Carotid Artery Balloon Injury Research Articles

MiR-1290 induces endothelial-to-mesenchymal transition and promotes vascular restenosis after angioplasty by targeting FGF2

Background and aimsEndothelial-to mesenchymal transition (EndMT) is an important reason for restenosis but the specific mechanisms need to be further explored. Therefore, the purpose of this study is to screen significantly different microRNAs (miRNAs) and explore its functions and downstream pathways. MethodsThis study screened several miRNAs with significant differences between human arterial segments from restenosis patients and healthy volunteers using whole transcriptome resequencing and real-time quantitative reverse transcription PCR (qRT-PCR). We explored the correlation between miR-1290 and EndMT using Western Blot, qRT-PCR, Pearson correlation analysis and further functional gain and loss experiments. Subsequently, we identified the direct downstream target of miR-1290 by bioinformatics analysis, RNA pull-down, double Luciferase reporter gene and other functional experiments. Finally, rat carotid artery balloon injury model demonstrated the therapeutic potential of miR-1290 regulator. ResultsWe screened 129 differentially expressed miRNAs. Among them, miR-1290 levels were significantly higher in restenosis arteries than in healthy arteries, and as expected, EndMT was functionally enhanced with miR-1290 overexpression but comparatively weakened when miR-1290 was knocked down. In addition, fibroblast growth factor-2 (FGF2) was established as the downstream target of miR-1290. Finally, we utilized an animal model and found that low miR-1290 levels could alleviate EndMT and the progression of restenosis. ConclusionsOur study demonstrated the strong regulatory effects of miR-1290 on EndMT, endometrial hyperplasia and restenosis, which could be useful as biomarkers and therapeutic targets for stent implantation in patients with arterial occlusive disease of the lower extremities.

Role of miRNAs regulation of Orai1 and store operated Ca2+ entry in vascular restenosis

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Agencia Estatal de Investigación Background The main cause of restenosis is attributed to the excessive proliferation and migration of vascular smooth muscle cells (VSMCs). Orai1 is one of the main molecular components of Store-Operated Calcium Entry (SOCE), playing a key role in VSMCs proliferation during neointima formation and vascular remodeling. Recent studies have demonstrated the implication of microRNAs (miRNAs) in VSMCs proliferation and migration, however, the role of these miRNAs as regulators of SOCE during vascular remodeling has not been described. Purpose This study aims to determine whether miRNAs regulate the intracellular Ca2+ handling, focusing on SOCE, during neointima formation. Methods Experiments were conducted in an animal rat model following the "Principles of laboratory animal care" and according to the Spanish law. The neointima development was assessed after balloon injury of carotid arteries and confirmed by haematoxylin and eosin staining, and αSMA staining of tissue sections up to 3 weeks after surgery. miRNAs array and RT-qPCR was used to examine the expression of miRNAs. Dual-Luciferase reporter assay was performed in A7R5 VSMCs line. Results Injured arteries showed significant increase in the expression of Orai1, as compared to non-injured arteries, indicating their possible participation in the neointima layer formation. Microarray of miRNAs analysis of the injured carotids compared to control carotids showed significant increase in the expression of miRNAs related to signalling pathways involving cell proliferation and migration, cell differentiation, or VSMCs contraction. Of these, we confirmed the significant increase of miR-18a-5p, miR-20a-5p, miR-20b-5p and miR-17-5p, which belong to widely studied miR-17-92 cluster involved in different cell proliferation. Moreover, we found that mimics of those miRNAs were able to regulate differentially the activity of the Orai1 promotor in A7R5 cell line, indicating that Orai1 can be targeted by miRNAs during in VSMCs. Conclusions In conclusion, our data confirms that Orai1 is upregulated during neointima formation associated with miRNAs dysregulation. Moreover, we found that Orai1-dependent SOCE can be finely regulated by miRNAs during restenosis.

Exosomal MALAT1 from macrophages treated with high levels of glucose upregulates LC3B expression via miR-204-5p downregulation.

