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Effects of pirfenidone on vascular smooth muscle cell proliferation and intimal hyperplasia following arterial balloon injury
Effects of Pirfenidone on Vascular Smooth Muscle Cell Proliferation and Intimal Hyperplasia Following Arterial Balloon Injury J.R. Waller, G.J. Murphy, M.S. Metcalfe, G.R. Bicknell, R.N. Saunders, S.B. Margolin, and M.L. Nicholson
A
LLOGRAFT VASCULOPATHY, a central feature of chronic rejection in all transplanted organs, remains the leading cause of graft loss and morbidity after the first postoperative year.1 In cardiac transplantation, it is the primary cause of late death2 and is angiographically detectable in 50% to 60% of patients by 2 years and 90% at 5 years.3,4 Allograft vasculopathy is characterized histologically by progressive intimal hyperplasia that affects both arteries and veins producing luminal encroachment, endorgan ischemia, and eventual graft failure. The mechanisms underlying this process are incompletely understood, but it is thought that numerous immunological and nonimmunological etiological factors are involved, leading to repeated endothelial damage and repair.5 This is mediated by a myriad of cytokines and growth factors including plateletderived growth factor (PDGF) and basic fibroblast growth factor,6 matrix metalloproteinases (MMPs), and their inhibitors (tissue metalloproteinases [TIMPs]).7 The response of the vessel wall is vascular smooth muscle cell proliferation, migration, and deposition of extracellular matrix proteins (ECM). Presently, no drugs have proven effective in the clinical setting, and the only effective treatment is retransplantation.8 The novel antifibrotic agent pirfenidone has been shown to inhibit PDGF,9 transforming growth factor-10 and collagen deposition11 in nonvascular inflammatory models: its effect on injured or stimulated vascular smooth muscle cells has not been previously investigated. METHODS Arterial Injury Four-month-old male Sprague-Dawley rats were obtained from Harland (Cambridge, UK) and cared for in accordance with the Animals (Scientific Procedures) Act of 1986. Rats were randomized to receive either standard powdered diet or diet supplemented with pirfenidone powder, 0.7% w/w. The left common carotid artery was surgically exposed and the endothelium denuded using a saline-filled 2F balloon catheter (Baxter Healthcare Corp), as described previously.12 Injured carotid vessels were surgically removed for histological and molecular analyses at 2 weeks.
Morphometry Carotid arteries were removed under terminal anesthesia and flushed clear of blood with PBS, ph 7.4, and fixed for 16 hours in
2.5% glutaraldehyde in 0.1M phosphate buffer. Vessels were then embedded in epoxy resin, cut into 4 m cross-sections, and stained with hemotoxylin and eosin. Intima and medial areas were calculated using computer-aided planimetry and expressed as intima: medial ratios. Intimal and medial areas were defined by the internal and external elastic laminae, respectively.
Molecular Analysis The methods used to quantify levels of messenger RNA (mRNA) expression using reverse transciptase-polymerase chain reaction (RT-PCR) have been described previously.13 Messenger RNA was isolated from vessels previously snap-frozen in liquid nitrogen. Vessels were cut into 4 m sections, ground in a mortar and pestle, and mRNA isolated by using Dynabeads (Dynal, Bromborough, UK). RT-PCR was used to amplify genes of interest and these were quantified using an enzyme-linked immunosorbent assay. Differences between tissue cellularity were corrected for by using the housekeeping gene -actin. Genes measured in this study were MMPs 2 and 9, TIMP-1, collagen III, and TGF-.
Statistical Analysis Differences in mean intima:medial ratios and mean gene expression were compared with Student’s t-test for independent samples. Data are presented as mean values with 95% confidence interval for difference of means (CI). Differences were considered statistically significant at P ⬍ .05.
RESULTS Morphometry
Control animals developed significant intimal thickening by 2 weeks post carotid balloon injury (mean intima:medial ratio 1.54). Pirfenidone significantly inhibited intimal hyperplasia at 2 weeks (mean ratio 0.49, CI 0.70 to 1.41, P ⬍ .001) (Fig 1).
