text:
Oxymatrine inhibits the migration of human colorectal carcinoma RKO cells via inhibition of PAI-1 and the TGF-β1/Smad signaling pathway

Transforming growth factor-β1 (TGF-β1) signaling has been shown to play a critical role in the development of epithelial-mesenchymal transition (EMT). PAI-1 is one of the most important target genes in the TGF-β/Smad signaling pathway, which can hinder the degradation of ECM composition and may promote cell invasion and migration. Oxymatrine (OM) is an alkaloid extracted from the Chinese herb Sophora flavescens Ait and has been demonstrated to inhibit the growth of various types of cancer cells including colorectal cancer. However, the anticancer effect of OM in colorectal cancer remains unclear. In the present study, we detected the expression of E-cadherin, α-SMA, FN, TGF-β1, PAI-1, Smad4, pP38 and pSmad2 in FHC, RKO and OM-treated RKO cells. We also detected pSmad2 and PAI-1 in RKO cells following the addition of SB203580 (a p38 MAPK inhibitor). The results showed that E-cadherin expression in RKO cells was significantly decreased, while PAI-1, TGF-β1, α-SMA, FN, Smad4, pSmad2 and pP38 expression levels were significantly increased in the RKO cells compared to levels in the FHC cells, which was almost completely reversed by OM. OM alleviated EMT induced in colorectal cancer via inhibition of TGF-β1/Smad signaling pathway activation by reducing P38-dependent increased expression of PAI-1. Hence, OM could be a novel therapeutic agent for colorectal cancer.

Introduction
Oxymatrine (OM) (structure shown in Fig. 1) is the principal component of Sophora flavescens Ait, which is frequently prescribed in traditional Chinese medicine. OM has been reported to have immune regulation, anti-inflammatory and diuretic effects (1)(2)(3)(4)(5)(6). Moreover, OM inhibits the growth of various types of cancer cells (7)(8)(9). Although there are some studies in regards to OM concerning the inhibition of malignant tumor cell growth, studies concerning the molecular mechanisms, signaling pathways, invasion and metastasis of the anticancer effects of OM are rare.
Colorectal carcinoma is one of the most common malignant tumors (10). Invasion and metastasis are fundamental properties of malignant colon cancer, which lead to a high recurrence rate after surgery and therapeutic approaches (11). In the process of tumor metastasis, epithelial-mesenchymal transition (EMT) is a key mediator (12). The molecular mechanisms of EMT involved in tumor metastasis remain unclear, and current treatments have limited effectiveness. Thus, in order to develop new effective therapeutic measures for colon cancer, further investigation of its molecular mechanisms is required.
Plasminogen activator inhibitor 1 (PAI-1) is a rapid and specific inhibitor of the plasminogen/plasmin system (13). In recent years, studies have demonstrated that PAI-1 is a potent regulator of tumor growth in vivo. Increased PAI-1 has been confirmed in many solid tumor types and was found to be associated with a poor prognosis (14)(15)(16). Thus, PAI-1 is regarded as a biochemical marker for poor prognosis and may serve as a therapeutic target for various types of cancers. There is evidence that plasma PAI-1 is closely correlated with rectal cancer metastasis, and tumor tissue PAI-1 is associated with the histopathology and outcome of rectal cancer (17). Whether PAI-1 may serve as a target in the antitumor therapy of colorectal carcinoma remains unclear.
EMT is a process by which epithelial cells lose their orientation and cell-cell contact, and acquire migratory and invasive properties of mesenchymal cells. Transforming growth factor-β1 (TGF-β1) is known as a key mediator of EMT (18). TGF-β1 promotes EMT via upregulation of ECM expression and downregulation of transcriptional activity of matrix-degrading enzyme genes. Increasing studies have shown that SBE (Smad binding element) exists in the promoter region of genes, such as PAI-1, regulated by TGF-β (19). SBE in the promoter region of these genes combines directly or indirectly with the Smad complex. PAI-1 is one of the most

Oxymatrine inhibits the migration of human colorectal carcinoma RKO cells via inhibition of PAI-1 and the TGF-β1/Smad signaling pathway
important target genes in the TGF-β/Smad signaling pathway, which can hinder the degradation of ECM composition and may promote cell invasion and migration (20). It is unclear how TGF-β/Smad controls PAI-1 in colorectal cancer and the relation between PAI-1 and colorectal cancer.
In the present study, we investigated the anti-metastatic and anti-invasive effects of OM in RKO cells. Simultaneously we detected the effects of OM on the TGF-β1/Smad signaling pathway and PAI-1 in RKO cells to investigate the underlying signaling molecular mechanisms by which OM inhibits the invasion and metastasis of RKO cells.

