Expression Of Macrophage Colony-stimulating Factor Research Articles

Macrophage colony-stimulating factor expressed in non-cancer tissues provides predictive powers for recurrence in hepatocellular carcinoma.

AIMTo investigate the role of macrophage colony-stimulating factor (M-CSF) in patients with hepatocellular carcinoma (HCC) after surgery.METHODSExpression of M-CSF, distribution of M2 macrophages (MΦs), and angiogenesis were assessed in the liver, including tumors and peritumoral liver tissues. The prognostic power of these factors was assessed. Mouse isolated hepatic MΦs or monocytes were cultured with media containing M-CSF. The concentration of vascular endothelial growth factor (VEGF) in media was assessed. Furthermore, the role of the M-CSF-matured hepatic MΦs on proliferation of the vascular endothelial cell (VEC) was investigated.RESULTSA strong correlation between the expressions of M-CSF and CD163 was observed in the peritumoral area. Also, groups with high density of M-CSF, CD163 or CD31 showed a significantly shorter time to recurrence (TTR) than low density groups. Multivariate analysis revealed the expression of M-CSF or hepatic M2MΦs in the peritumoral area as the most crucial factor responsible for shorter TTR. Moreover, the expression of M-CSF and hepatic M2MΦs in the peritumoral area had better predictable power of overall survival. Values of VEGF in culture media were significantly greater in the hepatic MΦs compared with the monocytes. Proliferation of the VEC was greatest in the cells co-cultured with hepatic MΦs when M-CSF was present in media.CONCLUSIONM-CSF increases hepatocarcinogenesis, most likely by enhancing an angiogenic factor derived from hepatic MΦ and could be a useful target for therapy against HCC.

M2-polarized macrophages in keratocystic odontogenic tumor: relation to tumor angiogenesis.

The purpose of this study was to evaluate the presence of M2-polarized macrophages and their relationships to angiogenesis in keratocystic odontogenic tumor (KCOT). M2-polarized macrophages were detected in KCOT samples by immunohistochemistry and immunofluorescence. Meanwhile, microvessel density measured with antibody against CD31 was closely correlated with the presence of M2-polarized macrophages. In addition, macrophage colony-stimulating factor (M-CSF) significantly contributed to the activation of M2-polarized macrophages. Moreover, the results of in vitro wound healing, cell migration and tube formation assays further revealed the pro-angiogenic function of M2-polarized macrophage-like cells. This function might be associated with secretion of angiogenic cytokines, such as vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β) and matrix metalloprotein-9 (MMP-9). This study demonstrates for the first time that M2-polarized macrophages are prevalent in KCOT, and their presence is dependent on M-CSF expression. More importantly, these tumor-supportive cells can also promote tumor angiogenesis by secreting angiogenic cytokines.

Effects of intermedin on proliferation, apoptosis and the expression of OPG/RANKL/M-CSF in the MC3T3-E1 osteoblast cell line.

Bone remodeling is a vital physiological process of healthy bone tissue in humans. It is characterized by the formation of bone by osteoblasts and its resorption by osteoclasts, and the bone resorbed by osteoclasts is replaced through the differentiation and activity of osteoblasts. Imbalances in this vital process lead to pathological conditions, including osteoporosis. Intermedin (IMD) as a newly discovered peptide in the calcitonin (CT) family of peptides, which shares similar functions with CT, calcitonin gene-related peptide and amylin in bone resorption. However, the mechanism underlying its effect remains to be elucidated. This was investigated in the present study using the osteoblastic MC3T3-E1 cell line, which was treated with different doses of IMD (0, 1, 10 and 100 nM). Cell proliferation, apoptosis and the expression of receptor activator of NF-κB ligand (RANKL), osteoprotegerin (OPG) and macrophage colony-stimulating factor (M-CSF) were measured following treatment using multiple detection techniques, including an MTT assay, flow cytometry, reverse transcription-quantitative polymerase chain reaction and western blot analysis. The resulting data demonstrated that IMD significantly inhibited the apoptosis of MC3T3-E1 cells induced by serum-free culture and dexamethasone, however, no significant effects on MC3T3-E1 cell proliferation were observed. IMD had additional functions on the MC3T3-E1 cells, including inhibition of the expression of RANKL and M-CSF, and promotion of the expression of OPG. Previous studies have also demonstrated that RANKL and M-CSF are two vital factor produced by osteoblasts to promote the maturation and differentiation of osteoclasts, and it has been reported that IMD can inhibit the osteoclast formation stimulated by RANKL and M-CSF. Together with these findings, the present study concluded that IMD reduces bone resorption by inhibiting osteoblast apoptosis, decreasing the RANKL/OPG ratio and the expression of M-CSF, and inhibiting osteoclast maturation and differentiation.

