Eliminating Senescent Cells Can Promote Pulmonary Hypertension Development and Progression.

BACKGROUND: Senescent cells (SCs) are involved in proliferative disorders, but their role in pulmonary hypertension remains undefined. We investigated SCs in patients with pulmonary arterial hypertension and the role of SCs in animal pulmonary hypertension models. METHODS: We investigated senescence (p16, p21) and DNA damage (γ-H2AX, 53BP1) markers in patients with pulmonary arterial hypertension and murine models. We monitored p16 activation by luminescence imaging in p16-luciferase (p16LUC/+) knock-in mice. SC clearance was obtained by a suicide gene (p16 promoter-driven killer gene construct in p16-ATTAC mice), senolytic drugs (ABT263 and cell-permeable FOXO4-p53 interfering peptide [FOXO4-DRI]), and p16 inactivation in p16LUC/LUC mice. We investigated pulmonary hypertension in mice exposed to normoxia, chronic hypoxia, or hypoxia+Sugen, mice overexpressing the serotonin transporter (SM22-5-HTT+), and rats given monocrotaline. RESULTS: Patients with pulmonary arterial hypertension compared with controls exhibited high lung p16, p21, and γ-H2AX protein levels, with abundant vascular cells costained for p16, γ-H2AX, and 53BP1. Hypoxia increased thoracic bioluminescence in p16LUC/+ mice. In wild-type mice, hypoxia increased lung levels of senescence and DNA-damage markers, senescence-associated secretory phenotype components, and p16 staining of pulmonary endothelial cells (P-ECs, 30% of lung SCs in normoxia), and pulmonary artery smooth muscle cells. SC elimination by suicide gene or ABT263 increased the right ventricular systolic pressure and hypertrophy index, increased vessel remodeling (higher dividing proliferating cell nuclear antigen-stained vascular cell counts during both normoxia and hypoxia), and markedly decreased lung P-ECs. Pulmonary hemodynamic alterations and lung P-EC loss occurred in older p16LUC/LUC mice, wild-type mice exposed to Sugen or hypoxia+Sugen, and SM22-5-HTT+ mice given either ABT263 or FOXO4-DRI, compared with relevant controls. The severity of monocrotaline-induced pulmonary hypertension in rats was decreased slightly by ABT263 for 1 week but was aggravated at 3 weeks, with loss of P-ECs. CONCLUSIONS: Elimination of senescent P-ECs by senolytic interventions may worsen pulmonary hemodynamics. These results invite consideration of the potential impact on pulmonary vessels of strategies aimed at controlling cell senescence in various contexts.

Phospholipase A2 receptor 1 promotes lung cell senescence and emphysema in obstructive lung disease.

BACKGROUND: Cell senescence is a key process in age-associated dysfunction and diseases, notably chronic obstructive pulmonary disease (COPD). We previously identified phospholipase A2 receptor 1 (PLA2R1) as a positive regulator of cell senescence acting via Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling. Its role in pathology, however, remains unknown. Here, we assessed PLA2R1-induced senescence in COPD and lung emphysema pathogenesis. METHODS: We assessed cell senescence in lungs and cultured lung cells from patients with COPD and controls subjected to PLA2R1 knockdown, PLA2R1 gene transduction and treatment with the JAK1/2 inhibitor ruxolitinib. To assess whether PLA2R1 upregulation caused lung lesions, we developed transgenic mice overexpressing PLA2R1 (PLA2R1-TG) and intratracheally injected wild-type mice with a lentiviral vector carrying the Pla2r1 gene (LV-PLA2R1 mice). RESULTS: We found that PLA2R1 was overexpressed in various cell types exhibiting senescence characteristics in COPD lungs. PLA2R1 knockdown extended the population doubling capacity of these cells and inhibited their pro-inflammatory senescence-associated secretory phenotype (SASP). PLA2R1-mediated cell senescence in COPD was largely reversed by treatment with the potent JAK1/2 inhibitor ruxolitinib. Five-month-old PLA2R1-TG mice exhibited lung cell senescence, and developed lung emphysema and lung fibrosis together with pulmonary hypertension. Treatment with ruxolitinib induced reversal of lung emphysema and fibrosis. LV-PLA2R1-treated mice developed lung emphysema within 4 weeks and this was markedly attenuated by concomitant ruxolitinib treatment. CONCLUSIONS: Our data support a major role for PLA2R1 activation in driving lung cell senescence and lung alterations in COPD. Targeting JAK1/2 may represent a promising therapeutic approach for COPD.

