Microbial ligand-independent regulation of lymphopoiesis by NOD1.

Aberrant differentiation of progenitor cells in the hematopoietic system is known to severely impact host immune responsiveness. Here we demonstrate that NOD1, a cytosolic innate sensor of bacterial peptidoglycan, also functions in murine hematopoietic cells as a major regulator of both the generation and differentiation of lymphoid progenitors as well as peripheral T lymphocyte homeostasis. We further show that NOD1 mediates these functions by facilitating STAT5 signaling downstream of hematopoietic cytokines. In steady-state, loss of NOD1 resulted in a modest but significant decrease in numbers of mature T, B and natural killer cells. During systemic protozoan infection this defect was markedly enhanced, leading to host mortality. Lack of functional NOD1 also impaired T cell-dependent anti-tumor immunity while preventing colitis. These findings reveal that, in addition to its classical role as a bacterial ligand receptor, NOD1 plays an important function in regulating adaptive immunity through interaction with a major host cytokine signaling pathway.

Iterative epigenomic analyses in the same single cell.

Gene expression in individual cells is epigenetically regulated by DNA modifications, histone modifications, transcription factors, and other DNA-binding proteins. It has been shown that multiple histone modifications can predict gene expression and reflect future responses of bulk cells to extracellular cues. However, the predictive ability of epigenomic analysis is still limited for mechanistic research at a single cell level. To overcome this limitation, it would be useful to acquire reliable signals from multiple epigenetic marks in the same single cell. Here, we propose a new approach and a new method for analysis of several components of the epigenome in the same single cell. The new method allows reanalysis of the same single cell. We found that reanalysis of the same single cell is feasible, provides confirmation of the epigenetic signals, and allows application of statistical analysis to identify reproduced reads using data sets generated only from the single cell. Reanalysis of the same single cell is also useful to acquire multiple epigenetic marks from the same single cells. The method can acquire at least five epigenetic marks: H3K27ac, H3K27me3, mediator complex subunit 1, a DNA modification, and a DNA-interacting protein. We can predict active signaling pathways in K562 single cells using the epigenetic data and confirm that the predicted results strongly correlate with actual active signaling pathways identified by RNA-seq results. These results suggest that the new method provides mechanistic insights for cellular phenotypes through multilayered epigenome analysis in the same single cells.

Optimization of the Lead Compound NVP-BHG712 as a Colorectal Cancer Inhibitor.

The ephrin type-A receptor 2 (EPHA2) kinase belongs to the largest family of receptor tyrosine kinases. There are several indications of an involvement of EPHA2 in the development of infectious diseases and cancer. Despite pharmacological potential, EPHA2 is an under-examined target protein. In this study, we synthesized a series of derivatives of the inhibitor NVP-BHG712 and triazine-based compounds. These compounds were evaluated to determine their potential as kinase inhibitors of EPHA2, including elucidation of their binding mode (X-ray crystallography), affinity (microscale thermophoresis), and selectivity (Kinobeads assay). Eight inhibitors showed affinities in the low-nanomolar regime (KD <10 nM). Testing in up to seven colon cancer cell lines that express EPHA2 reveals that several derivatives feature promising effects for the control of human colon carcinoma. Thus, we have developed a set of powerful tool compounds for fundamental new research on the interplay of EPH receptors in a cellular context.

Inactivation of axon guidance molecule netrin-1 in human colorectal cancer by an epigenetic mechanism.

Netrin-1, the protein product of the NTN1 gene, is an axon guidance molecule implicated in regulation of cell survival and tumorigenesis. Expression of the netrin-1 receptors deleted in colorectal cancer (DCC) and uncoordinated 5 homolog (UNC5H) is frequently silenced in colorectal cancer (CRC) by either loss of heterozygosity or epigenetic mechanisms. However, netrin-1 expression and regulation in CRC are mostly unknown. Here, we report that NTN1 expression is significantly reduced in most CRC tissues compared to the adjacent normal intestinal mucosa, and that NTN1 DNA methylation is significantly higher in CRCs (24.6%) than in the adjacent normal intestinal mucosa (4.0%). In 6 CRC cell lines, NTN1 expression is low. Treatment with 5-Aza-2'-deoxycytidine increased expression of NTN1 in CRC cell lines, indicating that DNA methylation represses NTN1 transcription in CRCs. NTN1 DNA hypermethylation was significantly associated with advanced CRC disease. Median netrin-1 serum levels were significantly decreased in CRC patients (330.1 pg/mL) compared with normal individuals (438.6 pg/mL). Our results suggest that netrin-1 is a candidate biomarker for CRC.

