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E2F decoy suppresses E-Selectin expression in murine cardiac allograft arteriopathy
E2F Decoy Suppresses E-Selectin Expression in Murine Cardiac Allograft Arteriopathy J. Suzuki, M. Isobe, R. Morishita, A. Izawa, S. Yamazaki, Y. Okubo, Y. Kaneda, and M. Sekiguchi
C
ARDIAC transplantation has been established as a treatment for end-stage cardiac diseases, however, accelerated graft coronary disease in long-term survivors is still a problem.1,2 Selectin-dependent cell adhesion is an initial step in the infiltration of leukocytes to the site of inflammation, which is one of the first pathologic signs of rejection.3 The inhibition of arterial neointimal formation by E2F decoy after balloon angioplasty in rat carotid injury model was reported.4 Hemagglutinating virus of Japan (HVJ)-liposome method has been established to increase the efficiency of cellular uptake of E2F decoy.5,6 To test the hypothesis that E2F decoy suppresses E-selectin expression in allograft arteries, we performed a single intraluminal delivery of E2F decoy using HVJ-liposome mediated transfer. MATERIALS AND METHODS E2F decoy used in this study was synthesized, and HVJ-liposome E2F decoy complex was prepared as previously described.4,7,8 Male DBA/2 mice and B10.D2 mice (age 4 to 6 weeks, 20 to 25 g) were obtained from Japan Charles River Laboratories (Tokyo, Japan). Hearts were removed from DBA/2 mice and infused with E2F decoy-HVJ-liposome complex from the aorta and incubated for 10 minutes on ice. After transfection, the donor hearts were heterotopically transplanted into the recipient B10.D2 mice using a microsurgical technique. Using this combination, hearts are chronically rejected without immunosuppressants.9 Cardiac allografts were removed at day 28 and kept frozen in OCT compound. For control study, isografts (DBA/2 to DBA/2) were also harvested at day 28. Serial frozen sections (6 to 8 m) were cut and incubated with primary antibodies against mouse E-selectin (10E9.6), kindly provided by Dr Shinichiro Tojo, for 12 hours at 4°C.3 After incubation the slides were treated with biotinylated anti-rat IgG, then reacted with peroxidase-conjugated avidin complex using a Vectastain Elite ABC Kit (Vector, Burlingame, CA). Scoring of the intensity of these expression was as follows: 0, no visible staining; 1, few cells with faint staining; 2, moderate staining; and 3, intense diffuse staining. All data are expressed as mean ⫾ SD. A value of P ⬍ .05 using the Student’s t test was considered statistically significant.
RESULTS
E-selectin was strongly and diffusely expressed (2.7 ⫾ 2.5) in the thickened intima of the allograft arteries from nontreated recipients (36 allografts in 6 allografts), while
E2F decoy treatment (36 allografts in 6 allografts) resulted in limited E-selectin expression (0.7 ⫾ 0.5) on endothelial cells in the faintly thickened intima of the allografts (P ⬍ .05). Isografts were as free from E-selectin expression in the endothelium as native hearts. DISCUSSION
Graft arteriopathy after cardiac transplantation is characterized by diffuse intimal thickening comprised of proliferative vascular smooth muscle cells (SMCs).10 Little is known about the etiology of neointimal formation, however, it is believed that an immune-mediated process is the primary cause of the proliferation of SMCs. Since the initial process of inflammatory cell emigration into tissues involves the expression of E-selectin on the endothelium, suppression of this molecule is crucial for prevention of SMC proliferation. The HVJ-liposome method of E2F decoy transfer presented in this study clearly suppressed E-selectin expression up to 4 weeks after transplantation. Therefore, this method promises clinical usefulness for prevention of transplanted heart graft vasculopathy by suppressing E-selectin expression. ACKNOWLEDGMENTS We thank Midori Oike and Rie Shiohara for their technical assistance.
REFERENCES 1. Hosenpud JD, Bennet LE, Keck BM, et al: J Heart Lung Transplant 16:691, 1997 2. Johnson DE, Alderman EL, Schroeder JS, et al: J Am Coll Cardiol 17:449, 1991 From the First Department of Internal Medicine, Shinshu University, Nagano, Japan and the Division of Gene Therapy Science, Osaka University, Osaka, Japan. Supported by Japan Heart Foundation Research Grant, Grantin-Aid from the Kanae Foundation for Life & Socio-Medical Science and Grant-in-Aid from the Tokyo Biochemical Research Foundation. Address reprint requests to Dr Jun-ichi Suzuki, The First Department of Internal Medicine, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 Japan.
