Bone Marrow Endothelial Cells Research Articles

Transplanted Human Bone Marrow Endothelial Progenitor Cells Prolong Functional Benefits and Extend Survival of ALS Mice Likely via Blood-Spinal Cord Barrier Repair.

Amyotrophic lateral sclerosis (ALS) is a multifactorial disease with one of these factors being an impaired blood-spinal cord barrier (BSCB). In order to block harmful components in systemic circulation from accessing the CNS, barrier damage needs alleviation. Recently, we found that symptomatic ALS animals treated with intravenously delivered human bone marrow-derived CD34+ (hBM34+) cells or endothelial progenitor cells (hBMEPCs) showed delayed disease progression for 4 weeks post-transplant via BSCB repair. However, despite noted benefits from transplanted human bone marrow-derived stem cells, long-term effects of transplanted cells in ALS mice remain undetermined. This study aimed to determine prolonged effects of single equal doses of hBM34+ cells and hBMEPCs systemically transplanted into symptomatic G93A SOD1 mice on behavioral disease outcomes and mouse lifespan. Results showed that transplanted hBMEPCs better ameliorated disease behavioral outcomes than hBM34 + cells until near end-stage disease and significantly increased lifespan vs. media-treated mice. These results provide important evidence that transplanted hBMEPCs prolonged functional benefits and extended survival of ALS mice, potentially by repairing the damaged BSCB. However, due to modestly increased lifespan of hBMEPC-treated mice, repeated cell transplants into symptomatic ALS mice may more effectively delay motor function deficit and extend lifespan by continuous reparative processes via replacement of damaged endothelial cells during disease progression.

In vivo bone marrow microenvironment siRNA delivery using lipid–polymer nanoparticles for multiple myeloma therapy

Multiple myeloma (MM), a hematologic malignancy that preferentially colonizes the bone marrow, remains incurable with a survival rate of 3 to 6 mo for those with advanced disease despite great efforts to develop effective therapies. Thus, there is an urgent clinical need for innovative and more effective MM therapeutics. Insights suggest that endothelial cells within the bone marrow microenvironment play a critical role. Specifically, cyclophilin A (CyPA), a homing factor secreted by bone marrow endothelial cells (BMECs), is critical to MM homing, progression, survival, and chemotherapeutic resistance. Thus, inhibition of CyPA provides a potential strategy to simultaneously inhibit MM progression and sensitize MM to chemotherapeutics, improving therapeutic response. However, inhibiting factors from the bone marrow endothelium remains challenging due to delivery barriers. Here, we utilize both RNA interference (RNAi) and lipid-polymer nanoparticles to engineer a potential MM therapy, which targets CyPA within blood vessels of the bone marrow. We used combinatorial chemistry and high-throughput in vivo screening methods to engineer a nanoparticle platform for small interfering RNA (siRNA) delivery to bone marrow endothelium. We demonstrate that our strategy inhibits CyPA in BMECs, preventing MM cell extravasation in vitro. Finally, we show that siRNA-based silencing of CyPA in a murine xenograft model of MM, either alone or in combination with the Food and Drug Administration (FDA)-approved MM therapeutic bortezomib, reduces tumor burden and extends survival. This nanoparticle platform may provide a broadly enabling technology to deliver nucleic acid therapeutics to other malignancies that home to bone marrow.

Repair of dysfunctional bone marrow endothelial cells alleviates aplastic anemia.

Aplastic anemia (AA) is a life-threatening disease characterized by bone marrow (BM) failure and pancytopenia. As an important component of the BM microenvironment, endothelial cells (ECs) play a crucial role in supporting hematopoiesis and regulating immunity. However, whether impaired BM ECs are involved in the occurrence of AA and whether repairing BM ECs could improve hematopoiesis and immune status in AA remain unknown. In this study, a classical AA mouse model and VE-cadherin blocking antibody that could antagonize the function of ECs were used to validate the role of BM ECs in the occurrence of AA. N-acetyl-L-cysteine (NAC, a reactive oxygen species scavenger) or exogenous EC infusion was administered to AA mice. Furthermore, the frequency and functions of BM ECs from AA patients and healthy donors were evaluated. BM ECs from AA patients were treated with NAC in vitro, and then the functions of BM ECs were evaluated. We found that BM ECs were significantly decreased and damaged in AA mice. Hematopoietic failure and immune imbalance became more severe when the function of BM ECs was antagonized, whereas NAC or EC infusion improved hematopoietic and immunological status by repairing BM ECs in AA mice. Consistently, BM ECs in AA patients were decreased and dysfunctional. Furthermore, dysfunctional BM ECs in AA patients led to their impaired ability to support hematopoiesis and dysregulate T cell differentiation toward proinflammatory phenotypes, which could be repaired by NAC in vitro. The reactive oxygen species pathway was activated, and hematopoiesis- and immune-related signaling pathways were enriched in BM ECs of AA patients. In conclusion, our data indicate that dysfunctional BM ECs with impaired hematopoiesis-supporting and immunomodulatory abilities are involved in the occurrence of AA, suggesting that repairing dysfunctional BM ECs may be a potential therapeutic approach for AA patients.

