Epigenetics and stroke: role of DNA methylation and effect of aging on blood-brain barrier recovery

Incomplete recovery of blood-brain barrier (BBB) function contributes to stroke outcomes. How the BBB recovers after stroke remains largely unknown. Emerging evidence suggests that epigenetic factors play a significant role in regulating post-stroke BBB recovery. This study aimed to evaluate the epigenetic and transcriptional profile of cerebral microvessels after thromboembolic (TE) stroke to define potential causes of limited BBB recovery. RNA-sequencing and reduced representation bisulfite sequencing (RRBS) analyses were performed using microvessels isolated from young (6 months) and old (18 months) mice seven days poststroke compared to age-matched sham controls. DNA methylation profiling of poststroke brain microvessels revealed 11287 differentially methylated regions (DMR) in old and 9818 DMR in young mice, corresponding to annotated genes. These DMR were enriched in genes encoding cell structural proteins (e.g., cell junction, and cell polarity, actin cytoskeleton, extracellular matrix), transporters and channels (e.g., potassium transmembrane transporter, organic anion and inorganic cation transporters, calcium ion transport), and proteins involved in endothelial cell processes (e.g., angiogenesis/vasculogenesis, cell signaling and transcription regulation). Integrated analysis of methylation and RNA sequencing identified changes in cell junctions (occludin), actin remodeling (ezrin) as well as signaling pathways like Rho GTPase (RhoA and Cdc42ep4). Aging as a hub of aberrant methylation affected BBB recovery processes by profound alterations (hypermethylation and repression) in structural protein expression (e.g., claudin-5) as well as activation of a set of genes involved in endothelial to mesenchymal transformation (e.g., Sox17, Snail1), repression of angiogenesis and epigenetic regulation. These findings revealed that DNA methylation plays an important role in regulating BBB repair after stroke, through regulating processes associated with BBB restoration and prevalently with processes enhancing BBB injury.


Introduction
Stroke is a major cause of death and long-term disability, particularly in the elderly (1,2). The average stroke survival time is 6 to 7 years with many patients enduring physical disability and late cognitive impairment (3)(4)(5). Neurological outcomes after stroke depend on numerous factors including age, infarct size and location, genetic factors, as well as the degree of brain repair (6-9). Poststroke brain repair involves many events including reestablishing blood-brain barrier (BBB) structure and function (9)(10)(11)(12).
BBB restoration after stroke is a tightly regulated process involving de novo synthesis of junctional proteins for regenerating barrier integrity, as well as rebuilding other BBB systems (e.g., transport) to institute BBB functionality (11,(13)(14)(15)(16). However, a growing body of clinical and experimental evidence indicates the BBB never fully recovers after stroke and that persistent BBB leakiness exists for days and months (14,(17)(18)(19)(20). Although small barrier leakage may be bene cial short-term through increases in waste product clearance and tissue nutrient supply, it is harmful in in the long-term. Often described as poststroke BBB dysfunction, this condition is characterized by junctional complex alterations, particularly tight junction (TJ) complex instability, induction of uid-phase or nonspeci c pinocytosis and transcytosis, formation of transendothelial channels, endothelial cell membrane disruption and intense neurovascular unit remodeling (activation of pericytes and astrocytes and vascular innervation) (12,17,(21)(22)(23)(24). The consequences are chronic in ammation, secondary neuronal injury, and recurrent stroke (11,(25)(26)(27). Although the potential causes of poststroke BBB leakiness/dysfunction are still largely unknown, incomplete angiogenesis, uncontrolled in ammation, and defects in structural repair are implied. Thus, identifying and characterizing the factors and processes that contribute to incomplete BBB recovery hold a key for successful BBB restoration and improving poststroke recovery.
Recently, epigenetics has emerged as an important contributor to stroke pathogenesis and post-stroke recovery, acting as a higher order regulatory mechanism of tissue repair. De ned as altered gene expression independent of primary changes to the DNA sequence, epigenetics depends upon interactions between environmental factors and the genome (28, 29). Three epigenetic factors are essential for organism function: DNA methylation, post-translational histone modi cations (e.g., acetylation, methylation, phosphorylation, and ubiquitination), and non-coding RNAs.
One of the best understood epigenetic processes is DNA methylation, which involves addition of methyl groups to cytosine residues, speci cally those preceding guanine residues in CpG islands or CpG sites (de ned as 500 bp in size, with a GC content > 55%) (30,31). DNA methyltransferases (DNMTs; including DNMT-1, -2, -3A, -3B, -3L) catalyze the covalent transfer of a methyl group from an S-adenosyl methionine to the cytosine residue (32,33). The DNA methylome is not only maintained by addition of methyl groups, but also through demethylation processes. DNA demethylation can be passive, via dysregulation of DNMT1 and passive incorporation of unmethylated cytosines into the genome, or active, through oxidation or deamination catalyzed by ten-eleven translocations (TETs) and activation-induced deaminase (AID) (27,34,35). The relationship between gene expression and DNA methylation is best understood within the context of gene promoter methylation. Addition of methyl groups (hypermethylation) to CpG islands within gene promoters is associated with transcriptional repression, as it prevents transcription factors and RNA polymerase II from binding to DNA (29,30,36). In contrast, promoter region hypomethylation is associated with transcriptional activation (27,36). Nevertheless gene body methylation is less understood, and may result in transcriptional activation or repression, dependent on context.
