Circadian Adrenergic Regulation of Bone Marrow Endothelial Adhesion Molecule Expression Impacts Progenitor Recruitment and Engraftment Efficiency

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 398-398
Author(s):  
Yuya Kunisaki ◽  
Christoph Scheiermann ◽  
Daniel Lucas ◽  
Andrew Chow ◽  
Paul S. Frenette
Keyword(s):  
Bone Marrow ◽  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Peripheral Blood ◽  
Myeloid Cells ◽  
Fold Increase ◽  
Adherent Cells ◽  
Adhesion Molecule Expression ◽  
Vessel Area

Abstract Abstract 398 Previous studies have revealed that hematopoietic stem cells (HSCs) are released into peripheral blood in a circadian manner in a process controlled by the sympathetic nervous system (SNS) through the regulation of CXCL12 levels in the bone marrow (BM) (Mendez-Ferrer et. al. Nature 2008;452:442). Here, we have evaluated the constitutive recruitment of hematopoietic cells back to the BM. We have observed using high-speed multichannel fluorescence intravital microscopy (MFIM) significant circadian oscillations in the number of adherent BM cells in sinusoids with a nadir in the morning (Zeitgeber time, ZT5: 0.97 ± 0.17 adherent cells/ 100 μm2 vessel area) and a peak at night (ZT13: 2.54 ± 0.53 adherent cells/100 μm2, p = 0.007) after adoptive transfer on a 12 hour light-12 hour darkness cycle. Flow cytometric analyses revealed that the majority (∼70 %) of homed BM cells were Gr-1+ Mac-1+ myeloid cells. To investigate the underlying mechanisms, we have examined the expression levels of P- and E-selectins and VCAM-1, essential homing receptors for progenitor cells in the BM, and found that their mRNA and protein expression on BM endothelium oscillated over the course of a day with the peak expression overlapping the time of the highest cell adhesion numbers (ZT13). To examine the role of the SNS in this process, we surgically sympathectomized mice by unilateral section of the superior cervical ganglion (SCGx) whose neurons project into the calvarial vasculature, while performing sham surgery on the contralateral side. Sympathectomy abolished circadian fluctuations in the number of adoptively transferred adherent cells to the denervated calvarial BM compared to the control side in the same animals (nerve-intact side: ZT5 / ZT13: 1.66 ± 0.10 / 2.41 ± 0.08 cells / 100 μm2, p<0.0001; SCGx: ZT5 / ZT13: 1.65 ± 0.09 vs 1.63 ± 0.09 / 100 μm2 vessel area, p=0.90). We then ascertained further the role of adrenergic signals by evaluating mice deficient in b-adrenergic receptors. We found that the oscillations in cell adhesion molecule expression were markedly reduced in β2 (Adrb2-/-) and β3 (Adrb3-/-) adrenergic receptor deficient mice. These results suggest that hematopoietic cell recruitment to the BM is under circadian control, which is dependent on oscillating expression of endothelial selectins and VCAM-1, and regulated by the SNS. To test the relevance of circadian leukocyte recruitment, we investigated whether isoproterenol, a pan-b-adrenergic agonist commonly used in the clinic, could promote hematopoietic progenitor recruitment and thus BM reconstitution after BM transplantation (BMT). Treatment with isoproterenol (5 mg/kg) for 5 days significantly up-regulated expression of P-selectin (1.2-fold increase; p = 0.027), E-selectin (1.5-fold increase; P = 0.003) and VCAM-1 (2.3-fold increase; P=0.006) on BM endothelium in irradiated recipients as determined by flow cytometry of Tie-2+ PECAM-1+ endothelial cells. Consequently, homing of BM cells was dramatically increased (control / isoproterenol: 2.4 ± 0.2 ×104/4.9 ± 0.4 × 104 donor cells/femur; p = 0.0002) as was the number of recruited hematopoietic progenitors (17.0 ± 3.5/74.1 ± 18.8 CFU-C/femur; p = 0.017). In addition, the recovery of mature myeloid cells in peripheral blood was significantly accelerated in 3 weeks after transplantation of 5 × 104 BM cells (0.38 ± 0.21 × 103/1.64 ± 0.50 ×103/μl; p = 0.024). Of importance, using limiting numbers of BM cells (2.5 × 104) for transplantation, isoproterenol treatment markedly improved the survival (median survival time 10 vs 18 days, percent survival at 4 weeks post-BMT 5.8 vs 35.2%; p = 0.0097). These results indicate that the circadian timing of donor cell infusion and/or manipulation of adrenergic signals in the BM microenvironment may improve transplantation outcome through enhanced engraftment efficiency. Disclosures: No relevant conflicts of interest to declare.

