Background
The latest epidemiological data show that the prevalence of diabetes is 11.6%, and pre-diabetes is 50.1%, in people over 18 years old in China [
1]. The macrovascular complications of diabetes mainly cause damage to the cardiovascular system. Diabetes is related to a higher risk of coronary heart disease, especially in patients who have had diabetes for more than 10 years [
2]. Patients with diabetes are also more prone to atherosclerosis and cardiovascular events (angina, myocardial infarction, heart failure) [
3,
4]. However, the pathogenesis of diabetes has not been fully elucidated. β
2-glycoprotein I (β
2GP I) is a phospholipid-binding plasma protein, and an autoantigen. In patients with type 2 diabetes or myocardial infarction, plasma β
2GP I and oxidized low-density lipoprotein (oxLDL)/β
2GP I complex levels are significantly increased; these are predicted adverse consequences of cardiovascular events [
5]. Previous studies have shown that β
2GP I/oxLDL/C reactive protein (CRP) complexes can up-regulate the expression of p38MAPK, increasing the generation of atherosclerosis in diabetic mice [
6]. Reduced β
2GP I, which has free sulfhydryl groups, is also present in plasma and serum; it can protect endothelial cells from damage due to oxidative stress [
7]. Matrix metalloproteinases (MMPs) can degrade all extracellular matrix components, resulting in their increased activity in the aortic plaque. They can then degrade collagen fibers, making the fibrous cap thin and plaques easily broken. Type IV collagen is an important component of the basement membrane in atherosclerotic plaques and fibrous caps. Gelatinases (MMP2, MMP9) responsible for its degradation encourage smooth muscle cells to migrate into the intima membrane and accelerate atherosclerosis, leading to unstable plaque formation [
8]. Oxidative stress is present in diabetes, and elevated levels of reactive oxygen species can lead to elevated MMP2 and MMP9 levels [
9]. Tissue inhibitors of matrix metalloproteinases (TIMPs) are natural inhibitors of MMPs; TIMP-1 can inhibit MMP9, while TIMP-2 can inhibit MMP2. The TIMPs are known suppressors of atherosclerosis [
10,
11]. Previous studies have shown that reduced β
2GPI can inhibit the formation of foam cells by macrophages and apoptosis
in vitro[
12]. The aim of our study was to investigate how reduced β
2GP I by MMPs/TIMPs affect the aorta
in vivo, and to determine any related mechanisms of action involved.
Methods
Animal models and groups
All animal experiments were approved by the Animal Care and Research Committee of Tianjin Medical University. All procedures were performed in accordance with the Guidelines of Animal Experiments from the Committee of Medical Ethics, the National Health Department of China (1998). We obtained 160 female Balb/c mice (8 weeks old) weighing 18–25 g were obtained from the Peking University Experimental Animal Center. We randomly selected 40 mice as the normal control group; these mice were given a standard chow diet for 8 weeks and injected with sodium citrate buffer. The remaining 120 mice were given a high sugar and high fat diet (10% sugar, 10% lard, 5% yolk, 1% cholesterol, and 0.2% bile salt by mg) for 8 weeks. These mice were then intraperitoneally injected with 80 mg/kg of 2% streptozotocin twice. Tail vein blood glucose levels were measured one week later; mice with a blood glucose concentration ≥ 16.7 mM were considered diabetes.
Diabetic mice were randomly divided into six groups (n = 20 mice per group). There were three mono-dose groups that were injected once in the tail vein on day 1: the β2GP I group (20 μg); the reduced β2GP I group (20 μg); and the diabetic control group treated with phosphate-buffered saline (PBS). We used PBS as the vehicle for β2GP I and reduced β2GP I. We also had three complex-dose groups that were injected twice in the tail vein on days 1 and 22: the β2GP I group (20 μg each injection), the reduced β2GP I group (20 μg each injection); and the diabetic control group (PBS). The 40 normal control mice were randomly divided into two groups (n = 20 mice per group), so that there were controls for the mono- and complex-dose groups, and injected with PBS.
Body weight and blood glucose
Body weight was assessed every week. Following injection with streptozotocin, blood glucose levels were monitored weekly.