Altered DNA methylation patterns are hallmarks of multitude diseases including cerebrovascular disease (37,38). For example, patients with a high risk for stroke occurrence have hypomethylated Long Interspersed Nucleotide Element-1 (LINE-1) repeats, associated with increased circulating vascular cell adhesion molecule-1 (VCAM-1) levels (39,40). Similarly, hypomethylation of TNF receptor-associated factor 3 (TRAF3), hypermethylation of thrombospondin-1 (THBS1), and increased DNA methyltransferase 3A (DNMT3A) activity are indicated as predictors of stroke outcome (41)(42)(43). Regarding BBB and stroke, DNA methylation is implicated in BBB permeability regulation. Hypermethylation of the tissue inhibitor of metalloproteinase 2 (TIMP2) promoter decreases TIMP2 expression/activity, diminishing the balance with metalloproteinase 2 and 9 and causing TJ protein and extracellular matrix degradation (44). Such limited ndings suggest a role of DNA methylation in stroke and regulating poststroke BBB recovery. However, the link between DNA methylation and post-stroke BBB functional and structural integrity is still largely unknown.
The present study addresses the transcriptional and methylome landscapes of poststroke BBB recovery that could de ne the plasticity and capacity for BBB restoration after stroke. As aging has critical roles in stroke occurrence, outcomes and recovery, this study examines the methylome and transcriptome signatures of poststroke BBB recovery in young and old mice, highlighting age-related processes and factors that could affect capacity for BBB restoration.

Animals
All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Michigan. Experiments used male C57BL/6 mice from the Jackson Laboratory (10-12 weeks of age) and aging C57BL/6 mice (18-20 months) from the National Institute of Aging colony.
During the surgery, core body temperature was maintained at 37 ± 0.5°C. Sham-operated mice underwent all procedures except embolization.

Neurological Score
Stroke-related neurological de cits were scored as described by Yamamoto et al., (47) on days one and ve post-surgery. Brie y, mice were scored on two parameters: body symmetry and forelimb symmetry. The grading was as follows: 0 = no de cit (no body twisting), 1 = mild de cit (asymmetric twisting tendency of the body), and 2 = severe de cit (consistently twisting). Limb motor function was graded: 0 = no de cit (no exion of forelimbs), 1 = mild de cit (intermittent asymmetrical exion of forelimbs), and 2 = severe de cit (forelimb exion was consistent).
Magnetic Resonance Imaging (Mri) Analysis MRI was performed on a 7.0T Agilent MR scanner (horizontal bore, Agilent, Palo Alto, CA, USA). Axial T2weighted images were acquired using a fast spin-echo sequence with the following parameters: repetition Microvessel Isolation: Brain microvessels were isolated from the ischemic hemispheres of young and old C57BL/6 mice at 7 days post-TE stroke, as well as corresponding hemispheres in sham operated mice using an established protocol (17,52). Brie y, brain tissue was minced in Dulbecco's Phosphate Buffered Saline and homogenized gently in a Dounce glass homogenizer. Myelin was removed by centrifugation in a 15% Dextran solution (Dextran MW ∼70,000, Sigma Aldrich, St. Louis, MO, USA). Isolated brain microvessels were further processed for paired-end mRNA-sequencing and reduced representation bisul te sequencing for transcriptome and DNA methylome analyses, respectively.

Global Methylation Assay
Global DNA methylation of genomic DNA from brain microvessels was measured using the Global DNA Methylation LINE-1 Kit (Diagenode Inc. Denville, NJ). Assay and data analysis were conducted according to the manufacturer's protocol, with analysis measuring the percent of 5-methylcytosines (% 5-mC) based RRBS data processing was completed by Diagenode. Brie y, Bismark was used to align reads to a murine reference genome (mm10/GRCm38). Bisul te conversion rates and e ciency were veri ed through the use of spike-in control sequences. Differential methylation analysis was conducted using Methylkit, an R/Bioconductor package. Pairwise comparisons were conducted to identify differentially methylated regions (DMRs), consisting of 1000bp regions. To correct for multiple comparisons, p values were converted to q-values using the sliding window model (SLIM). Pairwise comparisons were conducted for the following comparisons: old stroke vs. old control and young stroke vs. young control. Signi cant DMRs are de ned as having a methylation difference greater than 25% and q-value < 0.01. For post-data processing, all data analysis was conducted in R. Gene name, genomic region, and location in relation to CpG islands were identi ed for each DMR using annotator, an R/Bioconductor package. DMRs were classi ed by their genomic location, resulting in promoter DMRs and non-promoter DMRs, comprised of gene body DMRs and other DMRs. Gene body was de ned as the 5'UTRs, 3'UTRs, introns, and exons, while other was de ned as 1-5kb upstream of transcription start sites or a lack of genomic location information. The following R packages were used for data visualization: ggplot2, VennDiagram and DOSE. The R/Bioconductor package clusterPro ler was used to conduct gene over-representation analysis with signi cant DMRs to identify signi cant gene ontology (GO) terms. The Benjamini-Hochberg procedure was used for p value correction for GO analysis. Pearson correlation was conducted to determine whether the percent change in methylation for common DMRs was signi cantly correlated across experimental groups.

RNA Sequencing
Total RNA was extracted from brain microvessels using TRIzol/chloroform, with concentration, purity, and integrity measured using agarose gel electrophoresis and an Agilent Bioanalyzer RNA 6000 Kit. RNA samples with RNA integrity number (RIN) score ≥ 5.8 were used to prepare libraries. RNA-seq library construction was generated using the Illumina NovaSeq 600 platform. Total RNA was depleted of rRNA, followed by enzymatic fragmentation, cDNA synthesis, and double-stranded cDNA puri cation. Each experimental group for RNA-seq consisted of three biological replicates.
