Long-term type 1 diabetes influences haematopoietic stem cells by reducing vascular repair potential and increasing inflammatory monocyte generation in a murine model.
ABSTRACT: AIMS/HYPOTHESIS:We sought to determine the impact of long-standing type 1 diabetes on haematopoietic stem/progenitor cell (HSC) number and function and to examine the impact of modulating glycoprotein (GP)130 receptor in these cells. METHODS:Wild-type, gp130(-/-) and GFP chimeric mice were treated with streptozotocin to induce type 1 diabetes. Bone marrow (BM)-derived cells were used for colony-formation assay, quantification of side population (SP) cells, examination of gene expression, nitric oxide measurement and migration studies. Endothelial progenitor cells (EPCs), a population of vascular precursors derived from HSCs, were compared in diabetic and control mice. Cytokines were measured in BM supernatant fractions by ELISA and protein array. Flow cytometry was performed on enzymatically dissociated retina from gfp(+) chimeric mice and used to assess BM cell recruitment to the retina, kidney and blood. RESULTS:BM cells from the 12-month-diabetic mice showed reduced colony-forming ability, depletion of SP-HSCs with a proportional increase in SP-HSCs residing in hypoxic regions of BM, decreased EPC numbers, and reduced eNos (also known as Nos3) but increased iNos (also known as Nos2) and oxidative stress-related genes. BM supernatant fraction showed increased cytokines, GP130 ligands and monocyte/macrophage stimulating factor. Retina, kidney and peripheral blood showed increased numbers of CD11b(+)/CD45(hi)/ CCR2(+)/Ly6C(hi) inflammatory monocytes. Diabetic gp130(-/-) mice were protected from development of diabetes-induced changes in their HSCs. CONCLUSIONS/INTERPRETATION:The BM microenvironment of type 1 diabetic mice can lead to changes in haematopoiesis, with generation of more monocytes and fewer EPCs contributing to development of microvascular complications. Inhibition of GP130 activation may serve as a therapeutic strategy to improve the key aspects of this dysfunction.
Project description:OBJECTIVES:Bone marrow derived endothelial progenitor cells (BM-EPCs) are increased in chronic liver disease (CLD). Their role in hepatic fibrosis and regeneration remains an area of intense studies. We investigated the migration and secretory functions of BM-EPCs in fibrotic mice liver. MATERIALS AND METHODS:Bone marrow cells from C57BL6-GFP mice were transplanted into the femur of irradiated C57BL6 mice, followed by CCl4 doses for 8 weeks, to develop hepatic fibrosis (n = 36). Transplanted C57BL6 mice without CCl4 treatment were used as controls. EPCs were analyzed in BM, blood and liver by flow cytometry and immunofluorescence. VEGF and TGF-? were analysed in the hepatic stellate cells (HSCs) and BM-EPCs co-cultures using ELISAs. RESULTS:There was a significant migration of EPCs from BM to blood and to the liver (P ? 0.01). Percentage of GFP+ CD31+ EPCs and collagen proportionate area was substantially increased in the liver at 4th week of CCl4 dosage compared to the controls (19.8% vs 1.9%, P ? 0.05). Levels of VEGF (533.6 pg/ml) and TGF-? (327.44 pg/ml) also increased significantly, when HSCs were treated with the EPC conditioned medium, as compared to controls (25.66 pg/ml and 5.87 pg/ml, respectively; P ? 0.001). CONCLUSIONS:Present findings suggest that BM-EPCs migrate to the liver during CCl4-induced liver injury and contribute to fibrosis.
Project description:Endothelial progenitor cells (EPCs) are essential in vasculogenesis and wound healing, but their circulating and wound level numbers are decreased in diabetes. This study aimed to determine mechanisms responsible for the diabetic defect in circulating and wound EPCs. Since mobilization of BM EPCs occurs via eNOS activation, we hypothesized that eNOS activation is impaired in diabetes, which results in reduced EPC mobilization. Since hyperoxia activates NOS in other tissues, we investigated whether hyperoxia restores EPC mobilization in diabetic mice through BM NOS activation. Additionally, we studied the hypothesis that impaired EPC homing in diabetes is due to decreased wound level stromal cell-derived factor-1alpha (SDF-1alpha), a chemokine that mediates EPC recruitment in ischemia. Diabetic mice showed impaired phosphorylation of BM eNOS, decreased circulating EPCs, and diminished SDF-1alpha expression in cutaneous wounds. Hyperoxia increased BM NO and circulating EPCs, effects inhibited by the NOS inhibitor N-nitro-L-arginine-methyl ester. Administration of SDF-1alpha into wounds reversed the EPC homing impairment and, with hyperoxia, synergistically enhanced EPC mobilization, homing, and wound healing. Thus, hyperoxia reversed the diabetic defect in EPC mobilization, and SDF-1alpha reversed the diabetic defect in EPC homing. The targets identified, which we believe to be novel, can significantly advance the field of diabetic wound healing.