Metastasis-associated lung adenocarcinoma transcript 1 ( MALAT1 ) plays a critical role in the pathophysiology of diabetes-related complications. However, whether macrophage-derived MALAT1 affects autophagic activity under hyperglycemic conditions is unclear. Therefore, we investigated the molecular regulatory mechanisms of macrophage-derived MALAT1 and autophagy under hyperglycemic conditions. Hyperglycemia was induced by culturing macrophages in 25 mM glucose for 1 hour. Exosomes were extracted from the culture media. A rat model of carotid artery balloon injury was established to assess the effect of MALAT1 on vascular injury. Reverse transcription, real-time quantitative polymerase chain reaction, western blotting, immunohistochemical staining, and luciferase activity assays were performed. Stimulation with high levels of glucose significantly enhanced MALAT1 expression in macrophage-derived exosomes. MALAT1 inhibited miR-204-5p expression in macrophage-derived exosomes under hyperglycemic conditions. siRNA-induced silencing of MALAT1 significantly reversed macrophage-derived exosome-induced miR-204-5p expression. Hyperglycemic treatment caused a significant, exosome-induced increase in the expression of the autophagy marker LC3B in macrophages. Silencing MALAT1 and overexpression of miR-204-5p significantly decreased LC3B expression induced by macrophage-derived exosomes. Overexpression of miR-204-5p significantly reduced LC3B luciferase activity induced by macrophage-derived exosomes. Balloon injury to the carotid artery in rats significantly enhanced MALAT1 and LC3B expression, and significantly reduced miR-204-5p expression in carotid artery tissue. Silencing MALAT1 significantly reversed miR-204-5p expression in carotid artery tissue after balloon injury. MALAT1 silencing or miR-204-5p overexpression significantly reduced LC3B expression after balloon injury. This study demonstrated that hyperglycemia upregulates MALAT1 . MALAT1 suppresses miR-204-5p expression and counteracts the inhibitory effect of miR-204-5p on LC3B expression in macrophages to promote vascular disease.

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.

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-β1 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.

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.

The role of calcium-sensing receptor in ginsenoside Rg1 promoting reendothelialization to inhibit intimal hyperplasia after balloon injury

Calcium-sensing receptor (CaSR) is a G protein-coupled receptor, widely distributed in various tissues, including vascular endothelial cells and smooth muscle cells, which plays an important role in the migration and homing of stem/progenitor cells and the proliferation of tissue cells. Restenosis after Percutaneous coronary intervention (PCI) seriously affects its prognosis and application. Our previous research has found that ginsenoside Rg1 (GS-Rg1) can inhibit the occurrence of restenosis after balloon injury of the common carotid artery in rats, but the mechanism is still unclear. In this study, it was found that GS-Rg1 (4, 8, 16 mg/kg) inhibited vascular restenosis caused by balloon injury, and mobilize endothelial progenitor cells (EPCs) to promote reendothelialization and inhibit intimal hyperplasia, which significantly reduced after administration of CaSR antagonist NPS 2143. Interestingly, CaSR and its downstream JNK, P38 were highly expressed in the proliferative intima and participated in the abnormal proliferation of vascular smooth muscle cells mediated by smooth muscle progenitor cells (SMPCs). GS-Rg1 inhibited intimal hyperplasia, while it decreased the expression of CaSR, JNK, and P38. This might relate to the distribution of CaSR and the facilitation of GS-Rg1 on the vascular endothelial repair. It is concluded that CaSR plays a key role in GS-Rg1 promoting reendothelialization to inhibit intimal hyperplasia after balloon 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.

Transcriptomic and physiological analyses reveal temporal changes contributing to the delayed healing response to arterial injury in diabetic rats

ObjectiveAtherosclerosis is a leading cause of mortality in the rapidly growing population with diabetes mellitus. Vascular interventions in patients with diabetes can lead to complications attributed to defective vascular remodeling and impaired healing response in the vessel wall. In this study, we aim to elucidate the molecular differences in the vascular healing response over time using a rat model of arterial injury applied to healthy and diabetic conditions. MethodsWistar (healthy) and Goto-Kakizaki (GK, diabetic) rats (n = 40 per strain) were subjected to left common carotid artery (CCA) balloon injury and euthanized at different timepoints: 0 and 20 hours, 5 days, and 2, 4, and 6 weeks. Noninvasive morphological and physiological assessment of the CCA was performed with ultrasound biomicroscopy (Vevo 2100) and corroborated with histology. Total RNA was isolated from the injured CCA at each timepoint, and microarray profiling was performed (n = 3 rats per timepoint; RaGene-1_0-st-v1 platform). Bioinformatic analyses were conducted using R software, DAVID bioinformatic tool, online STRING database, and Cytoscape software. ResultsSignificant increase in the neointimal thickness (P < .01; two-way analysis of variance) as well as exaggerated negative remodeling was observed after 2 weeks of injury in GK rats compared with heathy rats, which was confirmed by histological analyses. Bioinformatic analyses showed defective expression patterns for smooth muscle cells and immune cell markers, along with reduced expression of key extracellular matrix-related genes and increased expression of pro-thrombotic genes, indicating potential faults on cell regulation level. Transcription factor–protein-protein interaction analysis provided mechanistic evidence with an array of transcription factors dysregulated in diabetic rats. ConclusionsIn this study, we have demonstrated that diabetic rats exhibit impaired arterial remodeling characterized by a delayed healing response. We show that increased contractile smooth muscle cell marker expression coincided with decreased matrix metalloproteinase expression, indicating a potential mechanism for a lack of extracellular matrix reorganization in the impaired vascular healing in GK rats. These results further corroborate the higher prevalence of restenosis in patients with diabetes and provide vital molecular insights into the mechanisms contributing to the impaired arterial healing response in diabetes. Moreover, the presented study provides the research community with the valuable longitudinal gene expression data bank for further exploration of diabetic vasculopathy.

