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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:309.)
© 2000 American Heart Association, Inc.

Vascular Biology

Vascular Endothelial Cells and Smooth Muscle Cells Differ in Expression of Fas and Fas Ligand and in Sensitivity to Fas Ligand–Induced Cell Death

Implications for Vascular Disease and Therapy

Masataka Sata; Toshimitsu Suhara; Kenneth Walsh

From the Division of Cardiovascular Research, St. Elizabeth’s Medical Center (M.S., T.S., K.W.), and the Program in Cell, Molecular, and Developmental Biology, Sackler School of Biomedical Sciences (K.W.), Tufts University, Boston, Mass; and the Department of Cardiovascular Medicine, Graduate School of Medicine (M.S.), University of Tokyo, Tokyo, Japan.

Correspondence to Dr Kenneth Walsh, Division of Cardiovascular Research, St. Elizabeth’s Medical Center, 736 Cambridge St, Boston, MA 02135. E-mail kwalsh{at}opal.tufts.edu

	Abstract

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Abstract—Fas ligand (FasL) is a death factor that induces apoptosis in cells bearing its receptor, Fas. Fas and FasL have been detected in the vessel wall, and it has been proposed that Fas-mediated apoptosis has a role in physiological and pathological cell turnover in the vasculature. Here, we evaluated the expression of Fas in the presence and absence of cytokines on both endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). We also examined the sensitivity of ECs and VSMCs to Fas-mediated apoptosis induced by exposure to multiple Fas agonists: soluble FasL, anti-Fas antibody, and membrane-bound FasL resulting from transduction with a replication-defective adenovirus expressing FasL (Adeno-FasL). Cell-surface FasL expression was detected on human ECs with the use of 4 anti-FasL antibodies, whereas cell-surface FasL expression was not detected on VSMCs. Unstimulated ECs expressed relatively low levels of Fas, but expression was upregulated after treatment with tumor necrosis factor- (TNF-) or interferon gamma (IFN-). In contrast, VSMCs expressed relatively high levels of Fas, and treatment with TNF- or IFN- induced little or no upregulation under the conditions of these assays. ECs were resistant to death after exposure to soluble FasL or agonist anti-Fas antibody and also after infection with Adeno-FasL in the presence or absence of cytokine treatment. In contrast, VSMCs remained viable in the presence of soluble FasL or agonist anti-Fas antibody, but they underwent apoptosis after infection with Adeno-FasL. IFN- enhanced Adeno-FasL-induced death of VSMCs, but TNF- did not. These findings provide insights about the potential role of Fas-mediated apoptosis in the vessel wall and suggest strategies to treat proliferative vascular diseases by exploiting the differential sensitivity of ECs and VSMCs to FasL-induced cell death.

Key Words: Fas • adenovirus • smooth muscle cells • endothelial cells • apoptosis

	Introduction

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Fas is a type I membrane protein belonging to the tumor necrosis factor (TNF) receptor family that induces a death signal when bound to its ligand.¹ Fas is ubiquitously expressed, whereas the expression of Fas ligand (FasL), a membrane-bound factor, is typically confined to inflammatory cells and tissues that routinely encounter inflammatory cells.^{2 3 4 5 6} Expression of both Fas and FasL has been detected in the normal and diseased vessel wall, and it has been proposed that Fas-mediated apoptosis is a feature of atherogenesis,^{7 8 9} atherosclerotic plaque instability,¹⁰ allograft arteriopathy,¹¹ and the acute inflammatory response to cytokines.¹²

