CCN3 Inhibits Neointimal Hyperplasia Through Modulation of Smooth Muscle Cell Growth and Migration
Objective— CCN3 belongs to the CCN family, which constitutes multifunctional secreted proteins that act as matrix cellular regulators. We investigated the pathophysiological roles of CCN3 in the vessels.
Methods and Results— We examined the effects of CCN3 on the proliferation and migration of rat vascular smooth muscle cells (VSMC). CCN3 knockout mice were created, and vascular phenotypes and neointimal hyperplasia induced by photochemically induced thrombosis were investigated. CCN3 suppressed the VSMC proliferation induced by fetal bovine serum. The neutralizing antibody for transforming growth factor-β did not affect the growth inhibitory effect of CCN3. Moreover, CCN3 enhanced the mRNA expression of cyclin-dependent kinase inhibitors, p21 and p15. Gamma secretase inhibitor, an inhibitor of Notch signaling, partially inhibited the enhanced expression of p21 induced by CCN3. CCN3 also inhibited the VSMC migration. Finally, the histopathologic evaluation of the arteries 21 days after the endothelial injury revealed a 6-fold enhancement of neointimal thickening in the null mice compared with the wild-type mice.
Conclusion— CCN3 suppresses neointimal thickening through the inhibition of VSMC migration and proliferation. Our findings indicate the involvement of CCN3 in vascular homeostasis, especially on injury, and the potential usefulness of this molecule in the modulation of atherosclerotic vascular disease.
CCN3/NOV belongs to a family of multicellular growth regulators originally referred to as the acronym CCN (cysteine-rich protein, Cyr 61/CCN1; connective tissue growth factor, CTGF/CCN2; and nephroblastoma overexpressed, NOV/CCN3), which now includes 3 additional genes (Wnt-1-induced secreted proteins 1 to 3; WISP1–3/CCN4–6).1,2 The CCN3 gene was first isolated from avian nephroblastomas induced by viral infections.3 Its antiproliferative activity in a variety of cells is now well-established, especially in tumor cells.4,5
See accompanying article on page 667
Although other members of the CCN family, such as CCN1 and CCN2, are strongly expressed in a wide range of tissues,6,7 CCN3 mRNA is highly and restrictively expressed in rat aortas and carotid arteries.8 This specific expression pattern in vessels indicates that CCN3 plays an important role in vascular homeostasis.
This article accompanies the DVT Series that was published in the March 2010 issue.
The knockdown of CCN1/Cyr61 in mice suppresses neointimal hyperplasia in a rat artery balloon injury model.9 CCN2/CTGF also accumulates in the shoulders of human rupture-prone atherosclerotic plaques.10 Because CTGF induces mononuclear cell chemotaxis in a dose-dependent manner in vitro, CTGF may also have a role in atherogenesis. However, despite the similarity of the amino acid sequence of CCN3 and CCN1 and CCN2,4 the pathophysiological roles of CCN3 in vessels has not been fully elucidated. The present study confirmed the expression of CCN3 in medial layer of mouse aortas. Therefore, the effects of CCN3 on vascular smooth muscle cell (VSMC) proliferation and migration were investigated. Finally, CCN3-null mice were created and the physiological and pathological roles of CCN3 in vessels were determined.
Materials and Methods
The reagents used are described in the expanded Supplementary Materials and Methods section (available online at http://atvb.ahajournals.org).
Primary cultures of rat aortic smooth muscle cells were isolated from 250- to 300-gram Wister Rats (QLEA Japan, Inc.) as described previously.11 See the Supplementary Materials and Methods section for details.
Semiquantitative Reverse-Transcription Polymerases Chain Reaction
Reverse-transcription polymerase chain reaction is described in the Supplementary Materials and Methods section.
VSMC proliferation was quantified by direct cell counting and by 5-bromo-2′-deoxyuridine (BrdU) incorporation using a commercially available enzyme-linked immunosorbent assay kit (Roche Diagnostics). See the Supplementary Materials and Methods section for details.
The ability of smooth muscle cells to migrate toward CCN3 was examined using a 96-well chemotaxis chamber (AB96; Neuro Probe).12 See the Supplementary Materials and Methods section for details.
Western blotting was performed essentially as described previously.13 See the Supplementary Materials and Methods section for details.
