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Published in final edited form as: Transpl Int. 2017 Aug 14;30(11):1181–1189. doi: 10.1111/tri.13004

THALIDOMIDE TREATMENT PREVENTS CHRONIC GRAFT REJECTION AFTER AORTIC TRANSPLANTATION IN RATS

Katharine K Miller a,b,c,d, Dong Wang a,b,c,d, Xiaomeng Hu a,b,c,d, Xiaoqin Hua a,c,d, Tobias Deuse a,b,c,d,e, Evgenios Neofytou f,g, Thomas Renne h,i, Joachim Velden j, Hermann Reichenspurner c,d,e, Sonja Schrepfer a,b,c,d,e,*, Daniel Bernstein k,l,*
PMCID: PMC5643223  NIHMSID: NIHMS890793  PMID: 28672061

Abstract

Background

Cardiac allograft vasculopathy (CAV) affects approximately 30% of cardiac transplant patients at five years post-transplantation. To date there are few CAV treatment or prevention options, none of which are highly effective. The aim of the study was to investigate the effect of thalidomide on the development of CAV.

Methods

The effect of thalidomide treatment on chronic rejection was assessed in rat orthotopic aortic transplants in allogeneic F344 or syngeneic Lew rats (n=6/group). Animals were left untreated or received thalidomide for 30 days post-transplant, and evidence of graft CAV was determined by histology (trichrome and immunohistochemistry) and intragraft cytokine measurements.

Results

Animals that received thalidomide treatment post-transplant showed markedly reduced luminal obliteration, with concomitant rescue of smooth muscle cells (SMCs) in the aortic media of grafts. Thalidomide counteracted neointimal hyperplasia by preventing dedifferentiation of vascular SMCs. Measurement of intragraft cytokine levels after thalidomide treatment revealed down-regulation of matrix metalloproteinase 8 (MMP-8) and monocyte chemotactic protein 1 (MCP-1), cytokines involved in tissue remodeling and inflammation, respectively. Importantly, no negative side effects of thalidomide were observed.

Conclusions

Thalidomide treatment prevents CAV development in a rodent model and is therefore potentially useful in clinical applications to prevent post-transplant heart rejection.

Keywords: Cardiac allograft vasculopathy, rats, thalidomide

INTRODUCTION

Over the past several decades, patient survival after heart transplantation has improved; however, most of this benefit has accrued during the early period post-transplantation while long-term survival is still compromised by complications such as chronic cardiac allograft rejection, otherwise known as cardiac allograft vasculopathy (CAV). This form of chronic rejection – occurring months to years post-transplant – affects more than 30% of patients at 5 years post-transplant1–3. CAV is a highly aggressive form of coronary artery disease4 caused by a combination of immune and non-immune responses that result in characteristic narrowing of donor coronary arteries, although the exact mechanisms remain unclear. Of the few treatment options available for CAV, most focus on prevention and none are particularly effective5. To this date, the only treatment option for patients with CAV associated with longer lifespan is re-transplantation, which carries a higher risk compared to the original transplant6.

Thalidomide was originally introduced as a therapy for morning sickness in pregnant women, but was soon removed from the market due to its teratogenic effects7. However, thalidomide has since been repurposed as an immunomodulator, and has been approved for use as a treatment for multiple diseases, including multiple myeloma and erythema nodosum leprosum8–10. Thalidomide’s anti-inflammatory and immunomodulatory functions as well as its beneficial effect on chronic graft-versus host disease after bone marrow transplantation make it an ideal candidate for preventing graft rejection11. Indeed, thalidomide alone or low levels of thalidomide and cyclosporine can be used to reduce rejection of cardiac allografts in rabbits12, and thalidomide has been found to lessen neotintimal thickness after aortic graft transplantation13. However, the mechanism by which thalidomide reduces rejection rates remains unknown, with reports conflicting regarding its effects on inflammatory cells, vascular pericytes, and SMCs.

