C Yan

ASSA14-03-25 Cellular repressor of E1A-stimulated genes antagonises inflammation and promotes autophagy via lysosome biogenesis in mouse macrophages

Objective Macrophage inflammation plays an important role in the pathogenesis of atherosclerosis. In this study, we investigated the involvement of cellular repressor of E1A-stimulated genes (CREG) in tumour necrosis factor-α (TNF-α)-induced macrophage inflammation, and explored its inhibitory capacity and mechanisms to assess its potential as a therapeutic reagent for atherosclerosis. Methods and results We confirmed that CREG played an important role in TNF-α-induced macrophage inflammation and had anti-inflammatory effects in RAW 264.7 mouse macrophages induced by TNF-α, using enzyme-linked immunosorbent assays and western blotting. Gain-of-function and loss-of-function experiments revealed that CREG promoted autophagy in TNF-α-induced RAW 264.7 cells. Using the autophagy inhibitors 3-methyladenine and bafilomycin A, we demonstrated that autophagy played an important role in attenuating TNF-α-induced inflammation. Immunofluorescence analysis and western blotting showed that CREG protein stimulated the expression and maturation of cathepsin B and cathepsin L and induced the biogenesis of lysosomes, while CREG deficiency reduced lysosome biogenesis. Exogenous CREG protein was located in lysosomes, as shown by confocal microscopy and immunoprecipitation analysis. CREG protein played a critical role in the distribution but not in the expression of mannose-6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIR), as demonstrated by western blotting and immunofluorescence analysis. In vivo experiments indicated that CREG protein alleviated the development of aortic atherosclerosis and affected inflammation and autophagy in aortas of ApoE−/− mice. Conclusion CREG inhibits macrophage inflammation and promotes autophagy mediated by lysosome biogenesis, which is related to the distribution of M6P/IGFIIR. CREG may represent a new therapeutic target against atherosclerosis.

ASSA14-03-31 CREG modulates Angiotensin II type 1 receptor to antagonise coronary oxidative stress and hypertension in salt-sensitive rats

Background This study examined whether CREG protected Sprague-Dawley (SD) rats against high salt-related hypertension and vascular endothelium oxidative stress, and explored the mechanisms and associated changes in components of the renin-angiotensin system and coronary artery injury. Methods In this study, SD rats received pure drinking water, or drinking water containing 1.5% NaCl for 16 weeks. Systolic blood pressure was measured before rats were divided into salt-sensitive and salt-resistant groups. After 16 weeks of treatment, NO, malondialdehyde (MDA), angiotensin II and IL-6 in plasma and angiotensin II in coronary arteries were detected, as was the expression of eNOS, AT1R, AT2R and CREG. Primary lung vascular endothelial cells were incubated in culture medium containing aldosterone (0.45 nmol/l). Salt-induced changes in eNOS and activation of NAD(P)H oxidase-gp91 were assessed. Supernatant ONOO- and NO, and the expression of AT1R, AT2R and CREG were measured. Losartan was used to block AT1R effects, and to investigate the biofunction of CREG on endothelial cells. Results The results showed that a long-term high salt intake significantly increased plasma IL-6 and MDA, angiotensin II in coronary arteries and AT1R expression, but decreased plasma NO, angiotensin II, AT2R and CREG expression in coronary arteries of salt-sensitive rats, which was prior to increase of blood pressure. Conversely, no obvious oxidative stress or vascular remodelling was observed in the salt-resistance group accompanying anti-hypertension. Mechanically, sodium concentrations significantly increased the expression of gp91 (a marker of oxidative stress) and AT1R, and reduced expression of eNOS and CREG in primary lung vascular endothelial cells from salt-sensitive rats. These effects were abrogated in cells with overexpression of CREG. Furthermore, Losartan blocking the biofunction of AT1R antagonised the NaCl-induced oxidative stress in CREG-silencing endothelial cells. Conclusions This is the first study to identify CREG-modulated AT1R activation as a novel target to block oxidative stress in blood vessels and prevent salt-related hypertension.

