Abstract
Background and aims:
Histone methyltransferases are emerging targets for epigenetic therapy. DOT1L (disruptor of telomeric silencing 1-like) is the only known methylation writer at histone 3 lysine 79 (H3K79). It is little explored for intervention of cardiovascular disease. We investigated the role of DOT1L in neointimal hyperplasia (IH), a basic etiology for occlusive vascular diseases.
Methods and Results:
IH was induced via balloon angioplasty in rat carotid arteries. DOT1L and its catalytic products H3K79me2 and H3K79me3 (immunostaining) increased by 4.69 ±0.34, 2.38 ±0.052, and 3.07 ±0.27 fold, respectively, in injured (versus uninjured) carotid arteries at post-injury day 7. DOT1L silencing via shRNA-lentivirus infusion in injured arteries reduced DOT1L, H3K79me2, and IH at day 14 by 54.5%, 37.1%, and 76.5%, respectively. Moreover, perivascular administration of a DOT1L-selective inhibitor (EPZ5676) reduced H3K79me2, H3K79me3, and IH by 56.1%, 58.6%, and 39.9%, respectively. In addition, DOT1L silencing and its inhibition (with EPZ5676) in vivo in injured arteries boosted smooth muscle α-actin immunostaining; pretreatment of smooth muscle cells with EPZ5676 in vitro reduced pro-proliferative marker proteins, including proliferating cell nuclear antigen (PCNA) and cyclin-D1.
Conclusion:
While DOT1L is upregulated in angioplasty-injured rat carotid arteries, either its genetic silencing or pharmacological inhibition diminishes injury-induced IH. As such, this study presents a strong rationale for extending mechanistic and translational investigation into DOT1L targeting for treatment of (re)stenotic vascular conditions.
Keywords: DOT1L, H3K79 methylation, epigenetic target, in vivo silencing, neointimal hyperplasia
Graphical Abstract
Introduction
Balloon angioplasty is a common procedure to treat occlusive cardiovascular diseases and accounts for over one million cases per year in the US alone1. Unfortunately, ~10%−50% of treated arteries (particularly peripheral) are re-occluded due to lesion formation termed neointimal hyperplasia (IH)2. To retard IH, drug-eluting stents are commonly implanted following angioplasty. However, the FDA recently warned health providers of a potential association of paclitaxel-eluting stents or balloons with increased mortality (2019, FDA website). As such, the flawed clinical method presses an urgent need for discovery of new targets and improved therapeutics3.
Epigenetic modulation emerges as new medicine4 − a potential not well explored for vascular diseases. DOT1L (disruptor of telomeric silencing 1-like) is an epigenetic writer that catalyzes methylation at histone 3 lysine 79 (H3K79)5. Unlike other histone methylation sites clustered on the exposed and unstructured histone tail, H3K79 is partially buried in the histone 3 globular domain and its methylation is structurally dependent5, 6. Erasers and readers specific for H3K79 methylation (if any) remain unidentified7. Moreover, DOT1L is the only identified writer specific for H3K79, and it lacks the SET catalytic domain found in essentially all other histone lysine methyltransferases5. As such, DOT1L is a seeming “maverick”, and interpretation of its behaviors in heath and disease has relatively lagged. Whether DOT1L plays a role in IH was not known.
On the other hand, thanks to the development of inhibitor drugs selective to DOT1L, research on targeting DOT1L for therapy has recently advanced to human tests. DOT1L was found to increase in tumors and linked to poor prognosis7. There is a strong correlation between H3K79 methylation and cancer cell proliferation7, 8. We hypothesized that DOT1L plays a role in IH, a pathology closely associated with proliferative cell states9.
In this study, we detected elevated DOT1L protein and function (typically measured as H3K79 dimethylation)10 in rat and human pathological arteries. Silencing DOT1L diminished IH in the authentic model of rat carotid artery injury. We were also able to reduce IH by pharmacologically inhibiting DOT1L. These results support an important promotive role of DOT1L in IH.
Materials and methods
Animals.
All animal studies conform to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health) and protocols have been approved by the Institutional Animal Care and Use Committee at The Ohio State University (Columbus, Ohio). Male Sprague-Dawley rats purchased from Charles River Laboratories (Wilmington, MA) were used for experiments (at 300–350 g of body weight).