Uptake-Dependent and -Independent Effects of Fibroblasts-Derived Extracellular Vesicles on Bone Marrow Endothelial Cells from Patients with Multiple Myeloma: Therapeutic and Clinical Implications.

Extracellular vesicles (EVs) have emerged as important players in cell-to-cell communication within the bone marrow (BM) of multiple myeloma (MM) patients, where they mediate several tumor-associated processes. Here, we investigate the contribution of fibroblasts-derived EVs (FBEVs) in supporting BM angiogenesis. We demonstrate that FBEVs' cargo contains several angiogenic cytokines (i.e., VEGF, HGF, and ANG-1) that promote an early over-angiogenic effect independent from EVs uptake. Interestingly, co-culture of endothelial cells from MM patients (MMECs) with FBEVs for 1 or 6 h activates the VEGF/VEGFR2, HGF/HGFR, and ANG-1/Tie2 axis, as well as the mTORC2 and Wnt/β-catenin pathways, suggesting that the early over-angiogenic effect is a cytokine-mediated process. FBEVs internalization occurs after longer exposure of MMECs to FBEVs (24 h) and induces a late over-angiogenic effect by increasing MMECs migration, chemotaxis, metalloproteases release, and capillarogenesis. FBEVs uptake activates mTORC1, MAPK, SRC, and STAT pathways that promote the release of pro-angiogenic cytokines, further supporting the pro-angiogenic milieu. Overall, our results demonstrate that FBEVs foster MM angiogenesis through dual time-related uptake-independent and uptake-dependent mechanisms that activate different intracellular pathways and transcriptional programs, providing the rationale for designing novel anti-angiogenic strategies.

Abstract 102: Hyperlipidemia-induced Hematopoiesis is Repressed by MLKL in Endothelial Cells of the Splenic Niche

The mixed lineage kinase domain-like protein (MLKL) is well-known for its role in the execution of necroptotic cell death. We showed that antisense oligonucleotide knockdown of MLKL ( Mlkl KD) in Apoe -knockout mice reduces plaque necrotic core, as predicted, but not total plaque area. Additionally, Mlkl KD expanded splenic hematopoietic stem and progenitor cells (HSPCs) and mature myeloid cells known to drive atherogenesis. Thus, we hypothesized that MLKL limits splenic hematopoiesis and thereby immune cell contributions to the growing atheroma. To test whether hematopoietic or non-hematopoietic MLKL expression was responsible for HSPC expansion, we transplanted Ldlr -knockout mice with Mlkl -knockout bone marrow and found no changes in hematopoiesis, suggesting that MLKL functions in other cells in the splenic niche. Flow cytometry and immunofluorescence staining during a time course of atherosclerosis revealed a decrease of splenic endothelial cells that was correlated with a reduction of MLKL endogenously or by Mlkl KD, suggesting that MLKL maintains splenic endothelial cells. Both in vivo and in vitro we observed increased lipid accumulation and concomitant defects in cell cycle in Mlkl KD splenic endothelial cells. Similar to our previous work in macrophages, Mlkl KD led to an accumulation of CHMP4B in cultured splenic endothelial cells, suggesting aberrant endocytic trafficking, which has also been linked to dysfunctional proliferation. Furthermore, there was greater production of immune cells derived from transplanted CD45.1 HSPCs in Mlkl KD recipient spleens. Co-culture with Mlkl KD splenic endothelial cells increased HSPC expression of pStat5, a marker of activation, which was coupled with an increase in myeloid colony formation, as compared to co-culture with control splenic endothelial cells. Remarkably no changes in HSPC regulation were found upon Mlkl KD in bone marrow endothelial cells. In conclusion, we demonstrate a novel role for MLKL in regulating the balance of HSPCs, specifically through preservation of splenic endothelial cells that repress hematopoiesis, highlighting the importance of spleen lipid metabolism and its impact on inflammation that potentiates atherogenesis.

Timing and regulation of hematopoietic stem cell colonization of the human fetal bone marrow by endothelial and CAR stromal cells during pregnancy

