Diabetes mellitus (DM) is a metabolic disease with a serious impact on the quality of life and longevity of patients among global population.1 Atherosclerosis (AS) is the most common and serious complication in type 2 DM (T2DM).2,3 Coronary heart disease and stroke based on AS are the major reasons of high mortality in patients with T2DM, while the treatment is still less effective.3,4 Clinically, it is vital to further investigate the relationship between T2DM and AS.5 Evidence has shown that AS is accelerated by chronic inflammation rather than DM.6 Therefore, it is significant to determine new therapeutic targets for the source of systemic inflammation in T2DM.
Matrix metalloproteinases (MMPs), a large family of zinc and calcium-dependent endopeptidases that degrade all components of extracellular matrix and basement membrane proteins, are known to be closely associated with AS in DM.7 Activity of MMPs is regulated by tissue inhibitors of metalloproteinases (TIMPs) and α-2-macroglobulin.8,9 Accumulated results showed that in animal models, MMP-11, MMP-2, and matrix metallopeptidase 9 (MMP-9) are associated with insulin resistance.10 In particular, plasma MMP-9 levels were significantly increased in patients with acute coronary syndrome with T2DM.11 Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) together form a complete overlapping system, which transports white blood cells to the vascular wall and plays an important role in the formation of atherosclerotic plaques.12
Carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1), a member of the immunoglobulin superfamily, is known as a transmembrane glycoprotein located on the cell surface.13 Currently, CEACAM1 has also been found to play a protective role by regulating inflammation in many diseases. Our previous study has confirmed that CEACAM1 impairs mitochondrial function and aggravates cardiac injury after myocardial infarction.14 Moreover, CEACAM1 protects the blood–brain barrier from damage in ischemic stroke by regulating the levels of MMP-9.15 Thus, we hypothesize that CEACAM1 can also participate in inflammation through the regulation of MMP-9 in AS in T2DM, and ultimately affect the occurrence and development of diabetic AS.
In this study, we aimed to elucidate potential molecular mechanism of CEACAM1 on the inflammatory response in atherosclerosis.
MATERIALS AND METHODS
The serum (approximately 5 mL) from 100 patients diagnosed as T2DM (47 men and 53 women, mean age at 51.87 ± 8.2) and 63 control subjects (28 men and 35 women, mean age at 53.41 ± 6.8) were harvested from the First Affiliated Hospital of Kunming Medical University. The T2DM patients were recruited according to carotid intima-media thickness (CIMT) of the stiff artery indicated by clinical arterial ultrasound. Clinical samples were divided into T2DM + CIMT normal group (CIMT <1.0 mm, n = 35), T2DM + CIMT hypertrophic group (1.0 mm < CIMT < 1.3 mm, n = 42), and T2DM + carotid atherosclerosis plaque formation (T2DM + AS) group (CIMT > 1.3 mm, n = 23). Informed consents were obtained from all patients, and the study was approved by the Research Ethics Committee of The First Affiliated Hospital of Kunming Medical University. The clinicopathological characteristics were shown in Table 1.
TABLE 1. -
||Diabetic Patients (n = 100)
||Control Subjects (n = 63)
|Sex, M/F, n
||51.87 ± 8.2
||53.41 ± 6.8
||27.9 ± 5+6.5
||28.1 ± 7.2
|Plasma glucose, mg/dL
||158 ± 38
||95 ± 14
|Plasma insulin, μU/mL
||16 ± 9
||11 ± 4
|Serum lipids, mg/dL
| Total cholesterol
||198 ± 32
||184 ± 28
| HDL cholesterol
||43 ± 13
||41 ± 12
||151 ± 74
||137 ± 14
| Systolic blood pressure, mm Hg
||145 ± 15
||134 ± 19
| Diastolic blood pressure, mm Hg
||89 ± 7
||83 ± 4
| CIMT, mm
||0.924 ± 0.318
||0.698 ± 0.173
Data are presented as mean ± SD.
Adult male Wistar rats (Weight 22.89 ± 0.5 g, aged 8 weeks, certification No. 1107271911000520) were purchased from Department of Zoology, Medical University (Kunming, China). The rats were housed in the Animal Experimental Center of Kunming Medical University, and allowed free access to food and water. Animals were maintained at room temperature 22 ± 1°C, relative humidity 55%–65% with 12 hours light/dark cycle. All experimental procedures involving animals were conducted in accordance with the guidelines of the Animal Experimental Center of Kunming Medical University (Approved No. KY970864-m10, Date 2019-09-10).
