Matrix metalloproteinases (MMPs) contribute to the destruction of the extracellular matrix at the shoulder regions of atherosclerotic plaques that leads to plaque destabilisation and triggers clinical cardiovascular disease. There is therefore considerable interest in establishing the mechanisms responsible for increased MMP production. MMPs-1, -3 and -9 are upregulated by inflammatory cytokines and growth factors that are produced by plaque resident macrophages and smooth muscle cells. Our present studies focused on NF-kappaB, which regulates numerous inflammatory genes, and is activated in plaque smooth muscle cells. Moreover, an NF-kappaB binding site is present in the promoter of the MMP-9 gene and an NF-kappaB-like element in the promoter of the MMP-1 gene. We used adenovirus mediated overexpression of its inhibitor, I kappaBalpha to investigate the role of NF-kappaB in regulation of MMP-1, -3 and -9 by isolated, cytokine stimulated rabbit aortic and human saphenous vein VSMC. IL-1alpha potently activated NF-kappa B in VSMCs and acted synergistically with growth factors to upregulate expression of MMP-1, -3 and -9. Overexpression of I kappaBalpha, almost completely inhibited expression of MMP-1, -3 and -9 in response to IL-1alpha alone or in combination with bFGF and PDGF. NF-kappaB is required for cytokine upregulation of MMP-1, -3 and -9 in VSMCs, which suggests that NF-kappaB inhibition may promote plaque stabilisation.

Metalloproteinase secretion by macrophages is believed to play a key role in the matrix degradation that underlies atherosclerotic plaque instability and aneurysm formation. We studied the hypothesis that nuclear factor-κB (NF-κB), a transcription factor, is necessary for metalloproteinase secretion and, hence, is a target for pharmacological intervention. Adenovirus-mediated gene transfer of the inhibitory NF-κB subunit, I-κ Bα, was achieved into human monocyte-derived macrophages in vitro and into foam cells produced in vivo in cholesterol-fed rabbits. Human macrophages and rabbit foam cells secreted matrix-degrading metalloproteinase (MMP)-9 without further stimulation, and this was not inhibited by I-κBα (11±16% and 8±10%, respectively;P> 0.05). MMP-1 secretion from human macrophages increased in response to recombinant human CD40 ligand and was inhibited 92±5% by I-κBα (n=3,P<0.05). Rabbit foam cells secreted MMP-1 and -3 without further stimulation, and this was inhibited 83±12% and 69±11%, respectively, by I-κBα (n=6 or 7,P<0.001). I-κBα did not significantly affect the expression or activity of tissue inhibitor of metalloproteinases-1 or -2. Overexpression of I-κBα inhibited collagenolytic and β-caseinolytic activity by 42±2% and 41±7%, respectively (n=3,P<0.05). Secretion of MMP-1 and MMP-3 from macrophages stimulated in vitro or in vivo depends on the activation of NF-κB. Because the inhibition of NF-κB reduces proteolytic activity, it appears to be an attractive pharmacological target in unstable atheromas.

Tissue inhibitors of metalloproteinases (TIMPs) are important regulators of matrix metalloproteinase (MMP) and adamalysin metalloproteinase activity. We previously reported that overexpression of TIMP-3 inhibits MMPs and induces apoptotic cell death in a variety of cell types and demonstrated that apoptosis is mediated through the N terminus of TIMP-3, which harbors the MMP inhibitory domain. However, little is known about the mechanisms underlying TIMP-3-induced apoptosis. Here we demonstrate that overexpression of TIMP-3 induced activation of initiator caspase-8 and -9 and promoted caspase-mediated cleavage of the death substrates poly(ADP-ribose) polymerase and focal adhesion kinase. Furthermore, TIMP-3 induced mitochondrial activation as demonstrated by loss of mitochondrial membrane potential and release of cytochrome c. Intervention studies demonstrated that overexpression of Bcl-2, the anti-apoptotic mitochondrial membrane protein, or CrmA, a viral serpin inhibitor of caspase-8, completely inhibited TIMP-3-induced apoptosis. Furthermore, a dominant-negative Fas-associated death domain mutant inhibited TIMP-3-induced death substrate cleavage and apoptotic death. Taken together, these results indicate that TIMP-3 overexpression induces a type II apoptotic pathway initiated via a Fas-associated death domain-dependent mechanism.

Cyclic AMP signalling promotes VSMC quiescence in healthy vessels and during vascular healing following injury. Cyclic AMP inhibits VSMC proliferation via mechanisms that are not fully understood. We investigated the role of PKA and Epac signalling on cAMP-induced inhibition of VSMC proliferation. cAMP-mediated growth arrest was PKA-dependent. However, selective PKA activation with 6-Benzoyl-cAMP did not inhibit VSMC proliferation, indicating a requirement for additional pathways. Epac activation using the selective cAMP analogue 8-CPT-2'-O-Me-cAMP, did not affect levels of hyperphosphorylated Retinoblastoma (Rb) protein, a marker of G1-S phase transition, or BrdU incorporation, despite activation of the Epac-effector Rap1. However, 6-Benzoyl-cAMP and 8-CPT-2'-O-Me-cAMP acted synergistically to inhibit Rb-hyperphosphorylation and BrdU incorporation, indicating that both pathways are required for growth inhibition. Consistent with this, constitutively active Epac increased Rap1 activity and synergised with 6-Benzoyl-cAMP to inhibit VSMC proliferation. PKA and Epac synergised to inhibit phosphorylation of ERK and JNK. Induction of stellate morphology, previously associated with cAMP-mediated growth arrest, was also dependent on activation of both PKA and Epac. Rap1 inhibition with Rap1GAP or siRNA silencing did not negate forskolin-induced inhibition of Rb-hyperphosphorylation, BrdU incorporation or stellate morphology. This data demonstrates for the first time that Epac synergises with PKA via a Rap1-independent mechanism to mediate cAMP-induced growth arrest in VSMC. This work highlights the role of Epac as a major player in cAMP-dependent growth arrest in VSMC.