CCN3 Inhibits Neointimal Hyperplasia Through Modulation of Smooth Muscle Cell Growth and Migration

Abstract

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-␤ 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 See accompanying article on page 667Although 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 ReagentsThe reagents used are described in the expanded Supplementary Materials and Methods section (available online at http://atvb.ahajournals.org). Cell CulturePrimary 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 ReactionReverse-transcription polymerase chain reaction is described in the Supplementary Materials and Methods section. Proliferation AssayVSMC proliferation was quantified by direct cell counting and by 5-bro...

NOV/CCN3: A New Adipocytokine Involved in Obesity-Associated Insulin Resistance

“…Indeed, two reports describing the phenotype of NOV 2/2 mice revealed that they are viable and apparently normal and fertile (22,40). However, after a lesion of the femoral artery, the null mice presented an enhancement of neointimal thickening compared with WT mice during vascular repair (22).…”

“…Mice genotypes were performed as previously described (22). The genotypes were further checked by NOV mRNA levels in epididymal white adipose tissue (eWAT) and subcutaneous WAT (scWAT), the plasma level of the protein and its expression in heart where NOV is normally highly expressed ( Supplementary Fig.…”

“…Thus, it is conceivable that a relationship could exist between NOV and carbohydrate metabolism. In this context, a possible link between NOV and insulin was suggested by Shimoyama et al (22), who reported that NOV expression was reduced in rat aortas after streptozotocin injection and was increased by insulin treatment. Moreover, the nov gene is localized on chromosome 8q24 (23), which is a susceptibility locus controlling b-cell function in linkage studies of patients with diabetes (24).…”

CCN4 Regulates Vascular Smooth Muscle Cell Migration and Proliferation

“…Knockdown of CCN1 by siRNA inhibits neointimal hyperplasia after balloon angioplasty (Matsumae et al, 2008). CCN3 could inhi-bit neointimal hyperplasia through modulation of SMC growth and migration (Shimoyama et al, 2010). According to their structural similarity, we speculated that CCN4 may play a role in cardiovascular disease.…”

Emerging roles of CCN proteins in vascular development and pathology

“…Recent studies have shown that CCN3 can affect vascular function by regulation of endothelial cell inflammation and neointimal hyperplasia (Lin et al 2010;Shimoyama et al 2010). CCN3 knockout mice are viable and develop to adulthood with a normal vasculature and ECM composition (Shimoyama et al 2010). CCN4 (WISP1) was initially identified as a gene in a mouse mammary epithelial cell line (Pennica et al 1998) with later studies restricting CCN4 protein expression to osteoblast and osteoblastic progenitor cells during development and bone fracture repair (French et al 2004).…”