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Activation of CD137 signaling accelerates vascular calcification in vivo and vitro
IJCA-24353; No of Pages 6 International Journal of Cardiology xxx (2016) xxx–xxx
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International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Activation of CD137 signaling accelerates vascular calcification in vivo and vitro Yao Chen a,1, Abdul Basit Bangash b,1, Juan Song a,1, Wei Zhong a, Cuiping Wang a, Chen Shao a, Zhongqun Wang a, Jinchuan Yan a,⁎ a b
Department of Cardiology, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu Province 212001, PR China The School of Clinical Medicine, Jiangsu University, Zhenjiang, Jiangsu Province 212001, PR China
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Article history: Received 23 August 2016 Received in revised form 18 December 2016 Accepted 25 December 2016 Available online xxxx Keywords: CD137 VSMC Calcification Osteogenic TNFRSF
a b s t r a c t Objectives: Vascular calcification is a characteristic feature of atherosclerosis and is considered as an independent predictor of cardiovascular risk. CD137 signaling has previously shown to be involved in atherosclerosis. However, the possible role of CD137 signaling in regulation of vascular calcification has not been reported. In the present study, we investigated the effect of CD137 signaling on vascular calcification in ApoE−/− mice and in vascular smooth muscle cells (VSMCs) of mice. Methods: Calcium deposition and muscle fibers in vivo or vitro were identified by von-Kossa and Masson's trichrome staining respectively. Alkaline phosphatase (ALP) activity was measured by the ALP assay Kit. The presence of bone morphogenic protein 2 (BMP2) and runt-related transcription factor 2 (Runx2) was detected by real-time PCR, Western blot and immunofluorescence in vitro or vivo. Results: Our data shows that activation of CD137 signaling by intraperitoneal injection of agonist-CD137 antibody increased the areas of vascular calcification. Activation of CD137 signaling also increased the expression of BMP2 and Runx2 in the atherosclerotic plaques. In vitro, activation of CD137 signaling also aggravated VSMC calcification, while blocking CD137 signaling could alleviate agonist-CD137 induced VSMC calcification. In addition, the levels of calcium, BMP2 and Runx2, indicators of calcification, were all significantly elevated in agonist-CD137 group in VSMCs. Conclusion: Our data revealed a previously unrecognized role of CD137 signaling in vascular calcification in vivo and vitro and provides a novel target for prevention and treatment of atherosclerosis in the future. © 2016 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Vascular calcification, which refers to the ectopic deposition of calcium phosphate crystal in cardiovascular tissue, is a common characteristic of advanced atherosclerotic lesion and a well-known independent predictive risk factor of subsequent cardiovascular morbidity and mortality [1]. Recent evidence suggested that vascular smooth muscle cell (VSMC) differentiation to osteogenic cell and express bone related proteins with concomitant down-regulation of SMC contractile protein play a pivotal role in vascular calcification [2,3]. However, the molecular mechanisms that regulate VSMC osteogenic transdifferentiation are complex and poorly understood. Accumulated evidence has demonstrated that inflammation may play an important role in VSMC osteogenic transdifferentiation [4]. CD137, a member of the tumor necrosis factor receptor superfamily (TNFRSF), is mainly expressed in a variety of immune cells which ⁎ Corresponding author. E-mail address: [email protected] (J. Yan). 1 These authors contributed equally to this work.
include natural killer (NK) cells, neutrophils, CD4+CD25+ regulatory T (Treg) cells, resting monocytes, and dendritic cells (DCs). However, under proinflammatory conditions, it is also expressed in some nonimmune cells, such as VSMCs and endothelial cells [5,6]. Olofsson and colleagues showed that CD137 is expressed in human atherosclerotic plaques and promotes the development of plaque [7]. Another study demonstrated that CD137 not only regulates T-cell activation as a costimulatory receptor, but also mediates atherosclerosis. Deficiency of CD137 reduces atherosclerosis in mice on both chow and high-fat diets [8]. In addition, our previous study showed that CD137-CD137L signaling pathway played a positive role in facilitating atheromatous plaque formation and progression. Furthermore, inhibition of CD137CD137L signaling significantly inhibited the formation of atherosclerotic lesions in apolipoprotein E-deficient (ApoE−/−) mice [9,10]. Based on these studies, we hypothesize that CD137-CD137L signaling pathway plays a critical role in the progression of atherosclerotic calcification. In this study, our study demonstrates that activation of CD137 signaling exacerbated vascular calcification in vivo and aggravated VSMC calcification in vitro. The mechanisms may involve increasing VSMC osteogenic differentiation.
http://dx.doi.org/10.1016/j.ijcard.2016.12.174 0167-5273/© 2016 Elsevier Ireland Ltd. All rights reserved.
Please cite this article as: Y. Chen, et al., Activation of CD137 signaling accelerates vascular calcification in vivo and vitro, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.12.174
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2. Materials and methods
2.3. Immunohistochemical analysis
2.1. Animals
We performed immunohistochemical staining on 5 μm thick paraffin-embedded sections, which were prepared from 4% formaldehyde-fixed tissue. The following primary antibodies were used: anti-Runx2 (immunoway) and anti-BMP2 (immunoway). After incubation with primary antibody overnight at 4 °C, horseradish peroxidase-conjugated secondary antibody was used in the second step followed by 3,3-diaminobenzidine to visualize the antigen. Nuclei were stained with hematoxylin. Negative controls were routinely employed.
15 ApoE−/− mice aged 8 weeks were purchased from vital river laboratories (Distributor of Jackson Laboratory, Beijing, China). All the animals were housed under a 12-h lightdark cycle, and 23 ± 2 °C under 55 ± 10% humidity, in normal cages with free access to water and provision of high fat foods. At the age of 13 weeks, the mice were randomly divided into the following groups: agonist-CD137 group, anti-CD137 group, and the control group. Mice in each group were intraperitoneally injected with 200 μg agonist-CD137 antibody (R&D), 200 μg anti-CD137 antibody + 200 μg agonist-CD137 antibody, and 200 μg IgG 2b (eBioscience) at 13, 15, and 17 weeks of age respectively. At 19 weeks of age, the mice were euthanized using 8% chlorate hydrate and aortas were pushed with phosphate-buffered saline through the left ventricle. Aortas, from the proximal ascending aorta to the bifurcation were freed from connective tissue under a dissection microscope. Aortas were fixed in 10% formaldehyde in PBS overnight, and further embedded in paraffin, and then the paraffin sections were cut out from the aortic arch to the thoracic artery. We chose the same position (2 mm above the aortic valve, Fig. 1A) of the short-axial slice among groups as the representative image. All animal experiments were reviewed and approved by the Animal Care and Use Committee of Jiangsu University.
2.4. VMSC culture Primary VSMCs were obtained from mouse thoracic aorta by Patch-attaching method as previously described [11] and identified by immunofluorescence staining for smooth muscle specific α-actin antibody (α-SMA, Sigma). Briefly, adventitia and intima were striped from segments of thoracic aorta, and the remaining tunica media was cut to 1–3 mm3 pieces. Then, the small pieces were cultured in Dulbecco's modified Eagle's medium (DMEM; high glucose, 4.5 g/L; Gibco) supplemented with 20% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere containing 5% CO2. After migrated from the explants, cells were collected and maintained in growth medium. Passage 4–6 VSMCs were used in experiments.
