The role of Gas6 in endothelial cell (EC) function remains incompletely characterized. Here we report that Gas6 amplifies EC activation in response to inflammatory stimuli in vitro. In vivo, Gas6 promotes and accelerates the sequestration of circulating platelets and leukocytes on activated endothelium as well as the formation and endothelial sequestration of circulating platelet-leukocyte conjugates. In addition, Gas6 promotes leukocyte extravasation, inflammation, and thrombosis in mouse models of inflammation (endotoxinemia, vasculitis, heart trans- IntroductionThe growth arrest-specific gene 6 (Gas6) binds to the receptor tyrosine kinases Axl, Tyro3, and Mer. 1 Gas6 is composed of a N-terminal gamma-carboxy-glutamic acid domain (Gla-domain), a loop region, 4 EGF-like repeats, and a C-terminal steroid hormone binding globulin-like (SHBG-like) domain). 1 Even though this molecule was discovered as a homologue of the anticoagulant protein S more than a decade ago, its role in vivo remains incompletely characterized. 2,3 Originally identified in fibroblasts, Gas6 is expressed in various cell types, including endothelial cells (ECs), 4 smooth muscle, 5 and bone marrow cells. 6 Gas6 and its receptors modify platelet activation and aggregation, 7-10 but the role of Gas6 in the interplay between platelets and other cell types, such as ECs and leukocytes, 11 during inflammatory conditions remains unclear.Several lines of evidence indeed suggest that Gas6 may affect ECs and leukocytes. ECs and leukocytes express Gas6 and its receptors, especially in conditions of inflammation and repair. 4,[12][13][14][15][16] Gas6 stimulates EC survival [17][18][19][20] and promotes angiogenesis by enforcing the adhesion of Axl-expressing ECs via homophilic interactions, 21-23 yet another study suggested that activation of Axl impairs tyrosine phosphorylation of vascular endothelial growth factor (VEGF) receptor-2. 24 The activity of Gas6 on leukocytes also remains incompletely understood. Indeed, genetic loss of Mer inhibits cytokine production by natural killer cells 25 while it stimulates tumor necrosis factor-␣ (TNF-␣) production by monocytes 14 and impairs clearance of apoptotic cells. 26 Loss of all 3 Gas6 receptors, on the other hand, induces lymphoproliferative disorders via hyperactivation of antigen-presenting cells, 27,28 but mice lacking Gas6 (Gas6 Ϫ/Ϫ ) do not develop autoimmune health problems (P.C., unpublished data, 2008). In humans, the plasma levels of Gas6 were found to be elevated during severe sepsis, a life-threatening condition involving increased interactions between ECs, leukocytes, and platelets. 29,30 However, exogenous Gas6 inhibits granulocyte adhesion to ECs, but only at very high doses. 31 Furthermore, the role of endogenous Gas6 in leukocyte extravasation in vivo was not studied. Here, by using our previously generated Gas6 Ϫ/Ϫ mice, 7 we studied whether Gas6 might play a role in EC activation and in the interactions between ECs, platelets, and leukocytes during inflammatory conditions. Methods MiceTh...

Diabetes compromises the bone marrow (BM) microenvironment and reduces the number of circulating CD34(+) cells. Diabetic autonomic neuropathy (DAN) may impact the BM, because the sympathetic nervous system is prominently involved in BM stem cell trafficking. We hypothesize that neuropathy of the BM affects stem cell mobilization and vascular recovery after ischemia in patients with diabetes. We report that, in patients, cardiovascular DAN was associated with fewer circulating CD34(+) cells. Experimental diabetes (streptozotocin-induced and ob/ob mice) or chemical sympathectomy in mice resulted in BM autonomic neuropathy, impaired Lin(-)cKit(+)Sca1(+) (LKS) cell and endothelial progenitor cell (EPC; CD34(+)Flk1(+)) mobilization, and vascular recovery after ischemia. DAN increased the expression of the 66-kDa protein from the src homology and collagen homology domain (p66Shc) and reduced the expression of sirtuin 1 (Sirt1) in mice and humans. p66Shc knockout (KO) in diabetic mice prevented DAN in the BM, and rescued defective LKS cell and EPC mobilization. Hematopoietic Sirt1 KO mimicked the diabetic mobilization defect, whereas hematopoietic Sirt1 overexpression in diabetes rescued defective mobilization and vascular repair. Through p66Shc and Sirt1, diabetes and sympathectomy elevated the expression of various adhesion molecules, including CD62L. CD62L KO partially rescued the defective stem/progenitor cell mobilization. In conclusion, autonomic neuropathy in the BM impairs stem cell mobilization in diabetes with dysregulation of the life-span regulators p66Shc and Sirt1.

