Aortic Smooth Muscle Cell Line Research Articles

Fluoride impairs vascular smooth muscle A7R5 cell lines via disrupting amino acids metabolism

Given the insidious and high-fatality nature of cardiovascular diseases (CVDs), the emergence of fluoride as a newly identified risk factor demands serious consideration alongside traditional risk factors. While vascular smooth muscle cells (VSMCs) play a pivotal role in the progression of CVDs, the toxicological impact of fluoride on VSMCs remains largely uncharted. In this study, we constructed fluorosis model in SD rats and A7R5 aortic smooth muscle cell lines to confirm fluoride impaired VSMCs. Fluoride aggravated the pathological damage of rat aorta in vivo. Then A7R5 were exposed to fluoride with concentration ranging from 0 to 1200 μmol/L over a 24-h period, revealing a dose-dependent inhibition of cell proliferation and migration. The further metabolomic analysis showed alterations in metabolite profiles induced by fluoride exposure, notably decreasing organic acids and lipid molecules level. Additionally, gene network analysis underscored the frequency of fluoride's interference with amino acids metabolism, potentially impacting the tricarboxylic acid (TCA) cycle. Our results also highlighted the ATP-binding cassette (ABC) transporters pathway as a central element in VSMC impairment. Moreover, we observed a dose-dependent increase in osteopontin (OPN) and α-smooth muscle actin (α-SMA) mRNA level and a dose-dependent decrease in ABC subfamily C member 1 (ABCC1) and bestrophin 1 (BEST1) mRNA level. These findings advance our understanding of fluoride as a CVD risk factor and its influence on VSMCs and metabolic pathways, warranting further investigation into this emerging risk factor.

Molecular Mechanism of Calycosin Inhibited Vascular Calcification.

Vascular calcification (VC) is a pathological condition frequently observed in cardiovascular diseases. Primary factors contributing to VC are osteogenic differentiation of vascular smooth muscle and hydroxyapatite deposition. Targeted autophagy (a lysosome-mediated mechanism for degradation/recycling of unnecessary cellular components) is a useful approach for inhibiting VC and promoting vascular cell health. Calycosin has been shown to alleviate atherosclerosis by enhancing macrophage autophagy, but its therapeutic effect on VC has not been demonstrated. Using an in vitro model (rat thoracic aortic smooth muscle cell line A7r5), we demonstrated effective inhibition of VC using calycosin (the primary flavonoid component of astragalus), based on the enhancement of autophagic flux. Calycosin treatment activated AMPK/mTOR signaling to induce initiation of autophagy and restored mTORC1-dependent autophagosome-lysosome fusion in late-stage autophagy by promoting soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation, thereby preventing stoppage of autophagy in calcified cells. Calycosin substantially reduced degrees of both osteogenic differentiation and calcium deposition in our VC cell model by enhancing autophagy. The present findings clarify the mechanism whereby calycosin mitigates autophagy stoppage in calcified smooth muscle cells and provide a basis for effective VC treatment via autophagy enhancement.

The mechanosensitive Piezo1 channels contribute to the arterial medial calcification.

Vascular calcification (VC) is associated with a number of cardiovascular diseases, as well as chronic kidney disease. The role of smooth muscle cells (SMC) has already been widely explored in VC, as has the role of intracellular Ca2+ in regulating SMC function. Increased intracellular calcium concentration ([Ca2+]i) in vascular SMC has been proposed to stimulate VC. However, the contribution of the non-selective Piezo1 mechanosensitive cation channels to the elevation of [Ca2+]i, and consequently to the process of VC has never been examined. In this work the essential contribution of Piezo1 channels to arterial medial calcification is demonstrated. The presence of Piezo1 was proved on human aortic smooth muscle samples using immunohistochemistry. Quantitative PCR and Western blot analysis confirmed the expression of the channel on the human aortic smooth muscle cell line (HAoSMC). Functional measurements were done on HAoSMC under control and calcifying condition. Calcification was induced by supplementing the growth medium with inorganic phosphate (1.5mmol/L, pH 7.4) and calcium (CaCl2, 0.6mmol/L) for 7days. Measurement of [Ca2+]i using fluorescent Fura-2 dye upon stimulation of Piezo1 channels (either by hypoosmolarity, or Yoda1) demonstrated significantly higher calcium transients in calcified as compared to control HAoSMCs. The expression of mechanosensitive Piezo1 channel is augmented in calcified arterial SMCs leading to a higher calcium influx upon stimulation. Activation of the channel by Yoda1 (10μmol/L) enhanced calcification of HAoSMCs, while Dooku1, which antagonizes the effect of Yoda1, reduced this amplification. Application of Dooku1 alone inhibited the calcification. Knockdown of Piezo1 by siRNA suppressed the calcification evoked by Yoda1 under calcifying conditions. Our results demonstrate the pivotal role of Piezo1 channels in arterial medial calcification.

