The Cytoskeleton of Arterial Smooth Muscle Cells During Development, Atheromatosis and Tissue Culture

Abstract

Arterial smooth muscle cells (SMC) assume cytoskeletal features of embryonic cells during human and experimental atheromatosis, as well as when placed in culture. The expression of α-smooth muscle (SM) actin can be used to monitor these changes. The synthesis of α-SM actin is decreased early after plating in cultured cells and early after endothelial lesion in animals. The amount of mRNA for α-SM actin is not affected in cultured cells, while it is drastically decreased in the carotid artery after endothelial injury. Heparin does not modify in vivo the early decrease of α-SM actin mRNA and synthesis, but, by inhibiting the entry of SMC into the S phase of the cell cycle, it produces an early reinduction of α-SM actin. When heparin is incubated with cultured SMC, it reduces SMC proliferation and increases α-SM actin expression. This action is not always dependent on SMC proliferation, suggesting that heparin may act directly on SMC differentiation.

Medial smooth muscle cells (SMC) proliferate and migrate towards the intima to form the bulk of the atheromatous plaque. Evaluation of cytoskeletal features of SMC during human early atheromatous plaques and rat experimental intimai thickenings has furnished new information concerning the changes of SMC phenotype during atheromatosis. Data were mainly obtained by means of immunofluorescent staining with affinity purified polyclonal antibodies against vimentin or desmin (1) and with a monoclonal antibody against α-smooth muscle actin (2). Actin isoforms as percentage of total actin were estimated by laser beam densitometric analysis of two-dimensional gel electrophoresis (3). To localize the levels of synthesis regulation, Northern blot hybridizations of total RNA were performed with rat total actin and specific α-SM actin mRNA probes (4).

Normal adult rat aortic medial SMC, despite an homogeneous morphology, are heterogeneous as far as intermediate filament pattern expression is concerned: 51% are vimentin positive, 48% are vimentin plus desmin positive and 1% are desmin positive (1). Another main feature is a pattern of actin isoforms with α-SM actin predominance (1). One of the most used experimental models for the atheromatous plaque is the removal of rat aortic endothelium with an inflated balloon catheter. In 15-day-old experimental rat aortic intimai thickening, before endothelial regeneration and while SMC are actively replicating, the proportion of SMC in the intima containing only vimentin increases (79%); moreover, a switch to a ß-actin predominance is observed, accompanied by an increase in γ-actin (1). Similar changes are seen in human fibrous atheromatous plaques (5) as well as in poorly differentiated fetal and newborn aortic SMC (3). Thus, pathological SMC assume a “dedifferentiated” phenotype, as far as their cytoskeleton is concerned; this cytoskeletal remodelling is related, at least in part, to cell replicative activity. Sixty days after experimental injury, when endothelium continuity is completely re-established and SMC have again stopped replicating, SMC switch back to α-SM actin predominance and 50% of them are positive to both vimentin and desmin (6). Thus, in vivo, rat SMC can redifferentiate despite their ectopical intimai location. In human atheromatosis, desmin positive SMC reappear in complicated plaques, however a ß-actin predominance is maintained (5); this is probably due to blood born cells infiltration which contain only ß and γ actins. In vitro, proliferating SMC develop cytoskeletal features similar to those observed in normal fetal or pathological SMC (7); this model being useful for understanding mechanisms leading to atheromatous lesions and SMC differentiation. A slight increase in α-SM actin can be seen in adult SMC kept in 10% FCS culture 7 days after confluence or kept in a supplemented serum-free medium. During these situations, SMC rarely proliferate (see below). However, the expression of α-SM actin isoform is never as great as in normal medial SMC sixty days after endothelial removal (4,7). During culture in non-proliferating conditions, the index of thymidine cellular labeling is greater (1-5%) than in normal media in vivo (0.06%) (8); thus, true quiescence is never obtained in vitro. This could in part explain why, in vitro, SMC do not reacquire an α-SM actin predominance. To better understand the mechanisms underlying the changes in actin isoform expression, expression of actin mRNAs was studied in vivo and in vitro with the help of the two specific probes previously described (4). α-SM actin mRNA expression increases during the development of the aorta, reaching about 90% of total actin mRNA in adult tissue (4,6). In experimental aortic thickening, α-SM actin mRNA expression decreases 15 days after injury (32%) and is reacquired 60 days after injury (87%) (4). This is similar to what is observed above for the protein. Thus, the expression of α-SM actin isoform in vivo follows the level of its mRNA, situating the regulation of protein synthesis at the transcriptional or post-transcriptional level. Aortic SMC in primary culture exhibit a decrease in α-SM actin expression (7) and synthesis (9) by 48 hours after plating (even in a serum-free medium); these features progressively decline up to confluence (4,7). Surprisingly, α-SM actin mRNA expression is still predominant of ter 6 days of primary culture, accounting for 83% of total actin mRNA. Thus, in primary culture, regulation of α-SM actin synthesis seems to take place, to a certain extent, at the level of mRNA translation. SMC grown to passage 5, however, express cytoplasmic actin mRNAs’ predominance; α-SM actin mRNA representing only 20% of total actin message. At this stage, the proportion of α-SM actin mRNA becomes comparable, as in vivo, to the level of protein expression.

