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Bves: prototype of a new class of cell adhesion molecules expressed during coronary artery development

Aya M. Wada^*, David E. Reese^* and David M. Bader

Stahlman Cardiovascular Laboratories, Program for Developmental Biology and The Division of Cardiovascular Medicine, Vanderbilt University, Nashville, TN, USA
^* These authors contributed equally to this study

View larger version (53K): [in a new window]   Fig. 1. Subcellular distribution of Bves in the epicardium and mesenchyme. (A) A section through the developing atrioventricular junction stained with D033 shows the localization of Bves (red, Cy3) in the developing epicardium (epi) and delaminated vasculogenic mesenchyme. MF20 (green, Cy2) shows the position of the myocardium. (B) A higher power view shows Bves localization in the epicardium (B846) in the periphery of epithelial cells (arrows) while Bves appears as a spot in mesenchyme (arrowhead, B,E). (C-E) Bves (red) localization in epicardial epithelium is seen co-labeled with PAN-cadherin antibody (green). DAPI (blue) marks nuclei. Localization of Bves at the cell-cell boundaries coincided with cadherin (C,D, arrows). Nuclear co-localization shows the ‘spot’ is in a perinuclear region of the delaminated mesenchyme (E, F-H, arrowhead). (F-H) Bves-positive mesenchymes in the subepicardial space. Colocalization of Golgi 58K protein antibody (green) with Bves antibody (red) reveals the Bves-positive ‘spot’ is confined the perinuclear region (F,G are merged in H).

View larger version (82K): [in a new window]   Fig. 2. Localization of Bves during coronary vessel development. Bves-positive mesenchymal cells accumulate in the tunica media of developing arteries. Anti-smooth muscle actin (green) shows differentiated smooth muscle cells. (A) Delaminated mesenchyme from the epicardium accumulates around a developing vessel (v). (B) Bves-positive cells differentiated into vascular smooth muscle (Reese et al., 1999) and additional Bves-positive cells continue to be recruited to the vessel shown in the 3D composed image. (C-E; C, Bves; D, merge; E, smooth muscle actin) Bves has two staining patterns in cells of the developing artery. Outer, Bves-positive cells have the characteristic punctate staining of mesenchymal cells, while Bves is broadly distributed in differentiated smooth muscle cells of the inner lamellae. Arrows distinguish the boundary between the two staining patterns. (F) Later, all smooth muscle cells of the tunica media of coronary vessels have a peripheral staining pattern.

View larger version (74K): [in a new window]   Fig. 3. Bves is an integral membrane protein. (A) Schematic representation of Bves protein structure shows the predicted structure of Bves and the relative positions of antibody epitopes. (B) TNT reactions were conducted in the presence and absence of dog pancreatic microsomes. Both full-length and truncated Bves (tm), which lacks the putative transmembrane domain, were used. The first lane shows that truncated Bves is produced and present in the lysate, but it is not captured in the microsomal fraction (lane 2). In contrast, full-length Bves, produced in the absence of microsomes (lane 3), is captured by microsomes (lane 4) and shows a slight increase in mobility. (C) Confocal analysis of Bves distribution in REC epithelium is presented. Analysis of the x-y axis shows the peripheral distribution of Bves. x-z axis analysis demonstrates that Bves is located in the lateral cell compartment. (D) Bves (red) and E-cadherin (green) staining show the colocalization (yellow) of the two proteins in the membrane.

View larger version (82K): [in a new window]   Fig. 4. Bves localization in the Golgi. Immunofluorescent microscopy for Bves (Cy3, red) and ß-COP (Cy2, green) localization in REC cells reveals extensive overlap in the Golgi and additional Bves staining at the membrane top. Treatment of cell lines with BFA diminishes Golgi staining but Bves at the membrane is not disturbed bottom.

View larger version (112K): [in a new window]   Fig. 5. Bves localization during epithelium formation. (A) REC culture during epithelial formation (A) and at confluence (B). Prominent staining at the periphery of single cells and at the free surfaces of cells is absent (A,C, arrowhead). As the cells become confluent and form the epithelial sheet, uniform cell membrane localization is observed (B). Higher power images (C-F) show the deposition of Bves during sheet formation. Note that Bves localization is detected at the point of cell contact or newly made cell-cell boundaries (D,E, arrows). As the epithelial sheet forms, Bves accumulates around the entire cell surface. In mature epithelial sheets, cytoplasmic staining is greatly diminished (B,F). (G-I) Blocking antibody alters the generation of epithelium in REC. REC form epithelial sheets in Ca2+-free conditions (G). When Bves blocking antibody was added at 1:100 dilution in the medium, semi-confluent REC cells were unable to form the epithelial sheet (H). Higher magnification of treated culture is given in I. (J-K) Bves accumulates at points of cell contact and cell borders prior to E-cadherin. The leading edges of REC epithelial sheets during wound healing in vitro are shown 8 hours after injury. (J) Anti-Bves (B846) is detected in cell processes, punctuate structures along the cell/cell boundaries and as continuous borders between cells (arrows, J,L). (K) E-cadherin in the same cells is broadly distributed in the cytoplasm and at low levels in processes and at cell edges. (L) Merged image of J and K.

View larger version (50K): [in a new window]   Fig. 6. Transfection of full-length Bves promotes aggregation in L-cells. L-cells were transfected with full-length Bves (B, L-F cells) or full-length Bves with an engineered Kozak sequence (C, L-K cells). Aggregation assays were performed on transfected and non-transfected cells as described in Materials and Methods and the degree of adhesion was calculated. Non-transfected cells did not aggregate (A). Graphs show that both L-F cells and L-K cells gained adhesive activity significantly over controls. (D-E) Bves-expressing cells are distinct from the non-transfected cells. Equal numbers of non-transfected L-cells were labeled with DiI, and Bves-transfected L-K cells were mixed and allowed to aggregate. Cells were stained with DAPI to visualize all cells. In the three examples given, note that aggregates are composed almost exclusively of transfected cells. (G,H) Promotion of aggregation in L-K cells was reduced in the presence of blocking antibody. When blocking antibody was added at 1:100 dilution to the cell suspension, reduction of aggregates were observed (see graph). Increase of antibody concentration (1:50 dilution) results in decrease in aggregates (H, graph) compared with aggregates without blocking antibody (G), suggesting that aggregation of L-cells results in expression of Bves. The ratio of aggregate to total cell is given with the s.e.m.

View larger version (105K): [in a new window]   Fig. 7. Bves antibody B846 blocks epithelial migration of the PEO. Control untreated PEOs show epithelial migration and mesenchyme formation from the explant (top left). Epithelial-mesenchymal transition is seen as the light fringe at the edge of the culture. Treatment with B846 blocks epithelial migration (top right). The number of mesenchymal cells that migrate out from the PEO was counted after DAPI staining (graph). Forty individual PEO samples were counted for control and treated groups.