Kengo Sato

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

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-α 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-α 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-α and increased by salusin-β. Salusin-α 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-α and increased by salusin-β; however, neither salusin-α 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-α via ACAT-1 regulation.

Preventive Effect of Dipeptidyl Peptidase-4 Inhibitor on Atherosclerosis Is Mainly Attributable to Incretin's Actions in Nondiabetic and Diabetic Apolipoprotein E-Null Mice

Aim Several recent reports have revealed that dipeptidyl peptidase (DPP)-4 inhibitors have suppressive effects on atherosclerosis in apolipoprotein E-null (Apoe −/−) mice. It remains to be seen, however, whether this effect stems from increased levels of the two active incretins, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Methods Nontreated Apoe −/− mice, streptozotocin-induced diabetic Apoe −/− mice, and db/db diabetic mice were administered the DPP-4 inhibitor vildagliptin in drinking water and co-infused with either saline, the GLP-1 receptor blocker, exendin(9–39), the GIP receptor blocker, (Pro3)GIP, or both via osmotic minipumps for 4 weeks. Aortic atherosclerosis and oxidized low-density lipoprotein-induced foam cell formation in exudate peritoneal macrophages were determined. Results Vildagliptin increased plasma GLP-1 and GIP levels without affecting food intake, body weight, blood pressure, or plasma lipid profile in any of the animals tested, though it reduced HbA1c in the diabetic mice. Diabetic Apoe −/− mice exhibited further-progressed atherosclerotic lesions and foam cell formation compared with nondiabetic counterparts. Nondiabetic and diabetic Apoe −/− mice showed a comparable response to vildagliptin, namely, remarkable suppression of atherosclerotic lesions with macrophage accumulation and foam cell formation in peritoneal macrophages. Exendin(9–39) or (Pro3)GIP partially attenuated the vildagliptin-induced suppression of atherosclerosis. The two blockers in combination abolished the anti-atherosclerotic effect of vildagliptin in nondiabetic mice but only partly attenuated it in diabetic mice. Vildagliptin suppressed macrophage foam cell formation in nondiabetic and diabetic mice, and this suppressive effect was abolished by infusions with exendin(9–39)+(Pro3)GIP. Incubation of DPP-4 or vildagliptin in vitro had no effect on macrophage foam cell formation. Conclusions Vildagliptin confers a substantial anti-atherosclerotic effect in both nondiabetic and diabetic mice, mainly via the action of the two incretins. However, the partial attenuation of atherosclerotic lesions by the dual incretin receptor antagonists in diabetic mice implies that vildagliptin confers a partial anti-atherogenic effect beyond that from the incretins.

Systemic Distribution of Salusin Expression in the Rat

Salusin-α and salusin-β are multifunctional bioactive peptides with hypotensive and bradycardic effects. They were originally identified from full-length human cDNAs by bioinformatics analyses. Salusin peptides are expressed in human tissues at the mRNA level, but no information is available about their systemic distributions in any species. We examined the distributions of preprosalusin mRNA and the salusin peptides in a variety of normal rat organs. Whereas preprosalusin mRNA was expressed ubiquitously, immunoreactive salusin-β was detected most strongly in the hypothalamus and posterior pituitary, and less abundantly in the anterior pituitary and gastrointestinal, immune, and hematopoietic systems. Salusin-β−positive cells appeared to be of either hematopoietic or endocrine origin, and many hematopoietic cells were also stained with anti-CD68, which specifically recognizes macrophages. Salusin-α−like immunoreactivity was not detected in any of the rat tissues. These results indicate that rat salusin is immunologically similar to human salusin-β and widely expressed, especially in the immune, gastrointestinal, and central nervous systems and mainly in endocrine- and hematopoietic-derived cells.

