PERK Inhibition Promotes Post-angioplasty Re-endothelialization via Modulating SMC Phenotype Changes.

BACKGROUND: Drug-eluting stents impair post-angioplasty re-endothelialization thus compromising restenosis prevention while heightening thrombotic risks. We recently found that inhibition of protein kinase RNA-like endoplasmic reticulum kinase (PERK) effectively mitigated both restenosis and thrombosis in rodent models. This motivated us to determine how PERK inhibition impacts re-endothelialization. METHODS: Re-endothelialization was evaluated in endothelial-denuded rat carotid arteries after balloon angioplasty and periadventitial administration of PERK inhibitor in a hydrogel. To study whether PERK in smooth muscle cells (SMCs) regulates re-endothelialization by paracrinally influencing endothelial cells (ECs), denuded arteries exposing SMCs were lentiviral-infected to silence PERK; in vitro, the extracellular vesicles isolated from the medium of PDGF-activated, PERK-upregulating human primary SMCs were transferred to human primary ECs. RESULTS: Treatment with PERK inhibitor versus vehicle control accelerated re-endothelialization in denuded arteries. PERK-specific silencing in the denuded arterial wall (mainly SMCs) also enhanced re-endothelialization compared to scrambled shRNA control. In vitro, while medium transfer from PDGF-activated SMCs impaired EC viability and increased the mRNA levels of dysfunctional EC markers, either PERK inhibition or silencing in donor SMCs mitigated these EC changes. Furthermore, CXCL10, a paracrine cytokine detrimental to ECs, was increased by PDGF activation and decreased after PERK inhibition or silencing in SMCs. CONCLUSIONS: Attenuating PERK activity pharmacologically or genetically provides an approach to accelerating post-angioplasty re-endothelialization in rats. The mechanism may involve paracrine factors regulated by PERK in SMCs that impact neighboring ECs. This study rationalizes future development of PERK-targeted endothelium-friendly vascular interventions.

Mass Spectrometric Imaging Reveals Temporal and Spatial Dynamics of Bioactive Lipids in Arteries Undergoing Restenosis.

Restenosis, or renarrowing of the arterial lumen, is a common recurrent disease following balloon angioplasty and stenting treatments for cardiovascular disease. A major technical barrier for deciphering restenotic mechanisms is the dynamic, spatial profiling of bioactive lipids in the arterial wall, especially in small animals. Here, applying matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI), we conducted the first lipidomic study of temporal-spatial profiling in a small animal model of angioplasty-induced restenosis. Cross sections were collected 3, 7, and 14 days after balloon angioplasty of rat carotid arteries. MALDI-MSI analyses showed that diacylglycerols (DAGs), signaling lipids associated with restenosis, and lysophosphatidylcholines (LysoPCs), whose function was uncharacterized in restenosis, dramatically increased at postangioplasty day 7 and day 14 in the neointimal layer of balloon-injured arteries compared to uninjured controls. In contrast, sphingomyelins (SMs) did not increase, but rather decreased at day 3, day 7, and day 14 in injured arteries versus the uninjured control arteries. These results revealed previously unexplored distinct temporal-spatial lipid dynamics in the restenotic arterial wall. Additionally, we employed time-of-flight secondary ion mass spectrometry (TOF-SIMS) tandem MS imaging for both molecular identification and imaging at high spatial resolution. These imaging modalities provide powerful tools for unraveling novel mechanisms of restenosis involving lipids or small signaling molecules.

Epigenetic intervention with a BET inhibitor ameliorates acute retinal ganglion cell death in mice.

