Inhibition of Experimental Intimal Thickening in Mice Lacking a Novel G-Protein–Coupled Receptor
From the Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, University of Tokyo (S.T., J.N., M. Itoh, Y.N., T.T.), Department of First Medical Biochemistry, Ehime University School of Medicine (M. Iwai, M.H.), and First Department of Internal Medicine, Yamagata University School of Medicine (M. Itoh, H.T.), Japan.3r, http://www.100md.com
Abstract3r, http://www.100md.com
Background— Vascular restenosis attributable to intimal thickening remains a major problem after percutaneous transluminal coronary angioplasty (PTCA).3r, http://www.100md.com
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.xed, 百拇医药
Key Words: angioplasty genes restenosisxed, 百拇医药
Introductionxed, 百拇医药
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.xed, 百拇医药
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.%., http://www.100md.com
Methods%., http://www.100md.com
Cloning of Full-Length cDNA%., http://www.100md.com
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).%., http://www.100md.com
RNA Expression of the Human ITR Gene%., http://www.100md.com
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?}, 百拇医药
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.?}, 百拇医药
Computer Analysis and Database Search?}, 百拇医药
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?}, 百拇医药
Generation of Mice Lacking ITR?}, 百拇医药
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 Antibody3p, 百拇医药
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.3p, 百拇医药
Cuff-Induced Intimal Thickening of the Murine Femoral Artery3p, 百拇医药
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.^|, 百拇医药
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 ITRi, 百拇医药
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.i, 百拇医药
Resultsi, 百拇医药
Cloning and Characterization of the ITR Genei, 百拇医药
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.ommitted60z?f#], 百拇医药
. 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.60z?f#], 百拇医药
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 their nucleotide sequences showed at least 80% identity among all 4 species. Each predicted amino acid sequence revealed a signal sequence at the amino-terminus and 7 transmembrane regions in the center. A search for motifs in the ITR protein revealed GPCRRHODOPSN4, a signature of the Rhodopsin-like GPCR superfamily ().60z?f#], 百拇医药
fig.ommitted60z?f#], 百拇医药
Nucleotide and predicted amino acid sequences of human ITR gene. Predicted signal sequence cleavage site is shown by a triangle, and 7 transmembrane regions are boxed. Three polyadenylation sites are indicated by boldface type. Rhodopsin-like GPCR superfamily signatures (GPCRRHODOPSN4 motif) are underlined. Kozak sequence adjacent to initiation codon is shown by italics.
The ITR gene was ubiquitously expressed in normal human tissues on Northern blots, where 2 transcripts (approximate molecular sizes, 1.9 and 3.8 kb) were detected in the tissues analyzed (A), owing probably to the different sites of polyadenylation seen in . We also examined expression pattern of this gene among cells constituting blood vessel and found that this gene was predominantly expressed in smooth muscle cells (B).str, 百拇医药
fig.ommittedstr, 百拇医药
A, Northern-blot analysis of human ITR mRNA. Blots from various adult human tissues containing 2 µg of poly(A) RNA in each lane were probed with human ITR cDNA. The bottom panel shows the same blot hybridized with ß-actin. B, Expression of ITR mRNA in each of the cell types that constitute blood vessel. HUVEC, HCAEC, and HSMC represent human umbilical vein endothelial cells, human coronary artery endothelial cells, and human coronary artery smooth muscle cells, respectively. Other types of tissues serve as controls to compare the results of Northern blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control.
