CLINICAL PERSPECTIVE

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(Circulation. 2008;117:638-648.)
© 2008 American Heart Association, Inc.

Molecular Cardiology

Impact of Salusin- and -β on Human Macrophage Foam Cell Formation and Coronary Atherosclerosis

Takuya Watanabe, MD, PhD; Kae Nishio, MS; Tomoko Kanome, MS; Taka-aki Matsuyama, MD, PhD; Shinji Koba, MD, PhD; Tetsuo Sakai, MD, PhD; Kengo Sato, MS; Shigeki Hongo, PhD; Kiyoshi Nose, PhD; Hidekazu Ota, MD, PhD; Youichi Kobayashi, MD, PhD; Takashi Katagiri, MD, PhD; Masayoshi Shichiri, MD, PhD; Akira Miyazaki, MD, PhD

From the Department of Biochemistry (T.W., K. Nishio, T. Kanome, S.H., A.M.), Second Department of Pathology (T.M., H.O.), and Third Department of Internal Medicine (S.K., T.S., Y.K., T. Katagiri), Showa University School of Medicine, Tokyo, Japan; Department of Microbiology, Showa University School of Pharmaceutical Sciences (K. Nose), Tokyo, Japan; and Tokyo Medical and Dental University (K.S., M.S.), Tokyo, Japan.

Correspondence to Takuya Watanabe, MD, PhD, Department of Biochemistry, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. E-mail watanabemd{at}med.showa-u.ac.jp

Received May 1, 2007; accepted November 27, 2007.

	Abstract

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Background— Human salusins, related bioactive polypeptides with mitogenic effects on vascular smooth muscle cells and fibroblasts and roles in hemodynamic homeostasis, may be involved in the origin of coronary atherosclerosis. Macrophage foam cell formation, characterized by cholesterol ester accumulation, is modulated by scavenger receptor (cholesterol influx), acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1; storage cholesterol ester converted from free cholesterol), and ATP-binding cassette transporter A1 (cholesterol efflux).

Methods and Results— Serum salusin- levels were decreased in 173 patients with angiographically proven coronary artery disease compared with 40 patients with mild hypertension and 55 healthy volunteers (4.9±0.6 versus 15.4±1.1 and 20.7±1.5 pmol/L, respectively; P<0.0001). Immunoreactive salusin- and -β were detected in human coronary atherosclerotic plaques, with dominance of salusin-β in vascular smooth muscle cells and fibroblasts. After 7 days in primary culture, acetylated low-density lipoprotein–induced cholesterol ester accumulation in human monocyte-derived macrophages was significantly decreased by salusin- and increased by salusin-β. Salusin- significantly reduced ACAT-1 expression in a concentration-dependent manner. In contrast, salusin-β significantly increased ACAT-1 expression by 2.1-fold, with a maximal effect at 0.6 nmol/L. These effects of salusins were abolished by G-protein, c-Src tyrosine kinase, protein kinase C, and mitogen-activated protein kinase kinase inhibitors. ACAT activity and ACAT-1 mRNA levels were also significantly decreased by salusin- and increased by salusin-β; however, neither salusin- nor salusin-β affected scavenger receptor A function assessed by [¹²⁵I]acetylated low-density lipoprotein endocytosis or scavenger receptor class A and ATP-binding cassette transporter A1 expression.

Conclusions— Our results indicate that the 2 salusin isoforms have opposite effects on foam cell formation in human monocyte-derived macrophages. Development of atherosclerosis may be accelerated by salusin-β and suppressed by salusin- via ACAT-1 regulation.

Key Words: atherosclerosis • coronary disease • peptides • plaque • salusin-, human • salusin-β, human

	Introduction

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Salusins are a new class of bioactive peptides discovered by bioinformatics analyses of a full-length cDNA library.¹ Shichiri et al¹ recently identified and characterized 2 related peptides of 28 and 20 amino acids designated salusin- and salusin-β, respectively. These peptides are considered to be biosynthesized from preprosalusin, an alternative-splicing product of the torsion dystonia-related gene (TOR2A), after frameshift reading and digestion at dibasic amino acids.¹ Salusins are expressed and synthesized ubiquitously within human tissues, including the vasculature, central nervous system, and the kidney, and salusin- is present in human plasma and urine.^1,2 Salusin-β is the most potent hypotensive peptide identified to date but has no direct vasodilatory effect.^1,3 This peptide rapidly induces hypotension, bradycardia, and cardiac dysfunction by a cholinergic mechanism³ and promotes cardiomyocyte growth and antiapoptosis.^4,5 Salusin-β also stimulates the proliferation of vascular smooth muscle cells (VSMCs) and fibroblasts and induces the expression of growth-associated genes, such as c-myc and c-fos.¹ These phenomena are regarded as important characteristics of atherosclerosis. Salusin-, which has the same effects as those discussed above for salusin-β, is less potent than salusin-β in rats.