Alignment options followed ENCODE standards for RNA-seq (53). To ensure usage of high-quality data for expression quantitation and differential expression, FastQC was used in an additional post-alignment step. After obtaining count estimates, all data analysis was conducted in R. Differentially expressed genes (DEGs) were identi ed using DESeq2, de ning cutoffs as adjusted p < 0.05 and absolute value of log2 fold change (Log2FC) > 0.58. Log2FC shrinkage of the RNA-seq data was conducted using the Ashr method. Data was visualized using the R packages ggplot2, VennDiagram and DOSE. Enriched GO terms were identi ed by gene over-representation analysis with signi cant DEGs in the R/Bioconductor package clusterPro ler. For gene over-representation analysis, p values were adjusted with the Benjamini-Hochberg procedure. Pearson correlation was conducted to determine whether the Log2FC for common DEGs was signi cantly correlated across experimental groups.

Statistics
Additional statistical analyses were conducted in GraphPad Prism. Unpaired, two-tailed Student's t-test was used to determine differences across experimental groups. The relationships between transcriptome and methylome pro le in young and aging poststroke mice as well between young and aging methylome pro le were evaluated using the Pearson correlation coe cient. Data are represented as mean ± SEM, with statistical signi cance de ned as p < 0.05.

Results
Persistent BBB dysfunction with increased paracellular barrier permeability is present in chronic phases after stroke, questioning the recovery of the BBB (Fig. 1). To investigate the molecular mechanisms contributing to BBB recovery and detect potential causes of the limited BBB recovery, we pro led the DNA methylome and transcriptome of isolated microvessels from ischemic hemispheres 7 days after the induction of thromboembolic (TE) stroke via injection of thromboembolic suspension in two experimental groups: young mice aged 6 months and old mice aged 18 months. A diagram of the experimental ow is shown in Fig. 1.
DNA methylome pro le of the poststroke BBB recovery Global DNA methylation, determined by the percentage of 5-mC content, was not signi cantly altered following TE stroke in 6-month-old mice, although the TE stroke group showed slight hypomethylation comparing with sham control (control) group (Fig. 2a). As global methylation only captures large-scale changes to the DNA methylome, CpG-level changes were investigated through reduced representation bisul te sequencing (RRBS), comparing the DNA methylome of brain microvessels from post-TE stroke mice to age-matched controls. During poststroke BBB recovery, we detected 9,818 differentially methylated regions (DMRs, 1000 bp in size) with a percent change in methylation greater than 25% and a q-value < 0.01, with 4,638 hypermethylated and 5,180 hypomethylated DMRs (Fig. 2b). DMRs were primarily intronic (48.2%) and exonic (18.7%), while only 8.9% of DMRs were located in promoter regions (Fig. 2c). Strikingly, less than 1% of DMRs resided within CpG islands (Fig. 2c).
Categories of differentially methylated genes in post-stroke BBB recovery were identi ed through gene over-representation analysis. Overall, post-TE stroke changes to the DNA methylome largely affected genes encoding cell structural proteins (e.g., cell junction, and cell polarity, actin cytoskeleton, extracellular matrix, membrane microdomain), transporters and channels (e.g., potassium, organic anion and inorganic cation, calcium ion transport), and proteins involved in endothelial cell processes (e.g., angiogenesis and vasculogenesis, cell signaling and transcription regulation) (Fig. 2d, 2e).
Due to their well-de ned function in the context of transcription, DMRs within gene promoters and nonpromoter regions were individually assessed, with non-promoter regions including the 5' and 3' untranslated regions (UTRs), introns, exons, and 1-5kb upstream of transcription start sites. Out of the 1,020 DMRs in gene promoters, 474 DMRs were hypermethylated and 546 DMRs were hypomethylated.
Prominent categories of differentially methylated genes in post-stroke BBB recovery were cell junction, polarity, and actin cytoskeleton. Signi cant cell junction GO terms included tight junction (q-value = 0.0025) and adherens junction organization (q-value = 0.00012). Notable differentially methylated genes with prevalent hypermethylation included junctional adhesion molecule-A (F11r), while prevalent hypomethylation occurred in occludin (Ocln) and claudin-5 (Cldn5) (Fig. 3c). Establishment or maintenance of cell polarity, a process required for proper localization of TJ complexes, was signi cant (q-value = 0.0025), with differential methylation observed in the Wnt signaling ligand genes, Wnt5a (hypomethylated mostly in promotor region) and Wnt7b (hypermethylated in gene body), the ARF6regulating genes Cyth3, (hypomethylated in promotor region) and Frmd4a (hypermethylated in promoter and gene body), the endothelial polarity regulator Amotl2, as well as a regulator for establishing cell polarity Prkcz, (hypermethylation in promoter and gene body). Among the signi cant methylation changes in genes encoding actin cytoskeleton included those involved in actin lament organization (qvalue = 0.0058) and positive regulation of cell projection organization (q-value = 2.85E-08), particularly within the actin-interacting genes Ezr and Myo3b (Fig. 3c).
