Ludger Fink

WNT1-inducible signaling protein–1 mediates pulmonary fibrosis in mice and is upregulated in humans with idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterized by distorted lung architecture and loss of respiratory function. Enhanced (myo)fibroblast activation, ECM deposition, and alveolar epithelial type II (ATII) cell dysfunction contribute to IPF pathogenesis. However, the molecular pathways linking ATII cell dysfunction with the development of fibrosis are poorly understood. Here, we demonstrate, in a mouse model of pulmonary fibrosis, increased proliferation and altered expression of components of the WNT/beta-catenin signaling pathway in ATII cells. Further analysis revealed that expression of WNT1-inducible signaling protein-1 (WISP1), which is encoded by a WNT target gene, was increased in ATII cells in both a mouse model of pulmonary fibrosis and patients with IPF. Treatment of mouse primary ATII cells with recombinant WISP1 led to increased proliferation and epithelial-mesenchymal transition (EMT), while treatment of mouse and human lung fibroblasts with recombinant WISP1 enhanced deposition of ECM components. In the mouse model of pulmonary fibrosis, neutralizing mAbs specific for WISP1 reduced the expression of genes characteristic of fibrosis and reversed the expression of genes associated with EMT. More importantly, these changes in gene expression were associated with marked attenuation of lung fibrosis, including decreased collagen deposition and improved lung function and survival. Our study thus identifies WISP1 as a key regulator of ATII cell hyperplasia and plasticity as well as a potential therapeutic target for attenuation of pulmonary fibrosis.

Epithelial Endoplasmic Reticulum Stress and Apoptosis in Sporadic Idiopathic Pulmonary Fibrosis

RATIONALE The molecular pathomechanisms underlying idiopathic pulmonary fibrosis (IPF) are elusive, but chronic epithelial injury has recently been suggested as key event. OBJECTIVES We investigated the possible implication of endoplasmic reticulum (ER) stress-mediated apoptosis in sporadic IPF. METHODS We analyzed peripheral explanted lung tissues from patients with sporadic IPF (n = 24), chronic obstructive pulmonary disease (COPD) (n = 9), and organ donors (n = 12) for expression of major ER stress mediators and apoptosis markers by means of immunoblotting, semiquantitative reverse transcription-polymerase chain reaction, immunohistochemistry, and the TUNEL method. MEASUREMENTS AND MAIN RESULTS Compared with COPD and donor lungs, protein levels of ER stress mediators, such as processed p50 activating transcription factor (ATF)-6 and ATF-4 and the apoptosis-inductor CHOP (C/EBP-homologous protein), as well as transcript levels of spliced X-box binding protein (XBP)-1, were significantly elevated in lung homogenates and type II alveolar epithelial cells (AECIIs) of IPF lungs. Proapoptotic, oligomeric forms of Bax, which play a key role in ER stress-mediated apoptosis downstream of CHOP induction, as well as caspase-3 cleavage, could be detected in IPF lungs. By means of immunohistochemistry, exclusive induction of active ATF-6, ATF-4, and CHOP in AECIIs was encountered in IPF but not in COPD or donor lungs. Immunoreactivity was most prominent in the epithelium near dense zones of fibrosis and fibroblast foci, where these ER stress markers colocalized with markers of apoptosis (TUNEL, cleaved caspase-3). CONCLUSIONS Severe ER stress response in the AECIIs of patients with sporadic IPF may underlie the apoptosis of this cell type and development of fibrosis in this disease.

Hypoxia-Dependent Regulation of Nonphagocytic NADPH Oxidase Subunit NOX4 in the Pulmonary Vasculature

Nonphagocytic NADPH oxidases have recently been suggested to play a major role in the regulation of physiological and pathophysiological processes, in particular, hypertrophy, remodeling, and angiogenesis in the systemic circulation. Moreover, NADPH oxidases have been suggested to serve as oxygen sensors in the lung. Chronic hypoxia induces vascular remodeling with medial hypertrophy leading to the development of pulmonary hypertension. We screened lung tissue for the expression of NADPH oxidase subunits. NOX1, NOXA1, NOXO1, p22phox, p47phox, p40phox, p67phox, NOX2, and NOX4 were present in mouse lung tissue. Comparing mice maintained for 21 days under hypoxic (10% O2) or normoxic (21% O2) conditions, an upregulation exclusively of NOX4 mRNA was observed under hypoxia in homogenized lung tissue, concomitant with increased levels in microdissected pulmonary arterial vessels. In situ hybridization and immunohistological staining for NOX4 in mouse lungs revealed a localization of NOX4 mRNA and protein predominantly in the media of small pulmonary arteries, with increased labeling intensities after chronic exposure to hypoxia. In isolated pulmonary arterial smooth muscle cells (PASMCs), NOX4 was localized primarily to the perinuclear space and its expression levels were increased after exposure to hypoxia. Treatment of PASMCs with siRNA directed against NOX4 decreased NOX4 mRNA levels and reduced PASMC proliferation as well as generation of reactive oxygen species. In lungs from patients with idiopathic pulmonary arterial hypertension (IPAH), expression levels of NOX4, which was localized in the vessel media, were 2.5-fold upregulated. These results support an important role for NOX4 in the vascular remodeling associated with development of pulmonary hypertension.

