AMPKα2 controls the anti-atherosclerotic effects of fish oils by modulating the SUMOylation of GPR120.

Consuming fish oils (FO) is linked to reduced risk of cardiovascular disease in certain populations. However, FO failed to exhibit therapeutic effects in some patients with cardiovascular disease. This study aimed to determine the possible reasons for the inconsistent effects of FO. AMP-activated protein kinase (AMPK) α2 is an important energy metabolic sensor, which was reported to involve in FO mediated regulation of lipid and glucose metabolism. In an in vivo study, FO administration significantly reduced the aortic lesions and inflammation in the Ldlr-/- mouse model of atherosclerosis, but not in Ldlr-/-/Prkaa2-/-and Ldlr-/-/Prkaa2-/-Sm22Cre mice. Mechanistically, inactivation of AMPKα2 increased the SUMOylation of the fatty acid receptor GPR120 to block FO-induced internalization and binding to ß-arrestin. In contrast, activation of AMPKα2 can phosphorylate the C-MYC at Serine 67 to inhibit its trans-localization into the nuclei and transcription of SUMO-conjugating E2 enzyme UBC9 and SUMO2/3 in vascular smooth muscle cells (VSMCs), which result in GPR120 SUMOylation. In human arteries, AMPKα2 levels were inversely correlated with UBC9 expression. In a cohort of patients with atherosclerosis, FO concentrations did not correlate with atherosclerotic severity, however, in a subgroup analysis a negative correlation between FO concentrations and atherosclerotic severity was found in patients with higher AMPKα2 levels. These data indicate that AMPKα2 is required for the anti-inflammatory and anti-atherosclerotic effects of FO.

Recombinant Cellular Repressor of E1A-Stimulated Genes Protects against Renal Fibrosis in Dahl Salt-Sensitive Rats.

BACKGROUND: Human cellular repressor of E1A-stimulated genes (CREG) is a secreted glycoprotein that attenuates angiotensin II-induced hypertension, alleviates myocardial fibrosis, and improves heart function. However, the role of CREG in high-salt (HS) diet-induced hypertensive nephropathy is unclear. METHODS: To determine the effects and molecular mechanisms of CREG in HS diet-induced hypertensive nephropathy, we established a hypertensive nephropathy animal model in Dahl salt-sensitive (SS) rats fed a HS diet (8% NaCl, n = 20) for 8 weeks. At week 4 of HS loading, these rats were administered recombinant CREG (reCREG; 35 µg/kg·day, n = 5) and saline (n = 5) via subcutaneously implanted pumps and were also administered the vasodilator hydralazine (20 mg/kg·day, n = 5) in drinking water. We used hematoxylin and eosin staining, Masson's trichrome staining, immunohistochemical labeling, western blotting, RT-PCR, and Tunel staining to determine the signaling pathways of CREG in HS diet-induced hypertensive nephropathy. RESULTS: After 8 weeks of HS intake, the Dahl SS rats developed renal dysfunction and severe renal fibrosis associated with reductions of 78 and 67% in CREG expression, respectively, at both mRNA and protein levels in the kidney. Administration of reCREG improved renal function and relieved renal fibrosis. Administration of CREG also inhibited monocyte infiltration and reduced apoptosis in the kidney cells. CREG overexpression upregulated forkhead box P1 expression and inhibited the transforming growth factor-ß1 signaling pathway. CONCLUSION: Our study shows that CREG protected the kidney against HS-diet-induced renal damage and provides new insights into the mechanisms underlying kidney injury.

The novel intracellular protein CREG inhibits hepatic steatosis, obesity, and insulin resistance.

