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Background: Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce cardiovascular events. However, the precise mechanisms beyond glycemic control are not fully understood. The objective of this study was to determine the role of PDGF (platelet-derived growth factor)-related signaling in empagliflozin-mediated inhibition of neointima formation., Methods and Results: Adult male nondiabetic Wistar rats were subjected to carotid artery balloon injury. Empagliflozin (30 mg/kg per day) was administered by oral gavage for 18 days beginning 4 days before surgery. The in vitro effects of empagliflozin on rat aortic vascular smooth muscle cell (VSMC) proliferation and migration were also determined. Empagliflozin attenuated balloon injury-induced neointima formation in carotid arteries. In VSMCs, empagliflozin attenuated PDGF-BB-induced proliferation and migration. Moreover, empagliflozin-treated VSMCs did not undergo apoptosis or cytotoxic death. Empagliflozin suppressed PDGF-related signaling, including phosphorylation of PDGF receptor β, Akt, and STAT3 (signal transducer and activator of transcription 3). Overactivation of PDGF signaling attenuated empagliflozin-mediated inhibition of VSMC function. SGLT2 mRNA levels in rat VSMCs were undetectable, and SGLT2 silencing did not alter the empagliflozin-mediated effects, supporting the SGLT2-independent effects of empagliflozin on VSMC., Conclusions: This study highlights the crucial role of suppressing PDGF-related signaling in mediating the beneficial effects of empagliflozin on neointima formation and VSMC function, which are independent of SGLT2 and glycemic control. Our study provides a novel mechanistic aspect of empagliflozin for the prevention of vascular stenosis disorders.

The primary challenge in percutaneous coronary interventions for vascular restenosis is the occurrence of restenosis, which is defined by the excessive proliferation of neointimal tissue. Herein, our research team suggests that exosomes obtained from PSC, when paired with quercetin (Q@PSC-E), successfully reduce neointimal hyperplasia in a Sprague-Dawley rat model. Furthermore, the physical properties of the synthesized Q@PSC-E were examined using UV-vis, DLS, and FT-IR characterization techniques. The rats were subjected to balloon injury (BI) utilizing a 2-Fr Fogarty arterial embolectomy balloon catheter. Intimal hyperplasia and the degree of VSMC proliferation were evaluated using histological analysis in the rat groups that received a dosage of Q@PSC-E at 30 mg/kg/d. Significantly, Q@PSC-E inhibited cell proliferation through a pathway that does not include lipoxygenase, as demonstrated by [ 3 H] thymidine incorporation, MTT, and flow cytometry studies. Additionally, the data indicate that Q@PSC-E hinders cell proliferation by targeting particular events that promote cell growth, including the activation of Akt and NF-κB, disruption of cell-cycle progression and also obstructs the ERK signaling pathway., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background and Aims: Sodium-glucose co-transporter 2 (SGLT2) inhibitors have been shown to reduce the risk of cardiovascular events independently of glycemic control. However, the possibility that SGLT2 inhibitors improve vascular restenosis is unknown. The aim of this study was to examine whether dapagliflozin could prevent neointima thickening following balloon injury and, if so, to determine the underlying mechanisms., Methods: Saline, dapagliflozin (1.5 mg/kg/day), or losartan (30 mg/kg/day) was administered orally for five weeks to male Wistar rats. Balloon injury of the left carotid artery was performed a week after starting the treatment and rats were sacrificed 4 weeks later. The extent of neointima was assessed by histomorphometric and immunofluorescence staining analyses. Vascular reactivity was assessed on injured and non-injured carotid artery rings, changes of target factors by immunofluorescence, RT-qPCR, and histochemistry., Results: Dapagliflozin and losartan treatments reduced neointima thickening by 32 % and 27 %, respectively. Blunted contractile responses to phenylephrine and relaxations to acetylcholine and down-regulation of eNOS were observed in the injured arteries. RT-qPCR investigations indicated an increased in gene expression of inflammatory (IL-1beta, VCAM-1), oxidative (p47phox, p22phox) and fibrotic (TGF-beta1) markers in the injured carotid. While these changes were not affected by dapagliflozin, increased levels of AT1R and NTPDase1 (CD39) and decreased levels of ENPP1 were observed in the restenotic carotid artery of the dapagliflozin group., Conclusions: Dapagliflozin effectively reduced neointimal thickening. The present data suggest that dapagliflozin prevents restenosis through interfering with angiotensin and/or extracellular nucleotides signaling. SGLT2 represents potential new target for limiting vascular restenosis., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Dysregulation of vascular smooth muscle cell (VSMC) function leads to a variety of diseases such as atherosclerosis and hyperplasia after injury. However, antiproliferative drug targeting VSMC exhibits poor specificity. Therefore, there is an urgent to develop highly specific antiproliferative drugs to prevention and treatment VSMC dedifferentiation associated arteriosclerosis. Kanglexin (KLX), a new anthraquinone compound designed by our team, has potential to regulate VSMC phenotype according to the physicochemical properties., Purpose: This project aims to evaluate the therapeutic role of KLX in VSMC dedifferentiation and atherosclerosis, neointimal formation and illustrates the underlying molecular mechanism., Methods: In vivo, the ApoE -/- mice were fed with high-fat diet (HFD) for a duration of 13 weeks to establish the atherosclerotic model. And rat carotid artery injury model was performed to establish the neointimal formation model. In vitro, PDGF-BB was used to induce VSMC dedifferentiation., Results: We found that KLX ameliorated the atherosclerotic progression including atherosclerotic lesion formation, lipid deposition and collagen deposition in aorta and aortic sinus in atherosclerotic mouse model. In addition, The administration of KLX effectively ameliorated neointimal formation in the carotid artery following balloon injury in SD rats. The findings derived from molecular docking and surface plasmon resonance (SPR) experiments unequivocally demonstrate that KLX had potential to bind PDGFR-β. Mechanism research work proved that KLX prevented VSMC proliferation, migration and dedifferentiation via activating the PDGFR-β-MEK -ERK-ELK-1/KLF4 signaling pathway., Conclusion: Collectively, we demonstrated that KLX effectively attenuated the progression of atherosclerosis in ApoE-/- mice and carotid arterial neointimal formation in SD rats by inhibiting VSMC phenotypic conversion via PDGFR-β-MEK-ERK-ELK-1/KLF4 signaling. KLX exhibits promising potential as a viable therapeutic agent for the treatment of VSMC phenotype conversion associated arteriosclerosis., Competing Interests: Declaration of competing interest All authors disclosed no relevant relationships., (Copyright © 2024. Published by Elsevier GmbH.)

