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Intimal hyperplasia (IH) is an innegligible issue for patients undergoing interventional therapy. The proliferation and migration of vascular smooth muscle cells (VSMCs) induced by platelet-derived growth factor-BB (PDGF-BB) are critical events in the development of IH. While the exact mechanism and effective target for IH needs further investigation. Metabolic disorders of arachidonic acid (ARA) are involved in the occurrence and progression of various diseases. In this study, we found that the expressions of soluble epoxide hydrolase (sEH) and cyclooxygenase-2 (COX-2) were significantly increased in the VSMCs during balloon injury-induced IH. Then, we employed a COX-2/sEH dual inhibitor PTUPB to increase the concentration of epoxyeicosatrienoic acids (EETs) while prevent the release of pro-inflammatory prostaglandins. Results showed that PTUPB treatment significantly reduced neointimal thickening induced by balloon injury in rats in vivo and inhibited PDGF-BB-induced proliferation and migration of VSMCs in vitro. Our results showed that PTUPB may reverse the phenotypic transition of VSMCs by inhibiting Pttg1 expression. In conclusion, we found that the dysfunction of ARA metabolism in VSMCs contributes to IH, and the COX-2/sEH dual inhibitor PTUPB attenuates IH progression by reversing the phenotypic switch in VSMC through the Sirt1/Pttg1 pathway., Competing Interests: Declaration of competing interest We would like to submit the enclosed manuscript entitled “Restoration of ARA metabolic disorders in vascular smooth muscle cells alleviates intimal hyperplasia”, which we wish to be considered for publication in “European Journal of Pharmacology”. I would like to declare on behalf of my co-authors that:, (Copyright © 2024 Elsevier B.V. All rights reserved.)

The proliferative and migratory abilities of vascular smooth muscle cells (VSMCs) play a crucial role in neointima formation following vascular injury. Skp2 facilitates proliferation and migration in cells through cell cycle regulation, presenting an important therapeutic target for atherosclerosis, pulmonary hypertension, and vascular restenosis. This study aimed to identify a natural product capable of inhibiting neointima formation post vascular injury. Here, we demonstrate that troxerutin, a flavonoid, significantly reduced viability and downregulated Skp2 in VSMCs. Moreover, troxerutin exhibited anti-proliferative effects on VSMCs and mitigated neointima formation. These findings collectively elucidate the intrinsic mechanism of troxerutin in treating atherosclerosis, pulmonary hypertension, and vascular restenosis by targeting the E3-linked enzyme Skp2., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Vascular smooth muscle cell (VSMC) excessive proliferation and migration are considered the main pathological process in in-stent restenosis (ISR) following vascular intervention. Certain long noncoding RNAs play vital roles in this process. Therefore, this study aimed to explore novel regulators for ISR and further uncover the mechanism. Using a rat abdominal aorta stent implantation model, we observed that NONRATT000538.2 (NR538.2) served as a positive regulator for VSMC proliferation and migration. By manipulating NR538.2 expression via adenoviral overexpression or siRNA knockdown, we noted that NR538.2 promoted VSMC phenotypic switching, thereby inducing proliferation and migration. Significantly, the local delivery of siRNA of NR538.2 via adeno-associated virus vector suppressed balloon injury-induced neointima formation. Our study demonstrated for the first time that NR538.2 positively influenced VSMC proliferation during ISR., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The aberrant proliferation and migration of vascular smooth muscle cells (VSMCs) contribute to the development of neointima formation in vascular restenosis. This study aims to explore the function of the long noncoding RNA H19 in neointima formation. A mouse carotid ligation model was established, and human vascular smooth muscle cells (VSMCs) were used as a cell model. lncRNA H19 overexpression promoted VSMC proliferation and migration. Moreover, miR-125a-3p potentially bound to lncRNA H19, and Fms-like tyrosine kinase-1 (FLT1) might be a direct target of miR-125a-3p in VSMCs. Upregulation of miR-125a-3p alleviated lncRNA H19-enhanced VSMC proliferation and migration. Furthermore, rescue experiments showed that enhanced expression of miR-125a-3p attenuated lncRNA H19-induced FLT1 expression in VSMCs. In addition, the overexpression of lncRNA H19 significantly exacerbated neointima formation in a mouse carotid ligation model. In summary, lncRNA H19 stimulates VSMC proliferation and migration by acting as a competing endogenous RNA (ceRNA) of miR-125a-3p. lncRNA H19 may be a therapeutic target for restenosis.

Aims: Asprosin, a newly discovered hormone, is linked to insulin resistance. This study shows the roles of asprosin in vascular smooth muscle cell (VSMC) proliferation, migration, oxidative stress, and neointima formation of vascular injury. Methods: Mouse aortic VSMCs were cultured, and platelet-derived growth factor-BB (PDGF-BB) was used to induce oxidative stress, proliferation, and migration in VSMCs. Vascular injury was induced by repeatedly moving a guidewire in the lumen of the carotid artery in mice. Results: Asprosin overexpression promoted VSMC oxidative stress, proliferation, and migration, which were attenuated by toll-like receptor 4 (TLR4) knockdown, antioxidant (N-Acetylcysteine, NAC), NADPH oxidase 1 (NOX1) inhibitor ML171, or NOX2 inhibitor GSK2795039. Asprosin overexpression increased NOX1/2 expressions, whereas asprosin knockdown increased heme oxygenase-1 (HO-1) and NADPH quinone oxidoreductase-1 (NQO-1) expressions. Asprosin inhibited nuclear factor E2-related factor 2 (Nrf2) nuclear translocation. Nrf2 activator sulforaphane increased HO-1 and NQO-1 expressions and prevented asprosin-induced NOX1/2 upregulation, oxidative stress, proliferation, and migration. Exogenous asprosin protein had similar roles to asprosin overexpression. PDGF-BB increased asprosin expressions. PDGF-BB-induced oxidative stress, proliferation, and migration were enhanced by Nrf2 inhibitor ML385 but attenuated by asprosin knockdown. Vascular injury increased asprosin expression. Local asprosin knockdown in the injured carotid artery promoted HO-1 and NQO-1 expressions but attenuated the NOX1 and NOX2 upregulation, oxidative stress, neointima formation, and vascular remodeling in mice. Innovation and Conclusion: Asprosin promotes oxidative stress, proliferation, and migration of VSMCs via TLR4-Nrf2-mediated redox imbalance. Inhibition of asprosin expression attenuates VSMC proliferation and migration, oxidative stress, and neointima formation in the injured artery. Asprosin might be a promising therapeutic target for vascular injury. Antioxid. Redox Signal. 41, 488-504.

Introduction: Vascular neointimal hyperplasia, a pathological process observed in cardiovascular diseases such as atherosclerosis and pulmonary hypertension, involves the abundant presence of vascular smooth muscle cells (VSMCs). The proliferation, migration, and autophagy of VSMCs are associated with the development of neointimal lesions. Circular RNAs (circRNAs) play critical roles in regulating VSMC proliferation and migration, thereby participating in neointimal hyperplasia. However, the regulatory roles of circRNAs in VSMC autophagy remain unclear., Objectives: We aimed to identify circRNAs that are involved in VSMC autophagy-mediated neointimal hyperplasia, as well as elucidate the underlying mechanisms., Methods: Dual-luciferase reporter gene assay was performed to validate two competing endogenous RNA axes, hsa_circ_0001402/miR-183-5p/FKBP prolyl isomerase like (FKBPL) and hsa_circ_0001402/miR-183-5p/beclin 1 (BECN1). Cell proliferation and migration analyses were employed to investigate the effects of hsa_circ_0001402, miR-183-5p, or FKBPL on VSMC proliferation and migration. Cell autophagy analysis was conducted to reveal the role of hsa_circ_0001402 or miR-183-5p on VSMC autophagy. The role of hsa_circ_0001402 or miR-183-5p on neointimal hyperplasia was evaluated using a mouse model of common carotid artery ligation., Results: Hsa_circ_0001402 acted as a sponge for miR-183-5p, leading to the suppression of miR-183-5p expression. Through direct interaction with the coding sequence (CDS) of FKBPL, miR-183-5p promoted VSMC proliferation and migration by decreasing FKBPL levels. Besides, miR-183-5p reduced BECN1 levels by targeting the 3'-untranslated region (UTR) of BECN1, thus inhibiting VSMC autophagy. By acting as a miR-183-5p sponge, overexpression of hsa_circ_0001402 increased FKBPL levels to inhibit VSMC proliferation and migration, while simultaneously elevating BECN1 levels to activate VSMC autophagy, thereby alleviating neointimal hyperplasia., Conclusion: Hsa_circ_0001402, acting as a miR-183-5p sponge, increases FKBPL levels to inhibit VSMC proliferation and migration, while enhancing BECN1 levels to activate VSMC autophagy, thus alleviating neointimal hyperplasia. The hsa_circ_0001402/miR-183-5p/FKBPL axis and hsa_circ_0001402/miR-183-5p/BECN1 axis may offer potential therapeutic targets for neointimal hyperplasia., 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 © 2024. Production and hosting by Elsevier B.V.)

