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Fas Ligand Overexpression on Allograft Endothelium Inhibits Inflammatory Cell Infiltration and Transplant-Associated Intimal Hyperplasia¹

Masataka Sata^2,*,, Zhengyu Luo^2,* and Kenneth Walsh^3,*,,

^* Division of Cardiovascular Research, St. Elizabeth’s Medical Center, Tufts University School of Medicine, Boston, MA 02135; Department of Cardiovascular Medicine, Graduate School of Medicine, University of Tokyo, Tokyo, Japan; and Program in Cell, Molecular, and Developmental Biology, Sackler School of Biomedical Sciences, Tufts University, Boston, MA 02111

	Abstract

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Despite recent advances in immunosuppressive therapy, accelerated coronary atherosclerosis remains a major problem in the long-term survival of transplant recipients. Chronic graft vasculopathy is believed to result from recipient inflammatory responses, and it is characterized by early mononuclear cell infiltration of the transplanted vessel. Here we show that endothelial cells can be genetically modified to overexpress functional, cell-surface Fas ligand (FasL) by adenovirus-mediated gene transfer without undergoing self-destruction. In a rodent model of transplant graft vasculopathy, endothelial overexpression of FasL attenuated T cell and macrophage infiltration at 1 wk posttransplantation. These vessels also displayed reduced neointima formation at one and 2 mo posttransplantation. These results indicate that inhibition of the early inflammatory response to allografted vessels by endothelial cell-specific overexpression of FasL may have utility in the treatment of transplant arteriosclerosis.

	Introduction

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Improvements in immunosuppression and in the management of acute rejection have dramatically enhanced the early survival of cardiac transplant recipients. However, transplant coronary artery disease, the major manifestation of chronic rejection, has emerged as a leading cause of graft failure and retransplantation after the first postoperative year (1). Despite these circumstances, the pathogenesis of the transplant-associated atherosclerotic is poorly understood. Compared with common atherosclerosis, transplant-associated arteriosclerotic lesions tend to be diffuse and concentric with less lipid and more T cell accumulation (1, 2). In animal models of chronic graft vasculopathy, infiltrating T lymphocytes and macrophages concentrate early in the subendothelial space of grafted coronary arteries, and persistent cytokine expression is evident (3, 4). The lesion expands with infiltrates followed by a gradual increase in the presence of vascular smooth muscle cells (VSMCs)⁴ in the intima (5). This temporal sequence of events suggests that early infiltration of mononuclear cells is an initiating event in the VSMC-proliferative response, which leads to graft occlusion.

Fas ligand (FasL) is a type II membrane protein that induces apoptosis in cells bearing its receptor, Fas (6). Fas is expressed by many cell types including leukocytes, but FasL expression is much more restricted in its expression pattern. FasL is expressed on T cells, where it functions as a cytotoxic agent to eliminate unwanted cells from the body and control T cell number. Immune-privileged tissues and some tumors express FasL, where it is thought to inhibit the immune response by inducing apoptosis in infiltrating inflammatory cells (7, 8, 9, 10, 11, 12, 13). Based on these observations, FasL gene transfer has been tested for its ability to promote allograft survival in a number of models (14, 15, 16, 17, 18, 19, 20, 21). Some of these studies reported positive results, but most found that ectopic FasL expression did not promote allograft survival. In many instances, direct FasL expression was found to be toxic, leading to the destruction of the transplanted tissue, cytokine release, and infiltration of granulocytic cells (22).

In contrast to many cell types, vascular endothelial cells are normally resistant to Fas-mediated apoptosis (23, 24, 25, 26). Although endothelial cells express an intact Fas pathway, death signals appear to be blocked by the coexpression of inhibitory molecules (27, 28). These observations suggest that it might be possible to genetically engineer endothelial cells on grafted vessels to constitutively overexpress FasL such that host immune response would be minimized while avoiding a cytotoxic response in the endothelium. Here, it is shown that FasL gene transfer to the endothelium inhibits early mononuclear cell infiltration of the medium and attenuates intimal lesion formation in a rodent transplant model. These data support the hypothesis that early leukocyte extravasation provokes the occlusive hyperplastic response of VSMCs, and suggest that the constitutive overexpression of FasL by the endothelium can preserve graft patency.

