Candesartan cilexetil reduces graft arteriosclerosis in aortic transplantation model in rat

Transplant Immunology 8 Ž2001. 245᎐251

Candesartan cilexetil reduces graft arteriosclerosis in aortic transplantation model in rat Lilija Zezinaa,U , Erik Larsson b,c , Bengt Fellstrom ¨ a a Department of Medical Sciences, Uppsala Uni¨ ersity, Uppsala, Sweden Department of Genetics and Pathology, Uppsala Uni¨ ersity, Uppsala, Sweden c Department of Internal and Laboratory Medicine, Uni¨ ersity of Tronheim, Tronheim, Norway b

Received 21 November 2000; accepted 5 January 2001

Abstract To date established treatment of transplant arteriosclerosis is basically missing and there is a need for new therapeutic approaches. Angiotensin II ŽAng II. and Ang II receptor type 1 ŽAT1 . are present in the vascular wall. Blocking of the AT1 receptor by pharmacological agents may inhibit damaging effects of Ang II on endothelial and smooth muscle cells. The purpose of the study was to evaluate the effect of the AT1 receptor blocker Candesartan cilexetil on the development of graft arteriosclerosis in a rat aortic transplant model. Two strain combinations were used for aortic transplantation: DA to PVG; and PVG to PVG. The animals received Candesartan cilexetil treatment Ž9.5" 1.4 mgrkgrday. for 8 weeks. Candesartan cilexetil treatment reduced neointimal formation both in allografts ŽQint 30.2" 8.8% vs. 22.1" 8.7%, P- 0.05. and in isografts ŽQint 15.5" 4.4% vs. 6.7" 3.3%, Ps 0.0001.. Blocking of the AT1 receptor signalling by Candesartan cilexetil was also associated with a reduced expression of TGF-␤1. Macrophage infiltration was not affected by the treatment. Candesartan cilexetil treatment leads to reduced neointimal formation in aortic transplant. The positive effect of the drug might be partly explained by a reduction of TGF-␤1 expression in the grafts. Candesartan treatment may provide another possibility for prevention of transplant arteriosclerosis and chronic rejection. 䊚 2001 Elsevier Science B.V. All rights reserved. Keywords: Angiotensin II receptor; Graft arteriosclerosis; Aorta transplantation

1. Introduction Graft arteriosclerosis is a common finding in chronic rejection; a multifactorial process leading to the loss of solid organ grafts. Because of lack of treatment of chronic graft dysfunction, we focused our efforts on animal models of chronic rejection to elucidate its’ pathology and develop new therapeutic approaches. The aortic transplant model has striking morphological U

Corresponding author. Research Dept 2, University Hospital, Uppsala SE 751 85, Sweden. Tel.: q46-18-6113982; fax: q46-18553601. E-mail address: [email protected] ŽL. Zezina..

similarities to vascular lesions of solid organ grafts in humans and allows standardised quantitative measurement of structural changes in the vessel wall as well as a convenient evaluation of the results for a particular intervention w1,2x. The renin᎐angiotensin system is involved in the control of vascular structure and function. Ang II and AT1 receptors are found in the vascular wall. Ang II via the AT1 receptor regulates vascular smooth muscle cell ŽVSMC. contraction, proliferation, migration and matrix production w3᎐5x. Ang II can also contribute to inflammatory activation of VSMC by stimulation of IL-6 production w6x. Blocking of AT1 receptor by pharmacological agents inhibits many of the damaging ef-

0966-3274r01r$ - see front matter 䊚 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 6 6 - 3 2 7 4 Ž 0 1 . 0 0 0 2 8 - 4

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L. Zezina et al. r Transplant Immunology 8 (2001) 245᎐251

fects of Ang II on endothelial cells w7x and VSMC w8x. Recent studies have demonstrated a beneficial influence of Ang II blocking of vascular repair processes in the balloon injury model w9x. Transforming growth factor-beta ŽTGF-␤. is found to be upregulated after transplantation w10x, and may be involved in the development of vascular remodelling leading to chronic graft dysfunction. As demonstrated by Liu et al. Ž1997., TGF-␤1 is upregulated by Ang II and is involved in, for example, the anti-proliferative effect of Ang II on VSMC w11x. AT1 receptor blockers have recently been introduced for blood pressure control in renal graft recipients. Investigation of their potential effects on graft arteriosclerosis may provide additional indications for their use in the post-transplant period.

