Rat Carotid Artery Balloon Injury Research Articles

MiR-1290 induces endothelial-to-mesenchymal transition and promotes vascular restenosis after angioplasty by targeting FGF2

Background and aimsEndothelial-to mesenchymal transition (EndMT) is an important reason for restenosis but the specific mechanisms need to be further explored. Therefore, the purpose of this study is to screen significantly different microRNAs (miRNAs) and explore its functions and downstream pathways. MethodsThis study screened several miRNAs with significant differences between human arterial segments from restenosis patients and healthy volunteers using whole transcriptome resequencing and real-time quantitative reverse transcription PCR (qRT-PCR). We explored the correlation between miR-1290 and EndMT using Western Blot, qRT-PCR, Pearson correlation analysis and further functional gain and loss experiments. Subsequently, we identified the direct downstream target of miR-1290 by bioinformatics analysis, RNA pull-down, double Luciferase reporter gene and other functional experiments. Finally, rat carotid artery balloon injury model demonstrated the therapeutic potential of miR-1290 regulator. ResultsWe screened 129 differentially expressed miRNAs. Among them, miR-1290 levels were significantly higher in restenosis arteries than in healthy arteries, and as expected, EndMT was functionally enhanced with miR-1290 overexpression but comparatively weakened when miR-1290 was knocked down. In addition, fibroblast growth factor-2 (FGF2) was established as the downstream target of miR-1290. Finally, we utilized an animal model and found that low miR-1290 levels could alleviate EndMT and the progression of restenosis. ConclusionsOur study demonstrated the strong regulatory effects of miR-1290 on EndMT, endometrial hyperplasia and restenosis, which could be useful as biomarkers and therapeutic targets for stent implantation in patients with arterial occlusive disease of the lower extremities.

Exosomes of endothelial progenitor cells repair injured vascular endothelial cells through the Bcl2/Bax/Caspase-3 pathway

The main objective of this study is to evaluate the influence of exosomes derived from endothelial progenitor cells (EPC-Exo) on neointimal formation induced by balloon injury in rats. Furthermore, the study aims to investigate the potential of EPC-Exo to promote proliferation, migration, and anti-apoptotic effects of vascular endothelial cells (VECs) in vitro. The underlying mechanisms responsible for these observed effects will also be thoroughly explored and analyzed. Endothelial progenitor cells (EPCs) was isolated aseptically from Sprague–Dawley (SD) rats and cultured in complete medium. The cells were then identified using immunofluorescence and flow cytometry. The EPC-Exo were isolated and confirmed the identities by western-blot, transmission electron microscope, and nanoparticle analysis. The effects of EPC-Exo on the rat carotid artery balloon injury (BI) were detected by hematoxylin and eosin (H&E) staining, ELISA, immunohistochemistry, immunofluorescence, western-blot and qPCR. LPS was used to establish an oxidative damage model of VECs. The mechanism of EPC-Exo repairing injured vascular endothelial cells was detected by measuring the proliferation, migration, and tube function of VECs, actin cytoskeleton staining, TUNEL staining, immunofluorescence, western-blot and qPCR. In vivo, EPC-Exo exhibit inhibitory effects on neointima formation following carotid artery injury and reduce the levels of inflammatory factors, including TNF-α and IL-6. Additionally, EPC-Exo downregulate the expression of adhesion molecules on the injured vascular wall. Notably, EPC-Exo can adhere to the injured vascular area, promoting enhanced endothelial function and inhibiting vascular endothelial hyperplasia Moreover, they regulate the expression of proteins and genes associated with apoptosis, including B-cell lymphoma-2 (Bcl2), Bcl2-associated x (Bax), and Caspase-3. In vitro, experiments further confirmed that EPC-Exo treatment significantly enhances the proliferation, migration, and tube formation of VECs. Furthermore, EPC-Exo effectively attenuate lipopolysaccharides (LPS)-induced apoptosis of VECs and regulate the Bcl2/Bax/Caspase-3 signaling pathway. This study demonstrates that exosomes derived from EPCs have the ability to inhibit excessive carotid intimal hyperplasia after BI, promote the repair of endothelial cells in the area of intimal injury, and enhance endothelial function. The underlying mechanism involves the suppression of inflammation and anti-apoptotic effects. The fundamental mechanism for this anti-apoptotic effect involves the regulation of the Bcl2/Bax/Caspase-3 signaling pathway.