Ginsenoside Rg-1 prevents elevated cytosolic Ca2+ via store-operated Ca2+ entry in high-glucose–stimulated vascular endothelial and smooth muscle cells

BackgroundGinsenoside Rg-1 (Rg-1), a triterpenoid saponin abundantly present in Panax ginseng, is a type of naturally occurring steroid with known anti-diabetic and anti-inflammatory effects. In this study, we sought to confirm the effects and mechanisms of action of Rg-1 on store-operated Ca2+ entry (SOCE) in human vascular endothelial cell line (EA) and murine aortic vascular smooth muscle cell line (MOVAS) cells exposed to high glucose.MethodsCytosolic Ca2+ concentrations in EA and MOVAS cells were measured by monitoring fluorescence of the ratiometric Ca2+-indicator, Fura-2 AM.ResultsHigh glucose significantly increased Ca2+ influx by abnormally activating SOCE in EA and MOVAS cells. Notably, this high glucose-induced increase in SOCE was restored to normal levels in EA and MOVAS cells by Rg-1. Moreover, Rg-1 induced reductions in SOCE in cells exposed to high glucose were significantly inhibited by the plasma membrane Ca2+ ATPase (PMCA) blocker lanthanum, the Na+/K+-ATPase blocker ouabain, or the Na+/Ca2+ exchanger (NCX) blockers Ni2+ and KB-R7943. These observations suggest that the mechanism of action of Rg-1 inhibition of SOCE involves PMCA and Na+/K+-ATPase, and an increase in Ca2+ efflux via NCXs in both EA and MOVAS cells exposed to high glucose.ConclusionsThese findings indicate that Rg-1 may protect vascular endothelial and smooth muscle cells from Ca2+ increases following exposure to hyperglycemic conditions.

Promotional effects of HIF1α and KDM3A interaction on vascular smooth muscle cells in thoracic aortic dissection.

Aim: The current study was performed to define the role of KDM3A in thoracic aortic dissection (TAD). Methods: The binding of HIF1α andKDM3A in HES1 was detected by ChIP and dual-luciferase reporter gene assay. Loss and gain-of function assays of HIF1α, KDM3A and HES1 were further performed in Ang-II-induced mouse aortic smooth muscle cell line(MOVAS) cells. Lastly, in vivo TAD models were established. Results: HIF1α was highly expressed in TAD. KDM3A promoted the transcription activation of HES1. HIF1α enhanced the proliferation and migration of Ang-II-induced MOVAS cells, in addition to increasing thoracic aorta dilation to induce TAD formation in vivo. Silencing of HES1 reversed the effects of HIF1α in vivo and in vitro. Conclusion: The findings indicated that interaction between HIF1α and KDM3A enhances the proliferation and migration of MOVAS cells to induce TAD.

Characterization of murine cytomegalovirus infection and induction of calcification in Murine Aortic Vascular Smooth Muscle Cells (MOVAS)

Human cytomegalovirus (HCMV) is a widespread pathogen that causes lifelong latent infection in the majority of the world population. HCMV is associated with increased incidence and severity of many cardiovascular diseases including myocarditis, atherosclerosis, and transplant vasculopathy. Due to the species-restricted nature of cytomegalovirus infection, murine cytomegalovirus (MCMV) is a useful model that recapitulates many of the features of HCMV infection of the cardiovascular system. While in vivo MCMV studies are able to answer many questions regarding pathogenesis of infection, in vitro experiments using cell lines are useful tools to further understand the potential underlying mechanisms. In this study, we characterize MCMV infection of the murine aortic smooth muscle cell line (MOVAS). Our findings demonstrate that MOVAS cells are permissive for MCMV infection, form plaques under carboxymethyl cellulose overlay, and produce progeny virus similar to NIH 3T3 murine embryonic fibroblasts. In addition, MCMV infection induces calcification in MOVAS cells similar to that seen in the epicardium of MCMV-infected hearts. We conclude that MOVAS cells are a useful in vitro tool for studying CMV-mediated cardiac calcification.

