Angptl4 is upregulated under inflammatory conditions in the bone marrow of mice, expands myeloid progenitors, and accelerates reconstitution of platelets after myelosuppressive therapy
ABSTRACT: Upon inflammation, myeloid cell generation in the bone marrow (BM) is broadly enhanced by the action of induced cytokines, which are produced locally or at multiple sites throughout the body. Using microarray studies, we show that Angptl4 is a predominantly upregulated cytokine in the BM during systemic inflammatory conditions. Functionally we found recombinant murine Angptl4 (rmAngptl4) to stimulate the proliferation of myeloid CFUs in vitro. Upon repeated in vivo injections, rmAngptl4 increased BM progenitor cell frequency, and this was paralleled by a relative increase in phenotypically defined granulocyte-macrophage progenitors (GMPs). Furthermore, in vivo treatment with rmAngptl4 resulted in elevated platelet counts in steady-state mice and a significantly accelerated reconstitution of platelets in post-transplanted mice after myelosuppressive therapy. In contrast, transplantation of BM cells from donor mice pre-treated with rmAngptl4 had no effect on the recovery of platelets. The administration of rmAngptl4 additionally increased the number of CD61+CD41low-expressing megakaryocytes (MKs) in the BM of steady-state and in the spleen of transplanted mice as well as after in vitro MK-differentiation from hematopoietic stem and progenitor cells. Furthermore, using a stat3 reporter knockin model, we show that rmAngptl4 induces de novo stat3 expression in immature MKs which could be important for the effective expansion of MKs after myelosuppressive therapy. In summary our data suggest that Angptl4 plays a complementary role in hematopoiesis during emergency situations like sepsis. The use of Angptl4 in the setting of autologous stem cell transplantation could represent a potential approach for accelerating the reconstitution of megakaryopoiesis. Overall design: in total 4 probes: 2 replica of BM cells isolated from PBS- and LPS-treated C57BL/6N mice
Project description:We hypothesized that megakaryocyte (MK) phosphoinositide signaling mediated by phosphatidylinositol transfer proteins (PITPs) contributes to hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) regulation. Conditional knockout mice lacking PITPs specifically in MKs and platelets (pitpα-/- and pitpα-/-/β-/-) bone marrow (BM) manifested decreased numbers of HSCs, MK-erythrocyte progenitors, and cycling HPCs. Further, pitpα-/-/β-/- BM had significantly reduced engrafting capability in competitive transplantation and limiting dilution analysis. Conditioned media (CM) from cultured pitpα-/- and pitpα-/-/β-/- BM MKs contained higher levels of transforming growth factor β1 (TGF-β1) and interleukin-4 (IL-4), among other myelosuppressive cytokines, than wild-type BM MKs. Correspondingly, BM flush fluid from pitpα-/- and pitpα-/-/β-/- mice had higher concentrations of TGF-β1. CM from pitpα-/- and pitpα-/-/β-/- MKs significantly suppressed HPC colony formation, which was completely extinguished in vitro by neutralizing anti-TGF-β antibody, and treatment of pitpα-/-/β-/- mice in vivo with anti-TGF-β antibodies completely reverted their defects in BM HSC and HPC numbers. TGF-β and IL-4 synergized to inhibit HPC colony formation in vitro. Electron microscopy analysis of pitpα-/-/β-/- MKs revealed ultrastructural defects with depleted α-granules and large, misshaped multivesicular bodies. Von Willebrand factor and thrombospondin-1, like TGF-β, are stored in MK α-granules and were also elevated in CM of cultured pitpα-/-/β-/- MKs. Altogether, these data show that ablating PITPs in MKs indirectly dysregulates hematopoiesis in the BM by disrupting α-granule physiology and secretion of TGF-β1.
