Injured Carotid Arteries Research Articles

Intervention of Asprosin Attenuates Oxidative Stress and Neointima Formation in Vascular Injury.

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

Inhibition of CIRBP represses the proliferation and migration of vascular smooth muscle cells via inhibiting Rheb/mTORC1 axis

The excessive migration and proliferation of vascular smooth muscle cells (VSMCs) plays a vital role in vascular intimal hyperplasia. CIRBP is involved in the proliferation of various cancer cells. This study was aimed to explore the role of CIRBP in the proliferation and migration of VSMCs. Adenovirus was used to interfere with cold-inducible RNA-binding protein (CIRBP) expression, while lentivirus was used to overexpress Ras homolog enriched in brain (Rheb). Western blotting and qRT-PCR were used to evaluate the expression of CIRBP, Rheb, and mechanistic target of rapamycin complex 1 (mTORC1) activity. The cell proliferation was determined by Ki67 immunofluorescence staining and CCK-8 assay. The wound healing assay was performed to assess cell migration. Additionally, immunohistochemistry was conducted to explore the role of CIRBP in intimal hyperplasia after vascular injury. We found that silencing CIRBP inhibited the proliferation and migration of VSMCs, decreased the expression of Rheb and mTORC1 activity. Restoration of mTORC1 activity via insulin or overexpression of Rheb via lentiviral transfection both attenuated the inhibitory effects of silencing CIRBP on the proliferation and migration of VSMCs. Moreover, Rheb overexpression abolished the inhibitory effect of silencing CIRBP on mTORC1 activity in VSMCs. CIRBP was upregulated in the injured carotid artery. Silencing CIRBP ameliorated intimal hyperplasia after vascular injury. In the summary, silencing CIRBP attenuates mTORC1 activity via reducing Rheb expression, thereby supressing the proliferation and migration of VSMCs and intimal hyperplasia after vascular injury.

Differential Distribution and Expression of α1-adrenergic Receptors in Low Shear Stress Induced Neointimal Formation

Low fluid shear stress in conduit arteries has emerged as a determinant of neointimal formation—an accumulation of vascular smooth muscle cells on the surface of the intima—the most challenging component of atherosclerosis to treat. α1-adrenergic receptor (α1-AR) antagonists have been well evaluated in vascular injury models yielding reduced neointimal formation. However, the α1-AR subtypes’ (α1A-AR, α1B-AR, α1D-AR) role in neointimal formation have not been determined under low shear stress conditions. α1-AR subtypes are expressed in various tissues and evoke different responses to the same ligand resulting in both adaptive and maladaptive responses. This study was designed to identify and characterize the α1-AR subtypes in low shear stress induced neointimal formation. An in vivo partial ligation mouse model was utilized to actuate low fluid shear stress and create neointimal formation in the left carotid artery in 14 days. The right carotid artery remained uninjured as the control. For tissue collection and processing, the left and right carotid arteries were harvested, snap-frozen in liquid nitrogen, and cryosectioned. Immunofluorescence staining was performed using primary antibodies for α1B-AR or α1A-AR and CD31 (endothelial cell marker). Confocal microscopy combined with processing and analysis was utilized for the detection, location, and distribution of cell markers. Histomorphometric analysis revealed significant structural changes, including the medial area and neointimal area, between the injured and non-injured arteries at the 14-day timepoint. Moreover, the injured arteries showed an increase in percent area stenosis, compared to the non-injured control. Histological observation of fluorescently labeled images revealed α1B-AR is densely distributed in the neointimal and medial area of the injured carotid arteries compared to the non-injured control. NucBlue (nuclear) staining indicated an increase in cell number in the medial and neointimal areas of the injured artery. Together, these results offer insight into changes in vascular α1-ARs under low shear stress conditions suggesting that α1B-AR may be upregulated in response to injury and contribute to neointimal formation. NIH HL155288. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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

IntroductionArterial 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. MethodsThe 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. ResultsThe 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. ConclusionPeriadventitial 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.

SMYD2 regulates vascular smooth muscle cell phenotypic switching and intimal hyperplasia via interaction with myocardin.