Clinical Perspective p 648

The development of atherosclerosis is influenced by abnormalities in cellular cholesterol homeostasis in subendothelial macrophages. Intracellular free cholesterol level is increased by the uptake of acetylated low-density lipoprotein (acetyl-LDL) via scavenger receptor class A (SR-A) and is decreased by the efflux of free cholesterol mediated by ATP-binding cassette transporter A1 (ABCA1).⁶ Because excessive accumulation of free cholesterol is toxic for cells, free cholesterol must either be removed through efflux to extracellular acceptors, such as HDL, or esterified to cholesterol ester (CE) by the microsomal enzyme acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1).⁷ ACAT-1 promotes CE accumulation in macrophages, thereby contributing to foam cell formation, a hallmark of early atherosclerosis^8–11; however, it has not been clarified whether salusins modulate human macrophage foam cell formation.

In the present study, we assessed the effects of salusin- and -β on acetyl-LDL–induced CE accumulation and its molecular mechanism in primary cultured human monocyte-derived macrophages. We also investigated the expression of salusin- and -β in coronary atherosclerotic lesions and serum levels of salusin- in patients with acute coronary syndrome (ACS).

	Methods

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Human Blood and Coronary Artery Sample Collection
Blood was collected from a total of 268 subjects: 60 ACS patients (12 with unstable angina pectoris, 48 with ST-elevation acute myocardial infarction) hospitalized for emergent coronary catheterization within 6 hours after onset (41 men and 19 women 47 to 89 years of age), 76 post–myocardial infarction patients at least 3 months after onset (61 men and 15 women 38 to 83 years old), 37 patients with stable effort angina pectoris (26 men and 11 women 46 to 80 years old), 40 patients with mild hypertension (140 to 159 over 90 to 99 mm Hg) without chest pain (28 men and 12 women 40 to 91 years old), and 55 healthy volunteers (35 men and 20 women 20 to 90 years of age) who were free from hypertension, diabetes mellitus, hyperlipidemia, and ischemic heart disease and were taking no medications. Patients with congestive heart failure were excluded from the study, because we have found decreased levels of serum salusin- in these patients. Human coronary artery samples were collected from 4 patients with ACS (2 men and 2 women 40 to 76 years of age) at autopsy. The study was approved by the Ethics Committee of Showa University.

Chemicals
Human salusin- and -β were obtained from Peptide Institute Inc (Osaka, Japan). GDP-β-S, rottlerin, PP2, PD98059, and anti-β-actin antibody were purchased from Sigma (St Louis, Mo). Human anti-SR-A1, anti-ABCA1, and anti-CD68 antibodies were purchased from R&D Systems (Minneapolis, Minn), Novus Biologicals (Littleton, Colo), and Santa Cruz Biotechnology (Santa Cruz, Calif), respectively. Human anti-ACAT-1 antibody was a gift from the Department of Biochemistry, Dartmouth Medical School (Hanover, NH). Human anti-salusin- and -β antibodies originally raised against N-terminal and C-terminal fragments of authentic salusin- and -β, respectively,¹ were affinity-purified for use in immunohistochemistry.

Cell Culture
Human peripheral mononuclear cells were isolated from the blood of healthy volunteers by Ficoll density gradient centrifugation.^9–11 Purified monocytes were suspended in RPMI-1640 medium and seeded onto 6-cm dishes (4x10⁶ cells/dish). After 1 hour of incubation (37°C, 5% CO₂) for adherence, the medium was replaced with RPMI-1640 medium supplemented with 10% pooled human sera, streptomycin (0.1 mg/mL), and penicillin G (100 U/mL).

Adherent monocytes were incubated at 37°C in 5% CO₂ for 7 days to induce differentiation into macrophages in the presence or absence of the indicated concentrations of salusin- or -β. To evaluate signal transduction pathways of salusin-– or salusin-β–induced ACAT-1 expression, anti-salusin- or -β antibody (2 µL/2-mL plate), the specific G-protein inactivator GDP-β-S (100 µmol/L), the specific c-Src tyrosine kinase inhibitor PP2 (1 µmol/L), the specific protein kinase C (PKC) inhibitor rottlerin (1 µmol/L), or the specific mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059 (1 µmol/L) was added along with salusin- or -β, and the signal blockade continued for 7 days.^10,11 To examine the inhibitory effect of salusin- on salusin-β–induced ACAT-1 expression, various concentrations of salusin- were added 1 hour before the addition of salusin-β, and then the simultaneous presence of both salusins was maintained for 7 days. Medium was replaced every 3 days with fresh medium that contained salusin- and/or -β in the presence or absence of the indicated inhibitors. To ensure that the blocking activities of inhibitors were not due to their cytotoxic effects, dose-response studies were performed with the inhibitors alone to select an appropriate concentration that was neither cytotoxic nor disturbed monocytic differentiation into macrophages.

Western Blotting Analysis
Cells were extracted with 100 µL of 10% SDS as described previously.^9–11 In a standard experiment, aliquots of 25 µg of protein were separated by 10% SDS-PAGE and subjected to Western blotting with a rabbit polyclonal antibody raised against human ACAT-1, SR-A, and ABCA1.

