DZNeP

Genetic or pharmacologic blockade of enhancer of zeste homolog 2 inhibits the progression of peritoneal fibrosis

Abstract
Dysregulation of histone methyltransferase enhancer of zeste homolog 2 (EZH2) has been implicated in the pathogenesis of many cancers. However, the role of EZH2 in peritoneal fibrosis remains unknown. We investigated EZH2 expression in peritoneal dialysis (PD) patients and assessed its role in peritoneal fibrosis in cultured human peritoneal mesothelial cells (HPMCs) and murine models of peritoneal fibrosis induced by chlorhexidine gluconate (CG) or high glucose peritoneal dialysis fluid (PDF) by using 3-deazaneplanocin A (3-DZNeP), and EZH2 conditional knockout mice. An abundance of EZH2 was detected in the peritoneum of patients with PD associated peritonitis and the dialysis effluent of long-term PD patients, which was positively correlated with expression of TGF-β1, vascular endothelial growth factor, and IL-6. EZH2 was found highly expressed in the peritoneum of mice following injury by CG or PDF. In both mouse models, treatment with 3-DZNeP attenuated peritoneal fibrosis and inhibited activation of several profibrotic signaling pathways, including TGF-β1/Smad3, Notch1, epidermal growth factor receptor and Src. EZH2 inhibition also inhibited STAT3 and nuclear factor-кB phosphorylation, and reduced lymphocyte and macrophage infiltration and angiogenesis in the injured peritoneum. 3-DZNeP effectively improved high glucose PDF-associated peritoneal dysfunction by decreasing the dialysate-to-plasma ratio of blood urea nitrogen and increasing the ratio of dialysate glucose at 2 h after PDF injection to initial dialysate glucose. Moreover, delayed administration of 3-DZNeP inhibited peritoneal fibrosis progression, reversed established peritoneal fibrosis and reduced expression of tissue inhibitor of metalloproteinase 2, and matrix metalloproteinase-2 and -9. Finally, EZH2-KO mice exhibited less peritoneal fibrosis than EZH2-WT mice. In HPMCs, treatment with EZH2 siRNA or 3-DZNeP suppressed TGF-β1-induced upregulation of α-SMA and Collagen I and preserved E-cadherin. These results indicate that EZH2 is a key epigenetic regulator that promotes peritoneal fibrosis. Targeting EZH2 may have the potential to prevent and treat peritoneal fibrosis.

Introduction
Continuous ambulatory peritoneal dialysis (CAPD) has been recognized as a renal replacement therapy since 1970s. It has become a major alternative treatment for patients with end-stage renal disease worldwide [1]. However, persistent exposure of peritoneum to bio-incompatible peritoneal dialysis fluid (PDF) leads to peritoneal fibrosis, which decreases ultrafiltration capac- ity of peritoneum and limits its clinic application [2,3]. To date, there is no available treatment to prevent peri- toneal fibrosis or halt its progression. Therefore, under- standing the mechanisms leading to peritoneal fibrosisand discovery of new agents to block its progression holds great significance for continuing PD long-term in patients.Peritoneal fibrosis is characterized by epithelial-to- mesenchymal transition (EMT) of mesothelial cells (MCs), activation of fibroblasts and deposition of ECM components as well as angiogenesis [4]. Many growth factors/cytokines, in particular, TGF-β1, mediate acti- vation of peritoneal fibroblasts and EMT, and play a key role in the pathogenesis of peritoneal fibrosis [5,6]. Activation of epidermal growth factor recep- tor (EGFR), Src and Notch signaling pathway also contributes to these processes [7–9]. Moreover, in the case of peritonitis, NF-κB and signal transducerand activator of transcription 3 (STAT3) are activated and induce expression and production of multiple pro-inflammatory cytokines/chemokines, including IL-6, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α) and IL-1β [5–13].

MCP-1 and other chemoattractants can induce accumu- lation of inflammatory cells, such as macrophages, in the peritoneum. Macrophages can also produce TGF-β and connective tissue growth factor, vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs), to promote progression of peritoneal fibrosis [13,14]. VEGF stimulates angiogenesis and vascu- lopathy, eventually leading to peritoneal ultrafiltration failure [4].Increasing evidence support that epigenetic modi- fication of gene expression mediates expression and activation of many transcriptional factors and signal- ing molecules associated with peritoneal fibrosis [15]. DNA methylation and histone modifications are two major types of epigenetic modifications. Among the possible ways of histone modification, mono-, di-, or tri-methylation can occur on lysine or arginine amine acids of histone H3 and some nonhistone proteins [16, 17]. Histone methylation can alter chromatin structure and affect transcriptional factor accessibility to DNA promoters, thereby regulating gene expression. In addi- tion, methylation of a nonhistone can also affect its acti- vation and expression [17,18]. Protein methylation is catalyzed by histone lysine and arginine methyltrans- ferases. Enhancer of zeste homolog 2 (EZH2) is one of the histone lysine methyltransferases that catalyzes trimethylation of histone H3 at lysine 27 (H3K27me3), and promotes transcriptional silencing of many genes, including E-cadherin [19,20].