Generation of CCN3 Mutant Mice
A knockout mouse line of the CCN3 gene was generated according to methods previously described.14 See the Supplementary Materials and Methods section for details.
Genotyping of Mutant Mice
Genomic southern hybridization and polymerase chain reaction were used to determined the CCN3 genotype (Supplementary Figure IVA, IVB available online at http://atvb.ahajournals.org) using genomic DNA extracted from embryo stem cells and tails, according to methods previously described.14 See the Supplementary Materials and Methods section for details.
Induction of Diabetes
Diabetes was induced by the injection of streptozotocin. See the Supplementary Materials and Methods section for details.
Immunohistochemistry and Immunocytochemistry
Immunohistochemistry and immunocytochemistry are described in the Supplementary Materials and Methods section.
Femoral Artery Injury
Mice femoral arteries were injured by means of a photochemically induced thrombosis method.15 See the Supplementary Materials and Methods section for details.
All values are expressed as the mean±SD. The statistical significance was evaluated using the unpaired Student t test. P<0.05 was considered to be significant.
Expression of CCN3 Protein in Mouse Aorta and Distribution of CCN3 Gene Expression in Mouse Tissues
First, we determined the expression of the CCN3 protein in the mouse aortas by immunohistochemistry using the anti-CCN3-specific antibody. The anti-alpha smooth muscle actin antibody was used as a marker for VSMC and anti-platelet endothelial cell adhesion molecule-1 (PECAM-1) antibody was used as a marker for endothelial cells. The CCN3 protein was localized in the medial layer of the mouse aorta and colocalized with anti-alpha smooth muscle actin-positive cells but not with anti-PECAM-1- positive cells (Figure 1A). Next, we examined the expression of CCN3 in different mouse tissues by using reverse-transcription polymerase chain reaction. As shown in Figure 1B, the expression of CCN3 mRNA was the highest in the heart, weak in the brain, lung, and muscle tissues, and absent in the spleen and intestine.
CCN3 Inhibits VSMC Proliferation Independent of TGF-β Signaling and VSMC Migration
The expression of CCN3 mRNA was examined in cultured rat VSMC. CCN3 mRNA was detected in rat VSMC and increased with time in culture (Supplementary Figure IA).
Next, the biological functions of CCN3 were examined in cultured VSMC. Because CCN3 has antiproliferative activities in several tumor cell lines, we used human recombinant CCN3 proteins to determine whether CCN3 also inhibited VSMC proliferation in vitro. VSMC were plated in culture dishes in the presence or absence of 100 ng/mL CCN3 and the number of cells was counted every day. The number of VSMC was significantly lower in the presence of CCN3 from day 2 until day 4 after the start of the culture. However, this difference disappeared once the VSMC reached confluence (Supplementary Figure IB). The antiproliferative activity of CCN3 was also evaluated using BrdU incorporation. CCN3 alone had no effect on VSMC proliferation. However, CCN3 significantly inhibited VSMC proliferation induced by 10% fetal bovine serum (FBS), as well as that by platelet-derived growth factor-BB (PDGF-BB), in a dose-dependent manner (Figure 2A). Further, we also examined the effects of CCN3 on the cell cycle by flow cytometric analysis. The VSMC were treated with FBS in the presence of CCN3 or TGF-b and labeled with propidium iodide (PI). The ratio of PI-labeled smooth muscle cells in the G2/M phase to those in the G0/G1 phase was significantly decreased after both CCN3 and TGF-b treatment (Supplementary Figure IC, ID). In addition, the Trypan blue exclusion test was used to confirm that CCN3 was not toxic to cells (data not shown).
TGF-β inhibits the proliferation of a variety of cell types.16 Therefore, we examined whether TGF-β is involved in the inhibition of VSMC proliferation by CCN3. A TGF-β-specific neutralizing antibody was used to inhibit TGF-β. Both CCN3 and TGF-β inhibited the VSMC proliferation induced by 10% fetal bovine serum. An anti-TGF-β-specific neutralizing antibody could reverse the antiproliferative effects induced by TGF-β, but not those induced by CCN3. The anti-TGF-β-specific neutralizing antibody alone had no effects on VSMC proliferation (Figure 2B). Moreover, CCN3 also inhibited the VSMC migration induced by FBS in a dose-dependent manner (Figure 2C).
