Apolipoprotein E (apoE) is a 34-kDa glycoprotein involved in lipoprotein transport through interaction with the low-density lipoprotein receptor and related receptors. Recently, it has become clear that apoE binding to its receptors plays a role both in development and in control of the immune system. In this study, we show that apoE modulates the rate of uptake of apoptotic cells by macrophages. In vitro, apoE-deficient macrophages ingest less apoptotic thymocytes (but not latex beads) than wild-type macrophages, and this defect can be corrected by addition of exogenous apoE protein. In vivo, the number of dying macrophages is increased in a range of tissues, including lung and brain. Possibly in response to the larger numbers of persistent apoptotic bodies, the number of live macrophages in these tissues are also increased compared with those of wild-type control mice. In addition to the significant changes in macrophage population dynamics we observed, levels of the proinflammatory cytokine TNF-α and the positive acute phase reactant fibrinogen are also elevated in the livers from apoE-deficient mice. In contrast, neither deletion of the gene encoding the LDL receptor nor cholesterol feeding of wild-type mice affected either the number of apoptotic bodies or the number of live macrophages. We conclude that apoE deficiency results in impaired clearance of apoptotic cell remnants and a functionally relevant systemic proinflammatory condition in mice, independent of its role in lipoprotein metabolism. Any similar reduction of apoE activity in humans may contribute to the pathogenesis of a wide range of chronic diseases including atherosclerosis, dementia, and osteoporosis.

Activin receptor-like kinase 1 (ALK1)-mediated endothelial cell signalling in response to bone morphogenetic protein 9 (BMP9) and BMP10 is of significant importance in cardiovascular disease and cancer. However, detailed molecular mechanisms of ALK1-mediated signalling remain unclear. Here, we report crystal structures of the BMP10:ALK1 complex at 2.3 Å and the prodomain-bound BMP9:ALK1 complex at 3.3 Å. Structural analyses reveal a tripartite recognition mechanism that defines BMP9 and BMP10 specificity for ALK1, and predict that crossveinless 2 is not an inhibitor of BMP9, which is confirmed by experimental evidence. Introduction of BMP10-specific residues into BMP9 yields BMP10-like ligands with diminished signalling activity in C2C12 cells, validating the tripartite mechanism. The loss of osteogenic signalling in C2C12 does not translate into non-osteogenic activity in vivo and BMP10 also induces bone-formation. Collectively, these data provide insight into ALK1-mediated BMP9 and BMP10 signalling, facilitating therapeutic targeting of this important pathway.

Summary Chemokines participate in the regulation of leucocyte recruitment in a wide variety of inflammatory processes, including host defence and diseases such as asthma, atherosclerosis and autoimmune disorders. We have previously described the properties of Peptide 3, the first broad‐specificity chemokine inhibitor in vitro. Here, we report the properties of NR58‐3.14.3, a retroinverso analogue of Peptide 3. NR58‐3.14.3 inhibited leucocyte migration induced by a range of chemokines, including monocyte chemoattractant protein‐1 (MCP‐1) (2·5 nm), macrophage inflammatory protein‐1α (MIP‐1α) (5 nm), regulated on activation, normal T‐cell expressed and presumably secreted (RANTES) (20 nm), stromal cell‐derived factor‐1α (SDF‐1α) (25 nm) and interleukin‐8 (IL‐8) (30 nm), but did not affect migration induced by N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) or complement C5a (> 100 µm). NR58‐3.14.3 is therefore ≈ 1000‐fold more potent than Peptide 3 but retains the broad‐spectrum chemokine inhibitory activity of the parent peptide. In vivo, pretreatment with a systemic dose of 10 mg of NR58‐3.14.3, but not the inactive derivative NR58‐3.14.4, abolished leucocyte recruitment in response to intradermal injection of 500 ng of MCP‐1 into rat skin. This suggests that NR58‐3.14.3 is a functional chemokine inhibitor in vivo as well as in vitro. We utilized NR58‐3.14.3 as a tool to investigate the role of chemokine activity during leucocyte recruitment in response to lipopolysaccharide (LPS) in vivo. NR58‐3.14.3, but not NR58‐3.14.4, abolished leucocyte recruitment in response to intradermal injection of 50 ng of LPS into rat skin. Furthermore, NR58‐3.14.3 completely inhibited LPS‐induced accumulation of tumour necrosis factor‐α (TNF‐α). This data is consistent with a model in which multiple chemokines act in parallel upstream of TNF‐α. NR58‐3.14.3 is therefore a powerful anti‐inflammatory agent in vivo, suppressing proinflammatory cytokine production and leucocyte recruitment in response to endotoxin stimulus in rat skin.