PPARγ agonist pioglitazone reverses pulmonary hypertension and prevents right heart failure via fatty acid oxidation.

Right ventricular (RV) heart failure is the leading cause of death in pulmonary arterial hypertension (PAH). Peroxisome proliferator-activated receptor γ (PPARγ) acts as a vasoprotective metabolic regulator in smooth muscle and endothelial cells; however, its role in the heart is unclear. We report that deletion of PPARγ in cardiomyocytes leads to biventricular systolic dysfunction and intramyocellular lipid accumulation in mice. In the SU5416/hypoxia (SuHx) rat model, oral treatment with the PPARγ agonist pioglitazone completely reverses severe PAH and vascular remodeling and prevents RV failure. Failing RV cardiomyocytes exhibited mitochondrial disarray and increased intramyocellular lipids (lipotoxicity) in the SuHx heart, which was prevented by pioglitazone. Unbiased ventricular microRNA (miRNA) arrays, mRNA sequencing, and lipid metabolism studies revealed dysregulation of cardiac hypertrophy, fibrosis, myocardial contractility, fatty acid transport/oxidation (FAO), and transforming growth factor-ß signaling in the failing RV. These epigenetic, transcriptional, and metabolic alterations were modulated by pioglitazone through miRNA/mRNA networks previously not associated with PAH/RV dysfunction. Consistently, pre-miR-197 and pre-miR-146b repressed genes that drive FAO (Cpt1b and Fabp4) in primary cardiomyocytes. We recapitulated our major pathogenic findings in human end-stage PAH: (i) in the pressure-overloaded failing RV (miR-197 and miR-146b up-regulated), (ii) in peripheral pulmonary arteries (miR-146b up-regulated, miR-133b down-regulated), and (iii) in plexiform vasculopathy (miR-133b up-regulated, miR-146b down-regulated). Together, PPARγ activation can normalize epigenetic and transcriptional regulation primarily related to disturbed lipid metabolism and mitochondrial morphology/function in the failing RV and the hypertensive pulmonary vasculature, representing a therapeutic approach for PAH and other cardiovascular/pulmonary diseases.

Novel mouse model of cardiopulmonary bypass.

OBJECTIVES: Cardiopulmonary bypass (CPB) is an essential component of many cardiac interventions, and therefore, there is an increasing critical demand to minimize organ damage resulting from prolonged extracorporeal circulation. Our goal was to develop the first clinically relevant mouse model of CPB and to examine the course of extracorporeal circulation by closely monitoring haemodynamic and oxygenation parameters. METHODS: Here, we report the optimization of device design, perfusion circuit and microsurgical techniques as well as validation of physiological functions during CPB in mice after circulatory arrest and reperfusion. Validation of the model required multiple blood gas analyses, and therefore, this initial report describes an acute model that is incompatible with survival due to the need of repetitive blood draws. RESULTS: Biochemical and histopathological assessment of organ damage revealed only mild changes in the heart and lungs and signs of the beginning of acute organ failure in the liver and kidneys. CONCLUSIONS: This new CPB mouse model will facilitate preclinical testing of therapeutic strategies in cardiovascular diseases and investigation of CPB in relation to different insults and pre-existing comorbidities. In combination with genetically modified mice, this model will be an important tool to dissect the molecular mechanisms involved in organ damage related to extracorporeal circulation.

Comparative analysis of morphological and molecular motifs in bronchiolitis obliterans and alveolar fibroelastosis after lung and stem cell transplantation.

