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Aloe-emodin inhibits osteogenic differentiation and calcification of mouse vascular smooth muscle cells
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Aloe-emodin inhibits osteogenic differentiation and calcification of mouse vascular smooth muscle cells Mahesh Sapkota, Saroj Kumar Shrestha, Ming Yang, Young Ran Park, Yunjo Soh∗ Department of Dental Pharmacology, School of Dentistry, Chonbuk National University, Jeon-Ju, 561-756, Republic of Korea
A R T I C LE I N FO
A B S T R A C T
Keywords: Aloe-emodin Vascular smooth muscle cells Vascular calcification BMP-2 RUNX-2
Vascular calcification increases the risk of morbidity and mortality in patients with cardiovascular diseases, chronic kidney diseases, and diabetes. However, viable therapeutic methods to target vascular calcification are limited. Aloe-emodin (AE), an anthraquinone is a natural compound found in the leaves of Aloe-vera. In this study, we investigated the underlying mechanism of AE in the calcification of vascular smooth muscle cells (VSMCs) and murine thoracic aorta. We demonstrate that AE repressed not only the phenotypes of Ca2+ induced calcification but also level of calcium in VSMCs. AE has no effect on cell viability in VSMC cells. Alizarin red, von Kossa stainings and calcium quantification showed that Ca2+ induced vascular calcification is significantly decreased by AE in a concentration-dependent manner. In contrast, AE attenuated Ca2+ induced calcification through inhibiting osteoblast differentiation genes such as SMAD4, collagen 1α, osteopontin (OPN), Runt-related transcription factor (RUNX-2) and Osterix. AE also suppressed Ca2+ induced osteoblast-related protein expression including collagen 1α, bone morphogenic protein 2 (BMP-2), RUNX-2 and smooth muscle actin (SMA). Furthermore, Alizarin red, von Kossa stainings and calcium quantification showed that AE significantly inhibited the calcification of ex vivo ring formation in murine thoracic aorta, and markedly inhibited vitamin D3 induced medial aorta calcification in vivo. Taken together, our findings suggest that AE may have therapeutic potential for the prevention of vascular calcification program.
1. Introduction Cardiovascular disease (CVD) is a class of disease that involves the heart or blood vessels. It includes coronary artery diseases such as angina pectoris and myocardial infarction (Badimon et al., 1991). Other CVD include stroke, heart failure, rheumatic heart disease and aortic aneurysms (Badimon et al., 1991). There are different kind of mechanisms depending on the disease. Coronary artery disease, stroke and peripheral artery disease involve atherosclerosis (Tinkov et al., 2018). CVD are caused by high blood pressure, smoking, diabetes, lack of exercise, obesity, high blood cholesterol, poor diet and excessive alcohol consumption (Hruska et al., 2005). Cardiovascular disease increases the risk of mortality and morbidity worldwide (Ruan et al., 2018). Vascular calcification (VC) is related with pathological conditions such as aging, atherosclerosis, diabetes, vascular endothelial injury, chronic Kidney diseases and hypertension (London et al., 2003; Vattikuti and Towler, 2004; Wallin et al., 2001). Calcium minerals deposition in arteries leads to the vascular stiffening, called as vascular calcification (Shroff and Shanahan, 2007). Vascular calcification is an active biological process similar to bone development, which is preventable, and reversible. In
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specific, the development and progression of VC involved the transformation of vascular smooth muscle cells (VSMCs) into an osteoblastlike phenotype (Abedin et al., 2004; Bostrom et al., 2011; Shao et al., 2006). Trans-differentiation of VSMCs from a fibroblastic phenotype to an osteoblastic phenotype was carried out by enhancing the osteochondrogenic transcription factor RUNX-2 and a series of osteoblast differentiation markers (Cui et al., 2012; Liang et al., 2012; Shan et al., 2011; Thompson and Towler, 2012; Yuan et al., 2011). Bone morphogenetic proteins (BMP) played crucial role in bone formation. BMP-2 is a super family of transforming growth factors-β (TGF-β) and secretory growth factors. Downstream effectors of BMP-2, osterix (OSX) and RUNX-2 played crucial role in vascular calcification and atherosclerosis (Derwall et al., 2012; Hruska et al., 2005; Johnson et al., 2006). BMP-2 is a mediator of vascular calcification; BMP-2 activates BMP-2 receptor and induced-receptor SMADs, which induce osteoblast differentiation by up-regulation of transcription factors such as RUNX-2, OPN, SMAD4 and collagen 1α. RUNX-2 is major transcription factor of vascular calcification, which induced osteoblast differentiation. Furthermore, expression of osteopontin (OPN) and type I collagen (COL 1α) are
Corresponding author. E-mail address: [email protected] (Y. Soh).
https://doi.org/10.1016/j.ejphar.2019.172772 Received 30 May 2019; Received in revised form 23 October 2019; Accepted 1 November 2019 0014-2999/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Mahesh Sapkota, et al., European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2019.172772
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necessary for preosteoblast and osteoblastogenic differentiation (Byon et al., 2008; Harada et al., 1999). Therefore, BMP-2 and RUNX-2 signaling play pivotal role in addressing the therapeutic target in vascular diseases. Vitamin D3 plays crucial role in mineralization, and stimulating absorption of calcium and phosphorous, as well as in enhancing osteoblast differentiation and bone modulators expression. (Inada et al., 1999; Yang et al., 2015). Aloe-emodin (AE) 1, 8-Dihydroxy-3-(hydroxymethyl) anthraquinone, is a natural compound found in the leaves of Aloe-vera. AE has been in various pharmacological activities and showed antiviral, antimicrobial, and hepato-protective roles and anti-inflammatory activities (Agarwal et al., 2000; Eshun and He, 2004; Hu et al., 2014; Li et al., 2014). Recent studies showed that, AE exhibit anti-cancer activity on hepatoma cancer cell growth (Zhang et al., 2015). Next, several study showed that, AE significantly repressed proliferation of MKN45 gastric cancer cells (Chihara et al., 2015). AE also exhibit anti-cancer effects on lung carcinoma, neuro-ectodermal tumors, and cervical cancer (Guo et al., 2007; Lee, 2001; Pecere et al., 2000). Those results suggest that AE acts as a cancer chemo-preventive agent. However, the role of AE in vascular calcification is unknown to date. In this study, we demonstrate that AE suppressed vascular calcification through inhibiting osteoblast differentiation genes and proteins in vitro.
