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.

Association of serum HMGB2 level with MACE at 1 mo of myocardial infarction: Aggravation of myocardial ischemic injury in rats 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.

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.

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-ß, 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.

Chromosome 9p21 polymorphism is associated with myocardial infarction but not with clinical outcome in Han Chinese.

BACKGROUND: rs1333049 polymorphism on chromosome 9p21 has been shown to affect susceptibility to coronary artery disease (CAD) in Caucasians. This study examined the association of rs1333049 with myocardial infarction (MI), angiographic severity of CAD and clinical outcome after a first acute MI in Han Chinese. METHODS: rs1333049 polymorphism was genotyped in 520 patients with a first acute MI and in 560 controls. The number of angiographically documented diseased coronary arteries (luminal diameter stenosis > or = 50%), echocardiographic left ventricular ejection fraction (LVEF), and major adverse cardiac events (MACE) during follow-up (mean, 29+/-15 months) were recorded. RESULTS: Patients with MI had higher frequencies of the CC genotype (30.0% vs. 20.7%) or C allele (55.5% vs. 46.2%) compared with controls (all p<0.01). rs1333049 polymorphism was strongly associated with MI [odds ratio (OR) 1.48, 95% confidence interval (CI) 1.22-1.79] after adjusting for traditional risk factors. Although longer hospitalization stay was observed in patients with the rs1333049-C allele, this polymorphism was not related to angiographic severity of CAD, LVEF, and occurrence of MACE after MI. CONCLUSIONS: This study demonstrates an association of rs1333049 polymorphism locus on chromosome 9p21 with risk for MI, but not with post-MI prognosis in Han Chinese.

Secretory vimentin is associated with coronary artery disease in patients and induces atherogenesis in ApoE-/- mice.

PURPOSE: The present study aimed to investigate the relationship between serum levels of secretory vimentin and coronary artery disease (CAD). The biological effect of secretory vimentin was ascertained by experiments. METHODS: We analysed serum levels of secretory vimentin in CAD patients (n = 288) and non-CAD controls (n = 195) by ELISA. To evaluate the pro-inflammatory effects of secreted vimentin, the human aortic endothelial cells (HAECs) and human peripheral blood mononuclear cells (PBMCs) were treated with recombinant vimentin or saline. Intraperitoneal injection of vimentin (1 µg/each) or saline was performed every other day for 12 weeks in ApoE-/- mice for assessment of atherogenic effect. RESULTS: Serum levels of secretory vimentin were significantly increased in CAD patients than in health controls (p < 0.05), and correlated with the number of diseased coronary arteries, Syntax and Gensini score (for all comparison, p < 0.01). Logistic regression analysis showed that vimentin level is an independent determinant of CAD. In experiments, recombinant vimentin protein enhanced the expression of adhesion molecules and inflammatory cytokines in both endothelial cells and macrophages. This protein also promoted macrophage-endothelial cells adhesion in vitro and the recruitment of leukocytes to mesenteric venules in C57BL/6 mice. Compared with saline, intraperitoneal injection of recombinant vimentin (1 µg/each) every other day induced atherogenesis in ApoE-/- mice at 12-weeks, with significant increase of inflammatory cytokine and adhesion molecules expression in aortic tissue (p < 0.05). CONCLUSION: Serum vimentin levels are associated with the presence and the severity of CAD. Vimentin protein promotes atherogenesis in ApoE-/- mice.

Dysregulation of matrix metalloproteinases and their tissue inhibitors is related to abnormality of left ventricular geometry and function in streptozotocin-induced diabetic minipigs.

This study aimed to characterize matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in relation to changes in left ventricle (LV) geometry and function in a porcine model with streptozotocin (STZ)-induced diabetes. In 15 Chinese Guizhou minipigs with STZ-induced diabetes (diabetic group) and 15 age-matched normal controls (control group), Doppler tissue imaging was performed at 6 months of diabetes. Serum MMP-2, -9, TIMP-1, -4 and B-type natriuretic peptide (BNP) were determined. Expression of MMPs, TIMPs, urokinase type-plasminogen activator (uPA), its receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1) in aortic intima and LV myocardium was evaluated, with gelatinolytic activities of tissue MMP-2, -9 accessed by zymography. Left ventricle end-diastolic septum thickness (P < 0.05) and mass (P < 0.05) were increased, whereas peak systolic mitral annulus velocity (Sm, P < 0.001), LV systolic (P = 0.01) and diastolic strain (P < 0.001) were significantly decreased in diabetic group than in controls. Diabetic group showed higher expression of TIMP-1, -4 in aortic intima and LV myocardium (P < 0.01 or P < 0.05), with increased collagen content and elevated serum BNP level (P = 0.004) and lower gelatinolytic activities of tissue MMP-2, -9 (all P < 0.05). Semi-quantitative RT-PCR of those diabetic tissues revealed elevated mRNA levels of major TIMPs, uPA, uPAR and PAI-1. Reduction of serum MMP-2 and -9 levels was observed in diabetic group vs. control group (both P < 0.05). This study features elevated levels of TIMP-1, -4, uPA, uPAR and PAI-1, and decreased activities of MMP-2, -9 in aorta and myocardium in STZ-induced diabetic minipigs, indicating that MMP-TIMP dysregulation is associated with LV hypertrophy, cardiac dysfunction and increased cardiovascular fibrosis in diabetes.