The SET and MYND domain-containing protein 2 (SMYD2) is a histone lysine methyltransferase that has been reported to regulate carcinogenesis and inflammation. However, its role in vascular smooth muscle cell (VSMC) homeostasis and vascular diseases has not been determined. Here, we investigated the role of SMYD2 in VSMC phenotypic modulation and vascular intimal hyperplasia and elucidated the underlying mechanism. We observed that SMYD2 expression was downregulated in injured carotid arteries in mice and phenotypically modulated VSMCs in vitro. Using an SMC-specific SMYD2 knockout mouse model, we found that SMYD2 ablation in VSMCs exacerbated neointima formation after vascular injury in vivo. Conversely, SMYD2 overexpression inhibited VSMC proliferation and migration in vitro and attenuated arterial narrowing in injured vessels in mice. SMYD2 downregulation promoted VSMC phenotypic switching accompanied with enhanced proliferation and migration. Mechanistically, genome-wide transcriptome analysis and loss/gain-of-function studies revealed that SMYD2 up-regulated VSMC contractile gene expression and suppressed VSMC proliferation and migration, in part, by promoting expression and transactivation of the master transcription cofactor myocardin. In addition, myocardin directly interacted with SMYD2, thereby facilitating SMYD2 recruitment to the CArG regions of SMC contractile gene promoters and leading to an open chromatin status around SMC contractile gene promoters via SMYD2-mediated H3K4 methylation. Hence, we conclude that SMYD2 is a novel regulator of VSMC contractile phenotype and intimal hyperplasia via a myocardin-dependent epigenetic regulatory mechanism.

Regulation of DNA damage and transcriptional output in the vasculature through a cytoglobin-HMGB2 axis

Identifying novel regulators of vascular smooth muscle cell function is necessary to further understand cardiovascular diseases. We previously identified cytoglobin, a hemoglobin homolog, with myogenic and cytoprotective roles in the vasculature. The specific mechanism of action of cytoglobin is unclear but does not seem to be related to oxygen transport or storage like hemoglobin. Herein, transcriptomic profiling of injured carotid arteries in cytoglobin global knockout mice revealed that cytoglobin deletion accelerated the loss of contractile genes and increased DNA damage. Overall, we show that cytoglobin is actively translocated into the nucleus of vascular smooth muscle cells through a redox signal driven by NOX4. We demonstrate that nuclear cytoglobin heterodimerizes with the non-histone chromatin structural protein HMGB2. Our results are consistent with a previously unknown function by which a non-erythrocytic hemoglobin inhibits DNA damage and regulates gene programs in the vasculature by modulating the genome-wide binding of HMGB2.

Empagliflozin inhibits neointimal hyperplasia through attenuating endothelial-to-mesenchymal transition via TAK-1/NF-κB pathway

ObjectiveTo investigate whether empagliflozin could prevent injury-induced vascular neointimal hyperplasia and to further explore its mechanism. MethodsMale C57BL/6J mice were divided into two groups with or without the empagliflozin treatment, and carotid ligation injury was performed to induce neointimal hyperplasia. The injured carotid arteries were collected for Western blotting (WB), histology and immunofluorescence analysis after four weeks. The inflammatory responses were analyzed by qRT-PCR to detect the inflammatory gene mRNA expression. To further explore its mechanism, HUVECs were treated with TGFβ-1 to induce EndMT followed by empagliflozin or vehicle treatment in vitro. A23187 (Calcimycin), an agonist of NF-κB signaling was used in the experiment. ResultsThe wall thickness and the neointima area was significantly reduced in the empagliflozin treatment group on day 28 after artery ligation. The Ki-67 positive cells were 28.33 ± 12.66% and 48.83 ± 10.41% in the empagliflozin-treated group and control group, respectively (P < 0.05). The mRNA expression levels of the inflammatory genes and inflammatory cells were decreased in the empagliflozin treatment group, as well as the MMP2 and MMP9. Meanwhile, empagliflozin can significantly reduce the migratory ability of inflammatory-treated HUVECs. The CD31 was increased in the TGFβ1+empagliflozin group, whereas the FSP-1, phosphorylation of TAK-1 (p-TAK-1) and phosphorylation of NF-κB (p- NF-κB) expression level were decreased, compared to the control group without empagliflozin treatment. However, the expression level of FSP-1 and p–NF–κB were reversed after co-treatment with A23187, whereas the p-TAK-1 expression level was without any significant difference. ConclusionEmpagliflozin inhibits the inflammation-induced EndMT via the TAK-1/NF-κB signaling pathway.

