POLYUBIQUITIN IS A NEW PHENOTYPIC MARKER OF CONTRACTILE VASCULAR SMOOTH-MUSCLE CELLS

Objective: Medial vascular smooth muscle cells (VSMCs) in healthy vess els are phenotypically distinct from their intimal counterparts in vas cular disease. To compare the genes expressed in these phenotypes we h ave previously performed a differential cDNA library screen on culture d rat VSMCs. The aim of this study was to identify and characterise a 2.8 kb transcript, 2E10, which was highly expressed in freshly dispers ed rat aortic VSMCs and downregulated in multiply passaged cultured VS MCs, Methods: Sequence analysis was used to identify the 2.8 kb rat cD NA. After trypsinisation of proliferating cultured rat and human VSMCs , or enzymatic digestion of aortic tunica media, total cytoplasmic RNA was isolated from VSMCs by lysis in Nonidet P-40 and extraction in ph enol; 15 mu g of total cytoplasmic RNA was used in Northern blot analy sis with a P-32-[dCTP]-labelled 2E10 cDNA probe. S-35-[dATP]-labelled 2E10 riboprobe was hybridised in situ to frozen sections of normal and diseased human coronary arteries. Results: DNA sequencing identified 2E10 as a rat polyubiquitin which is homologous to the human polyubiqu itin, UbC. Northern blot analysis showed that this polyubiquitin was m ore highly expressed in differentiated, freshly dispersed rat and huma n aortic VSMCs compared with their dedifferentiated proliferating coun terparts. This also identified a 3.2 kb transcript cross-reaching with the polyubiquitin probe which is specific to differentiated rat VSMCs only. However, expression in growth arrested and proliferating VSMCs was identical, suggesting that UbC does not have a role in VSMC growth arrest. In situ hybridisation of the polyubiquitin riboprobe to secti ons of diseased human coronary arteries indicated much higher expressi on in medial than in intimal VSMCs. Northern blot analysis of RNA from the developing rat aorta showed that polyubiquitin expression increas ed substantially after week 2 of neonatal life, coincident with expres sion of VSMC-specific contractile proteins. Conclusions: The greater e xpression of a UbC polyubiquitin transcript in contractile, differenti ated VSMCs compared with proliferating, synthetic VSMCs provides a new gene marker for the phenotypic characterisation of VSMCs in vivo. Thi s, and the finding that the developmental induction of expression of p olyubiquitin (UbC) mirrors that of VSMC contractile proteins, suggests that ubiquitin, a protein known to associate with and degrade contrac tile proteins in skeletal muscle, is involved in the function or maint enance of the contractile phenotype of VSMCs.