Human Aortic Smooth Muscle Cells Proliferation Research Articles

Different Effects of Endothelial Extracellular Vesicles and LPS-Induced Endothelial Extracellular Vesicles on Vascular Smooth Muscle Cells: Role of Curcumin and Its Derivatives.

Background: During the progression of atherosclerosis (AS), the vascular endothelial and smooth muscle cells are reciprocally regulated by extracellular vesicles (EVs). EVs have different effects on pathological and physiological processes due to the different cargoes contained in EVs.Purpose: To study the effects of endothelial cells-derived EVs on normal and inflammatory conditions. To investigate the effects of curcumin and curcumin derivatives (Nicotinic-curcumin) on endothelial EVs.Methods: EVs were isolated from human umbilical vein endothelial cells (HUVECs) by ultracentrifugation. To examined the effect of normal and LPS-induced endothelial cells-derived EVs on the proliferation of human aortic smooth muscle cells (HASMCs), the CCK-8 assay was performed. Transwell and wound healing assays were conducted to assess cell migration. The effects of EVs on lipid accumulation following treatment with oxidized low-density lipoprotein (Ox-LDL) were evaluated with the oil red O staining assay and HPLC. The number of EVs was calculated using the nanoparticle tracking analysis (NTA) and BCA. The expression levels of Rab27a and Rab27b that regulate the EVs secretion were measured by Western blotting assay. The differential expression of miRNAs in endothelial EVs and LPS-induced endothelial EVs was analyzed using miRNA-Sequencing (miRNA-Seq) and RT-PCR.Results: Treatment with endothelial EVs reduced the proliferation and migration of HASMCs as well as lipid accumulation in HASMCs. However, treatment with LPS-induced endothelial EVs did not inhibit the migration of HASMCs or lipid accumulation, instead it promoted the proliferation of HASMCs. Treatment with the two types of EVs induced differential expression of several miRNAs, including miR-92a-3p, miR-126-5p, miR-125a-3p, miR-143-3p, etc. Moreover, 1 μg/mL LPS induction greatly increased secretion of endothelial EVs. Treatment with curcumin and nicotinic-curcumin reduced endothelial EVs secretion, possibly by inhibiting inflammation.Conclusion: Endothelial EVs may confer beneficial effects on atherosclerosis by regulating vascular smooth muscle cell (VSMCs), whereas pro-inflammatory factors may disrupt this effect.

Silencing a disintegrin and metalloproteinase‑33 attenuates the proliferation of vascular smooth muscle cells via PI3K/AKT pathway: Implications in the pathogenesis of airway vascular remodeling.

Accumulating evidence suggests that pulmonary expression of a disintegrin and metalloproteinase-33 (ADAM33) serves a key role in the pathogenesis of airway remodeling-related diseases, including asthma. Airway vascular proliferation has been recognized as a key feature of airway remodeling. Our previous study showed that ADAM33 is constitutively expressed in airway vascular smooth muscle cells in patients with asthma, suggesting a potential role of ADAM33 in regulating airway vascular remodeling. Using in vitro human aortic smooth muscle cells (HASMCs) and lentiviral vector carrying short hairpin RNA for ADAM33, the present study aimed to evaluate the influence of ADAM33 silencing on the proliferation and apoptosis of HASMCs and the underlying molecular pathways. Cellular proliferation was observed using the Cell Counting Kit-8 method. Cellular apoptosis was evaluated with Annexin V-PE/7-AAD staining and flow cytometry. Reverse transcription-quantitative PCR and western blotting were used to evaluate the changes in mRNA and protein levels of involved signaling molecules. It was found that silencing of ADAM33 expression in HASMCs significantly inhibited proliferation, but induced the apoptosis of HASMCs. These changes were accompanied by inhibition of the PI3K/AKT/ERK pathway and Bcl-2, but an increase in Bax expression. These results suggested that constitutive expression of ADAM33 may be important to maintain a proliferative phenotype in HASMCs. The influences of ADAM33 on proliferation and apoptosis of HASMCs may involve regulation of PI3K/AKT/ERK and Bax/Bcl-2 pathways. These findings suggested an important role of ADAM33 in airway vascular remodeling and potential therapeutic significance of ADAM33 inhibition in airway remodeling-related diseases.

MiR-634 inhibits human vascular smooth muscle cell proliferation and migration in hypertension through Wnt4/β-catenin pathway.