2. Objective The aim of this study was to evaluate the effect of AT1 receptor blocker Candesartan cilexetil on the development of graft arteriosclerosis in a rat aortic transplantation model. For this purpose, syngeneic and allogeneic aortic transplantations were performed between two rat strains. In our model, the development of graft arteriosclerosis in case of syngeneic transplantation is dependent mostly on the severity of ischemiarreperfusion injury, whilst in the case of allogeneic transplantation, it is influenced by immunological conflict w12x.

3. Material and methods 3.1. Experimental animals Male PVG and DA rats Žfrom Mollegaard, Skensved, Denmark. aged 3᎐4 months, weighing 200᎐270 g, were used in the study. Allogeneic transplantation was performed from DA donors to PVG recipients and syngeneic transplantation was performed from PVG to PVG rats. The study protocol was approved by the local ethical committee for animal experiments. Animal care and experiments were performed in accordance with international guidelines. 3.2. Aorta transplantation Animals were anaesthetised by an intraperitoneal injection of Eqviticin Ža mixture of Chloral hydrate, Phenobarbital and Magnesium sulfate .. The infrarenal aorta was exposed from the renal arteries to the aortic bifurcation via a medline laparotomy. One microclip was placed below the renal arteries and the other above the aortic bifurcation. A 10᎐15-mm aorta segment was removed from the donor animal and stored

in a histidine-buffered solution ŽFrodin ¨ solution. for 1 h in allogeneic transplantation, and 18 h in syngeneic transplantation. The recipient’s own aortic fragment was removed immediately prior to transplantation. The graft was placed in the recipient animal from below the renal arteries down to the bifurcation of the abdominal aorta using 9᎐0 non-absorbable monofilament nylon sutures. The abdominal incision was closed and the animal was returned to the cage. Candesartan cilexetil ŽAstra Hassle, Molndal, Sweden. was administered by ¨ ¨ diluting in drinking water ᎏ mean dose 9.5" 1.4 mgrkgrday ᎏ during the whole post-transplant period. After 8 weeks the rats were anaesthetised and the grafts were excised. From each recipient a 5᎐10-mm long non-transplanted portions of thoracic and abdominal aorta were harvested to serve as control samples of normal aorta for image analysis and immunohistochemical stains. Each transplant and non-transplanted portion was divided into three parts; one part was frozen in a mixture of cold isopentane and dry ice and stored at y70⬚C until processed for immunohistochemistry, and the other two were fixed in 4% buffered formaldehyde at room temperature and embedded in paraffin. 3.3. Morphometric analysis Five-micron cross-sections, taken from different levels of the paraffin imbedded grafts, underwent morphometrical evaluation. Morphometry was performed on sections stained with van Gieson and Mayer’s hematoxylin and eosin, and on sections stained for elastin. Separate areas of the vessel were measured, namely the lumen, intima and media using a computer digital image analysis system wLeica Qwin ŽLeica Imaging Systems, Cambridge, UK.x. The relative thickness Ž%. of intima wQint s intimarŽlumen q intima q media. = 100x and of media wQmeds mediarŽlumen q intima q media. = 100x were calculated. Three measurements were performed on each part of the graft to obtain the mean value for Qint and Qmed. 3.4. Immunohistochemistry Sections from the frozen graft segment and from the own aorta, were used for immunohistochemical evaluation of VSMC population, macrophage infiltration and the expression of TGF-␤1. The primary antibodies used were mouse anti-alpha smooth muscle actin ŽSMA. ŽM0851, Dako, Dakopattis AB, Sweden., mouse anti-rat ED-1 ŽMCA341R, Serotec, UK., rabbit anti-TGF-␤1 Žsc-146, Santa Cruz Biotechnology, Inc., USA.. Secondary antibodies were horseradish peroxidase conjugated goat anti-mouse IgG ŽSerotec, UK., rabbit antimouse link antibody, mouse PAP, swine anti-rabbit,

