Abnormal Vascular Smooth Muscle Cell Research Articles

A novel protein encoded by circLARP1B promotes the proliferation and migration of vascular smooth muscle cells by suppressing cAMP signaling

Background and aimsCircular RNA (circRNA) is closely related to atherosclerosis (AS) incidence and progression, but its regulatory mechanism in AS needs further elucidation. AS development is significantly influenced by abnormal vascular smooth muscle cells (VSMCs) growth and migration. This study explored the potential protein role of circLARP1B in VSMC proliferation and migration. MethodsWe performed whole-transcriptome sequencing in human normal arterial intima and advanced atherosclerotic plaques to screen for differentially expressed circRNAs. The sequencing results were combined with database analysis to screen for circRNAs with coding ability. Real-time quantitative polymerase chain reaction was utilized to assess circLARP1B expression levels in atherosclerotic plaque tissues and cells. circLARP1B-243aa function and pathway in VSMCs growth and migration were studied by scratch, transwell, 5-ethynyl-2′-deoxyuridine, cell counting kit-8, and Western blot experiments. ResultsWe found that circLARP1B was downregulated in atherosclerotic plaque tissue and promoted the proliferation and migration of VSMCs. circLARP1B encodes a novel protein with a length of 243 amino acids. Through functional experiments, we confirmed the role of circLARP1B-243aa in enhancing VSMCs migration and proliferation. Mechanistically, circLARP1B-243aa promotes VSMCs migration and growth by upregulating phosphodiesterase 4C to inhibit the cyclic adenosine monophosphate signaling pathway. ConclusionsOur results suggested that circLARP1B could promote VSMCs growth and migration through the encoded protein circLARP1B-243aa. Therefore, it could be a treatment target and biomarker for AS.

Pers reverse angiotensin II -induced vascular smooth muscle cell proliferation by targeting cyclin E expression via inhibition of the MAPK signaling pathway

ABSTRACT The circadian rhythm of blood pressure (BP) is believed to be regulated by the clock system, which is closely linked to levels of angiotensin II (Ang II). This study aimed to investigate whether Ang II mediates the proliferation of vascular smooth muscle cells (VSMCs) through the interaction between the clock system and the mitogen-activated protein kinase (MAPK) signaling pathway. Primary rat aortic VSMCs were treated with Ang II, with or without MAPK inhibitors. VSMC proliferation, expression of clock genes, CYCLIN E, and MAPK pathways were assessed. Ang II treatment resulted in increased VSMC proliferation and rapid upregulation of clock gene Periods (Pers) expression. Compared to the non-diseased control (NC) group, VSMCs incubated with Ang II displayed a noticeable delay in the G1/S phase transition and downregulation of CYCLIN E upon silencing of Per1 and Per2 genes. Importantly, silencing Per1 or Per2 in VSMCs led to decreased expression of key MAPK pathway proteins, including RAS, phosphorylated mitogen-activated protein kinase (P-MEK), and phosphorylated extracellular signal-regulated protein kinase (P-ERK). Moreover, the MEK and ERK inhibitors, U0126 and SCH772986, significantly attenuated the Ang II-induced proliferation of VSMCs, as evidenced by an increased G1/S phase transition and decreased CYCLIN E expression. The MAPK pathway plays a critical role in regulating VSMC proliferation in response to Ang II stimulation. This regulation is controlled by the expression of circadian clock genes involved in the cell cycle. These findings provide novel insights for further research on diseases associated with abnormal VSMC proliferation.

C1q/TNF-related protein 4 mediates proliferation and migration of vascular smooth muscle cells during vascular remodelling.

