Levon M. Khachigian

Egr-1-Induced Endothelial Gene Expression: A Common Theme in Vascular Injury

A number of pathophysiologically relevant genes, including platelet-derived growth factor B-chain (PDGF-B), are induced in the vasculature after acute mechanical injury. In rat aorta, the activated expression of these genes was preceded by a marked increase in the amount of the early-growth-response gene product Egr-1 at the endothelial wound edge. Egr-1 interacts with a novel element in the proximal PDGF-B promoter, as well as with consensus elements in the promoters of other genes induced by endothelial injury. This interaction is crucial for injury-induced PDGF-B promoter-dependent expression. Sp1, whose binding site in the PDGF-B promoter overlaps that of Egr-1, occupies this element in unstimulated cells and is displaced by increasing amounts of Egr-1. These findings implicate Egr-1 in the up-regulated expression of PDGF-B and other potent mediators in mechanically injured arterial endothelial cells.

Coronary in-stent restenosis: Current status and future strategies

In-stent restenosis (ISR) is a novel pathobiologic process, histologically distinct from restenosis after balloon angioplasty and comprised largely of neointima formation. As percutaneous coronary intervention increasingly involves the use of stents, ISR is also becoming correspondingly more frequent. In this review, we examine the available studies of the histology and pathogenesis of ISR, with particular reference to porcine and other animal models. An overview of mechanical treatments is then provided, which includes PTCA, directional coronary atherectomy and high speed rotational atherectomy. Radiation-based therapies are discussed, including a summary of current problems associated with this modality of treatment. Finally, novel strategies for the prevention of ISR are addressed, including novel developments in stents and stent coatings, conventional drugs, nucleic acid-based drugs and gene transfer. Until recently, limited pharmacologic and mechanical treatment options have been available for both treatment and prevention of ISR. However, recent advances in gene modification and gene transfer therapies and, more particularly, in local stent-based drug delivery systems make it conceivable that the incidence of ISR will now be seriously challenged.

Transcription factor Egr-1 supports FGF-dependent angiogenesis during neovascularization and tumor growth

Current understanding of key transcription factors regulating angiogenesis is limited. Here we show that RNA-cleaving phosphodiester-linked DNA-based enzymes (DNAzymes), targeting a specific motif in the 5′ untranslated region of early growth response (Egr-1) mRNA, inhibit Egr-1 protein expression, microvascular endothelial cell replication and migration, and microtubule network formation on basement membrane matrices. Egr-1 DNAzymes blocked angiogenesis in subcutaneous Matrigel plugs in mice, an observation that was independently confirmed by plug analysis in Egr-1-deficient animals, and inhibited MCF-7 human breast carcinoma growth in nude mice. Egr-1 DNAzymes suppressed tumor growth without influencing body weight, wound healing, blood coagulation or other hematological parameters. These agents inhibited endothelial expression of fibroblast growth factor (FGF)-2, a proangiogenic factor downstream of Egr-1, but not that of vascular endothelial growth factor (VEGF). Egr-1 DNAzymes also repressed neovascularization of rat cornea. Thus, microvascular endothelial cell growth, neovascularization, tumor angiogenesis and tumor growth are processes that are critically dependent on Egr-1.

New DNA enzyme targeting Egr-1 mRNA inhibits vascular smooth muscle proliferation and regrowth after injury

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.

Sp1 Phosphorylation and Its Regulation of Gene Transcription

Sp1 is a ubiquitously expressed, prototypic C2H2-type zinc finger-containing DNA binding protein that can activate or repress transcription in response to physiologic and pathological stimuli. It was originally found to selectively transactivate the early and late simian virus 40 promoters without influencing numerous other promoters (16) and has since been shown to regulate the expression of thousands of genes implicated in the control of a diverse array of cellular processes, such as cell growth (26, 57), differentiation (48), apoptosis (26), angiogenesis (43), and immune response (25), to name just a few. Sp1 is a 785-amino-acid, 100- to 110-kDa nuclear transcription factor which regulates gene expression via multiple mechanisms. It binds GC-rich motifs (such as 5′-G/T-GGGCGG-G/A-G/A-C/T-3′ or 5′-G/T-G/A-GGCG-G/T-G/A-G/A-C/T-3′) with high affinity (4, 27, 28) and can regulate the expression of TATA-containing and TATA-less genes via protein-protein interactions or interplay with other transcription factors (47), such as Ets-1 (56), c-myc (51), c-Jun (44), Stat1 (6), and Egr-1 (31), and/or components of the basal transcriptional machinery. Sp1 has been linked to chromatin remodeling through interactions with chromatin-modifying factors such as p300 (62) and histone deacetylases (HDACs) (71). Sp1 was once thought to serve mainly as a constitutive activator of housekeeping genes. However, growing evidence indicates that phosphorylation, acetylation, sumoylation, ubiquitylation, and glycosylation are among the posttranslational modifications that can influence the transcriptional activity and stability of Sp1. Here we will discuss recent developments in our understanding of the role of posttranslational modifications influencing Sp1-dependent transcription, focusing mainly on phosphorylation.