CCN3 Increases the Expression of the CDK Inhibitors p15 and p21 Through the Notch Signaling Pathway
We examined the expression of cell cycle regulators to determine the molecular mechanism underlying the antiproliferative activity of CCN3. Among the factors studied, both CCN3 and TGF-β upregulated the cyclin-dependent kinase inhibitors p15 and p21 within 12 hours (Figure 3A). A time-course study revealed that CCN3 treatment increased the expression of p21 mRNA after 12 hours, but the expression returned to the baseline within 24 hours, whereas TGF-β treatment increased the expression of p21 mRNA in a time-dependent manner (Supplementary Figure IE). This result supported the idea that CCN3 increases the expression of cell-cycle regulators independently of TGF-β signaling. The effects of CCN3 on TGF-β signaling were also examined. CCN3 had little effect on either the phosphorylation of Smad2 (Figure 3B) or the activation of plasminogen activator inhibitor-1 (PAI-1) promoter, which contains the TGF-β-responsive element (Supplementary Figure IF). Collectively, these data indicated that CCN3 inhibited VSMC proliferation independent of TGF-β signaling.
CCN3 activates the Notch signaling pathway through its carboxyl-terminal cysteine-rich domain and inhibits osteogenic differentiation and cell proliferation in Kusa-A1 cells.17 Therefore, the effects of CCN3 on the Notch pathway in VSMC were examined. First, we examined the effect of both TGF-β and CCN3 on the expression of the intracellular domain of Notch1 (ICN1). This domain is proteolytically cleaved from the plasma membrane in conjunction with the activation of the Notch receptor. The antibody (Val 1774) that we used could recognize cleaved ICN1 but could not recognize uncleaved ICN1. Therefore, once the Notch pathway was activated, we were able to observe the ICN1 protein in the cells by immunocytochemistry. CCN3 increased ICN1 expression in the VSMC within 30 minutes (Figure 3C), whereas TGF-β had little effect (data not shown). Next, we investigated the effect of a γ-secretase inhibitor, which blocks the Notch signaling pathway not only during VSMC proliferation but also during p21 regulation. γ-Secretase inhibitor reversed the CCN3-dependent antiproliferative effects of VSMC, which had been evaluated using BrdU incorporation (Supplementary Figure IIA). γ-Secretase inhibitor alone slightly increased the expression of p21. However, the upregulation of p21 expression by both CCN3 and TGF-β was partially but significantly suppressed by γ-secretase inhibitor treatment (Figure 3D). We also examined the effects of RPMS-1, a known blocker of the RBP-J-mediated Notch signaling pathway, on the effects of CCN3. As shown in Supplementary Figure IIB, the expression of Hey1, a gene targeted by the Notch signal, increased in the presence of CCN3. The overexpression of RPMS-1 in the VSMC inhibited the CCN3-induced expression of Hey1. The increase in the expression of p21 mRNA induced by CCN3 was suppressed by the overexpression of RPMS-1. These results indicated that CCN3/Notch is a signaling pathway that increases the expression of the cell-cycle regulators. Recently it has been reported that PDGF receptor-β is a direct target of the activated Notch1 and Notch3 receptors.18 Therefore, we examined the effects of CCN3 on the expression of PDGF receptor-β and its tyrosine phosphorylation and ERK activation in VSMC. None of these responded to CCN3, as shown in Supplementary Figure IIIA and IIIB.
