Levels In Vascular Smooth Muscle Cells Research Articles

Nuclear receptor subfamily 4 group a member 1 eases angiotensin II-arose oxidative stress in vascular smooth muscle cell by boosting nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing 3 transcription

Objective: Hypertension significantly contributes to morbidity and mortality. Nuclear receptor subfamily 4 group a member 1 (Nur77) participates in regulating oxidative stress, but the mechanism in hypertension remains unclear. This study aimed to explore the function of Nur77 in oxidative stress induced by Angiotensin II (Ang II) in vascular smooth muscle cells (VSMCs) in hypertension. Material and Methods: First, models of VSMC with Nur77, nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing 3 (NLRC3) and tumor necrosis factor receptor-associated factor 6 (TRAF6) knockdown or overexpression were constructed using Short Hairpin RNA (Nur77) or pcDNA3.1 vector, respectively. Next, the putative-binding motifs between Nur77 and NLRC3 promoters were detected by dual luciferase assay. We conducted reverse transcription quantitative polymerase chain reaction (qPCR) and Western blot (WB) analysis to detect Nur77, NLRC3, and TRAF6 levels in VSMCs. Then, cell counting kit-8 assay, 5-ethynyl-2’-deoxyuridine assay, wound-healing assay, enzyme-linked immunosorbent assay, and 2’,7’-dichlorofluorescin diacetate were employed to examine the impact of the knockdown or overexpression of Nur77, NLRC3, and TRAF6 on VSMCs treated with Ang II. The assays measured cell viability and proliferation, cell migration, malondialdehyde levels, and reactive oxygen species levels. Results: The overexpression of Nur77 repressed cell growth (P < 0.001), migration (P < 0.01), and oxidative stress (P < 0.01) induced by Ang II in VSMCs. Nur77 transcriptionally promoted the expression of NLRC3 (P < 0.001), and the upregulation of NLRC3 suppressed cell proliferation (P < 0.05) and oxidative stress (P < 0.001) mediated by Ang II. Furthermore, NLRC3 negatively regulated the TRAF6/nuclear factor-kappa B (NF-κB) axis activated by Ang II, which resulted in the repression of hyperproliferation of VSMCs (P < 0.01) and oxidative stress (P < 0.001). Conclusion: Nur77 suppressed growth and oxidative stress induced by Ang II in VSMCs by promoting NLRC3 transcription, which, further, repressed the TRAF6/NF-κB axis. This understanding provides novel insights into the pathogenesis of hypertension.

Inhibition of poly(ADP-Ribosyl)ation reduced vascular smooth muscle cells loss and improves aortic disease in a mouse model of human accelerated aging syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disorder associated with features of accelerated aging. HGPS is an autosomal dominant disease caused by a de novo mutation of LMNA gene, encoding A-type lamins, resulting in the truncated form of pre-lamin A called progerin. While asymptomatic at birth, patients develop symptoms within the first year of life when they begin to display accelerated aging and suffer from growth retardation, and severe cardiovascular complications including loss of vascular smooth muscle cells (VSMCs). Recent works reported the loss of VSMCs as a major factor triggering atherosclerosis in HGPS. Here, we investigated the mechanisms by which progerin expression leads to massive VSMCs loss. Using aorta tissue and primary cultures of murine VSMCs from a mouse model of HGPS, we showed increased VSMCs death associated with increased poly(ADP-Ribosyl)ation. Poly(ADP-Ribosyl)ation is recognized as a post-translational protein modification that coordinates the repair at DNA damage sites. Poly-ADP-ribose polymerase (PARP) catalyzes protein poly(ADP-Ribosyl)ation by utilizing nicotinamide adenine dinucleotide (NAD+). Our results provided the first demonstration linking progerin accumulation, augmented poly(ADP-Ribosyl)ation and decreased nicotinamide adenine dinucleotide (NAD+) level in VSMCs. Using high-throughput screening on VSMCs differentiated from iPSCs from HGPS patients, we identified a new compound, trifluridine able to increase NAD+ levels through decrease of PARP-1 activity. Lastly, we demonstrate that trifluridine treatment in vivo was able to alleviate aortic VSMCs loss and clinical sign of progeria, suggesting a novel therapeutic approach of cardiovascular disease in progeria.

PANoptosis in vascular smooth muscle cells regulated by TNF-α/IL-1β can be a new target for alleviating the progression of abdominal aortic aneurysm.