2.2. Analysis of vascular lesions in ApoE−/− mice
2.5. In vitro calcification and quantification of VSMCs
Hematoxylin/eosin (H&E) was used to assess tissue architecture. Additionally, sections were stained with the following staining kits according to supplied protocol: vonKossa, Masson's trichrome, and ALP. Digital images of arterials were captured using an Olympus microscope, and quantitative analyses of indicated stains were performed using image-pro plus 6.0 (IPP6.0) software.
Calcification of VSMCs was induced as previously described. Briefly, VSMCs were calcified with medium containing DMEM, 6% FBS, and 10 mmol/L β-glycerophosphate (β-GP). To induce CD137 expression, cells were treated with TNFɑ (10 ng/ml) for 24 h [11]. Then, the following incubations were performed: agonist-CD137 (10 μg/ml), antiCD137 (10 μg/ml) + agonist-CD137 (10 μg/ml), and IgG 2b (10 μg/ml). The calcification
Fig. 1. Activation of CD137 signaling promotes atherosclerotic calcification and accelerates osteogenic cell formation in ApoE−/− mice. (A) En face aorta with oil red O staining: from the proximal ascending aorta to the thoracic aorta. Short-axial slice of the aorta (yellow box area), 2 mm above the aortic valve (white arrows), red oil o staining positive area (yellow triangle). Histological analysis of aortas, 200× magnification: Hematoxylin/eosin (H&E) staining is used to assess tissue architecture; black arrow denotes osteogenic-like cells. The calcification was checked by von-Kossa staining. For further characterization of the calcified lesions, the lesions were stained for alkaline phosphatase (ALP) and fibers. Masson's trichrome staining shows that muscle fibers and collagen fibers are stained red and blue, respectively. The expression of BMP2 and Runx2 was stained by immunohistochemistry. Scale bar: 100 μm. The quantization bar graph of the plaque areas was below the pictures (D). (B) Quantitation of von-Kossa staining. (C) Quantitation of BMP2 and Runx2 expression. IOD: integrated optical density. Data are shown as mean ± SD, n = 5. In all panels, ⁎ and ⁎⁎ denote significance with p b 0.05 and p b 0.01 respectively, by Tukey's procedure. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Y. Chen, et al., Activation of CD137 signaling accelerates vascular calcification in vivo and vitro, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.12.174
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Fig. 1 (continued).
medium was replaced every 3 days for a total 15 days. VSMCs were washed with PBS for 3 times followed by treatment with 0.6 mol/L HCl overnight at 4 °C. The calcium content of HCl supernatants was determined colorimetrically by the Methylthymol Blue complexone method. The remaining cell layers were then dissolved in 0.1 mol/L NaOH and 0.1% SDS for protein concentration analysis. Calcium content was normalized by protein content. 2.6. Von-Kossa staining Calcium deposition in atherosclerosis plaque and VSMCs was identified by von-Kossa staining. VSMCs and paraffin-embedded sections were incubated with 5% and 2% silver nitrate solution respectively, and were placed under ultraviolet light for 30 min. Un-reacted silver was removed with 5% sodium thiosulfate for 5 min and counterstained with nuclear fast red for 3 min. Images were collected using an Olympus microscope. 2.7. Alkaline phosphatase activity assay Proteins were extracted from VSMCs by freeze-thawing the cells in 0.1% Triton X-100 in PBS. Alkaline phosphatase (ALP) activity was measured by the ALP assay Kit (Nanjing Jiancheng Bioengineering Research Institute, China) following the manufacturer's instructions. Culture medium was collected and centrifuged at 2500 rpm for 10 min; the supernatant of the medium was harvested for subsequent assay. Briefly, after adding buffer solution and matrix solution, water bathing, and coloring, the absorbance value was measured at 520 nm with the microplate reader. Subsequently, the activity values with a computational formula were calculated. Results were normalized to the protein content. 2.8. Quantitative real-time reverse-transcription PCR (qRT-PCR) The effect of CD137 on the expression of bone-related gene markers in VSMCs was determined by quantitative real-time RT-PCR using a Roche Molecular LightCycler. Total RNA was isolated with the Trizol reagent (Invitrogen) and reverse transcribed into cDNA. Quantitative real-time PCR (SYBR green; Invitrogen) was performed with primers against mouse BMP2, Runx2, OPN, and GAPDH (Sangon Biotech, China). The primer sequences were as follows: BMP2, 5′-TGAGGATTAGCAGGTCTTTGC-3′ and 5′-GCTGTTTGTGTTTGGC TTGA-3′; Runx2, 5′-CCTCTGACTTCTGCCTCTGG-3′ and 5′-ATGAAATGCTTGGGAACTGC-3′; OPN, 5′-GAGGAAACCAGCCAAGGTAA-3′ and 5′-GCAAATCACTGCCAATCTCA-3′; and GAPDH, 5′-GGCATTGCTCTCAATGACAA-3′ and 5′-TGTGAGGGAGATGCTCAGTG-3′.
2.9. Western blot Protein samples were extracted using a protein extraction reagent. Proteins were then separated by electrophoresis on an SDS-PAGE polyacrylamide gel and transferred to PVDF membranes. The membranes were incubated at 4 °C overnight with antibodies against BMP2 (1:1000), Runx2 (1:1000), OPN (1:1000) and GAPDH (1:2000) respectively. After that, membranes were incubated with appreciate HRP-conjugated secondary antibodies for 1 h. Band detection was performed according to the manufacturer's protocol (ECL; Pierce Biotechnology Inc., Rockford, USA). 2.10. Statistical analysis The results were expressed as means ± SD. Comparisons between groups were performed using ANOVA and the Tukey posttest procedure with the SPSS 17.0 software package. A p value b0.05 was considered statistically significant.
