Promoted Vascular Smooth Muscle Cell Calcification Research Articles

The novel role of complement 3 in Wnt5a-mediated vascular smooth muscle cell calcification

Abstract Background Although vascular calcification (VC) is a risk factor for cardiovascular and all-cause mortality, a therapy is not yet available. VC involves the transdifferentiation of vascular smooth muscle cells (VSMC) towards an osteochondrogenic lineage that results in calcium phosphate salts deposition in the arterial wall. We have previously shown an association between plasma WNT5A levels and new onset and progression of coronary artery calcification. Purpose We aimed to study the poorly understood role of Wnt5a in VSMC calcification. Methods Study 1 included 18 patients with coronary artery disease (CAD) undergoing cardiac surgery; VSMC were isolated from their saphenous veins to perform in vitro assays and microarrays. Study 2 included 11 patients with CAD; IMA were treated ex vivo with Wnt5a recombinant protein (rWnt5a) to perform western blot (WB) analyses. Study 3 included 647 patients with CAD; RNA sequencing was performed on samples from their internal mammary arteries (IMA), IMA perivascular adipose (PVAT), and thoracic adipose tissue (ThAT). VSMC and human aortic smooth muscle cells (HASMC) from healthy donors were grown in osteogenic medium in the presence or absence of rWnt5a with or without specific protein inhibitors. Calcium deposition and expression of VC markers (i.e. RUNX2, BMP2, and MSX2) were assessed with a colorimetric assay and by RT-qPCR, respectively. Phosphorylation/activation of non-canonical Wnt pathway effectors was evaluated by WB analyses. Results rWnt5a treatment significantly increased calcium deposition in both VSMC and HASMC at a concentration that activates both JNK and CaMKII, that are effectors of the non-canonical planar cell polarity pathway and the calcium-dependent pathway, respectively. Inhibition of either JNK or CaMKII could not counteract rWnt5a-mediated increase in VSMC calcium deposition; whereas, inhibition of both non-canonical pathway effectors (JNK-i and CaMKII-i) prevented rWnt5a-mediated increase in calcium deposition by VSMC (A) and HASMC. Inhibition of JNK and CaMKII also prevented rWnt5a-induced gene expression of VC markers, such as RUNX2 (B). rWnt5a treatment in IMA increased the activation of JNK and CaMKII. Patients with higher WNT5A levels in IMA had significantly higher RUNX2 levels. By intersecting RNA sequencing and microarray data complement 3 (C3) resulted as the most upregulated gene in rWnt5a-treated VSMC among those genes whose levels positively correlated in IMA with both WNT5A and RUNX2 expression (C). Patients with higher WNT5A levels in IMA, their PVAT, and ThAT had significantly higher C3 levels (D), and patients with higher levels of C3AR1, that is the gene coding for the C3a receptor, had higher VC markers expression. C3 inhibition (C3-i) with a C3AR1 antagonist counteracted Wnt5a-induced VSMC calcium deposition (E). Conclusions Non-canonical Wnt signalling promotes VSMC calcification through C3. Wnt5a may be a therapeutic target in VC.Abstract Figure

Endothelial cells derived extracellular vesicles promote diabetic arterial calcification via circ_0008362/miR-1251-5p/Runx2 axial

IntroductionArterial calcification, an independent predictor of cardiovascular events, increases morbidity and mortality in patients with diabetes mellitus (DM), but its mechanisms remain unclear. Extracellular vesicles (EVs) play an important role in intercellular communication. The study investigates the role and potential mechanisms of EVs derived from endothelial cells (ECs) in regulating vascular smooth muscle cell (VSMC) calcification under high glucose (HG) condition, with a goal of developing effective prevention and treatment strategies for diabetic arterial calcification.ResultsThe results showed that EVs derived from HG induced ECs (ECHG-EVs) exhibited a bilayer structure morphology with a mean diameter of 74.08 ± 31.78 nm, expressing EVs markers including CD9, CD63 and TSG101, but not express calnexin. ECHG-EVs was internalized by VSMCs and induced VSMC calcification by increasing Runx2 expression and mineralized nodule formation. The circ_0008362 was enriched in ECHG-EVs, and it can be transmitted to VSMCs to promote VSMC calcification both in vitro and in vivo. Mechanistically, miR-1251-5p might be one of the targets of circ_0008362 and they were co-localization in the cytoplasm of VSMCs. Runx2 was identified as the downstream target of miR-1251-5p, and circ_0008362 acted as a sponge, enhancing Runx2 expression and then promoted VSMC calcification. Besides, circ_0008362 could directly interact with Runx2 to aggravate VSMC calcification. Notably, DiR-labelled ECHG-EVs was detected in the vessels of mice. Meanwhile, the level of circ_0008362 and Runx2 were increased significantly, while the expression of miR-1251-5p was decreased significantly in calcified artery tissues of mice. However, inhibiting the release of EVs by GW4869 attenuated arterial calcification in diabetic mice. Finally, the level of circulation of plasma EVs circ_0008362 was significantly higher in patients with DM compared with normal controls. Elevated levels of plasma EVs circ_0008362 were associated with more severe coronary and aorta artery calcification in patients with DM.ConclusionsOur findings suggested that circ_0008362 was enriched in EVs derived from ECs and promoted VSMC calcification under HG conditions, both by sponging miR-1251-5p to upregulate Runx2 expression and through direct interaction with Runx2. Furthermore, elevated levels of plasma EVs circ_0008362 were associated with more severe coronary and aorta artery calcification in patients with DM. These results may serve as a potential prevention and therapeutic target for diabetic arterial calcification.Graphical abstract

