Salusin-β induces foam cell formation and monocyte adhesion in human vascular smooth muscle cells via miR155/NOX2/NFκB pathway

Vascular smooth muscle cells (VSMCs) are indispensible components in foam cell formation. Salusin-β is a stimulator in the progression of atherosclerosis. Here, we showed that salusin-β increased foam cell formation evidenced by accumulation of lipid droplets and intracellular cholesterol content, and promoted monocyte adhesion in human VSMCs. Salusin-β increased the expressions and activity of acyl coenzyme A:cholesterol acyltransferase-1 (ACAT-1) and vascular cell adhesion molecule-1 (VCAM-1) in VSMCs. Silencing of ACAT-1 abolished the salusin-β-induced lipid accumulation, and silencing of VCAM-1 prevented the salusin-β-induced monocyte adhesion in VSMCs. Salusin-β caused p65-NFκB nuclear translocation and increased p65 occupancy at the ACAT-1 and VCAM-1 promoter. Inhibition of NFκB with Bay 11-7082 prevented the salusin-β-induced ACAT-1 and VCAM-1 upregulation, foam cell formation and monocyte adhesion in VSMCs. Scavenging ROS, inhibiting NADPH oxidase or knockdown of NOX2 abolished the effects of salusin-β on ACAT-1 and VCAM-1 expressions, p65-NFκB nuclear translocation, lipid accumulation and monocyte adhesion in VSMCs. Salusin-β increased miR155 expression, and knockdown of miR155 prevented the effects of salusin-β on ACAT-1 and VCAM-1 expressions, p65-NFκB nuclear translocation, lipid accumulation, monocyte adhesion and ROS production in VSMCs. These results indicate that salusin-β induces foam formation and monocyte adhesion via miR155/NOX2/NFκB-mediated ACAT-1 and VCAM-1 expressions in VSMCs.

Atherosclerosis is positively associated with the formation of foam cells characterized by cholesterol esters and triglycerides accumulation in the cytoplasm 1,2 . The foam cell formation is a critical step in the atherosclerosis and plays essential role in the destabilization, rupture and erosion of atherosclerotic plaque 3 . Most of foam cells have been found to be derived from macrophages, but VSMCs give rise to a considerable number of foam cells as well 4 . Simultaneous thymidine autoradiography and immunostaining with cell type-specific monoclonal antibodies showed that about 30% of the labeled cells were macrophages and 45% were VSMCs in advanced atherosclerosis lesions in both Watanabe heritable hyperlipidemic rabbits and hypercholesterolemic fat-fed rabbits 5 . On the other hand, leukocyte recruitment from blood stream to the intima of vessels is a crucial event in the pathogenesis of atherosclerosis and a potential candidate for therapeutic approaches of atherosclerosis 6 . Cell adhesion molecules including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are important for the initiating event of leukocyte recruitment to vascular endothelium and VSMCs in the development of atherosclerosis 7 . Although several adhesive mechanisms have been provided to be responsible for leukocyte recruitment in atherosclerosis, the mechanisms for the foam cell formation from VSMCs and the leukocyte recruitment in atherosclerosis are poorly elucidated. Salusin-β is a peptide with 20 amino acids, which is translated from an alternatively spliced mRNA of TOR2A, a gene encoding a protein of the torsion dystonia family 8 . The initial 18 amino acids of human salusin-β have high homology with the N-terminal sequence of rat salusin 9 . Salusin-β is widely expressed within vasculature and central nervous system 10 . Our previous studies have showed that central salusin-β is involved in sympathetic activation and hypertension in hypertensive rats [11][12][13] , and peripheral salusin-β contributes to vascular remodeling associated with hypertension by promoting human VSMC proliferation via cAMP-PKA-EGFR-CREB/ERK pathway and vascular fibrosis via TGF-β 1-Smad pathway 14 . Salusin-β is dominantly detected within VSMCs and fibroblasts in the human coronary atherosclerotic plaques 15 . Atherosclerotic lesions are deteriorated in apolipoprotein E-deficient mice with chronic salusin-β infusion 16 . Plasma salusin-β levels in subjects with diabetes mellitus, coronary artery disease, and cerebrovascular disease showed distinctly higher levels than healthy controls, and it may contribute to the pathogenesis of atherosclerosis 17 . However, the roles of salusin-β in the foam formation and monocyte adhesion in human VSMCs are rarely known. Thus, fully elucidating the roles of salusin-β and its underlying mechanisms in the foam cell formation and monocyte adhesion in VSMCs may be useful for the prevention and treatment of vascular lesions in atherosclerosis.

