Received: 15 September 2019 | Revised: 24 October 2019 | Accepted: 11 November 2019 DOI: 10.1111/jcmm.14865 ORIGINAL ARTICLE Up-regulation of HDACs, a harbinger of uraemic endothelial dysfunction, is prevented by defibrotide Marta Palomo1,2,3 | Manel Vera4 | Susana Martin1 | Sergi Torramadé-Moix1 | Julia Martinez-Sanchez1,2,3 | Ana Belen Moreno1 | Enric Carreras2,3 | Ginés Escolar1 | Aleix Cases4 | Maribel Díaz-Ricart1,3 1 Hematopathology, Centre Diagnòstic Biomèdic (CDB), Hospital Clinic, Institut Abstract d'Investigacions Biomèdiques August Pi i Endothelial dysfunction is an earlier contributor to the development of atherosclero- Sunyer (IDIBAPS), Universitat de Barcelona (UB), Barcelona, Spain sis in chronic kidney disease (CKD), in which the role of epigenetic triggers cannot be 2 Josep Carreras Leukaemia Research ruled out. Endothelial protective strategies, such as defibrotide (DF), may be useful Institute, Hospital Clinic/University of in this scenario. We evaluated changes induced by CKD on endothelial cell proteome Barcelona Campus, Barcelona, Spain 3 Barcelona Endothelium Team (BET), and explored the effect of DF and the mechanisms involved. Human umbilical cord Barcelona, Spain vein endothelial cells were exposed to sera from healthy donors (n = 20) and patients 4 Nephrology Department, Hospital Clinic, with end-stage renal disease on haemodialysis (n = 20). Differential protein expres- Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de sion was investigated by using a proteomic approach, Western blot and immuno- Barcelona (UB), Barcelona, Spain fluorescence. HDAC1 and HDAC2 overexpression was detected. Increased HDAC1 Correspondence expression occurred at both cytoplasm and nucleus. These effects were dose-de- Marta Palomo, Josep Carreras Leukaemia pendently inhibited by DF. Both the HDACs inhibitor trichostatin A and DF prevented Research Institute, Hospital Clinic/ University of Barcelona Campus, C/ the up-regulation of the endothelial dysfunction markers induced by the uraemic Villarroel 170, 08036 Barcelona, Spain. milieu: intercellular adhesion molecule-1, surface Toll-like receptor-4, von Willebrand Email: mpalomo@carrerasresearch.org Factor and reactive oxygen species. Moreover, DF down-regulated HDACs expres- Funding information sion through the PI3/AKT signalling pathway. HDACs appear as key modulators of Jazz Pharmaceuticals, Grant/Award Number: IST-16-10355; Germán José the CKD-induced endothelial dysfunction as specific blockade by trichostatin A or by Carreras Leukaemia Fundation, Grant/ DF prevents endothelial dysfunction responses to the CKD insult. Moreover, DF ex- Award Number: 03R/2019 and 11R/2016; Generalitat de Catalunya, Grant/Award erts its endothelial protective effect by inhibiting HDAC up-regulation likely through Number: 2017-SGR675 and CERCA PI3K/AKT. Programme; Instituto de Salud Carlos III Spanish Government, Grant/Award Number: DTS16/00133 and PIE15/0027 KEYWORDS chronic kidney disease, defibrotide, endothelial dysfunction, HDAC, HDAC1, HDAC2, inflammation, oxidative stress 1 | I NTRO D U C TI O N costs, poorer quality of life and reduced life expectancy.1 CKD is as- sociated with an increased cardiovascular (CV) risk, and CV disease Chronic kidney disease (CKD) is a major public health concern due to accounts for nearly half of all deaths.2 The increased incidence of CV its increased prevalence, high morbidity and mortality associated, high disease (CVD) in CKD cannot be explained by the high prevalence of This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2019 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd. J Cell Mol Med. 2020;24:1713–1723.  wileyonlinelibrary.com/journal/jcmm | 1713 1714 | PALOMO et al. traditional risk factors in this setting, and classical CV risk scores, such 2 | M E TH O DS as the Framingham risk score, underestimate the CV risk in this popu- lation.3 Further, the results of pharmacological interventions that have 2.1 | Patients and sample collection proven a cardio-protective benefit in the general population or other high risk populations (eg in diabetes or in patients with established Sera from patients with end-stage renal disease on maintenance cardiovascular disease), such as statins or inhibitors of the renin-an- haemodialysis (HD, n = 20) were included. Patient characteristics are giotensin system, have failed to show a benefit in patients with end- detailed in Table 1. In order to exclude causes other than uraemia in stage renal disease.4-7 Because of that, new or uraemia-related CV risk endothelial dysfunction, patients with diabetes, dyslipidaemia and/ factors have been postulated to play a role in the CV risk of these