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Fragmentation/loss of the structural protein elastin represents the precipitating event translating to aortic expansion and subsequent aneurysm formation. The present study tested the hypothesis that greater protein expression of tissue inhibitor of matrix metalloproteinase‐1 (TIMP‐1) and neointimal growth secondary to a reduction of medial elastin content represent sex‐dependent events limiting aortic vessel expansion in females. TIMP‐1 protein levels were higher in the ascending aorta of female versus male patients diagnosed with a bicuspid aortic valve (BAV). The latter paradigm was recapitulated in the aorta of adult male and female rats complemented by greater TIMP‐2 expression in females. CaCl2 (0.5 M) treatment of the infrarenal aorta of adult male and female rats increased the in situ vessel diameter and expansion was significantly smaller in females despite a comparable reduction of medial elastin content. The preferential appearance of a neointimal region of the CaCl2‐treated infrarenal aorta of female rats may explain in part the smaller in situ expansion and neointimal growth correlated positively with the % change of the in situ diameter. Neointimal formation was secondary to a significant increase in the density of medial/neointimal vascular smooth muscle cells (VSMCs) that re‐entered the G2‐M phase whereas VSMC cell cycle re‐entry was attenuated in the CaCl2‐treated infrarenal aorta of male rats. Thus, greater TIMP‐1 expression in the aorta of female BAV patients may prevent excessive elastin fragmentation and preferential neointimal growth following CaCl2‐treatment of the infrarenal aorta of female rats represents a sex‐dependent biological event limiting vessel expansion secondary to a significant loss of the structural protein. [ABSTRACT FROM AUTHOR]

Abstract Background Bioresorbable stents are designed to provide temporary mechanical support to the coronary arteries and then slowly degrade in vivo to avoid chronic inflammation. Zinc (Zn) is a promising material for bioresorbable stents; However, it can cause inflammation and neointimal formation after being implanted into blood vessels. Methods To improve biocompatibility of Zn, we first coated it with polydopamine (PDA), followed by immobilization of endothelial vascular growth factor (VEGF) onto the PDA coatings. Adhesion, proliferation, and phenotype maintenance of endothelial cells (ECs) on the coated Zn were evaluated in vitro. Then, a wire aortic implantation model in rats mimicking endovascular stent implantation in humans was used to assess vascular responses to the coated Zn wires in vivo. Thrombosis in aortas post Zn wire implantation, degradation of Zn wires in vivo, neointimal formation surrounding Zn wires, and macrophage infiltration and extracellular matrix (ECM) remodeling in the neointimas were examined. Results In vitro data showed that the PDA-coated Zn encouraged EC adhesion, spreading, proliferation, and phenotype maintenance on its surfaces. VEGF functionalization on PDA coatings further enhanced the biocompatibility of Zn to ECs. Implantation of PDA-coated Zn wires into rat aortas didn’t cause thrombosis and showed a faster blood flow than pure Zn or the Zn wires coated with VEGF alone. In addition, the PDA coating didn’t affect the degradation of Zn wires in vivo. Besides, the PDA-coated Zn wires reduced neointimal formation, increased EC coverage, decreased macrophage infiltration, and declined aggrecan accumulation in ECM. VEGF immobilization onto PDA coatings didn’t cause thrombosis and affect Zn degradation in vivo as well, and further increased the endothelization percentage as compared to PDA coating alone, thus resulting in thinner neointimas. Conclusion These results indicate that PDA coatings with VEGF immobilization would be a promising approach to functionalize Zn surfaces to increase biocompatibility, reduce inflammation, and inhibit neointimal formation after Zn implantation in vivo. Graphical Abstract

Neointimal hyperplasia is a major clinical complication of coronary artery bypass graft and percutaneous coronary intervention. Smooth muscle cells (SMCs) play a vital roles in neointimal hyperplasia development and undergo complex phenotype switching. Previous studies have linked glucose transporter member 10(Glut10) to the phenotypic transformation of SMCs. In this research, we reported that Glut10 helps maintain the contractile phenotype of SMCs. The Glut10-TET2/3 signaling axis can arrest neointimal hyperplasia progression by improving mitochondrial function via promotion of mtDNA demethylation in SMCs. Glut10 is significantly downregulated in both human and mouse restenotic arteries. Global Glut10 deletion or SMC-specific Glut10 ablation in the carotid artery of mice accelerated neointimal hyperplasia, while Glut10 overexpression in the carotid artery triggered the opposite effects. All of these changes were accompanied by a significant increase in vascular SMCs migration and proliferation. Mechanistically, Glut10 is expressed primarily in the mitochondria after platelet-derived growth factor-BB (PDGF-BB) treatment. Glut10 ablation induced a reduction in ascorbic acid (VitC) concentrations in mitochondria and mitochondrial DNA (mtDNA) hypermethylation by decreasing the activity and expression of the Ten-eleven translocation (TET) protein family. We also observed that Glut10 deficiency aggravated mitochondrial dysfunction and decreased the adenosinetriphosphate (ATP) content and the oxygen consumption rate, which also caused SMCs to switch their phenotype from contractile to synthetic phenotype. Furthermore, mitochondria-specific TET family inhibition partially reversed these effects. These results suggested that Glut10 helps maintain the contractile phenotype of SMCs. The Glut10-TET2/3 signaling axis can arrest neointimal hyperplasia progression by improving mitochondrial function via the promotion of mtDNA demethylation in SMCs. [ABSTRACT FROM AUTHOR]

Abstract Background Restenosis after percutaneous coronary intervention (PCI) limits therapeutic revascularization. Neuropeptide Y (NPY), co-stored and co-released with the sympathetic nervous system, is involved in this process, but its exact role and underlying mechanisms remain to be fully understood. This study aimed to investigate the role of NPY in neointima formation after vascular injury. Methods Using the left carotid arteries of wild-type (WT, NPY-intact) and NPY-deficient (NPY−/−) mice, ferric chloride-mediated carotid artery injury induced neointima formation. Three weeks after injury, the left injured carotid artery and contralateral uninjured carotid artery were collected for histological analysis and immunohistochemical staining. RT-qPCR was used to detect the mRNA expression of several key inflammatory markers and cell adhesion molecules in vascular samples. Raw264.7 cells were treated with NPY, lipopolysaccharide (LPS), and lipopolysaccharide-free, respectively, and RT-qPCR was used to detect the expression of these inflammatory mediators. Results Compared with WT mice, NPY−/− mice had significantly reduced neointimal formation three weeks after injury. Mechanistically, immunohistochemical analysis showed there were fewer macrophages and more vascular smooth muscle cells in the neointima of NPY−/− mice. Moreover, the mRNA expression of key inflammatory markers such as interleukin-6 (IL-6), transforming growth factor-β1 (TGF-β1), and intercellular adhesion molecule-1 (ICAM-1) was significantly lower in the injured carotid arteries of NPY−/− mice, compared to that in the injured carotid arteries of WT mice. In RAW264.7 macrophages, NPY significantly promoted TGF-β1 mRNA expression under unactivated but not LPS-stimulated condition. Conclusions Deletion of NPY attenuated neointima formation after artery injury, at least partly, through reducing the local inflammatory response, suggesting that NPY pathway may provide new insights into the mechanism of restenosis.