Curcumin-mediated Photodynamic Therapy Inhibits the Phenotypic Transformation, Migration, and Foaming of Oxidized Low-density Lipoprotein-treated Vascular Smooth Muscle Cells by Promoting Autophagy

Vascular smooth muscle cells (VSMCs) are becoming a hot spot and target of atherosclerosis research. This study aimed to observe the specific effects of curcumin (CUR)-mediated photodynamic therapy (CUR-PDT) on oxidized low-density lipoprotein (ox-LDL)-treated VSMCs and confirm whether these effects are mediated by autophagy. In this study, the mouse aortic smooth muscle cell line and A7r5 cell lines were used for parallel experiments. VSMC viability was evaluated by Cell Counting Kit-8 assay. VSMCs were treated with ox-LDL to establish a model of atherosclerosis in vitro. The autophagy level and the expression of proteins related to phenotypic transformation were detected by western blotting. The migration ability of the cells was detected by using transwell assay. The presence of intracellular lipid droplets was detected by Oil Red O staining. The results showed that VSMCs transformed from the contraction phenotype to the synthetic phenotype when stimulated by ox-LDL, during which autophagy was inhibited. However, CUR-PDT treatment significantly promoted the level of autophagy and inhibited the process of phenotypic transformation induced by ox-LDL. In addition, ox-LDL significantly promoted VSMC migration and increased the number of lipid droplets, whereas CUR-PDT treatment significantly reduced the ox-LDL-induced increase in the migration ability of, and lipid droplet numbers in, VSMCs. When the VSMCs were pretreated with the autophagy inhibitor 3-methyladenine for 24 hours, the effects of CUR-PDT were reversed. Therefore, our study indicated that CUR-PDT can inhibit the phenotypic transformation, migration, and foaming of ox-LDL-treated VSMCs by inducing autophagy.

Hydrolysis of Extracellular ATP by Vascular Smooth Muscle Cells Transdifferentiated into Chondrocytes Generates Pi but Not PPi.

(1) Background: Tissue non-specific alkaline phosphatase (TNAP) is suspected to induce atherosclerosis plaque calcification. TNAP, during physiological mineralization, hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PPi). Since atherosclerosis plaques are characterized by the presence of necrotic cells that probably release supraphysiological concentrations of ATP, we explored whether this extracellular adenosine triphosphate (ATP) is hydrolyzed into the mineralization inhibitor PPi or the mineralization stimulator inorganic phosphate (Pi), and whether TNAP is involved. (2) Methods: Murine aortic smooth muscle cell line (MOVAS cells) were transdifferentiated into chondrocyte-like cells in calcifying medium, containing ascorbic acid and β-glycerophosphate. ATP hydrolysis rates were determined in extracellular medium extracted from MOVAS cultures during their transdifferentiation, using 31P-NMR and IR spectroscopy. (3) Results: ATP and PPi hydrolysis by MOVAS cells increased during transdifferentiation. ATP hydrolysis was sequential, yielding adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine without any detectable PPi. The addition of levamisole partially inhibited ATP hydrolysis, indicating that TNAP and other types of ectonucleoside triphoshatediphosphohydrolases contributed to ATP hydrolysis. (4) Conclusions: Our findings suggest that high ATP levels released by cells in proximity to vascular smooth muscle cells (VSMCs) in atherosclerosis plaques generate Pi and not PPi, which may exacerbate plaque calcification.

Cyclin G2 promotes the formation of smooth muscle cells derived foam cells in atherosclerosis via PP2A/NF-κB/LOX-1 pathway.

