Biochemical and Biophysical Research Communications xxx (2016) 1e7
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Gelatinases promote calcification of vascular smooth muscle cells by up-regulating bone morphogenetic protein-2 Yong-Gang Zhao, Fan-Xing Meng, Bing-Wei Li, You-Ming Sheng, Ming-Ming Liu, Bing Wang, Hong-Wei Li, Rui-Juan Xiu* Institute of Microcirculation and Key Laboratory, Ministry of Health, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 December 2015 Accepted 10 January 2016 Available online xxx
Matrix metalloproteinase-2 (MMP-2), also known as gelatinase A, is involved in vascular calcification. Another member of gelatinases is MMP-9 (gelatinase B). However, the role of gelatinases in the pathogenesis of vascular calcification is not well understood. The current study aims to clarify the relationship between gelatinases and vascular calcification and to elucidate the underlying mechanism. Betaglycerophosphate (b-GP) was used to induce calcification of vascular smooth muscle cells (VSMCs) with or without 2-[[(4-Phenoxyphenyl)sulfonyl]methyl]-thiirane (SB-3CT), a specific gelatinases inhibitor. Levels of calcification were determined by assessing calcium content and calcification area of VSMCs. Phenotype transition of VSMCs was observed by assessing expressions of alkaline phosphatase (ALP), smooth muscle a-actin (SM-a-actin) and desmin. Gelatin zymography was applied to determine the activities of gelatinases, and western blot was applied to determine expressions of gelatinases, bone morphogenetic protein-2 (BMP-2), Runt-related transcription factor 2 (RUNX2) and msh homeobox homolog 2 (Msx-2). Gelatinases inhibition by SB-3CT alleviated calcification and phenotype transition of VSMCs induced by b-GP. Increased gelatinases expression and active MMP-2 were observed in calcifying VSMCs. Gelatinases inhibition reduced expression of RUNX2, Msx-2 and BMP-2. BMP-2 treatment increased expressions of RUNX2 and Msx-2, while noggin, an antagonist of BMP-2, decreased expressions of RUNX2 and Msx-2. Gelatinases promote vascular calcification by upregulating BMP-2 which induces expression of RUNX2 and Msx-2, two proteins associated with phenotype transition of VSMCs in vascular calcification. Interventions targeting gelatinases inhibition might be a proper candidate for ameliorating vascular calcification. © 2016 Elsevier Inc. All rights reserved.
Keywords: Vascular calcification Vascular smooth muscle cell Matrix metalloproteinase-2 Bone morphogenetic protein-2 Runt-related transcription factor 2
1. Introduction
Abbreviations: ALP, alkaline phosphatase; BMP-2, bone morphogenetic protein2; DMEM, dulbecco's modified eagle medium; ECM, extracellular matrix; FBS, fetal bovine serum; MMP-2, matrix metalloproteinase-2; Msx-2, msh homeobox homolog 2; PBS, phosphate-buffered saline; PVDF, polyvinylidene fluoride; RUNX2, runt-related transcription factor 2; SB-3CT, 2-[[(4-Phenoxyphenyl)sulfonyl] methyl]-thiirane; SDS-PAGE, sodium dodecyl sulfate-polyacrilamide gel electrophoresis; TBST, tris-buffered saline and Tween 20; VSMCs, vascular smooth muscle cells; b-GP, beta-glycerophosphate. * Corresponding author. Institute of Microcirculation and key laboratory, Ministry of Health, Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, China. E-mail addresses: [email protected] (Y.-G. Zhao), [email protected] (F.-X. Meng), [email protected] (B.-W. Li), [email protected] (Y.-M. Sheng), [email protected] (M.-M. Liu), [email protected] (B. Wang), [email protected] (H.-W. Li), [email protected] (R.-J. Xiu).
Vascular calcification is a common consequence of aging, hypertension, diabetes, atherosclerosis and chronic renal failure. Vascular calcification is associated with shorter survival for patients with renal failure and is a strong predictor of mortality and lowerextremity amputation in patients with diabetes [1,2]. Vascular calcification is generally divided into intimal calcification and medial calcification. The mechanisms of vascular calcification remain to be established. Previously it was considered as a passive process. Recent studies show that medial calcification is an actively regulated process resembling bone formation and is characterized by phenotype transition of vascular smooth muscle cells (VSMCs) into osteoblast-like cells [3]. Osteoblast-like cells secrete such proteins as osteocalcin, alkaline phosphatase (ALP) and osteopontin which accelerate calcium deposit.
http://dx.doi.org/10.1016/j.bbrc.2016.01.067 0006-291X/© 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: Y.-G. Zhao, et al., Gelatinases promote calcification of vascular smooth muscle cells by up-regulating bone morphogenetic protein-2, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.01.067
2
Y.-G. Zhao et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e7
The plasticity of VSMCs phenotype is well documented and its response is mediated, in part, by the composition of the extracellular matrix (ECM) [4]. As two important enzymes in matrix degradation, gelatinases (MMP-2 and MMP-9) are involved in the pathogenesis of vascular calcification. Active MMP-2 is present in calcified aortic stenosis cusps in human [5], and increased expression of MMP-2 is present in areas of aortic calcification [6]. GM 6001, a MMPs inhibitor, suppresses vascular calcification both in vivo and in organ culture [7]. Moreover, aortas from both MMP-2and MMP-9- knockout mice do not develop calcification after perivascular application of CaCl2 [8], while deficiency of MMP-2 alone impairs aortic atherosclerotic calcification in ApoE-deficient mice [9]. Efforts to elucidate the role of gelatinases in vascular calcification have been primarily made on the ability of MMP-2 to degrade elastin, an ECM protein abundant in the arterial wall [10]. MMP-2 binds and degrades elastin to produce elastin-derived peptides which binds to the elastin laminin receptor (ELR) on the surface of VSMCs. Interplay between elastin-derived peptides and ELR induces the transition of VSMCs into osteoblast-like cells and calcification gradually arises [4]. Bone morphogenetic protein-2 (BMP-2) is critical in bone formation and vascular calcification. BMP-2 expression was observed in calcified arteries of low-density lipoprotein receptor deficient mice [11]. Treatment of human VSMCs with BMP-2 increases calcification in vitro [12]. However, the relationship between gelatinases and BMP-2 in vascular calcification is scarcely investigated with a latest report showing that MMP-2 deficiency reduces BMP-2 expression in aortae of ApoE-deficient mice [9]. In the present study, we investigated the mechanism by which gelatinases are involved in vascular calcification using SB-3CT, a specific gelatinases inhibitor [13]. We report that gelatinases contribute to vascular calcification by a way independent of their ability to degrade elastin, i.e. gelatinases up-regulate expression of BMP-2 and thereby increase expressions of Runx2 and Msx-2 which are stimulations for osteoblastic differentiation of VSMCs. 2. Materials and methods 2.1. Animals Male Wistar rats (8 weeks) weighing 180e200 g with fasting and free access to water were used in this study. All experimental procedures involving use and care of animals conformed to guidelines approved by Experimental Animal Committee of Institute of Microcirculation, Chinese Academy of Medical Sciences & Peking Union Medical College. 2.2. VSMCs culture and in vitro calcification The explant culture method of VSMCs was used as described previously [14]. VSMCs of 5th to 8th passages were used for the experiments. All procedures were conducted in accordance with protocol guidelines approved by Experimental Animal Committee of Institute of Microcirculation, Chinese Academy of Medical Sciences & Peking Union Medical College. b-GP was used to induce in vitro calcification of VSMCs [15]. After confluence, the cells were inoculated in 24-well plates (1 104 cells/mL) and divided into 3 groups. The control group was treated with growing medium (DMEM containing 15% FBS) while bGP group was treated with calcification medium (DMEM containing 15% FBS in the presence of 10 mM b-GP). In the SB-3CT group, 10 mM SB-3CT was added to the calcification medium. The medium was replaced every 3 days. After 12 days, the medium was collected and the cell layer was washed with phosphate-buffered saline (PBS).