MicroRNAs (miRNAs) have been regarded as modulators in vascular pathologies, including hypertension. Dysregulated proliferation and migration of VSMCs (vascular smooth muscle cells) contributes to vascular remodeling during hypertension. miR-634 was reported to be dysregulated in hypertensive patients. The involvement of miR-634 in hypertension and the role of miR-634 on VSMCs proliferation and migration were then evaluated. Firstly, HASMCs (human aortic smooth muscle cells) were incubated with 2 μM angiotensin (Ang) II for 12 hours to establish the cell model of Ang II-induced hypertension. Results showed that Ang II treatment promoted proliferation and migration of HASMCs. Secondly, miR-634 was down-regulated in the hypertensive patients, and reduced in Ang II-induced HASMCs in a time dependent manner. Functional assays revealed that Ang II promoted proliferation and migration of HASMCs were suppressed by miR-634 mimic. Lastly, miR-634 targeted 3' untranslated region (UTR) of Wnt4, and reduced Wnt4 expression in HASMCs. miR-634 inhibited β-catenin nuclear translocation. Over-expression of Wnt4 counteracted the suppressive effects of miR-634 on Ang II-induced proliferation and migration of HASMCs. In conclusion, miR-634 inhibited HASMCs proliferation and migration through inactivation of Wnt4/β-catenin pathway.

MiR-223/NFAT5 signaling suppresses arterial smooth muscle cell proliferation and motility in vitro.

Aberrant proliferation and migration of vascular smooth muscle cells contributes to cardiovascular diseases (CVDs), including atherosclerosis. MicroRNA-223 (miR-223) protects against atherosclerotic CVDs. We investigated the contribution of miR-223 to platelet-derived growth factor-BB (PDGF-BB)-induced proliferation and migration of human aortic smooth muscle cells (HASMCs). We found that miR-223 was downregulated in PDGF-BB-treated HASMCs in a dose- and time-dependent manner, while nuclear factor of activated T cells 5 (NFAT5) was upregulated. Gain- and loss-of-function studies demonstrated that miR-223 treatment reduced PDGF-BB-induced HASMC proliferation and motility, whereas miR-223 inhibitor enhanced these processes. Moreover, NFAT5 was identified as a direct target of miR-223 in HASMC. The inhibitory effects of miR-223 on HASMC proliferation and migration were partly rescued by NFAT5 restoration. Overall, these findings suggest that miR-223 inhibits the PDGF-BB-induced proliferation and motility of HASMCs by targeting NFAT5 and that miR-223 and NFAT5 may be potential therapeutic targets for atherosclerosis.

T cell co-stimulator inducible co-stimulatory (ICOS) exerts potential anti-atherosclerotic roles through downregulation of vascular smooth muscle phagocytosis and proliferation.

BackgroundAtherosclerosis (AS) is a chronic inflammatory disease. The role of the immune system in the etiology of the disease, particularly T cells, has been widely studied and is well established. T cell activation directly regulates co-signaling molecules present in immune synapses. Targeting one or several of these co-signaling molecules can inhibit T cell-mediated inflammation and delay or reduce AS. In recent years, this strategy has increasingly become a research focus. As such, we explored the role and therapeutic potential of the T cell co-stimulatory molecule inducible co-stimulatory (ICOS) in AS.MethodsWe compared the expression of ICOS in early AS lesions occurring in ApoE-deficient (ApoE-KO) rats fed a fat-diet and wild type (WT) rats fed the same diet. Eight-week old ApoE-KO and WT rats [ApoE-KO(0) and WT(0)] were fed a high-fat diet for 16 weeks [ApoE-KO(16) and WT(16)]. ICOS expression in aortic tissues was analyzed by quantitative real-time PCR, western blot, and confocal microscopy. The effect of ICOS overexpression in a transfected human T cell line on the phagocytosis and proliferation of co-cultured human aortic smooth muscle cells (HASMCs) was studied in vitro.ResultsCompared with WT(0), ApoE-KO(0), and WT(16) rats, ICOS expression in ApoE-KO(16) rats was significantly down-regulated both at the mRNA and protein levels. In vitro experiments indicated that ICOS overexpression reduces phagocytosis and proliferation by HASMCs, and may therefore produce an anti-atherosclerotic effect.ConclusionsThe immune synaptic co-signaling molecule ICOS has an anti-atherosclerotic effect through inhibition of HASMC phagocytosis and proliferation, and can be used to delay plaque formation during the early stages of AS.

LncRNA BANCR induced vascular smooth muscle cell proliferation by downregulating miR-34c methylation in atherosclerosis.