Cellular repressor of E1A-stimulated genes (CREG), a novel cellular glycoprotein, has been identified as a suppressor of various cardiovascular diseases because of its capacity to reduce hyperplasia, maintain vascular homeostasis, and promote endothelial restoration. However, the effects and mechanism of CREG in metabolic disorder and hepatic steatosis remain unknown. Here, we report that hepatocyte-specific CREG deletion dramatically exacerbates high-fat diet and leptin deficiency-induced (ob/ob) adverse effects such as obesity, hepatic steatosis, and metabolic disorders, whereas a beneficial effect is conferred by CREG overexpression. Additional experiments demonstrated that c-Jun N-terminal kinase 1 (JNK1) but not JNK2 is largely responsible for the protective effect of CREG on the aforementioned pathologies. Notably, JNK1 inhibition strongly prevents the adverse effects of CREG deletion on steatosis and related metabolic disorders. Mechanistically, CREG interacts directly with apoptosis signal-regulating kinase 1 (ASK1) and inhibits its phosphorylation, thereby blocking the downstream MKK4/7-JNK1 signaling pathway and leading to significantly alleviated obesity, insulin resistance, and hepatic steatosis. Importantly, dramatically reduced CREG expression and hyperactivated JNK1 signaling was observed in the livers of nonalcoholic fatty liver disease (NAFLD) patients, suggesting that CREG might be a promising therapeutic target for NAFLD and related metabolic diseases. CONCLUSION: The results of our study provides evidence that CREG is a robust suppressor of hepatic steatosis and metabolic disorders through its direct interaction with ASK1 and the resultant inactivation of ASK1-JNK1 signaling. This study offers insights into NAFLD pathogenesis and its complicated pathologies, such as obesity and insulin resistance, and paves the way for disease treatment through targeting CREG. (Hepatology 2017;66:834-854).

CREG1 ameliorates myocardial fibrosis associated with autophagy activation and Rab7 expression.

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 tissues homeostasis that has been shown to antagonize injury of tissues or cells. In the present study, we aimed to investigate the regulatory role of CREG1 in cardiac autophagy, and to clarify autophagy activation mechanisms. First, we generated a CREG1 haploinsufficiency (Creg1(+/-)) mouse model, and identified that CREG1 deficiency aggravates myocardial fibrosis in response to aging or angiotensin II (Ang II). Conversely, exogenous infusion of recombinant CREG1 protein complete reversed cardiac damage. CERG1 deficiency in Creg1(+/-) mouse heart showed a market accumulation of autophagosome that acquired LC3II and beclin-1, and a decrease in autophagic flux clearance as indicated by upregulating the level of p62. Inversely, restoration of CREG1 activates 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 cardiomyocytes, overexpression of CREG1 with concurrent resveratrol treatment significantly increased autophagy, while silencing CREG1 blocked the resveratrol-induced autophagy. These results suggest that CREG1-induced autophagy is required to maintain heart function in the face of stress-induced myocardiac damage. Both in vitro and in vivo studies identified that CREG1 deficiency influenced the maturation of lysosomes and reduced the espression of Rab7, which might be involved in CREG1-induced cardiomyocyte autophagy. These findings suggest that autophagy activation via CREG1 may be a viable therapeutic strategy autophagy for improving cardiac performance under pathologic conditions. This article is part of a Special Issue entitled: autophagy and protein quality control in cardiometabolic diseases.

MicroRNA-495 regulates the proliferation and apoptosis of human umbilical vein endothelial cells by targeting chemokine CCL2.

INTRODUCTION: Endothelium dysfunction plays a critical role in atherosclerosis. MicroRNAs are endogenous non-coding RNAs that suppress gene expression by binding to the 3' untranslated regions of target genes. MiR-495 can regulate the proliferation and apoptosis of cancer cells, however, the roles of miR-495 in endothelial cells (ECs) remain unclear. Therefore, this study aims to investigate the roles and mechanisms of miR-495 on ECs proliferation and apoptosis. MATERIALS AND METHODS: MiR-495 and CCL2 expressions were examined using quantitative RT-PCR, ELISA assay and western blot. Bioinformatics analysis and luciferase reporter assay were used to examine the regulatory relationship between miR-495 and CCL2. CCK8 assay, BrdU incorporation assay and flow cytometry were used to analyze the roles of miR-495 and CCL2 on the proliferation of human umbilical vein endothelial cells (HUVECs). The effects of miR-495 and CCL2 on HUVECs apoptosis were examined by tunnel staining and western blot. RESULTS: MiR-495 was down-regulated in patients with coronary artery disease compared with healthy controls. CCL2 was a novel target gene of miR-495. MiR-495 significantly promoted HUVECs proliferation by altering cell cycle distribution, and it also inhibited HUVECs apoptosis by affecting the expression of cleaved caspase 3. Effects of miR-495 on HUVECs proliferation and apoptosis were significantly reversed by overexpression of CCL2. CONCLUSIONS: MiR-495 could affect HUVECs proliferation and apoptosis by directly targeting CCL2. This is the first report to disclose the roles and mechanisms of miR-495 on HUVECs proliferation and apoptosis, which may provide a theoretical basis for clarifying the mechanisms of atherosclerosis.