Specimen collection
Blood from mice in the mono-dose groups were sampled at day 22, and at day 43 for those in the complex-dose groups. Blood was obtained via retro-orbital plexus and mice were sacrificed by cervical dislocation. Aortas were carefully dissected from the iliac bifurcation to the aortic arch and external fatty deposits were removed. Complete aortas were then collected.
Determination of serum lipids
Blood samples were centrifuged (3500 rpm, 5 min, room temperature) and the plasma concentration of triglycerides, total cholesterol, low density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c) were determined by enzymatic colorimetric assays using an Automatic Biochemical Analyzer (Hitachi Co., Japan).
Aortic lipid analysis
Aortas were fixed in 4% (w/v) paraformaldehyde overnight and cut open longitudinally. After rinsing in 70% (v/v) ethanol, specimens were stained with Sudan IV solution for 15 min and decolorized in 80% (v/v) ethanol for 20 min until the normal tissue turned white. Neutral resins were used to block specimens. Images were acquired using a PowerShot S70 camera (Canon, Japan) and analyzed with ImageJ 2.1.4.7 (National Institutes of Health, USA). We then calculated the percentage of plaque coverage.
Histopathology analysis
Paraffin-embedded aortas were serially sectioned (5 μm thickness) and deparaffinized with dimethylbenzene (2 × 15 min), then treated with absolute ethanol (2 × 5 min), washed with distilled water, and stained with hematoxylin and 0.5% eosin. Aortas were washed with distilled water, then placed through a graded series of ethanol [80, 95 and 100% (v/v)], incubated with dimethylbenzene (2 × 2 min), and blocked with neutral resins. Sections were observed using microscopy to compare histopathological alterations in the various groups.
Quantitative polymerase chain reaction assays
The oligonucleotide primer sequences for amplification and quantitation of TIMP-1, TIMP-2, MMP2, MMP9, p38MAPK, and glyceraldehyde-3-phosphate dehydrogenase (GADPH) are presented in Table
1. Total RNA was isolated using Trizol (Sigma-Aldrich, USA). We used a reverse transcription kit to synthesize RNA into cDNA. For quantitative polymerase chain reaction assays, each reaction comprised 5 μL of SYBR® Green II, 0.4 μL of each downstream and upstream, 1 μL of cDNA, and 3.2 μL of diethylpyrocarbonate-treated water. Thermal cycling conditions involved incubation at 50°C for 2 min followed by 94°C for 3 min, then 45 cycles of 94°C for 30 s, 30 s at the appropriate annealing temperature (64.5°C for MMP2 and MMP9; 61.4°C for TIMP-1, TIMP-2, and p38MAPK; and 58°C for GADPH), and 75°C for 45 s. After the 45th cycle, samples were incubated at 72°C for 10 s, then at 65°C for 5 s, and the temperature raised to 95°C to complete the assay. The fold change in mRNA expression levels were assessed using the 2
-ΔΔCt method [ΔΔCt = (Ct1 - Ct2) - (Ct3 - Ct4)]. Ct1 and Ct2 represent the critical cycle numbers for the target gene and GADPH, respectively, in the β
2GP I, reduced β
2GP I and diabetic control groups, respectively. Ct3 and Ct4 represent the critical cycle numbers for the target gene and GADPH, respectively, in the normal control group.
