[Layer-specific strain assessment on left ventricular function before and after PCI in patients with ST segment elevation myocardial infarction].

Objective: To evaluate the changes of left ventricular function in patients with ST segment elevation myocardial infarction (STEMI) before PCI and within 24 hours after PCI by layer-specific strain, and to explore the value of this new assessment method for quantitative monitoring on the myocardial function in STEMI patients. Methods: A total of 40 patients with acute anterior wall myocardial infarction, who underwent PCI in Affiliated Hospital of Jiangsu University during July 2017 to July 2018, were included in this prospective cohort study. According to the symptom to balloon time (STB), the patients were divided into STB ≤6 hours group (26 cases) and STB 6-12 hours group (14 cases). Echocardiography was performed before, immediately, 3 hours and 24 hours after PCI. Echocardiographic indexes including endocardial myocardial longitudinal strain (LS-endo), 18-segment full-thickness myocardial longitudinal strain (LS) of left ventricle and left ventricular global longitudinal strain (GLS) were measured. The mean LS-endo and LS values of myocardial segments in infarcted area (IALS-endo, IALS) and the mean LS-endo and LS values of myocardial segments in non-infarcted area (NIALS-endo, NIALS) were calculated. Results: There were 34 males and 6 females in this cohort and age was (62±10) years. In STB≤6 hours group, the IALS-endo value ((13.7±4.9)% vs. (10.0±2.7)%, P<0.05) and NIALS-endo value ((17.0±2.9)% vs. (14.6±2.9)%, P<0.05) were significantly higher at 24 hours after PCI than those before PCI. In the group of STB 6-12 hours, IALS-endo decreased immediately after PCI ((6.7±3.3)% vs. (11.9±6.5)%, P<0.05), and there was a rising trend at 3 hours after PCI (P>0.05). At 24 hours after PCI, the index was higher than that immediately after PCI ((13.6±8.4)% vs. (6.7±3.3)%, P<0.05). The NIALS-endo value was significantly higher at 24 hours after PCI than that before PCI ((17.1±2.1)% vs. (14.5±3.2)%, P<0.05). In the STB 6-12 hours group, the decrease rate of IALS-endo immediately after PCI was higher than that in the STB ≤6 hours group (93% (13/14) vs. 35% (9/26), P<0.001). In STB ≤6 hours group, the NIALS value at 24 hours after PCI was higher than that before PCI (P<0.05), and there was no significant difference in IALS, NIALS and GLS at other time points (P>0.05). Conclusions: Layered LS is superior to full-thickness LS and GLS in evaluating left ventricular function in STEMI patients. LS measured by echocardiography can continuously and quantitatively evaluate the changes of left ventricular myocardial function in STEMI patients before and after PCI.

[CD137 signaling promotes angiogenesis through regulating macrophage M1/M2 polarization].

Objective: To investigate whether CD137 signaling can promote angiogenesis via regulating macrophage M1/M2 polarization. Methods: (1) The primary peritoneal macrophages in mice induced by 3% thiglycollate broth were divided into three groups: control group, CD137 signaling activated group and CD137 signaling inhibited group. Various specific markers of M1 and M2 macrophages were detected to observe the phenotype change of macrophages, and the macrophages protein expression of CD137, CD86 and CD206 was detected by flow cytometry (FCM). The protein and mRNA expression of induced nitric oxide synthase (iNOS), arginase Ⅰ(Arg-1) was determined by Western blot and RT-PCR, respectively. The secretion levels of IL-12 and IL-10 in culture supernatant of macrophages were detected by ELISA. (2) Macrophages were co-cultured with the endothelial cells (bEnd.3), and macrophages were implanted in the upper chamber, endothelial cells were implanted in stromal glue of the lower chamber. The experiment was divided into three groups: the control group, CD137 signaling activated group and peroxisome proliferator-activated receptor-γ (PPAR-γ) inhibited group, and tube formation ability of endothelial cells in each group was determined. Results: (1) The purity of primary peritoneal macrophages in mice was (97.93±1.31)%. The expression of CD137 on the surface of macrophages was (97.40±2.70)%. (2) Compared with control group, the mRNA and protein expression levels of Arg-1 were significantly increased and the mRNA and protein expression of iNOS were significantly decreased in CD137 signaling activated group (all P<0.05). Compared with CD137 signaling activated group, the mRNA and protein expression of Arg-1 were significantly lower and the mRNA and protein expression levels of iNOS were significantly higher in CD137 signaling inhibited group (all P<0.05). FCM results showed that the average fluorescence intensity of CD206 was higher, while the average fluorescence intensity of CD86 was lower in CD137 signaling activated group than in control group (P<0.05, P<0.01, respectively); the expression of CD206 was significantly lower, while the expression of CD86 was higher, in the CD137 signaling inhibited group than in CD137 signaling activated group (P<0.05, P<0.01, respectively). ELISA results showed that the secretion of IL-10 was higher, and the secretion level of IL-12 was significantly lower in CD137 signaling activated group than in control group (both P<0.01); the secretion of IL-10 was significantly lower and the secretion of IL-12 was significantly higher in CD137 signaling inhibited group than in CD137 signaling activated group (both P<0.05). (3) Values of the formation of tube length and branch number were both longer in CD137 signaling activated group than control group (P<0.05). The formation of the tube length and branch number were less in PPAR-γ inhibited group than in CD137 signaling activated group (P<0.05). Conclusion: CD137 signaling can promote angiogenesis by regulating macrophage M1/M2 polarization.

