial hypertension

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00717.2019 ◽

2020 ◽

Vol 319 (2) ◽

pp. H377-H391 ◽

Cited By ~ 1

Author(s):

Si Lei ◽

Fei Peng ◽

Mei-Lei Li ◽

Wen-Bing Duan ◽

Cai-Qin Peng ◽

...

Keyword(s):

Smooth Muscle ◽

Pulmonary Arterial Hypertension ◽

Arterial Hypertension ◽

Long Noncoding Rna ◽

Vascular Remodeling ◽

Noncoding Rna ◽

In Vivo Models ◽

Pulmonary Arterial

Smooth muscle-enriched long noncoding RNA (SMILR), as a long noncoding RNA (lncRNA), was increased in pulmonary arterial hypertension (PAH) patients and in vitro and in vivo models. SMILR activated RhoA/ROCK signaling by targeting miR-141 to disinhibit its downstream target RhoA. SMILR knockdown or miR-141 overexpression inhibited hypoxia-induced cell proliferation and migration via repressing RhoA/ROCK signaling in pulmonary arterial smooth muscle cells (PASMCs), which was confirmed in vivo experiments that knockdown of SMILR inhibited vascular remodeling and alleviated PAH in rats. SMILR may be a promising and novel therapeutic target for the treatment and drug development of PAH.

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Epigenetic Inheritance Underlying Pulmonary Arterial Hypertension

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.118.312262 ◽

2019 ◽

Vol 39 (4) ◽

pp. 653-664 ◽

Cited By ~ 23

Author(s):

Claudio Napoli ◽

Giuditta Benincasa ◽

Joseph Loscalzo

Keyword(s):

Pulmonary Arterial Hypertension ◽

Arterial Hypertension ◽

Vascular Remodeling ◽

Disease Onset ◽

Later Life ◽

Epigenetic Changes ◽

Apoptosis Resistance ◽

Integrated Network ◽

Mesenchymal Transition ◽

Pulmonary Arterial

In pulmonary arterial hypertension (PAH), the Warburg effect (glycolytic shift) and mitochondrial fission are determinants of phenotype alterations characteristic of the disease, such as proliferation, apoptosis resistance, migration, endothelial-mesenchymal transition, and extracellular matrix stiffness. Current therapies, focusing largely on vasodilation and antithrombotic protection, do not restore these aberrant phenotypes suggesting that additional pathways need be targeted. The multifactorial nature of PAH suggests epigenetic changes as potential determinants of vascular remodeling. Transgenerational epigenetic changes induced by hypoxia can result in permanent changes early in fetal development increasing PAH risk in adulthood. Unlike genetic mutations, epigenetic changes are pharmacologically reversible, making them an attractive target as therapeutic strategies for PAH. This review offers a landscape of the most current clinical, epigenetic-sensitive changes contributing to PAH vascular remodeling both in early and later life, with a focus on a network medicine strategy. Furthermore, we discuss the importance of the application (from morphogenesis to disease onset) of molecular network-based algorithms to dissect PAH molecular pathobiology. Additionally, we suggest an integrated network-based program for clinical disease gene discovery that may reveal novel biomarkers and novel disease targets, thus offering a truly innovative path toward redefining and treating PAH, as well as facilitating the trajectory of a comprehensive precision medicine approach to PAH.

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Abstract 16770: The Epigenetic Modifier EP300: A New Corner Stone in the Regulation of Both Vascular Remodeling and Right Ventricle Failure in Pulmonary Arterial Hypertension

Circulation ◽

10.1161/circ.142.suppl_3.16770 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Alice Bourgeois ◽

Sarah-Eve Lemay ◽

Yann Grobs ◽

Charlotte romanet ◽

Junichi Omura ◽

...

