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Background and aims Circulating monocytes have been proven to be critical mediators in the propagation and progression of atherosclerosis and myocardial infarction. The present study was designed to characterise a new transmembrane protein—NFAT activating protein with ITAM motif 1 (NFAM1)—on monocytes and uncover the potential effects and underlying mechanisms in coronary artery disease. Methods Monocytes from a population of four controls, five stable coronary artery disease patients and five acute coronary syndrome patients were isolated for RNA sequencing. A potential monocyte biomarker molecule was discovered and then validated with a cohort of 79 controls, 70 stable coronary artery disease patients and 183 acute coronary syndrome patients. A stable cell line was generated as an in vitro model to determine chemotaxis migration and chemokine receptor expression. Results NFAM1 was identified through RNA sequencing analysis. The validation results confirmed that NFAM1 expression on monocytes was significantly increased by coronary artery disease status. A higher expression level of NFAM1 on classical and intermediate monocytes was observed compared with that on nonclassical monocytes. As shown in the in vitro cell model, knockdown of NFAM1 significantly attenuated chemotactic migration of monocytes by downregulating chemokine receptor expression and the p38 MAPK signalling pathway. Multivariable regression analysis of a group of 16 individuals suggested that NFAM1 was positively correlated with CCR2 expression. Conclusions The present study reported for the first time that distinctive alterations of NFAM1 expression on monocytes may correlate with atherosclerosis pathobiology and serve as a potential monocyte biomarker and therapeutic target for coronary artery disease.

Summary Objectives Monocyte-derived macrophages, as the predominant immune cell type that is increased in inflamed synovium, play a vital role during knee osteoarthritis (KOA) progression. However, the mechanisms underlying the recruitment of circulating monocytes to osteoarthritic knees remain uncertain. Based on previous data obtained from plasma, we investigated the contributions of CCL2, CCL3, CCL4 and their cognate receptors in circulating monocyte chemotaxis and KOA development. Methods Using flow cytometry staining, we characterized the expression patterns of the chemokine receptors in CD14+CD16− circulating monocytes from KOA patients and healthy volunteers. The expression of chemokines in synovial fluids, synovium and cartilage was investigated in KOA patients and in patients without KOA. The role of chemokines and their cognate receptors in the chemotaxis of CD14+CD16− circulating monocytes was assessed using chemokine neutralizing antibodies (NA) and receptor antagonists in vitro and in vivo. Results The majority of CD14+CD16− circulating monocytes were CCR1-and CCR2-positive. CCL2, CCL3 and CCL4 were elevated in synovial fluid of KOA patients compared with that of controls. The most likely source of these chemokines is inflamed synovium and cartilage in the osteoarthritic knee. The CCL3/CCR1 and CCL2/CCR2 axes showed substantial ability to recruit CD14+CD16− monocytes in transwell assays. Similar results were confirmed in a mouse model of collagenase-induced KOA (CIA) in which blocking either the CCL3/CCR1 axis or the CCL2/CCR2 axis reduced synovial hyperplasia and F4/80+ macrophage infiltration. Conclusions Our findings suggested that, analogous to the CCL2/CCR2 axis, CCL3 produced in osteoarthritic knees can chemoattract circulating monocytes to the inflamed synovium through CCR1.

The lung is the entry site for Bacillus anthracis in inhalation anthrax, the most deadly form of the disease. Spores must escape through the alveolar epithelial cell (AEC) barrier and migrate to regional lymph nodes, germinate and enter the circulatory system to cause disease. Several mechanisms to explain alveolar escape have been postulated, and all these tacitly involve the AEC barrier. In this study, we incorporate our primary human type I AEC model, microarray and gene enrichment analysis, qRT-PCR, multiplex ELISA, and neutrophil and monocyte chemotaxis assays to study the response of AEC to B. anthracis, (Sterne) spores at 4 and 24 hours post-exposure. Spore exposure altered gene expression in AEC after 4 and 24 hours and differentially expressed genes (±1.3 fold, p ≤ 0.05) included CCL4/MIP-1β (4 hours), CXCL8/IL-8 (4 and 24 hours) and CXCL5/ENA-78 (24 hours). Gene enrichment analysis revealed that pathways involving cytokine or chemokine activity, receptor binding, and innate immune responses to infection were prominent. Microarray results were confirmed by qRT-PCR and multiplex ELISA assays. Chemotaxis assays demonstrated that spores induced the release of biologically active neutrophil and monocyte chemokines, and that CXCL8/IL-8 was the major neutrophil chemokine. The small or sub-chemotactic doses of CXCL5/ENA-78, CXCL2/GROβ and CCL20/MIP-3α may contribute to chemotaxis by priming effects. These data provide the first whole transcriptomic description of the human type I AEC initial response to B. anthracis spore exposure. Taken together, our findings contribute to an increased understanding of the role of AEC in the pathogenesis of inhalational anthrax.

Recent studies have revealed considerable dysfunction of vascular endothelial cells (VECs) and abnormal expression of microRNA (miR)-137 in women with gestational diabetes mellitus (GDM), and the aim of this study was to clarify the underlying mechanism and possible role of microRNA (miR)-137 in dysfunction of VECs during GDM. We found increased levels of miR-137 in the plasma of GDM women and high-glucose (HG)-exposed HUVECs. Upregulating miR-137 in HUVECs elevated the chemokine (C-C motif) ligand 2 (CCL2) secretion and enhanced the chemotaxis and adhesion of U937 and THP-1 (two human acute monocytic leukemia cell lines) cells to HUVECs in a co-culture system. Moreover, HG stimulation and/or overexpression of miR-137 inhibited the viability, upregulated the expression levels of vascular cell adhesion molecule-1 (VCAM-1), intercellular cell adhesion molecule-1 (ICAM-1), E-selectin, and inflammatory cytokine interleukin (IL)-6, and downregulated the production of IL-8, vascular endothelial growth factor (VEGF), and angiogenesis of HUVECs in vitro. These results imply that up-regulated miR-137 by HG can restrict the viability and angiogenesis, promote the activation and inflammatory cytokine secretion of VECs, and stimulate the monocyte chemotaxis and adhesion to VECs. Ultimately, we have concluded that miR-137 is crucial to HG-induced VEC dysfunction and may be involved in pathology of GDM.

Spinal cord injury (SCI) is a serious devastating condition and it has a high mortality rate and morbidity rate. The early pathological changes in the immediate phase of SCI may play a major part in the development of secondary injury. Alterations in the expression of many long noncoding RNAs (lncRNAs) have been shown to play fundamental roles in the diseases of the central nervous system. However, the roles of lncRNAs and messenger RNAs (mRNAs) in the immediate phase of SCI are not clear. We examined the expression of mRNAs and lncRNAs in a rat model at 2 h after SCI and identified the differentially expressed lncRNAs (DE lncRNAs) and differentially expressed mRNAs (DE mRNAs) using microarray analysis. 772 DE lncRNAs and 992 DE mRNAs were identified in spinal cord samples in the immediate phase following SCI compared with control samples. Moreover, Gene Ontology (GO) term annotation results showed that CXCR chemokine receptor binding, neutrophil apoptotic process, neutrophil migration, neutrophil extravasation, macrophage differentiation, monocyte chemotaxis and cellular response to interleukin-1 (IL-1) were the main significantly enriched GO terms. The results of Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the DEGs were enriched in toll−like receptor signaling pathway, p53 signaling pathway, MAPK signaling pathway and Jak–STAT signaling pathway. IL6, MBOAT4, FOS, TNF, JUN, STAT3, CSF2, MYC, CCL2 and FGF2 were the top 10 high-degree hub nodes and may be important targets in the immediate phase of SCI. The current study on provides novel insights into how lncRNAs and mRNAs regulate the pathogenesis of the immediate phase after SCI.

IFN-β directly downregulates NRP-1 expression in monocytes, inhibiting monocyte chemotaxis toward a VEGF enriched environment. Type I interferons (IFNs) inhibit angiogenesis, the sprouting of new blood vessels, during tissue development, remodeling, and tumor growth. One of the major targets type I IFNs inhibit are circulating monocytes, which promote vascular development by secreting growth factors, chemokines, and proteases. This study tested the hypothesis that IFN-β directly inhibits monocyte chemotaxis towards VEGF. We were interested in looking at chemotaxis towards VEGF because VEGF is known to create a pro-angiogenesis environment by acting as a stimulator and chemotactic factor for endothelial cells and monocytes. Here, we demonstrate that IFN-β, a type I IFN, downregulates neuropilin-1 (NRP-1) expression by human monocytes and inhibits chemotaxis induced by vascular endothelial growth factor (VEGF), a NRP-1 ligand. Together, the data suggest that IFN-β directly downregulates NRP-1 expression in monocytes, thus inhibiting monocyte chemotaxis toward a VEGF enriched environment.

