DNA enzyme ED5 depletes egr-1 and inhibits neointimal hyperplasia in rats.

OBJECTIVES: Depletion of early growth response factor-1 (Egr-1) by a DNA enzyme, ED5, inhibits neointimal hyperplasia (NH) following vascular injury by an unknown mechanism. The aim of this study was to characterize the effects of ED5 in a rat carotid injury model in order to elucidate the mechanism by which ED5 inhibits NH. METHODS: ED5 was transfected into the arterial wall of Wistar rats using FuGENE6 transfection reagent following artery balloon injury. Hematoxylin and eosin staining, immunohistochemistry, real-time reverse transcription polymerase chain reaction and Western blotting analysis were used to characterize the response to ED5. RESULTS: NH decreased significantly in the ED5- plus FuGENE6-treated rats (p < 0.05) compared with the control groups, and this was accompanied by a reduced inflammatory response. Egr-1 mRNA and protein levels were significantly decreased in the ED5-treated group, as expected. The decrease in Egr-1 was accompanied by decreases in the mRNA and protein levels of PDGF-BB, Cyclin D1, CDK4, MCP-1, and ICAM-1 (p < 0.05). CONCLUSIONS: Transfection of the Egr-1-specific synthetic DNA enzyme ED5 significantly reduced NH after injury in rats, at least in part, as a result of decreased expression of downstream proliferative genes such as PDGF-BB, Cyclin D1, CDK4, and the inflammatory factors MCP-1 and ICAM-1.

Positive regulation of the Egr-1/osteopontin positive feedback loop in rat vascular smooth muscle cells by TGF-beta, ERK, JNK, and p38 MAPK signaling.

Previous studies identified a positive feedback loop in rat vascular smooth muscle cells (VSMCs) in which early growth response factor-1 (Egr-1) binds to the osteopontin (OPN) promoter and upregulates OPN expression, and OPN upregulates Egr-1 expression via the extracellular signal-regulated protein kinase (ERK) signaling pathway. The current study examined whether transforming growth factor-beta (TGF-beta) activity contributes to Egr-1 binding to the OPN promoter, and whether other signaling pathways act downstream of OPN to regulate Egr-1 expression. ChIP assays using an anti-Egr-1 antibody showed that amplification of the OPN promoter sequence decreased in TGF-beta DNA enzyme-transfected VSMCs relative to control VSMCs. Treatment of VSMCs with PD98059 (ERK inhibitor), SP600125 (JNK inhibitor), or SB203580 (p38 MAPK inhibitor) significantly inhibited OPN-induced Egr-1 expression, and PD98059 treatment was associated with the most significant decrease in Egr-1 expression. OPN-stimulated VSMC cell migration was inhibited by SP600125 or SB203580, but not by PD98059. Furthermore, MTT assays showed that OPN-mediated cell proliferation was inhibited by PD98059, but not by SP600125 or SB203580. Taken together, the results of the current study show that Egr-1 binding to the OPN promoter is positively regulated by TGF-beta, and that the p38 MAPK, JNK, and ERK pathways are involved in OPN-mediated Egr-1 upregulation.

EGR-1 decoy ODNs inhibit vascular smooth muscle cell proliferation and neointimal hyperplasia of balloon-injured arteries in rat.

AIMS: Early growth response factor-1 (EGR-1) plays a master regulatory role in multiple cardiovascular pathological processes, such as atherosclerosis and restenosis. For investigating the possibility of using "decoy" strategy to prevent and cure vascular hyperplasia disease, we synthesized the double-stranded, cis-element, decoy oligodeoxynucleotides (ODNs) targeting EGR-1. MAIN METHODS: EGR-1 decoy ODNs were transfected into the balloon-injured arteria carotis of rat as well as primary cultures of vascular smooth muscle cells (VSMC). Changes in the thickness of the arterial intima were evaluated by hematoxylin-eosin (HE) staining. VSMC proliferation, DNA synthesis, cell cycle and apoptosis were observed via MTT assay, bromodeoxyuridine (BrdU) incorporation and flow cytometry (FCM). Changes in the expression of EGR-1, and cell cycle related genes, were detected by reverse transcriptase polymerase chain reaction (PT-PCR) and western blot. KEY FINDINGS: As a result of specific binding to EGR-1 protein, transfected EGR-1 decoy ODNs can reduce EGR-1 promoter affinity, hamper the transcriptional activation of EGR-1-dependent genes, block cell cycle progression of VSMCs, and inhibit neointimal hyperplasia. SIGNIFICANCE: Through regulating the cell cycle progression and transcription of target gene, this new "decoy" strategy targeting EGR-1 provides further experimental evidence demonstrating the effectiveness of gene therapy in the treatment of restenosis following percutaneous coronary interventions.