	Materials and Methods

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Cells and reagents

Human aortic endothelial cells (HAECs) were obtained from Clonetics (San Diego, CA) and cultured in EGM medium (Clonetics). Mastocytoma cell line P815 stably transfected with human Fas (P815-huFas) was provided by Douglas Green. The human T cell leukemia line, Jurkat clone E6-1, was obtained from American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 with 10% FBS. Replication-defective adenovirus vectors encoding murine FasL (Adeno-FasL) or -galactosidase (Adeno-gal) were described previously (16, 29). Viral titer was measured by standard plaque assay using 293 cells.

Flow cytometric detection of apoptosis

HAECs and P815huFas cells were treated with either soluble FasL (0.1 µg/ml; Alexis) for 24 h, an agonistic anti-Fas mAb (0.5 µg/ml, clone CH11; MBL, Nagoya, Japan) for 24 h, or Adeno-FasL at a multiplicity of infection of 300 for 48 h. Cells were harvested, fixed with 70% ethanol, and stained with propidium iodide as described (24). DNA content was analyzed by flow cytometry (BD Biosciences, Mountain View, CA).

Flow cytometric analysis of FasL

HAECs were infected with Adeno-FasL at a multiplicity of infection of 300 for 24 h. HAECs were detached from the culture plate with 0.5% EDTA and incubated with anti-FasL mAb (A11; Alexis) or with rat IgM. Cells were then washed and stained with FITC-conjugated anti-rat IgM Ab (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Immunofluorescence staining was analyzed by flow cytometry.

Cytotoxicity assay

The ability of FasL on endothelial cells to induce apoptosis in Fas-positive target cells was assessed by coincubating Jurkat cells with HAECs using a technique described previously (30). HAECs were plated in triplicate in 24-multiwell culture plates and allowed to reach 90% confluence as a monolayer. HAECs were infected with Adeno-FasL at a multiplicity of infection of 300 for 24 h, after which HAECs were washed twice with PBS. Jurkat cells were labeled with 10 µCi/ml [³H]thymidine (DuPont-NEN, Boston, MA) for 24 h, washed twice in PBS, and then incubated at 37°C for 8 h to minimize spontaneous release. Jurkat cells were applied to HAECs at a ratio of 1:4 (HAECs/Jurkat cells). The 24-multiwell culture plates were centrifuged at 200 x g for 2 min and incubated at 37°C for 18 h. Cells were harvested by trypsinization. The amounts of the fragmented and retained DNA were measured by liquid scintillation counting as described (31). The percentage of the specific DNA fragmentation was calculated as 100 x (spontaneous - experimental)/spontaneous. "Experimental" is cpm of the retained DNA in the presence of HAECs and "spontaneous" is cpm of the retained DNA in the absence of HAECs. All values are presented as mean ± SEM.

Transplantation

Male Wistar Furth (WF, RT1A^u) and ACI (RT1A^a) rats were purchased from Harlan Sprague Dawley (Indianapolis, IN). Rats weighing 300–350 g were used as donors and recipients. Rats were anesthetized with i.p. injection of pentobarbital. Common carotid arteries of ACI rats were temporarily isolated and incubated with either saline, Adeno-gal (1 x 10⁸ PFU), or Adeno-FasL (1 x 10⁸ PFU) without positive pressure via a 24-gauge i.v. catheter inserted just proximal to the carotid bifurcation. After 15 min, the viral solution was withdrawn and the cannula was removed. The common carotid arteries were excised from the donor rats and used as arterial grafts (1 cm in length). The native left common carotid arteries of recipient rats were excised and replaced with the arterial graft. All anastomoses were performed using continuous 8–0 nylon suture in an end-to-end fashion. Recipient rats were sacrificed at 3 days or 1, 4, or 8 wk after transplantation.