[Platelet derived growth factor-BB regulates phenotype transformation of pulmonary artery smooth muscle cells via SIRT3 affecting glycolytic pathway].

Objective: To investigate whether platelet-derived growth factor-BB (PDGF-BB) can regulate phenotypic transformation of pulmonary artery smooth muscle cells (PASMCs) via SIRT3 affecting glycolytic pathway. Methods: The PASMCs were isolated from Sprague Dawley rats. PASMCs were divided into 3 groups by using 2-deoxyglucose (2-DG), an inhibitor of the glycolytic pathway: normal control group, PDGF-BB group(30 ng/ml) and PDGF-BB (30 ng/ml)+2-DG (10 mmol/L) group. In lentivirus-mediated overexpression assay, cells were divided into control group, PDGF-BB group(30 ng/ml), PDGF-BB+deacetylase sirtuin-3 (SIRT3) overexpression group and PDGF-BB+empty vector group. The expression levels of phenotype related index such as α-smooth muscle actin (α-SMA), smooth muscle myosin heavy chain (SM-MHC), calponin, vimentin were detected by qRT-PCR and Western blot. Meanwhile, the expression of α-SMA was detected by cellular immunofluorescence staining. EDU staining was used to detect the proliferation of PASMCs. The expression of SIRT3 was detected by Western blot. The expressions of glucose transporter 1 and aerobic glycolytic enzymes were detected by qRT-PCR and Western blot in lentivirus-mediated overexpression assay. Results: (1) PDGF-BB affects PASMCs phenotypic transformation through glycolytic pathway: compared with normal control group, PDGF-BB significantly decreased the expressions of contractile phenotype markers such as α-SMA, SM-MHC, calponin mRNA and protein (all P<0.05), but it increased the expressions of the synthetic phenotype marker vimentin mRNA and protein (both P<0.05). Cellular immunofluorescence assay showed that PDGF-BB significantly decreased the number of α-SMA positive cells, while 2-DG reversed the process. (2) PDGF-BB promoted cell proliferation through glycolytic pathway: the proliferation of PASMCs was significantly higher in PDGF-BB group than in control group (P<0.05), and which could be significantly reduced by 2-DG (P<0.05). (3) PDGF-BB inhibited the expression of SIRT3 protein in PASMCs: the expression of SIRT3 protein in PDGF-BB group was lower than that in control group (P<0.05). (4) PDGF-BB affected glycolytic pathway through SIRT3:compared with the control group, PDGF-BB significantly increased the expression levels of glucose transporter 1 (Glut1), hexokinase 2 (HK2) and 6-phosphfructo-2-kinase 3 (PFKFB3) mRNA (all P<0.05), which was reserved by over-expression of SIRT3. There were no significant difference in mRNA expression levels between PDGF-BB group and PDGF-BB+empty vector group (P>0.05).Compared with the control group, PDGF-BB significantly increased the expression levels of Glut1, HK2 and PFKFB3 protein(all P<0.05), which was reserved by over-expression of SIRT3. There were no significant differences in protein expression levels between PDGF-BB group and PDGF-BB+empty vector group (all P>0.05). Conclusion: PDGF-BB regulates phenotypic transformation of PASMCs via SIRT3 affecting glycolytic pathway.

[Impact of CD137-CD137L signaling on secretion of mouse vascular smooth muscle cells-derived exosomes: role of Rab7 pathway].

