Jochen Wilhelm

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.

Real-Time Polymerase Chain Reaction

Real‐time PCR is the state‐of‐the‐art technique to quantify nucleic acids for mutation detection, genotyping and chimerism analysis. Since its development in the 1990s, many different assay formats have been developed and the number of real‐time PCR machines of different design is continuously increasing. This review provides a survey of the instruments and assay formats available and discusses the pros and cons of each. The principles of quantitative real‐time PCR and melting curve analysis are explained. The quantification algorithms with internal and external standardization are derived mathematically, and potential pitfalls for the data analysis are discussed. Finally, examples of applications of this extremely versatile technique are given that demonstrate the enormous impact of real‐time PCR on life sciences and molecular medicine.

Impact of TASK-1 in Human Pulmonary Artery Smooth Muscle Cells

The excitability of pulmonary artery smooth muscle cells (PASMC) is regulated by potassium (K+) conductances. Although studies suggest that background K+ currents carried by 2-pore domain K+ channels are important regulators of resting membrane potential in PASMC, their role in human PASMC is unknown. Our study tested the hypothesis that TASK-1 leak K+ channels contribute to the K+ current and resting membrane potential in human PASMC. We used the whole-cell patch-clamp technique and TASK-1 small interfering RNA (siRNA). Noninactivating K+ current performed by TASK-1 K+ channels were identified by current characteristics and inhibition by anandamide and acidosis (pH 6.3), each resulting in significant membrane depolarization. Moreover, we showed that TASK-1 is blocked by moderate hypoxia and activated by treprostinil at clinically relevant concentrations. This is mediated via protein kinase A (PKA)-dependent phosphorylation of TASK-1. To further confirm the role of TASK-1 channels in regulation of resting membrane potential, we knocked down TASK-1 expression using TASK-1 siRNA. The knockdown of TASK-1 was reflected by a significant depolarization of resting membrane potential. Treatment of human PASMC with TASK-1 siRNA resulted in loss of sensitivity to anandamide, acidosis, alkalosis, hypoxia, and treprostinil. These results suggest that (1) TASK-1 is expressed in human PASMC; (2) TASK-1 is hypoxia-sensitive and controls the resting membrane potential, thus implicating an important role for TASK-1 K+ channels in the regulation of pulmonary vascular tone; and (3) treprostinil activates TASK-1 at clinically relevant concentrations via PKA, which might represent an important mechanism underlying the vasorelaxing properties of prostanoids and their beneficial effect in vivo.

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.

The Inflammatory versus Constitutive Trafficking of Mononuclear Phagocytes into the Alveolar Space of Mice Is Associated with Drastic Changes in Their Gene Expression Profiles

Mononuclear phagocytes enter the lungs both constitutively to maintain alveolar macrophage and dendritic cell homeostasis, as well as during lung inflammation, where the role of these cells is less well defined. We used a transgenic mouse strain (CX3CR1+/GFP) that harbors a GFP label in circulating monocytes to identify and sort these cells from the vascular and alveolar compartments under both constitutive and acute lung inflammatory conditions. Using nylon arrays combined with real-time RT-PCR for gene expression profiling, we found that flow-sorted, highly purified mononuclear phagocytes recruited to acutely inflamed mouse lungs showed strongly up-regulated mRNA levels of the neutrophil chemoattractants KC, MIP-2, and IP-10, which contrasted with alveolar mononuclear phagocytes that immigrated in steady state. Similar observations were made for the lysosomal cathepsins B, L, and K being strongly up-regulated in mononuclear phagocytes upon recruitment to inflamed lungs but not during constitutive alveolar immigration. Inflammatory elicited mononuclear phagocytes also demonstrated significantly increased mRNA levels of the cytokine TNF-α and the PRR-associated molecules CD14, TLR4, and syndecan-4. Together, inflammatory elicited mononuclear phagocytes exhibit strongly increased neutrophil chemoattractants, lysosomal proteases, and LPS signaling mRNA transcripts, suggesting that these cells may play a major role in acute lung inflammatory processes.