EZH2 can also directly methylate some intracellular signaling molecules, such as STAT3, and regulate their phosphorylation and acti- vation [21].Numerous studies have shown that EZH2 is highlyexpressed in many cancers and closely related to tumor invasion, metastasis, and poor prognosis [19,22]. Inhibition of EZH2 activity has become a new strategy for anti-tumor therapy. A variety of EZH2 inhibitors have been used for antitumor preclinical studies and have achieved encouraging results [23,24]. 3-Deazaneplanocin A (3-DZNeP) is an inhibitor of S-adenosylhomocysteine hydrolase that inhibits the activity of EZH2 and downregulates the methylation level of H3K27 [25]. It has been reported that 3-DZNeP can inhibit activation of hepatic stellate cells, and pre- vent the progression of liver fibrosis [26]. Our recent studies also showed that EZH2 is highly expressed in the diseased human kidney with fibrosis and the mouse kid- ney after unilateral ureteral obstruction (UUO) and that inhibition of EZH2 with 3-DZNeP blocks renal myofi- broblast activation, EMT, and relieves UUO-induced renal fibrosis [11,27]. However, it remains unknown whether EZH2 is involved in peritoneal fibrosis.In this study, we examined the expression of EZH2in the peritoneum of patients with PD-related peritoni- tis and in murine models of peritoneal fibrosis inducedby 4.25% PDF and 0.1% chlorhexidine gluconate (CG); we also evaluated the effect on peritoneal fibrosis of early or late administration of 3-DZNeP and specific deletion of EZH2 in peritoneal fibrosis animal model to learn the mechanisms involved.

Furthermore, we exam- ined pharmacological and genetic inhibition of EZH2 on TGF-β1-induced EMT in cultured human peritoneal mesothelial cells (HPMCs).HPMCs (kind gifts from Haiping Mao at Sun Yat-Sen University, Guangzhou, PR China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with nutrient mixture F12 and containing 10% FBS and 1% penicillin and streptomycin stock solution in an atmo-sphere of 5% CO2, and 95% air at 37 ∘C. To exam-ine the anti-fibrotic effect of 3-DZNeP (Selleckchem,Houston, TX, USA) in TGF-β1-induced EMT in vitro, HPMCs were starved for 24 h in DMEM/F12 contain- ing 0.5% FBS and then exposed to TGF-β1 (2 ng/ml) (R&D Systems, Minneapolis, MN, USA) in the presence of 3-DZNeP (0, 1, 5, and 10 μM) for 36 h. In addition, serum-starved HPMCs were cultured in PDF contain- ing 4.25% glucose for 36 h with different concentrations of 3-DZNeP (0, 1, 5, and 10 μM), in order to verify the anti-apoptotic effect of 3-DZNeP. Cells were harvested for immunoblotting analyses. All of the in vitro experi- ments were repeated at least three times.The small interfering (si) RNA oligonucleotides tar- geted to EZH2 (GenePharma, Shanghai, PR China) were used to downregulate EZH2 more specifically in vitro. HPMCs were seeded at 30 – 40% confluence in antibiotic-free medium and grown for 24 h then trans- fected with EZH2 siRNA (100 pmol) with lipofectamine 2000 (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. In parallel, scram- bled siRNA (100 pmol) was used as control for off-target changes in HPMCs.

At 24 h following transfection, the original antibiotic-free medium was changed and cells were further treated with TGF-β1 (2 ng/ml) for an addi- tional 36 h before being harvested for the experiments.HPMCs were seeded in a 6-well plate and allowed to reach 90% confluence. A scratch wound was created on the cell surface using a micropipette tip. Then, cells were washed with PBS in three times and incubated in serum-free DMEM/F12 with TGF-β1 (2 ng/ml) in the presence or absence of 3-DZNeP (10 μM). Photomicro- graphs (×40 objective magnification) of migrating cells were taken at 0 and 24 h. The width of the wound was measured using ImageJ software (National Institutes ofHealth, Bethesda, MD, USA). The migratory rate was calculated as (A − B)/A × 100%, where A and B reflect the width of the wound at 0 and 24 h respectively.All the experiments were conducted in accordance with the animal experimentation guidelines of Tongji University School of Medicine, PR China. Male C57/black mice (Shanghai Super-B&K Laboratory Animal Corp. Ltd., Shanghai, PR China) that weighed 20 – 25 g were housed under a 12 h light – dark cycle with food and water supplied ad libitum. Two mouse models of peritoneal fibrosis were established. The first peritoneal fibrosis model was created by dailyi.p. injection of 100 ml/kg peritoneal dialysis solution with 4.25% glucose (Baxter Healthcare, Guangzhou, PR China) for 28 days [10]. The second peritoneal fibrosis model was established by i.p. injection of 0.1% CG (Sigma-Aldrich, St. Louis, MO, USA) dis- solved in saline every other day for 21 days [28,29].

To investigate the effect of 3-DZNeP in peritoneal fibrosis, mice were injected i.p. with a single dose of 3-DZNeP (1 mg/kg) in DMSO (Sigma-Aldrich) every day. Mice were randomly allocated into four groups for each model: (1) mice injected with an equivalent amount of saline i.p. and DMSO (n = 6), defined as the sham group, (2) mice injected an equivalent amount of saline i.p. and 3-DZNeP (n = 6), defined as sham+3-DZNeP group, (3) mice injected with PDF or CGi.p. and DMSO (n = 6), defined as PDF/CG group, and(4) mice injected PDF or CG i.p. and 3-DZNeP, defined as PDF/CG + 3-DZNeP group (n = 6). Animals were sacrificed, and parietal peritoneum was collected from each mouse for further protein analysis and histological examination at the end of 28 days for the PDF model or 21 days for the CG model. To examine the therapeutic effect on established peritoneal fibrosis, 3-DZNeP was administered starting at 28 days and given daily for 14 days to mice injected with PDF. At the end of 42 days, all mice were euthanized for collection of peritoneal tissue.Mice in each group received a peritoneal equilibra- tion test before euthanasia in the last day. Mice were injected with 2 ml 4.25% PDF for 2 h and then eutha- nized for collection of blood and dialysate. Glucose in dialysate and blood urea nitrogen (BUN) in plasma and dialysate were determined using BUN (#C013-2-1) and glucose (F006-1-1) biochemical reagent kits according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, PR China).

