Inducible NOS inhibition reverses tobacco-smoke-induced emphysema and pulmonary hypertension in mice.

Chronic obstructive pulmonary disease (COPD) is one of the most common causes of death worldwide. We report in an emphysema model of mice chronically exposed to tobacco smoke that pulmonary vascular dysfunction, vascular remodeling, and pulmonary hypertension (PH) precede development of alveolar destruction. We provide evidence for a causative role of inducible nitric oxide synthase (iNOS) and peroxynitrite in this context. Mice lacking iNOS were protected against emphysema and PH. Treatment of wild-type mice with the iNOS inhibitor N(6)-(1-iminoethyl)-L-lysine (L-NIL) prevented structural and functional alterations of both the lung vasculature and alveoli and also reversed established disease. In chimeric mice lacking iNOS in bone marrow (BM)-derived cells, PH was dependent on iNOS from BM-derived cells, whereas emphysema development was dependent on iNOS from non-BM-derived cells. Similar regulatory and structural alterations as seen in mouse lungs were found in lung tissue from humans with end-stage COPD.

CEACAM6 as a Novel Therapeutic Target to Boost HO-1-mediated Antioxidant Defense in COPD.

Rationale: Tobacco smoking and air pollution are primary causes of chronic obstructive pulmonary disease (COPD). However, only a minority of smokers develop COPD. The mechanisms underlying the defense against nitrosative/oxidative stress in nonsusceptible smokers to COPD remain largely unresolved. Objectives: To investigate the defense mechanisms against nitrosative/oxidative stress that possibly prevent COPD development or progression. Methods: Four cohorts were investigated: 1) sputum samples (healthy, n = 4; COPD, n = 37), 2) lung tissue samples (healthy, n = 13; smokers without COPD, n = 10; smoker+COPD, n = 17), 3) pulmonary lobectomy tissue samples (no/mild emphysema, n = 6), and 4) blood samples (healthy, n = 6; COPD, n = 18). We screened 3-nitrotyrosine (3-NT) levels, as indication of nitrosative/oxidative stress, in human samples. We established a novel in vitro model of a cigarette smoke extract (CSE)-resistant cell line and studied 3-NT formation, antioxidant capacity, and transcriptomic profiles. Results were validated in lung tissue, isolated primary cells, and an ex vivo model using adeno-associated virus-mediated gene transduction and human precision-cut lung slices. Measurements and Main Results: 3-NT levels correlate with COPD severity of patients. In CSE-resistant cells, nitrosative/oxidative stress upon CSE treatment was attenuated, paralleled by profound upregulation of heme oxygenase-1 (HO-1). We identified carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) as a negative regulator of HO-1-mediated nitrosative/oxidative stress defense in human alveolar type 2 epithelial cells (hAEC2s). Consistently, inhibition of HO-1 activity in hAEC2s increased the susceptibility toward CSE-induced damage. Epithelium-specific CEACAM6 overexpression increased nitrosative/oxidative stress and cell death in human precision-cut lung slices on CSE treatment. Conclusions: CEACAM6 expression determines the hAEC2 sensitivity to nitrosative/oxidative stress triggering emphysema development/progression in susceptible smokers.

Validation of the German Version of Functional Oral Intake Scale (FOIS-G) for Flexible Endoscopic Evaluation of Swallowing (FEES).

The Functional Oral Intake Scale (FOIS) is the most frequently used scale for the evaluation of functional oral intake by dysphagia patients. FOIS was validated using data from Videofluoroscopic Swallowing Study (VFSS). Until now, a validated German version of FOIS for Flexible Endoscopic Evaluation of Swallowing (FEES) is lacking. The aim of this study was a cross-cultural validation of the German version of FOIS (FOIS-G) for FEES. The translation of the original FOIS was carried out according to the Translation, Review, Adjudication, Pretesting, Documentation (TRAPD) translation methodology. For the validation process, six experienced language therapists (SLT) retrospectively analyzed charts of 93 stroke patients. Inclusion criteria were comprised of stroke, clinical examination by an SLT within 24 h of admission, and FEES within 72 h of admission. The validity was calculated by comparison with Modified Rankin Scale (MRS), Barthel Index (BI), the Penetration-Aspiration-Scale (PAS), and a water swallow test. Spearman rank correlation of all paired raters ranged from rs = 0.96 to rs = 0.99, and percentage agreement ranged from 81 to 94%. The overall agreement between all raters was calculated by Fleiss kappa (0.83) (s.e. 0.02). There is a significant correlation between the BI and the MRS with the FOIS-G (rs = 0.301, p = 0.003 for BI; rs = - 0.366, p < 0.001 for MRS), between the PAS and the FOIS-G (rs = - 0.758, p < 0.001), as well as between the 70 ml-water-test and the FOIS-G (rs = 0.470, p < 0.001). FOIS-G is a valid instrument for the evaluation of the functional oral intake of food and liquids in dysphagia patients.

