Adenosine A2A Receptor in Bone Marrow-Derived Cells Mediated Macrophages M2 Polarization via PPARγ-P65 Pathway in Chronic Hypoperfusion Situation

Background: The role of adenosine A2A receptor (A2AR) in the ischemic white matter damage induced by chronic cerebral hypoperfusion remains obscure. Here we investigated the role of A2AR in the process of macrophage polarizations in the white matter damage induced by chronic cerebral hypoperfusion and explored the involved signaling pathways.Methods: We combined mouse model and macrophage cell line for our study. White matter lesions were induced in A2AR knockout mice, wild-type mice, and chimeric mice generated by bone marrow cells transplantation through bilateral common carotid artery stenosis. Microglial/macrophage polarization in the corpus callosum was detected by immunofluorescence. For the cell line experiments, RAW264.7 macrophages were treated with the A2AR agonist CHS21680 or A2AR antagonist SCH58261 for 30 min and cultured under low-glucose and hypoxic conditions. Macrophage polarization was examined by immunofluorescence. The expression of peroxisome proliferator activated receptor gamma (PPARγ) and transcription factor P65 was examined by western blotting and real-time polymerase chain reaction (RT-PCR). Inflammatory cytokine factors were assessed by enzyme-linked immunosorbent assay (ELISA) and RT-PCR.Results: Both global A2AR knockout and inactivation of A2AR in bone marrow-derived cells enhanced M1 marker expression in chronic ischemic white matter lesions. Under low-glucose and hypoxic conditions, CGS21680 treatment promoted macrophage M2 polarization, increased the expression of PPARγ, P65, and interleukin-10 (IL-10) and suppressed the expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). The CGS21680-induced upregulation of P65 and IL-10 was abolished in macrophages upon PPARγ knockdown. The downregulation of TNF-α and IL-1β by CGS21680 was less affected by PPARγ knockdown.Conclusions: In the cerebral hypoperfusion induced white matter damage, A2AR signaling in bone marrow-derived cells induces macrophage M2 polarization and increases the expression of the anti-inflammatory factor IL-10 via the PPARγ-P65 pathway, both of which might explain its neuroprotective effect.

Cancer-Associated Stromal Cells Promote the Contribution of MMP2-Positive Bone Marrow-Derived Cells to Oral Squamous Cell Carcinoma Invasion

Tumor stromal components contribute to tumor development and invasion. However, the role of stromal cells in the contribution of bone marrow-derived cells (BMDCs) in oral squamous cell carcinoma (OSCC) invasion is unclear. In the present study, we created two different invasive OSCC patient-derived stroma xenografts (PDSXs) and analyzed and compared the effects of stromal cells on the relation of BMDCs and tumor invasion. We isolated stromal cells from two OSCC patients: less invasive verrucous OSCC (VSCC) and highly invasive conventional OSCC (SCC) and co-xenografted with the OSCC cell line (HSC-2) on green fluorescent protein (GFP)-positive bone marrow (BM) cells transplanted mice. We traced the GFP-positive BM cells by immunohistochemistry (IHC) and detected matrix metalloproteinase 2 (MMP2) expression on BM cells by double fluorescent IHC. The results indicated that the SCC-PDSX promotes MMP2-positive BMDCs recruitment to the invasive front line of the tumor. Furthermore, microarray analysis revealed that the expressions of interleukin 6; IL-6 mRNA and interleukin 1 beta; IL1B mRNA were higher in SCC stromal cells than in VSCC stromal cells. Thus, our study first reports that IL-6 and IL1B might be the potential stromal factors promoting the contribution of MMP2-positive BMDCs to OSCC invasion.

Donor Bone Marrow Derived Macrophage Engraftment into the Central Nervous System of Allogeneic Transplant Patients

