Jörg Hänze

Publications of Jörg Hänze

• Comparison of the effects of carbon ion and photon irradiation on the angiogenic response in human lung adenocarcinoma cells.

  Authors: Florentine Kamlah, Jörg Hänze, Andrea Arenz, Ulrike Seay, Diya Hasan, Janko Juricko, Birgit Bischoff, Oana R Gottschald, Claudia Fournier, Gisela Taucher-Scholz, Michael Scholz, Werner Seeger, Rita Engenhart-Cabillic, Frank Rose

  International journal of radiation oncology, biology, physics. 05/2011; 80(5):1541-9.

  Radiotherapy resistance is a commonly encountered problem in cancer treatment. In this regard, stabilization of endothelial cells and release of angiogenic factors by cancer cells contribute to thisRadiotherapy resistance is a commonly encountered problem in cancer treatment. In this regard, stabilization of endothelial cells and release of angiogenic factors by cancer cells contribute to this problem. In this study, we used human lung adenocarcinoma (A549) cells to compare the effects of carbon ion and X-ray irradiation on the cells' angiogenic response. A549 cells were irradiated with biologically equivalent doses for cell survival of either carbon ions (linear energy transfer, 170 keV/μm; energy of 9.8 MeV/u on target) or X-rays and injected with basement membrane matrix into BALB/c nu/nu mice to generate a plug, allowing quantification of angiogenesis by blood vessel enumeration. The expression of angiogenic factors (VEGF, PlGF, SDF-1, and SCF) was assessed at the mRNA and secreted protein levels by using real-time reverse transcription-PCR and enzyme-linked immunosorbent assay. Signal transduction mediated by stem cell factor (SCF) was assessed by phosphorylation of its receptor c-Kit. For inhibition of SCF/c-Kit signaling, a specific SCF/c-Kit inhibitor (ISCK03) was used. Irradiation of A549 cells with X-rays (6 Gy) but not carbon ions (2 Gy) resulted in a significant increase in blood vessel density (control, 20.71 ± 1.55; X-ray, 36.44 ± 3.44; carbon ion, 16.33 ± 1.03; number per microscopic field). Concordantly, irradiation with X-rays but not with carbon ions increased the expression of SCF and subsequently caused phosphorylation of c-Kit in endothelial cells. ISCK03 treatment of A549 cells irradiated with X-rays (6 Gy) resulted in a significant decrease in blood vessel density (X-ray, 36.44 ± 3.44; X-ray and ISCK03, 4.33 ± 0.71; number of microscopic field). These data indicate that irradiation of A549 cells with X-rays but not with carbon ions promotes angiogenesis. The present study provides evidence that SCF is an X-ray-induced mediator of angiogenesis in A549 cells, a phenomenon that could not be observed with carbon ion irradiation. Thus, in this model system evaluating angiogenesis, carbon ion irradiation may have a therapeutic advantage. This observation should be confirmed in orthotopic lung tumor models.

• TIAR and TIA-1 mRNA-binding proteins co-aggregate under conditions of rapid oxygen decline and extreme hypoxia and suppress the HIF-1α pathway.

  Authors: Oana R Gottschald, Viktor Malec, Gabriela Krasteva, Diya Hasan, Florentine Kamlah, Susanne Herold, Frank Rose, Werner Seeger, Jörg Hänze

  Journal of molecular cell biology. 10/2010; 2(6):345-56.

  T-cell intracellular antigen (TIA)-1 and TIA-1-related protein (TIAR) are mRNA-binding proteins that can aggregate within granules under specific stress conditions. In this study, we analyzedT-cell intracellular antigen (TIA)-1 and TIA-1-related protein (TIAR) are mRNA-binding proteins that can aggregate within granules under specific stress conditions. In this study, we analyzed TIAR/TIA-1 aggregation under different hypoxic conditions, and studied the effects on the hypoxia-inducible factor (HIF)-1α in different cancer cell lines. Under acute and pronounced hypoxic conditions TIAR/TIA-1 co-aggregated to granules and positive co-staining with eIF3η marker suggested these to represent stress granules. In parallel, HIF-1α expression was blocked in cells displaying TIAR/TIA-1 granules. Silencing of TIAR and TIA-1 caused upregulation of HIF-1α expression, as demonstrated by western blot, immunocytochemistry and HIF-1-dependent reporter gene expression. Additionally, a critical region of the 3' end of the untranslated HIF-1α mRNA with possible adenosine-uridine-rich elements (AREs) was coupled to the luciferase reporter gene, causing downregulation of expression. Employing this reporter construct, inhibition of TIAR by siRNA attenuated the inhibitory cis-effect of this ARE-sequence. Furthermore, immunohistochemical analysis of A549 cell tumor xenografts revealed a nearly complementary expression of HIF-1α and TIAR reflecting the control of HIF-1α expression by TIAR as revealed in the cell culture studies. In sum, rapid and severe hypoxia caused co-aggregation of TIAR/TIA-1 and these proteins suppressed HIF-1α expression.

