8 The Genomics of Restenosis

The Genomics of Restenosis

Thomas W. Johnson, ^BS c, ^MBBS , ^MRCP and Karl R. Karsch, ^MD

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INTRODUCTION

Interventional cardiology took off in 1977, with the development of percutaneous coronary balloon angioplasty (1). Despite immediate success in regaining vessel patency, long-term results were undermined by luminal loss secondary to the vessel injury induced by the balloon (2,3). A decade later a new technique involving stent deployment had been designed to overcome vessel recoil (4), initially, considered the major contributor to the loss of lumen diameter. Rather than ridding interventional cardiology of resteno- sis, a purely iatrogenic process, stents have shifted the focus of attention toward the phenomenon of intimal hyperplasia (5).

Now, a further 10 yr on and great excitement has been generated by the discovery of sirolimus’and paclitaxel’s capacity to inhibit the intimal response encountered follow- ing the deployment of intracoronary stents (6,7). A sceptic might speculate that in 10-yr time the focus will have shifted again, and further novel therapies will be in production targeting the latest culprit of restenosis unmasked by today’s technology. However, major changes in molecular biology technology have occurred in the last decade giving us new insight into pathophysiological mechanisms and thus offering us novel methods of diagnosis and therapy (8). It is quite obvious that the phenomenon of restenosis is secondary to a complex interaction between extrinsic factors of predisposition (e.g., diabetes, small vessels, long lesions, and so on) and a multiplicity of genes.

Without an idea of the hierarchy of communication, between genes and the extrin- sic factors, attempts at halting the process can only be governed by best guess, thus

From: Contemporary Cardiology: Essentials of Restenosis: For the Interventional Cardiologist Edited by: H. J. Duckers, E. G. Nabel, and P. W. Serruys © Humana Press Inc., Totowa, NJ

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diminishing any likelihood of abolishing restenosis. The advent of genomics has changed this, allowing investigators to observe gene function and to interrogate disease mechanisms at the molecular level. Consequently, the next decade will be an exciting time of new discovery. The purpose of this chapter is to introduce the new technologies available within the field of genomics and to relate this to how they might impact on the understanding and therapy of restenosis.

AN INTRODUCTION TO GENOMICS

Following its proposal in the 1980s, the human genome project has accelerated expo- nentially. The first major landmark came in June 2000 with the release of a “first draft”

of the genome, by which time “Genomics,” the scientific discipline of mapping, sequencing and analyzing genomes, was well established. Three years on and more than 100 organisms have had their genomes completely sequenced (see list www.ncbi.

nlm.nih.gov/entrez/query.fcgi?db=Genome), completion of the final draft of the human genome was announced in April 2003. Numerous subspecialities have evolved within this relatively immature field; structural, comparative, and functional genomics, and all are reliant on highly advanced computational analysis, which itself has evolved into the subspeciality of Bioinformatics (9). Figure 1 graphically demonstrates the interrela- tionship between the subspecialties of genomics.

In brief, structural genomics (10) represents the sequencing of the genome and ulti- mately will culminate in the construction of genetic, physical, and transcript maps of organisms. Mapping of genes using tandem-repeat polymorphisms allows comparison of the human genome against the genomes of model organisms and other vertebrates—

comparative genomics. Comparison with model organism genomes, for example, the

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fruit fly (Drosophila melanogaster) or nematode worm (Caenorhabditis elegans), both with well-characterized genomes, can aid the interrogation of distant, yet related human genes with unknown function.

Additionally, comparison of the human genome with those from other vertebrates and mammals demonstrates the highly conserved nature of many genes. Subsequently, the gene map from one species can be used to reveal the location of a target gene in a related species. Furthermore, comparison of nontranscriptional domains can highlight important sequences, conserved in many species, attributed with roles in the regulation of gene expression. Functional genomics relies on the data gathered from structural and comparative analysis of the genome; it focuses on gaining an understanding of the function of genes and gene products.

Therefore, genomics—the interrogation of the genome—potentially allows the link- ing of gene expression with biological behavior. The shift in emphasis from studying single genes to assessing the whole genome simultaneously has led to the creation of vast databases of information, allowing collaboration in the generation of gene struc- ture, the mapping of genes within the genome, assessment in gene functionality, and the postulation of the interrelations of “clusters” of genes. Linking of gene expression to biological behavior requires more than just an explicit understanding of the genome, as ultimately it is the gene products that act as effectors. The mere presence of a gene does not reflect functionality therefore much energy has been invested in the assess- ment of both the transcriptome, transcriptomics, and the eventual pool of effector pro- teins, proteomics.

