Effects of reduced β2-glycoprotein I on the expression of aortic matrix metalloproteinases and tissue inhibitor matrix metalloproteinases in diabetic mice

Jun Xu 0 Penghua Wang 0 Tong Wang 0 Meijun Wang 0 Sisi Chen 0 Pei Yu 0 Demin Yu 0 0 2011 Collaborative Innovation Center of Tianjin for Medical Epigenetics, the Key Laboratory of Hormones and Development (Ministry of Health), Metabolic Diseases Hospital & Tianjin Institute of Endocrinology, Tianjin Medical University , No.66 Tong-An Road, Heping district, 300070 Tianjin , China Background: Reduced 2-glycoprotein I (reduced 2GP I), which has free sulfhydryl groups, is present in plasma and serum; it can protect vascular endothelial cells from damage due to oxidative stress in vitro. We investigated the effects of reduced 2GP I on the expression of various matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in the aortas of diabetic mice. Methods: We provided 120 female 8-week-old Balb/c mice with a high sugar, high fat diet. After 8 weeks they were injected with streptozotocin to induce diabetes. We treated mice in the mono dose groups with 2GP I, reduced 2GP I, or phosphate-buffered saline (PBS) on day 1 and fed them for 3 weeks. The mice in the complex dose groups were treated with 2GP I, reduced 2GP I, or PBS on days 1 and 22 and fed for 6 weeks. Control mice were given a standard chow diet. Blood lipids were measured at the end of 3 or 6 weeks, and aortas removed to observe morphological and molecular biological changes. Results: The low-density lipoprotein cholesterol levels in mice of the reduced 2GP I group were lower than those in the diabetic group. Aortic lipid deposition in the reduced 2GP I group was significantly less than in the diabetic control group. In the aortas, reduced 2GP I decreased MMP2/TIMP2 mRNA and protein expression levels, and MMP9/TIMP1 expression levels compared with those in diabetic controls. Reduced 2GP I down-regulated p38 mitogen-activated protein kinase (p38MAPK) mRNA expression and phosphorylated p38MAPK protein expression compared with those in diabetic controls of the complex dose group. Conclusions: Reduced 2GP I plays a role in diabetic mice related to vascular protection, inhibiting vascular lipid deposition, and plaque formation by reducing MMPs/TIMPs expression through down-regulation of the p38MAPK signaling pathway. - 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. 2-glycoprotein I (2GP I) is a phospholipid-binding plasma protein, and an autoantigen. In patients with type 2 diabetes or myocardial infarction, plasma 2GP I and oxidized low-density lipoprotein (oxLDL)/2GP I complex levels are significantly increased; these are predicted adverse consequences of Table 1 Primer sequence of TIMP-1, 2, MMP2, MMP9, p38MAPK and GAPDH for real time PCR cardiovascular events [5]. Previous studies have shown that 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 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 2GPI can inhibit the formation of foam cells by macrophages and apoptosis in vitro [12]. The aim of Table 2 Changes in blood glucose and body weight *P < 0.05 vs. normal control. our study was to investigate how reduced 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 1825 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 2GP I group (20 g); the reduced 2GP I group (20 g); and the diabetic control group treated with phosphate-buffered saline (PBS). We used PBS as the vehicle for 2GP I and reduced 2GP I. We also had three complex-dose groups that were injected twice in the tail vein on days 1 and 22: the 2GP I group (20 g each injection), the reduced 2GP I group (20 g 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. Figure 1 Blood lipid changes in each group. We obtained 160 female Balb/c mice (8 weeks old) and randomly selected 40 mice as the normal control group, which were given a standard chow diet for 8 weeks. The remaining 120 mice were given a high sugar and high fat diet for 8 weeks. These mice were then intraperitoneally injected with 80 mg/kg of 2% streptozotocin twice. Mice with a blood glucose concentra (...truncated)