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Cardiovascular Research 2001 51(4):749-761; doi:10.1016/S0008-6363(01)00329-7
© 2001 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

Fas ligand/Fas-mediated apoptosis in human coronary artery smooth muscle cells: therapeutic implications of fratricidal mode of action

Adam J. Belanger, Abraham Scaria, Hsienwie Lu, Jennifer A. Sullivan, Seng H. Cheng, Richard J. Gregory and Canwen Jiang*

Genzyme Corporation, 31 New York Avenue, Framingham, MA 01701, USA

* Corresponding author. Tel.: +1-508-270-2418; fax: +1-508-872-4091 canwen.jiang{at}genzyme.com

Received 28 December 2000; accepted 19 April 2001


   Abstract
Top
 Abstract
1 Introduction
 2 Methods and materials
 3 Results
 4 Discussion
 References
 
Objective: This study aimed to determine the mode of action of Fas ligand (FasL)/Fas at mediating apoptosis so as to evaluate the potential of FasL in gene therapy for restenosis. Methods: Passaged human coronary artery smooth muscle (HCASM) cells were infected with recombinant adenoviral vectors expressing murine FasL. Various parameters of FasL expression and apoptosis were measured using FACS, immunofluorescence, calorimetric, and cytotoxicity assays. Results: Most HCASM cells under normal growth conditions expressed Fas and were shown to be susceptible to membrane bound but not soluble FasL. However, some FasL expressing cells survived for up to 7 days. These surviving cells were observed to be spatially distributed and were not in direct physical contact with each other. Upon examination, it was determined that although the majority of the surviving cells expressed FasL, only 30% expressed both Fas and FasL. These cells were capable of inducing apoptosis of target cells and some were also susceptible to FasL expressing cells, provided that the effector and target cells were in close physical contact. FasL/Fas-mediated apoptosis was inhibited by p35, a baculovirus gene that inhibits caspases. Additionally, in contrast to HCASM cells, neither membrane-bound nor soluble FasL induced apoptosis in coronary artery endothelial cells. Conclusions: FasL expressing HCASM cells do not undergo FasL/Fas mediated "suicide" but kill neighboring cells bearing Fas in a "fratricidal" manner. A small population of HCASM cells expresses no surface Fas. These results imply that HCASM cells transduced in vivo with FasL may serve as "scavengers" and exert a bystander effect on surrounding cells that may be enhanced by co-expression of p35. As FasL-mediated apoptosis occurs in coronary arterial smooth muscle but not endothelial cells, FasL may also offer an advantage over other genes for use in restenosis since the latter may indiscriminately delay re-endothelialization at the sites of gene.

KEYWORDS Apoptosis; Gene therapy; Restenosis; Signal transduction; Smooth muscle


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods and materials
 3 Results
 4 Discussion
 References
 
Fas is a member of the tumor necrosis factor receptor superfamily and mediates the initiation of apoptotic signaling [1]. While Fas is expressed in all cell types examined, physiologic expression of Fas ligand (FasL) is found predominantly in activated T-lymphocytes and natural killer cells, as well as vascular endothelial cells and "immune-privileged" tissues such as eye and testis [2,3]. Cells of the "immune privileged" organs immediately kill activated inflammatory cells that enter these organs by FasL/Fas-mediated apoptosis and therefore avoid inflammation. Cytotoxic T-lymphocytes and natural killer cells eliminate their target cells in part through FasL/Fas mediated apoptosis. In addition, antigen-activated apoptosis of T-cells is FasL-dependent, providing a mechanism for the maintenance of homeostasis of the immune system, for suppression of immune response and for peripheral tolerance [3].