Glycated Apolipoprotein A-IV Induces Atherogenesis in Patients With CAD in Type 2 Diabetes.

BACKGROUND: Nonenzymatic glycation of apolipoproteins plays a role in the pathogenesis of the vascular complications of diabetes. OBJECTIVES: This study investigated whether apolipoprotein (apo) A-IV was glycated in patients with type 2 diabetes mellitus (T2DM) and whether apoA-IV glycation was related to coronary artery disease (CAD). The study also determined the biological effects of glycated apoA-IV. METHODS: The authors consecutively enrolled 204 patients with T2DM without CAD (Group I), 515 patients with T2DM with CAD (Group II), and 176 healthy subjects (control group) in this study. ApoA-IV was precipitated from ultracentrifugally isolated high-density lipoprotein, and its glycation level was determined based on Western blotting densitometry (relative intensity of apoA-IV glycation). ApoA-IV NƐ-(carboxylmethyl) lysine (CML) modification sites were identified by mass spectrometry in 37 control subjects, 63 patients in Group I, and 138 patients in Group II. Saline or glycated apoA-IV (g-apoA-IV) generated by glyoxal culture was injected into apoE-/- mice to evaluate atherogenesis, and was also used for the cell experiments. RESULTS: The relative intensity and the abundance of apoA-IV glycation were associated with the presence and severity of CAD in patients with T2DM (all p < 0.05). The experiments showed that g-apoA-IV induced proinflammatory reactions in vitro and promoted atherogenesis in apoE-/- mice through the nuclear receptor NR4A3. G-apoA-IV with mutations (K-A) at high-frequency glycation sites exhibited more weakened proinflammatory and atherogenic effects than did g-apoA-IV both in vitro and in vivo. CONCLUSIONS: ApoA-IV glycation is associated with CAD severity in patients with T2DM, and g-apoA-IV induces atherogenesis through NR4A3 in apoE-/- mice.

Association of decreased serum vasostatin-2 level with ischemic chronic heart failure and with MACE in 3-year follow-up: Vasostatin-2 prevents heart failure in myocardial infarction rats.

BACKGROUND: We investigated whether serum vasostatin-2 level is related to chronic heart failure (CHF) in patients with previous myocardial infarction (MI) and MACE in 3-year follow-up. The biological effect of vasostatin-2 on ischemic HF was evaluated in animal experiments. METHODS: After exclusion of the subjects not eligible, this study included 450 patients with CHF and previous MI, and 149 healthy controls. Serum vasostatin-2 level was analyzed. CHF patients were followed up for three years and major adverse cardiac events (MACE) were recorded, defined as reinfarction, target-vessel revascularization, cardiovascular death and refractory HF requiring hospitalizations. RESULTS: Notably, serum vasostatin-2 level was decreased in CHF patients than in controls, and significant difference was observed between CHF patients with MACE and those without (both P<0.05). Vasostatin-2 level was correlated with HF stages (Spearman's r=-0.288, P<0.05), LVEF (r=0.377, P<0.05) and pro-BNP level (r=-0.294, P<0.05). Multivariable logistic regression analysis suggested that vasostatin-2, conventional risk factors, severity of HF stages and LVEF were independently associated with MACE in CHF patients. Vasostatin-2 (100µg) or PBS was injected intraperitoneally every other day in MI rats, follow by echocardiography, hemodynamic analysis after 2months. Compared with PBS, vasostatin-2 treatment prevented ischemic HF in MI rats, accompanied with reduction of infarct size, remodeling, fibrosis and inflammation, mainly through inhibition of Rho, Wnt and TLR-4 pathways and modulation of renin-angiotensin system. CONCLUSION: Decreased serum vasostatin-2 level is associated with ischemic CHF and with MACE in three-year follow-up. Intraperitoneal injection of vasostatin-2 protects against ischemic HF in MI rats.

Increased serum HMGB1 level is associated with coronary artery disease in nondiabetic and type 2 diabetic patients.

OBJECTIVE: This cross-sectional study tested the hypothesis that increased serum level of high mobility group box-1 protein (HMGB1), a pro-inflammatory ligand of receptor for advanced glycation end products (RAGE), is associated with coronary artery disease (CAD) in nondiabetic and type 2 diabetic patients. METHODS: Serum levels of HMGB1, endogenous secretory RAGE (esRAGE), soluble RAGE (sRAGE) and inflammatory cytokines were determined in 512 patients categorized as Group I (n=132, without diabetes and CAD), Group II (n=149, with CAD but no diabetes), Group III (n=80, with diabetes but no CAD) and Group IV (n=151, with diabetes and CAD). RESULTS: Serum levels of HMGB1 and hsCRP were higher in Group II than in Group I, and in Group IV than in Group III (all P<0.001). HMGB1 was positively related to hsCRP, TNF-alpha and IL-6 levels in the whole subjects (all P<0.01). Group II patients had lower sRAGE (P=0.058) and esRAGE (P<0.001) levels versus those in Group I. However, in the diabetic patients, those in Group IV had lower esRAGE (P<0.001) but higher sRAGE (P=0.002) levels compared to those in Group III. In multivariable regression analysis, HMGB1, esRAGE and conventional risk factors (age, smoking, hypertension, HDL-C, hsCRP, TNF-alpha) were independent determinants of CAD in nondiabetic patients. Moreover, HMGB1 and esRAGE consistently remained to be independently associated with CAD in diabetic patients, so did other major conventional risk factors. CONCLUSION: This study demonstrates that increased serum HMGB1 level is associated with CAD in nondiabetic and type 2 diabetic patients.