Materials and methods
Reagents and medicine. OM was obtained from National Institutes for Food and Drug Control (Beijing, China). The relative molecular mass of OM is 264.367, and the molecular structural formula is C 15 H 24 N 2 O 2 . The purity of OM was determined by high-performance liquid chromatography (HPLC) as 98%. OM, a colorless columnar crystal, was dissolved in PBS and prepared for 20 mg/ml mother solution. The solution was stored at -20˚C in a refrigerator and stored away from light. Serum-free and antibiotic-free RPMI-1640 medium was diluted to the required concentration. OM was diluted to the required concentration by serum-free and antibiotic-free RPMI-1640 medium for use.
Cell culture. Colorectal carcinoma RKO cells, obtained from ATCC Biosciences Center, were grown in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Carlsbad, CA, uSA), 100 µg/ml streptomycin and 100 U/ml penicillin in a humidified atmosphere containing 5% CO 2 at 37˚C. Cells grew as a single cell layer attached to specially treated plastic surfaces. The human fetal colon FHC cell line was cultured in basal medium (cat. no. 30-2006) supplemented with 10 mM HEPES, 10 ng/ml cholera toxin, 0.005 mg/ml insulin, 0.005 mg/ml transferrin, 100 ng/ ml hydrocortisone and 10% fetal bovine serum. In order to maintain the viability and active growth of adherent cells, it is necessary to subculture them at regular intervals (2 or 3 times a week). Cells during an exponential growth phase were used in the experiments. However, in the invasion and migration experiments, the cells were cultured in a serum-free medium. The cells were randomly divided into three groups: FHC cell control group (FHC group), untreated RKO cell group (uR group), and OM-treated RKO cell group (OR group).
Cell Counting Kit-8 assay. The antiproliferative effects of different concentrations of OM on the RKO cell line were detected by CCK-8 assay. Cells were seeded at a density of 1x10 4 per well in 100 µl of RPMI-1640 medium with 10% FBS in 96-well plates. On the next day, the medium was replaced with serum-free RPMI-1640 with OM (from 0-20 mg/ml) for the RKO cells. Each concentration was used in 4 wells. After incubation for 24, 48, 72 h in a humidified atmosphere containing 5% CO 2 at 37˚C, respectively, 10 µl CCK (5 mg/l) was added each well, and the plates were incubated for an additional 4 h. Then the medium was discarded, and the absorbance (A) of the CCK-8 solution was determined at 450 nm using a multiwell spectrophotometer (Bio-Tek Instruments, XL-808, Winooski, VT, uSA). The proliferation rate of the cells was calculated as follows: Proliferation rate (%) = A of the treated wells/A of the control wells (OM 0 mg/ml) x 100. The assay was performed in triplicate.
Wound healing assay. The cells were seeded into 6-well plates and when the cells were grown to confluency, a wound was created by manually scraping the cells with a pipette tip. Debris was removed from the culture by washing it twice with PBS. Then the cells were incubated with serum-free medium to exclude the effect of cell growth facilitated by serum and the cells were then allowed to migrate into the wounded area at 37˚C. Images were immediately acquired at 0 and 24 h after wounding and measured using Image-Pro Plus 6.0 software. The cell migration rate (%) = (wound width at 0 h -wound width at 24 h)/wound width at 0 h x 100. The assay was performed in triplicate.
Western blot analysis. Cells were plated onto culture flasks at a density of 2x10 5 cells/ml and cultured at 37˚C in 5% CO 2 . On the next day, different concentrations of OM (0, 0.25, 0.5, 1 and 2 mg/ml) were added. After 24 h, the levels of proteins were quantified through western blot analysis. Proteins were extracted by the addition of 200 µl of lysis buffer [50 mmol/l Tris-HCl (pH 7.4), 5 mmol/l EDTA, 1% Triton X-100, 150 mmol/l NaCl, 5 mmol/l MgCl 2 , 2 mmol/l Na 3 VO 4 , 1X Complete™ protease inhibitor] to each well. The cell lysates were incubated on ice for 30 min vortexing every 10 min, followed by centrifugation at 12000 x g for 30 min at 4˚C. An amount 40 µg/µl of protein of the cell lysate was mixed equally with 2X electrophoresis buffer [50% glycerol, 25% mercaptoethanol, 10% SDS, 0.3 M Tris (pH 6.8), 0.025% bromphenol blue] and boiled for 10 min. The samples (40 µg of protein) of total cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 75 V (the voltage was changed to 100 V when the indicator reached 1.5 cm from the edge of the 10% separation gel) and electrophoretically transferred onto a polyvinylidene difluoride membrane (Millipore) in transfer buffer containing 25 mM Tris, 150 mM glycine and 20% methanol. The membranes were blocked using 5% skim milk at 37˚C for 2 h. The membranes were then incubated with primary antibodies for 16-18 h at 4˚C. The membranes were washed in 1X TBST 3 times (10 min each time) at room temperature. The membranes were subsequently probed with an anti-rabbit IgG antibody with the HRP-conjugated secondary antibody (1:1000) for 2 h. Control blots were performed using anti-actin antibodies. The membranes were washed in 1X TBST for 3 times (10 min each time) at room temperature, and detection was achieved by measuring the chemiluminescence of the blotting agent after exposure of the filters to X-omat films. The densities of the bands were quantified using a computerized densitometer (ImageJ Launcher, Broken Symmetry Software).
Reverse transcription PCR. Different concentrations of OM (0, 0.25, 0.5, 1 and 2 mg/ml) were added into the cells, respectively. After 24 h, the levels of mRNA were quantified through reverse transcription-PCR (RT-PCR). RNA was isolated from the treated RKO cells using TRIzol reagent. Following the manufacturer's protocol, cDNA was generated from the total RNA using the GoScript™ Reverse Transcription system kit. Subsequently, PCR was conducted using the following primer sequences: sense primer: β-actin (used as the input control), forward primer, 5'-ACCACCATGTACCCAGGCAT-3' and reverse primer 5'-CCGGACTCATCGTACTCCTG-3'; TGF-β1 (400 bp NM 021578.2), forward primer 5'-AAGGCTCGCCAG TCCCCCGA-3' and reverse primer 5'-AGTGGGGGTCAG CAGCCGT-3'; PAI (1400 bp), forward primer 5'-CGGAGC ACGGTCAAGCAAGTG-3' and reverse primer 5'-GGTGAG GGCAGAGAGAGGCAC-3' . Reaction conditions consisted of: 95˚C for 5 min (initial denaturation), followed by 35 cycles of 95˚C for 30 sec, 58˚C for 60 sec and 72 ˚C for 60 sec. This was followed by a final 8-min extension period at 72˚C. The 5 µl amplified fragments, to which 1 µl 6X loading buffer was added, were visualized on 1.5% agarose gel electrophoresis at 120 V for 20 min. The results were investigated under a transilluminator. The PCR product was subjected to electrophoresis and the absorbance (A) was analyzed by gel imaging and an analysis system. The relative mRNA level (%) = A of the treated PCR product/A of the β-actin PCR product.
Statistical analysis. All assays were performed in triplicate. Statistical analyses were performed using the SPSS 19.0 software program (SPSS, uSA). All data are presented as the mean ± standard deviation (SD). Statistical differences were determined by the Student's t-test. Statistical significance of differences was accepted at P<0.05.
Statement of ethics. The present study was approved by the Institutional Review Board (CWO) of Liaoning university of Traditional Chinese Medicine.

Results
Effects of oxymatrine on the proliferation of RKO cells. The cytotoxicity of OM was evaluated using the Cell Counting Kit-8 assay. As illustrated in Fig. 2 and Table I, after RKO   Table I. Effects of OM on the proliferation of RKO cells.   OM inhibits the migration of RKO cells. OM was used to examine the effect on the migratory ability of RKO cells by wound healing assay. As a result, treatment of OM obviously reduced the migratory potential of the RKO cells (Fig. 3). The treated cells survived up to 2 days, indicating that the inhibitory changes were not due to the cytotoxic effect of OM. These findings suggest a critical role of OM in inhibiting the migration of RKO cells.

Effect of OM on the EMT of colorectal cancer cells.
To evaluate the effect of OM on RKO cells, the results of the CCK-8 assay, in dose-dependent and time-dependent trials, confirmed that a dose of 0.50 mg/ml for 24 h was unlikely to exert a significant toxic effect on the cells. Therefore, 0.50 mg/ml OM was used in the subsequent tests. To determine whether OM affected EMT, related marker expression levels of E-cadherin (marker for epithelial cells) and α-SMA (marker for mesenchymal cells) were examined by western blotting. The results showed that E-cadherin was significantly reduced and α-SMA was markedly increased in the RKO cells as compared to the FHC cells. After the RKO cells were treated with 0.50 mg/ml OM, OM obviously upregulated E-cadherin expression and downregulated α-SMA expression (Fig. 4). Taken together, these results demonstrated that OM reversed the EMT process in the colorectal cancer cells.