Intimal hyperplasia (IH) is a common pathological feature of vascular proliferative diseases, such as atherosclerosis and restenosis after angioplasty. Urotensin II (UII) and its receptor (UTR) are widely expressed in cardiovascular tissues. However, it remains unclear whether the UII/UTR system is involved in IH. Right unilateral common carotid artery ligation was performed and maintained for 21 days to induce IH in UTR knockout (UTR -/- ) and wild-type (WT) mice. Histological analysis revealed that compared with WT mice, UTR-deficient mice exhibited a decreased neointimal area, angiostenosis and intima-media ratio. Immunostaining revealed fewer smooth muscle cells (SMCs), endothelial cells and macrophages in the lesions of UTR -/- mice than in those of WT mice. Protein interaction analysis suggested that the UTR may affect cell proliferation by regulating YAP and its downstream target genes. In vitro experiments revealed that UII can promote the proliferation and migration of SMCs, and western blotting also revealed that UII increased the protein expression of RhoA, CTGF, Cyclin D1 and PCNA and downregulated p-YAP protein expression, while these effects could be partly reversed by urantide. To evaluate the translational value of UTRs in IH management, WT mice were also treated with two doses of urantide, a UTR antagonist, to confirm the benefit of UTR blockade in IH progression. A high dose of urantide (600 μg/kg/day), rather than a low dose (60 μg/kg/day), successfully improved ligation-induced IH compared with that in mice receiving vehicle. The results of the present study suggested that the UII/UTR system may regulate IH partly through the RhoA-YAP signaling pathway., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest with respect to the publication of this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Krüppel-like factor 13 (KLF13), a zinc finger transcription factor, is considered as a potential regulator of cardiomyocyte differentiation and proliferation during heart morphogenesis. However, its precise role in the dedifferentiation of vascular smooth muscle cells (VSMCs) during atherosclerosis and neointimal formation after injury remains poorly understood. In this study, we investigated the relationship between KLF13 and SM22α expression in normal and atherosclerotic plaques by bioanalysis, and observed a significant increase in KLF13 levels in the atherosclerotic plaques of both human patients and ApoE -/- mice. Knockdown of KLF13 was found to ameliorate intimal hyperplasia following carotid artery injury. Furthermore, we discovered that KLF13 directly binds to the SM22α promoter, leading to the phenotypic dedifferentiation of VSMCs. Remarkably, we observed a significant inhibition of platelet-derived growth factor BB-induced VSMCs dedifferentiation, proliferation, and migration when knocked down KLF13 in VSMCs. This inhibitory effect of KLF13 knockdown on VCMC function was, at least in part, mediated by the inactivation of p-AKT signaling in VSMCs. Overall, our findings shed light on a potential therapeutic target for treating atherosclerotic lesions and restenosis after vascular injury., (© 2024 Wiley Periodicals LLC.)

Background:  Smooth muscle cells (SMCs) are the main driver of neointima formation and restenosis following vascular injury. In animal models, endothelial progenitor cells (EPCs) accelerate endothelial regeneration and reduce neointima formation after arterial injury; however, EPC-capture stents do not reduce target vessel failure compared with conventional stents. Here we examined the influence of EPCs on features of SMCs pivotal for their impact on injury-induced neointima formation including proliferation, migration, and phenotype switch., Methods and Results:  EPCs, their conditioned medium, and EPC-derived microparticles induced proliferation of SMCs while limiting their apoptosis. In transwell membrane experiments and scratch assays, EPCs stimulated migration of SMCs and accelerated their recovery from scratch-induced injury. Treatment of SMCs with an EPC-derived conditioned medium or microparticles triggered transformation of SMCs toward a synthetic phenotype. However, co-cultivation of EPCs and SMCs enabling direct cell-cell contacts preserved their original phenotype and protected from the transformative effect of SMC cholesterol loading. Adhesion of EPCs to SMCs was stimulated by SMC injury and reduced by blocking CXCR2 and CCR5. Interaction of EPCs with SMCs modulated their secretory products and synergistically increased the release of selected chemokines. Following carotid wire injury in athymic mice, injection of EPCs resulted not only in reduced neointima formation but also in altered cellular composition of the neointima with augmented accumulation of SMCs., Conclusion:  EPCs stimulate proliferation and migration of SMCs and increase their neointimal accumulation following vascular injury. Furthermore, EPCs context-dependently modify the SMC phenotype with protection from the transformative effect of cholesterol when a direct cell-cell contact is established., Competing Interests: None declared., (Thieme. All rights reserved.)

Background: Arteriovenous fistula (AVF) stenosis remains an important cause of AVF maturation failure, for which there are currently no effective therapies. We examined the pattern and phenotype of cellular proliferation at different timepoints in a mouse model characterized by a peri-anastomotic AVF stenosis., Methods: Standard immunohistochemical analyses for cellular proliferation and macrophage infiltration were performed at 2, 7 and 14 d on our validated mouse model of AVF stenosis to study the temporal profile, geographical location and cellular phenotype of proliferating and infiltrating cells in this model., Results: Adventitial proliferation and macrophage infiltration (into the adventitia) began at 2 d, peaked at 7 d and then declined over time. Surprisingly, there was minimal macrophage infiltration or proliferation in the neointimal region at either 7 or 14 d, although endothelial cell proliferation increased rapidly between 2 d and 7 d, and peaked at 14 d., Conclusions: Early and rapid macrophage infiltration and cellular proliferation within the adventitia could play an important role in the downstream pathways of both neointimal hyperplasia and inward or outward remodelling.

Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) and excessive accumulation of dysfunctional PVAT are hallmarks of pathogenesis after angioplasty. Recent genome-wide association studies reveal that single-nucleotide polymorphism (SNP) in MIA3 is associated with atherosclerosis-relevant VSMC phenotypes. However, the role of MIA3 in the vascular remodeling response to injury remains unknown. Here, we found that expression of MIA3 is increased in proliferative VSMCs and knockdown of MIA3 reduces VSMCs proliferation, migration, and inflammation, whereas MIA3 overexpression promoted VSMC migration and proliferation. Moreover, knockdown of MIA3 ameliorates femoral artery wire injury-induced neointimal hyperplasia and increases brown-like perivascular adipocytes. Collectively, the data suggest that MIA3 deficiency prevents neointimal formation by decreasing VSMC proliferation, migration, and inflammation and maintaining BAT-like perivascular adipocytes in PVAT during injury-induced vascular remodeling, which provide a potential therapeutic target for preventing neointimal hyperplasia in proliferative vascular diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lei, Xu, Li, Meng, Chen, Yang, Li, Zhuang and Zuo.)

Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) contribute to the development of atherosclerosis and restenosis. Glycolysis and glutaminolysis are increased in rapidly proliferating VSMCs to support their increased energy requirements and biomass production. Thus, it is essential to develop new pharmacological tools that regulate metabolic reprogramming in VSMCs for treatment of atherosclerosis. The effects of 6-diazo-5-oxo-L-norleucine (DON), a glutamine antagonist, have been broadly investigated in highly proliferative cells; however, it is unclear whether DON inhibits proliferation of VSMCs and neointima formation. Here, we investigated the effects of DON on neointima formation in vivo as well as proliferation and migration of VSMCs in vitro. DON simultaneously inhibited FBS- or PDGF-stimulated glycolysis and glutaminolysis as well as mammalian target of rapamycin complex I activity in growth factor-stimulated VSMCs, and thereby suppressed their proliferation and migration. Furthermore, a DON-derived prodrug, named JHU-083, significantly attenuated carotid artery ligation-induced neointima formation in mice. Our results suggest that treatment with a glutamine antagonist is a promising approach to prevent progression of atherosclerosis and restenosis.

