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-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.

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.

[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.

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.

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.

[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.

[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.

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.

[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.

[Association of C1019T polymorphism in the connexin 37 gene and coronary artery disease in Chinese Han population].

OBJECTIVE: To investigate the association between the connexin 37 (CX37) C1019T polymorphism and the susceptibility to coronary artery disease (CAD) in northern Han population of China. METHODS: A total of 514 CAD patients and 400 healthy controls diagnosed by angiography were genotyped by using polymerase chain reaction-restriction fragment length polymorphism and polyacrylamide gel electrophoresis. RESULTS: The genotype frequencies of CC, TC and TT in the CX37 C1019T polymorphism was 22.37%, 53.31% and 24.32% in CAD patients, 17.75%, 46.50% and 35.75% in the controls respectively (P = 0.0007). The frequency of the CX37 C allele in CAD patients was significantly higher than that of the control group (49.03% vs 41.00%, OR = 1.38, 95% CI = 1.15 - 1.66, P = 0.0006). The frequency of the C allele carriers (CC + TC) was 75.68% in the CAD group and 64.25% in the control group (P = 0.0002). Compared with the TT homozygote, the CAD risk was significantly increased in the carriers of C allele (CC + TC) (OR = 1.73, 95% CI = 1.30 - 2.30). Subsequent stratified analysis revealed that the frequency of C allele was significantly higher in the male CAD patients than in the male controls (49.37% vs 39.60%, OR = 1.49, 95% CI = 1.18 - 1.89, P = 0.0009). The CAD risk was nearly two-fold increased in the carriers of C allele (CC + TC) than in the TT homozygote (95% CI = 1.38 - 2.78). However in the female population, there was no difference in the CAD risk between the carriers of (CC + TC) type and the TT homozygote (P = 0.24). CONCLUSION: The C allele in the CX37 gene might be associated with the susceptibility to CAD and potentially plays an important role in the manifestation of coronary atherosclerosis among Chinese.

[The antiplatelet effect of clopidogrel is not attenuated by statin treatment in patients with acute coronary syndromes undergone coronary stenting].

OBJECTIVE: To evaluate the interaction of atorvastatin or pravastatin with clopidogrel on platelet activation and aggregation function in patients with acute coronary syndromes (ACS) undergoing coronary stenting. METHODS: Between April and December 2006, a total of 150 hospitalized ACS patients undergoing coronary stenting were randomized to receive atorvastatin (n = 50), pravastatin (n = 50) or no statin (n = 50) one day post procedure. All patients received standard antiplatelet treatment including aspirin 300 mg/d and loading dose 300 mg of clopidogrel followed by maintenance dose 75 mg/d. The expressions of CD62P and PAC-1 and the maximal platelet aggregation rate (MPAR) induced by 20 micromol/L ADP were measured at day 1 before statin therapy (baseline) and day 3 after procedure. RESULTS: Baseline clinical characteristics and levels of CD62P, PAC-1 and MPAR at the baseline were comparable among three groups. After 3-day statin treatment, the changes of CD62P [(4.69 +/- 16.78)% vs. (1.35 +/- 10.86)% vs. (2.97 +/- 10.21)%], PAC-1 [(12.78 +/- 22.07)% vs. (8.01 +/- 21.23)% vs. (10.65 +/- 21.39)%] and MPAR [(5.44 +/- 18.68)% vs. (7.15 +/- 19.59)% vs. (3.76 +/- 23.42)%] among three groups were not significantly different (all P > 0.05). Subgroup analysis showed that DeltaCD62P [(7.50 +/- 19.35)% vs. (3.24 +/- 11.18)% vs. (2.53 +/- 8.87)%], DeltaPAC-1 [(13.40 +/- 24.62)% vs. (11.28 +/- 19.90)% vs. (10.11 +/- 21.29)%] and DeltaMPAR [(7.56 +/- 19.11)% vs. (7.87 +/- 23.60)% vs. (6.75 +/- 23.30)%] in ACS patients were also similar among three groups (all P > 0.05). CONCLUSION: Neither atorvastatin nor pravastatin attenuates the antiplatelet function of clopidogrel in ACS patients early post coronary stenting.

[Over-expression of the cellular repressor of E1A-stimulated genes inhibits the apoptosis of human vascular smooth muscle cells in vitro.].