This report demonstrates the successful treatment of a carotid artery pseudoaneurysm using percutaneous thrombin injection. The patient, a 62-year-old woman with multiple comorbidities, experienced a pseudoaneurysm following an unintentional carotid artery puncture during a failed attempt to place a triple lumen catheter in the right jugular vein. Percutaneous thrombin injection was chosen as the treatment method, with Doppler ultrasound monitoring. Follow-up examinations showed no signs of recurrence, and the patient was discharged after nine days without complications., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Purpose: Activation of mitogen-activated protein kinases (MAPKs) by pathological stimuli participates in cardiovascular diseases. Dysfunction of adventitial fibroblast has emerged as a critical regulator in vascular remodeling, while the potential mechanism remains unclear. In this study, we sought to determine the effect of different activation of MAPKs in adventitial fibroblast contributing to neointima formation., Methods: Balloon injury procedure was performed in male 12-week-old Sprague-Dawley rats. After injury, MAPK inhibitors were applied to the adventitia of injured arteries to suppress MAPK activation. Adventitial fibroblasts were stimulated by platelet-derived growth factor-BB (PDGF-BB) with or without MAPK inhibitors. RNA sequencing was performed to investigate the change of pathway and cell function. Wound healing, transwell assay, and flow cytometry were used to analyze adventitial fibroblast function., Results: Phosphorylation of p38, c-Jun N-terminal kinase (JNK), and extracellular regulated kinases 1/2 (ERK1/2) was increased in injured arteries after balloon injury. In primary culture of adventitial fibroblasts, PDGF-BB increased phosphorylation of p38, JNK, ERK1/2, and extracellular regulated kinase 5 (ERK5) in a short time, which was normalized by their inhibitors respectively. Compared with the injury group, perivascular administration of four MAPK inhibitors significantly attenuated neointima formation by quantitative analysis of neointimal area, intima to media (I/M) ratio, and lumen area. RNA sequencing of adventitial fibroblasts treated with PDGF-BB with or without four inhibitors demonstrated differentially expressed genes involved in multiple biological processes, including cell adhesion, proliferation, migration, and inflammatory response. Wound healing and transwell assays showed that four inhibitors suppressed PDGF-BB-induced adventitial fibroblast migration. Cell cycle analysis by flow cytometry demonstrated that JNK, ERK1/2, and ERK5 but not p38 inhibitor blocked PDGF-BB-induced G1 phase release associated with decrease expression of cell cycle protein Cyclin D1 and transcription factor GATA4. Moreover, four inhibitors decreased macrophage infiltration into adventitia and monocyte chemoattractant protein-1 (MCP-1) expression., Conclusion: These results suggest that MAPKs differentially regulate activation of adventitial fibroblast through GATA4/Cyclin D1 axis that participates in neointima formation., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Vascular lesions frequently arise as complication in patients diagnosed with diabetes mellitus (DM). Presently, percutaneous coronary intervention (PCI) and antithrombotic therapy serve as primary treatments. However, in-stent restenosis persists as a challenging clinical issue following PCI, lacking sustained and effective treatment. Linarin (LN) exhibits diverse pharmacological activities and is regarded as a potential drug for treating various diseases, including DM. But its specific role in restenosis after vascular injury in DM patients remains unclear. A rat model of diabetes-related restenosis was established to evaluate the role of LN on neointimal hyperplasia. Vascular smooth muscle cells (VSMCs) stimulated by high glucose (HG, 30 mM) underwent LN treatment. Additionally, an overexpression plasmid of A disintegrin and metalloproteinases (ADAM10) was constructed to transfect VSMCs. We employed CCK-8, Brdu, wound-healing scratch, and transwell migration assays to evaluate the proliferation and migration of VSMCs. Furthermore, western blot and immunofluorescence assays were utilized to investigate the expressions of ADAM10 and the downstream Notch signaling pathway in vivo and in vitro models. LN notably alleviated intimal hyperplasia after vascular injury in DM rats and reduced the protein expression of ADAM10, alongside its downstream Notch1 signaling pathway-related proteins (Notch1, NICD and Hes1) in rat carotid artery tissues. LN effectively suppressed the proliferation and migration of VSMCs induced by HG, downregulating the protein expression of ADAM10, Notch1, NICD and Hes1. Moreover, our findings indicated that ADAM10 overexpression significantly reversed LN's effects on proliferation, migration, and the expression of Notch1 signaling pathway-related proteins in HG-treated VSMCs. LN demonstrates potential therapeutic efficacy in addressing restenosis after diabetic-related vascular injury, with the ADAM10 mediated Notch signaling pathway playing a pivotal role., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: Arterial restenosis caused by intimal hyperplasia (IH) is a serious complication after vascular interventions. In the rat carotid balloon injury model, we injected phosphate buffer saline (PBS), rapamycin-phosphate buffer saline suspension (RPM-PBS), blank fibrin glue (FG) and rapamycin-fibrin glue (RPM-FG) around the injured carotid artery under ultrasound guidance and observed the inhibitory effect on IH., Methods: The properties of RPM-FG in vitro were verified by scanning electron microscopy (SEM) and determination of the drug release rate. FG metabolism in vivo was observed by fluorescence imaging. The rat carotid balloon injury models were randomly classified into 4 groups: PBS group (control group), RPM-PBS group, FG group, and RPM-FG group. Periadventitial administration was performed by ultrasound-guided percutaneous puncture on the first day after angioplasty. Carotid artery specimens were analyzed by immunostaining, Evans blue staining and hematoxylin-eosin staining., Results: The RPM particles showed clustered distributions in the FG block. The glue was maintained for a longer time in vivo (> 14 days) than in vitro (approximately 7 days). Two-component liquid FG administered by ultrasound-guided injection completely encapsulated the injured artery before coagulation. The RPM-FG inhibited IH after carotid angioplasty vs. control and other groups. The proliferation of vascular smooth muscle cells (VSMCs) was significantly inhibited during neointima formation, whereas endothelial cell (EC) repair was not affected., Conclusion: Periadventitial delivery of RPM-FG contributed to inhibiting IH in the rat carotid artery injury model without compromising re-endothelialization. Additionally, FG provided a promising platform for the future development of a safe, effective, and minimally invasive perivascular drug delivery method to treat vascular disease., Competing Interests: Declaration of Competing Interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled., (Copyright © 2023. Published by Elsevier B.V.)

Objective: Neointimal hyperplasia is the primary mechanism underlying atherosclerosis and restenosis after percutaneous coronary intervention. Ketogenic diet (KD) exerts beneficial effects in various diseases, but whether it could serve as non-drug therapy for neointimal hyperplasia remains unknown. This study aimed to investigate the effect of KD on neointimal hyperplasia and the potential mechanisms., Methods and Results: Carotid artery balloon-injury model was employed in adult Sprague-Dawley rats to induce neointimal hyperplasia. Then, animals were subjected to either standard rodent chow or KD. For in-vitro experiment, impacts of β-hydroxybutyrate (β-HB), the main mediator of KD effects, on platelet-derived growth factor BB (PDGF-BB) induced vascular smooth muscle cell (VSMC) migration and proliferation were determined. Balloon injury induced event intimal hyperplasia and upregulation of protein expression of proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA), and these changes were significantly ameliorated by KD. In addition, β-HB could markedly inhibit PDGF-BB induced VMSC migration and proliferation, as well as inhibiting expressions of PCNA and α-SMC. Furthermore, KD inhibited balloon-injury induced oxidative stress in carotid artery, indicated by reduced ROS level, malondialdehyde (MDA) and myeloperoxidase (MPO) activities, and increased superoxide dismutase (SOD) activity. We also found balloon-injury induced inflammation in carotid artery was suppressed by KD, indicated by decreased expressions of proinflammatory cytokines IL-1β and TNF-α, and increased expression of anti-inflammatory cytokine IL-10., Conclusion: KD attenuates neointimal hyperplasia through suppressing oxidative stress and inflammation to inhibit VSMC proliferation and migration. KD may represent a promising non-drug therapy for neointimal hyperplasia associated diseases.

Aims: Endothelial progenitor cells (EPCs) play an important role in vascular repair. However, they are dysfunctional in the inflammatory microenvironment during restenosis. In this study, we investigated whether omentin-1, an anti-inflammatory factor, could reduce neointima formation after carotid artery injury (CAI) in rats by improving EPC functions that were damaged by inflammation and the underlying mechanisms., Main Methods: EPCs were transfected with adenoviral vectors expressing human omentin-1 or green fluorescent protein (GFP). Then, the rats received 2 × 10 6 EPCs expressing omentin-1 or GFP by tail vein injection directly after CAI and again 24 h later. Hematoxylin-eosin staining and immunohistochemistry were used for analyzing neointimal hyperplasia. Besides, EPCs were treated with omentin-1 and TNF-α to examine the underlying mechanism., Key Findings: Our results showed that omentin-1 could significantly improve EPC functions, including proliferation, apoptosis and tube formation. Meanwhile, EPCs overexpressed with omentin-1 could significantly reduce neointimal hyperplasia and tumor necrosis factor-α (TNF-α) expression after CAI in rats. TNF-α could notably induce EPC dysfunction, which could be markedly reversed by omentin-1 through the inhibition of the p38 MAPK/CREB pathway. Furthermore, a p38 MAPK agonist (anisomycin) significantly abrogated the protective effects of omentin-1 on EPCs damaged by TNF-α., Significance: Our results indicated that genetically modifying EPC with omentin-1 could be an alternative strategy for the treatment of restenosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Purpose: Dysfunction of endothelium is associated with multiple pathological vascular diseases. However, how to regulate reendothelialization after vascular injury is not well defined. This study aims to determine whether and how Paeonol controls reendothelialization following artery injury., Methods: The endothelium of murine carotid artery was denuded by catheter guide wires injury. H&E staining and IF staining were performed to determine whether Paeonol is critical for reendothelialization. BRDU Incorporation Assay, Boyden Chamber Migration Assay, Tube Formation Assay, and Spheroid Sprouting Assay were used to investigate whether Paeonol is involved in regulating proliferation and migration of endothelial cells. The underlying mechanism of how Paeonol regulates reendothelialization was determined by Molecular docking simulation and CO-IP Assay., Results: Paeonol treatment significantly inhibits neointima formation in carotid artery ligation model by promoting proliferation and migration of endothelial cells. Mechanistically, Paeonol enhances c-Myc expression, consequently interacts with VEGFR2 results in activating VEGF signaling pathway, and eventually promotes reendothelialization after vascular injury., Conclusion: Our data demonstrated that Paeonol plays a critical role in regulating vascular reendothelialization, which may be therapeutically used for treatment of pathological vascular diseases., Competing Interests: The authors report no conflicts of interest in this work., (© 2023 Wang et al.)

