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...

Werner syndrome (WS) is an autosomal recessive progeroid disorder caused by mutations in RecQ DNA helicase. Ectopic soft tissue calcification is one of the well known symptoms in WS. However, the prevalence, clinical outcome, and mechanism of such calcification remain to be elucidated. The clinical features and mechanism of ectopic calcification were examined in seven patients with WS whose diagnosis were confirmed by a genomic DNA analysis. X-ray examinations revealed subcutaneous calcification in 35 of 41 major joints (85.3%). The patients complained of dermal pain at 23 joints among 35 joints (65.7%) with calcification. Refractory skin ulcers were found at the area of the skin overlaying the calcification in 16 joints (45.7%). In contrast, no pain or ulcers were observed in the joints without calcification. The presence of ectopic calcification could not be explained by a systemic hormonal abnormality. Cultured fibroblasts from WS patients underwent spontaneous mineralization in vitro in the normal phosphate condition, and overexpressed Pit-1, a transmembrane type III Na-Pi cotransporter both at the mRNA and protein levels. Phosphonophormic acid, a specific inhibitor for Pit-1, inhibited mineralization in the WS fibroblasts. Both calcification and Pit-1 overexpression were detected in the skin of WS in situ. WS showed a high prevalence of ectopic calcification, which was associated with dermal pain and refractory skin ulcers. An overexpression of Pit-1 therefore seems to play a key role in the formation of soft tissue calcification in this syndrome.

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