A7r5 Cells Research Articles

Chicken Muscle-Derived ACE2 Upregulating Peptide VVHPKESF Inhibits Angiotensin II-Stimulated Inflammation in Vascular Smooth Muscle Cells via the ACE2/Ang (1–7)/MasR Axis

This study aimed to evaluate the modulatory effects of four chicken muscle-derived peptides [VRP, LKY, VRY, and VVHPKESF (V-F)] on angiotensin II (Ang II)-induced inflammation in rat vascular smooth muscle A7r5 cells. Only V-F could significantly attenuate Ang II-stimulated inflammation via the inhibition of NF-κB and p38 MAPK signaling, being dependent on the Mas receptor (MasR) not on the Ang II type 1 or type 2 receptor (AT1R or AT2R). V-F accelerated Ang II degradation by enhancing cellular ACE2 activity, which was due to ACE2 upregulation other than a direct ACE2 activation. These findings demonstrated that V-F ameliorated Ang II-induced inflammation in A7r5 cells via the ACE2/Ang (1-7)/MasR axis. Three peptide metabolites of V-F─VHPKESF, PKESF, and SF─were identified but were not considered major contributors to V-F's bioactivity. The regulation of peptide V-F on vascular inflammation supported its functional food or nutraceutical application in the prevention and treatment of hypertension and cardiovascular diseases.

Hydroxyapatite nanoparticles promote mitochondrial-based pyroptosis via activating calcium homeostasis and redox imbalance in vascular smooth muscle cells

Hydroxyapatite nanoparticles (HAP) have been widely used in various fields because of their natural biological origin and functional properties. The emerging evidence on their toxicities has attracted research interest. HAP-induced vascular smooth muscle cell (VSMC) damage is a key step in vascular calcification (VC), particularly in patients with chronic kidney disease. However, the injury effects and mechanism of action of HAP on VSMCs have not been extensively investigated. This study comprehensively characterized commercially available HAP and investigated its adverse biological effects in cultured A7R5 cells. In vitro experiments revealed that internalized HAP was localized in lysosomes, followed by the release of Ca2+ owing to the low pH microenvironment. Upon Ca2+ homeostasis, Ca2+ enters the mitochondria, leading to the simultaneous generation of reactive oxygen species (ROS). ROS subsequently attack mitochondrial transmembrane potentials, promote mitochondrial ROS production, and oxidize mitochondrial DNA (Ox-mtDNA). Mitochondrial permeability-transition pores open, followed by the release of more Ox-mtDNA from the mitochondria into the cytosol due to the redox imbalance. This activates NLRP3/caspase-1/gasdermin D-dependent pyroptosis and finally excretes inflammatory factors to induce VC; an antioxidant could rescue this process. It has been suggested that HAP could induce an imbalance in intracellular Ca2+ homeostasis in A7R5 cells, followed by the promotion of mitochondrial dysfunction and cell pyroptosis, finally enhancing VC. To detect the in vivo toxicity of HAP, mice were treated with Cy7-labelled HAP NPs for 24 h. In vivo results also demonstrated that HAP accumulated in the kidneys, accompined with increased Ca concentration, upregulated oxidative stress-related factor and kidney damage. Overall, our research elucidates the mechanism of calcium homeostasis and redox imbalance, providing insights into the prevention of HAP-induced cell death.

Rosmarinic Acid Attenuates the Lipopolysaccharide-Provoked Inflammatory Response of Vascular Smooth Muscle Cell via Inhibition of MAPK/NF-κB Cascade.

