A Smooth Muscle Cell-Based Ferroptosis Model to Evaluate Iron-Chelating Molecules for Cardiovascular Disease Treatment.

Dysregulation of iron homeostasis causes iron-mediated cell death, recently described as ferroptosis. Ferroptosis is reported in many chronic diseases, such as hepatic cancer, renal, and cardiovascular diseases (heart failure, atherosclerosis). However, there is a notable scarcity of research studies in the existing literature that explore treatments capable of preventing ferroptosis. Additionally, as far as the author is aware, there is currently no established model for studying ferroptosis within cardiovascular cells, which would be essential for assessing metal-chelating molecules with the potential ability to inhibit ferroptosis and their application in the treatment of cardiovascular diseases. In this study, a smooth muscle cell-based ferroptosis model is developed upon the inhibition of the system Xc- transporter by erastin associated or not with Fe(III) overload, and its rescue upon the introduction of well-known iron chelators, deferoxamine and deferiprone. We showed that erastin alone decreased the intracellular concentration of glutathione (GSH) without affecting peroxidized lipid concentrations. Erastin with ferric citrate was able to decrease intracellular GSH and induce lipid peroxidation after overnight incubation. Only deferiprone was able to rescue the cells from ferroptosis by decreasing lipid peroxidation via iron ion chelation in a 3:1 molar ratio.

Neutrophil Elastase Inhibition by Sivelestat (ONO-5046) Attenuates AngII-Induced Abdominal Aortic Aneurysms in Apolipoprotein E-Deficient Mice.

BACKGROUND: Abdominal aortic aneurysm (AAA) is an arterial disease characterized by dilatation of the aortic wall. It has been suggested that neutrophil counts and neutrophil elastase activity are associated with AAA. We investigated whether a neutrophil elastase (NE) inhibitor, sivelestat (Siv), had a protective effect against angiotensin II (AngII)-induced AAAs. METHODS: Male apolipoprotein E-deficient mice were assigned into three groups: Vehicle + saline, AngII + saline, and AngII + Siv. All mice were administered intraperitoneally with either Siv or vehicle twice daily after AngII infusion. RESULTS: In the 4-week AngII infusion study, plasma NE concentration (P = 0.041) and its activity (P = 0.011) were elevated by AngII. These increases were attenuated by Siv (concentration:P = 0.010, activity:P = 0.027). Further, plasma elastase activity was closely correlated with aortic width (R = 0.6976, P < 0.001). In the 1-week AngII infusion study, plasma and tissue elastase activity increased by AngII (plasma:P = 0.034, tissue:P < 0.001), but were reduced by Siv (plasma:P = 0.014, tissue:P = 0.024). AngII increased aortic width (P = 0.011) but was attenuated by co-administration of Siv (P = 0.022). Moreover, Siv decreased the incidence of AAAs (P = 0.009). Elastin fragmentation induced by AngII was reduced by Siv. Many inflammatory cells that were either CD68 or Gr-1 positive were observed in the AngII + saline group, whereas few inflammatory cells were accumulated in the AngII + Siv group. MMP-2 and MMP-9 were enhanced by AngII, but were reduced by Siv. In vitro, MMP-2 activity was induced by human NE (medium:P < 0.001, cells:P = 0.001), which was attenuated by co-incubation of Siv in medium (P < 0.001) and protein of human aortic smooth muscle cells (P = 0.001). CONCLUSIONS: Siv attenuated AngII-induced AAA through the inhibition of NE.

Single Nucleotide Polymorphisms Mutation of Tropoelastin Gene Affects Tropoelastin mRNA and Elastin Expressions in Human Aortic Smooth Muscle Cells.

