- Email: [email protected]
Effect of AP-1 Decoy Using Hemagglutinating Virus of Japan-Liposome on the Intimal Hyperplasia of the Autogenous Vein Graft in Mongrel Dogs
Effect of AP-1 Decoy Using Hemagglutinating Virus of JapanLiposome on the Intimal Hyperplasia of the Autogenous Vein Graft in Mongrel Dogs W.H. Cho, H.T. Kim, J.H. Koo, and I.K. Lee ABSTRACT Intimal hyperplasia is the leading cause of late vein graft failure. Smooth muscle cell proliferation and migration is the underlying mechanism. Pharmacological approaches to prolong vein graft patency have produced limited results. AP-1 proteins play a role in the expression of many genes involved in cellular proliferation and cell cycle progression. Previously we reported inhibition of vascular smooth muscle cell migration, proliferation, and intimal hyperplasia in the balloon-injured rat carotid artery using an AP-1 decoy with HVJ-liposomes. In this report, we evaluated the effect of the AP-1 decoy on intimal hyperplasia in a large animal model. The jugular vein was transfected with hemagglutinating virus of Japan-liposomes containing the AP-1 decoy or scrambled oligonucleotides. An interposition graft was performed with the pretreated jugular vein between the transected femoral arteries. The graft was harvested at 16 weeks after the procedure. The intimal area was compared: the intimal area of the AP-1 decoy-treated versus control group was 47.3 ⫾ 15.2 versus 102.3 ⫾ 15.9 (P ⬍ .05), respectively. In conclusion, AP-1 decoy using HVJ-liposomes effectively prevented intimal hyperplasia of an autogenous vein graft in mongrel dogs.
I
NTIMAL HYPERPLASIA is the leading cause of the late vein graft failure. Smooth muscle cell migration and proliferation is the underlying mechanism of intimal hyperplasia. Pharmacological approaches to prolong vein graft patency have produced limited results. AP-1 proteins play a role in the expression of many genes involved in cellular proliferation and cell cycle progression. Previously, we showed effective inhibition of vascular smooth cell proliferation in vitro and neointima formation in vivo with AP-1 decoy using the hemagglutinating virus of Japan-liposome method.1 Here we have reported our data about the effect of an AP-1 decoy in a large animal vein graft model. MATERIALS AND METHODS Reconstruction of our HVJ-liposome and AP-1 decoy have been described in detail in a previous report.1,2
BASE SEQUENCE OF AP-1 DECOY ODN Oligodeoxynucleotide (ODN) used in our research is dumbbellshaped ODN, which is inocuous and resistant to the exonuclease.3 The base sequences of the AP-1 decoy ODN (AODN) and mismatched ODN (MODN) are AODN, 5=-GGATCCATGACTCAGAAGACACACGTCTTCTGAGTCAT-3=; MODN, 5=-GGA-
TCCAAATCTCAGAAGACGACACACGTCTTCTGAGATTT-3= (underlined sequences are binding sites).
Preparation of HVJ-Liposomes Preparation of the liposomes was described previously.2 Purified HVJ was ultraviolet light inactivated for 3 minutes. The mixture of inactivated HVJ and liposomes was incubated then HVJ-liposomes isolated by sucrose density gradient centrifugation.
Vascular Bypass Eight male mongrel dogs weighing 18 to 25 kg included four for the AP-1 decoy group and the other four for the control group. Under general anesthesia, the right jugular vein was obtained for transfection with AP-1 decoy or a scrambled ODN during nondistending From the Dept. of Surgery, Dongsan Medical Center, Keimyung University (W.H.C., H.T.K., J.H.K.), and Dept. of Internal Medicine, Kyungpook National University Hospital (I.K.L.), Daegu, South Korea. This work was supported by a research promoting grant from the Keimyung University Dongsan Medical Center in 2002-2003. Address reprint requests to W.H. Cho, Department of Surgery, Dongsan Medical Center, Keimyung University 194, Dongsandong, Jung-gu, Daegu, S. Korea. E-mail: [email protected]
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.06.103
Transplantation Proceedings, 38, 2161–2163 (2006)
2161
2162
CHO, KIM, KOO ET AL
Fig 1. Effect of AP-1 decoy ODN on neointimal formation in the autogenous vein graft. Light microscopic appearance of autogenous vein graft in dog 16 weeks after interposition graft (a, b: H&E stain, ⫻50; c, d: elastic stain ⫻ 100); control group (b, d) and AP-1 treated group (a, c). e: Bars represents intimal area of autogenous vein graft 16 weeks after interposition graft; intimal area was measured by computerized planimetry (Leica Q500MC system, Leica Cambridge, England). inflation for 15 minutes at room temperature. An interposition graft was performed with the pretreated jugular vein in transected femoral arteries using 8-0 prolene interrupted sutures. Antibiotics were administered at the induction of anesthesia and for 3 days thereafter.