Context: Curcumin has antitumor, antioxidative, anti-inflammatory, and anti-proliferative properties. Objective: To investigate the role of miR-22 during curcumin-induced changes in vascular smooth muscle cells (VSMC) and neointima formation in balloon-injured rat abdominal aorta. Materials and methods: Sprague-Dawley rats were randomised to the sham-operated ( n  = 10), operated control (injured, n  = 10), and curcumin treatment ( n  = 10) groups. miR-22 expression was determined by real-time PCR. SP1 was assessed by western blot and real-time PCR. Rat aortic smooth muscle A7r5 cells were used to determine VSMC proliferation and migration, which were measured by the MTS, EdU staining, Transwell, and wound healing assays. Results: miR-22 levels declined following arterial balloon injury in vivo (48% at 3d, p  < 0.05) and serum stimulation in vitro (45% at 24 h, p  < 0.01). Functional studies revealed that miR-22 negatively regulated the proliferation and migration of VSMCs by directly targeting the SP1 transcription factor in VSMCs. Curcumin increased the expression of miR-22 (81%, p  < 0.05) and decreased the protein expression of SP1 in VSMCs (25%, p  < 0.05). miR-22 inhibition was found to attenuate the effects of curcumin on VSMC functions. Curcumin increased miR-22 (46%, p  < 0.01), decreased the SP1 protein (19%, p  < 0.05), and inhibited vascular neointimal area (48%, p  < 0.01) in vivo . Discussion: The miR-22/SP1 pathway is involved in the protective role of curcumin during arterial balloon injury, but the mechanisms remain unclear. Conclusion: miR-22 is involved in the inhibitory effects of curcumin on VSMCs' proliferation, migration and neointima hyperplasia after arterial balloon injury in rats. Curcumin could be used to prevent neointimal hyperplasia after angioplasty.

The excessive proliferation and migration of vascular smooth muscle cells (VSMCs) are mainly responsible for vascular occlusion diseases, such as pulmonary arterial hypertension and restenosis. Our previous study demonstrated thymoquinone (TQ) attenuated monocrotaline-induced pulmonary arterial hypertension. The aim of the present study is to systematically examine inhibitory effects of TQ on platelet-derived growth factor-BB (PDGF-BB)-induced proliferation and migration of VSMCs in vitro and neointimal formation in vivo and elucidate the potential mechanisms. Vascular smooth muscle cells were isolated from the aorta in rats. Cell viability and proliferation were measured in VSMCs using the MTT assay. Cell migration was detected by wound healing assay and Transwell assay. Alpha-smooth muscle actin (α-SMA) and Ki-67-positive cells were examined by immunofluorescence staining. Reactive oxygen species (ROS) generation and apoptosis were measured by flow cytometry and terminal deoxyribonucleotide transferase-mediated dUTP nick end labelling (TUNEL) staining, respectively. Molecules including the mitochondria-dependent apoptosis factors, matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), PTEN/AKT and mitogen-activated protein kinases (MAPKs) were determined by Western blot. Neointimal formation was induced by ligation in male Sprague Dawley rats and evaluated by HE staining. Thymoquinone inhibited PDGF-BB-induced VSMC proliferation and the increase in α-SMA and Ki-67-positive cells. Thymoquinone also induced apoptosis via mitochondria-dependent apoptosis pathway and p38MAPK. Thymoquinone blocked VSMC migration by inhibiting MMP2. Finally, TQ reversed neointimal formation induced by ligation in rats. Thus, TQ is a potential candidate for the prevention and treatment of occlusive vascular diseases., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is a hallmark of vascular restenosis. We investigated whether polypyrimidine tract-binding protein 1 (PTBP1), a novel regulator of cell proliferation and differentiation, is implicated in VSMC proliferation and neointima hyperplasia responding to injury. C57BL/6 J mice of 10-12 weeks old were randomly divided into sham and carotid artery injury group. Primary VSMCs obtained from thoracic aortas of 10- to 12-week-old mice were treated with physiological saline and platelet derived growth factor-BB (PDGF-BB). Adenovirus expressing shCon, shPTBP1 or shYY2 were transfected into the injured common carotid artery or VSMCs. qRT-PCR and immunoblotting were used to determine the mRNA and protein expression levels, respectively. Immunohistochemical staining of H&E and Ki-67 were used to evaluate restenosis of vessels. Cell counting kit-8 assay and Ki-67 immunofluorescent staining were utilized to evaluate the rate of VSMC proliferation. The expression of PTBP1 were upregulated both in injured arteries and in PDGF-BB-treated VSMCs. PTBP1 inhibition significantly attenuated neointima hyperplasia and Ki-67 positive area induced by injury. Knockdown of PTBP1 in vitro also suppressed VSMC proliferation after PDGF-BB treatment. The effects of PTBP1 inhibition mentioned above were all abolished by knockdown of YY2. Finally, we identified four cell cycle regulators (p53, p21, Cdkn1c, Cdkn2b) that were regulated by PTBP1/YY2 axis both in vitro and in vivo. These findings demonstrated that PTBP1 is a critical regulator of VSMC proliferation and neointima hyperplasia via modulating the expression of YY2., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background/Aims : Vascular smooth muscle cell (VSMC) hyperplasia plays important roles in the pathogenesis of many vascular diseases, such as atherosclerosis and restenosis. Many microRNAs (miRs) have recently been reported to regulate the proliferation and migration of VSMC. In the current study, we aimed to explore the function of miR-93 in VSMCs and its molecular mechanism. Methods : First, qRT-PCR and immunofluorescence assays were performed to determine miR-93 expression in rat VSMCs following carotid artery injury in vivo and platelet-derived growth factor-BB (PDGF-BB) stimulation in vitro. Next, the biological role of miR-93 in rat VSMC proliferation and migration was examined in vivo and vitro. EdU incorporation assay and MTT assay for measuring cell proliferation, Transwell cell invasion assay and Cell scratch wound assay for measuring cell migration. Then, the targets of miR-93 were identified. Finally, the expression levels of proteins in the Raf-ERK1/2 pathway were measured by western blot. Results : MiR-93 was upregulated in rat VSMCs following carotid artery injury in vivo. Similar results were observed in ex vivo cultured VSMCs after PDGF-BB treatment. MiR-93 inhibition suppressed neointimal formation after carotid artery injury. Moreover, our results demonstrated that a miR-93 inhibitor suppressed the PDGF-BB induced proliferation and migration of in VSMC. This inhibitor also decreased the expression levels of MMP2 and cyclin D1. Mechanistically, we discovered that mitofusin 2(Mfn2) is a direct target of miR-93. Furthermore, an analysis of the signaling events revealed that miR-93-mediated VSMC proliferation and migration occurred via the Raf-ERK1/2 pathway. Conclusions : Our findings suggest that miR-93 promotes VSMCs proliferation and migration by targeting Mfn2. MiR-93 may be a new target for treating in-stent restenosis., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Sildenafil is known to reduce cardiac hypertrophy through cGMP-dependent protein kinase (cGK) activation. Studies have demonstrated that cGK has a central switching role in modulating vascular smooth muscle cell (VSMC) phenotype in response to vascular injury. Here, we aimed to examine the effects of cGK activation by sildenafil on neointimal formation and platelet aggregation. After vascular injury, neointimal hyperplasia in rat carotid arteries was significantly reduced in the sildenafil-treated group. This effect of sildenafil was accompanied by the reduction of viability and migration of VSMCs. Further experiments showed that the increased cGK activity by sildenafil inhibited platelet-derived growth factor-induced phenotype change of VSMCs from a contractile form to a synthetic one. Conversely, the use of cGK inhibitor or gene transfer of dominant-negative cGK reversed the effects of sildenafil, increasing viability of VSMCs and neointimal formation. Interestingly, sildenafil significantly inhibited platelet aggregation induced by ADP or thrombin. This effect was reversed by cGK inhibitor, suggesting that sildenafil inhibits platelet aggregation via cGK pathway. This study demonstrated that sildenafil inhibited neointimal formation and platelet aggregation via cGK pathway. These results suggest that sildenafil could be a promising candidate for drug-eluting stents for the prevention of both restenosis and stent thrombosis.

Rationale: TEAD (TEA domain transcription factor) 1-a major effector of the Hippo signaling pathway-acts as an oncoprotein in a variety of tumors. However, the function of TEAD1 in vascular smooth muscle cells (VSMCs) remains unclear., Objective: To assess the role of TEAD1 in vascular injury-induced smooth muscle proliferation and delineate the mechanisms underlying its action., Methods and Results: We found that TEAD1 expression is enhanced in mouse femoral artery after wire injury and correlates with the activation of mTORC1 (mechanistic target of rapamycin complex 1) signaling in vivo. Using an inducible smooth muscle-specific Tead1 KO (knockout) mouse model, we found that specific deletion of Tead1 in adult VSMCs is sufficient to attenuate arterial injury-induced neointima formation due to inhibition of mTORC1 activation and VSMC proliferation. Furthermore, we found that TEAD1 plays a unique role in VSMCs, where it not only downregulates VSMC differentiation markers but also activates mTORC1 signaling, leading to enhanced VSMC proliferation. Using whole-transcriptome sequencing analysis, we identified Slc1a5 (solute carrier family 1 member 5)-a key glutamine transporter-as a novel TEAD1 target gene. SLC1A5 overexpression mimicked TEAD1 in promoting mTORC1 activation and VSMC proliferation. Moreover, depletion of SLC1A5 by silencing RNA or blocking SLC1A5-mediated glutamine uptake attenuated TEAD1-dependent mTORC1 activation and VSMC proliferation., Conclusions: Our study unravels a novel mechanism by which TEAD1 promotes VSMC proliferation via transcriptional induction of SLC1A5, thereby activating mTORC1 signaling and promoting neointima formation.

