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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, 10–12 weeks) and middle-old (O, 12–13 months) 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 3 h of low tidal volume (8 mL/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.

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. ispartof: EUROPEAN RESPIRATORY JOURNAL vol:54 issue:4 ispartof: location:England status: published

International audience; 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 8weeks 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 hypoxicmice 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.

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.

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.

Pulmonary artery smooth muscle cell (PA-SMC) proliferation and inflammation are key components of pulmonary arterial hypertension (PAH). Interleukin (IL)-1β 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-1β, 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-1β-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 MyD88fl/flmice) decreased pulmonary hypertension severityversuscontrols, suggesting IL-1β-mediated effects on PA-SMCs and macrophages. The growth-promoting effect of media conditioned by M1 or M2 macrophages from M.lys-Cre MyD88fl/flmice was attenuated.Pulmonary vessel remodelling and inflammation during pulmonary hypertension require IL-1R1/MyD88 signalling. Targeting the IL-1β/IL-1R1 pathway may hold promise for treating human PAH.

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.

Chronic obstructive pulmonary disease (COPD) is a highly prevalent and devastating condition for which no curative treatment is available. Exaggerated lung-cell senescence may contribute substantially to the pathogenesis of COPD. Here, we explored the potential role for the mTOR signaling pathway in cell senescence and lung alterations in COPD, using lung specimens 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 pro-inflammatory senescence-associated secretory phenotype. To assess whether mTOR activation was causal in lung pathology, we generated mice with constitutive or conditional deletion of the tuberous sclerosis complex heterodimer TSC1 (a negative regulator of mTOR complex 1), in smooth muscle cells (SMCs), endothelial cells (ECs), or alveolar epithelial cells (AECs). In this model, mTOR activation was sufficient to induce early-onset replicative cell senescence in cultured pulmonary artery SMCs from mice with TSC1 deletion. Mice with conditional TSC1 deletion in ECs or AECs developed lung emphysema, pulmonary hypertension, and inflammation in the absence of any other stimuli and concomitantly with the accumulation of senescent lung cells. Rapamycin prevented all these effects. These results support causal links between mTOR activation, cell senescence, and lung alterations in COPD, thereby identifying the mTOR pathway as a new therapeutic target for COPD. Keywords: mTOR, senescence, COPD

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.

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.

International audience; 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.

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 β-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.

Objective: Constitutive activation of the mammalian target of rapamycin (mTOR) 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. Approach and Results: 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 pulmonary-artery muscularization and Ki67-positive PA-SMC counts, without systemic hypertension or cardiac dysfunction. Marked activation of the mTORC1 substrates S6K and 4EBP and of the mTORC2 substrates p-Akt Ser473 and GSK3 was found in lungs and pulmonary vessels from SM22-TSC1 -/- mice compared to controls. 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 PA-SMCs 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 S6K1/2 in mice also activated mTOR signaling and induced PH. Conclusion: Activation of mTORC1 signaling leads to increased PA-SMC proliferation and subsequent PH development.

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.

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.

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.

Background Pulmonary hypertension (PH) results from complex vessel remodeling involving both pulmonary artery smooth muscle cell (PA-SMC) proliferation and inflammatory processes. Both toll-like receptors (TLR) and interleukine-1 receptor (IL1R1) are involved in innate immunity and they share common signaling pathways: their activation results in recruitment of the molecular adaptor Myd88, leading to the activation of NFKB and subsequently synthesis and release of many cytokines including IL-1s, IL-6 and TNFα. In this study we question whether reducing PH, it is efficient to target IL1R1 or Myd88. Methods We first examined expression and localization of IL1R1 and Myd88 in lungs from patient with iPAH or controls, as well as in mice with hypoxic PH. Secondly we evaluated the role for IL1R1 and Myd88 in PH by studying IL1R1-/-, MyD88-/− and WT mice treated daily by Anakinra (selective IL1R1 antagonist) exposed to chronic hypoxia, in comparison with WT mice. PA-SMCs from these mice were studied in vitro. Results Marked increase expression of IL1R1 and Myd88 were observed in patients with iPAH and in mice exposed to chronic hypoxia. In contrast to IL1R1, which was widely distributed in the lung, Myd88 was preferentially express in remodeled vessels and in particular in PA-SMC. IL1R1-/-, MyD88-/− and WT mice treated with anakinra were similarly protected against hypoxic PH development, as shown by decreases in right ventricular systolic pressure, Fulton index, muscularization of vessels and proliferative ki67-positive cells in arteries compared to untreated WT mice. The increase in lung perivascular macrophages, IL-1s and IL-6 associated with hypoxia exposure was abrogated in anakinra-treated WT mice and in IL1R1-/− and MyD88-/− mice. In vitro, the potent IL-1s-mediated growth-promoting activity on PA-SMCs was abolished by anakinra and by genetic deletion of IL1R1 or MyD88. Interestingly, the growth-promoting effect of IL-1s was stronger in cells from hypoxic WT mice than control. PA-SMC proliferation induced by conditioned media from alveolar macrophages was potentiated by previous macrophage treatment with IL-1s. Conclusion The IL-1s/IL1R1/Myd88 axis contributes to PH development, by stimulating PA-SMC growth through both direct and indirect macrophage-mediated effects. IL1R1 or Myd88 inhibition may represent a new option for treating PH.

