Laminin α4 Deficiency Results in Impaired Hematopoietic Recovery Accompanied with Altered Bone Marrow Niche after Myelosuppression

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 27-27
Author(s):  
Makoto Kondo ◽  
Pingnan Xiao ◽  
Lakshmi Sandhow ◽  
Monika Dolinska ◽  
Thibault Bouderlique ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Steady State ◽  
Progenitor Cells ◽  
Wild Type ◽  
Post Irradiation ◽  
Colony Forming Unit ◽  
Hematopoietic Recovery

Abstract Myelosuppression is a life-threatening complication of anti-cancer therapy including irradiation. Rapid and complete hematopoietic recovery after therapy-induced myelosuppression is required for a successful treatment outcome. This process relies on efficient regeneration of hematopoietic stem cells (HSCs) and is tightly controlled by bone marrow (BM) microenvironment consisting of mesenchymal stem/progenitor cells, endothelial cells as well as secreted factors including cytokines and extracellular matrix proteins (ECM). However, the extrinsic factors critical for promoting the hematopoietic recovery remain poorly understood. Laminins are heterotrimetric ECM composed of α, β, and γ chains. Laminin α4 chain (LAMA4) is an active component for laminin-411 and -421, which are located in vascular basement membrane. LAMA4 plays an important role for HSC homing after transplantation via interaction with laminin receptor integrin α6 (Qian H et al., Blood 2006). However, the role of LAMA4 in normal hematopoiesis and HSC reconstitution after irradiation-induced myelosuppression is not known. In this study, we first detected Lama4 gene expression in BM endothelial cells (CD31+), mesenchymal stem cells (MSC: CD45-Ter119-CD31-CD44-Sca1+CD51+), and mesenchymal progenitor cells (MPC: CD45-Ter119-CD31-CD44-Sca1-CD51+) in young adult mice. By using Lama4 deficient (Lama4-/-) mice, we analyzed the functional role of LAMA4 on hematopoietic activity at steady state. We found the lower number of platelets (PLTs) (p = 0.03), and neutrophils (Gr1+CD11b+) (p = 0.03) in the peripheral blood (PB) of Lama4-/- mice, but a higher frequency of common myeloid progenitor (Lin-Sca1-Kit+CD34+FcRlow) (p < 0.01) in the Lama4-/- BM at steady state, indicating that LAMA4 plays a role in the maintenance of physiological hematopoiesis. The important role of LAMA4 in hematopoietic recovery was demonstrated by delayed and incomplete recoveries of mature red blood cells, PLTs, and Gr1+CD11b+ cells in PB following sublethal irradiation (7Gy). The impaired recovery of erythropoiesis was also indicated by the higher values of mean corpuscular hemoglobin and mean corpuscular volume in PB as well as the higher frequency of megakaryocyte-erythrocyte progenitor (Lin-Sca1-Kit+CD34-FcR-) (p < 0.01) and colony-forming unit-erythrocyte (CFU-E) (p = 0.03) in the BM of the Lama4-/- mice at 6 weeks after irradiation, suggesting blocked erythrocyte maturation. In keeping with the refractory neutropenia, the frequency of colony-forming unit-granulocyte-macrophage (CFU-GM) was lower in the Lama4-/- BM compared to that in the age- and gender-matched wild type mice (p = 0.04). These data indicate that LAMA4 is critical for multiple hematopoietic lineage reconstitution post irradiation. To investigate the cellular and molecular mechanisms underlying the critical role of LAMA4 in hematopoietic recovery after the irradiation, we characterized the BM niche by colony assay, flow cytometry immunophenotyping, quantitative real time PCR (qPCR), and histological analysis. The number of colony-forming unit-fibroblast (CFU-F) was comparable between wild type and Lama4-/- in steady state. Interestingly, the proportion of BM MPCs, a population containing osteoblast progenitors, was significantly lower in the Lama4-/- mice compared to that in the wild type controls at steady state (p < 0.01). qPCR analysis showed downregulation of Il6 (p < 0.05) in the MSC and Angpt1 (p = 0.02) in the MPC of the Lama4-/-mice post irradiation. These data suggest that Lama4 deficiency alters BM stromal cell composition and gene expressions, which may be related to the impaired hematopoietic reconstitution. The recovery of BM vascular structure is essential for efficient reconstitution of hematopoiesis. We observed uniquely dilated blood vessels in Lama4-/- BM at 6-week post irradiation. This might be caused by the lower Angpt1 expression in Lama4-/- MPC since Angpt1/Tie2 signaling is required for vascular regeneration (Kopp HG et al., Blood 2005, Zhou BO et al., eLife 2015). The functional consequences of this phenotype are still under investigation. Altogether, LAMA4 is required for rapid and complete hematopoietic recovery post irradiation-induced myelosuppression. Therapeutic strategies to upregulate Lama4 may facilitate the recovery of hematopoiesis following HSC transplantation under preconditioning using irradiation. Disclosures No relevant conflicts of interest to declare.

