The Role of gas6 in Venous Thrombosis In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3057-3057
Author(s):  
Richard Robins ◽  
Peter Carmeliet ◽  
Mark Blostein
Keyword(s):  
Bone Marrow ◽  
Venous Thrombosis ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Recovery Period ◽  
Vena Cava ◽  
Specific Gene ◽  
Combined Genotypes

Abstract Abstract 3057 Poster Board II-1033 Gas6 is the vitamin-K dependent protein product of growth arrest specific gene 6. A genetic deficiency of this protein protects mice against experimentally induced thrombosis without causing a bleeding diathesis. Protection from thrombosis results from a deficiency in platelet aggregation and secretion. In addition to being expressed by platelets, Gas6 and its receptors are also expressed by vascular cells including the endothelium, an organ known to play a role in the hemostatic balance. While endothelial Gas6 has been shown to promote inflammation and cell survival, it remains unknown if it contributes to the pathophysiology of venous thrombosis. To answer this question, we employed a bone marrow transplantation (BMT) strategy using wild type and Gas6 null mice to create chimeric mice with combined genotypes in the vascular and platelet compartments. Mice were exposed to a dose of radiation optimized to maximize both survival and ablation of recipient marrow. Irradiated mice were then infused with bone marrow cells isolated from the femurs and tibias of donor mice and were allowed a one month recovery period for hematologic reconstitution. Success of marrow uptake was confirmed by PCR. They were then subjected to the Ferric Chloride model of venous thrombosis in the Inferior Vena Cava (IVC). Four groups of transplanted mice were studied. Results from these BMT experiment show a contributing effect by both endothelial as well as platelet Gas6 to thrombus formation (n=8, p<0.01). Mice with combined genotypes (Gas6-/- into WT and WT into Gas6 -/-) show an intermediate thrombus weight suggesting that both vascular and platelet derived Gas6 are both responsible for thrombosis pathology. Therefore, Gas6 at both sites could be potential targets in treating venous thrombosis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Divisional Memory Drives Hematopoietic Stem Cell Functional Diversity

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 20-20
Author(s):  
James Bartram ◽  
Baobao (Annie) Song ◽  
Juying Xu ◽  
Nathan Salomonis ◽  
H. Leighton Grimes ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Functional Decline ◽  
Functional Heterogeneity ◽  
Hematopoietic Stem ◽  
Donor Cells ◽  
Marrow Cells

Abstract Hematopoietic stem cells are endowed with high regenerative potential but their actual self-renewal capacity is limited. Studies using the H2B-retention labeling system show HSC functional decline at each round of division (Qiu, Stem Cell Reports 2014). We have shown that mitochondria drive HSC functional decline with division history after transplantation (Cell Stem Cell 2020). Here we examined the link between mitochondrial metabolism, in vivo division at steady state, and HSC functions using the GFP label-Histone 2B (GFP-H2B) mouse model driven by a doxycycline-inducible promoter. Five months after doxycycline removal, mitochondrial membrane potential (MMP) was examined using TMRE in HSC with varying GFP intensity. HSC were separated into an H2B-labeled retention population and an H2B-labeled population. Interestingly, within the H2B-labeled retention population, HSC could be further subdivided into GFP high, medium, and low. MMP increased in a stepwise fashion with GFP dilution in HSC. We noted the presence of 2 TMRE peaks within each GFP high and medium populations leading to 5 populations: GFP-high;MMP-low (G1), GFP-high;MMP-high (G2), GFP-medium;MMP-low (G3), GFP-medium;MMP-high (G4), GFP-low;MMP-high (G5). We examined the repopulation activity of each population in a serial competitive transplant assay. G1 and G2 maintained higher peripheral blood chimerism up to 24 weeks post-transplant than G3 and G4. G5 did not engraft at all. However, only G1 reconstituted high frequency of HSC in primary recipients. In secondary recipients, G1, G2, G3 but not G4 gave rise to positive engraftment. Interestingly, G1 and G2 grafts showed myeloid/lymphoid balanced engraftment whereas the G3 graft was myeloid-bias, suggesting that myeloid skewing can be acquired upon HSC division. We further examined lineage fate maps of bone marrow cells derived from G1 or G3 population in vivo, using single cell RNA sequencing, 10X genomics. Surprisingly, G3-derived bone marrow cells displayed a distinct myeloid cell trajectory from G1-derived bone marrow cells, in which G3 gave rise to increased immature neutrophils but fewer myeloid precursors. Remarkably, each lineage population derived from G3 donor cells had different gene expression signatures than those derived from G1 donor cells. Therefore, HSC that have divided in vivo in the same bone marrow microenvironment are intrinsically and molecularly different such that not only do they exhibit lineage potential differences but they also produce progeny that are transcriptionally different. These findings imply that cellular division rewires HSC and that this rewiring is passed down to their fully differentiated progeny. When G1 and G3 single HSC were cultured in-vitro, G1 had a slower entry into cell-cycle which has been associated with increased stemness. Additionally, when single HSC from G1 and G3 were assessed for their multipotency in a lineage differentiation assay, G1 HSC had a higher propensity to produce all four myeloid lineages (megakaryocytes, neutrophils, macrophages, and erythroid), further supporting increased stemness in G1 compared to G3 HSC. Finally, HSC from G1, G2, G3 and G4 populations carried mitochondria that were morphologically different, and express distinct levels of Sca-1, CD34 and EPCR, with Sca-1 high, CD34-, EPCR+ cells more enriched in G1. In summary, this study suggests that HSC transition into distinct metabolic and functional states with division history that may contribute to HSC diversity and functional heterogeneity. It also suggests the existence of a cell-autonomous mechanism that confers HSC divisional memory to actively drive HSC functional heterogeneity and aging. Disclosures No relevant conflicts of interest to declare.