Numerous recent studies have examined the role of Fas-mediated cell death in the vasculature.^{7 8 9 10 11 12 13 14 15 16 17 18 19} These studies have led to conflicting hypotheses regarding the susceptibility of vascular cells to Fas-mediated cell death and the expression of Fas-regulatory components by vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). For example, it has been shown that VSMCs undergo apoptosis both in vitro and in vivo after infection with a replication-defective adenovirus expressing FasL.^{13 18} However, another study found that VSMCs were resistant to agonist anti-Fas antibody and that Fas-mediated VSMC death occurred only after treatment with interferon gamma (IFN), interleukin-1ß, or TNF-.¹⁰ Many of these discrepancies may result from differences in experimental conditions, the use of Fas agonists that may differ in their ability to transmit death signals,^{20 21} or the use of commercial anti-FasL antibody reagents that lack specificity for FasL.^{22 23 24 25} Owing to the potential importance of Fas-mediated cell death in vascular disorders, we systematically examined Fas and FasL expression by ECs and VSMCs and characterized the sensitivity of these cells to Fas-mediated apoptosis by using multiple reagents. These results document that ECs and VSMCs differ markedly in their expression of Fas and FasL and in their sensitivity to FasL-induced cell death. It might be possible to exploit these differences to develop strategies to treat inflammatory-fibroproliferative disorders of the vessel wall.

	Methods

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Cells and Reagents
Human umbilical vein endothelial cells (HUVECs) were isolated as described²⁶ and grown in endothelial growth medium (EGM, Clonetics). The human T-cell leukemia cell line, Jurkat clone E6-1, was obtained from the American Type Culture Collection (Rockville, Md) and maintained in RPMI 1640 medium with 10% fetal bovine serum (FBS). Jurkat cells were activated by treatment with phorbol 12-myristate 13-acetate (PMA,10 ng/mL) and ionomycin (400 ng/mL) for 4 hours. Human VSMCs (hVSMCs) were isolated from an internal mammary artery obtained during coronary bypass surgery and cultured as described previously.²⁷ P815huFas cells, the mouse mastocytoma cell line (P815) stably transfected with human Fas, were kindly provided by Dr Douglas Green (La Jolla Institute of Allergy and Immunology, San Diego, CA). Soluble FasL (sFasL) was purchased from Alexis. Agonistic anti-Fas mouse monoclonal antibody (clone CH11) was purchased from MBL.²⁸ Human TNF- and human IFN- were purchased from R&D Systems and Gibco BRL, respectively.

Adenoviral Constructs
A replication-defective adenoviral vector expressing murine FasL from the cytomegalovirus promoter (Adeno-FasL) was constructed as described.²⁹ The Adeno-FasL lacks functional E1 and E3 regions. Viral titer was measured by standard plaque assay with 293 cells.

Flow Cytometry to Detect Fas or FasL Expression
To detect cell-surface expression of FasL, cells were detached from the culture plate with 0.5% EDTA in PBS and washed with 10% FBS in PBS. Cells were incubated with antibody (20 µg/mL) diluted with 1% BSA and 0.1% NaN₃ in PBS at 4°C for 60 minutes. C-20 (rabbit IgG, Santa Cruz), clone 4H9 (hamster IgG, MBL), clone A11 (rat IgM, Alexis), or clone Mike-1 (rat IgG, Alexis) was used as an anti-FasL antibody. Isotype-matched nonspecific immunoglobulins were used as negative controls. Cells were washed with 10 mmol/L EDTA and 0.1% NaN₃ in PBS and incubated with FITC-conjugated secondary antibodies appropriate to the first antibodies at 4°C for 60 minutes (1:100). Immunofluorescence staining was analyzed by a fluorescence-activated cell sorter (FACS, Becton Dickinson). Cells were washed and fixed in freshly prepared 1% paraformaldehyde. To analyze cell-surface expression of Fas, cells were detached from the plates with 0.5% EDTA and incubated with an FITC-conjugated anti-Fas mouse monoclonal antibody (1:10, UB2, MBL) or an FITC-conjugated mouse IgG (Pharmingen). Immunofluorescence staining was analyzed by flow cytometry.

