1Division of Nephrology, Department of Internal Medicine, 2Immunology Graduate Group, and 4Cancer Center, University of California, Davis; 3Department of Pathology, University of California, San Francisco; and 5Department of Veterans Affairs Northern California Health Care System, Sacramento, California
Submitted 28 September 2004 ; accepted in final form 10 February 2005
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ABSTRACT |
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atherosclerosis; inflammation; plaque
Because apoptosis is a major contributor to the process of plaque rupture, research into the mechanism and mediators of this process in vascular smooth muscle (VSM) cells is an area of pivotal importance in atherosclerosis research. TNF- is a 17-kDa proinflammatory cytokine that acts as a homotrimer on two distinct membrane bound receptors, TNF-R1 and TNF-R2 (4). This cytokine is detected at elevated levels in atherosclerotic vessels in rabbits (19), as well as in human atheroma (1, 30), and it may contribute to rupture of these plaques. In vitro studies have shown that TNF-
stimulates VSM cell migration (14) and, perhaps more importantly to its role in atherosclerosis, that intimal smooth muscle cells exposed to TNF-
undergo apoptosis at a higher rate than unexposed medial smooth muscle cells (25).
p73 is a family of proteins that are closely related in sequence, as well as in some functions, to the tumor suppressor p53. As such, p73 activates many p53 target genes and, similarly to p53, causes growth suppression and apoptosis in a variety of cell lines (13, 15) including VSM cells (6). Our earlier findings that p73 is present in human atherosclerotic plaque lysate (40) and that overexpression of p73 causes apoptosis in rat thoracic aorta smooth muscle cells (6) led us to investigate the role of p73 in VSM cell apoptosis.
At present, six alternatively spliced p73 mRNAs have been identified in normal cells, named ,
,
,
,
, and
, but the function of the various isoforms in VSM cells is just beginning to be investigated. We have shown (6) that overexpression of p73
in VSM cells results in apoptosis, but the
-isoform of p73 may be more important than other isoforms in apoptosis (28) and growth suppression (9, 16). Indeed, in several assays p73
has been shown to have stronger transcriptional activity than p73
, possibly because the longer COOH-terminal region of full-length p73 is inhibitory (7). For this reason, we examined primarily this isoform in the current study. We now show that, on serum stimulation, both human and rat VSM cells express p73
. In addition, treatment of VSM cells with TNF-
in the presence of serum-containing media further upregulates p73
levels in these cells, and p73 is in fact required for the full effect of TNF-
on apoptosis. In light of the data presented in this study, p73 can be considered a target for further research into the apoptotic pathways, and thus fibrous cap stability, of atherosclerotic lesions.
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MATERIALS AND METHODS |
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Cell culture. Rat VSM cells, passages 1930, were cultured in growth medium containing DMEM, 10% FBS, and 50 U/ml penicillin-streptomycin. Human VSM cells, passages 58, were grown in Clonetics proprietary growth medium. To serum starve the cells, they were placed in serum-free medium containing DMEM, 20 mM HEPES (pH 7.4), 5 mg/ml transferrin, 0.5 mg/ml BSA, and 50 U/ml penicillin-streptomycin for 48 h. Serum-starved cells were stimulated with either growth medium or 40 ng/ml PDGF-BB for the times indicated.
Western blotting.
A10 cells were grown to 6070% confluence. The cells were then treated with 10 ng/ml rat or human TNF- for the times indicated. Media and washes were collected and centrifuged to collect the dead cells. Cells were lysed, and then equal protein amounts were loaded and electrophoresed and immunoblotted with the Bio-Rad DC protein assay (Hercules, CA) as previously described (41). The membranes were blocked in 510% nonfat dry milk for 1 h at room temperature and probed with appropriate antibodies in 5% nonfat dry milk overnight at 4°C. The membranes were then probed with horseradish peroxidase-tagged anti-mouse or anti-rabbit IgG antibodies (Bio-Rad) diluted 1:15,000 in 2.55% nonfat dry milk for 1 h at room temperature. Chemiluminescence was detected by enhanced chemiluminescence (Amersham Biosciences).
Transfections.
VSM cells were seeded into 35-mm dishes at 5070% confluence 1 day before transfection. Transient transfection using Gene Juice transfection reagent with no plasmid, pcDNA-green fluorescent protein, pcDNA hemagglutinin (HA)-tagged p73 (7), and pcDNA HA-tagged
Np73
(24) was performed according to the manufacturer's protocol. Fresh medium was added after 6-h incubation with the transfection complex. Cells were lysed 2448 h after transfection.
Caspase activity.
A10 cells were grown in 100-mm dishes until 5070% confluence. Rat TNF-
(10 ng/ml) was added for the times indicated. The media and washes were collected and centrifuged to collect the dead cells. Both cells and media, as well as washes (containing dead cells), were combined and then lysed. Caspase activity was detected colorimetrically on equal quantities of lysate protein with the CaspACE assay system according to the manufacturer's protocol.