Rat carotid artery balloon angioplasty
The neointimal hyperplasia (IH) model of rat carotid artery balloon angioplasty was performed as we previously described with minor modifications11. Briefly, rats were kept anesthetized (throughout the procedure) with 2–2.5% isoflurane via inhaling at a flow rate of 2 L per min. A neck skin incision was made and the left common carotid artery was dissected out. A 2-F balloon catheter (Edwards Lifesciences, Irvine, CA) was inserted into the common carotid artery through an arteriotomy on the left external carotid artery. The balloon was inflated to a pressure of 1.5 atm, withdrawn to the carotid bifurcation, and then deflated. This action was repeated four times, with the last withdrawal accompanied with balloon rotation (~3/4 round). These procedures cause injury to the artery (overstretch and endothelium denudation) that induces IH. DOT1L inhibition in injured arteries via gene silencing or a small molecule inhibitor was performed immediately following balloon angioplasty as described below. Blood flow was finally resumed and the neck incision was closed. The animal was kept on a 37°C warm pad to recover. For postoperative analgesia, in addition to carprofen and bupivacaine, buprenorphine (0.03 mg/kg) was subcutaneously injected.
Lentiviral vector construction for DOT1L silencing in vivo
To construct a lentiviral vector for the expression of DOT1L-specific shRNAs, the pLKO.1-puro empty vector was purchased from Addgene (Watertown, MA). A scrambled shRNA control and shRNAs specific for the rat DOT1L gene were designed by RNAi Central (http://cancan.cshl.edu/RNAi_central/step2.cgi). Three most efficient sequences are listed in Supplementary Table 1. The corresponding shRNA-expressing lentivectors were constructed by using the pLKO.1-puro vector as a template. Lentiviruses were packaged in Lenti-X 293T cells (cat#632180, Clontech, Mountain View, CA) using a three-plasmid expression system (pLKO.1-shRNAs-puro, psPAX2 and pMD2.G) as described in our recent reports11, 12, and used in combination (1:1:1) for carotid artery luminal infusion.
DOT1L silencing in vivo via shRNA expression in balloon injured rat carotid arteries
In vivo DOT1L silencing was performed via luminal infusion of lentiviruses (into the denuded artery wall) to express DOT1L-specific shRNAs or a scrambled shRNA. We followed the infusion procedures described in our recent report13. Briefly, immediately following balloon angioplasty, a BD Insyte Autoguard IV catheter (24GA, BD, Franklin Lakes, NJ) was inserted into the injured segment of common carotid artery and suture-ligated together with the external carotid artery to prevent liquid leaking. The catheter was stabilized using a flexible magnetic mount arm platform (Quadhands, Charleston, SC). The lentivirus (>2.0 × 105 IFU/ml) was injected with a syringe into the common carotid artery through the catheter, and the luminal infusion lasted for 25 min. The common carotid artery lumen was first flushed by temporally unclamping the proximal common carotid artery and internal carotid artery and then flushed with a 20 USP/ml heparin saline solution. The external carotid artery was permanently ligated and blood flow in both the common and internal carotid arteries was resumed. The surgery was finished as described above. To prevent thrombosis possibly caused by luminal infusion, 50 USP of heparin was subcutaneously administered prior to neck skin incision.
Perivascular administration of a DOT1L-selective inhibitor to injured rat carotid arteries
A selective DOT1L methyltransferase inhibitor (EPZ5676)14 was periadventitially administered, as described in our previous report11. Briefly, balloon angioplasty was performed as described above except that the 4th balloon withdrawal was not included in this experiment which was performed at a different time than the lentivirus infusion experiment. After the surgery, the external carotid artery was permanently ligated and blood flow was resumed in the common and internal carotid arteries. EPZ5676 (10 mg/rat) or an equal amount of DMSO (vehicle control) was dispersed in mixed thermosensitive hydrogels (200 μl Triblock gel, AK12, Akina Inc., IN, and 200 μl Pluronic gel, Sigma, St. Louis, MO). The mix was then applied around the balloon-injured carotid artery. Surgery was finished as described above for the angioplasty model.