PANoptosis is an inflammatory programmed cell death (PCD) regulated by multifaceted PANoptosome complexes with major features of pyroptosis, apoptosis, and/or necroptosis that cannot be accounted for by any of these PCD pathways alone. The aim of this study was to investigate the role of PANoptosis on the occurrence and development of abdominal aortic aneurysm (AAA). Clinical samples of patients with AAA, angiotensin II (ANG II)-induced AAA mouse model, and ANG II-induced vascular smooth muscle cells (VSMCs) in vitro model were used for investigation on PANoptosis features. The expressions of ZBP1, AIM2, and other markers related to pyroptosis, apoptosis, and necroptosis elevated obviously in aortic wall tissues of patients with AAA, mice with AAA, and ANG II-treated VSMCs. ANG II treatment increased inflammatory cytokines levels in VSMCs. The stimulation of tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β) alone promoted VSMCs death, and the effect of TNF-α combined with IL-1β is more obvious. The expressions of ZBP1, AIM2, and related markers of pyroptosis, apoptosis, and necroptosis were increased by TNF-α and IL-1β combined treatment. Inhibition of TNF-α and/or IL-1β in mice with AAA improved the AAA pathology, reduced the loss of VSMCs, decreased the expression of ZBP1 and AIM2, and markers associated with pyroptosis, apoptosis, and necroptosis. PANoptosis features were observed in aortic wall tissues of patients with AAA, mice with AAA, and ANG II-treated VSMCs. The inhibition of TNF-α and IL-1β can alleviate PANoptosis in mice with AAA, which provides a new strategy for the prevention and treatment of AAA.NEW & NOTEWORTHY Early detection, diagnosis, and treatment are very important to improve the quality of life and prognosis of patients with abdominal aortic aneurysm (AAA). Based on the findings of apoptosis, necroptosis, and pyroptosis (PANoptosis) in AAA clinical samples, this study further explored the molecular mechanism in vivo and in vitro. Specifically, inhibition of tumor necrosis factor-α and interleukin-1β can reduce PANoptosis in vascular smooth muscle cell and thus alleviate the process of AAA.

Angiotensin II reduces glyoxalase 1 activity and expression in vascular smooth muscle cells: Implications for diabetic vascular complications.

Angiotensin II (Ang II), a key mediator of vascular diseases, is linked to methylglyoxal (MGO) formation, a by-product of glucose metabolism implicated in vascular complications. The glyoxalase system, consisting of glyoxalase 1 (Glo1) and reduced glutathione (GSH), is responsible for detoxifying MGO. This study investigated the effect of Ang II on Glo1 activity and expression in vascular smooth muscle cells (VSMCs). Primary VSMCs were isolated from rat aortas and exposed to Ang II under standard or high glucose conditions. We examined Glo1 activity, expression, intracellular GSH, and methylglyoxal-derived hydroimidazolone 1 (MG-H1) levels. We also analyzed the expressions of nuclear factor-κB (NF-κB) p65 and nuclear factor erythroid 2-related factor 2 (Nrf2) as potential regulators of Glo1 expression. The results demonstrated that Ang II reduced Glo1 activity, expression, and GSH levels while increasing MG-H1 levels in VSMCs. Telmisartan and irbesartan, AT1R blockers, restored Glo1 activity, expression, and GSH levels and alleviated MG-H1 levels. Treatment with AT1R blockers or inhibitors targeting signaling pathways involved in Ang II-induced responses mitigated these effects. High glucose exacerbated the reduction in Glo1 activity and expression. In conclusion, this study provides evidence that Ang II reduces Glo1 activity and expression in VSMCs, which may contribute to developing vascular complications in diabetes. AT1R blockers and inhibitors targeting specific signaling pathways show potential in restoring Glo1 function and mitigating MGO-associated damage. These findings highlight the complex interactions between RAS, MGO, and vascular diseases, highlighting potential therapeutic targets for diabetic vascular complications.

Wogonin inhibits oxidative stress and vascular calcification via modulation of heme oxygenase-1

Vascular calcification (VC) is highly prevalent and increases the morbidity and mortality of cardiovascular diseases. However, the underlying mechanism remains unclear and there is no effective treatment so far. Interestingly, using systems pharmacology approach, we have predicted that Wogonin (Wog) exhibited potential activity against VC. Then we validated the effect of Wog on VC using human and rat vascular smooth muscle cells (VSMCs), rat arterial rings and vitamin D3-overloaded mouse models. Our results showed that Wog dose-dependently inhibited calcification of VSMCs and rat arterial rings. Consistently, alizarin red staining and calcium content assay confirmed that Wog inhibited aortic calcification in vitamin D3-overloaded mice. Moreover, by constructing the protein regulating network of Wog in suppressing VC, we found heme oxygenase-1 (HMOX-1) was regulated by Wog. Additionally, pathway enrichment analysis revealed that inhibition of reactive oxygen species (ROS) pathway participated in the inhibitory role of Wog in VC and HMOX-1 was also involved in this process. Notably, our study revealed that Wog treatment promoted HMOX-1 expression, and reduced ROS levels in VSMCs. Interestingly, both inhibition of HMOX-1 by ZnPP9 and knockdown of HMOX-1 by siRNA independently eliminated the inhibitory effect of Wog on VC. Finally, administration of Wog suppressed aortic calcification in vitamin D3-overloaded mice and this effect was counteracted by ZnPP9,suggesting the crucial role of HMOX-1 in the inhibitory effect of Wog on VC. Collectively, this study combines systems pharmacology-based strategy and experiments to identify the therapeutic potential of Wog for VC via upregulating HMOX-1 and reducing oxidative stress.

