Abstract-Smooth muscle cells (SMCs) are called on to proliferate during vascular restructuring but must return to a nonproliferative state if remodeling is to appropriately terminate. To identify mediators of the reacquisition of replicative quiescence, we undertook gene expression screening in a uniquely plastic human SMC line. As proliferating SMCs shifted to a contractile and nonproliferative state, expression of TIMP-3, Axl, and KIAA0098 decreased whereas expression of complement C1s, cathepsin B, cellular repressor of E1A-activated genes increased. Wilms' tumor 1-associating protein (WTAP), a nuclear constituent of unknown function, was also upregulated as SMCs became nonproliferative. Furthermore, WTAP in the intima of injured arteries was substantially upregulated in the late stages of repair. Introduction of WTAP complementary DNA into human SMCs inhibited their proliferation, with a corresponding decrease in DNA synthesis and an increase in apoptosis. Knocking down endogenous WTAP increased SMC proliferation, because of increased DNA synthesis and G 1 /S phase transition, together with reduced apoptosis. WTAP was found to associate with the Wilms' tumor-1 protein in human SMCs and WTAP overexpression inhibited the binding of WT1 to an oligonucleotide containing a consensus WT1 binding site, whereas WTAP knockdown accentuated this interaction. Expression of the WT1 target genes, amphiregulin and Bcl-2, was suppressed in WTAP-overexpressing SMCs and increased in WTAP-deficient SMCs. Moreover, exogenous amphiregulin rescued the antiproliferative effect of WTAP. These findings identify WTAP as a novel regulator of the cell cycle and cell survival and implicate a WTAP-WT1 axis as a novel pathway for controlling vascular SMC phenotype. (Circ Res. 2006;99:1338-1346.)Key Words: amphiregulin Ⅲ smooth muscle cells Ⅲ Wilms' tumor 1-associating protein Ⅲ vascular smooth muscle cell proliferation P henotype plasticity is a feature of adult vascular smooth muscle cells (SMCs). A widely studied example of this is the dedifferentiation of mature, nonproliferative SMCs into proliferative SMCs, a process central to vascular remodeling. 1,2 Although less well studied, an equally important manifestation of SMC plasticity is the reverse shift, whereby proliferative adult SMCs convert back to a nonproliferative state. This particular phenotype switch is essential for limiting SMC accumulation and for terminating vascular remodeling. As such, the regulatory factors that drive proliferative SMCs into a nonproliferative state, and hold them in that state, are critical for effective vascular remodeling and for limiting vascular disease.We have generated unique lines of nonimmortalized human SMCs that are capable of converting between proliferative and nonproliferative states. 2,3 In the presence of serum, these SMCs proliferate, migrate, and elaborate extracellular matrix similar to primary SMCs. On withdrawal of serum however they undergo a reproducible program of cellular maturation whereby they exit the cell cycle, migrate...

Background-The production of collagen is fundamental to atherosclerosis and critically dependent on posttranslational modification by prolyl 4-hydroxylase. Methods and Results-We report the cloning of a novel prolyl 4-hydroxylase catalytic (␣) subunit from human vascular smooth muscle cells. The peptide displayed conservation of critical residues for interacting with Fe 2ϩ and 2-oxoglutarate, essential cosubstrates for prolyl 4-hydroxylase activity. Furthermore, when the recombinant protein was expressed in cells, it associated with the ␤-subunit of prolyl 4-hydroxylase and could catalyze prolyl 4-hydroxylation of a collagen-like peptide. The tissue distribution was dissimilar from that of the 2 previously cloned ␣-subunits, suggesting a role beyond redundancy. Importantly, the novel gene was expressed in the fibrous cap of human carotid atherosclerotic lesions. Conclusions-The discovery of a novel prolyl 4-hydroxylase ␣-subunit, here termed the ␣(III)-subunit, suggests a new participant in collagen synthesis that, in view of the expression findings, may be relevant to atherosclerotic disease.