L. Zezina et al. r Transplant Immunology 8 (2001) 245᎐251

rabbit PAP Žall from Dako, Dakopattis AB, Sweden.. The staining procedure was performed as follows. Frozen sections were fixed in cold acetone, air dried, immersed in methanol with 1% hydrogen peroxide Žto quench the internal peroxidase activity. and incubated overnight with the primary antibody wdiluted in 0.1% BSA 1:200 ŽED-1., 1:50 ŽSMA., and 1:50 ŽTGF-␤1.x. Sections were rinsed in PBS, and incubated with the secondary antibody, or link antibody followed by a respective PAP antibody. Incubation at room temperature was 30᎐60 min long depending on the protocol for a particular antibody. The staining was developed with DAB and counterstained with hematoxylin. Image analysis was conducted with the help of the Leica Qwin software ŽLeica Imaging Systems, Cambridge, UK., and the results were expressed as area of positive staining per 1000 ␮m2 of the respective vessel layer.

247

Mann᎐Whitney U test was used for statistical comparison of immunohistochemical stainings between groups, since these data were non-parametrically-distributed. Results show the median and range Žin brackets.. A P value below 0.05 was considered statistically significant.

4. Results 4.1. Blood pressure Candesartan cilexetil treatment resulted in a decrease of blood pressure: SBP, 58 " 14 mmHg vs. 103 " 30 mmHg Ž P- 0.005. in the control group; DBP, 42 " 8 mmHg vs. 72 " 16 mmHg Ž Ps 0.005.; and MBP, 51 " 11 mmHg vs. 77 " 21 mmHg Ž P- 0.005.; respectively. 4.2. Vascular structural changes

3.5. Blood pressure Direct blood pressure measurement was performed on the last day of the experiment. A vascular catheter filled with saline was inserted into arteria femoralis under general anaesthesia. Blood pressure recordings were performed after a 10-min stabilisation period via P50 transducer ŽGould Statham, UK. attached to a Siemens amplifier ŽErbe, Tubingen, Germany., and a Mingocard 7 printer ŽSiemens, Elema, Sweden.. 3.6. Statistical analysis The mean values for Qint and Qmed for each animal were used for statistical analysis with the help of Student’s t test; results are expressed as mean " S.D.

Blocking of AT1 receptors with Candesartan cilexetil resulted in a reduction of intimal thickening both in allogeneic and syngeneic grafts ŽFig. 1.. Massive intimal hyperplasia detected in the control group allografts ŽQint s 30.2" 8.8%. was reduced by Candesartan cilexetil treatment ŽQint s 22.1" 8.7%. ᎏ P- 0.05 between allografts. Isografts initially had moderate intimal thickening ŽQint 15.5" 4.4%.. AT1 receptor blocking led to further reduction of intimal area ŽQint 6.7" 3.3%. ᎏ Ps 0.0001 between isografts. The proportion of SMA-positive cells in the control allograft neointima w153 Ž60᎐224.%x was lower compared to that of control isografts w275 Ž87᎐659.%x, P- 0.05. Candesartan cilexetil treatment had no influence on isograft intima, but led to an increase in the proportion of

Fig. 1. Effect of Candesartan cilexetil on neointima formation. Results presented as mean " S.E.M. of Qint s intimarŽlumen q intima q media.%. Control isografts-white bars, control allografts-grey bars, Candesartan cilexetil treated-striped white resp. grey bars.

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Fig. 2. Effect of Candesartan cilexetil on media preservation. Results presented as mean " S.E.M. of Qmeds mediarŽlumen q intima q media.%. Control isografts-white bars, control allografts-grey bars, Candesartan cilexetil treated-striped white resp. grey bars. Normal aorta-black bars.

SMA-positive cells w234 Ž45᎐641.%x in allograft neointima, P- 0.05. No improvement of the medial thickness by Candesartan cilexetil treatment could be observed in the allografts ᎏ Qmed 28.9" 4.6% vs. 31.4" 7.1% in the control group ŽFig. 2.. Media was better preserved in the isografts ŽQmed 38.3" 12.5% in the control animals., and the thickness of media in the Candesartan cilexetil treated group ŽQmed 54.4" 4.3%. was comparable to that of the normal aorta ŽQmed 46 " 10%.. Allograft media was essentially depleted of cells, SMA-positive area occupied only 46 Ž5᎐86.% compared to 291 Ž179᎐439.% in the control isografts Ž P0.05. and 650 Ž237᎐791.% in the normal aorta Ž P0.05.. Candesartan cilexetil treatment had no significant effect on SMA-positive cell population in the media.

