Survivin inhibition with YM155 ameliorates experimental pulmonary arterial hypertension.

Background: Imbalance between cell proliferation and apoptosis underlies the development of pulmonary arterial hypertension (PAH). Current vasodilator treatment of PAH does not target the uncontrolled proliferative process in pulmonary arteries. Proteins involved in the apoptosis pathway may play a role in PAH and their inhibition might represent a potential therapeutic target. Survivin is a member of the apoptosis inhibitor protein family involved in cell proliferation. Objectives: This study aimed to explore the potential role of survivin in the pathogenesis of PAH and the effects of its inhibition. Methods: In SU5416/hypoxia-induced PAH mice we assessed the expression of survivin by immunohistochemistry, western-blot analysis, and RT-PCR; the expression of proliferation-related genes (Bcl2 and Mki67); and the effects of the survivin inhibitor YM155. In explanted lungs from patients with PAH we assessed the expression of survivin, BCL2 and MKI67. Results: SU5416/hypoxia mice showed increased expression of survivin in pulmonary arteries and lung tissue extract, and upregulation of survivin, Bcl2 and Mki67 genes. Treatment with YM155 reduced right ventricle (RV) systolic pressure, RV thickness, pulmonary vascular remodeling, and the expression of survivin, Bcl2, and Mki67 to values similar to those in control animals. Lungs of patients with PAH also showed increased expression of survivin in pulmonary arteries and lung extract, and also that of BCL2 and MKI67 genes, compared with control lungs. Conclusion: We conclude that survivin might be involved in the pathogenesis of PAH and that its inhibition with YM155 might represent a novel therapeutic approach that warrants further evaluation.

Pre-exposure prophylaxis with hydroxychloroquine for COVID-19: a double-blind, placebo-controlled randomized clinical trial.

BACKGROUND: Pre-exposure prophylaxis (PrEP) is a promising strategy to break COVID-19 transmission. Although hydroxychloroquine was evaluated for treatment and post-exposure prophylaxis, it is not evaluated for COVID-19 PrEP yet. The aim of this study was to evaluate the efficacy and safety of PrEP with hydroxychloroquine against placebo in healthcare workers at high risk of SARS-CoV-2 infection during an epidemic period. METHODS: We conducted a double-blind placebo-controlled randomized clinical trial in three hospitals in Barcelona, Spain. From 350 adult healthcare workers screened, we included 269 participants with no active or past SARS-CoV-2 infection (determined by a negative nasopharyngeal SARS-CoV-2 PCR and a negative serology against SARS-CoV-2). Participants allocated in the intervention arm (PrEP) received 400 mg of hydroxychloroquine daily for the first four consecutive days and subsequently, 400 mg weekly during the study period. Participants in the control group followed the same treatment schedule with placebo tablets. RESULTS: 52.8% (142/269) of participants were in the hydroxychloroquine arm and 47.2% (127/269) in the placebo arm. Given the national epidemic incidence decay, only one participant in each group was diagnosed with COVID-19. The trial was stopped due to futility and our study design was deemed underpowered to evaluate any benefit regarding PrEP efficacy. Both groups showed a similar proportion of participants experiencing at least one adverse event (AE) (p=0.548). No serious AEs were reported. Almost all AEs (96.4%, 106/110) were mild. Only mild gastrointestinal symptoms were significantly higher in the hydroxychloroquine arm compared to the placebo arm (27.4% (39/142) vs 15.7% (20/127), p=0.041). CONCLUSIONS: Although the efficacy of PrEP with hydroxychloroquine for preventing COVID-19 could not be evaluated, our study showed that PrEP with hydroxychloroquine at low doses is safe. TRIAL REGISTRATION: ClinicalTrials.gov NCT04331834 . Registered on April 2, 2020.

Circulating Cell Biomarkers in Pulmonary Arterial Hypertension: Relationship with Clinical Heterogeneity and Therapeutic Response.