Effect of OM on fibronectin (FN) in colorectal cancer cells.
One key indicator of tumor cell migration is the accumulation of ECM proteins such as FN. EMT mainly leads to the deposition of ECM. To confirm whether OM affects ECM expression, we examined the expression of FN by western blotting, which showed that FN was significantly increased in the RKO cells as compared to the FHC cells. After the RKO cells were treated with 0.50 mg/ml OM, the expression of FN was significantly decreased in the RKO cells as compared to the untreated control group (Fig. 4)  used western blotting and RT-PCR to examine the protein and mRNA expression levels of TGF-β1 and PAI-1. In addition, Smad4, pSmad2 and pP38 were detected by western blotting. Western blotting showed that the protein levels of TGF-β1, pP38 and PAI-1 were significantly increased in the RKO cells as compared to the FHC cells. Meanwhile, the protein expression levels of TGF-β1 and PAI-1 were significantly decreased in the OR group as compared to the uR group (Fig. 5). RT-PCR results showed that TGF-β1 and PAI-1 mRNA levels were markedly increased in the uR group as compared to the FHC group. After RKO cells were treated with OM, OM was able to decrease TGF-β1 and PAI-1 mRNA expression as compared to the uR group (Fig. 6). Of note, the protein levels of Smad2/3/4 and pSmad2 were significantly increased in the RKO cells as compared to the FHC cells, which implied that TGF-β1 markedly induced Smad2 phosphorylation of RKO cells. Meanwhile, the protein levels of Smad2/3/4 and pSmad2 were significantly decreased in the OR group as compared to the uR group, which demonstrated that OM could reverse Smad2 phosphorylation via downregulation of TGF-β1 expression.
Taken together, these results demonstrated that OM markedly inhibited the Smad2 phosphorylation and the formation of Smad2/3/4 induced by TGF-β1 and eventually reduced the protein and mRNA levels of PAI-1.

Effect of OM on the expression of P38 in RKO cells.
Previous studies have found that P38 is associated with PAI-1 proteins in their free forms, as well as when they are bound to TGF-β1. P38 had a positive correlation with PAI-1 between the uR group and FHC group, which suggests that the RKO cells exhibited increased PAI-1 expression perhaps through the P38 and OM-mediated downregulated PAI-1 expression perhaps by inhibiting P38 expression. To further investigate the molecular mechanisms involved in OM-inhibited EMT in RKO cells, we treated the RKO cells with SB203580 (a p38 MAPK inhibitor). Then western blotting was used to detect the protein levels of pSmad2 and PAI-1. Western blotting showed that SB203580 inhibited pSmad2 and the expression of PAI-1 induced by  TGF-β1 as compared to the RKO cells (Fig. 7). The results suggest that P38 regulates the phosphorylation of Smad2 and the synthesis of the Smad2/3/4 compound. Taken together, these results demonstrated that the protein expression and transcriptional activity of PAI-1 were regulated by the TGF-β1 signaling pathway via P38. Moreover, these results suggest that OM inhibits the protein expression and transcriptional activity of PAI-1 via inhibiting the increase in P38 induced by the TGF-β1 signaling pathway.

Discussion
Tumor metastasis is the principal cause of the mortality of colorectal cancer patients, and is also a key factor determining the extent of colorectal cancer progression (10). TGF-β1, as a fundamental mediator of ECM, plays a critical role in the EMT process by a Smad-dependent pathway, resulting in tumor metastasis (18). Smad2 proteins are phosphorylated by the TGF-β1 receptor and mediate the intracellular signal transduction of TGF-β1 (21). PAI-1 is not only a rapid and specific inhibitor of the plasminogen/plasmin system, but is a potent regulator of tumor growth in vivo. Increased PAI-1 has been confirmed in many solid tumors and was found to be associated with a poor prognosis in several human cancers including colorectal cancer. Thus, PAI-1 has been an attractive potential target for various cancers. Recent studies have demonstrated that the expression of TGF-β1 was significantly increased, while the expression of PAI-1 was significantly increased in colorectal tissues from patients with colorectal cancer, resulting in the persistent activation of the TGF-β1/Smad pathway (19,20). This activation leads to the induction and promotion of EMT in colorectal cancer cells and the subsequent onset of tumor metastasis. Similarly, these studies have shown that the expression of E-cadherin (a marker for epithelial cells) was significantly reduced, while the expression of α-SMA (a marker for mesenchymal cells) and FN (an important component of ECM) were significantly increased. The mRNA and protein levels of TGF-β1 and PAI-1 were markedly increased in the colorectal cancer cells. These results suggest that EMT was induced and promoted in colorectal cancer by elevating the expression of TGF-β1 and PAI-1.
In the present study, the protein and mRNA levels of PAI-1 were upregulation in the colorectal cancer cells, which implied that the expression of PAI-1 was regulated at the transcriptional level, including the P38 gene. P38 is recognized as an important positive regulating factor of the TGF-β1/Smad signaling pathway, which promotes activation of TGF-β1 target genes  and enhances the biological effects of the TGF-β1/Smad signaling pathway by interacting with Smad proteins (22). Furthermore, we performed in vitro western blotting assay and found that SB203580, a p38 MAPK inhibitor, markedly inhibited the expression of PAI-1.
As an important regulator, P38 modulates signaling pathways. P38 promotes activation of the TGF-β1 signaling pathway. Once TGF-β1 signaling is activated, P38 binds to pSmad2/3 and induces upregulation of Smad2/3/4. In the present study, the expression of P38, pSmad2 and PAI-1 was significantly increased in the RKO cells, demonstrating that TGF-β1/Smad promotes the P38-mediated increased expression of PAI-1 mRNA mediated by p-Smad2/Smad3.
OM is a traditional Chinese herbal product. As the main active component of Sophora flavescens Ait, OM has multiple pharmacological effects and functions. OM was found to attenuate EMT of hepatocellular carcinoma and pulmonary carcinoma via inhibiting the TGF-β1/Smad signaling pathway (23). However, it is not known whether OM can attenuate EMT of colorectal carcinoma in the development of tumor metastasis. Our findings showed that OM reversed the marked decrease in E-cadherin and significantly increased α-SMA, FN, TGF-β1, pSmad2, Smad2/3/4 and P38 and attenuated the P38-dependent increased expression of PAI-1 induced in colorectal cancer, which indicates that OM can inhibit EMT in colorectal cancer via inhibiting the TGF-β1/ Smad signaling pathway by reducing P38-dependent increased expression of PAI-1. Hence, OM could be a novel therapeutic agent for colorectal cancer.


s2fieldsofstudy: Biology Medicine

text:
Chronic senolytic treatment alleviates established vasomotor dysfunction in aged or atherosclerotic mice

Summary While reports suggest a single dose of senolytics may improve vasomotor function, the structural and functional impact of long‐term senolytic treatment is unknown. To determine whether long‐term senolytic treatment improves vasomotor function, vascular stiffness, and intimal plaque size and composition in aged or hypercholesterolemic mice with established disease. Senolytic treatment (intermittent treatment with Dasatinib + Quercetin via oral gavage) resulted in significant reductions in senescent cell markers (TAF + cells) in the medial layer of aorta from aged and hypercholesterolemic mice, but not in intimal atherosclerotic plaques. While senolytic treatment significantly improved vasomotor function (isolated organ chamber baths) in both groups of mice, this was due to increases in nitric oxide bioavailability in aged mice and increases in sensitivity to NO donors in hypercholesterolemic mice. Genetic clearance of senescent cells in aged normocholesterolemic INK‐ATTAC mice phenocopied changes elicited by D+Q. Senolytics tended to reduce aortic calcification (alizarin red) and osteogenic signaling (qRT–PCR, immunohistochemistry) in aged mice, but both were significantly reduced by senolytic treatment in hypercholesterolemic mice. Intimal plaque fibrosis (picrosirius red) was not changed appreciably by chronic senolytic treatment. This is the first study to demonstrate that chronic clearance of senescent cells improves established vascular phenotypes associated with aging and chronic hypercholesterolemia, and may be a viable therapeutic intervention to reduce morbidity and mortality from cardiovascular diseases.

