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Cartilage oligomeric matrix protein prevents vascular aging and vascular smooth muscle cells senescence
Biochemical and Biophysical Research Communications xxx (2016) 1e8
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Cartilage oligomeric matrix protein prevents vascular aging and vascular smooth muscle cells senescence Meili Wang a, b, Yi Fu a, b, Cheng Gao a, b, Yiting Jia a, b, Yaqian Huang c, Limei Liu a, Xian Wang a, b, Wengong Wang d, Wei Kong a, b, * a
Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing, China Department of Pediatrics, Peking University First Hospital, Beijing, China d Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 July 2016 Accepted 2 August 2016 Available online xxx
Aging-related vascular dysfunction contributes to cardiovascular morbidity and mortality. Cartilage oligomeric matrix protein (COMP), a vascular extracellular matrix protein, has been described as a negative regulatory factor for the vascular aging-related processes including atherosclerosis and vascular calcification. However, whether COMP is implicated in the process of vascular aging remains unclear. Here, we identified a novel function of COMP in preventing vascular aging and vascular smooth muscle cells (VSMCs) senescence. Firstly, vascular COMP expression was decreased in three different senescenceaccelerated mouse models and was also declining with age. COMP/ mice displayed elevated senescence-associated markers expression, including p53, p21 and p16, in the aortas compared with their wild type (WT) littermates. In accordance, COMP deficiency induced aging-related vascular dysfunction as evidenced by the significantly reduced phenylephrine-induced contraction and increased vascular stiffness as evaluated by pulse wave velocity. The aortic wall of COMP/ mice was susceptible to senescence by displaying senescence-associated b-galactosidase (SA b-gal) activity induced by periadventitial application of CaCl2 to the abdominal aorta. In vitro, COMP knockdown by small interfering (si) RNA led to the elevation of p53, p21 and p16 as well as SA b-gal activity in VSMCs after H2O2 stimulation. VSMCs isolated from COMP/ mice showed elevated senescence-associated markers expression and supplement of COMP adenovirus to COMP-deficient VSMCs greatly rescued cellular senescence. Taken together, these findings revealed the essential role of COMP in retarding the development of vascular aging and VSMC senescence. © 2016 Elsevier Inc. All rights reserved.
Keywords: Vascular extracellular matrix protein COMP Vascular aging VSMC senescence
1. Introduction Vascular aging is a major risk factor for a variety of cardiovascular diseases including atherosclerosis, vascular calcification and hypertension, etc [1]. Aging vasculature exhibits the reduced arterial compliance and increased stiffness, as well as the impaired function of contraction as a result of senescence-related arterial alterations in cells, matrix, and biomolecules [1]. The occurrence of cellular senescence, particularly in vascular smooth muscle cells (VSMCs) upon stress, has been demonstrated in human
* Corresponding author. Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University Health Science Center, 38 Xueyuan Road, Haidian District, Beijing, 100191, China. E-mail address: [email protected] (W. Kong).
atherosclerotic, calcifying and hypertensive arteries [2], as evidenced by the positive staining of senescence-associated b-galactosidase (SA b-gal) and the expression of senescence-related signaling hallmarks, such as high levels of p53, p21, p16 and phosphorylated p38. Consequently, VSMC senescence promotes pathogenesis of atherosclerosis, vascular calcification and other aging-related vascular diseases [3]. Multiple triggers have been identified nowadays to induce VSMC senescence, such as DNA damage, telomere shortening, oxidative stress, oncogene activation, the loss of tumor suppressors, epigenetic stress, angiotensin II and mitochondrial dysfunction [2,4]. However, the mechanism by which VSMCs counteract the multiple triggers of senescence and maintain cellular homeostasis following environmental stimuli remains less understood. An investigation of the endogenous modulators of vascular aging is favorable for the prevention and
http://dx.doi.org/10.1016/j.bbrc.2016.08.004 0006-291X/© 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: M. Wang, et al., Cartilage oligomeric matrix protein prevents vascular aging and vascular smooth muscle cells senescence, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.004
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M. Wang et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e8
treatment of vascular aging and aging-related vascular diseases. The extracellular matrix (ECM) constitutes an active and dynamic microenvironment for VSMCs and plays a fundamental role in the regulation of vascular function in normal and pathophysiological conditions. The collagen content increases, whereas elastin decreases with age, giving rise to a corresponding elastin fragmentation and progressive medial degeneration, aortic stiffening and aging-related reduction of vascular complications [5]. Nevertheless, a limited number of reports have recognized the effects of proteoglycans and glycoproteins, which constitute more than half of the human aortic ECM as demonstrated by recent proteomics analysis, on vascular aging [6e8]. Cartilage oligomeric matrix protein (COMP), a 524 kDa pentameric noncollagenous glycoprotein, is a matricellular protein found in both the musculoskeletal and cardiovascular systems. Our recent studies have demonstrated that COMP plays critical roles in the maintenance of vascular homeostasis. COMP maintains the contractile phenotype of VSMCs via integrin a7b1 and prevents osteochondrogenic transdifferentiation of VSMCs by directly binding to BMP-2, which inhibits post injury neointima formation and vascular calcification [9,10]. We recently also demonstrated that COMP negatively regulates atherosclerosis and lesional calcification formation via direct interaction with integrin b3 [11]. As COMP is involved in aging-related vascular diseases including atherosclerosis and vascular calcification, we asked whether COMP is implicated in vascular aging. 2. Materials and methods 2.1. Reagents Antibodies against COMP, p53, p21, p16 and b-actin were purchased from Abcam (Cambridge, UK). IRDye-conjugated secondary antibodies for western blotting were purchased from Rockland, Inc. (Gilbertsville, PA, USA). Other reagents were obtained from SigmaAldrich (St. Louis, MO, USA) unless specified.