Ephrin ligands and Eph receptors contribution to hematopoiesis.

Hematopoietic stem and progenitor cells reside predominantly in the bone marrow. They supply billions of mature blood cells every day during life through maturation into multilineage progenitors and self-renewal. Newly produced mature cells serve to replenish the pool of circulating blood cells at the end of their life-span. These mature blood cells and a few hematopoietic progenitors normally exit the bone marrow through the sinusoidal vessels, a specialized venous vascular system that spreads throughout the bone marrow. Many signals regulate the coordinated mobilization of hematopoietic cells from the bone marrow to the circulation. In this review, we present recent advances on hematopoiesis and hematopoietic cell mobilization with a focus on the role of Ephrin ligands and their Eph receptors. These constitute a large family of transmembrane ligands and receptors that play critical roles in development and postnatally. New insights point to distinct roles of ephrin and Eph in different aspects of hematopoiesis.

Burkitt lymphoma expresses oncofetal chondroitin sulfate without being a reservoir for placental malaria sequestration.

Burkitt lymphoma (BL) is a malignant disease, which is frequently found in areas with holoendemic Plasmodium falciparum malaria. We have previously found that the VAR2CSA protein is present on malaria-infected erythrocytes and facilitates a highly specific binding to the placenta. ofCS is absent in other non-malignant tissues and thus VAR2CSA generally facilitates parasite sequestration and accumulation in pregnant women. In this study, we show that the specific receptor for VAR2CSA, the oncofetal chondroitin sulfate (ofCS), is likewise present in BL tissue and cell lines. We therefore explored whether ofCS in BL could act as anchor site for VAR2CSA-expressing infected erythrocytes. In contrast to the placenta, we found no evidence of in vivo sequestering of infected erythrocytes in the BL tissue. Furthermore, we found VAR2CSA-specific antibody titers in children with endemic BL to be lower than in control children from the same malaria endemic region. The abundant presence of ofCS in BL tissue and the absence of ofCS in non-malignant tissue encouraged us to examine whether recombinant VAR2CSA could be used to target BL. We confirmed the binding of VAR2CSA to BL-derived cells and showed that a VAR2CSA drug conjugate efficiently killed the BL-derived cell lines in vitro. These results identify ofCS as a novel therapeutic BL target and highlight how VAR2CSA could be used as a tool for the discovery of novel approaches for directing BL therapy.

Induction of Kaposi's Sarcoma-Associated Herpesvirus-Encoded Viral Interleukin-6 by X-Box Binding Protein 1.

UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent for Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and a subset of multicentric Castleman disease (MCD). The KSHV life cycle has two principal gene repertoires, latent and lytic. KSHV viral interleukin-6 (vIL-6), an analog of human IL-6, is usually lytic; production of vIL-6 by involved plasmablasts is a central feature of KSHV-MCD. vIL-6 also plays a role in PEL and KS. We show that a number of plasmablasts from lymph nodes of patients with KSHV-MCD express vIL-6 but not ORF45, a KSHV lytic gene. We further show that vIL-6 is directly induced by the spliced (active) X-box binding protein-1 (XBP-1s), a transcription factor activated by endoplasmic reticulum (ER) stress and differentiation of B cells in lymph nodes. The promoter region of vIL-6 contains several potential XBP-response elements (XREs), and two of these elements in particular mediate the effect of XBP-1s. Mutation of these elements abrogates the response to XBP-1s but not to the KSHV replication and transcription activator (RTA). Also, XBP-1s binds to the vIL-6 promoter in the region of these XREs. Exposure of PEL cells to a chemical inducer of XBP-1s can induce vIL-6. Patient-derived PEL tumor cells that produce vIL-6 frequently coexpress XBP-1, and immunofluorescence staining of involved KSHV-MCD lymph nodes reveals that most plasmablasts expressing vIL-6 also coexpress XBP-1. These results provide evidence that XBP-1s is a direct activator of KSHV vIL-6 and that this is an important step in the pathogenesis of KSHV-MCD and PEL. IMPORTANCE: Kaposi sarcoma herpesvirus (KSHV)-associated multicentric Castleman disease (KSHV-MCD) is characterized by severe inflammatory symptoms caused by an excess of cytokines, particularly KSHV-encoded viral interleukin-6 (vIL-6) produced by lymph node plasmablasts. vIL-6 is usually a lytic gene. We show that a number of KSHV-MCD lymph node plasmablasts express vIL-6 but do not have full lytic KSHV replication. Differentiating lymph node B cells express spliced (active) X-box binding protein-1 (XBP-1s). We show that XBP-1s binds to the promoter of vIL-6 and can directly induce production of vIL-6 through X-box protein response elements on the vIL-6 promoter region. We further show that chemical inducers of XBP-1s can upregulate production of vIL-6. Finally, we show that most vIL-6-producing plasmablasts from lymph nodes of KSHV-MCD patients coexpress XBP-1s. These results demonstrate that XBP-1s can directly induce vIL-6 and provide evidence that this is a key step in the pathogenesis of KSHV-MCD and other KSHV-induced diseases.

Clinical Features and Outcomes of Patients With Symptomatic Kaposi Sarcoma Herpesvirus (KSHV)-associated Inflammation: Prospective Characterization of KSHV Inflammatory Cytokine Syndrome (KICS).

BACKGROUND: Kaposi sarcoma herpesvirus (KSHV) is the cause of Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and a form of Castleman disease (KSHV-MCD). Recently a KSHV-associated inflammatory cytokine syndrome (KICS) distinct from KSHV-MCD was reported. METHODS: We prospectively characterized the clinical, laboratory, virologic and immunologic features of KICS by evaluating symptomatic adults with KSHV using a prespecified definition. These features and overall survival were compared with controls from 2 prospectively characterized human immunodeficiency virus (HIV)-infected cohorts, including 1 with KSHV coinfection. RESULTS: All 10 KICS subjects were HIV infected males; 5 had HIV viral load (VL) suppressed <50 copies mL (median 72, range <50-74 375); all had KS and 2 also had PEL. All had multiple severe symptoms attributable to KICS: median number of symptoms 8 (6-11), median grade of worst symptom 3 (2-4). These included gastrointestinal disturbance (present in 9); edema (9); respiratory (6); and effusions (5). Laboratory abnormalities included anemia (all); hypoalbuminemia (all) and thrombocytopenia (6). None developed KSHV-MCD; 6 died with median survival from KICS diagnosis 13.6 months. KICS subjects compared with controls had more severe symptoms; lower hemoglobin and albumin; higher C-reactive protein; higher KSHV VL; elevated interleukin (IL)-6 and IL-10; and an increased risk of death (all P < .05). Anemia and hypoalbuminemia at presentation were independently associated with early death. CONCLUSIONS: KICS subjects demonstrated diverse severe symptoms, a high rate of KSHV-associated tumors, high mortality, and a distinct IL-6/IL-10 signature. KICS may be an important unrecognized cause of morbidity and mortality, including symptoms previously ascribed to HIV. Exploration of KSHV-directed therapy is warranted.

Human and viral interleukin-6 and other cytokines in Kaposi sarcoma herpesvirus-associated multicentric Castleman disease.

