Study of Early Growth Response Gene-1 DNA Enzyme on Preventing Stenosis and Occlusion of Autogenous Vein Graft: An Experiment with Rats_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

LIU Chengwei ¹ , ZHANG Xuesong ¹ , WANG Shi ¹ , TIAN Hao ¹ , CHENG Mingxun ¹ , WANG Shuqing ¹ , HU Xinhua ² , ZHANG Qiang ² , XIN Shijie ² , DUAN Zhiquan. ²

1.Department of General Surgery, The First Affiliated Hospital, Jiamusi University, Jiamusi 154003, Heilongjiang Province, China;;
2.Department of Vascular Surgery, The First Affiliated Hospital, China Medical University, Shenyang 110001, Liaoning Province, China;

Corresponding author：

Keywords：

DNA enzyme; Vein graft; Intimal hyperplasia

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ObjectiveTo detect the inhibitory effect of early growth response gene-1 DNA enzyme (EDRz) on proliferation of vascular smooth muscle cell (VSMC) and intimal hyperplasia, and confirm the effect of gene therapy on stenosis and occlusion after vein transplantation. MethodsEDRz was constructed, and autogenous vein graft model was established with Wistar rats, transplanting the right jugular vein to infra renal abdominal aorta by microsurgical technique. EDRz was transfected to the graft veins and the vein graft samples were harvested at hour 1, 2, 6, 24 and on day 3, 7, 14, 28, 42 after grafting, 10 Wistar rats were randomly selected in every time. The expression of EDRz in transfected vein graft was detected by fluorescent microscope. Egr-1 mRNA was measured by reverse transcription-PCR (RT-PCR) and in situ hybridization, respectively. The protein expression of Egr-1 was detected by Western blot and immunohistochemistry, respectively. HE stained vein grafts were observed under microscope. Results① The results of EDRz transfected vein graft: At hour 1 after grafting, EDRz was mainly located in adventitia, tunica media, and partial endothelial cells of vein graft; At hour 2, 6, and 24, EDRz was located in tunica media of vein graft; and on day 7, it was mainly located in intima of vein graft. There wasn’t EDRz in vein grafts on day 14, 28, and 42. ② The results of expression of Egr-1 mRNA: Detection by RT-PCR: At hour 1 after transfecting, the expression of Egr-1 mRNA arrived at the peak, and declined at hour 2, 6, and 24. The expression was tenuity on day 3. Egr-1 mRNA expression was not found on day 7, 14, 28, and 42. The expression of Egr-1 mRNA at hour 1 was significantly higher than that of the other time point (P lt;0.01). The result of in situ hybridization was coincident with RT-PCR. ③ The results of expression of Egr-1 protein: The result of Western blot: There was no expression of Egr-1 protein in normal veins. At hour 2 after grafting, expression of Egr-1 protein was found, and declined at hour 6, 24, and on day 3. There was no expression of Egr-1 protein at hour 1, and on day 7, 14, 28, and 42. The expression of Egr-1 protein at hour 2 was significantly higher than that of the other time point (P lt;0.01). The result of immunohistochemistry was coincident with Western blot. ④The degree of VSMC hyperplasia and intimal thickness were lighter in EDRz transfected vein grafts than that in nottransfected vein grafts contemporarily. ConclusionsEDRz could reduce the expression of Egr-1 in autogenous vein graft, and could effectively restrain VSMC proliferation and intimal hyperplasia, and prevent vascular stenosis and occlusion after vein grafting.

Citation： LIU Chengwei,ZHANG Xuesong,WANG Shi,TIAN Hao,CHENG Mingxun,WANG Shuqing,HU Xinhua,ZHANG Qiang,XIN Shijie,DUAN Zhiquan.. Study of Early Growth Response Gene-1 DNA Enzyme on Preventing Stenosis and Occlusion of Autogenous Vein Graft: An Experiment with Rats. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2011, 18(10): 1043-1048. doi: Copy