Vascular remodelling is an essential pathophysiological state in many circulatory diseases. Abnormal vascular smooth muscle cell (VSMC) behaviour leads to neointimal formation and may eventually results in major adverse cardiovascular events. The C1q/TNF-related protein (C1QTNF) family is closely associated with cardiovascular disease. Notably, C1QTNF4 has unique two C1q domains. However, the role of C1QTNF4 in vascular diseases remains unclear. C1QTNF4 expression was detected in human serum and artery tissues using ELISA and multiplex immunofluorescence (mIF) staining. Scratch assay, transwell assay and confocal microscopy were used to investigate C1QTNF4 effects on VSMC migration. EdU incorporation, MTT assay and cell counting experiment revealed C1QTNF4 effects on VSMC proliferation. C1QTNF4-transgenic, C1QTNF4-/- and AAV9-mediated VSMC-specific C1QTNF4 restoration C1QTNF4-/- mouse and rat disease models were generated. RNA-seq, quantitative real-time PCR, western blot, mIF, proliferation and migration assays were used to investigate the phenotypic characteristics and underlying mechanisms. Serum C1QTNF4 levels were decreased in patients with arterial stenosis. C1QTNF4 shows colocalisation with VSMC in human renal arteries. In vitro, C1QTNF4 inhibits VSMC proliferation and migration and alters VSMC phenotype. In vivo, an adenovirus-infected rat balloon injury model, C1QTNF4-transgenic and C1QTNF4-/- mouse wire-injury models with or without VSMC-specific C1QTNF4 restoration were established to mimic the VSMC repair and remodelling. The results show that C1QTNF4 decreases intimal hyperplasia. Especially, we displayed the rescue effect of C1QTNF4 in vascular remodelling using AAV vectors. Next, transcriptome analysis of artery tissue identified the potential mechanism. In vitro and in vivo experiments confirm that C1QTNF4 ameliorates neointimal formation and maintains vascular morphology by downregulating the FAK/PI3K/AKT pathway. Our study demonstrated that C1QTNF4 is a novel inhibitor of VSMC proliferation and migration that acts by downregulating the FAK/PI3K/AKT pathway, thus protecting blood vessels from abnormal neointima formation. These results provide new insights into promising potent treatments for vascular stenosis diseases.

PS-B01-8: MALAYSIAN TUALANG HONEY INHIBITS PDGF-INDUCED HUMAN VASCULAR SMOOTH MUSCLE CELLS PROLIFERATION AND MIGRATION

Introduction and Objective: Cardiovascular diseases (CVD) are the leading cause of death globally. Atherosclerosis is a common CVD which can lead to the deadliest heart failure. Increasing evidence showed that abnormal vascular smooth muscle cells (VSMC) proliferation and migration constitute the main pathological basis of atherosclerosis. These events are potently stimulated by platelet-derived growth factor (PDGF), causing phenotypic switching of VSMC from a mature, quiescent phenotype to a proliferative, migratory phenotype which is commonly observed in the development of atherosclerosis. Furthermore, study have revealed that PDGF elevates the concentration of reactive oxygen species (ROS) which stimulates VSMC proliferation and migration. Pharmacological treatment for atherosclerosis is often an ongoing, long-term regimen that may have adverse effect to an individual. Thus, alternative therapy based on natural products can potentially be the frontier in atherosclerosis treatment. Interestingly, previous study revealed that Malaysian Tualang Honey (MTH) is capable of protecting vascular endothelial cells from inflammation. However, little is known on the effect of MTH on the PDGF-induced VSMC proliferation and migration. Therefore, the present study aimed to determine the antioxidant, anti-proliferative and anti-migratory effects of MTH in PDGF-induced VSMC. Methodology: VSMC were treated with increasing concentrations of MTH (0.001–10%) for 24 hours and the cell viability were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were then be treated with non-cytotoxic concentrations of MTH and induced with 20ng/mL PDGF for 72 hours to determine the proliferation rate via MTT assay. The intracellular ROS were measured in these cultured cells by dichlorodihydrofluorescein diacetate (DCFH-DA) assay. Wound healing assay were used to determine the anti-migration activities on PDGF-induced VSMC treated with MTH. Results: Our data showed that MTH concentrations ranging from 0.01% to 1.00% showed no cytotoxic effect to VSMC. Co-culture of the cells with MTH significantly inhibits the proliferation and migration of PDGF-stimulated VSMC in a concentration-dependent manner. Pre-treatment of cells with MTH prior to culturing in 20ng/mL PDGF showed a significant decrease in the production of ROS. Conclusion: This study demonstrates that MTH inhibits VSMC proliferation and migration by suppressing the production of ROS which constitute the pharmacological basis of anti-atherosclerotic action. Therefore, MTH potentially can be a promising therapeutic agent in the treatment of atherosclerosis.

