Linarin Ameliorates Restenosis After Vascular Injury in Type 2 Diabetes Mellitus via Regulating ADAM10-Mediated Notch Signaling Pathway.

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

KLF13 promotes VSMCs phenotypic dedifferentiation by directly binding to the SM22α promoter.

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.

Small nucleolar RNA host gene 18 controls vascular smooth muscle cell contractile phenotype and neointimal hyperplasia.

AIMS: Long non-coding RNA (LncRNA) small nucleolar RNA host gene 18 (SNHG18) has been widely implicated in cancers. However, little is known about its functional involvement in vascular diseases. Herein, we attempted to explore a role for SNHG18 in modulating vascular smooth muscle cell (VSMC) contractile phenotype and injury-induced neointima formation. METHODS AND RESULTS: Analysis of single-cell RNA sequencing and transcriptomic datasets showed decreased levels of SNHG18 in injured and atherosclerotic murine and human arteries, which is positively associated with VSMC contractile genes. SNHG18 was upregulated in VSMCs by TGFß1 through transcription factors Sp1 and SMAD3. SNHG18 gene gain/loss-of-function studies revealed that VSMC contractile phenotype was positively regulated by SNHG18. Mechanistic studies showed that SNHG18 promotes a contractile VSMC phenotype by up-regulating miR-22-3p. SNHG18 up-regulates miR-22 biogenesis and miR-22-3p production by competitive binding with the A-to-I RNA editing enzyme, adenosine deaminase acting on RNA-2 (ADAR2). Surprisingly, we observed that ADAR2 inhibited miR-22 biogenesis not through increasing A-to-I editing within primary miR-22, but by interfering with the binding of microprocessor complex subunit DGCR8 to primary miR-22. Importantly, perivascular SNHG18 overexpression in the injured vessels dramatically up-regulated the expression levels of miR-22-3p and VSMC contractile genes, and prevented injury-induced neointimal hyperplasia. Such modulatory effects were reverted by miR-22-3p inhibition in the injured arteries. Finally, we observed a similar regulator role for SNHG18 in human VSMCs and a decreased expression level of both SNHG18 and miR-22-3p in diseased human arteries; and we found that the expression level of SNHG18 was positively associated with that of miR-22-3p in both healthy and diseased human arteries. CONCLUSION: We demonstrate that SNHG18 is a novel regulator in governing VSMC contractile phenotype and preventing injury-induced neointimal hyperplasia. Our findings have important implications for therapeutic targeting snhg18/miR-22-3p signalling in vascular diseases.

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

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

Atlas of Cell Repertoire Within Neointimal Lesions Is Metabolically Altered in Hypertensive Rats.

BACKGROUND: High blood pressure has been suggested to accelerate vascular injury-induced neointimal formation and progression. However, little is known about the intricate relationships between vascular injury and hypertension in the context of arterial remodeling. METHODS: Single-cell RNA-sequencing analysis was used to depict the cell atlas of carotid arteries of Wistar Kyoto rats and spontaneously hypertensive rats with or without balloon injury. RESULTS: We found that hypertension significantly aggravated balloon injury-induced arterial stenosis. A total of 36 202 cells from carotid arteries with or without balloon injury were included in single-cell RNA-sequencing analysis. Cell composition analysis showed that vascular injury and hypertension independently induced distinct aortic cell phenotypic alterations including immune cells, endothelial cells (ECs), and smooth muscle cells. Specifically, our data showed that injury and hypertension-induced specific EC phenotypic alterations, and revealed a transition from functional ECs to hypermetabolic, and eventually dysfunctional ECs in hypertensive rats upon balloon injury. Importantly, our data also showed that vascular injury and hypertension-induced different smooth muscle cell phenotypic alterations, characterized by deferential expression of synthetic signatures. Interestingly, pathway analysis showed that dysregulated metabolic pathways were a common feature in monocytes/macrophages, ECs, and smooth muscle cells in response to injury and hypertension. Functionally, we demonstrate that inhibition of mitochondrial respiration significantly ameliorated injury-induced neointimal formation in spontaneously hypertensive rats. CONCLUSIONS: This study provides the cell landscape changes of the main aortic cell phenotypic alterations in response to injury and hypertension. Our findings suggest that targeting cellular mitochondrial respiration could be a novel therapeutic for patients with hypertension undergoing vascular angioplasty.