The bone marrow (BM) contains the major reservoir of immature and maturing hematopoietic and immune cells throughout adult life while also harboring most hematopoietic stem and progenitor cells (HSPCs). BM-retained hematopoietic stem cells (HSCs) are mostly maintained in a quiescent, non-motile mode. A small fraction of BM-retained HSPC daily proliferate, differentiate, and migrate to the circulation, to replenish the blood with new immature and maturing blood and (all) immune (both myeloid and lymphoid) cells with a finite life span.1 The anti-coagulation and anti-inflammatory receptor EPCR is also functionally expressed by primitive BM-retained HSCs which are endowed with the highest competitive long-term repopulation potential (LT-HSC). Only BM-retained, quiescent EPCR-positive LT-HSCs are protected from DNA damaging insults including clinical chemotherapy and radiation treatments. Primitive EPCR-positive LT-HSC chemotherapy resistance requires the CXCL12-CXCR4 axis that also regulates HSC quiescence, cell cycle, and directional migration as well as the aPC/EPCR/PAR1 axis.2,3 The chemokine CXCL12 is highly expressed by many BM endothelial and stromal cells types including HSC niche supporting osteoprogenitor cells termed CXCL12 abundant reticular cells (CAR cells), while primitive EPCR-positive fetal liver and adult BM HSC functionally express its major receptor CXCR4.4 Most functional HSC studies involve mice experimental preclinical models as well as results obtained from clinical BM transplantation protocols. During fetal development, HSCs migrate from the fetal liver to the fetal BM and spleen for their lodgment and repopulation. Stem cell homing to the BM during development and in functional experimental transplantation assays including with human HSC in transplanted immune deficient mice is CXCL12/CXCR4-dependent.5,6 The precise timepoint of HSC colonization of the human fetal BM and spleen during pregnancy, as well as the identity of BM stromal niche cells and ligand–receptor interactions which regulate human BM HSC lodgment during fetal development are poorly understood. The group of Zheng et al7 have recently reported on the emergence of HSC in the human fetal BM and spleen and their landscape microenvironment during pregnancy by single-cell RNAseq analysis. This work revealed that functional, human fetal LT-HSC as assayed in transplanted immune deficient mice do not emerge before week 12 post conception. By careful examination of various types of fetal BM stromal and endothelial cells during weeks 10–14 post conception and their transcriptome, this study revealed that fetal BM CAR-like cells endowed with the high levels of CXCL12 production together with fetal endothelial cells which also highly express CXCL12 are the major LT-HSC niche supporting cells. Interestingly, the human fetal spleen was not repopulated with functional human LT-HSC before week 14 post conception, revealing that fetal BM is superior in providing critical signals for LT-HSC homing and lodgment over the fetal spleen. Importantly, self-renewing human CD146 positive osteoprogenitor CAR-like cells were detected in adult human BM and were found to provide a niche for adult human LT-HSC as well as in a miniature human bone implanted in immune deficient mice.8 In conclusion, this study provides evidence for the first functional fetal BM LT-HSC as well as their BM supporting microenvironment and niches.

N-acetyl-L-cysteine attenuates oxidative stress-induced bone marrow endothelial cells apoptosis by inhibiting BAX/caspase 3 pathway

Bone marrow endothelial cells (BMECs) play a crucial role in the maintenance of bone homeostasis. The decline in BMECs is associated with abnormal bone development and loss. At present, the mechanism of age-related oxidative stress enhancement in BMEC dysfunction remains unclear. Our experiment explored injury caused by oxidative stress enhancement in BMECs both in vivo and in vitro. The BMECs, indicators of oxidative stress, bone mass, and apoptosis-related proteins were analyzed in different age groups. We also evaluated the ability of N-Acetyl-L-cysteine (NAC) attenuate oxidative stress injury in BMECs. NAC treatment attenuated reactive oxygen species (ROS) overgeneration and apoptosis in BMECs in vitro and alleviated the loss of BMECs and bone mass in vivo. In conclusion, this study could improve our understanding of the mechanism of oxidative stress-induced BMECs injury and whether NAC has therapeutic potential in senile osteoporosis.

Regulation of endothelial cells on the osteogenic ability of bone marrow mesenchymal stem cells in peri-implantitis

ObjectivesThe relationship between bone resorption and angiogenesis in peri-implantitis remains to be studied. We constructed a Beagle dog model of peri-implantitis, and extracted bone marrow mesenchymal stem cells (BMSCs) and endothelial cells (ECs) for culture. The osteogenic ability of BMSCs in the presence of ECs was investigated through an in vitro osteogenic induction model, and its mechanism was initially explored. Subjects and methodsThe peri-implantitis model was verified by ligation, bone loss was observed by micro-CT, and cytokines were detected by ELISA. The isolated BMSCs and ECs were cultured to detect the expression of angiogenesis, osteogenesis-related proteins, and NF-κB signaling pathway-related proteins. Results8 weeks after surgery, the peri-implant gums were swollen, and micro-CT showed bone resorption. Compared with the control group, IL-1β, TNF-α, ANGII and VEGF were markedly increased in the peri-implantitis group. In vitro studies found that the osteogenic differentiation ability of BMSCs co-cultured with IECs was decreased, and the expression of NF-κB signaling pathway-related cytokines was increased. ConclusionEndothelial cells inhibit the osteogenic differentiation of bone marrow mesenchymal stem cells through NF-κB signaling in the environment of peri-implantitis, which may become a new target for the treatment of peri-implantitis.