Table 1 Primer sequences and conditions for RT-PCR. Target genes (Accession number)
Primer (Forward, Reverse)
Annealing Tm (°C)
PCR cycles
SMAD4 (AY493561)
5′-atctatgcccgtctgtggag-3′ 5′-aacattcctgtggcttccac-3′ 5′-actttctccaggaagactgc-3′ 5′-acagcaacagcaacaacagc-3′ 5′-actacccacccttccctcac-3′ 5′-ccttaacccagctccctacc-3′ 5′-ttctcctggtaaagatggtgc-3′ 5′-tgttaaaggtgatgctggtcc-3′ 5′-gaccaccatggacgacgatg-3′ 5′-tggaacttgcttgactatcga-3′ 5′-ttctacaatgagctgcgtgt-3′ 5′-ctcatagctcttctccaggg-3′
50
30
50
30
55
35
50
35
58
30
50
26
RUNX-2 (NM_009820) Osterix (AF184902) Col 1α (NM_007742) OPN (BC002113.1) β-actin (NM_007393)
On every 48 h media was changed. On day 7, both alizarin red staining and von Kossa staining were performed. Briefly, VSMCs was washed with 1 × PBS for 3 times and fixed with 1 × paraformaldehyde for 1 h at 16 °C. Then 2% alizarin red (AR) was added next for 1 h. Then, plate was washed with 1 × PBS and image was captured by the digital camera. Similarly, for Von Kossa staining, VSMCs were washed with distilled water for several times, and then 5% silver nitrite solution was added and exposed in UV rays for 1 h. Now, silver nitrate solution was washed with distilled water and image was captured by digital camera.
2. Material and methods 2.1. Reagents, antibodies and chemicals
2.5. Reverse transcriptase – PCR
Aloe-emodin (AE) was purchased from Tokyo Chemical industry (TCI, Tokyo, Japan). Anti-rabbit RUNX-2 antibody was obtained from Bio world (Bioworld Technology, MN, USA). Anti-mouse smooth muscle actin (SMA), anti-goat collagen 1α antibody, anti-goat osteoprotegerin (OPG) antibody and anti-mouse β-actin antibody were obtained from Santa Cruz biotechnology Inc (Santa Cruz, CA, USA). Antirabbit BMP-2 antibody was purchased from Abcam (Cambridge, MA). TRIzol and superscript II Reverse Transcriptase was obtained from Invitrogen (Carlsbad, CA, USA). AgNO3, CaCl2, NaH2PO4, Na2HPO4 and other chemicals were bought from sigma (St. Louis, MO, USA) and other details are previously described (Sapkota et al., 2015).
Total RNA was isolated from cell culture using TRizol (Invitrogen) and cDNA synthesis was performed with super script II reverse transcriptase (Invitrogen) according to manufacturer's protocol and stored at −80 °C. All PCR primers were purchased from Bioneer (Daejon, Korea). Specific primer sequences are listed in Table 1. After initialization and denaturation, PCR was performed for various cycles (30 s at 94 °C, 1 min at annealing temperature and 2 min at 72 °C) using Taq polymerase (Promega, Madison, WI, USA). Reaction products were separated in 1% agarose gel, stained with ethidium bromide, and analyzed by densitometry using a Phosphoimager and Quantity One software (Version 4.3.1) (Bio-Rad, Hercules, CA, USA).
2.2. Vascular smooth muscle cell culture
2.6. Western blotting
Mouse VSMCs were isolated from the thoracic aorta of 6 week-old ICR mice (n = 5, approximately 30 g body weight). Animal handling was approved by ethics committee of Chonbuk National University, South Korea (Approval no. CBNU 2018-094). Enzyme solution was prepared by mixing collagenase II, elastase and soybean trypsin inhibitor, according to protocol. VSMCs were treated with enzyme solution for 30 min and washed with DMEM/F12 for three times. Then, VSMCs were seeded in 100 mm plate for 5–6 days, and media was changed every 48 h. Cells were incubated at 37 °C, 5% CO2 humidified condition. VSMCs of 5–8 passages were used for further experiments.
VSMCs were harvested, lysed in lysis buffer [20 mM Tris-HCl (pH = 7.5), 137 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM Na3VO4, 1 mM phenylmethylsulfonylfluoride (PMSF), and 1 × protease inhibitor cocktail]. Cell lysates were centrifuged at 13,000×g for 15 min, supernatant were used as cell extracts and stored at −70 °C until use. Proteins were quantified by bicinchoninic acid (BCA) protein assay. Proteins were resolved by SDS-PAGE on 8–10% gels and then transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad) with a glycine transfer buffer [192 mM glycine, 25 mM Tris-HCl (pH = 8.8), 20% MeOH (v/v)]. Membranes were blocked with 5% nonfat skim milk in Tris-buffered saline (TBS) containing 0.25% Tween – 20 (TBST) 16 °C for 1 h. Then incubated for 16 h at 4 °C with rabbit anti -collagen Iα (Santa Cruz Biotechnology), anti – rabbit BMP-2 (Abcam), anti-rabbit RUNX-2 (Bio world), Anti-mouse smooth muscle actin (SMA) (Santa Cruz Biotechnology), anti-goat osteoprotegerin (OPG) (Santa Cruz Biotechnology) or anti – mouse β-actin (Santa Cruz Biotechnology) antibodies diluted 1:1000 in 5% nonfat skim milk in TTBS. Antibody-antigen complexes were visualized with Clarity ™ Western ECL Substrate (Bio-Rad).
2.3. Cell viability MTT assay was used to observe the cell viability in VSMCs. VSMCs were seeded with or without Ca2+ and various doses of AE for 48 h. After 48 h, VSMCs cell were washed with 1 × PBS and 100 μg/ml of MTT was added for 2 h at 37 °C and again washed with 1 × PBS, then VSMCs was dissolved with 200 μl dimethyl sulfoxide (DMSO) and quantified with a spectrophotometer at 540 nm. 2.4. Alizarin red staining and von Kossa staining
2.7. Ex-vivo cell culture
VSMCs were seeded in 48 well plate in the presence of complete media DMEM/F12. VSMCs were treated with or without 3.6 mM Ca2+ as a calcification media with different concentration of AE for 7 days.
In aseptic condition, VSMCs were obtained from thoracic aorta of 6 2
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(oCPC). The calcium level increased in Ca2+ induced VSMCs compared to without calcium group whereas significantly suppressed by AE in a dose dependent manner in Ca2+ induced VSMCs (Fig. 1B). As we observed, AE inhibits VSMCs calcification in various concentration, which may be due to cytotoxic effects. To examine the effects of AE on cell viability in Ca2+ induced VSMCs, we performed MTT assay. We showed that there was no effect on viability of VSMCs at given concentrations and 20 μM of AE was selected as working concentration in VSMCs (Fig. 1C). These results demonstrate that AE prevents vascular calcification of VSMCs by inhibiting mineralization.
week-old ICR mice (n = 5, approximately 30 gm). All the procedures were performed with some modification as described in (Price et al., 2006). Mice thoracic aorta were chopped into 3 mm in length and placed in 48 well plates with DMEM/F12 for 7 days in presence or absence of Ca2+ and AE. After 7 days, thoracic aorta was washed with 1 × PBS. 5% formalin was used to fixed, and thoracic aorta was vacuumed overnight at 60 °C, embedded in paraffin. Histologic sections of 5 μm slices were made for alizarin red staining, von Kossa staining and hematoxylin and eosin (H&E) staining. 2.8. Calcium deposition quantification
3.2. AE attenuates osteogenic genes expression during vascular calcification induction
For the quantification of calcium deposition in VSMCs, the cells were washed with 1 × PBS for several times, then 0.6 N HCL was used for overnight to decalcified calcium. The calcium contents were measured by using colorimetric reagents, the o-cresolphthalein complexone (oCPC) method (Bioassay System, Cat.DICA500). Briefly, the remaining cells were washed with 1 × PBS and the cells were lysed with lysis buffer, bicinchoninic acid (BCA) method (Pierce) was used to determined total protein contents. For each culture, total calcium content was normalized by total protein content. Further, calcium quantification in aortic tissues, segment of aorta was weighed and decalcified with 0.6 N HCL. According to above procedure calcium contents were determined and the data were expressed as calcium per mg of dry aortic tissues.
