Midkine Promotes Atherosclerotic Plaque Formation Through Its Pro-Inflammatory, Angiogenic and Anti-Apoptotic Functions in Apolipoprotein E-Knockout Mice

Abstract

Background: A recent study suggested that midkine (MK), a heparin-binding growth factor, is associated with atherosclerosis progression in patients with artery disease. It has previously been reported that MK plays a critical role in neointima formation in a restenosis model, whereas the role of MK in the development of atherosclerosis has not been investigated. The present study assessed the effect of MK administration on the process of atherosclerotic plaque formation in apolipoprotein E-knockout (ApoE^−/−) mice.

Methods and Results: Using an osmotic pump, human recombinant MK protein was intraperitoneally administered for 12 weeks in C57BL/6 ApoE^−/− (ApoE^−/−-MK) and ApoE^+/+ mice fed a high-fat diet. Saline was administered to the control groups of ApoE^−/− (ApoE^−/−-saline) and ApoE^+/+ mice. The atherosclerotic lesion areas in longitudinal aortic sections were significantly larger in ApoE^−/−-MK mice than in ApoE^−/−-saline mice. The aortic mRNA levels of pro-inflammatory and angiogenic factors, and the percentage of macrophages in aortic root lesions, were significantly higher in ApoE^−/−-MK mice than in ApoE^−/−-saline mice, whereas the percentage of apoptotic cells was significantly lower in ApoE^−/−-MK mice than in ApoE^−/−-saline mice.

Conclusions: The systemic administration of MK in ApoE^−/− mice promoted atherosclerotic plaque formation through pro-inflammatory, angiogenic, and anti-apoptotic effects. MK may serve as a potential therapeutic target for the prevention of atherosclerosis under atherogenic conditions.

Cardiovascular diseases are currently the leading cause of death in the world.¹ The most common cause of cardiovascular diseases is atherosclerosis,¹ a process mediated by chronic inflammation,^2–5 during which the accumulation of inflammatory cells within the arterial wall leads to the local production of chemokines, interleukins, and proteases, contributing to changes in the function of endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). EC dysfunction triggers lipid accumulation and promotes leukocyte invasion into the arterial walls.⁶ The aberrant proliferation of VSMCs also promotes atherosclerotic plaque formation, although VSMCs in advanced plaques are entirely beneficial because they can inhibit the rupture of the fibrous cap.⁷ In addition to ECs and VSMCs, macrophages produce pro-inflammatory cytokines and also participate in lipid retention and vascular cell remodeling during atherosclerosis.⁸ Importantly, reduction of macrophage apoptotic activity promotes the development of atherosclerosis, suggesting that macrophage apoptosis provides a critical self-defense mechanism in suppressing atherosclerosis.⁹

Midkine (MK) is a basic, low molecular-weight protein that belongs to the family of growth factors and cytokines.^10–12 MK expression is not only strongly induced during the process of inflammation and repair, but it also promotes cell survival and cell migration.¹³ MK is closely associated with cancer progression, the onset of inflammatory diseases, and the repair of injured tissues.¹⁴ Recent studies have shown that obesity induces chronic inflammation within adipose tissue,¹⁵ leading to metabolic abnormalities and inflammation in distant tissues.¹⁶ MK expression is increased in the adipose tissue of obese mice, and, more importantly, serum MK levels are significantly increased in overweight/obese human subjects than in controls, suggesting an association between MK and obesity.¹⁷ Moreover, MK is present in the thickened intima of fatty streaks containing VSMCs, ECs, and inflammatory cells, as well as in advanced atherosclerotic regions in human atheromatous plaques.¹⁸ Serum MK levels were significantly higher in patients with severe peripheral artery disease than in healthy controls.¹⁹ Remarkably, a recently published study demonstrated that MK could be used as a coronary atherosclerosis diagnostic tool in humans to predict the presence of anatomically significant coronary artery disease.²⁰ Taken together, these findings suggest that MK mediates atherosclerotic plaque formation under atherogenic conditions.

The other important role of MK relies on its anti-apoptotic activity, as shown in our recent study reporting that MK prevented the progression of cardiac remodeling in a chronic heart failure rabbit model, probably through the activation of anti-apoptotic signaling.²¹ The central role of cardiac myocyte apoptosis in the pathophysiology of cardiac diseases has been experimentally demonstrated for ischemia/reperfusion injury,²² coronary embolization,²³ myocardial infarction,²⁴ ventricular pacing,²⁵ and cardiac aging.²⁶ In addition to its anti-apoptotic effect, we reported that MK plays a critical role in neointima formation in a rat carotid artery mechanical injury model and activates VSMC migration.²⁷ Despite these findings, the role of MK in the development of atherosclerosis has not yet been investigated. Here, we hypothesized that MK promotes atherosclerotic plaque formation via the enhancement of VSMC and macrophage migration, as well as the inhibition of macrophage apoptosis in atheromatous plaques. In the present study, we used apolipoprotein E-knockout (ApoE^−/−) mice to investigate the chronic effect of continuous systemic administration of MK on spontaneously developing atherosclerosis without mechanical injury.