We have seen above that quiescent SMC in normal artery express predominantly the α-SM actin isoform. This pattern is altered to ß-actin predominance during development, pathological conditions as well as culture, when SMC proliferate. From these data, however, the relationships between entry into the cell cycle and the changes in the expression of actin isoforms in SMC are not clear. To study these relationships, the model of the rat carotid artery injury was chosen (10). In this model, after the injury, 30% of SMC enter the cell cycle as a synchronous wave. Protein synthesis experiments were performed by 35S-methionine infusion and in vitro translation of total mRNA experiments were performed by using rabbit reticulocyte lysate. In the first 8–24 hours after carotid injury, before cells had left the G0/G1 phase of cell cycle, a decrease of α-SM actin mRNA expression and an increase of ß and γ-actin mRNAs were seen (11). α-SM actin in vitro translation and in vivo synthesis also declined in the first 24 hours, indicating a decrease in the level of functional α-SM actin mRNA, its amount being proportional to the amount of mRNA present. Synthesis of ß and γ-actin isoforms increased. DNA synthesis starts around 27 hours after injury (12) and, at 36 hours, only the S/G2 SMC population showed a small decrease in α-SM actin isoform, accompanied by a major decrease in α-SM actin mRNA (11). α-SM actin isoform in the whole SMC population declined only at 5 days after injury (11).During this period, total actin content remained the same. To determine whether the changes in actin isoforms are related to entry into the S phase of the cell cycle, heparin, which blocks SMC in late G1 (13), was administered with an osmotic pump. Heparin did not prevent the early (8–24 hours) changes in actin mRNAs and actin isoform synthesis. However, α-SM mRNA and α-SM actin expression were reinduced at 5 days in heparin treated animals. These findings suggest that actin isoform changes after injury follow variation in the mRNA levels. These variations in mRNAs take place during G0/G1 phase, whether or not the cell will subsequently enter the S phase of the cell cycle. They become greater in the S/G2 population compared to those remaining in GO/G1. These early changes are not prevented by heparin; however, heparin blocks SMC proliferation and reinduces a quiescent phenotype with α-SM actin predominance earlier after injury than in heparin untreated animals. Heparin or related proteoglycans could have a role in maintaining in vivo SMC in a quiescent state, by controlling α-SM actin mRNA and α-SM actin expression.

In order to better understand the possible mechanisms of heparin action on SMC, we have investigated the effect of heparin on proliferation and actin isoform expression in cultured rat SMC (14). Heparin treated primary and passage 5 SMC showed, in the presence of 10% fetal calf serum, a decrease of proliferation and an increase of α-SM actin (measured by Western blots or two-dimensional gel electrophoresis) compared to untreated SMC. When SMC were cultured in the presence of 10% plasma derived serum, no proliferation occurred and heparin did not modify α-SM actin expression. This suggests that the action of heparin is related to its anti-proliferative activity. SMC cultured in the presence of 10% FCS plus heparin had the same level of proliferation as SMC cultured in 5% FCS, but a higher content of α-SM actin. SMC cultured in 20% rat whole blood serum had a similar proliferation rate to that observed in SMC cultured in 10% FCS, but a higher content of α-SM actin. Moreover, in SMC cultured in 20% whole blood serum, heparin inhibited SMC proliferation but did not modify α-SM actin expression. Thus, the action of heparin on α-SM actin expression appears to be partially independent of proliferation and related to culture conditions. The proportion of α-SM actin mRNA, as measured by Northern blots with an α-SM actin mRNA specific probe, was increased by heparin compared to cells cultured in 10% FCS; this suggests that heparin acts at the transcriptional or post-transcriptional levels. Our results show that heparin acts not only on SMC proliferation but also on SMC differentiation; work on these lines may help in the understanding of the mechanisms of SMC adaptation during the atheromatous process.