Evodiamine Attenuates PDGF-BB-Induced

Article

Evodiamine Attenuates PDGF-BB-Induced Migration of Rat Vascular Smooth Muscle Cells through Activating PPARγ Xie Ge, Siyu Chen, Mei Liu, Tingming Liang and Chang Liu * Received: 21 September 2015; Accepted: 18 November 2015; Published: 26 November 2015 Academic Editor: Julian Borejdo Jiangsu Key Laboratory for Molecular and Medical Biotechnology and College of Life Sciences, Nanjing Normal University, Nanjing 210023, China; [email protected] (X.G.); [email protected] (S.C.); [email protected] (M.L.); [email protected] (T.L.) * Correspondence: [email protected]; Tel.: +86-25-8589-1870; Fax: +86-25-8589-1702

Abstract: The uncontrolled migration of vascular smooth muscle cells (VSMCs) into the intima is a critical process in the development of atherosclerosis. Evodiamine, an indole alkaloid extracted from the Chinese medicine evodia, has been shown to inhibit tumor cell invasion and protect the cardiovascular system, but its effects on VSMCs remain unknown. In the present study, we investigated the inhibitory effects of evodiamine on the platelet-derived growth factor-BB (PDGF-BB)-induced VSMC migration using wound healing and transwell assays, and assessed its role in decreasing the protein levels of matrix metalloproteinases and cell adhesion molecules. More importantly, we found that evodiamine activated the expression and nuclear translocation of peroxisome proliferator-activated receptor γ (PPARγ). Inhibition of PPARγ activity by using its antagonist T0070907 and its specific siRNA oligonucleotides significantly attenuated the inhibitory effects of evodiamine on VSMC migration. Taken together, our results indicate a promising anti-atherogenic effect of evodiamine through attenuation of VSMC migration by activating PPARγ. Keywords: evodiamine; vascular smooth muscle cells; migration; peroxisome proliferator-activated receptor γ

1. Introduction The migration of vascular smooth muscle cells (VSMCs) occurs under both physiological (e.g., vascular development) and pathological (e.g., atherogenesis responsive to vascular injury) circumstances. During vascular development, chemoattractants such as platelet-derived growth factor-BB (PDGF-BB) stimulate migration of pericytes or other smooth muscle precursors, leading to the correct formation of vessel wall structure [1]. In contrast, accelerated VSMC migration contributes importantly to neo-intimal hyperplasia observed in atherosclerosis and restenosis. Usually, balloon catheterization or stent placement triggers the endothelial injury and subsequent abberant VSMC migration, and then the cells migrate from the adventitial and the medial layers to the intimal layer of the vessel wall. Followed by their abnormal migration, VSMC proliferation starts and causes arterial thickening [2]. Migration of VSMCs is a very complicated process and is finely regulated by multiple factors. One of the most important mechanisms involves the crosstalk between inflammation and oxidative stress. Inflammatory cytokines, which are involved in the development and progression of inflammation, function robustly in the regulation of VSMC migration [3]. In addition, chronic and mild inflammation leads to the increased generation of reactive oxygen species (ROS), which induces VSMC migration through activating a series of signal molecules, including protein tyrosine