4.3. TGF-␤ 1 expression Expression of TGF-␤1 was most intensive in the control allografts ŽTable 1., present in all vascular layers ŽFig. 3.. AT1 receptor blocking with Candesartan cilexetil reduced TGF-␤1 expression throughout the allograft vessel wall so that the intensity was comparable to the control isografts. Due to relatively low expression of TGF-␤1, no significant difference could be detected between the control and Candesartan cilexetil-treated isografts. No positive staining for TGF-␤1 could be detected in normal aortas. 4.4. Macrophage infiltration The most intense macrophage infiltration was found in the control allografts ŽTable 2. and macrophages

Table 1 TGF-␤1 expression in aortic grafts Control group

Candesartan cilexetil-treated

Allografts Intima Media Adventitia

ns7 6.17a Ž3.88᎐25.13.a 3.44 Ž3.04᎐48.24. 3.17 Ž1.17᎐13.96.

ns7 0.02 Ž0᎐1.26. 0.03 Ž0᎐2.45. 0 Ž0᎐2.75.

P - 0.01 P - 0.01 P - 0.05

Isografts Intima Media Adventitia

1.72 Ž0.01᎐7.89. 0.18 Ž0.02᎐5.54. 6.44 Ž0.15᎐12.73.

0.26b Ž0᎐0.83. 0.19b Ž0.02᎐0.38. 0.07b Ž0᎐0.65.

P - 0.05 NS P - 0.05

a b

Results presented as the median and range Žin brackets. of TGF-␤1 positive area per 1000 ␮m2 of respective vascular layer. Note that n s 6.

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249

Fig. 3. TGF-␤1 expression in the grafts: top left-control allograft; top right-candesartan cilexetil-treated allograft; below left-control isograft; and below right-candesartan cilexetil-treated isograft.

were present in all vascular layers. A trend to a decrease in macrophage infiltration was observed in Candesartan cilexetil-treated allografts. Isografts had a weak staining with the macrophage marker, which was not affected by Candesartan cilexetil treatment. No positive staining was observed in the media of normal aortas and, occasionally, macrophage could be detected in the adventitia.

5. Discussion In the present study we have evaluated the effect of AT1 receptor blocker Candesartan cilexetil on vascular remodelling in aortic transplantation in rat. It is rare for non-immunosuppressive medication to have a sig-

nificant beneficial effect on neointimal formation in allografts. Previously, we and other groups used various pharmacological agents in an attempt to reduce development of graft arteriosclerosis. However, strong immunological attack of the allograft did not allow full prevention of vascular remodelling w13x. Candesartan cilexetil reduced neointimal formation as reflected by the decrease of Qint. Simultaneously, the area of SMA-positive staining per 1000 ␮m2 of allograft neointima increased. This increase of the proportion of SMA-positive cells could be a result of decreased matrix accumulation, which agrees with studies showing that AT1 receptor blockade reduces matrix formation Žespecially for fibronectin synthesis . in the neointima of balloon-injured rat artery w5x. TGF-␤1 ᎏ a growth factor participating in tissue

L. Zezina et al. r Transplant Immunology 8 (2001) 245᎐251

250 Table 2 Macrophage infiltration in aortic grafts Control group

Candesartan cilexetil-treated

Allografts Intima Media Adventitia

ns9 7.2a Ž3.2᎐96.0.a 37.4 Ž10.1᎐287.7. 13.9 Ž2.5᎐13.9.

ns7 3.9 Ž0᎐10.9. 6.0 Ž1.9᎐35.6. 7.1 Ž2.0᎐45.1.

NS P- 0.05 NS

Isografts Intima Media Adventitia

ns5 0.03 Ž0᎐1.4. 0.01 Ž0᎐0.1. 0 Ž0᎐0.1.

ns7 0.02 Ž0᎐0.3. 0.05 Ž0᎐0.4. 0.04 Ž0᎐0.4.