Objective: To investigate whether CD137-CD137L signaling could affect the secretion of mouse vascular smooth muscle cells (VSMCs) -derived exosomes through autophagy mediated Rab7 pathway. Methods: Primary thoracic aorta VSMCs from C57BL/6J mouse were obtained by tissue block adherence method. VSMCs between the third to fifth passages were used and VSMCs were divided into 4 groups: control group, CD137 agonist group, lentivirus control group, Rab7 lentiviral interference group. VSMCs in CD137 agonist group were treated with recombinant protein of CD137L (10 µg/ml), VSMCs in lentivirus control group were treated with lentiviral followed by recombinant protein of CD137L (10 µg/ml), VSMCs in Rab7 lentiviral interference group were treated with Rab7 lentiviral intervention followed by recombinant protein of CD137L (10 µg/ml). Western blot was used to detect the protein expression of LC3Ⅱ, p62, Rab7, CD9, CD81 and Hsc70. Fluorescence microscopy was used to track the changes of autophagy in cells infected with mRFP-GFP-LC3. Transmission electron microscope was used to observe the morphology and size of VSMCs-derived exosomes. The nanoparticle tracking analysis(NTA) was used to detect the concentration and size of exosomes in each group. Results: (1) The expressions of Rab7, LC3Ⅱ and p62 protein in VSMCs of CD137 activation group were significantly higher than those in control group (all P<0.05). The expressions of Rab7, LC3Ⅱ and p62 protein in Rab7 lentivirus interference group was lower than in CD137 activation group (all P<0.05), while the expressions were similar between the lentivirus control group and the CD137 activation group (all P>0.05). (2) The total number of fluorescent spots and yellow fluorescent spots in the VSMCs of the CD137 activation group were higher than those in the control group (all P<0.05), and the number of yellow fluorescent spots was higher than that of the red fluorescent spots in the VSMCs of the CD137 activation group ((50.3±0.9) vs. (10.3±1.5)/cell). The total numbers of fluorescent spots and yellow fluorescent spots in VSMCs of Rab7 lentivirus interference group were lower than those of CD137 activation group (both P<0.05), and the number of red fluorescent spots in VSMCs was higher than that of yellow fluorescent spots ((40.7±4.0) and (10.7±1.2)/cell) in the Rab7 lentiviral interference group. The total numbers of fluorescent spots and yellow fluorescent spots in the VSMCs were similar between the lentivirus control group and the CD137 activation group (all P>0.05). (3) Under transmission electron microscopy, the size of the VSMCs-derived exosomes was about 30-150 nm. The exosome markers (CD9, CD81) could be detected in vesicles by Western blot. NTA results showed that the concentration of VSMCs-derived exosomes was significantly higher in the CD137-activated group than in the control group (P<0.05), which was significantly lower in the Rab7 lentiviral interference group than in the CD137-activation group (P<0.05) and was similar between the lentivirus control group and the CD137 activation group (P>0.05). The expression of Hsc70 protein in exosomes secreted by CD137 activation group was higher than that in the control group (P<0.05). The expression of Hsc70 protein in exosomes was lower in Rab7 lentivirus interference group than in the CD137 activation group (P<0.05), which was similar between the lentivirus control group and the CD137 activation group (P>0.05). The expression of LC3Ⅱ protein in exosome was higher in CD137 activation group than in control group (P<0.05), which was lower in Rab7 lentivirus interference group than in CD137 activation group (P<0.05), which was similar between the lentivirus control group and the CD137 activation group (P>0.05). Conclusion: The CD137-CD137L signaling may affect the secretion of mouse VSMCs-derived exosomes through modulating the Rab7 pathway mediated autophagy.

Deep ultraviolet light-emitting diodes with improved performance via nanoporous AlGaN template.

We report the performance enhancement of AlGaN-based deep ultraviolet light-emitting diodes (DUV LEDs) using AlGaN nanoporous template (NPT). The NPT was fabricated by the electrochemical etching method and served as the dislocation filtering layer and strain relieving layer. The n-AlGaN laterally regrown on NPT showed reduced dislocation density and mitigated compressive strain comparing with that on the as-grown template (AGT). A 23% improvement of internal quantum efficiency was achieved for the multiple quantum wells thereon. Moreover, the nanopores in the NPT transformed into elongated air voids during high temperature growth process, which could facilitate the escaping of photons by scattering and thus improve the light extraction efficiency. As a consequence, the DUV LED based on NPT demonstrated an increase of the light outpower by 50% at 20 mA than that on AGT.

[CD137-CD137L interaction induced the calcification of mouse smooth muscle cells via P38 MAPK signaling].

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 ß-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].

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.

Homogeneous (Cu, Ni)6Sn5 intermetallic compound joints rapidly formed in asymmetrical Ni/Sn/Cu system using ultrasound-induced transient liquid phase soldering process.

