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, 159 (3), 875-83

Members of the Jagged/Notch Gene Families Are Expressed in Injured Arteries and Regulate Cell Phenotype via Alterations in Cell Matrix and Cell-Cell Interaction

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Members of the Jagged/Notch Gene Families Are Expressed in Injured Arteries and Regulate Cell Phenotype via Alterations in Cell Matrix and Cell-Cell Interaction

V Lindner et al. Am J Pathol.

Abstract

The Jagged/Notch signaling pathways control cell fate determination and differentiation, and their dysfunction is associated with human pathologies involving cardiovascular abnormalities. To determine the presence of these genes during vascular response to injury, we analyzed expression of Jagged1, Jagged2, and Notch1 through 4 after balloon catheter denudation of the rat carotid artery. Although low levels of Jagged1, Jagged2, and constitutive expression of Notch1 were seen in uninjured endothelium, expression of all was significantly increased in injured vascular cells. High Jagged1 expression was restricted to the regenerating endothelial wound edge, whereas Notch transcripts were abundant in endothelial and smooth muscle cells. To understand the basis for Jagged/Notch control of cellular phenotype, we studied an in vitro model of NIH3T3 cells transfected with a secreted form of the extracellular domain of Jagged1. We report that the soluble Jagged1 protein caused decreased cell-matrix adhesion and cell migration defects. Cadherin-mediated intercellular junctions as well as focal adhesions were modified in soluble Jagged1 transfectants, demonstrating that cell-cell contacts and adhesion plaques may be targets of Jagged/Notch activity. We suggest that Jagged regulation of cell-cell and cell-matrix interactions may contribute to the control of cell migration in situations of tissue remodeling in vivo.

Figures

Figure 1.
Figure 1.
Expression of Jagged in normal and injured vessels in vivo. In situ hybridization with the sense (A) or antisense probes (B–H) for Jagged1 (B, D, and G) and Jagged2 (C, E, F, and H) was performed on en face preparations of vessels as indicated. Although normal expression for both genes in uninjured vessels was present (B and C), transcripts were up-regulated in both injured endothelium (EC, D–F) and smooth muscle cells (SMC, G and H). Transcripts were again reduced to background levels in a stable lesion (F). Original magnification, ×400. I: Western blot analysis was performed using an anti-Jagged1 antibody with tissue lysates from normal vessel (nor) or carotid arteries 7 days after balloon catheter injury. The Jagged1 protein (arrowhead) was found to be present in normal, and more abundant in injured carotid arteries.
Figure 2.
Figure 2.
Notch expression in uninjured endothelial cells in vivo. In situ hybridization was performed on en face preparations of normal endothelium using antisense probes for Notch1 through 4. Constitutive but low expression for Notch1 transcript was detected, whereas background levels of Notch2–4 were seen (B–D). Original magnification, ×200.
Figure 3.
Figure 3.
Expression of Notch in injured SMC in vivo. In situ hybridization was performed using antisense probes for Notch1 through 4 on en face preparations of vessels 8 days after injury. Depending on the area of the vessel examined, SMC were either alone (A–D) or adjacent to the endothelial wound edge (E–H). Notch1 (A), Notch2 (B), and Notch3 (C and D) expressions were seen in SMC. However, both Notch3 (E and F) and Notch4 (G and H) expression in SMC was increased in regions adjacent to the endothelial monolayer (EC). C, E, and G show bright-field images of corresponding dark-field images in D, F, and H, respectively. Note that G and H demonstrate SMC both directly adjacent to endothelial cells (EC), as well as SMC not in contact with the endothelium (right). All Notch genes were found to be abundantly expressed in injured SMC at 8 days as well as 2 weeks after injury. Original magnification, ×200.
Figure 4.
Figure 4.
Production of soluble Jagged1 inhibits NIH3T3 cell migration. A: Stable transfectants of vector control or soluble Jagged1-expressing clones were assayed for the expression and secretion of the soluble Jagged1 protein as described. Cell lysates or conditioned media from metabolically labeled cells were immunoprecipitated with anti-myc antibody, and subjected to SDS-PAGE. Arrow indicates the soluble Jagged1 protein. The identity of this band in cell lysates was confirmed by Western blot analysis. B–E: Migratory ability of clones was assessed using a scrape assay and measuring the migration of cells onto the denuded surface as described. B: Photomicrographs show representative fields of the clones at 24 and 48 hours after the scrape injury. Original magnification, ×100. C: Quantitation of the denuded area reflected a 24- to 48-hour lag in the repopulation of the denuded area in soluble Jagged1 transfectants compared to vector controls. D: When the assay was performed on fibronectin (FN)-coated plates (10 μg/ml), the migration defect of the soluble Jagged1 cells was partially rescued, with migration intermediate between migration of vector control and soluble Jagged1 cells plated on plastic. E: Inclusion of 100 mmol/L SM256, a peptide integrin inhibitor, into the assay caused a reduction in the migration of both vector control and soluble Jagged1 cells.
Figure 5.
Figure 5.
Delayed cell spreading and FAK phosphorylation in soluble Jagged1 cells. A: Cells were plated at equal cell densities on plastic and photographed after 1 hour, 3 hours, and 5 hours. Note delayed cell spreading in soluble Jagged1 cells. Original magnification, ×200. B: Cell lysates were collected as described from vector control or soluble Jagged1 transfectants at 2 and 24 hours after plating on plastic. Lysates were immunoprecipitated using anti-FAK antibodies and subjected to SDS-PAGE and Western blot analysis with an anti-phosphotyrosine antibody (top). Blots were stripped and reprobed with anti-FAK antibodies (bottom).
Figure 6.
Figure 6.
Alterations in focal adhesions and cell-cell adhesions in soluble Jagged1 cells. Immunofluorescent staining was performed for the proteins indicated on vector control (left) and soluble Jagged1 (right) transfectants. Focal adhesions were demonstrated by anti-vinculin staining of cells on plastic (A and B) or plated on collagen (C and D). Arrowheads show focal adhesion plaques. Using confocal fluorescence microscopy, cell-cell contacts were visualized using a pan-cadherin antibody (E and F) and an anti-β-catenin antibody (G and H). Original magnifications, ×1000.

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