Alternative Oxidase Attenuates Cigarette Smoke-induced Lung Dysfunction and Tissue Damage.

Cigarette smoke (CS) exposure is the predominant risk factor for the development of chronic obstructive pulmonary disease (COPD) and the third leading cause of death worldwide. We aimed to elucidate whether mitochondrial respiratory inhibition and oxidative stress are triggers in its etiology. In different models of CS exposure, we investigated the effect on lung remodeling and cell signaling of restoring mitochondrial respiratory electron flow using alternative oxidase (AOX), which bypasses the cytochrome segment of the respiratory chain. AOX attenuated CS-induced lung tissue destruction and loss of function in mice exposed chronically to CS for 9 months. It preserved the cell viability of isolated mouse embryonic fibroblasts treated with CS condensate, limited the induction of apoptosis, and decreased the production of reactive oxygen species (ROS). In contrast, the early-phase inflammatory response induced by acute CS exposure of mouse lung, i.e., infiltration by macrophages and neutrophils and adverse signaling, was unaffected. The use of AOX allowed us to obtain novel pathomechanistic insights into CS-induced cell damage, mitochondrial ROS production, and lung remodeling. Our findings implicate mitochondrial respiratory inhibition as a key pathogenic mechanism of CS toxicity in the lung. We propose AOX as a novel tool to study CS-related lung remodeling and potentially to counteract CS-induced ROS production and cell damage.

Resident cell lineages are preserved in pulmonary vascular remodeling.

Pulmonary vascular remodeling is the main pathological hallmark of pulmonary hypertension disease. We undertook a comprehensive and multilevel approach to investigate the origin of smooth muscle actin-expressing cells in remodeled vessels. Transgenic mice that allow for specific, inducible, and permanent labeling of endothelial (Cdh5-tdTomato), smooth muscle (Acta2-, Myh11-tdTomato), pericyte (Cspg4-tdTomato), and fibroblast (Pdgfra-tdTomato) lineages were used to delineate the cellular origins of pulmonary vascular remodeling. Mapping the fate of major lung resident cell types revealed smooth muscle cells (SMCs) as the predominant source of cells that populate remodeled pulmonary vessels in chronic hypoxia and allergen-induced murine models. Combining in vivo cell type-specific, time-controlled labeling of proliferating cells with a pulmonary artery phenotypic explant assay, we identified proliferation of SMCs as an underlying remodeling pathomechanism. Multicolor immunofluorescence analysis showed a preserved pattern of cell type marker localization in murine and human pulmonary arteries, in both donors and idiopathic pulmonary arterial hypertension (IPAH) patients. Whilst neural glial antigen 2 (chondroitin sulfate proteoglycan 4) labeled mostly vascular supportive cells with partial overlap with SMC markers, PDGFRα-expressing cells were observed in the perivascular compartment. The luminal vessel side was lined by a single cell layer expressing endothelial markers followed by an adjacent and distinct layer defined by SMC marker expression and pronounced thickening in remodeled vessels. Quantitative flow cytometric analysis of single cell digests of diverse pulmonary artery layers showed the preserved separation into two discrete cell populations expressing either endothelial cell (EC) or SMC markers in human remodeled vessels. Additionally, we found no evidence of overlap between EC and SMC ultrastructural characteristics using electron microscopy in either donor or IPAH arteries. Lineage-specific marker expression profiles are retained during pulmonary vascular remodeling without any indication of cell type conversion. The expansion of resident SMCs is the major underlying and evolutionarily conserved paradigm of pulmonary vascular disease pathogenesis. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

A novel mouse Cre-driver line targeting Perilipin 2-expressing cells in the neonatal lung.