The effect of AE on the osteogenic differentiation of VSMCs was investigated by using reverse transcriptase - PCR (RT-PCR). We showed that the expression of osteogenic differentiation genes such as SMAD4, Collagen 1α, OPN, RUNX-2 and Osterix markedly increased in Ca2+ treated group and those expressions were significantly inhibited by AE in a dose dependent manner (Fig. 2A and B). Pi also increased the calcification of osteogenic genes such as SMAD4, RUNX-2 and OPN, expression of those genes were markedly decreased by AE in dose dependent manner (Fig. 3A and B). Together, these results suggested that AE modulates osteogenic differentiation of VSMC by inhibiting various genes, which are important for VSMCs calcification.
2.9. Vitamin D3-induced calcification in mice aorta 3.3. AE suppresses osteogenic-protein expression in VSMCs
Vitamin D3-induced calcification of aorta in 6 weeks ICR male mice were used for in vivo study. Briefly, 6 weeks ICR mice were randomly selected into 4 groups with (n = 5) mice in each group; 1) Control, 2) Vitamin D3, 3) Vitamin D3 + AE (5 mg/kg), Vitamin D3 + AE (25 mg/ kg). 25 mg/kg AE do not cause any mortality or clinical signs (Fabrice et al., 2009). All mice were subjected to gavage daily with corn oil (control) or 5 mg/kg or 25 mg/kg AE at once per day for 13 days. Three days after AE treatment, the mice were subcutaneously administrated Vitamin D3 (cholecalciferol, 5.5 × 105 U/kg per day) for 3 following days. After six days of final exposure to vitamin D3, mice were anesthetized and entire aorta was taken out by dissection to observe calcium deposition (Ha et al., 2014).
Ca2+ plays crucial role in calcium deposition in VSMCs (Demer and Tintut, 2008). Osteogenic transcription factors including Collagen 1α, BMP-2, RUNX-2 and SMA are representative markers (Du et al., 2011; Leopold, 2014; Steitz et al., 2001; Wallin et al., 2001). We investigate whether AE regulates the bone-formation associated protein in calcification. Ca2+ significantly increased the protein expressions of Collagen 1α, BMP-2, RUNX-2 and SMA whereas increased protein expression was significantly decreased by AE in a concentration dependent manner (Fig. 4A and B). These results suggest that AE controlled different osteogenic protein, which plays pivotal role in vascular calcification.
2.10. Statistical analysis
3.4. AE represses calcification of VSMC in an ex-vivo model
Data are presented as mean ± S.D. of at least three experiments was performed. One-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test was performed in SPSS ver. 12.0 software to compare among the various dose groups and *P < 0.05, **P < 0.001 was considered as statistical significant.
As we found that AE inhibits the calcification of VSMCs by regulating the expression of osteogenic gene and protein expression in vitro. Next, we used AE in ex-vivo calcification model by extracting thoracic aorta from the mouse. Thoracic aorta was cultured in media in presence or absence of Ca2+ and AE. VSMCs calcification was observed by alizarin red staining and von Kossa staining. We found that Ca2+ increased mouse aortic calcification while AE markedly reduced calcification area (Fig. 5A). Furthermore, AE decreased the deposition of calcium in mouse aorta dose dependently (Fig. 5B). These results suggest that AE plays an important role in suppressing aortic calcification.
3. Results 3.1. AE inhibits Ca2+ induced calcification in VSMCs Previous studies have shown that vascular calcification is occurred by Ca2+, which is well known in both in vitro and in vivo model (Demer and Tintut, 2008). We investigated whether the AE modulated on mouse thoracic aorta in VSMCs. VSMCs were treated with or without Ca2+ and AE followed by culturing for 7 days. The results of alizarin red staining showed that Ca2+ treated group markedly increased calcium phosphate deposition compared to the group without Ca2+ treatment and these effects were significantly inhibited by co-treatment with Ca2+ and AE in a dose dependent manner. Moreover, von Kossa staining showed that Ca2+ induced VSMCs calcium phosphate and those VSMCs calcium phosphates were significantly inhibited by AE in a concentration dependent manner (Fig. 1A). Furthermore, we explored the calcium level in VSMCs by using o-cresolphthalein complexone
3.5. AE attenuates vitamin D3-induced calcification in-vivo Treatment with Ca2+ in in-vitro and ex-vivo model induced vascular calcification. Vitamin D3 was subcutaneously injected in mice to create an aortic calcified model. Vitamin D3 intoxication has been studied as a model of arterial calcification (Neven and D’Haese, 2011). We found that AE markedly repressed Vitamin D3 induced calcium deposition in the aorta of mice (Fig. 6A). Furthermore, AE decreased calcium level in a concentration dependent manner (Fig. 6B). Taken together, these results suggest that AE plays crucial role to suppress vascular calcification by suppressing bone-related VSMCs calcification. 3
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Fig. 1. AE suppresses the calcification in vascular smooth muscle cells. VSMCs were cultured with DMEM/F12 (growth media) or Ca2+ (3.6 mM) with or without 10 μM and 20 μM concentration of AE for 7 days. (A) Alizarin Red staining and von Kossa staining and (B) calcium level in mice was observed. Results are expressed as mean ± SDs (n = 3). *P < 0.05 indicates significant differences from the Ca2+ (3.6 mM)-induced group. (C) VSMCs were cultured with DMEM/F12 or Ca2+ (3.6 mM) in the presence or absence of various dose (5, 10, 20, 40 and 80 μM) of AE for 48 h. Cell viability in VSMCs was observed by MTT assay. Results are presented as the mean ± S.D. (n = 3). Ca2+ (3.6 mM) act as control.
