Bone Marrow Endothelial Progenitor Research Articles

Activation of PPARδ in bone marrow endothelial progenitor cells improves their hematopoiesis-supporting ability after myelosuppressive injury

Dysfunctional bone marrow (BM) endothelial progenitor cells (EPCs) with high levels of reactive oxygen species (ROS) are responsible for defective hematopoiesis in poor graft function (PGF) patients with acute leukemia or myelodysplastic neoplasms post-allotransplant. However, the underlying mechanism by which BM EPCs regulate their intracellular ROS levels and the capacity to support hematopoiesis have not been well clarified. Herein, we demonstrated decreased levels of peroxisome proliferator-activated receptor delta (PPARδ), a lipid-activated nuclear receptor, in BM EPCs of PGF patients compared with those with good graft function (GGF). In vitro assays further identified that PPARδ knockdown contributed to reduced and dysfunctional BM EPCs, characterized by the impaired ability to support hematopoiesis, which were restored by PPARδ overexpression. Moreover, GW501516, an agonist of PPARδ, repaired the damaged BM EPCs triggered by 5-fluorouracil (5FU) in vitro and in vivo. Clinically, activation of PPARδ by GW501516 benefited the damaged BM EPCs from PGF patients or acute leukemia patients in complete remission (CR) post-chemotherapy. Mechanistically, we found that increased expression of NADPH oxidases (NOXs), the main ROS-generating enzymes, may lead to elevated ROS level in BM EPCs, and insufficient PPARδ may trigger BM EPC damage via ROS/p53 pathway. Collectively, we found that defective PPARδ contributes to BM EPC dysfunction, whereas activation of PPARδ in BM EPCs improves their hematopoiesis-supporting ability after myelosuppressive therapy, which may provide a potential therapeutic target not only for patients with leukemia but also for those with other cancers.

The bone marrow endothelial progenitor cell response to septic infection.

Early increase in the level of endothelial progenitor cells (EPCs) in the systemic circulation occurs in patients with septic infection/sepsis. The significance and underlying mechanisms of this response remain unclear. This study investigated the bone marrow EPC response in adult mice with septic infection induced by intravenous injection (i.v.) of Escherichia coli. For in vitro experiments, sorted marrow stem/progenitor cells (SPCs) including lineage(lin)-stem cell factor receptor (c-kit)+stem cell antigen-1 (Sca-1)-, lin-c-kit+, and lin- cells were cultured with or without lipopolysaccharides (LPSs) and recombinant murine vascular endothelial growth factor (VEGF) in the absence and presence of anti-Sca-1 crosslinking antibodies. In a separate set of experiments, marrow lin-c-kit+ cells from green fluorescence protein (GFP)+ mice, i.v. challenged with heat-inactivated E. coli or saline for 24h, were subcutaneously implanted in Matrigel plugs for 5 weeks. Marrow lin-c-kit+ cells from Sca-1 knockout (KO) mice challenged with heat-inactivated E. coli for 24h were cultured in the Matrigel medium for 8 weeks. The marrow pool of EPCs bearing the lin-c-kit+Sca-1+VEGF receptor 2 (VEGFR2)+ (LKS VEGFR2+) and LKS CD133+VEGFR2+ surface markers expanded rapidly following septic infection, which was supported by both proliferative activation and phenotypic conversion of marrow stem/progenitor cells. Increase in marrow EPCs and their reprogramming for enhancing angiogenic activity correlated with cell-marked upregulation of Sca-1 expression. Sca-1 was coupled with Ras-related C3 botulinum toxin substrate 2 (Rac2) in signaling the marrow EPC response. Septic infection caused a substantial increase in plasma levels of IFN-γ, VEGF, G-CSF, and SDF-1. The early increase in circulating EPCs was accompanied by their active homing and incorporation into pulmonary microvasculature. These results demonstrate that the marrow EPC response is a critical component of the host defense system. Sca-1 signaling plays a pivotal role in the regulation of EPC response in mice with septic infection.

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.

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.

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.

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.

Abstract 5626: Acquisition of lymphatic phenotype in bone marrow endothelial progenitors is regulated by immunosuppressive Th2 cytokines