“Transcriptomics” falls within the auspices of functional genomics and is covered within this review, however the vast discipline of proteomics is now established as a research field in its own right, the proteomics of restenosis is covered separately within this book. The combination of genomics, transcript profiling, and proteomics will give insight into the mechanisms of disease, allow identification of diagnostic and prognostic markers and highlight suitable targets for therapy. These disciplines remain relative new- comers to the field of molecular biology and consequently the associated technology and the understanding of the principles underpinning these processes are rapidly advan- cing—new molecular biology techniques, reliant on the colossal processing power of computers, have allowed the profiling of 100–1000s of genes/transcripts concurrently and therefore opened up this exciting field of science to widespread application.

GENOMIC TOOLS

The tools available for genomic and transcriptomic study have been revolutionized since the first suggestions of sequencing the human genome in the 1980s.

Sequencing Tools

As the relative importance of genomic study and the suggested uses of genomic infor- mation escalated so too did commercial interest. The creation of the first commercial genomics company, Celera Genomics, in 1998, potentially jeopardized the international, and cooperative approach taken by the human genome project. Instead, commercial competition galvanized the public sector and resulted in a boost in public spending and triggered an unprecedented acceleration in the production of raw sequence. Until 1998 approx 6% of the human genome had been sequenced within 3 yr 90% was complete.

Sequencing is still achieved using methods first described by Sanger in the 1970s (11).

The genome’s size prevents analysis in its entirety; therefore it is broken into smaller pieces for sequencing. Genome fragments of about 150 kB are inserted into bacterial artificial chromosomes for cloning. The clones are then further fragmented or “shot- gunned” to produce manageable segments of DNA for sequencing, i.e., about 1500 base pairs (bps). Repeated shotgun fragmentation of the clones allows piecing together of the entire clone following sequencing (see Fig. 2).

Sequence Polymorphisms and Association Studies

Following completion of the sequencing of the human genome, mapping of genes onto chromosomes has been facilitated by detection of sequence polymorphisms (poly- morphic DNA markers) (12). The presence of polymorphic DNA markers located throughout the human genome has facilitated the mapping of genes onto other individ- uals and species genomes. Rare Mendelian disorders, attributable to single gene defects can be highlighted by careful analysis of short-tandem repeat polymorphisms located close to the target gene in unison with detection of disease phenotype, thus permitting identification of causative mutations.

However, restenosis like atherosclerosis and hypertension is a complex disorder involving the interaction of several genes, and consequently harder to decipher. Single nucleotide polymorphisms (SNPs), frequently occurring inherited sequence polymor- phisms (about every 300–500 bp) might possibly reveal genetic associations with more

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Fig. 2. A flow diagram, illustrating the principle of “shot-gun” cleavage, used to facilitate genomic sequencing.

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complex diseases. It has been postulated that a subset of SNPs contribute to the genetic risk of complex disorders (13). Genes of relevance to a particular disorder can be sequenced and the SNPs present within the region noted—SNP association study.

Comparison of the same chromosomal region in numerous individuals demonstrates patterns of SNP distribution—individuals with similar clustering of SNPs share the same haplotype. Haplotypes represent descendence from a common ancestral chromo- some. For example, it is possible that a particular haplotype is more likely to associate with in-stent restenosis, thus detection would be a useful marker of risk and thus facil- itate extra measures of precaution.

Gene Function Assessment

The combination of new high-throughput technologies and a complete draft of the human genome enable investigators to interrogate gene function in two ways. Either, through manipulation of a specific gene, by mutation or altered expression, to assess the effect on phenotype, or, the genetic profile of various phenotypes can be compared to reveal genes involved in characterizing the phenotype (14). The tools available to assess function either through gene manipulation or profiling of phenotypes have advanced rap- idly since the first suggestion of sequencing the human genome in the 1980s. The funda- mental techniques for functional genomics are described in the following subheadings.

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Chemical Mutagenesis remains an important method of elucidating gene function.

By its nature it is obviously limited to animal models, commonly the zebra fish, a vertebrate, and the mouse, a mammal. Commonly, males are exposed to a mutagen that induces mutations at high frequency throughout the genome, before mating with a wild-type female. The first generation progeny are then phenotypically assessed for dominant mutations, this may include simple observation, morphological analysis, bio- chemical tests, and genetic sequencing. Homozygotes for mutations can be generated by mating first-generation males with wild-type females and then with the female prog- eny of this cross. See Fig. 3, taken from Rubin and Tall’s review entitled “Perspectives for vascular genomics” (12).

