WO2010130295A1 - Procédés d'évaluation de la prédisposition d'un sujet humain au rétrécissement des vaisseaux sanguins après une opération vasculaire - Google Patents

Procédés d'évaluation de la prédisposition d'un sujet humain au rétrécissement des vaisseaux sanguins après une opération vasculaire Download PDF

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WO2010130295A1
WO2010130295A1 PCT/EP2009/055938 EP2009055938W WO2010130295A1 WO 2010130295 A1 WO2010130295 A1 WO 2010130295A1 EP 2009055938 W EP2009055938 W EP 2009055938W WO 2010130295 A1 WO2010130295 A1 WO 2010130295A1
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encoding gene
nurrl
susceptibility
kipl
narrowing
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PCT/EP2009/055938
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Caroline Jacoba Maria De Vries
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Academisch Medisch Centrum
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Priority to PCT/EP2010/056665 priority patent/WO2010133520A2/fr
Publication of WO2010130295A1 publication Critical patent/WO2010130295A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • the present invention relates to methods for assessing the susceptibility of a human individual for narrowing of blood vessels after vascular intervention, such as transplant arteriopathy, graft disease including arterial, venous, artificial, allo- and homo-auto graft, stenting, balloon dilation, and especially in-stent restenosis.
  • vascular intervention such as transplant arteriopathy, graft disease including arterial, venous, artificial, allo- and homo-auto graft, stenting, balloon dilation, and especially in-stent restenosis.
  • the present invention further relates to the use of genotyping for assessing the susceptibility of a human individual for narrowing of blood vessels after vascular intervention, and especially in-stent restenosis, and kits of parts comprising means for said genotyping.
  • PCI percutaneous coronary intervention
  • drug-eluting- stents have been shown to significantly reduce restenosis, as compared to bare-metal stents, these stents may not be necessary in all lesions and patient subsets, because some individuals are at low risk to develop in-stent restenosis when treated with a bare-metal stent.
  • Drug-eluting stents have a long term risk of late and very late stent thrombosis and require prolonged uninterrupted dual anti -platelet therapy.
  • sirolimus or paclitaxel drug-eluting stents has considerably reduced the incidence of in-stent restenosis.
  • These drugs effectively inhibit smooth muscle cell (SMC) proliferation, but also hinder effective re-endothelialization and delay arterial healing, pathophysiological processes implicated in (late) in-stent thrombosis, a potentially fatal complication.
  • SMC smooth muscle cell
  • SNPs single nucleotide polymorphisms
  • p27 kipl a cyclin-dependent kinase (CDK) inhibitor located on human chromosome 12, positions 12,761,576 to
  • mice deficient in both p27 kipl and Apolipoprotein E display increased arterial cell proliferation and accelerated atherogenesis as compared to ApoE-null mice with an intact p27 kipl gene. Furthermore, reconstitution of sublethally irradiated ApoE-null mice with p27 kipl -deficient bone marrow was sufficient to enhance arterial macrophage proliferation and atherosclerosis.
  • a significantly lower level of p27 kipl has been detected in primary atherosclerotic and restenotic tissue versus non-diseased arterial tissue and p27 kipl expression levels and proliferation rates of vascular SMCs and leukocytes are inversely correlated in human atheroma.
  • SNP single nucleotide polymorphism
  • the nuclear "orphan” receptor Nurrl (also denoted NR4A2 or NOT; database accession GI: 226493744), located on human chromosome 2, is classified into the NR4A subfamily of nuclear receptors that, in addition, comprises Nur77 and NOR-I.
  • the nuclear receptor superfamily comprises both ligand-activated receptors, such as the estrogen receptor, liver X receptors (LXR) and peroxisome-proliferator- activated receptors (PPAR), as well as receptors like Nurrl, for which ligands have not been identified.
  • Nurrl contains a two zinc-finger DNA-binding domain, a C-terminal ligand- binding domain, and an N-terminal activation domain.
  • Nurrl interacts as a monomer, or homodimer, with consensus response elements in promoters of target genes, but can also form heterodimers with retinoid X receptors (RXR) via its C- terminal domain to mediate retinoid responses.
  • RXR retinoid X receptors
  • Nurrl modulates gene transcription by transrepression of other transcription factors.
  • the transcription factor Nurrl has mainly been associated with brain development, which is in line with the absence of dopaminergic neurons in Nurrl knockout mice. Nurrl also regulates inflammatory responses in macrophages and microglia and is expressed in human atherosclerosis and rheumatoid arthritis.
