CD137 signaling promotes angiogenesis through regulating macrophage M1/M2 polarization / 中华心血管病杂志

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.

Evaluation of plasma soluble CD137 level in relapsing-remitting multiple sclerosis patients in comparison with healthy controls in Isfahan Province, Iran

@#Objective: Multiple sclerosis (MS) is a chronic neuroinflammatory disease, characterizes by demyelination in the central nervous system (CNS). Co-stimulatory molecules such as CD137 (4-1 BB) play a major role in the activation of lymphocytes in CNS. The exact immunopathogenesis of MS is unknown. Hence, detection of specific biomarkers in the process of MS disease can lead to new therapeutic approaches. This study aimed to compare plasma sCD137 levels in relapsing-remitting multiple sclerosis (RRMS) patients with healthy controls in Isfahan province. Methods: Plasma sCD137 level was measured by enzyme-linked immune sorbent assays (ELISA) in 36 RRMS patients as well as 52 (age and sex-matched) healthy controls and the results were compared. Results: The plasma sCD137 level in studied RRMS patients was significantly higher in the patient group compared to the healthy controls (P- value=0.027). In addition, there was no significant association between age, sex, job and education level, with plasma sCD137 level in both the control and the case groups (P value>0.05). There was no correlation between mean of sCD137 and EDSS score, age of onset, duration of disease as well as serum 25 (OH) D concentrations of the patients. Conclusion: High plasma sCD137 level was detected in RRMS patients when compared with the controls, which may indicate the possible role of this biomarker in the immunopathogenesis of MS. Since CD137 can affect T lymphocytes activation and apoptosis, further studies are needed to elucidate its exact role in the pathogenesis of MS.

Impact of CD137-CD137L signaling on secretion of mouse vascular smooth muscle cells-derived exosomes: role of Rab7 pathway / 中华心血管病杂志

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.

Effective and persistent antitumor activity of HER2-directed CAR-T cells against gastric cancer cells in vitro and xenotransplanted tumors in vivo

Human epidermal growth factor receptor 2 (HER2) proteins are overexpressed in a high proportion of gastric cancer (GC) cases and affect the maintenance of cancer stem cell (CSC) subpopulations, which are used as targets for the clinical treatment of patients with HER2-positive GC. Despite improvements in survival, numerous HER2-positive patients fail treatment with trastuzumab, highlighting the need for more effective therapies. In this study, we generated a novel type of genetically modified human T cells, expressing a chimeric antigen receptor (CAR), and targeting the GC cell antigen HER2, which harbors the CD137 and CD3ζ moieties. Our findings show that the expanded CAR-T cells, expressing an increased central memory phenotype, were activated by the specific recognition of HER2 antigens in an MHC-independent manner, and effectively killed patient-derived HER2-positive GC cells. In HER2-positive xenograft tumors, CAR-T cells exhibited considerably enhanced tumor inhibition ability, long-term survival, and homing to targets, compared with those of non-transduced T cells. The sphere-forming ability and in vivo tumorigenicity of patient-derived gastric cancer stem-like cells, expressing HER2 and the CD44 protein, were also inhibited. Our results support the future development and clinical application of this adoptive immunotherapy in patients with HER2-positive advanced GC.

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.

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.

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.

The Roles of CD137 Signaling in Atherosclerosis

The tumor necrosis factor receptor superfamily (TNFRSF), which includes CD40, LIGHT, and OX40, plays important roles in the initiation and progression of cardiovascular diseases, involving atherosclerosis. CD137, a member of TNFRSF, is a well-known activation-induced T cell co-stimulatory molecule and has been reported to be expressed in human atherosclerotic plaque lesions, and plays pivotal roles in mediating disease processes. In this review, we focus on and summarize recent advances in mouse studies on the involvement of CD137 signaling in the pathogenesis and plaque stability of atherosclerosis, thereby highlighting a valuable therapeutic target in atherosclerosis.