Abstract-Thrombospondin (TSP)1 is implicated in various inflammatory processes, but its role in atherosclerotic plaque formation and progression is unclear. Therefore, the development of atherosclerosis was compared in ApoE Ϫ/Ϫ and Tsp1 Ϫ/Ϫ ApoE Ϫ/Ϫ mice kept on a normocholesterolemic diet. At 6 months, morphometric analysis of the aortic root of both mouse genotypes showed comparable lesion areas. Even when plaque burden increased Ϸ5-fold in ApoE Ϫ/Ϫ and 10-fold in Tsp1 Ϫ/Ϫ ApoE Ϫ/Ϫ mice, during the subsequent 3 months, total plaque areas were comparable at 9 months. In contrast, plaque composition differed substantially between genotypes: smooth muscle cell areas, mostly located in the fibrous cap of ApoE Ϫ/Ϫ plaques, both at 6 and 9 months, were 3-fold smaller in Tsp1 Ϫ/Ϫ ApoE Ϫ/Ϫ plaques, which, in addition, were also more fibrotic. Moreover, inflammation by macrophages was twice as high in Tsp1plaques. This correlated with a 30-fold elevated incidence of elastic lamina degradation, with matrix metalloproteinase-9 accumulation, underneath plaques and manifestation of ectasia, exclusively in Tsp1 Ϫ/Ϫ ApoE Ϫ/Ϫ mice. At 9 months, the necrotic core was 1.4-fold larger and 4-fold higher numbers of undigested disintegrated apoptotic cells were found in Tsp1 Ϫ/Ϫ ApoE Ϫ/Ϫ plaques. Phagocytosis of platelets by cultured Tsp1 Ϫ/Ϫ macrophages revealed the instrumental role of TSP1 in phagocytosis, corroborating the defective intraplaque phagocytosis of apoptotic cells. Hence, the altered smooth muscle cell phenotype in Tsp1 Ϫ/Ϫ ApoE Ϫ/Ϫ mice has limited quantitative impact on atherosclerosis, but defective TSP1-mediated phagocytosis enhanced plaque necrotic core formation, accelerating inflammation and macrophageinduced elastin degradation by metalloproteinases, speeding up plaque maturation and vessel wall degeneration.

The role of the fragile X mental retardation protein (FMRP) is well established in brain, where its absence leads to the fragile X syndrome (FXS). FMRP is almost ubiquitously expressed, suggesting that, in addition to its effects in brain, it may have fundamental roles in other organs. There is evidence that FMRP expression can be linked to cancer. FMR1 mRNA, encoding FMRP, is overexpressed in hepatocellular carcinoma cells. A decreased risk of cancer has been reported in patients with FXS while a patient-case with FXS showed an unusual decrease of tumour brain invasiveness. However, a role for FMRP in regulating cancer biology, if any, remains unknown. We show here that FMRP and FMR1 mRNA levels correlate with prognostic indicators of aggressive breast cancer, lung metastases probability and triple negative breast cancer (TNBC). We establish that FMRP overexpression in murine breast primary tumours enhances lung metastasis while its reduction has the opposite effect regulating cell spreading and invasion. FMRP binds mRNAs involved in epithelial mesenchymal transition (EMT) and invasion including E-cadherin and Vimentin mRNAs, hallmarks of EMT and cancer progression.