Histologic Study of Vein Graft Sixteen weeks after the bypass procedure, the dogs were anesthetized and the grafts fixed in situ with 4% paraformaldehyde solution under a pressure of 100 mm Hg for 15 minutes. Thereafter, they were removed with a short segment of adjacent femoral artery. Four longitudinal sections of the grafts were evaluated using hematoxylin and eosin (H&E) and elastin staining. The intimal area was measured by computerized planimetry (Leica Q500MC System, Leica, Cambridge, England). Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) was performed with anti-PCNA antibody (Santa Cruz, Santa Cruz, Calif, USA) in one dog of each group at 4 weeks after the bypass.
Statistical Analysis Intimal areas of the two groups were compared using paired Student’s t test.
RESULTS
All animals were alive until 16 weeks after the procedure. One vein graft of the control group was occluded due to thrombus. All six vein grafts of the AP-1 decoy-treated group were patent. Intimal areas of AP-1 decoy-treated group versus control group were 47.3 ⫾ 15.2 versus 102.3 ⫾ 15.9 (P ⬍ .05; Fig 1). Immunohistochemical staining for PCNA showed marked suppression in the AP-1 decoytreated group compared with the control group (data not shown).
DISCUSSION
Vein graft bypass is the standard, effective treatment for patients with chronic arterial occlusive disease. But its effectiveness is limited by intimal hyperplasia. Intimal hyperplasia is the leading cause of vein graft occlusion at 1 month to 2 years after the bypass procedure. The intimal hyperplasia is initiated by mechanical injury, such as balloon angioplasty, thrombectomy, and surgical anastomosis, and electric or radiation injury. It is known that 24 hours
AP-1 DECOY AND HVJ-LIPOSOME EFFECT
after intimal injury vascular smooth muscle cells (VSMCs) of the media proliferate and migrate to the intima.4,5 Mitogen-activated protein kinase (MAP kinase) and AP-1 cascades play central roles in proliferation and growth of VSMC.6,7 Among the MAP kinases, JNK bind and activate c-jun, which comprise the AP-1 complex. AP-1 is a diverse peptide composed of fos (v-fos, c-fos, fosB, Fra-1, Fra-2), jun (v-jun, c-jun, junB, junD), or activating transcription factor protein family.8 According to the composition of these peptide, AP-1 binds to specific DNA sequences expressing various genes involved in cell proliferation or construction of the extracellular matrix. A decoy strategy has been applied in a variety of situations, such as tumor growth and infiltration, angiogenesis, myocardial infarction, and hypertension. Mann et al9 used an E2F decoy to achieve significant inhibition of intimal hyperplasia of vein grafts in a high-risk group. The AP-1 decoy inhibits many genes involved in intimal hyperplasia, such as plasminogen activator inhibitor-1 (PAI-1),10 TGF-,11 and endothelin-1 (ET-1).12 The success of a transcription factor decoy strategy is dependent on the specificity, stability, and efficacy of intracellular transfection of decoy ODN. Chemical transformations, such as phosphothioation or methylphosphonation, to augment the stability of decoy ODN have shown immunogenicity and a poor response to RNaseH. The decoy ODN we used is circular dumbbell shaped, showing high resistance to exonucleases. So it is active for a longer duration, showing greater inhibition of intimal hyperplasia. Since gene delivery by endocytosis is subject to destruction by nucleases, its efficacy was low. Our delivery system, HVJliposomes, acted by fusion with cell membranes. The HVJliposomes method was first introduced by Kaneda13 and Nakanishi.14 Fusigenic F-protein of the HVJ viral coat allowed fusion with cell membranes under neutral pH and body temperature. The safety of the HVJ-liposomes was documented by intraocular injection, continuous intravenous infusion, or intraventricular injection in primates. Our previous data also showed 10 to 100 times greater transfection efficacy than LipopectAMINE.1 Vein grafts for arterial occlusive disease are ideal candidates for gene therapy because the vein for the graft is taken out of the body in usual practice. Therefore it can be treated with minimal systemic introduction of genetic material. Our data showed effective inhibition of intimal hyperplasia of a vein graft in a large animal model. Since Mann et al9 showed significant inhibition of intimal hyper-
