Yan Chenghui

ASSA14-11-03 Dietary Salt Intake and Coronary Atherosclerosis in Patients with Prehypertension

Background The epidemic data shows that heavy dietary salt intake could improve the developing of hypertension, cardiovascular and cerebrovascular disease. We detected the mechanism of effect on coronary atherosclerosis of prehypertensive patients with different levels of dietary salt intake. Methods In total, 243 patients aged 45 to 75 with prehypertension were followed-up from 2003 to 2009. They were all diagnosed with coronary heart disease and had no history of myocardial infarction, coronary revascularisation, congenital heart disease or cerebrovascular disease. Results The results showed that compared with normal-salt group, the patients who took heavy salt daily were younger (60.8 ± 7.4 vs 58.7 ± 7.5 years), with higher body mass indexes, and patients who had a history of coronary heart disease were more in high-salt group, especially in men. (p < 0.05). After a median follow-up period of 4.53 years, there were 71 (29.2%) patients who experienced the end point events (fatal and nonfatal myocardial infarction, or coronary revascularisation). The odds radios of the patients with high-salt diet was 2.659 (95% CI: 1.438–4.919). Meanwhile, among the patients with progression to hypertension, heavy salt intake was in a high risk of cardiovascular disease, which did not performed in the patients normotensive or prehypertensive. Conclusions Heavy salt intake is a risk factor of the developing of coronary atherosclerosis in the patients with prehypertensive. The results of our study suggest that the mechanisms of the influence of salt intake to coronary atherosclerosis may be associated with obesity, hypertension, high triglycerides and total cholesterol.

ASSA14-03-19 The change of cellular repressor of E1A-stimulated genes during vascular remodelling in a mouse model of arterial injury

Background Cellular repressor of E1A-stimulated genes (CREG) has been shown to be ubiquitously expressed in human and mouse tissues. However, its physiological functions and possible involvement in pathological processes remain unknown. Method To explore pathophysiology pathogenesis of vascular remodelling and possible role of CREG, we established an injury model of the mouse carotid artery in the present study. High-resolution small-animal ultrasound, Masson staining, immunohistochemistry, RT-PCR and western-blot were used to detect the intima-media thickness, collagen content, the change of collagen type I and CREG expression of arterial wall at different time after aterial injury. Results CREG was expressed in normal arteries. The expression of CREG mRNA and protein of the arterial wall was marked down-regulated after injury of mouse carotid artery, and reached its lowest value on 3rd day after arterial injury, with close correlation to the process of vascular remodelling (increase in mRNA level of collagen type I). CREG expression gradually restored on the 7th day, and almost returned to normal levels on 14th day and 28th day after arterial injury. In contrast, injured arteries developed marked vascular remodelling after 7 days as manifested by increase in intima-media thickness, narrowing of the vascular lumen, collagen content as well as mRNA and protein level of collagen type I. There were negative relationships between CREG expression and vascular remodelling at the early time of artery injuries. The expression of CREG was decreased at beginning and then increased, but the degree of vascular remodelling was continued to exacerbate. Conclusion These data strongly suggest that CREG is involved in the development of vascular remodelling after arterial injury, and that injury-induced CREG down-regulation may contribute to the progression of vascular remodelling.

ASSA14-03-28 Cellular repressor of E1A-stimulated genes improves heart function in a mouse model of MI

