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Reports demonstrate the role of M-CSF (CSF1) in tumor progression in mouse models as well as the prognostic value of macrophage numbers in breast cancer patients. Recently, a subset of CD14+ monocytes expressing the Tie2 receptor, once thought to be predominantly expressed on endothelial cells, has been characterized. We hypothesized that increased levels of CSF1 in breast tumors can regulate differentiation of Tie2- monocytes to a Tie2+ phenotype. We treated CD14+ human monocytes with CSF1 and found a significant increase in CD14+/Tie2+ positivity. To understand if CSF1-induced Tie2 expression on these cells improved their migratory ability, we pre-treated CD14+ monocytes with CSF1 and used Boyden chemotaxis chambers to observe enhanced response to angiopoietin-2 (ANG2), the chemotactic ligand for the Tie2 receptor. We found that CSF1 pre-treatment significantly augmented chemotaxis and that Tie2 receptor upregulation was responsible as siRNA targeting Tie2 receptor abrogated this effect. To understand any augmented angiogenic effect produced by treating these cells with CSF1, we cultured human umbilical vein endothelial cells (HUVECs) with conditioned supernatants from CSF1-pre-treated CD14+ monocytes for a tube formation assay. While supernatants from CSF1-pre-treated TEMs increased HUVEC branching, a neutralizing antibody against the CSF1R abrogated this activity, as did siRNA against the Tie2 receptor. To test our hypothesis in vivo, we treated PyMT tumor-bearing mice with CSF1 and observed an expansion in the TEM population relative to total F4/80+ cells, which resulted in increased angiogenesis. Investigation into the mechanism of Tie2 receptor upregulation on CD14+ monocytes by CSF1 revealed a synergistic contribution from the PI3 kinase and HIF pathways as the PI3 kinase inhibitor LY294002, as well as HIF-1α-deficient macrophages differentiated from the bone marrow of HIF-1αfl/fl/LysMcre mice, diminished CSF1-stimulated Tie2 receptor expression.

A variable radio frequency proton-electron double-resonance imaging (VRF PEDRI) approach for pH mapping of aqueous samples has been recently developed (Efimova et al. J. Magn. Reson. 2011, 209, 227-232). A pH map is extracted from two PEDRI acquisitions performed at electron paramagnetic resonance (EPR) frequencies of protonated and unprotonated forms of a pH-sensitive probe. To translate VRF PEDRI to an in vivo setting, an advanced pH probe was synthesized. Probe deuteration resulted in a narrow spectral line of 1.2 G compared to a nondeuterated analogue line width of 2.1 G allowing for an increase of Overhauser enhancements and reduction in rf power deposition. Binding of the probe to the cell-impermeable tripeptide, glutathione (GSH), allows for targeting to extracellular tissue space for monitoring extracellular tumor acidosis, a prognostic factor in tumor pathophysiology. The probe demonstrated pH sensitivity in the 5.8-7.8 range, optimum for measurement of acidic extracellular tumor pH (pH(e)). In vivo VRF PEDRI was performed on Met-1 tumor-bearing mice. Compared to normal mammary glands with a neutral mean pH(e) (7.1 ± 0.1), we observed broader pH distribution with acidic mean pH(e) (6.8 ± 0.1) in tumor tissue. In summary, VRF PEDRI in combination with a newly developed pH probe provides an analytical approach for spatially resolved noninvasive pHe monitoring, in vivo.

In response to elevated glucocorticoid levels, erythroid progenitors rapidly expand to produce large numbers of young erythrocytes. Previous work demonstrates hematopoietic changes in rodents exposed to various physical and psychological stressors, however, the effects of chronic psychological stress on erythropoiesis has not be delineated. We employed laboratory, clinical and genomic analyses of a murine model of chronic restraint stress (RST) to examine the influence of psychological stress on erythropoiesis. Mice exposed to RST demonstrated markers of early erythroid expansion involving the glucocorticoid receptor. In addition, these RST-exposed mice had increased numbers of circulating reticulocytes and increased erythropoiesis in primary and secondary erythroid tissues. Mice also showed increases in erythroid progenitor populations and elevated expression of the erythroid transcription factor KLF1 in these cells. Together this work reports some of the first evidence of psychological stress affecting erythroid homeostasis through glucocorticoid stimulation.

Monocytes/macrophages are potent mediators of antitumor antibody therapy, where they engage target cells via Fcγ receptors (FcγR). Binding of these cells to opsonized tumor targets elicits cytokine production, phagocytosis, and antibody-mediated cellular cytotoxicity. Here we show for the first time that activation of monocyte FcγR results in the secretion of soluble vascular endothelial growth factor receptor-1 (VEGFR-1/sFlt-1), which serves to antagonize VEGF-mediated angiogenesis and tumor growth. Consistent with this, using a murine solid tumor model of antibody therapy, we show that sFlt-1 is involved in restricting tumor growth. Analyzing the mechanism of induction of sFlt-1, we found that the Erk and PI3K pathways were required for transcription, and NF-κB was required for translation. Upon closer examination of the role of NF-κB, we found that a microRNA, miR181a, negatively regulates FcγR-mediated sFlt-1 production and that NF-κB serves to antagonize this microRNA. Taken together, these results demonstrate a novel and biologically important function of monocytes and macrophages during antibody therapy.