TLR7 Ligands Induce Higher IFN-α Production in Females

IFN-α exercises multiple immune modulatory and antiviral activities and has been suggested to play a critical role in the pathogenesis of systemic lupus erythematosus (SLE). Plasmacytoid dendritic cells (pDCs) release IFN-α upon TLR7 and TLR9 ligation. With respect to the nine times higher incidence of SLE in women and the clinical use of synthetic TLR ligands as novel immune adjuvants, we analyzed IFN-α and TNF-α production in healthy human individuals. Blood samples were incubated with synthetic TLR7 and TLR9 ligands. In three independent groups (n1 = 120, n2 = 101, and n3 = 123), analysis revealed a capacity of female PBLs to produce significantly higher IFN-α levels after TLR7 stimulation (p1 < 0.0000001, p2 < 0.0000001, and p3 < 0.0001) compared with male PBLs. In contrast, no sex differences were evident after TLR9 stimulation. TNF-α production after TLR7 stimulation and also total pDC numbers were not different between females and males. X-inactivation escape of the TLR7 gene was investigated in monoclonal B cell lines and, independently, in pDCs after cell sorting and single-cell picking, indicating regular silencing of one TLR7 allele in females. Additionally, exogenous 17β-estrogen and estrogen receptor antagonism did not indicate a significant role on TLR7-induced IFN-α production. Our data reveal for the first time a profound sex-dependent pathway of TLR7-induced IFN-α with higher production in females. These findings may explain the higher prevalence of SLE in females and the reported decreased therapeutic efficacy of synthetic TLR7 ligands in male individuals.

Increased levels and reduced catabolism of asymmetric and symmetric dimethylarginines in pulmonary hypertension

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS) and has been implicated in endothelial dysfunction. ADMA is metabolized by the enzyme dimethylarginine dimethylaminohydrolase (DDAH), with DDAH2 representing the predominant endothelial DDAH isoform. Symmetric dimethylarginine (SDMA), also originating from arginine methylation by protein arginine methyltransferases, is an inhibitor of intracellular arginine uptake. In both chronic pulmonary hypertensive rats and patients suffering from idiopathic pulmonary arterial hypertension (IPAH; NYHA class III and IV), a marked increase in plasma ADMA and SDMA levels, as well as tissue levels of asymmetric and symmetric dimethylated proteins, was observed. Moreover, when comparing lung tissue from pulmonary hypertensive rats and IPAH patients to corresponding normal lung tissue, expression of DDAH2 was found to be reduced at both the mRNA and the protein level with no significant changes in DDAH1 expression. These findings were further supported by demonstrating a decrease in DDAH2 function in the experimental pulmonary hypertension model. Immunohistochemistry in human and rat control tissue demonstrated both isoforms of DDAH in the endothelial layer and in the alveolar epithelium. In contrast, in pulmonary hypertensive tissue, the immunoreactivity of DDAH2 in pulmonary endothelium was significantly decreased compared with DDAH1. Therefore, altogether we can conclude that enhanced dimethylarginine levels may contribute to vascular abnormalities in pulmonary arterial hypertension. Suppression of endothelial DDAH2 expression and function represents an important underlying mechanism.

