ABSTRACT
Objective To investigate whether chemokine CC motif 2 (CCL2) is involved in the high residual platelet response,and the mechanism of CCL2 being involved in the regulation of platelets.Methods Forty patients with ST elevation myocardial infarction (STEMI) were admitted.P2Y12 reaction unit (PRU) was detected by VerifyNow.Forty patients were divided into high platelet reactivity group (high reactivity group,n=24) and normal platelet reactivity group (normal reactivity group,n=16) according to the results of PRU detection.Plasma CCL2 concentration of the STEMI patients was examined by ELISA.The expressions of CCL2 and CCR2 in the platelets were detected by Western blotting.After CCL2 stimulation,the kinases of which phosphorylation was changed in the platelets were screened by ARY003B protein chips.The phosphorylation of p38MAPK and HSP27 in the platelets was tested by Western blotting after CCL2 stimulation in the presence or absence of CCR2 antagonist (RS 102895) or p38MAPK signal pathway inhibitor (SB 203580).Results The plasma CCL2 concentration of high reactivity group was markedly higher than that of normal reactivity group.Moreover,compared with normal reactivity group,the expressions of CCL2 and CCR2 in the platelets of high reactivity group significantly increased.After the platelets were stimulated by CCL2,the phosphorylation of p38α and HSP27 enhanced in the platelets by protein chips screening.When RS 102895 or SB 203580 was treated before CCL2 stimulation,the phosphorylation of p38MAPK and HSP27 decreased.Conclusions CCL2 participates in high residual platelet response in an autocrine/paracrine way.CCL2/CCR2 might affect the function ofplatelets through p38MAPKHSP27 signal pathway.
ABSTRACT
Objective:To investigate the influence of different salt concentration on the renal fibrosis and macrophages infiltration in salt sensitive hypertension.Methods:Dahl salt sensitive rats were randomly divided into the normal salt (0.3 ％ nacl) group,4 ％ high salt,8 ％ high salt groups at six weeks continuously feeding for 8 weeks,each group contained 15 rats.Tail-cuffmethod was used to value rat blood pressure at 8 weeks,Masson trichromatic method was used to detect renal fibrosis of the three groups at 8 week.Immunohistochemistry and Western blot method were used to depict the renal macrophage infiltration at 8 week.Results:1) The blood pressure of 4 ％ salt and 8％ high salt group rats were significantly higher than those of the normal salt group at 8week,meanwhile the blood pressure of 8 ％ high salt was further increased than that of 4 ％ high salt group at 8 week.2) The relative kidney weight and renal fibrosis of 4 ％ salt and 8 ％ high salt group rats were obviously higher than that of normal salt group at 8week,meanwhile the relative kidney weight and renal fibrosis of 8 ％ high salt were further increased than those of 4 ％ high salt group at 8 week.3) The macrophage infiltration of 4 ％ salt and 8％ high salt group rats were higher than that of the normal salt group at 8week,meanwhile the macrophage infiltration of 8 ％ high salt was further increased than that of 4 ％ high salt group at 8 week.Conclusion:Different high salt concentrations had different effect on the renal fibrosis and macrophage infiltration in the salt sensitive hypertension,high salt concentration could exacerbate the renal fibrosis and macrophage infiltration.
ABSTRACT
BACKGROUND:It has been found that cel ular repressor of E1A-stimulated genes (CREG) is a lysosomal protein binding directly to the mannose-6-phosphate (M6P)/insulin-like growth factor II receptor (IGFIIR) and depends on the interaction with M6P receptors for efficient delivery to lysosomes OBJECTIVE:To study the interactions between the exogenous CREG protein and cathepsins and M6P/IGFIIR and to confirm the effect of CREG protein on expression and distribution of M6P/IGFIIR. METHODS:Double-stained immunofluorescence and coimmunoprecipitation were applied to observe the interactions between the exogenous CREG protein and cathepsin B, cathepsin L and M6P/IGFIIR. Using gain-of-function and loss-of-function approaches, the effect of CREG on expression and distribution of M6P/IGFIIR were studied by western blot assay and immunofluorescence staining. RESULTS AND CONCLUSION:Double-stained immunofluorescence and coimmunoprecipitation analyses confirmed the direct interactions between the exogenous CREG protein and cathepsin B, cathepsin L and M6P/IGFIIR. It was verified that CREG plays a critical role not in the expression but in the distribution of M6P/IGFIIR using gain-of-function and loss-of-function approaches. These findings provide evidence that exogenous CREG protein is located in lysosomes and has interactions with cathepsins and M6P/IGFIIR, also CREG plays a critical role in the distribution of M6P/IGFIIR.