In Vivo Monitoring of Venous Thrombosis In Mice Using Ultrasonography

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4214-4214
Author(s):  
Meghedi Aghourian ◽  
Catherine Lemarie ◽  
Mark Blostein
Keyword(s):  
Venous Thrombosis ◽  
Murine Model ◽  
Conflicts Of Interest ◽  
Genetically Modify ◽  
Vena Cava ◽  
Venous Stasis ◽  
Clinical Aspects ◽  
Reliable Technique ◽  
Over Time

Abstract Abstract 4214 Deep venous thrombosis is an important cause of morbidity and mortality in clinical medicine. There has been extensive research dedicated to the clinical aspects of venous thrombosis, especially with regards to its diagnosis and treatment. However, animal models studying this phenomenon are scarce and, in most cases, very crude, relying on sacrificing animals to excise the formed thrombi. Developing an in vivo murine model of venous thrombosis, detecting and monitoring thrombi non-invasively as is done in humans can be a powerful tool given our ability to genetically modify the murine genome. Therefore, we developed such a murine model using the Vevo770®, a microimaging ultrasound system previously developed to study the arterial circulation of mice. Two different thrombosis models were employed to generate clots in the inferior vena cava (IVC) of wild type C57Bl6 mice: 1) ligation of the IVC to generate venous stasis and 2) application of Ferric Chloride (FeCl3) to the outer layer of the IVC to injure the endothelium. Using both of these techniques, adequate thromboses were generated in the IVCs of mice as determined pathologically. Other mice were allowed to recover after surgery, and the development of venous thrombosis was assessed by ultrasonography using the Vevo 770®. In order to assess the precision of clot measurements using this novel technique, we then sacrificed the mice and excised the clots. In both models, the measurement of the clot pathologically correlates favorably (R2= 0, 9116 for the ligation model, and R2 = 0,905 for the FeCl3 model) with measurements done by ultrasonography (n=20 for the ligation model, and n=5 for the FeCl3 injury model). In the ligation model, a thrombus develops less than an hour after ligation of the IVC, and the size of the clot increases over time. For example, five hours after the ligation of the IVC, a clot develops and has a cross sectional area of 4,5 mm2. The clot size increases significantly (p=0.001) over time to 6.2 mm2 at 24 hours post ligation (n=20). Treatment of these mice with an anticoagulant (dalteparin at a dose of 200 u/kg) prior to the procedure prevented the development of IVC thrombosis as determined by ultrasonagraphy. These data suggest that the Vevo770® can be used as a reliable technique for the non-invasive assessment of venous thrombosis in mice. Developing a murine model for thrombosis using more accurate, and clinically more relevant techniques such as ultrasonography, is a step towards better understanding the pathophysiology of venous thromboembolism. Figure 1. Clot length correlation using histology and ultrasonography, 24 hrs post ligation of the IVC in 20 mice. R2= 0,9116. Figure 1. Clot length correlation using histology and ultrasonography, 24 hrs post ligation of the IVC in 20 mice. R2= 0,9116. Disclosures: No relevant conflicts of interest to declare.