BackgroundTo investigate the role and underlying mechanism of cyclin G2 (G2-type cyclin) in the formation of vascular smooth muscle cells (VSMCs) derived foam cells.MethodsThe levels of α-SMA (alpha-SM-actin), p-NF-κB (phosphorylation nuclear transcription factors kappa B), and LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) were measured by immunohistochemistry and western blotting. The mouse aortic root smooth muscle cell line MOVAS was transfected to over-express cyclin G2, which were then stimulated with 80 µg/mL ox-LDL (oxidized low-density lipoprotein) to induce foam cell formation. DT-061 an activator of PP2A (protein phosphatase 2A) agonist was used to verify the role of PP2A in the process.ResultsKnocking out the Ccng2 gene in Apoe-/- mice alleviated aortic lipid plaque, foam cell formulation, ameliorative body weight, and LDL-cholesterol. We observed that the number of α-SMA positive cells was significantly decreased in Apoe-/-Ccng2-/- mice compared to Apoe-/- mice. Also, the protein levels of p-NF-κB and LOX-1 were markedly reduced in the aortic root of Apoe-/-Ccng2-/- mice. Upon stimulation with ox-LDL, upregulated cyclin G2 increased the intracellular lipid accumulation in MOVAS cells. Also, it suppressed the activity of PP2A but up-regulated LOX-1. Additionally, the cell nuclear translocation of p-NF-κB was increased. Interestingly, DT-061 intervention, re-activating the activity of PP2A, reduced the levels of nuclear p-NF-κB and LOX-1. This led to decreased lipid endocytosis reducing the formation of VSMCs- derived foam cells.ConclusionsCyclin G2 increases the nuclear translocation of p-NF-κB by reducing the enzymatic activity of PP2A and upregulating LOX-1, thereby promotes the formation of VSMCs -derived foam cells in atherosclerosis.

Oxidized Low-Density Lipoprotein Induces WNT5A Signaling Activation in THP-1 Derived Macrophages and a Human Aortic Vascular Smooth Muscle Cell Line.

The pathogenesis of atherosclerosis is complex, evolves, and involves many cell types. Macrophages and vascular smooth muscle cells (VSMCs) are critically involved in atherosclerosis development and progression. Several studies have shown that WNT5A protein is abundantly expressed in human atherosclerotic lesions; however, the mechanism and role of WNT signaling pathway activation is not clearly known. Using THP-1 derived macrophages, and human aortic VSMC cells, we evaluated in vitro how oxidized low-density lipoprotein (oxLDL) and WNT5A signaling interact in these two cell lines. We used western blot, scratch assay, metabolic proliferation assay, as well as immunostaining to analyze the effect of Wnt signaling activation. The results demonstrated that oxLDL, as well as WNT5A (control), induced Disheveled-2 (DVL2) activation and Kif26b degradation, indicating activation of non-canonical Wnt signaling. We found that oxLDL and WNT5A induced FZD5-ROR2 co-localization at the cellular membrane in vitro in THP-1 derived macrophages. Box5 (FZD5 receptor antagonist) inhibited oxLDL-induced DVL2/JNK activation secondary to newly secreted WNT protein from THP-1 derived macrophages. We found that WNT3A (canonical Wnt) and WNT5A showed different roles in this VSMC cell line. These findings indicate that WNT5A is upregulated by oxLDL, promotes foam cell formation, and affects VSMC phenotype and migration in these two cell lines. Also, in these cell lines FZD5 signaling seems to be necessary for lipid accumulation and, through this mechanism, WNT5A could modulate foam cell formation. Thus, our results suggest that WNT5A may contribute to the pathogenesis of vascular disease through modulating macrophage and VSMC behavior.

On the biocompatibility of graphene oxide towards vascular smooth muscle cells

Graphene and its derivatives have shown fascinating potential in biomedical applications. However, the biocompatibility of graphene with vascular smooth muscle cells (VSMCs) and applications to vascular engineering have not been explored extensively. Using a rat aortic smooth muscle cell line, A7r5, as a VSMC model, we have explored the effects of graphene oxide (GO) on the growth and behaviours of VSMCs. Results demonstrated that GO had no obvious toxicity to VSMCs. Cells cultured on GO retained the expression of smooth muscle cell-specific markers CNN1, ACTA2 and SMTN, on both mRNA and protein levels. A wound healing assay demonstrated no effect of GO on cell migration. We also found that small-flaked GO favoured the proliferation of VSMCs, suggesting a potential of using surface chemistry or physical properties of GO to influence cell growth behaviour. These results provide insight into the suitability of GO as a scaffold for vascular tissue engineering.