4. Discussion
differentiation during vascular calcification (Shimizu et al., 2011; Tyson et al., 2003). BMP-2 and SMAD4 are closely associated with each other during vascular calcification. Various studies showed that BMP-2 canonical SMAD4 pathway is included in bone remodeling with osteogenic activity (Broege et al., 2013; Zhu et al., 2015). AE is a variety of emodin present in aloe latex, and an exudate from aloe plant. Emodin activates the mRNA and protein expressions of BMP-2 in the differentiation process of mouse osteoblastic MC3T3-E1 subclone 4 cells (Lee et al., 2007). Emodin prevents intima thickness in balloon-injured carotoid artery of rats via Wnt4/Dvl-1/β-catenin signaling pathway mediated by miR-126 (Jun et al., 2015). The expressions of proliferating cell nuclear antigen (PCNA) mRNA and protein were prominently decreased by the addition of AE in VSMC after arterial injury (Chengbin et al., 2001). Our study showed that AE markedly suppressed Ca2+ induced BMP-2 and SMAD4 expression during VSMCs calcification (Figs. 2 and 5). Next, VSMCs osteogenic differentiation is characterized by up regulation of bone related molecule RUNX-2 (Steitz et al., 2001). We found that AE attenuated Ca2+ induced RUNX-2 expression of gene and protein throughout VSMCs calcification (Figs. 2 and 5). Likewise, vascular calcification is an active process as osteogenesis, in which vascular smooth muscle cell acquire the osteogenic phenotype with the increase of collagen 1. Collagen 1 play an important role in the formation of calcified structures and osteoblast differentiation (Chen et al., 2015). We observed that AE prevents Ca2+ induced Collagen 1α expression during VSMCs calcification both mRNA and
Several studies shown that vascular smooth muscle cells (VSMCs) calcification is an active, highly mediated and controlled route. Those processes may be associated with various mechanism including disorder of calcium-phosphorous mechanism, osteoblast phenotype transformation and imbalance of inhibitor/promoter of vascular calcification. AE is an anthroquinone from the Aloe vera (Aloe barbadensis miller). AE has been reported to have functions in anti-inflammatory agent, anti-immunomodulator, and anti-tumor effects (Wang et al., 2014). However, the effects of AE on vascular calcification in VSMC have not been identified. In this study, we initially detected effects of AE on mouse thoracic aorta in VSMCs. We found that AE significantly suppressed Ca2+ induced calcification as decided by alizarin red staining, von Kossa staining and calcium quantities in in-vitro (Fig. 1A and B). A series of experiments were conducted to identify the toxicological effect of AE on vascular smooth muscle cells. AE had no cytotoxic effect till 20 μM in the Ca2+ containing media treated to VSMCs (Fig. 1C). In the present study, Osteoblast alteration to VSMCs was known as a key process of calcification. During calcification, VSMCs acquire bone related phenotype and categorized by an increased in BMPs, such as BMP-2, OPN, and collagen 1α (Du et al., 2011; Leopold, 2014; Wallin et al., 2001). BMP-2 plays pivotal role in osteoblastic cells 4
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Fig. 2. AE inhibits osteoblastic genes during vascular calcification. VSMCs were cultured with DMEM/F12 (growth media) or Ca2+ (3.6 mM) with or without 10 μM and 20 μM concentration of AE for 7 days. (A) RT-PCR was performed to observe the gene expression of osteoblastic related genes. (B) Quantitative analysis of mRNA (%) compared with Ca2+ (3.6 mM). Results are presented as means ± S.D. (n = 3). *P < 0.05, **P < 0.001 indicates significant differences from the Ca2+ (3.6 mM) - induced group.
addition, osterix regulated the expression of osteoblast related genes during the mineralization of VSMCs by activating the downstream of RUNX-2 (Nakashima et al., 2002). We found that AE attenuates Ca2+ induced mRNA expression of osterix during VSMCs calcification (Fig. 2). Smooth muscle actin is found in VSMCs enhances VSMCs
protein expression (Figs. 2 and 5). Similarly, OPN involved in regulation of embryonic bone formation as well as skeleton bone remodeling and also plays pivotal role against vascular calcification (Dhore et al., 2001; Speer et al., 2002). In this study, we found that AE prevents Ca2+ induced OPN gene expression during VSMCs calcification (Fig. 2). In
Fig. 3. AE inhibits osteoblastic marker genes during vascular calcification. (A) VSMCs were cultured with DMEM/F12 (growth media) or Pi (3.6 mM) with or without 10 μM and 20 μM concentration of AE for 7 days. RT-PCR was performed to observed osteoblastic marker genes expression. (B) Quantitative analysis of mRNA (%) compared with Pi (3.6 mM). Results are presented as means ± S.D. (n = 3). *P < 0.05 indicates significant differences from the Pi (3.6 mM) - induced group. 5
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Fig. 4. AE attenuates BMP-2 pathways. VSMCs were cultured with DMEM/F12 (growth media) or Ca2+ (3.6 mM) with or without 10 μM and 20 μM concentration of AE for 7 days. (A) Western blot analysis was performed to determine the expression of osteoblastic related proteins. (B) Expression of collagen 1α, BMP-2, RUNX-2 and SMA quantified and compared with Ca2+ (3.6 mM). Results are presented as means ± S.D. (n = 3). *P < 0.05, **P < 0.001 indicates significant differences from the Ca2+ (3.6 mM) - induced group.
could induce cardiovascular abnormalities such as intima-media thickness and coronary artery calcification (Briese et al., 2006; Goldsmith et al., 1997). Vitamin D3 induced aortic calcification was enhanced by up-regulation of osteogenic marker gene expression in medial aorta region (Han et al., 2013). We investigated whether AE treatment could inhibit vitamin D3 induced aortic calcification in vivo. We found that AE markedly reduced vitamin D3 induced aortic calcification in vivo (Fig. 6). Taken together, our study suggest that AE attenuates VSMCs calcification through BMP-2 dependent SMAD4 signaling pathways (Fig. 5C). Furthermore, this results shown that AE inhibits vascular calcification in vitro and in vivo. Finally, our finding suggest that AE may
during vascular calcification (Du et al., 2011; Leopold, 2014). In our study, we found that AE decreases Ca2+ induced protein expression of SMA during VSMCs calcification protein expression (Fig. 4). Together these results support the suggestion that AE significantly inhibits the vascular calcification and osteoblast differentiation related-genes and proteins in VSMCs which suggest that AE could prevents vascular calcification in-vitro. Next, we explored the effects of AE on vascular calcification in us ex vivo thoracic aortic ring calcification model. We found that AE markedly decreased aortic calcification and calcium deposition, which suggest that AE could suppress phenotypic conversion of VSMCs to osteoblast. Furthermore, various study showed that vitamin D therapy
Fig. 5. AE suppresses vascular calcification in an ex-vivo condition. The 3 mm aortic ring was isolated from mouse and incubated with DMEM/F12 (growth media) or Ca2+ (3.6 mM) in the presence or absence of 10 μM and 20 μM concentration of AE for 7 days. (A) Alizarin red staining and von Kossa staining were used to detect calcified region. (B) Amount of calcium deposition level in aortic ring was observed. Results are presented as means ± S.D. (n = 5). *P < 0.05, indicates significant differences from the Ca2+ (3.6 mM) - induced group. 6
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Fig. 6. AE protects vitamin D3-induced calcium level in mice aorta. Von Kossa staining was performed to identify calcium deposition (A) and quantified the calcium deposition level (B) in aorta. Results are presented as means ± S.D. (n = 5). (C) The schematic diagram of the effects of AE on Ca2+ induced vascular calcification in VSMCs. *P < 0.05, indicates significant differences from the Ca2+ (3.6 mM)-induced group.
be a possible therapeutic phytochemical in inhibiting diseases related to vascular calcification.