Aims Accumulating evidence supports the indispensable role of protein arginine methyltransferase 5 (PRMT5) in the pathological progression of several human cancers. As an important enzyme-regulating protein methylation, how PRMT5 participates in vascular remodelling remains unknown. The aim of this study was to investigate the role and underlying mechanism of PRMT5 in neointimal formation and to evaluate its potential as an effective therapeutic target for the condition. Methods and results Aberrant PRMT5 overexpression was positively correlated with clinical carotid arterial stenosis. Vascular smooth muscle cell (SMC)-specific PRMT5 knockout inhibited intimal hyperplasia with an enhanced expression of contractile markers in mice. Conversely, PRMT5 overexpression inhibited SMC contractile markers and promoted intimal hyperplasia. Furthermore, we showed that PRMT5 promoted SMC phenotypic switching by stabilizing Kruppel-like factor 4 (KLF4). Mechanistically, PRMT5-mediated KLF4 methylation inhibited ubiquitin-dependent proteolysis of KLF4, leading to a disruption of myocardin (MYOCD)–serum response factor (SRF) interaction and MYOCD–SRF-mediated the transcription of SMC contractile markers. Conclusion Our data demonstrated that PRMT5 critically mediated vascular remodelling by promoting KLF4-mediated SMC phenotypic conversion and consequently the progression of intimal hyperplasia. Therefore, PRMT5 may represent a potential therapeutic target for intimal hyperplasia–associated vascular diseases. [ABSTRACT FROM AUTHOR]

Background: Stent apposition to the vessel wall and in-stent neointimal formation after stent-assisted coil embolization for intracranial aneurysm are important factors associated with postoperative thromboembolic complications. No assessment methods have been established to depict 3-dimensional (3D) allround in-stent neointimal formation. Objective: To demonstrate the superiority of Dyna-3D imaging assessment as a modality for all-round ISNF in comparison with conventional two-dimensional digital subtraction angiography (2D-DSA). Methods: Consecutive patients who underwent braided stent-assisted coil embolization for unruptured aneurysm between November 2016 and September 2021 were enrolled. Radiological assessments for stent apposition to the parent vessel after stent deployment and in-stent neointimal formation after 3 months were obtained. Dyna-3D was reconstructed by overlapping a plain image showing stent struts with a rotational DSA image showing the vessel lumen. Reconstructed Dyna-3D images can be rotated to any angle on the screen to evaluate to stent apposition around the vessel and in-stent neointimal formation in 3D, for comparison with 2D-DSA evaluations. Results: Among the 73 patients enrolled, 70 patients (96%) showed complete stent wall apposition on Dyna-3D. Higher intra-rater agreement was confirmed on assessment of in-stent neointimal formation with Dyna-3D (Cohen's κ = 0.811) than with conventional 2D-DSA (Cohen's κ = 0.517). in-stent neointimal formation could not be confirmed on conventional imaging in 9 cases (16%) and on Dyna-3D in 2 cases (3%). The number of in-stent neointimal formations rated as stent wire completely outside the endothelial line was significantly higher with Dyna-3D than with 2D-DSA (p = 0.0001). Conclusion: All-round 3D evaluation by Dyna-3D imaging appears useful for confirming in-stent neointimal formation after braided stent deployment in patients after stent-assisted coil embolization. [ABSTRACT FROM AUTHOR]

Background: Restenosis after percutaneous coronary intervention (PCI) limits therapeutic revascularization. Neuropeptide Y (NPY), co-stored and co-released with the sympathetic nervous system, is involved in this process, but its exact role and underlying mechanisms remain to be fully understood. This study aimed to investigate the role of NPY in neointima formation after vascular injury. Methods: Using the left carotid arteries of wild-type (WT, NPY-intact) and NPY-deficient (NPY−/−) mice, ferric chloride-mediated carotid artery injury induced neointima formation. Three weeks after injury, the left injured carotid artery and contralateral uninjured carotid artery were collected for histological analysis and immunohistochemical staining. RT-qPCR was used to detect the mRNA expression of several key inflammatory markers and cell adhesion molecules in vascular samples. Raw264.7 cells were treated with NPY, lipopolysaccharide (LPS), and lipopolysaccharide-free, respectively, and RT-qPCR was used to detect the expression of these inflammatory mediators. Results: Compared with WT mice, NPY−/− mice had significantly reduced neointimal formation three weeks after injury. Mechanistically, immunohistochemical analysis showed there were fewer macrophages and more vascular smooth muscle cells in the neointima of NPY−/− mice. Moreover, the mRNA expression of key inflammatory markers such as interleukin-6 (IL-6), transforming growth factor-β1 (TGF-β1), and intercellular adhesion molecule-1 (ICAM-1) was significantly lower in the injured carotid arteries of NPY−/− mice, compared to that in the injured carotid arteries of WT mice. In RAW264.7 macrophages, NPY significantly promoted TGF-β1 mRNA expression under unactivated but not LPS-stimulated condition. Conclusions: Deletion of NPY attenuated neointima formation after artery injury, at least partly, through reducing the local inflammatory response, suggesting that NPY pathway may provide new insights into the mechanism of restenosis. [ABSTRACT FROM AUTHOR]

(1) Background: Coronary artery stenting leads to local inflammation, disturbs vasomotion, and slows endothelialization, increasing vascular thrombus risk. We used a pig stenting coronary artery model to assess how peri-interventional triple therapy with dabigatran ameliorates these effects. (2) Methods: In a total of 28 pigs bare-metal stents were implanted. Four days before the percutaneous coronary intervention (PCI), we started 16 of the animals on dabigatran, maintained through 4 days after the procedure. As controls, the remaining 12 pigs received no therapy. In both groups, dual antiplatelet therapy (DAPT) (clopidogrel, 75 mg plus aspirin, 100 mg) was administered until animals were euthanized. Just after the PCI and on day 3 after the procedure, we performed optical coherence tomography (OCT) in eight animals in the dabigatran group and four controls and euthanized them. We followed the eight remaining animals in each group with OCT and angiography for one month before euthanizing them and performed in vitro myometry and histology on harvested coronary arteries from all animals. (3) Results: The dabigatran group showed a significantly increased vasoconstriction at 3 days after PCI (10.97 ± 3.85 mN vs. 7.32 ± 5.41 mN, p = 0.03), but we found no differences between endothelium-dependent and -independent vasodilatation. We also found no group differences in OCT, quantitative angiography, or histomorphometry findings. (4) Conclusions: Starting a short course of dabigatran just before PCI and continuing for a 3-day window along with usual post-PCI DAPT is associated with enhanced vasoconstriction after bare-metal stent implantation without reducing neointimal formation at one month. [ABSTRACT FROM AUTHOR]

Vascular smooth muscle cells (VSMCs) play an important role in the pathogenesis of vascular remolding, such as atherosclerosis and restenosis. Solute carrier family 6 member 6 (SLC6A6) is a transmembrane transporter that maintains a variety of physiological functions and is highly expressed in VSMCs. However, its role on VSMCs during neointimal formation remains unknown. In this study, mRNA and protein levels of SLC6A6 were examined using models of VSMC phenotype switching in vivo and in vitro and human artery samples with or without atherosclerosis. SLC6A6 gain- and loss-of-function approaches were performed by adenovirus infection or small interfering RNA (siRNA) transfection, respectively. Reactive oxygen species (ROS), proliferation, migration, and phenotype-related proteins of VSMCs were measured. Vascular stenosis rate and related genes were assessed in a rat vascular balloon injury model overexpressing SLC6A6. SLC6A6 was downregulated in dedifferentiated VSMCs, atherosclerotic vascular tissues, and injured vascular tissues. SLC6A6 suppressed VSMC proliferation and migration, while increasing contractile VSMC proteins. Mechanistically, SLC6A6 overexpression reduced ROS production and inhibited the Wnt/β-catenin pathway. Furthermore, SLC6A6 overexpression suppressed neointimal formation in vivo. Collectively, overexpression of SLC6A6 suppresses neointimal formation by inhibiting VSMC proliferation and migration via Wnt/β-catenin signaling and maintaining the VSMC contractile phenotype. [ABSTRACT FROM AUTHOR]