Interaction of Sp1 and Setd8 promotes vascular smooth muscle cells apoptosis by activating Mark4 in vascular calcification.

Vascular calcification (VC) is directly related to high mortality in chronic kidney disease (CKD), and cellular apoptosis of vascular smooth muscle cells (VSMCs) is a crucial process in the initiation of VC. Microtubule affinity-regulating kinase 4 (Mark4), known as a serine/threonine protein kinase, can induce cell apoptosis and autophagy by modulating Akt phosphorylation. However, the potential functions and molecular mechanisms of Mark4 in VSMCs apoptosis and calcification need to be further explored. Initially, our data indicated that the mRNA expression of Mark4 was prominently elevated in high phosphorus-stimulated human VSMCs compared with the other members in Marks. Consistently, Mark4 expression was found to be significantly increased in the calcified arteries of both CKD patients and rats. In vitro, silencing Mark4 suppressed apoptosis-specific marker expression by promoting Akt phosphorylation, finally attenuating VSMCs calcification induced by high phosphate. Mechanically, the transcription factor Sp1 was enriched in the Mark4 promoter region and modulated Mark4 transcription. Moreover, SET domain-containing protein 8 (Setd8) was proved to interact with Sp1 and jointly participated in the transcriptional regulation of Mark4. Finally, rescue experiments revealed that Setd8 contributed to VSMCs apoptosis and calcification by modulating Mark4 expression. In conclusion, these findings reveal that Mark4 is transcriptionally activated by Sp1, which is interacted with Setd8, to promote VSMCs calcification through Akt-mediated antiapoptotic effects, suggesting that Mark4 represents a potent and promising therapeutic target for VC in CKD.

Long non-coding RNA MALAT1 sponges miR-30c to promote the calcification of human vascular smooth muscle cells by regulating Runx2

Objectives Recent evidence suggested that long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) play critical roles in the pathogenesis of vascular calcification (VC). In this study, we tried to explore the expression and role of a lncRNA, i.e., metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), and a miRNA, i.e., miR-30c, in VC. Methods In vitro VC model was induced in human vascular smooth muscle cells (VSMCs) after 10 days culture in calcifying medium containing 2 mM Na2HPO4. Alizarin red S staining, calcium assay and western blot analysis of runt-related transcription factor 2 (Runx2) and alpha smooth muscle actin (α-SMA) were performed to evaluate VC. Knockdown of MALAT1 and up-regulation of MALAT1, miR-30c and Runx2 was performed to determine the impact of these molecules on VSMCs calcification. Dual-luciferase report assay was performed to confirm the relationship between MALAT1 and miR-30c or miR-30c and Runx2. In addition, quantitative reverse transcription PCR and western blot were used to determine gene and protein expression. Results MALAT1 was increased, while miR-30c was decreased in calcified VSMCs. Knockdown of MALAT1 suppressed VSMCs calcification; on the contrary, up-regulation of MALAT1 promoted VSMCs calcification. The effect of MALAT1 over-expression on VSMCs calcification was reversed by upregulation of miR-30c, which was reversed again by upregulation of Runx2. Dual-luciferase report assay confirmed that there is a direct interaction between MALAT1 and miR-30c, and Runx2 is a direct target of miR-30c. Conclusion MALAT1 over-expression promoted VSMCs calcification, which was at least partially through regulating the miR-30c/Runx2 axis.