Polymerase delta-interacting protein 2 (Poldip2) is a multi-functional protein with numerous roles in the vasculature, including the regulation of cell apoptosis and migration, as well as extracellular matrix deposition; however, its role in VSMC proliferation and neointimal formation is unknown. In this study, we investigated the role of Poldip2 in intraluminal wire-injury induced neointima formation and proliferation of vascular smooth muscle cells in vitro and in vivo. Poldip2 expression was observed in the intima and media of human atherosclerotic arteries, where it colocalized with proliferating cell nuclear antigen (PCNA). Wire injury of femoral arteries of Poldip2 +/+ mice induced robust neointimal formation after 2 weeks, which was impaired in Poldip2 +/‒ mice. PCNA expression was significantly reduced and expression of the cell cycle inhibitor p21 was significantly increased in wire-injured arteries of Poldip2 +/‒ animals compared to wild-type controls. No difference was observed in apoptosis. Downregulation of Poldip2 in rat aortic smooth muscle cells significantly reduced serum-induced proliferation and PCNA expression, but upregulated p21 expression. Downregulation of p21 using siRNA reversed the inhibition of proliferation induced by knockdown of Poldip2. These results indicate that Poldip2 plays a critical role in the proliferation of VSMCs.

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&#95;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.

The present study investigated the effects of formononetin (FMN) against balloon injury‑induced neointima formation in vivo and platelet‑derived growth factor (PDGF)‑BB‑induced proliferation and migration of vascular smooth muscle cells (VSMCs) in vitro, and explored the underlying mechanisms. A rat model of carotid artery injury was established, in order to examine the effects of FMN on balloon injury‑induced neointima formation. Histological observation of the carotid artery tissues was conducted by hematoxylin and eosin staining. VSMC proliferation during neointima formation was observed by proliferating cell nuclear antigen staining. Subsequently, rat aortic VSMCs were isolated, and the effects of FMN on PDGF‑BB‑induced VSMC proliferation and migration were determined using Cell Counting Kit‑8 and Transwell/wound healing assays, respectively. Immunohistochemical and immunocytochemical staining was applied to measure the expression of transforming growth factor (TGF)‑β in carotid artery tissues and VSMCs, respectively. SMAD family member 3 (Smad3)/phosphorylated (p)‑Smad3 expression was examined by western blotting. FMN treatment significantly inhibited the abnormal proliferation of smooth muscle cells in neointima, and alterations to the vascular structure were attenuated. In addition, pretreatment with FMN effectively inhibited the proliferation of PDGF‑BB‑stimulated VSMCs (P<0.05). FMN also reduced the number of cells that migrated to the lower surface of the Transwell chamber and decreased wound‑healing percentage (P<0.05). The expression levels of TGF‑β were decreased by FMN treatment in vivo and in vitro, and Smad3/p‑Smad3 expression was also markedly inhibited. In conclusion, FMN significantly protected against balloon injury‑induced neointima formation in the carotid artery of a rat model; this effect may be associated with the regulation of VSMC proliferation and migration through altered TGF‑β1/Smad3 signaling.

Atherosclerosis remains the number one cause of death and disability worldwide. Atherosclerosis is treated by revascularization procedures to restore blood flow to distal tissue, but these procedures often fail due to restenosis secondary to neointimal hyperplasia. Diabetes mellitus is a metabolic disorder that accelerates both atherosclerosis development and onset of restenosis. Strategies to inhibit restenosis aim at reducing neointimal hyperplasia by inhibiting vascular smooth muscle cell (VSMC) proliferation and migration. Since increased production of reactive oxygen species promotes VSMC proliferation and migration, redox intervention to maintain vascular wall redox homeostasis holds the potential to inhibit arterial restenosis. Cinnamic aldehyde (CA) is an electrophilic Nrf2 activator that has shown therapeutic promise in diabetic rodent models. Nrf2 is a transcription factor that regulates the antioxidant response. Therefore, we hypothesized that CA would activate Nrf2 and would inhibit neointimal hyperplasia after carotid artery balloon injury in the Zucker Diabetic Fatty (ZDF) rat. In primary ZDF VSMC, CA inhibited cell growth by MTT with an EC 50 of 118 ± 7 μM. At a therapeutic dose of 100 μM, CA inhibited proliferation of ZDF VSMC in vitro and reduced the proliferative index within the injured artery in vivo, as well as migration of ZDF VSMC in vitro. CA activated the Nrf2 pathway in both ZDF VSMC and injured carotid arteries while also increasing antioxidant defenses and reducing markers of redox dysfunction. Additionally, we noted a significant reduction of neutrophils (69%) and macrophages (78%) within the injured carotid arteries after CA treatment. Lastly, CA inhibited neointimal hyperplasia evidenced by a 53% reduction in the intima:media ratio and a 61% reduction in vessel occlusion compared to arteries treated with vehicle alone. Overall CA was capable of activating Nrf2, and inhibiting neointimal hyperplasia after balloon injury in a rat model of diabetic restenosis., (Copyright © 2018. Published by Elsevier B.V.)

Lysophosphatidic acid (LPA), which is one of the intermediate products of membrane phospholipid metabolism, is a bioactive phospholipid that possesses diverse activities. In the present study, the effects of LPA on neointimal formation following vascular injury were investigated. A carotid artery balloon injury model was employed in the present study, and following vascular injury, rats received an intraperitoneal injection of 1 mg/kg LPA. Subsequently, histopathological alterations were assessed by hematoxylin and eosin staining, the expression levels of proliferating cell nuclear antigen (PCNA) were detected by immunohistochemistry, apoptosis was assessed via a terminal deoxynucleotidyl transferase‑mediated dUTP nick end labeling assay, and the expression levels of apoptosis‑associated and autophagy‑associated proteins were detected by western blotting. In addition, inflammatory and oxidative stress‑associated factors were assessed by reverse transcription‑quantitative polymerase chain reaction or corresponding kits. The results of the present study demonstrated that LPA enhanced vascular injury‑induced neointimal hyperplasia. LPA further elevated the expression levels of PCNA in the injured carotid artery tissues. LPA exhibited no effect on apoptosis in carotid artery tissues, whereas it modulated autophagy in the injured carotid artery tissues. Furthermore, LPA enhanced vascular injury‑induced inflammation and oxidative stress. The present study demonstrated that LPA may enhance neointimal hyperplasia following vascular injury by modulating proliferation, autophagy, inflammation and oxidative stress, but not apoptosis. Furthermore LPA may contribute to the pathology of atherosclerosis and may be considered a promising therapeutic target for the treatment of atherosclerosis.

As a potent and selective allosteric inhibitor of MEK, TAK-733 has been shown to exert anti-cancer effects for a wide range of cancers both in vitro and in vivo. However, its effects on inhibiting growth have never been investigated in the cardiovascular system, where regulation of abnormal vascular smooth muscle cell growth in neointimal hyperplasia is an important area of focus. Angiotensin II was used to mimic inflammatory neointimal hyperplasia in an in vitro environment, and balloon catheter-induced injury with an infusion of angiotensin II was used to generate an in vivo rat restenosis model under inflammatory conditions. TAK-733 exerted anti-proliferative and anti-migratory effects on human vascular smooth muscle cells. These multiple effects of TAK-733 were evaluated using various assays, such as cell cycle analysis and wound healing. Interestingly, TAK-733 did not induce apoptosis in smooth muscle cells but only reduced the proliferation rate; additionally, it did not affect EC viability. TAK-733 also exhibited anti-inflammatory activity, as observed by attenuated monocyte adhesion to smooth muscle cells via inhibition of ICAM1 and VCAM1 overexpression. The in vivo study demonstrated that neointimal hyperplasia after balloon injury and angiotensin II stimulation was suppressed by TAK-733, and downregulation of the inflammatory signal and enhanced re-endothelialization were observed. TAK-733 may have therapeutic potential for treating neointimal hyperplasia by attenuating smooth muscle cell proliferation, migration, and inflammation. Thus, TAK-733 could be a promising drug candidate for treating patients with restenosis.

