Abstract 1719: Tissue Factor Pathway Inhibitor-2 Gene Hypermethylation is Associated with Low Expression in Carotid Atherosclerotic Plaques
Background. In atherosclerotic plaques, matrix metalloproteinase (MMP) activity could be regulated by Tissue Factor Pathway Inhibitor-2 (TFPI-2), a Kunitz-type serine-protease inhibitor. Several CpG islands are present in the TFPI-2 gene and methylation frequently down-regulates TFPI-2 expression in cancer. We hypothesized that TFPI-2 gene methylation could regulate TFPI-2 expression and MMP-9 activity in atherosclerotic plaques and normal vessels.
Methods We used 3 systems of Methylation Specific PCR (MSP1, MSP2, MSP3) in order to identify 17 of the 29 CpG located in the promoter of TFPI-2 gene, in DNA from 59 carotid atherosclerotic plaques and 13 control mammary arteries.
Results MSP1 showed methylation of the TFPI-2 gene in 13 plaques (22%), MSP2 in no plaque and MSP3 in 3 plaques (5%). No methylation was found in control arteries, whatever the MSP system. The TFPI-2 mRNA levels were significantly lower in plaques with methylated TFPI-2 gene than in unmethylated ones and than in control arteries (p=0.03 and 0.009 respectively). Several CpG nucleotides recognized by the MSP1 system were located in a Sp1 and egr-1/Sp1 transcription factor binding sites at -214 and -118 bp respectively, which may be involved in TFPI-2 down-regulation. MMP-9 activity was not significantly different according to the TFPI-2 methylation status, but was higher in plaques with low than in plaques with high levels of TFPI-2 mRNA (p=0.01). The prevalence of cardiovascular risk factors and ischemic cerebral events was not different between methylated and unmethylated samples.
Conclusion These results demonstrate that TFPI-2 gene methylation process takes place in atherosclerotic plaques and may regulate TFPI-2 expression. The role of this specific regulation in plaques on metalloproteinases activity and cardiovascular events warrants further investigations.