In this Issue

HomeCirculation ResearchVol. 132, No. 4In this Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn this Issue Originally published16 Feb 2023https://doi.org/10.1161/RES.0000000000000595Circulation Research. 2023;132:397is related toADAR1 Non-Editing Function in Macrophage Activation and Abdominal Aortic AneurysmCirculating Extracellular Vesicle-Propagated microRNA Signature as a Vascular Calcification Factor in Chronic Kidney DiseaseCNP Promotes Antiarrhythmic Effects via Phosphodiesterase 2CNP Promotes Anti-Arrhythmic Effects via Phosphodiesterase 2 (p 400)Download figureDownload PowerPointCachorro et al suggest new targets for treating arrhythmia in heart failure.If a person survives a myocardial infarction they remain at risk of eventual death from heart failure, with arrhythmia and sudden cardiac death being major causes. β-blockers are often used to mitigate arrhythmia in heart failure but have side effects such as hypotension, dizziness and brachycardia due to the heart’s reduced contractile force. In the search for novel targets and treatments, it was discovered that overexpression of the enzyme phosphodiesterase 2 (PDE2), involved in cardiomyocyte contractility among other things, protected mice from arrhythmia and death after infarction. Furthermore, in rats with acute myocardial ischemia, treatment with the vasoactive molecule c-type natriuretic protein (CNP) also reduced arrhythmia. Cachorro and colleagues now show that these two separate findings are in fact linked. Indeed, CNP treatment of mice induced PDE2 to reduce arrhythmia, they found. And, pharmaceutical or genetic inhibition of PDE2 activity, prevented CNP’s antiarrhythmic effects. Similar results were found in cultured cells, where CNP suppressed arrhythmogenic activity in mouse and human cardiomyocytes but not if PDE2 was inhibited. Together the results point to the CNP-PDE2 pathway as a possible target for new heart failure medications, say the team.ADAR1 Non-Editing Function in Macrophage Activation and Abdominal Aortic Aneurysm (p e78)Download figureDownload PowerPointMacrophage ADAR1 is a key driver of abdominal aortic aneurysm, report Cai et al.Abdominal aortic aneurysm (AAA) is a life-threatening chronic inflammatory disorder in which the wall of the vessel becomes progressively weakened leading to its localized bulging and potential rupture. It causes around 10,000 deaths annually in the US alone and with the exception of surgery there are no targeted treatments. An RNA processing protein called ADAR1, with RNA-editing and non-editing functions, has been linked to the disease, being found at raised levels in mouse and patient AAA lesions. Deletion of ADAR1 from smooth muscle cells (SMCs)—key cells of blood vessel walls—has also been shown to reduce AAA formation in mice. Now Cai and colleagues show that SMCs are not the only cells in which ADAR1 plays a role in AAA. Mice whose macrophages lacked ADAR1, were more resistant to AAA induction than were their wildtype counterparts. And the ADAR1-lacking macrophages were less inflammatory. Mechanistically, the team showed that ADAR1 normally enhances inflammation not by its RNA editing activity but by degrading another RNA protein called Drosha, which triggers a molecular cascade to inflammatory signaling. Finally, the team showed this ADAR1-Drosha interaction also exists in human AAA tissue, thus confirming the clinical relevance of their discoveries.Circulating Extracellular Vesicle-Propagated microRNA Signature a A Vascular Calcification Factor in Chronic Kidney Disease (p 415)Download figureDownload PowerPointExtracellular vesicles drive vascular calcification in chronic kidney disease, say Koide et al.Declining renal function in chronic kidney disease (CKD) is associated with the risk of cardiovascular issues such as vascular calcification. This link to calcification is partly due to the body’s altered handling of minerals. But, as Koide and colleagues show, extracellular vesicles (EVs), also play a role. EVs are membrane-bound packets released from cells that act as an inter-cellular and inter-organ communication system. Previously, the serum of CKD patients, but not healthy subjects, was shown to promote calcification of cultured vascular smooth muscle cells (VSMCs), but the agent responsible was unknown. Now the team show, serum from rats with CKD also promotes VSMC calcification, but not if EVs are depleted. Furthermore, injection of an EV inhibitor into CKD-model mice prevented vascular calcification. Analyses of EVs from CKD rodents revealed four microRNAs that were less abundant than in control EVs. And these miRNAs were found to target and suppress the pro-calcification activity of growth factor VEGFA. The same four miRNAs were reduced in human CKD patient EVs where levels of the RNAs correlated with kidney function. The authors thus conclude, these EV RNAs have the potential to be biomarkers of disease severity as well as targets for new therapies. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesADAR1 Non-Editing Function in Macrophage Activation and Abdominal Aortic AneurysmDunpeng Cai, et al. Circulation Research. 2023;132:e78-e93Circulating Extracellular Vesicle-Propagated microRNA Signature as a Vascular Calcification Factor in Chronic Kidney DiseaseTakaaki Koide, et al. Circulation Research. 2023;132:415-431CNP Promotes Antiarrhythmic Effects via Phosphodiesterase 2Eleder Cachorro, et al. Circulation Research. 2023;132:400-414 February 17, 2023Vol 132, Issue 4 Advertisement Article InformationMetrics © 2023 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000595 Originally publishedFebruary 16, 2023 PDF download Advertisement

Exosomal STAT1 derived from high phosphorus‑stimulated vascular endothelial cells induces vascular smooth muscle cell calcification via the Wnt/β‑catenin signaling pathway.