Mice Lacking CCN3 Show Enhanced Neointimal Hyperplasia in Response to Injury
CCN3−/− mice were generated by conventional homologous recombination to investigate the physiological and pathological roles of CCN3 in vivo. Bacterial artificial chromosome-based targeting vectors were used to specifically replace exons 1, 2, and a part of exon 3 of the CCN3 gene with a neomycin selection cassette (Figure 4A). Chimeric mice derived from embryo stem cell clones were bred to C57BL/6 males to produce F1 offspring and CCN3+/− mice were intercrossed to produce homozygous offspring (Supplementary Figure IVA, IVB). The deletion in the mRNA corresponding to the genomic deletion of the CCN3 locus was confirmed using reverse-transcription polymerase chain reaction with cDNA generated from heart RNA that had been extracted from 28-day-old mice (Supplementary Figure IVC). CCN3 protein was not detected in the aortas from CCN3−/− mice by immunohistochemistry (Figure 4B). Intercrosses between CCN3+/− animals produced viable offspring at the expected Mendelian frequency (data not shown). CCN3−/− animals developed normally to adulthood and both the males and females were fertile. The Table shows the background data for the 2-month-old CCN3−/− mice in comparison to their littermate controls. There were no obvious differences in body weight, systolic blood pressure, plasma glucose concentrations, and HbA1c levels between the 2 groups. Hematoxylin and eosin staining and Masson trichrome staining of the aortas dissected from the 2-month-old mice showed that the vasculature appeared to have developed normally (Figure 5A). The amount of type III collagen, which is one of the main components of the extracellular matrix in the aorta,19 also showed that there were no obvious changes in the amount of extracellular matrix because of the lack of CCN3 (Figure 5A). Next, to evaluate the role of CCN3 in the pathogenesis of neointimal hyperplasia, we injured the femoral arteries of the wild-type (n=6) and CCN3-null (n=6) male mice, using photochemically induced thrombosis. Histopathologic examination of the arteries 21 days after the injury revealed markedly enhanced neointimal thickening in the CCN3-null mice in comparison to the wild-type mice (Figure 5B). The mean intima-to-media ratios at 21 days after the injury were significantly higher in the arteries of the CCN3-null mice (1.193±0.071) than those of the wild-type mice (0.205±0.011; P<0.01; Figure 5C).
To understand how intimal thickening is enhanced in the CCN3-null mice, we studied cell proliferation and endothelialization after the vascular injury (Supplementary Figure VA, VB). We investigated the BrdU incorporated into the vascular wall 1 week after photochemically induced thrombosis. The number of BrdU-positive cells was higher in the aortas of the CCN3-null mice than in those of the control mice, indicating that the VSMC proliferated even in the absence of CCN3. We also investigated the endothelialization after antiplatelet endothelial cell adhesion molecule staining and found that, compared to the controls, endothelialization was reduced in the aortas of the CCN3-null mice.
We also examined platelet function by measuring the tail bleeding time (data not shown) and adventitial neovascularization by anti-PECAM-1 staining (Supplementary Figure VB). However, there were no differences in either the tail bleeding time or the adventitial neovascularization.
CCN3 Expression Decreases in the Aorta of Streptozotocin-Induced Diabetic Mice
Finally, to determine the relevance of CCN3 expression to disease conditions, we examined the CCN3 expression in diabetic mice and found that it was significantly lower in the aorta of diabetic rats in comparison to control rats (Supplementary Figure VIA, VIB). Insulin treatment for reducing the blood glucose level increased the CCN3 expression in vessels, excluding the possibility that CCN3 expression decreased because of the toxic effects of streptozotocin (Supplementary Figure VIC).
This article reports 5 novel findings. First, the CCN3 protein was expressed in the medial layer of the mouse aorta, especially in VSMC in vivo. Second, CCN3 inhibited VSMC proliferation and migration. Third, CCN3 inhibited VSMC proliferation independent of TGF-β signaling. Fourth, CCN3 increased the expression of the cyclin-dependent kinase inhibitor, p21, at least partly through Notch signaling in VSMC. Fifth, CCN3−/− mice had a normal vascular phenotype under normal conditions; however, CCN3 enhanced the neointimal hyperplasia in femoral arteries induced by endothelial injury.
Atherosclerosis is a leading cause of cardiovascular diseases around the world. VSMC migration and proliferation in the subendothelial space are pivotal steps in atherosclerosis development20 and in restenosis formation after vascular intervention.21 The recent development of drug-eluting stents, which release small amounts of antiproliferative agents locally, is an effective strategy for reducing restenosis,22 which is primarily caused by the proliferation of VSMC. However, reducing the incidence of restenosis remains a considerable burden for the medical community. Therefore, the biological stimuli that inhibit VSMC proliferation and migration must be completely understood to address various clinical problems.