Aims: Trimethylamine-N-oxide (TMAO) is a metabolite generated from dietary choline, betaine, and l-carnitine, after their oxidization in the liver. TMAO has been identified as a novel independent risk factor for atherosclerosis through the induction of vascular inflammation. However, the effect of TMAO on neointimal formation in response to vascular injury remains unclear. Results: This study was conducted using a murine model of acutely disturbed flow-induced atherosclerosis induced by partial carotid artery ligation. 3,3-Dimethyl-1-butanol (DMB) was used to reduce TMAO concentrations. Wild-type mice were divided into four groups [regular diet, high-TMAO diet, high-choline diet, and high-choline diet+DMB] to investigate the effects of TMAO elevation and its inhibition by DMB. Mice fed high-TMAO and high-choline diets had significantly enhanced neointimal hyperplasia and advanced plaques, elevated arterial elastin fragmentation, increased macrophage infiltration and inflammatory cytokine secretion, and enhanced activation of nuclear factor (NF)-κB, the NLRP3 inflammasome, and endoplasmic reticulum (ER) stress relative to the control group. Mice fed high-choline diets with DMB treatment exhibited attenuated flow-induced atherosclerosis, inflammasome expression, ER stress, and reactive oxygen species expression. Human aortic smooth muscle cells (HASMCs) were used to investigate the mechanism of TMAO-induced injury. The HASMCs were treated with TMAO with or without an ER stress inhibitor to determine whether inhibition of ER stress modulates the TMAO-induced inflammatory response. Innovation: This study demonstrates that TMAO regulates vascular remodeling via ER stress. Conclusion: Our findings demonstrate that TMAO elevation promotes disturbed flow-induced atherosclerosis and that DMB administration mitigates vascular remodeling, suggesting a rationale for a TMAO-targeted strategy for the treatment of atherosclerosis. Antioxid. Redox Signal. 38, 215–233. [ABSTRACT FROM AUTHOR]

Background and aimtRNA-derived fragments (tRFs) are a new class of non-coding RNAs involved in a variety of pathological processes, but their biological functions and mechanisms in human aortic smooth muscle cells (HASMCs) phenotype transition and vascular intimal hyperplasia are unclear.Methods/resultstiRNA-Gly-GCC is upregulated in synthetic HASMCs, atherosclerotic arteries, plasma, and the balloon injured carotid artery of rats. Functionally, the inhibition of tiRNA-Gly-GCC represses HASMCs proliferation, migration, and reversed dedifferentiation, whereas the overexpression of tiRNA- Gly-GCC have contrary effects. Mechanistically, tiRNA-Gly-GCC performs these functions on HASMCs via downregulating chromobox protein homolog 3 (CBX3). Finally, the inhibition of tiRNA-Gly-GCC could ameliorate neointimal formation after vascular injury in vivo.ConclusionstiRNA-Gly-GCC is a mediator of HASMCs phenotypic switching by targeting CBX3 and inhibition of tiRNA-Gly-GCC suppresses neointimal formation.

Abstract Background This study examined whether BI113823, a novel selective kinin B1 receptor antagonist can reverse established pulmonary arterial hypertension (PAH), prevent right heart failure and death, which is critical for clinical translation. Methods Left pneumonectomized male Wistar rats were injected with monocrotaline to induce PAH. Three weeks later, when PAH was well established, the rats received daily treatment of BI113823 or vehicle for 3 weeks. Results Treatment with BI113823 from day 21 to day 42 after monocrotaline injection reversed established PAH as shown by normalized values of mean pulmonary arterial pressure (mPAP). BI113823 therapy reversed pulmonary vascular remodeling, pulmonary arterial neointimal formation, and heart and lung fibrosis, reduced right ventricular pressure, right heart hypertrophy, improved cardiac output, and prevented right heart failure and death. Treatment with BI113823 reduced TNF-α and IL-1β, and macrophages recruitment in bronchoalveolar lavage, reduced CD-68 positive macrophages and expression of proliferating cell nuclear antigen (PCNA) in the perivascular areas, and reduced expression of iNOS, B1 receptors, matrix metalloproteinase (MMP)-2 and MMP-9 proteins, and the phosphorylation of ERK1/2 and AKT in lung. Treatment with BI113823 reduced mRNA expression of ANP, BNP, βMHC, CGTF, collange-I and IV in right heart, compared to vehicle treated controls. In human monocytes cultures, BI113823 reduced LPS-induced TNF-α production, MMP-2 and MMP-9 expression, and reduced TNF-α-induced monocyte migration. Conclusions We conclude that BI113823 reverses preexisting severe experimental pulmonary hypertension via inhibition of macrophage infiltration, cytokine production, as well as down regulation of matrix metalloproteinase proteins.

Yohimbine (YOH) has antiproliferative effects against breast cancer and pancreatic cancer; however, its effects on vascular proliferative diseases such as atherosclerosis remain unknown. Accordingly, we investigated the inhibitory mechanisms of YOH in vascular smooth muscle cells (VSMCs) stimulated by platelet-derived growth factor (PDGF)-BB, a major mitogenic factor in vascular diseases. YOH (5–20 μM) suppressed PDGF-BB-stimulated a mouse VSMC line (MOVAS-1 cell) proliferation without inducing cytotoxicity. YOH also exhibited antimigratory effects and downregulated matrix metalloproteinase-2 and -9 expression in PDGF-BB-stimulated MOVAS-1 cells. It also promoted cell cycle arrest in the initial gap/first gap phase by upregulating p27Kip1 and p53 expression and reducing cyclin-dependent kinase 2 and proliferating cell nuclear antigen expression. We noted phospholipase C-γ1 (PLCγ1) but not ERK1/2, AKT, or p38 kinase phosphorylation attenuation in YOH-modulated PDGF-BB-propagated signaling pathways in the MOVAS-1 cells. Furthermore, YOH still inhibited PDGF-BB-induced cell proliferation and PLCγ1 phosphorylation in MOVAS-1 cells with α2B-adrenergic receptor knockdown. YOH (5 and 10 mg/kg) substantially suppressed neointimal hyperplasia in mice subjected to CCA ligation for 21 days. Overall, our results reveal that YOH attenuates PDGF-BB-stimulated VSMC proliferation and migration by downregulating a α2B-adrenergic receptor–independent PLCγ1 pathway and reduces neointimal formation in vivo. Therefore, YOH has potential for repurposing for treating atherosclerosis and other vascular proliferative diseases. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of electrical stimulation of vagus nerve on migration and differentiation of c-kit+ vascular wall cells in rats under carotid artery injury. Methods A total of 24 SD rats were randomly divided into three groups: the sham-operation group, the operation group and the vagus nerve stimulation group, with 8 rats in each group. After the common carotid artery balloon injury model was carried out, the left vagus nerve was electrically stimulated in the vagus nerve stimulation group. HE staining and immunohistochemical staining were used to observe the thickness of vascular intima and the distribution of c-kit+ vascular wall cells. The serum levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured by enzyme-linked immunosorbent assay (ELISA). After isolating c-kit+ cells from the vessel wall, the c-kit+ cells were divided into three groups: the control group (no acetylcholine), the 10-5 mol/L acetylcholine group and the 10-5 mol/L acetylcholine group with the + alpha 7 nicotinic acetylcholine receptor (α7nAChR) agonist group. Transwell assay was applied to detect the effect of acetylcholine on c-kit+ migration, and the expression of smooth muscle 22 alpha (SM22α) was detected by Western blot assay. Results (1) Compared with operation group, the thickness of vascular neointima was reduced, and the number of c-kit+ cells in vascular neointima was decreased in the vagus nerve stimulation group (P＜0.05), and the serum levels of TNF-α and IL-6 were also reduced (P＜0.01). (2) Acetylcholine inhibited the ckit+ migration and increased the expression of SM22α, which could be reversed by α7nAChR agonist. Conclusion Acetylcholine can mitigate the neointimal formation through inhibiting the migration of c-kit+ and accelerating the differentiation of c-kit+ to vascular smooth muscle cells, the mechanism of which may be related to acetylcholine combined with α7nAChR that activating anti-inflammatory system. [ABSTRACT FROM AUTHOR]