We aimed to explore the effects of single nucleotide polymorphisms (SNPs) in tropoelastin gene on tropoelastin mRNA and elastin expressions in human aortic smooth muscle cells (HASMCs). Two SNP loci, rs2071307 (G/A) and rs1785598 (G/C), were selected to construct recombinant lentivirus vectors carrying wild-type and mutant tropoelastin gene. Recombinant plasmids including pWSLV-02-ELN, pWSLV-02-ELN-mut1, and pWSLV-02-ELN-mut2 were constructed, before being amplified by polymerase chain reaction (PCR) and sequenced. The prepared plasmids and the packaging plasmids (pVSV-G and psPAX2) were cotransfected into HEK293T cells to obtain recombinant lentiviruses carrying tropoelastin gene. Afterward, HASMCs were infected with recombinant lentiviruses, and the positive cells sorted by flow cytometry were amplified. Four stable HASMCs cell lines including pWSLV-02-ELN, pWSLV-02-ELN-mut1, pWSLV-02-ELN-mut2, and pWSLV-02 vector were constructed. The expressions of tropoelastin mRNA and elastin in HASMCs were detected by real-time quantitative reverse transcription-PCR and western blot, respectively. Recombinant plasmids including pWSLV-02-ELN-mut1, pWSLV-02-ELN-mut2, and pWSLV-02-ELN were successfully constructed. Recombinant lentiviruses carrying tropoelastin gene were obtained via lentivirus packaging. After infection for 24 h, 3 days and 5 days in HASMCs, tropoelastin mRNA expressions in pWSLV-02-ELN-mut1 and pWSLV-02-ELN-mut2 groups were significantly lower than that of pWSLV-02-ELN group. Besides, after infection for 24 h, 3 days, and 5 days, elastin levels in pWSLV-02-ELN-mut1 and pWSLV-02-ELN-mut2 groups were significantly lower than that in pWSLV-02-ELN group. In conclusion, SNPs mutation of tropoelastin gene affected the expression of tropoelastin mRNA and elastin, suggesting that the polymorphisms of rs2071307 and rs17855988 in tropoelastin gene might be important factors for AD development.

Human aortic smooth muscle cell regulation by METTL3 via upregulation of m6A NOTCH1 modification and inhibition of NOTCH1.

Background: Thoracic aortic dissection (TAD) is a very serious vascular condition that requires immediate treatment. Phenotypic conversion of human aortic smooth muscle cells (HASMCs) has been reported to be a causal factor for TAD development. Genetic variations affecting RNA modification may play a functional role in TAD. In this study, we aimed to explore the potential role of the methyltransferase like 3 (METTL3) and notch homolog 1 (NOTCH1) N6-methyladenosine (m6A) modification mechanisms in HASMCs. Methods: HASMCs were cultured. METTL3 was knocked down and overexpressed. Then, both METTL3 and NOTCH1 were simultaneously knocked down in HASMCs. HASMC proliferation was determined using Cell Counting Kit-8 (CCK-8). METTL3, NOTCH1, α-smooth muscle actin (α-SMA), smooth muscle protein 22-alpha (SM22α), and calponin expressions were monitored with quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. An m6A dot blot assay was used to examine the m6A modification levels. The NOTCH1 3' untranslated region (3'UTR) m6A modification was analyzed using SRAMP and RMBase v. 2.0. A methylated RNA immunoprecipitation (MeRIP) assay was used to evaluate the METTL3 overexpression effect on m6A modification of NOTCH1 messenger RNA (mRNA). A dual-luciferase assay was used to investigate the effect of METTL3 binding of the NOTCH1 mRNA m6A modification site. YTH domain family 2 (YTHDF2)-RNA immunoprecipitation (RIP) was used to detect the change in YTHDF2's ability to bind to NOTCH1 mRNA after METTL3 overexpression. Results: Overexpression of METTL3 inhibited α-SMA, SM22α, calponin, and NOTCH1 expressions and promoted HASMC proliferation. Knocking down METTL3 had the opposite effect. The cointerference of the METTL3 and NOTCH1 results suggested that METTL3 regulated NOTCH1, contributing to HASMC phenotypic changes. The MeRIP assay showed that the m6A modification of NOTCH1 mRNA increased after METTL3 overexpression. The dual-luciferase assay indicated that the NOTCH1 mRNA m6A modification site and METTL3 overexpression promoted NOTCH1 mRNA degradation. YTHDF2-RIP further demonstrated that the binding ability of YTHDF2 and NOTCH1 mRNA was enhanced after METTL3 overexpression. Conclusions: METTL3 regulated the phenotypic changes of HASMC by upregulating m6A modification of NOTCH1 and inhibiting NOTCH1.