Morphometric analysis of neointimal hyperplasia
At post-injury day 3, 7, and 14, the injured left and uninjured contralateral common carotid arteries were excised from anesthetized animals following perfusion fixation at a physiological pressure of 100 mm Hg. Animal euthanasia immediately followed in a chamber gradually filled with CO2. Cross-sections preparation, Verhoeff-Van Gieson (VVG) or Haemotoxylin and Eosin (H&E) staining, and morphometric analysis were performed as we previously descibed11. To assess neointimal hyperplasia (IH = intima/media area ratio), we measured a series of planimetric parameters. EEL area, inside external elastic lamina; IEL area, inside internal elastic lamina; lumen area, inside lumen; intima area = IEL area- lumen area; media area = EEL area – IEL area. ImageJ was used for all measurements by a student blinded to treatment groups. The data from all 3–5 sections were pooled to generate the mean for each animal. The means from all the animals in each treatment group were then averaged, and the standard error of the mean (SEM) was calculated.
Immunofluorescence staining on artery tissue sections
Fluorescent immunostaining was performed following our published protocol13. Briefly, artery sections were incubated without (negative staining) or with a primary antibody for 12 h and rinsed at least 3 times. The sections were then incubated with an anti-rabbit/mouse secondary antibody conjugated with Alexa Fluor 594 (A-11037/A-21203, Invitrogen, Carlsbad, CA) and rinsed. The specific antigen was then visualized with fluorescence microscopy. Detailed information of antibodies is included in Supplementary Table 2. For quantification, 5 immunostained sections from each animal were used. Fluorescence intensity in each image field was quantified by using an ImageJ software and normalized to the number of DAPI– stained nuclei in the media and neointima layers. The values from all 5 sections were pooled to generate the mean for each animal. The means from all animals in each group were then averaged, and the final mean (± SEM) was calculated.
DOT1L silencing in vitro via siRNA transfection of vascular smooth muscle cells
MOVAS cells (mouse aortic smooth muscle cell line, ATCC, Manassas, VA) were cultured to ~70% confluence in full medium (DMEM with 10% FBS), starved overnight in basal medium (DMEM with 0.25% FBS), and then in the same medium transfected overnight with DOT1L-specific siRNA or scrambled siRNA together with the RNAi Max reagent (Cat.13778150, Thermo Fisher). The culture continued for 48h in fresh basal medium (no transfection reagents) followed by proliferative stimulation with 20 ng/ml PDGF-BB (solvent as control) for another 24h. The cells were then harvested for microarray or Western blot analysis15. Sequences of the siRNA for DOT1L: sense, CAGCCUACCUGUUAGCAUUTT; antisense, AAUGCUAACAGGUAGGCUGAT. The scrambled siRNA is the same as previously reported15.
Western blot analysis
The harvested cells were lysed in RIPA buffer. After quantifying (Pierce BCA Protein Assay kit, Thermo Fisher Scientific, 23227), equal amount of protein was loaded to and separated in 12% SDS PAGE gel and then transferred to PVDF membrane. The membrane was incubated overnight at 4°C with anti-DOT1L, anti-PCNA, anti-cyclin-D1, or anti-β-actin (for antibody information, see Supplementary Table 3), rinsed 3x, and then incubated with a peroxidase-conjugated secondary antibody for 1h at room temperature. Specific proteins bands were illuminated using ECL detection reagents and recorded by Azure C600 imager (Azure Biosystems, Dublin, CA). Band intensity was quantified using the ImageJ 64 software (https://imagej.nih.gov/ij/).
Microarray and analysis
To investigate the impact of DOT1L silencing on gene expression, MOVAS cells transfected with DOT1L-specific siRNA or scrambled siRNA (control) were used for total RNA isolation with TRIzol reagent following the manufacturer’s instruction (Thermo Fisher Scientific, 15596026). Microarray (100 ng RNA/sample) was performed using Affymetrix Microarrays at the Ohio State University Comprehensive Cancer Center – James Hospital Shared Resources and Core Facilities. The data was quantified using Transcriptome Analysis Console, and then filtered and plotted using R Statistical computing software. Gene annotation was performed with ToppGene.