Project description:Hematopoietic stem cells (HSCs) reside in association with bone marrow (BM) sinusoidal vessels in vivo, but the function of BM endothelial cells (ECs) in regulating hematopoiesis is unclear. We hypothesized that hematopoietic regeneration following injury is regulated by BM ECs. BALB/c mice were treated with total body irradiation (TBI) and then infused with C57Bl6-derived endothelial progenitor cells (EPCs) to augment endogenous BM EC activity. TBI caused pronounced disruption of the BM vasculature, BM hypocellularity, ablation of HSCs, and pancytopenia in control mice, whereas irradiated, EPC-treated mice displayed accelerated recovery of BM sinusoidal vessels, BM cellularity, peripheral blood white blood cells (WBCs), neutrophils, and platelets, and a 4.4-fold increase in BM HSCs. Systemic administration of anti-VE-cadherin antibody significantly delayed hematologic recovery in both EPC-treated mice and irradiated, non-EPC-treated mice compared with irradiated controls. These data demonstrate that allogeneic EPC infusions can augment hematopoiesis and suggest a relationship between BM microvascular recovery and hematopoietic reconstitution in vivo.
Project description:Background & Aims:Endothelial precursor cell (EPC) dysfunction is one of the risk factors for diabetes mellitus (DM) which results in delayed wound healing. Rosiglitazone (RSG) is a frequently prescribed oral glucose-lowering drug. Previous studies have shown the positive effects of RSG on ameliorating EPC dysfunction in diabetic patients. Interestingly, knowledge about RSG with regard to the wound healing process caused by DM is scarce. Therefore, in this study, we investigated the possible actions of RSG on wound healing and the related mechanisms involved in db/db diabetic mice. Methods:Db/db mice with spontaneous glucose metabolic disorder were used as a type 2 DM model. RSG (20 mg/kg/d, i.g.,) was administered for 4 weeks before wound creation and bone marrow derived EPC (BM-EPC) isolation. Wound closure was assessed by wound area and CD31 staining. Tubule formation and migration assays were used to judge the function of the BM-EPCs. The level of vascular endothelial growth factor (VEGF), stromal cell derived factor-1? (SDF-1?) and insulin signaling was determined by ELISA. Cell viability of the BM-EPCs was measured by CCK-8 assay. Results:RSG significantly accelerated wound healing and improved angiogenesis in db/db mice. Bioactivities of tube formation and migration were decreased in db/db mice but were elevated by RSG. Level of both VEGF and SDF-1? was increased by RSG in the BM-EPCs of db/db mice. Insulin signaling was elevated by RSG reflected in the phosphorylated-to-total AKT in the BM-EPCs. In vitro, RSG improved impaired cell viability and tube formation of BM-EPCs induced by high glucose, but this was prevented by the VEGF inhibitor avastin. Conclusion:Our data demonstrates that RSG has benefits for wound healing and angiogenesis in diabetic mice, and was partially associated with improvement of EPC function through activation of VEGF and stimulation of SDF-1? in db/db mice.
Project description:The aim of this study was to evaluate the angiogenicity of a combination of BM-EPCs and BM-MSCs in vitro in the presence of SP and its working mechanism.BM-MSCs and BM-EPCs were cocultured with or without SP. ELISA and RT-PCR were performed to detect angiogenic factors such as VEGF and PDGF-BB. N-cadherin was detected by Western blot analysis. The tubular network-forming ability was evaluated by a Matrigel tube-forming assay.BM-EPCs coculture with BM-MSCs strongly stimulated the recruitment of BM-MSCs onto the BM-EPC-generated endothelial tubular network. Upon SP treatment, endothelial branching point, tubule length, and tubular recruitment of BM-MSCs were further increased and stabilized. The coculture of BM-EPCs and BM-MSCs synergistically stimulated expression of VEGF, VEGF receptor, N-cadherin, and PDGF-BB, all of which were further enhanced by SP treatment. Blockade of PDGF-BB by its functional blocking antibodies markedly reduced the BM-MSC incorporation into the endothelial tubules. SP-pretreated BM-MSCs were preferentially incorporated into the preformed BM-EPC tubular network.BM-EPCs along with SP promote the pericyte-like coverage of BM-MSCs on endothelial tubules possibly through the induction of PDGF-BB.