The Novel gem-Dihydroperoxide 12AC3O Suppresses High Phosphate-Induced Calcification via Antioxidant Effects in p53LMAco1 Smooth Muscle Cells.

The excessive intake of phosphate (Pi), or chronic kidney disease (CKD), can cause hyperphosphatemia and eventually lead to ectopic calcification, resulting in cerebrovascular diseases. It has been reported that reactive oxygen species (ROS), induced by high concentrations of Pi loading, play a key role in vascular calcification. Therefore, ROS suppression may be a useful treatment strategy for vascular calcification. 12AC3O is a newly synthesized gem-dihydroperoxide (DHP) that has potent antioxidant effects. In the present study, we investigated whether 12AC3O inhibited vascular calcification via its antioxidative capacity. To examine whether 12AC3O prevents vascular calcification under high Pi conditions, we performed Alizarin red and von Kossa staining, using the mouse aortic smooth muscle cell line p53LMAco1. Additionally, the effect of 12AC3O against oxidative stress, induced by high concentrations of Pi loading, was investigated using redox- sensitive dyes. Further, the direct trapping effect of 12AC3O on reactive oxygen species (ROS) was investigated by ESR analysis. Although high concentrations of Pi loading exacerbated vascular smooth muscle calcification, calcium deposition was suppressed by the treatment of both antioxidants and 12AC3O, suggesting that the suppression of ROS may be a candidate therapeutic approach for treating vascular calcification induced by high concentrations of Pi loading. Importantly, 12AC3O also attenuated oxidative stress. Furthermore, 12AC3O directly trapped superoxide anion and hydroxyl radical. These results suggest that ROS are closely involved in high concentrations of Pi-induced vascular calcification and that 12AC3O inhibits vascular calcification by directly trapping ROS.

FNDC5 Attenuates Oxidative Stress and NLRP3 Inflammasome Activation in Vascular Smooth Muscle Cells via Activating the AMPK-SIRT1 Signal Pathway.

Vascular oxidative stress and inflammation play a major role in vascular diseases. This study was aimed at determining the protective roles of fibronectin type III domain-containing 5 (FNDC5) in angiotensin II- (Ang II-) induced vascular oxidative stress and inflammation and underlying mechanisms. Wild-type (WT) and FNDC5−/− mice, primary mouse vascular smooth muscle cells (VSMCs), and the rat aortic smooth muscle cell line (A7R5) were used in the present study. Subcutaneous infusion of Ang II caused more serious hypertension, vascular remodeling, oxidative stress, NLRP3 inflammasome activation, AMPK phosphorylation inhibition, and SIRT1 downregulation in the aorta of FNDC5−/− mice than those of WT mice. Exogenous FNDC5 attenuated Ang II-induced superoxide generation, NADPH oxidase 2 (NOX2) and NLRP3 upregulation, mature caspase-1, and interleukin-1β (IL-1β) production in A7R5 cells. The protective roles of FNDC5 were prevented by SIRT-1 inhibitor EX527, AMPK inhibitor compound C, or integrin receptor inhibitor GLPG0187. FNDC5 attenuated the Ang II-induced inhibition in SIRT1 activity, SIRT1 protein expression, and AMPKα phosphorylation in A7R5 cells, which were prevented by compound C, EX527, and GLPG0187. FNDC5 deficiency deteriorated Ang II-induced oxidative stress, NLRP3 inflammasome activation, AMPK phosphorylation inhibition, and SIRT1 downregulation in primary aortic VSMCs of mice, which were prevented by exogenous FNDC5. These results indicate that FNDC5 deficiency aggravates while exogenous FNDC5 alleviates the Ang II-induced vascular oxidative stress and NLRP3 inflammasome activation via the AMPK-SIRT1 signal pathway in VSMCs.

Lymphotoxin beta receptor is associated with regulation of microRNAs expression and nuclear factor-kappa B activation in lipopolysaccharides (LPS)-stimulated vascular smooth muscle cells.

