Migration Of Bone Marrow-derived Cells Research Articles

Abstract A047: Fibroblast-specific IL1 receptor inhibition reprograms the inflammatory stroma to overcome the immunosuppressive tumor microenvironment in pancreatic cancer

Abstract OBJECTIVE: KRAS-driven inflammatory signaling in pancreatic ductal adenocarcinoma (PDAC) promotes cancer-associated fibroblast (CAF) polarization and an immunosuppressive tumor microenvironment (iTME), driving therapeutic resistance. IL1α, a downstream product of KRAS signaling, activates the pro-inflammatory CAF (iCAF) phenotype and correlates with worse survival. Using patient and PDAC mouse model single-cell RNA sequencing (scRNAseq) data, we have shown that the IL1 Receptor (IL1R1) is highly enriched in the fibroblast compartment. However, the role of IL1R1 in modulating iCAFs and the iTME remains poorly understood. We hypothesize that CAF-specific disruption of IL1R1 signaling will reprogram cellular components of the PDAC TME to overcome the immunosuppressive milieu and therapeutic resistance. METHODS: The effects of IL1R1 inhibition on iCAF modulation in vivo were determined in Ptf1aCre/+, LSL-KrasG12D/+, Tgfbr2flox/flox (PKT) mice treated with IL1R1 antagonist. To determine if CAF-specific silencing of IL1R1 recapitulates the TME remodeling after IL1R1 inhibition, we generated a fibroblast-specific IL1R1KO mouse model Col1a2Cre/+;Il1r1flox/flox (CAF-Il1r1KO) and orthotopically injected KPC tumor cells for endpoint analysis. Flow cytometry was performed in primary tumors of CAF-Il1r1KO and littermate controls. Functional experiments evaluating myeloid-derived suppressor cell (MDSC) trafficking were performed using ex vivo bone marrow-derived cells (BMDCs) co-cultured with CAF/tumor-conditioned media (CM) with/without IL1R1 inhibition in a transwell system. MDSC contribution to iTME was assessed by co-culturing MDSCs from CAF-Il1r1KO tumors and splenic T cells from naïve C57BL/6 mice. To investigate the effects of IL1R1 deletion in overcoming therapeutic resistance, survival studies in CAF-Il1r1KO mice with chemotherapy were conducted. RESULTS: Treatment of PKT mice with IL1R1 antagonist diminished transcriptional levels of Il6 and Cxcl1, iCAF-associated cytokines, in the CAF compartment. Compared to littermate controls, orthotopic tumors in CAF-Il1r1KO showed significant reduction in tumor weights, increased CD8+ T cell infiltration, and overall reduction in polymorphonuclear (PMN) MDSC trafficking by flow cytometry. Moreover, migration of BMDCs was reduced when co-cultured with CAF/tumor CM and IL1R1 antagonist. In addition, co-culture of MDSCs from CAF-Il1r1KO mice with naïve T cells showed diminished inhibition of T cell functional activity. Chemotherapy treatment of orthotopic tumors in CAF-Il1r1KO mice showed significantly decreased tumor size and improved survival compared to tumors in littermate controls. CONCLUSION: Our findings illustrate a novel mechanism of CAF-specific disruption of IL1R1 signaling inhibiting trafficking of immunosuppressive MDSCs and increased infiltration of cytotoxic CD8+ T cells in the PDAC TME. These data pave the way for novel combinatorial treatment strategies targeting IL1R1 to overcome chemotherapeutic resistance through reprogramming of the stromal compartment in PDAC. Citation Format: Camille Acevedo, Samara Singh, Austin Dosch, Edmond W Box, Iago C Silva, Anna Bianchi, Siddharth Mehra, Haleh Amirian, Andrew Adams, Nagaraj Nagathihalli, Jashodeep Datta, Nipun B Merchant. Fibroblast-specific IL1 receptor inhibition reprograms the inflammatory stroma to overcome the immunosuppressive tumor microenvironment in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A047.

CXCL12 Attracts Bone Marrow-Derived Cells to Uterine Leiomyomas.