Vascular endothelial cells (ECs) at arterial branches and curvatures experience disturbed blood flow and induce a quiescent-to-activated phenotypic transition of the adjacent smooth muscle cells (SMCs) and a subsequent smooth muscle hyperplasia. However, the mechanism underlying the flow pattern-specific initiation of EC-to-SMC signaling remains elusive. Our previous study demonstrated that endothelial microRNA-126-3p (miR-126-3p) acts as a key intercellular molecule to increase turnover of the recipient SMCs, and that its release is reduced by atheroprotective laminar shear (12 dynes/cm2) to ECs. Here we provide evidence that atherogenic oscillatory shear (0.5 ± 4 dynes/cm2), but not atheroprotective pulsatile shear (12 ± 4 dynes/cm2), increases the endothelial secretion of nonmembrane-bound miR-126-3p and other microRNAs (miRNAs) via the activation of SNAREs, vesicle-associated membrane protein 3 (VAMP3) and synaptosomal-associated protein 23 (SNAP23). Knockdown of VAMP3 and SNAP23 reduces endothelial secretion of miR-126-3p and miR-200a-3p, as well as the proliferation, migration, and suppression of contractile markers in SMCs caused by EC-coculture. Pharmacological intervention of mammalian target of rapamycin complex 1 in ECs blocks endothelial secretion and EC-to-SMC transfer of miR-126-3p through transcriptional inhibition of VAMP3 and SNAP23. Systemic inhibition of VAMP3 and SNAP23 by rapamycin or periadventitial application of the endocytosis inhibitor dynasore ameliorates the disturbed flow-induced neointimal formation, whereas intraluminal overexpression of SNAP23 aggravates it. Our findings demonstrate the flow-pattern-specificity of SNARE activation and its contribution to the miRNA-mediated EC-SMC communication.

Restenosis after angioplasty is a serious clinical problem that can result in re-occlusion of the coronary artery. Although current drug-eluting stents have proved to be more effective in reducing restenosis, they have drawbacks of inhibiting reendothelialization to promote thrombosis. New treatment options are in urgent need. We have shown that netrin-1, an axon-guiding protein, promotes angiogenesis and cardioprotection via production of nitric oxide (NO). The present study examined whether and how netrin-1 attenuates neointimal formation in a femoral wire injury model. Infusion of netrin-1 into C57BL/6 mice markedly attenuated neointimal formation following wire injury of femoral arteries, measured by intimal to media ratio (from 1.94 ± 0.55 to 0.45 ± 0.86 at 4 weeks). Proliferation of VSMC in situ was largely reduced. This protective effect was absent in DCC+/- animals. NO production was increased by netrin-1 in both intact and injured femoral arteries, indicating netrin-1 stimulation of endogenous NO production from intact endothelium and remaining endothelial cells post-injury. VSMC migration was abrogated by netrin-1 via a NO/cGMP/p38 MAPK pathway, while timely EPC homing was induced. Injection of netrin-1 preconditioned wild-type EPCs, but not EPCs of DCC+/- animals, substantially attenuated neointimal formation. EPC proliferation, NO production, and resistance to oxidative stress induced apoptosis were augmented by netrin-1 treatment. In conclusion, our data for the first time demonstrate that netrin-1 is highly effective in reducing neointimal formation following vascular endothelial injury, which is dependent on DCC, and attributed to inhibition of VSMC proliferation and migration, as well as improved EPC function. These data may support usage of netrin-1 and netrin-1 preconditioned EPCs as novel therapies for post angioplasty restenosis.Key messageNetrin-1 attenuates neointimal formation following post endothelial injury via DCC and NO. Netrin-1 inhibits VSMC proliferation in situ following endothelial injury. Netrin-1 inhibits VSMC migration via a NO/cGMP/p38 MAPK pathway. Netrin-1 augments proliferation of endothelial progenitor cells (EPCs) and EPC eNOS/NO activation. Netrin-1 enhances resistance of EPCs to oxidative stress, improving re-endothelialization following injury.

Background The endothelium is essential for maintaining vascular physiological homeostasis and the endothelial injury leads to the neointimal hyperplasia because of the excessive proliferation of vascular smooth muscle cells. Endothelial Foxp1 (forkhead box P1) has been shown to control endothelial cell (EC) proliferation and migration in vitro. However, whether EC‐Foxp1 participates in neointimal formation in vivo is not clear. Our study aimed to investigate the roles and mechanisms of EC‐Foxp1 in neointimal hyperplasia. Methods and Results The wire injury femoral artery neointimal hyperplasia model was performed in Foxp1 EC‐specific loss‐of‐function and gain‐of‐function mice. EC‐Foxp1 deletion mice displayed the increased neointimal formation through elevation of vascular smooth muscle cell proliferation and migration, and the reduction of EC proliferation hence reendothelialization after injury. In contrast, EC‐Foxp1 overexpression inhibited the neointimal formation. EC‐Foxp1 paracrine regulated vascular smooth muscle cell proliferation and migration via targeting matrix metalloproteinase‐9. Also, EC‐Foxp1 deletion impaired EC repair through reduction of EC proliferation via increasing cyclin dependent kinase inhibitor 1B expression. Delivery of cyclin dependent kinase inhibitor 1B‐siRNA to ECs using RGD (Arg‐Gly‐Asp)‐peptide magnetic nanoparticle normalized the EC‐Foxp1 deletion‐mediated impaired EC repair and attenuated the neointimal formation. EC‐Foxp1 regulates matrix metalloproteinase‐9/cyclin dependent kinase inhibitor 1B signaling pathway to control injury induced neointimal formation. Conclusions Our study reveals that targeting EC‐Foxp1‐matrix metalloproteinase‐9/cyclin dependent kinase inhibitor 1B pathway might provide future novel therapeutic interventions for restenosis.

Vascular smooth muscle cells (VSMCs) play an important role in the pathogenesis of vascular remolding, such as atherosclerosis and restenosis. Solute carrier family 6 member 6 (SLC6A6) is a transmembrane transporter that maintains a variety of physiological functions and is highly expressed in VSMCs. However, its role on VSMCs during neointimal formation remains unknown. In this study, mRNA and protein levels of SLC6A6 were examined using models of VSMC phenotype switching in vivo and in vitro and human artery samples with or without atherosclerosis. SLC6A6 gain- and loss-of-function approaches were performed by adenovirus infection or small interfering RNA (siRNA) transfection, respectively. Reactive oxygen species (ROS), proliferation, migration, and phenotype-related proteins of VSMCs were measured. Vascular stenosis rate and related genes were assessed in a rat vascular balloon injury model overexpressing SLC6A6. SLC6A6 was downregulated in dedifferentiated VSMCs, atherosclerotic vascular tissues, and injured vascular tissues. SLC6A6 suppressed VSMC proliferation and migration, while increasing contractile VSMC proteins. Mechanistically, SLC6A6 overexpression reduced ROS production and inhibited the Wnt/β-catenin pathway. Furthermore, SLC6A6 overexpression suppressed neointimal formation in vivo. Collectively, overexpression of SLC6A6 suppresses neointimal formation by inhibiting VSMC proliferation and migration via Wnt/β-catenin signaling and maintaining the VSMC contractile phenotype.