Kaposi sarcoma herpesvirus (KSHV)-associated multicentric Castleman disease (MCD) is a polyclonal B-cell lymphoproliferative disorder. Human (h) IL-6 and a KSHV-encoded homolog, viral IL-6, have been hypothesized to contribute to its pathogenesis, but their relative contributions to disease activity is not well understood. We prospectively characterized KSHV viral load (VL), viral (v) and hIL-6, and other cytokines during KSHV-MCD flare and remission in 21 patients with 34 flares and 20 remissions. KSHV-VL, vIL-6, hIL-6, IL-10, and to a lesser extent TNF-α, and IL-1ß were each elevated during initial flares compared with remission. Flares fell into 3 distinct IL-6 profiles: those associated with elevations of vIL6-only (2 flares, 6%), hIL-6 elevations only (17 flares, 50%), and elevations in both hIL-6 and vIL-6 (13 flares, 38%). Compared with hIL-6-only flares, flares with elevated hIL-6 plus vIL-6 exhibited higher C-reactive protein (CRP) (P = .0009); worse hyponatremia (P = .02); higher KSHV VL (P = .016), and higher IL-10 (P = .012). This analysis shows vIL-6 and hIL-6 can independently or together lead to KSHV-MCD flares, and suggests that vIL-6 and hIL-6 may jointly contribute to disease severity. These findings have implications for the development of novel KSHV-MCD therapies targeting IL-6 and its downstream signaling. This trial was registered at clinicaltrials.gov as #NCT099073.

Investigation of the interactions between the EphB2 receptor and SNEW peptide variants.

EphB2 interacts with cell surface-bound ephrin ligands to relay bidirectional signals. Overexpression of the EphB2 receptor protein has been linked to different types of cancer. The SNEW (SNEWIQPRLPQH) peptide binds with high selectivity and moderate affinity to EphB2, inhibiting Eph-ephrin interactions by competing with ephrin ligands for the EphB2 high-affinity pocket. We used rigorous free energy perturbation (FEP) calculations to re-evaluate the binding interactions of SNEW peptide with the EphB2 receptor, followed by experimental testing of the computational results. Our results provide insight into dynamic interactions of EphB2 with SNEW peptide. While the first four residues of the SNEW peptide are already highly optimized, change of the C-terminal end of the peptide has the potential to improve SNEW-binding affinity. We identified a PXSPY motif that can be similarly aligned with several other EphB2-binding peptides.

Semaphorin 6A regulates angiogenesis by modulating VEGF signaling.

Formation of new vessels during development and in the mature mammal generally proceeds through angiogenesis. Although a variety of molecules and signaling pathways are known to underlie endothelial cell sprouting and remodeling during angiogenesis, many aspects of this complex process remain unexplained. Here we show that the transmembrane semaphorin6A (Sema6A) is expressed in endothelial cells, and regulates endothelial cell survival and growth by modulating the expression and signaling of VEGFR2, which is known to maintain endothelial cell viability by autocrine VEGFR signaling. The silencing of Sema6A in primary endothelial cells promotes cell death that is not rescued by exogenous VEGF-A or FGF2, attributable to the loss of prosurvival signaling from endogenous VEGF. Analyses of mouse tissues demonstrate that Sema6A is expressed in angiogenic and remodeling vessels. Mice with null mutations of Sema6A exhibit significant defects in hyaloid vessels complexity associated with increased endothelial cell death, and in retinal vessels development that is abnormally reduced. Adult Sema6A-null mice exhibit reduced tumor, matrigel, and choroidal angiogenesis compared with controls. Sema6A plays important roles in development of the nervous system. Here we show that it also regulates vascular development and adult angiogenesis.

HHV-8-encoded viral IL-6 collaborates with mouse IL-6 in the development of multicentric Castleman disease in mice.

Human herpes virus 8 (HHV-8) or Kaposi sarcoma-associated herpes virus is the etiologic agent of Kaposi sarcoma, primary effusion lymphoma, and plasma cell-type multicentric Castleman disease (MCD). HHV-8 encodes a viral homolog of human IL-6, called viral IL-6 (vIL-6), which does not require the cellular IL-6 receptor for binding to the ubiquitously expressed gp130 receptor subunit and subsequent JAK-STAT signaling. Thus, in contrast to IL-6, vIL-6 can stimulate virtually all cells in the body. To elucidate the mechanism by which vIL-6 drives human diseases, we generated transgenic mice that constitutively express vIL-6 under control of the MHC class I promoter. The mice were found to exhibit vIL-6 serum levels comparable with those observed in HHV-8-infected patients, to contain elevated amounts of phosphorylated STAT3 in spleen and lymph nodes, where vIL-6 was produced, and to spontaneously develop key features of human plasma cell-type MCD, including splenomegaly, multifocal lymphadenopathy, hypergammaglobulinemia, and plasmacytosis. Transfer of the vIL-6 transgene onto an IL-6-deficient genetic background abrogated MCD-like phenotypes, indicating that endogenous mouse IL-6 is a crucial cofactor in the natural history of the disease. Our results in mice suggest that human IL-6 plays an important role in the pathogenesis of HHV-8-associated MCD.

