Vasostatin-1, a chromogranin A (CgA)-derived peptide (76 amino acids), is known to suppress vasoconstriction and angiogenesis. A recent study has shown that vasostatin-1 suppresses the adhesion of human U937 monocytes to human endothelial cells (HECs) via adhesion molecule down-regulation. The present study evaluated the expression of vasostatin-1 in human atherosclerotic lesions and its effects on inflammatory responses in HECs and human THP-1 monocyte-derived macrophages, macrophage foam cell formation, migration and proliferation of human aortic smooth muscle cells (HASMCs) and extracellular matrix (ECM) production by HASMCs, and atherogenesis in apolipoprotein E-deficient (ApoE−/−) mice. Vasostatin-1 was expressed around Monckeberg’s medial calcific sclerosis in human radial arteries. Vasostatin-1 suppressed lipopolysaccharide (LPS)-induced up-regulation of monocyte chemotactic protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin in HECs. Vasostatin-1 suppressed inflammatory M1 phenotype and LPS-induced interleukin-6 (IL-6) secretion via nuclear factor-κB (NF-κB) down-regulation in macrophages. Vasostatin-1 suppressed oxidized low-density lipoprotein (oxLDL)-induced foam cell formation associated with acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) and CD36 down-regulation and ATP-binding cassette transporter A1 (ABCA1) up-regulation in macrophages. In HASMCs, vasostatin-1 suppressed angiotensin II (AngII)-induced migration and collagen-3 and fibronectin expression via decreasing ERK1/2 and p38 phosphorylation, but increased elastin expression and matrix metalloproteinase (MMP)-2 and MMP-9 activities via increasing Akt and JNK phosphorylation. Vasostatin-1 did not affect the proliferation and apoptosis in HASMCs. Four-week infusion of vasostatin-1 suppressed the development of aortic atherosclerotic lesions with reductions in intra-plaque inflammation, macrophage infiltration, and SMC content, and plasma glucose level in ApoE−/− mice. These results indicate the inhibitory effects of vasostatin-1 against atherogenesis. The present study provided the first evidence that vasostatin-1 may serve as a novel therapeutic target for atherosclerosis.

Introduction

Atherosclerosis is characterized by a complex interaction of vascular injury, inflammation with monocyte adhesion to endothelial cells (ECs), lipid deposition with macrophage foam cells, vascular smooth muscle cell (VSMC) proliferation, and extracellular matrix (ECM) remodeling [1]. Inflammation is characterized by up-regulation of monocyte chemotactic protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin in ECs and production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in monocytes/macrophages, and macrophage phenotypes classified as pro-inflammatory M1 and anti-inflammatory M2 [2,3]. Macrophage foam cell formation is characterized by cholesterol ester accumulation that depends on the homeostatic balance amongst the uptake of oxidized low-density lipoprotein (oxLDL) via a scavenger receptor CD36, the efflux of free cholesterol controlled by ATP-binding cassette transporter A1 (ABCA1), and cholesterol esterification from excess free cholesterol by acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) [4]. The migration, proliferation, and production of ECM components, such as collagens, matrix metalloproteinases (MMPs), fibronectin, and elastin, in VSMCs together with EC proliferation accelerate the development of atherosclerotic lesions [1,5].

Vasostatin-1 represents the N-terminal fragment of chromogranin A (CgA) containing 76-amino acid residue [6], and is cleaved from CgA by plasmin [7]. The sympathoadrenal system is the most abundant source of CgA/vasostatin-1 in vivo [6]. CgA and vasostatin-1 are also known to be abundantly expressed in lung carcinoma and pancreas, respectively [8,9]. Specific receptors for vasostatin-1 have not been identified so far. Several lines of evidence have shown the endothelium-dependent cardiovasoactive effects of vasostatin-1 [10–14]. Vasostatin-1 activates endothelial nitric oxide synthase in ECs [15], and suppresses tension in isolated human blood vessel segments [16]. Vasostatin-1 inhibits vascular endothelial growth factor (VEGF)-induced migration and proliferation in human umbilical vein ECs (HUVECs) [17,18], and inhibits angiogenesis [19]. Vasostatin-1 inhibits TNF-α-, angiotensin II (AngII)-, and oxLDL-induced expression of ICAM-1, VCAM-1, and E-selectin, and attenuates TNF-α-induced adhesion of human U937 monocytes to human arterial ECs [20]. Recent clinical studies have shown the increased plasma levels of vasostatin-1 in patients with carotid and coronary atherosclerosis and Takayasu arteritis [21–23]. However, the effects of vasostatin-1 on atherogenesis have not yet been clarified.

In the present study, we assessed the expression of vasostatin-1 in human atherosclerotic lesions and the suppressive effects of vasostatin-1 on the inflammatory response in HUVECs, the inflammatory response and foam cell formation in human THP-1 monocyte-derived macrophages, and the migration, proliferation, and ECM production in human aortic smooth muscle cells (HASMCs) in vitro. In addition, in vivo experiments focussed on the inhibitory effects of vasostatin-1 against atherogenesis in apolipoprotein E-deficient (ApoE−/−) mice, an animal model of atherosclerosis. The present study was performed as a translational research in an attempt to search a candidate therapeutic target for atherosclerosis.

Methods

Ethics statement

The present study was approved by the Ethics Committees and the Institutional Animal Care and Use Committee of Tokyo University of Pharmacy and Life Sciences.

