Conditional deletion of Krüppel-like factor 4 delays downregulation of smooth muscle cell differentiation markers but accelerates neointimal formation following vascular injury

Abstract

Phenotypic switching of smooth muscle cells (SMCs) plays a key role in vascular proliferative diseases. We previously showed that Krüppel-like factor 4 (Klf4) suppressed SMC differentiation markers in cultured SMCs. Here, we derive mice deficient for Klf4 by conditional gene ablation and analyze their vascular phenotype following carotid injury. Klf4 expression was rapidly induced in SMCs of control mice after vascular injury but not in Klf4-deficient mice. Injury-induced repression of SMC differentiation markers was transiently delayed in Klf4-deficient mice. Klf4 mutant mice exhibited enhanced neointimal formation in response to vascular injury caused by increased cellular proliferation in the media but not an altered apoptotic rate. Consistent with these findings, cultured SMCs overexpressing Klf4 showed reduced cellular proliferation, in part, through the induction of the cell cycle inhibitor, p21(WAF1/Cip1) via increased binding of Klf4 and p53 to the p21(WAF1/Cip1) promoter/enhancer. In vivo chromatin immunoprecipitation assays also showed increased Klf4 binding to the promoter/enhancer regions of the p21(WAF1/Cip1) gene and SMC differentiation marker genes following vascular injury. Taken together, we have demonstrated that Klf4 plays a critical role in regulating expression of SMC differentiation markers and proliferation of SMCs in vivo in response to vascular injury.

Figure 1

Induction of Klf4 expression following vascular injury was abolished in conditional Klf4-deficient mice. A, Experimental protocol showing the timing of 4-OHT injection, ligation injury, and harvesting tissues. B, After completion of 4-OHT injection IP 10 times, recombination of the Klf4^loxP allele was examined by Southern blotting in the aorta of ERT-Cre⁺/Klf4^loxP/loxP (Klf4 KO) mice and ERT-Cre⁻/Klf4^loxP/loxP (control) mice. C, Expression of Klf4 and GAPDH protein was determined by Western blotting in the lung of Klf4 KO and control mice. *P<0.05 compared with control (n=4). D, Expression of Klf4 mRNA was determined by real-time RT-PCR in the colon of Klf4 KO and control mice. *P<0.05 compared with control (n=5). E, Injury-induced changes in expression of SM α-actin and Klf4 were determined by real-time RT-PCR in the carotid arteries of wild-type mice. The ratios of the right injured artery to the left uninjured artery are shown (n=3). *P<0.05 compared with Klf4 expression at day 0, #P<0.05 compared with SM α-actin expression at day 0. F, Klf4 expression was examined by immunohistochemistry in the carotid arteries of Klf4 KO and control mice at day 3 after injury. Representative pictures are shown from 4 mice analyzed per each genotype. Bar: 20 μm.

Figure 2

Injury-induced downregulation of SMC differentiation markers was delayed in conditional Klf4-deficient mice. A, Expression of SM α-actin and SM22α was examined by immunohisto-chemistry in the carotid arteries in Klf4 KO and control mice at day 3 and day 7 after injury. Representative pictures are shown from 4 to 6 mice analyzed per each genotype and each time point. Bar: 50 μm. B, In vivo ChIP assays were performed to determine the binding of Klf4 with the TGF-β control element–containing regions of the SM α-actin promoter and the SM22α promoter, as well as the first intronic region of the SM22α gene, in carotid arteries of wild-type mice at day 3 and day 7 after injury. Un indicates uninjured; Inj, injured. *P<0.05 compared with uninjured arteries (n=2).

Figure 3

Neointimal formation was accelerated in conditional Klf4-deficient mice following vascular injury. Carotid arteries were harvested from Klf4 KO and control mice at days 7, 14, and 21 after injury. Six mice were analyzed per each genotype and each time point. A, Verhoeff–van Gieson staining was performed in cross-sections from the injured and uninjured carotid arteries. Bar: 100 μm. B and C, Areas of the media (B) and the intima (C) were determined using ImagePro software. Ct indicates control; KO, Klf4 KO; Un, uninjured; Inj, injured. *P<0.05 compared with uninjured arteries, #P<0.05 compared with the arteries in corresponding control mice.