NS NS NS

a

Results presented as the median and range Žin brackets. of ED-1 positive area per 1000 ␮m2 of the respective vascular layer. Note that n s 5.

repair and scarring ᎏ is upregulated during chronic rejection w14,15x. Recent studies have demonstrated that the effects of Ang II on VSMC, including migration and proliferation, involve autocrine TGF-␤1 loop w4,16x. Therefore, we have evaluated TGF-␤1 expression in the vascular layers of aortic grafts. Blocking of AT1 receptor signalling by Candesartan cilexetil was associated with reduced expression of TGF-␤1 in all allograft layers. We also observed a tendency to decreased TGF-␤1 expression in isografts, significant only in the neointima. It has been shown that TGF-␤1 may stimulate formation of neointima w17x, while inhibition of TGF-␤1 by antibodies prevents intimal thickening after balloon injury w18x. Thus we suppose that the positive effect of Candesartan cilexetil on neointimal formation might be explained by the reduction of TGF␤1 expression in the grafts. Recent studies have demonstrated that AT1 blockade not only prevents VSMC proliferation and matrix synthesis, but is also capable of reducing the inflammatory response w6x. Ang II is implicated to be involved in macrophage recruitment into the vessel wall w18x by induction of MCP-1 expression in VSMC. Administration of ACE inhibitor reduced both MCP-1 expression and the number of macrophages invading neointima in a rabbit model of early atherosclerosis w19x. Macrophage infiltration is a typical finding in aortic graft arteriosclerosis. Similarly, we observed strong expression of the macrophage marker ED-1 in the control aortic allografts as described previously w14x. However, Candesartan cilexetil had only a borderline influence on macrophage infiltration in our model. These findings agree with the fact that modification in the repair process rather than reduction of acute inflammation by AT1 receptor blockade or ACE inhibition is important in preventing graft arteriosclerosis w20x. We suppose that the immunological incompatibility in allogeneic transplantation, absent in mechanical endothelial damage models, contributes to macrophage recruitment

into the vascular wall and counteracts possible effects of Candesartan cilexetil. Our findings suggest that the use of AT1 receptor blockers could be an option for treating chronic rejection of solid organ grafts to prevent deterioration of their function, since this group of drugs provides beneficial effect of hemodynamics and prevents the damaging action of Ang II on vascular cells, effects which have been demonstrated to have a pivotal role in the progression of chronic graft dysfunction w21x. In conclusion, our study demonstrated reduction in graft arteriosclerosis by AT1 receptor blockade by Candesartan cilexetil in a rat aortic transplant model. The beneficial effect of the drug might, in part, be explained by the reduction of TGF-␤1 expression in the grafts.

Acknowledgements This study was supported by grants from the Swedish Heart-Lung Foundation, Swedish Medical Research Council and the Kidney Disease Association of the CUWX Counties. The excellent technical assistance of Ulla Svensson is gratefully acknowledged. References w1x Isik FF, Ferguson M, Yamanaka E, Gordon D. Transplant arteriosclerosis in a rat aortic model. Am J Pathol 1992;141Ž5.:1139᎐1149. w2x Mennander A, Tiisala S, Halttunen J, Yilmaz S, Paavonen T, Hayry P. Chronic rejection in rat aortic grafts. An experimental model for transplant arteriosclerosis. Arterioscler Thromb 1991;11Ž3.:671᎐680. w3x Su EJ, Lombardi DM, Siegal J, Schwartz SM. Angiotensin II induces vascular smooth muscle replication independent of blood pressure. Hypertension 1998;13Ž6.:1331᎐1337. w4x Ford CM, Li S, Pickering G. Angiotensin II stimulates collagen synthesis in human vascular smooth muscle cells. Involvement of the ATŽ1. receptor, transforming growth factor-beta, and tyrosine phosphorylation. Arterioscler Thromb Vasc Biol 1999;19Ž8.:1843᎐1851. w5x Kim S, Kawamura M, Wanibuchi H et al. Angiotensin II type-1 receptor blockade inhibits the expression of immediate-early genes and fibronectin in rat injured artery. Circulation 1995;92Ž1.:88᎐95. w6x Kranzhofer R, Schmidt J, Pfeiffer CAH, Hagl S, Libby P, Kubler W. Angiotensin induces inflammatory activation of hu¨ man vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 1999;19Ž7.:1623᎐1629. w7x Pratt RE. Angiotensin II and the control of cardiovascular structure. J Am Soc Nephrol 1999;10ŽSuppl 11.:S120᎐S128. w8x Miyazaki M, Wada T, Shiota N, Takai S. Effect of angiotensin II receptor antagonist Candesartan cilexetil on canine intima hyperplasia after balloon injury. J Human Hypertens 1999;13ŽSuppl 1.:S21᎐S25.