Int. J. Mol. Sci. 2015, 16, 28180–28193; doi:10.3390/ijms161226093

www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2015, 16, 28180–28193

phosphatases/kinases, extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK), and transcription factors [4]. Once VSMC migration is initiated, the expression and secretion of matrix metalloproteinases (MMPs) are upregulated, which facilitates the degradation of vascular basement membrane and extracellular matrix (ECM) proteins [3]. Given the importance of VSMC migration in the pathogenesis of cardiovascular diseases, inhibiting this pathological process may be beneficial for the prevention or alleviation of angiogenesis and other kinds of vascular injury. In fact, retardment of excess smooth muscle cell migration through targeted disruption of MMP-9 leads to reduced intimal hyperplasia and improves arterial remodeling [5]. In addition, the inhibition of VSMC migration by rapamycin and paclitaxel is a contributing factor in their beneficial effects of incorporation into vascular stents [1]. Similarly, as the inhibitors of VSMC migration, peroxisome proliferator-activated receptor γ (PPARγ) activators can also be used for the treatment of cardiovascular diseases [6–8]. For example, troglitazone, as a PPARγ agonist, inhibits the activation of ERK, the degradation of p27 (Kip1), the phosphorylation of retinoblastoma protein, and the progression of cell cycle from G1 to S, and thus collectively leads to the reduction of VSMC migration [9]. However, the compounds mentioned above have serious side-effects. Rapamycin (an inhibitor of mammalian target of rapamycin (mTOR)) and paclitaxel (a tubulin-binding agent) encounter unresolved issues such as impaired re-endothelialization and subsequent thrombosis induction [10]. On the other hand, the clinic use of strong PPARγ-activating drugs were either suspended or restricted because of their adverse effects including weight gain, liver injury and the increased risk of cancer, and cardiovascular events [11]. Such problems make the characterization of other compounds able to suppress VSMC migration highly clinically relevant. To this end, the implication of natural plant-derived compounds in controlling the migration of VSMCs in diseased arteries has been widely studied in the last decade. Evodia rutaecarpa (Chinese name: Wu-Chu-Yu) has been used to make spicy sauce for a long history, and is a traditional Chinese medicine used for the treatment of headache, hypertension, dyspepsia, gastrointestinal dysfunction, and so on. Evodiamine (Figure 1A) is the major ingredient isolated from the fruit of E. rutaecarpa. As a quinolone alkaloid, it possesses many biological effects, such as anti-inflammatory, anti-tumor, anti-obesity, vascular relaxing, thermoregulatory and uterine contracting effects [12]. Of note, evodiamine exhibits anti-tumor properties by inhibiting motility of various cancer cell lines. For exapmle, it has been reported that evodiamine suppresses migration of several tumor cell lines induced by hepatocyte growth factor partly through retardation of cell spreading [13]. In addition, evodiamine represses the migration and invasion of human breast cancer cells by downregulating the expression of MMP-9, urokinase-type plasminogen activator (uPA), and uPA receptor (uPAR) [14]. More interestingly, evodiamine shows a protective effect on the cardiovascular system. For example, in isolated rat mesenteric arteries, evodiamine exhibits a vasodilative effect [15]. Evodiamine also acts as a diuretic through inhibiting the releasing of aldosterone, which plays a role in blood volume controlling [16]. Moreover, evodiamine inhibits LIGHT-induced production of ROS and pro-inflammatory cytokines, the phosphorylation of MAPKs (p38 and ERKs), and the activation of NADPH oxidase in human monocytes [17]. These findings suggest that evodiamine has the potential to treat cardiovascular diseases. Although evodiamine has been proven to retard the metastasis of tumor cells and is beneficial for the cardiovascular system, whether evodiamine possesses similar actions in VSMCs remains unknown. Therefore, in the present study, we sought to investigate the anti-migrative activity and the potential target of evodiamine in PDGF-BB-stimulated VSMCs.

28181

Int. J. Mol. Sci. 2015, 16, 28180–28193

2. Results 2.1. Evodiamine Inhibits Platelet-Derived Growth Factor (PDGF)-BB-Induced Vascular Smooth Muscle Cell (VSMC) Migration We first assessed the cytotoxicity of evodiamine prior to investigating its effect on VSMC migration. The Cell Counting Kit-8 (CCK-8) assay results revealed that evodiamine at the concentrations lower than 2 µM did not inhibit cell viability over 30 h (Figure 1B). We therefore selected 0.1 and 0.5 µM of evodiamine as the safe doses for VSMCs and used them throughout Int. J. Mol. Sci. 2015, 16, page–page our study. We then used a wound healing migration assay to evaluate the effect of evodiamine on concentrations lower than 2 μM did inhibit cell viability over 30 h (Figure 1B). Werate therefore PDGF-BB-induced VSMC migration. In not contrast to the unchanged migration of VSMCs with selected 0.1 and 0.5 μM of evodiamine as the safe doses for VSMCs and used them throughout our the treatment of evodiamine alone, the wound closure rate reached to approximately a four-fold study. We then used a wound healing migration assay to evaluate the effect of evodiamine on higher level upon stimulation with 10 ng/mL PDGF-BB for 24 migration h, compared to thewith untreated control. PDGF-BB-induced VSMC migration. In contrast to the unchanged rate of VSMCs the treatment of evodiamine alone, theatwound rate inhibited reached to approximately a four-fold higher However, treatment with evodiamine 0.1 orclosure 0.5 µM PDGF-BB-induced VSMC migration level upon stimulation with 10 ng/mL PDGF-BB for 24 h, compared to the untreated control. in a dose-dependent manner (Figure 1C). The inhibitory effect of evodiamine on VSMC migration However, treatment with evodiamine at 0.1 or 0.5 μM inhibited PDGF-BB-induced VSMC migration was further confirmed by a transwell migration assay. Figure showson that themigration number of VSMCs in a dose-dependent manner (Figure 1C). The inhibitory effect of 1D evodiamine VSMC was further confirmed by a transwell migration assay. Figure 1D shows that the number of VSMCs percentage able to move across the membrane was significantly reduced by evodiamine. Inhibition able to move across the membrane was significantly reduced by evodiamine. Inhibition percentage of migrated cells treated with evodiamine (0.1 or 0.5 µM) was 40% and 60%, respectively, compared of migrated cells treated with evodiamine (0.1 or 0.5 μM) was 40% and 60%, respectively, compared with the PDGF-BB-stimulated cells. cells. with the PDGF-BB-stimulated