Rosmarinic acid (RA) is a phenolic compound that has several bioactivities, such as anti-inflammatory and antioxidant activities. Here, we further investigate the anti-inflammatory effect of RA on rat A7r5 aortic smooth muscle cells with exposure to lipopolysaccharide (LPS). Our findings showed that low-dose RA (10–25 μM) did not influence the cell viability and morphology of A7r5 cells and significantly inhibited LPS-induced mRNA expression of the pro-inflammatory mediators TNFα, IL-8, and inducible NO synthase (iNOS). Consistently, RA reduced the production of TNFα, IL-8, and NO by A7r5 cells with exposure to LPS. Signaling cascade analysis showed that LPS induced activation of Erk, JNK, p38 mitogen-activated protein kinase (MAPK), and NF-κB, and RA treatments attenuated the activation of the three MAPKs and NF-κB. Moreover, cotreatment with RA and Erk, JNK, p38 MAPK, or NF-κB inhibitors further downregulated the mRNA expression of TNFα, IL-8, and iNOS, and decreased the production of TNFα, IL-8, and NO by A7r5 cells. Taken together, these findings indicate that RA may ameliorate the LPS-provoked inflammatory response of vascular smooth muscle cells by inhibition of MAPK/NF-κB signaling.

Ageing causes an aortic contractile dysfunction phenotype by targeting the expression of members of the extracellular signal-regulated kinase pathway.

The extracellular signal‐regulated kinase (ERK) pathway is a well‐known regulator of vascular smooth muscle cell proliferation, but it also serves as a regulator of caldesmon, which negatively regulates vascular contractility. This study examined whether aortic contractile function requires ERK activation and if this activation is regulated by ageing. Biomechanical experiments revealed that contractile responses to the alpha1‐adrenergic agonist phenylephrine are attenuated specifically in aged mice, which is associated with downregulation of ERK phosphorylation. ERK inhibition attenuates phenylephrine‐induced contractility, indicating that the contractile tone is at least partially ERK‐dependent. To explore the mechanisms of this age‐related downregulation of ERK phosphorylation, we transfected microRNAs, miR‐34a and miR‐137 we have previously shown to increase with ageing and demonstrated that in A7r5 cells, both miRs downregulate the expression of Src and paxillin, known regulators of ERK signalling, as well as ERK phosphorylation. Further studies in aortic tissues transfected with miRs show that miR‐34a but not miR‐137 has a negative effect on mRNA levels of Src and paxillin. Furthermore, ERK phosphorylation is decreased in aortic tissue treated with the Src inhibitor PP2. Increases in miR‐34a and miR‐137 with ageing downregulate the expression of Src and paxillin, leading to impaired ERK signalling and aortic contractile dysfunction.

Sdox, a H2S releasing anthracycline, with a safer profile than doxorubicin toward vasculature

Sdox is a synthetic H2S-releasing doxorubicin (Dox) less cardiotoxic and more effective than Dox in pre-clinical, Dox-resistant tumour models. The well-known anthracycline vascular toxicity, however, might limit Sdox clinical use. This study aimed at evaluating Sdox vascular toxicity in vitro, using Dox as reference compound.Both vascular smooth muscle A7r5 and endothelial EA.hy926 cells were more sensitive to Dox than Sdox, although both drugs equally increased intracellular free radical levels. Sdox released H2S in both cell lines. The H2S scavenger hydroxocobalamin partially reverted Sdox-induced cytotoxicity in A7r5, but not in EA.hy926 cells, suggesting a role for H2S in smooth muscle cell death. Markers of Sdox-induced apoptosis were significantly lower than, in A7r5 cells, and comparable to those of Dox in EA.hy926 cells. In A7r5 cells, Dox increased the activity of caspase 3, 8, and 9, Sdox affecting only that of caspase 3. Moreover, both drugs induced comparable DNA damage in A7r5 cells, while Sdox was less toxic than Dox in Ea.hy926 cells.In fresh aorta rings, only Dox weakly increased phenylephrine-induced contraction when endothelium was present. In rings cultured with both drugs for 7 days, Sdox blunted phenylephrine- and high K+-induced contractions though at a concentration 10-fold higher than that of Dox.In conclusion, Sdox may represent the prototype of an innovative anthracycline, effective against Dox-resistant tumours, displaying a more favourable vascular toxicity profile compared to the parent compound.