Inhibitory effects of 6′-sialyllactose on angiotensin II-induced proliferation, migration, and osteogenic switching in vascular smooth muscle cells

Excessive production and migration of vascular smooth muscle cells (VSMCs) are associated with vascular remodeling that causes vascular diseases, such as restenosis and hypertension. Angiotensin II (Ang II) stimulation is a key factor in inducing abnormal VSMC function. This study aimed to investigate the effects of 6'-sialyllactose (6'SL), a human milk oligosaccharide, on Ang II-stimulated cell proliferation, migration and osteogenic switching in rat aortic smooth muscle cells (RASMCs) and human aortic smooth muscle cells (HASMCs). Compared with the control group, Ang II increased cell proliferation by activating MAPKs, including ERK1/2/p90RSK/Akt/mTOR and JNK pathways. However, 6'SL reversed Ang II-stimulated cell proliferation and the ERK1/2/p90RSK/Akt/mTOR pathways in RASMCs and HASMCs. Moreover, 6'SL suppressed Ang II-stimulated cell cycle progression from G0/G1 to S and G2/M phases in RASMCs. Furthermore, 6'SL effectively inhibited cell migration by downregulating NF-κB-mediated MMP2/9 and VCAM-1 expression levels. Interestingly, in RASMCs, 6'SL attenuated Ang II-induced osteogenic switching by reducing the production of p90RSK-mediated c-fos and JNK-mediated c-jun, leading to the downregulation of AP-1-mediated osteopontin production. Taken together, our data suggest that 6'SL inhibits Ang II-induced VSMC proliferation and migration by abolishing the ERK1/2/p90RSK-mediated Akt and NF-κB signaling pathways, respectively, and osteogenic switching by suppressing p90RSK- and JNK-mediated AP-1 activity.

Abstract 135: Sex-specific Differences In The Role Of TSP1 On Metabolic Syndrome-induced Atherosclerosis

Metabolic syndrome (MetS) amplifies risks of atherosclerotic complications. MetS induces abnormal vascular smooth muscle cell (VSMC) migration and proliferation, hallmark of SMC de-differentiation, crucial in the pathogenesis of atherosclerosis. Despite multiple studies revealing sexual dimorphism in atherosclerosis, underlying sex-specific mechanisms are poorly understood. We previously reported a protective role of a potent proatherogenic protein, thrombospondin-1 (TSP1), in hyperglycemia- or hyperleptinemia-induced atherosclerosis. The goal of the present study was to interrogate sex-specific differences in the role of TSP1 on MetS-induced atherosclerosis. We generated a mouse model of combined MetS and atherosclerosis (KKAy +/- ApoE -/- ) by crossing obese hyperglycemic agouti KKAy +/- mice with atherosclerotic ApoE -/- . Male and female age-matched MetS KKAy +/- ApoE -/- and non-MetS KKAy -/- ApoE -/- mice were placed on standard lab diet from 4-24 wks age. After overnight fasting, mice were harvested; plasma, aorta and heart were collected for various studies. In male MetS KKAy +/- ApoE -/- , increased aortic root ORO-positive lipid burden, reduced LMOD (SM contractile marker) and SRF (transcriptional activator of SM differentiation) expression in aortic vessels associated with augmented TSP1 expression vs. non-MetS KKAy -/- ApoE -/- mice. In contrast, no significant differences in lesion lipid burden, TSP1, LMOD and SRF expression were detected between the female genotypes. To delineate whether TSP1 plays a direct role in SMC de-differentiation in MetS, we next crossed KKAy +/- with TSP1 -/- to generate MetS mice with and without global TSP1 deletion. Male and female KKAy +/- TSP1 +/+ (with intact TSP1) and KKAy +/- TSP1 -/- (lacking TSP1) on standard lab diet from 4-18 wks age were harvested at endpoint for aortic tissue collection. In male KKAy +/- TSP1 -/- aortic vessels, SM contractile marker expression (LMOD, calponin) was markedly increased vs. KKAy +/- TSP1 +/+ , suggesting reduced SMC de-differentiation. In contrast, TSP1 deletion had no effect on SM contractile marker expression in female MetS KKAy +/- . Together, these data suggest a sex-specific role of TSP1 on SMC de-differentiation and atherosclerotic lesion formation in MetS.