Pulmonary diseases such as chronic obstructive pulmonary disease, lung fibrosis, and bronchopulmonary dysplasia are characterized by the destruction or malformation of the alveolar regions of the lung. The underlying pathomechanisms at play are an area of intense interest since these mechanisms may reveal pathways suitable for interventions to drive reparative processes. Lipid-laden fibroblasts (lipofibroblasts) express the Perilipin 2 (Plin2) gene-product, PLIN2, commonly called adipose-differentiation related protein (ADRP). These cells are also thought to play a role in alveolarization and repair after injury to the alveolus. Progress in defining the functional contribution of lipofibroblasts to alveolar generation and repair is hampered by a lack of in vivo tools. The present study reports the generation of an inducible mouse Cre-driver line to target cells of the ADRP lineage. Robust Cre-mediated recombination in this mouse line was detected in mesenchymal cells of the postnatal lung, and in additional organs including the heart, liver, and spleen. The generation and validation of this valuable new tool to genetically target, manipulate, and trace cells of the ADRP lineage is critical for assessing the functional contribution of lipofibroblasts to lung development and repair.

Survival with sildenafil and inhaled iloprost in a cohort with pulmonary hypertension: an observational study.

BACKGROUND: Combination therapy is frequently used to treat patients with pulmonary hypertension but few studies have compared treatment regimens. This study examined the long-term effect of different combination regimens of inhaled iloprost and oral sildenafil on survival and disease progression. METHODS: This was a retrospective study of patients in the Giessen Pulmonary Hypertension Registry who received iloprost monotherapy followed by addition of sildenafil (iloprost/sildenafil), sildenafil monotherapy followed by addition of iloprost (sildenafil/iloprost), or upfront combination therapy (iloprost + sildenafil). The primary outcome was transplant-free survival (Kaplan-Meier analysis). When available, haemodynamic parameters and 6-minute-walk distance were evaluated. RESULTS: Overall, 148 patients were included. Baseline characteristics were similar across treatment groups; however, the iloprost + sildenafil cohort had higher mean pulmonary vascular resistance and pulmonary arterial pressure than the others. Transplant-free survival differed significantly between groups (P = 0.007, log-rank test). Cumulative transplant-free survival was highest for patients who received iloprost/sildenafil (1 year survival: iloprost/sildenafil, 95.1%; sildenafil/iloprost, 91.8%; iloprost + sildenafil, 62.9%); this group also remained on monotherapy significantly longer than the sildenafil/iloprost group (median 17.0 months vs 7.0 months, respectively; P = 0.004). Compared with pre-treatment values, mean 6-minute-walk distance increased significantly for all groups 3 months after beginning combination therapy. CONCLUSIONS: In this observational study of patients with pulmonary hypertension receiving combination therapy with iloprost and sildenafil, cumulative transplant-free survival was highest in those who received iloprost monotherapy initially. However, owing to the size and retrospective design of this study, further research is needed before making firm treatment recommendations.

Characterization of the platelet-derived growth factor receptor-α-positive cell lineage during murine late lung development.

A reduced number of alveoli is the structural hallmark of diseases of the neonatal and adult lung, where alveoli either fail to develop (as in bronchopulmonary dysplasia), or are progressively destroyed (as in chronic obstructive pulmonary disease). To correct the loss of alveolar septa through therapeutic regeneration, the mechanisms of septa formation must first be understood. The present study characterized platelet-derived growth factor receptor-α-positive (PDGFRα(+)) cell populations during late lung development in mice. PDGFRα(+) cells (detected using a PDGFRα(GFP) reporter line) were noted around the proximal airways during the pseudoglandular stage. In the canalicular stage, PDGFRα(+) cells appeared in the more distal mesenchyme, and labeled α-smooth muscle actin-positive tip cells in the secondary crests and lipofibroblasts in the primary septa during alveolarization. Some PDGFRα(+) cells appeared in the mesenchyme of the adult lung. Over the course of late lung development, PDGFRα(+) cells consistently expressed collagen I, and transiently expressed markers of mesenchymal stem cells. With the use of both, a constitutive and a conditional PDGFRα(Cre) line, it was observed that PDGFRα(+) cells generated alveolar myofibroblasts including tip cells of the secondary crests, and lipofibroblasts. These lineages were committed before secondary septation. The present study provides new insights into the time-dependent commitment of the PDGFRα(+) cell lineage to lipofibroblasts and myofibroblasts during late lung development that is needed to better understand the cellular contribution to the process of alveolarization.