Aims : The proliferation and migration of vascular smooth muscle cells (VSMCs) play vital roles in the pathological process of neointima formation after vascular injury. Galangin, an extract of the ginger plant galangal, is involved in numerous biological activities, including inhibiting the proliferation and migration of tumor cells, but its effect on VSMCs is unknown. This study focused on the role and mechanism of galangin in the neointima formation induced by vascular injury. Methods and results : In this study, we found that galangin restrained the PDGF-BB-induced proliferation, migration and phenotypic switching of VSMCs in a concentration-dependent manner. In vivo , we established a model of carotid artery balloon injury in rats, followed by intragastric administration of galangin (40 mg kg -1 day -1 or 80 mg kg -1 day -1 ) for 14 or 28 consecutive days. Then, the degree of neointima hyperplasia was evaluated by H&E staining, and the level of relevant protein expression was assessed by immunofluorescence and western blotting. In vitro , we isolated and grew primary rat aortic smooth muscle cells, which were treated with PDGF-BB and different doses of galangin, and then CCK-8 assay, wound healing assay, transwell assay, western blotting and immunofluorescence assays were performed. We found that galangin significantly inhibited PDGF-BB-induced proliferation, migration, and phenotypic switching of VSMCs and promoted autophagy in VSMCs in vitro , and galangin significantly inhibited neointimal hyperplasia after the common carotid artery balloon injury in rats. In terms of mechanisms, galangin inhibited the PI3K/AKT/mTOR pathway, thereby suppressing VSMC's switch from a contractile to a synthetic phenotype, inhibiting VSMC proliferation, migration and phenotypic switching and upregulating the Beclin1 protein expression levels and the ratio of LC3BII/I, promoting VSMC autophagy, and thereby inhibiting neointimal hyperplasia after vascular injury. Conclusion : Our study suggests that galangin inhibits neointimal hyperplasia after vascular injury by inhibiting smooth muscle cell proliferation, migration and phenotypic switching and by promoting autophagy, and that galangin may be a promising drug for the prevention and treatment of vascular restenosis after PCI.

Abstract: Angioplasty often fails due to the abnormal proliferation of vascular smooth muscle cells (VSMCs). Success rates of angioplasty may increase following the administration of an agent that effectively ameliorates aberrant vascular remodeling. Icariside II (ICS-II) is a natural flavonol glycoside extract from the Chinese herbal medicine Epimedii that possesses several medicinal qualities that are beneficial in humans. Nevertheless, the role of ICS-II in addressing aberrant vascular remodeling have yet to be clarified. The current investigation studies the molecular effects of ICS-Ⅱ on balloon-inflicted neointimal hyperplasia in rats in vivo and on platelet-derived growth factor-induced vascular proliferation in primary rat aortic smooth muscle cells (VSMCs) in vitro. ICS-II was found to be as effective as rapamycin, the positive control used in this study. ICS-II inhibited neointimal formation in injured rat carotid arteries and notably reduced the expression of Wnt7b. ICS-Ⅱ significantly counteracted platelet-derived growth factor-induced VSMCs proliferation. Cell cycle analysis showed that ICS-II triggered cell cycle arrest during the G1/S transition. Western blot analysis further indicated that this cell cycle arrest was likely through Wnt7b suppression that led to CCND1 inhibition. In conclusion, our findings demonstrate that ICS-II possesses significant antiproliferative qualities that counteracts aberrant vascular neointimal hyperplasia. This phenomenon most likely occurs due to the suppression of the Wnt7b/CCND1 axis., Competing Interests: The authors report no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Objective: Smooth muscle cell (SMC) phenotypic switching is associated with development of a variety of occlusive vascular diseases. Paeonol has been reported to be involved in suppressing SMC proliferation. However, it is still unknown whether paeonol can regulate SMC phenotypic switching, and which eventually result in suppressing vasculogenesis., Methods: Murine left common carotid artery was injured by completely ligation, and paeonol was administrated by intraperitoneal injection. Hematoxylin and eosin (H&E) staining was performed to visualize vascular neointima formation. Rat aortic SMCs were used to determine whether paeonol suppresses cell proliferation and migration. And murine hind limb ischemia model was performed to confirm the function role of paeonol in suppressing vasculogenesis., Results: Complete ligation of murine common carotid artery successfully induced neointima formation. Paeonol treatment dramatically reduced the size of injury-induced neointima. Using rat aortic primary SMC, we identified that paeonol strongly suppressed cell proliferation, migration, and decreased extracellular matrix deposition. And paeonol treatment dramatically suppressed vasculogenesis after hind limb ischemia injury., Conclusion: Paeonol could regulate SMC phenotypic switching through inhibiting proliferation and migration of SMC, which results in inhibiting ischemia-induced vasculogenesis.

The injury of endothelial cells is one of the initiating factors in restenosis after endovascular treatment. Human urinary kallidinogenase (HUK) is a tissue kallikrein which is used for ischemia-reperfusion injury treatment. Studies have shown that HUK may be a potential therapeutic agent to prevent stenosis after vascular injury, however, the precise mechanisms have not been fully established. This study is to investigate whether HUK can protect endothelial cells after balloon injury or H 2 O 2 -induced endothelial cell damage through the proline-rich tyrosine kinase 2 (Pyk2)/mitochondrial calcium uniporter (MCU) pathway. Intimal hyperplasia, a decrease of pinocytotic vesicles and cell apoptosis were found in the common carotid artery balloon injury and H 2 O 2 -induced endothelial cell damage, Pyk2/MCU was also up-regulated in such pathological process. HUK could prevent these injuries partially via the bradykinin B2 receptor by inhibiting Pyk2/MCU pathway, which prevented the mitochondrial damage, maintained calcium balance, and eventually inhibited cell apoptosis. Furthermore, MCU expression was not markedly increased if Pyk2 was suppressed by shRNA technique in the H 2 O 2 treatment group, and cell viability was significantly better than H 2 O 2 -treated only. In short, our results indicate that the Pyk2/MCU pathway is involved in endothelial injury induced by balloon injury or H 2 O 2 -induced endothelial cell damage. HUK plays an protective role by inhibiting the Pyk2/MCU pathway in the endothelial injury., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Vascular intimal hyperplasia is a hallmark event in vascular restenosis. The excessive proliferation, migration and phenotypic transformation of vascular smooth muscle cells (VSMCs) play important roles in the pathological mechanism of vascular intimal hyperplasia. Physalin B is an alcoholate isolated from Physalis (Solanaceae) that has a wide range of biological activities. However, the effect of physalin B on VSMCs is currently unclear. In this study, we demonstrated that physalin B significantly inhibited the proliferation, migration and phenotypic transformation of VSMCs induced by PDGF-BB. Physalin B also reduced inflammation and oxidative stress in VSMCs induced by PDGF-BB. Mechanistic studies showed that physalin B plays a role mainly by activating Nrf2. After Nrf2 activation, physalin B mitigates oxidative stress by enhancing the expression of the antioxidant gene HO-1 ; on the other hand, physalin B inhibits the NF-κB pathway to alleviate the inflammatory response. These two effects ultimately reduce the proliferation, migration and phenotypic transformation of VSMCs induced by PDGF-BB. In addition, in the mouse carotid artery ligation model, physalin B prevented intimal hyperplasia and inhibited the proliferation, migration and phenotypic transformation of cells in the hyperplastic intima. In conclusion, we provided significant evidence that physalin B abrogates PDGF-BB-induced VSMC proliferation, migration, phenotypic transformation and intimal hyperplasia by activating Nrf2-mediated signal transduction. Therefore, physalin B may be a potential therapeutic agent for preventing or treating restenosis.

