Balloon-injured Rat Carotid Arteries Research Articles

Matrix-Binding, N-Cadherin-Targeting Chimeric Peptide Inhibits Intimal Thickening but Not Endothelial Repair in Balloon-Injured Carotid Arteries.

Treatment of occluded vessels can involve angioplasty, stenting, and bypass grafting, which can be limited by restenosis and thrombosis. Drug-eluting stents attenuate restenosis, but the current drugs used are cytotoxic, causing smooth muscle cell (SMC) and endothelial cell (EC) death that may lead to late thrombosis. N-cadherin is a junctional protein expressed by SMCs, which promotes directional SMC migration contributing to restenosis. We propose that engaging N-cadherin with mimetic peptides can act as a cell type-selective therapeutic strategy to inhibit polarization and directional migration of SMCs without negatively impacting ECs. We designed a novel N-cadherin-targeting chimeric peptide with a histidine-alanine-valine cadherin-binding motif, combined with a fibronectin-binding motif from Staphylococcus aureus. This peptide was tested in SMC and EC culture assays of migration, viability, and apoptosis. Rat carotid arteries were balloon injured and treated with the N-cadherin peptide. Treating scratch-wounded SMCs with the N-cadherin-targeting peptide inhibited migration and reduced polarization of wound-edge cells. The peptide colocalized with fibronectin. Importantly, EC junction, permeability, or migration was not impacted by peptide treatment in vitro. We also demonstrated that the chimeric peptide persisted for 24 hours after transient delivery in the balloon-injured rat carotid artery. Treatment with the N-cadherin-targeting chimeric peptide reduced intimal thickening in balloon-injured rat carotid arteries at 1 and 2 weeks after injury. Reendothelialization of injured vessels after 2 weeks was unimpaired by peptide treatment. These studies show that an N-cadherin-binding and fibronectin-binding chimeric peptide is effective in inhibiting SMC migration in vitro and in vivo and limiting neointimal hyperplasia after balloon angioplasty without affecting EC repair. These results establish the potential of an advantageous SMC-selective strategy for antirestenosis therapy.

Tissue-Engineered Endothelial Cells Induce Sustained Vascular Healing Through Early Induction of Vascular Repair

BackgroundPerivascular implantation of tissue-engineered endothelial cells (TEEC) after vascular injury profoundly inhibits neointimal hyperplasia. However, the time course and mechanism by which this effect occurs remain unknown. By developing genetically modified TEEC that express a “suicide gene,” we can control the time during which the TEEC could exert their effect and determine the length of time TEEC need to be present following vascular injury to exert their inhibitory effect on long-term neointimal hyperplasia.MethodsBovine aortic endothelial cells (BAE) were transfected with the human herpes simplex virus thymidine kinase (tk) gene to render them sensitive to ganciclovir (GCV). These BAE+tk were grown to confluence on Gelfoam and shown to have the same growth kinetics and biologic potency as control cells but were sensitive to GCV at low concentrations. The BAE+tk were grown on Gelfoam and placed in the perivascular space around balloon-injured rat carotid arteries. Rats randomly received BAE-tk, BAE+tk, or nothing (control) after balloon injury. GCV was administered early (days 1–7), late (days 5–11), or not at all.ResultsTwo weeks after injury, extensive neointimal hyperplasia was observed in control animals with an intima:media (I:M) area ratio of 0.80 ± 0.19. Early administration of GCV killed the BAE in constructs with TK sensitivity and eliminated the impact of TEEC regulation of intimal hyperplasia (0.45 ± 0.06). Intimal hyperplasia was still effectively reduced in animals with implants containing BAE-tk or BAE+tk which received GCV late (0.11 ± 0.04 and 0.19 ± 0.05). Immunohistochemistry demonstrated the lethal effect of GCV on TK-sensitive cells.ConclusionsThe application of perivascular TEEC for only the first few days after injury had a significant inhibitory effect on intimal hyperplasia. This is in contrast to the results obtained in this same animal model with the infusion of isolated anti-smooth muscle cell proliferative agents. This suggests that the mechanism of action of TEEC may be upstream from smooth muscle cell proliferation. Moreover, the use of this technique to further elucidate biologic mechanisms will prove invaluable in the tissue engineering field.Lay SummaryWe report a novel, genetically altered tissue-engineered endothelial cell (TEEC) implant that inhibits neointimal hyperplasia after experimental vascular injury. The viability of these implants can be carefully controlled and suggest a putative mechanism by which TEEC recapitulate control over the vascular response to injury.