Aberrant vascular smooth muscle cell (VSMCs) proliferation involves in the development of atherosclerosis. It reported that Long noncoding BRAF-activated noncoding RNA (BANCR) and miR-34c played opposite roles in the regulation of the proliferation of VSMCs, indicating that there might be a potential interaction between them. This study was to investigate the relationship between BANCR and miR-34c in atherosclerosis. Blood (5ml) was obtained from 56 patients with atherosclerosis and 56 healthy volunteers after they were fasted overnight, and plasma was extracted from the blood. Human Aortic Smooth Muscle Cells (HASMCs) were used to perform in vitro cell experiments. RT-qPCR was performed to measure the expression of BANCR and miR-34c in plasma and HASMCs. Dual luciferase reporter assay detected the interaction between BANCR and miR-34c. CCK-8 assay was used to assess the effects of BANCR and miR-34c overexpression on the proliferation of HASMCs. Western blotting was used to assess the effects of BANCR and miR-34c overexpression on the protein expression of HMGB1, TNF-ɑ and Bcl-2. In this study, we found that BANCR was upregulated, while miR-34c was downregulated in atherosclerosis. Bioinformatics analysis showed that BANCR and miR-34c could directly interact with each other. Moreover, overexpression of BANCR could decrease the expression of miR-34c in HASMCs, but overexpression of miR-34c could not affect the expression of BANCR. Furthermore, overexpression of BACNR increased miR-34c methylation, and knockdown of endogenous BANCR decreased miR-34c methylation. In addition, overexpression of BANCR reduced the effects of miR-34c on HASMCs proliferation and reversed the effects of miR-34c on HMGB1, TNF-ɑ and Bcl-2 expression. BANCR overexpression could induce HASMCs proliferation by downregulating the miR-34c methylation. Therefore given BANCR upregulation in atherosclerosis, its expression may be considered as a novel and useful biomarker for atherosclerosis prevention and prognosis. However considering the possible effects of other underlying diseases on both BANCR expression and miR-34c in atherosclerosis, further investigation is suggested for future research.

Downregulating long non-coding RNA PVT1 expression inhibited the viability, migration and phenotypic switch of PDGF-BB-treated human aortic smooth muscle cells via targeting miR-27b-3p

Long non-coding RNA Plasmacytoma Variant Translocation 1 (LncRNA PVT1) was involved in various human diseases, but its role in aortic dissection (AD) remained to be fully examined. In this study, the viability and migration of human aortic smooth muscle cells (HASMCs) were respectively measured by MTT assay and wound-healing assay. Relative phenotypic switch-related protein expressions were measured with quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot as needed. An AD model was established in animals and hematoxylin-eosin (H&E) staining was used for pathological examination. We found that, in HASMCs, microRNA (miR)-27b-3p could competitively bind with PVT1. In AD, PVT1 expression was upregulated, yet that of miR-27b-3p was downregulated. Downregulating PVT1 reversed the effects of growth factor-BB (PDGF-BB) treatment on PVT1, miR-27b-3p and expressions of phenotypic switch-related markers, and cell viability and migration, while downregulating miR-27b-3p reversed the effects of downregulating PVT1. Moreover, downregulating PVT1 suppressed the effects of upregulated PVT1 and downregulated miR-27b-3p induced by AD as well as media degeneration in vivo. In conclusion, downregulating PVT1 expression suppressed the proliferation, migration and phenotypic switch of HASMCs treated by PDGF-BB via targeting miR-27b-3p.

Inhibitory effect of CP-25 on intimal formation and vascular hyperplasia via suppression of GRK2/ERK1/2/EVI1 signaling

Excessive proliferation, migration and dedifferentiation of vascular smooth muscle cells (VSMCs) are the center of intimal formation during in-stent restenosis and vein graft disease. Paeoniflorin-6′-O-benzene sulfonate (CP-25) is known to suppress inflammation and atherogenesis. However, the potential effect of CP-25 on intimal formation remains elusive. In the present study, we found that CP-25 significantly attenuated wire injury-induced intimal formation in C57BL/6 mice (intimal area: 2.64 ± 0.25 × 104 μm2 vs. 1.53 ± 0.21 × 104 μm2, P < 0.05) and vascular hyperplasia indicated by PCNA staining. In vitro experiments showed that CP-25 significantly alleviated human aortic smooth muscle cell (HASMC) proliferation, migration and dedifferentiation induced by PDGF-BB. Mechanistically, CP-25 inhibited GRK2 phosphorylation through PDGF receptor in the presence of PDGF-BB. In accordance with these results, CP-25 disrupted the interaction of GRK2 with ERK1/2 and suppressed the activation of ERK1/2 signaling in HASMCs. EVI1, which is considered as a downstream of ERK1/2 signaling and a novel transcription factor for VSMC differentiation, was also downregulated by CP-25 treatment. Moreover, overexpression of EVI1 partly restored the decreased proliferation and dedifferentiation of HASMCs treated by CP-25. Collectively, these findings suggested that CP-25 could alleviate intimal formation in response to wire injury via suppression of the interaction of GRK2 and ERK1/2 and EVI1 activation, indicating CP-25 might serve as a potent pharmaceutical for intimal formation.