Functional alteration of peritoneal membranes was evaluated by the urea nitrogen transport rate from plasma with the dialysate-to-plasma (D/P) ratio of blood urea nitrogen, and the glucose absorption rate from dialysate with the ratio of dialysate glucose at 2 h after PDF injection to dialysate glucose at 0 h (D/D0).EZH2loxP/loxP (Stock No 022616) and tamoxifen- inducible Col1a2-Cre mice (Col1a2-CreER+/−) (Stock No 016237) were purchased from The Jackson Labora- tory (Bar Harbor, ME, USA). All mice were backcrossed six times to C57BL/6J before starting this experiment. EZH2 knockout mice were generated by breeding EZH2loxP/loxP mice with Col1a2-CreER+/− to obtain Col1a2-Cre+: EZH2loxP/loxP mice (EZH2-KO) and Col1a2-Cre−: EZH2loxP/loxP mice (EZH2-WT). Both of EZH2-KO and EZH2-WT mice received a dailyi.p. injection of 100 ml/kg peritoneal dialysis solution with 4.25% glucose for 28 days to establish peri- toneal fibrosis and given tamoxifen (75 mg/kg body weight) (Sigma-Aldrich) by i.p. injection for seven consecutive days.Immunoblotting analysis was conducted as described previously [11] (primary antibodies are listed in sup- plementary material, Table S1). Densitometry analysis of immunoblot results was conducted by using ImageJ software.Formalin-fixed peritoneum was embedded in paraffin and cut into 3-μm-thick sections. For evaluation of peritoneal fibrosis, Masson trichrome staining was per- formed according to the protocol provided by the sup- plier (Sigma-Aldrich). The thickness of the submesothe- lial tissue was measured (in μm), and the average of 10 independent measurements was calculated for each section (original magnification, ×200).

Immunofluorescence staining was carried out accord- ing to the procedure described in our previous study [30]. FFPE sections (3 μm) were rehydrated and incu- bated with primary antibodies against α-SMA or EZH2 (primary antibodies are listed in supplementary mate- rial, Table S2) and then Texas Red- or FITC-labeled sec- ondary antibodies (Invitrogen).Sections cut at 3 μm thick were de-paraffinized and rehydrated, quenched with 3% H2O2, immersed in cit- rate buffer and heated in a microwave for retrieval of antigens as described in our previous study [30] (pri- mary antibodies are listed in supplementary material, Table S2).ELISAs were used to measure concentrations of TNF-α, IL-1β, TGF-β1, MCP-1, IL-6, EZH2, VEGF, and CA125protein, which was performed in accordance with the manufacturer’s instructions (R&D Systems).Chromatin immunoprecipitation (ChIP) was performed by using a ChIP assay Kit (Millipore, MA, USA) according to the manufacturer’s instructions and a ChIP antibody against H3K27me3 (#9733) purchased from Cell Signaling Technology (Danvers, MA, USA). The precipitated DNA fragments were quantified by real-time qPCR and normalized using the internal con- trol IgG. The ChIP data was presented as a percentage relative to the input DNA amount by the equation: 2[Input Ct – Target Ct] × 100.This study was approved by the Medical Ethics Com- mittee of Shanghai East Hospital and was conducted in accordance with the Declaration of Helsinki. Writ- ten informed consent was obtained from each patient, and we obtained a registration number from the Chinese Clinical Trial Register (ChiCTR): ChiCTR1800015272.