New potential diagnostic biomarkers for pulmonary hypertension.

This study aimed to determine whether the vascular endothelial growth factor (VEGF) family members soluble VEGF receptor 1 (also called soluble fms-like tyrosine kinase 1 (sFlt-1)) and placental growth factor (PlGF) could be used as biomarkers for pulmonary hypertension (PH). Consecutive patients undergoing right heart catheterisation were enrolled (those with mean pulmonary arterial pressure ≥25 mmHg were classed as having PH; those with mean pulmonary arterial pressure <25 mmHg acted as non-PH controls). Plasma from the time of PH diagnosis was analysed for PlGF and sFlt-1 using enzyme immunoassays. In total, 247 patients with PH were enrolled: 62 with idiopathic pulmonary arterial hypertension (IPAH), 14 with associated pulmonary arterial hypertension (APAH), 21 with collagen vascular disease (CVD), 26 with pulmonary venous hypertension, 67 with lung disease-associated PH and 57 with chronic thromboembolic PH. The non-PH control group consisted of 40 patients. sFlt-1 plasma levels were significantly higher in patients with IPAH, APAH, CVD and lung disease-associated PH versus controls; PlGF levels were significantly higher in all PH groups versus controls. The combination of sFlt-1 and PlGF resulted in a sensitivity of 83.7% with specificity of 100% for pulmonary arterial hypertension. There was no association between sFlt-1 or PlGF and haemodynamic parameters, 6-min walking distance or survival. In summary, PlGF and sFlt-1 are promising diagnostic biomarkers for PH.

Defective angiogenesis delays thrombus resolution: a potential pathogenetic mechanism underlying chronic thromboembolic pulmonary hypertension.

OBJECTIVE: Restoration of patency is a natural target of vascular remodeling after venous thrombosis that involves vascular endothelial cells and smooth muscle cells, as well as leukocytes. Acute pulmonary emboli usually resolve <6 months. However, in some instances, thrombi transform into fibrous vascular obstructions, resulting in occlusion of the deep veins, or in chronic thromboembolic pulmonary hypertension (CTEPH). We proposed that dysregulated thrombus angiogenesis may contribute to thrombus persistence. APPROACH AND RESULTS: Mice with an endothelial cell-specific conditional deletion of vascular endothelial growth factor receptor 2/kinase insert domain protein receptor were used in a model of stagnant flow venous thrombosis closely resembling human deep vein thrombosis. Biochemical and functional analyses were performed on pulmonary endarterectomy specimens from patients with CTEPH, a human model of nonresolving venous thromboembolism. Endothelial cell-specific deletion of kinase insert domain protein receptor and subsequent ablation of thrombus vascularization delayed thrombus resolution. In accordance with these findings, organized human CTEPH thrombi were largely devoid of vascular structures. Several vessel-specific genes, such as kinase insert domain protein receptor, vascular endothelial cadherin, and podoplanin, were expressed at lower levels in white CTEPH thrombi than in organizing deep vein thrombi and organizing thrombi from aortic aneurysms. In addition, red CTEPH thrombi attenuated the angiogenic response induced by vascular endothelial growth factor. CONCLUSIONS: In the present work, we propose a mechanism of thrombus nonresolution demonstrating that endothelial cell-specific deletion of kinase insert domain protein receptor abates thrombus vessel formation, misguiding thrombus resolution. Medical conditions associated with the development of CTEPH may be compromising early thrombus angiogenesis.

Pulmonary Hemodynamic Response to Exercise in Chronic Thromboembolic Pulmonary Hypertension before and after Pulmonary Endarterectomy.