Transfer RNA-derived small RNA tRF-Glu-CTC attenuates neointimal formation via inhibition of fibromodulin.

Neointimal hyperplasia is a pathological vascular remodeling caused by abnormal proliferation and migration of subintimal vascular smooth muscle cells (VSMCs) following intimal injury. There is increasing evidence that tRNA-derived small RNA (tsRNA) plays an important role in vascular remodeling. The purpose of this study is to search for tsRNAs signature of neointima formation and to explore their potential functions. The balloon injury model of rat common carotid artery was replicated to induce intimal hyperplasia, and the differentially expressed tsRNAs (DE-tsRNAs) in arteries with intimal hyperplasia were screened by small RNA sequencing and tsRNA library. A total of 24 DE-tsRNAs were found in the vessels with intimal hyperplasia by small RNA sequencing. In vitro, tRF-Glu-CTC inhibited the expression of fibromodulin (FMOD) in VSMCs, which is a negative modulator of TGF-ß1 activity. tRF-Glu-CTC also increased VSMC proliferation and migration. In vivo experiments showed that inhibition of tRF-Glu-CTC expression after balloon injury of rat carotid artery can reduce the neointimal area. In conclusion, tRF-Glu-CTC expression is increased after vascular injury and inhibits FMOD expression in VSMCs, which influences neointima formation. On the other hand, reducing the expression of tRF-Glu-CTC after vascular injury may be a potential approach to prevent vascular stenosis.

Ultrasound-guided periadventitial administration of rapamycin-fibrin glue attenuates neointimal hyperplasia in the rat carotid artery injury model.

INTRODUCTION: Arterial restenosis caused by intimal hyperplasia (IH) is a serious complication after vascular interventions. In the rat carotid balloon injury model, we injected phosphate buffer saline (PBS), rapamycin-phosphate buffer saline suspension (RPM-PBS), blank fibrin glue (FG) and rapamycin-fibrin glue (RPM-FG) around the injured carotid artery under ultrasound guidance and observed the inhibitory effect on IH. METHODS: The properties of RPM-FG in vitro were verified by scanning electron microscopy (SEM) and determination of the drug release rate. FG metabolism in vivo was observed by fluorescence imaging. The rat carotid balloon injury models were randomly classified into 4 groups: PBS group (control group), RPM-PBS group, FG group, and RPM-FG group. Periadventitial administration was performed by ultrasound-guided percutaneous puncture on the first day after angioplasty. Carotid artery specimens were analyzed by immunostaining, Evans blue staining and hematoxylin-eosin staining. RESULTS: The RPM particles showed clustered distributions in the FG block. The glue was maintained for a longer time in vivo (> 14 days) than in vitro (approximately 7 days). Two-component liquid FG administered by ultrasound-guided injection completely encapsulated the injured artery before coagulation. The RPM-FG inhibited IH after carotid angioplasty vs. control and other groups. The proliferation of vascular smooth muscle cells (VSMCs) was significantly inhibited during neointima formation, whereas endothelial cell (EC) repair was not affected. CONCLUSION: Periadventitial delivery of RPM-FG contributed to inhibiting IH in the rat carotid artery injury model without compromising re-endothelialization. Additionally, FG provided a promising platform for the future development of a safe, effective, and minimally invasive perivascular drug delivery method to treat vascular disease.

Methyl donor diet attenuates intimal hyperplasia after vascular injury in rats.