BACKGROUND: Endothelial dysfunction is central to PAH. In this study, we simultaneously analysed circulating levels of endothelial microvesicles (EMVs) and progenitor cells (PCs) in PAH and in controls, as biomarkers of pulmonary endothelial integrity and evaluated differences among PAH subtypes and as a response to treatment. METHODS: Forty-seven controls and 144 patients with PAH (52 idiopathic, 9 heritable, 31 associated with systemic sclerosis, 15 associated with other connective tissue diseases, 20 associated with HIV and 17 associated with portal hypertension) were evaluated. Forty-four patients with scleroderma and 22 with HIV infection, but without PAH, were also studied. Circulating levels of EMVs, total (CD31+CD42b-) and activated (CD31+CD42b-CD62E+), as well as circulating PCs (CD34+CD133+CD45low) were measured by flow cytometry and the EMVs/PCs ratio was computed. In treatment-naïve patients, measurements were repeated after 3 months of PAH therapy. RESULTS: Patients with PAH showed higher numbers of EMVs and a lower percentage of PCs, compared with healthy controls. The EMV/PC ratio was increased in PAH patients, and in patients with SSc or HIV without PAH. After starting PAH therapy, individual changes in EMVs and PCs were variable, without significant differences being observed as a group. Conclusion: PAH patients present disturbed vascular homeostasis, reflected in changes in circulating EMV and PC levels, which are not restored with PAH targeted therapy. Combined measurement of circulating EMVs and PCs could be foreseen as a potential biomarker of endothelial dysfunction in PAH.

A Potential Role for Exosomal Translationally Controlled Tumor Protein Export in Vascular Remodeling in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is characterized by increased proliferation and resistance to apoptosis of pulmonary vascular cells. Increased expression of translationally controlled tumor protein (TCTP), a prosurvival and antiapoptotic mediator, has recently been demonstrated in patients with heritable PAH; however, its role in the pathobiology of PAH remains unclear. Silencing of TCTP in blood outgrowth endothelial cells (BOECs) isolated from control subjects led to significant changes in morphology, cytoskeletal organization, increased apoptosis, and decreased directionality during migration. Because TCTP is also localized in extracellular vesicles, we isolated BOEC-derived extracellular vesicles (exosomes and microparticles) by sequential ultracentrifugation. BOECs isolated from patients harboring BMPR2 mutations released more exosomes than those derived from control subjects in proapoptotic conditions. Furthermore, TCTP expression was significantly higher in exosomes than in microparticles, indicating that TCTP is mainly exported via exosomes. Coculture assays demonstrated that exosomes transferred TCTP from ECs to pulmonary artery smooth muscle cells, suggesting a role for endothelial-derived TCTP in conferring proliferation and apoptotic resistance. In an experimental model of PAH, rats treated with monocrotaline demonstrated increased concentrations of TCTP in the lung and plasma. Consistent with this finding, we observed increased circulating TCTP levels in patients with idiopathic PAH compared with control subjects. Therefore, our data suggest an important role for TCTP in regulating the critical vascular cell phenotypes that have been implicated in the pathobiology of PAH. In addition, this research implicates TCTP as a potential biomarker for the onset and development of PAH.

Effect of targeted therapy on circulating progenitor cells in precapillary pulmonary hypertension.

BACKGROUND: Endothelial dysfunction is key in the development of pulmonary hypertension (PH) and is associated with reduced number of circulating progenitor cells. Studies to date evaluating levels of circulating progenitor cells in PH have provided conflicting results. Current treatment of pulmonary arterial hypertension (PAH) and medical treatment of chronic thromboembolic pulmonary hypertension (CTEPH) targets endothelium dependent signalling pathways. The effect of PAH-targeted therapy on circulating progenitor cells has not been clearly established. OBJECTIVES: To investigate whether levels of circulating progenitor cells in treatment-naïve patients with PAH or CTEPH differ from healthy subjects and to assess the effect of PAH-targeted therapy on the circulating levels of these progenitors. METHODS: Thirty controls, 33 PAH and 11 CTEPH treatment-naïve patients were studied. Eighteen patients with PAH and 9 with CTEPH were re-evaluated 6-12months after starting PAH-targeted therapy. Levels of progenitors were measured by flow cytometry as CD45+CD34+ and CD45+CD34+CD133+ cells. RESULTS: Compared with controls, the number of circulating progenitor cells was reduced in PAH but not in CTEPH. After 6-12months of treatment, levels of circulating progenitors increased in PAH and remained unchanged in CTEPH. Patients with lower exercise tolerance presented lower levels of circulating progenitors. No other relation was found between levels of progenitors and clinical or hemodynamic parameters. CONCLUSIONS: Patients with PAH, but not those with CTEPH, present reduced levels of circulating progenitor cells. PAH-targeted therapy increases levels of progenitors in PAH but not in CTEPH, suggesting different involvement of progenitor cells in the pathobiology of these pulmonary hypertensive disorders.