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.

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.

Recombinant Hirudin Protects the Vasculature and Myocardium in Atherosclerotic Rats by Inhibiting the p38 MAPK/NF-kB Signaling Pathway

Recombinant hirudin (r-hirudin) has a good anticoagulant effect and has a certain inhibitory effect on atherosclerosis (AS), however, its intrinsic mechanism of inhibiting AS is still unclear. In this study, we investigated the mechanism underlying the vascular and myocardial protective effects of r-hirudin in AS rats through animal experiments. A rat AS model was established by high-fat diet feeding combined with common carotid artery balloon injury. The model rats were given low, medium, or high doses of r-hirudin (0.05, 0.1, or 0.2 mg/kg/ day), simvastatin tablets (1 mg/kg/day) and p38 mitogen-activated protein kinase (MAPK) pathway inhibitors (SB203580, 100 mg/kg/day) by gavage for 8 weeks. The results showed that in AS rats, r-hirudin significantly alleviated pathological changes in the common carotid artery and myocardial tissue; decreased serum total cholesterol (TC), triglyceride (TG) and low-density lipoprotein-cholesterol (LDL-C) levels; increased highdensity lipoprotein-cholesterol (HDL-C) levels; decreased serum oxidized low-density lipoprotein (ox-LDL), tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, and endothelin (ET)-1 levels; increased nitric oxide (NO) levels; and decreased p38 MAPK, nuclear factor-kappa B (NF-κB), caspase-9, caspase-3 mRNA and protein expression. This study showed that r-hirudin may protect blood vessels and the myocardium in AS rats by adjusting blood lipid levels and inhibiting the p38 MAPK/NF-κB signaling pathway to exert anti-inflammatory and anti-apoptotic effects and protect the vascular endothelium.

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.

MicroRNA-17-5p, a novel endothelial cell modulator, controls vascular re-endothelialization and neointimal lesion formation.

The functions of miR-17-5p in tumorigenesis have been explored. However, their functionalities in arterial endothelial cells (ECs) have not been investigated. Besides, the issue of vascular remodelling is barely addressed. The study aimed to determine the effect of overexpression or inhibition of miR-17-5p on arterial endothelial cells' (ECs) function and vascular remodelling in vitro and the rat carotid arteries model. Quantitative RT-PCR analysis was performed to examine the expression of miR-17-5p. Then, gain-of-function and loss-of-function approaches were employed to investigate the functional roles of miR-17-5p in cultured human coronary artery endothelial cells (HCAECs); further, TargetScan software analysis and luciferase reporter activity assay were performed to investigate the potential mechanism. Lastly, the results of the cell segment were verified in a rat carotid artery balloon injury model by Western blot analysis, measurement of the vascular cGMP level and plasma 8-iso-prostaglandin F2 (8-iso-PGF2) testing. Moreover, morphometric analysis was implemented to detect the re-endothelialization and neointimal formation in rat carotid artery after balloon injury. This study firstly found that miR-17-5p expression was upregulated in the injured vascular walls and highly expressive in ECs; overexpression of miR-17-5p inhibited HCAECs' proliferation and migration, whereas miR-17-5p knockdown strengthened its proliferative and migratory roles, influenced inflammatory response, through regulating VEGRA and VEGFR2. It was found that miR-17-5p bind to VEGFA and VEGFR2 at the 3'UTR. Next, downregulation of miR-17-5p promotes re-endothelialization, and attenuates neointimal formation as measured by the I/M ratio (0.63±0.05 vs 1.45±0.06, antagomiR-17-5p vs. Lenti-NC, p < 0.05). In addition, the functional recovery of the endothelium was also accelerated by miR-17-5p knockdown. Our study suggests that miR-17-5p is a feasible strategy for the selective modulation of endothelialization and vascular remodelling through regulating VEGFA and VEGFR2.