The rats were randomly divided into 4 groups: control group (n = 10), DM model group (n = 10), atherosclerosis (AS) model group, and CEACAM1 group. In the control group (n = 10), rats that served as control group were fed standard animal chew. The rats that remained were fed with a high-fat diet for 8 weeks. Then, rats were injected with streptozotocin (STZ) (50 mg/kg, STZ was dissolved in 0.1 mmol/L citrate buffer, pH 4.5) through the tail vein,16 rats in control group were injected with the same dose of citrate buffer. The blood sample was harvested from tail vein and blood glucose was measured by a blood glycosometer after 72 hours of STZ injection. The rats with BG ≥ 16.7 mmol/L were confirmed as diabetic rats and kept feeding high-fat diet for another 8 weeks. The formation of atherosclerotic plaque was confirmed with transabdominal aortic ultrasound and the diabetic rats with atherosclerotic plaque served as the AS group. Rats in CEACAM1 group received intraperitoneal injections of 2 mg/kg CEACAM1 recombinant protein mixed with saline (1 mg/mL), and rats in the other 3 groups received intraperitoneal injection of saline in the same dose. Mice were returned to their cages after treatment and were given free access to food and water.
Hematoxylin & Eosin (HE) Staining
Mice were anesthetized with 2% chloral hydrate injection (0.2 mL/10 g) anesthesia. The chest was opened to expose heart, dissect aortic root and aorta. Tissue samples were fixed with 4% polyformaldehyde and embedded with paraffin. Then, the tissue samples were cut into slices. After being dried, the tissue slices were deparaffinaged using dimethylbenzene and dehydrated with water-free 95% and 80% ethanol. The slices were washed with phosphate buffer saline (PH 7.4). Subsequently, the slices were immersed in the hematoxylin and hydrochloride alcohol and washed with running water. Afterward, slices were dehydrated with ethanol at gradient degree, and then made transparent with dimethylbenzene. The tissue slices were observed using the microscope.
Oil Red O Staining
The tissue slices were treated with sodium oleate for 24 hours, and were washed with phosphate buffer saline twice. Then, 10% paraformaldehyde was added into slices. After being washed with H2O, slices were treated with 0.05%-TritonX-100 for 10 minutes 60% Isopropyl alcohol was used to soak the slices. Sixty percent oil red O solution was used to stain for 30 minutes and then slices were washed with 60% isopropyl alcohol. The tissue slices were observed using the microscope.
RAECs and RASMCs Culture
Rat aorta vascular endothelial cells (RAECs) were isolated from the thoracic aortas of 8-week-old Wistar male rats according to the previous protocol.16 Rat aorta smooth muscle cells (RASMCs) were obtained as previously described.17 RAECs were cultured in endothelial cell medium (ScienCell, Shanghai, China) and Dulbecco's modified eagle medium (Gibco, Beijing, China) containing 10% fetal bovine serum at 37°C under humid conditions with 5% CO2 and 95% air. RASMCs were cultured in Dulbecco's modified eagle medium containing 10% fetal bovine serum and 100 U/mL of penicillin and streptomycin at 37°C under humid conditions with 5% CO2 and 95% air. RAECs and RASMCs were inoculated into 24-well plates (104/mL), and labeled with von Willebrand factor, platelet endothelial cell adhesion molecule-1 (CD31), and α-smooth muscle actin (Abcam, Shanghai, China), and photographed under fluorescence microscope (Olympus, Tokyo, Japan).
The small interfering RNA (siRNA) targeting CEACAM1 (si-CEACAM1) and control siRNA (si-NC) were purchased from Ribo (Guangzhou, China). si-CEACAM1 and si-NC were transfected in RAECs and RASMCs using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The concentration of si-CEACAM1 (50 nM) and CEACAM1 recombinant protein (10 μg/mL) were selected in our previous experiments.
Enzyme-Linked Immunosorbent Assay
The release of CEACAM1, MMP-9, TIMP-1, TNF-α, VCAM-1, monocyte chemotactic protein 1 (MCP-1), and ICAM-1 were detected by enzyme-linked immunosorbent assay (ELISA). According to the instructions of the ELISA Kit, the supernatant was collected by centrifugation of the homogenate (5000 rpm for 15 minutes at 4°C). The activities of CEACAM1, MMP-9, TIMP-1, TNF-α, VCAM-1, MCP-1, and ICAM-1 were determined by the ELISA Kit. Quantification of ELISA results were performed at 450 nm using an ELISA plate reader (Spectra Max M5; Molecular Devices, San Jose, CA).
Western Blot Analysis
Total proteins were extracted using a Total Protein Extraction Kit following the instructions. BCA Protein Assay Kit was used to detect the protein concentration. The total protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked using 5% skimmed milk at room temperature for 2 hours. Primary antibodies shown in Table 2 were incubated overnight at 4°C and the secondary antibodies were incubated for 1 h at room temperature. Enhanced chemiluminescence assay was used and captured through Imager. The optical density of the bands was analyzed using NIH Image J software.
