T2D-Associated Carotid Plaque Burden and Retinopathy
T2D-Associated Carotid Plaque Burden and Retinopathy
T2D patients were selected to participate in this single-centre study, which aimed to investigate the association between carotid atherosclerosis and DR in T2D. All study participants were recruited from the outpatient clinic at our centre, and potential candidates were identified by screening patients enrolled in the diabetic eye disease programme of our centre as well as those visiting the diabetic retinopathy unit. The inclusion criteria for both groups were as follows: age range, 40–75 yrs; absence of established impaired renal function (calculated glomerular filtration rate (eGFR) <60 ml/min); and known CVD. From the total number of subjects who were initially recruited, 60 subjects were excluded after the screening visit because of the presence of an exclusion criterion (previous CVD, impaired renal function, increased urine albumin excretion, or not having T2D), and 15 additional patients withdrew from the study after the initial screening visit (consented withdrawal before completing the study assessment). A total of 312 participants with (n = 153) or without (n = 159) DR were included in the study. To achieve a uniform age distribution, patients with and without DR were selected to have representative and similar numbers of patients by gender and age (stratified according to 5-yr age intervals). However, we could not recruit enough patients with retinopathy in the age range between 40 and 50 yrs. For the purpose of this study, a patient was arbitrarily considered to have previous hypertension or dyslipidaemia if the patient was taking medication for the given condition. The weight, height and waist circumference of the subjects were measured using standardised methods, and the blood pressure (mean of 2 measurements, 5 min apart) of the subjects was measured after 10 min in the seated position using a blood pressure monitor (HEM-7001E, Omron, Barcelona, Spain). The patients underwent a complete eye evaluation by experienced ophthalmologists (AT and CJ) to assess the presence or absence of diabetic retinopathy according to an international clinical DR consensus. Retinopathy was evaluated using multifield stereoscopic retinal photography with the following definitions: a) mild nonproliferative DR - microaneurysms only; b) moderate nonproliferative DR - more than just microaneurysms but less than severe nonproliferative DR; c) severe nonproliferative DR by any of the following - more than 20 intraretinal haemorrhages in each of 4 quadrants, definite venous beading in 2+ quadrants, prominent intraretinal microvascular abnormalities in 1+ quadrant, and no signs of proliferative retinopathy; and d) proliferative DR, by neovascularisation and/or vitreous/preretinal haemorrhage. In addition to the anamnestic evaluation and physical examination, the patients' clinical records were reviewed to rule out any previously known CV events or associated revascularisation procedures, including coronary heart disease, cerebrovascular disease, or peripheral vascular disease (including the diagnosis of diabetic foot disease). Ours is the only reference hospital for CVDs and procedures in the region, but we also had access to reports from three small hospitals in the region. Therefore, along with the anamnestic evaluation, we could ascertain any CV events that occurred in the health-care area. Heart failure and macroalbuminuria (urine albumin/creatinine ratio >299 mg/g) were also considered exclusion criteria. Serum and spot urine samples were collected in the fasting state, and all serum and urine tests were performed using standard laboratory methods. Low-density lipoprotein cholesterol was estimated using the Friedewald formula, and eGFR was estimated using the diet modification in renal disease (MDRD-4) formula. Haemoglobin A1c (HbA1c) levels were determined using HPLC (Variant, Bio-Rad Laboratories SA, Spain), and its concentrations are expressed in National Glycohemoglobin Standardization Program/Diabetes Control and Complications Trial units. Urine albumin was measured using an immunoturbidimetric method and a Roche/Hitachi Modular P analyser (Roche Diagnostics, Spain).
All of the study participants underwent the same carotid ultrasound protocol. All measures and ultrasound studies were assessed by the same researcher (ER), who was blinded to the conditions of the participants and did not have access to the study data. B-mode ultrasound imaging was performed using a Siemens Sequoia 512 and a 15-Mhz linear array probe. Moreover, a standardised imaging protocol was performed to evaluate intima-media-adventitia thickness (IMT), defined as the distance between the lumen-intima and the media-adventitia ultrasound interfaces (intima-media complex), and plaque presence in the carotid arteries. The patients were examined from their back; they were placed in the supine position with the head turned 45° contralateral to the side of scanning. Images were obtained in longitudinal sections, with a single lateral angle of insonation and optimisation of the image to the far wall. The B-mode images of the left and right segments were recorded and electronically stored. The last (previous to the bulb) and first (starting at the flow divider) centimetres of the common and internal carotid arteries, respectively, and the total length of the bulb were used for IMT measurements, which were performed off-line using semiautomatic software. The data (mean IMT and mean-maximum IMT) from each segment were provided, and the right- and left-side values were averaged to obtain the mean and mean-maximum common carotid (CC), carotid bulb, and internal carotid (ICA) measurements. Plaques were identified using B-mode and colour Doppler examinations in both the longitudinal and transverse planes to consider circumferential asymmetry and were defined as a "focal structure that encroaches into the arterial lumen of at least 0.5 mm or 50% of the surrounding cIMT value or demonstrates a thickness of 1.5 mm, as measured from the media adventitia interference to the intima-lumen surface" according to the Mannheim consensus.
