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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Hsp47 is a heat stress protein that interacts with procollagen in the lumen of the endoplasmic reticulum, which is vital for collagen elaboration and embryonic viability. The precise actions of Hsp47 remain unclear, however. To evaluate the effects of Hsp47 on collagen production we infected human vascular smooth muscle cells (SMCs) with a retrovirus containing Hsp47 cDNA. SMCs overexpressing Hsp47 secreted type I procollagen faster than SMCs transduced with empty vector, yielding a greater accumulation of pro␣1(I) collagen in the extracellular milieu. Interestingly, the amount of intracellular pro␣1(I) collagen was also increased. This was associated with an unexpected increase in the rate of pro␣1(I) collagen chain synthesis and 2.5-fold increase in pro␣1(I) collagen mRNA expression, without a change in fibronectin expression. This amplification of procollagen expression, synthesis, and secretion by Hsp47 imparted SMCs with an enhanced capacity to elaborate a fibrillar collagen network. The effects of Hsp47 were qualitatively distinct from, and independent of, those of ascorbate and the combination of both factors yielded an even more intricate fibril network. Given the in vitro impact of altered Hsp47 expression on procollagen production, we sought evidence for interindividual variability in Hsp47 expression and identified a common, single nucleotide polymorphism in the Hsp47 gene promoter among African Americans that significantly reduced promoter activity. Together, these findings indicate a novel means by which type I collagen production is regulated by the endoplasmic reticulum constituent, Hsp47, and suggest a potential basis for inherent differences in collagen production within the population.

Background-Heat shock protein 47 (Hsp47) is a stress protein that may act as a chaperone for procollagen. Its involvement in atherosclerosis is unknown. Methods and Results-Hsp47 expression in human coronary arteries was assessed by immunostaining. Strong focal expression was evident in atherosclerotic, but not normal, arteries and was prevalent in the collagenous regions. Double immunostaining revealed that all cells expressing type I procollagen also expressed Hsp47. Moreover, parallel regulation of pro␣1(I)collagen and Hsp47 mRNA expression occurred with cultured human smooth muscle cells stimulated with transforming growth factor-␤1 or fibroblast growth factor-2. However, a proportion of Hsp47-expressing cells in plaque did not express type I procollagen, and this pattern could be reproduced in culture. Heat shock and oxidized LDL stimulated the expression of Hsp47 mRNA by smooth muscle cells, without a concomitant rise in pro␣1(I)collagen expression. Conclusions-These See p 1224The major collagen species in human atherosclerotic plaque is type I collagen. The synthesis of type I collagen involves the productive association, within the endoplasmic reticulum, of 2 pro␣1(I)collagen chains and one pro␣2(I)collagen chain. The sorting and processing of procollagen chains into a native triple helix is not spontaneous; instead, it likely depends on the participation of molecular chaperones. 2 Heat shock protein 47 (Hsp47) is a heat shockinducible glycoprotein that binds nascent type I procollagen chains as they translocate into the endoplasmic reticulum. 3 The duration of binding is longer if stable triple helix formation is inhibited, 4 and the inhibition of Hsp47 expression has been associated with the decreased production of type I collagen. 3 These findings suggest that Hsp47 may be a chaperone for type I procollagen.Factors that control the efficiency and fidelity of procollagen folding could be critical to the clinical course of atherosclerosis. Therefore, we determined whether Hsp47 was expressed in human atheromas, and we ascertained its spatial and regulatory interactions with type I procollagen. Methods ReagentsGene expression was assessed using a mouse monoclonal antibody to rat Hsp47, 5 a mouse monoclonal antibody to sheep pro␣1(I)collagen (SP1.D8, Developmental Studies Hybridoma Bank, University of Iowa), and partial cDNA clones for rat Hsp47 (pIP1) and human pro␣1(I)collagen (Hf677, ATCC). Human recombinant transforming growth factor (TGF)-␤1 and fibroblast growth factor (FGF)-2 were purchased from R&D Systems and Gibco/BRL, respectively. Copper-oxidized LDL was kindly provided by Dr M. Huff of the Robarts Research Institute. Human TissuesA total of 25 segments of the right coronary artery from 25 patients were obtained from postmortem tissue or native hearts harvested from cardiac transplant recipients at the London Health Sciences Center, London, Canada. The arteries were from patients aged 18 to 76 years. Specimens were fixed in 10% neutral-buffered formalin and embedded in paraffin. Sections of 4...

Abstract-HSP47 is a heat-shock protein that interacts with intracellular procollagen. It has been found in fibrous atherosclerotic plaque, but its involvement in acute vascular restructuring is unknown. We analyzed the expression of HSP47 and its regulation in the developing rat aorta and after balloon injury to the adult rat carotid artery. HSP47 was strongly expressed in each layer of the maturing fetal aorta (embryonic day 17 to birth). Expression declined during the first 4 postnatal days but persisted at low abundance into adulthood. HSP47 expression was substantially upregulated in the injured carotid artery, with intense immunostaining in neointimal smooth muscle cells (SMCs). HSP47 expression in SMCs was correlated with the emergence of a less mature phenotype and with expression of type I procollagen. Interestingly, a precipitous decline in HSP47 expression was evident during aortic development and after carotid artery injury, in association with the appearance of collagen fibrils in the local extracellular matrix. Furthermore, type I collagen fibrils, but not collagen monomers, inhibited expression of HSP47 by SMCs. These findings indicate that upregulation of HSP47 is a feature of vascular restructuring, including acute neointimal formation, and that the constituents of the extracellular matrix regulate the duration of expression. This feedback control may be important for self-termination of vascular development and lesion growth. (SMCs) is vital to the structural and functional integrity of the artery wall. Collagen provides mechanical strength to the artery wall, sufficient to withstand the large hemodynamic loads imposed on it, and also serves as an important ligand that regulates SMC proliferation and migration. [1][2][3] The major collagen species in both normal and diseased human arteries is type I collagen, a heterotrimeric, fibril-forming collagen that comprises 2 ␣1(I) collagen chains and 1 ␣2(I) collagen chain. The component ␣-chains are derived from precursors, namely pro␣1(I) collagen and pro␣2(I) collagen chains, that associate with each other in the lumen of the endoplasmic reticulum (ER). This assembly process requires precise sorting and folding of the pro␣ collagen chains within the ER and must occur before procollagen can be transported out of the cell. Quality control for procollagen assembly is dependent on a number of ER-resident enzymes and molecular chaperones. For example, prolyl 4-hydroxylase hydroxylates proline residues, the presence of which are necessary for winding of the long triple helical domain. The molecular chaperones, protein disulfide isomerase and immunoglobulin heavy-chain binding protein, transiently bind to a target region of the propeptide and facilitate either physiological folding or degradation of misfolded protein. 4,5 HSP47 is a 47-kDa heat-shock-inducible glycoprotein that has also been found to associate with procollagen in the ER. 6,7 Although its exact role in procollagen processing is unclear, data suggest that it acts as a collagen-specific chape...

S-chimeric oligonucleotides are stable and can specifically inhibit gene expression in human VSMCs. Nuclear transport is a feature of oligonucleotide processing by human VSMCs, indicating a potential influence at the nuclear level rather than with cytoplasmic mRNA. Cationic liposomes increased oligonucleotide uptake but not intracellular bioavailability, and S-chimeric oligonucleotides can be incorporated into cells within human atherosclerotic plaque, despite the presence of a dense extracellular matrix.