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.

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.

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.

International audience; 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-1β, insulin-like growth factor-1, or fetal calf serum (FCS), with increases in phosphorylated (Ser-473)Akt, (Thr-308)Akt, glycogen synthase kinase (GSK)3, and S6K reflecting activated mTORC1 and mTORC2 signaling. Treatment with rapamycin (0.5 μM) or the Akt inhibitor, A-443654 (0.5 μM), reduced FCS-stimulated growth of PA-SMCs from MCT-PH rats to the level in control rats while inhibiting Akt, GSK3, and S6K activation. Neither the tyrosine kinase inhibitor, imatinib (0.1 μM), nor the 5-hydroxytryptophan transporter inhibitor, fluoxetine (5 μM), normalized the increased PA-SMC growth response to FCS. Rapamycin treatment (5 mg/kg/d) of MCT-PH rats from Day 21 to Day 28 markedly reduced phoshop (p)-Aky, p-GSK3, and p-S6K in PAs, and normalized growth of derived PA-SMCs. This effect was not observed after 1 week of imatinib (100 mg/kg/d) or fluoxetine (20 mg/kg/d). Rapamycin given preventively (Days 1-21) or curatively (Days 21-42) inhibited MCT-PH to a greater extent than did imatinib or fluoxetine. Experimental PH in rats is associated with a sustained proliferative PA-SMC phenotype linked to activation of both mTORC1 and mTORC2 signaling and is suppressed by rapamycin treatment.

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.

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.

Background— Pulmonary hypertension (PH) is among the complications of HIV infection. Combination antiretroviral therapy may influence the progression of HIV-related PH. Because Akt signaling is a potential molecular target of HIV protease inhibitors (HPIs), we hypothesized that these drugs altered monocrotaline- and hypoxia-induced PH in rats by downregulating the Akt pathway, thereby inhibiting pulmonary artery smooth muscle cell proliferation. Methods and Results— Daily treatment with each of 3 first-generation HPIs (ritonavir 30 mg/kg, amprenavir 100 mg/kg, and nelfinavir 500 mg/kg) started 3 weeks after a subcutaneous monocrotaline injection (60 mg/kg) substantially diminished pulmonary artery pressure, right ventricular hypertrophy, number of muscularized pulmonary vessels, pulmonary arterial wall thickness, and proliferating pulmonary vascular Ki67-labeled cells without affecting vessel caspase 3 staining. HPI treatment partially prevented the development of hypoxia- and monocrotaline-induced PH. Monocrotaline-induced PH was associated with marked activation of Akt signaling in the lungs and proximal pulmonary arteries, with increases in phosphorylated Akt, phosphorylated glycogen-synthase-kinase-3β (GSK3), and phosphorylated endothelial nitric oxide synthase, all of which decreased markedly after treatment with each HPI. In contrast, PH-associated increases in phosphorylated extracellular signal-related kinase 1/2 and myosin light-chain phosphatase were unaltered by the HPIs. The 3 HPIs and the phosphatidylinositol 3-kinase inhibitor LY294002 inhibited platelet-derived growth factor–induced phosphorylation of Akt and GSK3 in cultured pulmonary artery smooth muscle cells and blocked cell proliferation; this last effect was abolished by the GSK3 inhibitor SB216763. Conclusion— These results support an effect of HPIs on pulmonary vascular remodeling mediated by inhibition of Akt phosphorylation and consequently of pulmonary artery smooth muscle cell proliferation.