In the vasculature, the FasL/Fas pathway has been implicated in atherogenesis [4–7], allograft arteriopathy [8], and the acute inflammatory response to cytokines [9]. Although endothelial cells express both Fas and FasL they are normally resistant to FasL/Fas-mediated apoptosis due to endogenous mechanisms that inhibit the downstream signaling pathways. It is speculated that FLIP, a FLICE inhibitory protein which is constitutively expressed in endothelial cells, is involved in the blockade of FasL/Fas signaling [10]. In contrast, vascular smooth muscle cells express only Fas and are susceptible to FasL/Fas mediated apoptosis [11]. To explore the therapeutic potential of FasL/Fas mediated apoptosis, FasL has been evaluated for the treatment of proliferative disorders of the vessel wall [11]. For example, in rat carotid arteries, local delivery of an adenoviral vector encoding the murine FasL significantly inhibits neointima formation following balloon catheter injury. In addition, transduction of vascular smooth muscle cells with FasL suppresses T-cell infiltration and possibly allows adenovirus-harboring cells to evade immune attack [11]. Furthermore, adenovirus-mediated gene transfer of FasL has been shown to reduce neointima formation in immunologically primed animals [12]. Together, these observations suggest that FasL gene therapy may represent an approach for the treatment of vascular smooth muscle proliferative disorders, such as restenosis after coronary angioplasty.

In this study we assessed several aspects of FasL/Fas-mediated apoptosis in human coronary artery smooth muscle (HCASM) cells. We established profiles of Fas expression in these cells prior to and after infection with adenoviral vectors. The fate of vascular smooth muscle cells expressing FasL was also examined. We then investigated whether FasL/Fas-mediated apoptosis in non-passaged HCASM cells was paracrine or "fratricidal" in nature, or if these cells committed "suicide" by autocrine signaling. We reasoned that if FasL/Fas-mediated apoptosis was fratricidal, some FasL transduced smooth muscle cells might be capable of evading the cytotoxic T-lymphocytes and thereby prolong the effect of FasL. However, this bystander effect might not extend beyond those cells that were in direct physical contact with these "scavenger" cells. On the other hand, if autocrine suicide could occur via soluble FasL shed from the FasL expressing cells, one might expect an extended bystander effect through diffusion of the ligand. If suicide occurred only via contact of the receptor and ligand during movements of the cell membrane, an extended bystander effect would be unlikely. Furthermore, we tested the feasibility of extending the presence of FasL expressing cells by co-expressing p35, a baculovirus gene that inhibits caspases [13,14]. Finally, we studied the cytotoxicity of soluble human FasL in several cell types, addressing concerns of potential systemic toxicity due to a soluble FasL cleaved from transduced cells.


   2 Methods and materials
Top
 Abstract
 1 Introduction
 2 Methods and materials
3 Results
 4 Discussion
 References
 
2.1 Cell culture
The investigation conforms with principles outlined in the Declaration of Helsinki (Cardiovascular Research 1997;35:2–3). HCASM cells, human coronary (HCAEC) and umbilical vein (HUVEC) endothelial cells, bronchial epithelial cells (NHBE), hepatocytes (NHeps), and skeletal muscle cells (SkMC) were obtained from Clonetics Corporation (San Diego, CA) and grown in the medium provided by the company. A20 and WR19L cells (mouse B-lymphoma cell lines) were obtained from American Type Culture Collection (Rockville, MD) and were grown in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum and Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, respectively.

2.2 Construction of recombinant adenoviral vectors
Ad2/CMVβgal-FasL is an Ad2 based vector in which the E1 region was replaced with a β-galactosidase expression cassette driven by the human CMV enhancer/promoter. The murine FasL cDNA was cloned by PCR from mouse testis cDNA (Clontech Laboratories, Palo Alto, CA). The E3 region was replaced with the murine FasL expression cassette driven by the human CMV enhancer/promoter. The FasL expression cassette was inserted in the opposite orientation to the E3 region. The E4 region except for ORF6 was deleted. Ad2/CMVEV was constructed in a similar fashion as Ad2/CMVβgal-FasL except that there was no transgene. Ad2/CMVβgal and Ad2/EGFP encoding β-galactosidase and green fluorescent protein (GFP) respectively, were also constructed in a similar fashion as Ad2/CMVβgal-FasL except that the adenovirus E3 region was unmodified. Ad2/CMVp35 contains the same E1 and E3 deletions as Ad2/CMVβgal-FasL. The p35 gene was cloned by PCR from baculovirus genomic DNA. The E1 region codes no transgene while the p35 expression cassette was inserted into the E3 region.

