HSP90-Dependent Upregulation of EZH2 Promotes Hypoxia/Reoxygenation-Induced Pyroptosis by Inhibiting miR-22 in Endothelial Cells

Introduction

Increasing evidence indicates that cardiovascular disease is the leading global cause of death.¹ Myocardial infarction (MI) is the major cause of death in cardiovascular disease and has attracted widespread attention due to its increasing incidence, complications, poor outcomes and limited treatment.² Vascular endothelial cells play a key role in maintaining and supporting coronary microvessels and adjacent cardiomyocytes under normal conditions and angiogenesis under pathophysiological conditions.³ Vascular endothelial cells have been shown to be sensitive to ischemic and reperfusion (I/R)-induced insult and I/R-induced vascular endothelial cell injury is a crucial and initial stage for the development of MI.^4–6 Therefore, exploring the molecular etiology of I/R-induced vascular endothelial cell injury of great significance to develop more effective treatment strategies for MI.

Pyroptosis, a form of proinflammatory cell death, is mediated by NOD-like receptor thermal protein domain associated protein 3 (NLRP3)-dependent inflammasome assembly, which is accompanied by gasdermin-D (GSDMD) cleavage and Interleukin-1β (IL-1β) and Interleukin-18 (IL-18) release.^7,8 Pyroptosis has been demonstrated to aggravate hypoxia/reoxygenation (H/R)-induced injury in various cells, including endothelial cells.^9,10 Recently, pyroptosis-associated endothelial cell death is significant in the development of MI.^11,12 Furthermore, by reducing vascular endothelial cell pyroptosis, chitosan hydrogel has been shown to increase the efficiency of bone marrow mesenchymal stem cells in the treatment of MI.¹³ These findings indicated that the inhibition of I/R-induced endothelial cell pyroptosis could be a potential strategy for the treatment of MI. However, the molecular mechanisms underlying H/R-induced pyroptosis in vascular endothelial cells remain largely unknown.

MicroRNAs (miRNAs) are a highly conserved group of small endogenous non-coding RNA molecules composed of 21–23 nucleotides.^14,15 They act as post-transcriptional regulators of gene expression via binding to the partial sequence homology of the 3′-untranslated region of their target mRNAs.^14,15 miRNAs have been shown to be involved in the regulation of many biological processes, such as cancer, metabolism and inflammation.^14,15 Accumulating evidence has suggested that miRNAs are key regulators of pyroptosis.^16–18 Recently, miR-22 was reported to play a key role in the regulation of pyroptosis in different types of cells, including endothelial cells.^19–22 However, whether miR-22 mediates pyroptosis in H/R-induced pyroptosis in endothelial cells and the underlying mechanism are unclear.

Enhancer of zeste homolog 2 (EZH2), the enzymatic catalytic subunit of polycomb repressive complex 2 (PRC2), regulates gene expression epigenetically by catalyzing histone H3 lysine 27 (H3K27) trimethylation (H3K27me3).^23–25 An extensive number of studies have shown that EZH2 functions in various biological processes. For example, EZH2 was shown to inhibit cellular senescence and promote DNA damage repair.²⁶ EZH2 was also demonstrated to work as a master regulator of apoptosis, autophagy, and cell cycle progression.^25,27 In addition, EZH2 played a critical role in cell lineage determination and relative signaling pathways.²⁸ Recently, EZH2 was shown to promote oxidative stress-mediated renal pyroptosis.²⁹ However, the role of EZH2 in H/R-induced endothelial cell pyroptosis still remains unclear.

In this study, we used an in vitro model of H/R-exposed human umbilical vein endothelial cells (HUVECs) to simulate the in vivo vascular endothelial I/R and explore the role of EZH2 in H/R-induced vascular endothelial cell pyroptosis as well as its link with miR-22.