Systemic single administration of anti-inflammatory microRNA 146a-5p loaded in polymeric nanomedicines with active targetability attenuates neointimal hyperplasia by controlling inflammation in injured arteries in a rat model.

Neointimal hyperplasia (NIH) after revascularization is a key unsolved clinical problem. Various studies have shown that attenuation of the acute inflammatory response on the vascular wall can prevent NIH. MicroRNA146a-5p (miR146a-5p) has been reported to show anti-inflammatory effects by inhibiting the NF-κB pathway, a well-known key player of inflammation of the vascular wall. Here, a nanomedicine, which can reach the vascular injury site, based on polymeric micelles was applied to deliver miR146a-5p in a rat carotid artery balloon injury model. In vitro studies using inflammation-induced vascular smooth muscle cell (VSMC) was performed. Results showed anti-inflammatory response as an inhibitor of the NF-κB pathway and VSMC migration, suppression of reactive oxygen species production, and proinflammatory cytokine gene expression in VSMCs. A single systemic administration of miR146a-5p attenuated NIH and vessel remodeling in a carotid artery balloon injury model in both male and female rats in vivo. MiR146a-5p reduced proinflammatory cytokine gene expression in injured arteries and monocyte/macrophage infiltration into the vascular wall. Therefore, miR146a-5p delivery to the injury site demonstrated therapeutic potential against NIH after revascularization.

MicroRNA-17-5p, a novel endothelial cell modulator, controls vascular re-endothelialization and neointimal lesion formation.

The functions of miR-17-5p in tumorigenesis have been explored. However, their functionalities in arterial endothelial cells (ECs) have not been investigated. Besides, the issue of vascular remodelling is barely addressed. The study aimed to determine the effect of overexpression or inhibition of miR-17-5p on arterial endothelial cells' (ECs) function and vascular remodelling in vitro and the rat carotid arteries model. Quantitative RT-PCR analysis was performed to examine the expression of miR-17-5p. Then, gain-of-function and loss-of-function approaches were employed to investigate the functional roles of miR-17-5p in cultured human coronary artery endothelial cells (HCAECs); further, TargetScan software analysis and luciferase reporter activity assay were performed to investigate the potential mechanism. Lastly, the results of the cell segment were verified in a rat carotid artery balloon injury model by Western blot analysis, measurement of the vascular cGMP level and plasma 8-iso-prostaglandin F2 (8-iso-PGF2) testing. Moreover, morphometric analysis was implemented to detect the re-endothelialization and neointimal formation in rat carotid artery after balloon injury. This study firstly found that miR-17-5p expression was upregulated in the injured vascular walls and highly expressive in ECs; overexpression of miR-17-5p inhibited HCAECs' proliferation and migration, whereas miR-17-5p knockdown strengthened its proliferative and migratory roles, influenced inflammatory response, through regulating VEGRA and VEGFR2. It was found that miR-17-5p bind to VEGFA and VEGFR2 at the 3'UTR. Next, downregulation of miR-17-5p promotes re-endothelialization, and attenuates neointimal formation as measured by the I/M ratio (0.63±0.05 vs 1.45±0.06, antagomiR-17-5p vs. Lenti-NC, p < 0.05). In addition, the functional recovery of the endothelium was also accelerated by miR-17-5p knockdown. Our study suggests that miR-17-5p is a feasible strategy for the selective modulation of endothelialization and vascular remodelling through regulating VEGFA and VEGFR2.

Sophocarpine Alleviates Injury-Induced Intima Hyperplasia of Carotid Arteries by Suppressing Inflammation in a Rat Model.

