Effects Of RhGM-CSF On The Expression Of CCR2 In THP-1 Monocytes

Background:Venous thrombosis is a common disease of vascular surgery in the clinical. Venous thromboembolism mainly includes deep vein thrombosis. The classic treatment strategy of this disease is non-operative therapy that mainly includes anticoagulation,preventing polymerization and thrombolysis. Anticoagulation and preventing polymerization aim at preventing new thrombus formation,but it carries a significant risk of severe hemorrhage when used for long term therapy. Although the thrombolytic therapy can dissolute formed thrombus, thrombolytic drugs can only act on the thrombus surface,it is difficult to enter the interior of thrombus and cause thrombolysis. Direct thrombolysis has not been established as a standard therapy strategy. Venous thrombosis often induce postthrombotic syndrome, cost expensive and unsatisfactory results.The resolution and absorption of venous thrombi mainly depend on body's own mechanisms, involving plasmin-mediated fibrinolysis ,as well as proteinase-mediated collagen breakdown. Growing into thrombi and replacing it of new granulation tissue is process of organization of thrombi. 1-2 days after thrombosis, monocytes,macrophages,fibroblast and vascular endothelial cells, enter into the thrombus under the role of chemokines. With the proliferation of granulation tissue, the new vessels appear within the body of the thrombus and coalesce and enlarge, and then blood flow is established through the thrombus. Complete resolution of venous thrombi would restore the recanalization, otherwise irregular or stenosed vein, and lead to postthrombotic syndrome accompanied by the destruction of venous valves and the formation of patent lumen but venous system with insufficient venous valves and reflux. Promoting angiogenesis and accelerating organization of thrombi may prevent postthrombotic complications by reducing valvular damage and residual obstruction.Current studies revealed that monocytes and play an important role in the resolution of venous thrombi. Monocytes release and direct smooth muscle cell proangiogenic release. The neovascularization in thrombi mostly exist at the area of monocyte and macrophage accumulation in human and animal studies. The resolution of thrombi was increased by the injection of monocyte and macrophage into thrombi. Updated studies have shown, through mobilization of bone marrow, the stem cells and progenitor cells derived from bone marrow involved in the resolution and absorption of venous thrombi.Prior studies have suggested an important role for chemokines in DVT resolution. Chemokines are ubiquitous peptide inflammatory mediators that act as leukocyte chemotactic agents as well as cell activators.Although four main chemokine families exist,the ones most relevant to DVT resolution are the cysteine-cysteine(CC) and cysteine-X-cysteine(CXC) chemokine types. Exogenous administration of MCP-1(a CC chemokine) and IL-8(a CXC chemokine) have been shown to accelerate thrombus resolution in experimental models. Chemokines and their receptors constitute the cell-specific signal transduction pathways, and play a chemotactic and activating role on target cells.CC chemokine primarily drive and activate monocytes. The role is required to achieve through its corresponding receptor CCR. MCP-1 and its specific receptor CCR2 constitute MCP-1/CCR2 signaling pathway, participating in migration of monocyte and macrophage, which is critical for monocytes mobilization. Genetic deletion of CCR2 was associated with fewer thrombus monocytes, reduction in both matrix metalloproteinase MMP-2 and MMP-9 activity, increased thrombus collagen, larger thrombi,and significantly impaired thrombi resolution.We hypothesized that enhancing the function of monocyte in thrombi would promote the thrombi resolution, realize the early recanalization of thrombi and reduce the postthrombotic syndrome. This study was to investigate through the use of cytokine to increase CCR2 expression in monocyte,thereby enhancing its function, in order to explore its mechanism in thrombolysis and lay a theoretical foundation and experimental basis for its use in treatment.Methods:1 Cell experimentHuman monocyte line THP-1 was grown in RPMI 1640 with 10% fetal bovine serum, 2.0mmol/L glutamine, 10.0mmol/L HEPES, 1.0 mmol/L sodium pyruvate, 5.0×10~-5mol/Lβ-Mercaptoethanol, 1.0×10~5U/L penicillin and 100mg/L streptomycin. Culture media contained the rhGM-CSF with the concentrations of 0,40,80,160ng/ml. THP-1 cells were seeded at a density of 1×10~5 cells/well into 24-well plates or 6×10~5 cells/ flask into 100ml culture flask.The influence of treatment on the secretion of MCP-1 by monocyte was determined by ELISA. The assay was conducted in accordance with the Human MCP-1 ELISA Kit manufacturer's instructions.The total RNA extract of cultured THP-1 cells was conducted in accordance with the instructions of small amount of total cellular RNA extraction kit. The RT-PCR was conducted in accordance with the instructions of TaKaRa RNA PCR Kit (AMV) Ver.3.0. PCR products were subjected to electrophoresis on 2% agarose gel, stained with Goldview, and photographed. The results were measured by Gel imaging analysis system.Protein from THP-1 cells was extracted according to manufacture's instructions with a total protein extraction kit. Equal amounts of protein were resolved on 40% Polyacrylamide / Bis Acrylamide gels under 120 V and transferred to PVDF blotting membrane. The membrane was blocked for one night in TBS-Tween. The membrane was incubated with anti-CCR2 (final dilution 1:400) followed by washes and incubation with HRP-conjugated goat anti- rabbit IgG antibody (final dilution 1:4,000). Detection of Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) antibody in the light of the above methods. The results were measured after the proteins were detected by Chemiluminescence.2 Statistical analysisSPSS11.5 was used to analyze the data. F test, Dunnett test and other testing methods were used according to the difference of data. Statistical significance was assumed for P < 0.05.Results:1 The growth of THP-1 cellsTHP-1 cells were cultured after 24 and 48h and counted in a counting chamber. The results showed that rhGM-CSF did not promote or inhibit the growth of THP-1 cells.2 Levels of MCP-1After 24h and 48h of culture, there were no statistical difference observed in MCP-1 levels in THP-1 cell culture medium between groups with different concentrations. It revealed that rhGM-CSF did not promote or inhibit THP-1 to secrete MCP-1.3 Expression of CCR2 mRNAIn different concentration groups, 80ng/ml rhGM-CSF stimulated the strongest expression of THP-1 CCR2 mRNA. Levels of CCR2 mRNA were continuously and highly expressed after 36 and 48h of 80ng/ml rhGM-CSF stimulation.4 Expression of CCR2 proteinIn different concentration groups, 80ng/ml rhGM-CSF stimulated the strongest expression of THP-1 CCR2 protein. Levels of CCR2 protein were continuously and highly expressed after 36 and 48h of 80ng/ml rhGM-CSF stimulation.Conclusion:1. rhGM-CSF has no effects on MCP-1 secrete of THP-1.2. rhGM-CSF enhances chemokine CCR2 expression in THP-1.