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Fir High t Dsearchpar Affair m High n Www High f Www Int Affair rnal Medicine, Yamagata University School of Medicine (M. Itoh, H.T.), Japan.Abstract
Background— Vascular restenosis attributable to intimal thickening remains a major problem after percutaneous transluminal coronary angioplasty (PTCA).
Methods and Results— Through differential-display analysis, we have identified a novel gene whose expression was increased after catheter injury of rabbit aorta. The gene that is expressed predominantly in vascular smooth muscle cells encodes a novel protein with 7 transmembrane domains, and we termed it ITR (intimal thickness–related receptor). The ITR sequence contains a motif common to the Rhodopsin-like GPCR (G-protein-coupled receptor) superfamily. In vivo analyses of this gene revealed that expression of ITR protein increased with intimal thickening induced by cuff placement around murine femoral artery. Furthermore, ITR-knockout mice were found to be resistant to this experimental intimal thickening.
Conclusions— ITR thus seems to be a novel receptor that may play a role in vascular remodeling and that may represent a good target for development of drugs in the prevention of vascular restenosis.
Key Words: angioplasty genes restenosis
Introduction
Percutaneous transluminal coronary angioplasty (PTCA) is a widely used therapeutic procedure for coronary artery diseases, including angina pectoris and myocardial infarction. However, restenosis, which occurs in 30% to 50% of patients after PTCA as a consequence of cellular hyperplasia within the neointima, is an issue of clinical urgency. Cell-cell interactions, cell-matrix interactions, redox state, ligand-receptor interactions, tyrosine kinases, and transcription factors are all involved in endothelial dysfunction and formation of neointima.1 However, only a few genes have been reported in relation to this highly complex phenomenon.
Genes encoding G-protein–coupled receptors (GPCRs) represent the largest superfamily yet identified; more than 800 of them have been discovered to date from a wide range of species. A characteristic motif of gene products in this superfamily is the presence of 7 distinct hydrophobic regions, each 20 to 30 amino acids in length, that are generally regarded as the transmembrane domains of these integral membrane proteins. There is little conservation of amino acid sequence across the entire superfamily of receptors, but key sequence motifs can be found within phylogenetically related subfamilies.2–6 GPCRs are excellent drug targets, and in fact several hundred drugs launched within the past 3 decades are directed to known GPCRs.7–10 Therefore, any GPCR whose expression level is changed when vascular smooth muscle cells (VSMCs) are multiplying would be a promising target molecule for prevention of restenosis.
We had performed differential mRNA display analyses11 of a rabbit model of vascular injury to identify genes that were upregulated or downregulated after denudation by a balloon catheter.12 In the present study, we report isolation and characterization of a novel gene, designated ITR, that was upregulated in the acute phase after balloon injury of the rabbit aorta.
Methods
Cloning of Full-Length cDNA
To isolate a full-length rabbit ITR gene, 5’-RACE was performed using the Marathon cDNA Amplification Kit (Clontech Laboratories). Full-length cDNAs of human, mouse, and rat orthologs were cloned by screening heart cDNA libraries of each species (Clontech).
RNA Expression of the Human ITR Gene
For Northern blot analysis, human multiple-tissue Northern blot (CLONTECH) was hybridized to [32P]dCTP-labeled full-length human ITR cDNA. All blots were washed at 0.1x SSC, 65°C, and exposed to autoradiography film overnight. Total RNAs were isolated from human umbilical vein endothelial cells, human coronary artery endothelial cells, and human coronary artery smooth muscle cells. Reverse transcriptase–polymerase chain reaction (RT-PCR) was performed using a set of primers, hITR-RTF (5’-TGGCATTGCAGTATTCATTG-3’) and hITR-RTR (5’-CAGACT- GGCAAAGGATAACAC-3’).
Chromosomal Localization of the Human ITR Gene
The human genomic cosmid clone was isolated from a human genomic DNA library (STRATAGENE, Gebouw) using the human full-length cDNA as a probe and used for the FISH experiment on a R-banded metaphase spreads, as described previously.13–15 More than 100 metaphase cells were examined after in situ hybridization.
Computer Analysis and Database Search
Using the FASTA program,16 we compared nucleotide and deduced amino acid sequences to the respective databases (nonredundant combination of GenBank, EMBL, and DDBJ databases for nucleotides and the nonredundant combination of Swiss-prot, PIR, and PRF databases plus translations of DNA sequences in GenBank for amino acids). A motif search was done at PRINTS.17
Generation of Mice Lacking ITR
An ITR-targeting plasmid was constructed according to a method based on inverse-PCR described previously.18 To isolate BAC clones containing the mouse ITR gene, "down to the well mouse ES BAC DNA pool" (Genome Systems Inc, St Louis, Mo) was screened by the primer pair ITR-BACF (5’-CCGATGATTGGTTACTGTCCCCG-3’) and ITR-BACR (5’-CCGATGATTGGTTACTGTCCCCG-3’), corresponding to DNA sequences in exon 1. A 2-µg aliquot of isolated BAC DNA was digested with HindIII, circularized by self-ligation at 4°C for 16 hours, and used as template for inverse-PCR. Amplification was performed by the primer pair ITR-KI5’ (5’-ACGCGTTTAATTAAGTCGGCAGCAGAGGGCGGG- GCGGGACTTAG-3’) and ITR-KI3’ (5’-ACGCGTGCGGCCGCGCAT- TCGAGCCCTGTGGTTATGAGTGGATG-3’). After digesting both ends of PCR product with NotI and PacI, the DNA fragment was cloned into p1108 vector and linearized by HindIII digestion before transfection. Targeting of the ITR gene and generation of knockout mice using E14.1 embryonic stem (ES) cells (129/SVJ) were performed by essentially standard techniques.19 Animals considered to be chimeric on the basis of their coat colors were mated with 129/SVJ or C57BL/6J mice. DNA samples were isolated from tails of ES cell-derived animals for genotyping. ES cells and F1 mice were analyzed for the presence of a disrupted ITR gene by Southern blot analysis. F2 mice genotyping was done by PCR using tail genomic DNA as a template and the following three primers: primer 1 specific for wild-type allele (5’-CCGTTTCCATTTCCCCGAC- AC-3’), primer 2 specific for recombinant allele (5’-CTCCAAAAAAGC- CTCCTCACT-3’), and primer 3 for both alleles (5’-CGGTCTTAC- AAACAACAGGGA-3’).