Immunohistochemistry. Internal Review Board approval was obtained from the University of California, Davis, and the University of California, San Francisco, and we searched the pathological records from the University of California, San Francisco, from 1995 to 2002 and selected representative sections from 20 different carotid endarterectomy specimens. Of these, 14 of 20 clearly demonstrated characteristic features of arterial intimal plaque on hematoxylin and eosin-stained sections. Routine histological staining and immunohistochemistry were performed on 5-µm-thick tissue sections cut from formalin-fixed paraffin-embedded tissue blocks and placed on Superfrost/Plus slides.
The immunohistochemical staining was performed using the avidin-biotin peroxidase method. -Smooth muscle actin (SMA; Dako, Carpinteria, CA) was used to evaluate for the presence of cells of muscular derivation, and a polyclonal antibody recognizing both p73
and p73
(Ab-4) was used to identify the presence of p73 in these same cells. After deparaffinization and blocking of endogenous peroxidases, tissue sections were steam treated and allowed to stand in buffer for 1 additional hour. p73 antibody diluted 1:3,500 and SMA antibody diluted 1:100 were incubated with the tissue sections at 4°C overnight. Biotinylated anti-mouse and avidin-biotin complex (Vector, Burlingame, CA) were applied to each section and developed with the peroxidase reaction, using diaminobenzidine as chromogen according to standard methods. Sections were counterstained with hematoxylin. An internal positive control for SMA included small, intact arteries present in the plaque material. Elastica-van Gieson (EVG) staining was used to identify elastic fibers normally present in the intima and media of vessels.
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RESULTS |
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TNF- causes apoptosis in VSM cells.
TNF-
has been shown to induce apoptosis in intimal VSM cells (25), which can lead to fibrous cap instability and plaque rupture as discussed above. To determine whether such an effect is mediated by p73, we incubated proliferating VSM cells (replicating the in vivo milieu) with TNF-
plus serum-containing medium for 2496 h and examined the lysate for PARP cleavage, an early event in the apoptosis cascade, by immunoblotting. Compared with serum-only stimulated cells, TNF-
-treated cells treated in the presence of serum showed an increased cleavage of the 113-kDa PARP band, an early event in apoptosis, with the maximal difference in PARP cleavage between TNF-
and serum-alone stimulation being at 96 h (Fig. 4A). There was some apoptosis seen in serum-only stimulated cells, probably due to the length of time in the absence of fresh serum, but in all cases PARP cleavage in cells incubated with serum + TNF-
was greater than with serum alone. To confirm this finding with another measure of apoptosis, we also examined caspase-3 activation in the same cell lysate. As expected, caspase-3 activity paralleled PARP cleavage in cells treated with TNF-
(Fig. 4B). Thus VSM cells undergo apoptosis on treatment with TNF-
.
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VSM cells were transfected with the Np73
plasmid before treatment with TNF-
in serum-containing medium. Transfection efficiency was determined with an antibody recognizing the COOH terminus of p73
and thus also the NH2-terminally truncated forms. Although TNF-
caused significant apoptosis in mock-transfected VSM cells, transfection of the cells with the dominant-negative
Np73 plasmid showed a DNA concentration-dependent decrease in apoptosis in response to TNF-
(Fig. 5A). Successful transfection of the
Np73
plasmid was confirmed by immunoblotting (Fig. 5B).
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DISCUSSION |
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Whereas before our earlier findings p73 had not been described to be present in vascular tissues, p53 induction has been associated with apoptotic changes in arterial aneurysms (reviewed in Ref. 23). p53, which is closely related in structure (but not in all functions) to p73, is also markedly increased after balloon angioplasty of the rabbit iliac artery in a manner that parallels apoptosis (32). Because p73 expression is often observed under similar situations when p53 is present, it is likely that p73 protein also plays a role in VSM cell growth and in the pathogenesis of atherosclerosis.
Although p73 and p53 have different ultimate effects, there is evidence that p73 can activate many, although not all, p53 target genes, including p21 (35). p73, in particular, has been shown to activate expression of the cyclin kinase inhibitor p57Kip2 in cancer cell lines, whereas p53 does not (2); however, the function of p57Kip2 in nontransformed and vascular cell lines, both stimulated by p73 and not, is not known. There are also situations in which p73 and p53 induce expression of the same proteins, such as p21waf1/cip1 (43), consistent with our data showing that dominant-negative p73 at high transfection levels attenuates the TNF-
(and thus presumably p73)-dependent increase in p21 (Fig. 5C). The p21 story is made more complicated by our work showing that this cyclin kinase inhibitor can act in both a pro- and an antiapoptotic manner (reviewed in Ref. 39), such that p21's function downstream of p73 is not obvious. Whether the differences in p73 function and tissue specificity are wholly dictated by these target genes remains to be established.