The aim of present study is to investigate the role of lymphotoxin beta receptor (Ltβr) in lipopolysaccharides (LPS)-induced inflammation in vascular smooth muscle cells (VSMCs) and whether its effects are mediated by modulating microRNAs (miRNAs) and nuclear factor-kappa B (NF-κB). Mouse aortic smooth muscle cell (SMC) line (MOVAS cells) were transduced with short hairpin Ltβr (shLtβr) and mRNA and protein expression level of Ltβr were measured by qPCR and Western blot in shLtβr-transduced cells. Lentiviral vector-transduced (control) and lentiviral vector/shLtβr-transduced MOVAS cells were stimulated with LPS (1 μg/mL) for 0, 16, or 24 h. Then the mRNA and protein levels of Ltβr, interleukin-18 (IL-18), p-p65, p65 and vascular cell adhesion molecule 1 (VCAM-1) were measured by real-time quantitative polymerase chain reaction (qPCR), Western blot and enzyme-linked immunosorbent assay (ELISA). Different miRNAs expression in LPS-stimulated normal and shLtβr-transduced cells were detected by small RNA sequencing (smRNA-seq). The mRNA and protein expression of Ltβr was significantly downregulated in shLtβr-transduced cells. LPS-increased the mRNA and protein levels of Ltβr, IL-18, p-p65 and VCAM-1 in were attenuated by shLtβr transducing compared with LPS-stimulated control group. Moreover, LPS treatment induced 10 upregulated and 64 downregulated miRNAs in shLtβr-transduced cells compared with control cells. Moreover, miR-146b-5p and miR-27a-5p levels were significantly decreased in shLtβr-transduced cells. Our results show for the first time that the role of Ltβr in regulating inflammatory response in LPS-stimulated VSMCs via modulating miRNAs and NF-κB pathway. Our findings might provide valuable information with respect to better understanding in the treatment of cardiovascular diseases, such as atherosclerosis.

Purification and identification of angiotensin II type I receptor downregulating peptide from egg white hydrolysate.

The vasoconstricting effect of angiotensin II (Ang II) is mediated by Ang II type I receptor (AT1R); blocking or blunting AT1R is a key strategy for the development of antihypertensive drugs. Our previous study showed that the blood pressure lowering the activity of egg white hydrolysate (EWH) in spontaneously hypertensive rats was due to the downregulation of aortic AT1R expression, which promoted us to further identify the AT1R downregulating peptides in the present study. The protein expression of AT1R in a rat aortic vascular smooth muscle cell line A7r5 cells was used for the activity test. The hydrolysate was fractionated stepwise by C18 Sep-Pack cartridge and reverse-phase chromatography, and the peptide sequences were characterized by LC-MS/MS. Peptides including ITKPNDVYS, VVGSAEAGVDAAS, AVHAAHAEINEAGRE, AGREVVGSAEAGVD, and VVGSAEAGVD, were identified. ITKPNDVYS showed the most potent peptide for lowering the level of AT1R in A7r5 cells. These results suggested that ITKPNDVYS is the responsible peptide for lowering the AT1R level in EWH. PRACTICAL APPLICATIONS: A new peptide ITKPNDVYS was identified as a downregulator of AT1R from egg white hydrolysate, which provides a new approach for the development of food protein-derived antihypertensive peptides as nutraceuticals or functional food ingredients.

Metformin inhibits aortic atherosclerosis in mice by regulating actin skeleton in vascular smooth muscle cells

ObjectiveInvestigate the effect and mechanism of metformin on the development of metabolic syndrome related atherosclerosis.MethodTransfecting EGFP-CLIP170 or EGFP-Pdlim5 plasmid to the mouse aortic smooth muscle cell line, to test the expression of p-AMPK, pCLIP-170 and pPdlim5, and observe the microtubule or the actin skeleton system by immunofluorescence staining. Scratch the cells to perform wound healing experiment, stimulating the cells with gradient metformin (0, 0.5, 1 mmol/L) for 8 h, and observe the change of the scratch size and the dynamic change of cell skeleton and migration in vitro. ApoE-/- mice were injected with streptozotocin and followed by 8 weeks of high fat diet to induce metabolic syndrome model. In the therapeutic group, mice were treated metformin (Met) instead of saline in control group (Control, CTL group). In the end, the whole aorta and its root were isolated and performed oil red O staining and immol/Lunostaining of α-SMA to evaluate the migration of smooth muscle cells and the accumulation of lipids in the aorta.ResultsMouse aortic smooth muscle cells showed an enhanced stress fiber and focal adhesion which representing the dynamic change of actin skeleton after Met stimulation, while the tubulin system rarely showed any change to Met. In animal model, The staining of α-SMA showed smooth muscle cells migrated to the intima or even to the lipid area from the media of aorta in CTL group compared to the Met group. Oil red O staining showed a reduced accumulation of lipids in the Met group than the controls (P < 0.05).ConclusionMetformin reduces the formation of atherosclerosis by inhibiting the migration of smooth muscle cells through modulating cellular actin skeleton system in mice.