Adult human circulating CD34⁻Lin⁻CD45⁻CD133⁻ cells can differentiate into hematopoietic and endothelial cells.

A precise identification of adult human hemangioblast is still lacking. To identify circulating precursors having the developmental potential of the hemangioblast, we established a new ex vivo long-term culture model supporting the differentiation of both hematopoietic and endothelial cell lineages. We identified from peripheral blood a population lacking the expression of CD34, lineage markers, CD45 and CD133 (CD34⁻Lin⁻CD45⁻CD133⁻ cells), endowed with the ability to differentiate after a 6-week culture into both hematopoietic and endothelial lineages. The bilineage potential of CD34⁻Lin⁻CD45⁻CD133⁻ cells was determined at the single-cell level in vitro and was confirmed by transplantation into NOD/SCID mice. In vivo, CD34⁻Lin⁻CD45⁻CD133⁻ cells showed the ability to reconstitute hematopoietic tissue and to generate functional endothelial cells that contribute to new vessel formation during tumor angiogenesis. Molecular characterization of CD34⁻Lin⁻D45⁻CD133⁻ cells unveiled a stem cell profile compatible with both hematopoietic and endothelial potentials, characterized by the expression of c-Kit and CXCR4 as well as EphB4, EphB2, and ephrinB2. Further molecular and functional characterization of CD34⁻Lin⁻CD45⁻CD133⁻ cells will help dissect their physiologic role in blood and blood vessel maintenance and repair in adult life.

High-dose zidovudine plus valganciclovir for Kaposi sarcoma herpesvirus-associated multicentric Castleman disease: a pilot study of virus-activated cytotoxic therapy.

Kaposi sarcoma herpesvirus (KSHV)-associated multicentric Castleman disease (MCD) is a lymphoproliferative disorder most commonly observed in HIV-infected patients. It is characterized by KSHV-infected plasmablasts that frequently express lytic genes. Patients manifest inflammatory symptoms attributed to overproduction of KSHV viral IL-6, human IL-6, and human IL-6. There is no standard therapy and no established response criteria. We investigated an approach targeting 2 KSHV lytic genes, ORF36 and ORF21, the protein of which, respectively, phosphorylate ganciclovir and zidovudine to toxic moieties. In a pilot study, 14 HIV-infected patients with symptomatic KSHV-MCD received high-dose zidovudine (600 mg orally every 6 hours) and the oral prodrug, valganciclovir (900 mg orally every 12 hours). Responses were evaluated using new response criteria. A total of 86% of patients attained major clinical responses and 50% attained major biochemical responses. Median progression-free survival was 6 months. With 43 months of median follow-up, overall survival was 86% at 12 months and beyond. At the time of best response, the patients showed significant improvements in C-reactive protein, albumin, platelets, human IL-6, IL-10, and KSHV viral load. The most common toxicities were hematologic. These observations provide evidence that therapy designed to target cells with lytic KSHV replication has activity in KSHV-MCD. This trial was registered at www.clinicaltrials.gov as #NCT00099073.

Human herpesvirus 8+ polyclonal IgMλ B-cell lymphocytosis mimicking plasmablastic leukemia/lymphoma in HIV-infected patients.