Introduction: Balloon angioplasty is a commonly applied procedure for treating atherosclerotic vascular diseases. However, the maintenance of long-term lumen patency is relatively difficult due to the occurrence of restenosis. Previous research has shown that the occurrence of vascular wall inflammation is associated with higher rates of restenosis. Sophocarpine (SPC) can exert various therapeutic effects such as anti-oxidation, anti-inflammation, anti-tumor, antivirus and immune regulation. This study aimed to investigate whether SPC can alleviate intimal hyperplasia following balloon injury in a rat carotid artery model. Methods: Twenty Sprague–Dawley rats were randomly assigned to four groups: (i) control, (ii) balloon injury, (iii) balloon injury followed by saline injection, and (iv) balloon injury followed by SPC administration. Each group contained five rats. A high-pressure balloon of 3 mm × 20 mm was placed in the carotid artery. The balloon was inflated to a pressure of 8 atmospheres to carry out rat carotid artery balloon injury model. The areas of neointimal and media were determined by Verhoeff_Van Gieson staining, and the intima-to-media (I:M) ratios were subsequently evaluated. After that, the protein levels of IL-6, IL-1β, MCP-1, NF-κB, TNF-α, VCAM-1, ICAM-1 and eNOS were measured. Results: The ratio of I:M was remarkably higher in the balloon injury group than in the control group (p < 0.01). SPC could significantly decrease the ratio of I:M compared with the balloon injury group (p < 0.01). Besides, the protein levels of IL-6, IL-1β, MCP-1, NF-κB, TNF-α, ICAM-1 and VCAM-1 were increased in rat carotid arteries exposed to balloon injury (p < 0.01), and treatment with SPC could attenuate these effects (p < 0.05). Furthermore, balloon injury inhibited the protein expression of eNOS (p < 0.01), and SPC could elevate its level (p < 0.05). Conclusions: SPC could alleviate an intimal hyperplasia in balloon-injured carotid artery, and the mechanisms underlying this protective effect might be due to its inhibitory potency against inflammation signals. Our study also implies the potential applicability of SPC in treating restenosis after balloon angioplasty.

CARMN Is an Evolutionarily Conserved Smooth Muscle Cell-Specific LncRNA That Maintains Contractile Phenotype by Binding Myocardin.

Vascular homeostasis is maintained by the differentiated phenotype of vascular smooth muscle cells (VSMCs). The landscape of protein coding genes comprising the transcriptome of differentiated VSMCs has been intensively investigated but many gaps remain including the emerging roles of noncoding genes. We reanalyzed large-scale, publicly available bulk and single-cell RNA sequencing datasets from multiple tissues and cell types to identify VSMC-enriched long noncoding RNAs. The in vivo expression pattern of a novel smooth muscle cell (SMC)-expressed long noncoding RNA, Carmn (cardiac mesoderm enhancer-associated noncoding RNA), was investigated using a novel Carmn green fluorescent protein knock-in reporter mouse model. Bioinformatics and quantitative real-time polymerase chain reaction analysis were used to assess CARMN expression changes during VSMC phenotypic modulation in human and murine vascular disease models. In vitro, functional assays were performed by knocking down CARMN with antisense oligonucleotides and overexpressing Carmn by adenovirus in human coronary artery SMCs. Carotid artery injury was performed in SMC-specific Carmn knockout mice to assess neointima formation and the therapeutic potential of reversing CARMN loss was tested in a rat carotid artery balloon injury model. The molecular mechanisms underlying CARMN function were investigated using RNA pull-down, RNA immunoprecipitation, and luciferase reporter assays. We identified CARMN, which was initially annotated as the host gene of the MIR143/145 cluster and recently reported to play a role in cardiac differentiation, as a highly abundant and conserved, SMC-specific long noncoding RNA. Analysis of the Carmn GFP knock-in mouse model confirmed that Carmn is transiently expressed in embryonic cardiomyocytes and thereafter becomes restricted to SMCs. We also found that Carmn is transcribed independently of Mir143/145. CARMN expression is dramatically decreased by vascular disease in humans and murine models and regulates the contractile phenotype of VSMCs in vitro. In vivo, SMC-specific deletion of Carmn significantly exacerbated, whereas overexpression of Carmn markedly attenuated, injury-induced neointima formation in mouse and rat, respectively. Mechanistically, we found that Carmn physically binds to the key transcriptional cofactor myocardin, facilitating its activity and thereby maintaining the contractile phenotype of VSMCs. CARMN is an evolutionarily conserved SMC-specific long noncoding RNA with a previously unappreciated role in maintaining the contractile phenotype of VSMCs and is the first noncoding RNA discovered to interact with myocardin.