From the Division of Transplant Surgery, Liecester General Hospital, Leicester, UK. Address reprint requests to JR Waller, Royal College of Surgeons of England Surgical Research Fellow, Division of Transplant Surgery, Liecester General Hospital, Gwendolen Road, Leicester, LE5 4PW, UK. E-mail: julian@waller79. fsnet.co.uk
0041-1345/01/$–see front matter PII S0041-1345(01)02615-X
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
3816
Transplantation Proceedings, 33, 3816–3818 (2001)
EFFECTS OF PIRFENIDONE
3817
Fig 1. Graph showing effect of pirfenidone on intimal:media ratios 14 days after balloon injury. Values are expressed as means with 95% confidence intervals shown; n ⫽ 6 rats for control and 500 mg.kg⫺1 pirfenidone (P ⬍ .001). Error bars show 95% CI of the mean; bars show means.
Profibrotic Gene Expression
Gene data were studied using RT-PCR. -actin was used as a housekeeping gene to correct for differences in tissue cellularity. There was no significant difference for -actin expression between control and pirfenidone-treated rats, indicating pirfenidone has no effect on -actin expression. Results are shown in Table 1 and are stated as 95% confidence intervals for differences of mean values. Pirfenidone significantly inhibited all profibrotic genes measured in this study, including transcription for collagen III (P ⬍ .05). DISCUSSION
The development of allograft vasculopathy following transplantation is multifaceted. The initial vascular injury to the Table 1. Histologic Data and Pro-fibrotic Gene Expression at 14 Days Control Pirfenidone
Intima:media MMP-2 mRNA expression MMP-9 mRNA expression TIMP-1 mRNA expression Collagen III mRNA expression TGF- mRNA expression
1.54 2.41 1.41 1.60 1.89 1.51
0.49 1.17 0.57 0.89 0.83 0.80
95% Confidence Intervals/P-value
0.70 0.15 0.30 0.31 0.23 0.75
to to to to to to
1.41/.001 2.34/.03 1.38/.006 1.12/.002 2.12/.05 1.38/.04
Data are presented as means with 95% confidence intervals of the difference; n ⫽ 6 for control and 500 mg 䡠 kg⫺1day⫺1 pirfenidone. The value of messenger (mRNA) expression is stated in arbitary units and expressed as a ratio to that of housekeeping gene -actin.
endothelium is mediated by humoral and cellular responses to HLA-antigens and vascular endothelial cell antigens,14 resulting in leucocyte adhesion, thrombosis, release of growth factors, and smooth muscle cell proliferation.15 Uninjured rat arteries do not contain smooth muscle cells in the intima,16 and thus migration of proliferating medial smooth muscle cells (SMC) requires the degradation of ECM proteins and the basement membrane. Continued administration of pirfenidone significantly inhibits intimal hyperplasia 2 weeks following vascular injury. Vascular smooth muscle cells constitutively express MMP-2 under basal conditions, while MMP-9 expression is induced by arterial injury.17 It is therefore likely that the observed reduction in early intimal thickening is attributable to inhibition of MMP-2 and MMP-9 in rats treated with pirfenidone. Previous studies using metalloproteinase inhibitors support this hypothesis.18,19 The reduction of TIMP-1 expression is more difficult to interpret, since the role of TIMP-1 in the development of intimal lesions is complex. TIMP-1 is the major endogenous inhibitor of MMP activity, forming a 1:1 stoichiometric complex with MMPs, inhibiting both latent and activated enzymes.20 For proliferating SMCs to migrate to the neointima, cells must free themselves from their surrounding extracellular architecture. This is facilitated by a shift in the “balance of forces” between synthesis and degradation of ECM proteins toward degradation.21 Previous studies have shown TIMP-1 expression is detectable by 24 hours following balloon injury but returns to baseline levels by day 7.22 We have shown pirfenidone to inhibit TIMP-1 production to 14 days. A reduction of TIMP-1 at this time-point favors degradation at a critical time-point when intimal lesion size is increased solely due to the deposition of ECM proteins.23 Furthermore, we have shown collagen III expression, a major component of the ECM in intimal lesions, is synchronously inhibited by pirfenidone. Pirfenidone attenuates collagen deposition in many animal models of fibrosis24 –26 and is currently under phase III trials for the treatment of idiopathic pulmonary fibrosis.27 Its effect in models of vascular disease has not been previously reported. Finally, TGF- expression is suppressed in pirfenidone-treated rats. TGF- is a polypeptide growth factor released by many different cell types facilitating cell proliferation and migration and augmenting ECM deposition in developing arteriosclerotic lesions.28 A reduction in TGF- and subsequent decrease in collagen deposition has been previously observed in a bleomycin hamster model of fibrosis and this also occurred at a transcriptional level.29 Pirfenidone attenuates intimal hyperplasia and significantly inhibits both metalloproteinase gene expression, governing medial smooth muscle cell proliferation and migration, and collagen deposition. Pirfenidone therefore may have a role in the prevention of intimal hyperplasia and attenuate the development of chronic rejection in solid organ allografts.
