On the receptor side, however, hypoxia induces upregulation of RAMP1 and RAMP3, but not RAMP2, Bicalutamide androgen receptor in the rat lung (32), suggesting preferential IMD signaling. Two mature IMD peptides of 47 and 40 amino acids, sharing in mice ~33% sequence homology to AM, were identified in 2004 by Roh et al. (33) and Takei et al. (37). Current knowledge on its vascular and pulmonary effects is limited but points toward a potential beneficial role in pathophysiological conditions: IMD is cardioprotective in ischemia-reperfusion injury (2, 43), its intravenous application decreases blood pressure (1, 14, 15), and it dilates the rat pulmonary vascular bed (5, 23). Hence, we reasoned that IMD might represent an endogenous pulmonary peptide being regulated by hypoxia and contributing to vascular homeostasis under inflammatory and hypoxic conditions.

To test this hypothesis, we conducted experiments to identify hypoxia-responsive elements in the IMD promoter, to characterize hypoxia-induced alterations in IMD expression, and to elucidate the role of IMD in regulating endothelial hyperpermeability. MATERIALS AND METHODS Animals. All experiments conformed to National Institutes of Health guidelines on the care and use of experimental animals and were approved by Regierungsp?sidium Giessen. FVB mice of either sex (n = 4 for mRNA analysis, n = 5 for immunofluorescence) were exposed to normobaric hypoxia [inspired O2 fraction (FiO2) = 0.10] in a ventilated chamber for 15 h. The level of hypoxia was held constant by an autoregulatory control unit (model 4010 O2 controller, Labotec, G?ttingen, Germany).

Control animals (n = 4 for mRNA analysis, n = 4 for immunofluorescence) were exposed to normobaric normoxia in a similar chamber at an FiO2 of 0.21. Antibodies. Monoclonal anti-IMD antibodies were obtained by screening of the MorphoSys Human Combinatorial Antibody Library (HuCAL) GOLD library (AbD Serotec, Martinsried, Germany). In this library, more than 15 billion functional human antibody specificities in Fab format are prefabricated, and they were screened by ELISA for reactivity against a synthetic peptide (RPAGRRDSAPVDPSSPHSY) corresponding to amino acid residues 131�C149 (accession no. “type”:”entrez-protein”,”attrs”:”text”:”NP_891558″,”term_id”:”33620732″,”term_text”:”NP_891558″NP_891558) of mouse IMD. At the same time, Carfilzomib they were tested for nonreactivity to the homologous peptide AM with the synthetic peptide TDKDKDGMAPRNKISPQGY, which corresponds to amino acid residues 126�C144 (accession no. “type”:”entrez-protein”,”attrs”:”text”:”NP_033757″,”term_id”:”6752988″,”term_text”:”NP_033757″NP_033757) of mouse AM.