• Irradiation-dependent effects on tumor perfusion and endogenous and exogenous hypoxia markers in an A549 xenograft model.

  Authors: Emmanouil Fokas, Jörg Hänze, Florentine Kamlah, Bastian G Eul, Nico Lang, Boris Keil, Johannes T Heverhagen, Rita Engenhart-Cabillic, Hanxiang An, Frank Rose

  International journal of radiation oncology, biology, physics. 08/2010; 77(5):1500-8.

  Hypoxia is a major determinant of tumor radiosensitivity, and microenvironmental changes in response to ionizing radiation (IR) are often heterogenous. We analyzed IR-dependent changes in hypoxia andHypoxia is a major determinant of tumor radiosensitivity, and microenvironmental changes in response to ionizing radiation (IR) are often heterogenous. We analyzed IR-dependent changes in hypoxia and perfusion in A549 human lung adenocarcinoma xenografts. Immunohistological analysis of two exogenously added chemical hypoxic markers, pimonidazole and CCI-103F, and of the endogenous marker Glut-1 was performed time dependently after IR. Tumor vessels and apoptosis were analyzed using CD31 and caspase-3 antibodies. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and fluorescent beads (Hoechst 33342) were used to monitor vascular perfusion. CCI-103F signals measuring the fraction of hypoxic areas after IR were significantly decreased by approximately 50% when compared with pimonidazole signals, representing the fraction of hypoxic areas from the same tumors before IR. Interestingly, Glut-1 signals were significantly decreased at early time point (6.5 h) after IR returning to the initial levels at 30.5 h. Vascular density showed no difference between irradiated and control groups, whereas apoptosis was significantly induced at 10.5 h post-IR. DCE-MRI indicated increased perfusion 1 h post-IR. The discrepancy between the hypoxic fractions of CCI-103F and Glut-1 forces us to consider the possibility that both markers reflect different metabolic alterations of tumor microenvironment. The reliability of endogenous markers such as Glut-1 to measure reoxygenation in irradiated tumors needs further consideration. Monitoring tumor microvascular response to IR by DCE-MRI and measuring tumor volume alterations should be encouraged.

• HIF-1 alpha signaling is augmented during intermittent hypoxia by induction of the Nrf2 pathway in NOX1-expressing adenocarcinoma A549 cells.

  Authors: Viktor Malec, Oana R Gottschald, Shu Li, Frank Rose, Werner Seeger, Jörg Hänze

  Free radical biology & medicine. 03/2010; 48(12):1626-35.

  Fluctuations in cellular oxygenation causing intermittent hypoxia and oxidative stress affect the regulation of hypoxia-inducible factor (HIF-1) and the nuclear factor erythroid 2-related factor 2Fluctuations in cellular oxygenation causing intermittent hypoxia and oxidative stress affect the regulation of hypoxia-inducible factor (HIF-1) and the nuclear factor erythroid 2-related factor 2 (Nrf2). HIF-1 is primarily induced in hypoxia, whereas Nrf2 is induced in response to oxidative stress. Whereas HIF-1 regulates the expression of genes important for the adaptation of cells to hypoxia, Nrf2 induces antioxidative enzymes such as thioredoxin 1 (Trx1), exerting a cytoprotective role. Here, we investigated the regulation and cross talk of HIF-1 alpha and Nrf2 in intermittent hypoxia in lung adenocarcinoma A549 cells expressing high levels of the NADPH oxidase subunit NOX1. Whereas continuous hypoxia induced only HIF-1 alpha, intermittent hypoxia induced both HIF-1 alpha and Nrf2, including its target Trx1. NOX1 was determined to be crucial for enhanced ROS production in intermittent hypoxia that in turn mediated induction of Nrf2 and Trx1. The regulation of Nrf2 and Trx1 by NOX1 was confirmed by both inhibition of endogenous NOX1 and overexpression of recombinant NOX1 protein. By using a proteasomal inhibitor, NOX1 was demonstrated to activate Nrf2 by protein stabilization. Subsequently, Nrf2-dependent Trx1 induction turned out to enhance HIF-1 alpha signaling in intermittent hypoxia.

• NOX4 Regulates ROS Levels under Normoxic and Hypoxic Conditions, Triggers Proliferation, and Inhibits Apoptosis in Pulmonary Artery Adventitial Fibroblasts.