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Aloe-emodin inhibits osteogenic differentiation and calcification of mouse vascular smooth muscle cells Mahesh Sapkota, Saroj Kumar Shrestha, Ming Yang, Young Ran Park, Yunjo Soh∗ Department of Dental Pharmacology, School of Dentistry, Chonbuk National University, Jeon-Ju, 561-756, Republic of Korea
A R T I C LE I N FO
A B S T R A C T
Keywords: Aloe-emodin Vascular smooth muscle cells Vascular calcification BMP-2 RUNX-2
Vascular calcification increases the risk of morbidity and mortality in patients with cardiovascular diseases, chronic kidney diseases, and diabetes. However, viable therapeutic methods to target vascular calcification are limited. Aloe-emodin (AE), an anthraquinone is a natural compound found in the leaves of Aloe-vera. In this study, we investigated the underlying mechanism of AE in the calcification of vascular smooth muscle cells (VSMCs) and murine thoracic aorta. We demonstrate that AE repressed not only the phenotypes of Ca2+ induced calcification but also level of calcium in VSMCs. AE has no effect on cell viability in VSMC cells. Alizarin red, von Kossa stainings and calcium quantification showed that Ca2+ induced vascular calcification is significantly decreased by AE in a concentration-dependent manner. In contrast, AE attenuated Ca2+ induced calcification through inhibiting osteoblast differentiation genes such as SMAD4, collagen 1α, osteopontin (OPN), Runt-related transcription factor (RUNX-2) and Osterix. AE also suppressed Ca2+ induced osteoblast-related protein expression including collagen 1α, bone morphogenic protein 2 (BMP-2), RUNX-2 and smooth muscle actin (SMA). Furthermore, Alizarin red, von Kossa stainings and calcium quantification showed that AE significantly inhibited the calcification of ex vivo ring formation in murine thoracic aorta, and markedly inhibited vitamin D3 induced medial aorta calcification in vivo. Taken together, our findings suggest that AE may have therapeutic potential for the prevention of vascular calcification program.
1. Introduction Cardiovascular disease (CVD) is a class of disease that involves the heart or blood vessels. It includes coronary artery diseases such as angina pectoris and myocardial infarction (Badimon et al., 1991). Other CVD include stroke, heart failure, rheumatic heart disease and aortic aneurysms (Badimon et al., 1991). There are different kind of mechanisms depending on the disease. Coronary artery disease, stroke and peripheral artery disease involve atherosclerosis (Tinkov et al., 2018). CVD are caused by high blood pressure, smoking, diabetes, lack of exercise, obesity, high blood cholesterol, poor diet and excessive alcohol consumption (Hruska et al., 2005). Cardiovascular disease increases the risk of mortality and morbidity worldwide (Ruan et al., 2018). Vascular calcification (VC) is related with pathological conditions such as aging, atherosclerosis, diabetes, vascular endothelial injury, chronic Kidney diseases and hypertension (London et al., 2003; Vattikuti and Towler, 2004; Wallin et al., 2001). Calcium minerals deposition in arteries leads to the vascular stiffening, called as vascular calcification (Shroff and Shanahan, 2007). Vascular calcification is an active biological process similar to bone development, which is preventable, and reversible. In
∗
specific, the development and progression of VC involved the transformation of vascular smooth muscle cells (VSMCs) into an osteoblastlike phenotype (Abedin et al., 2004; Bostrom et al., 2011; Shao et al., 2006). Trans-differentiation of VSMCs from a fibroblastic phenotype to an osteoblastic phenotype was carried out by enhancing the osteochondrogenic transcription factor RUNX-2 and a series of osteoblast differentiation markers (Cui et al., 2012; Liang et al., 2012; Shan et al., 2011; Thompson and Towler, 2012; Yuan et al., 2011). Bone morphogenetic proteins (BMP) played crucial role in bone formation. BMP-2 is a super family of transforming growth factors-β (TGF-β) and secretory growth factors. Downstream effectors of BMP-2, osterix (OSX) and RUNX-2 played crucial role in vascular calcification and atherosclerosis (Derwall et al., 2012; Hruska et al., 2005; Johnson et al., 2006). BMP-2 is a mediator of vascular calcification; BMP-2 activates BMP-2 receptor and induced-receptor SMADs, which induce osteoblast differentiation by up-regulation of transcription factors such as RUNX-2, OPN, SMAD4 and collagen 1α. RUNX-2 is major transcription factor of vascular calcification, which induced osteoblast differentiation. Furthermore, expression of osteopontin (OPN) and type I collagen (COL 1α) are
Corresponding author. E-mail address: [email protected] (Y. Soh).
https://doi.org/10.1016/j.ejphar.2019.172772 Received 30 May 2019; Received in revised form 23 October 2019; Accepted 1 November 2019 0014-2999/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Mahesh Sapkota, et al., European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2019.172772
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necessary for preosteoblast and osteoblastogenic differentiation (Byon et al., 2008; Harada et al., 1999). Therefore, BMP-2 and RUNX-2 signaling play pivotal role in addressing the therapeutic target in vascular diseases. Vitamin D3 plays crucial role in mineralization, and stimulating absorption of calcium and phosphorous, as well as in enhancing osteoblast differentiation and bone modulators expression. (Inada et al., 1999; Yang et al., 2015). Aloe-emodin (AE) 1, 8-Dihydroxy-3-(hydroxymethyl) anthraquinone, is a natural compound found in the leaves of Aloe-vera. AE has been in various pharmacological activities and showed antiviral, antimicrobial, and hepato-protective roles and anti-inflammatory activities (Agarwal et al., 2000; Eshun and He, 2004; Hu et al., 2014; Li et al., 2014). Recent studies showed that, AE exhibit anti-cancer activity on hepatoma cancer cell growth (Zhang et al., 2015). Next, several study showed that, AE significantly repressed proliferation of MKN45 gastric cancer cells (Chihara et al., 2015). AE also exhibit anti-cancer effects on lung carcinoma, neuro-ectodermal tumors, and cervical cancer (Guo et al., 2007; Lee, 2001; Pecere et al., 2000). Those results suggest that AE acts as a cancer chemo-preventive agent. However, the role of AE in vascular calcification is unknown to date. In this study, we demonstrate that AE suppressed vascular calcification through inhibiting osteoblast differentiation genes and proteins in vitro.