Chronic lung allograft dysfunction (CLAD) remains the major obstacle to long-term survival following lung transplantation (LuTx). Morphologically CLAD is defined by obliterative remodelling of the small airways (bronchiolitis obliterans, BO) as well as a more recently described collagenous obliteration of alveoli with elastosis summarised as alveolar fibroelastosis (AFE). Both patterns are not restricted to pulmonary allografts, but have also been reported following haematopoietic stem cell transplantation (HSCT) and radio chemotherapy (RC). In this study we performed compartment-specific morphological and molecular analysis of BO and AFE lesions in human CLAD (n = 22), HSCT (n = 29) and RC (n = 6) lung explants, utilising conventional histopathology, laser-microdissection, PCR techniques and immunohistochemistry to assess fibrosis-associated gene and protein expression. Three key results emerged from our analysis of fibrosis-associated genes: (i) generally speaking, "BO is BO". Despite the varying clinical backgrounds, the molecular characteristics of BO lesions were found to be alike in all groups. (ii) "AFE is AFE". In all groups of patients suffering from restrictive changes to lung physiology due to AFE there were largely - but not absolutely - identical gene expression patterns. iii) BO concomitant to AFE after LuTx is characterised by an AFE-like molecular microenvironment, representing the only exception to (i). Additionally, we describe an evolutionary model for the AFE pattern: a non-specific fibrin-rich reaction to injury pattern triggers a misguided resolution attempt and eventual progression towards manifest AFE. Our data point towards an absence of classical fibrinolytic enzymes and an alternative fibrin degrading mechanism via macrophages, resulting in fibrous remodelling and restrictive functional changes. These data may serve as diagnostic adjuncts and help to predict the clinical course of respiratory dysfunction in LuTx and HSCT patients. Moreover, analysis of the mechanism of fibrinolysis and fibrogenesis may unveil potential therapeutic targets to alter the course of the eventually fatal lung remodelling.

Airway remodelling in the transplanted lung.

Following lung transplantation, fibrotic remodelling of the small airways has been recognized for almost 5 decades as the main correlate of chronic graft failure and a major obstacle to long-term survival. Mainly due to airway fibrosis, pulmonary allografts currently show the highest attrition rate of all solid organ transplants, with a 5-year survival rate of 58 % on a worldwide scale. The observation that these morphological changes are not just the hallmark of chronic rejection but rather represent a manifestation of a multitude of alloimmune-dependent and -independent injuries was made more recently, as was the discovery that chronic lung allograft dysfunction manifests in different clinical phenotypes of respiratory impairment and corresponding morphological subentities. Although recent years have seen considerable advances in identifying and categorizing these subgroups on the basis of clinical, functional and histomorphological changes, as well as susceptibility to medicinal treatment, this process is far from over. Since the actual pathophysiological mechanisms governing airway remodelling are still only poorly understood, diagnosis and therapy of chronic lung allograft dysfunction presents a major challenge to clinicians, radiologists and pathologists alike. Here, we review and discuss the current state of the literature on chronic lung allograft dysfunction and shed light on classification systems, corresponding clinical and morphological changes, key cellular players and underlying molecular pathways, as well as on emerging diagnostic and therapeutic approaches.

Adhesion Prevention Efficacy of Composite Meshes Parietex®, Proceed® and 4DryField® PH Covered Polypropylene Meshes in an IPOM Rat Model.

Background: Adhesions to intraperitoneally implanted meshes (IPOM) are a common problem following hernia surgery and may cause severe complications. Recently, we showed that missing peritoneal coverage of the intestine is a decisive factor for adhesion formation and 4DryField® PH (4DF) gel significantly prevents intestine-to-mesh adhesions even with use of uncoated Ultrapro® polypropylene mesh (UPM). The present study investigates adhesion prevention capability of coated Parietex® mesh (PTM) and Proceed® mesh (PCM) in comparison to 4DF treated UPM. Methods: 20 rats were randomized into two groups. A 1.5 x 2 cm patch of PTM or PCM was attached to the abdominal wall and the cecum was depleted from peritoneum by abrasion. After seven days incidence of intestine-to-mesh adhesions was evaluated using Lauder and Hoffmann adhesion scores. Histological specimens were evaluated; statistics were performed using student's t-test. The data were compared with recently published data of 4DF treated uncoated UPM. Results: Use of PTM or PCM did not significantly diminish development of intestine-to-mesh adhesions (adhesion reduction rate PTM: 29%, p = 0.069 and PCM: 25%, p = 0.078). Histological results confirmed macroscopic finding of agglutination of intestine and abdominal wall with the mesh in between. Compared to these data, the use of UPM combined with 4DF gel reveals significantly better adhesion prevention capability (p < 0.0001) as shown in earlier studies. However, in clinical situation interindividual differences in adhesion induction mechanisms cannot be excluded by this experimental approach as healing responses towards the different materials might vary. Conclusion: This study shows that in case of impaired intestinal peritoneum coated PTM and PCM do not provide significant adhesion prevention. In contrast, use of UPM combined with 4DF gel achieved a significant reduction of adhesions. Hence, in case of injury of the visceral peritoneum, application of a polysaccharide barrier device such as 4DF gel might be considered more effective in reducing intestine-to-mesh adhesions than coated mesh devices.

