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.

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.

Middle age exacerbates acute respiratory distress syndrome in a double hit murine model.

RATIONALE: In a recent systematic review, aging has been identified as the only factor independently associated with mortality during human acute respiratory distress syndrome (ARDS). We explored this age-dependent severity in a clinically relevant double hit murine ARDS model. METHODS: Young adult (Y, 10-12weeks) and middle-old (O, 12-13months) male C57BL6 mice underwent an aspiration of Escherichia coli lipopolysaccharide (LPS) or control saline vehicle. Twenty hours later, four groups of mice were sacrificed [Y(control), O(control), Y(LPS) and O(LPS)]. Four other groups of mice underwent 3h of low tidal volume (8mL/kg) mechanical ventilation (MV) [Y(MV), O(MV), Y(LPS+MV) and O(LPS+MV)]. Lung mechanics were assessed hourly during MV. Right ventricular pressure and cardiac output were measured at the end of the MV. After sacrifice, lung inflammation, edema and injury were explored with bronchoalveolar lavage (BAL) and histology. RESULTS: After saline aspiration, middle-old mice had a higher respiratory system compliance than young adult mice. LPS aspiration dramatically altered the baseline compliance in middle-old (O(LPS)), but not in young adult (Y(LPS)) mice. Middle-old mice had a more pronounced alteration in lungs mechanics during MV as compared to young adult mice. Lung inflammation (as assessed by the total cell count, IL-6, TNFα and MIP-2 concentrations in BAL fluid), systemic inflammation (as assessed by plasma IL-6 concentration) and alveolocapillary leak (as assessed by the total protein concentration of BAL fluid) were higher in O(LPS) and O(LPS+MV) mice as compared to Y(LPS) and Y(LPS+MV) mice, respectively. The combination of LPS+MV induced a higher lung injury as compared to LPS alone in middle-old mice but not in young adult mice. Hemodynamics (systemic blood pressure, cardiac output and pulmonary vascular resistances) were similar between Y(MV) and O(MV) on the one hand and between Y(LPS+MV) and O(LPS+MV) on the other hand. CONCLUSION: Middle-old mice were more susceptible to both LPS alone and the combination of LPS and low tidal volume MV as compared to their young adult counterparts. The synergism between LPS and MV was amplified in middle-old mice.

Interleukin-6 displays lung anti-inflammatory properties and exerts protective hemodynamic effects in a double-hit murine acute lung injury.

BACKGROUND: Interleukin 6 (IL-6) is a predictive factor of poor prognosis in patients with acute respiratory distress syndrome (ARDS). However, its acute pulmonary hemodynamic effects and role in lung injury have not been investigated in a clinically relevant murine model of ARDS. METHODS: We used adult C57Bl6 wild-type (WT) and IL-6 knock-out (IL-6KO) mice. The animals received intravenous recombinant human IL-6 (rHuIL-6) or vehicle followed by intratracheal lipopolysaccharide (LPS) or saline before undergoing low tidal volume mechanical ventilation (MV) for 5 h. Before sacrifice, right ventricular systolic pressure and cardiac output were measured and total pulmonary resistance was calculated. After sacrifice, lung inflammation, edema and injury were assessed with bronchoalveolar lavage (BAL) and histology. In other experiments, right ventricular systolic pressure was recorded during hypoxic challenges in uninjured WT mice pretreated with rHuIL-6 or rHuIL-6 + non-selective nitric oxide synthase inhibitor L-NAME or vehicle. RESULTS: IL-6KO(LPS+MV) mice showed a faster deterioration of lung elastic properties and more severe bronchoalveolar cellular inflammation as compared to WT(LPS+MV). Treatment with rHuIL-6 partially prevented this lung deterioration. Total pulmonary resistance was higher in IL-6KO(LPS+MV) mice and this increase was abolished in rHuIL-6-treated IL-6KO mice. Finally, rHuIL-6 reduced hypoxic pulmonary vasoconstriction in uninjured WT mice, an effect that was abolished by co-treatment with L-NAME. CONCLUSIONS: In a double-hit murine model of ARDS, IL-6 deficient mice experienced more severe bronchoalveolar cellular inflammation as compared to wild-type littermates. Furthermore, IL-6 deficiency caused marked acute pulmonary hypertension, which may be, at least partially, due to vasoactive mechanisms. A dysregulation of nitric oxide synthase may account for this observation, a hypothesis that will need to be investigated in future studies.