Methods

Mouse Models of Atherosclerosis and Study Protocol

All animal experiments were performed in accordance with the regulations of the National Institute of Health and approved by the Animal Care and Use Committee of Nagoya University. ApoE^−/− mice were supplied by the Department of Cardiology, Nagoya University Graduate School of Medicine, Nagoya, Japan. Eight-week-old male wild-type C57BL/6 (ApoE^+/+) and ApoE^−/− mice with a C57BL/6 genetic background were fed a high-fat diet (24.2% protein, 13.6% fat, 46.5% calories; CLEA JAPAN, Japan) for 12 weeks (Figure 1A). Either saline or human recombinant MK protein in saline (1.0 mg/mL) was continuously infused into ApoE^+/+ and ApoE^−/− mice using an osmotic pump (Alzet; DURECT Corporation, Cupertino, CA, USA), as shown in Figure 1B. The MK protein was purified from yeast by anion exchange chromatography and affinity chromatography on a heparin column, as previously reported.²⁷ Surgery to implant the osmotic pumps was performed under anesthesia with pentobarbital (100 mg/kg, intraperitoneally). The pumps infused a total of 90 μL continuously over a period of 2 weeks. The pumps were implanted under the abdominal skin and were exchanged every 2 weeks for a total of 12 weeks. After the 12-week treatment period, wild-type (ApoE^+/+) mice treated with saline (WT-saline, non-treatment, n=10), wild-type (ApoE^+/+) mice treated with MK (WT-MK, n=10), ApoE^−/− mice treated with saline (ApoE^−/−-saline, n=15), and ApoE^−/− mice treated with MK (ApoE^−/−-MK, n=15) were euthanized and analyzed.

Figure 1.

Scheme showing the administration of saline or midkine (MK) to wild type (ApoE^+/+) or apolipoprotein E-knockout (ApoE^−/−) mice. (A,B) Saline or MK protein in saline (1.0 mg/mL) was continuously infused using an osmotic pump into ApoE^+/+ or ApoE^−/− mice fed a high-fat diet from 8 weeks of age until 20 weeks of age. The pumps were implanted under the abdominal skin and were exchanged every 2 weeks for a total of 12 weeks.

Serum Cholesterol and MK Levels, and Lipoprotein Profile Measurements

The serum cholesterol levels and lipoprotein profile were determined by using kits for clinical use, which were supplied by SRL Inc. (Tokyo, Japan). The serum MK levels were evaluated using an ELISA kit for MK (Wuhan USCN Business Co., Ltd., Hubei, China).²⁸

Lesion Assessment

After anesthesia with pentobarbital, perfusion fixation was performed by infusing 4% paraformaldehyde through the left ventricular apex, and the entire aorta, from the ascending aorta to the abdominal aorta and up to the bifurcation of the common iliac arteries, was removed and fixed in 4% paraformaldehyde for 12–16 h at 4℃. Adventitial fat and extraneous tissue were dissected, and the aorta was split open longitudinally and stained with Sudan IV to visualize the extent of atherosclerosis. The percentage of the aortic surface covered by atheroma was quantified using Image Pro 6.1 (Media Cybernetics, Inc., MD, USA). The stained areas are presented as the percentage of the total plaque area.

Immunohistochemistry

After the mice were euthanized and perfused with ice-cold phosphate-buffered saline (PBS), perfusion fixation was performed prior to histological and immunohistological analyses, by infusing 4% paraformaldehyde through the left ventricular apex. Serial 6-μm-thick paraffin sections of the aortic root were prepared as described previously,²⁹ dewaxed in xylene, rehydrated in alcohol, and washed in PBS. The sections were then incubated in 0.3% hydrogen peroxide in methanol for 30 min to block endogenous peroxidase activity. For immunohistological analysis of macrophages, the sections were incubated for 12–16 h at 4℃ with a monoclonal rat anti-mouse Mac-3 antibody (BD Biosciences, Co, USA; 1:50 dilution). Then, the sections were washed with PBS, incubated with a biotinylated mouse anti-rat IgG1/2a antibody (BD Biosciences, Co, USA) for 15 min at 20–22℃, and finally, incubated with horseradish peroxidase (HRP)-conjugated streptavidin (BD Biosciences, Co, USA) for 10 min at 20–22℃. For the immunohistological analysis of smooth muscle actin, the sections were incubated for 12–16 h at 4℃ with a polyclonal rabbit anti-mouse α-smooth muscle actin antibody (Abcam, MA, USA; 1:200 dilution). Then, the sections were washed with PBS, incubated with a biotinylated goat anti-rabbit IgG (Abcam, MA, USA) for 30 min at room temperature, and finally, incubated with HRP-conjugated streptavidin (BD Biosciences, Co, USA) for 10 min at room temperature. Each incubation was followed by washing in PBS. Finally, the sections were stained using the DAB (3, 3’-diaminobenzidine) substrate kit (Vector Laboratories Inc., Co., USA) and counterstained with hematoxylin. The control sections were incubated with non-immune rat IgG or goat IgG (1:50 dilution). Non-specific binding of rat serum was prevented by pre-incubating the sections with 3.0% bovine serum.

Apoptosis Analysis (TUNEL Staining)

Apoptotic myocardial cells were identified by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) staining. For the TUNEL assay, deparaffinized sections of the aortic root, fixed with 4% paraformaldehyde in PBS, were incubated with proteinase K, and the DNA fragments were labeled with fluorescein-conjugated dUTP by TdT (Roche Diagnostics Corp., IN, USA). The total cell population was estimated by counting the DAPI-stained nuclei (a total of 400–1,000 nuclei were counted per specimen), and the ratio of TUNEL-positive cells to DAPI-stained nuclei was calculated.

