Atherosclerotic Calcification Research Articles

Sodium-glucose cotransporter 2 inhibitors attenuate vascular calcification by suppressing endoplasmic reticulum protein thioredoxin domain containing 5 dependent osteogenic reprogramming

AimsVascular calcification is strongly linked to the development of major adverse cardiovascular events, but effective treatments are lacking. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are an emerging category of oral hypoglycemic drugs that have displayed marked effects on metabolic and cardiovascular diseases, including recently reported vascular medial calcification. However, the roles and underlying mechanisms of SGLT2 inhibitors in vascular calcification have not been fully elucidated. Thus, we aimed to further determine whether SGLT2 inhibitors protect against vascular calcification and to investigate the mechanisms involved. Methods and ResultsA computed tomography angiography investigation of coronary arteries from 1554 patients with type 2 diabetes revealed that SGLT2 inhibitor use was correlated with a lower Agatston calcification score. In the vitamin D3 overdose, 5/6 nephrectomy chronic kidney disease -induced medial calcification and Western diet-induced atherosclerotic intimal calcification models, dapagliflozin (DAPA) substantially alleviated vascular calcification in the aorta. Furthermore, we showed that DAPA reduced vascular calcification via Runx2-dependent osteogenic transdifferentiation in vascular smooth muscle cells (VSMCs). Transcriptome profiling revealed that thioredoxin domain containing 5 (TXNDC5) was involved in the attenuation of vascular calcification by DAPA. Rescue experiments showed that DAPA-induced TXNDC5 downregulation in VSMCs blocked the protective effect on vascular calcification. Furthermore, TXNDC5 downregulation disrupted protein folding-dependent Runx2 stability and promoted subsequent proteasomal degradation. Moreover, DAPA downregulated TXNDC5 expression via amelioration of oxidative stress and ATF6-dependent endoplasmic reticulum stress. Consistently, the class effects of SGLT2 inhibitors on vascular calcification were validated with empagliflozin in intimal and medial calcification models. ConclusionsSGLT2 inhibitors ameliorate vascular calcification through blocking endoplasmic reticulum stress-dependent TXNDC5 upregulation and promoting subsequent Runx2 proteasomal degradation, suggesting that SGLT2 inhibitors are potentially beneficial for vascular calcification treatment and prevention.

Bilateral Carotid Calcification Correlates with Regional Cerebral Glucose Metabolism: Insights from PET/CT Imaging of Patients with Cardiovascular Risk Factors

Background: Cardiovascular disease is a leading cause of illness and death globally, primarily due to atherosclerosis. This disease reduces blood flow and oxygen delivery to organs, and when it affects the carotid arteries, it can lead to cognitive impairment and dementia. In a population of 104 individuals, comprising both healthy controls and individuals at elevated risk for developing cardiovascular diseases (CVD) due to identified risk factors, we used PET imaging with 18F-fluorodeoxyglucose (FDG) to assess cerebral glucose metabolism and 18F-sodium fluoride (NaF) to detect atherosclerotic calcification. Our statistical analysis revealed significant differences in metabolic activity between healthy and at-risk individuals in specific brain regions. 18F-FDG uptake in the brain varied inversely with respect to the clinical assessment of cardiovascular risk in regions such as the cuneus (β = −0.030, SE = 0.014, p = 0.035), middle occipital gyrus (β = −0.032, SE = 0.011, p = 0.005), and posterior cingulate gyrus (β = −0.032, SE = 0.015, p = 0.044). In contrast, areas including the basis pontis (β = 0.025, SE = 0.012, p = 0.038) and the pons (β = 0.034, SE = 0.013, p = 0.008) exhibited direct correlations. Notably, carotid 18F-NaF uptake had inverse associations with 18F-FDG uptake in the cerebellum (β = −0.825, SE = 0.354, p = 0.021), medulla (β = −0.888, SE = 0.405, p = 0.029), and posterior cingulate gyrus (β = −1.253, SE = 0.567, p = 0.028), while increased carotid calcification influenced metabolic activity in the fusiform gyrus (β = 1.660, SE = 0.498, p = 0.001) and globus pallidus (β = 1.505, SE = 0.571, p = 0.009). We observed that atherosclerotic plaque accumulation, especially in the carotid arteries, has potential implications for metabolic changes in brain regions governing cognition, emotion, sensory perception, and motor activities. Our findings underscore the possible early interventions that can be used to preempt or delay cognitive deterioration linked with cardiovascular ailments.