Maximakinin reduced intracellular Ca2+ level in vascular smooth muscle cells through AMPK/ERK1/2 signaling pathways.

We detect the antihypertensive effects of maximakinin (MK) on renal hypertensive rats (RHRs) and further research the influence of MK on vascular smooth muscle cells (VSMCs) to explore its hypotensive mechanism. The effects of MK on arterial blood pressure were observed in RHRs. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assays were performed to detect the effect of MK on VSMC viability. Western blot and flow cytometry were used to investigate the influence of MK on intracellular Ca2+ levels and protein expression changes in VSMCs. In addition, specific protein inhibitors were applied to confirm the involvement of Ca2+-related signaling pathways induced by MK in VSMCs. MK showed a more significant antihypertensive effect than bradykinin in RHRs. MK significantly decreased intracellular Ca2+ concentrations. Furthermore, MK significantly induced the phosphorylation of signaling molecules, including extracellular signal-regulated kinase 1/2 (ERK1/2), P38, AMP-activated protein kinase (AMPK) and Akt in VSMCs. Moreover, only ERK1/2 inhibitor U0126 and AMPK inhibitor Compound C completely restored the decreased intracellular Ca2+ level induced by MK, and further research demonstrated that AMPK functioned upstream of ERK1/2 following exposure to MK. Finally, HOE-140, an inhibitor of the bradykinin B2 receptors (B2Rs), was applied to investigate the potential targets of MK in VSMCs. HOE-140 significantly blocked the AMPK/ERK1/2 pathway induced by MK, suggesting that the B2Rs might play an important role in MK-induced AMPK and ERK1/2 activation. MK significantly reduces blood pressure in RHRs. MK exerts its antihypertensive effect by activating the B2Rs and downstream AMPK/ERK1/2 pathways, leading to significantly reduced Ca2+ levels in VSMCs.

Betulinaldehyde inhibits vascular remodeling by regulating the microenvironment through the PLCγ1/Ca2+/MMP9 pathway

BackgroundVascular remodeling plays a crucial role in the pathogenesis of several cardiovascular diseases (CVDs). Unfortunately, current drug therapies offer limited relief for vascular remodeling. Therefore, the development of innovative therapeutic strategies or drugs that target vascular remodeling is imperative. Betulinaldehyde (BA) is a triterpenoid with diverse biological activities, but its effects on vascular remodeling remain unclear. ObjectiveThis study aimed to investigate the role of BA in vascular remodeling and its mechanism of action, providing valuable information for future applications of BA in the treatment of CVDs. MethodsNetwork pharmacology was used to predict the key targets of BA in vascular remodeling. The effect of BA on vascular remodeling was assessed in a rat model of balloon injury using hematoxylin and eosin staining, Masson staining, immunohistochemistry staining, and Western blotting. A phenotypic transformation model of vascular smooth muscle cells (VSMCs) was induced by platelet-derived growth factor-BB, and the functional impacts of BA on VSMCs were assessed via CCK-8, EdU, Wound healing, Transwell, and Western blotting. Finally, after manipulation of phospholipase C gamma1 (PLCγ1) expression, Western blotting and Ca2+ levels determination were performed to investigate the potential mechanism of action of BA. ResultsThe most key target of BA in vascular remodeling, matrix metalloproteinase 9 (MMP9), was identified through network pharmacology screening. Vascular remodeling was alleviated by BA in vivo and its effects were associated with decreased MMP9 expression. In vitro studies indicated that BA inhibited VSMC proliferation, migration, phenotypic transformation, and downregulated MMP9 expression. Additionally, BA decreased PLCγ1 expression and Ca2+ levels in VSMCs. However, after pretreatment with a phospholipase C agonist, BA's effects on down-regulating the expression of PLCγ1 and Ca2+ levels were inhibited, while the expression of MMP9 increased compared to that in the BA treatment group. ConclusionThis study demonstrated the critical role of BA in vascular remodeling. These findings revealed a novel mechanism whereby BA mediates its protective effects through MMP9 regulation by inhibiting the PLCγ1/Ca2+/MMP9 signaling pathway. Overall, BA may potentially be developed into a novel medication for CVDs and may serve as a promising therapeutic strategy for improving recovery from CVDs by targeting MMP9.