Statistical analysis
Data are presented as mean ± SEM. Normality of the data was assessed based on Shapiro-Wilk normality test prior to statistical calculation using Prism 6.0 software (GraphPad). p < 0.05 was considered significant. One-way ANOVA followed by post-hoc Tukey’s test was applied to multi-group comparison; both unpaired Student t-test and Mann–Whitney non-parametric test were used for 2-group comparison, as specified in each figure legend.
Results
To investigate a possible role of DOT1L in neointimal development, we used an authentic model of IH whereby balloon angioplasty injures the rat common carotid artery inducing neointima formation11. While neointima typically initiates at post-angioplasty day 3, accelerates around day 7, and maximizes at day 1411, 13, 16, we observed that immunostaining of DOT1L (Figure 1A and D, Supplementary Fig. 1) dramatically increased at day 3 (3.53 ±0.36 fold), peaked at day 7 (4.69 ±0.34 fold), and remained high at day 14 (3.62 ±0.36 fold), in injured arteries vs uninjured control.
Indicative of elevated DOT1L methyltransferase function after injury, staining of H3K79me2 and H3K79me3 exhibited temporal and spatial changes similar to that of the DOT1L protein (Figure 1B/E and C/F). Importantly, increased staining of DOT1L and H3K79me2 was also observed in human restenotic and atherosclerotic (vs normal) artery tissues (Figure 2) where neointima typically develops11.
To determine the functional specificity of DOT1L in IH, we expressed shRNAs to silence DOT1L in the injured (endothelial-denuded) artery wall via luminal infusion with lentivirus. This genetic manipulation effectively reduced DOT1L protein and H3K79me2 (immunostaining) by 54.5% and 37.1%, respectively, as compared to scrambled shRNA control (Figure 3A and B). DOT1L silencing led to a 76.5% decrease of IH and nearly doubled lumen size (Figure 3C) which became similar to that of uninjured arteries (Supplementary Fig. 2A). The total vessel area and media area were not significantly changed (Supplementary Fig. 2B and C).
To test a pharmacotherapy effect, we administered around the injured artery the clinical-trial drug Pinometostat (EPZ5676, NCT03701295 for leukemia) that is selective to DOT1L14. We found that both H3K79me2 and H3K79me3 were markedly reduced (by 56.1% and 58.6%, respectively) in EPZ5676-treated arteries compared to vehicle control (Figure 4D and E). These changes were accompanied by a 39.9% decrease of IH and 38.6% increase of lumen size (Figure 4F).
To explore the cellular events that may have accounted for the observed positive role for DOT1L in IH, we detected (via immunostaining) levels of smooth muscle α-actin (α-SMA), a commonly used marker for vascular smooth muscle cells (SMCs). Decrease of α-SMA is often indicative of pro-IH SMC phenotypic switching15, 17. We found that silencing or inhibiting DOT1L substantially raised α-SMA levels in the injured artery wall (Figure 5). Moreover, boosted α-SMA staining predominantly localized in the medial layer where normal SMCs reside, suggesting treatment effectivity in preserving favorable SMC phenotypes. Indeed, silencing DOT1L, or its pharmacological inhibition with EPZ5676, curtailed the upregulation of SMC pro-proliferative marker proteins (including PCNA and cyclin-D1) that was stimulated by platelet-derived growth factor (PDGF-BB) in vitro (Figure 6 and Supplementary Fig. 3). In addition, preliminary evidence from microarray (Figure 7) implicates a possible function of DOT1L in SMC expression of genes involved in hormonal signaling (Spx, Adm2), protein synthesis (Wars), and cell cycle (Slx), whose regulation by DOT1L has not been specifically reported, while a report exists regarding Bcl324. The data also points to possible DOT1L influence on extracellular matrix signaling (e.g. Adamts1, Tgfbr2, Rab38). Though promising, the preliminary microarray result needs to be re-evaluated in more rigorous biochemical and cellular studies.
Discussion
To the best of our knowledge this is the first study addressing the importance of the epigenetic writer DOT1L in the development of IH. Our data18 show that DOT1L (protein and methylation function) markedly increase in rat common carotid arteries following balloon injury; either genetically silencing or pharmacologically inhibiting DOT1L effectively abates IH.