Plantamajoside Attenuates Neointima Formation via Upregulation of Tissue Inhibitor of Metalloproteinases in Balloon-Injured Rats.

The abnormal change of vascular smooth muscle cell (VSMC) behavior is an important cellular event leading to neointimal hyperplasia in atherosclerosis and restenosis. Plantamajoside (PMS), a phenylethanoid glycoside compound of the Plantago asiatica, has been reported to have anti-inflammatory, antioxidative, and anticancer activities. In this study, the protective effects of PMS against intimal hyperplasia and the mechanisms underlying the regulation of VSMC behavior were investigated. MTT and BrdU assays were performed to evaluate the cytotoxicity and cell proliferative activity of PMS, respectively. Rat aortic VSMC migrations after treatment with the determined concentration of PMS (50 and 150 μM) were evaluated using wound healing and Boyden chamber assays. The inhibitory effects of PMS on intimal hyperplasia were evaluated in balloon-injured (BI) rat carotid artery. PMS suppressed the proliferation in platelet-derived growth factor-BB-induced VSMC, as confirmed from the decrease in cyclin-dependent kinase (CDK)-2, CDK-4, cyclin D1, and proliferating cell nuclear antigen levels. PMS also inhibited VSMC migration, consistent with the downregulated expression and zymolytic activities of matrix metalloproteinase (MMP)2, MMP9, and MMP13. PMS specifically regulated MMP expression through p38 mitogen-activated protein kinase and focal adhesion kinase pathways. Tissue inhibitor of metalloproteinase (TIMP)1 and TIMP2 levels were upregulated via Smad1. TIMPs inhibited the conversion of pro-MMPs to active MMPs. PMS significantly inhibited neointimal formation in BI rat carotid arteries. In conclusion, PMS inhibits VSMC proliferation and migration by upregulating TIMP1 and TIMP2 expression. Therefore, PMS could be a potential therapeutic agent for vascular atherosclerosis and restenosis treatment.

CircSOD2: A Novel Regulator for Smooth Muscle Proliferation and Neointima Formation.

Vascular smooth muscle cell (SMC) proliferation contributes to neointima formation following vascular injury. Circular RNA-a novel type of noncoding RNA with closed-loop structure-exhibits cell- and tissue-specific expression patterns. However, the role of circular RNA in SMC proliferation and neointima formation is largely unknown. The objective of this study is to investigate the role and mechanism of circSOD2 in SMC proliferation and neointima formation. Approach and Results: Circular RNA profiling of human aortic SMCs revealed that PDGF (platelet-derived growth factor)-BB up- and downregulated numerous circular RNAs. Among them, circSOD2, derived from back-splicing event of SOD2 (superoxide dismutase 2), was significantly enriched. Knockdown of circSOD2 by short hairpin RNA blocked PDGF-BB-induced SMC proliferation. Inversely, circSOD2 ectopic expression promoted SMC proliferation. Mechanistically, circSOD2 acted as a sponge for miR-206, leading to upregulation of NOTCH3 (notch receptor 3) and NOTCH3 signaling, which regulates cyclin D1 and CDK (cyclin-dependent kinase) 4/6. In vivo studies showed that circSOD2 was induced in neointima SMCs in balloon-injured rat carotid arteries. Importantly, knockdown of circSOD2 attenuated injury-induced neointima formation along with decreased neointimal SMC proliferation. CircSOD2 is a novel regulator mediating SMC proliferation and neointima formation following vascular injury. Therefore, circSOD2 could be a potential therapeutic target for inhibiting the development of proliferative vascular diseases.