Chronic infusion of salusin-α and -β exerts opposite effects on atherosclerotic lesion development in apolipoprotein E-deficient mice

OBJECTIVE Human salusin-alpha and -beta are two-related peptides processed from the same precursor, preprosalusin. Our previous in vitro studies have shown that human macrophage foam cell formation is stimulated by salusin-beta but suppressed by salusin-alpha. Thus we investigated the effects of salusin-alpha and -beta on atherosclerotic plaque formation in vivo in apolipoprotein E-deficient (ApoE-/-) mice. METHODS Saline (vehicle), salusin-alpha or -beta (0.6 nmol/kg/h) was continuously infused through osmotic mini-pumps into 13-week-old ApoE-/- mice for 8 weeks. Aortic atherosclerosis, oxidized LDL-induced cholesterol ester accumulation (foam cell formation), and its related gene expression in exudate peritoneal macrophages were determined. RESULTS After 4-week infusion of salusin-beta, atherosclerotic lesions were 2.6 times greater than vehicle controls, which paralleled 1.9-fold increase in foam cell formation and up-regulation of scavenger receptors (CD36, scavenger receptor class A) and acyl-CoA: cholesterol acyltransferase-1 (ACAT1). In contrast, salusin-alpha decreased serum total cholesterol levels by 15% and foam cell formation by 68% associated with ACAT1 down-regulation. After 8-week infusion of salusin-alpha, atherosclerotic lesions were significantly suppressed by 54% compared with vehicle controls. CONCLUSIONS Our study provided the first evidence that salusin-beta accelerates the development of atherosclerotic lesions associated with up-regulation of scavenger receptors and ACAT1 in ApoE-/- mice. Whilst, salusin-alpha exerts anti-atherosclerotic effects by suppressing serum total cholesterol levels and ACAT1 expression.

Serum Salusin-α Levels Are Decreased and Correlated Negatively with Carotid Atherosclerosis in Essential Hypertensive Patients

Salusin-α is a new bioactive peptide with mild hypotensive and bradycardic effects. Our recent study showed that salusin-α suppresses foam cell formation in human monocyte-derived macrophages by down-regulating acyl-CoA:cholesterol acyltransferase-1, contributing to its anti-atherosclerotic effect. To clarify the clinical implications of salusin-α in hypertension and its complications, we examined the relationship between serum salusin-α levels and carotid atherosclerosis in hypertensive patients. The intima-media thickness (IMT) and plaque score in the carotid artery, blood pressure, serum levels of salusin-α, and atherosclerotic parameters were determined in 70 patients with essential hypertension and in 20 normotensive controls. There were no significant differences in age, gender, body mass index, fasting plasma glucose level, or serum levels of high-sensitive C-reactive protein, high- or low-density lipoprotein (LDL) cholesterol, small dense LDL, triglycerides, lipoprotein(a), or insulin between the two groups. Serum salusin-α levels were significantly lower in hypertensive patients than in normotensive controls. The plasma urotensin-II level, maximal IMT, plaque score, systolic and diastolic blood pressure, and homeostasis model assessment for insulin resistance (HOMA-IR) were significantly greater in hypertensive patients than in normotensive controls. In all subjects, maximal IMT was significantly correlated with age, systolic blood pressure, LDL cholesterol, urotensin-II, salusin-α, and HOMA-IR. Forward stepwise multiple linear regression analysis revealed that salusin-α levels had a significantly independent and negative association with maximal IMT. Serum salusin-α levels were significantly lower in accordance with the severity of plaque score. Our results suggest that the decrease in serum salusin-α, an anti-atherogenic peptide, may be associated with carotid atherosclerosis in hypertensive patients.