Uterine leiomyomas, also known as fibroids or myomas, are a common benign gynecologic tumor found in women of reproductive age. Though advances have been made in understanding leiomyomas, the etiology and pathogenesis of this disease are not fully characterized. Current evidence supports a role of putative human uterine stem/progenitor cells in the onset of uterine disease such as uterine myomas. In this study, we report that increased expression of CXCL12 in leiomyomas recruits bone marrow-derived cells (BMDCs) that may contribute to leiomyoma growth. Tissue was collected from leiomyomas or control myometrium from women with or without leiomyomas. qRT-PCR analysis showed increased expression of CXCL12 and decreased CXCR4 expression in the leiomyoma and myometrium of women with leiomyoma compared with normal myometrium. Increased CXCL12 protein secretion from cultured myoma cells was confirmed by ELISA. Further, we found that BMDCs migration was increased toward leiomyoma conditioned medium compared with conditioned medium from normal myometrium. CXCR4 antagonist AMD3100 completely blocked this migration. Engraftment of BMDCs significantly increased in myoma of mouse uteri treated with CXCL12 compared with placebo. We conclude that CXCL12 may play a role in leiomyomas growth by attracting bone marrow-derived cells to leiomyoma. Therefore, CXCL12 and its receptors are novel targets for leiomyoma therapy.

Protumoral bone marrow-derived cells migrate via Gβγ-dependent signaling pathways and exhibit a complex repertoire of RhoGEFs.

Reciprocal communication among cells of the tumor microenvironment contributes to cancer progression. Here, we show that a protumoral population of cultured bone marrow-derived cells (BMDC) containing Tie2+/CD45+/CD11b + cells responded to lung carcinoma cells and reciprocally stimulated them. These cells migrated via heterotrimeric G protein-dependent signaling pathways and strongly activated the PI3K/AKT, ERK and mTOR signaling cascades in response to conditioned media and chemotactic agonists. To get insight into the molecular machinery involved in BMDC migration, we revealed their repertoire of guanine nucleotide exchange factors for Rho GTPases (RhoGEFs) and G proteins in comparison with fresh bone marrow cells, proven that these cell populations had contrasting effects on tumor growth. BMDC exhibited a higher expression of G protein regulated RhoGEFs including P-Rex1, PDZ-RhoGEF, LARG, Trio and some less well characterized RhoGEFs such as ARHGEF5, ARHGEF17 and PLEKHG6. G proteins such as Gα12/13, Gαq, and the small GTPase RhoJ were also highly expressed in BMDC. Our results indicate that Tie2+/CD45+/CD11b + BMDC express a unique variety of chemotactic transducers and effectors potentially linked to their protumoral effect, warranting further studies to their characterization as molecular targets.

MicroRNA-188-5p regulates contribution of bone marrow-derived cells to choroidal neovascularization development by targeting MMP-2/13

Our previous investigations have shown that bone marrow-derived cells (BMCs), including mesenchymal stem cells (MSCs), contribute to the development of choroidal neovascularization (CNV) as sources of cells and angiogenic factors. Two main steps for circulating BMCs to integrate into CNV lesions are extracellular matrix remodeling and consequential cell migration. MicroRNAs (miRNAs) were found to be involved in CNV development; however, little is known about whether miRNAs regulate the contribution of BMCs to CNV. In the present study, we found that the expression of miR-188-5p was decreased in cultured hypoxic MSCs and BMCs within laser-induced CNV in mice. Matrix metalloproteinase 2 (MMP-2) and MMP-13 were both discovered as targets of miR-188-5p by bioinformatics predictions and dual-luciferase reporter system. Accordingly, increased expression of MMP-2/13 was found in hypoxic MSCs and BMCs in CNV lesions. Furthermore, miR-188-5p mimic transfection caused downregulation of MMP-2/13 in hypoxic MSCs and decreased tube formation of co-cultured vascular endothelial cells. Intravitreal injections of a miR-188-5p agomir attenuated the severity of CNV and inhibited the migration of BMCs into CNV lesions in mice. Our study suggests that miR-188-5p regulates the contribution of BMCs to CNV development by targeting MMP-2/13-mediated extracellular matrix degeneration, and miR-188-5p serves as a therapeutic target to treat CNV-related diseases.

Abstract A59: A rodent in situ glioma allograft model to study bone marrow-derived cell migration