PURPOSE: The bromo and extraterminal (BET) epigenetic "reader" family is becoming an appealing new therapeutic target for several common diseases, yet little is known of its role in retinal neurodegeneration. We explored the potential of BET inhibition in the protection of retinal ganglion cells (RGCs). METHODS: To test the therapeutic effect of JQ1, an inhibitor highly selective for the BET family of proteins, we used an acute RGC damage model induced by N-methyl-D-aspartic acid (NMDA) excitotoxicity. Adult C57BL/6 mice received an intravitreal injection of NMDA with (or without) JQ1 in one eye and vehicle control in the contralateral eye; RGC loss was assessed on retinal sections and whole mounts. Gene expression and apoptosis were analyzed by quantitative real time (RT)-PCR and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), respectively. For counting RGCs, immunostaining of the marker protein BRN3A was performed on whole mounts. RESULTS: NMDA treatment eliminated RGCs (day 7 and day 14 post injection) and diminished the expression (mRNAs) of RGC-selective genes, including Thy1, Nrn1, Sncg, and Nfl (day 3 and day 7). In contrast, co-injection with JQ1 maintained the number and gene expression of RGCs at ~2 fold of the control (NMDA only, no JQ1), and it decreased NMDA-induced TUNEL-positive cells in the RGC layer by 35%. While NMDA treatment dramatically upregulated mRNAs of inflammatory cytokines (TNFα, IL-1ß, MCP-1, RANTES) in retinal homogenates, co-injection with JQ1 suppressed their upregulation by ~50%. CONCLUSIONS: Intravitreal injection of a BET inhibitor (JQ1) ameliorates NMDA-induced RGC death, revealing the RGC-protective potential of pharmacological blockage of the BET family. This new strategy of epigenetic intervention may be extended to other retinal degenerative conditions.

Structural analysis of adventitial collagen to feature aging and aneurysm formation in human aorta.

OBJECTIVE: Adventitial collagen structure provides the aorta with tensile strength. Like other collagen-rich tissues, it can be affected by internal factors including aging and location. We determined whether the structural characteristics of human aortic adventitial collagen change with aging, location, and aneurysm formation. METHODS: Nonatherosclerotic nonaneurysmal (NANA) human abdominal aortas were collected from 15 individuals who had died of noncardiovascular diseases (<40 years old, NANA young, n = 5; >60 years old, NANA old, n = 5). The architecture of adventitial collagen in the aortas was assessed by scanning electron microscopy, and fiber orientation was assessed by polarized microscopy with two-dimensional fast Fourier transform. We then analyzed retardation as an anisotropic property of adventitial collagen by polarized light microscopy. The orientation and retardation of NANA aortas were compared with those of abdominal aortic specimens from patients who were surgically treated for abdominal aortic aneurysm (AAA) (>60 years old, n = 11). RESULTS: Adventitial collagen of the abdominal aortas on scanning electron microscopy images appeared as wavy, ropy fibers in aortas from young individuals (NANA young, n = 5) and were essentially flattened in those from older patents (NANA old, n = 5) and from those with AAA. Collagen fibers were thicker but sparser in the adventitia of aortas with AAA. Orientation maintained in the collagen fibers of NANA aortas (n = 15) on two-dimensional fast Fourier transform analysis was unrelated to either location or age and did not differ between NANA aortas and those with AAA. However, collagen fibrils in NANA aortas (n = 15) were significantly less retarded only at the level of the inferior mesenteric artery compared with other aortic locations. In addition, retardation was significantly reduced in abdominal aortas with AAA at the level of the inferior mesenteric artery. CONCLUSIONS: The basic structure of adventitial collagen fiber was maintained in abdominal aortas regardless of location or age. Because the molecular structure at the subfibril level changed at abdominal aorta and enhanced in aortas with AAA, alterations in the molecular structure of adventitial collagen might be associated with aneurysmal formation.

Neointima abating and endothelium preserving - An adventitia-localized nanoformulation to inhibit the epigenetic writer DOT1L.

