Intracoronary delivery of DNAzymes targeting human EGR-1 reduces infarct size following myocardial ischaemia reperfusion.

Despite improvements in treatment, myocardial infarction (MI) remains an important cause of morbidity and mortality. Inflammation arising from ischaemic and reperfusion injury is a key mechanism which underpins myocardial damage and impairment of cardiac function. Early growth response-1 (Egr-1) is an early immediate gene and a master regulator that has been implicated in the pathogenesis of ischaemia-reperfusion (IR) injury. This study sought to examine the effect of selective inhibition of Egr-1 using catalytic deoxyribonucleic acid molecules (DNAzymes, DZs) delivered via the clinically relevant coronary route in a large animal model of myocardial IR. It was hypothesized that Egr-1 inhibition with intracoronary DZ would reduce infarction size by modulating its downstream effector molecules. Egr-1 DZs inhibited the adherence of THP-1 monocytes to IL-1ß-activated endothelial cells in vitro and retained its catalytic activity up to 225 min after in vivo administration. In a porcine model of myocardial IR (45 min ischaemia/3 h reperfusion), DZ was taken up in the cytoplasm and nuclei of cardiomyocytes and endothelial cells in the myocardium after intracoronary delivery. Egr-1 DZs reduced infarct size and improved cardiac functional recovery following intracoronary delivery at the initiation of IR in this large animal model of MI. This was associated with inhibition of pro-inflammatory Egr-1 and ICAM-1 expression, and the reduced expression of TNF-α, PAI-1, TF, and myocardial MPO activity in tissue derived from the border zone of the infarct. Taken together, these data suggest that strategies targeting Egr-1 via the intracoronary route after IR injury in pigs have potential therapeutic implications in human MI.

Albendazole inhibits endothelial cell migration, tube formation, vasopermeability, VEGF receptor-2 expression and suppresses retinal neovascularization in ROP model of angiogenesis.

The angiogenic process begins with the cell proliferation and migration into the primary vascular network, and leads to vascularization of previously avascular tissues and organs as well to growth and remodeling of the initially homogeneous capillary plexus to form a new microcirculation. Additionally, an increase in microvascular permeability is a crucial step in angiogenesis. Vascular endothelial growth factor (VEGF) plays a central role in angiogenesis. We have previously reported that albendazole suppresses VEGF levels and inhibits malignant ascites formation, suggesting a possible effect on angiogenesis. This study was therefore designed to investigate the antiangiogenic effect of albendazole in non-cancerous models of angiogenesis. In vitro, treatment of human umbilical vein endothelial cells (HUVECs) with albendazole led to inhibition of tube formation, migration, permeability and down-regulation of the VEGF type 2 receptor (VEGFR-2). In vivo albendazole profoundly inhibited hyperoxia-induced retinal angiogenesis in mice. These results provide new insights into the antiangiogenic effects of albendazole.

Angiotensin II-inducible platelet-derived growth factor-D transcription requires specific Ser/Thr residues in the second zinc finger region of Sp1.

Sp1, the first identified and cloned transcription factor, regulates gene expression via multiple mechanisms including direct protein-DNA interactions, protein-protein interactions, chromatin remodeling, and maintenance of methylation-free CpG islands. Sp1 is itself regulated at different levels, for example, by glycosylation, acetylation, and phosphorylation by kinases such as the atypical protein kinase C-zeta. Although Sp1 controls the basal and inducible regulation of many genes, the posttranslational processes regulating its function and their relevance to pathology are not well understood. Here we have used a variety of approaches to identify 3 amino acids (Thr668, Ser670, and Thr681) in the zinc finger domain of Sp1 that are modified by PKC-zeta and have generated novel anti-peptide antibodies recognizing the PKC-zeta-phosphorylated form of Sp1. Angiotensin II, which activates PKC-zeta phosphorylation (at Thr410) via the angiotensin II type 1 receptor, stimulates Sp1 phosphorylation and increases Sp1 binding to the platelet-derived growth factor-D promoter. All 3 residues in Sp1 (Thr668, Ser670, and Thr681) are required for Sp1-dependent platelet-derived growth factor-D activation in response to angiotensin II. Immunohistochemical analysis revealed that phosphorylated Sp1 is expressed in smooth muscle cells of human atherosclerotic plaques and is dynamically expressed together with platelet-derived growth factor-D in smooth muscle cells of the injured rat carotid artery wall. This study provides new insights into the regulatory mechanisms controlling the PKC-zeta-phospho-Sp1 axis and angiotensin II-inducible gene expression.

Brothers in arms: DNA enzymes, short interfering RNA, and the emerging wave of small-molecule nucleic acid-based gene-silencing strategies.

