Mouse Vascular Smooth Muscle Research Articles

#1299 The SGLT2 inhibitor canagliflozin, but not dapagliflozin or empagliflozin, ameliorates vascular calcification in a mouse model of nephrocalcinosis

Abstract Background and Aims Vascular calcification, defined as the abnormal deposition of minerals in blood vessels, is a major cardiovascular risk factor in patients with kidney failure. Sodium-glucose co-transporter 2 (SGLT2) inhibitors, commonly known as gliflozins, are a class of pharmaceutical compounds that have revolutionised the therapeutic management of type 2 diabetes. These drugs target the SGLT2 transporter, increasing glucose elimination through the urine, effectively lowering hyperglycaemia. In addition to their effect on blood sugar control, SGLT2 inhibitors have recently gained attention for potential cardiovascular benefits in patients with type 2 diabetes, chronic kidney disease (CKD) or established cardiovascular disease. Initially thought to be secondary to the promotion of urinary sodium and glucose excretion and weight loss, recent studies have also reported direct effects of gliflozins on the vascular compartment. Given the significant impact of vascular calcification on cardiovascular health in patients with kidney failure, the aim of this study was to investigate whether the three different SGLT2 inhibitors (canagliflozin, empagliflozin and dapagliflozin) could reduce the development of vascular calcification. Method Western blotting was used to investigate SGLT2 expression in mouse and human vascular smooth muscle cells (VSMC) and vascular explants. Human and mouse aortic VSMC and mouse aorta and human epigastric and iliac artery segments were treated with calcification medium supplemented with 1 or 5 µM of gliflozins. A nephrocalcinosis mouse model consisting of 2 weeks of high phosphorus diet (2%) in female DBA/2 mice was used in in vivo experiments. The animals were treated daily with the different gliflozins (3 mg/kg/j) by oral gavage. Calcium deposits were quantified using a quantitative colorimetric assay and alizarin red staining, blood and urine glucose levels were measured using a quantitative colorimetric assay and glomerular filtration rate (GFR) was measured using sinistrin-FITC excretion. Results We observed that SGLT2 is expressed in mouse and human vessels and vascular smooth muscle cells (VSMC). Treatment with canagliflozin, but not dapagliflozin or empagliflozin, reduced calcification in vitro, in human and mouse VSMC and ex vivo, in mouse aorta and human epigastric and iliac artery segments. The high phosphorus diet in mice led to nephrocalcinosis associated with impaired renal function as well as vascular calcification development in the aorta. In this model, although all 3 gliflozins showed efficacy in increasing urinary glucose excretion, blood glucose levels were not affected by gliflozins and renal function was not improved. However, canagliflozin, but not dapagliflozin or empagliflozin, significantly reduced vascular calcification in this nephrocalcinosis mouse model. Conclusion Our study demonstrated that canagliflozin reduces vascular calcification development, highlighting its potential as a therapeutic strategy. This protective effect was found to be drug-dependent and not class-dependent, as the other two SGLT2 inhibitors, dapagliflozin and empagliflozin, showed no significant effect on vascular calcification. Further studies are needed to understand the mechanisms and pathways involved and to determine whether the differential effect of the gliflozins is due to an off-target action of canagliflozin on other SGLT transporters.

Abstract 1026: Apolipoprotein A1 On High-density Lipoprotein Interferes With Low-density Lipoprotein Binding To Proteoglycans

Background: The entrapment of apolipoprotein (APO) B-100 containing low density lipoproteins (LDL) by proteoglycans (PGs) in the extracellular matrix (ECM) of the arterial intima is a key initial step in the development of atherosclerotic vascular lesions. High density lipoproteins (HDL) can interfere with this process, but the underlying mechanism is not fully understood and is thought to be due to APOE. The aim of this study was to utilize our recently developed quantitative assay that measures the binding of LDL to PGs in the ECM and to investigate how HDL and other apolipoproteins may influence this process. Methods: An in-cell ELISA was used to measure the binding of LDL to PGs in the ECM synthesized by mouse vascular smooth muscle cells. Fast Protein Liquid Chromatography, immunoprecipitation, and immunoblotting analysis were performed to identify how HDL interacts with LDL to prevent binding to ECM-PGs. Results: LDL binding to PGs was inhibited by HDL and APOA1 in a dose dependent manner. Competition experiments showed that HDL does not compete directly with LDL for ECM-PG binding. Instead, our data revealed an association between LDL with APOA1 that diminishes LDL ability to bind ECM-PGs. Conclusion: HDL inhibits LDL binding to ECM-PG through an interaction with its main apolipoprotein, APOA1 that is different from APOE. Further investigation is warranted to understand the role of this interaction in the atherosclerosis process.