Materials

Human vasostatin-1 (CgA17-76), with the purity of ≥95% on HPLC, was purchased from Phoenix Pharmaceuticals (Burlingame, CA, U.S.A.). Rabbit polyclonal antibodies against human vasostatin-1 and human CgA were purchased from Aviscera Bioscience (Santa Clara, CA, U.S.A.) and Abgent (San Diego, CA, U.S.A.), respectively. Lipopolysaccharide (LPS) and AngII were purchased from Sigma (St. Louis, MO, U.S.A.), and PMA was from Wako (Osaka, Japan). HUVECs and HASMCs were purchased from Lonza (Walkersville, MD, U.S.A.) and human monocytic leukemia cell line THP-1 was from Health Science Research Resources Bank (Osaka, Japan). EA.hy926 ECs were derived by the fusion of HUVECs with the continuous human lung carcinoma cell line A549. EC growth media-2 (EGM-2; 5% FBS, 0.1% epidermal growth factor (EGF), 0.4% fibroblast growth factor (FGF), 0.1% VEGF, 0.1% heparin, 0.04% hydrocortisone, 0.1% R3-insulin-like growth factor-1, and 0.1% gentamicin–amphotericin B mixture) and smooth muscle growth medium-2 (SmGM-2; 5% FBS, 0.1% EGF, 0.2% FGF, 0.1% insulin, and 0.1% gentamicin–amphotericin B mixture) were purchased from Lonza.

Reverse transcription-PCR

HUVECs were seeded on to 3.5-cm dishes, and incubated at 37°C in 5% CO2 for 24 h in EGM-2 (Lonza). When cells reached 60–70% confluence, they were incubated for 30 min with 440 nM vasostatin-1 and then for 2 h with 1 μg/ml LPS and 440 nM vasostatin-1 [24–26]. Vasostatin-1 was used at 440 nM (3 μg/ml), since at this concentration it showed the maximal effect in HUVECs [17] with a significant inhibitory effect on TNF-α-induced ICAM-1 expression in human arterial ECs [20]. The mRNAs of MCP-1, ICAM-1, VCAM-1, E-selectin, and glyceraldehyde-3-dehydrogenase (GAPDH) were detected as described previously [24–28].

Proliferation assay

EA.hy926 ECs or HASMCs were seeded on to 96-well plates (1 × 104 cells/100 μl/well) and incubated at 37°C in 5% CO2 for 24 h in EGM-2 or SmGM-2 (Lonza), respectively. Cells were further incubated for 48 h with the indicated concentrations of vasostatin-1 with renewal of each medium. Then, 10 μl of WST-8 solution (Cell Count Reagent SF; Nacalai Tesque, Kyoto, Japan) was added to each well [24–31]. After 1 h of incubation, the amount of formazan product was determined by measuring the absorbance at 450 nm using a Sunrise Remote R™-micro plate reader (Tecan, Kawasaki, Japan) [24–31].

Apoptosis assay

HASMCs were seeded into 12-well plates (3 × 105 cells/1 ml/well) and incubated at 37°C in 5% CO2 for 24 h in SmGM-2, followed by a 48-h incubation with 440 nM vasostatin-1. Vasostatin-1 was used at 440 nM, since it showed the significant effects at this concentration in various types of cells [17,32]. Cells were fixed with 4% paraformaldehyde. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining was then performed using an In Situ Apoptosis Detection Kit (Takara Bio, Kusatsu, Japan) as described previously [25–28].

Migration assay

HASMCs were seeded on to eight-well culture slide (3 × 103 cells/200 μl/well). Cells were incubated at 37°C in 5% CO2 for 3–5 h in SmGM-2, and were then incubated for 24 h in serum-free SmGM-2 for starvation. Subsequently, while cells were incubated for 15 h in serum-free SmBM with 500 nM AngII and/or 440 nM vasostatin-1, photographs of cells were taken for the last 5 h at 10-min intervals. Vasostatin-1 was used at 440 nM, since it showed the significant effects at this concentration in various types of cells [17,32]. The average migration distance of ten cells randomly selected in each well was measured using a BIOREVO BZ-9000 microscope (Keyence, Osaka, Japan) [24–31].

Foam cell formation assay

THP-1 monocytes were seeded on to 3.5-cm dishes (1 × 106 cells/1 ml/dish). Cells were incubated at 37°C in 5% CO2 for 3 days in RPMI-1640 medium (Sigma) supplemented with 10% FBS, 0.05 mg/ml streptomycin, and 50 U/ml penicillin (monocyte/macrophage-conditioned medium (MCM)) and the indicated concentrations of vasostatin-1 in the presence of 150 ng/ml PMA to induce differentiation into macrophages [25,26]. After then, THP-1 monocyte-derived macrophages were incubated for 3 days in MCM with vasostatin-1, and further incubated for 2 days in the fresh MCM supplemented with vasostatin-1, 50 μg/ml human oxLDL, and 100 μM [3H]oleate (PerkinElmer, Yokohama, Japan) conjugated with BSA [24–31]. Cellular lipids were extracted and the radioactivity of cholesterol-[3H]oleate was determined by TLC.

Inflammatory cytokine measurement

THP-1 monocyte-derived macrophages cultured in MCM were incubated for 2 h in serum-free MCM with 50 nM vasostatin-1 and then for 24 h with 1 μg/ml LPS and 50 nM vasostatin-1. Vasostatin-1 was used at 50 nM, since it showed the maximal anti-inflammatory responses at this concentration in THP-1 monocyte-derived macrophages in the present study. In culture supernatants, the concentrations of IL-6 and TNF-α were measured by ELISA (human IL-6 and human TNF-α ELISA Kits, R&D Systems) [24–26].

Western blotting

Aliquots of protein extracts (20 μg) derived from THP-1 monocytes, derived macrophages, HASMCs, HUVECs, and EA.hy926 ECs were separated by SDS/PAGE (10% gel), and then immunoblotted with specific antibodies raised against CgA or vasostatin-1, and others as described previously [24–31]. Rabbit polyclonal CgA antibody used in the present study was more useful for detecting clearly bands compared with that in our previous report [25].

Gelatin zymography

The activities of MMP-2 and MMP-9 in culture supernatants of HASMCs incubated with the indicated concentrations of vasostatin-1 for 48 h were determined using a gelatin-zymography kit (Cosmo Bio, Tokyo, Japan) as described previously [24–28].