Sophocarpine Alleviates Injury-Induced Intima Hyperplasia of Carotid Arteries by Suppressing Inflammation in a Rat Model.

Introduction: Balloon angioplasty is a commonly applied procedure for treating atherosclerotic vascular diseases. However, the maintenance of long-term lumen patency is relatively difficult due to the occurrence of restenosis. Previous research has shown that the occurrence of vascular wall inflammation is associated with higher rates of restenosis. Sophocarpine (SPC) can exert various therapeutic effects such as anti-oxidation, anti-inflammation, anti-tumor, antivirus and immune regulation. This study aimed to investigate whether SPC can alleviate intimal hyperplasia following balloon injury in a rat carotid artery model. Methods: Twenty Sprague–Dawley rats were randomly assigned to four groups: (i) control, (ii) balloon injury, (iii) balloon injury followed by saline injection, and (iv) balloon injury followed by SPC administration. Each group contained five rats. A high-pressure balloon of 3 mm × 20 mm was placed in the carotid artery. The balloon was inflated to a pressure of 8 atmospheres to carry out rat carotid artery balloon injury model. The areas of neointimal and media were determined by Verhoeff_Van Gieson staining, and the intima-to-media (I:M) ratios were subsequently evaluated. After that, the protein levels of IL-6, IL-1β, MCP-1, NF-κB, TNF-α, VCAM-1, ICAM-1 and eNOS were measured. Results: The ratio of I:M was remarkably higher in the balloon injury group than in the control group (p < 0.01). SPC could significantly decrease the ratio of I:M compared with the balloon injury group (p < 0.01). Besides, the protein levels of IL-6, IL-1β, MCP-1, NF-κB, TNF-α, ICAM-1 and VCAM-1 were increased in rat carotid arteries exposed to balloon injury (p < 0.01), and treatment with SPC could attenuate these effects (p < 0.05). Furthermore, balloon injury inhibited the protein expression of eNOS (p < 0.01), and SPC could elevate its level (p < 0.05). Conclusions: SPC could alleviate an intimal hyperplasia in balloon-injured carotid artery, and the mechanisms underlying this protective effect might be due to its inhibitory potency against inflammation signals. Our study also implies the potential applicability of SPC in treating restenosis after balloon angioplasty.

Essential role of Orai1 and SARAF in vascular remodeling

Orai1 and STIM1, molecular components of store-operated calcium entry (SOCE), have been associated with vascular smooth muscle cell (VSMC) proliferation in vascular remodeling. Nevertheless, the role of SARAF (SOCE-associated regulatory factor), a regulatory protein involved in STIM1 inhibition, in vascular remodeling has not been examined. The aim of this study is to examine the role of SARAF and Orai1 in VSMC proliferation and neointima formation after balloon injury of rat carotid arteries. Experiments were conducted in an animal model of rat carotid angioplasty to characterize neointima formation. VSMC isolated from rat coronary arteries was also used to examine cell proliferation. The formation of neointima after balloon injury of rat carotid arteries was confirmed by hematoxylin and eosin staining of tissue sections up to 3 wk after surgery. Injured arteries showed significantly higher expression of SARAF, STIM1, and Orai1 compared with control tissues, corroborating the presence of these regulatory proteins in the neointima layer. Proximity ligation and coimmunoprecipitation assays revealed that SARAF interacts with Orai1 in the neointima. Furthermore, selective silencing of SARAF and Orai1 by small interfering RNA (siRNA) inhibited IGF-1-induced VSMC proliferation. Our data suggest that SARAF interacts with Orai1 to modulate SOCE and VSMC proliferation after vascular injury.

How to Establish a Minimal Invasive and Stable Carotid Artery Stenosis Rabbit Model? A Simple and Effective Carotid Artery Balloon Strain Technique.