Background: This study examined whether BI113823, a novel selective kinin B1 receptor antagonist can reverse established pulmonary arterial hypertension (PAH), prevent right heart failure and death, which is critical for clinical translation.Methods: Left pneumonectomized male Wistar rats were injected with monocrotaline to induce PAH. Three weeks later, when PAH was well established, the rats received daily treatment of BI113823 or vehicle for 3 weeks.Results: Treatment with BI113823 from day 21 to day 42 after monocrotaline injection reversed established PAH as shown by normalized values of mean pulmonary arterial pressure (mPAP). BI113823 therapy reversed pulmonary vascular remodeling, pulmonary arterial neointimal formation, and heart and lung fibrosis, reduced right ventricular pressure, right heart hypertrophy, improved cardiac output, and prevented right heart failure and death. Treatment with BI113823 reduced TNF-α and IL-1β, and macrophages recruitment in bronchoalveolar lavage, reduced CD-68 positive macrophages and expression of proliferating cell nuclear antigen (PCNA) in the perivascular areas, and reduced expression of iNOS, B1 receptors, matrix metalloproteinase (MMP)-2 and MMP-9 proteins, and the phosphorylation of ERK1/2 and AKT in lung. Treatment with BI113823 reduced mRNA expression of ANP, BNP, βMHC, CGTF, collange-I and IV in right heart, compared to vehicle treated controls. In human monocytes cultures, BI113823 reduced LPS-induced TNF-α production, MMP-2 and MMP-9 expression, and reduced TNF-α-induced monocyte migration.Conclusions: We conclude that BI113823 reverses preexisting severe experimental pulmonary hypertension via inhibition of macrophage infiltration, cytokine production, as well as down regulation of matrix metalloproteinase proteins. [ABSTRACT FROM AUTHOR]

Objective: We evaluated the short and long-term effect of peri-interventional dabigatran therapy on vasomotion, endothelialization, and neointimal formation in a porcine coronary artery stenting model.Background: Stenting of coronary arteries induces local inflammation, impairs vasomotion and delays endothelialization.Methods: Twenty-eight animals underwent percutaneous coronary intervention (PCI) with drug eluting stents. Sixteen pigs started dabigatran therapy 4 days prior to PCI and continued for 4 days post-stenting, while 12 animals served as controls. Post-stenting dual antiplatelet therapy (75 mg clopidogrel and 100 mg aspirin) was continued in both groups until termination. Immediately post-stenting and at day 3 optical coherence tomography (OCT) was performed in all animals, followed by euthanasia of 8 dabigatran and 4 control animals. The remaining pigs (8 of each group) were followed up for 1 month, with control angiography and OCT. Tissue burden (degree of peri-strut structure—thrombus and/or fibrin) was evaluated. After euthanasia coronary arteries were harvested for in-vitro myometry and histology.Results: Thrombin generation was lower (p < 0.001) and tissue burden (0.83 ± 0.98 vs. 3.0 ± 2.45; p = 0.031) was significantly decreased in dabigatran treated animals. After 3 days post-PCI endothelium-dependent vasodilation was significantly improved (77 ± 40% vs. 41 ± 31%, p = 0.02) in dabigatran animals. Neither quantitative angiography nor histomorphometry showed differences between the groups. Endothelialization was faster in the dabigatran group as compared with controls (p = 0.045).Conclusion: Short-term peri-interventional triple therapy with dabigatran, aspirin, and clopidogrel led to an enhanced endothelium dependent vasodilation and faster endothelialization. However, neointimal formation 1-month after stent implantation was comparable between groups.

Background Migration of vascular smooth muscle cells (VSMCs) is the main contributor to neointimal formation. The Arp2/3 (actin‐related proteins 2 and 3) complex activates actin polymerization and is involved in lamellipodia formation during VSMC migration. Milk fat globule‐epidermal growth factor 8 (MFG‐E8) is a glycoprotein expressed in VSMCs. We hypothesized that MFG‐E8 regulates VSMC migration through modulation of Arp2/3‐mediated actin polymerization. Methods and Results To determine whether MFG‐E8 is essential for VSMC migration, a model of neointimal hyperplasia was induced in the common carotid artery of wild‐type and MFG‐E8 knockout mice, and the extent of neointimal formation was evaluated. Genetic deletion of MFG‐E8 in mice attenuated injury‐induced neointimal hyperplasia. Cultured VSMCs deficient in MFG‐E8 exhibited decreased cell migration. Immunofluorescence and immunoblotting revealed decreased Arp2 but not Arp3 expression in the common carotid arteries and VSMCs deficient in MFG‐E8. Exogenous administration of recombinant MFG‐E8 biphasically and dose‐dependently regulated the cultured VSMCs. At a low concentration, MFG‐E8 upregulated Arp2 expression. By contrast, MFG‐E8 at a high concentration reduced the Arp2 level and significantly attenuated actin assembly. Arp2 upregulation mediated by low‐dose MFG‐E8 was abolished by treating cultured VSMCs with β1 integrin function‐blocking antibody and Rac1 inhibitors. Moreover, treatment of the artery with a high dose of recombinant MFG‐E8 diminished injury‐induced neointimal hyperplasia and reduced VSMC migration. Conclusions MFG‐E8 plays a critical role in VSMC migration through dose‐dependent regulation of Arp2‐mediated actin polymerization. These findings suggest that high doses of MFG‐E8 may have therapeutic potential for treating vascular occlusive diseases.

The excessive proliferation and migration of vascular smooth muscle cells (VSMCs) play vital roles in neointimal hyperplasia and vascular restenosis. In the present study, we aimed to investigate the function and mechanism of octamer‐binding transcription factor 4 (OCT4, a key transcription factor for maintaining stem cells in de‐differentiated state) on neointima formation in response to vascular injury. Quantitative reverse‐transcription polymerase chain reaction and western blot results displayed a significant increase of OCT4 levels in injured carotid arteries. Immunohistochemistry and immunofluorescence assays confirmed that the increased OCT4 expression was primarily localized in α‐SMA‐positive VSMCs from neointima, and colocalized with PCNA in the nuclei of VSMCs. Adenovirus‐mediated OCT4 overexpression in injured carotid arteries exacerbated intimal thickening, while OCT4 knockdown significantly inhibited intimal thickening. In‐vitro experiments confirmed that the increased OCT4 expression in VMSCs could be induced by platelet‐derived growth factor‐BB (PDGF‐BB) in a time‐dependent manner. Overexpression of OCT4 greatly promoted VSMCs proliferation and migration, while OCT4 knockdown significantly retarded the PDGF‐BB‐induced excessive proliferation and migration of VSMCs. Bioinformatics analysis, dual‐luciferase reporter assay, and chromatin immunoprecipitation assay confirmed that OCT4 could upregulate matrix metalloproteinases 2 (MMP2) expression through promoting its transcription. Moreover, knockdown of MMP2 significantly attenuated OCT4‐mediated VSMCs proliferation and migration. These results indicated that OCT4 facilitated neointimal formation in response to vascular injury by MMP2‐mediated VSMCs proliferation and migration, and targeting OCT4 in VSMCs might be a novel therapeutic strategy for vascular restenosis. [ABSTRACT FROM AUTHOR]

Yohimbine (YOH) has antiproliferative effects against breast cancer and pancreatic cancer; however, its effects on vascular proliferative diseases such as atherosclerosis remain unknown. Accordingly, we investigated the inhibitory mechanisms of YOH in vascular smooth muscle cells (VSMCs) stimulated by platelet-derived growth factor (PDGF)-BB, a major mitogenic factor in vascular diseases. YOH (5–20 μM) suppressed PDGF-BB-stimulated a mouse VSMC line (MOVAS-1 cell) proliferation without inducing cytotoxicity. YOH also exhibited antimigratory effects and downregulated matrix metalloproteinase-2 and -9 expression in PDGF-BB-stimulated MOVAS-1 cells. It also promoted cell cycle arrest in the initial gap/first gap phase by upregulating p27Kip1 and p53 expression and reducing cyclin-dependent kinase 2 and proliferating cell nuclear antigen expression. We noted phospholipase C-γ1 (PLCγ1) but not ERK1/2, AKT, or p38 kinase phosphorylation attenuation in YOH-modulated PDGF-BB-propagated signaling pathways in the MOVAS-1 cells. Furthermore, YOH still inhibited PDGF-BB-induced cell proliferation and PLCγ1 phosphorylation in MOVAS-1 cells with α2B-adrenergic receptor knockdown. YOH (5 and 10 mg/kg) substantially suppressed neointimal hyperplasia in mice subjected to CCA ligation for 21 days. Overall, our results reveal that YOH attenuates PDGF-BB-stimulated VSMC proliferation and migration by downregulating a α2B-adrenergic receptor–independent PLCγ1 pathway and reduces neointimal formation in vivo. Therefore, YOH has potential for repurposing for treating atherosclerosis and other vascular proliferative diseases.