Notch1 signalling regulates endothelial proliferation and apoptosis in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is characterised by excessive pulmonary vascular remodelling involving deregulated proliferation of cells in intima, media as well as adventitia. Pulmonary arterial endothelial cell (PAEC) hyperproliferation and survival underlies the endothelial pathobiology of the disease.The indispensable involvement of Notch1 in the arterial endothelial phenotype and angiogenesis provides intriguing prospects for its involvement in the pathogenesis of PAH.We observed an increased expression of Notch1 in lungs of idiopathic PAH (IPAH) patients and hypoxia/SU5416 (SUHx) rats compared with healthy subjects. In vitro loss- and gain-of-function studies demonstrated that Notch1 increased proliferation of human PAECs (hPAECs) via downregulation of p21 and inhibited apoptosis via Bcl-2 and Survivin. Inhibition of Notch signalling using the γ-secretase inhibitor dibenzazepine dose-dependently decreased proliferation and migration of hPAECs. Notably, Notch1 expression and transcriptional activity were increased under hypoxia in hPAECs and knockdown of Notch1 inhibited hypoxia-induced proliferation of the cells. Furthermore, in vivo treatment with a γ-secretase inhibitor (AMG2008827) significantly reduced the right ventricular systolic pressure and right heart hypertrophy in SUHx rats.Here, we conclude that Notch1 plays a critical role in PAH and Notch inhibitors may be a promising therapeutic option for PAH.

CXCL13 in idiopathic pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension.

BACKGROUND: Chemokine CXC ligand 13 (CXCL13) has been implicated in perivascular inflammation and pulmonary vascular remodeling in patients with idiopathic pulmonary artery hypertension (IPAH). We wondered whether CXCL13 may also play a role in chronic thromboembolic pulmonary hypertension (CTEPH) and whether serum levels of CXCL13 might serve as biomarkers in these conditions. METHODS: Lung tissue from patients with IPAH or CTEPH was immunostained for CXCL13. Serum samples were obtained from patients with IPAH (n = 42) or CTEPH (n = 50) and from healthy controls (n = 13). Serum CXCL13 concentrations were measured by enzyme-linked immunosorbent assay technology and were evaluated for associations with markers of disease severity and survival. RESULTS: CXCL13 was expressed in pulmonary vascular lesions and lymphocytes of patients with IPAH and inoperable CTEPH, respectively. Serum CXCL13 was elevated in patients compared to healthy controls [median, interquartile range, 83 (55,114) pg/ml versus 40 (28, 48) pg/ml; p < 0.001]. Serum CXCL13 showed only weak and inconsistent correlations with markers of inflammation or disease severity. In both populations, patients with serum CXCL13 above the median of the respective groups did not have a higher risk of death than patients with lower serum CXCL13. CONCLUSIONS: CXCL13 was overexpressed in pulmonary vascular lesions of patients with IPAH and CTEPH, and increased serum concentrations were found in patients with IPAH and CTEPH, suggesting a potential pathogenic role of CXCL13 in both diseases. However, given the weak associations between serum CXCL13 and markers of disease severity and outcome, CXCL13 is unlikely to become a promising biomarker in these patient populations.

Treatment of de-peritonealized intestine with 4DryField® PH prevents adhesions between non-resorbable intra-peritoneal hernia mesh and bowel.