Dual Role of Cyanidin-3-glucoside on the Differentiation of Bone Cells

Cyanidin-3-glucoside (C3G) is one of the major components of anthocyanin, a water-soluble phytochemical. Recent studies demonstrated the chemopreventive and chemotherapeutic activities of C3G in various conditions, including cancer, although the precise effects of C3G on osteoclast and osteoblast differentiation remain unclear. Here, we investigated the role of C3G in the differentiation of bone-associated cells and its underlying mechanism. C3G inhibited receptor activator of nuclear factor kappa-B ligand (RANKL)–mediated osteoclast differentiation and formation in a dose-dependent manner and downregulated the expression of osteoclast differentiation marker genes. Pretreatment with C3G considerably reduced the induction of extracellular signal–regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated kinases activation by RANKL in osteoclast precursor cells. Furthermore, C3G dramatically inhibited the expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1, which are important transcription factors for osteoclast differentiation and activation. The formation of osteoclasts in coculture of bone marrow cells and calvaria-derived osteoblasts was also inhibited by C3G treatment, although the expression of macrophage colony-stimulating factor and RANKL (master factors for osteoclast differentiation and formation) and osteoprotegerin (a decoy receptor for RANKL) on osteoblasts was unaffected. The inhibitory effect of C3G on osteoclastogenesis is therefore targeted specifically to osteoclasts but not osteoblasts. Moreover, analysis of the expression levels of osteoblast differentiation marker genes and alizarin red staining showed that osteoblast differentiation and matrix formation increased after C3G treatment. Taken together, these results strongly suggest that C3G has a dual role in bone metabolism, as an effective inhibitor of osteoclast differentiation but also as an activator of osteoblast differentiation. Therefore, C3G may be used as a potent preventive or therapeutic agent for bone-related diseases, such as osteoporosis, rheumatoid arthritis, and periodontitis.

Glioblastoma-derived Macrophage Colony-stimulating Factor (MCSF) Induces Microglial Release of Insulin-like Growth Factor-binding Protein 1 (IGFBP1) to Promote Angiogenesis

Glioblastoma (grade IV glioma/GBM) is the most common primary adult malignant brain tumor with poor prognosis. To characterize molecular determinants of tumor-stroma interaction in GBM, we profiled 48 serum cytokines and identified macrophage colony-stimulating factor (MCSF) as one of the elevated cytokines in sera from GBM patients. Both MCSF transcript and protein were up-regulated in GBM tissue samples through a spleen tyrosine kinase (SYK)-dependent activation of the PI3K-NFκB pathway. Ectopic overexpression and silencing experiments revealed that glioma-secreted MCSF has no role in autocrine functions and M2 polarization of macrophages. In contrast, silencing expression of MCSF in glioma cells prevented tube formation of human umbilical vein endothelial cells elicited by the supernatant from monocytes/microglial cells treated with conditioned medium from glioma cells. Quantitative proteomics based on stable isotope labeling by amino acids in cell culture showed that glioma-derived MCSF induces changes in microglial secretome and identified insulin-like growth factor-binding protein 1 (IGFBP1) as one of the MCSF-regulated proteins secreted by microglia. Silencing IGFBP1 expression in microglial cells or its neutralization by an antibody reduced the ability of supernatants derived from microglial cells treated with glioma cell-conditioned medium to induce angiogenesis. In conclusion, this study shows up-regulation of MCSF in GBM via a SYK-PI3K-NFκB-dependent mechanism and identifies IGFBP1 released by microglial cells as a novel mediator of MCSF-induced angiogenesis, of potential interest for developing targeted therapy to prevent GBM progression.

MicroRNA-26a suppresses recruitment of macrophages by down-regulating macrophage colony-stimulating factor expression through the PI3K/Akt pathway in hepatocellular carcinoma.

