Early growth response factor-1 (Egr-1) binds to the promoters of many genes whose products influence cell movement and replication in the artery wall. Here we targeted Egr-1 using a new class of DNA-based enzyme that specifically cleaved Egr-1 mRNA, blocked induction of Egr-1 protein, and inhibited cell proliferation and wound repair in culture. The DNA enzyme also inhibited Egr-1 induction and neointima formation after balloon injury to the rat carotid artery wall. These findings demonstrate the utility of DNA enzymes as biological tools to delineate the specific functions of a given gene, and implicate catalytic nucleic acid molecules composed entirely of DNA as potential therapeutic agents.

Aims/hypothesis TNF-related apoptosis-inducing ligand (TRAIL) is implicated in the regulation of diabetes and is reduced in patients with cardiovascular disease. Although TRAIL receptors are widespread, and TRAIL can promote cell proliferation and apoptosis, it is not known how TRAIL might protect against diabetes and atherosclerosis. Methods We examined the development of atherosclerosis and diabetes in Apoe −/−

Fas ligand (FasL), a member of the tumor necrosis factor family, initiates apoptosis by binding to its surface receptor Fas. As a consequence, there is sequential activation of caspases and the release of cytochrome c from the mitochondria, with additional caspase activation followed by cellular degradation and death. Recent studies have shed important insight into the molecular mechanisms controlling FasL gene expression at the level of transcription. Nuclear factors such as nuclear factor in activated T cells, nuclear factor-kappa B, specificity protein-1, early growth response factor, interferon regulatory factor, c-Myc and the forkhead transcriptional regulator, alone or cooperatively, activate FasL expression. These factors are often coexpressed with FasL in pathophysiologic settings including human atherosclerotic lesions. Here, we review these important advances in our understanding of the signaling and transcriptional mechanisms controlling FasL gene expression.

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Rationale: TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is well reported as an inducer of apoptosis in tumor models; however, its role and function in vivo in atherosclerosis and vascular injury has not been established. Objective: We sought to study the function of TRAIL in cardiovascular pathology and its regulation in vivo. Methods and Results: Here, we show that TRAIL was upregulated in medial vascular smooth muscle cells (VSMCs) 24 hours following perivascular cuff placement around femoral arteries of mice. We also show that TRAIL mRNA and promoter activity was induced in VSMCs following in vitro mechanical injury. Intimal thickening 15 days after cuff placement was reduced 2-to 3-fold in TRAIL ؊/؊ compared to wild-type mice and was reversible by administration of recombinant TRAIL. Additionally, reduced VSMC proliferation was observed in injured arteries of TRAIL ؊/؊ mice. Fibroblast growth factor (FGF)-2, a potent growth factor released following vascular injury, was also reduced in arteries of TRAIL ؊/؊ mice, and VSMCs isolated from these animals did not respond to FGF-2 in vitro. Injury and FGF-2 regulated TRAIL transcriptional activity via 2 specificity protein (Sp)1 elements in the proximal TRAIL promoter, a binding site also shared by nuclear factor (NF)B. Mutational studies confirmed a role for Sp1 in injury-and FGF-2-inducible TRAIL transcription. Furthermore, increased NFB expression after injury transactivated the TRAIL promoter. Interestingly, following mechanical injury, Sp1 phosphorylation (Thr453) and an increase in the physical interaction of p-Sp1(Thr453) with NFB was observed. Conclusions: We conclude that TRAIL induction involves FGF-2, Sp1-phosphorylation and NFB and that TRAIL promotes VSMC proliferation and neointima formation after arterial injury. (Circ Res. 2010;106: 1061-1071.)Key Words: TRAIL Ⅲ VSMC proliferation Ⅲ injury Ⅲ transcriptional regulation T RAIL (tumor necrosis factor-related apoptosis inducing ligand) is expressed by a variety of cells, traditionally known to induce apoptosis via the death receptor pathway analogous to Fas ligand (FasL). Unlike FasL, however, TRAIL apoptotic signaling occurs following its engagement with 2 death domain-containing receptors, death receptors 4 and 5 (DR4 and DR5). Additional receptors are also present in humans, including decoy receptors 1 and 2 (DCR1 and DCR2) and osteoprotegerin, which may compete with DR4 and/or DR5 for TRAIL binding, thereby protecting cells from apoptosis. 1,2 TRAIL expression is regulated by insulin, interferons type I and II, and transcription factors Stat1, nuclear factor (NF)B, Egr2/3 (early growth response factor 2/3), and IRF-1/3/7 (interferon regulatory factor 1/3/7). 3 In the vessel wall, TRAIL is expressed in vascular smooth muscle cells (VSMCs), endothelial cells (ECs), macrophages, and T cells (reviewed 3 ); however, the role and regulation of TRAIL in cardiovascular disorders is currently controversial. Under certain conditions TRAIL can induce apoptosis of vascular cells which may...