In Vivo Monitoring of Venous Thrombosis in Mice.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5061-5061
Author(s):  
Meghedi Aghourian ◽  
Mark Blostein
Keyword(s):  
Venous Thromboembolism ◽  
Venous Thrombosis ◽  
Murine Model ◽  
Conflicts Of Interest ◽  
Vena Cava ◽  
Reliable Technique ◽  
Non Invasive ◽  
Post Surgery ◽  
Ultrasound Technology

Abstract Abstract 5061 Venous thromboembolism afflicts 117 people per 100,000 each year and is an important cause of morbidity and mortality. There has been extensive research dedicated to the clinical aspect of venous thrombosis, especially with regards to its diagnosis and treatment. However, animal models studying this phenomenon are scarce and, in most cases, very crude. Developing a murine model of venous thrombosis using techniques similar to the ones used to detect thrombosis in humans can be a constructive step in studying this phenomenon in more detail. The model developed in our lab uses ultrasound imaging to visualize venous clots in the Inferior Vena Cava (IVC) of mice, allowing for precise measurements of the formed clot. Ligation of the IVC is one of the well established models for studying thrombosis in mice. We ligated the IVC of wild type C57B6 mice, and allowed them to recover. We then followed clot formation at several time points after the operation using micro-ultrasonography, the Vevo 770®, a novel imaging ultrasound technology designed to monitor murine vasculature. To assess the precision of the clot measurements, we then sacrificed the mice, and dissected out the thrombi in order to precisely measure and weigh them. A thrombosis develops only after 5 hours of ligation post surgery when a clot is visualized in the IVC. The clot increases slightly over the next 24 hours. The measurements of the clot after dissection correlates favourably with the measurements done by ultrasonagraphy using the Vevo770®. These data suggest that the Vevo770® can be used as a reliable technique for non-invasive assessment of venous thromboembolism in mice. Developing a murine model for thrombosis using more accurate, and clinically more relevant techniques such as ultrasonography, is a step towards better understanding and treatment of venous thromboembolism. Disclosures No relevant conflicts of interest to declare.

JAK2V617F Promotes Stem Cell Amplification Driving MPN Clonal Dominance in Mice and Treatment by IFNa Prevents This Effect

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 616-616 ◽  
Author(s):  
Caroline Marty ◽  
Catherine Lacout ◽  
Marie Cuingnet ◽  
Salma Hasan ◽  
Eric Solary ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasm ◽  
Jak2 V617f ◽  
Lymphoid Cells ◽  
Hematopoietic Stem ◽  
Endogenous Expression ◽  
Long Term Treatment

Abstract Abstract 616 JAK2V617F is the major mutation involved in classic myeloproliferative neoplasm (MPN). It promotes growth factor independent cell growth and is able to recapitulate MPN features in retroviral, transgenic (TG) or knock-in (KI) mouse models. Several mutations implicated in epigenetic modifications or leukemic transformations have been also identified in MPN and several reports have questioned the particular role of JAK2V617F on hematopoietic stem cells (HSC) proliferation thus as a driver of MPN emergence. Therefore, we investigated the in vivo effect of an endogenous expression of JAK2V617F on early stages of differentiation and their ability to compete for normal cells in a repopulation assay. For this study, we develop a novel mouse conditional JAK2V617F KI model based on the “FLEX switch” strategy. These KI mice were crossed with TG mice expressing the Cre recombinase under the control of the vav promoter in order to restrict JAK2V617F expression to hematopoietic and some endothelial tissues. VavCre/JAK2+/V617F KI mice developed high hematocrit (70 ± 2 %, control values 49 ± 1 % n=13), platelet (2.3 ± 0.1 × 109 / mL, control values 0.84 ± 0.04 × 109 / mL n=20) and white blood cell (20-40 × 106/mL, control values between 6–10 × 106 / mL) values and a splenomegaly at 2–3 months of age but after 6 months of age an anemia and a thrombocytopenia appeared. This model mimics human polycythemia vera with secondary myelofibrosis. At 2–3 months of age, cumulative numbers in bone marrow (BM) and spleen of CFU-E, BFU-E and GM-CFC were increased 15-, 3-, 1.2–fold, respectively, compared to control. Most CFU-E grew without the addition of erythropoietin. A 6-fold amplification of total early progenitors LSK and a tendency toward SLAM (LSK/CD48−/CD150+) cell amplification, mainly due to a significant 9-fold increase in the spleen, were also observed. Competitive repopulation assays using 30% KI and 70% WT bone marrow cells demonstrated 17 weeks after BM transplantation (BMT) a rapid and strong amplification, from 30% to > 80%, of blood myeloid cells (Gr-1+/Mac1+) from KI origin. Late after transplantation (35 weeks), Lin-, LSK and SLAM cell compartments from KI origin raised from the initial 30% to almost 100% in the BM and even KI blood lymphoid cells (B220+ and CD3+) demonstrated a significant amplification compared to control. This shows that endogenous expression of JAK2V617F gives an advantage to HSC, promoting clonal dominance in mice. Then, we analyzed at which levels of differentiation acts IFNα, a drug promoting cycling of dormant cells and proven efficacious in PV treatment in human. In a chimeric model, we demonstrated that IFNα could prevent the development of MPN induced in vavCre/JAK2+/V617F KI recipient mice by inhibiting the amplification of KI cells. Secondary BMT from treated animals demonstrated the eradication of disease-initiating cells after long-term treatment. This study shows that IFNα acts at the level of the disease-initating cell by reverting the HSC promoting clonal dominance induced by JAK2V617F. Disclosures: No relevant conflicts of interest to declare.