2163
plasia of a vein graft with an E2F decoy using simple pressure delivery in normal saline, our more effective delivery system is expected to show better results in the clinical setting. REFERENCES 1. Ahn JD, Morishita R, Kaneda Y, et al: Inhibitory effects of novel AP-1 decoy oligodeoxynucleotides on vascular smooth muscle cell proliferation in vitro and neointimal formation in vivo. Circ Res 90:1325, 2002 2. Ahn JD, Morishita R, Kaneda Y, et al: Novel E2F decoy oligodeoxynucleotides inhibit in vitro vascular smooth muscle cell proliferation and in vivo neointimal hyperplasia. Gene Ther 9:1682, 2002 3. Lee I-K, Ahn JD, Kim HS, et al: Advantages of the circular dumbbell decoy in gene therapy and studies of gene regulation. Curr Drug Targ 4:619, 2003 4. Clowes AW, Clowes MM, Reidy MA: Kinetics of cellular proliferation after arterial injury, I: smooth muscle growth in the abscence of endothelium. Lab Invest 49:327, 1983 5. Clowes AW, Schwartz SM: Significance of quiescent smooth muscle cell migration in the injured rat carotid artery. Circ Res 56:139, 1985 6. Hu Y, Cheng L, Hochleitner BW, et al: Activation of mitogenactivated protein kinases (ERK/JNK) and AP-1 transcription factor in rat carotid arteries after balloon injury. Arterioscler Thromb Vasc Biol 17:2808, 1997 7. Pyles JM, March KL, Franklin M, et al: Activation of MAP kinase in vivo follows balloon overstretch injury of porcine coronary and carotid arteries. Circ Res 81:904, 1997 8. Karin M, Liu Z, Zandi E: AP-1 function and regulation. Curr Opin Cell Biol 9:240, 1997 9. Mann MJ, Whittemore AD, Donaldson MC, et al: Ex-vivo gene therapy of human vascular bypass grafts with E2F decoy: The PREVENT single-centre, randomised, controlled trial. Lancet 354:1493, 1999 10. Ahn JD, Morishita R, Kaneda Y, et al: Transcription factor decoy for activator protein-1 (AP-1) inhibits high glucose- and angiotensin II-induced type 1 plasminogen activator inhibitor (PAI-1) gene expression in cultured human vascular smooth muscle cells. Diabetologia 44:713, 2001 11. Morishita R, Gibbons GH, Horiuchi M, et al: Role of AP-1 complex in angiotensin II-mediated transforming growth factor- expression and growth of smooth muscle cells: using decoy approach against AP-1 binding site. Biochem Biophys Res Commun 243:361, 1998 12. Lauth M, Wagner AH, Cattaruzza M, et al: Transcriptional control of deformation-induced preproendothelin-1 gene expression in endothelial cells. J Mol Med 78:441, 2000 13. Kaneda Y, Uchida T, Kim J, et al: The improved efficient method for introducing macromolecules into cells using HVJ (Sendai virus) liposomes with gangliosides. Exp Cell Res 173:56, 1987 14. Nakanishi M, Uchida T, Sugawa H, et al: Efficient introduction of contents of liposomes into cells using HVJ (Sendai virus). Exp Cell Res 159:399, 1985
I
NTIMAL HYPERPLASIA is the leading cause of the late vein graft failure. Smooth muscle cell migration and proliferation is the underlying mechanism of intimal hyperplasia. Pharmacological approaches to prolong vein graft patency have produced limited results. AP-1 proteins play a role in the expression of many genes involved in cellular proliferation and cell cycle progression. Previously, we showed effective inhibition of vascular smooth cell proliferation in vitro and neointima formation in vivo with AP-1 decoy using the hemagglutinating virus of Japan-liposome method.1 Here we have reported our data about the effect of an AP-1 decoy in a large animal vein graft model. MATERIALS AND METHODS Reconstruction of our HVJ-liposome and AP-1 decoy have been described in detail in a previous report.1,2
BASE SEQUENCE OF AP-1 DECOY ODN Oligodeoxynucleotide (ODN) used in our research is dumbbellshaped ODN, which is inocuous and resistant to the exonuclease.3 The base sequences of the AP-1 decoy ODN (AODN) and mismatched ODN (MODN) are AODN, 5=-GGATCCATGACTCAGAAGACACACGTCTTCTGAGTCAT-3=; MODN, 5=-GGA-
TCCAAATCTCAGAAGACGACACACGTCTTCTGAGATTT-3= (underlined sequences are binding sites).
Preparation of HVJ-Liposomes Preparation of the liposomes was described previously.2 Purified HVJ was ultraviolet light inactivated for 3 minutes. The mixture of inactivated HVJ and liposomes was incubated then HVJ-liposomes isolated by sucrose density gradient centrifugation.
