Although high-density lipoprotein (HDL) can exhibit anti-inflammatory properties, these potent activities can become deficient and even transform into proinflammatory effects under various pathophysiological states. We investigated the effect of diabetic HDL on the inflammatory response in human monocytes and its relation to the existence of coronary artery disease (CAD) in patients with type 2 diabetes mellitus (DM). HDL was isolated from DM patients with (n = 61) or without (n = 31) CAD (diameter stenosis ≥50%) and healthy controls (n = 40). Human peripheral blood mononuclear cells were incubated with HDL and the proinflammatory ability of HDL was determined by tumor necrosis factor-α (TNF-α) secretion in peripheral blood mononuclear cells. Secretion of TNF-α in human monocytes in response to diabetic HDL was significantly increased compared with that of the control HDL. Of note, HDL from DM patients with CAD stimulated the release of TNF-α in monocytes to a greater extent than that of HDL from those without CAD. Multiple linear regression analysis showed that the proinflammatory ability of HDL was independently associated with diabetes duration, hemoglobin A1c, serum levels of high-sensitivity C-reactive protein (hs-CRP) and reduced glomerular filtration rate (GFR). Furthermore, the proinflammatory ability of HDL was a significant predictor for the presence of CAD in patients with DM.
Emerging studies have focused on the anti-inflammatory properties of high-density lipoprotein (HDL). In vitro studies indicate that HDL from healthy volunteers effectively blunts the inflammatory response of macrophages and endothelial cells to stimuli such as lipopolysaccharide or tumor necrosis factor-α (TNF-α). In vivo infusion of reconstituted HDL also effectively inhibits inflammation induced by periarterial collar in rabbits. This antiatherogenic property of HDL is highly variable, however, and can even transform into proinflammatory effects in both acute phase responses and chronic inflammatory diseases. In patients with type 2 diabetes mellitus (DM), increased level of chronic inflammation, such as C-reactive protein (CRP) and TNF-α, might be implicated in diabetic HDL conversion from anti-inflammatory to proinflammatory. Previous research provided data that diabetic HDL can exhibit impaired anti-inflammatory properties, which is measured by its ability to inhibit cytokine release induced by stimuli. However, whether diabetic HDL displays a proinflammatory ability still remains unknown. In this study, we directly incubated diabetic HDL with human monocytes to test its role in inflammatory responses and its relation to the presence of coronary artery disease (CAD) in patients with DM.
Methods
A total of 140 consecutive patients with type 2 DM undergoing coronary angiography with chest pain from November 2014 to September 2015 at Shanghai Rui Jin Hospital were recruited in the study. The diagnosis of type 2 DM was made according to the criteria of the American Diabetes Association, including increased fasting glucose concentration (≥7.0 mmol/L) or 2-hour postprandial plasma glucose (≥11.1 mmol/L), or receiving insulin treatment or antiiabetic medicine. Diabetes onset was defined as the point in time when any of the aforementioned criteria were first met. The information on diabetes onset in patients was obtained from medical charts or patient interviews. Diabetes duration was calculated as the difference between current age and the age at diabetes onset. For the purpose of avoiding the confounding influence of other diseases, we excluded patients with acute coronary syndrome within 7 days (n = 5) or a history of coronary revascularization (percutaneous coronary intervention, n = 15 and coronary artery bypass grafting, n = 5); New York Heart Association class IV, n = 7; end-stage renal disease, n = 5; chronic viral or bacterial infection, n = 4; immune system disorders or tumors, n = 4; and pulmonary heart disease, n = 3. Patients with type 1 diabetes were also excluded by measurement of C-peptide. The remaining 92 eligible patients were enrolled in the study as the DM group. We also included 40 age- and gender-matched subjects to serve as the control group who were receiving an annual physical check-up in our hospital. They had normal lipid profiles, serum glucose concentration, and renal function. None had any evidence of cardiovascular disease (including history of angina/myocardial infarction). The study protocol was approved by the Hospital Ethics Committee and was in accordance with the Code of Ethics of the World Medical Association. Written informed consent was obtained from all participants.
Blood samples were collected from all participants after overnight fasting. Serum levels of glucose, hemoglobin A1c (HbA1c), total cholesterol, HDL cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides, and creatinine were measured using standard laboratory techniques on a Hitachi 912 Analyzer (Roche Diagnostics, Germany). Serum levels of high-sensitivity CRP (hs-CRP) were assessed by enzyme-linked immunosorbent assay (Biocheck Laboratories, Toledo, Ohio). Glomerular filtration rate (GFR) was calculated by the Chronic Kidney Disease Epidemiology Collaboration equation. Human HDL was isolated and purified from fresh plasma by density-gradient ultracentrifugation (density 1.063 to 1.21 g/ml), as described previously. Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy subjects by Ficoll-Hypaque density-gradient centrifugation, as previously described. Monocytes were then exposed to either HDL from healthy subjects (control HDL group), HDL from patients with DM (diabetic HDL group; 100 μg/ml), or sodium chloride (basal group) for 24 hours. Supernatants were then collected and measured by enzyme-linked immunosorbent assay to determine TNF-α levels (Anogen, Mississauga, Canada).
Coronary angiography was performed through radial or femoral approach by experienced interventional cardiologists blinded to the study protocol. CAD was diagnosed if luminal diameter narrowing was estimated visually as ≥50% in a major epicardial coronary artery.
Data were expressed as percentages or frequencies for categorical variables and summarized as mean ± SD for continuous ones. Chi-square tests were used to analyze differences in clinical categorical variables. Differences among groups were determined by one-way analysis of variance. Multiple linear regression analysis was performed to evaluate the relation between the proinflammatory ability of HDL and the following variables: age, gender, body mass index, hypertension, fasting glucose, HbA1c, diabetes duration, triglycerides, total cholesterol, HDL, LDL, creatinine, GFR, hs-CRP, and statin treatment. The diagnostic values of the proinflammatory ability of HDL and the serum hs-CRP levels for detecting CAD were calculated by receiver-operating characteristic (ROC) curves. A value of p <0.05 was considered statistically significant. SPSS software version 20.0 (IBM, New York) was used for statistical analyses.
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