Vascular restenosis remains a major problem in patients with coronary artery disease (CAD) and peripheral artery disease (PAD). Neointimal hyperplasia, defined by post-procedure proliferation and migration of vascular smooth muscle cells (VSMCs) is a key underlying pathology. Here we investigated the role of Interleukin 11 (IL-11) in a mouse model of injury-related plaque development. Apoe -/- mice were fed a hyperlipidaemic diet and subjected to carotid wire injury of the right carotid. Mice were injected with an anti-IL11 antibody (X203), IgG control antibody or buffer. We performed ultrasound analysis to assess vessel wall thickness and blood velocity. Using histology and immunofluorescence approaches, we determined the effects of IL-11 inhibition on VSMC and macrophages phenotypes and fibrosis. Treatment of mice with carotid wire injury using X203 significantly reduced post-endothelial injury vessel wall thickness, and injury-related plaque, when compared to control. Immunofluorescence staining of the injury-related plaque showed that X203 treatment did not reduce macrophage numbers, but reduced the number of VSMCs and lowered matrix metalloproteinase 2 (MMP2) levels and collagen content in comparison to control. X203 treatment was associated with a significant increase in smooth muscle protein 22α (SM22α) positive cells in injury-related plaque compared to control, suggesting preservation of the contractile VSMC phenotype. Interestingly, X203 also reduced the collagen content of uninjured carotid arteries as compared to IgG, showing an additional effect on hyperlipidemia-induced arterial remodeling in the absence of mechanical injury. Therapeutic inhibition of IL-11 reduced vessel wall thickness, attenuated neointimal hyperplasia, and has favorable effects on vascular remodeling following wire-induced endothelial injury. This suggests IL-11 inhibition as a potential novel therapeutic approach to reduce arterial stenosis following revascularization in CAD and PAD patients., (© 2021. The Author(s).)

Uncontrolled bleeding is the main cause of mortality from trauma. Collagen has been developed as an important hemostatic material due to its platelet affinity function. A bath sponge skeleton is rich in collagen, also known as spongin. To understand the hemostatic effect of spongin, spongin materials, SX, SFM and SR were prepared from the bath sponge Spongia officinalis , and hemostatic experiments were performed. The SX, SFM and SR were significantly better than the positive control, type I collagen, in shortening the whole blood clotting time in vitro and hemostasis upon rat tail amputation. In a hemostatic experiment of rabbit common carotid artery injury, the hemostatic time and 3 h survival rate of the SFM group were 3.00 ± 1.53 min and 100%, respectively, which are significantly better than those of the commercial hemostat CELOX-A (10.33 ± 1.37 min and 67%, respectively). Additionally, the SFM showed good coagulation effects in platelet-deficient blood and defibrinated blood, while also showing good biocompatibility. Through a variety of tests, we speculated that the hemostatic activity of the SFM is mainly caused by its hyperabsorbency, high affinity to platelets and high effective concentration. Overall, the SFM and spongin derivates could be potential hemostatic agents for uncontrolled bleeding and hemorrhagic diseases caused by deficiency or dysfunction of coagulation factors.

Rationale: Tamoxifen prevents the recurrence of breast cancer and is also beneficial against bone demineralization and arterial diseases. It acts as an ER (estrogen receptor) α antagonist in ER-positive breast cancers, whereas it mimics the protective action of 17β-estradiol in other tissues such as arteries. However, the mechanisms of these tissue-specific actions remain unclear., Objective: Here, we tested whether tamoxifen is able to accelerate endothelial healing and analyzed the underlying mechanisms., Methods and Results: Using 3 complementary mouse models of carotid artery injury, we demonstrated that both tamoxifen and estradiol accelerated endothelial healing, but only tamoxifen required the presence of the underlying medial smooth muscle cells. Chronic treatment with 17β-estradiol and tamoxifen elicited differential gene expression profiles in the carotid artery. The use of transgenic mouse models targeting either whole ERα in a cell-specific manner or ERα subfunctions (membrane/extranuclear versus genomic/transcriptional) demonstrated that 17β-estradiol-induced acceleration of endothelial healing is mediated by membrane ERα in endothelial cells, while the effect of tamoxifen is mediated by the nuclear actions of ERα in smooth muscle cells., Conclusions: Whereas tamoxifen acts as an antiestrogen and ERα antagonist in breast cancer but also on the membrane ERα of endothelial cells, it accelerates endothelial healing through activation of nuclear ERα in smooth muscle cells, inviting to revisit the mechanisms of action of selective modulation of ERα.

Context: Clinically, Pinellia ternata (Thunb.) Breit. (Araceae) ( PT ) has been widely used in the treatment of atherosclerosis and hyperlipidaemia, but the underlying mechanisms are still not clearly understood., Objective: This research was conducted to confirm the mechanism by which PT affects carotid artery intimal hyperplasia., Materials and Methods: An intestinal hyperplasia Sprague-Dawley rat model was established by carotid artery injury. The rats were randomly divided into five groups ( n  = 8): sham, model, PT (with daily intragastric administration of 10 g/mL/kg PT tubers water extract), PT +LY294002 (with intraperitoneal injection of 50 mg/kg LY294002 + 10 g/mL/kg PT ) and endothelial progenitor cells (EPCs) (with injection of 5 × 10 5 /cells), and treated for 4 or 8 weeks., Results: HE staining showed that PT attenuated intimal hyperplasia. RT-PCR, Western blotting and immunohistochemistry showed that PT increased the expression of vascular endothelial growth factor (VEGF) and eNOS in the atherosclerotic carotid artery. PT increased the Dil-acLDL + /FITC-UEA-1 + population (from 0.41 ± 0.085% to 0.60 ± 0.092%) in the blood, decreased TCHO, TG, LDL-C, IL-6 and TNF-α levels, and increased HDL-C and IL-10 levels in the blood. However, these changes were reversed by the PI3K/Akt pathway inhibitor LY294002., Discussion and Conclusions: PT can be developed as an atherosclerosis and carotid intimal hyperplasia treatment drug. Therefore, further study will focus on the effects of PT on intimal hyperplasia in wire-injured atherosclerosis patients and explore in depth some other relevant molecular mechanisms.

Thrombus formation at athero-thrombotic sites is initiated by the exposure of collagen followed by platelet adhesion mediated by the platelet-specific collagen receptor glycoprotein VI (GPVI). Here, dimeric GPVI was used as a targeting motif to functionalize polymeric nanoparticle-based drug carriers and to show that with proper design, such GPVI-coated nanoparticles (GPNs) can efficiently and specifically target arterial injury sites while withstanding physiological flow. In a microfluidic model, under physiological shear levels (1-40 dyne/cm 2 ), 200 nm and 2 μm GPNs exhibited a >60 and >10-fold increase in binding to collagen compared to control particles, respectively. In vitro experiments in an arterial stenosis injury model, subjected to physiological pulsatile flow, showed shear-enhanced adhesion of 200 nm GPNs at the stenosis region which was confirmed in vivo in a mice ligation carotid injury model using intravital microscopy. Altogether, our results illustrate how engineering tools can be harnessed to design nano-carriers that efficiently target cardiovascular disease sites., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The present study was conducted to assess the effects of AMD3100 and stromal cell-derived factor 1 (SDF-1) on cellular functions and endothelial regeneration of endothelial progenitor cells (EPCs). The cell proliferation and adhesion capacity of EPCs were evaluated in vitro following treatment with AMD3100 and SDF‑1 using a Cell Counting Kit‑8 assay. Furthermore, the expression levels of C‑X‑C motif chemokine receptor 4 (CXCR4) and C‑X‑C motif chemokine receptor 7 (CXCR7) were detected before and after treatment with AMD3100 and SDF‑1 to elucidate their possible role in regulating the cellular function of EPCs. A rat carotid artery injury model was established to assess the influences of AMD3100 and SDF‑1 on endothelial regeneration. AMD3100 reduced the proliferation and adhesion capacity of EPCs to fibronectin (FN), whereas it increased the adhesion capacity of EPCs to human umbilical vein endothelial cells (HUVECs). However, SDF‑1 stimulated the proliferation and cell adhesion capacity of EPCs to HUVECs and FN. Additionally, the expression levels of CXCR7 but not CXCR4 were upregulated following AMD3100 treatment, whereas the expression levels of both CXCR4 and CXCR7 were upregulated after SDF‑1 treatment. In vivo results demonstrated that AMD3100 increased the number of EPCs in the peripheral blood and facilitated endothelial repair at 7 days after treatment. However, local administration of SDF‑1 alone did not enhance reendothelialization 7 and 14 days after treatment. Importantly, the combination of AMD3100 with SDF‑1 exhibited superior therapeutic effects compared with AMD3100 treatment alone, accelerated reendothelialization 7 days after treatment, and attenuated neointimal hyperplasia at day 7 and 14 by recruiting more EPCs to the injury site. In conclusion, AMD3100 could positively regulate the adhesion capacity of EPCs to HUVECs via elevation of the expression levels of CXCR7 but not CXCR4, whereas SDF‑1 could stimulate the proliferation and adhesion capacity of EPCs to FN and HUVECs by elevating the expression levels of CXCR4 and CXCR7. AMD3100 combined with SDF‑1 outperformed AMD3100 alone, promoted early reendothelialization and inhibited neointimal hyperplasia, indicating that early reendothelialization attenuated neointimal hypoplasia following endothelial injury.

