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While Staphylococcus aureus has classically been considered an extracellular pathogen, these bacteria are also capable of being taken up by host cells, including nonprofessional phagocytes such as endothelial cells, epithelial cells, or osteoblasts. The intracellular S. aureus lifestyle contributes to infection development. The predominant recognition and internalization pathway appears to be the binding of the bacteria via a fibronectin bridge to the α5β1-integrin on the host cell membrane, followed by phagocytosis. Although osteoblasts showed high expression of α5β1-integrin and fibronectin, and bacteria adhered to osteoblasts to a high proportion, here we demonstrate by internalization assays and immunofluorescence microscopy that S. aureus was less engulfed in osteoblasts than in epithelial cells. The addition of exogenous fibronectin during the infection of cells with S. aureus resulted in an increased uptake by epithelial cells but not by osteoblasts. This contrasts with the previous conception of the uptake mechanism, where high expression of integrin and fibronectin would promote the bacterial uptake into host cells. Extracellular fibronectin surrounding osteoblasts, but not epithelial cells, is organized in a fibrillary network. The inhibition of fibril formation, the short interfering RNA-mediated reduction of fibronectin expression, and the disruption of the fibronectin-fibril meshwork all resulted in a significant increase in S. aureus uptake by osteoblasts. Thus, the network of fibronectin fibrils appears to strongly reduce the uptake of S. aureus into a given host cell, indicating that the supramolecular structure of fibronectin determines the capacity of particular host cells to internalize the pathogen. IMPORTANCE Traditionally, Staphylococcus aureus has been considered an extracellular pathogen. However, among other factors, the frequent failure of antimicrobial therapy and the ability of the pathogen to cause recurrent disease have established the concept of eukaryotic invasion of the pathogen, thereby evading the host's immune system. In the current model of host cell invasion, bacteria initially bind to α5β1 integrin on the host cell side via a fibronectin bridge, which eventually leads to phagocytosis of S. aureus by host cells. However, in this study, we demonstrate that not the crude amount but the supramolecular structure of fibronectin molecules deposited on the eukaryotic cell surface plays an essential role in bacterial uptake by host cells. Our findings explain the large differences of S. aureus uptake efficacy in different host cell types as well as in vivo differences between courses of bacterial infections and the localization of bacteria in different clinical settings.

Arteriogenesis is an intricate process in which increased shear stress in pre-existing arteriolar collaterals induces blood vessel expansion, mediated via endothelial cell activation, leukocyte recruitment and subsequent endothelial and smooth muscle cell proliferation. Extracellular RNA (eRNA), released from stressed cells or damaged tissue under pathological conditions, has recently been discovered to be liberated from endothelial cells in response to increased shear stress and to promote collateral growth. Until now, eRNA has been shown to enhance coagulation and inflammation by inducing cytokine release, leukocyte recruitment, and endothelial permeability, the latter being mediated by vascular endothelial growth factor (VEGF) signaling. In the context of arteriogenesis, however, eRNA has emerged as a transmitter of shear stress into endothelial activation, mediating the sterile inflammatory process essential for collateral remodeling, whereby the stimulatory effects of eRNA on the VEGF signaling axis seem to be pivotal. In addition, eRNA might influence subsequent steps of the arteriogenesis cascade as well. This article provides a comprehensive overview of the beneficial effects of eRNA during arteriogenesis, laying the foundation for further exploration of the connection between the damaging and non-damaging effects of eRNA in the context of cardiovascular occlusive diseases and of sterile inflammation.

