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Islet transplantation provides an approach to compensate for loss of insulin-producing cells in patients with type 1 diabetes. However, the intraportal route of transplantation is associated with instant inflammatory reactions to the graft and subsequent islet destruction as well. Although matrix metalloprotease (MMP)-2 and -9 are involved in both remodeling of extracellular matrix and leukocyte migration, their influence on the outcome of islet transplantation has not been characterized. We observed comparable MMP-2 mRNA expressions in control and transplanted groups of mice, whereas MMP-9 mRNA and protein expression levels increased after islet transplantation. Immunostaining for CD11b (Mac-1)-expressing leukocytes (macrophage, neutrophils) and Ly6G (neutrophils) revealed substantially reduced inflammatory cell migration into islet-transplanted liver in MMP-9 knockout recipients. Moreover, gelatinase inhibition resulted in a significant increase in the insulin content of transplanted pancreatic islets and reduced macrophage and neutrophil influx compared with the control group. These results indicate that the increase of MMP-9 expression and activity after islet transplantation is directly related to enhanced leukocyte migration and that early islet graft survival can be improved by inhibiting MMP-9 (gelatinase B) activity. [ABSTRACT FROM AUTHOR]

Rationale: Acute lung injury (ALI) causes high mortality, but its molecular mechanisms and therapeutic options remain ill-defined. Gram-negative bacterial infections are the main cause of ALI, leading to lung neutrophil infiltration, permeability increases, deterioration of gas exchange, and lung damage. Platelets are activated during ALI, but insights into their mechanistic contribution to neutrophil accumulation in the lung are elusive.Objectives: To determine mechanisms of platelet-mediated neutrophil recruitment in ALI.Methods: Interference with platelet-neutrophil interactions using antagonists to P-selectin and glycoprotein IIb/IIIa or a small peptide antagonist disrupting platelet chemokine heteromer formation in mouse models of ALI.Measurements and Main Results: In a murine model of LPS-induced ALI, we uncover important roles for neutrophils and platelets in permeability changes and subsequent lung damage. Furthermore, platelet depletion abrogated lung neutrophil infiltration, suggesting a sequential participation of platelets and neutrophils. Whereas antagonists to P-selectin and glycoprotein IIb/IIIa had no effects on LPS-mediated ALI, antibodies to the platelet-derived chemokines CCL5 and CXCL4 strongly diminished neutrophil eflux and permeability changes. The two chemokines were found to form heteromers in human and murine ALI samples, positively correlating with leukocyte influx into the lung. Disruption of CCL5-CXCL4 heteromers in LPS-, acid-, and sepsis-induced ALI abolished lung edema, neutrophil infiltration, and tissue damage, thereby revealing a causal contribution.Conclusions: Taken together, our data identify a novel function of platelet-derived chemokine heteromers during ALI and demonstrate means for therapeutic interference. [ABSTRACT FROM AUTHOR]

RATIONALE: Low-density lipoprotein receptor-related protein-1 (LRP-1) mediates the endocytic clearance of various proteinases, including matrix metalloproteinases (MMPs). The ectodomain of LRP-1 can be shed from the cell surface, releasing a soluble form of this receptor (sLRP-1), which antagonizes ligand endocytosis by cellular LRP-1. OBJECTIVES: To assess if increased LRP-1 shedding occurs in the lungs of patients with acute respiratory distress syndrome (ARDS) and may lead to the accumulation of MMPs and subsequent tissue injury. METHODS: We determined sLRP-1 levels in bronchoalveolar lavage fluids (BALF) from 46 patients with ARDS and their correlation with MMP concentration and disease severity. In complementary in vitro studies, we investigated the mechanisms underlying the LRP-1 release from the cell surface and its impact on MMP cellular uptake. MEASUREMENTS AND MAIN RESULTS: sLRP-1 levels were significantly elevated in BALF but not in plasma from patients with ARDS compared with control subjects and further increased in the later course of the disease. Baseline BALF sLRP-1 concentration was positively correlated with disease severity and significantly higher in nonsurvivors compared with survivors. The presence of ARDS BALF enhanced LRP-1 shedding from cultured lung fibroblasts but not from alveolar type II cells or macrophages. This process was blocked when ARDS BALF was supplemented with metalloproteinase inhibitor resulting in enhanced cellular uptake and degradation of MMP-2 and -9. Accordingly, sLRP-1 BALF concentration in patients with ARDS was positively correlated with MMP levels and laminin, a marker of basement membrane disruption. CONCLUSIONS: Increased LRP-1 shedding prevents the cellular clearance of MMPs and might thereby contribute to tissue destruction in ARDS lungs. [ABSTRACT FROM AUTHOR]

Rationale: The pivotal role of phosphoinositide 3-kinase gamma (PI3Kgamma) in leukocyte recruitment makes it an attractive target for immunomodulatory therapy. However, interfering with PI3Kgamma signaling might increase the risk of bacterial infections in humans. Objectives: We hypothesized that deletion or pharmacologic inhibition of PI3Kgamma would impair the lung inflammatory response to the prototypic gram-positive bacterial pathogen Streptococcus pneumoniae. Methods: PI3Kgamma knockout (KO) and wild-type mice were infected with S. pneumoniae or challenged with the pneumococcal virulence factor pneumolysin (PLY), and inflammatory leukocyte recruitment, bacterial pathogen elimination, and resolution/repair processes were determined. Measurements and Main Results: PI3Kgamma KO mice challenged with PLY responded with lung edema and neutrophilic alveolitis, but showed a drop in alveolar macrophages and failed to recruit exudate macrophages when compared with wild-type mice. S. pneumoniae-infected PI3Kgamma KO mice and wild-type mice pretreated with the pharmacologic inhibitor AS-605240 recruited similar numbers of neutrophils but substantially fewer exudate macrophages into their lungs than control animals. They also displayed a significantly reduced lung pneumococcal clearance and showed an impaired resolution/repair process, leading to progressive pneumococcal pneumonia. Conclusions: PI3Kgamma gene deletion or pharmacologic inhibition of PI3Kgamma leads to perturbations of critical innate immune responses of the lung to challenge with S. pneumoniae. These data are of clinical relevance for the treatment of chronic inflammatory diseases where pharmacologic inhibition of PI3Kgamma signaling to attenuate effector cell recruitment may have implications for innate immune surveillance of remote organ systems. [ABSTRACT FROM AUTHOR]