INKILN is a Novel Long Noncoding RNA Promoting Vascular Smooth Muscle Inflammation via Scaffolding MKL1 and USP10.

Activation of vascular smooth muscle cell (VSMC) inflammation is vital to initiate vascular disease. The role of human-specific long noncoding RNAs in VSMC inflammation is poorly understood. Bulk RNA sequencing in differentiated human VSMCs revealed a novel human-specific long noncoding RNA called inflammatory MKL1 (megakaryoblastic leukemia 1) interacting long noncoding RNA (INKILN). INKILN expression was assessed in multiple in vitro and ex vivo models of VSMC phenotypic modulation as well as human atherosclerosis and abdominal aortic aneurysm. The transcriptional regulation of INKILN was verified through luciferase reporter and chromatin immunoprecipitation assays. Loss-of-function and gain-of-function studies and multiple RNA-protein and protein-protein interaction assays were used to uncover a mechanistic role of INKILN in the VSMC proinflammatory gene program. Bacterial artificial chromosome transgenic mice were used to study INKILN expression and function in ligation injury-induced neointimal formation. INKILN expression is downregulated in contractile VSMCs and induced in human atherosclerosis and abdominal aortic aneurysm. INKILN is transcriptionally activated by the p65 pathway, partially through a predicted NF-κB (nuclear factor kappa B) site within its proximal promoter. INKILN activates proinflammatory gene expression in cultured human VSMCs and ex vivo cultured vessels. INKILN physically interacts with and stabilizes MKL1, a key activator of VSMC inflammation through the p65/NF-κB pathway. INKILN depletion blocks interleukin-1β-induced nuclear localization of both p65 and MKL1. Knockdown of INKILN abolishes the physical interaction between p65 and MKL1 and the luciferase activity of an NF-κB reporter. Furthermore, INKILN knockdown enhances MKL1 ubiquitination through reduced physical interaction with the deubiquitinating enzyme USP10 (ubiquitin-specific peptidase 10). INKILN is induced in injured carotid arteries and exacerbates ligation injury-induced neointimal formation in bacterial artificial chromosome transgenic mice. These findings elucidate an important pathway of VSMC inflammation involving an INKILN/MKL1/USP10 regulatory axis. Human bacterial artificial chromosome transgenic mice offer a novel and physiologically relevant approach for investigating human-specific long noncoding RNAs under vascular disease conditions.

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

BackgroundRestenosis 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.MethodsUsing 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.ResultsCompared 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.ConclusionsDeletion 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 496: Inhibition Of Tirna-gly-gcc Ameliorates Neointimal Formation Via Cbx3-mediated Vascular Smooth Muscle Cells Phenotypic Switching

Introduction: Vascular smooth muscle cells (VSMCs) are the main component of blood vessels and synthetic VSMCs play an essential role in vascular diseases. tRNA-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 VSMCs phenotype transition and vascular intimal hyperplasia are unclear. Hypothesis: We assessed the hypothesis that tRFs play important role in VSMCs phenotype transition, regulation of tRFs could ameliorates neointimal formation. Methods: Differential expression profiles of tRFs from different phenotype of VSMCs was acquired using small-RNA sequence. The expression of tiRNA-Gly-GCC was confirmed in VSMCs, vascular tissues and plasma from healthy and atherosclerosis using quantitative real-time PCR and RNA fluorescence in situ hybridization. Gain-and loss- of function were performed using mimics and inhibitors to investigate the role of tiRNA-Gly-GCC in VSMCs phenotypic transition and neointimal formation after vascular injury. Dual-luciferase reporter gene and rescue experiments were performed to demonstrate the relationship between tiRNA-Gly-GCC and CBX3. Results: tiRNA-Gly-GCC is upregulated in synthetic VSMCs, atherosclerotic arteries, plasma and the balloon injured carotid artery of rats. Functionally, the inhibition of tiRNA-Gly-GCC represses VSMCs proliferation, migration, and reversed dedifferentiation, whereas the overexpression of tiRNA-Gly- GCC have contrary effects. Mechanistically, tiRNA-Gly-GCC performs these functions on VSMCs via downregulating the expression of CBX3. Finally, the inhibition of tiRNA-Gly-GCC could ameliorate neointimal formation after vascular injury in vivo . Conclusions: In conclusion, tiRNA-Gly-GCC is a mediator of VSMCs phenotypic switching by targeting CBX3, inhibition of tiRNA-Gly-GCC suppresses neointimal formation.