CircTMEM165 Facilitates Vascular Endothelial Repair by Modulating Mitochondrial Fission via miR-192/SCP2 in vitro and in vivo

Constitutive explorations have demonstrated that circular RNAs (circRNAs) are closely related to cardiovascular diseases. However, few studies have identified regulatory roles of circRNAs in endothelial repair. In particular, circTMEM165 has first been reported to be highly expressed in hypoxic human umbilical vein endothelial cells (HUVECs), indicating it to be closely linked with endothelial functions and atherosclerosis. Here, we explored the main biological functions of circTMEM165 and its regulatory mechanism in atherosclerosis. We identified that circTMEM165 was downregulated in patients with atherosclerosis and negatively associated with atherosclerosis progression. Functionally, circTMEM165 was found to be abundant in endothelial cells, inhibiting inflammation and adhesion in HUVECs. Particularly, we first observed that lipopolysaccharide (LPS) could induce HUVEC apoptosis and mitochondrial fission, and circTMEM165 was later found to alleviate these effects. Mechanistically, circTMEM165, as a miR-192-3p sponge, upregulated the downstream expression of SCP2, which serves as critical regulator of biological functions of HUVECs. Moreover, the classic rat carotid artery balloon injury model demonstrated that circTMEM165 reduced apoptosis and intimal hyperplasia in vivo. These findings demonstrated the specific role of the circTMEM165/miR-192-3p/SCP2 pathway in regulating endothelial function and indicated that these molecules served as promising targets for the prediction and diagnosis of vascular diseases. Funding Information: This work was supported by The National Natural Science Foundation of China (grant no. 81870331), and The Qingdao municipal science and technology bureau project (grant no. 21-1-4-rkjk-12-nsh). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: This study was approved by the Ethical Committee of Qingdao University and conducted in accordance with the Declaration of Helsinki. All human subjects provided signed informed consent. And the animal experiments approved by Qingdao University Laboratory Animal Welfare Ethics Committee (No. 201809SD18202012014).

Transcriptome-wide N6-methyladenosine methylation landscape of coronary artery disease.

Aim: To reveal transcriptome-wide N6-methyladenosine (m6A) methylome of coronary artery disease (CAD). Materials & methods: The m6A levels of RNA from peripheral blood mononuclear cellsmeasured by colorimetry were significantly decreased in CAD cases. Transcriptome-wide m6A methylome profiled bymethylated RNA immunoprecipitation sequencing (MeRIP-seq) identified differentially methylated m6A sites within both mRNAs and lncRNAs between CADand control group. Results: Bioinformatic analysis indicated that differentially methylated genes were involved in the pathogenesis of atherosclerosis. MeRIP-quantitative real-time PCRassay confirmed the reliability of MeRIP-seq data. Finally, the rat carotid artery balloon injury model was performed to confirm the role of m6A demethylase FTO in neointima formation. Conclusion: Our study provided a resource of differentially methylated m6A profile for uncovering m6A biological functions in the pathogenesis of CAD.

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application.

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

Natural History of the Impaired Arterial Wall Healing Under Diabetic Conditions in the Rat Carotid Artery Balloon Injury Model

Patients with type 2 diabetes mellitus have an increased cardiovascular morbidity and mortality and amplified risk of postoperative complications following vascular interventions. In this study, we aim to explore the molecular and physiological mechanisms behind the impaired arterial wall healing in type 2 diabetes mellitus.

LncRNA GAS5 regulates vascular smooth muscle cell cycle arrest and apoptosis via p53 pathway

Vascular remodeling is a pathological process following cardiovascular intervention. Vascular smooth muscle cells (VSMC) play a critical role in the vascular remodeling. Long noncoding RNAs (lncRNA) are a class of gene regulators functioning through various mechanisms in physiological and pathological conditions. By using cultured VSMC and rat carotid artery balloon injury model, we found that lncRNA growth arrest specific 5 (GAS5) serves as a negative regulator for VSMC survival in vascular remodeling. By manipulating GAS5 expression via adenoviral overexpression or short hairpin RNA knockdown, we found that GAS5 suppresses VSMC proliferation while promoting cell cycle arrest and inducing cell apoptosis. Mechanistically, GAS5 directly binds to p53 and p300, stabilizes p53-p300 interaction, and thus regulates VSMC cell survival via induction of p53-downstream target genes. Importantly, local delivery of GAS5 via adenoviral vector suppresses balloon injury-induced neointima formation along with an increased expression of p53 and apoptosis in neointimal SMCs. Our study demonstrated for the first time that GAS5 negatively impacts VSMC survival via activation the p53 pathway during vascular remodeling.