Purification and functional assessment of smooth muscle cells derived from mouse embryonic stem cells.

OBJECTIVE: To obtain a pure population of smooth muscle cells (SMC) derived from mouse embryonic stem cells (ESC) and further assess their functions. METHODS: A vector, expressing both puromycin resistance gene (puro(r) ) and enhanced green fluorescent protein (EGFP) gene driven by smooth muscle 22α (SM22α) promoter, named pSM22α-puro(r)-IRES2-EGFP was constructed and used to transfect ESC. Transgenic ESC (Tg-ESC) clones were selected by G418 and identified by PCR amplification of puro(r) gene. The characteristics of Tg-ESC were detected by alkaline phosphatase (ALP) staining, SSEA-1 immunofluorescence and teratoma formation test in vivo. After induction of SMC differentiation by all-trans retinoic acid, differentiated Tg-ESC were treated with 10 µg/mL puromycin for three days to obtain purified SMC (P-SMC). Percentage of EGFP(+) cells in P-SMC was assessed by flow cytometer. Expressions of smooth muscle specific markers were detected by immunostaining and Western blotting. Proliferation, migration and contractility of P-SMC were analyzed by growth curve, trans-well migration assay, and carbachol treatment, respectively. Finally, both P-SMC and unpurified SMC (unP-SMC) were injected into syngeneic mouse to see teratoma development. RESULTS: Tg-ESC clone was successfully established and confirmed by PCR detection of puro(r) gene in its genomic DNA. The Tg-ESC was positive for ALP staining, SSEA-1 staining and formed teratoma containing tissues derived from three germ layers. After retinoic acid induction, large amount of EGFP positive cells outgrew from differentiated Tg-ESC. Three days of puromycin treatment produced a population of P-SMC with an EGFP(+) percentage as high as 98.2% in contrast to 29.47% of unP-SMC. Compared with primary mouse vascular smooth muscle cells (VSMC), P-SMC displayed positive, but lowered expression of SMC-specific markers including SM α-actin and myosin heavy chain (SM-MHC) detected either, by immunostaining, or immunoblotting, accelerated proliferation, improved migration (99.33 ± 2.04 vs. 44.00 ± 2.08 migrated cells/field, P < 0.05), and decreased contractility in response to carbachol (7.75 ± 1.19 % vs. 16.50 ± 3.76 % in cell area reduction, P < 0.05). In vivo injection of unP-SMC developed apparent teratoma while P-SMC did not. CONCLUSIONS: We obtained a pure population of ESC derived SMC with less mature (differentiated) phenotypes, which will be of great use in research of vascular diseases and in bio-engineered vascular grafts for regenerative medicine.

The impact of gene polymorphism and high on-treatment platelet reactivity on clinical follow-up: outcomes in patients with acute coronary syndrome after drug-eluting stent implantation.

AIMS: The current study sought to evaluate the clinical impact of newly reported genetic variations and their association with clopidogrel high on-treatment platelet reactivity (HTPR) in acute coronary syndrome (ACS) patients after drug-eluting stent (DES) implantation. METHODS AND RESULTS: The study enrolled 1,016 consecutive patients with ACS undergoing DES implantation. A total of 19 tag single nucleotide polymorphisms (SNPs) were selected from CYP3A4/5, CYP2C19, P2Y12 and ABCB1 genes. ADP-induced light transmittance aggregometry (LTA) was performed to test the post-procedure maximum platelet agglutination (MPA). The primary endpoint was a composite of cardiovascular death, non-fatal myocardial infarction (MI), stent thrombosis, and ischaemic stroke at one-year follow-up after DES placement. The secondary endpoint was the incidence of bleeding events. The post-procedure MPA was calculated and the cut-off point was determined for the HTPR. Using multivariate logistic regression analysis, the carriage of two CYP2C19 LOF alleles was an independent predictor of the post-procedure HTPR (OR: 2.8, 95% CI: 1.70-7.23, p<0.001). Through multivariate Cox regression analysis, the carriage of two CYP2C19 LOF alleles and the post-procedure HTPR were independent predictors of the primary endpoint (HR: 2.3, 95% CI: 1.40-4.97, p<0.001; HR: 2.9, 95% CI: 1.52-5.57, p<0.001, respectively). However, post-procedure MPA did not predict a bleeding event (HR: 0.9, 95% CI: 0.44-1.59, p=0.532). CONCLUSIONS: In patients with ACS, the CYP2C19 LOF allele was associated with post-procedure HTPR and a subsequently increased risk of adverse clinical events at one-year follow-up following DES implantation and clopidogrel administration.