Cord Blood–Derived Endothelial Progenitor Cells Promote In Vivo Regeneration of Human Hematopoietic Bone Marrow

Victims of acute radiation exposure are susceptible to hematopoietic toxicity due to bone marrow damage and loss of mature blood elements. Here, we evaluated cord blood-derived endothelial progenitor cells (CB-EPCs) as a potential cellular therapy for mitigation of hematologic acute radiation syndrome. CB-EPCs express endothelial cell markers and maintain their growth characteristics beyond 10+ passages without diminishing their doubling capacity. Further, CB-EPCs can be cryopreserved in vapor-phase liquid nitrogen and easily recovered for propagation, making them an attractive nonimmunogenic cellular therapy for off-the-shelf use. Importantly, we show CB-EPCs have the capacity to potently expand adult human bone marrow hematopoietic progenitor cells both in vitro and in vivo. To demonstrate the role of CB-EPCs in promoting in vivo human immune reconstitution after irradiation, we employed a novel humanized mouse model established by transplant of CD34+ bone marrow cells from 9 unique adult organ donors into immunocompromised NSG-SGM3 mice. The response of the humanized immune system to ionizing irradiation was then tested by exposure to 1 Gy followed by subcutaneous treatment of CB-EPCs, Food and Drug Administration-approved growth factor pegfilgrastim (0.3 mg/kg), or saline. At day 7, total human bone marrow was decreased by 80% in irradiated controls. However, treatment with either growth factor pegfilgrastim or CB-EPCs increased recovery of total human bone marrow by 2.5-fold compared with saline. Notably, CB-EPCs also increased recovery of both human CD34+ progenitors by 5-fold and colony-forming capacity by 3-fold versus saline. Additionally, CB-EPCs promoted recovery of endogenous bone marrow endothelial cells as observed by both increased vessel area and length compared with saline. These findings indicate the feasibility of using humanized mice engrafted with adult bone marrow for radiation research and the development of CB-EPCs as an off-the-shelf cellular therapy for mitigation of hematologic acute radiation syndrome.

Alteration of the Essential Role of Endothelial Cells for Promoting Self-Renewal and Lineage Differentiation by Glucosylceramide and 16,16-dimethyl Prostaglandin E2

Bone marrow endothelial cells (BMECs) play an essential role for supporting hematopoietic stem cell expansion, self-renewal, and lineage differentiation. It has been known that BMECs receiving chronic constitutive inflammatory signals play a significant role in promoting functional alterations in hematopoietic stem cells (HSCs). Among many types of Inflammatory agents, bioactive lipids, including glucosylceramide (GlcCer) and dimethyl-Prostaglandin (dmPGE2) has been recently shown to be the potent mediator of inflammation through their own cognate receptor, CLEC4E (Mincle) and PGE receptors, respectively. Despite their biological significance, however, it is not known if GlcCer and dmPGE2, directly induce gene expression of self-renewal and lineage differentiation factors in BMECs or any other endothelial cells in general. We attempt to model the effect of GlcCer and dmPGE2 in vitro by employing primary human vein endothelial cells (HUVECs), and modified HUVECs transduced with E4ORF1 enhancing glucose uptake pathway. We hypothesize that GlcCer and dmPGE2 treatment will trigger reduction in the expression of ANGPTL2, JAG1, and PTN, while an increase in KITLG, CCL5, M-CSF, and GM-CSF in HUVEC. The significance of this study could be identifying a major pathway that contributes to the loss of self-renewal and increased myeloid lineage differentiated mediated by endothelial cells upon exposure to GlcCer. Results showed varied expressions of self-renewal and maintenance genes by GlcCer and dmPGE2. In conclusion, HUVEC may not be a good model for in vitro study as there was no expression seen in the lineage differentiation gene M-CSF and varied GM-CSF expression. However, the results of our study could potentially be utilized for in vitro expansion of HSCs for autologous bone marrow transfer, further genetic manipulation of endothelial cells, and characterization of the signal transduction pathway.

Engineered periosteum-diaphysis substitutes with biomimetic structure and composition promote the repair of large segmental bone defects

The repair of large segmental bone defects remains a big challenge due to limited self-healing capacity of bone. Digging into the structure and composition of natural long bone let us realize that the periosteum cambium on the surface of diaphysis plays a crucial role in bone repair. In this study, we explored the feasibility of using a tissue-engineered periosteum-diaphysis substitute to repair the large segmental bone defects. To create an artificial periosteum cambium, bone marrow mesenchymal stem cells (BMSCs) and endothelial progenitor cells (EPCs) were co-cultured on electrospun silk fibroin (SF) fibrous membranes for mimicking the cellular composition and microstructure of cambium layer of the native periosteum. These SF membranes supported the adhesion and proliferation of both BMSCs and EPCs. In addition, we found that a 1:1 ratio of BMSCs and EPCs supported osteogenesis and angiogenesis optimally. This biomimetic periosteum layer was integrated with artificial diaphysis made of tubular SF scaffolds to construct a biomimetic periosteum-diaphysis substitute. Animal studies confirmed that the biomimetic periosteum-diaphysis substitutes promoted the repair of critical-size bone defects of rabbit radius. Furthermore, the transplanted biomimetic periosteum-diaphysis substitute could prevent the growth of fibrous tissues in the bone defect, and thus reduce the occurrence of nonunion. This study described a promising tissue engineering strategy for the repair of large segmental bone defects.

