Molecular docking in Nεcarboxymethyl lysine targeting scavenger receptor CD36 / 中华老年心脑血管病杂志

Objective To study whether Nεcarboxymethyl lysine(CML)can form a good molecular docking with the scavenger receptor CD36and induce a stable interaction.Methods The interaction between CML and CD36was studied by co-immunoprecipitation.The binding mode and affinity of CD36to CML were tested using AutoDock 4.2,iBabel and XQuartz-2.7.7software respectively. Results Co-immunoprecipitation showed that anti-CD36antibody magnetic bead could precipitate CD36from the total protein in RAW264.7cells and anti-CML could detect CD36 binding CML.CD36had a good molecular docking with CML,CD36and CML interacted stably with each other.The affinity of CML to 4Q4Bprotein structure of CD4extracellular domain was -29.62kJ/mol.ARG82,ASN71and THR70were the products of amino acid receptor interaction. Further docking analysis showed that CML could form 3interacting hydrogen bonds with 4Q4B,and the docking prediction inhibition constant was 6.92with a root mean square deviation of 2.54.Conclusion A good molecular docking between CML and 4Q4Bprotein structure of CD36extracellular domain can induce a stable interaction between CML and CD36.Hydrogen bonding is the main interaction mode.

CD137-CD137L interaction induced the calcification of mouse smooth muscle cells via P38 MAPK signaling / 中华心血管病杂志

Objective@#To explore whether CD137-CD137L interaction could induce mouse vascular smooth muscle cells(VSMCs) calcification via P38 MAPK signaling.@*Methods@#(1) Mouse VSMCs obtained from 8-week old male C57 mice were cultured by using method of tissue piece inoculation.The cells from 3 to 8 passage were divided into 4 groups: control group, agonist-CD137 group(recombinant CD137L protein), anti-P38 group(agonist-CD137 group+P38 inhibitor), single anti-P38 group(P38 inhibitor). The calcification was induced by adding a mixture of 10 mmol/L β-glycerophosphate+10-8 mol/L dexamethasone+10-7 mol/L insulin in the culture medium.Immunofluorescence was used to observe the changes of VSMCs markers(α-SMA and OPN).Real time-PCR was used to observe the mRNA expression of OPN and RUNX-2. Western blot was used to observe the protein expression of p-P38, OPN and RUNX-2. The level of cell calcification was observed by detecting alkaline phosphatase activity and calcium concentration. (2) The degeree of local calcium deposition was also tested on Von Kossa staining and Alizarin red staining methods in following 5 mouse VSMCs groups: control group, agonist-CD137 group(recombinant CD137L protein), anti-P38 group (agonist-CD137 group+P38 inhibitor), anti-CD137 group (agonist-CD137 group+CD137 inhibitor),agonist-P38 group(anti-CD137 group+P38 agonist).@*Results@#(1) Compared with the control group, the fluorescence intensity of α-SMA was lower in the agonist-CD137 group(2.79±0.25 vs. 5.42±0.47,P<0.05), while the fluorescence intensity of OPN was higher(4.91±0.23 vs. 1.63±0.26, P<0.05). The fluorescence intensity of α-SMA was partly recovered after adding P38 inhibitor(4.48±0.27 vs. 2.79±0.25,P<0.05),but it was still lower than the control group (4.48±0.27 vs. 5.42±0.47, P<0.05),the fluorescence intensity of OPN decreased(2.66±0.15 vs. 4.91±0.23,P<0.05),but it was still higher than that in the control group (2.66±0.15 vs. 1.63±0.26,P<0.05).The fluorescence intensity of α-SMA and OPN(5.32±0.67 vs. 5.42±0.47,1.82±0.30 vs.1.63±0.26,both P>0.05) was similar between the control group and single anti-P38 group.(2) Compared with the control group, the protein level of p-P38(4.15±0.24 vs. 3.48±0.26, P<0.05), OPN(2.43±0.21 vs. 1.53±0.08, P<0.05), RUNX-2(3.20±0.23 vs. 1.13±0.10, P<0.05) was significantly increased in agonist-CD137 group,the above effects were blocked by adding specific P38 inhibitor SB203580(1.16±0.12 vs. 4.15±0.24, 0.50±0.02 vs. 2.43±0.21,and 1.74±0.14 vs. 3.20±0.23,all P<0.05);the protein level of p-P38(2.93±0.60 vs. 3.48±0.26,P>0.05),OPN (1.4±0.64 vs. 1.53±0.08,P>0.05),RUNX-2(1.26±0.26 vs.1.13±0.10, P>0.05) was similar between single anti-P38 group and the control group. (3) Compared with the control group, the mRNA level of OPN (1.51±0.34 vs. 1, P<0.05) and RUNX-2(2.67±0.19 vs. 1, P<0.05) was significantly upregulated in agonist-CD137 group, and these effects were blocked by adding specific P38 inhibitor SB203580(0.33±0.14 vs. 1 and 0.45±0.03 vs. 1,P<0.05);the mRNA level of OPN (1.05±0.09 vs. 1, P>0.05) and RUNX-2(1.18±0.10 vs. 1, P>0.05) was similar between the single anti-P38 group and the control group.(4) Compared with the control group,the ALP activity and calcium concentration(2.40±0.25 vs. 1.40±0.21,5.51±0.33 vs. 3.15±0.31,both P<0.05) were significantly increased in agonist-CD137 group,while the effects could be blocked by adding specific P38 inhibitor SB203580((1.99±0.07) king unit/gprot vs. (2.40±0.25) king unit/gprot, (3.74±0.20) mmol/gprot vs. (5.51±0.33) mmol/gprot, both P<0.05).The ALP activity and calcium concentration was similar between single anti-P38 group and the control group((1.60±0.25) king unit/gprot vs. (1.40±0.21)king unit/gprot, (2.66±0.28) mmol/gprot vs. (3.15±0.31) mmol/gprot, both P>0.05). (5) Compared with the control group,the calcification of VSMCs in the agonist-CD137 group was significantly increased,while the calcification in the anti-P38 group was significantly reduced.Compared with the agonist-CD137 group,the level of calcification in the anti-CD137 group was obviously increased,and the calcification in the agonist-P38 group was significantly higher than that in the anti-CD137 group and the control group.@*Conclusion@#These findings suggest that CD137-CD137L signaling may regulate VSMCs calcification via modulating P38 pathway.

