Edith Speir, Toren Finkel, and Stephen E. Epstein (Cardiology Branch, NHLBI)
One of the major breakthroughs in cardiovascular therapeutics over the past two decades has been the development of catheter-based angioplasty to open constricted coronary and peripheral arteries without surgery. This is accomplished by passing a catheter with a deflated balloon at its tip into the obstructed artery and then inflating the balloon. Atherosclerotic plaque is compressed and remodeled by the expanded balloon, thereby relieving the obstruction. Although the initial success rate of balloon angioplasty in opening stenotic coronary arteries approaches 95%, recurrent narrowing, or restenosis, occurs in 25 - 50% of patients within six months.
The causes of restenosis are complex and undoubtedly multifactorial; however, one of the dominant mechanisms involves injury-induced activation of the smooth muscle cells (SMCs) located in the media of the vessel wall. Activiating the SMCs results in their proliferation and migration to the subintima, where they continue to proliferate and secrete extracellular matrix. As this neointima expands, it obstructs the vessel lumen and diminishes blood flow, thereby causing myocardial ischemia. The SMC proliferative response to angioplasty is a normal healing response to injury, and hypothetically, the development of restenosis might well be due to individual differences in the magnitude of a response that has a normal bell-curve distribution. Although this is a plausible explanation, we were intrigued by an alternative hypothesis.
More than 20 years ago, Benditt and Benditt (1) published a seminal but still-controversial paper in which they postulated that atherosclerotic plaques might be a form of benign neoplasia. Their hypothesis was based on the studies of atherosclerotic plaques of women who were heterozygous for glucose-6-phosphate-dehydrogenase and who, therefore, expressed both of the two major isoforms of the allele. Instead of finding the expected normal mosaic pattern of expression for these two isoforms, the large majority of atherosclerotic plaques contained SMCs expressing only one isoform. This finding was compatible with the idea that each plaque contained SMCs derived from the clonal expansion of a single cell. The authors postulated that the SMCs of an atherosclerotic plaque were the progeny of a single cell that had acquired a genetic mutation conveying a selective growth advantage, thereby leading to clonal expansion to form the plaque. They further suggested that the mutational genetic event could be due to a virus.
To examine further the basic tenets of the Benditts' hypotheses about atherosclerosis, we explored the possibility that restenosis may, at least in a subset of patients, be caused by some mutational process that conveys to an SMC, or group of SMCs, a selective growth advantage, such that when the cells are activated, as by injury, they will proliferate excessively and contribute to the development of restenosis.
We focused on p53 as the candidate gene in initializing the growth response. Wild-type p53 is a tumor-suppressor gene; its gene product is a nuclear protein that, in its hyperphosphorylated state, blocks progression of cells through the cell cycle. Mutations of this gene eliminate the suppressor function and constitute the most common genetic defect associated with a large number of human cancers (2). Cell transformation and the development of malignancies associated with p53 mutation require multiple genetic defects. We wondered whether, in at least some patients undergoing angioplasty, an isolated defect in p53 function in a subset of vascular-wall SMCs might contribute to excessive proliferation (without transformation) and, thereby, to restenosis.
Wild-type p53 protein has a very short half-life (about 20 minutes) (3). Partly as a result of this, its steady state concentrations are so low that the protein cannot be detected in normal cells by conventional immunohistochemical methods. In contrast, many missense mutations that impair the suppressor function of p53 and are associated with malignant transformation also prolong p53's half-life, leading to elevated protein concentrations and p53 immunopositivity. We, therefore, first determined whether restenosis tissue, obtained by atherectomy, contains SMCs that are p53-immunopositive. (Atherectomy involves advancing a catheter with a cutting implement at its end into a stenotic coronary segment. The lesion is then resected, and the catheter allows retrieval of the atherosclerotic tissue. Atherectomy can be used as an alternative to balloon angioplasty and is the procedure of choice when morphological characteristics of the lesion suggest balloon angioplasty will not successfully open the stenosis.)
Analysis of the lesions of 60 patients who had restenosis showed almost 40% of the lesions were immunopositive for p53. When we made this observation, it was commonly believed that p53 immunopositivity of cells in a malignant lesion was synonymous with p53 mutation. Indeed, David Lane of the University of Dundee in Scotland and one of the world's experts in the immunohistochemistry of p53, wrote, "Overexpression of p53 is synonymous with mutation." (4). So at this point in our studies, we really thought we had come upon an extremely important linkage between mutations in the p53 gene and an atherosclerotic-related process. On a molecular level, this observation would have connected the mechanisms responsible for cancer with those involved in atherogenesis.
Our next task was sequencing the genomic p53 DNA present in the restenotic tissue, and the original research team, Speir and Epstein, enlisted the aid of Rama Modali, who had previously studied p53 with Curt Harris at NCI. (We also received extremely helpful advice from Harris as well as from his associate Bill Bennett.) Expecting to find a mutant p53 gene in the atherosclerotic tissue, we were dismayed when sequencing revealed only normal p53.
This left us initially at a loss to explain the p53 immunopositivity. At about this time, however, we became aware of the work of several labs, including those of Peter Howley, then at NIH (5), and Arthur Levine at Princeton University (6), demonstrating that occasionally tumors are immunopositive for p53 despite the absence of mutations. Both Howley and Levine found that some of these p53 immunopositive-but-nonmutant tumors were triggered when wild-type p53 formed complexes with cellular (7) or viral oncoproteins (5), prolonging p53's half-life and steady state levels. These complexes, which also inactivate p53's suppressor function, are formed with oncoproteins encoded by DNA tumor viruses such as adenovirus, SV40, and Epstein-Barr virus. We wondered whether the p53 immunopositivity we found in the restenosis lesions was caused by a similar mechanism.