BackgroundmicroRNAs (miRNAs) have been reported to modulate macrophage colony-stimulating factor (M-CSF) and macrophages. The aim of this study was to find whether miR-26a can suppress M-CSF expression and the recruitment of macrophages.MethodsHepatocellular carcinoma (HCC) cell lines with decreased or increased expression of miR-26a were established in a previous study. M-CSF expression by tumor cells was measured by enzyme-linked immunosorbent assay, and cell migration assays were used to explore the effect of HCC cell lines on macrophage recruitment in vitro. Real-time PCR measured a panel of mRNAs expressed by macrophages. Xenograft models were used to observe tumor growth. Immunohistochemistry was conducted to study the relation between miR-26a expression and M-CSF expression and macrophage recruitment in patients with HCC.ResultsEctopic expression of miR-26a reduced expression of M-CSF. The conditioned medium (CM) from HepG2 cells that overexpressed miR-26a reduced the migration ability of THP-1 cells stimulated by phorbol myristate acetate (PMA) increased expression of interleukin (IL)-12b or IL-23 mRNA and decreased expression of chemokine (C-C motif) ligand (CCL)22, CCL17, and IL-10 mRNA, in comparison to the medium from the parental HepG2 cells. These effects could be interrupted by the PI3K/Akt pathway inhibitor LY294002. Ectopic expression of miR-26a in HCC cells suppressed tumor growth, M-CSF expression, and infiltration of macrophages in tumors. Similar results were also found when using HCCLM3 cells. Furthermore, the expression of miR-26a was inversely correlated with M-CSF expression and macrophage infiltration in tumor tissues from patients with HCC.ConclusionsmiR-26a expression reduced M-CSF expression and recruitment of macrophages in HCC.

Multiple anti-inflammatory and anti-atherosclerotic properties of red wine polyphenolic extracts: differential role of hydroxycinnamic acids, flavonols and stilbenes on endothelial inflammatory gene expression.

The aim of the study was to evaluate the vascular anti-inflammatory effects of polyphenolic extracts from two typical South Italy red wines, the specific contribution of individual polyphenols and the underlying mechanisms of action. Human endothelial cells were incubated with increasing concentrations (1-50 μg/mL) of Primitivo and Negroamaro polyphenolic extracts (PWPE and NWPE, respectively) or pure polyphenols (1-25 μmol/L), including hydroxycinnamic acids (p-coumaric, caffeic and caftaric acids), flavonols (kaempferol, quercetin, myricetin) or stilbenes (trans-resveratrol, trans-piceid) before stimulation with lipopolysaccharide. Through multiple assays, we analyzed the endothelial-monocyte adhesion, the endothelial expression of adhesion molecules (ICAM-1, VCAM-1 and E-Selectin), monocyte chemoattractant protein-1 (MCP-1) and macrophage colony-stimulating factor (M-CSF), as well as ROS intracellular levels and the activation of NF-κB and AP-1. Both PWPE and NWPE, already at 1 μg/mL, inhibited monocyte adhesion to stimulated endothelial cells, a key event in triggering vascular inflammation. They down-regulated the expression of adhesion molecules, ICAM-1, VCAM-1, E-Selectin, as well as MCP-1 and M-CSF, at mRNA and protein levels. All polyphenols reduced intracellular ROS, and everything, except caftaric acid, inhibited the endothelial expression of adhesion molecules and MCP-1, although with different potency. Flavonols and resveratrol significantly reduced also the endothelial expression and release of M-CSF. The decrease in endothelial inflammatory gene expression was related to the inhibition of NF-κB and AP-1 activation but not to intracellular oxidative stress. This study showed multiple anti-inflammatory and anti-atherosclerotic properties of red wine polyphenolic extracts and indentified specific bioactive polyphenols which could counteract inflammatory diseases including atherosclerosis.

Maternal high-fat diet exaggerates atherosclerosis in adult offspring by augmenting periaortic adipose tissue-specific proinflammatory response.