The antioxidant N-acetylcysteine protects from lung emphysema but induces lung adenocarcinoma in mice.

Oxidative stress is a major contributor to chronic lung diseases. Antioxidants such as N-acetylcysteine (NAC) are broadly viewed as protective molecules that prevent the mutagenic effects of reactive oxygen species. Antioxidants may, however, increase the risk of some forms of cancer and accelerate lung cancer progression in murine models. Here, we investigated chronic NAC treatment in aging mice displaying lung oxidative stress and cell senescence due to inactivation of the transcription factor JunD, which is downregulated in diseased human lungs. NAC treatment decreased lung oxidative damage and cell senescence and protected from lung emphysema but concomitantly induced the development of lung adenocarcinoma in 50% of JunD-deficient mice and 10% of aged control mice. This finding constitutes the first evidence to our knowledge of a carcinogenic effect of antioxidant therapy in the lungs of aged mice with chronic lung oxidative stress and warrants the utmost caution when considering the therapeutic use of antioxidants.

CCR2/CCR5-mediated macrophage-smooth muscle cell crosstalk in pulmonary hypertension.

Macrophages are major players in the pathogenesis of pulmonary arterial hypertension (PAH).To investigate whether lung macrophages and pulmonary-artery smooth muscle cells (PASMCs) collaborate to stimulate PASMC growth and whether the CCL2-CCR2 and CCL5-CCR5 pathways inhibited macrophage-PASMC interactions and PAH development, we used human CCR5-knock-in mice and PASMCs from patients with PAH and controls.Conditioned media from murine M1 or M2 macrophages stimulated PASMC growth. This effect was markedly amplified with conditioned media from M2 macrophage/PASMC co-cultures. CCR2, CCR5, CCL2 and CCL5 were upregulated in macrophage/PASMC co-cultures. Compared to inhibiting either receptor, dual CCR2 and CCR5 inhibition more strongly attenuated the growth-promoting effect of conditioned media from M2-macrophage/PASMC co-cultures. Deleting either CCR2 or CCR5 in macrophages or PASMCs attenuated the growth response. In mice with hypoxia- or SUGEN/hypoxia-induced PH, targeting both CCR2 and CCR5 prevented or reversed PH more efficiently than targeting either receptor alone. Patients with PAH exhibited CCR2 and CCR5 upregulation in PASMCs and perivascular macrophages compared to controls. The PASMC growth-promoting effect of conditioned media from M2-macrophage/PASMC co-cultures was greater when PASMCs from PAH patients were used in the co-cultures or as the target cells and was dependent on CCR2 and CCR5. PASMC migration toward M2-macrophages was greater with PASMCs from PAH patients and was attenuated by blocking CCR2 and CCR5.CCR2 and CCR5 are required for collaboration between macrophages and PASMCs to initiate and amplify PASMC migration and proliferation during PAH development. Dual targeting of CCR2 and CCR5 may hold promise for treating human PAH.

Cardiac adenylyl cyclase overexpression precipitates and aggravates age-related myocardial dysfunction.