TABLE 2. -
||Cell Signaling Technology
||Cell Signaling Technology
Statistical analyses were performed using SPSS13.0 (SPSS Inc., Chicago, IL). Data were presented as means ± SD. Comparison between 2 groups was analyzed using Student's t-test. Comparison among 3 or more groups was analyzed using one-way ANOVA with a Tukey test. P values <0.05 indicate statistically significant differences. All experiments were repeated at least 3 times.
Changes of CEACAM1, MMP-9, and TIMP-1 in Serum of Patients With T2DM
We collected blood samples from T2DM patients according to the CIMT of the stiff artery indicated by clinical arterial ultrasound. ELISA results showed that the contents of CEACAM1 and TIMP-1 was significantly decreased in T2DM + carotid atherosclerotic plaque formation (T2DM + AS) group, whereas the content of MMP-9 was increased, which indicated that the contents of CEACAM1, MMP-9, and TIMP-1 were associated with CIMT (Fig. 1) (P < 0.01, P < 0.001).
CEACAM1 Ameliorated Atherosclerosis
Oil red O staining was used to observe the effect of CEACAM1 recombinant protein on the atherosclerotic plaque size in the rats. Oil red O staining of aorta specimens was shown in Figure 2A, and the proportion of oil red O staining area in the artery area was shown in Figure 2B (P < 0.05). Almost no plaque was observed in the control group (0.1 ± 0.02%). In the DM group, atheromatous plaques stained by oil red O were observed in the aortic arch, accounting for (15.254 ± 1.245%). However, the atheromatous plaques accounted for (30.143 ± 2.521%) in AS group. Moreover, the plaque area was reduced to (16.352 ± 1.875%) in the injection of CEACAM1 recombinant protein group, which was similar to that in DM rats. Then, we performed HE staining for aorta sections (Figs. 2C, D) (P < 0.05). The result showed that there was almost no arterial plaque in control group (0.115 ± 0.021 in area ratio of plaque/aortic cross section). In the AS group, the area of atherosclerotic plaque was larger than that in DM group (0.306 ± 0.019 vs. 0.254 ± 0.021, P < 0.05). In the AS group, the fibrous cap of arterial plaque was thinner. However, the result was revised by injection with CEACAM1 recombinant protein (0.226 ± 0.016, P < 0.05). CEACAM1 recombinant protein reduced the size and composition of atherosclerotic plaques in DM + AS group. The results showed that a large amount of lipid deposition could be seen in the artery of rats in the DM + AS group, while lipid deposition was improved by CEACAM1 recombinant protein.
CEACAM1 Recombinant Protein Ameliorated the Stability of Arterial Plaque
In the DM group, the expression of CEACAM1 was significantly decreased, whereas the expression change of MMP-9 and TIMP-1 induced by T2DM was reversed by CEACAM1 recombinant protein (Figs. 3A, B) (P < 0.01, P < 0.001). As we all know, the production of inflammation is believed to be an important development process of atherosclerosis. We also detected contents of TNF-α, MCP-1, ICAM-1, and VCAM-1 by ELISA (Fig. 3C) (P < 0.05, P < 0.01, P < 0.001). The level of CEACAM1 was significantly decreased in the CEACAM1-siRNA group. The result showed that injection of CEACAM1 recombinant protein could significantly reduce the contents of TNF-α, MCP-1, ICAM-1, and VCAM-1. The plaque stability mainly depends on the content of lipids.17 Therefore, atherosclerosis was accelerated in STZ-induced rats, and atherosclerotic plaques were more vulnerable. However, the stability of atherosclerotic plaque was increased by CEACAM1 recombinant protein.
CEACAM1 Recombinant Protein Decreased the Release of Vascular Inflammatory Factors
The cultured rat thoracic aorta endothelial cells (RAECs) and RASMCs was identified by immunofluorescence (Figs. 4A, B). Based on our preliminary experiments, we explored the concentration of CECAM1 and the condition of CECAM1-siRNA. Then, the contents of TNF-α, MCP-1, ICAM-1, and VCAM-1 in the medium were detected by ELISA. As a result, high glucose could significantly induce the secretion of inflammatory factors in the medium, which was reversed by CEACAM1 (Figs. 4C, D) (P < 0.05, P < 0.01, P < 0.001). In RAECs and RASMCs, knockdown of CEACAM1 aggravated the pro-inflammatory effect of high glucose.
