Mechanisms Of High Glucose Inducing Atherosclerosis Via Dendritic Cells And Changes Of Dendritic Cells Subsets In Type 2 Diabetic Patients With Or Without Coronary Artery Disease

Atherosclerosis and its clinic consequence, such as conorary atherosclerotic disease, have become the leading health problem worldwide. Type 2 diabetes, a presently rapidly expanding disease, is a major risk factor for cardiovascular morbidity and mortality. Diabetics have a two-to fourfold increased incidence of coronary heart disease (CAD) and stroke. Established diabetes is associated with impaired prognosis after myocardial infarction (Ml). Newly detected abnormal glucose tolerance is one of the strongest prognostic factors following an Ml. In addition, the Euro Heart Survey on Diabetes and the Heart demonstrated that only 29% of people with acute CAD and 34% of people with stable CAD were normoglycaemic. However, the mechanism of diabetes-accelerated atherosclerosis is still not well understood. Hyperglycemia, the hallmark of diabetes mellitus, is a major independent risk factor for diabetic macrovascular disease and plays a key pathogenic role in the development of diabetes-associated atherosclerosis. The precise molecular mechanism of how hyperglycemia induces these damages is not known till now.Inflammation and immune reactions might play a crucial role in the development of atherosclerosis and diabetes associated vascular complications. Atherosclerotic lesions contain large numbers of immune cells, such as macrophages, activated T cells, and dendritic cells (DCs). Additionally, a systemic proinflammatory state with elevated serum levels of C-reactive protein (CRP) or interleukin (IL)-6 is detectable in patients with atherosclerotic vascular diseases. In atherosclerotic plaques DCs form contacts with T cells and NKT cells, suggesting that DCs may play a curial role in T cell activation in atherosclerosis. The immature DCs take up antigens and pathogens, generate MHC-peptide complexes, migrate from the site of antigen acquisition to secondary lymphoid organs, and fulfill the maturation; mature DCs then physically interact with and activate T lymphocytes. In the arterial wall, DCs are present in their immature forms and become activated during atherogenesis. Recent studies have shown that DCs were markedly increased in atherosclerotic lesions, and they might play a crucial role in the immunogenesis of atherosclerosis. Diabetes is also associated with accelerated atherosclerotic macrovascular disease affecting arteries that supply the heart, brain and lower extremities. The role of DCs in human type 2 diabetes with or without coronary heart disease remains unstudied. We hypothezid that microenvironment in type 2 diabetic mellitus might result in changes of DCs numeration and function, leading to the pathogenesis of diabetes-associated atherosclerosis.Thus, the aim of this research is to explore the role of dendritic cells in the immunologic mechanism of typy 2 diabetes associated atherosclerosis. It is made up of two parts:Part I : High Glucose Stimulates Immune Maturation of Human Dendritic Cells: Potential Implication for Progression of Atherosclerotic LesionsObjective: Hyperglycemia is the hallmark of diabetes meliitus and plays a key pathogenic role in the development of diabetes-associated atherosclerosis. Recent findings suggested that dendritic cells (DCs) may play a crucial role in T cell activation in atherosclerosis. However, whether they function interactively in the process remains uncertain. We therefore studied the effects of high glucose on the maturation and immune function of monocyte-derived dendritic cells (MDC) and its underlying mechanisms.Methods: Human monocytes were purified (over 98%) using anti-CD¹⁴ microbeads. After 6 days' culture in RPMI1640 medium containing recombinated human granulocyte-macrophage colony stimulating factor (rhGM-CSF, 100ng/ml) and recombinated human interleukin-4(rhlL-4, 20ng/ml), immature MDC were derived. MDC then were cultured in above medium with either 5.5mM (NG, as a control treatment), 25mM D-glucose (HG, as a high concentration treatment) or 5.5mM D-glucose+19.5mM mannitol(HM, as a high osmolarity control) in the absence or presence of N-acetyleysteine(NAC) or p38 MAPK inhibitor SB203580 for further 48 hours. FACS was used to investigate the immunophenotypic