AIMS: Increase of cardiac cAMP bioavailability and PKA activity through adenylyl-cyclase 8 (AC8) overexpression enhances contractile function in young transgenic mice (AC8TG). Ageing is associated with decline of cardiac contraction partly by the desensitization of ß-adrenergic/cAMP signalling. Our objective was to evaluate cardiac cAMP signalling as age increases between 2 months and 12 months and to explore whether increasing the bioavailability of cAMP by overexpression of AC8 could prevent cardiac dysfunction related to age. METHODS AND RESULTS: Cardiac cAMP pathway and contractile function were evaluated in AC8TG and their non-transgenic littermates (NTG) at 2- and 12 months old. AC8TG demonstrated increased AC8, PDE1, 3B and 4D expression at both ages, resulting in increased phosphodiesterase and PKA activity, and increased phosphorylation of several PKA targets including sarco(endo)plasmic-reticulum-calcium-ATPase (SERCA2a) cofactor phospholamban (PLN) and GSK3α/ß a main regulator of hypertrophic growth and ageing. Confocal immunofluorescence revealed that the major phospho-PKA substrates were co-localized with Z-line in 2-month-old NTG but with Z-line interspace in AC8TG, confirming the increase of PKA activity in the compartment of PLN/SERCA2a. In both 12-month-old NTG and AC8TG, PLN and GSK3α/ß phosphorylation was increased together with main localization of phospho-PKA substrates in Z-line interspaces. Haemodynamics demonstrated an increased contractile function in 2- and 12-month-old AC8TG, but not in NTG. In contrast, echocardiography and tissue Doppler imaging (TDI) performed in conscious mice unmasked myocardial dysfunction with a decrease of systolic strain rate in both old AC8TG and NTG. In AC8TG TDI showed a reduced strain rate even in 2-month-old animals. Development of age-related cardiac dysfunction was accelerated in AC8TG, leading to heart failure (HF) and premature death. Histological analysis confirmed early cardiomyocyte hypertrophy and interstitial fibrosis in AC8TG when compared with NTG. CONCLUSION: Our data demonstrated an early and accelerated cardiac remodelling in AC8TG mice, leading to the development of HF and reduced lifespan. Age-related reorganization of cAMP/PKA signalling can accelerate cardiac ageing, partly through GSK3α/ß phosphorylation.

mTOR pathway activation drives lung cell senescence and emphysema.

Chronic obstructive pulmonary disease (COPD) is a highly prevalent and devastating condition for which no curative treatment is available. Exaggerated lung cell senescence may be a major pathogenic factor. Here, we investigated the potential role for mTOR signaling in lung cell senescence and alterations in COPD using lung tissue and derived cultured cells from patients with COPD and from age- and sex-matched control smokers. Cell senescence in COPD was linked to mTOR activation, and mTOR inhibition by low-dose rapamycin prevented cell senescence and inhibited the proinflammatory senescence-associated secretory phenotype. To explore whether mTOR activation was a causal pathogenic factor, we developed transgenic mice exhibiting mTOR overactivity in lung vascular cells or alveolar epithelial cells. In this model, mTOR activation was sufficient to induce lung cell senescence and to mimic COPD lung alterations, with the rapid development of lung emphysema, pulmonary hypertension, and inflammation. These findings support a causal relationship between mTOR activation, lung cell senescence, and lung alterations in COPD, thereby identifying the mTOR pathway as a potentially new therapeutic target in COPD.

New Nitric Oxide Donor NCX 1443: Therapeutic Effects on Pulmonary Hypertension in the SAD Mouse Model of Sickle Cell Disease.

Nitric oxide (NO) donors may be useful for treating pulmonary hypertension (PH) complicating sickle cell disease (SCD), as endogenous NO is inactivated by hemoglobin released by intravascular hemolysis. Here, we investigated the effects of the new NO donor NCX1443 on PH in transgenic SAD mice, which exhibit mild SCD without severe hemolytic anemia. In SAD and wild-type (WT) mice, the pulmonary pressure response to acute hypoxia was similar and was abolished by 100 mg/kg NCX1443. The level of PH was also similar in SAD and WT mice exposed to chronic hypoxia (9% O2) alone or with SU5416 and was similarly reduced by daily NCX1443 gavage. Compared with WT mice, SAD mice exhibited higher levels of HO-1, endothelial NO synthase, and PDE5 but similar levels of lung cyclic guanosine monophosphate. Cultured pulmonary artery smooth muscle cells from SAD mice grew faster than those from WT mice and had higher PDE5 protein levels. Combining NCX1443 and a PDE5 inhibitor suppressed the growth rate difference between SAD and WT cells and induced a larger reduction in hypoxic PH severity in SAD than in WT mice. By amplifying endogenous protective mechanisms, NCX1443 in combination with PDE5 inhibition may prove useful for treating PH complicating SCD.