Maternal obesity elicits offspring's metabolic disorders via developmental modifications of visceral adipose tissue; however, its effect on atherogenesis remains undefined. Perivascular adipose tissue has recently been implicated in vascular remodeling and vasoreactivity. We hypothesize that developmental modifications of perivascular adipose tissue by maternal high-fat diet (HFD) exposure promotes atherosclerosis in adult offspring. Eight-week-old female apolipoprotein E-deficient mice were fed an HFD or normal diet (ND) during gestation and lactation. Offspring were fed a high-cholesterol diet from 8 weeks of age. Twenty-week-old male offspring of HFD-fed dams (O-HFD) showed a 2.1-fold increase in atherosclerotic lesion of the entire aorta compared with those of ND-fed dams (O-ND). Although mRNA expressions of interleukin-6, tumor necrosis factor, and monocyte chemotactic protein-1 and accumulation of macrophages in epididymal white adipose tissue were less in O-HFD than in O-ND, thoracic periaortic adipose tissue (tPAT) showed an exaggerated inflammatory response in O-HFD. Intra-abdominal transplantation of tPAT from 8-week-old O-HFD alongside the distal abdominal aorta exaggerated atherosclerosis development of the infrarenal aorta in recipient apolipoprotein E-deficient mice compared with tPAT from O-ND (210%, P<0.01). Although macrophage accumulation was rarely detected in tPAT of 8-week-old offspring, mRNA expression and protein levels of macrophage colony-stimulating factor were markedly elevated in O-HFD (2.3-fold, 3.3-fold, respectively, P<0.05), suggesting that increased macrophage colony-stimulating factor expression contributes to the augmented accumulation of macrophages, followed by the enhanced proinflammatory response. Our findings demonstrate that maternal HFD exaggerates atherosclerosis development in offspring by augmenting tPAT-specific inflammatory response proceeded by an increased expression of macrophage colony-stimulating factor.

TOA02, a recombinant adenovirus with tumor-specific granulocyte macrophage colony-stimulating factor expression, has limited biodistribution and low toxicity in rhesus monkeys.

TOA02 is a genetically modified oncolytic adenovirus that contains human granulocyte macrophage colony-stimulating factor (hGM-CSF). It has been verified in vitro that TOA02 can specifically replicate in tumor cells that possess high telomerase reverse transcriptase activity and Rb pathway deficiency. However, the replication specificity, hGM-CSF expression, and toxicity of TOA02 in vivo are still unknown. Therefore, the biosafety of TOA02 remains a critical issue before its potential clinical use. In this study, viral replication and hGM-CSF expression levels were investigated in both xenograft nude mouse models and rhesus monkeys, and chronic toxicity was evaluated in rhesus monkeys. Our results show that (1) the replication and hGM-CSF expression of TOA02 are high in tumor model, (2) there are no hGM-CSF expression and continuous viral replication in rhesus monkeys except in pancreas and epididymis, and (3) the antiadenovirus antibody was positive in the chronic toxicity experiment, but pathological change of blood cytology and blood biochemistry were not found. There were no other histopathology lesions apart from skin inflammation of the administration region, lymphadenitis of draining lymph nodes. Our findings suggest that TOA02 is relatively safe for in vivo application, thus laying the foundation for future clinical trials with TOA02.

The Difference between Growth Factor Expression after Single and Multiple Fractures: Preliminary Results in Human Fracture Healing.

Objectives. Circulating levels of VEGF-A (Vascular Endothelia Growth Factor-A), TGF-β1 (Transforming Growth Factor-beta 1), and M-CSF (Macrophage-Colony Stimulating Factor) were found to be predictors of bone healing and therefore prognostic criteria of delayed bone healing or nonunion. The aim of this study was to evaluate a potential rise of these markers in patients with multiple fractures of long bones compared to patients with single fractured long bone. Methods. 92 patients were included in the study and finally after excluding all female patients 45 male patients were left for final analysis and divided into the single or multiple fracture group. TGF-β1, M-CSF, and VEGF-A serum levels were analysed over a time period of two weeks. Results. MCSF serum concentrations were higher in the group with multiple fractures as also TGF-β1 serum concentrations were at one and two weeks after trauma. No statistically significant difference was observed in the VEGF-A serum concentrations of both groups at either measurement point. Conclusion. We did observe a correlation between the quantity of the M-CSF and TGF-β1 expressions in serum and the number of fractured bones; surprisingly there was no statistically significant difference in the serum levels between patients with single and multiple fractures of long bones.

TLR4-dependent immune response promotes radiation-induced liver disease by changing the liver tissue interstitial microenvironment during liver cancer radiotherapy.