Assay for ACAT Activity
Enzyme activity was determined by reconstituted assay as described previously.^9–11 Cell extracts (80 µg/20 µL) were reconstituted with 140 µL of sodium taurocholate–cholesterol-phosphatidylcholine mixed micelles. The enzyme reaction was initiated by the addition of 20 µL of the substrate mixture containing 250 µmol/L [¹⁴C]oleoyl-coenzyme A (20 disintegrations per minute per picomole), followed by incubation for 30 minutes at 37°C. Lipids were extracted, and the amount of radioactive cholesterol-[¹⁴C]oleate was determined by thin-layer chromatography.

Quantitative Real-Time Reverse-Transcription Polymerase Chain Reaction
Total RNA and first-strand cDNA were prepared from samples (4x10⁶ cells). mRNA was quantified by real-time reverse-transcription polymerase chain reaction with qPCR MasterMix Plus SYBR Green I (Eurogentec, San Diego, Calif) in a GeneAmp 5700 Sequence Detection System (Applied Biosystems Japan, Ltd, Tokyo). Amplification was performed in a total volume of 25 µL for 40 cycles of 20 seconds at 94°C, 20 seconds at 55°C, and 30 seconds at 72°C. These mRNA levels were determined after normalization of RNA concentration with human β-actin, and values were expressed as fold changes against control. All samples were run in triplicate. The primers used in the present study were as follows: β-actin, 5'-CCTGGCACCCAGCACAAT (forward) and 5'-GGGCCGGACTCGTCATAC (reverse); ACAT-1, 5'-TTCGGAATATCAAACAGGAGCC (forward) and 5'-CACACCTGGCAAGATGGAGTT (reverse).

Assay for Cholesterol Esterification
Human LDL (d=1.019 to 1.063 g/mL) and acetyl-LDL were prepared as described previously.¹⁰ Monocytes were incubated for 7 days with or without the different concentrations of salusin- and -β, followed by incubation for 24 hours with the indicated concentrations of acetyl-LDL in the presence of 0.1 mmol/L [³H]oleate conjugated with bovine serum albumin.¹⁰ Cellular lipids were extracted, and the radioactivity of cholesterol-[³H]oleate was determined by thin-layer chromatography.

Cellular Assay for Endocytic Uptake of [¹²⁵I]Acetyl-LDL
Monocytes were incubated for 7 days with or without indicated concentrations of salusin- and -β. The cells in each dish were incubated for 18 hours with 5 or 10 µg/mL [¹²⁵I]acetyl-LDL.¹⁰ Aliquots (0.75 mL) of the culture medium were mixed with 0.25 mL of 40% trichloroacetic acid. To this solution was added 0.2 mL of 0.7 mol/L AgNO₃, followed by centrifugation at 2500 rpm for 10 minutes. Trichloroacetic acid–soluble radioactivity in the supernatant and cell-associated radioactivity were determined as described previously.¹⁰

Immunohistochemistry
Immunohistochemical analyses were performed to identify localization of salusin- and -β in human coronary arteries. Snap-frozen samples obtained at autopsy were stained with rabbit polyclonal antibodies raised against salusin- or -β after fixation with acetaldehyde. A dextran-based method (EnVision system; DakoCytomation A/S, Glostrup, Denmark) was used to detect antigens. Horseradish peroxidase activity was visualized with 3,3'-diaminobenzidine tetrahydrochloride, and hematoxylin was used for nuclear staining.

Radioimmunoassay for Salusin-
Salusin- concentrations in human monocytic culture media and sera from 268 subjects were measured by radioimmunoassay as described previously.² Measurements of salusin-β concentration in biological fluids are currently unavailable because of technical difficulties that arise due to the unexpected physical properties of this peptide.¹²

Statistical Analysis
All values are expressed as mean±SEM. Data were compared by 2-tailed unpaired Student t test between 2 groups and by 1-way ANOVA followed by Bonferroni post hoc test when >2 groups were involved. Differences were considered statistically significant at P<0.05.

The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

	Results

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Effects of Salusin- and -β on Acetyl-LDL–Induced CE Accumulation in Human Monocyte-Derived Macrophages
Figure 1A shows the effects of salusin- and -β on foam cell formation as assessed by acetyl-LDL–induced CE accumulation in human monocyte-derived macrophages. Coincubation with acetyl-LDL (5 µg/mL) significantly increased CE accumulation in macrophages (P<0.0001). Furthermore, salusin-β (0.6 nmol/L) significantly increased CE accumulation (P<0.005). In contrast, coincubation with salusin- (0.6 nmol/L) resulted in a significant decrease in CE accumulation (P<0.05). As shown in Figure 1B, CE accumulation in macrophages induced by acetyl-LDL (5 µg/mL) was maximized to 1.5-fold by salusin-β at 0.6 nmol/L (P<0.001), whereas it was decreased significantly by salusin-, with the maximal effect observed at 0.6 nmol/L (P<0.001).