3. Result 3.1. Body weight and survival All 15 ApoE−/− mice survived before the animals were killed and there were no significant differences in body weight among the 3 groups (control group: 31.60 ± 1.82 g; agonist-CD137 group: 31.20 ± 2.39 g; anti-CD137 group: 30 ± 1.87 g; p N 0.05). 3.2. Activation of CD137 signaling promotes atherosclerotic calcification in ApoE−/− mice To probe the potential role of CD137 signaling involvement in atherosclerotic calcification, we activated the CD137 signaling by agonistCD137 antibody and inhibited it by anti-CD137 antibody. H&E staining shows that activation of CD137 signaling significantly increased the
Please cite this article as: Y. Chen, et al., Activation of CD137 signaling accelerates vascular calcification in vivo and vitro, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.12.174
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plaque areas, while inhibition of CD137 signaling decreased the plaque areas (Fig. 1A and D). Atherosclerotic calcification was verified by vonKossa staining. Our data shows that activation of CD137 signaling increased mean calcification compared to control (p b 0.01), In contrast, additional anti-CD137 treatment decreased the agonist-CD137 antibody induced atherosclerotic calcification (p b 0.05) (Fig. 1A and B). This data identifies that activation of CD137 signaling promotes atherosclerotic calcification, and this effect is independent from the lesion size extending. 3.3. Activation of CD137 signaling increase osteogenic cell formation in the plaque To further illustrate the calcification in the plaque. We then examined the effect of CD137 signaling on osteogenic transdifferentiation. Masson's trichrome staining shows that activation of CD137 signaling significantly decreased the number of muscle fibers in the vascular wall, while inhibition of CD137 signaling slightly decreased muscle fibers compare to control (Fig. 1A). Immunohistochemical staining showed that the ALP is located near the areas of calcification, and the expression of BMP2, Runx2 and ALP is significantly increased after treated with agonist-CD137 antibody, and this effect was partly blocked when treated with anti-CD137 antibody (Fig. 1A and C). 3.4. Activation of CD137signaling promotes VSMC calcification To elucidate the mechanisms by which CD137 regulates the vascular calcification, we performed in vitro experiments using VSMCs. vonKossa staining shows that there is no apparent difference between the IgG group and the normal control group. VSMCs treated with agonistCD137 antibody appear more seriously calcified, but the anti-CD137 antibody treated VSMCs partly inhibited the appearance of calcification (Fig. 2A). Similar results were observed in calcium content and ALP activation (Fig. 2B and C). 3.5. Activation of CD137 signaling accelerates VSMC osteogenic transdifferentiation VSMC osteogenic transdifferentiation is one of the most important procedures in vascular calcification. Therefore, we examined the effect of CD137 signaling on VSMC osteogenic transdifferentiation. As shown in Fig. 3, the mRNA levels of BMP2 and Runx2 were all significantly increased after the VSMCs were incubated with agonist-CD137 antibody and decreased while VSMCs were incubated with additional antiCD137 antibody (Fig. 3A). Similar results were obtained from Western blot analysis (Fig. 3B). Notably, compared with the control group, the mRNA and protein levels of osteopontin (OPN) were also increased in the agonist-CD137 group, however, blocking CD137 with additional anti-CD137, the expression of OPN was further increased. 4. Discussion Most individuals aged N60 years have progressively enlarging deposits of calcium mineral in their major arteries. This vascular calcification reduces aortic and arterial elastance, which impairs cardiovascular hemodynamics, resulting in substantial morbidity and mortality in the form of hypertension, cardiac hypertrophy, and myocardial ischemia [12]. The severity and extent of calcification reflect atherosclerotic plaque burden and strongly and independently predict cardiovascular events. In this study, we demonstrated that, for the first time to our knowledge, CD137 signaling contributes to the calcification of atherosclerotic lesion and VSMCs. The mechanisms involve an increase in VSMC osteogenic differentiation. As such, this proof-of concept study provides a novel target for prevention and treatment of atherosclerosis in the future.
CD137, known as a costimulator of T cells, activates nuclear factorκB and promotes T cell proliferation and cytokine production [7]. A large number of studies have indicated an important role for CD137 signaling in autoimmune disease processes [13]. Since CD137 molecule was first detected in human atherosclerotic lesions, slowly the potential relationships between CD137 molecule and atherosclerosis were discovered. Previous study showed that CD137 deficiency reduced atherosclerosis in hyperlipidemic mice and CD137 signaling was involved in the development and quality of atherosclerotic plaques [8]. Another study demonstrates that activation of CD137 signaling decreases the stability of advanced atherosclerotic plaques [6]. Vascular calcification is the progression of pathological mineralization of the arteries, and often been observed in advanced atheromatous plaque. We found that activation of CD137 increased the areas of atherosclerotic plaque calcification. Activation of CD137 promotes the atherosclerotic plaque development, and the areas of vascular calcification are associated with the size of atheromatous plaques. To ensure the positive effect of CD137 on vascular calcification, we further normalized the calcification area by plaque size, our data show that activation of CD137 signaling still enlarged the area of calcification even though it was normalized by plaque area. These results indicate that CD137 that accelerates vascular calcification is independent from its positive effect on the size of atherosclerotic plaque. Studies in the last decade suggest that vascular calcification is an active process, regulated in a manner similar to orthotopic bone formation [14]. Previous studies have suggested that some substances, such as TNF and inflammatory cytokines, are capable of inducing osteogenic genes, transforming osteoblasts and secreting some bone matrix proteins in the walls of blood vessels [15]. Consistent with these previous studies, data from our study shows that osteogenic-like cells were identified at the areas beside calcified lesions in the plaques. We further showed that activation of CD137 signaling with agonist-CD137 antibody promotes the formation of osteogenic cells in atherosclerotic lesions. BMP2 is known as a powerful bone morphogenic protein and its expression triggers osteogenic transcriptional, while Runx2 is thought to be a key regulator of osteoblast differentiation [16]. ALP is one of the osteoblastic phenotype markers and is considered essential in the vascular calcification process [2]. Therefore, we focused on the effect of CD137 signaling on expression of BMP2 and Runx2. It has been reported that CD137 could activate nuclear factor-κB and promotes T cell proliferation and cytokine production [7]. Others have demonstrated that NF-κB activation is implicated in receptor activator of NF-κB ligand (RANKL)-mediated osteogenic differentiation of smooth muscle cells and activation of NF-κB promotes calcification and increased the expression of BMP2 and Runx2 in aortic smooth muscle cells [17]. Thus, it is possible that CD137 signaling pathways have an essential role in mediating the expression of BMP2, Runx2 and ALP. Indeed, our data showed that treatment with agonist-CD137 antibody increased the expression of both BMP2 and Runx2, and inhibition CD137 signaling with anti-CD137 antibody decreased the expression of BMP2 and Runx2. This means that CD137 signaling has the ability to increase osteogenic cell formation through promoting vascular calcification. Several cells are involved in the procedure of vascular calcification, including endothelial cells, VSMCs, monocytes and macrophages [18]. A series of in-vitro studies demonstrate that monocytes and macrophages release inflammatory cytokines that promote cardiovascular calcification by regulating the differentiation of calcifying vascular cells, which express a number of phenotypic markers synonymous with osteogenic cells [19]. We and others have previously reported that NF-κB is involved in CD137 signaling [11]. NF-κB activation is implicated in receptor activator of NF-κB ligand (RANKL)-mediated osteogenic differentiation of smooth muscle cells. Moreover, researchers found that activation of NF-κB promotes calcification in aortic smooth muscle cells [20]. Thus, it is possible that CD137 signaling regulates VSMC calcification. Consistent with the present study in vivo, we provide evidence that activation of CD137 signaling aggravated VSMC
Please cite this article as: Y. Chen, et al., Activation of CD137 signaling accelerates vascular calcification in vivo and vitro, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.12.174
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Fig. 2. Activation of CD137 signaling promoted β-glycerophosphate (β-GP) induced vascular smooth muscle cell (VSMC) calcification. (A) VSMC mineralization was stained by von-Kossa staining. The calcification was also quantified by HCL extraction followed by colorimetric assay (B). Alkaline phosphatase (ALP) activity was measured by ALP assay (C). Scale bar: 200 μm. Data are shown as mean ± SD, n = 5, ⁎p b 0.05, ⁎⁎p b 0.01; NS: no significance.
calcification, upregulated the expression of BMP2 and Runx2, and increased the ALP activity, while blocking CD137 signaling can partly inhibit VSMC calcification and reduce the expression of these calcification indicators. This result indicates that CD137 signaling can promote the transformation of VSMCs into osteogenic cells. OPN is another biomarker of osteogenic cells which acts as a regulator of mineralization by inhibiting apatite crystals and promoting osteoclast function, not normally found within the vasculature, it is seen at high levels in calcified arteries and is potentially upregulated to counteract the advancement of vascular calcification [21]. Our data from
the present study showed that compared with the agonist-CD137 group, the level of OPN is up-regulated while blocking CD137 signaling with anti-CD137 antibody. This means that activation of CD137 signaling probably directly inhibits OPN expression and promotes VSMC calcification. In conclusion, the present study demonstrates that CD137 signaling promotes vascular calcification in vivo and vitro, and the following two mechanisms are probably involved in this process: 1) activation of CD137 signaling accelerates the transformation of VSMCs to osteogenic cells; 2) activation of CD137 signaling breaks the balance of calcification
Fig. 3. Activation of CD137 signaling induces osteogenic differentiation of vascular smooth muscle cells (VSMCs) in vitro. VSMCs were treated with control, IgG, agonist-CD137 antibody, or anti-CD137 antibody + agonist-CD137 antibody respectively, and incubated with 10 mmol/l β-glycerophosphate (β-GP). The mRNA levels of osteogenic marker genes (bone morphogenetic protein 2, BMP2; runt-related transcription factor 2, Runx2; osteopontin, OPN) were determined by reverse transcription-polymerase chain reaction and normalized to that of GAPDH (A, n = 4), and the protein levels were examined by Western blot (B, n = 5). Data are shown as mean ± SD, ⁎p b 0.01; NS: no significance. (1: Control; 2: Ig G; 3: Agonist-CD137; 4: Anti-CD137).