Vascular Bypass Eight male mongrel dogs weighing 18 to 25 kg included four for the AP-1 decoy group and the other four for the control group. Under general anesthesia, the right jugular vein was obtained for transfection with AP-1 decoy or a scrambled ODN during nondistending From the Dept. of Surgery, Dongsan Medical Center, Keimyung University (W.H.C., H.T.K., J.H.K.), and Dept. of Internal Medicine, Kyungpook National University Hospital (I.K.L.), Daegu, South Korea. This work was supported by a research promoting grant from the Keimyung University Dongsan Medical Center in 2002-2003. Address reprint requests to W.H. Cho, Department of Surgery, Dongsan Medical Center, Keimyung University 194, Dongsandong, Jung-gu, Daegu, S. Korea. E-mail: [email protected]
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.06.103
Transplantation Proceedings, 38, 2161–2163 (2006)
2161
2162
CHO, KIM, KOO ET AL
Fig 1. Effect of AP-1 decoy ODN on neointimal formation in the autogenous vein graft. Light microscopic appearance of autogenous vein graft in dog 16 weeks after interposition graft (a, b: H&E stain, ⫻50; c, d: elastic stain ⫻ 100); control group (b, d) and AP-1 treated group (a, c). e: Bars represents intimal area of autogenous vein graft 16 weeks after interposition graft; intimal area was measured by computerized planimetry (Leica Q500MC system, Leica Cambridge, England). inflation for 15 minutes at room temperature. An interposition graft was performed with the pretreated jugular vein in transected femoral arteries using 8-0 prolene interrupted sutures. Antibiotics were administered at the induction of anesthesia and for 3 days thereafter.
Histologic Study of Vein Graft Sixteen weeks after the bypass procedure, the dogs were anesthetized and the grafts fixed in situ with 4% paraformaldehyde solution under a pressure of 100 mm Hg for 15 minutes. Thereafter, they were removed with a short segment of adjacent femoral artery. Four longitudinal sections of the grafts were evaluated using hematoxylin and eosin (H&E) and elastin staining. The intimal area was measured by computerized planimetry (Leica Q500MC System, Leica, Cambridge, England). Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) was performed with anti-PCNA antibody (Santa Cruz, Santa Cruz, Calif, USA) in one dog of each group at 4 weeks after the bypass.
Statistical Analysis Intimal areas of the two groups were compared using paired Student’s t test.
RESULTS
All animals were alive until 16 weeks after the procedure. One vein graft of the control group was occluded due to thrombus. All six vein grafts of the AP-1 decoy-treated group were patent. Intimal areas of AP-1 decoy-treated group versus control group were 47.3 ⫾ 15.2 versus 102.3 ⫾ 15.9 (P ⬍ .05; Fig 1). Immunohistochemical staining for PCNA showed marked suppression in the AP-1 decoytreated group compared with the control group (data not shown).
DISCUSSION
Vein graft bypass is the standard, effective treatment for patients with chronic arterial occlusive disease. But its effectiveness is limited by intimal hyperplasia. Intimal hyperplasia is the leading cause of vein graft occlusion at 1 month to 2 years after the bypass procedure. The intimal hyperplasia is initiated by mechanical injury, such as balloon angioplasty, thrombectomy, and surgical anastomosis, and electric or radiation injury. It is known that 24 hours
AP-1 DECOY AND HVJ-LIPOSOME EFFECT
after intimal injury vascular smooth muscle cells (VSMCs) of the media proliferate and migrate to the intima.4,5 Mitogen-activated protein kinase (MAP kinase) and AP-1 cascades play central roles in proliferation and growth of VSMC.6,7 Among the MAP kinases, JNK bind and activate c-jun, which comprise the AP-1 complex. AP-1 is a diverse peptide composed of fos (v-fos, c-fos, fosB, Fra-1, Fra-2), jun (v-jun, c-jun, junB, junD), or activating transcription factor protein family.8 According to the composition of these peptide, AP-1 binds to specific DNA sequences expressing various genes involved in cell proliferation or construction of the extracellular matrix. A decoy strategy has been applied in a variety of situations, such as tumor growth and infiltration, angiogenesis, myocardial infarction, and hypertension. Mann et al9 used an E2F decoy to achieve significant inhibition of intimal hyperplasia of vein grafts in a high-risk group. The AP-1 decoy inhibits many genes involved in intimal