Keyword(s):

Pulmonary Arterial Hypertension ◽

Animal Models ◽

Pulmonary Artery ◽

Arterial Hypertension ◽

Vascular Remodeling ◽

Annexin V ◽

Right Heart Catheterization ◽

Heart Catheterization ◽

Pulmonary Arterial

Introduction: Pulmonary Arterial Hypertension (PAH) is characterized by excessive proliferation and resistance to apoptosis of pulmonary artery (PA) smooth muscle cells (PASMCs), leading to progressive increases in pulmonary vascular resistance, and ultimately right ventricular (RV) failure and death. Thanks to omics technologies, we made tremendous progress in understanding gene misregulation during disease processes and identified the epigenetic factor EP300 as a critical player in pathological processes like proliferation/apoptosis and hypertrophy/fibrosis all of which are critical features of both PA remodeling and RV failure in PAH. We hypothesized that EP300 is upregulated in PAH and contributes to both PA remodeling and RV failure. Methods and Results: By Western blot (WB) and immunofluorescence (IF), we found that EP300 is up-regulated in isolated PASMCs and distal PAs from PAH patients (n=11-14) compared to controls (n=8-10) (p<0.01). Similar results were observed in 3 PAH animal models, namely the monocrotaline (MCT), the Sugen/Hypoxia (Su/Hx) and the Fawn-Hooded rat (FHR) (p<0.05). In vitro, pharmacological inhibition of EP300 using CCS-1477 reduces PAH-PASMC proliferation (Ki67 labeling & WB PCNA; p<0.05) and resistance to apoptosis (Annexin V assay & WB Survivin; p<0.05). These effects were confirmed at the molecular level by RNA-Seq analysis. In addition, increased EP300 expression was observed in hypertrophied and failed RV from PAH patients, as well as in rats injected with MCT or subjected to pulmonary artery banding (WB, p<0.05). In animal models, EP300 negatively correlates with CO and positively correlates with RVEDP, cardiomyocyte surface area and fibrosis. Finally, we demonstrated that inhibition of EP300 using CCS-1477 or SGC-CBP30 significantly improved established PAH (right heart catheterization) in two animal models (MCT and FHR). Conclusion: EP300 upregulation contributes to both pulmonary vascular remodeling and RV dysfunction seen in PAH and its inhibition represents a promising therapeutic avenue.

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Dual ETA/ETB blockade with macitentan improves both vascular remodeling and angiogenesis in pulmonary arterial hypertension

Pulmonary Circulation ◽

10.1177/2045893217741429 ◽

2017 ◽

Vol 8 (1) ◽

pp. 204589321774142 ◽

Cited By ~ 12

Author(s):

Valerie Nadeau ◽

Francois Potus ◽

Olivier Boucherat ◽

Renee Paradis ◽

Eve Tremblay ◽

...

Keyword(s):

Pulmonary Arterial Hypertension ◽

Arterial Hypertension ◽

Vascular Remodeling ◽

Skeletal Muscles ◽

Cell Metabolism ◽

Critical Role ◽

Capillary Density ◽

Pulmonary Hemodynamics ◽

Pulmonary Arterial

Dysregulated metabolism and rarefaction of the capillary network play a critical role in pulmonary arterial hypertension (PAH) etiology. They are associated with a decrease in perfusion of the lungs, skeletal muscles, and right ventricle (RV). Previous studies suggested that endothelin-1 (ET-1) modulates both metabolism and angiogenesis. We hypothesized that dual ETA/ETB receptors blockade improves PAH by improving cell metabolism and promoting angiogenesis. Five weeks after disease induction, Sugen/hypoxic rats presented severe PAH with pulmonary artery (PA) remodeling, RV hypertrophy and capillary rarefaction in the lungs, RV, and skeletal muscles (microCT angiogram, lectin perfusion, CD31 staining). Two-week treatment with dual ETA/ETB receptors antagonist macitentan (30 mg/kg/d) significantly improved pulmonary hemodynamics, PA vascular remodeling, and RV function and hypertrophy compared to vehicle-treated animals (all P = 0.05). Moreover, macitentan markedly increased lung, RV and quadriceps perfusion, and microvascular density (all P = 0.05). In vitro, these effects were associated with increases in oxidative phosphorylation (oxPhox) and markedly reduced cell proliferation of PAH-PA smooth muscle cells (PASMCs) treated with macitentan without affecting apoptosis. While macitentan did not affect oxPhox, proliferation, and apoptosis of PAH–PA endothelial cells (PAECs), it significantly improved their angiogenic capacity (tube formation assay). Exposure of control PASMC and PAEC to ET-1 fully mimicked the PAH cells phenotype, thus confirming that ET-1 is implicated in both metabolism and angiogenesis abnormalities in PAH. Dual ETA/ETB receptor blockade improved the metabolic changes involved in PAH-PASMCs’ proliferation and the angiogenic capacity of PAH-PAEC leading to an increased capillary density in lungs, RV, and skeletal muscles.

