Publications

Gelatinase A (matrix metalloproteinase-2) plays a prominent role in multiple biologic processes. Prior studies have established critical roles for gelatinase A transcriptional regulation by defined enhancer elements. To determine possible functional single nucleotide polymorphisms within these elements, we determined the single nucleotide polymorphism distribution within 1,665 bp of the gelatinase A 5'-flanking region, using a healthy homogeneous Caucasian study group of 463 individuals. Among the polymorphisms detected, a G --> A transition at bp -1575 was located immediately 5' to a half-palindromic potential estrogen receptor binding site. In estrogen receptor-positive MCF-7 cells the -1575G allele functioned as an enhancer, whereas the -1575A allele reduced transcription activity significantly. Gel shift assays confirmed that the differences in allelic expression affected binding of the estrogen receptor-alpha to this region. Cotransfection experiments with an estrogen receptor-alpha expression vector in MDA-MB-231 cells, which do not constitutively express an estrogen receptor, revealed that estrogen receptor is absolutely required for enhancing activity. Allelic distribution analysis indicated that a previously reported C --> T transition within an Sp1 binding site at -1306 was in linkage disequilibrium with the -1575G --> A transition. Luciferase reporter studies of the linked variant -1575A -1306T allele versus the wild type -1575G -1306C allele demonstrated an additive reduction in estrogen-dependent reporter activity. The frequency of the -1575G --> A transition deviated significantly from the expected Hardy-Weinberg distribution in two independently assembled study populations consisting of healthy adult blood donors and newborns of Caucasian origin, both with a calculated 21% reduction in genetic fitness. Gelatinase A is a known estrogen-responsive gene and the demonstration of a loss of function polymorphism within an operational estrogen receptor binding site associated with a decrease in genetic fitness underscores the biologic significance of promoter polymorphism analyses.

OBJECTIVES

We previously showed that an angiotensin-converting enzyme inhibitor (captopril) or an angiotensin receptor blocker (losartan) reduced infarct size and improved endothelial function in a rat model of ischaemia-reperfusion. The present study was undertaken to see if aspirin (ASA) antagonised the beneficial effects of captopril or losartan.

METHODS

One hundred and fourteen Sprague-Dawley rats were randomised into six groups; Control, ASA, captopril, losartan, ASA+captopril, and ASA+losartan. ASA, captopril or losartan were given at a concentration of 40 mg/kg/day in drinking water. After six weeks of pre-treatment, the rats were subjected to 17 minutes of left anterior descending coronary artery occlusion and 120 minutes of reperfusion, with haemodynamic and ECG monitoring. During the reperfusion period, the effective refractory period (ERP), ventricular fibrillation threshold (VFT) and bleeding time (BT) were measured. In fresh aortic rings precontracted with phenylephrine, endothelium-dependent and -independent relaxations were assessed using acetylcholine and nitroglycerin.

RESULTS

Haemodynamic changes were not different between the groups. Serum ASA concentrations were 0.5, 1.1 and 0.6 mg/dl in the ASA, ASA+captopril and ASA+losartan groups, respectively, and BT was prolonged (p<0.01). ASA alone reduced endothelium-dependent relaxation (-29+8 vs. -69+11%, p<0.01), but did not change endothelium-independent relaxation. ASA did not affect endothelial relaxation induced by acetylcholine in the presence of either captopril or losartan. Angiotensin I and ERP were elevated by captopril and losartan. Angiotensin II and VFT were elevated by losartan. ASA with captopril, captopril and losartan equally reduced infarct size, compared with control (39+3, 39+4, and 39+5 vs. 53+3%, all p<0.05).

CONCLUSIONS

Captopril and losartan had similar cardiovascular protective effects in a rat model of ischaemia-reperfusion. Aspirin did not attenuate the cardiovascular protective effects of captopril or losartan.