ASSA14-03-22 Lack of association between PON1 gene polymorphisms and high platelet reactivity in patients undergoing selective coronary stent placement in Chinese Han population

Background There is a wide variation in the clinical therapeutic activity of clopidogrel, which has mainly been attributed to genetic effects. Recent a study reported that Paraoxonase-1 (PON1) as a key enzyme plays a vital part in clopidogrel bioactivation. The aim of our study was to assess whether PON1 gene polymorphisms are correlation with the low rates of clopidogrel bioactivation and responsible for negative clopidogrel efficacy in patients undergoing selective coronary stent implantation. Methods A total of 2069 patients undergoing DES placement were enrolled in this study, all the subjects were pre-treatment with 600 mg clopidogrel and post-treatment with 75 mg/day for at least 12 months. Five tagSNPs (the coding polymorphisms Q192R (rs662) and L55M (rs854560); the promoter polymorphisms -108C/T (rs705379), -162A/G (rs705381) and -909G/C (rs854572) of PON1 gene were selected in this study. Genotypes were identified by polymerase chain reaction (PCR) direct sequencing of genomic DNA, and platelet function was assessed by 20 μmol/l ADP-induced platelet aggregation and by flow-cytometric analysis of platelet surface CD62-P and PAC-1 protein expression before and after clopidogrel. In addition, activity level of PON1 towards phenylacetate was measured by at 270 nm using a UV-2550 spectrophotometer. PON1 concentration was measured by human Elisa kit. Results Among all of the studied polymorphisms, none of these was related to individuals with high platelet reactivity (HPR). The variations of CD62-P, PAC-1 protein expression were not statistically significant in HPR and no-HPR groups (p = 0.258 and p = 0.574, respectively). The mean values for serum PON1 activity and concentration were 98.83 ± 22.02 and 81.48 ± 17.82 for HPR patients and 105.12 ± 27.76 and 90.67 ± 16.33 for no-HPR patients, and no significant differences can be observed in the two groups. Conclusion Based on these results, no statistically significant difference was detected between PON1 gene polymorphisms and clopidogrel on-treatment platelet reactivity in patients undergoing selective coronary stent placement in Chinese Han population.

ASSA14-03-33 Over restrictive salt diet promotes atherosclerotic plaque growth and instability through PCSK9 in ApoE knockout mice

Background Recently, over restrictive salt diet have shown to increase cardiovascular events, including atherosclerosis in normotensive individuals or in individuals with pre-hypertension. However, the surprised view attracted extensive attention and query. The authenticity and possible mechanism still should be given a deep discussion. Proprotein convertase subtilisin/kexin 9 (PCSK9), which is expressed in the wall of aorta and atherosclerotic plaque, has a closely relationship with atherosclerosis. Here, we investigated whether restrictive salt could accelerate development of atherosclerotic lesions via PCSK9 in ApoE-/- mice. Method Male mice aged 10-weeks and weighting between 20 to 25 g were allocated to receive an isocaloric diet with low-salt content (0.03%), low-salt diet (0.03%) combined with siRNA tail injection or normal salt content (0.3%) containing 6% fat. After 12 weeks of study, the basic metabolic index, including weight, blood pressure, urine sodiumand urine potassiumwere evaluated. The gross aorta was stained by oil red-O, and the quantification of plaque in cross section was detected by immunohistochemistry. The expression of PCSK9 was measured by Western blot. Results Plaque accumulation of salt restriction diet for 12-weeks was 4.3 times higher than mice receiving normal salt in ApoE-/-mice. Meanwhile, the mortality rate was extreme higher in the low salt group. Quantification of staining demonstrated a marked increasing inflammatory cells and larger necrotic core areas those could lead plaque instability in the low salt diet group. And the expression of PCSK9 in aorta showed that an increase of PCSK9 in a low salt group than normal salt diet. Group of low salt diet combined with PCSK9 siRNA could reduce the plaque development and the instability in ApoE-/- mice. Conclusions Over restrictive salt diet causes a high mortality, activates aortic PCSK9 expression to improve atherosclerotic plaque formation and leads to plaque instability as well. This finding provides mechanistic insights on low salt diet associated atherosclerosis.