Essential role of CCN2 in the tgf-Β pathway regulation in vascular smooth muscle cells by enhancing TGF-Β receptor type II expression

Background and Aims: Besides the traditional role attributed to the cellular communication network factor 2 (CCN2/CTGF) as a main mediator of transforming growth factor β (TGF-β) induced fibrotic responses, recent evidences have demonstrated a direct role of CCN2 acting as a growth factor in vascular smooth muscle cells (VSMCs) inducing oxidative and proinflammatory responses. However, presence of both, CCN2 and TGF-β, are needed to induce a persistent fibrotic response in some tissues. Aims: Considering the main role of TGF-β receptors (TβR) in the TGF-β pathway activation, our aim was to explore the effects of CCN2 in the regulation of TβRI and TβRII levels in VSMCs.

Adropin Inhibits Vascular Smooth Muscle Cell Osteogenic Differentiation to Alleviate Vascular Calcification via the JAK2/STAT3 Signaling Pathway.

Vascular calcification is characterized as the deposition of hydroxyapatite mineral in the form of calcium-phosphate complexes in the vasculature. Transdifferentiation between vascular smooth muscle cells (VSMCs) and osteoblast-like cells is considered essential in the progression of vascular calcification. The pathophysiological mechanisms underlying vascular calcification and VSMC osteogenic differentiation remain to be fully elucidated, and the development of novel therapies is required. In the present study, PCR and western blot analysis were conducted to quantify the mRNA and protein expression levels of calcification-associated markers (bone morphogenetic protein 2, alkaline phosphatase, osteoprotegerin, osteocalcin, and runt-related transcription factor 2) and adropin in VSMCs and rat vascular tissues. The calcification of VSMCs was assessed using alizarin red staining. Moreover, adropin expression levels in VSMCs were analyzed using immunofluorescence. Lentiviral transfection and small interfering RNA were used for overexpression and knockdown of adropin in VSMCs, respectively. The results demonstrated that adropin alleviated vascular calcification in vivo. Moreover, adropin also inhibited osteogenic differentiation and the calcification of VSMCs in vitro. Notably, results of the present study revealed that the tyrosine protein kinase JAK2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway played a key role in the aforementioned inhibition. In conclusion, the results of the present study demonstrated that adropin inhibited VSMC osteogenic differentiation to alleviate vascular calcification via the JAK2/STAT3 signaling pathway.

Morin Inhibits Dox-Induced Vascular Inflammation By Regulating PTEN/AKT/NF-κB Pathway

The side effects of doxorubicin (Dox) may influence the long-term survival of patients with malignancies. Therefore, it is necessary to clarify the mechanisms generating these side effects induced by Dox and identify effective therapeutic strategies. Here, we found that interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-alpha (TNF-α) levels were significantly increased in vascular tissues of Dox-treated mice and Dox-treated vascular smooth muscle cells (VSMCs). Furthermore, we revealed that Dox downregulated the phosphatase and tension homology deleted on chromosome 10 (PTEN) level while upregulated p-AKT and p65 level in VSMCs in vitro. Overexpression of PTEN in VSMCs partly reversed Dox-induced inflammation. Importantly, we demonstrated that Morin could inhibit Dox-induced inflammation by facilitating an increase of PTEN, thus inhibiting the activation of protein kinase B (AKT)/nuclear factor kappa B (NF-κB)/pathway. Additionally, we showed that Morin could reduce the miR-188-5p level, which was increased in Dox-treated VSMCs. Inhibition of miR-188-5p suppressed Dox-induced vascular inflammation in vitro. In conclusion, Morin reduced the Dox-induced vascular inflammatory by moderating the miR-188-5p/PTEN/AKT/NF-κB pathway, indicating that Morin might be a therapeutic agent for overcoming the Dox-induced vascular inflammation.

Therapeutic Response of miR-145 Micelles on Patient-Derived Vascular Smooth Muscle Cells.