L. Zezina et al. r Transplant Immunology 8 (2001) 245᎐251 w9x Tazawa S, Nakane T, Chiba S. Angiotensin II type 1 receptor blockade prevents upregulation of angiotensin II type 1 receptors in rat injured artery. J Pharmacol Exp Therap 1999;288Ž2.:898᎐904. w10x Little DM, Haynes LD, Alam T, Geraghty JG, Sollinger HW, Hullett DA. Does transforming growth factor-1 play a role in the pathogenesis of chronic allograft rejection? Transpl Int 1999;12Ž6.:393᎐401. w11x Liu Q, Espinosa E, Oemar BS, Luscher FT. Bimodal effects of ¨ angiotensin II on migration of human and rat smooth muscle cells. Direct stimulation and indirect inhibition via transforming growth factor. Arterioscl Thromb Vasc Biol 1997;17Ž7.:1251᎐1257. w12x Wanders A, Akyurek ML, Waltenberger J et al. Ischemia¨ induced transplant arteriosclerosis in the rat. Arterioscler Thromb Vasc Biol 1995;15Ž1.:145᎐155. w13x Akyurek LM, Funa K, Wanders A, Larsson E, Fellstrom ¨ ¨ BC. Inhibition of transplant arteriosclerosis in rat aortic grafts by non- -anticoagulant heparin derivatives. Transplantation 1995;59Ž11.:1517᎐1524. w14x Waltenberger J, Akyurek LM, Aurvillius M et al. Ischemia¨ induced transplant arteriosclerosis. Induction of peptide growth factor expression. Arterioscler Thromb Vasc Biol 1996;16Ž12.:1516᎐1523. w15x Waltenberger J, Wanders A, Fellstrom ¨ B, Miyazono K, Heldin

w16x

w17x

w18x w19x

w20x

w21x

251

CH, Funa K. Induction of transforming growth factor-beta during cardiac allograft rejection. J Immunol 1993;151Ž2.: 1147᎐1157. Gibbons GH, Pratt RE, Dzau VJ. Vascular smooth muscle cell hypertrophy vs. hyperplasia. Autocrine transforming growth factor-beta 1 expression determines growth response to angiotensin II. J Clin Invest 1992;90Ž2.:456᎐461. Kanzaki T, Tamura K, Takahashi K et al. In vivo effect of TGF-1. Enhanced intimal thickening by administration of TGF-1 in rabbit arteries injured with a balloon catheter. Arterioscl Thromb Vasc Biol 1995;15Ž11.:1951᎐1957. Wolf YG, Rasmussen LM, Ruoslahti E. Antibodies against transforming growth factor-1 suppress intimal hyperplasia in a rat model. J Clin Invest 1994;93Ž3.:1172᎐1178. Hernandez-Presa M, Bustos C, Ortego M et al. Angiotensinconverting enzyme inhibition prevents arterial nuclear factor-kB activation, monocyte chemoattractant protein-1 expression and macrophage infiltration in rabbit model of early-accelerated atherosclerosis. Circulation 1997;95Ž6.:1532᎐1541. Furukawa Y, Matsumori A, Hirozane T, Sasayama S. Angiotensin II receptor antagonist TCV-116 reduces graft coronary artery disease and preserves graft status in murine model. Circulation 1996;93Ž2.:333᎐339. Paul LC, Benediktsson H. Post-transplant hypertension and chronic allograft failure. Kidney Int 1994;48ŽSuppl 52.:S34᎐S37.