Please cite this article as: Y. Chen, et al., Activation of CD137 signaling accelerates vascular calcification in vivo and vitro, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.12.174
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biomarkers by increasing the levels of calcification promoters (BMP2, Runx2) or directly decreasing the expression of calcification inhibitor (OPN). Our data revealed a previously unrecognized role of CD137 signaling in vascular calcification, and suggest that CD137 signaling takes part in osteogenic differentiation of VSMCs. However, the underlying mechanisms are still unclear and require our further study.
[8]
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Sources of funding
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This project was supported by the Natural Foundation of Jiangsu Province (BK20161355) and National Natural Science Foundation of China (81670405, 81370408, and 81370409).
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Disclosure statement
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The authors have no conflicts of interest to declare. References [1] K. Lee, H. Kim, D. Jeong, Microtubule stabilization attenuates vascular calcification through the inhibition of osteogenic signaling and matrix vesicle release, Biochem. Biophys. Res. Commun. 451 (2014) 436–441. [2] J.M. Valdivielso, Vascular calcification: types and mechanisms, Nefrologia 31 (2011) 142–147. [3] L. Deng, L. Huang, Y. Sun, J.M. Heath, H. Wu, Y. Chen, Inhibition of FOXO1/3 promotes vascular calcification, Arterioscler. Thromb. Vasc. Biol. 35 (2015) 175–183. [4] C.M. Shanahan, Inflammation ushers in calcification: a cycle of damage and protection? Circulation 116 (2007) 2782–2785. [5] M.W. Anderson, S. Zhao, A.G. Freud, D.K. Czerwinski, H. Kohrt, A.A. Alizadeh, R. Houot, D. Azambuja, I. Biasoli, J.C. Morais, N. Spector, H.F. Molina-Kirsch, R.A. Warnke, R. Levy, Y. Natkunam, CD137 is expressed in follicular dendritic cell tumors and in classical Hodgkin and T-cell lymphomas: diagnostic and therapeutic implications, Am. J. Pathol. 181 (2012) 795–803. [6] I.H. Jung, J.H. Choi, J. Jin, S.J. Jeong, S. Jeon, C. Lim, M.R. Lee, J.Y. Yoo, S.K. Sonn, Y.H. Kim, B.K. Choi, B.S. Kwon, J.Y. Seoh, C.W. Lee, D.Y. Kim, G.T. Oh, CD137-inducing factors from T cells and macrophages accelerate the destabilization of atherosclerotic plaques in hyperlipidemic mice, FASEB J. 28 (2014) 4779–4791. [7] P.S. Olofsson, L.A. Soderstrom, D. Wagsater, Y. Sheikine, P. Ocaya, F. Lang, C. Rabu, L. Chen, M. Rudling, P. Aukrust, U. Hedin, G. Paulsson-Berne, A. Sirsjo, G.K. Hansson,
[15] [16]
[17]
[18] [19] [20]
[21]
CD137 is expressed in human atherosclerosis and promotes development of plaque inflammation in hypercholesterolemic mice, Circulation 117 (2008) 1292–1301. H.J. Jeon, J.H. Choi, I.H. Jung, J.G. Park, M.R. Lee, M.N. Lee, B. Kim, J.Y. Yoo, S.J. Jeong, D.Y. Kim, J.E. Park, H.Y. Park, K. Kwack, B.K. Choi, B.S. Kwon, G.T. Oh, CD137 (41BB) deficiency reduces atherosclerosis in hyperlipidemic mice, Circulation 121 (2010) 1124–1133. Y. Li, J. Yan, C. Wu, Z. Wang, W. Yuan, D. Wang, CD137-CD137L interaction regulates atherosclerosis via cyclophilin A in apolipoprotein E-deficient mice, PLoS One 9 (2014), e88563. J. Yan, H. Yang, W. Yuan, C. Wang, The effect of CD137-CD137 ligand interaction on the expression of NFATC1 in apolipoprotein E-deficient mice, Int. J. Cardiol. 157 (2012) 134–137. J. Yan, Y. Yin, W. Zhong, C. Wang, Z. Wang, CD137 regulates nfatc1 expression in mouse VSMCs through TRAF6/NF-kappaB p65 signaling pathway, Mediat. Inflamm. 2015 (2015) 639780. J.A. Leopold, Vascular calcification: an age-old problem of old age, Circulation 127 (2013) 2380–2382. D.S. Vinay, B.S. Kwon, Therapeutic potential of anti-CD137 (4-1BB) monoclonal antibodies, Expert Opin. Ther. Targets 20 (2016) 361–373. L. Tian, K. Chen, J. Cao, Z. Han, L. Gao, Y. Wang, Y. Fan, C. Wang, Galectin-3-induced oxidized low-density lipoprotein promotes the phenotypic transformation of vascular smooth muscle cells, Mol. Med. Rep. 12 (2015) 4995–5002. G. Pugliese, C. Iacobini, C. Blasetti Fantauzzi, S. Menini, The dark and bright side of atherosclerotic calcification, Atherosclerosis 238 (2015) 220–230. S. Evrard, P. Delanaye, S. Kamel, J.P. Cristol, E. Cavalier, calcifications SSjwgov, Vascular calcification: from pathophysiology to biomarkers, Clin. Chim. Acta 438 (2015) 401–414. C.P. Lin, P.H. Huang, C.F. Lai, J.W. Chen, S.J. Lin, J.S. Chen, Simvastatin attenuates oxidative stress, NF-kappaB activation, and artery calcification in LDLR−/− mice fed with high fat diet via down-regulation of tumor necrosis factor-alpha and TNF receptor 1, PLoS One 10 (2015), e0143686. L.L. Demer, Y. Tintut, Inflammatory, metabolic, and genetic mechanisms of vascular calcification, Arterioscler. Thromb. Vasc. Biol. 34 (2014) 715–723. J.A. Leopold, Vascular calcification: mechanisms of vascular smooth muscle cell calcification, Trends Cardiovasc. Med. 25 (2015) 267–274. Y. Wang, Z.Y. Zhang, X.Q. Chen, X. Wang, H. Cao, S.W. Liu, Advanced glycation end products promote human aortic smooth muscle cell calcification in vitro via activating NF-kappaB and down-regulating IGF1R expression, Acta Pharmacol. Sin. 34 (2013) 480–486. N.J. Paloian, C.M. Giachelli, A current understanding of vascular calcification in CKD, Am. J. Physiol. Ren. Physiol. 307 (2014) F891–F900.