CEACAM1 Recombinant Protein Inhibited Diabetic Atherosclerosis Through MMP-9/TIMP-1 and IkB-α/NF-kB
CEACAM1 recombinant protein increased the content of TIMP-1 and decreased the content of MMP-9 in the medium, whereas the result is opposite when CEACAM1 was knocked down (Fig. 5) (P < 0.05, P < 0.01, P < 0.001). To further confirm the molecular mechanism of CEACAM1 regulating vascular inflammation, we analyzed the expression levels of p65, P-p65, IκB-α, P-IκB-α by western blot. As a result, high glucose significantly increased the expression of P-p65, but decreased the expression of P-IκB-α, which could be reversed by CEACAM1. However, knockdown of CEACAM1 aggravated the effect of glucose (Fig. 5). These data confirmed that CEACAM1 attenuates diabetic atherosclerosis by inhibition of IκB/NF-κB signal pathway via MMP-9/TIMP-1 axis.
DM is not only the main cause of macrovascular disease and its complications, but also an independent risk factor for atherosclerosis (AS).18 The previous studies on CEACAM1 were mainly focused on cancer.19–21 Accumulated evidences have shown that CEACAM1 alleviates the inflammatory response and protects the BBB by regulating MMP-9.22 Therefore, we hypothesized that CEACAM1 may affect atherosclerosis in T2DM by regulating the expression of MMP-9/TIMP-1. Here, we first collected clinical samples of patients with T2DM, and then detected the expression of CEACAM1 and MMP-9 in serum. We found that CEACAM1 was significantly decreased during the development of AS, whereas the secretion of MMP-9 was increased in serum. HE and oil red O staining showed that CEACAM1 decreased intima-media thickness and the area of AS plaque in aortic root in atherosclerotic rats with T2DM. In STZ-treated rats, the expression of CEACAM1 was significantly decreased, whereas the expression of MMP-9 and TIMP-1 induced by T2DM could be reversed by CEACAM1. These results suggested that CEACAM1 may be involved in the pathophysiological process of diabetic atherosclerosis.
Epidemiological studies have shown that the global incidence of diabetes is closely related to obesity.23 Obesity is associated with the pathogenesis of diabetic inflammation.24 Relevant studies have confirmed that cytokines play an important role in regulating the function of islet cells, among which TNF-α and MCP-1 play an important role in the pathogenesis of DM.25 TNF-α is produced by macrophages and adipocytes, which promotes lipid breakdown and free fatty acid release and finally leads to insulin resistance.26 Hyperglycemia promotes the activation of endothelial cells and the expression of MCP-1 and VCAM-1 in cultured endothelial cells and MCP-1, which play a key role in T2DM. Therefore, endothelial function is impaired from the onset of disease in patients with T2DM and is closely related to adverse outcomes.27 In the present study, we detected the expression of TNF-α, MCP-1, ICAM-1, and VCAM-1 in vivo and in vitro. The results showed that the expression of TNF-α, MCP-1, ICAM-1, and VCAM-1 was decreased significantly in the AS group, and the change was reversed significantly after CEACAM1 treatment, further demonstrated that the regulatory role of CEACAM1 is important in AS caused by DM.
It is reported that there are many signaling pathways involved in the pathological process of vascular injury in atherosclerosis.28,29 NF-κB is a major transcription factor regulating immune inflammation. As a key regulator of inflammatory response, p65 directly or indirectly promotes the secretion of inflammatory cytokines. Generally speaking, nuclear translocation of NF-κB, whose localization is masked by IκB-α, results in its presence in cytosol.30 NF-κB was no longer inhibited after atherosclerotic plaque formation.4 In the current study, our data confirmed that CEACAM1 recombinant protein reduced the phosphorylation of NF-κB signaling pathway after treatment, which is consistent with previous reports.31 However, CEACAM1 reduced the phosphorylation of p65 and IκB-α in diabetic-induced atherosclerosis model. The results suggest that CEACAM1 recombinant protein regulates inflammatory immune response by inhibiting NF-κB signaling pathway. Thus, CEACAM1 may regulate MMP-9/TIMP-1 axis by inhibiting IκB-α/NF-κB signal pathways in atherosclerosis in T2DM. Thus, CEACAM1 recombinant protein may become a new therapeutic target for atherosclerosis. In addition to inflammation, oxidative stress also accelerates the progression of atherosclerosis. However, the role of CEACAM1 in oxidative stress of AS caused by DM still needs to be further explored.
CEACAM1 is downregulated in atherosclerosis in T2DM, suggesting that CEACAM1 is involved in the pathophysiological process of the disease. CEACAM1 inhibited IκB/NF-κB signal pathway via MMP-9/TIMP-1 axis in vascular inflammation T2DM atherosclerosis.
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