Materials and Methods

Cell Culture and Treatment

HUVECs (Cat. No. BNCC342247) were purchased from BeNa culture collection (Henan, China) and cultured in endothelial cell medium (ECM, Cat. No.1001, Sciencell, USA) containing 10% fetal bovine serum (FBS, Cat. No. 0025, Sciencell), 1% endothelial cell growth supplement (ECGS, Cat. No. 1052, Sciencell) and 1% penicillin/streptomycin (P/S, Cat. No. 0503, Sciencell) at 37°C, 5% CO₂. For H/R induction, HUVECs were cultured in glucose-free and serum-free medium and then transferred to the AnaeroPack^TM container (Mitsubishi Gas Chemical, Tokyo, Japan) containing a mixture of 1% O₂, 94% N₂ and 5% CO₂ at 37 °C for 12 h to establish hypoxia. Subsequently, the cells were cultured in normal DMEM with 10% FBS for 4 h to establish reoxygenation.

Cell Viability Assay

The viability of HUVECs was evaluated by using a CCK-8 assay kit (Cat. No. C0037, Beyotime, Shanghai, China) according to the manufacturer’s instructions. HUVECs were treated as described in the text and then cultured into 96-well plates at a density of 5×10³ cells/well. CCK-8 reagent (10 μL/well) was then added to the culture medium and continuously incubated for 2 h at 37 °C. The absorbance was determined at 450 nm using a microplate reader (ELx800, BioTek Instruments, Inc., Vermont, USA).

Calcein-AM/Propidium Iodide (PI) Staining

The treated HUVECs were analyzed with a Calcein-AM/PI cell viability/cytotoxicity assay kit (Cat. No. C2015M, Beyotime) according to the manufacturer’s instructions. Briefly, after different stimulation, HUVECs were stained with Calcein-AM/PI at 37 °C for 30 min in the dark. Subsequently, the fluorescence images were acquired by using a fluorescence microscope (Leica DM2500). Image Pro advanced software was used to analyze the average fluorescence intensity.

ELISA Assay

After different stimulation, culture supernatants of HUVECs were collected and the concentrations of IL-1β and IL-18 in the supernatants were quantified by using commercial ELISA kits (IL-18, Cat. No. EMC011.96; IL-1β, Cat. No. EMC001b.96.2, NeoBioscience, Shenzhen, China) according to the manufacturer’s instructions.

Western Blot

After different treatment, total protein was extracted from HUVECs using RIPA lysis buffer (Cat. No. P0013C, Beyotime). The protein concentrations were detected with an enhanced BCA protein assay kit (Cat. No. P0010, Beyotime). 30 μg of proteins was separated by SDS-PAGE, and subsequently transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% fat-free milk for 1 h at room temperature. The membranes were then incubated overnight at 4 °C with primary antibodies. The primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and the dilution ratios of antibodies were as follows: NLRP3 (Cat. No. #15101, dilution 1/1000), cleaved caspase-1 (Cat. No. #4199, dilution 1/2000), GSDMD-N (Cat. No. #36425, dilution 1/2000), HSP90 (Cat. No. #4877, dilution 1/1000), EZH2 (Cat. No. #5246, dilution 1/1000), H3K27me3 (Cat. No. #9733, dilution 1/1000) and GAPDH (Cat. No. #5174, dilution 1/3000). After being washed three times with TBST, the membranes were incubated with secondary antibodies (Cat. No. sc-2004, Santa Cruz Technology, USA) for 1 h at room temperature. Protein bands were detected using enhanced chemiluminescence (ECL) reagent (Cat. No. 34577, Thermo Fisher Scientific, USA).

Immunofluorescence Staining

HUVECs were cultured under normal conditions or subjected to H/R exposure. After that cells were washed three times by PBS and then fixed with 4% paraformaldehyde for 30 min at room temperature. After being washed three times by PBS, the cells were treated with 0.2% Triton X-100 for 5 min at room temperature. The cells were washed three times by PBS followed by blocking in 1% BSA for 1 h at room temperature. Subsequently, the cells were incubated with antibody against EZH2 (Cat. No. #5246, dilution 1/200) or caspase-1 (Cat. No. #4199, dilution 1/200) overnight at 4° C. After that, the cells were incubated with the secondary antibodies Alex Fluor^®488-labeled secondary antibody (green, A11008, 1:1000; Invitrogen, USA) at 37° C for 1 h. Finally, the cells were stained with DAPI blue (1:1000; cat. 0100–20, Southern Biotech, USA) for 5 min. Slides were observed by a fluorescent microscope (Leica DM2500).