Role of interleukin-1 receptor 1/MyD88 signalling in the development and progression of pulmonary hypertension.

Pulmonary artery smooth muscle cell (PA-SMC) proliferation and inflammation are key components of pulmonary arterial hypertension (PAH). Interleukin (IL)-1ß binds to IL-1 receptor (R)1, thereby recruiting the molecular adaptor myeloid differentiation primary response protein 88 (MyD88) (involved in IL-1R1 and Toll-like receptor signal transduction) and inducing IL-1, IL-6 and tumour necrosis factor-α synthesis through nuclear factor-κB activation.We investigated the IL-1R1/MyD88 pathway in the pathogenesis of pulmonary hypertension.Marked IL-1R1 and MyD88 expression with predominant PA-SMC immunostaining was found in lungs from patients with idiopathic PAH, mice with hypoxia-induced pulmonary hypertension and SM22-5-HTT(+) mice. Elevations in lung IL-1ß, IL-1R1, MyD88 and IL-6 preceded pulmonary hypertension in hypoxic mice. IL-1R1(-/-), MyD88(-/-) and control mice given the IL-1R1 antagonist anakinra were protected similarly against hypoxic pulmonary hypertension and perivascular macrophage recruitment. Anakinra reversed pulmonary hypertension partially in SM22-5-HTT(+) mice and markedly in monocrotaline-treated rats. IL-1ß-mediated stimulation of mouse PA-SMC growth was abolished by anakinra and absent in IL-1R1(-/-) and MyD88(-/-) mice. Gene deletion confined to the myeloid lineage (M.lys-Cre MyD88(fl/fl) mice) decreased pulmonary hypertension severity versus controls, suggesting IL-1ß-mediated effects on PA-SMCs and macrophages. The growth-promoting effect of media conditioned by M1 or M2 macrophages from M.lys-Cre MyD88(fl/fl) mice was attenuated.Pulmonary vessel remodelling and inflammation during pulmonary hypertension require IL-1R1/MyD88 signalling. Targeting the IL-1ß/IL-1R1 pathway may hold promise for treating human PAH.

Osteopontin, a Key Mediator Expressed by Senescent Pulmonary Vascular Cells in Pulmonary Hypertension.

OBJECTIVE: Senescent pulmonary artery smooth muscle cells (PA-SMCs) may contribute to the pathogenesis of pulmonary hypertension by producing secreted factors. The aim of this study was to explore the role in pulmonary hypertension of extracellular matrix proteins released by senescent PA-SMCs. APPROACH AND RESULTS: Polymerase chain reaction array analysis of human PA-SMCs undergoing replicative senescence revealed osteopontin upregulation, which mediated the stimulatory effect of senescent PA-SMC media and matrix on PA-SMC growth and migration. Osteopontin was upregulated in lungs from patients with chronic obstructive pulmonary disease or idiopathic pulmonary arterial hypertension. Prominent osteopontin immunostaining was noted in PA-SMCs that also stained for p16 at sites of vascular hypertrophy, and lung osteopontin levels correlated closely with age. Compared with younger mice, 1-year-old mice displayed higher lung osteopontin levels, right ventricular systolic pressure, pulmonary vessel muscularization, and numbers of PA-SMCs stained for p16 or p21 and also for osteopontin. No such changes with age were observed in osteopontin(-/-) mice, which developed attenuated pulmonary hypertension during hypoxia. Compared with cultured PA-SMCs from young mice, PA-SMCs from 1-year-old mice grew faster; a similar fast growth rate was seen with PA-SMCs from young mice stimulated by matrix or media from old mice. Differences between old/young mouse PA-SMC growth rates were suppressed by antiosteopontin antibodies. PA-SMCs from osteopontin(-/-) mice grew more slowly than did wild-type PA-SMCs; they were stimulated by wild-type PA-SMCs media and matrix, and this effect was stronger with PA-SMCs from older versus younger mice. CONCLUSIONS: Osteopontin is a key mediator released by senescent PA-SMCs and contributing to pulmonary hypertension progression.