Quantitative Real-Time Polymerase Chain Reaction

Total RNA was extracted from the entire aorta and was purified using an RNeasy Fibrous Tissue Mini Kit (QIAGEN Inc., CA, USA). Briefly, RNA was first converted to cDNA using the High Capacity RNA-to-cDNA kit (Applied Biosystems Inc., CA, USA). Then, quantitative polymerase chain reaction (PCR) was performed using an ABI 7700 instrument (Applied Biosystems Inc.) with fluorescent TaqMan probes.¹⁴ The primers and assay ID information for quantitative real-time PCR are listed in Table B. ∆Ct was calculated as: (gene of interest Ct)−(GAPDH RNA Ct), using the Sequence Detector (Applied Biosystems Inc.) and Microsoft Excel (Microsoft Corp., WA, USA) software. The relative quantity of mRNA for each gene of interest was calculated using a ∆∆Ct calculation as: 2^{-([∆Ct of treated sample]-[∆Ct of control sample])}.

Table. (A) Serum Cholesterol Levels, Lipoprotein Profiles, and MK Levels at the Time of Mouse Sacrifice (20 Weeks Old), (B) List of Primers and Assay ID Information for Quantitative Real-Time Polymerase Chain Reaction

A
	WT-saline	WT-MK	ApoE−/−-saline	ApoE−/−-MK
TC (mg/dL)	137.0±4.8	136.6±11.2	984.0±53.7**,#	1043.6±87.4**,#
TG (mg/dL)	21.5±1.5	21.2±2.0	100.3±17.2**,#	93.6±13.4**,#
LDL (mg/dL)	10.5±1.0	10.7±1.0	300.3±22.2**,#	335.5±18.1**,#
MK (ng/mL)	0.274±0.044	0.515±0.044*	0.391±0.049	0.774±0.12**,†
B
Abbreviation	mRNA	Assay ID
IL1A	Interleukin-1α	Mm00439620_m1
IL1B	Interleukin-1β	Mm01336189_m1
CCL-2	Chemokine ligand 2	Mm00441242_m1
IFNG	Interferon-γ	Mm00801778_m1
bFGF	Basic fibroblast growth factor	Mm00433587_m1
HGF	Hepatocyte growth factor	Mm01135185_m1
Bax	BCL2-associated X protein	Mm00432050_m1
Bcl-2	B-cell lymphoma 2	Mm00477631_m1
GAPDH	Glyceraldehyde 3-phosphate dehydrogenase	Mm9999915_m1

*P<0.05 and **P<0.01 vs. WT-saline. ^#P<0.01 vs. WT-MK. ^†P<0.01 vs. ApoE^−/−-saline. n=15 for ApoE^−/−-saline and ApoE^−/−-MK; n=10 for WT-saline and WT-MK. WT, wild-type; ApoE^−/−, apolipoprotein E-knockout; TC, total cholesterol; TG, triglyceride; LDL, low-density lipoprotein; MD, midkine.

Statistical Analyses

All the values are presented as mean±standard error of the mean (S.E.M.). Multi-group comparisons were analyzed by analysis of variance (ANOVA) followed by the Bonferroni post-hoc test. Direct 2-group comparisons were performed using the 2-sided Student’s t-test. Differences with a P<0.05 were considered significant.

Results

MK Increases Atherosclerosis

Serum MK levels increased significantly after MK administration in both ApoE^+/+ and ApoE^−/− mice (Table A). MK increased the area of atherosclerotic lesions in ApoE^−/− mice in en face preparations of the aortic root, as determined by assessing lipid deposition (Figure 2A). Compared with ApoE^−/−-saline mice, ApoE^−/−-MK mice had a significantly larger percent area of atherosclerotic lesions (26.3±0.6% vs. 6.8±1.8%, Figure 2B). In ApoE^+/+ mice, no significant difference in the percent area of atherosclerotic lesions was observed between WT-saline and WT-MK mice. Specifically, aortas from the four different mouse groups were divided into 4 sections (arch, upper, middle, and lower aorta), and each section was analyzed separately (Figure 2C). In ApoE^−/− mice, MK substantially increased the atherosclerotic lesions at the lower aortic sections, including the common iliac artery. There was no significant difference in atherosclerotic areas between WT-saline, WT-MK, and ApoE^−/−-saline mice, indicating that under atherogenic conditions, MK specifically accelerates the progression of atherosclerosis in the iliac bifurcation. Although atherosclerotic lesions at the aortic arch were significantly increased in ApoE^−/−-saline mice than in WT-saline and WT-MK mice, MK significantly increased the atherosclerotic lesions in ApoE^−/− mice. The cholesterol levels in ApoE^−/− mice were significantly higher than those in ApoE^+/+ mice, as previously reported,^30,31 whereas MK administration did not induce lipid profile changes in either ApoE^+/+ or ApoE^−/− mice (Table A).

Figure 2.

