Zhu Hui Liu

C1q/TNF-related protein-1: an adipokine marking and promoting atherosclerosis

AIMS We investigated the association of the adipokine C1q/TNF-related protein (CTRP) 1 with coronary artery disease (CAD), and the biological vascular effects of CTRP1. METHODS AND RESULTS We analysed CTRP1 levels in sera of CAD patients (n = 451) and non-CAD controls (n = 686), and in coronary endarterectomy specimens (n = 32), non-atherosclerotic internal mammary arteries (n = 26), aortic atherosclerotic plaques (n = 15), and non-atherosclerotic aortic samples (n = 10). C1q/TNF-related protein-levels were higher in sera, endarterectomy specimens, aortic atherosclerotic plaques, and peripheral blood mononuclear cells (PBMCs) from CAD patients compared with controls, and were related to CAD severity. The production of CTRP1 was profusely induced by inflammatory cytokines and itself caused a concentration-dependent expression of adhesion molecules and inflammatory markers in human endothelial cells, human peripheral blood monocytes, and THP-1 cells. C1q/TNF-related protein-1 induced p38-dependent monocyte-endothelium adhesion in vitro and the recruitment of leucocytes to mesenteric venules in C57BL/6 mice. Immunohistochemistry of atherosclerotic femoral arteries exhibited CD68 and VE-cadherin loci-associated increased CTRP1 expression in plaques. Compared with saline, intraperitoneal injection of recombinant CTRP1 protein (200 μg/kg) every other day promoted atherogenesis in apoE(-/-) mice at 24 weeks. However, pro-atherogenic effects were significantly attenuated in CTRP1(-/-)/apoE(-/-) double-knockout mice compared with apoE(-/-) mice, with a consistent decrease in vascular adhesion molecule, phospho-p38 and TNF-α expression and macrophage infiltration in plaque in CTRP1(-/-) and double-knockout mice. Tumour necrosis factor-α-induced expression of adhesion molecules and cytokines were lower in primary endothelial cells and macrophages from CTRP(-/-) mice than in those from C57BL/6 mice. CONCLUSION C1q/TNF-related protein-1 is a marker of atherosclerosis in humans and promotes atherogenesis in mice.

Toll-like receptor 4 mediates inflammatory cytokine secretion in smooth muscle cells induced by oxidized low-density lipoprotein.

Oxidized low-density lipoprotein (oxLDL)-regulated secretion of inflammatory cytokines in smooth muscle cells (SMCs) is regarded as an important step in the progression of atherosclerosis; however, its underlying mechanism remains unclear. This study investigated the role of toll-like receptor 4 (TLR4) in oxLDL-induced expression of inflammatory cytokines in SMCs both in vivo and in vitro. We found that the levels of TLR4, interleukin 1-β (IL1-β), tumor necrosis factor-α (TNFα), monocyte chemoattractant protein 1 (MCP-1) and matrix metalloproteinase-2 (MMP-2) expression were increased in the SMCs of atherosclerotic plaques in patients with femoral artery stenosis. In cultured primary arterial SMCs from wild type mice, oxLDL caused dose- and time-dependent increase in the expression levels of TLR4 and cytokines. These effects were significantly weakened in arterial SMCs derived from TLR4 knockout mice (TLR4−/−). Moreover, the secretion of inflammatory cytokines was blocked by TLR4-specific antibodies in primary SMCs. Ox-LDL induced activation of p38 and NFκB was also inhibited in TLR4−/− primary SMCs or when treated with TLR4-specific antibodies. These results demonstrated that TLR4 is a crucial mediator in oxLDL-induced inflammatory cytokine expression and secretion, and p38 and NFκB activation.