Potential Therapeutic Role of the Selective Adhesion Molecule (SAM) Inhibitor Natalizumab in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1850-1850 ◽  
Author(s):  
Klaus Podar ◽  
Alexander Zimmerhackl ◽  
Ursula Hainz ◽  
Mariateresa Fulciniti ◽  
Sonia Vallet ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Migration ◽  
Cell Adhesion ◽  
Cell Surface ◽  
Adhesion Molecule ◽  
Research Funding ◽  
Therapeutic Role ◽  
Potential Therapeutic Role

Abstract Abstract 1850 Poster Board I-876 Multiple Myeloma (MM) is characterized by the clonal proliferation of malignant plasma cells in the bone marrow. Despite current therapeutic approach and prolongation of the median survival, new therapies are urgently needed. Integrins are cell surface receptors which mediate both cell-cell adhesion and cell-extracellular matrix (ECM) protein adhesion. beta1-integrins, including very-late antigen-4 (VLA-4;á4β1), are typically expressed on MM cells. In MM, VLA-4-mediated binding to ECMS and bone marrow stromal cells (BMSCs) confers protection against drug-induced apoptosis and triggers transcription and secretion of IL-6, the major MM growth and survival factor. In addition to up-regulation of cell surface-clustering, integrin activity can also be triggered by multiple agonists through ‘inside-out’ signaling, independent of changes in integrin expression levels. Importantly, VEGF-induced migration of MM cells on fibronectin is also associated with β1-integrin- and PI3-kinase- dependent PKC activation. Targeting VLA-4 is therefore of potential high therapeutic interest in MM. Indeed, an antibody against murine á4 induces inhibition of MM growth in a murine model. Natalizumab is a recombinant humanized IgG4 monoclonal antibody, which belongs to a new class of molecules known as selective adhesion molecule (SAM) inhibitors and binds to á4-integrin. Clinically, Natalizumab has demonstrated activity in patients with multiple sclerosis and Crohn's disease. Here we tested the potential therapeutic role of Natalizumab on MM cell survival, and migration in the BM microenvironment. VLA-4 is expressed by all MM cell lines investigated (NCIH929, RPMI8226, INA-6, MM.1S, and OPM2). Functionally, Natalizumab but not a control antibody, triggered dose-dependent inhibition of MM cell adhesion to fibronectin, BMSCs, and endothelial cells (ECs). Importantly, inhibition of adhesion to fibronectin, BMSCs, or ECs was observed in MM cells pretreated with Natalizumab. Moreover, inhibition of MM cell adhesion to fibronectin, BMSCs, or ECs was also observed when Natalizumab was added to already adherent MM cells. Taken together, Natalizumab decreases adhesion of non-adherent MM cells as well as binding of already adherent MM cells to non-cellular and cellular components of the microenvironment. Given the protective role of the microenvironment on MM cell survival, we next sought to evaluate the chemosensitizing activity of Natalizumab. Specifically, we investigated dose- and time- dependent effects of Natalizumab, alone and when combined with conventional and novel therapies, on MM cells. Our results show that Natalizumab alone did not inhibit growth or survival of MM cells when cultured without components of the microenvironment. However, Natalizumab enhanced sensitivity of tumor cells to both bortezomib and dexamethasone in MM-BMSC and, MM-EC co-cultures. These data indicate a potential role of Natalizumab in bortezomib- and dexamethasone-containing treatment regimens including MPV. Moreover, Natalizumab decreases IL-6 and VEGF secretion triggered in MM-BMSC co-cultures. Consequently, angiogenesis triggered by supernatants of Natalizumab- treated MM-BMSC co-cultures was inhibited. Moreover, Natalizumab blocked MM cell migration on fibronectin triggered by both VEGF and IGF-1. Finally, our previous results implicate an PKC signaling in MM cell migration on fibronectin, and our current results show that Natalizumab inhibits phosphorylation of á4 integrins and PKC induced by co-stimulation with VEGF/ fibronectin, IGF-1/ fibronectin, and patient serum. Taken together, our data indicate a potential therapeutic role of Natalizumab in MM. Ongoing studies evaluating the effect of Natalizumab in a SCID-hu murine model of MM will also be reported. Disclosures: Podar: Biogen Idec: Research Funding. Off Label Use: natalizumab, integrin inhibitor. Zimmerhackl:Biogen Idec: Research Funding. Olsen:Biogen Idec: Employment.