Administration of vasostatin-1 into mice

Animal experiments were performed in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals, with protocols approved by the Institutional Animal Care and Use Committee of Tokyo University of Pharmacy and Life Sciences. A total of 25 male spontaneously hyperlipidemic ApoE−/− mice (BALB/c. KOR/StmSlc-Apoeshl mice) at the age of 9 weeks were purchased from Japan SLC and maintained on a normal diet until 13 weeks of age. Subsequently, these mice were fed a high-cholesterol diet containing 1.25% cholesterol, 3.0% lard, and 1.625% glucose (F2HFD1; Oriental Yeast, Tokyo, Japan). At 17 weeks of age, six mice were killed as pre-infusion controls. The remaining 19 were divided into three groups of seven, five, and seven mice, which were infused with three doses of vasostatin-1 (0, 45, 90 ng/kg/min), respectively, for 4 weeks using osmotic mini-pumps (Alzet Model 1002; Durect, Cupertino, CA, U.S.A.). Doses of vasostatin-1 were selected on the basis of previous data [32,33]. Once every 2 weeks, the mini-pumps were implanted subcutaneously into the dorsum under medetomidine-midazolam-butorphanol anesthesia [26].

At the experimental end point (before and 4 weeks after infusion), the ApoE−/− mice were killed by exsanguination (total blood collection) under medetomidine-midazolam-butorphanol anesthesia [26]. The whole aorta was immediately washed by perfusion with PBS and fixed with 4% formaldehyde. The aorta was excised from the aortic sinus to the abdominal area and connective and adipose tissues were carefully removed [24–30].

Parameter measurements in mice

Body weight and food intake were measured through the study. Systolic and diastolic blood pressures were measured using the indirect tail-cuff method (Kent Scientific, Torrington, CT, U.S.A.). At the experimental end point, blood samples were collected after a 4-h fast. Plasma levels of glucose, total cholesterol, triglyceride, and free fatty acid were measured by enzymatic methods (Denka Seiken, Tokyo, Japan) [24–30]. Plasma insulin level was measured by ELISA (Ultra-sensitive mouse insulin ELISA kit; Morinaga, Yokohama, Japan) [26]. The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as fasting plasma insulin (pM) × 0.139 (conversion into μU/ml) × fasting plasma glucose (mg/dl)/405 [26].

Assessment of atherosclerotic lesions in mice

The entire aorta lumen surface and cross-sections of the aortic sinus were stained with Oil Red O for assessment of atherosclerotic lesion area and plaque burden, respectively [24–30]. In aortic sinus wall, vascular inflammation and monocyte/macrophage, VSMC, and collagen-3 contents were visualized by staining with specific antibodies for pentraxin-3 (Bioss, Woburn, MA, U.S.A.), MOMA-2 (Millipore, Billerica, MA, U.S.A.), α-SMA (Sigma), or collagen-3 (GeneTex, Irvine, CA, U.S.A.), respectively [24–30]. Collagen fibers (total collagen) were stained with Masson’s Trichrome [24–26,30]. The positive stained areas were traced by an investigator blind to the treatment and quantitated by image analysis (Adobe Photoshop and NIH ImageJ). The atherosclerotic lesions in the aortic tree were expressed as a percentage of the area of lesions, relative to the surface area of the entire aorta [34]. Plaque burden was expressed as a percentage relative to the entire cross-section of the aortic sinus wall [28].

Human artery immunostaining

Human samples were selected from archive autopsy collections from 2000 to 2010 of National Cardiovascular Center. The written informed consent to medical study use was obtained from families. Serial cross-sections of buffered 10% formalin-fixed paraffin-embedded human radial, internal mammary, and coronary arteries and pancreas specimens were stained with rabbit polyclonal anti-human vasostatin-1 antibody. Immunodetection (as a secondary antibody) was performed with the Bond Polymer Refine Detection kit (Leica Biosystems, Newcastle, U.K.) [30,31]. All processes of immunostaining of human samples were performed in National Cardiovascular Center.

Statistical analysis

All values are expressed as means ± S.E.M. The data were compared by the unpaired Student’s t test between two groups and one-way ANOVA followed by Bonferroni’s post hoc test amongst more than three groups using Statview-J 5.0 (SAS Institute, Cary, NC, U.S.A.). Statistical significance was defined as P<0.05.

Results

Expression of vasostatin-1 in human normal and stenotic arteries

The expression of vasostatin-1 was not observed in human normal radial, internal mammary, and coronary arteries (Figure 1A–C). Vasostatin-1 was abundantly expressed around Monckeberg’s medial calcific sclerosis in human radial arteries (Figure 1D–F), but not intimal and atheromatous lesions in human coronary arteries (Figure 1G,H). The intensity of the expression of vasostatin-1 was equivalent to that of human pancreatic islets (positive control, Figure 1I).

Immunohistological expression of vasostatin-1 in human normal and stenotic arteries

Figure 1
Immunohistological expression of vasostatin-1 in human normal and stenotic arteries

Serial cross-sections of human normal radial (A), internal mammary (B), and coronary arteries (C), Monckeberg’s lesions in radial arteries (DF), atheromatous plaques in coronary arteries (G,H), and pancreatic islet tissue (I) were stained with rabbit polyclonal anti-human vasostatin-1 antibody. Hematoxylin was used for nuclear staining. (E) is a higher magnification image of (D). Vasostatin-1 is expressed around Monckeberg’s medial calcific sclerosis in human radial arteries as well as pancreatic islet cells (positive control). Pictures were provided from the National Cerebral and Cardiovascular Center.

Figure 1
Immunohistological expression of vasostatin-1 in human normal and stenotic arteries

Serial cross-sections of human normal radial (A), internal mammary (B), and coronary arteries (C), Monckeberg’s lesions in radial arteries (DF), atheromatous plaques in coronary arteries (G,H), and pancreatic islet tissue (I) were stained with rabbit polyclonal anti-human vasostatin-1 antibody. Hematoxylin was used for nuclear staining. (E) is a higher magnification image of (D). Vasostatin-1 is expressed around Monckeberg’s medial calcific sclerosis in human radial arteries as well as pancreatic islet cells (positive control). Pictures were provided from the National Cerebral and Cardiovascular Center.

