BET Bromodomain Blockade Mitigates Intimal Hyperplasia in Rat Carotid Arteries

Abstract

BackgroundIntimal 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.MethodsWe investigated the effect of BET inhibition on intimal hyperplasia using a rat balloon angioplasty model.ResultsWhile 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.ConclusionsBlocking BET bromodomains mitigates neointima formation, suggesting an epigenetic approach for effective prevention of intimal hyperplasia and associated vascular diseases.

Photoreceptor protection via blockade of BET epigenetic readers in a murine model of inherited retinal degeneration

“…Eyeballs were immediately enucleated and dissected. For morphometric and immunohistochemistry analyses, eyeballs were fixed in 4% paraformaldehyde for 7 h at 4 °C, and then used for preparation of paraffin-embedded sections or cryosections, according to our published methods [19]. Briefly, for cryosections, eyeballs were soaked in 30% sucrose in PBS for 14 h at 4 °C and 10-μm sections were cut from the eyeballs frozen in an optimum cutting temperature (OCT) embedding medium (Sakura Finetek 4583; Sakura Finetek USA, Inc., Torrance, CA).…”

“…Immunostaining was performed on paraffin-embedded retinal sections following our published method [19]. Briefly, sections were first incubated with each of the primary antibodies for 1 h: rabbit anti-BET2 (1:150, Abcam, 139690, Cambridge, MA); rabbit anti-BET4 (1:200, Abcam, 128874); mouse anti-BET3 (1:200, Abcam, 56342).…”

“…Purified messenger RNA (mRNA) (1 μg) was used for the first-strand complementary DNA (cDNA) synthesis using iScript cDNA synthesis kit (Bio-Rad) and quantitative RT-PCR was performed using the 7500 Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA), as described in our previous report [22]. Real-time quantitative PCR (qRT-PCR) data with a high cycle number (e.g., >35) was not considered.…”

“…To identify appropriate concentrations of BET inhibitors for various experiments, we performed dose response pilot studies. N9 cells were pre-treated with BET inhibitors at various concentrations for 12 h, and then subjected to CellTiterGlo viability assay, as described in our previous report [22]. We chose 0.5, 30, and 30 μM for JQ1, Olinone, and RVX208, respectively, as these represent the maximal concentrations that did not compromise N9 cell viability (Additional file 1: Figure S6).…”

“…To study the effect of BET inhibitors on the proliferation of N9 microglial cells, we used a Cell Proliferation BrdU ELISA (colorimetric) Kit (Roche Applied Science, Indianapolis, IN) following manufacturer instructions, as described in our previous study [22] with minor modifications. Briefly, N9 cells were seeded in 96-well plates at a density of 4000 cells per well with a final volume of 200 μl, in DMEM containing 0.5% FBS.…”