Monocyte migration is a key element in atherosclerosis. LDL-C facilitates monocyte migration via induction of CCR2. PCSK9 regulates cell surface expression of the LDL-R and is expressed in vascular smooth muscle cells (VSMCs). The present study was done to investigate the regulation of PCSK9 in VSMCs and its impact on monocyte function. Methods and results PCSK9 mRNA and protein levels were upregulated in VSMCs by the TLR-4 ligand LPS, whereas TGF-β or angiotensin II had no effect. Induction of PCSK9 was selectively inhibited by TLR-4 blockade and further downstream by the SAPK/JNK-inhibitor SP600125, whereas inhibitors of ERK1/2, p38 or PI3-kinase pathways had no effect. Incubation of monocytes in conditioned media from LPS-stimulated VSMCs resulted in a significant reduction of LDL-R levels on monocytes, comparable to the effects of recombinant PCSK9. LDL-C increased monocyte CCR2 expression, which augmented monocyte migration towards MCP-1. This LDL-C dependent monocyte chemotaxis was inhibited by supernatants from LPS-stimulated VSMCs, similar to recombinant PCSK9 and a specific LDL-R blocking antibody. Conclusion PCSK9 is regulated in VSMCs by TLR-4 – SAPK/JNK signaling, a pathway important in inflammation and metabolism. VSMC-derived PCSK9 reduces monocyte LDL-R expression, affecting LDL-C/LDL-R-mediated CCR2-expression on monocytes, which is crucial to cell motility and atherogenesis.

Monocyte migration requires the dynamic redistribution of integrins through a regulated endo-exocytosis cycle, but the complex molecular mechanisms underlying this process have not been fully elucidated. Glia maturation factor-γ (GMFG), a novel regulator of the Arp2/3 complex, has been shown to regulate directional migration of neutrophils and T-lymphocytes. In this study, we explored the important role of GMFG in monocyte chemotaxis, adhesion, and β1-integrin turnover. We found that knockdown of GMFG in monocytes resulted in impaired chemotactic migration toward formyl-Met-Leu-Phe (fMLP) and stromal cell-derived factor 1α (SDF-1α) as well as decreased α5β1-integrin-mediated chemoattractant-stimulated adhesion. These GMFG knockdown impaired effects could be reversed by cotransfection of GFP-tagged full-length GMFG. GMFG knockdown cells reduced the cell surface and total protein levels of α5β1-integrin and increased its degradation. Importantly, we demonstrate that GMFG mediates the ubiquitination of β1-integrin through knockdown or overexpression of GMFG. Moreover, GMFG knockdown retarded the efficient recycling of β1-integrin back to the plasma membrane following normal endocytosis of α5β1-integrin, suggesting that the involvement of GMFG in maintaining α5β1-integrin stability may occur in part by preventing ubiquitin-mediated degradation and promoting β1-integrin recycling. Furthermore, we observed that GMFG interacted with syntaxin 4 (STX4) and syntaxin-binding protein 4 (STXBP4); however, only knockdown of STXBP4, but not STX4, reduced monocyte migration and decreased β1-integrin cell surface expression. Knockdown of STXBP4 also substantially inhibited β1-integrin recycling in human monocytes. These results indicate that the effects of GMFG on monocyte migration and adhesion probably occur through preventing ubiquitin-mediated proteasome degradation of α5β1-integrin and facilitating effective β1-integrin recycling back to the plasma membrane.

Objective To investigate the effects of Ca 2 + activated potassium channel K Ca 3.1 and voltage-gated potassium channel Kv1.3 of B lymphocyte on inflammatory monocytes chemotaxis and the potential mechanisms. Materials and methods Thanswell test was used to detect the inflammatory monocyte (Ly-6C hi ) chemotaxis caused by the B lymphocyte. Enzyme-linked immunosorbent assay (ELISA) was applied to detecting the C-C motif ligand 7 (CCL7) in cultured media. Cell counting kit-8 (CCK) was used to detect the proliferation of B lymphocytes after activation and blockage of both K Ca 3.1 and Kv1.3 channels. Western blot was used to detect the expression of phosphorylated extracellular signal-regulated kinase (P-ERK) of the B lymphocytes. Results When activated, B lymphocytes significantly proliferated. After application of K Ca 3.1 channel-specific inhibitor TRAM-34 and potent Kv1.3 channel inhibitor ShK, both B lymphocytes proliferation and Ly-6C hi monocyte chemotaxis were significantly inhibited. The expression of chemotaxis related factor CCL7 decreased remarkably. Conclusion The opening of K Ca 3.1 and Kv1.3 channels promote B lymphocyte activation, proliferation and Ly-6C hi monocyte chemotaxis. The increase of CCL7 secretion by B lymphocyte may explain the pro migration effects.

BACKGROUND: Increased hemoglobin (Hb) accumulation was reported in abdominal aortic aneurysms (AAAs). CD163 is a macrophage receptor involved in tissue Hb clearance, however its role in AAA has not been reported. We investigated the role of Hb on monocyte recruitment and differentiation towards CD163 expressing macrophages ex vivo, in vitro and in human AAA.METHODS AND RESULTS: CD163 mRNA and protein expression was significantly higher in human AAA (n=7) vs. healthy wall (n=6). CD163 was predominantly found in adventitia of AAA, coinciding with areas rich in hemosiderin and adjacent to neoangiogenic microvessels. Dual CD14/CD163 expression was observed in recently infiltrated monocytes surrounding microvessels. A higher release of soluble CD163 was observed in the conditioned medium from AAA (AAA-CM, n=10), mainly in the adventitial layer. Similar to Hb, AAA-CM induced CD163-dependent monocyte chemotaxis, especially on circulating monocytes from AAA patients. Hb or AAA-CM promoted differentiation towards CD163(high)/HLA-DR(low)-expressing macrophages, with enhanced Hb uptake, increased anti-inflammatory IL-10 secretion and decreased pro-inflammatory IL-12p40 release. All these effects were partially suppressed when Hb was removed from AAA-CM. Separate analysis on circulating monocytes reported increased percentage of pre-infiltrating CD14(++)CD16(+) monocytes in patients with AAA (n=21), as compared to controls (n=14). A significant increase in CD163 expression in CD14(++)CD16(+) monocyte subpopulation was observed in AAA patients.CONCLUSIONS: The presence of Hb in the adventitial AAA-wall promotes the migration and differentiation of activated circulating monocytes in AAA patients, explaining the existence of a protective CD163-macrophage phenotype that could take up the Hb present in the AAA-wall, avoiding its injurious effects.

Objective Monocytes and macrophages, together with vascular smooth muscle cells (VSMCs), play key roles at all stages of atherogenesis. There is also growing evidence that BMP signaling is involved in vascular diseases, including atherosclerosis. Here we evaluate the role played by the BMP agonist/antagonist axis in monocyte recruitment during atherogenesis. Methods and results Using ApoE−/− mice and BMPs, Gremlin and BMPRII siRNAs we show that BMPs (2 and 4) and their antagonist Gremlin are co-expressed in murine and human atherosclerotic vessels. Additionally, those genes are co-expressed and upregulated in cultured vascular smooth muscle cells early in atherosclerosis formation in ApoE−/− mice. Furthermore, we demonstrate that BMP-2 and -4 produced in atherosclerotic VSMCs promote, whereas Gremlin inhibits, monocyte chemoattraction. Finally, we demonstrate that chemotaxis induction occurs through direct BMP receptor II (BMPRII) activation. Conclusion These findings suggest that the balance between BMPs (2 and 4) and Gremlin levels modulate crosstalk processes between vascular and immune cells and ultimately the homeostasis in normal vasculature. They also indicate that under pro-atherogenic conditions, BMP signaling prevails, favoring monocyte recruitment and inflammation. Manipulation of BMP signaling may enable the identification of novel molecular approaches for preventing, stabilizing, and reverting atherosclerosis.

The cytokine resistin and the chemokine fractalkine (FKN) were found at increased levels in human atherosclerotic plaque, in the subendothelium, but their role in this location still needs to be characterized. Recently, high local resistin in the arterial vessel wall was shown to contribute to an enhanced accumulation of macrophages by mechanisms that need to be clarified. Our recent data showed that resistin activated smooth muscle cells (SMC) by up-regulating FKN and MCP-1 expression and monocyte chemotaxis by activating toll-like receptor 4 (TLR4) and Gi/o proteins. Since in the vessel wall both endothelial cells (EC) and SMC respond to cytokines and promote atherosclerosis, we questioned whether subendothelial resistin (sR) has a role in vascular cells cross-talk leading to enhanced monocyte transmigration and we investigated the mechanisms involved. To this purpose we used an in vitro system of co-cultured SMC and EC activated by sR and we analyzed monocyte transmigration. Our results indicated that: (1) sR enhanced monocyte transmigration in EC/SMC system compared to EC cultured alone; (2) sR activated TLR4 and Gi/o signaling in EC/SMC system and induced the secretion of more FKN and MCP-1 compared to EC cultured alone and used both chemokines to specifically recruit monocytes by CX3CR1 and CCR2 receptors. Moreover, FKN produced by resistin in EC/SMC system, by acting on CX3CR1 on EC/SMC specifically contributes to MCP-1 secretion in the system and to the enhanced monocyte transmigration. Our study indicates new possible targets for therapy to reduce resistin-dependent enhanced macrophage infiltration in the atherosclerotic arterial wall.

