AP-1 decoy oligodeoxynucleotide inhibition of matrix metalloproteinase expression in vascular smooth muscle cells of fibrillin-1-deficient mgR/mgR-mice

Objectives: Vascular changes associated with the Marfan syndrome are characterised by an abnormally high activity of matrix metalloproteinases (MMPs) in aortic smooth muscle cells leading to destruction of collagen fibres. It has been shown that the transcription factor activating factor-1 (AP-1) plays a pivotal role in the regulation of MMP expression. In the present study we aimed to decrease MMP-2 and MMP-9-expression and subsequent activity in cultured cells by a decoy deoxyoligoribonucleotide (dODN)-mediated neutralization of AP-1.

Methods: To analyze dODN function, murine aortal smooth muscle cells (mAoSMC) from fibrillin-1 deficient marfan mice (mgR/mgR), mAoSMC from wildtype (wt) -mice and human umbilical arterial smooth muscle cells (HUASMC) were used. Interleukin-1β (IL-1β)/fMLP- and non-stimulated cells were compared to dODN pretreated stimulated cells. We rated the MMP-2 and MMP-9 expression and subsequent activity by using western blot, gelatine-zymography and immonoprecipitation combined with gelatine-zymography. In addition, we tested the penetration depth of a ATTO-590 fluorescent dODN (fODN) in the aorta of mgR/mgR-and wt-mice.

Results: MAoSMC of Marfan mice expressed significantly more MMP-2 and MMP-9 than mAoSMC from wild type mice (p = 0.016). In all cultured cells reduction of basal MMP-2 and MMP-9 expression could be observed after AP-1 dODN application. After stimulation of mAoSMC-wt with IL1β, treatment with dODN resulted in reduction of MMP-9 expression by 87% compared to untreated controls (p = 0.016). Similarly, MMP-2 activity was reduced by 50% (p = 0.025). Comparably in mgR/mgR-mAoSMC the MMP-2 activity was reduced by 70% (p = 0.002).This downregulation of MMPs by dODN was also observed in human cells (HUASMC), resulting in reduction of expression of MMP-9 by 80% (p = 0.025) and reduction of activity of MMP-2 by 190% (p = 0.025). Moreover, fluorescent microscopy identified the fODN in the SMC of the aortic media of mgR/mgR but not in SMC of the aortic media of wt-mice, demonstrating that dODN can overcome the endothelial cell barrier only in mgR/mgR-mice.

Conclusion: Neutralization of the AP-1 transcription factor resulted in a significant inhibition of MMP expression and activity. This effect was more pronounced in Marfan mice compared to wt- animals due to elevated MMP-levels. Increased permeability for fODN may be related to an endothelial cell dysfunction in mgR/mgR aortas, offering new potential therapeutic strategies using dODNs.