CXCR4-SDF-1 Signaling Mediated up-Regulation of Survivin Expression In Mesenchymal Progenitor Cells Is Critical for Their Proliferation and Maintenance of Osteoblastic Niche

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 941-941
Author(s):  
Pratibha Singh ◽  
Jennifer Speth ◽  
Peirong Hu ◽  
Louis M. Pelus
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Survivin Expression ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cxcr4 Signaling

Abstract Abstract 941 Hematopoietic stem cells reside in osteoblastic and vascular niches within the bone marrow. The osteoblastic niche is composed of mesenchymal stem cell derived progenitor cells (MPC) and osteoblasts and are the main sources of the CXC chemokine CXCL12/SDF-1 in the bone marrow microenvironment. Several published studies suggest that the interaction between CXCR4 expressed on hematopoietic stem cells with SDF-1 produced in the bone marrow microenvironment is important for their retention in the bone-marrow. However, the role of SDF-CXCR4 signaling in formation and maintenance of osteoblastic niches in the bone marrow is not known. In this study, we examined the role of CXCR4 signaling in MPC proliferation and differentiation and its effects on hematopoietic stem cell (HSC) function. Flow cytometry analysis demonstrated that CXCR4 is expressed on the phenotypically defined MPC. Deletion of CXCR4 in tamoxifen cre inducible CXCR4flox-flox mice (verified by PCR and flow cytometry; 90% gene deletion and surface CXCR4 expression) results in significantly decreased numbers of Lin- CD45- CD31- Sca-1+ ALCAM- MPC (39±4.2%) and Lin- CD45- CD31- Sca-1-CD51+ osteoblasts (25±2.6%) in bone marrow 15 days after tamoxifen treatment. SDF-1 induced proliferation of CXCR4 deficient MPC was decreased by 4-fold compared to control, measured by the colony forming unit-fibroblast (CFU-F) assay. To determine, whether CXCR4 deficiency in bone marrow stromal cells affects SDF-1 induced HSC proliferation, we cultured FACS sorted wild-type SLAM SKL (103 cells) on CXCR4 deficient stroma for 5 days and total SLAM SKL cell numbers were counted by flow-cytometey analysis. CXCR4 deficient stroma failed to support optimal HSC proliferation and 48±5.2% less SLAM KSL cells was observed on CXCR4 deficient stroma compared to wild-type stroma. To investigate the mechanisms through which CXCR4-SDF-1 signaling regulates MPC proliferation, we evaluated the effect of SDF-1 treatment on expression of the anti-apoptotic and cell-cycle regulator protein, Survivin, in MPC. Multivariate intracellular flow cytometry demonstrated that Survivin expression increased by 23±4.2% in wild-type MPC after SDF-1 treatment (50ng/ml), however no significant increased was demonstrated in CXCR4 deficient MPC cells. CFU-F formation was reduced by 2.5 fold when the Survivin gene was conditionally deleted in MPC. Moreover, fewer SLAM SKL cells were detected on Survivin deficient stroma compared to wild-type stroma after SDF-1 treatment for 5 days. In conclusion, our data suggest that CXCR4-SDF-1 signaling mediated Survivin expression in MPC is important for their proliferation and maintenance of the bone-marrow hematopoietic niche. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The critical role of T cells in glucocorticoid-induced osteoporosis

2020 ◽  
Vol 12 (1) ◽  
Author(s):  
Lin Song ◽  
Lijuan Cao ◽  
Rui Liu ◽  
Hui Ma ◽  
Yanan Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Steady State ◽  
Side Effects ◽  
Critical Role ◽  
Wild Type ◽  
Receptor Activator ◽  
Steady State Levels ◽  
Induced Apoptosis

