9 Gene-Expression Profiling and Restenosis

Gene-Expression Profiling and Restenosis

Dietlind Zohlnhöfer, ^MD

and Franz-Josef Neumann, ^MD

C

ONTENTS

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NTRODUCTION

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ECHNICAL

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PPROACH TO

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ENE

-E

XPRESSION

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ROFILING

PCR-B

ASED

A

PPROACHES

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LUCIDATION OF

M

ECHANISMS OF

R

ESTENOSIS

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N

V

ITRO

E

XPERIMENTS

A

NIMAL

E

XPERIMENTS

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TUDIES IN

P

ATIENTS

T

HERAPEUTIC

D

EVELOPMENT

P

ERSPECTIVES

R

EFERENCES

9

INTRODUCTION

A central goal of molecular cardiology is the characterization of molecular mecha- nisms regulating complex cardiovascular functions and the identification of disease- associated gene-expression patterns. Gene transcription is the most important regulatory mechanism by which a phenotype and functional state of a cell and a tissue is deter- mined. Therefore, qualitative and quantitative assessment is the first step into the nature of biological processes. When small cell numbers are used, messenger RNA (mRNA) abundance is easier to investigate in a comprehensive manner than protein expression.

The traditional approach to the elucidation of mechanisms of the disease was to develop a pathophysiological hypothesis and to identify genes, whose dysregulation might be involved in the pathogenesis of the disease. The importance of these kinds of candidate genes was verified further in in vitro and in vivo experiments. This kind of analyses led to identification of biochemical or cellular interactions, which are essen- tial for the homeostasis of the organ or cell. So far, the approach has been unsystem- atic and was based on existing pathophysiological concepts. New advanced methods of molecular biology allow the retrograde approach. Instead of testing if the dysregula- tion of single candidate genes result in the disease, it is now possible to analyze the

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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whole genome to identify known or so far unknown genes (and their variants) that are related to the disease.

TECHNICAL APPROACH TO GENE-EXPRESSION PROFILING The cluster of genes that are transcribed from the genomic DNA in a tissue or cell—

the so called transcriptome—and the changes of gene expression under certain physio- logical or pathophysiological circumstances give insight into the biological function of the encoded proteins. Measurements of gene expression have the advantage of provid- ing all available sequence information for any given experimental design and date inter- pretation in pursuit of biological understanding. By comprehensive gene-expression profiling, the identification of new therapeutic options—and maybe an individual ther- apeutic management of patients is possible in the future. Similarly, transcriptome analy- sis might lead to new diagnostic indicators and prognostic markers. Additionally, investigating the effect of drugs on the gene-expression pattern of vascular cells can provide information about regulatory mechanisms and bioactivity. Therefore, this research tool has the potential to revolutionize the understanding of pathophysiological mechanisms of cardiovascular diseases.

With the completion of the human genome project and the introduction of technolo- gies such as DNA microarrays and laser microdissection, many fields in biology and medicine await the application of comprehensive gene-expression analyses of specific cell types isolated from defined tissues. There are various methods available to study differential gene expression including subtractive hybridization, differentential dis- play, sequential analysis of gene expression, representional difference analysis, DNA array technology, and polymerase chain reaction (PCR)-based approaches (Fig. 1).

Currently, PCR-based approaches in conjunction with array technology are most com- monly used.

PCR-BASED APPROACHES

Small volumes (<300 cells) such as atherectomy specimens from patients can be successfully analyzed by the PCR-based approach (1,2). The authors used a method that had been optimized to determine gene-expression profiles of single disseminated tumor cells (3). Briefly, tissue specimens or cells are lysed and Dynabeads Oligo (dT)

₂₅

were added to extract mRNA. Afterwards mRNA is reverse transcribed using a random primer [5 ′-(CCC)

₅

GTC TAG A (NNN)

₂

-3 ′] and a tailing reaction is performed using dGTP. Complementary DNA (cDNA) can then be amplified by PCR with the so called CP2-Primer [5 ′-TCA GAA TTC ATG (CCC)

₅

-3 ′]. Aliquots of 25 ng of each amplified cDNA are labeled with digoxigenin-11-dUTP during PCR.