Objective: ERα (estrogen receptor alpha) exerts nuclear genomic actions and also rapid membrane-initiated steroid signaling. The mutation of the cysteine 451 into alanine in vivo has recently revealed the key role of this ERα palmitoylation site on some vasculoprotective actions of 17β-estradiol (E2) and fertility. Here, we studied the in vivo role of the arginine 260 of ERα which has also been described to be involved in its E2-induced rapid signaling with PI-3K (phosphoinositide 3-kinase) as well as G protein in cultured cell lines. Approach and Results: We generated a mouse model harboring a point mutation of the murine counterpart of this arginine into alanine (R264A-ERα). In contrast to the C451A-ERα , the R264A-ERα females are fertile with standard hormonal serum levels and normal control of hypothalamus-pituitary ovarian axis. Although R264A-ERα protein abundance was normal, the well-described membrane ERα-dependent actions of estradiol, such as the rapid dilation of mesenteric arteries and the acceleration of endothelial repair of carotid, were abrogated in R264A-ERα mice. In striking contrast, E2-regulated gene expression was highly preserved in the uterus and the aorta, revealing intact nuclear/genomic actions in response to E2. Consistently, 2 recognized nuclear ERα-dependent actions of E2, namely atheroma prevention and flow-mediated arterial remodeling were totally preserved., Conclusions: These data underline the exquisite role of arginine 264 of ERα for endothelial membrane-initiated steroid signaling effects of E2 but not for nuclear/genomic actions. This provides the first model of fertile mouse with no overt endocrine abnormalities with specific loss-of-function of rapid ERα signaling in vascular functions.

Carotid artery stenting has been a widely used interventional treatment method for the last 3 decades in the treatment of carotid artery stenosis. In the current literature, unlike major cardiovascular complications, less emphasis has been placed on carotid pseudoaneurysm (PA). A carotid artery PA can be caused by trauma, spontaneous infection, vasculitis, or it may be iatrogenic. However, the incidence of PA secondary to carotid stenting is extremely rare. Although it may be completely asymptomatic in rare instances, it usually progresses symptomatically (neck swelling, nerve compression, respiratory distress, hoarseness, dysphagia, and ischemic cerebrovascular events). Doppler ultrasound, contrast-enhanced computed tomography, and conventional angiography are the main diagnostic tools. Primary closure, including graft interposition, has been described as a surgical therapeutic option. An endovascular approach with placement of a covered or bare metal stent is an alternative treatment method to surgery.

Apatinib (YN968D1) is a small-molecule tyrosine kinase inhibitor（TKI）which can inhibit the activity of vascular endothelial growth factor receptor-2 (VEGFR-2). It has been reported that apatinib has anti-tumour effect of inhibiting proliferation and inducing apoptosis of a variety of solid tumour cells, whereas its effect on vascular smooth muscle cells (VSMC) remains unclear. This study investigated the effect of apatinib on phenotypic switching of arterial smooth muscle cells in vascular remodelling. Compared to the vehicle groups, mice that were performed carotid artery ligation injury and treated with apatinib produced a reduction in abnormal neointimal area. For in vitro experiment, apatinib administration inhibited VSMC proliferation, migration and reversed VSMC dedifferentiation with the stimulation of platelet-derived growth factor type BB (PDGF-BB).In terms of mechanism, with the preincubation of apatinib, the activations of PDGF receptor-β (PDGFR-β) and phosphoinositide-specific phospholipase C-γ1 (PLC-γ1) induced by PDGF-BB were inhibited in VSMCs. With the preincubation of apatinib, the phosphorylation of PDGFR-β, extracellular signal-related kinases (ERK1/2) and Jun amino-terminal kinases (JNK) induced by PDGF-BB were also inhibited in rat vascular smooth muscle cell line A7r5. Herein, we found that apatinib attenuates phenotypic switching of arterial smooth muscle cells induced by PDGF-BB in vitro and vascular remodelling in vivo. Therefore, apatinib is a potential candidate to treat vascular proliferative diseases., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Vascular remodeling is an adaptive response to various stimuli, including mechanical forces, inflammatory cytokines and hormones. In the present study, we investigated the role of angiotensin II type 1 receptor (AT1R) and calcium channel in carotid artery remodeling in response to increased biomechanical forces by using the transverse aortic constriction (TAC) rat model. TAC was induced on ten-week-old male Sprague-Dawley rats and these models were treated with AT1R blocker olmesartan (1 mg/kg/day) or/and calcium channel blocker (CCB) amlodipine (0.5 mg/kg/day) for 14 days. After the treatment, the right common carotid artery proximal to the band (RCCA-B) was collected for further assay. Results showed that olmesartan, but not amlodipine, significantly prevented TAC-induced adventitial hyperplasia. Similarly, olmesartan, but not amlodipine, signifcantly prevented vascular infammation, as indicated by increased tumor necrosis factor α (TNF-α) and increased p65 phosphorylation, an indicator of nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) activation in RCCA-B. In contrast, both olmesartan and amlodipine reversed the decreased expression of endothelial nitric oxidase synthase (eNOS) and improved endothelium-dependent vasodilation, whereas combination of olmesartan and amlodipine showed no further synergistic protective effects. These results suggest that AT1R was involved in vascular remodeling and inflammation in response to pressure overload, whereas AT1R and subsequent calcium channel were involved in endothelial dysfunction.

Intimal hyperplasia, the key event of arterial restenosis, is a result of vascular smooth muscle cell (VSMC) proliferation and migration. Previous studies have demonstrated that total Panax notoginseng saponin (TPNS) represses intimal hyperplasia and inhibits the proliferation of VSMCs following balloon injury. However, the underlying roles of TPNS in intimal hyperplasia remain unclear. In this study, we first found that TPNS inhibited the intimal hyperplasia and reversed the reduced m 6 A quantity in balloon catheter-injured rat carotid artery. Then, we measured the expression profiles of m 6 A "writers" (i.e., methyltransferase like 3 (METTL3), methyltransferase like 14 (METTL14), and WT1 associated protein (WTAP)) and "erasers" (i.e., FTO alpha-ketoglutarate dependent dioxygenase (FTO) and alkB homolog 5, RNA demethylase (ALKBH5)) in vivo and found that TPNS up-regulated the reduced the WTAP expression in balloon catheter-injured rat carotid artery. Furthermore, we illustrated that TPNS inhibited the viability, proliferation, and migration potential of VSMCs via promotion of WTAP expression and suppression of WTAP restored the TPNS-induced inhibition of cell viability, proliferation and migration potential of VSMCs. In addition, we found that p16 was up-regulated in VSMCs treated with TPNS and repression of p16 restored the TPNS-induced inhibition of cell viability, proliferation and migration potential of VSMCs. Finally, we elucidated that, mechanistically, WTAP exerted its role by regulating p16 via m 6 A modification. Collectively, our results reveal the WTAP-p16 signaling axis and highlight the critical roles of m6A modification in intimal hyperplasia. Thus, this study provided a potential biomarker for the assessment of intimal hyperplasia risk following angioplasty as well as a novel therapeutic target for this disease., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interests., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Objective: The present study aimed to assess the effect of curcumin (Cur) on carotid artery restenosis following carotid endarterectomy (CEA) and its associated mechanism in vivo and in vitro., Methods: Ang II was used to induce excessive proliferation of rabbit aortic smooth muscle cells (CCC-SMC-1) in order to establish a hemadostenosis cell model. Similarly, the animal model of carotid artery restenosis was established by carotid artery gas drying injury combined with high-fat feed prior to CEA. CCC-SMC-1 cells and animals were treated by Cur and its effects on neointimal hyperplasia, inflammation and oxidative stress were detected and observed. The proteins that were associated with the Raf/MEK/ERK pathway were detected in cells and rabbit carotid artery tissues., Results: Cur inhibited the proliferation of smooth muscle cells and neointimal formation and reduced the inflammation and oxidative stress indices. Concomitantly, Cur reduced the phosphorylation of the Raf/MEK/ERK pathway proteins., Conclusion: Cur could inhibit carotid restenosis following CEA by inhibiting the activation of the Raf/MEK/ERK pathway., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Zhang et al.)

