1. CEA-Related Cell Adhesion Molecule 1
Süleyman Ergün, Nerbil Kilic, Gudrun Ziegeler, Arne Hansen, Peter Nollau, Julica Götze, Jan-Henner Wurmbach, Andrea Horst, Joachim Weil, Malkanthi Fernando, Christoph Wagener 
Molecular Cell 
Volume 5, Issue 2, Pages (February 2000)
DOI: /S (00)

2. Figure 1 Purification and Detection of CEACAM1
(A) SDS-PAGE of CEACAM1 purified from human granulocyte membranes. Silver staining of an 8% polyacrylamide gel electrophoresis reveals a band of 160 kDa corresponding to CEACAM1. Molecular weight markers are as indicated.
(B) Western blot of recombinant CEACAM1 produced in HEK293 cells (lane 1) using the CEACAM1-specific monoclonal antibody 4D1/C2. The Mr of 160 kDa corresponds to the Mr of CEACAM1 purified from granulocytes. The purification scheme for recombinant CEACAM1 was applied to HEK293 cells lacking the CEACAM1 gene. No specific band can be observed in this preparation (lane 2).
(C) CEACAM1 purified from conditioned media of endothelial cells stimulated by VEGF165 is composed of two major fractions of 120 kDa and 50 kDa.
Molecular Cell 2000 5, DOI: ( /S (00) )

3. Figure 2
Proliferative and Migrative Effects of CEACAM1 on Endothelial Cells
CEACAM1 preparations at concentrations indicated were applied to cultured HDMECs with 1 ng/ml FGF-2 in a 72 hr proliferation assay. Both CEACAM1 purified from granulocytes (A) and recombinant CEACAM1 (rCEACAM1) (B) stimulate endothelial cell proliferation in a dose-dependent manner. As negative controls, the base media (basal media) or elutes without CEACAM1 (control) after immunoaffinity chromatography were applied. The chemotactic response of HDMECs to CEACAM1 is studied using the Boyden chamber (C). rCEACAM1 increases the chemotaxis of HDMECs in a dose-dependent manner in comparison with the controls and, in addition, potentiates the chemotactic effect of VEGF165. CEACAM1 from granulocytes (gCEACAM1) has comparable chemotactic effect as rCEACAM1. Note that this effect is inhibited significantly when rCEACAM1 is applied together with the monoclonal CEACAM1 antibody 4D1/C2. Basal media or elute fractions without CEACAM1 (control) after immunoaffinity chromatography have no chemotactic effect on HDMECs. Each bar represents the mean ± SEM.
Molecular Cell 2000 5, DOI: ( /S (00) )

4. Figure 3 Endothelial Tube Formation Assay
(A–F) Capillary-like endothelial tube formation in three-dimensional collagen gels. gCEACAM1 induces endothelial tube formation (A) after two applications within 6 days at concentrations of 300 ng/ml per application. VEGF165 (50 ng/ml) (B) was used as positive control. The combination of gCEACAM1 (300 ng/ml) with VEGF165 (50 ng/ml) enhances the number of endothelial cells involved in tube formation and branching of tubes (C) when compared with the factors applied alone. Similarly to gCEACAM1, rCEACAM1 (D) also induces endothelial tube formation. Endothelial tube formation induced by VEGF165 is blocked completely by the CEACAM1 specific monoclonal antibody 4D1/C2 (E). In contrast, tubes induced by VEGF165 are not blocked when mouse immunoglobulin is used instead of the CEACAM1 antibody (F).
(G–J) Semithin sections of collagen: examples are shown for gCEACAM1 (G) and VEGF165 (H). No endothelial tubes are observed in HDMECs cultures treated by a combination of VEGF165 and FGF-2 in the presence of the CEACAM1 antibody 4D1/C2 (I). Similarly, tubes are absent and the HDMECs remain on top of the gel in the control performed with basal medium (J).
Magnifications: (A–F), ×90; (G–J), ×180.
Molecular Cell 2000 5, DOI: ( /S (00) )

5. Figure 4 CEACAM1 Expression and Production in Endothelial Cells
Northern (A) and Western blot (B) analyses of CEACAM1 expression after stimulation with VEGF165. VEGF165 upregulates CEACAM1 mRNA (A) and protein (B) level. Western blots were performed using the CEACAM1 specific monoclonal antibody 4D1/C2. In comparison with nonstimulated HDMECs (lane NS), the intensity of two bands corresponding to 120 kDa and 50 kDa is increased significantly after stimulation of HDMECs by VEGF165 (lane S). mRNA from normal human colonic tissue served as positive control (lane C).
Molecular Cell 2000 5, DOI: ( /S (00) )