Liver tissue interstitial fluid (TIF) a special microenvironment around liver cells, which may play a vital role in cell communication during liver injury. Moreover, toll-like receptor 4 (TLR4) is an important trigger of the immune response that may also play a role in liver injuries, including radiation-induced liver disease (RILD). Therefore, the purpose of this study was to identify the roles of the TLR4-dependent immune response and TIFs in RILD after radiation therapy (RT) for liver cancer. This study consisted of two phases, and in the primary phase, the livers of TLR4 mutant (TLR4(-)) and normal (TLR4(+)) mice were irradiated with 30 Gy. TIF was then obtained from mouse livers and assessed by cytokine array analysis 20 days after irradiation, and cytokines in the TIFs, TLR4 and RILD were analyzed. In the second or validation phase, hepatocytes were isolated from TLR4(+) or TLR4(-) mice irradiated with 8 Gy and were co-cultured with TIFs from mouse livers, apoptosis of the hepatocytes was then measured using flow cytometry. We found that severe RILD was accompanied by higher expression of granulocyte macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-related apoptosis inducing ligand (TRAIL) and vascular endothelial growth factor receptor 2(VEGFR-2) in liver TIFs, from in TLR4(+) mice compared with TLR4(-) mice (P < 0.05). In both TLR4(+) and TLR4(-) hepatocytes, apoptosis after irradiaton was increased significantly after co-culture in TIFs from TLR4(+) mice that had their livers irradiated, compared with TIFs from TLR4(-) mice that had their livers irradiated or TIFs from unirradiated mice (P < 0.05). In summary, these findings indicate that the TLR4-dependent immune response may promote RILD by enhancing the expression of GM-CSF, VEGFR-2 and TRAIL in liver TIFs.

Oxyresveratrol suppresses lipopolysaccharide-induced inflammatory responses in murine macrophages.

Excessive inflammation is considered a critical factor in many human diseases. Oxyresveratrol(trans-2,3',4,5'-tetrahydroxystilbene), a natural hydroxystilbene, has been shown to possess antioxidant and free radical-scavenging activity. In this study, we investigated the effects of oxyresveratrol (OxyR) on the lipopolysaccharide (LPS)-induced production of inflammatory cytokines and mediators and further explored the mechanism of action in RAW264.7 murine macrophage cell line. Production of nitric oxide (NO), prostaglandin E2 (PGE2), messenger RNA (mRNA) and protein expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin 6 (IL-6), and granulocyte macrophage colony-stimulating factor (GM-CSF), phosphorylation of mitogen-activated protein kinases (MAPKs; extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38), and the activation of nuclear factor κ-light chain enhancer of activated B cells (NFκB) with OxyR were assayed in LPS-stimulated RAW264.7 cells. OxyR inhibited the productions of NO, PGE2, IL-6, and GM-CSF significantly in LPS-stimulated RAW264.7 cells. OxyR suppressed mRNA and protein expressions of iNOS, COX-2, IL-6, and GM-CSF in LPS-stimulated RAW264.7 cells. OxyR suppressed the phosphorylation of Akt and JNK and p38 MAPKs and the translocation of NFκB p65 subunit into the nucleus. These results indicate that OxyR inhibits LPS-stimulated inflammatory responses though the blocking of MAPK and NFκB signaling pathway in macrophages, and suggest that OxyR possesses anti-inflammatory effects.

Semiquinone glucoside derivative provides protection against γ-radiation by modulation of immune response in murine model.

Present study was undertaken to evaluate radioprotective and immunomodulatory activities of a novel semiquinone glucoside derivative (SQGD) isolated from Bacillus sp. INM-1 in C57 BL/6 mice. Whole body survival study was performed to evaluate in vivo radioprotective efficacy of SQGD. To observe effect of SQGD on immunostimulation, Circulatory cytokine (i.e., interleukin-2 (IL-2), IFN-γ, IL-10, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), and macrophage colony stimulating factor (M-CSF) expression was analyzed in serum of irradiated and SQGD treated mice at different time intervals using ELISA assay. Results of the present investigation indicated that SQGD pre-treatment (-2 h) to lethally irradiated mice provide ∼ 83% whole body survival compared with irradiated mice where no survival was observed at 30(th) post irradiation day. Significant (p < 0.05) induction in IL-2 and IFN-γ expression was observed at all tested time intervals with SQGD pre-treated irradiated mice as compared with irradiated mice alone. However, sharp increase in IL-10 expression was observed in irradiated mice which were found to be subsidized in irradiated mice pre-treated with SQGD. Similarly, significant (p < 0.05%) induction in G-CSF, M-CSF and GM-CSF expression was observed in irradiated mice treated with SQGD as compared with irradiated control mice at tested time intervals. In conclusion, SQGD pre-treatment to irradiated mice enhanced expression of IL-12 and IFN-γ while down-regulated IL-10 expression and thus modulates cytoprotective pro-inflammatory TH1 type immune response in irradiated mice. Further, SQGD pre-treatment to irradiated mice accelerate G-CSF, GM-CSF and M-CSF expression suggesting improved haematopoiesis and enhanced cellular immune response in immuno-compromised irradiated mice that may contribute to in vivo radiation protection.

