Pj. Adam et al., POLYUBIQUITIN IS A NEW PHENOTYPIC MARKER OF CONTRACTILE VASCULAR SMOOTH-MUSCLE CELLS, Cardiovascular Research, 33(2), 1997, pp. 416-421
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.