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2.	Stephenson ML, Zamecnik PC. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide [J]. Proc Natl Acad Sci USA, 1978, 75(1): 285288.
3.	Pavri R, Gazumyan A, Jankovic M, et al. Activationinduced cytidine deaminase targets DNA at sites of RNA polymerase Ⅱ stalling by interaction with Spt5 [J]. Cell, 2010, 143(1): 122133.
4.	刘程伟, 胡新华, 张雪松, 等. Egr1在自体移植静脉中的表达及意义 [J]. 中国普外基础与临床杂志, 2006, 13(6): 642646.
5.	Breaker RR, Joyce GF. A DNA enzyme that cleaves RNA [J]. Chem Biol, 1994, 1(4): 223229.
6.	Tell G, Wilson DM, Lee CH. Intrusion of a DNA repair protein in the rnome world: is this the beginning of a new era? [J]. Mol Cell Biol, 2010, 30(2): 366371.
7.	Lam CH, Perrin DM. Introduction of guanidiniummodified deoxyuridine into the substrate binding regions of dnazyme 10－23 to enhance target affinity: implications for dnazyme design [J]. Bioorg Med Chem Lett, 2010, 20(17): 51195122.
8.	Li J, Wang N, Luo Q, et al. The 10－23 DNA enzyme generated by a novel expression vector mediate inhibition of taco expression in macrophage [J]. Oligonucleotides, 2010, 20(2): 6168.
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10.	Patel N, Kalra VK. Placenta growth factorinduced early growth response 1 (Egr1) regulates hypoxiainducible factor1alpha (HIF1alpha) in endothelial cells [J]. J Biol Chem, 2010, 285(27): 2057020579.
11.	Mckinney KA, AlRawi N, Maciag PC, et al. Effect of a novel DNA vaccine on angiogenesis and tumor growth in vivo [J]. Arch Otolaryngol Head Neck Surg, 2010, 136(9): 859864.
12.	Lockney D, Franzen S, Lommel S. Viruses as nanomaterials for drug delivery [J]. Methods Mol Biol, 2011, 726: 207221.
13.	Yu HW, Liu QF, Liu GN. 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 [J]. Biochem Biophys Res Commun, 2010, 396(2): 451456.
14.	Wang B, Cao L, Chiuman W, et al. Probing the function of nucleotides in the catalytic cores of the 8-17 and 10-23 DNAzymes by abasic nucleotide and C3 spacer substitutions [J]. Biochemistry, 2010, 49(35): 75537562.
15.	Deindl E, Fischer S, Preissner KT. New directions in inflammation and immunity: the multifunctional role of the extracellular RNA/RNase system [J]. Indian J Biochem Biophys, 2009, 46(6): 461466.
16.	Ni J, Waldman A, Khachigian LM. cJun regulates shear and injuryinducible Egr1 expression, vein graft stenosis after autologous endtoside transplantation in rabbits, and intimal hyperplasia in human saphenous veins [J]. J Biol Chem, 2010, 285(6): 40384048.
17.	Robaldo L, Montserrat JM, Iribarren AM. 10-23 DNA zyme modified with (2′R)and (2′S)2′deoxy2′Cmethyluridine in the catalytic core [J]. Bioorg Med Chem Lett, 2010, 20(15): 43674370.
18.	Dyson OF, Traylen CM, Akula SM. Cell membranebound Kaposi’s sarcomaassociated herpesvirusencoded glycoprotein B promotes virus latency by regulating expression of cellular Egr1 [J]. J Biol Chem, 2010, 285(48): 3749137502.
19.	Liu GN, Teng YX, Yan W. Transfected synthetic DNA enzyme gene specifically inhibits Egr1 gene expression and reduces neointimal hyperplasia following balloon injury in rats [J]. Int J Cardiol, 2008, 129(1): 118124.
20.	杨军, 胡新华, 刘程伟, 等. 反义寡脱氧核苷酸抑制survivin基因表达对移植静脉内膜增生的影响 [J]. 中国普外基础与临床杂志, 2006, 13(6): 647652.