Clontech cDNA arrays (www.clontech.com) are prehybridized overnight and dena- tured, labeled probes are added to the hybridization solution and incubated for 48 h.

Detection of filter bound probes is performed according to the Digoxigenin detection system (Roche Diagnostics Gmbh, Germany). Developed films are scanned and ana- lyzed using the array vision™ software (Imaging Research Inc. Great Britain).

Background is subtracted and signals are normalized to nine housekeeping genes pres- ent on each filter, whereby the average of the signals of the housekeeping genes is set to one and the background to zero.

Differences between the two groups can be analyzed by a diversity of approaches

ranging from descriptive analyses (e.g., difference in median densitometric signal

intensity by a factor of 2.5 at a descriptive p value ≤0.05) to more complex statistical methods for cluster identification (4).

There are numerous modifications of this technique, which share the essential steps of the experimental setup, but are different in many details of mRNA preparation and amplification as well as array technololgy. The latest generation of arrays, such as the Affymetrix

^TM

(Santa Clara, CA) arrays, are miniaturized and can assay more than 60,000 oligonucleotides on one chip.

ELUCIDATION OF MECHANISMS OF RESTENOSIS

Restenosis is the most important limitation of percutaneous angioplasty procedures.

Although stent implantation reduces the risk of restenosis compared with other percu- taneous treatment modalities, angiographic restenosis rates continue to range around 30% (5,6). More than 90% of the late lumen loss after stent implantation is caused by neointima formation (7). Neointima formation is considered an arterial healing response, which is initiated by dedifferentiation of vascular smooth muscle cells (SMCs), followed by emigration and proliferation with subsequent elaboration of abun- dant extracellular matrix (8–10). The current knowledge is mainly based on

1. Histological analysis of vessel tissue from patients that have passed away;

2. Animal models in general;

3. The use of specifically designed “knockout mice;” and 4. In vitro experiments, for example, with cell cultures.

Therefore, gene-expression analyses of SMCs cultured under pathophysiological con- ditions or of animal or human samples from neointima after vascular injury were needed.

Fig. 1. Protocol for PCR-based gene-expression profiling. The basic goal of the protocol is to intro- duce two binding sites of the PCR primers into all transcripts allowing alike amplification of each transcript. The first primer-binding site consists in a flanking region that lies at the 5′-end of the ran- dom cDNA synthesis primer, the second primer-binding site is introduced trough a tailing step. After lysis of frozen tissue, mRNA is isolated and reverse transcribed. Subsequent to the tailing reaction, cDNA was amplified and labeled with digoxigenin by PCR. Denatured, labeled probes are hybridized to cDNA arrays and filter bound probes are detected by chemiluminescence.

IN VITRO EXPERIMENTS

In vitro, mechanical stress leads to responses in SMCs that are similar to those after balloon injury (11). The first study that analyzed the molecular response of SMCs to mechanical stress systematically by gene-expression profiling using cDNA arrays was reported by Feng et al. (11). This group investigated the expression of 5000 genes with putative relevance to neointima formation. They found that only three transcripts were induced more than 2.5-fold: cyclooxygenase-1, tenascin-C, and plasminogen activator inhibitor-1. Downregulated genes were P-selectin ligand, interleukin (IL)-1 β, matrix metalloproteinase-1, and thrombomodulin (11). Schaub et al. (12) demonstrated that induction of apoptosis by Fas/FADD-activation mediates a specific mRNA expression program of inflammatory genes in SMCs. This included upregulation of IL-8, mono- cyte chemolactic protein-1, plasminogen activator inhibitor type 2, IL-6 growth- regulated protein-1, and IL-1a. The inflammatory responses increased the recruitment of macrophages to the site of vessel injury in a rat model (12).