Detection of Apoptosis by Flow Cytometry
Cells were treated with either sFasL (500 ng/mL) for 24 hours, an agonistic anti-Fas antibody (500 ng/mL, CH11, MBL) for 24 hours, or Adeno-FasL (multiplicity of infection [MOI] 300) for 48 hours. Adherent cells were harvested by trypsinization and combined with floating cells. Cells were fixed with 70% ethanol and stained with propidium iodide, and DNA content was analyzed by flow cytometry.¹² Gating was set to exclude debris and cellular aggregates, and 10 000 or more events were counted for each analysis. All flow cytometric analyses were repeated between 2 and 4 times with different preparations of hVSMCs and HUVECs. Analyses of the effects of Adeno-FasL infection on VSMC apoptosis and the cell cycle profile in 0.5% and 10% serum was performed on parallel cultures at 40% confluence.

RT-PCR Analyses
Reverse transcriptase–polymerase chain reaction (RT-PCR) assays were used to qualitatively assess FasL expression in HUVECs, hVSMCs, and activated and nonactivated Jurkat cells. Total RNA was isolated from HUVECs, hVSMCs, and activated and nonactivated Jurkat cells. The RNA was then reverse-transcribed to cDNA and amplified by PCR (TaKaRa RNA PCR kit), generating a 534-bp fragment of FasL (forward primer: 5'-GTTCTGGTTGC-CTTGGTAGG-3'; reverse primer: 5'-GACCAGAGAGAGCTC-AGATACG-3') or a 983-bp fragment of GAPDH (forward primer: 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3'; reverse primer: 5'-CATGTGGGCCATGAGGTCCACCAC-3'; Clontech). The following conditions were used: 1 cycle at 94°C for 5 minutes and then 35 cycles at 95°C for 40 seconds, 58°C for 1 minute, and 72°C for 1.5 minutes. The PCR products were electrophoresed in a 2% agarose gel. A plasmid containing the human FasL cDNA was used as a positive control.

	Results

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FasL Expression by ECs and VSMCs
Previously we reported that functional FasL is expressed on vascular ECs¹² whereas others have reported FasL expression by VSMCs.¹⁵ Because questions have been raised on the sensitivity and specificity of some anti-FasL antibodies^{22 23 24 25} and FasL expression can be regulated by translocation from intracellular stores,^{30 31} cell-surface expression of FasL was examined by flow cytometric analysis by using 4 different anti-FasL antibodies (C-20, 4H9, A11, or Mike-1). With each reagent, cell-surface FasL expression was detected on HUVECs (Figure 1A). Preinfection of HUVECs with a replication-defective adenovirus that expresses FasL (Adeno-FasL) increased signal on the cell surface. Untreated Jurkat cells, which do not express FasL,^{23 32} were negative for cell-surface FasL expression, indicating antibody specificity. FasL expression was not detected on the cell surface of hVSMCs, but cell-surface FasL signal could be detected when hVSMCs were preinfected with Adeno-FasL (Figure 1A). The specificity of the FasL signal on ECs was confirmed by incubating the antibody with HUVECs in the presence of an excess amount of purified, recombinant sFasL (40 µg/mL), which led to the complete competition of the FasL signal (Figure 1B).

View larger version (48K): [in this window] [in a new window]   Figure 1. FasL expression by HUVECs. A, Cell-surface expression of FasL on HUVECs. To detect cell-surface expression of FasL, cells were detached from the culture plate with 0.5% EDTA and stained with anti-human FasL rabbit polyclonal IgG C-20, hamster IgG 4H9, rat IgG Mike-1, or rat IgM A11. Isotype-matched nonimmune immunoglobulins are used as negative controls (open curve). Cells were incubated with FITC-conjugated appropriate secondary antibodies. Immunofluorescence staining was analyzed by FACS. Cultured HUVECs or hVSMCs were infected with Adeno-FasL at an MOI of 300. After 24 hours’ incubation, cells were harvested. The harvested cells were stained with anti-FasL monoclonal antibody (A11, filled curve) or rat IgM (open curve) followed by FITC-conjugated anti-rat IgM antibody. Immunofluorescence staining was analyzed by flow cytometry. B, FasL signal is competed by recombinant FasL. C-20 antibody was incubated with HUVECs in the absence (filled curve) or presence (shaded curve) of purified, recombinant, sFasL (40 µg/mL) followed by incubation with FITC-conjugated anti-rabbit IgG antibody. C, RT-PCR analysis of FasL (534-bp) and GAPDH (983-bp) transcripts in HUVECs, hVSMCs, and Jurkat cells. Jurkat cells were activated with PMA (10 ng/mL) and ionomycin (400 ng/mL) for 4 hours. A plasmid containing human FasL cDNA served as a positive control for the FasL RT-PCR signal.

