Subjects

Subjects with Type 2 diabetes and without known coronary artery disease (n = 22) were recruited from the Elsie Bertram Diabetes Centre, Norwich if they had Type 2 diabetes based on the American Diabetes Association criteria [12] and no clinical or electrocardiographic evidence of ischaemic heart disease. Coronary angiography or stress testing were not undertaken as part of this protocol, but patients with a history of previous confirmed myocardial infarction, or 12 – lead or exercise electrocardiogram evidence of myocardial infarction or ischaemic heart disease were excluded. Controls without Type 2 diabetes (n = 18) had no clinical history or electrocardiographic evidence of vascular disease, and all had a fasting plasma glucose below 6.1 mmol/l [12] and no patient with impaired fasting glucose was included in the control or diabetes group. No patient or control was taking aspirin or lipid lowering therapy. No subjects with Type 2 diabetes had macroproteinuria or microalbuminuria as determined by an elevated early morning albumin : creatinine ratio, and active smokers or subjects taking insulin were excluded. Thirteen of the 22 patients with Type 2 diabetes were taking a sulphonylurea (either alone in combination), of whom 7 were taking gliclazide. No patient was taking rosiglitazone or pioglitazone.

Primary human monocyte isolation

Monocytes were purified from plasma by density gradient centrifugation after collection of peripheral blood into EDTA-containing tubes as previously described [13]. Monocyte purity was greater than 85% and cell viability (assessed by trypan blue exclusion) was normally greater than 95% [13].

Monocyte culture

The monocytes were resuspended in RPMI 1640 medium (Gibco) containing 25 mmol/L glucose, 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, non-essential amino acids, penicillin, streptomycin. Polymixin B was also included in the medium to avoid possible stimulation by endotoxin. The cells were cultured in duplicate for 48 hours, 37°C, 5% CO₂, at a density of 250 000/well of 24-well tissue culture dish (Costar) in a total volume of 0.5 ml. After 48 hours cell viability was assessed by measuring lactate dehydrogenase release using a cytotoxicity detection kit (Boehringer-Mannheim). Cell viability at the end of the culture period was normally 70%. Conditioned media was collected, centrifuged to remove cell debris and frozen at -20°C.

MMP analysis

Total MMP-3, total TIMP-1, pro-and complexed MMP-9 levels in monocyte conditioned media were measured using the Biotrak human ELISA systems (Amersham Pharmacia Biotech UK) according to the manufacturers instructions. Total MMP-1 was assessed using an in-house double antibody sandwich ELISA employing the monoclonal capture antibody RRU-CL1 and a biotinylated polyclonal anticollagenase IgG [14]. To detect the conversion of pro-to active MMP-9 gelatin zymography was carried out as previously described [15]. Conditioned media diluted in non-reducing sample buffer was subjected to SDS-PAGE on 10% (w/v) polyacrylamide gels incorporating 0.1% (w/v) gelatin. After electrophoresis the gel was washed in 2 × 15 minute washes of 2.5% (v/v) Triton X-100 followed by overnight incubation at room temperature in 50 mM Tris, pH 7.5, 0.2 M NaCl, 10 mM CaCl₂. The gel was stained with 0.25% (w/v) Coomassie blue R-250, 50% (v/v) methanol, 10% (v/v) acetic acid, and destained in 30% (v/v) methanol, 1% (v/v) acetic acid. Enzyme activity appeared as cleared bands where the substrate had been degraded.

Statistical analysis

Data for MMP-1, MMP-3, MMP-9 and TIMP-1 were not normally distributed and are expressed as a median with interquartile range. Differences between groups were analysed by two-tailed Mann-Whitney U tests. Relationships between variables were assessed using simple linear regression. < 0.05 was taken as significant.

Although no available data is available to allow definitive power calculations, sample sizes were selected to offer more than 80 % power at the 5 % level to detect one standard deviation difference between group means assuming normal and equal variance between groups.

Clinical features (Table 1)

Clinical features of the groups are shown in Table 1.

Basal monocyte MMP expression (Table 1)

MMP-9 and TIMP-1 were abundantly expressed in the cultured primary human monocytes when compared to MMP-1, and MMP-3, production was negligible (median 0.0 ng/ml). Using gelatin zymography it was shown that MMP-9 was expressed in the proform and little active enzyme was found (not shown). MMP-2 was not found in any sample (not shown). In the diabetes group, there was no significant difference in MMP-9 or TIMP-1 expression between those who were treated with diet and those receiving oral agents (p > 0.5 for both MMP-9 and TIMP-1), or between those taking gliclazide or other sulphonylureas (p > 0.5 for both MMP-9 and TIMP-1).

Comparison between groups (Table 1)

Basal median monocyte MMP-1, MMP-3, MMP-9 and TIMP-1 expression did not differ significantly between controls and Type 2 diabetes groups. Age, fasting plasma glucose and HbA1c were not significantly related to MMP or TIMP-1 production in either group (all p > 0.1)