Abstract TP143: Longitudinal Evaluation of Vessel Wall MRI Demonstrates Bilaterally Symmetric Evolution of Carotid Atherosclerosis

Introduction: Carotid atherosclerosis is a leading cause of stroke worldwide. Previous studies demonstrated bilateral symmetry in atherosclerotic plaque burden and calcification scores. By leveraging longitudinal 3D vessel wall MRI, this study aims to investigate if there is a symmetrical trend of bilateral plaque progression. Methods: Asymptomatic patients with confirmed carotid stenosis were prospectively recruited in the study. Subjects underwent baseline and follow-up 3D vessel wall imaging for a time span up to 8 years. A deep learning-based pipeline was used to bilaterally segment the carotid lumen and vessel wall on axial image slices. Within each artery, the range of slices with wall thickness > 2mm on any scan was included as potential atherosclerotic plaque. Plaque burden, positively associated with stroke risk, was defined as the percent wall volume (wall volume / total vessel volume х 100%). For each plaque, a linear model was fitted across all available time points to determine the growth rate of plaque burden. We used generalized estimating equation-based linear regression models to evaluate associations of the growth rate of each side with the contralateral side, adjusted for clinical risk factors and medications. Effect sizes were summarized using the partial correlation coefficient. Results: 20 subjects (mean age 73.5 years, 3 females, 14 on statins) who underwent at least three scans with bilateral carotid plaque were included in the analysis. Each subject underwent an average of 4.8 scans over 5.2 years. Overall, the plaque growth rate of one side was moderately correlated with the contralateral side (r=0.50, p=0.004), even after adjusting for age, sex and statins use. Then, for each subject, the larger of the two plaques at baseline (left or right side) was identified. The growth rate of the larger plaque was also predictive of the growth rate of the smaller plaque over the same time period (r=0.66, p=0.004). Conclusion: The systemic nature of atherosclerotic disease entails an association in symmetrical disease evolution for left and right carotid arteries. In bilaterally diseased patients, the longitudinal monitoring of the more advanced carotid lesion may provide prognostic information on the less advanced contralateral plaque.

The Identification Potential of Atherosclerotic Calcifications in the Context of Forensic Anthropology.

Atherosclerosis is an inflammatory disease that, in its more developed stages, can lead to the calcification of fatty plaques on the walls of arteries, resulting in the appearance of new bone elements. It is a condition that has been studied and documented little in the context of paleopathology, especially in the framework of forensic anthropology. This article analyzed the skeletal remains of 71 individuals (35 females and 36 males) from the Luís Lopes Identified Collection of the National Museum of Natural History and Science in Lisbon, 31 of whom had an autopsy report. An attempt was made to ascertain whether these bone elements resulting from atherosclerotic calcification would resist cadaveric decomposition and whether they would be recoverable several years after burial, and a survey was carried out of their distribution according to sex and age, as well as their association with other pathologies, such as osteoporosis and cardiac and renal pathologies. An imaging analysis of an atherosclerotic plaque was also carried out to complement the macroscopic analysis and present other methods of identifying plaques. It was concluded that each atherosclerotic calcification has a unique profile, which can be useful for identification, especially in cases where the individual shows a severe condition. In terms of identification potential, the analysis of calcified atherosclerotic plaques can be useful, as they can corroborate or reject an identification. However, it always requires the existence of ante-mortem imaging exams and must always be used in addition to other identification methods. Regardless of the identification, these plaques are bone elements resulting from a pathology and should, therefore, be known and recognized by the scientific community.

Visceral and Ectopic Abdominal Fat Effect on the Calcification of the Abdominal Aorta and Its Branches-An MSCT Study.