WNT/β-Catenin Signaling Induces IL-1β Expression by Alveolar Epithelial Cells in Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease of unknown etiology. It is characterized by alterations of the alveolar epithelium, myofibroblast activation, and increased extracellular matrix deposition. Recently, reactivation of the developmental WNT/β-catenin pathway has been linked with pulmonary fibrosis. The cell-specific mechanisms and mediators of WNT/β-catenin signaling in the lung, however, remain elusive. Here, we applied an unbiased gene expression screen to identify epithelial cell-specific mediators of WNT/β-catenin signaling. We found the proinflammatory cytokine IL-1β to be one of the most up-regulated genes in primary murine alveolar epithelial Type II (ATII) cells after WNT3a treatment. Increased transcript and protein expression of IL-1β upon WNT3a treatment was further detected in primary ATII cells by quantitative RT-PCR (log fold change, 2.0 ± 0.5) and ELISA (1.8-fold increase). We observed significant up-regulation of IL-1β and IL-6 in bronchoalveolar lavage fluid (BALF) in bleomycin-induced lung fibrosis in vivo. Importantly, primary fibrotic ATII cells isolated from lungs subjected to bleomycin secreted enhanced IL-1β and IL-6 in vitro. Furthermore, the orotracheal application of recombinant WNT protein in the Tcf optimal promoter (TOP)-β-galactosidase reporter animals led to WNT/β-catenin activation in epithelial cells, along with significant increases in IL-1β and IL-6 in vivo (2.7-fold and 6.0-fold increases, respectively). Finally, we found increased WNT3a protein in fibrotic alveolar epithelia, accompanied by enhanced IL-1β and IL-6 concentrations in BALF from patients with IPF. Taken together, our findings reveal that the alveolar epithelium is a relevant source of proinflammatory cytokines induced by active WNT/β-catenin in pulmonary fibrosis. Thus, WNT/interleukin signaling represents a novel link between developmental pathway reactivation and inflammation in the development of pulmonary fibrosis.

Fhl-1, a New Key Protein in Pulmonary Hypertension

Background— Pulmonary hypertension (PH) is a severe disease with a poor prognosis. Different forms of PH are characterized by pronounced vascular remodeling, resulting in increased vascular resistance and subsequent right heart failure. The molecular pathways triggering the remodeling process are poorly understood. We hypothesized that underlying key factors can be identified at the onset of the disease. Thus, we screened for alterations to protein expression in lung tissue at the onset of PH in a mouse model of hypoxia-induced PH. Methods and Results— Using 2-dimensional polyacrylamide gel electrophoresis in combination with matrix-assisted laser desorption/ionization time-of-flight analysis, we identified 36 proteins that exhibited significantly altered expression after short-term hypoxic exposure. Among these, Fhl-1, which is known to be involved in muscle development, was one of the most prominently upregulated proteins. Further analysis by immunohistochemistry, Western blot, and laser-assisted microdissection followed by quantitative polymerase chain reaction confirmed the upregulation of Fhl-1, particularly in the pulmonary vasculature. Comparable upregulation was confirmed (1) after full establishment of hypoxia-induced PH, (2) in 2 rat models of PH (monocrotaline-treated and hypoxic rats treated with the vascular endothelial growth factor receptor antagonist SU5416), and (3) in lungs from patients with idiopathic pulmonary arterial hypertension. Furthermore, we demonstrated that regulation of Fhl-1 was hypoxia-inducible transcription factor dependent. Abrogation of Fhl-1 expression in primary human pulmonary artery smooth muscle cells by small-interfering RNA suppressed, whereas Fhl-1 overexpression increased, migration and proliferation. Coimmunoprecipitation experiments identified Talin1 as a new interacting partner of Fhl-1. Conclusions— Protein screening identified Fhl-1 as a novel protein regulated in various forms of PH, including idiopathic pulmonary arterial hypertension.

The Noncanonical WNT Pathway Is Operative in Idiopathic Pulmonary Arterial Hypertension

Idiopathic pulmonary arterial hypertension (IPAH) is a fatal disease that comprises sustained vasoconstriction, enhanced proliferation of pulmonary vascular cells, and in situ thrombosis. The discovery of several contributing signaling pathways in recent years has resulted in an expanding array of novel therapies; however, IPAH remains a progressive disease with poor outcome in most instances. To identify new regulatory pathways of vascular remodeling in IPAH, we performed transcriptome-wide expression profiling of laser-microdissected pulmonary arterial resistance vessels derived from explanted IPAH and nontransplanted donor lung tissues. Statistical analysis of the data derived from six individuals in each group showed significant regulation of several mediators of the canonical and noncanonical WNT pathway. As to the noncanonical WNT pathway, the planar cell polarity (PCP) pathway, the ras homolog gene family member A (RHOA), and ras-related C3 botulinum toxin substrate-1 (RAC1) were strongly up-regulated. Real-time PCR of laser-microdissected pulmonary arteries confirmed these array results and showed in addition significant up-regulation of further PCP mediators wingless member 11 (WNT11), disheveled associated activator of morphogenesis-1 (DAAM1), disheveled (DSV), and RHO-kinase (ROCK). Immunohistochemical staining and semiquantitative expression analysis confirmed the markedly enhanced expression of the PCP mediators in the pulmonary resistance vessels, in particular in the endothelial layer in IPAH. Therefore we propose the PCP pathway to be critically involved in the regulation of vascular remodeling in IPAH.