Roles for the CX3CL1/CX3CR1 and CCL2/CCR2 Chemokine Systems in Hypoxic Pulmonary Hypertension.

Monocytes/macrophages are major effectors of lung inflammation associated with various forms of pulmonary hypertension (PH). Interactions between the CCL2/CCR2 and CX3CL1/CX3CR1 chemokine systems that guide phagocyte infiltration are incompletely understood. Our objective was to explore the individual and combined actions of CCL2/CCR2 and CX3CL1/CX3CR1 in hypoxia-induced PH in mice; particularly their roles in monocyte trafficking, macrophage polarization, and pulmonary vascular remodeling. The development of hypoxia-induced PH was associated with marked increases in lung levels of CX3CR1, CCR2, and their respective ligands, CX3CL1 and CCL2. Flow cytometry revealed that both inflammatory Ly6Chi and resident Ly6Clo monocyte subsets exhibited sustained increases in blood and a transient peak in lung tissue, and that lung perivascular and alveolar macrophage counts showed sustained elevations. CX3CR1-/- mice were protected against hypoxic PH compared with wild-type mice, whereas CCL2-/- mice and double CX3CR1-/-/CCL2-/- mice exhibited similar PH severity, as did wild-type mice. The protective effects of CX3CR1 deficiency occurred concomitantly with increases in lung monocyte and macrophage counts and with a change from M2 to M1 macrophage polarization that markedly diminished the ability of conditioned media to induce pulmonary artery smooth muscle cell (PA-SMC) proliferation, which was partly dependent on CX3CL1 secretion. Results in mice given the CX3CR1 inhibitor F1 were similar to those in CX3CR1-/- mice. In conclusion, CX3CR1 deficiency protects against hypoxia-induced PH by modulating monocyte recruitment, macrophage polarization, and PA-SMC cell proliferation. Targeting CX3CR1 may hold promise for treating PH.

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.

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.

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.

ICAM-2 regulates vascular permeability and N-cadherin localization through ezrin-radixin-moesin (ERM) proteins and Rac-1 signalling.

BACKGROUND: Endothelial junctions control functions such as permeability, angiogenesis and contact inhibition. VE-Cadherin (VECad) is essential for the maintenance of intercellular contacts. In confluent endothelial monolayers, N-Cadherin (NCad) is mostly expressed on the apical and basal membrane, but in the absence of VECad it localizes at junctions. Both cadherins are required for vascular development. The intercellular adhesion molecule (ICAM)-2, also localized at endothelial junctions, is involved in leukocyte recruitment and angiogenesis. RESULTS: In human umbilical vein endothelial cells (HUVEC), both VECad and NCad were found at nascent cell contacts of sub-confluent monolayers, but only VECad localized at the mature junctions of confluent monolayers. Inhibition of ICAM-2 expression by siRNA caused the appearance of small gaps at the junctions and a decrease in NCad junctional staining in sub-confluent monolayers. Endothelioma lines derived from WT or ICAM-2-deficient mice (IC2neg) lacked VECad and failed to form junctions, with loss of contact inhibition. Re-expression of full-length ICAM-2 (IC2 FL) in IC2neg cells restored contact inhibition through recruitment of NCad at the junctions. Mutant ICAM-2 lacking the binding site for ERM proteins (IC2 ΔERM) or the cytoplasmic tail (IC2 ΔTAIL) failed to restore junctions. ICAM-2-dependent Rac-1 activation was also decreased in these mutant cell lines. Barrier function, measured in vitro via transendothelial electrical resistance, was decreased in IC2neg cells, both in resting conditions and after thrombin stimulation. This was dependent on ICAM-2 signalling to the small GTPase Rac-1, since transendothelial electrical resistance of IC2neg cells was restored by constitutively active Rac-1. In vivo, thrombin-induced extravasation of FITC-labeled albumin measured by intravital fluorescence microscopy in the mouse cremaster muscle showed that permeability was increased in ICAM-2-deficient mice compared to controls. CONCLUSIONS: These results indicate that ICAM-2 regulates endothelial barrier function and permeability through a pathway involving N-Cadherin, ERMs and Rac-1.