CD137-CD137L signaling influences the autophagy via JNK pathway in mouse vascular smooth muscle cells / 中华心血管病杂志

Objective@#To investigate whether CD137-CD137L signaling can affect the autophagy of mouse vascular smooth muscle cells(VSMCs) through JNK signal pathway.@*Methods@#Primary culture of C57BL/6J mouse thoracic aorta VSMCs was performed by tissue block adherence method. VSMCs between the third to fifth passages were isolated and cultured. VSMCs were divided into 4 groups: control group, CD137 agonist group, JNK inhibition group, and DMSO group. VSMCs in CD137 agonist group were treated with recombinant protein of CD137L (10 μg/ml), VSMCs in JNK inhibition group were treated with JNK inhibitor SP600125 (10 μmol/L) for 30 minutes followed by recombinant protein of CD137L (10 μg/ml) and DMSO group was treated with the same amount of DMSO in JNK inhibition group for 30 minutes, then added recombinant protein of CD137L (10 μg/ml). Western blot was used to detect the protein expression of p-JNK, LCⅡ and p62 in each group. Fluorescence microscopy was used to track the changes of autophagy in cells which was infected with adenovirus expressing tandem mRFP-GFP-LC3. Transmission electron microscope (TEM) was used to observe intracellular autophagosomes and autolysosomes.@*Results@#(1) Compared with the control group, stimulating CD137-CD137L axis by recombinant protein of CD137L significantly upregulated the expression of p-JNK, LCⅡ and p62 (1.15±0.19 vs. 0.72±0.21, P<0.05;1.03±0.13 vs. 0.59±0.15, P<0.05, and 1.10±0.19 vs. 0.76±0.15, P<0.05). These effects could be reduced by JNK inhibitor (0.61±0.21 vs. 1.15±0.19, P<0.05;0.74±0.11 vs. 1.03±0.13, P<0.05, and 0.21±0.12 vs. 1.10±0.19, P<0.05). The expression of these proteins in DMSO group remained unchanged compared with CD137 agonist group (P>0.05). (2) Changes of autophagy in cells of various group: the number of total fluorescent spots and yellow fluorescent spots in CD137 agonist group was significantly increased compared to control group (total fluorescent spots:(93.00±14.11)/cell vs. (52.33±9.61)/cell, P<0.05, and (64.33±6.81)/cell vs. (25.67±3.51)/cell, P<0.05), moreover, the number of yellow fluorescent spots was higher than the red fluorescent spots fluorescent spots in CD137 agonist group. Compared with CD137 agonist group, pretreatment with JNK inhibitor significantly reduced the number of total fluorescent spots and yellow fluorescent spots ((53.00±3.17)/cell vs. (93.00±14.11)/cell, P<0.05,and (15.33±4.51)/cell vs. (64.33±6.81)/cell, P<0.05). The red fluorescent spots were higher than the yellow fluorescent spots in JNK inhibition group. The number of total fluorescent spots and yellow fluorescent spots in CD137 agonist group was not affected by pretreatment with DMSO (P>0.05). (3) The number of intracellular autophagosomes and autolysosomes was significantly higher in CD137 agonist group than in control group((17.67±6.03)/cell vs. (5.67±2.52)/cell, P<0.05), and the number of autophagosomes was higher than that of autolysosomes in CD137 agonist group((14.00±4.00)/cell vs. (3.67±2.08)/cell, P<0.05). The number of intracellular autophagosomes and autolysosomes was significantly lower in JNK inhibition group compared to CD137 agonist group((5.67±4.04)/cell vs. (17.67±6.03)/cell, P<0.05) and the number of autophagosomes was lower than that of autolysosomes in JNK inhibition group((1.33±1.53)/cell vs. (4.33±2.52)/cell, P<0.05). The number of intracellular autophagosomes and autolysosomes was similar between DMSO group and CD137 agonist group (P>0.05).@*Conclusion@#CD137-CD137L signal may influence autophagy of mouse VSMCs via JNK pathway.