Corilagin relieves atherosclerosis via the toll-like receptor 4 signaling pathway in vascular smooth muscle cells.

Corilagin possesses a diverse range of pharmacologic bioactivities. However, the specific protective effects and mechanisms of action of corilagin in the context of atherosclerosis remain unclear. In this study, we investigated the impact of corilagin on the toll-like receptor (TLR)4 signaling pathway in a mouse vascular smooth muscle cell line (MOVAS) stimulated by oxidized low-density lipoprotein (ox-LDL). Additionally, we examined the effects of corilagin in Sprague-Dawley rats experiencing atherosclerosis. The cytotoxicity of corilagin was assessed using the CCK8 assay. MOVAS cells, pre-incubated with ox-LDL, underwent treatment with varying concentrations of corilagin. TLR4 expression was modulated by either downregulation through small interfering (si)RNA or upregulation via lentivirus transfection. Molecular expression within the TLR4 signaling pathway was analyzed using real-time polymerase chain reaction (PCR) and Western blotting. The proliferation capacity of MOVAS cells was determined through cell counting. In a rat model, atherosclerosis was induced in femoral arteries using an improved guidewire injury method, and TLR4 expression in plaque areas was assessed using immunofluorescence. Pathological changes were examined through hematoxylin and eosin staining, as well as Oil-Red-O staining. Corilagin demonstrated inhibitory effects on the TLR4 signaling pathway in MOVAS cells pre-stimulated with ox-LDL, consequently impeding the proliferative impact of ox-LDL. The modulation of TLR4 expression, either through downregulation or upregulation, similarly influenced the expression of downstream molecules. In an in vivo context, corilagin exhibited the ability to suppress TLR4 and MyD88 expression in the plaque lesion areas of rat femoral arteries, thereby alleviating the formation of atherosclerotic plaques. Corilagin can inhibit the TLR4 signaling pathway in VSMCs, possibly by downregulating TLR4 expression and, consequently, relieving atherosclerosis.

A novel assay to measure low-density lipoproteins binding to proteoglycans.

The binding of low-density lipoprotein (LDL) to proteoglycans (PGs) in the extracellular matrix (ECM) of the arterial intima is a key initial step in the development of atherosclerosis. Although many techniques have been developed to assess this binding, most of the methods are labor-intensive and technically challenging to standardize across research laboratories. Thus, sensitive, and reproducible assay to detect LDL binding to PGs is needed to screen clinical populations for atherosclerosis risk. The aim of this study was to develop a quantitative, and reproducible assay to evaluate the affinity of LDL towards PGs and to replicate previously published results on LDL-PG binding. Immunofluorescence microscopy was performed to visualize the binding of LDL to PGs using mouse vascular smooth muscle (MOVAS) cells. An in-cell ELISA (ICE) was also developed and optimized to quantitatively measure LDL-PG binding using fixed MOVAS cells cultured in a 96-well format. We used the ICE assay to show that, despite equal APOB concentrations, LDL isolated from adults with cardiovascular disease bound to PG to a greater extent than LDL isolated from adults without cardiovascular disease (p<0.05). We have developed an LDL-PG binding assay that is capable of detecting differences in PG binding affinities despite equal APOB concentrations. Future work will focus on candidate apolipoproteins that enhance or diminish this interaction.