Vascular calcification is commonly observed in chronic kidney disease. The mechanism of how the calcification signal from endothelial cells is transmitted to vascular smooth muscle cells (VSMCs) remains unknown. The aim of the present study was to investigate whether exosomes from HUVECs (HUVEC‑Exos) could regulate VSMC calcification and its potential signaling pathway. HUVEC‑Exos were isolated from HUVECs under no phosphorus (NP) and high phosphorus (HP) conditions. Alizarin Red S staining and calcium (Ca) content analysis were carried out to detect calcification in VSMCs. Proteomics analysis was carried out to detect the differential expression of exosomal proteins. Protein and mRNA levels were measured by western blot analysis and reverse transcription‑quantitative PCR (RT‑qPCR). Exosomes derived from HP‑HUVECs promoted the calcification of VSMCs, as assessed by Alizarin Red S staining, alkaline phosphatase activity assays, Ca content measurements and the increased expression of runt‑related transcription factor 2 and osteopontin. Proteomic analysis detected the upregulation of STAT1 in HP‑exosomes from HUVECs (HUVEC‑Exos) compared with NP‑HUVEC‑Exos, which was also confirmed by western blot analysis and RT‑qPCR. Inhibition of STAT1 expression in VSMCs using fludarabine or knockdown of STAT1 expression using small interfering RNA alleviated the calcification of VSMCs. Furthermore, lithium chloride (Wnt activator) reversed the protective effect of STAT1 inhibition on VSMC calcification, while Dickkopf‑1 (Wnt inhibitor) exerted the opposite effect, suggesting that activation of the Wnt/β‑catenin signaling pathway was involved in STAT1‑mediated VSMC calcification. In conclusion, the present results indicated that exosomal STAT1 derived from HP‑treated HUVECs could promote VSMC calcification, and activation of the Wnt/β‑catenin pathway may be a potential mechanism of the VSMC calcification promoted by exosomes.

Repression of the antiporter SLC7A11/glutathione/glutathione peroxidase 4 axis drives ferroptosis of vascular smooth muscle cells to facilitate vascular calcification

Vascular calcification is a common pathologic condition in patients with chronic kidney disease (CKD). Cell death such as apoptosis plays a critical role in vascular calcification. Ferroptosis is a type of iron-catalyzed and regulated cell death resulting from excessive iron-dependent reactive oxygen species and lipid peroxidation. However, it is unclear whether ferroptosis of vascular smooth muscle cells (VSMCs) regulates vascular calcification in CKD. Our results showed that high calcium and phosphate concentrations induced ferroptosis in rat VSMCs invitro. Inhibition of ferroptosis by ferrostatin-1 dose-dependently reduced mineral deposition in rat VSMCs under pro-osteogenic conditions, as indicated by alizarin red staining and quantification of calcium content. In addition, gene expression analysis revealed that ferrostatin-1 inhibited osteogenic differentiation of rat VSMCs. Similarly, ferrostatin-1 remarkably attenuated calcification of rat and human arterial rings exvivo and aortic calcification in vitamin D3-overloaded mice invivo. Moreover, inhibition of ferroptosis by either ferrostatin-1 or deferoxamine attenuated aortic calcification in rats with CKD. Mechanistically, high calcium and phosphate downregulated expression of SLC7A11 (a cystine-glutamate antiporter), and reduced glutathione (GSH) content in VSMCs. Additionally, GSH depletion induced by erastin (a small molecule initiating ferroptotic cell death) significantly promoted calcification of VSMCs under pro-osteogenic conditions, whereas GSH supplement by N-acetylcysteine reduced calcification of VSMCs. Consistently, knockdown of SLC7A11 by siRNA markedly promoted VSMC calcification. Furthermore, high calcium and phosphate downregulated glutathione peroxidase 4 (GPX4) expression, and reduced glutathione peroxidase activity. Inhibition of GPX4 by RSL3 promoted VSMC calcification. Thus, repression of the SLC7A11/GSH/GPX4 axis triggers ferroptosis of VSMCs to promote vascular calcification under CKD conditions, providing a novel targeting strategy for vascular calcification.

Periostin Augments Vascular Smooth Muscle Cell Calcification via β-Catenin Signaling.

Medial vascular calcification is common in chronic kidney disease (CKD) and is closely linked to hyperphosphatemia. Vascular smooth muscle cells (VSMCs) can take up pro-calcific properties and actively augment vascular calcification. Various pro-inflammatory mediators are able to promote VSMC calcification. In this study, we investigated the effects and mechanisms of periostin, a matricellular signaling protein, in calcifying human VSMCs and human serum samples. As a result, periostin induced the mRNA expression of pro-calcific markers in VSMCs. Furthermore, periostin augmented the effects of β-glycerophosphate on the expression of pro-calcific markers and aggravated the calcification of VSMCs. A periostin treatment was associated with an increased β-catenin abundance as well as the expression of target genes. The pro-calcific effects of periostin were ameliorated by WNT/β-catenin pathway inhibitors. Moreover, a co-treatment with an integrin αvβ3-blocking antibody blunted the pro-calcific effects of periostin. The silencing of periostin reduced the effects of β-glycerophosphate on the expression of pro-calcific markers and the calcification of VSMCs. Elevated serum periostin levels were observed in hemodialysis patients compared with healthy controls. These observations identified periostin as an augmentative factor in VSMC calcification. The pro-calcific effects of periostin involve integrin αvβ3 and the activation of the WNT/β-catenin pathway. Thus, the inhibition of periostin may be beneficial to reduce the burden of vascular calcification in CKD patients.