  • vascular intervention such as transplant arteriopathy, graft disease including arterial, venous, artificial, allo- and homo-auto graft, stenting, and balloon dilation.
  • transplant arteriopathy graft disease including arterial, venous, artificial, allo- and homo-auto graft, stenting, and balloon dilation.
  • the above objects, amongst other objects, are met by a method as described in the appended claim 1.
  • a method for assessing the susceptibility of a human individual for narrowing of blood vessels after vascular intervention such as transplant arteriopathy, graft disease including arterial, venous, artificial, allo- and homo-auto graft, stenting, and balloon dilation, preferably percutaneous coronary intervention, comprising : determining, in a sample originating from said human individual, the genotype of the cyclin-dependent kinase inhibitor p27 kipl encoding gene and/or nuclear receptor Nurrl encoding gene,- and establishing the susceptibility of a human individual for narrowing of blood vessels after vascular intervention based on said determination .
  • Narrowing of blood vessel after vascular intervention is also known as restenosis, a term common in vascular and cardiac intervention and angioplasty.
  • Restenosis means the reoccurrence of stenosis, a narrowing of a blood vessel, leading to restricted blood flow. Restenosis usually pertains to an artery or other large blood vessel that has become narrowed, received treatment to clear the blockage and subsequently becomes narrowed again. This is usually restenosis of an artery, or another blood vessel, or possibly a vessel within an organ. Restenosis can be defined as a reduction in the circumference of the lumen of a blood vessel of approximately 50% or more. The incidence of restenosis is high in patients who had undergone balloon angioplasty, with the majority of patients needing further angioplasty within 6 months .
  • the susceptibility of patient, receiving vascular intervention, for the development of restenosis can be assessed, before intervention, by determining the genotype, or the nucleic acid sequence, of the cyclin-dependent kinase inhibitor p27 kipl encoding gene and/or nuclear receptor Nurrl encoding gene of said patient. This is surprising because these genes were not previously identified as genetic risk factors in restenosis . Methods for determining a genotype, or nucleic acid sequence, of an individual are commonly know in the art.
  • Genomic material can be isolated from, for example, an already available blood, biopsy or saliva sample, and subsequently subjected to, for example, nucleic acid amplification or hybridization generally using primers based on a known nucleic acid sequence of a gene.
  • the amplification products can then be sequenced thereby providing the genotype of the gene.
  • primer design the presence, or absence, of an amplification or hybridization product, or the size of an amplification product can already be indicative of the genotype thereby obviating the sequencing step.
  • susceptibility is a relative term providing an indication of the risk of developing restenosis. Individuals having an increased susceptibility for the development of restenosis are in general more likely to be diagnosed by a trained physician, also based on other criteria such as age, sex and other medical conditions, to benefit from a drug- eluting stent. Individuals having a decreased susceptibility for the development of restenosis are in general more likely to be diagnosed by a trained physician, also based on other criteria such as age, sex and other medical conditions, to benefit from a bare-metal stent.
  • susceptibility is a relative term allowing discrimination within patient groups requiring vascular intervention.
  • patients requiring vascular intervention can be divided in two patient groups, i.e., a bare-metal stent benefitting group and a drug-eluting stent benefitting group.
  • the beneficial contribution to therapy by the present invention is not restricted to stents.
  • an individual having a specific genotype of p27 kipl and/or Nurrl could benefit from a pluronic gel loaded with other medicaments, or other concentrations or release profiles, other coatings thereof, as compared to an individual with a different genotype, thereby allowing tailor made, or personalized, therapy.
  • an individual having a specific genotype of p27 kipl and/or Nurrl could benefit from an artificial or tissue engineered graft loaded with other medicaments, or other concentrations or release profiles, other coatings thereof, as compared to an individual with a different genotype, thereby allowing tailor made, or personalized, therapy.
  • the present invention is not restricted to discriminating between two patient groups requiring vascular intervention. It is contemplated within the context of the present invention that the genotypes determined allow for a further, or alternative, discrimination within patient groups.
  • patients requiring vascular intervention can be divided in three patient groups, i.e., a patient group having decreased susceptibility p27 kipl and Nurrl genotypes, a patient group having increased susceptibility p27 kipl and Nurrl genotypes, and a patient group having intermediate susceptibility p27 kipl and Nurrl genotypes, i.e., patients having an in creased susceptibility p27 kipl or Nurrl genotype in combination with a decreased susceptibility Nurrl or p27 kipl genotype, respectively.