Large Extracellular Vesicle-Associated Rap1 Accumulates in Atherosclerotic Plaques, Correlates With Vascular Risks and Is Involved in Atherosclerosis

Metabolic syndrome (MetS) is a cluster of interrelated risk factors for cardiovascular diseases and atherosclerosis. Circulating levels of large extracellular vesicles (lEVs), submicrometer-sized vesicles released from plasma membrane, from MetS patients were shown to induce endothelial dysfunction, but their role in early stage of atherosclerosis and on vascular smooth muscle cells (SMC) remain to be fully elucidated. To determine the mechanisms by which lEVs lead to the progression of atherosclerosis in the setting of MetS. Proteomic analysis revealed that the small GTPase, Rap1 was overexpressed in lEVs from MetS patients compared with those from non-MetS subjects. Rap1 was in GTP-associated active state in both types of lEVs, and Rap1-lEVs levels correlated with increased cardiovascular risks, including stenosis. MetS-lEVs, but not non-MetS-lEVs, increased Rap1-dependent endothelial cell permeability. MetS-lEVs significantly promoted migration and proliferation of human aortic SMC and increased expression of proinflammatory molecules and activation of ERK (extracellular signal-regulated kinase) 5/p38 pathways. Neutralization of Rap1 by specific antibody or pharmacological inhibition of Rap1 completely prevented the effects of lEVs from MetS patients. High-fat diet-fed ApoE-/- mice displayed an increased expression of Rap1 both in aortas and circulating lEVs. lEVs accumulated in plaque atherosclerotic lesions depending on the progression of atherosclerosis. lEVs from high-fat diet-fed ApoE-/- mice, but not those from mice fed with a standard diet, enhanced SMC proliferation. Human atherosclerotic lesions were enriched in lEVs expressing Rap1. These data demonstrate that Rap1 carried by MetS-lEVs participates in the enhanced SMC proliferation, migration, proinflammatory profile, and activation of ERK5/p38 pathways leading to vascular inflammation and remodeling, and atherosclerosis. These results highlight that Rap1 carried by MetS-lEVs may be a novel determinant of diagnostic value for cardiometabolic risk factors and suggest Rap1 as a promising therapeutic target against the development of atherosclerosis. Graphical Abstract: A graphical abstract is available for this article.

Silencing CCL8 inhibited the proliferation and migration of PDGF-BB-stimulated human aortic smooth muscle cells.

C-C motif Chemokine ligand 8 (CCL8) has been found in diseases' pathogenesis. But its molecular mechanism in atherosclerosis (AS) remains to be elucidated. Human aortic smooth muscle cells (HASMCs) were stimulated by PDGF-BB to establish cell model. α-SMA in PDGF-BB-stimulated HASMCs was measured by immunofluorescence staining. Relative gene expressions in PDGF-BB-stimulated HASMCs were detected by quantitative real-time polymerase chain reaction and western blot. HASMCs proliferation, migration, and cell cycle were assessed by cell counting kit-8, wound-healing assay, and flow cytometry. HASMCs viability was increased after PDGF-BB stimulation, with α-SMA downregulation yet CCL8 upregulation. Silencing CCL8 inhibited PDGF-BB-stimulated HASMCs proliferation and migration, and increased cells percentage in G1 phases but decreased those in S phase. Also, silencing CCL8 decreased OPN and cyclinD1 expressions and AKT and ERK1/2 phosphorylation while increased those of α-SMA and Sm22α. However, upregulating CCL8 led to opposite effects, suggesting CCL8 could be an atherosclerosis therapeutic target.