Cells were stored at
70 C until use.
2.3. Von Kossa staining Cells were fixed in 4% paraformaldehyde and rinsed with PBS twice. Then cells were exposed to 5% silver nitrate under a 75-W lamp for 30 min, then to 5% sodium thiosulfate for 3 min to remove unreduced silver. After rinsing, stained cells were examined by light microscope and photographed for black areas indicating deposited mineral. Black areas were analyzed colorimetrically using the Image J 1.48 software (National Institutes of Health (NIH), Bethesda, MD). 2.4. Measurement of calcium content in VSMCs Cellular calcium was extracted with 0.6 mM HCl overnight. After removing the HCl supernatant, the remaining cell layers on the plates were treated with lysis buffer containing 0.1 M NaOH and 0.1% SDS. Calcium content in the HCl supernatant was determined by the O-cresolphthalein complexone method. Calcium content was normalized to total protein content of the same well. 2.5. Measurement of alkaline phosphatase (ALP) activity in VSMCs Cells were rinsed three times with PBS and sonicated in 100 mL of lysis buffer. After centrifugation at 10,000 g for 10 min at 4 C, ALP activity in the supernatant was measured with the ALP assay kit (Beyotime Institute of Biotechnology). ALP activity was calculated using p-nitrophenol as a standard. One unit was defined as the activity producing l nM of p-nitrophenol in 30 min. ALP values were normalized to protein content. 2.6. Western blot analysis Western blot analysis was performed as follows. Briefly, samples were prepared and run on a 10% sodium dodecyl sulfateepolyacrilamide gel electrophoresis (SDS-PAGE) before being transferred onto polyvinylidene fluoride (PVDF) membranes (BioRad, Hercules, CA, USA). The membranes were washed with TBST, blocked with 5% nonfat dry milk in TBST for 1 h at room temperature and then incubated overnight at 4 C with primary antibodies. The membranes were then incubated for 1 h at room temperature with appropriate secondary antibodies. Chemiluminescent reagents (Beyotime Institute of Biotechnology) were used to detect protein bands. Quantification of bands was carried out by densitometry using Image J 1.48 software and the protein values were normalized with b-actin. 2.7. Gelatin zymography The conditioned medium was collected and centrifuged and the supernatant was analyzed for gelatin zymography of MMP-2 and MMP-9 [16]. Briefly, twenty micrograms of protein from each sample was subjected to electrophoresis on 8% SDS-PAGE using gelatin (0.1%) as the substrate. After electrophoresis, the gel was washed twice and gently shaken in 2.5% Triton X-100 for 30 min. The gel was then incubated overnight at 37 C in a 50 mM TriseHCl buffer, containing 5 mM CaCl2 and 0.2 M NaCl. The gel was stained with Coomassie brilliant blue R for 1 h. Unstained bands indicating gelatinolytic activity were seen after slight destaining with 30% ethanol and 10% acetic acid. Band intensity was quantitatively assessed using Image J 1.48.
Please cite this article in press as: Y.-G. Zhao, et al., Gelatinases promote calcification of vascular smooth muscle cells by up-regulating bone morphogenetic protein-2, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.01.067
Y.-G. Zhao et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e7
2.8. Statistical analysis All experiments were performed in triplicate. All data were expressed as mean ± S.D. One-way ANOVA, followed by StudenteNewmaneKeuls test for significance was carried out among the groups. The threshold for significance was P < 0.05.
3. Results 3.1. Gelatinases inhibition alleviated calcification of VSMCs Calcification area (Fig. 1A and B) and calcium content (Fig. 1C) in
b-GP group were significantly higher than that of control group. Gelatinases inhibition by SB-3CT caused reductions in calcification area (Fig. 1A and B) and calcium content (Fig. 1C) compared with bGP group. Вeta-GP group had an increased expression of both MMP-2 and MMP-9 compared with control group, and SB-3CT decreased expressions of MMP-2 and MMP-9 (Fig. 2A). The levels of pro-MMP-9 and pro-MMP-2 were almost the same among the three groups, and no active MMP-9 was detected by gelatin zymography (Fig. 2B). However, the level of active MMP-2 in b-GP group was higher than that of control group, and SB-3CT decreased it (Fig. 2B).
3.2. Effects of gelatinases inhibition on the expressions of tissue inhibitor of metalloproteinase-2 (TIMP-2), tissue inhibitor of metalloproteinase-1 (TIMP-1), MMP-2/TIMP-2 ratio and MMP-9/ TIMP-1 ratio Вeta-GP group had an increased expression of TIMP-1 compared with the control group, and SB-3CT decreased its expression (Fig. 2C). Compared with the control group, b-GP group had an increased MMP-2/TIMP-2 ratio, which was decreased by SB-3CT (Fig. 2D). In addition, there were no significant differences in the expression of TIMP-2 and MMP-9/TIMP-1 ratio among the three groups (Fig. 2C and D).
3
3.3. Gelatinases inhibition suppressed phenotype transition of VSMCs and expressions of RUNX2 and Msx-2 in VSMCs The present study found increased expression of ALP and decreased expressions of smooth muscle a-actin (SM a-actin) and desmin in b-GP group, demonstrating a transition of VSMCs phenotype into calcifying phenotype which was attenuated by SB3CT (Fig. 3AeD). Additionally, ALP activity in the b-GP group was significantly higher than that of control group, demonstrating an activity of calcium deposit which was inhibited by SB-3CT (Fig. 3E). Increased expressions of RUNX2 and Msx-2 were found in b-GP group compared with that of control group, and this increase was inhibited by SB-3CT (Fig. 3F and H). 3.4. Gelatinases inhibition suppressed BMP-2 expression BMP-2 expression in b-GP group was increased compared with that of control group, and gelatinases inhibition decreased its level (Fig. 3I). 3.5. Gelatinases inhibition decreased expressions of RUNX2 and Msx-2 via suppressing BMP-2 The group treated with BMP-2 or noggin was nominated as BMP-2 group or noggin group, while the group treated with neither BMP-2 nor noggin was nominated as control group. BMP-2 group had increased expressions of RUNX2 and Msx-2 compared with control group, while noggin decreased their expressions compared with control group (Fig. 4). 4. Discussion Medial calcification is characterized by phenotype transition of VSMCs which can be mediated by the composition of the ECM. In view of the important role of ECM alteration in phenotype transition of VSMCs, it is reasonable to hypothesize that gelatinases, critical enzymes in ECM degradation, may be involved in vascular calcification. In the present study, b-GP was used to induce in vitro calcification of VSMCs. And SB-3CT was used to inhibit MMP-2 and
Fig. 1. Gelatinases inhibition suppressed calcification induced by b-GP in cultured VSMCs. VSMCs were treated with b-GP in the absence or presence of SB-3CT for 12 days. Von kossa stainging was made to show black areas indicating deposited mineral, and the black areas were analyzed colorimetrically. Calcium contents were normalized to total protein content of the same well. A, Von kossa stainging of VSMCs at day 12. B, Colorimetric measurement of calcification area for von kossa staining. ***P < 0.001 vs control group, ### P < 0.001 vs b-GP group. C, Calcium content of VSMCs at day 12. ***P < 0.001 vs control group, ###P < 0.001 vs b-GP group. b-GP: beta-glycerophosphate; SB-3CT: 2-[[(4Phenoxyphenyl)sulfonyl]methyl]-thiirane.