Abstract Introduction: Myeloid-lymphatic endothelial progenitors (M-LECPs) significantly contribute to expansion of tumor lymphatics and metastasis to lymph nodes. Bone marrow (BM) generated M-LECPs are a subset of M2-type of tumor-associated macrophages (TAMs) that heavily infiltrate breast and other human cancers. The M2-type of TAMs is primarily induced by immunosuppressive Th2 cytokines IL-4, IL-13, and IL-10. The goal of this study was to determine whether Th2 factors also promote the lymphatic phenotype in precursors of M-LECPs. Methods: Mouse BM cells were differentiated into M-LECPs by priming with CSF-1 followed by activation of LPS-TLR4 pathway. Transcripts of IL-4, IL-13, and IL-10 were measured by qPCR, and their proteins secreted into conditioned medium were detected by ELISA. The levels of receptors for Th2 cytokines IL-4, IL-13, and IL-10 as well as for stem cells (CD117, Sca-1), M2-TAMs (CD204, CD206) and lymphatic endothelial cells (Lyve-1, podoplanin, integrin-9a, collectin-12 and stabilin-1) were quantified by qPCR and flow cytometry. Impact of TLR4 induced autocrine IL-10 pathway was determined by analyzing the surface markers and gene profiles of differentiated M-LECPs in the presence of IL-10 blocking or control antibodies. Tumor levels of Th2 cytokines and expression of Th2 receptors in Lyve-1+ progenitors were detected by ELISA and immunofluorescence, respectively. Results: More than 95% of myeloid precursors treated with CSF-1 and LPS upregulated all three Th2 receptors and IL-10 but not IL-4 or IL-13 ligands. All receptors were functional as indicated by upregulation of M2, immunosuppressive, lymphatic and stem markers in cells co-treated with exogenous Th2 ligands as compared with CSF-1 alone. Blocking autocrine IL-10 significantly suppressed both M2-specific and pro-lymphatic differentiation. Tumor-infiltrating M-LECPs expressed Th2 receptors as well as all corresponding ligands including IL-4 and IL-13 that were absent in the BM cells. Conclusion: These data present a novel evidence that activation of immunosuppressive Th2 pathways in BM myeloid precursors induces both the M2-type and lymphatic phenotype. Expansion of lymphatics can enhance tumor immunosuppression by physical removal of immunostimulatory cells. These findings suggest that targeting Th2 pathways can simultaneously relieve immunosuppression and inhibit differentiation of pro-lymphatic progenitors that promote metastasis. Citation Format: Maria Espinoza Gonzalez, Lisa Volk-Draper, Nihit Bhattarai, Sophia Ran. Acquisition of lymphatic phenotype in bone marrow endothelial progenitors is regulated by immunosuppressive Th2 cytokines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5626.

The glycolytic enzyme PFKFB3 determines bone marrow endothelial progenitor cell damage after chemotherapy and irradiation.

Bone marrow (BM) endothelial progenitor cell (EPC) damage of unknown mechanism delays the repair of endothelial cells (EC) and recovery of hematopoiesis after chemo-radiotherapy. We found increased levels of the glycolytic enzyme PFKFB3 in the damaged BM EPC of patients with poor graft function, a clinical model of EPC damage-associated poor hematopoiesis after allogeneic hematopoietic stem cell transplantation. Moreover, in vitro the glycolysis inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) alleviated the damaged BM EPC from patients with poor graft function. Consistently, PFKFB3 overexpression triggered BM EPC damage after 5-fluorouracil treatment and impaired hematopoiesis-supporting ability in vitro. Mechanistically, PFKFB3 facilitated pro-apoptotic transcription factor FOXO3A and expression of its downstream genes, including p21, p27, and FAS, after 5-fluorouracil treatment in vitro. Moreover, PFKFB3 induced activation of NF-κB and expression of its downstream adhesion molecule E-selectin, while it reduced hematopoietic factor SDF-1 expression, which could be rescued by FOXO3A silencing. High expression of PFKFB3 was found in damaged BM EC of murine models of chemo-radiotherapy-induced myelosuppression. Furthermore, a murine model of BM EC-specific PFKFB3 over-expression demonstrated that PFKFB3 aggravated BM EC damage, and impaired the recovery of hematopoiesis after chemotherapy in vivo, effects which could be mitigated by 3PO, indicating a critical role of PFKFB3 in regulating BM EC damage. Clinically, PFKFB3-induced FOXO3A expression and NF-κB activation were confirmed to contribute to the damaged BM EPC of patients with acute leukemia after chemotherapy. 3PO repaired the damaged BM EPC by reducing FOXO3A expression and phospho-NF-κB p65 in patients after chemotherapy. In summary, our results reveal a critical role of PFKFB3 in triggering BM EPC damage and indicate that endothelial-PFKFB3 may be a potential therapeutic target for myelosup-pressive injury.