Open vascular reconstructions such as bypass are common treatments for cardiovascular disease. Unfortunately, neointimal hyperplasia (IH) follows, leading to treatment failure for which there is no approved therapy. Here we combined the strengths of tailoring nanoplatforms for open vascular reconstructions and targeting new epigenetic mechanisms. We produced adhesive nanoparticles (ahNP) that could be pen-brushed and immobilized on the adventitia to sustainably release pinometostat, an inhibitor drug selective to the epigenetic writer DOT1L that catalyzes histone-3 lysine-79 dimethylation (H3K79me2). This treatment not only reduced IH by 76.8% in injured arteries mimicking open reconstructions in obese Zucker rats with human-like diseases but also avoided the shortcoming of endothelial impairment in IH management. In mechanistic studies, chromatin immunoprecipitation (ChIP) sequencing revealed co-enrichment of the histone mark H3K27ac(acetyl) and its reader BRD4 at the gene of aurora kinase B (AURKB), where H3K79me2 was also enriched as indicated by ChIP-qPCR. Accordingly, DOT1L co-immunoprecipitated with H3K27ac. Furthermore, the known IH driver BRD4 governed the expression of DOT1L which controlled AURKB's protein level, revealing a BRD4- > DOT1L- > AURKB axis. Consistently, AURKB-selective inhibition reduced IH. Thus, this study presents a prototype nanoformulation suited for open vascular reconstructions, and the new insights into chromatin modulators may aid future translational advances.

Gene-repressing epigenetic reader EED unexpectedly enhances cyclinD1 gene activation.

Epigenetically switched, proliferative vascular smooth muscle cells (SMCs) form neointima, engendering stenotic diseases. Histone-3 lysine-27 trimethylation (H3K27me3) and acetylation (H3K27ac) marks are associated with gene repression and activation, respectively. The polycomb protein embryonic ectoderm development (EED) reads H3K27me3 and also enhances its deposition, hence is a canonical gene repressor. However, herein we found an unexpected role for EED in activating the bona fide pro-proliferative gene Ccnd1 (cyclinD1). EED overexpression in SMCs increased Ccnd1 mRNA, seemingly contradicting its gene-repressing function. However, consistently, EED co-immunoprecipitated with gene-activating H3K27ac reader BRD4, and they co-occupied at both mitogen-activated Ccnd1 and mitogen-repressed P57 (bona fide anti-proliferative gene), as indicated by chromatin immunoprecipitation qPCR. These results were abolished by an inhibitor of either the EED/H3K27me3 or BRD4/H3K27ac reader function. In accordance, elevating BRD4 increased H3K27me3. In vivo, while EED was upregulated in rat and human neointimal lesions, selective EED inhibition abated angioplasty-induced neointima and reduced cyclinD1 in rat carotid arteries. Thus, results uncover a previously unknown role for EED in Ccnd1 activation, likely via its cooperativity with BRD4 that enhances each other's reader function; i.e., activating pro-proliferative Ccnd1 while repressing anti-proliferative P57. As such, this study confers mechanistic implications for the epigenetic intervention of neointimal pathology.

Angioplasty induces epigenomic remodeling in injured arteries.

Neointimal hyperplasia/proliferation (IH) is the primary etiology of vascular stenosis. Epigenomic studies concerning IH have been largely confined to in vitro models, and IH-underlying epigenetic mechanisms remain poorly understood. This study integrates information from in vivo epigenomic mapping, conditional knockout, gene transfer and pharmacology in rodent models of IH. The data from injured (IH-prone) rat arteries revealed a surge of genome-wide occupancy by histone-3 lysine-27 trimethylation (H3K27me3), a gene-repression mark. This was unexpected in the traditional view of prevailing post-injury gene activation rather than repression. Further analysis illustrated a shift of H3K27me3 enrichment to anti-proliferative genes, from pro-proliferative genes where gene-activation mark H3K27ac(acetylation) accumulated instead. H3K27ac and its reader BRD4 (bromodomain protein) co-enriched at Ezh2; conditional BRD4 knockout in injured mouse arteries reduced H3K27me3 and its writer EZH2, which positively regulated another pro-IH chromatin modulator UHRF1. Thus, results uncover injury-induced loci-specific H3K27me3 redistribution in the epigenomic landscape entailing BRD4→EZH2→UHRF1 hierarchical regulations. Given that these players are pharmaceutical targets, further research may help improve treatments of IH.

