Abstract-Collagen triple helix repeat containing 1 (Cthrc1) was identified in a screen for differentially expressed sequences in balloon-injured versus normal arteries. Cthrc1 expression was not detectable in normal arteries. However, on injury it was transiently expressed by fibroblasts of the remodeling adventitia and by smooth muscle cells of the neointima. It was also found in the matrix of calcifying human atherosclerotic plaques. CTHRC1 is a secreted 28-kDa protein that is glycosylated and highly conserved from lower chordates to mammals. A short collagen motif with 12 Gly-X-Y repeats appears to be responsible for trimerization of the protein and this renders the molecule susceptible to cleavage by collagenase. Cthrc1 mRNA expression levels are increased in response to transforming growth factor-␤ and bone morphogenetic protein-4. Cell migration assays performed with CTHRC1-overexpressing fibroblasts and smooth muscle cells demonstrate that increased CTHRC1 levels are associated with enhanced migratory ability. Furthermore, CTHRC1 overexpression caused a dramatic reduction in collagen type I mRNA and protein levels.

Abstract-We identified collagen triple helix repeat containing-1 (Cthrc1) as a novel gene expressed in the adventitia and neointima on arterial injury and found that it functionally increases cell migration while reducing collagen deposition.To address the in vivo role of Cthrc1, we generated transgenic mouse lines that constitutively overexpress Cthrc1. An intercross of 2 transgenic lines produced offspring with brittle bones caused by a reduction in collagenous bone matrix. Hemizygous Cthrc1 transgenic mice developed normally but neointimal lesion formation and adventitial collagen deposition in response to carotid artery ligation were significantly reduced compared with wild-type littermates. In 75% of Cthrc1 transgenic mice, cartilaginous metaplasia of medial smooth muscle cells was observed as assessed by Alcian blue staining and expression of the chondrocyte marker collagen type II. Transforming growth factor-␤ signaling was reduced in smooth muscle cells of Cthrc1 transgenic arteries, as demonstrated by reduced phospho-Smad2/3 immunoreactivity, whereas Smad signaling related to bone morphogenetic proteins was unaffected. Similarly, primary smooth muscle cells and PAC1 smooth muscle cells overexpressing Cthrc1 had reduced levels of phospho-Smad2/3 as well as procollagen. Furthermore, Cthrc1 inhibited transforming growth factor-␤-sensitive reporter constructs in smooth muscle but not endothelial cells. These data indicate that Cthrc1 is a cell-type-specific inhibitor of transforming growth factor-␤, which in turn impacts collagen type I and III deposition, neointimal formation, and dedifferentiation of smooth muscle cells. Key Words: intimal hyperplasia Ⅲ fibrosis Ⅲ bone morphogenetic protein Ⅲ TGF-␤ Ⅲ SMC differentiation W e originally identified collagen triple helix repeat containing-1 (Cthrc1) in a screen for differentially expressed genes in normal versus balloon-injured arteries. 1 In the vasculature, Cthrc1 expression is limited to injured tissue, where it is induced abundantly in adventitial fibroblasts and neointimal smooth muscle cells (SMCs). Expression of Cthrc1 in the injured artery is transient, with Cthrc1 levels becoming undetectable 4 weeks following injury. Thus Cthrc1 expression coincides and colocalizes with adventitial fibrosis, which is responsible for constrictive remodeling following arterial injury and contributes to the failure of angioplasty procedures. 2 Cthrc1 was also found in the matrix of calcifying human atherosclerotic plaques and in mineralized bone of skeletal tissues. 3 The injury response of tissues involves the activated fibroblast termed myofibroblasts based on its characteristic expression of the SMC marker smooth muscle ␣-actin (␣-SMA). 4 As part of the general wound healing process, these myofibroblasts exhibit proliferation, migration, production of collagen types I and III, and other extracellular matrix (ECM) molecules. 5 Cthrc1 enhances migration of SMCs and fibroblasts in a scratch wound assay. 1 We also demonstrated that Cthrc1 reduces collagen type I mR...