Please cite this article in press as: Y.-G. Zhao, et al., Gelatinases promote calcification of vascular smooth muscle cells by up-regulating bone morphogenetic protein-2, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.01.067
4
Y.-G. Zhao et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e7
Fig. 2. Effects of SB-3CT on expressions of MMP-2, MMP-9, TIMP-2 and TIMP-1 and activity of MMP-2 and MMP-9 in VSMCs. VSMCs were treated with b-GP in the absence or presence of SB-3CT for 12 days. The conditioned medium was collected and analyzed for expressions of MMP-2, MMP-9, TIMP-2 and TIMP-1 and activity of MMP-2 and MMP-9. A, Expressions of MMP-2 and MMP-9 in the conditioned medium of VSMCs at day 12. **P < 0.01 vs control group, ##P < 0.01 vs b-GP group. B, Gelatin zymography of conditioned medium of VSMCs and comparison of active MMP-2 at day 12. *P < 0.05 vs control group, #P < 0.05 vs b-GP group. C, Expressions of TIMP-2 and TIMP-1 in the conditioned medium of VSMCs at day 12. **P < 0.01 vs control group, ##P < 0.01 vs b-GP group. D, MMP-2/TIMP-2 ratio and MMP-9/TIMP-1 ratio in each group. **P < 0.01 vs control group, ##P < 0.01 vs b-GP group. b-GP: beta-glycerophosphate; SB-3CT: 2-[[(4-Phenoxyphenyl)sulfonyl]methyl]-thiirane; MMP-2: matrix metalloproteinase-2; MMP-9: matrix metalloproteinase-9; TIMP-2: tissue inhibitors of metalloproteinase-2; TIMP-1: tissue inhibitors of metalloproteinase-1.
Please cite this article in press as: Y.-G. Zhao, et al., Gelatinases promote calcification of vascular smooth muscle cells by up-regulating bone morphogenetic protein-2, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.01.067
Y.-G. Zhao et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e7
5
Fig. 3. Effects of gelatinases inhibition on phenotype transition of VSMCs and expressions of RUNX2, Msx-2 and BMP-2. VSMCs were treated with b-GP in the absence or presence of SB-3CT for 12 days. Expressions of ALP, SM a-actin, desmin, RUNX2, Msx-2 and BMP-2 and ALP activity were analyzed. A, VSMCs transition into calcifying phenotype in the b-GP group was characterized by increased expression of ALP and decreased expressions of SM a-actin and desmin, and gelatinases inhibition by SB-3CT attenuated this process. B-D, Relative expressions of ALP, SM a-actin and desmin, respectively. E, Effect of gelatinases inhibition on ALP activity. **P < 0.01, ***P < 0.001 vs control group, #P < 0.05, ###P < 0.001 vs b-GP group. FeI, Gelatinases inhibition by SB-3CT suppressed expressions of RUNX2, Msx-2 and BMP-2. **P < 0.01, ***P < 0.001 vs control group; #P < 0.05, ##P < 0.01 vs b-GP group. b-GP: beta-glycerophosphate; SB-3CT: 2-[[(4-Phenoxyphenyl)sulfonyl]methyl]-thiirane; ALP: alkaline phosphatase; SM a-actin: smooth muscle a-actin; RUNX2: Runt-related transcription factor 2; Msx-2: msh homeobox homolog 2; BMP-2: bone morphogenetic protein-2.
MMP-9. The calcification area in b-GP group was 7.4-fold of the control group, while SB-3CT inhibited the increase in calcification area induced by b-GP. And gelatinases inhibition attenuated the increase of VSMCs calcium content induced by b-GP. These results indicate gelatinases promote VSMCs calcification. The b-GP group had increased gelatinases expression and active MMP-2, which were reduced by SB-3CT, suggesting up-regulation and activation of gelatinases are involved in pathogenesis of VSMCs calcification. These results confirmed findings of previous reports which found enhanced MMP-2 activity and increased MMP-2 expression in calcified aorta [5,6]. As tissue inhibitors of metalloproteinases (TIMPs) are endogenous regulator of MMPs, we detected the expressions of TIMP-2 and TIMP-1, and calculated the MMP-2/TIMP-2 ratio and MMP-9/TIMP-1 ratio. The b-GP group had an increased MMP-2/TIMP-2 ratio, which was decreased by SB-3CT. In addition, there were no significant differences in the expression of TIMP-2 and MMP-9/TIMP-1 ratio among the three groups. Therefore, it may be postulated that the one gelatinase exerting more effects may be MMP-2 rather than MMP-9. However, verification is needed. Involvement of gelatinases in vascular calcification is further supported by the observations that mice deficient of MMP2 alone or in combination with MMP-9 had lesser or no vascular calcification [8,9]. However, it has not been answered how osteogenic stimulation induces MMP-2. It may be explained in two aspects. Firstly, it is due to the alternation of ECM. As an ostoegenic
stimulator, b-GP is degraded by ALP, raising the local concentration of phosphorus [15]. High phosphorus levels causes accumulation of ECM [17], and this alternation of ECM activates MMP-2. Secondly, bGP treatment caused an increase in MMP-2/TIMP-2 ratio in the present study, which is an activator of MMP-2. Consistent with the occurrence of calcification, we found phenotype transition of VSMCs as well as increased expressions of RUNX2 and Msx-2 in b-GP group which were attenuated by gelatinases inhibition. BMP-2, a crucial factor in bone formation, is also involved in vascular calcification. BMP-2 was expressed in human atherosclerotic lesions [18]. Treatment of human VSMCs with BMP-2 increases calcification in vitro [12]. BMP-2 induces expressions of RUNX2 and Msx-2 in VSMCs [19]. RUNX2 is crucial for phenotype transition of SMCs [20], while Msx-2 is critical for osteoblast differentiation and bone formation [21]. However, the relationship between gelatinases and BMP-2 in vascular calcification is scarcely investigated with a latest report showing that MMP-2 deficiency reduces BMP-2 expression in aortae of ApoE-deficient mice [9]. We, therefore, hypothesize that gelatinases promotes BMP-2 expression to induce expressions of RUNX2 and Msx-2 which are crucial for phenotype transition of VSMCs. To investigate the relationship between gelatinases and BMP-2 in VSMCs calcification, the expressions of BMP-2 were analyzed 12 days after treatment with bGP and SB-3CT. The results showed that b-GP increased expressions
Please cite this article in press as: Y.-G. Zhao, et al., Gelatinases promote calcification of vascular smooth muscle cells by up-regulating bone morphogenetic protein-2, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.01.067
6
Y.-G. Zhao et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e7
Fig. 4. Effects of BMP-2 and noggin on expressions of RUNX2 and Msx-2 in VSMCs. Cells were treated with BMP-2 (100 ng/mL) or noggin (100 ng/mL) for 4 h and incubated with calcification medium in the presence of SB-3CT for 24 h, and then expressions of RUNX2 and Msx-2 were analyzed by western blot. The group treated with BMP-2 or noggin was nominated as BMP-2 group or noggin group, while the group treated with neither BMP-2 nor noggin was nominated as control group. *P < 0.05, **P < 0.01 vs control group. b-GP: beta-glycerophosphate; SB-3CT: 2-[[(4-Phenoxyphenyl)sulfonyl]methyl]-thiirane; BMP-2: bone morphogenetic protein-2; RUNX2: Runt-related transcription factor 2; Msx-2: msh homeobox homolog 2.
