Adam P J, Weissberg P L, Cary N R, Shanahan C M
Department of Medicine, Addenbrookes Hospital, University of Cambridge, UK.
Cardiovasc Res. 1997 Feb;33(2):416-21. doi: 10.1016/s0008-6363(96)00220-9.
Medial vascular smooth muscle cells (VSMCs) in healthy vessels are phenotypically distinct from their intimal counterparts in vascular disease. To compare the genes expressed in these phenotypes we have previously performed a differential cDNA library screen on cultured rat VSMCs. The aim of this study was to identify and characterise a 2.8 kb transcript, 2E10, which was highly expressed in freshly dispersed rat aortic VSMCs and downregulated in multiply passaged cultured VSMCs.
Sequence analysis was used to identify the 2.8 kb rat cDNA. 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 phenol; 15 micrograms of total cytoplasmic RNA was used in Northern blot analysis with a 32P-[dCTP]-labelled 2E10 cDNA probe. 35S-[dATP]-labelled 2E10 riboprobe was hybridised in situ to frozen sections of normal and diseased human coronary arteries.
DNA sequencing identified 2E10 as a rat polyubiquitin which is homologous to the human polyubiquitin, UbC. Northern blot analysis showed that this polyubiquitin was more highly expressed in differentiated, freshly dispersed rat and human aortic VSMCs compared with their dedifferentiated proliferating counterparts. This also identified a 3.2 kb transcript cross-reacting 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 sections of diseased human coronary arteries indicated much higher expression in medial than in intimal VSMCs. Northern blot analysis of RNA from the developing rat aorta showed that polyubiquitin expression increased substantially after week 2 of neonatal life, coincident with expression of VSMC-specific contractile proteins.
The greater expression of a UbC polyubiquitin transcript in contractile, differentiated VSMCs compared with proliferating, synthetic VSMCs provides a new gene marker for the phenotypic characterisation of VSMCs in vivo. This, and the finding that the developmental induction of expression of polyubiquitin (UbC) mirrors that of VSMC contractile proteins, suggests that ubiquitin, a protein known to associate with and degrade contractile proteins in skeletal muscle, is involved in the function or maintenance of the contractile phenotype of VSMCs.
健康血管中的中膜血管平滑肌细胞（VSMC）在表型上与其在血管疾病中的内膜对应细胞不同。为了比较这些表型中表达的基因，我们之前对培养的大鼠VSMC进行了差异cDNA文库筛选。本研究的目的是鉴定和表征一个2.8 kb的转录本2E10，它在新鲜分离的大鼠主动脉VSMC中高表达，而在多次传代培养的VSMC中表达下调。
采用序列分析鉴定2.8 kb的大鼠cDNA。在对增殖的培养大鼠和人VSMC进行胰蛋白酶消化，或对主动脉中膜进行酶消化后，通过在Nonidet P - 40中裂解并在苯酚中提取，从VSMC中分离总细胞质RNA；15微克总细胞质RNA用于用32P - [dCTP]标记的2E10 cDNA探针进行Northern印迹分析。用35S - [dATP]标记的2E10核糖探针原位杂交至正常和患病的人冠状动脉冰冻切片。
DNA测序鉴定2E10为大鼠多聚泛素，与人多聚泛素UbC同源。Northern印迹分析表明，与去分化的增殖对应细胞相比，这种多聚泛素在分化的、新鲜分离的大鼠和人主动脉VSMC中表达更高。这还鉴定了一个与多聚泛素探针交叉反应的3.2 kb转录本，它仅对分化的大鼠VSMC特异。然而，在生长停滞和增殖的VSMC中的表达是相同的，表明UbC在VSMC生长停滞中不起作用。多聚泛素核糖探针与患病的人冠状动脉切片的原位杂交表明，中膜VSMC中的表达远高于内膜VSMC。对发育中的大鼠主动脉RNA的Northern印迹分析表明，多聚泛素表达在新生生命第2周后大幅增加，与VSMC特异性收缩蛋白的表达一致。
与增殖的、合成型VSMC相比，UbC多聚泛素转录本在收缩性、分化的VSMC中表达更高，为体内VSMC的表型特征提供了一种新的基因标记。这一点，以及多聚泛素（UbC）表达的发育诱导与VSMC收缩蛋白的发育诱导相似这一发现，表明泛素（一种已知在骨骼肌中与收缩蛋白结合并降解收缩蛋白的蛋白质）参与VSMC收缩表型的功能或维持。