BACKGROUND: Intraperitoneal onlay meshes (IPOM) can be associated with intestine-to-mesh adhesion formation, implementing risks like pain, enterocutaneous fistula, infection, and female infertility. This study investigates, whether a treatment of impaired intestinum with the anti-adhesive and hemostyptic agent 4DryField® PH prevents adhesion formation. METHODS: In 20 male LEWIS rats uncoated polypropylene meshes were sewn to the inner abdominal wall and the cecum of the respective animal was de-peritonealized by peritoneal abrasion by a gauze swap, and meso-sutures ensured a constant contact of injured areas. Rats were treated with 4DryField® PH gel either premixed or applied as a powder with in-situ transformation (100 mg powder plus 0.4 ml 0.9% saline solution). One week postoperatively, the extent of intestine-to-mesh adhesions and the quality of mesh ingrowth were evaluated macroscopically by two independent investigators using two scoring systems. Furthermore, specimens were analysed microscopically. All data were compared with control animals without 4DryField® PH treatment and analysed statistically using student's t-test. RESULTS: Treatment of de-peritonealised cecum with 4DryField® PH significantly reduced intestine-to-mesh adhesions in both treatment groups as compared to controls without 4DryField® PH treatment (68% reduction with premixed gel, P<0.0001; 80% reduction with in-situ gel, P<0.0001). There was no impact on the quality of mesh ingrowth, confirmed histologically by a single-layer mesothelial coverage. CONCLUSION: These experiments mimick clinical IPOM implantation scenarios with adjacent bowel depleted from peritoneum. 4DryField® PH gel treatment resulted in intestinal mesothelial surface recovering without development of bowel-to-mesh adhesions. Concurrently, integration of mesh into the abdominal wall is undisturbed by 4DryField® PH treatment.

Evaluation of the biological tolerability of the starch-based medical device 4DryField® PH in vitro and in vivo a rat model.

PURPOSE: To evaluate in vitro cytotoxicity/biocompatibility as well as in vivo tolerability of the novel polysaccharide 4DryField® PH, certified for haemostasis and adhesion prevention. METHODS: In vitro cytotoxicity/viability testing according to ISO EN 10,993 using murine and human tumour cell lines incubated with 4DryField® PH (PlantTec Medical GmbH). Using a rat model the impact of 4DryField® PH on animals viability and in vivo effects were macro- and micropathologically assessed. RESULTS: In vitro testing revealed no cytotoxic effect of 4DryField® PH nor enhancement of viability to tumour cell lines. In vivo viability of rats was unimpaired by 4DryField® PH. Bodyweight loss in animals with abdominal injury plus treatment with 4DryField® PH was in the range of controls and less than in injured rats without treatment. At day 7 after surgery no formation of adhesions, neither macroscopic nor histological remnants nor signs of foreign body reaction were present in animals without injury. In animals with peritoneal injury and 4DryField® PH application, histopathological observation revealed minor residuals of polysaccharide in the depth of wound cavity embedded in a thickened subperitoneal layer; however, with a suggested intact neoperitoneum. The presence of mononuclear cells surrounding polysaccharide particles in varying states of degradation was observable as well. CONCLUSION: 4DryField® PH is not cytotoxic and does not enhance viability of tumour cell lines. High dose of 4DryField® PH of 1.09 g/kg bodyweight is well tolerated and reduces weight loss in animals with peritoneal injury. The biocompatibility of 4DryField® PH can be rated as being excellent.

Tumour angiogenesis in Epstein-Barr virus-associated post-transplant smooth muscle tumours.