During atherosclerosis, vascular smooth muscle cells (VSMCs) undergo a phenotypic transition from a healthy contractile state into pathological phenotypes including a proliferative and migratory, synthetic phenotype and osteochondrogenic-like phenotype that exacerbate plaques. Thus, inhibiting the transition of healthy, quiescent VSMCs to atherogenic cell types has the potential to mitigate atherosclerosis. To that end, previously, we reported that delivery of microRNA-145 (miR-145, a potent gatekeeper of the contractile VSMC phenotype) using nanoparticle micelles limited atherosclerotic plaque growth in murine models of atherosclerosis. Building on this preclinical data and toward clinical application, in this study, we tested the therapeutic viability of miR-145 micelles on patient-derived VSMCs and evaluated their effects based on disease severity. We collected vascular tissues from 11 patients with healthy, moderate, or severe stages of atherosclerosis that were discarded following vascular surgery or organ transplant, and isolated VSMCs from these tissues. We found that with increasing disease severity, patient-derived VSMCs had decreasing levels of contractile markers (miR-145, ACTA2, MYH11) and increasing levels of synthetic markers (KLF4, KLF5, and ELK1). Treatment with miR-145 micelles showed that an increase in disease severity correlated with a more robust response to therapy in VSMCs. Notably, miR-145 micelle therapy rescued contractile marker expression to baseline contractile levels in VSMCs derived from the most severely diseased tissues. As such, we demonstrate the use of miR-145 micelles across different stages of atherosclerosis disease and present further evidence of the translatability of miR-145 micelle treatment for atherosclerosis.

Ox-LDL regulates proliferation and apoptosis in VSMCs by controlling the miR-183-5p/FOXO1.

microRNA-mRNA axes that are involved in oxidized low-density lipoprotein (ox-LDL)-induced vascular smooth muscle cells (VSMCs) proliferation/apoptosis imbalance need to be further investigated. To investigate the functional role of miR-183-5p/FOXO1 in VSMCs and its interaction with ox-LDL. RNA sequencing was used to detect transcriptome changes of VSMCs treated with ox-LDL. miR-183-5p and FOXO1 expression levels in VSMCs after ox-LDL treatment were assessed using qRT-PCR and western blotting. The regulatory effect of miR-183-5p on FOXO1 has been tried to prove using a dual-luciferase reporter assay. The functions of miR-183-5p, and FOXO1 were analyzed by CCK-8 assay and flow cytometry assay. The tissue samples or serum samples of high fat-feeding mice and carotid atherosclerosis patients were collected, and the levels of miR-183-5p/FOXO1 were analyzed. RNA sequencing data showed 81 miRNAs including miR-183-5p was significantly changed after ox-LDL treatment in VSMCs. FOXO1, a miR-183-5p's potential target, was also down-regulated in ox-LDL treated cells. qRT-PCR and western blot found that expression of FOXO1 mRNA and protein significantly reduced in VSMCs treated with ox-LDL, accompanied by overexpression of miR-183-5p. miR-183-5p inhibited FOXO1 mRNA by binding to its 3' UTR. Interference miR-183-5p/FOXO1 could change proliferation/apoptosis imbalance in VSMCs under ox-LDL stimulation. Higher levels of miR-183-5p but reduced FOXO1 can be found in the thoracic aorta tissues of high fat-feeding mice. In serum samples from individuals with carotid atherosclerosis, Higher levels of miR-183-5p were observed. the miR-183-5p level was positively related to the level of serum ox-LDL in patients. Aberrant expression of miR-183-5p/FOXO1 pathway mediated ox-LDL-induced proliferation/apoptosis imbalance in VSMCs. The miR-183-5p/FOXO1 axis can potentially be utilized as the target in the treatment of patients with atherosclerosis.

Knockdown of NCK1-AS1 inhibits the development of atherosclerosis by targeting miR-1197/COX10 axis

BackgroundAlthough long non-coding RNA (lncRNA) NCK1-AS1 plays important roles in human cancer, its function in atherosclerosis (AS) remains unclear.MethodThe expression of NCK1-AS1 in AS blood samples was detected by qRT-PCR. Oxidized low-density lipoprotein (ox-LDL) was used to construct the AS cell model, and quantitative real-time polymerase chain reaction (qRT-PCR) assay was used to evaluate NCK1-AS1 level. Cell phenotypes including proliferation and apoptosis were assessed by Cell Counting Kit-8 (CCK-8) assay and flow cytometer, respectively. The malondialdehyde level was measured to evaluate oxidative stress. The expression of apoptosis-related proteins was evaluated by western blot. The expression of inflammatory cytokines (IL-1β, IL-6 and TNK-α) was measured by qRT-PCR and ELISA assays. The relationship among NCK1-AS1, miR-1197 and COX10 was determined by bioinformatic analysis and luciferase reporter assay.ResultsNCK1-AS1 was significantly upregulated in AS blood samples and ox-LDL stimulated vascular smooth muscle cells (VSMCs). Knockdown of NCK1-AS1 increased cell viability, reduced cell apoptosis and MDA level, and also inhibited the expression of inflammatory cytokines (IL-1β, IL-6 and TNK-α) in ox-LDL stimulated VSMCs. NCK1-AS1 could positively regulate COX10 expression by directly sponging miR-1197. Moreover, co-transfection of sh-NCK1-AS1 and miR-1197 inhibitor, or co-transfection of sh-NCK1-AS1 and pc-COX10 (COX10 overexpressing plasmid) obviously reduced cell viability, promoted cell apoptosis, and increased MDA level in VSMCs followed by ox-LDL treatment for 24 h compared to that in sh-NCK1-AS1 transfected VSMCs.ConclusionOur study revealed that knockdown of NCK1-AS1 attenuated the development of AS by regulating miR-1197/COX10 axis, suggesting that this lncRNA might be a potential therapeutic target for AS.