  • the identification of further subsets of patients requiring vascular intervention is contemplated.
  • the present invention is not limited to determining a susceptibility to restenosis providing an indication, or tool, for a trained physician to be useful in the decision to treat a patient with a bare-metal or drug- eluting stent.
  • the present determined susceptibility for restenosis is also contemplated to be beneficial for other treatments of conditions comprising vascular intervention, in general resulting in arterial wall injury, such as transplant arteriopathy, graft disease including arterial, venous, artificial, allo- and homo-auto graft, stenting, balloon dilation, and especially in-stent restenosis.
  • determining the genotype (s) of p27 kipl and/or Nurrl comprises establishing the identity of one or more nucleic acids present at nucleic acid positions chosen from the group consisting of -838 of the cyclin-dependent kinase inhibitor p27 kipl encoding gene,- -15906 of the nuclear receptor Nurrl encoding gene,- -3413 of the nuclear receptor Nurrl encoding gene,- and +7275 of the nuclear receptor Nurrl encoding gene.
  • the differences in susceptibility observed in patient groups were especially correlated with the identity of the nucleic acids present at the indicated positions.
  • an identification of the nucleic acid position is used which is common in the field.
  • the first nucleic acid of the coding sequence in a gene is assigned the numeral 0 and the position of upstream nucleic acids are counted starting from this first coding nucleic acid and the numeral characterizing the position is preceded by "-" to indicate the upstream position.
  • downstream nucleic acids are counted starting from this first coding nucleic acid and the numeral indicating the position is preceded by "+" to indicate the downstream position.
  • the position -838 indicates the 838 th nucleic acid upstream of the first coding nucleic acid (position 0) of p27 kipl (NM_004064.3, GI: 207113192).
  • the positions -15906 and -3413 indicate the 15906 th and 3413 th position upstream of the first coding nucleic acid (position 0) of the Nurrl gene (NG_011821.1 , GI: 226493744), respectively.
  • Position +7275 indicates the 7275 th nucleic acid downstream of the first coding nucleic acid (position 0) of the Nurrl gene (NG_011821.1 , GI: 226493744).
  • determining according to the present invention comprises establishing the identity of the nucleic acid present at position-838 of the cyclin-dependent kinase inhibitor p27 kipl encoding gene.
  • the identity A/A at position -838 of the cyclin-dependent kinase inhibitor p27 kipl encoding gene establishes a decreased susceptibility for restenosis and the identity A/C or C/C establishes an increased susceptibility.
  • an identity A/A indicates the presence of the nucleic acid adenine on both chromosomes 12 at the indicated position.
  • the identity A/C indicates that one chromosome 12 comprises the nucleic acid adenine and one chromosome 12 comprises the nucleic acid cytosine at the indicated position.
  • C/C indicates the presence of the nucleic acid cytosine on both chromosomes 12.
  • the present inventors found that individuals, carrying in their genome the present identity A/A, showed a remarkably increased cumulative in- stent restenosis-free survival as compared to the identities A/C and C/C after percutaneous coronary intervention.
  • determining according to the present invention comprises establishing the identity of the nucleic acids present at positions -15906, -3413 and +7275 of the nuclear receptor Nurrl encoding gene.
  • the haplotype TCT or ATT at the indicated positions of the nuclear receptor Nurrl encoding gene establishes an increased susceptibility and the haplotype TTG or TTT establishes a decreased susceptibility.
  • a haplotype TCT indicates the presence of at least one thymine at position -15906, the presence of at least one cytosine at position -3413, and the presence of at least one thymine at position +7275.
  • Haplotype ATT indicates the presence of at least one adenine at position -15906 and the presence of at least one thymine at positions -3413 and +7275.
  • Haplotype TTG indicates the presence of at least one thymine at positions -15906 and -3413 and the presence of at least one guanine at position +7275.
  • Haplotype TTT indicates the presence of at least one thymine at positions -15906, -3413 and +7275.
  • the present inventors found that individuals carrying in their genome the present haplotypes TTG and TTT showed a remarkably increased cumulative in- stent restenosis-free survival as compared to the haplotypes TCT and ATT after percutaneous coronary intervention.
  • the present narrowing of blood vessels after vascular intervention is in-stent restenosis, i.e., restenosis occurring after placing a stent.
  • the present method is an in vitro method indicating that the present method is not a diagnostic method practised on the human or animal body.
  • the present genotyping comprises single nucleotide polymorphism (SNP) genotyping.
  • SNP single nucleotide polymorphism
  • single nucleotide polymorphism (SNP) genotyping can be performed using Single-base extension (SBE) .