In vivo pharmacokinetics, tissue distribution and underlying mechanisms of various PEI(–PEG)/siRNA complexes

BACKGROUND RNA interference (RNAi) represents a novel therapeutic strategy allowing the knockdown of any pathologically relevant target gene. Since it relies on the action of small interfering RNAs (siRNAs), the in vivo delivery of siRNAs is instrumental. Polyethylenimines (PEIs) and PEGylated PEIs have been shown previously to complex siRNAs, thus mediating siRNA protection against nucleolytic degradation, cellular uptake and intracellular release. PURPOSE The present study determines in vivo pharmacokinetics, tissue distribution/efficacy of siRNA delivery and adverse effects of a broad panel of PEI(-PEG)-based siRNA complexes. The aim is to systematically evaluate the effects of different degrees and patterns of PEGylation in PEI-PEG copolymers on the in vivo behavior of PEI(-PEG)/siRNA complexes in mice. RESULTS Upon i.v. injection of radioactively labeled, PEI(-PEG) complexed siRNAs, marked differences in the pharmacokinetics and biodistribution of the complexes are observed, with the fate of the PEI(-PEG)/siRNA complexes being mainly dependent on the degree of uptake in liver, spleen, lung and kidney. Thus, the role of these tissues is investigated in greater detail using representative PEI(-PEG)/siRNA complexes. The induction of erythrocyte aggregation and hemorrhage is dependent on the degree and pattern of PEGylation as well as on the PEI/siRNA (N/P) ratio, and represents one important effect in the lung. Furthermore, siRNA uptake in liver and spleen, but not in lung or kidney, is mediated by macrophage and is dependent on macrophage activity. In the kidney PEI(-PEG)/siRNA uptake is mostly passive and reflects the total stability of the complexes. CONCLUSION Liver, lung, spleen and kidney are the major players determining the in vivo biodistribution of PEI(-PEG)/siRNA complexes. Beyond their physicochemical and in vitro bioactivity characteristics, PEI(-PEG)/siRNA complexes show marked differences in vivo which can be explained by distinct effects in different tissues. Based on these data, our study also identifies which PEGylated PEIs are promising tools for in vivo siRNA delivery in future therapeutic studies and which major determinants require further investigation.

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.

Endotoxin-Induced Myocardial Tumor Necrosis Factor-α Synthesis Depresses Contractility of Isolated Rat Hearts Evidence for a Role of Sphingosine and Cyclooxygenase-2–Derived Thromboxane Production

BackgroundAlthough endotoxin (lipopolysaccharides, LPS) is recognized as a mediator of septic cardiodepression, its cardiac effects are still not fully elucidated. Methods and ResultsPerfusion of isolated rat hearts with LPS for 180 minutes resulted in a decline of left ventricular contractility after 90 minutes, whereas coronary perfusion pressure remained unaffected. This cardiodepression was paralleled by a release of tumor necrosis factor (TNF)-&agr; into the perfusate and preceded by myocardial TNF-&agr; mRNA upregulation as quantified by real-time polymerase chain reaction. The cardiodepression was abrogated when LPS was perfused with a TNF-&agr; antiserum or the ceramidase inhibitor N-oleoylethanolamine. In contrast, the cardiac release of nitric oxide (NO) was not augmented by LPS. Immunohistochemical studies of LPS-perfused hearts revealed a positive staining for the constitutive (NOSIII) but not for the inducible NO synthase (NOSII). Accordingly, NOSII mRNA levels commenced to increase only at the very end of the LPS perfusion period. Progressive liberation of thromboxane (Tx) A2 and prostacyclin was induced by LPS together with myocardial cyclooxygenase (Cox)-2 mRNA expression. Both nonselective inhibition of Cox by indomethacin and selective inhibition of the inducible Cox-2 by NS-398 abolished prostanoid release. Interestingly, the generation of TNF-&agr; and the associated cardiodepression caused by LPS were reduced by indomethacin, NS-398 and the Tx-receptor antagonist daltroban. ConclusionsLPS depresses contractility of isolated rat hearts by inducing TNF-&agr; synthesis and subsequently activating the sphingomyelinase pathway, whereas no evidence for a role of NOSII- or NOSIII-generated NO was found. Moreover, Cox-2–derived TxA2 appears to facilitate TNF-&agr; synthesis in response to LPS.Background —Although endotoxin (lipopolysaccharides, LPS) is recognized as a mediator of septic cardiodepression, its cardiac effects are still not fully elucidated. Methods and Results —Perfusion of isolated rat hearts with LPS for 180 minutes resulted in a decline of left ventricular contractility after 90 minutes, whereas coronary perfusion pressure remained unaffected. This cardiodepression was paralleled by a release of tumor necrosis factor (TNF)-α into the perfusate and preceded by myocardial TNF-α mRNA upregulation as quantified by real-time polymerase chain reaction. The cardiodepression was abrogated when LPS was perfused with a TNF-α antiserum or the ceramidase inhibitor N -oleoylethanolamine. In contrast, the cardiac release of nitric oxide (NO) was not augmented by LPS. Immunohistochemical studies of LPS-perfused hearts revealed a positive staining for the constitutive (NOSIII) but not for the inducible NO synthase (NOSII). Accordingly, NOSII mRNA levels commenced to increase only at the very end of the LPS perfusion period. Progressive liberation of thromboxane (Tx) A2 and prostacyclin was induced by LPS together with myocardial cyclooxygenase (Cox)-2 mRNA expression. Both nonselective inhibition of Cox by indomethacin and selective inhibition of the inducible Cox-2 by NS-398 abolished prostanoid release. Interestingly, the generation of TNF-α and the associated cardiodepression caused by LPS were reduced by indomethacin, NS-398 and the Tx-receptor antagonist daltroban. Conclusions —LPS depresses contractility of isolated rat hearts by inducing TNF-α synthesis and subsequently activating the sphingomyelinase pathway, whereas no evidence for a role of NOSII- or NOSIII-generated NO was found. Moreover, Cox-2–derived TxA2 appears to facilitate TNF-α synthesis in response to LPS.