Background: Mechanisms of coronary occlusion in ST-elevation acute coronary syndrome (STE-ACS) are poorly understood. We have previously reported accumulation of neutrophils (polymorphonuclear cells [PMNs]) in culprit lesion site thrombi. The goal of the present study was to quantify PMNs, their formation of neutrophil extracellular traps (NETs), and to examine the relationships of extracellular DNA, DNase and clinical outcomes.Methods and Results: We analyzed coronary thrombectomy aspirates from 112 patients undergoing primary percutaneous coronary intervention. Compared to systemic PMNs, coronary thrombus PMNs were characterized by high expression of activation markers and by the formation of aggregates with platelets. Nucleosomes, neutrophil elastase, myeloperoxidase and myeloid-related protein 8/14 were increased in coronary plasma, and NETs significantly contributed to the scaffolds of particulate coronary thrombi. Thrombus NET burden was directly correlated with infarct size, while culprit site DNase activity showed a reverse correlation with infarct size. Recombinant DNase accelerated lysis of coronary thrombi ex vivo.Conclusion: PMNs are highly activated in STE-ACS and undergo NETosis at the culprit lesion site. Coronary NET burden and DNase activity are predictors of myocardial infarct size. [ABSTRACT FROM PUBLISHER]

Despite optimal therapy, the morbidity and mortality of patients presenting with an acute myocardial infarction (MI) remain significant. Extracellular RNA (eRNA), exposed after cell damage, serves as cofactor for coagulation proteases and cytokines thereby promoting their procoagulant and proinflammatory functions in vivo. Following myocardial ischemia/reperfusion (I/R) in mice or I/R induced in the isolated Langendorff heart, increased eRNA levels were found together with cell injury markers. Likewise, eRNA was released from cardiomyocytes under hypoxia and subsequently induced tumor-necrosis-factor-a (TNF-α) liberation by activation of TNF-α converting enzyme (TACE) and provoked cardiomyocyte death. Conversely, TNF-a promoted eRNA release especially under hypoxia, feeding a vicious cell damaging cycle during I/R. Administration of RNase1 or TAPI (TACE-inhibitor) prevented cell death and myocardial infarction. Likewise, RNase1 significantly reduced I/R-mediated energy exhaustion, opening of mitochondrial permeability transition pores (mPTP) as well as oxidative damage in cardiomyocytes. Together, RNase1 as well as inhibition of TACE provide novel therapeutic regimen to interfere with the adverse eRNA-TNF-a interplay and significantly reduce or prevent the pathological outcome of ischemic heart injury. [ABSTRACT FROM PUBLISHER]

Macrophages respond to external stimuli with rapid changes in their expression of many inflammation-related genes to undergo polarization towards the M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotype. This unique property of macrophages allows these cells to modulate chronic inflammatory processes such as atherogenesis. We have previously shown that extracellular RNA (eRNA) can exert prothrombotic and inflammatory properties in the vasculature as a cofactor in protease auto-activation and cytokine mobilization. Given the association of eRNA with macrophages within atherosclerotic lesions, we assessed whether eRNA generated by the macrophages themselves may induce inflammatory responses within macrophages, independent of Toll-like receptor signaling. Recombinant mouse macrophage CSF-driven bone marrow-derived macrophage (BMDM) differentiation was skewed towards the M1 phenotype by exposure of cells to eRNA. This resulted in the overexpression of inflammatory markers such as Tnf-α, Arg2, Il-1β, Il-6, or Ifn-Γ together with Il-12 and iNOS, whereas anti-inflammatory genes such as Il-10 and Il-4 together with Arg1 and macrophage mannose receptor-2 (Cd206) were significantly down-regulated by eRNA. Accordingly, the release of TNF-α and IL-6 proteins into the cell supernatant was significantly elevated by eRNA stimulation. Moreover, the capacity of granulocyte macrophage CSF-driven BMDM differentiation (already representing M1 phenotype) towards further M1 polarization in response to eRNA was moderate. Nevertheless, a significant down-regulation of M2 markers was found. Sialoadhesin-1 (SN-1) is a membrane-anchored basic protein, predominantly expressed on macrophages and involved in cellular interactions via recognition of sialylated glycoconjugates. Interestingly, exposure of SN-1-/- BMDM towards eRNA resulted in prominent down-regulation of pro-inflammatory cytokines together with M1 phenotype markers, while anti-inflammatory cytokines together with M2 phenotype mediators were significantly raised. In accordance with our proposal that eRNA serves as pro-inflammatory "alarm signal" during physiologically distressed conditions, these data may shed light on the role of eRNA and macrophages in chronic inflammatory environment such as atherosclerosis. The self-perpetuating process of atherosclerosis as a largely inflammation-driven pathology may be controlled by SN-1 expression on lesional macrophages as well as the injury-mediated release of eRNA as a master trigger in inflammation. [ABSTRACT FROM PUBLISHER]

Arteriogenesis is an inflammatory driven mechanism, describing the growth of a natural bypass from pre-existing collateral arteries to compensate for an occluded artery. The complement system component C3 is a potent natural inflammatory activator. Here, we investigated its impact on the process of collateral artery growth using C3-deficient (C3 -/-) and wildtype control mice in a murine hindlimb model of arteriogenesis. Induction of arteriogenesis by unilateral femoral artery ligation resulted in decreased perfusion recovery in C3 -/- mice on day 7 as shown by Laser Doppler imaging. Immunofluorescence staining revealed a reduced vascular cell proliferation in C3 -/- mice. Gene expression analysis displayed a significant reduction in monocyte chemoattractant protein-1 (MCP-1) expression in C3 -/- mice. Interestingly, 3 days after induction of arteriogenesis, the number of macrophages (CD68 + ) recruited to growing collaterals was not affected by C3 deficiency. However, a significant reduction in inflammatory M1-like polarized macrophages (CD68 + /MRC1 - ) was noted. Forced mast cell activation by Compound 48/80 as well as exogenous MCP-1 application rescued the number of M1-like polarized macrophages along with perfusion recovery in C3 -/- mice. In summary, this study demonstrates that complement C3 influences arteriogenesis by mediating MCP-1 expression, which is essential for the induction and enhancement of sterile inflammation.

