To determine key enzymes enabling treprostinil palmitil (TP) conversion to treprostinil and the main converting sites in the respiratory system. We performed in vitro activity assays to identify lung Show more
To determine key enzymes enabling treprostinil palmitil (TP) conversion to treprostinil and the main converting sites in the respiratory system. We performed in vitro activity assays to identify lung enzymes hydrolyzing TP, and cell-based assays and immunostainings to establish the likely locations within the lung. Lipoprotein lipase (LPL) had greater activity than the other tested lung enzymes. Excess LPL activity was present both in vitro and at the target TP dose in vivo. LPL is likely the key enzyme enabling TP conversion. The rate-limiting step is likely the accessibility of TP and not the enzyme activity. Show less
CD55 or decay accelerating factor (DAF), a ubiquitously expressed glycosylphosphatidylinositol (GPI)-anchored protein, confers a protective threshold against complement dysregulation which is linked t Show more
CD55 or decay accelerating factor (DAF), a ubiquitously expressed glycosylphosphatidylinositol (GPI)-anchored protein, confers a protective threshold against complement dysregulation which is linked to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Since lung fibrosis is associated with downregulation of DAF, we hypothesize that overexpression of DAF in fibrosed lungs will limit fibrotic injury by restraining complement dysregulation. Normal primary human alveolar type II epithelial cells (AECs) exposed to exogenous complement 3a or 5a, and primary AECs purified from IPF lungs demonstrated decreased membrane-bound DAF expression with concurrent increase in the endoplasmic reticulum (ER) stress protein, ATF6. Increased loss of extracellular cleaved DAF fragments was detected in normal human AECs exposed to complement 3a or 5a, and in lungs of IPF patients. C3a-induced ATF6 expression and DAF loss was inhibited using pertussis toxin (an enzymatic inactivator of G-protein coupled receptors), in murine AECs. Treatment with soluble DAF abrogated tunicamycin-induced C3a secretion and ER stress (ATF6 and BiP expression) and restored epithelial cadherin. Bleomycin-injured fibrotic mice subjected to lentiviral overexpression of DAF demonstrated diminished levels of local collagen deposition and complement activation. Further analyses showed diminished release of DAF fragments, as well as reduction in apoptosis (TUNEL and caspase 3/7 activity), and ER stress-related transcripts. Loss-of-function studies using Show less