Abstract Radiotherapy (RT) induced hypoxia recruits bone marrow-derived cells (BMDCs) to tumors through the upregulation of Stromal Derived Factor-1, a chemokine that binds to receptor CXCR4 present on BMDCs. The infiltration of BMDCs, which includes endothelial progenitors, pericyte progenitors, tumor-associated macrophages, immature monocytes, VEGFR+ hemangiocytes, and CD11b+ myeloid cells has been implicated in supporting tumor establishment by contributing to tumor growth, angiogenesis, vasculogenesis, inflammation, and immune suppression. In this work, we have used a rodent glioma in situ allograft model to study the recruitment/migration of BMDCs post RT. Female albino C57 (8-10 weeks old, Jackson Laboratories) animals underwent +DsRed bone marrow transplant (B6.Cg-Tg(CAG-DsRed*MST)1Nagy/J donor mouse). Following engraftment, glioma cells were cranially implanted with 1x105 glioma cells (GL261-Luc, Perkin Elmer), at an average size of 26.4mm3 indicated by a bioluminescence signal of 5.28x108 photons/sec/cm2/sr, animals underwent 6Gy (RadSource 2000, 250kVp, 25mA) of irradiation limited to a 1cm wide slit centered on the tumor. Bioluminescence (tumor size) and fluorescence imaging (+DsRed BMDCs) was performed prior to and at 1- and 2-weeks post-RT to assess tumor growth and measure the influx of +DsRed BMDCs. In addition, flow cytometry was used to measure the number of +DsRed BMDCs in the tumor and brain. To study the effect of BMDCs in tumor regression, tumors were irradiated at a smaller size (9.3mm3) with 10Gy irradiation. At 1-week post-RT, flow cytometry was used to measure the number of BMDCs (CD45+) in the tumor. In our model 6Gy radiation was able to stall the growth of the tumor. Irradiated tumors did not significantly change in tumor size at either 1- or 2-weeks post irradiation compared to pre-RT size. In addition, irradiated tumors were significantly smaller than untreated tumors (p=0.0008) 1-week post-RT. Using flow cytometry 1-week post irradiation, we observed a significant increase in the number of +DsRed BMDCs in the irradiated tumor (23.5% of cells +DsRed BMDCs) compared to the irradiated brain (6.17% cells +DsRed BMDCs, p=0.03) as well as a significant increase in the tumors (16.6% cells +DsRed BMDCs) compared to the brain (11.1% cells +DsRed BMDCs p=0.02). There was no difference in the number of +DsRed BMDCS in tumors ± irradiation or brains ± irradiation. We found similar results in our tumor regression model using flow cytometry 1-week post-RT; we observed an increase in the number of BMDCs CD45+ in the tumor (~20% of cells CD45+) compared to control tumors (~6% of cells CD45+). Indicating that tumor size at the onset of RT did not effect the migration of BMDCs. Fluorescence imaging did not measure any significant change in the DsRed signal at 1- or 2-weeks post-irradiation. We have successfully shown that post-RT there is an increase in BMDCs migration at 1-week post RT regardless of tumor size at RT. Further experimentation is needed to better elucidate the role of BMDCs in tumor inflammation and immune suppression as well as angiogenesis and vasculogenesis. Our next step is to perform immunohistochemistry on our collected tumor tissue to map out the spatial location of migrated BMDCs within the tumor. Citation Format: Janice A. Zawaski, Tien T. Tang, M Waleed Gaber. A rodent in situ glioma allograft model to study bone marrow-derived cell migration. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A59.

Effect of Irradiation on Tumor Microenvironment and Bone Marrow Cell Migration in a Preclinical Tumor Model

To characterize the tumor microenvironment after standard radiation therapy (SRT) and pulsed radiation therapy (PRT) in Lewis lung carcinoma (LLC) allografts. Subcutaneous LLC tumors were established in C57BL/6 mice. Standard RT or PRT was given at 2Gy/d for a total dose of 20Gy using a 5days on, 2days off schedule to mimic clinical delivery. Radiation-induced tumor microenvironment changes were examined after treatment using flow cytometry and antibody-specific histopathology. Normal tissue effects were measured using noninvasive (18)F-fluorodeoxyglucose positron emission tomography/computed tomography after naïve animals were given whole-lung irradiation to 40Gy in 4weeks using the same 2-Gy/d regimens. Over the 2weeks of therapy, PRT was more effective than SRT at reducing tumor growth rate (0.31±0.02mm(3)/d and 0.55±0.04mm(3)/d, respectively; P<.007). Histopathology showed a significant comparative reduction in the levels of Ki-67 (14.5%±3%), hypoxia (10%±3.5%), vascular endothelial growth factor (2.3%±1%), and stromal-derived factor-1α (2.5%±1.4%), as well as a concomitant decrease in CD45(+) bone marrow-derived cell (BMDC) migration (7.8%±2.2%) after PRT. The addition of AMD3100 also decreased CD45(+) BMDC migration in treated tumors (0.6%±0.1%). Higher vessel density was observed in treated tumors. No differences were observed in normal lung tissue after PRT or SRT. Pulsed RT-treated tumors exhibited slower growth and reduced hypoxia. Pulsed RT eliminated initiation of supportive mechanisms utilized by tumors in low oxygen microenvironments, including angiogenesis and recruitment of BMDCs.