Background: The objective of this study is to establish a minimally invasive technique to create a stable carotid artery stenosis rabbit model. This article summarizes the specific methods and key points of this technology.Methods: The experiment studied a rabbit that was anesthetized through the vein. After the femoral artery was exposed, a minimally invasive needle was used to puncture the femoral artery, then the sheath was placed into the artery. We primarily put a catheter in the ascending aorta for angiography and then used a PT2 guidewire for super-selection. The PT2 guidewire was retained, and a balloon was placed in the right common carotid artery (CCA) through a guidewire to inflate it three times. Six rabbits in the 2- (2W) and 4-week (4W) groups were examined at 14 and 28 days, respectively. The rabbits in the control group received angiography at the beginning and 28 days later but without balloon injury. After angiography assessment, specimens of right CCA were dissected. Pathological and immunohistochemical examinations were performed on the collected specimens, and iFlow analysis was performed as well.Results: All the 18 animals which survived were observed. The rabbits in the 2W and 4W groups showed stenosis of the right CCA. Digital subtraction angiography showed the diameter was lower than that in the control group (1.04 ± 0.1, 0.71 ± 0.12, and 1.83 ± 0.08 mm in 2W, 4W, and control group, P < 0.05). Pathology also suggested carotid stenosis and obvious intimal hyperplasia. The results of immunohistochemistry showed that α-smooth muscle actin was highly expressed in the 2W and 4W groups, and the integrated optical density (IOD) value was higher than that in the control group (14,807.11 ± 1,822.3, 22,245.96 ± 1,212.82, and 6,537.16 ± 1,186.62 in the 2W, 4W, and control group, P < 0.05). Meanwhile, a cluster of differentiation 31 (CD31) was low expressed in the 2W and 4W groups, and the IOD value was lower than that in the control group (519.14 ± 44.4, 1,029.64 ± 98.48, and 1,502.05 ± 88.79 in the 2W, 4W, and control group, P < 0.05), which suggested endothelial damage and partial repair. The analysis by iFlow showed that the time-to-peak after balloon strain in the 2W and 4W groups were longer than that in the control group.Conclusion: We established a minimally invasive, effective, and safe method to establish a carotid artery stenosis rabbit model. The highlights of this technology were the application of minimally invasive methods, reducing surgical bleeding, infection, and related complications. This technology avoided the influence of tissue around CCA in the traditional carotid artery balloon injury model, which might lead to more accurate treatment outcomes.

CARMN Is an Evolutionarily Conserved Smooth Muscle Cell-Specific LncRNA That Maintains Contractile Phenotype by Binding Myocardin.

Vascular homeostasis is maintained by the differentiated phenotype of vascular smooth muscle cells (VSMCs). The landscape of protein coding genes comprising the transcriptome of differentiated VSMCs has been intensively investigated but many gaps remain including the emerging roles of noncoding genes. We reanalyzed large-scale, publicly available bulk and single-cell RNA sequencing datasets from multiple tissues and cell types to identify VSMC-enriched long noncoding RNAs. The in vivo expression pattern of a novel smooth muscle cell (SMC)-expressed long noncoding RNA, Carmn (cardiac mesoderm enhancer-associated noncoding RNA), was investigated using a novel Carmn green fluorescent protein knock-in reporter mouse model. Bioinformatics and quantitative real-time polymerase chain reaction analysis were used to assess CARMN expression changes during VSMC phenotypic modulation in human and murine vascular disease models. In vitro, functional assays were performed by knocking down CARMN with antisense oligonucleotides and overexpressing Carmn by adenovirus in human coronary artery SMCs. Carotid artery injury was performed in SMC-specific Carmn knockout mice to assess neointima formation and the therapeutic potential of reversing CARMN loss was tested in a rat carotid artery balloon injury model. The molecular mechanisms underlying CARMN function were investigated using RNA pull-down, RNA immunoprecipitation, and luciferase reporter assays. We identified CARMN, which was initially annotated as the host gene of the MIR143/145 cluster and recently reported to play a role in cardiac differentiation, as a highly abundant and conserved, SMC-specific long noncoding RNA. Analysis of the Carmn GFP knock-in mouse model confirmed that Carmn is transiently expressed in embryonic cardiomyocytes and thereafter becomes restricted to SMCs. We also found that Carmn is transcribed independently of Mir143/145. CARMN expression is dramatically decreased by vascular disease in humans and murine models and regulates the contractile phenotype of VSMCs in vitro. In vivo, SMC-specific deletion of Carmn significantly exacerbated, whereas overexpression of Carmn markedly attenuated, injury-induced neointima formation in mouse and rat, respectively. Mechanistically, we found that Carmn physically binds to the key transcriptional cofactor myocardin, facilitating its activity and thereby maintaining the contractile phenotype of VSMCs. CARMN is an evolutionarily conserved SMC-specific long noncoding RNA with a previously unappreciated role in maintaining the contractile phenotype of VSMCs and is the first noncoding RNA discovered to interact with myocardin.