Downregulation of miR-4284 can inhibit the apoptosis of Human Arterial Smooth Muscle Cells (HASMCs) in arteriosclerosis obliterans (ASO).

Introduction The disease arteriosclerosis obliterans (ASO) affects the lower extremities. ASO's mechanism involves the proliferation and migration of vascular smooth muscle cells (VSMCs). The miR-4284 is involved in several biological processes of the cardiovascular system, including VSMC proliferation, migration, and death. However, it is unknown if the miR-4284 gene is involved in the control of ASO. Furthermore, the molecular processes behind the contribution of human arterial smooth muscle cells (HASMCs), one of the most significant components of the arterial wall, to arteriosclerosis obliterans (ASO) pathogenesis remain unknown. Previously, we explored the alterations of miRNAs in the blood of ASO patients, and now we wanted to test further whether these changes also take place in the HASMCs that are responsible for the pathogenesis of ASO. Methods The expression levels of miR-29a in arterial walls were analyzed via a real-time polymerase chain reaction. An ASO cell model was established to investigate the expression of miR-4284 on HASMCs. The Transwell system and CCK-8 detection were used to assess the migration and proliferation of HASMCs. The proportion of apoptotic cells as well as the concentrations of apoptotic signal protein production were assessed using flow cytometry. A Western blot technique was used to identify B cell lymphoma-2 (Bcl2), Bcl2-associated X protein (BAX), as well as X-linked inhibitors of apoptosis protein (XIAP). Results The results showed that PCR confirmed that the qualified production or expression of miR-4284 was significantly reduced in HASMCs after they were cultured without FBS and in an atmosphere of 1% O2 + 5% CO2 + 94% N2 and that glucose had no effect on its expression. MiR-4284 has no effect on migration and proliferation, but downregulation of miR-4284 can decrease the apoptotic rate of HASMCs, as revealed by flow cytometry. Furthermore, western blot experiments showed that the expression of BAX was low, while the expression of the other two proteins, viz., Bcl2 and XIAP, was over-expressed. Conclusion We found that miR-4284 downregulation enhanced Bcl2, as well as XIAP, and decreased Bax. This shows that downregulated miR-4284 regulates apoptosis-related protein expression in HASMCs. The mechanism is not clear, and we need further study to confirm it.

Stanniocalcin 2 Is Upregulated by Calcium-Sensing Receptor and Protects Human Vascular Smooth Muscle Cells from High-Phosphate-Induced Apoptosis.

INTRODUCTION: Apoptosis of vascular smooth muscle cells induced by hyperphosphatemia is a critical mechanism of chronic kidney disease-related vascular disorders. The present study investigated whether extracellular calcium-sensing receptor (CaSR) regulates stanniocalcin 2 (STC2) expression in HAoSMCs and subsequently protects HAoSMCs from high-phosphate-induced apoptosis. METHODS: HAoSMCs were cultured, and STC2 expression was determined by qPCR. A calcimimetic (NPS R-568) or calcilytic (NPS-2143) was applied to HAoSMCs. STC2 mRNA and protein levels were measured by qPCR and Western blot, respectively, and confocal microscopy was employed to investigate subcellular localization. STC2 overexpression and silencing were induced to assess the effects of STC2 on high-phosphate-induced apoptosis, which was determined by caspase-3 levels and TUNEL staining. The anti-apoptotic effect of CaSR-induced STC2 was confirmed by interfering with STC2 expression in the presence of NPS R-568. RESULTS: The constitutive expression of STC2 was confirmed. STC2 mRNA and protein levels were increased by NPS R-568 with or without high phosphate. NPS-2143 resulted in decreased STC2 mRNA levels, but decreased STC2 protein levels were only found under the high-phosphate condition. Confocal microscopy demonstrated the colocalization of STC2 and plasma membrane or endoplasmic reticulum markers. STC2 overexpression reduced HAoSMCs apoptosis, which were reversed with STC2 silencing. NPS R-568 treatment reduced HAoSMCs apoptosis, but STC2 silencing abolished the protective effect. CONCLUSION: This is the first evidence that STC2 is regulated by CaSR in HAoSMCs. CaSR activation-induced STC2 has putative anti-apoptotic effects against high phosphate. Calcimimetics are promising agents to treat uremic vascular injury.