  • SNP single nucleotide polymorphism
  • SBE Single-base extension
  • an oligonucleotide primer hybridizes to a complementary region along the nucleic acid, to form a duplex, with the primer's terminal 3' end directly adjacent to the nucleotide base to be identified.
  • the oligonucleotide primer is enzymatically extended a single base by a nucleotide terminator complementary to the nucleotide being identified. The terminator prevents additional nucleotides from being incorporated.
  • SNP genotyping methods is the use of TaqMan probes consisting of a fluorophore attached to the 5' -end of the oligonucleotide probe and a quencher at the 3' -end.
  • the quencher molecule quenches the fluorescence emitted by the fluorophore when excited by a light source via FRET (Fluorescence Resonance Energy Transfer) .
  • FRET Fluorescence Resonance Energy Transfer
  • the present invention also relates to the use of the present identity of the nucleic acid present at position-838 of the cyclin-dependent kinase inhibitor p27 kipl encoding gene and/or the present identities of the nucleic acids present at positions -15906, -3413 and +7275 of the nuclear receptor Nurrl encoding gene for assessing the susceptibility of a human individual for narrowing of blood vessels after vascular intervention, preferably percutaneous coronary intervention.
  • the present use is preferably for assessing the susceptibility of human individuals for in-stent restenosis.
  • kits of parts for assessing the susceptibility of a human individual for narrowing of blood vessels after vascular intervention, preferably percutaneous coronary intervention comprising: means for determining the present identity of the nucleic acid present at position-838 of the cyclin-dependent kinase inhibitor p27 kipl encoding gene,- and/or means for determining the present identities of the nucleic acids present at positions - 15906, -3413 and +7275 of the nuclear receptor Nurrl encoding gene,- and - instructions for use.
  • the present means comprise means for genotyping single nucleotide polymorphisms (SNPs) .
  • the present kit of parts is preferably for assessing the susceptibility of human individuals for in-stent restenosis.
  • the present invention also relates to methods for assessing the susceptibility of a human individual for narrowing of blood vessels, or stenosis, involving excessive smooth muscle cell (SMC) activation and proliferation, comprising: determining, in a sample originating from said human individual, the genotype, as defined above, of the cyclin-dependent kinase inhibitor p27 kipl encoding gene and/or nuclear receptor Nurrl encoding gene, as defined above ; and - establishing the susceptibility of a human individual for narrowing of blood vessels, or stenosis, involving excessive smooth muscle cell (SMC) activation and proliferation , based on said determination.
  • SMC smooth muscle cell
  • kits of parts for assessing the susceptibility of a human individual for narrowing of blood vessels, or stenosis, involving excessive smooth muscle cell (SMC) activation and proliferation comprising: - means for determining the identity, as defined above, of the nucleic acid present at position-838 of the cyclin-dependent kinase inhibitor p27 kipl encoding gene,- and/or means for determining the identity, as defined above, of the nucleic acids present at positions -15906, -3413 and +7275 of the nuclear receptor Nurrl encoding gene,- and instructions for use.
  • Figure 1 shows the cumulative frequency distribution of late luminal loss (mm) in patients with a p27 kipl -838AA genotype compared to the combined p27 kipl -838 CA and CC genotypes
  • Figures 2-4 show the Kaplan-Meier estimates of event-free survival in patients with a p27 kipl -838AA genotype compared to the combined heterozygous and p27 kipl -838CC genotypes.
  • Figure 5 shows the effect of p27 kipl -838C>A polymorphism on the promoter transcriptional activity.
  • Figure 6 shows the differences in potential transcription factor binding sites on the p27 kipl promoter between -838A and -838C alleles determined by using the Genomatix Suite (http://www.genomatix.com).
  • STAT signal transducer and activator of transcription
  • EBFl early B-cell factorl
  • GABP GA-binding protein
  • MAZ myc-associated zinc finger protein.
  • Figure 7 shows a schematic depiction of the linkage disequilibrium (LD) block including the Nurrl gene, individual ht-SNPs in the Nurrl gene and composition and frequencies of the Nurrl haplotypes in the study population.
  • A Visualization of the location of the Nurrl gene (8.3kb) in the LD block (36kb) in a genome stretch (100kb) with a total of 3 LD blocks according to HapMap .
  • B The Nurrl gene including 5'- and 3'-UTRs (untranslated exons, dotted boxes), translated exons (black boxes) and the location of the three ht-SNPs.