Increased serum level of CTRP1 is associated with low coronary collateralization in stable angina patients with chronic total occlusion

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Two-Dimensional Fluorescence In-Gel Electrophoresis of Coronary Restenosis Tissues in Minipigs Increased Adipocyte Fatty Acid Binding Protein Induces Reactive Oxygen Species–Mediated Growth and Migration in Smooth Muscle Cells

Objective—We aimed to uncover the protein changes of coronary artery in-stent restenosis (ISR) tissue in minipigs with and without streptozotocin-induced diabetes mellitus by quantitative 2-dimensional fluorescence in-gel electrophoresis (2D-DIGE), and to investigate the influences of crucial proteins identified, particularly adipocyte fatty acid binding protein (AFABP), in human arterial smooth muscle cells. Methods and Results—Sirolimus-eluting stents were implanted in the coronary arteries of 15 diabetic and 26 nondiabetic minipigs, and angiography was repeated after 6 months. The intima tissue of significant ISR and non-ISR segments in both diabetic and nondiabetic minipigs was analyzed by 2D-DIGE and MALDI-TOF/TOF mass spectrometry. AFABP level was significantly increased in ISR tissue than in non-ISR tissue in both diabetic and nondiabetic minipigs, with level being higher in diabetic ISR than in nondiabetic ISR tissue. In human arterial smooth muscle cells, overexpression of AFABP significantly altered phenotype and promoted growth and migration, with effects more prominent in high-glucose than in low-glucose medium, whereas AFABP knockdown inhibited these effects. AFABP overexpression increased reactive oxygen species production by upregulating the expression of NADPH oxidase subunits Nox1, Nox4, and P22 through multiple pathways, with elevation of downstream gene cyclin D1, matrix metalloproteinase-2, and monocyte chemoattractant protein-1. However, AFABP-induced effects were inhibited by diphenyleneiodonium, pathway inhibitors, and small interfering RNA. In addition, the supernatant from AFABP-expressing human arterial smooth muscle cells and recombinant AFABP also promoted cellular growth and migration. Conclusion—This study has demonstrated that AFABP is significantly increased in coronary artery ISR segments of both diabetic and nondiabetic minipigs. Increased AFABP expression and secretory AFABP of human arterial smooth muscle cells promote growth and migration via reactive oxygen species-mediated activation.

C1q/TNF-related protein 1 links macrophage lipid metabolism to inflammation and atherosclerosis

BACKGROUND AND AIMS Macrophage is a major contributor to the development of atherosclerosis by taking up deposited lipoprotein and eliciting local inflammation. Previously, we and others have shown C1q/TNF-related proteins (CTRPs) play diverse roles in vascular functions. In this study, we sought to investigate the changes in CTRP expression levels during vital biological processes in macrophages and their relation to inflammatory responses. METHODS Western blot and real-time PCR were performed to analyze CTRPs expression levels in human peripheral blood mononuclear cells, primary macrophages and lipid-laden foam cells. Mechanisms that regulate CTPR1 expression were further investigated by bioinformatic analysis and chromatin immunoprecipitation. Enzyme-linked immunosorbent assay was performed to measure the concentration of inflammatory cytokines. RESULTS We found that almost all CTRPs were significantly increased in primary human macrophages after differentiation from peripheral blood mononuclear cells. In particular, CTRP1 was further up-regulated upon exposure to oxidized low-density lipoprotein (oxLDL) in a peroxisome proliferator-activated receptor (PPAR)-dependent manner. Chromatin immunoprecipitation also confirmed the presence of PPAR-γ in the CTRP1 promoter after oxLDL treatment. Stimulation of CTRP1 led to markedly enhanced secretion of pro-atherogenic factors, including MCP-1, TNF-α, IL-1β, and IL-6, whereas oxLDL-induced inflammatory cytokine production was significantly attenuated after the treatment with CTRP1 neutralizing antibody. CONCLUSIONS These data suggest an essential role of CTRP1 in linking dysregulation of lipid metabolism and inflammatory responses in macrophages.