Table 1 Primer sequences and conditions for RT-PCR. Target genes (Accession number)
Primer (Forward, Reverse)
Annealing Tm (°C)
PCR cycles
SMAD4 (AY493561)
5′-atctatgcccgtctgtggag-3′ 5′-aacattcctgtggcttccac-3′ 5′-actttctccaggaagactgc-3′ 5′-acagcaacagcaacaacagc-3′ 5′-actacccacccttccctcac-3′ 5′-ccttaacccagctccctacc-3′ 5′-ttctcctggtaaagatggtgc-3′ 5′-tgttaaaggtgatgctggtcc-3′ 5′-gaccaccatggacgacgatg-3′ 5′-tggaacttgcttgactatcga-3′ 5′-ttctacaatgagctgcgtgt-3′ 5′-ctcatagctcttctccaggg-3′
50
30
50
30
55
35
50
35
58
30
50
26
RUNX-2 (NM_009820) Osterix (AF184902) Col 1α (NM_007742) OPN (BC002113.1) β-actin (NM_007393)
On every 48 h media was changed. On day 7, both alizarin red staining and von Kossa staining were performed. Briefly, VSMCs was washed with 1 × PBS for 3 times and fixed with 1 × paraformaldehyde for 1 h at 16 °C. Then 2% alizarin red (AR) was added next for 1 h. Then, plate was washed with 1 × PBS and image was captured by the digital camera. Similarly, for Von Kossa staining, VSMCs were washed with distilled water for several times, and then 5% silver nitrite solution was added and exposed in UV rays for 1 h. Now, silver nitrate solution was washed with distilled water and image was captured by digital camera.
2. Material and methods 2.1. Reagents, antibodies and chemicals
2.5. Reverse transcriptase – PCR
Aloe-emodin (AE) was purchased from Tokyo Chemical industry (TCI, Tokyo, Japan). Anti-rabbit RUNX-2 antibody was obtained from Bio world (Bioworld Technology, MN, USA). Anti-mouse smooth muscle actin (SMA), anti-goat collagen 1α antibody, anti-goat osteoprotegerin (OPG) antibody and anti-mouse β-actin antibody were obtained from Santa Cruz biotechnology Inc (Santa Cruz, CA, USA). Antirabbit BMP-2 antibody was purchased from Abcam (Cambridge, MA). TRIzol and superscript II Reverse Transcriptase was obtained from Invitrogen (Carlsbad, CA, USA). AgNO3, CaCl2, NaH2PO4, Na2HPO4 and other chemicals were bought from sigma (St. Louis, MO, USA) and other details are previously described (Sapkota et al., 2015).
Total RNA was isolated from cell culture using TRizol (Invitrogen) and cDNA synthesis was performed with super script II reverse transcriptase (Invitrogen) according to manufacturer's protocol and stored at −80 °C. All PCR primers were purchased from Bioneer (Daejon, Korea). Specific primer sequences are listed in Table 1. After initialization and denaturation, PCR was performed for various cycles (30 s at 94 °C, 1 min at annealing temperature and 2 min at 72 °C) using Taq polymerase (Promega, Madison, WI, USA). Reaction products were separated in 1% agarose gel, stained with ethidium bromide, and analyzed by densitometry using a Phosphoimager and Quantity One software (Version 4.3.1) (Bio-Rad, Hercules, CA, USA).
2.2. Vascular smooth muscle cell culture
2.6. Western blotting
Mouse VSMCs were isolated from the thoracic aorta of 6 week-old ICR mice (n = 5, approximately 30 g body weight). Animal handling was approved by ethics committee of Chonbuk National University, South Korea (Approval no. CBNU 2018-094). Enzyme solution was prepared by mixing collagenase II, elastase and soybean trypsin inhibitor, according to protocol. VSMCs were treated with enzyme solution for 30 min and washed with DMEM/F12 for three times. Then, VSMCs were seeded in 100 mm plate for 5–6 days, and media was changed every 48 h. Cells were incubated at 37 °C, 5% CO2 humidified condition. VSMCs of 5–8 passages were used for further experiments.
VSMCs were harvested, lysed in lysis buffer [20 mM Tris-HCl (pH = 7.5), 137 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM Na3VO4, 1 mM phenylmethylsulfonylfluoride (PMSF), and 1 × protease inhibitor cocktail]. Cell lysates were centrifuged at 13,000×g for 15 min, supernatant were used as cell extracts and stored at −70 °C until use. Proteins were quantified by bicinchoninic acid (BCA) protein assay. Proteins were resolved by SDS-PAGE on 8–10% gels and then transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad) with a glycine transfer buffer [192 mM glycine, 25 mM Tris-HCl (pH = 8.8), 20% MeOH (v/v)]. Membranes were blocked with 5% nonfat skim milk in Tris-buffered saline (TBS) containing 0.25% Tween – 20 (TBST) 16 °C for 1 h. Then incubated for 16 h at 4 °C with rabbit anti -collagen Iα (Santa Cruz Biotechnology), anti – rabbit BMP-2 (Abcam), anti-rabbit RUNX-2 (Bio world), Anti-mouse smooth muscle actin (SMA) (Santa Cruz Biotechnology), anti-goat osteoprotegerin (OPG) (Santa Cruz Biotechnology) or anti – mouse β-actin (Santa Cruz Biotechnology) antibodies diluted 1:1000 in 5% nonfat skim milk in TTBS. Antibody-antigen complexes were visualized with Clarity ™ Western ECL Substrate (Bio-Rad).
2.3. Cell viability MTT assay was used to observe the cell viability in VSMCs. VSMCs were seeded with or without Ca2+ and various doses of AE for 48 h. After 48 h, VSMCs cell were washed with 1 × PBS and 100 μg/ml of MTT was added for 2 h at 37 °C and again washed with 1 × PBS, then VSMCs was dissolved with 200 μl dimethyl sulfoxide (DMSO) and quantified with a spectrophotometer at 540 nm. 2.4. Alizarin red staining and von Kossa staining
2.7. Ex-vivo cell culture
VSMCs were seeded in 48 well plate in the presence of complete media DMEM/F12. VSMCs were treated with or without 3.6 mM Ca2+ as a calcification media with different concentration of AE for 7 days.
In aseptic condition, VSMCs were obtained from thoracic aorta of 6 2
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(oCPC). The calcium level increased in Ca2+ induced VSMCs compared to without calcium group whereas significantly suppressed by AE in a dose dependent manner in Ca2+ induced VSMCs (Fig. 1B). As we observed, AE inhibits VSMCs calcification in various concentration, which may be due to cytotoxic effects. To examine the effects of AE on cell viability in Ca2+ induced VSMCs, we performed MTT assay. We showed that there was no effect on viability of VSMCs at given concentrations and 20 μM of AE was selected as working concentration in VSMCs (Fig. 1C). These results demonstrate that AE prevents vascular calcification of VSMCs by inhibiting mineralization.
week-old ICR mice (n = 5, approximately 30 gm). All the procedures were performed with some modification as described in (Price et al., 2006). Mice thoracic aorta were chopped into 3 mm in length and placed in 48 well plates with DMEM/F12 for 7 days in presence or absence of Ca2+ and AE. After 7 days, thoracic aorta was washed with 1 × PBS. 5% formalin was used to fixed, and thoracic aorta was vacuumed overnight at 60 °C, embedded in paraffin. Histologic sections of 5 μm slices were made for alizarin red staining, von Kossa staining and hematoxylin and eosin (H&E) staining. 2.8. Calcium deposition quantification
3.2. AE attenuates osteogenic genes expression during vascular calcification induction
For the quantification of calcium deposition in VSMCs, the cells were washed with 1 × PBS for several times, then 0.6 N HCL was used for overnight to decalcified calcium. The calcium contents were measured by using colorimetric reagents, the o-cresolphthalein complexone (oCPC) method (Bioassay System, Cat.DICA500). Briefly, the remaining cells were washed with 1 × PBS and the cells were lysed with lysis buffer, bicinchoninic acid (BCA) method (Pierce) was used to determined total protein contents. For each culture, total calcium content was normalized by total protein content. Further, calcium quantification in aortic tissues, segment of aorta was weighed and decalcified with 0.6 N HCL. According to above procedure calcium contents were determined and the data were expressed as calcium per mg of dry aortic tissues.