Results
Effects of salusin-β on foam cell formation and monocyte recruitment. Salusin-β dose-and time-relatedly increased the accumulation of lipid droplets in VSMCs and the adhesion of monocyte to VSMCs, and the maximal effects were observed at the dose of 30 nM of salusin-β for 48 h (Fig. 1A,B). Total intracellular cholesterol quantification and the number of adherent cells further confirmed that salusin-β stimulated VSMC foam cell formation and monocyte recruitment (Fig. 1C,D). Effects of salusin-β on ACAT-1 and VCAM-1. It is known that ACAT-1 promotes foam cell formation by promoting intracellular cholesteryl ester synthesis 18 , and VCAM-1 are an important cell adhesion molecule for leukocyte recruitment in the development of atherosclerosis 7 . Salusin-β upregulated the ACAT-1 and VCAM-1 protein levels in VSMCs in concentration-and time-dependent manner, and the maximal effects were found at the concentration of 30 nM of salusin-β for 48 h ( Fig. 2A,B). Furthermore, it increased ACAT-1 and VCAM-1 mRNA levels, which were parallel to those of protein expressions (Fig. 2C). Luciferase reporter genes assay showed that salusin-β enhanced the promoter activities in the ACAT-1 and VCAM-1 truncated forms containing proximal NF-κ B binding sites (Fig. 2D).

Discussion
Atherosclerosis is a crucial underlying pathology of cardiovascular diseases. Foam cell formation is a major hallmark of early stage atherosclerotic lesions 19 . ACAT-1 is a key enzyme for intracellular cholesteryl ester synthesis 18 . Excessive cholesterol esterification resulted in accumulation of cholesterol ester stored as cytoplasmic lipid droplets and subsequently trigger the formation of foam cells 19 . Foam cells are not only derived from macrophages, but also from VSMCs 4 . It is known that circulating salusin-β levels is increased in coronary artery disease 20 and salusin-β increased ACAT-1 expression in human monocyte-derived macrophages 15 . We found that salusin-β increased the accumulation of lipid droplets and intracellular total cholesterol content indicating that salusin-β  promoted VSMCs-derived foam cell formation. In support of this notion, we found that ACAT-1 mRNA and protein expressions as well as the promoter activity in the ACAT-1 were upregulated in salusin-β -treated VSMCs, and knockdown of ACAT-1 with siRNA diminished salusin-β -induced increases in lipid droplets and intracellular total cholesterol contents in VSMCs. The results indicate the importance of ACAT-1 in salusin-β -induced foam cell formation from VSMCs.
Recruitments of monocytes and monocyte-derived phagocytes into the wall of large arteries accelerate the chronic inflammation and atherosclerosis 21 . ICAM-1 and VCAM-1 are important cell adhesion molecules and are merged as functional mediators in monocyte extravasation 22 . VCAM-1 expression is found in VSMCs, which promotes the migration of monocytes and lymphocytes into the vessel wall 23 . We found that salusin-β promoted the monocyte adhesion to VSMCs, and increased VCAM-1 mRNA and protein expressions as well as the promoter activity in the VCAM-1 in VSMCs. Knockdown of VCAM-1 with siRNA prevented the salusin-β -induced  Values are mean ± S.E.M. * P < 0.05 and ** P < 0.01 vs. PBS or Veh; † P < 0.05 and † † P < 0.01 vs. PBS+ Salusin-β or Veh+ Salusin-β . n = 4 for each group. monocyte adhesion. These results indicate that salusin-β promotes monocyte recruitment to VSMCs and VCAM-1 is pivotal for the monocyte recruitment in VSMCs.
Transcription factor NFκ B is implicated in inflammatory activation associated with the onset of atherosclerosis 24 . Translocation of p65 of NFκ B from cytoplasm to nucleus is recognized as a prerequisite for the   25 . VCAM-1 and ACAT-1 expressions at sites of atherosclerotic lesion formation are majorly controlled by NFκ B system in early atherosclerotic lesions 26 . We found that salusin-β induced p65-NFκ B nuclear translocation and recruitment of p65 to promoters of ACAT-1 and VCAM-1 in VSMCs. Inhibition of NFκ B suppressed the salusin-β -induced ACAT-1 and VCAM-1 expressions, lipid accumulation in VSMCs and monocyte adhesion to VSMCs. These results indicate that p65-NFκ B nucleus translocation is necessary for salusin-β -induced ACAT-1 and VCAM-1 expressions and the following foam cell formation from VSMCs and monocyte recruitment to VSMCs.
ROS is a critical contributor in the initiation and progression of atherosclerosis 27 . NFκ B is a redox-sensitive transcription factor and can be regulated by intracellular redox status 28 . A major source for ROS in cardiovascular system is a family of NOX 29,30 . Our recent studies have shown that NAD(P)H oxidase-derived ROS in the brain is responsible for salusin-β -induced sympathetic activation 12,13 . In the present study, we found that salusin-β increased NOX2 expression and ROS production in VSMCs. Scavenging ROS, inhibiting NADPH oxidase or downregulating NOX2 abolished salusin-β -induced p65-NFκ B nuclear translocation, ACAT-1 and VCAM-1 expressions, foam cell formation and monocyte adhesion in VSMCs. These results indicate that NOX2-derived ROS is a major downstream mediator of salusin-β in the ROS production and the subsequent p65-NFκ B nuclear translocation, ACAT-1 and VCAM-1 expressions, foam cell formation and monocyte adhesion in VSMCs. MicroRNAs are small RNA molecules that regulate gene expression at post-transcriptional level 31 . MicroRNA-155 (miR155) is dominantly expressed in the atherosclerotic plaques and proinflammatory macrophages, and loss of miR155 reduces the proinflammatory NF-κ B signaling associated with decreased recruitment of monocytes to atherosclerotic plaques 32 . Deficiency of miR155 attenuates atherogenesis in apolipoprotein E-deficient mice 33 . We found that salusin-β up-regulated miR155 level in VSMCs. Knockdown of miR155 abolished the salusin-β -induced NOX2 upregulation, ROS production, p65-NFκ B nuclear translocation, ACAT-1 and VCAM-1 expressions, foam cell formation and monocyte adhesion in VSMCs. These results indicate that miR155 is crucial in salusin-β -induced oxidative stress and subsequent cascade process. A limitation in the present study was that the results were not examined in vivo, which need further investigation.
In conclusion, salusin-β upregulates miR155 in VSMCs, which increases NOX2-derived ROS production and subsequent nucleus translocation of p65-NFκ B. The activation of NFκ B promotes ACAT-1 and VCAM-1 expressions, and then causes foam cell formation from VSMCs and monocyte adhesion to VSMCs. Intervention of salusin-β may be a promising strategy for retarding the vascular lesions in atherosclerosis.