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Notoginsenoside R1 improves monocrotaline-induced pulmonary arterial hypertension via modulation NF-κB signaling in rats

Tropical Journal of Pharmaceutical Research ◽

10.4314/tjpr.v18i6.7 ◽

2021 ◽

Vol 18 (6) ◽

pp. 1191-1196

Author(s):

Yinglu Feng ◽

Na Hu ◽

Min Tang ◽

Shanglong Yao

Keyword(s):

B Cells ◽

Pulmonary Arterial Hypertension ◽

Arterial Hypertension ◽

Vascular Remodeling ◽

Associated Factors ◽

Nuclear Factor ◽

Sprague Dawley ◽

Pulmonary Hemodynamics ◽

Pulmonary Vascular Remodeling ◽

Pulmonary Arterial

Purpose: To investigate the potentials of notoginsenoside R1 (NGR1) in ameliorating inflammation and pulmonary vascular remodeling in rats with pulmonary arterial hypertension (PAH) induced by monocrotaline (MCT), and to examine the mechanisms underlying such effects. Methods: Eight-week-old male Sprague Dawley rats were randomly divided into groups: control, MCT, MCT+5mg/kg NGR1, MCT+12.5mg/kg NGR1, and MCT + 25 mg/kg NGR1. Right cardiac catheterization was used to measure pulmonary hemodynamics. Pulmonary morphology was evaluated with the aid of H & E staining. Serum levels of inflammatory cytokines were measured using ELISA, while levels of inflammation-associated factors in the lung were measured using RT-PCR. NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and IκBα (nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha) protein levels were determined by western blot. Results: Pulmonary hemodynamics and pulmonary morphology worsened following MCT injection and were accompanied by NF-κB pathway activation and elevated levels of inflammation-associated factors. In contrast, MCT treatment followed by NGR1 treatment ameliorated MCT-induced PAH by improving pulmonary hemodynamics and pulmonary vascular remodeling while reducing NF-κB activation and levels of inflammation-associated factors. Conclusion: NGR1 exerts ameliorative effects on MCT-induced PAH by inhibiting NF-κB pathway. Therefore, NGR1 may be a new potential therapy for PAH.

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MicroCT analysis of vascular morphometry: a comparison of right lung lobes in the SUGEN/hypoxic rat model of pulmonary arterial hypertension

Pulmonary Circulation ◽

10.1177/2045893217709001 ◽

2017 ◽

Vol 7 (2) ◽

pp. 522-530 ◽

Cited By ~ 9

Author(s):

Erin M. Faight ◽

Kostas Verdelis ◽

Lee Zourelias ◽

Rong Chong ◽

Raymond L. Benza ◽

...

Keyword(s):