Egr-1 upregulates OPN through direct binding to its promoter and OPN upregulates Egr-1 via the ERK pathway.

Early growth response factor-1 (Egr-1) and osteopontin (OPN) play important roles in the migration and proliferation of vascular smooth muscle cells (VSMC), but little is known about their relationship. Therefore, we transfected VSMCs with either Egr-1 cDNA, Opn cDNA, a DNA enzyme designed to target Egr-1 (ED5), or antisense Opn oligodeoxynucleotides and examined changes in Egr-1 and OPN expression at the mRNA and protein levels. OPN expression levels were increased in cells that were stably transfected with Egr-1 cDNA. By contrast, both Egr-1 and OPN expression were reduced when ED5 was transfected into Egr-1-expressing cells. Similarly, Opn transfection upregulated Egr-1 levels, while Opn anti-sense oligodeoxynucleotide transfection decreased Egr-1 expression. ChIP analysis showed that Egr-1 binds to the Opn gene promoter. Furthermore, treatment with the extracellular-regulated protein kinase (ERK) inhibitor PD98059 inhibited the upregulation of Egr-1 by OPN. We find that Egr-1 and OPN positively regulate each other in VSMCs.

[Upregulated PGC-NRF-mtTFA expressions contributed to the development of atherosclerosis in rabbits fed with a high fat diet].

OBJECTIVE: To investigate the relationship between PPAR coactivator 1 (PGC-1), nuclear respiratory factor (NRF), mitochondrial transcription factor A (mtTFA) expressions of vascular smooth muscle cells (VSMC) and development of atherosclerosis in a rabbit model. METHODS: Atherosclerotic model was established by feeding the rabbits with high-fat diet for 4, 8 and 12 weeks (n = 10 each). Another 8 rabbits fed with normal diet served as normal controls. Intima-media ratio, mRNA and protein expressions of PGC-1, NRF, mtTFA and SMemb, a marker for synthetic VSMC, were detected on aorta specimens. RESULTS: With the blood lipid increased, the intima-media ratio rose from (0.031 +/- 0.010) microm up to (0.814 +/- 0.258) microm during 12 weeks. Increasing SMemb means that synthetic VSMC grew more and more. The expressions of PGC-1 became significant after 4 weeks (P < 0.01), while that of NRF-1 and mtTFA rose significantly after 8 weeks (P < 0.01). CONCLUSIONS: The PGC-NRF-mtTFA pathway might play a critical role in VSMC proliferation and development of atherosclerosis.

Transfected synthetic DNA enzyme gene specifically inhibits Egr-1 gene expression and reduces neointimal hyperplasia following balloon injury in rats.

BACKGROUND: Early growth response factor-1 (Egr-1) controls the gene expression involved in postangioplasty restenosis. In the present study we synthesized specific catalytic DNA targeting sequences in human Egr-1 mRNA to investigate the effects on artery balloon injury. METHODS: The catalytic DNA, ED5, was synthesized and transfected into the arterial wall of Wistar rats using the FuGENE6 transfection reagent. The animals were euthanized at day 3, 7, 14 and 21 following artery balloon injury. Serum nitric oxide (NO), nitric oxide synthase (NOS), and endothelin (ET) levels were measured before sacrifice. Histopathological changes to the arterial tissue were evaluated by H&E staining and observed via transmission electromicroscopy. Egr-1, PCNA and TGF-beta(1) expression was detected by immunohistochemistry, RT-PCR, and western-blot. RESULTS: Compared with the control groups, ED5-treated rats exhibited increased levels of both NO and NOS (p<0.05); by contrast, plasma ET levels were decreased relative to controls (p<0.05). Neointimal hyperplasia (NH) was significantly reduced and vascular smooth muscle cells (VSMCs) in the neointima exhibited a general contractile phenotype. Both protein and mRNA expression of Egr-1, PCNA, and TGF-beta(1) in the ED5-treated group were decreased at each time point (p<0.001). CONCLUSIONS: ED5 may specifically inhibit Egr-1 gene expression and reduce NH after balloon injury in rats; the latter effect may be mediated by a down-regulation of TGF-beta(1) and up-regulation of NOS to inhibit NH following balloon injury.