We then began to search for a candidate virus and settled on human cytomegalovirus (HCMV) as the most likely suspect because the literature was replete with studies suggesting that this herpesvirus plays a potential causal role in the genesis of atherosclerosis. A large percentage of individuals over age 50 have been infected with HCMV. Several studies have demonstrated HCMV sequences in the wall of atherosclerotic vessels. Marek's disease virus, a herpesvirus, produces lesions in chickens and Japanese quail very similar to those seen in human atherosclerosis (8). Evidence implicates a causal role of HCMV in the development of accelerated coronary atherosclerosis in cardiac transplant patients (9). Finally, other studies have shown cellular effects of HCMV that predispose infected cells to processes identified with atherogenesis, including potentiating DNA replication and mitotic activity (10); inducing the secretion of growth factors and the expression of cell adhesion molecules; and inducing defects mechanisms responsible for removal of cholesterol from cells.
At this critical juncture of our studies, we sought the advice and collaboration of Eng-Shang Huang, who was working at the University of North Carolina at Chapel Hill and who is one of the world's experts in HCMV. Also, Toren Finkel, a cardiologist and molecular biologist, joined the lab and began to play an important collaborative role in the project.
Our expanded team then started a series of studies designed to test the hypothesis that HCMV was playing a role in restenosis. We first analyzed the tissue on which we had performed our immunohistochemistry to determine whether HCMV sequences were present. We found a highly significant concordance between p53 immunopositivity and the presence of HCMV sequences, as determined by the polymerase chain reaction (PCR): 85% of the p53 immunopositive lesions contained HCMV sequences, whereas only 27% of the p53 immunonegative lesions contained HCMV sequences. The correlation between p53 immunopositivity and HCMV suggested that HCMV was not just an innocent bystander, present only because of the high prevalence of HCMV in the adult population. To test whether HCMV plays causative role, we performed an additional series of studies.
We first cultured smooth muscle cells derived from the lesions to determine whether the HCMV sequences present in the lesions were capable of expressing viral protein products. Immunohistochemical evidence indicated that IE84, one of the two major immediate-early gene products of HCMV, could be expressed. Moreover, whenever SMCs expressed IE84, they also were immunopositive for p53.
We next demonstrated that when cultured normal human SMCs were infected with HCMV, infection caused p53 immunopositivity, with remarkable temporal concordance between the appearance of IE84 and p53; moreover, the two proteins co-localized within cells. Then, searching for a functional interaction between IE84 and p53, we co-transfected expression vectors containing the genes encoding each of these proteins and a reporter-gene construct. We wished to determine whether IE84 could inhibit the demonstrated capacity of p53 to transactivate a promoter containing p53 binding elements placed upstream from the CAT reporter gene. We found such an interaction; the transactivational effects of p53 were markedly inhibited by co-expression of IE84. We also found that the two proteins were capable of physical association, demonstrating that they co-immunoprecipitated in a baculovirus-insect cell system.
These findings, in addition to the fact that herpesviruses can remain in a latent state in infected host cells for decades, suggested to us a new pathophysiologic model for restenosis development: in that subgroup of patients who have been exposed to HCMV, angioplasty-induced injury to the vessel wall reactivates latent HCMV, which in turn causes p53 inhibition and other cellular changes that predispose SMCs to proliferate. If HCMV plays such a role, the mechanisms underlying its actions must be complex because the virus is large and has more than 200 open-reading frames. Our results suggest that one key mechanism is the interaction between HCMV-encoded protein(s) and p53. And because of the many similarities between restenosis and atherogenesis itself, it is also possible that similar HCMV-mediated mechanisms might contribute to the initial development of arteriosclerosis.
If additional studies confirm this working hypothesis, we would speculate that the most likely role for HCMV would be as an additional, and perhaps potent, risk factor for the development of restenosis and atherosclerosis. It might play a role analogous to that of hypercholesterolemia -- not everyone with elevated cholesterol concentrations develops atherosclerosis, but the elevated concentrations predispose individuals to its development in the presence of additional risk factors.
We are currently pursuing several lines of research. Zhou Yi-Fu in our lab has just found that CMV-infected rats undergoing balloon injury of the carotid artery show greater neointimal response to the injury than do uninfected controls -- a finding that supports the hypothesis that HCMV contributes to the development of restenosis in patients. We are also gathering data in a collaborative, prospective study with Martin Leon at the Washington Hospital Center to determine whether patients with previous HCMV infection have an increased incidence of restenosis following successful angioplasty. Zhou and Tom Johnson are exploring other cellular mechanisms by which HCMV might contribute to atherogenesis. Esther Guetta is studying the molecular mechanisms by which the virus is reactivated from latency, and, with Tomoko Shibutani, we are exploring the role of free radicals in viral gene expression and trying to develop clinically useful inhibitors of such expression.Fig. Hypothetical model of human cytomegalovirus in atherosclersis and restenosis. Upper left: Blood vessel wall containing smooth muscle cells (SMCs), some of which are latently infected with HCMV. Upper right: Following exposure of the vessel wall to prologed hypertension, elevated cholesterol, substances contained in cigarette smoke, or other risk factors or injury, as in angioplasty, HCMV is reactivated and expresses many of its genes -- including immediate-early genes. Through multiple mechanisms, including inactivation of the tumor-supressor gene product p53, the proteins encoded by these genes augment the SMC response to injury, encouraging extensive SMC proliferation. The resulting mass of tissue compromises blood flow.
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