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, synthetic 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, proliferate, and secrete extracellular matrix components. In vivo, this phenotypic shift is a chief factor behind the involvement of smooth muscle cells in formation of atherosclerotic and restenotic lesions. Here, the effects of the proteasome inhibitors carbobenzoxy-leucyl-leucyl-leucinal, N-acetyl-leucyl-leucyl-norleucinal, and lactacystin on the morphologic structure and growth of rat aortic smooth muscle cells in primary culture were examined. Electron microscopic analysis revealed that the volume density of myofilaments was higher and the volume density of the endoplasmic reticulum and the Golgi complex was lower in cells exposed to these drugs than in solvent-treated 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-protein conjugates and proteasomes, whereas the material collecting in lysosomes reacted only with those against ubiquitin-protein conjugates. Moreover, weak staining for smooth muscle alpha-actin was noted in the cytoplasmic aggregates. Otherwise, reactivity for this protein was concentrated in myofilaments. In addition to the effects on cell structure described above, the proteasome inhibitors blocked cell multiplication. This was probably due to a decreased rate of transition into a synthetic state as well as direct interference with cell cycle progression in synthetic cells. These observations suggest 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 accumulates in the cytoplasm and is only partially taken up into lysosomes for digestion. These findings raise the possibility that proteasome inhibitors may have a beneficial effect on vascular pathologies associated with phenotypic modulation and proliferation of smooth muscle cells.
Thyberg
J
J
Department of Cell and Molecular Biology, Karolinska Institut, Stockholm, Sweden. Johan.Thyberg@cmb.ki.se
Blomgren
K
K
D000199:Actins / Q000032:analysis; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors*; D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors*; D002455:Cell Division / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D007150:Immunohistochemistry; D008297:Male; D008854:Microscopy, Electron; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014452:Ubiquitins / Q000032:analysis
D000199:Actins; D015853:Cysteine Proteinase Inhibitors; D009097:Multienzyme Complexes; D014452:Ubiquitins; D002154:Calpain; D000251:Adenosine Triphosphatases