Flow-dependent epigenetic regulation of IGFBP5 expression by H3K27me3 contributes to endothelial anti-inflammatory effects

Abstract

Rationale: Atherosclerosis is a chronic inflammatory and epigenetic disease that is influenced by different patterns of blood flow. However, the epigenetic mechanism whereby atheroprotective flow controls endothelial gene programming remains elusive. Here, we investigated the possibility that flow alters endothelial gene expression through epigenetic mechanisms. Methods: En face staining and western blot were used to detect protein expression. Real-time PCR was used to determine relative gene expression. RNA-sequencing of human umbilical vein endothelial cells treated with siRNA of enhancer of zeste homolog 2 (EZH2) or laminar flow was used for transcriptional profiling. Results: We found that trimethylation of histone 3 lysine 27 (H3K27me3), a repressive epigenetic mark that orchestrates gene repression, was reduced in laminar flow areas of mouse aorta and flow-treated human endothelial cells. The decrease of H3K27me3 paralleled a reduction in the epigenetic "writer"-EZH2, the catalytic subunit of the polycomb repressive complex 2 (PRC2). Moreover, laminar flow decreased expression of EZH2 via mechanosensitive miR101. Genome-wide transcriptome profiling studies in endothelial cells treated with EZH2 siRNA and flow revealed the upregulation of novel mechanosensitive gene IGFBP5 (insulin-like growth factor-binding protein 5), which is epigenetically silenced by H3K27me3. Functionally, inhibition of H3K27me3 by EZH2 siRNA or GSK126 (a specific EZH2 inhibitor) reduced H3K27me3 levels and monocyte adhesion to endothelial cells. Adenoviral overexpression of IGFBP5 also recapitulated the anti-inflammatory effects of H3K27me3 inhibition. More importantly, we observed EZH2 upregulation, and IGFBP5 downregulation, in advanced atherosclerotic plaques from human patients. Conclusion: Taken together, our findings reveal that atheroprotective flow reduces H3K27me3 as a chromatin-based mechanism to augment the expression of genes that confer an anti-inflammatory response in the endothelium. Our study exemplifies flow-dependent epigenetic regulation of endothelial gene expression, and also suggests that targeting the EZH2/H3K27me3/IGFBP5 pathway may offer novel therapeutics for inflammatory disorders such as atherosclerosis.

Keywords: EZH2; H3K27me3; IGFBP5; atherosclerosis; endothelial cells; epigenetic.

Figure 1

Laminar flow specifically reduces repressive epigenetic mark H3K27me3 in endothelial cells in vitro and in vivo. (A) Diagram of PRC2. PRC2 is composed of EED, SUZ12, RbAp46/48, and EZH2, which imposes the H3K27me3 epigenetic mark to the target gene promoter, leading to gene silencing. (B) Laminar flow decreases global H3K27me3 levels. HUVECs were exposed to laminar flow for 48 h, and then total histones were purified as described in the methods section. GSK126, a specific inhibitor of EZH2 activity was used as positive control (n=4-5, **P<0.01, ***P<0.001 vs. static control or DMSO). (C) Laminar flow does not affect the level of H3K9me3 or H3K9ac. HUVECs were exposed to laminar flow. Cell alignment in response to flow was observed to ensure successful induction of laminar flow. Then, epigenetic marks H3K9me3 and H3K9ac were determined by Western blot. The same membranes were stripped and incubated with histone 3 (H3) as loading controls (n=3). (D) En face immunofluorescent staining of H3K27me3 in mouse aorta. The aortic arch and thoracic aorta were collected from 3-month-old ApoE^-/- mice fed a normal chow diet for en face staining. Red, H3K27me3; green, VE-cadherin; blue, DAPI; scale bar=20 µm (n=5).

Figure 2

Laminar flow decreases the expression of chromatin modifier EZH2 in vitro and in vivo. (A) HUVECs were exposed to laminar flow for the indicated time points, and then EZH2 protein expression was determined by Western blot (*P<0.05, ***P<0.001 vs. static control, n=6). (B) HCAECs were exposed to laminar flow for 24 h, and then EZH2 protein expression was determined by Western blot (**P<0.01, n=3). (C) Illustration of atheroprone (inner curvature of aortic arch, #1) and atheroprotective (thoracic aorta, #2) regions of mouse aorta. The picture was manually drawn by powerpoint. (D) EZH2 mRNA expression in aortic endothelium from atheroprone and atheroprotective regions of mouse aorta. Intima-enriched RNA was isolated from the aortic arch and thoracic aorta of ApoE^-/- mice fed a normal chow diet for 3 months. RNA was reverse transcribed and cDNA was used for real-time PCR quantification of EZH2 using GAPDH as internal control (**P<0.01 vs. aortic arch, n=8). (E) En face immunofluorescent staining of EZH2 in mouse aorta. The aortic arch and thoracic aorta were collected from 3-month-old ApoE^-/- mice fed a normal chow diet for en face staining. Red, EZH2; green, VE-cadherin; blue, DAPI; scale bar=20 µm (n=5).

