CYR61 (CCN1) is a member of the CCN family of secreted matricellular proteins that includes connective tissue growth factor (CCN2), NOV (CCN3), WISP-1 (CCN4), WISP-2 (CCN5), and WISP-3 (CCN6). First identified as the product of a growth factor-inducible immediate-early gene, CYR61 is an extracellular matrixassociated angiogenic inducer that functions as a ligand of integrin receptors to promote cell adhesion, migration, and proliferation. Aberrant expression of Cyr61 is associated with breast cancer, wound healing, and vascular diseases such as atherosclerosis and restenosis. To understand the functions of CYR61 during development, we have disrupted the Cyr61 gene in mice. We show here that Cyr61-null mice suffer embryonic death: ϳ30% succumbed to a failure in chorioallantoic fusion, and the reminder perished due to placental vascular insufficiency and compromised vessel integrity. These findings establish CYR61 as a novel and essential regulator of vascular development. CYR61 deficiency results in a specific defect in vessel bifurcation (nonsprouting angiogenesis) at the chorioallantoic junction, leading to an undervascularization of the placenta without affecting differentiation of the labyrinthine syncytiotrophoblasts. This unique phenotype is correlated with impaired Vegf-C expression in the allantoic mesoderm, suggesting that CYR61-regulated expression of Vegf-C plays a role in vessel bifurcation. The genetic and molecular basis of vessel bifurcation is presently unknown, and these findings provide new insight into this aspect of angiogenesis.

cyr61 was first identified as a growth factor-inducible immediate-early gene in mouse fibroblasts. The encoded Cyr61 protein is a secreted, cystein-rich heparin-binding protein that associates with the cell surface and the extracellular matrix, and in these aspects it resembles the Wnt-1 protein and a number of known growth factors. During embryogenesis, cyr61 is expressed most notably in mesenchymal cells that are differentiating into chondrocytes and in the vessel walls of the developing circulatory system. cyr61 is a member of an emerging gene family that encodes growth regulators, including the connective tissue growth factor and an avian proto-oncoprotein, Nov cyr61 also shares sequence similarities with two Drosophila genes, twisted gastrulation and short gastrulation, which interact with decapentaplegic to regulate dorsal-ventral patterning. In this report we describe the purification of the Cyr61 protein in a biologically active form, and we show that purified Cyr61 has the following activities: (i) it promotes the attachment and spreading of endothelial cells in a manner similar to that of fibronectin; (ii) it enhances the effects of basic fibroblast growth factor and platelet-derived growth factor on the rate of DNA synthesis of fibroblasts and vascular endothelial cells, although it has no detectable mitogenic activity by itself; and (iii) it acts as a chemotactic factor for fibroblasts. Taken together, these activities indicate that Cyr61 is likely to function as an extracellular matrix signaling molecule rather than as a classical growth factor and may regulate processes of cell proliferation, migration, adhesion, and differentiation during development.

Cyr61 is a heparin-binding, extracellular matrix-associated protein of the CCN family, which also includes connective tissue growth factor, Nov, WISP-1, WISP-2, and WISP-3. Cyr61 is capable of multiple functions, including induction of angiogenesis in vivo. Purified Cyr61 mediates cell adhesion and induces adhesive signaling, stimulates cell migration, enhances cell proliferation, and promotes cell survival in both fibroblasts and endothelial cells. In this study, we have used cDNA array hybridization to identify genes regulated by Cyr61 in primary human skin fibroblasts. The Cyr61-regulated genes fall into several groups known to participate in processes important for cutaneous wound healing, including: 1) angiogenesis and lymphogenesis (VEGF-A and VEGF-C); 2) inflammation (interleukin-1␤); 3) extracellular matrix remodeling (MMP1, MMP3, TIMP1, uPA, and PAI-1); and 4) cell-matrix interactions (Col1␣1, Col1␣2, and integrins ␣ 3 and ␣ 5 ). Cyr61-mediated gene expression requires heparin binding activity of Cyr61, cellular de novo transcription, and protein synthesis and is largely dependent on the activation of p42/p44 MAPKs. Cyr61 regulates gene expression not only in serum-free medium but also in fibroblasts cultured on various matrix proteins or in the presence of 10% serum. These effects of Cyr61 can be sustained for at least 5 days, consistent with the time course of wound healing in vivo. Interestingly, Cyr61 can interact with transforming growth factor-␤1 to regulate expression of specific genes in an antagonistic, additive, or synergistic manner. Furthermore, we show that the Cyr61 gene is highly induced in dermal fibroblasts of granulation tissue during cutaneous wound repair. Together, these results show that Cyr61 is inducibly expressed in granulation tissues after wounding and that Cyr61 activates a genetic program for wound repair in skin fibroblasts. We propose a model in which Cyr61 integrates its activities on endothelial cells, fibroblasts, and macrophages to regulate the processes of angiogenesis, inflammation, and matrix remodeling in the context of cutaneous wound healing.