Fluid shear stress in the vasculature is the driving force for natural bypass growth, a fundamental endogenous mechanism to counteract the detrimental consequences of vascular occlusive disease, such as stroke or myocardial infarction. This process, referred to as "arteriogenesis," relies on local recruitment of leukocytes, which supply growth factors to preexisting collateral arterioles enabling them to grow. Although several mechanosensing proteins have been identified, the series of mechanotransduction events resulting in local leukocyte recruitment is not understood. In a mouse model of arteriogenesis (femoral artery ligation), we found that endothelial cells release RNA in response to increased fluid shear stress and that administration of RNase inhibitor blocking plasma RNases improved perfusion recovery. In contrast, treatment with bovine pancreatic RNase A or human recombinant RNase1 interfered with leukocyte recruitment and collateral artery growth. Our results indicated that extracellular RNA (eRNA) regulated leukocyte recruitment by engaging vascular endothelial growth factor receptor 2 (VEGFR2), which was confirmed by intravital microscopic studies in a murine cremaster model of inflammation. Moreover, we found that release of von Willebrand factor (VWF) as a result of shear stress is dependent on VEGFR2. Blocking VEGFR2, RNase application, or VWF deficiency interfered with platelet-neutrophil aggregate formation, which is essential for initiating the inflammatory process in arteriogenesis. Taken together, the results show that eRNA is released from endothelial cells in response to shear stress. We demonstrate this extracellular nucleic acid as a critical mediator of mechanotransduction by inducing the liberation of VWF, thereby initiating the multistep inflammatory process responsible for arteriogenesis., (© 2019 by The American Society of Hematology.)

Tissue-resident mast cells (MCs) are well known for their role in inflammatory responses and allergic and anaphylactic reactions, but they also contribute to processes of arterial remodeling. Although ribosomes and cytosolic RNAs are located around secretory granules in mature MCs, their functional role in MC responses remains unexplored. Previous studies by our group characterized extracellular RNA (eRNA) as an inflammatory and pathogenetic factor in vitro and in vivo. In the present study, RNA-containing MCs and eRNA were located in close proximity to growing collateral arteries in vivo. In vitro, various agonists were found to induce the degranulation of MCs and the concomitant release of eRNA in association with microvesicles (MVs). The liberation of eRNA from MCs was abolished by MC stabilizers or by preventing the increase of intracellular Ca 2+ in MCs. eRNA was found to be mainly contained inside MVs, as demonstrated by electron microscopy and immunocytochemistry. The exposure to and the uptake of MC-released MVs by cultured endothelial cells increased their expression of cytokines, such as monocyte chemoattractant protein or IL-6, in a dose- and time-dependent manner. These results indicate that RNA-containing MC-derived MVs are likely to be involved in inflammatory responses, relevant, for example, to processes of vascular remodeling.-Elsemüller, A.-K., Tomalla, V., Gärtner, U., Troidl, K., Jeratsch, S., Graumann, J., Baal, N., Hackstein, H., Lasch, M., Deindl, E., Preissner, K. T., Fischer, S. Characterization of mast cell-derived rRNA-containing microvesicles and their inflammatory impact on endothelial cells.

Mechanical forces in blood circulation such as shear stress play a predominant role in many physiological and pathophysiological processes related to vascular responses or vessel remodeling. Arteriogenesis, defined as the growth of pre-existing arterioles into functional collateral arteries compensating for stenosed or occluded arteries, is such a process. Midkine, a pleiotropic protein and growth factor, has originally been identified to orchestrate embryonic development. In the adult organism its expression is restricted to distinct tissues (including tumors), whereby midkine is strongly expressed in inflamed tissue and has been shown to promote inflammation. Recent investigations conferred midkine an important function in vascular remodeling and growth. In this review, we introduce the midkine gene and protein along with its cognate receptors, and highlight its role in inflammation and the vascular system with special emphasis on arteriogenesis, particularly focusing on shear stress-mediated vascular cell proliferation and vasodilatation.