Here we show effects of thalidomide treatment in a chronic rat orthotopic (Male Fischer 344:Male Lewis) aortic transplantation model. Thalidomide dramatically reduces the development of intimal thickening by rescuing SMC numbers, differentiation, and localization. Intragraft cytokine levels were measured to further determine thalidomide’s possible mechanisms of action.

MATERIALS AND METHODS

Animals

All animals were purchased from Charles River Laboratories, Germany, and received care in compliance with the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1996). Studies were approved by the local ethical committee “Hamburg Amt für Gesundheit und Verbraucherschutz”.

Orthotopic Aortic Transplantation (Chronic CAV Study)

Orthotopic aortic transplantation was performed as previously described14. Briefly, a section of the thoracic aorta from F344 (allogeneic) or Lew (syngeneic) was orthotopically transplanted into the infra-renal abdominal aorta of Lew rats via an end-to-end anastomosis. Three groups (n=6 animals per group) were randomly assigned to receive either 100mg/kg/d of thalidomide p.o., or vehicle control.

Heterotopic Heart Transplantation (Acute Study)

Heterotopic heart transplantation was performed as previously described15. Briefly, BN (allogeneic) and Lew (syngeneic) hearts were heterotopically transplanted to the abdominal great vessels of Lew rats. Three groups (n=6 animals per group) were randomly assigned to receive either 100mg/kg/d of thalidomide p.o., or vehicle control. Six days post transplant, animals were euthanized, the apical half of the graft was snap frozen for cytokine quantification, and the caudal part was fixed (4% paraformaldehyde) for further tissue processing.

Histopathology

Harvested grafts were fixed (4% paraformaldehyde), dehydrated and embedded in paraffin. Each block was sectioned into five-micrometer sections followed by trichrome staining using the manufacturer’s protocol (Sigma Aldrich) for evaluating luminal occlusion and immunofluorescence. Luminal obliteration was quantified, using Image J (Bethesda, MD) as follows: Vascular occlusion (%)=[Area of intima/(Area of intima+Vascular lumen)]×100. Three sections were analyzed from each aortic allograft, and results were averaged.

Immunofluorescence Staining

Paraffin sections underwent heat-induced antigen retrieval with Dako antigen retrieval solution (Dako, Glostrup, Denmark) followed by blocking with Image-iT FX signal enhancer (Invitrogen). Primary antibodies were used as appropriate: Smooth Muscle Actin (SMA) (ab5694), Smooth Muscle Heavy Chain (SMHC) (ab124679), or embryonic smooth muscle heavy chain (SmemB) (Yamasa 7602). A mouse irrelevant IgG1 (Abcam) was used as negative control. Secondary antibodies were Alexa Fluor 488 or Alexa Fluor 555 (Invitrogen). Imaging was performed using a Nikon Eclipse TiE microscope equipped with the Perkin Elmer UltraVIEW VoX confocal imaging system. Analysis was carried out with Volocity 6.1.1 (Perkin Elmer).

Side Effect Screening

Serum was obtained prior to animal sacrifice in order to investigate the effect of thalidomide on blood cholesterol, triglycerides, kidney, liver, and blood count.

Cytokine Antibody Array

Cytokine antibody arrays of homogenized grafts were performed according to the manufacturer’s protocol (Raybiotech, Norcross, Georgia, USA). Membranes were digitized using bioluminescence imaging and quantified using Image J (Bethesda, Maryland). Cytokine concentrations are expressed in arbitrary units (AU).

Unidirectional ELISpot Assay

Recipient splenocytes (responder cells) were isolated from fresh spleen six days after heart transplantation. 1x106 donor stimulator cells were incubated with 1x106 responder cells for 24h. IFNγ spot frequencies were assessed in quadruplicate using an automatic counted ELISpot plate reader (CTL, Cincinnati, OH).