Expression of vasostatin-1 in human vascular cells

CgA was expressed at high levels in HASMCs, HUVECs, and EA.hy926 ECs (positive control), but not THP-1 monocytes and their derived macrophages (Figure 2A). Vasostatin-1 was expressed at infinitesimal levels in HASMCs, HUVECs, and EA.hy926 ECs, but not THP-1 monocytes and macrophages (Figure 2A).

Expression of vasostatin-1 and its effects on EC and macrophage responses

Figure 2
Expression of vasostatin-1 and its effects on EC and macrophage responses

(A) Expression of CgA and vasostatin-1 in human vascular cells, such as THP-1 monocytes, THP-1 monocyte-derived macrophages, HASMCs, HUVECs, and EA.hy926 ECs (positive control) were assessed by immunoblotting. β-actin served as a loading control. Independent experiments were repeated minimum twice to assure reproducibility. (B) Effect of vasostatin-1 on EA.hy926 EC proliferation was determined by WST-8 assay (n=5). (C) Effect of vasostatin-1 (440 nM) on LPS (1 μg/ml)-induced mRNA expression of MCP-1, ICAM-1, VCAM-1, and E-selectin in HUVECs was assessed by reverse transcription-PCR. (D) The graph shows densitometric data of each molecule after normalization relative to GAPDH (n=3). *P<0.005, P<0.05, P<0.0001, §P<0.001 compared with control; #P<0.0001, P<0.001, P<0.005 compared with LPS. (E) Effects of vasostatin-1 on inflammatory phenotypes and signal transductions in THP-1 monocyte-derived macrophages were assessed by immunoblotting. (F) Effects of vasostatin-1 on the secretion of inflammatory cytokines, such as IL-6 and TNF-α, from THP-1 monocyte-derived macrophages were assessed by ELISA (n=4). **P<0.001, ††P<0.05, ‡‡P<0.005.

Figure 2
Expression of vasostatin-1 and its effects on EC and macrophage responses

(A) Expression of CgA and vasostatin-1 in human vascular cells, such as THP-1 monocytes, THP-1 monocyte-derived macrophages, HASMCs, HUVECs, and EA.hy926 ECs (positive control) were assessed by immunoblotting. β-actin served as a loading control. Independent experiments were repeated minimum twice to assure reproducibility. (B) Effect of vasostatin-1 on EA.hy926 EC proliferation was determined by WST-8 assay (n=5). (C) Effect of vasostatin-1 (440 nM) on LPS (1 μg/ml)-induced mRNA expression of MCP-1, ICAM-1, VCAM-1, and E-selectin in HUVECs was assessed by reverse transcription-PCR. (D) The graph shows densitometric data of each molecule after normalization relative to GAPDH (n=3). *P<0.005, P<0.05, P<0.0001, §P<0.001 compared with control; #P<0.0001, P<0.001, P<0.005 compared with LPS. (E) Effects of vasostatin-1 on inflammatory phenotypes and signal transductions in THP-1 monocyte-derived macrophages were assessed by immunoblotting. (F) Effects of vasostatin-1 on the secretion of inflammatory cytokines, such as IL-6 and TNF-α, from THP-1 monocyte-derived macrophages were assessed by ELISA (n=4). **P<0.001, ††P<0.05, ‡‡P<0.005.

Effects of vasostatin-1 on proliferation and inflammatory response in human ECs

Vasostatin-1 had no significant effects on the proliferation of EA.hy926 ECs at any concentrations tested (Figure 2B).

Vasostatin-1 at 440 nM had no significant effects on mRNA expression of MCP-1, ICAM-1, VCAM-1, and E-selectin, but LPS significantly stimulated these expressions in HUVECs (Figure 2C,D). However, vasostatin-1 at 440 nM significantly suppressed LPS-induced mRNA expression of MCP-1, VCAM-1, and E-selectin in HUVECs (Figure 2C,D). Vasostatin-1 tended to suppress LPS-induced ICAM-1 mRNA expression (Figure 2C,D).

Effects of vasostatin-1 on inflammatory phenotype and cytokine secretion in human macrophages

After 1–6 days of culture, the differentiation of THP-1 monocytes to macrophages was confirmed by increased expression of CD68, a macrophage differentiation marker (Figure 2E). Vasostatin-1 did not affect the differentiation from monocytes to macrophages. However, vasostatin-1 at 50 nM decreased the expression of MARCO, an M1 marker, but not MRC1, an M2 marker, through differentiation (Figure 2E). Vasostatin-1 suppressed ERK1/2 and nuclear factor-κB (NF-κB) phosphorylation with peroxisome proliferator-activated receptor-γ (PPAR-γ) up-regulation.

As shown in Figure 2F, secretions of IL-6 and TNF-α from THP-1 monocyte-derived macrophages were significantly increased by LPS (P<0.001, P<0.005). However, vasostatin-1 significantly decreased the LPS-induced IL-6 secretion (P<0.05) and tended to decrease the LPS-induced TNF-α secretion (P=NS).

Effects of vasostatin-1 on human macrophage foam cell formation

Vasostatin-1 significantly suppressed oxLDL-induced foam cell formation by 20% at 25 nM in THP-1 monocyte-derived macrophages (P<0.05; Figure 3A). Vasostatin-1 significantly suppressed protein expression of CD36 and ACAT-1 by 26 and 41% maximum at 250 nM, respectively, and enhanced ABCA1 expression by two-fold at 100 and 250 nM in THP-1 monocyte-derived macrophages (all at least P<0.05; Figure 3B–D).

