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Ru Adviceporn R Dating Szh 1 Advice Porn 关于Inhibition of Experimental Intimal Thickening in Mice Lacking a Novel G-Proteinǖ奈侍�

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    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 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 ().

    fig.ommitted

     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).

    fig.ommitted

     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).

    Role of ITR in Vascular Remodeling

    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 {}

    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).

    fig.ommitted

     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).

    fig.ommitted

     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

     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.

    Discussion

    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.

    The deduced amino acid sequence of ITR contains a secondary structure of 7 transmembrane {}

    -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.

    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.

    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.

    Received July 2, 2002; revision received September 6, 2002; accepted September 13, 2002.

    References

    Gibbons GH, Dzau VJ. Molecular therapies for vascular diseases. Science. 1996; 272: 689–693.
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