The past decade has seen the rapid evolution of small-molecule gene-silencing strategies, driven largely by enhanced understanding of gene function in the pathogenesis of disease. Over this time, many genes have been targeted by specifically engineered agents from different classes of nucleic acid-based drugs in experimental models of disease to probe, dissect, and characterize further the complex processes that underpin molecular signaling. Arising from this, a number of molecules have been examined in the setting of clinical trials, and several have recently made the successful transition from the bench to the clinic, heralding an exciting era of gene-specific treatments. This is particularly important because clear inadequacies in present therapies account for significant morbidity, mortality, and cost. The broad umbrella of gene-silencing therapeutics encompasses a range of agents that include DNA enzymes, short interfering RNA, antisense oligonucleotides, decoys, ribozymes, and aptamers. This review tracks current movements in these technologies, focusing mainly on DNA enzymes and short interfering RNA, because these are poised to play an integral role in antigene therapies in the future.

Suppression of growth factor expression and human vascular smooth muscle cell growth by small interfering RNA targeting EGR-1.

Smooth muscle cell (SMC) proliferation and migration are key processes that occur in the reparative response to injury after percutaneous coronary intervention and in failed bypass grafts for the treatment of atherosclerosis. In the present study, we generated novel synthetic small interfering RNA (siRNA) molecules targeting the coding region of human early growth response-1 (EGR-1) mRNA that attenuate the expression of EGR-1 and that of fibroblast growth factor-2 (FGF-2) and granulocyte-colony stimulating factor (G-CSF). These agents suppressed SMC proliferation in a dose-dependent and non-toxic manner and blocked SMC regrowth from the wound edge following mechanical injury in vitro. In contrast, the scrambled counterpart did not inhibit SMC proliferation, EGR-1 protein expression or SMC regrowth after injury. These findings demonstrate that EGR-1 siRNA can serve as inhibitors of SMC proliferation and wound repair suggesting that these agents may potentially be useful in the control of vascular proliferative disorders.

JUN siRNA regulates matrix metalloproteinase-2 expression, microvascular endothelial growth and retinal neovascularisation.

Transcription factors link changes in the extracellular environment with alterations in gene expression. As such, these molecules serve as attractive targets for intervention in pathological settings. Since JUN has been linked with microvascular disease in humans, we hypothesised that small interfering RNA (siRNA) targeting this immediate-early gene may be useful agents that suppress endothelial growth and neovascularisation. Here we show that Jun siRNA inhibits Jun mRNA and protein expression in murine microvascular endothelial cells, blocks cell proliferation and suppresses migration in a scratch-wound assay. It also inhibits three-dimensional tubular formation on basement membrane extracts and reduces angiogenesis in mice bearing Matrigel plugs as subcutaneous implants. Single intravitreal administration of Jun siRNA reduces neovascularisation in a murine model of proliferative retinopathy, and suppresses endothelial JUN and matrix metalloproteinase-2 (MMP-2) immunoreactivity in retinal vessels, data supported by its repression of MMP-2 expression and gelatinolytic activity in vitro. Co-administration of TGFbeta with the siRNA reverses this neovascular inhibitory effect, which is in turn abrogated by cis-9-octadecenoyl-N-hydroxylamide, consistent with the involvement of a metalloproteinase such as MMP-2. Thus, JUN siRNA can serve as a specific inhibitor of aberrant endothelial and neovascular growth.

Suppression of vascular permeability and inflammation by targeting of the transcription factor c-Jun.

Conventional anti-inflammatory strategies induce multiple side effects, highlighting the need for novel targeted therapies. Here we show that knockdown of the basic-region leucine zipper protein, c-Jun, by a catalytic DNA molecule, Dz13, suppresses vascular permeability and transendothelial emigration of leukocytes in murine models of vascular permeability, inflammation, acute inflammation and rheumatoid arthritis. Treatment with Dz13 reduced vascular permeability due to cutaneous anaphylactic challenge or VEGF administration in mice. Dz13 also abrogated monocyte-endothelial cell adhesion in vitro and abolished leukocyte rolling, adhesion and extravasation in a rat model of inflammation. Dz13 suppressed neutrophil infiltration in the lungs of mice challenged with endotoxin, a model of acute inflammation. Finally, Dz13 reduced joint swelling, inflammatory cell infiltration and bone erosion in a mouse model of rheumatoid arthritis. Mechanistic studies showed that Dz13 blocks cytokine-inducible endothelial c-Jun, E-selectin, ICAM-1, VCAM-1 and VE-cadherin expression but has no effect on JAM-1, PECAM-1, p-JNK-1 or c-Fos. These findings implicate c-Jun as a useful target for anti-inflammatory therapies.