Epstein-Barr virus (EBV)-associated post-transplant smooth muscle tumours (PTSMT), are rare complications following organ/stem cell transplantation. Despite the mainly benign behaviour of PTSMT, alternative therapies are needed for those patients with progressive tumours. In tumours not approachable by surgery or reduction of immunosuppression, the angiogenic microenvironment might be a potential target of therapy, an approach that is well utilised in other soft tissue neoplasms. In a previous study, we evaluated the expression of EBV-related genes and the microRNA profile in PTSMT, but so far the characteristics of angiogenesis in PTSMT are not known. Therefore, the aim of this study was to evaluate the expression pattern of angiogenesis-related genes in PTSMT, in order to identify potential target molecules for anti-angiogenic therapy.PTSMT (n = 5 tumours) were compared with uterine leiomyomas (n = 7). Analyses included real-time PCR of 45 angiogenesis-associated genes, immunohistochemistry (CD31, prostaglandin endoperoxide synthase 1/PTGS1) and assessment of tumour vascularisation by conventional histopathology.PTSMT showed similar or fewer vessels than leiomyomas. Of the genes under investigation, 23 were down-deregulated (pro-angiogenic and some anti-angiogenic factors) and five were up-regulated (e.g. PTGS1 which is expressed at very low levels in leiomyomas but moderately higher levels in PTSMT).In summary, no particular target molecule could be identified, because tumour angiogenesis in PTSMT is characterised by low levels of major pro-angiogenic factors and there is no prominent increase in tumour vascularisation. EBV can induce angiogenesis via its viral late membrane protein 1 (LMP1) but PTSMT frequently do not express LMP1, which could be an explanation why, despite EBV infection, PTSMT show no exaggerated tumour angiogenesis.

Anti-inflammatory and immunomodulatory properties of α1-antitrypsin without inhibition of elastase.

The rationale of α1-antitrypsin (AAT) augmentation therapy to treat progressive emphysema in AAT-deficient patients is based on inhibition of neutrophil elastase; however, the benefit of this treatment remains unclear. Here we show that clinical grade AAT (with elastase inhibitory activity) and a recombinant form of AAT (rAAT) without anti-elastase activity reduces lung inflammatory responses to LPS in elastase-deficient mice. WT and elastase-deficient mice treated with either native AAT or rAAT exhibited significant reductions in infiltrating neutrophils (23% and 68%), lavage fluid levels of TNF-α (70% and 80%), and the neutrophil chemokine KC (CXCL1) (64% and 90%), respectively. Lung parenchyma TNF-α, DNA damage-inducible transcript 3 and X-box binding protein-1 mRNA levels were reduced in both mouse strains treated with AAT; significantly lower levels of these genes, as well as IL-1ß gene expression, were observed in lungs of AAT-deficient patients treated with AAT therapy compared with untreated patients. In vitro, LPS-induced cytokines from WT and elastase-deficient mouse neutrophils, as well as neutrophils of healthy humans, were similarly reduced by AAT or rAAT; human neutrophils adhering to endothelial cells were decreased by 60-80% (P < 0.001) with either AAT or rAAT. In mouse pancreatic islet macrophages, LPS-induced surface expression of MHC II, Toll-like receptor-2 and -4 were markedly lower (80%, P < 0.001) when exposed to either AAT or rAAT. Consistently, in vivo and in vitro, rAAT reduced inflammatory responses at concentrations 40- to 100-fold lower than native plasma-derived AAT. These data provide evidence that the anti-inflammatory and immunomodulatory properties of AAT can be independent of elastase inhibition.

MicroRNA expression in Epstein-Barr virus-associated post-transplant smooth muscle tumours is related to leiomyomatous phenotype.

Epstein-Barr virus (EBV)-associated post-transplant smooth muscle tumours (PTSMT) are rare complications. In our previous molecular analysis, we have evaluated the expression of regulatory microRNA which are known to be EBV-related (miR-146a and miR-155) but found no deregulation in PTSMT. In this current analysis, we aimed to characterize the expression profiles of several hundred microRNA. Tissue samples from PTSMT and uterine leiomyomas were analysed by quantitative real-time PCR for the expression of 365 mature microRNA. PTSMT and leiomyomas share a highly similar microRNA profile, e.g. strong expression of miR-143/miR-145 cluster and low expression of miR-200c. Among EBV-related microRNA (miR-10b, miR-21, miR-29b, miR-34a, miR-127, miR-146a, miR-155, miR-200b, miR-203 and miR-429) only miR-10b and miR-203 were significantly deregulated. The expression pattern of microRNA in PTSMT is not associated with EBV infection but reflects the leiomyomatous differentiation of the tumour cells.