The crosstalk between endothelial cells and vascular smooth muscle cells aggravates high phosphorus-induced arterial calcification

Arterial calcification is highly prevalent, particularly in patients with end-stage renal disease (ESRD). The osteogenic differentiation of vascular smooth muscle cells (VSMCs) is the critical process for the development of arterial calcification. However, the detailed mechanism of VSMCs calcification remains to be elucidated. Here, we investigated the role of exosomes (Exos) derived from endothelial cells (ECs) in arterial calcification and its potential mechanisms in ESRD. Accelerated VSMCs calcification was observed when VSMCs were exposed to ECs culture media stimulated by uremic serum or high concentration of inorganic phosphate (3.5 mM Pi). and the pro-calcification effect of the ECs culture media was attenuated by exosome depletion. Exosomes derived from high concentrations of inorganic phosphate-induced ECs (ECsHPi-Exos) could be uptaken by VSMCs and promoted VSMCs calcification. Microarray analysis showed that miR-670-3p was dramatically increased in ECsHPi-Exos compared with exosomes derived from normal concentrations of inorganic phosphate (0.9 mM Pi) induced ECs (ECsNPi-Exos). Mechanistically, insulin-like growth factor 1 (IGF-1) was identified as the downstream target of miR-670-3p in regulating VSMCs calcification. Notably, ECs-specific knock-in of miR-670-3p of the 5/6 nephrectomy with a high-phosphate diet (miR-670-3pEC-KI + NTP) mice that upregulated the level of miR-670-3p in artery tissues and significantly increased artery calcification. Finally, we validated that the level of circulation of plasma exosomal miR-670-3p was much higher in patients with ESRD compared with healthy controls. Elevated levels of plasma exosomal miR-670-3p were associated with a decline in IGF-1 and more severe artery calcification in patients with ESRD. Collectively, these findings suggested that ECs-derived exosomal miR-670-3p could promote arterial calcification by targeting IGF-1, which may serve as a potential therapeutic target for arterial calcification in ESRD patients.

Dual-specificity Phosphatase 1 Suppresses Vascular Smooth Muscle Cell Calcification by Optical Atrophy 1-related Pathway

Objective To investigate the effect of dual-specificity phosphatase 1/optical atrophy 1 (DUSP1/OPA1) signaling pathway on vascular smooth muscle cell (VSMC) calcification.Methods An in vitro model of VSMC calcification was induced by exposure to β-glycerophosphate and calcium chloride.VSMC calcification was assessed by Alizarin Red S staining and calcium content by ELISA.Apoptosis was detected by TUNEL.Western blotting was employed to determine the protein levels of DUSP1,OPA1,Runt-related transcription factor 2 (Runx-2),bone morphogenetic protein 2 (BMP-2),and cysteinyl aspartate-specific proteinase-3 (Caspase-3).The effects of DUSP1 overexpression and OPA1 knockdown on cell calcification were investigated.Results Calcium chloride and β-glycerolphosphate induced VSMC calcification and down-regulated the expression levels of DUSP1 (t=11.951,P<0.001) and OPA1 (t=8.487,P<0.001).DUSP1 overexpression promoted OPA1 expression (t=-8.921,P<0.001),attenuated VSMC calcification,reduced calcium content and apoptosis rate,and down-regulated the expression of Runx-2,BMP-2,and active Caspase-3 (all P<0.001).OPA1 knockdown increased calcium content and apoptosis rate,up-regulated the expression of Runx-2,BMP-2,and active Caspase-3,and promoted VSMC calcification (all P<0.001).Conclusion DUSP1 may inhibit the VSMC calcification through the OPA1 signaling pathway.

Microparticles from Hyperphosphatemia-Stimulated Endothelial Cells Promote Vascular Calcification Through Astrocyte-Elevated Gene-1.

Endothelial microparticles (EMPs) can be released in chronic kidney disease (CKD). Plasma concentration of high inorganic phosphate (HP) is considered as a decisive determinant of vascular calcification in CKD. We therefore explored the role of HP-induced EMPs (HP-EMPs) in the vascular calcification and its potential mechanism. We observed the shape of HP-EMPs captured by vascular smooth muscle cells (VSMCs) dynamically changed from rare dots, rosettes, to semicircle or circle. Our results demonstrated that HP-EMPs could directly promote VSMC calcification, or accelerate HP-induced calcification through signal transducers and activators of transcription 3 (STAT3)/bone morphogenetic protein-2 (BMP2) signaling pathway. AEG-1 activity was increased through HP-EMPs-induced VSMC calcification, in arteries from uremic rats, or from uremic rats treated with HP-EMPs. AEG-1 deficiency blocked, whereas AEG-1 overexpression exacerbated, the calcium deposition of VSMCs. AEG-1, a target of miR-153-3p, could be suppressed by agomiR-153-3p. Notably, VSMC-specific enhance of miR-153-3p by tail vein injection of aptamer-agomiR-153-3p decreased calcium deposition in both uremia rats treated with HP-EMPs or not. HP-EMPs could directly induce VSMCs calcification and accelerate Pi-induced calcification, and AEG-1 may act as crucial regulator of HP-EMPs-induced vascular calcification. This study sheds light on the therapeutic agents that influence HP-EMPs production or AEG-1 activity, which may be of benefit to treat vascular calcification.