2.3 Measurement of β-galactosidase expression
HCASM cells were grown to >95% confluence and infected at a multiplicity of infection (MOI) of approximately 100 with either Ad2/CMVβgal or Ad2/CMVβgal-FasL. Levels of β-galactosidase were measured using the Galacto-Light Plus chemiluminescent reporter assay (Tropix, Bedford, MA). In some experiments, β-galactosidase expression was detected by light microscopy as nuclear-localized blue staining using the β-galactosidase substrate, X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) as described previously [15].

2.4 Detection of apoptosis
HCASM cells were grown to >95% confluence on chamber slides and infected with Ad2/CMVβgal or Ad2/CMVβgal-FasL (100 MOI). Cells were fixed in 4% formaldehyde 18 h after infection. Terminal deoxynucletidyl transferase nick end labeling (TUNEL) was performed using the ApoTACS in situ–TACS Blue TUNEL assay kit (R&D Systems, Minneapolis, MN). Apoptotic nuclei were visualized and photographed at 100x magnification on an Olympus AX70 inverted microscope with video camera.

2.5 Measurement of cell viability
HCASM cells were grown to >95% confluence in 96-well plates. Forty-eight h following infection with either Ad2/CMVβgal or Ad2/CMVβgal-FasL, cell viability was measured using the CellTiter96 Aqueous One kit (Promega, Madison, WI).

2.6 51Cr release assay
Twenty-four h after infection with Ad2/CMVβgal-FasL (approximately 400 MOI), some HCASM cells were labeled with 51Cr (40 µCi/cm) for an additional 14 h and then trypsinized and resuspended at a concentration of 105 cells/ml. Equal volume (100 µl) of cell suspensions in various combinations were added together sequentially to a 96-well culture plate. 51Cr-labeled non-infected cells were plated in either regular culture medium or 1% Triton X-100 in water (200 µl) to estimate spontaneous release and total cell lysis, respectively. The cells were immediately spun down and incubated for an additional 8 h at 37°C. Cell-free supernatant (25 µM/well) was transferred to a 96-well counter plate containing 125 µl scintillation fluid/well, allowed to diffuse in the fluid overnight, and then read in a micro-scintillation counter (Wallac, Gaithersburg, MD). Counts for average spontaneous release were subtracted from averages of total counts, which were then expressed as a percentage of average counts from total cell lysis.

2.7 Immunofluorescence staining
HCASM cells grown on glass slides were infected with Ad2/CMVβgal or Ad2/CMVβgal-FasL (approximately 100 MOI) and stained 1, 2, 4 and 8 days post-infection. The cells were fixed with cold methanol for 5 min, acetone for 30 s, and air-dried. They were washed with phosphate buffered saline (PBS), blocked with 3% horse serum for 1 h, and then labeled with the monoclonal anti-mouse/human FasL clone H11 (Alexis, San Diego, CA) or isotope control antibody (Chemicon, Temacula, CA) for 1 h. The cells were washed further with PBS and stained with PE-conjugated anti-rat/human Fas IgG (Pharmingen, San Diego, CA) for 1 h. Finally, the cells were mounted with Immumount (Shandon, Pittsburg, PA) and visualized by fluorescence microscopy.

2.8 Flow cytometry
Twenty-four hours post-infection with Ad2/CMVβgal or Ad2/CMVβgal-FasL, HCASM cells were harvested and labeled either with a PE-conjugated monoclonal antibody against human Fas, a FITC-conjugated monoclonal antibody against human Fas, a PE-conjugated monoclonal antibody against human FasL, or appropriate isotope controls (Pharmingen). Cells were fixed in 1.5% paraformaldehyde, counted within 24 h on a FACS Calibur flow cytometer, and analyzed using CellQuest software (Becton-Dickinson, San Diego, CA).

2.9 Co-culture studies
HCASM cells were grown to approximately 90% confluence and then infected with various vectors or their combinations. The cells were harvested 5 h post-infection. The appropriate groups of cells were mixed, seeded, and allowed to incubate for an additional 24 h. GFP expression was then assessed by both light microscopy and flow cytometry.