RT-qPCR

Total RNA was extracted from HUVECs by using TRIzol (Invitrogen, USA) and was reverse-transcribed into cDNA using a reverse transcriptase kit (Cat. No. RR037A, TaKaRa, Tokyo, Japan) according to the manufacturer’s instructions. Real-time qPCR was then carried out using SYBR premix Ex Taq II kit (Cat. No. RR390A, Tokyo, Japan) on an ABI 7300 Real-Time PCR system (Applied Biosystems, CA, USA). U6 was served as internal references and the relative expression level of miR-22 was calculated by 2^−ΔΔCt method. Primer sequences used were listed as follows: miR-22, forward: 5’-GGTTAAGCTGCCAGTTGAA-3’ and reverse: 5’-CAGTGCGTGTCGTGGAGT-3’; U6 snRNA, forward: 5’- GCTTCGGCAGCACATATACTAAAAT-3’ and reverse: 5’-CGCTTCACGAATTTGCGTGTCAT-3’.

siRNAs, Plasmid and Cell Transfection

Specific siRNAs for EZH2 and HSP90, miR-22 mimic, miR-22 inhibitor, control mimic, and control inhibitor were obtained from GenePharm (Shanghai, China). pcDNA3.1-HA plasmid with EZH2 overexpression (pcDNA3.1-HA-EZH2) was bought from Shanghai Genechem Co., Ltd. (Shanghai, China). Lipo6000^™ (Cat. No. D2107, Beyotime) was used to transfect these oligonucleotides or plasmid into HUVECs following the protocol of the manufacturer. The corresponding sequences were as follows: siEZH2, 5′-CCAUGUUUACAACUAUCAA-3′; siHSP90-sense 5’-TCGTCAGAGCTGATGATGAAGT-3’; siNC, 5′-UUCUCCGAACGUGUCACGU-3’. The efficiency of transfection was validated by comparing the levels of EZH2 and HSP90 between transfected and controlled cells by Western blot.

Chromatin Immunoprecipitation (ChIP) Assay

ChIP assay was performed with ChIP assay kit (Cat. No. P2078, Beyotime, Shanghai, China) according to the manufacturer’s instructions. Briefly, HUVECs were crosslinked with 1% formaldehyde for 10 minutes, and the reaction was quenched with glycine (0.125 mol/L) for 5 minutes. The cross-linked chromatin was then sonicated followed by immunoprecipitation with 10 mg of anti-EZH2 (Cat. No. #5246, CST), H3K27me3 (Cat. No. #9733, CST) or rabbit anti-IgG (Cat. No. A7016, Beyotime) overnight at 4 °C. After being purified, the enrichment of the miR-22 promoter was determined by qPCR. The corresponding primer sequences used were as follows: forward, 5’-ACTCTCGTTTGACGTAGCGCTT-3’, and reverse, 5’- CGCCCTGGCTCTGATTGGCAAG-3’. The amount of immunoprecipitated DNA was calculated and then normalized to the amount of input DNA.

Dual Luciferase Assay

Double luciferase assay was used to detect the binding of miR-22 to NLRP3. The wild-type (WT) untranslated region or mutant (Mut) of NLRP3 was amplified and subcloned into a pGLuc-Dura-miR basic luciferase reporter vector (Cat. No. C0526, Beyotime). HUVECs were then cotransfected with the above two vectors and the miR-22 mimics or control mimic (mi-NC) using Lipo6000^™ (Cat. No. D2107, Beyotime) according to the manufacturer’s protocol. After 48 h, luciferase assays were performed using dual luciferase reporter gene assay kit (Cat. No. RG027, Beyotime). The luciferase activity was normalized to values of Renilla luciferase activity.