Innate myeloid cells especially neutrophils and their extracellular traps are known to promote intravascular coagulation and thrombosis formation in infections and various other conditions. Innate myeloid cell-dependent fibrin formation can support systemic immunity while its dysregulation enhances the severity of infectious diseases. Less is known about the immune mechanisms preventing dysregulation of fibrin homeostasis in infection. During experimental systemic infections local fibrin deposits in the liver microcirculation cause rapid arrest of CD4+ T cells. Arrested T-helper cells mostly represent Th17 cells that partially originate from the small intestine. Intravascular fibrin deposits activate mouse and human CD4+ T cells which can be mediated by direct fibrin-CD4+ T-cell interactions. Activated CD4+ T cells suppress fibrin deposition and microvascular thrombosis by directly counteracting coagulation activation by neutrophils and classical monocytes. T-cell activation, which is initially triggered by IL-12p40- and MHC-II-dependent mechanisms, enhances intravascular fibrinolysis via LFA-1. Moreover, CD4+ T cells disfavor the association of the thrombin-activatable fibrinolysis inhibitor (TAFI) with fibrin whereby fibrin deposition is increased by TAFI in the absence but not in the presence of T cells. In human infections thrombosis development is inversely related to microvascular levels of CD4+ T cells. Thus, fibrin promotes LFA-1-dependent T-helper cell activation in infections which drives a negative feedback cycle that rapidly restricts intravascular fibrin and thrombosis development.

Tissue ischemia, caused by the blockage of blood vessels, can result in substantial damage and impaired tissue performance. Information regarding the functional contribution of the complement system in the context of ischemia and angiogenesis is lacking. To investigate the influence of complement activation and depletion upon femoral artery ligation (FAL), Cobra venom factor (CVF) (that functionally resembles C3b, the activated form of complement component C3) was applied in mice in comparison to control mice. Seven days after induction of muscle ischemia through FAL, gastrocnemius muscles of mice were excised and subjected to (immuno-)histological analyses. H&E and apoptotic cell staining (TUNEL) staining revealed a significant reduction in ischemic tissue damage in CVF-treated mice compared to controls. The control mice, however, exhibited a significantly higher capillary-to-muscle fiber ratio and a higher number of proliferating endothelial cells (CD31 + /CD45 - /BrdU + ). The total number of leukocytes (CD45 + ) substantially decreased in CVF-treated mice versus control mice. Moreover, the CVF-treated group displayed a shift towards the M2-like anti-inflammatory and regenerative macrophage phenotype (CD68 + /MRC1 + ). In conclusion, our findings suggest that treatment with CVF leads to reduced ischemic tissue damage along with decreased leukocyte recruitment but increased numbers of M2-like polarized macrophages, thereby enhancing tissue regeneration, repair, and healing.

Neurological disorders such as stroke, multiple sclerosis, as well as the neurodegenerative diseases Parkinson's or Alzheimer's disease are accompanied or even powered by danger associated molecular patterns (DAMPs), defined as endogenous molecules released from stressed or damaged tissue. Besides protein-related DAMPs or "alarmins", numerous nucleic acid DAMPs exist in body fluids, such as cell-free nuclear and mitochondrial DNA as well as different species of extracellular RNA, collectively termed as self-extracellular nucleic acids (SENAs). Among these, microRNA, long non-coding RNAs, circular RNAs and extracellular ribosomal RNA constitute the majority of RNA-based DAMPs. Upon tissue injury, necrosis or apoptosis, such SENAs are released from neuronal, immune and other cells predominantly in association with extracellular vesicles and may be translocated to target cells where they can induce intracellular regulatory pathways in gene transcription and translation. The majority of SENA-induced signaling reactions in the brain appear to be related to neuroinflammatory processes, often causally associated with the onset or progression of the respective disease. In this review, the impact of the diverse types of SENAs on neuroinflammatory and neurodegenerative diseases will be discussed. Based on the accumulating knowledge in this field, several specific antagonistic approaches are presented that could serve as therapeutic interventions to lower the pathological outcome of the indicated brain disorders., (© 2023. National Science Council of the Republic of China (Taiwan).)