Murine double minute 2-mediated estrogen receptor 1 degradation activates macrophage migration inhibitory factor to promote vascular smooth muscle cell dedifferentiation and oxidative stress during thoracic aortic aneurysm progression

Estrogen receptor 1 (ESR1) has been recently demonstrated as a potential diagnostic biomarker for thoracic aortic aneurysm (TAA). However, its precise role in the progression of TAA remains unclear. In this study, TAA models were established in ApoE-knockout mice and primary mouse vascular smooth muscle cells (VSMCs) through treatment with angiotensin (Ang) II. Our findings revealed a downregulation of ESR1 in Ang II-induced TAA mice and VSMCs. Upregulation of ESR1 mitigated expansion and cell apoptosis in the mouse aorta, reduced pathogenetic transformation of VSMCs, and reduced inflammatory infiltration and oxidative stress both in vitro and in vivo. Furthermore, we identified macrophage migration inhibitory factor (MIF) as a biological target of ESR1. ESR1 bound to the MIF promoter to suppress its transcription. Artificial MIF restoration negated the mitigating effects of ESR1 on TAA. Additionally, we discovered that murine double minute 2 (MDM2) was highly expressed in TAA models and mediated protein degradation of ESR1 through ubiquitination modification. Silencing of MDM2 reduced VSMC dedifferentiation and suppressed oxidative stress. However, these effects were reversed upon further silencing of ESR1. In conclusion, this study demonstrates that MDM2 activates MIF by mediating ESR1 degradation, thus promoting VSMC dedifferentiation and oxidative stress during TAA progression.

Zic family member 3 attenuates oxidative stress-induced vascular smooth muscle cell apoptosis in patients with chronic kidney disease

Neointimal hyperplasia is reportedly essential for arteriovenous fistulas (AVF) in patients undergoing hemodialysis. Oxidative stress is vital in the progression of uremic venous intimal hyperplasia. Studies have suggested that zinc ions obstruct vascular calcification in patients with chronic kidney disease (CKD). Recent studies have shown that the zinc finger protein, Zic family member 3 (ZIC3), is crucial for the earliest cardiovascular progenitors. ZIC3 mutations are associated with congenital heart disease. However, the mechanism of action of ZIC3 in vascular intimal hyperplasia in CKD remains unelucidated. Venous specimens were collected during primary AVF surgery and traumatic amputation, and serum samples were collected from patients with CKD and healthy controls. Mouse vascular smooth muscle cells (VSMCs) were treated with hydrogen peroxide (H2O2) to clarify the role of ZIC3 in CKD. ZIC3 expression was reduced in the veins of patients with uremia and the serum of those with CKD. Zic3 and Bcl2 levels were significantly decreased in mouse VSMCs treated with H2O2·H2O2 inhibited mouse VSMC activity, upregulated Bax, and cleaved caspase 3 expression. Following Zic3 overexpression, Bcl2 expression level and cell viability were elevated, whereas Bax and cleaved caspase 3 expression levels were downregulated. In contrast, Zic3 knockdown yielded the opposite results. Therefore, ZIC3 could be a new therapeutic target in venous neointimal hyperplasia of CKD.

Brazil nut treatment attenuates vascular calcification in mouse vascular smooth muscle cells

Brazil nut treatment attenuates vascular calcification in mouse vascular smooth muscle cells

SIRT6 is an epigenetic repressor of thoracic aortic aneurysms via inhibiting inflammation and senescence

Thoracic aortic aneurysms (TAAs) develop asymptomatically and are characterized by dilatation of the aorta. This is considered a life-threating vascular disease due to the risk of aortic rupture and without effective treatments. The current understanding of the pathogenesis of TAA is still limited, especially for sporadic TAAs without known genetic mutation. Sirtuin 6 (SIRT6) expression was significantly decreased in the tunica media of sporadic human TAA tissues. Genetic knockout of Sirt6 in mouse vascular smooth muscle cells accelerated TAA formation and rupture, reduced survival, and increased vascular inflammation and senescence after angiotensin II infusion. Transcriptome analysis identified interleukin (IL)-1β as a pivotal target of SIRT6, and increased IL-1β levels correlated with vascular inflammation and senescence in human and mouse TAA samples. Chromatin immunoprecipitation revealed that SIRT6 bound to the Il1b promoter to repress expression partly by reducing the H3K9 and H3K56 acetylation. Genetic knockout of Il1b or pharmacological inhibition of IL-1β signaling with the receptor antagonist anakinra rescued Sirt6 deficiency mediated aggravation of vascular inflammation, senescence, TAA formation and survival in mice. The findings reveal that SIRT6 protects against TAA by epigenetically inhibiting vascular inflammation and senescence, providing insight into potential epigenetic strategies for TAA treatment.