The effect of AE on the osteogenic differentiation of VSMCs was investigated by using reverse transcriptase - PCR (RT-PCR). We showed that the expression of osteogenic differentiation genes such as SMAD4, Collagen 1α, OPN, RUNX-2 and Osterix markedly increased in Ca2+ treated group and those expressions were significantly inhibited by AE in a dose dependent manner (Fig. 2A and B). Pi also increased the calcification of osteogenic genes such as SMAD4, RUNX-2 and OPN, expression of those genes were markedly decreased by AE in dose dependent manner (Fig. 3A and B). Together, these results suggested that AE modulates osteogenic differentiation of VSMC by inhibiting various genes, which are important for VSMCs calcification.
2.9. Vitamin D3-induced calcification in mice aorta 3.3. AE suppresses osteogenic-protein expression in VSMCs
Vitamin D3-induced calcification of aorta in 6 weeks ICR male mice were used for in vivo study. Briefly, 6 weeks ICR mice were randomly selected into 4 groups with (n = 5) mice in each group; 1) Control, 2) Vitamin D3, 3) Vitamin D3 + AE (5 mg/kg), Vitamin D3 + AE (25 mg/ kg). 25 mg/kg AE do not cause any mortality or clinical signs (Fabrice et al., 2009). All mice were subjected to gavage daily with corn oil (control) or 5 mg/kg or 25 mg/kg AE at once per day for 13 days. Three days after AE treatment, the mice were subcutaneously administrated Vitamin D3 (cholecalciferol, 5.5 × 105 U/kg per day) for 3 following days. After six days of final exposure to vitamin D3, mice were anesthetized and entire aorta was taken out by dissection to observe calcium deposition (Ha et al., 2014).
Ca2+ plays crucial role in calcium deposition in VSMCs (Demer and Tintut, 2008). Osteogenic transcription factors including Collagen 1α, BMP-2, RUNX-2 and SMA are representative markers (Du et al., 2011; Leopold, 2014; Steitz et al., 2001; Wallin et al., 2001). We investigate whether AE regulates the bone-formation associated protein in calcification. Ca2+ significantly increased the protein expressions of Collagen 1α, BMP-2, RUNX-2 and SMA whereas increased protein expression was significantly decreased by AE in a concentration dependent manner (Fig. 4A and B). These results suggest that AE controlled different osteogenic protein, which plays pivotal role in vascular calcification.
2.10. Statistical analysis
3.4. AE represses calcification of VSMC in an ex-vivo model
Data are presented as mean ± S.D. of at least three experiments was performed. One-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test was performed in SPSS ver. 12.0 software to compare among the various dose groups and *P < 0.05, **P < 0.001 was considered as statistical significant.
As we found that AE inhibits the calcification of VSMCs by regulating the expression of osteogenic gene and protein expression in vitro. Next, we used AE in ex-vivo calcification model by extracting thoracic aorta from the mouse. Thoracic aorta was cultured in media in presence or absence of Ca2+ and AE. VSMCs calcification was observed by alizarin red staining and von Kossa staining. We found that Ca2+ increased mouse aortic calcification while AE markedly reduced calcification area (Fig. 5A). Furthermore, AE decreased the deposition of calcium in mouse aorta dose dependently (Fig. 5B). These results suggest that AE plays an important role in suppressing aortic calcification.
3. Results 3.1. AE inhibits Ca2+ induced calcification in VSMCs Previous studies have shown that vascular calcification is occurred by Ca2+, which is well known in both in vitro and in vivo model (Demer and Tintut, 2008). We investigated whether the AE modulated on mouse thoracic aorta in VSMCs. VSMCs were treated with or without Ca2+ and AE followed by culturing for 7 days. The results of alizarin red staining showed that Ca2+ treated group markedly increased calcium phosphate deposition compared to the group without Ca2+ treatment and these effects were significantly inhibited by co-treatment with Ca2+ and AE in a dose dependent manner. Moreover, von Kossa staining showed that Ca2+ induced VSMCs calcium phosphate and those VSMCs calcium phosphates were significantly inhibited by AE in a concentration dependent manner (Fig. 1A). Furthermore, we explored the calcium level in VSMCs by using o-cresolphthalein complexone
3.5. AE attenuates vitamin D3-induced calcification in-vivo Treatment with Ca2+ in in-vitro and ex-vivo model induced vascular calcification. Vitamin D3 was subcutaneously injected in mice to create an aortic calcified model. Vitamin D3 intoxication has been studied as a model of arterial calcification (Neven and D’Haese, 2011). We found that AE markedly repressed Vitamin D3 induced calcium deposition in the aorta of mice (Fig. 6A). Furthermore, AE decreased calcium level in a concentration dependent manner (Fig. 6B). Taken together, these results suggest that AE plays crucial role to suppress vascular calcification by suppressing bone-related VSMCs calcification. 3
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Fig. 1. AE suppresses the calcification in vascular smooth muscle cells. VSMCs were cultured with DMEM/F12 (growth media) or Ca2+ (3.6 mM) with or without 10 μM and 20 μM concentration of AE for 7 days. (A) Alizarin Red staining and von Kossa staining and (B) calcium level in mice was observed. Results are expressed as mean ± SDs (n = 3). *P < 0.05 indicates significant differences from the Ca2+ (3.6 mM)-induced group. (C) VSMCs were cultured with DMEM/F12 or Ca2+ (3.6 mM) in the presence or absence of various dose (5, 10, 20, 40 and 80 μM) of AE for 48 h. Cell viability in VSMCs was observed by MTT assay. Results are presented as the mean ± S.D. (n = 3). Ca2+ (3.6 mM) act as control.