MicroRNA-31 controls phenotypic modulation of human vascular smooth muscle cells by regulating its target gene cellular repressor of E1A-stimulated genes.

Phenotypic modulation of vascular smooth muscle cells (VSMCs) plays a critical role in the pathogenesis of a variety of proliferative vascular diseases. The cellular repressor of E1A-stimulated genes (CREG) has been shown to play an important role in phenotypic modulation of VSMCs. However, the mechanism regulating CREG upstream signaling remains unclear. MicroRNAs (miRNAs) have recently been found to play a critical role in cell differentiation via target-gene regulation. This study aimed to identify a miRNA that binds directly to CREG, and may thus be involved in CREG-mediated VSMC phenotypic modulation. Computational analysis indicated that miR-31 bound to the CREG mRNA 3' untranslated region (3'-UTR). miR-31 was upregulated in quiescent differentiated VSMCs and downregulated in proliferative cells stimulated by platelet-derived growth factor and serum starvation, demonstrating a negative relationship with the VSMC differentiation marker genes, smooth muscle α-actin, calponin and CREG. Using gain-of-function and loss-of-function approaches, CREG and VSMC differentiation marker gene expression levels were shown to be suppressed by a miR-31 mimic, but increased by a miR-31 inhibitor at both protein and mRNA levels. Notably, miR-31 overexpression or inhibition affected luciferase expression driven by the CREG 3'-UTR containing the miR-31 binding site. Furthermore, miR-31-mediated VSMC phenotypic modulation was inhibited in CREG-knockdown human VSMCs. We also determined miR-31 levels in the serum of patients with coronary artery disease (CAD), with or without in stent restenosis and in healthy controls. miR-31 levels were higher in the serum of CAD patients with restenosis compared to CAD patients without restenosis and in healthy controls. In summary, these data demonstrate that miR-31 not only directly binds to its target gene CREG and modulates the VSMC phenotype through this interaction, but also can be an important biomarker in diseases involving VSMC phenotypic modulation. These novel findings may have extensive implications for the diagnosis and therapy of a variety of proliferative vascular diseases.

[Effects of astragali radix extract on matrix metalloproteinase 9 expression and atherosclerotic plaque formation in aorta of apolipoprotein E deficient mice fed with high fat diet].

OBJECTIVE: To explore the effects of astragali radix extract on the expressions of matrix metalloproteinase 9 (MMP-9) and the formation of atherosclerotic plaque in aortic atherosclerotic plaques of apolipoprotein E-deficient mice (ApoE-/-). METHODS: Male 8-week-old ApoE-/- mice fed with high fat diet were randomly divided into four groups (n=12 each): control group (saline 0.2 ml/d), atorvastatin group (atorvastatin 10 mg×kg(-1)×d(-1)), low-dose astragali radix extract group (1.25 g×kg(-1)×d(-1)) and high-dose astragali radix extract group (5 g×kg(-1)×d(-1)). After 12 weeks, serum oxLDL was measured by the method of ELISA. The formation of atherosclerotic plaque was determined in HE and oil red O stained aortic slice. The expressions of macrophage and MMP-9 in the aortic plaque were detected by immune fluorescence and immunohistochemistry staining method. RESULTS: Similarly as atorvastatin, astragali radix extract significantly decreased the level of serum oxLDL in ApoE-/-1 mice in a dose-dependent manner. The level of oxLDL in the high-dose astragali radix extract group [(5.2±6.1) µg/ml] was significantly lower than that in the control group [(15.8±5.4) µg/ml, P<0.01]. The area of atherosclerosis plaques was smaller (17.24%±4.22% vs. 49.87%±9.37%, P<0.01) and the penetration degree of plaques in the arterial wall was relieved in the high-dose astragali radix extract group compared to those in the control group (P<0.01). The expressions of Mac3 in atherosclerosis plaques of the high-dose astragali radix extract group was also significantly lower than in the control group (P<0.01). The mean absorbance value of the expression of MMP-9 in the high-dose astragali radix extract group (0.0154±0.0014)was significantly lower than that in the control group (0.0263±0.0065) (P<0.01). CONCLUSIONS: Similar as atorvastatin, astragali radix extract can dose-dependently inhibit the expression of MMP-9 and the formation of the atherosclerotic plaque in ApoE-/- mouse, probably by reducing the serum oxLDL, inhibiting macrophage infiltration, migration and secretion of MMP-9.