The Scaffolding Protein NEDD9 Regulates Chronic Lymphocytic Leukemia Cell Migration Via the CXCR4 - CXCL12 Axis and Promotes Disease Progression

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 2-2
Author(s):  
Lisa Rusyn ◽  
Sebastian Reinartz ◽  
Anastasia Nikiforov ◽  
Nelly Mikhael ◽  
Christian Pallasch ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Adhesion ◽  
B Cells ◽  
Peripheral Blood ◽  
Treatment Strategies ◽  
Scaffold Protein ◽  
Lower Proportion ◽  
Dramatic Reduction

Despite the undeniable vast improvement in CLL treatment strategies, resistance to novel compounds such as ibrutinib and venetoclax already emerged and posed a challenge in many aggressive cases. The fundamental role of the homing process in CLL progression and presumably relapse prompted us to analyze the impact of a crucial regulator of chemokine response, migration and lymphocyte homing - namely NEDD9 - on CLL pathogenesis. The scaffold protein NEDD9 is frequently upregulated and hyperphosphorylated in different cancer entities, with its deregulation being associated with poor clinical outcome and therapy resistance. In B cells, activation of integrin- and the B cell receptor signaling pathways leads to hyperphosphorylation of NEDD9, predominantly by Src family kinases, promoting cell adhesion, migration and chemotaxis. To elucidate the functional relevance of NEDD9 in CLL pathogenesis in vivo,Eµ-TCL1 transgenic mice were crossbred with Nedd9 deficient mice. CLL burden was monitored in the peripheral blood of Nedd9-proficient (TCL1tg/wt Nedd9wt/wt)versus Nedd9-deficient (TCL1tg/wt Nedd9-/-) mice every two months over a year, revealing a significantly lower proportion of CLL per total B cells (CLL/B cells) in the peripheral blood in TCL1tg/wt Nedd9-/- mice at four and six months of age. CLL onset was clearly delayed in TCL1tg/wt Nedd9-/- mice in comparison to TCL1tg/wt Nedd9wt/wt mice. The infiltration of CLL cells into the spleen and bone marrow was significantly reduced in TCL1tg/wt Nedd9-/- mice at three and ten months, accompanied by significantly longer overall survival of the TCL1tg/wt Nedd9-/- group. Particularly, this eminent role of Nedd9 in CLL pathogenesis could be largely attributed to Nedd9 expression in B cells. Using a conditional Nedd9 knockout mouse exclusively in B cells (TCL1tg/wt CD19Cretg/wt Nedd9flfl (TCN)), we observed a highly similar phenotype of TCN mice to the TCL1tg/wt Nedd9-/- mice, including significantly delayed CLL onset, lower proportion of CLL per total B cells (CLL/B cells) in the peripheral blood, and reduced hepatosplenomegaly in TCN mice compared to the control TCL1tg/wt CD19Crewt/wt Nedd9flfl (TN) mice. In summary, our mouse data suggest that Nedd9 deficiency significantly delayed CLL onset and progression, particularly in the early stages of CLL. Moreover, Nedd9 deficiency significantly decreased the accumulation of CLL cells both in typical leukemic homing organs such as spleen and bone marrow as well as in the peripheral blood in two independent mouse models and significantly prolonged survival of the TCL1tg/wt Nedd9-/- mice. The strongly reduced capacity of Nedd9-deficient CLL cells to migrate and home to the lymphoid niche prompted us to investigate the underlying mechanistic signaling pathway upon Nedd9 loss. For this purpose, we examined surface expression levels of prominent cell trafficking mediators on Nedd9-proficient and -deficient CLL cells, and found a consistently reduced level of CXCR4 on the surface of TCL1tg/wt Nedd9-/-cells. In the transwell assay, CLL cells isolated from TCL1tg/wt Nedd9-/- mice showed a dramatic reduction in migration towards CXCL12 compared to their wild type counter parts. In line with the results of the murine CLL cells, NEDD9-depleted MEC1 cells (shNedd9) also showed decreased CXCR4 levels and dramatic reduction in migration towards CXCL12 compared to the control cell line (shNT). Collectively, we provide the first direct evidence that genetic targeting of Nedd9 in vivo impairs CLL cell adhesion, migration and chemotaxis, resulting in decreased CLL cell infiltration into secondary lymphoid organs and the bone marrow. These observations could serve as basis for the development of new treatment strategies, targeting a scaffold protein to impair the homing process of CLL cells, a prerequisite for their survival and expansion within the microenvironment of protective niches. Disclosures No relevant conflicts of interest to declare.