3818
ACKNOWLEDGMENTS We would like to thank the Royal College of Surgeons of England for their financial support and awarding this project, a College Fellowship, and Dr S.B. Margolin (President, Marnac Inc) for generously supplying pirfenidone powder.
REFERENCES 1. Tilney NL, Whitely WD, Diamond JR, et al: Transplantation 52:389, 1991 2. Schroeder JS, Gao SZ, Hunt SA, et al: J Heart Lung Transplant 11:S258, 1992 3. Keogh AM, Valantine HA, Hunt SA, et al: J Heart Lung Transplant 11:892, 1992 4. Gao SZ, Alderman EL, Schroeder JS, et al: J Am Coll Cardiol 12:334, 1988 5. Labarrere CA, Pitts D, Nelson DR, et al: Transplant Proc 27:1939, 1995 6. Duquesnoy RJ, Demetris AJ: Curr Opin Cardiol 10:193, 1995 7. Galis Z, Muszynski M, Sukhova G, et al: Circ Res 75:181, 1994 8. Ensly RD, Hunt S, Taylor DO, et al: J Heart Lung Transplant 11:S142, 1992 9. Gurujeyalakshmi G, Hollinger SM, Giri SN: Am J Physiol 276:L311, 1999 10. Iyer SN, Gurujeyalakshmi G, Giri SN: Pharm Exp Ther 291:367, 1999 11. Kaneko M, Inoue H, Nakazawa R, et al: Clin Exp Immunol 113:72, 1998 12. Clowes AW, Clowes MM, Reidy MA: Circulation 49:327, 1983
WALLER, MURPHY, METCALFE ET AL 13. Hall LL, Bicknell GR, Primrose L, et al: Biotechniques 24:652, 1998 14. Libby P, Swanson SJ, Tanaka H: J Heart Lung Transplant 11:5, 1992 15. Treasure CB, Alexander RW: Cardiovasc Drugs Ther 9:13, 1995 16. Tada T, Reidy MA: Am J Pathol 129:429, 1987 17. Dollery C, McEwan J, Henney A: Circ Res 77:863, 1995 18. Southgate KM, Davies M, Booth RFG, et al: Biochem J 288:93, 1992 19. Bendeck MP, Irvin C, Reidy MA: Circ Res 78:38, 1996 20. Murphy G, Willnebrock F: Methods Enzymol 248:496, 1995 21. Montesano R, Pepper MS, Mohle-Steinlein U, et al: Cell 62:435, 1990 22. Clowes MM, Lynch CM, Miller AD, et al: J Clin Invest 93:644, 1994 23. Schwartz RS, Holmes DR, Topol EJ: J Am Coll Cardiol 20:1284, 1992 24. Dosanjh AK, Wan B, Throndset W, et al: Transplant Proc 30:1910, 1998 25. Schimizu T, Fukagawa M, Kuroda T, et al: Kidney Int 52(suppl 63):S-239, 1997 26. Byung-Seok L, Margolin SB, Nowak RA: J Clin Endocrinol Metab 83:219, 1998 27. Nicod LP: Lancet 354:268, 1999 28. Nabel EG, Shum L, Pompili VJ, et al: Proc Natl Acad Sci USA 90:10759, 1993 29. Iyer SN, Gurujeyalakshmi G, Giri SN: J Pharmacol Exp Ther 291:367, 1999
A
LLOGRAFT VASCULOPATHY, a central feature of chronic rejection in all transplanted organs, remains the leading cause of graft loss and morbidity after the first postoperative year.1 In cardiac transplantation, it is the primary cause of late death2 and is angiographically detectable in 50% to 60% of patients by 2 years and 90% at 5 years.3,4 Allograft vasculopathy is characterized histologically by progressive intimal hyperplasia that affects both arteries and veins producing luminal encroachment, endorgan ischemia, and eventual graft failure. The mechanisms underlying this process are incompletely understood, but it is thought that numerous immunological and nonimmunological etiological factors are involved, leading to repeated endothelial damage and repair.5 This is mediated by a myriad of cytokines