CD137 signaling promotes the formation of plaque calcification via inhibiting the fusion of autophagy and lysosomal in Apo E-/- mice / 中华心血管病杂志

Objective@#To investigate whether CD137 signaling promoted the formation of atherosclerotic plaque calcification by inhibiting the fusion of autophagosome and lysosome.@*Methods@#(1) In vivo, CD137 agonist antibody and anti-CD137 antibody were used to stimulate and inhibit the CD137 signaling, respectively. Fifteen Apo E-/- mice were randomly divided into three groups: control group (intraperitoneal injection of IgG2b 200 µg) , CD137 agonist group (intraperitoneal injection of CD137 agonist antibody 200 µg) , anti-CD137 group (pretreatment with 200 µg anti-CD137 antibody for 24 hours, then injection of CD137 agonist antibody) . (2) In vitro, primary culture of mouse aortic VSMCs obtained through adherence methods for tissues explants. The cells was divided into three groups: control group, agonist-CD137 group (CD137 agonist antibody 10 μg/ml) , and anti-CD137 group (pretreatment with 10 μg/ml anti-CD137 antibody for 60 minutes, then incubated with 10 μg/ml CD137 agonist antibody) . Von kossa staining was used to detect the calcification in the cell and plaque. Immunohistochemical staining was used to observe the expression of LC3B, Beclin 1 and p62 which are associated with autophagy. The levels of autophagy related protein (LC3) , Beclin 1, p62, and the expression of Runx2 and bone morphogenetic protein 2, which is associated with osteogenic differentiation in the VSMCs, were determined by Western blot. The autophagy flow of each group was detected by fluorescence microscopy. The autophagy was observed by transmission electron microscope in vivo and in vitro.@*Results@#(1) In vivo, the calcified plaque area in CD137 agonist group was significantly larger than that in the control group (3.01%±0.45% vs. 0.27%±0.06%, P<0.01) , and calcified plaque area in anti-CD137 group was significantly smaller compared with that in the CD137 agonist group (1.23%±0.39% vs. 3.01%±0.45%, P<0.05) . Immunohistochemical staining showed that the expression of early autophagy marker protein LC3B and Beclin 1 were significantly upregulated in CD137 agonist group and anti-CD137 group than in control group, and the highest expression was observed in CD137 agonist group (P<0.05) . The expression of advanced autophagy marker protein p62 was higher in the CD137 agonist group than in the anti-CD137 group (P<0.05) . (2) In vitro, the ratio of autophagy related protein LC3 Ⅱ/Ⅰ and p62 protein expression were significantly higher in CD137 agonist group and anti-CD137 group than in control group (P<0.01) , while the expression of p62 protein was significantly higher in CD137 agonist group than that in anti-CD137 group (P<0.05) . In the cell calcification inducing experiment, the expression of BMP-2 and Runx2 protein was significantly higher in CD137 agonist group than that in control group (P<0.01) , but the levels of BMP-2 and Runx2 protein were lower in anti-CD137 group than in CD137 agonist group (P<0.05) .@*Conclusion@#Our results indicate that activation of CD137 signaling can promote the formation of atherosclerotic plaque calcification by inhibiting the fusion of autophagosome and lysosome.

Association between serum Nε-carboxymethyl-lysine level and anterior tibial arterial plaque calcification in patients with diabetic foot post foot amputation / 中华心血管病杂志

Objective@#To observe the correlation between Nε-carboxymethyl-Lysine (CML), the main component of advanced glycation end products and the calcification of the anterior tibial artery plaque in patients with diabetic foot post foot amputation.@*Methods@#Sixty patients hospitalized for foot amputation operation due to diabetic foot from June 2012 to June 2016 in the Department of Orthopedics, Affiliated Hospital of Jiangsu University were prospectively recruited.The patients were categorized into mild stenosis (0<stenosis<50%, n=20), moderate stenosis (50%≤stenosis<70%, n=20) and severe stenosis (70%≤stenosis≤100%, n=20) based on the color Doppler ultrasound assessed severity of anterior tibial artery stenosis.The baseline clinical data of patients were collected and anterior tibial artery was isolated.Then, HE staining, O-Cresolphthalein Complexone method, enzymic method and ELASA analysis were then performed to detect the evolution of calcification, arterial calcium content, alkaline phosphatase activity and serum CML concentration, respectively.@*Results@#The results from both color Doppler ultrasound scan before amputation and HE staining after amputation showed that echo intensity as well as spotty blue calcium particles of anterior tibial artery plaque increased significantly in proportion to degree of stenosis and destructed elastic plate of the arterial wall was evidenced in patients with severest stenosis.The content of calcium ((2.3±0.9), (3.9±1.3), (6.6±1.7) μmol/mg, respectively, P<0.001), ALP activity ((102.4±39.4), (202.3±73.4), (483.7±117.9) U/mg, respectively, P<0.001) and serum CML level ((28.9±4.4), (37.9±5.3), (57.3±7.1)μg/L, respectively, P<0.001) increased significantly in proportion to stenosis severity.Pearson correlation analysis showed that serum CML level was positively correlated with the content of calcium (r=0.749, P<0.001) and ALP activity (r=0.923, P<0.001), respectively.@*Conclusions@#Serum CML level is positively correlated with the calcification of anterior tibial arterial plaque in patients with diabetic foot and could be used to evaluate the calcification of anterior tibial arterial plaque and stenosis degree of anterior tibial arterial in these patients.