Additional arterial transplants were performed to test the effect of Adeno-FasL in immune-suppressed animals. DA (RT1A^a) rats (Harlan Sprague Dawley) were used as donors. Common carotid arteries of DA rats were incubated with either saline or Adeno-FasL (1 x 10⁸ PFU) and transplanted into WF rats as described above. Either saline or cyclosporin A (CyA; 5 mg/kg/day) were administered into recipient rats s.c. Recipient rats were sacrificed at 4 wk after transplantation.

The grafted artery was excised and snap-frozen (1 wk) or fixed in methanol (4 and 8 wk). The cross-sections (5 µm) from four separate segments of each arterial graft were stained with hematoxylin and eosin or with Richardson’s combination elastic tissue trichrome stain. The intimal, medial, and luminal areas were measured by quantitative morphometric analysis using a computerized sketching program and expressed as mean ± SEM. The experimental protocol was approved by the Institutional Animal Care and Use Committee and complied with the "Guide for the Care and Use of Laboratory Animals".

Histological examination

Cryosections (5 µm thick) were mounted on microscope slides and fixed in 4% paraformaldehyde for 10 min. Paraffin-embedded sections were deparaffinized. Sections were washed in PBS and blocked with 5% normal goat serum and 0.01% Triton X-100 in PBS for 1 h. To detect FasL expression, adjacent sections were incubated with three different anti-FasL Abs (clone MFL3, PharMingen, San Diego, CA; clone Kay10, PharMingen; N-20, Santa Cruz Biotechnology, Santa Cruz, CA), independently, because questions have been raised against specificity of some anti-FasL Abs (32). Sections were also stained with anti-CD3 polyclonal Ab (T cells, C-7930; Sigma, St. Louis, MO), anti-macrophage Ab (clone ED1; Serotec, Oxford, U.K.), or anti-TGF-1 Ab (sc-146; Santa Cruz Biotechnology) was added to the sections for 1 h at room temperature. Ab distribution was revealed using appropriate secondary Abs, biotin-streptavidin complex system, and Fast Red substrate (BioGenex Laboratories, San Ramon, CA). Sections were counterstained with hematoxylin. Neutrophils were identified by their appearance on the sections stained with hematoxylin and eosin. Distribution of neutrophils was also analyzed using anti-rat granulocytes mAb (clone MOM/3F12/F2; Serotec) on the frozen sections harvested 1 wk after transplantation. To detect -galactosidase expression, the arterial graft was harvested 3 days posttransplantation, fixed in 2% paraformaldehyde, and stained with 5-bromo-4-chloro-3-indolyl -D-galactopyranoside (X-gal; Sigma) for 16 h at room temperature followed by counterstaining with hematoxylin.

Statistics

All data were reported as mean ± SEM. The mean values were compared by one-way ANOVA as well as unpaired t test. A value of p < 0.05 was considered to be significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Overexpression of FasL on aortic endothelial cells does not lead to cytotoxicity

A previous study documented that HUVECs do not undergo apoptosis when exposed to an agonistic anti-Fas Ab (23). Thus we tested whether human endothelial cells could be genetically engineered to overexpress cell surface FasL by adenovirus-mediated gene transfer, yet remain impervious to Fas-mediated self-destruction. FACS analyses of hypodiploid DNA, a marker of apoptosis-induced chromatin fragmentation, revealed HAECs remained viable following exposure to soluble FasL, agonistic anti-Fas Ab, or a replication-defective adenoviral vector expressing murine FasL (Adeno-FasL) (Fig. 1A). In contrast, each of these Fas agonists induced apoptosis in the P815huFas cell line as well as other cell types (24, 29). Twenty-four hours after Adeno-FasL infection, augmentation of cell surface FasL expression on HUVECs (Fig. 1B) and HAECs (data not shown) was shown by FACS analysis. A coculture DNA fragmentation assay was used to assess the functional significance of exogenous FasL expression on Adeno-FasL-infected HAECs (Fig. 1C). Marked DNA fragmentation of Jurkat cell DNA (83.0 ± 3.7%) was detected after 18 h of coincubation with Adeno-FasL-infected HAECs, whereas only 5.8 ± 0.3 or 5.5 ± 1.0% DNA fragmentation was detected in target cells when incubated with either uninfected or Adeno-gal-infected HAECs, respectively. Collectively, these in vitro data suggest that aortic endothelial cells can be genetically modified by adenovirus-mediated gene transfer to overexpress functional FasL on their cell surface without undergoing self-destruction.