ANIMAL EXPERIMENTS

In a systematic approach, Geary et al. (13), analyzed the gene-expression pattern of neointimal SMCs 4 wk after aortic grafting in cynomolgus monkeys. They compared these expression patterns with those of SMCs from healthy aorta and vena cava (13). Thereby, the authors were able to characterize gene expression events associated with neointima formation and to provide the basis for novel hypotheses. Only 13 genes were upregulated in neointimal SMCs compared with aortic SMCs, whereas 134 genes were downregulated.

For example, specific collagen subtypes, like collagen-I, -III, -VI, and -V and other matrix genes like versican were upregulated in neointimal SMCs emphasizing the pivotal role of extracellular matrix synthesis in the pathogenesis of neointima formation (13).

STUDIES IN PATIENTS

Despite abundant animal data, molecular mechanisms of neointima formation had been investigated on only a limited basis in patients. Therefore, the authors sought to establish a method for profiling gene expression in human in-stent neointima (2). As an initial step, the authors were able to demonstrate that gene-expression patterns of human neointima retrieved by helix-cutter atherectomy can be reliably analyzed by cDNA array technology. The expression of 2435 genes in atherectomy specimens and blood cells of patients with in-stent restenosis, normal coronary artery specimens, and cul- tured human SMCs were investigated (1). The tissue retrieved from in-stent restenosis by helix-cutter atherectomy exhibited known gene-expression patterns of neointima, such as the upregulation of cyclooxygenase-1 or the downregulation of desmin (2).

Additionally, previously unknown gene expression events of neointima, including downregulation of mammary-derived growth inhibitor and upregulation of FK506 bind- ing protein 12 were discovered. Of the 223 differentially expressed genes, 37 genes indicated activation of interferon (IFN)- γ signaling in neointimal SMCs (1). In cultured SMCs, IFN- γ inhibited apoptosis. Genetic disruption of IFN-γ signaling in a mouse model of restenosis significantly reduced the vascular proliferative response. Therefore, the data suggest an important role of IFN- γ in the control of neointima proliferation (1).

Zhang et al. (14) analyzed differential gene expression in cultured SMCs explanted

from neointima from human in-stent restenosis and compared it with the gene-expression

pattern of cultured SMCs from aorta and coronary artery from patients undergoing renal or cardiac transplantation. They identified 32 genes that were upregulated in SMCs from human neointima. Consistent with the authors gene-expression analysis of human neointima from in-stent restenosis (2), they also described upregulation of thrombospondin-1.

THERAPEUTIC DEVELOPMENT

Gene-expression profiling in conjunction with current knowledge of pathophysio- logical role of the genes analyzed may help identify novel therapeutic targets. For exam- ple, thrombospondin-1 that was found to be upregulated in human neointima is involved in SMC proliferation and migration (15). Accordingly, blockade of thrombosponsin-1 by specific antibodies reduced neointima formation in balloon-injured rat carotid arteries by decreasing the number of proliferating SMCs (16). Thus, the consistent results from the independent gene-expression analyses provide a rationale to test antibody blockade of thrombospondin-1 in humans.

Another example of this paradigm are the findings regarding upregulation of FKBP12 in human neointima (2). FKBP12 is the receptor for sirolimus, which in ani- mal models reduced neointima formation. By the same time, the authors found upregu- lation of FKBP12 in human neointima by gene-expression profiling, the first clinical study showed that sirolimus-coated stents dramatically decrease the risk of in-stent restenosis in patients without increasing the risk of other major adverse cardiac events (17,18). Additionally, it has been recently shown that sirolimus treatment with a high oral loading dose regimen before coronary intervention resulted in a significant reduc- tion of restenosis after placement of bare stents. In this study, the plasma concentration of sirolimus on the day of the procedure correlated inversely with the late lumen loss at follow-up (19).