RNA Interference Technology

A new technique of RNA interference (RNAi) allows silencing of individual genes by post-transcriptional disruption of gene function. Loss of all messenger RNA (mRNA) produced by a gene effectively silences it, a project is underway to produce human cell lines each with a silenced gene, the first phase has focused on approx 8000 genes but the intention is to have a library of cell lines for all 30,000 + genes within the genome by mid-2004. An obvious drive for this new technique is the ability to silence genes required for the proliferation of cancerous cell lines. Restenosis has often been likened to a mitotic process and current best therapy utilizes antimitotic agents, there- fore RNAi technology may be of use in cell lines obtained from restenotic tissue sam- ples. However, it is well known that only limited information can be gleaned from isolated cell lines and consequently RNAi technology cannot give a complete answer.

The disadvantage of mutational methods for functional gene assessment is the

reliance on the investigator to select an individual gene of interest. As mentioned ear-

lier, the complex, and as yet not fully understood, nature of restenosis in addition to the

existence of more than 30,000 genes within the human genome makes gene selection

more of a lottery than a scientific process. If the understanding of the disease process was complete, then a definitive therapy would be rapidly achievable. However, these techniques have arrived before the full understanding of the complex process of in-stent restenosis, consequently investigators have utilized the technology to assess their favored targets. It is possible that a cure for restenosis might be found using these technologies alone but the chance is slim.

The emergence of high-throughput technologies capable of gene, transcript or protein profiling enable assessment of phenotype at a molecular level, offering up the opportunity to define mechanisms of disease process, markers of risk, disease progression or regres- sion, and most importantly specific targets for therapy and possibly cure. Therefore, the use of mutational models, RNAi silencing, “knockout” models and vector-driven gene over- and under-expression would be best used in confirming effect detected by these more widespread assessments of the entire genetic milieu within its environment.

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The genome varies very little between cells, however, levels of gene transcription within cells are sensitive to multiple intrinsic and extrinsic factors, thus study of the transcriptome acts as a good meter of gene expression. Transcriptomics, a component of functional genomics allows investigation of the complex interrelationships between genes and facili- tates the generation of expression profiles for various phenotypes that can be compared with revealed putative mechanisms of physiological and pathological functions.

Differential display and subtractive hybridization, both well-established techniques in molecular biology can be utilized to assess expression (15), however, they have

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Fig. 3. A schematic, illustrating the theoretical use of genome-wide mutagenesis in the elucidation of gene function.

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been superseded by newer high-throughput technologies facilitating rapid, repro- ducible, and widespread comparison of gene expression, for example, complementary DNA (cDNA) and oligonucleotide microarray systems, and serial analysis of gene expression (SAGE) (16).

Microarrays

Microarrays (15,17,18) require the selection of a known library of genes for profil- ing. Fragments of double-stranded cDNA or shorter single-stranded oligonucleotide sequences are spotted onto a solid support, usually glass, silicon, or nylon, using pins or an ink-jet printer. Advancing technology has allowed increasingly dense array pro- duction, currently more than 20,000 sequences can be displayed per array, and in the near future it is possible that the entire genome will be displayed on a single chip.

Microarray analysis requires labeling of RNA extracted from sample tissue with a fluo- rescent or radioactive probe. If the quantity of RNA extracted from the test tissue is limited then polymerase chain reaction (PCR) amplification is permitted, however, it can potentially distort subsequent profile analysis.

Following labeling of the test RNA/cDNA, the sample is hybridized to the micro- array—complementary sequences bind to the solid support whereas nonmatching sequences are washed off (see Fig. 4). An expression profile can be assessed by the use of two individual probes for test and control RNA extracts and subsequent comparison of fluorescence between the two samples. Therefore, genes can be assessed as over- expressed, repressed, or equivalent.

Serial Analysis of Gene Expression

SAGE (15,19,20) requires no previous knowledge of genetic sequence, and is there- fore capable of detecting novel genes. An initial PCR amplification step allows the analysis of small samples (50–500 ng of mRNA or 5–50 µg of total RNA). SAGE relies on the ability to identify mRNA transcripts by a unique short oligonucleotide sequence located close to its 3 ′ poly-A tail, see Fig. 5.

Briefly, mRNA is purified from total cellular RNA by passage through solid phase oligo(dT) magnetic beads. Subsequently, cDNA is synthesized from the mRNA and digested with a restriction enzyme, the resulting 3 ′-most fragments (anchored to the dT beads) are then ligated with two different linkers containing PCR primer sites. The linkers possess a recognition site for a type IIS restriction enzyme, which cuts 10-bp 3 ′ from the anchoring enzyme recognition site, thus producing unique oligonucleotide sequences for each mRNA transcript. The 10-bp tags or SAGE tags, bound to linkers are then ligated to form “ditags” and PCR amplified.