CREG mediated adventitial fibroblast phenotype modulation: a possible therapeutic target for proliferative vascular disease.

Proliferative vascular diseases, of which neointimal formation is a key pathological feature, cause significant morbidity and mortality worldwide. Adventitia is the outermost connective tissue that surrounds an artery. In recent years, accumulating data indicate that adventitial fibroblasts participate in the formation of neointimal lesions. Our previous studies have demonstrated that cellular repressor of E1A-stimulated genes (CREG) plays critical roles in reducing neointimal hyperplasia by promoting vascular smooth muscle cell (VSMC) differentiation and grow arrest and inhibiting migration. Hence, it is plausible that genetical modification with CREG gene in adventitial fibroblasts might inhibit angiotensin II-induced transdifferentiation to myofibroblasts, proliferation and migration, as well as adventitial thickening, finally decreasing neointimal formation. Possible mechanisms may include CREG direct attenuation of reactive oxygen species derived from reduced cathepsin-dependent activation of NADPH oxidase and indirect suppression of the downstream of NADPH oxidase including ERK1/2, JNK and p38 MAPK pathways in adventitial fibroblasts. Therefore, CREG may be a potential therapeutic target for proliferative vascular diseases.

Anti-atherosclerotic function of Astragali Radix extract: downregulation of adhesion molecules in vitro and in vivo.

BACKGROUND: Atherosclerosis is considered to be a chronic inflammatory disease. Astragali Radix extract (ARE) is one of the major active ingredients extracted from the root of Astragalus membranaceus Bge. Although ARE has an anti-inflammatory function, its anti-atherosclerotic effects and mechanisms have not yet been elucidated. METHODS: Murine endothelial SVEC4-10 cells were pretreated with different doses of ARE at different times prior to induction with tumor necrosis factor (TNF)-α. Cell adhesion assays were performed using THP-1 cells and assessed by enzyme-linked immunosorbent assay, western blotting and immunofluorescence analyses to detect the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), phosphorylated inhibitor of κB (p-iκB) and nuclear factor (NF)-κB. We also examined the effect of ARE on atherosclerosis in the aortic endothelium of apolipoprotein E-deficient (apoE(-/-)) mice. RESULTS: TNF-α strongly increased the expression of VCAM-1 and ICAM-1 accompanied by increased expression of p-iκB and NF-κB proteins. However, the expression levels of VCAM-1 and ICAM-1 were reduced by ARE in dose- and time-dependent manners, with the strongest effect at a dose of 120 µg/ml incubated for 4 h. This was accompanied by significantly decreased expression of p-iκB and inhibited activation of NF-κB. Immunofluorescence analysis also revealed that oral administration of ARE resulted in downregulation of adhesion molecules and decreased expression of macrophages in the aortic endothelium of apoE(-/-) mice. ARE could suppress the inflammatory reaction and inhibit the progression of atherosclerotic lesions in apoE(-/-) mice. CONCLUSION: This study demonstrated that ARE might be an effective anti-inflammatory agent for the treatment of atherosclerosis, possibly acting via the decreased expression of adhesion molecules.

[Tumor necrosis factor-α promote permeability of human umbilical vein endothelial cells via activating RhoA-ERK1/2 pathway].

