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...

Methods and Results: Compared to Apoe؊/؊ mice, Cramp ؊/؊ Apoe ؊/؊ mice exhibit reduced lesion sizes with lower macrophage numbers. In atherosclerotic aortas, we could detect CRAMP specifically in neutrophils, but not in monocytes or macrophages. By use of intravital microscopy, CRAMP was found to be deposited by activated neutrophils on inflamed endothelium of large arteries. In this location cathelicidins promote adhesion of classical monocytes and neutrophils, but not nonclassical monocytes in a formyl-peptide receptor-dependent manner. Key Words: atherosclerosis Ⅲ monocyte recruitment Ⅲ neutrophil A therosclerosis is a chronic inflammation of the arterial vessel wall with relatively well-defined roles for leukocytes such as macrophages and lymphocytes. 1,2 Recent studies, however, have revealed that neutrophils infiltrate atherosclerotic lesions at various time points, 3-5 and depletion studies provide evidence for a proatherogenic role of neutrophils. 5,6 Nevertheless, mechanistic insights into how neutrophils promote early atherosclerotic lesion formation remain elusive. Neutrophils contain granules with more than 300 different proteins that undergo limited exocytosis on neutrophil extravasation. 7 Some of these proteins are able to activate and recruit immune cells and thus have been coined alarmins. 8 Cathelicidins (CRAMP in mouse, LL37 in humans) residing in neutrophil secondary granules were shown to potently activate and recruit monocytes and macrophages, 9,10 thus fulfilling alarmin criteria. Because cathelicidins were identified in atherosclerotic lesions, 11 we investigated their role in a mouse model of atherosclerosis. Conclusions: Editorial, see p 1036 In This Issue, see p 1035 MethodsDetailed Methods are provided in the Online Supplement. Plaque StudiesCramp Ϫ/Ϫ mice 12 were crossed with Apoe Ϫ/Ϫ mice. Atherosclerotic lesion size as well as lesional neutrophil and macrophage content were assessed by histology and immunohistochemistry. Intravital MicroscopyLeukocyte adhesion to the carotid artery was studied by intravital fluorescence microscopy as described previously. 5 ResultsTo investigate the role of CRAMP in early atherosclerotic lesion formation, we fed Apoe Ϫ/Ϫ and CrampOriginal received January 29, 2012; revision received February 15, 2012; accepted February 27, 2012. In January 2012, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.88 days.Brief UltraRapid Communications are designed to be a format for manuscripts that are of outstanding interest to the readership, report definitive observations, but have a relatively narrow scope.

Rationale The leukocyte response in acute inflammation is characterized by an initial recruitment of neutrophils preceding a second wave of monocytes. Neutrophil-derived granule proteins were suggested to hold an important role in this cellular switch. The exact mechanisms by which neutrophils mediate these processes are only partially understood. Objective To investigate the role of neutrophils and their granule contents in the adhesion of monocyte subpopulations in acute inflammation. Methods and Results Here, we show that neutrophil-derived cathelicidins (human: LL37, mouse: CRAMP) induce adhesion of classical monocytes but not of non-classical monocytes in the mouse cremaster muscle and in in vitro flow chamber assays. CRAMP is released from emigrated neutrophils and then transported across the endothelium where it is presented to rolling leukocytes. Endothelial-bound cathelicidin activates FPR2 on classical monocytes, resulting in monocytic β1- and β2-integrin conformational change towards an extended, active conformation that allows for adhesion to their respective ligands VCAM-1 and ICAM-1. Conclusions These data elucidate a novel mechanism of neutrophil-mediated monocyte recruitment, which could be targeted in conditions where recruitment of classical monocytes plays an unfavorable role.

Percutaneous transluminal angioplasty with stent implantation is used to dilate of arteries narrowed by atherosclerotic plaques and to revascularize coronary arteries occluded by atherothrombosis in myocardial infarction. Commonly applied drug-eluting stents release antiproliferative or anti-inflammatory agents to reduce the incidence of in-stent stenosis. However, these stents may lead to in-stent stenosis and increase the rate late stent thrombosis, an obstacle to Correspondence: Oliver Soehnlein, MD, PhD or Christian Weber, MD, Institute for Cardiovascular Prevention, Pettenkoferstr. 9, 80336 Munich, Phone +49-(0)89-5160-4350, Fax +49-(0)89-5160-4352, oliver.soehnlein@med.uni-muenchen.de or christian.weber@med.uni-muenchen.de. * These authors contributed equally. Competing interests:The authors do not declare any competing financial interests. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript optimal revascularization possibly related to endothelial recovery. Here we examined the contribution of neutrophils and neutrophilic granule proteins to arterial healing after injury. We found that neutrophil-born cathelicidin (mouse CRAMP, human LL-37) promoted reendothelization and thereby limited neointima formation after stent implantation. We then translated these findings, generating a neutrophil-instructing biofunctionalized miniaturized Nitinol stent coated with LL-37. This stent reduced in-stent stenosis in a mouse model of atherosclerosis, suggesting that LL-37 may promote vascular healing after interventional therapy.

Aims Atherosclerosis is a chronic inflammatory disease of the arteries leading to the formation of atheromatous plaques. Human mesenchymal stem cells (hMSCs) are recruited from the circulation into plaques where in response to their environment they adopt a phenotype with immunomodulatory properties. However, the mechanisms underlying hMSC function in these processes are unclear. Recently, we described that miRNA let-7f controls hMSC invasion guided by inflammatory cytokines and chemokines. Here, we investigated the role of let-7f in hMSC tropism to human atheromas and the effects of the plaque microenvironment on cell fate and release of soluble factors. Methods and results Incubation of hMSCs with LL-37, an antimicrobial peptide abundantly found in plaques, increased biosynthesis of let-7f and N-formyl peptide receptor 2 (FPR2), enabling chemotactic invasion of the cells towards LL-37, as determined by qRT-PCR, flow cytometry, and cell invasion assay analysis. In an Apoe  -/- mouse model of atherosclerosis, circulating hMSCs preferentially adhered to athero-prone endothelium. This property was facilitated by elevated levels of let-7f in the hMSCs, as assayed by ex vivo artery perfusion and 2-photon laser scanning microscopy. Exposure of hMSCs to homogenized human atheromatous plaque material considerably induced the production of various cytokines, chemokines, matrix metalloproteinases, and tissue inhibitors of metalloproteinases, as studied by PCR array and Western blot analysis. Moreover, exposure to human plaque extracts elicited differentiation of hMSCs into cells of the myogenic lineage, suggesting a potentially plaque-stabilizing effect. Conclusions Our findings indicate that let-7f promotes hMSC tropism toward atheromas through the LL-37/FPR2 axis and demonstrate that hMSCs upon contact with human plaque environment develop a potentially athero-protective signature impacting the pathophysiology of atherosclerosis. TRANSLATIONAL PERSPECTIVE Human mesenchymal stem cells (hMSCs) represent a promising therapeutic approach in various pathophysiological processes associated with inflammation including atherosclerosis. The current knowledge about the mechanisms of hMSC tropism towards human atherosclerotic plaques and their beneficial effects at the site is poor. Bridging this gap is essential for clinical application of hMSCs. Our work provides insight into the contribution of microRNA let-7f in hMSC recruitment to atheroprone areas, where hMSCs display athero-protective potential by releasing immunomodulatory factors and differentiating towards plaque-stabilizing cells. Our findings highlight circulating hMSCs as a possible therapeutic strategy for the stabilization of atherosclerotic plaques.