Myristoylation of LMCD1 leads to its species‐specific derepression of E2F1 and NFATc1 in the modulation of CDC6 and IL‐33 expression during development of vascular lesions

Majority of translational research is focused on the use of rodent models to understand the pathophysiology of human disease. However, many experimental findings of rodent models fail to translate to human. In this regard, our previous studies show that LIM and cysteine‐rich domains 1 (LMCD1) plays a role in thrombin‐induced vascular smooth muscle cell (VSMC) proliferation and migration only in human but not rodents. Based on these findings, we asked the question whether LMCD1 exhibits any species‐specific role in the development of vascular lesions. A combination of genetic, molecular, cellular and disease models were used to test species‐specific role of LMCD1 in the pathogenesis of vascular lesions. Here, we report species‐specific regulation of LMCD1 expression in mediating VSMC proliferation and migration during vascular wall remodeling in human versus mice. Thrombin induced LMCD1 expression in human aortic smooth muscle cells (HASMCs) but not mouse aortic smooth muscle cells (MASMCs). We found association of ‘rapid promoter evolution’ in this expression divergence of LMCD1 in human versus mice. In exploring the mechanism, we found that Par1‐Gαq/11‐PLCβ3‐NFATc1 signaling axis mediates thrombin‐induced LMCD1 expression in HASMCs. Furthermore, although LMCD1 mediates thrombin‐induced proliferation and migration of both HASMCs and MASMCs via influencing CDC6 and IL‐33 expression, respectively, in humans it acts as an activator and in mice it acts as a repressor of their promoters. Interestingly, its repressor activity was revoked by its myristoylation in mouse. Besides, we found increased expression of LMCD1 in human stenotic arteries as compared to non‐stenotic arteries. On the other hand, LMCD1 expression was decreased in neointimal lesions of mouse injured arteries as compared to non‐injured arteries. Together, these observations reveal that LMCD1 acts as an activator and repressor of E2F1 and NFATc1 in human and mice, respectively, in the induction of CDC6 and IL‐33 expression during development of vascular lesions. Based on these findings, LMCD could be a potential target for drug development against restenosis and atherosclerosis in humans.Support or Funding InformationThis work was supported by a grant HL069908 from NIH to Dr. Gadiparthi N. Rao

Candesartan ameliorates vascular smooth muscle cell proliferation via regulating miR-301b/STAT3 axis.

Excessive vascular smooth muscle cell (VSMC) proliferation contributes to vascular remodeling and stroke during hypertension. Blockade of Angiotensin (AngII) type 1 receptor (AT1R) is shown to effectively attenuate VSMC proliferation and vascular remodeling, while the mechanisms underlying these protective effects are unclear. Here, we investigated whether the amelioration of VSMC proliferation mediated by candesartan, an AT1R blocker, could be associated with miRNA regulation. Based on the published data in rat aortic smooth muscle cells (RASMCs), we discovered that candesartan specifically reversed the AngII-induced decrease of miR-301b level in RASMCs and human aortic smooth muscle cells (HASMCs). Knockdown of miR-301b abolished candesartan-mediated inhibition of HASMC proliferation via promoting cell cycle transition. Computational analysis showed that miR-301b targets at 3'UTR of STAT3. MiR-301b upregulation inhibited the luciferase activity and protein expression of STAT3, whereas miR-301b knockdown increased STAT3 luciferase activity and expression. Furthermore, downregulation of STAT3 markedly abrogated the effects of miR-301b inhibition on candesartan-mediated HASMC proliferation, invasion, and migration. Collectively, this study suggests that miR-301b may be a novel molecular target of candesartan and provides a new understanding for the mechanisms underlying the cardiovascular effects of candesartan.

Screening and functional prediction of differentially expressed circRNAs in proliferative human aortic smooth muscle cells.

Vascular smooth muscle cell (VSMC) proliferation is the pathological base of vascular remodelling diseases. Circular RNAs (circRNAs) are important regulators involved in various biological processes. However, the function of circRNAs in VSMC proliferation regulation remains largely unknown. This study was conducted to identify the key differentially expressed circRNAs (DEcircRNAs) and predict their functions in human aortic smooth muscle cell (HASMC) proliferation. To achieve this, DEcircRNAs between proliferative and quiescent HASMCs were detected using a microarray, followed by quantitative real‐time RT‐PCR validation. A DEcircRNA‐miRNA‐DEmRNA network was constructed, and functional annotation was performed using Gene Ontology (GO) and KEGG pathway analysis. The function of hsa_circ_0002579 in HASMC proliferation was analysed by Western blot. The functional annotation of the DEcircRNA‐miRNA‐DEmRNA network indicated that the four DEcircRNAs might play roles in the TGF‐β receptor signalling pathway, Ras signalling pathway, AMPK signalling pathway and Wnt signalling pathway. Twenty‐seven DEcircRNAs with coding potential were screened. Hsa_circ_0002579 might be a pro‐proliferation factor of HASMC. Overall, our study identified the key DEcircRNAs between proliferative and quiescent HASMCs, which might provide new important clues for exploring the functions of circRNAs in vascular remodelling diseases.