MiR579-3p is an inhibitory modulator of neointimal hyperplasia and transcription factors c-MYB and KLF4

Neointimal hyperplasia (IH) is a common vascular pathology that typically manifests in in-stent restenosis and bypass vein graft failure. Smooth muscle cell (SMC) phenotypic switching is central to IH, both regulated by some microRNAs, yet the role of miR579-3p, a scarcely studied microRNA, is not known. Unbiased bioinformatic analysis suggested that miR579-3p was repressed in human primary SMCs treated with different pro-IH cytokines. Moreover, miR579-3p was software-predicted to target both c-MYB and KLF4 − two master transcription factors known to promote SMC phenotypic switching. Interestingly, treating injured rat carotid arteries via local infusion of miR579-3p-expressing lentivirus reduced IH 14 days after injury. In cultured human SMCs, transfection with miR579-3p inhibited SMC phenotypic switching, as indicated by decreased proliferation/migration and increased SMC contractile proteins. miR579-3p transfection downregulated c-MYB and KLF4, and luciferase assays indicated miR579-3p’s targeting of the 3′UTRs of the c-MYB and KLF4 mRNAs. In vivo, immunohistochemistry showed that treatment of injured rat arteries with the miR579-3p lentivirus reduced c-MYB and KLF4 and increased SMC contractile proteins. Thus, this study identifies miR579-3p as a previously unrecognized small-RNA inhibitor of IH and SMC phenotypic switch involving its targeting of c-MYB and KLF4. Further studies on miR579-3p may provide an opportunity for translation to develop IH-mitigating new therapeutics.

Conserved miR-370-3p/BMP-7 axis regulates the phenotypic change of human vascular smooth muscle cells

Endothelial dysfunction and inflammatory immune response trigger dedifferentiation of vascular smooth muscle cells (SMCs) from contractile to synthetic phenotype and initiate arterial occlusion. However, the complex vascular remodeling process playing roles in arterial occlusion initiation is largely unknown. We performed bulk sequencing of small and messenger RNAs in a rodent arterial injury model. Bioinformatic data analyses reveal that six miRNAs are overexpressed in injured rat carotids as well as synthetic-type human vascular SMCs. In vitro cell-based assays show that four miRNAs (miR-130b-5p, miR-132-3p, miR-370-3p, and miR-410-3p) distinctly regulate the proliferation of and monocyte adhesion to the vascular SMCs. Individual inhibition of the four selected miRNAs strongly prevents the neointimal hyperplasia in the injured rat carotid arteries. Mechanistically, miR-132-3p and miR-370-3p direct the cell cycle progression, triggering SMC proliferation. Gene ontology analysis of mRNA sequencing data consistently reveal that the miRNA targets include gene clusters that direct proliferation, differentiation, and inflammation. Notably, bone morphogenic protein (BMP)-7 is a prominent target gene of miR-370-3p, and it regulates vascular SMC proliferation in cellular and animal models. Overall, this study first reports that the miR-370-3p/BMP-7 axis determines the vascular SMC phenotype in both rodent and human systems.

Inhibition of tiRNA-Gly-GCC ameliorates neointimal formation via CBX3-mediated VSMCs phenotypic switching.

tRNA-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. tiRNA-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. tiRNA-Gly-GCC is a mediator of HASMCs phenotypic switching by targeting CBX3 and inhibition of tiRNA-Gly-GCC suppresses neointimal formation.

The Autotaxin-LPA Axis Emerges as a Novel Regulator of Smooth Muscle Cell Phenotypic Modulation during Intimal Hyperplasia.