  • the composition and frequencies of the four Nurrl haplotypes with frequencies over 1% detected in the study population are indicated.
  • Figures 8-9 show Nurrl haplotypes and the risk of ISR, target-lesion revascularization (TLR) , repeat PCI or MACE.
  • Figure 8 Hazard ratios of Nurrl haplotypes and ISR, target-lesion revascularization, repeat PCI or MACE risk.
  • Haplotype 1 (TTG) is used as reference. Hazard ratios are adjusted for age, gender, hypertension, diabetes, current smoking, statin use, stent length and length of stenosis by Cox regression analysis.
  • Figure 9 Nurrl haplotypes and Kaplan Meier estimates of event-free survival. The Kaplan Meier curves represent the cumulative proportion of patients free of ISR, TLR, repeat PCI or MACE during the follow up of 12 months. Patients were grouped having no copies of the risk haplotypes (haplotypes 3 and 4) or having at least one copy of a risk haplotype .
  • Figure 10 shows Nurrl expression in human in- stent restenosis atherectomy specimens. Serial sections were analyzed to detect (A) SMCs and
  • Figure 11 shows that Nurrl inhibits proliferation of human SMCs.
  • A Endogenous Nurrl expression is induced by serum treatment of SMCs. Nuclear localization of endogenous Nurrl 4 hours after stimulation is visualized by immunofluorescence. Nuclei are counterstained with Hoechst staining.
  • B left panel
  • Overexpression of Nurrl enhances p27 kipl protein expression as analyzed by Western blotting (specific band indicated with an arrow, nonspecific bands indicated by asterisk (*) .
  • B right panel
  • Quantification of p27 kipl protein showed a 1.8 fold increase in Nurrl overexpressing SMCs as compared to control.
  • (C) Nurrl overexpression results in inhibition of proliferation of SMCs as compared to control as demonstrated by DNA synthesis (BrdU- incorporation assay) .
  • (D) shRNA-mediated knockdown of endogenous Nurrl increased proliferation of SMCs as compared to control (n 3 ⁇ SD, Student t-test; p ⁇ 0.05).
  • Figure 12 shows that Nurrl inhibits inflammatory gene expression in SMCs.
  • A, B TNF ⁇ treatment of SMCs results in enhanced Nurrl, ILl ⁇ , TNF ⁇ and MCP-I mRNA expression.
  • (C) Nurrl overexpression inhibits expression of ILl ⁇ , TNF ⁇ and MCP-I at 6 hrs after TNF ⁇ treatment.
  • Figure 13 shows that Nurrl inhibits neointima formation in the wire-injury model in ApoE-/- mice.
  • A Human Nurrl transgene expression was detected in medial SMCs by radioactive in situ hybridization. The internal elastic lamina is marked (dashed line) .
  • B Analysis of the neointima in control mice revealed a SMC-rich lesion. SMCs were detected by immunohistochemistry (red) and Lawson (gray) was used to visualize the elastic laminae. All sections were counterstained with hematoxylin (blue) . Arrows indicate the internal elastic laminae.
  • C Morphometric analyses revealed that medial surface areas were comparable between all groups.
  • Example 1 The p27 kipl -838C>A single nucleotide polymorphism is associated with restenosis risk after coronary stenting and modulates p27 kipl promoter activity
  • the present example discloses p27 kipl polymorphisms that predict human clinical restenosis after stent- placement. It is demonstrated that the homozygote AA genotype of the genetic variation -838C>A located in the promoter of the p27 kipl gene is associated with reduced angiographic restenosis and target vessel revascularization risk in two independent cohort studies. Subsequently, the function of this genetic variation was evaluated on the promoter activity of p27 kipl and in line with the clinical data observed an increased promoter activity of the cell- cycle inhibitor p27 kipl was observed when the protective -838A genetic variation was present.
  • Indications for stent placement were bail-out or unsatisfactory results after balloon angioplasty alone, chronic total occlusion, ostial disease and restenosis after balloon angioplasty.
  • Patients with in-stent restenosis and complex lesions such as saphenous vein graft lesions, bifurcated lesions, lesions longer than 25 mm, reference vessel diameter ⁇ 2.5 mm and primary percutaneous coronary intervention (PCI) for myocardial infarction were excluded.
  • PCI primary percutaneous coronary intervention
  • Statin therapy was recorded at the time of the procedure and at follow-up. Patients were treated with 100 mg aspirin and 250 mg of Ticlopidine bid or 75 mg of clopidogrel daily for 1 month after PCI and 100 mg of aspirin thereafter. Blood from the participants was collected and genomic DNA was isolated from leukocytes.
