The underlying mechanisms for the far greater severity of vascular disease in diabetics remain to be fully defined [3
]. Endothelial dysfunction, characterized by increased adhesion molecules, increased proinflammatory markers, increased pro-thrombotic factors, increased ROS and loss of normal regulation of vascular tone, occurs in diabetes, likely a result of the convergence of multiple proinflammatory mechanisms [2
]. Endothelial dysfunction is accompanied by the development of an autoimmune reaction that appears to play an important role in the inflammatory evolution of atherosclerosis [9
]. There are a number of molecules, which are thought to contribute to this process, including oxidized(ox) LDL, advanced glycation end-products, and HSP 60, with oxLDL and HSP60 acting as autoantigens [9
]. The oxLDL likely are a result of ROS action on LDL, which have penetrated the subendothelial space. Activation of the immune system through chronic infection or exposure of intracellular proteins, such as HSP60, which are then recognized as danger associated molecular patterns (DAMPs), can lead to inflammation and cell apoptosis and contribute to the development and progression of vascular disease [3
HSP60 is primarily a mitochondrial protein, where it is critical for proper folding of key metabolic proteins, but HSP60 is also found in the cytosol, where in the heart it has an antiapoptotic role [14
]. Diabetes has been reported to be associated with very high levels of HSP60 in blood [15
]. HSP60 associates with the cell membrane under stress conditions, and in heart failure localized to the surface of cardiac myocytes, which correlated with myocyte apoptosis [10
]. HSP60 has been identified as a potential ligand for TLR4. We have recently shown that TNFα
and HSP60 each drives the expression of the other, which could explain the correlation between HSP60 and TNFα
]. HSP60 has been shown to activate endothelium, smooth muscle cells, and macrophages [18
]. TLR4 activation and expression increase in diabetes in response to hyperglycemia, and this was not prevented by insulin treatment [19
]. TLR4 mediates vascular inflammation and insulin resistance in diet induced obesity. We have previously reported that extracellular HSP60 causes cell apoptosis via TLR4 activation and production of TNFα
]. Chlamydial and other bacterial HSP60 (HSP65), which have high homology to human HSP60, have also been implicated in vascular disease, but because these infectious agents are so commonplace, it has been difficult to irrefutably prove [18
]. Cellular (T cells) and humoral (anti-HSP60 antibodies) immune responses potentially also play a fundamental role in triggering the inflammatory process that fuels atherosclerosis [13
]. Thus, serum HSP60, which is usually an intracellular protein, can contribute to the inflammatory state seen in diabetes through multiple mechanisms.
Clinically, elevated HSP60 plasma levels have been found to correlate with increased carotid stiffness in middle-aged individuals [33
]. Most significantly, antibodies to HSP60, either mammalian or bacterial (HSP65) were found to correspond to increased intima-media thickness (IMT) in young males (17-18 years.) [34
]. These studies support a role for HSP60 and anti-HSP60 antibodies in atherosclerotic disease in the nondiabetic. Correlation may be more difficult to identify in older individuals as a multitude of factors, which increase with age, and contribute to the progression of established atherosclerotic disease.
4.1. HSP60 Antibodies
A very high titer of HSP60 antibodies was present in our patients, and it did not change with optimization of metabolic control. A number of studies have implicated HSP60 antibodies in endothelial apoptosis and dysfunction [35
]. A recent study demonstrated using a randomized double-blind, placebo-controlled cross-over design that simvastatin could lower anti-HSP60 antibody titers [37
]. Only 15% of these patients were diabetic, and the anti-inflammatory properties of simvastatin may have been a critical attribute for reduction in anti-HSP60 antibodies [38
]. High titers of anti-HSP60 antibody were still present after months of treatment, and these antibodies can interact with HSP60 on the surface of endothelial cells, leading to monocyte recruitment and further inflammation.
Six different cytokines were measured, and of these only IL-6 was significantly reduced after intensive treatment. This could be secondary to the effect of reduced glucose levels on the inflammatory state of endothelium. However, IL-6 also reflects the low-grade chronic inflammatory state that characterizes diabetes per se, the increased level of insulin resistance, and the increase in visceral adipose tissue, all of them present in the metabolic syndrome [39
]. The observed decrease in IL-6 could be due to the reduction of blood glucose as well as of BMI, waist circumference and insulin resistance reached by our patients. This study is one of the few that show this effect in diabetic patients in a context of metabolic syndrome. IL-8 levels were increased, but the decrease in IL-8 with treatment did not reach significance. Other cytokines including TNFα
, IL-10, and IL-12 did not change with diabetes control. Growing evidence supports a cause-effect relationship between systemic inflammation related to diabetes or obesity, and endothelial inflammation. This is important, as systemic inflammation may make plaques unstable and rupture prone [1
]. Therefore, any reduction of the systemic inflammation could have a positive effect on atherosclerosis-related disease.
compares serum cytokine levels measured at baseline in the current study with recent studies in the literature. These studies covered different populations from different countries. All patients were type-2 diabetics, but the duration of diabetes varied from newly diagnosed to 10 years. Little to no IL-1β
was detected in any of the studies [24
]. IL-6 levels ranged from undetectable in one study to over 38
]. The IL-6 levels for the patients in the current study were in the middle of this range. IL-8 levels in the literature were split with two groups finding levels less than 10
pg/mL, and two other studies, including the current one, finding levels of just over 100
]. Similarly, IL-10, which was only measured in three studies, was very low in one, 3.78 (current study) and 8.13
pg/mL in the other studies [27
]. IL-12 levels were only measured by Mishra et al. with these investigators finding 100 times the levels found in the patients in the current study [28
levels also had a wide range, but most of the studies, including the current one, detected levels between 0.065 to 6.6
pg/mL, while the remaining 3 had average serum values as high as 50.8
]. Thus, blood cytokine levels vary widely in diabetic populations.
Summary of serum cytokine values reported from recent studies.