Neointimal hyperplasia is characterized by a loss of the contractile phenotype of vascular smooth muscle cells (VSMCs). Our group has recently shown that VSMC proliferation and migration are mediated by lysophosphatidic acid (LPA) during restenosis, but the role of autotaxin (ATX; lysophospholipase D), which produces LPA, remains unclear. Endothelial denudation of the mouse carotid artery was performed to induce neointimal hyperplasia, and the extent of damage caused by the ATX-LPA axis was assessed in VSMCs. We observed the upregulation of ATX activity (p < 0.0002) in the injured carotid artery using an AR2 probe fluorescence assay. Further, the tissue carotid LPA levels were elevated 2.7-fold in carotid vessels, augmenting neointimal hyperplasia. We used an electrical cell-substrate impedance sensor (ECIS) to measure VSMC proliferation and migration. Treatment with an ATX inhibitor (PF8380) or LPA receptor inhibitor (Ki16425) attenuated VSMC proliferation (extracellular signal-regulated kinases) activity and migration in response to recombinant ATX. Indeed, PF8380 treatment rescued the aggravated post-wire injury neointima formation of carotid arteries. The upregulation of ATX following vessel injury leads to LPA production in VSMCs, favoring restenosis. Our observations suggest that inhibition of the ATX-LPA axis could be therapeutically targeted in restenosis to minimize VSMC phenotypic modulation and inflammation after vascular injury.

Effect of local unstable atherosclerotic plaque on plaque formation in the carotid artery and abdominal aorta of rabbits

To analyze the effect of local unstable atherosclerotic plaque on plaque formation in the carotid and aortic arteries of rabbits. Thirty New Zealand white rabbits were randomly divided into atherosclerosis model group, highfat diet feeding group, and normal chow feeding group (blank control group). In the model group, carotid artery balloon injury was induced after 4 weeks of high-fat diet feeding. Eight weeks later, all the rabbits were euthanized for histopathological examination of the carotid artery and abdominal aorta, and the mean intimal thickness and plaque to lumen area ratio were measured using image analysis software. Venous blood samples were collected from the rabbits for blood lipid analysis. At the ends of 4, 8 and 12 weeks, the rabbits in the model group and high-fat feeding group, but not those in the blank control group, all showed significant weight gain compared with their body weight at 0 week (P < 0.05). The mean intimal thickness was significantly greater in atherosclerosis model group than in the other two groups (P < 0.05). In atherosclerosis model group, the mean intimal thickness and plaque to lumen area ratio in the injured carotid artery were significantly greater than those in the contralateral carotid artery and abdominal aorta (P < 0.05). At the end of the 12 weeks, the levels of triglyceride (TG), total cholesterol (TC), low-density cholesterol (LDL-C), high-density cholesterol (HDL-C) and highsensitivity C-reactive protein (CRP) were all significantly higher in the model group and high-fat feeding group than in the blank control group (P < 0.05); the levels of TG, TC, LDL-C, or HDL-C did not differ significantly between the model group and high-fat feeding group (P>0.05), but the level of CRP was significantly higher in arteriosclerosis model group (P < 0.05). Local unstable atherosclerotic plaque can increase the level of CRP and promote the formation of atherosclerotic plaques in the carotid artery and abdominal aorta in rabbits.

MiR-7-5p attenuates vascular smooth muscle cell migration and intimal hyperplasia after vascular injury by NF-kB signaling

BackgroundAtherosclerosis (AS) is the primary cause of coronary artery disease, which is featured by aberrant proliferation, differentiation, and migration of vascular smooth muscle cells (VSMCs). MicroRNAs play crucial roles in AS, but the function of miR-7-5p in AS remains unclear. Here, we aimed to explore the effect of miR-7-5p on AS and VSMCs in vitro and in vivo. MethodsThe in vivo rat AS model and apoE−/− mouse model were established. The carotid artery injury was checked by immunohistochemistry staining. The RNA levels of miR-7-5p and p65 were measured by qPCR assay. Protein levels were checked by western blotting. Cell apoptosis was evaluated by flow cytometry. Cell migration was checked by Transwell assay and wound healing assay. The potential interaction between miR-7-5p with p65 was checked by luciferase reporter gene assay. ResultsMiR-7-5p was downregulated and NF-κB p65 was upregulated in injured carotid arteries in rat model. The carotid artery injury in the AS rats and the treatment of miR-7-5p attenuated the phenotype in the model. Immunohistochemistry staining and Western blot analysis revealed that PCNA levels were increased in injured carotid arteries of the model rats and miR-7-5p could reverse the levels. The cell viability of VSMCs was induced by PDGF-BB but miR-7-5p blocked the phenotype. PDGF-BB decreased apoptosis of VSMCs, while miR-7-5p was able to restore the cell apoptosis in the model. PDGF-BB-induced migration of VSMCs was attenuated by miR-7-5p. miR-7-5p mimic remarkably repressed the luciferase activity of p65 in VSMCs. The levels of p65 were inhibited by miR-7-5p in the cells. The PDGF-BB-promoted cell viability and migration of VSMCs was repressed by miR-7-5p and p65 overexpression reversed the phenotype. ConclusionWe concluded that miR-7-5p attenuates vascular smooth muscle cell migration and intimal hyperplasia after vascular injury by NF-kB signaling.