In several animal models of restenosis after angioplasty, it has been shown that sirolimus inhibits the proliferative response causing neointima formation by enhance- ment of the level of p27

^KIP1

protein and activation of retinoblastoma protein (pRb) (8,20). Nevertheless, this pathway does not appear to be the only mechanism involved in the beneficial action of sirolimus. Sirolimus also inhibits neointima formation in p27

^KIP1

-knockout mice through p27

^KIP1

-independent mechanisms (21).

Sirolimus, an immunosuppressive, binds to cytosolic FKBP-12 and this complex inhibits the protein kinase mammalian target of rapomycin (mTOR) (22). The mTOR kinase is essential for viability and regulates translation initiation and cell-cycle progression by altering the phosphorylation state of downstream targets like the p70 S6 kinase (p70S6K) (23). In T lymphocytes, inhibition of mTOR by sirolimus leads to dephosphorylation and thereby inactivation of p70S6K and to hypophosphorylation and thereby activation of pRb (24). The p70S6K is an important regulator of cell- cycle progression in response to mitogens that play pivotal roles in neointima for- mation, like placelet-derived growth factor and angiotensin (25,26).

In the gene-expression analysis of human neointima from in-stent restenosis study, the

authors showed that p70S6K-II and FKBP-12 were upregulated in cells from human in-

stent neointima (1). However, a systematic analysis of the effect of sirolimus on neoin-

tima formation in vivo has not been carried out yet. Therefore, the authors investigate the

regulation of mammalian target of rapomycin kinase and the effect of sirolimus on

p70S6K, pRb activity, and on global gene-expression patterns in coronary artery SMCs

(27). mTOR was nuclear translocated and phosphorylated in coronary artery smooth mus- cle cells (CASMCs) and T cells from human neointima from the coronary in-stent resteno- sis indicative for an activation of mTOR during neointima formation in humans.

Comparative gene-expression analysis of CASMCs treated with sirolimus revealed downregulation of the transcription factor E2F-1, a key regulator of G

₁

/S-phase entry, and of various pRb/E2F-1-regulated genes. In addition, changes in the expression of genes associated with replication, apoptosis, and inflammation were found. Furthermore, sirolimus decreased the gene expression of endothelial monocyte-activating polypeptide (EMAP)-II. This decrease of EMAP-II expression was reflected in a reduced adhesiveness of CASMCs for monocytic cells. Addition of EMAP-II counteracted the antiadhesive effect of sirolimus. Therefore, EMAP-II may include a mechanism of sirolimus-mediated reduction of the proinflammatory activation of CASMCs (27).

The effects of sirolimus on the downregulation of genes involved in cell-cycle pro- gression, apoptosis, proliferation, and extracellular matrix formation in CASMCs pro- vide further insight into how sirolimus reduces CASMC proliferation. The data suggest an additional antiadhesive and thereby anti-inflammatory effect of sirolimus: by inhibit- ing the expression of the monocyte chemoattractant EMAP-II, sirolimus leads to a decrease in CASMC adhesiveness and thereby may block the early recruitment of inflammatory cells to the injured arteries. This effect may further reduce the vascular proliferative response in humans.

PERSPECTIVES

Major progress has been made in the availability of technologies to analyze global gene-expression patterns in clinical samples (28). Although gene-expression profiling can provide important information about molecular mechanisms of disease, such as neointima formation, one has to be aware of the pitfalls and limitations of these analyses.

Gene-expression profiling represents an effective and powerful tool to generate hypotheses of potential pathophysiological relevance and to identify novel therapeutic targets. On the other hand, a careful validation and verification of the gene-expression data is essential. Therefore, the new approach of gene-expression profiling cannot replace conventional methods such as validation by Northern or Western blotting or proof of concept in experimental animals, such as knockout or transgenic mice.

Nevertheless, increased availability of this technology will bring into light some of the thus far ill-understood features of cardiovascular disease and will help identify new, potentially more individualized therapeutic options.

REFERENCES

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