Aims: The migration and proliferation of vascular smooth muscle cells (VSMCs) are crucial events in the neointimal formation, a hallmark of atherosclerosis and restenosis. Semaphorin3E (Sema3E) has been found to be a critical regulator of cell migration and proliferation in many scenarios. However, its role on VSMCs migration and proliferation is unclear. This study aimed to investigate the effect of Sema3E on VSMCs migration, proliferation and neointimal formation, and explore possible mechanisms., Methods and Results: We found that the expression of Sema3E was progressively decreased during neointimal formation in a carotid ligation model. H&E-staining showed lentivirus-mediated overexpression of Sema3E in carotid ligation area attenuated neointimal formation. Immunofluorescence staining showed that the receptor (PlexinD1) of Sema3E was expressed in vascular walls. In cultured mouse VSMCs, Sema3E inhibited VSMCs migration and proliferation via plexinD1 receptor. The inhibitory effect was mediated, at least in part, by inactivating Rap1-AKT signalling pathways in VSMCs. Moreover, we found that PDGFBB down-regulated the expression of Sema3E in VSMCs and Sema3E notably inhibited the expression of PDGFB in endothelial cells. In addition, the number of Sema3E-positive VSMCs was diminished in plaques of atherosclerotic patients. Results from a public GEO microarray database showed a negative correlation between Sema3E and PDGFB transcriptional levels in the human plaques examined., Conclusion: Our study demonstrates that Sema3E/plexinD1 inhibits proliferation and migration of VSMCs via inactivation of Rap1-AKT signalling pathways. The mutual inhibition between PDGF-BB and Sema3E after vascular injury plays a critical role in the process of neointimal formation., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions please email: journals.permissions@oup.com.)

Hypoadiponectinemia is associated with an increased risk of coronary artery disease. Although adiponectin replenishment mitigates neointimal hyperplasia and atherosclerosis in mouse models, adiponectin therapy has been hampered in a clinical setting due to its large molecular size. Recent studies demonstrate that AdipoRon (a small-molecule adiponectin receptor agonist) improves glycemic control in type 2 diabetic mice and attenuates postischemic cardiac injury in adiponectin-deficient mice, in part, through activation of AMP-activated protein kinase (AMPK). To date, it remains unknown as to whether AdipoRon regulates vascular smooth muscle cell (VSMC) proliferation, which plays a major role in neointima formation. In the present study, oral administration of AdipoRon (50mg/kg) in C57BL/6J mice significantly diminished arterial injury-induced neointima formation by ∼57%. Under in vitro conditions, AdipoRon treatment led to significant inhibition of platelet-derived growth factor (PDGF)-induced VSMC proliferation, DNA synthesis, and cyclin D1 expression. While AdipoRon induced a rapid and sustained activation of AMPK, it also diminished basal and PDGF-induced phosphorylation of mTOR and its downstream targets, including p70S6K/S6 and 4E-BP1. However, siRNA-mediated AMPK downregulation showed persistent inhibition of p70S6K/S6 and 4E-BP1 phosphorylation, indicating AMPK-independent effects for AdipoRon inhibition of mTOR signaling. In addition, AdipoRon treatment resulted in a sustained and transient decrease in PDGF-induced phosphorylation of Akt and ERK, respectively. Furthermore, PDGF receptor-β tyrosine phosphorylation, which controls the phosphorylation state of Akt and ERK, was diminished upon AdipoRon treatment. Together, the present findings suggest that orally-administered AdipoRon has the potential to limit restenosis after angioplasty by targeting mTOR signaling independent of AMPK activation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Neointima formation is the major reason for vein graft failure. However, the underlying mechanism is unclear. The aim of this study was to determine the role of miR-26a in the development of neointimal hyperplasia of autogenous vein grafts. Using autologous jugular vein grafts in the rat carotid artery as a model, we found that miR-26a was significantly downregulated in grafted veins as well as proliferating vascular smooth muscle cells (VSMCs) stimulated with platelet-derived growth factor-BB (PDGF-BB). Overexpression of miR-26a reduced the proliferation and migration of VSMCs. Further analysis revealed that the effects of miR-26a in VSMCs were mediated by targeting MAPK6 at the mRNA and protein levels. Luciferase assays showed that miR-26a repressed wild type (WT) MAPK6-3'-UTR-luciferase activity but not mutant MAPK6-3'-UTR-luciferease reporter. MAPK6 deficiency reduced proliferation and migration; in contrast, overexpression of MAPK6 enhanced the proliferation and migration of VSMCs. This study confirmed that neointimal hyperplasia in vein grafts was reduced in vivo by up-regulated miR-26a expression. In conclusion, our results showed that miR-26a is an important regulator of VSMC functions and neointimal hyperplasia, suggesting that miR-26a may be a potential therapeutic target for autologous vein graft diseases.

Obesity is a strong risk factor for the development of cardiovascular diseases and is associated with a marked increase in circulating leptin concentration. Leptin is a peptide hormone mainly produced by adipose tissue and is regulated by energy level, hormones and various inflammatory mediators. Genistein is an isoflavone that exhibits diverse health-promoting effects. Here, we investigated whether genistein suppressed the atherogenic effect induced by leptin. The A10 cells were treated with leptin and/or genistein, and then the cell proliferation and migration were analysed. The reactive oxygen species (ROS) and proteins levels were also measured, such as p44/42MAPK, cell cycle-related protein (cyclin D1 and p21) and matrix metalloproteinase-2 (MMP-2). Immunohistochemistry and morphometric analysis were used for the neointima formation in a rat carotid artery injury model. Genistein (5 μM) significantly inhibited both the proliferation and migration of leptin (10 ng/ml)-stimulated A10 cells. In accordance with these finding, genistein decreased the leptin-stimulated ROS production and phosphorylation of the p44/42MAPK signal transduction pathway. Meanwhile, genistein reversed the leptin-induced expression of cyclin D1, and cyclin-dependent kinase inhibitor, p21. Genistein attenuated leptin-induced A10 cell migration by inhibiting MMP-2 activity. Furthermore, the leptin (0.25 mg/kg)-augmented neointima formation in a rat carotid artery injury model was attenuated in the genistein (5 mg/kg body weight)-treated group when compared with the balloon injury plus leptin group. Genistein was capable of suppressing the atherogenic effects of leptin in vitro and in vivo, and may be a promising candidate drug in the clinical setting., (© 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

3-hydroxybenzaldehyde (3-HBA) is a precursor compound for phenolic compounds like Protocatechuic aldehyde (PCA). From recent reports, PCA has shown vasculoprotective potency, but the effects of 3-HBA remain unclear. The aim of this study is to investigate the vasculoprotective effects of 3-HBA in endothelial cells, vascular smooth muscle cells and various animal models. We tested effects of 3-HBA in both vitro and vivo. 3-HBA showed that it prevents PDGF-induced vascular smooth muscle cells (VSMCs) migration and proliferation from MTS, BrdU assays and inhibition of AKT phosphorylation. It arrested S and G0/G1 phase of VSMC cell cycle in PI staining and it also showed inhibited expression levels of Rb1 and CD1. In human umbilical vein endothelial cells (HUVECs), 3-HBA inhibited inflammatory markers and signaling molecules (VCAM-1, ICAM-1, p-NF-κB and p-p38). For ex vivo, 3-HBA has shown dramatic effects in suppressing the sprouting from aortic ring of Spargue Dawley (SD) rats. In vivo data supported the vasculoprotective effects of 3-HBA as it inhibited angiogenesis from Matrigel Plug assay in C57BL6 mouse, prevented ADP-induced thrombus generation, increased blood circulation after formation of thrombus, and attenuated neointima formation induced by common carotid artery balloon injury of SD rats. 3-HBA, a novel therapeutic agent, has shown vasculoprotective potency in both in vitro and in vivo.

Vascular smooth muscle (VSM) expresses calcium/calmodulin-dependent protein kinase II (CaMKII)-δ and -γ isoforms. CaMKIIδ promotes VSM proliferation and vascular remodeling. We tested CaMKIIγ function in vascular remodeling after injury. CaMKIIγ protein decreased 90% 14 d after balloon injury in rat carotid artery. Intraluminal transduction of adenovirus encoding CaMKIIγC rescued expression to 35% of uninjured controls, inhibited neointima formation (>70%), inhibited VSM proliferation (>60%), and increased expression of the cell-cycle inhibitor p21 (>2-fold). Comparable doses of CaMKIIδ2 adenovirus had no effect. Similar dynamics in CaMKIIγ mRNA and protein expression were observed in ligated mouse carotid arteries, correlating closely with expression of VSM differentiation markers. Targeted deletion of CaMKIIγ in smooth muscle resulted in a 20-fold increase in neointimal area, with a 3-fold increase in the cell proliferation index, no change in apoptosis, and a 60% decrease in p21 expression. In cultured VSM, CaMKIIγ overexpression induced p53 mRNA (1.7 fold) and protein (1.8-fold) expression; induced the p53 target gene p21 (3-fold); decreased VSM cell proliferation (>50%); and had no effect on expression of apoptosis markers. We conclude that regulated CaMKII isoform composition is an important determinant of the injury-induced vasculoproliferative response and that CaMKIIγ and -δ isoforms have nonequivalent, opposing functions., (© FASEB.)