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.

P.059 Identifying the role of trace amine-associated receptor 9 in behaviour, brain neurochemistry and blood biochemistry

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.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.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 β-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.FOXM1 is an important molecular mediator of IH that contributes to the proliferation and survival of SMCs following vascular injury.

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

BackgroundAccelerated 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. ObjectiveWe 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 resultsFOXM1 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 β-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. ConclusionsFOXM1 is an important molecular mediator of IH that contributes to the proliferation and survival of SMCs following vascular injury.

Dendropanax morbifera Extract Inhibits Intimal Hyperplasia in Balloon-Injured Rat Carotid Arteries by Modulating Phenotypic Changes in Vascular Smooth Muscle Cells

Dendropanax morbifera Extract Inhibits Intimal Hyperplasia in Balloon-Injured Rat Carotid Arteries by Modulating Phenotypic Changes in Vascular Smooth Muscle Cells

Chronological Change of Vascular Reactivity to cGMP Generators in the Balloon-Injured Rat Carotid Artery

Background/Aims: Soluble guanylate cyclase (sGC) exists as reduced, oxidized, and heme-free forms. Currently, it is unclear whether endovascular mechanical stenosis has an impact on vascular tone control by drugs targeting sGC, namely cGMP generators. Methods: Pharmacological responses to acidified sodium nitrite (reduced sGC stimulant) and BAY 60-2770 (oxidized/heme-free sGC stimulant) were studied in balloon-injured rat carotid arteries at several time points. In addition, sGC expression was detected by immunohistochemistry. Results: At 1 day after injury, acidified sodium nitrite-induced relaxation was attenuated in the injured artery, whereas BAY 60-2770-induced relaxation was augmented. Similar attenuation of response to acidified sodium nitrite was seen at 7 and 14 days after injury. On the other hand, the augmentation of response to BAY 60-2770 disappeared at 7 and 14 days after injury. At 1 day after injury, the immunohistochemical expression pattern of sGC in the smooth muscle layer of the injured artery was not different from that of the uninjured artery. However, in the injured artery, the intensity of sGC staining was weak at 7 and 14 days after injury. Conclusion: Balloon injury alters vascular responsiveness to cGMP generators, which seems to be associated with the form and/or expression of sGC.

Protective Role of RNA Helicase DEAD-Box Protein 5 in Smooth Muscle Cell Proliferation and Vascular Remodeling.

RNA helicases, highly conserved enzymes, are currently believed to be not only involved in RNA modulation, but also in other biological processes. We recently reported that RNA helicase DDX (DEAD-box protein)-5 is required for maintaining the homeostasis of vascular smooth muscle cells (SMCs). However, the expression and function of RNA helicase in vascular physiology and disease is unknown. To investigate the role of RNA helicase in vascular diseases. We showed here that DDX-5 was the most abundant DEAD-box protein expressed in human and rodent artery, which mainly located in SMCs. It was demonstrated that DDX-5 levels were reduced in cytokine-stimulated SMCs and vascular lesions. DDX-5 knocking down or deficiency increased SMC proliferation and migration, whereas overexpression of DDX-5 prevented aberrant proliferation and migration of SMCs. Mechanistic studies revealed transcription factor GATA (GATA-binding protein)-6 as a novel downstream target of DDX-5, which directly interacted with GATA-6 and protected it from MDM (mouse double minute)-2-mediated degradation. Our ChIP assay identified a previously unreported binding of p27Kip1 promoter to GATA-6. DDX-5 increased the recruitment of GATA-6 to p27Kip1 promoter, which enhanced p27Kip1 expression and maintained SMC quiescence. Finally, we showed exacerbated neointima formation in DDX-5 SMC-deficient mice after femoral artery injury, whereas overexpression of DDX-5 potently inhibited vascular remodeling in balloon-injured rat carotid artery. These findings provide the first evidence for a role of RNA helicase DDX-5 in the protection against SMC proliferation, migration, and neointimal hyperplasia. Our data extend the fundamental role of RNA helicase beyond RNA modulation, which provides the basic information for new therapeutic strategies for vascular diseases.