CD137 induces vascular muscle cells phenotype transformation through activating nuclear factor of activated T-cells 1 signaling / 中华心血管病杂志

Objective@#To investigate whether CD137 induces primary vascular muscle cells (VSMCs) phenotype transformation through activating nuclear factor of activated T-cells 1(NFATc1) signaling.@*Methods@#VSMCs were obtained from aorta of C57BL/6J mice (8 weeks, male) through tissue-piece inoculating. Cells were divided into control group, CD137 agonist group (treated with CD137L recombinant protein) and anti-CD137 group (treated with anti-CD137 antibody). In si-RNA transfection assay, cells were divided into si-control group and si-NFATc1 group which were transfected with control or si-NFATc1 sequence respectively. The levels of NFATc1 and other phenotype related protein such as α-smooth muscle actin (α-SMA), smooth muscle myosin heavy chain (SM-MHC), vimentin were detected by Q-PCR and Western blot. Nuclear protein expression and activity of NFATc1 were detected by immunofluorescence and Western blot. Transwell assay was performed to measure the migration of VSMCs.@*Results@#According to Western blot, the expression of NFATc1 and vimentin was significantly upregulated (5.07±0.36 vs. 1.00±0.00, P<0.05; 3.23±0.27 vs. 1.00±0.00, P<0.05) while α-SMA and SM-MHC expressions was significantly downregulated (0.73±0.15 vs. 1.00±0.00, P<0.05; 0.45±0.05 vs. 1.00±0.00, P<0.05) in CD137 agonist group compare to control group. Compared with CD137 agonist group, the expression of NFATc1 and vimentin was significantly downregulated (1.56±0.27 vs. 5.07±0.36, P<0.05; 1.21±0.17 vs. 3.23±0.27, P<0.05), but the levels of α-SMA and SM-MHC were significantly upregulated (2.01±0.43 vs. 0.73±0.15, P<0.05; 2.85 ±0.32 vs. 0.45±0.05, P<0.05) in anti-CD137 group. Compared with si-con group, the expression of SM-MHC and α-SMA was significantly upregulated while the expression of vimentin was significantly downregulated in si-NFATc1 group. Transwell assay results demonstrated that migration cell numbers was significantly higher in CD137L group compared with control group(3.85±0.31 vs. 1.00±0.00, P<0.05), this effect was significantly attenuated by inhibiting NFATc1.@*Conclusion@#CD137 could induce VSMC phenotype transformation through activating NFATc1 signaling.

Feasibility and efficacy of the regional cooperative ST-segment elevation myocardial infarction rescue network among the prefectural-level city hospitals / 中华心血管病杂志

Objective@#To investigate the feasibility and efficacy of the establishment of regional cooperative acute ST-segment elevation myocardial infarction (STEMI) rescue network among the prefectural-level city hospitals in China.@*Methods@#Based on real-time remote electrocardiogram transmission and "120" emergency systems, we established a regional collaborative STEMI treatment network with our hospital as the network unclears including 8 second-class affiliated hospitals of Jiangsu University in 2013. STEMI treatment time, therapeutic effects and economic indexes were compared before (from January 2010 to December 2012, 180 cases, pre-network) and after (From January 2013 to December 2015, 374 cases, post-network) the establishment of the regional collaborative STEMI treatment network.@*Results@#Post establishment of the rescue network, mean first medical contact (FMC) to balloon (FMC-to-B) time, referral time and obtaining informed consent time were all significantly decreased from (191±41), (94±18), (25±9) minutes to (93±19), (53±18), (7±5) minutes, respectively, in comparison with the pre-network era(all P<0.05). There was a trend of prolonged FMC-to-B time in proportion to aging of STEMI patients(trend P<0.05). Three months post discharge, LVEF was higher (55.3%±10.7% vs. 48.8%±12.1%, P<0.05) and LVEDd was lower ((49.1±10.8)mm vs.(51.8±9.2)mm, P<0.05) in the post-network group compared to pre-network group.In-hospital mortality was also significantly reduced post the establishment of the rescue network (2.14%(8/374) vs. 3.89%(7/180), P<0.05). The results also showed that the total costs (42 017(25 069, 75 148)yuan vs.51 030(28 137, 105 861)yuan), days of hospitalization ((9.1±4.5) days vs. (15.3±4.8)days) and percentage of medicine and consumables were all significantly decreased in the post-network group compared to pre-network group(all P<0.05).@*Conclusion@#Establishment of the regional cooperative rescue network is feasible among the prefectural-level city hospitals in China. Establishment of such network can improve the prognosis and decrease the FMC-to-B time, the rate of in-hospital mortality and financial burden of patients with STEMI, and serves as an effective strategy to improve the rescue ability for STEMI patients.

Docosahexaenoic acid inhibits hypoxia-induced pulmonary arterial smooth muscle cells phenotype switching by inhibiting NFATc1 signaling / 中华心血管病杂志

Objective@#To explore the molecular mechanism of docosahexaenoic acid (DHA) on regulating the phenotype switching of hypoxia-induced pulmonary arterial smooth muscle cells (PASMCs).@*Methods@#The PASMCs were isolated from Sprague Dawley rats. PASMCs were divided into five groups: normal control group, hypoxia group (1%O2, 94%N2, 5% CO2 stimulation for 12 hours), hypoxia+ DHA group (10 μmol/L DHA pretreatment followed by 12 hours hypoxia), hypoxia+ DHA+ NFATc1 overexpression group (transfection of the NFATc1 lentivirus for 24 hours, followed by hypoxia stimulation for 12 hours after 10 μmol/L DHA treatment), and hypoxia+ DHA+ siNFATc1 group (transfection the siNFATc1 for 24 hours, followed by hypoxia stimulation for 12 hours after 10 μmol/L DHA treatment). The hypoxia stimulation was achieved by use of a special hypoxia incubator (1%O2, 94%N2, 5%CO2). The expressions of NFATc1 of various groups were determined by qRT-PCR and Western blot. The expression of α-SMA was determined by immunofluorescence staining, qRT-PCR and Western blot. The expression of SM22 was determined by qRT-PCR. The proliferation of PASMC was determined by EDU staining.@*Results@#The mRNA and protein expression levels of NFATc1 were significantly upregulated in hypoxia group compared with the normal control group (P<0.05), while hypoxia-induced upregulation of NFTAc1 could be significantly downregulated by DHA treatment (P<0.05). The α-SMA positive cell number, protein and mRNA levels of α-SMA and the mRNA level of SM22 were significantly lower in the hypoxia group than in normal control group, which could be significantly reversed by DHA, the protective effects could then be abolished by NFATc1 overexpression. Above indices were significantly lower in the hypoxia+ DHA+ siNFATc1 group than in hypoxia+ DHA+ NFATc1 overexpression group (P<0.05). The proliferation of PASMCs was significantly higher in the hypoxia group than in the control group (P<0.05), and which could be significantly reduced by DHA (P<0.05), and the protective effect of DHA could be significantly abolished by overexpression of NFATc1 (P<0.05). The proliferation of PASMCs was significantly lower in the hypoxia+ DHA+ siNFATc1 group than in the hypoxia+ DHA+ overexpression NFATc1 group (P<0.05).@*Conclusion@#DHA could prevent hypoxia-induced PASMCs phenotype switching and proliferation by inhibiting NFATc1 signaling.