Selective Tuberous Sclerosis Complex 1 Gene Deletion in Smooth Muscle Activates Mammalian Target of Rapamycin Signaling and Induces Pulmonary Hypertension.

Constitutive activation of the mammalian target of rapamycin (mTOR) complexes mTORC1 and mTORC2 is associated with pulmonary hypertension (PH) and sustained growth of pulmonary artery (PA) smooth muscle cells (SMCs). We investigated whether selective mTORC1 activation in SMCs induced by deleting the negative mTORC1 regulator tuberous sclerosis complex 1 gene (TSC1) was sufficient to produce PH in mice. Mice expressing Cre recombinase under SM22 promoter control were crossed with TSC1(LoxP/LoxP) mice to generate SM22-TSC1(-/-) mice. At 8 weeks of age, SM22-TSC1(-/-) mice exhibited PH with marked increases in distal PA muscularization and Ki67-positive PASMC counts, without systemic hypertension or cardiac dysfunction. Marked activation of the mTORC1 substrates S6 kinase and 4E-BP and the mTORC2 substrates p-Akt(Ser473) and glycogen synthase kinase 3 was found in the lungs and pulmonary vessels of SM22-TSC1(-/-) mice when compared with control mice. Treatment with 5 mg/kg rapamycin for 3 weeks to inhibit mTORC1 and mTORC2 fully reversed PH in SM22-TSC1(-/-) mice. In chronically hypoxic mice and SM22-5HTT(+) mice exhibiting PH associated with mTORC1 and mTORC2 activation, PH was maximally attenuated by low-dose rapamycin associated with selective mTORC1 inhibition. Cultured PASMCs from SM22-TSC1(-/-), SM22-5HTT(+), and chronically hypoxic mice exhibited similar sustained growth-rate enhancement and constitutive mTORC1 and mTORC2 activation; both effects were abolished by rapamycin. Deletion of the downstream mTORC1 effectors S6 kinase 1/2 in mice also activated mTOR signaling and induced PH. We concluded that activation of mTORC1 signaling leads to increased PASMC proliferation and subsequent PH development.

Extracellular Calpain/Calpastatin Balance Is Involved in the Progression of Pulmonary Hypertension.

Excessive growth of pulmonary arterial (PA) smooth muscle cells (SMCs) is a major component of PA hypertension (PAH). The calcium-activated neutral cysteine proteases calpains 1 and 2, expressed by PASMCs, contribute to PH but are tightly controlled by a single specific inhibitor, calpastatin. Our objective was to investigate calpastatin during pulmonary hypertension (PH) progression and its potential role as an intracellular and/or extracellular effector. We assessed calpains and calpastatin in patients with idiopathic PAH and mice with hypoxic or spontaneous (SM22-5HTT(+) strain) PH. To assess intracellular and extracellular roles for calpastatin, we studied effects of the calpain inhibitor PD150606 on hypoxic PH in mice with calpastatin overexpression driven by the cytomegalovirus promoter (CMV-Cast) or C-reactive protein (CRP) promoter (CRP-Cast), inducing increased calpastatin production ubiquitously and in the liver, respectively. Chronically hypoxic and SM22-5HTT(+) mice exhibited increased lung calpastatin and calpain 1 and 2 protein levels and activity, both intracellularly and extracellularly. Prominent calpastatin and calpain immunostaining was found in PASMCs of remodeled vessels in mice and patients with PAH, who also exhibited increased plasma calpastatin levels. CMV-Cast and CRP-Cast mice showed similarly decreased PH severity compared with wild-type mice, with no additional effect of PD150606 treatment. In cultured PASMCs from wild-type and CMV-Cast mice, exogenous calpastatin decreased cell proliferation and migration with similar potency as PD150606 and suppressed fibronectin-induced potentiation. These results indicate that calpastatin limits PH severity via extracellular mechanisms. They suggest a new approach to the development of treatments for PH.