Downregulation of the transcriptional co-activator PCAF inhibits the proliferation and migration of vascular smooth muscle cells and attenuates NF-κB-mediated inflammatory responses

P300/CBP-associated factor (PCAF) regulates vascular inflammation. This study was to explore the effect of PCAF on the proliferation and migrationof vascular smooth muscle cells (VSMCs) and neointimal hyperplasia in balloon-injured rat carotid artery. Downregulation of PCAF remarkably suppressed VSMCs proliferation and migration induced by lipopolysaccharide, and also significantly inhibit the nuclear translocation of nuclear factor-kappaB p65. Meanwhile, downregulation of PCAF inhibited the mRNA expression of tumor necrosis factor-α and interleukin-6, and also the levels in culture supernatants. Moreover, downregulation of PCAF profoundly reduced the intima area and the ratio of intima area to media area in balloon-injured rat carotid artery. In addition, the expression of PCNA and NF-κB p65 in intima were decreased by downregulation of PCAF. These results highlight that PCAF may be a potential target for prevention and treatment of neointimal hyperplasia and restenosis after angioplasty.

CircDiaph3 regulates rat vascular smooth muscle cell differentiation, proliferation, and migration.

Intimal hyperplasia is a reaction to vascular injury, which is the primary reason for vascular restenosis caused by the diagnostic or therapeutic procedure for cardiovascular diseases. Circular RNAs (circRNAs) are known to be associated with several cardiovascular conditions, but the expression of circRNAs in the neointima has not been reported in detail. In this study, we established the balloon-injured rat carotid artery model and detected the expression of circRNAs in the carotid arteries with a microarray. We found that the circRNA expression profile of the healthy carotid arteries and the injured arteries were significantly different. We investigated the role of rno-circ_005717 ( circDiaph3) in the differentiation of rat vascular smooth muscle cells (VSMCs). We found that knockdown of circDiaph3 up-regulated the level of diaphanous-related formin-3 and promoted the differentiation of VSMCs to contractile type. In addition, circDiaph3 up-regulated the transcription of Igf1r and supported the proliferation and migration of VSMCs. circDiaph3 could be a molecular target to combat intimal hyperplasia.-Xu, J.-Y., Chang, N.-B., Rong, Z.-H., Li, T., Xiao, L., Yao, Q.-P., Jiang, R., Jiang, J. circDiaph3 regulates rat vascular smooth muscle cell differentiation, proliferation, and migration.

GW29-e1749 Delayed reendothelialization with rapamycin is rescued by the addition of nicorandil in balloon-injured rat carotid arteries

We build the hypothesis that nicorandil may promote reendothelialization impaired by rapamycin through inhibiting XO-generated ROS formation and promoting eNOS expression. In vivo carotid balloon injury model and in vitro CMECs were used to indentify this hypothesis. Sprague-Dawley (SD) rats (male

Xenobiotic pregnane X receptor promotes neointimal formation in balloon-injured rat carotid arteries.

Pregnane X receptor (PXR) is a member of nuclear receptor superfamily and responsible for the detoxification of xenobiotics. Recent studies demonstrated that PXR was also expressed in the vasculature and protected the vessels from endogenous and exogenous insults, thus representing a novel gatekeeper in vascular defense. In this study, we examined the potential function of PXR in the neointimal formation following vascular injury. In the rat carotid artery after balloon injury, overexpression of a constitutively active PXR increased the intima-to-media ratio in the injured region. PXR increased cell proliferation and migration in cultured rat aortic smooth muscle cells (SMCs) by inducing the expressions of cyclins (cyclin A, D1, and E) and cyclin-dependent kinase 2. In addition, PXR increased the phosphorylation and activation of extracellular-signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). Inactivation of ERK1/2 and p38 MAPK pathways using selective inhibitors (U0126 and SB203580) abrogated PXR-induced SMC proliferation and migration. Furthermore, cigarette smoke particles (CSP) activated PXR in SMCs. Knockdown of PXR by small interfering RNA suppressed the cell proliferation, migration, and activation of the MAPK pathways by CSP. These findings suggested a novel role for PXR in promoting SMC proliferation and migration, and neointimal hyperplasia. Therefore, PXR may be a potential therapeutic target for vascular disease related to xenobiotics such as cigarette smoking and other environmental pollutants.