RT-PCR analysis was performed to assay for FasL transcripts in vascular cells. Amplification products corresponding to FasL were detected in HUVECs and, surprisingly, in hVSMCs (Figure 1C). In contrast, Jurkat cells were devoid of mRNA under nonstimulatory conditions, but FasL mRNA expression could be detected in Jurkat cells after stimulation with PMA and ionomycin as described previously.^{23 32}

Regulation of Fas Expression
Fas expression was analyzed on cultured HUVECs, hVSMCs, and Jurkat cells with the monoclonal antibody UB2 that recognizes human Fas (Figure 2). Like rat VSMCs,¹³ Fas was abundantly expressed on hVSMCs. Abundant Fas expression was also detected on Jurkat cells, as reported by others.²⁰ Consistent with previous studies,^{12 14} HUVECs expressed little or no Fas when compared with hVSMCs and Jurkat cells.

View larger version (36K): [in this window] [in a new window]   Figure 2. Cytokine regulation of Fas expression on ECs, VSMCs, and Jurkat cells. HUVECs, hVSMCs, or Jurkat cells were incubated with TNF- (25 ng/mL) or IFN- (100 ng/mL) for 24 hours and stained with FITC-conjugated anti-human Fas antibody. Fluorescence intensity was analyzed by FACS.

Previous reports have documented that inflammatory cytokines regulate Fas expression on VSMCs and ECs,^{8 10 14} and in some cases, it has been proposed that upregulation of Fas will promote vascular cell apoptosis.^{8 10 15 16 17} Therefore, to understand the potential role of Fas expression in the differential sensitivity of ECs and VSMCs to Fas-mediated apoptosis, we systematically compared the effects of TNF- and IFN- on Fas expression in HUVECs, hVSMCs, and Jurkat cells (Figure 2). Under the conditions of our assays, TNF- modestly upregulated Fas expression on HUVECs but had little or no effect on Fas expression by hVSMCs or Jurkat cells. IFN- markedly upregulated Fas expression on ECs, consistent with a previous report,¹⁴ but IFN- had little or no effect on Fas expression by hVSMCs or Jurkat cells.

Fas-mediated VSMC apoptosis has been implicated in cell turnover in atherosclerotic plaque,^{8 10} and it has been reported that plaque VSMCs express more Fas than do the underlying medial VSMCs.¹⁰ Therefore, we tested whether Fas expression might be different between quiescent hVSMCs cultured in low-serum medium (0.5% FBS) and in proliferating hVSMCs cultured in high-serum medium (10% FBS). As shown in Figure 3A, quiescent and proliferating hVSMCs expressed similar levels of cell-surface Fas.

View larger version (40K): [in this window] [in a new window]   Figure 3. Fas expression on VSMCs is not affected by serum or adenovirus infection. A, Human VSMCs at 50% confluence cultured in low-serum (0.5% FBS) or high-serum (10% FBS) medium were harvested with 0.5% EDTA and stained with an FITC-conjugated anti-Fas antibody. Under conditions of these assays, the cell cycle profile for hVSMCs in low serum was G0/G1, 93.3±0.5%; S, 2.0±0.3%; and G2/M, 4.7±0.3%. The cell cycle profile for hVSMCs in high serum was G0/G1, 68.0±2.9%; S, 12.5±0.8%; and G2/M, 19.5±3.2%. B, hVSMCs were infected with Adeno-FasL at an MOI of 300. After 24 hours’ incubation, cells were detached from the culture plate with 0.5% EDTA. The harvested cells were stained with an FITC-conjugated anti-Fas mouse monoclonal antibody (filled curve) or an FITC-conjugated mouse IgG (open curve).

