118 Oxidative Processing of Latent Fas in the Endoplasmic Reticulum Controls the Strength of Apoptosis 1
Yvonne MW Janssen-Heininger 1 University of Vermont We recently demonstrated that S-glutathionylation of the death receptor Fas (Fas-SSG) amplifies apoptosis. In the present study we demonstrate that distinct pools of Fas exist in cells. Upon ligation of surface Fas, a separate pool of latent Fas in the ER underwent rapid oxidative processing characterized by loss of free sulfhydryl content (Fas-SH), and resultant increases in Sglutathionylation of Cys 294, leading to increases of surface Fas. Stimulation with FasL rapidly induced associations between Fas, ERp57, and Glutathione S-transferase π (GSTP), a protein disulfide isomerase, and catalyst of S-glutathionylation, respectively, in the ER. Knock down or inhibition of ERp57 and GSTP1 substantially decreased FasL-induced oxidative processing and S-glutathionylation of Fas, resulting in decreased DISC formation, caspase activity and enhanced survival. FasERp57-GSTP1 interactions and S-glutathionylation of Fas were increased in a mouse model of pulmonary fibrosis, which depends on functional Fas. Collectively, these findings illuminate a regulatory switch, ligand-initiated oxidative processing of latent Fas- to control the strength of apoptosis.
119 Myeloperoxidase Aggravates Pulmonary Arterial Hypertension in Mice 1
Anna Klinke , Matti Adam2, Kai Friedrichs1, Tanja Katharina Rudolph1, Thorben Ravekes1, Denise Lau1, Ralph Theo Schermuly3, Volker Rudolph1, and Stephan Baldus1 1 2 University Heart Center Hamburg,Germany, Stanford University 3 School of Medicine, Lung Center University of Giessen, Germany Background: Myeloperoxidase (MPO), a heme protein abundantly expressed by polymorphonuclear neutrophils (PMN) generating reactive oxygen species, has been identified as a powerful prognostic indicator for heart failure and coronary artery disease. We have observed recently, that MPO plasma levels were elevated in patients with pulmonary hypertension with increased MPO concentrations correlating with increased mortality. Here we sought to investigate MPO’s mechanistic impact on pulmonary arterial hypertension (PAH) in mice. Methods and Results: Pulmonary arterial hypertension was -/induced in wild-type C57bl/6J (WT) and MPO-deficient (Mpo ) mice by maintaining the animals under normoxic (21% O2) or hypoxic conditions (10% O2) for either 1 or 4 weeks. Hypoxia led to marked activation of PMN reflected by increased MPO plasma levels in WT mice. Whereas no difference was observed in right -/ventricular systolic pressure (RVPsys) between WT and Mpo mice under normoxic conditions (22.81 ± 1.61 vs. 20.19 ± 3.18 mmHg), the increase in RVPsys under hypoxic conditions in WT -/animals was attenuated in Mpo mice (1 week hypoxia: 33.27 ± 0.76 vs. 31.05 vs. 0.76 mmHg, p<0.05; 4 weeks hypoxia: 41.23 ± 1.81 vs. 30.89 ± 1.31 mmHg, p<0.01). Right ventricular/left ventricular mass ratio, an indicator of RV hypertrophy, was profoundly enhanced in hypoxia-treated WT as compared to Mpo /mice upon 4 weeks (0.372 ± 0.018 vs. 0.317 ± 0.013, p<0.05). Immunoblot analysis of lung tissue revealed markedly increased -/activity of RhoA-kinase in WT but not Mpo mice upon hypoxia. Activation of Rho-kinase is an established pathophysiological component of PAH, which mediates augmented vasoconstriction
of pulmonary arteries. Accordingly, Prostaglandin-F2-alpha induced vasoconstriction of explanted pulmonary arteries was profoundly increased in WT mice upon 1 week of hypoxia as -/compared to Mpo mice (0.89 ± 0.03 vs. 0.67 ± 0.06 mN, p<0.01), whereas the enhanced constriction was attenuated upon administration of the Rho-kinase inhibitor Y-27632 (0.71 ± 0.02 mN, p<0.05). Conclusions: The current data reveal that MPO is causally linked to the formation of PAH via activation of the Rho-kinase pathway. These data not only call for revisiting the role of leukocyte activation in the pathophysiology of PAH but also suggest that MPO may evolve as a novel therapeutic target.
120 PTEN Inactivation by Peroxynitrite Induces Vascular Remodeling in Pulmonary Hypertension Secondary to Left-Heart Failure
Yazhini Ravi1, Karuppaiyah Selvendiran1, Shan K. Naidu1, Periannan 1 Kuppusamy , and Chittoor B. Sai-Sudhakar1 1 Ohio State University Pulmonary hypertension secondary to left-heart failure (LHF-PH; Group 2 PH) originates from LV dysfunction and elevated afterload. The pathogenesis of LHF-PH is characterized by progressive pulmonary vascular remodeling involving smooth muscle cell (SMC) proliferation. However, the mechanism is unknown. We hypothesized that peroxynitrite (PN), a potent oxidant, is a key mediator of SMC proliferation in the pulmonary vasculature associated with left-heart failure. Despite an abundance of reports implicating the involvement of ROS in the pathogenesis of myocardial infarction and heart failure, little is known about the involvement of ROS in the progression of PH associated with heart failure. The objective of this study was to determine the mechanism of involvement of PN in vascular SMC proliferation in LHF-PH. LHF was induced by permanent ligation of LAD coronary artery in rats. Echocardiography and hemodynamic measurements were performed to monitor LHFPH. Histopathology, western-blot, and RT-PCR analyses were used to identify vascular remodeling and key signaling proteins. Human pulmonary artery SMC (PASMC) was used for in vitro studies. In vitro studies demonstrated low doses of PN (0.5 -1 μM) to induce PASMC proliferation. PTEN cDNA and PTEN siRNA transfection studies clearly implicated PTEN in SMC proliferation. Western-blot analysis showed a dose-dependent inactivation of PTEN by PN. LHF-PH was confirmed by significant elevation of pulmonary artery systolic pressure (57% increase vs control) and vascular remodeling at 4 weeks post-LAD ligation. Nitrotyrosine-staining of lung tissues showed a 6-fold increase of PN generation in LHF rats vs control). Activity of PTEN, a dualfunction phosphatase known to modulate cell proliferation, was significantly lower (38% vs control) in lung tissues. Taken together, the results, for the first time, established the involvement of peroxynitrite in vascular remodeling via PTEN inactivation. The results showed the involvement of peroxynitrite and PTEN in the development of LHF-PH and demonstrated a potential therapeutic approach for the management of PH associated with left-heart failure.