Requirement of Na+/H+ Exchanger NHE1 for Vasopressin-Induced Osteogenic Signaling and Calcification in Human Aortic Smooth Muscle Cells.

BACKGROUND/AIMS: Vasopressin is a powerful stimulator of vascular calcification, augmenting osteogenic signaling in vascular smooth muscle cells (VSMCs) including upregulation of transcription factors such as core-binding factor α-1 (CBFA1), msh homeobox 2 (MSX2), and SRY-Box 9 (SOX9), as well as of tissue-nonspecific alkaline phosphatase (ALPL). Vasopressin-induced osteogenic signaling and calcification require the serum- and glucocorticoid-inducible kinase 1 (SGK1). Known effects of SGK1 include upregulation of Na+/H+ exchanger 1 (NHE1). NHE1 further participates in the regulation of reactive oxygen species (ROS). NHE1 has been shown to participate in the orchestration of bone mineralization. The present study, thus, explored whether vasopressin modifies NHE1 expression and ROS generation, as well as whether pharmacological inhibition of NHE1 disrupts vasopressin-induced osteogenic signaling and calcification in VSMCs. METHODS: Human aortic smooth muscle cells (HAoSMCs) were treated with vasopressin in the absence or presence of SGK1 silencing, SGK1 inhibitor GSK-650394, and NHE1 blocker cariporide. Transcript levels were determined by using quantitative real-time polymerase chain reaction, protein abundance by Western blotting, ROS generation with 2',7'-dichlorofluorescein diacetate fluorescence, and ALP activity and calcium content by using colorimetric assays. RESULTS: Vasopressin significantly enhanced the NHE1 transcript and protein levels in HAoSMCs, effects significantly blunted by SGK1 inhibition with GSK-650394 or SGK1 silencing. Vasopressin increased ROS accumulation, an effect significantly blocked by the NHE1 inhibitor cariporide. Vasopressin further significantly increased osteogenic markers CBFA1, MSX2, SOX9, and ALPL transcript levels, as well as ALP activity and calcium content in HAoSMCs, all effects significantly blunted by SGK1 silencing or in the presence of GSK-650394 or cariporide. CONCLUSION: Vasopressin stimulates NHE1 expression and ROS generation, an effect dependent on SGK1 and required for vasopressin-induced stimulation of osteogenic signaling and calcification of VSMCs.

Inhibition of IL17A Using an Affibody Molecule Attenuates Inflammation in ApoE-Deficient Mice.

The balance between pro- and anti-inflammatory cytokines released by immune and non-immune cells plays a decisive role in the progression of atherosclerosis. Interleukin (IL)-17A has been shown to accelerate atherosclerosis. In this study, we investigated the effect on pro-inflammatory mediators and atherosclerosis development of an Affibody molecule that targets IL17A. Affibody molecule neutralizing IL17A, or sham were administered in vitro to human aortic smooth muscle cells (HAoSMCs) and murine NIH/3T3 fibroblasts and in vivo to atherosclerosis-prone, hyperlipidaemic ApoE-/- mice. Levels of mediators of inflammation and development of atherosclerosis were compared between treatments. Exposure of human smooth muscle cells and murine NIH/3T3 fibroblasts in vitro to αIL-17A Affibody molecule markedly reduced IL6 and CXCL1 release in supernatants compared with sham exposure. Treatment of ApoE-/- mice with αIL-17A Affibody molecule significantly reduced plasma protein levels of CXCL1, CCL2, CCL3, HGF, PDGFB, MAP2K6, QDPR, and splenocyte mRNA levels of Ccxl1, Il6, and Ccl20 compared with sham exposure. There was no significant difference in atherosclerosis burden between the groups. In conclusion, administration of αIL17A Affibody molecule reduced levels of pro-inflammatory mediators and attenuated inflammation in ApoE-/- mice.

Circular RNA circLMF1 regulates PDGF-BB-induced proliferation and migration of human aortic smooth muscle cells by regulating the miR-125a-3p/VEGFA or FGF1 axis.

