Participants and study design
The study has a cross sectional design and included 268 (65%) patients out of 415 eligible patients with T1D. Inclusion criteria were T1D patients aged 18–59 years, diabetes duration ≥ 1-year, and performed measurements of MMP-14. Exclusion criteria were pregnancy, severe somatic and psychiatric disorders such as cancer, hepatic failure, end-stage renal disease, Cushing’s disease, severe autoimmune disorders, psychotic, bipolar or severe personality disorders, severe substance abuse, cognitive deficiency (due to stroke, dementia or intellectual disability), use of systemic corticosteroids, and inadequate knowledge of the Swedish language. The patients were consecutively recruited during a 9-month period, from 25 March to 28 December 2009, from the largest out of two hospital diabetes outpatient clinics in Region Kronoberg, Sweden. The catchment population was 1,25,000. A self-report questionnaire was used to assess depression. Blood samples, anthropometrics and blood pressure were collected, supplemented with data from electronic health records.
Cardiovascular disorder
CVD was defined as ischemic heart disease [angina pectoris, previous myocardial infarction, performed percutaneous transluminal coronary angioplasty (PTCA), and/or coronary artery bypass graft (CABP) surgery], heart failure, stroke, and/or transient ischemic attack (TIA).
Depression
Self-reported depression was assessed by Hospital Anxiety and Depression Scale- the depression subscale (HADS-D), and was defined as HADS-D ≥ 8 points [25, 26].
Thyroid disease
Patients treated for hypothyroidism or hyperthyroidism were identified, but not excluded.
Biochemical analyses
Plasma levels of MMP-2, MMP-14, TIMP-2, and TIMP-3, were analysed by using commercial human DuoSet enzyme linked immunosorbent assays (ELISAs) and supplementary ancillary kit (R&D Systems, Minneapolis, MN, USA). Before the analyses, the plasma samples were diluted in phosphate-buffered saline (PBS) supplemented with 1% bovine serum albumin (BSA), and were run in duplicates. The dilution factors were for MMP-2: 1:30; MMP-9: 1:400; MMP-14: 1:3 (1:25/1:50); TIMP-2: 1:400; TIMP-3: 1:16; and Galectin-3: 1:2. The ELISA analyses were performed according to the manufacturer’s instructions. Absorbance was measured at 450–580 nm in a FLOUstar optima plate reader (BMG Labtech Gmbh, Ortenberg, Germany). Concentrations of unknown samples were calculated using a 4-parameter logistic regression curve. The intra-assay coefficients of variation were for MMP-2: 3.7%; MMP-9: 2.2%; MMP-14: 2.8%; TIMP-2: 2.0%; TIMP-3: 1.6%; and galectin-3: 4.3%. The analyses were performed at the Diabetes Laboratory, BMC, Lund University, Lund.
For MMP-14 there were exact values for 173 patients, but for 95 patients the MMP-14 values were under the detection limit of 0.154 ng/mL. The undetected values were approximated by “zero”.
Hemoglobin A1c (HbA1c) and serum (s)-lipids were collected after overnight fasting and analysed with an Olympus automated clinical chemistry analyser with high specificity (Olympus AU®, Tokyo, Japan). The intra-coefficients of variation were for HbA1c < 1.2%; total cholesterol < 2.1%; High-density lipoprotein (HDL)-cholesterol < 3.0%; low-density lipoprotein (LDL)-cholesterol < 2.6%; and for triglycerides < 2.2%.
S-creatinine was assayed by an AU2700® instrument (Beckman Coulter, Brea, CA, USA). The intra-coefficient of variation was < 3%. HbA1c, s-lipids and s-creatinine were analysed at the department of Clinical Chemistry, Växjö Central Hospital.
CRP was assayed by spectrophotometry on a Roche Cobas C501® at the diabetes laboratory, Lund University Hospital, Lund.