The increased proliferation and migration of vascular smooth muscle cells (VSMCs) are key events in the development of artery restenosis following percutaneous coronary intervention. Digoxin has long been used in the treatment of heart failure and has been shown to inhibit the proliferation of cancer cells through multiple pathways. However, the potential role of digoxin in the regulation of VSMC proliferation and migration and its effectiveness in the treatment of cardiovascular diseases, such as restenosis, remains unexplored. In the present study, we demonstrate that digoxin-induced growth inhibition is associated with the downregulation of CDK activation and the restoration of p27Kip1 levels in platelet-derived growth factor (PDGF)-stimulated VSMCs. In addition, we found that digoxin restored the PDGF‑BB-induced inhibition of integrin linked kinase (ILK) expression and prevented the PDGF‑BB-induced activation of glycogen synthase kinase (GSK)-3β. Furthermore, digoxin inhibited adhesion molecule and extracellular matrix relative protein expression. Finally, we found that digoxin significantly inhibited neointima formation, accompanied by a decrease in cell proliferation following vascular injury in rats. These effects of digoxin were shown to be mediated, at least in part, through an increase in ILK/Akt signaling and a decrease in GSK-3β signaling in PDGF‑BB-stimulated VSMCs. In conclusion, our data demonstrate that digoxin exerts an inhibitory effect on the PDGF‑BB-induced proliferation, migration and phenotypic modulation of VSMCs, and prevents neointima formation in rats. These observations indicate the potential therapeutic application of digoxin in the treatment of cardiovascular diseases, such as restenosis.

Neointimal formation and cell proliferation resulting into in-stent restenosis is a major pathophysiological event following the deployment of stents in the coronary arteries. In this study, we assessed the degree of injury, based on damage to internal elastic lamina, media, external elastic lamina, and adventitia following the intravascular stenting, and its relationship with the degree of smooth muscle cell proliferation. We examined the smooth muscle cell proliferation and their phenotype at different levels of stent injury in the coronary arteries of domestic swine fed a normal swine diet. Five weeks after stent implantation, swine with and without stents were euthanized and coronaries were excised. Arteries were embedded in methyl methacrylate and sections were stained with H&E, trichrome, and Movat's pentachrome. The expression of Ki67, α-smooth muscle actin (SMA), vimentin, and HMGB1 was evaluated by immunofluorescence. There was a positive correlation between percent area stenosis and injury score. The distribution of SMA and vimentin was correlated with the degree of arterial injury such that arteries that had an injury score >2 did not have immunoreactivity to SMA in the neointimal cells near the stent struts, but these neointimal cells were positive for vimentin, suggesting a change in the smooth muscle cell phenotype. The Ki67 and HMGB1 immunoreactivity was highly correlated with the fragmentation of the IEL and injury in the tunica media. Thus, the extent of coronary arterial injury during interventional procedure will dictate the degree of neointimal hyperplasia, in-stent restenosis, and smooth muscle cell phenotype.

Quiescin sulfhydryl oxidase 1 (QSOX1) is a flavoenzyme largely present in the extracellular milieu whose physiological functions and substrates are not known. QSOX1 has been implicated in the regulation of tumor cell survival, proliferation and migration, in addition to extracellular matrix (ECM) remodeling. However, data regarding other pathophysiological conditions are still lacking. Arterial injury by balloon catheter is an established model of post-angioplasty restenosis. This technique induces neointima formation due to migration and proliferation of vascular smooth muscle cells (VSMC), followed by ECM synthesis and remodeling. Here, we show that QSOX1 knockdown inhibited VSMC migration and proliferation in vitro. In contrast, QSOX1 overexpression stimulated these processes. While migration could be induced by the incubation of cells with the active recombinant QSOX1, proliferation was induced by addition of the active and also of an inactive mutant QSOX1 protein. The proliferation induced by both recombinants was independent of intracellular hydrogen peroxide and dependent of the MEK/ERK pathway. To recapitulate in vivo VSMC pathophysiology, balloon-induced arterial injury was performed. The expression of QSOX1 in the neointimal layer of balloon-injured rat carotids was high and peaked at 14 days post-injury. In vivo QSOX1 knockdown led to a significant decrease in PCNA expression at day 14 post-injury and a decreased intima/media area ratio at day 21 post-injury, compared with scrambled siRNA transfection. In summary, our findings demonstrate that QSOX1 induces VSMC migration and proliferation in vitro and contributes to neointima thickening in balloon-injured rat carotids., (Copyright © 2015. Published by Elsevier B.V.)

In response to vascular injury, inflammation, oxidative stress, and cell proliferation often occur simultaneously in vascular tissues. We previously observed that microRNA-155 (miR-155), which is implicated in proliferation and inflammation is involved in neointimal hyperplasia; however, the molecular mechanisms by which it regulates these processes remain largely unknown. In this study, we observed that vascular smooth muscle cell (VSMC) proliferation and neointimal formation in wire-injured femoral arteries were reduced by the loss of miR-155 and increased by the gain of miR-155. The proliferative effect of miR-155 was also observed in cultured VSMCs. Notably, expression of the miR-155-target protein mammalian sterile 20-like kinase 2 (MST2) was increased in the injured arteries of miR-155-/- mice. miR-155 directly repressed MST2 and thus activated the extracellular signal-regulated kinase (ERK) pathway by promoting an interaction between RAF proto-oncogene serine/threonine-protein kinase (Raf-1) and mitogen-activated protein kinase kinase (MEK) and stimulating inflammatory and oxidative stress responses; together, these effects lead to VSMC proliferation and vascular remodeling. Our data reveal that MST2 mediates miR-155-promoted inflammatory and oxidative stress responses by altering the interaction of MEK with Raf-1 and MST2 in response to vascular injury. Therefore, suppression of endogenous miR-155 might be a novel therapeutic strategy for vascular injury and remodeling., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Dihydroaustrasulfone alcohol (DA), an active compound firstly isolated from marine corals, has been reported to reveal anti-cancer and anti-inflammation activities. These reported activities of DA raised a possible application in anti-restenosis. Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) and the stimulation of platelet-derived growth factor (PDGF)-BB play major pathological processes involved in the development of restenosis. Experimental results showed that DA markedly reduced balloon injury-induced neointima formation in the rat carotid artery model and significantly inhibited PDGF-BB-stimulated proliferation and migration of VSMCs. Our data further demonstrated that translational and active levels of several critical signaling cascades involved in VSMC proliferation, such as extracellular signal-regulated kinase/ mitogen-activated protein kinases (ERK/MAPK), phosphatidylinositol 3-kinase (PI3K)/AKT, and signal transducer and activator of transcription (STAT), were obviously inhibited. In addition, DA also decreased the activation and expression levels of gelatinases (matrix metalloproteinase (MMP)-2 and MMP-9) involved in cell migration. In conclusion, our findings indicate that DA can reduce balloon injury-neointimal hyperplasia, the effect of which may be modulated through suppression of VSMC proliferation and migration. These results suggest that DA has potential application as an anti-restenotic agent for the prevention of restenosis.

Aims: In-stent restenosis remains a serious problem after the implantation of drug-eluting stents, which is attributable to neointima formation and re-endothelialization. Here, we tried to find a new method which aims at selectively inhibiting proliferation of vascular smooth muscle cells (VSMC) proliferation without inhibition of re-endothelialization., Methods and Results: We used the smooth muscle-specific SM22alpha promoter in a recombinant lentiviral vector to drive overexpression of cell-cycle inhibitor, p27, in VSMCs. p27 effectively inhibited VSMC proliferation mediated by cell cycle arrest at the G0/G1 checkpoint. The SM22alpha-p27 lentiviral vector inhibited VSMC proliferation more effectively than paclitaxel. Rats infected with Lenti-SM22alpha-p27 had a significantly lower intima/media (I/M) ratio and also showed inhibition of restenosis on day 28 after balloon injury. Moreover, the repair of injured endothelium, and re-endothelialization of the carotid artery wall, was not affected by the smooth muscle cell-specific expression of p27., Conclusion: A recombinant lentiviral vector carrying the SM22alpha promoter was used to effectively infect and selectively overexpress p27 protein in VSMCs, leading to inhibition of intimal hyperplasia without compromising endothelial repair.

Aim: Pentraxin 3 (PTX3) is a novel marker for the primary local activation of innate immunity and inflammatory responses. Although clinical and experimental evidence suggests that PTX3 is associated with atherosclerosis, the relationship between PTX3 and vascular remodeling after wall injury remains to be determined. We investigated the effects of PTX3 on neointimal hyperplasia following wire vascular injury., Methods: PTX3 systemic knockout (PTX3-KO) mice and wild-type littermate (WT) mice were subjected to wire-mediated endovascular injury. At four weeks after wire-mediated injury, the areas of neointimal and medial hyperplasia were evaluated., Results: The PTX3-KO mice exhibited higher hyperplasia/media ratios than the WT mice after wire injury, and the degree of Mac-3-positive macrophage accumulation was significantly higher in the PTX3-KO mice than in the WT mice. Furthermore, the PTX3-KO mice showed a much greater increase in the number of PCNA-stained cells in the vascular wall than that observed in the WT mice., Conclusions: A deficiency of PTX3 results in deteriorated neointimal hyperplasia after vascular injury via the effects of macrophage accumulation and vascular smooth muscle cell proliferation and migration.