Atherosclerosis is a chronic, inflammatory disease of the vessel wall where triggered immune cells bind to inflamed endothelium, extravasate and sustain local inflammation. Leukocyte adhesion and extravasation are mediated by adhesion molecules expressed by activated endothelial cells, like intercellular adhesion molecule 1 (ICAM-1). Extracellular adherence protein (Eap) from Staphylococcus aureus binds to a plethora of extracellular matrix proteins, including ICAM-1 and its ligands macrophage-1 antigen (Mac-1, α M β 2 ) and lymphocyte function-associated antigen 1 (LFA-1, α L β 2 ), thereby disrupting the interaction between leukocytes and endothelial cells. We aimed to use Eap to inhibit the interaction of leukocytes with activated endothelial cells in settings of developing and established atherosclerosis in apolipoprotein E (ApoE) deficient mice on high-fat diet. In developing atherosclerosis, Eap treatment reduced circulating platelet-neutrophil aggregates as well as infiltration of T cells and neutrophils into the growing plaque, accompanied by reduced formation of neutrophil extracellular traps (NETs). However, plaque size did not change. Intervention treatment with Eap of already established plaques did not result in cellular or morphological plaque changes, whereas T cell infiltration was increased and thereby again modulated by Eap. We conclude that although Eap leads to cellular changes in developing plaques, clinical implications might be limited as patients are usually treated at a more advanced stage of disease progression. Hence, usage of Eap might be an interesting mechanistic tool for cellular infiltration during plaque development in basic research but not a clinical target., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Upon microbial infections with the subsequent host response of innate immunity, a variety of fragmented RNA- and DNA-based "Pathogen-associated molecular patterns" (PAMPs) are recognized mainly by endosomal or cytoplasmic host cell "Pattern recognition receptors" (PRRs), particularly "Toll-like receptors" (TLRs). Concomitantly, various self-extracellular RNA species (exRNAs) are present in extracellular body fluids where they contribute to diverse physiological and homeostatic processes. In principle, such exRNAs, including the most abundant one, ribosomal exRNA (rexRNA), are designated as "Danger-associated molecular patterns" (DAMPs) and are prevented by e.g. natural modifications from uncontrolled signaling via TLRs to avoid hyper-inflammatory responses or autoimmunity. Upon cellular stress or tissue damage/necrosis, the levels and composition of released self-exRNA species, either in free form, in complex with proteins or in association with extracellular vesicles (EVs), can change considerably. Among the self-exRNAs, rexRNA is considered as a non-typical DAMP, since it may induce inflammatory responses by cell membrane receptors, both in the absence or presence of PAMPs. Yet, its mode of receptor activation to mount inflammatory responses remains obscure. RexRNA also serves as a universal damaging factor in cardiovascular and other diseases independent of PRRs. In general, RNase1 provides a profound antagonist in these pathologies and in rexRNA-mediated inflammatory cell responses. Based on the extrapolation of the here described aspects of rexRNA-biology, further activities of this molecular entity are hypothesized that may stimulate additional research in this area., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Arteriogenesis, the growth of natural bypass blood vessels, can compensate for the loss of arteries caused by vascular occlusive diseases. Accordingly, it is a major goal to identify the drugs promoting this innate immune system-driven process in patients aiming to save their tissues and life. Here, we studied the impact of the Cobra venom factor (CVF), which is a C3-like complement-activating protein that induces depletion of the complement in the circulation in a murine hind limb model of arteriogenesis. Arteriogenesis was induced in C57BL/6J mice by femoral artery ligation (FAL). The administration of a single dose of CVF (12.5 µg) 24 h prior to FAL significantly enhanced the perfusion recovery 7 days after FAL, as shown by Laser Doppler imaging. Immunofluorescence analyses demonstrated an elevated number of proliferating (BrdU + ) vascular cells, along with an increased luminal diameter of the grown collateral vessels. Flow cytometric analyses of the blood samples isolated 3 h after FAL revealed an elevated number of neutrophils and platelet-neutrophil aggregates. Giemsa stains displayed augmented mast cell recruitment and activation in the perivascular space of the growing collaterals 8 h after FAL. Seven days after FAL, we found more CD68 + /MRC-1 + M2-like polarized pro-arteriogenic macrophages around growing collaterals. These data indicate that a single dose of CVF boosts arteriogenesis by catalyzing the innate immune reactions, relevant for collateral vessel growth.

Self-extracellular RNA (eRNA), which is released under pathological conditions from damaged tissue, has recently been identified as a new alarmin and synergistic agent together with toll-like receptor (TLR)2 ligands to induce proinflammatory activities of immune cells. In this study, a detailed investigation of these interactions is reported. The macrophage cell line J774 A.1 or C57 BL/6 J wild-type mice were treated with 18S rRNA and different TLR2 agonists. Gene and protein expression of tumor necrosis factor (Tnf)-α ; interleukin (Il)-1β, Il-6 ; or monocyte chemoattractant protein (Mcp)-1 were analyzed and furthermore in vitro binding studies to TLR2 were performed. The TLR2/TLR6-agonist Pam 2 CSK 4 (Pam2) together with 18S rRNA significantly increased the mRNA expression of inflammatory genes and the release of TNF-α from macrophages in a TLR2- and nuclear factor kappa B (NF-κB)-dependent manner. The injection of 18S rRNA/Pam2 into mice increased the cytokine levels of TNF-α, IL-6, and MCP-1 in the peritoneal lavage. Mechanistically, 18S rRNA built complexes with Pam2 and thus enhanced the affinity of Pam2 to TLR2. These results indicate that the alarmin eRNA, mainly consisting of rRNA, sensitizes TLR2 to enhance the innate immune response under pathological conditions. Thus, rRNA might serve as a new target for the treatments of bacterial and viral infections.

The high incidence of thrombotic events suggests a possible role of the contact system pathway in COVID-19 pathology. In this study, we determined the altered levels of factor XII (FXII) and its activation products in critically ill patients with COVID-19 in comparison with patients with severe acute respiratory distress syndrome related to the influenza virus (acute respiratory distress syndrome [ARDS]-influenza). Compatible with those data, we found rapid consumption of FXII in COVID-19 but not in ARDS-influenza plasma. Interestingly, the lag phase in fibrin formation, triggered by the FXII activator kaolin, was not prolonged in COVID-19, as opposed to that in ARDS-influenza. Confocal and electron microscopy showed that increased FXII activation rate, in conjunction with elevated fibrinogen levels, triggered formation of fibrinolysis-resistant, compact clots with thin fibers and small pores in COVID-19. Accordingly, clot lysis was markedly impaired in COVID-19 as opposed to that in ARDS-influenza. Dysregulated fibrinolytic system, as evidenced by elevated levels of thrombin-activatable fibrinolysis inhibitor, tissue-plasminogen activator, and plasminogen activator inhibitor-1 in COVID-19 potentiated this effect. Analysis of lung tissue sections revealed widespread extra- and intravascular compact fibrin deposits in patients with COVID-19. A compact fibrin network structure and dysregulated fibrinolysis may collectively contribute to a high incidence of thrombotic events in COVID-19., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Objective: Astrocytes participate in the local innate immune response of the central nervous system. In response to stress such as ischemia, activated cells release endogenous factors known as damage-associated molecular patterns (DAMPs). Self-extracellular RNA (eRNA) is such a ubiquitous alarm signal. However, it is unclear whether eRNA is involved in the early acute phase of cerebral ischemia and is sufficient to sensitize astrocytes towards a DAMP or PAMP (pathogen-associated molecular pattern) reaction., Methods: Pro-inflammatory activation upon eRNA stimulation was characterized in primary murine astrocyte cultures. In vivo, an experimental stroke model was used to localize and quantify eRNA in murine brain sections. Using primary cortical neurons and the mouse hippocampal neuronal cell line HT-22, neuronal RNA release upon stress conditions related to cerebral hypoxia/ischemia was analyzed., Results: While low-dose eRNA alone did not promote pro-inflammatory activation of astrocytes in culture, it strongly enhanced the expression of pro-inflammatory cytokines in the presence of either Pam2CSK4, a synthetic PAMP molecule that mimics bacterial infection, or high mobility group box 1 (HMGB1), a prominent DAMP. Synergism of eRNA/Pam2CSK4 and eRNA/HMGB1 was prevented by blockage of the astroglial toll-like receptor (TLR)-2. Inhibition of NF-κB- and mitogen-activated protein kinase-dependent signaling pathways hampered eRNA/Pam2CSK4-mediated pro-inflammatory activation of astrocytes. In vivo, the amount of non-nuclear, presumably extracellular ribosomal RNA in close proximity to neurons significantly accumulated across the infarct core and peri-infarct areas that was accompanied by transcriptional up-regulation of various pro-inflammatory factors. Accordingly, the exposure of neurons to hypoxic/ischemic stress in vitro resulted in the release of eRNA, partly mediated by active cellular processes dependent on the cytosolic calcium level., Conclusion: The DAMP signal eRNA can sensitize astrocytes as active players in cerebral innate immunity towards exogenous and endogenous activators of inflammation (PAMPs and DAMPs) in a synergistic manner via TLR2-NF-κB-dependent signaling mechanisms. These findings provide new insights into the pathogenesis of ischemic stroke and other inflammatory neurological disorders. Further studies will clarify whether administration of RNase in vivo may serve as an effective treatment for inflammatory brain pathologies., (© 2021. The Author(s).)