  Authors: Shu Li, Sarvenaz Shafiei Tabar, Viktor Malec, Bastian G Eul, Walter Klepetko, Norbert Weissmann, Friedrich Grimminger, Werner Seeger, Frank Rose, Jörg Hänze

  Antioxidants & redox signaling. 08/2008;

  The NADPH oxidases are involved in vascular remodeling processes and oxygen sensing. Hypoxia-induced pulmonary arterial remodeling results in thickening of the vessel wall and reduction of the areaThe NADPH oxidases are involved in vascular remodeling processes and oxygen sensing. Hypoxia-induced pulmonary arterial remodeling results in thickening of the vessel wall and reduction of the area of vessel lumen, leading to pulmonary hypertension and cor pulmonale. The proliferation of pulmonary artery adventitial fibroblasts (PAFB) is critically involved in this process. In this study, we analyzed the role of the non-phagocytic NADPH oxidase subunits NOX1 and NOX4 in PAFB. NOX4 was predominantly expressed in comparison to NOX1 at mRNA levels. Under hypoxic conditions, NOX4 was significantly upregulated at mRNA and protein levels. Silencing of NOX4 by siRNA caused reduction of ROS levels under both normoxic and hypoxic (24 h) conditions and suppressed the significant hypoxic-induced ROS increase. PAFB proliferation was significantly decreased in cells transfected with NOX4 siRNA, whereas apoptosis was enhanced. Also, the expression of NOX4 was studied in PAFB isolated from the lungs of patients with idiopathic pulmonary arterial hypertension (IPAH). Interestingly, a significant increase of NOX4 mRNA expression was observed under hypoxic conditions in PAFB from the lungs with IPAH compared to healthy donors. In conclusion, NOX4 maintains ROS levels under normoxic and hypoxic conditions and enhances proliferation and inhibits apoptosis of PAFB.

• Hypoxia-dependent regulation of nonphagocytic NADPH oxidase subunit NOX4 in the pulmonary vasculature.

  Authors: Manish Mittal, Markus Roth, Peter König, Simone Hofmann, Eva Dony, Parag Goyal, Anne-Christin Selbitz, Ralph Theo Schermuly, Hossein Ardeschir Ghofrani, Grazyna Kwapiszewska, Wolfgang Kummer, Walter Klepetko, Mir Ali Reza Hoda, Ludger Fink, Jörg Hänze, Werner Seeger, Friedrich Grimminger, Harald H H W Schmidt, Norbert Weissmann

  Circulation research. 09/2007; 101(3):258-67.

  Nonphagocytic NADPH oxidases have recently been suggested to play a major role in the regulation of physiological and pathophysiological processes, in particular, hypertrophy, remodeling, andNonphagocytic 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% O(2)) or normoxic (21% O(2)) 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.

• Cellular and molecular mechanisms of hypoxia-inducible factor driven vascular remodeling.

  Authors: Jörg Hänze, Norbert Weissmann, Friedrich Grimminger, Werner Seeger, Frank Rose

  Thrombosis and haemostasis. 06/2007; 97(5):774-87.

  Hypoxia-inducible factor (HIF) is an oxygen-dependent transcription factor that activates a diverse set of target genes, the products of which are involved in adaptive processes to hypoxia. EmployingHypoxia-inducible factor (HIF) is an oxygen-dependent transcription factor that activates a diverse set of target genes, the products of which are involved in adaptive processes to hypoxia. Employing genetic manipulation of HIF expression, in-vivo and cellular studies have focused on HIF as a crucial factor affecting hypoxia-induced vascular remodeling. Vascular remodeling comprises processes which establish and improve blood vessel supply such as vasculogenesis, angiogenesis and arteriogenesis. These processes are observed during ontogenesis, tumor progression, ischemic disease or physical training. Furthermore, under hypoxic conditions, a pulmonary-specific type of vascular remodeling called pulmonary arterial remodeling occurs that is characterized by thickening of the vessel wall with a concomitant reduction in the vessel lumen area, thereby limiting blood flow. This response results in pulmonary hypertension with right ventricular hypertrophy, a lethal disease. In this review, we summarize and discuss mechanisms by which HIF interferes with the different vascular remodeling processes.

• Human RELMbeta is a mitogenic factor in lung cells and induced in hypoxia.

  Authors: Aparna Renigunta, Christiane Hild, Frank Rose, Walter Klepetko, Friedrich Grimminger, Werner Seeger, Jörg Hänze

  FEBS letters. 03/2006; 580(3):900-3.

  RELMbeta (resistin-like molecule) represents the most related human homologue of mouse RELMalpha, also known as hypoxic-induced mitogenic factor (HIMF). In this study, we isolated RELMbeta cDNA fromRELMbeta (resistin-like molecule) represents the most related human homologue of mouse RELMalpha, also known as hypoxic-induced mitogenic factor (HIMF). In this study, we isolated RELMbeta cDNA from human lung tissue and performed regulatory and functional expression studies. RELMbeta mRNA was upregulated in hypoxia in human lung A549 cell line as well as primary cultured adventitial fibroblasts and smooth muscle cells (SMC) of pulmonary arteries. Upon transfection of a RELMbeta encoding expression plasmid into these cells, we observed significant induction of proliferation particularly in SMC and A549 cells, which could be blocked by phosphatidyl-inositol 3-kinase (PI3K) inhibitors LY294002 and wortmannin. The results suggest that human RELMbeta may contribute to hypoxic-induced pulmonary vascular remodeling processes or hypoxia related fibrotic lung disease.