View larger version (37K): [in this window] [in a new window]   FIGURE 1. Endothelial cells can overexpress functional FasL but are resistant to Fas-mediated cell death. A, HAECs or P815huFas cells were treated with either soluble FasL (0.1 µg/ml; Alexis) for 24 h, an agonistic anti-Fas monoclonal Ab (0.5 µg/ml, clone CH11; MBL) for 24 h, or Adeno-FasL at a multiplicity of infection of 300 for 48 h. Cells were harvested, fixed with 70% ethanol, and stained with propidium iodide as described (29 ). DNA content was analyzed by flow cytometry. B, FasL overexpression on human endothelial cells. HUVECs were infected by Adeno-FasL at a multiplicity of infection of 300 for 24 h. Twenty-four hours after Adeno-FasL infection, cell surface FasL expression on endothelial cells was documented by flow cytometry as described in Materials and Methods. C, FasL-overexpressing endothelial cells can induce apoptosis in cocultured Jurkat cells. Jurkat cells labeled with [3H]thymidine were coincubated with Adeno-gal-infected, or mock-infected Adeno-FasL-infected HAECs for 18 h. The percentage of specific DNA fragmentation was calculated as described in Materials and Methods. Values are presented as mean ± SEM.

A rat model of transplant arteriosclerosis was developed where carotid arteries from ACI rats were transplanted to WF rats. Before excision, the donor artery from the ACI rat was temporarily isolated and incubated with saline, Adeno-gal, or Adeno-FasL (1 x 10⁸ PFU) for 15 min, after which the grafts were transplanted to recipient rats. Adeno-gal-infected vessels harvested at 3 days posttransplantation revealed that transgene expression was confined exclusively to luminal endothelial cells (Fig. 2A).

View larger version (67K): [in this window] [in a new window]   FIGURE 2. Adenovirus-mediated FasL overexpression on graft endothelium inhibits mononuclear cell infiltration at 1 wk. A, Adenoviral vectors efficiently and selectively transduce the endothelium of the transplanted vessel. A common carotid artery graft from an ACI rat was infected with Adeno-gal (1 x 108 PFU) and transplanted in a WF rat. The arterial graft was harvested at 3 days posttransplantation and stained with X-gal. B, Histological sections indicating reduced inflammatory cell infiltrates in Adeno-FasL-treated vessels. Common carotid arteries from ACI rats were transplanted into WF rats. Rats were sacrificed 7 days after the surgery. The grafted artery was snap-frozen in OCT compound. Cryosections were stained for FasL (clone MFL3; PharMingen), T cell (anti-CD3 polyclonal Ab; Sigma), or macrophage (clone ED1; Serotec). FasL expression was also confirmed by using the mAb clone Kay10 and polyclonal Ab N-20 (data not shown). C, Histograms summarizing the number of infiltrating T cells (left) and macrophages (right) per cross-section (n = 4 arteries for each group). M = macrophage. *, Significant difference (p < 0.05) from the saline- or Adeno-gal-treated grafts.

It has been reported that ectopic overexpression of FasL in allografts results in neutrophil accumulation (17, 18, 21, 22). In this study, we observed little or no granulocytic infiltration into the grafts the endothelia of which overexpressed ectopic FasL, based on morphologic examination of hematoxylin and eosin staining (data not shown). We also confirmed the absence of neutrophil infiltration by immunohistochemical study using an Ab specific for rat granulocytes (clone MOM/3F12/F2) on the cryosections of the grafts harvested 1 wk after the transplantation (Fig. 3A). Fewer neutrophils were seen in the FasL-treated allografts than in control allografts. Because TGF- has been shown to regulate proinflammatory effects of FasL (33), we analyzed the expression of TGF-1 in the vascular grafts. As reported by others (34), TGF-1 was abundantly expressed in the vascular grafts pretreated with saline or Adeno-FasL at 1 wk posttransplantation (Fig. 3B). TGF-1 expression in the arterial grafts could also be detected 2 mo after transplantation, but the immunostain signal was less intense.