It is also reported that the adenovirus-encoded proteins E1B-19K or RID (receptor internalizing and degradation) modulate sensitivity to Fas-mediated apoptosis by affecting Fas expression on the surface.³³ Thus, cell-surface Fas levels were determined after infection with Adeno-FasL to ascertain whether the previously reported cytotoxic effects of Adeno-FasL on VSMCs^{13 18} is modulated by a confounding effect of the viral genome on Fas expression. Infection with Adeno-FasL produced little or no change in Fas expression on hVSMCs (Figure 3B). Similarly, Adeno-FasL had no effect on HUVEC Fas expression (not shown).

Differential Sensitivity of Vascular ECs and VSMCs to Fas-Mediated Apoptosis
The sensitivity of HUVECs, hVSMCs, Jurkat cells, or P815-huFas mastocytoma cells to various anti-Fas agonists was analyzed (Figure 4). Cells were incubated with sFasL, agonistic anti-Fas antibody (CH11), or Adeno-FasL (MOI 300). Hypodiploid DNA content was analyzed by flow cytometry to assess the extent of apoptotic cell death. No death was evident when HUVECs were treated with any of these Fas agonists. In contrast, P815-huFas or Jurkat cells readily underwent apoptosis after treatment with sFasL, anti-Fas antibody, or Adeno-FasL. Despite abundant Fas expression (Figures 2 and 3), hVSMCs did not undergo apoptosis on exposure to sFasL or anti-Fas antibody, consistent with a previous report.¹⁰ However, VSMCs underwent apoptosis after infection with Adeno-FasL (Figure 4), which produces membrane-bound FasL (Figure 1A). Infection with a control adenovirus encoding ß-galactosidase (Adeno–ß-gal) had no effect on the DNA profile (Table 1). These results indicate that membrane-bound FasL, encoded by Adeno-FasL, is sufficient to trigger an apoptotic signal in VSMCs, whereas Fas stimulation by recombinant sFasL or agonistic anti-Fas antibody is not.

View larger version (42K): [in this window] [in a new window]   Figure 4. Differential sensitivity of HUVECs, hVSMCs, Jurkat cells, and P815-huFas cells to Fas-mediated apoptosis. HUVECs, hVSMCs, or Jurkat cells were treated with sFasL (500 ng/mL) for 24 hours, agonistic anti-Fas antibody (CH11, 500 ng/mL) for 24 hours, or Adeno-FasL (MOI of 300) for 48 hours. A longer incubation with Adeno-FasL was performed to allow for infection and expression. Cells were fixed with 70% ethanol and stained with propidium iodide as described.13 Arrows indicate hypodiploid DNA, an established indicator of apoptosis.46

View this table: [in this window] [in a new window]   Table 1. Adeno-FasL–Induced VSMC Apoptosis Under Different Growth Conditions

Because cytokines can modulate the expression of Fas and FasL^{10 12 14} and are reported to promote VSMC cell death by agonist anti-Fas antibody,¹⁰ we examined the effects of IFN- or TNF- treatment on the viability of HUVECs and hVSMCs that were infected with Adeno-FasL. HUVECs failed to undergo apoptosis with any treatment regimen tested (Figure 5). Of note, HUVECs did not undergo apoptosis after incubation with IFN- and infection with Adeno-FasL, which markedly upregulates cell-surface Fas (Figure 2) and increases the expression of cell-surface FasL (Figure 1A), respectively. TNF- by itself appeared to induce hypodiploid DNA formation in HUVECs, consistent with a previous report on the cytotoxic action of this cytokine on ECs.³⁴ However, HUVEC apoptosis in the presence of TNF- was not enhanced by infection with Adeno-FasL (Figure 5). In contrast to HUVECs, Adeno-FasL induced apoptosis in hVSMCs in the presence or absence of IFN- or TNF- (Figure 5). Although these cytokines had no effect on hVSMC viability alone, IFN- sensitized the cells toward the cytotoxic action of Adeno-FasL, whereas TNF- reduced Adeno-FasL cytotoxicity.

