Diabetes impairs cardioprotective function of endothelial progenitor cell-derived extracellular vesicles via H3K9Ac inhibition

Background and Purpose: Myocardial infarction (MI) in diabetic patients results in higher mortality and morbidity. We and others have previously shown that bone marrow-endothelial progenitor cells (EPCs) promote cardiac neovascularization and attenuate ischemic injury. Lately, small extracellular vesicles (EVs) have emerged as major paracrine effectors mediating the benefits of stem cell therapy. Modest clinical outcomes of autologous cell-based therapies suggest diabetes-induced EPC dysfunction and may also reflect their EV derivatives. Moreover, studies suggest that post-translational histone modifications promote diabetes-induced vascular dysfunctions. Therefore, we tested the hypothesis that diabetic EPC-EVs may lose their post-injury cardiac reparative function by modulating histone modification in endothelial cells (ECs). Methods: We collected EVs from the culture medium of EPCs isolated from non-diabetic (db/+) and diabetic (db/db) mice and examined their effects on recipient ECs and cardiomyocytes in vitro, and their reparative function in permanent ligation of left anterior descending (LAD) coronary artery and ischemia/reperfusion (I/R) myocardial ischemic injuries in vivo. Results: Compared to db/+ EPC-EVs, db/db EPC-EVs promoted EC and cardiomyocyte apoptosis and repressed tube-forming capacity of ECs. In vivo, db/db EPC-EVs depressed cardiac function, reduced capillary density, and increased fibrosis compared to db/+ EPC-EV treatments after MI. Moreover, in the I/R MI model, db/+ EPC-EV-mediated acute cardio-protection was lost with db/db EPC-EVs, and db/db EPC-EVs increased immune cell infiltration, infarct area, and plasma cardiac troponin-I. Mechanistically, histone 3 lysine 9 acetylation (H3K9Ac) was significantly decreased in cardiac ECs treated with db/db EPC-EVs compared to db/+ EPC-EVs. The H3K9Ac chromatin immunoprecipitation sequencing (ChIP-Seq) results further revealed that db/db EPC-EVs reduced H3K9Ac level on angiogenic, cell survival, and proliferative genes in cardiac ECs. We found that the histone deacetylase (HDAC) inhibitor, valproic acid (VPA), partly restored diabetic EPC-EV-impaired H3K9Ac levels, tube formation and viability of ECs, and enhanced cell survival and proliferative genes, Pdgfd and Sox12, expression. Moreover, we observed that VPA treatment improved db/db EPC-mediated post-MI cardiac repair and functions. Conclusions: Our findings unravel that diabetes impairs EPC-EV reparative function in the ischemic heart, at least partially, through HDACs-mediated H3K9Ac downregulation leading to transcriptional suppression of angiogenic, proliferative and cell survival genes in recipient cardiac ECs. Thus, HDAC inhibitors may potentially be used to restore the function of diabetic EPC and other stem cells for autologous cell therapy applications.


Chromatin immunoprecipitation sequencing (ChIP-seq)
MCECs were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine after db/+ or db/db EPC-EV treatment for 24 hours and then subjected to ChIP-seq (Active Motif, Inc.) In brief, chromatin was isolated by adding lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated, and the DNA sheared to an average length of 300-500 bp with Active Motif's EpiShear probe sonicator (53051) and cooled sonication platform (53080). Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K, and heat for de-crosslinking, followed by SPRl beads clean up. Clariostar quantified eluted DNA. Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield. An aliquot of chromatin (30 mg) was precleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using 5 ul antibody against H3K9Ac (Active Motif cat# 39917).
Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65°C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation.
Illumina sequencing libraries were prepared from the ChIP and Input DNAs using the standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation using Active Motif's custom liquid handling robotics pipeline. After the final 18 cycle PCR amplification step, the resulting DNA libraries were quantified and sequenced on Illumina NexSeq 500. Sequences (75 bp, single-end) were aligned to the mouse genome (mm10) using the BWA algorithm (default settings). Duplicate reads were removed, and only uniquely mapped reads (mapping quality ≥ 25) were used for further analysis. Alignments were extended in silico at their 3'-ends to a length of 200 bp, which is the average genomic fragment length in the size-selected library, and assigned to 32-nt bins along the genome.
The resulting histograms (genomic "signal maps") were stored in BigWig files. Peaks were identified using the MACS 2.1.0 algorithm at a cutoff of p-value 1e-7, without control file, and with the -nomodel option. Peaks that were on the ENCODE blacklist of known false ChIP-Seq peaks were removed. Signal maps and peak locations were used as input data to Active Motifs proprietary analysis program, which creates Excel tables containing detailed information on sample comparison, peak metrics, peak locations, and gene annotations. Figure S1: Physiological parameters and EPC-EVs characterization from diabetic and non-diabetic mice. (A) The bodyweight of 10-week-old mice was measured before EPC isolation. (B) Whole blood was collected without pre-fasting for blood glucose measurement using the OneTouch ULTRA2 glucose meter. (C-D) EV particle number of db/+ and db/db EPCs is in average of 5X10^7 from 3X10^5 number of cells and (C-E) with a diameter of approximately 150 nm measured by Nanosight. (F). Identification of EVs by EV marker proteins Alix and HSP70. Both db/+ and db/db EPC-EVs expressed Alix and HSP70. All Data shown as mean ± SEM. n > 3. **** P<0.0001.

Figure S2: Uptake of PKH26-labled EPC-EVs by MCECs (A) and AC16 cells (B). EPC-
EVs from db/+ mice were resuspended in 1 ml Diluent C and mixed with 1 ml stain solution (1 ml Diluent C + 4 µL PKH26) and incubated for 4 min on ice. Then equal volume of 1% BSA was added to stop the labeling reaction. Then the EVs were diluted with 1xPBS andcollected on a 30% sucrose-D2O solution with ultracentrifugation (100,000g for 1 hour). Following separation on the sucrose gradient, the EVs were washed in 1xPBS and the pelleted EVs were suspended in desired volume of PBS and added to MCECs (A) and AC16 cardiomyocytes (B) cells. After 1.5 hrs, the EV-treated MCECs and AC16 cells were washed with 1xPBS and fixed with 4% PFA for 10 min on ice. Images were acquired using the Niko Eclipse Ti Florescence microscope using 20x objectives. were treated with vehicle, db/+ or db/db EPC-EVs for 24 hours. Cells were collected, lysed, and subjected to Western blotting using HDAC1, HDAC2, and HDAC3 antibodies.
(B) Quantification analysis showed that db/db-EPC-EV trend to increase HDAC1-3 protein but did not achieve statistical significance in protein expression. All Data are shown as mean ± SEM. n=3 for each group. HDAC, histone deacetylases. Vehi., vehicle. Data are presented as bar graphs representing the mean. n=2 for each group. Vehi., vehicle.