The Ryk Receptor Regulates Hematopoietic Stem and Progenitor Cell Proliferation and Their Sensitivity To Myeloablative Agents

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 796-796
Author(s):  
Benjamin Povinelli ◽  
Michael Nemeth
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Molecular Mechanisms ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Peripheral Blood Cells ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract The molecular mechanisms that control the balance between quiescence and proliferation of hematopoietic stem and progenitor cells (HSPCs) are critical for maintaining life-long hematopoiesis. In a recent study (Povinelli, et al. Stem Cells, In Press, 2013) we demonstrated that the Wnt5a ligand inhibits HSPC proliferation through a functional interaction with a non-canonical Wnt ligand receptor termed Related to Receptor Tyrosine Kinase (Ryk). Expression of Ryk on HSPCs in vivo was associated with a decreased rate of proliferation. Following treatment with fluorouracil (5-FU), the percentage of Ryk+ HSPCs increased at the expense of Ryk-/low HSPCs. Based on these data, we hypothesized that one function of the Ryk receptor is to protect HSPCs from the effects of myeloablative agents. To test this hypothesis, we injected 6-8 week old C57BL/6 mice with 150 mg/kg of 5-FU and analyzed bone marrow 48 hours later for the presence of apoptotic HSPCs, defined as lineage negative (Lin-), Sca-1+, CD48- cells positive for active caspase-3. There was a 2.5-fold decrease in the percentage of apoptotic Ryk+ HSPCs (12.9 ± 1.7%) compared to Ryk-/low HSPCs (32.4 ± 5.3%, p < 0.001, n = 3). To test whether this effect was limited to 5-FU, we performed a similar study in which we irradiated C57BL/6 mice with 3 cGy of total body irradiation (TBI) and analyzed bone marrow 72 hours later for apoptotic HSPCs (for this experiment, defined by a Lin-, c-kit+, Sca-1+, CD150+, CD48- immunophenotype or LSK, SLAM). Comparable to the effects of 5-FU, there was a significant 3.0-fold reduction in the percentage of apoptotic Ryk+ HSPCs (3.1 ± 0.2%) compared to Ryk-/low HSPCs (9.2 ± 1.5%, p < 0. 001, n = 3) in mice receiving 3 cGy TBI. These results demonstrated an association between Ryk expression and survival of HSPCs following myeloablative injury. To determine whether in vivo targeting of the Ryk receptor would increase the sensitivity of HSPCs to myeloablative injury, we utilized a neutralizing rabbit anti-Ryk antibody (α-Ryk). We injected C57BL/6 mice with 5 mg/kg α-Ryk or rabbit IgG isotype for 2 consecutive days. Twenty-four hours after the second dose, we determined the frequency and cell cycle status of LSK SLAM cells. Treatment with α-Ryk significantly increased the percentage of LSK SLAM cells in the S/G2/M phases compared to control (α-Ryk: 17.8 ± 2.2%; isotype IgG: 11.6 ± 2.7%, p < 0.05, n = 3). This was associated with a decrease in the percentage of LSK, SLAM cells in G1 following treatment with α-Ryk (α-Ryk: 40.5 ± 3.2%, isotype IgG: 51.3 ± 2.2; p < 0.01, n = 3). The percentage of G0 LSK SLAM cells was unchanged (α-Ryk: 37.9 ± 2.6, isotype IgG: 35.7 ± 3.1% n = 3) indicating that inhibiting Ryk promoted the exit of LSK SLAM cells from G1. Treatment with α-Ryk also increased the percentage of whole bone marrow cells expressing the LSK SLAM phenotype by 1.4-fold compared to controls (p < 0.05, n = 3). To determine if α-Ryk treatment altered HSPC function, we transplanted whole bone marrow cells from C57BL/6 mice treated with two days of α-Ryk or isotype IgG at a 1:1 ratio with whole bone marrow from untreated Ubc-GFP transgenic mice into lethally irradiated B6.SJL mice. Four weeks after transplant, we analyzed peripheral blood cells for the percentage of CD45.2+ GFP- cells. There was no difference in engraftment by transplanted bone marrow cells from mice treated with α-Ryk or isotype IgG (α-Ryk: 61.6 ± 6.1% n = 4, isotype IgG: 52.8 ± 13.6%, n = 5), indicating that the neutralizing antibody does not inhibit short-term HSPC function on its own. We then tested whether blocking Ryk function resulted in greater sensitivity of HSPCs to 5-FU. We treated B6.SJL mice with 5 mg/kg α-Ryk or isotype IgG for 2 consecutive days, followed by 150 mg/kg of 5-FU. Forty-eight hours after 5-FU treatment, we transplanted 2x106 C57BL/6 whole bone marrow cells into treated B6.SJL mice without additional conditioning. Four weeks after transplant, we determined the percentage of donor-derived CD45.2+ peripheral blood cells. Treatment of recipient mice with α-Ryk prior to 5-FU treatment resulted in increased engraftment of donor bone marrow by 3.6-fold compared to isotype (p < 0.05, n = 5), suggesting that inhibition of Ryk resulted in increased elimination of host HSPCs by 5-FU. Collectively, these data suggest a model in which inhibition of the Ryk receptor results in increased proliferation of HSPCs, rendering them more sensitive to the effects of myeloablative agents such as chemotherapy or TBI. Disclosures: No relevant conflicts of interest to declare.