Biological evaluation of linalool on the function of blood vessels.

Long-term hypertension leads to alterations in the structure and function of blood vessels, and abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are important factors for these changes. Linalool is a natural compound extracted from plants. The present study aimed to explore the role and underlying mechanism of linalool in the physiological behavior of VSMCs. Angiotensin II (Ang II) was utilized to treat VSMCs, and MTT and western blotting assays were then employed to detect the effect of linalool on the induced proliferation and migration of VSMCs. The target gene of linalool was predicted by the SwissTargetPrediction website, and its expression level in VSMCs was determined using reverse transcription-quantitative PCR and western blotting. Next, the role of the target gene in the physiological behavior of VSMCs treated with linalool was examined, and the signaling pathway was explored. The results revealed that the proliferation and migration of VSMCs treated with Ang II were significantly promoted, and linalool could alleviate these effects in a dose-dependent manner. Cholinergic receptor muscarinic 3 (CHRM3), as a predicted target, was found to be highly expressed in Ang II-induced VSMCs, and CHRM3 overexpression could prevent the inhibitory effect of linalool on cell proliferation and migration. In addition, its overexpression caused an increase in the expression of proteins related to the MAPK signaling pathway. In conclusion, linalool inhibited the proliferation and migration of Ang II-induced VSMCs and blocked the MAPK signaling pathway by downregulating CHRM3.

Abstract 36: High Salt Induces Vascular Damage Through Redox-sensitive Parp/trpm2-induced Calcium Influx And Inflammasome Modulation Without Influencing Blood Pressure

High salt diet (HSD) has deleterious effects on the vasculature by mechanisms not fully elucidated. We demonstrated tight coupling between Na + and Ca 2+ levels in VSMCs, where PARP-regulated TRPM2, a redox-sensitive Ca 2+ channel, plays an important role. Increased [Ca 2+ ]i also contributes to inflammasome assembly, which dysregulates vascular function. We hypothesized that HSD induces a pro-oxidant environment that contributes to PARP-induced TRPM2-activation, Ca 2+ influx and inflammasome assembly, leading to vascular damage. WKY rats were treated with 1% HSD (3 weeks). Blood pressure was assessed by tail-cuff methodology, vascular reactivity was assessed in mesenteric arteries and calcium influx, ROS generation, inflammasome and pro-contractile marker in vascular smooth muscle cells (VSMCs) in presence and absence of HS medium (HSM-140mM). HSD did not increase blood pressure (BP), but vascular contractility was exaggerated (Emax(mN): WT 10.20 ± 0.70 vs 15.17 ± 1.74), effect reversed by PARP (Emax: 11.14 ± 1.24) and TRPM2 (8-br) (Emax: 11.45 ± 1.11) inhibitors. In VSMCs, HSM behaves as a pro-oxidant agent (ROS-AU: Control 77.51 ± 2.80 vs 130.04 ± 13.89 HSM), leading to increased [Ca 2+ ]i (AUC: Control 25562.45 ± 880.48 vs 30924.8 ± 1263.85 HSM) and activation of myosin light chain (pMLC-AU: Control 99.27 ± 1.01 vs 626.87 ±71.28 HSM), by mechanisms dependent on ROS and PARP/TRPM2 activation. HSM also increased expression of inflammasome components NLRP3 (2 ΔΔCt : Control 1.20 ± 0.01 vs 1.85 ± 0.19 HSM), ASC (2 ΔΔCt : Control 1.02 ± 0.01 vs 1.49 ± 0.17 HSM) and Caspase 1 (2 ΔΔCt : Control 1.06 ± 0.03 vs 2.35 ± 0.46 HSM), which was prevented by ROS scavenger Tiron and PARP inhibitor. In conclusion, HSD-induced vascular hypercontractility involves ROS activation of PARP/TRPM2 signalling. Activation of PARP/TRPM2 was associated with increased [Ca 2+ ]i and activation of pro-contractile signaling and inflammasome assembly. Normal BP in HSD-fed rats in the presence of vascular damage suggests that ROS/PARP/TRPM2 signaling induced by salt influences vascular function independently of BP elevation. We identify a novel pathway that underlies salt-induced vascular damage and suggest a potential therapeutic role for PARP/TRPM2 inhibitors in vascular dysfunction.