Congenital phimosis in patients with and without lichen sclerosus: distinct expression patterns of tissue remodeling associated genes.

PURPOSE: Lichen sclerosus is a potentially important factor in the ongoing debate concerning the pathology of persistent congenital phimosis. We assessed the molecular differences of congenital phimosis in boys with and without lichen sclerosus compared to age matched boys with fully retractable foreskins to gain more insight into the pathogenesis of fibrotic remodeling of the prepuce. MATERIALS AND METHODS: A total of 150 boys were circumcised in a prospective study between 2007 and 2009. Using target gene specific preamplification and quantitative real-time polymerase chain reaction based low density arrays, we measured the mRNA expression of 45 tissue remodeling associated genes in foreskins of boys with absolute phimosis and lichen sclerosus (8 patients) and those of an age matched group of boys with phimosis but no lichen sclerosus (8), as well as a control group with foreskins without delimitable changes (6). Complementary protein expression and inflammatory infiltrates were assessed by immunohistochemical analysis. RESULTS: Cellular composition, inflammatory infiltrate and microenvironment as seen in histologically proven lichen sclerosis differed significantly from the other groups. In particular, lichen sclerosis was characterized by over expression of bone morphogenetic protein 2 and its corresponding receptor, matrix metalloproteinases 1 and 9 and tissue inhibitor of metalloproteinases 1, cytokine chemokine ligands 5 (RANTES) and interleukin 4, and transforming growth factor-ß2 and its corresponding receptor. There were no major molecular differences between specimens from boys with congenital phimosis without signs of lichen sclerosis and controls. CONCLUSIONS: Distinct expression patterns of tissue remodeling associated genes are evident in boys with congenital phimosis and lichen sclerosis, while congenital phimosis without lichen sclerosis represents a physiological condition.

Plexiform vasculopathy of severe pulmonary arterial hypertension and microRNA expression.

BACKGROUND: Recent studies have revealed that microRNAs (miRNAs) play a key role in the control of angiogenesis and vascular remodeling. Specific miRNAs in plexiform vasculopathy of severe pulmonary arterial hypertension (PAH) in humans have not yet been investigated. METHODS: We analyzed expression of miR-143/145 (vascular smooth muscle-specific), miR-126 (endothelial-specific) and related mRNAs in plexiform (PLs) and concentric lesions (CLs), which had been laser-microdissected from specimens of formalin-fixed, paraffin-embedded, explanted lungs of PAH patients (n = 12) and unaffected controls (n = 8). Samples were analyzed by real-time polymerase chain reaction, and protein expression was determined by immunohistochemistry. RESULTS: Expression levels of miR-143/145 and its target proteins (e.g., myocardin, smooth muscle myosin heavy chain) were found to be significantly higher in CLs than in PLs, whereas miR-126 and VEGF-A were significantly up-regulated in PLs when compared with CLs, indicating a more prominent angiogenic phenotype of PL. This correlates with a down-regulation of miR-204 as well as an up-regulation of miR-21 in PLs, which in turn corresponds to enhanced cell proliferation. CONCLUSIONS: Our findings show that morphologic changes of plexiform vasculopathy in the end-stage PAH lung are reflected by alterations at the miRNA level.

Plexiform lesions in pulmonary arterial hypertension composition, architecture, and microenvironment.