Impact of CD137-CD137L signaling mediated exocytosis of autophagosome within vascular smooth muscle cells on the formation of atherosclerotic calcification / 中华心血管病杂志

Objective@#To explore whether CD137-CD137L signaling mediated exocytosis of autophagosome within vascular smooth muscle cells (VSMCs) could influence the formation of atherosclerotic calcification.@*Methods@#Fifteen 8-week-old male ApoE-/-(C57BL/6J-KO) mice fed with high fat diet for 5 weeks were randomly divided into three groups by using stochastic indicator method as follows: control group, n=5; agonist-CD137 group: agonist-CD137 antibody 200 μg/2 weeks for 4 weeks, ip, n=5; anti-CD137 group: 200 μg anti-CD137 antibody+ 200 μg agonist-CD137 antibody/2 weeks for 4 weeks, ip, n=5. Von Kossa staining was applied to observe the calcification of the thoracic aortic atherosclerotic plaque in each group. Immunohistochemistry was used to detect the expression of LC3 and Beclin1 which were the autophage markers of early-to-mid stage; Western blot was adopted to quantify protein level of microtubule-associated proteins 1 light chain 3B(LC3B) and mammalian ortholog of the yeast autophagy-related gene 6 (Beclin1). Transmission electron microscope (TME) was used to observe the formation of autophagosome in plaque. C57BL/6J mouse VSMCs were cultured by using tissue piece inoculation method. Groups of in vitro studies were the same as in vivo study: control group, agonist-CD137 group, anti-CD137 group, the agonist-CD137 groups was treated with agonist-CD137 antibody (10 μg/ml) and anti-CD137 group was treated with anti-CD137 antibody (10 μg/ml) for 30 minutes, followed by agonist-CD137 antibody (10 μg/ml). Von Kossa staining and osteogenesis phenotypic alkaline phosphatase (ALP) activity detection were adopted to observe calcification in VSMCs. Autophagosomes were separated from the supernatant of the agonist-CD137 group with density gradient centrifugation method. VSMCs were divided into two groups: positive group (containing complete medium with above autophagosomes to a final concentration 15 μg/ml) and negative group (only complete medium) after being pretreated with mixed inflammatory cytokines (IL-1β、IFN-γ and TNF-α, final concentration was 25 ng/ml respectively) for 24 hours and calcium deposition and osteogenesis phenotypic marker bone morphogenetic protein 2(BMP2) were then detected.@*Results@#(1) Compared with the control group, activation of the CD137-CD137L signal significantly increased the formation of calcification area in thoracic aortic atherosclerotic plaque of ApoE-/- mice((1.82±0.15)×104 μm2 vs. (0.34±0.08)×104 μm2, P<0.01), this effect was significantly attenuated by inhibiting this signal ((0.83±0.30)×104 μm2 vs. (1.82±0.15)×104 μm2, P<0.05); positive autophagy makers LC3B and Beclin1 were detected in both agonist-CD137 group and anti-CD137 groups and the expression of LC3B and Beclin1 was substantially higher in anti-CD137 group. Western blot analysis indicated that the expression of LC3B and Beclin1 in agonist-CD137 group was significantly upregulated compared with the control group (0.17±0.01 vs. 0.03±0.08, P<0.05, and 0.12±0.02 vs. 0.06±0.02, P<0.05), which could be significantly downregulated in anti-CD137 group (0.28±0.09 vs. 0.17±0.01, P<0.05 and 0.17±0.02 vs. 0.12±0.02, P<0.05). TME showed that the number (QTY /HP) of autophagosome of agonist-CD137 group and anti-CD137 group in plaque were both increased (14.67±2.52 vs. 3.67±1.53, P<0.01, and 15.33±2.08 vs. 3.67±1.53, P<0.01), while in the agonist-CD137 group, the number of extracellular autophagosome within thoracic aortic atherosclerotic plaque of ApoE-/- mice increased more substantially (5.33±1.53 vs. 1.33±0.58, P<0.01). (2) In vitro study showed that activating CD137-CD137L signal could promote calcium deposition in extracellular matrix and the activity of osteogenesis phenotypic ALP((6.73±0.02) μmol/mg protein vs. (1.07±0.03) μmol/mg protein, P<0.05), and ((563.20±0.72) U/mg protein vs. (117.50±0.64) U/mg protein, P<0.05), while these effects were significantly blunted in anti-CD137 group ((1.94±0.05) μmol/mg protein vs. (6.73±0.02) μmol/mg protein, P<0.05, and (236.10±0.14) U/mg protein vs. (563.20±0.72) U/mg protein, P<0.05). TME showed that the number of intracellular autophagosome in agonist-CD137 group and anti-CD137 group was both significantly higher than in control group ((21.65±1.34) μg/ml vs. (8.32±1.58) μg/ml, P<0.01, and (15.42±1.65) μg/ml vs. (8.32±1.58) μg/ml, P<0.05). After the density gradient centrifugation, exocytotic autophagosome in the medium of agonist-CD137 group was markedly higher than in control group ((14.67±1.53) μg/ml vs. (2.33±1.15) μg/ml, P<0.01). (3) Compared with the control group, autophagosomes isolated from culture supernatant (final concentration: 15 μg/ml) could significantly stimulate calcium deposition((2.30±0.10) μmol/mg protein vs. (0.15±0.40) μmol/mg protein, P<0.05) and enhance the expression of bone morphogenetic protein 2 (2.10±0.04 vs. 0.30±0.01, P<0.05).@*Conclusion@#CD137-CD137L signaling could mediate exocytosis of autophagosome within VSMCs, thus influence the formation of atherosclerotic calcification.