With the intention to modulate gene expression in vascular mural cells of remodeling vessels, we generated and characterized transgenic mouse lines with Cre recombinase under the control of the platelet-derived growth factor receptor-β promoter, referred to as Tg(Pdgfrb-Cre)35Vli. Transgenic mice were crossed with the Gt(ROSA)26Sortm1Sor strain and examined for Cre activation by β-galactosidase activity, which was compared with endogenous Pdgfrb expression. In addition, Pdgfrb-Cre mice were used to drive expression of a conditional myc-tagged Cthrc1 transgene. There was good overlap of β-galactosidase activity with endogenous Pdgfrb immunoreactivity. However, dedifferentiation of vascular mural cells induced by carotid artery ligation revealed a dramatic discrepancy between ROSA26 reporter activity and Pdgfrb promoter driven Cre dependent myc-tagged Cthrc1 transgene expression. Our studies demonstrate the capability of the Pdgfrb-Cre mouse to drive conditional transgene expression as a result of prior Cre mediated recombination in tissues known to express endogenous Pdgfrb. In addition, the study shows that ROSA26 promoter driven reporter mice are not suitable for lineage marking of smooth muscle in remodeling blood vessels.

T issue remodeling after injury involves the coordinate regulation of numerous genes, including those involved with extracellular matrix (ECM) synthesis and turnover, cell adhesion, and cell migration. Major components of the ECM include structural proteins and a number of soluble ECMassociated proteins. The soluble ECM-associated proteins include SPARC, lumican, and the fasciclin domain-containing proteins ig-H3 and periostin. Periostin, originally named osteoblast-specific factor-2 (Genbank D13664) was identified as a soluble ECM protein that was expressed in bone and, to a lesser extent, lung.1 The periostin mRNA is alternatively spliced, resulting in polypeptides that differ in the length of their C-terminal domains and in their tissue-specific patterns of expression. 2Periostin is a 90-kDa heparin-binding N-glycosylated protein that was proposed to associate with bone ECM, and it is highly homologous to ig-h3, previously known as the 68-kDa transforming growth factor (TGF)-1-inducible protein. Both periostin and ig-h3 contain 4 tandem fasciclin (Fas1) domains homologous to the insect protein fasciclin. 3 In the case of ig-h3, the second and fourth fasciclin domains interact with the 31 integrin, and all 4 fasciclin domains can interact with the v5 integrin, thus mediating adhesion.4 Periostin also mediates cell adhesion by binding to v3 and v5, and increases cell motility. 5 The localization of recombinant periostin with v5 integrin at sites of focal adhesion 5 suggests the contribution of periostin to cell adhesion and motility. Despite these recent observations of periostin interacting with cell surface receptors, its functional roles are not clear. More recently, periostin expression was found in embryonic hearts by embryonic day E10.5, and increased to a plateau by E14. Periostin mRNA expression was localized to the endocardial cushions that ultimately divide the primitive heart into the mature 4-chambered heart. 6 In addition, periostin expression is increased in pulmonary aortic smooth muscle cells (SMCs)in response to hypoxia. 7The goal of the present study was to examine the role of periostin in SMCs. Our data indicate that periostin is associated with SMC differentiation and functions as a migratory stimulus in vitro. After balloon catheter injury, periostin expression coincides with the proliferative phase of SMCs in the neointima. Methods AnimalsSprague-Dawley rats were used and the left carotid artery and the aorta were denuded with a 2-French balloon catheter as described. Vascular Injury Induces Expression of Periostin Implications for Vascular Cell Differentiation and MigrationVolkhard Lindner, Qiaozeng Wang, Barbara A. Conley, Robert E. Friesel, Calvin P.H. VaryObjective-Periostin mRNA is among the most strongly upregulated transcripts in rat carotid arteries after balloon injury.The goal of the present study was to gain insight into the significance of periostin in the vasculature. Methods and Results-Periostin expression after injury was localized to smooth muscle cells of ...