Pulmonary Center and Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts 02118
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ABSTRACT |
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TRAIL is a cell-associated tumor necrosis factor-related apoptosis-inducing ligand originally identified in immune cells. The ligand has the capacity to induce apoptosis after binding to cell surface receptors. To examine TRAIL expression in murine vascular tissue, we employed in situ hybridization and immunohistochemistry. In these studies, we found that TRAIL mRNA and protein were specifically localized throughout the medial smooth muscle cell layer of the pulmonary artery. Notably, a similar pattern of expression was observed in the mouse aorta. Consistent with these findings, we found that cultures of primary human aorta and pulmonary artery smooth muscle cells express abundant TRAIL mRNA and protein. We also found that these cells and endothelial cells undergo cell lysis in response to exogenous addition of TRAIL. Last, we confirmed that TRAIL specifically activated a death program by confirming poly(ADP ribose) polymerase cleavage. Overall, we believe that these findings are relevant to understanding the factors that regulate cell turnover in the vessel wall.
apoptosis; smooth muscle cell; endothelial cell; tumor necrosis factor-related apoptosis-inducing ligand
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INTRODUCTION |
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TUMOR NECROSIS FACTOR (TNF)-related apoptosis-inducing ligand (TRAIL) is a 33- to 34-kDa member of the tumor necrosis family of ligands with the closest homology to Fas ligand (33). This family of ligands regulates fundamental aspects of immune cell activity, including immunoglobulin isotype switching, B-cell maturation, lymph node development, and establishment of peripheral tolerance (14, 18). The inducible expression of TRAIL in T cells (14) during antigenic stimulation suggests that like Fas ligand, TRAIL mediates antigen-induced cell death. TRAIL has the ability to transduce an apoptotic signal after binding to specific cell surface receptors that contain intracytoplasmic death domains (2, 7, 17, 32, 33). Studies identified the presence of two different TRAIL signaling receptors in addition to at least two so-called decoy receptors that lack functional death domains (7, 17, 21, 32, 33). These receptors are widely expressed in all inflammatory cell types and in various lymphoid and nonlymphoid organs (22, 29, 30).
Abundant TRAIL mRNA has been localized to nonlymphoid tissues; the precise cell types that express TRAIL at these sites in vivo, however, have not been identified (33). Constitutively expressed sources of Fas ligand in nonlymphoid cells, such as the corneal endothelium (11) and the Sertoli cells of the testis (3), are thought to modulate immune activity through induction of apoptosis in influxing inflammatory cells, thereby shielding tissues from immune-mediated injury. In this regard, we found that Fas ligand is specifically localized to the Clara cell in the airway and may regulate local immune activity in the bronchial wall (10). Whether nonlymphoid sources of TRAIL expression serve a similar function is uncertain at this time.
Importantly, Fas ligand-induced apoptosis is not restricted to immune cell targets. For example, the cyclical involution of ovarian follicles is likely controlled by the regulated expression of the Fas ligand receptor (13). Similarly, the rapid turnover of dermal epithelium during inflammation may be Fas ligand dependent (27). It is noteworthy therefore that targeted disruption of the Fas gene in mice is associated with spontaneous development of hepatic hyperplasia (1). Taken together, these findings suggest that members of the TNF family of ligands may have a key role in regulating cell turnover in nonlymphoid tissues.
The precise factors controlling vascular cell turnover in the vessel wall, particularly in the homeostatic state, are poorly understood. During vessel injury, enhanced smooth muscle cell proliferation is thought to result from action of specific mitogenic ligands derived from influxing inflammatory cells, serum, and local sources (24). Increasingly, a role for apoptosis in controlling vascular smooth muscle cell number has been supported by experimental data (5). Pathological conditions associated with increased smooth muscle cell mass, such as pulmonary hypertension, thus may not only involve alterations in the control of proliferation but may also involve alterations in the control of cell death (5, 24).
In this study, we show that TRAIL is selectively expressed in the vascular smooth muscle cells of the pulmonary artery and aorta. We provide further evidence that TRAIL regulates smooth muscle and vascular endothelial cell apoptosis. Overall, we believe that these findings have significant implications for understanding mechanisms that control cell turnover in the vessel wall.
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METHODS |
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Processing of mouse lung and aortic tissue. Adult C57BL/6 mice (19-21 g; Charles River Laboratories) were killed by cervical dislocation followed by intra-abdominal aortic exsanguination. The lungs were fixed by intratracheal inflation at a constant pressure of 20 cm H2O, processed, embedded, and sectioned at 6 µm as previously described (8). In other animals, the entire abdominal aorta was fixed, processed, embedded, and also sectioned at 6 µm. All animal protocols conformed to institutional standards.