Methods
Cell culture. Human aortic VSMCs were obtained from American Type Culture Collection (Rockville, MD, USA). VSMCs were cultured as we previously reported 14 . In brief, VSMCs were maintained F12K Kaighn's modification medium containing 10% fetal bovine serum (FBS) supplemented with 1× penicillin and streptomycin in a humidified cell incubator with standard cell culture condition (37 °C at 5% CO 2 humidified atmosphere). Cells between passages 3 and 8 were used for the experiments. When cells reached 70-80% confluence, the cells were starved for 24 h in serum-free medium prior to use 34 .
Oil red O staining. Oil red O staining for VSMCs was conducted according to previous study 35 . Simply, cultured VSMCs were plated on the 24-well plates and reached to 70-80% confluence, and then incubated with or without different doses of salusin-β for 12, 24 or 48 h in serum-free media. Afterwards, VSMCs were washed with 0.01 M PBS for three times, and stained with Oil Red O and Harris' hematoxylin. The images were captured under a microscope at × 400 magnification. Intracellular total cholesterol measurement. Intracellular total cholesterol level was determined by enzymatic assay (Applygen Technologies, Beijing, China). Briefly, VSMCs were harvested into a centrifuge tube and then washed with PBS three times. Isopropylalcohol was employed to extract the intracellular lipids by ultrasonication. After centrifugation for 5 min at 2,000 g, the supernatant was used to determine the total cholesterol. The total cellular protein levels were quantified by Bradford assay method. The results in each sample were expressed in nmol of cholesterol per milligram of cellular protein and finally normalized to those of the control 36 . In vitro monocyte adhesion assay. Monocyte-VSMC binding assays were carried out as described previously 37 . U937 cells (monocytes) were purchased from FuDan IBS Cell Center (Shanghai, China) and cultured in RPMI-1640 Medium supplemented with 10% FBS. The VSMCs were incubated with or without salusin-β for 12, 24 or 48 h, respectively and followed by two rinses with PBS. Then, U937 cells (1 × 10 6 cells/well) were added to VSMC culture and incubated for 1 h at 10 rpm at 37 °C. The medium was then drained and washed twice with FBS for removing the non-attached cells, the VSMC layers with attached monocytes were fixed with 4% paraformaldehyde and measured with a microscope at × 200 filed. The number of attached monocyte was randomly counted in five areas per well from three independent experiments and averaged. siRNA transfection. Human acyl coenzyme A:cholesterol acyltransferase-1 (ACAT-1) siRNA or human VCAM-1 siRNA (Santa Cruz, CA, USA) was transfected to human VSMCs as previously described 38,39 . Recommended scrambled siRNA (Santa Cruz, CA, USA) was used as a negative control. The human VSMCs were transfected with 100 nM of indicated siRNA with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) for 24 h, the cells were plated for foam formation and monocyte adhesion assay after the 48 h of salusin-β treatment.
Reporter gene transfection and luciferase activity assay. The forms of human VCAM-1 luciferase plasmids spanning − 1350 to + 45 bp or the core regions (− 125 to + 34) of human ACAT-1 proximal promoter was cloned into pGL3 basic luciferase reporter vector (Promega, WI, USA) as previous report 18,40 . VSMCs were transfected with 1 μg of luciferase plasmids or control pCMV-β -gal plasmid with lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) for 24 h. VSMCs were harvested and luciferase activity was measured using a dual luciferase reported gene assay kit (Beyotime Biotechnology, Shanghai, China). The luciferase activity was normalized to the internal control.
Western Blot. VSMCs were lysed in lysis buffer and incubated for 30 min on the ice, the supernatant was retained after centrifugation. The total protein concentration in the supernatant was quantified with the Bradford assay (Santa Cruz, CA, USA). Equal amounts of protein in each sample were separated by SDS-PAGE electrophoresis and transferred to PVDF membrane. The membrane was blocked with 5% milk blocking buffer and incubated with the designed primary antibodies overnight at 4 °C. Horseradish peroxidase-conjugated secondary antibodies were used for detection. The protein bands were visualized with Enhanced Chemiluminescence Detection Kit (Thermo Scientific, Rockford, IL, USA).
Preparation of cytoplasmic fractions and nuclear extracts. Nuclear and cytoplasmic extracts were prepared and obtained as previous report 43 . The purity of nuclear extracts was evaluated by immunoblotting with incubation of primary antibodies against lamin B1 (Santa Cruz, CA, USA). The purity of cytoplasmic fractions was confirmed by immunoblotting with primary antibodies against GAPDH (Santa Cruz Biotechnology, CA, USA).

