Study design and Patients: This is an observational study including outpatients with T1D who were monitoring glucose by CGM or by meters uploading glucose data into the Accu-Chek Connect diabetes management system (DMS). Exclusion criteria were: known cardiovascular disease (myocardial infarction, angina, stroke, TIA, peripheral arterial disease), use of vasoactive drugs as calcium channel blocker, nitrates, beta and alfa blockers, use of hybrid closed-loop system or (predictive) low glucose suspend, concomitance of diseases such as infection, surgery, and any acute event requiring intensification of insulin treatment, treatment with corticosteroids, and pregnancy. The research was approved by the local Ethical Committee, and only patients who gave their informed consent were enrolled in the study. Fasting plasma glucose (FPG) and HbA1c were measured at the time of the recruitment. HbA1c was measured by high-performance liquid chromatography aligned with DCCT (HbA1c-DCCT); FPG was measured by commercially available kits. Disease duration and ongoing treatment were obtained from clinical files. As controls, age, and sex, comparable healthy subjects without diabetes were recruited.
Novel glucose parameters TIR, TAR, TBR, mean daily glucose, and standard deviation were collected 2 weeks and 3 months before the vascular study by DMS uploading and analyzing CGM glucose data. The most recent 2 weeks are a sufficient collection interval evaluating the overall time spent in the target range. A more extended collection period offers a more robust evaluation of time spent in hypoglycemia and glycemic variability (17).
Vascular study: The study was performed in the morning using an echo-Doppler Philips HD 11XE (Royal Philips Electronics, the Netherlands) equipped with a 12–3 MHz linear array transducer and simultaneous ECG recording, in a temperature-controlled (20-24°C) room, and after the patient was at rest for 10 minutes in the supine position. Participants were asked to abstain from exercise, alcohol, caffeine, food, and smoking for 12 h before the study.
Participants were invited to extend the neck to visualize the common carotid artery (CCA) 1 cm below the carotid bulb in three different projections, anterior, lateral, and posterior. The transducer position was then adjusted to obtain an angle between the ultrasound beam and the longitudinal axis of the vessel at 90°. The gain was fine-tuned to improve the image of the intima plus media complex of the far wall defined as the distance between the leading edge of the lumen-intima interface and the inner edge of the media-adventitia interface. Images were then recorded for the offline measurement of intima-media thickness (IMT) using a dedicated software (Autodesk® Design Review, BSA Italy) (23).
IMT was measured at the right and left CCA as the mean of the three projections. The maximal value between those measured at the right and left CCA was used for statistical analyses and defined as mean maximal IMT of CCA.
Eleven participants (6 with T1D and 5 healthy subjects) were studied twice apart to estimate the intra-operator reproducibility of the IMT measurement expressed as the coefficient of correlation between two measurements (0.98).
The endothelial function of the brachial artery was evaluated in the non-dominant arm by two different tests using ischemia and exercise as stimuli (24,25). Both stimuli cause a reduction of peripheral resistance downstream to the brachial artery that, in turn, causes an increase of brachial artery blood flow velocity. This phenomenon, defined as reactive hyperemia, is responsible for the following brachial artery dilation known as flow-mediated dilation (FMD). The magnitude of FMD is proportional to the ability of the endothelium to dilate in response to stimuli. FMD was expressed as the percentage change of brachial artery diameter from baseline to the end of the test. Brachial artery internal diameter (ID) was defined as the distance between the intima-lumen interface of the near-wall and lumen-intima interface of the far wall. The two tests were performed on the same day at least 30 min apart, and the second test was not performed until the baseline brachial artery diameter was restored. In both tests, the brachial artery was imaged ~3-4 cm above the elbow in the longitudinal section on the anterior side of the biceps muscle keeping the angle between the ultrasound beam and the vessel at 90°. The gain was fine-tuned until the lumen-intima interface was clear. Images were recorded for offline measurement by the software Autodesk1 Design Review (13).
Ischemic test was carried out by inflating a pneumatic cuff around the forearm up to 250 mmHg and maintaining inflation for 5 min. Brachial artery ID was recorded at baseline, 1, 2 min, and 3 min after cuff release.
An exercise test was performed using the handgrip manometer. Participants were instructed on how to perform a maximal voluntary contraction (MVC) and a short bout of isometric handgrip exercise in a 2s contraction:3s relaxation ratio with the non-dominant arm. The exercise test consisted of 12 contractions min-1 for a total of 6 min of handgrip exercise at 30% intensity of MVC. MVC was evaluated at the beginning of the visit to avoid any interference with vascular tests. The duration and the intensity of the exercise were preliminarily established in healthy volunteers. Six min and 30% of MVC represented the maximal contraction, ensuring an adequate vasodilation without systemic hemodynamic changes (heart rate and blood pressure) and pain or fatigue.
FMD after ischemia was calculated using the following formula: [(after ischemia ID)-(baseline ID)/(baseline ID)] ×100. The highest FMD among those calculated at 1, 2, and 3 min after cuff deflation was defined as peak FMD. FMD during exercise was expressed as percentage change from baseline and calculated using the following formula: [(1-2-3-4-5-6- min exercise ID)-(baseline ID)/(baseline ID)] ×100.
The reproducibility of the FMD test after ischemia and exercise was calculated in 6 patients with T1D and 5 healthy subjects. Vascular studies were performed on two different days and in the same condition. The coefficient of correlation between the first and the second ischemic test was 0.99, and between the first and the second exercise test 0.98.
Statistical analyses: All tests were performed using SPSS 23 for Microsoft (SPSS, Inc., Chicago, IL). Variables not normally distributed were time spent in hypoglycemia, and GV; non-parametric tests or 2-Step Rank transformation before applying parametric tests were used. The sample size was calculated using the correlation sample size formula for one group: N=[(Za+Zb)/C]2+3, where Za=1.96, Zb=0.842, a=0.05 and b=0.20. Currently, no data are evaluating the association between glucose metrics and preclinical atherosclerosis. Therefore, the expected correlation between glucose metrics and IMT, FMD in T1D patients we used in the formula was 0.35 and set according to data concerning the association between IMT, FMD, and age or disease duration (12). The total calculated number with an anticipated drop-out=20% was 78.
Parameters were analyzed in T1D patients and patients divided according to the instrument used to monitor blood glucose (SMBG and CGM group) or according to ongoing treatment [continuous subcutaneous insulin infusion (CSII) and multiple daily insulin injection (MDI) group]. TIR, TAR, mean daily glucose, and standard deviation (SD) were collected from the diabetes management software. GV was expressed as the coefficient of variation (CV) calculated using the following formula: CV=(SD/mean daily glucose). The t-test for paired data and Mann-Whitney U for unpaired data were used to compare continuous variables between T1D and control group and between SMBG and CGM group, and between CSII and MDI group. The Chi-squared test was used to compare the percentage between groups. Repeated measures ANOVA test was applied to evaluate differences among arterial dilation detected for every min up to 6 min of handgrip exercise in T1D and control group. The one-way ANOVA was used to compare arterial dilation during exercise between SMBG and CGM groups, and between CSII and MDI groups. The simple regression analysis was performed to evaluate the association between mean maximal IMT, FMD (peak FMD and FMD at the 6th min of exercise) and age, disease duration, and glucometric parameters; results have been shown as coefficient of correlation and square of correlation.