Environmental factors, particularly dietary habits, play an important role in cardiovascular disease susceptibility and progression through epigenetic modification. Previous studies have shown that hyperplastic vascular intima after endarterectomy is characterized by genome-wide hypomethylation. The purpose of this study was to investigate whether methyl donor diet affects intimal hyperplasia and the possible mechanisms involved. Intimal hyperplasia was induced in SD rats by carotid artery balloon injury. From 8 d before surgery to 28 d after surgery, the animals were fed a normal diet (ND) or a methyl donor diet (MD) supplemented with folic acid, vitamin B12, choline, betaine, and zinc. Carotid artery intimal hyperplasia was observed by histology, the effect of MD on carotid protein expression was analyzed by proteomics, functional clustering, signaling pathway, and upstream-downstream relationship of differentially expressed proteins were analyzed by bioinformatics. Results showed that MD attenuated balloon injury-induced intimal hyperplasia in rat carotid arteries. Proteomic analysis showed that there were many differentially expressed proteins in the common carotid arteries of rats fed with two different diets. The differentially expressed proteins are mainly related to the composition and function of the extracellular matrix (EMC), and changes in the EMC can lead to vascular remodeling by affecting fibrosis and stiffness of the blood vessel wall. Changes in the levels of vasculotropic proteins such as S100A9, ILF3, Serpinh1, Fbln5, LOX, HSPG2, and Fmod may be the reason why MD attenuates intimal hyperplasia. Supplementation with methyl donor nutrients may be a beneficial measure to prevent pathological vascular remodeling after injury.

Ketogenic diet inhibits neointimal hyperplasia by suppressing oxidative stress and inflammation.

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

Exosomes Derived from Endothelial Cells Inhibit Neointimal Hyperplasia Induced by Carotid Artery Injury in Rats via ROS-NLRP3 Inflammasome Pathway.

This study attempted to investigate whether exosomes derived from rat endothelial cells (EC-Exo) attenuate intimal hyperplasia after balloon injury using hematoxylin and eosin staining, immunohistochemistry, immunofluorescence staining, Evans blue staining, and Western blotting. The results indicated that EC-Exo inhibited intimal hyperplasia in the carotid artery after balloon injury, promoted re-endothelialization, and reduced vascular inflammation and ROS-NLRP3-mediated cell pyroptosis. Thus, EC-Exo can inhibit neointimal hyperplasia after carotid artery injury in rats presumably by inhibiting the ROS-NLRP3 inflammasome and phenotypic transformation of vascular smooth muscle cells.

Deficiency of neuropeptide Y attenuates neointima formation after vascular injury in mice.

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-ß1 (TGF-ß1), 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-ß1 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.

Ant-Neointimal Formation Effects of SLC6A6 in Preventing Vascular Smooth Muscle Cell Proliferation and Migration via Wnt/ß-Catenin Signaling.

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/ß-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/ß-catenin signaling and maintaining the VSMC contractile phenotype.

Galangin inhibits neointima formation induced by vascular injury via regulating the PI3K/AKT/mTOR pathway.

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

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

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

Sex differences in arterial identity correlate with neointimal hyperplasia after balloon injury.