CFSE-MLR Proliferation Assay

The carboxyfluorescein succinimidyl ester–mixed lymphocyte reaction (CFSE-MLR) was performed with the acute heterotopic transplantation study animals. Briefly, Responder and stimulator cells were co-cultured at equal ratios (3 x106/ml) in a 48 well plate for five days at 37°C, 5% CO2 in RPMI 1640 medium (Gibco) supplemented with 1% penicillin/streptomycin solution, 10% FCS and 50μM β-mercaptoethanol (Millipore, ES-007-E). Cells were harvested and analyzed by flow cytometry, gating for intensity of CFSE fluorescence. Stimulation index (SI) was calculated based on the mean value of each animal.

Statistics

Data are presented as mean ± standard deviation. Comparisons within groups used analysis of variance with Bonferroni or LSD Post-hoc tests, as appropriate. Probability values (P) less than 0.05 were considered significant. Statistical analysis was performed using SPSS statistical software package 15.0 for Windows (SPSS Inc., Chicago, IL).

RESULTS

Thalidomide treatment reduces luminal obliteration in chronic rejection model

We measured the effect of thalidomide on intimal thickening in a chronic low responder model using an accepted orthotopic aortic transplantation model for CAV14,16. Syngeneic transplants were performed as a procedural control. Allogeneic transplants were given either vehicle control or 100mg/kg thalidomide by oral gavage for 30 days post-transplant (Figure 1A).

Figure 1. Treatment with thalidomide reduces luminal obliteration.

Figure 1

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(A) Overview of experimental protocol. Rats underwent either syngeneic (blue) or allogeneic (green and red) orthotopic aortic transplants. Allogeneic transplant recipients received either no medication (vehicle control, green) or thalidomide (100 mg/kg; red) by oral gavage for 30d post-transplant. (B) Representative Masson’s trichrome-stained images of aortic luminae after treatment regime show that the increased luminal obliteration after allogeneic transplant is drastically reduced by thalidomide treatment. (Top row: 50x magnification; Bottom row: 400x magnification). (C) Quantification of luminal obliteration confirms levels of hyperplasia. The syngeneic model shows low base levels of obliteration (mean=1.34%), which is strongly increased in the allogeneic no medication control (mean=21.27%) and strikingly mitigated upon treatment with thalidomide (mean=6.76%; p<0.001). Error bars indicate mean ± SD. *P<0.05.

As expected, syngeneic grafts showed no signs of cellular rejection in histology and exhibited minimal levels of luminal obliteration (1.32%) as compared to vehicle-treated allogeneic grafts (21.27%). Strikingly, animals treated with thalidomide showed marked reduction in luminal occlusion (6.76%, p<0.001) (Figure 1B/C), suggesting that thalidomide may be a viable option for preventing CAV.

SMCs of the aortic media are maintained by thalidomide in CAV model

Staining for smooth muscle actin (SMA) confirms that the aortic media of syngeneic grafts is largely comprised of mature SMCs (Figure 2A). A strong reduction in medial SMA intensity was found in vehicle-treated allogeneic grafts, an effect that was prevented by thalidomide-treatment. Quantification of immunohistochemical images confirmed these observations (Figure 2C).

Figure 2. SMC number, differentiation, and localization are maintained by thalidomide treatment.

Figure 2

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(A) Mature SMCs stained with SMA (green) are shown organized at the aortic media in syngeneic model. While allogeneic no medication control treated animals show a disorganized media with a reduction in the number of SMCs, treatment with thalidomide shows a clear retention of both number and localization of SMCs (400x magnification). (B) Allogeneic transplant shows an increase in dedifferentiated SMCs (Smemb; Red) which correlates with a parallel loss of differentiated SMCs (SMHC; Green). Thalidomide treatment maintains differentiation and localization of SMCs. (400x magnification). (C) Quantification of fluorescence further confirms the retention of mature SMC by thalidomide treatment (SMA, SMHC) and dedifferentiated SMC (SMemb) levels. Error bars indicate mean ± SD. *P<0.05.