Empagliflozin Attenuates Vascular Calcification in Mice with Chronic Kidney Disease by Regulating the NFR2/HO-1 Anti-Inflammatory Pathway through AMPK Activation.

Vascular calcification (VC) is associated with increased cardiovascular risks in patients with chronic kidney disease (CKD). Sodium-glucose cotransporter 2 inhibitors, such as empagliflozin, can improve cardiovascular and renal outcomes. We assessed the expression of Runt-related transcription factor 2 (Runx2), interleukin (IL)-1β, IL-6, AMP-activated protein kinase (AMPK), nuclear factor erythroid-2-related factor (Nrf2), and heme oxygenase 1 (HO-1) in inorganic phosphate-induced VC in mouse vascular smooth muscle cells (VSMCs) to investigate the mechanisms underlying empagliflozin's therapeutic effects. We evaluated biochemical parameters, mean artery pressure (MAP), pulse wave velocity (PWV), transcutaneous glomerular filtration rate (GFR), and histology in an in vivo mouse model with VC induced by an oral high-phosphorus diet following a 5/6 nephrectomy in ApoE-/- mice. Compared to the control group, empagliflozin-treated mice showed significant reductions in blood glucose, MAP, PWV, and calcification, as well as increased calcium and GFR levels. Empagliflozin inhibited osteogenic trans-differentiation by decreasing inflammatory cytokine expression and increasing AMPK, Nrf2, and HO-1 levels. Empagliflozin mitigates high phosphate-induced calcification in mouse VSMCs through the Nrf2/HO-1 anti-inflammatory pathway by activating AMPK. Animal experiments suggested that empagliflozin reduces VC in CKD ApoE-/- mice on a high-phosphate diet.

Murine Cytomegalovirus Infection Induced miR-1929-3p Down-Regulation Promotes the Proliferation and Apoptosis of Vascular Smooth Muscle Cells in Mice by Targeting Endothelin A Receptor and Downstream NLRP3 Activation Pathway.

Our previous study demonstrated in vivo that mouse cytomegalovirus (MCMV) infection promoted vascular remodeling after downregulation of miR-1929-3p. This study aimed to investigate the role of miR-1929-3p/ETAR/NLRP3 pathway in mouse vascular smooth muscle cells (MOVAS) after MCMV infection. First, PCR was used to detect the success of the infection. Second, MOVAS were transfected with the miR-1929-3p mimic, inhibitor, and ETAR overexpressed adenovirus vector. Cell proliferation was detected using EdU, whereas apoptosis was detected using flow cytometry. The expression of miR-1929-3p and ETAR were detected using qRT-PCR. Western blot detected proteins of cell proliferation, apoptosis, and the NLRP3 inflammasome. Interleukin-1β and interleukin-18 were determined using ELISA. The results revealed that after 48h, MCMV infection promoted the proliferation of MOVAS when the MOI was 0.01. MCMV infection increased ETAR by downregulating miR-1929-3p. The miR-1929-3p mimic reversed the proliferation and apoptosis, whereas the miR-1929-3p inhibitor promoted this effect. ETAR overexpression further promoted MCMV infection by downregulating miR-1929-3p-mediated proliferation and apoptosis. MCMV infection mediates the downregulation of miR-1929-3p and the upregulation of ETAR, which activates NLRP3 inflammasome. In conclusion, MCMV infection promoted the proliferation of MOVAS, possibly by downregulating miR-1929-3p, promoting the upregulation of the target gene ETAR and activating NLRP3 inflammasome.

Influence of FOSL1 Inhibition on Vascular Calcification and ROS Generation through Ferroptosis via P53-SLC7A11 Axis.