Introduction The development of hypoxia-induced pulmonary hypertension (PH) is associated with lung inflammation and vessel infiltration by monocytes. Two major monocyte subsets showing differential expression of CCR2 and CX3CR1 receptors have been identified. Ly6C hi monocytes are involved in inflammation and express high levels of CCR2 but low levels of CX3CR1 (Ly6C hi CCR2 hi /CX3CR1 lo ). Ly6C lo monocytes are the noninflammatory subtype and express high levels of CX3CR1 (Ly6C l °CCR2 lo /CX3CR1 hi ). Methods To determine the roles for Ly6C hi and Ly6C lo monocytes in hypoxic PH in mice, we studied CX3CR1 -/- , CCL2 -/− or double CX3CR1/CCL2 -/− mice compared to wild type (WT) mice. Results Development of hypoxia-induced PH was associated with a marked increase in lung mRNA levels of CX3CR1, CCR2 and of their respective ligands CX3CL1 and CCL2. FACs analysis revealed sustained increases in both monocyte subsets in blood but only a transient peak on day 3 of hypoxia exposure in lung tissue. Total lung macrophage counts remained unchanged. CX3CR1 -/− mice showed protection against hypoxic PH compared to WT mice, whereas CCL2 -/− mice and double CX3CR1/CCL2 -/− mice exhibited similar PH severity as did WT mice. Chronically hypoxic WT and mutant mice did not differ regarding lung Ly6C lo monocytes, whereas Ly6C hi counts were reduced in CCL2 -/− or double CX3CR1/CCL2 -/− mice compared to WT or CX3CR1 -/− mice. Total lung macrophage counts did not differ between WT and CCL2 -/− or double CX3CR1/CCL2 -/− mice but were markedly increased in CX3CR1 -/− mice. These results are consistent with previous studies showing that CCR2 deficiency exacerbates PH in hypoxic mice, and they support a prominent effect of the CX3CL1/CX3CR1 axis on pulmonary vascular remodeling. Accordingly, we found that CX3CL1 potentiated proliferation of cultured PA-SMCs from WT mice but not from CX3CR1 -/− mice. Conclusion CX3CR1 deficiency protects against hypoxia-induced PH by modulating monocyte recruitment and PA-SMC cell proliferation.

Background Telomere dysfunction and exaggerated lung cell senescence now appear as major pathological processes leading to lung alterations in COPD. Pulmonary vascular endothelial cells (P-ECs) and smooth muscle cells (PA-SMCs) derived from lungs of patients with COPD exhibit increased susceptibility to replicative senescence compared to controls. However, the mechanisms underlying lung cell senescence in COPD remain poorly understood. Recent studies suggest that cell senescence and aging occur in response to overactivation of the mammalian target of rapamycin (mTOR) signaling pathway. Methods To investigate whether lung cell senescence is related to overactivation of the mTOR signaling pathway in COPD, we studied lung specimens and derived cultured PA-SMCs and P-ECs from patients with COPD and age- and sex-matched control smokers. Results Marked activation of the mTOR-Akt signaling pathway was found in lungs from patients with COPD compared to controls, with a marked PTEN decrease and activation of mTOR complex 1 (mTORC1) substrates S6K and 4EBP and of mTORC2 substrates P-Akt 473 and GSK3, together with an increase in p21 protein levels. In-situ analysis of lung specimens revealed prominent P-Akt 473 , P-S6K, and P-GSK3 staining in PA-SMCs and P-ECs from patients with COPD compared to controls, most notably the cells exhibiting staining for p16 and p21. In cultured PA-SMCs from patients and controls that were subjected to repeated passages, the onset of replicative cell senescence was associated with similar alterations, including decreased PTEN and activation of mTORC1 and mTORC2 substrates. Cell exposure to low-dose rapamycin, which selectively inhibits mTORC1 but not mTORC2, delayed the early onset of senescence, increasing the cell-population doublings and decreasing the percentage of beta-galactosidase-positive cells; and suppressed the senescence-associated secretory phenotype associated with COPD. Cell exposure to sulforaphane, which stimulates nff2, or bortezomib, which inhibits the proteasome, did not alter or potentiated the cell senescence process. Conclusion These results show that the increased propensity of lung vascular cells to senescence in COPD is related to overactivation of the mTOR/Akt signaling pathway and can be suppressed by rapamycin treatment.