MicroRNA-132 targeting PTEN contributes to cilostazol-promoted vascular smooth muscle cell differentiation

Background and aimsCilostazol, beyond its antiplatelet effect, is also capable of promoting vascular smooth muscle cell (VSMC) differentiation. The aim of this study was to explore the potential role of PTEN, known to associate with VSMC differentiation, and its related microRNA (miRNA) in cilostazol-dependent effects. Methods and resultsMicroarray analysis in balloon-injured rat carotid arteries comparing with and without balloon injury revealed that miR-132 was differentially expressed. Bioinformatic analysis predicts PTEN as a novel target of miR-132. Western blot and quantitative real-time reverse transcription-polymerase chain reaction along with in situ hybridization documented that cilostazol treatment enhanced PTEN and reduced miR-132 expression in the neointima of balloon-injured arteries. Treatment of cultured rat VSMCs with cilostazol resulted in the up-regulation of PTEN mRNA and the down-regulation of miR-132, supporting an in vitro relevance. Co-transfection experiments showed that transfection of miR-132 mimic into VSMCs suppressed PTEN 3′UTR activities, further reflecting that PTEN is the direct target of miR-132. Over-expression of miR-132 in VSMCs led to an attenuation of cilostazol-induced PTEN and its downstream VSMC differentiation marker (calponin) expression, confirming the critical role of miR-132 in VSMC differentiation. Transient transfection studies demonstrated that cilostazol reduced the activity of miR-132 promoter, which was mediated via cyclic AMP response element-binding protein. Notably, the use of lentivirus to over-express miR-132 in the neointima of balloon-injured arteries could reverse the effect of cilostazol in vivo. ConclusionsThese results suggest that miR-132 by targeting PTEN may be an important regulator in mediating cilostazol actions on VSMC differentiation.

O-Linked β-N-Acetylglucosamine Modification of A20 Enhances the Inhibition of NF-κB (Nuclear Factor-κB) Activation and Elicits Vascular Protection After Acute Endoluminal Arterial Injury.

Recently, we have demonstrated that acute glucosamine-induced augmentation of protein O-linked β-N-acetylglucosamine (O-GlcNAc) levels inhibits inflammation in isolated vascular smooth muscle cells and neointimal formation in a rat model of carotid injury by interfering with NF-κB (nuclear factor-κB) signaling. However, the specific molecular target for O-GlcNAcylation that is responsible for glucosamine-induced vascular protection remains unclear. In this study, we test the hypothesis that increased A20 (also known as TNFAIP3 [tumor necrosis factor α-induced protein 3]) O-GlcNAcylation is required for glucosamine-mediated inhibition of inflammation and vascular protection. In cultured rat vascular smooth muscle cells, both glucosamine and the selective O-linked N-acetylglucosaminidase inhibitor thiamet G significantly increased A20 O-GlcNAcylation. Thiamet G treatment did not increase A20 protein expression but did significantly enhance binding to TAX1BP1 (Tax1-binding protein 1), a key regulatory protein for A20 activity. Adenovirus-mediated A20 overexpression further enhanced the effects of thiamet G on prevention of TNF-α (tumor necrosis factor-α)-induced IκB (inhibitor of κB) degradation, p65 phosphorylation, and increases in DNA-binding activity. A20 overexpression enhanced the inhibitory effects of thiamet G on TNF-α-induced proinflammatory cytokine expression and vascular smooth muscle cell migration and proliferation, whereas silencing endogenous A20 by transfection of specific A20 shRNA significantly attenuated these inhibitory effects. In balloon-injured rat carotid arteries, glucosamine treatment markedly inhibited neointimal formation and p65 activation compared with vehicle treatment. Adenoviral delivery of A20 shRNA to the injured arteries dramatically reduced balloon injury-induced A20 expression and inflammatory response compared with scramble shRNA and completely abolished the vascular protection of glucosamine. These results suggest that O-GlcNAcylation of A20 plays a key role in the negative regulation of NF-κB signaling cascades in TNF-α-treated vascular smooth muscle cells in culture and in acutely injured arteries, thus protecting against inflammation-induced vascular injury.