Generation of digoxigenin-labeled TRAIL probes for in situ hybridization. To synthesize riboprobes, a 218-bp murine TRAIL cDNA sequence corresponding to a unique region of the TRAIL molecule (33) was generated by RT-PCR and subcloned into pGEM-T (Promega). Before generation of riboprobes, the orientation and fidelity of the TRAIL cDNA construct were assessed by DNA sequencing. For in vitro transcription, linearized plasmid was incubated with T3 (5 U/µl) or SP6 (5 U/µl) RNA polymerase (Boehringer Mannheim), transcription buffer, digoxigenin-labeled UTP, and other precursor nucleotides at 37°C for 2 h. Unincorporated nucleotides were removed after ethanol precipitation with glycogen carrier. The relative concentrations of antisense and sense probes were estimated by dot blot analysis using a standard curve of known concentrations of digoxigenin substrate, as previously described (8).
Nonisotopic in situ hybridization. The methods are modifications of those of Panoskaltsis-Mortari and Bucy (23) as described by Fine et al. (8). Briefly, rehydrated tissue sections were first incubated with proteinase K (10 µg/ml) for 30 min at 37°C followed by treatment with 1 M triethanolamine-0.25% acetic anhydride. Hybridization was subsequently performed overnight in a humidified chamber at 42°C in buffer (8, 23) containing various concentrations of antisense or sense digoxigenin-labeled riboprobes. After hybridization, sections were washed with 50% formamide-2× saline-sodium citrate and then with a NaCl (0.5 M)-Tris (10 mM, pH 7.5)-EDTA (1 mM) buffer before digestion with RNase A (20 µg/ml) and RNase T1 (1 U/ml). Sections were rewashed before localization of digoxigenin-labeled cells with a sheep polyclonal anti-digoxigenin-alkaline phosphatase conjugate (Boehringer Mannheim). For this, nonspecific binding sites were first blocked by incubation with 2% rabbit serum for 30 min. Subsequently, the anti-digoxigenin antibody-alkaline phosphatase conjugate was applied (1:250) for an overnight exposure at 4°C. After washing, color developing solution (337.5 µg/ml of 4-nitro blue tetrazolium chloride and 175 µg/ml of 5-bromo-4-chloro-3-indolyl phosphate) containing levamisole (2 mM) was added for an overnight incubation in a light-free container at 4°C. Color development was terminated by immersion in stop solution (Tris-EDTA) before counterstaining, dehydration with graded alcohols, and mounting with immersion oil.
Immunohistochemistry. Tissue sections were sequentially incubated with a primary goat anti-mouse TRAIL polyclonal antibody at 2 µg/ml (Santa Cruz) followed by a secondary biotinylated polyclonal horse anti-goat rabbit IgG conjugate. For localization of primary antibody staining, slides were subsequently exposed to chromogenic substrates in accordance with instructions for the Vectastain Elite ABC Kit (Vector Laboratories). Control tissue sections were developed after incubation with only the secondary antibody. Importantly, the primary antibody employed in these studies specifically recognizes an amino-terminal epitope expressed by mouse TRAIL and is non-cross-reactive with other TNF family members.
Cell culture. Primary human pulmonary artery or aortic smooth muscle cells and human umbilical vein endothelial cells (HUVEC, Clonetics) were cultured according to the manufacturer's recommendations. All experiments were performed on cells before the fifth passage.
Northern analysis. Total RNA was extracted by employing a kit according to the manufacturer's recommendations (Qiagen). RNA was quantified by absorbance at 260 nm before electrophoresis through a 1% agarose-6% formaldehyde gel and transfer to a nylon filter. For determination of TRAIL mRNA levels, the filter was prehybridized with Rapid-hyb buffer (Amersham) and then incubated with a radiolabeled human TRAIL cDNA probe (33) for 2 h before washing. Filters were then exposed to X-ray film.
Protein isolation and Western analysis. Cells were washed with cold PBS before scraping into RIPA buffer [50 mM Tris · HCl, pH 7.4, 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 mM NaF, and 1 mM NaV3O4]. Subsequently, lysates were sheared by passing through a 21-gauge needle before centrifugation. The pellets were discarded before measurement of protein concentration in the supernatants by the Bradford assay (4). For Western analysis, equal amounts (15 µg) of total protein were separated through a 7.5% SDS-polyacrylamide gel before electrophoretic transfer to a nitrocellulose membrane. Membranes were stained briefly with Ponceau S to grossly visualize successful transfer of proteins. To probe for TRAIL or poly(ADP ribose) polymerase (PARP) protein, nonspecific binding sites on the membrane were first blocked by incubation with PBS with 0.1% Tween containing 5 g nonfat dry milk per 100 ml. After washing, membranes were incubated for 1 h with a mouse monoclonal anti-human TRAIL (1.5 µg/ml) or anti-human PARP antibody (0.5 µg/ml, PharMingen). After extensive washing, proteins bound to the primary antibody were identified by incubating with a secondary polyclonal goat anti-mouse antibody-horseradish peroxidase (HRP) conjugate (Promega) at a dilution of 1:10,000 and then developed after exposure to reagents (Dupont-NEN) that generate a chemiluminescent signal.