Introduction
Risk factors for ischemic heart disease include hypercholesterolemia, arterial stiffness, chronic inflammation, hypertension, metabolic syndrome, and aging (Eckel et al., 2013). Importantly, these risk factors contribute to impaired endothelial function (Feletou & Vanhoutte, 2006), which can contribute to arterial remodeling and accelerate atherosclerotic plaque formation and expansion (Landmesser et al., 2004).
Can chronic senolytic treatment improve age-related vascular pathology?
To determine whether senolytic treatment with dasatinib and quercetin (D+Q) reduces senescent cell burden and improves vascular function in aged mice, we maintained C57BL/6J mice on standard chow for 24 months, and then initiated vehicle or D+Q once monthly for 3 months (i.e., oral gavage with a dasatinib (5 mg kg À1 )/quercetin (10 mg kg À1 ) cocktail once per month for months 24-27). TAF + (telomere-associated foci) nuclei were used as a marker of senescent cell burden (Hewitt et al., 2012). TAF + nuclei were readily evident in subpopulations of cells in aorta (and localized in both endothelial and smooth muscle layers in the vessel wall) from vehicle-treated mice ( Fig. 1A-B and S1) and markedly reduced by D+Q (Fig. 1A-B and S1). Reductions in TAF + cells were also associated with reductions in DNA damage (number of c-H2AX foci per cell) following D+Q (Fig. 1C).
In vehicle-treated mice, carotid arteries showed minimal relaxation to acetylcholine assessed with an isolated organ chamber bath system (Miller et al., 2010), which was significantly improved in mice receiving intermittent D+Q for 3 months (Fig. 1D). In contrast to our previous study using a single dose of D+Q in aged mice, vascular relaxation to sodium nitroprusside was not changed by clearance of senescent cells in aged mice (Fig. 1E), and peak contractile responses increased following D+Q (Fig. S5, Supporting information). Similar senolytic efficacy and phenotypic changes were observed following genetic clearance of senescent cells in a subgroup of INK-ATTAC mice [which allow for clearance of p16 ink4a -positive senescent cells via a caspase-dependent mechanism (Baker et al., 2011)], thus confirming the effectiveness and phenotypic consequences of senescent cell clearance (Figs S2-S4). Collectively, these data suggest that acute senolytic treatment improves vasomotor function by increasing VSMC sensitivity to NO, whereas chronic, intermittent senolytic treatment improves vasomotor function by increasing NO bioavailability.
To probe mechanisms underlying improvements in vasomotor function, we assayed endothelial nitric oxide synthase phosphorylation levels (p-eNOS ser1177 , which are indicative of eNOS activation), but did not find significant differences between vehicle-and D+Q-treated groups Fig. S6). Despite this finding, p-VASP 239 (Fig. 1F), a target of NO-activated, cGMP-dependent kinases (Sporbert et al., 1999), was significantly higher in D+Q-treated aged mice. Although previous reports implicated reduced NOS cofactor production and increased Nox2derived free radicals in age-associated endothelial dysfunction (Turgeon et al., 2012;Roos et al., 2013), we did not find significant changes in mRNA levels of NOS-related or NADPH oxidase-related enzymes following senolytic treatment (Figs S7-S9). Collectively, while our data suggest that senolytic treatment improves NO signaling in aged mice, precise molecular mechanisms underlying this phenomenon-including the potential contributions of other endothelium-derived relaxing factorshave yet to be elucidated. Effects of senolytic treatment on senescent cell burden, DNA damage, vasomotor function, nitric oxide signaling, calcification, and osteogenic signaling in chronologically aged mice. Chronic treatment with Dasatinib + Quercetin reduced senescent cell burden (A-B) and DNA damage (C) in aorta (Panel A pseudocolor legend for high magnification lower panels: DAPI/nuclei (blue), cH2A.X (green), telomeres (red). Low magnification micrographs in upper images are provided as an anatomic frame of reference/origin). White squares mark regions that are magnified in subsequent micrographs, and telomere-associated foci are numbered in the bottom panels. Chronic senescent cell clearance improved vascular relaxation to acetylcholine (D) independently from changes in sodium nitroprusside (E) and significantly increased p-VASP 239 levels (F). Chronic senolytic treatment with Dasatinib + Quercetin also tended to reduce vascular calcification (G), which was associated with modest reductions in osterix immunofluorescence (H). In all panels, asterisk denotes p < 0.05.
To determine whether senolytic treatment elicited structural changes in conduit arteries, we evaluated pressure-diameter relationships from carotid arteries in a pressurized organ chamber system (Matsumoto et al., 2007). No changes in carotid artery cross-sectional compliance or distensibility were observed following pharmacological senescent cell clearance (Fig. S10). Medial vascular calcification in aorta, however, tended to be reduced by pharmacological cell clearance (Fig. 1G) and was paralleled by changes in protein levels of osterix (Fig. 1H). The modest effects of senolytic treatment on structural aspects of large arteries may be due to the period of time required to regress/reverse age-related structural changes or the efficacy of senescent cell clearance (e.g., higher and/or more frequent dosing with senolytic agents) required to elicit regression of calcification or deleterious changes in vascular compliance in conduit vessels. as an anatomic frame of reference/origin). Chronic senescent cell clearance modestly improved vascular relaxation to acetylcholine (D) but markedly improved relaxation to sodium nitroprusside (E), and significantly increased levels of p-eNOS ser1177 (F) and p-VASP 239 (G) in both the media and intima of atherosclerotic vessels. While chronic intermittent senolytic treatment with Dasatinib + Quercetin did not alter lipid composition of plaques (H), senolytic treatment did significantly reduce vascular calcification (I), which was associated with marked reductions in osterix immunofluorescence (J). In all panels, asterisk denotes p < 0.05.
Can chronic senolytic treatment improve hypercholesterolemia-induced vascular pathology?
To determine whether senolytic treatment with D+Q reduces senescent cell burden and improves vascular function and structure in a model of atherosclerosis, ApoE À/À mice were fed a Western diet (TD88137; Harlan Teklad) for 4 months to allow development of established atherosclerosis (Dansky et al., 1999). Vehicle or D+Q was then administered once weekly (i.e., oral gavage with a dasatinib (5 mg kg À1 )/quercetin (10 mg kg À1 ) cocktail) for the next 2 months (i.e., months 4-6). Similarly to aged mice, TAF-positive nuclei were abundant in the media and in intimal plaques of aorta from vehicle-treated mice ( Fig. 2A-B). Interestingly, senolytic treatment reduced senescent cell burden in medial segments of the vessel ( Fig. 2A-B), but not in regions with established intimal atherosclerotic plaques ( Fig. 2A-B). Indices of DNA damage paralleled reductions in senescent cell burden: reductions in DNA damage were evident only in medial segments of atherosclerotic vessels (Fig. 2C).
Unlike chronologically aged mice, D+Q did not improve responses to acetylcholine in isolated carotid arterial rings in an organ chamber bath system (Fig. 2D). We did, however, observe improved relaxation to sodium nitroprusside (Fig. 2E). Peak tension elicited by the thromboxane A2 agonist U46619 did not differ between treatment groups (Fig. S11).
Despite a lack of improvement in vasomotor responses to acetylcholine, we found that D+Q significantly increased levels of p-eNOS ser1177 (Fig. 2F). Similarly to aging mice, we observed increases in p-VASP 239 in aorta (Fig. 2G), suggesting that basal NO signaling was improved in both medial and intimal regions of atherosclerotic vessels. Although we observed relatively large increase in p-eNOS ser1177 levels, increases in p-VASP 239 levels remained small (particularly when compared to the relative reduction in p-VASP 239 levels in the intimal plaque region itself). When taken with the observation that senolytic treatment increased NADPH oxidase-related protein in the intima (Figs S13-S14) in the absence of changes in enzymes related to NOS cofactor generation (Fig. S12), we conclude that changes in vasomotor function are likely the result of a complex interplay between nitric oxide bioavailability originating from the intima and oxidation state of sGC or downstream signal transduction molecules within the media (Landmesser et al., 2004;Feletou & Vanhoutte, 2006).
Regarding effects of senolytics on atherosclerotic plaque burden and composition in aorta, we found intimal plaque size was not affected in mice receiving D+Q for 2 months (Fig. S15), and analysis of Oil Red O-stained sections suggested the overall lipid content of the plaque was also unaffected by senolytic treatment (Fig. 2H). Furthermore, expression of F4/80 in atherosclerotic vessels did not change appreciably (Fig. S16), suggesting negligible changes in overall macrophage burden. Among other possibilities, this could be related to intimal plaque penetrance by D+Q. Effects of newer senolytics, such as navitoclax (Zhu et al., 2015a,b), remain to be determined. We did, however, find that intimal plaque calcification was significantly reduced in D+Q-vs. vehicletreated mice (Fig. 2I). This was associated with reductions in protein levels of osterix (Fig. 2J). Intimal plaque fibrosis was not appreciably changed by senescent cell clearance (Fig. S17). This combination of findings is remarkable given the observations that calcified lesions are typically refractory to classic lipid-lowering interventions and occurred in the absence of changes in plasma cholesterol levels (Fig. S18). These changes in plaque morphology are consistent, however, with previous reports demonstrating that senescent cells and cells overexpressing genes associated with progeroid syndromes have greater propensity to undergo osteogenic differentiation in vitro (Roos et al., 2013). Critically, unlike previous studies leveraging chronic treatment with compounds such as quercetin (e.g., administered in drinking water) (Hayek et al., 1997;Shen et al., 2013), our study shows that weekly, intermittent treatment is sufficient to elicit lasting molecular and functional consequences in the vasculature of hypercholesterolemic mice. Altogether, we believe these observations lay the groundwork for future studies aimed at understanding effects of senolytics on plaque composition and stability both in isolation and as a complementary therapy to lipidlowering strategies.