Presence of immunoreactive salusin-α in human serum and urine

Salusins, identified from a full-length enriched human cDNA library by bioinformatics analyses, show mitogenic, neuromodulatory and hemodynamic activities in rats. They are expressed in a wide variety of human tissues, but their precise structures and levels in human body fluids remain unknown. We developed a radioimmunoassay suitable for the detection of immunoreactive human salusin-alpha and characterized the molecular forms and concentrations of salusin-alpha in human serum and urine. The assay allowed for measurement of immunoreactive salusin-alpha concentrations as low as 1 fmol/tube after extraction of serum with an octyl-silica column, and the concentration required for 50% inhibition of binding was 40 fmol/tube. Cross-reactivities with salusin-beta and other bioactive peptides were negligible. Salusin-alpha-like immunoreactivity in normal human serum and urine ranged from 11.0 to 40.4 pmol/l (mean+/-S.D., 23.3+/-8.1 pmol/l, n=31) and from 18.6 to 367.3 pmol/l (mean+/-S.D., 156.8+/-95.8 pmol/l), respectively. Reverse-phase high performance liquid chromatography coupled with radioimmunoassay detection revealed a major immunoreactive component that coeluted with authentic salusin-alpha. These data indicate the presence of salusin-alpha in human serum and urine, thereby verifying the initially predicted processing sites for salusin-alpha in humans.

A glucagon-like peptide-1 analog liraglutide suppresses macrophage foam cell formation and atherosclerosis

Macrophage foam cell formation, characterized by cholesterol ester accumulation catalyzed by acyl-CoA:cholesterol acyltransferase 1 (ACAT1), is the hallmark of early atherogenesis. We previously demonstrated the suppressive effects of incretins, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), on the development of atherosclerotic lesions in apolipoprotein E-deficient (apoE(-/-)) mice. The present study was performed to evaluate the suppressive effects of these incretins and GLP-1 analogs, such as exendin-4 and liraglutide, on human macrophage foam cell formation in vitro and those of liraglutide on atherosclerotic lesion development in apoE(-/-) mice. We investigated the suppressive effects of GLP-1, GIP, exendin-4, and liraglutide against oxidized low-density lipoprotein (oxLDL)-induced foam cell formation in primary cultured human monocyte-derived macrophages. Seventeen-week-old apoE(-/-) mice were administered a long-acting GLP-1 analog liraglutide by osmotic mini-pumps for 4 weeks. Aortic atherosclerosis, oxLDL-induced foam cell formation, and related gene expression in exudate peritoneal macrophages were determined in vivo and ex vivo. Receptors for GLP-1 and GIP were expressed at high levels in human aortic smooth muscle cells and monocytes, but at relatively low levels in human macrophages and foam cells. GLP-1, GIP, exendin-4, and liraglutide significantly suppressed oxLDL-induced foam cell formation mainly associated with ACAT1 down-regulation in human monocyte-derived macrophages. The infusion of liraglutide into apoE(-/-) mice significantly retarded atherosclerotic lesions with monocyte/macrophage infiltration in the aortic wall and suppressed foam cell formation and ACAT1 expression in macrophages. These findings indicate that liraglutide could prevent the development of atherosclerotic lesions by suppressing macrophage foam cell formation mainly associated with ACAT1 down-regulation.

Counteractive effects of omentin-1 against atherogenesis

AIMS Omentin-1, a novel adipocytokine expressed in visceral fat tissue, is negatively correlated with obesity, insulin resistance, and stable coronary artery disease (CAD). However, there have been no previous reports regarding the effects of omentin-1 on atherogenesis. METHODS AND RESULTS This study was performed to evaluate the atheroprotective effects of omentin-1 on human monocyte-derived macrophages, human aortic smooth muscle cells (HASMCs) in vitro, and aortic lesions in Apoe(-/-) mice in vivo. The histological expression of omentin-1 in coronary artery lesions and epicardial adipose tissues and its plasma levels were compared between CAD and non-CAD patients. Omentin-1 was abundantly expressed in human umbilical vein endothelial cells, macrophages, HASMCs, and human coronary artery SMCs in vitro. Omentin-1 promoted anti-inflammatory M2 phenotype during differentiation of human monocytes into macrophages. Omentin-1 suppressed oxidized low-density lipoprotein-induced foam cell formation associated with down-regulation of CD36, scavenger receptor class A, and acyl-CoA:cholesterol acyltransferase-1 and up-regulation of neutral cholesterol ester hydrolase in human macrophages. Omentin-1 suppressed angiotensin II-induced migration and platelet-derived growth factor-BB-induced proliferation, and collagen-1 and -3 expression in HASMCs. Four-week infusion of omentin-1 into Apoe(-/-) mice retarded the development of aortic atherosclerotic lesions with reduced contents of monocytes/macrophages, SMCs, and collagen fibres along with peritoneal M2-activated macrophages with inflammasome down-regulation and lowered plasma total cholesterol levels. Omentin-1 levels were markedly reduced in coronary endothelium and epicardial fat but increased in plasma and atheromatous plaques (macrophages/SMCs) in CAD patients compared with non-CAD patients. CONCLUSION This study provided the first evidence that omentin-1 may serve as a novel therapeutic target for atherosclerosis and CAD.