CCN3 plays potent roles in tumorigenesis,2 chondrogenesis, skeletal and cardiac development,23 and hematopoietic stem cell regulation.24 However, the roles of CCN3 in vessels are not yet fully understood. Ellis et al8 precisely examined the expression of CCN3 in the rat aorta and in the carotid artery. They localized CCN3 mRNA expression to the smooth muscle cells in both the aorta and carotid artery. Further, they showed 7 days after injury to the carotid artery CCN3 expression in the media is substantially reduced but that at the edge of the intima increases. At 14 days after the injury, the CCN3 expression in the media remains low, but it is substantially increased throughout the intima in the VSMC. These observations strongly indicate that CCN3 is involved in vascular homeostasis, especially after vascular injury. Using immunohistology, we established that the CCN3 protein is expressed in the smooth muscle cell layer in the mouse aorta. To our knowledge, this is the first in vivo report to confirm CCN3 protein expression in aortic smooth muscle cells.
In agreement with the antiproliferative effects of CCN3 in a large variety of cell types, we could also show that CCN3 inhibited VSMC proliferation in vitro (Figure 2A, 2B). However, Ellis et al8 reported that CCN3 has no effect on VSMC proliferation. However, there were some significant differences in the experimental procedures used. The current study used human recombinant CCN3, which shares 80% amino acid identity with that of mouse CCN3. In contrast, Ellis et al used recombinant CCN3 prepared using a baculovirus system. They also used a higher concentration of PDGF-BB and a different kind of fetal bovine serum, which induced higher proliferation rates than those seen in the current study. Therefore, the antiproliferative effects of CCN3 may have been masked by the potent mitogenic stimulation. Furthermore, the antiproliferative effects of CCN3 seemed transient (Supplementary Figure IB). Therefore, it might be difficult to determine these antiproliferative effects if the incubation time is longer than that used in this experiment.
The next series of experiments examined how CCN3 inhibits VSMC proliferation. CCN3 physically interacts with fibulin C,25 integrins,5 and connexin 43.26 In addition, CCN3 inhibits proliferation and differentiation through the Notch pathway.17,27 Notch signaling dictates the cell fate and critically influences cell proliferation, differentiation, and apoptosis. It is observed in various tissues, including vessels. For instance, Notch ligands,28 receptors,29 and effectors30 are expressed in VSMC in vivo. Furthermore, Notch signaling also plays a critical role in the correct architecture of vascular systems.31 Therefore, we investigated the involvement of the Notch signaling pathway in the antiproliferative activity of CCN3. CCN3 increased the expression of ICN1, whereas γ-secretase inhibitor reversed the antiproliferative effects of CCN3 and partially inhibited the CCN3-induced upregulation of p21 (Figure 3C, 3D, and Supplementary Figure II); this indicated that the Notch signaling pathway was at least partially involved in the antiproliferative activity of CCN3. In addition, we examined the participation of TGF-β signaling in the CCN3-induced antiproliferative activity of VSMC, because TGF-β inhibits cell growth in a variety of cell types. Moreover, TGF-β and the Notch pathway cooperatively suppress epithelial cell growth.32 However, anti-TGF-β neutralizing antibody could not reverse the CCN3-induced antiproliferative activity in VSMC (Figure 2B). Furthermore, CCN3 had no effect on the phosphorylation of Smad2, which mediates TGF-β signaling and the activity of the plasminogen activator inhibitor-1 promoter, which contains the TGF-β-responsive element. CCN3 also did not have any effect on the expression of the mRNA of either TGF-β or its receptors (data not shown). These results indicate that the inhibition of VSMC proliferation by CCN3 is partially dependent on the Notch pathway but independent of TGF-β signaling.
Despite the growth inhibitory effects of CCN3 in a wide variety of cell types, CCN3−/− mice were surprisingly viable and fertile. CCN3−/− mice could not be distinguished from their littermate controls by their gross appearance or vascular structure (Figure 5A).