Conclusions
Collectively, this study shows that chronic pharmacological clearance of senescent cells alleviates vasomotor dysfunction in naturally aging mice and mice with established atherosclerosis. Furthermore, senescent cell clearance reduces markers of osteogenesis in advanced intimal plaques, ultimately reducing intimal plaque calcification. Based on these findings, we conclude that senescent cell clearance may be an effective complementary therapy to classical risk factor management to reduce morbidity and mortality associated with age-related cardiovascular diseases.

Supporting Information
Additional Supporting Information may be found in the online version of this article at the publisher's web-site.                  Data S1. Methods. Senolytic treatment in aging and atherosclerosis, C. M. Roos et al. 977


extfieldsofstudy: Biology Medicine

text:
MicroRNA-16 suppresses the activation of inflammatory macrophages in atherosclerosis by targeting PDCD4

Programmed cell death 4 (PDCD4) is involved in a number of bioprocesses, such as apoptosis and inflammation. However, its regulatory mechanisms in atherosclerosis remain unclear. In this study, we investigated the role and mechanisms of action of PDCD4 in high-fat diet-induced atherosclerosis in mice and in foam cells (characteristic pathological cells in atherosclerotic lesions) derived from ox-LDL-stimulated macrophages. MicroRNA (miR)-16 was predicted to bind PDCD4 by bioinformatics analysis. In the mice with atherosclerosis and in the foam cells, PDCD4 protein expression (but not the mRNA expression) was enhanced, while that of miR-16 was reduced. Transfection with miR-16 mimic decreased the activity of a luciferase reporter containing the 3′ untranslated region (3′UTR) of PDCD4 in the macrophage-derived foam cells. Conversely, treatment with miR-16 inhibitor enhanced the luciferase activity. However, by introducing mutations in the predicted binding site located in the 3′UTR of PDCD4, the miR-16 mimic and inhibitor were unable to alter the level of PDCD4, suggesting that miR-16 is a direct negative regulator of PDCD4 in atherosclerosis. Furthermore, transfection wtih miR-16 mimic and siRNA targeting PDCD4 suppressed the secretion and mRNA expression of pro-inflammatory factors, such as interleukin (IL)-6 and tumor necrosis factor-α (TNF-α), whereas it enhanced the secretion and mRNA expression of the anti-inflammatory factor, IL-10. Treatment with miR-16 inhibitor exerted the opposite effects. In addition, the phosphorylation of p38 and extracellular signal-regulated kinase (ERK), and nuclear factor-κB (NF-κB) expression were altered by miR-16. In conclusion, our data demonstrate that the targeting of PDCD4 by miR-16 may suppress the activation of inflammatory macrophages though mitogen-activated protein kinase (MAPK) and NF-κB signaling in atherosclerosis; thus, PDCD4 may prove to be a potential therapeutic target in the treatment of atherosclerosis.