Atherosclerosis is a major cause of cardiovascular disease, in which vascular smooth muscle cells (VSMCs) proliferation and migration play a vital role. Circular RNAs (circRNAs) have been reported to be correlated with the VSMCs function. Therefore, this study is designed to explore the role and mechanism of circRNA lipase maturation factor 1 (circLMF1) in Human aortic VSMCs (HASMCs). The microarray was used for detecting the expression of circLMF1 in proliferative and quiescent HASMCs. Levels of circLMF1, microRNA-125a-3p (miR-125a-3p), vascular endothelial growth factor A (VEGFA), and fibroblast growth factor 1 (FGF1) were determined by real-time quantitative polymerase chain reaction (RT-qPCR). Cell viability, cell cycle progression, and migration were assessed by Cell Counting Kit-8 (CCK-8), flow cytometry, wound healing, and transwell assays, respectively. Western blot assay determined proliferating cell nuclear antigen (PCNA), Cyclin D1, matrix metalloproteinase (MMP2), osteopontin (OPN), VEGFA, and FGF1 protein levels. The possible interactions between miR-125a-3p and circLMF1, and miR-125a-3p and VEGFA or FGF1 were predicted by circbank or targetscan, and then verified by a dual-luciferase reporter, RNA Immunoprecipitation (RIP), RNA pull-down assays. CircLMF1, VEGFA, and FGF1 were increased, and miR-125a-3p was decreased in platelet-derived growth factor-BB (PDGF-BB)-inducted HASMCs. Functionally, circLMF1 knockdown hindered cell viability, cell cycle progression, and migration in PDGF-BB-treated HASMCs. Mechanically, circLMF1 could regulate VEGFA or FGF1 expression through sponging miR-125a-3p. Our findings revealed that circLMF1 deficiency could inhibit cell viability, cell cycle progression, and migration of PDGF-BB stimulated atherosclerosis model partly through the miR-125a-3p/VEGFA or FGF1 axis, suggesting that targeting circLMF1 can be a feasible therapeutic strategy for atherosclerosis.

Effect of fibulin-5 on aldosterone-induced apoptosis in human ascending aortic smooth muscle cells.

The aim of the current study was to investigate the effect of aldosterone on apoptosis in human aortic smooth muscle cells (HA-VSMC) and to determine the role of fibulin-5 in the aldosterone-induced apoptosis of HA-VSMC cells. Through the construction of a fibulin-5 eukaryotic overexpression vector and a short hairpin RNA interference plasmid, fibulin-5 was overexpressed and silenced, respectively. The role of fibulin-5 in the aldosterone-induced apoptosis of HA-VSMC was subsequently determined. The overexpression of fibulin-5 inhibited the apoptosis of cells, particularly at low concentrations of aldosterone; a smaller effect on apoptosis was induced by high concentrations of aldosterone. fibulin-5 knockdown promoted the apoptosis of cells induced by high concentrations of aldosterone but had a smaller effect on the apoptosis of cells induced by low concentrations of aldosterone. Therefore, the results of the current study indicate that fibulin-5 inhibits the aldosterone-induced apoptosis of HA-VSMC cells and that this effect may be altered by changing the aldosterone concentration.

Silencing a disintegrin and metalloproteinase­33 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 metalloproteinase­33 (ADAM33) serves a key role in the pathogenesis of airway remodeling­related 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 Kit­8 method. Cellular apoptosis was evaluated with Annexin V­PE/7­AAD staining and flow cytometry. Reverse transcription­quantitative 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 Bcl­2, 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/Bcl­2 pathways. These findings suggested an important role of ADAM33 in airway vascular remodeling and potential therapeutic significance of ADAM33 inhibition in airway remodeling­related diseases.

Avenanthramide C Suppresses Matrix Metalloproteinase-9 Expression and Migration Through the MAPK/NF- κB Signaling Pathway in TNF-α-Activated HASMC Cells.