Cytotoxicity assay. Smooth muscle cells or HUVEC were labeled
overnight with 51Cr at 37°C and washed before addition
of recombinant human TRAIL at 100 ng/ml (Research Diagnostics) or
interleukin-1 (IL-1
at 5 ng/ml, R&D Systems). In one
experiment, smooth muscle cells were also coincubated with an
inhibitory soluble TRAIL chimeric R2 receptor protein (R&D Systems).
Quadruplicate wells were set up for all experimental groups to measure
spontaneous release and to determine total counts (1% NP-40 treated).
Specific lysis was computed by the formula (25)
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RESULTS |
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Expression of TRAIL in vascular smooth muscle cells in vivo and in
vitro. Having found that Fas ligand expression is specifically localized to the airway epithelium (10), we initially sought to examine
whether the related molecule TRAIL had a similar pattern of expression.
To identify cell types expressing TRAIL in the normal mouse lung, we
generated nonradioactive sense and antisense mouse TRAIL RNA probes for
in situ hybridization. As shown in the representative field in Fig.
1A, we found that TRAIL mRNA was
restricted to smooth muscle cells contained within the muscularized medial layer of large and small pulmonary vessels. Close
inspection indicated that vascular smooth muscle TRAIL expression was
homogeneous throughout the entire medial layer. Furthermore, we found
that bronchial smooth muscle did not contain positive signals. A
higher-power photomicrograph demonstrates that TRAIL mRNA is not
expressed in the intima (Fig. 1B). Sections incubated with
sense probe had no signal (Fig. 1C).
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To determine whether systemic arteries display a similar pattern of TRAIL expression, nonisotopic in situ hybridization was performed on fixed sections of the normal mouse abdominal aorta. As shown in Fig. 1D, we found that TRAIL mRNA was also expressed in a homogeneous pattern in the smooth muscle cells of the medium of the aorta.
To extend these findings, we performed immunohistochemistry with an
affinity-purified specific polyclonal anti-TRAIL antibody. Localization
of signal was achieved by incubating sections with a HRP-labeled
secondary antibody and chromogenic substrate. Consistent with our
results in Fig. 1, TRAIL protein expression was also restricted to
pulmonary vascular smooth muscle (Fig. 2).
Interestingly, in some cells, staining appeared punctate in appearance.
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In view of our results with the mouse lung and aorta, we examined TRAIL
expression in cultures of primary human vascular smooth muscle cells
derived from the pulmonary artery and aorta. We found that human
pulmonary vascular smooth muscle cells express ample TRAIL mRNA as
determined by Northern analysis of total RNA (Fig. 3); the size of the transcript (~1.6 kb)
is consistent with a previous study (33). In contrast and also
consistent with prior work (16), we found that these cells do not
express Fas ligand mRNA as determined by RT-PCR (data not shown).
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We consequently determined the expression of TRAIL protein in primary
human smooth muscle cells in culture. For this, we employed a
monoclonal antibody specifically directed to an extracellular region of
the human TRAIL protein in Western analyses of total protein lysates
(15 µg). As shown in Fig. 4A, we
found that both aorta and pulmonary artery smooth muscle cells express
abundant levels of TRAIL protein. In agreement with the histological
data, cultures of HUVEC do not express appreciable levels of
TRAIL protein (Fig. 4B).
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TRAIL induces cytoxicity of smooth muscle cells and endothelial
cells. Having demonstrated that primary human smooth muscle cells
express cell-associated TRAIL, we went on to assess the capacity of
these cells to respond to addition of soluble TRAIL. By RT-PCR, we
first confirmed that human smooth muscle cell cultures express death
domain-containing signaling TRAIL receptors (data not shown). After
this, we examined the effect of adding recombinant human TRAIL to
51Cr-labeled smooth muscle cell cultures. After an
overnight incubation with TRAIL, specific lysis (25) was induced at a
level ranging from 10 to 20%. A representative experiment is shown
in Fig. 5. In this study, we found that
TRAIL-mediated cytotoxicity could be inhibited by coincubation with the
soluble R2 TRAIL signaling receptor protein (32). Moreover, although
HUVEC do not express endogenous TRAIL (Fig. 4B), exposure to
exogenous TRAIL also induced cell lysis.
