Abstract 347: Glutathione-S-Transferase P Regulates Bone Marrow Derived Endothelial Progenitor Cell (EPC) Function and Neovascularization in the Infarcted Heart

Glutathione-S-transferase P (GSTP) modulates proliferation of bone marrow (BM) myeloid progenitors via non-catalytic inhibition of c-Jun N-terminal kinase (JNK); however, its effects on BM-derived EPCs are unknown. We hypothesized that GSTP supports EPC mobilization while suppressing inflammatory cells, thereby promoting tissue neovascularization. We generated chimeric mice using BM-ablated wild-type (WT) mice reconstituted with either GSTP-/- (GSTP-/-c) or WT (WTc) BM. GSTP-/-c and WTc mice underwent coronary ligation or sham operation (n = 8-15/group). Compared to WTc sham, 4 w after surgery, WTc HF mice exhibited significant (p < 0.05): LV dilatation, dysfunction, and fibrosis; increased mortality; and augmented circulating pro-inflammatory CD11b+Ly6Chi monocytes, but comparable circulating CD34+VEGFR2+ EPCs. In contrast, compared to WTc HF, GSTP-/-c HF hearts had significant (p < 0.01): 1) worsening of LV dilatation (LVEDV 118 ± 30 vs 88 ± 13 μL), dysfunction (LVEF 21 ± 4 vs 32 ± 9 %), wall thinning (anterior wall thickness 0.41 ± 0.06 vs 0.49 ± 0.1 mm), and LV hypertrophy (LV/tibia length 4.9 ± 0.5 vs 4.5 ± 0.6 mg/mm); 2) increased remote zone fibrosis by trichrome staining (1.3 ± 0.2 vs 0.61 ± 0.3 %); 3) reduced capillary density (476 ± 60 vs 544 ± 37 capillary/mm2) and increased capillary area (14 ± 1.1 vs 12 ± 1.2 μm2); 4) diminished gene expression of VEGF A, VEGF B, and VEGF C; 5) and increased IL-1β, IL-6 gene expression. GSTP-/-c mice also exhibited (p < 0.01) increased BM JNK activation, reduced circulating EPCs (0.18±0.07 vs 0.24±0.06%), increased CD11b+Ly6Chi monocytes (2.25±0.5 vs 1.8±0.6%), but comparable anti-inflammatory CD11b+Ly6Clow monocytes at baseline as compared with WTc mice. Moreover, at 3 and 7 d after ligation, GSTP-/-c HF mice exhibited fewer circulating EPCs and anti-inflammatory monocytes, and significantly higher pro-inflammatory monocytes as compared with WTc HF mice. We conclude that GSTP promotes EPC and reparative monocyte mobilization, thereby improving angiogenic gene expression, neovascularization and LV remodeling after myocardial infarction. This suggests that GSTP polymorphisms can impact cardiac reparative capacity in humans, and that enhancing GSTP function after infarction may yield therapeutic benefit.

Download Full-text

Inflammasome and Autophagy Activation In Symptomatic Multiple Myeloma Could Predict Response To Thalidomide-Based Chemotherapy

Blood ◽

10.1182/blood.v122.21.3131.3131 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3131-3131

Author(s):