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.

Extracellular Cold-inducible RNA-binding protein (eCIRP), a damage-associated molecular pattern, is released from cells upon hypoxia and cold-stress. The overall absence of extra- and intracellular CIRP is associated with increased angiogenesis, most likely induced through influencing leukocyte accumulation. The aim of the present study was to specifically characterize the role of eCIRP in ischemia-induced angiogenesis together with the associated leukocyte recruitment. For analyzing eCIRPs impact, we induced muscle ischemia via femoral artery ligation (FAL) in mice in the presence or absence of an anti-CIRP antibody and isolated the gastrocnemius muscle for immunohistological analyses. Upon eCIRP-depletion, mice showed increased capillary/muscle fiber ratio and numbers of proliferating endothelial cells (CD31 + /CD45 - /BrdU + ). This was accompanied by a reduction of total leukocyte count (CD45 + ), neutrophils (MPO + ), neutrophil extracellular traps (NETs) (MPO + CitH3 + ), apoptotic area (ascertained via TUNEL assay), and pro-inflammatory M1-like polarized macrophages (CD68 + /MRC1 - ) in ischemic muscle tissue. Conversely, the number of regenerative M2-like polarized macrophages (CD68 + /MRC1 + ) was elevated. Altogether, we observed that eCIRP depletion similarly affected angiogenesis and leukocyte recruitment as described for the overall absence of CIRP. Thus, we propose that eCIRP is mainly responsible for modulating angiogenesis via promoting pro-angiogenic microenvironmental conditions in muscle ischemia.

Platelet activation and thrombus formation have been implicated to be detrimental for intraportal pancreatic islet transplants. The platelet-specific collagen receptor glycoprotein VI (GPVI) plays a key role in thrombosis through cellular activation and the subsequent release of secondary mediators. In aggregometry and in a microfluidic dynamic assay system modeling flow in the portal vein, pancreatic islets promoted platelet aggregation and triggered thrombus formation, respectively. While platelet GPVI deficiency did not affect the initiation of these events, it was found to destabilize platelet aggregates and thrombi in this process. Interestingly, while no major difference was detected in early thrombus formation after intraportal islet transplantation, genetic GPVI deficiency or acute anti-GPVI treatment led to an inferior graft survival and function in both syngeneic mouse islet transplantation and xenogeneic human islet transplantation models. These results demonstrate that platelet GPVI signaling is indispensable in stable thrombus formation induced by pancreatic islets. GPVI deficiency resulted in thrombus destabilization and inferior islet engraftment indicating that thrombus formation is necessary for a successful intraportal islet transplantation in which platelets are active modulators., (© 2020 The Authors. American Journal of Transplantation published by Wiley Periodicals LLC on behalf of The American Society of Transplantation and the American Society of Transplant Surgeons.)

Acute pulmonary embolism generally resolves within 6 months. However, if the thrombus is infected, venous thrombi transform into fibrotic vascular obstructions leading to chronic deep vein thrombosis and/or chronic thromboembolic pulmonary hypertension (CTEPH), but precise mechanisms remain unclear. Neutrophils are crucial in sequestering pathogens; therefore, we investigated the role of neutrophil extracellular traps (NETs) in chronic thrombosis. Because chronic pulmonary thrombotic obstructions are biologically identical to chronic deep venous thrombi, the murine inferior vena cava ligation model was used to study the transformation of acute to chronic thrombus. Mice with staphylococcal infection presented with larger thrombi containing more neutrophils and NETs but less resolution. Targeting NETs with DNase1 diminished fibrosis and promoted thrombus resolution. For translational studies in humans, we focused on patients with CTEPH, a severe type of deep venous and pulmonary artery fibrotic obstruction after thrombosis. Neutrophils, markers of neutrophil activation, and NET formation were increased in CTEPH patients. NETs promoted the differentiation of monocytes to activated fibroblasts with the same cellular phenotype as fibroblasts from CTEPH vascular occlusions. RNA sequencing of fibroblasts isolated from thrombo-endarterectomy specimens and pulmonary artery biopsies revealed transforming growth factor-β (TGF-β) as the central regulator, a phenotype which was replicated in mice with fibroblast-specific TGF-β overactivity. Our findings uncover a role of neutrophil-mediated inflammation to enhance TGF-β signaling, which leads to fibrotic thrombus remodeling. Targeting thrombus NETs with DNases may serve as a new therapeutic concept to treat thrombosis and prevent its sequelae., (© 2021 by The American Society of Hematology.)