Stressor exposure has been shown to enhance host susceptibility and the severity of a plethora of illnesses, including gastrointestinal disease. In mice, susceptibility to Citrobacter rodentium has been shown to be dependent on host genetics as well as the composition of the intestinal microbiota, but the effects of stressor exposure on this gastrointestinal pathogen have not been elucidated fully. Previously, our lab showed that exposure to the prolonged-restraint stressor prior to a challenge with C. rodentium alters the intestinal microbiota community structure, including a reduction of beneficial genera such as Lactobacillus, which may contribute to stressor-enhanced C. rodentium-induced infectious colitis. To test the effects of stressor exposure on C. rodentium infection, we exposed resistant mice to a prolonged-restraint stressor concurrent with pathogen challenge. Exposure to prolonged restraint significantly enhanced C. rodentium-induced infectious colitis in resistant mice, as measured by increases in colonic histopathology, colonic inflammatory mediator gene production, and pathogen translocation from the colon to the spleen. It was further tested if the beneficial bacterium Lactobacillus reuteri could reduce the stressor-enhanced susceptibility to C. rodentium-enhanced infectious colitis. While L. reuteri treatment did not reduce all aspects of stressor-enhanced infectious colitis, it did significantly reduce pathogen translocation from the colon to the spleen. Taken together, these data demonstrate the deleterious effects that prolonged stressor exposure can have at the onset of a gastrointestinal infection by its ability to render a resistant mouse highly susceptible to C. rodentium. Probiotic treatment ameliorated the systemic manifestations of stress on colonic infection.

Aim: Peri-implant gingival healing following one-stage implant placement was investigated and compared to periodontal healing., Methods: Healing at surgical sites [implant (I) and adjacent teeth (T+)] was compared to non-operated tooth (T-) in non-smokers receiving one-stage implant. Periodontal Indices (PI, GI) were recorded at surgery and up to 12 weeks post-operatively. Peri-implant (PICF) and gingival crevicular fluids (GCF) were analysed for cytokines, collagenases and inhibitors. Data were analysed by linear mixed model regression analysis and repeated measures anova., Results: Forty patients (22 females; 21-74 years old) completed the study. Surgical site GI, increased at week 1, decreased significantly during early healing (weeks 1-3; p = 0.0003) and continually decreased during late healing (weeks 6-12) for I (p < 0.01). PICF volume decreased threefold by week 12 (p = 0.0003). IL-6, IL-8, MIP-1β and TIMP-1 levels significantly increased at surgical sites at week one, significantly decreasing thereafter (p < 0.016). Week one IL-6, IL-8 and MIP-1β levels were ~threefold higher and TIMP-1 levels 63% higher, at I compared to T+ (p = 0.001)., Conclusion: Peri-implant gingival healing, as determined by crevicular fluid molecular composition, differs from periodontal healing. The observed differences suggest that peri-implant tissues, compared to periodontal tissues, represent a higher pro-inflammatory state., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Pancreatic stellate cells (PSC) are a subset of pancreatic cancer-associated fibroblasts. These cells provide prosurvival signals to tumors; however, little is known regarding their interactions with immune cells within the tumor microenvironment. We hypothesized that factors produced by human PSC could enhance myeloid-derived suppressor cell (MDSC) differentiation and function, which promotes an immunosuppressive microenvironment. Primary PSC cell lines (n = 7) were generated from human specimens and phenotypically confirmed via expression of vimentin, α-smooth muscle actin (α-SMA), and glial fibrillary acidic protein (GFAP). Luminex analysis indicated that PSC but not human fetal primary pancreatic fibroblast cells (HPF; negative controls) produced MDSC-promoting cytokines [interleukin (IL-6), VEGF, macrophage colony-stimulating factor (M-CSF) ] and chemokines (SDF-1, MCP-1). Culture of peripheral blood mononuclear cells [peripheral blood mononuclear cell (PBMC), n = 3 donors] with PSC supernatants or IL-6/granulocyte macrophage colony-stimulating factor (GM-CSF; positive control) for 7 days promoted PBMC differentiation into an MDSC (CD11b+CD33+) phenotype and a subpopulation of polymorphonuclear CD11b+CD33+CD15+ cells. The resulting CD11b+CD33+ cells functionally suppressed autologous T-lymphocyte proliferation. In contrast, supernatants from HPF did not induce an MDSC phenotype in PBMCs. Culture of normal PBMCs with PSC supernatants led to STAT3 but not STAT1 or STAT5 phosphorylation. IL-6 was an important mediator as its neutralization inhibited PSC supernatant-mediated STAT3 phosphorylation and MDSC differentiation. Finally, the FLLL32 STAT3 inhibitor abrogated PSC supernatant-mediated MDSC differentiation, PSC viability, and reduced autocrine IL-6 production indicating these processes are STAT3 dependent. These results identify a novel role for PSC in driving immune escape in pancreatic cancer and extend the evidence that STAT3 acts as a driver of stromal immunosuppression to enhance its interest as a therapeutic target., (©2013 AACR.)

