Redox Status of β 2GPI in Different Stages of Diabetic Angiopathy

We explored the redox status of beta 2 glycoprotein I (β 2GPI) in different stages of diabetic angiopathy. Type 2 diabetes mellitus (T2DM) had a significantly lower proportion of reduced β 2GPI as compared to healthy controls (p < 0.05). There was a trend that the mild coronal atherosclerosis heart disease (CAD) had higher proportion of reduced β 2GPI than non-CAD and severe-CAD groups, however without significances (p > 0.05). The mild-A-stenosis group and mild-diabetic retinopathy (DR) groups had higher proportion of reduced β 2GPI than their severely affected counterparts. The mild-slow nerve conduction velocity (NCVS) group had higher proportion of reduced β 2GPI than normal nerve conduction velocity (NCVN group) and severe-NCVS groups. The proportion of reduced β 2GPI was in positive correlation with 24 h urine microalbumin and total urine protein, and the proportion of reduced β 2GPI was in negative correlation with serum and skin advanced glycation end products (AGEs). Taken together, our data implicate that the proportion of reduced β 2GPI increased in the early stage of angiopathy and decreased with the aggravation of angiopathy.


Introduction
Cardiovascular disease (CVD), blindness, renal failure, and amputation caused by diabetic angiopathy contribute to healthy burden in modern society. The pathological mechanism of diabetic angiopathy is still unclear, although oxidative stress (ROS) is one of the critical initiating factors in diabetic complications [1]. Beta 2 glycoprotein I ( 2 GPI) is a type of single-strand protein that contains five structural domains (DI-DV). As the major autoantigen of antiphospholipid syndrome (APS), 2 GPI is closely associated with thrombotic events in patients with APS [2]. The Cys288 to Cys326 disulfide bond in domain V can be reduced by oxidoreductase enzymes thioredoxin (TRX-1) and protein disulfide isomerase (PDI). Free thiolcontaining 2 GPI was discovered in 2010 [3] and this reduced 2 GPI, in contrast to oxidized 2 GPI, can protect endothelial cells from oxidative stress [4]. Research shows that the APS patients have significantly higher serum oxidized 2 GPI level than that in healthy controls, and reduced 2 GPI level is significantly reduced in APS patients. Our previous work has shown that reduced 2 GPI could inhibit the formation of foam cells and macrophage apoptosis [5], protect endothelial cells from oxidative stress-induced cell injury [4], inhibit retinal angiogenesis in diabetic rats, and reduce expression of collagen type IV in diabetic kidney. Conversely, an imbalanced redox state of 2 GPI may be important to increase thrombotic events in APS, and 2 GPI levels could form the basis of thrombosis risk [2].
Diabetics tend to be exposed to high oxidative stress state due to elevated radical ROS production caused by increased advanced glycation end products (AGEs), polyol pathway, and other factors. However, there is no report regarding the redox balance of 2 GPI in diabetic patients. Therefore, this study aimed to explore the relationship between the redox states of 2 GPI in diabetics and at different stages of angiopathy.  [2,8], acute inflammation, pregnancy or had surgery recently [9][10][11]. Diabetic ketoacidosis, hyperosmolar hyperglycemia, acute lactic acidosis, and the use of drugs that could change redox state of the body were also excluded from the study [12].

Materials and Methods
Age, sex, Body Mass Index (BMI), Waist-Hip Ratio (WHR), systolic blood pressure (SBP), diastolic blood pressure (DBP), history of cardiovascular disease and hypertension, diabetic history, and diabetic duration were obtained from the patients' medical records. Fasting blood glucose (FBG), P2BG, HbA1c, platelet (PLT), red blood cell (RBC), hemoglobin, FIB, D-Dimer, blood urea nitrogen (BUN), creatinine (SCR), hepatic function, total cholesterol (CHO), triglyceride (TG), high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), 24 h urine microalbumin (UMA), and 24 h urine total protein (UTP) were all determined by standard clinical biochemical assays. Nerve Conduction Velocity Test (NCVT) was done using Electromyography/Evoked Potentials Equipment (NDI-200P+, Shanghai). Lower limb arteries were tested by Color Doppler Ultrasound (JYQ TCD-2000). The Ocular Fundus Test was performed using an ophthalmoscope (TOP-CON TRC-NW7SF), and skin AGEs were measured by AGEs Reader (The Netherlands). The relations between complications of CAD, artery stenosis, abnormal nerve conduction velocity, diabetic retinopathy, and abnormal urinary albumin excretory rate and 2 GPI were analyzed. CAD was diagnosed based on one or more of the following criteria: electrocardiogram, echocardiogram, and myocardial perfusion imaging showing myocardial ischemia or infarction. Coronary angiogram or CTA revealed one or more main brunches of coronary artery with more than 50% stenosis.