Polymorphisms of MMP-3 and TIMP-4 genes affect angiographic coronary plaque progression in non-diabetic and type 2 diabetic patients.

BACKGROUND: This study examined whether genetic variants of matrix metallopeptidases (MMPs) and their tissue inhibitors (TIMPs) were associated with angiographic coronary plaque progression (PP) in type 2 diabetic and non-diabetic patients. METHODS: Four hundred and ninety-nine patients were grouped, who underwent coronary angiography and received repeat examinations after 1-y follow-up. Twelve functional polymorphisms of MMPs and TIMPs were characterized. RESULTS: Genotype distribution and allele frequency of -1612 5A/6A MMP-3 and 3'UTR C/T TIMP-4 differed between patients with PP and those without in both diabetic and non-diabetic groups after Bonferroni's correction (all P<0.0041667, except for allele frequency of MMP-3 [P=0.007] and genotype/allele frequency of TIMP-4 [P=0.04 and P=0.016, respectively] in diabetes). MMP-3 and TIMP-4 polymorphisms were associated with changes in percent diameter stenosis and minimal lumen diameter in diabetic patients, and changes in cumulative coronary obstruction in both diabetic and non-diabetic patients (all P<0.05). Multivariable regression analysis revealed that hypertension, low HDL-C and genotypes of MMP-3 and TIMP-4 were independent determinants of PP in the whole patients, with these 2 genetic factors being associated with PP in diabetic and non-diabetic subgroups. CONCLUSION: This study demonstrated that MMP-3 and TIMP-4 polymorphisms affect angiographic coronary PP in type 2 diabetic and non-diabetic patients.

Decreased serum esRAGE level is associated with angiographically determined coronary plaque progression in diabetic patients.

OBJECTIVE: This study evaluated the relationship between serum levels of endogenous secretory receptor for advanced glycation endproducts (esRAGE) and coronary plaque progression in diabetic and nondiabetic patients. DESIGN AND METHODS: Serum esRAGE level was measured and quantitative coronary angiography (QCA) was performed in 265 consecutive patients at baseline and 1-year follow-up. RESULTS: Comparing to baseline, serum esRAGE level was significantly increased during follow-up in nondiabetic patients without plaque progression (p=0.014), unchanged in nondiabetic patients with plaque progression and diabetic patients without plaque progression, and decreased in diabetic patients with plaque progression (p=0.011). Moreover, change of esRAGE levels correlated with change of QCA measurements. Multivariable regression analyses revealed that high-density lipoprotein cholesterol (OR=0.214, p=0.037), hypertension (OR=2.755, p=0.011), high-sensitivity C-reactive protein (OR=1.083, p<0.001) and change of esRAGE (OR=23.477, p<0.001) were independent risk factors for plaque progression in diabetic patients. CONCLUSIONS: This study demonstrated an association of decreased serum esRAGE level with coronary plaque progression in patients with diabetes.

RAGE gene polymorphisms are associated with circulating levels of endogenous secretory RAGE but not with coronary artery disease in Chinese patients with type 2 diabetes mellitus.

BACKGROUND AND AIMS: Engagement of the receptor for advanced glycation end products (RAGE) with advanced glycation end products and subsequent signaling play an important role in the development of diabetic complications. This pathophysiological effect was mitigated partially by endogenous secretory RAGE (esRAGE). The present study aimed to explore the possible association of RAGE polymorphism with serum esRAGE level and coronary artery disease (CAD) in Chinese patients with type 2 diabetes mellitus (T2DM). METHODS: A total of 740 consecutive patients with T2DM undergoing coronary angiography were enrolled. The severity of coronary atherosclerosis was defined as the number of diseased vessels; 69 bp insertion/deletion was determined by polymerase chain reactions, and -429 T/C, -374A/T and G82S variants were assessed by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: Patients with genotypes carrying C allele of -429 T/C and G allele of G82S had significantly higher esRAGE levels. 82S allele was also associated with increased tumor necrosis factor-alpha and interleukin-6 levels in diabetic patients with CAD (all p <0.05), but none of the polymorphisms or haplotypes was related to the presence and severity of CAD. CONCLUSIONS: G82S and -429 T/C polymorphisms of RAGE were associated with the circulating levels of esRAGE but not with CAD in Chinese patients with T2DM.