CD137 molecule permits single cell sorting and expansion of HIV-specific T cells in vitro / 中华微生物学和免疫学杂志

Objective To isolate HIV-specific T cell clone and to expand them in vitro through the activation-induced expression of CD137 molecule.Methods The peripheral blood mononuclear cells were isolated from HIV-infected patients and HIV Gag specific CD3+CD8+CD137+T cell subset were sorted to 96-well plate in 1 cell/well by multicolor flowcytometry and single cell sorting.After 14 days in vitro culture with feeder cells and cytokines, the numbers and phenotypes of the cultured HIV-specific T cells were calcu-lated and identified.Results The CD137 expression was low on rested T cells but up regulated by the stim-ulation with Gag peptide pool.The CD8+CD137+T cells could secret IFN-γ.The number of CD8 T cells reached to 106 after 14 days in culture and expanded to 107-108 cells after 28 days of culture in vitro 100%of the cells remained activated upon Gag stimulation.Conclusion In stead of using IFN-γ, CD137 could be utilized as a novel molecule to isolate and expand HIV specific T cells in vitro.The expanded antigen spe-cific T cell clones could maintain good activation status.

Is CD137 Ligand (CD137L) Signaling a Fine Tuner of Immune Responses?

Now, it has been being accepted that reverse signaling through CD137 ligand (CD137L) plays an important role in vivo during hematopoiesis and in immune regulation. However, due to technical difficulty in dissecting both directional signaling events simultaneously in vivo, most biological activities caused by CD137-CD137L interactions are considered as results from signaling events of the CD137 receptor. To make the story more complex, CD137-/- and CD137L-/- mice have increased or decreased immune responses in a context-dependent manner. In this Mini review, I will try to provide a plausible explanation for how CD137L signaling is controlled during immune responses.

Effects of Danhong on the serum levels of CD137, high-sensitivity C-reactive protein (hs-CRP) and homocysteine in patients with non-ST elevation acute myocardial infarction complicating metabolic syndrome / 国际中医中药杂志

Objective To investigate effects of Danhong on the serum levels of CD137, high-sensitivity C-reactive protein (hs-CRP) and homocysteine (Hcy) in patients with non-ST elevation acute myocardial infarction complicating metabolic syndrome. Methods A total of 126 patients with non-ST elevation acute myocardial infarction complicating metabolic syndrome were enrolled and randomly divided into a conventional treatment group and a Danhong treatment group using a random-digit table, with 63 patients in each group. All patients underwent angiography or percutaneous coronary intervention. The patients in the Danhong treatment group treated with intravenous Danhong 20 ml on the basis of conventional treatment for 1 week. The serum levels of CD137, hs-CRP and Hcy were measured at hospital admission and 10 days after treatment. The severity of coronary artery disease was assessed by the Gensini-score. Results The levels of CD137, hs-CRP and Hcy in both groups after treatment were significantly lower than before treatment (conventional treatment group: t 12.393, 17.408 and 9.458; Danhong treatment group: t 16.110, 17.573 and 13.481; all P<0.01), and the Danhong treatment group were significantly decreased than the conventional treatment group (t 2.815, 3.224 and 3.157, all P<0.01). The serum levels of CD137 and hs-CRP before treatment were significantly correlated with Gensini scores in 126 patients (r 0.720 and 0.562,all P<0.01). Conclusions The serum levels of CD137 and hs-CRP are significantly correlated with the severity of coronary artery disease, intravenous Danhong may has protective effect for coronary artery disease via decreasing CD137 and hs-CRP.

Integration of the Innate and Adaptive Immunity by CD137-CD137L Bidirectional Signals: Implications in Allograft Rejection / 대한이식학회지

Two-signal models are useful in explaining various types of immune responses. In particular, secondary, so-called costimulatory, signals are critically required for the process of T-cell activation, survival, differentiation, and memory formation. Early studies in rodent models showed that targeting T-cell costimulatory pathways elicits immunological tolerance, providing a basis for development of costimulatory therapeutics in allograft rejection. However, as the classic definition of T-cell costimulation continues to evolve, simple blockade of costimulatory pathways has limitations in prevention of allograft rejection. Furthermore, functions of costimulatory molecules are much more diverse than initially anticipated and beyond T cells. In this mini-review, we will discuss CD137-CD137L bidirectional signals as examples showing that two-signals can be applicable to multiple phases of immune responses.