Bone marrow-derived cells in ocular neovascularization: contribution and mechanisms

Ocular neovascularization often leads to severe vision loss. The role of bone marrow-derived cells (BMCs) in the development of ocular neovascularization, and its significance, is increasingly being recognized. In this review, we discuss their contribution and the potential mechanisms that mediate the effect of BMCs on the progression of ocular neovascularization. The sequence of events by which BMCs participate in ocular neovascularization can be roughly divided into four phases, i.e., mobilization, migration, adhesion and differentiation. This process is delicately regulated and liable to be affected by multiple factors. Cytokines such as vascular endothelial growth factor, granulocyte colony-stimulating factor and erythropoietin are involved in the mobilization of BMCs. Studies have also demonstrated a key role of cytokines such as stromal cell-derived factor-1, tumor necrosis factor-α, as well as vascular endothelial growth factor, in regulating the migration of BMCs. The adhesion of BMCs is mainly regulated by vascular cell adhesion molecule-1, intercellular adhesion molecule-1 and vascular endothelial cadherin. However, the mechanisms regulating the differentiation of BMCs are largely unknown at present. In addition, BMCs secrete cytokines that interact with the microenvironment of ocular neovascularization; their contribution to ocular neovascularization, especially choroidal neovascularization, can be aggravated by several risk factors. An extensive regulatory network is thought to modulate the role of BMCs in the development of ocular neovascularization. A comprehensive understanding of the involved mechanisms will help in the development of novel therapeutic strategies related to BMCs. In this review, we have limited the discussion to the recent progress in this field, especially the research conducted at our laboratory.

Hypoxia-Inducible Factor 1α Is a Critical Downstream Mediator for Hypoxia-Induced Mitogenic Factor (FIZZ1/RELMα)–Induced Pulmonary Hypertension

Pulmonary hypertension (PH) is characterized by progressive elevation of pulmonary vascular resistance, right ventricular failure, and ultimately death. We have shown that in rodents, hypoxia-induced mitogenic factor (HIMF; also known as FIZZ1 or resistin-like molecule-β) causes PH by initiating lung vascular inflammation. We hypothesized that hypoxia-inducible factor-1 (HIF-1) is a critical downstream signal mediator of HIMF during PH development. In this study, we compared the degree of HIMF-induced pulmonary vascular remodeling and PH development in wild-type (HIF-1α(+/+)) and HIF-1α heterozygous null (HIF-1α(+/-)) mice. HIMF-induced PH was significantly diminished in HIF-1α(+/-) mice and was accompanied by a dysregulated vascular endothelial growth factor-A-vascular endothelial growth factor receptor 2 pathway. HIF-1α was critical for bone marrow-derived cell migration and vascular tube formation in response to HIMF. Furthermore, HIMF and its human homolog, resistin-like molecule-β, significantly increased interleukin (IL)-6 in macrophages and lung resident cells through a mechanism dependent on HIF-1α and, at least to some extent, on nuclear factor κB. Our results suggest that HIF-1α is a critical downstream transcription factor for HIMF-induced pulmonary vascular remodeling and PH development. Importantly, both HIMF and human resistin-like molecule-β significantly increased IL-6 in lung resident cells and increased perivascular accumulation of IL-6-expressing macrophages in the lungs of mice. These data suggest that HIMF can induce HIF-1, vascular endothelial growth factor-A, and interleukin-6, which are critical mediators of both hypoxic inflammation and PH pathophysiology.

Migration of bone marrow–derived cells for endogenous repair in a new tail-looping disc degeneration model in the mouse: a pilot study

Background contextMobilization and homing of bone marrow–derived cells (BMCs) play a pivotal role in healing and regeneration of various tissues. However, the cellular response of BMCs in avascular tissue such as the intervertebral disc (IVD) has not been studied in detail. One of the main obstacles to this is a lack of a suitable mouse disc degeneration model. PurposeThe purpose of this study was to establish a reproducible disc degeneration mouse model suitable for analyzing the cellular response of the disc microenvironment and to determine whether BMCs are recruited into the IVD. Study designAn experimental animal study of disc degeneration investigating the potential of BMCs in an endogenous repair of the IVD. MethodsWe transplanted whole bone marrow cells from mice ubiquitously expressing enhanced green fluorescent protein into lethally irradiated mice. Intervertebral disc degeneration was induced through uneven loading by creating a loop in the tail of these mice. The vertebral bone-disc-vertebral bone units were harvested, and BMCs were identified by immunohistochemistry. ResultsA new disc degeneration model was established in the mouse. Applying this model in the bone marrow chimeric mice increased the number of BMCs in the peripheral bone marrow and vascular canals in the endplate, and some were found in the IVD. The migration of BMCs was related to the severity of IVD degeneration. ConclusionsAlthough providing a new disc degeneration model in mice, the present study provides evidence to suggest that although BMCs are recruited during disc degeneration, only a limited number of BMCs migrate to the IVD, presumably because of its avascular nature. This fact provides important elements for developing new treatments as many growth factors and compounds are being tested, both in investigational levels and clinical trials to nourish resident endogenous cells during the degenerative process.

