Circulating endothelial progenitor cells and large artery structure and function in young subjects with uncomplicated Type 1 Diabetes

Type 1 diabetes mellitus (T1DM) is a major risk factor for cardiovascular disease as the incidence of cardiovascular complications in T1DM patients is reported to be 2- to 10-fold higher than in normal population [1]. Clinically overt diabetes-related vascular complications are rare in childhood and adolescence, and an optimal glycemic control at earlier age and stage of the disease may attenuate the development and progression of functional and structural alterations in the arterial tree [2, 3]. It is therefore important to identify preclinical vascular changes at a very early stage in order to improve glycemic control and reduce the risk of later cardiovascular complications. A number of noninvasive measurements, such as endothelium-dependent flow-mediated vasodilation (FMD), common carotid artery intima-media thickness (C-IMT), carotid-femoral pulse velocity (PWV, an index of aortic stiffness) and carotid augmentation index (AIx, an index of arterial pressure wave reflection) have been proposed as "tissue biomarkers" capable to improve risk stratification and tracking early atherosclerotic disease beyond the simple determination of established risk factors [4].

Endothelial dysfunction has been suggested to anticipate structural changes in large artery wall in young T1DM patients [5], in keeping with the hypothesis that functional abnormalities of the endothelium represent not only an early marker of atherosclerosis but above all a pathophysiologic mechanism promoting the development of arterial wall thickening and stiffening [6]. This hypothesis was further confirmed in a recent study on young T1DM patients [7] that demonstrated a severe impairment of FMD, important reduction in bone marrow derived circulating endothelial progenitor cells (EPCs), moderate increase in C-IMT and an inverse relationship between FMD and C-IMT. C-IMT has been extensively evaluated in children and adolescents with T1DM, and an increased C-IMT has been reported not only in subjects with suboptimal metabolic control [8] but also in those with satisfactory insulin treatment and with a relatively short diabetes duration (5.5 years) [9–11]. On the other hand, reports on large artery stiffness in young diabetic patients are still limited and indirect, based mainly on applanation tonometry [12, 13].

Various metabolic abnormalities with atherogenic potential, like fasting and post-prandial high plasma glucose levels, increased advanced glycated endproducts (AGEs) and post-secretory modified LDL particles, may be accountable for accelerated development and progression of vascular organ damage in T1DM [14–17]. The possible role of adiponectin in T1DM-related vascular changes [18] requires further elucidation, as plasma levels of this atheroprotective adipokine have been reported increased in young peoples with T1DM [19, 20].

To identify a sensitive and early marker of vascular organ damage in T1DM and to provide some insight on mechanisms underlying these early vascular changes, the present study compared several different indices of vascular structure and function between T1DM adolescents and healthy controls of similar age, and it also tested the relationships between the vascular measures and circulating EPCs, glyco-metabolic control, serum levels of AGEs, soluble receptors for AGEs (sRAGE) and adiponectin.

Vascular parameters in T1DM and controls are reported in Figure 1. After adjustment for sex, age and mean BP, T1DM adolescents had higher central PP, AIx, carotid-femoral PWV as well as local carotid WS and C-IMT. Peripheral endothelium-dependent vasodilator capacity (RHI score) did not differ significantly between the 2 groups, although it was lower in T1DM patients with HbA1c ≥7.5% as compared to those with HbA1c < 7.5% (1.5 ± 0.4 vs. 2.2 ± 0.8, p < 0.05) and decreased with diabetes duration (r = -0.54, p < 0.05).

In univariate analysis, with increasing fasting plasma glucose increased central PP, AIx, C-F PWV and carotid WS. With increasing HDL-cholesterol decreased central PP and C-IMT, whereas with increasing LDL-cholesterol increased C-F PWV. With increasing plasma adiponectin decreased AIx and C-IMT, which was also inversely related to EPCs (Table 2). Triglycerides, AGEs and sRAGEs did not correlate with any parameter of vascular structure or function. In successive multivariate models, fasting plasma glucose resulted as an independent determinant of central PP, AIx and carotid WS. LDL-cholesterol was an independent determinant of C-F PWV. Adiponectin was an inverse and independent correlate of AIx and C-IMT, and EPCs were inversely and independently correlated with C-IMT (Table 3). When diagnosis of T1DM was included into the multiple regression model of central PP, AIx and carotid WS, it replaced fasting plasma glucose as an independent determinant of these vascular measures. In regression model of C-IMT, T1DM diagnosis replaced EPCs and adiponectin.

We also evaluated the possible inter-relationships between structural and functional vascular measures. In the entire study population, C-IMT was directly related to AIx, C-F PWV and carotid WS (r = 0.51, 0.30 and 0.31, respectively, p at least < 0.05), and inversely to RHI score (r = -0.34, p < 0.05)

In recent years, particular attention has been paid to the preclinical alterations in the vascular system in diabetic children and adolescents in order to prevent development of overt atherosclerosis and clinical events in the adulthood. The high risk of atherosclerosis in diabetes is mainly attributed to endothelial dysfunction that results both from endothelial cell damage and impaired endothelial repair [29]. Bone-marrow derived circulating EPCs are believed to contribute to endothelial repair, to vascular homeostasis and to compensatory angiogenesis [30], and their decrease in diabetes was shown to closely correlate with the severity of carotid atherosclerosis and progression of diabetic complications [31, 32]. Endothelial dysfunction is considered not only the first marker of vascular damage but also a pathophysiologic mechanism promoting further structural changes within the arterial wall [6].