To investigate the effects and molecular mechanisms of the cellular repressor of E1A-stimulated genes (CREG) on the apoptosis of vascular smooth muscle cells (VSMCs), the human internal thoracic artery-Shenyang (HITASY) cells were infected with sense-CREG [pLNCX(2)(+)/CREG] and antisense-CREG [pLXSN(-)/CREG] retrovirus respectively. The stably infected cells were obtained by screening the G418-resistant clones. DAPI nuclei staining and Annexin V/PI FASC assay indicated that over-expression of CREG in HITASY cells infected with pLNCX(2) (+)/CREG inhibited VSMC apoptosis induced by serum deprivation, accompanied with decreased expression of caspase-9 mRNA detected by RT-PCR. Furthermore, Western blot analysis showed that p38 mitogen activated protein kinase (p38 MAPK) expression and activation were significantly enhanced in HITASY cells infected with pLNCX(2) (+)/CREG. The inhibition of CREG protein expression in cells infected with pLXSN(-)/CREG promoted the VSMC spontaneous apoptosis, as well as down-regulated p38 MAPK expression and activation, when cells were cultured with 10% fetal bovine serum (FBS) mediums. These results implicate that the CREG protein has the ability to regulate VSMC apoptosis in which the activation of p38 MAPK is possibly involved. To further identify the role of p38 MAPK in VSMC apoptosis, SB203580, a specific inhibitor of p38 MAPK, was used to inhibit p38 MAPK activity. When p38 MAPK signaling pathway was blocked, the effects that over-expression of CREG protein inhibited VSMC apoptosis disappeared. Taken together, the present work indicates that over-expression of CREG protein inhibits VSMC apoptosis, and this inhibitory effect is partly mediated by p38 MAPK signaling pathway.

[Rac1 accelerates endothelial cell senescence induced by hypoxia in vitro].

To investigate the role and mechanism of Rac1 protein in the process of the human umbilical vein endothelial cell (HUVEC) senescence, we used hypoxia as a model for modulating HUVECs entering replicative senescence in vitro. Premature senescence of HUVECs was evidenced by detecting the SA-beta-Gal activity and PAI-1 expression. Meanwhile, cell cycle distribution and cell proliferation rate were investigated by flow cytometry assay and BrdU staining. The results indicated that the HUVECs became enlarged and flattened, both SA-beta-Gal activity and PAI-1 expression increased obviously, while cell proliferation was inhibited and G(1) phase cell cycle arresting occurred when HUVECs were treated with continued hypoxia for 96 h. Accompanied with these changes, the expression of activated Rac1 increased obviously in cells after hypoxia. All these observations suggested that endothelial senescence could be induced by continued hypoxia and it might correlate with the activity of Rac1. To further define the relationship between Rac1 and HUVEC senescence, HUVECs were transiently infected with the constitutively active form of Rac1 (V12Rac1) or dominant negative form of Rac1 (N17Rac1) using retrovirus vector pLNCX-V12Rac1 or pLNCX-N17Rac1. We observed the changes of these three kinds of HUVECs (HUVECs, N17Rac1-HUVECs, V12Rac1-HUVECs) after hypoxia for 48 h and 96 h, the expression and localization of serum response factor (SRF), which is one of the downstream signal molecules of Rac1, were also investigated. The results obtained indicated that after continued hypoxia for 48 h, HUVECs infected by V12Rac1 showed obvious senescence accompanied with SA-beta-Gal activation, PAI-1 expression increase, G(1) phase arrest and cell proliferation inhibition which were similar to HUVECs after continued 96-hour hypoxia treatment, while the senescence of HUVECs infected by N17Rac1 was significantly inhibited even if the cells were exposed to hypoxia for more than 96 h. All the results identified that the activation of Rac1 might accelerate HUVEC senescence induced by hypoxia and that inactivation of Rac1 could partly block the cell senescence. To further investigate the mechanism of HUVEC senescence induced by Rac1, we detected the expression of total SRF (tSRF) and nuclear SRF (nSRF) in these three kinds of HUVECs by immunofluorescent analysis and Western blot assay after hypoxia. The results showed that the expression of nSRF decreased obviously and the nuclear translocation of SRF was inhibited in HUVECs infected by V12Rac1 compared with those in the normal HUVECs. In contrast, the expression of nSRF increased obviously in the HUVECs infected by N17Rac1. These results suggest that activation of Rac1 accelerates endothelial cell senescence and inhibition of Rac1 activity prevents HUVECs from entering senescence induced by hypoxia, while the nuclear translocation of SRF regulated by Rac1 might play an important role in the process of senescence.