H19 Promotes Osteoblastic Transition by Acting as ceRNA of miR-140-5p in Vascular Smooth Muscle Cells.

Arterial medial calcification is a common disease in patients with type 2 diabetes, end-stage renal disease and hypertension, resulting in high incidence and mortality of cardiovascular event. H19 has been demonstrated to be involved in cardiovascular diseases like aortic valve diseases. However, role of H19 in arterial medial calcification remains largely unknown. We identified that H19 was upregulated in ß-glycerophosphate (β-GP) induced vascular smooth muscle cells (VSMCs), a cellular calcification model in vitro. Overexpression of H19 potentiated while knockdown of H19 inhibited osteogenic differentiation of VSMCs, as demonstrated by changes of osteogenic genes Runx2 and ALP as well as ALP activity. Notably, H19 interacted with miR-140-5p directly, as demonstrated by luciferase report system and RIP analysis. Mechanistically, miR-140-5p attenuated osteoblastic differentiation of VSMCs by targeting Satb2 and overexpression of miR-140-5p blocked H19 induced elevation of Satb2 as well as the promotion of osteoblastic differentiation of VSMCs. Interestingly, over-expression of Satb2 induced phosphorylation of ERK1/2 and p38MAPK. In conclusion, H19 promotes VSMC calcification by acting as competing endogenous RNA of miR-140-5p and at least partially by activating Satb2-induced ERK1/2 and p38MAPK signaling.

Plasma Exosomes Derived From Patients With End-Stage Renal Disease and Renal Transplant Recipients Have Different Effects on Vascular Calcification.

End-stage renal disease (ESRD) patients usually develop extensive and progressive vascular calcification, and lots of calcification inhibitors as well as procalcifying factors are involved in the process. However, the mechanisms of vascular calcification in ESRD patients are still ill-defined. In the present study, we found that the plasma exosomes derived from ESRD patients (ESRD-Ex) promoted calcification of vascular smooth muscle cells (VSMCs) significantly, while plasma exosomes from renal transplant recipients (RTR-Ex) could partially attenuate VSMCs calcification. Moreover, the protein concentration of ESRD-Ex was significantly higher than plasma exosomes from the normal health control group (Nor-Ex) and RTR-Ex, and the content of both matrix gla protein (MGP) and Fetuin-A, the calcification inhibitors, were prominently lower in ESRD-Ex than those in Nor-Ex. The content of Annexin-A2, one of the calcification promoters, was significantly higher in ESRD-Ex and RTR-Ex than that in Nor-Ex. However, bone morphogenetic protein (BMP-2) and receptor activator for nuclear factor-κB ligand (Rankl) had no significant difference among the three groups. In addition, the content of Fetuin-A in RTR-Ex was higher than that in ESRD-Ex, although it was still lower than that in Nor-Ex. Furthermore, the levels of both Fetuin-A and MGP in plasma exosomes were negatively while the levels of Annexin-A2 in plasma exosomes was positively correlated to coronary artery calcification scores (CACS). These results indicated that ESRD-Ex significantly promoted VSMCs calcification, while renal transplantation could partially attenuate the procalcification effect of exosomes. Fetuin-A and MGP were decreased, but Annexin-A2 was increased in ESRD-Ex, and renal transplantation could increase the level of Fetuin-A rather than MGP.

MiR155 modulates vascular calcification by regulating Akt-FOXO3a signalling and apoptosis in vascular smooth muscle cells.

microRNA‐155 (miR155) is pro‐atherogenic; however, its role in vascular calcification is unknown. In this study, we aim to examine whether miR155 regulates vascular calcification and to understand the underlying mechanism. Quantitative real‐time PCR showed that miR155 is highly expressed in human calcific carotid tissue and positively correlated with the expression of osteogenic genes. Wound‐healing assay and TUNEL staining showed deletion of miR155 inhibited vascular smooth muscle cell (VSMC) migration and apoptosis. miR155 deficiency attenuated calcification of cultured mouse VSMCs and aortic rings induced by calcification medium, whereas miR155 overexpression promoted VSMC calcification. Compared with wild‐type mice, miR155−/− mice showed significant resistance to vitamin D3 induced vascular calcification. Protein analysis showed that miR155 deficiency alleviated the reduction of Rictor, increased phosphorylation of Akt at S473 and accelerated phosphorylation and degradation of FOXO3a in cultured VSMCs and in the aortas of vitamin D3‐treated mice. A PI3K inhibitor that suppresses Akt phosphorylation increased, whereas a pan‐caspase inhibitor that suppresses apoptosis reduced VSMC calcification; and both inhibitors diminished the protective effects of miR155 deficiency on VSMC calcification. In conclusion, miR155 deficiency attenuates vascular calcification by increasing Akt phosphorylation and FOXO3a degradation, and thus reducing VSMC apoptosis induced by calcification medium.