Dysfunctional bone marrow endothelial progenitor cells are involved in patients with myelodysplastic syndromes

BackgroundMyelodysplastic syndromes (MDS) are a group of heterogeneous myeloid clonal disorders characterized by ineffective haematopoiesis and immune deregulation. Emerging evidence has shown the effect of bone marrow (BM) endothelial progenitor cells (EPCs) in regulating haematopoiesis and immune balance. However, the number and functions of BM EPCs in patients with different stages of MDS remain largely unknown.MethodsPatients with MDS (N = 30), de novo acute myeloid leukaemia (AML) (N = 15), and healthy donors (HDs) (N = 15) were enrolled. MDS patients were divided into lower-risk MDS (N = 15) and higher-risk MDS (N = 15) groups according to the dichotomization of the Revised International Prognostic Scoring System. Flow cytometry was performed to analyse the number of BM EPCs. Tube formation and migration assays were performed to evaluate the functions of BM EPCs. In order to assess the gene expression profiles of BM EPCs, RNA sequencing (RNA-seq) were performed. BM EPC supporting abilities of haematopoietic stem cells (HSCs), leukaemia cells and T cells were assessed by in vitro coculture experiments.ResultsIncreased but dysfunctional BM EPCs were found in MDS patients compared with HDs, especially in patients with higher-risk MDS. RNA-seq indicated the progressive change and differences of haematopoiesis- and immune-related pathways and genes in MDS BM EPCs. In vitro coculture experiments verified that BM EPCs from HDs, lower-risk MDS, and higher-risk MDS to AML exhibited a progressively decreased ability to support HSCs, manifested as elevated apoptosis rates and intracellular reactive oxygen species (ROS) levels and decreased colony-forming unit plating efficiencies of HSCs. Moreover, BM EPCs from higher-risk MDS patients demonstrated an increased ability to support leukaemia cells, characterized by increased proliferation, leukaemia colony-forming unit plating efficiencies, decreased apoptosis rates and apoptosis-related genes. Furthermore, BM EPCs induced T cell differentiation towards more immune-tolerant cells in higher-risk MDS patients in vitro. In addition, the levels of intracellular ROS and the apoptosis ratios were increased in BM EPCs from MDS patients, especially in higher-risk MDS patients, which may be therapeutic candidates for MDS patients.ConclusionOur results suggest that dysfunctional BM EPCs are involved in MDS patients, which indicates that improving haematopoiesis supporting ability and immuneregulation ability of BM EPCs may represent a promising therapeutic approach for MDS patients.

Diabetes impairs cardioprotective function of endothelial progenitor cell-derived extracellular vesicles via H3K9Ac inhibition.

Background and Purpose: Myocardial infarction (MI) in diabetic patients results in higher mortality and morbidity. We and others have previously shown that bone marrow-endothelial progenitor cells (EPCs) promote cardiac neovascularization and attenuate ischemic injury. Lately, small extracellular vesicles (EVs) have emerged as major paracrine effectors mediating the benefits of stem cell therapy. Modest clinical outcomes of autologous cell-based therapies suggest diabetes-induced EPC dysfunction and may also reflect their EV derivatives. Moreover, studies suggest that post-translational histone modifications promote diabetes-induced vascular dysfunctions. Therefore, we tested the hypothesis that diabetic EPC-EVs may lose their post-injury cardiac reparative function by modulating histone modification in endothelial cells (ECs).Methods: We collected EVs from the culture medium of EPCs isolated from non-diabetic (db/+) and diabetic (db/db) mice and examined their effects on recipient ECs and cardiomyocytes in vitro, and their reparative function in permanent ligation of left anterior descending (LAD) coronary artery and ischemia/reperfusion (I/R) myocardial ischemic injuries in vivo.Results: Compared to db/+ EPC-EVs, db/db EPC-EVs promoted EC and cardiomyocyte apoptosis and repressed tube-forming capacity of ECs. In vivo, db/db EPC-EVs depressed cardiac function, reduced capillary density, and increased fibrosis compared to db/+ EPC-EV treatments after MI. Moreover, in the I/R MI model, db/+ EPC-EV-mediated acute cardio-protection was lost with db/db EPC-EVs, and db/db EPC-EVs increased immune cell infiltration, infarct area, and plasma cardiac troponin-I. Mechanistically, histone 3 lysine 9 acetylation (H3K9Ac) was significantly decreased in cardiac ECs treated with db/db EPC-EVs compared to db/+ EPC-EVs. The H3K9Ac chromatin immunoprecipitation sequencing (ChIP-Seq) results further revealed that db/db EPC-EVs reduced H3K9Ac level on angiogenic, cell survival, and proliferative genes in cardiac ECs. We found that the histone deacetylase (HDAC) inhibitor, valproic acid (VPA), partly restored diabetic EPC-EV-impaired H3K9Ac levels, tube formation and viability of ECs, and enhanced cell survival and proliferative genes, Pdgfd and Sox12, expression. Moreover, we observed that VPA treatment improved db/db EPC-mediated post-MI cardiac repair and functions.Conclusions: Our findings unravel that diabetes impairs EPC-EV reparative function in the ischemic heart, at least partially, through HDACs-mediated H3K9Ac downregulation leading to transcriptional suppression of angiogenic, proliferative and cell survival genes in recipient cardiac ECs. Thus, HDAC inhibitors may potentially be used to restore the function of diabetic EPC and other stem cells for autologous cell therapy applications.

