FSAP (Factor VII-activating protease) is a new plasma-derived serine protease with putative dual functions in haemostasis, including activation of coagulation Factor VII and generation of urinary-type plasminogen activator (urokinase). The (auto-)activation of FSAP is facilitated by polyanionic glycosaminoglycans, such as heparin or dextran sulphate, whereas calcium ions stabilize the active form of FSAP. In the present study, extracellular RNA was identified and characterized as a novel FSAP cofactor. The conditioned medium derived from various cell types such as smooth muscle cells, endothelial cells, osteosarcoma cells or CHO (Chinese-hamster ovary) cells contained an acidic factor that initiated (auto-)activation of FSAP. RNase A, but not other hydrolytic enzymes (proteases, glycanases and DNase), abolished the FSAP cofactor activity, which was subsequently isolated by anionexchange chromatography and unequivocally identified as RNA. In purified systems, as well as in plasma, different forms of natural RNA (rRNA, tRNA, viral RNA and artificial RNA) were able to (auto-)activate FSAP into the two-chain enzyme form. The specific binding of FSAP to RNA (but not to DNA) was shown by mobility-shift assays and UV crosslinking, thereby identifying FSAP as a new extracellular RNA-binding protein, the K D estimated to be 170-350 nM. Activation of FSAP occurred through an RNA-dependent template mechanism involving a nucleic acid size of at least 100 nt. In a purified system, natural RNA augmented the FSAP-dependent Factor VII activation severalfold (as shown by subsequent Factor Xa generation), as well as the FSAP-mediated generation of urokinase. Our results provide evidence for the first time that extracellular RNA, present at sites of cell damage or vascular injury, can serve an important as yet unrecognized cofactor function in haemostasis by inducing (auto-)activation of FSAP through a novel surface-dependent mechanism.
Angiogenesis, the growth of new blood vessels, is a complex biological process that is orchestrated by several growth factors and components of the extracellular matrix, including fibronectin (FN) and its receptor the integrin alpha5beta1. Angiogenesis is a critical part of inflammation and wound repair, but the mechanism by which vascular proliferation and migration is regulated by inflammatory cells is not completely understood. We have previously shown that human neutrophil peptides (HNPs), also known as alpha-defensins, which are secreted in high concentrations when neutrophils are activated, bind specifically to FN in the extracellular matrix and inhibit plasminogen activation. Therefore, we asked whether HNPs act as a link between inflammation and angiogenesis. Alpha5beta1-mediated endothelial cell adhesion and migration to FN, both under control conditions and under stimulation by vascular endothelial growth factor (VEGF), were inhibited specifically and in a dose-dependent manner by HNPs, whereas endothelial cell adhesion and migration to other components of the extracellular matrix, such as vitronectin, collagen, or fibrinogen/fibrin were not. Consistent with this finding, HNPs bound to and promoted the binding of fibronectin to alpha5beta1 integrin in arginine-glycine-aspartic acid (RGD)-independent manner. HNPs also completely inhibited VEGF-induced proliferation and induced apoptosis of endothelial cells in a dose-dependent manner. Moreover, HNPs inhibited capillary tube formation in three-dimensional fibrin-matrices as well as neovascularization in vivo in the chicken chorioallantoic membrane assay. Taken together, these data indicate that HNPs can regulate angiogenesis by affecting endothelial cell adhesion and migration in an FN-dependent manner as well as endothelial cell proliferation. These findings provide new insight into the role of inflammatory cells in angiogenesis and might provide a platform for developing a novel class of anti-angiogenesis drugs.
Staphylococcus aureus is a major human pathogen interfering with host-cell functions. Impaired wound healing is often observed in S aureus-infected wounds, yet, the underlying mechanisms are poorly defined. Here, we identify the extracellular adherence protein (Eap) of S aureus to be responsible for impaired wound healing. In a mouse wound-healing model wound closure was inhibited in the presence of wild-type S aureus and this effect was reversible when the wounds were incubated with an isogenic Eap-deficient strain. Isolated Eap also delayed wound closure. In the presence of Eap, recruitment of inflammatory cells to the wound site as well as neovascularization of the wound were prevented. In vitro, Eap significantly reduced intercellular adhesion molecule 1 (ICAM-1)-dependent leukocyte-endothelial interactions and diminished the consequent activation of the proinflammatory transcription factor nuclear factor B (NFB) in leukocytes associated with a decrease in expression of tissue factor. Moreover, Eap blocked ␣ v -integrin-mediated endothelial-cell migration and capillary tube formation, and neovascularization in matrigels in vivo. Collectively, the potent anti-inflammatory and antiangiogenic properties of Eap provide an underlying mechanism that may explain the impaired wound healing in S aureus-infected wounds. Eap may also serve as a lead compound for new antiinflammatory and antiangiogenic therapies in several pathologies. IntroductionStaphylococcus aureus, and especially strains with resistance to antimicrobial agents, is an unabated challenge in communityacquired and nosocomial infections ranging from wound infections or osteomyelitis to life-threatening endocarditis, or septic shock. 1,2 Wound infection with S aureus is frequently associated with impaired healing; yet, interference of S aureus with wound-healing mechanisms is poorly understood. 3,4 S aureus expresses a number of bacterial cell wall-anchored adhesins mediating its adherence to host extracellular matrix (ECM) components. 5 Moreover, S aureus produces and secretes proteins with ECM binding properties, such as coagulase, 6 the extracellular fibrinogen binding protein, 5 as well as Eap, also designated Map (major histocompatibility [MHC] class II analogous protein), a 60-kDa protein with a broad repertoire of interactions to host ECM components. [7][8][9] Recently, we demonstrated direct interactions of Eap with the host adhesive proteins intercellular adhesion molecule 1 (ICAM-1), fibrinogen (FBG), and vitronectin (VN), resulting in the disruption of integrin-dependent leukocyte recruitment in vitro and in vivo, thereby serving as a potent anti-inflammatory factor. 10 While Eap was also shown to exert immunomodulatory actions by interfering with T-cell functions, 11 a possible interference of Eap with host wound healing is not defined.Wound healing is a well-organized sequence of events involving inflammatory, proliferative, and maturation phases. 12,13 (1) The inflammatory phase requires initial recruitment of neutrophils and later of ma...
The receptor for advanced glycation end products (RAGE) is a transmembrane receptor of the Ig superfamily. While vascular RAGE expression is associated with kidney and liver fibrosis, high expression levels of RAGE are found under physiological conditions in the lung. In this study, RAGE expression in idiopathic pulmonary fibrosis was assessed, and the relationship of the receptor to functional changes of epithelial cells and pulmonary fibroblasts in the pathogenesis of the disease was investigated. Significant down-regulation of RAGE was observed in lung homogenate and alveolar epithelial type II cells from patients with idiopathic pulmonary fibrosis, as well as in bleomycin-treated mice, demonstrated by RT-PCR, Western blotting, and immunohistochemistry. In vitro, RAGE down-regulation was provoked by stimulation of primary human lung fibroblasts and A549 epithelial cells with the proinflammatory cytokines, transforming growth factor-beta1 or TNF-alpha. Blockade of RAGE resulted in impaired cell adhesion, and small interfering RNA-induced knockdown of RAGE increased cell proliferation and migration of A549 cells and human primary fibroblast in vitro. These results indicate that RAGE serves a protective role in the lung, and that loss of the receptor is related to functional changes of pulmonary cell types, with the consequences of fibrotic disease.
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