OBJECTIVE: Tumor necrosis factor-α (TNF-α) is known to induce changes in endothelial cell morphology and permeability. The aim of this study is to determine the underlying signaling mechanisms involved in these responses. METHODS: Cultured human umbilical vein endothelial cells (HUVECs) were exposed to TNF-α, and HUVEC cytoskeletal changes were evaluated by observing fluorescence of F-actin following ligation with labeled antibodies. Endothelial permeability was detected by measuring the flux of horseradish peroxidase (HRP)-albumin across the EC monolayers. To explore the signaling pathways behind TNF-α-induced changes in HUVEC morphology and permeability, HUVECs were treated with either the Rho GTPase inhibitor Y27632 or the mitogen-activated protein kinases (MAPK) inhibitors PD98059 and SB203580 before TNF-α administration. To further elucidate possible involvement of the RhoA and ERK pathways in TNF-α-induced HUVEC changes, retrovirus-carried recombinant dominant-negative forms and constitutive-activative forms of RhoA, namely T19NRhoA and Q63LRhoA, were pre-infected into HUVECs prior to TNF-α exposure. RESULTS: TNF-α induced F-actin cytoskeleton rearrangement and increased HUVEC permeability in a dose and time-dependent manner. The maximal increase in the HRP-BSA flux (40 ng/ml) was seen in cells exposed to TNF-α at 100 ng/ml after 2 h. Preconditioning of HUVEC monolayer with Y27632 or PD98059 significantly reduced TNF-α induced permeability increase (HRP concentration from 40 ng/ml decreased to 12.5 ng/ml, P < 0.05) and F-actin cytoskeleton rearrangement, HUVEC pre-infection with activated forms of Q63LRhoA increased HUVEC permeability and upregulated pERK compared to GFP infection, while HUVEC pre-infection with inhibited forms of T19NRhoA attenuated the effects of TNF-α on HUVEC permeability. CONCLUSION: These results indicate that TNF-α-induced EC barrier dysfunction and morphological changes of the F-actin via activating RhoA-ERK/MAPK signal pathway.

Overexpression of cellular repressor of E1A-stimulated genes inhibits TNF-α-induced apoptosis via NF-κB in mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (MSCs) show great potential for therapeutic repair after myocardial infarction. However, poor viability of transplanted MSCs in the ischemic heart has limited their use. Cellular repressor of E1A-stimulated genes (CREG) has been identified as a potent inhibitor of apoptosis. This study therefore aimed to determine if rat bone marrow MSCs transfected with CREG-were able to effectively resist apoptosis induced by inflammatory mediators, and to demonstrate the mechanism of CREG action. Apoptosis was determined by flow cytometric and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assays. The pathways mediating these apoptotic effects were investigated by Western blotting. Overexpression of CREG markedly protected MSCs from tumor necrosis factor-α (TNF-α) induced apoptosis by 50% after 10 h, through inhibition of the death-receptor-mediated apoptotic pathway, leading to attenuation of caspase-8 and caspase-3. Moreover, CREG resisted the serine phosphorylation of IκBα and prevented the nuclear translocation of the transcription factor nuclear factor-κB (NF-κB) under TNF-α stimulation. Treatment of cells with the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) significantly increased the transcription of pro-apoptosis proteins (p53 and Fas) by NF-κB, and attenuated the anti-apoptotic effects of CREG on MSCs. The results of this study indicate that CREG acts as a novel and potent survival factor in MSCs, and may therefore be a useful therapeutic adjunct for transplanting MSCs into the damaged heart after myocardial infarction.

[Effect of percutaneous coronary intervention timing and cilostazol use on left ventricular remodeling in patients with non-ST elevation myocardial infarction].

OBJECTIVE: To observe the dynamic changes of plasma matrix metalloproteinases (MMPs) and investigate the effect of early or delayed percutaneous coronary intervention (PCI) in the presence or absence cilostazol on left ventricle (LV) remodeling in patients with non-ST elevation myocardial infarction (NSTEMI). METHODS: One hundred and sixty-four patients undergoing PCI with NSTEMI were randomized to early PCI (PCI within 24 h) group or delayed PCI group (PCI after 36 h), and patients in both group were further assigned to cilostazol or no cilostazol group. Plasma MMP-2 and MMP-9 concentrations were measured at 2, 4 days and 2 and 4 weeks after PCI. Left ventricular end-diastolic volume (LVEDV), left ventricle ejection fraction (LVEF), left ventricle posterior wall (LVPW) and interventricular septum (IVS) were measured by echocardiography at baseline and 1 year after PCI. RESULTS: MMP-2 concentration at 2 weeks after PCI is higher than that at 2, 4 days and 4 weeks after PCI. MMP-9 concentration at 4 days is higher than that at 2 days, 2 weeks and 4 weeks after PCI. MMP-2 and MMP-9 were significantly lower in cilostazol group compared with that in non-cilostazol group at 4 days, 2 weeks and 4 weeks after NSTEMI (all P < 0.05). Changes of LVEDV and LVEF were significantly less in cilostazol group and early PCI group than that in no cilostazol group and delay PCI group (P < 0.05 or P < 0.01) at 1 year after NSTEMI. CONCLUSIONS: Early PCI and Cilostazol use are associated with less LV remodeling in patients with NSTEMI. Cilostazol attenuated LV remodeling possibly by reducing concentration of MMP-2 and MMP-9 after PCI.