euthanasia. The dissected vessels were immediately placed in Krebs-Henseleit buffer, and then cleaned of additional connective tissues. The aorta was sectioned into 1.5e1.8 mm rings and subsequently subjected to vascular tension experiments. The cumulative dose responses to phenylephrine were obtained to characterize vasocontraction. Data are expressed as percentages to KCl-induced contraction. 2.5. Measurement of pulse wave velocity The in vivo pulse wave velocity (PWV) was measured using an ECG-triggered 10-MHz Doppler probe [16]. The animals were anesthetized with isoflurane and maintained during the measurement by mask ventilation of 1.5% isoflurane with a coupled charcoal scavenging system. The animals were positioned supine with their limbs taped to electrocardiogram electrodes, which were incorporated into a temperature-controlled printed circuit board. The aortic arch blood flow and the abdominal aorta blood flow were captured, and the separation distance between them was measured. The PWV was calculated as a quotient of the separation distance and time difference between pulse arrivals, as measured from the ECG R-peaks. 2.6. CaCl2-induced mouse abdominal aortic calcification Mice were anesthetized with isoflurane and the infrarenal abdominal aortas were treated with periadventitial application of 0.2 mol/L CaCl2 for 15 min as described previously [10]. Control mice were treated with 0.2 mol/L NaCl. After 7 days, the abdominal aorta was harvested and sectioned for SA b-gal staining. Analysis of calcium content and von Kossa staining were used to assess the efficiency of the model as described previously [10]. 2.7. Senescence-associated b-galactosidase (SA b-gal) staining
COMP/ mice with a C57BL/6J background were kindly pro€ Oldberg (Department of Cell and Molecular vided by Professor Ake Biology, University of Lund, Sweden) [12]. Aortas of Zmpste24/ mice and their WT littermates were provided by Professor Zhongjun Zhou (Department of Biochemistry, University of Hong Kong, Hong Kong) [13]. ApoE/, SAM-R1 and SAM-P8 mice were purchased from the Department of Laboratory Animal Science, Peking University Health Science Center. All animal studies followed the guidelines of the Animal Care and Use Committee of Peking University.
SA b-gal staining was performed using a Senescence Detection Kit (Medical & Biological Laboratories CO., LTD). For the abdominal aortas, mice were perfused via the left ventricle with phosphatebuffered saline (PBS). Aortas were harvested and incubated in fixative solution for 1 h and in SA b-gal staining solution (prepared according to the manufacture's protocol) at 37 C for 16 h and then cryosectioned at 10 mm. SA b-gal activity was identified based on positively stained blue cells. Cells were washed with PBS and fixed with fixative solution for 15 min at room temperature. Then cells were incubated with staining solution at 37 C for 16 h. Senescent cells were bluestained as observed under light microcopy. Cells were counterstained with Hoechst 33342 to count the total number.
2.3. Western blotting
2.8. Cell culture
Western blot was performed as previously described [14]. Cells and mouse tissue extracts that contained equal amounts of total protein were resolved by 10% or 15% SDS-PAGE and subsequently transferred onto nitrocellulose membranes. The membranes were blocked with 5% milk in TBST, followed by incubation with primary antibody at 4 C overnight. Following 1 h of incubation with IRDyeconjugated secondary antibodies, the membranes were subjected to an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA) to detect the immunofluorescence signal.
The rat smooth muscle embryonic thoracic aorta cell line A7r5 was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum at 37 C in a humidified atmosphere containing 5% CO2. H2O2 treatment was performed by adding 10 mM H2O2 for 2 days. Primary mouse thoracic VSMCs were isolated by collagenase digestion as described previously [17]. Briefly, following mouse euthanasia, thoracic aortas were harvested and cut into small pieces. To remove the endothelial cells, these pieces were digested with 1 mg/ml trypsin (Hyclone) at 37 C for 10 min. After centrifugation, the precipitate was resuspended with 10 mg/ml collagenase type I (Gibco) for 6e8 h. Gelatin was pre-laid onto the culture dishes before the cells were seeded. The cell cultures contained >95%
2.2. Animals
2.4. Aortic ring myograph A myograph was performed as previously described [15]. Briefly, the aortic rings were isolated and removed from mice following
Please cite this article in press as: M. Wang, et al., Cartilage oligomeric matrix protein prevents vascular aging and vascular smooth muscle cells senescence, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.004
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Fig. 1. Protein level of COMP was decreased in aortas of aging mice models. Representative western blot analysis and quantification of the protein level of COMP in the aortas from Zmpste24/ mice and their WT littermates (A), ApoE/ mice and their WT littermates (B), SAM-R1 and SAM-P8 mice (C). Data are presented as mean ± SEM. *P < 0.05. (D) Representative western blot analysis and quantification of the protein level of COMP in the aortas from different aged C57BL mice. Error bars represent SEM. n ¼ 3 for each group, *P < 0.05.
VSMCs as determined by SM22 staining. The cells were cultured in complete DMEM that contained 10% fetal bovine serum (FBS, Hyclone) and were passaged by 0.125% trypsin digestion. The cells of passages 3e8 were used for experiments.
2.9. Transfection of small interfering RNA Small interfering RNA (siRNA) was purchased from GenePharma Co., Ltd (Shanghai). The sequences that corresponded to the siRNA of rat COMP were sense, 50 -AGAAACUUGAGCUGUGUUGAUGCC-30 , and anti-sense, 50 -GGCUAUCAAGACAGCUCAAGUUUCU-30 . A scramble stealth RNAi duplex served as a negative control. In vitro siRNA transfection (50 nM) of rat VSMCs was performed using RNAi MAX (Invitrogen, CA, USA). The transfection procedures followed the manufacturers' instructions.
2.10. Recombinant adenovirus construction and infection The adenovirus for full-length mouse COMP (Ad-COMP; NM_016685.2) was constructed and amplified according to the manufacturer's protocol (BD Biosciences). An adenovirus that carried b-galactosidase (Ad-LacZ) was used as a negative control. Cells cultured at approximately 80% confluence were infected with recombinant adenovirus (50 multiplicity of infection) for 48 h. 2.11. Statistical analysis Data are expressed as the mean ± SEM. Comparisons of the aortic gene expression and PWV between two genotypic mice were analyzed using unpaired two-tailed Student's t-tests. Comparisons of the aortic contraction between the WT and COMP/ mice were analyzed via two-way ANOVA followed by the Bonferroni test. The
Please cite this article in press as: M. Wang, et al., Cartilage oligomeric matrix protein prevents vascular aging and vascular smooth muscle cells senescence, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.004
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Fig. 2. COMP¡/¡ mice exhibited aging-related vascular dysfunction. (A) Representative western blot analysis and quantification of the protein levels of p53, p21 and p16 in the aortas from WT and COMP/- mice. n ¼ 3, *P < 0.05. (B) Contraction in response to the vasoconstrictor phenylephrine of isolated aortic rings from WT and COMP/ mice. Error bars represent SEM. n ¼ 5 for each group, *P < 0.05. (C) Pulse wave velocity measurements of the aortas from WT and COMP/ mice. Error bars represent SEM. n ¼ 5 for each group, *P < 0.05.
comparisons of the protein expression, calcium deposition or the percentages of SA b-gal-positive cells among more than 2 groups were analyzed using one-way ANOVA followed by the StudentNewman-Keuls test for post hoc comparison. In all cases, statistical significance was indicated when the two-tailed probability was less than 0.05.