Regulation of total LC3 levels by angiotensin II in vascular smooth muscle cells

Hypertension is associated with high circulating angiotensin II (Ang II). We have reported that autophagy regulates Ang II‐induced vascular smooth muscle cell (VSMC) hypertrophy, but the mechanism mediating this effect is still unknown. Therefore, we studied how Ang II regulates LC3 levels in VSMCs and whether Bag3, a co‐chaperone known to regulate LC3 total levels, may be involved in the effects elicited by Ang II. A7r5 cell line or rat aortic smooth muscle cell (RASMC) primary culture were stimulated with Ang II 100 nM for 24 h and LC3 I, LC3 II and Bag3 protein levels were determined by Western blot. MAP1LC3B mRNA levels were assessed by RT‐qPCR. Ang II increased MAP1LC3B mRNA levels and protein levels of LC3 I, LC3 II and total LC3 (LC3 I + LC3 II). Cycloheximide, but not actinomycin D, abolished LC3 II and total LC3 increase elicited by Ang II in RASMCs. In A7r5 cells, cycloheximide prevented the Ang II‐mediated increase of LC3 I and total LC3, but not LC3 II. Moreover, Ang II increased Bag3 levels, but this increase was not observed upon co‐administration with either losartan 1 μM (AT1R antagonist) or Y‐27632 10 μM (ROCK inhibitor). These results suggest that Ang II may regulate total LC3 content through transcriptional and translational mechanisms. Moreover, Bag3 is increased in response to Ang II by a AT1R/ROCK signalling pathway. These data provide preliminary evidence suggesting that Ang II may stimulate autophagy in VSMCs by increasing total LC3 content and LC3 processing.

MiR-129-5p inhibits oxidized low-density lipoprotein-induced A7r5 cell viability and migration by targeting HMGB1 and the PI3k/Akt signaling pathway.

The mechanisms underlying gene therapy for the treatment of cardiovascular diseases remain to be elucidated. microRNAs (miRs) have been recognized as key regulators in vascular smooth muscle cells, which are involved in the formation of atherosclerosis. The present study aimed to explore the role of miR-129-5p in the regulation of high-mobility group box 1 protein (HMGB1) and the PI3k/Akt signaling pathway, and further explore the role of miR-129-5p in the viability and migration of A7r5 cells induced by oxidized low-density lipoprotein (ox-LDL). Cell viability, viability and migration were determined using Cell Counting Kit-8, colony formation, wound healing and Transwell assays. The expression levels of miR-129-5p and HMGB1 were detected using reverse transcription-quantitative PCR and western blotting. A dual-luciferase assay was used to confirm the association between miR-129-5p and HMGB1. RT-qPCR results in the present study demonstrated that the expression levels of miR-129-5p in A7r5 cells induced by ox-LDL were significantly decreased, compared with the control cells. Moreover, the viability and migration of A7r5 cells induced by ox-LDL were increased compared with control group. Western blot and RT-qPCR results showed that miR-129-5p decreased the expression of HMGB1 in A7r5 cells compared with control group. The present results demonstrated that miR-129-5p inhibited the viability, viability and migration of A7r5 cells induced by ox-LDL, and directly targeted HMGB1 to regulate the PI3k/Akt signaling pathway. In conclusion, miR-129-5p inhibited the PI3k/Akt signaling pathway by directly targeting HMGB1, and reduced the viability, viability and migration of A7r5 cells induced by ox-LDL.

Spatial distribution analysis of intracellular flows

Retrograde flow is the intracellular flow of the cytoskeleton, and it allows for transportation of the structure from the cell edge towards the cell center. It is important to understand the retrograde flow since it is closely related to biological functions, such as contraction forces, metabolisms and turnovers. In this work, we analyze the retrograde flow in immobile cell types given that most of previous studies focused on mobile ones. We obtain fluorescence images of α-actinin, associated with the actin cytoskeleton, in individual A7r5 cells with a fluorescence microscope. We quantified and visualized the retrograde flow in the cells with particle image velocimetry (PIV) and discussed spatial distribution of the intracellular flows. By calculating the divergence of the retrograde flow, we found that the cells have a source of the field not only in the cell periphery but also at the central region of the cells. Since some of the fluid sources are located at the topological defects, we also hypothesize that the topology of cytoskeletons might be related with the cell functions.

Mulberry polyphenols ameliorate atherogenic migration and proliferation by degradation of K-Ras and downregulation of its signals in vascular smooth muscle cell.