Abstract P375: Gender-based Cardio-protective Functional Dimorphism Of Bone Marrow Endothelial Progenitor Cells And Their Exosomes: Estrogen-independent Epigenetic Mechanisms

Introduction: Estrogen or estrogen receptor-dependent mechanisms in enhancing the cardioprotective efficacy of bone marrow endothelial progenitor cells (BM-EPC) is well-established in preclinical studies. However, the efficacy of estrogen does not reflect in the data from randomized cardiovascular clinical trials, suggesting an estrogen-independent role of female BM-EPC in eliciting enhanced cardiac protection compared to males. Hypothesis: Epigenetic mechanisms may contribute to the sex-specific dimorphism of Sca-1 + /CD31 + BM-EPC in regulating cell-homing, pro-angiogenic and anti-inflammatory functions in the ischemic myocardium leading to enhanced reparative function of female progenitor cells. Methods &amp; Results: Transplantation of GFP-BM-mononuclear cells from male and female GFP transgenic mice into the BM of lethally irradiated recipient male C57BL/6 mice resulted in the enhanced mobilization of female Sca-1 + CD31 + /GFP + BM-EPC into circulation post-MI. A higher number of female BM-EPC homed to the ischemic myocardium and significantly improved LV functions and capillary density post-MI compared to male BM-EPC. Female BM-EPC showed increased expression of bFGF, VEGFR2, SDF-1α, and IL-10 genes, thereby efficiently promoted endothelial tube formation in vitro compared to male BM-EPC. Transplantation of female BM-EPC and their exosomes into post-MI male mice improved LV cardiac function, reduced scar size, and improved capillary density compared to male BM-EPC and exosomes. Male BM-EPC showed an increased expression of G9a/Ehmt2, an H3K9me3 methyltransferase, and Dnmt3a DNA methyltransferase compared to female BM-EPC. In contrast, Kdm6b/JMJD3, H3K27me3 demethylase was highly expressed in female BM-EPC compared to males. Treatment of BM-EPC of both sexes with 17-β-estradiol did not alter the expression of Kdm6b/JMJD3. Male BM-EPC highly expressed repressive gene marks, H3K9me3, and H3K27me3 compared to females. Compared to the male, BM-EPC from female and ovariectomized (OXV) female mice showed equally high expression of angiogenic genes ANGPT-1, MDK, PLAU, Tie-2, and VEGFR2 and lower levels of inflammatory cytokines, TNFα, IFNγ, IL-1β, and CCL3. Conditioned medium from female and OVX BM-EPC equally promoted enhanced migration and tube formation of HUVEC in vitro, compared to male BM-EPC. Conclusions: An estrogen-independent epigenetic mechanism likely governs the enhanced cardiac reparative properties of female BM-EPC.

Beneficial Effects of Transplanted Human Bone Marrow Endothelial Progenitors on Functional and Cellular Components of Blood-Spinal Cord Barrier in ALS Mice.

Convincing evidence of blood-spinal cord barrier (BSCB) alterations has been demonstrated in amyotrophic lateral sclerosis (ALS) and barrier repair is imperative to prevent motor neuron dysfunction. We showed benefits of human bone marrow-derived CD34+ cells (hBM34+) and endothelial progenitor cells (hBM-EPCs) intravenous transplantation into symptomatic G93A SOD1 mutant mice on barrier reparative processes. These gains likely occurred by replacement of damaged endothelial cells, prolonging motor neuron survival. However, additional investigations are needed to confirm the effects of administered cells on integrity of the microvascular endothelium. The aim of this study was to determine tight junction protein levels, capillary pericyte coverage, microvascular basement membrane, and endothelial filamentous actin (F-actin) status in spinal cord capillaries of G93A SOD1 mutant mice treated with human bone marrow-derived stem cells. Tight junction proteins were detected in the spinal cords of cell-treated versus non-treated mice via Western blotting at four weeks after transplant. Capillary pericyte, basement membrane laminin, and endothelial F-actin magnitudes were determined in cervical/lumbar spinal cord tissues in ALS mice, including controls, by immunohistochemistry and fluorescent staining. Results showed that cell-treated versus media-treated ALS mice substantially increased tight junction protein levels, capillary pericyte coverage, basement membrane laminin immunoexpressions, and endothelial cytoskeletal F-actin fluorescent expressions. The greatest benefits were detected in mice receiving hBM-EPCs versus hBM34+ cells. These study results support treatment with a specific cell type derived from human bone marrow toward BSCB repair in ALS. Thus, hBM-EPCs may be advanced for clinical applications as a cell-specific approach for ALS therapy through restored barrier integrity.