ASSA14-03-21 Polymorphisms of PON1 gene are associated with coronary artery disease

Background Paraoxonase 1 (PON1) is a plasma enzyme that is capable of inhibiting the progression of atherosclerosis, and is significantly associated with the susceptibility of coronary artery disease (CAD). Recently, PON total protein expression was found in human aortic tissue and it played an important role in progression of atherosclerosis. For studying its crucial role in preventing CAD, we performed the PON1 immunohistochemical analysis in human coronary arteries, explored its polymorphisms and plasma status, and analysed its association with the risk of CAD. Methods The expression of PON1 in human coronary arterial tissues was detected by immunohistochemical staining analysis. PON1 gene polymorphisms were determined by polymerase chain reaction (PCR) direct sequencing in 2456 unrelated Chinese Han individuals. Serum PON1 level was reflected indirectly by PON1 activity towards paraoxon and phenylacetate by spectrophotometry separately and directly by its concentration using human enzyme linked immunosorbent kit (ELISA). Results Immunohistochemical analysis showed that PON1 expression in human coronary arteries was lower in atherosclerotic arteries than in normal arteries. Among all of the studied polymorphisms, only PON1 Q192R (rs662) had a significant effect on the risk of CAD (p = 0.001). In a logistic regression model, after adjusting the conventional risk factors of CAD, R allele carriers had a significantly higher risk of CAD than other allele carriers. Both serum PON1 activity and concentration were significantly reduced in CAD patients compared with the controls (p < 0.05), and highly associated with Q192R polymorphisms. Conclusion Decreased PON1 expression in human atherosclerotic coronary arteries was associated with CAD. Moreover, our finding indicated that PON1 Q192R polymorphism might be a genetic risk factor for the susceptibility of CAD in Chinese Han population, and the R allele might be an independent predictor for CAD.

ASSA14-03-26 Cellular repressor of E1A stimulated genes antagonise inflammation in RAW 264.7 cells via autophagy-lysosome pathway

Background Macrophage inflammation plays an important role in the pathogenesis of atherosclerosis. In this study, we investigated the involvement of cellular repressor of E1A-stimulated genes (CREG) in tumour necrosis factor-α (TNF-α)-induced macrophage inflammation, and explored its inhibitory capacity and mechanisms to assess its potential as a therapeutic reagent for atherosclerosis. Methods RAW 264.7 mouse macrophage-like cells were transfected with CREG siRNA to down-regulate the expression of CREG. The expression of CREG, cathepsin B, cathepsin L, LAMP1, IGFIIR, LC3, Beclin 1, p62 and Tubulin was identified by Western blot. The amounts of IL-6 and MCP-1 secreted into the cell culture supernate and expressed in the tissue lysates were determined using ELISA kits. Colocalization of anti-His and anti-cathepsin B/ cathepsin L/ M6P/IGFIIR/ LAMP1 was determined by immunofluorescence analysis and confocal microscopy. Lysosomal visualisation was carried out using the Lysotracker Red staining. Accumulation of autophagosomes and autolysosomes were detected in CREG-treated and CREG down-regulated cells using electron microscopy. The interaction between the exogenous recombinant CREG protein and cathepsin B, cathepsin L and IGFIIR were studied with immunoprecipitation analysis. Male ApoE−/− mice (n = 40, age 10 weeks, 20–25 g) were fed a high-fat, high-cholesterol diet containing 21% fat and 1.3% cholesterol for 16 weeks. The mice underwent sham operation or were infused with CREG protein (Abcam, UK) at a rate of 30 μg/kg/d during the 4–12 weeks by an osmotic mini-pump. Aortas were collected for oil red O and hematoxylin-eosin staining to assess atherosclerotic plaques. The expression of CD68 and MCP-1 in the atherosclerotic plaques was detected by the immunohistochemical staining. Results We confirmed that CREG played an important role in TNF-α-induced macrophage inflammation and had anti-inflammatory effects in RAW 264.7 mouse macrophages induced by TNF-α. Results of ELISA assays showed that the amount of IL-6 and MCP-1 secreted into the cell culture supernate decreased in a dose-dependent manner in the RAW 264.7 cells supplemented with exogenous recombinant CREG protein and increased in those knocking down of CREG expression by siRNA. Gain-of-function and loss-of-function experiments revealed that CREG promoted autophagy in TNF-α-induced RAW 264.7 cells. The numbers of autolysosomes increased in RAW 264.7 cells following treatment with CREG protein while less autolysosome were seen in those transfected with CREG siRNA. Meanwhile, exogenous CREG protein induced autophagy with significant increased autophagosome-bound LC3-II and Beclin 1 abundance and decreased the level of p62. Increment of LC3-II and Beclin 1 and accumulation of p62 in CREG down-regulated cells suggested accumulation of autophagosomes and impairment of autophagy. Using the autophagy inhibitors 3-methyladenine and bafilomycin A, we demonstrated that autophagy played an important role in attenuating TNF-α-induced inflammation. Immunofluorescence analysis and western blotting showed that CREG protein stimulated the expression and maturation of cathepsin B and cathepsin L and induced the biogenesis of lysosomes, while CREG deficiency reduced lysosome biogenesis. Since lysosomal activity is essential to autophagy, we deduce that CREG may mediate the regulation of autophagy via its effects on lysosomes. Exogenous CREG protein was located in lysosomes, as shown by confocal microscopy and immunoprecipitation analysis. CREG protein played a critical role in the distribution but not in the expression of mannose-6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIR), as demonstrated by western blotting and immunofluorescence analysis. In vivo experiments indicated that CREG protein alleviated the development of aortic atherosclerosis and affected inflammation and autophagy in aortas of ApoE−/− mice. Haematoxylin-eosin staining showed that the aortic lesion area was significantly reduced in the CREG-treated mice compared with those in the control group. The expression of MCP-1 in arterial tissue detected by ELISA assays and CD68, a marker of active macrophages and MCP-1 in atherosclerotic lesions of aortic root sections determined by immunohistochemical staining were all decreased in CREG-treated group. Furthermore, Western analysis of aorta lysates showed increased autophagosome-bound LC3-II abundance and p62 consumption in CREG-treated group. Conclusion CREG inhibits macrophage inflammation and promotes autophagy mediated by lysosome biogenesis, which is related to the distribution of M6P/IGFIIR. CREG may represent a new therapeutic target against atherosclerosis.