AbstractGlucocorticoids (GC) are widely used clinically, despite the presence of significant side effects, including glucocorticoid-induced osteoporosis (GIOP). While GC are believed to act directly on osteoblasts and osteoclasts to promote osteoporosis, the detailed underlying molecular mechanism of GC-induced osteoporosis is still not fully elucidated. Here, we show that lymphocytes play a pivotal role in regulating GC-induced osteoporosis. We show that GIOP could not be induced in SCID mice that lack T cells, but it could be re-established by adoptive transfer of splenic T cells from wild-type mice. As expected, T cells in the periphery are greatly reduced by GC; instead, they accumulate in the bone marrow where they are protected from GC-induced apoptosis. These bone marrow T cells in GC-treated mice express high steady-state levels of NF-κB receptor activator ligand (RANKL), which promotes the formation and maturation of osteoclasts and induces osteoporosis. Taken together, these findings reveal a critical role for T cells in GIOP.

scholarly journals CXCR4-SDF-1 Signaling in Nestin+ Mesenchymal Stem Cell Is Required for HSC Maintenance during Homeostasis and Regeneration after Irradiation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3883-3883 ◽  
Author(s):  
Pratibha Singh ◽  
Louis M. Pelus
Keyword(s):  
Stem Cells ◽  
Stromal Cell ◽  
Cxcr4 Expression ◽  
Wild Type ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Cxcr4 Signaling ◽  
Hematopoietic Recovery ◽  
Hematopoietic Reconstitution

Hematopoietic stem cells (HSC) reside in a complex microenvironment (niche) within the bone marrow (BM), where multiple populations of microenvironmental stromal cells regulate and finely tune their proliferation, differentiation and trafficking. Recent studies have shown that mesenchymal stem cells (MSC) are an essential component of the HSC niche. Intrinsic HSC CXCR4-SDF-1 signaling has been implicated in self-renewal and quiescence; however, the role of microenvironment CXCR4-SDF-1 signaling in supporting HSC function remains unclear. We previously demonstrated that microenvironmental stromal cell-derived CXCR4 is important for HSC recovery, as transplantation of wild-type HSC into CXCR4 deficient recipients showed reduced HSC engraftment. In this study, we now show that CXCR4-SDF-1 signaling in nestin+ MSC regulates HSC maintenance under normal homeostatic conditions and promotes hematopoietic regeneration after irradiation. Multivariate flow cytometry analysis of marrow stroma cells revealed that mouse BM MSCs identified as CD45-Ter119-CD31-Nestin+PDGFR+CD51+ express the CXCR4 receptor, which was confirmed by RT-PCR analysis. To investigate the role of MSC CXCR4 signaling in niche maintenance and support of HSC function, we utilized genetic mouse models, in which CXCR4 could be deleted in specific stromal cell types. Selective deletion of CXCR4 from nestin+ MSC in adult tamoxifen inducible nestin-cre CXCR4flox/flox mice resulted in reduced total MSC in BM (Control vs. Deleted: 647±128 vs. 209±51/femur, respectively, n=5, p<0.05), which was associated with a significant reduction in Lineage-Sca-1+c-Kit+ (LSK) cells (Control vs. Deleted: 18,033±439 vs. 4523±358/femur, respectively n=5, p<0.05). Selective CXCR4 deletion in nestin+ MSC also resulted in enhanced LSK cell egress to the peripheral circulation (Control vs. Deleted: 1022±106 vs. 2690±757/ml blood, respectively n=5, p<0.05), with no detectable difference in HSC cell cycle or apoptosis. However, the repopulation ability of HSC obtained from mice where CXCR4 was deleted in nestin+ MSC was reduced by >2 fold. In contrast, deletion of CXCR4 from osteoblasts using osteocalcin cre CXCR4flox/flox mice had no effect on HSC numbers in BM and blood.To investigate the role of nestin+ MSC CXCR4 signaling in BM niche reconstruction and hematopoietic recovery, we transplanted BM cells from wild-type mice into syngeneic wild-type or nestin+ MSC CXCR4 deleted recipients after lethal irradiation (950 rad) and analyzed HSC homing, niche recovery and hematopoietic reconstitution. Deletion of CXCR4 from nestin expressing MSC resulted in significantly reduced LSK cell homing at 16 hrs post transplantation (Control vs. Deleted: 8643±1371 vs. 3004±1044/ mouse, respectively, n=5, p<0.05). Robust apoptosis and senescence after total body irradiation was observed in nestin expressing MSCs lacking CXCR4 expression. At 15 days post-transplantation, chimeric mice with nestin+ MSC lacking CXCR4 expression displayed attenuated niche recovery and hematopoietic reconstitution compared to mice with wild-type stroma. In conclusion, our study suggests that CXCR4-SDF-1 signaling in nestin+ MSC is critical for the maintenance and retention of HSC in BM during homeostasis and promotes niche regeneration and hematopoietic recovery after transplantation. Furthermore, our data suggest the modulating CXCR4 signaling in the hematopoietic niche could be beneficial as a means to enhance HSC recovery following clinical hematopoietic transplantation or radiation/chemotherapy injury. Disclosures No relevant conflicts of interest to declare.