4. Discussion
differentiation during vascular calcification (Shimizu et al., 2011; Tyson et al., 2003). BMP-2 and SMAD4 are closely associated with each other during vascular calcification. Various studies showed that BMP-2 canonical SMAD4 pathway is included in bone remodeling with osteogenic activity (Broege et al., 2013; Zhu et al., 2015). AE is a variety of emodin present in aloe latex, and an exudate from aloe plant. Emodin activates the mRNA and protein expressions of BMP-2 in the differentiation process of mouse osteoblastic MC3T3-E1 subclone 4 cells (Lee et al., 2007). Emodin prevents intima thickness in balloon-injured carotoid artery of rats via Wnt4/Dvl-1/β-catenin signaling pathway mediated by miR-126 (Jun et al., 2015). The expressions of proliferating cell nuclear antigen (PCNA) mRNA and protein were prominently decreased by the addition of AE in VSMC after arterial injury (Chengbin et al., 2001). Our study showed that AE markedly suppressed Ca2+ induced BMP-2 and SMAD4 expression during VSMCs calcification (Figs. 2 and 5). Next, VSMCs osteogenic differentiation is characterized by up regulation of bone related molecule RUNX-2 (Steitz et al., 2001). We found that AE attenuated Ca2+ induced RUNX-2 expression of gene and protein throughout VSMCs calcification (Figs. 2 and 5). Likewise, vascular calcification is an active process as osteogenesis, in which vascular smooth muscle cell acquire the osteogenic phenotype with the increase of collagen 1. Collagen 1 play an important role in the formation of calcified structures and osteoblast differentiation (Chen et al., 2015). We observed that AE prevents Ca2+ induced Collagen 1α expression during VSMCs calcification both mRNA and
Several studies shown that vascular smooth muscle cells (VSMCs) calcification is an active, highly mediated and controlled route. Those processes may be associated with various mechanism including disorder of calcium-phosphorous mechanism, osteoblast phenotype transformation and imbalance of inhibitor/promoter of vascular calcification. AE is an anthroquinone from the Aloe vera (Aloe barbadensis miller). AE has been reported to have functions in anti-inflammatory agent, anti-immunomodulator, and anti-tumor effects (Wang et al., 2014). However, the effects of AE on vascular calcification in VSMC have not been identified. In this study, we initially detected effects of AE on mouse thoracic aorta in VSMCs. We found that AE significantly suppressed Ca2+ induced calcification as decided by alizarin red staining, von Kossa staining and calcium quantities in in-vitro (Fig. 1A and B). A series of experiments were conducted to identify the toxicological effect of AE on vascular smooth muscle cells. AE had no cytotoxic effect till 20 μM in the Ca2+ containing media treated to VSMCs (Fig. 1C). In the present study, Osteoblast alteration to VSMCs was known as a key process of calcification. During calcification, VSMCs acquire bone related phenotype and categorized by an increased in BMPs, such as BMP-2, OPN, and collagen 1α (Du et al., 2011; Leopold, 2014; Wallin et al., 2001). BMP-2 plays pivotal role in osteoblastic cells 4
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Fig. 2. AE inhibits osteoblastic genes during vascular calcification. VSMCs were cultured with DMEM/F12 (growth media) or Ca2+ (3.6 mM) with or without 10 μM and 20 μM concentration of AE for 7 days. (A) RT-PCR was performed to observe the gene expression of osteoblastic related genes. (B) Quantitative analysis of mRNA (%) compared with Ca2+ (3.6 mM). Results are presented as means ± S.D. (n = 3). *P < 0.05, **P < 0.001 indicates significant differences from the Ca2+ (3.6 mM) - induced group.
addition, osterix regulated the expression of osteoblast related genes during the mineralization of VSMCs by activating the downstream of RUNX-2 (Nakashima et al., 2002). We found that AE attenuates Ca2+ induced mRNA expression of osterix during VSMCs calcification (Fig. 2). Smooth muscle actin is found in VSMCs enhances VSMCs
protein expression (Figs. 2 and 5). Similarly, OPN involved in regulation of embryonic bone formation as well as skeleton bone remodeling and also plays pivotal role against vascular calcification (Dhore et al., 2001; Speer et al., 2002). In this study, we found that AE prevents Ca2+ induced OPN gene expression during VSMCs calcification (Fig. 2). In
Fig. 3. AE inhibits osteoblastic marker genes during vascular calcification. (A) VSMCs were cultured with DMEM/F12 (growth media) or Pi (3.6 mM) with or without 10 μM and 20 μM concentration of AE for 7 days. RT-PCR was performed to observed osteoblastic marker genes expression. (B) Quantitative analysis of mRNA (%) compared with Pi (3.6 mM). Results are presented as means ± S.D. (n = 3). *P < 0.05 indicates significant differences from the Pi (3.6 mM) - induced group. 5
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Fig. 4. AE attenuates BMP-2 pathways. VSMCs were cultured with DMEM/F12 (growth media) or Ca2+ (3.6 mM) with or without 10 μM and 20 μM concentration of AE for 7 days. (A) Western blot analysis was performed to determine the expression of osteoblastic related proteins. (B) Expression of collagen 1α, BMP-2, RUNX-2 and SMA quantified and compared with Ca2+ (3.6 mM). Results are presented as means ± S.D. (n = 3). *P < 0.05, **P < 0.001 indicates significant differences from the Ca2+ (3.6 mM) - induced group.
could induce cardiovascular abnormalities such as intima-media thickness and coronary artery calcification (Briese et al., 2006; Goldsmith et al., 1997). Vitamin D3 induced aortic calcification was enhanced by up-regulation of osteogenic marker gene expression in medial aorta region (Han et al., 2013). We investigated whether AE treatment could inhibit vitamin D3 induced aortic calcification in vivo. We found that AE markedly reduced vitamin D3 induced aortic calcification in vivo (Fig. 6). Taken together, our study suggest that AE attenuates VSMCs calcification through BMP-2 dependent SMAD4 signaling pathways (Fig. 5C). Furthermore, this results shown that AE inhibits vascular calcification in vitro and in vivo. Finally, our finding suggest that AE may
during vascular calcification (Du et al., 2011; Leopold, 2014). In our study, we found that AE decreases Ca2+ induced protein expression of SMA during VSMCs calcification protein expression (Fig. 4). Together these results support the suggestion that AE significantly inhibits the vascular calcification and osteoblast differentiation related-genes and proteins in VSMCs which suggest that AE could prevents vascular calcification in-vitro. Next, we explored the effects of AE on vascular calcification in us ex vivo thoracic aortic ring calcification model. We found that AE markedly decreased aortic calcification and calcium deposition, which suggest that AE could suppress phenotypic conversion of VSMCs to osteoblast. Furthermore, various study showed that vitamin D therapy
Fig. 5. AE suppresses vascular calcification in an ex-vivo condition. The 3 mm aortic ring was isolated from mouse and incubated with DMEM/F12 (growth media) or Ca2+ (3.6 mM) in the presence or absence of 10 μM and 20 μM concentration of AE for 7 days. (A) Alizarin red staining and von Kossa staining were used to detect calcified region. (B) Amount of calcium deposition level in aortic ring was observed. Results are presented as means ± S.D. (n = 5). *P < 0.05, indicates significant differences from the Ca2+ (3.6 mM) - induced group. 6
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Fig. 6. AE protects vitamin D3-induced calcium level in mice aorta. Von Kossa staining was performed to identify calcium deposition (A) and quantified the calcium deposition level (B) in aorta. Results are presented as means ± S.D. (n = 5). (C) The schematic diagram of the effects of AE on Ca2+ induced vascular calcification in VSMCs. *P < 0.05, indicates significant differences from the Ca2+ (3.6 mM)-induced group.
be a possible therapeutic phytochemical in inhibiting diseases related to vascular calcification.
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