  • GENDER GENetic DEterminants of Restenosis project
  • Target vessel revascularization was defined as revascularization of the stented segment or within 5 mm margins proximal or distal to the stent by either repeat PCI or coronary artery bypass grafting.
  • the primary endpoints of this study were angiographic late luminal loss in minimal lumen diameter and binary in-stent restenosis (>50% diameter stenosis) at 6-10 month follow-up.
  • the secondary endpoint was the occurrence of major adverse cardiac events (death, non- fatal myocardial infarction, coronary artery bypass grafting, repeat PCI and TVR) .
  • Quantitative coronary analysis was performed as described and was performed off-line on images obtained before, immediately after stent placement and at follow-up using a computerized quantitative analysis system (QCA-CMS, version 5.0, MEDIS, Leiden, The Netherlands).
  • QCA-CMS computerized quantitative analysis system
  • the p27 kipl -838C>A SNP was genotyped using a Custom TaqMan Genotyping Assay (Applied Biosystems, Fortster City CA) . Reactions were performed using the LC480 (Roche Diagnostics, Pemzburg, Germany) .
  • the -79C>T and +326G>T SNPs were determined as described using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer from Bruker Daltonics.
  • the pRL-TK renilla reporter contains the complete thymidine kinase promoter and was used to correct for transfection efficiency and cell number.
  • Gl arrest was induced by growth factor deprivation.
  • Growth medium was removed 16 h after transfection and the cells were washed with PBS and incubated with serum-free DMEM containing 0.1% bovine serum albumin. After 48 h luciferase activity was determined using the dual-luciferase reporter assay system (Promega) according to the manufacturer's protocol.
  • Angiographic follow-up was performed in 598 out of 688 cases, since 90 patients were either lost to follow-up or refused follow-up angiography. Of the patients with angiographic follow-up, 105 developed in-stent restenosis (18%) .
  • the p27 kipl -838C>A SNP is associated with clinical in-stent restenosis, where the -838AA genotype gives rise to a ⁇ 2-4-fold decreased risk of target vessel revascularisation. Importantly, this polymorphism is associated with differences in basal p27 kipl promoter activity.
  • this risk stratification may support the intervention cardiologist to triage patients with low risk of restenosis for treatment with a bare-metal stents rather than with a drug-eluting-stent .
  • Nurrl haplotype-defined variation of the Nurrl gene is reported to be associated with coronary in-stent restenosis risk in PCI patients and that Nurrl is expressed in human coronary in-stent restenosis lesions. It was demonstrated that Nurrl has an anti-proliferative and anti- inflammatory function in human cultured SMCs and protects against arterial wire-injury induced neointima formation in ApoE-/- mice.
  • the primary endpoint of this study was angiographic binary in-stent restenosis (ISR, >50% diameter stenosis) at follow-up.
  • the secondary endpoints were target lesion revascularization, repeat PCI, non-fatal myocardial infarction, coronary artery bypass grafting (CABG) or death or the combined endpoint of major adverse cardiac events (MACE) .
  • Target lesion revascularization was defined as repeat revascularization of the stented segment or within 5 mm margins proximal or distal to the stent by either repeat PCI or CABG.
  • Genotypes were determined in 2 ng genomic DNA with the Taqman allelic discrimination assay (Applied Biosystems, Foster City, California) . Reactions were performed with the Taqman Prism 7900HT in 384 -wells format. Association between individual SNPs and ISR, PCI, target-lesion revascularization or MACE were examined using Cox proportional hazard models in SPSS 16.0 for Windows (SPSS Inc, Chicago, IL) . From the obtained unphased SNP genotype data, haplotype frequencies and their effect on risk of ISR, repeat PCI, target-lesion revascularization or MACE were estimated using weighted Cox regression as described.
  • Variables depicted by univariate linear regression analysis to be predictive for ISR, PCI, target-lesion revascularization or MACE were entered into the model (control of confounding) .
  • Covariates included into the model were age, gender, hypertension, diabetes mellitus, current smoking, statin use, stent length and lesion length.
  • Event-free survival curves were calculated by Kaplan-Meier analysis, differences between the groups were calculated with the log-rank statistic. A p-value ⁇ 0.05 was considered to be statistically significant.