CCN3 knockout mice were recently generated by another group.23 Exon 3 of the CCN3 gene was targeted (NOVdel3−/−) and some, but not all, of the NOVdel3−/− mice were embryonically lethal and exhibited defects in both skeletal and cardiovascular development. However, NOVdel3−/− mice produce a mutant form of the CCN3 protein, which can be secreted and lacks only the von Willebrand factor (VWC) domain but contains 3 other intact functional domains. Furthermore, this phenotype is partly transmitted by dominant inheritance. Therefore, this protein is strongly suspected to result in the phenotypes observed in NOVdel3−/− mice. In contrast, the mRNA in CCN3−/− mice lacked a region encoding 127 amino acids from the N-terminal end. This may result in a peptide that is translated from the fourth Methionine in complete CCN3 mRNA and is not secreted because of the absence of the presequence required for secretion. Therefore, we believe that the presence of the mutant CCN3 peptide only in NOVdel3−/− leads to phenotypic differences between both types of mutant mice.
Although the vascular phenotype was normal under normal conditions, the enhancement of the intimal thickening in the CCN3-null mice after endothelial injury suggested that CCN3 plays important roles in pathological situations and also indicated that CCN3 inhibited VSMC proliferation and migration. Among the several methods that could induce the intimal thickening, such as flexible wire, balloon catheter, and laser injury, we chose the photochemically induced thrombosis method, which could induce intimal thickening constantly and relatively milder than the others.
It has also been reported that CCN3 induces neovascularization. Further, we observed reduced endothelialization in CCN3-null mice. Therefore, increased VSMC proliferation and reduced endothelialization can affect the increased intimal thickening seen in the CCN3-null mice.
Finally, we studied CCN3 expression in diabetic conditions and found that this expression was reduced in diabetic aortas as compared to the control aortas. Diabetic patients are prone to atherosclerotic vascular disease33 and diabetic vascular lesions are prone to restenosis after angioplasty.34 However, the mechanism by which atherogenesis is accelerated in patients with diabetes mellitus has not yet been determined. Because CCN3 inhibited VSMC proliferation and migration, the reduced expression of CCN3 might be linked with the accelerated atherogenesis in diabetes. So far, there have been few reports describing CCN3 gene regulation. For instance, 1 report35 describes that Wilms tumor 1, a transcriptional factor, binds to the CCN3 promoter region and regulates its expression. In addition, the expression of the tumor suppressor gene p53 depends on CCN3 expression.36 It therefore would be useful to investigate the precise transcriptional regulation of CCN3 and seek pharmacological agents to prevent the downregulation of CCN3 in diabetic vessels to reduce the risk of diabetic vascular complications in the future.
In summary, this study demonstrated that CCN3 is a regulator of VSMC proliferation and migration and of neointimal hyperplasia. Therefore, these results indicate the potential usefulness of this molecule in the modulation of atherosclerotic vascular disease.
The authors thank Reiko Kimura (Department of Clinical Cell Biology and Medicine, Chiba University Graduate School of Medicine) for her valuable technical assistance.
Sources of Funding
This study is supported by grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology; Ministry of Health, Labor, and Welfare; a grant from Mitsubishi Pharma Research Foundation; and a grant from the Takeda Scientific foundation.
T.S. and S.H. contributed equally to this work.
Received on: November 29, 2009; final version accepted on: January 14, 2009.
Brigstock DR, Goldschmeding R, Katsube KI, Lam SC, Lau LF, Lyons K, Naus C, Perbal B, Riser B, Takigawa M, Yeger H. Proposal for a unified CCN nomenclature. Mol Pathol. 2003; 56: 127–128.
Gupta N, Wang H, McLeod TL, Naus CC, Kyurkchiev S, Advani S, Yu J, Perbal B, Weichselbaum RR. Inhibition of glioma cell growth and tumorigenic potential by CCN3 (NOV). Mol Pathol. 2001; 54: 293–299.
Ryseck RP, Macdonald-Bravo H, Mattei MG, Bravo R. Structure, mapping, and expression of fisp-12, a growth factor-inducible gene encoding a secreted cysteine-rich protein. Cell Growth Differ. 1991; 2: 225–233.
Ellis PD, Chen Q, Barker PJ, Metcalfe JC, Kemp PR. Nov gene encodes adhesion factor for vascular smooth muscle cells and is dynamically regulated in response to vascular injury. Arterioscler Thromb Vasc Biol. 2000; 20: 1912–1919.
Matsumae H, Yoshida Y, Ono K, Togi K, Inoue K, Furukawa Y, Nakashima Y, Kojima Y, Nobuyoshi M, Kita T, Tanaka M. CCN1 knockdown suppresses neointimal hyperplasia in a rat artery balloon injury model. Arterioscler Thromb Vasc Biol. 2008; 28: 1077–1083.