Previously we proposed the concept of dual function pH and oxygen paramagnetic probes based on the incorporation of ionizable groups into the structure of persistent triarylmethyl radicals, TAMs (J. Am. Chem. Soc.2007, 129, 7240-7241). In this paper, we synthesized an asymmetric monophosphonated TAM probe with the simplest doublet hfs pattern ideally suited for dual function electron paramagnetic resonance (EPR)-based applications. An extraordinary low line width of the synthesized deuterated derivative, p1TAM-D (ΔHpp ≤ 50 mG, Lorentz line width, ≤20 mG) results in high sensitivity to pO2 due to oxygen-induced line broadening (ΔLW/ΔpO2 ≈ 0.5 mG/mmHg or ≈400 mG/mM); accuracy of pO2 measurement, ≈1 mmHg). The presence of a phosphono group in the p1TAM-D structure provides pH sensitivity to its EPR spectra in the physiological range of pH from 5.9 to 8.2 with the ratio of signal intensities of protonated and deprotonated states being a reliable pH marker (accuracy of pH measurements, ± 0.05). The independent character of pH and [O2] effects on the EPR spectra of p1TAM-D provides dual functionality to this probe. The L-band EPR studies performed in breast tumor-bearing mice show a significant difference in extracellular pH and pO2 between tumor and normal mammary gland tissues, as well as the effect of animal breathing with 100% O2 on tissue oxygenation. The developed dual function phosphonated p1TAM-D probe provides a unique tool for in vivo concurrent tissue oxygen and pH monitoring.

Microvesicles are small membrane-bound particles comprised of exosomes and various-sized extracellular vesicles. These are released by several cell types. Microvesicles have a variety of cellular functions from communication to mediating growth and differentiation. Microvesicles contain proteins and nucleic acids. Previously, we showed that plasma microvesicles contain microRNAs (miRNAs). Based on our previous report, the majority of peripheral blood microvesicles are derived from platelets, while mononuclear phagocytes, including macrophages, are the second most abundant population. Here, we characterized macrophage-derived microvesicles and explored their role in the differentiation of naive monocytes. We also identified the miRNA content of the macrophage-derived microvesicles. We found that RNA molecules contained in the macrophage-derived microvesicles were transported to target cells, including mono cytes, endothelial cells, epithelial cells, and fibroblasts. Furthermore, we found that miR-223 was transported to target cells and was functionally active. Based on our observations, we hypothesize that microvesicles bind to and activate target cells. Furthermore, we find that microvesicles induce the differentiation of macrophages. Thus, defining key components of this response may identify novel targets to regulate host defense and inflammation.

Studies suggest that psychosocial factors can impact cancer progression. Parallel work in the fields of psychoneuroimmunology and developmental neuroscience have led to the implication of catecholamine hormones (norepinephrine and epinephrine) and their receptors (the β-adrenergic receptors; β-ARs) in regulating cancer progression. In this review we discuss studies that describe the effects of psychological stress as mediated by factors including the catecholamines norepinephrine and epinephrine on various aspects of tumor progression including proliferation, angiogenesis, and metastasis. We discuss the role of β-ARs in facilitating these effects and the potential use of β-blockers in adjuvant cancer therapy.

Altered inflammatory cytokine profiles are often observed in individuals suffering from major depression. Recent clinical work reports on elevated IL-6 and decreased IL-10 in depression. Elevated IL-6 has served as a consistent biomarker of depression and IL-10 is proposed to influence depressive behavior through its ability to counterbalance pro-inflammatory cytokine expression. Clinical and animal studies suggest a role for IL-10 in modifying depressive behavior. Murine restraint stress (RST) is regularly employed in the study of behavioral and biological symptoms associated with depressive disorders. While responses to acute RST exposure have been widely characterized, few studies have examined the ongoing and longitudinal effects of extended RST and fewer still have examined the lasting impact during the post-stress period. Consistent with clinical data, we report that a protocol of prolonged murine RST produced altered cytokine profiles similar to those observed in major depressive disorder. Parallel to these changes in circulating cytokines, IL-10 mRNA expression was diminished in the cortex and hippocampus throughout the stress period and following cessation of RST. Moreover, chronic RST promoted depressive-like behavior throughout the 28-day stress period and these depressive-like complications were maintained weeks after cessation of RST. Because of the correlation between IL-10 suppression and depressive behavior and because many successful antidepressant therapies yield increases in IL-10, we examined the effects of IL-10 treatment on RST-induced behavioral changes. Behavioral deficits induced by RST were reversed by exogenous administration of recombinant IL-10. This work provides one of the first reports describing the biological and behavioral impact following prolonged RST and, taken together, this study provides details on the correlation between responses to chronic RST and those seen in depressive disorders.