Figure 1. Evodiamine cytotoxicity test and its effect on platelet-derived growth factor (PDGF)

Figure 1. Evodiamine cytotoxicity test and its effect on platelet-derived growth factor (PDGF) -BB-induced vascular smooth muscle cell (VSMC) migration. (A) Chemical structure of evodiamine; -BB-induced (B) vascular smooth muscle cell (VSMC) migration. Chemical structure of evodiamine; Cytotoxicity test using evodiamine. VSMCs were cultured (A) in medium containing 10% fetal bovinetest serum, and treated with evodiamine at thewere indicated concentrations for 30 h. Cell viability (B) Cytotoxicity using evodiamine. VSMCs cultured in medium containing 10% fetal was measured using the Cell Counting Kit-8 (CCK-8) assay. Results are expressed as cell viability bovine serum, and treated with evodiamine at the indicated concentrations for 30 h. Cell viability relative to untreated controls; (C,D) Evodiamine inhibits PDGF-BB-induced VSMC migration; (C) was measured using thecells Cell Counting Kit-8 (CCK-8)forassay. Results are expressed Serum-starved were pretreated with evodiamine 6 h before the stimulation of PDGF-BBas forcell viability 24 h. Thereafter, cell migration was measured by usinginhibits a wound healing assay; (D) Cells seeded into migration; relative to untreated controls; (C,D) Evodiamine PDGF-BB-induced VSMC the inner chamber in serum-free medium were exposed to evodiamine with PDGF-BB for 6 h. Cell (C) Serum-starved cells were pretreated with evodiamine for 6 h before the stimulation of PDGF-BB migration was thereafter measured by using a Boyden chamber assay. Blue color indicates cell nuclei for 24 h. Thereafter, cell migration was measured byEvo, using a wound assay; (D) Cells seeded stained with 4′,6-diamidino-2-phenylindole (DAPI). evodiamine. Datahealing are represented as mean values ± SD ofin three independently preparedwere samples each with measurements. ** pPDGF-BB < 0.01 into the inner chamber serum-free medium exposed tofive evodiamine with for 6 h. compared with the control group; ## p < 0.01 compared with the PDGF-BB-stimulated group. Cell migration was thereafter measured by using a Boyden chamber assay. Blue color indicates cell nuclei stained with 41 ,6-diamidino-2-phenylindole (DAPI). Evo, evodiamine. Data are represented as mean values ˘ SD of three independently prepared samples each with five measurements. ** p < 0.01 3 compared with the control group; ## p < 0.01 compared with the PDGF-BB-stimulated group.

28182

Int. J. Mol. Sci. 2015, 16, 28180–28193 Int. J. Mol. Sci. 2015, 16, page–page

Migration-Associated Regulators Regulators 2.2. Evodiamine Inhibits the Expression of Migration-Associated previous studies, studies, the the increased increased expression expression of of MMPs MMPs induced induced by by PDGF-BB PDGF-BB plays According to previous promoting VSMC motility, motility, which which is tightly an important role in facilitating ECM degradation and promoting linked to to the the development development of of atherosclerosis atherosclerosis [18]. [18]. On On the the other hand, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and osteopontin (OPN) are inflammation-associated proteins, proteins, and and are are also also key regulators involved in cell migration [19,20]. inflammation-associated Western blot results showed that PDGF-BB treatment consistently increased the protein expression Western blot results showed ofallallthese these factors in VSMCs (Figure and evodiamine wastofound inhibit such levels of factors in VSMCs (Figure 2A,B)2A,B) and evodiamine was found inhibittosuch increases increases in a dose-dependent manner. Enzyme Linked Immunosorbent Assays (ELISA) that also in a dose-dependent manner. Enzyme Linked Immunosorbent Assays (ELISA) also indicated indicated that evodiamine suppressed the concentrations of MMP-2 and MMP-9 released to the evodiamine suppressed the concentrations of MMP-2 and MMP-9 released to the medium, which medium, which was originally upregulated PDGF-BB. The extents of suppression were 40% and was originally upregulated by PDGF-BB. Theby extents of suppression were 40% and 16%, respectively, 16%, respectively, compared to the PDGF-BB-stimulated group (Figure 2C). We also used Biotrak compared to the PDGF-BB-stimulated group (Figure 2C). We also used Biotrak Activity Assay Kits Activity Assay Kits (GE Healthcare) to quantifyactivities actual enzymatic of MMP-2 andactive MMP-9 in (GE Healthcare) to quantify actual enzymatic of MMP-2activities and MMP-9 in their form theirour active formindicated and our results indicated that evodiamine indeed suppressed PDGF-BB-induced and results that evodiamine indeed suppressed PDGF-BB-induced upregulation in upregulation in their2D). activities (Figure indicate 2D). These findings indicate evodiamine inhibits their activities (Figure These findings that evodiamine inhibits that PDGF-BB-induced VSMC PDGF-BB-induced VSMC migrationmigration-related through downregulating migration through downregulating proteins.migration-related proteins.