Introduction
The aging of the population and the increasing incidence of obesity and type 2 diabetes have led to the global burden of atherosclerosis (1), which is a chronic inflammatory disease of the blood vessel wall driven by the subendothelial retention of macrophages (2). The excessive production of oxidized lowdensity lipoprotein (ox-LDL) in the blood leads to the adequate removal of fats and cholesterol from macrophages, and consequently promotes the formation of multiple atheromatous plaques within the arteries (3). In addition to macrophages, dendritic cells, inflammatory smooth muscle cells, lymphocytes and other inflammatory cells are found in atherosclerotic plaques, indicating the major involvement of immune and inflammatory mechanisms in the pathogenesis of atherosclerosis (4,5). In addition, apoptosis, vascular smooth muscle cell (VSMC) apoptosis in particular, is a critical cellular event responsible for the pathogenesis of atherosclerosis and plaque stability (6).
Programmed cell death 4 (PDCD4) expression is markedly induced by apoptosis and PDCD4 was initially recognized as a tumor suppressor (7). However, the biological functions of PDCD4 in other diseases have been discussed. Apart from tumor cells, PDCD4 has been found to be expressed in cardiac muscle cells and VSMCs (8). In VSMCs, PDCD4 acts as a regulator of apoptosis via activator protein-1 (AP-1) (9), demonstrating the vital role of PDCD4 in cardiovascular disease, particularly in atherosclerosis-related diseases. Furthermore, PDCD4 promotes the inflammatory response by activating nuclear factor-κB (NF-κB) and suppressing interleukin (IL)-10 expression (10,11). These findings underline the potential role of PDCD4 as a novel therapeutic target in the clinical treatment of atherosclerosis.
MicroRNAs (miRNAs or miRs) are small non-encoding RNAs, which play an important role in the fine regulation of multiple physiological processes (12). Both the transcriptional silencing and translational disruption of genes are regulated by miRNAs via direct binding to the 3' or 5' untranslated region (UTR) (13). To date, a number of atherosclerosis-related miRNAs have been identified, such as miR-33, miR-155, miR-342-5p and miR-126-5p (14,15).
Although PDCD4 has been proven to be a pro-inflammatory protein, the involvement of PDCD4 in atherosclerosis has not been reported until recently. Jiang et al found that a deficiency in the PDCD4 gene had a positive regulatory effect in atherosclerosis by increasing the expression of IL-10 (16). Nevertheless, to the best of our knowledge, miRNAs targeting PDCD4 in atherosclerosis have not been discovered to date. Hence, in this study, we firstly validated miR-16 as a regulator of PDCD4 miRNA and examined the alterations in the expression levels of both miR-16 and PDCD4 in high-fat diet induced atherosclerosis in mice and in foam cells derived from ox-LDL-stimulated macrophages. Moreover, the underlying mechanisms were subsequently investigated.
Animal models of atherosclerosis. ApoE -/mice with a C57BL/6 background were purchased from Beijing University (Beijing, China). The mice used in this study were male, 6-8 weeks old, weighing 20-25 g, and were housed at the Second Hospital of Tianjin Medical University Animal Care Facility (Tianjin, China) under pathogen-free conditions, according to institutional guidelines. All animal study protocols were approved by the Animal Care and Utilization Committee of Tianjin Medical University. At 8 weeks of age, the ApoE -/mice were randomly divided into 2 groups (n=10 in each group). The mice in the control group received a standard diet, while the mice in the model of atherosclerosis group (AS model group) received a high-fat diet (0.25% cholesterol and 15% cocoa butter) for 12 weeks in order to induce the development of atherosclerotic plaques. The animals were then sacrificed by cervical dislocation. The thoracic aorta was immediately harvested after phosphatebuffered saline (PBS) perfusion. Part of the aorta was immersed in 4% paraformaldehyde overnight for immunohistochemical analysis, and the other part of the aorta was stored in liquid nitrogen for the isolation of RNA and protein.
Hematoxylin and eosin (H&E) staining was used to evaluate the atherosclerotic lesions. The mRNA levels of PDCD4 and miR-16 were measured by reverse transcription-quantita-tive PCR (RT-qPCR), and the protein epxression levels were measured by western blot analysis and immunohistochemistry.
Macrophage-derived foam cells. The murine macrophage cell line, RAW264.7, was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were maintained in RPMI-1640 (supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin G and 100 µg/ml streptomycin) and cultured at 37˚C in a humidified incubator with 5% CO 2 . To induce the differentiation of macrophages into foam cells, the RAW264.7 cells were seeded in a 6-well plate and cultured with 50 nM ox-LDL for 24 h. Luciferase constructs and reporter gene assays. To obtain recombinant luciferase mRNAs, the DNA segment of the 3'UTR of mouse PDCD4 mRNA or its mutant, with the changing of the 7 nt binding site for miR-16, was amplified by PCR using modified primers (sense, PDCD4UTR-F-X and antisense, PDCD4UTR-R-N for both the wild-type (WT) and mutant 3'UTRs. As regards the mutated one, 2 additional primers were required to generate the mutant site from the original WT template. One was paired with the aforementioned sense primer, PDCD4UTR-m-F, and the other was used together with the antisense one, PDCD4UTR-m-R. The 2 types of products were then mixed to perform an overlapping PCR experiment and the final product was the expected mutated 3'UTR, digested with XhoI and NotI. Each of them was then respectively cloned into the vector, siCHECK™-II (Promega, Shanghai, China), downstream of a luciferase CDS. The validity of these constructs was verified by sequencing.
293T cells (ATCC) which were grown in DMEM with high glucose, were used to examine the regulatory effects of miR-16 on PDCD4 mRNA and protein expression. The cells were seeded in 24-well plates and co-transfected with the WT or mutated PDCD4 3'UTR combined with dsRNA mimic or inhibitor of miR-16 using Lipofectamine 2000 reagent (Invitrogen). After 48 h of incubation, the cells were harvested and lysates were measured for application in the Luciferase Reporter system (Promega) following the manufacturer's instructions. All the luciferase reporter assays were repeated 3 times within each experiment.
RNA extraction and RT-qPCR. Total RNA from the cultured foam cells and thoracic aorta tissues was isolated using TRIzol reagent (Invitrogen). Reverse transcription was carried out using the M-MLV Reverse Transcription system (Takara, Dalian, China). qPCR was performed using the SYBR-Green PCR kit as described by the manufacture (TransGen, Beijing, China). The primers used for PCR are listed in Table I. Amplification was performed by denaturation at 95˚C for 10 min, followed by 40 cycles of 95˚C for 30 sec, 58˚C for 30 sec and 72˚C for 30 sec. qPCR was carried out on an Applied Biosystems 7500 Fast Real-Time PCR system. The relative mRNA expression levels were normalized to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene according to the 2 -ΔΔCt method, and the miRNA expression levels were normalized to U6. All the PCR experiments were carried out in triplicate within each experiment, and the experiments were replicated at least 3 times.
Immunohistochemistry. The paraffin-embedded sections (4 ml) were dewaxed and rehydrated in a graded ethanol series. For antigen retrieval, the sections were autoclaved in 0.01 M citrate buffer (pH 6.0) for 30 min, followed by immersion in 3% H 2 O 2 methanol for 15 min to block endogenous peroxidase. The sections were then blocked in 15% normal goat serum in phosphate-buffered saline, followed by incubation with polyclonal anti-PDCD4 antibody (1:500) overnight at 4˚C. The sections were then incubated with biotinylated goat antirabbit IgG diluted at 1:100 in 15% normal goat serum for 30 min Table I. Primers used in this study.
at room temperature. The standard ABC process was then performed according to the instructions provided with the goat ABC Staining System (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Diaminobenzidine was used as a chromogen, followed by counterstaining with hematoxylin.
Enzyme-linked immunosorbent assay (ELISA). The secretion of inflammatory factors, including IL-6, tumor necrosis factor-α (TNF-α), IL-10 and transforming growth factor-β (TGF-β) was determined using ELISA. Following transfection and stimulation with ox-LDL for the indicated time periods, the cell supernatants were collected and the amounts of the cytokines in the supernatants were quantified using commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA).
Statistical analysis. All the data are expressed as the means ± SEM and analyzed using SPSS 19.0 software. Differences between 2 groups were analyzed using the Student's t-test, while differences among many groups were analyzed using one-way analysis of variance, followed by Bonferroni's multiple comparison test. Values of P<0.05 were considered to represent statistically significant differences.

miR-16 is predicted as a regulator of PDCD4.
To determine the regulator of PDCD4 miRNA, the potential miRNA binding site to the 3'UTR was identified using TargetScan with the 3'UTR of mouse PDCD4 as the query sequence. As a result, PDCD4 3'UTR possessed a nucleotide sequence (456-462 nt) complementary to the miR-16 seed sequence, which indicated a potential binding pattern of miR-16 to PDCD4 (Fig. 1A). In addition, the nucleotide sequence was highly conserved among different mammalian species (Fig. 1B), demonstrating the conserved function of the miR-16/PDCD4 regulatory pattern in the evolution of higher animals.