A hierarchical and collaborative BRD4/CEBPD partnership governs vascular smooth muscle cell inflammation.

Bromodomain protein BRD4 reads histone acetylation (H3K27ac), an epigenomic mark of transcription enhancers. CCAAT enhancer binding protein delta (CEBPD) is a transcription factor typically studied in metabolism. While both are potent effectors and potential therapeutic targets, their relationship was previously unknown. Here we investigated their interplay in vascular smooth muscle cell (SMC) inflammation. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) revealed H3K27ac/BRD4 enrichment at Cebpd in injured rat carotid arteries. While genomic deletion of BRD4-associated enhancer in SMCs in vitro decreased Cebpd transcripts, BRD4 gene silencing also diminished Cebpd mRNA and protein, indicative of a BRD4 control over CEBPD expression. Bromodomain-1, but not bromodomain-2, accounted for this BRD4 function. Moreover, endogenous BRD4 protein co-immunoprecipitated with CEBPD, and both proteins co-immunoprecipitated the Cebpd promoter and enhancer DNA fragments. These co-immunoprecipitations (coIPs) were all abolished by the BRD4-bromodomain blocker JQ1, suggesting a BRD4/CEBPD /promoter/enhancer complex. While BRD4 and CEBPD were both upregulated upon tumor necrosis factor alpha (TNF-α) stimulation of SMC inflammation (increased interleukin [IL]-1b, IL-6, and MCP-1), they mediated this stimulation via preferentially elevated expression of platelet-derived growth factor receptor alpha (PDGFRα, versus PDGFRß), as indicated by loss- and gain-of-function experiments. Taken together, our study unravels a hierarchical yet collaborative BRD4/CEBPD relationship, a previously unrecognized mechanism that prompts SMC inflammation and may underlie other pathophysiological processes as well.

A Role for Polo-Like Kinase 4 in Vascular Fibroblast Cell-Type Transition.

Polo-like kinase 4 (PLK4) is canonically known for its cytoplasmic function in centriole duplication. Here we show a noncanonical PLK4 function of regulating the transcription factor SRF's nuclear activity and associated myofibroblast-like cell-type transition. In this context, we have further found that PLK4's phosphorylation and transcription are respectively regulated by PDGF receptor and epigenetic factor BRD4. Furthermore, in vivo experiments suggest PLK4 inhibition as a potential approach to mitigating vascular fibrosis.

An adventitial painting modality of local drug delivery to abate intimal hyperplasia.

A major medical problem is the persistent lack of approved therapeutic methods to prevent postoperative intimal hyperplasia (IH) which leads to high-rate failure of open vascular reconstructions such as bypass grafting. Hydrogel has been widely used in preclinical trials for perivascular drug administration to mitigate postoperative IH. However, bulky hydrogel is potentially pro-inflammatory, posing a significant hurdle to clinical translation. Here we developed a new modality of directly "painting" drug-loaded unimolecular micelles (UM) to the adventitia thus obviating the need for a hydrogel. To render tissue adhesion, we generated amine-reactive unimolecular micelles with N-hydroxysuccinimide ester (UM-NHS) terminal groups to form stable amide bonds with the adventitia. To test periadventitial application, we either soaked balloon-injured rat carotid arteries in crosslinked UM-NHS (Mode-1) or non-crosslinked UM-NHS (Mode-2), or painted the vessel surface with non-crosslinked UM-NHS (Mode-3). The UM-NHS were loaded with or without a model drug (rapamycin) known to be IH inhibitory. We found that Mode-1 produced a marked IH-mitigating drug effect but also caused severe tissue damage. Mode-2 resulted in lower tissue toxicity yet less drug effect on IH. However, the painting method, Mode-3, demonstrated a pronounced therapeutic effect (75% inhibition of IH) without obvious toxicity. In summary, we present a simple painting modality of periadventitial local drug delivery using tissue-adhesive UM. Given the robust IH-abating efficacy and low tissue toxicity, this prototype merits further development towards an effective anti-stenosis therapy suitable for open vascular reconstructions.