RNA may be released from vascular cells including endothelial cells in the event of injury and in vascular disease. Extracellular RNAs have been recognized as novel procoagulant and permeability-increasing factors. Extracellular RNA may function as inflammatory host alarm signals that serve to amplify the defense mechanism, but it may provide important links to thrombus formation. Extracellular RNA is degraded by RNase. We propose that RNase and its inhibitor RNase inhibitor (RI) act as modulators of homeostasis in the vasculature to control the functions of extracellular RNA. We aimed to investigate the expression and localization of RNase 1 and RI in cells that contact blood, such as platelets, mononuclear cells, polymorphonuclear cells, and red blood cells. RNase 1 and RI expression and localization in blood cells were compared with those in the human umbilical vein endothelial cell line, EAhy926. Additionally, we further investigated the effect of thrombin on the expression of RNase 1 and RI in platelets. We used an RNase activity assay, reverse transcription-polymerase chain reaction, western blot, immunocytochemistry, transmission electron microscopy, and immunoelectron microscopy (pre- and post-embedding methods). RNase activity in the supernatant from EAhy926 cells was 50 times than in blood cells (after 60 min). RNase 1 mRNA and protein expression in EAhy926 cells was highest among the cells examined. However, RI mRNA and protein expression was similar in most cell types examined. Furthermore, we observed that RNase 1 and von Willebrand factor were partially colocalized in EAhy926 cells and platelets. In conclusion, we propose that high RNase activity is ordinarily released from endothelial cells to support anticoagulation in the vasculature. On the other hand, platelets and leukocytes within thrombi at sites of vascular injury show very low RNase activity, which may support hemostatic thrombus formation. However, activated platelets and leukocytes may accelerate pathologic thrombus formation.

Streptococcus pneumoniae is the most frequent cause of community-acquired pneumonia. The infection process involves bacterial cell surface receptors, which interact with host extracellular matrix components to facilitate colonization and dissemination of bacteria. Here, we investigated the role of host-derived extracellular RNA (eRNA) in the process of pneumococcal alveolar epithelial cell infection. Our study demonstrates that eRNA dose-dependently increased S. pneumoniae invasion of alveolar epithelial cells. Extracellular enolase (Eno), a plasminogen (Plg) receptor, was identified as a novel eRNA-binding protein on S. pneumoniae surface, and six Eno eRNA-binding sites including a C-terminal 15 amino acid motif containing lysine residue 434 were characterized. Although the substitution of lysine 434 for glycine (K434G) markedly diminished the binding of eRNA to Eno, the adherence to and internalization into alveolar epithelial cells of S. pneumoniae strain carrying the C-terminal lysine deletion and the mutation of internal Plg-binding motif were only marginally impaired. Accordingly, using a mass spectrometric approach, we identified seven novel eRNA-binding proteins in pneumococcal cell wall. Given the high number of eRNA-interacting proteins on pneumococci, treatment with RNase1 completely inhibited eRNA-mediated pneumococcal alveolar epithelial cell infection. Our data support further efforts to employ RNAse1 as an antimicrobial agent to combat pneumococcal infectious diseases.

The body has the capacity to compensate for an occluded artery by creating a natural bypass upon increased fluid shear stress. How this mechanical force is translated into collateral artery growth (arteriogenesis) is unresolved. We show that extravasation of neutrophils mediated by the platelet receptor GPIbα and uPA results in Nox2-derived reactive oxygen radicals, which activate perivascular mast cells. These c-kit(+)/CXCR-4(+) cells stimulate arteriogenesis by recruiting additional neutrophils as well as growth-promoting monocytes and T cells. Additionally, mast cells may directly contribute to vascular remodeling and vascular cell proliferation through increased MMP activity and by supplying growth-promoting factors. Boosting mast cell recruitment and activation effectively promotes arteriogenesis, thereby protecting tissue from severe ischemic damage. We thus find that perivascular mast cells are central regulators of shear stress-induced arteriogenesis by orchestrating leukocyte function and growth factor/cytokine release, thus providing a therapeutic target for treatment of vascular occlusive diseases., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Different types of high and low molecular weight extracellular RNA (eRNA) are liberated from cells upon conditions of tissue damage or vascular diseases and have been demonstrated in vivo and in vitro to influence the integrity and barrier function of the vascular endothelium. Among the types of self eRNA studied in this respect, ribosomal RNA appears to engage cytokines to promote hyperpermeability, while counteracting RNase1 serves as a potent vessel-protective factor. Different microRNAs may change the expression program of endothelial cells with consequences for cellular contacts and stability. Non-self viral RNAs are recognized by Toll-like receptors that transmit intracellular inflammation signals to disturb the vascular barrier function, largely in connection with infectious diseases. Although derived from the same nucleotide building blocks, the various forms of eRNA exhibit a multitude of molecular interactions with the endothelium that may drastically change its phenotypical characteristics. The impact of eRNA on vascular integrity in health and disease is summarized in this concise review.

