High rates of vascular smooth muscle cell (SMC) replication are observed, at least transiently, after injury to the arterial wall and contribute to the formation of a neointima. Neutralizing antibodies designed to inhibit growth of SMC have only been variably successful in inhibiting neointima formation, raising the possibility that neointimal cell proliferation involves unique growth mechanisms. This study examined the possibility that SMC isolated from injured rat carotid arteries would express an autonomous, mitogen-independent growth phenotype similar to that utilized by embryonic vascular SMC during periods of rapid growth. We found that primary cultures of SMC isolated 7 and 14 days after injury, times at which high in vivo replication rates were observed, demonstrated high intrinsic DNA synthetic rates compared to SMC isolated from uninjured arteries or at 2, 4, 21, and 28 days after injury where in vivo replication rates were far less. Subcultured SMC isolated from 7-day injured vessels (Neo7 SMC) exhibited a stable, autonomous growth phenotype, did not secrete detectable mitogenic activity, and had decreased alpha-actin and myosin expression compared to mitogen-dependent SMC. Heterokaryons constructed between autonomous Neo7 SMC and mitogen-dependent SMC exhibited a mitogen-dependent growth phenotype suggesting that nonautonomous SMC produce factors that actively inhibit autonomous growth. In contrast, heterokaryons constructed between Neo7 SMC and autonomous embryonic SMC retained an autonomous growth phenotype. We examined the expression of known tumor suppressors to determine if any of these factors played a role in inhibiting SMC autonomous growth. p27, p53, pRb, and PTEN were abundantly expressed by Neo7 SMC and e17 SMC under both basal and serum stimulated conditions. The data suggest that the mechanisms driving SMC replication during neointimal formation are self-driven and self-regulated, and that at specific times after injury, SMC escape normal growth suppressive mechanisms through the loss of intracellular growth suppressor activity.

Abstract-We sought to identify and characterize the expression pattern of genes expressed by smooth muscle cells (SMCs) during periods of self-driven replication during vascular development and after vascular injury. Primary screening of a rat embryonic aortic SMC-specific cDNA library was accomplished with an autonomous embryonic SMC-enriched, nonautonomous adult SMC-subtracted cDNA probe. Positive clones were rescreened in parallel with embryonic SMC-specific and adult SMC-specific cDNA probes. We identified 14 clones that hybridized only with the embryonic cDNA ("emb" clones), 11 of which did not share significant homology with sequences in any of the databases. Five of these novel emb genes (emb7, emb8, emb20, emb37, and emb41) were selectively and only transiently reexpressed in vivo by neointimal SMCs during periods of rapid replication. The emb8:embryonic growth-associated protein (EGAP), which was studied the most extensively, was expressed at high levels by cultured, autonomously replicating embryonic and neointimal SMCs but was detected only at low levels even in mitogenically stimulated adult SMCs. Finally, the administration of antisense EGAP oligonucleotides markedly attenuated embryonic and neointimal SMC replication rates. We suggest that autonomous replication of SMCs may be essential for normal vascular morphogenesis and for the vascular response to injury and that these newly identified "embryonic" genes may be part of the molecular machinery that drives this unique growth phenotype. (Circ Res. 2000;87:608-615.)Key Words: arteries Ⅲ vasculature Ⅲ restenosis Ⅲ muscle, smooth Ⅲ clones D uring vascular development, aortic smooth muscle cells (SMCs) in vivo undergo a distinct phase of rapid proliferation, a time period during which the vessel wall acquires its complement of SMCs, followed by a period of extensive extracellular matrix production that contributes to the structural maturation of the vessel wall. [1][2][3][4][5] We have documented in the rat that aortic SMCs replicate at a high rate (Ͼ80%/d) throughout the embryonic period of life, demonstrate dramatic decreases in replication at the embryonic-tofetal transition of intrauterine life (rates decrease to Ͻ40%/d), and gradually acquire a quiescent phenotype, reaching replication rates of Ͻ0.06%/d in the adult. 1 Significant proliferation of SMCs in the adult animal is observed only during certain stages in the development of vascular fibroproliferative diseases such as atherosclerosis, in restenosis after angioplasty, and in transplant arteriopathies. 6 -9 For instance, Clowes et al 10 have shown that after experimental arterial injury, neointimal SMCs demonstrate large increases in replication rates that reach levels similar to those observed during embryonic life. Furthermore, data from this laboratory and others suggest that after vascular injury, as during development, adult SMCs proceed through a period of rapid cell division followed by a period of extracellular matrix production. 4,[11][12][13] However, the factors that regulate SMC...

High rates of vascular smooth muscle cell (SMC) replication are observed, at least transiently, after injury to the arterial wall and contribute to the formation of a neointima. Neutralizing antibodies designed to inhibit growth of SMC have only been variably successful in inhibiting neointima formation, raising the possibility that neointimal cell proliferation involves unique growth mechanisms. This study examined the possibility that SMC isolated from injured rat carotid arteries would express an autonomous, mitogen-independent growth phenotype similar to that utilized by embryonic vascular SMC during periods of rapid growth. We found that primary cultures of SMC isolated 7 and 14 days after injury, times at which high in vivo replication rates were observed, demonstrated high intrinsic DNA synthetic rates compared to SMC isolated from uninjured arteries or at 2, 4, 21, and 28 days after injury where in vivo replication rates were far less. Subcultured SMC isolated from 7-day injured vessels (Neo7 SMC) exhibited a stable, autonomous growth phenotype, did not secrete detectable mitogenic activity, and had decreased alpha-actin and myosin expression compared to mitogen-dependent SMC. Heterokaryons constructed between autonomous Neo7 SMC and mitogen-dependent SMC exhibited a mitogen-dependent growth phenotype suggesting that nonautonomous SMC produce factors that actively inhibit autonomous growth. In contrast, heterokaryons constructed between Neo7 SMC and autonomous embryonic SMC retained an autonomous growth phenotype. We examined the expression of known tumor suppressors to determine if any of these factors played a role in inhibiting SMC autonomous growth. p27, p53, pRb, and PTEN were abundantly expressed by Neo7 SMC and e17 SMC under both basal and serum stimulated conditions. The data suggest that the mechanisms driving SMC replication during neointimal formation are self-driven and self-regulated, and that at specific times after injury, SMC escape normal growth suppressive mechanisms through the loss of intracellular growth suppressor activity.