expression (CD1a, CD83, CD86, and HLA-DR). Allogeneic mixed T lymphocytes reaction and supernatants' cytokine measurements were used for immune functional assays. Intracellular production of reactive oxygen species(ROS) in MDC was measured by 2' ,7' -dichlorodihydrofluorescin (DCF, 10μM) fluorescence using confocal laser-scanning microscopy techniques. Phosphorylation of cellular p38 MAPK was determined by western blot analysis. Results: HG treatment induced DCs maturation accompanied with increased expressions of costimulatory molecule CD86, DCs maturation marker CD83, CD1a and MHC class II molecule HLA-DR. Moreover, the capacity of DCs to stimulate T cell proliferation and secretion of cytokines (interferon-γ, interleukin-10 and -12) was significantly increased by 25mM D-Glucose independent of osmolality. These effects of HG were primarily mediated by reactive oxygen species and p38 MAPK pathway, because HG could upregulate the production of ROS as well as phosphorylation of p38 MAPK in DCs and NAC or SB 203580(a specific inhibitor of p38 MAPK) partly reversed these stimulatory effects of HG on these cells.Conclusion: High glucose promote the maturation of DCs and augment their capacity to stimulate T-cell proliferation and cytokine secretion at least in part through enhancing intracellular ROS and signaling via p38 MAPK pathway. These interactive roles of high glucose and DCs may contribute to the immunopathogenesis of atherosclerosis. Part II: Changes of Peripheral Blood Dendritic Cells Subsets in Type 2 Diabetic Patients with or without Coronary Artery Disease.Objective: Inflammation and immune reations are implicated in atherosclerosis and coronary artery disease (CAD), whose complications represent the main cause of death in diabetic patients. Recent findings suggest dendritic cells might play an important role in the development of atherosclerosis. The role of DCs in human type 2 diabetes with or without coronary heart disease remains unstudied. We hypothesised that diabetic patients with or without CAD had different profile of circulating DCs or its subsets which may be an index of the risk of developing atherosclerosis.Methods: Twenty-Four patients with type 2 diabetes were divided into two groups based on presence (DM+CAD, n=13) or absence (DM) of angiographically-documented CAD (lumen diameter narrowing≥50%) or 99mTc-sestamibi SPECT stress imaging (n=11). Age and sex matched health were as control (Normal, n=16). The percentage and absolute number of peripheral blood DCs and DCs subsets were measured by three color flow cytometry of whole blood cells with specific blood dendritic cell antigens: HLA - DR-PerCP, Lin-1(CD3, CD16, CD14, CD19 and CD56 )-FITC, CD11C-PE and CD123-PE. lgG1-PE and lgG2a-PE were used as isotype-matched controls. DCs were defined as Lin1^- HLA-DR⁺, and mDC as Linr1^- HLA-DR⁺CD11c⁺, PDC as Lin1^- HLA-DR⁺CD123⁺, Serum levels of Glycated hemoglobin (HbA1c), hs-CRP, IL-6 as well as TNF—αwere measured in all patients.Results: The percentage and absolute numbers of DCs (Lin1^- HLA-DR⁺ cells) within the total leukocyte population was similar for Normal (1.73±0.43×10⁷/L, 0.25±0.06%) and DM (1.57±0.32×10⁷/L, 0.22±0.05%), while significantly lower in DM+CAD group (1.19±0.54×10⁷/L, p=0.045, 0.006 vs DM and Normal respectively; 0.17±0.07%, p=0.029, 0.002 vs DM and Normal respectively). The percent and absolute number of the mDC was significantly decreased in CAD+DM (0.91±0.44×10⁷/L, 0.13±0.06%) compared with DM (1.31±0.29×10⁷/L, 0.19±0.05%) and Normal (1.40±0.37×10⁷/L, 0.21±0.05%). In contrast, pDC numbers were not significantly altered. The ratio of mDC:pDC with mDC comprising the vast majority of DCs tended to decrease more in DM+CAD, although differences were not significantly different (p= 0.283) . Furthermore, circuculating mDC inversely correlated with serum hs -CRP(r=-0.750, p<0.001), TNF-α(r= -0.716, p<0.001) level and the number of disease vessle(r=-0.633, p=0.020). No statistically significant Spearman's correlation coefficients were found between mDC and IL-6 level.Conclusion: The present study shows a significant decrease of circulating blood DCs and mDC in type 2 diabetes with coronary heart disease which maybe the cause of enhenced Th1 response in local coronary plaque and might useful for screening coronary heart disease in patients with type 2 diabetes.