Effects of MK treatment for 12 weeks on atherosclerotic lesions. (A) Representative macroscopic findings of the entire aorta from wild-type mice treated with saline (WT-saline), wild-type mice treated with MK (WT-MK), ApoE^−/−-saline, and ApoE^−/−-MK mice at 20 weeks of age. The aortas were stained with Sudan IV. (B) Comparison of the percentages of atherosclerotic lesion areas to the entire aorta areas between the 4 groups (n=5 for each group). (C) Comparison of the percentages of atherosclerotic lesion areas in the 4 divided sections of the aorta between the 4 groups (n=5 for each group). *P<0.01 vs. WT-saline, ^†P<0.01 vs. WT-MK, ^‡P<0.01 vs. ApoE^−/−-saline mice. Abbreviations as in Figure 1.

MK Increases the mRNA Levels of Pro-Inflammatory and Angiogenic Factors

The mRNA levels of different factors were examined in extracts from the entire aorta. In ApoE^−/− mice, MK administration was associated with significantly higher levels of pro-inflammatory factors (interleukin 1 α [IL1A], interleukin 1 β [IL1B], C-C motif chemokine ligand 2 [CCL2], and interferon γ [IFNG]) and angiogenic factors (basic fibroblast growth factor [bFGF], and hepatocyte growth factor [HGF]), as shown in Figure 3. Such MK treatment-induced pro-inflammatory and angiogenic effects were not observed in wild-type mice.

Figure 3.

MK increases the mRNA levels of pro-inflammatory and angiogenic factors. The normalized gene expression of interleukin 1 α (IL1A), interleukin 1 β (IL1B), chemokine ligand 2 (CCL-2), interferon γ (IFNG), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF) in entire aorta extracts is shown for the 4 groups (n=5 for each group) at 20 weeks of age using quantitative real-time polymerase chain reaction (PCR) analysis. *P<0.01 vs. WT-saline, ^†P<0.01 vs. WT-MK, ^‡P<0.01 vs. ApoE^−/−-saline mice. Abbreviations as in Figure 1.

Macrophages and VSMCs Migrate Into Atherosclerotic Lesions

Representative images of macrophages and macrophage-foam cells detected using the Mac-3 antibody showed significantly larger Mac-3-positive areas within the lesions of aortic roots from ApoE^−/−-MK mice, compared with the corresponding areas in ApoE^−/−-saline mice (Figure 4A). Statistical analysis revealed that these differences were statistically significant (Figure 4B), suggesting that the increase in the atherosclerotic areas as a result of MK administration is most likely caused by an increase in the number of macrophages localized in these lesions. In addition to the effect of MK on macrophages, we also investigated the effect of MK on VSMCs in atherosclerotic lesions. VSMCs in atherosclerotic areas within the lesions in aortic roots were detected using an α-smooth muscle actin antibody. α-smooth-muscle actin-positive lesions tended to be more abundant in ApoE^−/−-MK mice than in ApoE^−/−-saline mice, but the difference was not statistically significant (12.7±1.2% vs. 10.4±1.3%, P=0.19).

Figure 4.

MK enhances macrophage migration into the atherosclerotic lesions. (A) Paraffin-embedded sections of aortic roots from 20-week-old ApoE^−/−-saline and ApoE^−/−-MK mice were stained with antibodies against Mac-3 (brown) and counterstained with hematoxylin (blue). Representative staining patterns for ApoE^−/−-saline and ApoE^−/−-MK mice are shown. (B) Percentage of Mac-3-positive areas relative to the total area of the aortic root (ApoE^−/−-saline and ApoE^−/−-MK mice, n=5 each). **P<0.01 vs. ApoE^−/−-saline mice. Abbreviations as in Figure 1.

MK Preferentially Inhibits Macrophage Apoptosis In Vivo

Apoptotic cells were detected by TUNEL staining (Figure 5A). The ratio of the TUNEL-positive area relative to the DAPI-positive area was significantly smaller in ApoE^−/−-MK lesions than in ApoE^−/−-saline lesions (0.9±0.2% in ApoE^−/−-MK mice vs. 2.6±0.8% in ApoE^−/−-saline mice; P<0.01). The majority of the TUNEL-positive areas were located in the central atheroma, rather than in the smooth muscle cell-rich lesion cap, implying that MK administration preferentially inhibited macrophage apoptosis. The mRNA levels of Bcl-2 and Bax, members of the Bcl-2 family of cell death regulatory proteins, extracted from the entire aorta were evaluated by using real-time PCR. In ApoE^−/− mice, MK treatment increased the mRNA levels of Bcl-2, whereas it decreased the mRNA levels of Bax, resulting in a decrease in the Bax/Bcl-2 ratio (Figure 6). In ApoE^+/+ mice, MK tended to decrease the mRNA levels of Bax, leading to a decrease in the Bax/Bcl-2 ratio, but there was no significant effect of MK on the Bax/Bcl-2 ratio in wild-type mice.

Figure 5.

MK decreases the number of apoptotic cells in atherosclerotic lesions in ApoE^−/− mice. (A) Apoptosis was quantified in ApoE^−/−-saline and ApoE^−/−-MK mice using a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling staining (TUNEL) assay. Co-staining with DAPI (blue) was performed to identify nuclei. The upper panel shows representative DAPI-stained nuclei (blue), the middle panel shows TUNEL-positive cells (green), and the bottom panel shows a merged image. (B) The TUNEL-positive nuclei/total nuclei percentage was used to quantify the apoptotic cells. The data presented are the means of 3 independent areas in the aortic valve region from 5 different ApoE^−/−-saline and ApoE^−/−-MK mice. *P<0.05 vs. ApoE^−/−-saline mice (n=5 each). Abbreviations as in Figure 1.