Co-Immunoprecipitation (Co-IP)

Co-IP was conducted by using immunoprecipitation kit (Cat. No. P2177S, Beyotime) according to the manufacturer’s instructions. Briefly, after being treated as described in the text, 500 µg of total cellular proteins were extracted and used for immunoprecipitation by adding 1 µg of anti-HSP90 antibody (Cat. No. #4877, CST) and incubating at 4 °C for overnight. Immunoprecipitated proteins were analyzed by Western blot analysis with anti-EZH2 antibody (Cat. No. #5246, CST).

Statistical Analysis

All data are represented as the mean ± SD. Statistical analyses were carried out by using GraphPad Prism (version 9.0, USA). An unpaired Student’s t-test was used to analyze the data between two groups. The differences among multiple groups were analyzed using one-way ANOVA followed by Tukey post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

Hypoxia/Reoxygenation (H/R) Evokes Pyroptosis in HUVECs

To investigate the effect of H/R on the pyroptotic cell death of endothelial cells, HUVECs were subjected to hypoxia for 12 h and reoxygenation for 4 h. The results of the CCK-8 assay showed that H/R significantly reduced the viability of HUVECs (Figure 1A). Calcein-AM/PI staining showed that the percentage of PI positive cells was increased after H/R exposure (Figure 1B). Immunofluorescence showed that the core component caspase-1 was increased after H/R exposure (Figure 1C). H/R also increased the protein expression levels of pyroptosis-related proteins, including NLRP3, c-caspase-1, and GSDMD-N (Figure 1D). The results of ELISA assay showed that H/R increased IL-1β and IL-18 levels in the cell supernatants (Figure 1E). Collectively, these data suggest that H/R evokes pyroptosis in HUVECs.

Figure 1 Effect of H/R on the pyroptotic cell death of endothelial cells. HUVECs were cultured under normal (control) conditions or subjected to hypoxia for 12 h and reoxygenation for 4 h (H/R). The viability of HUVECs was determined by the CCK-8 assay (A), the pyroptotic cell death was detected by calcein-AM/PI staining, the viable cells were stained with calcein-AM (green), whereas the dead cells were stained with PI (red), the dead cells (PI positive) were counted, Scale bar=100 μm (B), Immunofluorescence was used for detection of caspase-1, Scale bar=50 μm (C), the protein expression levels of, NLRP3, c-caspase-1, and GSDMD-N were examined by Western blot (D), the levels of secreted IL-1β and IL-18 were evaluated by ELISA (E). Data are presented as the means ± SD. **P<0.01, n=3.

Increased EZH2 Expression Promotes H/R-Induced Pyroptosis in HUVECs

To analyze the potential role of EZH2 in H/R-induced pyroptosis in HUVECs, the expression level of EZH2 in H/R-exposed HUVECs was firstly detected. As shown in Figure 2A, H/R significantly upregulated the expression level of EZH2 in HUVECs. The effect of EZH2 upregulation on H/R-induced pyroptosis was then investigated by transfecting HUVECs with EZH2 siRNA (siEZH2). As shown in Figure 2B, H/R-induced EZH2 upregulation was obviously inhibited by transfection with siEZH2. SiEZH2 also significantly reversed H/R-induced reduction in cell viability (Figure 2C) and increases in protein expression levels of NLRP3, c-caspase-1, and GSDMD-N (Figure 2B). The secreted protein levels of IL-1β and IL-18 (Figure 2D) and PI-positive cells (Figure 2E) were significantly decreased by siEZH2 in H/R-exposed HUVECs. Taken together, these results indicate that H/R-induced EZH2 promotes pyroptosis in HUVECs.