Introduction: Hematopoietic stem cell transplantation (HSCT) has had a major impact on the treatment of hematologic malignancies. Recent studies have shown the role HSCT can have in gene therapy by providing long-lived genetically modified cells to treat a variety of human diseases. It is well known that HSC and bone marrow-derived cells can differentiate into long-lived tissue macrophages and populate a wide spectrum of tissues including the brain. These cells are termed bone marrow derived macrophages and are akin to microglial cells in both morphology and function. There is an expanding literature of preclinical animal studies focused on the potential benefits of bone marrow derived-macrophage engraftment into the central nervous system (CNS). In this study we report the detection and characterization of donor bone marrow-derived macrophages in the cerebral cortex of allogeneic transplant patients. Methods: To determine the frequency of donor cell engraftment in post-transplant patients, we selected a cohort of 20 patients who had undergone a sex-mismatched transplant. Formalin fixed paraffin embedded cerebral cortex samples were obtained from the Fred Hutch tissue repository. Samples from male and female autologous transplants were used as controls. Tissue sections were stained by XY fluorescent in situ hybridization (FISH) to identify male and female cells. The XY FISH-stained slides were imaged at 40X magnification on a TissueFAX system. Scanned images were analyzed in blinded fashion using TissueQuest software. Male donor cells were defined by the presence of the Y chromosome within DAPI stained nuclei. Parameters were established using a small area and then applied to a larger area covering 10,000-15,000 cells. Identified donors were confirmed by manual inspection. Adjacent sections were used in Iba1 immunohistochemistry (IHC) studies to quantify the microglia/macrophage population. Select cases were used in double fluorescent Iba1 IHC (tyramide signal amplification)/XY-FISH studies to identify the donor cell type. Results: Intraparenchymal donor bone marrow derived cells were identified in all cerebral cortex sex mismatched samples. To determine the identity of donor cells, select cases were stained with fluorescent tyramide based Iba1 IHC, imaged, stained with XY FISH and re-imaged. The majority of donor cells (>80%) showed strong expression of Iba1, confirming them to be bone marrow-derived macrophages. In parallel Iba1 IHC studies we showed that microglial cells constitute ~12% of the scanned cell population. Thus, when computed as a percentage of the macrophage/microglial population, donor cells from myeloablative transplants range from 4.2-25%. The bone marrow derived cells are stable over time since length of the post-transplant period did not have a major impact on the number of donor cells. Prior animal studies have demonstrated the importance of conditioning (total body irradiation (TBI) or Busulfan) in providing access to the CNS and stimulating engraftment. Consistently, we found that the strength of the conditioning regimen had a significant impact on donor cell engraftment into the CNS. Donor cells in myeloablative cases (>1,000cGy) averaged 8.0% (4.2-14.9%) of microglial cells, while those in non-myeloablative cases (<300cGy) averaged 1.3% (1.2-1.3%). In agreement with preclinical studies, we also noted that myeloablative cases from Busulfan or Treosulfan based conditioning had similar levels of donor-derived cells as cases with TBI myeloablative conditioning, averaging 6.6% (4.4-8.3%) of microglial cells. Although only a limited number of samples were available for analysis, the highest level of donor engraftment was observed in patients who had received 2 separate transplants; on average they comprised 16.3% (12.2-25.1%) of microglial cells. Conclusion: This, the largest study of bone marrow-derived macrophages in post-transplant patients, shows that donor derived cells from myeloablative transplants account for 4.1-25.1% of microglial cells. Donor engraftment is highest following myeloablative conditioning or in patients receiving multiple transplants, and lowest in non-myeloablative cases. Our studies document the magnitude of donor-derived macrophages in the CNS following a bone marrow transplant and serve as a basis for future gene therapy studies targeting neurodegenerative disorders. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

MiR-153 Enriched in Bone Marrow Mesenchymal Stem Cell-Derived Exosomes Promotes Chemotherapy Sensitivity of Papillary Thyroid Carcinoma

Our study assesses the effect of bone marrow mesenchymal stem cells (BMSCs) exosomes miR-153 on papillary thyroid carcinoma (PTC). Adipogenesis and osteogenic induction of MSCs was performed and labeled with Cy5 labeled miR inhibitor. Cells were transfected followed by analysis of miR-153 level by real-time PCR, P-gp level by immunoblotting, and cell viability. MSCs are non-hematopoietic bone marrow-derived cells and symmetrical fibroblasts have the same characteristics as MSCs. MSCs have the potential for adipogenesis and osteogenic differentiation; miR-Cy5 can only enter PTC cells through vesicle transfer. TMZ treatment upregulated miR-153 in exosomes; MSC-derived exosomes can be directly transferred to PTC cells. miR-153-inhibitor-Cy5 can effectively inhibit miR-153 transcription and expression of resistance-related proteins. miR-153-inhibitor can promote TMZ’s effect and lead to cell death as demonstrated by increased level of active caspase-3. Inhibiting the endogenous transcription of miR-153 by miR-153 inhibitor can significantly down-regulate cell resistance protein, thereby promoting cell apoptosis under the action of TMZ.

Pre-metastatic Niche Formation in Different Organs Induced by Tumor Extracellular Vesicles

Primary tumors selectively modify the microenvironment of distant organs such as the lung, liver, brain, bone marrow, and lymph nodes to facilitate metastasis. This supportive metastatic microenvironment in distant organs was termed the pre-metastatic niche (PMN) that is characterized by increased vascular permeability, extracellular matrix remodeling, bone marrow-derived cells recruitment, angiogenesis, and immunosuppression. Extracellular vesicles (EVs) are a group of cell-derived membranous structures that carry various functional molecules. EVs play a critical role in PMN formation by delivering their cargos to recipient cells in target organs. We provide an overview of the characteristics of the PMN in different organs promoted by cancer EVs and the underlying mechanisms in this review.