Under hypoxia, tumor cells, and tumor-associated macrophages produce VEGF (vascular endothelial growth factor), a signaling molecule that induces angiogenesis. The same macrophages, when treated with GM-CSF (granulocyte/macrophage colony-stimulating factor), produce sVEGFR-1 (soluble VEGF receptor-1), a soluble protein that binds with VEGF and inactivates its function. The production of VEGF by macrophages is regulated by HIF-1α (hypoxia inducible factor-1α), and the production of sVEGFR-1 is mediated by HIF-2α. Recent experiments measured the effect of inhibiting tumor growth by GM-CSF treatment in mice with HIF-1α-deficient or HIF-2α-deficient macrophages. In the present paper, we represent these experiments by a mathematical model based on a system of partial differential equations. We show that the model simulations agree with the above experiments. The model can then be used to suggest strategies for inhibiting tumor growth. For example, the model qualitatively predicts the extent to which GM-CSF treatment in combination with a small molecule inhibitor that stabilizes HIF-2α will reduce tumor volume and angiogenesis.

Macrophage secretion of vascular endothelial growth factor (VEGF) in response to hypoxia contributes to tumor growth and angiogenesis. In addition to VEGF, hypoxic macrophages stimulated with GM-CSF secrete high levels of a soluble form of the VEGF receptor (sVEGFR-1), which neutralizes VEGF and inhibits its biological activity. Using mice with a monocyte/macrophage-selective deletion of hypoxia-inducible factor (HIF)-1α or HIF-2α, we recently demonstrated that the antitumor response to GM-CSF was dependent on HIF-2α-driven sVEGFR-1 production by tumor-associated macrophages, whereas HIF-1α specifically regulated VEGF production. We therefore hypothesized that chemical stabilization of HIF-2α using an inhibitor of prolyl hydroxylase domain 3 (an upstream inhibitor of HIF-2α activation) would increase sVEGFR-1 production from GM-CSF-stimulated macrophages. Treatment of macrophages with the prolyl hydroxylase domain 3 inhibitor AKB-6899 stabilized HIF-2α and increased sVEGFR-1 production from GM-CSF-treated macrophages, with no effect on HIF-1α accumulation or VEGF production. Treatment of B16F10 melanoma-bearing mice with GM-CSF and AKB-6899 significantly reduced tumor growth compared with either drug alone. Increased levels of sVEGFR-1 mRNA, but not VEGF mRNA, were detected within the tumors of GM-CSF- and AKB-6899-treated mice, correlating with decreased tumor vascularity. Finally, the antitumor and antiangiogenic effects of AKB-6899 were abrogated when mice were simultaneously treated with a sVEGFR-1 neutralizing Ab. These results demonstrate that AKB-6899 decreases tumor growth and angiogenesis in response to GM-CSF by increasing sVEGFR-1 production from tumor-associated macrophages. Specific activation of HIF-2α can therefore decrease tumor growth and angiogenesis.

M-CSF is overexpressed in breast cancer and is known to stimulate macrophages to produce VEGF resulting in angiogenesis. It has recently been shown that the growth factor GM-CSF injected into murine breast tumors slowed tumor growth by secreting soluble VEGF receptor-1 (sVEGFR-1) that binds and inactivates VEGF. This study presents a mathematical model that includes all the components above, as well as MCP-1, tumor cells, and oxygen. The model simulations are representative of the in vivo data through predictions of tumor growth using different protocol strategies for GM-CSF for the purpose of predicting higher degrees of treatment success. For example, our model predicts that once a week dosing of GM-CSF would be less effective than daily, twice a week, or three times a week treatment because of the presence of essential factors required for the anti-tumor effect of GM-CSF., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Approach for in vivo real-time assessment of tumor tissue extracellular pH (pH(e)), redox, and intracellular glutathione based on L-band EPR spectroscopy using dual function pH and redox nitroxide probe and disulfide nitroxide biradical, is described. These parameters were monitored in PyMT mice bearing breast cancer tumors during treatment with granulocyte macrophage colony-stimulating factor. It was observed that tumor pH(e) is about 0.4 pH units lower than that in normal mammary gland tissue. Treatment with granulocyte macrophage colony-stimulating factor decreased the value of pH(e) by 0.3 units compared with PBS control treatment. Tumor tissue reducing capacity and intracellular glutathione were elevated compared with normal mammary gland tissue. Granulocyte macrophage colony-stimulating factor treatment resulted in a decrease of the tumor tissue reducing capacity and intracellular glutathione content. In addition to spectroscopic studies, pH(e) mapping was performed using recently proposed variable frequency proton-electron double-resonance imaging. The pH mapping superimposed with MRI image supports probe localization in mammary gland/tumor tissue, shows high heterogeneity of tumor tissue pH(e) and a difference of about 0.4 pH units between average pH(e) values in tumor and normal mammary gland. In summary, the developed multifunctional approach allows for in vivo, noninvasive pH(e), extracellular redox, and intracellular glutathione content monitoring during investigation of various therapeutic strategies for solid tumors., (Copyright © 2011 Wiley Periodicals, Inc.)