• Hypoxic pulmonary vasoconstriction--triggered by an increase in reactive oxygen species?

  Authors: Norbert Weissmann, Ralph T Schermuly, Hossein A Ghofrani, Jörg Hänze, Parag Goyal, Friedrich Grimminger, Werner Seeger

  Novartis Foundation symposium. 02/2006; 272:196-208; discussion 208-17.

  Hypoxic pulmonary vasoconstriction (HPV) is an essential mechanism of the lung that matches perfusion to ventilation in order to optimize pulmonary gas exchange. Despite intensive research, theHypoxic pulmonary vasoconstriction (HPV) is an essential mechanism of the lung that matches perfusion to ventilation in order to optimize pulmonary gas exchange. Despite intensive research, the underlying mechanism has not yet been fully elucidated. Reactive oxygen species (ROS) have been proposed as key mediators of HPV. However, there is ongoing discussion as to whether ROS really contribute to HPV regulation and if so, whether an increase or a decrease in ROS occurs during alveolar hypoxia. In this overview, we summarize our data that have led us to conclude that alveolar hypoxia induces an increase in superoxide and subsequently H2O2, and thus elicits HPV. This conclusion is drawn from investigations employing various inhibitors that interfere with ROS in isolated buffer-perfused rabbit lungs challenged with 10-minute periods of alveolar hypoxia. Targeting possible sources of a hypoxia-induced increase in ROS, our data are only partially in accordance with the hypothesis that mitochondria are the hypoxia-dependent ROS generators, and suggest NADPH oxidases as an alternative source. From measurements of intracellular and exhaled H2O2, we hypothesize that total lung ROS release is reduced in alveolar hypoxia, but that in specialized cells or sub-cellular structures an increased ROS release may occur, triggering HPV.

• Impact of HIF-1alpha and HIF-2alpha on proliferation and migration of human pulmonary artery fibroblasts in hypoxia.

  Authors: Bastian Eul, Frank Rose, Stefanie Krick, Rajkumar Savai, Parag Goyal, Walter Klepetko, Friedrich Grimminger, Norbert Weissmann, Werner Seeger, Jörg Hänze

  The FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 02/2006; 20(1):163-5.

  Proliferation of adventitial fibroblasts of small intrapulmonary arteries (FBPA) has been disclosed as an early event in the development of pulmonary hypertension and cor pulmonale in response toProliferation of adventitial fibroblasts of small intrapulmonary arteries (FBPA) has been disclosed as an early event in the development of pulmonary hypertension and cor pulmonale in response to hypoxia. We investigated the role of hypoxia-inducible transcription factors (HIF) in human FBPA exposed to hypoxia. Primary cultures of FBPA displayed a strong mitogenic response to 24 h hypoxia, whereas the rate of apoptosis was significantly suppressed. In addition, the migration of FBPA was strongly increased under hypoxic conditions but not the expression of alpha-smooth muscle actin. Hypoxia induced a marked up-regulation (protein level) of both HIF-1alpha and HIF-2alpha, alongside with nuclear translocation of these transcription factors. Specific inhibition of either HIF-1alpha or HIF-2alpha was achieved by RNA interference technology, as proven by HIF-1alpha and HIF-2alpha mRNA and protein analysis and expression analysis of HIF downstream target genes. With the use of this approach, the hypoxia-induced proliferative response of the FBPA was found to be solely HIF-2alpha dependent, whereas the migratory response was significantly reduced by both HIF-1alpha and HIF-2alpha interference. In conclusion, HIF up-regulation is essential for hypoxic cellular responses in human pulmonary artery adventitial fibroblasts such as proliferation and migration, mimicking the pulmonary hypertensive phenotype in vivo. Differential HIF subtype dependency was noted, with HIF-2alpha playing a predominant role, which may offer future intervention strategies.

• Analysis of tumor vessel supply in Lewis lung carcinoma in mice by fluorescent microsphere distribution and imaging with micro- and flat-panel computed tomography.

  Authors: Rajkumar Savai, Joachim C Wolf, Susanne Greschus, Bastian G Eul, Ralph T Schermuly, Jörg Hänze, Robert Voswinckel, Alexander C Langheinrich, Friedrich Grimminger, Horst Traupe, Werner Seeger, Frank Rose

  The American journal of pathology. 11/2005; 167(4):937-46.