ABSTRACT
AIM:To investigate the effect of potassium treatment on coronary arterial impairment induced by high salt intake.METHODS:Sprague-Dawley rats (4-week-old, n=10 in each group) received distilled water (NS), water containing 1.5%NaCl (HS), or 1.5% NaCl and 0.5% KCl (HS+HP) for 16 weeks.Systolic blood pressure (SBP) was determined by tail plethysmography every 2 weeks.After 16 weeks of treatment, vascular remodeling, superox-ide production, malondialdehyde (MDA) content, and endothelial nitric oxide synthase (eNOS) and gp91 expression in the coronary arteries were detected .RESULTS:After 16 weeks of salt loading , the rats in HS group was divided into salt sensitive subgroup and salt resistance subgroup according to the tail-cuff blood pressure .In this experiment , the salt-sensi-tive rats were selected as HS group .In HS group, salt loading significantly increased SBP , serum MDA and gp91 expres-sion, decreased serum NO and eNOS expression in the coronary arteries , and induced the coronary artery remodeling com-pared with NS group .In salt-loaded SD rats , 16-week potassium treatment abrogated the effects induced by salt loading . CONCLUSION:High salt may affect structural and functional changes in coronary arteries by activating oxidative stress . Potassium treatment antagonizes the effect of high salt intake .
ABSTRACT
Objective To explore possible role of cellular repressor of E1A-stimulated genes(CREG) in the process of phenotypic switching of adventitial fibroblasts(AFs). Methods Immunofluorescent staining was performed with tissue sections from mouse carotid arteries to evaluate the relationship between the expression of CREG and smooth muscle actin-α(α-SMA) in injured arteries, especially in the adventitia. Tissue block pasted culture method was used to isolate and culture AFs. RT-PCR and Western-blot were used to detect the change of CREG andα-SMA mRNA and protein expression in AFs in the presence of different concentrations of AngⅡfor 12 h/24 h or in the presence of 100 nmol/L Ang Ⅱ for different times. Results Normal mouse carotid arteries had little α-SMA expression throughout the tunica adventitia. Arteries at day 1 and day 3 post-injury exhibited significantly higher immunofluorescence of α-SMA compared with non-injured arteries. Alpha-SMA expression began to decrease on day 7 and progressively declined on day 14. In contrast, immunofluorescent staining revealed that CREG was expressed in the adventitia of normal arteries. Expression of CREG in the adventitia of injured arteries was decreased on the 1st day, reached its lowest value on the 3rd day, and increased gradually from the 7th day, and was higher compared with that in non-injured arteries on the 14th day after injury. Similarly, the expression of CREG in AFs was very high, and AngⅡremarkably decreased mRNA and protein expression levels of CREG in a dose-dependent and time-dependent manner. Conclusions The changes in CREG expression correlate with AF phenotypic modulation, and CREG down-regulation may facilitate AF phenotypic switching into myofibroblasts (MFs).