Please cite this article as: Y. Chen, et al., Activation of CD137 signaling accelerates vascular calcification in vivo and vitro, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.12.174
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International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Activation of CD137 signaling accelerates vascular calcification in vivo and vitro Yao Chen a,1, Abdul Basit Bangash b,1, Juan Song a,1, Wei Zhong a, Cuiping Wang a, Chen Shao a, Zhongqun Wang a, Jinchuan Yan a,⁎ a b
Department of Cardiology, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu Province 212001, PR China The School of Clinical Medicine, Jiangsu University, Zhenjiang, Jiangsu Province 212001, PR China
a r t i c l e
i n f o
Article history: Received 23 August 2016 Received in revised form 18 December 2016 Accepted 25 December 2016 Available online xxxx Keywords: CD137 VSMC Calcification Osteogenic TNFRSF
a b s t r a c t Objectives: Vascular calcification is a characteristic feature of atherosclerosis and is considered as an independent predictor of cardiovascular risk. CD137 signaling has previously shown to be involved in atherosclerosis. However, the possible role of CD137 signaling in regulation of vascular calcification has not been reported. In the present study, we investigated the effect of CD137 signaling on vascular calcification in ApoE−/− mice and in vascular smooth muscle cells (VSMCs) of mice. Methods: Calcium deposition and muscle fibers in vivo or vitro were identified by von-Kossa and Masson's trichrome staining respectively. Alkaline phosphatase (ALP) activity was measured by the ALP assay Kit. The presence of bone morphogenic protein 2 (BMP2) and runt-related transcription factor 2 (Runx2) was detected by real-time PCR, Western blot and immunofluorescence in vitro or vivo. Results: Our data shows that activation of CD137 signaling by intraperitoneal injection of agonist-CD137 antibody increased the areas of vascular calcification. Activation of CD137 signaling also increased the expression of BMP2 and Runx2 in the atherosclerotic plaques. In vitro, activation of CD137 signaling also aggravated VSMC calcification, while blocking CD137 signaling could alleviate agonist-CD137 induced VSMC calcification. In addition, the levels of calcium, BMP2 and Runx2, indicators of calcification, were all significantly elevated in agonist-CD137 group in VSMCs. Conclusion: Our data revealed a previously unrecognized role of CD137 signaling in vascular calcification in vivo and vitro and provides a novel target for prevention and treatment of atherosclerosis in the future. © 2016 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Vascular calcification, which refers to the ectopic deposition of calcium phosphate crystal in cardiovascular tissue, is a common characteristic of advanced atherosclerotic lesion and a well-known independent predictive risk factor of subsequent cardiovascular morbidity and mortality [1]. Recent evidence suggested that vascular smooth muscle cell (VSMC) differentiation to osteogenic cell and express bone related proteins with concomitant down-regulation of SMC contractile protein play a pivotal role in vascular calcification [2,3]. However, the molecular mechanisms that regulate VSMC osteogenic transdifferentiation are complex and poorly understood. Accumulated evidence has demonstrated that inflammation may play an important role in VSMC osteogenic transdifferentiation [4]. CD137, a member of the tumor necrosis factor receptor superfamily (TNFRSF), is mainly expressed in a variety of immune cells which ⁎ Corresponding author. E-mail address: [email protected] (J. Yan). 1 These authors contributed equally to this work.
include natural killer (NK) cells, neutrophils, CD4+CD25+ regulatory T (Treg) cells, resting monocytes, and dendritic cells (DCs). However, under proinflammatory conditions, it is also expressed in some nonimmune cells, such as VSMCs and endothelial cells [5,6]. Olofsson and colleagues showed that CD137 is expressed in human atherosclerotic plaques and promotes the development of plaque [7]. Another study demonstrated that CD137 not only regulates T-cell activation as a costimulatory receptor, but also mediates atherosclerosis. Deficiency of CD137 reduces atherosclerosis in mice on both chow and high-fat diets [8]. In addition, our previous study showed that CD137-CD137L signaling pathway played a positive role in facilitating atheromatous plaque formation and progression. Furthermore, inhibition of CD137CD137L signaling significantly inhibited the formation of atherosclerotic lesions in apolipoprotein E-deficient (ApoE−/−) mice [9,10]. Based on these studies, we hypothesize that CD137-CD137L signaling pathway plays a critical role in the progression of atherosclerotic calcification. In this study, our study demonstrates that activation of CD137 signaling exacerbated vascular calcification in vivo and aggravated VSMC calcification in vitro. The mechanisms may involve increasing VSMC osteogenic differentiation.
http://dx.doi.org/10.1016/j.ijcard.2016.12.174 0167-5273/© 2016 Elsevier Ireland Ltd. All rights reserved.
Please cite this article as: Y. Chen, et al., Activation of CD137 signaling accelerates vascular calcification in vivo and vitro, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.12.174
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2. Materials and methods
2.3. Immunohistochemical analysis
2.1. Animals
We performed immunohistochemical staining on 5 μm thick paraffin-embedded sections, which were prepared from 4% formaldehyde-fixed tissue. The following primary antibodies were used: anti-Runx2 (immunoway) and anti-BMP2 (immunoway). After incubation with primary antibody overnight at 4 °C, horseradish peroxidase-conjugated secondary antibody was used in the second step followed by 3,3-diaminobenzidine to visualize the antigen. Nuclei were stained with hematoxylin. Negative controls were routinely employed.
15 ApoE−/− mice aged 8 weeks were purchased from vital river laboratories (Distributor of Jackson Laboratory, Beijing, China). All the animals were housed under a 12-h lightdark cycle, and 23 ± 2 °C under 55 ± 10% humidity, in normal cages with free access to water and provision of high fat foods. At the age of 13 weeks, the mice were randomly divided into the following groups: agonist-CD137 group, anti-CD137 group, and the control group. Mice in each group were intraperitoneally injected with 200 μg agonist-CD137 antibody (R&D), 200 μg anti-CD137 antibody + 200 μg agonist-CD137 antibody, and 200 μg IgG 2b (eBioscience) at 13, 15, and 17 weeks of age respectively. At 19 weeks of age, the mice were euthanized using 8% chlorate hydrate and aortas were pushed with phosphate-buffered saline through the left ventricle. Aortas, from the proximal ascending aorta to the bifurcation were freed from connective tissue under a dissection microscope. Aortas were fixed in 10% formaldehyde in PBS overnight, and further embedded in paraffin, and then the paraffin sections were cut out from the aortic arch to the thoracic artery. We chose the same position (2 mm above the aortic valve, Fig. 1A) of the short-axial slice among groups as the representative image. All animal experiments were reviewed and approved by the Animal Care and Use Committee of Jiangsu University.
2.4. VMSC culture Primary VSMCs were obtained from mouse thoracic aorta by Patch-attaching method as previously described [11] and identified by immunofluorescence staining for smooth muscle specific α-actin antibody (α-SMA, Sigma). Briefly, adventitia and intima were striped from segments of thoracic aorta, and the remaining tunica media was cut to 1–3 mm3 pieces. Then, the small pieces were cultured in Dulbecco's modified Eagle's medium (DMEM; high glucose, 4.5 g/L; Gibco) supplemented with 20% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere containing 5% CO2. After migrated from the explants, cells were collected and maintained in growth medium. Passage 4–6 VSMCs were used in experiments.