Integration of single-cell transcriptomes and biological function reveals distinct behavioral patterns in bone marrow endothelium

Heterogeneity of endothelial cell (EC) populations reflects their diverse functions in maintaining tissue’s homeostasis. However, their phenotypic, molecular, and functional properties are not entirely mapped. We use the Tie2-CreERT2;Rosa26-tdTomato reporter mouse to trace, profile, and cultivate primary ECs from different organs. As paradigm platform, we use this strategy to study bone marrow endothelial cells (BMECs). Single-cell mRNA sequencing of primary BMECs reveals that their diversity and native molecular signatures is transitorily preserved in an ex vivo culture that conserves key cell-to-cell microenvironment interactions. Macrophages sustain BMEC cellular diversity and expansion and preserve sinusoidal-like BMECs ex vivo. Endomucin expression discriminates BMECs in populations exhibiting mutually exclusive properties and distinct sinusoidal/arterial and tip/stalk signatures. In contrast to arterial-like, sinusoidal-like BMECs are short-lived, form 2D-networks, contribute to in vivo angiogenesis, and support hematopoietic stem/progenitor cells in vitro. This platform can be extended to other organs’ ECs to decode mechanistic information and explore therapeutics.

Tantalum and magnesium nanoparticles enhance the biomimetic properties and osteo-angiogenic effects of PCL membranes.

Segmental bone defects, accompanied by periosteum stripping or injury, usually lead to delayed bone union or nonunion, which have challenged orthopedic surgeons. The periosteum, which provides essential blood supply and initial stem cells for bone tissue, plays an important role in the repair of bone defects. The reconstruction of the destroyed periosteum has attracted the attention of researchers exploring more satisfactory therapies to repair bone defects. However, periosteum-like biomaterials have yet to meet the clinical requirements and resolve this challenging problem. In this study, we manufactured a nanofiber periosteum replacement based on poly-ε-caprolactone (PCL), in which tantalum nanoparticles (TaNPs) and nanoscale magnesium oxide (MgO) were introduced to enhance its osteogenic and angiogenic ability. The results of in vitro experiments indicated that the PCL/Ta/MgO periosteum replacement, with excellent cytocompatibility, promoted the proliferation of both bone marrow mesenchymal stem cells (BMSCs) and endothelial progenitor cells (EPCs). Furthermore, the incorporation of TaNPs and nano-MgO synergistically enhanced the osteogenic differentiation of BMSCs and the angiogenic properties of EPCs. Similarly, the results of in vivo experiments from subcutaneous implantation and critical-sized calvarial defect models showed that the PCL/Ta/MgO periosteum replacement combined the osteogenesis and angiogenesis abilities, promoting vascularized bone formation to repair critical-sized calvarial defects. The results of our study suggest that the strategy of stimulating repairing bone defects can be achieved with the periosteum repaired in situ and that the proposed periosteum replacement can act as a bioactive medium to accelerate bone healing.

Jagged1+ Myeloid-Derived Suppressor Cells Are Required for Recovery of the Hematopoietic Bone Marrow Niche after Myelosuppression

Myelosuppressive treatments such as chemotherapy and radiotherapy are widely used to treat a variety of hematopoietic diseases and disorders, however current treatment regiments induce broad cytotoxicity illustrated by off-target tissue damage. In order for timely recovery of vascular and hematopoietic function, bone marrow (BM) endothelial cells must regenerate following myelosuppression. The identity and mechanism by which myelosuppressive-resistant cells that trigger endothelial regeneration in the damaged BM microenvironment are unknown. In the present study, we show that Jagged1 (Jag1) expression was significantly upregulated in various hematopoietic populations, particularly in myeloid-derived suppressor cells (MDSCs), following myelosuppression. By crossing Jag1conditional knockoutmice with Vav1-Cre driver mice, we generated the Jag1f/fVavCre+ strain that specific deletes Jag1 in hematopoietic cells. We demonstrate that loss of hematopoietic Jag1 does not affect numbers and function of hematopoietic stem and progenitor cells (HSPCs) under homeostatic condition, but Jag1f/fVavCre+ mice rapidly succumb to both chemo- and radio-therapeutic stress exhibited by severe pancytopenia, very limited endothelial regeneration, and rapid weight loss and morbidity. Full recovery can be achieved by adoptive transfer of WT bone marrow MDSCs, which we show can efficiently rescue the severe phenotypes of Jag1f/fVavCre+ by improving endothelial cell recovery and hematopoietic niche regeneration. Using reporter models, we show that robust Notch pathway activation in endothelial cells is mediated by Jag1+MDSCs. This leads to hematopoietic recovery due to proliferation and elevated numbers of HSPCs in Jag1f/fVavCre+ mice. Treatment with Jag1-coated nanoparticles similarly rescues viability of the BM endothelial cells. Our findings indicate that hematopoietic Jag1 is essential in accelerating niche regeneration and hematopoietic recovery and is a potential therapeutic target in ameliorating damage from multiple myelosuppressive treatments.