Abstract 204: Sex-specific Differences In The Role Of TSP1 On Metabolic Syndrome-induced Atherosclerosis

Metabolic syndrome (MetS) amplifies risks of atherosclerotic complications. MetS induces abnormal vascular smooth muscle cell (VSMC) migration and proliferation, hallmark of SMC de-differentiation, crucial in the pathogenesis of atherosclerosis. Despite multiple studies revealing sexual dimorphism in atherosclerosis, underlying sex-specific mechanisms are poorly understood. We previously reported a protective role of a potent proatherogenic protein, thrombospondin-1 (TSP1), in hyperglycemia- or hyperleptinemia-induced atherosclerosis. The goal of the present study was to interrogate sex-specific differences in the role of TSP1 on MetS-induced atherosclerosis. We generated a mouse model of combined MetS and atherosclerosis (KKAy +/- ApoE -/- ) by crossing obese hyperglycemic agouti KKAy +/- mice with atherosclerotic ApoE -/- . Male and female age-matched MetS KKAy +/- ApoE -/- and non-MetS KKAy -/- ApoE -/- mice were placed on standard lab diet from 4-24 wks age. After overnight fasting, mice were harvested; plasma, aorta and heart were collected for various studies. In male MetS KKAy +/- ApoE -/- , increased aortic root ORO-positive lipid burden, reduced LMOD (SM contractile marker) and SRF (transcriptional activator of SM differentiation) expression in aortic vessels associated with augmented TSP1 expression vs. non-MetS KKAy -/- ApoE -/- mice. In contrast, no significant differences in lesion lipid burden, TSP1, LMOD and SRF expression were detected between the female genotypes. To delineate whether TSP1 plays a direct role in SMC de-differentiation in MetS, we next crossed KKAy +/- with TSP1 -/- to generate MetS mice with and without global TSP1 deletion. Male and female KKAy +/- TSP1 +/+ (with intact TSP1) and KKAy +/- TSP1 -/- (lacking TSP1) on standard lab diet from 4-18 wks age were harvested at endpoint for aortic tissue collection. In male KKAy +/- TSP1 -/- aortic vessels, SM contractile marker expression (LMOD, calponin) was markedly increased vs. KKAy +/- TSP1 +/+ , suggesting reduced SMC de-differentiation. In contrast, TSP1 deletion had no effect on SM contractile marker expression in female MetS KKAy +/- . Together, these data suggest a sex-specific role of TSP1 on SMC de-differentiation and atherosclerotic lesion formation in MetS.

Circular RNAs: regulators of vascular smooth muscle cells in cardiovascular diseases.

The aberrant expansion and dysfunction of vascular smooth muscle cells (VSMCs) contribute to the occurrence and development of many cardiovascular diseases. Circular RNAs, a new class of non-coding RNAs with the 3' and 5' ends covalently linked together due to back-splicing, have recently been revealed to function as new regulators of VSMCs. These circular RNAs mainly act as RNA sponge to downregulate other regulatory non-coding RNAs such as microRNAs, influencing the overgrowth and transformation of VSMCs under pathogenic conditions. The purpose of this review is to summarize how circular RNAs fluctuate their own expression in response to multiple stimuli in vitro and in vivo and how they modulate the phenotypic adaptation, proliferation, migration, apoptosis, and senescence of VSMCs, which in turn affects the progression of cardiovascular diseases. Finally, we highlight the potential of circular RNAs as the biomarkers and therapeutic targets for abnormal VSMCs and cardiovascular diseases.

Inhibition of mitochondrial fission alters neo-intimal hyperplasia via PI3K/Akt signaling in arteriovenous fistulas.