Figure 2. 2. Cont. Cont. Figure

28183 4


Figure 2.Figure Evodiamine inhibits inhibits the expression of migration-associated regulators. (A) VSMCs 2. Evodiamine the expression of migration-associated regulators. (A) were VSMCs were pretreated with evodiamine for 6 h and then stimulated with PDGF-BB for another 24 h. Western pretreated with evodiamine for 6 h and then stimulated with PDGF-BB for another 24 h. Western blot blot was was usedused to measure the protein expression levels of regulators involved in the VSMC to measure the protein expression levels of regulators involved in themigration, VSMC migration, includingincluding matrix metalloproteinase (MMP)-2, MMP-9, intercellular adhesion molecule-1 (ICAM-1), matrix metalloproteinase (MMP)-2, MMP-9, intercellular adhesion molecule-1 (ICAM-1), vascular vascular cell adhesion molecule-1 (VCAM-1), and osteopontin (OPN). For bothFor MMP-2 MMP-9, cell adhesion molecule-1 (VCAM-1), and osteopontin (OPN). both and MMP-2 and MMP-9, their latent and (72 92 kDa, respectively) were detected; (B) The signal of examined theirforms latent(72forms and 92 kDa, respectively) were detected; (B) Theratio signal ratio of examined protein to the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was quantified protein to the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was quantified by densitometric scanning;scanning; (C) The (C) concentrations of MMP-2 -9 inand the-9culture were by densitometric The concentrations of and MMP-2 in the medium culture medium were detected by Enzyme Linked Immunosorbent Assay (ELISA); (D) The activities of MMP-2 and MMP-9 detected by Enzyme Linked Immunosorbent Assay (ELISA); (D) The activities of MMP-2 and were determined by using their respective Biotrak Activity Assay Systems. Evo, evodiamine. Data MMP-9 were determined by using their respective Biotrak Activity Assay Systems. Evo, evodiamine. are presented as mean values ˘ SD of three independent experiments. * p < 0.05, ** p < 0.01 compared Data are presented as mean values ± SD of three independent experiments. * p < 0.05, ** p < 0.01 with the control group; # p < 0.05, ## p < 0.01 compared with the PDGF-BB-stimulated group. compared with the control group; # p < 0.05, ## p < 0.01 compared with the PDGF-BB-stimulated group.

2.3. Evodiamine Increases Peroxisome Proliferator-Activated Receptor γ (PPARγ) Expression and Nuclear Translocation in VSMCs Increases Peroxisome Proliferator-Activated Receptor γ (PPARγ) Expression and Nuclear 2.3. Evodiamine

Translocation VSMCs Evodiamine hasin been proven to be a PPARγ activator [21,22]. Therefore, we examined whether the expression of PPARγhas was also affected bya evodiamine in VSMCs. We found we thatexamined althoughwhether Evodiamine been proven to be PPARγ activator [21,22]. Therefore, rosiglitazone, a well-known PPARγ agonist, significantly induced PPARγ expression both at the the expression of PPARγ was also affected by evodiamine in VSMCs. We found that although mRNA rosiglitazone, and protein levels, evodiamine alone did not regulate PPARγ expression. In contrast, an a well-known PPARγ agonist, significantly induced PPARγ expression both at the obviousmRNA decrease wasprotein observed in VSMCs when treated withnot PDGF-BB, wasexpression. dose-dependently and levels, evodiamine alone did regulatewhich PPARγ In contrast, an reversedobvious by evodiamine (Figure 3A,B). decrease was observed in VSMCs when treated with PDGF-BB, which was Ondose-dependently the other hand, reversed since theby increased nuclear translocation from cytosol is an important evodiamine (Figure 3A,B). characteristicOn of the activated PPARγ, we also analyzed the distribution of PPARγ in cytosol the nucleus other hand, since the increased nuclear translocation from is anand important cytoplasm of VSMCs. We found that PDGF-BB inhibited, while evodiamine promoted, the ratio characteristic of activated PPARγ, we also analyzed the distribution of PPARγ in the nucleus and of nuclear/cytoplasmic PPARγ All these findings that PPARγpromoted, is one of the the ratio of cytoplasm of VSMCs. We(Figure found 3C). that PDGF-BB inhibited,suggest while evodiamine pharmaceutical targets of evodiamine. nuclear/cytoplasmic PPARγ (Figure 3C). All these findings suggest that PPARγ is one of the pharmaceutical targets of evodiamine.