2.10 Soluble FasL mediated cytotoxicity assay
Various types of cells grown to confluence were treated overnight with increasing concentrations of soluble human FasL in the presence or absence of an enhancer (Alexis). Cell viability was then measured by using the MTS assay (Promega, Madison, WI).


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods and materials
 3 Results
4 Discussion
 References
 
3.1 Mode of FasL-induced cell death in HCASM cells
Expression of β-galactosidase in HCASM cells was assessed by X-Gal staining 24 h following infection with adenoviral vectors. Infection with Ad2/CMVβgal caused no cell death compared to time-matched non-infected cells while a dose-dependent increase in the number of positively stained cells was observed (Fig. 1). At a dose of 100 MOI, approximately half of the cells stained positively with X-Gal. When the dose was increased to 500 MOI, 90% of the cells were stained positively, indicating highly efficient transgene expression. By contrast, infection with Ad2/CMVβgal-FasL resulted in massive cell death. At a dose of 100 MOI, approximately 25% of the cells survived. These surviving cells were mainly distributed as individual cells having little or no physical contact with each other. Approximately 65% of the surviving cells stained positively with X-Gal, suggesting FasL co-expression. Infection of the cells at greater MOIs did not enhance the extent of cell death or generate a different pattern of distribution of the surviving cells. However, approximately 90% of the surviving cells stained positively with X-Gal at a dose of 500 MOI. No further increase in the number of positively stained cells was observed at a MOI of 1000, though staining intensity was somewhat higher (Fig. 1).


Figure 1
Figure 1
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  		Fig. 1 Pattern of surviving cells following infection with Ad2/CMVβgal orAd2/CMVβgal-FasL. (A) HCASM cells were stained with X-Gal 24 h after infection with Ad2/CMVβgal (solid columns) or Ad2/CMVβgal-FasL (open columns) at 10, 100, 500, or 1000 MOI. The percentage of the cells stained positively with X-Gal (B) and the percentage of surviving cells (C) are expressed as mean±S.E.M. (n=3).

 
To examine the nature of FasL/Fas-mediated cell death in HCASM cells, the TUNEL assay was performed to detect apoptotic nuclei following infection with Ad2/CMVβgal or Ad2/CMVβgal-FasL (100 MOI). In cells infected with Ad2/CMVβgal, neither cell death nor TUNEL positive nuclei was observed. In contrast, massive cell death and detachment were detected following infection with Ad2/CMVβgal-FasL. The nuclei of some remaining cells were TUNEL positive (Fig. 2) and showed a punctate pattern of DNA condensation and fragmentation characteristic of apoptosis, indicating FasL/Fas-mediated apoptosis in HCASM cells. However, it should be noted that positive TUNEL staining may occur in either necrotic cells containing fragmented DNA strands or non-apoptotic cells undergoing active RNA and DNA synthesis/repair. Thus, our results cannot be interpreted to exclude non-apoptotic cell death.


Figure 2
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  		Fig. 2 Apoptosis of HCASM cells following infection with either Ad2/CMVβgal or Ad2/CMVβgal-FasL (100 MOI). HCASM cells were stained using a TUNEL kit 18 h after adenoviral infection. Apoptotic nuclei (blue) were visualized and photographed at 100x magnification on an inverted microscope with video camera.

 
FasL can be cleaved by a metalloproteinase to yield a soluble form, which can mediate autocrine suicide and paracrine fratricide in activated T-lymphocytes [16]. However, supernatants from the HCASM cells infected with Ad2/CMVβgal-FasL failed to kill HCASM cells. Assuming the viability of the time-matched untreated cells was 100%, the viability of the cells 24 h following treatment with supernatants from cells infected with Ad2/CMVβgal and Ad2/CMVβgal-FasL (100 MOI) was 99±2% and 98±1% (mean±S.E.M., n=6), respectively. These results suggest that in HCASM cells, soluble mouse FasL shed from FasL expressing cells does not mediate apoptotic signaling, as reported previously in other cell types [17]. Furthermore, recombinant soluble human FasL did not cause death of HCASM cells even at extremely high concentrations (Fig. 8).