Hepatic Shunting of Eggs and Pulmonary Vascular Remodeling in Bmpr2(+/-) Mice with Schistosomiasis.

RATIONALE: Schistosomiasis is a major cause of pulmonary arterial hypertension (PAH). Mutations in the bone morphogenetic protein type-II receptor (BMPR-II) are the commonest genetic cause of PAH. OBJECTIVES: To determine whether Bmpr2(+/-) mice are more susceptible to schistosomiasis-induced pulmonary vascular remodeling. METHODS: Wild-type (WT) and Bmpr2(+/-) mice were infected percutaneously with Schistosoma mansoni. At 17 weeks postinfection, right ventricular systolic pressure and liver and lung egg counts were measured. Serum, lung and liver cytokine, pulmonary vascular remodeling, and liver histology were assessed. MEASUREMENTS AND MAIN RESULTS: By 17 weeks postinfection, there was a significant increase in pulmonary vascular remodeling in infected mice. This was greater in Bmpr2(+/-) mice and was associated with an increase in egg deposition and cytokine expression, which induced pulmonary arterial smooth muscle cell proliferation, in the lungs of these mice. Interestingly, Bmpr2(+/-) mice demonstrated dilatation of the hepatic central vein at baseline and postinfection, compared with WT. Bmpr2(+/-) mice also showed significant dilatation of the liver sinusoids and an increase in inflammatory cells surrounding the central hepatic vein, compared with WT. This is consistent with an increase in the transhepatic passage of eggs. CONCLUSIONS: This study has shown that levels of BMPR-II expression modify the pulmonary vascular response to chronic schistosomiasis. The likely mechanism involves the increased passage of eggs to the lungs, caused by altered diameter of the hepatic veins and sinusoids in Bmpr2(+/-) mice. Genetically determined differences in the remodeling of hepatic vessels may represent a new risk factor for PAH associated with schistosomiasis.

Sildenafil in a cigarette smoke-induced model of COPD in the guinea-pig.

Sildenafil, a phosphodiesterase-5 inhibitor used to treat pulmonary hypertension, may have effects on pulmonary vessel structure and function. We evaluated the effects of sildenafil in a cigarette smoke (CS)-exposed model of chronic obstructive pulmonary disease (COPD).42 guinea-pigs were exposed to cigarette smoke or sham-exposed and treated with sildenafil or vehicle for 12 weeks, divided into four groups. Assessments included respiratory resistance, pulmonary artery pressure (PAP), right ventricle (RV) hypertrophy, endothelial function of the pulmonary artery and lung vessel and parenchymal morphometry.CS-exposed animals showed increased PAP, RV hypertrophy, raised respiratory resistance, airspace enlargement and intrapulmonary vessel remodelling. CS exposure also produced wall thickening, increased contractility and endothelial dysfunction in the main pulmonary artery. CS-exposed animals treated with sildenafil showed lower PAP and a trend to less RV hypertrophy than CS-exposed only animals. Furthermore, sildenafil preserved the intrapulmonary vessel density and attenuated the airspace enlargement induced by CS. No differences in gas exchange, respiratory resistance, endothelial function and vessel remodelling were observed.We conclude that in this experimental model of COPD, sildenafil prevents the development of pulmonary hypertension and contributes to preserve the parenchymal and vascular integrity, reinforcing the notion that the nitric oxide-cyclic guanosine monophosphate axis is perturbed by CS exposure.