Calpastatin overexpression impairs postinfarct scar healing in mice by compromising reparative immune cell recruitment and activation.

The activation of the calpain system is involved in the repair process following myocardial infarction (MI). However, the impact of the inhibition of calpain by calpastatin, its natural inhibitor, on scar healing and left ventricular (LV) remodeling is elusive. Male mice ubiquitously overexpressing calpastatin (TG) and wild-type (WT) controls were subjected to an anterior coronary artery ligation. Mortality at 6 wk was higher in TG mice (24% in WT vs. 44% in TG, P < 0.05) driven by a significantly higher incidence of cardiac rupture during the first week post-MI, despite comparable infarct size and LV dysfunction and dilatation. Calpain activation post-MI was blunted in TG myocardium. In TG mice, inflammatory cell infiltration and activation were reduced in the infarct zone (IZ), particularly affecting M2 macrophages and CD4(+) T cells, which are crucial for scar healing. To elucidate the role of calpastatin overexpression in macrophages, we stimulated peritoneal macrophages obtained from TG and WT mice in vitro with IL-4, yielding an abrogated M2 polarization in TG but not in WT cells. Lymphopenic Rag1(-/-) mice receiving TG splenocytes before MI demonstrated decreased T-cell recruitment and M2 macrophage activation in the IZ day 5 after MI compared with those receiving WT splenocytes. Calpastatin overexpression prevented the activation of the calpain system after MI. It also impaired scar healing, promoted LV rupture, and increased mortality. Defective scar formation was associated with blunted CD4(+) T-cell and M2-macrophage recruitment.

Telomere Dysfunction and Cell Senescence in Chronic Lung Diseases: Therapeutic Potential.

Cellular senescence--defined as a stable cell-cycle arrest combined with stereotyped phenotypic changes--might play a causal role in various lung diseases, including chronic obstructive pulmonary disease (COPD), which is predicted to become the third leading cause of death worldwide by 2020. COPD is characterized by slowly progressive airflow obstruction and emphysema due to destruction of the lung parenchyma and is often complicated by pulmonary hypertension (PH). No curative treatment is available. Senescent cells, which accumulate with age, are increased in lungs from patients with COPD and express a robust senescence-associated secretory phenotype (SASP), which is proinflammatory. The aim of this review is to show how senescent cells can drive the lung alterations seen in COPD, which mechanisms may be involved, and whether therapeutic interventions may slow or delay the in vitro cell-senescence process and in vivo lung alterations.

Role for telomerase in pulmonary hypertension.

BACKGROUND: Cells exhibiting dysregulated growth may express telomerase reverse transcriptase (TERT), the dual function of which consists of 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. METHODS AND RESULTS: Marked TERT expression or activity was found in lungs from patients with idiopathic PH and from mice with PH induced by hypoxia or serotonin-transporter overexpression (SM22-5HTT(+) mice), chiefly within PA-SMCs. In cultured mouse PA-SMCs, TERT was expressed on growth stimulation by serum. The TERT inhibitor imetelstat and the TERT activator TA65 abrogated and stimulated PA-SMC growth, respectively. PA-SMCs from PH mice showed a heightened proliferative phenotype associated with increased TERT expression, which was suppressed by imetelstat treatment. TERC(-/-) mice at generation 2 and TERT(-/-) mice at generations 2, 3, and 4 developed less severe PH than did wild-type mice exposed to chronic hypoxia, with less distal pulmonary artery muscularization and fewer Ki67-stained proliferating PA-SMCs. Telomere length differed between TERC(-/-) and TERT(-/-) mice, whereas PH severity was similar in the 2 strains and across generations. Chronic imetelstat treatment reduced hypoxia-induced PH in wild-type mice or partially reversed established PH in SM22-5HTT(+) mice while simultaneously decreasing TERT expression. Opposite effects occurred in mice treated with TA65. CONCLUSIONS: Telomerase exerts telomere-independent effects on PA-SMC growth in PH and may constitute a treatment target for PH.

