We previously reported that 3T3-L1 and rat primary adipocytes secreted microvesicles, known as adipocyte-derived microvesicles (ADMs). In the present study, we further characterized the 3T3-L1 ADMs and found that they exhibited angiogenic activity in vivo. Antibody arrays and gelatin zymography analyses revealed that several angiogenic and antiangiogenic proteins, including leptin, TNFalpha, acidic fibroblast growth factor (FGFa), interferon-gamma, and matrix metalloprotease (MMP)-2 and MMP-9, were present in the ADMs. Gene expression of most of these angiogenic factors was induced in the adipose tissue of diet-induced obese mice. Furthermore, leptin, TNFalpha, and MMP-2 were up-regulated at the protein level in the adipocyte fractions prepared from epididymal adipose tissues of high-fat-diet-induced obese mice. ADMs induced cell migration and tube formation of human umbilical vein endothelial cells, which were partially suppressed by neutralizing antibodies to leptin, TNFalpha, or FGFa but not to interferon-gamma. Supporting these data, a mixture of leptin, TNFalpha, and FGFa induced tube formation. ADMs also promoted cell invasion of human umbilical vein endothelial cells through Matrigel, which was suppressed by the addition of the MMP inhibitor 1,10'-phenanthroline and a neutralizing antibody to MMP-2 but not to MMP-9. These results suggest that ADMs are associated with multiple angiogenic factors and play a role in angiogenesis in adipose tissue.

HSC fate decisions are regulated by cellintrinsic and cell-extrinsic cues. The latter cues are derived from the BM niche. Membrane-type 1 matrix metalloproteinase (MT1-MMP), which is best known for its proteolytic role in pericellular matrix remodeling, is highly expressed in HSCs and stromal/niche cells. We found that, in MT1-MMP ؊/؊ mice, in addition to a stem cell defect, the transcription and release of kit ligand (KitL), stromal cell-derived factor-1 (SDF-1/CXCL12), erythropoietin (Epo), and IL-7 was impaired, resulting in a trilineage hematopoietic differentiation block, while addition of exogenous KitL and SDF-1 restored hematopoiesis. Further mechanistic studies revealed that MT1-MMP activates the hypoxia-inducible factor-1 (HIF-1) pathway via factor inhibiting HIF-1 (FIH- 1 IntroductionThe adult hematopoietic system is maintained by a small number of HSCs that reside in the BM in a specialized microenvironment (the niche). 1,2 Here, HSCs undertake fate decisions including differentiation to progenitor cells and self-renewal, which ensures a lifelong supply of terminally differentiated blood cells. Intrinsic cellular programming and external stimuli such as adhesive interactions with the microenvironmental stroma and cytokine activities regulate HSC fate. However, it is unclear how niche factor production is controlled to adjust to external demand with a fine-tuned response.Hypoxia-inducible factors (HIFs) consist of an ␣ (HIF-␣) and a ␤ (HIF-␤, or ARNT) subunit and activate the expression of genes encoding proteins that regulate cell metabolism, motility, angiogenesis, hematopoiesis, and other functions. HSCs maintain cell-cycle quiescence by regulating HIF-1␣ levels. 3,4 Mice with mutations in the heterodimeric transcription factor HIF develop extensive hematopoietic pathologies: embryos lacking Arnt have defects in primitive hematopoiesis. 5 Mice lacking endothelial PAS domain protein 1 (EPAS1, also known as HIF-2alpha/HRF/HLF/MOP3), a second HIF family member, exhibited pancytopenia, and it was shown that EPAS1 is necessary to maintain a functional microenvironment in the BM for effective hematopoiesis. 6 HIFs bind to canonical DNA sequences in the promoters or enhancers of target genes such as erythropoietin (Epo), vascular endothelial growth factor-A, SDF-1␣/CXCL12, angiopoietin-2, platelet-derived growth factor-B and Kit Ligand (KitL)/stem cell factor, which are involved in HSC maintenance within the BM niche. 7-10 The chemokine SDF-1␣/CXCL12 (SDF-1␣) is expressed by perivascular, endosteal, mesenchymal stem and progenitor cells as well as by osteoblasts. 11,12 SDF-1␣ deficiency leads to a reduction in HSCs and impaired B-cell development in mice. 13,14 IL-7 is another stromal cell-derived niche factor, which, in cooperation with CXCL12, functions at sequential stages of B-cell development. 15,16 IL-7 or IL-7R deficiency results in impaired B-cell development. 17,18 Proteases such as matrix metalloproteinase-9 (MMP-9) and the serine proteinase plasmin(ogen) regulate HSC fate through KitL release i...