Pulmonary arterial hypertension (PAH) is a debilitating disease with a high mortality rate. A hallmark of PAH is plexiform lesions (PLs), complex vascular formations originating from remodeled pulmonary arteries. The development and significance of these lesions have been debated and are not yet fully understood. Some features of PLs resemble neoplastic disorders, and there is a striking resemblance to glomeruloid-like lesions (GLLs) in glioblastomas. To further elucidate PLs, we used in situ methods, such as (fluorescent) IHC staining, three-dimensional reconstruction, and laser microdissection, followed by mRNA expression analysis. We generated compartment-specific expression patterns in the lungs of 25 patients (11 with PAH associated with systemic shunts, 6 with idiopathic PAH, and 8 controls) and GLLs from 5 glioblastomas. PLs consisted of vascular channels lined by a continuously proliferating endothelium and backed by a uniform myogenic interstitium. They also showed up-regulation of remodeling-associated genes, such as HIF1a, TGF-ß1, VEGF-α, VEGFR-1/-2, Ang-1, Tie-2, and THBS1, but also of cKIT and sprouting-associated markers, such as NOTCH and matrix metalloproteinases. The cellular composition and signaling seen in GLLs in neural neoplasms differed significantly from those in PLs. In conclusion, PLs show a distinct cellular composition and microenvironment, which contribute to the plexiform phenotype and set them apart from other processes of vascular remodeling in patients with PAH. Neoplastic models of angiogenesis seem to be of limited use in further study of plexiform vasculopathy.

GDF-15 is abundantly expressed in plexiform lesions in patients with pulmonary arterial hypertension and affects proliferation and apoptosis of pulmonary endothelial cells.

BACKGROUND: Growth-differentiation factor-15 (GDF-15) is a stress-responsive, transforming growth factor-ß-related cytokine, which has recently been reported to be elevated in serum of patients with idiopathic pulmonary arterial hypertension (IPAH). The aim of the study was to examine the expression and biological roles of GDF-15 in the lung of patients with pulmonary arterial hypertension (PAH). METHODS: GDF-15 expression in normal lungs and lung specimens of PAH patients were studied by real-time RT-PCR and immunohistochemistry. Using laser-assisted micro-dissection, GDF-15 expression was further analyzed within vascular compartments of PAH lungs. To elucidate the role of GDF-15 on endothelial cells, human pulmonary microvascular endothelial cells (HPMEC) were exposed to hypoxia and laminar shear stress. The effects of GDF-15 on the proliferation and cell death of HPMEC were studied using recombinant GDF-15 protein. RESULTS: GDF-15 expression was found to be increased in lung specimens from PAH patients, compared to normal lungs. GDF-15 was abundantly expressed in pulmonary vascular endothelial cells with a strong signal in the core of plexiform lesions. HPMEC responded with marked upregulation of GDF-15 to hypoxia and laminar shear stress. Apoptotic cell death of HPMEC was diminished, whereas HPMEC proliferation was either increased or decreased depending of the concentration of recombinant GDF-15 protein. CONCLUSIONS: GDF-15 expression is increased in PAH lungs and appears predominantly located in vascular endothelial cells. The expression pattern as well as the observed effects on proliferation and apoptosis of pulmonary endothelial cells suggest a role of GDF-15 in the homeostasis of endothelial cells in PAH patients.

Obliterative airway remodeling: molecular evidence for shared pathways in transplanted and native lungs.

Obliteration of the small airways is a largely unresolved challenge in pulmonary medicine. It represents either the irreversible cause of functional impairment or a morphologic disorder of limited importance in a multitude of diseases. Bronchiolitis obliterans is a key complication of lung transplantation. No predictive markers for the onset of obliterative remodeling are currently available. To further elucidate the molecular mechanisms of airway remodeling, compartment-specific expression patterns were analyzed in patients. For this purpose, remodeled and nonremodeled bronchioli were isolated from transplanted and nontransplanted lung explants using laser-assisted microdissection (n = 24). mRNA expression of 45 fibrosis-associated genes was measured using quantitative real-time RT-PCR. For 20 genes, protein expression was also analyzed by immunohistochemistry. Infiltrating cells were characterized at conventional histology and immunohistochemistry. Obliterative remodeling of the small airways in transplanted and nontransplanted lungs shared similar grades of chronic inflammation and pivotal fibrotic pathways such as transforming growth factor ß signaling and increased collagen expression. Bone morphogenetic protein and thrombospondin signaling, and also matrix metalloproteinases and tissue inhibitor of metalloproteinases, were primarily up-regulated in obliterative airway remodeling in nontransplanted lungs. In transplanted lungs, clinical remodeled bone morphogenetic protein but nonremodeled bronchioli were characterized by a concordant up-regulation of matrix metalloproteinase-9, RANTES, and tissue inhibitor of metalloproteinase-1. These distinct expression patterns warrant further investigation as potential markers of impending airway remodeling, especially for prospective longitudinal molecular profiling.