Figure 3

miR101 is involved in laminar flow-induced EZH2 downregulation (A) Laminar flow decreases EZH2 mRNA expression. HUVECs were exposed to laminar flow for 24 h, and then RNA was isolated for real-time PCR analysis using GAPDH as loading control (**P<0.01 vs. static control, n=3). (B-C) HUVECs were transfected with miR101 inhibitor (miR101 inh) or control (100 nM) for 48 h and then exposed to laminar flow (L-flow) for 24 h before Western blot was performed to determine EZH2 protein expression (**P<0.01 static+anti-miR control, ##P<0.01 vs. laminar flow+anti-miR control, n=3).

Figure 4

siRNA depletion and pharmacological inhibition of EZH2 reduces monocyte adhesion to endothelial cells. (A) HUVECs were transfected with non-targeting control siRNA (siNC, 20 nM) or two independent EZH2 siRNAs (#1, #2, 20 nM). Then, EZH2 and H3K27me3 protein expression was determined by Western blot using tubulin and Histone 3 (H3) as the loading controls, respectively (n=4). (B) Quantification of (A) (**P<0.01, ***P<0.001 vs. siNC, n=4). (C) HUVECs were transfected with non-targeting control siRNA (siNC) or two independent EZH2 siRNAs. Then, a monocyte adhesion assay was performed, original magnification X20. (D) Quantification of (C) (***P<0.001 vs. siNC+vehicle (veh), ###P<0.001 vs. siNC+TNFα, n=4). (E) HUVECs were incubated with different doses of GSK126, then, a monocyte adhesion assay was performed (n=4). Representative data from one experiment is shown in the quantification data (*P<0.05, **P<0.01 vs. TNFα).

Figure 5

Identification of IGFBP5 as a flow-sensitive and EZH2/H3K27me3-target gene in endothelial cells. (A) Venn diagram analysis of overlapping genes revealed by RNA-seq of HUVECs exposed to laminar flow or EZH2 depletion: a total of 293 genes were identified. A complete list of overlapping genes was presented in Table S7. The RNA-seq data were deposited in NCBI Gene Omnibus (http://www.ncbi.nlm.nih.gov/geo/) with the accession number GEO: GSE87534. (B) Laminar flow upregulates IGFBP5 mRNA expression (n=3). NOS3 gene was used as a positive control for flow treatment (*P<0.05, **P<0.01 vs. static control). (C) Laminar flow upregulates IGFBP5 protein expression in culture supernatant. After exposure to laminar flow for 24 h, culture supernatant was adjusted by cell number, then IGFBP5 protein expression in the media was determined (n=3). (D) Laminar flow reduces H3K27me3 binding to IGFBP5 gene promoter. Chromatin immunoprecipitation (ChIP) coupled with quantitative real-time PCR (ChIP-qPCR) for IGFBP5 was performed in HUVECs exposed to static conditions or laminar flow for 24 h. Representative data from one experiment are presented (as % input; **P<0.01 vs. IgG control, #P<0.05 vs static with H3K27me3 ChIP, n=2). (E) EZH2 overexpression (M.O.I.=2, 48 h) decreases IGFBP5 gene expression, as determined by qRT-PCR (= *P<0.05 vs. Adcon, n=3). (F) EZH2 siRNA depletion (20 nM, 48 h) increases IGFBP5 gene expression, as determined by qRT-PCR (**P<0.01, ***P<0.001 vs. siNC, n=3). (G) Pharmacological inhibition of EZH2 by GSK126 (10 μM, 72 h) increases IGFBP5 gene expression without affecting EZH2 expression, as determined by qRT-PCR (*P<0.05 vs. veh (DMSO), n=4). (H) COS7 cells were transfected with pGL3-IGFBP5-luc (-1000-+45bp). 4 h after transfection, cells were infected with control adenovirus (AdNC) or AdEZH2 (M.O.I=2) for an additional 24 h before luciferase activity assay was performed (*P<0.05 vs. AdNC, n=3).

Figure 6

IGFBP5 inhibits endothelial inflammation. (A-B) HUVECs were infected with increasing doses (M.O.I.=1-10) of IGFBP5 adenovirus for 48 h, then a monocyte adhesion assay was performed in the absence (vehicle) or presence of TNFα. Adherent monocytes were counted and quantified (***P<0.001 vs. veh plus control adenovirus, #P<0.05 vs. TNFα plus control adenovirus, n=4), original magnification X20. (C-D) HUVECs received the same treatments as described in (A), then, whole cell lysates were collected for Western blot analysis using GAPDH as the loading control. Densitometric analysis of (C) is provided in (D) (**P<0.01, ***P<0.001 vs. veh plus control adenovirus, #P<0.05, ##P<0.01, ###P<0.001 vs. TNFα plus control adenovirus, n=3).

Figure 7

EZH2 and IGFBP5 expression in human atherosclerotic plaques. (A) Hematoxylin and eosin (H&E) staining of healthy control and atherosclerotic carotid arteries (n=3, scale bar=600 µm). (B) mRNA expression levels of EZH2 and IGFBP5 determined in human carotid atherosclerotic plaques and healthy controls using real-time PCR (**P<0.01, ***P<0.001 vs. healthy control, n=9-10).