of BMP-2, whereas gelatinases inhibition decreased it. These results demonstrated gelatinases can regulate expression of BMP-2. The above-mentioned results showed gelatinases inhibition decreased expressions of RUNX2 and Msx-2 and down-regulated BMP-2. And previous study showed BMP-2 induces expressions of RUNX2 and Msx-2 [19]. To clarify whether the decreases in expressions of RUNX2 and Msx-2 in SB-3CT group were dependent on BMP-2 down-regulation induced by gelatinases inhibition, cells were treated with BMP-2 (100 ng/mL) or noggin (100 ng/mL) for 4 h and incubated with calcification medium in the presence of SB-3CT for 24 h, and then expressions of RUNX2 and Msx-2 were analyzed by western blot. Noggin, a BMP antagonist, binds to BMP-2 with high affinity and blocks its interaction with BMP-2 receptors, thereby inhibiting BMP signaling [22,23]. Our results demonstrated BMP-2 treatment increased expressions of RUNX2 and Msx-2 in VSMCs, while noggin decreased their expressions, demonstrating the decrease in expressions of RUNX2 and Msx-2 in VSMCs were dependent on BMP-2 down-regulation induced by gelatinases inhibition. However, more researches are needed to figure out which gelatinase is more important in the initiation and progression of vascular calcification, and target inhibition of this gelatinase may alleviate the deleterious effects of vascular calcification more effectively. The present study find gelatinases contribute to vascular calcification by a new way independent of their property of degrading ECM, i.e. gelatinases promote VSMCs calcification by up-regulating BMP-2 which induces expressions of RUNX2 and Msx-2, two proteins associated with phenotype transition of VSMCs. Therefore, interventions targeting gelatinases may be a proper candidate for ameliorating the devastating cardiovascular consequences of medial artery calcification in diabetes and end-stage renal disease.
Competing interests The authors declare that they have no competing interests.
Authors' contributions YZ and FM carried out the design of the study, performed the experiments, analyzed the data, and wrote the manuscript. BW, BL, ML, YS participated in the experimental procedures. HL contributed to reagents and tools. RX participated in experimental instructions, manuscript revision and coordination. All authors read and approved the final manuscript. Authors' information Institute of Microcirculation and key laboratory, Ministry of Health, China; Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing, 100005, China Acknowledgments This study was funded by the Postgraduate Innovation Fund of Peking Union Medical Colloge (2013-1001-05). References rin, S.J. Marchais, F. Me tivier, B. Pannier, H. Adda, [1] G.M. London, A.P. Gue Arterial media calcification in end-stage renal disease: impact on all-cause and cardiovascular mortality, Nephrol. Dial. Transpl. 18 (2003) 1731e1740. [2] R. Vattikuti, D.A. Towler, Osteogenic regulation of vascular calcification: an early perspective, Am. J. Physiol. Endocrinol. Metab. 286 (2004) E686eE696. [3] R.J. Rennenberg, L.J. Schurgers, A.A. Kroon, C.D. Stehouwer, Arterial calcifications, J. Cell Mol. Med. 14 (2010) 2203e2210. [4] A. Simionescu, K. Philips, N. Vyavahare, Elastin-derived peptides and TGFbeta1 induce osteogenic responses in smooth muscle cells, Biochem. Biophys. Res. Commun. 334 (2005) 524e532. [5] B. Jian, P.L. Jones, Q. Li, E.R. Mohler, F.J. Schoen, R.J. Levy, Matrix metalloproteinase-2 is associated with tenascin-C in calcific aortic stenosis, Am. J. Pathol. 159 (2001) 321e327. [6] N.X. Chen, K.D. O'Neill, X. Chen, K. Kiattisunthorn, V.H. Gattone, S.M. Moe, Activation of arterial matrix metalloproteinases leads to vascular calcification in chronic kidney disease, Am. J. Nephrol. 34 (2011) 211e219. [7] X. Qin, M.A. Corriere, L.M. Matrisian, R.J. Guzman, Matrix metalloproteinase inhibition attenuates aortic calcification, Arterioscler. Thromb. Vasc. Biol. 26
Please cite this article in press as: Y.-G. Zhao, et al., Gelatinases promote calcification of vascular smooth muscle cells by up-regulating bone morphogenetic protein-2, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.01.067
Y.-G. Zhao et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e7 (2006) 1510e1516. [8] D.M. Basalyga, D.T. Simionescu, W. Xiong, B.T. Baxter, B.C. Starcher, N.R. Vyavahare, Elastin degradation and calcification in an abdominal aorta injury model: role of matrix metalloproteinases, Circulation 110 (2004) 3480e3487. [9] T. Sasaki, K. Nakamura, K. Sasada, et al., Matrix metalloproteinase-2 deficiency impairs aortic atherosclerotic calcification in ApoE-deficient mice, Atherosclerosis 227 (2013) 43e50. [10] C. Kumata, M. Mizobuchi, H. Ogata, et al., Involvement of matrix metalloproteinase-2 in the development of medial layer vascular calcification in uremic rats, Ther. Apher. Dial. 15 (2011) 18e22. [11] S.L. Cheng, J.S. Shao, N. Charlton-Kachigian, A.P. Loewy, D.A. Towler, MSX2 promotes osteogenesis and suppresses adipogenic differentiation of multipotent mesenchymal progenitors, J. Biol. Chem. 278 (2003) 45969e45977. [12] X. Li, H.Y. Yang, C.M. Giachelli, BMP-2 promotes phosphate uptake, phenotypic modulation, and calcification of human vascular smooth muscle cells, Atherosclerosis 199 (2008) 271e277. [13] P. Tao, J.F. Fisher, S. Mobashery, H.B. Schlegel, DFT studies of the ring-opening mechanism of SB-3CT, a potent inhibitor of matrix metalloproteinase 2, Org. Lett. 11 (2009) 2559e2562. [14] S.Y. Wu, B.H. Zhang, C.S. Pan, et al., Endothelin-1 is a potent regulator in vivo in vascular calcification and in vitro in calcification of vascular smooth muscle cells, Peptides 24 (2003) 1149e1156. [15] A. Shioi, Y. Nishizawa, S. Jono, H. Koyama, M. Hosoi, H. Morii, Beta-glycerophosphate accelerates calcification in cultured bovine vascular smooth
7
muscle cells, Arterioscler. Thromb. Vasc. Biol. 15 (1995) 2003e2009. [16] S.A. Silva, M.G. Gobbo, M.E. Pinto-Fochi, et al., Prostate hyperplasia caused by long-term obesity is characterized by high deposition of extracellular matrix and increased content of MMP-9 and VEGF, Int. J. Exp. Pathol. 96 (2015) 21e30. [17] A. Papadimitriou, E.B. Peixoto, K.C. Silva, J.M. Lopes de Faria, J.B. Lopes de Faria, Inactivation of AMPK mediates high phosphate-induced extracellular matrix accumulation via NOX4/TGFß-1 signaling in human mesangial cells, Cell Physiol. Biochem. 34 (2014) 1260e1272. €m, K.E. Watson, S. Horn, C. Wortham, I.M. Herman, L.L. Demer, Bone [18] K. Bostro morphogenetic protein expression in human atherosclerotic lesions, J. Clin. Invest. 91 (1993) 1800e1809. [19] C. Sirard, S. Kim, C. Mirtsos, et al., Targeted disruption in murine cells reveals variable requirement for Smad4 in transforming growth factor beta-related signaling, J. Biol. Chem. 275 (2000) 2063e2070. [20] J.A. Balderman, H.Y. Lee, C.E. Mahoney, et al., Bone morphogenetic protein-2 decreases microRNA-30b and microRNA-30c to promote vascular smooth muscle cell calcification, J. Am. Heart Assoc. 1 (2012) e003905. [21] I. Satokata, L. Ma, H. Ohshima, et al., Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation, Nat. Genet. 24 (2000) 391e395. nchez-Duffhues, P. ten Dijke, BMP signaling in vascular [22] J. Cai, E. Pardali, G. Sa diseases, FEBS Lett. 586 (2012) 1993e2002. [23] J.W. Lowery, M.P. de Caestecker, BMP signaling in vascular development and disease, Cytokine Growth Factor Rev. 21 (2010) 287e298.