Obliterative airway remodelling in transplanted and non-transplanted lungs.

Obliterative airway remodelling is a morphological sequence in a variety of pulmonary diseases. Notably, bronchiolitis obliterans represents one of the key complications of lung transplantation, induced by (immigrating) myofibroblasts. A comparative expression analysis of obliterative airway remodelling in transplanted and non-transplanted patients has not been reported so far. Obliterated and unremodelled airways from explanted lungs (n = 19) from patients suffering from chronic allograft dysfunction, infection, graft-versus-host disease and toxic exposure were isolated by laser-assisted microdissection. Airways from lung allografts harvested shortly before and after transplantation (n = 4) as well as fibroblastic foci from lungs with interstitial pulmonary fibrosis (n = 4) served as references. Pre-amplified cDNA was analysed by quantitative real-time RT-PCR for expression of fibrosis, inflammation and apoptosis-associated genes. Composition of infiltrating cells and protein expression were assessed by conventional histology and immunohistochemistry. Bronchiolitis obliterans in transplanted patients showed a significant increase of BMP-7 expression (p = 0.0141 compared with controls), while TGF-beta1 and FGF-2 as well as BMP-4 and BMP-7 were up-regulated in fibroblastic foci in interstitial pulmonary fibrosis (p < 0.0424 compared with controls). Regarding other fibrosis-associated genes (BMP-6, SMAD-3, CASP-3 and CASP-9, FASLG, NF-KB1, IL-1 and IL-2) as well as cellularity and cellular composition, no significant differences between obliterative airway remodelling in transplanted and non-transplanted patients could be shown. Obliterative airway remodelling in lung allografts and in non-transplanted patients share many morphological and genetic traits. BMPs, especially BMP-7, warrant further investigation as possible markers for the aggravation of airway remodelling.

Circulating angiopoietins in idiopathic pulmonary arterial hypertension.

AIMS: To determine the diagnostic utility of circulating angiopoietin-1 (Ang-1) and its antagonist angiopoietin-2 (Ang-2) as potential biomarkers of disease severity or response to treatment in idiopathic pulmonary arterial hypertension (IPAH). Imbalances in angiogenic factors including vascular endothelial cell growth factor (VEGF) and the angiopoetin-Tie2 receptor system have been implicated in the pathogenesis of IPAH. METHODS AND RESULTS: Plasma Ang-1, Ang-2, soluble Tie2 (sTie2), and VEGF were determined by in-house immunoassays in two cohorts of IPAH patients: a retrospective cohort (n = 81) and a prospective cohort (n = 25). Ten patients with normal pulmonary artery pressures and 14 apparently healthy subjects served as controls. Plasma levels of all angiogenic factors were elevated in IPAH patients compared with controls (all P < 0.005). Angiopoietin-2, but not Ang-1, sTie2, and VEGF correlated with cardiac index (r = -0.53, P < 0.001), pulmonary vascular resistance (PVR) (r= 0.60, P < 0.001), and mixed venous oxygen saturation (SvO(2)) (r= -0.63, P < 0.001). In multivariate analysis, elevated Ang-2 was an independent risk factor of mortality (P = 0.004). The patients in the prospective cohort were studied longitudinally at baseline and 3 months after initiation of therapy. Changes in Ang-2 after initiation of therapy correlated with changes in mean right atrial pressure (r = 0.6, P = 0.008), PVR (r = 0.51, P = 0.04), and inversely related to changes in SvO(2) (r = -0.75, P < 0.001). Histological studies showed that the expression of Ang-2 mRNA and protein was up-regulated in plexiform lesions from IPAH lung tissue samples. CONCLUSION: Ang-2 may be involved in the pathogenesis of IPAH, and plasma Ang-2 might serve as a promising new biomarker of disease severity and response to treatment in patients with IPAH.