or previous CVD and/or current smokers were excluded. All CKD 8 patients. Thus, there is a need for the search of new therapies able to patients were dialysed with biocompatible membranes, ultrapure reduce the high CV risk and the associated mortality in this population. water, for ≥4 hours, three times a week with a Kt/V ≥ 1.3. None of the There is wide evidence indicating that endothelial dysfunction patients were dialysed through an HD catheter as a vascular access. is an initial stage for the development of atherosclerosis, and in vivo Blood samples were collected before the 2nd or 3rd HD session of and in vitro studies have demonstrated that endothelial dysfunction the week. Sex and age-matched healthy donors (n = 20), with no pre- is present in CKD.9 In previous in vitro studies, endothelial dysfunc- vious history of CVD, and preserved renal function (eGFR > 90 mL/ tion induced by the CKD milieu has been characterized. Exposure min/1.73 m2, CKD-EPI formula) were enrolled as controls (Table 1). of endothelial cells (ECs) to sera from CKD patients results in mor- Serum samples from CKD patients and from healthy donors were phological alterations, enhanced thrombogenicity of the extracel- obtained by centrifugation of non-anticoagulated blood (3000 g, lular matrix and changes towards a pro-inflammatory phenotype, 15 minutes) and stored at −80°C until used. Pools of serum were with increased expression of adhesion receptors and activation of signalling pathways, such as nuclear factor-kappa B (NFkB),10 or the innate immunity Toll-like receptor 4 (TLR4) and the NALP3 inflam- TA B L E 1 Clinical characteristics of patients and controls 11 masome. Moreover, the endothelial response to the CKD insult is Control HD also characterized by an enhanced oxidative stress and changes in (n = 20) (n = 20) the expression of related proteins.12-15 Age, years, mean ± SD 48.3 ± 9.3 55.4 ± 15.7 Our previous in vitro results suggest that the CKD setting induces Female/male 12/8 9/11 activation of specific genes in the endothelium that promotes a proo- Estimated filtration rate CKD- >90 0 xidant, pro-inflammatory, prothrombotic and proproliferative state. In EPI (mL/min/1.73 m2) this regard, HDACs enzymes are important epigenetic factors that reg- Mean time on dialysis, 23 ± 5.7 ulate pro-inflammatory gene expression, in ECs and in other tissues, months ± SD and also deacetylate non-histone proteins that regulate inflamma- Dialysis adequacy, Kt/V, mean, 2.3 ± 0.4 tory signalling.16 Therefore, they appear to be promising therapeutic ±SD targets for the treatment of atherosclerosis and other cardiovascular Serum albumin (g/L), 44 ± 4 39 ± 6 diseases.17,18 Furthermore, HDAC activity is linked to a number of car- mean ± SD dio-renal pathologies, including heart failure,19 hypertension,20 and CRP (mg/dL), mean ± SD 0.2 ± 0.1 0.4 ± 0.2 diabetes or diabetic kidney disease.21,22 Thus, the dysfunctional en- Haemoglobin (g/L), mean ± SD 12.6 ± 1.6 11.3 ± 0.4 dothelium and its epigenetic regulation arise as an attractive target 9 Leucocytes (10 /L), mean ± SD 7.4 ± 1.6 6.8 ± 1.2 for interventions designed to reduce the risk and burden of CVD in Causes of CKD n (%) CKD patients.23,24 Defibrotide (Defitelio®) (DF) is a drug composed Glomerulonephritis 4 (20%) by a complex single-stranded oligodeoxyribonucleotides derived from Polycystic kidney disease 3 (15%) porcine intestinal mucosal DNA that has demonstrated profibrinolytic, Interstitial nephropathy 4 (20%) antithrombotic-thrombolytic, antiischaemic, antishock, antiatheroscle- Obstructive kidney disease 2 (10%) rotic, antirejection and anti-angiogenic effects.25 The endothelial pro- Nephrosclerosis 3 (15%) tective activity of this drug has been demonstrated in different clinical settings.26-28 This drug interacts specifically with the membrane of ECs Unknown 4 (20%) where it displays its anti-inflammatory and anti-oxidant effects. 29 Hypertension n (%) 0 14 (70%) 12 In the present study, we applied a translational approach to iden- Use of statins n (%) 0 8 (40%) tify proteins differentially expressed in an endothelium exposed to the Use of vitamin D n (%) 0 18 (90%) CKD milieu and the effect of DF. Functional assays were performed to Use of erythropoiesis stimu- 0 17 (85%) confirm the role of the most physiologically relevant identified proteins lating agents n (%) and their implication in the development of endothelial dysfunction. Use of inhibitors of the renin- 0 6 (30%) Moreover, we aimed at identifying the mechanisms involved in the pro- angiotensin system n (%) tective effect exerted by DF in this setting. Abbreviation: CRP, C-Reactive protein. PALOMO et al. | 1715 prepared containing serum samples from different healthy donors Fisher Scientific) coupled to a nanoelectrospray ion source or HD patients, which were used as reagent for the different experi- (Thermo Fisher Scientific). MS/MS data acquisition was completed ments performed. using Xcalibur 2.1 (Thermo Fisher Scientific). 