Expression of PDCD4 and miR-16 in a mouse model of atherosclerosis and in foam cells.
The association of the expression of PDCD4 and miR-16 with atherosclerosis was assessed using ApoE -/mice fed a high-fat diet (AS model group) compared with mice fed a standard diet (control group). H&E staining of the cross section of the aorta revealed that large plaques were formed after the mice were fed a high-fat diet (indicated by arrows in Fig. 2A), thus indicating that the mouse model of atherosclerosis was successfully developed in the ApoE -/mice. No significant difference in the relative PDCD4 mRNA expression was observed between the 2 groups (P>0.05; Fig. 2B). However, compared with the mice in the control group, the highfat diet-fed ApoE -/mice exhibited a greater accumulation of PDCD4 protein in the aorta, which was detected by both western blot analysis (Fig. 2C) and immunohistochemistry (Fig. 2D). Conversely, the results of stem-loop RT-qPCR revealed that mature miR-16 was expressed at a markedly lower level in the AS model group than in the control group (P<0.001; Fig. 2E).
Additionally, the expression of PDCD4 mRNA and protein and miR-16 was examined in foam cells, characteristic pathological cells in atherosclerotic lesions, which were derived from macrophages stimulated with ox-LDL. Similar to the findings observed in our mouse model of atherosclerosis, the mRNA level of PDCD4 was not altered (P>0.05; Fig. 2F), whereas its protein expression was notably increased in the foam cells (Fig. 2G) and a decreased level of mature miR-16 (P<0.001; Fig. 2H) was also observed in the foam cells as compared to the controls (RAW264.7 cells not stimulated with ox-LDL).
PDCD4 is validated as a direct target gene of miR-16. To investigate the role of mature miR-16 in PDCD4 expression, the RAW264.7 cells were transfected with miR-16 mimic or inhibitor. Subsequently, the PDCD4 mRNA and protein levels were detected in the foam cells (ox-LDL-stimulated cells). Neither the miR-16 mimic nor the inhibitor affected the mRNA expression of PDCD4 (P=0.413; Fig. 3A). In addi- tion, various concentrations (1, 10 and 100 nM) of miR-16 mimic (Fig. 3B) or inhibitor (Fig. 3C) did not alter PDCD4 gene expression (P= 0.064 and 0.110, respectively). Nevertheless, transfection with miR-16 mimic markedly inhibited the protein expression of PDCD4, while treatment with miR-16 inhibitor led to a 1.6-fold induction in the PDCD4 protein level (Fig. 3D). Moreover, a concentration-dependent influence of the miR-16 mimic and inhibitor on the production of PDCD4 protein was observed ( Fig. 3D and F). These findings suggest the post-transcriptional regulation of PDCD4 by miR-16; miR-16 suppresses PDCD4 expression by restraining mRNA translation rather than affecting mRNA stability.
Furthermore, in order to validate the direct regulation of PDCD4 by miR-16, luciferase reporter plasmids carrying WT or mutated PDCD4 3'UTR ( Fig. 3G and H) were co-transfected into 293T cells with the miR-16 mimic or inhibitor. The activity of the luciferase reporter in which the WT PDCD4 3'UTR was fused to the downstream of the report gene was intensively suppressed by co-transfection with miR-16 mimic (P=0.003), while the activity of the luciferase reporter containing the mutated PDCD4 3'UTR was not significantly altered (P>0.05), suggesting that miR-16 suppressed the expression of PDCD4 by directly binding to the 3'UTR region (Fig. 3I). In addition, no effect of the miR-16 inhibitor was detected on the luciferase reporter carrying the WT or mutated PDCD4 3'UTR (P>0.05; Fig. 3I).

Targeting of PDCD4 by miR-16 affects the expression of inflammatory cytokines in atherosclerosis.
To address the possible role of miR-16 and PDCD4 in the inflammatory response in athero-sclerosis, the RAW264.7 cells were transfected with miR-16 mimic, miR-16 inhibitor or PDCD4 siRNA and subsequently stimulated with ox-LDL over a 24-h time period to induce the development of foam cells.
The secretion levels of inflammatory factors were then determined by ELISA. The levels of pro-inflammatory cytokines, including IL-6 and TNF-α were enhanced, rapidly reaching peak levels at 6 h and then decreasing ( Fig. 4A and B). Compared with the NC oligonucleotide duplex, foam cells harboring the miR-16 mimic or PDCD4 siRNA exhibited much lower levels of IL-6 and TNF-α (IL-6, P<0.001; TNF-α, P= 0.005), while the miR-16 inhibitor promoted the secretion of IL-6 and TNF-α (IL-6, P<0.001; TNF-α, P<0.001). By contrast, the levels of anti-inflammatory cytokines, including IL-10 and TGF-β were suppressed following stimulation with ox-LDL. The level of IL-10 continued to increase during the 24-h time course, while the TGF-β level reached a peak at 12 h. Transfection wtih miR-16 mimic and PDCD4 siRNA enhanced the secretion of IL-10, whereas treatment with the miR-16 inhibitor exerted the opposite effect (P= 0.009; Fig. 4C and D). However, no effects of miR-16 mimic, miR-16 inhibitor or PDCD4 siRNA on the secretion of TGF-β were detected (P>0.005).
In addition, we determined the mRNA levels of these inflammation factors in foam cells by RT-qPCR. Consistently, transfection with miR-16 mimic and PDCD4 siRNA exerted an inhibitory effect on the relative mRNA expression levels of IL-6 (P<0.001 and P<0.001, respectively; Fig. 4E) and TNF-α (P=0.003 and P<0.001, respectively; Fig. 4F); however, miR-16 mimic and PDCD4 siRNA induced the mRNA expres-    -β1 (TGF-β1). Differences among groups were analyzed by one-way analysis of variance, followed by Bonferroni's multiple comparison test. Values with different letters indicate significant differences with a P-value cut-off of 0.05. NC, negative control (oligonucleotide duplex that did not target any gene). sion of IL-10 (P<0.001 and P<0.001, respectively; Fig. 4G). The miR-16 inhibitor increased the IL-6 (P= 0.001; Fig. 4E) and TNF-α mRNA levels in the foam cells (P<0.001; Fig. 4F) and decreased IL-10 mRNA expression (P<0.001; Fig. 4G). However, the mRNA level of TGF-β in the foam cells was not significantly altered by miR-16 mimic, miR-16 inhibitor or PDCD4 siRNA (P>0.05; Fig. 4H).

Targeting of PDCD4 by miR-16 mediates ox-LDL-induced mitogen-activated protein kinase (MAPK) and NF-κB signaling.
Considering that MAPK and NF-κB signaling have been implicated in inflammatory cytokine expression (17)(18)(19), we thus wished to determine whether these two pathways are involved in the miR-16 induced anti-inflammatory response in atherosclerosis. No significant effects of miR-16 mimic or inhibitor on the protein expression levels of p38, JNK and ERK were observed (Fig. 5A). Nevertheless, the phosphorylation of p38 and ERK was induced, and that of JNK was inhibited by miR-16 mimic. Conversely, the miR-16 inhibitor lowered the phosphorylation level of p38 and ERK, but increased that of JNK (Fig. 5A).
Additionally, NF-κB mRNA expression was prominently suppressed by treatment with miR-16 mimic or siRNA targeting PDCD4, but it was increased to approximately 6-fold by the miR-16 inhibitor compared with the NC oligonucleotide duplex (P<0.001; Fig. 5B). Estimates with regard to NF-κB protein expression were consistent with the mRNA results (Fig. 5C).