To detect EZH2 expression in the peritoneum samples of PD patients, we collected peritoneal tissue during oper- ations to insert (n = 10) or remove (n = 6) PD catheters at Shanghai East Hospital affiliated with Tongji Univer- sity and conducted immunohistochemistry. We also col- lected PD effluents from patients using PD for diverse times: 1 month (n = 9), 24 months (n = 8) and 48 months (n = 8) at Shanghai East Hospital, Shanghai Wusong Central Hospital and Shanghai Songjiang District Cen- tral Hospital from January 2017 to February 2019.All the experiments were conducted at least three times. Data depicted in graphs represent the means ± SEM for each group. Intergroup comparison was made using one-way ANOVA. Multiple means were compared using Tukey’s test. The differences between two groups were determined by Student’s t-test. Statistically significant differences between mean values were marked in each graph. p < 0.05 was considered significant. The statisti- cal analyses were conducted by using IBM SPSS Statis- tics 20.0 (Version X; IBM, Armonk, NY, USA). Results To examine EZH2 expression in patients with PD, we collected the peritoneum from both non-PD patients (n = 10) and PD patients associated with peritonitis (n = 6). As shown in Figure 1A, Masson trichrome stain- ing illustrated that the peritoneum of patients with PD associated peritonitis had severe peritoneal fibrosis as characterized with the increased thickness of the sub- mesothelial area, a layer of mature fibrous tissue con- taining collagen and elastin fibers. The thickness and positive area of the submesothelial area in patients’ peri- toneum with PD associated peritonitis was much greaterthan in non-PD patients. A high expression level of EZH2 was also observed in the peritoneum of patients with PD associated peritonitis compared with non-PD (Figure 1B).Dialysate CA125 and IL-6 has been reported to be a marker for evaluating the peritoneal membrane in noninfected patients to predict peritoneal fibrosis [31, 32]. Thus, we also collected PD effluent from patients on PD for different lengths of time: 1 month (n = 9), 24 months (n = 8), and 48 months (n = 8), respectively. After centrifugation, the pellet from PD effluent was submitted to immunoblotting analyses. We found elevated expression levels of EZH2 and Collagen I in samples collected from PD samples from patients on PD for 24 months and 48 months compared to effluent from patients on PD for only 1 month (Figure 1C,D). In line with this observation, ELISA assays of PD effluent showed an increase in the expression of EZH2 and mul- tiple growth factors and cytokines, including TGF-β1, VEGF, IL-6, and a decrease in the expression of CA125. Further analysis indicated that EZH2 expression was positively correlated with expression of TGF-β1, VEGF, IL-6 and negatively with CA125 in human PD efflu- ent (Figure 1E– M). Therefore, EZH2 may be used as a biomarker of peritoneal fibrosis in PD patients.To elucidate the role of EZH2 in mediating peri- toneal fibrosis, we examined the effect of 3-DZNeP on the development of peritoneal fibrosis induced by high glucose peritoneal dialysate or CG. As shown in Figure 2A– C and see supplementary material, Figure S1A – C, Masson trichrome staining illustrated that we successfully established two mouse models of peritoneal fibrosis induced by 4.25% glucose PDF or 0.1% CG, as characterized by increased thickness of the submesothelial area and a layer of mature fibrous tissue containing collagen and elastin fibers. The thickness and positive area of the submesothelial area in PDF- or CG- injured mice were evidently greater than sham peritoneum with/without administration of 3-DZNeP. Administration of 3-DZNeP attenuated these patho- logical changes. These data indicate that exposure to high glucose PDF or CG contributes to peritoneal fibrosis, while 3-DZNeP is a potent agent for preventing development of peritoneal fibrosis. Low levels of EZH2 and H3K27me3 were detected in sham peritoneum with/without administration of 3-DZNeP, and expression of each was predominantly increased after 4.25% glucose PDF or CG injection as indicated in Figure 2D– F and see supplementary material, Figure S1D,F,G. Administration of 3-DZNeP remarkably inhibited PDF or CG induced upregulation of EZH2 and H3K27me3. However, 3-DZNeP had no inhibitory effect on trimethylation of histone H3 at lysine 9 (H3K9me3) (Figure 2D,F). Furthermore, immunofluorescent co-staining of α-SMA and EZH2 showed that EZH2 is abundantly expressed in theα-SMA-positive cells (Figure 2G). Thus, these results suggest that EZH2-induced histone methylation is associated with peritoneal fibrosis.and improves dysfunction of peritoneal membrane in the fibrotic peritoneum of miceTo assess the anti-fibrotic effect and mechanisms of 3-DZNeP, we examined the expression levels of α-SMA and Collagen I, two hallmarks of EMT and myofibroblasts, in two peritoneal fibrosis mouse models. Immunoblotting and immunohistochemistry analyses showed that α-SMA was barely detected insham peritoneum with/without 3-DZNeP treatment; its expression level was dramatically increased in the peritoneum after continuous exposure to PDF or CG. Administration of 3-DZNeP decreased its upregulation (Figure 2D,E and see supplementary material, Figure S1D,E). 3-DZNeP also inhibited excessive expression of Collagen I in injured peri- toneum (Figure 2D,E and see supplementary material, Figure S1D,E).Loss of E-cadherin, one of the selected adhesion molecules, contributes to separation of intercellular junctions of MCs, resulting in reorganization and migra- tion of MCs [33,34]. In normal peritoneum, high level of E-cadherin was detected by Western blot analysis, whileinjury to the peritoneum reduced E-cadherin expression. Inhibition of EZH2 with 3-DZNeP restored E-cadherin expression to normal levels (Figure 2D,E and see sup- plementary material, Figure S1D,E). Taken together, these results suggest that 3-DZNeP may inhibit progres- sion of peritoneal fibrosis by suppressing EMT. Fur- thermore, we also evaluated the functional alteration of peritoneal membrane by the urea nitrogen transport rate from plasma with the D/P ratio of BUN and the glucose absorption rate from dialysate with the ratio of D/D0. 