Increased numbers of bone marrow-derived cells in parathyroid adenoma.

In primary hyperparathyroidism (PHPT), the increased levels of parathyroid hormone (PTH) result in mobilisation of bone-marrow-derived cells (BMCs) into peripheral blood. However, the fate of these cells is still unknown. In this study, we sought to investigate cells with typical surface markers of BMCs within parathyroid adenomas (PA) of patients with primary hyperparathyroidism. We therefore investigated PA and normal parathyroid glands (NPG) of 15 patients with PHPT by immunohistochemistry and PCR. mRNA levels for CD31, CD34 and CD45 were significantly increased in PA compared to NPG. Immunohistochemical staining for CD31 and CD34 revealed a significantly higher vessel density in PA. Furthermore, scattered single cells expressing CD31, CD34 or CD45 were significantly augmented compared to normal parathyroid glands and directly correlated with vessel density. mRNA levels of SDF-1 was increased whereas its major inhibitor dipeptidylpeptidase IV (DPP IV) is decreased in PA, suggesting that the SDF-1 axis plays a role in the migration of BMCs into PA. These data indicate a possible role of BMCs in the pathophysiology of PA of patients with PHPT.

Spontaneous Development of Autoimmune Uveitis Is CCR2 Dependent

Development of novel strategies to treat noninfectious posterior uveitis is an ongoing challenge, in part because of limited availability of animal models that mimic the naturally occurring disease in humans. Mice deficient in the autoimmune regulatory gene Aire develop a spontaneous T-cell and macrophage-mediated autoimmune uveitis that closely recapitulates human endogenous uveitis and thus provide a useful model for mechanistic and therapeutic investigations. Lymphocytic and mononuclear infiltration of the retina in Aire knockout (KO) mice triggers the onset of uveitis from initial retinal inflammation to eventual destruction of the neuroretina with loss of photoreceptors. The C-C chemokine receptor type 2 protein (CCR2) functions in directing monocyte and macrophage migration to inflamed tissues via interaction with monocyte chemotactic proteins. Using the Aire KO mouse model, we demonstrated an essential role for CCR2 in the pathogenesis of autoimmune-mediated uveitis. Loss of functional CCR2 effectively reduced immune cell infiltration and rescued the retina from destruction. CCR2-dependent migration of bone marrow-derived cells provided the driving force for retinal inflammation, with CCR2-expressing mononuclear cells contributing to retinal damage via recruitment of CD4(+) T cells. These studies identify the CCR2 pathway as a promising therapeutic target that may prove an effective approach to treat uveitis associated with autoimmunity.

Ethanol and liver: Recent insights into the mechanisms of ethanol-induced fatty liver

Alcoholic fatty liver disease (AFLD), a potentially pathologic condition, can progress to steatohepatitis, fibrosis, and cirrhosis, leading to an increased probability of hepatic failure and death. Alcohol induces fatty liver by increasing the ratio of reduced form of nicotinamide adenine dinucleotide to oxidized form of nicotinamide adenine dinucleotide in hepatocytes; increasing hepatic sterol regulatory element-binding protein (SREBP)-1, plasminogen activator inhibitor (PAI)-1, and early growth response-1 activity; and decreasing hepatic peroxisome proliferator-activated receptor-α activity. Alcohol activates the innate immune system and induces an imbalance of the immune response, which is followed by activated Kupffer cell-derived tumor necrosis factor (TNF)-α overproduction, which is in turn responsible for the changes in the hepatic SREBP-1 and PAI-1 activity. Alcohol abuse promotes the migration of bone marrow-derived cells (BMDCs) to the liver and then reprograms TNF-α expression from BMDCs. Chronic alcohol intake triggers the sympathetic hyperactivity-activated hepatic stellate cell (HSC) feedback loop that in turn activates the HSCs, resulting in HSC-derived TNF-α overproduction. Carvedilol may block this feedback loop by suppressing sympathetic activity, which attenuates the progression of AFLD. Clinical studies evaluating combination therapy of carvedilol with a TNF-α inhibitor to treat patients with AFLD are warranted to prevent the development of alcoholic liver disease.