ABSTRACT
BACKGROUND:Embryonic stem cells (ESCs) serve as a major cell source for smooth muscle cells,but the heterogeneity of cells derived from ESCs result in difficulty to obtain high purity smooth muscle cells.OBJECTIVE:To construct a double expression vector of puromycin resistance (pac) gene and enhanced green fluorescence protein (EGFP) gene driven by smooth muscle specific SM22α promoter (pSM22α-PAC-IRES2-EGFP),in addition,to detect its availability and specificity in ESCs.DESIGN,TIME AND SETTING:The observational experiment of gene level was performed at the Cardiovascular Institute,General Hospital of Shenyang Military Region from April 2007 to September 2008.MATERIALS:ESCs line R1 with number SCRC-1011TM was purchased from American ATCC Company.The pSM22α-EGFP vector was constructed by our laboratory.And the pIRES2-EGFP,pSM2C and pSuper.basic vectors were purchased from Invitrogen Company.METHODS:SM22α promoter was cloned from pSM22α-EGFP by polymerase chain reaction.CMV promoter of pIRES2-EGFP vector was replaced by SM22 promoter to establish pSM22α-IRES2-EGFP.Pac gene,excised from pSM2C by HindⅢ/Clal digestion,was sub-cloned into pSuper.basic to establish pSuper-PAC.After BgⅢ/Accl enzyme digestion of pSuper-PAC,pac gene fragment was obtained,which was further sub-cloned into pSM22α-IRES2-EGFP to produce pSM22α-PAC-IRES2-EGFP.ESCs were transfected with pSM22α-PAC-IRES2-EGFP using lipofectamine.Positive clones were selected by G418 and induced to differentiate and further identified by amplification of pac gene by RT-PCR.Differentiated cells were immunostained by SM α-actin,and expression of SM α-actin and EGFP was observed simultaneously under fluorescence microscope.MAIN OUTCOME MEASURES:Sequencing result of pSM22α-PAC-IRES2-EGFP;Amplification of pac gene;EGFP expression;as well as SM α-actin immunostaining.RESULTS:Three segments of 261 bp,664 bp,and 5000 bp were obtained by HindⅢ/Clal digestion,which was coincident with expectation,and the sequencing results showed that pSM22α-PAC-IRES2-EGFP vector was successfully constructed.Amplification of pac gene identified 4 ESCs clones successfully transfected.After induction of differentiation,partial portion of differentiated cells expressed EGFP,accompanied by positively stained by SM α-actin antibody.CONCLUSION:pSM22α-PAC-IRES2-EGFP vector was successfully constructed.ESCs clones transfected with this vector expressed pac gene and EGFP gene,and the expression of EGFP is smooth muscle specific.
ABSTRACT
OBJECTIVE:To investigate the possible association between the gene ALOX5AP encoding 5-lipoxygenase activating protein (FLAP)and coronary artery disease(CAD)in the Han population of North China.METHODS:A total of 680 cases underwent selective coronary angiography(SCA)from Shenyang General Hospital of Chinese PLA was recruited from January 2006 to September 2007.According to the results of SCA.680 cases were divided into CAD group with angiography positive(n=336)and control group with angiography negative or the stenosis of coronary arteries<50%(n=344)without evidence of cardiac ischemia.Single nucleotide polymorphisms of ALOX5AP gene was screened in 48 unrelated Han individuals of North China by polymerase chain reaction fPCR)-Re-sequencing method and 7 polymorphisms were found.The genotype and allele distribution of T(-1340)G polymorphism between two groups was determined by polymerase chain reaction and restriction fragment Iength polymorphism(PCR-RFLP)analysis in CAD and controI subjects.RESULTS:The genotype frequencies of TT,TG and GG in the ALOX5AP T(-1 340)G polymorphism were 26.79%,51 179%and 21.43%in CAD patients,33.72%,47.38%and 18.90%in the controls,respectively(x~2=3.90,P>0.06).The genotype distribution between two groups was in accordance with hardy-weinberg equilibrium.There are no significant differences in the distribution of three genotypes between the two groups.The frequencies of ALOX5AP G allele in cases and controls were 47.32%,42.59%,respectively(x~2=3.08,P>0.05).Subsequent stratified analysis by gender also showed no statistical significance in the genotype frequencies and allele frequencies between the two groups.CONCLUSION:The result suggests that T(-1340)G polymorphism of the ALOX5AP gene might not be associated with CAD in the Han population of North China.