[The effects of post coronary stenting triple antiplatelet therapies on platelet functions].

OBJECTIVE: To explore the effects of triple antiplatelet therapy on platelet aggregation and activation in patients who underwent coronary stenting. METHODS: 120 in-hospital coronary heart disease patients with coronary stenting were randomized to two groups receiving either triple antiplatelet drugs of aspirin and clopidogrel combined with cilostazol or dual antiplatelet drugs of aspirin and clopidogrel. On the first day after stenting cilostazol was added to the triple group patients who were previously administered aspirin and clopidogrel. Expressions of PAC-1 and CD62p which indicate platelet activation were assessed with flow cytometry and the maximal platelet aggregation rate (MPAR) induced by 5 and 20 micromol/L ADP was measured at the day before receiving cilostazol and the fifth day after stenting, respectively. RESULTS: The baseline clinical characteristics did not differ significantly between the two groups. There were no significant differences in the baseline level of MPAR CD62p and PAC-1 at the first day after stenting between the two groups. The margins between the two measurements were [(6.44 +/- 14.44)% vs (5.41 +/- 13.77)%, P > 0.05] for DeltaMPAR induced by 5 micromol/L ADP, [(8.50 +/- 15.50)% vs (7.84 +/- 14.21)%, P > 0.05] for DeltaMPAR induced by 20 micromol/L ADP, [(5.12 +/- 11.25)% vs (1.08 +/- 4.97)%, P < 0.05] for DeltaCD(62)p and [(12.12 +/- 12.30)% vs (2.22 +/- 15.15)%, P < 0.01] for DeltaPAC-1 in the triple and dual group patients, respectively. Among the above measurements, DeltaCD62p and DeltaPAC-1 in the triple group patients were significantly higher than those in the dual group patients although DeltaMPAR did not significantly differ between the two groups at the fifth day after stenting. Subgroup analysis for patients with acute coronary syndrome (ACS) showed that DeltaMPAR induced by 5 micromol/L ADP [(8.68 +/- 10.35)% vs (2.92 +/- 13.06)%, P = 0.018], DeltaMPAR induced by 20 micromol/L [(11.05 +/- 11.14)% vs (5.16 +/- 13.27)%, P = 0.019], DeltaCD62p [(5.57 +/- 12.08)% vs (1.35 +/- 4.42)%, P = 0.028] and DeltaPAC-1 [(11.62 +/- 12.73)% vs (1.29 +/- 15.73)%, P = 0.001] in triple group were significantly higher than that in dual group. At 3-month clinical follow-up, the rate of major adverse cardiac and cerebral events was 0 in the triple group and 3.3% (2/60) in the dual group, and the rate of hemorrhage was 5% (3/60) in the triple group and 3.3% (2/60) in the dual group, the differences were not statistically significant. CONCLUSIONS: Compared with dual antiplatelet regimen with aspirin plus clopidogrel, triple antiplatelet therapy with aspirin and clopidogrel combined with cilostazol is more efficient in inhibiting platelet activation and aggregation after coronary stent implantation. Large scale clinical trials are needed to confirm efficacy and safety of the triple antiplatelet regimen.

[Serum response factor participates in RhoA-induced endothelial cell F-actin rearrangements].