Fernando V Pericole ◽

Adriana Silva Santos Duarte ◽

Mariana Lazarini ◽

Sara Teresinha Olalla Saad

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Bone Marrow ◽

Chronic Inflammation ◽

Clinical Data ◽

Conflicts Of Interest ◽

Inflammatory Cells ◽

Inflammasome Activation ◽

Caspase 1 ◽

Inflammatory Monocytes

Abstract The inflammasome is a protein complex activated by signals of cellular danger to trigger the innate immune defenses through the maturation of proinflammatory cytokines such as interleukin (IL)1β and IL18. Inflammasomes consist of pattern-recognition sensors (NLR family), adaptors (ASC), and effectors (caspase 1). IL1β initiates feedback loops through the IL1R-MyD88-NFκB pathway. Inflammasome activation promotes adaptive responses and limits reactive oxygen species (ROS) production. Autophagy is a cytoprotective pathway by which the cell sequesters damaged proteins and organelles for lysosomal degradation. The inflammasome is negatively regulated by autophagy and inflammasome activates autophagy. Inflammasome activity may play an important role in several nonmicrobial diseases with chronic inflammation, such as obesity, type 2 diabetes and cancer. We collected a cohort of diagnostic samples of total bone marrow (BM) from 31 MM patients, 3 plasma cell leukemia (PCL) patients and 9 healthy bone marrow donors (HD), together with clinical data. Four smoldering MM, 27 symptomatic MM and 3 PCL (2 primary and 1 secondary) were included. According to ISS, six patients were ISS I, 9 patients were ISS II and 16 were ISS III. We also stratified 18 of our symptomatic MM into groups according to response (CR/VGPR, PR, SD/PD). The offered treatment was thalidomide-based triplets. Essential genes from inflammasome (NLRP3, PYCARD, CASP1), pro-inflammatory interleukins (IL1B, IL18) and autophagy (BECN1, SQSTM1, MAP1LC3B) were verified by q-PCR. Gene expression was compared among subgroups and correlated with clinical data. CASP1 and PYCARD were increased in MM compared with HD, without differences among ISS subgroups. In PCL cases, CASP1 and PYCARD had lower expression when compared with MM, but did not differ from HD, confirming that PCL did not have inflammasome activation as MM did. NLRP3 was not different among all diagnostic subgroups. IL18 had decreased expression in PCL patients, compared with HD and MM. IL1B was not different among subgroups. PCL and MM had higher BECN1 expression compared with HD and these differences were also highlighted among ISS subgroups. SQSTM1 had increased expression in PCL, compared with HD and MM, but MM did not differ from HD. MAP1LC3B gene expression was similar among groups. A positive correlation was observed between CASP1 and PYCARD, IL1B and IL18, and between the three autophagic genes (BECN1, SQSTM1 and MAP1LC3B) as expected. BECN1 also correlated with IL1B and with CASP1; CASP1 and PYCARD correlated with BECN1 and SQSTM1, reinforcing the relation between autophagy and inflammasome. When the clinical data were analyzed, monocyte counts correlated with CASP1 and PYCARD; beta-2-microglobulin levels correlated with CASP1 and with NLRP3 and, finally, leukocyte and neutrophil counts correlated with IL1B, BECN1 and SQSTM1 BM expression levels. Analyzing response to thalidomide-based first line chemotherapy, VGPR/CR responders showed higher diagnostic NLRP3 expression (unpaired t test, P=0.03). The intricate interplay between autophagy and inflammasome has only recently been elucidated. Multiple myeloma is a B-cell neoplasm with great dependence of BM microenvironment. Inflammatory cells, especially monocytes and macrophages, are the main inflammasome activators, reducing ROS in the BM microenvironment. Autophagy is essential for plasmocytes, due to their high levels of intracellular proteins. Basal levels of autophagy in myeloma are high and are upregulated in response to ER stress and proteasome inhibition, protecting myeloma cells against apoptosis. Our results confirm the essential role of autophagy activation in myeloma. Interestingly, inflammasome activation in our cohort predicted a better response to thalidomide-based regimens and could be a novel response biomarker. Reinforcing our theory, recently thalidomide was described as an inhibitor of caspase 1 activation. Chronic inflammation is an established cancer hallmark and its role in myeloma pathogenesis seems to be important through inflammasome and autophagy dysfunction during disease progression, possibly creating dependency loops between inflammatory monocytes and neoplastic plasmocyte within the BM microenvironment. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Fibrin, Bone Marrow Cells and macrophages interactively modulate cardiomyoblast fate.

10.1101/2022.01.06.475189 ◽

2022 ◽

Author(s):

Ines Borrego ◽

Aurelien FROBERT ◽

Guillaume AJALBERT ◽

Jeremy VALENTIN ◽

Cyrielle KALTENRIEDER ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Cardiac Function ◽