QiShenYiQi Pills (QSYQ) is a compound Chinese medicine widely used in China for treatment of cardiovascular disease. However, limited data are available regarding the anti-fibrotic role of QSYQ after ischemia/reperfusion (I/R) injury. This study aimed to investigate the effect of post-treatment with QSYQ on myocardial fibrosis after I/R-induced myocardium injury, and the role of different compounds of QSYQ, focusing especially on the involvement of chemokine ribosomal protein S19 (RP S19) dimer and monocyte migration. Male Sprague-Dawley rats were subjected to left anterior descending coronary artery occlusion for 30 min followed by reperfusion with or without administration of QSYQ (0.6, 1.2, or 1.8 g/kg) once daily by gavage for 6 days. Post-treatment with QSYQ diminished I/R-induced infarct size, alleviated myocardium injury, attenuated myocardial fibrosis after 6 days of reperfusion, and restored heart function and myocardial blood flow after I/R. In addition, the drug significantly inhibited monocyte infiltration and macrophage polarization towards M2, which was attributable to chemokine RP S19 dimer. Moreover, Western blots revealed that QSYQ blocked I/R-induced increase in TGFβ1 and TGFβRⅡ and reversed its relevant gene expression, such as Smad3,4,6,7, and inhibited the increase of MMP 2,9 expression. As the major components of QSYQ, astragaloside IV (AsIV), 3,4-dihydroxy-phenyl lactic acid (DLA), and notoginsenoside R1 (R1) were assessed as to the contribution of each of them to the expression of the proteins concerned. The results showed that the effect of AsIV was similar to QSYQ, while DLA and R1 only partly simulated the effect of QSYQ. The results provide evidence for the potential role of QSYQ in treating myocardial fibrosis following I/R injury. This effect may be associated with QSYQ’s inhibition effect on monocyte chemotaxis and TGFβ1/Smads signaling pathway with different component targeting distinct link (s) of the signaling.

Objective Monocytes are motile cells which sense inflammatory stimuli and subsequently migrate to sites of inflammation. Key players in host defense, monocytes have nevertheless been implicated as requisite mediators of several chronic inflammatory diseases. Inhibition of monocyte chemotaxis is therefore an attractive anti-inflammatory strategy. Oxidized phospholipids (OxPL) are native regulators of inflammation, yet their direct effect on monocyte chemotaxis is poorly defined. In this study, we investigated the direct effect of natural and synthetic phospholipids on monocyte chemotaxis. Methods Exploring various phospholipids using in vitro chemotaxis assays, we found that the natural phospholipid 1-palmitoyl-2-glutaryl phosphatidylcholine (PGPC) can decrease monocyte chemotaxis by 50%, while other tested OxPL had no effect. We generated a library of synthetic OxPL designated lecinoxoids, which was screened for anti-inflammatory properties. Results and conclusions VB-201, a small-molecule lecinoxoid, exhibited up to 90% inhibition of monocyte chemotaxis in vitro . Molecular analysis revealed that the effect of VB-201 was not restricted to a specific chemotactic ligand or receptor, and resulted from inhibition of signaling pathways required for monocyte chemotaxis. Interestingly, VB-201 did not inhibit monocyte adhesion or phagocytosis and had no effect on chemotaxis of CD4 + T-cells or neutrophils. In vivo , oral treatment with VB-201 reduced monocyte migration in a peritonitis model and inhibited atheroma development in ApoE −/− mice, without affecting cholesterol or triglyceride levels. Our findings highlight a novel role played by native and synthetic phospholipids in regulation of monocyte chemotaxis. The data strengthen the involvement of phospholipids as key signaling molecules in inflammatory settings and demonstrate their potential therapeutic applicability.

Background The controlled recruitment of monocytes from the circulation to the site of injury and their differentiation into tissue macrophages are critical events in the reconstitution of tissue integrity. Subsets of monocytes/macrophages have been implicated in the pathogenesis of atherosclerosis and tumor vascularity; however, the significance of monocyte heterogeneity in physiologic neovascularization is just emerging. Materials and methods A cranial-based, peninsular-shaped myocutaneous flap was surgically created on the dorsum of wild-type mice (C57BL6) and populations of mice with genetic deletion of subset-specific chemokine ligand-receptor axes important in monocyte trafficking and function (CCL2−/− and CX3CR1−/−) (n = 36 total; 12 mice per group, nine with flap and three unoperated controls). Planimetric analysis of digital photographic images was utilized to determine flap surface viability in wild-type and knockout mice. Real-time myocutaneous flap perfusion and functional revascularization was determined by laser speckle contrast imaging. Image analysis of CD-31 immunostained sections confirmed flap microvascular density and anatomy. Macrophage quantification and localization in flap tissues was determined by F4/80 gene and protein expression. Quantitative reverse transcription-polymerase chain reaction was performed on nonoperative back skin and postoperative flap tissue specimens to determine local gene expression. Results Myocutaneous flaps created on wild type and CX3CR1−/− mice were engrafted to the recipient site, resulting in viability. In contrast, distal full thickness cutaneous necrosis and resultant flap dehiscence was evident by d 10 in CCL2−/− mice. Over 10 d, laser speckle contrast imaging documented immediate graded flap ischemia in all three groups of mice, functional flap revascularization in wild type and CX3CR1−/− mice, and lack of distal flap reperfusion in CCL2−/− mice. Immunostaining of serial histologic specimens confirmed marked increases in microvascular density and number of macrophages in wild type mice, intermediate increases in CX3CR1−/− mice, and no significant change in vessel count or macrophage quantity in CCL2−/− mice over the study interval. Finally, quantitative reverse transcriptase polymerase chain reaction demonstrated that the loss of function of chemokine ligand and receptor genes influenced the transcription of local genes involved in monocyte chemotaxis and wound angiogenesis. Conclusions In a graded-ischemia wound healing model, monocyte recruitment was severely impaired in CCL2−/− mice, resulting in failure of flap revascularization and concomitant cutaneous necrosis. Analysis of CX3CR1-deficient mice revealed adequate monocyte recruitment and revascularization for flap survival; however, the myeloid cell response and magnitude of neovascularization were dampened compared with wild type mice.

Background: Neutrophil-specific membrane-type 6 matrix metalloproteinase (MT6-MMP)/leukolysin has seven known substrates. Results: We identified 72 new MT6-MMP substrates by proteomics and family-wide chemokine screens. Cell membrane-bound vimentin chemoattracts macrophages, whereas MT6-MMP-cleaved vimentin is an “eat-me” signal greatly increasing phagocytosis. Conclusion: MT6-MMP substrates indicate a role for clearance of apoptotic neutrophils. Significance: MT6-MMP cleaves many bioactive proteins important in innate immunity., The neutrophil-specific protease membrane-type 6 matrix metalloproteinase (MT6-MMP)/MMP-25/leukolysin is implicated in multiple sclerosis and cancer yet remains poorly characterized. To characterize the biological roles of MT6-MMP, it is critical to identify its substrates for which only seven are currently known. Here, we biochemically characterized MT6-MMP, profiled its tissue inhibitor of metalloproteinase inhibitory spectrum, performed degradomics analyses, and screened 26 chemokines for cleavage using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. MT6-MMP processes seven each of the CXC and CC chemokine subfamilies. Notably, cleavage of the neutrophil chemoattractant CXCL5 activates the chemokine, thereby increasing its agonist activity, indicating a feed-forward mechanism for neutrophil recruitment. Likewise, cleavage also activated CCL15 and CCL23 to increase monocyte recruitment. Utilizing the proteomics approach proteomic identification of cleavage site specificity (PICS), we identified 286 peptidic cleavage sites spanning from P6 to P6′ from which an unusual glutamate preference in P1 was identified. The degradomics screen terminal amine isotopic labeling of substrates (TAILS), which enriches for neo-N-terminal peptides of cleaved substrates, was used to identify 58 new native substrates in fibroblast secretomes after incubation with MT6-MMP. Vimentin, cystatin C, galectin-1, IGFBP-7, and secreted protein, acidic and rich in cysteine (SPARC) were among those substrates we biochemically confirmed. An extracellular “moonlighting” form of vimentin is a chemoattractant for THP-1 cells, but MT6-MMP cleavage abolished monocyte recruitment. Unexpectedly, the MT6-MMP-cleaved vimentin potently stimulated phagocytosis, which was not a property of the full-length protein. Hence, MT6-MMP regulates neutrophil and monocyte chemotaxis and by generating “eat-me” signals upon vimentin cleavage potentially increases phagocytic removal of neutrophils to resolve inflammation.