Invasive pneumococcal infections due to Streptococcus pneumoniae lead to inflammatory infiltration of leucocytes into lung alveolus, meninges and to septic dissemination within the vascular system. The lung microvasculature is covered by pulmonary endothelial cells containing Weibel-Palade bodies (WPB) releasing procoagulant von Willebrand factor (vWF) and other proteins in response to inflammatory stimuli. The influence of pathogenic pneumococci on secretion of WPB proteins is unknown. Here, we report that adherence of S. pneumoniae to primary human pulmonary microvascular endothelial cells (HPMEC) stimulates the WPB exocytosis and the secretion of vWF and interleukin 8 (IL-8). Moreover, infection analyses performed with pneumococcal mutants deficient in the expression of cytotoxic pneumolysin demonstrated that, in addition to direct bacterial adherence, sublytic concentrations of pneumolysin stimulated vWF secretion. The release of vWF was induced after infection with pneumococci from both the apical and the basal cell surfaces, which implies a stimulation of WPB exocytosis in both directions: from inside the vasculature and also following invasive pneumococcal transmigration from pulmonary tissue into the bloodstream. In conclusion, this study demonstrates that the most relevant pulmonary pathogen S. pneumoniae induces release of proinflammatory and procoagulative components directly contributing to pathophysiological processes leading to fatal tissue injury during course of infection., (© 2011 Blackwell Publishing Ltd.)

Neutrophils play an important role in innate immunity by defending the host organism against invading microorganisms. Antimicrobial activity of neutrophils is mediated by release of antimicrobial peptides, phagocytosis as well as formation of neutrophil extracellular traps (NET). These structures are composed of DNA, histones and granular proteins such as neutrophil elastase and myeloperoxidase. This study focused on the influence of NET on the host cell functions, particularly on human alveolar epithelial cells as the major cells responsible for gas exchange in the lung. Upon direct interaction with epithelial and endothelial cells, NET induced cytotoxic effects in a dose-dependent manner, and digestion of DNA in NET did not change NET-mediated cytotoxicity. Pre-incubation of NET with antibodies against histones, with polysialic acid or with myeloperoxidase inhibitor but not with elastase inhibitor reduced NET-mediated cytotoxicity, suggesting that histones and myeloperoxidase are responsible for NET-mediated cytotoxicity. Although activated protein C (APC) did decrease the histone-induced cytotoxicity in a purified system, it did not change NET-induced cytotoxicity, indicating that histone-dependent cytotoxicity of NET is protected against APC degradation. Moreover, in LPS-induced acute lung injury mouse model, NET formation was documented in the lung tissue as well as in the bronchoalveolar lavage fluid. These data reveal the important role of protein components in NET, particularly histones, which may lead to host cell cytotoxicity and may be involved in lung tissue destruction.