Migration of Bone Marrow‐Derived Cells Into the Central Nervous System in Models of Neurodegeneration

Microglia are the brain-resident macrophages tasked with the defense and maintenance of the central nervous system (CNS). The hematopoietic origin of microglia has warranted a therapeutic potential for the hematopoietic system in treating diseases of the CNS. However, migration of bone marrow-derived cells (BMDC) into the CNS is a marginal event under normal, healthy conditions. A busulfan-based chemotherapy regimen was used for bone marrow transplantation in wild-type mice before subjecting them to a hypoxic-ischemic brain injury or in APP/PS1 mice prior to the formation of amyloid plaques. The cells were tracked and analyzed throughout the development of the pathology. The efficacy of a preventive macrophage colony-stimulating factor (M-CSF) treatment was also studied to highlight the effects of circulating monocytes in hypoxic-ischemic brain injury. Such an injury induces a strong migration of BMDC into the CNS, without the need for irradiation. These migrating cells do not replace the entire microglial pool but rather are confined to the sites of injury for several weeks, suggesting that they could perform specific functions. M-CSF showed neuroprotective effects as a preventive treatment. In APP/PS1 mice, the formation of amyloid plaques was sufficient to induce the entry of cells into the parenchyma, though in low numbers. This study confirms that BMDC infiltrate the CNS in animal models for stroke and Alzheimer's disease and that peripheral cells can be targeted to treat affected regions of the CNS.

Relationship Between the Chemokine Receptor CCR5 and Microglia in Neurological Disorders: Consequences of Targeting CCR5 on Neuroinflammation, Neuronal Death and Regeneration in a Model of Epilepsy

Chemokines may play a role in leukocyte migration across the blood-brain barrier (BBB) during neuroinflammation and other neuropathological processes, such as epilepsy. The CC chemokine receptor 5 (CCR5) is a member of CC-chemokine receptor family that binds several chemokines, including CCL3 (macrophage inflammatory protein-1alpha, MIP-1alpha), CCL4 (macrophage inflammatory protein-1beta, MIP-1beta) and CCL5 (RANTES). The current review examines the relationship between CCR5 and the microglia in different neurological disorders and models of CNS injury. CCR5 expression is upregulated in different neurological diseases, where it is often immunolocalized in microglial cells. A multistep cascade couples CCR5 activation by chemokines to Ca(2+) increases in human microglia. Because changes in [Ca(2+)] (i) affect chemotaxis, secretion, and gene expression, pharmacologic modulation of this pathway may alter inflammatory and degenerative processes in the CNS. Consequently, targeting CCR5 by using CCR5 antagonists may attenuate critical aspects of neuroinflammation in different models of neurological disorders. To illustrate the interaction between CCR5 and microglia in the CNS, we used a model of excitotoxicity, and demonstrate the intimate involvement of CCR5 in neuron injury and inflammation attendant to kainic acid (KA)-induced neurotoxicity. CCR5 participates in neuronal injury caused by the excitotoxin, KA, brings inflammatory cells to the sites of KA-induced CNS injury, defines the extent of tissue loss after KA exposure and limits reparative responses. We used a SV40-derived vector carrying an interfering RNA (RNAi) that targets CCR5. Delivered directly to the bone marrow, this vector decreased CCR5 expression in circulating cells. Animals so treated showed greatly reduced expression of CCR5 and its ligands (MIP-1alpha and RANTES) in the CNS, including in the brain vasculature, decreased BBB leakage, demonstrated greater KA-stimulated neurogenesis and increased migration of bone marrow-derived cells to the brain to become neurons. Thus, therapeutic targeting of CCR5 may allow control of potentially injurious neuroinflammatory responses, including decrease in microglial cells activation and proliferation, and facilitate neurogenic repair in seizure-induced and, potentially, other forms of CNS injury.