BACKGROUND: Endovascular treatment of atherosclerotic arterial disease exhibits sex differences in clinical outcomes including restenosis. However, sex-specific differences in arterial identity during arterial remodeling have not been described. We hypothesized that sex differences in expression of the arterial determinant erythropoietin-producing hepatocellular receptor interacting protein (Ephrin)-B2 occur during neointimal proliferation and arterial remodeling. METHODS AND RESULTS: Carotid balloon injury was performed in female and male Sprague-Dawley rats without or 14 days after gonadectomy; the left common carotid artery was injured and the right carotid artery in the same animal was used as an uninjured control. Arterial hemodynamics were evaluated in vivo using ultrasonography pre-procedure and post-procedure at 7 and 14 days and wall composition examined using histology, immunofluorescence and Western blot at 14 days after balloon injury. There were no significant baseline sex differences. 14 days after balloon injury, there was decreased neointimal thickness in female rats with decreased smooth muscle cell proliferation and decreased type I and III collagen deposition, as well as decreased TNFα- or iNOS-positive CD68+ cells and increased CD206- or TGM2-positive CD68+ cells. Female rats also showed less immunoreactivity of VEGF-A, NRP1, phosphorylated EphrinB2, and increased Notch1, as well as decreased phosphorylated Akt1, p38 and ERK1/2. These differences were not present in rats pretreated with gonadectomy. CONCLUSIONS: Decreased neointimal thickness in female rats after carotid balloon injury is associated with altered arterial identity that is dependent on intact sex hormones. Alteration of arterial identity may be a mechanism of sex differences in neointimal proliferation after arterial injury.

BMP-4 impedes endothelial cell migration in neointimal hyperplasia via FoXO-3 specific modulation of reactive oxygen species.

BACKGROUND AND AIMS: Endothelial cell injury causes vascular barrier dysfunction and leukocyte recruitment to the underlying tissue. Bone morphogenetic protein 4 (BMP-4) is a transforming growth factor that exerts pro-inflammatory effects on the endothelium. Here, we investigated the effects of BMP-4 on endothelial cell (EC) migration following balloon injury in SD rats. METHODS: An intimal hyperplasia model was established using balloon injury. Hematoxylin-eosin staining (HE) and silver staining were used to detect the alteration of endothelial cells recovery after balloon injury. Serum BMP-4 levels were assessed by ELISA. Human umbilical vein endothelial cells (HUVECs) were cultured. MTT assay was used to measure cell viability. Protein expression was detected by Western blot. Intracellular reactive oxygen species (ROS) was detected by dichloro-dihydro-fluorescein diacetate (DCFH-DA). HUVECs migration was measured via transwell assay and scratch wound assay. RESULTS: The results indicated that BMP-4 inhibition significantly decreased total plasma activity of BMP-4 and reduced neointimal hyperplasia by stimulating endothelial cell migration, but did not affect the medial area following balloon injury. BMP-4 suppressed the formation of ROS via forkhead box O3 (FoXO-3)/superoxide dismutase 1 (SOD-1). In vitro, a high level of ROS induced by BMP-4 impeded HUVECs migration. CONCLUSIONS: The results suggest that BMP-4 inhibition is a potential means of preventing intimal hyperplasia formation after balloon injury.

Apolipoprotein J Attenuates Vascular Restenosis by Promoting Autophagy and Inhibiting the Proliferation and Migration of Vascular Smooth Muscle Cells.

This research investigated the mechanism of CLU in vascular restenosis by regulating vascular smooth muscle cell (VSMC) proliferation and migration. Firstly, rat models of balloon injury (BI) were established, followed by the assessment of the injury to the common carotid artery. The effect of CLU on the intimal hyperplasia of BI rats was measured after the intervention in CLU, in addition to the evaluation of proliferation, migration, and autophagy of VSMCs. Moreover, the interaction between ATG and LC3 was analyzed, followed by validation of the role of autophagy in CLU's regulation on the proliferation and migration of VSMCs. It was found that CLU was highly expressed in BI rats. Altogether, our findings indicated that CLU was highly expressed in vascular restenosis, and CLU over-expression promoted the binding between ATG3 and LC3, thus facilitating VSMC autophagy and eventually attenuating intimal hyperplasia and vascular restenosis.

CFTR Suppresses Neointimal Formation Through Attenuating Proliferation and Migration of Aortic Smooth Muscle Cells.

