Introduction:

Accumulating evidence suggests a major role of the ubiquitin-proteasome-system in atherosclerosis. Pharmacological studies targeting proteasomes in atherosclerosis models indicate differential impact depending on the type and dose of inhibitor. However, the precise composition of proteasomes in vascular cells is not known.

Aim

of the study was to characterize vascular proteasomes in detail.

Methods and Results:

Proteasome complexes were analyzed based on covalent binding of fluorescent inhibitors, which specifically bind to proteolytic subunits of intact proteasome complexes. By this, an unanticipated low biological variance was detected among the same type of vascular cells derived from human umbilical cords. In contrast, comparing endothelial and smooth muscle cells revealed characteristic differences in the number and quantity of labeled subunits visualized by SDS-PAGE. Bands were identified by competitive assays using site specific proteasome inhibitors as well as subunit specific antibodies. Accordingly, there was a higher incorporation of subunits β1 and β2 in proteasome complexes derived from endothelial compared to smooth muscle cells. Subunits β1, β2 and β5 of proteasome complexes can be replaced by β1i, β2i and β5i under conditions of cellular stress or inflammation. Interestingly, all six subunits were identified in vascular cells without prior stimulation. Since the vascular cells were derived from human origin, a potential stimulation might have occurred prior to isolation. Therefore, aortae derived from unstimulated mice were analyzed in a similar manner. Again, all proteolytic subunits were co-expressed and incorporated in functional proteasomes.

Conclusion:

Endothelial and smooth muscle cells of the vasculature contain a distinct composition of proteasome subpopulations as measured by assembly and proteolytic function, enabling differential drug targeting of proteasomes in these cells.