PURPOSE: Multicentric Castleman disease (MCD) is a distinct lymphoproliferative disorder characterized by inflammatory symptoms, lymphadenopathy, splenomegaly, and cytopenia. Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus-8 (HHV-8), is the cause of virtually all cases of MCD occurring in patients with HIV infection. MCD lesions characteristically contain HHV-8-infected polyclonal IgMλ plasmablasts. A high frequency of HHV-8-related non-Hodgkin lymphoma has been reported in these patients. PATIENTS AND METHODS: We now report on three patients who presented with severe symptoms of MCD, extreme splenomegaly, and rapid expansion of B-cell lymphocytosis (44-81%) attributable to circulating HHV-8 positive plasmablasts. RESULTS: The circulating plasmablastic cells shared the phenotype (IgMλ, CD19+, CD20- CD138-) of HHV-8-infected cells from MCD lesions, mimicking the leukemic phase of large B-cell lymphoma occurring in HHV-8-related MCD. These patients displayed a very high HHV-8 viral load in blood (>7 logs HHV-8 DNA copies/ml) and high levels of serum vIL-6, the viral homolog of human interleukin 6. Serum IL-6 and IL-10 were also abnormally elevated. HHV-8-infected cells were demonstrated by immunoglobulin gene rearrangement analysis, to be polyclonal and likely represent an expansion of HHV-8-infected cells similar to those found in MCD lesions. CONCLUSION: Thus, the spectrum of HHV-8-related plasmablastic lymphoproliferative disorders in patients with HIV infection is expanded to include HHV-8+ polyclonal IgMλ B-cell lymphocytosis. At onset, this lymphoproliferative disorder may mimic plasmablastic leukemia/lymphoma. Recognizing this unusual complication may have important implications in treatment decision avoiding unnecessary toxicity to the patients.

Targeting EPHA2 with Kinase Inhibitors in Colorectal Cancer.

The ephrin type-A 2 receptor tyrosine kinase (EPHA2) is involved in the development and progression of various cancer types, including colorectal cancer (CRC). There is also evidence that EPHA2 plays a key role in the development of resistance to the endothelial growth factor receptor (EGFR) monoclonal antibody Cetuximab used clinically in CRC. Despite the promising pharmacological potential of EPHA2, only a handful of specific inhibitors are currently available. In this concept paper, general strategies for EPHA2 inhibition with molecules of low molecular weight (small molecules) are described. Furthermore, available examples of inhibiting EPHA2 in CRC using small molecules are summarized, highlighting the potential of this approach.

NF-kappaB activation stimulates transcription and replication of retrovirus XMRV in human B-lineage and prostate carcinoma cells.

Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus linked to prostate carcinoma and chronic fatigue syndrome. Here we report that NF-κB activation can markedly increase XMRV production. The inflammatory cytokine tumor necrosis factor alpha (TNF-α), which activates NF-κB, significantly augmented viral Gag protein production in XMRV-infected cells. Reporter assays showed that TNF-α and Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), an intrinsic NF-κB activator, increased long terminal repeat (LTR)-dependent XMRV transcription. We identified two NF-κB binding sites (designated κB-1 and κB-2) in the LTR U3 region of XMRV and demonstrated that both sites bind to the NF-κB component p65/RelA. Mutation of the κB-1 site, but not the κB-2 site, impaired responsiveness to TNF-α and LMP1 in reporter assays. A mutant XMRV with a mutation at the κB-1 site replicated significantly less efficiently than the wild-type XMRV in the prostate carcinoma LNCaP, DU145, and PC-3 cell lines, HEK293 cells, the EBV-immortalized cell line IB4, and the Burkitt's lymphoma cell line BJAB. These results demonstrate that TNF-α and EBV LMP1 enhance XMRV replication in prostate carcinoma and B-lineage cells through the κB-1 site in the XMRV LTR, suggesting that inflammation, EBV infection, and other conditions leading to NF-κB activation may promote XMRV spread in humans.

Oligo-guanosine nucleotide induces neuropilin-1 internalization in endothelial cells and inhibits angiogenesis.