ASSOCIATION OF SERUM HMGB2 LEVEL WITH MACE AT 1 MONTH OF MYOCARDIAL INFARCTION: AGGRAVATION OF MYOCARDIAL ISCHEMIC INJURY IN RAT BY HMGB2 VIA ROS

High-mobility group box (HMGB) family is related to inflammatory diseases. We investigated whether serum HMGB2 levels are related to myocardial infarction (MI) severity and major adverse cardiac events (MACE) during MI. We included 432 consecutive patients with ST-segment elevation myocardial infarction and 312 controls. Serum HMGB2 levels were significantly higher in MI patients than in controls. Increased HMGB2 levels were associated with MACE and negatively with ejection fraction in MI patients. HMGB2 was an independent determinant of MACE in logistic regression analysis. HMGB2 protein (10 μg) or saline was injected intramyocardially in MI rats, with or without coadministration of the NADPH oxidase inhibitor apocynin. After 72 h, pathological, echocardiographic, and hemodynamic examinations showed that HMGB2 increased infarct size and worsened cardiac function in MI rats. Moreover, HMGB2 administration enhanced reactive oxygen species (ROS) production, cell apoptosis, inflammation, and autophagosome clearance impairment, which were attenuated by coadministration of apocynin or knock down of receptor for advanced glycation end products (RAGE). In conclusion, increased serum HMGB2 levels are associated with MI severity and MACE at 1 mo. HMGB2 promotes myocardial ischemic injury in rats and hypoxic H9C2 cell damage via ROS provoked by RAGE.NEW & NOTEWORTHY We demonstrate that serum high-mobility group box 2 is associated with major adverse cardiac events at 1 mo in myocardial infarction patients. Mechanistically, high-mobility group box 2 promotes reactive oxygen species production via receptor for advanced glycation end products signaling in ischemic myocardium, thereby aggravating cell apoptosis, inflammation, and autophagosome clearance impairment. This study reveals that high-mobility group box 2 is a novel factor enhancing ischemic injury in myocardial infarction.

Increased serum TREM-1 level is associated with in-stent restenosis, and activation of TREM-1 promotes inflammation, proliferation and migration in vascular smooth muscle cells.

BACKGROUND AND AIMS In-stent restenosis (ISR) remains a major limitation of percutaneous coronary intervention despite improvements in stent design and pharmacological agents, whereas the mechanism of ISR has not been fully clarified. In the present study, we sought to investigate the potential association of serum soluble TREM-1 (sTREM-1) levels with the incidence of ISR. The role of TREM-1 was evaluated in cultured vascular smooth muscle cells (VSMCs). METHODS Out of 1683 patients undergoing coronary intervention and follow-up coronary angiography after approximately one year, 130 patients were diagnosed with ISR, and 150 gender- and age-matched patients with no ISR were randomly included as controls. Levels of sTREM-1 were determined by ELISA. The role of TREM-1 signaling in the activation of VSMCs was tested. RESULTS Serum sTREM-1 concentrations were significantly elevated in patients with than without ISR. Multivariable logistic regression analysis showed that sTREM-1, besides conventional factors, was independently associated with the incidence of ISR. Evident expression of TREM-1 in VSMCs was detected in the neointimal and medial layers of stenotic lesions of mouse carotid ligation models. In cultured VSMCs, expression of TREM-1 was significantly induced upon exposure to lipopolysaccharide. Blocking of TREM-1 with a synthetic inhibitory peptide LP17 dramatically inhibited, whereas TREM-1-activating antibody promoted cellular inflammation, proliferation and migration in VSMCs. CONCLUSIONS These data suggest that TREM-1 is a predictive biomarker of ISR and an important mediator of cellular inflammation, migration, and proliferation in VSMCs. Pharmacological inhibition of TREM-1 may serve as a promising approach to attenuate the progression of ISR.

C1q/TNF-related protein 1 promotes endothelial barrier dysfunction under disturbed flow

Endothelial hyper-permeability is a major determinant factor that contributes to the accelerated development of atherosclerotic lesions at hemodynamically disturbed sites. Previously, we showed that C1q/TNF related protein (CTRP) 1 promotes endothelium-leukocyte interactions and inflammatory responses in vascular cells. Here, we sought to investigate the role of CTRP1 in modulation of endothelial permeability under disturbed flow condition. By using en face staining of mouse aorta, we found CTRP1 expression was significantly increased in vascular endothelial cells under disturbed flow as compared to steady laminar flow. Vascular permeability to Evans blue dye was notably enhanced in CTRP1 knockin mice as compared to wild type animals, whereas aortic hyper-permeability at disturbed sites was remarkably restored after deletion of CTRP1. In cultured endothelial cells, treatment of CTRP1 led to increased permeability to fluorescent-labelled dextran and apparent formation of paracellular holes as observed after disturbed flow exposure, which was evidently reduced in the presence of a CTRP1-specific neutralizing antibody. Mechanistically, we found activation of VEGFR2 by CTRP1 might be involved in vascular hyper-permeability under disturbed flow condition. Taken together, this study suggests that CTRP1 is a mechano-sensitive proinflammatory factor that mediates disturbed flow-induced vascular barrier dysfunction. Inhibition of CTRP1 may inhibit the pathogenesis of atherosclerosis at early stage.

