ABSTRACT
BACKGROUND: Venous grafts are commonly used as conduits to bypass occluded arteries. Unfortunately, patency rates are limited by vein graft disease (VGD). Toll like receptors (TLRs) can be activated in vein grafts by endogenous ligands. This study aims to investigate the role of TLR3 in VGD. METHODS: Vein graft surgery was performed by donor caval vein interpositioning in the carotid artery of recipient Tlr2-/-, Tlr3-/-, Tlr4-/- and control mice. Vein grafts were harvested 7, 14 and 28d after surgery to perform immunohistochemical analysis. Expression of TLR-responsive genes in vein grafts was analysed using a RT2-profiler PCR Array. mRNA expression of type-I IFN inducible genes was measured with qPCR in bone marrow-derived macrophages (BMM). RESULTS: TLR2, TLR3 and TLR4 were observed on vein graft endothelial cells, smooth muscle cells and macrophages. Tlr3-/- vein grafts demonstrated no differences in vessel wall thickening after 7d, but after 14d a 2.0-fold increase (pâ¯=â¯0.02) and 28d a 1.8-fold increase (pâ¯=â¯0.009) compared to control vein grafts was observed, with an increased number of macrophages (pâ¯=â¯0.002) in the vein graft. Vessel wall thickening in Tlr4-/- decreased 0.6-fold (pâ¯=â¯0.04) and showed no differences in Tlr2-/- compared to control vein grafts. RT2-profiler array revealed a down-regulation of type-I IFN inducible genes in Tlr3-/- vein grafts. PolyI:C stimulated BMM of Tlr3-/- mice showed a reduction of Ifit1 (pâ¯=â¯0.003) and Mx1 (pâ¯<â¯0.0001) mRNA compared to control. CONCLUSIONS: We here demonstrate that TLR3 can play a protective role in VGD development, possibly regulated via type-I IFNs and a reduced inflammatory response.
Subject(s)
Carrier Proteins/genetics , Toll-Like Receptor 3/genetics , Transplants/metabolism , Veins/growth & development , Adaptor Proteins, Signal Transducing , Animals , Bone Marrow Cells/metabolism , Bone Marrow Cells/pathology , Carotid Arteries/growth & development , Carotid Arteries/metabolism , Cell Differentiation/genetics , Endothelial Cells/metabolism , Endothelial Cells/pathology , Gene Expression Regulation/genetics , Humans , Interferon Type I/genetics , Ligands , Macrophages/metabolism , Mice , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , RNA-Binding Proteins , Signal Transduction/genetics , Toll-Like Receptor 2/genetics , Toll-Like Receptor 4/genetics , Transplants/growth & development , Transplants/pathology , Veins/metabolismABSTRACT
BACKGROUND: T cells have a distinctive role in neovascularization, which consists of arteriogenesis and angiogenesis under pathological conditions and vasculogenesis under physiological conditions. However, the role of co-stimulation in T cell activation in neovascularization has yet to be established. The aim of this study was to investigate the role T cell co-stimulation and inhibition in angiogenesis, arteriogenesis and vasculogenesis. METHODS AND RESULTS: Hind limb ischemia was induced by double ligation of the left femoral artery in mice and blood flow recovery was measured with Laser Doppler Perfusion Imaging in control, CD70-/-, CD80/86-/-, CD70/80/86-/- and CTLA4+/- mice. Blood flow recovery was significantly impaired in mice lacking CD70 compared to control mice, but was similar in CD80/86-/-, CTLA4+/- and control mice. Mice lacking CD70 showed impaired vasculogenesis, since the number of pre-existing collaterals was reduced as observed in the pia mater compared to control mice. In vitro an impaired capability of vascular smooth muscle cells (VSMC) to activate T cells was observed in VSMC lacking CD70. Furthermore, CD70-/-, CD80/86-/- and CD70/80/86-/- mice showed reduced angiogenesis in the soleus muscle 10â¯days after ligation. Arteriogenesis was also decreased in CD70-/- compared to control mice 10 and 28â¯days after surgery. CONCLUSIONS: The present study is the first to describe an important role for T cell activation via co-stimulation in angiogenesis, arteriogenesis and vasculogenesis, where the CD27-CD70 T cell co-stimulation pathway appears to be the most important co-stimulation pathway in pre-existing collateral formation and post-ischemic blood flow recovery, by arteriogenesis and angiogenesis.
Subject(s)
CD27 Ligand/physiology , Hindlimb/diagnostic imaging , Ischemia/diagnostic imaging , Neovascularization, Pathologic/diagnostic imaging , T-Lymphocytes/physiology , Tumor Necrosis Factor Receptor Superfamily, Member 7/physiology , Animals , CD27 Ligand/deficiency , Hindlimb/blood supply , Ischemia/physiopathology , Laser-Doppler Flowmetry/methods , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neovascularization, Pathologic/physiopathology , Neovascularization, Physiologic/physiology , Tumor Necrosis Factor Receptor Superfamily, Member 7/deficiencyABSTRACT
BACKGROUND: Toll like receptors (TLR) play an important role in vein graft disease (VGD). Interferon regulatory factors (IRF) 3 and 7 are the transcriptional regulators of type I interferons (IFN) and type I IFN responsive genes and are downstream factors of TLRs. Relatively little is known with regard to the interplay of IRFs and TLRs in VGD development. The aim of this study was to investigate the role of IRF3 and IRF7 signaling downstream TLRs and the effect of IRF3 and IRF7 in VGD. METHODS AND RESULTS: In vitro activation of TLR3 induced IRF3 and IRF7 dependent IFNß expression in bone marrow macrophages and vascular smooth muscle cells. Activation of TLR4 showed to regulate pro-inflammatory cytokines via IRF3. Vein graft surgery was performed in Irf3-/- , Irf7-/- and control mice. After 14 days Irf3-/- vein grafts had an increased vessel wall thickness compared to both control (P = 0.01) and Irf7-/- (P = 0.02) vein grafts. After 28 days, vessel wall thickness increased in Irf3-/- (P = 0.0003) and Irf7-/- (P = 0.04) compared to control vein grafts and also increased in Irf7-/- compared to Irf3-/- vein grafts (P = 0.02). Immunohistochemical analysis showed a significant higher influx of macrophages after 14 days in Irf3-/- vein grafts and after 28 days in Irf7-/- vein grafts compared to control vein grafts. CONCLUSIONS: The present study is the first to describe a protective role of both IRF3 and IRF7 in VGD. IRFs regulate VGD downstream TLRs since Irf3-/- and Irf7-/- vein grafts show increased vessel wall thickening after respectively 14 and 28 days after surgery.