The functions of extracellular RNA in the vascular system as new procoagulatory and permeability-increasing factor in vivo and in vitro were shown to be counteracted by pancreatic type RNase1. Based on the identification of RNase1 in plasma and serum, it is proposed that the enzyme is expressed by vascular cells to contribute in the regulation of extracellular RNA. It is demonstrated that RNase1 and RNase5 (also termed angiogenin) were differentially expressed in various types of endothelial cells, whereby human umbilical vein endothelial cells (HUVEC) expressed and released the highest concentration of active RNase1. Expression and release of RNase5 were similar in all types of endothelial cells tested. Both RNases were constitutively produced and secreted, whereby a portion of RNase1, but not RNase5, was stored in Weibel-Palade bodies, co-localising with von Willlebrand factor and P-selectin. Accordingly, immediate release of RNase1 from these granules was demonstrated in vitro and in vivo using Weibel-Palade body exocytosis-inducing agents. Additionally, extracellular RNA or poly:IC (but not DNA) induced this short-term release of RNase1. Our results indicate that vascular RNase1 and RNase5 are mainly produced by vascular endothelial cells and can serve, depending on the vascular bed, different functions in vascular homeostasis and endothelial cell responses.

Objective: The ubiquitin-proteasome system is the main degradation machinery for intracellularly altered proteins. Hyperglycemia has been shown to increase intracellular levels of the reactive dicarbonyl methylglyoxal (MGO) in cells damaged by diabetes, resulting in modification of proteins and alterations of their function. In this study, the influence of MGO-derived advanced glycation end product (AGE) formation on the activity of the proteasome was investigated in vitro and in vivo., Research Design and Methods: MGO-derived AGE modification of proteasome subunits was analyzed by mass spectrometry, immunoprecipitation, and Western blots. Proteasome activity was analyzed using proteasome-specific fluorogenic substrates. Experimental models included bovine retinal endothelial cells, diabetic Ins2(Akita) mice, glyoxalase 1 (GLO1) knockdown mice, and streptozotocin (STZ)-injected diabetic mice., Results: In vitro incubation with MGO caused adduct formation on several 20S proteasomal subunit proteins. In cultured endothelial cells, the expression level of the catalytic 20S proteasome subunit was not altered but proteasomal chymotrypsin-like activity was significantly reduced. In contrast, levels of regulatory 19S proteasomal proteins were decreased. In diabetic Ins2(Akita), STZ diabetic, and nondiabetic and diabetic G101 knockdown mice, chymotrypsin-like activity was also reduced and MGO modification of the 20S-beta2 subunit was increased., Conclusions: Hyperglycemia-induced formation of MGO covalently modifies the 20S proteasome, decreasing its activity in the diabetic kidney and reducing the polyubiquitin receptor 19S-S5a. The results indicate a new link between hyperglycemia and impairment of cell functions.

Tumor-derived exosomes containing the tetraspanin Tspan8 can efficiently induce angiogenesis in tumors and tumor-free tissues. However, little information exists on exosome-endothelial cell (EC) interactions or the proangiogenic role of tetraspanins, which are a constitutive component of exosomes. In this study, we used a rat adenocarcinoma model (AS-Tspan8) to explore the effects of exosomal Tspan8 on angiogenesis. Tspan8 contributed to a selective recruitment of proteins and mRNA into exosomes, including CD106 and CD49d, which were implicated in exosome-EC binding and EC internalization. We found that EC internalized Tspan8-CD49d complex-containing exosomes. Exosome uptake induced vascular endothelial growth factor (VEGF)-independent regulation of several angiogenesis-related genes, including von Willebrand factor, Tspan8, chemokines CXCL5 and MIF, chemokine receptor CCR1, and, together with VEGF, VEGF receptor 2. EC uptake of Tspan8-CD49d complex-containing exosomes was accompanied by enhanced EC proliferation, migration, sprouting, and maturation of EC progenitors. Unraveling these new pathways of exosome-initiated EC regulation could provide new options for therapeutic interference with tumor-induced angiogenesis.