Backgroud Cellular repressor of E1A-stimulated genes (CREG) is a mannose-6-phosphate-containing secreted glycoprotein of 220 amino acids. It has been proposed that CREG acts as a ligand that enhances differentiation and/or reduces cell proliferation. In humans, the potential therapeutic role of embryonic stem cells (ESCs) in ischaemic heart disease is subject to intense investigation. Particularly, the contribution of ESCs to angiogenesis and cardiomyogenesis in myocardial ischemia is not well established. In our studies, we induced myocardial infarct (MI) in mouse model, and monitored the effects of ESCs transplantation of overexpression of CREG on cardiac function. Methods pCXN2-Flag-wtCREG, pCXN2-Flag-mutCREG and pCXN2-Flag-EGFP plasmids were transfected into ESCs by lipofectamine 2000. Coronary artery ligation to induce MI model in seven- to nine-week-old mice was developed by a novel and rapid surgical method. wtCREG, mutCREG and EGFP ESCs or DMEM were then injected into the peri-ischaemic area. Four groups of mice were analysed for haemodynamic and pathologic parameters 1 and 2 months after MI and injection. Results The heart weight to body weight ratio was also significantly decreased at day 28 (5.9 ± 0.5 and 5.5 ± 0.4) in comparison with control hearts (7.0 ± 0.5, p < 0.05). The heart weight to tibia length ratio of EGFP ESCs or wtCREG ESCs was decreased at day 28 (1.5 ± 0.1 and 1.4 ± 0.1) in comparison with age-matched control hearts (1.6 ± 0.2, p < 0.05). TTC analysis performed at 28 days after MI shows a highly significant reduction of the infarct size in the EGFP ESCs and wtCREG ESCs groups, as compared with the DMEM and sham mice (p < 0.05). Haemodynamic assessment by Millar catheterization demonstrated a significant increase in +dP/dt and –dP/dt, as well as a significant drop of LVEDP at 21 days after MI in mice transplanted with wtCREG ESCs, as compared with mice transplanted with EGFP ESCs or DMEM (p < 0.05). Intriguingly, animals transplanted with EGFP ESCs also showed a significant improvement of these cardiac functional parameters, as compared with those transplanted with DMEM (p < 0.05). WtCREG animals had a significantly greater decrease in end-diastolic and end-systolic dimensions in comparison with DMEM and EGFP animals, reflecting decelerated dilatative remodelling in the wtCREG. This was associated with marked LV function improvement as reflected by increaced LVEF and LVFS. Results from our experiments show a remarkable decrease of both myocardial fibrosis and cardiomyocyte and noncardiomyocyte apoptosis in wtCREG ESCs-injected mice. A significant release of VEGF was observed not only in the wtCREG ESCs animals (41.3 ± 12.6 pg/ml), but also in the EGFP ESCs animals (50.0 ± 4.1). Over-expression of CREG (wtCREG ESCs) decreased ASK1 protein level by 48% (p < 0.05). Upon elevation of CREG levels, also decreased JNK1/2 and p38 expression levels in the cardiomyocytes. Here, we found increased Bax and decreased bcl-2 expression at 2 weeks post-MI. Conclusions Therefore, the expression of CREG improves cardiac functions and inhibits birosis and apoptosis. These data suggest that a key mechanism of the protective effects of ASK1 in reducing ischaemic injury is via maintaining the classic proapoptotic factor Bax in an inactive state.

ASSA13-10-10 CREG Promotes Vasculogenesis of Embryonic Stem Cells by Activating PI3K/Akt/VEGF Pathway

Background Vasculogenesis plays an important role under both physiological and pathological conditions. Factors controlling vasculogenesis is still far to be fully elucidated. Cellular repressor of E1A activating gene (CREG) has been reported to be highly expressed in endothelium and prevent injured endothelial cells from apoptosis, suggesting its potential role in regulating vasculogenesis. Objective The aim of this study was to investigate the role and mechanism of CREG in regulating vasculogenesis of embryonic (ES) stem cells. Methods CREG over-expression (wtCREG) and knock-down (shCREG) ES cell lines were established by transfection of wild type ES cell R1 (wtR1) with pCXN2-Flag-CREG-IRES-EGFP and mouse CREG shRNA vectors respectively, using ES cells expressing EGFP as a control (ctlR1). We first detected CREG expression during wtR1 and ctlR1 differentiation by Western Blot. Then embryoid bodies (EB) derived from 4 groups of ES cells were plated on fibronectin coated cover slips and cultured for 10 days to make an in vitro vasculogenesis model. Endothelial network formation was detected by CD31 immunofluorescence. Transcription of CD31, VEGFR2 and VEGF were measured by real time polymerase chain reaction (RT-PCR). Protein involved in several signalling pathways, including JNK, ERK1/2, PI3K/Akt and VEGFR2, VEGF were detected by Western Blot. PI3K/Akt inhibitor and VEGF neutralising antibody were used for blocking study in wtCREG group. VEGF were supplemented for rescue study in shCREG group. Results Expression of CREG increased with differentiation of ES cells in control group (wtR1 and ctlR1). wtCREG had significantly higher density of endothelial network formation identified by CD31 immunofluorescence in contrast to shCREG, which barely had endothelial network formation. RT-PCR also showed that transcription level of CD31, VEGFR2 and VEGF are up-regulated in wtCREG and down-regulated in shCREG. Western blot showed no difference in JNK, ERK1/2, but significant change of PI3K/Akt, VEGFR2, VEGF parallel to CREG expression in 4 groups. Blocking assay showed that PI3K/Akt inhibitor wortmannin and VEGF neutralising antibody could effectively eliminate the CREG induced vasculogenesis, and VEGF could successfully rescue failed vasculogenesis due to CREG gene silence. Conclusions CREG promotes vasculogenesis of ES cells by activating PI3K/Akt/VEGF pathway.