Figure 6.

MK decreases the apoptosis-related Bax/Bcl-2 ratio. Normalized gene expression of Bax and Bcl-2 and the Bax/Bcl-2 ratio were determined by quantitative real-time PCR analysis in entire aorta extracts from 20-week-old mice from the 4 groups (n=5 for each group). Values are the means±S.E.M. *P<0.05. Abbreviations as in Figures 1–3.

Discussion

In the present study, we assessed the effects of long-term systemic administration of MK on atherosclerosis in ApoE^−/− and wild-type mice. Whether MK enhances or inhibits the progression of atherosclerosis in atherosclerotic models without mechanical injury has been uncertain because MK has contradictory effects: it enhances VSMC migration and proliferation in addition to inhibiting macrophage apoptosis (promoting atherosclerosis), whereas it repairs EC dysfunction (inhibiting atherosclerosis). Importantly, its recovery effects on ECs are critical for the early stage of atherosclerosis, because EC deterioration in spontaneously developed lesions can greatly influence the subsequent progression of atherosclerosis. This is the first study, to our knowledge, that investigated the effects of continuous MK administration in ApoE^−/− and wild-type mice. We found that under atherogenic conditions, MK administration enhanced early-stage atherosclerosis in an ApoE^−/− mouse model without mechanical injury, by increasing pro-inflammatory and angiogenic factors. MK also enhanced the migration of macrophages into atherosclerotic lesions and inhibited apoptosis in those lesions, leading to plaque formation.

Pro-Inflammatory Factors (IL1A, IL1B, IFNG) and Angiogenic Factors (bFGF, HGF)

Experimental data from mouse atherosclerosis models have shown that IL1A and IL1B have pro-atherogenic properties associated with the upregulation of endothelial adhesion molecules and the activation of macrophages and vascular cells.^32–34 In the present study, the mRNA levels of IL1A and IL1B in entire aorta extracts were significantly increased upon MK administration in ApoE^−/− mice, indicating that MK directly or indirectly affected the activities of the various cytokines that participate in inflammation and regulate inflammatory cells, leading to the release of IL1A and IL1B. Concerning IFNG, atherosclerosis is reportedly inhibited by the deletion of IFNG or the IFNG receptor,³⁵ and exogenous IFNG enhances atherosclerosis in ApoE^−/− mice.³⁶ Moreover, postnatal inhibition of IFNG prevents the progression of existing atherosclerotic plaques and stabilizes lipid and macrophage-rich advanced plaques,³⁷ suggesting that IFNG has athero-progressive effects. This result indicates that MK may have paracrine and/or direct effects on immunoreactive cells resulting in the production of IFNG.

Local administration of bFGF in the adventitia induces the development of vasa vasorum and accelerates plaque progression in ApoE^−/− mice, supporting the notion that vasa vasorum formation plays a crucial role in the pathogenesis of atherosclerosis.³⁸ HGF induces EC migration and proliferation, and angiogenesis.³⁹ In the present study, MK significantly increased the mRNA levels of bFGF and HGF in inflammatory cells, supporting the hypothesis that MK enhances atherosclerosis in ApoE^−/− mice. Interestingly, the MK-induced significant increase in the mRNA levels of pro-inflammatory (IL1A, IL1B and IFNG) and angiogenic (bFGF and HGF) factors was observed only in ApoE^−/− mice and not in wild-type mice. Thus, the role of MK under atherogenic conditions might be different from that under normal conditions, and further investigations are required to show the underlying mechanism.

Macrophage and VSMC Migration

Macrophage migration into atherosclerotic plaques is critical for the pathogenesis of atherosclerosis. In vitro studies using a Boyden chamber assay have demonstrated that MK enhances macrophage migration.²⁷ In the present in vivo study, immunohistochemistry analysis showed that MK enhanced macrophage migration in the atherosclerotic lesions (Figure 4). In the in vitro study by Horiba et al,²⁷ MK had a direct effect on macrophage migration. However, in in vivo studies, many factors can influence macrophage migration into atherosclerotic lesions, and cross-talk between macrophages and other cell types can also occur. For example, ECs release the chemokines monocyte chemotactic protein-1 (MCP-1) and macrophage colony-stimulating factor (M-CSF), which recruit monocytes into atherosclerotic lesions and differentiate monocytes into macrophages.^40,41 Taken together, the results of these previous reports and our present study results that found that the mRNA level of CCL2 (MCP-1) in entire aorta extracts was elevated, and this suggests that MK might have a direct effect on ECs, causing the release of CCL2.

VSMC migration is also important for the formation of atherosclerotic lesions. According to a well-known theory, VSMCs migrate into the sub-endothelial space in response to injury or inflammatory stimuli, and produce extracellular matrix, forming the tunica intima of the artery. Following their migration to the intima, VSMCs gradually resume a quiescent phenotype.^42,43 The effect of MK on the enhancement of VSMC migration in vitro has been published.²⁷ Taken together, these results suggest that in ApoE^−/− mice, MK activates macrophage and VSMC migration, leading to the progression of atherosclerosis (Figure 7).