Computed Tomography ◽

Pulmonary Arterial Hypertension ◽

Arterial Hypertension ◽

Rat Model ◽

Vascular Remodeling ◽

Vascular Density ◽

Sprague Dawley ◽

Micro Computed Tomography ◽

Pulmonary Arterial ◽

Lung Lobes

Pulmonary arterial hypertension (PAH) is a rare disease characterized by significant vascular remodeling within the lung. Clinical computed tomography (CT) scans are routinely used to aid in PAH diagnosis. Animal models, including the Sugen-hypoxic rat model (SU/hyp), of PAH closely mimic human PAH development. We have previously used micro-computed tomography (microCT) to find extensive right lung vascular remodeling in the SU/hyp. We hypothesized that the individual right lung lobes may not contribute equally to overall lung vascular remodeling. Sprague-Dawley rats were subjected to a subcutaneous injection of vascular endothelial growth factor receptor blocker (Sugen 5416) and subsequently exposed to chronic hypoxic conditions (10% O2) for three weeks. Following perfusion of the lung vasculature with an opaque resin (Microfil), the right lung lobes were microCT-imaged with a 10-µm voxel resolution and 3D morphometry analysis was performed separately on each lobe. As expected, we found a significantly lower ratio of vascular volume to total lobe volume in the SU/hyp compared with the control, but only in the distal lobes (inferior: 0.23 [0.21–0.30] versus 0.35 [0.27–0.43], P = 0.02; accessory: 0.27 [0.25–0.33] versus 0.37 [0.29–0.43], P = 0.06). Overall, we observed significantly fewer continuous blood vessels and reduced vascular density while having greater vascular lumen diameters in the distal lobes of both groups ( P < 0.05). In addition, the vascular separation within the SU/hyp lobes and the vascular surface area to volume ratio were significantly greater in the SU/hyp lobes compared with controls ( P < 0.03). Results for the examined parameters support the overall extensive vascular remodeling in the SU/hyp model and suggest this may be lobe-dependent.

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MiR-193-3p attenuates the vascular remodeling in pulmonary arterial hypertension by targeting PAK4

Pulmonary Circulation ◽

10.1177/2045894020974919 ◽

2020 ◽

Vol 10 (4) ◽

pp. 204589402097491

Author(s):

Zhenhua Wu ◽

Jie Geng ◽

Yujuan Qi ◽

Jian Li ◽

Yaobang Bai ◽

...

Keyword(s):

Cell Proliferation ◽

Pulmonary Arterial Hypertension ◽

Pulmonary Artery ◽

Arterial Hypertension ◽

Vascular Remodeling ◽

Luciferase Reporter ◽

Pulmonary Vascular Remodeling ◽

Pulmonary Arterial

Pulmonary arterial hypertension (PAH) is a progressive pulmonary vascular disease associated with dysfunction of pulmonary artery endothelial cells and pulmonary artery smooth muscle cells (PASMCs). To explore the potential mechanism of miR-193-3p in pulmonary arterial hypertension, human PASMCs and rats were respectively stimulated by hypoxia and monocrotaline to establish PAH model in vivo and in vitro. The expressions of miR-193-3p and p21-activated protein kinase 4 (PAK4) in the lung samples of PAH patients and paired healthy samples from the healthy subjects in PHA cells and rats were detected by quantitative reverse transcriptase-PCR. Morphological changes in lung tissues were determined using hematoxylin and eosin staining. Right ventricular systolic pressure (RVSP) and ratio of right ventricle to left ventricle plus septum (RV/LV p S) were measured. The binding relationship between miR-193-3p and PAK4 was analyzed by TargetScan and verified by luciferase reporter assay. Cell viability, apoptosis, and migration were detected by 3-(4, 5-Dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) flow cytometry, and wound-healing assays, respectively. The protein expressions of PAK4, proliferating cell nuclear antigen (PCNA), P21, p-AKT, and AKT in vivo or in vitro were determined by Western blot. In this study, we found that in pulmonary arterial hypertension, miR-193-3p expression was downregulated and PAK4 expression was up-regulated. MiR-193-3p directly targeted PAK4 and negatively regulated its expression. Hypoxia condition promoted cell proliferation, migration, and inhibited apoptosis accompanied with increased expressions of PCNA and p-AKT/AKT and decreased expression of P21 in PASMCs. MiR-193-3p overexpression attenuated the effects of hypoxia on PASMCs via downregulating PAK4. Monocrotaline treatment increased p-AKT/AKT and decreased P21 expression and caused pulmonary vascular remodeling in the model rats. MiR-193-3p overexpression attenuated pulmonary vascular remodeling, decreased p-AKT/AKT, and increased P21 levels via downregulating PAK4 in monocrotaline-induced rats. The results in this study demonstrated that upregulation of miR-193-3p reduced cell proliferation, migration, and apoptosis of PAH in vitro and pulmonary vascular remodeling in PAH in vivo through downregulating PAK4.