View larger version (23K): [in this window] [in a new window]   Figure 5. Cytokine regulation of sensitivity to membrane-bound FasL-induced apoptosis. HUVECs or hVSMCs were infected with Adeno-FasL in the absence or presence of TNF- (25 ng/mL) or IFN- (100 ng/mL) for 48 hours. Cells were harvested by trypsinization, stained with propidium iodide, and analyzed by flow cytometry. M1 indicates the cell population with hypodiploid DNA.

Finally, hVSMCs cultured in 0.5% and 10% serum were compared for their sensitivity to Adeno-FasL–induced cell death. The dose responsiveness of VSMCs to Adeno-FasL is shown in Table 1. Quiescent, mitogen-deprived, and proliferating VSMCs were similar in their sensitivity to Fas-mediated apoptosis resulting from infection with Adeno-FasL. Analysis of cell cycle profiles revealed that hVSMCs cultured in a high-serum medium displayed an increased proportion of cells in G2/M phases when treated with low, subtoxic doses of Adeno-FasL (Table 2). However, toxic doses of Adeno-FasL increased the fraction of VSMCs in G0/G1 relative to controls. Under low-serum growth conditions, infection with Adeno-FasL did not appear to alter the cell cycle profile of hVSMCs. Furthermore, Adeno-FasL did not affect the cell cycle distribution of HUVECs at any dose examined (data not shown).

View this table: [in this window] [in a new window]   Table 2. Ad-FasL–Induced Changes in the VSMC Cell Cycle Under Different Growth Conditions

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Previous studies have led to conflicting hypotheses regarding the role of Fas-mediated apoptosis in the vasculature. Owing to the potential importance of Fas and FasL in vessel wall homeostasis and disease, we systematically analyzed Fas and FasL expression on human VSMCs and ECs and compared the susceptibility of these cells to Fas-mediated apoptosis by using multiple Fas agonists.

Previously, we reported that ECs express cell-surface FasL.¹² Because the specificity of some commercial anti-FasL antibodies has come into question,^{22 23 24 25} FasL expression on HUVECs was analyzed by using 4 separate anti-FasL antibodies. All 4 antibodies detected cell-surface FasL expression on HUVECs by FACS analysis, but little or no signal was detected in cultures of hVSMCs or nonactivated Jurkat cells. Moreover, the FasL signal on HUVECs was increased after infection with Adeno-FasL and abrogated when anti-FasL antibody was incubated with excess, recombinant sFasL. In contrast to HUVECs, FasL expression was not detected on the cell surface of hVSMCs. Expression of FasL by HUVECs was also documented by RT-PCR analysis. Surprisingly, FasL mRNA was also detected in hVSMCs, suggesting that posttranscriptional control mechanisms may inhibit cell-surface expression of FasL protein in this cell type.

In contrast to FasL, Fas was abundantly expressed on the cell surface of hVSMCs, but HUVECs expressed little or no cell-surface Fas compared with VSMCs or Jurkat cells. Consistent with a previous report,¹⁴ IFN- markedly upregulated Fas expression on HUVECs, whereas IFN- treatment had relatively little effect on Fas expression in hVSMCs or Jurkat cells. Treatment with TNF- modestly upregulated Fas expression on HUVECs but had little or no effect on Fas expression by hVSMCs.