Smad3 Regulates Neuropilin 2 Transcription by Binding to its 5' Untranslated Region.

Background Vascular smooth muscle cell phenotypic change and consequential intimal hyperplasia (IH) cause arterial stenosis and posttreatment restenosis. Smad3 is a master transcription factor, yet its underlying functional mechanisms in this disease context are not well defined. Methods and Results In cultured smooth muscle cells, Smad3 silencing and overexpression respectively reduced and increased the mRNA and protein of NRP2 (neuropilin 2), a recently reported pro-IH signaling factor. Smad3 silencing attenuated pro-IH smooth muscle cell phenotypes including proliferation, migration, and dedifferentiation (reduced smooth muscle α-actin). While increased Smad3 enhanced these phenotypes, NRP2 silencing abolished this enhancement. Interestingly, the 5' untranslated region but not the promoter of NRP2 was indispensable for Smad3-enhanced transcriptional activity (luciferase assay); both chromatin immunoprecipitation and electrophoretic mobility shift assay showed predominant Smad3 binding in the +51 to +78 bp region of NRP2's 5' untranslated region. In vivo, Smad3 haploinsufficiency reduced NRP2 (immunostaining) and IH (by 47%) in wire-injured mouse femoral arteries. Conclusions Smad3 controls NRP2 expression by occupying its 5' untranslated region in promoting smooth muscle cell phenotypic change in vitro. This and in vivo results shed new light on the long-debated role of Smad3 in IH.

Nullifying epigenetic writer DOT1L attenuates neointimal hyperplasia.

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 of 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. CONCLUSIONS: 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 continued mechanistic and translational investigation into DOT1L targeting for treatment of (re)stenotic vascular conditions.

PERK Inhibition Mitigates Restenosis and Thrombosis: A Potential Low-Thrombogenic Antirestenotic Paradigm.

Developing endothelial-protective, nonthrombogenic antirestenotic treatments has been a challenge. A major hurdle to this has been the identification of a common molecular target in both smooth muscle cells and endothelial cells, inhibition of which blocks dysfunction of both cell types. The authors' findings suggest that the PERK kinase could be such a target. Importantly, PERK inhibition mitigated both restenosis and thrombosis in preclinical models, implicating a low-thrombogenic antirestenotic paradigm.

Role of FOXM1 in vascular smooth muscle cell survival and neointima formation following vascular injury.

BACKGROUND: Accelerated smooth muscle cell (SMC) proliferation is the primary cause of intimal hyperplasia (IH) following vascular interventions. Forkhead Box M1 (FOXM1) is considered a proliferation-associated transcription factor. However, the presence and role of FOXM1 in IH following vascular injury have not been determined. OBJECTIVE: We examined the expression of FOXM1 in balloon-injured rat carotid arteries and investigated the effect of FOXM1 inhibition in SMCs and on the development of IH. METHODS AND RESULTS: FOXM1 was detected by immunofluorescent staining in balloon-injured rat carotid arteries where we observed an upregulation at day 7, 14, and 28 compared to uninjured controls. Immunofluorescence staining revealed FOXM1 coincided with proliferating cell nuclear antigen (PCNA). FOXM1 was also detectable in human carotid plaque samples. Western blot showed an upregulation of FOXM1 protein in serum-stimulated SMCs. Inhibition of FOXM1 using siRNA or chemical inhibition led to the induction of apoptosis as measured by flow cytometry and western blot for cleaved caspase 3. Perturbations in survival signaling were measured by western blot following FOXM1 inhibition, which showed a decrease in phosphorylated AKT and ß-catenin. The chemical inhibitor thiostrepton was delivered by intraperitoneal injection in rats that underwent balloon injury and led to reduced intimal thickening compared to DMSO controls. CONCLUSIONS: FOXM1 is an important molecular mediator of IH that contributes to the proliferation and survival of SMCs following vascular injury.