The results of our study confirm and extend this hypothesis. Our young T1DM patients free of overt clinical complications had, as compared to controls of similar age, a significantly reduced number of specific CD34⁺KDR⁺ EPCs, and, in entire study population, EPCs count was inversely and independently related to C-IMT. These findings are in agreement with previous data in adults [7, 33–35], as well as with assumptions that the abnormalities of endothelial function precede the development of clinically evident arterial damage and that an impaired regenerative capacity of the endothelium yields an increased risk of developing early atherosclerotic changes [35]. Together with a low EPCs count, a low plasma adiponectin was another independent determinant of increased C-IMT in our young population. An inhibitory effect of adiponectin against excessive intimal growth was previously reported in experimental carotid injury [36], and a large cross-sectional study in healthy subjects has proposed a low adiponectin as a risk factor for early atherosclerosis [37]. Although our data confirmed a protective role of adiponectin against early carotid atherosclerosis, they did not confirm the increase in plasma adiponectin levels in T1DM patient that has been described by other authors [19, 20]. This discrepancy can be explained by a better glycemic control of our patients, since Barnes el al. [20] have demonstrated, in a large population of 440 young T1DM subjects, a positive correlation between adiponectin and HbA1c. Mean HbA1c of our patients (7.7%) was lower that that of young T1DM subjects (9.8%), in which an increase in plasma adiponectin levels has been demonstrated [19].

Chronic glycemic control might also affect endothelial function. Although RHI score, an index of peripheral endothelium-dependent vasodilator capacity, was not decreased in our T1DM patients as compared to controls, it was significantly lower in patients with HbA1c ≥7.5% than in those with HbA1c < 7.5%. Furthermore, the glycemic control of T1DM children and adolescents in studies that described an impairment of brachial artery flow-mediated dilation and microvascular flow-mediated vasodilatation [7, 38, 39] was worse than in our group (HbA1c 8.9, 8.5 and 9.4 vs. 7.7%).

Data on large artery stiffness in children and adolescents with T1DM are more sporadic and report above all an increase in AIx [12, 13]. AIx is an integrated measure of stiffness and reflection from the periphery, and thus it does not represent a proper estimate of large artery function [26]. It has been suggested that an augmented pressure wave reflection in diabetes is a consequence of combined changes of both small resistance arteries and large arteries [40]. Increased radial artery AIx has been demonstrated in T1DM children as young as 10 years of age [12], and increased carotid artery AIx has been observed in children and adolescents with relatively short duration of diabetes (5.4 ± 3.4 years) and poor glycemic control (HbA1c 9.8%) [41]. Recent data from the SEARCH of Diabetes in Youth Study have confirmed an increased AIx in T1DM children together with a decrease of brachial artery distensibility [42]. In our study, young T1DM patients showed increased local carotid stiffness, aortic stiffness and wave reflection, which together resulted in a significantly higher central pulse pressure despite similar blood pressure values at brachial artery level. In addition, in the entire study population, local carotid stiffness, AIx and pulse pressure were independently associated with fasting plasma glucose, a finding that confirms data regarding the effect of acute hyperglycemia on arterial stiffness of T1DM patients [43] and emphasizes the importance of a strict glycemic control. Interestingly, carotid-femoral PWV was not independently related to plasma glucose but to LDL-cholesterol. The different impact of metabolic factors on aortic and carotid stiffness implies that the two vascular districts may be not interchangeable in predicting cardiometabolic risk [44].

It must be also taken in consideration that increased local carotid thickness, stiffness and wave reflection may ultimately results in a positive feedback loop, promoting further progression of cardiovascular disease. The altered local mechanical properties of the carotid artery could play a role in a baroreflex gain reduction that may increase the susceptibility to develop hypertension [45], and an increased central pulse pressure may represent a mechanism accelerating intima-media thickening over the time [46].

The results of this study suggest that young subjects with relatively long-lasting T1DM and free of overt clinical complications, have a significantly lower count of circulating EPCs and a generalized preclinical involvement of large artery structure and function as compared to healthy controls. An increase in C-IMT is independently related to decrease in circulating EPCs and to lower plasma adiponectin, whereas impairment of peripheral flow-mediated dilation seems to be associated with worse chronic glycemic control. Local carotid stiffness and pressure wave reflection are mainly determined by fasting plasma glucose, while aortic stiffness is depending on plasma LDL-cholesterol. From a clinical point of view, our findings highlight the need of a more strict control of glycemic levels, considering also the fact that hyperglycemia represents a primary contributor to the reduced number and impaired function of EPCs in diabetes [18, 30]. Although our T1DM young patients had a better glycemic control than that found in the adolescent cohort of the Diabetes Control and Complications Trial (a mean HbA1c of 8.1%,) [3], various patients showed an average HbA1c still above the recently indicated target of HbA1c below 7.5% [47]. Finally the radiofrequency-based tracking of arterial wall, which allows an accurate real-time determination of the basic blood vessel wall properties, may facilitate and anticipate the detection of early vascular organ damage in young T1DM subjects [48].