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.

Photodynamic therapy of balloon-injured rat carotid arteries using indocyanine green.

Photodynamic therapy (PDT) has been used to inhibit intimal hyperplasia in injured arteries. Because of the limited tissue penetration of visible light, an endovascular light source with a guided wire is often required for effective treatment. Indocyanine green (ICG), a near-infrared (NIR) photosensitizer, has been used in PDT for cancers. An extracorporeal light source may be used for shallow tissue because of the better tissue penetration of NIR light. The aim of this study was to evaluate the effect of ICG-PDT using extracorporeal NIR light on the inhibition of intimal hyperplasia in balloon-injured carotid arteries. A balloon injury (BI) model was used to induce intimal hyperplasia of carotid artery. Sprague-Dawley rats were divided into control, BI, BI + 1 × PDT, and BI + 2 × PDT groups. The control group underwent a sham procedure. PDT was performed 7days after BI. In the BI + 1 × PDT group, ICG was administered 1h before light irradiation. External illumination with 780-nm light-emitting diode light at a fluence of 4J/cm2 was applied. For the BI + 2 × PDT group, PDT was performed again at day 7, following the first PDT. Hematoxylin and eosin (H & E) staining was performed to assess vessel morphology. Arterial wall thickness was significantly larger in the BI group compared with the control group. ICG-PDT significantly reduced arterial wall thickness compared with the BI group. Repeated PDT further decreased arterial wall thickness to the level of the control group. These findings indicate a promising approach for the treatment of restenosis of carotid arteries.

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.

Desalted Salicornia europaea extract attenuated vascular neointima formation by inhibiting the MAPK pathway-mediated migration and proliferation in vascular smooth muscle cells

Salicornia europaea L. (SE) has been used as folk medicine for the treatment of various diseases such as obesity, diabetes, and cancer. However, its effects on atherosclerotic events in vascular smooth muscle cells (VSMCs) remain unknown. The present study explored the effects of the ethyl acetate fraction of desalted SE hot water extract (SEWEAF) on atherosclerotic responses (especially migration and proliferation) in VSMCs and vascular neointima formation. Treatment with the SEWEAF significantly suppressed the platelet-derived growth factor (PDGF)-BB-induced VSMC migration and proliferation as well the phosphorylation of mitogen-activated protein kinases (MAPKs) such as the p38 MAPK and extracellular signal-regulated kinase (ERK) 1/2. Moreover, oral administration of the SEWEAF resulted in the attenuation of neointima formation in balloon-injured rat carotid arteries. Additionally, HPLC analysis showed that the major components in the two subfractions of the SEWEAF were five phenolic acids and four flavonols. In the SEWEAF components, for which atherosclerosis-linked responses in VSMCs have not been known, p-coumaric acid, quercetin-3-β-d-glucoside, and isorhamnetin-3-β-d-glucoside inhibited both PDGF-BB-induced migration and proliferation and isorhamnetin attenuated only PDGF-BB-stimulated VSMC proliferation. These results suggest that the SEWEAF may suppress PDGF-BB-induced VSMC migration by downregulating the phosphorylation of p38 MAPK and ERK1/2, thus leading to the reduction of neointimal hyperplasia during vascular remodeling. Therefore, the desalted SE extract, SEWEAF may be a potential ingredient for dietary supplements or nutraceuticals to ameliorate and/or prevent vascular remodeling-related disorders.