ALDH1A3 Regulations of Matricellular Proteins Promote Vascular Smooth Muscle Cell Proliferation.

Vascular smooth muscle cell (VSMC) proliferation promotes intimal hyperplasia (IH) in occluding vascular diseases. Here we identified a positive role of ALDH1A3 (an aldehyde dehydrogenase) in this pro-IH process. The expression of ALDH1A3, but not that of 18 other isoforms of the ALDH family, was substantially increased in cytokine-stimulated VSMCs. PDGF(BB) stimulated VSMC total ALDH activity and proliferation, whereas ALDH1A3 silencing abolished this effect. ALDH1A3 silencing also diminished the expression of two matricellular proteins (TNC1 and ESM1), revealing a previously unrecognized ALDH1A3 function. Loss-of-function experiments demonstrated that TNC1 and ESM1 mediated ALDH1A3's pro-proliferative function via activation of AKT/mTOR and/or MEK/ERK pathways. Furthermore, ALDH inhibition with disulfiram blocked VSMC proliferation/migration in vitro and decreased TNC1 and ESM1 and IH in angioplasty-injured rat carotid arteries. Thus, ALDH1A3 promotes VSMC proliferation at least partially through TNC1/ESM1 upregulation; dampening excessive ALDH1A3 activity represents a potential approach to IH mitigation.

The BD2 domain of BRD4 is a determinant in EndoMT and vein graft neointima formation.

BACKGROUND: Vein-graft bypass is commonly performed to overcome atherosclerosis but is limited by high failure rates, principally due to neointimal wall thickening. Recent studies reveal that endothelial-mesenchymal transition (EndoMT) is critical for vein-graft neointima formation. BETs are a family of Bromo/ExtraTerminal domains-containing epigenetic reader proteins (BRD2, BRD3, BRD4). They bind acetylated histones through their unique tandem bromodomains (BD1, BD2), facilitating transcriptional complex formation and cell-state transitions. The role for BETs, including individual BRDs and their unique BDs, is not well understood in EndoMT and neointimal formation. METHODS AND RESULTS: Repression of BRD4 expression abrogated TGFß1-induced EndoMT, with greater effects than BRD2 or BRD3 knockdown. An inhibitor selective for BD2 in all BETs, but not that for BD1, blocked EndoMT. Moreover, expression of a dominant-negative BRD4-specific BD2 fully abolished EndoMT. Concordantly, BRD4 knockdown repressed TGFß1-stimulated increase of ZEB1 protein - a transcription factor integral in EndoMT. In vivo, lentiviral gene transfer of either BRD4 shRNA or dominant negative BRD4-specific BD2 mitigated neointimal development in rat jugular veins grafted to carotid arteries. CONCLUSIONS: Our data reveal the BD2 domain of BRD4 as a determinant driving EndoMT in vitro and neointimal formation in vivo. These findings provide new insight into BET biology, while offering prospects of specific BET domain targeting as an approach to limiting neointima and extending vein graft patency.

HDAC6 Regulates the MRTF-A/SRF Axis and Vascular Smooth Muscle Cell Plasticity.