34 Data were processed using Proteome Discoverer 1.2 (Thermo Fisher Scientific). For database searching, processed data were 2.2 | Endothelial cell cultures submitted to the in-house Homo sapiens UniProtKB/Swiss-Prot database (released June 2011; 20 211 protein entries) using ECs were isolated from human umbilical cord veins.30 Cells were SEQUEST, version 28.0 (Thermo Fisher Scientific). The follow- maintained and subcultured at 37°C in a 5% CO2 humidified in- ing search parameters were used: two maximum missed cleav- cubator in EGM-2 Endothelial Cell Growth Medium-2 BulletKit ages for trypsin; carbamidomethylation as a fixed modification; (Lonza, Cultek SLU). Culture medium was replaced every 48 hours. methionine oxidation as a variable modification; 20 pp peptide To perform the experiments, ECs were used between the 2nd mass tolerance; and 0.8 Da fragment ion tolerance. Criteria used and 3rd passage. To study the CKD-induced endothelial dam- to accept identification included a false discovery rate (FDR) of age, we used an established in vitro model of uraemic endothelial 0.05, and five minimum spectra (and at least one unique pep- dysfunction.11,13-15,24 This model consists of exposing endothelial tide) matched per protein. Proteins identified were classified ac- cells to Medium 199 (Gibco BRL, Life Technologies) supplemented cording to biological function(s) using the information available with 100 U/mL penicillin, 100 g/mL streptomycin (Gibco BRL, Life at the UniProt Knowledge base (UniProtKB/Swiss-Prot) website Technologies) and 20% of pooled sera from patients (n = 20) or from (http://www.unipr​ot.org) and checked according to previous de- matched healthy donors (n = 20), for different periods depending on scriptions found in PubMed (http://www.ncbi.nlm.nih.gov/pubme​ the endothelial damage marker to be evaluated. d/; National Center for Biotechnology Information, US National Library of Medicine, National Institutes of Health, Bethesda, MD). Spectral counts were normalized using the average of the sum of 2.3 | Differential proteomic analysis raw counts across all samples. Differentially expressed proteins were identified using stringent selection criteria: fold change ≥3 ECs were isolated from human umbilical cord veins (n = 6) and cul- for up-regulated proteins, detected in all samples and a mean nor- tured as previously described. After reaching confluence during the malized count in positive samples of ≥2. first passage, cells were mixed, seeded in 75-cm2 flasks and exposed to media containing 20% of pooled sera of controls (n = 20 distributed in 6 different pools) or CKD patients (n = 20 distributed in 6 different 2.4 | Confirmation of proteins by Western blot and pools) with or without DF (100 μg/mL, a dose selected from our previ- immunofluorescence studies ous studies)27,29 during 24 hours. Then ECs were washed, trypsinized, counted, pelleted and stored at –80°C until proteomic analysis. From the differentially up-regulated proteins found comparing the an- Each sample was then solubilized in Laemmli lysis buffer alysed settings, both HDAC1 and HDAC2 were selected to be deeply (60 mmol/L Tris HCl, pH 6.8, 2.2% (w/v) SDS, 5% (v/v) glycerol, studied. First, protein lysates from ECs exposed to the same condi- 0.1 mol/L DTT), incubated for 3 minutes at 99°C, sonicated and tions studied in the proteomic assay were analysed by Western blot centrifuged for 15 minutes. Protein concentration was determined (WB, n = 4) and immunofluorescence (IF, n = 6) with specific primary using the Quick Start Bradford Protein Assay (Bio-Rad) following antibodies (Abcam) to confirm the proteomic results. 31 the manufacturer's recommendation. Then, 100 µg of proteins For WB analysis, ECs were exposed to media containing 20% of was separated by using SDS-PAGE 12% acrylamide gel. The gel pooled sera of controls (4 different pools) or CKD patients (4 different was fixed with 30% ethanol and 10% acetic acid and precondi- pools) with or without DF (100 μg/mL, a dose selected from our previ- tioned with sodium thiosulphate, before silver nitrate staining ous studies)27,29 during 24 hours. Then, ECs were lysed with Laemmli's 32 treatment with the modified Blum et al silver staining proto- buffer, sonicated to shear DNA and reduce viscosity (15 seconds), and col. Gels were cut into 3 mm slices prior to trypsin digestion and heated to 90°C (5 minutes). Protein concentration in the supernatants peptide extraction. 