Extra-proliferation and increased migration of vascular smooth cells con-tribute to the formation of atherosclerosis. Ras small G proteins play a critical role in the prolif-eration and migration of a wide range of cells. Mulberry, an economic fruit in Asia, exhibits anti-inflammation, anti-migration, and anti-oxidant properties. The mechanisms of action of mulberry extracts on K-Ras small G protein-induced proliferation and migration of vascular smooth muscle cell have not been extensively investigated. In this study, we explored the effects of mulberry polyphenol extracts (MPE) on the proliferation and migration of K-Ras-overexpressing A7r5 smooth muscle cells. The overexpression of K-Ras enhanced the ex-pression and activity of matrix metalloproteinase (MMP)-2, promoted vascular endothelial growth factor (VEGF) production, and eventually triggered the migration of A7r5 cells. Treatment with MPE attenuated K-Ras-induced phenomenon. In addition, MPE blocked K-Ras-induced actin fibril stress. MPE dose-dependently diminished K-Ras-induced Rho A, Rac1, CDC42, and phosphorylated focal adhesion kinase (FAK) expression. MPE elevated Rho B ex-pression. Phosphorylated AKT and glycogen synthase kinase (GSK) induced by K-Ras were also repressed by MPE treatment. MPE enhanced the interaction of IκB with NFκB. MPE restored the G0/G1 population and p21 and p27 expressions, which were repressed by K-Ras. Finally, MPE triggered the degradation of K-Ras by ubiquitination. MPE inhibited the migration and proliferation of vascular smooth cell through K-Ras-induced pathways and eventually pre-vented atherosclerosis.

Mulberry polyphenol extracts attenuated senescence through inhibition of Ras/ERK via promoting Ras degradation in VSMC.

Ageing is one of the major risk factors of human diseases, including cancer, diabetes, and cardiovascular disease. Mulberry exhibits a wide range of functions, such as anti-oxidant, anti-inflammation, and anti-diabetes. In this study, we investigated the role of mulberry polyphenol extract (MPE) in K-Ras-induced senescence of smooth muscle cells. Forced expression of K-Ras enhanced senescence of smooth muscle A7r5 cells as shown by the elevation of β-galactosidase activity. Treatment with MPE significantly repressed the Ras, phosphorylated ERK, and β-galactosidase level. MPE triggered the association of cyclins with their corresponding cyclin-dependent protein kinases and hyperphosphorylated retinoblastoma (RB). MPE also down-regulated the levels of K-Ras-induced CDK inhibitors. MPE enhanced the phosphorylated AMP-dependent protein kinase (AMPK) and inducible nitric oxide synthase (iNOS) level in the presence of K-Ras. Pretreatment with either L-NAME or AMPK inhibitor reversed the effects of MPE. In addition, L-NAME and AMPK inhibitor repressed the MPE-induced total and phosphorylated 3-hydroxy-3-methylglutaryl coenzyme A (HMG-Co A) level. MPE repressed K-Ras-induced G0/G1 arrest, whereas L-NAME and AMPK inhibitor blocked the effects of MPE. Our results indicated that MPE recovered the K-Ras-induced senescence of vascular smooth muscle cells through iNOS and AMPK-dependent pathway. Our findings suggested that MPE may prevent ageing-induced atherosclerosis.

Pharmacodynamic Mechanism of Kuanxiong Aerosol for Vasodilation and Improvement of Myocardial Ischemia.