TNF- is known to cause apoptosis in VSM cells, and this has been postulated to be a contributor to plaque rupture (1). It has also been hypothesized that apoptosis caused by activation of endogenous TNF receptors in VSM cells (25) may be a mechanism by which injured arteries limit accumulation of these cells. Our finding of the requirement for TNF-
to be added to VSM cells in conjunction with serum for p73
to be increased (and presumably for apoptosis to occur) is consistent with other reports showing minimal effect alone but a synergistic effect of TNF-
when added to VSM cells 1) overexpressing truncated I
B (26) and 2) in combination with a proteosome inhibitor (17) or interferon-
(11).
The p53 class of tumor suppressor proteins are responsible for mediating the cellular response to DNA damage, through the cyclin kinase inhibitors as well as other downstream proteins (reviewed in Ref. 39). Although p73 is dissimilar to p53 in a variety of its effects, the two cousins both have the ability to cause apoptosis under certain conditions. The mechanisms of the proapoptotic effects of p53 and p73 may be different, especially in light of the complex pattern of both pro- and antiapoptotic isoforms generated by differential splicing and the presence of alternative promoters on the p73 gene (reviewed in Ref. 34).
Although there is abundant information concerning the mechanism and tissue specificity of apoptosis by p73, data on the apoptotic function of p73 in the human vasculature are sparse. Using a conditional expression system, we showed previously (6) that overexpression of p73 leads to apoptosis in rat VSM cells, yet others have shown that apoptosis induced by E2F1 is independent of p73 in human VSM cells (33). p73 may also be important in tumor progression via its effect on tumor vasculature, as evidenced by the finding that overexpression of this protein increases VEGF (38), a result consistent with the work of other investigators showing a close association between VEGF and p73 expression in colorectal carcinoma (12). Because both VEGF and TNF-
are vasoactive and inflammatory mediators, whether a proapoptotic effect of TNF-
through p73, as shown by our data, is important in countering tumor angiogenesis remains to be seen.
Our previous reported findings of increased p73 in VSM cells exposed to serum may have been due to the recognition of both p73
and p73
by the antibody in the cells used at earlier passage (40) or by the lack of specificity of the antibody used in the earlier studies. Now that we have a positive control for p73
(6), we are able to determine that p73
is the predominant species in this cell line in response to a mitogenic stimulus (Fig. 1). On the basis of the known proapoptotic roles of both p73
and p73
, the potential function of p73
in VSM cell growth likely relates to growth suppression and/or apoptosis. As mentioned above, the ability of p73 to affect both cyclin kinase inhibitors p21, as we confirm here, and p57 (in cancer cells; Ref. 2) may actually result in either cell cycle arrest or apoptosis similar to p53. However, the lack of tumor formation in p73-knockout models and in primary human tumor data argues against p73 functioning as a classic tumor suppressor (34). Until further data are obtained showing the effect of overexpressed p73
in VSM cells, as we have done for p73
(6), this remains an open question in this cell type.
Our finding that the apoptotic function of a pluripotent mediator of inflammation, TNF-, is mediated by p73 is consistent with the known function of p73 in growth suppression and apoptosis. Our data are also consistent with a recent study in a human B cell lymphoblastoid cell line (Ramos cells) in which TNF-
also increased p73 levels (5), suggesting that this phenomenon may be more generally applicable. Although we were unable to completely attenuate the TNF-
-induced apoptotic effect with a dominant-negative plasmid approach, our work indicates that the effect of TNF-
specific to the vasculature requires functional p73. Thus artificial regulation of p73 may be a viable approach to improving the stability of a plaque or fibrous cap that may occur after angioplasty, for example. This is especially important, given the high rate of angioplasty failure, which in some cases may be due to plaque rupture.
Consistent with our findings, activation of TNF- receptors has been shown to increase the rate of intimal VSM cell apoptosis (25). In that study, TNF-
mRNA, but not VSM cell growth rate, was increased in response to other inflammatory mediators, interferon-
and interleukin-1
. However, VSM cells that have been induced to proliferate by balloon injury express TNF-
(37). Other investigators have shown that treatment of VSM cells with TNF-
alone did not result in apoptosis (11), a disparity that may be due to cell type, passage number, or other conditions. Although our data are consistent with other studies and support a definite role of TNF-
in VSM cell apoptosis, the nature of the effect of this cytokine on cell proliferation, also important for atherosclerotic lesion pathogenesis, is not known and was not addressed in this study.
The location of p73 within the nuclei of musclelike cells within plaque tissue, as described in our study, suggests that this protein may be playing a growth- or apoptosis-modulatory function in this tissue. Should p73 be directing these cells into an apoptotic pathway, ultimate targeting of this protein for attenuation by techniques such as antisense or small interfering RNA may lead to a salutary effect by decreasing plaque rupture. On the other hand, p73 may be mediating an antiproliferative effect on the VSM cells, which in the case of plaque growth may indeed be a desired outcome. In either case, further research into the role of p73 in the atherosclerotic process is likely to yield data that may be used in future therapy of this devastating disease.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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