View larger version (70K): [in this window] [in a new window]   FIGURE 3. Absence of neutrophil infiltration into the adeno-FasL-infected vessels and TGF- expression in the arterial grafts. A, Frozen sections of the vascular grafts harvested at 1 wk posttransplantation were stained for granulocytes. Arrows indicate positive cells. Bar, 25 µm. B, Untreated rat carotid artery and the vascular grafts were stained for TGF-1 as described in Materials and Methods. Arrowheads indicate the internal elastic lamina. Bar, 25 µm.

Allografted arteries developed concentric neointimal lesions by 4 wk (Fig. 4A). The neointima was mainly composed of -smooth muscle actin-positive cells (Fig. 4B), whereas the medial layer was primarily composed of macrophages as reported by others (Ref. 1, 5 ; and data not shown). The luminal surface was coated by endothelium as determined by CD31 staining, but the endothelium of the Ad-FasL-transduced vessels no longer expressed detectable FasL (data not shown). The intima-media ratio of Adeno-FasL-infected graft was significantly smaller than that of saline-treated or Adeno-gal-treated grafts (Fig. 4C). At 4 wk posttransplantation, the FasL-transduced vessels exhibited 63% less intimal hyperplasia than the -galactosidase-transduced vessels. At 8 wk posttransplantation, FasL-transduced vessels had 57% less intimal hyperplasia than the -galactosidase-transduced vessels (Fig. 4C). Of note, comparisons of the 4 and 8 wk time points did not reveal any statistically significant increases in lesion size within each of the three experimental groups (Fig. 4C), nor did it reveal detectable differences in the cellular composition of the graft (data not shown), suggesting that the lesions within the grafted vessels stabilize by 4 wk.

View larger version (97K): [in this window] [in a new window]   FIGURE 4. FasL overexpression on graft endothelium inhibits transplant-associated intimal hyperplasia. A, Representative cross-sections of transplanted arterial grafts at 4 wk. Paraffin-embedded sections (5 µm) were stained with Richardson’s combination elastic tissue trichrome stain (top). Arrows indicates internal elastic lamina. B, Sections of transplanted grafts at 4 wk were stained for -smooth muscle actin (clone 1A4) followed by counterstaining with hematoxylin. C, Histograms summarizing the intima-media ratio of the transplanted arterial grafts at 4 (left) and 8 wk (right) (n = 5 arteries for each group). *, Significant difference (p < 0.05) from the saline- or Adeno-gal-treated grafts.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

It is generally believed that graft vasculopathy is caused by cellular and humoral immune responses to the transplanted grafts (4, 40, 41). It has been demonstrated that ectopic expression of FasL on tumor cells inhibits the generation of allo-Abs in addition to the T cell-mediated immune response (42), suggesting that local expression of FasL functions to eliminate monocytic Ag-presenting cells. In this study we observed that endothelial FasL overexpression inhibits both T cell and monocyte/macrophage infiltration into the subendothelial space, and it is possible that FasL gene transfer to the endothelium inhibits both T cell-mediated cellular immunity and humoral responses to the graft. In our experiments FasL was transduced to the endothelium using a replication-defective adenoviral vector. Because adenovirus Ags can provoke immunoreaction against the transduced cells (43), the adenovirus vector could make the grafts more susceptible to acute and chronic rejections. However, the Adeno-gal-transduced vessels did not differ significantly from saline control in the number of infiltrating cells at 1 wk and in the intimal hyperplasia at 1 and 2 mo posttransplantation. The lack of a detectable vector-mediated immune response in this model may be due to the low dose of adenovirus used in these experiments (1 x 10⁸ PFU).