Visceral and ectopic fat accumulation might have an impact on the atherosclerotic calcification of abdominal arteries. The pattern of calcification of the abdominal aorta and its branches is not fully investigated. We retrospectively analyzed the abdominopelvic MSCT images and calculated calcification volumes of the abdominal aorta, celiac trunk, superior and inferior mesenteric arteries, and both common and external iliac arteries. On the same MSCT scans, a visceral fat volume and ectopic fat deposits (liver-to-spleen ratio (L/S) and pancreas-to-spleen (P/S) ratio) were also measured. The results showed that calcifications of the abdominal aorta and its branches were associated with visceral fat volume, less strongly associated with L/S, and not associated with the P/S ratio. The abdominal aorta, the common iliac and external iliac arteries were more calcified arteries compared to the celiac trunk and superior and mesenterial arteries. In conclusion, visceral fat has a stronger effect on abdominopelvic arteries' calcification than ectopic fat. Visceral aortic branches are generally less calcified than iliac arteries.

The effect of osteoprotegerin nanocomplex on osteoprotegerin and arteriosclerosis in mice

Osteoprotegerin (OPG), as one of the tumor necrosis factor receptors, is believed to be related with Osteoprotegerin (OP) and arteriosclerosis (AS). This study aims to explore the effect of OPG on osteoblasts (OB) and AS. The nanocomplex Poly(ethylene glycol)-Poly(L-Lysine)-Osteoprotegerin (PPO) was prepared by introducing OPG plasmid to Poly(ethylene glycol)-Poly(L-Lysine). Healthy group, AS group, AS combined with OP group were designed to measure apoptosis, activity, adhesion, and calcification of OB in Polycaprolactone (PCL) by flow cytometry, MTT method, alizarin red staining, scanning electron microscope and other methods. The effect of PPO on bone mineral density and arteriosclerosis of ApoE−/−/RANKL+/+ mice was observed. Bone Mineral Density (BMD) was positively correlated with ABI while negatively correlated with pulse wave velocity. OPG in AS combined with OP group was higher than healthy group, and AS group was also higher than healthy group. PPO-administered mice had dense bone trabeculae and higher bone density while the control group was the opposite. The effect of PPO on the stable expression of OPG in mice reduced the plaque area and the degree of vascular calcification. PPO can enhance OB activity in vitro, inhibit cell apoptosis, promote cell calcification and PCL adhesion, decrease the area of atherosclerotic plaque and calcification, and increase the BMD of the femoral neck. PPO can promote the adhesion and calcification of MC3T3-E1 on PCL, which is of great significance for maintaining sufficient bone strength and reducing blood calcium. In addition, PPO compound drugs can increase bone density, reduce arterial plaque area and vascular calcification.

Empagliflozin alleviates atherosclerotic calcification by inhibiting osteogenic differentiation of vascular smooth muscle cells

SGLT-2 inhibitors, such as empagliflozin, have been shown to reduce the occurrence of cardiovascular events and delay the progression of atherosclerosis. However, its role in atherosclerotic calcification remains unclear. In this research, ApoE−/− mice were fed with western diet and empagliflozin was added to the drinking water for 24 weeks. Empagliflozin treatment significantly alleviated arterial calcification assessed by alizarin red and von kossa staining in aortic roots and reduced the lipid levels, while had little effect on body weight and blood glucose levels in ApoE−/− mice. In vitro studies, empagliflozin significantly inhibits calcification of primary vascular smooth muscle cells (VSMCs) and aortic rings induced by osteogenic media (OM) or inorganic phosphorus (Pi). RNA sequencing of VSMCs cultured in OM with or without empagliflozin showed that empagliflozin negatively regulated the osteogenic differentiation of VSMCs. And further studies confirmed that empagliflozin significantly inhibited osteogenic differentiation of VSMCs via qRT-PCR. Our study demonstrates that empagliflozin alleviates atherosclerotic calcification by inhibiting osteogenic differentiation of VSMCs, which addressed a critical need for the discovery of a drug-based therapeutic approach in the treatment of atherosclerotic calcification.

The Role of Osteopontin in Atherosclerosis and Its Clinical Manifestations (Atherosclerotic Cardiovascular Diseases)-A Narrative Review.