Figure 2 Knockdown of EZH2 represses H/R-induced pyroptosis in HUVECs. (A) The expression level of EZH2 in HUVECs was examined by Western blot under normal (control) and H/R conditions. (B–E) HUVECs were transfected with control siRNA (siNC) or EZH2 siRNA (siEZH2) and then cultured under normal (control) conditions or subjected to H/R exposure. The protein expression levels of EZH2, NLRP3, c-caspase-1, and GSDMD-N were examined by Western blot (B), the viability of HUVECs was determined by the CCK-8 assay (C), and the levels of secreted IL-1β and IL-18 were evaluated by ELISA (D), the pyroptotic cell death was detected by calcein-AM/PI staining, the viable cells were stained with calcein-AM (green), whereas the dead cells were stained with PI (red), the dead cells (PI positive) were counted, Scale bar=100 μm (E), Data are presented as the means ± SD.**P<0.01, n=3.

EZH2 Promotes H/R-Induced Pyroptosis by Inhibiting miR-22 in HUVECs

The mechanism by which EZH2 promotes H/R-induced pyroptosis was then investigated. Recent studies revealed that EZH2 could regulate the expression level of miR-22,^30–32 which plays a key role in regulation of pyroptosis in various cells, including endothelial cells.^19,20,22 Thus, we investigated whether EZH2 promotes H/R-induced pyroptosis by modulating the expression level of miR-22. The results showed a significant decrease in miR-22 expression under H/R conditions (Figure 3A). EZH2 siRNA (siEZH2) significantly upregulated miR-22 expression in H/R-exposed HUVECs (Figure 3A). EZH2 knockdown cells that were treated with miR-22 inhibitor (In-miR-22) showed obvious decreases in miR-22 expression (Figure 3B) and viability of HUVECs under H/R conditions (Figure 3C). In-miR-22 also led to significant increases in expression levels of NLRP3, c-caspase-1 and GSDMD-N (Figure 3D), releases of IL-1β and IL-18 (Figure 3E), and PI-positive cells (Figure 3F) in siEZH2-transfected cells under H/R conditions.

Figure 3 Inhibition of miR-22 suppresses EZH2 siRNA-induced pyroptosis inhibition in H/R-exposed HUVECs. (A) HUVECs were transfected with control siRNA (siNC) or EZH2 siRNA (siEZH2) and then cultured under normal (control) conditions or subjected to H/R exposure. The expression level of miR-22 in HUVECs was examined by RT-qPCR. (B–F) HUVECs were transfected with siNC or siEZH2 followed by treating with negative control inhibitor (In-NC) or miR-22 inhibitor (In-miR-22), and then subjected to H/R exposure. The expression level of miR-22 in HUVECs was examined by RT-qPCR (B), the viability of HUVECs was determined by the CCK-8 assay (C), the protein expression levels of EZH2, NLRP3, c-caspase-1, and GSDMD-N were examined by Western blot (D) and the levels of secreted IL-1β and IL-18 were evaluated by ELISA (E), the pyroptotic cell death was detected by calcein-AM/PI staining, the viable cells were stained with calcein-AM (green), whereas the dead cells were stained with PI (red), the dead cells (PI positive) were counted, Scale bar=100 μm (F), Data are presented as the means ± SD. *P<0.05, **P<0.01, n=3.

The effect of miR-22 on EZH2-mediated pyroptosis were also investigated by treating EZH2- overexpressed HUVECs with miR-22 mimic (miR-22) under H/R conditions. As shown in Figure 4A, H/R exposure induced a significant decrease in miR-22 expression, which was further enhanced by EZH2 overexpression. miR-22 mimic (miR-22) remarkably increased the expression of miR-22 in EZH2-overexpressed HUVECs under H/R conditions (Figure 4B). miR-22 also reversed EZH2 overexpression-induced decrease in cell viability (Figure 4C) and increases in expression levels of NLRP3, c-caspase-1, GSDMD-N (Figure 4D), releases of IL-1β and IL-18 (Figure 4E) as well as PI-positive cells (Figure 4F) in H/R-exposed cells. Collectively, the above results indicate that EZH2 promotes H/R-induced pyroptosis by inhibiting miR-22 in HUVECs.