Leukocyte XII Regulates Venous Thrombosis Risk

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 238-238
Author(s):  
Evi X. Stavrou ◽  
Chao Fang ◽  
Alona A. Merkulova ◽  
Lalitha V. Nayak ◽  
Howard Meyerson ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Venous Thrombosis ◽  
Conflicts Of Interest ◽  
Human Peripheral Blood ◽  
Vena Cava ◽  
Leukocyte Migration ◽  
Venous Stasis ◽  
Factor Xii ◽  
Fibrin Network

Abstract Introduction: Previous studies show that Factor XII (XII) participates in the inflammatory response. XII regulates the expression of monocyte FcγII receptor and stimulates monocytes and macrophages to release interleukin (IL)-1 and IL-6. XII deficient patients have reduced leukocyte migration into skin windows. In vitro, purified XIIa corrects neutrophil aggregation and degranulation defects in XII-deficient plasma. Recent studies show that leukocytes initiate and propagate venous thrombosis in vivo. We examined the contribution of XII in the inflammatory response and venous thrombosis. Methods & Results: Sterile punch biopsy wounds were created on wild type (WT) and F12-/- mice. On Days 2 and 5, there was a ~3-fold decrease in CD11b-stained cells in F12-/- woundsvs. WT. On the thioglycolate (TG)-induced sterile peritonitis assay, lavage fluid from F12-/- mice contained significantly less peritoneal exudative cells (PEC)] on days 1 and 7, (p<0.008). To determine the contribution of XII in WBC function, we used XII siRNA (Alnylam Pharmaceuticals) to create plasma XII deficiency in WT mice. After tail vein injection, plasma XII was reduced to < 5% within 24 h (T1/2 plasma XII: 6.7 h). In the TG assay, even though plasma XII is decreased to less than 5%, PEC migration is the same as in WT mice. These data suggest that the reduced leukocyte migration observed in F12-/- mice is related to altered leukocyte function. On adoptive bone marrow (BM) transplantation (BMT) experiments, WT BM transplanted into KO hosts corrects the leukocyte migration defect on the TG assay. These data suggest that there is a pool of XII associated with BM cells that is functionally distinct than plasma-derived, hepatic XII. F12 cDNA is found in leukocytes and shares sequence homology to hepatic XII. Immunofluorescence confirms XII antigen on murine BM-derived and human peripheral blood WBCs. No XII antigen is observed in BM-derived leukocytes from F12-/- mice. When WBC are activated with fMLF, XII antigen translocates to the external membrane. F12-/- PMNs have reduced chemotaxis to fMLF and adherence to several integrin-binding glycoproteins. pAktS473 mediates neutrophil cell migration, integrin activation, and cytoskeletal assembly. Normal and F12-/- PMNs exhibit pAktS473 in response to fMLF and XII. Histologically, F12-/- wounds show a smaller wound gap and a greater percentage of wound re-epithelialization than WT controls. Inferior vena cava (IVC) thrombosis induced by 90% restriction to flow at 24h contains a smaller thrombus in F12-/- than WT mice (p<0.04). Histologically, IVC thrombi from WT mice contain abundant neutrophils that are adherent to the wall and trapped within a dense fibrin network (Fig 1). siRNA treatment results in less-occlusive thrombi (n.s) with an adequate neutrophil content but a finer fibrin network (Fig 1). F12-/- thrombi are non-occlusive and contain significantly less adherent neutrophils (Fig 1). XII itself is integrally a part of neutrophil extracellular traps (NETs) in the forming thrombus and F12-/- mice have reduced NETs at sites of occlusion. WT BM transplanted into F12-/- hosts corrects the thrombus weight and degree of inflammation in F12-/- mice to normal. Likewise, F12-/- BM into WT hosts, reduces thrombus weight and degree of inflammation. Conclusions: Leukocyte XII has a dual role in neutrophil function. We hypothesize that signaling by leukocyte XII contributes to neutrophil trafficking in sites of inflammation and venous stasis. At these sites, neutrophils become indispensable for activation of both the extrinsic and intrinsic pathways of coagulation during the early formation of intraluminal fibrin and for subsequent thrombus propagation by NETs and the activation of circulating XII. Defining the signaling pathway of XII in leukocytes will further our understanding as to the mechanism(s) by which these cells cooperate to initiate and propagate venous thrombosis in vivo. Disclosures No relevant conflicts of interest to declare.