View larger version (21K): [in this window] [in a new window]   Figure 1. Effects of salusin- and -β on acetyl-LDL–induced CE accumulation (foam cell formation) and endocytic uptake of [125I]acetyl-LDL (SR-A function) by human monocyte-derived macrophages. Human macrophages differentiated by 7-day culture with indicated concentrations of salusin- or -β were incubated for 24 hours with 5 µg/mL acetyl-LDL in the presence of 0.1 mmol/L [14C]oleate, and cellular CE accumulation was determined from the radioactivity of cholesterol-[14C]oleate (A and B). Otherwise, human macrophages differentiated by 7-day culture with 0.6 nmol/L salusin- or -β were incubated for 18 hours with the indicated concentrations of [125I]acetyl-LDL, followed by determination of cell association (C) and degradation (D) of [125I]acetyl-LDL. Data are expressed as mean± SEM from 6 independent experiments with monocytes from 6 different donors. A, *P<0.0001 vs 0 µg/mL acetyl-LDL of control; P<0.005, P<0.05 vs 5 µg/mL acetyl-LDL of control. B, Baseline of acetyl-LDL–induced CE accumulation at 0 nmol/L salusins= 4.61±0.31 nmol/mg cell protein; #P<0.05, P<0.01, ¶P<0.001 vs 0 nmol/L salusins. C, Control of 5 µg/mL [125I]acetyl-LDL=0.2±0.1 µg/mg cell protein per 18 hours. D, Control of 5 µg/mL [125I]acetyl-LDL=9.6±4.5 µg/mg cell protein per 18 hours.

Effects of Salusin- and -β on Endocytic Uptake of [¹²⁵I]Acetyl-LDL in Human Monocyte- Derived Macrophages
Figure 1C and 1D show the effects of salusin- and -β on SR-A function as assessed by the endocytic uptake of [¹²⁵I]acetyl-LDL by monocyte-derived macrophages. Neither salusin-β nor salusin- (0.6 nmol/L) showed significant effects on cell association or degradation of [¹²⁵I]acetyl-LDL (Figure 1C and 1D). Significant effects of salusin- and -β on endocytic uptake of [¹²⁵I]acetyl-LDL were not observed at other concentrations tested (data not shown).

Dose-Dependent Effects of Salusin- and -β on SR-A, ABCA1, and ACAT-1 Expression in Human Monocyte-Derived Macrophages
The concentration-dependent effects of salusin- and -β on SR-A and ABCA1 protein expression in human monocyte-derived macrophages are shown in Figure 2A and 2B. Neither salusin-β nor salusin- at any concentrations tested showed significant effects on SR-A and ABCA1 expression in human monocyte-derived macrophages on day 7 in culture. Figure 3 shows the concentration-dependent effects of salusin- and -β on ACAT-1 protein expression under identical conditions. Salusin-β significantly increased ACAT-1 expression by 2.1-fold, with the maximal effect observed at 0.6 nmol/L (Figure 3A and 3C). In contrast, salusin- significantly decreased ACAT-1 expression in a concentration-dependent manner (Figure 3B and 3C).

View larger version (23K): [in this window] [in a new window]   Figure 2. Dose-dependent effects of salusin- and -β on SR-A and ABCA1 expression in human monocyte-derived macrophages. Human monocytes incubated for 7 days with the indicated concentrations of salusin-β (A) or salusin- (B) were subjected to immunoblotting analysis for SR-A, ABCA1, and β-actin. β-Actin was used as an internal loading control. A and B, Top, Representative results of protein expression of each of these molecules; Bottom, densitometric data of SR-A and ABCA1 after normalization with β-actin. Data are expressed as mean±SEM from 6 independent experiments with monocytes from 6 different donors.

View larger version (22K): [in this window] [in a new window]   Figure 3. Dose-dependent effects of salusin- and -β on ACAT-1 expression in human monocyte-derived macrophages. Human monocytes incubated for 7 days with the indicated concentrations of salusin-β (A) or salusin- (B) were subjected to immunoblotting analysis for ACAT-1 and β-actin. A and B, Top: representative results of protein expression of each molecule; bottom: densitometric data of ACAT-1 after normalization with β-actin. A, B, and C, Data are expressed as mean±SEM from 6 independent experiments with monocytes from 6 different donors. *P<0.05, P<0.0005, P<0.005, P<0.01 vs control (0 nmol/L salusin- or -β).

Time-Dependent Effects of Salusin- and -β on SR-A, ABCA1, and ACAT-1 Expression in Human Monocytes/Macrophages
Figure 4 shows the time-dependent effects of salusin- and -β (0.6 nmol/L) on protein expression of SR-A, ABCA1, and ACAT-1 compared with a macrophage differentiation marker, CD68,¹³ during differentiation from human monocytes into mature macrophages (up to 7 days in culture). Expression of SR-A, ABCA1, and ACAT-1, as well as CD68, increased with monocytic differentiation into macrophages in the absence of salusin- or -β (as a control). In the presence of salusin- or -β, the expression of SR-A, ABCA1, and CD68 increased at similar rates relative to the corresponding controls; however, notable differences in ACAT-1 expression were present between salusin-– and salusin-β–treated monocytes/macrophages on day 7. ACAT-1 expression was significantly enhanced by salusin-β (P<0.0001), whereas it was reduced by salusin- (P<0.05).