In addition to nucleosomes, chromatin contains non-histone chromatin-associated proteins, of which the high-mobility group proteins are the most abundant. Chromatin-mediated regulation of transcription involves DNA methylation and histone modifications. However, the order of events and the precise function of high-mobility group proteins during transcription initiation remain unclear. Here we show that high-mobility group AT-hook 2 protein (HMGA2) induces DNA nicks at the transcription start site, which are required by the histone chaperone FACT complex to incorporate nucleosomes containing the histone variant H2A.X. Further, phosphorylation of H2A.X at S139 (γ-H2AX) is required for repair-mediated DNA demethylation and transcription activation. The relevance of these findings is demonstrated within the context of TGFB1 signaling and idiopathic pulmonary fibrosis, suggesting therapies against this lethal disease. Our data support the concept that chromatin opening during transcriptional initiation involves intermediates with DNA breaks that subsequently require DNA repair mechanisms to ensure genome integrity.

Upon vascular injury, tissue damage, ischemia, or microbial infection, intracellular material such as nucleic acids and histones is liberated and comes into contact with the vessel wall and circulating blood cells. Such "Danger-associated molecular patterns" (DAMPs) may thus have an enduring influence on the inflammatory defense process that involves leukocyte recruitment and wound healing reactions. While different species of extracellular RNA (exRNA), including microRNAs and long non-coding RNAs, have been implicated to influence inflammatory processes at different levels, recent in vitro and in vivo work has demonstrated a major impact of ribosomal exRNA as a prominent DAMP on various steps of leukocyte recruitment within the innate immune response. This includes the induction of vascular hyper-permeability and vasogenic edema by exRNA via the activation of the "vascular endothelial growth factor" (VEGF) receptor-2 system, as well as the recruitment of leukocytes to the inflamed endothelium, the M1-type polarization of inflammatory macrophages, or the role of exRNA as a pro-thrombotic cofactor to promote thrombosis. Beyond sterile inflammation, exRNA also augments the docking of bacteria to host cells and the subsequent microbial invasion. Moreover, upon vessel occlusion and ischemia, the shear stress-induced release of exRNA initiates arteriogenesis (i.e., formation of natural vessel bypasses) in a multistep process that resembles leukocyte recruitment. Although exRNA can be counteracted for by natural circulating RNase1, under the conditions mentioned, only the administration of exogenous, thermostable, non-toxic RNase1 provides an effective and safe therapeutic regimen for treating the damaging activities of exRNA. It remains to be investigated whether exRNA may also influence viral infections (including COVID-19), e.g., by supporting the interaction of host cells with viral particles and their subsequent invasion. In fact, as a consequence of the viral infection cycle, massive amounts of exRNA are liberated, which can provoke further tissue damage and enhance virus dissemination. Whether the application of RNase1 in this scenario may help to limit the extent of viral infections like COVID-19 and impact on leukocyte recruitment and emigration steps in immune defense in order to limit the extent of associated cardiovascular diseases remains to be studied., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Preissner, Fischer and Deindl.)

Objective: Arteriogenesis, describing the process of collateral artery growth, is activated by fluid shear stress (FSS). Since this vascular mechanotransduction may involve microRNAs (miRNAs), we investigated the FSS-induced expression of vascular cell miRNAs and their functional impact on collateral artery growth during arteriogenesis. Approach and Results: To this end, rats underwent femoral artery ligation and arteriovenous anastomosis to increase collateral blood flow to maximize FSS and trigger collateral vessel remodeling. Five days after surgery, a miRNA expression profile was obtained from collateral tissue, and upregulation of 4 miRNAs (miR-24-3p, miR-143-3p, miR-146a-5p, and miR-195-5p) was verified by quantitative polymerase chain reaction. Knockdown of miRNAs at the same time of the surgery in an in vivo mouse ligation and recovery model demonstrated that inhibition of miR-143-3p only severely impaired blood flow recovery due to decreased arteriogenesis. In situ hybridization revealed distinct localization of miR-143-3p in the vessel wall of growing collateral arteries predominantly in smooth muscle cells. To investigate the mechanotransduction of FSS leading to the increased miR-143-3p expression, cultured endothelial cells were exposed to FSS. This provoked the expression and release of TGF-β (transforming growth factor-β), which increased the expression of miR-143-3p in smooth muscle cells in the presence of SRF (serum response factor) and myocardin. COL5A2 (collagen type V-α2)-a target gene of miR-143-3p predicted by in silico analysis-was found to be downregulated in growing collaterals., Conclusions: These results indicate that the increased miR-143-3p expression in response to FSS might contribute to the reorganization of the extracellular matrix, which is important for vascular remodeling processes, by inhibiting collagen V-α2 biosynthesis.

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.)

The discovery of extracellular RNA (exRNA) has shifted our understanding of the role of RNA in complex cellular functions such as cell-to-cell communication and a variety of pathologies. ExRNAs constitute a heterogenous group of RNAs ranging from small (such as microRNAs) and long non-coding to coding RNAs or ribosomal RNAs. ExRNAs can be liberated from cells in a free form or bound to proteins as well as in association with microvesicles (MVs), exosomes, or apoptotic bodies. Their composition and quantity depend heavily on the cellular or non-cellular component, the origin, and the RNA species being investigated; ribosomal RNA provides the majority of exRNA and miRNAs are predominantly associated with exosomes or MVs. Several studies showed that ribosomal exRNA (rexRNA) constitutes a proinflammatory and prothrombotic alarmin. It is released by various cell types upon inflammatory stimulation and by damaged cells undergoing necrosis or apoptosis and contributes to innate immunity responses. This exRNA has the potential to directly promote the release of cytokines such as tumor necrosis factor factor-α (TNF-α) or interleukin-6 from immune cells, thereby leading to a proinflammatory environment and promoting cardiovascular pathologies. The potential role of exRNA in different pathologies of the central nervous system (CNS) has become of increasing interest in recent years. Although various exRNA species including both ribosomal exRNA as well as miRNAs have been associated with CNS pathologies, their precise roles remain to be further elucidated. In this review, the different entities of exRNA and their postulated roles in CNS pathologies including tumors, vascular pathologies and neuroinflammatory diseases will be discussed. Furthermore, the potential role of exRNAs as diagnostic markers for specific CNS diseases will be outlined, as well as possible treatment strategies addressing exRNA inhibition or interference., (Copyright © 2019 Tielking, Fischer, Preissner, Vajkoczy and Xu.)