ASSA14-03-24 CREG1 upregulates Rab7 expression to activate autophagy and ameliorate cardiac damage

Background In cardiomyocytes subjected to stress, autophagy activation is a critical survival mechanism that preserves cellular energy status while degrading damaged proteins and organelles. However, little is known about the mechanisms that govern this autophagic response. Cellular repressor of E1A genes (Creg1) is an evolutionarily conserved lysosomal protein, and an important new factor in regulating tissue homeostasis that has been shown to antagonise injury of tissues or cells. Recent studies showed that CREG1 is a lysosomal protein that undergoes proteolytic maturation by lysosomal cysteine proteases in the course of its biosynthesis. CREG1 contains a mannose 6-phosphate (M6P) recognition marker, and depends on interactions with M6P receptors for efficient delivery to lysosomes. CREG1 is implicated in the regulation of lysosome functions. In the present study, we aimed to investigate the regulatory role of CREG1 in cardiac autophagy, and to clarify autophagy activation mechanisms. Methods Male heterozygous Creg1+/− mice were generated and bred at the Southern model animal centre. Age-matched male transgene-negative wild-type (WT) littermates were used as controls. Two- to six-week-old Creg1+/− and WT mice were subjected to an infusion of Ang II (2.5 g/kg per day) for 28 days. In another series of experiments (reversal experiment), 4-week-old male Creg1+/− mice were subjected to an infusion of AngII for 28 days, meanwhile was treated with recombinant CREG1 protein (5 mg/kg per day, IP injections) or with chloroquine (5 or 10 mg/kg per day) for 14 days (starting day 1 and continuing until day 14). To measure fibrosis, Masson’s trichrome staining was performed on paraffin sections for all experimental animals. To measure autophagic flux, western blot for analyses of LC3-II and p62 levels was employed on the myocardium samples. RT-PCR was adopted for the total RNA. Primary culture of cardiac myocytes and Adenoviral Infection were all used in this study. Results We generated a Creg1 haploinsufficiency (Creg1+/−) mouse model, and identified that Creg1 deficiency aggravates myocardiac fibrosis in response to ageing or angiotensin II (Ang II). Conversely, exogenous infusion of recombinant Creg1 protein completely reversed cardiac damage. Creg1 deficiency in Creg1+/− mouse hearts showed a marked accumulation of autophagesomes that acquired LC3II and beclin-1, and a decrease in autophagic flux as indicated by the level of p62. Inversely, restoration of Creg1 activity activated cardiac autophagy. Furthermore, chloroquine, an inhibitor of lysosomal acidification, was used to confirm that Creg1 protected the heart tissue against Ang II-induced fibrosis by activating autophagy. Using adenoviral infection of primary cardiacmyocytes, overexpression of Creg1 with concurrent resveratrol treatment significantly increased autophagy, while silencing Creg1 blocked the resveratrol-induced autophagy. Conclusions These results suggest that rapid, Creg1-induced activation of autophagy is required to maintain heart function in the face of stress-induced myocardial damage. Both in vitro and in vivo studies identified that Creg1 deficiency reduced the expression of Rab7, thereby influencing the maturation of lysosomes and the formation of autophagolysosomes and imparing autophagy. These findings also suggest that activating autophagy via Creg1 may be a viable therapeutic strategy for improving cardiac performance under pathologic conditions.