R-Ras Regulates Hematopoietic Stem/Progenitor Cell Adhesion, Migration, and Mobilization through Rac GTPase-Mediated Signals.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 221-221
Author(s):  
Xun Shang ◽  
Lina Li ◽  
Jose Concelas ◽  
Fukun Guo ◽  
Deidre Daria ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Adhesion ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Wild Type ◽  
Rac Gtpase ◽  
Hematopoietic Stem ◽  
And Migration ◽  
Marrow Cells

Abstract Hematopoietic stem/progenitor cells (HSPCs) are maintained by strictly regulated signals in the bone marrow microenvironment. One challenge in understanding the complex mode of HSPC regulation is to link intracellular signal components with extracellular stimuli. R-Ras is a member of the Ras family small GTPases. Previous mouse genetic studies suggest that R-Ras mRNA is primarily expressed in endothelial cells and R-Ras is involved in vascular angiogenesis. In clonal cell lines, although dominant mutant overexpression studies suggest a possible role of R-Ras in regulating cell adhesion and spreading, proliferation and/or differentiation in a cell-type dependent manner, it remains controversial whether R-Ras activity may promote or inhibit cell adhesion and migration. Here, in a mouse knockout model, we have examined the role of R-Ras in HSPC regulation by a combined in vivo and in vitro approach. Firstly, we found that R-Ras is expressed in the Lin− low density bone marrow cells of wild-type mice, and R-Ras activity in the cells is downregulated by cytokines and chemokines such as SCF and SDF-1a (∼ 20% and 40% of unstimulated control, respectively). Secondly, R-Ras deficiency did not significantly affect peripheral blood CBC, nor alter the frequency or distribution of long-term and short-term hematopoietic stem cells (defined by IL7Ra−Lin−Sca-1+c-Kit+CD34− and IL7Ra−Lin−Sca-1+c-Kit+CD34+ genotypes, respectively) in the bone marrow, peripheral blood and spleen. Competitive repopulation experiments using the wild-type and R-Ras−/− bone marrow cells at 1:1 ratio in lethally irradiated recipient mice showed no significant difference of blood cells of the two genotypes in the recipients up to 6 months post-transplantation. R-Ras−/− bone marrow cells did not show a detectable difference in colony forming unit activities assayed in the presence of various combinations of SCF, TPO, EPO, IL3, G-CSF and serum, compared with the matching wild-type cells. Thirdly, upon challenge with G-CSF, a HSPC mobilizing agent, R-Ras−/− mice demonstrated a markedly enhanced ability to mobilize HSPCs from bone marrow to peripheral blood as revealed by genotypic and colony-forming unit analyses (WT: 150 vs. KO: 320 per 200uL blood, p=0.018), and R-Ras−/− HSPCs exhibit significantly decreased homing activity (WT: 4.3% vs. KO: 2.8%, p&lt;0.001). Fourthly, isolated R-Ras−/− HSPCs displayed a constitutively assembled cortical actin cytoskeleton structure in the absence of cytokine or chemokine stimulation, similar to that of activated wild-type HSPCs. The R-Ras−/− HSPCs were defective in adhesion of cobblestone area-forming cells to a bone marrow-derived stroma cell line (FBMD-1) and in adhesion to fibronectin CH296 fragment, and showed a drastically increased ability to migrate toward a SDF-1a gradient (WT: 16% vs. KO: 38%, p&lt;0.001). These data point to a HSPC-intrinsic role of R-Ras in adhesion and migration. Finally, the functional changes of R-Ras−/− cells were associated with a ∼3 fold increase in Rac-GTP species and constitutively elevated Rac downstream signals of phsopho-PAK1 and phospho-myosin light chain. Partial inhibition of Rac activity by NSC23766, a Rac GTPase-specific inhibitor, readily reversed the migration phenotype under SDF-1a stimulation. Taken together, these studies demonstrate that R-Ras is a critical signal regulator for HSPC adhesion, homing, migration, and mobilization through a mechanism involving Rac GTPase-regulated cytoskeleton and adhesion machinery.