scholarly journals Epigenetic Targeting of the Myeloma-Bone Microenvironment in 3D

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 246-246 ◽  
Author(s):  
Juraj Adamik ◽  
Yerneni Saigopalakrishna ◽  
Sree Pulugulla ◽  
Quanhong Sun ◽  
Philip E Auron ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Inflammatory Responses ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Marrow Cell ◽  
Critical Role ◽  
Physical Interaction ◽  
Bone Microenvironment ◽  
Selective Protection

Abstract EZH2, the methyltransferase subunit of Polycomb Repressive Complex 2 (PRC2), catalyzes H3K27me3 histone modifications and epigenetically regulates genes involved in cellular pluripotency and differentiation. EZH2 plays a role in myeloma (MM) cell proliferation, survival, stemness and its elevated expression correlates with poor prognosis in MM patients. We have previously shown that EZH2 plays a critical role in preventing osteoblast differentiation of myeloma-exposed bone marrow stromal cells (BMSCs). Here we show that GSK126 blocks MM-induced hyperactivation of osteoclast precursors (OCLp). RNA-seq profiling revealed that inhibition of EZH2 prevented RANKL-induced repression of genes associated with bivalent and/or H3K27me3 promoter signatures including OCL inhibitory factors MafB, Irf8, Bcl6b and Arg1. In contrast, we found that OCLp expansion in MM1.S-conditioned media induced significant gene expression changes, which correlated with TNF and IKK signaling, inflammatory responses and CXC-chemokine receptor pathways. Several classes of small molecule EZH2 inhibitors exhibited anti-MM effects, but their efficacy has been primarily studied in 2-dimensional (2D) cell culture systems or subcutaneous MM-tumor models in vivo. Therefore, we evaluated the effectiveness of GSK126 in the context of the bone microenvironment in a novel 3D model of MM co-cultures (3D-MM). We combined basement membrane extract (BME) hydrogels with devitalized bone slices to mimic the 3D setting of MM with the OCL-resorbing endosteal surface. This enabled us to test GSK126 alone or in combination with bortezomib simultaneously on MM survival and OCLp differentiation and resorption. Differentiating OCLp did not protect MM cells from GSK126 anti-MM effects, nor did the MM cells prevent GSK126 from blocking OCL differentiation. However, mature OCL added to 3D-MM co-cultures increased the IC50 MM inhibition dose of both bortezomib and GSK126 by 40% and 50%, respectively. Further their synergy on MM cells was reduced by 70%. 3D-MM co-cultures with total bone marrows harvested from mice of different ages (1-12 months old) showed selective protection from GSK126, but not bortezomib, on MM viability. Furthermore, the resistance to GSK126 was age-dependent. Cultures of bone marrows from older mice exhibited enhanced protection of MM cells from GSK126 as compared to younger marrows. Using confocal microscopy, we found that in addition to soluble factors, physical interaction between MM and bone marrow cells reduced the effectiveness of the epigenetic drug targeting against MM. Depletion of the BMSC population from the total bone marrows using CD45+ selection before establishing the 3D MM co-cultures resulted in diminished protection of MM cell survival from GSK126. Consistent with this, addition of both primary murine and MM-patient derived BMSCs to MM cultures significantly protected MM cells from EZH2 inhibition. In addition to cell-cell contacts, the pro-survival factor IL6 released by mature OCL and BMSCs, has been implicated in mediating chemo-resistance of MM cells. In agreement with this, addition of soluble IL6 to MM-3D cultures significantly protected MM cells from GSK126 inhibition. Here we show that various cell compartments of the bone microenvironment exhibit differential and drug-specific protection for MM cells from EZH2 inhibition. In addition to direct bone marrow-MM cell interactions, soluble IL6 also exhibits resistance to GSK126. Our novel 3D-MM system enables us to rapidly screen drug combinations, and simultaneously evaluate the influence of bone-microenvironmental interactions on MM drug resistance and bone marrow cell responses to the drugs. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