Electronegative low-density lipoprotein of patients with metabolic syndrome induces pathogenesis of aorta through disruption of the stimulated by retinoic acid6 cascade.

Aims/IntroductionElectronegative low‐density lipoprotein (L5) is the most atherogenic fraction of low‐density lipoprotein and is elevated in people with metabolic syndrome (MetS), whereas the retinol‐binding protein 4 receptor (stimulated by retinoic acid 6 [STRA6]) cascade is disrupted in various organs of patients with obesity‐related diseases. Our objective was to investigate whether L5 from MetS patients capably induces pathogenesis of aorta through disrupting the STRA6 cascade.Material and MethodsWe examined the in vivo and in vitro effects of L5 on the STRA6 cascade and aortic atherogenic markers. To investigate the role of this cascade on atherosclerotic formation, crbp1 transfection was carried out in vitro.ResultsThis study shows that L5 activates atherogenic markers (p38 mitogen‐activated protein kinases, pSmad2 and matrix metallopeptidase 9) and simultaneously suppresses STRA6 signals (STRA6, cellular retinol‐binding protein 1, lecithin‐retinol acyltransferase, retinoic acid receptor‐α and retinoid X receptor‐α) in aortas of L5‐injected mice and L5‐treated human aortic endothelial cell lines and human aortic smooth muscle cell lines. These L5‐induced changes of the STRA6 cascade and atherogenic markers were reversed in aortas of LOX1−/− mice and in LOX1 ribonucleic acid‐silenced human aortic endothelial cell lines and human aortic smooth muscle cell lines. Furthermore, crbp1 gene transfection reversed the disruption of the STRA6 cascade, the phosphorylation of p38 mitogen‐activated protein kinases and Smad2, and the elevation of matrix metallopeptidase 9 in L5‐treated human aortic endothelial cell lines.ConclusionsThis study shows that L5 from MetS patients induces atherogenic markers by disrupting STRA6 signaling. Suppression of STRA6 might be one novel pathogenesis of aorta in patients with MetS.

Genotoxic and Apoptotic Effects of Heavy Metal Mixture on Human Aortic Vascular Smooth Muscle Cell Line

Objective: Heavy metals are the most common pollutants with specific gravity greater than 5 g cm-3. Human exposure to heavy metals occurs mostly through the consumption of food and water containing heavy metals. Metals that enter the body via drinking water may cause genotoxicity and various diseases. Therefore, permissible limits of heavy metals in water for human consumption have been determined by laws and regulations. Our study aimed to investigate the effects of a mix of metals (Cu, Zn, Pb and Fe) at permissible limits in drinking water on aortic smooth muscle cells (TG-HA-VSMC) with at molecular level. Material-Method: A metal mix at the permissible concentration for drinking water and 10-fold higher concentration were exposed to human aortic smooth muscle for 24 and 48 hours, and gene expression of antioxidant enzymes, heat-shock proteins and apoptosis pathway were investigated in addition to RAPD-DNA polymorphism. Results: It was determined that heavy metal exposure caused increase in the DNA polymorphism, apoptotic cell ratios and enhanced antioxidant and apoptosis pathway gene expression in cells. Conclusion: In conclusion, the mix consisting of heavy metals at permissible limits in drinking water has been shown to cause oxidative stress, genotoxicity and apoptosis in human aortic smooth muscle cells. Considering that apoptosis in vascular smooth muscle cells plays a role in the pathophysiology of diseases such as arteriosclerosis and hypertension, we believe it would be beneficial to assess these limits through extensive investigations in further studies.

Egg White-Derived Tripeptide IRW (Ile-Arg-Trp) Is an Activator of Angiotensin Converting Enzyme 2.