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate if there are microsatellite loci in the long arm of chromosome 6 that have close relationship with non-small cell lung cancer.</p><p><b>METHODS</b>Multiple PCR approach was used to analyze the 18 loci in the long arm of chromosome 6. The PCR products were analyzed in PAGE, and then the electrophoresis maps were analyzed with Gene Scan(TM) and Genotyper(TM).</p><p><b>RESULTS</b>There were different frequencies of loss of heterozygosity (LOH) in different loci (varying from 3.85% to 38.45%). The total frequency of LOH in 41 gastric cancers was 58.5%(24/41). Eight loci with the LOH frequency higher than 20% were mainly located in 2 regions: 6q24 and 6q27. The accurate location is 6q24-6q25.3 [D6S1699(35%), D6S409(23.33%), D6S441(33.33%)] and 6q26-27 [D6S1550(38.45%), D6S264(20%), D6S1585(25%), D6S446(33.33%), D6S281(30.77%)].</p><p><b>CONCLUSION</b>There may be tumor suppressor genes located in the region of 6q24 and 6q27, which have close relationship with non-small cell lung cancer.</p>
Subject(s)
Humans , Carcinoma, Non-Small-Cell Lung , Genetics , Chromosome Mapping , Chromosomes, Human, Pair 6 , Genes, Tumor Suppressor , Loss of Heterozygosity , Genetics , Lung Neoplasms , Genetics , Polymerase Chain Reaction , MethodsABSTRACT
Objective To analyze the human cellular repressor of E1A-stimulated genes (hCREG)using bioinformatics tools and predict the promoter position and transciption factor binding sites.Methods Complete coding hCREG sequences were obtained from Genbank.hCREG was analyzed with First EF software in order to obtain the promoter sequence of hCREG.Moreover,transcription factor binding sites of hCREG was convinced by Motif software.Subsequently,the transcription factor of hCREG by bioinformatics tools was related between hCREG and smooth muscle cells differentiation observed by both Western blot and immunoflourescence.Results The promoter of hCREG was located in -109~-359 bp of up-stream of transcriptional site,which was predicted with 945bp length.Transciption factor binding sites of hCREG was predicted by Motif software which was found with 80 transciption factors.The expression of hCREG and smooth muscle ?-actin(SM ?-actin)increased in HITASY after 72 hours with serum deprivation detected by both Western blot and immunoflourescence.Meanwhile,the results showed that the expression of wtp53 increased significantly.These results suggested that transcription factor wtp53 might regulate the expression of hCREG and promoted dedifferentiated phenotype of VSMCs.Conclusion The transcriptional information of the proximal promoter of hCREG obtained.As up-stream regulational factor of hCREG,wtp53 can up-regulate the expression of hCREG and may play a vital role in the process of VSMCs differentiation and phenotype modulation.
ABSTRACT
AIM: To evaluate the effects of over-expression of cellular repressor of E1A-stimulated genes(CREG) mediated by retrovirus on neointima formation in injured rat carotid.METHODS: The pluronic F127 containing pLNCX/CREG or pLNCX/GFP retroviral vectors was placed around the injured rat carotid.The neointima,media areas and the intima to media ratio were calculated.Expressions of CREG,SM ?-actin and Ki-67 were detected.RESULTS: The GFP expression was observed at day 2 in pLNCX/GFP groups.The expression of exogenous CREG was also significantly increased in arteries at day 2 after pLNCX-CREG infection.Over-expression of CREG significantly suppressed neointima formation,attenuated the expression of Ki-67 and up-regulated SM ?-actin expression.CONCLUSION: Over-expression of CREG inhibits VSMCs proliferation and promotes VSMCs differentiation after vascular injury.It suggests that modulation of CREG expression or activity may be a viable approach to treat neointimal restenosis after percutaneous coronary intervention.