Chromatin immunoprecipitation (ChIP).
ChIP assays were conducted as described previously 44 .
The ChIP-enriched DNA fragments were immunoprecipitated in the presence or absence of p65 and Pol II antibodies, and a rabbit monoclonal control IgG (Santa Cruz Biotechnology, CA, USA). qPCR was used to assess the NFκ B and Pol II binding site on the ACAT-1 and VCAM-1 promoters. The data were quantified and normalized with input samples and expressed as fold change of control. Precipitated DNA fragments were amplified by PCR. The primers encompassing the p65 site were as follows. In ACAT-1 promoter: 5′ -CTTAACCTGGGGACCACCAATAG-3′ (forward), 5′ -CTTCTATTGGTGGTCCCCAGGTT-3 (reverse) 40 ; in VCAM-1 promoter: 5′ -AAATCAATTCACATGGCATA-3′ (forward), 5′ -AAGGGTCTTGTTGCAGAGG-3′ (reverse) 45 .

Lentiviral vector transduction in VSMCs.
VSMCs were seeded at 10 5 cells/well in 6-well plates 24 h prior to transfection. The VSMCs were grown to a 40-60% confluence and transfected with recombinant lentivirus vector harboring ACAT-1 (sc-402300-LAC, Santa Cruz, CA, USA) or VCAM-1 (sc-400135-LAC, Santa Cruz, CA, USA) in serum-free growth medium overnight. The viruses contained medium was replaced with 3 ml of standard medium. Measurements were conducted 2 days after transfection 46 . Measurement of ROS generation. DHE fluorescent dye was used to determine intracellular superoxide anion generation in VSMCs as previous report 47 . VSMCs were fixed and loaded with DHE (10 μmol/L) for 30 min in a light-protected humidified chamber. The fluorescence (488/525 nm) were obtained with fluorescence microscope.

Chemicals and antibodies.
Human salusin-β was obtained from Phoenix Pharmaceuticals (Belmont, CA, USA). Cell culture supplies were purchased from Costar (Corning Inc., Cypress, CA, USA). Antibodies against ACAT-1, VCAM-1, GAPDH, Lamin B1 and indicated horseradish peroxidase (HRP) conjugated secondary antibodies were purchased from Santa Cruz Biotechnology Co. (Santa Cruz, CA, USA). Antibody against p65 was obtained from Cell Signaling Technology (Beverly, MA, USA). Antibody against NOX2 was purchased from Abcam (Cambridge, MA, USA). Sequence of human miR155 inhibitor is 5′ -ACCCCUAUCACAAUUAGCAUUAA-3′ and the sequence of its control is 5′ -CAGUACUUUUGUGUAGUACAA-3′ (GenePharma, Shanghai, China). Bay11-7082, N-acetyl-L-cysteine (NAC), deihydroethidium and the reporter plasmid pGL6-NFκ B-Luc for measurement of NFκ B activity were obtained from Beyotime Biotechnology (Shanghai, China). Apocynin and dimethyl sulfoxide (DMSO) were obtained from Sigma Chemical (St. Louis, MO, USA). Statistical analysis. Data were expressed as mean ± S.E.M. One-way or two-way ANOVA followed by post hoc Bonferroni test was used for multiple comparisons. A value of P < 0.05 was considered statistically significant.