3. Results 3.1. The expression of aortic COMP was inversely correlated with aging process Considering the protective role of COMP against cardiovascular diseases and to examine the involvement of COMP in vascular
aging, we measured the COMP expression in aortas obtained from three aging-accelerated rodent models, including Zmpste24/ mice, ApoE/ mice and senescence accelerated mice/prone substrain 8 (SAM-P8). Deletion of the Zmpste24 gene, which encodes the prelamin A processing enzyme, has been demonstrated to lead to an abnormal accumulation of prelamin A and thus a deficiency of lamin A, which plays an important role in the maintenance of chromatin organization and thus induces premature-aging phenotypes in mice [18]. Of interest, COMP was greatly reduced in the aortas of 5-month-old Zmpste24/ mice as evidenced by western blot analysis (Fig. 1A). Apolipoprotein E (ApoE) has been the most replicated longevity-associated gene, and ApoE/ mice exhibit a shorter life-span and many features of premature-aging [19,20]. A similar decrease of COMP protein was observed in aortas of 10-
Please cite this article in press as: M. Wang, et al., Cartilage oligomeric matrix protein prevents vascular aging and vascular smooth muscle cells senescence, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.004
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Fig. 3. Aortas of COMP¡/¡ mice were more susceptible to senescence. (A) Quantitative analysis of calcium deposition of abdominal aortas at 0 days, 7 days or 14 days after 0.2 mol/L NaCl or CaCl2 treatment. n ¼ 3 for each group, *P < 0.05. (B) Representative von Kossa staining of abdominal aortas at 14 days after 0.2 mol/L NaCl or CaCl2 treatment. Scale bar ¼ 100 mm. (C) Light micrographs of the abdominal aortas of 18-month-old WT and COMP/ mice, harvested after 7 days of perivascular application of 0.2 mol/L NaCl or CaCl2 to the infrarenal abdominal aortas and stained for SA b-gal activity. Scale bar ¼ 20 mm.
month-old ApoE/ mice (Fig. 1B). As while, SAM-P8 mice have been demonstrated to exhibit vascular dysfunction during aging [21]. Senescence accelerated mouse/resistance substrain 1 (SAMR1) was used as a control group. In accordance, a repressed COMP expression level was identified in the aortas of 6-month-old SAMP8 mice compared with their littermates (Fig. 1C). Furthermore, we compared the aortic COMP expression at young (2-month, 5month), middle-aged (12-month) and aged (20-month old) normal C57BL/6J mice, respectively. As shown in Fig. 1D, a gradual decline in the COMP level with aging was identified. Together, these findings indicated that COMP may be inversely correlated with vascular aging process. 3.2. COMP/ mice exhibited vascular aging and aging-related vascular dysfunction To further investigate the association between COMP and vascular aging, we applied COMP/ mice to examine whether COMP deficiency would lead to aging-related vascular dysfunction. The levels of p53, p21, and p16 were compared between aortas from the 10-month-old WT and COMP/ mice via western blot (Fig. 2A). Notably, COMP/- mice displayed markedly enhanced protein levels of above aging-related molecules. These data indicate that COMP deficiency may cause vascular aging in mice. To examine whether COMP deficiency affects vascular function, we performed isometric tension studies to evaluate vascular contraction in response to phenylephrine. The vascular contractility was detected ex vivo using aortic rings obtained from 10-month-old WT and COMP/ mice. Compared with those of WT, the COMP/ aortic rings exhibited a reduced contractility in response to phenylephrine and a significantly impaired maximal response (Fig. 2B). This finding implies that COMP deficiency abates the aortic
contractility, which is the major characteristic of aged arteries. To further reveal the functional significance of COMP deficiency, we subsequently measured arterial compliance via the pulse wave velocity (PWV) to assess the arterial stiffness, which represents a functional indicator of vascular aging [22]. 10 month-old COMP/ mice showed increased PWV compared with the WT (Fig. 2C), suggesting greater aortic stiffness. 3.3. Aortas of COMP/ mice were more susceptible to senescence Having identified aging phenotype in COMP/ mice, we were interested in determining whether COMP deficiency led to vascular aging. Calcification in tunica media (medical calcification) is often observed in the elderly population and increases throughout aging. Senescent vascular cells could play a major role in the pathogenesis of age-related vascular calcification [23]. As thus, senescence is thought to be a characteristic feature of the calcification process. We have reported that in vivo medial calcification model can be induced by perivascular application of 0.2 mol/L CaCl2 to the infrarenal abdominal aortas; COMP is an endogenous inhibitor of vascular calcification [10]. In this study, 18-month-old WT and COMP/ mice were subjected to in vivo medical calcification and vascular calcification was successfully induced as evidenced by calcium deposition and von Kossa staining (Fig. 3A, B). The infrarenal abdominal aortas were assessed for senescenceassociated b-galactosidase (SA b-gal) staining, which is a classic biochemical marker for senescence. The SA b-gal activity was increased in the CaCl2 group and was significantly evident in the aortic wall of COMP/ mice (Fig. 3C). These data indicate that vascular senescence is an important manifestation of calcification and COMP deficiency renders blood vessels susceptible to premature vascular aging.
Please cite this article in press as: M. Wang, et al., Cartilage oligomeric matrix protein prevents vascular aging and vascular smooth muscle cells senescence, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.004
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Fig. 4. COMP deficiency induced VSMCs senescence in vitro. (A) VSMCs were transfected with scramble siRNA or COMP siRNA. Protein level of COMP was determined by western blot and quantified. (B) Representative western blot analysis and quantification of the protein levels of p53, p21 and p16 in cell lysates from scramble or COMP siRNA-transfected VSMCs, with or without H2O2 treatment. (C) Light micrographs and quantification of the cells stained for SA b-gal activity. Scale bar ¼ 100 mm. Mean ± SEM values are shown for n ¼ 500 cells counted in 3 independent groups. *P < 0.05. (D) Western blot analysis and quantification of the protein levels of p53, p21, and p16 in cell lysates from the WT and COMP/ mouse VSMCs infected with Ad-lacZ or Ad-COMP. Data are from three independent experiments and error bars represent SEM. *P < 0.05.