Atherosclerotic cardiovascular diseases (ASCVDs) are the most common and severe public health problem nowadays. Osteopontin (OPN) is a multifunctional glycoprotein highly expressed at atherosclerotic plaque, which has emerged as a potential biomarker of ASCVDs. OPN may act as an inflammatory mediator and/or a vascular calcification (VC) mediator, contributing to atherosclerosis progression and eventual plaque destabilization. In this article, we discuss the complex role of OPN in ASCVD pathophysiology, since many in vitro and in vivo experimental data indicate that OPN contributes to macrophage activation and differentiation, monocyte infiltration, vascular smooth muscle cell (VSMC) migration and proliferation and lipid core formation within atherosclerotic plaques. Most but not all studies reported that OPN may inhibit atherosclerotic plaque calcification, making it "vulnerable". Regarding clinical evidence, serum OPN levels may become a biomarker of coronary artery disease (CAD) presence and severity. Significantly higher OPN levels have been found in patients with acute coronary syndromes than those with stable CAD. In limited studies of patients with peripheral artery disease, circulating OPN concentrations may be predictive of future major adverse cardiovascular events. Overall, the current literature search suggests the contribution of OPN to atherosclerosis development and progression, but more robust evidence is required.

Abstract 150: Arterial Calcification and Neural Metabolic Activity: Assessing Neurovascular Status Through FDG and NaF PET/CT Imaging

Introduction Atherosclerosis, the occlusion of arteries due to the accumulation of fatty substances, cholesterol, calcium, and fibrin in the arterial walls, can result in reduced blood flow and a subsequent decrease in oxygen delivery to the organs supplied by the affected artery, including the brain. Positron emission tomography‐computed tomography (PET‐CT) utilizing 18F‐fluorodeoxyglucose (FDG) and 18F‐Sodium Fluoride (NaF) have demonstrated efficacy in the assessment of cerebral glucose metabolism and atherosclerotic calcification, respectively. However, the reported literature has not clearly correlated arterial disease throughout the body with cerebral hypometabolism. Utilizing a novel integration of FDG and NaF PET/CT imaging, our study explores the link between cardiovascular risk factors and regional cerebral metabolism. Methods We compared 79 healthy controls (mean age 44.5 ± 13.8 years, 53.2% males) and 40 individuals (mean age 55.9 ± 11.9 years, 50% males) at increased risk for cardiovascular disease (CVD) as assessed by the systematic coronary risk evaluation (SCORE) tool. All subjects underwent whole‐body FDG‐PET/CT imaging. [18F]fluoride PET/CT imaging was performed using hybrid PET/CT scanners with image correction and reconstruction processes following standard protocols. The quantitative regional analysis of PET images was performed using MIMneuro version 7.1.5 (MIM Software, Inc., Cleveland, Ohio). 70 whole brain structures were analyzed in each subject. MIMneuro provided region‐based analysis, with z‐scores generated by comparing the patient to the selected age‐matched set of normal controls. Using OsiriX MD software, [18F]fluoride PET/CT scans were analyzed with regions of interest (ROIs) manually drawn to assess carotid calcification and standardized uptake value mean (SUVmean), along with ROI volume values. Correlations between the degree of molecular calcification and cerebral FDG uptake in patients with cardiovascular risk factors were evaluated using Pearson’s R, with the significance threshold set at P < 0.05. Results Unhealthy patients demonstrated significant negative correlations between increased calcification in the carotid arteries, as assessed by NaF‐PET, and metabolic activity in the anterior cingulate (r = ‐0.42, p = 0.008) and cingulate regions (r = ‐0.34, p = 0.036). Conversely, healthy patients exhibited significant positive correlations in the pons (r = 0.25, p = 0.042) and pontine tegmentum (r = 0.34, p = 0.005), suggestive of a compensatory mechanism to maintain autonomic and motor functions. Conclusion The present study provides insight regarding the interplay between arterial calcification and neural metabolic activity, particularly as it pertains to regions supplied by the anterior cerebral artery. Our investigation suggests that calcification in the carotid arteries, which give rise to the ACA, has a deleterious impact on the function of the cingulate gyrus. Additionally, the observed positive correlation in the pons and pontine tegmentum in healthy individuals raises intriguing questions regarding potential compensatory mechanisms that may help maintain autonomic and motor functions despite vascular impediments. Understanding the temporal progression of vascular calcification allows for the optimization of surgical strategy and timing, potentially reducing stroke risk and improving patient outcomes. These findings underscore the necessity of persistent surveillance and targeted management strategies concerning carotid calcification.