In relation to poststroke BBB recovery, there were changes in a spectrum of genes associated with angiogenesis (q-value = 7.29E-06), endothelium development (q-value = 0.0084), regulation of endothelial cell migration (q-value = 0.0086) and endothelial cell differentiation (q-value = 0.0091). Signi cant differential methylation was present on the promoter and gene body of endothelial transcription factor genes include Sox17, Gata2 and Gata3 (all hypomethylated) while Robo4, involved in angiogenesis and endothelial barrier maintenance, had hypomethylated promoter DMRs. Strikingly, hypermethylation of the angiogenic inhibitor Angptl4 was present but not Hmgb2, a promoter of endothelial cell proliferation and migration (Fig. 3d).
Altered methylation patterns were also found in genes encoding proteins involved in endothelial cell processes, such as intracellular signaling cascades and transcription (Fig. 2e). Examples include canonical Wnt signaling pathway (q-value = 8.74E-06), with differential methylation of the inhibitor Gsk3b but not the Wnt ligand receptor Fzd4, as well as Ras protein signal transduction (q-value = 1.74E-05), with differentially methylated (hypomethylated) genes including the GTPase Cdc42ep4 and Rap2a, in both promotor and gene body DMRs. Other notable signaling cascade genes with hypomethylation include Mapk4, which regulates proin ammtory cytokines expression. Regarding transcriptional regulation, the DNA-binding transcription activator activity (q-value = 0.0018) and transcription regulator complex (qvalue = 0.005) genes, such as Foxo3 and Grhl2, were hypomethylated predominately in their gene bodies (Fig. 3e).
Transporter, receptor, and channel genes also had striking changes in methylation. For example, differentially methylated genes involved in passive transmembrane transporter activity (q-value = 0.00028) included the ion channel-encoding genes Glra1, Kcnt2 and Scn3a. Lrp1b and Lrp5, low-density lipoprotein receptor family members, were hypomethylated predominantly in their gene bodies (Fig. 3f).
Effect of methylome changes on the transcriptome pro le of the poststroke BBB recovery The methylomic changes are best understood within the context of transcription, manifested as transcriptional repression due to promoter region methylation, while gene body methylation can lead to either transcriptional activation or repression (29,30). Thus, we performed a parallel analysis of transcriptomic changes in brain microvessels during post-stroke BBB recovery in young (6-months old) mice. Compared to age-matched controls, post-stroke BBB recovery resulted in 2,740 differentially expressed genes (DEGs), de ned as log2 fold change > 0.58 and an adjusted p-value < 0.05, with 1,546 upregulated and 1,194 downregulated (Fig. 4a). Gene ontology analysis revealed upregulated DEGs were involved in cell junction organization, such as cell-substrate junction (q-value = 0.001), establishment or maintenance of cell polarity (q-value = 0.009), ATP metabolic process (q-value = 5.66E-07), mitochondrial transport (q-value = 0.0010) and signaling cascades, such as ERK1 and ERK2 cascade (q-value = 0.0026) and small GTPase mediated signal transduction (q-value = 0.0028). Other enriched GO terms for upregulated DEGs included angiogenesis (q-value = 9.10E-06) and regulation of DNA-binding transcription factor activity (q-value = 0.0091). Downregulated DEGs demonstrated an enrichment of GO terms related to cell junctions, such as cell junction assembly (q-value = 0.0011) and cell-cell adhesion via plasma membrane adhesion molecules (q-value = 0.0012), ion channel activity (q-value = 1.63E-08), passive transmembrane transporter activity (q-value = 3.84E-08), and signaling pathways, such as G protein-coupled receptor activity (q-value = 1.62E-10) (Fig. 4b).
As promoter and gene body methylation directly affect the transcriptome, we assessed the overlap between changes to the DNA methylome and the transcriptome. Only 94 DEGs had altered promoter methylation, while 588 DEGs had differential methylation of non-promoter regions (Fig. 4c). However, the correlation between promoter methylation and gene expression demonstrated a non-signi cant trend toward a negative correlation (R = -0.045, p = 0.67). Despite that, there are groups of genes with signi cant promoter hypomethylation (q-value) and transcript upregulation (p.adjust), like the RhoGTPases, Cdc42ep4 (p.adjust = 0.0004, q-value = 7.94E-09) and Rhoa (p.adjust = 0.0236, q-value = 4.28E-12), focal adhesion molecule Tns2 (p.adjust = 0.00255, q-value = 3.74E-20), the regulator of angiogenesis and endothelial barrier establishment Robo4 (p.adjust = 0.0051, q-value = 3.15E-09), integrin β5 (Itgb5, p.adjust = 0.032, q-value = 0.00014), extracellular matrix protein bronectin (Fn1, p.adjust = 0.032, q-value = 3.34E-29) and the angiogenic factors thrombospondin 4 (Thbs4, p.adjust = 0.04, q-value = 2.14E-17) and Alkbh5 (p.adjust = 6.56E-05, q-value = 1.66E-13). Promoter hypermethylation and transcript downregulation was observed in the regulator of VEGF-induced angiogenesis and glycine- Effect of aging on the BBB DNA methylome and transcriptome pro le in poststroke recovery Aging plays a critical role in the epigenetic alteration of the brain endothelial cells function and consequently on barrier properties (55). Aged mice (18 months) had larger infarct sizes with profound BBB leakiness 7 days after TE stroke onset (Fig. 1). Analyzing DNA methylome pro le in post-stroke BBB recovery in aged mice, we found no changes in the global methylation level (Fig. 5a). However, RRBS analysis revealed that aging post-TE stroke brain microvessels had 11,287 DMRs, 5,005 hypermethylated and 6,282 hypomethylated, compared to age matched controls (Fig. 5b). The genomic regions containing the highest percentages of DMRs were introns, exons, and 1-5 kb upstream of the transcriptional start site, respectively while only 8.9% of DMRs were located within promoter regions (4.0% hypermethylated and 4.9% hypomethylated). When investigating DMR location in relationship to CpG islands, most (86.2%) were located within the open sea, while only 1.1% of DMRs were located within CpG islands ( Supplementary Fig. 1a, b).