Dioscin inhibits intimal hyperplasia in rat carotid artery balloon injury model through inhibition of the MAPK-FoxM1 pathway

Vascular smooth muscle cell (VSMC) proliferation and migration are crucial events in the pathological course of atherosclerosis and restenosis after percutaneous coronary intervention (PCI). Dioscin has been shown to exhibit powerful cardiovascular protective effects and potent therapeutic potential in cancer owing to the inhibition of cell proliferation and migration. However, its effects on arterial wall hypertrophy-related diseases caused by VSMC proliferation and migration remain unclear. In this study, we investigated the effects of dioscin on intimal hyperplasia after balloon injury in vivo, its effects on VSMC proliferation and migration in vitro, and the mechanisms underlying these effects. Results showed that dioscin treatment significantly inhibited VSMC proliferation and intimal thickening after balloon injury. In cultured VSMCs, treatment with dioscin significantly decreased fetal bovine serum or platelet-derived growth factor-induced cell proliferation and migration. Moreover, dioscin inhibited the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and expression of Forkhead box M1 (FoxM1) and its downstream target genes. FoxM1 knockdown with shRNA partially counteracted the inhibitory effects of dioscin on cell proliferation and migration. In conclusion, we demonstrated that dioscin attenuated neointima formation in response to balloon injury by suppressing VSMC proliferation and migration through MAPK-FoxM1 pathway. Our data suggested that dioscin might be a potential therapeutic agent for atherosclerosis and restenosis after PCI.

Noninvasive in vivo Assessment of the Re-endothelialization Process Using Ultrasound Biomicroscopy in the Rat Carotid Artery Balloon Injury Model.

Ultrasound biomicroscopy (UBM), or ultra high-frequency ultrasound, is a technique used to assess the anatomy of small research animals. In this study, UBM was used to assess differences in intimal hyperplasia thickness as a surrogate measurement of the re-endothelialization process after carotid artery balloon injury in rats. Ultrasound biomicroscopic data from 3 different experiments and rat strains (Sprague Dawley, Wistar, and diabetic Goto-Kakizaki) were analyzed. All animals were subjected to carotid artery balloon injury and examined with UBM (30-70 MHz) 2 and 4 weeks after injury. Re-endothelialization on UBM was defined as the length from the carotid bifurcation to the most distal visible edge of the intimal hyperplasia. En face staining with Evans blue dye was performed at euthanasia 4 weeks after injury, followed by tissue harvesting for histochemical and immunohistochemical evaluations. A significant correlation (Spearman r = 0.63; P < .0001) was identified when comparing all measurements of re-endothelialization obtained from UBM and en face staining. The findings revealed a similar pattern for all rat strains: Sprague Dawley (Spearman r = 0.70; P < .0001), Wistar (Spearman r = 0.36; P < .081), and Goto-Kakizaki (Spearman r = 0.70; P < .05). A Bland-Altman test showed agreement between en face staining and UBM. Immunohistochemical staining confirmed the presence of the endothelium in the areas detected as re-endothelialized by the UBM assessment. Ultrasound biomicroscopy can be used for repeated in vivo assessment of re-endothelialization after carotid artery balloon injury in rats.