  In lung carcinomas the blood supply varies depending on tumor type and stage and can develop from pulmonary or bronchial circulation, or both. To examine this in vivo, primary bronchogenic Lewis lungIn lung carcinomas the blood supply varies depending on tumor type and stage and can develop from pulmonary or bronchial circulation, or both. To examine this in vivo, primary bronchogenic Lewis lung carcinoma cells were intratracheally instilled in C57BL/6 mice. Within 7 days, histological examinations showed progressive tumor growth at the peripheral parenchymal region. The relative contribution of tumor blood supply via the pulmonary and systemic arteries was studied in detail using fluorescent microspheres (10 microm). When compared to healthy lung parenchyma (13:1), Lewis lung carcinoma tumor tissue (52:1) showed a fourfold increase in pulmonary to systemic microspheres, indicating that the pulmonary arteries are the predominant tumor-feeding vessels. After filling the vessels with a vascular cast, the microanatomy of vessels being derived from the pulmonary artery was visualized with micro computed tomography. Flat-panel volumetric computed tomography provided longitudinal visualization of tissue bridges between the growing tumor and the pulmonary vasculature. In this model of peripheral parenchymal malignancy, new imaging techniques allowed effective visualization of lung tumor growth and vascularization in living mice, demonstrating a pulmonary blood supply for lung tumors.

• HIF-1alpha attenuates tumor growth in spite of augmented vascularization in an A549 adenocarcinoma mouse model.

  Authors: Rajkumar Savai, Ralph Theo Schermuly, Robert Voswinckel, Aparna Renigunta, Bernd Reichmann, Bastian Eul, Friedrich Grimminger, Werner Seeger, Frank Rose, Jörg Hänze

  International journal of oncology. 09/2005; 27(2):393-400.

  The hypoxia-inducible transcription factor, HIF-1, adapts cells to low oxygen tension. In addition to the activation of angiogenesis by induction of VEGF, HIF-1 may trigger hypoxia-induced growthThe hypoxia-inducible transcription factor, HIF-1, adapts cells to low oxygen tension. In addition to the activation of angiogenesis by induction of VEGF, HIF-1 may trigger hypoxia-induced growth arrest and apoptosis. The aim of this study was to analyze the overall effect of HIF-1 on tumor growth in a mouse model, employing human lung adenocarcinoma A549 cells. The A549 cells were transfected to overexpress HIF-1alpha. These cells displayed decreased proliferation, cell-cycle entry, colony formation in soft agar and elevated levels of apoptosis, when compared to control cells transfected with empty vector. The cells were employed in a tumor model by subcutaneous injection into CD-1 nude mice. Persistent overexpression of HIF1alpha and VEGF was demonstrated in these tumors. The HIF-1alpha-overexpressing tumors displayed less tumor growth when compared to tumors formed by control cells. Tumors derived from HIF-1alpha-overexpressing cells revealed an increase in apoptosis when compared to control tumors, in spite of a marked increase in vascular density. We conclude that in lung A549 cells, overexpression of HIF-1alpha negatively affects tumor growth due to decreased proliferation and increased apoptosis, despite augmented angiogenesis.

• Role of hypoxia-inducible factor-1alpha in hypoxia-induced apoptosis of primary alveolar epithelial type II cells.

  Authors: Stefanie Krick, Bastian G Eul, Jörg Hänze, Rajkumar Savai, Friedrich Grimminger, Werner Seeger, Frank Rose

  American journal of respiratory cell and molecular biology. 06/2005; 32(5):395-403.

  Hypoxia affects alveolar homeostasis and may induce epithelial injury, which has been implicated in lung diseases such as fibrosis. The underlying cellular and molecular mechanisms are, however,Hypoxia affects alveolar homeostasis and may induce epithelial injury, which has been implicated in lung diseases such as fibrosis. The underlying cellular and molecular mechanisms are, however, largely unknown. Primary rat alveolar epithelial type II cells (ATII) exposed to graded hypoxia for 24 and 48 h caused a dose-dependent induction of cell cycle arrest and suppression of proliferation, which were comparable to the effects of angiotensin II, a potent inducer of ATII cell death. Hypoxia-induced changes in ATII homeostasis are thought to proceed primarily via activation of hypoxia inducible-factor (HIF)-1alpha, because hypoxia increased HIF-1alpha protein expression, nuclear translocation, and transactivation of its specific DNA binding domain, the hypoxia responsive element (HRE). Under hypoxic conditions, expression of the proapoptotic protein Bnip3L, which belongs to the Bcl 2 family and is known to be one of the HIF-1-dependent target genes, was upregulated. Suppression of HIF-1alpha or Bnip-3L with small interfering RNA (siRNA) fully blocked the hypoxia-induced apoptosis and Bnip3L expression. In line with these data, overexpression of HIF-1alpha by transient transfection enhanced the hypoxia-induced apoptosis. Thus, we conclude that hypoxia suppresses alveolar epithelial cell proliferation and enhances ATII apoptosis through activation of the HIF-1alpha/HRE axis and a mechanism that involves Bnip3L. Targeting HIF-1alpha may represent a new strategy that could impede the alveolar denudation that is observed in several lung diseases.