We have shown that both insulin and resveratrol (RSV) decrease neointimal hyperplasia in chow-fed rodents via mechanisms that are in part overlapping and involve the activation of endothelial nitric oxide synthase (eNOS). However, this vasculoprotective effect of insulin is abolished in high-fat-fed insulin-resistant rats. Since RSV, in addition to increasing insulin sensitivity, can activate eNOS via pathways that are independent of insulin signaling, such as the activation of sirtuin 1 (SIRT1) and AMP-activated kinase (AMPK), we speculated that unlike insulin, the vasculoprotective effect of RSV would be retained in high-fat-fed rats. We found that high-fat feeding decreased insulin sensitivity and increased neointimal area and that RSV improved insulin sensitivity (p < 0.05) and decreased neointimal area in high-fat-fed rats (p < 0.05). We investigated the role of SIRT1 in the effect of RSV using two genetic mouse models. We found that RSV decreased neointimal area in high-fat-fed wild-type mice (p < 0.05), an effect that was retained in mice with catalytically inactive SIRT1 (p < 0.05) and in heterozygous SIRT1-null mice. In contrast, the effect of RSV was abolished in AMKPα2-null mice. Thus, RSV decreased neointimal hyperplasia after arterial injury in both high-fat-fed rats and mice, an effect likely not mediated by SIRT1 but by AMPKα2., (© 2020 The Author(s) Published by S. Karger AG, Basel.)

Background: Optimal management of patients with extracranial blunt cerebrovascular injury (BCVI) remains controversial, with both anticoagulation and antiplatelet therapy being recommended. The purpose of this study was to evaluate the efficacy and safety of using acetylsalicylic acid (ASA) in the management of BCVI., Methods: Patients with BCVI were identified from the registry of a Level 1 trauma center between 2010 and 2017. Digital imaging and electronic medical records were reviewed for patient information including demographic characteristics, injury type, therapy, outcomes, and follow-up., Results: Over the study period, 13,578 patients were admitted following blunt trauma, with 94 (0.7%) having confirmed BCVI (mean age, 42 years; 72% male). Mean Injury Severity Score and Glasgow Coma Score were 27 and 10, respectively. BCVI was identified in 130 vessels with Biffl grade I (38%) and grade II injury (29%) being most common. Twelve (13%) patients experienced an ischemic event, but only 3 events occurred after diagnosis. ASA was primary treatment for 56 (60%) patients. Thirty patients (32%) received no treatment; 21 patients died within 24 hours of primary injury. Only 4 patients had ASA contraindications. Four patients (7%) had ASA-related complications; there were 2 cases of intracranial hemorrhage progression and 2 cases of gastrointestinal bleeding. Follow-up vascular imaging at a mean of 36 days demonstrated stable or improved levels of BCVI in 94% of patients., Conclusions: An ASA-based management strategy for BCVI was efficacious and relatively safe in this study. This approach may be the preferred treatment for BCVI, but confirmation is needed., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background and Purpose: The increased proliferation and migration of vascular smooth muscle cells (VSMCs) after arterial injury contributes greatly to the pathogenesis of neointimal hyperplasia. As a major component of epigenetics, histone methylation plays an important role in several cardiovascular diseases. However, its role in restenosis is still unclear., Experimental Approach: Human aortic VSMCs were challenged with PDGF-BB, and total histones were extracted and analysed by HPLC/MS. For the in vivo study, rats were subjected to wire-guided common carotid injury., Key Results: PDGF-BB markedly increased the H3K27me3 level, as demonstrated by use of HPLC/MS and confirmed by western blot analysis. Enhancer of zeste homologue 2 (EZH2), the histone H3K27 methyltransferase component of polycomb repressive complex 2, was also up-regulated by PDGF-BB in VSMCs, and in the neointimal hyperplasia induced by wire injury of the rat carotid artery. Furthermore, inhibiting H3K27me3 by treatment with 3-μM UNC1999, an EZH2/1 inhibitor, significantly suppressed PDGF-BB-induced VSMC proliferation compared with the PDGF-BB-treated group. Consistently, neointimal formation was significantly attenuated by oral or perivascular administration of UNC1999 compared with the sham group. Mechanistically, the increase in H3K27me3 inhibited the transcription of the cyclin-dependent kinase inhibitor p16 INK4A and thus promoted VSMC proliferation., Conclusions and Implications: Vascular injury elevated the expression of EZH2 and the downstream target H3K27me3, which suppressed p16 INK4A expression in VSMCs and promoted VSMC proliferation and neointimal hyperplasia. EZH2 inhibition might be a potential therapeutic target for restenosis., (© 2019 The British Pharmacological Society.)

Excessive bleeding due to trauma, surgery and diseases may cause severe mortalities. Here, an emerging chitin nanogel/rectorite nanocomposite is developed for effective hemorrhage control. Chitin chains are intercalated into rectorite and subsequent mechanical high speed stirring generates chitin nanogels, which assemble on the surface of the rectorite nanoplates through electrostatic interactions to form a sandwich structure. The in vitro experiments reveal that the nanocomposite exhibits favorable biocompatibility and negligible hemolysis (<3.5%) as compared to rectorite (40%). The nanocomposite stops bleeding in 121 s in rat tail incision and exhibits higher hemostatic activity in the rabbit artery injury model as compared to a commercialized chitosan hemostat, Celox. The efficient blood clotting activity is attributed to the induction of a coagulation cascade by rectorite and the quick adsorption and aggregation of platelets and red blood cells by chitin. The enhanced biocompatibility and hemostatic activity of the chitin/rectorite nanocomposite make it a safe and cost effective hemostat to control bleeding.

Background and Purpose: Sacubitril/valsartan (Sac/val) is more effective than valsartan in lowering BP and mortality in patients with heart failure. Here, we proposed that Sac/val treatment would be more effective in preventing pathological vascular remodelling in 129X1/SvJ (129X1), than in C57BL/6J (B6) inbred mice., Experimental Approach: Sac/val (60 mg·kg -1 ·day -1 ) and valsartan (27 mg·kg -1 ·day -1 ) were given as prophylactic or therapeutic treatments, to 129X1 or B6 mice with carotid artery ligation for 14 days. Blood flow was measured by ultrasound. Ex vivo, carotid tissue was analysed with histological and morphometric techniques, together with RNA sequencing and gene ontology., Key Results: Sac/val was more effective than valsartan in lowering BP in 129X1 compared with B6 mice. Liver expression of CYP2C9 and plasma cGMP levels were similar across treatments. A reduction in carotid thickening after prophylactic treatment with valsartan or Sac/val also resulted in significant arterial shrinkage in B6 mice. In 129X1 mice, Sac/val and prophylactic treatment with valsartan had no effect on carotid thickening but preserved carotid size. BP lowering significantly correlated with a decline in carotid stiffness (R 2  = .37, P = .0096) in 129X1 but not in B6 mice. The gene expression signature associated with hyalurononglucosaminidase activity was down-regulated in injured arteries after both regimens of Sac/val only in 129X1 mice. Administration of Sac/val but not valsartan significantly reduced deposition of hyaluronic acid and carotid fibrosis in 129X1 mice., Conclusion and Implications: These results underscore the importance of the genetic background in the efficacy of the Sac/val on vascular fibrosis., (© 2019 The British Pharmacological Society.)

Background: The beneficial effects of angiotensin II type 1 receptor blockers (ARBs) on atherosclerosis have been demonstrated in numerous studies. We investigated the effects of fimasartan on reducing neointimal formation and systemic inflammation after carotid artery (CA) injury in Apolipoprotein E knockout (ApoE KO) mice., Methods: ApoE KO mice were randomly allocated to Group I (without CA injury), Group II (without CA injury + Fimasartan), Group III (CA injury), and Group IV (CA injury + Fimasartan). Fimasartan was orally administered everyday starting 3 days before iatrogenic left CA injury., Results: At 28 days, neointimal hyperplasia and the inflammatory cytokines including TNFα, IL-6, ICAM, and MMP-9 in the peripheral blood were significantly reduced in Groups II and IV compared to Groups I and III, respectively. All fimasartan-administered groups revealed significant increases of CD4 + CD25 + Foxp3 + regulatory T (Treg) cells with increased plasma levels of IL-10 and TGFβ. In addition, increased CD8 + T cells by fimasartan were correlated with reduced smooth muscle cell (SMC) proliferation in the neointima in Groups II and IV. Furthermore, the populations of Treg and CD8 + T cells in total splenocytes were increased in Groups II and IV compared to Groups I and III, respectively. The enlargement of spleens due to CA injury in the Group III was attenuated by fimasartan, as shown in the Group IV. These data indicate that fimasartan significantly reduced SMC proliferation in neointima and increased Treg cells in ApoE KO CA injury mice., Conclusions: This study suggests fimasartan could be an efficient strategy for reduction of atherosclerotic progression, with a decrease in immune response and systemic inflammation.