A systems biology approach reveals a link between systemic cytokines and skeletal muscle energy metabolism in a rodent smoking model and human COPD.

BACKGROUND: A relatively large percentage of patients with chronic obstructive pulmonary disease (COPD) develop systemic co-morbidities that affect prognosis, among which muscle wasting is particularly debilitating. Despite significant research effort, the pathophysiology of this important extrapulmonary manifestation is still unclear. A key question that remains unanswered is to what extent systemic inflammatory mediators might play a role in this pathology. Cigarette smoke (CS) is the main risk factor for developing COPD and therefore animal models chronically exposed to CS have been proposed for mechanistic studies and biomarker discovery. Although mice have been successfully used as a pre-clinical in vivo model to study the pulmonary effects of acute and chronic CS exposure, data suggest that they may be inadequate models for studying the effects of CS on peripheral muscle function. In contrast, recent findings indicate that the guinea pig model (Cavia porcellus) may better mimic muscle wasting. METHODS: We have used a systems biology approach to compare the transcriptional profile of hindlimb skeletal muscles from a Guinea pig rodent model exposed to CS and/or chronic hypoxia to COPD patients with muscle wasting. RESULTS: We show that guinea pigs exposed to long-term CS accurately reflect most of the transcriptional changes observed in dysfunctional limb muscle of severe COPD patients when compared to matched controls. Using network inference, we could then show that the expression profile in whole lung of genes encoding for soluble inflammatory mediators is informative of the molecular state of skeletal muscles in the guinea pig smoking model. Finally, we show that CXCL10 and CXCL9, two of the candidate systemic cytokines identified using this pre-clinical model, are indeed detected at significantly higher levels in serum of COPD patients, and that their serum protein level is inversely correlated with the expression of aerobic energy metabolism genes in skeletal muscle. CONCLUSIONS: We conclude that CXCL10 and CXCL9 are promising candidate inflammatory signals linked to the regulation of central metabolism genes in skeletal muscles. On a methodological level, our work also shows that a system level analysis of animal models of diseases can be very effective to generate clinically relevant hypothesis.

Stimulation of soluble guanylate cyclase prevents cigarette smoke-induced pulmonary hypertension and emphysema.

RATIONALE: Chronic obstructive pulmonary disease (COPD) is a major cause of death worldwide. No therapy stopping progress of the disease is available. OBJECTIVES: To investigate the role of the soluble guanylate cyclase (sGC)-cGMP axis in development of lung emphysema and pulmonary hypertension (PH) and to test whether the sGC-cGMP axis is a treatment target for these conditions. METHODS: Investigations were performed in human lung tissue from patients with COPD, healthy donors, mice, and guinea pigs. Mice were exposed to cigarette smoke (CS) for 6 hours per day, 5 days per week for up to 6 months and treated with BAY 63-2521. Guinea pigs were exposed to CS from six cigarettes per day for 3 months, 5 days per week and treated with BAY 41-2272. Both BAY compounds are sGC stimulators. Gene and protein expression analysis were performed by quantitative real-time polymerase chain reaction and Western blotting. Lung compliance, hemodynamics, right ventricular heart mass alterations, and alveolar and vascular morphometry were performed, as well as inflammatory cell infiltrate assessment. In vitro assays of cell adhesion, proliferation, and apoptosis have been done. MEASUREMENTS AND MAIN RESULTS: The functionally essential sGC ß1-subunit was down-regulated in patients with COPD and in CS-exposed mice. sGC stimulators prevented the development of PH and emphysema in the two different CS-exposed animal models. sGC stimulation prevented peroxynitrite-induced apoptosis of alveolar and endothelial cells, reduced CS-induced inflammatory cell infiltrate in lung parenchyma, and inhibited adhesion of CS-stimulated neutrophils. CONCLUSIONS: The sGC-cGMP axis is perturbed by chronic exposure to CS. Treatment of COPD animal models with sGC stimulators can prevent CS-induced PH and emphysema.

