Figure 2. Cx37 and Cx43 are on opposite sides of the venous valve leaflets.
(A) Cx37 immunostaining at a brachial vein valve (arrow) where the section presents an en face view of the relatively flat valve leaflet. (B) The same section as in panel A immunostained for Cx43, shows that Cx43, like Cx37, is expressed exclusively at the valve. (C) An overlay of Cx37 and Cx43 staining at the valve shows little or no overlap between the two immunosignals. (D) DIC image of the valve (arrow) shown in panels A–C and E. The box shows the area imaged by confocal microscopy in A–C. (E) Confocal z-stack imaging of the flattest part of the valve shown in A–D, which extends into the vessel lumen, colabeled for Cx37 and Cx43. In the center is the x–y projection through the z-stack. At the top is the x–z projection and on the right is the y–z projection, both showing strong separation of the Cx37 and Cx43 signals. The spatial separation of the signals in the x–z and y–z projections is not perfect perhaps because the valve is not exactly flat in the section, and there may also be a few Cx43-expressing leukocytes adhered to the valve leaflets on either side (isolated blotches of Cx43 signal). The orientation of this and other vein specimens was recorded, therefore we could determine that Cx43 is on the upstream side of the valve leaflet and Cx37 is on the downstream side. (F) A transverse section of a different brachial vein valve, where the valve is viewed in cross-section (end-on), shows in a different way that Cx37 and Cx43 are expressed on opposite sides of the valve leaflets. The Cx43 signal appears as short linear stretches because Cx43 is localized to endothelial cell-cell appositions. The Cx37 signal in this example was very strong and there may be some Cx37 present in the non-junctional membranes as well as at cell-cell appositions. (G) A lower magnification phase contrast image showing both leaflets (arrows) of the brachial vein valve immunostained in F. The boxed region shows the small area imaged in F. Blood flow is perpendicular to the plane of section in F and G. (H,I) Additional examples of the polarized expression of Cx37 and Cx43 on the two sides of venous valve leaflets (end-on views). Flow is from left to right. Taken together, these images show that Cx43 is present on the central, lumenal side of the valve leaflets (u, upstream side) and Cx37 is found on the sides of the leaflets facing the valve sinus or pocket (d, downstream side). A schematic illustration of the polarized expression of Cx43 and Cx37 on the upstream and downstream sides of venous valve leaflets is shown in J. Note that the illustration shows an idealized longitudinal section of a vein whereas the actual sections shown in the figure have differing orientations. Scale bars: 20 µm.