scholarly journals Circulating stem and progenitor cells as markers of inflammation, endothelial regeneration, and prediction of vascular complications in metabolic syndrome and type 1 and 2 diabetes mellitus

2018 ◽  
pp. 58-67
Author(s):  
О.В. Першина ◽  
А.В. Пахомова ◽  
Н.Н. Ермакова ◽  
О.Ю. Рыбалкина ◽  
В.А. Крупин ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Metabolic Syndrome ◽  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Progenitor Cells ◽  
Vascular Complications ◽  
Endothelial Regeneration

Цель исследования состояла в выявлении информативных клеточных маркеров сосудистых осложнений, регенерации микрососудистой сети и воспаления в венозной крови здоровых волонтеров, больных с метаболическим синдромом, сахарным диабетом 1 и 2 типа. Методы. Обследованы больные с метаболическим синдромом (МС), диабетом 2 типа без осложнений, диабетом 1 типа средней степени тяжести и здоровые волонтеры. Диагноз пациентов подтвержден общеклиническими, биохимическими, коагулометрическими и иммуноферментными методами исследования, для оценки экспрессии антигенов использовался многопараметрический цитометрический анализ. Результаты. При анализе экспрессии маркеров показано изменение числа эндотелиальных клеток, мезенхимальных стволовых клеток (МСК) и гемопоэтических стволовых клеток (ГСК) в крови в зависимости от патологии. Эндотелиальные клетки миелоидного (CD45CD14CD34CD309CD144CD31) и немиелоидного (CD45CD14CD34CD309CD144CD31) происхождения, CD309-эндотелиальные клетки и МСК (CD44CD73CD90CD105) предлагаются в качестве маркеров повреждения эндотелия при диабетической симптоматике. При этом ГСК (CD45CD34) могут выступать ценным диагностическим и прогностическим маркером воспаления. Заключение. Для подтверждения сосудистых повреждений и прогноза развития осложнений при диабете 1 и 2 типа в венозной крови пациентов целесообразно оценивать эндотелиальные прогениторные клетки (ЭПК) не костномозговой локализации (CD31CD309CD144) и костномозговой локализации (CD34CD309), и ЭПК c высоким регенеративным потенциалом (CD45CD34CD31CD144). Циркулирующие ЭПК, формирующие колонии in vitro (CD45CD34CD31), рекомендуется использовать в качестве дифференциального маркера состояния регенерации эндотелия при диабете 2 типа. The aim of this study was to identify mesenchymal stem cells (MSC), hematopoietic stem cells (HSC), mature endothelial cells, and endothelial progenitor cells (EPC) in the blood of healthy volunteers, patients with metabolic syndrome, and type 1 and 2 diabetes mellitus as new, informative cellular markers of vascular complications, endothelial regeneration, and inflammation. Methods. The diagnosis was confirmed by general clinical, biochemical, coagulometeric and ELISA studies; multi-parameter cytometric assay was used for evaluation of antigen expression. Results. Changes in the count of MSC, HSC, mature endothelial cells, and endothelial progenitor cells in blood of patients with metabolic syndrome and type 1 and 2 diabetes depended on the type of pathology. We propose using endothelial cells of myeloid (CD45CD14CD34CD309CD144CD31) and non-myeloid origin (CD45CD14CD34CD309CD144CD31), CD309-endothelial cells, and MSCs with the CD44CD73CD90CD105 phenotype as nonspecific markers of endothelial damage in presence of diabetic symptoms. Furthermore, HSCs (CD45CD34) can be used as a valuable diagnostic and prognostic marker of inflammation. Conclusions. It is relevant to evaluate EPCs of non-bone marrow localization (CD31CD309CD144) and bone marrow localization (CD34CD309) and EPCs with a high regenerative potential (CD45CD34CD31CD144) in the blood of patients with type 1 and 2 diabetes to confirm the presence of vascular damage and predict development of complications. Circulating, in vitro colony-forming EPCs (CD45CD34CD31) are recommended as a differential marker for inhibition of endothelial regeneration in type 2 diabetes.