Proteomic analysis implicates translationally controlled tumor protein as a novel mediator of occlusive vascular remodeling in pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a lethal disease characterized by excessive proliferation of pulmonary vascular endothelial cells (ECs). Hereditary PAH (HPAH) is often caused by mutations in the bone morphogenetic protein receptor type 2 gene (BMPR2). However, the mechanisms by which these mutations cause PAH remain unclear. Therefore, we screened for dysregulated proteins in blood-outgrowth ECs of HPAH patients with BMPR2 mutations compared with healthy control subjects. METHODS AND RESULTS: A total of 416 proteins were detected with 2-dimensional PAGE in combination with liquid chromatography/tandem mass spectrometry analysis, of which 22 exhibited significantly altered abundance in blood-outgrowth ECs from patients with HPAH. One of these proteins, translationally controlled tumor protein (TCTP), was selected for further study because of its well-established role in promoting tumor cell growth and survival. Immunostaining showed marked upregulation of TCTP in lungs from patients with HPAH and idiopathic PAH, associated with remodeled vessels of complex lesions. Increased TCTP expression was also evident in the SU5416 rat model of severe and irreversible PAH, associated with intimal lesions, colocalizing with proliferating ECs and the adventitia of remodeled vessels but not in the vascular media. Furthermore, silencing of TCTP expression increased apoptosis and abrogated the hyperproliferative phenotype of blood-outgrowth ECs from patients with HPAH, raising the possibility that TCTP may be a link in the emergence of apoptosis-resistant, hyperproliferative vascular cells after EC apoptosis. CONCLUSION: Proteomic screening identified TCTP as a novel mediator of endothelial prosurvival and growth signaling in PAH, possibly contributing to occlusive pulmonary vascular remodeling triggered by EC apoptosis.

Effects of aclidinium bromide in a cigarette smoke-exposed Guinea pig model of chronic obstructive pulmonary disease.

Long-acting muscarinic antagonists are widely used to treat chronic obstructive pulmonary disease (COPD). In addition to bronchodilation, muscarinic antagonism may affect pulmonary histopathological changes. The effects of long-acting muscarinic antagonists have not been thoroughly evaluated in experimental models of COPD induced by chronic exposure to cigarette smoke (CS). We investigated the effects of aclidinium bromide on pulmonary function, airway remodeling, and lung inflammation in a CS-exposed model of COPD. A total of 36 guinea pigs were exposed to CS and 22 were sham exposed for 24 weeks. Animals were nebulized daily with vehicle, 10 µg/ml, or 30 µg/ml aclidinium, resulting in six experimental groups. Pulmonary function was assessed weekly by whole-body plethysmography, determining the enhanced pause (Penh) at baseline, after treatment, and after CS/sham exposure. Lung changes were evaluated by morphometry and immunohistochemistry. CS exposure increased Penh in all conditions. CS-exposed animals treated with aclidinium showed lower baseline Penh than untreated animals (P = 0.02). CS induced thickening of all bronchial wall layers, airspace enlargement, and inflammatory cell infiltrate in airways and septa. Treatment with aclidinium abrogated the CS-induced smooth muscle enlargement in small airways (P = 0.001), and tended to reduce airspace enlargement (P = 0.054). Aclidinium also attenuated CS-induced neutrophilia in alveolar septa (P = 0.04). We conclude that, in guinea pigs chronically exposed to CS, aclidinium has an antiremodeling effect on small airways, which is associated with improved respiratory function, and attenuates neutrophilic infiltration in alveolar septa. These results indicate that, in COPD, aclidinium may exert beneficial effects on lung structure in addition to its bronchodilator action.

Effects of cigarette smoke and chronic hypoxia on ventilation in guinea pigs. Clinical significance.