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.

Activation of lung p53 by Nutlin-3a prevents and reverses experimental pulmonary hypertension.

BACKGROUND: 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.

Rapamycin reverses pulmonary artery smooth muscle cell proliferation in pulmonary hypertension.

Pulmonary 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-1ß, 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.

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

Decreasing 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.

Telomere dysfunction causes sustained inflammation in chronic obstructive pulmonary disease.

RATIONALE: Chronic obstructive pulmonary disease (COPD) is associated with chronic inflammation of unknown pathogenesis. OBJECTIVES: To investigate whether telomere dysfunction and senescence of pulmonary vascular endothelial cells (P-ECs) induce inflammation in COPD. METHODS: Prospective comparison of patients with COPD and age- and sex-matched control smokers. Investigation of mice null for telomerase reverse transcriptase (Tert) or telomerase RNA component (Terc) genes. MEASUREMENTS AND MAIN RESULTS: In situ lung specimen studies showed a higher percentage of senescent P-ECs stained for p16 and p21 in patients with COPD than in control subjects. Cultured P-ECs from patients with COPD exhibited early replicative senescence, with decreased cell-population doublings, a higher percentage of ß-galactosidase-positive cells, reduced telomerase activity, shorter telomeres, and higher p16 and p21 mRNA levels at an early cell passage compared with control subjects. Senescent P-ECs released cytokines and mediators: the levels of IL-6, IL-8, monocyte chemotactic protein (MCP)-1, Hu-GRO, and soluble intercellular adhesion molecule (sICAM)-1 were elevated in the media of P-ECs from patients compared with control subjects at an early cell passage, in proportion to the senescent P-EC increase and telomere shortening. Up-regulation of MCP-1 and sICAM-1 led to increased monocyte adherence and migration. The elevated MCP-1, IL-8, Hu-GROα, and ICAM-1 levels measured in lungs from patients compared with control subjects correlated with P-EC senescence criteria and telomere length. In Tert(-/-) and/or Terc(-/-) mouse lungs, levels of the corresponding cytokines (MCP-1, IL-8, Hu-GROα, and ICAM-1) were also altered, despite the absence of external stimuli and in proportion to telomere dysfunction. CONCLUSIONS: Telomere dysfunction and premature P-EC senescence are major processes perpetuating lung inflammation in COPD.

Pulmonary artery smooth muscle cell senescence is a pathogenic mechanism for pulmonary hypertension in chronic lung disease.

RATIONALE: Senescence of pulmonary artery smooth muscle cells (PA-SMCs) caused by telomere shortening or oxidative stress may contribute to pulmonary hypertension associated with chronic lung diseases. OBJECTIVE: To investigate whether cell senescence contributes to pulmonary vessel remodeling and pulmonary hypertension in chronic obstructive pulmonary disease (COPD). METHODS AND RESULTS: In 124 patients with COPD investigated by right heart catheterization, we found a negative correlation between leukocyte telomere length and pulmonary hypertension severity. In-depth investigations of lung vessels and derived cultured PA-SMCs showed greater severity of remodeling and increases in senescent p16-positive and p21-positive PA-SMCs and proliferating Ki67-stained cells in 14 patients with COPD compared to 13 age-matched and sex-matched control subjects who smoke. Cultured PA-SMCs from COPD patients displayed accelerated senescence, with fewer cell population doublings, an increased percentage of ß-galactosidase-positive cells, shorter telomeres, and higher p16 protein levels at an early cell passage compared to PA-SMCs from controls. Both in situ and in vitro PA-SMC senescence criteria correlated closely with the degree of pulmonary vessel wall hypertrophy. Because senescent PA-SMCs stained for p16 and p21 were virtually confined to the media near the Ki67-positive cells, which predominated in the neointima and hypertrophied media, we evaluated whether senescent cells affected normal PA-SMC functions. We found that senescent PA-SMCs stimulated the growth and migration of normal target PA-SMCs through the production and release of paracrine soluble and insoluble factors. CONCLUSION: PA-SMC senescence is an important contributor to the process of pulmonary vascular remodeling that underlies pulmonary hypertension in chronic lung disease.