Effects of vasostatin-1 on macrophage foam cell formation and VSMC responses

Figure 3
Effects of vasostatin-1 on macrophage foam cell formation and VSMC responses

(AD) Effects of vasostatin-1 on oxLDL-induced foam cell formation and related protein expression in THP-1 monocyte-derived macrophages were assessed by cholesterol esterification assay and immunoblotting, respectively. (A) One-fold = 1.16 ± 0.31 nmol/mg cell protein. n=6. (B–D) Top: Representative results of protein expression of each molecule. Bottom: Densitometric data of each molecule after normalization relative to β-actin (n=5–7). *P<0.05, P<0.01, P<0.0001 compared with 0 nM of vasostatin-1. (E) Effect of vasostatin-1 on HASMC migration was determined in 10 cells per well using a BIOREVO BZ-9000 microscope. The experiments were repeated independently three times (n=30). §P<0.0001. (F) Effect of vasostatin-1 on HASMC proliferation was determined by WST-8 assay (n=3). (G) Effect of vasostatin-1 on HASMC apoptosis was evaluated by detecting apoptotic cells (green) using the TUNEL method. Nuclei were co-stained with DAPI (blue). Bar = 100 μm. The graph indicates the percentage of apoptotic cells in four independent experiments.

Figure 3
Effects of vasostatin-1 on macrophage foam cell formation and VSMC responses

(AD) Effects of vasostatin-1 on oxLDL-induced foam cell formation and related protein expression in THP-1 monocyte-derived macrophages were assessed by cholesterol esterification assay and immunoblotting, respectively. (A) One-fold = 1.16 ± 0.31 nmol/mg cell protein. n=6. (B–D) Top: Representative results of protein expression of each molecule. Bottom: Densitometric data of each molecule after normalization relative to β-actin (n=5–7). *P<0.05, P<0.01, P<0.0001 compared with 0 nM of vasostatin-1. (E) Effect of vasostatin-1 on HASMC migration was determined in 10 cells per well using a BIOREVO BZ-9000 microscope. The experiments were repeated independently three times (n=30). §P<0.0001. (F) Effect of vasostatin-1 on HASMC proliferation was determined by WST-8 assay (n=3). (G) Effect of vasostatin-1 on HASMC apoptosis was evaluated by detecting apoptotic cells (green) using the TUNEL method. Nuclei were co-stained with DAPI (blue). Bar = 100 μm. The graph indicates the percentage of apoptotic cells in four independent experiments.

Effects of vasostatin-1 on migration and proliferation of HASMCs

AngII (500 nM) significantly increased the migration of HASMCs (P<0.0001; Figure 3E). Vasostatin-1 (440 nM) significantly suppressed the AngII-induced migration of HASMCs (P<0.0001; Figure 3E). However, vasostatin-1 did not affect significantly the proliferation and apoptosis in HASMCs (Figure 3F,G).

Effects of vasostatin-1 on ECM expression in HASMCs

Vasostatin-1 significantly suppressed protein expression of collagen-3 and fibronectin (both P<0.05; Figure 4B,D), but increased that of MMP-2 and elastin in HASMCs (both P<0.05; Figure 4C,E). However, vasostatin-1 had no significant effects on protein expression of collagen-1 and MMP-9 in HASMCs (both P=NS; Figure 4A,C). However, gelatin zymography revealed significant increases in both MMP-2 and MMP-9 activities by vasostatin-1 (both P<0.05; Figure 4F).

Effects of vasostatin-1 on ECM production in HASMCs

Figure 4
Effects of vasostatin-1 on ECM production in HASMCs

(AE) Effects of vasostatin-1 on ECM expression in HASMCs were assessed by immunoblotting for collagen-1, collagen-3, fibronectin, elastin, MMP-2, MMP-9, and α-tubulin (n=3–6). Top: Representative results of protein expression of each molecule. Bottom: Densitometric data of each molecule after normalization relative to α-tubulin. *P<0.05 compared with 0 nM of vasostatin-1. (F) Effects of vasostatin-1 on MMP-2 and MMP-9 activities in culture supernatants of HASMCs were assessed by gelatin zymography (n=4–5). P<0.05 compared with 0 nM of vasostatin-1.

Figure 4
Effects of vasostatin-1 on ECM production in HASMCs

(AE) Effects of vasostatin-1 on ECM expression in HASMCs were assessed by immunoblotting for collagen-1, collagen-3, fibronectin, elastin, MMP-2, MMP-9, and α-tubulin (n=3–6). Top: Representative results of protein expression of each molecule. Bottom: Densitometric data of each molecule after normalization relative to α-tubulin. *P<0.05 compared with 0 nM of vasostatin-1. (F) Effects of vasostatin-1 on MMP-2 and MMP-9 activities in culture supernatants of HASMCs were assessed by gelatin zymography (n=4–5). P<0.05 compared with 0 nM of vasostatin-1.

Effects of vasostatin-1 on intracellular signaling in HASMCs

Vasostatin-1 significantly suppressed c-Src protein expression and ERK1/2 and p38 phosphorylation, but increased JNK and Akt phosphorylation (Figure 5A–E). Vasostatin-1 did not alter significantly NF-κB phosphorylation (Figure 5F).

Effects of vasostatin-1 on intracellular signal transductions in HASMCs

Figure 5
Effects of vasostatin-1 on intracellular signal transductions in HASMCs

Relevant HASMC signals in response to vasostatin-1 were assessed by immunoblotting. Top: Representative results of protein expression/phosphorylation of c-Src (A), ERK1/2 (B), p38 (C), JNK (D), Akt (E), and NF-κB (F). Bottom: Densitometric data of each molecule after normalization relative to α-tubulin (n=3–5). *P<0.05, P<0.005 compared with 0 nM of vasostatin-1.