ASSA13-10-13 Cellular Repressor E1A-Stimulated Genes Controls Phenotypic Switching of Adventitial Fibroblasts by Blocking p38MAPK Activation

Background Phenotypic modulation of adventitial fibroblasts (AFs) plays an important role in the pathogenesis of proliferative vascular diseases. The current study aimed to identify the role of cellular repressor E1A-stimulated genes (CREG), a critical mediator in the maintenance of vascular homeostasis, in AF phenotypic modulation and adventitial remodelling. Methods Two models of AF phenotypic modulation were used: the in vivo injury-induced adventitial remodelling and the in vitro Ang II-induced AF phenotypic modulation. CREG’s function in AFs was investigated after over-expression of CREG with infection with adenovirus expressing full-length human CREG cDNA. Results Using in situ double-immunofluorescence staining, we ascertained that CREG expression was significantly downregulated in the adventitia after vascular injury, and its expression pattern was conversely correlated with the expression of smooth muscle α-actin (α-SMA), a marker for differentiation of AFs into myofibroblasts. In vitro data confirmed the association of CREG in angiotensin II (Ang II)-induced AF differentiation. Additionally, over-expression of CREG attenuated Ang II-induced α-SMA expression in AFs. CREG over-expressing AFs showed decreased levels of proliferation on days 2 to 5 following stimulation by Ang II compared with controls, with changes in the cell cycle profile as shown by BrdU incorporation assay and fluorescence activated cell sorting analysis. Moreover, wound healing assay and transwell migration model demonstrated that upregulation of CREG expression inhibited Ang II-induced AF migration. We found that CREG mediated its counterbalancing effects in Ang II-induced phenotypic modulation, proliferation and migration by inhibition of the p38MAPK signalling pathway, validated by pharmacological blockade of p38MAPK with SB 203580 and by over-expression of p38MAPK with transfectants expressing constitutively active p38αMAPK. Conclusions Our findings suggest that CREG is a novel AF phenotypic modulator in a p38MAPK-dependent manner. Modulating CREG on the local vascular wall may become a new therapeutic target against proliferative vascular diseases.

ASSA13-10-6 Relationship Between Paraoxonase 1 (PON1) Gene Polymorphisms, Haplotypes, Concentration, Activity and Immunohistochemical Analysis with Coronary Artery Disease Risk in Chinese Han Population