Arteriovenous fistulas (AVFs) are the preferred vascular access for hemodialysis of patients with end-stage renal disease. However, there is a high incidence of AVF failures caused by insufficient outward remodeling or venous neo-intimal hyperplasia formation. Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) play an important role in many cardiovascular diseases. Abnormal VSMC proliferation and migration could be abolished by inhibition of mitochondrial division. We found that abnormal proliferation and migration of VSMCs and increased mitochondrial fission were associated with AVF stenosis in patients. We also investigated the mechanisms, particularly the role of mitochondrial dynamics, underlying these VSMC behaviors. In vitro, we observed that inhibition of mitochondrial fission and Akt phosphorylation can diminish proliferation and migration of VSMCs induced by platelet-derived growth factor-BB (PDGF-BB). In vivo, daily intraperitoneal injections of mitochondrial division inhibitor 1 (Mdivi-1) decreased VSMC proliferation and reduced AVF wall thickness in a rat AVF model. Our results suggest that inhibition of mitochondrial fission improves AVF patency by reducing wall thickening through the PI3K/Akt signaling pathway. Therefore, inhibition of mitochondrial fission has the clinical potential to improve AVF patency.

Anti-Atherosclerotic Effect of Gossypetin on Abnormal Vascular Smooth Muscle Cell Proliferation and Migration.

Gossypetin (GTIN), known as 3,5,7,8,3′,4′-hexahydroxyflavone, has been demonstrated to exert anti-atherosclerotic potential against apoptotic injury in oxidized low-density lipoprotein-incubated endothelial cells, and atherosclerotic lesions of cholesterol-fed rabbits. However, the effect and underlying mechanism of GTIN on abnormal vascular smooth muscle cells (VSMCs) proliferation and migration, a major event in the pathogenesis of atherosclerosis, is still unknown. In this study, non-cytotoxic doses of GTIN abolished the VSMCs A7r5 proliferation and cell-cycle S phase distribution. The GTIN-arrested G0/G1 phase might be performed by increasing the expressions of phosphorylated p53 and its downstream molecules that inhibit the activation of cyclin E/cyclin-dependent kinase (cdk)-2, blocking retinoblastoma protein (Rb) phosphorylation and the subsequent dissociation of Rb/transcription factor E2F1 complex. In addition, the results indicated that GTIN inhibited VSMCs wound-healing and migratory abilities through reducing matrix metalloproteinase (MMP)-9 activity and expression, as well as down-regulating protein kinase B (PKB)/nuclear factor-kappaB (NF-κB) signaling. GTIN also revealed potential in diminishing reactive oxygen species (ROS) generation. These findings suggested the inhibitory effects of GTIN on VSMCs dysfunction could likely lead to the containment of atherosclerosis and other cardiovascular illness.

Cytokine-induced apoptosis inhibitor 1 (CIAPIN1) accelerates vascular remodelling via p53 and JAK2-STAT3 regulation in vascular smooth muscle cells.

Abnormal vascular smooth muscle cell (VSMC) proliferation and migration lead to neointima formation, which eventually results in cardiovascular hyperplastic diseases. The molecular mechanisms underlying these cellular processes have not been fully understood. Cytokine-induced apoptosis inhibitor 1 (CIAPIN1) has been identified as an anti-apoptotic molecule, but little is known about its target genes and related pathways in VSMC dysfunction or its clinical implication in neointima formation following vascular injury. Determination, using loss/gain-of-function approaches by gene delivery, of whether CIAPIN1 modulates VSMC proliferation, migration and neointima formation and the underlying mechanisms was carried out. Balloon injury or ligation and local delivery of lentivirus were performed on rat or mouse carotid arteries. Rat aortic smooth muscle cells, the primary cell, was used as the model to evaluate the effect of CIAPIN1 on proliferation and migration. CIAPIN1 was overexpressed in the neointimal region of rat arteries. CIAPIN1 deficiency markedly inhibited injury-induced or ligation-induced intimal hyperplasia and suppressed PDGF-BB-induced VSMC proliferation, migration and cell cycle progression, while overexpression promoted proliferation, migration and neointima formation. CIAPIN1 negatively regulated Tp53 transcription, which promoted cell cycle progression and migration via cyclin E1-CDK2/pRb/PCNA and the MMP2 pathway. CIAPIN1 also increased JAK2 expression, enhancing JAK2 and STAT3 phosphorylation by vascular injury, which forced phenotypic switching from contractile to synthetic state in injured arteries. These findings provide new insights into the mechanism by which CIAPIN1 regulates vascular remodelling and suggest a novel therapeutic target for treating vascular proliferative diseases.

