BET Bromodomain Blockade Mitigates Intimal Hyperplasia in Rat Carotid Arteries

Abstract

Background: Intimal hyperplasia is a common cause of many vasculopathies. There has been a recent surge of interest in the bromo and extra-terminal (BET) epigenetic "readers" including BRD4 since the serendipitous discovery of JQ1(+), an inhibitor specific to the seemingly undruggable BET bromodomains. The role of the BET family in the development of intimal hyperplasia is not known.

Methods: We investigated the effect of BET inhibition on intimal hyperplasia using a rat balloon angioplasty model.

Results: While BRD4 was dramatically up-regulated in the rat and human hyperplastic neointima, blocking BET bromodomains with JQ1(+) diminished neointima in rats. Knocking down BRD4 with siRNA, or treatment with JQ1(+) but not the inactive enantiomer JQ1(-), abrogated platelet-derived growth factor (PDGF-BB)-stimulated proliferation and migration of primary rat aortic smooth muscle cells. This inhibitory effect of JQ1(+) was reproducible in primary human aortic smooth muscle cells. In human aortic endothelial cells, JQ1(+) prevented cytokine-induced apoptosis and impairment of cell migration. Furthermore, either BRD4 siRNA or JQ1(+) but not JQ1(-), substantially down-regulated PDGF receptor-α which, in JQ1(+)-treated arteries versus vehicle control, was also reduced.

Conclusions: Blocking BET bromodomains mitigates neointima formation, suggesting an epigenetic approach for effective prevention of intimal hyperplasia and associated vascular diseases.

Keywords: BET; BET, bromo- and extra-terminal domain family of epigenetic readers; BRD4; BRD4, bromodomain-containing protein 4, a BET family member; EC, vascular endothelial cells; Epigenetic reader; IH, intimal hyperplasia; Intimal hyperplasia; JQ1(+), a BET-specific bromodomain inhibitor; JQ1(−), inactive enantiomer; PDGF, platelet-derived growth factor; SMC, vascular smooth muscle cell; Smooth muscle cell.

Fig. 1

Up-regulation of BRD4 in the neointima of injured rat carotid arteries and in human vein and artery grafts. A. Immunostaining of rat carotid artery sections showing increase of BRD4 positive cells at days 3, 7, and 14 after balloon angioplasty versus uninjured arteries. “Negative staining” refers to background control without using a primary antibody. Quantification: mean ± SEM; n = 3 animals at each time point; *P < 0.05 compared to uninjured control. Arrowhead: internal elastic lamina (IEL); arrow: external elastic lamina (EEL). Neointima is defined between lumen and IEL. Scale bar: 50 μm. B. Immunohistochemistry showing increase of BRD4 in the neointima of human saphenous vein and internal mammary artery grafts versus normal vessels. Arrowhead: IEL. Scale bar: 50 μm.

Fig. 2

Inhibition of intimal hyperplasia by treatment with JQ1(+) in balloon-injured rat carotid arteries. A. Van Gieson-stained sections showing reduced neointima in JQ1(+)-treated rat carotid arteries compared to vehicle control 14 days after injury. Arrowhead: IEL; arrow: EEL. B. Quantification of IH (intima/media area ratio), lumen area, and EEL length is presented as mean ± SEM; n = 4 animals; *P < 0.05 versus vehicle control.

Fig. 3

Inhibition of rat and human aortic smooth muscle cell proliferation by treatment with JQ1(+). A and B. Dose responses to JQ1 enantiomers showing inhibition of proliferation of primary rat (A) and human (B) aortic SMCs, by JQ1(+) but not JQ1(−). Proliferation was measured by CellTiter-Glo assay or BrdU assay 24 h after PDGF-BB stimulation. Quantification: mean ± SEM; n = 3 independent experiments. C. Immunostaining of PCNA showing reduced proliferating cells in JQ1(+)-treated (500 μg per rat) carotid arteries versus vehicle control 14 days after injury. Arrowhead: IEL; arrow: EEL. “Negative staining” refers to the background control without using a primary antibody. Quantification: mean ± SEM; n = 4 animals; *P < 0.05 versus vehicle control. HPF: high power field. Scale bar: 50 μm.