• Hypoxia-driven proliferation of human pulmonary artery fibroblasts: cross-talk between HIF-1alpha and an autocrine angiotensin system.

  Authors: Stefanie Krick, Jörg Hänze, Bastian Eul, Rajkumar Savai, Ulrike Seay, Friedrich Grimminger, Jürgen Lohmeyer, Walter Klepetko, Werner Seeger, Frank Rose

  The FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 06/2005; 19(7):857-9.

  Pulmonary artery adventitial fibroblasts (FBPA) may play a central role in lung vascular remodeling under conditions of hypoxia and inflammation, the result being pulmonary hypertension and corPulmonary artery adventitial fibroblasts (FBPA) may play a central role in lung vascular remodeling under conditions of hypoxia and inflammation, the result being pulmonary hypertension and cor pulmonale. In cultured human FBPA, both angiotensin II (Ang II) and hypoxia promoted cell cycle progression and cell proliferation and suppressed apoptosis. These effects were further enhanced when both stimuli were applied simultaneously. Hypoxia elevated the expression of hypoxia-inducible factor 1alpha (HIF-1alpha) and increased the expression of genes regulated by the hypoxia-responsive element (HRE). Up-regulation of both angiotensin-converting enzyme (ACE) and Ang II receptor type 1 (AT1) was also observed. Exogenous Ang II further increased HIF/HRE-dependent signaling in FBPA, whereas suppression of the autocrine ACE-Ang II-AT1 loop with inhibitors of ACE, AT1, and phosphatidylinositol 3-kinase (PI3K) reduced the proliferative response to both hypoxia and exogenous Ang II. Overexpression of HIF-1alpha by transient transfection caused the same proliferative effect and up-regulation of AT1 expression that were observed under hypoxic conditions. In contrast, small interfering RNA targeting HIF-1alpha inhibited hypoxia-induced ACE and AT1 expression. Our studies indicate that the ACE-Ang II-AT1 system serves as a positive feedback loop and fosters FBPA proliferation under hypoxic conditions, with the PI3K-HIF-HRE axis as the central effector pathway. This pathway may thus facilitate vascular remodeling under hypoxic conditions.

• Identification of novel Nox4 splice variants with impact on ROS levels in A549 cells.

  Authors: Parag Goyal, Norbert Weissmann, Frank Rose, Friedrich Grimminger, Hans J Schäfers, Werner Seeger, Jörg Hänze

  Biochemical and biophysical research communications. 05/2005; 329(1):32-9.

  NAD(P)H oxidases (Nox) generate reactive oxygen species (ROS) that function in host defense and cellular signaling. While analyzing the expression of Nox4 at the protein and the mRNA levels, weNAD(P)H oxidases (Nox) generate reactive oxygen species (ROS) that function in host defense and cellular signaling. While analyzing the expression of Nox4 at the protein and the mRNA levels, we identified four novel Nox4 splice-variants Nox4B, Nox4C, Nox4D, and Nox4E, which are expressed in human lung A549 cell line and lung tissues. One Nox4 isoform lacks the first NAD(P)H binding site (Nox4B) while another lacks all FADH and NAD(P)H binding sites (Nox4C). Cells over-expressing NoxB or Nox4C exhibited a decrease in ROS levels. Thus, these isoforms have dominant negative characteristics for ROS generation. Two other splice-variants (Nox4D, Nox4E) lack the transmembrane domains, suggesting these as non-membrane associated isoforms. Nox4D contains all FADH and NAD(P)H binding domains and shows the same rate of ROS generation as Nox4 prototype. Taken together, we suggest that Nox4 exists as several isoforms that may have different functions in ROS-related cell signaling.

• Upregulation of NAD(P)H oxidase 1 in hypoxia activates hypoxia-inducible factor 1 via increase in reactive oxygen species.

  Authors: Parag Goyal, Norbert Weissmann, Friedrich Grimminger, Cornelia Hegel, Lucius Bader, Frank Rose, Ludger Fink, Hossein A Ghofrani, Ralph T Schermuly, Harald H H W Schmidt, Werner Seeger, Jörg Hänze

  Free radical biology & medicine. 06/2004; 36(10):1279-88.