and growth factors including plateletderived growth factor (PDGF) and basic fibroblast growth factor,6 matrix metalloproteinases (MMPs), and their inhibitors (tissue metalloproteinases [TIMPs]).7 The response of the vessel wall is vascular smooth muscle cell proliferation, migration, and deposition of extracellular matrix proteins (ECM). Presently, no drugs have proven effective in the clinical setting, and the only effective treatment is retransplantation.8 The novel antifibrotic agent pirfenidone has been shown to inhibit PDGF,9 transforming growth factor-10 and collagen deposition11 in nonvascular inflammatory models: its effect on injured or stimulated vascular smooth muscle cells has not been previously investigated. METHODS Arterial Injury Four-month-old male Sprague-Dawley rats were obtained from Harland (Cambridge, UK) and cared for in accordance with the Animals (Scientific Procedures) Act of 1986. Rats were randomized to receive either standard powdered diet or diet supplemented with pirfenidone powder, 0.7% w/w. The left common carotid artery was surgically exposed and the endothelium denuded using a saline-filled 2F balloon catheter (Baxter Healthcare Corp), as described previously.12 Injured carotid vessels were surgically removed for histological and molecular analyses at 2 weeks.
Morphometry Carotid arteries were removed under terminal anesthesia and flushed clear of blood with PBS, ph 7.4, and fixed for 16 hours in
2.5% glutaraldehyde in 0.1M phosphate buffer. Vessels were then embedded in epoxy resin, cut into 4 m cross-sections, and stained with hemotoxylin and eosin. Intima and medial areas were calculated using computer-aided planimetry and expressed as intima: medial ratios. Intimal and medial areas were defined by the internal and external elastic laminae, respectively.
Molecular Analysis The methods used to quantify levels of messenger RNA (mRNA) expression using reverse transciptase-polymerase chain reaction (RT-PCR) have been described previously.13 Messenger RNA was isolated from vessels previously snap-frozen in liquid nitrogen. Vessels were cut into 4 m sections, ground in a mortar and pestle, and mRNA isolated by using Dynabeads (Dynal, Bromborough, UK). RT-PCR was used to amplify genes of interest and these were quantified using an enzyme-linked immunosorbent assay. Differences between tissue cellularity were corrected for by using the housekeeping gene -actin. Genes measured in this study were MMPs 2 and 9, TIMP-1, collagen III, and TGF-.
Statistical Analysis Differences in mean intima:medial ratios and mean gene expression were compared with Student’s t-test for independent samples. Data are presented as mean values with 95% confidence interval for difference of means (CI). Differences were considered statistically significant at P ⬍ .05.
RESULTS Morphometry
Control animals developed significant intimal thickening by 2 weeks post carotid balloon injury (mean intima:medial ratio 1.54). Pirfenidone significantly inhibited intimal hyperplasia at 2 weeks (mean ratio 0.49, CI 0.70 to 1.41, P ⬍ .001) (Fig 1).