ENDOTHELIAL GAS6 Contributes to Thrombogenesis and Promotes TISSUE FACTOR Upregulation DURING Vessel Injury

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 702-702
Author(s):  
Richard Robins ◽  
Catherine A Lemarie ◽  
Jianqiu Wu ◽  
Mark Blostein
Keyword(s):  
Tissue Factor ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Vena Cava ◽  
Functional Assay ◽  
Mrna Induction ◽  
Vessel Injury ◽  
Platelet Depletion

Abstract Abstract 702 Gas6 is a vitamin K-dependent, secreted protein that amplifies platelet aggregation and secretion in response to platelet agonists. Gas6−/− mice are protected from experimentally induced lethal venous and arterial thromboembolism. This protection has been attributed to defective aggregation in platelets from gas6−/− mice. However, this platelet phenotype was only observed when platelets were challenged by only one agonist, ADP, and only at a concentrations of 5.0μM. This subtle platelet abnormality resulting in a rather dramatic clinical phenotype raise the possibility that gas6 from a source other than platelets contributes to thrombus formation. We hypothesize that gas6 derived from the endothelium plays a role in venous thrombus formation. Gas6−/− mice are protected against venous thrombosis induced by 0.37 M FeCl3 in the inferior vena cava (IVC). Bone marrow transplantation experiments generating mice with selective ablations of gas6 from either the hematopoietic or non-hematopoietic compartments demonstrate an approximately equal contribution by gas6 from both compartments to thrombus formation. Platelet depletion in wild type or gas6−/− mice followed by reconstitution with platelets from either WT or gas6−/− mice confirm that gas6 from compartments other than the platelet contribute to thrombosis development. Furthermore, gas6−/− mice are hyporesponsive to FeCl3 mediated tissue factor induction in venous endothelium, as observed by immunofluorescence staining and later validated by a functional assay. In addition, in vitro, gas6−/− endothelial cells are hyporesponsive to thrombin mediated tissue factor mRNA induction. Taken together, these results suggest that non-hematopoietic gas6, possibly from the endothelium, contributes to thrombus formation in vivo and can be explained by the ability of gas6 to promote endothelial tissue factor induction. These findings support the notion that endothelial gas6 may play a pathophysiologic role in venous thromboembolism. Disclosures: No relevant conflicts of interest to declare.

Potent Cooperation Between NUP98-NSD1 and FLT3-ITD In AML Induction

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3797-3797
Author(s):  
Angeliki Thanasopoulou ◽  
Alexandar Tzankov ◽  
Juerg Schwaller
Keyword(s):  
Bone Marrow ◽  
Fusion Protein ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Latency Period ◽  
Histopathological Analysis ◽  
Murine Bone Marrow ◽  
Marrow Cells