ASSA14-03-32 Over restrictive salt diet promotes dyslipidemia via up-regulating PCSK9 in ApoE-deficient mice

Background Hyperlipidemia is one of most important detrimental risk factors in leading cardiovascular disease, but the underlying mechanism remains in heated discussion. Recently, studies reporting a low salt intake could lead to dyslipidemia attracted extensive attention and query. The authenticity and possible aetiology need to be discussed in-depth. Proprotein convertase subtilisin/kexin 9(PCSK9), a vital regulator of low-density lipoprotein (LDL)-cholesterol levels, is closely related to hyperlipidemia. Here, we investigated whether restrictive salt could lead to dyslipidemia and role of PCSK9 in regulating this process in ApoE-/- mice. Method Male mice aged 10-weeks between 20 to 25 g were allocated to receive an isocaloric diet with low-salt content (0.03%), low-salt diet (0.03%) combined with PCSK9 siRNA tail injection, normal salt content (0.3%) or a high-salt content (3%) containing 20% fat. After 8 weeks of feeding, the basic metabolic index, including weight, blood pressure, urine sodiumand urine potassiumwere evaluated. After anaesthetised by chloral hydrate, blood was collected by exsanguination via carotid artery. Total cholesterol, triglycerides and LDL-c were measured in fasting plasma samples using an auto-analyzer. The expression of PCSK9 in liver was measured by immunohistochemistry and Western blot. Results Eight-weeks of a low-salt diet resulted in increased cholesterol, LDL-c and triglyceride in ApoE-/- when compared with mice receiving normal salt, while a high-salt diet attenuated the dyslipidemia. Then the expression of PCSK9 was detected by immunohistochemistry and western blot in liver, the result showed that a low salt diet increased PCSK9 in liver, by contrast, a high-salt diet decreased PCSK9 level compared to normal salt diet group. A low salt diet combined PCSK9 siRNA could suppress the lipid increasing. Conclusions Over restrictive salt could cause dyslipidemia, and one of the possible mechanisms is up-regulating the expression of PCSK9 in ApoE-/- mice. This finding provides mechanistic insights on low salt diet associated dyslipidemia.

ASSA14-03-20 Cellular repressor of E1A-stimulated genes protects against angiotensin II-induced vascular remodelling via degradation of angiotensin II type 1 receptor