33 Peptides were separated by means of nano was determined using Coomassie Plus (Pierce). Samples were resolved liquid chromatography using a Proxeon EASY-nLC (Thermo Fisher by 8% SDS-PAGE, proteins transferred to nitrocellulose membranes Scientific) with a flow rate of 300 nL/min, a C18 trap column and probed with specific antibodies against HDAC1, HDAC2 (Abcam) (5 µm, 120 Å, 100 µm inner diameter 2 cm in length), an EASY and B-actin (Cell Signaling). Membranes were incubated with a per- C18 analytical column (3 µm, 120 Å, 75 µm inner diameter 10 cm oxidase-conjugated anti-rabbit IgG and developed by chemilumines- in length). The following linear gradient, using solvent B (97% ace- cence.29 Densitometric assay of protein bands was performed using tonitrile, 0.1% formic acid) and solvent A (3% acetonitrile, 0.1% Fiji (ImageJ, National Institutes of Health), and all values were normal- formic acid), was employed: 5%-40% buffer B (90 minutes); 40%- ized with their respective B-actin bands. 100% buffer B (5 minutes); 100% buffer B (15 minutes) finally. MS/ For IF analysis, ECs were exposed to the same conditions pre- MS analysis was performed using an LTQ Orbitrap Velos (Thermo viously described, fixed with 4% paraformaldehyde in 0.15 mol/L 1716 | PALOMO et al. PBS, pH 7.4 (4°C, 10 minutes), blocked with 1% BSA, permeabilized 2.7 | Study approval with triton X-100 (Sigma) and incubated with a primary antibody against HDAC1 and a secondary antibody anti-rabbit IgG conju- Written informed consent was obtained from all participants, and gated with Alexa 488 (Molecular probes). Nuclei were stained with the study was approved by the Ethical Committee of the Hospital DAPI (Sigma). Samples were evaluated by a fluorescence micros- Clinic (HCB/2014/0302) and carried out according to the principles copy (Leica DM4000B), images were captured through a video cam- of World Medical Association Declaration of Helsinki. era (Leica DFC310FX), and the percentage of the area positive for staining was calculated by computerized establishing a threshold through morphometric analysis (Fiji, ImageJ, National Institutes of 2.8 | Statistics Health). Moreover, with the aim of analysing the location of HDAC1, a colour threshold tool defining blue colour was used to differenti- Results are expressed as mean ± standard error of the mean (SEM) ate nuclei (blue) and cytoplasm (not blue). Then, HDAC1 expression and correspond to the % of labelled area/% of nuclei area for IF as- was differentially quantified in these two compartments. Moreover, says when analysing total HDAC1 expression, in % of labelled area to evaluate the specificity of HDAC1 inhibition by DF, a dose-de- when analysing HDAC1 expression in nuclei or cytoplasm, and in fold pendent study (0, 50 and 100 µg/mL) was performed by using an increase vs control for the values obtained from densitometric analysis IF assay. of the protein bands normalized with their respective B-actin bands for WB. N corresponds to different experiments performed with dif- ferent pools (from healthy donors or CKD patients) for each value ana- 2.5 | Roles of HDAC1 and HDAC2 in endothelial lysed. Statistical analysis was performed using SPSS statistical package damage induced by CKD sera 17.0.0 (SPSS Inc, Chicago, IL) with raw data using t test. Results were considered statistically significant when P < .05. To evaluate the role of HDAC1 and HDAC2 in uraemic endothe- lial dysfunction, a pan-HDAC inhibitor trichostatin A (TSA) (Sigma Aldrich) and DF were used as HDAC inhibitors. ECs on 6-well mi- 3 | R E S U LT S croplates were pretreated (24 hours) with TSA (50 nmol/L) or DF (100 µg/mL) and exposed to media containing 20% of pooled sera 3.1 | Endothelial dysfunction in CKD is from the uraemic patients or healthy donors, as previously described characterized by an increase in HDAC1 and HDAC2, for the differential proteomic analysis. Cells were then fixed and among other proteins labelled for intercellular adhesion molecule-1 (ICAM-1) (Millipore, Merck), Toll-like receptor 4 (TLR4) (Abcam) and von Willebrand To study the proteomic changes involved in endothelial uraemic dys- Factor (vWF) (DAKO) using IF, as previously described. Reactive oxy- function and the potential protective role of DF in this setting, a pro- gen species (ROS) production was explored by using CM-H2DCFDA. teomic approach was performed on ECs exposed to sera of healthy This compound can passively diffuse into cells being oxidized by ROS controls or CKD patients in the absence and presence of DF. After to the highly fluorescent dichlorofluorescein. ROS production