Thalidomide Attenuates Graft Arteriosclerosis of Aortic Transplant in a Rat Model

Thalidomide Attenuates Graft Arteriosclerosis of Aortic Transplant in a Rat Model Y. Zhang, M. Yang, Y. Yang S.L. Zheng, Y. Cai, P. Xia, W.W. Chen, B.C. Chen, and Y.R. Yang ABSTRACT Objective. The purpose of the current study was to evaluate the effects of thalidomide on graft arteriosclerosis. Materials and Methods. Male Lewis rats received abdominal aorta grafts from male Brown-Norway rats. The animals were divided into 4 groups: no treatment controls, a low-dose group that received thalidomide (50 mg/kg per day), a middle dose group that received thalidomide (100 mg/kg per day), and a high-dose group that received thalidomide (200 mg/kg per day) by daily intragastric administration. Rats were humanely killed at 60 days after surgery. The grafted aortas were analyzed by histology, immunohistochemistry, and Western blot analysis. The serum was analyzed by an enzyme-linked immunosorbent assay (ELISA). Results. The neointimal thickness of the thalidomide treated aortas was significantly thinner compared with that of no treatment aortas (P ⬍ .05). Vascular endothelial growth factor (VEGF), platelet-derived growth factor, and intracellular adhesian molecule (ICAM-1) protein expression in the treatment group were significantly lower than those in the control group (P ⬍ .05). Moreover, thalidomide significantly inhibited the production of VEGF and ICAM-1 in serum (P ⬍ .05). Conclusion. Our data suggested that thalidomide can attenuate graft arteriosclerosis so as to protect aortic grafts. LTHOUGH graft loss owing to acute rejection has been steadily reduced to ⬍10%, the development of graft arteriosclerosis remained a major obstacle to long-term allograft survival and a major cause of late allograft dysfunction and late death in some patients.1 Thalidomide was first introduced in the late 1950s for the prevention of morning sickness in pregnant women, but it was withdrawn from the market in the 1960s because of its well-known teratogenicity.2 Recently, thalidomide has been found to have anti-angiogenic, anticancer, and antiinflammatory properties. Based on these observations, thalidomide has been used as a therapeutic reagent in chronic graft-versus-host disease (GVHD) of bone marrow transplantation and some malignant tumors, including multiple myeloma, liver cancer, gliomas, renal cell carcinoma, and breast cancer.3 Graft arteriosclerosis is a leading cause of chronic transplant dysfunction and is characterized by occlusive neointima formation in intragraft arteries. Development of graft arteriosclerosis is refractory to conventional immunosuppressive drugs and adequate therapy is not available.

A

A number of studies have demonstrated that the action of thalidomide as an immunosuppressive and antiinflammatory drug in bone marrow transplant rejection,4,5 and current study showed that thalidomide could prolong allograft survival of animals that underwent heterotopic heart transplantation.6 The present study was designed to determine the effect of thalidomide on graft arteriosclerosis in a rat model, as well to determine its possible molecular mechanism.

From the Transplantation Center, The First Affiliate Hospital of Wenzhou Medical College, Wenzhou, Zhejiang, China. This Work Was Supported by the Zhejiang Provincial Top Key Discipline in Surgery and Nature Science Foundation of Zhejiang Province (Y2110944). Address correspondence to Dr. Y.R. Yang and B.C. Chen, The First Affiliate Hospital of Wenzhou Medical College, Transplantation Center, 1#Fuxuexiang, Wenzhou, Zhejiang 325000, China. E-mail: [email protected]

0041-1345/11/$–see front matter doi:10.1016/j.transproceed.2011.03.086

© 2011 Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710

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Transplantation Proceedings, 43, 2022–2026 (2011)