To explore the effect of Kuanxiong Aerosol (KXA) on isoproterenol (ISO)-induced myocardial injury in rat models. Totally 24 rats were radomly divided into control, ISO, KXA low-dose and high-dose groups according to the randomized block design method, and were administered by intragastric administration for 10 consecutive days, and on the 9th and 10th days, rats were injected with ISO for 2 consecutive days to construct an acute myocardial ischemia model to evaluate the improvement of myocardial ischemia by KXA. In addition, the diastolic effect of KXA on rat thoracic aorta and its regulation of ion channels were tested by in vitro vascular tension test. The influence of KXA on the expression of calcium-CaM-dependent protein kinase II (CaMK II)/extracellular regulated protein kinases (ERK) signaling pathway has also been tested. KXA significantly reduced the ISO-induced increase in ST-segment, interventricular septal thickness, cardiac mass index and cardiac tissue pathological changes in rats. Moreover, the relaxation of isolated thoracic arterial rings that had been precontracted using norepinephrine (NE) or potassium chloride (KCl) was increased after KXA treatment in an endothelium-independent manner, and was attenuated by preincubation with verapamil, but not with tetraethylammonium chloride, 4-aminopyridine, glibenclamide, or barium chloride. KXA pretreatment attenuated vasoconstriction induced by CaCl2 in Ca2+-free solutions containing K+ or NE. In addition, KXA pretreatment inhibited accumulation of Ca2+ in A7r5 cells mediated by KCl and NE and significantly decreased p-CaMK II and p-ERK levels. KXA may inhibit influx and release of calcium and activate the CaMK II/ERK signaling pathway to produce vasodilatory effects, thereby improving myocardial injury.

PDGF-BB promotes vascular smooth muscle cell migration by enhancing Pim-1 expression via inhibiting miR-214.

BackgroundSeveral studies have indicated that the platelet-derived growth factor/platelet-derived growth factor receptor (PDGF/PDGFR) pathway is involved in the process of atherosclerosis. However, its underlying mechanism remains to be further elucidated. Serine/threonine-protein kinase pim-1 (Pim-1), a member of serine/threonine-specific kinases, is a pro-oncogene published to be related to cell proliferation, apoptosis, and metastasis of cancer cells. Whether Pim-1 is involved in PDGF/PDGFR pathway-mediated coronary atherosclerotic heart disease remains to be elucidated.MethodsWe established a cell model of PDGF-BB-stimulated smooth muscle cells using A7r5 cells. Transwell assay was used to detect the potential of cell migration and invasion. The targeted regulation of Pim-1 by miR-214 was confirmed by luciferase assay. Rescue experiments were performed to determine the role of the PDGF-BB/miR-214/Pim-1 axis on the cell migration of smooth muscle cells by including PDGF-BB treatment, and the overexpression of miR-214 and Pim-1. Quantitative polymerase chain reaction (qPCR) was used to examine the gene expression and western blot was performed to detect the protein expression.ResultsOur data indicated that PDGF-BB could effectively enhance smooth muscle cell migration. We also showed Pim-1 was a target of miR-214 in A7r5 cells. The expression of Pim-1 was shown to be upregulated by PDGF-BB via suppression of the expression of miR-214. Moreover, overexpression miR-214 inhibited PDGF-BB-stimulated Pim-1 expression and smooth muscle cell migration via modulating epithelial-mesenchymal transition (EMT), but no change on cell cycle. However, overexpression of Pim-1 reversed miR-214-blocked cell migration by promoting the activation of the STAT3, AKT, and ERK signaling pathways.ConclusionsOur data suggested that the PDGF/miR-214/Pim-1 axis could be a potential target for coronary atherosclerotic heart disease.

Calcium/calmodulin-dependent protein kinase IV regulates vascular autophagy and insulin signaling through Akt/mTOR/CREB pathway in ob/ob mice

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) has recently emerged as an important regulator of glucose metabolism and vascular function, but the underlying mechanism is not fully understood. Recently, we revealed that CaMKIV limits metabolic disorder and liver insulin resistance and regulates autophagy in high-fat diet-induced obese mice. In the present study, we demonstrated that CaMKIV was not only associated with improvement of glucose tolerance and insulin sensitivity in ob/ob mice but also involved in the regulation of vascular autophagy and mitochondrial biogenesis. Our in vitro data indicated that CaMKIV reversed autophagic imbalance and restored insulin sensitivity in palmitate-induced A7r5 cells with insulin resistance. However, the protective effects of CaMKIV were nullified by suppression of Akt, mTOR, or CREB, suggesting that CaMKIV inhibits autophagy and improves insulin signaling in insulin resistance cell models in an Akt/mTOR/CREB-dependent manner. CaMKIV reversed autophagic imbalance and insulin sensitivity in vascular tissues and vascular cells through Akt/mTOR/CREB signaling, which could be regarded as a novel opportunity for the treatment of insulin resistance.