The chromosomal localization of the human ITR gene was determined by fluorescence in situ hybridization using a full-length human ITR cDNA as the probe. Metaphase cells showed specific hybridization signals with twin spots at chromosomal band 13q31 (data not shown).g, 百拇医药
Role of ITR in Vascular Remodelingg, 百拇医药
To investigate the in vivo role of ITR, we generated mice lacking this gene (see for targeting scheme). Under normal conditions, ITR-null mice were indistinguishable from wild-type mice in appearance, growth rate, reproduction, and histology of major organs, including liver, heart, lung, brain, aorta, kidney, thymus, testis, muscle, pancreas, or spleen. To elucidate the in vivo role of ITR in intimal thickening, we performed cuff placement experiment around mouse femoral artery. As shown in A, intimal thickening was observed 14 days after surgery in wild-type mouse. Immunohistochemical staining revealed that expression of ITR protein was induced in media and intima of the injured artery at 7 days after cuff placement, when intimal thickening was not yet overt (). On the other hand, ITR-null mice hardly showed any of the neointima that were formed in wild-type mice during the experimental period (A). The neointimal area was {approx}
200% greater in wild-type mouse than in ITR-null mouse 14 days after cuff placement (P<0.01, D). There was no significant difference in the medial area between wild-type and ITR-null mice (C). As a marker of DNA synthesis in vascular smooth muscle cell (VSMC), BrdU incorporation into VSMC after cuff placement was significantly suppressed in ITR-null mice (P<0.001, E).n, 百拇医药
fig.ommittedn, 百拇医药
Generation of ITR-deficient mice by gene targeting. A, ITR gene was disrupted by replacing part of exon 1 and intron 1 with the neomycin-resistance cassette. B, Southern blot analysis of genomic DNA from ES cells and F1 mice using probes outside the homologous recombination region, as indicated in panel A. C, Genotyping of F2 mice by means of PCR. For the presence of recombinant allele, primer 2 and primer 3 were used (top), and for wild-type allele, primer 1 and primer 2 were used (bottom).n, 百拇医药
fig.ommittedn, 百拇医药
Morphometric analysis of injured artery after placement of polyethylene-cuff in wild-type (WT) and ITR-null (ITR KO) mice. A, Elastica van Gieson staining of cuffed femoral artery 14 days after cuff placement. Magnification x50. Artery samples were obtained 7 days and 14 days after cuff placement. B, Intimal area; C, Medial area; D, Intima/media ratio. *P<0.01 versus WT. Values are mean±SEM. E, Bromodeoxyuridine (BrdU) incorporation in media and neointima of cuffed femoral artery. BrdU index (BrdU-positive nuclei/total nuclei) was analyzed 7 days after surgery. **P<0.001 vs WT. Values are mean±SEM.
fig.ommitted\7[, 百拇医药
Immunohistochemical staining of ITR in injured artery 7 days after cuff placement. Artery samples were obtained from wild-type (WT) and ITR-null (ITR KO) mice, and paraffin-sections of arteries were stained with monoclonal antibody for ITR.\7[, 百拇医药
Discussion\7[, 百拇医药
We have described here the cloning and characterization of a putative G-protein–coupled receptor, called ITR, that was isolated as a novel gene differentially expressed at an early phase of vascular injury in rabbits.\7[, 百拇医药
The deduced amino acid sequence of ITR contains a secondary structure of 7 transmembrane {alpha}\7[, 百拇医药
-helical domains characteristic of the Rhodopsin-like GPCR superfamily. GPCRs represent an increasingly large and functionally diverse superfamily of receptors whose intracellular actions are mediated by signaling pathways involving G proteins and downstream secondary messengers.2–4 Receptors of this class respond to a variety of extracellular signals, including peptide hormones, lipid-derived messengers, and neurotransmitters. Because 7 transmembrane domains are present in all GPCRs, most of these receptors bear sequence similarity to one another, primarily in the transmembrane regions.5,6,24,25 However, ITR has little sequence homology to any known proteins, although its putative transmembrane domains do contain signatures of the Rhodopsin-like GPCR superfamily. Therefore, ITR protein may be an orphan GPCR rather than a member of any known GPCR subfamily. The lack of sequence homology to other known GPCRs makes it difficult to predict the specific ligand for this receptor or the identities of its coupled G protein and second messengers.