2.2. Analysis of vascular lesions in ApoE−/− mice
2.5. In vitro calcification and quantification of VSMCs
Hematoxylin/eosin (H&E) was used to assess tissue architecture. Additionally, sections were stained with the following staining kits according to supplied protocol: vonKossa, Masson's trichrome, and ALP. Digital images of arterials were captured using an Olympus microscope, and quantitative analyses of indicated stains were performed using image-pro plus 6.0 (IPP6.0) software.
Calcification of VSMCs was induced as previously described. Briefly, VSMCs were calcified with medium containing DMEM, 6% FBS, and 10 mmol/L β-glycerophosphate (β-GP). To induce CD137 expression, cells were treated with TNFɑ (10 ng/ml) for 24 h [11]. Then, the following incubations were performed: agonist-CD137 (10 μg/ml), antiCD137 (10 μg/ml) + agonist-CD137 (10 μg/ml), and IgG 2b (10 μg/ml). The calcification
Fig. 1. Activation of CD137 signaling promotes atherosclerotic calcification and accelerates osteogenic cell formation in ApoE−/− mice. (A) En face aorta with oil red O staining: from the proximal ascending aorta to the thoracic aorta. Short-axial slice of the aorta (yellow box area), 2 mm above the aortic valve (white arrows), red oil o staining positive area (yellow triangle). Histological analysis of aortas, 200× magnification: Hematoxylin/eosin (H&E) staining is used to assess tissue architecture; black arrow denotes osteogenic-like cells. The calcification was checked by von-Kossa staining. For further characterization of the calcified lesions, the lesions were stained for alkaline phosphatase (ALP) and fibers. Masson's trichrome staining shows that muscle fibers and collagen fibers are stained red and blue, respectively. The expression of BMP2 and Runx2 was stained by immunohistochemistry. Scale bar: 100 μm. The quantization bar graph of the plaque areas was below the pictures (D). (B) Quantitation of von-Kossa staining. (C) Quantitation of BMP2 and Runx2 expression. IOD: integrated optical density. Data are shown as mean ± SD, n = 5. In all panels, ⁎ and ⁎⁎ denote significance with p b 0.05 and p b 0.01 respectively, by Tukey's procedure. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Y. Chen, et al., Activation of CD137 signaling accelerates vascular calcification in vivo and vitro, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.12.174
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Fig. 1 (continued).
medium was replaced every 3 days for a total 15 days. VSMCs were washed with PBS for 3 times followed by treatment with 0.6 mol/L HCl overnight at 4 °C. The calcium content of HCl supernatants was determined colorimetrically by the Methylthymol Blue complexone method. The remaining cell layers were then dissolved in 0.1 mol/L NaOH and 0.1% SDS for protein concentration analysis. Calcium content was normalized by protein content. 2.6. Von-Kossa staining Calcium deposition in atherosclerosis plaque and VSMCs was identified by von-Kossa staining. VSMCs and paraffin-embedded sections were incubated with 5% and 2% silver nitrate solution respectively, and were placed under ultraviolet light for 30 min. Un-reacted silver was removed with 5% sodium thiosulfate for 5 min and counterstained with nuclear fast red for 3 min. Images were collected using an Olympus microscope. 2.7. Alkaline phosphatase activity assay Proteins were extracted from VSMCs by freeze-thawing the cells in 0.1% Triton X-100 in PBS. Alkaline phosphatase (ALP) activity was measured by the ALP assay Kit (Nanjing Jiancheng Bioengineering Research Institute, China) following the manufacturer's instructions. Culture medium was collected and centrifuged at 2500 rpm for 10 min; the supernatant of the medium was harvested for subsequent assay. Briefly, after adding buffer solution and matrix solution, water bathing, and coloring, the absorbance value was measured at 520 nm with the microplate reader. Subsequently, the activity values with a computational formula were calculated. Results were normalized to the protein content. 2.8. Quantitative real-time reverse-transcription PCR (qRT-PCR) The effect of CD137 on the expression of bone-related gene markers in VSMCs was determined by quantitative real-time RT-PCR using a Roche Molecular LightCycler. Total RNA was isolated with the Trizol reagent (Invitrogen) and reverse transcribed into cDNA. Quantitative real-time PCR (SYBR green; Invitrogen) was performed with primers against mouse BMP2, Runx2, OPN, and GAPDH (Sangon Biotech, China). The primer sequences were as follows: BMP2, 5′-TGAGGATTAGCAGGTCTTTGC-3′ and 5′-GCTGTTTGTGTTTGGC TTGA-3′; Runx2, 5′-CCTCTGACTTCTGCCTCTGG-3′ and 5′-ATGAAATGCTTGGGAACTGC-3′; OPN, 5′-GAGGAAACCAGCCAAGGTAA-3′ and 5′-GCAAATCACTGCCAATCTCA-3′; and GAPDH, 5′-GGCATTGCTCTCAATGACAA-3′ and 5′-TGTGAGGGAGATGCTCAGTG-3′.
2.9. Western blot Protein samples were extracted using a protein extraction reagent. Proteins were then separated by electrophoresis on an SDS-PAGE polyacrylamide gel and transferred to PVDF membranes. The membranes were incubated at 4 °C overnight with antibodies against BMP2 (1:1000), Runx2 (1:1000), OPN (1:1000) and GAPDH (1:2000) respectively. After that, membranes were incubated with appreciate HRP-conjugated secondary antibodies for 1 h. Band detection was performed according to the manufacturer's protocol (ECL; Pierce Biotechnology Inc., Rockford, USA). 2.10. Statistical analysis The results were expressed as means ± SD. Comparisons between groups were performed using ANOVA and the Tukey posttest procedure with the SPSS 17.0 software package. A p value b0.05 was considered statistically significant.