BACKGROUND: Pulmonary endarterectomy (PEA) is the treatment of choice in surgically accessible chronic thromboembolic pulmonary hypertension (CTEPH). An important predictor of outcome is postsurgical residual pulmonary hypertension. OBJECTIVE: We aimed to use the hemodynamic response during exercise before PEA as a measurement for the hemodynamic outcome 1 year after PEA. METHODS: Between January 2011 and December 2013, 299 patients underwent PEA in our center. A total of 16 patients who were assessed by means of invasive hemodynamic measurements during exercise both at baseline and 1 year after PEA were retrospectively analyzed. RESULTS: Pre-PEA mean pulmonary arterial pressure (mPAP) increased during exercise from 35.8 ± 7.6 to 53.8 ± 5.1 mm Hg, diastolic pulmonary arterial pressure (dPAP) from 21.5 ± 5.6 to 30.3 ± 9.6 mm Hg, cardiac output (CO) from 4.4 ± 0.8 to 6.5 ± 1.9 l/min and diastolic pulmonary gradient (DPG) from 14.6 ± 4.9 to 20.7 ± 12.7 mm Hg. Post-PEA mPAP increased from 23.7 ± 6.6 at rest to 43.2 ± 7.1 mm Hg, while CO increased to a higher extent from 5.1 ± 0.9 to 8.4 ± 1.9 l/min. There were significant correlations between pre-PEA DPG/CO and dPAP/CO slopes with the pulmonary vascular resistance (Spearman r = 0.578, p = 0.019, and r = 0.547, p = 0.028) and mPAP at rest after PEA (Spearman r = 0.581, p = 0.018, and r = 0.546, p = 0.028). CONCLUSIONS: In CTEPH, the presurgical dynamic DPG/CO and dPAP/CO slopes during submaximal exercise are associated with the hemodynamic outcome 1 year after PEA.

Senescence-associated secretory phenotype and its possible role in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a major disease of the lungs. It primarily occurs after a prolonged period of cigarette smoking. Chronic inflammation of airways and the alveolar space as well as lung tissue destruction are the hallmarks of COPD. Recently it has been shown that cellular senescence might play a role in the pathogenesis of COPD. Cellular senescence comprises signal transduction program, leading to irreversible cell cycle arrest. The growth arrest in senescence can be triggered by many different mechanisms, including DNA damage and its recognition by cellular sensors, leading to the activation of cell cycle checkpoint responses and activation of DNA repair machinery. Senescence can be induced by several genotoxic factors apart from telomere attrition. When senescence induction is based on DNA damage, senescent cells display a unique phenotype, which has been termed "senescence-associated secretory phenotype" (SASP). SASP may be an important driver of chronic inflammation and therefore may be part of a vicious cycle of inflammation, DNA damage, and senescence. This research perspective aims to showcase cellular senescence with relevance to COPD and the striking similarities between the mediators and secretory phenotype in COPD and SASP.

Phenotypical and ultrastructural features of Oct4-positive cells in the adult mouse lung.

Octamer binding trascription factor 4 (Oct4) is a transcription factor of POU family specifically expressed in embryonic stem cells (ESCs). A role for maintaining pluripotency and self-renewal of ESCs is assigned to Oct4 as a pluripotency marker. Oct4 can also be detected in adult stem cells such as bone marrow-derived mesenchymal stem cells. Several studies suggest a role for Oct4 in sustaining self-renewal capacity of adult stem cells. However, Oct4 gene ablation in adult stem cells revealed no abnormalities in tissue turnover or regenerative capacity. In the present study we have conspicuously found pulmonary Oct4-positive cells closely resembling the morphology of telocytes (TCs). These cells were found in the perivascular and peribronchial areas and their presence and location were confirmed by electron microscopy. Moreover, we have used Oct4-GFP transgenic mice which revealed a similar localization of the Oct4-GFP signal. We also found that Oct4 co-localized with several described TC markers such as vimentin, Sca-1, platelet-derived growth factor receptor-beta C-kit and VEGF. By flow cytometry analyses carried out with Oct4-GFP reporter mice, we described a population of EpCAM(neg) /CD45(neg) /Oct4-GFP(pos) that in culture displayed TC features. These results were supported by qRT-PCR with mRNA isolated from lungs by using laser capture microdissection. In addition, Oct4-positive cells were found to express Nanog and Klf4 mRNA. It is concluded for the first time that TCs in adult lung mouse tissue comprise Oct4-positive cells, which express pluripotency-related genes and represent therefore a population of adult stem cells which might contribute to lung regeneration.

The number of cardiac myocytes in the hypertrophic and hypotrophic left ventricle of the obese and calorie-restricted mouse heart.