Please cite this article in press as: M. Wang, et al., Cartilage oligomeric matrix protein prevents vascular aging and vascular smooth muscle cells senescence, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.004
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3.4. COMP deficiency induces VSMCs senescence in vitro Compelling evidence indicates that VSMC senescence promotes vascular calcification, atherosclerosis and features of plaque vulnerability [3,24,25]. We have previously demonstrated that COMP was mainly expressed in VSMCs, with no expression in endothelial cells and limited expression in adventitial fibroblast cells [11]. Therefore, the question arose as to whether COMP would affect VSMC senescence. We first determined the activation status of the p53/p21 and p16 axes. Specific siRNA targeted to COMP markedly decreased expression of COMP protein (Fig. 4A). As a consequence, the expression of senescence-associated markers (p53, p21 and p16) was significantly increased following COMP knockdown by siRNA in rat VSMCs, and further elevated after treatment with H2O2, an inducing agent of stress induced premature senescence. (Fig. 4B). We then performed SA b-gal staining. There was no significant change of the percentage of SA b-gal positive cells between the VSMCs transfected with scramble or COMP siRNA. However, after 48 h treatment with 10 mM H2O2, 22.20% ± 2.68 were SA b-gal positive in the COMP siRNA transfected VSMCs, whereas the number was only 7.10% ± 0.92 in the scramble siRAN treated group (n ¼ 3) (Fig. 4C), indicating that VSMCs undergo stress induced premature senescence by COMP silencing. 3.5. COMP overexpression reversed COMP-deficiency-induced VSMC senescence To further clarify the role of COMP in VSMC senescence, we isolated primary aortic VSMCs from 1-month-old WT and COMP/ mice, respectively. The COMP-deficient VSMCs exhibited enhanced senescence-associated markers, which were rescued by overexpression with Adenovirus-COMP infection (Fig. 4D). Together, our data reinforced that COMP plays pivotal role to prevent VSMCs senescence.
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degeneration of artery compliance. Our data is in accordance with our previous reports that first, COMP is essential for maintaining VSMC contractile phenotype and VSMCs switch from a contractile/ differentiated to a synthetic/dedifferentiated phenotype upon COMP deficiency [9], and secondly, COMP/ mice are more susceptible to restenosis, atherosclerosis and vascular calcification [10,11,14]. The cellular senescence of nearly all vascular cells is involved in vascular aging; however, endothelial cells and VSMCs are the major two cell types accountable for vascular aging [27]. Our finding that COMP/ VSMCs exhibited senescence features is consistent with spontaneous vascular aging in COMP/ mice. VSMCs undergo telomere shortening dependent replicative senescence or telomere length independent senescence referred to as stress-induced premature senescence (SIPS) [28]. The latter may be particularly relevant to vascular aging, in which vessel wall-resident VSMCs may not be able to sufficiently replicate in vivo [29,30]. Low doses of H2O2 treatment induced significant increase in senescenceassociated marker (pH 6.0 optimum b-galactosidase (SA b-gal) positivity) in COMP siRNA-transfected VSMCs, which suggested COMP-deficiency may induce SIPS upon H2O2-treatment. Knockdown of COMP activates senescence-associated molecules p53/p21 and p16 signals and COMP supplement blocks these axes. How COMP deficiency causes the activation of aging signals needs to be further explored. In conclusion, our study displayed a novel finding that COMP deficiency leads to vascular aging and VSMC senescence. Increasing of endogenous COMP may protect blood vessels from dysfunction and ameliorate aging-related vascular diseases. Beyond that, these findings suggest a new model that extracellular matrix protein maintains vascular homeostasis via against vascular aging process. Conflicts of interest All authors have no conflicts of interest.
4. Discussion In this study, we demonstrate for the first time that extracellular matrix protein COMP retards the development of vascular aging and VSMC senescence. Our study identified a novel endogenous guardian of vascular aging during chronological and biological aging. Compelling evidence indicates that vascular diseases, including atherosclerosis, vascular calcification and hypertension, are highly related to vascular aging. The slowing of vascular cell senescence is emerging as an exciting possibility for controlling vascular diseases [2]. Our findings that vascular COMP expression gradually declines with age and COMP/ mice exhibited aging-related vascular dysfunction even in the absence of environmental injury stimuli are in accordance with our previous reports that COMP deficiency deteriorates post-injury restenosis, atherosclerosis and vascular calcification [10,11,14]. Of interest, the COMP level was substantially reduced in the vessels of three different premature aging syndrome models (Zmpste24/, ApoE/ and SAM-P8 mice), suggesting COMP is an endogenous regulator of vascular aging and may be a potential therapeutic target for aging-associated cardiovascular diseases. Aging is accompanied by remodeling of the vascular wall and altered function. In the current study, we demonstrated reduced vascular contraction in the COMP/ artery, which is a key functional indicator of vascular aging. Physical stiffening of the large arteries is the central paradigm of vascular aging and significantly contributes to cardiovascular diseases in elder individuals [26]. In this study, we determined that COMP/ mice show enhanced conduit artery stiffness assessed by PWV, which means the
Acknowledgments This research was supported by funding from the National Natural Science Foundation of the P. R. China (91539203); the National Program on Key Basic Research Projects (973 Program) (2012CB518002); the National Science Fund for distinguished Young Scholars (81225002); International Cooperation and Exchanges NSFC (81220108004); the 111 Project of Chinese Ministry of Education (no. B07001). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.08.004. References [1] B.J. North, D.A. Sinclair, The intersection between aging and cardiovascular disease, Circ. Res. 110 (2012) 1097e1108. [2] C. Matthews, I. Gorenne, S. Scott, et al., Vascular smooth muscle cells undergo telomere-based senescence in human atherosclerosis: effects of telomerase and oxidative stress, Circ. Res. 99 (2006) 156e164. [3] T. Kunieda, T. Minamino, J. Nishi, et al., Angiotensin II induces premature senescence of vascular smooth muscle cells and accelerates the development of atherosclerosis via a p21-dependent pathway, Circulation 114 (2006) 953e960. [4] J.C. Wang, M. Bennett, Aging and atherosclerosis: mechanisms, functional consequences, and potential therapeutics for cellular senescence, Circ. Res. 111 (2012) 245e259. [5] A.U. Ferrari, A. Radaelli, M. Centola, Invited review: aging and the cardiovascular system, J. Appl. Physiol. 95 (2003) (1985) 2591e2597. [6] K.A. Williamson, A. Hamilton, J.A. Reynolds, et al., Age-related impairment of
