Role of chronic hyperglycemia in the pathogenesis of coronary microvascular dysfunction in diabetes

1.1 Study population

We studied 35 subjects with DM (18 with type 1 and 17 with type 2 diabetes) (age 42 ± 7 years), who were free of overt cardiovascular complications (age 24 to 52 years) (Table 1). Patients were classified as either type 1 or 2 DM based on standard criteria used including C-peptide levels, age at onset of diabetes, and a history of ketosis (7). The average duration of diabetes for the group was 14 ± 12 years. Eleven age-matched subjects without diabetes served as healthy controls. Each subject was evaluated with a careful history, physical examination, resting electrocardiogram (ECG), and maximal treadmill exercise test echocardiography. We included diabetic subjects fulfilling the following inclusion criteria: 1) no clinical evidence of heart disease (i.e., angina or heart failure symptoms); 2) a negative maximal treadmill exercise test; 3) no ischemic changes or left ventricular hypertrophy on resting ECG or two-dimensional echocardiography; 4) no evidence of cerebrovascular or peripheral vascular disease; 5) no history of more than mild hypertension (blood pressure [BP] <160/95 mm Hg); 6) no overt nephropathy (serum creatinine <1.4 mg/dl); and 7) a glycohemoglobin level <12%. Subjects with a history of cardiomyopathy or valvular heart disease or active smoking were excluded. Ten diabetics had evidence of mild-to-moderate retinopathy. Eight subjects with type 2 diabetes were receiving insulin treatment. All subjects in the study had a low probability of significant obstructive CAD based on the absence of cardiovascular symptoms, a normal resting and maximal exercise ECG, and echocardiography.

1.2 Study design

The Human Investigation Committee of Wayne State University approved the study protocol, and all participants gave written informed consent. Each subject made two visits to the study hospital, during which time cardiac sympathetic nerve function and myocardial blood flow (MBF) were assessed. Cardiac sympathetic innervation and MBF were evaluated using a whole-body positron emission tomography (PET) (Siemens/CTI EXACT HR, Knoxville, Tennessee).

All subjects refrained from caffeine-containing beverages or theophylline-containing medications for 24 h before each hospital visit. Two diabetic subjects were receiving a calcium channel blocker for mild hypertension, and eight subjects were receiving a low-dose angiotensin-converting enzyme inhibitor. None of the subjects were using nitrates or beta-blockers. These medications were discontinued 24 h prior to PET imaging. None of the subjects received medications known to interfere with catecholamine uptake in presynaptic nerve terminals. All subjects were studied in the fasted state.

1.3 PET imaging

1.4 Laboratory analyses

Venous plasma and serum samples were taken after an overnight fast. Plasma glucose was measured by the glucose oxidase method. Serum cholesterol and triglyceride concentrations were measured using standard enzymatic methods. High-density lipoprotein (HDL) cholesterol was measured with the Equal HDL Direct Method and the Technicon DAX System (Bayer, Tarrytown, New York). Low-density lipoprotein cholesterol was calculated using the Friedewald formula (8). The von Willebrand factor (vWF) antigen (a marker of endothelial cell damage) was measured by immunoelectrophoresis. Glycohemoglobin level was measured by high-performance liquid chromatography (4% to 8%).

1.5 Statistical analysis

Data are presented as mean ± SD. Baseline characteristics of patients between groups were compared using single factor analysis of variance (ANOVA). Significant ANOVAs were followed with Tukey post-hoc tests to identify differences between groups. To compare systemic hemodynamics, MBF, and myocardial vasodilator reserve across subject groups (i.e., type 1 diabetics, type 2 diabetics, and controls) and conditions (i.e., baseline, CPT, and hyperemia), separate 3×3 mixed design ANOVAs were run where group was the between subjects factor and condition was the repeated measures factor. For the outcomes of systolic BP and mean aortic BP a doubly multivariate 3×3 mixed design ANOVA was run due to the high intercorrelation of these two measures. Multivariate tests are reported for main effects and interactions involving the repeated measures factor. When the sphericity assumption was violated the Greenhouse-Geisser correction was used for the error term and degrees of freedom for the post-hoc tests.