Project description:Muscle satellite cells are essential for muscle regeneration. However, efficient regeneration does not occur without muscle-resident mesenchymal progenitor cells. We show here that bone marrow-derived mesenchymal stromal cells (Bm-MSCs) also facilitate muscle regeneration in Duchenne muscular dystrophy (DMD) model mice. Bm-MSCs transplanted into peritoneal cavities of DMD model mice with severe muscle degeneration strongly suppressed dystrophic pathology and improved death-related symptoms, which resulted in dramatic lifespan extension. Isolated single myofibers from Bm-MSC-transplanted mice manifested considerably less myofiber splitting compared with myofibers from non-transplanted mice, which indicated that transplantation significantly ameliorated abnormal regeneration. With regard to the number of satellite cells, several cells remained on myofibers from Bm-MSC-transplanted model mice, but satellite cells rarely occurred on myofibers from non-transplanted mice. Also, CXCL12 was crucial for muscle regeneration. CXCL12 facilitated muscle regeneration and paired box protein-7 (PAX7) expression after cardiotoxin-related muscle injury in vivo. The majority of primary muscle satellite cells sorted by integrin-?7 and CD34 expressed CXCR4, a receptor specific for CXCL12. CXCL12 strongly suppressed p-STAT3 expression in these sorted cells in vitro. CXCL12 may therefore influence muscle regeneration through STAT3 signaling in satellite cells. Targeting these proteins in or on muscle satellite cells may improve many degenerative muscle diseases.
Project description:Millions of platelets are produced each hour by bone marrow (BM) megakaryocytes (MKs). MKs extend transendothelial proplatelet (PP) extensions into BM sinusoids and shed new platelets into the blood. The mechanisms that control platelet generation remain incompletely understood. Using conditional mutants and intravital multiphoton microscopy, we show here that the lipid mediator sphingosine 1-phosphate (S1P) serves as a critical directional cue guiding the elongation of megakaryocytic PP extensions from the interstitium into BM sinusoids and triggering the subsequent shedding of PPs into the blood. Correspondingly, mice lacking the S1P receptor S1pr1 develop severe thrombocytopenia caused by both formation of aberrant extravascular PPs and defective intravascular PP shedding. In contrast, activation of S1pr1 signaling leads to the prompt release of new platelets into the circulating blood. Collectively, our findings uncover a novel function of the S1P-S1pr1 axis as master regulator of efficient thrombopoiesis and might raise new therapeutic options for patients with thrombocytopenia.
Project description:The JAK2V617F mutation commonly found in myeloproliferative neoplasms (MPNs) induces constitutive phosphorylation/activation of the signal transducer and activator of transcription 3 (Stat3). However, the contribution of Stat3 in MPN evoked by JAK2V617F remains unknown. To determine the role of Stat3 in JAK2V617F-induced MPN, we generated Stat3-deficient Jak2V617F-expressing mice. Whereas expression of Jak2V617F resulted in a polycythemia vera-like disease characterized by increased red blood cells (RBCs), hematocrit, neutrophils and platelets in the peripheral blood of Jak2V617F knock-in mice, deletion of Stat3 slightly reduced RBC and hematocrit parameters and modestly increased platelet numbers in Jak2V617F knock-in mice. Moreover, deletion of Stat3 significantly increased the neutrophil counts/percentages and markedly reduced the survival of mice expressing Jak2V617F. These phenotypic manifestations were reproduced upon bone marrow (BM) transplantation into wild-type animals. Flow cytometric analysis showed increased hematopoietic stem cell and granulocyte-macrophage progenitor populations in the BM and spleens of Stat3-deficient Jak2V617F mice. Stat3 deficiency also caused a marked expansion of Gr-1+/Mac-1+ myeloid cells in Jak2V617F knock-in mice. Histopathologic analysis revealed marked increase in granulocytes in the BM, spleens and livers of Stat3-deficient Jak2V617F-expressing mice. Together, these results suggest that deletion of Stat3 increases the severity of MPN induced by Jak2V617F.
