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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]

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Humboldt foundation Background Calcium-Sensing Receptor (CaSR) is a cell surface G-protein coupled receptor that senses calcium in the extracellular environment. This receptor is widely studied in mineral homeostasis due to its presence in varied calcitropic tissues. However, CaSR is also present on the surface of vascular and hematopoietic cells and recent studies suggest that this receptor can also have pathological consequences related to cardiovascular diseases, like atherosclerosis. Since endothelial cells are crucial players in atherogenesis, we aimed to investigate which role endothelial CaSR plays in the development and progression of atherosclerosis. Methods We cross-bred CaSRflox/flox Apoe-/- mice with BmxCreERT2 Apoe-/- (endothelial cell specific Cre driver) mice to obtain tamoxifen-inducible mice which have an endothelial cell specific deletion of CaSR. The mice were then fed with a high fat diet (HFD) for 4 or 12 weeks, reflecting early and late atherosclerosis, respectively. We used histological and immuno-fluorescent stainings to analyse the atherosclerotic lesion size and its cellular and acellular composition. Systemic effects on leukocytes were evaluated using flow-cytometry, while intra-vital microscopy was used to study leukocyte adhesion to activated endothelium in-vivo. Further mechanistic studies were performed in human coronary artery endothelial cells (HCAECs) in-vitro. Results Endothelial CaSR deficiency in mice resulted in significantly smaller lesion size in early atherosclerotic stages (4 weeks HFD), whereas late atherosclerotic lesions (12 weeks HFD) were not affected by the lack of endothelial CaSR. Moreover, the phenotype of atherosclerotic plaques, characterized by macrophage, smooth muscle cell, collagen and necrotic core content remained unaltered between wild-type and endothelial cell specific CaSR knockout mice. Interestingly, leukocyte adhesion in-vivo, especially neutrophil adhesion, was significantly reduced in endothelial cell CaSR deficient mice. In line with this, overexpression of CaSR in HCAECs in-vitro increased the production of inflammatory cytokines and expression of adhesion molecules ICAM and VCAM. Conclusion Our results indicate that while endothelial cell specific CaSR is involved in the induction of atherosclerosis, it does not play a major role in its progression. CaSR has a major influence on endothelium-leukocyte interaction and endothelial inflammation. The increased expression of ICAM/VCAM suggest that CaSR mainly mediates such adhesive effects by modulating integrin expression. Thereby this study highlights that CaSR plays a crucial role in atherosclerosis development, rendering it a novel, so far unexplored therapeutic target. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft SFB1123 European Research Council AdG_692511 Background Genome wide association studies revealed a strong association between the CXCL12 gene locus and cardiovascular diseases, highlighting its receptors CXCR4 and atypical chemokine receptor-3 (ACKR3) as targets for CVD research. Although vascular CXCR4 is well studied in atherosclerosis, the exact role of ACKR3 remains elusive. Therefore, the aim of this project is to decipher the role of vascular ACKR3 in atherosclerosis. Methods Vascular endothelium (Bmx-cre) specific Ackr3 deficient mice on Apoe-/- background were fed with a cholesterol-rich diet. Lesions in the aortic roots and arches were analyzed via H&E staining. The phenotype was further characterized employing ex-vivo perfusion and intravital microscopy for cell adhesion and Evans-blue injection for endothelial permeability. Signaling studies are performed in human coronary artery endothelial cells (HCAECs). Results Endothelial Ackr3 deficiency resulted in significantly smaller atherosclerotic lesions, accompanied by a more stable plaque phenotypes (more lesional collagen and smooth muscle cell content, smaller necrotic cores). Endothelial-Ackr3-/- mice revealed a reduction in plaque macrophage content and ICAM positive endothelial cells in atherosclerotic lesions. Moreover, immune cell adhesion on carotid arteries lacking endothelial ACKR3 was decreased whilst endothelial permeability was not affected. ACKR3 silencing in TNF-stimulated HCAECs decreased adhesion molecule expression and downregulated MAPK signaling and NF-kB p65 phosphorylation. Endothelial cells in atherosclerotic lesions also revealed decreased phospho-NF-kB p65 expression in Ackr3 deficient mice. Conclusion Collectively, our findings indicate that arterial endothelial ACKR3 fuels atherosclerosis by mediating endothelium-immune cell adhesion, most likely through inflammatory MAPK and NF-kB pathways resulting in increased adhesion molecule expression. [ABSTRACT FROM AUTHOR]