To compare coronary flow reserve across the subject groups a single factor ANOVA was performed. All significant main effects for condition were followed with Tukey post-hoc tests to assess changes (from baseline) during the CPT and peak hyperemia. Significant main effects for group were followed with Tukey post-hoc tests comparing each of the groups. Significant group by condition interactions were followed with simple effects tests using a Tukey correction to assess differences across the conditions within each subject group and differences across the subject groups within each condition (9). Independent predictors of changes in MBF in response to adenosine and the CPT were investigated using multiple regression analysis. For all analyses an alpha of 0.05 was used to define statistical significance. Values of p < 0.1 also are reported as they were taken to indicate trends toward significance.

2.1 Baseline characteristics

Table 1summarizes the baseline characteristics of the study patients. As expected, the disease duration was longer in the subjects with type 1 than among those with type 2 diabetes. Body mass index (BMI) was higher in the subjects with type 2 compared with those with type 1 diabetes and the controls. Baseline glucose was lower in the controls as compared with both groups of diabetics. However, glycemic control was comparable in both groups of diabetics. Additionally, HDL cholesterol was lower and cholesterol/HDL cholesterol ratio was higher in the subjects with type 2 diabetes.

2.2 Systemic hemodynamics

Analyses of the systemic hemodynamics revealed significant main effects by condition (adenosine or CPT) for all measures (heart rate F[2, 42] = 218.13, p < 0.001; overall systolic BP and mean aortic BP F[4, 172] = 20.35, p < 0.001; and the rate-pressure product F[2, 42] = 89.44, p < 0.001), and main effects by subject group for three of the measures (systolic BP F[2, 43] = 7.94, p = 0.001; mean aortic BP F[2, 43] = 4.76, p = 0.014; and the rate-pressure product F[2, 43] = 3.41, p = 0.042) (Table 2). Across all three conditions (baseline, adenosine, and CPT), type 2 diabetics had significantly higher systolic BP, mean aortic BP, and rate-pressure product as compared with controls. Likewise, type 1 diabetics had significantly higher systolic BP and marginally higher mean aortic BP and rate-pressure product as compared with controls.

2.3 Predictors of changes in MBF in response to adenosine and the CPT

2.4 Regional MBF and coronary vascular resistance

2.4.2 MBF response to adenosine infusion

During hyperemia, MBF increased and coronary vascular resistance decreased significantly in the three groups (Table 3). However, the increase (from baseline) in MBF with adenosine was similar in the subjects with type 1 (161 ± 18%) and type 2 (185 ± 19%) diabetes, but significantly lower than in the controls (351 ± 43%) (p < 0.001 for the comparison with both groups of diabetics) (Fig. 1). Consequently, coronary vasodilator reserve was lower in the diabetics than in the controls. These differences persisted after adjusting for the duration of diabetes, blood glucose, BMI, insulin treatment, and lipid profile.

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Figure 1

Percent change in myocardial blood flow in response to the infusion of adenosine and the cold pressor test in the subjects with type 1 and type 2 diabetes and in the healthy controls. All p values are based on Tukey post-hoc tests.

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Table 3

MBF and Coronary Vascular Resistance in the Subjects With Diabetes and in the Healthy Controls

3.1 Comparison with previous studies

Our findings demonstrate striking similarities in the magnitude of sympathetically mediated coronary vasodilation between the subjects with type 1 and type 2 diabetes. The results of multivariable analysis assigned an important role to the degree of cardiac sympathetic dysfunction and the duration of diabetes in predicting the MBF response to the CPT, consistent with previous results from our laboratory (6). Importantly, plasma glucose was a statistically significant predictor of the flow response to the CPT that provided incremental information above and beyond that provided by the HED retention data and the duration of diabetes. Furthermore, the presence of type 1 (insulin deficiency) or type 2 (insulin resistance) diabetes had no significant effect on the MBF response to cold, even among those without cardiac sympathetic dysfunction. Together, these findings agree and extend previous studies in healthy volunteers (10)and in subjects with impaired glucose tolerance (11). Williams et al. (10)demonstrated that acute local hyperglycemia (achieved by infusion of 50% dextrose in the brachial artery) significantly attenuated the forearm blood flow response to methacholine in healthy nondiabetic humans, a finding that was independent of the systemic insulin concentration. Kawano et al. (11)showed impaired flow-mediated brachial artery dilation after an oral glucose loading in subjects with normal and impaired glucose tolerance.