Subsequently, the linkers are cleaved and the ditags are then serially ligated into concatemers, cloned, and sequenced using an automated sequencer. Software allows identification of sequences and measurement of their relative frequency. Obviously, SAGE is limited by the accuracy of sequencing; misplacement of a single base will result in a loss of data. Equally, any variations in “ditag” amplification between linker sets will affect any data relating to frequency of expression.

Although a 10-bp sequence tag should theoretically give rise to more than one million

different sequence combinations and thus be adequate to discriminate between the

approx 35,000 genes within the human genome, not all genes within the unannotated

genome can be detected. Therefore a modification that extends the length of the SAGE

tag has been introduced to overcome this problem—”LongSAGE.” LongSAGE utilizes

a type IIS restriction enzyme capable of cleaving cDNA 17-bp 3 ′ of the anchoring

site—these lengthened tags heighten sensitivity and consequently enable matching to the unannotated genome.

Currently, only sample availability and the ethical issues surrounding tissue retention limit profiling of genes, transcripts, and proteins. As the technology advances sample size is becoming less of an issue, it is now possible to profile genetic material from a single cell! Thus, phenotypes can be defined at a molecular level using profiles derived from human tissue, the major limitation now lies with the vast quantity of data generated by such high-throughput techniques, therefore much time is being spent advancing compu- tational power—bioinformatics has become a separate field within molecular biological science (9), and will not be covered within this review.

APPLICATION OF GENOMICS TO RESTENOSIS

Hundreds of studies have been undertaken in an attempt to find methods to sup- press the response to experimental vascular injury, most commonly by balloon over- stretch injury to rodent, rabbit, or pig arteries (21–23). Although many have succeeded in the suppression of an injury response in animal models, very few have translated into viable clinical therapies. This discrepancy highlights the existence of fundamen- tal differences between animal models and clinical reality (24). First, there is great

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Fig. 4. A flow diagram, illustrating the processes involved in microarray systems. Sample RNA is labeled with a biotin probe (B) before hybridization to the microarray, which carries thousands of known nucleic acid probes. The quantity of label associated with each probe location on the array permits the study of gene expression.

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disparity between the use of therapies in juvenile animals, undergoing injury of healthy arteries, against application in an aged human population with severely diseased ves- sels. Second, the injury induced in small animals varies markedly from that elicited in humans, i.e., endothelial denudation rather than extensive endothelial and medial dis- ruption. Use of swine and nonhuman primates combined with coronary overstretch injury has reduced the gap between animal models and reality; however, size, cost, and ethical issues prevent their widespread use. Genomics offers new uses for such animal models and potentially may refine and reduce the scientific communities reliance on nonhuman experimentation. Rather than being the “test bed” for best guess therapy, genomic interrogation of the human restenotic process may provide specific targets for treatment; these can then be tested in well-established animal models of restenosis before human trailing.

Genomics offers enumerable avenues of new biomedical research, however, within the field of restenosis most work, to-date, has concentrated on the development of risk markers, through assessment of SNP and haplotype associations within populations susceptible to in-stent restenosis and the probing for genes involved in the restenotic process, using methods of profiling gene and transcript expression, from which plat- form further exploration of target genes or clusters of genes can be undertaken.

EXISTING DATA WITHIN THE FIELD OF RESTENOSIS GENOMICS Unlike other cardiovascular pathologies, a small animal genetic model of restenosis cannot be readily produced. As previously discussed, great disparity exists between the nature of injury caused by balloon inflation in small animal and human arteries, and

Fig. 5. A schematic representation of the SAGE protocol. (Please see insert for color version.)

expense, and ethics prevent large-scale experimentation in large animal/nonhuman primate models. Therefore, early investigation of restenosis at the genomic level has con- centrated on finding who is the most susceptible and which genes are responsible in those individuals with restenosis. Gene profiling of tissue extracted from restenotic lesions has generated vast amounts of data and offered up numerous targets for therapy, this is dis- cussed at length in the Chapter 9.

Much interest has been shown in attempting to highlight individuals at risk of restenosis before percutaneous coronary intervention. Numerous haplotype and SNP association studies have been performed, only a few associations have been found.