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Epigenetic Targets for Oligonucleotide Therapies of Pulmonary Arterial Hypertension

International Journal of Molecular Sciences ◽

10.3390/ijms21239222 ◽

2020 ◽

Vol 21 (23) ◽

pp. 9222

Author(s):

William Gerthoffer

Keyword(s):

Pulmonary Arterial Hypertension ◽

Arterial Hypertension ◽

Small Molecules ◽

Vascular Remodeling ◽

Arterial Wall ◽

Vascular Wall ◽

Wall Structure ◽

Wall Thickening ◽

Pulmonary Arterial

Arterial wall remodeling underlies increased pulmonary vascular resistance and right heart failure in pulmonary arterial hypertension (PAH). None of the established vasodilator drug therapies for PAH prevents or reverse established arterial wall thickening, stiffening, and hypercontractility. Therefore, new approaches are needed to achieve long-acting prevention and reversal of occlusive pulmonary vascular remodeling. Several promising new drug classes are emerging from a better understanding of pulmonary vascular gene expression programs. In this review, potential epigenetic targets for small molecules and oligonucleotides will be described. Most are in preclinical studies aimed at modifying the growth of vascular wall cells in vitro or normalizing vascular remodeling in PAH animal models. Initial success with lung-directed delivery of oligonucleotides targeting microRNAs suggests other epigenetic mechanisms might also be suitable drug targets. Those targets include DNA methylation, proteins of the chromatin remodeling machinery, and long noncoding RNAs, all of which act as epigenetic regulators of vascular wall structure and function. The progress in testing small molecules and oligonucleotide-based drugs in PAH models is summarized.

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Abstract 17034: The AKT/AMPK/FOXO3 Axis in Pulmonary Arterial Hypertension

Circulation ◽

10.1161/circ.142.suppl_3.17034 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Yann Grobs ◽

Charlotte romanet ◽

Valerie Nadeau ◽

Junichi Omura ◽

Mark Orcholski ◽

...

Keyword(s):

Pulmonary Arterial Hypertension ◽

Arterial Hypertension ◽

Vascular Remodeling ◽

Pulmonary Arteries ◽

Right Heart Failure ◽

Metabolic Reprogramming ◽

Nuclear Exclusion ◽

Pulmonary Arterial

Like cancer, pulmonary arterial hypertension (PAH) is characterized by exaggerated proliferation and resistance to apoptosis related to metabolic alterations (Warburg effect) of pulmonary smooth muscle cells (PASMCs). These anomalies result in a progressive narrowing of the pulmonary arteries, increasing pulmonary resistance and leading to right heart failure and premature death. In cancer cells, unphosphorylated and nuclear FOXO3 has been extensively studied as a crucial protein that functions as a tumor suppressor by regulating expression of genes involved in apoptosis and cell cycle arrest. These functions combined with other FOXO3 attributes, including its key role in communicating mitochondrial-nuclear signals, make the FOXO3 a suitable candidate for controlling the cancer-like phenotype of PAH-PASMCs. Interestingly, AKT and AMPK known to be implicated in PAH exert antagonistic effects on FOXO3; AKT promoting its nuclear exclusion while AMPK favors its nuclear and mitochondrial accumulation. The thus made the hypothesis that FOXO3’s nuclear exclusion (secondary to AKT/AMPK imbalance) promotes metabolic reprogramming towards glycolysis leading to enhanced proliferation/resistance to apoptosis of PAH-PASMCs and vascular remodeling. Using Western blot and immunofluorescence in isolated PASMCs from both PAH and control patients (n=10), we found that nuclear and mitochondrial exclusion of FOXO3 due to its phosphorylation is a feature of PAH-PASMCs. In vitro, we demonstrated that nuclear localization of FOXO3 using an adenovirus expressing a constitutively active, non-phosphorylable form of FOXO3 or trifluoperazine (TFP) resulted in reduced PAH-PASMC proliferation (Ki67 labeling, p<0,0005) and resistance to apoptosis (Annexin V assay, p<0,05). These effects were accompanied by increased expression of P27 and SOD2 and diminished expression of Survivin (p<0,05). In vivo, we showed that FOXO3 activation using TPF improved established PAH in the monocrotaline rats (reduced RVSP and increased Sv and CO, by right catheterization, p<0,01, n=29) without any sign of toxicity. We showed that FOXO3 is implicated in pulmonary vascular remodeling. Pharmacological activation of FOXO3 may represent a novel avenue to improve PAH.

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