6. Figure 5 Immunolocalization of CEACAM1 in Angiogenic Blood Vessels
Immunohistochemical localization of CEACAM1 in blood vessels using the monoclonal antibody 4D1/C2. CEACAM1 immunostaining is found in capillaries (arrowhead) of Leydig cell tumor (A) and of bladder carcinoma (B). Note that CEACAM1 immunostaining is stronger at the basal side (arrowhead) when compared to the luminal side of endothelium. In prostate carcinoma, small tumor blood vessels (arrowhead) are labeled for CEACAM1, whereas large tumor blood vessels (arrow) consisting of at least one layer of smooth muscle cells in their wall are negative (C). A strong immunostaining for CEACAM1 is present in blood vessels of hemangioblastoma of human brain (D). Microvessels (arrowhead) of human renal cell carcinoma are CEACAM1 positive (E). In human endometrium, CEACAM1 staining is visible in endothelial cells of microvessels (arrowhead) surrounding tubular glands and at the apical poles of epithelial cells (arrow) (F). No specific staining is observed in the control section of endometrium (G). Endothelial cells of newly formed small blood vessels (arrowhead) during wound healing 3 weeks after a biopsy exhibit a strong CEACAM1 immunostaining (H). In all panels except (C), the sections have been counterstained with Calcium Red. Immune electron microscopic studies of human testicular tumors show that, in tumor blood vessels without basement membrane, CEACAM1 immunostaining (arrowhead) is confined to endothelial cells (EC). More mature tumor vessels exhibit CEACAM1 immunoreaction also in their basement membrane ([J and K], arrowhead) and around some periendothelial cells ([K], PEC). No specific immunostaining is detectable in the control (L). Er, erythrocytes; TC, tumor cell.
Magnifications: (A and B), ×650; (C–H), ×350; (I), ×1000; (J–L), ×2200.
Molecular Cell 2000 5, DOI: ( /S (00) )

7. Figure 6 Angiogenic Effects of CEACAM1 in CAM Assay
(A) Overview of an opened chicken egg demonstrating the CAM blood vessels.
(B–F) The application of gCEACAM1 (300 ng/ml) (B), VEGF165 (50 ng/ml) (C), and of gCEACAM1 (300 ng/ml) plus VEGF165 (50 ng/ml) (D) significantly increases vascular density when compared to the negative control performed without factors added (E). Note that the capillary density and the formation of brush-like blood vessels indicating active angiogenesis is strongest when CEACAM1 plus VEGF165 is applied (D). The angiogenic effect of CEACAM1 is blocked in the presence of the monoclonal CEACAM1 antibody (4D1/C2) (F).
(G–J) Semithin sections of CAM areas treated with various agents. gCEACAM1 (G) induces a high vascularization of the CAM comparable to the vascularization induced by VEGF165 (H). Controls performed by application of basal solution without any factor added (I) reveal no increased vascularization. When gCEACAM1 and VEGF165 are combined, a higher vascular density is obtained (J) when compared to the application of the factors alone.
(K) The number of blood vessels observed in the semithin sections of the CAM assay is shown. In comparison with the negative control (no factor added), a significantly higher vascular density in the CAMs is observed after treatment with gCEACAM1. The effect of gCEACAM1 is stronger than the effect of VEGF165 used as positive control. The simultaneous application of gCEACAM1 and VEGF acts synergistically. The angiogenic activity of gCEACAM1 is inhibited significantly by the monoclonal CEACAM1 antibody 4D1/C2 (K).
Magnifications: (A), ×1; (B–F), ×10; (G–J), ×350.
Molecular Cell 2000 5, DOI: ( /S (00) )

8. Figure 7 The Role of CEACAM1 in Vascular Formation
This graphic summarizes the role of CEACAM1 in angiogenesis as derived from the results presented here. VEGF-activated endothelial cells produce and secrete CEACAM1, which stimulates proliferation and migration of endothelial cells and for this reason functions as an autocrine factor. In a further step of blood vessel formation, transmembrane forms of CEACAM1 may act as membrane-bound cell adhesion molecules in endothelial tube formation. Finally, through binding to components of the basal lamina, CEACAM1 may also be involved in the deposition of the vascular basement membrane and, in this way, may contribute to the maturation of newly formed blood vessels.
Molecular Cell 2000 5, DOI: ( /S (00) )