The local Ethics Committee of Hospital Arnau de Vilanova (Lleida, Spain) approved the protocol, and all of the patients signed written informed consent forms.
To determine the sample size, we used preliminary data from a previous study of our group in which the plaque frequency was 69% in patients with retinopathy or 52% in patients without retinopathy. We calculated a sample size of 126 subjects for each study group, which would allow an 80% power to detect differences between groups with a significance level of <0.05. Thus, the number of patients included in each group was sufficient to test the initial hypothesis.
The data are presented as the median values and 25th and 75th percentiles, means ± standard deviations (SDs), and n (%), as appropriate. Non-normally distributed variables were log transformed to reduce skewness, and normality was re-evaluated. Between-group (DR vs. non-DR, male vs. female, and with hypertension vs. without hypertension, or dyslipidaemia, or smoking habit) differences in anthropometric, clinical, cIMT, and laboratory variables were evaluated using the chi-squared test (for categorical variables), Wilcoxon test (for continuous, non-normally distributed variables), or Student's t-test (for continuous, normally distributed variables). Unadjusted and adjusted general linear models (PROC GLM in SAS) were used to test whether cIMT values differed between diabetic patients with retinopathy vs. those without retinopathy. Univariate or multiple logistic regression models were used to evaluate the differences in plaque presence between the groups. The following factors were entered into the adjusted general linear and multiple logistic regression models: age, sex, body mass index, presence of hypertension, presence of dyslipidaemia, smoking habit, urinary albumin excretion rate and eGFR. The significance level was defined as p ≤ 0.05, and all analyses were performed using SAS software, v. 9.2 (SAS Institute Inc., USA).
Methods
Subjects
T2D patients were selected to participate in this single-centre study, which aimed to investigate the association between carotid atherosclerosis and DR in T2D. All study participants were recruited from the outpatient clinic at our centre, and potential candidates were identified by screening patients enrolled in the diabetic eye disease programme of our centre as well as those visiting the diabetic retinopathy unit. The inclusion criteria for both groups were as follows: age range, 40–75 yrs; absence of established impaired renal function (calculated glomerular filtration rate (eGFR) <60 ml/min); and known CVD. From the total number of subjects who were initially recruited, 60 subjects were excluded after the screening visit because of the presence of an exclusion criterion (previous CVD, impaired renal function, increased urine albumin excretion, or not having T2D), and 15 additional patients withdrew from the study after the initial screening visit (consented withdrawal before completing the study assessment). A total of 312 participants with (n = 153) or without (n = 159) DR were included in the study. To achieve a uniform age distribution, patients with and without DR were selected to have representative and similar numbers of patients by gender and age (stratified according to 5-yr age intervals). However, we could not recruit enough patients with retinopathy in the age range between 40 and 50 yrs. For the purpose of this study, a patient was arbitrarily considered to have previous hypertension or dyslipidaemia if the patient was taking medication for the given condition. The weight, height and waist circumference of the subjects were measured using standardised methods, and the blood pressure (mean of 2 measurements, 5 min apart) of the subjects was measured after 10 min in the seated position using a blood pressure monitor (HEM-7001E, Omron, Barcelona, Spain). The patients underwent a complete eye evaluation by experienced ophthalmologists (AT and CJ) to assess the presence or absence of diabetic retinopathy according to an international clinical DR consensus. Retinopathy was evaluated using multifield stereoscopic retinal photography with the following definitions: a) mild nonproliferative DR - microaneurysms only; b) moderate nonproliferative DR - more than just microaneurysms but less than severe nonproliferative DR; c) severe nonproliferative DR by any of the following - more than 20 intraretinal haemorrhages in each of 4 quadrants, definite venous beading in 2+ quadrants, prominent intraretinal microvascular abnormalities in 1+ quadrant, and no signs of proliferative retinopathy; and d) proliferative DR, by neovascularisation and/or vitreous/preretinal haemorrhage. In addition to the anamnestic evaluation and physical examination, the patients' clinical records were reviewed to rule out any previously known CV events or associated revascularisation procedures, including coronary heart disease, cerebrovascular disease, or peripheral vascular disease (including the diagnosis of diabetic foot disease). Ours is the only reference hospital for CVDs and procedures in the region, but we also had access to reports from three small hospitals in the region. Therefore, along with the anamnestic evaluation, we could ascertain any CV events that occurred in the health-care area. Heart failure and macroalbuminuria (urine albumin/creatinine ratio >299 mg/g) were also considered exclusion criteria. Serum and spot urine samples were collected in the fasting state, and all serum and urine tests were performed using standard laboratory methods. Low-density lipoprotein cholesterol was estimated using the Friedewald formula, and eGFR was estimated using the diet modification in renal disease (MDRD-4) formula. Haemoglobin A1c (HbA1c) levels were determined using HPLC (Variant, Bio-Rad Laboratories SA, Spain), and its concentrations are expressed in National Glycohemoglobin Standardization Program/Diabetes Control and Complications Trial units. Urine albumin was measured using an immunoturbidimetric method and a Roche/Hitachi Modular P analyser (Roche Diagnostics, Spain).