Clinical implications of elevated serum soluble CD137 levels in patients with acute coronary syndrome

OBJECTIVES: Atherosclerosis is a chronic inflammatory disease. Research has focused on identifying specific serum biomarkers to detect vulnerable plaques. These markers serve as diagnostic tools for acute coronary syndrome and assist in identifying high-risk patients. However, the existing data are limited and conflicting. This study tested the hypothesis that CD137 levels identify patients with acute coronary syndrome who are at a heightened risk for recurrent cardiac events. METHODS: The levels of soluble CD137 (sCD137) were measured using ELISA in 180 patients with acute coronary syndrome and 120 patients with acute chest pain. Platelet activation was assessed by flow cytometry. Receiver operating characteristic curve analysis was performed to evaluate the prognostic characteristics of sCD137. RESULTS: The levels of sCD137 were elevated in 75 patients with acute coronary syndromes and 20 patients with acute chest pain (>35.0 ng/ml). In patients with acute coronary syndrome, elevated sCD137 levels (>35.0 ng/ml) indicated an increased risk for major adverse cardiovascular events (OR =1.93, 95% CI: 1.39-2.54). Elevated serum levels of sCD137 and cTnT were correlated with a significantly increased risk of major adverse cardiovascular events in both groups after 30 days, six months and nine months of follow-up. The increased sCD137 levels were significantly correlated with the levels of troponin I (r = 0.4799, p<0.001). Importantly, 26 patients with normal cTnI levels had acute coronary syndrome. However, elevated sCD137 levels identified these patients as a being high-risk subgroup (OR = 2.14, 95% CI: 1.25-4.13). CONCLUSIONS: Elevated sCD137 levels indicate an increased risk of cardiovascular events in patients with acute coronary syndrome. Soluble CD137 may be a useful prognostic marker or indicator for adverse events in patients with acute coronary syndrome.

Regulation of Inflammation by Bidirectional Signaling through CD137 and Its Ligand

Although the majority of research on CD137 has been directed to T cells, it is becoming clear that this molecule has distinct functions in other lineages of cells, including non-hematopoietic cells. In particular, emerging evidence suggests that the CD137-its ligand (CD137L) network involving immune cells and non-immune cells, directly or indirectly regulates inflammation in both positive and negative manners. Bidirectional signaling through both CD137 and CD137L is critical in the evolution of inflammation: 1) CD137L signaling plays an indispensible role in the activation and recruitment of neutrophils by inducing the production of proinflammatory cytokines and chemokines in hematopoietic and non-hematopoietic cells such as macrophages, endothelial cells and epithelial cells; 2) CD137 signaling in NK cells and T cells is required for their activation and can influence other cells participating in inflammation via either their production of proinflammatory cytokines or engagement of CD137L by their cell surface CD137: 3) CD137 signaling can suppress inflammation by controlling regulatory activities of dendritic cells and regulatory T cells. As recognition grows of the role of dysregulated CD137 or CD137L stimulation in inflammatory diseases, significant efforts will be needed to develop antagonists to CD137 or CD137L.

Hu.4-1BB-Fc fusion protein inhibits allergic inflammation and airway hyperresponsiveness in a murine model of asthma / 소아과

PURPOSE: 4-1BB (CD 137) is a costimulatory molecule expressed on activated T-cells. Repression by 4-1BB is thought to attenuate Th2-mediated allergic reactions. The aim of this study was to investigate the effect of 4-1BB on allergic airway inflammation in a murine asthma model. METHODS: BALB/c mice were sensitized to and challenged with ovalbumin (OVA). Hu.4-1BB-Fc was administered 1 day before the first OVA sensitization or 1 day after the second OVA sensitization. Following antigen challenge, airway responsiveness to methacholine was assessed and bronchoalveolar lavage (BAL) fluid was analyzed. Total immunoglobulin (Ig) E, OVA-specific IgE, IgG1, and IgG2a levels in sera were measured by enzyme-linked immunosorbent assay. Lung pathology was also evaluated. RESULTS: In mice treated with Hu.4-1BB-Fc before the first OVA sensitization, there was a marked decrease in airway hyperresponsiveness, total cell count, and eosinophil count in the BAL fluid. In addition, Hu.4-1BB-Fc treatment decreased serum OVA-specific IgG1 levels and increased serum IgG2a level significantly compared with the corresponding levels in mice sensitized to and challenged with OVA. Hu.4-1BB-Fc-treated mice also showed suppressed peribronchial and perivascular inflammatory cell infiltration. In contrast, treatment with Hu.4-1BB-Fc 1 day after sensitization had no effect on airway hyperresponsiveness and showed less suppression of inflammation in lung tissue. CONCLUSION: Administration of Hu.4-1BB-Fc can attenuate airway inflammation and hyperreactivity in a mouse model of allergic airway inflammation. In addition, administration before sensitization may be more effective. These findings suggest that 4-1BB may be a useful therapeutic molecule against asthma.