Cicha I, Yilmaz A, Klein M, Raithel D, Brigstock DR, Daniel WG, Goppelt-Struebe M, Garlichs CD. Connective tissue growth factor is overexpressed in complicated atherosclerotic plaques and induces mononuclear cell chemotaxis in vitro. Arterioscler Thromb Vasc Biol. 2005; 25: 1008–1013.
Yokote K, Mori S, Hansen K, McGlade J, Pawson T, Heldin CH, Claesson-Welsh L. Direct interaction between Shc and the platelet-derived growth factor beta-receptor. J Biol Chem. 1994; 269: 15337–15343.
Joyner AL. Gene Targeting. Oxford, UK: Oxford University Press; 1992.
Kikuchi S, Umemura K, Kondo K, Saniabadi AR, Nakashima M. Photochemically induced endothelial injury in the mouse as a screening model for inhibitors of vascular intimal thickening. Arterioscler Thromb Vasc Biol. 1998; 18: 1069–1078.
Katsuki Y, Sakamoto K, Minamizato T, Makino H, Umezawa A, Ikeda MA, Perbal B, Amagasa T, Yamaguchi A, Katsube K. Inhibitory effect of CT domain of CCN3/NOV on proliferation and differentiation of osteogenic mesenchymal stem cells, Kusa-A1. Biochem Biophys Res Commun. 2008; 368: 808–814.
Jin S, Hansson EM, Tikka S, Lanner F, Sahlgren C, Farnebo F, Baumann M, Kalimo H, Lendahl U. Notch signaling regulates platelet-derived growth factor receptor-beta expression in vascular smooth muscle cells. Circ Res. 2008; 102: 1483–1491.
Gupta R, Hong D, Iborra F, Sarno S, Enver T. NOV (CCN3) functions as a regulator of human hematopoietic stem or progenitor cells. Science. 2007; 316: 590–593.
Perbal B, Martinerie C, Sainson R, Werner M, He B, Roizman B. The C-terminal domain of the regulatory protein NOVH is sufficient to promote interaction with fibulin 1C: a clue for a role of NOVH in cell-adhesion signaling. Proc Natl Acad Sci U S A. 1999; 96: 869–874.
Fu CT, Bechberger JF, Ozog MA, Perbal B, Naus CC. CCN3 (NOV) interacts with connexin43 in C6 glioma cells: possible mechanism of connexin-mediated growth suppression. J Biol Chem. 2004; 279: 36943–36950.
Sakamoto K, Yamaguchi S, Ando R, Miyawaki A, Kabasawa Y, Takagi M, Li CL, Perbal B, Katsube K. The nephroblastoma overexpressed gene (NOV/CCN3) protein associates with Notch1 extracellular domain and inhibits myoblast differentiation via Notch signaling pathway. J Biol Chem. 2002; 277: 29399–29405.
Chin MT, Maemura K, Fukumoto S, Jain MK, Layne MD, Watanabe M, Hsieh CM, Lee ME. Cardiovascular basic helix loop helix factor 1, a novel transcriptional repressor expressed preferentially in the developing and adult cardiovascular system. J Biol Chem. 2000; 275: 6381–6387.
Roca C, Adams RH. Regulation of vascular morphogenesis by Notch signaling. Genes Dev. 2007; 21: 2511–2524.
Niimi H, Pardali K, Vanlandewijck M, Heldin CH, Moustakas A. Notch signaling is necessary for epithelial growth arrest by TGF-beta. J Cell Biol. 2007; 176: 695–707.
Andresen JL, Rasmussen LM, Ledet T. Diabetic macroangiopathy and atherosclerosis. Diabetes. 1996; 45 (Suppl 3): S91–S94.
Rozenman Y, Sapoznikov D, Mosseri M, Gilon D, Lotan C, Nassar H, Weiss AT, Hasin Y, Gotsman MS. Long-term angiographic follow-up of coronary balloon angioplasty in patients with diabetes mellitus: a clue to the explanation of the results of the BARI study. Balloon Angioplasty Revascularization Investigation. J Am Coll Cardiol. 1997; 30: 1420–1425.