Proresolving Lipid Mediators Resolvin D1 and Protectin D1 Isomer Attenuate Neointimal Hyperplasia in the Rat Carotid Artery Balloon Injury Model

BackgroundSpecialized proresolving mediators from ω-3 polyunsaturated fatty acid may control resolution of inflammation. We evaluated the influence of two specialized proresolving mediators, resolvin D1 (RvD1) and protectin D1 isomer (PD1 iso) on neointimal hyperplasia after balloon injury. Materials and methodsSprague Dawley male rats at 12-14 wk of age were injured as a model of balloon angioplasty. Then, 1 μg/rat of RvD1 or PD1 iso was administered intravenously via the tail vein immediately and 2 d after angioplasty. The proliferation of injured artery and the infiltration of leukocytes, monocytes, and macrophages at 3 d after injury were evaluated by immunostaining. The activity of the inflammatory transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) in the injured artery at 3 d after injury was evaluated using an enzyme-linked immuno sorbent assay kit. The proliferation of the neointima was evaluated by calculating the ratio of the neointimal and medial areas using specimens at 14 d after injury. ResultsRvD1 and PD1 iso attenuated proliferation of medial cells (P < 0.05) and infiltration of leukocytes (P < 0.05) and monocytes/macrophages (P < 0.01). Although both RvD1 and PD1 iso mitigated NFκB activity (P < 0.01), RvD1 attenuated this activity more strongly (P < 0.01). RvD1 decreased neointimal hyperplasia by 37.3% (P < 0.01), whereas PD1 iso decreased neointimal hyperplasia by 31.8% (P < 0.05) (RvD1 versus PD1 iso: P = 0.51). ConclusionsRvD1 and PD1 iso reduced the activity of inflammatory transcription factor NFκB within the injured artery and attenuated inflammatory cell infiltration, leading to a reduction in early inflammation and subsequent neointimal hyperplasia.

Exosomes of Endothelial Progenitor Cells Inhibit Neointima Formation After Carotid Artery Injury

BackgroundExosomes released from endothelial progenitor cells (EPCs) play a protective role in various disease models. Both endothelial cell (EC) damage and smooth muscle cell (SMC) proliferation are involved in the pathological process of restenosis after angioplasty and stenting. Few studies have focused on the therapeutic role of exosomes in EC damage and SMC proliferation. In this study, we sought to investigate the effect of exosomes released by human fetal aorta-derived EPCs on the rat carotid artery balloon injury model in vivo. We also sought to determine the effect of exosomes on both ECs and SMCs in vitro. MethodsExosomes (Exo group) or saline (Con group) were injected in rat carotid balloon injury model animals. The rats were sacrificed after 2, 4, 14, and 28 d, and injured carotid specimens were collected for Evans blue staining, hematoxylin-eosin staining, and immunohistochemistry. ResultsWhen the Con group and the Exo group were compared, the reendothelialized areas were not significantly different after 2 or 4 d, as shown by Evans blue staining. The hematoxylin-eosin results showed that the intimal to medial area ratio was slightly but not significantly higher in the Exo group after 2 and 4 d. The immunohistochemistry results showed that the proliferation of SMCs was slightly higher in the Exo group after 2 and 4 d, but the difference was not significant. The reendothelialization area of the Con group was significantly smaller than that of the Exo group at day 14. Both the intimal to medial area ratio and SMC proliferation in the Exo group were significantly smaller than those of the Con group at 14 or 28 d. In the in vitro study, exosome treatment significantly enhanced the proliferation and migration of both ECs and SMCs. ConclusionsExosomes derived from EPCs could inhibit neointimal hyperplasia after carotid artery injury in rats. The protective effect of exosomes may manifest through the promotion of EC repair rather than direct suppression of proliferation and migration of smooth muscles cells.

Abstract 505: Targeting FOXM1 in Vascular Smooth Muscle Cells Induces Apoptotic Cell Death