Macrophage secretion of vascular endothelial growth factor (VEGF) in response to the hypoxic tumor microenvironment contributes to tumor growth, angiogenesis, and metastasis. We have recently demonstrated that macrophages stimulated with GM-CSF at low O(2) secrete high levels of a soluble form of the VEGF receptor 1 (sVEGFR-1), which neutralizes VEGF and inhibits its biological activity. Using small interfering RNA targeting to deplete hypoxia-inducible factor (HIF)-1α or HIF-2α in murine macrophages, we found that macrophage production of sVEGFR-1 in response to low O(2) was dependent on HIF-2α, whereas HIF-1α specifically regulated VEGF production. In our current report, we evaluated the growth of B16F10 malignant melanoma in mice with a monocyte/macrophage-selective deletion of HIF-1α or HIF-2α (HIF-1α(flox/flox)- or HIF-2α(flox/+)/LysMcre mice). GM-CSF treatment increased intratumoral VEGF and sVEGFR-1 in control mice, an effect that was associated with a decrease in microvessel density. GM-CSF treatment of HIF-1α(flox/flox)/LysMcre mice induced sVEGFR-1 but not VEGF, resulting in an overall greater reduction in tumor growth and angiogenesis compared with control mice. In addition, real-time PCR for melanoma-specific genes revealed a significantly reduced presence of lung micrometastases in HIF-1α(flox/flox)/LysMcre mice treated with GM-CSF. Conversely, GM-CSF treatment induced VEGF but not sVEGFR-1 in HIF-2α(flox/+)/LysMcre mice, and, correspondingly, GM-CSF did not decrease tumor growth, angiogenesis, or lung metastasis in these mice. This study reveals opposing roles for the HIFs in the regulation of angiogenesis by tumor-associated macrophages and suggests that administration of GM-CSF might be an effective means of inducing sVEGFR-1 and inhibiting tumor growth and angiogenesis in patients with melanoma.

Macrophages contribute to tumor growth through the secretion of the proangiogenic molecule vascular endothelial growth factor (VEGF). We previously observed that monocytes treated with the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) produce a soluble form of the VEGF receptor-1 (sVEGFR-1), which neutralizes VEGF biologic activity. The VEGF and VEGFR-1 promoters both contain a hypoxia regulatory element, which binds the hypoxia-inducible factor (HIF) transcription factors under hypoxic conditions. Based on this observation, we examined VEGF and sVEGFR-1 production from monocytes cultured at various O(2) concentrations. The amount of sVEGFR-1 production observed from GM-CSF-treated monocytes increased with decreasing levels of O(2). This sVEGFR-1 was biologically active and sequestered VEGF. To evaluate the role of the HIFs in sVEGFR-1 production, we used macrophages with a genetic deletion of HIF-1α. HIF-1α(-/-) macrophages cultured with GM-CSF at hypoxia secreted diminished amounts of VEGF compared with HIF-1α(+/+) macrophages, whereas sVEGFR-1 secretion was unaffected. In contrast, siRNA-mediated knockdown of HIF-2α inhibited the production of sVEGFR-1 in response to GM-CSF and low O(2), whereas VEGF production was unaffected. These studies suggest that hypoxia, generally thought to promote angiogenesis, can induce antiangiogenic behavior from macrophages within a GM-CSF-rich environment. Furthermore, these results suggest specific and independent roles for HIF-1α and HIF-2α in hypoxic macrophages.

Monocytes and macrophages are central cells of the innate immune system, responsible for defending against diverse pathogens. While they originate from a common myeloid precursor and share functions in innate immunity, each has a very distinct life span finely tuned by the apoptotic caspases. Normally, circulating monocytes are short-lived and undergo spontaneous apoptosis on a daily basis. Macrophages, however, have a longer life span. In chronic inflammatory diseases and, as recently recognized, in the tumor microenvironment, the inhibition of the apoptotic program promotes monocyte survival contributing to the accumulation of macrophages and the persistence of an inflammatory milieu. A complex network of differentiation factors and inflammatory stimuli determine monocyte/macrophage life span by blocking the apoptotic pathway and activating a myriad of survival pathways. Our understanding of apoptosis has flourished over the last decade, and its relevance in the regulation of the immune system is now indisputable. Nevertheless, how the complicated networks of survival and apoptotic regulators are integrated to determine cellular life span remains elusive. This review summarizes the contribution of the caspases and their regulators in monocyte/macrophage cell fate and discusses how these molecules orchestrate the initiation, maintenance, and resolution of inflammation. More provocatively, we discuss possible strategies to control inflammation by manipulating leukocyte life span., ((c) 2010 S. Karger AG, Basel.)