Project description:Myelosuppression damages the bone marrow (BM) vascular niche, but it is unclear how regeneration of bone marrow vessels contributes to engraftment of transplanted hematopoietic stem and progenitor cells (HSPCs) and restoration of hematopoiesis. We found that chemotherapy and sublethal irradiation induced minor regression of BM sinusoidal endothelial cells (SECs), while lethal irradiation induced severe regression of SECs and required BM transplantation (BMT) for regeneration. Within the BM, VEGFR2 expression specifically demarcated a continuous network of arterioles and SECs, with arterioles uniquely expressing Sca1 and SECs uniquely expressing VEGFR3. Conditional deletion of VEGFR2 in adult mice blocked regeneration of SECs in sublethally irradiated animals and prevented hematopoietic reconstitution. Similarly, inhibition of VEGFR2 signaling in lethally irradiated wild-type mice rescued with BMT severely impaired SEC reconstruction and prevented engraftment and reconstitution of HSPCs. Therefore, regeneration of SECs via VEGFR2 signaling is essential for engraftment of HSPCs and restoration of hematopoiesis.
Project description:Although the unique role of hematopoietic stem cell (HSC) niche in hematopoiesis has long been recognized, unsuccessful isolation of intact niche units limited their in vitro study, manipulation, and therapeutic application. Here, we isolated cell complexes based on size fractionation from mouse bone marrow (BM), characterized the derived cells, and transplanted them to irradiated mice. These cell complexes were the origin of both BM mesenchymal stem cells and various hematopoietic lineages when kept in appropriate culture conditions. They also had the potential of recruiting circulating HSC. Intraperitoneal transplantation of these structures into irradiated mice not only showed long-lasting hematopoietic multilineage reconstitution, but also could recover the stromal cells of BM. In conclusion, this study for the first time provides evidences on the feasibility and efficacy of transplantation of HSC in association with their native specialized microenvironment. As the molecular cross-talk between HSC and niche is crucial for their proper function, the proposed method could be considered as a novel hematopoietic transplantation strategy.
Project description:The development of cell therapies to treat peripheral vascular disease has proven difficult because of the contribution of multiple cell types that coordinate revascularization. We characterized the vascular regenerative potential of transplanted human bone marrow (BM) cells purified by high aldehyde dehydrogenase (ALDH(hi)) activity, a progenitor cell function conserved between several lineages. BM ALDH(hi) cells were enriched for myelo-erythroid progenitors that produced multipotent hematopoietic reconstitution after transplantation and contained nonhematopoietic precursors that established colonies in mesenchymal-stromal and endothelial culture conditions. The regenerative capacity of human ALDH(hi) cells was assessed by intravenous transplantation into immune-deficient mice with limb ischemia induced by femoral artery ligation/transection. Compared with recipients injected with unpurified nucleated cells containing the equivalent of 2- to 4-fold more ALDH(hi) cells, mice transplanted with purified ALDH(hi) cells showed augmented recovery of perfusion and increased blood vessel density in ischemic limbs. ALDH(hi) cells transiently recruited to ischemic regions but did not significantly integrate into ischemic tissue, suggesting that transient ALDH(hi) cell engraftment stimulated endogenous revascularization. Thus, human BM ALDH(hi) cells represent a progenitor-enriched population of several cell lineages that improves perfusion in ischemic limbs after transplantation. These clinically relevant cells may prove useful in the treatment of critical ischemia in humans.