Endothelial-to-mesenchymal transition in pulmonary hypertension.

BACKGROUND: The vascular remodeling responsible for pulmonary arterial hypertension (PAH) involves predominantly the accumulation of α-smooth muscle actin-expressing mesenchymal-like cells in obstructive pulmonary vascular lesions. Endothelial-to-mesenchymal transition (EndoMT) may be a source of those α-smooth muscle actin-expressing cells. METHODS AND RESULTS: In situ evidence of EndoMT in human PAH was obtained by using confocal microscopy of multiple fluorescent stainings at the arterial level, and by using transmission electron microscopy and correlative light and electron microscopy at the ultrastructural level. Findings were confirmed by in vitro analyses of human PAH and control cultured pulmonary artery endothelial cells. In addition, the mRNA and protein signature of EndoMT was recognized at the arterial and lung level by quantitative real-time polymerase chain reaction and Western blot analyses. We confirmed our human observations in established animal models of pulmonary hypertension (monocrotaline and SuHx). After establishing the first genetically modified rat model linked to BMPR2 mutations (BMPR2(Δ140Ex1/+) rats), we demonstrated that EndoMT is linked to alterations in signaling of BMPR2, a gene that is mutated in 70% of cases of familial PAH and in 10% to 40% of cases of idiopathic PAH. We identified molecular actors of this pathological transition, including twist overexpression and vimentin phosphorylation. We demonstrated that rapamycin partially reversed the protein expression patterns of EndoMT, improved experimental PAH, and decreased the migration of human pulmonary artery endothelial cells, providing the proof of concept that EndoMT is druggable. CONCLUSIONS: EndoMT is linked to alterations in BPMR2 signaling and is involved in the occlusive vas cular remodeling of PAH, findings that may have therapeutic implications.

CCR5 as a treatment target in pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PH), whether idiopathic or related to underlying diseases such as HIV infection, results from complex vessel remodeling involving both pulmonary artery smooth muscle cell (PA-SMC) proliferation and inflammation. CCR5, a coreceptor for cellular HIV-1 entry expressed on macrophages and vascular cells, may be involved in the pathogenesis of PH. Maraviroc is a new CCR5 antagonist designed to block HIV entry. METHODS AND RESULTS: Marked CCR5 expression was found in lungs from patients with idiopathic PH, in mice with hypoxia-induced PH, and in Simian immunodeficiency virus-infected macaques, in which it was localized chiefly in the PA-SMCs. To assess the role for CCR5 in experimental PH, we used both gene disruption and pharmacological CCR5 inactivation in mice. Because maraviroc does not bind to murine CCR5, we used human-CCR5ki mice for pharmacological and immunohistochemical studies. Compared with wild-type mice, CCR5-/- mice or human-CCR5ki mice treated with maraviroc exhibited decreased PA-SMC proliferation and recruitment of perivascular and alveolar macrophages during hypoxia exposure. CCR5-/- mice reconstituted with wild-type bone marrow cells and wild-type mice reconstituted with CCR5-/- bone marrow cells were protected against PH, suggesting CCR5-mediated effects on PA-SMCs and macrophage involvement. The CCR5 ligands CCL5 and the HIV-1 gp120 protein increased intracellular calcium and induced growth of human and human-CCR5ki mouse PA-SMCs; maraviroc inhibited both effects. Maraviroc also reduced the growth-promoting effects of conditioned media from CCL5-activated macrophages derived from human-CCR5ki mice on PA-SMCs from wild-type mice. CONCLUSION: The CCL5-CCR5 pathway represents a new therapeutic target in PH associated with HIV or with other conditions.