Please cite this article in press as: Y.-G. Zhao, et al., Gelatinases promote calcification of vascular smooth muscle cells by up-regulating bone morphogenetic protein-2, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.01.067
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Gelatinases promote calcification of vascular smooth muscle cells by up-regulating bone morphogenetic protein-2 Yong-Gang Zhao, Fan-Xing Meng, Bing-Wei Li, You-Ming Sheng, Ming-Ming Liu, Bing Wang, Hong-Wei Li, Rui-Juan Xiu* Institute of Microcirculation and Key Laboratory, Ministry of Health, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 December 2015 Accepted 10 January 2016 Available online xxx
Matrix metalloproteinase-2 (MMP-2), also known as gelatinase A, is involved in vascular calcification. Another member of gelatinases is MMP-9 (gelatinase B). However, the role of gelatinases in the pathogenesis of vascular calcification is not well understood. The current study aims to clarify the relationship between gelatinases and vascular calcification and to elucidate the underlying mechanism. Betaglycerophosphate (b-GP) was used to induce calcification of vascular smooth muscle cells (VSMCs) with or without 2-[[(4-Phenoxyphenyl)sulfonyl]methyl]-thiirane (SB-3CT), a specific gelatinases inhibitor. Levels of calcification were determined by assessing calcium content and calcification area of VSMCs. Phenotype transition of VSMCs was observed by assessing expressions of alkaline phosphatase (ALP), smooth muscle a-actin (SM-a-actin) and desmin. Gelatin zymography was applied to determine the activities of gelatinases, and western blot was applied to determine expressions of gelatinases, bone morphogenetic protein-2 (BMP-2), Runt-related transcription factor 2 (RUNX2) and msh homeobox homolog 2 (Msx-2). Gelatinases inhibition by SB-3CT alleviated calcification and phenotype transition of VSMCs induced by b-GP. Increased gelatinases expression and active MMP-2 were observed in calcifying VSMCs. Gelatinases inhibition reduced expression of RUNX2, Msx-2 and BMP-2. BMP-2 treatment increased expressions of RUNX2 and Msx-2, while noggin, an antagonist of BMP-2, decreased expressions of RUNX2 and Msx-2. Gelatinases promote vascular calcification by upregulating BMP-2 which induces expression of RUNX2 and Msx-2, two proteins associated with phenotype transition of VSMCs in vascular calcification. Interventions targeting gelatinases inhibition might be a proper candidate for ameliorating vascular calcification. © 2016 Elsevier Inc. All rights reserved.
Keywords: Vascular calcification Vascular smooth muscle cell Matrix metalloproteinase-2 Bone morphogenetic protein-2 Runt-related transcription factor 2
1. Introduction
Abbreviations: ALP, alkaline phosphatase; BMP-2, bone morphogenetic protein2; DMEM, dulbecco's modified eagle medium; ECM, extracellular matrix; FBS, fetal bovine serum; MMP-2, matrix metalloproteinase-2; Msx-2, msh homeobox homolog 2; PBS, phosphate-buffered saline; PVDF, polyvinylidene fluoride; RUNX2, runt-related transcription factor 2; SB-3CT, 2-[[(4-Phenoxyphenyl)sulfonyl] methyl]-thiirane; SDS-PAGE, sodium dodecyl sulfate-polyacrilamide gel electrophoresis; TBST, tris-buffered saline and Tween 20; VSMCs, vascular smooth muscle cells; b-GP, beta-glycerophosphate. * Corresponding author. Institute of Microcirculation and key laboratory, Ministry of Health, Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, China. E-mail addresses: [email protected] (Y.-G. Zhao), [email protected] (F.-X. Meng), [email protected] (B.-W. Li), [email protected] (Y.-M. Sheng), [email protected] (M.-M. Liu), [email protected] (B. Wang), [email protected] (H.-W. Li), [email protected] (R.-J. Xiu).
Vascular calcification is a common consequence of aging, hypertension, diabetes, atherosclerosis and chronic renal failure. Vascular calcification is associated with shorter survival for patients with renal failure and is a strong predictor of mortality and lowerextremity amputation in patients with diabetes [1,2]. Vascular calcification is generally divided into intimal calcification and medial calcification. The mechanisms of vascular calcification remain to be established. Previously it was considered as a passive process. Recent studies show that medial calcification is an actively regulated process resembling bone formation and is characterized by phenotype transition of vascular smooth muscle cells (VSMCs) into osteoblast-like cells [3]. Osteoblast-like cells secrete such proteins as osteocalcin, alkaline phosphatase (ALP) and osteopontin which accelerate calcium deposit.
http://dx.doi.org/10.1016/j.bbrc.2016.01.067 0006-291X/© 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: Y.-G. Zhao, et al., Gelatinases promote calcification of vascular smooth muscle cells by up-regulating bone morphogenetic protein-2, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.01.067
2
Y.-G. Zhao et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e7
The plasticity of VSMCs phenotype is well documented and its response is mediated, in part, by the composition of the extracellular matrix (ECM) [4]. As two important enzymes in matrix degradation, gelatinases (MMP-2 and MMP-9) are involved in the pathogenesis of vascular calcification. Active MMP-2 is present in calcified aortic stenosis cusps in human [5], and increased expression of MMP-2 is present in areas of aortic calcification [6]. GM 6001, a MMPs inhibitor, suppresses vascular calcification both in vivo and in organ culture [7]. Moreover, aortas from both MMP-2and MMP-9- knockout mice do not develop calcification after perivascular application of CaCl2 [8], while deficiency of MMP-2 alone impairs aortic atherosclerotic calcification in ApoE-deficient mice [9]. Efforts to elucidate the role of gelatinases in vascular calcification have been primarily made on the ability of MMP-2 to degrade elastin, an ECM protein abundant in the arterial wall [10]. MMP-2 binds and degrades elastin to produce elastin-derived peptides which binds to the elastin laminin receptor (ELR) on the surface of VSMCs. Interplay between elastin-derived peptides and ELR induces the transition of VSMCs into osteoblast-like cells and calcification gradually arises [4]. Bone morphogenetic protein-2 (BMP-2) is critical in bone formation and vascular calcification. BMP-2 expression was observed in calcified arteries of low-density lipoprotein receptor deficient mice [11]. Treatment of human VSMCs with BMP-2 increases calcification in vitro [12]. However, the relationship between gelatinases and BMP-2 in vascular calcification is scarcely investigated with a latest report showing that MMP-2 deficiency reduces BMP-2 expression in aortae of ApoE-deficient mice [9]. In the present study, we investigated the mechanism by which gelatinases are involved in vascular calcification using SB-3CT, a specific gelatinases inhibitor [13]. We report that gelatinases contribute to vascular calcification by a way independent of their ability to degrade elastin, i.e. gelatinases up-regulate expression of BMP-2 and thereby increase expressions of Runx2 and Msx-2 which are stimulations for osteoblastic differentiation of VSMCs. 2. Materials and methods 2.1. Animals Male Wistar rats (8 weeks) weighing 180e200 g with fasting and free access to water were used in this study. All experimental procedures involving use and care of animals conformed to guidelines approved by Experimental Animal Committee of Institute of Microcirculation, Chinese Academy of Medical Sciences & Peking Union Medical College. 2.2. VSMCs culture and in vitro calcification The explant culture method of VSMCs was used as described previously [14]. VSMCs of 5th to 8th passages were used for the experiments. All procedures were conducted in accordance with protocol guidelines approved by Experimental Animal Committee of Institute of Microcirculation, Chinese Academy of Medical Sciences & Peking Union Medical College. b-GP was used to induce in vitro calcification of VSMCs [15]. After confluence, the cells were inoculated in 24-well plates (1 104 cells/mL) and divided into 3 groups. The control group was treated with growing medium (DMEM containing 15% FBS) while bGP group was treated with calcification medium (DMEM containing 15% FBS in the presence of 10 mM b-GP). In the SB-3CT group, 10 mM SB-3CT was added to the calcification medium. The medium was replaced every 3 days. After 12 days, the medium was collected and the cell layer was washed with phosphate-buffered saline (PBS).