Following creation, an arteriovenous fistula (AVF) must mature (i.e., enlarge lumen to allow high blood flow) before being used for hemodialysis. AVF maturation failure rates are high, and currently, there are no effective therapy to treat this problem. The maturation process is likely affected by the integrity of the vascular extracellular matrix (ECM). Natural Vascular Scaffolding (NVS) Therapy is a new technology that interlinks collagen and elastin via photoactivation of a locally delivered small molecule (4-amino-1,8-naphtalamide). We hypothesized that NVS Therapy may improve AVF remodeling by preserving ECM integrity. AVFs were created in Wistar male rats by connecting the femoral vein (end) to femoral artery (side) in the same limb. Immediately after blood flow was restored to dilate the femoral vein by arterial pressure, a 10 μl-drop of the NVS compound (2 mg/ml) was placed on the anastomosis perivascularly. Following 5-min incubation, the NVS treated area was exposed to 1-min illumination by 450-nm light. The control group received 10 μl-drop of phosphate buffered saline (PBS) and the same light activation. The skin was closed, and rats were euthanized 4 weeks (n = 6–9 per group) post-AVF creation for histology, morphometry, immunohistochemistry (IHC), and multiphoton microscopy for second-harmonic-generation evaluation of collagen fibers. The vascular thickness was similar in both groups. The AVF vein’s open lumen area and % open lumen area in NVS-treated rats were significantly larger than in PBS-treated rats (4.2-fold p = 0.014 and 2-fold p = 0.009, respectively). The inflammatory markers IL-6 and MMP-9 in the AVF walls were significantly decreased in the NVS group than the PBS group. Collagen fibers in the vascular wall trended toward perpendicular alignment to the lumen circumference in the NVS-treated AVFs, with more defined shape but less area than in the PBS-treated AVFs. These results indicate that the NVS Therapy exerted changes in collagen, which may influence AVF maturation. Rats tolerated the NVS treatment well, and the lack of cell death by the treatment was confirmed in cell culture experiments. These results suggest that NVS treatment is safe and may have therapeutic potential by facilitating lumen expansion to enhanced AVF maturation in patients.

Uremic patients are characterized by an increased risk of atherosclerotic cardiovascular diseases. Vascular smooth muscle cell (VSMC) proliferation contributes to neointimal formation, a main pathological feature in atherosclerosis. Activation of CREB/ATF3 signaling is pivotal in VSMC proliferation, yet its role in uremic atherosclerosis is unknown. This study aimed to explore whether CREB/ATF3 signaling is involved in the molecular mechanism underlying neointimal formation in uremia. Treatment of VSMCs with uremic toxin (indoxyl sulfate [IS]) activated cAMP/CREB/ATF3/cyclin D signaling, which was reflected by increased VSMC proliferation. Blocking cAMP/PKA/CREB/ATF3 signaling attenuated the promoting effect of IS on cyclin D1 expression and VSMC proliferation. Loss-of-function and time-dependent experiments showed that ATF3 lies downstream of the CREB signaling. Mutational analysis of cyclin D1 promoter along with chromatin immunoprecipitation assays showed that CREB/ATF3 signaling participated in IS-induced cyclin D transcription. In vivo , phosphorylated CREB (an active form of CREB) and ATF3 were prominently upregulated in the neointima of experimental uremic rats, the atherosclerotic plaques of uremic ApoE −/− mice, and the iliac arteries of uremic patients. Notably, the use of lentivirus to knock down ATF3 in the neointima of balloon-injured arteries could suppress the effect of uremia in vivo , including neointimal formation and cyclin D expression. In this study, we demonstrated that CREB/ATF3-related signaling may be involved in IS-induced VSMC proliferation and the pathogenesis of neointimal formation during uremia. Image 1 • Indoxyl sulfate (IS), a uremic toxin, activated cAMP/CREB/ATF3/cyclin D signaling in VSMCs. • Blocking CREB/ATF3 signaling attenuated the promoting effect of IS on cyclin D expression and VSMC proliferation. • CREB/ATF3 signaling participated in IS-induced cyclin D transcription. • CREB/ATF3 signaling is upregulated in the atherosclerotic plaques of ApoE −/ − mice and the iliac arteries of uremic patients. • Silencing of ATF3 in the neointima of balloon-injured arteries could suppress the effect of uremia in vivo. [ABSTRACT FROM AUTHOR]

The demand for cardiac implantable electronic devices for arrhythmia therapy is still unabated and rising. Despite onward optimizations, lead-related problems such as infections or fractures often necessitate lead extraction. Due to adhesive thrombo-fibrotic lead encapsulations (TFLE) transvenous lead extraction is challenging and risky. However, knowledge on TFLEs and possible correlations with technical lead parameters and dwelling time (DT) were hitherto insufficiently studied. Therefore, we analyzed TFLEs of 62 lead from 35 body donor corpses to gain information for a potential lead design optimization. We examined both TFLE topography on the basis on anatomical landmarks and histo-morphological TFLE characteristics by means of histological paraffin sections and scanning electron microscopy of decellularized samples. The macroscopic analysis revealed that all leads were affected by TFLEs, mainly in the lead bearing veins. Half (47.2%) of the right-ventricular leads possessed adhesions to the tricuspid valve. On average, 49.9 ± 21.8% of the intravascular lead length was covered by TFLE of which 82.8 ± 16.2% were adhesive wall bindings (WB). The discrete TFLEs with at least one WB portion had a mean length of 95.0 ± 64.3 mm and a maximum of 200 mm. Neither sex, DT nor certain technical lead parameters showed distinct tendencies to promote or prevent TFLE. TFLE formation seems to start early in the first 1–2 weeks after implantation. The degree of fibrotization of the TFLE, starting with a thrombus, was reflected by the amount of compacted collagenous fibers and likewise largely independent from DT. TFLE thickness often reached several hundred micrometers. Calcifications were occasionally seen and appeared irregularly along the TFLE sheath. Leadless pacemaker systems have the advantage to overcome the problem with TFLEs but hold their own specific risks and limitations which are not fully known yet.

Activated endothelial cells (ECs) play an important role in the development of atherosclerosis (AS) because they form neointima resulting from abnormal vascular smooth muscle cell (VSMC) hyperplasia. TET2 was recently reported as a major regulator of the phenotypic switch of VSMCs, and it also protects ECs from noxious stimuli and represses inflammation in AS. The purpose of the present study is to determine whether activation of CD137 signaling can regulate the function of VSMCs by altering endothelial TET2 expression and to explore the potential mechanisms. VSMCs were isolated from C57BL/6 J mice, and the influence of ECs on VSMCs was analyzed using a co-culture system. Western blotting was used to detect the protein expression levels of TET2 and CD9, CD81, and CD63 as well as VSMC phenotypic markers. RT-PCR was performed to detect the TET2 mRNA levels. EdU proliferation assay and transwell migration assay were employed to assess the functions of VSMCs. EXO-spin kits were used to extract and purify exosomes. Transmission electron microscopy and nanoparticle tracking analysis were applied to characterize exosomes. ECs transduced with the TET2-overexpression lentivirus showed that the activation of endothelial CD137 signaling decreased TET2 expression and the TET2 content in exosomes. EC-derived exosomes were internalized by VSMCs, which inhibited phenotypic switching in vitro and neointimal formation in vivo. However, exosomes derived from CD137-activated ECs exhibited weakened protective effects on VSMCs. In conclusion, activation of endothelial CD137 signaling attenuated the repressive effects of EC-derived exosomes on platelet-derived growth factor (PDGF)-BB-induced phenotypic switching of VSMCs and neointimal formation after carotid injury.