Figure 4 Upregulation of miR-22 suppresses EZH2 overexpression-enhanced pyroptosis in H/R-exposed HUVECs. (A) HUVECs were transfected with control plasmid (vector) or EZH2 overexpressed plasmid (EZH2) and then cultured under normal (control) conditions or subjected to H/R exposure. The expression level of miR-22 in HUVECs was examined by RT-qPCR. (B–F) HUVECs were transfected with vector or EZH2 followed by treating with control mimic (mi-NC) or miR-22 mimic (miR-22), and then subjected to H/R exposure. The expression level of miR-22 in HUVECs was examined by RT-qPCR (B), the viability of HUVECs was determined by the CCK-8 assay (C), the protein expression levels of EZH2, NLRP3, c-caspase-1, and GSDMD-N were examined by Western blot (D), the levels of secreted IL-1β and IL-18 were evaluated by ELISA (E), the pyroptotic cell death was detected by calcein-AM/PI staining, the viable cells were stained with calcein-AM (green), whereas the dead cells were stained with PI (red), the dead cells (PI positive) were counted, Scale bar=100 μm (F), Data are presented as the means ± SD. **P<0.01, n=3.

EZH2 Suppresses miR-22 Expression Through H3K27 Trimethylation in H/R-Exposed HUVECs

EZH2 was shown to inhibit many miRNAs expression through H3K27me3-mediated promoter histone methylation, including miR-22.^31–35 We then investigated whether EZH2 inhibits miR-22 expression through H3K27me3 in HUVECs. As shown in Figure 5A, H/R significantly increased the nuclear expression of EZH2. Chromatin immunoprecipitation (ChIP) assay showed that enrichment of EZH2 and H3K27me3 in the miR-22 promoter region was significantly increased after H/R exposure (Figure 5B). When the methyltransferase activity of EZH2 was silenced by its specific inhibitor, GSK126,^36,37 the expression level of H3K27me3 was obviously decreased, whereas the expression of EZH2 remained unchanged (Figure 5C). Notably, GSK126 treatment led to a significant increase in miR-22 expression in H/R-exposed HUVECs (Figure 5D). Furthermore, GSK126 treatment also significantly reduced the association of EZH2 with the promoter of miR-22 in H/R-exposed HUVECs (Figure 5E). Collectively, these results indicate that EZH2 suppresses miR-22 expression through H3K27me3 in H/R-exposed HUVECs.

Figure 5 EZH2 represses miR-22 expression through H3K27me3 trimethylation in H/R-exposed HUVECs. (A and B) HUVECs were cultured under control or H/R conditions, and the intracellular localization of EZH2 was determined by IF (A), and the enrichments of EZH2 and H3K27me3 in miR-22 promoter were detected by chromatin immunoprecipitation (ChIP) assay (B). (C–E) HUVECs were treated with vehicle or GSK126 (5µM), and then cultured under H/R conditions. The expression levels of EZH2 and H3K27me3 were measured by Western blot (C), the expression level of miR-22 was determined by RT-qPCR (D), and the enrichment of EZH2 in miR-22 promoter was detected by ChIP assay (E). Scale bar: 100µm. Data are presented as the means ± SD. **P<0.01, ns, no significant, n=3.

miR-22 Directly Targets NLRP3 in HUVECs

It has been demonstrated that miR-22 suppressed NLRP3 expression through directly binding to the 3’-UTR of NLRP3 mRNA in various cells.^38,39 To confirm binding between miR-22 and NLRP3 in HUVECs, a dual luciferase assay was conducted. The results showed that miR-22 mimic (miR-22) significantly inhibited the luciferase activity of NLRP3 harboring wild type (WT) 3’UTR, but it had no effect on the luciferase activity of NLRP3 with mutant type (Mut) 3’UTR of NLRP3 (Figure 6A and B). Furthermore, the expression level of NLRP3 was obviously downregulated in H/R-exposed HUVECs after miR-22 mimic transfection (Figure 6C). In addition, miR-22 mimic obviously suppressed NLRP3 expression, whereas miR-22 inhibitor remarkably increased NLRP3 expression in HUVECs (Figure 6D). Collectively, these results demonstrate that miR-22 negatively regulates NLRP3 expression by directly targeting it in HUVECs.