Role of Focal Adhesion Kinase in Myelotoxicity, Inflammatory Stress Response and Myeloid Cell Functions.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3615-3615
Author(s):  
Sasidhar Vemula ◽  
Baskar Ramdas ◽  
Philip Hanneman ◽  
Hillary Beggs ◽  
Reuben Kapur
Keyword(s):  
Bone Marrow ◽  
Stress Response ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Peripheral Blood ◽  
Hematopoietic Cells ◽  
Myeloid Cells ◽  
Inflammatory Stress

Abstract Abstract 3615 Poster Board III-551 Focal adhesion kinase (FAK) is a key signaling molecule in focal adhesion signaling and a potential integrator of integrin and growth factor receptor mediated signals. FAK has been implicated in various cellular functions such as growth, survival, migration, adhesion and cytoskeletal reorganization in fibroblasts but its role in hematopoietic stem and progenitors is unknown. To demonstrate the role of FAK in normal and stress-induced hematopoiesis, we generated FAK deficient mice by using a Cre/loxP method from here on termed FAKflox/flox (WT) mice. FAK deletion was induced by injecting poly (I)-poly(C) to FAK flox/flox mice containing the Mx.Cre transgene for one month (FAK-/-). PCR and western blot analysis revealed that after one month of poly (I)-poly(C) induction, hematopoietic cells failed to express detectable levels of FAK in bone marrow (BM), spleen and thymus. To determine the effect of FAK deletion on the development of hematopoietic cells a thorough analysis of the hematopoietic compartment in FAK-/- mice was performed. Total and differential cell counts of peripheral blood revealed significantly high red blood cell distribution width {RDW (%)} and mean cell volume (MCV) in FAK-/- mice compared to WT (n=13, WT; 18.6, 47.2 vs. FAK-/-; 20.06, 48.7, *p<0.05), respectively. In addition, differential basophil counts were significantly less in FAK-/- mice compared to WT (n=13, WT; 0.68 vs. FAK-/-; 0.3 *p<0.04) but all leukocyte populations were present at normal frequencies. Furthermore, platelet counts were significantly higher in FAK-/- peripheral blood compared to WT controls (n=13, WT; 759 vs FAK-/-; 978, *p<0.01). Under basal steady-state conditions, granulopoiesis appeared to be significantly altered in FAK deficient bone marrow (BM), as frequency of granulocytes, but not of other myeloid cells was reduced (n=10, WT; 44.14% vs. FAK-/-; 34.4%, *p<0.0001). Interestingly the frequency of Lin-, c-Kit+, Sca-1+ was also impaired in FAK deficient BM compared to controls (n=9, *p<0.05). FAK deficient BM progenitors displayed significantly lower frequency of colony-forming units compared to WT controls in response to various cytokine combinations (n=6, *p<0.01), which was associated with higher apoptosis in vitro (n=9, *p<0.006). Under conditions of stress, recovery of BM myeloid compartment and Lin−,c-Kit+, Sca-1+ cells following 5-Fluorouracil myeloablation was much slower in FAK-/- mice compared to WT controls (n=3, *p<0.05). Furthermore, the response of myeloid cells to acute inflammatory stress inflicted by intraperitoneal injection of thioglycollate was impaired in FAK-/- mice compared to WT mice (Macrophages: WT; 7.47 × 106 vs. FAK−/−; 3.1 × 106, n=8, *p <0.01. Neutrophils: WT; 5.47 × 106 vs. FAK−/−; 2.1 × 106, n=3, *p <0.05). These results led us to more closely examine the myeloid compartment in these