Translocator protein imaging with 18F-FEDAC-positron emission tomography in rabbit atherosclerosis and its presence in human coronary vulnerable plaques

Background and aimsThis study aimed to investigate whether N-benzyl-N-methyl-2-[7,8-dihydro-7-(2-[18F]fluoroethyl)-8-oxo-2-phenyl-9H-purin-9-yl]acetamide (18F-FEDAC), a probe for translocator protein (TSPO), can visualize atherosclerotic lesions in rabbits and whether TSPO is localized in human coronary plaques. Methods18F-FEDAC-PET of a rabbit model of atherosclerosis induced by a 0.5% cholesterol diet and balloon injury of the left carotid artery (n = 7) was performed eight weeks after the injury. The autoradiography intensity of 18F-FEDAC in carotid artery tissue sections was measured, and TSPO expression was evaluated immunohistochemically. TSPO expression was examined in human coronary arteries obtained from autopsy cases (n = 16), and in human coronary plaques (n = 12) aspirated from patients with acute myocardial infarction (AMI). Results18F-FEDAC-PET visualized the atherosclerotic lesions in rabbits as high-uptake areas, and the standard uptake value was higher in injured arteries (0.574 ± 0.24) than in uninjured arteries (0.277 ± 0.13, p < 0.05) or myocardium (0.189 ± 0.07, p < 0.05). Immunostaining showed more macrophages and more TSPO expression in atherosclerotic lesions than in uninjured arteries. TSPO was localized in macrophages, and arterial autoradiography intensity was positively correlated with macrophage concentration (r = 0.64) and TSPO (r = 0.67). TSPO expression in human coronary arteries was higher in AMI cases than in non-cardiac death, or in the vulnerable plaques than in early or stable lesions, respectively. TSPO was localized in macrophages in all aspirated coronary plaques with thrombi. Conclusions18F-FEDAC-PET can visualize atherosclerotic lesions, and TSPO-expression may be a marker of high-risk coronary plaques.

Protective effects of dapagliflozin on vascular remodeling in the carotid artery following balloon injury – potential role of angiotensin and purinergic signaling

Abstract Introduction Sodium-glucose co-transporter 2 (SGLT2) inhibitors have been shown to reduce the risk of cardiovascular events independently of glycemic control. The possibility that SGLT2 inhibitors improve endothelial regeneration and vascular restenosis is unknown. Purpose To examine whether dapagliflozin, a selective SGLT2 inhibitor, could prevent neointima thickening induced by balloon injury and, if so, to determine the underlying mechanisms. The effect of dapagliflozin was compared to that of losartan, an angiotensin type 1 receptor (AT1R) antagonist. Methods Saline, dapagliflozin (1.5 mg/kg/day), or losartan (30 mg/kg/day) were administered orally for 5 weeks to male Wistar rats. Balloon injury of the left carotid artery was performed 1 week after starting the treatment and sacrificed 4 weeks later. Vascular reactivity was assessed on left (injured) and right (healthy) carotid artery rings. The extent of neointima was assessed by histomorphometric analysis, changes of target factors by immunofluorescence, RT-qPCR and histochemistry. Results Dapagliflozin and losartan treatments reduced neointima thickening by 32% and 27%, respectively. Blunted contractile responses to phenylephrine and relaxations to acetylcholine and down-regulation of eNOS were observed in the injured artery. These effects were not modified by the dapagliflozin or the losartan treatments. RT-qPCR investigations indicated an increased in gene expression of inflammatory (IL-1beta, ITGAM, VCAM-1), oxidative (p47phox, p22phox) and fibrotic (TGF-beta1) markers and a decreased of eNOS in the injured carotid. However, these changes were not affected by the pharmacological treatments. By contrast, significant increased levels of AT1R angiotensin receptor and NTPDase1 (CD39) ectonucleotidase were observed in the restenotic carotid artery of the dapagliflozin group. Histochemical analysis evidenced important NTPDase1 activity in the neointima. Conclusions Dapagliflozin effectively reduced neointimal thickening. As the contribution of AT1R and P2Y2 ATP receptor in smooth muscle cell proliferation and neointima formation has been reported in the literature, the present data suggest that dapagliflozin prevents restenosis through interfering with angiotensin and/or extracellular nucleotides signaling. SGLT2 transporter represent potential new target for limiting vascular restenosis. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): This work was supported by AstraZeneca