Collectively, these findings indicate that early control of inflammatory cell infiltration by FasL overexpression on the graft endothelium can have a sustained beneficial effect on the allografted vessel. This is consistent with the observations that a blockade of CD28 and CD40, or depletion of CD8⁺ T cells by systemic administration of neutralizing Abs, inhibit cardiac allograft vasculopathy (44, 45). In addition, attenuated intimal lesion formation is reported in recipient mice that are deficient in T lymphocytes or macrophages (46, 47). The goal of this study was to genetically alter the endothelium to achieve a reduction in inflammatory cell extravasation, and thereby inhibit VSMC proliferation. In contrast, other strategies have sought to directly inhibit VSMC proliferation, a late component of transplant arteriosclerosis (5, 48). It has been shown that receptor antagonists for VSMC mitogens (49, 50) and rapamycin (51, 52, 53), a potent VSMC growth inhibitor, are effective at inhibiting chronic graft vasculopathy in animal models. Molecular strategies that target VSMC growth have also been examined in models of chronic graft vasculopathy. In these studies, access to medial VSMCs was achieved either by the administration of phosphorothioate oligonucleotide to cdk2 packaged in HVJ-liposomes (54) or by lengthy incubation of an adenoviral vector expressing thymidine kinase within distended vessels (55).

Constitutive FasL expression by transplanted cells and tissues has been examined by several groups. In many instances, ectopic FasL expression can lead to inflammatory responses marked by graft destruction, IL-1 release, and neutrophil recruitment (18, 21, 22). In contrast to other cell types that have been used as a vehicle for expression of recombinant FasL, cultured vascular endothelial cells are highly resistant to Fas-mediated apoptosis (23, 24, 25) and no endothelial cell apoptosis was observed in vivo following administration of Adeno-FasL in these or previous experiments examining constitutive FasL expression in the rabbit ear central artery (24). In our experiments FasL-dependent neutrophil accumulation in grafted vessels was not observed under any treatment condition nor at any time point examined. Furthermore, neutrophil accumulation was not detected when FasL was transduced in vessel wall of normal rabbit ear central artery (24) or balloon-denuded rat carotid arteries (29, 56). Suppression of neutrophil accumulation in the vessel wall may be due to the production of TGF- (33, 57, 58). This pleiotropic peptide is detected in human vascular lesions and is markedly up-regulated in arteries following allogenic transplantation (34, 59) and balloon injury (60, 61, 62), perhaps creating a microenvironment that promotes FasL-mediated immunologic tolerance (33). In this study TGF- expression was detected in the allograft preinfected with Adeno-FasL 1 wk and 2 mo posttransplantation. Furthermore, it is now recognized that neutrophil infiltration is a consequence of Fas-mediated cellular destruction of the allograft, rather than a direct effect of FasL on neutrophils themselves (63). Thus, neutrophil recruitment would be minimized in this system because endothelial cells are resistant to Fas-mediated apoptosis (23, 24, 25, 26) Collectively, these characteristics suggest that endothelial cells can be uniquely suited to accommodate high levels of exogenous FasL expression, leading to conditions that suppress the immune response to allografts.

In conclusion, our finding supports the idea that cellular infiltrates into subendothelial space contribute to neointima development in allografted vessels. Our data also suggest that inhibition of this early inflammatory response at the endothelial surface is sufficient to inhibit intimal hyperplasia in transplanted vessels, and they suggest that FasL may have utility for the prevention of transplant arteriosclerosis.

	Footnotes

 
¹ This study was supported by National Institutes of Health Grants AG-15052, HL-50692, and AR-40197 (to K.W.).

² M.S. and Z.L. contributed equally to this study.

³ Address correspondence and reprint requests to Dr. Kenneth Walsh, Division of Cardiovascular Research, St. Elizabeth’s Medical Center, 736 Cambridge Street, Boston, MA 02135. E-mail address: kwalsh{at}opal.tufts.edu

⁴ Abbreviations used in this paper: VSMC, vascular smooth muscle cell; FasL, Fas ligand; HAEC, human aortic endothelial cell; WF, Wistar Furth; CyA, cyclosporin A.

Received for publication November 3, 2000. Accepted for publication March 26, 2001.

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