WNT5B promotes vascular smooth muscle cell dedifferentiation via mitochondrial dynamics regulation in chronic thromboembolic pulmonary hypertension.

Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by proliferative vascular remodeling. Abnormal vascular smooth muscle cell (VSMC) phenotype switching is crucial to this process, highlighting the need for VSMC metabolic changes to cover cellular energy demand in CTEPH. We report that elevated Wnt family member 5B (WNT5B) expression is associated with vascular remodeling and promotes VSMC phenotype switching via mitochondrial dynamics regulation in CTEPH. Using primary culture of pulmonary artery smooth muscle cells, we show that high WNT5B expression activates VSMC proliferation and migration and results in mitochondrial fission via noncanonical Wnt signaling in CTEPH. Abnormal VSMC proliferation and migration were abolished by mitochondrial division inhibitor 1, an inhibitor of mitochondrial fission. Secreted frizzled-related protein 2, a soluble scavenger of Wnt signaling, attenuates VSMC proliferation and migration by accelerating mitochondrial fusion. These findings indicate that WNT5B is an essential regulator of mitochondrial dynamics, contributing to VSMC phenotype switching in CTEPH.

Programmable dual responsive system reconstructing nerve interaction with small-diameter tissue-engineered vascular grafts and inhibiting intimal hyperplasia in diabetes

Small-diameter tissue-engineered vascular grafts (sdTEVGs) with hyperglycemia resistance have not been constructed. The intimal hyperplasia caused by hyperglycemia remains problem to hinder the patency of sdTEVGs. Here, inspired by bionic regulation of nerve on vascular, we found the released neural exosomes could inhibit the abnormal phenotype transformation of vascular smooth muscle cells (VSMCs). The transformation was a prime culprit causing the intimal hyperplasia of sdTEVGs. To address this concern, sdTEVGs were modified with an on-demand programmable dual-responsive system of ultrathin hydrogels. An external primary Reactive Oxygen Species (ROS)-responsive Netrin-1 system was initially triggered by local inflammation to induce nerve remolding of the sdTEVGs overcoming the difficulty of nerve regeneration under hyperglycemia. Then, the internal secondary ATP-responsive DENND1A (guanine nucleotide exchange factor) system was turned on by the neurotransmitter ATP from the immigrated nerve fibers to stimulate effective release of neural exosomes. The results showed nerve fibers grow into the sdTEVGs in diabetic rats 30 days after transplantation. At day 90, the abnormal VSMCs phenotype was not detected in the sdTEVGs, which maintained long-time patency without intima hyperplasia. Our study provides new insights to construct vascular grafts resisting hyperglycemia damage.

Circular RNA UVRAG Mediated by Alternative Splicing Factor NOVA1 Regulates Adhesion and Migration of Vascular Smooth Muscle Cells.

The movement of abnormal vascular smooth muscle cells (VSMCs) contributes to intimal hyperplasia in vein graft disease. Circular RNAs (circRNAs) are single stranded RNAs with 3’ and 5’ ends covalently joined together. They have been shown to regulate cell function in many diseases. NOVA1 is considered to be a brain-specific splicing factor that plays an important role in the nervous system and cancer. The role of NOVA1 in VSMCs remains unclear. In the present study, transcriptome sequencing was used to identify differentially expressed circRNAs in the rat vein graft model. A novel circRNA, circUVRAG, was decreased in the grafted vein and stably located in the cytoplasm. Knockdown of circUVRAG suppressed VSMC adhesion and migration. In addition, we demonstrated that the alternative splicing factor NOVA1 co-located with UVRAG pre-mRNA in the nucleus and modulated the production of circUVRAG. These new discoveries may serve as a potential means to treat intimal hyperplasia after vein grafts.

LncRNA-modulated autophagy in plaque cells: a new paradigm of gene regulation in atherosclerosis?