3. Result 3.1. Body weight and survival All 15 ApoE−/− mice survived before the animals were killed and there were no significant differences in body weight among the 3 groups (control group: 31.60 ± 1.82 g; agonist-CD137 group: 31.20 ± 2.39 g; anti-CD137 group: 30 ± 1.87 g; p N 0.05). 3.2. Activation of CD137 signaling promotes atherosclerotic calcification in ApoE−/− mice To probe the potential role of CD137 signaling involvement in atherosclerotic calcification, we activated the CD137 signaling by agonistCD137 antibody and inhibited it by anti-CD137 antibody. H&E staining shows that activation of CD137 signaling significantly increased the
Please cite this article as: Y. Chen, et al., Activation of CD137 signaling accelerates vascular calcification in vivo and vitro, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.12.174
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plaque areas, while inhibition of CD137 signaling decreased the plaque areas (Fig. 1A and D). Atherosclerotic calcification was verified by vonKossa staining. Our data shows that activation of CD137 signaling increased mean calcification compared to control (p b 0.01), In contrast, additional anti-CD137 treatment decreased the agonist-CD137 antibody induced atherosclerotic calcification (p b 0.05) (Fig. 1A and B). This data identifies that activation of CD137 signaling promotes atherosclerotic calcification, and this effect is independent from the lesion size extending. 3.3. Activation of CD137 signaling increase osteogenic cell formation in the plaque To further illustrate the calcification in the plaque. We then examined the effect of CD137 signaling on osteogenic transdifferentiation. Masson's trichrome staining shows that activation of CD137 signaling significantly decreased the number of muscle fibers in the vascular wall, while inhibition of CD137 signaling slightly decreased muscle fibers compare to control (Fig. 1A). Immunohistochemical staining showed that the ALP is located near the areas of calcification, and the expression of BMP2, Runx2 and ALP is significantly increased after treated with agonist-CD137 antibody, and this effect was partly blocked when treated with anti-CD137 antibody (Fig. 1A and C). 3.4. Activation of CD137signaling promotes VSMC calcification To elucidate the mechanisms by which CD137 regulates the vascular calcification, we performed in vitro experiments using VSMCs. vonKossa staining shows that there is no apparent difference between the IgG group and the normal control group. VSMCs treated with agonistCD137 antibody appear more seriously calcified, but the anti-CD137 antibody treated VSMCs partly inhibited the appearance of calcification (Fig. 2A). Similar results were observed in calcium content and ALP activation (Fig. 2B and C). 3.5. Activation of CD137 signaling accelerates VSMC osteogenic transdifferentiation VSMC osteogenic transdifferentiation is one of the most important procedures in vascular calcification. Therefore, we examined the effect of CD137 signaling on VSMC osteogenic transdifferentiation. As shown in Fig. 3, the mRNA levels of BMP2 and Runx2 were all significantly increased after the VSMCs were incubated with agonist-CD137 antibody and decreased while VSMCs were incubated with additional antiCD137 antibody (Fig. 3A). Similar results were obtained from Western blot analysis (Fig. 3B). Notably, compared with the control group, the mRNA and protein levels of osteopontin (OPN) were also increased in the agonist-CD137 group, however, blocking CD137 with additional anti-CD137, the expression of OPN was further increased. 4. Discussion Most individuals aged N60 years have progressively enlarging deposits of calcium mineral in their major arteries. This vascular calcification reduces aortic and arterial elastance, which impairs cardiovascular hemodynamics, resulting in substantial morbidity and mortality in the form of hypertension, cardiac hypertrophy, and myocardial ischemia [12]. The severity and extent of calcification reflect atherosclerotic plaque burden and strongly and independently predict cardiovascular events. In this study, we demonstrated that, for the first time to our knowledge, CD137 signaling contributes to the calcification of atherosclerotic lesion and VSMCs. The mechanisms involve an increase in VSMC osteogenic differentiation. As such, this proof-of concept study provides a novel target for prevention and treatment of atherosclerosis in the future.
CD137, known as a costimulator of T cells, activates nuclear factorκB and promotes T cell proliferation and cytokine production [7]. A large number of studies have indicated an important role for CD137 signaling in autoimmune disease processes [13]. Since CD137 molecule was first detected in human atherosclerotic lesions, slowly the potential relationships between CD137 molecule and atherosclerosis were discovered. Previous study showed that CD137 deficiency reduced atherosclerosis in hyperlipidemic mice and CD137 signaling was involved in the development and quality of atherosclerotic plaques [8]. Another study demonstrates that activation of CD137 signaling decreases the stability of advanced atherosclerotic plaques [6]. Vascular calcification is the progression of pathological mineralization of the arteries, and often been observed in advanced atheromatous plaque. We found that activation of CD137 increased the areas of atherosclerotic plaque calcification. Activation of CD137 promotes the atherosclerotic plaque development, and the areas of vascular calcification are associated with the size of atheromatous plaques. To ensure the positive effect of CD137 on vascular calcification, we further normalized the calcification area by plaque size, our data show that activation of CD137 signaling still enlarged the area of calcification even though it was normalized by plaque area. These results indicate that CD137 that accelerates vascular calcification is independent from its positive effect on the size of atherosclerotic plaque. Studies in the last decade suggest that vascular calcification is an active process, regulated in a manner similar to orthotopic bone formation [14]. Previous studies have suggested that some substances, such as TNF and inflammatory cytokines, are capable of inducing osteogenic genes, transforming osteoblasts and secreting some bone matrix proteins in the walls of blood vessels [15]. Consistent with these previous studies, data from our study shows that osteogenic-like cells were identified at the areas beside calcified lesions in the plaques. We further showed that activation of CD137 signaling with agonist-CD137 antibody promotes the formation of osteogenic cells in atherosclerotic lesions. BMP2 is known as a powerful bone morphogenic protein and its expression triggers osteogenic transcriptional, while Runx2 is thought to be a key regulator of osteoblast differentiation [16]. ALP is one of the osteoblastic phenotype markers and is considered essential in the vascular calcification process [2]. Therefore, we focused on the effect of CD137 signaling on expression of BMP2 and Runx2. It has been reported that CD137 could activate nuclear factor-κB and promotes T cell proliferation and cytokine production [7]. Others have demonstrated that NF-κB activation is implicated in receptor activator of NF-κB ligand (RANKL)-mediated osteogenic differentiation of smooth muscle cells and activation of NF-κB promotes calcification and increased the expression of BMP2 and Runx2 in aortic smooth muscle cells [17]. Thus, it is possible that CD137 signaling pathways have an essential role in mediating the expression of BMP2, Runx2 and ALP. Indeed, our data showed that treatment with agonist-CD137 antibody increased the expression of both BMP2 and Runx2, and inhibition CD137 signaling with anti-CD137 antibody decreased the expression of BMP2 and Runx2. This means that CD137 signaling has the ability to increase osteogenic cell formation through promoting vascular calcification. Several cells are involved in the procedure of vascular calcification, including endothelial cells, VSMCs, monocytes and macrophages [18]. A series of in-vitro studies demonstrate that monocytes and macrophages release inflammatory cytokines that promote cardiovascular calcification by regulating the differentiation of calcifying vascular cells, which express a number of phenotypic markers synonymous with osteogenic cells [19]. We and others have previously reported that NF-κB is involved in CD137 signaling [11]. NF-κB activation is implicated in receptor activator of NF-κB ligand (RANKL)-mediated osteogenic differentiation of smooth muscle cells. Moreover, researchers found that activation of NF-κB promotes calcification in aortic smooth muscle cells [20]. Thus, it is possible that CD137 signaling regulates VSMC calcification. Consistent with the present study in vivo, we provide evidence that activation of CD137 signaling aggravated VSMC
Please cite this article as: Y. Chen, et al., Activation of CD137 signaling accelerates vascular calcification in vivo and vitro, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.12.174
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Fig. 2. Activation of CD137 signaling promoted β-glycerophosphate (β-GP) induced vascular smooth muscle cell (VSMC) calcification. (A) VSMC mineralization was stained by von-Kossa staining. The calcification was also quantified by HCL extraction followed by colorimetric assay (B). Alkaline phosphatase (ALP) activity was measured by ALP assay (C). Scale bar: 200 μm. Data are shown as mean ± SD, n = 5, ⁎p b 0.05, ⁎⁎p b 0.01; NS: no significance.
calcification, upregulated the expression of BMP2 and Runx2, and increased the ALP activity, while blocking CD137 signaling can partly inhibit VSMC calcification and reduce the expression of these calcification indicators. This result indicates that CD137 signaling can promote the transformation of VSMCs into osteogenic cells. OPN is another biomarker of osteogenic cells which acts as a regulator of mineralization by inhibiting apatite crystals and promoting osteoclast function, not normally found within the vasculature, it is seen at high levels in calcified arteries and is potentially upregulated to counteract the advancement of vascular calcification [21]. Our data from
the present study showed that compared with the agonist-CD137 group, the level of OPN is up-regulated while blocking CD137 signaling with anti-CD137 antibody. This means that activation of CD137 signaling probably directly inhibits OPN expression and promotes VSMC calcification. In conclusion, the present study demonstrates that CD137 signaling promotes vascular calcification in vivo and vitro, and the following two mechanisms are probably involved in this process: 1) activation of CD137 signaling accelerates the transformation of VSMCs to osteogenic cells; 2) activation of CD137 signaling breaks the balance of calcification
Fig. 3. Activation of CD137 signaling induces osteogenic differentiation of vascular smooth muscle cells (VSMCs) in vitro. VSMCs were treated with control, IgG, agonist-CD137 antibody, or anti-CD137 antibody + agonist-CD137 antibody respectively, and incubated with 10 mmol/l β-glycerophosphate (β-GP). The mRNA levels of osteogenic marker genes (bone morphogenetic protein 2, BMP2; runt-related transcription factor 2, Runx2; osteopontin, OPN) were determined by reverse transcription-polymerase chain reaction and normalized to that of GAPDH (A, n = 4), and the protein levels were examined by Western blot (B, n = 5). Data are shown as mean ± SD, ⁎p b 0.01; NS: no significance. (1: Control; 2: Ig G; 3: Agonist-CD137; 4: Anti-CD137).
