Email : Constance.Barazzone @ hcuge.ch
Tel: +41 22 3795758 (+41 22 3725466) / FAX: +41 22 3794546
Addresses

Research areas: Role of oxidative stress in acute and chronic lung disease

In most pulmonary diseases, such as asthma, tobacco exposure, cystic fibrosis and respiratory distress syndrome (RDS), oxidant burden is central resulting from the concomitant direct effects of oxidants in the extracellular space as well as the increased release of reactive oxygen species (ROS) from inflammatory cells. We developed several in vivo and in vitro models to study the molecular and physiological mechanisms underlying acute and chronic lung diseases. The goal of our ongoing research is to identify the role of oxidative stress in alveolar cell death and repair and in particular to define whether apoptosis or/and necrosis occur in the same cells (epithelial and endothelial cells) and which cells are crucial in maintaining the alveolo-capillary barrier integrity.
At the cellular level, mitochondria and the NOX family are major source of ROS induced by external stimuli. We recently determined that NOX1, an NADPH oxidase isoform, is an important contributor to ROS production and cell death of the alveolo-capillary barrier in acute lung injury. To determine the mechanisms of NOX1-mediated cell death in lung epithelial cells, we generated NOX1 specific shRNA expressed from lentivirus transduction in mouse lung epithelial cells (MLE12). We found that NOX1 silencing prevented ROS generation and cell death in these cells. We will further explore the pathways involved in NOX1-generated ROS and a possible cross-talk between mitochondria and NOX family during oxidative-stress induced by hyperoxia.
 Mitochondria are the main targets of oxidative damage and we recently generated transgenic mice overexpressing Bcl-2 specifically in alveolar type II cells. Bcl-2 is a mitochondrial antiapoptotic protein. We concomitantly used an in vitro complementary approach by studying transfected cells with Bcl-2 gene and found that Bcl-2 overexpression significantly protected the mitochondria of transfected cells from ROS-induced cell death, as observed by the decreased release of cytochrome c. Indeed, cytochrome c is a proapoptotic factor located between the outer and inner membranes of the mitochondria. This suggests that, by modulating the degree of mitochondrial damage and consequently the intracellular level of ATP, it is possible to control cell death and thereby minimize oxidative stress-induced lesions.
We are also interested in the role of ROS in the development of chronic lung disease. We analyzed lungs of patients with idiopathic pulmonary fibrosis and found that there was a strong expression of NOX4, another NADPH isoform, in hyperplastic alveolar type II cells and to a lesser extent in myofibroblasts and vascular smooth muscle cells, which express smooth muscle actin (SMA) as well as in the musculature of hyperplastic lung arteries (ref). We used NOX4-deficient mice generated by Prof. KH Krause. Wild-type mice developed massive fibrosis in response to bleomycin while NOX4-deficient mice displayed almost normal lung histology. Interestingly, the protective effects of NOX4 deficiency preceded the fibrotic stage. Indeed, at day 7 after bleomycin, lungs of wild-type mice showed massive increase in epithelial cell apoptosis and inflammation. In NOX4-deficient mice, no apoptosis and less Smad2 phosphorylation and accumulation of myofibroblasts were observed, whereas inflammation was comparable to wild-type. In vitro, NOX4-deficient primary alveolar epithelial cells exposed to TGF-b did not generate ROS and were protected from apoptosis. We demonstrated that NOX4 is also a key player in epithelial cell death, an early step of lung fibrosis.
These strategies should help us first, to define the role of ROS in the signalling pathways damaging specifically the epithelial side of alveolo-capillary barrier. Secondly, this study could open new fields in finding biological markers of oxidative stress which might reflect lung damage and lead to development of therapeutic targets to prevent acute and chronic lung disease.

Double immunostaining of mouse lung : Surfactant protein C (marker of type II epithelial cells) and DiHydroEthidium (superoxide production