RETRACTED: Linc-ROR targets FGF2 to regulate HASMC proliferation and migration via sponging miR-195-5p

Atherosclerosis is a common cardiovascular disorder and is characterized by damage of endothelial cells, cell inflammation, hyper-proliferation of vascular smooth muscle cells and the accumulation of extracellular lipids and fibrous tissues. In this study, we firstly examined the expression level of long intergenic non-protein coding RNA, regulator of reprogramming (linc-ROR) in homocysteine (Hcy)-stimulated human aortic smooth muscle cells (HASMCs), and then looked into the potential molecular signaling axis of linc-ROR in regulating the proliferation and migration of HASMCs. Hcy promoted HASMC proliferation and up-regulated linc-ROR expression. Functional studies showed that linc-ROR exerted enhanced actions on the proliferation and migration of HASMCs. In addition, linc-ROR acted as a competing endogenous RNA for miR-195-5p and repressed the miR-195-5p expression in HASMCs. Linc-ROR was up-regulated the miR-195-3p was down-regulated in the plasma from CAD patients when compared to normal controls. Furthermore, fibroblast growth factor 2 (FGF2) was identified as a target of miR-195-5p and was negatively regulated by miR-195-5p in HASMCs. The rescue experiments revealed that linc-ROR-mediated HASMC proliferation and migration may be via regulating miR-195-5p/FGF2 axis. Linc-ROR inhibition blocked the miR-195-5p/FGF2 signaling in Hcy-treated HASMCs, and this effect may also involve in the miR-195-5p/FGF2 axis. To summarize, the data of the present study identified the up-regulation of linc-ROR in Hcy-stimulated HASMCs, and further mechanistic functional studies revealed that linc-ROR promoted HASMC proliferation and migration via regulating miR-195-5p/FGF2 axis. The present study provided the novel actions of linc-ROR in regulating HASMC proliferation and migration, which may be related to the pathophysiology of atherosclerosis.

Angiotensin II-Induced vascular remodeling and hypertension involves cathepsin L/V- MEK/ERK mediated mechanism

The development of hypertension involves extensive arterial wall remodeling, in which cysteine proteases play an essential role. Cathepsin L/V has been reported to be a cysteine protease that is closely intertwined with the tissue inflammatory response and extracellular matrix accumulation, allowing it to regulate arterial remodeling. The aim of this study was to determine the role of cathepsin L/V and its regulatory pathway in vascular remodeling in hypertension. We showed that cathepsin L/V and its endogenous inhibitor cystatin C, as well as mitogen-activated protein kinase (MEK) phosphorylation, were upregulated in the mesenteric arteries and serum of hypertensive patients. Using a model of angiotensin II-induced hypertension, we observed an increase in aortic artery media thickness, cathepsin L activity, blood pressure and MEK phosphorylation. Treatment with the cathepsin L inhibitor Z-FF-FMK or the genetic deletion of cathepsin L significantly attenuated angiotensin II-induced hypertension, arterial remodeling, cathepsin L activation and MEK phosphorylation. In addition, siRNA-cathepsin V significantly blocked angiotensin II-induced MEK and extracellular signal–regulated kinase (ERK) phosphorylation but not cell proliferation in human aortic smooth muscle cells (HASMCs). Further treatment with the ERK phosphorylation inhibitor U0126 also blocked angiotensin II-induced HASMCs proliferation. The results indicate that the inhibition of cathepsin L prevents arterial remodeling and hypertension, in part by inhibiting the MEK-ERK signaling-mediated regulation of smooth muscle cell proliferation in the vessel wall.

LncRNA TNK2-AS1 regulated ox-LDL-stimulated HASMC proliferation and migration via modulating VEGFA and FGF1 expression by sponging miR-150-5p.