Locked nucleic acid modified DNA enzymes targeting early growth response-1 inhibit human vascular smooth muscle cell growth.

Smooth muscle cell (SMC) proliferation and migration are key processes that occur in the pathogenesis of atherosclerosis and post-angioplasty restenosis. In the present study, we designed locked nucleic acid (LNA)-modified DNAzymes targeting a specific region spanning the translational start site of human EGR-1, an immediate-early gene, wherein two of the nucleotides in each of the 9+9 hybridizing arms of the DNAzyme were substituted with LNA monomers. In vitro cleavage experiments revealed that the LNA- modified DNAzyme (LzF4) cleaved a 32P-labelled 388 nt EGR-1 transcript with greater efficacy than its native unmodified phosphodiester counterpart, DzF. The scrambled versions of these molecules, LzF4SCR and DzFSCR, did not display any ability to cleave the transcript. Western blot analysis revealed that both active molecules abrogated serum-inducible EGR-1 protein expression in primary human aortic SMCs and inhibited serum-inducible SMC proliferation in a dose-dependent and non-toxic manner. SMC proliferation was inhibited by >50% with LzF4 at concentrations as low as 20 nM, whereas inhibition by DzF at this concentration was not evident. Finally, LzF4 and DzF inhibited SMC regrowth from the wound edge after mechanical injury in vitro. In contrast, neither DzFSCR nor LzF4SCR had any influence on EGR-1 protein expression, SMC proliferation or regrowth. These findings provide the first functional demonstration of LNA-modified DNAzyme efficacy in a biological setting of any kind. These studies also demonstrate that LNA modification increases DNAzyme potency without necessarily compromising specificity.

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.

c-Jun regulates vascular smooth muscle cell growth and neointima formation after arterial injury. Inhibition by a novel DNA enzyme targeting c-Jun.

Neointima formation is a characteristic feature of common vascular pathologies, such as atherosclerosis and post-angioplasty restenosis, and involves smooth muscle cell proliferation. Determination of whether the bZIP transcription factor c-Jun plays a direct regulatory role in arterial lesion formation, or indeed in other disease, has been hampered by the lack of a potent and specific pharmacological inhibitor. c-Jun is poorly expressed in the uninjured artery wall and transiently induced following arterial injury in animal models. Here we generated a gene-specific DNAzyme-targeting c-Jun. We show that c-Jun protein is expressed in human atherosclerotic lesions. Dz13, a catalytically active c-Jun DNAzyme, cleaved c-Jun RNA and inhibited inducible c-Jun protein expression in vascular smooth muscle cells. Dz13 blocked vascular smooth muscle cell proliferation with potency exceeding its exact non-catalytic antisense oligodeoxynucleotide equivalent. Moreover, Dz13 abrogated smooth muscle cell repair following scraping injury in vitro and intimal thickening in injured rat carotid arteries in vivo. These studies demonstrate the positive influence on neointima formation by c-Jun and the therapeutic potential of a DNAzyme controlling its expression.

Antisense Egr-1 RNA driven by the CMV promoter is an inhibitor of vascular smooth muscle cell proliferation and regrowth after injury.

Smooth muscle cell (SMC) migration and proliferation are key events in the pathogenesis of atherosclerotic and post-angioplasty restenotic lesions. Mechanical injury to the artery wall induces the SMC expression of the zinc finger transcription factor, early growth response factor-1 (Egr-1). Egr-1 in turn can bind and activate the promoters of many genes, whose products influence vascular repair. Here, a 127-bp cDNA fragment corresponding to the 5' region of murine Egr-1 mRNA was cloned into a CMV-driven expression vector, in the sense or antisense orientation. We demonstrate that antisense Egr-1 RNA inhibited rat vascular SMC proliferation, whereas the sense counterpart produced only a modest effect. By semi-quantitative reverse-transcription PCR, antisense Egr-1 RNA blocked serum-inducible Egr-1 mRNA expression. Western blot analysis demonstrated that antisense RNA overexpression inhibited Egr-1 protein synthesis, without affecting levels of the immediate early gene product, c-fos. Finally, antisense Egr-1 RNA overexpression inhibited SMC regrowth after mechanical injury in vitro. In contrast, sense Egr-1 RNA had no effect on SMC repair, Egr-1 mRNA expression or protein synthesis. Analysis of transfection efficiencies revealed that both CMV-driven constructs (sense and antisense) were taken up by the SMCs with equivalent efficiency. These findings provide the first demonstration of antisense RNA strategies targeting Egr-1 as inhibitors of Egr-1 and Egr-1-dependent cellular processes. The antisense RNA approach may be potentially useful in gene therapeutic efforts to control SMC growth in the injured artery wall.