USP10 exacerbates neointima formation by stabilizing Skp2 protein in vascular smooth muscle cells

The underlying mechanism of neointima formation remains unclear. Ubiquitin-specific peptidase 10 (USP10) is a deubiquitinase that plays a major role in cancer development and progression. However, the function of USP10 in arterial restenosis is unknown. Herein, USP10 expression was detected in mouse arteries and increased after carotid ligation. The inhibition of USP10 exhibited thinner neointima in the model of mouse carotid ligation. In vitro data showed that USP10 deficiency reduced proliferation and migration of rat thoracic aorta smooth muscle cells (A7r5) and human aortic smooth muscle cells (HASMCs). Mechanically, USP10 can bind to Skp2 and stabilize its protein level by removing polyubiquitin on Skp2 in the cytoplasm. The overexpression of Skp2 abrogated cell cycle arrest induced by USP10 inhibition. Overall, the current study demonstrated that USP10 is involved in vascular remodeling by directly promoting VSMC proliferation and migration via stabilization of Skp2 protein expression.

Study on Optimal Parameter and Target for Pulsed-Field Ablation of Atrial Fibrillation.

Pulsed-field ablation (PFA) had potential advantages in atrial fibrillation ablation, and we aim to confirm the optimal parameter and target of PFA for atrial fibrillation. Two ablation modes in vitro of single-cell system (ablation in electrode cup) and monolayer cell system (ablation in inserts with electrode tips) were established to perform PFA for myocardial cell H9C2 and smooth muscle cell A7r5. Ablation effect, calcium ion influx, the expression of Cx45, and surface morphological change were observed. Three Bama minipigs were used to verify the in vivo ablation effect of PFA. In monolayer cell system, H9C2 was significantly sensitive to PFA compared with A7r5, with shrinking of the whole monolayer. The ablation effect of bidirectional pulse was weaker than that of the two mono-polar pulses. Expressed Cx45 proteins were increased in H9C2 but decreased in A7r5 cells. Bidirectional PFA performed on Bama minipigs was able to effectively block electrical activity from the pulmonary vein to the atrium with week muscle contraction, not generating pulmonary vein stenosis. Bidirectional PFA was able to significantly ablate myocardial cells, maintain cell–cell connection, and reduce muscle contraction, which was a kind of optimized PFA strategy for atrial fibrillation.

The promoting role of Cx43 on the proliferation and migration of arterial smooth muscle cells for angiotensin II-dependent hypertension

BackgroundRecent studies have shown that endothelin-1 and angiotensin II (AngII) can increase gap junctional intercellular communication (GJIC) by activating Mitogen-activated protein kinases (MAPKs) pathway. However, not only the precise interaction of AngII with Connexin43(Cx43) and the associated functions remain unclear, but also the regulatory role of Cx43 on the AngII-mediated promotion proliferation and migration of VSMCs is poorly understood. Material and methodsOur research applicated pressure myography measurements, immunofluorescence and Western blot analyses to investigate the changes in physiological indicators in spontaneously hypertensive rats (SHRs) and AngII-stimulated proliferation and migration of A7r5 SMCs(Rat vascular smooth muscle cells). The aim was to elucidate the role of CX43 in hypertension induced by AngII. ResultsChronic ramipril (angiotensin converting enzyme inhibitor) management for SHRs significantly attenuated blood pressure and blood vessel wall thickness, also reduced contraction rate in the cerebral artery. The cerebral artery contraction rates, mRNA and protein expression of Cx43, osteopontin (OPN) and proliferating cell nuclear antigen (PCNA) protein expression in the SHR + ramipril and SHR + ramipril + carbenoxolone (CBX, Cx43 specific blocker) groups were significantly lower than those in the SHR group. Cx43 protein expression and Ser368 phosphorylated Cx43 protein levels increased significantly in AngII-stimulated A7r5 cells. However, the levels of phosphorylated Cx43 decreased after pre-treatment with candesartan (AT1 receptor blocker), GF109203X (protein kinase C (PKC) blocker) and U0126 (mitogen-activated protein kinases/extracellular signal-regulated kinase1/2(MEK/ERK1/2)-specific blocker) in AngII-stimulated A7r5 cells. Cx43 was widely distributed in the cell membrane, nucleus, and cytoplasm of the SMCs. Furthermore, pre-treatment of the AngII- stimulated A7r5 cells with Gap26 (Cx43 blocker) significantly inhibited cell migration and decreased the expression levels of MEK1/2, ERK1/2, P-MEK1/2, and P-ERK1/2. ConclusionOur research confirms that Cx43 plays an important role in the regulation of proliferation and migration of VSMCs via MEK/ERK and PKC signal pathway in AngII-dependent hypertension.