Long non‐coding RNAs (lncRNAs) have been indicated for the regulatory roles in cardiovascular diseases. This study determined the expression of lncRNA TNK2 antisense RNA 1 (TNK2‐AS1) in oxidized low‐density lipoprotein (ox‐LDL)‐stimulated human aortic smooth muscle cells (HASMCs) and examined the mechanistic role of TNK2‐AS1 in the proliferation and migration of HASMCs. Our results demonstrated that ox‐LDL promoted HASMC proliferation and migration, and the enhanced proliferation and migration in ox‐LDL‐treated HASMCs were accompanied by the up‐regulation of TNK2‐AS1. In vitro functional studies showed that TNK2‐AS1 knockdown suppressed cell proliferation and migration of ox‐LDL‐stimulated HASMCs, while TNK2‐AS1 overexpression enhanced HASMC proliferation and migration. Additionally, TNK2‐AS1 inversely regulated miR‐150‐5p expression via acting as a competing endogenous RNA (ceRNA), and the enhanced effects of TNK2‐AS1 overexpression on HASMC proliferation and migration were attenuated by miR‐150‐5p overexpression. Moreover, miR‐150‐5p could target the 3’ untranslated regions of vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 1 (FGF1) to regulate FGF1 and VEGFA expression in HASMCs, and the inhibitory effects of miR‐150‐5p overexpression in ox‐LDL‐stimulated HASMCs were attenuated by enforced expression of VEGFA and FGF1. Enforced expression of VEGFA and FGF1 also partially restored the suppressed cell proliferation and migration induced by TNK2‐AS1 knockdown in ox‐LDL‐stimulated HASMCs, while the enhanced effects of TNK2‐AS1 overexpression on HASMC proliferation and migration were attenuated by the knockdown of VEGFA and FGF1. Collectively, our findings showed that TNK2‐AS1 exerted its action in ox‐LDL‐stimulated HASMCs via regulating VEGFA and FGF1 expression by acting as a ceRNA for miR‐150‐5p.

Long non-coding RNA SNHG16 regulates human aortic smooth muscle cell proliferation and migration via sponging miR-205 and modulating Smad2.

The present study investigated the role of long non‐coding RNA (lncRNA) small nucleolar RNA host gene 16 (SNHG16) in the human aortic smooth muscle cell (HASMC) proliferation and migration and explored the potential link between SNHG16 and atherosclerosis. Our results showed that platelet‐derived growth factor (PDGF)‐bb treatment promoted cell proliferation and migration with concurrent up‐regulation of SNHG16 in HASMCs. Small nucleolar RNA host gene 16 overexpression promoted HASMC proliferation and migration, while SNHG16 knockdown suppressed cell proliferation and migration in PDGF‐bb‐stimulated HASMCs. The bioinformatic analyses showed that SNHG16 possessed the complementary binding sequence with miR‐205, where the interaction was confirmed by luciferase reporter assay and RNA pull‐down assay in HASMCs, and SNHG16 inversely regulated miR‐205 expression. MiR‐205 overexpression attenuated the enhanced effects of PDGF‐bb treatment on HASMC proliferation and migration. Moreover, Smad2 was targeted and inversely regulated by miR‐205, while being positively regulated by SNHG16 in HASMCs. Smad2 knockdown attenuated PDGF‐bb‐mediated actions on HASMC proliferation and migration. Both miR‐205 overexpression and Smad2 knockdown partially reversed the effects of SNHG16 overexpression on HASMC proliferation and migration. Moreover, SNHG16 and Smad2 mRNA were up‐regulated, while miR‐205 was down‐regulated in the plasma from patients with atherosclerosis. Small nucleolar RNA host gene 16 expression was inversely correlated with miR‐205 expression and positively correlated with Smad2 expression in the plasma from atherosclerotic patients. In conclusion, our data showed the up‐regulation of SNHG16 in pathogenic‐stimulated HASMCs and clinical samples from atherosclerotic patients. Small nucleolar RNA host gene 16 regulated HASMC proliferation and migration possibly via regulating Smad2 expression by acting as a competing endogenous RNA for miR‐205.

Inhibitory effects of herbal decoction Ojeoksan on proliferation and migration in vascular smooth muscle cells.