Figure 8
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  		Fig. 8 Differential cytotoxicity of soluble FasL on different types of human cell: HCASM, HCAEC, NHeps, NHBE, SkMC, A20 cells, and WR19L cells were treated with soluble human FasL in the presence of an enhancer protein (10 µg/ml) for 16 h. Cell viability was estimated by assuming the time-matched, untreated samples as 1. Data are expressed as mean±S.E.M. (n=3).

 
3.2 Fate of HCASM cells expressing murine FasL
We next examined the time course of β-galactosidase expression in cells infected with either Ad2/CMVβgal or Ad2/CMVβgal-FasL. The levels of β-galactosidase expression in the cells infected with Ad2/CMVβgal persisted through day 4, which was consistent with our previous reports in other cell types in vitro [15]. A similar profile of persistence was also observed in the surviving cells following infection with Ad2/CMVβgal-FasL (Fig. 3). Because of detectable β-galactosidase expression at day 4, these surviving cells were speculated to express FasL.


Figure 3
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  		Fig. 3 Persistence of β-galactosidase expression. β-Galactosidase expression in HCASM cells was measured 1, 2, and 4 days after infection with Ad2/CMVβgal (A) or Ad2/CMVβgal-FasL (B) at 100 MOI. Data are expressed as mean±S.E.M. (n=3).

 
More direct evidence that the surviving HCASM cells expressed FasL was obtained from immunofluorescence staining experiments. Neither cell death nor cell stained positively for FasL was detected following infection with Ad2/CMVβgal (100 MOI). In contrast, 24 h following infection with Ad2/CMVβgal-FasL (100 MOI), the majority (approximately 70%) of the surviving cells stained positively with the PE conjugated FasL antibody. Although the total number of surviving cells at day 2 was reduced, the percentage of FasL expressing cells was similar to that observed at 24 h. No further decrease in either the total number of surviving cells or the percentage of FasL expressing cells was detected at day 4 and 8 following infection (Fig. 4). These results suggest that HCASM cells expressing FasL can survive for up to 8 days, the longest time point examined.


Figure 4
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  		Fig. 4 Persistence of FasL expression in HCASM cells infected with Ad2/CMVβgal-FasL. HCASM cells were stained with an FITC conjugated FasL antibody 1, 2, 4, and 8 days after infection with Ad2/CMVβgal or Ad2/CMVβgal-FasL (100 MOI).

 
We next utilized a modified cytotoxicity assay to further assess FasL/Fas-mediated cell death. Cells infected with Ad2/CMVβgal-FasL (100 MOI) but unlabeled with 51Cr (effectors) were harvested and then mixed with cells that were labeled with 51Cr but not infected with viral vectors (targets). Lysis of the target cells was estimated at approximately 50% at a 1:1 ratio of effector to target cells, and decreased to a baseline level (zero) at a ratio of 1:64 (Fig. 5). These results suggest that the surviving cells were still capable of inducing death of target cells. When the effector cells alone, instead of a mixture of effector and target cells, were re-suspended and incubated together in a similar fashion, approximately 30% of the cells were lysed. These results suggest that some surviving cells expressing FasL were either less or not susceptible to paracrine signaling by membrane-bound FasL.


Figure 5
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  		Fig. 5 Cytotoxicity exerted by surviving cells following infection with Ad2/CMVβgal-FasL. HCASM cells that were infected with Ad2/CMVβgal-FasL (approximately 100 MOI) 24 h earlier as well as uninfected cells were labeled with 51Cr (40 µCi/ml) for 14 h before they were harvested. Equal volume (100 µl) of cell suspensions at different ratios of infected but unlabeled cells (effector) and their labeled but uninfected counterparts (target) were mixed and incubated to estimate the percentage of total cell lysis (panel A). Labeled uninfected cells were plated in either regular culture medium or 1% Triton X100 (200 µl) to estimate spontaneous release and total cell lysis, respectively. Suspensions of infected and labeled cells were also cultured in a similar fashion (panel B). Data are expressed as mean±S.E.M. (n=3).