Project description:INTRODUCTION: Bone marrow transplantation (BMT) is a complex process regulated by different cytokines and growth factors. The pleiotropic cytokine IL-6 (Interleukin-6) and related cytokines of the same family acting on the common signal transducer gp130 are known to play a key role in bone marrow (BM) engraftment. In contrast, the exact signalling events that control IL-6/gp130-driven haematopoietic stem cell development during BMT remain unresolved. METHODS: Conditional gp130 knockout and knockin mice were used to delete gp130 expression (gp130(?Mx)), or to selectively disrupt gp130-dependent Ras (gp130(?MxRas)) or STAT signalling (gp130(?MxSTAT)) in BM cells. BM derived from the respective strains was transplanted into irradiated wildtype hosts and repopulation of various haematopoietic lineages was monitored by flow cytometry. RESULTS: BM derived from gp130 deficient donor mice (gp130(?Mx)) displayed a delayed engraftment, as evidenced by reduced total white blood cells (WBC), marked thrombocytopenia and anaemia in the early phase after BMT. Lineage analysis unravelled a restricted development of CD4(+) and CD8(+) T-cells, CD19(+) B-cells and CD11b(+) myeloid cells after transplantation of gp130-deficient BM grafts. To further delineate the two major gp130-induced signalling cascades, Ras-MAPK and STAT1/3-signalling respectively, we used gp130(?MxRas) and gp130(?MxSTAT) donor BM. BMT of gp130(?MxSTAT) cells significantly impaired engraftment of CD4(+), CD8(+), CD19(+) and CD11b(+) cells, whereas gp130(?MxRas) BM displayed a selective impairment in early thrombopoiesis. Importantly, gp130-STAT1/3 signalling deficiency in BM grafts severely impaired survival of transplanted mice, thus demonstrating a pivotal role for this pathway in BM graft survival and function. CONCLUSION: Our data unravel a vital function of IL-6/gp130-STAT1/3 signals for BM engraftment and haematopoiesis, as well as for host survival after transplantation. STAT1/3 and ras-dependent pathways thereby exert distinct functions on individual bone-marrow-lineages.
Project description:The role of bone marrow (BM) cells in modulating pulmonary hypertensive responses is not well understood. Determine if BM-derived endothelial progenitor cells (EPCs) induce pulmonary hypertension (PH) and if this is attenuated by mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs). Three BM populations were studied: (a) BM from vehicle and monocrotaline (MCT)-treated mice (PH induction), (b) BM from vehicle-, MCT-treated mice that received MSC-EV infusion after vehicle, MCT treatment (PH reversal, in vivo), (c) BM from vehicle-, MCT-treated mice cultured with MSC-EVs (PH reversal, in vitro). BM was separated into EPCs (sca-1+/c-kit+/VEGFR2+) and non-EPCs (sca-1-/c-kit-/VEGFR2-) and transplanted into healthy mice. Right ventricular (RV) hypertrophy was assessed by RV-to-left ventricle+septum (RV/LV+S) ratio and pulmonary vascular remodeling by blood vessel wall thickness-to-diameter (WT/D) ratio. EPCs but not non-EPCs from mice with MCT-induced PH (MCT-PH) increased RV/LV+S, WT/D ratios in healthy mice (PH induction). EPCs from MCT-PH mice treated with MSC-EVs did not increase RV/LV+S, WT/D ratios in healthy mice (PH reversal, in vivo). Similarly, EPCs from MCT-PH mice treated with MSC-EVs pre-transplantation did not increase RV/LV+S, WT/D ratios in healthy mice (PH reversal, in vitro). MSC-EV infusion reversed increases in BM-EPCs and increased lung tissue expression of EPC genes and their receptors/ligands in MCT-PH mice. These findings suggest that the pulmonary hypertensive effects of BM are mediated by EPCs and those MSC-EVs attenuate these effects. These findings provide new insights into the pathogenesis of PH and offer a potential target for development of novel PH therapies. Stem Cells Translational Medicine 2017;6:1595-1606.