Differential Role for VLA-5 in Mobilization of Heamotopoieic Stem Cells Toward Peripheral Blood and Homing to Bone Marrow and Spleen.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2190-2190 ◽  
Author(s):  
Pieter K. Wierenga ◽  
Ellen Weersing ◽  
Bert Dontje ◽  
Gerald de Haan ◽  
Ronald P. van Os
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Late Antigen ◽  
Cell Mobilization

Abstract Adhesion molecules have been implicated in the interactions of hematopoietic stem and progenitor cells with the bone marrow extracellular matrix and stromal cells. In this study we examined the role of very late antigen-5 (VLA-5) in the process of stem cell mobilization and homing after stem cell transplantation. In normal bone marrow (BM) from CBA/H mice 79±3 % of the cells in the lineage negative fraction express VLA-5. After mobilization with cyclophosphamide/G-CSF, the number of VLA-5 expressing cells in mobilized peripheral blood cells (MPB) decreases to 36±4%. The lineage negative fraction of MPB cells migrating in vitro towards SDF-1α (M-MPB) demonstrated a further decrease to 3±1% of VLA-5 expressing cells. These data are suggestive for a downregulation of VLA-5 on hematopoietic cells during mobilization. Next, MPB cells were labelled with PKH67-GL and transplanted in lethally irradiated recipients. Three hours after transplantation an increase in VLA-5 expressing cells was observed which remained stable until 24 hours post-transplant. When MPB cells were used the percentage PKH-67GL+ Lin− VLA-5+ cells increased from 36% to 88±4%. In the case of M-MPB cells the number increased from 3% to 33±5%. Although the increase might implicate an upregulation of VLA-5, we could not exclude selective homing of VLA-5+ cells as a possible explanation. Moreover, we determined the percentage of VLA-5 expressing cells immediately after transplantation in the peripheral blood of the recipients and were not able to observe any increase in VLA-5+ cells in the first three hours post-tranpslant. Finally, we separated the MPB cells in VLA-5+ and VLA-5− cells and plated these cells out in clonogenic assays for progenitor (CFU-GM) and stem cells (CAFC-day35). It could be demonstared that 98.8±0.5% of the progenitor cells and 99.4±0.7% of the stem cells were present in the VLA-5+ fraction. Hence, VLA-5 is not downregulated during the process of mobilization and the observed increase in VLA-5 expressing cells after transplantation is indeed caused by selective homing of VLA-5+ cells. To shed more light on the role of VLA-5 in the process of homing, BM and MPB cells were treated with an antibody to VLA-5. After VLA-5 blocking of MPB cells an inhibition of 59±7% in the homing of progenitor cells in bone marrow could be found, whereas homing of these subsets in the spleen of the recipients was only inhibited by 11±4%. For BM cells an inhibition of 60±12% in the bone marrow was observed. Homing of BM cells in the spleen was not affected at all after VLA-5 blocking. Based on these data we conclude that mobilization of hematopoietic progenitor/stem cells does not coincide with a downregulation of VLA-5. The observed increase in VLA-5 expressing cells after transplantation is caused by preferential homing of VLA-5+ cells. Homing of progenitor/stem cells to the bone marrow after transplantation apparantly requires adhesion interactions that can be inhibited by blocking VLA-5 expression. Homing to the spleen seems to be independent of VLA-5 expression. These data are indicative for different adhesive pathways in the process of homing to bone marrow or spleen.

Hemangiogenic Role of ELF4 in Bone Marrow Post Myeloablation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1783-1783
Author(s):  
Mariela Sivina ◽  
Takeshi Yamada ◽  
Natalie Dang ◽  
H. Daniel Lacorazza
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Hematopoietic Stem Cells ◽  
Blood Vessels ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Huvec Cells