Figure 5
Effects of vasostatin-1 on intracellular signal transductions in HASMCs

Relevant HASMC signals in response to vasostatin-1 were assessed by immunoblotting. Top: Representative results of protein expression/phosphorylation of c-Src (A), ERK1/2 (B), p38 (C), JNK (D), Akt (E), and NF-κB (F). Bottom: Densitometric data of each molecule after normalization relative to α-tubulin (n=3–5). *P<0.05, P<0.005 compared with 0 nM of vasostatin-1.

Effects of vasostatin-1 on atherosclerotic lesion development in ApoE−/− mice

Both the entire atherosclerotic lesions in aortic lumen surface and plaque burden in aortic sinus wall significantly increased with age (17–21 weeks old) in ApoE−/− mice (Figure 6A–D). These developments were accompanied with vascular inflammation, macrophage infiltration, VSMC content, and collagen fibers in atheromatous plaques (Figure 6B,E–H). However, high-dose infusion of vasostatin-1 significantly decreased the entire atherosclerotic lesions in aortic lumen surface (Figure 6A,C) and tended to reduce plaque burden in aortic sinus wall (Figure 6B,D). High-dose infusion of vasostatin-1 significantly decreased macrophage infiltration and tended to decrease vascular inflammation, VSMC content, and the ratio of collagen-3/total collagen within atheromatous plaques (Figure 6B,E–H). Low-dose infusion of vasostatin-1 significantly decreased macrophage infiltration and vascular inflammation (Figure 6B,E,G).

Effects of vasostatin-1 on atherosclerotic lesion development in ApoE−/− mice

Figure 6
Effects of vasostatin-1 on atherosclerotic lesion development in ApoE−/− mice

Six mice were killed before infusion (17 weeks old), and seven, five, and seven mice were killed after a 4-week infusion of three doses of vasostatin-1 (0, 45, 90 ng/kg/min), respectively. (A) Atherosclerotic lesions were stained with Oil Red O on the aortic lumen surface. (B) Cross-sections of the aortic sinus wall were stained with Oil Red O, anti-MOMA-2 (monocytes/macrophages), anti-α-SMA (VSMCs), anti-pentraxin-3 (vascular inflammation), anti-collagen-3 antibody, or Masson’s Trichrome (total collagen). Hematoxylin was used for nuclear staining. Bar = 500 μm. (CH) Comparisons of these parameters were performed amongst four groups. Data are expressed as means ± S.E.M. *P<0.0001, P<0.05 compared with before infusion of vasostatin-1 (17 weeks old); P<0.005, §P<0.05 compared with 0 ng/kg/min of vasostatin-1 (21 weeks old).

Figure 6
Effects of vasostatin-1 on atherosclerotic lesion development in ApoE−/− mice

Six mice were killed before infusion (17 weeks old), and seven, five, and seven mice were killed after a 4-week infusion of three doses of vasostatin-1 (0, 45, 90 ng/kg/min), respectively. (A) Atherosclerotic lesions were stained with Oil Red O on the aortic lumen surface. (B) Cross-sections of the aortic sinus wall were stained with Oil Red O, anti-MOMA-2 (monocytes/macrophages), anti-α-SMA (VSMCs), anti-pentraxin-3 (vascular inflammation), anti-collagen-3 antibody, or Masson’s Trichrome (total collagen). Hematoxylin was used for nuclear staining. Bar = 500 μm. (CH) Comparisons of these parameters were performed amongst four groups. Data are expressed as means ± S.E.M. *P<0.0001, P<0.05 compared with before infusion of vasostatin-1 (17 weeks old); P<0.005, §P<0.05 compared with 0 ng/kg/min of vasostatin-1 (21 weeks old).

There were no significant differences in body weight, food intake, systolic and diastolic blood pressures, or plasma levels of total cholesterol and HDL cholesterol amongst four groups of ApoE−/− mice (Table 1). However, low- and high-dose infusions of vasostatin-1 significantly decreased fasting plasma glucose level and tended to decrease plasma insulin level and HOMA-IR (Table 1). Further, high-dose infusion of vasostatin-1 significantly lowered plasma levels of triglyceride and free fatty acid (Table 1).