View larger version (23K): [in this window] [in a new window]   Figure 4. Time-dependent effects of salusin- and -β on SR-A, ABCA1, and ACAT-1 expression during differentiation of human monocytes into macrophages. Human monocytes were incubated for the indicated times with or without 0.6 nmol/L salusin- or salusin-β. Cells were subjected to immunoblotting analysis for SR-A, ABCA1, ACAT-1, differentiation marker CD68, and β-actin. Each upper panel shows a representative result of protein expression of these molecules. Each lower panel shows densitometric data of SR-A, ABCA1, and ACAT-1 after normalization with β-actin. Data are expressed as mean±SEM from 4 independent experiments with monocytes from 4 different donors. *P<0.0001, P<0.05 vs corresponding day of control.

Effects of Salusin- and -β on ACAT Activity and ACAT-1 mRNA in Human Monocyte- Derived Macrophages
We examined the effects of salusin- and -β on ACAT activity and ACAT-1 mRNA level in monocyte-derived macrophages on day 7. As shown in Figure 5A and 5B, salusin-β (0.6 nmol/L) significantly increased ACAT activity and ACAT-1 mRNA level (both P<0.0001), whereas salusin- (0.6 nmol/L) significantly decreased both parameters (both P<0.05). In the treatment of monocyte-derived macrophages with salusin- or -β, the changes in ACAT activity and ACAT-1 mRNA were consistent with those in ACAT-1 protein expression.

View larger version (28K): [in this window] [in a new window]   Figure 5. Effects of salusin- and -β on ACAT activity (A) and ACAT-1 mRNA level (B), signal transduction pathways of salusin-β– or salusin-–induced ACAT-1 protein expression (C and D), and the inhibitory effect of salusin- on salusin-β–induced ACAT-1 expression (E) in human monocyte-derived macrophages. Human monocytes were incubated for 7 days with or without salusin- or -β (0.6 nmol/L) in the presence or absence of anti-salusin- or anti-salusin-β antibody (2 µL/2-mL plate), G-protein inactivator GDP-β-S (100 µmol/L), c-Src tyrosine kinase inhibitor PP2 (1 µmol/L), PKC inhibitor rottlerin (1 µmol/L), or MAPK kinase inhibitor PD98059 (1 µmol/L). Otherwise, human monocytes were treated with various concentrations of salusin- 1 hour before the addition of salusin-β (0.6 nmol/L) and incubated in the simultaneous presence of both salusins for 7 days. Medium in each dish was replaced with fresh medium with salusin- and/or -β in the presence or absence of inhibitors every 3 days. A, Cell homogenates were solubilized, and ACAT activity was determined with the reconstituted assay. B, Total RNA was extracted, and ACAT-1 mRNA level was quantified by real-time reverse-transcription polymerase chain reaction. C, D, and E, Cells were subjected to Western blotting analyses for ACAT-1. A and B, Data are expressed as mean±SEM from 5 independent experiments with monocytes from 5 different donors. A and B, *P<0.0001, P<0.05 vs each control.

Signal Transduction Pathways of Salusin-– or -β–Induced ACAT-1 Expression in Human Monocyte-Derived Macrophages
To determine how salusin- downregulates or salusin-β upregulates ACAT-1 expression, we examined the effects of anti-salusin- or -β antibody and specific inhibitors of G protein (GDP-β-S), c-Src tyrosine kinase (PP2), PKC (rottlerin), or MAPK kinase (PD98059) on salusin-– or -β–induced ACAT-1 expression. As shown in Figure 5C, the increase in ACAT-1 expression by salusin-β (0.6 nmol/L) was completely inhibited by anti-salusin-β antibody (2 µL/2-mL plate), PP2 (1 µmol/L), rottlerin (1 µmol/L), or PD98059 (1 µmol/L) and partially inhibited by GDP-β-S (100 µmol/L). As shown in Figure 5D, decreased ACAT-1 expression by salusin- (0.6 nmol/L) was also abolished by the same inhibitors.

Combined Effect of Salusin- and -β on ACAT-1 Expression in Human Monocyte- Derived Macrophages
We examined the inhibitory effect of various concentrations of salusin- on salusin-β–induced ACAT-1 protein expression in human monocyte-derived macrophages. As shown in Figure 5E, salusin-β (0.6 nmol/L) alone increased ACAT-1 expression by 1.9-fold. Salusin- inhibited the stimulatory effect of salusin-β (0.6 nmol/L) on ACAT-1 expression in a concentration-dependent manner.