Cells were stored at
70 C until use.
2.3. Von Kossa staining Cells were fixed in 4% paraformaldehyde and rinsed with PBS twice. Then cells were exposed to 5% silver nitrate under a 75-W lamp for 30 min, then to 5% sodium thiosulfate for 3 min to remove unreduced silver. After rinsing, stained cells were examined by light microscope and photographed for black areas indicating deposited mineral. Black areas were analyzed colorimetrically using the Image J 1.48 software (National Institutes of Health (NIH), Bethesda, MD). 2.4. Measurement of calcium content in VSMCs Cellular calcium was extracted with 0.6 mM HCl overnight. After removing the HCl supernatant, the remaining cell layers on the plates were treated with lysis buffer containing 0.1 M NaOH and 0.1% SDS. Calcium content in the HCl supernatant was determined by the O-cresolphthalein complexone method. Calcium content was normalized to total protein content of the same well. 2.5. Measurement of alkaline phosphatase (ALP) activity in VSMCs Cells were rinsed three times with PBS and sonicated in 100 mL of lysis buffer. After centrifugation at 10,000 g for 10 min at 4 C, ALP activity in the supernatant was measured with the ALP assay kit (Beyotime Institute of Biotechnology). ALP activity was calculated using p-nitrophenol as a standard. One unit was defined as the activity producing l nM of p-nitrophenol in 30 min. ALP values were normalized to protein content. 2.6. Western blot analysis Western blot analysis was performed as follows. Briefly, samples were prepared and run on a 10% sodium dodecyl sulfateepolyacrilamide gel electrophoresis (SDS-PAGE) before being transferred onto polyvinylidene fluoride (PVDF) membranes (BioRad, Hercules, CA, USA). The membranes were washed with TBST, blocked with 5% nonfat dry milk in TBST for 1 h at room temperature and then incubated overnight at 4 C with primary antibodies. The membranes were then incubated for 1 h at room temperature with appropriate secondary antibodies. Chemiluminescent reagents (Beyotime Institute of Biotechnology) were used to detect protein bands. Quantification of bands was carried out by densitometry using Image J 1.48 software and the protein values were normalized with b-actin. 2.7. Gelatin zymography The conditioned medium was collected and centrifuged and the supernatant was analyzed for gelatin zymography of MMP-2 and MMP-9 [16]. Briefly, twenty micrograms of protein from each sample was subjected to electrophoresis on 8% SDS-PAGE using gelatin (0.1%) as the substrate. After electrophoresis, the gel was washed twice and gently shaken in 2.5% Triton X-100 for 30 min. The gel was then incubated overnight at 37 C in a 50 mM TriseHCl buffer, containing 5 mM CaCl2 and 0.2 M NaCl. The gel was stained with Coomassie brilliant blue R for 1 h. Unstained bands indicating gelatinolytic activity were seen after slight destaining with 30% ethanol and 10% acetic acid. Band intensity was quantitatively assessed using Image J 1.48.
Please cite this article in press as: Y.-G. Zhao, et al., Gelatinases promote calcification of vascular smooth muscle cells by up-regulating bone morphogenetic protein-2, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.01.067
Y.-G. Zhao et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e7
2.8. Statistical analysis All experiments were performed in triplicate. All data were expressed as mean ± S.D. One-way ANOVA, followed by StudenteNewmaneKeuls test for significance was carried out among the groups. The threshold for significance was P < 0.05.
3. Results 3.1. Gelatinases inhibition alleviated calcification of VSMCs Calcification area (Fig. 1A and B) and calcium content (Fig. 1C) in
b-GP group were significantly higher than that of control group. Gelatinases inhibition by SB-3CT caused reductions in calcification area (Fig. 1A and B) and calcium content (Fig. 1C) compared with bGP group. Вeta-GP group had an increased expression of both MMP-2 and MMP-9 compared with control group, and SB-3CT decreased expressions of MMP-2 and MMP-9 (Fig. 2A). The levels of pro-MMP-9 and pro-MMP-2 were almost the same among the three groups, and no active MMP-9 was detected by gelatin zymography (Fig. 2B). However, the level of active MMP-2 in b-GP group was higher than that of control group, and SB-3CT decreased it (Fig. 2B).
3.2. Effects of gelatinases inhibition on the expressions of tissue inhibitor of metalloproteinase-2 (TIMP-2), tissue inhibitor of metalloproteinase-1 (TIMP-1), MMP-2/TIMP-2 ratio and MMP-9/ TIMP-1 ratio Вeta-GP group had an increased expression of TIMP-1 compared with the control group, and SB-3CT decreased its expression (Fig. 2C). Compared with the control group, b-GP group had an increased MMP-2/TIMP-2 ratio, which was decreased by SB-3CT (Fig. 2D). In addition, there were no significant differences in the expression of TIMP-2 and MMP-9/TIMP-1 ratio among the three groups (Fig. 2C and D).
3
3.3. Gelatinases inhibition suppressed phenotype transition of VSMCs and expressions of RUNX2 and Msx-2 in VSMCs The present study found increased expression of ALP and decreased expressions of smooth muscle a-actin (SM a-actin) and desmin in b-GP group, demonstrating a transition of VSMCs phenotype into calcifying phenotype which was attenuated by SB3CT (Fig. 3AeD). Additionally, ALP activity in the b-GP group was significantly higher than that of control group, demonstrating an activity of calcium deposit which was inhibited by SB-3CT (Fig. 3E). Increased expressions of RUNX2 and Msx-2 were found in b-GP group compared with that of control group, and this increase was inhibited by SB-3CT (Fig. 3F and H). 3.4. Gelatinases inhibition suppressed BMP-2 expression BMP-2 expression in b-GP group was increased compared with that of control group, and gelatinases inhibition decreased its level (Fig. 3I). 3.5. Gelatinases inhibition decreased expressions of RUNX2 and Msx-2 via suppressing BMP-2 The group treated with BMP-2 or noggin was nominated as BMP-2 group or noggin group, while the group treated with neither BMP-2 nor noggin was nominated as control group. BMP-2 group had increased expressions of RUNX2 and Msx-2 compared with control group, while noggin decreased their expressions compared with control group (Fig. 4). 4. Discussion Medial calcification is characterized by phenotype transition of VSMCs which can be mediated by the composition of the ECM. In view of the important role of ECM alteration in phenotype transition of VSMCs, it is reasonable to hypothesize that gelatinases, critical enzymes in ECM degradation, may be involved in vascular calcification. In the present study, b-GP was used to induce in vitro calcification of VSMCs. And SB-3CT was used to inhibit MMP-2 and
Fig. 1. Gelatinases inhibition suppressed calcification induced by b-GP in cultured VSMCs. VSMCs were treated with b-GP in the absence or presence of SB-3CT for 12 days. Von kossa stainging was made to show black areas indicating deposited mineral, and the black areas were analyzed colorimetrically. Calcium contents were normalized to total protein content of the same well. A, Von kossa stainging of VSMCs at day 12. B, Colorimetric measurement of calcification area for von kossa staining. ***P < 0.001 vs control group, ### P < 0.001 vs b-GP group. C, Calcium content of VSMCs at day 12. ***P < 0.001 vs control group, ###P < 0.001 vs b-GP group. b-GP: beta-glycerophosphate; SB-3CT: 2-[[(4Phenoxyphenyl)sulfonyl]methyl]-thiirane.
