Parag Goyal

Publications of Parag Goyal

• 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.

• 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.

• 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.

• 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.

• 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.

  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 and Medicine.

  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-1α 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.