Background Ubiquitous calpains 1 and 2 are calcium-activated neutral cysteine proteases that were shown to be involved in the progression of pulmonary hypertension (PH). Whether calpains contribute to the pulmonary vascular remodeling process through intracellular- or extracellular-mediated effects remains unclear. Here, we investigated whether overexpression of calpastatin, the specific endogenous calpain inhibitor, affected PH progression in mice through intracellular and/or extracellular mechanisms. Methods To specify the role for intracellular versus extracellular calpains during PH progression, we used transgenic mice ubiquitously overexpressing intracellular calpastatin under the control of the CMV promoter (CMV-cast) and transgenic mice with hepatic specific/inflammation-inducible expression of secreted calpastatin under the control of the human CRP promoter (CRP-cast). Results Hypoxia exposure of wild-type (WT) mice led to marked increases in lung calpain 1 and 2 protein levels and activity, both intracellularly (as assessed by the spectrin breakdown product) and extracellularly (as assessed by fibronectin breakdown product), which peaked on day 8 and remained elevated until day 18. Calpastatin protein increased from day 1 to day 3 and returned to basal levels by day 18. Immunofluorescence studies revealed prominent calpain 1 and 2 staining in pulmonary-artery smooth muscle cells (PA-SMCs) of remodeled vessels. Both CRP-cast and CMV-cast mice showed decreased PH severity compared to WT mice, with similar decreases in right ventricular systolic pressure (RVSP), RV hypertrophy, and distal pulmonary-artery muscularization. Extracellular calpain activity was decreased only in CRP-cast mice. A selective calpain inhibitor, PD150606, protected WT mice against hypoxic PH but did not produce additional effects in CMV-cast or CRP-cast mice. PD150606 also partially reversed PH in SM22-5HTT+ mice, which spontaneously develop PH. The growth rate of PA-SMCs from CMV-cast mice was decreased compared to PA-SMCs from CRP-cast and WT mice. Treatment of PA-SMCs with PD150606 or calpastatin decreased PA-SMC proliferation and abolished the difference in cell growth rate across the three groups of mice. A role for extracellular calpastatin was shown in patients with idiopathic pulmonary arterial hypertension, who exhibited increased calpastatin pulmonary vessel immunostaining and increased circulating calpastatin levels compared to healthy controls. Conclusion These data indicate that both intracellular and extracellular calpains play an important role in the pathogenesis of PH and that calpastatin protects against PH by counteracting these effects.

International audience; Changes in cell architecture, essentially linked to profound cytoskeleton rearrangements, are common features accompanying cell transformation. Supporting the involvement of the microfilament network in tumor cell behavior, several actin-binding proteins, including zyxin, a potential regulator of actin polymerization, may play a role in oncogenesis. In this work, we investigate the status of zyxin in Ewing tumors, a family of pediatric malignancies of bone and soft tissues, which are mainly associated with a t(11;22) chromosomal translocation encoding the EWS-FLI1 oncoprotein. We observe that EWS-FLI1-transformed murine fibroblasts, as well as human Ewing tumor-derived SK-N-MC cells, exhibit a complete disruption of their actin cytoskeleton, retaining very few stress fibers, focal adhesions and cell-to-cell contacts. We show that within these cells, zyxin is expressed at very low levels and remains diffusely distributed throughout the cytoplasm, instead of concentrating in actin-rich dynamic structures. We demonstrate that zyxin gene transfer into EWS-FLI1-transformed fibroblasts elicits reconstitution of zyxin-rich focal adhesions and intercellular junctions, dramatic reorganization of the actin cytoskeleton, decreased cell motility, inhibition of anchorage-independent growth and impairment of tumor formation in athymic mice. We observe similar phenotypic changes after zyxin gene transfer in SK-N-MC cells, suggesting that zyxin has tumor suppressor activity in Ewing tumor cells.