Figure 6 miR-22 directly targets NLRP3 in HUVECs. (A) Predicted binding sites between miR-22 and NLRP3. (B) Relative luciferase activity for NLRP3 WT and NLRP3 Mut. (C) Protein levels of NLRP3 in HUVECs transfected with miR-22 mimics (miR-22) or control mimics (mi-NC) under H/R conditions. (D) Protein levels of NLRP3 in HUVECs transfected with miR-22 mimics (miR-22), miR-22 inhibitor (In-miR-22), control mimics (mi-NC), or control inhibitor (In-NC). Data are presented as the means ± SD. **P<0.01, ns, no significant, n=3.

HSP90 Contributes to H/R-Induced EZH2 Upregulation and Pyroptosis in HUVECs

Previous studies revealed that the protein stability and function of EZH2 were maintained by heat shock protein 90 (HSP90),^40,41 which also plays a critical role in the regulation of pyroptosis under different oxidative stress.^42,43 Thus, we investigated whether HSP90 contributes to H/R-induced EZH2 up-regulation and pyroptosis in endothelial cells. As shown in Figure 7A, H/R exposure significantly increased HSP90 expression in HUVECs. HSP90 siRNA (siHSP90) significantly reduced H/R-induced EZH2 upregulation in HUVECs (Figure 7A). Reciprocal coimmunoprecipitation revealed that HSP90 and EZH2 directly interacted with each other in H/R-exposed HUVECs (Figure 7B). Furthermore, siHSP90 significantly reversed H/R-induced downregulation of mi-22 (Figure 7C), decreased cell viability (Figure 7D), increased expression levels of NLRP3, c-caspase-1, GSDMD-N (Figure 7A), secreted protein levels of IL-1β and IL-18 (Figure 7E) as well as PI-positive cells (Figure 7F) in HUVECs. Collectively, these data indicate that HSP90 contributes to H/R-induced EZH2 upregulation and pyroptosis in HUVECs.

Figure 7 HSP90 is required for H/R-induced EZH2 upregulation and pyroptosis in HUVECs. (A) HUVECs were transfected with control siRNA (siNC) or HSP90 siRNA (siHSP90) and then cultured under control conditions or subjected to H/R exposure. The expression levels of HSP90, EZH2, NLRP3, c-caspase-1, and GSDMD-N were measured by Western blot. (B) The direct interaction between EZH2 and HSP90 in HUVECs after H/R exposure was determined by immunoprecipitation with anti-HSP90 antibody and analyzed by Western blot with anti-EZH2 antibody or anti-HSP90 antibody. (C–F) HUVECs were transfected with control siNC or siHSP90 and then cultured under control conditions or subjected to H/R exposure, the expression level of miR-22 was determined by RT-qPCR (C), the viability of HUVECs was determined by the CCK-8 assay (D), the levels of secreted IL-1β and IL-18 were evaluated by ELISA (E), the pyroptotic cell death was detected by calcein-AM/PI staining, the viable cells were stained with calcein-AM (green), whereas the dead cells were stained with PI (red), the dead cells (PI positive) were counted, Scale bar=100 μm (F), Data are presented as the means ± SD. *P<0.05, **P<0.01, n=3.

Discussion

Although EZH2 has been shown to play a crucial role in oxidative stress-mediated pyroptosis in human renal proximal tubular epithelial cells,²⁹ little is known about its role in H/R-induced endothelial cell pyroptosis. In the current study, we showed that H/R-induced EZH2 upregulation contributed to pyroptosis in endothelial cells. To our knowledge, the present study firstly reported the key role of EZH2 in endothelial cell pyroptosis under H/R conditions. Based on this, the mechanism by which EZH2 promotes H/R-induced pyroptosis was investigated.