Table 1
Characteristics and laboratory data of ApoE−/− mice
Parameter 17 weeks old 21 weeks old 
 Before Vasostatin-1 0 ng/kg/min Vasostatin-1 45 ng/kg/min Vasostatin-1 90 ng/kg/min 
n 
Body weight (g) 27.5 ± 0.5 28.9 ± 0.8 29.3 ± 1.2 30.7 ± 0.9 
Food intake (g/day) 4.3 ± 0.3 3.9 ± 0.4 3.9 ± 0.3 4.3 ± 0.4 
Systolic blood pressure (mmHg) 92.1 ± 3.3 92.8 ± 5.2 94.7 ± 2.2 97.2 ± 3.4 
Diastolic blood pressure (mmHg) 66.0 ± 4.9 70.7 ± 5.5 72.9 ± 1.4 73.9 ± 2.9 
Total cholesterol (mg/dl) 1983.9 ± 71.1 (n=5) 2122.4 ± 116.9 1822.8 ± 46.3 2007.8 ± 64.2 
HDL cholesterol (mg/dl) 7.91 ± 0.79 (n=3) 9.56 ± 3.11 12.9 ± 2.13 8.39 ± 0.82 
Triglyceride (mg/dl) 138.1 (n=1) 181.4 ± 36.0 158.8 ± 40.2 109.5 ± 8.0* 
Free fatty acid (mEq/l) 1.39 ± 0.31 (n=3) 1.64 ± 0.51 0.93 ± 0.15 0.58 ± 0.07* 
Glucose (mg/dl) 285.8 ± 30.0 280.8 ± 24.6 229.9 ± 44.7* 218.6 ± 24.4* 
Insulin (pM) 6.98 ± 1.19 (n=4) 34.6 ± 17.2 7.52 ± 3.13 18.0 ± 5.28 
HOMA-IR 0.73 ± 0.10 (n=4) 3.58 ± 1.90 0.74 ± 0.39 1.39 ± 0.40 
Parameter 17 weeks old 21 weeks old 
 Before Vasostatin-1 0 ng/kg/min Vasostatin-1 45 ng/kg/min Vasostatin-1 90 ng/kg/min 
n 
Body weight (g) 27.5 ± 0.5 28.9 ± 0.8 29.3 ± 1.2 30.7 ± 0.9 
Food intake (g/day) 4.3 ± 0.3 3.9 ± 0.4 3.9 ± 0.3 4.3 ± 0.4 
Systolic blood pressure (mmHg) 92.1 ± 3.3 92.8 ± 5.2 94.7 ± 2.2 97.2 ± 3.4 
Diastolic blood pressure (mmHg) 66.0 ± 4.9 70.7 ± 5.5 72.9 ± 1.4 73.9 ± 2.9 
Total cholesterol (mg/dl) 1983.9 ± 71.1 (n=5) 2122.4 ± 116.9 1822.8 ± 46.3 2007.8 ± 64.2 
HDL cholesterol (mg/dl) 7.91 ± 0.79 (n=3) 9.56 ± 3.11 12.9 ± 2.13 8.39 ± 0.82 
Triglyceride (mg/dl) 138.1 (n=1) 181.4 ± 36.0 158.8 ± 40.2 109.5 ± 8.0* 
Free fatty acid (mEq/l) 1.39 ± 0.31 (n=3) 1.64 ± 0.51 0.93 ± 0.15 0.58 ± 0.07* 
Glucose (mg/dl) 285.8 ± 30.0 280.8 ± 24.6 229.9 ± 44.7* 218.6 ± 24.4* 
Insulin (pM) 6.98 ± 1.19 (n=4) 34.6 ± 17.2 7.52 ± 3.13 18.0 ± 5.28 
HOMA-IR 0.73 ± 0.10 (n=4) 3.58 ± 1.90 0.74 ± 0.39 1.39 ± 0.40 

Data are represented as mean ± S.E.M. Since a sufficient volume of blood was not collected from 17-week-old mice, plasma concentrations of total cholesterol, HDL cholesterol, triglyceride, free fatty acid, and insulin were unable to be measured in all samples. Therefore, HOMA-IR could not be calculated in all 17-week-old mice. Abbreviation: HDL, high-density lipoprotein.

*P<0.05 compared with 0 ng/kg/min of vasostatin-1.

Discussion

This is the first demonstration that vasostatin-1 suppresses the inflammatory responses in both ECs and macrophages, macrophage foam cell formation, VSMC migration, and collagen-3 and fibronectin production by VSMCs in vitro, and retards the development of atherosclerotic lesions with reduced intra-plaque inflammation (pentraxin-3) and macrophage, VSMC, and collagen-3 contents in ApoE−/− mice in vivo. We need to discuss the reason why vasostatin-1 infusion into ApoE−/− mice did not dose-dependently suppress pentraxin-3 expression in the aortic wall. High-dose of vasostatin-1 is known to induce the release of noradrenaline from nerve terminals [35]. Since there is a positive correlation between plasma pentraxin-3 and noradrenaline levels [36], it is possible that noradrenalin may induce somewhat pentraxin-3 production in the aorta of ApoE−/− mice. The present study also suggests that increases in elastin expression and MMP-2 and MMP-9 activities with vasostatin-1 in VSMCs may be associated with vascular elasticity and remodeling. Other studies have shown that vasostatin-1 inhibits VEGF-A-induced permeability and TNF-α-induced gap formation in ECs [37,38]. Vasostatin-1 inhibits the infiltration of CD8+ T cells, neutrophils, and macrophages in vessel walls [39]. Vasostatin-1 is produced from CgA by plasmin which is also known to be associated with atherosclerosis and vascular remodeling [40,41], and modulates cell–ECM interaction [42]. These findings indicate that vasostatin-1 plays a key role in inflammation-associated vascular remodeling.

Compared with HUVECs, EA.hy926 ECs were more convenient for our WST-8 assay to determine cell proliferation without inducing apoptosis [43]. In contrast, HUVECs were more adequate to analyze the changes in inflammatory molecules’ expression with vasostatin-1, because HUVECs may involve the abundant cyclooxygenase-2 [44]. In addition, the receptor and signal transduction pathways for vasostatin-1 are yet to be identified. Our study suggests that vasostatin-1 suppresses the migration and collagen-3 and fibronectin expression associated with the down-regulation of c-Src expression as well as ERK1/2 and p38 phosphorylation, and increases elastin expression and MMP-2 and MMP-9 activities associated with the up-regulation of JNK and Akt phosphorylation in VSMCs.

Previous immunoblotting analyses showed that vasostatin-1 was abundantly expressed in human internal mammary arteries but not human coronary arteries and blood mononuclear cells [20]. In the present study, vasostatin-1 is expressed in cultured human VSMCs and around Monckeberg’s medial calcific sclerosis in human radial arteries, but not in cultured human monocytes/macrophages and intimal and atheromatous lesions in human coronary arteries. Our present and previous studies showed that its precursor CgA is expressed in human VSMCs and around Monckeberg’s medial calcific sclerosis in human coronary arteries, but not cultured human monocytes/macrophages and atheromatous plaques in human coronary arteries [25]. Further studies are needed to clarify the association between CgA/vasostatin-1 and vascular calcification, as their relationship has not been investigated at all.