Please cite this article in press as: Y.-G. Zhao, et al., Gelatinases promote calcification of vascular smooth muscle cells by up-regulating bone morphogenetic protein-2, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.01.067
4
Y.-G. Zhao et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e7
Fig. 2. Effects of SB-3CT on expressions of MMP-2, MMP-9, TIMP-2 and TIMP-1 and activity of MMP-2 and MMP-9 in VSMCs. VSMCs were treated with b-GP in the absence or presence of SB-3CT for 12 days. The conditioned medium was collected and analyzed for expressions of MMP-2, MMP-9, TIMP-2 and TIMP-1 and activity of MMP-2 and MMP-9. A, Expressions of MMP-2 and MMP-9 in the conditioned medium of VSMCs at day 12. **P < 0.01 vs control group, ##P < 0.01 vs b-GP group. B, Gelatin zymography of conditioned medium of VSMCs and comparison of active MMP-2 at day 12. *P < 0.05 vs control group, #P < 0.05 vs b-GP group. C, Expressions of TIMP-2 and TIMP-1 in the conditioned medium of VSMCs at day 12. **P < 0.01 vs control group, ##P < 0.01 vs b-GP group. D, MMP-2/TIMP-2 ratio and MMP-9/TIMP-1 ratio in each group. **P < 0.01 vs control group, ##P < 0.01 vs b-GP group. b-GP: beta-glycerophosphate; SB-3CT: 2-[[(4-Phenoxyphenyl)sulfonyl]methyl]-thiirane; MMP-2: matrix metalloproteinase-2; MMP-9: matrix metalloproteinase-9; TIMP-2: tissue inhibitors of metalloproteinase-2; TIMP-1: tissue inhibitors of metalloproteinase-1.
Please cite this article in press as: Y.-G. Zhao, et al., Gelatinases promote calcification of vascular smooth muscle cells by up-regulating bone morphogenetic protein-2, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.01.067
Y.-G. Zhao et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e7
5
Fig. 3. Effects of gelatinases inhibition on phenotype transition of VSMCs and expressions of RUNX2, Msx-2 and BMP-2. VSMCs were treated with b-GP in the absence or presence of SB-3CT for 12 days. Expressions of ALP, SM a-actin, desmin, RUNX2, Msx-2 and BMP-2 and ALP activity were analyzed. A, VSMCs transition into calcifying phenotype in the b-GP group was characterized by increased expression of ALP and decreased expressions of SM a-actin and desmin, and gelatinases inhibition by SB-3CT attenuated this process. B-D, Relative expressions of ALP, SM a-actin and desmin, respectively. E, Effect of gelatinases inhibition on ALP activity. **P < 0.01, ***P < 0.001 vs control group, #P < 0.05, ###P < 0.001 vs b-GP group. FeI, Gelatinases inhibition by SB-3CT suppressed expressions of RUNX2, Msx-2 and BMP-2. **P < 0.01, ***P < 0.001 vs control group; #P < 0.05, ##P < 0.01 vs b-GP group. b-GP: beta-glycerophosphate; SB-3CT: 2-[[(4-Phenoxyphenyl)sulfonyl]methyl]-thiirane; ALP: alkaline phosphatase; SM a-actin: smooth muscle a-actin; RUNX2: Runt-related transcription factor 2; Msx-2: msh homeobox homolog 2; BMP-2: bone morphogenetic protein-2.
MMP-9. The calcification area in b-GP group was 7.4-fold of the control group, while SB-3CT inhibited the increase in calcification area induced by b-GP. And gelatinases inhibition attenuated the increase of VSMCs calcium content induced by b-GP. These results indicate gelatinases promote VSMCs calcification. The b-GP group had increased gelatinases expression and active MMP-2, which were reduced by SB-3CT, suggesting up-regulation and activation of gelatinases are involved in pathogenesis of VSMCs calcification. These results confirmed findings of previous reports which found enhanced MMP-2 activity and increased MMP-2 expression in calcified aorta [5,6]. As tissue inhibitors of metalloproteinases (TIMPs) are endogenous regulator of MMPs, we detected the expressions of TIMP-2 and TIMP-1, and calculated the MMP-2/TIMP-2 ratio and MMP-9/TIMP-1 ratio. The b-GP group had an increased MMP-2/TIMP-2 ratio, which was decreased by SB-3CT. In addition, there were no significant differences in the expression of TIMP-2 and MMP-9/TIMP-1 ratio among the three groups. Therefore, it may be postulated that the one gelatinase exerting more effects may be MMP-2 rather than MMP-9. However, verification is needed. Involvement of gelatinases in vascular calcification is further supported by the observations that mice deficient of MMP2 alone or in combination with MMP-9 had lesser or no vascular calcification [8,9]. However, it has not been answered how osteogenic stimulation induces MMP-2. It may be explained in two aspects. Firstly, it is due to the alternation of ECM. As an ostoegenic
stimulator, b-GP is degraded by ALP, raising the local concentration of phosphorus [15]. High phosphorus levels causes accumulation of ECM [17], and this alternation of ECM activates MMP-2. Secondly, bGP treatment caused an increase in MMP-2/TIMP-2 ratio in the present study, which is an activator of MMP-2. Consistent with the occurrence of calcification, we found phenotype transition of VSMCs as well as increased expressions of RUNX2 and Msx-2 in b-GP group which were attenuated by gelatinases inhibition. BMP-2, a crucial factor in bone formation, is also involved in vascular calcification. BMP-2 was expressed in human atherosclerotic lesions [18]. Treatment of human VSMCs with BMP-2 increases calcification in vitro [12]. BMP-2 induces expressions of RUNX2 and Msx-2 in VSMCs [19]. RUNX2 is crucial for phenotype transition of SMCs [20], while Msx-2 is critical for osteoblast differentiation and bone formation [21]. However, the relationship between gelatinases and BMP-2 in vascular calcification is scarcely investigated with a latest report showing that MMP-2 deficiency reduces BMP-2 expression in aortae of ApoE-deficient mice [9]. We, therefore, hypothesize that gelatinases promotes BMP-2 expression to induce expressions of RUNX2 and Msx-2 which are crucial for phenotype transition of VSMCs. To investigate the relationship between gelatinases and BMP-2 in VSMCs calcification, the expressions of BMP-2 were analyzed 12 days after treatment with bGP and SB-3CT. The results showed that b-GP increased expressions
Please cite this article in press as: Y.-G. Zhao, et al., Gelatinases promote calcification of vascular smooth muscle cells by up-regulating bone morphogenetic protein-2, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.01.067
6
Y.-G. Zhao et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e7
Fig. 4. Effects of BMP-2 and noggin on expressions of RUNX2 and Msx-2 in VSMCs. Cells were treated with BMP-2 (100 ng/mL) or noggin (100 ng/mL) for 4 h and incubated with calcification medium in the presence of SB-3CT for 24 h, and then expressions of RUNX2 and Msx-2 were analyzed by western blot. The group treated with BMP-2 or noggin was nominated as BMP-2 group or noggin group, while the group treated with neither BMP-2 nor noggin was nominated as control group. *P < 0.05, **P < 0.01 vs control group. b-GP: beta-glycerophosphate; SB-3CT: 2-[[(4-Phenoxyphenyl)sulfonyl]methyl]-thiirane; BMP-2: bone morphogenetic protein-2; RUNX2: Runt-related transcription factor 2; Msx-2: msh homeobox homolog 2.