From the Division of Transplant Surgery, Liecester General Hospital, Leicester, UK. Address reprint requests to JR Waller, Royal College of Surgeons of England Surgical Research Fellow, Division of Transplant Surgery, Liecester General Hospital, Gwendolen Road, Leicester, LE5 4PW, UK. E-mail: julian@waller79. fsnet.co.uk
0041-1345/01/$–see front matter PII S0041-1345(01)02615-X
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
3816
Transplantation Proceedings, 33, 3816–3818 (2001)
EFFECTS OF PIRFENIDONE
3817
Fig 1. Graph showing effect of pirfenidone on intimal:media ratios 14 days after balloon injury. Values are expressed as means with 95% confidence intervals shown; n ⫽ 6 rats for control and 500 mg.kg⫺1 pirfenidone (P ⬍ .001). Error bars show 95% CI of the mean; bars show means.
Profibrotic Gene Expression
Gene data were studied using RT-PCR. -actin was used as a housekeeping gene to correct for differences in tissue cellularity. There was no significant difference for -actin expression between control and pirfenidone-treated rats, indicating pirfenidone has no effect on -actin expression. Results are shown in Table 1 and are stated as 95% confidence intervals for differences of mean values. Pirfenidone significantly inhibited all profibrotic genes measured in this study, including transcription for collagen III (P ⬍ .05). DISCUSSION
The development of allograft vasculopathy following transplantation is multifaceted. The initial vascular injury to the Table 1. Histologic Data and Pro-fibrotic Gene Expression at 14 Days Control Pirfenidone
Intima:media MMP-2 mRNA expression MMP-9 mRNA expression TIMP-1 mRNA expression Collagen III mRNA expression TGF- mRNA expression
1.54 2.41 1.41 1.60 1.89 1.51
0.49 1.17 0.57 0.89 0.83 0.80
95% Confidence Intervals/P-value
0.70 0.15 0.30 0.31 0.23 0.75
to to to to to to
1.41/.001 2.34/.03 1.38/.006 1.12/.002 2.12/.05 1.38/.04
Data are presented as means with 95% confidence intervals of the difference; n ⫽ 6 for control and 500 mg 䡠 kg⫺1day⫺1 pirfenidone. The value of messenger (mRNA) expression is stated in arbitary units and expressed as a ratio to that of housekeeping gene -actin.
endothelium is mediated by humoral and cellular responses to HLA-antigens and vascular endothelial cell antigens,14 resulting in leucocyte adhesion, thrombosis, release of growth factors, and smooth muscle cell proliferation.15 Uninjured rat arteries do not contain smooth muscle cells in the intima,16 and thus migration of proliferating medial smooth muscle cells (SMC) requires the degradation of ECM proteins and the basement membrane. Continued administration of pirfenidone significantly inhibits intimal hyperplasia 2 weeks following vascular injury. Vascular smooth muscle cells constitutively express MMP-2 under basal conditions, while MMP-9 expression is induced by arterial injury.17 It is therefore likely that the observed reduction in early intimal thickening is attributable to inhibition of MMP-2 and MMP-9 in rats treated with pirfenidone. Previous studies using metalloproteinase inhibitors support this hypothesis.18,19 The reduction of TIMP-1 expression is more difficult to interpret, since the role of TIMP-1 in the development of intimal lesions is complex. TIMP-1 is the major endogenous inhibitor of MMP activity, forming a 1:1 stoichiometric complex with MMPs, inhibiting both latent and activated enzymes.20 For proliferating SMCs to migrate to the neointima, cells must free themselves from their surrounding extracellular architecture. This is facilitated by a shift in the “balance of forces” between synthesis and degradation of ECM proteins toward degradation.21 Previous studies have shown TIMP-1 expression is detectable by 24 hours following balloon injury but returns to baseline levels by day 7.22 We have shown pirfenidone to inhibit TIMP-1 production to 14 days. A reduction of TIMP-1 at this time-point favors degradation at a critical time-point when intimal lesion size is increased solely due to the deposition of ECM proteins.23 Furthermore, we have shown collagen III expression, a major component of the ECM in intimal lesions, is synchronously inhibited by pirfenidone. Pirfenidone attenuates collagen deposition in many animal models of fibrosis24 –26 and is currently under phase III trials for the treatment of idiopathic pulmonary fibrosis.27 Its effect in models of vascular disease has not been previously reported. Finally, TGF- expression is suppressed in pirfenidone-treated rats. TGF- is a polypeptide growth factor released by many different cell types facilitating cell proliferation and migration and augmenting ECM deposition in developing arteriosclerotic lesions.28 A reduction in TGF- and subsequent decrease in collagen deposition has been previously observed in a bleomycin hamster model of fibrosis and this also occurred at a transcriptional level.29 Pirfenidone attenuates intimal hyperplasia and significantly inhibits both metalloproteinase gene expression, governing medial smooth muscle cell proliferation and migration, and collagen deposition. Pirfenidone therefore may have a role in the prevention of intimal hyperplasia and attenuate the development of chronic rejection in solid organ allografts.