3409 Stem cells reside in a physical niche, a particular microenvironment. The organization of cellular niches has been shown to play a key role in regulating normal stem cell differentiation, stem cell maintenance and regeneration. Various stem cell niches have been shown to be hypoxic, thereby maintaining the stem cell phenotype, e.g. for hematopoietic stem cells (HSCs) or cancer stem cells. The bone marrow (BM) niche is a rich reservoir for tissue-specific pluripotent HSCs. Proteases, such as matrix metalloproteinases (MMPs) can modulate stem cell fate due to their proteolytic or non-proteolytic functions (abilities). We have investigated the role of membrane-type1 matrix metalloproteinase (MT1-MMP), known for its role in pericellular matrix remodeling and cell migration, in hematopoiesis. MT1-MMP is highly expressed in HSCs and stromal cells. In MT1-MMP−/− mice, release of kit ligand (KitL), stromal cell derived factor-1 (SDF-1/CXCL12), erythropoietin (Epo) and interleukin-7 were impaired resulting in erythroid, myeloid and T and B lymphoid differentiation. Addition of exogenous rec. KitL and rec. SDF-1 restored hematopoiesis in vivo and in vitro. Further mechanistic studies revealed that MT1-MMP in a non-proteolytic manner activates the HIF-1 pathway, thereby inducing the transcription of the HIF-responsive genes KitL, SDF-1 and Epo. These results suggested MT1-MMP as a critical regulator of postnatal hematopoiesis, which as a modulator of the HIF pathway alters critical hematopoietic niche factors necessary for terminal differentiation and or migration. Thus, our results indicate that MT1-MMP as a key molecular link between hypoxia and the regulation of vital HSC niche factors. Disclosures: No relevant conflicts of interest to declare.

1897 Proinflammatory cytokines released upon cell damage can cause excessive tissue destruction in acute graft-versus-host disease (GVHD). Certain key cytokines for GVHD like TNF-α and Fas-ligand, are secreted by ectodomain shedding from matrix metalloproteinases (MMPs). Many MMP inhibitors have been developed and used over the last three or four decades, but its widespread use is hampered by severe side effects. We reported that the fibrinolytic system, above all plasmin formation promotes the activation of several MMPs. Therefore we hypothesized that plasmin inhibition might be a novel means to control acute GVHD in part by suppressing MMP and inflammatory cytokine secretion. We found that the fibrinolytic system is activated during the development of acute GVHD in mice and in humans. Here, we examined a novel plasmin inhibitor (YO-2) that inhibits MMP activation and inflammatory cytokines release for lethal acute GVHD model in mice. Administration of YO-2 into (BALB/c × C57BL/6) F1 that received C57BL/6 spleen cells showed markedly reduced mortality and weight loss in association with minimal signs of GVHD pathology in the liver, intestine, and hematopoietic tissues. Furthermore, our results suggest that administaration of YO-2 blocked the processing of inflammatory cytokines through MMP inhibition in vitro and in vivo. These data have major implications for transplantation medicine, as pharmacological inhibition of plasmin does not require the need for intensive immunosuppression. Our results suggest that YO-2 could be a novel molecular therapeutic agent for GVHD. Disclosures: No relevant conflicts of interest to declare.