OGT-Mediated KEAP1 Glycosylation Accelerates NRF2 Degradation Leading to High Phosphate-Induced Vascular Calcification in Chronic Kidney Disease.

Unraveling the complex regulatory pathways that mediate the effects of phosphate on vascular smooth muscle cells (VSMCs) may provide novel targets and therapies to limit the destructive effects of vascular calcification (VC) in patients with chronic kidney disease (CKD). Our previous studies have highlighted several signaling networks associated with VSMC autophagy, but the underlying mechanisms remain poorly understood. Thereafter, the current study was performed to characterize the functional relevance of O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) in high phosphate-induced VC in CKD settings. We generated VC models in 5/6 nephrectomized rats in vivo and VSMC calcification models in vitro. Artificial modulation of OGT (knockdown and overexpression) was performed to explore the role of OGT in VSMC autophagy and VC in thoracic aorta, and in vivo experiments were used to substantiate in vitro findings. Mechanistically, co-immunoprecipitation (Co-IP) assay was performed to examine interaction between OGT and kelch like ECH associated protein 1 (KEAP1), and in vivo ubiquitination assay was performed to examine ubiquitination extent of nuclear factor erythroid 2-related factor 2 (NRF2). OGT was highly expressed in high phosphate-induced 5/6 nephrectomized rats and VSMCs. OGT silencing was shown to suppress high phosphate-induced calcification of VSMCs. OGT enhances KEAP1 glycosylation and thereby results in degradation and ubiquitination of NRF2, concurrently inhibiting VSMC autophagy to promote VSMC calcification in 5/6 nephrectomized rats. OGT inhibits VSMC autophagy through the KEAP1/NRF2 axis and thus accelerates high phosphate-induced VC in CKD.

MiR32-5p promoted vascular smooth muscle cell calcification by upregulating TNF\u03b1 in the microenvironment

BackgroundVascular calcification is often associated with chronic inflammation and is a risk factor for brain arterial stiffness. Our previous results showed that miR32-5p was positively correlated with vascular smooth muscle cells (VSMC) calcification, but it is unclear whether miR32-5p promoted VSMC calcification by regulating inflammatory factor production.ResultsIn this study, bioinformatics analysis was used to select tumour necrosis factor α (TNFα) as a candidate inflammatory factor associated with calcification. Moreover, alizarin red staining and qRT-PCR analysis revealed that TNFα produced by BV2 cells was the key promoting factor of VSMC calcification. Interestingly, the expression of TNFα was significantly increased at the mRNA and protein levels after miR32-5p mimic treatment but significantly decreased after miR32-5p antagomir treatment. To explore the mechanism of the regulation of TNFα expression by miR32-5p, bioinformatics analysis indicated that PIKfyve was a candidate target gene of miR32-5p, and luciferase assays verified that the expression of PIKfyve was significantly repressed by miR32-5p mimics. Importantly, rescue experiments showed that the expression of TNFα in BV2 cells treated with miR32-5p antagomir and the PIKfyve inhibitor YM201636 was significantly increased.ConclusionsThe production of TNFα in microglia could be affected by miR32-5p targeting PIKfyve, and these results will be beneficial to reveal the mechanism of brain arterial calcification.

JAK2/STAT3/BMP-2 axis and NF-κB pathway are involved in erythropoietin-induced calcification in rat vascular smooth muscle cells.

Vascular calcification is common in chronic kidney disease (CKD) patients, while erythropoietin (EPO) is widely used in the treatment of renal anemia in CKD patients, whether there is a link between the two is still not clear. The primary rat vascular smooth muscle cells (VSMCs) and CKD rats were treated with EPO and the calcium deposition was observed by alizarin red staining, von Kossa staining and calcium quantification. Activation of JAK2/STAT3/BMP-2 axis and NF-κB signaling pathways was investigated by Western blotting. EPO-induced calcium deposition in VSMCs and significantly potentiated calcification in CKD rats. Furthermore, EPO activated JAK2/STAT3/BMP-2 axis, NF-κB pathway and the pro-calcification effect of EPO was partially blocked by the STAT3 inhibitor (Cryptotanshinone) or NF-κB inhibitor (BAY 11-7082), respectively, in vitro. EPO could promote VSMCs calcification in vitro and in vivo and this effect may be achieved through the JAK2/STAT3/BMP-2 axis and NF-κB pathway.