t-PA negatively regulating p75NRT for involving in atherosclerosis by changing reconstruction of vascular outer membrane autonomic nerve / 重庆医学

Objective To study the effect of tissue plasminogen activator (t-PA) on p57NTR ,inflammatory reaction ,immune regulation and oxidative stress and its effect on intimal hyperplasia .Methods The vascular injury treatment was performed in the diabetic rabbit model with carotid arterial adventitia stripping ,meanwhile t-PA controlled release microspheres were given ,the nerve distribution in the local blood vessels was observed by immunohistochemical staining .The change of nerve remodeling in the control group and treatment group was observed ,meanwhile the effect of giving t-PA controlled release microspheres on the release of ace-tylcholine and norepinephrine was detected .RT-PCR was used to detect local vascular tissue inflammation ,immune effects and oxi-dative stress .The sympathetic neurons and smooth muscle cell co-culture was adopted ,then giving glyoxal treatment as the athero-sclerosis cell model .With the t-PA treatment group as the intervention group ,the effect of t-PA on the number of cholinergic neu-ron ,and synaptic connections between the smooth muscle cells and acetylcholine secretion was observed .The change of t-PA-MMP-p75NTR and NF-kappa B signaling pathway were detected by RT-PCR .Results The vascular injury treatment was performed in the diabetic rabbit model with carotid arterial adventitia stripping ,meanwhile t-PA controlled release microspheres were given ,the nerve distribution in the local blood vessels was observed by immunohistochemical staining .The change of nerve remodeling in the control group and treatment group was observed ,meanwhile the effect of giving t-PA controlled release microspheres on the release of acetylcholine and norepinephrine was detected .RT-PCR was used to detect local vascular tissue inflammation ,immune effects and oxidative stress .The sympathetic neurons and smooth muscle cell co-culture was adopted ,then giving glyoxal treatment as the ath-erosclerosis cell model .With the t-PA treatment group as the intervention group ,the effect of t-PA on the number of cholinergic neuron ,and synaptic connections between the smooth muscle cells and acetylcholine secretion was observed .The change of t-PA-MMP-p75NTR and NF-kappa B signaling pathway were detected by RT-PCR .Conclusion t-PA activates MMPs and feedback in-hibits p75NTR-NF-kappa B signaling pathway to increase vascular adventitia autonomic nerve reconstruction and delay the occur-rence and development of atherosclerosis disease .

The effect of implementing regional cooperative rescue on patients with acute coronary syndrome / 中华急诊医学杂志

Objective To evaluated the effect of the regional cooperative rescue model implemented on the length of time from first medical contact (FMC) to balloon dilation (B),economic expense and prognosis in patients with acute coronary syndrome (ACS).Methods Patients with ACS (including ST-segment elevation and non-ST-segment elevation) selected from other hospitals within 24 hours after onset were treated with emergency percutaneous coronary intervention.Patients were divided into two groups, regional cooperative rescue group and control group without the regional cooperative rescue model approved.The lengths of FMC-to-B time and Door-to-B time (from arrival at emergency department or OPD to balloon dilation),time required for patients referred to our hospital,cardiac function,averaged hospital costs,average hospital stay,percentage of medication used and a major adverse cardiac event (MACE) were analyzed.Results Mean FMC-to-B time,Door-to-B time,referral time and time consumed to obtain informed consent were significantly shorter [(106±33) min,(31 ±8) min,(62 ±18,8 ±3) min] vs.[(231 ±35) min,(109 ±26) min,(98 ±31) min,(28 ±11) min,respectively] by implementing the regional cooperative rescue compared with control group,and LVEF was increased,and LVED was deceased inregional cooperative rescue group.The mean costs [(44 123.0 ±3 427.0) yuan vs.(51 587.0 ±5 621.0)] yuan,days of hospital stay [(8.7 ±4.1) vs.(13.2 ±6.4)] and percentage of medication used were significantly decreased in the regional cooperative rescue group.The incidence of MACE inregional cooperative rescue group was 6.2％,whereas the incidence in control group was 16.8％.Conclusions The regional cooperative rescue model can improve the prognosis and decrease the FMC-to-B time,the rate of MACE and financial burden in patients with ACS.