  • lentiviruses that contain human Nurrl cDNA or short hairpin (sh) human or mouse Nurrl sequences were generated. Lentiviral transduction efficiency in human SMCs was determined by immunofluorescence. Efficiency of endogenous Nurrl knockdown by shNurrl encoding virus was determined using semiquantitative real-time RT-PCR (qRT-PCR) . SMC Culture, DNA Synthesis and Western Blotting
  • SMCs were explanted from human umbilical cord arteries or mouse aortas and were cultured in Dulbecco's Modified Eagle's Medium (Invitrogen, Breda, The Netherlands) with 10% (v/v) fetal calf serum (FCS) and penicillin and streptomycin (Invitrogen) . SMCs were transduced and experiments were performed.
  • mice were anaesthetized and transluminal wire injury of the left common carotid artery was performed as described previously.
  • the lumen of the carotid artery was incubated for 15 minutes (108 IU/mouse supplemented with 10 ⁇ g/ml DEAE-dextran) directly after wire injury.
  • Animals were euthanized at 4 weeks after injury and the carotids were taken out and embedded in paraffin.
  • Nurrl Geno- and Haplotypes Associate with Angiographic and Clinical In-Stent Restenosis Risk A total of 601 patients were included in the study and DNA was obtained. Clinical follow-up was obtained from 600 patients and angiographic follow-up was performed in 597 out of these 600 cases. Failure rates of genotyping for Nurrl SNPs A; -15906T>A (rsl466408) , B; ⁇ 3413T>C (rsl3428968) and C; +7275G>T (rsl2803) were 3.7%, 6.2% and 1.2%, respectively.
  • ISR angiographic primary endpoint in- stent restenosis
  • MACE major adverse cardiac events
  • Haplotype 1 was most frequently found in the present study population (52.4%) and served as reference haplotype.
  • haplotype 3 which contains the minor alleles of SNP B and C (TCT)
  • haplotype 4 which contains the minor alleles of SNP A and C (ATT) were associated with a significantly increased ISR, target lesion revascularization, repeat PCI or MACE risk as compared to the reference haplotype 1.
  • Nurrl in human in-stent restenosis was analyzed. Coronary in-stent restenosis specimens of nine patients were obtained and analyzed for Nurrl expression by radioactive in situ hybridization.
  • Nurrl mRNA is induced by FCS with optimal mRNA expression levels 2 hours after treatment and after 4 hrs endogenous Nurrl protein expression is observed in the nucleus (Figure HA) .
  • Nurrl overexpression results in enhanced nuclear staining of Nurrl protein as shown by immunofluorescence and lentiviral delivery of shRNA directed against Nurrl mRNA resulted in over 80% knockdown of endogenous Nurrl mRNA.
  • Western blot analyses revealed that Nurrl overexpression in SMCs causes increased protein levels of p27 Kipl , a crucial cell-cycle inhibitor in SMCs ( Figure HB) .
  • SMCs showed a strong induction of several proinflammatory cytokines and chemokines in response to TNF ⁇ , most significantly interleukin (IL) l ⁇ , TNF ⁇ and monocyte chemoattractant protein- 1 (MCP-I) ( Figure 12B) and IL6 and IL8 (data not shown) .
  • IL interleukin
  • MCP-I monocyte chemoattractant protein- 1
  • Nurrl overexpression substantially inhibits TNF ⁇ - induced expression of ILl ⁇ (2.5 fold), TNF ⁇ (1.5 fold) and MCP-I (1.7 fold) (Figure 12C), but does not change IL6 and IL8 expression (data not shown) .
  • shRNA-mediated knockdown of endogenous Nurrl revealed a 4.5 fold increased expression of TNF ⁇ , while a non-significant 1.3 fold increase of ILl ⁇ and MCP-I expression was observed (Figure 12D) .
  • Nurrl is a Key Regulator of Neointima Formation after Arterial Injury in ApoE-/- mice
  • Nurrl transgene was detected in medial SMCs by radioactive in situ hybridization with a human cDNA- specific probe in the mice transduced with lentivirus encoding human Nurrl ( Figure 13A) .
  • ShRNA directed against murine Nurrl resulted in a 5.4 fold reduction of endogenous Nurrl expression in murine SMCs.
  • Nurrl knockdown resulted in a trend towards increase in neointimal area as compared to control (42.2+9.OxIO 3 ⁇ m 2 vs. 16.9 ⁇ 3.3xlO 3 ⁇ m 2 ; p ⁇ 0.05) and a 2.3 fold reduction in neointima/media ratio (0.75+0.17 vs. 0.32+0.05; p ⁇ 0.05) .