Abstract-The matricellular protein CCN1 (formerly named CYR61) regulates cell adhesion, migration, proliferation, survival, and differentiation through binding to integrin receptors and heparan sulfate proteoglycans. Here we show that Ccn1-null mice are impaired in cardiac valvuloseptal morphogenesis, resulting in severe atrioventricular septal defects (AVSD). Remarkably, haploinsufficiency for Ccn1 also results in delayed formation of the ventricular septum in the embryo and persistent ostium primum atrial septal defects (ASD) in Ϸ20% of adults. Mechanistically, Ccn1 is not required for epithelial-to-mesenchymal transformation or cell proliferation and differentiation in the endocardial cushion tissue. However, Ccn1 deficiency leads to precocious apoptosis in the atrial junction of the cushion tissue and impaired gelatinase activities in the muscular component of the interventricular septum at embryonic day 12.5, when fusion between the endocardial cushion tissue and the atrial and ventricular septa occurs, indicating that these defects may underlie the observed AVSD. Moreover, human CCN1 maps to 1p21-p31, the chromosomal location of an AVSD susceptibility gene. Together, these results provide evidence that deficiency in matrix signaling can lead to autosomal dominant AVSD, identify Ccn1 ϩ/Ϫ mice as a genetic model for ostium primum ASD, and implicate CCN1 as a candidate gene for AVSD in humans. Key Words: apoptosis Ⅲ cardiac development Ⅲ cardiovascular disease Ⅲ integrin Ⅲ matricellular genes Ⅲ matrix metalloproteinases Ⅲ transgenic mice A trioventricular septal defect (AVSD) is a common family of genetic disorders, accounting for Ϸ7.5% of the recognized congenital heart disease (CHD) in humans. 1 During cardiac development, the endocardial cushion tissue expands through formation of the cardiac jelly, a specialized extracellular matrix (ECM) between the endocardium and myocardium, into which endocardial cells that have undergone epithelial-to-mesenchymal transformation (EMT) invade. Subsequent growth and remodeling of the cushion tissue, coupled with its fusion with the developing atrial septum and muscular interventricular septum, lead to formation of the definitive atrioventricular (AV) septa and valves. 2,3 Defects in this process are manifested in a spectrum of abnormalities with varying severities, ranging from partial forms of AVSD with ostium primum atrial septal defects (ASD) to complete forms with absence of the AV septa and lack of partitioning of the AV valve into separate mitral (left) and tricuspid (right) valves. Although AVSD is frequently associated with Down's syndrome (trisomy 21), nonsyndromic AVSD also occur. Although nonsyndromic AVSD may be a sporadic trait or the result of multifactorial inheritance, strong evidence also support the presence of susceptibility genes for AVSD that are autosomal dominant and incompletely penetrant. 4 -6 CCN1 (formerly named CYR61 [CYsteine-Rich angiogenic inducer 61; NM_001554) is a secreted, cysteine-rich protein associated with the ECM. A dynamically ...