Alveolar type II epithelial cell (ATII) apoptosis and proliferation of mesenchymal cells are the hallmarks of idiopathic pulmonary fibrosis, a devastating disease of unknown cause characterized by alveolar epithelial injury and progressive fibrosis. We used a mouse model of bleomycin (BLM)-induced lung injury to understand the involvement of p53-mediated changes in urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) levels in the regulation of alveolar epithelial injury. We found marked induction of p53 in ATII cells from mice exposed to BLM. Transgenic mice expressing transcriptionally inactive dominant negative p53 in ATII cells showed augmented apoptosis, whereas those deficient in p53 resisted BLM-induced ATII cell apoptosis. Inhibition of p53 transcription failed to suppress PAI-1 or induce uPA mRNA in BLM-treated ATII cells. ATII cells from mice with BLM injury showed augmented binding of p53 to uPA, uPA receptor (uPAR), and PAI-1 mRNA. p53-binding sequences from uPA, uPAR, and PAI-1 mRNA 3' untranslated regions neither interfered with p53 DNA binding activity nor p53-mediated promoter transactivation. However, increased expression of p53-binding sequences from uPA, uPAR, and PAI-1 mRNA 3' untranslated regions in ATII cells suppressed PAI-1 and induced uPA after BLM treatment, leading to inhibition of ATII cell apoptosis and pulmonary fibrosis. Our findings indicate that disruption of p53-fibrinolytic system cross talk may serve as a novel intervention strategy to prevent lung injury and pulmonary fibrosis.

The endothelial cell protein C receptor (EPCR) functions as an important regulator of the protein C anticoagulant pathway by binding protein C and enhancing activation by the thrombin-thrombomodulin complex. EPCR binds to both protein C and activated protein C (APC) with high affinity. A soluble form of EPCR (sEPCR) circulates in plasma and inhibits APC anticoagulant activity. In this study, we investigate the mechanisms by which sEPCR modulates APC function. Soluble EPCR inhibited the inactivation of factor Va by APC only in the presence of phospholipid vesicles. By using flow cytometric analysis in the presence of 3 mM CaCl 2 and 0.6 mM MgCl 2 , sEPCR inhibited the binding of protein C and APC to phospholipid vesicles (K i ‫؍‬ 40 ؎ 7 and 33 ؎ 4 nM, respectively). Without MgCl 2 , the K i values increased approximately 4-fold. Double label flow cytometric analysis using fluorescein-APC and Texas RedsEPCR indicated that the APC⅐sEPCR complex does not interact with phospholipid vesicles. By using surface plasmon resonance, we found that sEPCR also inhibited binding of protein C to phospholipid in a dose-dependent fashion (K i ‫؍‬ 32 nM). To explore the possibility that sEPCR evokes structural changes in APC, fluorescence spectroscopy studies were performed to monitor sEPCR/Fl-APC interactions. sEPCR binds saturably to Fl-APC (K d ‫؍‬ 27 ؎ 13 nM) with a maximum decrease in Fl-APC fluorescence of 10.8 ؎ 0.6%. sEPCR also stimulated the amidolytic activity of APC toward synthetic substrates. We conclude that sEPCR binding to APC blocks phospholipid interaction and alters the active site of APC.

The effect of phosphatidylethanolamine (PE) on the activity of the factor VIIa-tissue factor complex (fVIIa-TF) has been examined with respect to plasma clotting activity and activation of factor X (fX) in a purified system. Vesicles prepared by relipidating membrane-anchored TF (dcTF; TF1-244, lacking the C-terminal cytoplasmic tail) into phospholipid vesicles containing 6 mol % phosphatidylserine (PS) and increasing levels of PE up to 40 mol % (the balance consisting of phosphatidylcholine) were found to progressively shorten TF-initiated clotting in normal human plasma to levels comparable to those observed using dcTF relipidated with cephalin. The shortened clotting times were at least in part due to the ability of PE-containing membranes to better support the activation of fX by the fVIIa.TF complex, as vesicles with increased PE content yielded progressively higher initial rates of fX activation. Surprisingly, PE substantially altered the sensitivity of fX activation to low levels of PS, yielding near-maximal rates of activation at only 3 mol % PS compared to 15-20 mol % PS required in the absence of PE. The effect of PE was not synergistic with that of PS since PE did not increase fX activation rates at high levels of PS (20 mol %). Examination of the kinetic parameters for fX activation revealed that the majority of the effect of PE was in decreasing the apparent Km for fX.(ABSTRACT TRUNCATED AT 250 WORDS)

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Wound healing is a complex tissue regeneration process that promotes the growth of new tissue to provide the body with the necessary barrier from the outside environment. In the class of non-healing wounds, diabetic wounds, and ulcers, dressing materials that are available clinically (e.g., gels and creams) have demonstrated only a slow improvement with current available technologies. Among all available current technologies, electrospun fibers exhibit several characteristics that may provide novel replacement dressing materials for the above-mentioned wounds. Therefore, in this review, we focus on recent achievements in electrospun drug-eluting fibers for wound healing applications. In particular, we review drug release, including small molecule drugs, proteins and peptides, and gene vectors from electrospun fibers with respect to wound healing. Furthermore, we provide an overview on multifunctional dressing materials based on electrospun fibers, including those that are capable of achieving wound debridement and wound healing simultaneously as well as multi-drugs loading/types suitable for various stages of the healing process. Our review provides important and sufficient information to inform the field in development of fiber-based dressing materials for clinical treatment of non-healing wounds.