THALI DOMIDE ATTENUATES GRAFT STENOSIS

MATERIALS AND METHODS Aortic transplantations were performed using Brow-Norway (BN) as donors and Lewis as recipients (purchased from Beijing Vital River). The rats were anesthetized by intraperitoneal injection of pentobarbital (60 mg/kg). The abdominal aorta from renal artery to the bifurcation (about 15 mm) was harvested from BN rats after intravenous injection of heparin. And then the aortic grafts were transplanted orthotopically into recipients with an end-to-end anastomosis technique, using non-interrupted 8-0 nylon sutures. After transplantation, the animals were randomly divided into 4 groups: Group 1 (n ⫽ 10) served as untreated controls; group 2

2023 (n ⫽ 10) received thalidomide (50 mg/kg per day) by daily intragastric administration; group 3 (n ⫽ 10) received thalidomide (100 mg/kg per day) by daily intragastric administration; and group 4 (n ⫽ 10) received thalidomide (200 mg/kg per day) by daily intragastric administration. All grafts were harvested 60 days after transplantation. After formaldehyde fixation, aortic segments were embedded in paraffin and 5-␮m sections stained with hematoxylin-eosin-saffron (HES) to examine the morphology, cytology, and neointimal thickness of the transplanted vessels. Immunohistochemistry was performed to localize the expression of vascular endothelial growth factor

Fig 1. HES staining and immunohistochemical staining in transplanted aortas. (A–D) Representative micrographs of HES staining (original magnification, ⫻100); (E–H) immunohistochemical staining for VEGF (original magnification, ⫻100); (I–L) immunohistochemical staining for PDGF (original magnification, ⫻ 100); and (M–P) immunohistochemical staining for ICAM-1 (original magnification, ⫻200); in transplanted aortas of the (A, E, I, M) no treatment, (B, F, J, N) thalidomide 50 mg/d treatment, (C, G, K, O) thalidomide 100 mg/d treatment, and (D, H, L, P) thalidomide 200 mg/d treatment groups.

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ZHANG, YANG, YANG ET AL nitrocellulose membranes, and probed with specific antibodies against VEGF (Santa Cruz Biotechnology), PDGF (Santa Cruz Biotechnology), and ICAM-1 (Abcam). ␤-Actin (Santa Cruz Biotechnology) was used as control. The results of each experimental group were expressed as relative integrated intensity compared with control normal arteries measured with the same batch. Statistical analysis was performed using 1-way analysis of variance. All data are presented as the mean values ⫾ standard error of the mean. Statistical significance was defined at P ⬍ .05.

RESULT Neointimal Thickness

HES staining shows that intimal cells of control group marked proliferation. The experimental group that received thalidomide resulted in a significant reduction in neointimal thickness (5.78 ⫾ 0.71 ␮m [50 mg/d]; 2.60 ⫾ 0.32 ␮m [100 mg/d]; or 2.54 ⫾ 0.29 ␮m [200 mg/d]) compared with the no treatment controls (9.28 ⫾ 0.33 ␮m; P ⬍ .05; Fig 1). Neointimal thickness in groups that received thalidomide 100 mg/d was significantly decreased compared with the 50 mg/d thalidomide group (P ⬍ .05). However, there was no additional beneficial effect in the 200-mg/d thalidomide group when compared with rats treated with 100 mg/d thalidomide (P ⬎ .05; Fig A–D). Inhibition of VEGF, PDGF, and ICAM-1 Expression in Aortic Allografts by Thalidomide

Fig 2. Western blotting showed thalidomide to down-regulate the expression of VEGF, PDGF, and ICAM-1 in transplanted aortas. The same blot was stripped and reprobed with actin to confirm equal loading. (VEGF; Santa Cruz Biotechnology, Santa Cruz, Calif), plateletderived growth factor PDGF (Santa Cruz Biotechnology), and intracellular adhesion molecule (ICAM-1; Abcam, Cambridge, Mass). The protein was quantified by scanning densitometry using a bio-image analysis system (Bio-Profile, Minneapolis, Minn). And the production of ICAM-1 and VEGF in serum was determined using an enzyme-linked immunosorbent assay (ELISA). Western blot analysis was conducted to evaluate the tissue expression of VEGF, PDGF, and ICAM-1 in the harvested transplanted artery. Vessel tissues were homogenized in lysis buffer and protein concentrations were determined using a Micro BCA Protein Assay kit (Pierce, Rock ford, Ill). Twenty micrograms of protein per specimen were separated on a SDS-polyacrylamide gel, blotted onto