Vascular calcification during aging is highly prevalent in patients with cardiovascular disease; however, there is still no improvement in clarifying the development of vascular calcification. FOSL1 is a transcription regulator belonging to the AP-1 family, which has a unique function in vascular senescence, but its role in vascular calcification needs to be further explored. Primary mouse vascular smooth muscle cells were isolated and used to construct a calcification model in vitro. Seven-week-old male C57BL/6 mice were used to build the vitD3-induced calcification model in vivo. qRT-PCR and western blot were used to verify the expression of FOSL1 and other genes expressed in vascular smooth muscle cells and aortas. The level of calcification was determined by Alizarin Red S (ARS) staining and the calcium content assay. The level of cellular GSH was detected by the GSH assay kit. Here, we report that FOSL1 was up-regulated after high-calcium/phosphate treatment in both the in vivo and in vitro vascular calcification models. Functional studies have shown that the reduction of FOSL1 attenuates ferroptosis and calcification in vascular smooth muscle cells, as indicated by ARS staining, calcium content assay, and western blot. The inhibition of FOSL1 downregulated the expression of bone-related molecules including Msh Homeobox 2 (MSX2) and tumor necrosis factor receptor superfamily, member 11b/osteoprotegerin (OPG), suggesting that FOSL1 promoted osteogenic differentiation of vascular smooth muscle cells. Furthermore, we found that the ferroptosis-inducing drug erastin can significantly accelerate calcification in the aortic ring while Ferrostatin-1 (fer-1), a drug to protect cells from ferroptosis, can alleviate calcification. Further experiments have shown that inhibiting FOSL1 can promote the expression of ferroptosis-related genes and attenuate calcification. Functionally, cellular GSH levels were increased after the reduction of FOSL1. In this study, we observed a significant protective effect when we reduced the expression of FOSL1 during vascular calcification, and this effect might regulate ferroptosis to a great extent.

Effects of Simvastatin on Endoplasmic Reticulum Stress-Mediated Apoptosis in Atherosclerotic Calcification

Objective: The effectiveness of statins in reducing atherosclerotic calcification remains controversial. The aim of this study was to confirm that simvastatin reduces atherosclerotic calcification and stabilizes plaque by restricting endoplasmic reticulum stress (ERS)-mediated apoptosis. Methods: Twenty-four 8-week-old male apolipoprotein E (ApoE)-/- mice (C57BL/6J genetic background) were selected and randomly divided into model (n = 12) and simvastatin (n = 12) groups. Twelve male C57BL/6J mice were selected as control group (n = 12). The mice were adaptively fed for 2 weeks and were put on a high-fat diet thereafter. After 9 weeks, they were treated with simvastatin (20 mg/kg) or phosphate-buffered saline daily for 8 weeks. Aortic sinus samples were obtained from ApoE-/- and C57BL/6J mice for hematoxylin and eosin, von Kossa, alizarin Red S, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and immunohistochemical staining after in vivo treatment with simvastatin. In addition, mouse vascular smooth muscle cells were analyzed after exposure to simvastatin in vitro. Results: Administration of simvastatin in vivo drastically attenuated the atherosclerosis, calcification, and apoptosis, and decreased the serum levels of triglycerides, total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol. The expression levels of glucose-regulated protein, 78 kDa (GRP78), C/EBP homologous protein (CHOP), and caspase 12 (CASP12) in the aortic sinus decreased in the simvastatin group compared with the model group. In vitro, simvastatin or simvastatin plus ERS inhibitor (taurine) attenuated calcification and apoptosis, and reduced the expression of ERS-related proteins GRP78, CHOP, and CASP12. Conclusion: Treatment with simvastatin suppressed atherosclerotic calcification. This effect may be mediated through the inhibition of ERS-related apoptosis.

Klotho Ameliorates Vascular Calcification via Promoting Autophagy.

Vascular calcification (VC) is regarded as a common feature of vascular aging. Klotho deficiency reportedly contributes to VC, which can be ameliorated by restoration of Klotho expression. However, the specific mechanisms involved remain unclear. Here, we investigated the role of autophagy in the process of Klotho-inhibiting VC. The clinical study results indicated that, based on Agatston score, serum Klotho level was negatively associated with aortic calcification. Then, Klotho-deficient mice exhibited aortic VC, which could be alleviated with the supplementation of Klotho protein. Moreover, autophagy increased in the aorta of Klotho-deficient mice and protected against VC. Finally, we found that Klotho ameliorated calcification by promoting autophagy both in the aorta of Klotho-deficient mice and in mouse vascular smooth muscle cells (MOVAS) under calcifying conditions. These findings indicate that Klotho deficiency induces increased autophagy to protect against VC and that Klotho expression further enhances autophagy to ameliorate calcification. This study is beneficial to exploring the underlying mechanisms of Klotho regulating VC, which has important guiding significance for future clinical studies in the treatment of VC.