We performed two distinct experiments to study pathogenesis of intimal thickening; one was the catheter injury of rabbit aorta and the other was the cuff placement around mouse femoral artery. In both models, expression of ITR was induced before intimal thickening became overt. Combining our results that ITR was predominantly expressed in vascular smooth muscle cell and that ITR-deficient mice were resistant to this experimental intimal thickening, it may be reasonable to suggest that ITR is essential in the early stage of intimal thickening, especially for vascular smooth muscle cells to receive one of the critical signals to migrate or proliferate.-jo, 百拇医药
Because ITR is a putative GPCR, it may be a good tar- get for drug development to encounter intimal thicken- ing, although the relevance of our findings in human remains to be clarified. Isolation of its ligand may provide an important clue for a better understanding of its pathogenesis.-jo, 百拇医药
Acknowledgments
This work was supported in part by Research for the Future Program Grant No. 00 L 01402 from the Japan Society for the Promotion of Science.wh, http://www.100md.com
Received July 2, 2002; revision received September 6, 2002; accepted September 13, 2002.wh, http://www.100md.com
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Abstract3r, http://www.100md.com
Background— Vascular restenosis attributable to intimal thickening remains a major problem after percutaneous transluminal coronary angioplasty (PTCA).3r, http://www.100md.com
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.xed, 百拇医药
Key Words: angioplasty genes restenosisxed, 百拇医药
Introductionxed, 百拇医药
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.xed, 百拇医药
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.%., http://www.100md.com
Methods%., http://www.100md.com
Cloning of Full-Length cDNA%., http://www.100md.com
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).%., http://www.100md.com
RNA Expression of the Human ITR Gene%., http://www.100md.com
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?}, 百拇医药
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.?}, 百拇医药
Computer Analysis and Database Search?}, 百拇医药
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?}, 百拇医药
Generation of Mice Lacking ITR?}, 百拇医药
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 Antibody3p, 百拇医药
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.3p, 百拇医药
Cuff-Induced Intimal Thickening of the Murine Femoral Artery3p, 百拇医药
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.^|, 百拇医药
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 ITRi, 百拇医药
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.i, 百拇医药
Resultsi, 百拇医药
Cloning and Characterization of the ITR Genei, 百拇医药
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.ommitted60z?f#], 百拇医药
. 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.60z?f#], 百拇医药
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 their nucleotide sequences showed at least 80% identity among all 4 species. Each predicted amino acid sequence revealed a signal sequence at the amino-terminus and 7 transmembrane regions in the center. A search for motifs in the ITR protein revealed GPCRRHODOPSN4, a signature of the Rhodopsin-like GPCR superfamily ().60z?f#], 百拇医药
fig.ommitted60z?f#], 百拇医药
Nucleotide and predicted amino acid sequences of human ITR gene. Predicted signal sequence cleavage site is shown by a triangle, and 7 transmembrane regions are boxed. Three polyadenylation sites are indicated by boldface type. Rhodopsin-like GPCR superfamily signatures (GPCRRHODOPSN4 motif) are underlined. Kozak sequence adjacent to initiation codon is shown by italics.
The ITR gene was ubiquitously expressed in normal human tissues on Northern blots, where 2 transcripts (approximate molecular sizes, 1.9 and 3.8 kb) were detected in the tissues analyzed (A), owing probably to the different sites of polyadenylation seen in . We also examined expression pattern of this gene among cells constituting blood vessel and found that this gene was predominantly expressed in smooth muscle cells (B).str, 百拇医药
fig.ommittedstr, 百拇医药
A, Northern-blot analysis of human ITR mRNA. Blots from various adult human tissues containing 2 µg of poly(A) RNA in each lane were probed with human ITR cDNA. The bottom panel shows the same blot hybridized with ß-actin. B, Expression of ITR mRNA in each of the cell types that constitute blood vessel. HUVEC, HCAEC, and HSMC represent human umbilical vein endothelial cells, human coronary artery endothelial cells, and human coronary artery smooth muscle cells, respectively. Other types of tissues serve as controls to compare the results of Northern blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control.