Figure 7.

Schematic summarization illustrating the possible effects of midkine (MK) on the process of atherosclerotic plaque formation. The solid lines illustrate the known pathways that give rise to lesion cells. The most important points of this study are: (1) MK promotes macrophage migration into atherosclerotic lesions;²⁷ (2) MK enhances vascular smooth muscle cell (VSMC) migration into the sub-endothelial space and produces extracellular matrix, forming the tunica intima of the artery;^42,43 and (3) MK inhibits macrophage and foam cell apoptosis.

Anti-Apoptotic Effect of MK

Apoptotic cell death occurs during the development and progression of atherosclerotic plaques. Among the various cell types that undergo apoptosis in atherosclerotic lesions, macrophages are the predominant (more than 40%) type.⁴⁴ VSMC apoptosis can destabilize the fibrous cap and induce rupture.⁴⁵ In such lesions, adequate efferocytosis is sufficient to protect against atherosclerosis.⁴⁶ Apoptotic cells and the phagocytes that engulf them produce anti-inflammatory cytokines, such as transforming growth factor-β and interleukin-10, which inhibit the early stages of atherosclerosis development.⁴⁶ In the present study, we found that the number of TUNEL-positive cells within the plaques was significantly lower in the MK-treatment group, and the apoptotic cells were estimated to be macrophages. In terms of mRNA levels, the ratio of the pro-apoptotic factor, Bax, to the anti-apoptotic factor, Bcl-2, was significantly lower in ApoE^−/−-MK mice than in ApoE^−/−-saline mice (Figure 6), suggesting that MK has an anti-apoptotic effect on atherosclerotic lesions under atherogenic conditions. These anti-apoptotic effects of MK are in agreement with recently reported findings in a congestive heart failure mouse model.^21,47

Regarding clinical manifestations of atherogenesis, serum MK levels are significantly increased in obese human subjects,¹⁷ and MK is present in the thickened intima of fatty streaks and advanced atherosclerotic regions in human atheromatous plaques.¹⁸ Moreover, serum MK levels were significantly higher in patients with severe peripheral artery disease than in healthy controls.¹⁹ A recent impressive clinical study demonstrated the use of MK as a diagnostic tool for coronary atherosclerosis in humans,²⁰ yet basic research investigating the direct effect of MK on atherosclerosis is missing. To the best of our knowledge, this is the first study demonstrating that long-term MK administration promotes atherosclerotic plaque formation in ApoE^−/− mice. Our data suggest that higher serum MK levels may play a role in atherogenesis, especially under atherogenic conditions, such as hyperlipidemia and/or obesity. Taken together, our results suggest that maintaining or reducing serum MK levels under atherogenic conditions would inhibit atherosclerotic plaque formation; therefore, MK inhibition could be used in upstream therapies targeting atherosclerosis. Further studies are needed to validate this hypothesis.

Future Aspects

The present study showed that MK had an athero-progressive effect in ApoE^−/− mice, indicating that MK might be a molecular target for the treatment of atherosclerosis. Further investigations are required to confirm the indications for the use of MK inhibitors (antisense oligoDNA, siRNA, and RNA aptamer) to prevent atherosclerosis progression.

Study Limitations

First, the mouse model used in the present study can be regarded as representative of an intermediate and not an early stage of atherosclerosis (the mice were fed a high-fat diet for 12 weeks). Second, and related to apoptosis, the TUNEL staining used in the present study is not specific for apoptosis. We did not directly show the co-existence of macrophages and apoptotic cells in the aortic lesions, and we did not identify the types of apoptotic cells. Third, we did not investigate the effect of MK on EC dysfunction in the present study. Moreover, we did not confirm the direct effect of MK by a genetic modification approach, such as double knockout of MK and ApoE. We did not investigate MK expression in atherosclerotic lesions in ApoE^−/− mice. Moreover, we did not investigate the time-course of blood MK levels in ApoE^−/− mice.

Conclusions

In conclusion, we found that the systemic administration of MK in apolipoprotein-E knockout mice increased atherosclerosis through its pro-inflammatory, angiogenic, and anti-apoptotic functions, suggesting that MK may be a therapeutic target for the prevention of atherosclerosis under atherogenic conditions.

Conflicts of Interest

The authors declare that they do not have anything to disclose regarding funding or conflicts of interest with respect to this manuscript.

Author Contributions

Y.T. designed the animal study, performed the animal surgeries, completed the experiments, analyzed the data, performed the statistical analyses, and wrote the manuscript. M. Horiba designed and supervised the project, interpreted the results, and wrote the manuscript. M. Harada designed and supervised the project. K.S. performed experiments and analyzed the data. K.T. and T.M. provided suggestions on the project and revised the manuscript. K. Kadomatsu and K. Kamiya supervised the project.

Acknowledgments

We would like to thank Mayumi Hojo, Mariko Ito, Minako Tatsumi, and Atsushi Horiba for their technical assistance.

Financial Support

This study was supported, in part, by grants from the Ministry of Education, Culture, Sports, Science, and Technology Japan (M. Horiba) and the Suzuken Memorial Foundation Grant-in-aid (M. Horiba and K. Kamiya).

