The perpetuation of inflammation is an important pathophysiological contributor to the global medical burden. Chronic inflammation is promoted by non-programmed cell death1,2; however, how inflammation is instigated, its cellular and molecular mediators, and its therapeutic value are poorly defined. Here we use mouse models of atherosclerosis—a major underlying cause of mortality worldwide—to demonstrate that extracellular histone H4-mediated membrane lysis of smooth muscle cells (SMCs) triggers arterial tissue damage and inflammation. We show that activated lesional SMCs attract neutrophils, triggering the ejection of neutrophil extracellular traps that contain nuclear proteins. Among them, histone H4 binds to and lyses SMCs, leading to the destabilization of plaques; conversely, the neutralization of histone H4 prevents cell death of SMCs and stabilizes atherosclerotic lesions. Our data identify a form of cell death found at the core of chronic vascular disease that is instigated by leukocytes and can be targeted therapeutically.

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 Pselectin 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.Keywords: neutrophil; platelet; chemokine; recruitment; acute lung injury Acute lung injury (ALI) is a life-threatening disease with an ageadjusted incidence of 86.2 per 100,000 person-years (1). Despite innovations in intensive care medicine, the mortality of ALI remains approximately 40%. ALI is characterized by an increased permeability of the alveolar-capillary barrier, resulting in lung edema with protein-rich fluid and consequently in impaired arterial oxygenation. A major cause for development of ALI is sepsis, wherein gram-negative bacteria are the dominating factor. LPS inhalation mimics human gram-negative ALI, leading to recruitment of neutrophils, pulmonary edema, and finally impairment of gas exchange (2).Recruitment of neutrophils is a key event in development of ALI (3) resulting in plasma leakage and deterioration of oxygenation. The importance of neutrophils in ALI is supported by studies, where lung injury was abolished or reversed by depletion of neutrophils (4,5). Much of the neutrophil-dependent ALI is thought to be mediated by granule proteins released from activated neutrophils. For example, azurocidin and a-defensins have been found to directly affect permeability changes (6, 7), whereas proteases of neutrophilic origin, such as neutrophil ela...

Tumor angiogenesis is a key event in cancer progression. Here, we report that tumors can stimulate tumor angiogenesis by secretion of galectin-1. Tumor growth and tumor angiogenesis of different tumor models are hampered in galectin-1-null (gal-1 −/− ) mice. However, tumor angiogenesis is less affected when tumor cells express and secrete high levels of galectin-1. Furthermore, tumor endothelial cells in gal-1 −/− mice take up galectin-1 that is secreted by tumor cells. Uptake of galectin-1 by cultured endothelial cells specifically promotes H-Ras signaling to the Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (Erk) kinase (Mek)/Erk cascade and stimulates endothelial cell proliferation and migration. Moreover, the activation can be blocked by galectin-1 inhibition as evidenced by hampered membrane translocation of H-Ras.GTP and impaired Raf/Mek/Erk phosphorylation after treatment with the galectin-1-targeting angiogenesis inhibitor anginex. Altogether, these data identify galectin-1 as a proangiogenic factor. These findings have direct implications for current efforts on galectin-1-targeted cancer therapies. Cancer Res; 70(15); 6216-24. ©2010 AACR.

Summary. Background: Matrix Gla protein (MGP) is a small vitamin K-dependent protein containing five c-carboxyglutamic acid (Gla) residues that are believed to be important in binding Ca 2+, calcium crystals and bone morphogenetic protein. In addition, MGP contains phosphorylated serine residues that may further regulate its activity. In vivo, MGP has been shown to be a potent inhibitor of vascular calcification; however, the precise molecular mechanism underlying the function of MGP is not yet fully understood. Methods and results: We investigated the effects of MGP in human vascular smooth muscle cell (VSMC) monolayers that undergo calcification after exposure to an increase in Ca 2+ concentration. Increased calcium salt deposition was found in cells treated with the vitamin K antagonist warfarin as compared to controls, whereas cells treated with vitamin K 1 showed decreased calcification as compared to controls. With conformationspecific antibodies, it was confirmed that warfarin treatment of VSMCs resulted in uncarboxylated (Gla-deficient) MGP. To specifically test the effects of MGP on VSMC calcification, we used full-length synthetic MGP and MGP-derived peptides representing various domains in MGP. Full length MGP, the ccarboxylated motif (Gla) (amino acids 35-54) and the phosphorylated serine motif (amino acids 3-15) inhibited calcification. Furthermore, we showed that the peptides were not taken up by VSMCs but bound to the cell surface and to vesicle-like structures. Conclusions: These data demonstrate that both cglutamyl carboxylation and serine phosphorylation of MGP contribute to its function as a calcification inhibitor and that MGP may inhibit calcification via binding to VSMC-derived vesicles.