Anthropometrics and blood pressure
Waist circumference, weight and length were measured by a nurse. Body mass index (BMI) (kg/m2) was calculated. Abdominal obesity was defined as WC ≥ 1.02 m for men and as WC ≥ 0.88 m for women [27]. Blood pressure was measured according to standard procedures in the sitting position by a nurse.
Smoking and physical activity
Smokers were defined as having smoked any amount of tobacco during the last year [27]. Information regarding levels of physical activity was collected by interviews performed by skilled nurses or physicians. Five levels were initially registered as in the Swedish National Diabetes Register (S-NDR) [28]: ≥ 30 min of moderate activities were performed either (1) never, (2) less than once a week, (3) 1–2 times a week, (4) 3–5 times a week, or (5) daily. In this study, level 1 and 2 were merged into one level: less than once a week.
Treatment for T1D
The patients used either multiple daily insulin injections (MDII) or continuous subcutaneous insulin infusion (CSII) administered by a pump.
Antihypertensive drugs and indications for treatment of hypertension
Antihypertensive drugs included ACE inhibitors (ATC codes C09AA-BA); (ARB) (ATC codes C09CA-DA); calcium antagonists (ATC codes C08CA01-02); diuretics (ATC codes C03AA03 or C03CA01); and/or selective beta-adrenoreceptor antagonists (ATC code C07AB). Indications for antihypertensive drugs were systolic BP > 130 mm Hg and/or BP > 80 mm Hg and/or CVD according to the Swedish national guidelines in 2009 [29]. The use of antihypertensive drugs was dichotomized into users and non-users.
Lipid-lowering drugs and indications for treatment of hyperlipidemia
Lipid-lowering drugs were hydroxy-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) (ATC-code C10AA). Indications for lipid-lowering drugs were total cholesterol > 4.5 mmol/L (> 1.74 mg/dL) and/or LDL-cholesterol > 2.5 mmol/L (> 97 mg/dL) or present CVD according to the Swedish national guidelines in 2009 [29]. The use of lipid-lowering drugs was dichotomized into users and non-users.
Antidepressants
Antidepressants were SSRIs, SNRIs and/or specific serotonergic antidepressants (N06AB, N06AX16, or N06AX11). The use of antidepressants was dichotomized into users and non-users.
Statistical analysis
Analysis of data distribution using histograms revealed that age, diabetes duration, and the inflammatory plasma biomarkers were not normally distributed. Data were presented as median [quartile (q)1, q3; min–max], and analyses were performed with Mann–Whitney U test. Fisher’s Exact Test (two-tailed) and Linear-by-linear association were used to analyse categorical data, and data were presented as N (%). Medians and prevalence rates for the included variables were compared between patients with high MMP-14 (≥ 5.81 ng/mL) and low MMP-14 (< 5.81 ng/mL), and between patients with and without CVD.
Variables with P values < 0.10 were included in the further analyses and crude odds ratios (CORs) were calculated. Variables with P values < 0.10 for the CORs were included in multiple logistic regression analyses (Backward: Wald) with high MMP-14 (one model) and CVD (three models) as dependent variables. In the first model, only baseline variables were included. In the second model, variables with P values < 0.10 in model 1 and MMP-14 used as a continuous variable were included. In the third model, continuous MMP-14 was exchanged by high MMP-14, otherwise the variables were the same as in model 2. The Hosmer and Lemeshow test for goodness-of-fit and Nagelkerke R2 were used to evaluate each multiple logistic regression analysis model. The area under the receiver operating characteristics curve (AUC of ROC) was performed for MMP-14 tried against CVD, in order to establish a cut-off value for MMP-14, based on the combined optimal sensitivity and specificity. To evaluate the statistical power, a post-hoc calculation was performed for high MMP-14 and CVD and showed a power of 79.7%: the result was calculated using the following data: group 1, 7 patients had CVD and the prevalence of high MMP-14 was 71%; group 2, 261 patients did not have CVD and the prevalence of high MMP-14 was 24% [30]. P values < 0.05 were considered statistically significant. SPSS® version 25 (IBM, Chicago, Il, USA) was used.