IL-34 and macrophage colony-stimulating factor are overexpressed in hepatitis C virus fibrosis and induce profibrotic macrophages that promote collagen synthesis by hepatic stellate cells.

Chronic hepatitis C virus (HCV) infection is characterized by progressive hepatic fibrosis, a process dependent on monocyte recruitment and accumulation into the liver. The mediators expressed in chronically injured liver that control the differentiation of human monocytes into profibrotic macrophages (Mφ) remain poorly defined. We report that chronically HCV-infected patients with high fibrosis stages have higher serum levels of macrophage colony-stimulating factor (M-CSF) and interleukin (IL)-34 than HCV-infected patients with lower fibrosis stages and healthy subjects. Immunohistochemistry reveals an intense expression of IL-34 and M-CSF by hepatocytes around liver lesions. In addition, HCV infection and inflammatory cytokines enhance the in vitro production of IL-34 and M-CSF by hepatocytes. We next analyzed the acquisition of profibrotic properties by Mφ generated with M-CSF (M-CSF-Mφ) or IL-34 (IL-34-Mφ). M-CSF and IL-34 up-regulate the expression, by differentiating monocytes, of chemokine (C-C motif) ligand (CCL)2, CCL4, C-C chemokine receptor (CCR)1, and CCR5, which are involved in monocyte recruitment/Mφ accumulation in liver lesions. M-CSF-Mφ and IL-34-Mφ also express the hepatic stellate cell (HSC) activators, platelet-derived growth factor, transforming growth factor beta, and galectin-3. IL-34-Mφ and M-CSF-Mφ induce type I collagen synthesis by HSCs, the main collagen-producing cells in liver fibrosis. IL-13, whose expression correlates with the fibrosis stage in HCV-infected patients, decreases the expression of the collagenase, matrix metalloproteinase 1, by IL-34-Mφ and M-CSF-Mφ, thereby enhancing collagen synthesis. By inhibiting the production of interferon-gamma (IFN-γ) by activated natural killer cells, IL-34-Mφ and M-CSF-Mφ prevent the IFN-γ-induced killing of HSCs. These results identify M-CSF and IL-34 as potent profibrotic factors in HCV liver fibrosis.

Increased expression of M-CSF and IL-13 in vitreous of patients with proliferative diabetic retinopathy: implications for M2 macrophage-involving fibrovascular membrane formation

PurposeWe recently demonstrated that M2 macrophages were involved in the development of fibrovascular membranes (FVM) associated with proliferative diabetic retinopathy (PDR) possibly through the induction of periostin. The purpose of...

Abstract 1172: Targeting c-Met and VEGFR2 in the stromal compartment of prostate cancer bone metastasis