Shang-Hung Chang,1 Cheng-Hung Lee,1 Yung-Hsin Yeh,1 Shih-Jung Liu,2 Chao-Jan Wang,3 Ming-Yi Hsu,3 Wei-Jan Chen1 1Cardiovascular Department, Chang Gung Memorial Hospital, Chang-Gung University College of Medicine, Tao-Yuan, Taiwan; 2Department of Mechanical Engineering, Chang Gung University, Tao-Yuan, Taiwan; 3Department of Medical Imaging and Intervention, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan Background: The drug-eluting stent is a standard approach for the treatment of coronary artery disease. Propylthiouracil (PTU), an antithyroid drug, has been proven to suppress neointimal formation after balloon injury. Materials and methods: This study used a biodegradable polymer coating with PTU to test its effects on platelet function, re-endothelialization, and neointimal formation after vascular injury. Electrospinning was used to fabricate hybrid stents and generate PTU-loaded nanofibers.Results: PTU-eluting stents maintained a stable release of PTU for 3 weeks. The PTU-coated stent markedly decreased the neointimal formation induced by vascular injury in the descending aorta of rabbits. Moreover, the PTU coating reduced platelet adhesion on the surface of the biodegradable membrane, which was reflected by the decreased expression of adhesion molecule in PTU-treated endothelial cells. The PTU coating enhanced re-endothelialization in injured aortas. In vitro, PTU exerted less suppressive effect on the proliferation and migration of endothelial cells than on those of vascular smooth muscle cells. Furthermore, treatment of endothelial cells with PTU induced phosphorylation (Ser1177) of endothelial nitric oxide synthase as well as its association with heat shock protein 90, supporting the protective role of PTU in endothelial function. The level of thyroid-stimulating hormone remained unchanged during the experimental period. Conclusion: This study indicates that PTU can be released locally and steadily in injured aortas, with some local effects but without systemic effects. Furthermore, PTU-coated stents may have beneficial effects on neointimal formation, endothelial cell, and platelet functions. Keywords: drug-eluting stent, propylthiouracil, neointimal formation, endothelial cell, platelet

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. [ABSTRACT FROM AUTHOR]

Restenosis is a common vascular complication after balloon angioplasty. Catheter balloon inflation-induced transient ischemia (hypoxia) of local arterial tissues plays a pathological role in neointima formation. Phosphoglycerate kinase 1 (PGK1), an adenosine triphosphate (ATP)-generating glycolytic enzyme, has been reported to associate with cell survival and can be triggered under hypoxia. The purposes of this study were to investigate the possible role and regulation of PGK1 in vascular smooth muscle cells (VSMCs) and balloon-injured arteries under hypoxia. Neointimal hyperplasia was induced by a rat carotid artery injury model. The cellular functions and regulatory mechanisms of PGK1 in VSMCs were investigated using small interfering RNAs (siRNAs), chemical inhibitors, or anaerobic cultivation. Our data indicated that protein expression of PGK1 can be rapidly induced at a very early stage after balloon angioplasty, and the silencing PGK1-induced low cellular energy circumstance resulted in the suppressions of VSMC proliferation and migration. Moreover, the experimental results demonstrated that blockage of PDGF receptor-β (PDGFRB) or its downstream pathway, the phosphoinositide 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) axis, effectively reduced hypoxia-induced factor-1 (HIF-1α) and PGK1 expressions in VSMCs. In vivo study evidenced that PGK1 knockdown significantly reduced neointima hyperplasia. PGK1 was expressed at the early stage of neointimal formation, and suppressing PGK1 has a potential beneficial effect for preventing restenosis.

Restenosis after angioplasty is caused usually by neointima formation characterized by aberrant vascular smooth muscle cell (VSMC) dedifferentiation. Myeloid-derived growth factor (MYDGF), secreted from bone marrow-derived monocytes and macrophages, has been found to have cardioprotective effects. In this study we investigated the effect of MYDGF to postinjury neointimal formation and the underlying mechanisms. Rat carotid arteries balloon-injured model was established. We found that plasma MYDGF content and the level of MYDGF in injured arteries were significantly decreased after balloon injury. Local application of exogenous MYDGF (50 μg/mL) around the injured vessel during balloon injury markedly ameliorated the development of neointimal formation evidenced by relieving the narrow endovascular diameter, improving hemodynamics, and reducing collagen deposition. In addition, local application of MYDGF inhibited VSMC dedifferentiation, which was proved by reversing the elevated levels of osteopontin (OPN) protein and decreased levels of α-smooth muscle actin (α-SMA) in the left carotid arteries. We showed that PDGF-BB (30 ng/mL) stimulated VSMC proliferation, migration and dedifferentiation in vitro; pretreatment with MYDGF (50-200 ng/mL) concentration-dependently eliminated PDGF-BB-induced cell proliferation, migration and dedifferentiation. Molecular docking revealed that MYDGF had the potential to bind with sphingosine-1-phosphate receptor 2 (S1PR2), which was confirmed by SPR assay and Co-IP analysis. Pretreatment with CCG-1423 (Rho signaling inhibitor), JTE-013 (S1PR2 antagonist) or Ripasudil (ROCK inhibitor) circumvented the inhibitory effects of MYDGF on VSMC phenotypic switching through inhibiting S1PR2 or its downstream RhoA-actin monomers (G-actin) /actin filaments (F-actin)-MRTF-A signaling. In summary, this study proves that MYDGF relieves neointimal formation of carotid arteries in response to balloon injury in rats, and suppresses VSMC dedifferentiation induced by PDGF-BB via S1PR2-RhoA-G/F-actin-MRTF-A signaling pathway. In addition, our results provide evidence for cross talk between bone marrow and vasculature., (© 2023. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.)

The high rate of autologous vein graft failure caused by neointimal hyperplasia remains an unresolved issue in the field of cardiovascular surgery; therefore, it is important to explore new methods for protecting against neointimal hyperplasia. MicroRNA‐365 has been reported to inhibit the proliferation of vascular smooth muscle cells (SMCs). This study aimed to test whether adenovirus‐mediated miR‐365 was able to attenuate neointimal formation in rat vein grafts. We found that miR‐365 expression was substantially reduced in vein grafts following engraftment. In vitro, overexpression of miR‐365 promoted smooth muscle‐specific gene expression and inhibited venous SMC proliferation and migration. Consistent with this, overexpression of miR‐365 in a rat vein graft model significantly reduced grafting‐induced neointimal formation and effectively improved the hemodynamics of the vein grafts. Mechanistically, we identified that cyclin D1 as a potential downstream target of miR‐365 in vein grafts. Specially, to increase the efficiency of miR‐365 gene transfection, a 30% poloxamer F‐127 gel containing 0.25% trypsin was mixed with adenovirus and spread around the vein grafts to increase the adenovirus contact time and penetration. We showed that adenovirus‐mediated miR‐365 attenuated venous SMC proliferation and migration in vitro and effectively inhibited neointimal formation in rat vein grafts. Restoring expression of miR‐365 is a potential therapeutic approach for the treatment of vein graft failure. © 2019 IUBMB Life, 2019 [ABSTRACT FROM AUTHOR]

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. Dihydroartemisinin (DHA) significantly inhibited neointimal formation in balloon‐induced rat carotid artery injury and the mechanism may be related to the inhibition of inhibitor of NF‐κB (IκB)/nuclear factor kappa B (NF‐κB) signaling, which alleviates the inflammatory response. [ABSTRACT FROM AUTHOR]

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

Abstract Aims The proliferation of VSMCs is the pathologic basis for intimal hyperplasia after angioplasty in diabetic patients. Translocator protein (TSPO), located in the outer mitochondrial membrane, has been found to regulate redox intermediate components in cell dysfunction. We hypothesized that TSPO may regulate VSMC proliferation and migration, and be involved in the intimal hyperplasia after angioplasty in diabetes. Materials and methods Cell proliferation was measured by cell counting and MTT assays. Cell migration was measured by Transwell® and scratch-wound assays. TSPO expression in arteries of rats and high glucose-treated A10 cells were detected by immunoblotting and immunofluorescence staining. Neointimal formation of carotid artery was induced by balloon injury in type 2 diabetic rat. Key findings TSPO expression was increased in the arterial samples from diabetic rats and A10 cells treated with high glucose. Down-regulation of TSPO expression by siRNA decreased the high-glucose-induced VSMC proliferation and migration in A10 cells. This phenomenon could be simulated by using TSPO ligands, PK 11195 and Ro5-4864. cGMP/PKG signals were involved in the TSPO ligand action, since in the presence of cGMP or PKG inhibitor ODQ or KT5823 respectively, the effect of PK 11195 on VSMC proliferation was blocked. Furthermore, PK 11195 significantly inhibited neointimal formation by the inhibition of VSMC proliferation. Significance This study suggests that TSPO inhibition suppresses the proliferation and migration of VSMCs induced by hyperglycemia, consequently, preventing atherosclerosis and restenosis after angioplasty in diabetic conditions. TSPO may be a potential therapeutic target to reduce arterial remodeling induced by angioplasty in diabetes. [ABSTRACT FROM AUTHOR]