GSK3β Inhibition Ameliorates Atherosclerotic Calcification.

Endothelial-mesenchymal transition (EndMT) drives endothelium to contribute to atherosclerotic calcification. In a previous study, we showed that glycogen synthase kinase-3β (GSK3β) inhibition induced β-catenin and reduced mothers against DPP homolog 1 (SMAD1) in order to redirect osteoblast-like cells towards endothelial lineage, thereby reducing vascular calcification in Matrix Gla Protein (Mgp) deficiency and diabetic Ins2Akita/wt mice. Here, we report that GSK3β inhibition or endothelial-specific deletion of GSK3β reduces atherosclerotic calcification. We also find that alterations in β-catenin and SMAD1 induced by GSK3β inhibition in the aortas of Apoe-/- mice are similar to Mgp-/- mice. Together, our results suggest that GSK3β inhibition reduces vascular calcification in atherosclerotic lesions through a similar mechanism to that in Mgp-/- mice.

M1-Type Macrophages Secrete TNF-α to Stimulate Vascular Calcification by Upregulating CA1 and CA2 Expression in VSMCs.

Vascular calcification is a hallmark of atherosclerosis (AS). We and others confirmed that carbonic anhydrase I (CA1) and CA2 increased expression and catalyzed calcium deposition in atherosclerotic aortas. Macrophages have been demonstrated to be strongly related to AS. This study aimed to clarify how and which macrophage subtypes regulate CA1 and CA2 expression to stimulate aortic calcification. THP-1 cells were induced to form M0, M1 and M2 macrophage subtypes. These cells and their culture supernatants were separately incubated with human vascular smooth muscle cells (VSMCs). Calcification was strongly increased in VSMCs treated with β-GP, a chemical inducer of cellular calcification, following incubation with M1 macrophages or their culture supernatants, and was much higher than that in VSMCs treated with β-GP alone. Meanwhile, the expression of CA1 and CA2, as well as calcification marker genes, including Runx2, BMP-2 and ALP, was increased in VSMCs during this process. TNF-α levels were also increased in the culture medium of M1 macrophages. M0 and M2 macrophages or their supernatants did not significantly stimulate calcification in VSMCs. Following transfection with anti-CA1 or CA2 siRNAs, β-GP-induced VSMCs showed decreased calcification, but the calcification level was partially increased when those VSMCs were incubated with the supernatants of M1 macrophages, while CA1 and CA2 expression as well as TNF-α levels were also elevated. When VSMCs were treated with TNF-α without β-GP induction, calcification and the expression of CA1 and CA2 were also significantly increased. The results of this study suggest that M1 macrophages can increase CA1 and CA2 expression to promote atherosclerotic calcification in VSMCs by secreting TNF-α.

Retraction of: Retraction of: Ablation of Adenosine Monophosphate-Activated Protein Kinase α1 in Vascular Smooth Muscle Cells Promotes Diet-Induced Atherosclerotic Calcification In Vivo.