3-DZNeP effectively improved high glucose PDF-associated peritoneal functional impairments by decreasing D/P ratio of BUN and increasing D/D0 ratio of glucose (Figure 2H,I).Blockade of EZH2 inhibits activationof TGF-β/Smad, EGFR/Src and Notch1/Jagged-1 signaling pathways in the peritoneum exposed to high glucose peritoneal dialysateWe further examined whether EZH2 plays a role in the regulation of three profibrotic signaling pathways (TGF-β1/Smad, EGFR/Src, and Notch1/Jagged-1) in peritoneal fibrosis [1,7,9,10,35–37]. We first exam- ined the expression levels of TGF-β1 in each group by ELISA. Figure 3B showed that TGF-β1 level in peritoneal fibrosis was higher than in the con- trol group and that 3-DZNeP treatment decreased this response. Immunoblotting results showed that3-DZNeP suppressed TGF-βRI and p-Smad3 levels, and preserved Smad7 expression (Figure 3A,C,D). Inhibition of EZH2 with 3-DZNeP also dramatically reduced EGFR and Src phosphorylation as well as the ratio of p-EGFR/EGFR and p-Src/Src (Figure 3E,F). Furthermore, as shown in Figure 3G,H, the base lev- els of Notch1 and Jagged-1 were negligible in the peritoneum of sham operated animals. However, their expression levels were remarkably upregulated in the injured peritoneum following 4.25% glucose PDF injection. Treatment with 3-DZNeP suppressed expres- sion of Notch1 and Jagged-1. These data suggest that 3-DZNeP may suppress peritoneal fibrosis by inhibiting activation of several profibrotic signaling pathways, including TGF-β1/Smad, EGFR/Src, and Notch1/Jagged-1.Blockade of EZH2 ameliorates peritoneal fibrosis by abrogating inflammation, reducing lymphocyte and macrophage infiltration, and inhibiting angiogenesis in the injured peritoneumIn vivo experiments showed that p-STAT3 was highly expressed in the peritoneum after PDF exposure, and treatment with 3-DZNeP reduced its expression level (Figure 4A,C). Figure 4B,C demonstrated that phospho- rylated NF-κB level remained at a high standard in PDF-induced peritoneal fibrosis group compared with sham group. The ratio between p-NF-κB and total NF-κB was further decreased with 3-DZNeP treat- ment, supporting the conclusion that 3-DZNeP effec- tively prevents the development of peritoneal fibrosis by inhibiting pro-inflammatory signaling pathways. NF-κBsignaling pathway activation is also essential for the pre- sentation of multiple pro-inflammatory cytokines and chemokines such as IL-6, MCP-1, TNF-α, and IL-1β. In Figure 4D, we examined the levels of those inflamma- tory factors by ELISA. Levels of four ELISA targets in the peritoneal fibrosis group were higher than in the con- trol group, especially for MCP-1 (Figure 4D). Their lev- els in peritoneal tissue trended down after treatment with 3-DZNeP in 4.25% glucose PDF model of peritoneal fibrosis. These data suggest that inflammation plays an important role in the initiation and progression of peri- toneal fibrosis, and 3-DZNeP can inhibit inflammation responses and prevent peritoneal fibrosis.We assessed the relationship between CD3+T cells and peritoneal fibrosis and the impact of EZH2inhibition on this immune regulatory mechanism. As shown in Figure 4E, CD3+ T cells were rarely expressed in sham peritoneum; PDF-induced peritoneal fibrosis led to an increase in the number of CD3+ T cells in the thickened submesothelial area. Blockade of EZH2 with 3-DZNeP mainly reduced the number of CD3 positive cells in the peritoneum (Figure 4E), suggesting that 3-DZNeP may prevent peritoneal fibrosis by reducing lymphocyte infiltration. We then used immunohisto- chemistry to examine the expression level of CD68, a bio-marker of macrophages, in each group. CD68 positive cells were rarely expressed in sham peritoneum with/without inhibitor. PDF-induced peritoneal fibrosis resulted in enhanced expression of CD68 positive cells in the thickened submesothelial area. Administration of3-DZNeP reduced the number of CD68 positive cells in the injured peritoneum group (Figure 4F).We also evaluated the effect of 3-DZNeP admin- istration on angiogenesis during peritoneal fibrosis by immunohistochemical staining of two angio- genetic markers, CD31 and VEGF. The number of CD31-positive vessels and VEGF-positive endothelial cells increased in fibrotic peritoneum. Inhibition of EZH2 with 3-DZNeP suppressed these popula- tions of vessels and cells (Figure 4G,H). In summary, 3-DZNeP significantly reduces CD31-positive vessels and VEGF-positive cells in the fibrotic peritoneum, which provides the evidence that EZH2 is predomi- nantly involved in peritoneal angiogenesis. To further understand the role of EZH2 in peritoneal fibrosis, we examined the effect of 3-DZNeP or siRNA specifically targeting EZH2 on TGF-β1-induced EMT in cultured HPMCs. As shown in supplementary mate- rial, Figure S2A,B,E,F, TGF-β1 significantly increased the expression of α-SMA and Collagen I and sup- pressed E-cadherin expression levels. Administration of 3-DZNeP inhibited upregulation of α-SMA and Col- lagen I and blocked downregulation of E-cadherin in a dose-dependent manner (see supplementary material, Figure S2A,B). EZH2 siRNA also had the similar inhibitory effects (see supplementary material, Figure S2E,F). ChIP assay results in supplemen- tary material, Figure S3A further demonstrated that EZH2 directly suppressed E-cadherin expression by H3K27me3. In parallel, TGF-β1 exposure resulted in increased expression of EZH2 and H3K27me3, which was blocked