SMOC-2 is a novel member of the SPARC family of matricellular proteins. The purpose of this study was to determine whether SMOC-2 can modulate angiogenic growth factor activity and angiogenesis. SMOC-2 was localized in the extracellular periphery of cultured human umbilical vein endothelial cells (HUVECs). Ectopically expressed SMOC-2 was also secreted into the tissue culture medium. In microarray profiling experiments, a recombinant SMOC-2 adenovirus induced the expression of transcripts required for cell cycle progression in HUVECs. Consistent with a growth-stimulatory role for SMOC-2, its overexpression stimulated DNA synthesis in a dosedependent manner. Overexpressed SMOC-2 also synergized with vascular endothelial growth factor or with basic fibroblast growth factor to stimulate DNA synthesis. Ectopically expressed SMOC-2 stimulated formation of network-like structures as determined by in vitro matrigel angiogenesis assays. Fetal calf serum enhanced the stimulatory effect of overexpressed SMOC-2 in this assay. Conversely, small interference RNA directed toward SMOC-2 inhibited network formation and proliferation. The angiogenic activity of SMOC-2 was also examined in experimental mice by subdermal implantation of Matrigel plugs containing SMOC-2 adenovirus. SMOC-2 adenovirus induced a 3-fold increase in the number of cells invading Matrigel plugs when compared with a control adenoviral vector. Basic fibroblast growth factor and SMOC-2 elicited a synergistic effect on cell invasion. Taken together, our results demonstrate that SMOC-2 is a novel angiogenic factor that potentiates angiogenic effects of growth factors.

Abstract-Glycogen-Synthase Kinase 3␤ (GSK3␤) has been shown to function as a nodal point of converging signaling pathways in endothelial cells to regulate vessel growth, but the signaling mechanisms downstream from GSK3␤ have not been identified. Here, we show that ␤-catenin is an important downstream target for GSK3␤ action in angiogenesis and dissect the signal transduction pathways involved in the angiogenic phenotype. Transduction of human umbilical vein endothelial cells (HUVECs) with a kinase-mutant form of the enzyme (KM-GSK3␤) increased cytosolic ␤-catenin levels, whereas constitutively active GSK3␤ (S9A-GSK3␤) reduced ␤-catenin levels. Lymphoid enhancer factor/T-cell factor promoter activity was upregulated by KM-GSK3␤ and diminished by S9A-GSK3␤, whereas manipulation of Akt signaling had no effect on this parameter. ␤-Catenin transduction induced capillary formation in a Matrigel-plug assay in vivo and promoted endothelial cell differentiation into network structures on Matrigel-coated plates in vitro. ␤-Catenin activated the expression of vascular endothelial growth factor (VEGF)-A and VEGF-C in endothelial cells, and these effects were mediated at the levels of protein, mRNA, and promoter activity. Consistent with these data, ␤-catenin increased the phosphorylation of the VEGF receptor 2 (VEGF-R2) and promoted its association with PI3-kinase, leading to a dose-dependent activation of the serine-threonine kinase Akt. Inhibition of PI3-kinase or Akt signaling led to a significant reduction in the pro-angiogenic activity of ␤-catenin. Collectively, these data show that the growth factor-PI3-kinase-Akt axis functions downstream of GSK3␤/␤-catenin signaling in endothelial cells to promote angiogenesis. (Circ Res. 2005;96:308-318.)Key Words: ␤-catenin Ⅲ Akt Ⅲ endothelial cells Ⅲ vacular endothelial growth factor Ⅲ VEGF receptor 2 Ⅲ angiogenesis G lycogen-synthase kinase 3␤ (GSK3␤) is a serine/threonine protein kinase that regulates differentiation and proliferation in diverse tissues. 1 Recently, GSK3␤ has been shown to play an important role in angiogenesis through its control of vascular cell migration and differentiation, 2 but the downstream targets that transmit these pro-angiogenic effects have not been elucidated.The serine/threonine kinase Akt/PKB is an upstream regulator of GSK3␤ that controls its activity in response to growth factor stimulation. Akt is an important regulator of angiogenic responses in endothelial cells through its ability to promote migration, differentiation, and nitric oxide production. 3 Phosphorylation of GSK3␤ at an amino-terminal Ser-9 residue by Akt results in the auto-inhibition of GSK3␤. 4 GSK3␤ activity can also be controlled by Wnts through a mechanism that generally differs from that used by mitogenic factor-mediated phosphorylation. 5,6 In the absence of Wnt signaling, ␤-catenin is associated within a cytosolic multiprotein complex consisting of adenomatous polyposis coli protein, GSK3␤, and axin. 1 GSK3␤ constitutively phosphorylates ␤-catenin at both serine and ...

Migration of smooth muscle cells (SMCs) and collagen synthesis by SMCs are central to the pathophysiology of vascular disease. Both processes can be induced shortly after vascular injury; however, a functional relationship between them has not been established. In this study, we determined if collagen synthesis was required for SMC migration, using ethyl-3,4-dihydroxybenzoate (EDHB), an inhibitor of prolyl-4-hydroxylase, and 3,4-DL-dehydroproline (DHP), a proline analogue, which we demonstrate inhibit collagen elaboration by porcine arterial SMCs. SMCs exposed to EDHB or DHP attached normally to collagen- and vitronectin-coated substrates; however, spreading on collagen but not vitronectin was inhibited. SMC migration speed, quantified by digital time-lapse video microscopy, was significantly and reversibly reduced by EDHB and DHP. Flow cytometry revealed that expression of beta1 integrins, through which SMCs interact with collagen, was unaffected by EDHB or DHP. However, both inhibitors prevented normal clustering of beta1 integrins on the surface of SMCs, consistent with a lack of appropriate matrix ligands for integrin engagement. Moreover, there was impaired recruitment of vinculin into focal adhesion complexes of spreading SMCs and disassembly of the smooth muscle alpha-actin-containing cytoskeleton. These findings suggest that de novo collagen synthesis plays a role in SMC migration and implicates a mechanism whereby newly synthesized collagen may be necessary to maintain the transcellular traction system required for effective locomotion.

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