It has been demonstrated that miRNAs act as key regulators of pyroptosis by modulating the expression levels of key genes in pyroptosis.^44–46 EZH2 was shown to suppress the expression of microRNAs under oxidative stress, such as miR-139,⁴⁷ miR-34a³⁵ and miR-451.⁴⁸ Recently, miR-22 was reported to promote endothelial cell pyroptosis.^19,20 Importantly, miR-22 was shown to be inhibited by EZH2 in various cells.^30–32 Consistent with these findings, we found that EZH2 promoted H/R-induced pyroptosis by inhibiting miR-22 in endothelial cells. However, it is undeniable that the pro-pyroptotic effects of EZH2 may be mediated by a variety of miRNAs in addition to miR-22 in H/R-exposed endothelial cells, and further investigation is required to uncover any other miRNAs involved.

Our results revealed that miR-22 mediates H/R-induced pyroptosis by directly targeting NLRP3 in HUVECs. This finding is consistent with previous studies that have shown NLRP3 is the direct target of miR-22 in other types of cells, such as neuronal cells, epithelial cells and cancer cells.^20,21,49 It is well known that miR-22 has many target genes, and the target gene that mediates the function of miR-22 depends on cell type and extracellular stress. To date, there is no other molecules are reported to be the targets of miR-22 and regulate pyroptosis in endothelial cells, except to NLRP3. Phosphatase and tensin homolog (PTEN), a tumor suppressor gene, has demonstrated to be the target gene of miR-22 in different types of cells.^50–52 Moreover, PTEN also plays a key role in pyroptosis in different types of cells, including endothelial cells.^53–55 Therefore, whether PTEN acts as the target of miR-22 during H/R-induced pyroptosis is another point for us to investigate in the near future.

H3K27me3 is an important epigenetic event associated with transcriptional silencing.^56,57 As a methyltransferase component of PRC2, EZH2 was reported to mediate epigenetic gene silencing through H3K27me3.^56,57 Emerging evidences suggested that EZH2 epigenetically silences miRNAs expression by trimethylating H3K27.^33–35 GSK126 is a highly selective inhibitor of EZH2 methyltransferase activity that influences H3K27me3 chromatin content and transcriptional control.^36,37 Our experimental data showed that GSK126 not only reversed H/R-induced miR-22 downregulation, but also inhibited the binding of EZH2 to the promoter of miR-22, indicating that EZH2 inhibits miR-22 through H3K27me3 in H/R-exposed endothelial cells. MiR-22 expression has been reported to be inactivated by aberrant DNA methylation.⁵⁸ Furthermore, H3K27me3 and DNA methylation have been linked to establish and maintain gene silencing.⁵⁹ Thus, it will be interesting to investigate whether there is an epigenetic cross-talk between DNA methylation and H3K27me3 in silencing of miR-22 expression in H/R-exposed endothelial cells.

HSP90 is a molecular chaperone that facilitates the correct folding and functionality of its client protein through the binding of ATP molecule.⁶⁰ Previous reports revealed that HSP90 plays a crucial role in various diseases, such as cancer, lung injury and cardiovascular diseases.⁶⁰ HSP90 has been shown to stabilize EZH2 protein expression through the direct interaction with EZH2.^40,41 In agreement with these previous studies, our experimental results showed that HSP90 and EZH2 directly interacted with each other in H/R-exposed HUVECs. Recently, inhibition of HSP90 was reported to suppress pyroptosis in THP-1 cells.⁴² Consistently, we found that HSP90 suppression increased miR-22 expression and inhibited pyroptosis in H/R-exposed HUVECs. These findings suggest that HSP90 facilitates H/R-induced pyroptosis in endothelial cells through EZH2-mediated miR-22 downregulation.

Conclusion

In conclusion, the current study demonstrated that EZH2, which is upregulated in H/R-exposed HUVECs in a HSP90-dependent manner, represses miR-22 through H3K27me3, thereby elevating NLRP3 to facilitate pyroptosis (Figure 8). These findings shed light on the molecular mechanisms of H/R-evoked pyroptosis of endothelial cells. However, an in vivo study is still needed to confirm our findings.

Figure 8 Schematic illustration of H/R-induced endothelial cell pyroptosis via the HSP90/EZH2/miR-22/NLRP3 signaling pathway.