Changes in body mass due to varying amounts of calorie intake occur frequently with obesity and anorexia/cachexia being at opposite sides of the scale. Here, we tested whether a high-fat diet or calorie restriction (CR) decreases the number of cardiac myocytes and affects their volume. Ten 6-8-week-old mice were randomly assigned to a normal (control group, n = 5) or high-fat diet (obesity group, n = 5) for 28 weeks. Ten 8-week-old mice were randomly assigned to a normal (control group, n = 5) or CR diet (CR group, n = 5) for 7 days. The left ventricles of the hearts were prepared for light and electron microscopy, and analysed by design-based stereology. In CR, neither the number of cardiac myocytes, the relationship between one- and multinucleate myocytes nor their mean volume were significantly different between the groups. In contrast, in the obese mice we observed a significant increase in cell size combined with a lower number of cardiomyocytes (P < 0.05 in the one-sided U-test) and an increase in the mean number of nuclei per myocyte. The mean volume of myofibrils and mitochondria per cardiac myocyte reflected the hypertrophic and hypotrophic remodelling in obesity and CR, respectively, but were only significant in the obese mice, indicating a more profound effect of the obesity protocol than in the CR experiments. Taken together, our data indicate that long-lasting obesity is associated with a loss of cardiomyocytes of the left ventricle, but that short-term CR does not alter the number of cardiomyocytes.

The prognostic significance of inspiratory capacity in pulmonary arterial hypertension.

BACKGROUND: Patients with pulmonary arterial hypertension (PAH) present with an altered inspiratory capacity (IC) reflecting dynamic hyperinflation (DH) that leads to mechanical constraints and excessive ventilatory demand, particularly during exercise, resulting in exertional dyspnea. OBJECTIVES: Assessment of the long-term consequences of altered IC and DH in PAH. METHODS: 50 patients with newly diagnosed PAH were prospectively recruited. All patients were assessed by means of right heart catheterization, 6-min walking distance (6MWD) test, lung function and cardiopulmonary exercise testing, including the assessment of IC. RESULTS: 37 patients with idiopathic PAH and 13 patients with conditions associated with PAH (29 female; mean age 51.6 ± 15.1 years; World Health Organization, WHO class, 2.7 ± 0.6) presented with a mean pulmonary arterial pressure of 42.8 ± 15.9 mm Hg and pulmonary vascular resistance (PVR) of 737.2 ± 592.8 dyn*s/cm(5). The mean IC at rest was 87.2 ± 17.3% pred. Kaplan-Meier analysis revealed that patients with an IC at rest >89% pred. had a significantly better 5-year survival than those with lower values (94.1 vs. 75.1%; log-rank p = 0.036). Univariate analysis identified IC at rest (% pred.) as a predictor of survival with a hazard ratio (HR) of 5.05 (95% confidence interval, CI, 0.97-26.24, p = 0.054). In multivariate analysis including PVR, WHO class, 6MWD and peak oxygen uptake as covariates, IC at rest remained an independent predictor of survival (HR: 8.06, 95% CI 0.92-70.34; p = 0.059). DH expressed as ΔIC or static hyperinflation expressed as IC/total lung capacity at rest revealed no prognostic significance. CONCLUSION: In patients with PAH, IC at rest is of prognostic significance at the time of diagnosis.

Quantitative proteome analysis of alveolar type-II cells reveals a connection of integrin receptor subunits beta 2/6 and WNT signaling.

Alveolar type-II cells (ATII cells) are lung progenitor cells responsible for regeneration of alveolar epithelium during homeostatic turnover and in response to injury. Characterization of ATII cells will have a profound impact on our understanding and treatment of lung disease. The identification of novel ATII cell-surface proteins can be used for sorting and enrichment of these cells for further characterization. Here we combined a high-resolution mass spectrometry-based membrane proteomic approach using lungs of the SILAC mice with an Affymetrix microarray-based transcriptome analysis of ATII cells. We identified 16 proteins that are enriched in the membrane fraction of ATII cells and whose genes are highly expressed in these cells. Interestingly, we confirmed our data for two of these genes, integrin beta 2 and 6 (Itgb2 and Itgb6), by qRT-PCR expression analysis and Western blot analysis of protein extracts. Moreover, flow cytometry and immunohistochemistry in adult lung revealed that ITGB2 and ITGB6 are present in subpopulations of surfactant-associated-protein-C-positive cells, suggesting the existence of different types of ATII cells. Furthermore, analysis of the Itgb2(-/-) mice showed that Itgb2 is required for proper WNT signaling regulation in the lung.