Enhanced migration of bone marrow-derived cells mediated by VCAM-1/VLA-4 interaction improves cardiac function in virus-induced dilated cardiomyopathy

Aims: Endogenous circulation of bone marrow-derived cells (BMCs) was observed in patients with dilated cardiomyopathy (DCM) who showed cardiac upregulation of Vascular Cell Adhesion Protein-1 (VCAM-1). However, the underlying pathophysiology is currently unknown. Thus, we aimed to analyze circulation, homing and G-CSF based mobilization of BMCs in a murine model of virus-induced DCM. Methods and results: Mice with coxsackievirus B3 (CVB3) induced DCM and healthy controls were analyzed regarding their myocardial homing factors by PCR. To determine cardiac VCAM-1 expression ELISA and immunohistochemistry was applied. Flow cytometry was performed to analyze BMCs and resident cardiac stem cells (CSCs). Heart function was evaluated by cardiac MRI before and 4 weeks after G-CSF treatment. In murine CVB3-induced DCM an increase of BMCs in peripheral blood and a decrease of BMCs in bone marrow was observed. We found an enhanced migration of Very Late Antigen-4 (VLA-4+) BMCs to the diseased heart overexpressing VCAM-1 and higher numbers of CSCs. Mobilization of BMCs by G-CSF boosted homing via VCAM-1/VLA-4 interaction and reduced apoptotic cardiomyocytes. Significant improvement of cardiac function was detected by MRI in G-CSF treated mice. Blocking VCAM-1 by a neutralizing antibody reduced the G-CSF dependent effects on stem cell homing and CSC proliferation. Conclusion: This is the first study showing that in virus-induced DCM VCAM-1/VLA-4 interaction is crucial for recruitment of circulating BMCs leading to a proliferation of CSCs and to beneficial antiapoptotic effects resulting in improved cardiac function after G-CSF induced mobilization.

Promotion of Transplanted Bone Marrow-derived Cell Migration into the Periodontal Tissues due to Orthodontic Mechanical Stress

Background: Bone marrow-derived cells (BMCs) have abilities of cell migration and differentiation into tissues/organs in the body and related with the differentiation of teeth or periodontal tissue including fibroblasts. Then, we examined the effect of orthodontic mechanical stress to the transplanted BMC migration into periodontal tissues using BMC transplantation model.Material and Method: BMC from green fluorescence protein (GFP) transgenic mice were transplanted into 8-week-old female C57BL/6 immunocompromised recipient mice, which had undergone 10 Gy of lethal whole-body-irradiation. Five mice as experimental group were received orthodontic mechanical stress using separator between first molar (M1) and second molar (M2) 1 time per week for 5 weeks and 5 mice as control group were not received mechanical stress. The maxilla with M1 and M2 was removed and was immunohistochemically analyzed using a Dako Envision + Kit-K4006 and a primary anti-GFP-polyclonal rabbit antibody. Immunohistochemically stained was defined as positive area and the pixel number of positive area in the periodontal tissue was compared with the previously calculated total pixel number of the periodontal tissue.Results: The immunohistochemistry revealed that GFP positive cells were detected in the periodontal tissues, both in the experimental and control specimens. The ratio of pixel number in the examination group showed 5.77 ± 3.24 % (mean ± SD); and that in the control group, 0.71±0.45 % (mean ± SD). The examination group was significantly greater than that of control group (Mann-Whitney U test: p<0.001).Conclusion: These results suggest that orthodontic mechanical stress accelerates transplanted BMC migration into periodontal tissues.

Transplanted Bone Marrow-Derived Cell Migration Into Periodontal Tissues Induced by Orthodontic Mechanical Stress