hyperplasia, such as plasminogen activator inhibitor-1 (PAI-1),10 TGF-,11 and endothelin-1 (ET-1).12 The success of a transcription factor decoy strategy is dependent on the specificity, stability, and efficacy of intracellular transfection of decoy ODN. Chemical transformations, such as phosphothioation or methylphosphonation, to augment the stability of decoy ODN have shown immunogenicity and a poor response to RNaseH. The decoy ODN we used is circular dumbbell shaped, showing high resistance to exonucleases. So it is active for a longer duration, showing greater inhibition of intimal hyperplasia. Since gene delivery by endocytosis is subject to destruction by nucleases, its efficacy was low. Our delivery system, HVJliposomes, acted by fusion with cell membranes. The HVJliposomes method was first introduced by Kaneda13 and Nakanishi.14 Fusigenic F-protein of the HVJ viral coat allowed fusion with cell membranes under neutral pH and body temperature. The safety of the HVJ-liposomes was documented by intraocular injection, continuous intravenous infusion, or intraventricular injection in primates. Our previous data also showed 10 to 100 times greater transfection efficacy than LipopectAMINE.1 Vein grafts for arterial occlusive disease are ideal candidates for gene therapy because the vein for the graft is taken out of the body in usual practice. Therefore it can be treated with minimal systemic introduction of genetic material. Our data showed effective inhibition of intimal hyperplasia of a vein graft in a large animal model. Since Mann et al9 showed significant inhibition of intimal hyper-
2163
plasia of a vein graft with an E2F decoy using simple pressure delivery in normal saline, our more effective delivery system is expected to show better results in the clinical setting. REFERENCES 1. Ahn JD, Morishita R, Kaneda Y, et al: Inhibitory effects of novel AP-1 decoy oligodeoxynucleotides on vascular smooth muscle cell proliferation in vitro and neointimal formation in vivo. Circ Res 90:1325, 2002 2. Ahn JD, Morishita R, Kaneda Y, et al: Novel E2F decoy oligodeoxynucleotides inhibit in vitro vascular smooth muscle cell proliferation and in vivo neointimal hyperplasia. Gene Ther 9:1682, 2002 3. Lee I-K, Ahn JD, Kim HS, et al: Advantages of the circular dumbbell decoy in gene therapy and studies of gene regulation. Curr Drug Targ 4:619, 2003 4. Clowes AW, Clowes MM, Reidy MA: Kinetics of cellular proliferation after arterial injury, I: smooth muscle growth in the abscence of endothelium. Lab Invest 49:327, 1983 5. Clowes AW, Schwartz SM: Significance of quiescent smooth muscle cell migration in the injured rat carotid artery. Circ Res 56:139, 1985 6. Hu Y, Cheng L, Hochleitner BW, et al: Activation of mitogenactivated protein kinases (ERK/JNK) and AP-1 transcription factor in rat carotid arteries after balloon injury. Arterioscler Thromb Vasc Biol 17:2808, 1997 7. Pyles JM, March KL, Franklin M, et al: Activation of MAP kinase in vivo follows balloon overstretch injury of porcine coronary and carotid arteries. Circ Res 81:904, 1997 8. Karin M, Liu Z, Zandi E: AP-1 function and regulation. Curr Opin Cell Biol 9:240, 1997 9. Mann MJ, Whittemore AD, Donaldson MC, et al: Ex-vivo gene therapy of human vascular bypass grafts with E2F decoy: The PREVENT single-centre, randomised, controlled trial. Lancet 354:1493, 1999 10. Ahn JD, Morishita R, Kaneda Y, et al: Transcription factor decoy for activator protein-1 (AP-1) inhibits high glucose- and angiotensin II-induced type 1 plasminogen activator inhibitor (PAI-1) gene expression in cultured human vascular smooth muscle cells. Diabetologia 44:713, 2001 11. Morishita R, Gibbons GH, Horiuchi M, et al: Role of AP-1 complex in angiotensin II-mediated transforming growth factor- expression and growth of smooth muscle cells: using decoy approach against AP-1 binding site. Biochem Biophys Res Commun 243:361, 1998 12. Lauth M, Wagner AH, Cattaruzza M, et al: Transcriptional control of deformation-induced preproendothelin-1 gene expression in endothelial cells. J Mol Med 78:441, 2000 13. Kaneda Y, Uchida T, Kim J, et al: The improved efficient method for introducing macromolecules into cells using HVJ (Sendai virus) liposomes with gangliosides. Exp Cell Res 173:56, 1987 14. Nakanishi M, Uchida T, Sugawa H, et al: Efficient introduction of contents of liposomes into cells using HVJ (Sendai virus). Exp Cell Res 159:399, 1985