To clarify SMC differentiation levels in each model, we compared smooth muscle cell heavy chain (SMHC; marker for differentiated SMCs) and embryonic smooth muscle heavy chain (SMemb; marker for dedifferentiated SMCs) intensities (Figure 2B/C). Our images visually and quantifiably revealed that syngeneic transplants have organized differentiated medial SMCs and low levels of dedifferentiated intimal SMCs. In contrast, vehicle-treated allogeneic transplants had decreased differentiated SMC levels and increased dedifferentiated SMCs. Thalidomide treatment strongly attenuated these effects, reducing dedifferentiated intimal cells to control levels with a corresponding increase in differentiated medial SMCs.

Thalidomide treatment alters intra-graft cytokine release profile

To investigate the mechanism by which thalidomide affects the proliferative and pathological features of CAV, we examined expression of key cytokines/chemokines induced in allogeneic graft transplantation (Figure 3). Cytokine protein arrays showed that several cytokines, including MMP-8, TIMP-1, MCP-1, and ICAM-1, were significantly upregulated in the vehicle-treated allogeneic group vs. controls. Thalidomide reduced expression levels of MMP-8 (p<0.05) and MCP-1 (p<0.01), cytokines associated with tissue remodeling and inflammation, respectively. Reduction of these cytokines may be indicative of thalidomide’s mechanism of action: decreased tissue remodeling, as seen in our histological results, as well as reduced post-transplant inflammation.

Figure 3. Cytokine profiles of grafts show thalidomide treatment reduces levels of specific cytokines.

Figure 3

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(A) Representative images of intragraft cytokine profiles for each graft type. (B) Quantification of cytokine profiles reveals that expression levels of MMP-8, TIMP-1, MCP-1 and ICAM-1 are upregulated after allogeneic transplant with no medication. MMP-8 and MCP-1 expression are reduced in the thalidomide treatment model. Error bars indicate mean ± SD. *P<0.05. †P<0.01.

No significant side effects caused by thalidomide treatment in rats

We tested the safety of thalidomide after allogeneic orthotopic aortic transplants. Animals treated with thalidomide showed no obvious signs of discomfort or distress. Blood samples from vehicle (n=3) or thalidomide (n=7) treated animals showed no significant differences in biomarkers of kidney function (BUN, creatinine), serum cholesterol and tryglyceride levels (cholesterol, triglyceride, LDL, HDL), liver function (ALT, AST), or whole blood count (RDW, Leukocyte, PLT, Hb, Hct, RBC, MCV, MCH, MCHC) (Supplementary Figure 1).

Thalidomide treatment does not attenuate lymphocyte activity in acute transplant rejection model

Since our data supports thalidomide as a potential therapy for CAV, we aimed to clarify whether the success of thalidomide treatment was due to a general reduction in graft rejection or whether it was specific to chronic rejection. Thus, we tested the effect of thalidomide treatment in heterotopic heart transplantation, an accepted rodent model for acute transplant rejection15. Syngeneic (Lew:Lew) or allogeneic (BN:Lew) transplantation was performed (Supplementary Figure 2A). Allogeneic transplant recipients received either vehicle control or thalidomide post-transplant. At six days post-operation (POD6), splenocytes were collected for ELISpot and mixed lymphocyte reaction (MLR) assays.

IFNγ levels – a proxy for systemic lymphocyte activation – were measured by ELISpot. In contrast to the dramatic effects of thalidomide in our chronic rejection model, thalidomide treatment did not appear to affect IFNγ production in our acute rejection model (Supplementary Figure 2B). Similarly, no alterations in lymphocyte proliferation were found by our MLR assay (Supplementary Figure 2C). These data suggest that thalidomide may be a specific treatment for chronic rejection, as it does not appear to be beneficial for reduction of acute rejection.