Ventilatory effects of chronic cigarette smoke (CS) alone or associated to chronic hypoxia (CH), as frequently occurs in chronic obstructive pulmonary disease (COPD), remain unknown. We have addressed this problem using whole-body plethysmography in guinea-pigs, common models to study harmful effects of CS on the respiratory system. Breathing frequencies (Bf) in control (2-5 months old) guinea pigs is 90-100 breaths/min, their tidal volume (TV) increased with age but lagged behind body weight gain and, as consequence, their minute volume (MV)/Kg decreased with age. MV did not change by acutely breathing 10% O(2) but doubled while breathing 5% CO(2) in air. Exposure to chronic sustained hypoxia (15 days, 12% O(2), CH) did not elicit ventilatory acclimatization nor adaptation. These findings confirm the unresponsiveness of the guinea pig CB to hypoxia. Exposure to CS (3 months) increased Bf and MV but association with CH blunted CS effects. We conclude that CS and CH association accelerates CS-induced respiratory system damage leading to a hypoventilation that can worsen the ongoing COPD process.

Pulmonary inflammatory reaction and structural changes induced by cigarette smoke exposure in the Guinea pig.

Cigarette smoke (CS) induces an inflammatory process in the lung that may underlie the development of chronic obstructive pulmonary disease (COPD). The nature and characteristics of this process have not been fully established in animal models. We aimed to evaluate the pulmonary inflammatory reaction and its involvement in structural changes in guinea pigs chronically exposed to CS. 19 Hartley guinea pigs were exposed to 7 cigarettes/day, during 3 or 6 months. 18 control guinea pigs were sham-exposed. Numbers of neutrophils, macrophages and eosinophils and lymphoid follicles were assessed in different lung structures. Airway and vessel morphometry, alveolar space size and collagen deposition were also quantified. After 6 months of exposure, CS-exposed guinea pigs showed increased numbers of neutrophils, macrophages and eosinophils in the airways, intrapulmonary vessels and alveolar septa, as well as lymphoid follicles. Increased numbers of muscularized intrapulmonary vessels were apparent at 3 months. After 6 months of exposure, the airway wall thickened and the alveolar space size increased. Collagen deposition was also apparent in airway walls and alveolar septa after 6 months' exposure. The magnitude of airway wall-thickening correlated with the number of infiltrating inflammatory cells, and the extension of collagen deposition correlated with alveolar space size. We conclude that in the guinea pig, 6 months of CS exposure induces inflammatory cell infiltrate in lung structures, at an intensity that correlates with airway remodelling. These changes resemble those observed in COPD, thus endorsing the pathogenic role of CS and the usefulness of this animal model for its study.

Effects of cigarette smoke and chronic hypoxia on airways remodeling and resistance. Clinical significance.

Previously we have reported that association of cigarette smoke (CS) and chronic hypoxia (CH) interact positively to physiopathologically remodel pulmonary circulation. In present study we have exposed guinea pigs to CS smoke (four cigarettes/day; 3 months; CS) and to chronic hypoxia (12% O(2), 15 days; CH) alone or in combination (CSCH animals) and evaluated airways remodeling and resistance assessed as Penh (enhance pause). We measured Penh while animals breathe air, 10% O(2) and 5% CO(2) and found that CS and CH animals have higher Penh than controls; Penh was even larger in CSCH animals. A rough parallelism between Penh and thickness of bronchiolar wall and muscular layer and Goblet cell number was noticed. We conclude that CS and CH association accelerates CS-induced respiratory system damage, evidenced by augmented airway resistance, bronchial wall thickness and muscularization and Goblet cell number. Our findings would suggest that appearance of hypoxia would aggravate any preexisting pulmonary pathology by increasing airways resistance and reactivity.

Mesenchymal stem cells restore lung function by recruiting resident and nonresident proteins.