Abstract Advanced-stage prostate cancer patients commonly develop bone metastasis, accounting for significant morbidity and mortality. Metastatic prostate cancer cells characteristically induce osteoblastic reactions, for which no targeted therapies are currently available. Recent clinical and experimental data showed that dual inhibition of c-Met and VEGF receptor (VEGFR)-2 kinase activity reduced prostate cancer growth in bone with indications for suppressing osteoblastic activities, while the specific functions of c-Met and VEGFR2 in osteoblasts remain unclear. In our in vitro studies using three types of osteoblasts (MC3T3-E1 subclone 4, hFOB1.19 and murine calvarial osteoblasts), hepatocyte growth factor (HGF, a ligand for c-Met) and VEGFA increased expression of receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony stimulating factor (M-CSF), two essential factors for osteoclastogenesis. In addition, siRNA-mediated knockdown of c-Met or VEGFR2 in osteoblasts suppressed HGF- or VEGFA-dependent gene expression, respectively. We demonstrate that insulin growth factor (IGF) increased RANKL and M-CSF expression in osteoblasts via c-Met transactivation. Furthermore, the conditioned media from IGF-, HGF-, or VEGFA-treated osteoblasts promoted in vitro osteoclastogenesis that was suppressed by inhibition of c-Met and VEGFR2 in osteoblasts. Subsequently, we examined whether cabozantinib, a dual kinase inhibitor of c-Met and VEGFR2 currently in clinical trials for metastatic prostate cancer, could suppress prostate cancer bone metastasis via inhibition of osteoblasts. To examine the effects of cabozantinib specifically in osteoblasts, cabozantinib-resistant PC-3 prostate cancer cells were generated and implanted in the tibiae of male nude mice. Cabozantinib (60mg/kg) or control diluent was administered via daily oral gavage for 3 weeks. In agreement with our in vitro data, cabozantinib suppressed tumor growth in bone (determined by in vivo bioluminescence) and tumor-induced osteolysis (determined by X-ray). Tumor cells were isolated and in vitro cultured to demonstrate that cabozantinib resistance was maintained during 3-week in vivo growth. Osteoblasts and osteoclasts numbers were reduced in the bones of cabozantinib-treated mice, and these histological changes were accompanied by significantly lower levels of RANKL and M-CSF levels in bone. Collectively, inhibition of c-Met and VEGFR2 in osteoblasts reduces RANKL and M-CSF expression, associated with decreased osteoclastogenesis and tumor-induced osteolysis. Therefore, we conclude that c-Met and VEGFR2 are promising therapeutic targets in the stromal compartment of prostate cancer bone metastasis, suggesting hat the effects of cabozantinib on skeletal-related events of prostate cancer are, at least in part, mediated by suppression of osteoblast function. Citation Format: Changki Lee, Preston Campbell, Young Mi Whang, Jamey D. Young, Florent Elefteriou, Serk In Park. Targeting c-Met and VEGFR2 in the stromal compartment of prostate cancer bone metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1172. doi:10.1158/1538-7445.AM2014-1172

Macrophage density and macrophage colony-stimulating factor expression predict the postoperative prognosis in patients with intrahepatic cholangiocarcinoma.

Macrophages are included in the stromal compartments in various neoplasms, and their behavior against tumors is diverse. The aim of this study was to examine the role of tumor-infiltrating macrophages and their main regulator, macrophage colony-stimulating factor (M-CSF), in intrahepatic cholangiocarcinoma (ICC). Macrophage density and M-CSF expression in 39 resected ICC specimens were immunohistochemically evaluated in the central and peripheral areas of tumors, which were defined as fields more than and within 500 μm from the invasive front, respectively. The number of CD68-positive macrophages was counted using an image-analyzing software program. The relationship between these results and other clinicopathological factors and the postoperative prognosis were evaluated. Sporadic M-CSF expression in cancer cells around the peripheral area was observed in fourteen patients. M-CSF-positive ICCs showed a higher macrophage density in the tumor-peripheral area than did M-CSF-negative ICCs. M-CSF expression and higher macrophage density in the tumor-peripheral area were related to a better postoperative prognosis, whereas a higher macrophage density in the central area was one of the significant risk factors for a poor prognosis in a univariate analysis. Tumor-peripheral macrophage infiltration, presumably dependent on M-CSF, and M-CSF-independent tumor-central macrophage infiltration are predictive factors for better and worse postoperative prognosis in ICC patients, respectively. The tumor microenvironment, such as the presence of hypoxia, may affect the behavior of infiltrating macrophages in ICC.

Rho-kinase regulation of TNF-α-induced nuclear translocation of NF-κB RelA/p65 and M-CSF expression via p38 MAPK in mesangial cells.