Vascular diseases such as atherosclerosis and restenosis artery angioplasty are associated with vascular smooth muscle cell (VSMC) proliferation and intimal thickening arterial walls. In the present study, we investigated the inhibitory effects of sulforaphane, an isothiocyanate produced in cruciferous vegetables, on VSMC proliferation and neointimal formation in a rat carotid artery injury model. Sulforaphane at the concentrations of 0.5, 1.0, and 2.0 μM significantly inhibited platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation in a concentration-dependent manner, determined by cell count. The IC50 value of sulforaphane-inhibited VSMC proliferation was 0.8 μM. Sulforaphane increased the cyclin-dependent kinase inhibitor p21 and p53 levels, while it decreased CDK2 and cyclin E expression. The effects of sulforaphane on vascular thickening were determined 14 days after the injury to the rat carotid artery. The angiographic mean luminary diameters of the group treated with 2 and 4 μM sulforaphane were 0.25±0.1 and 0.09±0.1 mm², respectively, while the value of the control groups was 0.40±0.1 mm², indicating that sulforaphane may inhibit neointimal formation. The expression of PCNA, maker for cell cycle arrest, was decreased, while that of p53 and p21 was increased, which showed the same pattern as one in in-vitro study. These results suggest that sulforaphane-inhibited VSMC proliferation may occur through the G1/S cell cycle arrest by up-regulation of p53 signaling pathway, and then lead to the decreased neointimal hyperplasia thickening. Thus, sulforaphane may be a promising candidate for the therapy of atherosclerosis and post-angiography restenosis., (© 2013.)

The Ca(2+)-sensing stromal interaction molecule (STIM) proteins are crucial Ca(2+) signal coordinators. Cre-lox technology was used to generate smooth muscle (sm)-targeted STIM1-, STIM2-, and double STIM1/STIM2-knockout (KO) mouse models, which reveal the essential role of STIM proteins in Ca(2+) homeostasis and their crucial role in controlling function, growth, and development of smooth muscle cells (SMCs). Compared to Cre(+/-) littermates, sm-STIM1-KO mice showed high mortality (50% by 30 d) and reduced bodyweight. While sm-STIM2-KO was without detectable phenotype, the STIM1/STIM double-KO was perinatally lethal, revealing an essential role of STIM1 partially rescued by STIM2. Vascular and intestinal smooth muscle tissues from sm-STIM1-KO mice developed abnormally with distended, thinned morphology. While depolarization-induced aortic contraction was unchanged in sm-STIM1-KO mice, α1-adrenergic-mediated contraction was 26% reduced, and store-dependent contraction almost eliminated. Neointimal formation induced by carotid artery ligation was suppressed by 54%, and in vitro PDGF-induced proliferation was greatly reduced (79%) in sm-STIM1-KO. Notably, the Ca(2+) store-refilling rate in STIM1-KO SMCs was substantially reduced, and sustained PDGF-induced Ca(2+) entry was abolished. This defective Ca(2+) homeostasis prevents PDGF-induced NFAT activation in both contractile and proliferating SMCs. We conclude that STIM1-regulated Ca(2+) homeostasis is crucial for NFAT-mediated transcriptional control required for induction of SMC proliferation, development, and growth responses to injury.-Mancarella, S., Potireddy, S., Wang, Y., Gao, H., Gandhirajan, K., Autieri, M., Scalia, R., Cheng, Z., Wang, H., Madesh, M., Houser, S. R., Gill, D. L. Targeted STIM deletion impairs calcium homeostasis, NFAT activation, and growth of smooth muscle.

Introduction: The role of hypoxia-inducible factor-1α (HIF-1α) in pulmonary vascular remodeling is still undetermined. The objective of this study is to investigate the expression of HIF-1α and its role in proliferating neointimal lesions in a rat model of pulmonary arterial hypertension induced by monocrotaline (MCT) administration after left pneumonectomy., Methods: The rats were subjected to MCT (60 mg/kg, subcutaneously) 7 days after left pneumonectomy or sham surgery; controls with vehicle treatment after left pneumonectomy or sham surgery were also studied. On day 35, hemodynamic parameters of the rats were measured. The right lower lobes of the lungs were fixed for morphometric analysis. The expression of proliferating cell nuclear antigen and survivin was detected with Western blot. The expressions of HIF-1α and hexokinase-2 (HK-2) were detected with Western blot and immunohistochemistry assay., Results: The rats treated with MCT after pneumonectomy developed severe pulmonary arterial hypertension and marked medial thickening on day 35. The neointimal lesions in pulmonary arterioles were observed only in MCT-treated pneumonectomized rats. The severely injured pulmonary arterioles (intimal proliferation causing greater than 50% luminal occlusion) accounted for 40% of all the measured arterioles in rats treated by MCT after pneumonectomy. The intriguing finding showed that HIF-1α was predominantly expressed in neointimal lesion areas, paralleled with the increased expression of HK-2 in MCT-treated pneumonectomized rats, which was not observed in rats undergoing MCT treatment alone., Conclusions: The activation of HIF-1α/HK-2 axis is probably the key mediator responsible for the neointimal lesion formation in MCT-treated pneumonectomized rats.

Dedifferentiation, migration, and proliferation of resident vascular smooth muscle cells (SMCs) are key components of neointima formation after vascular injury. Activation of signal transducer and activator of transcription-3 (STAT3) is suggested to be critically involved in this process, but the complex regulation of STAT3-dependent genes and the functional significance of inhibiting this pathway during the development of vascular proliferative diseases remain elusive. In this study, we demonstrate that STAT3 was activated in neointimal lesions following wire-induced injury in mice. Phosphorylation of STAT3 induced trans-activation of cyclin D1 and survivin in SMCs in vitro and in neointimal cells in vivo, thus promoting proliferation and migration of SMCs as well as reducing apoptotic cell death. WP1066, a highly potent inhibitor of STAT3 signaling, abrogated phosphorylation of STAT3 and dose-dependently inhibited the functional effects of activated STAT3 in stimulated SMCs. The local application of WP1066 via a thermosensitive pluronic F-127 gel around the dilated arteries significantly inhibited proliferation of neointimal cells and decreased the neointimal lesion size at 3 weeks after injury. Even though WP1066 application attenuated the injury-induced up-regulation of the chemokine RANTES at 6 h after injury, there was no significant effect on the accumulation of circulating cells at 1 week after injury. In conclusion, these data identify STAT3 as a key molecule for the proliferative response of SMC and neointima formation. Moreover, inhibition of STAT3 by the potent and specific compound WP1066 might represent a novel and attractive approach for the local treatment of vascular proliferative diseases.

Background/aims: Quinone reductase 2 (NQO2) is a flavoprotein that catalyzes the metabolic reduction of quinines, but its biological mechanism in vascular smooth muscle cells (VSMCs) is unclear. The aim of this study was to evaluate the role of NQO2 on VSMCs proliferation and the neointimal formation in balloon injured rat carotid artery., Methods: Left common carotid arteries from Sprague-Dawley rats were injured by a balloon catheter, and the injured arteries were incubated with 50 μL solution of NQO2-siRNA-GFP lentiviral vectors, NC-siRNA-GFP lentiviral vectors or PBS for 1 h. The rats were euthanized for morphometric and immunohistochemical analysis, real-time PCR and western blot analysis at 2 weeks after balloon injury and gene transfer. The cultured rat VSMCs transduced with NQO2-siRNA-GFP or NC-siRNA-GFP lentiviral vectors were used for cell proliferation assay, real-time PCR and western blot analysis. In order to detect the vascular or intracellular ROS level, the lentiviral vectors without GFP were used to transfect the injured common carotid arteries and the cultured rat VSMCs., Results: Lentiviral vectors bearing NQO2 siRNA could reduce NQO2 protein level and suppress NQO2 mRNA expression in balloon injured artery walls and cultured rat VSMCs. Downregulation of NQO2 significantly suppressed VSMCs proliferation and intimal formation. NQO2 siRNA treatment could reduce vascular or intracellular ROS level and decrease the phosphorylation of the ERK1/2 in balloon injured artery walls and cultured rat VSMCs., Conclusion: Our study suggests that downregulation of NQO2 significantly suppresses VSMCs proliferation and progression of neointimal formation after vascular injury., (Copyright © 2012 S. Karger AG, Basel.)