3D printing of dual-cell delivery titanium alloy scaffolds for improving osseointegration through enhancing angiogenesis and osteogenesis

BackgroundThe repair of large bone defects is a great challenge for orthopedics. Although the development of three-dimensional (3D) printed titanium alloy (Ti6Al4V) implants with optimized the pore structure have effectively promoted the osseointegration. However, due to the biological inertia of Ti6Al4Vsurface and the neglect of angiogenesis, some patients still suffer from postoperative complications such as dislocation or loosening of the prosthesis.MethodsThe purpose of this study was to construct 3D printed porous Ti6Al4V scaffolds filled with bone marrow mesenchymal stem cells (BMSC) and endothelial progenitor cells (EPC) loaded hydrogel and evaluate the efficacy of this composite implants on osteogenesis and angiogenesis, thus promoting osseointegration.ResultsThe porosity and pore size of prepared 3D printed porous Ti6Al4V scaffolds were 69.2 ± 0.9 % and 593.4 ± 16.9 μm, respectively, which parameters were beneficial to bone ingrowth and blood vessel formation. The BMSC and EPC filled into the pores of the scaffolds after being encapsulated by hydrogels can maintain high viability. As a cell containing composite implant, BMSC and EPC loaded hydrogel incorporated into 3D printed porous Ti6Al4V scaffolds enhancing osteogenesis and angiogenesis to repair bone defects efficiently. At the transcriptional level, the composite implant up-regulated the expression levels of the osteogenesis-related genes alkaline phosphatase (ALP) and osteocalcin (OCN), and angiogenesis-related genes hypoxia-inducible factor 1 alpha (HIF-1α), and vascular endothelial growth factor (VEGF).ConclusionsOverall, the strategy of loading porous Ti6Al4V scaffolds to incorporate cells is a promising treatment for improving osseointegration.

Silicified collagen scaffold induces semaphorin 3A secretion by sensory nerves to improve in-situ bone regeneration

Sensory nerves promote osteogenesis through the release of neuropeptides. However, the potential application and mechanism in which sensory nerves promote healing of bone defects in the presence of biomaterials remain elusive. The present study identified that new bone formation was more abundantly produced after implantation of silicified collagen scaffolds into defects created in the distal femur of rats. The wound sites were accompanied by extensive nerve innervation and angiogenesis. Sensory nerve dysfunction by capsaicin injection resulted in significant inhibition of silicon-induced osteogenesis in the aforementioned rodent model. Application of extracellular silicon in vitro induced axon outgrowth and increased expression of semaphorin 3 A (Sema3A) and semaphorin 4D (Sema4D) in the dorsal root ganglion (DRG), as detected by the upregulation of signaling molecules. Culture medium derived from silicon-stimulated DRG cells promoted proliferation and differentiation of bone marrow mesenchymal stem cells and endothelial progenitor cells. These effects were inhibited by the use of Sema3A neutralizing antibodies but not by Sema4D neutralizing antibodies. Knockdown of Sema3A in DRG blocked silicon-induced osteogenesis and angiogenesis almost completely in a femoral defect rat model, whereas overexpression of Sema3A promoted the silicon-induced phenomena. Activation of “mechanistic target of rapamycin” (mTOR) pathway and increase of Sema3A production were identified in the DRG of rats that were implanted with silicified collagen scaffolds. These findings support the role of silicon in inducing Sema3A production by sensory nerves, which, in turn, stimulates osteogenesis and angiogenesis. Taken together, silicon has therapeutic potential in orthopedic rehabilitation.

Atorvastatin improves bone marrow endothelial progenitor cell function from patients with immune-related hemocytopenia.

BackgroundImmune-related hemocytopenia (IRH) is a type of autoimmune disease that targets bone marrow hematopoietic cells. This study investigated the influence of atorvastatin on the functional aspects of bone marrow endothelial progenitor cells (BM EPCs) in IRH patients.MethodsBM EPCs were isolated from 15 patients with IRH and 20 normal controls. The isolated BM EPCs were characterized by flow cytometry. Cell counting kit-8, flow cytometry, and Transwell migration assays were used to determine the proliferation, apoptosis, and migration of BM EPCs, respectively. Protein levels were determined by western blot assay.ResultsThe BM EPCs isolated from IRH patients showed reduced proliferation, increased apoptosis, and attenuated migratory ability compared to those from normal controls. Western blot analysis showed that the protein level of p-p38 was significantly increased, while that of Phosphorylated protein kinase B (p-AKT) was significantly decreased in the BM EPCs from IRH patients, compared to BM EPCs from healthy subjects. Cell proliferation and migration were significantly enhanced by atorvastatin, recombinant human thrombopoietin, and SB20358 compared to the untreated BM EPCs from IRH patients. Atorvastatin, Recombinant human thrombopoietin (TPO), and SB20358 treatment significantly suppressed the protein levels of p-p38 protein, but increased those of p-AKT in BM EPCS from IRH patients.ConclusionsIn summary, atorvastatin increases the number and function of BM EPCs in IRH patients by regulating the p38 and AKT signaling pathways.