 
3.3 Expression of Fas and FasL in surviving cells
We assessed Fas expression in HCASM cells 24 h following infection with Ad2/CMVβgal or Ad2/CMVβgal-FasL. Assessed by flow cytometry, 94±3%, 93±2%, and 24±4% (mean±S.E.M., n=3) in uninfected cells, cells infected with Ad2/CMVβgal, and cells infected with Ad2/CMVβgal-FasL were labeled positive for Fas, respectively. However, the vast majority (<95%) of the surviving cells following infection with Ad2/CMVβgal-FasL were stained positively for FasL. One representative experiment (Fig. 6) shows that approximately 30 and 95% of the surviving cells were stained positively for Fas and FasL, respectively. These results suggest that the majority of the cells expressing Fas also co-expressed FasL. Importantly, the cells that expressed both FasL and Fas could survive for at least 24 h.


Figure 6
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  		Fig. 6 FACS analysis of Fas and FasL expression. HCASM cells were infected with Ad2/CMVβgal or Ad2/CMVβgal-FasL (approximately 100 MOI). Twenty-four h post-infection, the surviving cells were labeled with both FITC-conjugated monoclonal anti-human Fas and PE-conjugated monoclonal anti-FasL or appropriate isotope controls. Cells in the lower left quadruple (LL) expressed neither Fas nor FasL. The cells in the upper left (UL), upper right (UR), or lower right (LR) quadruples expressed FasL only, both Fas and FasL, or Fas only, respectively. Please note in this particular sample that 88% of the cells infected with Ad2/CMVβgal expressed Fas (LR, % gated). By contrast, 2 and 30% of the cells infected with Ad2/CMVβgal-FasL expressed Fas (LR, % gated) and both Fas and FasL (UR, % gated), respectively, giving a total of 32% of the cells expressing Fas. Since an additional 65% of the cells expressed FasL only (UL), a total of 95% of these cells expressed FasL.

 
3.4 Protection of FasL expressing cells by p35
To examine the effect of p35 on FasL/Fas signaling, we performed co-culture studies. The cells infected with various vectors or their combinations were harvested 5 h post-infection and utilized as either target or effector cells. Target and effector cells in various pairs were mixed and then co-cultured for an additional 24 h. The first co-culture consisted of two groups of cells. Both groups were infected with Ad2/CMVβgal-FasL while only one group was also infected with Ad2/EGFP. The surviving cells were evenly distributed with the majority being not in direct physical contact with other cells. Approximately 50% of the surviving cells expressed GFP (Fig. 7A). In the second co-culture, one group of cells was infected with Ad2/CMVβgal-FasL while the other was infected with Ad2/EGFP. More cells survived and a higher percentage of the surviving cells expressed GFP in the second than in the first co-culture. We also observed surviving cells that were distributed as "patches", possibly due to the heterogeneity in the distribution of FasL expressing cells (Fig. 7B). In the third co-culture, cells co-expressing FasL, p35 and GFP were mixed with their counterparts expressing FasL only. There were far more surviving cells here than observed in the second co-culture. The majority of the surviving cells expressed GFP. These observations indicate that p35 protected the cells from FasL/Fas mediated apoptosis while FasL was co-expressed in the same cells or was presented as membrane-bound FasL by the adjacent cells (Fig. 7C). The fourth co-culture consisted of one group of cells infected with Ad2/EGFP alone and another group of cells co-expressing FasL and p35. The total number of surviving cells was similar to that observed in the third co-culture but the percentage of GFP positive cells was much lower than that observed in the second and third co-culture (Fig. 7D). These observations suggest that the FasL expressing cells exerted the same effect on the GFP positive cells as in the second group, but were protected from FasL/Fas-mediated apoptosis by p35 in the fourth group. The surviving cells of different co-cultures were collected for FACS analysis (Fig. 7E). The quantitative analysis was consistent with the microscopic observations.


