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Additional file 1. Raw immunoblot image of HBV core protein (HBc) from extracts of HepAD38 and HepG2 cells expressing whole HBV genome and the HBV 1.3mer plasmid respectively. GAPDH was used as internal control.

Table S3. List of rare homozygous variants in III:5 and III:15 located within the shared homozygous segment on chr12. (DOCX 13 kb)

Figure S1. Sanger sequencing results of affected family. Both parents (II:3 and II:4) (right upper and lower panels) of Patient 1 were heterozygous carriers, arrows indicate the double peaks (heterozygosity). Patient 1 (III:9) (left lower panel) result indicates homozygous change, arrow is indicating the change. While an unaffected individual (III:11) (left upper panel) showed both correct/wild type alleles. (DOCX 106 kb)

Table S1. Clinical findings in the nine affected family members with a homozygous WNT1 mutation. The “+” signs indicates the presence and severity of the symptom and “-” sign indicating the absence of the symptom. (DOCX 15 kb)

Objective— Cell migration is central to multiple physiological and pathologic processes and involves interactions between integrins on the cell surface and the extracellular matrix. The Leu33Pro (Pl A ) polymorphism of integrin β 3 has been reported to be associated with a greater rate of restenosis after angioplasty, a process involving endothelial and smooth muscle cell migration. We have addressed the possibility that the Leu33Pro polymorphism could modify the migratory behavior of Chinese hamster ovary (CHO) cells expressing the β 3 -containing integrin complexes. Methods and Results— Haptotactic migratory responses of CHO α IIb β 3 cells to fibronectin and vitronectin were not statistically different between the Leu33 and Pro33 cells. However, CHO cells with the Pro33 (Pl A2 ) polymorphism had an enhanced haptotactic migratory response to fibrinogen and von Willebrand Factor. This enhanced migration (1) could be blocked by the α IIb β 3 -complex–specific neutralizing mAb 10E5, by 7E3, a neutralizing mAb specific for the β 3 integrin, and by the α IIb β 3 -blocking peptide Integrilin; (2) was not observed with a CHO cell line expressing an activating β 3 Cys435 to Ala mutation; and (3) was attributable to increased activity of mitogen-activated protein kinase and cyclooxygenase. CHO cell lines expressing the Pro33 isoform of α v β 3 had an enhanced haptotactic migratory response to vitronectin and osteopontin but not fibrinogen. Conclusions— The Leu33Pro polymorphism alters the migratory behavior of cells on extracellular matrix substrates, and the α subunit influences the substrate specificity of this genetic effect.

Background Integrin-associated protein (IAP) is a thrombospondin (TSP)-binding, Gi protein-coupled cell surface receptor. The vascular function of IAP has not been defined, and it is not known whether TSP and IAP are expressed at the same time in injured arteries. Methods Left brachial arteries of baboons were injured using balloon withdrawal technique. Arteries were harvested 1 week after injury, and immunohistochemistry and in situ hybridization were performed using standard techniques. Uninjured right brachial arteries served as a control. Proliferation studies were performed using cultured human aortic smooth muscle cells (SMC). Results We found significant IAP expression in the media and neointima 7 days after injury using BRIC-126, an immunoglobulin (Ig) G2b monoclonal antibody that recognizes IAP with high specificity. In contrast, IAP staining in the uninjured vessel was only observed in the endothelium. Concurrent with IAP expression, TSP mRNA and protein expression in the neointima and media was enhanced 1 week after injury. In cultured SMC, activation of IAP was sufficient to elicit a proliferative response. TSP-induced proliferation was inhibited by antibodies that block TSP binding to IAP and mimicked by 4N1K, a 10-amino acid peptide derived from the IAP binding site within the carboxyl terminus of TSP. Conclusions Vascular expression of IAP and TSP increased after mechanical injury and activation of IAP elicited a proliferative response in cultured SMC. These findings support the hypothesis that IAP participates in vascular healing responses.

Background —Treatment with an antibody that binds β 3 integrins (abciximab; c7E3 Fab) at the time of coronary angioplasty decreases the need for repeat revascularization. Two potential mechanisms have been proposed to explain this effect: (1) inhibition of platelet aggregation or (2) interruption of ligand binding to β 3 integrins on the smooth muscle cell (SMC) surface. We examined the latter hypothesis by determining (1) if β 3 integrin expression is upregulated after vascular injury in the baboon, (2) if 7E3 binds β 3 integrins on cultured SMC, and (3) if β 3 integrin activation plays a role in proliferation of cultured SMC. Methods and Results —Results demonstrated that immunostaining for β 3 integrins was present in the neointima 1 week after balloon withdrawal injury of baboon brachial arteries and that β 3 integrin expression colocalized with α-actin–positive cells. In contrast, staining for β 3 integrins was undetectable in contralateral uninjured brachial arteries. 7E3 bound to cultured human aortic SMC with an affinity ( K D =3.3 nmol/L) similar to 7E3 binding to endothelial cells or platelets. Cotreatment with 7E3 partially inhibited thrombospondin-induced or α-thrombin–induced proliferation but not PDGF-induced or serum-induced proliferation. Conclusions —In summary, these studies demonstrate that vascular cell β 3 integrin expression is increased after injury, that 7E3 binds to cultured SMC with high affinity, and that β 3 activation is important for thrombospondin-induced or α-thrombin–induced proliferation. These results support the hypothesis that β 3 integrins play a role in SMC growth responses after balloon injury.