[Purification and functional identification of the recombinant human CREG/myc-His glycoprotein].

OBJECTIVE: To purify the recombinant human cellular repressor of EIA stimulated gene (hCREG)/myc-His glycoprotein and confirm the biological function of hCREG/myc-His which could inhibit the proliferation of human internal thoracic artery smooth muscle cells (HITASY) cultured in vitro. METHODS: The recombinant hCREG/myc-His protein was purified with Ni-NTA column according to 6 x His affinity chromatographic theory. The recombinant hCREG/myc-His protein was desalted by HiTrap Desalting Column. The effect of recombinant hCREG/myc-His glycoprotein of different concentration (0.5 microg/ml, 1 microg/ml and 2 microg/ml) on proliferation of HITASY cells was studied by flow cytometric analysis and the effect of recombinant protein on proliferation of HITASY cells was confirmed by BrdU incorporation method. RESULTS: The recombinant hCREG protein was purified with Ni-NTA column according to 6 x His affinity chromatographic theory. The concentration of recombinant hCREG protein which has been concentrated and desalted was determined to be 1.6 mg/ml and the purity of recombinant protein reached 92%. The protein was identified to be glycosylated. The recombinant hCREG protein was identified to inhibit the proliferation of HITASY cells cultured in vitro and the inhibition effect was stronger in low-dosage group than that in high-dosage group by flow cytometric analysis. The proliferation of HITASY cells cultured in vitro with 2 microg/ml recombinant hCREG protein was inhibited significantly compared with that in control group according to the BrdU incorporation result. There was statistical difference among the groups (P < 0.05). CONCLUSION: The purification of recombinant hCREG/myc-His glycoprotein with biological activity provides an experiment platform for function study and engineering production of hCREG protein.

[Construction and assessment of short-hairpin RNA eukaryotic expression vector targeting TGF-beta1 labeled by GFP].

AIM: To construct short hairpin RNA (shRNA) eukaryotic expression vectors targeting TGF-beta1 for further research on the effects of TGF-beta1 on vasculogenesis and angiogenesis. METHODS: Three pairs of siRNA target sequences coding from the mRNA of TGF-beta1 gene were designed and three pairs of nucleotides were synthesized. After annealing, the double-strand DNA products were ligated into the pEN_mH1c entry vector, and in turn into the shRNA eukaryotic expression vector pDS_hpEy labled by GFP through the LR recombination reaction. After sequencing successfully, the three resulting TGF-beta1 shRNA expression vectors were transfected into the mouse fibroblast cell line (NIH/3T3), and then cell clones stably expressing TGF-beta1 shRNA were screened. Reverse Transcript-Polymerase Chain Reaction (RT-PCR) and Western blot were used to detect the mRNA and protein expression. RESULTS: RT-PCR and Western blot showed that one of the TGF-beta1 shRNA expression vectors pDS_Tc downregulated TGF-pl mRNA and protein expression markedly in NIH/3T3 cells. CONCLUSION: ShRNA eukaryotic expression vectors targeting TGF-beta1 are successfully constructed which can be used for further investigation on the mechanism through which TGF-beta1 regulates vasculogenesis and angiogenesis.

[Matrix metalloproteinase-1 gene -519A/G polymorphism and the risk of coronary heart disease in Northern Chinese Han population].

OBJECTIVE: To investigate the relationship between matrix metalloproteinase (MMP) 1 gene -519A/G polymorphism and the risk of coronary heart disease (CHD) in Northern Chinese Han population. METHODS: A total of 517 patients with CHD and 380 healthy adults diagnosed by coronary angiography were genotyped by polymerase chain reaction-restriction fragment length polymorphism and DNA sequence technology for the -519A/G polymorphism in MMP1 gene. RESULTS: (1) The frequency of AA genotype was significantly higher in patients with CHD than that in controls [67.70% (350/517) vs. 40.26% (153/380), OR = 1.64, P < 0.001, 95%CI: 1.44 - 1.86]. People carrying A allele had increased risk for CHD (OR = 1.49, P < 0.001, 95%CI: 1.33 - 1.69). (2) The frequency of AA genotype was higher in patients with acute coronary syndrome (ACS) than patients with stable angina pectoris [68.81% (278/404) vs. 51.76% (44/85), P < 0.01, 95%CI: 1.04 - 1.27]. The A allele carriers were more likely to develop ACS (OR = 1.11, 95%CI: 1.01 - 1.21, P < 0.05). CONCLUSION: Our data shows MMP1 gene -519A/G polymorphism is associated with the risk of CHD, and A allele carriers are more susceptible for CHD in Northern Chinese Han population.