Immunohistochemical staining showed that VEGF and PDGF staining was on the surface of endothelial cells (Figure 1 E–L), ICAM-1 staining was in the cytoplasm of endothelial cells as well as neointimas (Fig 1M–P). Western blot analysis of the same aortic grafts was performed to evaluate the expression of VEGF, PDGF, and ICAM-1 between the 4 groups (Fig 2). The experimental group that received thalidomide demonstrated markedly downregulated expression of VEGF (1.15 ⫾ 0.09 [50 mg/d], 0.49 ⫾ 0.11 [100 mg/d], 0.48 ⫾ 0.12 [200 mg/d]) compared with the no treatment controls (1.49 ⫾ 0.19; P ⬍ .05), PDGF (1.54 ⫾ 0.17 [50 mg/d], 0.71 ⫾ 0.09 [100 mg/d], 0.70 ⫾ 0.10 [200 mg/d]) compared with the no treatment controls (2.18 ⫾ 0.22; P ⬍ .05), and ICAM-1 (0.46 ⫾ 0.38 [50 mg/d], 0.29 ⫾ 0.20 [100 mg/d], 0.31 ⫾ 0.21 [200 mg/d]) compared with the no treatment controls (1.16 ⫾ 0.08; P ⬍ .05). The expression of VEGF, PDGF, and ICAM-1 in group that received thalidomide 100 mg/d was significantly decreased compared with 50-mg/d thalidomide group (P ⬍ .05). However, there was no additional beneficial effect in the 200-mg/d thalidomide group

Table 1. Comparison of the Densitometry for VEGF, PDGF, and ICAM-1 Expression Between the Treatment and Control Groups

VEGF PDGF ICAM-1

Control

Thalidomide 50 mg/d

Thalidomide 100 mg/d

Thalidomide 200 mg/d

1.49 ⫾ 0.19 2.18 ⫾ 0.22 1.16 ⫾ 0.08

1.15 ⫾ 0.09* 1.54 ⫾ 0.17* 0.46 ⫾ 0.38*

0.49 ⫾ 0.11*† 0.71 ⫾ 0.09*† 0.29 ⫾ 0.20*†

0.48 ⫾ 0.12*† 0.70 ⫾ 0.10*† 0.31 ⫾ 0.21*†

*P ⬍ .05 compared with the control group. † P ⬍ .05 compared with the thalidomide 50 mg/d treatment group.

THALI DOMIDE ATTENUATES GRAFT STENOSIS

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Table 2. Comparison of the Production of VEGF and ICAM-1 in Serum Between the Treatment and Control Groups

VEGF ICAM-1

Control

Thalidomide 50 mg/d

Thalidomide 100 mg/d

Thalidomide 200 mg/d

55.96 ⫾ 8.42 45.96 ⫾ 13.08

36.59 ⫾ 5.27* 37.52 ⫾ 11.23*

11.96 ⫾ 3.04* 29.61 ⫾ 9.28*†

11.38 ⫾ 3.25*† 28.98 ⫾ 9.31*†

†

*P ⬍ .05 versus the control group. † P ⬍ .05 compared with the thalidomide 50 mg/d treatment group.

when compared with rats treated with the 100-mg/d thalidomide (P ⬎ .05; Table 1). Effect of Thalidomide Treatment on the Production of ICAM-1 and VEGF in Serum