28184

5


Figure 3. Evodiamine Evodiamineactivates activatesperoxisome peroxisome proliferator-activated proliferator-activated receptor receptor γγ (PPARγ) (PPARγ) in PDGF-BB-stimulated VSMCs. (A) (A) VSMCs VSMCs were were pretreated pretreated with with evodiamine evodiamine or or rosiglitazone rosiglitazone for 6 h RT-qPCR was was used used to to measure measure the mRNA and then stimulated with PDGF-BB for another 24 h. RT-qPCR expression levels levels of ofPPARγ; PPARγ;(B) (B)Western Westernblot blot was used to measure protein levels of PPARγ. expression was used to measure the the protein levels of PPARγ. The The signal ratio of PPARγ to the internal control GAPDH is shown at the bottom; (C) Nuclear signal ratio of PPARγ to the internal control GAPDH is shown at the bottom; (C) Nuclear and and cytoplasmic proteins extracted separately, and Western blot wastoused to measure the cytoplasmic proteins were were extracted separately, and Western blot was used measure the protein protein levels of PPARγ in both fractions. The ratio of nuclear/cytoplasmic PPARγ is shown at the levels of PPARγ in both fractions. The ratio of nuclear/cytoplasmic PPARγ is shown at the bottom. bottom. Evo, evodiamine. RSG, rosiglitazone. Data areaspresented as mean SD of three Evo, evodiamine. RSG, rosiglitazone. Data are presented mean values ± SD values of three˘independent ## p < 0.01 compared with the independent experiments. ** p < 0.01 compared with the control group; ## p < 0.01 compared with the experiments. ** p < 0.01 compared with the control group; PDGF-BB-stimulated group. group. PDGF-BB-stimulated

PPARγ Mediates the Actions of Evodiamine in Regulating VSMC Migration 2.4. PPARγ PPARγ and PPARγ has been shown to inhibit VSMC abnormal Since evodiamine activates PPARγ activation including excess proliferation and migration [6,23], we wonder whether the inhibitory effect of migration is PPARγ-dependent. To explore this possibility, we treated of evodiamine evodiamineononVSMC VSMC migration is PPARγ-dependent. To explore this possibility, we VSMCsVSMCs with 10 with µM T0070907, which is which a PPARγ decreasing the phosphorylation of PPARγ treated 10 μM T0070907, is aantagonist PPARγ antagonist decreasing the phosphorylation and impairing its DNA-binding ability. As shown Figurein4A, the 4A, wound healing healing assay results of PPARγ and impairing its DNA-binding ability. Asinshown Figure the wound assay demonstrated that 0.5that µM0.5 of μM evodiamine retarded PDGF-BB-induced VSMCVSMC migration by 60%. results demonstrated of evodiamine retarded PDGF-BB-induced migration by Interestingly, pretreatment of cellsofwith partiallypartially released released the blockthe of VSMC migration 60%. Interestingly, pretreatment cellsT0070907 with T0070907 block of VSMC by evodiamine. Results from the transwell showed a similar (Figure 4B).(Figure 4B). migration by evodiamine. Results from the assay transwell assay showedtrend a similar trend

28185 6


Figure 4. PPARγ PPARγ antagonist antagonist alleviated alleviated the the inhibitory inhibitory effects of evodiamine on PDGF-BB-stimulated VSMC migration. VSMCs were pretreated with 0.5 µM μM evodiamine for 6 h and then stimulated with 10 ng/mL PDGF-BB for for 24 24 hh (wound (wound healing) healing) or or 6 h (transwell). 10 10 μM µM T0070907 was given 0.5 h in ng/mL PDGF-BB advance of evodiamine treatment when required. Cell migration was measured by using a wound required. healing assay (A) or a Boyden chamber transwell assay (B). In Panel B, blue color indicates cell nuclei stained with DAPI. Evo, evodiamine. evodiamine. T, T, T0070907. T0070907. Data Data are are represented represented as asmean meanvalues values˘ ± SD of three independently prepared samples samples each with with five five measurements. measurements. ** p < 0.01 compared with the control ## p $$ p group; ## p