The primary pathology in CAV is coronary vessel intimal thickening; thus, reducing luminal obliteration would be highly advantageous to the long-term survival of transplant recipients.

DISCUSSION

CAV is a leading cause of late death for cardiac transplant recipients, and has remained resistant to therapeutic interventions. The identification of thalidomide as an immunomodulatory agent suggests its potential use in preventing graft rejection. Thalidomide has been described as having anti-cytokine, anti-integrin, and anti-angiogenic properties17, and has beneficial effects in other proliferative diseases, including as a treatment for idiopathic pulmonary fibrosis, bone marrow transplantation, skin allograft transplantation, and heterotopic heart transplantation12,18,19. Since thalidomide’s complete mechanism of action is yet unclear, we have investigated the pathophysiological effects of thalidomide in a rat aortic model of CAV. In this model, analysis of histopathological specimens is drastically facilitated since transplant vasculopathy may be examined in one single vessel of a sole defined diameter instead of the exploration of numerous small cardiac vessels showing a vast variety in size in the heterotopic heart transplant model. Indeed, the immune response generated by an aortic allograft is sufficient to trigger chronic alterations in the transplant20.

Our data suggest that thalidomide dramatically reduces luminal narrowing in an established rat model of CAV. Immunohistochemical analysis showed that loss of medial differentiated SMCs – a hallmark of CAV – was prevented by thalidomide treatment. Furthermore, thalidomide prevented dedifferentiation and intimal proliferation of SMCs. Our results are supported by recent literature showing beneficial effects of thalidomide in rat models of transplant arteriosclerosis and hereditary hemorrhagic telangiectasia13,21.

Because the molecular mechanism of thalidomide is not yet clear, we generated an intragraft cytokine profile, showing that thalidomide significantly reduced expression of MMP-8 and MCP-1. Zhang et al. used immunohistochemistry and western blot to suggest that thalidomide alters VEGF, PDGF, and ICAM-1 levels, whereas we found no significant alteration in these molecules using the more accurate ELISpot and MLR assays13. This difference in observation is likely due to the fact that Zhang et al. used a high responder transplantation model (BN:Lew) in their assays, which does not accurately represent CA, while we used a more appropriate low responder model (F344:Lew) to simulate chronic rejection.

Matrix metalloproteases (MMPs) are zinc enzymes involved in extracellular matrix turnover. Interestingly, increased MMP expression levels have been connected with intimal thickening22 and increased levels of MMP-2 and MMP-9 have been associated with chronic graft rejection22. MMP-8 itself appears to largely be produced by neutrophils, and is upregulated within the first few weeks post-transplantation23. Furthermore, general inhibition of MMPs prevents migration and proliferation of SMCs in CAV24. An MMP inhibitor that has been shown to reduce levels of MMP-8, was also linked to attenuation of CAV24. This is consistent with our findings, which show downregulation of MMP-8 associated with thalidomide treatment and reduced SMC proliferation and dedifferentiation.

Intragraft cytokine MCP-1 levels were also significantly reduced by thalidomide treatment. The literature surrounding MCP-1’s function supports this data. Monocyte chemotactic protein-1 (MCP-1) is associated with inflammatory responses, namely recruiting monocytes, memory T cells, and dendritic cells to places of inflammation. Reduced levels of MCP-1 have been correlated with reduction of neointimal hyperplasia25,26 and decreased chronic cardiac rejection27.

Because of thalidomide’s previously identified teratogenic effects7, it was vital that we examine the potential side effects of thalidomide treatment. As seen with previous approval of thalidomide for treatment of erythema nodosum leprosum, we found no significant side effects to our thalidomide treatment in rats. However, the study period was only 28 days, thus lacking long-term data for safety and toxicity of thalidomide.