Nuclear factor-κB pathway mediates the effects of CD137 signaling on NFATc1 expression in mice vascular smooth muscle cells / 中华心血管病杂志

<p><b>OBJECTIVE</b>To observe whether CD137 signaling could affect the nuclear factor of activated T cells c1 (NFATc1) expression through nuclear factor-κB (NF-κB) pathway in mice aortic vascular smooth muscle cells (VSMCs).</p><p><b>METHODS</b>Adherence methods for tissues explants were used for primary culture of mouse aortic VSMCs. The mRNA expression of CD137 and NFATc1 was detected by real-time quantitative PCR (RT-qPCR). The VSMCs protein expression of IκB-α, NF-κB p65, phospo-p65 and NFATc1 was determined by Western blot. The level of CD137 was measured by Flow Cytometry (FCM).</p><p><b>RESULTS</b>(1) The mRNA and protein expression of CD137 in VSMCs was significantly upregulated at 24 h after co-culture with TNF-α (10 ng/ml, all P < 0.05). (2) Compared with the control group, the level of p-NF-κB p65 in cytoplasm and nucleus was significantly increased (8.34 ± 0.28 vs. 1, P < 0.05, and 2.64 ± 0.42 vs. 1, P < 0.05) while the level of IκB-α was reduced (1 vs. 2.70 ± 0.28, P < 0.05) after co-treatment with agonist-CD137 mAb, above effects were partly blocked by adding specific NF-κB inhibitor PDTC (30 µmol/L: 1.15 ± 0.14 vs. 8.34 ± 0.28, P < 0.05, and 2.09 ± 0.12 vs. 2.64 ± 0.42, P < 0.05, and 1.78 ± 0.74 vs. 1, P < 0.05). (3) The mRNA (2.07 ± 0.09 vs. 1, P < 0.05) and protein (1.75 ± 0.07 vs. 1, P < 0.05) expression of NFATc1 was significantly upregulated by agonist CD137mAb compared with the control group, and these effects could be reversed by PDTC (1.15 ± 0.07 vs. 2.07 ± 0.09, P < 0.05, and 0.90 ± 0.11 vs. 1.75 ± 0.07, P < 0.05).</p><p><b>CONCLUSION</b>CD137 signaling could affect the NFATc1expression in VSMCs through NF-kappaB pathway.</p>

CD137 signaling regulates the expression of nuclear factor of activated T cells c1 through miR-145a-5p in ApoE(-)/(-) mice / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate if miR-145a-5p participates the modulation process of CD137 signaling on the expression of nuclear factor of activated T cells c1 (NFATc1) in ApoE(-)/(-) mice.</p><p><b>METHODS</b>Atherosclerotic plaque model was produced by perivascular carotid collar placement in ApoE(-)/(-) mice. After surgery, the mice were randomly divided into the following groups: CD137 activated group (CD137 group, n = 6), CD137 inhibited group (anti-CD137 group, n = 6) and control group (n = 6). The mRNA expression of miR-145a-5p in plaque and cells was measured by real-time quantitative PCR (RT-PCR). Immunofluorescence was used to observe the distribution of NFATc1 in plaque and the expression of NFATc1 at mRNA and protein levels were detected by qRT-PCR, Western blot, respectively. The mouse vascular smooth muscle cells (VSMCs) were isolated and transfected with miR-145a-5p mimics or inhibitors by Lipofectamine. The eukaryotic expression vector and luciferase vector including p3xFLAG-NFATc1, p3xFLAG-NFATc1-3'UTR, psicheck2-NFATc1, psicheck2-NFATc1-Mut were constructed through molecular cloning and homologous recombination techniques, 293T cells were transfected with the miR-145a-5p mimics or inhibitors and the protein level and fluorescence intensity were then measured, respectively.</p><p><b>RESULTS</b>In vivo or in vitro, the level of miR-145a-5p was significantly decreased (0.21 ± 0.06 vs. 1.00 ± 0.00, P < 0.05, 0.22 ± 0.07 vs. 0.50 ± 0.12, P < 0.05) while the opposite effects were observed in anti-CD137 group. NFATc1 expression was decreased or increased in VSMCs transfected with miR-145a-5p mimics or inhibitors, respectively (all P < 0.05). miR-145a-5p mimics decreased the expression of p3xFLAG-NFATc1-3'UTR and the fluorescence intensity (0.56 ± 0.08 vs. 1.00 ± 0.00, P < 0.05).</p><p><b>CONCLUSION</b>CD137 signaling participates the regulation process on the expression of NFATc1 through miR-145a-5p in ApoE(-)/(-) mice.</p>