The proliferation of vascular smooth muscle cells plays a crucial role in pathogenesis of cardiovascular disease. The principal objective of this study was to determine the effects of Ojeoksan (OJS) on human aortic smooth muscle cell (HASMC) proliferation induced by tumor necrosis factor α (TNF-aα). Thymidine incorporation after TNF-α treatment was increased and this effect was inhibited significantly by OJS treatment. HASMC proliferation and migration by kinetic live cell imaging were also reduced by treatment with OJS. TNF-α induced the expression of cyclins/cyclin-dependent kinases (CDKs) and reduced the expression of p21waf1/cip1/p27kip1. However, OJS also attenuated the expression of TNF-α-induced cell-cycle regulatory proteins. The results of Western blot analysis demonstrated that the TNF-α treated HASMC secreted gelatinases, probably including MMP-2/-9, which may be involved in the invasion and migration of HASMC. Additionally, OJS suppressed the mRNA expression levels of matrix metalloproteinase-2/-9 (MMP-2/-9) in a dose-dependent manner. OJS inhibited the production of TNF-α-induced hydrogen peroxide (H2O2) and the formation of DCF-sensitive intracellular reactive oxygen species (ROS). Further, OJS suppressed the nuclear translocation and phosphorylation of inhibitor of kappa B-α (IκB-α) of nuclear factor κB (NF-κB) under TNF-α conditions. Our results demonstrate that OJS exerts inhibitory effects on TNF-α-induced HASMC proliferation and migration, suggesting the involvement of the inhibition of both MMP-2 and MMP-9 expressions, and the downregulation of ROS/NF-κB signaling. Thus, herbal decoction OJS may be a possible therapeutic approach to the inhibition of cardiovascular disease including atherosclerosis.

Knockdown of TRIM28 inhibits PDGF-BB-induced vascular smooth muscle cell proliferation and migration

Atherosclerosis is a common type of cardiovascular disease (CVD), remaining one of the leading causes of global death. Tripartite motif-containing 28 (TRIM28) is a member of TRIM family that has been found to be involved in atherosclerosis. However, the role of TRIM28 in atherosclerosis remains unknown. This study aimed to investigate the effects of TRIM28 on the phenotypic switching of human aortic smooth muscle cells (HASMCs), which is considered as a fundamental event during the development of atherosclerosis. The results showed that TRIM28 was highly expressed in human atherosclerotic tissues, as well in cultured HASMCs stimulated by platelet-derived growth factor subunit B homodimer (PDGF-BB). Knockdown of TRIM28 by transfection with siRNA targeting TRIM28 (si-TRIM28) significantly suppressed the PDGF-BB-induced cell proliferation and migration of HASMCs. Besides, knockdown of TRIM28 inhibited the expressions of matrix metalloproteinase (MMP)-2 and MMP-9. The VSMC markers including α-smooth muscle actin (α-SMA), calponin and SM22α were upregulated in TRIM28 knocked down HASMCs. Furthermore, knockdown of TRIM28 blocked PDGF-BB-induced NF-κB activation in HASMCs. Collectively, knockdown of TRIM28 prevented PDGF-BB-induced phenotypic switching of HASMCs, which might be mediated by the regulation of NF-κB signaling pathway.

The pseudogene PTENP1 regulates smooth muscle cells as a competing endogenous RNA.

The long non-coding RNA (lncRNA) PTENP1 is a pseudogene of phosphatase and tensin homologue deleted on chromosome ten (PTEN), has been implicated in smooth muscle cell (SMC) proliferation and apoptosis. PTENP1 is the pseudogene of PTEN. However, it is unclear whether and how PTENP1 functions in the proliferation and apoptosis of human aortic SMCs (HASMCs). Here, we hypothesised that PTENP1 inhibits HASMC proliferation and enhances apoptosis by promoting PTEN expression. PCR analysis and Western blot assays respectively showed that both PTENP1 and PTEN were up-regulated in human aortic dissection (AD) samples. PTENP1 overexpression significantly increased the protein expression of PTEN, promoted apoptosis and inhibited the proliferation of HASMCs. PTENP1 silencing exhibited the opposite effects and mitigated H2O2-induced apoptosis of HASMCs. In an angiotensin II (Ang II)-induced mouse aortic aneurysm (AA) model, PTENP1 overexpression potentiated aortic SMC apoptosis, exacerbated aneurysm formation. Mechanistically, RNA pull-down assay and a series of luciferase reporter assays using miR-21 mimics or inhibitors identified PTENP1 as a molecular sponge for miR-21 to endogenously compete for the binding between miR-21 and the PTEN transcript, releasing PTEN expression. This finding was further supported by in vitro immunofluorescent evidence showing decreased cell apoptosis upon miR-21 mimic administration under baseline PTENP1 overexpression. Ex vivo rescue of PTEN significantly mitigated the SMC apoptosis induced by PTENP1 overexpression. Finally, Western blot assays showed substantially reduced Akt phosphorylation and cyclin D1 and cyclin E levels with up-regulated PTENP1 in HASMCs. Our study identified PTENP1 as a mediator of HASMC homeostasis and suggests that PTENP1 is a potential target in AD or AA intervention.