The progression of diabetic nephropathy is associated with an infiltration of macrophages expressing different phenotypes. As classically activated chemokine receptor CCR2+ macrophages are thought to drive tissue inflammation and remodeling, we tested whether blocking CCR2 could reduce intrarenal inflammation and prevent glomerulosclerosis in type 2 diabetes. This was achieved with RO5234444, an orally active small-molecule CCR2 antagonist that blocks ligand binding, its internalization, and monocyte chemotaxis. Male type 2 diabetic db/db mice were uninephrectomized to increase glomerular hyperfiltration to accelerate the development of glomerulosclerosis. From 16 weeks until killing at 24 weeks of age, mice were chow fed with or without admixed antagonist to achieve a trough plasma concentration above IC50 for binding in the mouse. CCR2 blockade reduced circulating monocyte levels, but did not affect total leukocyte or neutrophil numbers, and was associated with a reduction in the number of macrophages and apoptotic podocytes in the glomerulus. This treatment resulted in a higher total number of podocytes, less glomerulosclerosis, reduced albuminuria, and a significantly improved glomerular filtration rate. This successful pre-clinical trial suggests that this antagonist may now be ready for testing in humans with the nephropathy of diabetes mellitus.

Summary. Background: Platelets release the immune-modulating lipid sphingosine-1-phosphate (S1P). However, the mechanisms of platelet S1P secretion are not fully understood. Objectives: The present study investigates the function of thromboxane (TX) for platelet S1P secretion during platelet activation and the consequences for monocyte chemotaxis. Methods: S1P was detected using thin-layer chromatography in [3H]sphingosine-labeled platelets and by mass spectrometry. Monocyte migration was measured in modified Boyden chamber chemotaxis assays. Results: Release of S1P from platelets was stimulated with protease-activated receptor-1-activating peptide (PAR-1-AP, 100 μm). Acetylsalicylic acid (ASA) and two structurally unrelated reversible cyclooxygenase inhibitors diclofenac and ibuprofen suppressed S1P release. Oral ASA (500-mg single dose or 100 mg over 3 days) attenuated S1P release from platelets in healthy human volunteers ex vivo. This was paralleled by inhibition of TX formation. S1P release was increased by the TX receptor (TP) agonist U-46619, and inhibited by the TP antagonist ramatroban and by inhibitors of ABC-transport. Furthermore, thrombin-induced release of S1P was attenuated in platelets from TP-deficient mice. Supernatants from PAR-1-AP-stimulated human platelets increased the chemotactic capacity of human peripheral monocytes in a S1P-dependent manner via S1P receptors-1 and -3. These effects were inhibited by ASA-pretreatment of platelets. Conclusions: TX synthesis and TP activation mediate S1P release after thrombin receptor activation. Inhibition of this pathway may contribute to the anti-inflammatory actions of ASA, for example by affecting activity of monocytes at sites of vascular injury.

Monocyte recruitment and their subsequent transformation to brain macrophages as well as microglial activation are key events in a variety of severe neurological diseases. Activated microglia and brain macrophages can have detrimental effects in brain pathology via the promotion of inflammatory processes and the release of neurotoxic substances. Therefore, it is of great interest to develop strategies aiming at i) the reduction of monocyte infiltration into the damaged brain and ii) specific inhibition of neurotoxic effects by activated microglia / brain macrophages. Recent evidence indicates an important role of ion channels in regulating monocyte chemotaxis and microglial activation. Thus, ion channels of monocytes and microglia / brain macrophages could represent good candidates for therapeutic interventions in neurological diseases.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder initiated by heparin administration. It is caused by the formation of pathogenic antibodies to complexes of platelet factor-4 (PF4) and heparin on platelet surfaces that cause platelet activation, aggregation and thrombosis. There has been intense research on this intriguing, drug-related thrombocytopenia explaining several characteristic aspects of this condition. However, prothrombotic potential of the key player, PF4 has not been investigated in many studies although it has been shown to be critical in monocyte chemotaxis, monocyte-platelet interaction, and megakaryocyte suppression, all of which can contribute to the pathophysiology of HIT. This article explains the important role of PF4 released during platelet activation with the administration of heparin in the pathogenesis of thrombocytopenia and thrombosis in HIT.

We showed that ursolic acid (UA), a pentacyclic triterpenoid with anti-inflammatory properties, reduces atherosclerotic lesion size and improves kidney function in diabetic mice. We proposed that the atheroprotective effects of UA are due to its ability to protect monocytes against metabolic stress-induced dysfunction or “priming”, i.e. the hyper-responsiveness to chemokine-induced cell adhesion and migration. Based on structure-function analyses of UA’s naturally occurring analogs, we identified and synthesized a compound, 23-hydroxy ursolic acid (23-OHUA), with a structural feature that we predicted to enhance both bioavailability and the protective effects of UA against monocyte dysfunction in vitro and in dyslipidemia atherosclerosis-prone mice. 23-OHUA protected monocytes from metabolic priming and dysfunction with a similar efficacy to UA. Both phytochemicals protected monocytes from metabolic stress by preventing the degradation of MAPK phosphatase-1 (MKP-1), a master regulator of monocyte chemotaxis and macrophage function. To test the efficacy of 23-OHUA in protecting mice against monocyte dysfunction and atherogenesis, we fed atherosclerosis-prone LDLR-/- mice a high fat diet (HFD) or a HFD supplemented with either 0.05% UA or 0.05% 23-OHUA. Monocytes were primed and hyper-sensitive to chemokines as early as 6 weeks after initiating HFD-feeding as shown by our in vivo chemotaxis assay. After 20 weeks, mice fed 23-OHUA showed 11% less weight gain compared to HFD-fed control mice and 40% reduced atherosclerotic plaque formation. Both dietary UA and 23-OHUA reduced metabolic priming and prevented the decrease in MKP-1 activity, suggesting that both compounds exert their anti-inflammatory, anti-obesogenic and anti-atherogenic effects through a novel but common mechanism involving the protection of blood monocytes against metabolic stress-induced priming and dysfunction. Our results suggest 23-OHUA and UA are viable candidates for dietary supplements for the prevention of chronic inflammatory diseases associated with metabolic disorders.

A significant portion of current medical research is devoted to the pursuit of genetic markers that can be used to identify disease or predict susceptibility to disease. In such a quest many investigators hypothesized that genetic variations that alter signalling pathways involved in atherosclerosis affect susceptibility to coronary artery disease (CAD). Fractalkine (FKN) is a small cytokine involved in monocyte chemotaxis and activation. Two single nucleotide polymorphisms, V249I and T280M, have been identified in the receptor coding sequence of FKN. The polymorphisms alter ligand-receptor affinity and are believed to influence an individual's susceptibility to atherosclerosis. Several investigators have tested the latter hypothesis with inconsistent results. In order to clarify the effect of the two polymorphisms on susceptibility to CAD we performed a meta-analysis, using pooled data retrieved from seven case-control studies. In total, 2000 CAD patients and 2841 subjects without evidence of cardiovascular disease were included in the meta-analysis. The 280M allele was associated with a reduced risk for CAD in the heterozygous state. Consequently, this effect was attributed to the only 280M-containing haplotype: I(249)M(280). The latter haplotype was found to be significantly more frequent in the control population's gene pool. Although we do not believe that the retrieved odds ratios render the T280M polymorphism a candidate genetic marker for clinical applications, we do believe that the above genotype-phenotype interaction is indicative of the strong associations between FKN-induced pathways and CAD.

Background: Monocytes/macrophages are crucially involved in the process of atherogenesis. Presence of an acute coronary syndrome (ACS) is associated with macrophage activation. We investigated whether ligand-induced monocyte chemotaxis can serve as biomarker in recent ACS and discriminate ACS from stable coronary artery disease (CAD). Methods: In a prospective study, the migratory response of monocytes towards the chemotactic ligands vascular endothelial growth factor-A (VEGF-A) and monocyte chemoattractant protein-1 (MCP-1) in patients with recent ACS (n = 29) (median time period since cardiovascular event, 11 days) and stable CAD patients (n = 41) was analysed. Furthermore, blood levels of C-reactive protein (CRP), VEGF-A and soluble vascular endothelial growth factor receptor-1 (sVEGRF-1) were determined. Results: Unexpectedly, VEGF-A-induced monocyte chemotaxis did not differ between ACS and CAD. The same was true for the chemotactic response of monocytes towards MCP-1. In addition, we could not find any difference in VEGF-A and sVEGFR-1 levels between recent ACS and stable CAD. CRP was significantly enhanced in the ACS group, but did not correlate with the VEGF-A- and MCP-1-induced chemotaxis. Conclusions: Recent ACS is not associated with enhanced monocyte chemotaxis towards VEGF-A and MCP-1. Therefore, VEGF-A- and MCP-1-induced monocyte chemotaxis as a potential novel biomarker remains unaffected by recent ACS.

We discovered that a seaweed sporophyll-derived polysaccharide of brown alga, Wakame (Undaria pinnatifida) bound to monocytes and attracted them in vitro and in vivo. Physicochemical properties, affinity to a lectin-bead column and sugar composition of the chemotactic polysaccharide indicated this molecule to be a highly sulfated fucogalactan. We then identified the monocyte receptor of the sulfated fucogalactan as the elastin peptide receptor by prophylactic inhibition of the binding and the chemoattraction with lactose and the synthetic elastin peptide, Val-Gly-Val-Ala-Pro-Gly. We assume that the galactose-binding lectin, which is a component of the elastin peptide receptor complex, would recognize a Gal residue of the sulfated fucogalactan. We also observed a similar chemoattracting polysaccharide in a pathogenic fungus, Candida albicans, although the content of it was much lower than in the case of seaweed sporophyll. We speculate that the chemotactic response of monocytes to the sulfated fucogalactan is part of the innate immune system to fungal infection.