Extracellular RNA has been shown to induce vascular endothelial growth factor (VEGF)-dependent hyperpermeability in vivo as well as in vitro. Studies were performed to investigate the mechanism of these effects. For permeability studies primary cultures of porcine brain-derived microvascular endothelial cells (BMECs) and for all other analytical studies the human brain endothelial cell line HCMEC/D3 or human umbilical vein endothelial cells (HUVECs) were used. RNA, but not DNA, initiated signaling events by binding of VEGF to neuropilin-1, followed by VEGF-R2 phosphorylation, activation of phospholipase C (PLC), and intracellular release of Ca(2+). Activation of these pathways by RNA also resulted in the release of von Willebrand Factor from Weibel-Palade bodies. Pretreatment of cells with heparinase totally abrogated the RNA-induced permeability changes, whereas RNA together with VEGF completely restored VEGF-R2-mediated hyperpermeability. Although poly:IC increased the interleukin-6 release via activation of toll-like receptor-3 (TLR-3), permeability changes mediated by poly:IC or RNA remained unchanged after blocking TLR-3 or NF-kB activation. These results indicate that extracellular RNA serves an important cofactor function to engage VEGF for VEGF-R2-dependent signal transduction, reminiscent of the coreceptor mechanism mediated by proteoglycans, which might be of relevance for the mobilization and cellular activities of RNA-binding cytokines in general.

Various virus infections cause dysfunctional hemostasis and in some instances lead to the development of viral hemorrhagic fever syndrome. How do diverse viruses induce the expression of tissue factor on vascular cells? We hypothesize that a direct stimulation of pattern recognition receptors (PRR) by viral nucleic acids may be the key. Double-stranded RNA (dsRNA) is produced by many viruses and is recognized by various PRR, including Toll-like receptor-3 (TLR3). We have investigated whether poly I:C, a model for viral dsRNA, can influence cellular hemostasis. Poly I:C could up-regulate tissue factor and down-regulate thrombomodulin expression on endothelial cells but not on monocytes. The response to poly I:C was diminished upon small interfering RNA (siRNA)-mediated inhibition of TLR3, but not other PRR. In vivo, application of poly I:C induced similar changes in the aortic endothelium of mice as determined by enface microscopy. D-dimer, a circulating marker for enhanced coagulation and fibrinolysis, and tissue fibrin deposition was elevated. All the hemostasis-related responses to poly I:C, but not cytokine secretion, were blunted in TLR3(-/-) mice. Hence, the activation of TLR3 can induce the procoagulant state in the endothelium, and this could be relevant for understanding the mechanisms of viral stimulation of hemostasis.

By interacting with components of the human host, including extracellular matrix (ECM) proteins, Streptococcus pneumoniae has evolved various strategies for colonization. Here, we characterized the interaction of pneumococci with the adhesive glycoprotein vitronectin and the contribution of this protein to pneumococcal uptake by host cells in an integrin-dependent manner. Specific interaction of S. pneumoniae with the heparin-binding sites of purified multimeric vitronectin was demonstrated by flow cytometry analysis. Host-cell-bound vitronectin promoted pneumococcal adherence to and invasion into human epithelial and endothelial cells. Pneumococci were trapped by microspike-like structures, which were induced upon contact of pneumococci with host-cell-bound vitronectin. Alphavbeta3 integrin was identified as the major cellular receptor for vitronectin-mediated adherence and uptake of pneumococci. Ingestion of pneumococci by host cells via vitronectin required a dynamic actin cytoskeleton and was dependent on integrin-linked kinase (ILK), phosphatidylinositol 3-kinase (PI3K), and protein kinase B (Akt), as demonstrated by gene silencing or in inhibition experiments. In conclusion, pneumococci exploit the vitronectin-alphavbeta3-integrin complex as a cellular receptor for invasion and this integrin-mediated internalization requires the cooperation between the host signalling molecules ILK, PI3K and Akt.