Cellular plasticity is fundamental in biology and disease. Vascular smooth muscle cell (SMC) dedifferentiation (loss of contractile proteins) initiates and perpetrates vascular pathologies such as restenosis. Contractile gene expression is governed by the master transcription factor, serum response factor (SRF). Unlike other histone deacetylases, histone deacetylase 6 (HDAC6) primarily resides in the cytosol. Whether HDAC6 regulates SRF nuclear activity was previously unknown in any cell type. This study found that selective inhibition of HDAC6 with tubastatin A preserved the contractile protein (alpha-smooth muscle actin) that was otherwise diminished by platelet-derived growth factor-BB. Tubastatin A also enhanced SRF transcriptional (luciferase) activity, and this effect was confirmed by HDAC6 knockdown. Interestingly, HDAC6 inhibition increased acetylation and total protein of myocardin-related transcription factor A (MRTF-A), a transcription co-activator known to translocate from the cytosol to the nucleus, thereby activating SRF. Consistently, HDAC6 co-immunoprecipitated with MRTF-A. In vivo studies showed that tubastatin A treatment of injured rat carotid arteries mitigated neointimal lesion, which is known to be formed largely by dedifferentiated SMCs. This report is the first to show HDAC6 regulation of the MRTF-A/SRF axis and SMC plasticity, thus opening a new perspective for interventions of vascular pathologies.

A paradigm of endothelium-protective and stent-free anti-restenotic therapy using biomimetic nanoclusters.

Drug-eluting stents are the most commonly employed method to control post-angioplasty restenosis. Unfortunately, they exacerbate life-threatening stent thrombosis because of endothelium damage caused by both drug and stenting. To solve this major medical problem, an endothelium-protective and stent-free anti-restenotic method is highly desirable. Here we have generated a biomimetic intravenous delivery system using dendritic polymer-based nanoclusters, which were coated with platelet membranes for targeting to the injured arterial wall where restenosis occurs. These nanoclusters were loaded with an endothelium-protective epigenetic inhibitor (JQ1) or an endothelium-toxic status quo drug (rapamycin), and compared for their ability to mitigate restenosis without hindering the process of re-endothelialization. Fluorescence imaging of Cy5-tagged biomimetic nanoclusters indicated their robust homing to injured, but not uninjured arteries. Two weeks after angioplasty, compared to no-drug control, both rapamycin- and JQ1-loaded biomimetic nanoclusters substantially reduced (by >60%) neointimal hyperplasia, the primary cause of restenosis. However, whereas the rapamycin formulation impaired the endothelial re-coverage of the denuded inner arterial wall, the JQ1 formulation preserved endothelial recovery. In summary, we have created an endothelium-protective anti-restenotic system with biomimetic nanoclusters containing an epigenetic inhibitor. This system warrants further development for a non-thrombogenic and stent-free method for clinical applications.

Electron-Transfer/Higher-Energy Collision Dissociation (EThcD)-Enabled Intact Glycopeptide/Glycoproteome Characterization.

Protein glycosylation, one of the most heterogeneous post-translational modifications, can play a major role in cellular signal transduction and disease progression. Traditional mass spectrometry (MS)-based large-scale glycoprotein sequencing studies heavily rely on identifying enzymatically released glycans and their original peptide backbone separately, as there is no efficient fragmentation method to produce unbiased glycan and peptide product ions simultaneously in a single spectrum, and that can be conveniently applied to high throughput glycoproteome characterization, especially for N-glycopeptides, which can have much more branched glycan side chains than relatively less complex O-linked glycans. In this study, a redefined electron-transfer/higher-energy collision dissociation (EThcD) fragmentation scheme is applied to incorporate both glycan and peptide fragments in one single spectrum, enabling complete information to be gathered and great microheterogeneity details to be revealed. Fetuin was first utilized to prove the applicability with 19 glycopeptides and corresponding five glycosylation sites identified. Subsequent experiments tested its utility for human plasma N-glycoproteins. Large-scale studies explored N-glycoproteomics in rat carotid arteries over the course of restenosis progression to investigate the potential role of glycosylation. The integrated fragmentation scheme provides a powerful tool for the analysis of intact N-glycopeptides and N-glycoproteomics. We also anticipate this approach can be readily applied to large-scale O-glycoproteome characterization. Graphical Abstract ᅟ.