References

• 1.   GBD 2013 Mortality and Causes of Death Collaborators. Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990–2013: A systematic analysis for the Global Burden of Disease Study 2013. Lancet 2015; 385: 117–171.
• 2.   Glass CK, Witztum JL. Atherosclerosis: The road ahead. Cell 2001; 104: 503–516.
• 3.   Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med 2005; 352: 1685–1695.
• 4.   Libby P. Inflammation in atherosclerosis. Nature 2002; 420: 868–874.
• 5.   Lusis AJ. Atherosclerosis. Nature 2000; 407: 233–241.
• 6.   Gimbrone MA Jr, Garcia-Cardena G. Endothelial cell dysfunction and the pathobiology of atherosclerosis. Circ Res 2016; 118: 620–636.
• 7.   Bennett MR, Sinha S, Owens GK. Vascular smooth muscle cells in atherosclerosis. Circ Res 2016; 118: 692–702.
• 8.   Galkina E, Ley K. Immune and inflammatory mechanisms of atherosclerosis (*). Annu Rev Immunol 2009; 27: 165–197.
• 9.   Liu J, Thewke DP, Su YR, Linton MF, Fazio S, Sinensky MS. Reduced macrophage apoptosis is associated with accelerated atherosclerosis in low-density lipoprotein receptor-null mice. Arterioscler Thromb Vasc Biol 2005; 25: 174–179.
• 10.   Muramatsu T. Midkine and pleiotrophin: Two related proteins involved in development, survival, inflammation and tumorigenesis. J Biochem 2002; 132: 359–371.
• 11.   Muramatsu T. Midkine (MK), the product of a retinoic acid responsive gene, and pleiotrophin constitute a new protein family regulating growth and differentiation. Int J Dev Biol 1993; 37: 183–188.
• 12.   Kadomatsu K, Muramatsu T. Midkine and pleiotrophin in neural development and cancer. Cancer Lett 2004; 204: 127–143.
• 13.   Muramatsu T. Midkine, a heparin-binding cytokine with multiple roles in development, repair and diseases. Proc Jpn Acad Ser B Phys Biol Sci 2010; 86: 410–425.
• 14.   Wang J, Takeuchi H, Sonobe Y, Jin S, Mizuno T, Miyakawa S, et al. Inhibition of midkine alleviates experimental autoimmune encephalomyelitis through the expansion of regulatory T cell population. Proc Natl Acad Sci U S A 2008; 105: 3915–3920.
• 15.   Wellen KE, Hotamisligil GS. Obesity-induced inflammatory changes in adipose tissue. J Clin Invest 2003; 112: 1785–1788.
• 16.   Johnson AR, Milner JJ, Makowski L. The inflammation highway: Metabolism accelerates inflammatory traffic in obesity. Immunol Rev 2012; 249: 218–238.
• 17.   Fan N, Sun H, Wang Y, Zhang L, Xia Z, Peng L, et al. Midkine, a potential link between obesity and insulin resistance. PLoS One 2014; 9: e88299.
• 18.   Salaru D, Arsenescu-Georgescu C, Halloul Z, Damerau M, Albert C, Mertens PR. Expression of midkine, a multifunctional cytokine, in human atherosclerotic plaques. Eur J Intern Med 2013; 24: e38.
• 19.   Salaru DL, Albert C, Konigsmark U, Brandt S, Halloul Z, Heller A, et al. Serum levels for midkine, a heparin-binding growth factor, inversely correlate with angiotensin and endothelin receptor autoantibody titers in patients with macroangiopathy. Int Angiol 2014; 33: 372–378.
• 20.   Ibrahim NE, Januzzi JL Jr, Magaret CA, Gaggin HK, Rhyne RF, Gandhi PU, et al. A clinical and biomarker scoring system to predict the presence of obstructive coronary artery disease. J Am Coll Cardiol 2017; 69: 1147–1156.
• 21.   Harada M, Hojo M, Kamiya K, Kadomatsu K, Murohara T, Kodama I, et al. Exogenous midkine administration prevents cardiac remodeling in pacing-induced congestive heart failure of rabbits. Heart Vessels 2016; 31: 96–104.
• 22.   Gottlieb RA, Burleson KO, Kloner RA, Babior BM, Engler RL. Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest 1994; 94: 1621–1628.
• 23.   Sharov VG, Sabbah HN, Shimoyama H, Goussev AV, Lesch M, Goldstein S. Evidence of cardiocyte apoptosis in myocardium of dogs with chronic heart failure. Am J Pathol 1996; 148: 141–149.
• 24.   Kajstura J, Cheng W, Reiss K, Clark WA, Sonnenblick EH, Krajewski S, et al. Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats. Lab Invest 1996; 74: 86–107.
• 25.   Liu Y, Cigola E, Cheng W, Kajstura J, Olivetti G, Hintze TH, et al. Myocyte nuclear mitotic division and programmed myocyte cell death characterize the cardiac myopathy induced by rapid ventricular pacing in dogs. Lab Invest 1995; 73: 771–787.
• 26.   Kajstura J, Cheng W, Sarangarajan R, Li P, Li B, Nitahara JA, et al. Necrotic and apoptotic myocyte cell death in the aging heart of Fischer 344 rats. Am J Physiol 1996; 271: H1215–H1228.