Several members of the neuropeptide family exert chemotactic actions on blood monocytes consistent with neurogenic inflammation. Furthermore, chromogranin A (CgA) containing Alzheimer plaques are characterized by extensive microglia activation and such activation induces neuronal damage. We therefore hypothesized that the catecholamine release inhibitory peptide catestatin (hCgA(352-372)) would induce directed monocyte migration. We demonstrate that catestatin dose-dependently stimulates chemotaxis of human peripheral blood monocytes, exhibiting its maximal effect at a concentration of 1 nM comparable to the established chemoattractant formylated peptide Met-Leu-Phe (fMLP). The naturally occurring catestatin variants differed in their chemotactic property insofar as that the Pro370Leu variant was even more potent than wild type, whereas the Gly364Ser variant was less effective. Specificity of this effect was shown by inhibition of catestatin-induced chemotaxis by a specific neutralizing antibody. In addition, catestatin mediated effect was blocked by dimethylsphingosine and treatment with endothelial differentiation gene (Edg)-1 and Edg-3 antisense RNA as well as by incubation with pertussis toxin and genistein indicating involvement of tyrosine kinase receptor-, G-protein- and sphingosine-1-phosphate signaling. Catestatin also stimulated Akt- and extracellular signal related kinase (ERK)-phosphorylation and catestatin-induced chemotaxis was blocked by blockers of phosphoinositide-3 (PI-3) kinase and nitric oxide as well as by inhibition of the mitogen-activated protein kinases (MAPK) system indicating involvement of these signal transduction pathways. In summary, our data indicate that catestatin induces monocyte chemotaxis by activation of a variety of signal transduction pathways suggesting a role of this peptide as an inflammatory cytokine.

A number of therapeutic strategies targeting high-density lipoprotein (HDL) cholesterol and reverse cholesterol transport are being developed to halt the progression of atherosclerosis or even induce regression. However, circulating HDL cholesterol levels alone represent an inadequate measure of therapeutic efficacy. Evaluation of the potential effects of HDL-targeted interventions on atherosclerosis requires reliable assays of HDL function and surrogate markers of efficacy. Promotion of macrophage cholesterol efflux and reverse cholesterol transport is thought to be one of the most important mechanisms by which HDL protects against atherosclerosis, and methods to assess this pathway in vivo are being developed. Indexes of monocyte chemotaxis, endothelial inflammation, oxidation, nitric oxide production, and thrombosis reveal other dimensions of HDL functionality. Robust, reproducible assays that can be performed widely are needed to move this field forward and permit effective assessment of the therapeutic potential of HDL-targeted therapies.

A new series of CCR2 antagonists has been discovered that incorporates intramolecular hydrogen bonding as a strategy for rigidifying the scaffold. The structure–activity relationship was established through initial systematic modification of substitution pattern and chain length, followed by independent optimization of three different substituents (benzylamine, carboxamide, and benzamide). Several of the acyclic compounds display 10–30 nM binding affinity for CCR2. Moreover, these antagonists are able to block both MCP-1-induced Ca2+ flux and monocyte chemotaxis, and are selective for binding to CCR2 over CCR1 and CCR3.

Although Polycystic Kidney Disease (PKD) is a leading cause of renal and hepatic pathologies, there are limited therapies available. PKD presents with cystic manifestations leading to organ enlargement, loss of functional parenchyma, progressive organ dysfunction and hypertension, ultimately leading to organ failure.Cardiac B-type natriuretic peptide (BNP) is central in the control of intravascular volume by regulating water and sodium homeostasis. Additionally, BNP has been characterized to counteract several key properties contributing to cystic disease progression such as; inhibiting fibroblast proliferation, preventing monocyte chemotaxis, antagonizing vasopressin (AVP)-mediated water reabsorption, and preventing renin-angiotensin-aldosterone activation. Along with its blood pressure-lowering propensity, BNP presents a promising therapeutic strategy in hindering the progression of PKD.We investigated the therapeutic effect of vector mediated BNP expression in the rat model of ARPKD (PCK). Littermate PCK neonates received placebo, or single intraperitoneal injection of adeno-associated virus 9 (AAV9) vectors at 1.0E+12 genome copies/kg, under the control of an internal CMV promoter.Three months post treatment, kidney weight was significantly reduced (TKW/BW 12.6±1.7* vs 14.5±2.0) in treated PCK, along with a significant reduction in renal cyst burden. Blood and urine analysis confirmed decreased disease with BNP treatment, with a nearly 50% reduction in 24 hour urine output, (8.8±3* vs 13.2±1 mL/24-hr), higher creatinine clearance (eGFR: 0.51±0.1* vs 0.26±0.2 mL/min), decreased proteinuria (490±270* vs 270±190 mg/dL), and increased urine concentrations of sodium (130±39* vs 85±20 mmol/L) and potassium (395.8±41* vs 282.1±75 mmol/L) in treated PCK, compared to controls, respectively. Excretion of renal injury markers, kidney injury molecule-1 and lipocalin-2, was lower in BNP-treated rats compared to controls. BNP treatment also preserved glomerular architecture, retarded basement membrane thickening and glomerular sclerosis in PCK. Real-time RT-PCR analysis demonstrated that BNP treatment significantly reduced renal and hepatic expression of the fibrosis-associated genes; Collagen, fibronectin, and transforming growth factor β. Furthermore, liver protection, in BNP-treated PCK, was observed through decreased expression of fibrosis marker desmin, and galectin-3, along with decreased cyst-index. Additionally, echocardiography demonstrated preserved cardiac function with treatment (ejection fraction: 78±5 * vs 73±5%) at three months, though both groups remained normotensive.Together, these observations indicate that a single intraperitoneal AAV9 injection elicits therapeutic benefit in protecting against declining hepatic and renal function, remodeling of healthy parenchyma, and increased cyst burden, while providing cardiac protection. The present study further suggests that sustained GC-A activation provides a novel opportunity for the treatment of polycystic kidney and liver diseases.±±

There has long been a need for biomarkers of disease activity in lupus nephritis (LN). Such markers ideally would be capable of detecting early sub-clinical disease and could be used to gauge response to therapy thus obviating the need for serial renal biopsies. Since urine can be readily obtained it lends itself as an obvious biological sample. Much of the focus has been on the measurement of urinary chemokines and cytokines in patients with LN. Elevations in urinary IL-6 and IL-10 had initially been reported to be associated with disease activity in LN but these markers have proven to be less reliable in larger studies. We and others have recently reported that MCP-1, a key chemokine involved in monocyte chemotaxis can be consistently found at high levels in the urine of patients with active LN. Moreover urinary MCP-1 levels decline with treatment of nephritis. In contrast urinary IL-8, a chemokine involved primarily in neutrophil chemotaxis is not a good predictor of disease activity in LN. Further longitudinal studies with larger numbers of patients are needed to determine the utility of urinary biomarkers such as MCP-1 which may act as surrogates of ongoing inflammation in LN.

Chronic hypoxia has been reported to be associated with macrophage infiltration in progressive forms of kidney disease. Here, we investigated the regulatory effects of hypoxia on constitutive and TNF-alpha-stimulated expression of monocyte chemoattractant protein-1 (MCP-1) in cultured human proximal renal tubular cells (HPTECs). Hypoxia reduced constitutive MCP-1 expression at the mRNA and protein levels in a time-dependent fashion for up to 48 h. Hypoxia also inhibited MCP-1 up-regulation by TNF-alpha. Treatment with actinomycin D showed that hypoxic down-regulation of MCP-1 expression resulted mainly from a decrease in the transcription but not the mRNA stability. Immunoblot and immunofluorescence analyses revealed that treatment with hypoxia or an iron chelator, desferrioxamine, induced nuclear accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) in HPTECs. Desferrioxamine mimicked hypoxia in the reduction of MCP-1 expression. However, overexpression of a dominant negative form of HIF-1alpha did not abolish the hypoxia-induced reduction of MCP-1 expression in HPTECs. These results suggest that hypoxia is an important negative regulator of monocyte chemotaxis to the renal inflamed interstitium, by reducing MCP-1 expression partly via hypoxia-activated signals other than the HIF-1 pathway.

