Abstract 310: Conditional Knockout of the Kruppel-Like Factor 4 Gene Delays Downregulation of Smooth Muscle Marker Genes but Accelerates Neointimal Formation in Response to Vascular Injury
Phenotypic switching of smooth muscle cells (SMCs) plays a key role in the development of vascular proliferative diseases. We previously showed that Krüppel-like factor 4 (KLF4) was a potent repressor of expression of SMC differentiation maker genes in cultured SMCs. To determine the role of KLF4 in SMC phenotypic switching in vivo, we generated Tamoxifen-inducible KLF4 knockout mice by breeding Tamoxifen-inducible Cre-recombinase expression (Tam-Cre+) mice and KLF4 floxed mice, and analyzed their phenotype following vascular injury. The KLF4 gene was deleted by activating Cre-recombinase by Tamoxifen injection in Tam-Cre+/KLF4fl/fl mice, and the right carotid artery was then ligation-injured. Tam-Cre−/KLF4fl/fl mice were also injected with Tamoxifen and used as controls. After the injury, KLF4 expression was rapidly induced in SMCs of control mice, but not in KLF4 knockout mice. Three days after the injury, expression of SMC differentiation marker genes was decreased in control mice, whereas it was not decreased in KLF4 knockout mice. Of major interest, KLF4 knockout mice exhibited the accelerated neointimal formation at day 7, 14, and 21 after the injury. Bromodeoxyuridine staining and TUNEL staining revealed that the enhanced formation of neointima was caused by the increased proliferation rate of SMCs rather than the altered apoptotic rate, suggesting that induction of KLF4 expression following the vascular injury plays a role in the attenuation of neointimal formation. To determine the mechanisms whereby KLF4 reduces the proliferation rate of SMCs, the effect of KLF4 on expression of p21, a cell cycle inhibitor, was examined in cultured SMCs. Results showed that KLF4 increased p21 expression through the direct binding to the promoter-enhancer region of the p21 gene, and also enhanced the binding of p53 to the p21 promoter-enhancer. Taken together, results of our present studies provide evidence that KLF4 inhibits downregulation of SMC differentiation marker genes as well as the proliferation of SMCs in response to vascular injury.