2,3-Diphospho-D-glyceric acid inhibits calciprotein particle growth and calcification in MOVAS cells but not in MC3T3-E1 cells

Anionic polyphosphate compounds are well-known to inhibit the formation of hydroxyapatite crystals. Vascular calcification (VC) is associated with an increase in crystalline calciprotein particles (CPPs) in the bloodstream. 2,3-Diphospho-D-glyceric acid (2,3-DPG) is a highly anionic Poly-P compound found in the concave center of erythrocytes where it binds to hemoglobin, thereby reducing oxygen affinity. Since 2.3-DPG exists in plasma, we hypothesized that 2,3-DPG may act as an endogenous inhibitor of VC. In comparison to widely recognized calcification inhibitors such as myo-inositol hexakisphosphate, 2,3-DPG significantly delayed the formation of crystalline CPPs. Additionally, 2,3-DPG inhibited calcification in a mouse vascular smooth muscle cell line (MOVAS) without cytotoxic effects. Taken together with the result that 2,3-DPG did not impact bone-like nodule formation in mouse osteoblast-like MC3T3-E1 cells, these results suggest that 2,3-DPG may inhibit VC selectively.

Local Ca2+ Signals within Caveolae Cause Nuclear Translocation of CaMK1α in Mouse Vascular Smooth Muscle Cells.

An increase in intracellular Ca2+ concentration ([Ca2+]i) activates Ca2+-sensitive enzymes such as Ca2+/calmodulin-dependent kinases (CaMK) and induces gene transcription in various types of cells. This signaling pathway is called excitation-transcription (E-T) coupling. Recently, we have revealed that a L-type Ca2+ channel/CaMK kinase (CaMKK) 2/CaMK1α complex located within caveolae in vascular smooth muscle cells (SMCs) can convert [Ca2+]i changes to gene transcription profiles that are related to chemotaxis. Although CaMK1α is expected to be the key molecular identity that can transport Ca2+ signals originated within caveolae to the nucleus, data sets directly proving this scheme are lacking. In this study, multicolor fluorescence imaging methods were utilized to address this question. Live cell imaging using mouse primary aortic SMCs revealed that CaMK1α can translocate from the cytosol to the nucleus; and that this movement was blocked by nifedipine or a CaMKK inhibitor, STO609. Experiments using two types of Ca2+ chelators, ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), combined with caveolin-1 knockout (cav1-KO) mice showed that local Ca2+ events within caveolae are required to trigger this CaMK1α nuclear translocation. Importantly, overexpression of cav1 in isolated cav1-KO myocytes recovered the CaMK1α translocation. In SMCs freshly isolated from mesenteric arteries, CaMK1α was localized mainly within caveolae in the resting state. Membrane depolarization induced both nuclear translocation and phosphorylation of CaMK1α. These responses were inhibited by nifedipine, STO609, cav1-KO, or BAPTA. These new findings strongly suggest that CaMK1α can transduce Ca2+ signaling generated within or very near caveolae to the nucleus and thus, promote E-T coupling.

Mmu_circ_0000271 regulated the growth of ox-LDL-stimulated mouse vascular smooth muscle cells via sponging miR-5123.