Endothelial surface and circulating glycoprotein von Willebrand factor (vWF) regulates platelet adhesion and is associated with thrombotic diseases, including ischemic stroke, myocardial infarction, and peripheral vascular disease. Thrombosis, as manifested in these diseases, is the leading cause of disability and death in the western world. Current parenteral antithrombotic and thrombolytic agents used to treat these conditions are limited by a short therapeutic window, irreversibility, and major risk of hemorrhage. To overcome these limitations, we developed a novel anti-vWF aptamer, called DTRI-031, that selectively binds and inhibits vWF-mediated platelet adhesion and arterial thrombosis while enabling rapid reversal of this antiplatelet activity by an antidote oligonucleotide (AO). Aptamer DTRI-031 exerts dose-dependent inhibition of platelet aggregation and thrombosis in whole blood and mice, respectively. Moreover, DTRI-031 can achieve potent vascular recanalization of platelet-rich thrombotic occlusions in murine and canine carotid arteries. Finally, DTRI-031 activity is rapidly (<5 min) and completely reversed by AO administration in a murine saphenous vein hemorrhage model, and murine toxicology studies indicate the aptamer is well tolerated. These findings suggest that targeting vWF with an antidote-controllable aptamer potentially represents an effective and safer treatment for thrombosis patients having platelet-rich arterial occlusions in the brain, heart, or periphery., (Copyright © 2019. Published by Elsevier Inc.)

Objectives: This study aims to evaluate the effects of dihydroartemisinin (DHA) on the balloon injury-induced neointimal formation in rats and to investigate the underlying mechanism., Methods: The balloon-induced carotid artery injury model was established in male Sprague-Dawley rats, immediately after which the DHA solution was injected into the tail vein of rats. In in vitro assays, primary rat vascular smooth muscle cells (VSMCs) were pretreated with DHA and then coincubated with LPS., Results: DHA ameliorated the induced neointimal formation and fibrosis but enhanced apoptosis in rat carotid artery after balloon injury. Furthermore, DHA suppressed migration and enhanced apoptosis of the lipopolysaccharide (LPS)-treated primary VSMCs in vitro. Moreover, in both the balloon injury-induced rat sera and the LPS-treated VSMCs, DHA significantly inhibited proinflammatory cytokines, including interleukin-1β, tumor necrosis factor-ɑ, and matrix metalloproteinase-1. Importantly, DHA significantly decreased the balloon injury-increased expression of nuclear factor kappa B (NF-κB) subunit NF-κB p65 expression, and increased the balloon injury-reduced expression of inhibitor of NF-κB-alpha, indicating the inhibition of the IκB/NF-κB pathway., Conclusion: DHA significantly inhibited neointimal formation in balloon-induced rat carotid artery injury and the mechanism may be related to the inhibition of IκB/NF-κB signaling, which alleviates the inflammatory response., (© 2018 Wiley Periodicals, Inc.)

Introduction: Oral statins reduce intimal hyperplasia (IH) after arterial injury by only ∼25%. Alternative drug delivery systems have gained attention as carriers for hydrophobic drugs. We studied the effects of simvastatin (free vs hyaluronic acid-tagged polysialic acid-polycaprolactone micelles) on vascular smooth muscle cell (VSMC) migration, VSMC proliferation and intimal hyperplasia. We hypothesized both free and micelle containing simvastatin would inhibit VSMC chemotaxis and proliferation, and local statin treatment would be more effective than oral in reducing IH in rats following carotid balloon injury., Methods: VSMCs pretreated with free simvastatin (20 minutes or 20 hours) or simvastatin-loaded micelles underwent chemotaxis and proliferation to platelet-derived growth factor. Next, rats that underwent balloon injury of the common carotid artery received statin therapy-intraluminal simvastatin-loaded micelles prior to injury, periadventitial pluronic gel following injury, or combinations of gel, micelle, and oral simvastatin. After 14 days, morphometric analysis determined the -intimal to medial ratio. Findings were compared to controls receiving oral simvastatin or no statin therapy. Statistical analysis was by analysis of variance for the in vitro experiments and a factorial general linear model for the in vivo experiments., Results: The simvastatin-loaded micelles and free simvastatin inhibited VSMC chemotaxis (54%-60%). IH was induced in all injured vessels. Simvastatin in pluronic gel or micelles reduced IH compared to untreated controls (0.208 ± 0.04 or 0.160 ± 0.03 vs 0.350 ± 0.03, respectively); however, neither gel nor simvastatin-loaded micelles were superior to oral statins (0.261 ± 0.03). Addition of oral statins or combining both local therapies did not provide additional benefit. Micelles were the single greatest contributing factor in IH attenuation., Conclusions: Intraluminally or topically delivered statins reduced IH. The efficacy of single-dose, locally delivered statin alone may lead to novel treatments to prevent IH. The different routes of administration may allow for treatment during endovascular procedures, without the need for systemic therapy.

Vascular endothelial growth factor (VEGF) is a promising molecule for cardiovascular diseases therapy. But lack of a targeted delivery system limits its translation into clinical application. This study aimed to develop stably overexpressing VEGF macrophages for targeted VEGF delivery to injured arteries and determine their potential for repairing of the damaged endothelium. Wire-induced carotid artery injury model was established in atherosclerosis-prone mice. It was observed that the VEGF-modified macrophages were recruited to the site of vascular injury and incorporated into new endothelium formation. VEGF-modified macrophages therapy accelerated reendothelialization and attenuated neointima formation. The VEGF protein level in tissues of injured arteries treated with VEGF-modified macrophages was increased. The upregulated C-C chemokine receptor type 5 (CCR5) and unaltered CCR2 protein levels were verified in VEGF-modified macrophages in vitro. Moreover, enhanced nitric oxide (NO) production in the culture medium of VEGF-modified macrophages was demonstrated. Our results indicated that VEGF-modified macrophages acted as vectors of VEGF targeting injured arteries, promoting the repairing directly by incorporating into new endothelium formation and indirectly by secreting sustainable VEGF and producing NO locally. This study represents a novel therapeutic application of targeted cell therapy with VEGF-modified macrophages for cardiovascular diseases., (© 2019 Wiley Periodicals, Inc.)

Background: Endothelial progenitor cells (EPCs) play a crucial role in endothelial repair after arterial injury. Hydrogen sulfide (H2S) is a novel gasotransmitter that regulates vascular homeostasis., Method: We investigated whether exogenous H2S could facilitate EPCs in repairing arterial injury., Results: Sodium hydrosulfide (NaHS), a precursor of H2S, promoted re-endothelialization and inhibited neointima formation in a rodent carotid artery injury model. Flow cytometric analysis revealed that NaHS treatment significantly increased the yield of EPCs after vascular injury. Furthermore, NaHS enhanced the capacity of EPCs to the luminal surface of injured arteries in wild-type mice, which had received a bone marrow transplantation from tie2-GFP donor mice. However, this enhancing effect was greatly attenuated in endothelial nitric oxide synthase knockout mice (eNOS). In-vitro incubation of human EPCs with NaHS not only increased the yield of EPCs, but also enhanced their adhesion and colony formation capacities. Treatment with an eNOS inhibitor (L-NAME) blocked the effects of NaHS on EPCs functions., Conclusion: H2S enhances eNOS-dependent mobilization of bone marrow-derived EPCs and facilitates re-endothelialization following vascular injury.