Regulation of SR-A, ABCA1, and ACAT-1 Expression by Salusin- and -β After Monocytic Differentiation Into Human Macrophages
When human macrophages differentiated by 7-day culture without salusin- or -β were incubated for an additional 3 days with the indicated concentrations of salusin- or -β, SR-A and ABCA1 protein expression in mature macrophages was not affected by salusins (Figure 6A and 6B); however, ACAT-1 protein expression was increased significantly by salusin-β (Figure 6A) and decreased by salusin- (Figure 6B). Salusins affected ACAT-1 expression during and after differentiation from human monocytes into macrophages.

View larger version (22K): [in this window] [in a new window]   Figure 6. Effects of salusin- and -β on SR-A, ABCA1, and ACAT-1 expression in mature macrophages after differentiation from human monocytes. Human macrophages differentiated by 7-day culture without salusin- or -β were incubated for an additional 3 days with the indicated concentrations of salusin-β (A) or - (B). Cells were harvested and subjected to immunoblotting analysis for SR-A, ABCA1, ACAT-1, CD68, and β-actin. CD68 was used as a macrophage differentiation marker. Densitometric data of SR-A, ABCA1, and ACAT-1 after normalization with β-actin are expressed as mean±SEM from 4 independent experiments with monocyte-derived macrophages from 4 different donors. *P<0.05 vs control (0 nmol/L salusin- or -β).

Immunohistochemical Analysis of Human Coronary Atherosclerotic Plaques in ACS
Immunohistochemical analysis of human coronary arteries with polyclonal antibodies raised against salusin- or -β showed positive staining in atheromatous plaque and fatty streak lesions (Figure 7A, 7C, and 7E). Salusin- was expressed in fibroblasts and macrophages (Figure 7B). Salusin-β was expressed at high levels in VSMCs, fibroblasts, and the cell membranes of macrophage foam cells (Figure 7D and 7F); however, salusins were absent from endothelial cells in both normal and atherosclerotic coronary arteries (data not shown).

View larger version (153K): [in this window] [in a new window]   Figure 7. Micrographs of immunohistochemical staining of human coronary artery with atheroma in a patient with ACS (A through D: left anterior descending coronary artery; E and F: right coronary artery, 72-year-old man). A, Section stained with anti-salusin- antibody. B, Some fibroblasts and macrophages showed positive staining in salusin- (area indicated by rectangle in high-magnification view in A). C, Section stained with anti-salusin-β antibody. VSMCs showed staining for salusin-β. D, Salusin-β was expressed on fibroblasts in the area of the atheroma lesion indicated by a square in the high-magnification view in C. E, Section stained with anti-salusin-β antibody in fatty streak lesion. F, Salusin-β was expressed on fibroblasts and the cell membrane of macrophage foam cells in the area indicated by a square in the high-magnification view in E.

Serum Levels of Salusin- in ACS Among Ischemic Heart Diseases
Serum salusin- levels were significantly lower in patients with angiographically proven coronary artery diseases, such as stable effort angina pectoris and ACS, and after myocardial infarction, than in mildly hypertensive patients and healthy volunteers (P<0.0001; Figure 8A). ACS patients had the lowest levels of serum salusin-. Among ACS patients, serum salusin- levels were reduced in accordance with the severity of atherosclerotic lesions on coronary arteriography (Figure 8B). In particular, salusin- levels were significantly lower in ACS patients with triple-vessel disease than in ACS patients with single-vessel disease (P<0.05; Figure 8B). Mildly hypertensive patients had significantly lower salusin- levels (P<0.005) and greater values of mean and maximal intima-media thickness and plaque score (the sum of all plaque thicknesses) in bilateral carotid arteries on ultrasonography (P<0.0001, P<0.005) than did healthy volunteers.

View larger version (24K): [in this window] [in a new window]   Figure 8. Relations between serum salusin- levels and severity of atherosclerosis. A, Serum salusin- levels were measured by radioimmunoassay in 60 ACS patients, 37 patients with stable effort angina pectoris (SAP), 76 post–myocardial infarction (PMI) patients, 40 patients with mild hypertension (HT), and 55 healthy volunteers. B, All ACS patients underwent emergent coronary arteriography within 6 hours after onset. VD indicates vessel disease. C, Mean and maximal intima-media thickness (IMT) and plaque score in carotid arteries on ultrasonography were significantly greater in 40 patients with mild HT than in 23 healthy volunteers. Data are expressed as mean±SEM. A, *P<0.005 vs healthy volunteers, P<0.0001 vs healthy volunteers and mild HT patients. B, P<0.05 vs patients with single-vessel disease (1-VD). C, #P<0.0001, P<0.005 vs healthy volunteers.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
To the best of our knowledge, this is the first study to demonstrate relationships between salusins and coronary atherosclerosis in humans. Salusins are expressed at high levels within atherosclerotic plaques in human coronary arteries. In human atherosclerotic lesions, large amounts of salusins are locally generated by VSMCs and fibroblasts and may modulate macrophage foam cell formation. The results of the present study clarified the molecular and cellular mechanisms of macrophage foam cell formation as regulated by salusins. It is noteworthy that salusin- and -β, which are processed from the same precursor peptide (prosalusin), have opposite effects on ACAT-1 expression during and after differentiation of human monocytes into macrophages: ACAT-1 expression is downregulated by salusin-, whereas it is upregulated by salusin-β. Expression of ACAT-1, SR-A, and ABCA1 is spontaneously increased during monocytic differentiation into macrophages.^8,14,15 Neither salusin- nor -β shows significant effects on SR-A and ABCA-1 expression during and after differentiation. These findings indicate that selective regulation of ACAT-1 by salusins is independent of monocyte/macrophage differentiation. Macrophage foam cell formation is suppressed by salusin- and accelerated by salusin-β via their opposite regulatory effects on ACAT-1.