Dysfunction of Bone Marrow Endothelial Progenitor Cells from Subjects with Prolonged Isolated Thrombocytopenia Following Allogeneic Hematopoietic Stem Cell Transplantation Can be Improved By N-Acetyl-L-Cysteine

BackgroundProlonged isolated thrombocytopenia (PT) is a frequent complication with low platelet count or dependence on platelet transfusions for more than 60 days after allogeneic hematopoietic stem cell transplantation (allo-HSCT). PT increased risk of bleeding and had poor overall survival. However, the exact mechanisms underlying PT remain unclear. Murine studies suggest that bone marrow (BM) endothelial progenitor cells (EPCs) not only support hematopoietic stem cells (HSCs) but also play an important role in differentiation and proliferation of megakaryocytes (MKs), thereby enhancing platelet production in BM microenvironment. In addition, our previous study found that a reduced number of BM EPCs was an independent risk factor for the occurrence of PT after allo-HSCT. However, little is known about the functional role of BM EPCs and BM HSCs in patients with PT after allo-HSCT. In addition, in vivo and in vitro studies reported that the anti-oxidant -N-acetyl-L-cysteine (NAC) repaired the endothelial functions and enhanced engraftment of HSCs through prevention of exogenous ROS accumulation.Nevertheless, whether in vitro treatment of NAC is able to improve the functions of BM EPCs from patients with PT has never been investigated.AimsTo evaluate the function of BM EPCs and HSCs in patients with PT. Moreover, to investigate the effect of NAC on the number and function of the cultivated BM EPCs in patients with PT and its underlying molecular mechanisms.MethodsThree cohorts were included: patients with PT (N=25), patients with good graft function (N=25), defined as persistent successful engraftment after allotransplant, and transplant donors as normal controls (N=25). BM mononuclear cells were cultivated in fibronectin pre-coated plates with EGM-2-MV-SingleQuots and 10% fetal bovine serum at 37°C for 7 days and characterized as EPCs by CD34(+)/CD133(+)/KDR(+) using flow cytometry. The number and functions of EPCs were evaluated by flow cytometry, cell counting, DiI-Ac-LDL and FITC-lectin-UEA-1 double staining,migration, tube formation test and apoptosis. ROS level was evaluated by flow cytometry. Protein expression for ERK,JNK, p38, Akt was measured by flow cytometry and western blots. 1mM NAC was administrated to cultivated BM EPCs from patients with PT 24 hours before tested on day 7. EPCs-MKs co-culture system and Colony-forming unit (CFU)-MKs assays were used to evaluate the supporting effect that NAC exposure on MKs in vitro.ResultsHuman BM EPCs was characterized by the spindle shape and expression of CD34, CD309 and CD133 at day 7 of cultivation. Dysfunctional BM EPCs, which were characterized by impaired migration, angiogenesis, and higher levels of reactive oxygen species(ROS) and apoptosis, were revealed in patients with PT. Activation of p38 was detected in BM EPCs from patients with PT. Furthermore, there was no significantly difference in functions of CD34+ cells between patients with PT and GGF, except higher level of ROS and lower number of CFU-GEMM, suggesting an impaired process of megakaryocytopoiesis in patients with PT. Furthermore, the number and function of BM EPCs derived from patients with PT were enhanced by NAC treatment in vitro through down-regulation of the p38 mitogen-activated protein kinase (MAPK) pathway. The platelet number and percentage and functions of MKs characterized by apoptosis, ploidy and CFU-MKs were further improved by NAC in BM MKs and EPCs co-culture system from patients with PT. NAC demonstrated similar improvement effect on the number and function of BM EPCs as p38 inhibitor from patients with PT.ConclusionThe dysfunction of BM EPCs in patients with PT was improved by in vitro treatment of NAC through down-regulation of the p38 MAPK pathway, which further augment megakaryocytopoiesis and platelet production in patients with PT. These data suggest that NAC may serve as an effective therapeutic option to improve platelet production through repair of BM EPCs in patients with PT. Further prospective clinical trials will be conducted in the future. DisclosuresNo relevant conflicts of interest to declare.