Myricetin suppresses the proliferation and migration of vascular smooth muscle cells and inhibits neointimal hyperplasia via suppressing TGFBR1 signaling pathways

BackgroundNeointimal formation, mediated by the proliferation and migration of vascular smooth muscle cells (VSMCs), is a common pathological basis for atherosclerosis and restenosis. Myricetin, a natural flavonoid, reportedly exerts anti-atherosclerotic effects. However, the effect and mechanism of myricetin on VSMCs proliferation and migration and neointimal hyperplasia (NIH) remain unknown. PurposeWe investigated myricetin's effect on NIH, as well as the potential involvement of transforming growth factor-beta receptor 1 (TGFBR1) signaling in mediating myricetin's anti-atherosclerotic and anti-restenotic actions. MethodsMyricetin's effects on the proliferation and migration of HASMCs and A7R5 cells were determined by CCK-8, EdU assays, wound healing, Transwell assays, and western blotting (WB).Molecular docking, molecular dynamics (MD) simulation, surface plasmon resonance (SPR) and TGFBR1 kinase activity assays were employed to investigate the interaction between myricetin and TGFBR1. An adenovirus vector encoding TGFBR1 was used to verify the effects of myricetin. In vivo, the left common carotid artery (LCCA) ligation mouse model was adopted to determine the impacts of myricetin on neointimal formation and TGFBR1 activation. ResultsMyricetin dose-dependently inhibited the migration and proliferation in VSMCs, suppressed the expression of CDK4, cyclin D3, MMP2, and MMP9. Molecular docking revealed that myricetin binds to key regions for TGFBR1 antagonist binding, and the binding energy was -9.61 kcal/mol. MD simulation indicated stable binding between TGFBR1 and myricetin. Additionally, SPR revealed an equilibrium dissociation constant of 4.35 × 10−5 M between myricetin and TGFBR1. According to the TGFBR1 kinase activity assay, myricetin directly inhibited TGFBR1 kinase activity (IC50 = 8.551 μM). Furthermore, myricetin suppressed the phosphorylation level of TGFBR1, Smad2, and Smad3 in a dose-dependent pattern, which was partially inhibited by TGFBR1 overexpression. Consistently, TGFBR1 overexpression partially rescued the suppressive roles of myricetin on VSMCs migration and proliferation. Moreover, myricetin dramatically inhibited NIH and reduced TGFBR1, Smad2, and Smad3 phosphorylation in the LCCA. ConclusionThis is the first study to demonstrate that myricetin suppresses NIH and VSMC proliferation and migration via inhibiting TGFBR1 signaling. Myricetin can be developed as a potential therapeutic candidate for treating atherosclerosis and vascular restenosis.

MiR-31-5p Promotes Oxidative Stress and Vascular Smooth Muscle Cell Migration in Spontaneously Hypertensive Rats via Inhibiting FNDC5 Expression.