Conditioned Medium ◽

Bone Marrow Cells ◽

Cardiac Repair ◽

Cardiac Cell ◽

Anti Inflammatory ◽

Marrow Cells

Interactions between macrophages, cardiac cells and the extracellular matrix are crucial for cardiac repair following myocardial infarction (MI). The paracrine effects of cell-based treatments of MI might modulate these interactions and impact cardiac repair. The immunomodulatory capacity of the therapeutic cells is therefore of interest and could be modulated by the use of biomaterials. We first showed that bone marrow cells (BMC) associated with fibrin could treat MI. Then, we interrogated the influence of fibrin, as a biologically active scaffold, on the secretome of BMC and the impact of their association on macrophage fate and cardiomyoblast proliferation. Methods: In vivo, two weeks post-MI, rats were treated with epicardial implantation of BMC and fibrin or sham-operated. High-resolution echocardiography was performed to evaluate the heart function and structure changes after 4 weeeks. Histology and immunostaining were performed on harvested hearts. In vitro, BMC were first primed with fibrin. Second, non-polarized macrophages were differentiated toward either pro-inflammatory or anti-inflammatory phenotypes and stimulated with the conditioned medium of fibrin-primed BMC (F-BMC). Proteomic, cytokine levels quantification, and RT-PCR were performed. EdU incorporation and real-time cell analysis assessed cell proliferation. Results: The epicardial implantation of fibrin and BMC reduced the loss of cardiac function induced by MI, increased wall thickness and prevented the fibrotic scar expansion. After 4 and 12 weeks, the infarct content of CD68+ and CD206+ was similar in control and treated animals. In vitro, we showed that fibrin profoundly influenced the gene expression and the secretome of BMC, simultaneously upregulating both pro- and anti-inflammatory mediators. Furthermore, the conditioned medium from F-BMC significantly increased the proliferation of macrophages in a subsets dependent manner and modulated their gene expression and cytokines secretion. For instance, F-BMC significantly downregulated the expression of Nos2, Il6 and Ccl2/Mcp1 while Arg1, Tgfb and IL10 were upregulated. Interestingly, macrophages educated by F-BMC increased cardiomyoblast proliferation. In conclusion, our study provides evidence that BMC/fibrin-based treatment lowered the infarct extent and improved cardiac function. The macrophage content was unmodified when measured at a chronic stage. Nevertheless, acutely and in vitro, the F-BMC secretome promotes an anti-inflammatory response that stimulates cardiac cell growth. Finally, our study emphases the acute impact of F-BMC educated macrophages on cardiac cell fate.

Download Full-text

129 Stimulating the Cholinergic Anti-inflammatory Pathway Alters Inflammatory Cell Mobilization after Burn Injury

Journal of Burn Care & Research ◽

10.1093/jbcr/irab032.133 ◽

2021 ◽

Vol 42 (Supplement_1) ◽

pp. S87-S87

Author(s):

Todd Costantini ◽

Jessica Weaver ◽

Brian Eliceiri

Keyword(s):

Bone Marrow ◽

Vagus Nerve ◽

Burn Injury ◽

Myeloid Cell ◽

Inflammatory Cells ◽

Severe Burn ◽

Inflammatory Pathway ◽

Anti Inflammatory ◽

Severe Burn Injury ◽

Patrolling Monocytes

Abstract Introduction Severe burn injury causes a systemic inflammatory response (SIRS) that is characterized by mobilization of inflammatory cells into the circulation and is associated with distant organ injury that can lead to significant morbidity and mortality. The cholinergic anti-inflammatory pathway, mediated by the vagus nerve, regulates the anti-inflammatory response to injury and infection. We have previously shown in models of burn injury that stimulating the vagus nerve may be a potential therapy aimed at limiting SIRS. Here, we hypothesized that stimulating the vagus nerve (VNS) would limit the SIRS response by altering the mobilization and trafficking of inflammatory cells after burn injury. Methods Wild type 10–12-week-old C57BL/6 mice were injured with a 30% total body surface area steam burn. A separate cohort of animals was treated with electrical stimulation of the cervical vagus nerve for 10 minutes immediately post-burn. Bone marrow, blood and lung tissue were collected 24 hours after burn injury. Flow cytometry of bone marrow was performed to measure Lineage- c-kit± Sca-1+ (LSK) hematopoietic stem cells (HSC), then further analyzed to quantify changes in Long-term (LT) HSC, short-term (ST) HSC, and Multipotential Progenitor (MPP) compartments. Bone marrow, blood and perfused lung tissue were analyzed by flow cytometry using a panel of myeloid cell markers. Results Severe burn injury decreased bone marrow LSK expression by 50% compared to sham, with LT-HSC and MPP expression decreasing to a greater degree than ST-HSCs. VNS did not alter burn-induced changes in any bone marrow HSC cell type. Burn injury was associated with increased mobilization of CD45+CD11b+ monocytes and CD11b+Ly6Chi inflammatory monocytes into the peripheral blood and lung, while increased CD11b+Ly6Clo patrolling monocytes and Gr1+Ly6C- neutrophils was seen in the lung only. VNS significantly prevented the burn-induced increase in CD45+ inflammatory cells, CD11b+Ly6Clo patrolling monocytes and Gr1+Ly6C- neutrophils in the lung (see Figure), reducing their expression to sham levels, despite only modest changes to myeloid cell expression in the blood. Conclusions VNS attenuates myeloid cell cell trafficking to the lung after severe burn injury despite having no effect on emergency myelopoiesis in the bone marrow. Further studies are needed to define the mechanism by which the cholinergic anti-inflammatory pathway attenuates the SIRS response to burn.