  Hypoxia sensing and related signaling events, including activation of hypoxia-inducible factor 1 (HIF-1), represent key features in cell physiology and lung function. Using cultured A549 cells, weHypoxia sensing and related signaling events, including activation of hypoxia-inducible factor 1 (HIF-1), represent key features in cell physiology and lung function. Using cultured A549 cells, we investigated the role of NAD(P)H oxidase 1 (Nox1), suggested to be a subunit of a low-output NAD(P)H oxidase complex, in hypoxia signaling. Nox1 expression was detected on both the mRNA and protein levels. Upregulation of Nox1 mRNA and protein occurred during hypoxia, accompanied by enhanced reactive oxygen species (ROS) generation. A549 cells, which were transfected with a Nox1 expression vector, revealed an increase in ROS generation accompanied by activation of HIF-1-dependent target gene expression (heme oxygenase 1 mRNA, hypoxia-responsive-element reporter gene activity). In A549 cells stably overexpressing Nox1, accumulation of HIF-1alpha in normoxia and an additional increase in hypoxia were noted. Interference with ROS metabolism by the flavoprotein inhibitor diphenylene iodonium (DPI) and catalase inhibited HIF-1 induction. This suggests that H2O2 links Nox1 and HIF-1 activation. We conclude that hypoxic upregulation of Nox1 and subsequently augmented ROS generation may activate HIF-1-dependent pathways.

• Basic features of hypoxic pulmonary vasoconstriction in mice.

  Authors: Norbert Weissmann, Ercan Akkayagil, Karin Quanz, Ralph Theo Schermuly, Hossein Ardeschir Ghofrani, Ludger Fink, Jörg Hänze, Frank Rose, Werner Seeger, Friedrich Grimminger

  Respiratory physiology & neurobiology. 02/2004; 139(2):191-202.

  Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion with ventilation which tends to optimize pulmonary gas exchange. Investigations using genetically engineered mice represent a promisingHypoxic pulmonary vasoconstriction (HPV) matches lung perfusion with ventilation which tends to optimize pulmonary gas exchange. Investigations using genetically engineered mice represent a promising approach to understand the underlying mechanisms. Our goal was to characterize basic features of HPV in the isolated buffer-perfused and ventilated mouse lung system. HPV was reproducible for several hours when ventilating the lungs with 1% O2 (10 min) alternated with normoxic ventilation periods (21% O2, 15 min). HPV was well elicitable and most constant using Krebs-Henseleit buffer with the addition of hydroxyethylamylopectin as an oncotic agent. Inhibition of both lung NO and prostanoid formation amplified HPV in an over-additive fashion. HPV was higher in BALB/c mive as compared to C57BL/6 mice, and was approximately threefold enhanced under positive pressure ventilation as compared to negative pressure ventilation. A three hour hypoxic ventilation period resulted in a biphasic vasoconstrictor response with loss of posthypoxic vasodilatation. In summary, we have characterised HPV and established an experimental set-up optimized for investigation of the basic mechanisms of HPV in mice.

• Effects of mitochondrial inhibitors and uncouplers on hypoxic vasoconstriction in rabbit lungs.

  Authors: Norbert Weissmann, Nadine Ebert, Marit Ahrens, Hossein A Ghofrani, Ralph T Schermuly, Jörg Hänze, Ludger Fink, Frank Rose, Jörg Conzen, Werner Seeger, Friedrich Grimminger

  American journal of respiratory cell and molecular biology. 01/2004; 29(6):721-32.

  Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion to ventilation for optimizing pulmonary gas exchange; however, the underlying mechanism has not yet been fully elucidated. Lung nitricHypoxic pulmonary vasoconstriction (HPV) matches lung perfusion to ventilation for optimizing pulmonary gas exchange; however, the underlying mechanism has not yet been fully elucidated. Lung nitric oxide (NO) generation appears to be involved in this process. Recently, mitochondria have been proposed as oxygen sensors, with HPV signaling via a hypoxia-induced increase in the generation of reactive oxygen species derived from mitochondrial complex III and escaping through an anion channel into the cytoplasm. In addition, complex II has been suggested to be specifically involved in hypoxia-dependent generation of reactive oxygen species in the lung. We investigated the effects of several mitochondrial inhibitors and uncouplers on the strength of HPV, and asked for their capacity to mimic HPV during normoxia in isolated buffer-perfused rabbit lungs. Specificity of the agents for HPV was tested by comparison of their effects on non-hypoxia-induced vasoconstriction, elicited by the thromboxane mimetic U46619. Interference with NO metabolism was determined by performing parallel studies with blocked lung NO generation and by measurement of exhaled NO. Rotenone, 3-nitroproprionic acid, and myxothiazol dose-dependently inhibited HPV without being mimics of HPV during normoxia. The inhibitory effect of these agents was only partly specific for HPV by comparison with U46619-induced vasoconstriction. During pre-blocked lung NO synthesis, the selectivity for HPV inhibition was increased for rotenone, but largely lost for myxothiazol. 2-tenoyltrifluoroacetone resulted in an unspecific inhibition of HPV as compared with U46619-induced vasoconstriction. 1-methyl-4-phenylpyridinium iodide and 2-heptyl-4-hydroxyquinoline-N-oxide specifically suppressed HPV and increased normoxic vascular tone. Antimycin A suppressed HPV, an effect being specific in lungs with intact NO synthesis and only partly specific while blocking NO. However, this agent did not mimic HPV during normoxia, as may be expected for interference with the mitochondrial electron transport downstream in complex III. The uncouplers 2,4-dinitrophenol (DNP, 10-200 microM) and carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, 1-3 microM) induced sustained vasoconstriction during normoxia, with enhancement of HPV by DNP at low and suppression of HPV for both agents at high concentrations. The anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid inhibited HPV and U46619-induced vasoconstriction with identical dose-response curves. These findings suggest that mitochondria are in some manner involved in the regulation of HPV in intact rabbit lungs. The hypothesis that enhanced superoxide leak at complex III of mitochondria represents the underlying mechanism of acute HPV is supported by the rotenone and 2-heptyl-4-hydroxyquinoline-N-oxide data, but partly contradicted by the findings with 1-methyl-4-phenylpyridinium iodide, antimycin A, DNP, and FCCP. Further studies are mandatory to clarify the link between mitochondrial respiratory chain and hypoxic pulmonary vasoconstriction.