Atherosclerosis (AS) is a complex inflammatory disease of the arterial wall. Mmu_circ_0000271 was found to be aberrantly expressed in AS, but its function and mechanism in AS have not been reported. This study investigated the role of mmu_circ_0000271 in AS. Mmu_circ_0000271 were identified using UCSC website, RNase R treatment, and nuclear‑cytoplasmic extraction. And mouse vascular smooth muscle cells (VSMCs) were used to establish the AS cell model by treating oxidized low-density lipoprotein (ox-LDL, 50μg/ml). Next, the expression of circ_0000271 in VSMCs was determined by quantitative real-time PCR. The effect of overexpressed or silenced circ_0000271 on ox-LDL-treated VSMCs viability, angiogenesis and invasion were detected by MTT, tube formation and transwell assays. The proteins expressions of proliferative and migratory markers were measured by western blot. After the target relationship predication between circ_0000271 and miR-5123, these experiments were performed again to evaluate the effect of miR-5123 inhibitor on AS model cells. Mmu_circ_0000271 was stably and highly expressed in ox-LDL-treated VSMCs, and its expression was enriched in the cell cytoplasm. Circ_0000271 overexpression further promoted the ox-LDL-induced viability, angiogenesis and invasion, as well as the proliferation- and migration-related proteins in VSMCs, whereas circ_0000271 knockdown had the opposite effect. Moreover, circ_0000271 was found to target miR-5123 and circ_0000271 overexpression suppressed its expression. And miR-5123 inhibitor was capable of reversing the effect of shcirc_0000271 on AS model cells. Mmu_circ_0000271 regulated ox-LDL-induced VSMC proliferation and migration through bind to miR-5123, and it could serve as a potential biomarker for AS.

Inhibition of XIST attenuates abdominal aortic aneurysm in mice by regulating apoptosis of vascular smooth muscle cells through miR-762/MAP2K4 axis.

Abdominal aortic aneurysm (AAA) is a common chronic aortic degenerative disease. Long non-coding RNA X-inactive specific transcript (XIST) is associated with the progression of AAA, while the underlying mechanism is still unclear. We investigated the functional role of XIST in AAA. AAA mouse model was established by administration of Angiotensin II (Ang II). Primary mouse vascular smooth muscle cells (VSMCs) were separated from the abdominal aorta of Ang II-induced AAA mice, and then treated with Ang II. XIST was highly expressed in Ang II-treated VSMCs. Cell proliferation ability was decreased and apoptosis was increased in VSMCs following Ang II treatment. XIST knockdown reversed the impact of Ang II on cell proliferation and apoptosis in VSMCs. XIST promoted mitogen-activated protein kinase kinase 4 (MAP2K4) expression by sponging miR-762. XIST overexpression suppressed cell proliferation and apoptosis of Ang II-treated VSMCs by regulating miR-762/MAP2K4 axis. Finally, Ang II-induced AAA mouse model was established to verify the function of XIST in AAA. Inhibition of XIST significantly attenuated the pathological changes of abdominal aorta tissues in Ang II-induced mice. The expression of miR-762 was inhibited, and MAP2K4 expression was enhanced by XIST knockdown in the abdominal aorta tissues of AAA mice. In conclusion, these data demonstrate that inhibition of XIST attenuates AAA in mice, which attributes to inhibit apoptosis of VSMCs by regulating miR-762/MAP2K4 axis. Thus, this study highlights a novel ceRNA circuitry involving key regulators in the pathogenesis of AAA.

Irisin protects against vascular calcification by activating autophagy and inhibiting NLRP3-mediated vascular smooth muscle cell pyroptosis in chronic kidney disease

Irisin protects the cardiovascular system against vascular diseases. However, its role in chronic kidney disease (CKD) -associated vascular calcification (VC) and the underlying mechanisms remain unclear. In the present study, we investigated the potential link among Irisin, pyroptosis, and VC under CKD conditions. During mouse vascular smooth muscle cell (VSMC) calcification induced by β-glycerophosphate (β-GP), the pyroptosis level was increased, as evidenced by the upregulated expression of pyroptosis-related proteins (cleaved CASP1, GSDMD-N, and IL1B) and pyroptotic cell death (increased numbers of PI-positive cells and LDH release). Reducing the pyroptosis levels by a CASP1 inhibitor remarkably decreased calcium deposition in β-GP-treated VSMCs. Further experiments revealed that the pyroptosis pathway was activated by excessive reactive oxygen species (ROS) production and subsequent NLR family pyrin domain containing 3 (NLRP3) inflammasome activation in calcified VSMCs. Importantly, Irisin effectively inhibited β-GP-induced calcium deposition in VSMCs in vitro and in mice aortic rings ex vivo. Overexpression of Nlrp3 attenuated the suppressive effect of Irisin on VSMC calcification. In addition, Irisin could induce autophagy and restore autophagic flux in calcified VSMCs. Adding the autophagy inhibitor 3-methyladenine or chloroquine attenuated the inhibitory effect of Irisin on β-GP-induced ROS production, NLRP3 inflammasome activation, pyroptosis, and calcification in VSMCs. Finally, our in vivo study showed that Irisin treatment promoted autophagy, downregulated ROS level and thereby suppressed pyroptosis and medial calcification in aortic tissues of adenine-induced CKD mice. Together, our findings for the first time demonstrated that Irisin protected against VC via inducing autophagy and inhibiting VSMC pyroptosis in CKD, and Irisin might serve as an effective therapeutic agent for CKD-associated VC.