Phosphodiesterase-4 promotes proliferation and angiogenesis of lung cancer by crosstalk with HIF

Lung cancer is the leading cause of cancer death worldwide. Recent data suggest that cyclic nucleotide phosphodiesterases (PDEs) are relevant in various cancer pathologies. Pathophysiological role of phosphodiesterase 4 (PDE4) with possible therapeutic prospects in lung cancer was investigated. We exposed 10 different lung cancer cell lines (adenocarcinoma, squamous and large cell carcinoma) to hypoxia and assessed expression and activity of PDE4 by real-time PCR, immunocytochemistry, western blotting and PDE activity assays. Expression and activity of distinct PDE4 isoforms (PDE4A and PDE4D) increased in response to hypoxia in eight of the studied cell lines. Furthermore, we analyzed various in silico predicted hypoxia-responsive elements (p-HREs) found in in PDE4A and PDE4D genes. Performing mutation analysis of the p-HRE in luciferase reporter constructs, we identified four functional HRE sites in the PDE4A gene and two functional HRE sites in the PDE4D gene that mediated hypoxic induction of the reporter. Silencing of hypoxia-inducible factor subunits (HIF1α and HIF2α) by small interfering RNA reduced hypoxic induction of PDE4A and PDE4D. Vice versa, using a PDE4 inhibitor (PDE4i) as a cyclic adenosine monophosphate (cAMP) -elevating agent, cAMP analogs or protein kinase A (PKA)-modulating drugs and an exchange protein directly activated by cAMP (EPAC) activator, we demonstrated that PDE4-cAMP-PKA/EPAC axis enhanced HIF signaling as measured by HRE reporter gene assay, HIF and HIF target genes expression ((lactate dehydrogenase A), LDHA, (pyruvate dehydrogenase kinase 1) PDK1 and (vascular endothelial growth factor A) VEGFA). Notably, inhibition of PDE4 by PDE4i or silencing of PDE4A and PDE4D reduced human lung tumor cell proliferation and colony formation. On the other hand, overexpression of PDE4A or PDE4D increased human lung cancer proliferation. Moreover, PDE4i treatment reduced hypoxia-induced VEGF secretion in human cells. In vivo, PDE4i inhibited tumor xenograft growth in nude mice by attenuating proliferation and angiogenesis. Our findings suggest that PDE4 is expressed in lung cancer, crosstalks with HIF signaling and promotes lung cancer progression. Thus, PDE4 may represent a therapeutic target for lung cancer therapy.

Macrophage Tumor Necrosis Factor-α Induces Epithelial Expression of Granulocyte–Macrophage Colony-stimulating Factor

RATIONALE Resident alveolar macrophages have been attributed a crucial role in host defense toward pulmonary infection. Their contribution to alveolar repair processes, however, remains elusive. OBJECTIVES We investigated whether activated resident alveolar macrophages contribute to alveolar epithelial repair on lipopolysaccharide (LPS) challenge in vitro and in vivo and analyzed the molecular interaction pathways involved. METHODS We evaluated macrophage-epithelial cross-talk mediators for epithelial cell proliferation in an in vitro coculture system and an in vivo model of LPS-induced acute lung injury comparing wild-type, granulocyte-macrophage colony-stimulating factor (GM-CSF)-deficient (GM(-/-)), and human SPC-GM mice (GM(-/-) mice expressing an SPC-promotor-regulated GM-CSF transgene). MEASUREMENTS AND MAIN RESULTS Using reverse transcription-polymerase chain reaction and ELISA we showed that LPS-activated alveolar macrophages stimulated alveolar epithelial cells (AEC) to express growth factors, particularly GM-CSF, in coculture. Antibody neutralization experiments revealed epithelial GM-CSF expression to be macrophage tumor necrosis factor (TNF)-alpha dependent. GM-CSF elicited proliferative signaling in AEC via autocrine stimulation. Notably, macrophage TNF-alpha induced epithelial proliferation in wild-type but not in GM-CSF-deficient AEC as shown by [(3)H]-thymidine incorporation and cell counting. Moreover, intraalveolar TNF-alpha neutralization impaired AEC proliferation in LPS-injured mice, as investigated by flow cytometric Ki-67 staining. Additionally, GM-CSF-deficient mice displayed reduced AEC proliferation and sustained alveolar barrier dysfunction on LPS treatment compared with wild-type mice. CONCLUSIONS Collectively, these findings indicate that TNF-alpha released from activated resident alveolar macrophages induces epithelial GM-CSF expression, which in turn initiates AEC proliferation and contributes to restoring alveolar barrier function.