Bone mineral density (BMD) is a major risk factor for osteoporosis. Circulating monocytes may serve as early progenitors of osteoclasts and produce a wide variety of factors important to bone metabolism. However, little is known about the roles of circulating monocytes in relation to the pathophysiology of osteoporosis. Using the Affymetrix HG-U133A GeneChip(R) array, we performed a comparative gene expression study of circulating monocytes in subjects with high and low BMD. We identified in total 66 differentially expressed genes including some novel as well as some already known to be relevant to bone metabolism. Three genes potentially contributing to bone metabolism, CCR3 (chemokine receptor 3), HDC (histidine decarboxylase, i.e. the histamine synthesis enzyme), and GCR (glucocorticoid receptor), were confirmed by quantitative real-time reverse transcriptase-PCR as up-regulated in subjects with lower BMD. In addition, significant negative correlation was observed between expression levels of the genes and BMD Z-scores. These three genes and/or their products mediate monocyte chemotaxis, histamine production, and/or sensitivity to glucocorticoids. Our results suggest a novel pathophysiological mechanism for osteoporosis that is characterized by increased recruitment of circulating monocyte into bone, enhanced monocyte differentiation into osteoclasts, as well as osteoclast stimulation via monocyte functional changes. This is the first in vivo microarray study of osteoporosis in humans. The results may contribute to identification of new genes and their functions for osteoporosis and suggest genetic markers to discern individuals at higher risk to osteoporosis with an aim for preventive intervention and treatment.

Chronic neuroinflammation plays a prominent role in the progression of Alzheimer's disease. Reactive microglia and astrocytes are observed within the hippocampus during the early stages of the disease. Epidemiological findings suggest that anti-inflammatory therapies may slow the onset of Alzheimer's disease. Chemokine receptor 5 (CCR5) up-regulation may influence the recruitment and accumulation of glia near senile plaques; activated microglia express CCR5 and reactive astrocytes express chemokines. We have previously shown that neuroinflammation induced by chronic infusion of lipopolysaccharide into the 4th ventricle reproduces many of the behavioral, neurochemical, electrophysiological and neuropathological changes associated with Alzheimer's disease. The current study investigated the ability of D-Ala-peptide T-amide (DAPTA), a chemokine receptor 5 chemokine receptor antagonist of monocyte chemotaxis, to influence the consequences of chronic infusion of lipopolysaccharide. DAPTA (0.01 mg/kg, s.c., for 14 days) dramatically reduced the number of activated microglia and astrocytes, as compared with lipopolysaccharide-infused rats treated with vehicle. DAPTA treatment also reduced the number of immunoreactive cells expressing nuclear factor kappa binding protein, a prominent component of the proinflammatory cytokine signaling pathway. The present study suggests that DAPTA and other CCR5 antagonists may attenuate critical aspects of the neuroinflammation associated with Alzheimer's disease.

The apolipoprotein A-I mimetic peptide 4F (Ac-DWFKAFYDKVAEKFKEAF-NH(2)), with four Phe residues on the nonpolar face of the amphipathic alpha-helix, is strongly anti-inflammatory, whereas two 3F analogs (3F(3) and 3F(14)) are not. To understand how changes in helix nonpolar face structure affect function, two additional 3F analogs, Ac-DKLKAFYDKVFEWAKEAF-NH(2) (3F-1) and Ac-DKWKAVYDKFAEAFKEFL-NH(2) (3F-2), were designed using the same amino acid composition as 3F(3) and 3F(14). The aromatic residues in 3F-1 and 3F-2 are near the polar-nonpolar interface and at the center of the nonpolar face of the helix, respectively. Like 4F, but in contrast to 3F(3) and 3F(14), these peptides effectively inhibited lytic peptide-induced hemolysis, oxidized phospholipid-induced monocyte chemotaxis, and scavenged lipid hydroperoxides from low density lipoprotein. High pressure liquid chromatography retention times and monolayer exclusion pressures indicated that there is no direct correlation of peptide function with lipid affinity. Fluorescence studies suggested that, although the peptides bind phospholipids similarly, the Trp residue in 4F, 3F-1, and 3F-2 is less motionally restricted than in 3F(3) and 3F(14). Based on these results and molecular modeling studies, we propose that the arrangement of aromatic residues in class A amphipathic helical molecules regulates entry of reactive oxygen species into peptide-phospholipid complexes, thereby reducing the extent of monocyte chemotaxis, an important step in atherosclerosis.

Monocyte chemoattractant protein-1 (MCP-1) is a potential therapeutic target for the treatment of several inflammatory conditions, including rheumatoid arthritis and chronic obstructive pulmonary disease. Current cell-based assays for MCP-1 use monocyte chemotaxis or calcium flux as a readout. Here, we describe an alternative bioassay based on MCP-1-induced phosphorylation of the mitogen-activated protein kinases (MAPK) p44 (ERK1) and p42 (ERK2). Adherent cells expressing the MCP-1 receptor CCR2B are treated with MCP-1 in 96-well plates in the presence or absence of inhibitors, fixed and permeabilized with methanol, and then probed with a monoclonal antibody that selectively recognizes the doubly phosphorylated form of p44/42 MAPK. Bound antibody is detected with a secondary antibody-peroxidase conjugate and a chromogenic substrate. The phosphorylation of p44/42 MAPK as detected in this assay peaks after 3-5 min of MCP-1 treatment, and the concentration of MCP-1 required for half-maximal p44/42 MAPK phosphorylation is 1-3 nM. MCP-1-induced phosphorylation of p44/42 MAPK is dependent upon the expression of CCR2B. The assay can be used for screening and characterization of small molecule inhibitors and antibodies blocking the binding of MCP-1 to its receptor. Since the assay is rapid and simple, it may represent a useful alternative to chemotaxis or calcium mobilization assays for the analysis of MCP-1 inhibitors.

Cardiovascular disease is significantly increased in patients with metabolic syndrome and type 2 diabetes. Several factors such as chronic hyperglycemia, lipId abnormalities, endothelium dysfunction, inflammation, oxIdative stress, increased thrombosis and decreased fibrinolysis are likely to promote cardiovascular events in these patients. Because of positive effects on glucose homeostasis, lipId metabolism, proteins involved in all stages of atherogenesis, endothelium function, inflammation, thrombosis and fibrinolysis, PPARS alpha (fibrates) and PPARs gamma (glitazones) agonists are good candIdates to reduce cardiovascular disease, more precisely in subjects with metabolic syndrome or type 2 diabetes. PPARS alpha agonists (fibrates) are potent hypolipIdemic agents increasing plasma HDL-cholesterol and reducing free fatty acIds, triglycerIdes, LDL-cholesterol and the number of small dense LDL pArticles. Moreover, they reduce vascular inflammation and thrombosis, promote fibrinolysis and inhibit the production of the vasoconstrictor factor, endothelin-1, by the endothelium. They have been shown, in clinical trials, to reduce cardiovascular disease, more particularly in patients displaying lipId abnormalities typical of metabolic syndrome and type 2 diabetes (high triglycerIdes, low HDL-cholesterol). PPARS gamma agonists (glitazones) have not only beneficial effects on glucose homeostasis, by increasing insulin sensitivity and reducing blood glucose level but also on lipId metabolism by elevating plasma HDL-cholesterol, decreasing free fatty acIds and the number of small dense LDL pArticles, and for pioglitazone by reducing plasma triglycerIdes. Furthermore, they diminish vascular inflammation and vasoconstriction, inhibit monocyte chemotaxis, proliferation and migration of smooth muscle cells, in the vascular wall and decrease the production of adhesion molecules and metalloproteinases. PPARs gamma agonists (glitazones) have been shown to reduce the development of atherosclerotic lesions in rats. The potential clinical benefit of PPARs gamma agonists on the reduction of cardiovascular disease, in type 2 diabetic patients, will be specified by the ongoing intervention studies.

Objective: Exposure of the subendothelium to flowing blood following rupture of atherosclerotic lesions or during balloon angioplasty initiates platelet adhesion to the vascular wall. Because activated platelets release proinflammatory mediators (e.g., interleukin (IL)-1β) and secrete growth factors, platelet adhesion to the subendothelial matrix might contribute to the recruitment of inflammatory cells and promote migration and proliferation of vascular smooth muscle cells (SMCs). Methods and Results: Here, we demonstrate that incubation of cultured monolayers of aortic SMCs with α-thrombin-activated platelets significantly enhances the secretion of monocyte chemoattractant protein-1 (MCP-1) (P

We examined the expression and participation of CCR6 and its ligand MIP-3alpha in rheumatoid arthritis (RA) by ELISA, RT-PCR, real-time PCR (TaqMan) analysis, monocyte chemotaxis, and two- and four-color flow cytometry. We found that RA synovial fluid (SF) contained significantly more MIP-3alpha than osteoarthritis (OA), indicating a potential role for MIP-3alpha in RA. IL-1beta, IL-18, and TNF-alpha stimulated RA fibroblast MIP-3alpha production at 48 hours of incubation in vitro. By TaqMan analysis, there were more CCR6 mRNA transcripts in RA synovial tissue (ST) than in OA or normal (NL) ST, and elevated MIP-3alpha mRNA expression in RA compared with NL ST. By FACS analysis, there were significantly elevated percentages of CD3+/CD4+/CD45RO+/CCR6+ memory lymphocytes found in RA peripheral blood (PB) compared with NL PB or RA SF. We also found that MIP-3alpha induced monocyte chemotactic activity at 1.25 pM, consistent with concentrations of MIP-3alpha found in RA SF. Furthermore, MIP-3alpha accounted for 40% of RA SF chemotactic activity for monocytes in modified Boyden chamber assays. We confirmed that MIP-3alpha may be binding a G-coupled protein receptor because in vitro monocyte chemotaxis was inhibited by preincubation of monocytes with pertussis toxin. RA patient clinical data revealed that CD3+ lymphocyte/CCR6 expression inversely correlated with the age of the patient, indicating that CCR6 expression may be important in the development of RA at a younger age. Overall, these studies indicate that MIP-3alpha and CCR6 may function to recruit monocytes and memory lymphocytes from the RA PB to the RA joint. These results further indicate that expression of CCR6, the receptor for MIP-3alpha, can be correlated with RA development.