P21-dependent protective effects of a carbon monoxide-releasing molecule-3 in pulmonary hypertension.

OBJECTIVE: Carbon monoxide-releasing molecules (CORMs) represent a pharmacological alternative to CO gas inhalation. Here, we questioned whether CORM-3, a well-characterized water-soluble CORM, could prevent and reverse pulmonary hypertension (PH) in chronically hypoxic mice and in smooth muscle promoter 22 serotonin transporter mice overexpressing the serotonin transporter in smooth muscle cells (SMCs). APPROACH AND RESULTS: Treatment with CORM-3 (50 mg/kg per day once daily) for 3 weeks prevented PH, right ventricular hypertrophy, and distal pulmonary artery muscularization in mice exposed to chronic hypoxia and partially reversed PH in smooth muscle promoter 22 serotonin transporter mice by reducing Ki67 dividing pulmonary artery SMCs (PA-SMCs). In these models, CORM-3 markedly increased lung p21 mRNA and protein levels and p21-stained PA-SMCs. These effects contrasted with the transient pulmonary vasodilatation and rise in lung cGMP levels induced by a single injection of CORM-3 in mice exposed to acute hypoxia. Studies in cultured rat PA-SMCs revealed that the inhibitory effects of CORM-3 on cell growth were independent of cGMP formation but associated with increased p21 mRNA and protein levels. Protection against PH by CORM-3 required increased lung expression of p21, as indicated by the inability of CORM-3 to prevent chronic hypoxia-induced PH in p21-deficient mice and to alter the growth of PA-SMCs derived from p21-deficient mice. CORM-3-induced p21 overexpression was linked to p53 activation as assessed by the inability of CORM-3 to prevent PH and induce p21 expression in p53-deficient mice and in PA-SMCs derived from p53-deficient mice. CONCLUSIONS: CORM-3 inhibits pulmonary vascular remodeling via p21, which may represent a useful approach for treating PH.

Serotonin transporter and serotonin receptors.

The nature of the primary defect responsible for triggering and maintaining pulmonary artery smooth muscle (PA-SMC) proliferation in pulmonary artery hypertension (PAH) is poorly understood but may be either an inherent characteristic of PA-SMCs or a secondary response to an external abnormality, such as upregulation of growth factors. The serotonin hypothesis of PAH originated in the 1960s after an outbreak of the disease was reported among patients taking the anorexigenic drugs aminorex. The anorexiant dexfenfluramine which inhibits 5-HT neuronal uptake, causes 5-HT platelet depletion, and increases plasma levels of 5-HT, was then shown to increase the relative risk of developing PAH in the adults. More recently, the incidence of persistent pulmonary hypertension of the newborn was shown to be increased by the use of selective 5-HT reuptake inhibitors taken in late pregnancy. Serotonin is a vasoconstrictor and a potent mitogen for pulmonary smooth muscle cells (PA-SMC), an effect which depends upon activity of both the 5-HT transporter (5-HTT) and the 5-HT receptors. Expression analysis of lung tissues from PAH patients undergoing lung transplantation revealed an increased expression of the 5-HT transporter (5-HTT) and an enhanced proliferative growth response of isolated pulmonary arterial smooth muscle cells (PASMC) to 5-HT. Serotonin is contained in platelets but is also synthesized by pulmonary endothelial cells which express tryptophan hydroxylase 1, the rate-limiting enzyme of 5-HT synthesis. While inhibitors of 5-HTT and of 5-HT2B receptors can reverse experimental PH, 5-HTT-overexpressing mice spontaneously develop PH. In patients with chronic lung disease, a close association has been found between a 5-HTT gene polymorphism and the severity of pulmonary hypertension. Agents capable of selectively inhibiting 5-HTT-mediated PA-SMC proliferation deserve to be investigated as potential treatments for pulmonary hypertension. However, the 5-HT pathway is still studied only on a preclinical level and the usefulness of drugs interacting with the 5-HT pathway remains to be established in human PAH.