Presence of immunoreactive salusin-β in human plasma and urine

Salusin-alpha and salusin-beta are multifunctional bioactive peptides originally identified using bioinformatics analyses. Salusin-beta has been shown to exert potent hypotensive, bradycardic, and pro-atherosclerotic effects. The form in which it exists in biological fluids remains undetermined due to technical difficulties originating from its unexpected physicochemical properties. Here we show that salusin-beta peptide adheres to polypropylene and polystyrene, so that the aliquoted peptide dissolved in distilled water may rapidly disappear from the solution. By circumventing these features and using an antibody against C-terminal portion of salusin-beta, we have successfully established a specific radioimmunoassay suitable for detection of immunoreactive human salusin-beta. We have characterized the molecular form of salusin-beta in human plasma and urine. The assay detected immunoreactive salusin-beta concentrations as low as 5 fmol/tube and the concentration required for 50% inhibition of binding was 122 fmol/tube. Cross-reactivities with salusin-alpha and other bioactive peptides were negligible. Reverse-phase high performance liquid chromatography coupled with the radioimmunoassay detection after extraction from plasma and urine and using an octyl-silica column, revealed a major immunoreactive component that co-eluted with authentic salusin-beta. Salusin-beta-like immunoreactivity in normal human urine ranged from 0.23 to 2.22 nmol/l (mean+/-SD, 1.16+/-0.84 nmol/l, n=10). These data present the first evidence that salusin-beta circulates and is excreted in its authentic form, thereby verifying the initially predicted processing sites for salusin-beta in humans.

Expression of prosalusin in human neuroblastoma cells

Salusins, which are derived from the prosalusin precursor molecule, regulate hemodynamics, mitogenesis and atherogenesis. The preprosalusin gene is ubiquitously expressed, while the salusin-beta peptide is present in systemic endocrine cells and the neuroendocrine system. However, the regulatory mechanisms for the preprosalusin gene and prosalusin expression remain to be investigated. Real-time quantitative RT-PCR and salusin-alpha radioimmunoassay revealed that the neuroblastoma cell line, SK-N-SH, exhibited marked upregulation of preprosalusin mRNA and salusin-alpha-like immunoreactivity (LI) when incubated under 2% serum condition. However, SK-N-SH cells released a limited amount of salusin-alpha-LI into the culture supernatant. Reverse-phase high performance liquid chromatography coupled with radioimmunoassay after extraction of proteins from the conditioned media using an octyl-silica column did not reveal a component that co-eluted with authentic salusin-alpha. Western blotting of the nuclear extracts from SK-N-SH showed the expression of prosalusin and its cleaved fragments, but not authentic salusin-alpha. Addition of Jak-2 inhibitors to growing SK-N-SH cells cultured under 10% serum condition resulted in increased salusin-alpha-LI expression. Suppression of Jak-2 mRNA using siRNAs upregulated intracellular salusin-alpha-LI, as detected by immunofluorescence. In summary, the preprosalusin gene and prosalusin protein are expressed in a neuroblastoma cell line and upregulated by reduced serum. The Jak-2 pathway may be involved in the regulation of salusin expression.