The development of atherosclerosis is accompanied by the functional deterioration of plaque cells, which leads to the escalation of endothelial inflammation, abnormal vascular smooth muscle cell phenotype switching and the accumulation of lipid-laden macrophages within vascular walls. Autophagy, a highly conserved homeostatic mechanism, is critical for the delivery of cytoplasmic substrates to lysosomes for degradation. Moderate levels of autophagy prevent atherosclerosis by safeguarding plaque cells against apoptosis, preventing inflammation, and limiting the lipid burden, whereas excessive autophagy exacerbates cell damage and inflammation and thereby accelerates the formation of atherosclerotic plaques. Increasing lines of evidence suggest that long noncoding RNAs can be either beneficial or detrimental to atherosclerosis development by regulating the autophagy level. This review summarizes the research progress related to 1) the significant role of autophagy in atherosclerosis and 2) the effects of the lncRNA-mediated modulation of autophagy on the plaque cell fate, inflammation levels, proliferative capacity, and cholesterol metabolism and subsequently on atherogenesis.

Differential Expression and Bioinformatics Analysis of CircRNA in PDGF-BB-Induced Vascular Smooth Muscle Cells.

Atherosclerosis is mediated by various factors and plays an important pathological foundation for cardiovascular and cerebrovascular diseases. Abnormal vascular smooth muscle cells (VSMCs) proliferation and migration have an essential role in atherosclerotic lesion formation. Circular RNAs (circRNA) have been widely detected in different species and are closely related to various diseases. However, the expression profiles and molecular regulatory mechanisms of circRNAs in VSMCs are still unknown. We used high-throughput RNA-seq as well as bioinformatics tools to systematically analyze circRNA expression profiles in samples from different VSMC phenotypes. Polymerase chain reaction (PCR), Sanger sequencing, and qRT-PCR were performed for circRNA validation. A total of 22191 circRNAs corresponding to 6273 genes (host genes) in the platelet-derived growth factor (PDGF-BB) treated group, the blank control group or both groups, were detected, and 112 differentially expressed circRNAs were identified between the PDGF-BB treated and control groups, of which 59 were upregulated, and 53 were downregulated. We selected 9 circRNAs for evaluation of specific head-to-tail splicing, and 10 differentially expressed circRNAs between the two groups for qRT-PCR validation. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses enrichment analyses revealed that the parental genes of the circRNAs mainly participated in cardiac myofibril assembly and positive regulation of DNA-templated transcription, indicating that they might be involved in cardiovascular diseases. Finally, we constructed a circRNA-miRNA network based on the dysregulated circRNAs and VSMC-related microRNAs. Our study is the first to show the differential expression of circRNAs in PDGF-BB-induced VSMCs and may provide new ideas and targets for the prevention and therapy of vascular diseases.

MiR-223 Promotes Smooth Muscle Cell Proliferation and Atherosclerotic Plaque Formation by Inhibiting Phosphatase and Tensin Homolog (PTEN)/ Phosphatidylinositol 3-Kinase (PI3K) and Protein Kinase B (AKT) Signaling Pathway

Abnormal vascular smooth muscle cells (VSMCs) proliferation is the pathological basis of atherosclerosis (AS) pathogenesis. miR-223 is abnormally expressed in AS plaques and affects the proliferation of VSMCs, but the mechanism of miR-223 affecting the proliferation of VSMCs is unclear. Our study intends to investigate the mechanism of miR-223 in VSMCs proliferation and AS formation. Healthy SD rats and miR-223 knockout SD rats took high-fat diet to induce AS model. Oil red O staining was done to observe AS formation. miR-223 mimics/NC was transferred to VSMCs followed by analysis of miR-214 expression by real-time PCR, cell proliferation by CCK8 assay, phosphatase and tensin homolog gene (PTEN) level by Western blot detection. Compared with control group, after knocking out miR-223, the AS level was significantly decreased and PTEN expression was significantly elevated (P < 0 05). After transfection of miR-223 mimics into VSMCs, PTEN expression protein was significantly decreased and the number of cells was increased (P < 0 05). In addition, the luciferase signal of miR-223 mimics and pmirGLO-PTEN-3 UTR-wt co-transfection group was significantly reduced (P < 0 05). miR-223 promotes VSMCs proliferation and AS plaque formation by targeting PTEN/PI3K/Akt signaling pathway.