Similar to poststroke BBB recovery in young mice, poststroke BBB in aging mice had gene enrichment in clusters of cell junctions, actin cytoskeleton, angiogenesis, signaling pathways and transcription factors, transporters, and channels, as well as the extracellular matrix (Fig. 5c, 5d). The promoter regions showed 1,154 DMRs (521 hypermethylated and 633 hypomethylated) (Fig. 6a), while non-promoter regions had 10,133 DMRs, 4,484 being hypermethylated and 5,649 being hypomethylated (Fig. 6b). The signi cant methylation pattern in promoter and non-promoter regions were present in the tight junction cluster (qvalue = 0.0015), with hypermethylation of genes that encode Cldn5, Tjp2, and Ocln and hypomethylation of F11r, actin cytoskeleton (e.g. regulation of actin lament organization, q-value = 0.0004; cell projection assembly, q-value = 0.0005; and actomyosin structure organization, q-value = 0.0031) with notable hypomethylation of actin cytoskeleton-related genes that encode Ezr, and lament-associated protein Cnn3. Two genes that regulate the BBB recovery and maintenance, Wnt5b and Wnt7a, showed hypermethylation and hypomethylation, respectively, predominantly in gene body ( Fig. 5c and 6c).
The altered methylation pattern was analyzed in the context of transcriptomic changes. RNA-seq revealed that poststroke BBB recovery in old mice have global transcriptomic changes compared to age-matched controls: out of the 6,999 total DEGs, 3,723 were upregulated, and 3,276 were downregulated (Fig. 7a).
In summary, the transcriptional and methylome pro le of poststroke BBB recovery in aging mice implies that angiogenesis and Wnt-related pathways drive the BBB recovery process, although the repair process is affected by the activation of genes involved in endothelial to mesenchymal transformation and angiogenic repression, limiting the nal outcomes.
Common and unique transcriptomic and DNA methylome pro les of BBB poststroke recovery in young and aging mice As the severity of poststroke BBB injury differs between young and old mice, we compared the DNA methylome and transcriptome changes to highlight cellular processes contributing to the discrepancy in poststroke BBB recovery. Remarkably, there are only 1,138 signi cant DMRs common to poststroke BBB recovery in both old and young mice (Fig. 8a). This lack of overlap is still observed when assessing methylation within speci c genomic regions, as there are 112 common DMRs within promoters and 1,026 DMRs within non-promoter regions ( Supplementary Fig. 2a, 2b). Common DMRs identi ed in poststroke BBB recovery of old and young mice do not signi cantly correlate (R = 0.0038, p = 0.134), which is also true when assessing the correlation of common non-promoter DMRs (R = 0.0018, p = 0.5227) (Fig. 8a,  Supplementary Fig. 2d). A signi cant correlation, however, is observed for the common promoter region DMRs (R = 0.0188, p-value = 0.00055) (Supplementary Fig. 2c). Common DMRs with methylation changes in the same direction (e.g. hypermethylated in both groups) belong to signaling pathways (e.g., positive regulation of MAPK cascade, q-value = 0.03; GTPase regulator activity, q-value = 0.015; and G proteincoupled receptor activity, q-value = 0.041) (Supplementary Fig. 2e). DMRs with methylation changes in opposite directions (e.g. hypermethylated in old and hypomethylated in young) are located within genes involved in endocytosis (e.g. clathrin-coated endocytic vesicle, q-value = 0.0187; endocytic vesicle, q-value = 0.039) and b-catenin binding (q-value = 0.038) (Supplementary Fig. 2f). Comparing the transcriptomic changes in BBB recovery across the young and old mice reveals 2,042 signi cant DEGs common to both experimental groups (Fig. 8b). The common DEGs between old and young poststroke BBB recovery have a strong positive correlation (R = 0.704, p < 2.2E-16), with only 8 DEGs regulated in the opposite direction (e.g., upregulated in old post-TE stroke mice and downregulated in young post-TE stroke mice) (Fig. 8b).
Furthermore, we identi ed genes of interest with similar changes in methylation (e.g., hypermethylated or hypomethylated in both experimental groups) for both young and old mice. These genes encode proteins involved in actin-binding signaling pathway activity, transcriptional regulation, and protection from oxidative stress. For example, actin-binding proteins Parva and Ezrin, and the Ets family transcription factor Erg were hypomethylated with increased transcript expression in the poststroke microvessels of both young and old mice. Other examples included the activator of MAPK signaling Alk (hypermethylated and decreased transcript expression) and the negative regulator of MAPK signaling Dusp3 (hypomethylated and increased transcript expression), indicating the same trend in the regulation of MAPK kinase in BBB recovery (Fig. 8c). Genes with differential methylation patterns (e.g. hypermethylated in old and hypomethylated in young) across the BBB of old and young poststroke mice include the signaling molecule Prkce, involved in actin cytoskeleton function (migration, adhesion) and actin cytoskeleton modulator Arf6 (both genes hypomethylated in old mice and hypermethylated in young mice), while Cdc42ep4 was hypermethylated in old and hypomethylated in the young post-stroke BBB. Conversely, increased transcript expression of Arf6 and Cdc42ep4 was observed in old and young post-stroke BBB (Fig. 8c).