Finally, to further elucidate thalidomide’s function, we wanted to clarify whether the effect we saw was specific to CAV or due to general immunogenic suppression. We examined whether thalidomide affected the pathogenic pathways associated with acute transplant rejection, namely T lymphocyte activation and proliferation. In contrast to the dramatic effects seen on CAV pathophysiology, we did not see significant inhibition of lymphocyte activation or proliferation. This leads us to conclude that thalidomide likely does not reduce CAV via prevention of generalized immune activation, but rather through a different, more specific mechanism of action on the vascular wall.

In conclusion, we demonstrate that thalidomide treatment dramatically reduces CAV in a well-established rat model of chronic rejection, preventing the intimal proliferation and the loss of medial differentiated SMCs that are hallmarks of CAV. Thalidomide is therefore potentially useful in clinical applications to prevent CAV after human heart transplantation.

Supplementary Material

Supp FigS1. Supplementary Figure 1: Thalidomide treatment did not cause significant side effects.

No significant side effects were identified through thorough biomarker screening after thalidomide treatment in rats. These screens included monitoring kidney function, serum cholesterol, liver function, and whole blood count. Error bars indicate mean ± SD.

Supp FigS2. Supplementary Figure 2. Thalidomide treatment does not mitigate lymphocyte activity in acute rejection model.

(A) Acute rejection was monitored in syngeneic (blue) and allogeneic (green and red) heterotopic transplantation models. Allogeneic transplant recipients received either no medication (vehicle control, green) or thalidomide (red) for 6 days post operation (POD6), when splenocytes were collected for analysis. No reduction in lymphocyte activation (IFNγ expression) was found in allogeneic grafts after thalidomide treatment as monitored by ELISpot (B). Similarly, no altered lymphocyte proliferation was measured through mixed lymphocyte reaction (MLR) (C). Error bars indicate mean ± SD. *P<0.05.

Acknowledgments

Funding sources:

This study was funded by the German Research Foundation (Deutsche Forschungsgemeinschaft; DFG: SCHR992/3-1 and SCHR992/4-1; S.S. and DE2133/2-1; T.D.), the International Society for Heart and Lung Transplantation (ISHLT; K.K.M., D.W. and S.S.), the Else Kröner Excellence Stipend from the Else-Kröner-Fresenius-Stiftung (2012_EKES.04), and the National Institutes of Health (NIH) grant R21 HL123655 (D.B.).

We thank Christiane Pahrmann for her technical assistance.

Footnotes

Authorship:

The concept was incepted by D.B. and S.S., project development and experiments were overseen by S.S., the manuscript was prepared by K.K.M. and E.N., additional scientific input and analysis was given by K.K.M., experiments were performed by D.W., X.H., X.H., T.D., T.R., and J.V., further scientific guidance was given by H.R., and final manuscript review was performed by D.B. and S.S.

Conflict of interest statement:

None of the authors has a conflict of interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp FigS1. Supplementary Figure 1: Thalidomide treatment did not cause significant side effects.

No significant side effects were identified through thorough biomarker screening after thalidomide treatment in rats. These screens included monitoring kidney function, serum cholesterol, liver function, and whole blood count. Error bars indicate mean ± SD.

NIHMS890793-supplement-Supp_FigS1.TIF (4.4MB, TIF)
Supp FigS2. Supplementary Figure 2. Thalidomide treatment does not mitigate lymphocyte activity in acute rejection model.

(A) Acute rejection was monitored in syngeneic (blue) and allogeneic (green and red) heterotopic transplantation models. Allogeneic transplant recipients received either no medication (vehicle control, green) or thalidomide (red) for 6 days post operation (POD6), when splenocytes were collected for analysis. No reduction in lymphocyte activation (IFNγ expression) was found in allogeneic grafts after thalidomide treatment as monitored by ELISpot (B). Similarly, no altered lymphocyte proliferation was measured through mixed lymphocyte reaction (MLR) (C). Error bars indicate mean ± SD. *P<0.05.

NIHMS890793-supplement-Supp_FigS2.png (84.4KB, png)

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