Background-Thrombin generation in vivo may be important in regulating atherosclerotic progression. In the present study, we examined for the first time the activity and presence of relevant coagulation proteins in relation to the progression of atherosclerosis. Methods and Results-Both early and stable advanced atherosclerotic lesions were collected pairwise from each individual (nϭ27) during autopsy. Tissue homogenates were prepared from both total plaques and isolated plaque layers, in which the activity of factors (F) II, X, and XII and tissue factor was determined. Microarray analysis was implemented to elucidate local messenger RNA synthesis of coagulation proteins. Part of each specimen was paraffin embedded, and histological sections were immunohistochemically stained for multiple coagulation markers with the use of commercial antibodies. Data are expressed as median (interquartile range [IQR] Key Words: atherosclerosis Ⅲ hypercoagulability Ⅲ immunohistochemistry Ⅲ plaque Ⅲ thrombosis A therosclerosis is widely recognized as a chronic inflammatory disease. 1 Rupture of an atherosclerotic plaque is considered the predominant underlying cause of acute atherothrombotic events such as myocardial infarction, ischemic stroke, and vascular death. A close relation between blood coagulation and atherosclerosis 2,3 is supported by studies revealing the presence of specific coagulation proteins within an atherosclerotic lesion. Tissue factor (TF) and factor (F) VII, of which the complex is the principal initiator of coagulation in vivo, are expressed on macrophages and vascular smooth muscle cells (SMC) within the arterial vessel wall and atherosclerotic lesion. 4,5 Both proteins potentially participate in multiple proatherogenic processes such as migration and proliferation of SMC, 6 inflammation, and angiogenesis. 7 In addition to the single effects of each protein, the local interaction between macrophage/SMC-derived TF and FVII may provide a catalytic complex for subsequent generation of thrombin and fibrin, of which the latter is also detectable in atherosclerotic lesions. 8,9 The procoagulant condition of the atherosclerotic lesion may be further enhanced by the presence of various proinflammatory cytokines (eg, tumor necrosis factor-␣, interleukin-1 10 ), which may downregulate local expression of anticoagulant proteins such as thrombomodulin and the endothelial protein C receptor on endothelial cells. 11 Received September 4, 2009; accepted June 28, 2010 Clinical Perspective on p 830Thrombin, a key enzyme in blood coagulation, may also play a critical role in many processes related to the development, progression, and atherothrombotic potential of atherosclerotic plaques. 12 Direct evidence for the role of thrombin in the atherogenic process comes from experiments showing reduced progression of atherosclerosis in apolipoprotein E Ϫ/Ϫ mice on pharmacological inhibition of thrombin. 13 Moreover, decreased expression of TF pathway inhibitor (TFPI) on an apolipoprotein E Ϫ/Ϫ background increased the atheroscle...

Objective: Matrix γ-carboxyglutamate (Gla) protein (MGP) is a vitamin K-dependent protein and a strong inhibitor of vascular calcification. Vitamin K deficiency leads to inactive uncarboxylated MGP (ucMGP), which accumulates at sites of arterial calcification. We hypothesized that as a result of ucMGP deposition around arterial calcification, the circulating fraction of ucMGP is decreased. Here we report on the development of an ucMGP assay and the potential diagnostic utility of monitoring serum ucMGP levels. Methods and Results: An ELISA-based assay was developed with which circulating ucMGP can be determined. Serum ucMGP levels were measured in healthy subjects (n = 165) and in four patient populations; patients who underwent angioplasty (n = 30), patients with aortic stenosis (n = 25), hemodialysis patients (n = 52), and calciphylaxis patients (n = 10). All four patient populations had significantly lower ucMGP levels. In angioplasty patients and in those with aortic stenosis, some overlap was observed with the control population. However, in the hemodialysis and calciphylaxis populations, virtually all subjects had ucMGP levels below the normal adult range. Conclusion: Serum ucMGP may be used as a biomarker to identify those at risk for developing vascular calcification. This assay may become an important tool in the diagnosis of cardiovascular calcification.