The small GTPase Rho and its downstream effector, Rho-associated coiled-coil containing protein kinase (Rho-kinase), regulate a number of cellular processes, including organization of the actin cytoskeleton, cell adhesion, and migration. While pharmacological inhibitors of Rho-kinase signaling are known to block renal inflammation, the molecular basis for this effect is unclear. Here, we provide evidence that proinflammatory TNF-α promotes mesangial expression of macrophage colony-stimulating factor (M-CSF), a key regulator for the growth and differentiation of mononuclear phagocytes, in a Rho-kinase-dependent manner. Consistent with this observation, TNF-α-mediated renal expression of M-CSF in insulin-resistant db/db mice was downregulated by Rho-kinase inhibition. Small interfering RNA-facilitated knockdown of Rho-kinase isoforms ROCK1 and ROCK2 indicated that both isoforms make comparable contributions to regulation of M-CSF expression in mesangial cells. From a mechanistic standpoint, Western blotting and EMSA showed that Rho-kinase and its downstream target p38 MAPK regulate nuclear translocation of NF-κB RelA/p65 and subsequent DNA binding activity, with no significant effects on IκBα degradation and RelA/p65 phosphorylation. Moreover, we showed that Rho-kinase-mediated cytoskeletal organization is required for the nuclear uptake of RelA/p65. Collectively, these findings identify Rho-kinase as a critical regulator of chemokine expression and macrophage proliferation.

M1/M2 macrophages and associated mechanisms in congenital bicuspid aortic valve stenosis

The aim of this study was to observe macrophage infiltration in congenital bicuspid aortic valve (CBAV) stenosis. M1/M2 macrophage distribution, inflammatory cytokine expression and the role of M1 macrophages during CBAV stenosis were also explored. The experimental and control groups comprised 30 severely stenotic CBAVs and 30 severely stenotic tricuspid aortic valves (TAVs), respectively. Histological and morphological changes were assessed using hematoxylin-eosin (HE) staining and mRNA levels of vascular endothelial growth factor (VEGF) were examined using the quantitative polymerase chain reaction. Nonspecific, M1 and M2 macrophages were monitored using cluster of differentiation (CD)68, inducible nitric oxide synthase (iNOS) and CD163 staining, respectively. Endothelial nitric oxide synthase (eNOS), interleukin (IL)-10, arginase (Arg)-1 and macrophage colony-stimulating factor (M-CSF) were also examined using immunohistochemical staining. Of note, HE staining revealed a higher cell density and neovascularization was more common in CBAVs than TAVs. At the mRNA level, VEGF expression was two-fold higher in CBAVs relative to that in TAVs (P=0.02). Furthermore, CD68 and iNOS were significantly higher in CBAVs compared with TAVs (P=0.029 and 0.021, respectively), while CD163 expression was lower in CBAVs (P=0.033). In addition, eNOS expression was higher and Arg-1, IL-10 and M-CSF expression were lower in CBAVs compared with TAVs (all P<0.0001). The present study suggested that CBAVs are associated with a higher total and M1 macrophage density and a lower M2 macrophage density than TAVs, and that M1 macrophage infiltration may contribute to calcification of CBAVs.

Key Role of MicroRNA in the Regulation of Granulocyte Macrophage Colony-stimulating Factor Expression in Murine Alveolar Epithelial Cells during Oxidative Stress

GM-CSF is an endogenous pulmonary cytokine produced by normal alveolar epithelial cells (AEC) that is a key defender of the alveolar space. AEC GM-CSF expression is suppressed by oxidative stress through alternations in mRNA turnover, an effect that is reversed by treatment with recombinant GM-CSF. We hypothesized that specific microRNA (miRNA) would play a key role in AEC GM-CSF regulation. A genome-wide miRNA microarray identified 19 candidate miRNA altered in primary AEC during oxidative stress with reversal by treatment with GM-CSF. Three of these miRNA (miR 133a, miR 133a*, and miR 133b) are also predicted to bind the GM-CSF 3'-untranslated region (UTR). PCR for the mature miRNA confirmed induction during oxidative stress that was reversed by treatment with GM-CSF. Experiments using a GM-CSF 3'-UTR reporter construct demonstrated that miR133a and miR133b effects on GM-CSF expression are through interactions with the GM-CSF 3'-UTR. Using lentiviral transduction of specific mimics and inhibitors in primary murine AEC, we determined that miR133a and miR133b suppress GM-CSF expression and that their inhibition both reverses oxidant-induced suppression of GM-CSF expression and increases basal expression of GM-CSF in cells in normoxia. In contrast, these miRNAs are not active in regulation of GM-CSF expression in murine EL4 T cells. Thus, members of the miR133 family play key roles in regulation of GM-CSF expression through effects on mRNA turnover in AEC during oxidative stress. Increased understanding of GM-CSF gene regulation may provide novel miRNA-based interventions to augment pulmonary innate immune defense in lung injury.