Download Full-text

Colchicine Attenuates Renal Ischemia-Reperfusion-Induced Liver Damage: Implication of TLR4/NF-κB, TGF-β, BAX and Bcl-2 gene expression

Canadian Journal of Physiology and Pharmacology ◽

10.1139/cjpp-2021-0007 ◽

2021 ◽

Author(s):

Azza Sayed Awad ◽

Hemat Abdel Fatah Elariny ◽

Amany Said Sallam

Keyword(s):

Gene Expression ◽

Liver Damage ◽

Transforming Growth Factor ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

B Cell Lymphoma ◽

Sham Operation ◽

Necrosis Factor Alpha ◽

Sham Operation Group ◽

Anti Inflammatory

Background: Ischemia/reperfusion injury (IRI) is typically associated with a vigorous inflammatory and oxidative stress response to hypoxia and reperfusion that disturbs the function of the organ. The remote effects of renal IRI on the liver, however, require further study. Renal damage associated with liver disease is a common clinical problem. Colchicine, a polymerization inhibitor of microtubules has been used as an anti-inflammatory and anti-fibrotic drug for liver diseases. Aims: The goal of the current study was to investigate the possible protective mechanisms of colchicine on liver injury following renal IRI. Methods: Forty rats were divided randomly into four groups; group of sham operation, group of colchicine treated, group of IRI, group of colchicine treated-IRI. Key findings: Treatment with colchicine significantly reduced hepatic toll-like receptor 4 {TLR4}, nuclear factor kappa B transcription factor {NF-κB}, myeloid differentiation factor 88 {MyD88}, and tumor necrosis factor-alpha {TNF-α} contents, down-regulated BCL2 Associated X Apoptosis Regulator {BAX} gene expression, transforming growth factor-β {TGF-β} content and upregulated hepatic B-cell lymphoma 2 {Bcl-2} gene expression as compared to the IRI group. Finally, hepatic histopathological examinations have confirmed the biochemical results. Significance: Renal IRI-induced liver damage in rats was alleviated by colchicine through its anti-inflammatory, anti-apoptotic, and anti-fibrotic actions.

Download Full-text

Phenylbutyrate facilitates homeostasis of non-resolving inflammatory macrophages

Innate Immunity ◽

10.1177/1753425919879503 ◽

2019 ◽

Vol 26 (1) ◽

pp. 62-72 ◽

Cited By ~ 1

Author(s):

Allison Rahtes ◽

Kisha Pradhan ◽

Mimosa Sarma ◽

David Xie ◽

Chang Lu ◽

...

