Effects of proteasome and calpain inhibitors on the structural reorganization and proliferation of vascular smooth muscle cells in primary culture

Vascular smooth muscle cells exhibit a striking plasticity and are able to change from a differentiated, contractile phenotype to a more immature, syn thetic phenotype. This includes a prominent structural reorganization with loss of myofilaments and construction of a large secretory apparatus. As a result, the cells lose their contractility and become able to migrate, prol iferate, and secrete extracellular matrix components. In vivo, this phenoty pic shift is a chief factor behind the involvement of smooth muscle cells i n formation of atherosclerotic and restenotic lesions. Here, the effects of the proteasome inhibitors carbobenzoxy-leucyl-leucyl-leucinal, N-acetyl-le ucyl-leucyl-norleucinal, and lactacystin on the morphologic structure and g rowth of rat aortic smooth muscle cells in primary culture were examined. E lectron microscopic analysis revealed that the volume density of myofilamen ts was higher and the volume density of the endoplasmic reticulum and the G olgi complex was lower in cells exposed to these drugs than in solvent-trea ted controls. Moreover, diffuse material representing incompletely degraded proteins gathered in the cytoplasm of exposed cells. Similar material was also found in lysosomes. Immunogold staining showed a positive reaction in the diffuse cytoplasmic aggregates with antibodies against ubiquitin-protei n conjugates and proteasomes, whereas the material collecting in lysosomes reacted only with those against ubiquitin-protein conjugates. Moreover, wea k staining for smooth muscle ol-actin was noted in the cytoplasmic aggregat es. Otherwise, reactivity for this protein was concentrated in myofilaments . In addition to the effects on cell structure described above, the proteas ome inhibitors blocked cell multiplication. This was probably due to a decr eased rate of transition into a synthetic state as well as direct interfere nce with cell cycle progression in synthetic cells. These observations sugg est that proteasomes have the major responsibility for protein degradation during transition of smooth muscle cells from a contractile to a synthetic phenotype. If proteasome activity is inhibited, undegraded material accumul ates in the cytoplasm and is only partially taken up into lysosomes for dig estion. These findings raise the possibility that proteasome inhibitors may have a beneficial effect on vascular pathologies associated with phenotypi c modulation and proliferation of smooth muscle cells.