Association of Serum HMGB2 Levels With In-Stent Restenosis: HMGB2 Promotes Neointimal Hyperplasia in Mice With Femoral Artery Injury and Proliferation and Migration of VSMCs

Objective— In a previous study, we established diabetic and nondiabetic minipig models with coronary artery in-stent restenosis (ISR). Mass spectrometry showed that high-mobility group box (HMGB) 2 level was higher in ISR than in non-ISR tissue from diabetic minipigs. We here investigated whether serum HMGB2 levels were related to ISR in coronary artery disease patients. The effect of HMGB2 was evaluated in mice with femoral artery wire injury and in human aortic smooth muscle cells. Approach and Results— From 2513 patients undergoing coronary artery intervention and follow-up angiography at ≈1 year, 262 patients were diagnosed with ISR, and 298 patients with no ISR were randomly included as controls. Serum HMGB2 levels were significantly higher in patients with ISR than in those without ISR and were associated with ISR severity. Multivariable logistic regression analysis showed that HMGB2 level was independently associated with ISR. In experiments, HMGB2 expression was increased in vascular tissue after injury. Perivascular HMGB2 administration promoted injury-induced neointimal hyperplasia in C57Bl/6 mice compared with in the control, whereas such pathophysiological features were attenuated in Hmgb2–/– mice. Mechanistically, HMGB2 enhanced neointimal hyperplasia in mice and proliferation and migration in human aortic smooth muscle cells by inducing reactive oxygen species through increased p47phox phosphorylation. Knocking down p47phox, however, inhibited HMGB2-induced effects in human aortic smooth muscle cells. Finally, HMGB2-induced effects were significantly declined in receptor of advanced glycation end products knockdown or deficient cells, but not in Toll-like receptor 4 knockdown or deficient cells. Conclusions— Serum HMGB2 levels were associated with ISR in patients. HMGB2 promoted neointimal hyperplasia in mice with arterial wire injury through reactive oxygen species activation.

Increment of HFABP Level in Coronary Artery In-Stent Restenosis Segments in Diabetic and Nondiabetic Minipigs: HFABP Overexpression Promotes Multiple Pathway-Related Inflammation, Growth and Migration in Human Vascular Smooth Muscle Cells.

Background: Our previous study suggested that heart-type fatty acid-binding protein (HFABP) levels were greatly elevated in the conditioned medium of explant culture of in-stent restenosis (ISR) tissue from diabetic minipigs compared with those of non-ISR tissue. We here verified this result in animal tissues and investigated the impact of HFABP overexpression in human aortic smooth muscle cells (hASMCs). Methods and Results: In Western blot and real-time RT-PCR, HFABP protein and mRNA levels were significantly higher in ISR than in non-ISR tissues from minipigs, and higher in the ISR tissue from diabetic minipigs than that from nondiabetic minipigs. The mRNA microarray and cellular effects of hASMC retroviral overexpression of HFABP and vector was analyzed. Compared with vector, HFABP transduction activates multiple signaling pathways (e.g. adipokine, TGF-β, Toll-like receptor, Wnt, Hedgehog, ErbB and Notch) and promotes inflammation, growth and migration in hASMCs whereas the knockdown of HFABP by small hairpin RNA attenuates these effects. Conclusion: HFABP expression is significantly higher in ISR tissue than in non-ISR tissue from diabetic and nondiabetic minipigs. Overexpression of HFABP induces multiple pathway-related promotion of inflammation, growth and migration in vascular SMCs, suggesting a potential role in coronary artery ISR.