Finally, there is a unique DNA methylome and transcriptome pro le for both experimental groups. These genes belong to categories relevant to endothelial cell biology, such as cell junctions, angiogenesis, and signaling pathways. The unique pattern for poststroke BBB recovery in young mice was characterized by alterations in methylation and transcriptome expression of cell junction and polarity complex regulators such as Ocln, Epha2, Tns2, and Cyth3; the angiogenic transcriptional regulators Sox14, Foxo3, Klf4, and Gata2 (Fig. 8d). Other unique patterns are observed in regulators of MAP kinase (e.g., Mapk4), transporters (e.g., Glr2), the extracellular matrix (e.g., Spock3), and in ammation (e.g., Il6) (Supplemental Fig. 3a).
The effects of aging on BBB recovery results in a unique transcriptome and DNA methylome pro le that includes the cell junction and polarity complex-encoding genes Cldn5, Cldn11, Tjp2 and Dlg5; the angiogenic transcription factors Ets2, Hes1, and Foxo1; the Wnt signaling pathway genes Fzd9, Wnt7a, and Wnt7b; the Rho kinase pathway gene Cdc42ep3; the integrins Itgb2, Itgb4 and Itgb1; and the transporters Lrp10 and Atp1a2. The intriguing pro le of aging poststroke BBB recovery is also characterized by a profound alteration in methylation and gene expression of regulators of endothelial to mesenchymal transformation (Snai1, Smad6, Tgfbr2, and Sox9) and epigenetic regulators (Sirt2 and Kdm6a) (Fig. 8e and Supplemental Fig. 3b).
Thus, DNA methylation has an important role in BBB recovery, directing some of the critical processes involved in restoring the structural and functional BBB, such as angiogenesis, junctional proteins, establishment of polarity, actin cytoskeleton reorganization, extracellular matrix, as well as transporter system reestablishment. Nevertheless, DNA methylation could also contribute to the limited BBB recovery in young mice, mostly through the activation of speci c signaling pathways (e.g., Rho GTPases), while in aging mice the limited BBB recovery could be due to the repression of structural protein expression (e.g., claudin-5), as well as activation of genes involved in endothelial to mesenchymal transformation, repression of angiogenesis, and epigenetic regulation.

Discussion
Poststroke BBB recovery has gained more attention recently due to accumulating evidence linking poststroke BBB "status" with stroke outcomes (17,19,21,23). Prolonged poststroke BBB leakage can increase the risk for stroke recurrence, limit stroke recovery, and lead to poststroke complications including cognitive decline (3)(4)(5). As the BBB possesses some capacity for recovery, it is critical to understand why the restoration process is limited and the role different pathways have in the recovery process. This study addresses two important questions regarding BBB poststroke recovery: a) what is the pro le of brain endothelial cells and the mechanisms involved in barrier recovery, and b) how do epigenetic modi cations induced by ischemic stroke direct the process of recovery? Our results highlighted that: a) DNA methylation contributes to the control of transcript expression associated with BBB recovery in brain endothelial cells, b) DNA methylation predominantly regulates genes that encode structural proteins (tight and adherens junction protein, actin cytoskeleton, cell polarity), extracellular matrix, transporter systems, angiogenesis (e.g., angiogenic transcription factors) and signaling pathways (Wnt/b-catenin and Rho GTPase), c) aging/senescence plays a prominent role in altering DNA methylation and the transcriptomic pro le in poststroke BBB recovery, d) the existence of the limited poststroke BBB recovery and prolonged BBB leakage is due in part to the aberrant methylation and increased expression and potential activation of Rho GTPase, as well as shifting the pro le of angiogenesis from physiological to pathological, and e) aging is a profound factor of poststroke BBB injury/leakage triggering aberrant methylation and upregulation of genes encoding endothelial to mesenchymal transformation as well angiogenic repressors, restricting full BBB recovery. These ndings are discussed below.
Ischemic stroke led to dynamic changes in DNA methylation, regulating widespread differential gene expression and modifying processes involved in injury and recovery (37)(38)(39). The regulatory potential of DNA methylation is shaped by two opposing processes, the addition and removal of a methyl group at position ve of cytosine in DNA (29,30,34,36). This results in transcriptional repression by preventing RNA polymerases from recognizing speci c DNA regions or transcription upregulation by removing the "DNA methylation breaks", respectively. The effect of DNA methylation on transcript expression is dependent on their location within the gene regions (promoter and/or gene bodies), with transcriptional changes typically dependent on methylation of multiple sites within a given gene (29,30). Global alterations in DNA methylation have been reported in stroke in several experimental and clinical studies, highlighting speci c gene dynamics. For example, patients with a high risk for ischemic heart disease and stroke have hypomethylated Long Interspersed Nucleotide Element-1 (LINE-1) repeats, associated with increased circulating vascular cell adhesion molecule-1 (VCAM-1) levels (39). Similarly, hypomethylation of TNF receptor-associated factor 3 (TRAF3), hypermethylation of thrombospondin-1 (THBS1), and increased DNMT3A activity are indicated as predictors of stroke outcome, as well degree of ischemic injury (38, 41). So far, within the BBB, it has been reported that TIMP2 promoter hypermethylation or VCAM-1 promoter hypomethylation control BBB permeability and leukocyte recruitment (37,40). Brain and BBB recovery poststroke are associated with speci c gene expression that controls the ongoing process of repair. DNA methylation appears to have important role, as the pattern of methylation changes at the global level. More important, however, are speci c alterations in methylation patterns of genes encoding proteins directly involved in processes for re-establishing the barrier function, as these changes in methylation could repress (hypermethylation) or activate (hypomethylation) transcription.