of BMP-2, whereas gelatinases inhibition decreased it. These results demonstrated gelatinases can regulate expression of BMP-2. The above-mentioned results showed gelatinases inhibition decreased expressions of RUNX2 and Msx-2 and down-regulated BMP-2. And previous study showed BMP-2 induces expressions of RUNX2 and Msx-2 [19]. To clarify whether the decreases in expressions of RUNX2 and Msx-2 in SB-3CT group were dependent on BMP-2 down-regulation induced by gelatinases inhibition, cells were treated with BMP-2 (100 ng/mL) or noggin (100 ng/mL) for 4 h and incubated with calcification medium in the presence of SB-3CT for 24 h, and then expressions of RUNX2 and Msx-2 were analyzed by western blot. Noggin, a BMP antagonist, binds to BMP-2 with high affinity and blocks its interaction with BMP-2 receptors, thereby inhibiting BMP signaling [22,23]. Our results demonstrated BMP-2 treatment increased expressions of RUNX2 and Msx-2 in VSMCs, while noggin decreased their expressions, demonstrating the decrease in expressions of RUNX2 and Msx-2 in VSMCs were dependent on BMP-2 down-regulation induced by gelatinases inhibition. However, more researches are needed to figure out which gelatinase is more important in the initiation and progression of vascular calcification, and target inhibition of this gelatinase may alleviate the deleterious effects of vascular calcification more effectively. The present study find gelatinases contribute to vascular calcification by a new way independent of their property of degrading ECM, i.e. gelatinases promote VSMCs calcification by up-regulating BMP-2 which induces expressions of RUNX2 and Msx-2, two proteins associated with phenotype transition of VSMCs. Therefore, interventions targeting gelatinases may be a proper candidate for ameliorating the devastating cardiovascular consequences of medial artery calcification in diabetes and end-stage renal disease.
Competing interests The authors declare that they have no competing interests.
Authors' contributions YZ and FM carried out the design of the study, performed the experiments, analyzed the data, and wrote the manuscript. BW, BL, ML, YS participated in the experimental procedures. HL contributed to reagents and tools. RX participated in experimental instructions, manuscript revision and coordination. All authors read and approved the final manuscript. Authors' information Institute of Microcirculation and key laboratory, Ministry of Health, China; Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing, 100005, China Acknowledgments This study was funded by the Postgraduate Innovation Fund of Peking Union Medical Colloge (2013-1001-05). References rin, S.J. Marchais, F. Me tivier, B. Pannier, H. Adda, [1] G.M. London, A.P. Gue Arterial media calcification in end-stage renal disease: impact on all-cause and cardiovascular mortality, Nephrol. Dial. Transpl. 18 (2003) 1731e1740. [2] R. Vattikuti, D.A. Towler, Osteogenic regulation of vascular calcification: an early perspective, Am. J. Physiol. Endocrinol. Metab. 286 (2004) E686eE696. [3] R.J. Rennenberg, L.J. Schurgers, A.A. Kroon, C.D. Stehouwer, Arterial calcifications, J. Cell Mol. Med. 14 (2010) 2203e2210. [4] A. Simionescu, K. Philips, N. Vyavahare, Elastin-derived peptides and TGFbeta1 induce osteogenic responses in smooth muscle cells, Biochem. Biophys. Res. Commun. 334 (2005) 524e532. [5] B. Jian, P.L. Jones, Q. Li, E.R. Mohler, F.J. Schoen, R.J. Levy, Matrix metalloproteinase-2 is associated with tenascin-C in calcific aortic stenosis, Am. J. Pathol. 159 (2001) 321e327. [6] N.X. Chen, K.D. O'Neill, X. Chen, K. Kiattisunthorn, V.H. Gattone, S.M. Moe, Activation of arterial matrix metalloproteinases leads to vascular calcification in chronic kidney disease, Am. J. Nephrol. 34 (2011) 211e219. [7] X. Qin, M.A. Corriere, L.M. Matrisian, R.J. Guzman, Matrix metalloproteinase inhibition attenuates aortic calcification, Arterioscler. Thromb. Vasc. Biol. 26
Please cite this article in press as: Y.-G. Zhao, et al., Gelatinases promote calcification of vascular smooth muscle cells by up-regulating bone morphogenetic protein-2, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.01.067
Y.-G. Zhao et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e7 (2006) 1510e1516. [8] D.M. Basalyga, D.T. Simionescu, W. Xiong, B.T. Baxter, B.C. Starcher, N.R. Vyavahare, Elastin degradation and calcification in an abdominal aorta injury model: role of matrix metalloproteinases, Circulation 110 (2004) 3480e3487. [9] T. Sasaki, K. Nakamura, K. Sasada, et al., Matrix metalloproteinase-2 deficiency impairs aortic atherosclerotic calcification in ApoE-deficient mice, Atherosclerosis 227 (2013) 43e50. [10] C. Kumata, M. Mizobuchi, H. Ogata, et al., Involvement of matrix metalloproteinase-2 in the development of medial layer vascular calcification in uremic rats, Ther. Apher. Dial. 15 (2011) 18e22. [11] S.L. Cheng, J.S. Shao, N. Charlton-Kachigian, A.P. Loewy, D.A. Towler, MSX2 promotes osteogenesis and suppresses adipogenic differentiation of multipotent mesenchymal progenitors, J. Biol. Chem. 278 (2003) 45969e45977. [12] X. Li, H.Y. Yang, C.M. Giachelli, BMP-2 promotes phosphate uptake, phenotypic modulation, and calcification of human vascular smooth muscle cells, Atherosclerosis 199 (2008) 271e277. [13] P. Tao, J.F. Fisher, S. Mobashery, H.B. Schlegel, DFT studies of the ring-opening mechanism of SB-3CT, a potent inhibitor of matrix metalloproteinase 2, Org. Lett. 11 (2009) 2559e2562. [14] S.Y. Wu, B.H. Zhang, C.S. Pan, et al., Endothelin-1 is a potent regulator in vivo in vascular calcification and in vitro in calcification of vascular smooth muscle cells, Peptides 24 (2003) 1149e1156. [15] A. Shioi, Y. Nishizawa, S. Jono, H. Koyama, M. Hosoi, H. Morii, Beta-glycerophosphate accelerates calcification in cultured bovine vascular smooth
7
muscle cells, Arterioscler. Thromb. Vasc. Biol. 15 (1995) 2003e2009. [16] S.A. Silva, M.G. Gobbo, M.E. Pinto-Fochi, et al., Prostate hyperplasia caused by long-term obesity is characterized by high deposition of extracellular matrix and increased content of MMP-9 and VEGF, Int. J. Exp. Pathol. 96 (2015) 21e30. [17] A. Papadimitriou, E.B. Peixoto, K.C. Silva, J.M. Lopes de Faria, J.B. Lopes de Faria, Inactivation of AMPK mediates high phosphate-induced extracellular matrix accumulation via NOX4/TGFß-1 signaling in human mesangial cells, Cell Physiol. Biochem. 34 (2014) 1260e1272. €m, K.E. Watson, S. Horn, C. Wortham, I.M. Herman, L.L. Demer, Bone [18] K. Bostro morphogenetic protein expression in human atherosclerotic lesions, J. Clin. Invest. 91 (1993) 1800e1809. [19] C. Sirard, S. Kim, C. Mirtsos, et al., Targeted disruption in murine cells reveals variable requirement for Smad4 in transforming growth factor beta-related signaling, J. Biol. Chem. 275 (2000) 2063e2070. [20] J.A. Balderman, H.Y. Lee, C.E. Mahoney, et al., Bone morphogenetic protein-2 decreases microRNA-30b and microRNA-30c to promote vascular smooth muscle cell calcification, J. Am. Heart Assoc. 1 (2012) e003905. [21] I. Satokata, L. Ma, H. Ohshima, et al., Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation, Nat. Genet. 24 (2000) 391e395. nchez-Duffhues, P. ten Dijke, BMP signaling in vascular [22] J. Cai, E. Pardali, G. Sa diseases, FEBS Lett. 586 (2012) 1993e2002. [23] J.W. Lowery, M.P. de Caestecker, BMP signaling in vascular development and disease, Cytokine Growth Factor Rev. 21 (2010) 287e298.
Please cite this article in press as: Y.-G. Zhao, et al., Gelatinases promote calcification of vascular smooth muscle cells by up-regulating bone morphogenetic protein-2, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.01.067