Ligand interaction with cognate cell-surface receptor often promotes receptor internalization, protecting cells from prolonged or excessive signaling from extracellular ligands. Compounds that induce internalization of surface receptors prevent ligand binding to cognate cell-surface receptors serving as inhibitors. Here, we show that synthetic polyriboguanosine (poly G) and oligo-deoxyriboguanosine (oligo G) reduce endothelial levels of surface neuropilin-1 (NRP1), a receptor shared by semaphorin 3A and vascular endothelial growth factor (VEGF), which plays critical roles in angiogenesis. Oligo G also reduces levels of cell-surface scavenger receptor expressed by endothelial cells I (SREC-I), but not levels of NRP2, gp130, CD31, VEGFR-1, or VEGFR-2. Poly or oligo A, T, and C do not promote NRP1 or SREC-I internalization. We find that oligo G binds to NRP1 with high affinity (Kd:1.3 ± 0.16 nM), bridges the extracellular domain of NRP1 to that of SREC-I, and induces coordinate internalization of NRP1 and SREC-I. In vitro, oligo G blocks the binding and function of VEGF(165) in endothelial cells. In vivo, intravitreal administration of oligo G reduces choroidal neovascularization in mice. These results demonstrate that synthetic oligo G is an inhibitor of pathologic angiogenesis that reduces cell-surface levels and function of NRP1 acting as an internalization inducer.

The transcription factor Gfi1 regulates G-CSF signaling and neutrophil development through the Ras activator RasGRP1.

The transcription factor growth factor independence 1 (Gfi1) and the growth factor granulocyte colony-stimulating factor (G-CSF) are individually essential for neutrophil differentiation from myeloid progenitors. Here, we provide evidence that the functions of Gfi1 and G-CSF are linked in the regulation of granulopoiesis. We report that Gfi1 promotes the expression of Ras guanine nucleotide releasing protein 1 (RasGRP1), an exchange factor that activates Ras, and that RasGRP1 is required for G-CSF signaling through the Ras/mitogen-activated protein/extracellular signal-regulated kinase (MEK/Erk) pathway. Gfi1-null mice have reduced levels of RasGRP1 mRNA and protein in thymus, spleen, and bone marrow, and Gfi1 transduction in myeloid cells promotes RasGRP1 expression. When stimulated with G-CSF, Gfi1-null myeloid cells are selectively defective at activating Erk1/2, but not signal transducer and activator of transcription 1 (STAT1) or STAT3, and fail to differentiate into neutrophils. Expression of RasGRP1 in Gfi1-deficient cells rescues Erk1/2 activation by G-CSF and allows neutrophil maturation by G-CSF. These results uncover a previously unknown function of Gfi1 as a regulator of RasGRP1 and link Gfi1 transcriptional control to G-CSF signaling and regulation of granulopoiesis.

MicroRNA126 contributes to granulocyte colony-stimulating factor-induced hematopoietic progenitor cell mobilization by reducing the expression of vascular cell adhesion molecule 1.

BACKGROUND: Mobilization of hematopoietic stem/progenitor cells from the bone marrow to the peripheral blood by granulocyte colony-stimulating factor is the primary means to acquire stem cell grafts for hematopoietic cell transplantation. Since hematopoietic stem/progenitor cells represent a minority of all blood cells mobilized by granulocyte colony-stimulating factor, the underlying mechanisms need to be understood in order to develop selective drugs. DESIGN AND METHODS: We analyzed phenotypic, biochemical and genetic changes in bone marrow cell populations from granulocyte colony-stimulating factor-mobilized and control mice, and linked such changes to effective mobilization of hematopoietic stem/progenitor cells. RESULTS: We show that granulocyte colony-stimulating factor indirectly reduces expression of surface vascular cell adhesion molecule 1 on bone marrow hematopoietic stem/progenitor cells, stromal cells and endothelial cells by promoting the accumulation of microRNA-126 (miR126)-containing microvescicles in the bone marrow extracellular compartment. We found that hematopoietic stem/progenitor cells, stromal cells and endothelial cells readily incorporate these miR126-loaded microvescicles, and that miR126 represses vascular cell adhesion molecule 1 expression on bone marrow hematopoietic stem/progenitor cells, stromal cells and endothelial cells. In line with this, miR126-null mice displayed a reduced mobilization response to granulocyte colony-stimulating factor. CONCLUSIONS: Our results implicate miR126 in the regulation of hematopoietic stem/progenitor cell trafficking between the bone marrow and peripheral sites, clarify the role of vascular cell adhesion molecule 1 in granulocyte colony-stimulating factor-mediated mobilization, and have important implications for improved approaches to selective mobilization of hematopoietic stem/progenitor cells.