0041-1345/99/$–see front matter PII S0041-1345(99)00253-5
© 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
2018
Transplantation Proceedings, 31, 2018–2019 (1999)
E2F DECOY SUPPRESSES E-SELECTIN EXPRESSION 3. Yamazaki S, Isobe M, Suzuki J, et al: J Heart Lung Transplant 17:1007, 1998 4. Morishita R, Gibbons GH, Ellison KE, et al: J Clin Invest 93:1458, 1994 5. Tsai LH, Harlow E, Meyerson M: Nature 353:174, 1991 6. Kaneda Y, Iwai K, Uchida T: Science 243:375, 1989
2019 7. Isobe M, Yagita H, Okumura K, et al: Science 255:1125, 1992 8. Suzuki J, Isobe M, Morishita R, et al: Nature Med 3:900, 1997 9. Furukawa Y, Matsumori A, Hirozane T, et al: Circulation 93:333, 1996 10. Hosenpud JD: Transplant Immunol 1:237, 1993
C
ARDIAC transplantation has been established as a treatment for end-stage cardiac diseases, however, accelerated graft coronary disease in long-term survivors is still a problem.1,2 Selectin-dependent cell adhesion is an initial step in the infiltration of leukocytes to the site of inflammation, which is one of the first pathologic signs of rejection.3 The inhibition of arterial neointimal formation by E2F decoy after balloon angioplasty in rat carotid injury model was reported.4 Hemagglutinating virus of Japan (HVJ)-liposome method has been established to increase the efficiency of cellular uptake of E2F decoy.5,6 To test the hypothesis that E2F decoy suppresses E-selectin expression in allograft arteries, we performed a single intraluminal delivery of E2F decoy using HVJ-liposome mediated transfer. MATERIALS AND METHODS E2F decoy used in this study was synthesized, and HVJ-liposome E2F decoy complex was prepared as previously described.4,7,8 Male DBA/2 mice and B10.D2 mice (age 4 to 6 weeks, 20 to 25 g) were obtained from Japan Charles River Laboratories (Tokyo, Japan). Hearts were removed from DBA/2 mice and infused with E2F decoy-HVJ-liposome complex from the aorta and incubated for 10 minutes on ice. After transfection, the donor hearts were heterotopically transplanted into the recipient B10.D2 mice using a microsurgical technique. Using this combination, hearts are chronically rejected without immunosuppressants.9 Cardiac allografts were removed at day 28 and kept frozen in OCT compound. For control study, isografts (DBA/2 to DBA/2) were also harvested at day 28. Serial frozen sections (6 to 8 m) were cut and incubated with primary antibodies against mouse E-selectin (10E9.6), kindly provided by Dr Shinichiro Tojo, for 12 hours at 4°C.3 After incubation the slides were treated with biotinylated anti-rat IgG, then reacted with peroxidase-conjugated avidin complex using a Vectastain Elite ABC Kit (Vector, Burlingame, CA). Scoring of the intensity of these expression was as follows: 0, no visible staining; 1, few cells with faint staining; 2, moderate staining; and 3, intense diffuse staining. All data are expressed as mean ⫾ SD. A value of P ⬍ .05 using the Student’s t test was considered statistically significant.
RESULTS
E-selectin was strongly and diffusely expressed (2.7 ⫾ 2.5) in the thickened intima of the allograft arteries from nontreated recipients (36 allografts in 6 allografts), while
E2F decoy treatment (36 allografts in 6 allografts) resulted in limited E-selectin expression (0.7 ⫾ 0.5) on endothelial cells in the faintly thickened intima of the allografts (P ⬍ .05). Isografts were as free from E-selectin expression in the endothelium as native hearts. DISCUSSION
Graft arteriopathy after cardiac transplantation is characterized by diffuse intimal thickening comprised of proliferative vascular smooth muscle cells (SMCs).10 Little is known about the etiology of neointimal formation, however, it is believed that an immune-mediated process is the primary cause of the proliferation of SMCs. Since the initial process of inflammatory cell emigration into tissues involves the expression of E-selectin on the endothelium, suppression of this molecule is crucial for prevention of SMC proliferation. The HVJ-liposome method of E2F decoy transfer presented in this study clearly suppressed E-selectin expression up to 4 weeks after transplantation. Therefore, this method promises clinical usefulness for prevention of transplanted heart graft vasculopathy by suppressing E-selectin expression. ACKNOWLEDGMENTS We thank Midori Oike and Rie Shiohara for their technical assistance.
REFERENCES 1. Hosenpud JD, Bennet LE, Keck BM, et al: J Heart Lung Transplant 16:691, 1997 2. Johnson DE, Alderman EL, Schroeder JS, et al: J Am Coll Cardiol 17:449, 1991 From the First Department of Internal Medicine, Shinshu University, Nagano, Japan and the Division of Gene Therapy Science, Osaka University, Osaka, Japan. Supported by Japan Heart Foundation Research Grant, Grantin-Aid from the Kanae Foundation for Life & Socio-Medical Science and Grant-in-Aid from the Tokyo Biochemical Research Foundation. Address reprint requests to Dr Jun-ichi Suzuki, The First Department of Internal Medicine, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 Japan.
0041-1345/99/$–see front matter PII S0041-1345(99)00253-5
© 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
2018
Transplantation Proceedings, 31, 2018–2019 (1999)
E2F DECOY SUPPRESSES E-SELECTIN EXPRESSION 3. Yamazaki S, Isobe M, Suzuki J, et al: J Heart Lung Transplant 17:1007, 1998 4. Morishita R, Gibbons GH, Ellison KE, et al: J Clin Invest 93:1458, 1994 5. Tsai LH, Harlow E, Meyerson M: Nature 353:174, 1991 6. Kaneda Y, Iwai K, Uchida T: Science 243:375, 1989
2019 7. Isobe M, Yagita H, Okumura K, et al: Science 255:1125, 1992 8. Suzuki J, Isobe M, Morishita R, et al: Nature Med 3:900, 1997 9. Furukawa Y, Matsumori A, Hirozane T, et al: Circulation 93:333, 1996 10. Hosenpud JD: Transplant Immunol 1:237, 1993