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Please cite this article in press as: M. Wang, et al., Cartilage oligomeric matrix protein prevents vascular aging and vascular smooth muscle cells senescence, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.004
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Cartilage oligomeric matrix protein prevents vascular aging and vascular smooth muscle cells senescence Meili Wang a, b, Yi Fu a, b, Cheng Gao a, b, Yiting Jia a, b, Yaqian Huang c, Limei Liu a, Xian Wang a, b, Wengong Wang d, Wei Kong a, b, * a
Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing, China Department of Pediatrics, Peking University First Hospital, Beijing, China d Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 July 2016 Accepted 2 August 2016 Available online xxx
Aging-related vascular dysfunction contributes to cardiovascular morbidity and mortality. Cartilage oligomeric matrix protein (COMP), a vascular extracellular matrix protein, has been described as a negative regulatory factor for the vascular aging-related processes including atherosclerosis and vascular calcification. However, whether COMP is implicated in the process of vascular aging remains unclear. Here, we identified a novel function of COMP in preventing vascular aging and vascular smooth muscle cells (VSMCs) senescence. Firstly, vascular COMP expression was decreased in three different senescenceaccelerated mouse models and was also declining with age. COMP/ mice displayed elevated senescence-associated markers expression, including p53, p21 and p16, in the aortas compared with their wild type (WT) littermates. In accordance, COMP deficiency induced aging-related vascular dysfunction as evidenced by the significantly reduced phenylephrine-induced contraction and increased vascular stiffness as evaluated by pulse wave velocity. The aortic wall of COMP/ mice was susceptible to senescence by displaying senescence-associated b-galactosidase (SA b-gal) activity induced by periadventitial application of CaCl2 to the abdominal aorta. In vitro, COMP knockdown by small interfering (si) RNA led to the elevation of p53, p21 and p16 as well as SA b-gal activity in VSMCs after H2O2 stimulation. VSMCs isolated from COMP/ mice showed elevated senescence-associated markers expression and supplement of COMP adenovirus to COMP-deficient VSMCs greatly rescued cellular senescence. Taken together, these findings revealed the essential role of COMP in retarding the development of vascular aging and VSMC senescence. © 2016 Elsevier Inc. All rights reserved.
Keywords: Vascular extracellular matrix protein COMP Vascular aging VSMC senescence
1. Introduction Vascular aging is a major risk factor for a variety of cardiovascular diseases including atherosclerosis, vascular calcification and hypertension, etc [1]. Aging vasculature exhibits the reduced arterial compliance and increased stiffness, as well as the impaired function of contraction as a result of senescence-related arterial alterations in cells, matrix, and biomolecules [1]. The occurrence of cellular senescence, particularly in vascular smooth muscle cells (VSMCs) upon stress, has been demonstrated in human
* Corresponding author. Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University Health Science Center, 38 Xueyuan Road, Haidian District, Beijing, 100191, China. E-mail address: [email protected] (W. Kong).
atherosclerotic, calcifying and hypertensive arteries [2], as evidenced by the positive staining of senescence-associated b-galactosidase (SA b-gal) and the expression of senescence-related signaling hallmarks, such as high levels of p53, p21, p16 and phosphorylated p38. Consequently, VSMC senescence promotes pathogenesis of atherosclerosis, vascular calcification and other aging-related vascular diseases [3]. Multiple triggers have been identified nowadays to induce VSMC senescence, such as DNA damage, telomere shortening, oxidative stress, oncogene activation, the loss of tumor suppressors, epigenetic stress, angiotensin II and mitochondrial dysfunction [2,4]. However, the mechanism by which VSMCs counteract the multiple triggers of senescence and maintain cellular homeostasis following environmental stimuli remains less understood. An investigation of the endogenous modulators of vascular aging is favorable for the prevention and
http://dx.doi.org/10.1016/j.bbrc.2016.08.004 0006-291X/© 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: M. Wang, et al., Cartilage oligomeric matrix protein prevents vascular aging and vascular smooth muscle cells senescence, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.004
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treatment of vascular aging and aging-related vascular diseases. The extracellular matrix (ECM) constitutes an active and dynamic microenvironment for VSMCs and plays a fundamental role in the regulation of vascular function in normal and pathophysiological conditions. The collagen content increases, whereas elastin decreases with age, giving rise to a corresponding elastin fragmentation and progressive medial degeneration, aortic stiffening and aging-related reduction of vascular complications [5]. Nevertheless, a limited number of reports have recognized the effects of proteoglycans and glycoproteins, which constitute more than half of the human aortic ECM as demonstrated by recent proteomics analysis, on vascular aging [6e8]. Cartilage oligomeric matrix protein (COMP), a 524 kDa pentameric noncollagenous glycoprotein, is a matricellular protein found in both the musculoskeletal and cardiovascular systems. Our recent studies have demonstrated that COMP plays critical roles in the maintenance of vascular homeostasis. COMP maintains the contractile phenotype of VSMCs via integrin a7b1 and prevents osteochondrogenic transdifferentiation of VSMCs by directly binding to BMP-2, which inhibits post injury neointima formation and vascular calcification [9,10]. We recently also demonstrated that COMP negatively regulates atherosclerosis and lesional calcification formation via direct interaction with integrin b3 [11]. As COMP is involved in aging-related vascular diseases including atherosclerosis and vascular calcification, we asked whether COMP is implicated in vascular aging. 2. Materials and methods 2.1. Reagents Antibodies against COMP, p53, p21, p16 and b-actin were purchased from Abcam (Cambridge, UK). IRDye-conjugated secondary antibodies for western blotting were purchased from Rockland, Inc. (Gilbertsville, PA, USA). Other reagents were obtained from SigmaAldrich (St. Louis, MO, USA) unless specified.