Background Cellular repressor of E1A-stimulated genes (CREG), a new cardiovascular homeostasis regulator, has been proposed to reduce the neointimal formation after vascular injury via the maintenance of the quiescent mature vascular smooth muscle cell (VSMC) phenotype. We hypothesised that CREG is a negative regulator of angiotensin (Ang) II-mediated vascular remodelling. Methods Ten-week-old male heterozygous CREG-deficient (CREG+/-) mice and their littermate wild-type (WT) mice were used. An osmotic minipump was implanted subcutaneously to infuse a dose of Ang II (1.5 mg/kg · d) or saline (vehicle) for 14 days. Ang II-infused WT mice were then treated with placebo or recombinant human CREG (rhCREG; 300 μg/kg · d). Systolic blood pressure (SBP); extent of vascular remodelling; protein level of collagen type I/III, angiotensin-converting enzyme-2 (ACE2), angiotensin receptor type 1 (AT1R) and Rab7 were evaluated. Primary VSMC culture was performed to assess AT1R and collagen protein expression before and after CREG gene transfection and knockdown. Results CREG levels are high in vascular media under basal conditions but rapidly decrease in response to Ang II. Ang II infusion for 14 days resulted that levels of SBP, intima medial thickness and vascular remodelling of the aorta and mesenteric artery were significantly greater in CREG+/- mice compared with the WT controls. Vascular gene expression level of CREG was lower in Ang II-treated CREG+/- mice than in WT mice. However, daily treatment of Ang II-infused WT mice with rhCREG blunted the increase of SBP. Ang II-mediated vascular remodelling and expression of collagen type I/III were also suppressed by rhCREG. Moreover, rhCREG treatment inhibited Ang II-mediated up-regulation of AT1R expression and down-regulation of ACE2 expression. Ang II-induced vascular remodelling was inhibited by rhCREG in association with reduced plasma Ang II and increased plasma Ang 1–7 levels. In adult VSMCs, CREG over-expression increased the expression of Rab7 by a regulatory mechanism of ubiquitination. Importantly, Ang II-mediated collagen production and AT1R expression was inhibited by CREG over-expression and augmented by CREG knock-down in a rab7-dependent manner. Conclusions Elevated Ang II induced hypertension and vascular remodelling, which were exacerbated by CREG deficiency, whereas rhCREG attenuated Ang II-induced adverse vascular remodelling. Hence, CREG is an important negative regulator of Ang II-induced vascular disease and suppresses adverse vascular remodelling.

ASSA14-03-18 Contribution of Homeostatic Chemokines CCL19 and CCL21 and their Receptor CCR7 to Coronary Artery Disease

Background Aim to identify the role of the homeostatic chemokines CCL19 and CCL21 and their common receptor CCR7 in atherogenesis, and to study the relationships between CCL19, CCL21 and CCR7 gene variants and coronary artery disease (CAD) in a Chinese Han population. Methods Collect atherosclerosis and normal human coronary arteries, and use the immunohistochemical staining to detect the experssion of CCL19, CCL21 and CCR7. Detect the role of CCL19, CCL21 and CCR7 in monocytes adhereing and migrating to the Human umbilical vein endothelial cells (HUVECs). Ten tagSNPs were retrieved from the Hapmap database for Han population samples in the CCL19, CCL21 and CCR7 genes and 2.0 KB regions on each side. In 1746 cases of the north group and 700 cases of the south group, the SNP genotypes were detected by polymerase chain reaction-direct sequencing method and the associations between every SNP genotypes and alleles and coronary heart disease, and associations between plasma CCL19 and CCL21 level and SNP genotypes were analysised. Results Immunohistochemical analysis of samples with atherosclerosis of various stages showed increased CCL19, CCL21 and CCR7 expression in atherosclerotic coronary plaques compared with non-atherosclerotic controls. Expression levels increased in positive correlation with the levels of the coronary lesions. Cell adhesion assays confirmed that CCL19 promoted monocyte adhesion, which was induced by CCR7, to human umbilical vein endothelial cells (HUVECs), and the increased adhesion could be partially antagonised by atorvastatin. The associations between genetic variants of CCL19, CCL21, CCR7 and CAD in a Chinese Han population were determined by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. The following single nucleotide polymorphisms were associated with CAD: CCL19 rs2227302, CCL21 rs2812377, and CCR7 rs588019. Individuals with the CCL19 rs2227302 T allele or CCL21 rs2812377 G allele had higher plasma CCL19 levels than those with C/C genotype, and higher CCL21 levels than those with T/T genotype in both case and control subjects. Conclusions CCL19/CCL21-CCR7 is a novel homeostatic chemokine system that modulates human monocyte adhesion and migration, promoting atherogenesis. It is associated with CAD risk in Chinese Han individuals. These data suggest that the CCL19/CCL21-CCR7 axis plays an important role in atherosclerosis progression.