by 3-DZNeP or EZH2 siRNA (see sup- plementary material, Figure S2C,D,G,H). Collectively, these data suggest that EZH2 activation is critically involved in the EMT of HPMCs.Blockade of EZH2 with 3-DZNeP inhibits apoptosis and migration of HPMCs and downregulates several profibrotic signaling pathways in vitroIn order to examine the anti-apoptotic ability of 3-DZNeP, serum-starved HPMCs were exposed to PDF containing 4.25% glucose for 36 h in the presence or absence of different concentrations of 3-DZNeP. High glucose exposure induced cell apoptosis of HPMCs, while co-treatment with 3-DZNeP signifi- cantly decreased Cleaved caspase 3 and Bax expression levels and increased Bcl-2, an important apopto- sis suppression gene (see supplementary material, Figure S3B – D). Wound-healing assay showed that 3-DZNeP has the ability to prevent cell migration and reduce the migratory rate of HPMCs (see supplemen- tary material, Figure S3E, F). Moreover, treatment with 3-DZNeP or EZH2 siRNA also suppressed phospho- rylation of Smad3 and EGFR, preserved Smad7 level and inhibited expression of Notch1 (see supplementary material, Figures S4 and S5). Collectively, these data suggest that EZH2 activation is critically involved in cell apoptosis and migration and above-mentioned profibrotic signaling pathways of HPMCs.To explore the therapeutic effect of 3-DZNeP on the progression of peritoneal fibrosis, we designed an ani- mal experiment with delayed treatment using 3-DZNeP (Figure 5A). 4.25% glucose PDF was injected daily for 42 days to establish a mouse model of peritoneal fibro- sis, and 3-DZNeP was used at the beginning of 28 days, when peritoneal fibrosis had been formed with obvi- ous thickened peritoneum tissue. At 42 days, animals were euthanized for collection of peritoneum and fur- ther analysis. The thickness of the submesothelial com- pact zone and Masson trichrome-positive area increased within 28 days and were further elevated at 42 days after continuous exposure to high glucose dialysate. In com- parison, peritoneal injury in mice treated with 3-DZNeP from day 28 onward was much milder than that of other two groups (Figure 5B, C).To confirm this observation, we further examined the expression levels of α-SMA, Collagen I, E-cadherin, EZH2, H3K27me3 and Histone H3 by immunoblot- ting analysis. 4.25% glucose PDF injection for the first 28 days led to increased expression of α-SMA, Colla- gen I, EZH2 and H3K27me3; continuous daily injection of PDF until 42 days led to further increased expression of α-SMA, Collagen I, EZH2 and H3K27me3. In contrast, late treatment with 3-DZNeP from day 28 to day 42 reduced their expression levels below their levels at day 28 (Figure 5D– F). Conversely, 4.25% glucose PDF injection decreased E-cadherin expression over time, whereas delayed administration of 3-DZNeP not only prevented further reduction of E-cadherin expression from day 28 to day 42, but also restored its expression to the normal level (Figure 5D, E).MMPs are a family of proteolytic enzymes that hydrolyze ECM components. Among them, MMP2 and MMP9, two specific type IV collagenases, have the ability to induce degradation of collagen IV and destroy the integrity of the basement membrane [38– 40]. A recent study reported that absence of MMP2 in MMP2 deficient mice protects obstructed kidney against hydronephrosis and renal fibrosis during UUO [41]. As demonstrated, the presence of tissue inhibitor of met- alloproteinase 2 (TIMP2) is essential for the cell sur- face activation of pro-MMP2 in the kidney upon injury [42]. TIMP2 can interact with membrane type 1 MMP and enhance the activation of pro-MMP2 [43]. TIMP2 increases MMP2/9 expression and regulates the degra- dation of ECM protein in fibrotic diseases [42,44,45]. Continuous daily injection of PDF until day 42 induced higher expression levels of TIMP2, MMP2 and MMP9 than at day 28. Delayed administration of 3-DZNeP attenuated TIMP2, MMP2 and MMP9 levels belowthose seen at day 28 (Figure 5G, H). Taken together, our results illustrate that inhibition of EZH2 with 3-DZNeP slows progression of peritoneal fibrosis, and downreg- ulation of TIMP2, MMP2 and MMP9 may become a key mechanism for 3-DZNeP to ameliorate peritoneal fibrosis.EZH2-KO mice develop less peritoneal fibrosis compared with EZH2-WT miceEZH2 knockout mice were generated by breeding our EZH2loxP/loxP mice with tamoxifen-inducible Col1a2-Cre mice (Col1a2-CreER+/−) to obtainCol1a2-Cre+: EZH2loxP/loxP mice (EZH2-KO) and Col1a2-Cre−: EZH2loxP/loxP mice (EZH2-WT). After daily i.p. injection of 100 ml/kg peritoneal dialysis solution with 4.25% glucose for 28 days, we found that the thickness and positive area of the submesothelial area in EZH2-WT mice were evidently greater than EZH2-KO mice as shown in Masson trichrome staining (Figure 6A– C). Immunoblotting analyses showed that α-SMA and Collagen I were barely detected in sham peritoneum of EZH2-WT and EZH2-KO mice, while their expression levels were dramatically increased in the EZH2-WT mice after continuous exposure to highglucose peritoneal dialysate compared with EZH2-KO (Figure 6D, E). We also assessed the levels of EZH2 and H3K27me3 in both EZH2-WT or KO mice with/without PDF injection. Results in Figure 6D– F showed that low levels of EZH2 and H3K27me3 were detected in sham peritoneum with/without EZH2 gene knockout, and their levels were remarkably increased after 4.25% PDF injection in EZH2-WT mice, but not obvious in EZH2-KO mice. Similarly, EZH2-KO mice showed lower expression levels of TIMP2, MMP2 and MMP9 than EZH2-WT group after 4.25% PDF injection for 28 days (Figure 6G, H). Taken together, these results further reiterate the importance of EZH2 in peritoneal fibrosis, and genetic blockage of EZH2 attenuates the development of peritoneal fibrosis. Discussion Peritoneal fibrosis is one of the most significant com- plications for CAPD patients, and yet there are no targeted solutions so far [2]. Here we demonstrated that pharmacological inhibition of EZH2 with 3-DZNeP or genetic deletion of EZH2 attenuated peritoneal fibrosis. 