Abstract Bone marrow suppression is an important cause of death in patients exposed to radiation or in cancer patients treated with conventional chemotherapeutic agents. Myeloablative treatments (i.e. 5-fluorouracil administration) lead to apoptosis of blood forming cells and to regression of blood vessels in bone marrow. It is well known that hematological recovery post-bone marrow insult depends on the capacity of hematopoietic stem cells to regenerate the entire hematopoietic system, however, the transcriptional machinery involved in the regeneration of sinusoidal blood vessels in bone marrow from endothelial progenitor cells is largely unknown. Endothelial cells express the Tie2 receptor tyrosine kinase (a.k.a. Tek), which is involved in the angiogenic remodeling and vessel stabilization. Gene targeting of Tie2 showed that it is not required for differentiation and proliferation of definitive hematopoietic lineages in the embryo although Tie2 is needed during postnatal bone marrow hematopoiesis. ELF is a subgroup of the ETS family of transcription factors composed by ELF1, ELF2 (a.k.a. NERF), ELF3, ELF4 (a.k.a. MEF) and ELF5. ELF1 and ELF2 have been shown to regulate Tie2 expression in vitro. Recently we showed that ELF4 modulates the exit of hematopoietic stem cells (HSC) from quiescence (Lacorazza et al., Cancer Cell2006, 9:175–187). Given the high homology between ELF1 and ELF4 and the same origin of HSC and endothelial progenitor cells, we hypothesize that ELF4 regulates proliferation and Tie2 expression of endothelial cells. We used a luciferase gene reporter system in COS-7 and HEK cells to examine the capacity of ELF proteins to activate Tie2. ELF4 is the strongest activator of Tie2 expression following the hierarchy ELF4>ELF1>ELF2 variant 1>ELF2 variant 2. Site directed mutagenesis of each of the five ETS-binding sites (EBS) present in the Tie2 promoter shows that ELF4 binds preferentially to EBS 1, 3 and 5. Binding of ELF4 to the Tie2 promoter was confirmed by chromatin immunoprecipitation and EMSA. Although Elf1 gene expression is essentially normal in Elf4−/− bone marrow cells collected after 5-FU treatment, we detected diminished Tie2 expression compared to Elf4+/+ bone marrow cells. The association of this effect to human endothelial cells derived from umbilical cord (HUVEC cells) was investigated. All-trans retinoic acid (ATRA) and vascular-endothelial growth factor (VEGF) induced ELF4 expression in HUVEC cells in a dose and time dependent manner which was followed by increased Tie2 expression, suggesting that expression of ELF4 is modulated by angiogenic signals. Moreover, endothelial cells treated with ATRA showed rapid wound colonization in a wound assay. Expression of the pan-endothelial marker MECA-32 was determined by immunohistochemistry to correlate Tie2 with the regeneration of blood vessels: myeloablated Elf4−/− femurs exhibited a reduction of MECA-32 positive arterioles. Finally, temporal and spatial expression of Tie2 during hematological recovery post ablation was measured in bone marrow using transgenic Tie2-LacZ mice crossed to Elf4−/− mice. Collectively, our data suggests that ELF4 regulates Tie2 expression in endothelial cells but most importantly their proliferative capacity in response to angiogenic signals.

Disturbed Endothelial Blood-Bone Marrow-Barrier In Nox4 Deficient Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1169-1169
Author(s):  
Maren Weisser ◽  
Kerstin B. Kaufmann ◽  
Tomer Itkin ◽  
Linping Chen-Wichmann ◽  
Tsvee Lapidot ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Stem Cell ◽  
Steady State ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Knock Out ◽  
Deficient Mice ◽  
Permeability State