Vascular endothelium is vulnerable to the attack of glucose-derived oxoaldehydes (glyoxal and methylglyoxal) during diabetes, through the formation of advanced glycation end products (AGEs). Although aminoguanidine (AG) has been shown to protect against the AGE-induced adverse effects, its protection against the glyoxal-induced alterations in vascular endothelial cells (ECs) such as cytotoxicity, barrier dysfunction, and inhibition of angiogenesis has not been reported and we investigated this in the bovine pulmonary artery ECs (BPAECs). The results showed that glyoxal (1-10 mM) significantly induced cytotoxicity and mitochondrial dysfunction in a dose- and time-dependent (4-12 h) fashion in ECs. Glyoxal was also observed to significantly inhibit EC proliferation. The study also revealed that glyoxal induced EC barrier dysfunction (loss of trans-endothelial electrical resistance), actin cytoskeletal rearrangement, and tight junction alterations in BPAECs. Furthermore, the results revealed that glyoxal significantly inhibited in vitro angiogenesis on the Matrigel. For the first time, this study demonstrated that AG significantly protected against the glyoxal-induced cytotoxicity, barrier dysfunction, cytoskeletal rearrangement, and inhibition of angiogenesis in BPAECs. Therefore, AG appears as a promising protective agent in the treatment of AGE-induced vascular endothelial alterations and dysfunction during diabetes, presumably by blocking the reactivity of the sugar-derived dicarbonyls such as glyoxal and preventing the formation of AGEs.

An asymmetric derivative of the triarylmethyl radical, TAM-H, containing one aldehyde and two carboxyl groups, was synthesized. The electron paramagnetic resonance (EPR) spectrum of TAM-H is characterized by a doublet of narrow lines with a linewidth of 105 mG under anoxic conditions and hyperfine interaction constant of 245 mG. The partial overlap of the components of the doublet results in enhanced sensitivity of the spectral amplitudes ratio to oxygen compared with oxygen-induced linewidth broadening of a single line. Application of the TAM-H probe allows for EPR measurements in an extended range of oxygen pressures from atmospheric to 1 mm Hg, whereas the EPR spectrum linewidth of the popular TAM-based oxygen sensor Oxo63 is practically insensitive to oxygen partial pressures below 20 mm Hg. Enhanced sensitivity of the TAM-H probe relative to Oxo63 was demonstrated in the detection of oxygen consumption by Met-1 cancer cells. The TAM-H probe allowed prolonged measurements of oxygen depletion during the hypoxia stage and down to true anoxia (

Tumor-educated macrophages facilitate tumor metastasis and angiogenesis. We discovered that granulocyte macrophage colony-stimulating factor (GM-CSF) blocked macrophages vascular endothelial growth factor (VEGF) activity by producing soluble VEGF receptor-1 (sVEGFR-1) and determined the effect on tumor-associated macrophage behavior and tumor growth. We show GM-CSF treatment of murine mammary tumors slowed tumor growth and slowed metastasis. These tumors had more macrophages, fewer blood vessels, and lower oxygen concentrations. This effect was sVEGFR-1 dependent. In situ hybridization and flow cytometry identified macrophages as the primary source of sVEGFR-1. These data suggest that GM-CSF can re-educate macrophages to reduce angiogenesis and metastases in murine breast cancer.

Latency Associated Peptide (LAP) binds TGF-beta1, forming a latent complex. Currently, LAP is presumed to function only as a sequestering agent for active TGF-beta1. Previous work shows that LAP can induce epithelial cell migration, but effects on leukocytes have not been reported. Because of the multiplicity of immunologic processes in which TGF-beta1 plays a role, we hypothesized that LAP could function independently to modulate immune responses. In separate experiments we found that LAP promoted chemotaxis of human monocytes and blocked inflammation in vivo in a murine model of the delayed-type hypersensitivity response (DTHR). These effects did not involve TGF-beta1 activity. Further studies revealed that disruption of specific LAP-thrombospondin-1 (TSP-1) interactions prevented LAP-induced responses. The effect of LAP on DTH inhibition depended on IL-10. These data support a novel role for LAP in regulating monocyte trafficking and immune modulation.