Preparation of Anti-ITR Monoclonal Antibody
COS7 cells were transfected with pFLAG-CMV-1 vector to design to express N-terminal portion of rat ITR (rN-ITR) corresponding to the extracellular domain, and the induced protein was purified with anti-FLAG affinity column. Anti-ITR monoclonal antibody (Mab) was prepared by immunizing female BALB/c mice (Shizuoka Laboratory Animal Center, Shizuoka, Japan) every seventh day with a footpad injection of purified rN-ITR. The booster injection was made into the footpad 2 days before fusion. Popliteal lymph node cells were fused with mouse myeloma cells, PAI, using PEG4000 (Life Technologies, Inc), and the resulting hybridomas were screened according to their ability to stain ITR transfectants.
Cuff-Induced Intimal Thickening of the Murine Femoral Artery
Adult male mice (n=10) lacking ITR and 10- to 12-week-old WT male mice (n=9) from the same genetic background were housed under climate-controlled conditions with a 12-hour light/dark cycle, with room temperature kept at 25°C. They were given a standard diet (MF, Oriental Yeast Co Ltd) and water ad libitum. The experimental protocol was approved by the Animal Studies Committee of Ehime University. Surgery to place polyethylene cuffs (2-mm long PE-90; Becton-Dickinson) and to obtain artery samples was performed according to methods described previously.20–23 Excised arterial tissues were fixed in 10% formalin overnight, dehydrated, and embedded in paraffin.
The middle segment of artery from each mouse was cut into 3 subserial cross sections of 5-µm thickness at intervals of 0.3 mm. The sections were stained by Elastica van Gieson staining to investigate overall morphology and photography. The areas covered by neointima and media were measured using image-analyzing software (NIH image). Neointima was defined as the area between the vessel lumen and the internal elastic lamina. Media was defined as the area between the internal and external elastic lamina. The average of 3 sections was taken as the value for each animal.
The artery samples were taken 7 days after cuff placement, and bromodeoxyuridine (BrdU, Sigma) was injected 100 mg/kg SC and 30 mg/kg IP at 18 hours before euthanasia and then 30 mg/kg IP at 12 hours before euthanasia.20,21 The sections were prepared in the same manner as morphometric analysis. Immunohistochemistry using anti-BrdU antibody was performed according to the manufacturer’s protocol (BrdU Staining Kit; Zymed Laboratories). After the sections were counterstained with hematoxylin, we calculated the BrdU index (the ratio of BrdU-positive nuclei versus total nuclei). The average index of 3 sections was taken as the value for each animal. Statistical analyses were performed using two-way ANOVA. When a significant effect was found, post-hoc analysis was done to detect the difference between the groups. P<0.05 was considered statistically significant.
Immunohistochemical Staining of ITR
ITR in the cuffed artery was stained with mouse-to-mouse staining kit (M.O.M. Immunodetection kit, Vector Laboratories) using formalin-fixed, paraffin-embedded sections. Endogenous peroxidase was blocked by 0.3% hydrogen peroxide in methanol. Primary antibody was applied to the sections and incubated for 16 to 24 hours at 4°C. Positive staining was visualized using diaminobenzidine, and counterstained nuclei with hematoxylin.
Results
Cloning and Characterization of the ITR Gene
Using a differential mRNA display method, we isolated a novel cDNA fragment that was upregulated early after injury of the rabbit aorta. Because the clone covered only part of the 3’-untranslated region, 5’ and 3’ rapid amplification of cDNA ends (RACE) experiments were performed to isolate the full-length cDNA, which we named ITR. RT-PCR showed that expression of this rabbit mRNA was significantly elevated 2 or 4 days after injury of the aorta and declined afterward (). This result was confirmed by a similar procedure using carotid arteries from rats (data not shown).
fig.ommitted
. Expression of ITR mRNA in rabbits after balloon injury, examined by RT-PCR. Numerals indicate days after injury, and C denotes intact aorta. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.
Human, mouse, and rat counterparts of the rabbit ITR were isolated from cDNA libraries of the respective species. Alignment of human, mouse, rabbit, and rat amino acid sequences deduced from aWww Sexydateclub Affair High Szh Sexy Date Club 关于Inhibition of Experimental Intimal Thickening in Mice Lacking a Novel G-Proteinǖ奈侍鈛 0 Sexual iWww Sexydateclub Affair High Szh Sexy Date Club 关于Inhibition of Experimental Intimal Thickening in Mice Lacking a Novel G-Proteinǖ奈侍鈗 b Home