Our preliminary study showed that serum salusin- levels were inversely correlated with maximum intima-media thickness in the carotid artery in 71 middle-aged men (r=–0.29, P<0.02). The present study demonstrated that serum salusin- levels were slightly reduced in patients with mild hypertension compared with healthy volunteers, because they had mild carotid atherosclerosis. Serum salusin- levels decrease even more in patients with ischemic heart disease, in particular ACS, compared with mildly hypertensive patients. Furthermore, among patients with ACS, serum salusin- levels are reduced in accordance with the severity of coronary artery lesions. Together with the results of our in vitro experiments, this indicates that salusin- may contribute to prevention of the progression of atherosclerosis followed by coronary events. In contrast, salusin-β stimulates human macrophage foam cell formation and proliferation of both VSMCs and fibroblasts. Salusin-β also induces rapid and marked hypotension and profound bradycardia and cardiac dysfunction via parasympathetic stimulation.^1,3 Thus, we speculate that salusin-β may play some role in the origin of atherosclerosis, severely decompensated heart failure, and cardiovascular collapse. Further studies with proatherogenic animals infused with salusins in the presence or absence of pretreatment with salusin- or -β antiserum and salusin-knockout or transgenic animals are required to investigate the precise roles of salusins in atherosclerosis.

Wang et al¹⁶ discovered that salusin-β increases [Ca²⁺]_i via mouse mas-like G-protein–coupled receptor (GPCR), MrgA1, with an EC₅₀ of 300 nmol/L, but it does not activate the corresponding human MrgA1 receptor, and they concluded that salusin-β serves as its surrogate ligand. Thus, the exact receptor for salusin peptides as natural ligands remains unidentified. In the original publication,¹ salusins caused mitogenesis of rat VSMCs and fibroblasts by increasing [Ca²⁺]_i. This suggests the possibility that salusins may activate unidentified GPCRs. On the other hand, it is difficult to link certain other biological effects of salusins, such as their potent hemodynamic effects, to an increase in [Ca²⁺]_i. Therefore, it is possible that salusin-β may also bind to non-GPCR types of receptors.

Previous studies have not provided convincing evidence for the presence of distinct receptors for salusin- and -β. The original publication demonstrated that pretreatment with salusin- did not block the binding of salusin-β to VSMCs.¹ Salusin- was shown to have the same but weaker biological effects as salusin-β,^1,3 which suggests that salusin- binds to a common receptor for salusin- and -β with lower affinity. In the present study, we first presented the evidence for multiple salusin receptors by demonstrating the opposite effects of salusin- and -β on human macrophage foam cell formation. On the basis of the distinct physicochemical properties of the 2 peptides (salusin-β but not salusin- has many hydrophobic amino acid residues), it is reasonable to hypothesize that each salusin peptide binds to specific binding sites of its own.

Yu and colleagues⁴ have shown that signal transduction pathways by which salusins promote protein synthesis in rat cardiomyocytes are related to Ca²⁺/calcineurin/PKC/MAPK. These pathways show cross talk and may have an intracellular network-regulation role. However, little information is available on the pathways of ACAT-1 expression in monocytes/macrophages. The results of the present study show that ACAT-1 regulation by salusins is abolished by specific inhibitors of G protein, c-Src tyrosine kinase, PKC, or MAPK, which indicates that the G-protein/c-Src/PKC/MAPK pathway may be involved in a possible pathway of salusin-induced ACAT-1 regulation in human monocyte-derived macrophages. The signal transduction pathways for ACAT-1 regulation by salusin- and -β, especially at downstream levels between MAPK and ACAT-1 gene transcription (Sp1),¹⁷ need to be addressed.