Hyperplastic neointimal lesion formation is etiologically central to the major occlusive vascular diseases, not only the primary disease of atherosclerosis, but also (re)stenosis following angioplasty or vein grafting to treat atherosclerosis9. In either case, the neointimal lesion is formed primarily by phenotypically transitioned SMCs17, 19. This SMC phenotypic switching (or state transitions) is not necessarily related to DNA sequence change but rather increasingly recognized as epigenetically driven17. Various, such as de-differentiating, proliferative, migratory, inflammatory, and synthetic (matrix producing) SMC state transitions, may occur depending on the extra- and/or intracellular micro-environmental cues20. It is highly complex as to how these transitions influence disease progression; e.g. SMC proliferation exacerbates lesion formation in early stages of atherosclerosis yet favorably contributes to late-stage plaque stability in forming the fibrous cap20, 21. Nonetheless, as for mechanical injury (e.g. angioplasty) induced restenosis in rodent models, however, mounting evidence supports that SMC proliferation is the chief factor detrimentally propelling IH19, 22. In accordance, nullifying DOT1L, whether by genetic silencing or pharmacological inhibition, tamped down the level of SMC proliferative marker proteins as observed here. Consistent with DOT1L’s epigenetic writer function7, DOT1L protein, H3K79me2, and H3K79me3 were all upregulated in the injured artery wall and were all confined in the nuclei, as illuminated by the overlap of immunostaining and DAPI staining (Figure 1). The molecular events responsible for DOT1L regulations of SMC state transitions await more in-depth investigation to reveal.
In a normal artery, the endothelial inner lining of the vessel wall stakes off a myriad of stimulants such as growth factors and cytokines rich in the circulation3, 9, thereby guarding an undisturbed medial layer “sanctuary” where SMCs are able to maintain their innate function of providing arterial strength and contractility. However, the endothelium is inevitably removed or damaged due to balloon angioplasty. Consequently, SMCs become abruptly exposed to blood-borne stimulants that trigger SMC state transitions and neointimal lesion formation in the peri-luminal subintimal layer. Interestingly, the cells positively stained for DOT1L, H3K79me2, and H3K79me3 mostly localized in the neointima (post-injury day-7 and day-14), more in the peri-luminal region. The staining intensity gradient (higher toward the lumen) likely reflects the artery wall permeability and accessibility by various stimulants in the blood.
This peri-luminal DOT1L staining pattern is reminiscent of that exhibited by the histone acetylation reader BRD4, another pro-IH epigenetic factor, as we recently reported11, 12. In accordance, functional inter-dependence of BRD4 and DOT1L was recently discovered in cancer cell lines where H3K79me2 facilitated histone H4 acetylation which in turn recruited BRD423. Moreover, synergistic effects resulted from small-molecule inhibition of BRD4 and DOT1L, both clinical trial targets for leukemia therapy25. Thus, our ongoing studies on the interplay of DOT1L and BRD4 in the context of IH should help understand the epigenetic mechanisms underlying the role for DOT1L in IH observed herein.
Conclusions
The combined genetic and pharmacological determinations reveal a clear promotive role for DOT1L in the development of IH and a translational potential of its molecular targeting. However, important questions need to be addressed, including those on DOT1L’s function in different vascular cell types, downstream target genes, and the interactive pathways that underlie DOT1L’s regulations of pro-IH SMC state transitions. In addition, it remains an open question as to how DOT1L regulates downstream genes (e.g. candidates in Figure 7, S4) through its H3K79 methylation writer function. Nevertheless, the new knowledge disclosed from the current study, together with the availability of clinical trial drugs selective to DOT1L, warrant further research at the levels of basic science and therapeutic translation.
Supplementary Material
Highlights.
DOT1L and its catalytic products H3K79me2 and H3K79me3 increased due to injury in rat carotid arteries after balloon angioplasty.
Silencing DOT1L in vivo inhibited H3K79 methylation and diminished injury-induced intimal hyperplasia.
Treatment with a DOT1L inhibitor reduced H3K79 methylation and intimal hyperplasia.
Acknowledgments
Financial support
This work was supported by NIH R01 grants HL133665, EY029809 (to L.-W.G.), HL143469, HL129785 (to K.C.K. and L.-W.G.), and an AHA post-doctoral fellowship award 20POST35210967 (to M.X.Z.).
Footnotes
Conflict of interest
The authors declared they do not have anything to disclose regarding conflict of interest with respect to this manuscript.
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