Non-muscle myosin isoforms and cell heterogeneity in developing rabbit vascular smooth muscle

Abstract

A panel of monoclonal antibodies specific for cytoskeletal and cytocontractile protein markers has been used to study the expression of vimentin, desmin and alpha-smooth muscle (SM) actin, as well as non-muscle (NM) and SM myosin isoforms, in developing rabbit aorta. Immunofluorescence experiments show that in the vascular smooth muscle cells (SMC): (1) vimentin and alpha-actin of SM-type are homogeneously expressed among SMC, since the early stage (day 19, in uterus) of development; (2) desmin is heterogeneously distributed throughout all the developmental stages examined (from day 19, foetal, to day 90, post-natal); and (3) myosin isoform content in pre- and post-natal vascular SM is different when analyzed by anti-SM myosin (SM-E7) and anti-NM myosin (NM-F6, NM-A9 and NM-G2) antibodies. SM myosin in vascular SM is present as early as day 19 in uterus, being especially evident in the region facing the lumen of aortic wall, but not in the outermost layer in which NM myosin is present exclusively. Western blotting and immunofluorescence assays indicate that the foetal aortic SM is specifically labeled by all the three anti-NM myosin antibodies. However, immunoreactivity of aortic SM with NM-F6 and NM-A9 disappears completely around birth. Conversely, NM-G2 binding is maintained during post-natal development up to day 45; between day 45 and day 90 immunoreactivity of aortic SMC with this antibody diminished progressively, without disappearing, in a small number of cells. In aortic SMC cultures from foetal and adult rabbits, NM myosin immunoreactivities appear to be differently distributed, i.e. according to the stress fiber system (NM-F6 and NM-G2), in a diffuse manner (NM-A9) or mainly localized at the level of the cortical cytoplasm (NM-G2). The fact that a different pattern of NM myosin antigenicity can also be shown in other cell types, such as in the endothelium and the cardiac pericytes as well as in the renal parenchyma, is consistent with the existence of multiple NM myosin in vascular SM isoforms whose expression is developmentally regulated.