RhoA is one of the main members of RhoGTPase family involved in cell morphology, smooth muscle contraction, cytoskeletal microfilaments and stress fiber formation. It has been demonstrated that RhoA modulates endothelial cell permeability by its effect on F-actin rearrangement, but the molecular mechanism of rearrangement of actin cytoskeleton remains unclear. Recent studies prove that RhoA/Rho kinase regulates smooth muscle specific actin dynamics by activating serum response factor (SRF)-dependent transcription. To further investigate the molecular mechanism of the rearrangement of vascular endothelial cell actin cytoskeleton, we explored the relationship between the activation of SRF and F-actin rearrangement induced by RhoA in human umbilical vein endothelial cells (HUVECs). HUVECs were infected with the constitutively active forms of RhoA (Q63LRhoA) or the dominant negative forms of RhoA(T19NRhoA) using retrovirus vector pLNCX-Q63LRhoA or pLNCX-T19NRhoA, the positive clone was obtained by G418 selection. The expression and distribution of SRF in normal and infected cells were evaluated by immunohistochemistry and Western blot in complete medium and in serum-free medium. The effect of F-actin polymerization was detected by Rhodamine-Phalloidine staining. Infection of PLNCX-Q63LRhoA induced F-actin rearrangement and stress fiber formation in HUVECs, as well as enhanced the expression of SRF in the nuclei. In contrast, the cells infected with T19NRhoA showed no distinct changes. With serum deprivation, the expression of SRF increased obviously in both normal and infected HUVECs, but the subcellular localization of SRF was evidently different. In HUVECs, the localization of SRF was in the nuclei after 3 d with serum deprivation, but it was redistributed outside the nuclei after 5 d with serum deprivation. In cells infected with Q63LRhoA, the immunolocalization of SRF was always in the nuclei compared with HUVECs infected with T19NRhoA, which was almost always localized in the cytoplasm. In HUVECs, the rearrangement of F-actin and formation of stress fiber increased after 3 d with serum deprivation, but appeared decreased and unpolymerized after 5 d with serum deprivation. The polymerization of F-actin and the formation of stress fiber in HUVECs infected with Q63LRhoA kept during the period of serum-free culture, whereas the rearrangement of F-actin in cells infected with T19NRhoA was not found. These results suggest that RhoA influences endothelial F-actin rearrangement in part by regulating the expression and subcellular localization of SRF.

[The cloning and expression of apoptosis associated gene ANNEXIN A2 induced by p53 gene].

OBJECTIVE: To identify the relationship between p53-dependent apoptosis associated genes and tumor metastasis. METHODS: mRNA differential display (mRNA DD) was adopted for gene cloning after the different metastatic potential lung cancer cell lines were infected by Adv-p53 (a reconstructed adenovirus encoding wild type p53 gene). RT-PCR, Northern blot and Western blot assays were used to confirm the result from mRNA DD. RESULTS: After induction by p53 gene, the ANNEXIN A2 gene had differential expression in the cell lines; its level was down regulated in all the cells infected by Adv-p53 gene, especially in the Anip973 cell lines with high metastatic potential. RT-PCR, Northern blot and Western blot assays confirmed the consequence. CONCLUSION: The experimental data suggest that the ANNEXIN A2 gene may relate to cellular apoptosis induced by p53 gene. The affirmative relationship between ANNEXIN A2 gene and p53 needs further investigation.

[Expression of cellular repressor of E1A-stimulated genes in vascular smooth muscle cells of different phenotypes].

OBJECTIVES: The phenotypic modulation of vascular smooth muscle cells (VSMC) plays a central role in the pathogenesis of arteriosclerosis. The purpose of this study was to investigate the expression of the cellular repressor of E1A-activated genes (CREG) at the transcriptional and protein level in human internal thoracic artery smooth muscle cells (HITASY), which express different patterns of differentiation markers after serum withdrawal. METHODS: After cloning and recombining the CREG vector, the antiserum against the CREG protein was produced from the rabbits immunized by the purification CREG protein. The specificity of purified polyclonal antibody was detected by Western blot assay. The DNA synthesis of HITASY cultured in serum-free and serum-supplemented medium was measured by the [(3)H]-thymidine incorporation. Western blot analysis detected the expression of smooth muscle-specific markers (smooth muscle alpha-actin, calponin). The localization of CREG in cells was examined with immunohistochemistry staining and expression of CREG mRNA and protein were analyzed by RT-PCR and Western blot in HITASY after serum withdrawal. RESULTS: The high specificity of polyclonal antibody against CREG obtained from rabbits was confirmed by Western blot assay. In response to serum withdrawal, cultured HITASY cells exhibited phenotypic conversion from synthetic into contractile one as evidenced by the data of [(3)H]-thymidine incorporation and Western blot. The DNA synthesis of HITASY precipitously dropped to background levels after serum withdrawal and nearly restored after reintroduction of serum to culture medium 2 days later. Western blot revealed a reversible upregulation of smooth muscle alpha-actin and calponin in HITASY after serum deprivation. Moreover, serum withdrawal also induced a prominent increase of CREG mRNA and protein expression which reached a peak on 3 days and decreased gradually on 5 approximately 7 days after serum withdrawal. Immunohistochemistry stain indicated the CREG protein mainly localizes in a perinuclear pattern in HITASY cells. CONCLUSIONS: Those data provide evidence that the coordinated changes in CREG gene and protein expression as well as smooth muscle-specific markers may take place in connection with the process of phenotypic modulation of VSMC in vitro.