The long‐term failure of vein grafts due to neointimal hyperplasia remains a difficult problem in cardiovascular surgery. Exploring novel approaches to prevent neointimal hyperplasia is important. MicroRNA‐146a (miR‐146a) plays an essential role in promoting vascular smooth muscle cell (VSMC) proliferation. Thus, the aim of the present study is to investigate whether adenovirus‐mediated miR‐146a sponge (Ad‐miR‐146a‐SP) gene therapy could attenuate neointimal formation in rat vein grafts. (Ad‐miR‐146a‐SP) was constructed to transfect cultured VSMCs and grafted veins. To improve the efficiency of transferring the miR‐146a sponge gene into the grafted veins, 20% poloxamer F‐127 gel incorporated with 0.25% trypsin was used to increase adenovirus contact time and penetration. miR‐146a‐SP transduction significantly reduced the expression of miR‐146a both in cultured VSMCs and vein grafts. miR‐146a sponge markedly attenuated VSMC proliferation and migration. Consistent with this, miR‐146a sponge gene therapy significantly attenuated neointimal formation and also improved blood flow in the vein grafts. Mechanistically, we identified the Krüppel‐like factor 4(KLF4) as a potential downstream target gene of miR‐146a in vein grafts. Our data show that miR‐146a sponge gene therapy could effectively reduce miR‐146a activity and attenuate neointimal formation in vein grafts, suggesting its potential therapeutic application for prevention of vein graft failure. © 2018 IUBMB Life, 71(1):125–133, 2019 [ABSTRACT FROM AUTHOR]

Neointimal hyperplasia is a major clinical complication of coronary artery bypass graft and percutaneous coronary intervention. Smooth muscle cells (SMCs) play a vital roles in neointimal hyperplasia development and undergo complex phenotype switching. Previous studies have linked glucose transporter member 10(Glut10) to the phenotypic transformation of SMCs. In this research, we reported that Glut10 helps maintain the contractile phenotype of SMCs. The Glut10-TET2/3 signaling axis can arrest neointimal hyperplasia progression by improving mitochondrial function via promotion of mtDNA demethylation in SMCs. Glut10 is significantly downregulated in both human and mouse restenotic arteries. Global Glut10 deletion or SMC-specific Glut10 ablation in the carotid artery of mice accelerated neointimal hyperplasia, while Glut10 overexpression in the carotid artery triggered the opposite effects. All of these changes were accompanied by a significant increase in vascular SMCs migration and proliferation. Mechanistically, Glut10 is expressed primarily in the mitochondria after platelet-derived growth factor-BB (PDGF-BB) treatment. Glut10 ablation induced a reduction in ascorbic acid (VitC) concentrations in mitochondria and mitochondrial DNA (mtDNA) hypermethylation by decreasing the activity and expression of the Ten-eleven translocation (TET) protein family. We also observed that Glut10 deficiency aggravated mitochondrial dysfunction and decreased the adenosinetriphosphate (ATP) content and the oxygen consumption rate, which also caused SMCs to switch their phenotype from contractile to synthetic phenotype. Furthermore, mitochondria-specific TET family inhibition partially reversed these effects. These results suggested that Glut10 helps maintain the contractile phenotype of SMCs. The Glut10-TET2/3 signaling axis can arrest neointimal hyperplasia progression by improving mitochondrial function via the promotion of mtDNA demethylation in SMCs., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Abstract Vine tea has been used as a medicinal herb in traditional Chinese medicine for hundreds of years. As the most abundant ingredient in vine tea, Dihydromyricetin (DHM) has been reported to exert anti-inflammatory, antioxidant, and anti-cardiovascular disease. However, the role of DHM in injury-induced neointimal formation remains poorly characterized. We determined the effects of DHM on ligation-induced carotid artery neointimal formation. We found that ligation-induced carotid artery neointimal formation could be significantly attenuated by DHM treatment. We provide evidence that DHM increases orphan nuclear receptor TR3 expression in smooth muscle cell (SMC) and carotid artery. Moreover, overexpression and loss-of-function strategies of TR3 were done to overexpression and knockdown of TR3, and demonstrate that DHM promotes SMC differentiation, however, inhibits SMC proliferation and migration, via regulating expression of TR3. Collectively, we reveal that DHM may be a therapeutic agent for the treatment of injury-induced vascular diseases. [ABSTRACT FROM AUTHOR]

MicroRNA 145 (miR-145) is a critical modulator of vascular smooth muscle cell (VSMC) phenotyping and proliferation. Flavonoids have been studied extensively due to their diverse pharmacological properties, including anti-inflammatory effects. The aims of this study is designed to evaluate the atheroprotective effects on angiotensin II (Ang II)-induced miR-145 and Klf4/myocardin expression in vitro and in vivo of flavonoids, including (-)-epigallocatechin gallate (EGCG), chrysin, wogonin, silibinin, and ferulic acid. Ang II significantly reduced the miR-145 compared with the control VSMC groups; all the tested flavonoids increased miR-145 in the 100 nM concentration. Among the test compounds, EGCG showed the strongest augmenting effect on miR-145 and myocardin, however, it also abolished Ang II-induced Klf4. A [3H]-thymidine incorporation proliferation assay demonstrated that EGCG inhibited Ang II-induced VSMC proliferation, and Klf4 siRNA presented with the similar results. Immunohistochemical analysis and confocal microscopy demonstrated increased Klf4 expression and the arterial lumen was narrowed after balloon injury 14 days. With the addition of EGCG (50 mg/kg) and Klf4 siRNA, neointimal formation was reduced by 40.7% and 50.5% compared with balloon injury 14 days; Klf4 expression also was attenuated. This study demonstrated EGCG increased miR-145 and attenuated Klf4, and ameliorated neointimal formation in vitro and in vivo. The novel suppressive effect was mediated through the miR-145 and Klf4/myocardin pathways. [ABSTRACT FROM AUTHOR]

Neointimal hyperplasia (or restenosis) is primarily attributed to excessive proliferation and migration of vascular smooth muscle cells (VSMCs). In this study, we investigated the inhibitory effects and mechanisms of ugonin J on VSMC proliferation and migration as well as neointimal formation. Cell viability and the cell-cycle distribution were, respectively, analyzed using an MTT assay and flow cytometry. Cell migration was examined using a wound-healing analysis and a transwell assay. Protein expressions and gelatinase activities were, respectively, measured using Western blot and gelatin zymography. Balloon angioplasty-induced neointimal formation was induced in a rat carotid artery model and then examined using immunohistochemical staining. Ugonin J induced cell-cycle arrest at the G0 /G1 phase and apoptosis to inhibit VSMC growth. Ugonin J also exhibited marked suppressive activity on VSMC migration. Ugonin J significantly reduced activations of focal adhesion kinase, phosphoinositide 3-kinase, v-akt murine thymoma viral oncogene homolog 1, and extracellular signal-regulated kinase 1/2 proteins. Moreover, ugonin J obviously reduced expressions and activity levels of matrix metalloproteinase-2 and matrix metalloproteinase-9. In vivo data indicated that ugonin J prevented balloon angioplasty-induced neointimal hyperplasia. Our study suggested that ugonin J has the potential for application in the prevention of balloon injury-induced neointimal formation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]