Immune and inflammatory cell involvement in the pathology of idiopathic pulmonary arterial hypertension.

RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by vasoconstriction and vascular remodeling. Recent studies have revealed that immune and inflammatory responses play a crucial role in pathogenesis of idiopathic PAH. OBJECTIVES: To systematically evaluate the number and cross-sectional distribution of inflammatory cells in different sizes of pulmonary arteries from explanted lungs of patients with idiopathic PAH versus healthy donor lungs and to demonstrate functional relevance by blocking stromal-derived factor-1 by the Spiegelmer NOX-A12 in monocrotaline-induced pulmonary hypertension in rats. METHODS: Immunohistochemistry was performed on lung tissue sections from patients with idiopathic PAH and healthy donors. All positively stained cells in whole-lung tissue sections, surrounding the vessels, and in the different compartments of the vessels were counted. To study the effects of blocking SDF-1, rats with monocrotaline-induced pulmonary hypertension were treated with NOX-A12 from Day 21 to Day 35 after monocrotaline administration. MEASUREMENTS AND MAIN RESULTS: We found a significant increase of the perivascular number of macrophages (CD68(+)), macrophages/monocytes (CD14(+)), mast cells (toluidine blue(+)), dendritic cells (CD209(+)), T cells (CD3(+)), cytotoxic T cells (CD8(+)), and helper T cells (CD4(+)) in vessels of idiopathic PAH lungs compared with control subjects. FoxP3(+) mononuclear cells were significantly decreased. In the monocrotaline model, the NOX-A12-induced reduction of mast cells, CD68(+) macrophages, and CD3(+) T cells was associated with improvement of hemodynamics and pulmonary vascular remodeling. CONCLUSIONS: Our findings reveal altered perivascular inflammatory cell infiltration in pulmonary vascular lesions of patients with idiopathic pulmonary arterial hypertension. Targeting attraction of inflammatory cells by blocking stromal-derived factor-1 may be a novel approach for treatment of PAH.

Bone marrow transplantation improves hepatic fibrosis in Abcb4-/- mice via Th1 response and matrix metalloproteinase activity.

OBJECTIVE: Reports on the effects of bone marrow-derived cells on hepatic fibrosis are contradictory. Impaired fibrosis but increased inflammation has recently been demonstrated 10 weeks after bone marrow transplantation (BM-Tx) in Abcb4-/- mice. It is hypothesised that BM-Tx might have long-term therapeutic potential by altering the immunological and matrix remodelling processes leading to hepatic regeneration. METHODS: After lethal irradiation of recipient mice, BM cells from GFP+ donor mice (allogeneic Tx) or Abcb4-/- mice (syngeneic Tx) were transplanted via tail vein injection. Readouts were performed 2, 10 and 20 weeks after Tx. Liver integrity was assessed serologically and histologically. Surrogate markers for fibrogenesis, T helper (Th) response, inflammation, graft-versus-host disease and fibrolysis were analysed by quantitative real-time PCR, zymography and immunohistology. RESULTS: 20 weeks after syngeneic and allogeneic BM-Tx, hepatic grading and staging were significantly improved. In contrast, 2 weeks after BM-Tx inflammatory grading, expression of inflammatory cell markers and associated chemokines and their receptors were increased and subsequently declined. In parallel, CD8+/GFP+ donor-derived T cells infiltrated the liver 2 weeks after BM-Tx. The Th1 cyokine interferon γ was increased 2 and 10 weeks after BM-Tx whereas the Th2 associated interleukin 13 was not altered. The gene expression of matrix metalloproteinases MMP-2, MMP-7, MMP-9 and MMP-13 was transiently upregulated and MMP-9 protein remained elevated 20 weeks after BM-Tx with enhanced gelatinase activity located within the fibrotic areas. Neutrophils were identified as major sources of MMP-9. CONCLUSION: These results show that BM-Tx causes an antifibrotic Th1 response combined with transient inflammatory effects and subsequently upregulated MMP activity. Antifibrotic Th polarisation and prolonged proteolytic activity, especially of MMP-9, might be responsible for long-term amelioration of hepatic fibrosis.