Oxidative stress and the migration of vascular smooth muscle cells (VSMCs) are important for vascular remodeling in a variety of vascular diseases. miR-31-5p promotes cell migration in colorectal cancer cells but inhibits cell migration in renal cell carcinoma. However, whether miR-31-5p is involved in oxidative stress and VSMC migration remains unknown. This study shows the crucial roles of miR-31-5p in oxidative stress and VSMC migration, as well as underlying mechanisms. Experiments were carried out in primary VSMCs from aortic media of Wistar–Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), as well as the A7r5 cell line. Oxidative stress was assessed by NADPH oxidase (NOX) expression, NOX activity, and reactive oxygen species (ROS) production. Cell migration was evaluated with a Boyden chamber assay and a wound healing assay. The miR-31-5p mimic and inhibitor promoted and attenuated oxidative stress and cell migration in the VSMCs of SHR, respectively. A dual-luciferase reporter assay indicated that miR-31-5p targeted the 3’UTR domain of FNDC5. The miR-31-5p level was raised and FNDC5 expression was reduced in the VSMCs of SHR compared with those of WKY. The miR-31-5p mimic reduced FNDC5 expression in the A7r5 cells and the VSMCs of both WKY and SHR, while the miR-31-5p inhibitor only increased FNDC5 expression in the VSMCs of SHR. Exogenous FNDC5 attenuated not only the oxidative stress and VSMC migration in SHR but also the roles of the miR-31-5p mimic in inducing oxidative stress and VSMC migration. These results indicate that miR-31-5p promotes oxidative stress and VSMC migration in SHR via inhibiting FNDC5 expression. The increased miR-31-5p and reduced FNDC5 in the VSMCs of SHR contribute to enhanced oxidative stress and cell migration.

MiR-133a is a potential target for arterial calcification in patients with end-stage renal disease.

Arterial calcification is an important risk factor for patients with end-stage renal disease. Despite substantial research efforts, the detailed mechanisms of the process of arterial calcification in end-stage renal disease remain unclear. miR-133a expression in radial artery samples was detected by FISH and Alizarin Red Staining. The expressions of miR-133a and RUNX2 in A7r5 cells with BMP2 induction were detected by qRT-PCR. In addition, qRT-PCR, Western blot, and ELISA assay were performed to detect changes in miR-133a levels in A7R5 cells after different treatments. Alizarin Red staining showed that red crystal deposition occurred in the tunica media. FISH analysis indicated that miR-133a was upregulated in the tunica media of the radial artery samples without calcificationwhen compared with those with calcification. We also found that expression of RUNX2 in A7r5 cells increased at day 7 and day 14 after BMP2 induction and decreased miR-133a expression decreased at day 14. In addition, RUNX2 protein and OCN expression were upregulated in A7r5 cells during BMP2-induced calcification. When miR-133a expression was suppressed, cell calcification aggravated, which led to upregulation of RUNX2 and OCN. When miR-133a was overexpressed, calcification of cells was inhibited, resulting in downregulation of RUNX2 and OCN. The present study reveals that miR-133a could indirectly regulate cell calcification through the RUNX2 gene expression. Our findings provide insight into the potential use of miR-133a as a molecular target for diagnosing vascular calcification in end-stage renal disease.

The inhibitory effect of trimetazidine on detrusor contractility - a potential repositioning of trimetazidine for the treatment of overactive bladder.

This study aimed to identify the effect of trimetazidine (TMZ), an antianginal drug, on detrusor smooth muscle (DSM) contractility and its possible mechanisms of action. We performed in-vitro contractility studies on isolated mouse DSM strips and investigated the effect of TMZ on Ca2+ levels in fura-2-loaded A7r5 cells. TMZ (300 or 1000 µM) inhibited carbachol (CCh)- and KCl-induced contractions and produced a concentration-dependent (10-1000 µM) relaxation in KCl-precontracted DSM strips. TMZ-induced relaxation was markedly decreased by BaCl2, an inward-rectifying K+ channel blocker, but was not altered by preincubation with tetraethylammonium, glibenclamide, 4-aminopyridine, propranolol, L-NAME or methylene blue. TMZ (300 or 1000 µM) reduced both the CaCl2-induced contraction of depolarized DSM strips under Ca2+-free conditions and the CCh-induced contraction of DSM strips preincubated with nifedipine in Ca2+-containing Krebs solution. Furthermore, TMZ (1000 µM) significantly decreased the Ca2+ levels in fura-2-loaded A7r5 cells. TMZ decreased DSM contractility and caused a concentration-dependent relaxation of the tissue possibly through its actions on Ca2+ transients and K+ channels. Our results provide preclinical evidence that TMZ would be a potential candidate to treat disorders related to the overactivity of the bladder.