Abstract-Early growth response factor-1 (Egr-1) controls the expression of a growing number of genes involved in the pathogenesis of atherosclerosis and postangioplasty restenosis. Egr-1 is activated by diverse proatherogenic stimuli. As such, this transcription factor represents a key molecular target in efforts to control vascular lesion formation in humans.In this study, we have generated DNAzymes targeting specific sequences in human EGR-1 mRNA. These molecules cleave in vitro transcribed EGR-1 mRNA efficiently at preselected sites, inhibit EGR-1 protein expression in human aortic smooth muscle cells, block serum-inducible cell proliferation, and abrogate cellular regrowth after mechanical injury in vitro. These DNAzymes also selectively inhibit EGR-1 expression and proliferation of porcine arterial smooth muscle cells and reduce intimal thickening after stenting pig coronary arteries in vivo. Key Words: catalytic DNA Ⅲ transcription factors Ⅲ early growth response factor-1 Ⅲ stenting Ⅲ gene therapy T he capacity to selectively target specific mRNA sequences with catalytic molecules composed of DNA provides immense potential to broaden our understanding of the roles of specific mediators in normal and pathologic settings. Catalytic DNA can be used to cleave the phosphodiester linkage between virtually any unpaired purine and paired pyrimidine, selectivity being conferred by the nucleotide sequences of the hybridizing arms. These next-generation antisense oligonucleotides are extremely specific and easy to synthesize and have low toxicity, because they do not require phosphorothioate or other backbone modifications to confer nuclease resistance. DNAzyme biotechnology has practical therapeutic implications as a new category of genesuppression agents in pathophysiological settings.Stenting of coronary atherosclerotic lesions has revolutionized the treatment of cardiovascular disease in the last 5 years, after two landmark trials demonstrating a reduction in restenosis relative to coronary balloon angioplasty in comparable vessels.

TRAIL/Apo2L (tumor necrosis factor-related apoptosis-inducing ligand) is a multifunctional protein regulating homeostasis of the immune system, infection, autoimmune diseases, and apoptosis. However, its function in normal, nontransformed tissues is not clear. Here we show that TRAIL increases vascular smooth muscle cell (VSMC) proliferation in vitro, effects that can be blocked with neutralizing antibodies to TRAIL receptors DR4 and DcR1. In aortocoronary saphenous vein bypass grafts in vivo, TRAIL co-localizes with VSMC, proliferating cell nuclear antigen, and insulin-like growth factor type 1 receptor (IGF1R) expression but not active caspase-3. TRAIL is required for serum-inducible IGF1R expression, and antisense IGF1R inhibits TRAIL-induced VSMC proliferation. At 1 ng/ml, TRAIL stimulates IGF1R mRNA expression greater than insulin-like growth factor-1 and also activates the IGF1R promoter 7-fold. TRAIL-inducible IGF1R expression requires NF-B activation. Consistent with this, ammonium pyrrolidine dithiocarbamate, a pharmacological inhibitor of NF-B, blocks TRAIL-induced IGF1R expression, and p65 overexpression increases IGF1R protein levels. In addition, NF-B binds a novel TRAIL-responsive element on the IGF1R promoter. Our findings suggest that the biological functions of TRAIL in VSMC extend beyond its role in promoting apoptosis. Thus, TRAIL may play an important role in atherosclerosis by regulating IGF1R expression in VSMC in an NF-B-dependent manner.

Apoptosis of smooth muscle cells (SMC) in atherosclerotic vessels can destabilize the atheromatus plaque and result in rupture, thrombosis, and sudden death. In efforts to understand the molecular processes regulating apoptosis in this cell type, we have defined a novel mechanism involving the ubiquitously expressed transcription factor Sp1. Subtypes of SMC expressing abundant levels of Sp1 produce the death agonist, Fas ligand (FasL) and undergo greater spontaneous apoptosis. Sp1 activates the FasL promoter via a distinct nucleotide recognition element whose integrity is crucial for inducible expression. Inducible FasL promoter activation is also inhibited by a dominant-negative form of Sp1. Increased SMC apoptosis is preceded by Sp1 phosphorylation, increased FasL transcription, and the autocrine/ paracrine engagement of FasL with its cell-surface receptor, Fas. Inducible FasL transcription and apoptosis are blocked by dominant-negative protein kinase C-, whose wild-type counterpart phosphorylates Sp1. Thus, Sp1 phosphorylation is a proapoptotic transcriptional event in vascular SMC and, given the wide distribution of this housekeeping transcription factor, may be a common regulatory theme in apoptotic signal transduction.