Objectives: Vascular smooth muscle cell (VSMC) proliferation and dedifferentiation are key contributors to the initiation and progression of vascular diseases including restenosis. Forkhead Box M1 (FOXM1) is a proliferation-associated transcription factor shown to play a role in a variety of biological processes including cell cycle progression, cell survival, and apoptosis in many cell types. However, the role of FOXM1 in VSMC phenotypic transformation as a result of vascular injury following balloon angioplasty has not been studied. We hypothesize that FOXM1 modulates VSMC response by enhancing pro-proliferative and pro-survival signaling following vascular injury. Methods &amp; Results: The rat carotid artery balloon injury was used to model vascular injury. Immunofluorescence staining was carried out on sections from injured carotid arteries or uninjured controls collected at 3, 7, or 14 days post injury (dpi). We observed that FOXM1 expression was upregulated in injured arteries at 7 and 14 dpi. Expression of FOXM1 in injured arteries co-localized with PCNA, a marker of proliferation. The upregulation of FOXM1 protein was replicated in cultured rat VSMCs by serum stimulation. Using thymidine synchronization, we observed that FOXM1 expression followed a cell cycle specific pattern in vitro . Chemical inhibition of FOXM1 using thiostrepton or FDI-6, or via siRNA resulted in decreased VSMC viability as measured by CCK-8, induction of apoptosis as measured by flow cytometry, and cleaved caspase 3 induction. More detailed immunocytochemistry analysis revealed that FDI-6 treated VSMCs exhibit disorganized microtubule networks. Furthermore, FDI-6 treatment decreased expression of Eg5, a motor protein essential for microtubule orientation and critical for mitosis execution. Additionally, FOXM1 inhibition decreased levels of β-catenin, a known activator of cell cycle-related genes and proliferation. Conclusions: In conclusion, our data suggest FOXM1 is critical to VSMC viability and may modulate VSMC proliferation and survival via regulation of important cell cycle and pro-proliferative proteins, and therefore may serve as a novel therapeutic target to prevent VSMC pathophysiology in the context of restenosis.

Niclosamide inhibits vascular smooth muscle cell proliferation and migration and attenuates neointimal hyperplasia in injured rat carotid arteries.

The anti-helminthic drug niclosamide regulates multiple cellular signals including STAT3, AMP-activated protein kinase (AMPK), Akt, Wnt/β-catenin and mitochondrial uncoupling which are involved in neointimal hyperplasia. Here we have examined the effects of niclosamide on vascular smooth muscle cell proliferation, migration and neointimal hyperplasia and assessed the potential mechanisms. Cell migration was measured by using wound-induced migration assay and Boyden chamber assay. Protein levels were measured by using Western blot technique. Neointimal hyperplasia in vivo was induced in rats by balloon injury to the carotid artery. Niclosamide treatment inhibited serum-induced (15% FBS) and PDGF-BB-induced proliferation and migration of vascular smooth muscle cells (A10 cells). Niclosamide showed no cytotoxicity at anti-proliferative concentrations, but induced cell apoptosis at higher concentrations. Niclosamide treatment inhibited serum-induced (15% FBS) and PDGF-BB-induced STAT3 activation (increased protein levels of p-STAT3 at Tyr705 ) but activated AMPK, in A10 cells. Niclosamide exerted no significant effects on β-catenin expression and the activities of ERK1/2 and Akt in A10 cells. Injection (i.p.) of soluble pegylated niclosamide (PEG5000-niclosamide) (equivalent to niclosamide 25mg·kg-1 ) attenuated neointimal hyperplasia following balloon-injury in rat carotid arteries in vivo. Niclosamide inhibited vascular smooth muscle cell proliferation and migration and attenuated neointimal hyperplasia in balloon-injured rat carotid arteries through a mechanism involving inhibition of STAT3.

Activation and Migration of Adventitial Fibroblasts Contributes to Vascular Remodeling.

The rat carotid artery balloon injury model was used to prove the activation and migration of adventitial fibroblasts. We found that at day 7 after injury, adventitial fibroblasts proliferated, transformed into myofibroblasts under transmission electron microscopy in the model group. Simultaneously, we proved that the adventitial cells migrated to the media and intima on seventh day after injury by directly labeled the adventitial cells by the in vivo gene transfer technique. Moreover, we captured the precise moment when the adventitial fibroblasts migrated from the adventitia to the media through the external elastic plate under transmission electron microscope. This study provides direct evidences that adventitial fibroblasts activate and migrate to the media and intima, then actively take part in revascularization. Anat Rec, 301:1216-1223, 2018. © 2018 Wiley Periodicals, Inc.