Please cite this article as: Y. Chen, et al., Activation of CD137 signaling accelerates vascular calcification in vivo and vitro, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.12.174
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biomarkers by increasing the levels of calcification promoters (BMP2, Runx2) or directly decreasing the expression of calcification inhibitor (OPN). Our data revealed a previously unrecognized role of CD137 signaling in vascular calcification, and suggest that CD137 signaling takes part in osteogenic differentiation of VSMCs. However, the underlying mechanisms are still unclear and require our further study.
[8]
[9]
Sources of funding
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This project was supported by the Natural Foundation of Jiangsu Province (BK20161355) and National Natural Science Foundation of China (81670405, 81370408, and 81370409).
[11]
Disclosure statement
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The authors have no conflicts of interest to declare. References [1] K. Lee, H. Kim, D. Jeong, Microtubule stabilization attenuates vascular calcification through the inhibition of osteogenic signaling and matrix vesicle release, Biochem. Biophys. Res. Commun. 451 (2014) 436–441. [2] J.M. Valdivielso, Vascular calcification: types and mechanisms, Nefrologia 31 (2011) 142–147. [3] L. Deng, L. Huang, Y. Sun, J.M. Heath, H. Wu, Y. Chen, Inhibition of FOXO1/3 promotes vascular calcification, Arterioscler. Thromb. Vasc. Biol. 35 (2015) 175–183. [4] C.M. Shanahan, Inflammation ushers in calcification: a cycle of damage and protection? Circulation 116 (2007) 2782–2785. [5] M.W. Anderson, S. Zhao, A.G. Freud, D.K. Czerwinski, H. Kohrt, A.A. Alizadeh, R. Houot, D. Azambuja, I. Biasoli, J.C. Morais, N. Spector, H.F. Molina-Kirsch, R.A. Warnke, R. Levy, Y. Natkunam, CD137 is expressed in follicular dendritic cell tumors and in classical Hodgkin and T-cell lymphomas: diagnostic and therapeutic implications, Am. J. Pathol. 181 (2012) 795–803. [6] I.H. Jung, J.H. Choi, J. Jin, S.J. Jeong, S. Jeon, C. Lim, M.R. Lee, J.Y. Yoo, S.K. Sonn, Y.H. Kim, B.K. Choi, B.S. Kwon, J.Y. Seoh, C.W. Lee, D.Y. Kim, G.T. Oh, CD137-inducing factors from T cells and macrophages accelerate the destabilization of atherosclerotic plaques in hyperlipidemic mice, FASEB J. 28 (2014) 4779–4791. [7] P.S. Olofsson, L.A. Soderstrom, D. Wagsater, Y. Sheikine, P. Ocaya, F. Lang, C. Rabu, L. Chen, M. Rudling, P. Aukrust, U. Hedin, G. Paulsson-Berne, A. Sirsjo, G.K. Hansson,
[15] [16]
[17]
[18] [19] [20]
[21]
CD137 is expressed in human atherosclerosis and promotes development of plaque inflammation in hypercholesterolemic mice, Circulation 117 (2008) 1292–1301. H.J. Jeon, J.H. Choi, I.H. Jung, J.G. Park, M.R. Lee, M.N. Lee, B. Kim, J.Y. Yoo, S.J. Jeong, D.Y. Kim, J.E. Park, H.Y. Park, K. Kwack, B.K. Choi, B.S. Kwon, G.T. Oh, CD137 (41BB) deficiency reduces atherosclerosis in hyperlipidemic mice, Circulation 121 (2010) 1124–1133. Y. Li, J. Yan, C. Wu, Z. Wang, W. Yuan, D. Wang, CD137-CD137L interaction regulates atherosclerosis via cyclophilin A in apolipoprotein E-deficient mice, PLoS One 9 (2014), e88563. J. Yan, H. Yang, W. Yuan, C. Wang, The effect of CD137-CD137 ligand interaction on the expression of NFATC1 in apolipoprotein E-deficient mice, Int. J. Cardiol. 157 (2012) 134–137. J. Yan, Y. Yin, W. Zhong, C. Wang, Z. Wang, CD137 regulates nfatc1 expression in mouse VSMCs through TRAF6/NF-kappaB p65 signaling pathway, Mediat. Inflamm. 2015 (2015) 639780. J.A. Leopold, Vascular calcification: an age-old problem of old age, Circulation 127 (2013) 2380–2382. D.S. Vinay, B.S. Kwon, Therapeutic potential of anti-CD137 (4-1BB) monoclonal antibodies, Expert Opin. Ther. Targets 20 (2016) 361–373. L. Tian, K. Chen, J. Cao, Z. Han, L. Gao, Y. Wang, Y. Fan, C. Wang, Galectin-3-induced oxidized low-density lipoprotein promotes the phenotypic transformation of vascular smooth muscle cells, Mol. Med. Rep. 12 (2015) 4995–5002. G. Pugliese, C. Iacobini, C. Blasetti Fantauzzi, S. Menini, The dark and bright side of atherosclerotic calcification, Atherosclerosis 238 (2015) 220–230. S. Evrard, P. Delanaye, S. Kamel, J.P. Cristol, E. Cavalier, calcifications SSjwgov, Vascular calcification: from pathophysiology to biomarkers, Clin. Chim. Acta 438 (2015) 401–414. C.P. Lin, P.H. Huang, C.F. Lai, J.W. Chen, S.J. Lin, J.S. Chen, Simvastatin attenuates oxidative stress, NF-kappaB activation, and artery calcification in LDLR−/− mice fed with high fat diet via down-regulation of tumor necrosis factor-alpha and TNF receptor 1, PLoS One 10 (2015), e0143686. L.L. Demer, Y. Tintut, Inflammatory, metabolic, and genetic mechanisms of vascular calcification, Arterioscler. Thromb. Vasc. Biol. 34 (2014) 715–723. J.A. Leopold, Vascular calcification: mechanisms of vascular smooth muscle cell calcification, Trends Cardiovasc. Med. 25 (2015) 267–274. Y. Wang, Z.Y. Zhang, X.Q. Chen, X. Wang, H. Cao, S.W. Liu, Advanced glycation end products promote human aortic smooth muscle cell calcification in vitro via activating NF-kappaB and down-regulating IGF1R expression, Acta Pharmacol. Sin. 34 (2013) 480–486. N.J. Paloian, C.M. Giachelli, A current understanding of vascular calcification in CKD, Am. J. Physiol. Ren. Physiol. 307 (2014) F891–F900.
Please cite this article as: Y. Chen, et al., Activation of CD137 signaling accelerates vascular calcification in vivo and vitro, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.12.174