• 27.   Horiba M, Kadomatsu K, Nakamura E, Muramatsu H, Ikematsu S, Sakuma S, et al. Neointima formation in a restenosis model is suppressed in midkine-deficient mice. J Clin Invest 2000; 105: 489–495.
• 28.   Honda Y, Shishido T, Takahashi T, Watanabe T, Netsu S, Kinoshita D, et al. Midkine deteriorates cardiac remodeling via epidermal growth factor receptor signaling in chronic kidney disease. Hypertension 2016; 67: 857–865.
• 29.   Ni W, Egashira K, Kitamoto S, Kataoka C, Koyanagi M, Inoue S, et al. New anti-monocyte chemoattractant protein-1 gene therapy attenuates atherosclerosis in apolipoprotein E-knockout mice. Circulation 2001; 103: 2096–2101.
• 30.   Zhang SH, Reddick RL, Piedrahita JA, Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science 1992; 258: 468–471.
• 31.   Plump AS, Smith JD, Hayek T, Aalto-Setala K, Walsh A, Verstuyft JG, et al. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells. Cell 1992; 71: 343–353.
• 32.   Kirii H, Niwa T, Yamada Y, Wada H, Saito K, Iwakura Y, et al. Lack of interleukin-1beta decreases the severity of atherosclerosis in ApoE-deficient mice. Arterioscler Thromb Vasc Biol 2003; 23: 656–660.
• 33.   Kamari Y, Werman-Venkert R, Shaish A, Werman A, Harari A, Gonen A, et al. Differential role and tissue specificity of interleukin-1alpha gene expression in atherogenesis and lipid metabolism. Atherosclerosis 2007; 195: 31–38.
• 34.   Clarke MC, Talib S, Figg NL, Bennett MR. Vascular smooth muscle cell apoptosis induces interleukin-1-directed inflammation: Effects of hyperlipidemia-mediated inhibition of phagocytosis. Circ Res 2010; 106: 363–372.
• 35.   Gupta S, Pablo AM, Jiang X, Wang N, Tall AR, Schindler C. IFN-gamma potentiates atherosclerosis in ApoE knock-out mice. J Clin Invest 1997; 99: 2752–2761.
• 36.   Whitman SC, Ravisankar P, Elam H, Daugherty A. Exogenous interferon-gamma enhances atherosclerosis in apolipoprotein E−/− mice. Am J Pathol 2000; 157: 1819–1824.
• 37.   Koga M, Kai H, Yasukawa H, Yamamoto T, Kawai Y, Kato S, et al. Inhibition of progression and stabilization of plaques by postnatal interferon-gamma function blocking in ApoE-knockout mice. Circ Res 2007; 101: 348–356.
• 38.   Tanaka K, Nagata D, Hirata Y, Tabata Y, Nagai R, Sata M. Augmented angiogenesis in adventitia promotes growth of atherosclerotic plaque in apolipoprotein E-deficient mice. Atherosclerosis 2011; 215: 366–373.
• 39.   Ma H, Calderon TM, Fallon JT, Berman JW. Hepatocyte growth factor is a survival factor for endothelial cells and is expressed in human atherosclerotic plaques. Atherosclerosis 2002; 164: 79–87.
• 40.   Cushing SD, Berliner JA, Valente AJ, Territo MC, Navab M, Parhami F, et al. Minimally modified low density lipoprotein induces monocyte chemotactic protein 1 in human endothelial cells and smooth muscle cells. Proc Natl Acad Sci U S A 1990; 87: 5134–5138.
• 41.   Liao F, Berliner JA, Mehrabian M, Navab M, Demer LL, Lusis AJ, et al. Minimally modified low density lipoprotein is biologically active in vivo in mice. J Clin Invest 1991; 87: 2253–2257.
• 42.   Aikawa M, Sakomura Y, Ueda M, Kimura K, Manabe I, Ishiwata S, et al. Redifferentiation of smooth muscle cells after coronary angioplasty determined via myosin heavy chain expression. Circulation 1997; 96: 82–90.
• 43.   Aikawa M, Rabkin E, Voglic SJ, Shing H, Nagai R, Schoen FJ, et al. Lipid lowering promotes accumulation of mature smooth muscle cells expressing smooth muscle myosin heavy chain isoforms in rabbit atheroma. Circ Res 1998; 83: 1015–1026.
• 44.   Kolodgie FD, Narula J, Burke AP, Haider N, Farb A, Hui-Liang Y, et al. Localization of apoptotic macrophages at the site of plaque rupture in sudden coronary death. Am J Pathol 2000; 157: 1259–1268.
• 45.   Clarke MC, Figg N, Maguire JJ, Davenport AP, Goddard M, Littlewood TD, et al. Apoptosis of vascular smooth muscle cells induces features of plaque vulnerability in atherosclerosis. Nat Med 2006; 12: 1075–1080.
• 46.   Van Vre EA, Ait-Oufella H, Tedgui A, Mallat Z. Apoptotic cell death and efferocytosis in atherosclerosis. Arterioscler Thromb Vasc Biol 2012; 32: 887–893.
• 47.   Horiba M, Kadomatsu K, Yasui K, Lee JK, Takenaka H, Sumida A, et al. Midkine plays a protective role against cardiac ischemia/reperfusion injury through a reduction of apoptotic reaction. Circulation 2006; 114: 1713–1720.