Keyword(s):

Gene Expression ◽

Low Dose ◽

Inflammatory Diseases ◽

Macrophage Polarization ◽

Related Factor ◽

Inflammatory Monocytes ◽

Inflammatory Protein ◽

Anti Inflammatory ◽

Inflammation Resolution ◽

Ccl2 Gene

Non-resolving inflammatory monocytes/macrophages are critically involved in the pathogenesis of chronic inflammatory diseases. However, mechanisms of macrophage polarization are not well understood, thus hindering the development of effective strategies to promote inflammation resolution. In this study, we report that macrophages polarized by subclinical super-low dose LPS preferentially expressed pro-inflammatory mediators such as ccl2 (which encodes the protein monocyte chemo attractant protein-1) with reduced expression of anti-inflammatory/homeostatic mediators such as slc40a1 (which encodes the protein ferroportin-1). We observed significantly elevated levels of the autophagy-associated and pro-inflammatory protein p62 in polarized macrophages, closely correlated with the inflammatory activation of ccl2 gene expression. In contrast, we noted a significant increase of ubiquitinated/inactive nuclear-erythroid-related factor 2 (NRF2), consistent with reduced slc40a1 gene expression in polarized macrophages. Addition of the homeostatic restorative agent phenylbutyrate (4-PBA) effectively reduced cellular levels of p62 as well as ccl2 gene induction by super-low dose LPS. On the other hand, application of 4-PBA also blocked the accumulation of ubiquitinated NRF2 and restored anti-inflammatory slc40a1 gene expression in macrophages. Together, our study provides novel insights with regard to macrophage polarization and reveals 4-PBA as a promising molecule in restoring macrophage homeostasis.

Download Full-text

The Inflammatory Subset of Monocytes Stimulates Angiogenesis at Sites of Ischemia by Altering the Balance of Pro- and Anti-Inflammatory Cytokines.

Blood ◽

10.1182/blood.v110.11.240.240 ◽

2007 ◽

Vol 110 (11) ◽

pp. 240-240

Author(s):

Alyssa D. Gregory ◽

Benjamin J. Capoccia ◽

Daniel C. Link

Keyword(s):

Bone Marrow ◽

Blood Flow ◽

Inflammatory Cytokines ◽

Adoptive Transfer ◽

Tissue Damage ◽

Doppler Imaging ◽

Saline Control ◽

Inflammatory Monocytes ◽

Ischemic Tissue ◽

Anti Inflammatory

Abstract Our laboratory has previously shown that early delivery of monocytes to ischemic tissue by mobilization from the bone marrow to blood or by adoptive transfer enhances revascularization after acute vascular injury. There is accumulating evidence that monocytes represent a heterogeneous population, containing subsets with distinct functional properties. Herein, we show that the inflammatory subset of monocytes mediates the angiogenic response in a murine model of hindlimb ischemia induced by femoral artery excision (FAE). Inflammatory (CX3CR1loGr-1+) and resident (CX3CR1hiGr-1−) monocytes were sorted from the bone marrow of CX3CR1+/GFP mice and injected intravenously into recipient mice 24 after FAE. Revascularization was monitored by laser Doppler imaging. A marked improvement in blood flow was observed after adoptive transfer of inflammatory but not resident monocytes [ratio of blood flow in ischemic to nonischemic limb 7 days after surgery: 0.75±0.05 (inflammatory monocytes); 0.40±0.07 (resident monocytes); 0.40±0.03 (saline control); P <0.01]. Consistent with this finding, inflammatory monocytes are selectively recruited to sites of ischemia. Finally, we show that the bone marrow is a rich reservoir of inflammatory monocytes that is readily mobilized into the blood by ischemia. To begin to elucidate the mechanisms by which inflammatory monocytes stimulate angiogenesis, we determined the effect of the adoptive transfer of monocytes on the local production of key cytokines/chemokines in the ischemic tissue. Specifically, a non-biased multiplex immunoassay-based screen for soluble factors present in the cell-free muscle homogenates was performed. Data revealed decreased levels of the pro-inflammatory cytokines IL-6, KC, and mip-1beta and increased levels of the anti-inflammatory cytokine IL-10 in mice receiving an adoptive transfer of monocytes. In keeping with this observation, levels of free myoglobin were dramatically decreased in mice receiving an adoptive transfer of monocytes, indicating a reduction in inflammation-mediated tissue damage [myoglobin levels at 28 hours post-ischemia: 18,580±879 ng/mL (no adoptive transfer); 158±112 ng/mL (adoptive transfer)]. Interestingly, known regulators of angiogenesis, MCP-1, MMP-9, VEGF, and FGF-basic were also significantly increased. Taken together, these data indicate that adoptive transfer of a small number of monocytes shifts the balance of pro- and anti-inflammatory signals in the ischemic environment, which limits tissue damage and enhances revascularization. This finding has important therapeutic implications, since deliver of inflammatory monocytes to sites of ischemia may polarize the microenviroment towards tissue regeneration and angiogenesis.