Discussion
Atherosclerotic vascular disease is the underlying cause of a number of diseases, including claudication, resulting from insufficient blood supply, myocardial infarction, ischemic heart disease and stroke (20). Notably, the two latter diseases are the top two causes of mortality worldwide (21). Thus, the pathogenesis of atherosclerosis is always a critical topic, but it is not yet fully understand. Substantial evidence has suggested that the inflammatory response is triggered in atherosclerosis, which was reflected by the leukocyte recruitment from blood into arterial intima (22,23), and many efforts have been done to investigate the involved molecules (24,25). The pro-inflammatory protein, PDCD4, was recently reported to promote atherosclerosis (16). In the present study, the production of PDCD4 protein was higher in the mice with atherosclerosis and in foam cells derived from ox-LDL-stimulated macrophages, further validating the positive involvement of PDCD4 in the development of atherosclerosis. Additionally, we found that miR-16 was the direct regulatory element of PDCD4 and played a vital role in atherosclerosis by regulating PDCD4 and the downstream NF-κB and MAPK pathways. Initially, miR-16 was well known as a tumor suppressor by inducing apoptosis. Some studies emerged to report the function of miR-16 in immune or inflammatory responses, but yielding inconsistent conclusions. For instance, as previousy demonstrated, in lipopolysaccharide (LPS)-stimulated H69 cells (SV40-transformed human biliary epithelial cells) and U937 cells (human macrophage cell line), the upregulation of miR-16 targets the silencing mediator for retinoid and thyroid hormone receptors (SMRT) and increases the NF-κB-regulated transactivation of IL-8 (26,27). By contrast, during monocyte differentiation into immature and mature dendritic cells, the downregulation of miR-16 has been shown to target the inhibitor of κB (IκB) kinase α (IKKα) and inhibit the activation of NF-κB target genes (28). However, miR-16 promotes the degradation of mRNAs encoding pro-inflammatory factors, such as TNF-α, IL-6 and IL-8 by binding to the AU-rich elements (AREs) in their 3'UTR (29). These findings demonstrate that miR-16 may play different roles in modulating inflammatory factors under different conditions by binding to its different target genes. In this study, we found that the expression of miR-16 was reduced in the mice with atherosclerosis and in foam cells, which indicated the negative role of miR-16 in atherosclerosis. Besides, miR-16 contained a highly conserved UGCUGCU sequence that was complementary to the 3'UTR of PDCD4. We did not detect a significant impact of miR-16 mimic or inhibitor on the mRNA expression level of PDCD4 in the foam cells (derived from RAW264.7 cells stimulated with ox-LDL). Instead, PDCD4 protein production was lowered by miR-16 mimic and increased by miR-16 inhibitor. By using a luciferase construct harboring the sequence of PDCD4 3'UTR that was predicted to bind to miR-16, the suppressed protein expression of PDCD4 was detected. By contrast, if mutations were introduced to the sequence of PDCD4 3'UTR, miR-16 was unable to affect the production of PDCD4 protein, which implied a translational prevention of PDCD4 by miR-16.
As miR-16 and PDCD4 have been proven to be associated with the inflammatory response (11,27), we examine their effects on inflammatory cytokine expression. Under the stimulation of ox-LDL, macrophages develop into foam cells that modify the production pro-inflammatory factors, such as IL-6 and TNF-α, as well as anti-inflammatory factors, such as IL-10 and TGF-β. Two recent studies demonstrated that IL-10 is suppressed by PDCD4 (11,16). Consistently, in this study, we found that the transfection of cells with miR-16 mimic or PDCD4 siRNA promoted the release and mRNA expression of IL-10, while miR-16 inhibitor exerted the opposite effect. However, the effect of PDCD4 on other inflammatory cytokines is debatable. For instance, the expression of IL-6 has been shown to be blocked by PDCD4 deficiency in the LPS-induced inflammatory response (11), while in another study, IL-6, TNF-α and TGF-β were not significantly altered between ox-LDL-treated macrophages from PDCD4 -/-ApoE -/and PDCD4 +/+ ApoE -/mice (16). In this study, transfection of the RAW264.7 cells with miR-16 inhibitor, miR-16 mimic or PDCD4 siRNA was used. Following stimulation with ox-LDL for various periods of time, miR-16 mimic inhibited the release and mRNA expression of IL-6 and TNF-α, which may be partly due to the direct regulation of miR-16 on their mRNA degradation (29). Besides, we also detected similar results in foam cells, which was inconsistent with the previous study (16). Although different cell sources and ox-LDL concentrations were employed, these incongruous results cannot be well explained and require further investigation.
The MAPK pathway has a critical function in inflammatory diseases, including atherosclerosis (30). It has been reported that MAPK signaling contributes to the regulation of pro-and anti-inflammatory cytokines (TNF-α and IL-10) in macrophages by the activation of p38 and JNK (31). Jiang et al found that the induction of IL-10 in mice deficient in PDCD4 was blocked by the inhibition of the p38/ERK-c-Maf pathway (16) Consistently, in the present study, miR-16 markedly increased the phosphorylation of p38 and ERK, suggesting that the p38/ ERK-c-Maf pathway may be involved in the miR-16 mediated inflammatory response in atherosclerosis. Besides, miR-16 suppressed the phosphorylation of JNK. JNK MAPK phosphorylation has been well-documented to activate NF-κB, which mediates vascular inflammation in the initiation and progression of atherosclerosis (1). Several miRNAs block or promote the activity or expression of NF-κB in atherosclerosis, such as miR-21 (32), miR-181b (33) and miR-29b (34). In this study, we also presented evidence that miR-16 inhibits the mRNA and protein expression of NF-κB in foam cells derived from ox-LDL-stimulated macrophages. The NF-κB pathway in endothelial cells leads to the elevated expression of downstream pro-inflammatory cytokines, including IL-6 and TNF-α (35). Hence, we assumed that our findings with regard to the effects of miR-16 and PDCD4 on IL-6 and TNF-α are a result of the altered phosphorylation levels of JNK and MAPK, as well as the expression level of NF-κB.
In conclusion, the findings of the present study suggest that miR-16 directly targets PDCD4 to suppress the activation of inflammatory macrophages in atherosclerosis via the MAPK and NF-κB pathways and downstream inflammatory cytokines. Four potential mechanisms of miR-16 are indicated in atherosclerosis: i) the decreased expression of miR-16 upregulates the mRNA level of pro-inflammatory factors by binding to the AREs in their 3'UTR; the decreased expression of miR-16 weakens its translational prevention role on PDCD4, which ii) reduces the IL-10 level by inhibiting the p38/ERK-c-Maf pathway and enhancing the expression of IL-6 and TNF-α by iii) activating NF-κB by JNK MAPK or iv) increasing NF-κB expression. In addition to PDCD4, miR-16 may be used as a potential therapeutic target in the treatment of atherosclerosis.