In oat ingredients, flavonoids and phenolic acids are known to be the most important phenolic compounds. In phenolic compounds, wide-ranging biological responses, including antioxidative, anti-inflammatory, anti-allergic, and anti-cancer properties, were reported. Avenanthramide C (Avn C), a component of the phenolic compound of oats, has been reported to be highly antioxidant and anti-inflammatory, but its role in an anti-atherosclerosis response is unknown. The aim of this research was to assess the effect of Avn C on expression of MMP-9 on TNF-α-activated human arterial smooth-muscle cells (HASMC) and signaling involved in its anti-atherosclerosis activity. HASMC cells are known to produce inflammatory cytokines involving IL-6, IL-1ß, and TNF-α during arteriosclerosis activity. Avn C specifically reduced IL-6 secretion in HASMC cells. Furthermore, we investigated whether Avn C could inhibit NF-κB nuclear protein translocation. Avn C suppressed nuclear protein translocation of NF-κB in TNF-α-stimulated HASMCs. The MMP-9 enzyme activity and expression are controlled through the MAPKs signaling path during the Avn C treatment. We confirmed that the levels of wound healing (p-value = 0.013, *p < 0.05) and migration (p-value = 0.007, **p < 0.01) are inhibited by 100 ng/ml TNF-α and 100 µM Avn C co-treated. Accordingly, Avn C inhibited the expression of MMP-9 and cell migration through the MAPK/NF-κB signaling pathway in TNF-α-activated HASMC. Therefore, Avn C can be identified and serve as disease prevention material and remedy for atherosclerosis.

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.

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.

Indoxyl sulfate promotes osteogenic differentiation of vascular smooth muscle cells by miR-155-5p-dependent downregulation of matrix Gla protein via ROS/NF-κB signaling.

Vascular calcification (VC) is a major risk factor for increasing cardiovascular morbidity and mortality in patients with chronic kidney disease (CKD). Indoxyl sulfate (IS), a representative uremic toxin, is closely associated with VC in CKD patients. Matrix Gla protein (MGP) plays pivotal role in VC as a calcification inhibitor. The aim of this work was to explore whether MGP was involved in IS-induced VC. Here, we demonstrated the role of MGP in the IS-induced osteogenic differentiation of human aortic smooth muscle cells (HASMCs). The methods included Von Kossa staining, immunohistochemistry, Alizarin Red staining, quantitative real-time PCR and western blotting. MGP was decreased in calcified arteries both in CKD patients and rats. In vitro, IS suppressed MGP expression in HASMCs by activating ROS/NF-κB signaling in parallel with osteogenic differentiation, which was mitigated by inhibiting ROS and NF-κB with diphenyleneiodonium and Bay11-7082. Further investigation showed that IS induced NF-κB-responsive microRNA (miR)-155-5p mediating MGP downregulation. Overexpression of miR-155-5p with mimics aggravated IS-induced MGP reduction and osteogenic differentiation. In contrast, these conditions were diminished by silencing miR-155-5p. We demonstrate that IS promotes the HASMCs phenotype switch by suppressing MGP expression via ROS/NF-κB/miR-155-5p signaling and provide a new insight for the pathogenesis of IS-induced VC.

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.

FOLFIRI-Mediated Toxicity in Human Aortic Smooth Muscle Cells and Possible Amelioration with Curcumin and Quercetin.

Systemic chemotherapy-mediated cell toxicity is a major risk factor for cardiovascular disease and atherosclerosis. Life-threatening acute events of the FOLFIRI (irinotecan, folinic acid and 5-fluorouracil) regimen are mainly due to DNA damage induced by antimetabolite and topoisomerase inhibition effects. However, the role of human aortic smooth muscle cells (HaVSMCs) in this process and the mechanisms of oxidative stress, DNA and protein damage and apoptosis have not been investigated. Therefore, the effects of curcumin and quercetin on HaVSMC survival in the generation of molecular and cellular toxicity by FOLFIRI treatment and the involvement of vital cellular signalling pathways were investigated. We analysed both FOLFIRI toxicity and the therapeutic potential of quercetin and curcumin in terms of HaVSMC damage using molecular probe and florescence staining, Random Amplified Polymorphic DNA (RAPD), qRT-PCR and Western blot assays. Our study presents two preliminary findings: (a) in HaVSMCs, FOLFIRI treatment significantly induces oxidative damage to both DNA and protein, leading to a dramatic increase in caspase-dependent apoptotic death through P53-mediated Caspase3-dependent mitochondrial apoptosis, and results in TNF-α/Caspase8-mediated necrotic death, and (b) flavonoids not only regulate the expression of genes encoding antioxidant enzymes and increase DNA damage but also limit programmed and necrotic cell death processes in HaVSMCs. Our results clearly indicate the potential for curcumin and, particularly, quercetin as preventative chemotherapeutic interventions for cardiovascular toxicity induced by the FOLFIRI regime in HaVSMCs.