Download Full-text

Lobeglitazone Exerts Anti-Inflammatory Effect in Lipopolysaccharide-Induced Bone-Marrow Derived Macrophages

Biomedicines ◽

10.3390/biomedicines9101432 ◽

2021 ◽

Vol 9 (10) ◽

pp. 1432

Author(s):

Dabin Jeong ◽

Wan-Kyu Ko ◽

Seong-Jun Kim ◽

Gong-Ho Han ◽

Daye Lee ◽

...

Keyword(s):

Gene Expression ◽

Nitric Oxide ◽

Bone Marrow ◽

Signaling Pathway ◽

Mapk Signaling ◽

No Production ◽

Inflammatory Gene Expression ◽

Inflammatory Gene ◽

Anti Inflammatory ◽

Inflammatory Effect

The purpose of this study is to elucidate the anti-inflammatory effect of lobeglitazone (LOBE) in lipopolysaccharide (LPS)-induced bone-marrow derived macrophages (BMDMs). We induced nitric oxide (NO) production and pro-inflammatory gene expression through LPS treatment in BMDMs. The changes of NO release and expression of pro-inflammatory mediators by LOBE were assessed via NO quantification assay and a real-time quantitative polymerase chain reaction (RT-qPCR), respectively. In addition, the regulatory effect of LOBE on activation of mitogen-activated protein kinase (MAPK) signaling pathway was investigated by measuring the phosphorylation state of extracellular regulatory protein (ERK) and c-Jun N-terminal kinase (JNK) proteins by Western blot. Our results show that LOBE significantly reduced LPS-induced NO production and pro-inflammatory gene expression of interleukin-1β (IL-1β), IL-6, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and monocyte chemoattractant protein-1 (MCP-1). Moreover, LOBE reduced phosphorylation levels of ERK and JNK of MAPK signaling pathway. In conclusion, LOBE exerts an anti-inflammatory effect in LPS-induced BMDMs by suppression of NO production and pro-inflammatory gene expression, and this effect is potentially through inhibition of the MARK signaling pathway.

Download Full-text

Bone Marrow Niche Attenuation by Non-Steroidal Anti-Inflammatory Drugs Mobilizes Hematopoietic Stem and Progenitor Cells by Differing Mechanisms

Blood ◽

10.1182/blood.v118.21.724.724 ◽

2011 ◽

Vol 118 (21) ◽

pp. 724-724

Author(s):

Jonathan Hoggatt ◽

Khalid S Mohammad ◽

Brahmananda Reddy Chitteti ◽

Pratibha Singh ◽

Jennifer M Speth ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Flow Cytometry ◽