Our preliminary study showed that diminishing of endogenous CgA with 4-week infusion of anti-CgA neutralizing antibody enhanced the development of aortic atherosclerosis in ApoE−/− mice (Supplementary Figure S1). This finding suggests that full length of CgA may suppress atherogenesis. Our recent study has shown that catestatin, a 21-amino acid residue (CgA344-364), suppresses atherogenesis [25]. In addition, the CgA N-terminal domain is also known to exert anti-atherosclerotic effects. There are two subtypes of vasostatins, vasostatin-1 (CgA1-76) and vasostatin-2 (CgA1-113) that are derived from cleavage within close pairs of basic amino acid residues in the CgA N-terminal domain. Several recent studies have shown the anti-atherosclerotic effects of vasostatin-2 [20,45,46]. The present study clarifies that its shorter fragment, vasostatin-1, has the essentially bioactive properties for anti-atherosclerosis. The form of vasostatin-1 in the circulating blood has not been clarified so far. Vasostatin-1 is enzymatically degraded by extracellular proteases, such as dipeptidyl peptidase-4 and trypsin [47,48]. It is possible that vasostatin-1-derived fragments may participate in the anti-atherogenic effects. CgA(29-42) and CgA(36-44) are the antigenic epitopes recognized by diabetogenic BDC2.5 CD4+ T cells and CD8+ T cells in NOD mice [49,50]. The mechanisms that vasostatin-1 lowers plasma glucose levels remain unclear. In contrast, CgA-derived pancreastatin (CgA250-301) inhibits glucose-stimulated insulin secretion [51]. CgA is processed to vasostatin-1 and pancreastatin in pancreatic islet β-cells [9,51]. Vasostatin-1 is the critical peptide for the formation of secretory granules that constitutively store insulin [52]. It is possible that both the peptides may exert opposing counter-regulatory effects on insulin secretion and glucose metabolism.

We discuss the integrity of vasostatin-1 concentrations in our experiments. Several studies have shown that plasma concentrations of vasostatin-1 are ∼0.3 nM in healthy subjects [23,53–55], and on an average 0.36 nM in patients with carotid atherosclerosis [21]. Plasma vasostatin-1 concentrations are increased up to on average 1.83 μM in CAD patients [22]. In our study, the concentrations of vasostatin-1 required for modulation of several responses of human macrophages, HUVECs, and HASMCs were 25–440 nM. These concentrations are within the range of plasma vasostatin-1 levels. In addition, the adequate concentrations of vasostatin-1 differed in inducing foam cell formation and related protein expression in macrophages and ECM expression in HASMCs. The former is mostly dependent on the difference in the presence or absence of oxLDL. The differences in cell type and the concerned signal transductions are also involved in the reason. According to our previous studies [24,27], the adequate concentrations of anti-inflammatory agents in inducing macrophage responses via NF-κB were lower compared with those in HUVEC and HASMC responses via ERK1/2. Vasostatin-1 at greater than adequate concentrations might lead to the down-regulation of its receptors, so that the effects of vasostatin-1 seem to be diminished occasionally.

In conclusion, the present study provided the first evidence that vasostatin-1 exerts anti-atherogenic effects by suppressing inflammatory responses in both ECs and macrophages, foam cell formation in macrophages, the migration of VSMCs, and collagen-3 and fibronectin production by VSMCs. In addition, chronic infusion of vasostatin-1 decreases fasting plasma glucose levels and ameliorates insulin resistance in ApoE−/− mice. Vasostatin-1-based treatments are expected to emerge as a new line of therapies against atherosclerosis and related diseases.

Clinical perspectives

  • Recent studies have shown the increased plasma levels of vasostatin-1 in patients with carotid and coronary atherosclerosis and Takayasu arteritis.

  • Our in vitro and in vivo experiments for the first time demonstrate the preventive effects of vasostatin-1 against atherogenesis.

  • Thus, vasostatin-1 may open up a new therapeutic window for combating atherosclerosis and related diseases.

We thank Dr Fumiko Itoh for her helpful support. Presented in part at the 49th Annual Scientific Meeting of the Japan Atherosclerosis Society, Hiroshima, Japan, 6–7 July 2017.

Author contribution

All authors reviewed the manuscript and agreed to submission. T.W. designed the project. Y.S., N.U., and R.W. planned the experimental design. Y.S., N.U., N.O., Y.T., R.S., K.S., Y.M., and T.H., planned and conducted the experiments. T.M. and H.I.-U. performed clinical sample collection and immunostaining. T.W. wrote the manuscript. Y.S., N.U., R.W., H.I.-U., T.H., and T.W. discussed the data.

Funding

This work was supported in part by grants-in-aid for scientific research (C) from the Japan Society for the Promotion of Science [grant number 17K08993 (to T.W.), 16K08943 (to K.S.)].

Competing interests

The authors declare that there are no competing interests associated with the manuscript.

Abbreviations

     
  • ABCA1

    ATP-binding cassette transporter A1

  •  
  • ACAT-1

    acyl-CoA:cholesterol acyltransferase-1

  •  
  • AngII

    angiotensin II

  •  
  • ApoE−/− mice

    apolipoprotein E-deficient mice

  •  
  • CgA

    chromogranin A

  •  
  • EC

    endothelial cell

  •  
  • ECM

    extracellular matrix

  •  
  • EGF

    epidermal growth factor

  •  
  • EGM-2

    EC growth media-2

  •  
  • FGF

    fibroblast growth factor

  •  
  • HASMC

    human aortic smooth muscle cell

  •  
  • HOMA-IR

    homeostasis model assessment of insulin resistance

  •  
  • HUVEC

    human umbilical vein EC

  •  
  • ICAM-1

    intercellular adhesion molecule-1

  •  
  • IL-6

    interleukin-6

  •  
  • LPS

    lipopolysaccharide

  •  
  • MCM

    monocyte/macrophage-conditioned medium

  •  
  • MCP-1

    monocyte chemotactic protein-1

  •  
  • MMP

    matrix metalloproteinase

  •  
  • NF-κB

    nuclear factor-κB

  •  
  • oxLDL

    oxidized low-density lipoprotein

  •  
  • SmGM-2

    smooth muscle growth medium-2

  •  
  • TNF-α

    tumor necrosis factor-α

  •  
  • VCAM-1

    vascular cell adhesion molecule-1

  •  
  • VSMC

    vascular smooth muscle cell

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Supplementary data