Table 1
Primer sequence of TIMP-1, -2, MMP2, MMP9, p38MAPK and GAPDH for real time PCR
TIMP-1 | Forward | 5′AGACACACCAGAGCAGATACC3′ |
| Reverse | 5′CAGCTACAGGCCTTACTGGAA3′ |
TIMP-2 | Forward | 5′GCTCCAACCCTGTCCTAACC3′ |
| Reverse | 5′GCACAACACGAAAATGCCCT3′ |
MMP2 | Forward | 5′TTTCTATGGCTGCCCCAAGG3′ |
| Reverse | 5′GTCAAGGTCACCTGTCTGGG3′ |
MMP9 | Forward | 5′CGGATCCCCAACCTTTTCCA3′ |
| Reverse | 5′GTGCCTGTCACAAAAGCCAG3′ |
p38 MAPK | Forward | 5′AAGACTCGTTGGAACCCCAG3′ |
| Reverse | 5′GGGTCGTGGTACTGAGCAAA3′ |
GADPH | Forward | 5′CAAGGTCATCCATGACAACTTG3′ |
| Reverse | 5′GTCCACCACCCTGTTGCTGTAG3′ |
Western blotting assays
Aortas were dissolved and the concentration of total protein was determined using BCA reagents (thermo scientific, USA) according to the manufacturer’s instructions. We added 30 μg of sample protein per 20 μL to a sodium dodecyl sulfate polyacrylamide gel electrophoresis buffer. The concentration of polyacrylamide gels used was dependent on the molecular weight of the protein examined. Gel concentrations were 10% for MMP2 (72 kDa, Proteintech, USA), p38MAPK and phosphorylated p38MAPK (38 kDa, Cell Signaling Technology, USA), β-tubulin (55 kDa, Sigma, USA), and 15% for TIMP-1 (28 kDa, Proteintech, USA), TIMP-2 (21 kDa, Santa Cruz, USA). Proteins were electrophoresed (110 V) and transferred to nitrocellulose membranes, then incubated at room temperature with 5% (w/v) skim milk in Tris-buffered saline with Tween 20 (TBST) for 1.5 h. Membranes were incubated overnight at 4°C with antibodies against MMP2, TIMP-1, p38MAPK, phosphorylated p38MAPK (all diluted 1:1000), TIMP-2 (1:100), and β-tubulin (1:5000) diluted in TBST. Membranes were washed with TBST (3 × 15 min) and incubated at room temperature with the appropriate secondary antibody (1:20,000) for 1 h. Membranes were washed with TBST (5 × 10 min) and immunoreactive bands were detected using enhanced chemiluminescence reagents followed by image analysis with ImageJ.
Statistical analysis
We used SPSS19.0 to analyze our data, with values expressed as mean ± standard deviation. Analysis of variance was used for mono- and complex-dose groups. If there was statistical significance then we conducted post-hoc analyses with the diabetic control group as a reference using Dunnett’s test. A P-value less than 0.05 was considered statistically significant.
Discussion
β
2GP I and oxLDL-c can form a complex in the body that acts as an antigen to promote arteriosclerosis. β
2GP I and CD4
+ lymphocytes and monocyte-derived macrophages co-localize to human atherosclerotic sites. This indicates that oxLDL-c/β
2GP I and β
2GP I antibodies are higher in acute coronary syndrome patients as adverse reactions increase [
13]. Our previous study results have shown that oxLDL-c/β
2GP I /CRP complexes promote macrophages that have internalized oxLDL-c to form foam cells. This accelerates the formation of atherosclerosis in diabetic mice [
6]. Results from our previous study also showed that endothelial cells can secrete thioredoxin-1 to change β
2GP I into reduced β
2GP I. This is generated when the functional disulfide (Cys288-Cys326) is opened and free sulfhydryl groups are present in domain V, playing a role in endothelial protection during oxidative stress
in vitro. We also found that β
2GP I and reduced β
2GP I are present in the plasma [
7]. Studies have shown elevated β
2GP I levels in the plasma of type 2 diabetic patients [
14]. However, another study showed that the level of β
2GP I was not different between diabetic and non-diabetic patients matched for age, sex and body mass index. These results indicated that β
2GP I levels rose in diabetic patients with obesity and metabolic syndromes [
15].
When plaques are present in blood vessels of diabetic patients, the greatest hazard is plaque rupture [
16]. A reason for plaque instability is its thin fibrous cap. MMPs are a class of widespread endopeptidases, whose main role is to break down the extracellular matrix. Collagen IV is an important component at the bottom of the plaque base and the fibrous cap. Collagen IV is degraded by MMP2 and MMP9, resulting in vascular smooth muscle cells moving from the intermediate membrane to the intimal membrane, causing fibrous cap thinning and plaque instability. Levels of MMP2, MMP9, TIMP-1 and TIMP-2 are all increased in diabetic patients with dyslipidemia or with acute coronary syndrome [
17‐
19].