was normalization, 11 proteins were differentially expressed comparing monitored by fluorescence microscopy. All samples were evaluated the analysed settings (Table 2). Proteins related to inflammation, such by microscopy (Leica DM4000B), and images were captured through as protein arginine N-methyltransferase 5 or histone arginine meth- a videocamera (Leica DFC310FX). Fluorescence micrographs were yltransferase; proteins involved in epigenetic regulation and proteins analysed using ImageJ (version 1.43m; NIH, http://rsb.info.nih.gov/ related to cell motility; proliferation and survival (HDAC1, HDAC2, nih-image/​ manua​ l/tech.html#). Cultured cells were selected from ATP-dependent DNA helicase Q1, TRIO and F-acting-binding pro- the background with the threshold tool, and the fluorescence inten- tein), among others were overexpressed in the uraemic setting. sity was measured. Results suggested that DF could re-establish the proper expression of proteins that were found overexpressed in the ECs exposed to CKD conditions. 2.6 | Activation of PI 3-kinase/AKT pathway as inducer of HDAC1 and HDAC2 expression and inhibitory effect of defibrotide 3.2 | DF prevents the overexpression of HDAC1 and HDAC2 induced by CKD sera, as well as increased 740 Y-P (Tocris Bioscience, Bio-Techne R&D Systems, S.LU), a cell- HDAC1 in both nucleic and cytoplasmic locations permeable PI 3-kinase/AKT activator, was used as reagent to inves- tigate the interaction between DF and HDAC1 and HDAC2. 740 Y-P WB and IF techniques were applied to confirm HDAC1 and HDAC2 was added (20 μmol/L, 5 hours) to ECs cultures in the presence or overexpression in ECs exposed to CKD sera and the effect of absence of DF and HDAC1 and HDAC2 expression was evaluated DF. WB results confirmed that CKD induced an overexpression through IF (n = 6) and WB (n = 4). of HDAC1 (1.9 ± 0.3 fold vs Control, n = 4, P < .05) and HDAC2 PALOMO et al. | 1717 TA B L E 2 Overexpressed proteins in endothelial cells exposed to CKD sera Swiss-Prot accession no. Protein name Gene Cellular location Molecular function Biological process P33121 Long-chain-fatty-acid- ACSL1 Mitochondrion, en- Ligase, activation of long-chain Fatty acid metabolism, lipid CoA ligase 1 doplasmic reticulum, fatty acids for both synthesis of metabolism and peroxisome cellular lipids, and degradation via beta-oxidation O14744 Protein arginine PRMT5 Golgi apparatus, nu- Chromatin regulator methyl- Biological N-methyltransferase 5 cleus, and cytoplasm transferase, repressor, and rhythms, transcription transferase Q03701 CCAAT/enhancer-bind- CEBPZ Nucleus Activator Transcription regulation ing protein zeta Q9UJW0 Dynactin subunit 4 DCTN4 Cytoskeleton, cell cor- Protein N-terminus binding Endoplasmic reticulum to Golgi tex, and sarcomere vesicle-mediated transport and nuclear migration Q9BY44 Eukaryotic translation EIF2A Cytosol, extracellular Initiation factor Protein biosynthesis and trans- initiation factor 2A region, and secreted lation regulation Q92556 Engulfment and cell ELMO1 Plasma membrane SH3 domain binding Actin cytoskeleton organiza- motility protein 1 tion, apoptotic process, and vascular endothelial growth factor receptor signalling pathway Q13451 Peptidyl-prolyl cis-trans FKBP5 Nucleus and Heat shock protein binding Chaperone-mediated protein isomerase FKBP5 cytoplasm folding Q96I24 Far upstream element- FUBP3 Nucleus DNA-binding transcription acti- Positive regulation of gene binding protein 3 vator activity, RNA polymerase expression II-specific, and RNA binding Q96PP9 Guanylate-binding GBP4 Golgi apparatus and GTPase activity Cellular response to protein 4 nucleus interferon-gamma Q13547 Histone deacetylase 1 HDAC1 Nucleus Chromatin regulator, Hydrolase, Biological rhythms and and repressor Transcription regulation Q92769 Histone deacetylase 2 HDAC2 Nucleus and Chromatin regulator, hydrolase, Biological rhythms and tran- cytoplasm and repressor scription regulation (1.5 ± 0.1 fold vs Control, n = 4, P < .05) that was normalized in the 3.3 | The inhibitory effect of DF on CKD-induced presence of DF (1.1 ± 0.2 for HDAC1, and 0.9 ± 0.2 for HDAC2, HDAC1 overexpression is dose-dependent n = 4 and P < .05 vs CKD for all experiments) (Panel A, Figure 1). Similarly, an increase in HDAC1 was detected by IF in ECs exposed Immunofluorescence assays were performed with different DF doses to to CKD from 3 ± 0.3% of labelled area/% nuclei area in controls to test the specificity of the reduction of HDAC1 expression previously de- 4.7 ± 0.2% of labelled area/% nuclei area in CKD (n = 6, P < .01). tected. In ECs exposed to CKD sera, HDAC1 total expression increased The effect of uraemic sera on endothelial