Homogeneous (Cu, Ni)6Sn5 intermetallic compound (IMC) joints were rapidly formed in asymmetrical Ni/Sn/Cu system by an ultrasound-induced transient liquid phase (TLP) soldering process. In the traditional TLP soldering process, the intermetallic joints formed in Ni/Sn/Cu system consisted of major (Cu, Ni)6Sn5 and minor Cu3Sn IMCs, and the grain morphology of (Cu, Ni)6Sn5 IMCs subsequently exhibited fine rounded, needlelike and coarse rounded shapes from the Ni side to the Cu side, which was highly in accordance with the Ni concentration gradient across the joints. However, in the ultrasound-induced TLP soldering process, the intermetallic joints formed in Ni/Sn/Cu system only consisted of the (Cu, Ni)6Sn5 IMCs which exhibited an uniform grain morphology of rounded shape with a remarkably narrowed Ni concentration gradient. The ultrasound-induced homogeneous intermetallic joints exhibited higher shear strength (61.6 MPa) than the traditional heterogeneous intermetallic joints (49.8 MPa).

[Association between serum Nε-carboxymethyl-lysine level and anterior tibial arterial plaque calcification in patients with diabetic foot post foot amputation].

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

[CD137 induces vascular muscle cells phenotype transformation through activating nuclear factor of activated T-cells 1 signaling].

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].

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].

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%O(2, )94%N(2, )5% CO(2) 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%O(2, )94%N(2, )5%CO(2)). 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].

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-1ß、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)×10(4) µm(2) vs. (0.34±0.08)×10(4) µm(2,) P<0.01), this effect was significantly attenuated by inhibiting this signal ((0.83±0.30)×10(4) µm(2) vs. (1.82±0.15)×10(4) µm(2,) 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.

[CD137 signaling promotes the formation of plaque calcification via inhibiting the fusion of autophagy and lysosomal in Apo E(-/-) mice].

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.

[In vivo and in vitro effects of CD137 stimulation on vascular calcification in high fat diet fed ApoE-/- mice].

Objective: To investigate the effect and related mechanism of CD137 stimulation on aortic atherosclerotic plaque calcification in high fat diet fed ApoE-/- mice and on calcification of vascular smooth muscle cells (VSMCs). Methods: (1) ApoE-/- mice fed with high fat diet were randomly divided into 3 groups: CD137 activated group (treated by 200 µg CD137 agonist i. p. once per week for 6 weeks, n=5); CD137 inhibited group (anti-CD137 group: 200 µg anti-CD137 antibody + 200 µg CD137 agonist, i. p., once per week for 6 weeks, n=5) and control group (n=5). Von kossa staining was used to observe the calcification of the aortic plaque and VSMCs. Immunohistochemistry was used to observe the expression of BMP-2 and Runx2 which are known mediators of osteogenic differentiation. (2) The mouse aortic VSMCs were obtained by Patch-attaching method. The calcium content was measured by Methylthymol Blue complexone method. The mRNA expressions of bone morphogenetic protein 2 (BMP-2) and Runx2 were measured by real-time fluorescent quantitative PCR (RT-PCR). The protein levels of BMP-2, Runx2 of the VSMCs were determined by Western blot. Results: (1) In vivo, the plaque calcified area in ApoE-/- mice was significantly larger in CD137-agonist group than that in control group ((1.75±0.33)×104 µm2 vs. (0.23±0.07)×104 µm2,P<0.01), and this effect was significantly reduced by cotreatment with CD137-antagonist ((0.83±0.30)×104 µm2 vs. (1.75 ±0.33)×104 µm2,P<0.05). The levels of BMP-2 and Runx2 were all significantly upregulated in CD137-agonist group than in control group (both P<0.01), again, this effect was blocked by cotreatment with CD137-antagonist (P<0.05). (2) Consistent with the in vivo results, VSMCs calcification was also more serious in CD137-agonist group than in control group, which could be significantly attenuated by cotreatment with CD137-antagonist. In VSMCs, calcium content level in CD137-agonist group was higher than in control group ((0.001 3±0.000 2) mmol/mg protein vs. (0.000 7±0.000 1) mmol/mg protein, P<0.01), which could be significantly reduced by co-treatment with CD137-antagonist ((0.000 9±0.000 2) mmol/mg protein vs. (0.001 3±0.000 2) mmol/mg protein, P<0.01). The mRNA and protein levels of BMP-2 and Runx2 were significantly upregulated in CD137-agonist group compared with the control group (P<0.05), which could be significantly down-regulated by cotreatment with CD-137 antagonist (P<0.05). Conclusion: CD137 activation can promote vascular calcification in high fat diet fed ApoE-/- mice both in vivo and in vitro.