Recombinant TAT-CD137 Ligand Cytoplasmic Domain Fusion Protein Induces the Production of IL-6 and TNF-alpha in Peritoneal Macrophages

BACKGROUND: The ligand for CD137 (CD137L; also called 4-1BBL) is mainly expressed on activated APCs such as dendritic cells, B cells and macrophages. Even though CD137L functions as a trigger of the CD137 signaling pathway for T cell activation and expansion, engagement of CD137L can deliver a signal leading to the production of proinflammatory cytokines in macrophages. METHODS: We generated cell-permeable TAT-CD137L cytoplasmic domain fusion protein (TAT-CD137Lct) and examined its ability to initiate the CD137L reverse signaling pathway. RESULTS: Treatment of TAT-CD137Lct induced the production of high levels of IL-6 and TNF-alpha mRNAs and proteins in peritoneal macrophages. TAT-CD137Lct increased phosphorylation of Erk, p38 MAPK and Jnk, and activated transcription factors C/EBP and CREB. However, TAT-CD137Lct did not visibly affect the degradation of the inhibitor of NF-kB (IkBalpha). We further demonstrated that JNK activation was required for TAT-CD137Lct-induced production of TNF-alpha, while activation of Erk and p38 MAPK were involved in IL-6 and TNF-alpha production. CONCLUSION: Our results suggest that TAT-CD137Lct is an effective activator for the CD137L reverse signaling pathway.

Clinicopathological significance of CD137L expression in laryngeal carcinoma / 临床耳鼻咽喉头颈外科杂志

Objective:To study the expression of CD137L in laryngeal carcinoma, and to analyze its clinicopathological significance.Method:The expression of CD137L in 50 laryngeal carcinoma specimens and 9 normal laryngeal mucous tissues were detected by immunohistochemical staining.Result:The positive CD137L staining were found in all 50 cases of laryngeal carcinomas (100%), while its staining were negative in normal laryngeal mucous. There was significant difference between two groups(P<0.01). The positive ratio of CD137L staining had no relationship with the factors such as age, sex and tumor site, while it had significant correlation with the pathological stage,T stage and lymph node metastasis.Conclusion:The expression of CD137L might play an important role in the development of laryngeal carcinomas.

Agonistic Anti-CD137 Monoclonal Antibody Treatment Induces CD11b+Gr-1+ Myeloid-derived Suppressor Cells

CD137 (4-1BB/tnfrsf9) has been shown to co-stimulate T cells. However, agonistic anti-CD137 monoclonal antibody (mAb) treatment can suppress CD4+ T cells, ameliorating autoimmune diseases, whereas it induces activation of CD8+ T cells, resulting in diverse therapeutic activity in cancer, viral infection. To investigate the CD137-mediated T cell suppression mechanism, we examined whether anti-CD137 mAb treatment could affect CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs). Intriguingly, anti-CD137 mAb injection significantly increased CD11b+Gr-1+ cells, peaking at days 5 to 10 and continuing for at least 25 days. Furthermore, this cell population could suppress both CD8+ T cells and CD4+ T cells. Thus, this study demonstrated that, for the first time, anti-CD137 mAb treatment could induce CD11b+Gr-1+ MDSCs under normal conditions, suggesting a possible relationship between myeloid cell induction and CD137-mediated immune suppression.