Diabetes-associated hypertension and dyslipidemia may contribute to abnormalities in coronary vascular function in diabetes. However, except for HDL cholesterol levels the diabetic subjects in our study were matched for these parameters. More importantly, none of the lipoprotein fractions measured in this study were independent predictors of the changes in MBF in the multivariable analysis. Although the duration of diabetes was a marginally significant predictor of the MBF response to cold, the similarities in MBF between subjects with type 1 and type 2 diabetes persisted after adjusting for the duration of diabetes.

Potential mechanisms by which hyperglycemia induces vascular dysfunction include hyperglycemia-mediated formation of oxygen-derived free radicals and activation of protein kinase C. Free radicals inactivate endothelium-derived nitric oxide, thereby interfering with endothelium-dependent vasodilation (12,13). Indeed, endothelial dysfunction resulting from hyperglycemia in type 1 and type 2 diabetics can be improved by the short-term administration of the antioxidant vitamin C (14,15). Activation of protein kinase C by hyperglycemia has also been implicated in the development of endothelial dysfunction in diabetes, thereby contributing to vascular dysfunction (16). Mechanisms proposed to account for the effect of protein kinase C on vascular dysfunction include the increased production of vasoconstrictor prostanoids, changes in endothelial cell muscarinic receptors (16), activation of nuclear factor kappa-B with subsequent alterations in gene transcription (17), and possibly increased formation of oxygen-derived free radicals (16).

We also demonstrated a similar impairment of myocardial vasodilator reserve in our subjects with type 1 and type 2 diabetes. One possibility is that occult atherosclerosis might have attenuated the maximal flow response to adenosine. However, we deliberately studied young asymptomatic diabetics, all of whom had normal maximal stress echocardiography, and none showed regional defects on rest-stress perfusion imaging. These findings argue against flow-limiting epicardial coronary stenoses in our diabetic subjects (18). Although structural abnormalities in the coronary microcirculation in the diabetics may have contributed to the impaired vasodilator response to adenosine (19), such abnormalities have not been universally observed (20). Alternatively, the impaired vasodilator response to adenosine in the diabetics may be related in part to the presence of endothelial dysfunction. Although the augmentation in MBF with adenosine is caused primarily by direct interaction with A₂receptors on vascular smooth muscle leading to direct vasodilation (a mechanism that is endothelium-independent), a relatively small proportion of the vasodilator response to adenosine is mediated by the endothelial release of nitric oxide (21). The fact that the vWF antigen level (a marker of endothelial cell damage) was the only independent predictor of the impaired MBF response to adenosine provides support for this hypothesis. Nevertheless, the relatively small contribution of endothelial function to the overall increase in MBF in response to adenosine may explain the lack of correlation between plasma glucose and the coronary vasodilator reserve.

3.2 Study limitations

Plasma insulin and free fatty acid levels were not measured routinely in the diabetic subjects. Thus, their role as possible predictors of the MBF responses to adenosine or the CPT cannot be determined from this study. However, the lack of differences in myocardial blood flow between our type 1 (insulin deficient) and type 2 (hyperinsulinemic, insulin resistant) diabetics suggest that in disease states including diabetes mellitus, insulin levels do not appear to contribute significantly to coronary vasodilation.

3.3 Conclusions

The results of this study demonstrate markedly reduced myocardial vasodilator reserve and sympathetically mediated changes in MBF in subjects with both type 1 and type 2 diabetes. Importantly, the similarities in coronary vascular dysfunction between the subjects with type 1 and type 2 diabetes persisted after adjusting for expected baseline differences in the duration of diabetes, BMI, insulin treatment, lipid profile, and autonomic neuropathy. Because patients with type 1 diabetes are insulin-deficient (rather than insulin-resistant, the hallmark of type 2 diabetes), these results provide further support for a key role of hyperglycemia in the pathogenesis of vascular dysfunction in diabetes.