Infection with Adeno-FasL is sufficient to kill hVSMCs (Figure 4) and rat VSMCs.^{13 35} In contrast, agonist anti-Fas antibody (CH11) or recombinant sFasL had no effect on hVSMC viability, but these compounds could induce apoptosis in Jurkat cells or the P815-huFas mastocytoma cell line that overexpresses Fas. The differential sensitivity of hVSMCs to adenovirus-encoded FasL versus anti-Fas antibody or sFasL agonists may result from the ability of cDNA delivery to produce membrane-bound FasL. It has been proposed that membrane-bound FasL is presented to target cells in a physiologically relevant manner, whereas anti-Fas antibody and sFas agonists do not efficiently induce apoptosis because of a lack of receptor clustering.^{20 36}

Geng et al¹⁰ reported that agonist anti-Fas antibody could trigger hVSMC apoptosis only in the presence of IFN- or TNF-. Under the conditions of our assays, IFN- increased hVSMC apoptosis induced by infection with Adeno-FasL, but TNF- protected hVSMCs from this death stimulus. The effects of these cytokines on the susceptibility of hVSMCs to Fas-mediated cell death is unlikely to result from alterations in receptor level, as IFN- or TNF- did not appreciably modulate cell-surface Fas expression in our assays. Presumably, these cytokines modulate Fas-mediated VSMC apoptosis by regulating downstream components of the death signaling pathway. Consistent with this hypothesis, TNF- induction of nuclear factor-B has been reported to protect cells from apoptosis.^{37 38} In related experiments, we also found that mitogen stimulation did not affect Fas-mediated apoptosis in hVSMCs. However, infection with Adeno-FasL appeared to alter cell cycle kinetics when VSMCs were cultured with high levels of serum.

Although ECs express Fas receptor on their cell surfaces, they do not undergo apoptosis when exposed to sFasL or agonist anti-Fas antibody,¹⁴ or when cell-surface Fas ligand is increased by adenovirus-mediated FasL gene delivery. It is also notable that infection with Adeno-FasL did not induce apoptosis in HUVECs in the presence of IFN-, which markedly increases Fas expression. Presumably, ECs are normally resistant to Fas-mediated cell death because they express endogenous cell-surface FasL and therefore possess endogenous mechanisms that block the transmission of the death signaling pathway after engagement of Fas by FasL. The factors that confer EC resistance to Fas-mediated apoptosis are currently unknown. One possibility is that the Fas-mediated death signal in ECs is blocked by FLICE (Fas-associated death domain–like interleukin-1ß–converting enzyme)-inhibitory proteins (FLIPs).^{39 40 41} Consistent with this notion, FLIPs have been detected in ECs⁴² and are downregulated by oxidized lipids, which sensitize ECs to Fas-mediated apoptosis.⁷

The differential sensitivity of ECs and SMCs to Fas-mediated apoptosis may lead to the development of novel strategies to treat proliferative diseases of the vessel wall. On one hand, the cytotoxic action of FasL on proliferating VSMCs may have utility for the treatment of injury-induced intimal hyperplasia.^{13 18} In this application, FasL gene transfer might function as a potent suppressor of intimal hyperplasia because it is expressed on the VSMC surface, leading to the induction of cell death in a paracrine manner.¹³ Furthermore, FasL may augment the endogenous immunosuppressive properties of the vessel wall,^{12 18} yet it should not inhibit the beneficial process of reendothelialization, as ECs are normally resistant to Fas-mediated cell death. The resistance of ECs to Fas-mediated apoptosis may also allow the use of these cells as vehicles for the overexpression of exogenous FasL to inhibit lesions that develop in the vessels of allografted organs.⁴³ These vascular lesions are associated with the early infiltration of T lymphocytes and macrophages in the subendothelial space,⁴⁴ followed by a gradual increase in the presence of VSMCs in the intima.⁴⁵ Therefore, inhibition of this early inflammatory response at the endothelial surface by the elevated expression of FasL could have utility in reducing the chronic vascular graft disease that develops within transplanted organs.

	Acknowledgments

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

Received April 1, 1999; accepted September 15, 1999.

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