1. Paterson BM, Roberts BE, Kuff EL. Structural gene identification and mapping by DNAmRNA hybridarrested cellfree translation [J]. Proc Natl Acad Sci USA, 1977, 74(10): 43704374.
2. Stephenson ML, Zamecnik PC. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide [J]. Proc Natl Acad Sci USA, 1978, 75(1): 285288.
3. Pavri R, Gazumyan A, Jankovic M, et al. Activationinduced cytidine deaminase targets DNA at sites of RNA polymerase Ⅱ stalling by interaction with Spt5 [J]. Cell, 2010, 143(1): 122133.
4. 刘程伟, 胡新华, 张雪松, 等. Egr1在自体移植静脉中的表达及意义 [J]. 中国普外基础与临床杂志, 2006, 13(6): 642646.
5. Breaker RR, Joyce GF. A DNA enzyme that cleaves RNA [J]. Chem Biol, 1994, 1(4): 223229.
6. Tell G, Wilson DM, Lee CH. Intrusion of a DNA repair protein in the rnome world: is this the beginning of a new era? [J]. Mol Cell Biol, 2010, 30(2): 366371.
7. Lam CH, Perrin DM. Introduction of guanidiniummodified deoxyuridine into the substrate binding regions of dnazyme 10－23 to enhance target affinity: implications for dnazyme design [J]. Bioorg Med Chem Lett, 2010, 20(17): 51195122.
8. Li J, Wang N, Luo Q, et al. The 10－23 DNA enzyme generated by a novel expression vector mediate inhibition of taco expression in macrophage [J]. Oligonucleotides, 2010, 20(2): 6168.
9. Maeda J, Nishida M, Takikawa H, et al. Inhibitory effects of sulfobacin B on DNA polymerase and inflammation [J]. Int J Mol Med, 2010, 26(5): 751758.
10. Patel N, Kalra VK. Placenta growth factorinduced early growth response 1 (Egr1) regulates hypoxiainducible factor1alpha (HIF1alpha) in endothelial cells [J]. J Biol Chem, 2010, 285(27): 2057020579.
11. Mckinney KA, AlRawi N, Maciag PC, et al. Effect of a novel DNA vaccine on angiogenesis and tumor growth in vivo [J]. Arch Otolaryngol Head Neck Surg, 2010, 136(9): 859864.
12. Lockney D, Franzen S, Lommel S. Viruses as nanomaterials for drug delivery [J]. Methods Mol Biol, 2011, 726: 207221.
13. Yu HW, Liu QF, Liu GN. 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 [J]. Biochem Biophys Res Commun, 2010, 396(2): 451456.
14. Wang B, Cao L, Chiuman W, et al. Probing the function of nucleotides in the catalytic cores of the 8-17 and 10-23 DNAzymes by abasic nucleotide and C3 spacer substitutions [J]. Biochemistry, 2010, 49(35): 75537562.
15. Deindl E, Fischer S, Preissner KT. New directions in inflammation and immunity: the multifunctional role of the extracellular RNA/RNase system [J]. Indian J Biochem Biophys, 2009, 46(6): 461466.
16. Ni J, Waldman A, Khachigian LM. cJun regulates shear and injuryinducible Egr1 expression, vein graft stenosis after autologous endtoside transplantation in rabbits, and intimal hyperplasia in human saphenous veins [J]. J Biol Chem, 2010, 285(6): 40384048.
17. Robaldo L, Montserrat JM, Iribarren AM. 10-23 DNA zyme modified with (2′R)and (2′S)2′deoxy2′Cmethyluridine in the catalytic core [J]. Bioorg Med Chem Lett, 2010, 20(15): 43674370.
18. Dyson OF, Traylen CM, Akula SM. Cell membranebound Kaposi’s sarcomaassociated herpesvirusencoded glycoprotein B promotes virus latency by regulating expression of cellular Egr1 [J]. J Biol Chem, 2010, 285(48): 3749137502.
19. Liu GN, Teng YX, Yan W. Transfected synthetic DNA enzyme gene specifically inhibits Egr1 gene expression and reduces neointimal hyperplasia following balloon injury in rats [J]. Int J Cardiol, 2008, 129(1): 118124.
20. 杨军, 胡新华, 刘程伟, 等. 反义寡脱氧核苷酸抑制survivin基因表达对移植静脉内膜增生的影响 [J]. 中国普外基础与临床杂志, 2006, 13(6): 647652.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Study of Early Growth Response Gene-1 DNA Enzyme on Preventing Stenosis and Occlusion of Autogenous Vein Graft: An Experiment with Rats

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