The chromosomal localization of the human ITR gene was determined by fluorescence in situ hybridization using a full-length human ITR cDNA as the probe. Metaphase cells showed specific hybridization signals with twin spots at chromosomal band 13q31 (data not shown).g, 百拇医药
Role of ITR in Vascular Remodelingg, 百拇医药
To investigate the in vivo role of ITR, we generated mice lacking this gene (see for targeting scheme). Under normal conditions, ITR-null mice were indistinguishable from wild-type mice in appearance, growth rate, reproduction, and histology of major organs, including liver, heart, lung, brain, aorta, kidney, thymus, testis, muscle, pancreas, or spleen. To elucidate the in vivo role of ITR in intimal thickening, we performed cuff placement experiment around mouse femoral artery. As shown in A, intimal thickening was observed 14 days after surgery in wild-type mouse. Immunohistochemical staining revealed that expression of ITR protein was induced in media and intima of the injured artery at 7 days after cuff placement, when intimal thickening was not yet overt (). On the other hand, ITR-null mice hardly showed any of the neointima that were formed in wild-type mice during the experimental period (A). The neointimal area was {approx}
200% greater in wild-type mouse than in ITR-null mouse 14 days after cuff placement (P<0.01, D). There was no significant difference in the medial area between wild-type and ITR-null mice (C). As a marker of DNA synthesis in vascular smooth muscle cell (VSMC), BrdU incorporation into VSMC after cuff placement was significantly suppressed in ITR-null mice (P<0.001, E).n, 百拇医药
fig.ommittedn, 百拇医药
Generation of ITR-deficient mice by gene targeting. A, ITR gene was disrupted by replacing part of exon 1 and intron 1 with the neomycin-resistance cassette. B, Southern blot analysis of genomic DNA from ES cells and F1 mice using probes outside the homologous recombination region, as indicated in panel A. C, Genotyping of F2 mice by means of PCR. For the presence of recombinant allele, primer 2 and primer 3 were used (top), and for wild-type allele, primer 1 and primer 2 were used (bottom).n, 百拇医药
fig.ommittedn, 百拇医药
Morphometric analysis of injured artery after placement of polyethylene-cuff in wild-type (WT) and ITR-null (ITR KO) mice. A, Elastica van Gieson staining of cuffed femoral artery 14 days after cuff placement. Magnification x50. Artery samples were obtained 7 days and 14 days after cuff placement. B, Intimal area; C, Medial area; D, Intima/media ratio. *P<0.01 versus WT. Values are mean±SEM. E, Bromodeoxyuridine (BrdU) incorporation in media and neointima of cuffed femoral artery. BrdU index (BrdU-positive nuclei/total nuclei) was analyzed 7 days after surgery. **P<0.001 vs WT. Values are mean±SEM.
fig.ommitted\7[, 百拇医药
Immunohistochemical staining of ITR in injured artery 7 days after cuff placement. Artery samples were obtained from wild-type (WT) and ITR-null (ITR KO) mice, and paraffin-sections of arteries were stained with monoclonal antibody for ITR.\7[, 百拇医药
Discussion\7[, 百拇医药
We have described here the cloning and characterization of a putative G-protein–coupled receptor, called ITR, that was isolated as a novel gene differentially expressed at an early phase of vascular injury in rabbits.\7[, 百拇医药
The deduced amino acid sequence of ITR contains a secondary structure of 7 transmembrane {alpha}\7[, 百拇医药
-helical domains characteristic of the Rhodopsin-like GPCR superfamily. GPCRs represent an increasingly large and functionally diverse superfamily of receptors whose intracellular actions are mediated by signaling pathways involving G proteins and downstream secondary messengers.2–4 Receptors of this class respond to a variety of extracellular signals, including peptide hormones, lipid-derived messengers, and neurotransmitters. Because 7 transmembrane domains are present in all GPCRs, most of these receptors bear sequence similarity to one another, primarily in the transmembrane regions.5,6,24,25 However, ITR has little sequence homology to any known proteins, although its putative transmembrane domains do contain signatures of the Rhodopsin-like GPCR superfamily. Therefore, ITR protein may be an orphan GPCR rather than a member of any known GPCR subfamily. The lack of sequence homology to other known GPCRs makes it difficult to predict the specific ligand for this receptor or the identities of its coupled G protein and second messengers.
We performed two distinct experiments to study pathogenesis of intimal thickening; one was the catheter injury of rabbit aorta and the other was the cuff placement around mouse femoral artery. In both models, expression of ITR was induced before intimal thickening became overt. Combining our results that ITR was predominantly expressed in vascular smooth muscle cell and that ITR-deficient mice were resistant to this experimental intimal thickening, it may be reasonable to suggest that ITR is essential in the early stage of intimal thickening, especially for vascular smooth muscle cells to receive one of the critical signals to migrate or proliferate.-jo, 百拇医药
Because ITR is a putative GPCR, it may be a good tar- get for drug development to encounter intimal thicken- ing, although the relevance of our findings in human remains to be clarified. Isolation of its ligand may provide an important clue for a better understanding of its pathogenesis.-jo, 百拇医药
Acknowledgments
This work was supported in part by Research for the Future Program Grant No. 00 L 01402 from the Japan Society for the Promotion of Science.wh, http://www.100md.com
Received July 2, 2002; revision received September 6, 2002; accepted September 13, 2002.wh, http://www.100md.com
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