3818
ACKNOWLEDGMENTS We would like to thank the Royal College of Surgeons of England for their financial support and awarding this project, a College Fellowship, and Dr S.B. Margolin (President, Marnac Inc) for generously supplying pirfenidone powder.
REFERENCES 1. Tilney NL, Whitely WD, Diamond JR, et al: Transplantation 52:389, 1991 2. Schroeder JS, Gao SZ, Hunt SA, et al: J Heart Lung Transplant 11:S258, 1992 3. Keogh AM, Valantine HA, Hunt SA, et al: J Heart Lung Transplant 11:892, 1992 4. Gao SZ, Alderman EL, Schroeder JS, et al: J Am Coll Cardiol 12:334, 1988 5. Labarrere CA, Pitts D, Nelson DR, et al: Transplant Proc 27:1939, 1995 6. Duquesnoy RJ, Demetris AJ: Curr Opin Cardiol 10:193, 1995 7. Galis Z, Muszynski M, Sukhova G, et al: Circ Res 75:181, 1994 8. Ensly RD, Hunt S, Taylor DO, et al: J Heart Lung Transplant 11:S142, 1992 9. Gurujeyalakshmi G, Hollinger SM, Giri SN: Am J Physiol 276:L311, 1999 10. Iyer SN, Gurujeyalakshmi G, Giri SN: Pharm Exp Ther 291:367, 1999 11. Kaneko M, Inoue H, Nakazawa R, et al: Clin Exp Immunol 113:72, 1998 12. Clowes AW, Clowes MM, Reidy MA: Circulation 49:327, 1983
WALLER, MURPHY, METCALFE ET AL 13. Hall LL, Bicknell GR, Primrose L, et al: Biotechniques 24:652, 1998 14. Libby P, Swanson SJ, Tanaka H: J Heart Lung Transplant 11:5, 1992 15. Treasure CB, Alexander RW: Cardiovasc Drugs Ther 9:13, 1995 16. Tada T, Reidy MA: Am J Pathol 129:429, 1987 17. Dollery C, McEwan J, Henney A: Circ Res 77:863, 1995 18. Southgate KM, Davies M, Booth RFG, et al: Biochem J 288:93, 1992 19. Bendeck MP, Irvin C, Reidy MA: Circ Res 78:38, 1996 20. Murphy G, Willnebrock F: Methods Enzymol 248:496, 1995 21. Montesano R, Pepper MS, Mohle-Steinlein U, et al: Cell 62:435, 1990 22. Clowes MM, Lynch CM, Miller AD, et al: J Clin Invest 93:644, 1994 23. Schwartz RS, Holmes DR, Topol EJ: J Am Coll Cardiol 20:1284, 1992 24. Dosanjh AK, Wan B, Throndset W, et al: Transplant Proc 30:1910, 1998 25. Schimizu T, Fukagawa M, Kuroda T, et al: Kidney Int 52(suppl 63):S-239, 1997 26. Byung-Seok L, Margolin SB, Nowak RA: J Clin Endocrinol Metab 83:219, 1998 27. Nicod LP: Lancet 354:268, 1999 28. Nabel EG, Shum L, Pompili VJ, et al: Proc Natl Acad Sci USA 90:10759, 1993 29. Iyer SN, Gurujeyalakshmi G, Giri SN: J Pharmacol Exp Ther 291:367, 1999