Background Paraoxonase 1 (PON1) is an high-density lipoprotein (HDL)-associated enzyme capable of inhibiting the progression of atherosclerosis, thus preventing the development of coronary artery disease (CAD). The polymorphisms of PON1 gene are known to affect the PON1 concentration and activity, thereby affect the CAD risk. As to its crucial role in preventing of CAD, we determined PON1 polymorphisms and haplotypes, concentration and activity, in addition to the immunohistochemical analysis of PON1 in this population and correlated them with CAD. Methods A total of 864 controls and 792 patients with CAD confirmed by angiography (≥ 70% stenosis) were recruited in Shenyang Northern Hospital. The concentration of PON1 was measured with Human PON1 Elisa Kit. PON1 activity towards phenylacetate was determined by spectro-photometrically at 270 nm. In addition, genotypes were determined by polymerase chain reaction (PCR). The genotypes and haplotypes were determined by SHEsis and SNPStats softwares respectively. PON1 expression in coronary and carotid arteries were detected by immunohistochemical analysis. Results Among all studied polymorphisms, only Q192R (rs662) had significant effect on the risk of CAD (Q192R, P < 0.001). In a logistic regression model, after adjustment for the conventional risk factors for CAD, QR and RR genotypes of Q192R had significantly higher CAD risk. Haplotypes Q-L-T-C-G (OR: 0.511, 95% CI : 0.401 ∼ 0.651) was also significantly associated with CAD. Both serum PON1 concentration and activity reduced significantly in CAD patients as compared to the controls (P < 0.001). Immunohistochemical analysis showed that during the atherosclerosis of coronary artery, smooth muscle cell staining for PON1 was greatly reduced as compared to the controls, so did in the external carotid artery. Conclusions The coding Q192R polymorphism and Q-L-T-C-G haplotype are all independently associated with CAD. Serum PON1 concentration and activity were lower in CAD patients than the controls. Additional with the evidence of immunohistochemical analysis, our data add support to the point that PON1 is a strong factor in predicting the risk of CAD.

ASSA13-10-14 Cellular Repressor of E1A-Stimulated Genes Accelerates Endothelial Angiogenesis Via Integrin-Linked Kinase-PINCH-CDC42 Activation

Background Cellular repressor of E1A-stimulated genes (CREG) is an important endothelial-protective gene, which is reported to modulate the mobility of endothelial cells. Objective The study aimed to investigate the effects of CREG on endothelial angiogenesis. Methods Aortic expression of CREG protein was detected by western blot and immnuostaining in CREG heterozygous (CREG+/-) mice and wild-type littermates. Perfusion recovery of hind limb was valued by laser Doppler in mice and wild-type littermates 14 days after the hind limb arterial ligation. The matrigel experiments in vivo and in vitro were performed after the human umbilical vein endothelial cells (HUVEC) were infected by adenovirus overexpressing CREG or adenovirus expressing GFP as control, and the filopodium formation was observed to evaluate the CREG function on neovascularization. Different integrin-linked kinase (ILK) subunit site mutant plasmids and small interfering RNA identified p-Cdc42 were transfected into HUVEC to explore the mechanism through which CREG-mediated endothelial angiogenesis. Results Aortic expression of CREG protein was detected to reduce remarkably in CREG+/- mice compared to that in wild-type littermates. Meanwhile, laser Doppler perfusion imaging showed that perfusion recovery was significantly impaired in CREG+/- mice than that in wild-type littermates after 14 days hind limb arterial ligation (78.23 ± 7.2% vs 12.15 ± 2.058% in calf; 58.23 ± 6.5% vs 32.15 ± 3. 514% in thigh; P < 0.001). Subsequently, overexpression of CREG in HUVEC increased endothelial cell network formation in vitro, enhanced neovascularization and improved limb perfusion in vivo, accompanied by filopodium formation and changes in cell shape. Mechanismly, integrin-linked kinase (ILK), a key adhesion plaque protein, participated in CREG-mediated endothelial angiogenesis. Furthermore, studies using small interfering RNA identified p-Cdc42 to be a key downstream molecule of ILK involved in CREG-mediated endothelial cell filopodium formation. Transfection with binding-site-mutant plasmids of ILK and co-immunoprecipitation revealed that CREG activated the ILK-PINCH complex, which is involved in the regulation of p-Cdc42 activation. Conclusions CREG overexpression stimulates the endothelial filopodium formation and regulates angiogenesis via the ILK/PINCH/p-Cdc42 signalling pathway, which provides the basis for future studies in the field of angiogenesis.