Impaired Bone Marrow Endothelial Progenitor Cells May Contribute to Acute Graft-Versus-Host Disease after Allotransplant

▪Background: Graft-versus-host disease (GVHD) is a major complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). While traditionally known as an immune cells-mediated immune injury, GVHD is also associated with endothelial injury, which determines the severity of GVHD. Moreover, emerging clinical and physiopathologic evidences suggest that vascular endothelium can be a target of aGVHD in the early phase and the increased circulating endothelial cells (ECs) represents a surrogate marker of endothelial damage. Bone marrow (BM) endothelial progenitor cells (EPCs), although the origin is not completely understood, are believed to be the precursors of ECs. Protective roles of EPCs in cardiovascular disorders have been extensively testified while their potential benefits in HSCT have rarely addressed. Moreover, BM suppression without infection is often observed in patients with acute GVHD (aGVHD) after allo-HSCT and the exact mechanism remains unclear. Therefore, it is imperative to evaluate whether the number and function of BM EPCs and hematopoietic stem cells (HSCs) in allo-HSCT patients with aGVHD differed from those without aGVHD. Furthermore, given that reactive oxygen species(ROS) exhibits the important role in maintaining endothelial function and vascular integrity, it will also be necessary to investigate whether aberrant levels of ROS in BM EPCs and HSCs play a role in the pathogenesis of aGVHD after allo-HSCT.Aims: To evaluate whether the number and function of BM EPCs and HSCs in allo-HSCT patients with aGVHD differed from those without aGVHD. Furthermore, to investigate whether aberrant levels of ROS in BM EPCs and HSCs play a role in the pathogenesis of aGVHD after allo-HSCT.Methods: In the current prospective case-pair study, 59 patients with aGVHD, 118 matched patients without aGVHD after allo-HSCT and 15 health donors as normal controls were enrolled. BM EPCs were cultured as previous reported. The number and function of BM EPCs were evaluated by cell counting, DiI-Ac-LDL and FITC-lectin-UEA-1 double staining, migration, tube formation, levels of ROS and apoptosis at 30 days, 60 days and 90 days after allo-HSCT. The number and function of BM HSCs were evaluated by cell counting, levels of ROS and apoptosis. Meanwhile, the quantity and function of synchronous peripheral blood (PB) ECs and HSCs were measured in patients with aGVHD at 15 days, 30 days, 60 days and 90 days after allo-HSCT.Results: Human BM EPCs were demonstrated as the spindle shape and the similar expression of CD34, VEGFR2 and CD133 at day 7 of cultivation among the enrolled subjects. Reduced and dysfunctional BM EPCs, characterized by decreased capacities of migration and angiogenesis, as well as higher levels of ROS and apoptosis, were observed in patients with the onset of aGVHD compared with those without aGVHD. Moreover, reduced and dysfunctional BM HSCs, characterized by higher levels of ROS and apoptosis, were identified in patients with aGVHD compared with those without aGVHD. In PB, increased frenquency of ECs and decreased HSCs with higher levels of ROS and apoptosis were detected in patients with aGVHD compared to those without aGVHD. In addition, the frequencies of BM EPCs and HSCs in patients with the onset of aGVHD ≥grade 2 were significantly lower than those with aGVHD <grade 2, while the levels of ROS and apoptosis in BM EPCs and HSCs were remarkably higher. At 30 days, 60 days and 90 days after allo-HSCT, the percentages of BM EPCs and HSCs were significant lower, whereas ROS levels in BM EPCs and HSCs were significant higher in patients with aGVHD than those without aGVHD. At 15 days, 30 days, 60 days and 90 days after allo-HSCT, the percentages of PB ECs and their ROS levels in patients with aGVHD were higher than those without aGVHD, while the percentages PB HSCs were lower than those without aGVHD at the different time points.Summary / Conclusion: These findings suggest that reduced and dysfunctional BM EPCs may be involved in the pathogenesis of aGVHD. Meanwhile, reduced and dysfunctional BM HSCs were also found in patients with aGVHD. Moreover, the impairment of EPCs and HSCs in BM and PB was associated with the elevated levels of ROS in patients with aGVHD. Therefore, our data indicate that it would be of value to investigate whether the improvement of BM EPCs would possibly ameliorate aGVHD, thereby providing a novel clinical intervention for aGVHD in the future. DisclosuresNo relevant conflicts of interest to declare.

Atorvastatin May Reverse Corticosteroid-Resistant Immune Thrombocytopenia Via Improving Impaired Bone Marrow Endothelial Cells