euthanasia. The dissected vessels were immediately placed in Krebs-Henseleit buffer, and then cleaned of additional connective tissues. The aorta was sectioned into 1.5e1.8 mm rings and subsequently subjected to vascular tension experiments. The cumulative dose responses to phenylephrine were obtained to characterize vasocontraction. Data are expressed as percentages to KCl-induced contraction. 2.5. Measurement of pulse wave velocity The in vivo pulse wave velocity (PWV) was measured using an ECG-triggered 10-MHz Doppler probe [16]. The animals were anesthetized with isoflurane and maintained during the measurement by mask ventilation of 1.5% isoflurane with a coupled charcoal scavenging system. The animals were positioned supine with their limbs taped to electrocardiogram electrodes, which were incorporated into a temperature-controlled printed circuit board. The aortic arch blood flow and the abdominal aorta blood flow were captured, and the separation distance between them was measured. The PWV was calculated as a quotient of the separation distance and time difference between pulse arrivals, as measured from the ECG R-peaks. 2.6. CaCl2-induced mouse abdominal aortic calcification Mice were anesthetized with isoflurane and the infrarenal abdominal aortas were treated with periadventitial application of 0.2 mol/L CaCl2 for 15 min as described previously [10]. Control mice were treated with 0.2 mol/L NaCl. After 7 days, the abdominal aorta was harvested and sectioned for SA b-gal staining. Analysis of calcium content and von Kossa staining were used to assess the efficiency of the model as described previously [10]. 2.7. Senescence-associated b-galactosidase (SA b-gal) staining
COMP/ mice with a C57BL/6J background were kindly pro€ Oldberg (Department of Cell and Molecular vided by Professor Ake Biology, University of Lund, Sweden) [12]. Aortas of Zmpste24/ mice and their WT littermates were provided by Professor Zhongjun Zhou (Department of Biochemistry, University of Hong Kong, Hong Kong) [13]. ApoE/, SAM-R1 and SAM-P8 mice were purchased from the Department of Laboratory Animal Science, Peking University Health Science Center. All animal studies followed the guidelines of the Animal Care and Use Committee of Peking University.
SA b-gal staining was performed using a Senescence Detection Kit (Medical & Biological Laboratories CO., LTD). For the abdominal aortas, mice were perfused via the left ventricle with phosphatebuffered saline (PBS). Aortas were harvested and incubated in fixative solution for 1 h and in SA b-gal staining solution (prepared according to the manufacture's protocol) at 37 C for 16 h and then cryosectioned at 10 mm. SA b-gal activity was identified based on positively stained blue cells. Cells were washed with PBS and fixed with fixative solution for 15 min at room temperature. Then cells were incubated with staining solution at 37 C for 16 h. Senescent cells were bluestained as observed under light microcopy. Cells were counterstained with Hoechst 33342 to count the total number.
2.3. Western blotting
2.8. Cell culture
Western blot was performed as previously described [14]. Cells and mouse tissue extracts that contained equal amounts of total protein were resolved by 10% or 15% SDS-PAGE and subsequently transferred onto nitrocellulose membranes. The membranes were blocked with 5% milk in TBST, followed by incubation with primary antibody at 4 C overnight. Following 1 h of incubation with IRDyeconjugated secondary antibodies, the membranes were subjected to an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA) to detect the immunofluorescence signal.
The rat smooth muscle embryonic thoracic aorta cell line A7r5 was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum at 37 C in a humidified atmosphere containing 5% CO2. H2O2 treatment was performed by adding 10 mM H2O2 for 2 days. Primary mouse thoracic VSMCs were isolated by collagenase digestion as described previously [17]. Briefly, following mouse euthanasia, thoracic aortas were harvested and cut into small pieces. To remove the endothelial cells, these pieces were digested with 1 mg/ml trypsin (Hyclone) at 37 C for 10 min. After centrifugation, the precipitate was resuspended with 10 mg/ml collagenase type I (Gibco) for 6e8 h. Gelatin was pre-laid onto the culture dishes before the cells were seeded. The cell cultures contained >95%
2.2. Animals
2.4. Aortic ring myograph A myograph was performed as previously described [15]. Briefly, the aortic rings were isolated and removed from mice following
Please cite this article in press as: M. Wang, et al., Cartilage oligomeric matrix protein prevents vascular aging and vascular smooth muscle cells senescence, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.004
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Fig. 1. Protein level of COMP was decreased in aortas of aging mice models. Representative western blot analysis and quantification of the protein level of COMP in the aortas from Zmpste24/ mice and their WT littermates (A), ApoE/ mice and their WT littermates (B), SAM-R1 and SAM-P8 mice (C). Data are presented as mean ± SEM. *P < 0.05. (D) Representative western blot analysis and quantification of the protein level of COMP in the aortas from different aged C57BL mice. Error bars represent SEM. n ¼ 3 for each group, *P < 0.05.
VSMCs as determined by SM22 staining. The cells were cultured in complete DMEM that contained 10% fetal bovine serum (FBS, Hyclone) and were passaged by 0.125% trypsin digestion. The cells of passages 3e8 were used for experiments.
2.9. Transfection of small interfering RNA Small interfering RNA (siRNA) was purchased from GenePharma Co., Ltd (Shanghai). The sequences that corresponded to the siRNA of rat COMP were sense, 50 -AGAAACUUGAGCUGUGUUGAUGCC-30 , and anti-sense, 50 -GGCUAUCAAGACAGCUCAAGUUUCU-30 . A scramble stealth RNAi duplex served as a negative control. In vitro siRNA transfection (50 nM) of rat VSMCs was performed using RNAi MAX (Invitrogen, CA, USA). The transfection procedures followed the manufacturers' instructions.
2.10. Recombinant adenovirus construction and infection The adenovirus for full-length mouse COMP (Ad-COMP; NM_016685.2) was constructed and amplified according to the manufacturer's protocol (BD Biosciences). An adenovirus that carried b-galactosidase (Ad-LacZ) was used as a negative control. Cells cultured at approximately 80% confluence were infected with recombinant adenovirus (50 multiplicity of infection) for 48 h. 2.11. Statistical analysis Data are expressed as the mean ± SEM. Comparisons of the aortic gene expression and PWV between two genotypic mice were analyzed using unpaired two-tailed Student's t-tests. Comparisons of the aortic contraction between the WT and COMP/ mice were analyzed via two-way ANOVA followed by the Bonferroni test. The
Please cite this article in press as: M. Wang, et al., Cartilage oligomeric matrix protein prevents vascular aging and vascular smooth muscle cells senescence, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.004
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Fig. 2. COMP¡/¡ mice exhibited aging-related vascular dysfunction. (A) Representative western blot analysis and quantification of the protein levels of p53, p21 and p16 in the aortas from WT and COMP/- mice. n ¼ 3, *P < 0.05. (B) Contraction in response to the vasoconstrictor phenylephrine of isolated aortic rings from WT and COMP/ mice. Error bars represent SEM. n ¼ 5 for each group, *P < 0.05. (C) Pulse wave velocity measurements of the aortas from WT and COMP/ mice. Error bars represent SEM. n ¼ 5 for each group, *P < 0.05.
comparisons of the protein expression, calcium deposition or the percentages of SA b-gal-positive cells among more than 2 groups were analyzed using one-way ANOVA followed by the StudentNewman-Keuls test for post hoc comparison. In all cases, statistical significance was indicated when the two-tailed probability was less than 0.05.
