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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of antigen-presenting cells, 27,28 but mice lacking Gas6 (Gas6 Ϫ/Ϫ ) do not develop autoimmune health problems (P.C., unpublished data, 2008).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...

Objective. Systemic juvenile idiopathic arthritis (JIA) is unique among the rheumatic diseases of childhood, given its distinctive systemic inflammatory character. Inappropriate control of innate immune responses following an initially harmless trigger is thought to account for the excessive inflammatory reaction. The aim of this study was to generate a similar systemic inflammatory syndrome in mice by injecting a relatively innocuous, yet persistent, immune system trigger: Freund's complete adjuvant (CFA), containing heat-killed mycobacteria.Methods. Given the central role of interferon-␥ (IFN␥) in immune regulation, we challenged wild-type (WT) and IFN␥-knockout (KO) BALB/c mice with CFA, and analyzed their clinical symptoms and biologic characteristics. The production of cytokines and the effects of anticytokine antibodies were investigated.Results. In WT mice, CFA injection resulted in splenomegaly, lymphadenopathy, neutrophilia, thrombocytosis, and increased cytokine expression. In the absence of IFN␥, these symptoms were more pro-

Microglial activation is a hallmark of all neurodegenerative diseases including amyotrophic lateral sclerosis (ALS; SOD1 G93A doubly transgenic mice had no effect on motor neuron degeneration. This suggests that proliferating microglia-expressing mutant SOD1 are not central contributors of the neurodegenerative process in ALS caused by mutant SOD1.

Despite progress in cardiovascular research, a cure for peripheral vascular disease has not been found. We compared the vascularization and tissue regeneration potential of murine and human undifferentiated multipotent adult progenitor cells (mMAPC-U and hMAPC-U), murine MAPC-derived vascular progenitors (mMAPC-VP), and unselected murine BM cells (mBMCs) in mice with moderate limb ischemia, reminiscent of intermittent claudication in human patients. mMAPC-U durably restored blood flow and muscle function and stimulated muscle regeneration, by direct and trophic contribution to vascular and skeletal muscle growth. This was in contrast to mBMCs and mMAPC-VP, which did not affect muscle regeneration and provided only limited and transient improvement. Moreover, mBMCs participated in a sustained inflammatory response in the lower limb, associated with progressive deterioration in muscle function. Importantly, mMAPC-U and hMAPC-U also remedied vascular and muscular deficiency in severe limb ischemia, representative of critical limb ischemia in humans. Thus, unlike BMCs or vascular-committed progenitors, undifferentiated multipotent adult progenitor cells offer the potential to durably repair ischemic damage in peripheral vascular disease patients.

Multipotent adult progenitor cells (MAPCs) are bone marrow-derived nonhematopoietic stem cells with a broad differentiation potential and extensive expansion capacity. A comparative study between human mesenchymal stem cells (hMSCs) and human MAPCs (hMAPCs) has shown that hMAPCs have clearly distinct phenotypical and functional characteristics from hMSCs. In particular, hMAPCs express lower levels of MHC class I than hMSCs and cannot only differentiate into typical mesenchymal cell types but can also differentiate in vitro and in vivo into functional endothelial cells. The use of hMSCs as cellular immunomodulatory stem cell products gained much interest since their immunomodulatory capacities in vitro became evident over the last decade. Currently, the clinical grade stem cell product of hMAPCs is already used in clinical trials to prevent graft-versus-host disease (GVHD), as well as for the treatment of acute myocardial infarct, ischemic stroke, and Crohn's disease. Therefore, we studied the immune phenotype, immunogenicity, and immunosuppressive effect of hMAPCs in vitro. We demonstrated that hMAPCs are nonimmunogenic for T-cell proliferation and cytokine production. In addition, hMAPCs exert strong immunosuppressive effects on T-cell alloreactivity and on T-cell proliferation induced by mitogens and recall antigens. This immunomodulatory effect was not MHC restricted, which makes off-the-shelf use promising. The immunosuppressive effect of hMAPCs is partially mediated via soluble factors and dependent on indoleamine 2,3-dioxygenase (IDO) activity. At last, we isolated hMAPCs, the clinical grade stem cell product of hMAPCs, named MultiStem, and hMSCs from one single donor and observed that both the immunogenicity and the immunosuppressive capacities of all three stem cell products are comparable in vitro. In conclusion, hMAPCs have potent immunomodulatory properties in vitro and can serve as a valuable cell source for the clinical use of immunomodulatory cellular stem cell product.

Enhanced Green Fluorescent Protein (EGFP) is the most commonly used live cell reporter despite a number of conflicting reports that it can affect cell physiology. Thus far, the precise mechanism of GFP-associated defects remained unclear. Here we demonstrate that EGFP and EGFP fusion proteins inhibit polyubiquitination, a posttranslational modification that controls a wide variety of cellular processes, like activation of kinase signalling or protein degradation by the proteasome. As a consequence, the NF-κB and JNK signalling pathways are less responsive to activation, and the stability of the p53 tumour suppressor is enhanced in cell lines and in vivo. In view of the emerging role of polyubiquitination in the regulation of numerous cellular processes, the use of EGFP as a live cell reporter should be carefully considered.

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