Figure 7
Figure 7
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  		Fig. 7 Protection of FasL expressing cells by p35. HCASM cells at ≤90% confluence were initially infected with Ad2/CMVEV, Ad2/EGFP, or Ad2/EVp35 (approximately 100 MOI and 24 h later were then infected with Ad2/CMVβgal-FasL (approximately 100 MOI). After an additional 5 h, the cells were harvested, re-suspended in different combinations, and co-cultured overnight. (A) Co-culture of cells infected with Ad2/CMVβgal-FasL and Ad2/CMVEV, and cells infected with Ad2/CMVβgal-FasL and Ad2/EGFP; (B) Co-culture of cells infected with Ad2/CMVβgal-FasL and Ad2/CMVEV, and cells infected with Ad2/EGFP only; (C) Co-culture of cells infected with Ad2/CMVβgal-FasL and Ad2/CMVEV, and cells infected with Ad2/CMVβgal-FasL, Ad2/EGFP, and Ad2/EV/P35; (D) Co-culture of cells infected with Ad2/EGFP, and cells infected with Ad2/CMVβgal-FasL and Ad2/EV/P35; (E) Quantitative measurements of GFP positive cells using FACS analysis in co-cultures shown in panels A, B, C, and D. These results were reproduced in two independent experiments.

 
3.5 Differential cytotoxicity of FasL on various cell types
We examined the effects of FasL expression on HCAEC cells. No cell death was observed 2 and 5 days following infection with Ad2/CMVβgal-FasL (100, 500 MOI). Assuming the viability of the time-matched uninfected cells was 100%, the viability of the cells 5 days following infection with Ad2/CMVβgal or Ad2/CMVβgal-FasL (500 MOI) was 97±1 and 99±1% (n=6), respectively. These results indicate that coronary artery endothelial cells are resistant to FasL/Fas-mediated apoptosis, similar to endothelial cells from other vasculatures [10,11].

Several types of human cells as well as A20 and WR19L cells were treated for 16 h with soluble human FasL in the presence of an enhancer protein. As expected [17], dose-dependent cell death was detected in A20 but not WR19L cells. The viability of the human cells was not affected by the treatment, except NHeps, which showed some toxicity at high doses (Fig. 8).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods and materials
 3 Results
 4 Discussion
References
 
4.1 Fas expression in HCASM cells
Fas expression in vascular smooth muscle cells may be regulated by cytokines. For example, Fas expression is more abundant in atherosclerotic plaques than in medial smooth muscle cells [6]. The majority of the HCASM cells expressed Fas at levels that were detectable by FACS analysis although there was potentially a small Fas negative population in culture. By contrast, the majority of the surviving cells following infection with Ad2/CMVβgal-FasL did not express Fas. The adenoviral RID (receptor internalization and degradation) protein complex has been proposed to induce forced internalization and degradation of Fas following adenoviral infection [18]. However, the fact that infection with Ad2/CMVβgal did not appreciably alter the profile of Fas expression contradicts this notion. Alternatively, over-expression of FasL might have down-regulated or masked Fas expression. The precise mechanism underlying this observation remains unclear. It was previously reported that infection with Ad5/FasL did not change Fas expression while vascular smooth muscle cells at 50% confluence were infected [19]. In our experiments, cells were >90% confluent at the time of infection. We performed independent experiments to examine the effects of cell density on cell survival following infection with Ad2/CMVβgal-FasL. Approximately the same number of cells survived when the cells were infected at either 50 or 100% confluence (data not shown), which suggests the importance of cell–cell contact in FasL/Fas signaling. Thus, differences in experimental conditions might explain this discrepancy.