Because human lungs are unlikely to repair or regenerate beyond the cellular level, cell therapy has not previously been considered for chronic irreversible obstructive lung diseases. To explore whether cell therapy can restore lung function, we administered allogenic intratracheal mesenchymal stem cells (MSCs) in the trachea of rats with chronic thromboembolic pulmonary hypertension (CTEPH), a disease characterized by single or recurrent pulmonary thromboembolic obliteration and progressive pulmonary vascular remodeling. MSCs were retrieved only in high pressure-exposed lungs recruited via a homing stromal derived factor-1α/CXCR4 pathway. After MSC administration, a marked and long-lasting improvement of all clinical parameters and a significant change of the proteome level were detected. Beside a variation of liver proteome, such as caspase-3, NF-κB, collagen1A1, and α-SMA, we also identified more than 300 resident and nonresident lung proteins [e.g., myosin light chain 3 (P16409) or mitochondrial ATP synthase subunit alpha (P15999)]. These results suggest that cell therapy restores lung function and the therapeutic effects of MSCs may be related to protein-based tissue reconstituting effects.

Endothelial progenitor cells undergo an endothelial-to-mesenchymal transition-like process mediated by TGFbetaRI.

AIMS: Endothelial progenitor cells (EPC) have been shown to repair pulmonary endothelium, although they can also migrate into the arterial intima and differentiate into smooth muscle-like (mesenchymal) cells contributing to intimal hyperplasia. The molecular mechanisms by which this process proceeds have not been fully elucidated. Here, we study whether genes involved in the endothelial-to-mesenchymal transition (EnMT) may contribute to the mesenchymal phenotype acquisition of EPC and we evaluate whether transforming growth factor ß1 (TGFß1) is involved in this process. METHODS AND RESULTS: Our results show that co-culture of EPC with smooth muscle cells (SMC) increases the expression of the mesenchymal cell markers α-smooth muscle actin, sm22-α, and myocardin, and decreases the expression of the endothelial cell marker CD31. In the same conditions, we also observed a concomitant increase in the gene expression of the EnMT-related transcription factors: slug, snail, zeb1, and endothelin-1. This indicates that mesenchymal phenotype acquisition occurred through an EnMT-like process. Inhibition of TGFß receptor I (TGFßRI) downregulated snail gene expression, blocked the EnMT, and facilitated the differentiation of EPC to the endothelial cell lineage. Furthermore, TGFßRI inhibition decreased migration of EPC stimulated by SMC without affecting their functionality and adhesion capacity. CONCLUSION: These results indicate that EPC may differentiate into SMC-like cells through an EnMT-like process and that TGFßI plays an important role in the fate of EPC.

Cigarette smoking exacerbates nonalcoholic fatty liver disease in obese rats.

UNLABELLED: The prevalence of cigarette smoking (CS) is increased among obese subjects, who are susceptible to develop nonalcoholic fatty liver disease (NAFLD). We investigated the hepatic effects of CS in control and obese rats. Control and obese Zucker rats were divided into smokers and nonsmokers (n = 12 per group). Smoker rats were exposed to 2 cigarettes/day, 5 days/week for 4 weeks. The effects of CS were assessed by biochemical analysis, hepatic histological examination, immunohistochemistry, and gene expression analysis. Phosphorylation of AKT and extracellular signal-regulated kinase (ERK) and quantification of carbonylated proteins were assessed by western blotting. As expected, obese rats showed hypercholesterolemia, insulin resistance, and histological features of NAFLD. Smoking did not modify the lipidic or glucidic serum profiles. Smoking increased alanine aminotransferase serum levels and the degree of liver injury in obese rats, whereas it only induced minor changes in control rats. Importantly, CS increased the histological severity of NAFLD in obese rats. We also explored the potential mechanisms involved in the deleterious effects of CS. Smoking increased the degree of oxidative stress and hepatocellular apoptosis in obese rats, but not in controls. Similarly, smoking increased the hepatic expression of tissue inhibitor of metalloproteinase-1 and procollagen-alpha2(I) in obese rats, but not in controls. Finally, smoking regulated ERK and AKT phosphorylation. The deleterious effects of CS were not observed after a short exposure (5 days). CONCLUSION: CS causes oxidative stress and worsens the severity of NAFLD in obese rats. Further studies should assess whether this finding also occurs in patients with obesity and NAFLD.