HDAC1 expression to 4.7 ± 0.2% of labelled area/% nuclei area compared to control, and was was prevented by DF (2.6 ± 0.1% of labelled area/% nuclei area, dose-dependently inhibited in the presence of 50 μg/mL (4.2 ± 0.3% of n = 6, P < .01 vs CKD) (Micrographs and left scatterplot, Panel B, labelled area/% nuclei area, n = 6,) and 100 μg/mL (3.8 ± 0.1% of labelled Figure 1). Specifically at the nucleus, CKD sera induced a signifi- area/% nuclei area, n = 6, P < .05 vs CKD) of DF (Figure 2). cant increase in HDAC1 (77.5 ± 2.9% vs 50.3 ± 2.4% of nuclei la- belled area in control samples, n = 6, P < .01), which was prevented by DF (41.8 ± 5.2% of nuclei labelled area, n = 6, P < .01 vs CKD). In 3.4 | CKD-induced endothelial dysfunction addition, the effect of DF was also notable in the cytoplasmic loca- is mediated through HDAC1 and HDAC2 tion in ECs exposed to CKD, as the overexpression of the protein overexpression in ECs exposed to uraemic sera vs controls was reduced to almost 75% of cytoplasmic labelled area (Micrographs and right scatter- ICAM-1 and TLR4 expression on cell surfaces and vWF con- plot, Panel B, Figure 1). tent were higher in ECs exposed to the CKD patients’ sera when 1718 | PALOMO et al. F I G U R E 1 Defibrotide prevents HDAC1 increased expression induced by CKD sera at both nucleic and cytoplasmatic locations. A, Immunoblot images show expression of HDAC1 (left) and HDAC2 (right) when endothelial cells were exposed to control or CKD sera (24 h) in the absence or presence of DF (100 µg/mL). B, Micrographs show HDAC1 expression (green) in endothelial cells exposed to control and CKD sera in the absence or presence of defibrotide (CKD + DF). Left column shows total presence of HDAC1 (green) and nucleic (blue) staining. The middle column shows HDAC1 expression only in the nuclei and the right column shows HDAC1 expression only in the cytoplasm (40× magnification). Left scatterplot (with median) represents HDAC1 expression in terms of the total percentage of labelled area/percentage of nuclei area, and right scatterplot represent HDAC1 expression in terms of the total percentage of labelled area in nuclei or cytoplasm, in endothelial cells exposed to healthy sera (Control) and sera from CKD patients with or without the presence of defibrotide (CKD and CKD + DF, respectively) (n = 6, being **P < .01 vs control and ##P < .01 vs CKD, n = number of independent experiments, statistical analysis was performed with raw data using t test) compared to control sera (1.5 ± 0.2%, 0.8 ± 0.1%, and 7.5 ± 0.9% vs effect on ROS generation in response to the uraemic sera, reduc- 0.6 ± 0.1%, 0.4 ± 0.1%, and 3.9 ± 0.2%, respectively, n = 6, P < .05). ing its production to control levels (18.1 ± 1.3 of mean fluorescence Exposure of ECs to CKD sera in the presence or absence of TSA intensity, n = 6, P < .05 vs CKD) (Figure 3). was performed to assess if HDAC1 and HDAC2 overexpression me- diated the increases in markers of endothelial damage induced by CKD. Our results point out that inhibition of HDACs overexpression 3.5 | Effect of DF on HDAC1 and HDAC2 reduced the increase in the markers of endothelial damage evalu- is potentially mediated through PI3K/AKT ated. In the presence of TSA, ICAM-1 expression was reduced from pathway inhibition 1.5 ± 0.2% to 0.9 ± 0.1%, TLR4 from 0.8 ± 0.1% to 0.4 ± 0.1%, vWF from 7.5 ± 0.9% to 3.6 ± 0.4% and ROS production from 4.1 ± 2.1 of ECs were exposed to P740-Y-P, a cell-permeable phosphopeptide mean fluorescence intensity to 21.8 ± 2.0% (P < .05 for all values) activator of the PI3K/AKT pathway in the presence or absence of (Figure 3). Furthermore, DF was also capable to inhibit the expres- DF (100 μg/mL). Then, HDAC1 expression was assessed by WB and sion of ICAM-1, TLR4 and vWF content on cells exposed to uraemic IF, and HDAC2 by WB (Figure 4). sera in a significant manner, decreasing their expression to control WB results revealed that the expression of HDAC1 and HDAC2 was levels (0.4 ± 0.1%, 0.4 ± 0.1% and 4.1 ± 0.2%, respectively, n = 6, increased in ECs incubated with P740-Y-P (5 hours) (fold of 1.9 ± 0.1 P < .05 vs CKD for ICAM and P < .05 vs CKD for TLR4 and vWF) and 1.4 ± 0.2, respectively vs control, n = 4, P < .05) and that these (Figure 3). DF also exhibited a remarkably and significant inhibitory increases were prevented by DF (0.9 ± 0.1 and 1.1 ± 0.2 fold vs control, PALOMO et al. | 1719 as a putative pathway through which DF modulates HDACs expres- sion (Figure 5). Thus, the results of the present study highlight the relevance of the epigenetic changes associated with endothelial dys- function in CKD and uncover the potential mechanisms of action by which DF exerts its