Angiotensin converting enzyme 2 (ACE2) plays beneficial roles in the renin angiotensin aldosterone system. Our previous studies indicated that egg white-derived antihypertensive peptide IRW (Ile-Arg-Trp) could upregulate ACE2 mRNA level in mesenteric artery of spontaneously hypertensive rats (SHRs), suggesting the potential of IRW as an in vivo ACE2 activator. In this study, the effects of IRW on activity and protein expression of ACE2 were investigated. Results indicated that IRW activated human recombinant ACE2 with an EC50 value of 7.24 × 10-5 M. In rat aortic vascular smooth muscle cell line A7r5 cells, IRW treatment (50 μM) significantly increased the expression and activity of ACE2. Oral administration of IRW to SHRs upregulated ACE2 protein levels in kidney and aorta. Molecular docking study suggested that IRW might activate ACE2 through interaction with the subdomain I near the active site through hydrogen bonds. Overall, this study established IRW as the first food-derived ACE2 activating peptide.

PDGFRβ Induces Ribosome Biogenesis in Aortic Smooth Muscle Cells

IntroductionArterial fibrosis (AF) is an important determinant of cardiovascular health. AF results in a thickened arterial wall and loss of compliance which is a direct result of excessive extracellular matrix (ECM) accumulation. Platelet Derived Growth Factor Receptor β (PDGFRβ) activation in vascular smooth muscle cells (VSMCs) results in AF that manifests with increased VSMC proliferation and increased collagen synthesis and deposition in the arteries. However, the mechanisms by which PDGFRβ activation results in AF are not fully elucidated.ObjectiveWe sought to investigate whether PDGFRβ regulates ribosome biogenesis (RB), and the extent to which increased RB downstream of PDGFRβ activation in VSMC mediates the AF‐associated structural and functional changes in mouse aorta.MethodsWe used Pdgfrb+/D849V mice (designated Bk) as a genetic model for AF where VSMC specific SM22Cre is used to drive expression of a conditional Pdgfrb knock‐in allele that encodes a constitutively active PDGFRβ in VSMC. In order to reduce ribosome content we crossed Bk mice with Rpl24Bst mice with a hypomorphic Rpl24 allele resulting in a 40% reduction in Ribosomal Protein L24. Arterial structure was evaluated in tissue sections. Ultrasound imaging was used to measure structural and functional arterial changes. We also serum‐starved rat aortic smooth muscle (A7r5) and 3T3 fibroblast cell lines and then stimulated with PDGF‐BB. RB was evaluated by measuring ribosomal proteins using Western Blot and assessing rRNA as RNA/DNA ratio using a fluorometric assay.ResultsCompared to wild type, Bk aortas were significantly larger with increased diameter and wall thickness and were significantly stiffer with reduced distension. Secondary to the arterial changes, Bk mice showed decreased cardiac contractility at 6 months of age that was not apparent at 3 months, but cardiac output did not decrease. Stimulating serum‐starved 3T3 fibroblasts and A7r5 smooth muscle cell lines with PDGF‐BB resulted in RB measured as increased ribosomal proteins and RNA/DNA ratio. In Bk mice, VSMC consisted of significantly more ribosomal proteins (RPL26, RPL10A, RPL17, RPS3 and RPS6, p&gt;0.05) and increased transcript and RNA/DNA ratio (150%, p&gt;0.05). Altering ribosomal proteins using Rpl24Bst partially attenuated some of these changes.ConclusionsThis study highlights the novel concept that RB is an important factor in AF. We found that RB and ribosome content is increased in VSMC in response to PDGFRβ activation and that altering ribosomes using Rpl24Bst mutation attenuated the arterial changes associated with PDGFRβ activation. It can be expected that RB will prove to be instrumental for AF in VSMC, however a few unanswered questions still remain. First, what are the downstream signaling events by which PDGFRβ stimulates RB? And second, what are the mechanisms by which increased ribosome content results in increased vascular ECM deposition? In my future research, I will investigate the signaling pathways downstream of PDGFRβ that result in RB. I will also identify specific transcripts that are targeted by ribosomes in response to PDGFRβ activation and will test the efficacy of ribosome‐modulating interventions in inhibiting PDGFRβ‐induced AF.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.