Abstract Reactive oxygen species (ROS) have been implicated in the regulation of stemness of hematopoietic stem cells (HSC). HSC with long-term repopulating capabilities are characterized by low ROS levels, whereas increased ROS levels correlate with lineage specification and differentiation. Several tightly regulated sources of ROS production are well known among which are the NADPH oxidases (Nox). HSC are known to express Nox1, Nox2 and Nox4, however, their role in maintenance of stem cell potential or in the activation of differentiation programs are poorly understood. While Nox2 is activated in response to various extrinsic and intrinsic stimuli, mainly during infection and inflammation, Nox4 is constitutively active and is considered to be responsible for steady-state ROS production. Consequently, Nox4 deficiency might lower ROS levels at steady-state hematopoiesis and thereby could have an impact on HSC physiology. In this work we studied HSC homeostasis in Nox4 knock-out mice. Analysis of the hematopoietic stem and progenitor cell (HSPC) pool in the bone marrow (BM) revealed no significant differences in the levels of Lineage marker negative (Lin-) Sca-1+ ckit+ (LSK) and LSK-SLAM (LSK CD150+ CD48-) cells in Nox4 deficient mice compared to wild type (WT) C57BL/6J mice. HSPC frequency upon primary and secondary BM transplantation was comparable between Nox4 deficient and WT mice. In addition, the frequency of colony forming cells in the BM under steady-state conditions did not differ between both mouse groups. However, Nox4 deficient mice possess more functional HSCs as observed in in vivo competitive repopulating unit (CRU) assays. Lin- cells derived from Nox4 knock out (KO) mice showed an increased CRU frequency and superior multilineage engraftment upon secondary transplantation. Surprisingly, ROS levels in different HSPC subsets of NOX4 KO mice were comparable to WT cells, implying that the absence of Nox4 in HSCs does not have a major intrinsic impact on HSC physiology via ROS. Therefore, the increased levels of functional HSCs observed in our studies may suggest a contribution of the BM microenvironment to steady-state hematopoiesis in the BM of Nox4 KO animals. Recent observations suggest a regulation of the BM stem cell pool by BM endothelial cells, in particular by the permeability state of the blood-bone marrow-barrier (Itkin T et al., ASH Annual Meeting Abstracts, 2012). Endothelial cells interact with HSCs predominantly via paracrine effects and control stem cell retention, egress and homing as well as stem cell activation. As Nox4 is highly expressed in endothelial cells and is involved in angiogenesis, we reasoned that the absence of NOX4 could affect HSC homeostasis through altered BM endothelium properties and barrier permeability state. Indeed, in preliminary assays we found reduced short-term homing of BM mononuclear cells into the BM of Nox4 deficient mice as compared to wild type hosts. Furthermore, in vivo administration of Evans Blue dye revealed reduced dye penetration into Nox4-/- BM compared to wild type mice upon intravenous injection. Taken together, these data indicate a reduced endothelial permeability in Nox4 KO mice. Ongoing experiments aim at further characterization of the Nox4-/- phenotype in BM sinusoidal and arteriolar endothelial cells, the impact of Nox4 deletion on BM hematopoietic and mesenchymal stem cells, and in deciphering the role of Nox4 in the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Role of bone marrow-derived stem cells, renal progenitor cells and stem cell factor in chronic renal allograft nephropathy

2013 ◽  
Vol 49 (3) ◽  
pp. 235-247
Author(s):  
Hayam Abdel Meguid El Aggan ◽  
Mona Abdel Kader Salem ◽  
Nahla Mohamed Gamal Farahat ◽  
Ahmad Fathy El-Koraie ◽  
Ghaly Abd Al-Rahim Mohammed Kotb
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Stem Cell Factor ◽  
Renal Allograft ◽  
Renal Progenitor Cells ◽  
Renal Progenitor

Endothelial cell P-selectin mediates a proinflammatory and prothrombogenic phenotype in cerebral venules of sickle cell transgenic mice

2004 ◽  
Vol 286 (5) ◽  
pp. H1608-H1614 ◽  
Author(s):  
Katherine C. Wood ◽  
Robert P. Hebbel ◽  
D. Neil Granger
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Sickle Cell ◽  
Leukocyte Adhesion ◽  
Chimeric Mice ◽  
Wild Type ◽  
Cell Disease ◽  
Adhesion Of Platelets

Whereas the adhesion of leukocytes and erythrocytes to vascular endothelium has been implicated in the vasooclusive events associated with sickle cell disease, the role of platelet-vessel wall interactions in this process remains undefined. The objectives of this study were to: 1) determine whether the adhesion of platelets and leukocytes in cerebral venules differs between sickle cell transgenic (βS) mice and their wild-type (WT) counterparts (C57Bl/6) under both resting and posthypoxic conditions, and 2) define the contributions of P-selectin to these adhesion processes. Animals were anesthetized, and platelet and leukocyte interactions with endothelial cells of cerebral postcapillary venules were monitored and quantified using intravital fluorescence microscopy in WT, βS, and chimeric mice produced by transplanting bone marrow from WT or βSmice into WT or P-selectin-deficient (P-sel–/–) mice. Platelet and leukocyte adhesion to endothelial cells in both unstimulated and posthypoxic βSmice were significantly elevated over WT levels. Chimeric mice involving bone marrow transfer from βSmice to P-sel–/–mice exhibited a profound attenuation of both platelet and leukocyte adhesion compared with βSbone marrow transfer to WT mice. These findings indicate that βSmice assume both an inflammatory and prothrombogenic phenotype, with endothelial cell P-selectin playing a major role in mediating these microvascular responses.

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