In poststroke BBB recovery, angiogenesis is a pivotal process (12,24,56). Starting 3-4 days after ischemic insult, intense remodeling of the vascular network occurs, involving brain endothelial cell proliferation and vessel sprouting that increases perfusion, predominantly in the peri-infarct area (12,57).
Angiogenesis occurs over days and weeks and, depending on the degree, is positively correlated with stroke survival (12,58). Our results revealed DNA methylation as an apparent modi er of angiogenesis by altering transcription and upregulating expression of different angiogenesis regulators. That includes the insulin-like growth factor 2 (IGF2BP2)/Ccnd1 axis important for the cell cycle and proliferation, and the transcription factors Sox17, which controls endothelial cell differentiation, and Erg, a regulator of VEGF angiogenic signaling pathways, along with the VEGF receptor Flt1 (59-62). Furthermore, altered DNA methylation regulates extracellular matrix remodeling, forming a more proangiogenic matrix by upregulating bronectin (Fn1) and thrombospondin-4 (Thbs4) expression, and increasing expression of integrin β5, which is responsible for the cell-matrix interaction (63, 64). As the balance between proangiogenic and anti-angiogenic factors is critical for the formation of mature vessels, hypomethylation of angiogenic repressors like Notch4 and its consequent upregulation is critical in part for limiting cell proliferation and vessel sprouting (65). The next important step where DNA methylation contributes is blood vessel maturation, speci cally in brain endothelial barrier formation. Promoter and gene body hypomethylation and subsequent transcript upregulation is seen in the group of genes regulating cell junction protein expression, such as Roundabout homolog 4 (Robo4) and the transcription factor SRY-Box 17 (Sox17), the structural tight junction protein and regulator of TJ complex stability occludin (Ocln), the actin cytoskeleton protein and stabilizer of TJ complex ezrin (Ezr), and the regulator of brain endothelial cell polarity cytohesin-3 (Cyth3), which controls Golgi complex structure and recruitment of junctional proteins (60, 66-68). These data pinpoint that DNA methylation directly or indirectly (e.g. forming proangiogenic conditions) could regulate the plasticity and capacity of brain endothelial cells for vascular remodeling and barrier recovery.
A remaining question, however, is why BBB recovery is limited when the transcriptional and methylome pro les indicate activation of bene cial restoration processes. There are several potential causes of limited BBB recovery. The methylome and transcriptome pro le showed that besides VEGF-mediated angiogenesis, there is also VEGF-independent angiogenesis. This is re ected by signi cant promoter hypomethylation and increased transcript expression of m6A demethylase alkB homolog 5 (Alkbh5) and thrombospondin-4 (TSP-4, Thb4), which acts as an accelerator of angiogenesis in response to TGF-β1, and contributes to cell-and disease-stage-speci c effects of TGF-β (63, 69). Shifting the angiogenic process to a more pathological one through VEGF-independent angiogenesis might affect the full differentiation of endothelial cells, and particularly barrier maturation. In addition, the poststroke BBB methylome pro le revealed promoter hypermethylation of glycine receptor 2 (Glra1) and repression of its expression. Repressing Glra1 expression, an essential regulator of neurovascular/cerebrovascular remodeling and protector against post-ischemic injury, could potentiate injury processes at the BBB (70).
Furthermore, hypomethylation within the exons of the ephrin type-A receptor (Epha2) is reported to mediate brain endothelial cell TJ disruption in in ammatory conditions (71). Other unfavorable factors for BBB recovery are the activation of the Rho GTPase RhoA and CDC42 effector protein 4 (Cdc42ep4), both indicated in regulating actin remodeling, stress ber formation, and TJ complex disassembly, as well as atypical Map kinase Mapk4, the overexpression of which shifts cells to a proin ammatory phenotype (72)(73)(74)(75). Presumably, the balance, or lack thereof, between favorable and unfavorable processes/factors may predict the nal outcomes of BBB poststroke recovery.
One important factor that predicts stroke and BBB recovery is aging. Aging causes a time-dependent decline in BBB properties, setting a variety of cellular functions at new levels. The epigenetic alterations largely re ect the aging-associated deleterious events. There is a loss of DNA methylation during aging and, with the advancement in age, genes such as estrogen receptor, insulin-like growth factor 2 (IGF2), and p16 are hypermethylated, with this abnormal DNA methylation leading to a heritable change (76).