Azacitidine and Panobinostat Overcome Bone Marrow Microenvironment-Induced Chemoprotection in KMT2A-rearranged Pediatric AML

KMT2A is the most common gene rearrangement in pediatric acute leukemias and is difficult to treat with a five-year overall survival rate of less than 50%, owing to frequent relapse and poor response to chemotherapy. Recent studies have investigated how interactions within the bone marrow microenvironment help AML cells evade chemotherapy and contribute to relapse by promoting the survival of leukemic blasts. Our group has previously identified that epigenetic drug combination of DNA hypomethylating agent azacitidine and histone deacetylase inhibitor panobinostat reduces cell adhesion and promotes mobilization of leukemic cells to peripheral blood (Weber et al., 132:2637, Blood, 2018). In this study, we used a multi cell coculture of pediatric AML cells with bone marrow stromal cells, endothelial cells and pediatric mesenchymal stem cells to better mimic the bone marrow microenvironment. We observed that the chemoprotection index in multi-cell coculture was much higher than individual cell type (Figure 1A). While 1.5 uM cytarabine reduced MV4;11 cell viability by 50%, there was less than 1% reduction in cell viability in multi-cell coculture. We further investigated if the bone marrow microenvironment induced chemoprotection was mediated by soluble factors, or via adhesive interactions between adherent cell layer and/or the extracellular matrix (ECM). Conditioned media failed to afford chemoprotection, while separation of AML cells from multi-cell layer via Transwells or culturing AML cells in Matrigel-coated dishes showed partial chemoprotection. These results indicated that direct cell-cell or cell-ECM contact was necessary for the chemoprotection. Pre-treatment with azacitidine and panobinostat sensitized five distinct patient-derived xenograft (PDX) lines of KMT2A-rearranged AML to both cytarabine and daunorubicin in multi-cell coculture. These data indicate that azacitidine and panobinostat combination can synergistically overcome chemoprotection mediated by stromal, endothelial and mesenchymal stem cells. We also observed improved survival with pre-treatment of azacitidine panobinostat (2.5 mg/Kg each, Qd5) followed by chemotherapy consisting of cytarabine (50 mg/Kg, Qd5) and daunorubcin (1.5 mg/Kg, Qd3) in a PDX model of KMT2A-rearranged AML (Figure 1B, **P<0.05). To determine if these epigenetic modifers alter the expression of cell adhesion molecules, cells treated with azacitidine panobinostat combination were subjected to a high throughput flow cytometry assay (Lyoplate, Human cell surface marker screening panel). This analysis showed reduced cell surface expression of integrins, ICAMs and chemokine receptors, indicating that epigenetic drug combination likely causes mobilization and chemosensitization via downregulation of cell adhesion molecules which not only mediate adhesive interactions but also promote cell survival. In summary, our data show azacitidine and panobinostat can overcome bone marrow microenvironment induced chemoprotection in vitro and in vivo in KMT2A rearranged AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Sickle Cell Disease Compromises the Hematopoietic Stem Cells Bone Marrow Niche with Age

Sickle cell disease (SCD) is characterized by chronic inflammation caused by red blood cell hemolysis, which can trigger oxidative stress and tissue injury that contribute to disease. Recent studies have shown that SCD pathophysiology reconstituted post-transplant in mouse models disrupts the architecture of bone marrow (BM) sinusoidal endothelium and causes a defect of mesenchymal stromal cells (MSCs) function. However, the impact of SCD on the ability MSCs and endothelial cells (ECs) to support of human and mouse hematopoiesis remains largely unknown. Using a mouse model of SCD, we now report that both sinusoidal (LepR+) and arteriolar MSCs (PαS) are more abundant in SCD BM, where they also display increased proliferation at 2-, 6- and 12-months of age. Additionally, 12-month-old SCD mice display a massive loss of sinusoidal ECs. Young SCD patient (n=9, age 2-18) BM also displayed increased MSC numbers relative to age-matched healthy controls (n=13). SCD MSCs isolated from 6- and 12-month old mice, maintained increased cell cycle when cultured ex vivo, showed decreased CFU-F potential, and impaired osteoblast differentiation. In ex vivo co-cultures of primary murine BM MSCs, ECs and HSCs, we found that 2-, 6-, and 12-month old SCD stroma (MSCs and ECs) failed to support non-SCD HSCs relative to controls. Phenotypic HSCs recovered from these co-cultures displayed a loss of CFU potential and transplantation activity. In tri-cultures, SCD MSCs, but not SCD ECs, drove increased HSC proliferation, apoptosis, and lymphoid-biased transplantation. In addition, MSCs isolated from BM of young SCD patients also displayed increased cell cycle, increased pro-inflammatory cytokine secretion, and decreased ability to support the expansion and CFU potential of co-cultured healthy human CD34+ cells, relative to MSCs isolated from young non-SCD donors. Bulk RNA-sequencing of 2-, 6-, and 12-month old murine SCD MSCs revealed gene signatures consistent with accelerated aging, increased proliferation, loss of extracellular matrix (ECM), impaired immune modulation, and reduced secretion of factors known to support HSCs. Genetic restoration of ECM rescued the ability of SCD MSCs to expand HSCs in ex vivo co-cultures, and support HSCs with robust CFU and transplantation potential. In sum, our data reveals a loss of HSPC supportive potential in BM MSCs in young individuals with SCD and that loss of ECM production by MSCs contributes to dysfunction in the HSC BM niche in both young and aged SCD mice.