Objective: Vascular smooth muscle cell (SMC) proliferation has an indispensable role in the pathogenesis of vascular disease, but the mechanism is not fully elucidated. The epigenetic enzyme histone deacetylase 7 (HDAC7) is involved in endothelial homeostasis and SMC differentiation and could have a role in SMC proliferation. In this study, we sought to examine the effect of 2 HDAC7 isoforms on SMC proliferation and neointima formation., Methods and Results: We demonstrated that overexpression of unspliced HDAC7 (HDAC7u) could suppress SMC proliferation through downregulation of cyclin D1 and cell cycle arrest, whereas spliced HDAC7 (HDAC7s) could not. Small interfering RNA (siRNA)-mediated knockdown of HDAC7 increased SMC proliferation and induced nuclear translocation of β-catenin. Additional experiments showed that only HDAC7u could bind to β-catenin and retain it in the cytoplasm. Reporter gene assay and reverse transcription polymerase chain reaction revealed a reduction of β-catenin activity in cells overexpressing HDAC7u but not HDAC7s. Deletion studies indicated that the C-terminal region of HDAC7u is responsible for the interaction with β-catenin. However, the addition of amino acids to the N terminus of HDAC7u disrupted the binding, further strengthening our hypothesis that HDAC7s does not interact with β-catenin. The growth factor platelet-derived growth factor-BB increased the splicing of HDAC7 while simultaneously decreasing the expression of HDAC7u. Importantly, in an animal model of femoral artery wire injury, we demonstrated that knockdown of HDAC7 by siRNA aggravates neointima formation in comparison with control siRNA., Conclusion: Our findings demonstrate that splicing of HDAC7 modulates SMC proliferation and neointima formation through β-catenin nuclear translocation, which provides a potential therapeutic target in vascular disease.

Objective: Although vascular smooth muscle cell (VSMC) apoptosis occurs after vessel injury and during remodeling, the direct role of VSMC death in determining final vessel structure is unclear. We sought to determine the role of VSMC apoptosis in vessel remodeling, medial repair, and neointima formation and to identify the mediators involved., Methods and Results: The left common carotid artery was ligated in SM22α-human diphtheria toxin receptor mice, in which diphtheria toxin treatment selectively induces VSMC apoptosis. Apoptosis induced from day 7 to day 14 after ligation significantly increased neointimal and medial areas, cell proliferation, migration, and vessel size. Neointima formation depended on VSMCs, as VSMC depletion before ligation significantly reduced neointimal area and cellularity. In culture, conditioned media from apoptotic VSMCs promoted VSMC migration, proliferation, and collagen synthesis. Interleukin-6 (IL-6) secretion increased 5-fold and IL-1α 1.5-fold after apoptosis, whereas IL-6 inhibition negated the effect of apoptotic VSMC supernatants on VSMC migration, proliferation, and matrix synthesis., Conclusion: Signaling from apoptotic VSMCs directly promotes vessel remodeling, medial repair, and neointima formation after flow reduction. Although lumen size appears to depend on flow, VSMC apoptosis is an important determinant of vessel, medial, and neointimal size after flow reduction.

Members of the NR4A subgroup of the nuclear hormone receptor superfamily have emerged as key transcriptional regulators of proliferation and inflammation. NOR1 constitutes a ligand-independent transcription factor of this subgroup and induces cell proliferation; however, the transcriptional mechanisms underlying this mitogenic role remain to be defined. Here, we demonstrate that the F-box protein SKP2 (S phase kinase-associated protein 2), the substrate-specific receptor of the ubiquitin ligase responsible for the degradation of p27(KIP1) through the proteasome pathway, constitutes a direct transcriptional target for NOR1. Mitogen-induced Skp2 expression is silenced in vascular smooth muscle cells (VSMC) isolated from Nor1-deficient mice or transfected with Nor1 siRNA. Conversely, adenovirus-mediated overexpression of NOR1 induces Skp2 expression in VSMC and decreases protein abundance of its target p27. Transient transfection experiments establish that NOR1 transactivates the Skp2 promoter through a nerve growth factor-induced clone B response element (NBRE). Electrophoretic mobility shift and chromatin immunoprecipitation assays further revealed that NOR1 is recruited to this NBRE site in the Skp2 promoter in response to mitogenic stimulation. In vivo Skp2 expression is increased during the proliferative response underlying neointima formation, and this transcriptional induction depends on the expression of NOR1. Finally, we demonstrate that overexpression of Skp2 rescues the proliferative arrest of Nor1-deficient VSMC. Collectively, these results characterize Skp2 as a novel NOR1-regulated target gene and detail a previously unrecognized transcriptional cascade regulating mitogen-induced VSMC proliferation.

Nitric oxide (NO) limits formation of neointimal hyperplasia in animal models of arterial injury in large part by inhibiting vascular smooth muscle cell (VSMC) proliferation through cell cycle arrest. The ubiquitin-conjugating enzyme UbcH10 is responsible for ubiquitinating cell cycle proteins for proper exit from mitosis. We hypothesize that NO prevents VSMC proliferation, and hence neointimal hyperplasia, by decreasing levels of UbcH10. Western blotting and immunofluorescent staining showed that NO reduced UbcH10 levels in a concentration-dependent manner in VSMC harvested from the abdominal aortas of Sprague-Dawley rats. Treatment with NO or siRNA to UbcH10 decreased both UbcH10 levels and VSMC proliferation (P<0.001), while increasing UbcH10 levels by plasmid transfection or angiotensin II stimulation increased VSMC proliferation to 150% (P=0.008) and 212% (P=0.002) of control, respectively. Immunofluorescent staining of balloon-injured rat carotid arteries showed a ~4-fold increase in UbcH10 levels, which was profoundly decreased following treatment with NO. Western blotting of carotid artery lysates showed no UbcH10 in uninjured vessels, a substantial increase in the injury alone group, and a significant decrease in the injury+NO group (~3-fold reduction versus injury alone). Importantly, in vitro and in vivo, a marked increase in polyubiquitinated UbcH10 was observed in the NO-treated VSMC and carotid arteries, respectively, indicating that NO may be decreasing unmodified UbcH10 levels by increasing its ubiquitination. Central to our hypothesis, we report that NO decreases UbcH10 levels in VSMC in vitro and following arterial injury in vivo in association with increasing polyubiquitinated-UbcH10 levels. These changes in UbcH10 levels correlate with VSMC proliferation and neointimal hyperplasia, making UbcH10 a promising therapeutic target for inhibiting this proliferative disease.

Objective: We have shown that the chloride-proton antiporter chloride channel-3 (ClC-3) is required for endosome-dependent signaling by the Nox1 NADPH oxidase in SMCs. In this study, we tested the hypothesis that ClC-3 is necessary for proliferation of smooth muscle cells (SMCs) and contributes to neointimal hyperplasia following vascular injury., Methods and Results: Studies were performed in SMCs isolated from the aorta of ClC-3-null and littermate control (wild-type [WT]) mice. Thrombin and tumor necrosis factor-α (TNF-α) each caused activation of both mitogen activated protein kinase extracellular signal-regulated kinases 1 and 2 and the matrix-degrading enzyme matrix metalloproteinase-9 and cell proliferation of WT SMCs. Whereas responses to thrombin were preserved in ClC-3-null SMCs, the responses to TNF-α were markedly impaired. These defects normalized following gene transfer of ClC-3. Carotid injury increased vascular ClC-3 expression, and compared with WT mice, ClC-3-null mice exhibited a reduction in neointimal area of the carotid artery 28 days after injury., Conclusions: ClC-3 is necessary for the activation of SMCs by TNF-α but not thrombin. Deficiency of ClC-3 markedly reduces neointimal hyperplasia following vascular injury. In view of our previous findings, this observation is consistent with a role for ClC-3 in endosomal Nox1-dependent signaling. These findings identify ClC-3 as a novel target for the prevention of inflammatory and proliferative vascular diseases.

MicroRNAs are phenotypic regulators of vascular smooth muscle cells (VSMCs). In this paper, we demonstrate that miR-146a targets the Krüppel-like factor 4 (KLF4) 3'-untranslated region and has an important role in promoting VSMC proliferation in vitro and vascular neointimal hyperplasia in vivo. Silencing of miR-146a in VSMCs increases KLF4 expression, whereas overexpression of miR-146a decreases KLF4 levels. Furthermore, we demonstrate that KLF4 competes with Krüppel-like factor 5 (KLF5) to bind to and regulate the miR-146a promoter, and that KLF4 and KLF5 exert opposing effects on the miR-146a promoter. Overexpression of KLF4 in VSMCs decreases miR-146a transcription levels. By using both gain-of-function and loss-of-function approaches, we found that miR-146a promotes VSMC proliferation in vitro. Transfection of antisense miR-146a oligonucleotide into balloon-injured rat carotid arteries markedly decreased neointimal hyperplasia. These findings suggest that miR-146a and KLF4 form a feedback loop to regulate each other's expression and VSMC proliferation.