Abstract The NUP98-NSD1 fusion protein, product of the t(5;11)(q35;p15.5) chromosomal translocation, is an AML-associated cytogenetically silent genetic aberration, recently identified as the most frequent fusion in pediatric AML, generally associated with aggressive disease and poor prognosis. Interestingly, the vast majority (>70%) of the reported NUP98-NSD1-positive cases also carried an activating FLT3-ITD mutation suggesting functional cooperation. The purpose of this study was to search for experimental evidence of a functional cooperation between NUP98-NSD1 and FLT3-ITD in the transformation of murine hematopoietic cells in vitro and in vivo. Lineage surface marker-depleted murine bone marrow cells were transduced with either pMSCV-NUP98-NSD1-neo or pMSCV-FLT3-ITD-GFP or both expression constructs on fibronectin-coated plates. Serial colony formation assays in myeloid favoring medium and immunophenotypic analysis by flow cytometry indicated that retroviral expression of NUP98-NSD1 provided increased self-renewal capacity and impaired differentiation of murine bone marrow stem and progenitor cells. NUP98–NSD1 expressing cells displayed a typical myeloblastic morphology and co-expressed myeloid and early stem cell surface markers (CD34low/c-kit+/FcgR+/Gr-1+/ Mac-I+/B220-). Co-expression of FLT3-ITD resulted in high rates of cell proliferation, showed a more differentiated phenotype and concomitantly impaired the in vitro clonogenic capacity in methylcellulose cultures. Bone marrow cells expressing NUP98-NSD1 with or without FLT3-ITD were harvested from methylcellulose cultures and transplanted into sub-lethally irradiated syngeneic mice. All mice receiving cells co-expressing NUP98-NSD1 and FLT3-ITD developed AML that was transplantable into all secondary recipients. Myeloid leukemic blasts that co-expressed NUP98-NSD1 and FLT3-ITD were present in abundance both in BM preparations and in blood smears, and histopathological analysis showed widespread infiltration into solid organs. By contrast, no AML ever developed in mice receiving cells expressing only NUP98-NSD1. These mice, similar to mice receiving cells expressing FLT3-ITD only, developed signs of a chronic myeloproliferative disorder, characterized by expansion of Mac-1+/Gr-1+ BM cells with granulocytic/monocytic differentiation that in some cases caused severe distress after a latency period of more than one year. Intriguingly, upon injection with double transduced NUP98-NSD1 and FLT3-ITD progenitors rather different latency periods of the AML development were observed between different experiments. Interestingly, the latency periods could be correlated to the ratio of expression levels of FLT3-ITD to wildtype FLT3, with higher FLT3-ITD levels associated with a shorter latency. To further investigate the significance of aberrant FLT3 signaling, in vitro and in vivo transformed NUP98-NSD1 and NUP98-NSD1/FLT3-ITD cells were treated with a selective FLT3 tyrosine kinase inhibitor (PKC412). The higher sensitivity of cells co-expressing NUP98-NSD1 and FLT3-ITD to PKC412, compared to cells expressing NUP98-NSD1 only, indicated that proliferation and survival were dependent on FLT3-derived signals. Taken together, these observations demonstrate a potent cooperation between NUP98-NSD1 fusion and FLT3-ITD in leukemic transformation. However, neither the NUP98-NSD1 fusion protein nor the FLT3-ITD mutation alone was sufficient to induce AML. Moreover, the high sensitivity of NUP98-NSD1 and FLT3-ITD co-expressing leukemic blasts to FLT3 signaling inhibition suggests a possible therapeutic strategy to be further explored in this AML subgroup. Disclosures: No relevant conflicts of interest to declare.

Angiotensin II enhances thrombosis development in renovascular hypertensive rats

2005 ◽  
Vol 93 (06) ◽  
pp. 1069-1076 ◽  
Author(s):  
Andrzej Mogielnicki ◽  
Ewa Chabielska ◽  
Robert Pawlak ◽  
Janusz Szemraj ◽  
Wlodzimierz Buczko
Keyword(s):  
Venous Thrombosis ◽  
Thrombus Formation ◽  
Vena Cava ◽  
At1 Receptor ◽  
Ang Ii ◽  
Hypertensive Rats ◽  
At1 Antagonist ◽  
Ang Iv

SummaryThere is an increased number of in vitro evidence that angioten-sin II (Ang II) may promote thrombosis. However there are no in vivo experiments exploring the effect of Ang II on thrombus formation. In the present study we have investigated the influence of Ang II on venous thrombosis in renovascular hypertensive rats. Furthermore, we examined the role of AT1 receptor and Ang II metabolites: angiotensin III (Ang III) and angioten-sin IV (Ang IV) in the mechanisms of Ang II action. The contribution of coagulation and fibrinolytic systems in the mode of Ang II action was also determined. Venous thrombosis was induced by ligation of vena cava. Ang II infused into rats developing venous thrombosis caused dose-dependent increase in thrombus weight, which was partially reversed by losartan, selective AT1 antagonist. Ang III did not influence the thrombus formation in hypertensive rats, while Ang IV caused a marked increase in thrombus weight only in one of the used doses. Our study shows that Ang II via AT1 receptor enhances thrombosis development. The prothrombotic effect of Ang II may partially depend on enhanced leukocytes adhesion to endothelial cells accompanied by accelerated fibrin formation and increased plasma level of PAI-1. Moreover, Ang II action is partially mediated by one of its metabolites – Ang IV.

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