DNAzyme targeting c-jun suppresses skin cancer growth

Catalytic DNA molecules that target the transcription factor c-jun inhibit skin cancer growth in mice. Getting Under Cancer’s Skin Summer brings to mind barbecues, baseball, and trips to the local pool. Yet, outdoor fun can be hazardous to one’s health—too much sun exposure can increase the risk of developing skin cancer. Indeed, one in three cancers worldwide is skin-related, and currently available treatments may induce scarring or other toxicities. Cai et al. now report that the DNAzyme Dz13—which targets an mRNA that encodes a cancer-associated transcription factor, c-Jun—inhibits the growth of two common types of skin cancers: basal cell and squamous cell carcinomas. DNAzymes are single-stranded, all-DNA, catalytic molecules that specifically bind and cleave their target RNAs. The authors examined the effects of Dz13, which destroys c-jun mRNA, on animal models of skin cancer. Dz13 inhibited tumor growth, blocked neovascularization, and prevented metastasis in mouse models of skin cancer—effects that were mediated, in part, through the induction of antitumor immunity. Minimal toxicity was observed in Dz13-treated cynomolgus monkeys, minipigs, and rodents, and there were no off-target effects in more than 70 in vitro bioassays. Thus, Dz13 may prove to be a safe, effective therapy for skin cancer. Nonetheless, one is advised to pack the sun block in preparation for extra innings—or a fifth set. Worldwide, one in three cancers is skin-related, with increasing incidence in many populations. Here, we demonstrate the capacity of a DNAzyme-targeting c-jun mRNA, Dz13, to inhibit growth of two common skin cancer types—basal cell and squamous cell carcinomas—in a therapeutic setting with established tumors. Dz13 inhibited tumor growth in both immunodeficient and immunocompetent syngeneic mice and reduced lung nodule formation in a model of metastasis. In addition, Dz13 suppressed neovascularization in tumor-bearing mice and zebrafish and increased apoptosis of tumor cells. Dz13 inhibition of tumor growth, which required an intact catalytic domain, was due in part to the induction of tumor immunity. In a series of good laboratory practice–compliant toxicology studies in cynomolgus monkeys, minipigs, and rodents, the DNAzyme was found to be safe and well tolerated. It also did not interfere in more than 70 physiologically relevant in vitro bioassays, suggesting a reduced propensity for off-target effects. If these findings hold true in clinical trials, Dz13 may provide a safe, effective therapy for human skin cancer.

Galectin-1 interacts with the {alpha}5{beta}1 fibronectin receptor to restrict carcinoma cell growth via induction of p21 and p27.

Surface binding of galectin family members has the potential to link distinct glycan structures to growth regulation. Therefore, we addressed the antiproliferative potential of galectin-1 (Gal-1) in a panel of carcinoma cell lines. We discovered growth inhibition by Gal-1 in epithelial tumor cell lines from different origins and provide evidence that this effect requires functional interaction with the α5β1 integrin. Antiproliferative effects result from inhibition of the Ras-MEK-ERK pathway and consecutive transcriptional induction of p27. We have further identified two Sp1-binding sites in the p27 promoter as crucial for Gal-1 responsiveness. Inhibition of the Ras-MEK-ERK cascade by Gal-1 increased Sp1 transactivation and DNA binding due to reduced threonine phosphorylation of Sp1. Furthermore, Gal-1 induced p21 transcription and selectively increased p27 protein stability. Gal-1-mediated accumulation of p27 and p21 inhibited cyclin-dependent kinase 2 activity and ultimately resulted in G1 cell cycle arrest and growth inhibition. These data define a novel mechanism whereby Gal-1 regulates epithelial tumor cell homeostasis via carbohydrate-dependent interaction with the α5β1 integrin.