Background: Intimal hyperplasia (IH) is the most common indicator for secondary intervention in peripheral vascular disease. Matrix metalloproteinases (MMPs) play a role in IH development due to their degradation of the extracellular matrix. Doxycycline (Doxy), a member of the tetracycline family of antibiotics, is a potent MMP inhibitor. We have previously shown that Doxy inhibits MMP activity and vascular smooth muscle cell migration in vitro. We hypothesized that Doxy would decrease MMP activity in vivo and inhibit the development of IH in a rodent model of vascular injury., Methods and Results: Doxy (400 mg/pellet) was delivered by a slow-release pellet implanted 3 days prior to or at the time of balloon angioplasty (BA) of the common carotid artery in female rats. At 14 days post-BA, intima-to-media (I:M) ratios were 0.77 ± 0.21 and 1.04 ± 0.32 in the Doxy treated groups, respectively, compared to 1.25 ± 0.26 in the control group (P = not significant; n = 3). Additionally, the tested dose of Doxy in either group had no inhibitory effect on membrane type 1-MMP or MMP-2 tissue levels, as measured by immunohistochemistry, or on systemic levels of MMP, as measured by total MMP serum levels using enzyme-linked immunosorbent assay. At 14 days post-BA, VSMC proliferation in the injured artery was increased to Doxy treatment prior to and at the time of surgery (23.5 ± 3.4 and 27.2 ± 3.9%, respectively), compared to control (11.4 ± 0.4%; n = 3), as measured by proliferating cellular nuclear antigen immunostaining., Conclusions: In our in vivo model of vascular injury, systemic Doxy administration prior to or at the time of vascular injury does not significantly hinder the progression of IH development. Additional doses and routes of administration could be examined in order to correlate therapeutic serum levels of Doxy with effective MMP inhibition in serum and arterial tissue. However, alternative drug delivery systems are needed in order to optimize therapeutic administration of targeted MMP inhibitors for the prevention of IH development., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Catalpol, an iridoid glycoside, is an isolated natural product of Rehmannia glutinosa , which has been reported to have antidiabetic properties. This study investigated the vascular protective effects of catalpol in hyperglycemic rats with balloon-injured carotid arteries. Balloon injury stress led to the upregulation of monocyte chemoattractant protein-1 expression in rats with streptozotocin-induced diabetes. Western blotting and real-time PCR were performed. In situ hybridization, immunohistochemistry, and confocal analyses were employed. Monocyte chemoattractant protein-1 levels were increased through streptozotocin induction or balloon injury. After treatment with catalpol, the neointimal hyperplasia area was reduced 2 weeks after balloon injury in hyperglycemic rats. Real-time PCR and immunohistochemical analysis demonstrated reduced levels of monocyte chemoattractant protein-1 2 weeks after the balloon injury. Monocyte chemoattractant protein-1 expression was significantly increased in balloon-injured rats compared with the control groups. Thus, treatment with catalpol affected monocyte chemoattractant protein-1 expression. This study demonstrated that catalpol downregulated monocyte chemoattractant protein-1 expression in carotid arteries and ameliorated neointimal hyperplasia in hyperglycemic rats. The suppressive effect of monocyte chemoattractant protein-1 suggests that it plays a key role in neointimal hyperplasia. The results imply that catalpol is potentially effective for preventing hyperglycemia-related ischemic cardiac diseases., Competing Interests: The authors have no conflicts of interest to declare., (Georg Thieme Verlag KG Stuttgart · New York.)

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., (© 2019 S. Karger AG, Basel.)

Background: Specialized 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 Methods: Sprague 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., Results: RvD1 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)., Conclusions: RvD1 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., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Antistasin, first identified as a potent inhibitor of the blood coagulation factor Xa, is a novel family of serine protease inhibitors. In this study, we purified a novel antistasin-type inhibitor from leech Poecilobdella manillensis called poecistasin. Amino acid sequencing of this 48-amino-acid protein revealed that poecistasin was an antistasin-type inhibitor known to consist of only one domain. Poecistasin inhibited factor XIIa, kallikrein, trypsin, and elastase, but had no inhibitory effect on factor Xa and thrombin. Poecistasin showed anticoagulant activities. It prolonged the activated partial thromboplastin time and inhibited FeCl₃-induced carotid artery thrombus formation, implying its potent function in helping Poecilobdella manillensis to take a blood meal from the host by inhibiting coagulation. Poecistasin also suppressed ischemic stroke symptoms in transient middle cerebral artery occlusion mice model. Our results suggest that poecistasin from the leech Poecilobdella manillensis plays a crucial role in blood-sucking and may be an excellent candidate for the development of clinical anti-thrombosis and anti-ischemic stroke medicines.

Background: Restenosis after percutaneous coronary intervention in coronary heart disease remains an unsolved problem. Clusterin (CLU) (or Apolipoprotein [Apo] J) levels have been reported to be elevated during the progression of postangioplasty restenosis and atherosclerosis. However, its role in neointimal hyperplasia is still controversial., Objective: To elucidate the role Apo J in neointimal hyperplasia in a rat carotid artery model in vivo with or without rosuvastatin administration., Methods: Male Wistar rats were randomly divided into three groups: the control group (n = 20), the model group (n = 20) and the statin intervention group (n = 32). The rats in the intervention group were given 10mg /kg dose of rosuvastatin. A 2F Fogarty catheter was introduced to induce vascular injury. Neointima formation was analyzed 1, 2, 3 and 4 weeks after balloon injury. The level of Apo J was measured by real-time PCR, immunohistochemistry and western blotting., Results: Intimal/medial area ratio (intimal/medial, I/M) was increased after balloon-injury and reached the maximum value at 4weeks in the model group; I/M was slightly increased at 2 weeks and stopped increasing after rosuvastatin administration. The mRNA and protein levels of Apo J in carotid arteries were significantly upregulated after rosuvastatin administration as compared with the model group, and reached maximum values at 2 weeks, which was earlier than in the model group (3 weeks)., Conclusion: Apo J served as an acute phase reactant after balloon injury in rat carotid arteries. Rosuvastatin may reduce the neointima formation through up-regulation of Apo J. Our results suggest that Apo J exerts a protective role in the restenosis after balloon-injury in rats.

Background and Aims: The phenotype switching of vascular smooth muscle cells (VSMCs) plays a key role during development and progression of vascular remodeling diseases. Recent studies show that GLP-1 can inhibit intima thickening to delay the progression of atherosclerotic plaques. The purpose of this study was to investigate the role of Exendin-4, a GLP-1 receptor agonist, in VSMCs phenotype switching and the related mechanisms., Methods: Immunohistochemistry and Western blot were used to detect the effect of Exendin-4 on expression of markers of contractile VSMCs. Phalloidin staining was performed to observe the effect of Exendin-4 on morphology of VSMCs., Results: Exendin-4 significantly increased the protein levels of contractile VSMCs markers like Calponin and SM22α. After treatment of Exendin-4, VSMCs showed more typical characteristic spindle shape. In addition, Exendin-4 significantly upregulated the phosphorylation of AMPK as well as the protein levels of Sirtuin1 (SIRT1) and FOXO3a in VSMCs. After inhibiting AMPK activity with compound C and SIRT1 activity with EX527, and knocking down FOXO3a expression through RNAi technique, Exendin-4 increased the protein levels of Calponin and SM22α and promoted the redifferentiation of VSMCs mainly through AMPK/SIRT1/FOXO3a signaling pathways., Conclusions: Exendin-4 can regulate the phenotype switching of VSMCs and promote redifferentiation of VSMCs through AMPK/SIRT1/FOXO3a signaling pathways., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The innate immunity system plays a critical role in vascular repair and restenosis development. Liposomes encapsulating bisphosphonates (LipBPs), but not free BPs, suppress neointima formation following vascular injury mediated in part by monocytes. The objective of this study was to elucidate the role of monocyte subpopulations on vascular healing following LipBP treatment. The potency- and dose-dependent treatment effect of clodronate (CLOD) and alendronate (ALN) liposomes on restenosis inhibition, total monocyte depletion, and monocytes subpopulation was studied. Rats subjected to carotid injury were treated by a single IV injection of LipBPs at the time of injury. Low- and high-dose LipALN treatment (3 and 10 mg/kg, respectively) resulted in a dose-dependent effect on restenosis development after 30 days. Both doses of LipALN resulted in a dose-dependent inhibition of restenosis, but only high dose of LipALN depleted monocytes (-60.1 ± 4.4%, 48 h post injury). Although LipCLOD treatment (at an equivalent potency to 3 mg/kg alendronate) significantly reduced monocyte levels (72.1 ± 6%), no restenosis inhibition was observed. The major finding of this study is the correlation found between monocyte subclasses and restenosis inhibition. Non-classical monocyte (NCM) levels were found higher in LipALN-treated rats, but lower in LipCLOD-treated rats, 24 h after injury and treatment. We suggest that the inhibition of circulating monocyte subpopulations is the predominant mechanism by which LipBPs prevent restenosis. The effect of LipBP treatment on the monocyte subpopulation correlates with the dose and potency of LipBPs.

Background and Aims: Cilostazol, 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 Results: Microarray 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., Conclusions: These results suggest that miR-132 by targeting PTEN may be an important regulator in mediating cilostazol actions on VSMC differentiation., (Copyright © 2018 Elsevier B.V. All rights reserved.)