Acute obstruction of a coronary artery causes myocardial ischaemia and if prolonged, may result in an ST-segment elevation myocardial infarction (STEMI). First-line treatment involves rapid reperfusion. However, a highly dynamic and co-ordinated inflammatory response is rapidly mounted to repair and remove the injured cells which, paradoxically, can further exacerbate myocardial injury. Furthermore, although cardiac remodelling may initially preserve some function to the heart, it can lead over time to adverse remodelling and eventually heart failure. Since the size of the infarct corresponds to the subsequent risk of developing heart failure, it is important to find ways to limit initial infarct development. In this review, we focus on the role of the innate immune system in the acute response to ischaemia-reperfusion (IR) and specifically its contribution to cell death and myocardial infarction. Numerous danger-associated molecular patterns are released from dying cells in the myocardium, which can stimulate pattern recognition receptors including toll like receptors and NOD-like receptors (NLRs) in resident cardiac and immune cells. Activation of the NLRP3 inflammasome, caspase 1, and pyroptosis may ensue, particularly when the myocardium has been previously aggravated by the presence of comorbidities. Evidence will be discussed that suggests agents targeting innate immunity may be a promising means of protecting the hearts of STEMI patients against acute IR injury. However, the dosing and timing of such agents should be carefully determined because innate immunity pathways may also be involved in cardioprotection. This article is part of a Cardiovascular Research Spotlight Issue entitled 'Cardioprotection Beyond the Cardiomyocyte', and emerged as part of the discussions of the European Union (EU)-CARDIOPROTECTION Cooperation in Science and Technology (COST) Action, CA16225., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.)

During an ST-elevation myocardial infarction (STEMI), the myocardium undergoes a prolonged period of ischaemia. Reperfusion therapy is essential to minimize cardiac injury but can paradoxically cause further damage. Experimental procedures to limit ischaemia and reperfusion (IR) injury have tended to focus on the cardiomyocytes since they are crucial for cardiac function. However, there is increasing evidence that non-cardiomyocyte resident cells in the heart (as discussed in a separate review in this Spotlight series) as well as circulating cells and factors play important roles in this pathology. For example, erythrocytes, in addition to their main oxygen-ferrying role, can protect the heart from IR injury via the export of nitric oxide bioactivity. Platelets are well-known to be involved in haemostasis and thrombosis, but beyond these roles, they secrete numerous factors including sphingosine-1 phosphate (S1P), platelet activating factor, and cytokines that can all strongly influence the development of IR injury. This is particularly relevant given that most STEMI patients receive at least one type of platelet inhibitor. Moreover, there are large numbers of circulating vesicles in the blood, including microvesicles and exosomes, which can exert both beneficial and detrimental effects on IR injury. Some of these effects are mediated by the transfer of microRNA (miRNA) to the heart. Synthetic miRNA molecules may offer an alternative approach to limiting the response to IR injury. We discuss these and other circulating factors, focussing on potential therapeutic targets relevant to IR injury. Given the prevalence of comorbidities such as diabetes in the target patient population, their influence will also be discussed. This article is part of a Cardiovascular Research Spotlight Issue entitled 'Cardioprotection Beyond the Cardiomyocyte', and emerged as part of the discussions of the European Union (EU)-CARDIOPROTECTION Cooperation in Science and Technology (COST) Action, CA16225., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.)

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and highly lethal lung disease with unknown etiology and poor prognosis. IPF patients die within 2 years after diagnosis mostly due to respiratory failure. Current treatments against IPF aim to ameliorate patient symptoms and to delay disease progression. Unfortunately, therapies targeting the causes of or reverting IPF have not yet been developed. Here we show that reduced levels of miRNA lethal 7d (MIRLET7D) in IPF compromise epigenetic gene silencing mediated by the ribonucleoprotein complex MiCEE. In addition, we find that hyperactive EP300 reduces nuclear HDAC activity and interferes with MiCEE function in IPF. Remarkably, EP300 inhibition reduces fibrotic hallmarks of in vitro (patient-derived primary fibroblast), in vivo (bleomycin mouse model), and ex vivo (precision-cut lung slices, PCLS) IPF models. Our work provides the molecular basis for therapies against IPF using EP300 inhibition.

To establish a method that allows for the reliable assessment of vascular endothelial growth factor (VEGF-A) concentrations in very small blood samples of preterm infants. Systemic VEGF measurements are important in view of the most appropriate Anti-VEGF drug to be used for the treatment of acute retinopathy of prematurity (ROP). Cord blood samples from preterm (n = 6) infants, blood samples from preterm infants with treatment requiring ROP (n = 12), and blood samples from healthy adults (n = 10) were collected. Serum, citrate plasma, and serum from recalcified citrate blood were obtained. Levels of VEGF-A and platelet factor-4 (PF-4) were quantified by ELISA or AlphaLISA immunoassay. VEGF-A levels could be detected by both assays, with the AlphaLISA generating slightly lower levels in healthy adults, but not in cord blood of preterm infants. In plasma samples, VEGF levels ranged from non detectable to 181 pg/ml. PF-4 concentrations were between 0.16-3.88 µg/ml. Values of VEGF-A and PF-4 in serum and recalcified serum were significantly higher compared to plasma through the release of these cytokines after platelet activation. In plasma samples of infants with ROP, VEGF-A could always be detected and its values ranged from 19.50 to 245.91 pg/ml and PF-4 concentrations were between 0.1 and 3.3 µg/ml. Using the AlphaLISA kit, we were able to detect VEGF in small sample volumes (5 µl plasma or serum/well) in premature infants with treatment requiring ROP and to monitor platelet activation by PF-4 detection. Minimal blood probe volumes reduce phlebotomy losses avoiding the risk of iatrogenic anemia, thus allowing close monitoring of the cytokine levels in these very fragile infants.