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For comparison, IL-1, which is known to induce vascular cell
apoptosis (9), was examined under these assay conditions in both
endothelial and smooth muscle cells. Because the bioactivities of
cloned proteins cannot be compared directly, we utilized a conventional
dose of IL-1
(2). In this assay, we found that IL-1
was more
effective than TRAIL in inducing endothelial cell apoptosis.
Interestingly, the effect of IL-1
and TRAIL were synergistic (Fig.
6.) As predicted, the soluble TRAIL
receptor inhibitor did not inhibit IL-1
-induced cell lysis (data not
shown). In contrast, IL-1
was less effective than TRAIL in inducing
apoptosis of smooth muscle cells (Fig. 6).
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To confirm that TRAIL induced apoptosis, we examined for the presence
of cleaved PARP. In the intact cell, this nuclear protein is 116 kDa
(16). During apoptosis, the cleavage of PARP by caspase-3 releases an
89-kDa COOH-terminal fragment. Thus the appearance of this fragment has
been utilized as a specific marker of cells undergoing apoptosis (16).
As shown in Fig. 7, in protein extracts derived from smooth muscle cells adherent to the culture dish and from
cells which lifted into medium, the cleaved COOH-terminal fragment of
PARP was only observed after TRAIL stimulation. As expected, the
predominant species in lifted smooth muscle cells was the 89-kDa
fragment. Taken together, these findings confirm that TRAIL-induced
cell lysis involves activation of apoptosis.
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DISCUSSION |
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In this study, we demonstrate that vascular smooth muscle cells express TRAIL in vivo and in vitro. Moreover, we found that exogenous soluble TRAIL induces cytotoxicity in smooth muscle cell and endothelial cell cultures. Although an immune regulatory role for TRAIL has been identified, we believe our data also support a role for this ligand in regulating cell turnover in the vessel wall.
The precise relationship between TRAIL-dependent apoptosis and the relative expression patterns of the various TRAIL receptor subtypes in vivo is currently undefined. Experimental data suggest that variations in the relative expression of specific TRAIL receptor subtypes modulates TRAIL action. Along these lines, increased expression of TRAIL decoy receptors attenuates engagement with death domain-containing receptors and, in turn, inhibits the transduction of cell death by caspase-dependent mechanisms (30). The factors facilitating TRAIL-induced apoptosis in an adherent cell such as the vascular smooth muscle cell are unclear. Whether TRAIL-induced cell death proceeds in a paracrine manner or involves autocrine signaling is not certain at this time. In the case of cytotoxic T cells, contact is probably facilitated by antigen recognition on targets (20, 28).
Similar to Fas ligand, a naturally occurring soluble form of TRAIL exists (15, 20, 31). Whereas cleavage of Fas ligand from the cell surface requires the action of zinc-dependent metalloproteinase (15, 31), generation of soluble TRAIL involves the action of a cysteine protease(s) (20). Notably, the vessel wall is a rich source of cysteine proteases (6). The site of TRAIL cleavage has not been identified but, based on the size of the released product, likely occurs at amino acids just distal to the membrane-spanning domain (28). It is important to note that the human soluble TRAIL protein we employed in the cytotoxicity assays is a cloned product derived from a foreshortened TRAIL cDNA. The functional role of soluble released ligand products is controversial; data supporting an ability to both induce and inhibit apoptosis have been generated (8, 20, 31, 33). Notably, endothelial cells, which do not express TRAIL, were found to be susceptible to TRAIL-induced apoptosis. Regardless, release of soluble TRAIL may be an important step in the regulation of TRAIL-dependent killing in the vessel wall.
Apoptotic smooth muscle and endothelial cells have been identified in
regions of vascular injury and inflammation (12). In this regard,
several distinct cytokines, including TNF and IL-1, have been shown
to induce smooth muscle cell death through caspase-dependent signaling
(9). Interestingly, we found that TRAIL and IL-1
synergistically
induce apoptosis. In contrast, the vasodilatory mediator nitric oxide
inhibits apoptosis (19), possibly through the S-nitrosylation
of the active site in caspase-3 (19). Taken together, the complex
interaction of these effector substances is likely an important factor
regulating the accumulation of vascular smooth muscle and determining
the outcome of fibroproliferative disorders of the vessel wall.
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ACKNOWLEDGEMENTS |
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This work was supported by the National Institutes of Health Grants AI-32531-05, AI-41994, and EY-12513.
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FOOTNOTES |
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Present address of B. R. Gochuico: Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, Bethesda, MD 20892.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. Fine, Pulmonary Center R-3, 80 E. Concord St., Boston Univ. School of Medicine, Boston, MA 02118.
Received 26 May 1999; accepted in final form 22 December 1999.
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