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    • AP plays a pivotal role in transcriptional regulation of MMP

    2023-02-20

    AP-1 plays a pivotal role in transcriptional regulation of MMP expression. The decoy technique allows the neutralization of AP-1 without any transfection reagents for cellular uptake that cause potential side effects or immune reactions. This energy-dependent transport mechanism of “naked” dODN is clearly distinct from the endocytotic pathway that has been described for antisense ODNs and involves both a saturable carrier-mediated component as well as a receptor-mediated podocytosis with subsequent intracellular release from caveloae vesicles. In addition, the length (i.e., uptake of decoy ODNs greater than 25-mer in size tends to be rather poor) but also certain sequence characteristics of the double-stranded DNA molecules seem to be important for the speed by which they are taken up by the target cell. Cationic lipids, such as the Lipofectamine that can greatly enhance plasmid DNA uptake, have only a modest impact on in vivo delivery of ODNs. ODN taken up via the Lipofectamine-mediated delivery could be exclusively detected in the cytosol and presumably ends in the lysosomal pathway for degradation, whereas the “naked” dODN was detectable in the cytosol and nucleus. This result is in line with Bene et al., demonstrating that lipid-complexed complexed AP-1 dODNs were readily transfectable into cells, but were consistently detectable only in the cytoplasm and not in the nucleus. In this context, it is particularly important that dODN become active immediately after N-Nonyldeoxynojirimycin into the target cells. By contrast, the efficacy of antisense or RNA-interference ODNs is primarily dependent upon the conversion of the protein in the cell and therefore upon its re-synthesis. Nevertheless, we previously demonstrated the same effectiveness of an antisense ODN targeting the transcription factor STAT1 (signal transducers and activators of transcription) and a STAT1 transcription factor dODN to reduce CD40 expression on an mRNA and protein level. Because of the need of a delivery aid for antisense ODN, which may induce cellular stress response and change gene expression pattern in transfected cells, this method has to be considered as critical in regard of therapeutic use. Treatment with decoys has been shown to limit restenosis after percutaneous transluminal coronary angioplasty in coronary arteries of hypercholesterolemic minipigs. Moreover, the AP-1 dODN treatment suppresses graft endothelial adhesion molecule expression, reducees graft infiltration, and in turn significantly delays acute rejection in different rat transplantation models and prevents the occurrence of cardiac allograft vasculopathy in a fully allogenic rat heart transplantation model. Therefore, single ex vivo perfusion of cardiac allografts with AP-1 dODN targeting allograft immunogenicity during the cardiac transplantation procedure might be an attractive adjunct or even alternative concept to systemic immunosuppressive therapy of the recipient. In the present study, we demonstrated an increased AP-1 abundance and consecutively elevated MMP activity in aortic smooth muscle cells of Marfan mice. Subsequently, both MMP expression and activity were significantly reduced using the AP-1 dODN approach in these cells. AP-1 is a key player in regulating inflammatory processes but also required for basal expression of several genes, thus constantly activated. We therefore assume that in contrast to HUASMCs used in Figure 3D, IL-1β presumably failed in murine SMCs to further increase AP-1 activation and MMP expression because of an already increased high basal AP-1 activation level. In line with this finding, we observed a reduced macrophage infiltration into the aortic adventitia of the AP-1 consensus dODN-treated grafts of mgR/mgR mice. Inflammatory infiltrates have already been described in the mgR/mgR mouse model and suggest that inflammation may represent a component of the complex pathogenesis of MFS. AP-1 dODN treatment also reduced ROS formation in smooth muscle cells, which is solely sufficient to create a pro-inflammatory environment and promote cell migration and MMP expression, subsequently leading to aneurysm formation. Indeed, a reduced IL-1β-induced migration of cultured smooth muscle cells could be demonstrated after AP-1 dODN treatment. It has been published previously that TGF-β can also increase vascular smooth muscle migration proliferation and also migration in patients with MFS.