• RNA interference for HIF-1alpha inhibits its downstream signalling and affects cellular proliferation.

  Authors: Jörg Hänze, Bastian G Eul, Rajkumar Savai, Stefanie Krick, Parag Goyal, Friedrich Grimminger, Werner Seeger, Frank Rose

  Biochemical and biophysical research communications. 01/2004; 312(3):571-7.

  Transcription factor HIF-1 is a key determinant of oxygen-dependent gene regulation. Suppression of HIF-1alpha is important for exploring HIF-1-dependent processes and for interfering withTranscription factor HIF-1 is a key determinant of oxygen-dependent gene regulation. Suppression of HIF-1alpha is important for exploring HIF-1-dependent processes and for interfering with hypoxia-induced pathophysiological events. This study applied RNA-interference targeting HIF-1alpha to the human lung A549 cell line. Transfection of HIF-1alpha-siRNA reduced HIF-1alpha synthesis as measured on mRNA and protein level by realtime RT-PCR, Western blot, and immuncytochemistry. A time kinetic for hypoxic stabilization of HIF-1alpha protein and its inhibition by HIF-1alpha-siRNA is included. Hypoxic induction of HIF-1-controlled target genes as heme oxygenase I (HO-1), phosphoglycerate kinase (PGK), and vascular endothelial growth factor (VEGF) was markedly attenuated by HIF-1alpha-siRNA treatment. Correspondingly, gene activation via hypoxia-responsive-element, as shown by reporter gene assay, was inhibited by HIF-1alpha-siRNA. Moreover, this approach was found to suppress the shift from from S-phase to G1-phase observed in A549 cells in response to hypoxia, supporting a role of HIF-1alpha in oxygen-dependent cell cycle regulation.

• Downregulation of hypoxic vasoconstriction by chronic hypoxia in rabbits: effects of nitric oxide.

  Authors: Norbert Weissmann, Matthias Nollen, Boris Gerigk, Hossein Ardeschir Ghofrani, Ralph Theo Schermuly, Andreas Gunther, Karin Quanz, Ludger Fink, Jörg Hänze, Frank Rose, Werner Seeger, Friedrich Grimminger

  American journal of physiology. Heart and circulatory physiology. 04/2003; 284(3):H931-8.

  Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion to ventilation for optimizing pulmonary gas exchange. Chronic alveolar hypoxia results in vascular remodeling and pulmonaryHypoxic pulmonary vasoconstriction (HPV) matches lung perfusion to ventilation for optimizing pulmonary gas exchange. Chronic alveolar hypoxia results in vascular remodeling and pulmonary hypertension. Previous studies have reported conflicting results of the effect of chronic alveolar hypoxia on pulmonary vasoreactivity and the contribution of nitric oxide (NO), which may be related to species and strain differences as well as to the duration of chronic hypoxia. Therefore, we investigated the impact of chronic hypoxia on HPV in rabbits, with a focus on lung NO synthesis. After exposure of the animals to normobaric hypoxia (10% O(2)) for 1 day to 10 wk, vascular reactivity was investigated in ex vivo perfused normoxic ventilated lungs. Chronic hypoxia induced right heart hypertrophy and increased normoxic vascular tone within weeks. The vasoconstrictor response to an acute hypoxic challenge was strongly downregulated within 5 days, whereas the vasoconstrictor response to the thromboxane mimetic U-46619 was maintained. The rapid downregulation of HPV was apparently not linked to changes in the lung vascular NO system, detectable in the exhaled gas and by pharmacological blockage of NO synthesis. Treatment of the animals with long-term inhaled NO reduced right heart hypertrophy and partially maintained the reactivity to acute hypoxia, without any impact on the endogenous NO system being noted. We conclude that chronic hypoxia causes rapid downregulation of acute HPV as a specific event, preceding the development of major pulmonary hypertension and being independent of the lung vascular NO system. Long-term NO inhalation partially maintains the strength of the hypoxic vasoconstrictor response.