LncRNA CDKN2B-AS1 hinders the proliferation and facilitates apoptosis of ox-LDL-induced vascular smooth muscle cells via the ceRNA network of CDKN2B-AS1/miR-126-5p/PTPN7

ObjectiveThe patterns of lncRNA CDKN2B-AS1 in coronary heart disease (CHD) have been extensively studied. This study investigated the competing endogenous RNA (ceRNA) network of CDKN2B-AS1 in coronary atherosclerosis (CAS). MethodsMicroarray analyses were performed to screen out the CHD-related lncRNAs (CDKN2B-AS1) and the downstream microRNAs (miR-126-5p). The expression of CDKN2B-AS1 in serum of patients with CHD and healthy volunteers was detected. Vascular smooth muscle cells (VSMCs) were treated with oxidized low density lipoprotein (ox-LDL) to establish the cell model. Then pcDNA-CDKN2B-AS1 and/or miR-126-5p mimic were transfected into ox-LDL-treated VSMCs to estimate cell proliferation, apoptosis and inflammation. The ceRNA network of CDKN2B-AS1 along with the possible pathway in CHD was testified. ResultsCDKN2B-AS1 expression was low in patients with CHD and ox-LDL-treated VSMCs. Upon CDKN2B-AS1 overexpression, TNF-α, NF-κB and IL-1β levels in VSMCs were decreased, the proliferation of VSMCs was inhibited and the apoptosis rate was increased. Overexpression of miR-126-5p could reverse these trends. CDKN2B-AS1 as a ceRNA competitively bound to miR-126-5p to upregulate PTPN7. CDKN2B-AS1 inhibited VSMC proliferation and accelerated apoptosis by inhibiting the PI3K-Akt pathway. ConclusionLncRNA CDKN2B-AS1 upregulates PTPN7 by absorbing miR-126-5p and inhibits the PI3K-Akt pathway, thus hindering the proliferation and accelerating apoptosis of VSMCs induced by ox-LDL, thus being a therapeutic approach for CAS.

MiR‑541‑3p inhibits the viability and migration of vascular smooth muscle cells via targeting STIM1.

The transformation of vascular smooth muscle cells(VSMCs) into the proliferative migratory phenotype in the plaque area contributes to stable plaque formation and facilitates the pathogenesis of atherosclerosis. Stromal interaction molecule1 (STIM1) has been identified to promote the proliferation of VSMCs, suggesting that STIM1 may be a potent target for the prevention and treatment of atherosclerosis. Bioinformatics analysis has previously predicted STIM1 as a target of microRNA (miR)‑541‑3p. The present study aimed to determine the effect of the miR‑541‑3p/STIM1 axis on the progression of atherosclerosis invitro. Oxidized low‑density lipoprotein (ox‑LDL)‑treated VSMCs were used as an invitro atherosclerosis model. Cell Counting Kit‑8 and Transwell migration assays were used to analyze cell viability and migration, respectively. Reverse transcription‑quantitative PCR and western blotting were applied to measure mRNA and protein expression levels, respectively. The association between miR‑541‑3p and STIM1 was detected using a dual luciferase gene reporter assay. The results of the present study revealed that ox‑LDL treatment significantly downregulated miR‑541‑3p expression levels and upregulated STIM1 expression levels in VSMCs. In addition, ox‑LDL stimulation enhanced cell viability and migration. The overexpression of miR‑541‑3p significantly reversed the ox‑LDL‑mediated increase in cell viability and migration, whereas the knockdown of miR‑541‑3p expression enhanced the ox‑LDL‑mediated effects. STIM1 was confirmed to be a target gene of miR‑541‑3p in VSMCs. The knockdown of STIM1 significantly impaired the stimulatory effects of miR‑541‑3p knockdown on cell viability and migration. In conclusion, the findings of the present study suggested that miR‑541‑3p may efficiently repress VSMC viability and migration by targeting STIM1 under the treatment of ox‑LDL. These results indicated that the miR‑541‑3p/STIM1 axis may represent a potent target to modulate VSMC viability and migration.