Bone Formation ◽

Progenitor Cells ◽

Surprising Result ◽

Treatment Results ◽

Bm Niche ◽

Inflammatory Drugs ◽

Anti Inflammatory

Abstract Abstract 724 Hematopoeitic stem (HSC) and progenitor cells (HPC) are localized in niches contained within the bone marrow (BM) microenvironment. To facilitate acquisition of HSC/HPC for hematopoietic transplantation, donors can be treated with granulocyte-colony stimulating factor (G-CSF) to “mobilize” HSC/HPC from the BM niche to the peripheral system. Our laboratory has recently discovered that short term administration of non-steroidal anti-inflammatory drugs (NSAIDs) also mobilizes HSC/HPC and works in synergy with G-CSF. To understand the mechanisms underlying this novel therapeutic utility of NSAIDs we analyzed the BM niche post-NSAID and G-CSF treatment. Femurs of mice were analyzed after a 4 day regimen of the NSAID Meloxicam (3mg/kg, bid) and compared to control or G-CSF treated mice. Gross histological analysis showed a remarkable osteoblast (OB) “flattening” similar to that seen with G-CSF. Goldners trichrome staining revealed significantly reduced osteoid bone surfaces, with ∼3-fold increase in quiescent surfaces. Similarly, dynamic bone formation analysis using calcein/tetracycline labeling demonstrated reductions in mineral apposition rate and bone formation rate. There were no significant alterations in trabecular bone as determined by MicroCT. TRAP+ osteoclasts (OCs) were slightly elevated in both Meloxicam and G-CSF treated groups. To further assess the role of OCs, mice were mobilized with or without zoledronic acid (ZA) treatment, which inhibits OC activity. Similar to a recent report (Winkler, Blood, 2010), ZA resulted in an increase in HSC/HPC mobilization by both Meloxicam and G-CSF, suggesting that increased OC activity is not a mitigating mechanism for NSAID-mediated mobilization. Immunohistochemical (IHC) staining showed marked reductions in osteopontin (OPN), stromal-derived factor-1 (SDF-1) and N-cadherin expression by Meloxicam and G-CSF treated mice. In a separate set of experiments, OBs and mesenchymal stem cells (MSCs) were sorted by flow cytometry and gene expression assessed by Taq-Man assay. Similar to IHC analysis, Meloxicam treatment resulted in reduced SDF-1, OPN, Jagged-1, Runx2 and VCAM-1 gene expression. While the interaction of SDF-1 with its hematopoietically expressed receptor CXCR4 is a well known mediator of niche retention, both HSC and HPC were significantly mobilized by Meloxicam in CXCR4 knockout (KO) mice, suggesting that while reduced SDF-1 expression may concurrently play a role in NSAID mediated mobilization, it is not the definitive mechanism. In contrast, when OPN KO mice were mobilized with Meloxicam or G-CSF, Meloxicam unexpectedly increased mobilization of HPC only, and not HSC, while both HPC and HSC were mobilized by G-CSF. This surprising result indicates that NSAID-mediated OPN reduction is specifically responsible for the observed HSC mobilization, while HPC mobilization appears to be mediated by another mechanism(s). Recently, it has been reported that G-CSF reduces resident F4/80+ monocytes/macrophages (MOs), which normally support niche OBs and MSCs, and this reduction is at least partially responsible for niche attenuation and hematopoietic mobilization. However, in contrast to G-CSF, IHC analysis showed no reduction in F4/80+ cells after Meloxicam treatment, nor was there a reduction in CD169+ BM macrophages as assessed by flow cytometry. Therefore, while NSAIDs and G-CSF attenuate the niche microenvironment similarly, these agents function through independent mechanisms, perhaps explaining the synergistic mobilization seen by the two. In conclusion, NSAID treatment results in significant attenuation of the BM niche, including the niche components SDF-1, OPN, and N-cadherin. NSAID-mediated mobilization is independent of reduced SDF-1/CXCR4 signaling, as CXCR4 KO mice can be mobilized by NSAID. OPN KO specifically blocks the NSAID mobilization of HSC, but not HPC, suggesting differing mechanisms, or possibly multiple niche locations for these two populations; an intriguing finding suggesting that other mobilization strategies may be able to specifically target HSC or HPC. While G-CSF treatment results in reduced MOs, NSAIDs do not alter BM levels of these supportive cells. These results define not only a novel strategy for mobilization of HSC/HPC, but also suggest a possible strategy for targeted niche attenuation for other therapeutic applications, such as reduced conditioning regimens. Disclosures: Hoggatt: Fate Therapeutics: Consultancy. Pelus:Fate Therapeutics: Consultancy.

Download Full-text

Abstract 11888: MyD88 Expressed by Myeloid Cells is Essential for Angiotensin II-Induced Vascular Dysfunction, Inflammation and Arterial Hypertension

Circulation ◽

10.1161/circ.130.suppl_2.11888 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Sabine Kossmann ◽

Tanja Schönfelder ◽

Stefanie Finger ◽

Melanie Brähler ◽

Daniel Minwegen ◽

...