  • Nurrl knockdown resulted in a trend towards increase in neointimal area as compared to control (42.2+9.OxIO 3 ⁇ m 2 vs.
  • Haplotypes 3 (TCT) and 4 (ATT) inferred by combining 3 individual ht-SNPs, are associated with increased angiographic restenosis, target-lesion revascularization, repeat PCI and MACE risk as compared to the reference haplotype 1 (TTG) , which contains the common alleles of all three ht-SNPs.
  • Haplotypes 3 and 4 contain the minor alleles of SNP B and A respectively and since both of these ht-SNPs are localized in the promoter region of the Nurrl gene, it may be reasoned that these genetic variations affect expression levels of Nurrl .

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Abstract

Cette invention concerne des procédés d'évaluation de la prédisposition d'un sujet humain au rétrécissement des vaisseaux sanguins après une opération vasculaire, et notamment, la resténose intrastent. En particulier, cette invention concerne un procédé d'évaluation de la prédisposition d'un sujet humain au rétrécissement des vaisseaux sanguins après une opération vasculaire, comprenant : 1) la détermination, dans un échantillon provenant dudit sujet humain, du génotype du gène codant pour l'inhibiteur de kinase dépendant de la cycline p27kip1 et/ou du gène codant pour le récepteur nucléaire Nurr1 ; et 2) l'établissement de la prédisposition d'un sujet humain au rétrécissement des vaisseaux sanguins après une opération vasculaire, en fonction de ladite détermination.
PCT/EP2009/055938 2009-05-15 2009-05-15 Procédés d'évaluation de la prédisposition d'un sujet humain au rétrécissement des vaisseaux sanguins après une opération vasculaire WO2010130295A1 (fr)

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PCT/EP2010/056665 WO2010133520A2 (fr) 2009-05-15 2010-05-14 Méthodes d'évaluation du risque de resténose vasculaire chez un sujet après une intervention chirurgicale

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Non-Patent Citations (7)

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Title
BRAUN-DULLAEUS RUEDIGER C ET AL: "Quantification of the cell-cycle inhibitors p27(Kip1) and p21(Cip1) in human atherectomy specimens: primary stenosis versus restenosis.", THE JOURNAL OF LABORATORY AND CLINICAL MEDICINE MAR 2003, vol. 141, no. 3, March 2003 (2003-03-01), pages 179 - 189, XP002537965, ISSN: 0022-2143 *
DÍEZ-JUAN ANTONIO ET AL: "Coordinate control of proliferation and migration by the p27Kip1/cyclin-dependent kinase/retinoblastoma pathway in vascular smooth muscle cells and fibroblasts.", CIRCULATION RESEARCH 7 MAR 2003, vol. 92, no. 4, 7 March 2003 (2003-03-07), pages 402 - 410, XP002537964, ISSN: 1524-4571 *
DÍEZ-JUAN ANTONIO ET AL: "Role of the growth suppressor p27Kip1 during vascular remodeling", CURRENT VASCULAR PHARMACOLOGY, BENTHAM SCIENCE PUBLISHER, HILVERSUM, NL, vol. 1, no. 1, 1 March 2003 (2003-03-01), pages 99 - 106, XP009120082, ISSN: 1570-1611 *
GONZÁLEZ PELAYO ET AL: "A single-nucleotide polymorphism in the human p27kip1 gene (-838C>A) affects basal promoter activity and the risk of myocardial infarction.", BMC BIOLOGY 2004, vol. 2, 2004, pages 5, XP002537963, ISSN: 1741-7007 *
MARKS A R: "Rapamycin: signaling in vascular smooth muscle.", TRANSPLANTATION PROCEEDINGS MAY 2003, vol. 35, no. 3 Suppl, May 2003 (2003-05-01), pages 231S - 233S, XP002537983, ISSN: 0041-1345 *
MONRAATS PASCALLE S ET AL: "Genetic inflammatory factors predict restenosis after percutaneous coronary interventions", CIRCULATION, LIPPINCOTT WILLIAMS & WILKINS, US, vol. 112, no. 16, 18 October 2005 (2005-10-18), pages 2417 - 2425, XP009096503, ISSN: 0009-7322 *
NIESSNER ALEXANDER ET AL: "Fractalkine receptor polymorphisms V249I and T280M as genetic risk factors for restenosis", THROMBOSIS AND HAEMOSTASIS, STUTTGART, DE, vol. 94, no. 6, 1 December 2005 (2005-12-01), pages 1251 - 1256, XP009096519, ISSN: 0340-6245 *

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