Acid sphingomyelinase promotes SGK1-dependent vascular calcification

In chronic kidney disease (CKD), hyperphosphatemia is a key factor promoting medial vascular calcification, a common complication associated with cardiovascular events and high mortality. Vascular calcification involves osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs), but the complex signaling events inducing pro-calcific pathways are incompletely understood. The present study investigated the role of acid sphingomyelinase (ASM)/ceramide as regulator of VSMC calcification. In vitro, both, bacterial sphingomyelinase and phosphate increased ceramide levels in VSMCs. Bacterial sphingomyelinase as well as ceramide supplementation stimulated osteo-/chondrogenic transdifferentiation during control and high phosphate conditions and augmented phosphate-induced calcification of VSMCs. Silencing of serum- and glucocorticoid-inducible kinase 1 (SGK1) blunted the pro-calcific effects of bacterial sphingomyelinase or ceramide. Asm deficiency blunted vascular calcification in a cholecalciferol-overload mouse model and ex vivo isolated-perfused arteries. In addition, Asm deficiency suppressed phosphate-induced osteo-/chondrogenic signaling and calcification of cultured VSMCs. Treatment with the functional ASM inhibitors amitriptyline or fendiline strongly blunted pro-calcific signaling pathways in vitro and in vivo. In conclusion, ASM/ceramide is a critical upstream regulator of vascular calcification, at least partly, through SGK1-dependent signaling. Thus, ASM inhibition by repurposing functional ASM inhibitors to reduce the progression of vascular calcification during CKD warrants further study.

Insulin enhances neointimal hyperplasia following arterial injury through the PI3K/Akt pathway in type 1 diabetic rats.

Although insulin is known to affect neointimal hyperplasia via distinct signaling pathways, how neointimal hyperplasia is affected in insulin‑deficient type1 diabetes remains unknown. The aim of the current study was to investigate two major signaling branches of insulin action regulating neointimal hyperplasia following arterial injury in type 1 diabetes with or without exogenous insulin administration. Rats were treated with vehicle (control group), streptozotocin(STZ) alone (STZ group; uncontrolled type1 diabetes) or STZ followed by insulin (STZ+I group; controlled type1 diabetes). Subsequently, a type 1 diabetic rat model of carotid artery balloon injury was established. Following this, the intima‑to‑media area ratios were examined for evidence of neointimal hyperplasia in the carotid arteries of the rats by performing hematoxylin‑eosin staining. Furthermore, the protein expression of extracellular signal‑regulated kinase (ERK), phosphorylated(p‑) ERK, protein kinaseB(Akt) and p‑Akt in the carotid arteries of the rats was determined via immunoblotting. Moreover, an invitro model of type1 diabetes was induced by incubation of primary vascular smooth muscle cells (VSMCs) with glucose and/or insulin. Cellular proliferation and signaling protein expression levels in VSMCs were determined by measuring the incorporation of tritiated thymidine and performing immunoblotting, respectively. The results demonstrated that compared with that in control rats, neointimal hyperplasia and expression of p‑Akt in uncontrolled type 1 diabetic rats were significantly decreased. This decrease was recovered in controlled type1 diabetes with insulin therapy. Furthermore, the difference in the expression of p‑ERK between groups was not significant. Additionally, the results of the cell experiments were consistent with those from the animal studies. In conclusion, the preferential signaling along the phosphatidylinositol3‑kinase/Akt pathway of insulin action in response to insulin restoration may contribute to neointimal hyperplasia. The present study provides a novel approach for the further treatment of neointimal hyperplasia in type1 diabetes.

Bazedoxifene inhibits PDGF-BB induced VSMC phenotypic switch via regulating the autophagy level

There is increasing evidence that Bazedoxifene, as an FDA-approved selective estrogen inhibitor, approved by FDA, not only inhibits estrogen receptors, but also has other pharmacological effects. The purpose of this study was to investigate the effects of Bazedoxifene on the functional changes of vascular smooth muscle cells (VSMCs) after PDGF-BB stimulation. VSMCs were divided into control group, PDGF-BB treatment group, and PDGF-BB treatment group with different concentrations of Bazedoxifene. CCK-8 and EdU staining were used to determine the VSMCs viability and proliferation. Western blot was used to detect the expressions of vimentin, SMA, ERK, p-ERK, STAT3, p-STAT3, AKT, p-AKT, and LC3 I/II. Wound healing method was used to detect the migration of VSMCs. PDGF-BB treatment significantly enhanced the viability and proliferation of VSMCs as indicated by CCK-8 and EdU assays (P < 0.01), while Bazedoxifene pretreatment could reduce the increased viability and proliferation of VSMCs caused by PDGF-BB (P < 0.05). Wound healing test also showed Bazedoxifene significantly attenuated the migration in the PDGF-BB stimulated VSMCs (P < 0.01). PDGF-BB also induced the phenotypic switch and decreased the autophagy level in VSMCs, manifested as a reduction in vimentin, SMA, and LC3 II (P < 0.01). These effects of PDGF-BB were partially reversed by Bazedoxifene (P < 0.05). Bazedoxifene may inhibit the proliferation and migration of VSMCs through up-regulate the autophagy level after PDGF-BB stimulation.