Figure 4

Enhanced neointimal formation in conditional Klf4-deficient mice was caused by increased cellular proliferation in the media. BrdUrd staining (A and C) and TUNEL staining (B and D) were performed in the carotid arteries of Klf4 KO and control mice at days 3, 7, 14, and 21 after injury. A and B, Representative pictures of BrdUrd staining at day 7 (A) and TUNEL staining at day 3 (B) are shown. Bars: 50 μm. C and D, The ratios of BrdUrd-positive cells (C) and TUNEL-positive cells (D) in the media and the intima were calculated (n=4 to 6). Ct indicates control; KO, Klf4 KO; Un, uninjured; Inj, injured. *P<0.05 compared with uninjured arteries, #P<0.05 compared with the arteries in corresponding control mice.

Figure 5

Klf4 reduced SMC proliferation. A, SMCs were isolated from the thoracic aorta of Klf4^loxP/loxP mice. Cells were infected with adenovirus-expressing Cre recombinase or empty adenovirus and designated as Klf4-null SMCs and control SMCs, respectively. Recombination of the Klf4^loxP alleles was confirmed by PCR. B and C, Cell numbers were counted in control SMCs (B) and Klf4-null SMCs (C). Cells were uninfected or infected with adenovirus expressing Klf4 (Ad/Klf4) or control (Ad/CMV) 1 day after plating (arrows). *P<0.05 compared with uninfected cells (n=4). D, BrdUrd-incorporation assays were performed in mouse SMCs infected with adenovi-rus expressing Klf4 (Ad/Klf4) or control (Ad/CMV). *P<0.05 compared with Ad/CMV-infected SMCs (n=3). E, Left, Mouse SMCs were transfected with FLAG-tagged Klf4 expression plasmid and stained with anti-FLAG antibody. Right, Magnified image of DAPI nuclear staining from the surrounded region in the left image. F, Mouse SMCs were uninfected or infected with adenovirus expressing Klf4 (Ad/Klf4) or control (Ad/CMV), and expression of caspase 3, Bax, Puma, and GAPDH was examined by Western blotting (caspase 3: 35 kDa; cleaved caspase 3: 17 and 19 kDa).

Figure 6

Klf4-induced growth suppression of SMCs was dependent on p21^WAF1/Cip1. A, p21^WAF1/Cip1 staining was performed in the carotid arteries of Klf4 KO and control mice at days 3, 7, 14, and 21 after injury. Representative pictures at day 7 are shown. The ratio of p21^WAF1/Cip1-positive cells in the media was calculated (n=4 to 6). Ct indicates control; KO, Klf4 KO; Un, uninjured; Inj, injured. Bar: 50 μm. *P<0.05 compared with uninjured arteries, #P<0.05 compared with the arteries in corresponding control mice. B, Expression of Klf4, p53, p21^WAF1/Cip1, and GAPDH was examined by Western blotting in cultured mouse SMCs that were uninfected or infected with adenovirus expressing Klf4 (Ad/Klf4) or empty virus (Ad/CMV). *P<0.05 compared with control (n=3). C, Cell numbers were counted in cultured p21^WAF1/Cip1 KO SMCs (n=4). Cells were uninfected or infected with adenovirus expressing Klf4 (Ad/Klf4) or control (Ad/CMV) 1 day after plating (arrows).

Figure 7

Klf4 induced p21^WAF1/Cip1 expression in concert with p53. A, Klf4 expression plasmid was cotransfected into rat aortic SMCs with the wild-type p21^WAF1/Cip1 promoter/enhancer–luciferase construct or its mutants, and luciferase activity was measured (n=3). B, Rat aortic SMCs were transfected with siRNA duplex for p53 or EGFP and infected with adenovirus expressing Klf4 (Ad/Klf4) or empty adenovirus (Ad/CMV). Expression of Klf4, p53, p21^WAF1/Cip1, and GAPDH was examined by Western blotting. *P<0.05 compared with control (n=3). C, Association of Klf4 and p53 with the proximal and the distal regions of the p21^WAF1/Cip1 promoter/enhancer was determined by ChIP assays in mouse cultured SMCs infected with adenovirus expressing Klf4 (Ad/Klf4) or empty adenovirus (Ad/CMV). *P<0.05 compared with Ad/CMV-infected SMCs (n=3). D, Association of Klf4 and p53 with the proximal and the distal regions of the p21^WAF1/Cip1 promoter/enhancer was determined by in vivo ChIP assays in carotid arteries of wild-type mice at day 3 after injury. Un indicates uninjured; Inj, injured. *P<0.05 compared with uninjured arteries (n=2).