ABSTRACT: Cystic fibrosis transmembrane conductance regulator (CFTR) plays important roles in arterial functions and the fate of cells. To further understand its function in vascular remodeling, we examined whether CFTR directly regulates platelet-derived growth factor-BB (PDGF-BB)-stimulated vascular smooth muscle cells (VSMCs) proliferation and migration, as well as the balloon injury-induced neointimal formation. The CFTR adenoviral gene delivery was used to evaluate the effects of CFTR on neointimal formation in a rat model of carotid artery balloon injury. The roles of CFTR in PDGF-BB-stimulated VSMC proliferation and migration were detected by mitochondrial tetrazolium assay, wound healing assay, transwell chamber method, western blot, and qPCR. We found that CFTR expression was declined in injured rat carotid arteries, while adenoviral overexpression of CFTR in vivo attenuated neointimal formation in carotid arteries. CFTR overexpression inhibited PDGF-BB-induced VSMC proliferation and migration, whereas CFTR silencing caused the opposite results. Mechanistically, CFTR suppressed the phosphorylation of PDGF receptor ß, serum and glucocorticoid-inducible kinase 1, JNK, p38 and ERK induced by PDGF-BB, and the increased mRNA expression of matrix metalloproteinase-9 and MMP2 induced by PDGF-BB. In conclusion, our results indicated that CFTR may attenuate neointimal formation by suppressing PDGF-BB-induced activation of serum and glucocorticoid-inducible kinase 1 and the JNK/p38/ERK signaling pathway.

Platelet-derived microvesicles regulate vascular smooth muscle cell energy metabolism via PRKAA after intimal injury.

Vascular intimal injury initiates various cardiovascular disease processes. Exposure to subendothelial collagen can cause platelet activation, leading to collagen-activated platelet-derived microvesicles (aPMVs) secretion. In addition, vascular smooth muscle cells (VSMCs) exposed to large amounts of aPMVs undergo abnormal energy metabolism; they proliferate excessively and migrate after the loss of endothelium, eventually contributing to neointimal hyperplasia. However, the roles of aPMVs in VSMC energy metabolism are still unknown. Our carotid artery intimal injury model indicated that platelets adhered to injured blood vessels. In vitro, phosphorylated Pka (cAMP-dependent protein kinase) content was increased in aPMVs. We also found that aPMVs significantly reduced VSMC glycolysis and increased oxidative phosphorylation, and promoted VSMC migration and proliferation by upregulating phosphorylated PRKAA (α catalytic subunit of AMP-activated protein kinase) and phosphorylated FoxO1. Compound C, an inhibitor of PRKAA, effectively reversed the enhancement of cellular function and energy metabolism triggered by aPMVs in vitro and neointimal formation in vivo. We show that aPMVs can affect VSMC energy metabolism through the Pka-PRKAA-FoxO1 signaling pathway and this ultimately affects VSMC function, indicating that the shift in VSMC metabolic phenotype by aPMVs can be considered a potential target for the inhibition of hyperplasia. This provides a new perspective for regulating the abnormal activity of VSMCs after injury.

LSD1 downregulates p21 expression in vascular smooth muscle cells and promotes neointima formation.

Neointima formation is characterized by the proliferation of vascular smooth muscle cells (VSMC). Although lysine-specific demethylase 1 (LSD1) has critical functions in several diseases, its role in neointima formation remains to be clarified. In this study, we aimed to explore the crucial role of LSD1 on neointima formation using a carotid artery injury model in mice. We observed that aberrant LSD1 expression was increased in human and mouse stenotic arteries and platelet-derived growth factor-BB (PDGF-BB)-treated VSMC. Furthermore, LSD1 knockdown significantly mitigated neointima formation in vivo and inhibited PDGF-BB-induced VSMC proliferation in vitro. We further uncovered that LSD1 overexpression exhibited opposite phenotypes in vivo and in vitro. Finally, LSD1 knockdown inhibited VSMC proliferation by increasing p21 expression, which is associated with LSD1 mediated di-methylated histone H3 on lysine 4 (H3K4me2) modification. Taken together, our data suggest that LSD1 may be a potential therapeutic target for the treatment of neointima formation.