Characterizing temporal and spatial recruitment of systemically administered RPE65-programmed bone marrow-derived cells to the retina in a mouse model of age-related macular degeneration

Purpose Previously, we reported that the intravenous injection of bone marrow-derived cells (BMDC) infected with lentivirus expressing the human RPE65 gene resulted in the programming of BMDC to promote visual recovery in a mouse model of age-related macular degeneration (AMD). The aim of this study was to characterize the spatial and temporal recruitment of these programmed BMDC to the retinal pigment epithelial (RPE) layer. Methods C57BL/6J female mice received a subretinal injection of AAV1-SOD2 ribozyme to knock down (KD) superoxide dismutase 2 (SOD2) and induce AMD-like pathology. BMDC were isolated from GFP+ mice and infected with a lentivirus expressing RPE65. One month after SOD2 KD, fifty thousand GFP+RPE65-BMDC were injected in the mouse tail vein. Animals were terminated at different time points up to 60 min following cell administration, and localization of GFP+ cells was determined by fluorescence microscopy of neural retina and RPE flat mounts and tissue sections. Results GFP+RPE65- BMDC were observed in SOD2 KD neural retina and RPE as early as 1 min following administration. With increasing time, the number of cells in the neural retina decreased, while those in the RPE increased. While the number of cells in peripheral and central retina remained similar at each time point, the number of BMDC recruited to the central RPE increased in a time-dependent manner up to a maximum by 60 min post administration. Immunohistochemistry of cross-sections of the RPE layer confirmed the incorporation of donor GFP+ BMDC into the RPE layer and that these GFP+ human RPE65 expressing cells co-localized with murine RPE65. No GFP+ cells were observed in the neural retina or RPE layer of normal uninjured control eyes. Conclusions Our study shows that systemically administered GFP+RPE65-BMDC can reach the retina within minutes and that the majority of these BMDC are recruited to the injured RPE layer by 60 min post injection.

Disruption of GPR35 Signaling in Bone Marrow-Derived Cells Does Not Influence Vascular Inflammation and Atherosclerosis in Hyperlipidemic Mice

G-protein-coupled receptor-35 (GPR35) has been identified as a receptor for the tryptophan metabolite kynurenic acid (KynA) and suggested to modulate macrophage polarization in metabolic tissues. Whether GPR35 can influence vascular inflammation and atherosclerosis has however never been tested. Lethally irradiated LdlrKO mice were randomized to receive GPR35KO or wild type (WT) bone marrow transplants and fed a high cholesterol diet for eight weeks to develop atherosclerosis. GPR35KO and WT chimeric mice presented no difference in the size of atherosclerotic lesions in the aortic arch (2.37 ± 0.58% vs. 1.95 ± 0.46%, respectively) or in the aortic roots (14.77 ± 3.33% vs. 11.57 ± 2.49%, respectively). In line with these data, no changes in the percentage of VCAM-1+, IAb + cells, and CD3+ T cells, as well as alpha smooth muscle cell actin expression, was observed between groups. Interestingly, the GPR35KO group presented a small but significant increase in CD68+ macrophage infiltration in the plaque. However, in vitro culture experiments using bone marrow-derived macrophages from both groups indicated that GPR35 plays no role in modulating the secretion of major inflammatory cytokines. Our study indicates that GPR35 expression does not play a direct role in macrophage activation, vascular inflammation, and the development of atherosclerosis.

Bone-Derived Modulators That Regulate Brain Function: Emerging Therapeutic Targets for Neurological Disorders

Bone has traditionally been regarded as a structural organ that supports and protects the various organs of the body. Recent studies suggest that bone also acts as an endocrine organ to regulate whole-body metabolism. Particularly, homeostasis of the bone is shown to be necessary for brain development and function. Abnormal bone metabolism is associated with the onset and progression of neurological disorders. Recently, multiple bone-derived modulators have been shown to participate in brain function and neurological disorders, including osteocalcin, lipocalin 2, and osteopontin, as have bone marrow-derived cells such as mesenchymal stem cells, hematopoietic stem cells, and microglia-like cells. This review summarizes current findings regarding the roles of these bone-derived modulators in the brain, and also follows their involvement in the pathogenesis of neurological disorders. The content of this review may aide in the development of promising therapeutic strategies for neurological disorders via targeting bone.