Uremic serum-induced calcification of human aortic smooth muscle cells is a regulated process involving Klotho and RUNX2.

Vascular calcification (VC) is common in subjects with chronic kidney disease (CKD) and is associated with increased cardiovascular risk. It is an active process involving transdifferentiation of arterial smooth muscle cells (SMCs) into osteogenic phenotype. We investigated the ability of serum from CKD subjects to induce calcification in human SMCs in vitro (calcific potential of sera: CP), and associated changes in expression of Runt-related transcription factor 2 (RUNX2), SM22α, and Klotho. Sera from subjects with CKD (18 stage 3, 17 stage 4/5, and 29 stage 5D) and 20 controls were added to human cultured SMCs and CP quantified. The CP of CKD sera was greater (P<0.01) than that of controls, though not influenced by CKD stage. Modification of diet in renal disease estimated glomerular filtration rate (MDRD-4 eGFR) (P<0.001), serum phosphate (P=0.042), receptor activator of nuclear factor κappa-B ligand (RANKL) (P=0.001), parathyroid hormone (PTH) (P=0.014), and high-density lipoprotein (HDL)/cholesterol ratio (P=0.026) were independent predictors of CP accounting for 45% of variation. Adding calcification buffer (CB: calcium chloride [7 mM] and ß-glycerophosphate [7 mM]) increased the CP of control sera to approximate that of CKD sera. CP of CKD sera was unchanged. CKD sera increased RUNX2 expression (P<0.01) in human SMCs and decreased SM22α expression (P<0.05). Co-incubating control but not CKD serum with CB further increased RUNX2 expression (P<0.01). Both SM22α and Klotho expression decreased significantly (P<0.01) in the presence of CKD serum, and were virtually abolished with stage 5D sera. These findings support active regulation by CKD serum of in vitro VC by induction of RUNX2 and suppression of SM22α and Klotho.

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.

MicroRNA-99a-5p alleviates atherosclerosis via regulating Homeobox A1.

AIMS: MicroRNAs have been demonstrated to be involved in the development of atherosclerosis. The present study aimed to evaluate the effect of miR-99a-5p and its target gene Homeobox A1 (HOXA1) in atherosclerosis. MAIN METHODS: The biological functions of miR-99a-5p on human aortic smooth muscle cells (ASMCs) were assessed by MTT, wound healing and transwell assays. The target genes of microRNAs were predicted by TargetScan and miRDB. The binding of miR-99a-5p and HOXA1 was confirmed by luciferase reporter assay. In the in vivo study, high-fat diet-induced atherosclerosis model was established in Apolipoprotein E knockout mice. Hematoxylin-eosin (H&E), oil Red O and Masson trichrome staining were performed for determination of atherosclerotic lesion. The levels of miR-99a-5p and HOXA1 mRNA were detected by real-time PCR. HOXA1 and migration-associated protein levels were detected by western blot or immunohistochemistry analysis. KEY FINDINGS: MiR-99a-5p inhibited HOXA1 expression by targeting 3'UTR of HOXA1 mRNA. Enforced HOXA1 significantly promoted the proliferation, migration, and invasion of ASMCs. Furthermore, miR-99a-5p overexpression inhibited the proliferation, migration, and invasion of ASMCs stimulated by HOXA1, whereas miR-99a-5p inhibition reversed the effects of HOXA1 knockdown on these behaviours of ASMCs. In vivo, the specific overexpression of miR-99a-5p significantly abated atherosclerotic lesions formatted, accompanied with a significant down-regulation of HOXA1 mRNA and protein expression levels. SIGNIFICANCE: We demonstrate for first time that miR-99a-5p may serve as a potential inhibitor of the atherosclerosis, and miR-99a-5p plays its role partially through targeting HOXA1.