protective effect on ECs in this setting. The involvement of vascular endothelium in the initiation and the progression of atherosclerosis in CKD patients has been progres- sively recognized. So far, the endothelial phenotype in CKD has been extensively characterized, but there is a lack of information regarding the mechanisms through which the uraemic milieu exerts an impact on the endothelial cell and its epigenome. To approach this knowl- edge, we applied an established translational methodology12 to look for the signature of endothelial dysfunction in CKD and find key fac- tors that may be susceptible to be regulated by DF. We were able to identify two proteins, HDAC1 and HDAC2, involved in epigenetic regulation, among other up-regulated proteins. Epigenetics refer to chromatin-based mechanisms important in the regulation of gene ex- pression that do not involve changes in the DNA sequence per se.35,36 Our proteomic results were confirmed by other techniques and revealed that the CKD milieu induces overexpression of both HDAC1 and 2 and the accumulation of HDAC1 in both the nucleus and the cytoplasm of ECs. These two enzymes are widely expressed in human tissues, belong to class I HDACs, which are considered the ‘classical’ HDACs, whose activities could be inhibited by TSA, and are involved in cell proliferation and cell survival.37 Although HDAC class I (HDAC 1, 2, 3 and 8) is classically described as being expressed in the nucleus, these proteins are also found in the cytosol. In this sense, several studies have shown that HDAC1 can also be expressed in the cytosol in different cell lines,38-40 and although its expression F I G U R E 2 Defibrotide prevents HDAC1 increased expression is mainly located in the nucleus, its expression is enhanced in the induced by CKD sera in a dose-dependent manner. Micrographs cytosol in pathological conditions.41 Further, it has been reported show endothelial cells exposed to CKD sera in absence (up) that the up-regulation of HDAC1 in ECs deacetylates nitric oxide and presence of defibrotide (50 µg/mL and 100 µg/mL). Nuclei are labelled with DAPI (blue) (40× magnification). Scatterplot synthase 3 (NOS3), directly resulting in a decrease of endothelial ni- (with median) represents quantification of the area positive for tric oxide production.42 HDAC overexpression in ECs in response to HDAC1 staining normalized by percentage of nuclei area, by CKD sera is in concordance with previous evidence by other authors immunofluorescence technique (n = 6, being *P < .05 vs control showing enhanced expression of these proteins in other cardio-renal and #P < .05 vs CKD, n = number of independent experiments, pathologies.19-22 The protective effect of DF in this setting concord statistical analysis was performed with raw data using t test) with its proved beneficial effect in other clinical entities associated respectively). Moreover, through an IF assay HDAC1 overexpression with endothelial dysfunction. 27-29 in the nuclei was confirmed after the incubation of ECs with P740- HDACs positively act on pro-inflammatory candidates, such as Y-P (from 32.6 ± 4.4% of covered area to 44.9 ± 6.4%, n = 6, P < .05). cytokines (IL-6, IL-12, TNF), chemokines (CCL2, CCL7 and CXCL) Defibrotide was able to prevent this increase (27.6 ± 5.9, n = 6, P < .05). and other inflammatory mediators (MMP-9 and endothelin-1).43,44 In this regard, HDACs inhibitors show broad anti-inflammatory ef- fects through different mechanisms. In our model of uraemic en- 4 | D I S CU S S I O N dothelium, both TSA and DF reduced ICAM-1 expression and vWF production. Moreover, there are many studies demonstrating the Our present study explored the protein signature of the endothe- important role of HDACs both in innate and adaptive immunity. In lium exposed to CKD sera in the presence and absence of DF, and particular, these enzymes seem to act on the expression of TLR tar- pointed out to HDACs as key molecules that mediate the endothelial get genes.45 In this regard, we show in the present study that TLR4 response to the CKD milieu. Both TSA and DF prevented the en- expression is regulated by HDACs in ECs exposed to a uraemic mi- dothelium from developing its pro-inflammatory, prooxidant, pro- lieu. In addition, the inhibition of ROS production by TSA and DF thrombotic and activated innate immunity phenotype induced by demonstrates that HDACs are also involved in the prooxidant endo- the CKD sera. Further, PI3K/AKT signalling pathway was identified thelial response to CKD. 1720 | PALOMO et al. F I G U R E 3 ICAM-1, TLR4, vWF and ROS increases in CKD are mediated through HDACs induction. Micrographs show enhanced expression of