Secreted CREG inhibits cell proliferation mediated by mannose 6-phosphate/insulin-like growth factor II receptor in NIH3T3 fibroblasts.

Cellular repressor of E1A-stimulated genes (CREG) is a recently described glycoprotein that plays a critical role in keeping cells or tissues in mature, homeostatic states. To understand the relationship between CREG and its membrane receptor, mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R), we first generated stable NIH3T3 fibroblasts by transfection of pDS_shCREGs vectors, which produced an approximately 80% decrease in CREG levels both in the lysate and in the media. We used fluorescence activated cell sorting and a bromide deoxyuridine incorporation assay to identify whether CREG knockdown promoted the cell proliferation associated with the increase of IGF-II in NIH3T3 fibroblasts. Proliferation was markedly inhibited in a concentration-dependent manner by re-addition of recombinant CREG protein into the media, and this was mediated by the membrane receptor M6P/IGF2R. We subsequently confirmed the direct interaction of CREG and M6P/IGF2R by both immunoprecipitation-Western blotting and immunofluorescence staining. We found that expression of CREG correlated with localization of the receptor in NIH3T3 fibroblasts but did not affect its expression. Our findings indicated that CREG might act as a functional regulator of M6P/IGF2R to facilitate binding and trafficking of IGF-II endocytosis, leading to growth inhibition.

[Changes of plasma high-sensitive C-reactive protein and monocyte chemotactic factor-1 following percutaneous coronary interventional procedures in patients with coronary artery disease].

OBJECTIVE: To investigate the changes of high-sensitive C-reactive protein (hs-CRP) and monocyte chemotactic factor-1 (MCP-1) following percutaneous coronary interventional procedures (PCI) in patients with coronary artery disease (CAD), and evaluate the impact of PCI on the inflammatory indices and postoperative vascular restenosis. METHODS: This study involved 80 patients undergoing PCI procedures for CAD compromising a single coronary artery. Forty healthy individuals with normal findings by coronary angiography were selected as the control group. Before and after PCI or coronary angiography, plasma hs-CRP and MCP-1 were measured in all the subjects by immunonephelometry and enzyme-linked immunosorbant assay (ELISA), respectively. RESULTS: In the CAD patients, the plasma hs-CRP level was significantly elevated after PCI as compared with the preoperative level (2.37-/+0.56 microg/L vs 1.59-/+0.41 microg/L, P<0.01), whereas in the control group, the hs-CRP level underwent no significant changes after coronary angiography (1.18-/+0.37 microg/L vs the preoperative level of 1.13-/+0.32 microg/L, P>0.05). PCI procedures also resulted in significant elevation of plasma MCP-1 level in the CAD patients (26.04-/+5.43 pg/L vs the preoperative level of 18.07-/+4.30 pg/L, P<0.01), but in the control group, MCP-1 showed no significant variation after coronary angiography (9.80-/+2.64 pg/L vs the preoperative level of 9.63-/+2.52 pg/L, P>0.05). CONCLUSION: Plasma hs-CRP and MCP-1 are elevated in CAD patients following PCI procedures, but their roles in the vascular restenosis following the procedures need further investigation.

Involvement of Annexin A2 in p53 induced apoptosis in lung cancer.

Tumor suppressor p53 plays important roles in cell cycle regulation, apoptosis and DNA repair in different cell types including lung cancer. There are different p53 apoptotic pathways in high and low metastatic ability lung cancer cells. However, the exactly mechanism in the pathway is still unclear. Here we found that Annexin A2, a Ca2+ -dependent phospholipid-binding protein, is involved in p53-mediated apoptosis. First, by using mRNA differential display technique, down-regulated Annexin A2 expression was found in all cell lines transfected of Ad-p53 (adenoviral expression construct encoding wild type p53 gene) especially in highly metastatic Anip973 lung cancer cells. Then, decreased expression of Annexin A2 was further confirmed by Northern blot and Western blot analysis. At last, knock down of Annexin A2 by siRNA inhibited cellular proliferation in BE1 cell line with highly metastatic ability. Taken together, our results suggested that Annexin A2 may play roles in p53 induced apoptosis and it is also involved in regulation of cell proliferation.