Cigarette smoke-induced oxidative stress: A role in chronic obstructive pulmonary disease skeletal muscle dysfunction.

RATIONALE: Inflammation and oxidative stress contribute to muscle dysfunction in patients with chronic obstructive pulmonary disease (COPD). Oxidants contained in cigarette smoke (CS) induce adverse effects on tissues through oxidative phenomena. OBJECTIVES: To explore oxidative stress and inflammation in quadriceps of human smokers and in diaphragm and limb muscles of guinea pigs chronically exposed to CS. METHODS: Muscle function, protein oxidation and nitration, antioxidants, oxidized proteins, inflammation, creatine kinase activity, and lung and muscle structures were investigated in vastus lateralis of smokers, patients with COPD, and healthy control subjects and in diaphragm and gastrocnemius of CS-exposed guinea pigs at 3, 4, and 6 months. MEASUREMENTS AND MAIN RESULTS: Compared with control subjects, quadriceps muscle force was mildly but significantly reduced in smokers; protein oxidation levels were increased in quadriceps of smokers and patients with COPD, and in respiratory and limb muscles of CS-exposed animals; glycolytic enzymes, creatine kinase, carbonic anydrase-3, and contractile proteins were significantly more carbonylated in quadriceps of smokers and patients with COPD, and in respiratory and limb muscles of CS-exposed guinea pigs. Chronic CS exposure induced no significant rise in muscle inflammation in either smokers or rodents. Muscle creatine kinase activity was reduced only in patients with COPD and in both diaphragm and gastrocnemius of CS-exposed animals. Guinea pigs developed bronchiolar abnormalities at 4 months of exposure and thereafter. CONCLUSIONS: CS exerts direct oxidative modifications on muscle proteins, without inducing any significant rise in muscle inflammation. The oxidative damage to muscle proteins, which precedes the characteristic respiratory changes, may contribute to muscle loss and dysfunction in smokers and patients with COPD.

Effects of cigarette smoke on endothelial function of pulmonary arteries in the guinea pig.

BACKGROUND: Cigarette smoking may contribute to pulmonary hypertension in chronic obstructive pulmonary disease by altering the structure and function of pulmonary vessels at early disease stages. The objectives of this study were to evaluate the effects of long-term exposure to cigarette smoke on endothelial function and smooth muscle-cell proliferation in pulmonary arteries of guinea pigs. METHODS: 19 male Hartley guinea pigs were exposed to the smoke of 7 cigarettes/day, 5 days/week, for 3 and 6 months. 17 control guinea pigs were sham-exposed for the same periods. Endothelial function was evaluated in rings of pulmonary artery and aorta as the relaxation induced by ADP. The proliferation of smooth muscle cells and their phenotype in small pulmonary vessels were evaluated by immunohistochemical expression of alpha-actin and desmin. Vessel wall thickness, arteriolar muscularization and emphysema were assessed morphometrically. The expression of endothelial nitric oxide synthase (eNOS) was evaluated by Real Time-PCR. RESULTS: Exposure to cigarette smoke reduced endothelium-dependent vasodilatation in pulmonary arteries (ANOVA p < 0.05) but not in the aorta. Endothelial dysfunction was apparent at 3 months of exposure and did not increase further after 6 months of exposure. Smoke-exposed animals showed proliferation of poorly differentiated smooth muscle cells in small vessels (p < 0.05) after 3 months of exposure. Prolonged exposure resulted in full muscularization of small pulmonary vessels (p < 0.05), wall thickening (p < 0.01) and increased contractility of the main pulmonary artery (p < 0.05), and enlargement of the alveolar spaces. Lung expression of eNOS was decreased in animals exposed to cigarette smoke. CONCLUSION: In the guinea pig, exposure to cigarette smoke induces selective endothelial dysfunction in pulmonary arteries, smooth muscle cell proliferation in small pulmonary vessels and reduced lung expression of eNOS. These changes appear after 3 months of exposure and precede the development of pulmonary emphysema.