Carotid Ultrasound Imaging Study
All of the study participants underwent the same carotid ultrasound protocol. All measures and ultrasound studies were assessed by the same researcher (ER), who was blinded to the conditions of the participants and did not have access to the study data. B-mode ultrasound imaging was performed using a Siemens Sequoia 512 and a 15-Mhz linear array probe. Moreover, a standardised imaging protocol was performed to evaluate intima-media-adventitia thickness (IMT), defined as the distance between the lumen-intima and the media-adventitia ultrasound interfaces (intima-media complex), and plaque presence in the carotid arteries. The patients were examined from their back; they were placed in the supine position with the head turned 45° contralateral to the side of scanning. Images were obtained in longitudinal sections, with a single lateral angle of insonation and optimisation of the image to the far wall. The B-mode images of the left and right segments were recorded and electronically stored. The last (previous to the bulb) and first (starting at the flow divider) centimetres of the common and internal carotid arteries, respectively, and the total length of the bulb were used for IMT measurements, which were performed off-line using semiautomatic software. The data (mean IMT and mean-maximum IMT) from each segment were provided, and the right- and left-side values were averaged to obtain the mean and mean-maximum common carotid (CC), carotid bulb, and internal carotid (ICA) measurements. Plaques were identified using B-mode and colour Doppler examinations in both the longitudinal and transverse planes to consider circumferential asymmetry and were defined as a "focal structure that encroaches into the arterial lumen of at least 0.5 mm or 50% of the surrounding cIMT value or demonstrates a thickness of 1.5 mm, as measured from the media adventitia interference to the intima-lumen surface" according to the Mannheim consensus.
The local Ethics Committee of Hospital Arnau de Vilanova (Lleida, Spain) approved the protocol, and all of the patients signed written informed consent forms.
Sample Size
To determine the sample size, we used preliminary data from a previous study of our group in which the plaque frequency was 69% in patients with retinopathy or 52% in patients without retinopathy. We calculated a sample size of 126 subjects for each study group, which would allow an 80% power to detect differences between groups with a significance level of <0.05. Thus, the number of patients included in each group was sufficient to test the initial hypothesis.
Statistical Analysis
The data are presented as the median values and 25th and 75th percentiles, means ± standard deviations (SDs), and n (%), as appropriate. Non-normally distributed variables were log transformed to reduce skewness, and normality was re-evaluated. Between-group (DR vs. non-DR, male vs. female, and with hypertension vs. without hypertension, or dyslipidaemia, or smoking habit) differences in anthropometric, clinical, cIMT, and laboratory variables were evaluated using the chi-squared test (for categorical variables), Wilcoxon test (for continuous, non-normally distributed variables), or Student's t-test (for continuous, normally distributed variables). Unadjusted and adjusted general linear models (PROC GLM in SAS) were used to test whether cIMT values differed between diabetic patients with retinopathy vs. those without retinopathy. Univariate or multiple logistic regression models were used to evaluate the differences in plaque presence between the groups. The following factors were entered into the adjusted general linear and multiple logistic regression models: age, sex, body mass index, presence of hypertension, presence of dyslipidaemia, smoking habit, urinary albumin excretion rate and eGFR. The significance level was defined as p ≤ 0.05, and all analyses were performed using SAS software, v. 9.2 (SAS Institute Inc., USA).
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