The main strategies used by pathogenic bacteria to infect eukaryotic tissue include their adherence to cells and the extracellular matrix (ECM), the subsequent colonization and invasion as well as the evasion of immune defences. A variety of structurally and functionally characterized adhesins and binding proteins of gram-positive bacteria facilitate these processes by specifically recognizing and interacting with various components of the host ECM, including different collagens, fibronectin and other macromolecules. The ECM affects the cellular physiology of our body and is critical for adhesion, migration, proliferation, and differentiation of many host cell types, but also provides the support for infiltrating pathogens, particularly under conditions of injury and trauma. Moreover, microbial binding to a variety of adhesive components in host tissue fluids leads to structural and/or functional alterations of host proteins and to the activation of cellular mechanisms that influence tissue and cell invasion of pathogens. Since the diverse interactions of gram-positive bacteria with the ECM represent important pathogenicity mechanisms, their characterization not only allows a better understanding of microbial invasion but also provides clues for the design of novel therapeutic strategies to manage infectious diseases.

Background: Sialic acids represent common terminal residues on numerous mammalian glycoconjugates, thereby influencing e.g. lumen formation in developing blood vessels. Interestingly, besides monosialylated also polysialylated glycoconjugates are produced by endothelial cells. Polysialic acid (polySia) is formed in several organs during embryonal and postnatal development influencing, for instance, cell migration processes. Furthermore, the function of cytokines like basic fibroblast growth factor (bFGF) is modulated by polySia., Results: In this study, we demonstrated that human umbilical vein endothelial cells (HUVEC) also secrete polysialylated glycoconjugates. Furthermore, an interaction between polySia and vascular endothelial growth factor (VEGF) was observed. VEGF modulates like bFGF the migration of HUVEC. Since both growth factors interact with polySia, we examined, if polySia modulates the migration of HUVEC. To this end scratch assays were performed showing that the migration of HUVEC is stimulated, when polySia was degraded., Conclusions: Since polySia can interact with bFGF as well as VEGF and the degradation of polySia resulted in an increased cell migration capacity in the applied scratch assay, we propose that polySia may trap these growth factors influencing their biological activity. Thus, polySia might also contribute to the fine regulation of physiological processes in 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.

One of the primary therapeutic goals of modern cardiology is to design strategies aimed at minimizing myocardial infarct size and optimizing cardiac function following acute myocardial infarction (AMI). Patients with AMI who underwent reperfusion therapy display dysfunction of the coronary endothelium. Consequently, ischemic endothelial cells become more permeable and weaken their natural anti-thrombotic and anti-inflammatory potential. Ischemia-reperfusion injury (IRI) is associated with activation of the humoral and cellular components of the hemostatic and innate immune system, and also with excessive production of reactive oxygen species (ROS), the inhibition of nitric oxide synthase, and with inflammatory processes. Given its essential role in the regulation of vascular homeostasis, involving platelets and leukocytes among others, dysfunctional endothelium can lead to increased risk of coronary vasospasm and thrombosis. Endothelial dysfunction can be prevented by ischemic conditioning with a protective intervention based on limited intermittent periods of ischemia and reperfusion. The molecular mechanisms and signal transduction pathways underlying conditioning phenomena in the coronary endothelium have been described as involving less ROS production, reduced adhesion of neutrophils to endothelial cells and diminished inflammatory reactions. This review summarizes our current understanding of the cellular and molecular mechanisms regulating IRI-affected and -damaged coronary endothelium, and how ischemic conditioning may preserve its function., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest.

The extracellular adherence protein (Eap) of Staphylococcus aureus is a secreted protein known to exert a number of adhesive and immunomodulatory properties. Here we describe the intrinsic DNA binding activity of this multifunctional secretory factor. By using atomic force microscopy, we provide evidence that Eap can bind and aggregate DNA. While the origin of the DNA substrate (e.g., eukaryotic, bacterial, phage, and artificial DNA) seems to not be of major importance, the DNA structure (e.g., linear or circular) plays a critical role with respect to the ability of Eap to bind and condense DNA. Further functional assays corroborated the nature of Eap as a DNA binding protein, since Eap suppressed the formation of "neutrophil extracellular traps" (NETs), composed of DNA-histone scaffolds, which are thought to function as a neutrophil-mediated extracellular trapping mechanism. The DNA binding and aggregation activity of Eap may thereby protect S. aureus against a specific anti-microbial defense reaction from the host.

Objectives: Enolase-1-dependent cell surface proteolysis plays an important role in cell invasion. Although enolase-1 (Eno-1), a glycolytic enzyme, has been found on the surface of various cells, the mechanism responsible for its exteriorization remains elusive. Here, we investigated the involvement of post-translational modifications (PTMs) of Eno-1 in its lipopolysaccharide (LPS)-triggered trafficking to the cell surface., Results: We found that stimulation of human lung adenocarcinoma cells with LPS triggered the monomethylation of arginine 50 (R50me) within Eno-1. The Eno-1R50me was confirmed by its interaction with the tudor domain (TD) from TD-containing 3 (TDRD3) protein recognizing methylarginines. Substitution of R50 with lysine (R50K) reduced Eno-1 association with epithelial caveolar domains, thereby diminishing its exteriorization. Similar effects were observed when pharmacological inhibitors of arginine methyltransferases were applied. Protein arginine methyltransferase 5 (PRMT5) was identified to be responsible for Eno-1 methylation. Overexpression of PRMT5 and caveolin-1 enhanced levels of membrane-bound extracellular Eno-1 and, conversely, pharmacological inhibition of PRMT5 attenuated Eno-1 cell-surface localization. Importantly, Eno-1R50me was essential for cancer cell motility since the replacement of Eno-1 R50 by lysine or the suppression of PRMT 5 activity diminished Eno-1-triggered cell invasion., Conclusions: LPS-triggered Eno-1R50me enhances Eno-1 cell surface levels and thus potentiates the invasive properties of cancer cells. Strategies to target Eno-1R50me may offer novel therapeutic approaches to attenuate tumor metastasis in cancer patients., (Copyright © 2018 Elsevier B.V. All rights reserved.)