Our previous
in vitro studies suggested reduced β
2GP I inhibits oxLDL-induced macrophages from forming foam cells and from inducing apoptosis, however β
2GP I did not have this effect [
12]. This raises the question whether early intervention with β
2GP I and reduced β
2GP I provides vascular protection in high glucose and high fat animals
in vivo, and whether β
2GP I and reduced β
2GP I can affect MMPs/TIMPs in the aorta. These mechanisms remain unknown and further research is required.
In the current study, diabetic mice models were successfully induced. Following treatment with β2GP I and reduced β2GP I for three weeks or six weeks, LDL-c in the reduced β2GP I group was lower than that in the diabetic control group. Our results suggest that long-term application of reduced β2GP I reduces plasma LDL-c levels.
Patients with diabetes are more prone to atherosclerosis and cardiovascular events [
3,
4]. In our study, lipid deposition in diabetic mice was demonstrated in the aorta along with atherosclerotic plaques. Total β
2GP I levels (oxidized and reduced form) in the plasma of stroke patients, and old patients with heart disease, were significantly decreased, and this did not alter after 6 weeks [
20]. Further studies have suggested high levels of total β
2GP I can reduce the risk of myocardial infarction in people older than 60 [
21]. In our study, treatment with β
2GP I resulted in arterial lipid deposition but no plaque formation in blood vessels. However, treatment with reduced β
2GP I showed that arterial lipid deposition was significantly decreased and plaques in blood vessels had not formed. These results suggest that reduced β
2GP I can prevent atherosclerosis in diabetic mice.
High glucose levels caused endothelial cells to express higher levels of MMP1, MMP2 and MMP9, however TIMP-1 levels were unaltered
in vitro[
22]. Plasma levels of MMPs and TIMPs change in diabetic patients, but these changes are inconsistent across different studies [
23‐
28]. Papazafiropoulou et al. reported that plasma concentrations of MMP-2 and MMP-9 were not different between diabetic and non-diabetic patients, while TIMP-1 levels were lower in diabetic patients. No significant associations were found between the expression of MMPs and TIMP-1 and arterial stiffness; duration of diabetes emerged as the strongest predictor of arterial stiffness [
29]. Uemura reported that diabetes increased the activity of MMP9 via oxidative stress, resulting in increased vascular complications. The probability of these vascular complications occurring was reduced by lowering the activity of MMP9 with antioxidants [
30]. In our study, MMP2, MMP9 and TIMP-1 expression in the aortas of diabetic mice were increased. After early intervention with β
2GP I and reduced β
2GP I, expression levels of MMP2, MMP9, TIMP-1, and TIMP-2 were decreased, with reduced β
2GP I having a more pronounced effect. TIMPs are natural inhibitors of MMPs
in vivo and have anti-atherogenic effects. TIMP-1 overexpression in ApoE-deficient atherosclerotic mice can prevent plaque rupture of vein grafts [
31]. The ratio of MMPs to TIMPs have an effect on atherosclerotic processes
in vivo; TIMP-1 mainly inhibits MMP9, while TIMP-2 mainly inhibits MMP2 [
10,
11]. The ratios of MMP2 to TIMP-2 and MMP9 to TIMP-1 are used to represent the total activities of MMPs. In our study, these ratios declined after intervention with reduced β
2GP I and β
2GP I.
To explore further vascular protective mechanisms of reduced β
2GP I, we investigated the p38MAPK signaling pathway. Results from our previous study confirmed that reduced β
2GP I plays a role in p38MAPK signaling [
6]. The promoter upstream of MMPs and TIMPs exists as a cis-acting element and is associated with signaling molecules of the p38MAPK signaling pathway. Activation or inhibition of MMP and TIMP expression is not always consistent [
32,
33]. Our findings showed that reduced β
2GP I down regulated the p38MAPK pathway in aortas.
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Competing interests
All authors declare that they have no competing interests.
Authors’ contributions
JX designed and performed the main experiments (Sudan IV and HE staining, real-time PCR, Western Blot) and drafted the manuscript. PHW participated in the design of this study and the data analysis. TW performed some parts of real-time PCR, SSC and MJW performed some parts of Western Blot. DMY and PY conceived the study, participated in its design and coordination and helped draft the manuscript. All authors read and approved the final manuscript.