The role of inflammation in idiopathic pulmonary fibrosis (IPF) is controversial. If inflammation were critical to the disease process, lung pathology would demonstrate an influx of inflammatory cells, and that the disease would respond to immunosuppression. Neither is true. The classic pathology does not display substantial inflammation, and no modulation of the immune system is effective as treatment. Recent data suggest that the pathophysiology of the disease is more a product of fibroblast dysfunction than of dysregulated inflammation. The role of inflammation in disease pathogenesis comes from pathology from atypical patients, biologic samples procured during exacerbations of the disease, and careful examination of biologic specimens from patients with stable disease. We suggest that inflammation is indeed a critical factor in IPF and propose five potential nontraditional mechanisms for the role of inflammation in the pathogenesis of IPF: the direct inflammatory hypothesis, the matrix hypothesis, the growth factor-receptor hypothesis, the plasticity hypothesis, and the vascular hypothesis. To address these, we review the literature exploring the differences in pathology, prognosis, and clinical course, as well as the role of cytokines, growth factors, and other mediators of inflammation, and last, the role of matrix and vascular supply in patients with IPF.

Background: M-CSF recruits mononuclear phagocytes which regulate processes such as angiogenesis and metastases in tumors. VEGF is a potent activator of angiogenesis as it promotes endothelial cell proliferation and new blood vessel formation. Previously, we reported that in vitro M-CSF induces the expression of biologically-active VEGF from human monocytes., Methodology and Results: In this study, we demonstrate the molecular mechanism of M-CSF-induced VEGF production. Using a construct containing the VEGF promoter linked to a luciferase reporter, we found that a mutation reducing HIF binding to the VEGF promoter had no significant effect on luciferase production induced by M-CSF stimulation. Further analysis revealed that M-CSF induced VEGF through the MAPK/ERK signaling pathway via the transcription factor, Sp1. Thus, inhibition of either ERK or Sp1 suppressed M-CSF-induced VEGF at the mRNA and protein level. M-CSF also induced the nuclear localization of Sp1, which was blocked by ERK inhibition. Finally, mutating the Sp1 binding sites within the VEGF promoter or inhibiting ERK decreased VEGF promoter activity in M-CSF-treated human monocytes. To evaluate the biological significance of M-CSF induced VEGF production, we used an in vivo angiogenesis model to illustrate the ability of M-CSF to recruit mononuclear phagocytes, increase VEGF levels, and enhance angiogenesis. Importantly, the addition of a neutralizing VEGF antibody abolished M-CSF-induced blood vessel formation., Conclusion: These data delineate an ERK- and Sp1-dependent mechanism of M-CSF induced VEGF production and demonstrate for the first time the ability of M-CSF to induce angiogenesis via VEGF in vivo.

We recently reported that NCX-4016, a derivative of aspirin containing a nitro moiety that releases nitric oxide (NO) in a sustained fashion in biologic systems, is a potent cytotoxic agent inhibiting the proliferation of cisplatin-resistant human ovarian cancer cells. Therefore, we hypothesize that NCX-4016 possesses antiangiogenic properties. Our study with the bovine lung microvascular endothelial cells (BLMVECs) revealed that NCX-4016 significantly induced the loss of redox-dependent cell viability in a dose- and time-dependent manner, as assayed by the redox-sensitive Alamar blue cell viability assay. Fluorescence microscopy of cells labeled with NO-specific fluorophore (DAF-FM) confirmed that NCX-4016 generated significant levels of intracellular NO. NO donors, including S-nitroso-N-acetylpenicillamine, spermine NONOate, and isosorbide dinitrite, were less effective in causing loss of cell viability. Thiol-protectant, N-acetylcysteine, significantly attenuated the NCX-4016-induced loss of cell viability, suggesting the role of alteration of thiol-redox status therein. NCX-4016 also suppressed oxygen consumption, decreased transendothelial electrical resistance (EC barrier dysfunction), and induced actin cytoskeletal reorganization in BLMVECs. The in vitro assay with human umbilical vein ECs and BLMVECs revealed that NCX-4016, in a dose-dependent manner, significantly inhibited angiogenesis with almost complete inhibition at a 100-microM concentration, suggesting that NCX-4016 can act as an antiangiogenic drug.

M-CSF induces PI 3-kinase activation, resulting in reactive oxygen species (ROS) production. Previously, we reported that ROS mediate macrophage colony-stimulating factor (M-CSF)-induced extracellular regulated kinase (Erk) activation and monocyte survival. In this work, we hypothesized that M-CSF-stimulated ROS products modulated Akt1 and p38 activation. Furthermore, we sought to clarify the source of these ROS and the role of ROS and Akt in monocyte/macrophage survival. Macrophages from p47(phox-/-) mice, lacking a key component of the NADPH oxidase complex required for ROS generation, had reduced cell survival and Akt1 and p38 mitogen-activated protein kinase (MAPK) phosphorylation compared with wild-type macrophages in response to M-CSF stimulation, but had no difference in M-CSF-stimulated Erk. To understand how ROS affected monocyte survival and signaling, we observed that NAC and DPI decreased cell survival and Akt1 and p38 MAPK phosphorylation. Using bone marrow-derived macrophages from mice expressing constitutively activated Akt1 (Myr-Akt1) or transfecting Myr-Akt1 constructs into human peripheral monocytes, we concluded that Akt is a positive regulator of monocyte survival. Moreover, the p38 MAPK inhibitor, SB203580, inhibited p38 activity and M-CSF-induced monocyte survival. These findings demonstrate that ROS generated from the NADPH oxidase complex contribute to monocyte/macrophage survival induced by M-CSF via regulation of Akt and p38 MAPK.