Qingkailing (QKL) and Shuanghuanglian (SHHL) are two commonly used Chinese herbal preparations with reported antiinflammatory activity. The effects of these two preparations on the capacity of staphylococcal toxic shock syndrome toxin 1 (TSST-1) to stimulate the production of cytokines (IL-1beta, IL-6, TNF-alpha, IFN-gamma) and chemokines (MIP-1alpha, MIP-1beta and MCP-1) by peripheral blood mononuclear cell (PBMC) was tested. We also evaluated their effect on LPS-stimulated NF-kappaB transcriptional activity in a THP-1 cell line, and on human monocyte chemotactic response to chemoattractants. Non-cytotoxic concentrations of QKL (0.1 to approximately 2%) and SHHL (6 to approximately 120 microg) significantly inhibited production of cytokines and chemokines in a dose-dependent manner (P < 0.05). Both, QKL at 1:100 and SHHL at 60 microg/ml, markedly inhibited RANTES, MIP-1alpha, SDF-1alpha and fMLP induced human monocyte migration (P < 0.05 or 0.01). QKL (1%) did not inhibit monocyte chemotaxis induced by super-or sub-optimal concentrations of fMLP (10(-5), 10(-6) and 10(-10) M), but only inhibited chemotaxis induced by optimal concentrations of fMLP at 10(-7), 10(-8) and 10(-9) M. QKL (0.1% or 1%) and SHHL (6 or 60 microg/ml) markedly inhibited LPS-induced NF-kappaB activity in THP-1 cells. The results suggested that the pharmacological basis for the antiinflammatory effects of QKL and SHHL is the result of suppression of NF-kappaB regulated gene transcription, leading to suppressed production of proinflammatory cytokine and chemokine. Interference with leukocyte chemotaxis also contributes to the antiinflammatory and immunomodulating effects of these medicinals. Identification of the responsible components in these two herbal preparations may yield compounds suitable for structural modification into potent novel drugs.

Background: Chronic inflammation is a characteristic feature of abdominal aortic aneurysms (AAAs), but the molecular signals responsible for recruiting monocytes into the outer aortic wall are unresolved. The purpose of this study was to examine whether AAA tissues elaborate chemotactic activity for mononuclear phagocytes and to determine whether this activity is attributable to interactions between elastin degradation peptides (EDPs) and their cell surface receptor, the 67-kD elastin binding protein (EBP). Material and Methods: Soluble proteins were extracted from human AAA tissues, and chemotactic activity for differentiated U937 mononuclear phagocytes was measured by use of a modified Boyden chamber. Chemotactic activity induced by N -formyl-Met-Leu-Phe was used as a positive control and checkerboard analysis was used to distinguish chemotaxis from chemokinesis. Inhibition of chemotaxis was tested by peptide competition, blocking antibodies and galactosugar-mediated dissociation of the 67-kD EBP. Results: AAA extracts stimulated a concentration-dependent increase in monocyte migration that reached up to 24% of the maximal effect induced by N -formyl-Met-Leu-Phe. Checkerboard analysis demonstrated that AAA extracts stimulated chemotaxis without a chemokinetic effect. AAA-derived chemotactic activity was eliminated by competition with Val-Gly-Val-Arg-Pro-Gly (VGVAPG), a repetitive peptide found in human elastin that binds to cellular elastin receptors, and decreased nearly 40% in the presence of BA-4, an antielastin monoclonal antibody that can block EDP-mediated chemotactic activity. Monocyte chemotaxis in response to both VGVAPG and AAA extracts was abolished in the presence of lactose, a galactosugar that specifically dissociates the 67-kD EBP, but it was unaffected by either glucose, fructose, or mannose. Conclusions: These findings indicate that soluble EDPs released within human AAA tissue can subsequently attract mononuclear phagocytes through ligand-receptor interactions with the 67-kD EBP, thereby providing a plausible molecular mechanism to explain the inflammatory response that accompanies aneurysmal degeneration. Better understanding of factors regulating inflammatory cell recruitment may lead to novel forms of therapy for early stages of aneurysmal degeneration. (J Vasc Surg 2002;35:254-61.)

Factor VIII-related antigen (FVIII-RA)-positive microvessel areas were measured by both immunohistochemistry and computerized image analysis in patients with active multiple myeloma (MM), nonactive MM, and monoclonal gammopathies of undetermined significance (MGUS). A five-to sixfold larger area was found in patients with active MM compared to the other two groups. Aquaporin 1 (AQP1)-positive microvessel areas, measured with the same techniques on adjacent tissue sections, were also increased in active MM, and tended to be larger than and closely correlated with the FVIII-RA areas. Numerous mast cells were found in the bone marrow of active MM patients, and counts were strictly correlated with the microvessel density. The conditioned medium (CM) of bone marrow plasma cells from active MM patients stimulated endothelial cell proliferation and chemotaxis, monocyte chemotaxis, and angiogenesis in vivo (assessed by the chick embryo chorioallantoic membrane [CAM] system) more strongly and frequently than the CM of patients with nonactive MM and MGUS. Immunoassay of plasma cell lysates gave significantly higher levels of fibroblast growth factor-2 (FGF-2) in patients with active MM than in the other two groups, and a neutralizing anti-FGF-2 antibody inhibited by 46% to 68% the biological activity exerted by the CM in vitro and in the CAM. In situ hybridization of bone marrow plasma cells and zymography of CM showed that patients with active MM express higher levels of matrix metalloproteinase-2 (MMP-2) mRNA and protein than those with nonactive MM and MGUS, whereas MMP-9 expression and secretion overlapped in all groups. Overall data indicate that patients with active MM represent the vascular phase of plasma cell tumors that is induced, at least partly, through FGF-2 and MMP-2. Mast cells possibly contribute to the vascular phase via angiogenic factors in their secretory granules. Both angiogenesis and MMP-2 secretion can account for intramedullary and extramedullary spreading of plasma cells in patients with active MM.

The oxidation of apolipoprotein B-containing lipoproteins and cell membrane lipids is believed to play an integral role in the development of fatty streak lesions, an initial step in atherogenesis. We have previously shown that two antioxidant-like enzymes, paraoxonase (PON)-1 and PON3, are high density lipoprotein-associated proteins capable of preventing the oxidative modification of low density lipoprotein (LDL) (Reddy, S. T., Wadleigh, D. J., Grijalva, V., Ng, C., Hama, S., Gangopadhyay, A., Shih, D. M., Lusis, A. J., Navab, M., and Fogelman, A. M. (2001)Arterioscler. Thromb. Vasc. Biol. 21, 542–547). In the present study, we demonstrate that PON2 (i) is not associated with high density lipoprotein; (ii) has antioxidant properties; and (iii) prevents LDL lipid peroxidation, reverses the oxidation of mildly oxidized LDL (MM-LDL), and inhibits the ability of MM-LDL to induce monocyte chemotaxis. The PON2 protein was overexpressed in HeLa cells using the tetracycline-inducible (“Tet-On”) system, and its antioxidant capacity was measured in a fluorometric assay. Cells that overexpressed PON2 showed significantly less intracellular oxidative stress following treatment with hydrogen peroxide or oxidized phospholipid. Moreover, cells that overexpressed PON2 were also less effective in oxidizing and modifying LDL and, in fact, were able to reverse the effects of preformed MM-LDL. Our results suggest that PON2 possesses antioxidant properties similar to those of PON1 and PON3. However, in contrast to PON1 and PON3, PON2 may exert its antioxidant functions at the cellular level, joining the host of intracellular antioxidant enzymes that protect cells from oxidative stress.