Hypoxia-induced miR-1260b regulates vascular smooth muscle cell proliferation by targeting GDF11

Vascular smooth muscle cells (VSMCs) are a unique cell type that has unusual plasticity controlled by environmental stimuli. As an abnormal increase of VSMC proliferation is associated with various vascular diseases, tight regulation of VSMC phenotypes is essential for maintaining vascular homeostasis. Hypoxia is one environmental stress that stimulates VSMC proliferation. Emerging evidence has indicated that microRNAs (miRNAs) are critical regulators in the hypoxic responses of VSMCs. Therefore, we previously investigated miRNAs modulated by hypoxia in VSMCs and found that miR-1260b is one of the most upregulated miRNAs under hypoxia. However, the mechanism that underlies the regulation of VSMCs via miR-1260b in response to hypoxia has not been explored. Here we demonstrated that hypoxia-induced miR-1260b promotes VSMC proliferation. We also identified growth differentiation factor 11 (GDF11), a member of the TGF-β superfamily, as a novel target of miR-1260b. miR-1260b directly targets the 3’UTR of GDF11. Downregulation of GDF11 inhibited Smad signaling and consequently enhanced the proliferation of VSMCs. Our findings suggest that miR-1260b-mediated GDF11-Smad-dependent signaling is an essential regulatory mechanism in the proliferation of VSMCs, and this axis is modulated by hypoxia to promote abnormal VSMC proliferation. Therefore, our study unveils a novel function of miR-1260b in the pathological proliferation of VSMCs under hypoxia.

MicroRNA-204-5p Participates in Atherosclerosis Via Targeting MMP-9.

The aim of the present study was to investigate the role and mechanism of microRNA-204-5p (miR-204-5p) in atherosclerosis (AS)-related abnormal human vascular smooth muscle cells (hVSMCs) function. Firstly, we analyzed the expression of miR-204-5p and found that the miR-204-5p expression level was clearly downregulated in atherosclerotic plaque tissues and blood samples compared to the normal controls. Then, matrix metallopeptidase-9 (MMP-9) was predicted to be the potential target of miR-204-5p by TargetScan and this prediction was confirmed by luciferase assays. Besides, we observed that miR-204-5p could negatively regulate the expression of MMP-9 in hVSMCs. Subsequently, Thiazolyl Blue Tetrazolium Bromide (MTT) assay, transwell assay and flow cytometry were performed to detect the proliferation, migration and apoptosis of hVSMCs. Down-expression of miR-204-5p led to the promotion of proliferation and migration accompanied with the suppression of apoptosis in hVSMCs, and these effects were reversed by MMP-9-siRNA. In addition, overexpressed miR-204-5p could inhibit hVSMC proliferation and migration and promote the apoptosis of hVSMCs. However, the effects were also abrogated by overexpressed MMP-9. Together, our findings showed that miR-204-5p plays an important role in the growth and migration of hVSMCs by targeting MMP-9, which might be a novel biomarker and promising therapeutic target for AS.

The Role of Ubiquitin E3 Ligase in Atherosclerosis.

Atherosclerosis is a chronic inflammatory vascular disease. Atherosclerotic cardiovascular disease is the main cause of death in both developed and developing countries. Many pathophysiological factors, including abnormal cholesterol metabolism, vascular inflammatory response, endothelial dysfunction and vascular smooth muscle cell proliferation and apoptosis, contribute to the development of atherosclerosis and the molecular mechanisms underlying the development of atherosclerosis are not fully understood. Ubiquitination is a multistep post-translational protein modification that participates in many important cellular processes. Emerging evidence suggests that ubiquitination plays important roles in the pathogenesis of atherosclerosis in many ways, including regulation of vascular inflammation, endothelial cell and vascular smooth muscle cell function, lipid metabolism and atherosclerotic plaque stability. This review summarizes important contributions of various E3 ligases to the development of atherosclerosis. Targeting ubiquitin E3 ligases may provide a novel strategy for the prevention of the progression of atherosclerosis.