4.2 Fate of smooth muscle cells expressing FasL
Autocrine FasL/Fas signaling can initiate apoptosis in activated T-cells and may play an important regulatory role in immune responses [16,20,21]. T-cell suicide provides a mechanism for the maintenance of homeostasis of the immune system, for suppression of immune response and for peripheral tolerance. Activation induces expression of both Fas and FasL in T-cells, and in such co-expressing cells, apoptosis soon follows, even in single-cell cultures [16,20,21]. It has been suggested that soluble FasL may be the active agent in Fas-dependant autocrine apoptosis. However, supernatants from activated T-cells, which presumably would contain this soluble FasL, did not induce apoptosis in other activated T-cells, though issues of soluble FasL stability were not directly addressed [16]. In this study, supernatants from FasL expressing HCASM cells also did not induce apoptosis. Neither did added recombinant soluble human FasL, even at very high concentrations (µg/ml) and in the presence of an "enhancer" protein [22]. These results suggest that it is unlikely that soluble FasL contributed to the overall effects of infection with Ad2/CMVβgal-FasL in HCASM cells. Furthermore, co-expression of Fas and FasL in surviving cells was detected by FACS analysis, while some surviving cells were stained positively for FasL up to 8 days post-infection. Finally, at 24 h post-infection with Ad2/CMVβgal-FasL HCASM cells were capable of inducing apoptosis of target cells, provided that the effector and target cells were in physical contact. Taken together, these results suggest that in HCASM cells the mode of FasL/Fas-mediated apoptosis is not "suicidal", in which a FasL expressing cell kills itself. Instead, it is likely to be "fratricidal", in which a FasL expressing cell kills neighboring cells bearing Fas. However, we observed that while nearly all cells expressed FasL at 24 h following infection with Ad2/CMVβgal-FasL, less than 30% expressed Fas. In addition, in a pellet of HCASM cells infected with Ad2/CMVβgal-FasL, FasL signaling could lyse only 30% of these cells, as indicated by 51Cr-release. These surviving cells were resistant to the membrane-bound FasL which was presented by the cells co-expressing FasL and p35. These results suggest that there is potentially a FasL-resistant population of HCASM cells in culture, possibly due to lack of Fas expression. The vessels in vivo may have a high proportion of Fas negative cells. It should also be noted that in vascular smooth muscle cells the sensitivity to FasL/Fas-mediated apoptosis was determined not only by expression of surface Fas but also by differential expression of specific death-signaling proteins such as caspase 3 [23].

4.3 Therapeutic potential of FasL for the treatment of vascular proliferative disease
There may be a delicate balance between cell proliferation and apoptosis in vascular smooth muscle cells. Shifts in this balance could account for the accumulation of vascular smooth muscle cells in response to arterial injury, a major feature of vascular proliferative disorders such as restenosis following angioplasty with or without stents [24,25]. In animal models, gene therapy strategies targeted at the smooth muscle cells have shown therapeutic potential. However, a fundamental challenge facing the current gene therapy approaches is inefficient gene transfer to the vessel wall, particularly in advanced human coronary lesions [26]. High doses of adenovirus vectors required for sufficient gene expression may induce inflammation and worsen neointima formation [26,27]. If some FasL expressing cells are capable of serving as "scavenger" cells in vivo a by-stander effect beyond the transduced cells may be achieved. In addition, if prolonged transgene expression is desired for therapeutic benefits, immune responses may present yet another barrier [26,27]. Although expression of FasL alone has been shown to reduce T-cell infiltration [11], co-expression of p35 and FasL may effectively prolong the presence of FasL expressing cells and enhance the effects of FasL on neointima formation. Another fundamental concern is systemic cytotoxicity. The fact that various types of human cells were not susceptible to extremely high concentrations of soluble human FasL suggests a tolerance to the potential leakage of soluble FasL from the sites of FasL gene transfer. The cytotoxicity of soluble FasL on hepatocytes has to be further investigated although such high concentrations are unlikely to result from the potential leakage. However, leakage of adenoviral vectors from gene transfer sites to the liver cannot be controlled until highly localized delivery systems or tissue-specific transgene expression vectors are developed. Finally, re-endothelialization is a hallmark of recovery of injured vessels. However, cytotoxic or cytostatic genes may indiscriminately inhibit endothelial cell growth. The differential sensitivity of vascular endothelial and smooth muscle cells to FasL/Fas signaling may provide a unique advantage over other genes since FasL/Fas-mediated apoptosis may affect smooth muscle but not endothelial cells [11,12].

Time for primary review 28 days.


   References
Top
 Abstract
 1 Introduction
 2 Methods and materials
 3 Results
 4 Discussion
 References
 

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