Background: Immune thrombocytopenia (ITP) is generally considered to be an autoimmune disorder characterized by increased peripheral platelet destruction and reduced platelet production. The pathogenesis of corticosteroid-resistant ITP, a clinically challenging condition in which patients exhibit either no response to corticosteroids or corticosteroid-dependent, remains poorly understood. Murine studies suggest that bone marrow (BM) endothelial progenitor cells (EPCs) play a crucial role in regulating thrombopoiesis. We recently reported that the impaired BM EPCs, which could be quantitatively and functionally improved by atorvastatin in vitro, induced the occurrence of poor graft function following allo-transplant (Blood, 2016,128:2988-2999). However, little is known regarding the number and function of BM EPCs or how to improve impaired BM EPCs in corticosteroid-resistant ITP.Aims: To determine whether quantitative and/or functional abnormalities of BM EPCs are involved in the occurrence of corticosteroid-resistant ITP. Moreover, to investigate the effects of atorvastatin and N-Acetyl-L-cysteine (NAC, a reactive oxygen species (ROS) scavenger) on the number and function of cultivated BM EPCs derived from patients with corticosteroid-resistant ITP and its underlying molecular mechanisms. Finally, to evaluate the efficacy and safety of atorvastatin and NAC to adult patients with corticosteroid-resistant ITP.Methods: Forty patients with corticosteroid-resistant ITP, 40 patients with newly diagnosed ITP and 30 healthy donors (age 18-60) were enrolled from September 1, 2016 to May 1, 2017 at Peking University Institute of Hematology. BM EPCs were cultured as previous reported. The 5-day cultivated BM EPCs were administrated to the atorvastatin and NAC until tested on day 7. The BM EPCs were evaluated pre- and post-treatment by cell counting, DiI-Ac-LDL and FITC-lectin-UEA-1 double staining, migration, cell proliferation, tube formation, levels of ROS and apoptosis or been cocultured with CD34+ cells for another 7 days. Proteins expressions for p38, ERK, JNK, Akt were measured by flow cytometry and western blot. Colony-forming unit megakaryocyte (CFU-MK) were counted and the quantity and maturation of megakaryocytes were determined by flow cytometry or cytology. Subsequently, a single-center pilot study was performed to evaluate the efficacy and safety of atorvastatin and/or NAC in corticosteroid-resistant ITP patients.Results: Human bone marrow EPCs were demonstrated as previous reported. Herein, reduced and dysfunctional BM EPCs, characterized by decreased capacities of proliferation, migration, and angiogenesis as well as higher levels of ROS and apoptosis, were observed in patients with corticosteroid-resistant ITP. Activation of phospho-p38 and deactivation of phospho-Akt were detected in BM EPCs from corticosteroid-resistant ITP patients. Furthermore, significant decreased and dysfunctional CD34+ cells with higher levels of ROS and apoptosis, while lower number of CFU-GEMM in patients of corticosteroid-resistant ITP. In vitro treatment with atorvastatin or NAC quantitatively and functionally improved BM EPCs derived from patients with corticosteroid-resistant ITP via down-regulating p38 MAPK and up-regulating Akt pathway and also partly rescued the impaired ability of BM EPCs to support megakaryocytopoiesis. In the single-center pilot study, a total of 13 corticosteroid-resistant ITP patients were recruited to receive either the combination of atorvastatin and NAC or alone. Although the results require further validation, the complete response (CR), response(R), and overall response (OR) rates were 23.1% (3/13), 46.2% (6/13) and 69.2% (9/13), respectively. In patients who achieved CR and R, the median (range) time to response(TTR) was 24 days (7-51 days), with no apparent adverse events.Summary / Conclusion: These findings suggest that impaired BM EPCs are involved in the pathogenesis of corticosteroid-resistant ITP. The effects of atorvastatin and NAC in patients with corticosteroid-resistant ITP are potentially mediated by improving the impaired ability of BM EPCs to support megakaryocytopoiesis. Further prospective multicenter randomized clinical trials are needed to validate the effects of atorvastatin and NAC, thereby providing a novel therapeutic avenue for corticosteroid-resistant ITP. DisclosuresNo relevant conflicts of interest to declare.

Extracellular vesicle-based therapy for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) stands as a neurodegenerative disorder characterized by the rapid progression of motor neuron loss in the brain and spinal cord. Unfortunately, treatment options for ALS are limited, and therefore, novel therapies that prevent further motor neuron degeneration are of dire need. In ALS, the infiltration of pathological elements from the blood to the central nervous system (CNS) compartment that spur motor neuron damage may be prevented via restoration of the impaired blood-CNS-barrier. Transplantation of human bone marrow endothelial progenitor cells (hBM-EPCs) demonstrated therapeutic promise in a mouse model of ALS due to their capacity to mitigate the altered blood-CNS-barrier by restoring endothelial cell (EC) integrity. Remarkably, the hBM-EPCs can release angiogenic factors that endogenously ameliorate impaired ECs. In addition, these cells may produce extracellular vesicles (EVs) that carry a wide range of vesicular factors, which aid in alleviating EC damage. In an in vitro study, hBM-EPC-derived EVs were effectively uptaken by the mouse brain endothelial cells (mBECs) and cell damage was significantly attenuated. Interestingly, the incorporation of EVs into mBECs was inhibited via β1 integrin hindrance. This review explores preclinical studies of the therapeutic potential of hBM-EPCs, specifically via hBM-EPC-derived EVs, for the repair of the damaged blood-CNS-barrier in ALS as a novel treatment approach.