3. Results 3.1. The expression of aortic COMP was inversely correlated with aging process Considering the protective role of COMP against cardiovascular diseases and to examine the involvement of COMP in vascular
aging, we measured the COMP expression in aortas obtained from three aging-accelerated rodent models, including Zmpste24/ mice, ApoE/ mice and senescence accelerated mice/prone substrain 8 (SAM-P8). Deletion of the Zmpste24 gene, which encodes the prelamin A processing enzyme, has been demonstrated to lead to an abnormal accumulation of prelamin A and thus a deficiency of lamin A, which plays an important role in the maintenance of chromatin organization and thus induces premature-aging phenotypes in mice [18]. Of interest, COMP was greatly reduced in the aortas of 5-month-old Zmpste24/ mice as evidenced by western blot analysis (Fig. 1A). Apolipoprotein E (ApoE) has been the most replicated longevity-associated gene, and ApoE/ mice exhibit a shorter life-span and many features of premature-aging [19,20]. A similar decrease of COMP protein was observed in aortas of 10-
Please cite this article in press as: M. Wang, et al., Cartilage oligomeric matrix protein prevents vascular aging and vascular smooth muscle cells senescence, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.004
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Fig. 3. Aortas of COMP¡/¡ mice were more susceptible to senescence. (A) Quantitative analysis of calcium deposition of abdominal aortas at 0 days, 7 days or 14 days after 0.2 mol/L NaCl or CaCl2 treatment. n ¼ 3 for each group, *P < 0.05. (B) Representative von Kossa staining of abdominal aortas at 14 days after 0.2 mol/L NaCl or CaCl2 treatment. Scale bar ¼ 100 mm. (C) Light micrographs of the abdominal aortas of 18-month-old WT and COMP/ mice, harvested after 7 days of perivascular application of 0.2 mol/L NaCl or CaCl2 to the infrarenal abdominal aortas and stained for SA b-gal activity. Scale bar ¼ 20 mm.
month-old ApoE/ mice (Fig. 1B). As while, SAM-P8 mice have been demonstrated to exhibit vascular dysfunction during aging [21]. Senescence accelerated mouse/resistance substrain 1 (SAMR1) was used as a control group. In accordance, a repressed COMP expression level was identified in the aortas of 6-month-old SAMP8 mice compared with their littermates (Fig. 1C). Furthermore, we compared the aortic COMP expression at young (2-month, 5month), middle-aged (12-month) and aged (20-month old) normal C57BL/6J mice, respectively. As shown in Fig. 1D, a gradual decline in the COMP level with aging was identified. Together, these findings indicated that COMP may be inversely correlated with vascular aging process. 3.2. COMP/ mice exhibited vascular aging and aging-related vascular dysfunction To further investigate the association between COMP and vascular aging, we applied COMP/ mice to examine whether COMP deficiency would lead to aging-related vascular dysfunction. The levels of p53, p21, and p16 were compared between aortas from the 10-month-old WT and COMP/ mice via western blot (Fig. 2A). Notably, COMP/- mice displayed markedly enhanced protein levels of above aging-related molecules. These data indicate that COMP deficiency may cause vascular aging in mice. To examine whether COMP deficiency affects vascular function, we performed isometric tension studies to evaluate vascular contraction in response to phenylephrine. The vascular contractility was detected ex vivo using aortic rings obtained from 10-month-old WT and COMP/ mice. Compared with those of WT, the COMP/ aortic rings exhibited a reduced contractility in response to phenylephrine and a significantly impaired maximal response (Fig. 2B). This finding implies that COMP deficiency abates the aortic
contractility, which is the major characteristic of aged arteries. To further reveal the functional significance of COMP deficiency, we subsequently measured arterial compliance via the pulse wave velocity (PWV) to assess the arterial stiffness, which represents a functional indicator of vascular aging [22]. 10 month-old COMP/ mice showed increased PWV compared with the WT (Fig. 2C), suggesting greater aortic stiffness. 3.3. Aortas of COMP/ mice were more susceptible to senescence Having identified aging phenotype in COMP/ mice, we were interested in determining whether COMP deficiency led to vascular aging. Calcification in tunica media (medical calcification) is often observed in the elderly population and increases throughout aging. Senescent vascular cells could play a major role in the pathogenesis of age-related vascular calcification [23]. As thus, senescence is thought to be a characteristic feature of the calcification process. We have reported that in vivo medial calcification model can be induced by perivascular application of 0.2 mol/L CaCl2 to the infrarenal abdominal aortas; COMP is an endogenous inhibitor of vascular calcification [10]. In this study, 18-month-old WT and COMP/ mice were subjected to in vivo medical calcification and vascular calcification was successfully induced as evidenced by calcium deposition and von Kossa staining (Fig. 3A, B). The infrarenal abdominal aortas were assessed for senescenceassociated b-galactosidase (SA b-gal) staining, which is a classic biochemical marker for senescence. The SA b-gal activity was increased in the CaCl2 group and was significantly evident in the aortic wall of COMP/ mice (Fig. 3C). These data indicate that vascular senescence is an important manifestation of calcification and COMP deficiency renders blood vessels susceptible to premature vascular aging.
Please cite this article in press as: M. Wang, et al., Cartilage oligomeric matrix protein prevents vascular aging and vascular smooth muscle cells senescence, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.004
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Fig. 4. COMP deficiency induced VSMCs senescence in vitro. (A) VSMCs were transfected with scramble siRNA or COMP siRNA. Protein level of COMP was determined by western blot and quantified. (B) Representative western blot analysis and quantification of the protein levels of p53, p21 and p16 in cell lysates from scramble or COMP siRNA-transfected VSMCs, with or without H2O2 treatment. (C) Light micrographs and quantification of the cells stained for SA b-gal activity. Scale bar ¼ 100 mm. Mean ± SEM values are shown for n ¼ 500 cells counted in 3 independent groups. *P < 0.05. (D) Western blot analysis and quantification of the protein levels of p53, p21, and p16 in cell lysates from the WT and COMP/ mouse VSMCs infected with Ad-lacZ or Ad-COMP. Data are from three independent experiments and error bars represent SEM. *P < 0.05.