Expression profiling of laser-microdissected intrapulmonary arteries in hypoxia-induced pulmonary hypertension.

BackgroundChronic hypoxia influences gene expression in the lung resulting in pulmonary hypertension and vascular remodelling. For specific investigation of the vascular compartment, laser-microdissection of intrapulmonary arteries was combined with array profiling.Methods and ResultsAnalysis was performed on mice subjected to 1, 7 and 21 days of hypoxia (FiO2 = 0.1) using nylon filters (1176 spots). Changes in the expression of 29, 38, and 42 genes were observed at day 1, 7, and 21, respectively. Genes were grouped into 5 different classes based on their time course of response. Gene regulation obtained by array analysis was confirmed by real-time PCR. Additionally, the expression of the growth mediators PDGF-B, TGF-β, TSP-1, SRF, FGF-2, TIE-2 receptor, and VEGF-R1 were determined by real-time PCR. At day 1, transcription modulators and ion-related proteins were predominantly regulated. However, at day 7 and 21 differential expression of matrix producing and degrading genes was observed, indicating ongoing structural alterations. Among the 21 genes upregulated at day 1, 15 genes were identified carrying potential hypoxia response elements (HREs) for hypoxia-induced transcription factors. Three differentially expressed genes (S100A4, CD36 and FKBP1a) were examined by immunohistochemistry confirming the regulation on protein level. While FKBP1a was restricted to the vessel adventitia, S100A4 and CD36 were localised in the vascular tunica media.ConclusionLaser-microdissection and array profiling has revealed several new genes involved in lung vascular remodelling in response to hypoxia. Immunohistochemistry confirmed regulation of three proteins and specified their localisation in vascular smooth muscle cells and fibroblasts indicating involvement of different cells types in the remodelling process. The approach allows deeper insight into hypoxic regulatory pathways specifically in the vascular compartment of this complex organ.

Hypoxic pulmonary artery fibroblasts trigger proliferation of vascular smooth muscle cells: role of hypoxia-inducible transcription factors

Chronic lung hypoxia causes vascular remodeling with pulmonary artery smooth muscle cell (SMCPA) hyperplasia, resulting in pulmonary hypertension and cor pulmonale. We investigated SMCPA and pulmonary artery adventitial fibroblasts (FBPA) for their proliferative response to hypoxia. Strong SMCPA growth occurred under hypoxic conditions in SMCPA/FBPA co‐cultures, but not in SMCPA monocultures. SMCPA growth was fully reproduced by transferring serum‐free supernatant from hypoxic cultured FBPA to normoxic SMCPA. Hypoxia‐inducible‐transcriptionfactor subtypes (HIF‐1α, HIF‐2α, HIF‐3α) and its dependent target genes, carrying the hypoxiaresponsive‐element as regulatory component, were strongly activated in both hypoxic FBPA and SMCPA. HIF‐transcription‐factor decoy technique, employed to FBPA during hypoxic culturing, blocked the mitogenic activity of FBPA conditioned medium on SMCPA. The data suggest that hypoxia‐driven gene regulation in pulmonary artery fibroblasts results in a mitogenic stimulus on adjacent pulmonary artery smooth muscle cells, and HIF‐transcription‐decoy may offer a new therapeutic approach to suppress these events.