αvβ3-Integrin antagonists reduced neointimal formation following vascular injury in eight different animal models. Because α-thrombin contributes to neointimal formation, we examined the hypothesis that αvβ3-integrins influence α-thrombin-induced signaling. Cultured rat aortic smooth muscle cells (RASMC) expressed αvβ3-integrins as demonstrated by immunofluorescence microscopy and fluorescence-activated cell sorting analysis. Proliferative responses to α-thrombin were partially inhibited by anti-β3-integrin monoclonal antibody F11 and by cyclic RGD peptides. Immunofluorescence microscopy showed that α-thrombin stimulated a rapid increase in the formation of focal adhesions as identified by vinculin staining and that this effect was partially inhibited by αvβ3antagonists. β3-Integrin staining was diffuse in quiescent RASMC and did not concentrate at sites of focal adhesions following thrombin treatment. α-Thrombin elicited a time-dependent increase in activation of c-Jun NH2-terminal kinase-1 (JNK1) and in tyrosine phosphorylation of focal adhesion kinase (FAK). αvβ3-Integrin antagonists partially inhibited increases in JNK1 activity but had no effect on FAK phosphorylation. In SMC isolated from β3-integrin-deficient mice, focal adhesion formation was impaired in response to thrombin but not sphingosine-1-phosphate, a potent activator of Rho. In summary, αvβ3-integrins play an important role in α-thrombin-induced proliferation and focal adhesion formation in RASMC.

Alpha(v)beta3 integrins play an important role in vascular healing. Vascular injury is a stimulus for expression of alpha(v)beta3 by vascular cells and, among other effects, alpha(v)beta3 integrins function in the adhesion of activated platelets to endothelium. white cell/endothelium interactions, platelet-mediated thrombin generation, fibrin clot retraction by nucleated cells, smooth muscle cell (SMC) migration and proliferation, vascular cell apoptosis, and vascular remodeling. There are ten different animal models in which treatment with alpha(v)beta3 antagonists reduced the vascular response, including (neo)intima formation, after mechanical injury. These studies, along with mechanistic data derived from cell culture studies, provide compelling evidence that alpha(v)beta3 integrins are involved in vascular repair processes. The challenge is to develop a therapeutic agent that will prove effective in reducing restenosis in humans following percutaneous coronary intervention (PCI).

BackgroundOur objective was to determine whether abciximab, eptifibatide, or tirofiban inhibited ligand binding to αvβ3integrins on human aortic smooth muscle cells (HASMCs) or human umbilical vein endothelial cells (HUVECs). Abciximab binds αIIbβ3on platelets and αvβ3on HUVECs with similar affinity, whereas eptifibatide and tirofiban are thought to be highly specific for αIIbβ3. The conclusion that eptifibatide does not bind vascular αvβ3integrins may be premature, however, because recent studies have demonstrated that the affinity of αvβ3for various ligands, including antagonists, is subject to modulation.Methods and ResultsAbciximab and 7E3, the anti–β3integrin monoclonal antibody from which abciximab was derived, bound αvβ3on HASMCs in a specific and saturable manner and with an affinity similar to binding to αIIbβ3on platelets. 7E3 and eptifibatide inhibited αvβ3-mediated attachment of HASMCs to thrombospondin (TSP) and prothrombin but had no effect on αvβ5- or β1-mediated HASMC attachment to vitronectin-, collagen-, or fibronectin-coated or noncoated tissue culture plates. The inhibitory effect of eptifibatide was similar in magnitude and not additive to that of 7E3. Eptifibatide and 7E3 inhibited αvβ3-mediated attachment of HUVECs. Tirofiban had only nonspecific effects on HASMC attachment to extracellular matrix proteins. In cell proliferation assays, eptifibatide inhibited αvβ3-mediated responses to soluble TSP by HASMCs and β3integrin–expressing HEK cells.ConclusionsEptifibatide and 7E3, but not tirofiban, specifically inhibit αvβ3-mediated binding of human smooth muscle and endothelial cells.

Transforming growth factor (TGF)-β1 has been implicated in vascular healing responses after mechanical injury. Using cultured rat aortic smooth muscle cells (RASMC), we examined the hypothesis that production and secretion of thrombospondin (TSP) contributes to TGF-β1-induced proliferation. We found that TGF-β1 enhanced production and secretion of TSP, with peak levels of secreted TSP observed 24 h after treatment. RASMC treated with TGF-β1 secreted a mitogenic activity that was transferable in conditioned media and partially inhibited by C6.7, a monoclonal anti-TSP antibody. Exogenous TSP stimulated a proliferative response, with maximal [3H]thymidine incorporation occurring 24 h earlier than maximal [3H]thymidine incorporation in response to TGF-β1-treatment. Pretreatment with C6.7 or polyclonal anti-TSP neutralizing antibodies inhibited TGF-β1-induced proliferation of RASMC. Proliferative responses to TGF-β1 were also inhibited by pretreatment with an anti-β3integrin monoclonal blocking antibody (F11), RGD peptides, and the anti-αvβ3disintegrin echistatin. Treatment with TSP and TGF-β1 increased c-Jun NH2-terminal kinase (JNK)1 activity, with peak effects observed at 15 min and 4 h, respectively. Treatment with C6.7 or F11 inhibited TGF-β-induced activation of JNK1. In summary, these studies support the hypothesis that TGF-β-induced JNK1 activation and proliferation of RASMC require secretion of TSP and ligation of αvβ3-integrins.