ROBO4 Inhibits Irradiation-Induced Microvascular Permeability, Endothelial to Mesenchymal Transition, and Improves Hematopoietic Reconstitution

Background: Hematopoietic Stem Cells Transplantation involves irradiation preconditioning which causes bone marrow endothelial cells dysfunction. While much emphasis is on the reconstitution of an adequate number of HSCs to repopulate the hematopoietic system after the death of malignant cells, EC preservation and normal functioning are indispensable to overcome the preconditioning damages. ROBO4 is an endothelial-specific receptor that is important in maintaining the vasculature. The purpose of this study is to ascertain ROBO4's role in regulating irradiation-induced damage of the endothelium and its subsequent effect on hematopoietic reconstruction. Method: Microvascular endothelial cells were treated with γ-radiation to establish an endothelial cell injury model. The expression of ROBO4 in the microvascular endothelial cells was manipulated employing lentiviral mediated RNAi (shRNA) and gene overexpression technology before irradiation treatment. The permeability of endothelial cells and its mesenchymal transition (Endo-MT) was measured using qPCR, immunocytochemistry, and immunoblotting to analyze the effect on the expression and distribution of junctional molecules (CD144, Connexin 43, and Occludin, etc.), endothelial cell-specific molecules (PECAM-1, VEGFR2 and TIE2, etc.), and specific mesenchymal cell markers (Fibronectin, Vimentin, S100A4, and ACTA2, etc.). Using Transwell and wound healing migration, Matrigel tube formation, and Transwell and FITC-Dextran permeability assays, we determined the changes in endothelial functions after ROBO4 gene manipulation and irradiation. In addition, a co-culture model of endothelial cells and hematopoietic stem cells isolated from C57BL/6 mice was constructed, and the effects of irradiated endothelial cells on the proliferation, differentiation and cell cycle of hematopoietic stem cells were analyzed using flow cytometry. Results: Ionizing radiation upregulates ROBO4 expression but disrupts endothelial junctional molecules. ROBO4 deletion enhances the irradiation-induced degradation of endothelial junctions hence increasing the permeability of the endothelium and also facilitating endothelial to mesenchymal transition (Endo-MT) by activating both the canonical (Smads) and non-canonical (PI3K/AKT/mTOR) pathways. Endothelial ROBO4 overexpression substantially reduces microvascular permeability and mitigates Endo-MT following irradiation. Even though ROBO4 overexpression does not dramatically affect endothelial functions, tube formation and migration potentials of endothelial cells significantly increase upon the knockdown of ROBO4 and irradiation. We also found that EC ROBO4 silencing inhibits the proliferation of HSPC and promotes HSC quiescence. On the other hand, ROBO4 overexpression lessened the inhibitory effects of the injured endothelium on hematopoietic stem cell expansion. Lastly, we saw that ROBO4 overexpression inhibits erythrocytosis and granulocytosis after irradiation compared to the control groups suggesting that EC ROBO4 may play a regulatory role in HSC differentiation. Conclusion: ROBO4 maintains microvascular integrity after radiation preconditioning treatment by regulating permeability and Endo-MT, protects endothelial function, and reduces the inhibitory effect of injured endothelium on hematopoietic reconstitution.

Super-Enhancer-Driven PIM Kinase Upregulation in Multiple Myeloma Maintains the Plasma Cell-Specific Oncogenic and Microenvironmental Circuits, and Can be Efficiently Targeted By the Pan-PIM Inhibitor MEN1703