Large-conductance Ca2 +-activated K+ channel β1-subunit maintains the contractile phenotype of vascular smooth muscle cells.

Vascular smooth muscle cells (VSMCs) phenotype switching is very important during the pathogenesis and progression of vascular diseases. However, it is not well understood how normal VSMCs maintain the differentiated state. The large-conductance Ca2+-activated K+ (BKCa) channels are widely expressed in VSMCs and regulate vascular tone. Nevertheless, there is limited understanding of the role of the BKCa channel in modulation of the VSMC phenotype. We assessed BKCa channel expression levels in normal and injured carotid arteries from rats of the balloon-injury model. A strong decrease of BKCa-β1 was seen in the injured carotid arteries, accompanied by a parallel decrease of the VSMC contractile markers. BKCa-β1 in primary rat aortic VSMCs was decreased with the increase of passage numbers and the stimulation of platelet-derived growth factor (PDGF)-BB. Conversely, transforming growth factor β upregulated BKCa-β1. Meanwhile, the BKCa-β1 level was positively associated with the levels of VSMC contractile proteins. Intravenous injection of PDGF-BB induced downregulation of BKCa-β1 expression in the carotid arteries. Knockdown of BKCa-β1 favored VSMC dedifferentiation, characterized by altered morphology, abnormal actin fiber organization, decreased contractile proteins expression and reduced contractile ability. Furthermore, the resultant VSMC dedifferentiated phenotype rendered increased proliferation, migration, enhanced inflammatory factors levels, and matrix metalloproteinases activity. Studies using primary cultured aortic VSMCs from human recapitulated key findings. Finally, protein level of BKCa-β1 was reduced in human atherosclerotic arteries. BKCa-β1 is important in the maintenance of the contractile phenotype of VSMCs. As a novel endogenous defender that prevents pathological VSMC phenotype switching, BKCa-β1 may serve as a potential therapeutic target for treating vascular diseases including post-injury restenosis and atherosclerosis.

Exosomes Derived from AT2R-Overexpressing BMSC Prevent Restenosis After Carotid Artery Injury by Attenuating the Injury-Induced Neointimal Hyperplasia

Restenosis is a severe complication after percutaneous transluminal coronary angioplasty which limits the long-term efficacy of the intervention. In this study, we investigated the efficiency of exosomes derived from AT2R-overexpressing bone mesenchymal stem cells on the prevention of restenosis after carotid artery injury. Our data showed that AT2R-EXO promoted the proliferation and migration of vascular endothelial cells and maintained the ratio of eNOS/iNOS. On the contrary, AT2R-EXO inhibited the proliferation and migration of vascular smooth muscle cells. In vivo study proved that AT2R-Exo were more effectively accumulated in the injured carotid artery than EXO and Vehicle-EXO controls. AT2R-EXO treatment could improve blood flow of the injured carotid artery site more effectively. Further analysis revealed that AT2REXO prevents restenosis after carotid artery injury by attenuating the injury-induced neointimal hyperplasia. Our study provides a novel and more efficient exosome for the treatment of restenosis diseases after intervention.

Pleiotropic Effects of Icariside II on the Cardiovascular System: Novel Applications of Ethnopharmacology in Targeting Vascular Remodeling.

Pleiotropic Effects of Icariside II on the Cardiovascular System: Novel Applications of Ethnopharmacology in Targeting Vascular Remodeling.

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

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 β, 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.