3-DZNeP effectively improved high glucose PDF-associated peritoneal functional impairments, as evidenced by decreasing D/P ratio of BUN and increasing D/D0 ratio of glucose. The antifibrotic effect of EZH2 inhibition is associated with block- ing several profibrotic signaling pathways, including, TGF-β/Smad, Notch, EGFR, and Src. Delayed admin- istration of 3-DZNeP, until 14 days subsequent to injury, also inhibited peritoneal fibrosis progression and partially reversed the established peritoneal fibrosis. Thus, to our knowledge, this is the first study showing that EZH2 plays a pivotal role in the development and progression of peritoneal fibrosis, suggesting that inhi- bition of EZH2 may be a potential therapeutic strategy for prevention and treatment of peritoneal fibrosis in patients on long-term PD. EZH2 has been recognized as a critical mediator of carcinogenesis [22]. Its overexpression was observed in many epithelial cancers [46,47], and mediated poor prognosis in several cancers including renal cell carci- noma [46]. This implicates that EZH2 is not only a ther- apeutic target, but also a tumor biomarker. In this study, we demonstrated that EZH2 was also highly present in the dialysis effluent of PD patients associated with inflammation and elevated over dialysis time. Mean- while, its expression levels were positively correlated with expression of several profibrotic growth factors, including TGF-β1, VEGF, and IL-6. In addition, EZH2 was also overexpressed in the peritoneum of peritoneal fibrosis induced by CG or PDF in mice. These data sug- gest that EZH2 may be a biomarker of peritoneal fibro- sis, but further investigation is needed. Currently, the source of EZH2 in the dialysis effluents remains unclear. We assume that it may come from detached peritoneal mesothelial and inflammatory cells. In support of this hypothesis, our immunostaining showed that EZH2 is highly expressed in the mesothelial layer of the injured peritoneum in two animal models and increased expres- sion of EZH2 is detected in cultured HPMCs.EZH2 may contribute to peritoneal fibrosis through induction of peritoneal EMT and activation of fibrob- lasts. Our data show that an abundance of EZH2 is not only expressed in the mesothelial layer, but also in sub- mesothelial zone in two models of peritoneal fibrosis. In support to the role of EZH2 in mediating renal fibrob- last activation, we found that EZH2 was co-stained with α-SMA, a hallmark of myofibroblasts in the submesothelial zone, and administration of 3-DZNeP and spe- cific depletion of EZH2 inhibited expression of α-SMA and deposition of ECM proteins in the peritoneum of those two models. In addition, we found that treatment with 3-DZNeP effectively restored E-cadherin levels toward the baseline in injured peritoneum as well as in cultured HPMCs exposed to TGF-β1. ChIP assay results also illustrated that EZH2 directly suppresses E-cadherin expression by H3K27me3. This provides evidence for the role of EZH2 in mediating peritoneal EMT. Since fibroblast activation and EMT are two pri- mary cellular events leading to production of ECM pro- tein, their effective inhibition by pharmacological and genetic inhibition of EZH2 suggest that EZH2 is a key epigenetic regulator promoting peritoneal fibrosis. How does EZH2 mediate activation of fibroblasts and EMT in peritoneal fibrosis? A number of studies demon- strated that TGF-β1 is a prototypical inducer of EMT and a key molecule in PD fluid-induced peritoneal mem- brane deterioration [4,48]. In this study, we demon- strated that TGF-βRI expression is subject to the epige- netic regulation of EZH2. EZH2-mediated H3K27me3 may not directly bind to the TGF-βRI promoter and increase its expression [11]. Because H3K27me3 tends to form a compact chromatin structure and leads to sup- pression rather than facilitation of gene expression [49]. Thus, we speculated that EZH2 may regulate the profi- brotic gene expression through a histone methyltrans- ferase independent mechanism or silencing of antifi- brotic genes, such as peroxisome proliferator-activated receptor γ [50]. On the other hand, the translocation of phosphorylated Smads from the cytoplasm into the nucleus is considered as a rate-limiting step in TGF-β signal transduction. It was reported that EZH2 inter- acted with nucleoporins would increase Smads accu- mulation in the nucleus, which promotes TGF-β sig- naling reversely [51]. Furthermore, EZH2 inhibition preserves Smad7 expression. Smad7 not only inhibits Smad3 phosphorylation [11], but also acts as a scaffold protein to recruit Smad ubiquitin regulatory factor 2, an E3 ubiquitin ligase, to the TGF-β receptor complex to facilitate its ubiquitination and change into a degrad- able state [52]. And several reports have shown that gene transfer of Smad7 prevents experimental peritoneal fibrosis [53–55]. Given the important role of EZH2 in the regulation of TGF-β/Smad signaling, we suggest a potential benefit from the clinical trial of treatment of peritoneal fibrosis by EZH2 inhibition. Furthermore, attenuation of peritoneal fibrosis by EZH2 suppression may also be associated with inhi- bition of EGFR signaling [8]. This is evidenced by our observations that inhibition of EZH2 by 3-DZNeP reduces EGFR and Src phosphorylation. Since EZH2 inhibition increases the expression of phosphatase and tensin homolog, a protein previously associated with dephosphorylation of tyrosine kinase receptors [11], it is speculated that EZH2 inhibition also suppresses EGFR signaling by facilitating PTEN expression. In addition, it has been documented that peritoneal fibrosis