ICAM-1 concentration in serum was significantly decreased in the thalidomide treatment groups (37.52 ⫾ 11.23 pg/mL [50 mg/d], 29.61 ⫾ 9.28 pg/mL [100 mg/d], 28.98 ⫾ 9.31 pg/mL [200 mg/d]) compared with no treatment controls (45.91 ⫾ 13.08 pg/mL; P ⬍ .05); meanwhile, the VEGF concentration in serum was significantly decreased in the thalidomide treatment group (36.59 ⫾ 5.27 pg/mL [50 mg/d], 11.96 ⫾ 3.04 pg/mL [100 mg/d], 11.38 ⫾ 3.25 pg/mL [200 mg/d]) compared with no treatment controls (55.96 ⫾ 8.42 pg/mL; P ⬍ .05). Moreover, rats that treated with 100 and 200 mg/d thalidomide showed significantly decreased the level of ICAM-1 and VEGF in serum compared with that of controls and the 50-mg/d thalidomide treatment group. There were no significant differences in the 100- and 200-mg/d thalidomide treatment groups (P ⬎ .05; Table 2). DISCUSSION

The development of transplant arteriosclerosis in allografts is a multifactorial process, with macrophages, T cells, proinflammatory cytokines, adhesion molecules, growth factors, and alloantibodies implicated in both the initiation and progression of this chronic inflammatory process.7,8 VEGF induces atherosclerotic plaque growth and allograft arteriosclerosis by recruiting macrophages and increasing intimal neovascularization.9,10 In clinical transplantation, VEGF induction occurs in both acute and chronic rejection.11–14 And some research showed that the functional role of VEGF has been linked to the development of allograft inflammation and neointimal development.15,16 PDGF, similar to VEGF, is a crucial growth factor in normal embryonic development, and it is a major migratory and proliferative stimulus for mesenchymal cells.17 PDGF regulates the recruitment and proliferation of pericytes and smooth muscle cells that form support for the endothelial cell layer in newly formed vessels.17,18 In addition to its physiologic functions, PDGF is induced in many pathologic conditions, such as atherosclerosis19 and allograft arteriosclerosis.20,21 In allograft arteriosclerosis, PDGF may serve as a migration and proliferation signal for smooth muscle cells and their progenitors invading the intima. ICAMs are also important in transplant arteriosclerosis because they mediate recipient inflammatory cell attachment to and migration into the donor vessel wall.22

In addition to their roles in leukocyte trafficking, adhesion molecules may also regulate the migration of smooth muscle cells within the vessel wall.23 Thalidomide has been used for the treatment of graftversus-host reaction in patients undergoing bone marrow transplantation for its antiinflammatory and immunosuppressive effects.24 In some research, thalidomide was effective in acute25,26 and chronic27 GVHD; moreover, thalidomide was found to be equally as effective as methylprednisolone,28 and appears to replace corticosteroids effectively in early postoperative immunosuppression after lung transplantation.29 The current study showed that thalidomide was effective against rejection, significantly increasing the survival of animals submitted to heterotopic heart transplantations in an abdominal position.30 And also thalidomide could inhibit melanoma tumor growth by decrease VEGF and PDGF expression in some experiments.31 In this study, we have demonstrated that thalidomide treatment exerts beneficial effects in a rat model of transplant arteriosclerosis. We have demonstrated herein that thalidomide could reduce the neointimal thickness, and inhibit VEGF, PDGF, and ICAM-1 protein expression in transplanted aortas. In addition, thalidomide could reduce the production of VEGF and ICAM-1 in serum. All of these findings support the thalidomide exert potent antiarteriosclerosis effects; what, then, is the mechanism by which thalidomide inhibits transplant arteriosclerosis? Our results indicate that thalidomide is effective in preventing the formation of transplant arteriosclerosis. In conclusion, the data presented in the present study demonstrate that thalidomide can effectively reduce the formation of transplant arteriosclerosis in a murine aortic transplant model. Furthermore, the financial cost of immunosuppression would also be considerably reduced because thalidomide is a less expensive medication. These findings therefore have important clinical implications, because patients who have transplant arteriosclerosis after transplantation might substantially benefit from treatment with this drug.

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