Please cite this article in press as: M. Wang, et al., Cartilage oligomeric matrix protein prevents vascular aging and vascular smooth muscle cells senescence, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.004
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3.4. COMP deficiency induces VSMCs senescence in vitro Compelling evidence indicates that VSMC senescence promotes vascular calcification, atherosclerosis and features of plaque vulnerability [3,24,25]. We have previously demonstrated that COMP was mainly expressed in VSMCs, with no expression in endothelial cells and limited expression in adventitial fibroblast cells [11]. Therefore, the question arose as to whether COMP would affect VSMC senescence. We first determined the activation status of the p53/p21 and p16 axes. Specific siRNA targeted to COMP markedly decreased expression of COMP protein (Fig. 4A). As a consequence, the expression of senescence-associated markers (p53, p21 and p16) was significantly increased following COMP knockdown by siRNA in rat VSMCs, and further elevated after treatment with H2O2, an inducing agent of stress induced premature senescence. (Fig. 4B). We then performed SA b-gal staining. There was no significant change of the percentage of SA b-gal positive cells between the VSMCs transfected with scramble or COMP siRNA. However, after 48 h treatment with 10 mM H2O2, 22.20% ± 2.68 were SA b-gal positive in the COMP siRNA transfected VSMCs, whereas the number was only 7.10% ± 0.92 in the scramble siRAN treated group (n ¼ 3) (Fig. 4C), indicating that VSMCs undergo stress induced premature senescence by COMP silencing. 3.5. COMP overexpression reversed COMP-deficiency-induced VSMC senescence To further clarify the role of COMP in VSMC senescence, we isolated primary aortic VSMCs from 1-month-old WT and COMP/ mice, respectively. The COMP-deficient VSMCs exhibited enhanced senescence-associated markers, which were rescued by overexpression with Adenovirus-COMP infection (Fig. 4D). Together, our data reinforced that COMP plays pivotal role to prevent VSMCs senescence.
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degeneration of artery compliance. Our data is in accordance with our previous reports that first, COMP is essential for maintaining VSMC contractile phenotype and VSMCs switch from a contractile/ differentiated to a synthetic/dedifferentiated phenotype upon COMP deficiency [9], and secondly, COMP/ mice are more susceptible to restenosis, atherosclerosis and vascular calcification [10,11,14]. The cellular senescence of nearly all vascular cells is involved in vascular aging; however, endothelial cells and VSMCs are the major two cell types accountable for vascular aging [27]. Our finding that COMP/ VSMCs exhibited senescence features is consistent with spontaneous vascular aging in COMP/ mice. VSMCs undergo telomere shortening dependent replicative senescence or telomere length independent senescence referred to as stress-induced premature senescence (SIPS) [28]. The latter may be particularly relevant to vascular aging, in which vessel wall-resident VSMCs may not be able to sufficiently replicate in vivo [29,30]. Low doses of H2O2 treatment induced significant increase in senescenceassociated marker (pH 6.0 optimum b-galactosidase (SA b-gal) positivity) in COMP siRNA-transfected VSMCs, which suggested COMP-deficiency may induce SIPS upon H2O2-treatment. Knockdown of COMP activates senescence-associated molecules p53/p21 and p16 signals and COMP supplement blocks these axes. How COMP deficiency causes the activation of aging signals needs to be further explored. In conclusion, our study displayed a novel finding that COMP deficiency leads to vascular aging and VSMC senescence. Increasing of endogenous COMP may protect blood vessels from dysfunction and ameliorate aging-related vascular diseases. Beyond that, these findings suggest a new model that extracellular matrix protein maintains vascular homeostasis via against vascular aging process. Conflicts of interest All authors have no conflicts of interest.
4. Discussion In this study, we demonstrate for the first time that extracellular matrix protein COMP retards the development of vascular aging and VSMC senescence. Our study identified a novel endogenous guardian of vascular aging during chronological and biological aging. Compelling evidence indicates that vascular diseases, including atherosclerosis, vascular calcification and hypertension, are highly related to vascular aging. The slowing of vascular cell senescence is emerging as an exciting possibility for controlling vascular diseases [2]. Our findings that vascular COMP expression gradually declines with age and COMP/ mice exhibited aging-related vascular dysfunction even in the absence of environmental injury stimuli are in accordance with our previous reports that COMP deficiency deteriorates post-injury restenosis, atherosclerosis and vascular calcification [10,11,14]. Of interest, the COMP level was substantially reduced in the vessels of three different premature aging syndrome models (Zmpste24/, ApoE/ and SAM-P8 mice), suggesting COMP is an endogenous regulator of vascular aging and may be a potential therapeutic target for aging-associated cardiovascular diseases. Aging is accompanied by remodeling of the vascular wall and altered function. In the current study, we demonstrated reduced vascular contraction in the COMP/ artery, which is a key functional indicator of vascular aging. Physical stiffening of the large arteries is the central paradigm of vascular aging and significantly contributes to cardiovascular diseases in elder individuals [26]. In this study, we determined that COMP/ mice show enhanced conduit artery stiffness assessed by PWV, which means the
Acknowledgments This research was supported by funding from the National Natural Science Foundation of the P. R. China (91539203); the National Program on Key Basic Research Projects (973 Program) (2012CB518002); the National Science Fund for distinguished Young Scholars (81225002); International Cooperation and Exchanges NSFC (81220108004); the 111 Project of Chinese Ministry of Education (no. B07001). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.08.004. References [1] B.J. North, D.A. Sinclair, The intersection between aging and cardiovascular disease, Circ. Res. 110 (2012) 1097e1108. [2] C. Matthews, I. Gorenne, S. Scott, et al., Vascular smooth muscle cells undergo telomere-based senescence in human atherosclerosis: effects of telomerase and oxidative stress, Circ. Res. 99 (2006) 156e164. [3] T. Kunieda, T. Minamino, J. Nishi, et al., Angiotensin II induces premature senescence of vascular smooth muscle cells and accelerates the development of atherosclerosis via a p21-dependent pathway, Circulation 114 (2006) 953e960. [4] J.C. Wang, M. Bennett, Aging and atherosclerosis: mechanisms, functional consequences, and potential therapeutics for cellular senescence, Circ. Res. 111 (2012) 245e259. [5] A.U. Ferrari, A. Radaelli, M. Centola, Invited review: aging and the cardiovascular system, J. Appl. Physiol. 95 (2003) (1985) 2591e2597. [6] K.A. Williamson, A. Hamilton, J.A. Reynolds, et al., Age-related impairment of
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Please cite this article in press as: M. Wang, et al., Cartilage oligomeric matrix protein prevents vascular aging and vascular smooth muscle cells senescence, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.004