© 2000 American Heart Association, Inc.
Editorial |
Genes, Matrix, and Restenosis
From COR Therapeutics Inc, South San Francisco, Calif, and the Cardiovascular Division, Department of Medicine, Stanford University School of Medicine, Stanford, Calif.
Correspondence to James N. Topper, MD, PhD, Vice President, Biology, COR Therapeutics Inc, 256 E Grand Ave, South San Francisco, CA 94080. E-mail jtopper{at}corr.com
Key Words: : genomics • vascular biology • extracellular matrix • quantitative RT-PCR
One of the major advances in vascular biology over the last 2 decades has been the realization that the vessel wall is an active, integrated tissue composed of multiple distinct cell types coupled to one another by complex regulated interactions. Blood vessels are capable of sensing their milieu and integrating information from diverse stimuli, both humoral and biomechanical. In response to alterations in these signals, such as with mechanical injury or sustained hemodynamic changes, the vessel is capable of adapting itself, an effect known as vascular remodeling. This process can result in a spectrum of changes within the vessel wall, ranging from alterations in the cellular and extracellular (matrix) content of the wall to changes in the reactivity and functional properties of the vessel. The advent of techniques that allow more efficient discovery of the genes involved in these processes as well as a description of their temporal patterns of expression is now providing a powerful opportunity to begin to understand vascular remodeling at the molecular level.
There are now multiple approaches aimed at understanding the
repertoire of gene expression in a given biological context. These
range from techniques such as differential display1 and
subtractive cloning strategies,2 which are designed to
identify unknown genes whose expression is modulated under the
conditions being examined, to transcriptional profiling
strategies,3 in which a set of known sequences are
prepared as an array and their patterns of expression are determined in
parallel. In general, the latter have the advantage of allowing one to
identify global patterns of gene expression for a defined cohort of
genes, whereas the former allow for the discovery of new species that
may not have been previously identified or are expressed at very low
levels. Despite the efficacy of these techniques when applied to
relatively uniform populations of cells in culture,4 5 6
they all present challenges when applied to the investigation of
complex tissues such as the vasculature. These include the need to
balance the sensitivity of the technique with the availability of
well-defined (both anatomically and pathologically) and high-quality
(ie, minimal necrolysis) tissue samples, as well as the desire to
assess the reproducibility of any findings encountered. That is, are
the effects seen consistent from individual to individual, a
question that necessarily requires multiple, well-matched samples. In
this issue of Arteriosclerosis, Thrombosis,
and Vascular Biology, Tai and colleagues7
describe a series of experiments in which they utilized real-time
quantitative reverse transcription–polymerase chain reaction (RT-PCR)
to examine the expression of 
80 genes in the rat model of carotid
injury. By choosing to examine an interrelated set of genes involved in
extracellular matrix biology in a well-defined and reproducible model
of vascular injury/remodeling, these investigators have created a
system that successfully addresses many of the challenges inherent in
these studies. Given the likely importance that extracellular matrix
(ECM) metabolism plays in vascular remodeling and the
relative paucity of data in this area,8 these studies
represent an important addition to our understanding of this
complex process.
These investigators have examined the detailed temporal patterns of expression of 68 of 81 genes that were chosen to represent a subset of genes involved in the biology of the ECM. These include genes encoding various proteoglycans, collagens, integrin subunits, extracellular proteases and their inhibitors, and components of the transforming growth factor-ß superfamily of growth factors. (The complete list can be found online at www.atvbaha.org) The highly quantitative and reproducible data generated by the real-time RT-PCR over the 28-day experimental protocol were then analyzed by a "clustering" algorithm that allowed them to discern 4 general, but definably discrete, patterns of gene expression manifested by the majority of these genes.
Interestingly, of all of the time points examined, the first day post injury (day 1) demonstrated the most profound (in terms of absolute number of genes) alterations in gene expression. This speaks to the severity of the original insult, a combination of mechanical and denudation injury that clearly rapidly alters the biology of the affected vessel. The nature of the genes unregulated at this time point indicates both the rapid recruitment of inflammatory cells to the injured vessel (eg, matrix metalloproteinase [MMP]-8, a neutrophil metalloproteinase) as well as the induction of genes known to be modulated by mechanical strain (eg, tenascin-C). However, it is particularly interesting that the predominant pattern of gene expression characterized was a rapid and profound downregulation at this early time point. This pattern was manifested by genes encoding some of the major ECM constituents, including various collagens, proteoglycans, and elastin. This striking finding suggests that cells of the vessel wall are normally actively expressing these genes at a significant level and that the immediate response to the acute injury is to shut these genes down. This initial phase is followed by "waves" of gene expression that appear to reflect both the coordinated upregulation of genes whose expression was not immediately altered by the injury and a return to baseline (or in some cases, a subsequent absolute induction) of the genes that were initially suppressed. This phenomenon of sequential, and presumably coordinated, waves of gene expression has been observed in a number of other biological systems responding to injurious or pathological stimuli.6 It seem likely that this is a manifestation of integrated transcriptional control mechanisms designed to orchestrate these patterns and suggests that interventions aimed at manipulating these transcriptional mechanisms may one day be utilized to therapeutically modulate vascular remodeling. Finally, at day 28 post injury, a time point when the gross morphological appearance of the vessel appears stable, the profile of gene expression is still markedly different from that seen at baseline. Although this is not particularly surprising, since both the cellular and extracellular makeup of the vessel and thus, the network of interactions and communications between cells, have been markedly altered, it may have important implications for the human condition. For many clinical angioplasties, the injury may in fact be more severe than that described here (eg, stent placement with subsequent high-pressure inflations or brachytherapy), and thus, the resulting alterations and remodeling responses may be significantly more prolonged than is currently appreciated.
Although this study has revealed important and novel information, there are a few limitations of this kind of approach that should be considered. The first, as the authors point out, is that the patterns of gene expression seen could be due to changes in the number of cells present or alterations in the number of transcripts per cell. For instance, it is hypothesized that upregulation of the presumed neutrophil protease MMP-8 is due to neutrophil infiltration and not to ectopic induction of this gene in a resident cell type, a fact that remains to be demonstrated. In addition, the question of precisely where in the vessel wall the changes in ECM gene expression described here are actually occurring is not addressed. Is the expression of these genes being altered in the neointima, media, or adventitia? These important questions need to be addressed by immunohistochemical or in situ hybridization approaches.
Although the limitations of the rat carotid model of restenosis/vascular injury as a paradigm for the human condition have been widely recognized,9 I believe that these data attest to its continued utility as a research tool. The defined (and novel) patterns of gene regulation described here would likely not have been discernible from human tissue specimens because of the inability to adequately match and control for variations in these tissues. In addition, the use of a highly sensitive technique (quantitative RT-PCR, which requires small amount of RNA input) allowed this sizeable group of genes to be evaluated as independent analyses in multiple independent individuals at each time point, thus ensuring the reproducibility of the patterns described. As a result, I believe that this system is now ideally suited to begin to analyze the effects of therapeutic interventions that may impact vascular remodeling and restenosis, such as growth factor or MMP inhibition.10 It is these types of studies that promise to identify the genes that are mechanistically linked to restenosis and/or its response to therapy. And it is precisely these genes that represent attractive targets for future diagnostic and therapeutic applications in human vascular disease.
Acknowledgments
The author would like to thank Dr Neill Giese and Dr David Phillips for helpful discussions in the preparation of this manuscript.
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