The three constitutively active PIM serine/threonine kinases have been previously reported to initiate and sustain lymphoid oncogenesis. In subsequent CRISPR/Cas9 drop-out screens, PIM kinases were highlighted as critical oncogenes in MM, which led to clinical trials of small-molecule PIM kinase inhibitors. Surprisingly, PIM inhibitors in MM patients exhibited limited activity due to their cytostatic, rather cytotoxic effects. These clinical trial results prompted us to further investigate the consequences of PIM kinase inhibition in MM using recently developed pan-PIM/FLT3 inhibitor MEN1703. Immunohistochemical staining revealed a markedly increased PIM1/2/3 levels in malignant plasma cells in comparison with normal plasma cells from healthy donors, with particularly abundant PIM2 expression. Through the integration of CRISPR/Cas9 dropout screen and genome-wide H3K27Ac profiling in MM cells, we identified PIM2 as a super-enhancer-driven oncogene in MM. BET inhibitor JQ1, which disrupts super-enhancer assembly, downregulated PIM kinase expression. Since super-enhancer associated genes are often the most essential nodes of key cellular pathways to be potentially targeted, these results further underscore the critical role of PIMs in MM. Next, we compared the activity of MEN1703 with all available clinical-stage PIM inhibitors in a panel of >20 different cell lines and found almost universal resistance to 4 out of 5 pan-PIM inhibitors administered at submicromolar concentrations achievable in vivo (Fig. 1A). In contrast, MEN1703 exhibited universal cytotoxic activity. We confirmed these results using patient-derived multiple myeloma cells cultured ex vivo and genetic knockdowns of PIM kinases. Mechanistically, MEN1703 decreased the activity of mTOR, JAK/STAT, and NFκB pathways essential for supporting myeloma cell survival. Consequently, we observed diminished expression of MYC and IRF4, two transcription factors crucial for supporting plasma cell-specific oncogenic program. In the IHC analysis of PIMs expression in MM tissue microarray, we also noted a distinctly high expression of PIM3 in myeloma-associated endothelial cells (ECs), but not in healthy bone marrow ECs. Since ECs play a significant role in shaping the bone marrow microenvironment and directly support the dissemination and survival of plasma cells, we studied the mechanism of PIM3 overexpression, PIM3 function, and the consequences of its inhibition in MM ECs. We found that myeloma-secreted VEGFA is responsible for PIM induction in ECs and that endothelial cells reciprocally induce PIMs expression in MM cells in a paracrine manner, thus increasing their proliferation and inhibiting cell death. PIM inhibition blocked these effects, which was confirmed using cocultures with primary MM endothelial cells. PIM inhibition decreased the MM EC vessel formation by decreasing the activity of endothelial GTPase RhoA, which is responsible for actin cytoskeleton formation during angiogenesis and cytokine secretion. Collectively, our data demonstrate that PIM inhibition with MEN1703 induces MM cell death and abolishes important tumor cell-ECs interactions (Fig. 1B), thus representing a promising therapeutic target in this disease. The clinical utility of next generation/more potent PIM inhibitor in MM should be re-investigated. Study supported by National Science Centre Grants PRELUDIUM 2018/31/N/NZ5/03214, ETIUDA 2020/36/T/NZ5/00610 and Ministry of Science and Higher Education Diamond Grant DI2015 007145. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Bone-targeting delivery of platelet lysate exosomes ameliorates glucocorticoid-induced osteoporosis by enhancing bone-vessel coupling

BackgroundGlucocorticoids (GCs) overuse is associated with decreased bone mass and osseous vasculature destruction, leading to severe osteoporosis. Platelet lysates (PL) as a pool of growth factors (GFs) were widely used in local bone repair by its potent pro-regeneration and pro-angiogenesis. However, it is still seldom applied for treating systemic osteopathia due to the lack of a suitable delivery strategy. The non-targeted distribution of GFs might cause tumorigenesis in other organs.ResultsIn this study, PL-derived exosomes (PL-exo) were isolated to enrich the platelet-derived GFs, followed by conjugating with alendronate (ALN) grafted PEGylated phospholipid (DSPE-PEG-ALN) to establish a bone-targeting PL-exo (PL-exo-ALN). The in vitro hydroxyapatite binding affinity and in vivo bone targeting aggregation of PL-exo were significantly enhanced after ALN modification. Besides directly modulating the osteogenic and angiogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and endothelial progenitor cells (EPCs), respectively, PL-exo-ALN also facilitate their coupling under GCs’ stimulation. Additionally, intravenous injection of PL-exo-ALN could successfully rescue GCs induced osteoporosis (GIOP) in vivo.ConclusionsPL-exo-ALN may be utilized as a novel nanoplatform for precise infusion of GFs to bone sites and exerts promising therapeutic potential for GIOP.Graphical

Hyperlipidemia attenuates the mobilization of endothelial progenitor cells induced by acute myocardial ischemia via VEGF/eNOS/NO/MMP-9 pathway.

This study aims to explore the role of hyperlipidemia in the mobilization of bone marrow (BM) endothelial progenitor cells (EPCs) induced by acute myocardial ischemia (AMI). To establish the hyperlipidemia complicated with AMI (HL-AMI) model, SD rats were intragastrically administered the high-fat emulsion for 4 weeks. Then their left anterior descending arteries were ligated. Rats in each group were randomly subdivided into seven subgroups. During 1st ~ 7th day following AMI modeling, rats in 1st ~ 7th subgroups were selected to be phlebotomized from their celiac artery after being anesthetized by pentobarbitone in turn. The quantity of circulating EPCs (CEPCs) was detected by flow cytometry, the expression of VEGF, eNOS, NO, MMP-9 in myocardial tissue was analyzed by western blot, and their plasma level was assayed by ELISA. Dynamic curves were plotted using these data. Within 7 days following AMI, compared with the AMI rats, in the HL-AMI rats, the myocardial infarct size, the plasma activity of CK, CK-MB, and the collagen deposition all remained at the higher levels; meanwhile, these rats showed more significant decreases in the count of CEPCs, the plasma level of VEGF etc., and their expression in myocardial tissue (P < 0.05 or P < 0.01). Our study showed that hyperlipidemia may attenuate the mobilization of BM EPCs induced by AMI via VEGF/eNOS/NO/MMP-9 signal pathway, which might partly account for hyperlipidemia hampering the repairs of AMI-induced cardiac injury.