Aims: Sphingosine-1-phosphate (S1P), a key regulator of vascular homeostasis and angiogenesis, promotes endothelial cell migration via stimulation of phosphoinositide 3-kinase (PI3K). The aim of this study was to identify the role of PI3Kbeta and gamma isoforms and their downstream effector pathways in mediating endothelial cell migration induced by S1P., Methods and Results: Experiments were performed in human umbilical vein endothelial cells (HUVEC) and murine lung endothelial cells (MLEC). A combination of specific inhibitors, RNA interference, and PI3Kgamma(-/-) mice were used to investigate the role of PI3Kbeta and gamma isoforms in endothelial cell migration. Both PI3Kbeta and gamma isoforms are required for full migration induced by S1P, with Rac1 being a major mediator downstream of both isoforms. In addition, PI3Kbeta but not PI3Kgamma mediates migration via Akt but independent of Rac1 and endothelial NO synthase (eNOS). Further, a S1P-mediated activation of extracellular signal-regulated kinases (Erk) 1/2 contributes to the chemotactic response independent of PI3Kbeta or PI3Kgamma., Conclusions: Our data demonstrate that both PI3Kbeta and PI3Kgamma isoforms are required for S1P-induced endothelial cell migration through activation of Rac1. In addition, PI3Kbeta initiates an Akt-sensitive chemotactic response which is independent of Rac1 and eNOS. Thus, PI3Kbeta and PI3Kgamma have both overlapping and distinct roles in regulating endothelial cell migration, which may underlie S1P-triggered angiogenic differentiation.

Cell injury leads to exposure of intracellular material and is associated with increased permeability of vessels in the vicinity of the damage. Here, we demonstrate that natural extracellular RNA as well as artificial RNA (poly-I:C), or single-stranded RNA but not DNA, significantly increased the permeability across brain microvascular endothelial cells in vitro and in vivo. RNA-induced hyperpermeability of tight monolayers of endothelial cells correlated with disintegration of tight junctions and was mediated through vascular endothelial growth factor (VEGF), reminiscent of heparin's activities. Antisense oligonucleotides against VEGF-receptor 2 (VEGF-R2) prevented the permeability-inducing activity of extracellular RNA and heparin completely. Hence, these polyanionic substances can lead to mobilization/stabilization of VEGF with the subsequent activation of VEGF-R2. In accordance with these functional data, strong binding of VEGF as well as other growth factors to RNA was demonstrable. In in vivo rat models of FeCl(3)-induced sinus sagittal is superior thrombosis and stroke/brain edema, pretreatment of animals with RNase (but not DNase) resulted in a significant reduction of vessel occlusion, infarct volume, and prevention of brain edema formation. Together, these results identify extracellular RNA as a novel natural permeability factor, upstream of VEGF, whereas counteracting RNase treatment may serve as new vessel-protective modality.

The extracellular adherence protein (Eap), a broad-spectrum adhesin secreted by Staphylococcus aureus, was previously shown to curb acute inflammatory responses, presumably through its binding to endothelial cell (EC) ICAM-1. Examining the effect of Eap on endothelial function in more detail, we here show that, in addition, Eap functions as a potent angiostatic agent. Concomitant treatment of EC with purified Eap resulted in the complete blockage of the mitogenic and sprouting responses elicited by vascular endothelial growth factor (VEGF)165 or basic fibroblast growth factor (bFGF). Moreover, the induction of tissue factor and decay-accelerating factor were repressed by Eap, as determined by qRT-polymerase chain reaction (qRT-PCR), with a corresponding reduction in Egr-1 protein up-regulation seen. This angiostatic activity was accompanied by a corresponding inhibition in ERK1/2 phosphorylation, while activation of p38 was not affected. Inhibition occurred downstream of tyrosine kinase receptor activation, as comparable effects were seen on TPA-induced ERK1/2 phosphorylation. Similar to previously described angiostatic agents like angiopoietin-1 or the 16-kDa prolactin fragment, Eap blockage of the Ras/Raf/MEK/ERK cascade was localized by pull-down assay at the level of Ras activation. Eap's combined anti-inflammatory and antiangiogenic properties render this bacterial protein not only an important virulence factor during S. aureus infection but open new perspectives for therapeutic applications in pathological neovascularization.