Coagulase-positive Staphylococcus aureus (S. aureus) is the major causal pathogen of acute endocarditis, a rapidly progressing, destructive infection of the heart valves. Bacterial colonization occurs at sites of endothelial damage, where (together with fibrin and platelets) it initiates the formation of abnormal growths known as vegetations. Here we report that an engineered analog of prothrombin detected S. aureus in endocarditic vegetations via noninvasive fluorescence or PET imaging. These prothrombin derivatives bound to staphylocoagulase and intercalated into growing bacterial vegetations. We also present evidence for bacterial quorum sensing in the regulation of staphylocoagulase expression by S. aureus. Staphylocoagulase expression was limited to the growing edge of mature vegetations, where it was exposed to the host and co-localized with the imaging probe. When endocarditis was induced with an S. aureus strain with genetic deletion of coagulases, survival of mice improved, highlighting the role of staphylocoagulase as a virulence factor.

Background The CXCL12/CXCR4 chemokine ligand/receptor axis controls (progenitor) cell homeostasis and trafficking. So far, an atheroprotective role of CXCL12/CXCR4 has only been implied through pharmacological intervention, particularly as the somatic deletion of the CXCR4 gene in mice is embryonically lethal. Moreover, cell-specific effects of CXCR4 in the arterial wall and underlying mechanisms remain elusive, prompting us to investigate the relevance of CXCR4 in vascular cell types for atheroprotection. Methods We examined the role of vascular CXCR4 in atherosclerosis and plaque composition by inducing an endothelial cell (EC, BmxCreERT2-driven)-specific or smooth muscle cell (SMC, SmmhcCreERT2- or TaglnCre-driven)-specific-deficiency of CXCR4 in an apolipoprotein E-deficient mouse model. To identify underlying mechanisms for effects of CXCR4, we studied endothelial permeability, intravital leukocyte adhesion, involvement of the Akt/WNT/β-catenin signaling pathway and relevant phosphatases in VE-cadherin expression and function, vascular tone in aortic rings, cholesterol efflux from macrophages, and expression of SMC phenotypic markers. Finally, we analyzed associations of common genetic variants at the CXCR4 locus with the risk for coronary heart disease, along with CXCR4 transcript expression in human atherosclerotic plaques. Results The cell-specific deletion of CXCR4 in arterial ECs (n=12-15) or SMCs (n=13-24) markedly increased atherosclerotic lesion formation in hyperlipidemic mice. Endothelial barrier function was promoted by CXCL12/CXCR4, which triggered Akt/WNT/β-catenin-signaling to drive VE-cadherin expression and stabilized junctional VE-cadherin complexes through associated phosphatases. Conversely, endothelial CXCR4-deficiency caused arterial leakage and inflammatory leukocyte recruitment during atherogenesis. In arterial SMCs, CXCR4 sustained normal vascular reactivity and contractile responses, whereas CXCR4-deficiency favored a synthetic phenotype, the occurrence of macrophage-like SMCs in the lesions, and impaired cholesterol efflux. Regression analyses in humans (n=259,796) identified the C-allele at rs2322864 within the CXCR4 locus to be associated with increased risk for coronary heart disease. In line, C/C risk genotype carriers showed reduced CXCR4 expression in carotid artery plaques (n=188), which was furthermore associated with symptomatic disease. Conclusions Our data clearly establish that vascular CXCR4 limits atherosclerosis by maintaining arterial integrity, preserving endothelial barrier function, and a normal contractile SMC phenotype. Enhancing these beneficial functions of arterial CXCR4 by selective modulators might open novel therapeutic options in atherosclerosis.