the adhesion receptor ICAM-1 (red), TLR4 (red), the adhesive protein vWF (red) and production of ROS (green) when endothelial cells were exposed to CKD patients' sera. The co-incubation of cells with CKD patients sera and TSA (CKD + TSA, middle row) or defibrotide (CKD + DF, lower row) induced a decrease in the expression of the markers of endothelial damage analysed. Cell nuclei are stained with DAPI (blue) (40× magnification). Scatterplots (with median) correspond to the quantification of the endothelial damage markers evaluated. All data correspond to relative expression compared to control levels (n = 6, being *P < .05 vs control and #P < .05 and ##P < .01 vs CKD, n = number of independent experiments, statistical analysis was performed with raw data using t test) The pharmacological inhibition of HDACs is a new target for sev- animal models of inflammatory diseases, attenuating the progres- eral disorders. These drugs have been approved for the treatment sion of renal fibrogenesis in obstructive nephropathy or reducing of some lymphomas, or epilepsy (valproic acid) and several others cyst formation in polycystic kidney disease.46 This property has been are in clinical trials. Further, these agents have shown its efficacy in successfully applied to ameliorate endothelial activation and vascular PALOMO et al. | 1721 F I G U R E 4 Defibrotide acts as a PI3/ AKT inhibitor to interact with HDACs. A, Immunoblot images show expression of HDAC1 (left) and HDAC2 (right) when endothelial cells were exposed to 740 Y-P in absence or presence of DF (100 µg/ mL). B, Micrographs show an increase in HDAC1 expression (green) in endothelial cells exposed to P740-Y-P (+P740-Y-P) and a decrease when DF was added (+740 Y-P + DF). Scatterplot (with median) represents the quantification of HDAC1 expression in the three situations (Control, +740 Y-P, +740 Y-P + DF) in terms of the labelled area (n = 6, being *P < .05 vs control and #P < .05 vs CKD, n = number of independent experiments, statistical analysis was performed with raw data using t test) pathophysiology in sickle cell anaemia in transgenic mice. In this ex- Once we demonstrated that DF is able to reduce HDACs expres- perimental animal model, and also in HUVECs,47,48 TSA significantly sion and their downstream effects in the CKD-induced endothelial reduced VCAM-1 and tissue factor expression.49 Although many of damage, we focused on the search of the signalling pathway po- the underlying mechanisms and functions of HDACs in human cells tentially used by the drug to modulate these epigenetic regulators. are poorly understood and remain unclear, they appear to be promis- There is evidence demonstrating that HDAC expression is regulated ing targets in the modulation of endothelial dysfunction. by the PI3K/AKT pathway.17,50 In addition, our group has previously F I G U R E 5 Visual abstract. A single, concise, pictorial and visual summary of the main findings of the present study in which we demonstrate that HDACs appear as key modulators of the CKD-induced endothelial dysfunction and that DF prevents endothelial dysfunction responses to the CKD insult likely through PI3K/AKT 1722 | PALOMO et al. demonstrated that DF inhibits the activation of PI3K/AKT. 27,51 Both patients, performed the images and reviewed the results and the facts prompted us to hypothesize that DF was regulating HDACs manuscript. SM contributed to the design and the conducting of the through this pathway. To prove this hypothesis, ECs were exposed experiments, acquired the data and analysed the results. ST and JMS to a PI3K/AKT inducer to stimulate HDAC expression, and then, the contributed to the conduction of the experiments and the mainte- effect of DF was explored. Our results indicate that DF could act as nance of cell cultures. ABM, EC, GE, and AC reviewed the results a PI3K/AKT pathway inhibitor to modulate HDACs expression. One and the manuscript. MDR. designed and supervised the project and of the limitations of our study is that we measured protein levels, reviewed the results and the manuscript. but not HDACs activity or other HDACs not appearing in our pro- teomic approach. Another limitation of our study is that we used a ORCID pan-HDAC inhibitor rather than specific HDAC1 or 2 inhibitor, and Manel Vera https://orcid.org/0000-0001-9937-7235 its effects on other HDACs cannot be ruled out. In conclusion, CKD is an example of a complex disease in which the DATA AVA I L A B I L I T Y S TAT E M E N T phenotype arises from a combination of environmental and inheritable The data that support the findings of this study are available from 52 factors. 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