Determination of Reduced 2 GPI and Total 2 GPI in
Serum. We adopted double-antibody sandwich ELISA to quantify total 2 GPI in serum samples [2]. Briefly, highbinding 96-well plates were coated overnight at 4 ∘ C with rabbit polyclonal anti-human 2 GPI (10 nM). Plates were washed 4 times with PBS-0.1% Tween and then blocked with 2% BSA/PBS-0.1% Tween for 1 hour at room temperature (RT). Following washing, 100 L of anti-human 2 GPI mouse mAb was added (10 nM) and then 100 L of the patient sample (diluted 4,000-fold in PBS-0.05% Tween) was coincubated for 1 hour at RT. After washing 4 times, AP-conjugated goat anti-mouse IgG was added (1 : 1,500) and incubated for 1 hour at RT. Five serum samples were randomly mixed to create a standard serum (internal control), which was used to construct an in-house standard curve for every ELISA. The mixture was aliquoted into Eppendorf tubes, snap frozen, and stored at −80 ∘ C. The level of total 2 GPI in standard serum was defined as 100%. Samples were read at 450 nm after addition of chromogenic substrate. Samples were assayed in duplicate.
The relative amount of reduced 2 GPI in patient samples was assayed as previously described [2]. MPB (4 mM) was added to 50 L of patient plasma or serum and incubated for 30 minutes at RT in dark and then the mixture was diluted 50-fold in 20 mM HEPES buffer (pH 7.4) and incubated for another 10 min at RT in dark. Proteins were then acetone precipitated. Protein pellets were resuspended in PBS-0.05% Tween. The samples were diluted 4000-fold and then added, in duplicate, to a streptavidin-coated 96-well plate (100 L/well), followed by incubation for 90 minutes at RT. Before adding MPB-labeled serum samples, streptavidincoated plates were washed 3 times with PBS-0.05% Tween and blocked with 2% BSA/PBS-0.1% Tween. After washing 3 times with PBS-0.1% Tween, the murine anti-2 GPI mAb was added (25 nM) and incubated for 1 hour at RT. After washing 3 times with PBS-0.1% Tween, alkaline phosphataseconjugated goat anti-mouse IgG (1 : 1,500) was added for 1 hour at RT and samples were read at 405 nm after adding chromogenic substrate. HAS, non-MPB-labeled serum and ox-2 GPI were used as controls. The pooled in-house standard used for the above-described 2 GPI quantification ELISA was used as an internal control and standard. The proportion of reduced 2 GPI was expressed as a percentage of that observed with the pooled in-house standard, after correction for the total amount of 2 GPI.

Statistical
Analyses. The data were presented as mean and standard deviation. Statistical analyses were performed using SPSS20.0. For normal distribution data, we used independent-samples -test and ANOVA. Nonnormal distribution variables were expressed as medians and interquartile ranges (IQRs) and analyzed by rank sum test. Differences in frequency of categorical variables were assessed by the chisquare test. All reported values were two-sided and values of < 0.05 were considered statistically significant. (severe-CAD group). According to Doppler ultrasonography, 108 patients were free of artery stenosis of lower limbs (non-A-stenosis group), 63 patients had less than 50% artery stenosis (mild-A-stenosis group), and 59 patients had more than 50% artery stenosis (severe-A-stenosis group

Type 2 Diabetes Mellitus and Redox State of 2 GPI.
The conformation and function of reduced 2 GPI are quite different from those of the oxidized form [13,14]. Therefore, ROS is likely to play a direct role in the regulation of 2 GPI status. APS, which is characterized by increased oxidative stress and vascular thrombosis, has elevated 2 GPI and a decreased proportion of reduced 2 GPI [2]. Similarly, diabetics are in a high oxidative stress state due to massive ROS production caused by increased AGEs, the polyol pathway, and other factors. Indeed, our study showed that T2DM patients had significantly lower proportion of reduced 2 GPI. Serum AGEs were in positive correlation with hs-CRP which is an important inflammatory marker and one of the strongest independent predictors of cardiovascular disease, and the proportion of reduced 2 GPI was in negative correlation with hs-CRP, serum and skin AGEs, D-Dimer, and FIB.

Reduced 2 GPI and Diabetes Macroangiopathy.
Atherosclerosis (AS) is a complex chronic disease caused by various factors. 2 GPI plasma concentrations are strongly associated to vascular disease in type 2 diabetic patients and have been proposed as a clinical marker of cardiovascular risk [15]. mediated by 2 GPI. Thus, it can be inferred that 2 GPI in its oxidized form has the potential to be one of the significant pathogenic factors of atherosclerosis. Reduced 2 GPI can inhibit the formation of foam cells, reduce macrophage apoptosis [5], and protect endothelial cells from oxidative stress-induced cell injury [4]. Our results implicated that the change of redox state of 2 GPI would affect its functions. Reduced 2 GPI could be a protective factor from atherosclerosis, while the oxidized 2 GPI could accelerate the disease. In the present study, we have found that the proportion of reduced 2 GPI appeared a rising trend in the early stage of diabetes macroangiopathy due to the compensatory mechanism and showed a decreasing trend in the late stage of CAD and arterial stenosis, although it was not statistically different. More samples are still needed to clarify the difference.   [21].
inhibiting phosphorylation of ERK1/2 and Akt through Ras/Raf/MEK/ERK and PI3K/Akt/Gsk3 pathways. In our previous study, reduced 2 GPI treated STZ-BALB/c mice had a lower urinary albumin excretion rate (UAER) and a less pronounced pathological damage of kidney compared with control mice [16]. Western blots showed that reduced 2 GPI treated group had less expression of phosphorylated p38MAPK, TGF-1, and type IV collagen. Reduced 2 GPI eliminates vWF, inhibits retinal angiogenesis, and inhibits glomerular fibrosis, thereby inhibiting the development of DR and DN. In our study, the proportion of reduced 2 GPI increased in the early stage of diabetes microangiopathy and then decreased with aggravating of diabetes microangiopathy. Therefore, reduced 2 GPI may be a protective factor in diabetes microangiopathy.
The redox state of 2 GPI changed in T2DM patients. The proportion of reduced 2 GPI increased in the early stage of angiopathy possibly as a compensatory mechanism and then decreased as angiopathy aggravated. This may implicate that reduced 2 GPI is a protective factor in diabetes angiopathy. Testing the amount and proportion of reduced B 2 GPI periodically in "at-risk" patients may offer the potential to better predict the occurrence and development of diabetic angiopathy.