Inducible Expression of Egr-1–Dependent Genes: A Paradigm of Transcriptional Activation in Vascular Endothelium

Throughout the vascular network, endothelium forms the continuous cellular interface between the circulating blood elements and the surrounding tissues. These cells provide a nonthrombogenic surface and a permeability barrier capable of modulating blood flow and vascular reactivity. As such, the integrity of the endothelium is a fundamental requirement for the maintenance of normal homeostasis. Injury to this lining and the subsequent inflammatory response are among the earliest cellular events in the pathogenesis of atherosclerosis.1 This can result in dramatic phenotypic changes that render otherwise quiescent endothelium adhesive and prothrombotic. Lesions of atherosclerosis and postangioplasty restenosis may develop under the influence of molecules inducibly expressed or simply released by activated or injured endothelium. Several lines of evidence over the last decade, based on ligand and receptor localization, overexpression, and infusion studies, have correlated PDGF with the development of vascular occlusive lesions. PDGF appears to play a regulatory role in the migratory, rather than proliferative, events associated with the response to arterial injury. PDGF expression is quite low in the quiescent vessel wall, but levels of the growth factor increase substantially after injury. This Mini Review will focus on the transcriptional mechanisms underlying inducible PDGF expression in vascular endothelium and smooth muscle cells, with particular emphasis on the role played by the early growth response gene product, Egr-1. The vessel wall responds to a changing local environment by modulating the expression of specific sets of genes. Transcriptional activation in response to these signals involves the regulated assembly of multiprotein complexes on promoters. The promoter regions of the PDGF-A and PDGF-B genes have been characterized, and some of the regulatory sequences and transcriptional activators have been defined. In endothelial cells, 5′ deletion analysis of both promoters has determined that the minimal promoters consist of ≈100 bp.2 3 The zinc-finger transcription factor, …

SP1 IS A COMPONENT OF THE CYTOKINE-INDUCIBLE ENHANCER IN THE PROMOTER OF VASCULAR CELL ADHESION MOLECULE-1

Transcription of the vascular cell adhesion molecule-1 (VCAM-1) gene in endothelial cells is induced by the inflammatory cytokines interleukin-1β, tumor necrosis factor-α, and lipopolysaccharide. Previous studies demonstrated that the cytokine-response region in the VCAM1 promoter contains binding sites for the transcription factors nuclear factor-κB (NF-κB) and interferon regulatory factor-1. Using a saturation mutagenesis approach, we report that the cytokine-inducible enhancer consists of these previously characterized elements and a novel region located 3′ of the NF-κB sites. Electrophoretic mobility shift assays and DNase I footprint studies with endothelial nuclear extracts and recombinant protein revealed that the transcriptional activator Sp1 interacts with this novel element in a specific manner. Transient transfection assays using vascular endothelial cells revealed that site-directed mutations in the Sp1 binding element decreased tumor necrosis factor-α-induced activity of the VCAM1 promoter. The cytokine-induced enhancer of the VCAM1 gene requires constitutively bound Sp1 and induced heterodimeric NF-κB for maximal promoter activity.

Hemodynamics, Endothelial Gene Expression, and Atherogenesisa

Vascular endothelium, the continuous lining of the cardiovascular system, forms a multifunctional interface between circulating blood and the various tissues and organs of the body. It constitutes a selectively permeable barrier for macromolecules, as well as non-thrombogenic container that actively maintains the fluidity of blood. It is a metabolically active tissue, serving as the source of multiple factors (peptides, proteins, lipids) that are critical for normal homeostasis. These include growth stimulators and inhibitors [e.g., platelet-derived growth factor, (PDGF), transforming growth factor-beta (TGF-beta), fibroblast growth factor (FGF), and heparin-like glycosaminoglycans]; vasoconstrictors and vasodilators [e.g., endothelin-1 (ET1 ), angiotensin 11, and endothelial-derived relaxing factors, such as nitric oxide]; the various proand anti-thrombotic factors (e.g., tissue factor, thrombomodulin, and von Willebrand factor); fibrinolytic activators and inhibitors (e.g., tissue plasminogen activator (tPA), urokinase, and plasminogen activator inhibitor1 , (PAIl)]; potent arachidonate metabolites (e.g., prostacyclin and PGI,); leukocyte adhesion molecules (e.g., E-Selectin, P-Selectin, ICAM-I, and VCAM-I) and multiple cytokines (e.g., IL-I, IL-6, IL-8, MCP-I, and GM-CSF). This partial list underscores the functional diversity of the endothelial interface in normal physiology and also illustrates its potential contributions to pathophysiological processes in vascular disease. It has been our laboratorys working concept that the vascular endothelium is a dynamically mutable interface, whose structural and functional properties are respon-