CD147 expression level and rs8259 T/A polymorphism of CD147 in patients with acute coronary syndrome / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the association between CD147 expression and its untranslated regions 3'UTR rs8259 T/A polymorphism and acute coronary syndrome (ACS).</p><p><b>METHODS</b>The genotypes of CD147 were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods in 182 ACS patients and 328 healthy controls. The plasma level of CD147 was determined by enzyme-linked immunosorbent assay (ELISA). CD147 mRNA and protein expression was detected by real-time fluorescent quantitative PCR (RT-qPCR) and Western blot.</p><p><b>RESULTS</b>The plasma CD147 level obtained from radial artery in ACS patients ((3.63 ± 0.70) pg/L) was significantly higher than in control ((2.45 ± 0.27) pg/L, P < 0.05), and highest in plasma obtained from the coronary artery ((4.28 ± 1.03) pg/L, P < 0.05) in ACS patients. Furthermore, the plasma CD147 level was higher in the ACS patients with rs8259 AA genotype than in the ACS patients with rs8259 TT genotype ((4.08 ± 0.41) pg/L vs. (3.05 ± 0.79) pg/L in radial artery and (5.29 ± 0.62) pg/L vs. (3.13 ± 0.52) pg/L in coronary artery, both P < 0.05). There are an enhanced expression of CD147 mRNA (2.45 times higher than control) and protein (3.66 ± 1.56 vs. 1.81 ± 1.29) in PBMCs from ACS patients than that from controls (both P < 0.05). The PBMCs CD147 mRNA and protein expression level were significantly higher in ACS patients with rs8259 AA genotype (mRNA:2.45 ± 0.35, protein:1.63 ± 0.16) compared to ACS patients with rs8259 TT genotype (mRNA:1.69 ± 0.15, protein: 0.88 ± 0.16, both P < 0.05). Multiple logistic analysis showed that CD147 T allele (AT+TT) was a protective factor to ACS (OR = 0.667, 95% CI 0.507-0.879, P < 0.05).</p><p><b>CONCLUSIONS</b>The over-expression of CD147 is involved in the pathogenesis of ACS. The CD147 3'UTR rs8259 T allele may be a protective factor for ACS, its polymorphism can affect the CD147 protein expression in ACS patients.</p>

The impact of cyclophilin A small interfering RNA on the formation of atherosclerotic plaque in apolipoprotein E-deficient mice / 中华心血管病杂志

<p><b>OBJECTIVE</b>The aim of this study was to determine whether inhibition of cyclophilin A by lentivirus-mediated RNA interference (RNAi) could inhibit progression of atherosclerotic plaques and increase collagen production.</p><p><b>METHODS</b>Atherosclerostic plaque model was induced by rapid perivascular carotid silicone collar placement in ApoE(-/-) mice. The recombinant CyPA-RNAi-Lentivirus (CyPA-RNAi-LV) or negative control-green fluorescent protein-Lentivirus (NC-GFP-LV) were constructed and transfected into right carotid plaques, respectively. Using the local injection method, ApoE(-/-) mice carotid artery plaque were intervened 10 min in the silicone collar placement with 10 µl (1.0 × 10⁸ TU/ml) lentivirus vector. The areas and CyPA expression of plaques were analyzed by morphological observation, real-time polymerase chain reaction (RT-PCR) and Western blot respectively.</p><p><b>RESULTS</b>CyPA-RNAi-LV not only prevented plaques progression ((9 085 ± 671) µm² to (18 021 ± 1 901) µm²), but also decreased plaque lipid content ((28.9 ± 6.3)% to (17.8 ± 4.5)%), increased plaque collagen content ((24.2 ± 4.8)% to (35.1 ± 5.2)%) at 6 weeks after lentivirus transfection. The intima/media ratio (0.36 ± 0.11 vs. 0.65 ± 0.12, P < 0.05) and degree of lumen stenosis (intima/lumen ratios, 0.18 ± 0.02 vs. 0.33 ± 0.03, P < 0.05) were also significantly reduced by CyPA-RNAi-LV. Moreover, RT-PCR analysis revealed downregulated expressions of proinflammatory cytokines and matrix metalloproteinases (MMP-9 -17.5%) in the CyPA-RNAi-LV group.</p><p><b>CONCLUSION</b>Lentivirus-mediated CyPA silencing by siRNA could inhibit plaques progression and reduce local inflammation through the anti-inflammatory effects in this model.</p>

Impact of new regional cooperative rescue model on first medical contact to balloon time and outcome in patients with ST-segment elevation myocardial infarction / 中华心血管病杂志

<p><b>OBJECTIVE</b>To evaluate the effect of new regional cooperative rescue model on the first medical contact-to-balloon time and outcome in patients with ST-elevation myocardial infarction.</p><p><b>METHOD</b>Patients with acute myocardial infraction (AMI) and onset time within 24 hours transferred from other hospitals to our clinic and underwent emergent percutaneous coronary intervention (PCI) between January 2010 and January 2013 were included in this study. Patients were divided into two groups: regional cooperative treatment group (n = 230) and control group (n = 168) according to whether the first contact clinic belongs to the regional cooperative rescue model or not. The first medical contact to balloon (FMC-to-B) time, door to balloon (D-to-B) time, referral time, cardiac function, mean cost, days of hospitalization, and major adverse cardiac event (MACE) during the 6 months follow up were compared.</p><p><b>RESULTS</b>Mean FMC-to-B time, D-to-B time and referral time were significantly decreased from (212 ± 37), (107 ± 18), (103 ± 23) min (control group) to (98 ± 23), (25 ± 7), (62 ± 12) min respectively in regional cooperative treatment group. Mean medical cost (42 221 (23 184, 77 768) RMB vs. 49 654 (25 126, 122 433) RMB) and days of hospitalization (7 (5, 13) days vs. 10 (6, 20) days) were also significantly lower in regional cooperative treatment group than in control group. At 6 months follow up, LVEF was significantly higher(54.9% ± 8.6% vs. 48.9% ± 9.1%, P = 0.01), LVEDD ((48.9 ± 5.7)mm vs.(51.4 ± 6.0) mm, P < 0.01) as well as MACE rate (7.4% (17/230) vs. 17.9% (30/168) , P < 0.05) were significantly lower in regional cooperative treatment group than in control group.</p><p><b>CONCLUSION</b>The regional cooperative rescue model can decrease the FMC-to-B time, improve cardiac function, and reduce both patients' financial burden and MACE in patients with acute myocardial infarction.</p>