Niclosamide downregulates LOX-1 expression in mouse vascular smooth muscle cells and changes the composition of atherosclerotic plaques in ApoE-/- mice.

Genetic lineage tracing studies have shown that phenotypic switching of vascular smooth muscle cells (VSMCs) results in less-differentiated cells, including macrophage-like cells that lack traditional VSMC markers. This switching contributes to the formation of necrotic core in plaques and promotes atherosclerosis, which is important for plaque stability. Niclosamide, a commonly used anti-helminthic drug, has recently attracted attention as an anti-cancer drug that inhibits multiple signaling pathways. The expression of the S100A4 protein is upregulated in synthetic VSMCs and inhibited by niclosamide on metastatic progression in colon cancer. We aimed to test the effect of niclosamide on VSMC phenotype switching and plaque stability. To examine murine atherosclerosis, we induced experimental lesions by blood flow cessation in apolipoprotein E knockout mice fed a high-fat diet. Oral administration of niclosamide changed 4-week-old plaques to collagen-rich and less-necrotic core phenotypes and downregulated the expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in vivo. In vitro analysis indicated that niclosamide reduced LOX-1 expression in VSMCs in a concentration-dependent and S100A4-independent manner. The inhibitory effect of niclosamide on LOX-1 and collagen type I was associated with the inactivation of the nuclear factor-κB signaling pathway. We demonstrated that the administration of niclosamide reduced LOX-1 expression and altered the composition of murine carotid plaques. Our results highlight the potential of niclosamide as an atheroprotective agent that enhances atherosclerotic plaque stability.

Gut microbiota dependent trimethylamine N-oxide aggravates angiotensin II–induced hypertension

Gut microbiota produce Trimethylamine N-oxide (TMAO) by metabolizing dietary phosphatidylcholine, choline, l-carnitine and betaine. TMAO is implicated in the pathogenesis of chronic kidney disease (CKD), diabetes, obesity and atherosclerosis. We test, whether TMAO augments angiotensin II (Ang II)-induced vasoconstriction and hence promotes Ang II-induced hypertension. Plasma TMAO levels were indeed elevated in hypertensive patients, thus the potential pathways by which TMAO mediates these effects were explored. Ang II (400 ng/kg−1min−1) was chronically infused for 14 days via osmotic minipumps in C57Bl/6 mice. TMAO (1%) or antibiotics were given via drinking water. Vasoconstriction of renal afferent arterioles and mesenteric arteries were assessed by microperfusion and wire myograph, respectively. In Ang II-induced hypertensive mice, TMAO elevated systolic blood pressure and caused vasoconstriction, which was alleviated by antibiotics. TMAO enhanced the Ang II-induced acute pressor responses (12.2 ± 1.9 versus 20.6 ± 1.4 mmHg; P < 0.05) and vasoconstriction (32.3 ± 2.6 versus 55.9 ± 7.0%, P < 0.001). Ang II-induced intracellular Ca2+ release in afferent arterioles (147 ± 7 versus 234 ± 26%; P < 0.001) and mouse vascular smooth muscle cells (VSMC, 123 ± 3 versus 157 ± 9%; P < 0.001) increased by TMAO treatment. Preincubation of VSMC with TMAO activated the PERK/ROS/CaMKII/PLCβ3 pathway. Pharmacological inhibition of PERK, ROS, CaMKII and PLCβ3 impaired the effect of TMAO on Ca2+ release. Thus, TMAO facilitates Ang II-induced vasoconstriction, thereby promoting Ang II-induced hypertension, which involves the PERK/ROS/CaMKII/PLCβ3 axis.