Activation of CD137 Signaling Enhances Vascular Calcification through c-Jun N-Terminal Kinase-Dependent Disruption of Autophagic Flux.

Background Vascular calcification is widespread and clinically significant, contributing to substantial morbidity and mortality. Calcifying vascular cells are partly derived from local vascular smooth muscle cells (VSMCs), which can undergo chondrogenic or osteogenic differentiation under inflammatory environment. Recently, we have found activation of CD137 signaling accelerated vascular calcification. However, the underlying mechanism remains unknown. This study aims to identify key mediators involved in CD137 signaling-induced vascular calcification in vivo and in vitro. Methods Autophagy flux was measured through mRFP-GFP-LC3 adenovirus and transmission electron microscopy. Von Kossa assay and alkaline phosphatase (ALP) activity were used to observe calcification in vivo and in vitro, respectively. Autophagosome-containing vesicles were collected and identified by flow cytometry and Western blot. Autophagy or calcification-associated targets were measured by Western blot, quantitative real-time PCR, and immunohistochemistry. Results Treatment with the agonist-CD137 displayed c-Jun N-terminal kinase- (JNK-) dependent increase in the expression of various markers of autophagy and the number of autophagosomes relative to the control group. Autophagy flux experiments suggested that agonist-CD137 blocked the fusion of autophagosomes with lysosomes in cultured VSMCs. Calcium deposition, ALP activity, and the expression of calcification-associated proteins also increased in agonist-CD137 group compared with anti-CD137 group, which could be recovered by autophagy stimulator rapamycin. Autophagosome-containing vesicles collected from agonist-CD137 VSMCs supernatant promoted VSMC calcification. Conclusion The present study identified a new pathway in which CD137 promotes VSMC calcification through the activation of JNK signaling, subsequently leading to the disruption of autophagic flux, which is responsible for CD137-induced acceleration of vascular calcification.

Quercetin attenuates Ox-LDL-induced calcification in vascular smooth muscle cells by regulating ROS-TLR4 signaling pathway

To determine whether quercetin inhibits oxidized low-density lipoprotein (Ox-LDL)-induced osteogenic differentiation and calcification of vascular smooth muscle cells (VSMCs) and understand the underlying mechanism. The calcification of human VSMCs following Ox-LDL treatment was assessed using alizarin red staining and by detecting ALP activity. The mRNA expressions of the bone-related genes including Msx2, BMP2 and Osterix, and the contractile proteins including SMA and SM22a were analyzed using qPCR. The effects of quercetin were investigated on OxLDL-induced VSMC calcification and changes in ALP activity, expressions of Msx2, BMP2, Osterix, SMA and SM22a, ROS levels and SOD activity. The effect of Toll like receptor 4 (TLR4) silencing mediated by siRNA transfection on cell calcification, ALP activity, gene expressions and ROS levels were investigated. Ox-LDL treatment promoted VSMC calcification and up-regulated TLR4 expression. Quercetin treatment significantly attenuated Ox-LDL-induced VSMC calcification, reduced ALP activity, down-regulated the expression levels of Msx2, BMP2 and Osterix, and up-regulated the expressions of vascular smooth muscle contractile proteins SMA and SM22a. In addition, Quercetin treatment markedly increased SOD activity, reduced ROS levels and TLR4 expression in VSMCs. Silencing TLR4 expression using TLR4 siRNA also significantly decreased calcification of the VSMCs. Quercetin inhibits Ox-LDL-induced VSMC calcification in VSMCs possibly by targeting the ROS/TLR4 signaling pathway.

Phosphate and Cardiovascular Disease beyond Chronic Kidney Disease and Vascular Calcification.

Phosphate is essential for life but its accumulation can be detrimental. In end-stage renal disease, widespread vascular calcification occurs as a result of chronic phosphate load. The accumulation of phosphate is likely to occur long before the rise in serum phosphate above the normal range since several observational studies in both general population and early-stage CKD patients have identified the relationship between high-normal serum phosphate and adverse cardiovascular outcomes. Consumption of food high in phosphate increases both fasting and postprandial serum phosphate and habitual intake of high phosphate diet is associated with aging, cardiac hypertrophy, endothelial dysfunction, and subclinical atherosclerosis. The decline in renal function and dietary phosphate load can increase circulating fibroblast growth factor-23 (FGF-23) which may have a direct impact on cardiomyocytes. Increased FGF-23 levels in both CKD and general populations are associated with left ventricular hypertrophy, congestive heart failure, atrial fibrillation, and mortality. Increased extracellular phosphate directly affects endothelial cells causing cell apoptosis and vascular smooth muscle cells (VSMCs) causing transformation to osteogenic phenotype. Excess of calcium and phosphate in the circulation can promote the formation of protein-mineral complex called calciprotein particles (CPPs). In CKD, these CPPs contain less calcification inhibitors, induce inflammation, and promote VSMC calcification.