Fig. 4

Inhibition of rat smooth muscle cell migration by treatment with JQ1(+). Scratch assay (A) and Transwell assay (B) showing inhibition of rat SMC migration by 1 μM JQ1(+) but not JQ1(−) at 24 h after PDGF-BB stimulation. Dashed line marks the cell-free gap at 0 h after scratch. Quantification: mean ± SEM; n = 3; *P < 0.05 versus vehicle control.

Fig. 5

Inhibition of rat smooth muscle cell proliferation and migration by siRNA knockdown of BRD4. A and B. Specific siRNA knockdown of BRD4 indicated by RT-PCR (A) and Western blot (B). C and D. Inhibition of PDGF-BB stimulated (for 24 h) rat SMC proliferation (BrdU assay)(C) and migration (scratch assay)(D), by BRD4 siRNA knockdown. Quantification: mean ± SEM; n = 3; *P < 0.05 versus scrambled siRNA control.

Fig. 6

Down-regulation of PDGFRα in vitro by treatment with JQ1(+) or siRNA knockdown of BRD4. A. Determination of PDGFRα mRNA (RT-PCR) and protein (Western blot). mRNA was determined at 6 h and protein was determined at 24 h after treatment with JQ1(+). Quantification: mean ± SEM; n = 3; *P < 0.05 versus vehicle control. B. Inhibition of ERK phosphorylation in SMCs treated with JQ1(+). In order to confirm the effect of JQ1(+) on PDGFR expression, phosphor-ERK, which is a well-established downstream effector and hence a surrogate of PDGFR activation, was determined. Rat aortic SMCs were pretreated with 1 μM JQ1(+), JQ1(−), or equal volume of DMSO for 48 h in starvation medium, as described in Methods, and then PDGF-BB was added (final 10 ng/ml) for stimulation of ERK phosphorylation. Cells were harvested after 15 min and used for Western blotting determinations. Quantification: mean ± SEM; n = 3; *P < 0.05. C. PDGFRα down-regulation by BRD4 siRNA. Quantification: mean ± SEM; n = 3; *P < 0.05 versus scrambled siRNA control.

Fig. 7

Down-regulation of PDGFRα in vivo by treatment with JQ1(+). A. Immunohistochemistry showing reduced PDGFRα in the media and neointima in JQ1(+)-treated (500 μg per rat) carotid arteries compared to vehicle control 14 days after balloon injury. Arrowhead: IEL. “Negative staining” refers to background control without using a primary antibody. Quantification: mean ± SEM; n = 4 animals; *P < 0.05. HPF: high power field. Scale bar: 50 μm. B. Immunohistochemistry showing increase of PDGFRα in the neointima of human saphenous vein and internal mammary artery grafts compared to normal vessels. Scale bar: 50 μm.

Fig. 8

Prevention of inflammatory cytokine-induced vascular endothelial cell dysfunction by JQ1(+). A. TNFα or IL-1β-induced (20 ng/ml, 4 h) human aortic EC apoptosis, measured as Caspase-3/7 activity, was prevented by pre-treatment with 100 nM JQ1(+) for 24 h. B. TNFα or IL-1β-induced (20 ng/ml, 4 h) inhibition of EC migration was prevented by pre-treatment with 100 nM JQ1(+) for 24 h. Quantification: mean ± SEM; n = 3; ^#P < 0.05 versus vehicle control; *P < 0.05 between the conditions in the absence and presence of JQ1(+). C. Images of human aortic ECs 24 h after scratch. Dashed line marks the cell-free gap at 0 h after scratch. Quantified data are presented in B.