Such studies require the investigator to select a gene of interest and assess the effect of known polymorphisms on phenotype. To date, positive associations include homo- zygosity for the T-allele of the PvuII polymorphism of the α-estrogen receptor in women (25), a 6A6A metalloproteinase-3 genotype (only with balloon angioplasty) (26), E- selectin Ser128Arg genotype (27), and angiotensin converting enzyme gene DD geno- type (28). However, negative associations include 677C/T and 1298A/C polymorphisms of methylenetetrahydrofolate reductase gene (29), a polymorphic marker of the gene encoding interleukin-1 receptor antagonist (30), and gene polymorphisms of tumor necrosis factor- α, lymphotoxin-α, and interleukin-10 (31).

FUTURE DIRECTIONS

Genomics potentially holds the key to the appropriate targeting of therapy at a molecular/genetic level. Cardiovascular genomics remains in its infancy, moreover, restenosis genomics is “embryonic”—in September 2003 only a handful of papers addressing in-stent restenosis genomics could be found on an extensive PubMed search. As SNP and haplotype association studies continue, increasingly sensitive screening tools will be developed enabling interventional cardiologists to tailor inter- vention to the individual not just the lesion type. The emergence of microarray tech- nology capable of screening the entire human genome will allow the temporal genetic profiling of the restenotic process to be realized. Subsequently, genes implicated in the process can be interrogated with regards their function and pathological role, thus developing the understanding of restenosis. In parallel with the advancing understand- ing of the pathological process, new targets for therapeutic intervention will arise.

Rather than seeking a cure for restenosis, genomics may offer a method of prevention.

However, amidst the excitement surrounding genomics and its potential, it must be remembered that assessment of the genome only reveals part of the story, it must not be considered on its own but instead in combination with analysis of the transcriptome and proteome. Gene expression and gene function cannot be used as direct surrogates of transcript or protein function.

SUMMARY

• SNP association studies in large cohorts of patients undergoing percutaneous coronary intervention, combined with phenotype may lead to the detection of markers of risk.

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High-throughput technologies have enabled investigators to interrogate restenotic gene expression without previous knowledge of the genes involved and despite small sample sizes.

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Expression profiling can highlight genes of importance in the restenotic process.

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Comparison genomics has an important role in deriving function for unknown genes through mapping onto model genomes or those of related species.

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Genomics must be assessed in combination with information from the transcrip- tome and proteome otherwise it is meaningless.

GLOSSARY Genome

The complete “library” of genetic information for an individual, containing more than three billion bp, distributed across 22 autosomal chromosomes (1–22) and two sex chromosomes (X and Y). The genome contains, in encrypted form (>30,000 genes), all the information required to create that individual.

Transcriptome

Despite almost all cells containing the required information to translate its DNA into mRNA transcripts for all human genes, mRNA production varies between cells and dif- fering environments. Gene expression, i.e., mRNA transcription from DNA is con- trolled by transcriptional factors present within the nucleus, which interact with regulatory regions bordering the coding regions genes (nontranscriptional domains).

Therefore, assessment of mRNA production from specific cell types in defined condi- tions results in the creation of a transcriptome.

Proteome

The mere presence of mRNA cannot predict the resulting pool of proteins. The mRNA transcribed from DNA needs modification before translation into protein, it contains coding sequences (exons), flanked by irrelevant sequences (introns). Splicing (removal of introns to join exons) can vary and thus result in the production of different protein products. Therefore, the proteome refers to the effective protein produced from a defined transcriptome.

Genotype

Generally refers to the complement of genes carried by an individual. Every individ- ual carries two copies of each gene, one inherited from their father and the other from their mother. Variations in genes exist (alleles) and these can be defined through inter- rogation of the genes sequence. Homozygosity relates to the carriage of two identical genes whereas heterozygosity refers to the presence of two variants of a single gene.

Phenotype

Variations in genes (alleles) and gene expression result in observable differences between individuals and can predispose to disease. The phenotype is defined as the observed characteristics of an individual. In part, the phenotype can be predicted by an individuals genotype.

Polymorphisms

Comparison of a single gene between two individuals will reveal variations in

nucleotide sequence, these can impact dramatically on protein formation if located

within coding regions of the gene. Sequence changes within the noncoding (intron)

regions are less likely to result in changes to protein production. Frequently occurring

variations in nucleotide sequence are called polymorphisms, alleles with a frequency of

less than 1% or sequence changes directly related to disease are known as mutations (32). Nucleotide sequence variations can be single or multiple.

Haplotype

Haplotype refers to patterns/clusters of polymorphisms within a gene sequence, detected in subsets of a population. It is a direct consequence of descendence from a common ancestral chromosome.

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