Aim: Bone marrow-derived cells have abilities of cell migration and differentiation into teeth and related tissues/organs, especially into periodontal ligament fibroblast cells (Tsujigiwa T. et al. J Hard Tissue Biol 20: 147-52, 2011; Muraoka R. et al. J Hard Tissue Biol 20: 301-6, 2011). In this examination using a bone marrow transplantation model, we examined the effect of orthodontic mechanical stress to the transplanted bone marrow-derived cell migration into periodontal tissues. Methods: Bone marrow derived cells from green fluorescence protein (GFP) transgenic mice were transplanted into 8 week-old female C57BL/6 immunocompromised recipient mice (n=10), which had undergone 10 Gy of lethal whole-body irradiation. After successful transplantation (about one month period), 5 mice received orthodontic mechanical stress using the Waldo method 5 times in 5 weeks; and 5 mice were compared as control without receiving orthodontic mechanical stress (Waldo CM. J Dent Res 32: 690-1, 1953). After that, the regional tissues were removed and fixed in formalin fixative. Paraffin-embedded sections were immunohistochemically analyzed using a Dako Envision + Kit-K4006 (Dako, Glostrup, Denmark) and a primary anti-GFP-polyclonal rabbit antibody (#598; 1/500; MBL, Nagoya, Japan). For semiquantitative evaluation of immunohistochemical staining, the following procedures were performed. First immunohistochemical images with same magnification from the periodontal tissues were prepared and pixel density was counted for each image. Then typical immunohistochemically positive staining part was defined as positive area. The pixel number of positive area in the periodontal tissue was compared with the previously calculated total pixel number of the periodontal tissue and the ratio was obtained. Results and discussion: We examined the transplanted bone marrow-derived cell migration into periodontal tissues. The immunohistochemistry revealed that GFP-positive cells were detected in the periodontal tissues, both in the experimental and control specimens. The GFP-positive cells histopathologically differentiated into some cell types. The fluorescence IHC and TRAP staining techniques demonstrated these cells were detected as osteoclasts and macrophages. Furthermore, GFP-positive cells gathered adjacent blood vessels. The data suggest that GFP-positive bone marrow-derived cell migrate into periodontal tissues and differentiate periodontal tissue component-cells. In the experimental group, there were numerous GFP-positive cells appearing in the experimental periodontal tissues which received intermittent stimulation of orthodontic mechanical stress, but there were few GFP-positive cells in the control specimens. As a results of the examination group specimens and control group, the ratio of pixel number in the examination group showed 5.77 ± 3.24 % (mean ± SD); and that in the control group, 0.71±0.45 % (mean ± SD). The examination group was significantly greater than that of control group (Mann-Whitney U test: p< 0.001). Thus, these data indicated that orthodontic mechanical stress acts as a possible promoting factor of transplanted bone marrow-derived cell migration into periodontal tissues, and of differentiation to fibroblasts. Conclusions: These results suggest that orthodontic mechanical stress induces transplanted bone marrow-derived cell migration and differentiation into periodontal tissue.

Novel anti‐fibrotic modalities for liver fibrosis: Molecular targeting and regenerative medicine in fibrosis therapy

Based on the cellular and molecular mechanisms underlying hepatic fibrogenesis, several kinds of approaches have been proposed to treat liver fibrosis. Among a number of growth factors and cytokines that regulate collagen metabolism, transforming growth factor (TGF)-β is the most potent factor to accelerate liver fibrosis by activating hepatic stellate cells, stimulating collagen gene transcription, and suppressing matrix metalloproteinases expression. Thus, TGF-β as well as its intracellular mediators, Smad proteins, can be potential therapeutic targets for liver fibrosis. Constitutive phosphorylation and nuclear accumulation of Smad3 is the common feature of activated stellate cells. We have synthesized a novel small compound that inhibits Smad3-dependent collagen gene transcription by promoting nuclear import of a transcriptional repressor, YB-1. Another insight into anti-fibrotic strategies is the contribution of bone marrow-derived cells to the regression of liver fibrosis. Administration of granulocyte-colony stimulating factor enhanced the migration of bone marrow-derived cells into fibrotic liver tissue and accelerated the regression of experimental liver fibrosis. We have recently identified novel unknown factors expressed by bone marrow-derived cells that not only ameliorate liver fibrosis but also accelerate regeneration of fibrotic liver.

Effects of hypoxia inducible factor-1α activator on the biological behavior of endothelial progenitor cells

Objective To investigate the effects of hypoxia inducible factor-1α （HIF-1α） on the proliferation, migration and angiogenesis of bone marrow-derived endothelial progenitor cells （EPCs）. Methods Bone marrow EPCs were interfered with the HIF-1α activator, dimethyloxalylglycine （DMOG） at different concentrations. Methyl thiazol tetrazolium （MTT） assay, CFU assay and Western blotting were employed. Nitric oxide （NO） production, vascular endothelial growth factor （ VEGF）, stromal cell derived factor 1 （SDF-1）, migratory capacity, cellular senescence levels and angiogenesis ability were evaluated. Results DMOG could significantly activate the expression of HIF-1α protein in EPCs and increase the cell viability in a dose-dependent fashion, which at peak proliferation at concentration of 500 μmol/L. DMOG could substantially improve the colony-forming ability （P 〈 0. 01 ） and significantly protect EPCs from cellular senescence （P 〈 0. 05 ）. Besides, DOMG ameliorated the migratory functions and angiogenesis ability of EPCs. Conclusion DMOG can activate HIF-1α and act on EPCs to improve the proliferation, migration capacities and angiogenesis ability. Key words: Endothelial progenitor ceils ; Dimethyloxalylglycine ; Hypoxia inducible factor-1α; Angiogenesis

Mechanical stress as a possible promoter of transplanted bone marrow-derived cell migration into periodontal tissues

Mechanical stress as a possible promoter of transplanted bone marrow-derived cell migration into periodontal tissues