MicroRNA-125b Affects Vascular Smooth Muscle Cell Function by Targeting Serum Response Factor

Background/Aims: Increasing evidence links microRNAs to the pathogenesis of peripheral vascular disease. We recently found microRNA-125b (miR-125b) to be one of the most significantly down‑regulated microRNAs in human arteries with arteriosclerosis obliterans (ASO) of the lower extremities. However, its function in the process of ASO remains unclear. This study aimed to investigate the expression, regulatory mechanisms, and functions of miR-125b in the process of ASO. Methods: Using the tissue explants adherent method, vascular smooth muscle cells (VSMCs) were prepared for this study. A rat carotid artery balloon injury model was constructed to simulate the development of vascular neointima, and a lentiviral transduction system was used to overexpress serum response factor (SRF) or miR-125b. Quantitative real‑time PCR (qRT‑PCR) was used to detect the expression levels of miR‑125b and SRF mRNA. Western blotting was performed to determine the expression levels of SRF and Ki67. In situ hybridization analysis was used to analyze the location and expression levels of miR-125b. CCK-8 and EdU assays were used to assess cell proliferation, and transwell and wound closure assays were performed to measure cell migration. Flow cytometry was used to evaluate cell apoptosis, and a dual-luciferase reporter assay was conducted to examine the effects of miR‑125b on SRF. Immunohistochemistry and immunofluorescence analyses were performed to analyze the location and expression levels of SRF and Ki67. Results: miR-125b expression was decreased in ASO arteries and platelet-derived growth factor (PDGF)-BB-stimulated VSMCs. miR-125b suppressed VSMC proliferation and migration but promoted VSMC apoptosis. SRF was determined to be a direct target of miR-125b. Exogenous miR-125b expression modulated SRF expression and inhibited vascular neointimal formation in balloon-injured rat carotid arteries. Conclusions: These findings demonstrate a specific role of the miR-125b/SRF pathway in regulating VSMC function and suggest that modulating miR-125b levels might be a novel approach for treating ASO.

Novel Zinc Finger Transcription Factor ZFP580 Facilitates All-Trans Retinoic Acid -Induced Vascular Smooth Muscle Cells Differentiation by Rarα-Mediated PI3K/Akt and ERK Signaling

Background/Aims: Phenotypic switching of vascular smooth muscle cells (VSMC) plays a vital role in the development of vascular diseases. All-trans retinoic acid (ATRA) is known to regulate VSMC phenotypes. However, the underlying mechanisms remain completely unknown. Here, we have investigated the probable roles and underlying mechanisms of the novel C2H2 zinc finger transcription factor ZFP580 on ATRA-induced VSMC differentiation. Methods: VSMCs were isolated, cultured, and identified. VSMCs were infected with an adenovirus encoding ZFP580 or Ad-siRNA to silence ZFP580. The expression levels of ZFP580, SMα-actin, SM22α, SMemb, RARα, RARβ, and RARγ were assayed by Q-PCR and western blot. A rat carotid artery injury model and morphometric analysis of intimal thickening were also used in this study. Results: ATRA caused a significant reduction of VSMC proliferation and migration in a doseand time-dependent manner. Moreover, it promoted VSMC differentiation by enhancing expression of differentiation markers and reducing expression of dedifferentiation markers. This ATRA activity was accompanied by up-regulation of ZFP580, with concomitant increases in RARα expression. In contrast, silencing of the RARα gene or inhibiting RARα with its antagonist Ro41-5253 abrogated the ATRA-induced ZFP580 expression. Furthermore, ATRA binding to RARα induced ZFP580 expression via the PI3K/Akt and ERK pathways. Adenovirusmediated overexpression of ZFP580 promoted VSMC differentiation by enhancing expression of SM22α and SMα-actin and reducing expression of SMemb. In contrast, silencing ZFP580 dramatically reduced the expression of differentiation markers and increased expression of dedifferentiation markers. The classic rat carotid artery balloon injury model demonstrated that ZFP580 inhibited proliferation and intimal hyperplasia in vivo. Conclusion: The novel zinc finger transcription factor ZFP580 facilitates ATRA-induced VSMC differentiation by the RARα-mediated PI3K/Akt and ERK signaling pathways. This might represent a novel mechanism of regulation of ZFP580 by ATRA and RARα, which is critical for understanding the biological functions of retinoids during VSMC phenotypic modulation.