Project description:Previous studies have shown that platelet-specific factor VIII (FVIII) expression (2bF8) restores hemostasis and induces immune tolerance in hemophilia A (HA) mice even with preexisting inhibitors. Here we investigated for the first time whether platelet FVIII expression can prevent severe spontaneous bleeding in rat HA, a model mimicking the frequent spontaneous bleeding in patients with severe HA. A novel FVIII-/- rat model in a Dahl inbred background (Dahl-FVIII-/-) with nearly the entire rat FVIII gene inverted was created by using a CRISPR/Cas9 strategy. There was no detectable FVIII in plasma. Spontaneous bleeding in the soft tissue, muscles, or joints occurred in 100% of FVIII-/- rats. Sixty-one percent developed anti-FVIII inhibitors after ?2 doses of recombinant human FVIII infusion. However, when 2bF8 transgene was crossed into the FVIII-/- background, none of the resulting 2bF8tg+FVIII-/- rats (with platelet FVIII levels of 28.26 ± 7.69 mU/108 platelets and undetectable plasma FVIII) ever had spontaneous bleeding. When 2bF8tg bone marrow (BM) was transplanted into FVIII-/- rats, only 1 of 7 recipients had a bruise at the early stage of BM reconstitution, but no other spontaneous bleeding was observed during the study period. To confirm that the bleeding diathesis in FVIII-/- rats was ameliorated after platelet FVIII expression, rotational thromboelastometry and whole-blood thrombin generation assay were performed. All parameters in 2bF8tg BM transplantation recipients were significantly improved compared with FVIII-/- control rats. Of note, neither detectable levels of plasma FVIII nor anti-FVIII inhibitors were detected in 2bF8tg BM transplantation recipients. Thus, platelet-specific FVIII expression can efficiently prevent severe spontaneous bleeding in FVIII-/- rats with no anti-FVIII antibody development.
Project description:B-domainless factor VIII (FVIII) ectopically expressed in megakaryocytes (MKs) is stored in α granules of platelets (pFVIII) and is capable of restoring hemostasis in FVIIInull mice, even in the presence of circulating inhibitors. However, our prior studies have shown that this ectopically expressed pFVIII can injure developing MKs. Moreover, the known risks of prolonged thrombocytopenia after bone marrow transplantation are significant challenges to the use of this strategy to treat individuals with severe hemophilia A and particularly those with intractable clinically relevant inhibitors. Because of these limitations, we now propose the alternative therapeutic pFVIII strategy of infusing pFVIII-expressing MKs or platelets derived from induced pluripotent stem cells (iPSCs). pFVIII-expressing iPSC-derived MKs, termed iMKs, release platelets that can contribute to improved hemostasis in problematic inhibitor patients with hemophilia A. As proof of principle, we demonstrate that hemostasis can be achieved in vitro and in vivo with pFVIII-expressing platelets and show prolonged efficacy. Notably, pFVIII-expressing platelets are also effective in the presence of inhibitors, and their effect was enhanced with recombinant FVIIa. Human pFVIII-expressing iMKs improved hemostasis in vitro, and derived platelets from infused human pFVIII-expressing iMKs improved hemostasis in FVIIInull mice. These studies indicate the potential therapeutic use of recurrent pFVIII-expressing MK or platelet infusions with prolonged hemostatic coverage that may be additive with bypassing agents in hemophilia A patients with neutralizing inhibitors.
Project description:Successful expansion of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) would benefit many HSPC transplantation and gene therapy/editing applications. However, current expansion technologies have been limited by a loss of multipotency and self-renewal properties ex vivo. We hypothesized that an ex vivo vascular niche would provide prohematopoietic signals to expand HSPCs while maintaining multipotency and self-renewal. To test this hypothesis, BM autologous CD34+ cells were expanded in endothelial cell (EC) coculture and transplanted in nonhuman primates. CD34+ C38- HSPCs cocultured with ECs expanded up to 17-fold, with a significant increase in hematopoietic colony-forming activity compared with cells cultured with cytokines alone (colony-forming unit-granulocyte-erythroid-macrophage-monocyte; p < .005). BM CD34+ cells that were transduced with green fluorescent protein lentivirus vector and expanded on ECs engrafted long term with multilineage polyclonal reconstitution. Gene marking was observed in granulocytes, lymphocytes, platelets, and erythrocytes. Whole transcriptome analysis indicated that EC coculture altered the expression profile of 75 genes in the BM CD34+ cells without impeding the long-term engraftment potential. These findings show that an ex vivo vascular niche is an effective platform for expansion of adult BM HSPCs. Stem Cells Translational Medicine 2017;6:864-876.