Project description:Hematopoietic stem cells (HSCs) reside in a specialized bone marrow (BM) microenvironment that supports the maintenance and functional integrity of long-term (LT)-HSCs throughout postnatal life. The objective of this work is to study the role of activated leukocyte cell adhesion molecule (Alcam) in HSC differentiation and self-renewal using an Alcam-null (Alcam(-/-) ) mouse model. We show here that Alcam is differentially regulated in adult hematopoiesis and is highly expressed in LT-HSCs where its level progressively increases with age. Young adult Alcam(-/-) mice had normal homeostatic hematopoiesis and normal numbers of phenotypic HSCs. However, Alcam(-/-) HSCs had reduced long-term replating capacity in vitro and reduced long-term engraftment potential upon transplantation. We show that Alcam(-/-) BM contain a markedly lower frequency of long-term repopulating cells than wild type. Further, the long-term repopulating potential and engraftment efficiency of Alcam(-/-) LT-HSCs was greatly compromised despite a progressive increase in phenotypic LT-HSC numbers during long-term serial transplantation. In addition, an age-associated increase in phenotypic LT-HSC cellularity was observed in Alcam(-/-) mice. This increase was predominately within the CD150(hi) fraction and was accompanied by significantly reduced leukocyte output. Consistent with an aging-like phenotype, older Alcam(-/-) LT-HSCs display myeloid-biased repopulation activity upon transplantation. Finally, Alcam(-/-) LT-HSCs display premature elevation of age-associated gene expression, including Selp, Clu, Cdc42, and Foxo3. Together, this study indicates that Alcam regulates functional integrity and self-renewal of LT-HSCs.
Project description:Angiogenesis is essential for cyclic endometrial growth, implantation, and pregnancy maintenance. Vasculogenesis, the formation of new blood vessels by bone marrow (BM)-derived endothelial progenitor cells (EPCs), has been shown to contribute to endometrial vasculature. However, it is unknown whether vasculogenesis occurs in neovascularization of the decidua during pregnancy. To investigate the contribution of BM-derived EPCs to vascularization of the pregnant uterus, we induced non-gonadotoxic submyeloablation by 5-fluorouracil administration to wild-type FVB/N female mice recipients followed by BM transplantation from transgenic mice expressing green fluorescent protein (GFP) under regulation of Tie2 endothelial-specific promoter. Following 1 month, Tie2-GFP BM-transplanted mice were bred and sacrificed at various gestational days (ED6.5, ED10.5, ED13.5, ED18.5, and postpartum). Bone-marrow-transplanted non-pregnant and saline-injected pregnant mice served as controls (n = 5-6/group). Implantation sites were analyzed by flow cytometry, immunohistochemistry, and immunofluorescence. While no GFP-positive EPCs were found in non-pregnant or early pregnant uteri of BM-transplanted mice, GFP-positive EPCs were first detected in pregnant uterus on ED10.5 (0.12%) and increased as the pregnancy progressed (1.14% on ED13.5), peaking on ED18.5 (1.42%) followed by decrease in the postpartum (0.9%). The percentage of endothelial cells that were BM-derived out of the total endothelial cell population in the implantation sites (GFP+CD31+/CD31+) were 9.3%, 15.8%, and 6.1% on ED13.5, ED18.5, and postpartum, respectively. Immunohistochemistry demonstrated that EPCs incorporated into decidual vasculature, and immunofluorescence showed that GFP-positive EPCs colocalized with CD31 in vascular endothelium of uterine implantation sites, confirming their endothelial lineage. Our findings indicate that BM-derived EPCs contribute to vasculogenesis of the pregnant mouse decidua.
Project description:Bone marrow (BM) is the major reservoir for endothelial progenitor cells (EPCs). Postnatal neovascularization depends on not only angiogenesis but also vasculogenesis, which is mediated through mobilization of EPCs from BM and their recruitment to the ischemic sites. Reactive oxygen species (ROS) derived from Nox2-based NADPH oxidase play an important role in postnatal neovascularization; however, their role in BM and EPC function is unknown. Here we show that hindlimb ischemia of mice significantly increases Nox2 expression and ROS production in BM-mononuclear cells (BMCs), which is associated with an increase in circulating EPC-like cells. Mice lacking Nox2 show reduction of ischemia-induced flow recovery, ROS levels in BMCs, as well as EPC mobilization from BM. Transplantation of wild-type (WT)-BM into Nox2-deficient mice rescues the defective neovascularization, whereas WT mice transplanted with Nox2-deficient BM show reduced flow recovery and capillary density compared to WT-BM transplanted control. Intravenous infusion of WT- and Nox2-deficient BMCs into WT mice reveals that neovascularization and homing capacity are impaired in Nox2-deficient BMCs in vivo. In vitro, Nox2-deficient c-kit+Lin- BM stem/progenitor cells show impaired chemotaxis and invasion as well as polarization of actins in response to stromal derived factor (SDF), which is associated with blunted SDF-1-mediated phosphorylation of Akt. In conclusion, Nox2-derived ROS in BM play a critical role in mobilization, homing, and angiogenic capacity of EPCs and BM stem/progenitor cells, thereby promoting revascularization of ischemic tissue. Thus, NADPH oxidase in BM and EPCs is potential therapeutic targets for promoting neovascularization in ischemic cardiovascular diseases.