Thus, the aged BBB could have its own pro le of BBB recovery that re ects greater poststroke BBB leakage. Our data pinpoints two important ndings. First, aberrant DNA methylation regulates some of the same transcripts as in young mice, like the upregulation of structural proteins ezrin (Ezr), actin related protein Cnn3, regulators of VEGF-associated angiogenesis (receptors Kdr and Flt1), the transcriptional factor Erg, as well as the Ccnd1/Igfbp3 axis, which regulates the cell cycle. There is also hypomethylated/upregulated/"activated" signaling pathways involved in re-establishment of the BBB, like the Wnt-beta-catenin signaling components, Wnt7b and Fzd9 (77)(78)(79). Furthermore, the aging BBB has similar limiting factors of the BBB recovery, such as the Rho GTPase Rhoa and Cdc42ep1 overexpression during the poststroke recovery. However, the aging poststroke BBB DNA methylome and transcriptome pro le is characterized by "unique" aberrant methylation in three categories: angiogenesis, structural proteins, and endothelial to mesenchymal transformation (EndMT). Although aging brain endothelial cells still possess angiogenic capacity, aging/senescence led to signi cant hypomethylation and upregulation of the angiogenic repressor transcriptional factor Foxo1 and repression of two angiogenic factor transcription factors, Klf8 and Epha6 (80-82). This could potentially induce more of an imbalance between pro-and anti-angiogenic factors and limit cerebrovascular network restoration. Recovery of the restrictive features of BBB is also disturbed, as aging brain endothelial cells have hypermethylation of the key TJ occlusion protein claudin-5, causing transcript downregulation. This nding is in agreement with some recent reports of decreased claudin-5 protein expression in aging mice (83). Finally, the most profound changes are in the activation of EndMT. Both chronic in ammation and aging/senescence can transform brain endothelial cells towards a mesenchymal phenotype, with a loss of speci c barrier endowed properties as well as functional/metabolic features (84, 85). Aberrant hypomethylation patterns associated with transcriptome upregulation occurred in some key players in EndMT, such as Snai1, Sox9, Tgfb1 and Tgfbr2, that lead to brain endothelial cell dedifferentiation and limit barrier plasticity and capacity for recovery. Thus, enhanced BBB poststroke injury with aging depends in part on epigenetic alterations that could limit BBB recovery.

Study Limitations
Designed to address the high cost and ine ciency of whole-genome bisul te sequencing (WGBS), RRBS enriches for CpG dinucleotide-containing regions through enzymatic DNA digestion, yielding small DNA fragments comprising ~ 1% of the genome (86). While other methylation pro ling methods, such as WGBS and MeDIP-Seq, recognize DMRs with ≥ 2 CpG/100 bp and < 5 CpG/100 bp, respectively, RRBS only recognizes DMRs with ≥ 3 CpG/100 bp (87). Comparatively, RRBS is biased toward CpG islands and promoter regions, with low coverage of repeat elements and enhancers (86-89). While the function of non-promoter methylation is still being studied, current research suggests that methylation of enhancers, insulators, and low-density CpG regions (termed CpG deserts) has functional signi cance, indicating a relationship between their methylation and transcription (30,90). Likewise, aberrant methylation of CpG shores, de ned as 2 kb from CpG islands, has been identi ed as a hallmark of speci c disease states (91). Ultimately, pro ling the poststroke BBB methylome with RRBS focuses our analysis on promoter methylation and neglects methylation changes in low-density CpG regions, which also potentially contribute to transcriptional regulation.

Conclusion
In conclusion, altered DNA methylation patterns are present in BBB poststroke recovery, regulating the processes that restore the BBB property, while parallelly activating and regulating some factors contributing to BBB injury. With the opportunity to assess genome-wide DNA methylation patterns in poststroke recovery in different ages, we have de ned the ongoing processes of BBB recovery (i.e., angiogenesis, barrier maintenance, signaling regulation via Rho GTPase and Wnt/beta catenin signaling, and EndMT) and their epigenetic (methylome) alterations that could limit the recovery process, resulting in profound BBB injury. This study offers new insights into the etiology of BBB and cerebrovascular decline after stroke and opens up new avenues for drug discovery and targeted therapies.

Declarations
The authors declare that the research was conducted in the absence of any commercial or nancial relationships that could be construed as a potential con ict of interest. RFK is a co-editor in chief of Fluids Barriers CNS.
Author Contributions CMP contribute to preparing samples for RBBS analysis, bioinformatic analysis, and manuscript writing.
SMS planned experiments, performed TE stroke, and contributed to manuscript writing. RFK and AVA contributed to planning experiments and manuscript writing.
Availability of Data and Materials Figure 1 Experimental ow chart. Young and old mice underwent thromboembolic (TE) stroke. At one and seven days they underwent T2 MRI and T1 MRI to determine infarct size and BBB permeability (in ux rate constant, Ki) before euthanasia and microvessel isolation, respectively. Those microvessels were used to determine the methylome and transcriptome changes. Graph represents means ± SD, n = 5, **p<0.01, ***p<0.001, ****p<0.0001 structural GO terms and (E) signaling GO terms, with the x-axis demonstrating gene count. GO term were selected by statistical signi cance (q-value < 0.01) and relevance to endothelial cell biology.

Figure 3
Genomic location of DMRs within poststroke BBB recovery in young (6 month) mice. Volcano plot of DMRs located within (A) gene promoters and (B) non-promoter regions, de ned as any DMR outside of a promoter region (e.g. exons, introns, untranslated regions, 1-5kb upstream of transcription start site). The x-axis is the percent methylation difference and the y-axis is the -log10(q-value). Signi cant DMRs (methylation difference > 25% and q-value < 0.01) are red, while DMRs lacking statistical signi cance are black. Violin plots demonstrate differential methylation of genes involved in (C) cell junctions, polarity and actin cytoskeleton, (D) angiogenesis and endothelial genes, (E) signaling pathways and transcription factors, (F)transporters and channels, and (G)cell adhesion and extracellular matrix. All DMRs, regardless of statistical signi cance, are represented for genes relevant to endothelial cell biology, with genes containing signi cant DMRs denoted with an asterisk (*). Gene promoter DMRs are red, and gene body DMRs are blue. Other DMRs, that are located 1-5kb upstream of transcription start site or no genomic location, are gray.