mice. In vitro, FAK-/- macrophage progenitors show reduced growth in response to M-CSF stimulation (n=4, *p <0.01). In addition, deficiency of FAK in macrophages resulted in significant reduction in haptotactic migration in response to M-CSF on extracellular matrix proteins such as fibronectin, laminin and collagen (n=4, *p <0.01). Consistently, a significant reduction in the migration of FAK-/- macrophages was also observed in a wound healing assay which was associated with reduced activation of Rho GTPases including Rac. The reduction in migration of FAK-/- macrophages was associated with a significant decrease in adhesion on fibronectin, laminin and collagen. The impaired migration and adhesion of FAK-/- macrophages was observed in spite of comparable levels of F4/80 as well as integrin (α4β1 & α5β1) expression. Consistent with enhanced neutrophil apoptosis and reduced frequency under basal conditions, FAK deficient BM derived neutrophil progenitors (BMNs) show reduced growth and cycling in response to G-CSF stimulation (n=4, *p <0.01). Deletion of FAK in BMNs led to increased apoptosis upon cytokine withdrawal, which was associated with reduced activation of AKT and increased caspase-3 cleavage compared to controls. Taken together, our findings indicate that FAK plays a vital role in modulating physiological stress response to myeloablation, inflammation as well as in regulating several functions in macrophages and neutrophils. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mesenchymal Stem Cells in Perichondrium Express Activated Leukocyte Cell Adhesion Molecule and Participate in Bone Marrow Formation

2002 ◽  
Vol 195 (12) ◽  
pp. 1549-1563 ◽  
Author(s):  
Fumio Arai ◽  
Osamu Ohneda ◽  
Takeshi Miyamoto ◽  
Xiu Qin Zhang ◽  
Toshio Suda
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Cell Adhesion Molecule ◽  
Hematopoietic Bone Marrow

Perichondrium in fetal limb is composed of undifferentiated mesenchymal cells. However, the multipotency of cells in this region and the role of perichondrium in bone marrow formation are not well understood. In this report, we purified and characterized perichondrial cells using a monoclonal antibody against activated leukocyte cell adhesion molecule (ALCAM) and investigated the role of perichondrial cells in hematopoietic bone marrow formation. ALCAM is expressed on hematopoietic cells, endothelial cells, bone marrow stromal cells, and mesenchymal stem cells and mediates homophilic (ALCAM–ALCAM)/heterophilic (ALCAM-CD6) cell adhesion. Here we show by immunohistochemical staining that ALCAM is expressed in perichondrium. ALCAM+ perichondrial cells isolated by FACS® exhibit the characteristics of mesenchymal stem cells. ALCAM+ cells can differentiate into osteoblasts, adipocytes, chondrocytes, and stromal cells, which can support osteoclastogenesis, hematopoiesis, and angiogenesis. Furthermore, the addition of ALCAM-Fc or CD6-Fc to the metatarsal culture, the invasion of the blood vessels to a cartilage was inhibited. Our findings indicate that ALCAM+ perichondrial cells participate in vascular invasion by recruiting osteoclasts and vessels. These findings suggest that perichondrium might serve as a stem cell reservoir and play an important role in the early development of a bone and bone marrow.

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