ACAT-1, an intracellular enzyme located in the rough endoplasmic reticulum, plays a crucial role in CE accumulation as lipid droplets within macrophages in atherosclerotic lesions. A previous immunohistochemical study demonstrated high levels of ACAT-1 expression in macrophage foam cells in human atherosclerotic lesions.⁸ In primary cultured human monocyte-derived macrophages, ACAT-1 expression is upregulated by dexamethasone,¹⁸ dehydroepiandrosterone,¹⁹ transforming growth factor-β₁ (2-fold),⁹ urotensin II (2.5-fold),¹⁰ serotonin (2-fold),¹¹ and angiotensin II (2-fold)²⁰ but is downregulated by adiponectin (0.3-fold).²¹ We regard the 2-fold increase in ACAT-1 expression by these vasoactive agents as a significant cellular event that accelerates human macrophage foam cell formation.^10,20

The present study has several potential limitations. The concentrations of salusin- required for suppression of macrophage foam cell formation and ACAT-1 expression were relatively high (10- to 30-fold) compared with serum salusin- concentrations in humans. In atherosclerotic lesions, it is mainly VSMCs and fibroblasts (among vascular wall cells) that may generate large amounts of salusins in an autocrine/paracrine manner. Animal and clinical studies showed that local levels of other vasoactive agents, such as serotonin, urotensin II, and angiotensin II, increase by 15- to 30-fold with coronary events or atherothrombosis formation in obstructive arteries and cardiac interstitial fluid,^22–25 which is comparable to the present results. Therefore, the concentrations of salusin- around monocytes/macrophages within atherosclerotic lesions may be close to those required for suppression of ACAT-1 and macrophage foam cell formation in the present study. However, 1 question raised was whether salusins endogenously produced by monocytes/macrophages in primary culture may have influenced the present results. We determined the salusin- concentrations in 7-day culture during differentiation of monocytes into macrophages in RPMI-1640 medium containing 10% human sera without addition of exogenous salusins. The levels of salusin- in the medium were below the limit of detection (ie, <0.1 pmol/L) by radioimmunoassay throughout the culture period. Thus, the concentrations of salusins produced endogenously by monocytes/macrophages in culture are negligible.

Salusin- is present in its authentic form in human serum and urine,² whereas salusin- and -β are concomitantly detected immunohistochemically in human tissues. These data at least suggest that the cleavage site between salusin- and -β is active in humans. Although definitive evidence that suggests N-terminal cleavage of salusin-β and the presence of authentic salusin-β is yet to be obtained owing to technical difficulties,¹² this site also contains dibasic amino acids, Arg and Lys, that easily receive digestion by the prohormone convertase. In addition to the processing of salusin-β, its release, degradation, and existing molecular forms remain to be elucidated. The difficulties appear to be caused in part by the unique physicochemical properties of salusin-β peptide, which prevent the establishment of highly sensitive assay systems.¹² However, determination of plasma salusin-β levels in normal and cardiovascular diseases would certainly add new information to our understanding of salusins and their impact on cardiovascular diseases.

In conclusion, the results of our present and previous studies indicate that salusin-β accelerates human macrophage foam cell formation by ACAT-1 upregulation and induces VSMC and fibroblast proliferation, probably contributing to the development of human coronary atherosclerotic lesions. Salusin- participates in antiatherogenesis by suppressing foam cell formation via ACAT-1 downregulation. Clinically, the results of the present study provide insights into the potential use of salusin- as a biomarker for atherosclerosis and a therapeutic target for the prevention of atherosclerotic cardiovascular diseases. Prospective studies of salusins that examine relationships with other risk factors and outcomes over time may be warranted.

	Acknowledgments

 
We thank Drs Takatoshi Koyama and Keiko Takahashi for their technical assistance and Professor Seiji Shioda for helpful discussion.

Sources of Funding

This work was supported in part by a grant-in-aid for scientific research (C; 18590824 to Dr Watanabe) from the Japan Society for the Promotion of Science and the High-Technology Research Center Project from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Disclosures

None.

	References

Top Abstract Introduction Methods Results Discussion References

 

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CLINICAL PERSPECTIVE

Human salusin- and -β are peptides processed from the same precursor peptide, prosalusin. The present study examined the potential roles of these peptides in atherosclerosis. Early events of atherosclerosis (foam cell formation) were oppositely influenced by salusin- and -β with in vitro assays of primary human monocyte-derived macrophages. Salusin- suppressed foam cell formation via downregulation of acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1), whereas salusin-β upregulated ACAT-1 to enhance foam cell formation. The in vitro observations have clinical relevance in that immunoreactive salusin- and -β were detected in human coronary atherosclerotic plaques, with dominance of salusin-β in vascular smooth muscle cells and fibroblasts. Serum salusin- levels were decreased in 173 patients with angiographically proven coronary artery disease compared with 40 patients with mild hypertension and 55 healthy volunteers (4.9±0.6 versus 15.4±1.1 and 20.7±1.5 pmol/L, respectively; P<0.0001). Furthermore, in 60 patients with acute coronary syndrome, serum salusin- levels were decreased in accordance with the severity of coronary atherosclerotic lesions. These data suggest that salusin-β may contribute to the pathogenesis of atherosclerosis. Salusin- could be a candidate biomarker for atherosclerosis and a therapeutic target for the prevention of atherosclerotic cardiovascular diseases. Prospective studies of salusins that examine relationships with other risk factors and outcomes over time may be warranted.

	Footnotes

 
Presented in part at the 80th Scientific Sessions of the American Heart Association, Orlando, Fla, November 3–7, 2007, and published in abstract form [Circulation. 2007;116(suppl):II-2].

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Clinical Summaries
Circulation 2008 117: 589-591. [Full Text]

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