Recent studies using ovarian cancer cells have shown that the catecholamine hormones norepinephrine (norepi) and epinephrine (epi) may influence cancer progression by modulating the expression of matrix metalloproteinases (MMP) and vascular endothelial growth factor (VEGF). The purpose of this study is to determine if the stress hormone norepi can influence the expression of MMP-2, MMP-9, and VEGF in nasopharyngeal carcinoma (NPC) tumors by using three NPC tumor cell lines. The NPC cell lines HONE-1, HNE-1, and CNE-1 were treated with norepi. The effects of norepi on MMP-2, MMP-9, and VEGF synthesis were measured by ELISA; functional MMP activity was measured by the invasive potential of the cells using a membrane invasion culture system whereas functional activity of VEGF was analyzed using a human umbilical vein endothelial cell tube formation assay. Norepi treatment increased MMP-2, MMP-9, and VEGF levels in culture supernatants of HONE-1 cells, which could be inhibited by the beta-blocker propranolol. Norepi induced the invasiveness of all NPC cell lines in a dose-dependent manner, which was blocked by CMT-3, an MMP inhibitor, and propranolol. Norepi stimulated the release of functional angiogenic VEGF by HONE-1 cells as well. Finally, HONE-1 cells were shown to express beta-adrenergic receptors as did seven of seven NPC biopsies examined. The data suggest that catecholamine hormones produced by the sympathetic-adrenal medullary axis may affect NPC tumor progression, in part, through modulation of key angiogenic cytokines.

GM-CSF promotes homeostasis of myeloid cells. We report that GM-CSF upregulates mRNA and protein production of the soluble form of membrane bound VEGF receptor-1 (sVEGFR-1) in human monocytes. This sVEGFR-1 was biologically active, as cell-free supernatants from GM-CSF-stimulated monocytes blocked detection of endogenously expressed VEGF and inhibited endothelial cell migration and tube formation, even in the presence of exogenous rhVEGF. VEGF activity was recovered by neutralizing sVEGFR-1. To determine whether these events were important in vivo, Matrigel plugs were incubated with rhVEGF, rhGM-CSF, or rhGM-CSF/rhVEGF and injected into mice. Plugs containing GM-CSF or GM-CSF/VEGF had less endothelial cell invasion than plugs containing rhVEGF and were similar to plugs incubated with PBS alone. Neutralizing antibodies specific for sVEGFR-1 injected in these plugs reversed the effects of GM-CSF or GM-CSF/VEGF, while an isogenic antibody did not. Thus, GM-CSF and monocytes play a vital role in angiogenesis through the regulation of VEGF and sVEGFR-1.

The impact of the immune response in malignancy is poorly understood. While immune cells can destroy transformed cells, the targeting and accumulation of monocytes and macrophages at tumor sites may promote tumor metastases. The growth factor M-CSF is important in promoting monocyte survival. Since M-CSF(-/-) mice are protected against tumor metastases, we hypothesized that M-CSF induced monocytes to produce angiogenic factors that facilitate metastases. In this study we demonstrate that recombinant human M-CSF induces freshly isolated normal human monocytes to produce and release the growth factor vascular endothelial growth factor (VEGF) in a dose-dependent manner, which peaked at 5 days in culture. VEGF released by these monocytes is biologically active, as cell-free supernatants from these M-CSF-stimulated monocytes induced tube formation in HUVEC. Network formation by these HUVECs after treatment with supernatants from monocytes stimulated with M-CSF were inhibited by anti-VEGF, but not by the isogenic control, Abs. Collectively, these data support an important role for M-CSF and monocytes in VEGF production and angiogenesis.

The potential of RNAs and RNA-protein (RNP) complexes as drug targets is currently being explored in various investigations. For example, a hexa-arginine derivative of neomycin (NeoR) and a tri-arginine derivative of gentamicin (R3G) were recently shown to disrupt essential RNP interactions between the trans-activator protein (Tat) and the Tat-responsive RNA (trans-activating region) in the human immunodeficiency virus (HIV) and also inhibit HIV replication in cell culture. Based on certain structural similarities, we postulated that NeoR and R3G might also be effective in disrupting RNP interactions and thereby inhibiting bacterial RNase P, an essential RNP complex involved in tRNA maturation. Our results indicate that indeed both NeoR and R3G inhibit RNase P activity from evolutionarily divergent pathogenic bacteria and do so more effectively than they inhibit partially purified human RNase P activity.