Keyword(s):

Bone Marrow ◽

Angiotensin Ii ◽

Arterial Hypertension ◽

Vessel Wall ◽

Vascular Dysfunction ◽

Radio Telemetry ◽

Flow Cytometric Analysis ◽

Inflammatory Cells ◽

Mrna Levels ◽

Inflammatory Monocytes

Background: Angiotensin II (ATII) causes hypertension and promotes infiltration of inflammatory cells into the vessel wall. Vascular superoxide formation and shear stress mediated inflammatory remodeling of conduit arteries was shown to be myeloid differentiation factor 88 (MyD88) dependent, but the exact mechanism are unknown. Objective: The goal of this study was to determine the mechanism, how MyD88 contributes to the development of ATII-induced vascular dysfunction and arterial hypertension. Methods and Results: MyD88 deficiency profoundly attenuated ATII-induced (1 mg/kg/d for 7 days) blood pressure increase (measured by radio telemetry) and vascular dysfunction (assessed by aortic ring relaxation studies). Additionally vascular superoxide levels as well as mRNA expression levels of VCAM-1, iNOS, Nox2 were decreased in ATII-infused MyD88 -/- mice compared to WT controls. Aortic flow cytometric analysis revealed that ATII-induced infiltration with CD11b + Ly6C high inflammatory monocytes was significantly dampened in MyD88 -/- mice. ATII led to an increased expression of inflammatory monocyte markers in blood and aorta, which was blocked in MyD88 -/- mice, indicating a role of MyD88 for myeloid cell differentiation. Additionally less IFN-gamma + NK cells were detected in the vessel wall of ATII-treated MyD88 -/- mice and aortic lysates showed reduced mRNA levels of several proinflammatory cytokines like IL-12 and IL-1beta. Bone marrow transfer experiments further revealed a protective effect of MyD88 deficiency in inflammatory cells represented by a reduction of vascular dysfunction and inflammation. Conclusion: We provide first evidence that MyD88 expressed by bone marrow-derived cells plays an essential role in ATII-induced vascular dysfunction and arterial hypertension. Our data indicate that MyD88 is important for cytokine production and might be required for ATII-induced differentiation of monocytes into an inflammatory phenotype.

Download Full-text

Genetically modified bone marrow continuously supplies anti-inflammatory cells and suppresses renal injury in mouse Goodpasture syndrome

Blood ◽

10.1182/blood.v98.1.57 ◽

2001 ◽

Vol 98 (1) ◽

pp. 57-64 ◽

Cited By ~ 34

Author(s):

Takashi Yokoo ◽

Toya Ohashi ◽

Yasunori Utsunomiya ◽

Jin Song Shen ◽

Yutaka Hisada ◽

...

Keyword(s):

Bone Marrow ◽

Chronic Inflammation ◽

Bone Marrow Cells ◽

Interleukin 1 ◽

Inflammatory Cells ◽

Therapeutic Effects ◽

Goodpasture Syndrome ◽

Chronic Nephritis ◽

Male Donor ◽

Anti Inflammatory

Abstract In chronic inflammation, macrophages and neutrophils, which are derived from bone marrow, play a pivotal role. Therefore, reconstitution of bone marrow with anti-inflammatory stem cells may modify inflammation. In this study, transplantation-based gene therapy was applied to glomerular inflammation for a long-lasting suppression of the glomerular damage seen in chronic nephritis. Bone marrow cells were harvested from male donor mice, which had received 5-fluorouracil 3 days previously, and transduced with an interleukin 1 (IL-1) receptor antagonist (IL-1Ra) or a mock gene using a retrovirus vector. After confirmation that transduced cells possessed the transgene at approximately 0.7 copies per cell and secreted recombinant IL-1Ra, these cells were infused into sublethally irradiated (6 Gy) female recipients once daily for 4 consecutive days. These female recipient mice had the male Y antigen in bone marrow, liver, and spleen, and 10% to 20% of their spleen cells possessed the transgene even 8 weeks after transplantation. Glomerulonephritis was then induced in these mice. Renal function and histology were retarded in the mice whose bone marrow was reconstituted with IL-1Ra–producing cells compared with mock transduced cells. In situ hybridization using a Y painting probe revealed that transplanted donor cells were recruited into the glomerulus upon induction of nephritis, suggesting therapeutic effects were channeled through the secretion of IL-1Ra from these cells. Furthermore, the survival rate after a second challenge with nephrotoxic antibody was significantly improved in the IL-1Ra chimera. These results suggest that reconstitution of bone marrow for continuous supply of anti-inflammatory cells may be a useful strategy for the treatment of chronic inflammation.

Download Full-text