Lipoprotein-X stimulates monocyte chemoattractant protein-1 expression in mesangial cells via nuclear factor-κB . Background Lipoprotein-X (Lp-X) is an abnormal lipoprotein found in the plasma of patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency. The majority of patients with this disorder develop progressive glomerulosclerosis. One key event in the pathogenesis of glomerulosclerosis is the infiltration of monocytes into affected glomeruli. Mesangial cells can synthesize and secrete monocyte chemoattractant protein-1 (MCP-1), an important chemoattractant for monocytes. The objective of the present study was to examine the effect of Lp-X on MCP-1 expression in mesangial cells leading to an enhanced monocyte chemotaxis and to elucidate the mechanisms involved in this process. Methods Lp-X was isolated from the plasma of a patient with familial LCAT deficiency. After rat mesangial cells were incubated with Lp-X for four or six hours, the expression of MCP-1 mRNA was determined by nuclease protection assay, and MCP-1 protein was measured by Western immunoblotting analysis. Monocyte chemotaxis was determined by using a Micro Chemotaxis Chamber. Results Lp-X (50 to 100nmol/mL) stimulated mesangial cell MCP-1 mRNA expression (137 to 220%) and MCP-1 protein levels (233 to 375%). Conditioned media collected from Lp-X–treated mesangial cells stimulated human acute monocytic leukemia (THP-1) monocyte chemotaxis (165 to 200%). The increase in MCP-1 expression in mesangial cells was associated with an elevation of intracellular diacylglycerol levels, and activation of protein kinase C (PKC) as well as nuclear factor-κB (NF-κB). Conclusion These results suggest that Lp-X participates in the pathogenesis of glomerulosclerosis and subsequent renal failure in familial LCAT deficient patients by stimulating monocyte infiltration via a mechanism involving mesangial MCP-1 expression.

Cellular adhesion molecules such as E-selectin function to recruit leukocytes into the inflammatory lesions of diseases such as rheumatoid arthritis (RA) and atherosclerosis. Monocytes are the key components of the cellular infiltrates present in these disorders. We hypothesized that soluble E-selectin (sE-selectin) might mediate the chemotaxis of monocytes. In this report, we show that sE-selectin induced normal human peripheral blood monocyte migration in the nanomolar range in a concentration-dependent manner. Neutralization studies using RA human joint synovial fluids and anti-E-selectin antibody showed a mean 31% reduction in RA synovial fluid-mediated monocyte chemotaxis (p0.05), indicating that sE-selectin is a major monocyte recruiter in RA. Next, we investigated the role of tyrosine phosphorylation pathways in sE-selectin-induced monocyte chemotaxis. Human peripheral blood monocytes stimulated with sE-selectin showed a time-dependent increase in the tyrosine phosphorylation of a broad range of cellular proteins, predominantly in the molecular size range of Src family kinases (50-60 kDa) and mitogen-activated protein kinases (MAPKs). Western blot analysis of Src family kinases showed a time-dependent increase in Src, Hck, and Lyn phosphorylation. The pretreatment of monocytes with the Src inhibitor AG1879: 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2) prior to stimulation with sE-selectin markedly inhibited Hck and Lyn phosphorylation, whereas the phosphorylation of Src was partially inhibited. In addition, the sE-selectin stimulation of monocytes resulted in the increased phosphorylation of extracellular signal-related kinase (ERK1/2) and p38 MAPK. The pretreatment of monocytes with PP2 showed 89 and 83% inhibition of ERK1/2 and p38 MAPK phosphorylation, respectively. sE-selectin also showed a time-dependent activation of Ras kinase. Furthermore, the pretreatment of monocytes with PP2 completely inhibited sE-selectin-mediated monocyte chemotaxis. Taken together, our data demonstrate a novel function for sE-selectin as a monocyte chemotactic agent and suggest that sE-selectin might be mediating its biological functions through the Src-MAPK pathway.

Human glycodelin is synthesized by endometrial cells in the late secretory phase and early pregnancy under hormonal regulation. Whereas the precise physiological functions of glycodelin are unknown, its expression during embryonic nidation and its inhibition of T cell proliferation suggest an immunomodulatory role. We purified human glycodelin from first trimester human decidual cytosol by using a rapid two-step high-performance liquid chromatography method and investigated its effects on human monocyte migration. Human U937 cells were used as a model of monocyte chemotaxis in Boyden chamber migration assays. N-Formyl-Met-Leu-Phe and the beta-chemokine RANTES (regulated on activation normal T cell expressed and secreted) were used as monocyte chemoattractants. Purified glycodelin inhibited monocyte migration in a dose-dependent fashion (IC50 = 550 nm). Glycodelin activity was totally reversed by heat inactivation (95 degrees C x 15 min) and neutralized by pretreatment with specific anti-glycodelin antibodies. Deglycosylated glycodelin was equipotent to intact glycodelin in the monocyte migration assay. 125I-Glycodelin binding to whole U937 cells revealed a single, saturable site with a Kd = 48 +/- 21 nm by Scatchard analysis. Cross-linking studies indicated that glycodelin binds to a high molecular mass (approximately 250 kDa) protein complex at the monocyte cell surface. Our findings support the hypothesis that glycodelin reduces the local maternal inflammatory response toward the implantation of a semiallogeneic conceptus.

Previous findings suggest that both the Tat polypeptide encoded by HIV-1 and Tat-derived peptides can induce angiogenesis via activation of the KDR receptor for Vascular Endothelial Growth Factor (VEGF). We identified 20 amino acids and 12 amino acid peptides corresponding to the cysteine-rich and basic domains of HIV-1 Tat which inhibited 125 I-VEGF165 binding to KDR and neuropilin-1 (NP-1) receptors in endothelial cells. Cysteine-rich and basic Tat peptides inhibited VEGFinduced ERK activation and mitogenesis in endothelial cells, and inhibited angiogenesis in vitro at concentrations similar to those which inhibited VEGF receptor binding. These peptides also inhibited proliferation, angiogenesis, and ERK activation induced by basic fibroblast growth factor with similar potency and efficacy. Surprisingly, we found that both cysteine-rich and basic domain Tat peptides strikingly induced apoptosis in endothelial cells, independent of their effects on VEGF and bFGF. Furthermore, we found no evidence for direct biological effects of recombinant Tat on VEGF receptor binding, ERK activation, endothelial cell survival, or mitogenesis. These findings demonstrate novel properties of Tat-derived peptides and indicate that their major effect in endothelial cells is apoptosis independent of specific inhibition of VEGF receptor activation. © 2001 Academic Press

The chemokine RANTES (regulated on activation normal T cell expressed and secreted; CCL5) binds selectively to glycosaminoglycans (GAGs) such as heparin, chondroitin sulfate, and dermatan sulfate. The primary sequence of RANTES contains two clusters of basic residues, (44)RKNR(47) and (55)KKWVR(59). The first is a BBXB motif common in heparin-binding proteins, and the second is located in the loop directly preceding the C-terminal helix. We have mutated these residues to alanine, both as point mutations as well as triple mutations of the 40s and 50s clusters. Using a binding assay to heparin beads with radiolabeled proteins, the (44)AANA(47) mutant demonstrated an 80% reduction in its capacity to bind heparin, whereas the (55)AAWVA(59) mutant retained full binding capacity. Mutation of the (44)RKNR(47) site reduced the selectivity of RANTES binding to different GAGs. The mutants were tested for their integrity by receptor binding assays on CCR1 and CCR5 as well as their ability to induce chemotaxis in vitro. In all assays the single point mutations and the triple 50s cluster mutation caused no significant difference in activity compared with the wild type sequence. However, the triple 40s mutant showed a 80-fold reduction in affinity for CCR1, despite normal binding to CCR5. It was only able to induce monocyte chemotaxis at micromolar concentrations. The triple 40s mutant was also able to inhibit HIV-1 infectivity, but consistent with its abrogated GAG binding capacity, it no longer induced enhanced infectivity at high concentrations.

We previously reported that glutaredoxin 1 (Grx1) protects monocytes from metabolic-stress induced priming, dysregulation and hypersensitization to chemokines. To address the role of monocytic Grx1 in mice and in the development of atherogenesis and obesity, we transplanted bone marrow (BM) from either wild-type (WT) or Grx1-/- (Grx1Leuko -/-) donor mice into atherosclerosis-prone LDLR-/- mice and fed these mice a high-fat diet (HFD) for up to 20 weeks. Grx1Leuko -/- mice showed accelerated weight gain after 9 weeks followed by early onset of hyperglycemia. After 6 weeks on HFD, atherosclerotic lesions were slightly larger in Grx1Leuko -/- mice than in WT, but the differences did not reach statistical significance. However, after 20 weeks, Grx1Leuko -/- mice showed 36% larger lesions than WT-BM recipients, and monocyte chemotaxis in vivo was increased 1.6-fold. Furthermore, compared to WT-BM recipients, adipose tissues and livers of Grx1Leuko -/- mice also showed increased macrophage content and elevated tissue inflammation as determined by IHC and qRT-PCR. Adipose tissue in particular, showed significant increases in the expression of proinflammatory genes in addition to an increased abundance proinflammatory “crown-like” structures. Gene expression analysis by qRT-PCR of peritoneal macrophages revealed that macrophages isolated from Grx1-/- mice also have increased expression of pro-inflammatory M1-associated genes and decreased M2-associated genes after activation with INFγ+TNFα and IL-4 respectively. Additionally, macrophages from Grx1-/- mice exposed to metabolic stress also display increased protein-S-glutathionylation, enhanced hypersensitization to chemokine, and impaired autophagy compared to wild-type cells. Taken together, our data show that loss of monocytic Grx1 worsens monocyte priming and accelerates the infiltration of dysfunctional monocyte-derived macrophages into tissues, such as aorta, liver and adipose tissues. We conclude that monocytic Grx1 is critical for maintaining metabolic homeostasis in mice and protects against obesity and atherosclerosis.