is regulated by Notch signaling pathways [7], and EZH2 can regu- late Notch1 transcriptional activation by directly binding to the Notch1 promoter and inhibiting Notch suppres- sor [56,57]. In line with this observation, we found that EZH2 inhibition suppressed expression of Notch1 and its ligand, Jagged-1 in the injured peritoneum. Activation of transcriptional factors such as STAT3 and NF-κB promotes inflammatory response during the pathogenesis of peritoneal fibrosis [58,59]. Our study indicated that EZH2 activity is required for phos- phorylation of STAT3. In support of this notion, Kim et al have reported that EZH2 can bind to and methy- late STAT3, leading to enhanced STAT3 activity by increasing its tyrosine phosphorylation [21]. However, the mechanism involved STAT3 methylation-induced phosphorylation increment is still obscure. Given the observation that STAT3 acetylation can prevent its dephosphorylation [60]. We speculated that methylation may protect STAT3 from dephosphorylation. EZH2 is also associated with activation of NF-κB signaling and expression of its target genes (IL-6, IL-8, and TNF-α) [61]. EZH2 can also control macrophage inflammatory polarization via PAK1-dependent NF-κB signaling [62]. Furthermore, EZH2 is required for lymphocyte development and promotion of T cell differentiation by inhibiting corresponding transcription factors [63,64]. In support of the role of EZH2 in the inflammatory response, we found that inhibition of EZH2 with 3-DZNeP suppresses phosphorylation of STAT3 and NF-κB brought on by high glucose dialysate, infiltra- tion of macrophages and lymphocytes and increased expression of multiple pro-inflammatory cytokines and chemokines. In line with these results, a high level of EZH2 was detected in the dialysis effluent of patients on long-term PD and positively correlated with expression of IL-6, a cytokine that links peritoneal inflammation to angiogenesis in the peritoneal membrane [65]. This sug- gests the importance of EZH2 in regulating peritoneal inflammation and angiogenesis. Angiogenesis is also an important mediator in the pro- cess of peritoneal fibrosis and associated with peritoneal ultrafiltration failure [66,67]. Our study demonstrated that EZH2 inhibition significantly reduced angiogenesis, as evidenced by decrease of CD31 and VEGF positive cells in the fibrotic peritoneum treated with 3-DZNeP. Mechanistically, EZH2 has been reported to increase H3K27me3 activity on regulatory regions of endothe- lial nitric oxide synthase (eNOS) and brain-derived neu- rotrophic factor (BDNF) promoters. Both eNOS and BDNF are highly expressed in endothelial cells and pro- mote vascular development and endothelial cell prolif- eration [68]. In addition, since IL-6 is able to promote VEGF expression and new vessel formation, EZH2 inhi- bition may also inhibit peritoneal angiogenesis through suppression of IL-6 production. In this regard, we observed that EZH2 expression is positively correlated with IL-6 levels in the dialysis effluents of long-term PD patients. Thus, EZH2 blockade with 3-DZNeP could be a potential therapeutic strategy for inhibiting vascu- lar proliferation and protecting the filtration function of peritoneum in patients on long-term PD. Our data showed that delayed administration of 3-DZNeP attenuates the progression of peritoneal fibrosis and partially reverses the established peritoneal fibrosis. The delayed administration of 3-DZNeP may elicit a peritoneal protective effect through its effects on MMPs. Of the MMPs, MMP2 and MMP9 are best known for their ability to degrade basement membrane components, laminin and type IV collagen, disrupt cell – cell, or cell – matrix attachment, facilitate the EMT and aggravate fibrosis [38,69]. Numerous investigations have proved that STAT3 is the upstream signaling molecule for MMP2 [70,71]. Intriguingly, we and other group have found that inhibition of EZH2 activity can suppress STAT3 phosphorylation [46]. Therefore, we speculated that EZH2 blockade suppressed the MMP2 by inhibiting the EZH2-STAT3 signaling axis. In addition, genome wide approaches suggest that EZH2 targets Fosl1 and Klf5, two activators of MMP9 [40]. In accordance with this observation, we found that EZH2 inhibition decreases MMP9 level. As such, downregulation of MMP2 and MMP9 caused by the delayed administration of 3-DZNeP would partially reverse peritoneal fibrosis. Increasing evidence has indicated that EZH2 is a crit- ical mediator in tumorgenesis and has become an effec- tive target for the treatment of some tumors [19,24,47, 72]. We and others have shown in previous studies that EZH2 plays an essential role in mediating tissue fibro- sis in several organs including liver, lung, and kidney [11,51,73]. In the current study, we provided strong evi- dence that EZH2 is critically involved in the develop- ment and progression of peritoneal fibrosis. Although 3-DZNeP was used to inhibit tissue fibrosis in most stud- ies, other EZH2 inhibitors have also been synthesized and used in the treatment of tumors in either preclinical studies or clinical trials [74,75]. We look forward to a possible benefit from a clinical trial of treatment of PD related to peritoneal fibrosis by EZH2 inhibition. In conclusion, we demonstrated that genetic or phar- macologic blockade of EZH2 can prevent and reverse peritoneal fibrosis. Inhibition of EZH2 with 3-DZNeP effectively improved high glucose PDF-associated peritoneal functional impairments. Mechanistically, EZH2 inhibition protected the peritoneal membrane from fibrosis by blocking EMT, fibroblast activation, inflammatory responses, and angiogenesis though inac- tivation of multiple profibrogenic signaling pathways. As such, DZNeP blocking EZH2 may have a potential thera- peutic benefit in preventing and treating patients with peritoneal fibrosis on long term PD.