Molecular Profiling of Vascular Remodeling in Chronic Pulmonary Disease.

Pulmonary hypertension and pulmonary vascular remodeling (PVR) are common in many lung diseases leading to right ventricular dysfunction and death. Differences in PVR result in significant prognostic divergences in both the pulmonary arterial and venous compartments, as in pulmonary arterial hypertension (PAH) and pulmonary veno-occlusive disease (PVOD), respectively. Our goal was to identify compartment-specific molecular hallmarks of PVR, considering the risk of life-threatening pulmonary edema in PVOD, if treated by conventional pulmonary hypertension therapy. Formalin-fixed and paraffin-embedded tissues from fresh explanted human lungs of patients with PVOD (n = 19), PAH (n = 20), idiopathic pulmonary fibrosis (n = 13), and chronic obstructive pulmonary disease (n = 15), were analyzed for inflammation and kinome-related gene regulation. The generated neuronal network differentiated PVOD from PAH samples with a sensitivity of 100% and a specificity of 92% in a randomly chosen validation set, a level far superior to established diagnostic algorithms. Further, various alterations were identified regarding the gene expression of explanted lungs with PVR, compared with controls. Specifically, the dysregulation of microtubule-associated serine/threonine kinase 2 and protein-o-mannose kinase SGK196 in all disease groups suggests a key role in pulmonary vasculopathy for the first time. Our findings promise to help develop novel target-specific interventions and innovative approaches to facilitate clinical diagnostics in an elusive group of diseases.

Morphomolecular motifs of pulmonary neoangiogenesis in interstitial lung diseases.

The pathogenetic role of angiogenesis in interstitial lung diseases (ILDs) is controversial. This study represents the first investigation of the spatial complexity and molecular motifs of microvascular architecture in important subsets of human ILD. The aim of our study was to identify specific variants of neoangiogenesis in three common pulmonary injury patterns in human ILD.We performed comprehensive and compartment-specific analysis of 24 human lung explants with usual intersitial pneumonia (UIP), nonspecific interstitial pneumonia (NSIP) and alveolar fibroelastosis (AFE) using histopathology, microvascular corrosion casting, micro-comupted tomography based volumetry and gene expression analysis using Nanostring as well as immunohistochemistry to assess remodelling-associated angiogenesis.Morphometrical assessment of vessel diameters and intervascular distances showed significant differences in neoangiogenesis in characteristically remodelled areas of UIP, NSIP and AFE lungs. Likewise, gene expression analysis revealed distinct and specific angiogenic profiles in UIP, NSIP and AFE lungs.Whereas UIP lungs showed a higher density of upstream vascularity and lower density in perifocal blood vessels, NSIP and AFE lungs revealed densely packed alveolar septal blood vessels. Vascular remodelling in NSIP and AFE is characterised by a prominent intussusceptive neoangiogenesis, in contrast to UIP, in which sprouting of new vessels into the fibrotic areas is characteristic. The molecular analyses of the gene expression provide a foundation for understanding these fundamental differences between AFE and UIP and give insight into the cellular functions involved.

Comparison of adhesion prevention capabilities of the modified starch powder-based medical devices 4DryField® PH and Arista™ AH in the Optimized Peritoneal Adhesion Model.

Adhesion barriers can be based on numerous substances. In the rat Optimized Peritoneal Adhesion Model (OPAM) the starch-based hemostats 4DryField and Arista were tested for their capability to act in a preventive manner against adhesion formation (applied as a powder that was mixed in situ with saline solution to form a barrier gel). Adhesions were scored using the established scoring systems by Lauder and Hoffmann, as well as histopathologically using the score by Zühlke. Animals receiving saline solution were used as controls. As previously published, 4DryField reduced peritoneal adhesions significantly. However, Arista did not lead to a statistically significant reduction of adhesion formation. When comparing 4DryField and Arista applied in the same manner, only 4DryField was significantly effective in preventing peritoneal adhesions. Histopathological evaluations confirmed the results of the macroscopic investigation, leading to the conclusion that starch-based hemostats do not generally have the capability to function as effective adhesion prevention devices.

Comprehensive three-dimensional morphology of neoangiogenesis in pulmonary veno-occlusive disease and pulmonary capillary hemangiomatosis.

Pulmonary veno-occlusive disease (PVOD) is a rare lung disease characterized by fibrotic narrowing of pulmonary veins leading to pulmonary hypertension (PH) and finally to death by right heart failure. PVOD is often accompanied by pulmonary capillary hemangiomatosis (PCH), a marked abnormal proliferation of pulmonary capillaries. Both morphological patterns often occur together and are thought to be distinct manifestations of the same disease process and accordingly are classified together in group 1' of the Nice classification of PH. The underlying mechanisms of these aberrant remodeling processes remain poorly understood. In this study, we investigated the three-dimensional structure of these vascular lesions in the lung explant of a patient diagnosed with PVOD by µ-computed tomography, microvascular corrosion casting, electron microscopy, immunohistochemistry, correlative light microscopy and gene expression analysis. We were able to describe multifocal intussusceptive neoangiogenesis and vascular sprouting as the three-dimensional correlate of progressive PCH, a process dividing pre-existing vessels by intravascular pillar formation previously only known from embryogenesis and tumor neoangiogenesis. Our findings suggest that venous occlusions in PVOD increase shear and stretching forces in the pulmonary capillary bloodstream and thereby induce intussusceptive neoangiogenesis. These findings can serve as a basis for novel approaches to the analysis of PVOD.

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.

PPARγ Links BMP2 and TGFß1 Pathways in Vascular Smooth Muscle Cells, Regulating Cell Proliferation and Glucose Metabolism.

BMP2 and TGFß1 are functional antagonists of pathological remodeling in the arteries, heart, and lung; however, the mechanisms in VSMCs, and their disturbance in pulmonary arterial hypertension (PAH), are unclear. We found a pro-proliferative TGFß1-Stat3-FoxO1 axis in VSMCs, and PPARγ as inhibitory regulator of TGFß1-Stat3-FoxO1 and TGFß1-Smad3/4, by physically interacting with Stat3 and Smad3. TGFß1 induces fibrosis-related genes and miR-130a/301b, suppressing PPARγ. Conversely, PPARγ inhibits TGFß1-induced mitochondrial activation and VSMC proliferation, and regulates two glucose metabolism-related enzymes, platelet isoform of phosphofructokinase (PFKP, a PPARγ target, via miR-331-5p) and protein phosphatase 1 regulatory subunit 3G (PPP1R3G, a Smad3 target). PPARγ knockdown/deletion in VSMCs activates TGFß1 signaling. The PPARγ agonist pioglitazone reverses PAH and inhibits the TGFß1-Stat3-FoxO1 axis in TGFß1-overexpressing mice. We identified PPARγ as a missing link between BMP2 and TGFß1 pathways in VSMCs. PPARγ activation can be beneficial in TGFß1-associated diseases, such as PAH, parenchymal lung diseases, and Marfan's syndrome.

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.

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.