HomeCirculation ResearchAhead of PrintRetraction of: Retraction of: Ablation of Adenosine Monophosphate-Activated Protein Kinase α1 in Vascular Smooth Muscle Cells Promotes Diet-Induced Atherosclerotic Calcification In Vivo Free AccessRetractionPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessRetractionPDF/EPUBRetraction of: Retraction of: Ablation of Adenosine Monophosphate-Activated Protein Kinase α1 in Vascular Smooth Muscle Cells Promotes Diet-Induced Atherosclerotic Calcification In Vivo Originally published22 Mar 2023https://doi.org/10.1161/RES.0000000000000606Circulation Research. 2023;0This article is retracted byRetraction of: Ablation of Adenosine Monophosphate-Activated Protein Kinase α1 in Vascular Smooth Muscle Cells Promotes Diet-Induced Atherosclerotic Calcification In VivoThe following Retraction Notice of a Circulation Research article is being retracted:Retraction of: Ablation of Adenosine Monophosphate-Activated Protein Kinase α1 in Vascular Smooth Muscle Cells Promotes Diet-Induced Atherosclerotic Calcification In Vivo. Circ Res. 2022;131:e151. doi: 10.1161/RES.0000000000000578In October 2022, the following Circulation Research article was retracted based on a request by the University of Oklahoma Health Sciences Center due to data irregularities and image reuse in Figure 3:Cai Z, Ding Y, Zhang M, Lu Q, Wu S, Zhu H, Song P, Zou MH. Ablation of Adenosine Monophosphate-Activated Protein Kinase α1 in Vascular Smooth Muscle Cells Promotes Diet-Induced Atherosclerotic Calcification In Vivo. Circ Res. 2016;119:422-33. doi: 10.1161/CIRCRESAHA.116.308301.Based on new information brought forward by Dr. Zhejun Cai, the University of Oklahoma Health Sciences Center revised its recommendation for the above article to correction instead of retraction.The editors are retracting this Retraction Notice in agreement with the University of Oklahoma. A separate Correction Notice has been published to update and correct Figure 3 in the original article. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesRetraction of: Ablation of Adenosine Monophosphate-Activated Protein Kinase α1 in Vascular Smooth Muscle Cells Promotes Diet-Induced Atherosclerotic Calcification In VivoCirculation Research. 2022;131:e151-e151 Advertisement Article InformationMetrics © 2023 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000606PMID: 36946882 Originally publishedMarch 22, 2023 PDF download Advertisement

Correction to: Ablation of Adenosine Monophosphate-Activated Protein Kinase α1 in Vascular Smooth Muscle Cells Promotes Diet-Induced Atherosclerotic Calcification In Vivo.

HomeCirculation ResearchAhead of PrintCorrection to: Ablation of Adenosine Monophosphate-Activated Protein Kinase α1 in Vascular Smooth Muscle Cells Promotes Diet-Induced Atherosclerotic Calcification In Vivo Free AccessCorrectionPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessCorrectionPDF/EPUBCorrection to: Ablation of Adenosine Monophosphate-Activated Protein Kinase α1 in Vascular Smooth Muscle Cells Promotes Diet-Induced Atherosclerotic Calcification In Vivo Originally published22 Mar 2023https://doi.org/10.1161/RES.0000000000000607Circulation Research. 2023;0This article corrects the followingAblation of Adenosine Monophosphate-Activated Protein Kinase α1 in Vascular Smooth Muscle Cells Promotes Diet-Induced Atherosclerotic Calcification In VivoIn the article by Cai Z., et al, “Ablation of Adenosine Monophosphate-Activated Protein Kinase α1 in Vascular Smooth Muscle Cells Promotes Diet-Induced Atherosclerotic Calcification In Vivo” which published in the July 22, 2016 issue of the journal (Circ Res. 2016;119:422-33. doi: 10.1161/CIRCRESAHA.116.308301), a correction is needed.Figure 3D, top left panel immunohistochemistry image labeled “ApoE-/-” with “Runx2” staining was mistakenly reused in Figure 4A, bottom left panel immunohistochemistry image labeled “ApoE-/-/AMPKα1f/f” with “Runx2” staining. This error does neither impact the main findings nor the main conclusion of this study.The correct version of Figure 3D has been updated with the corresponding original data.The authors apologize for the error. This correction has been made to the current online version of the article, which is available at https://www.ahajournals.org/doi/10.1161/CIRCRESAHA.116.308301. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesAblation of Adenosine Monophosphate-Activated Protein Kinase α1 in Vascular Smooth Muscle Cells Promotes Diet-Induced Atherosclerotic Calcification In VivoZhejun Cai, et al. Circulation Research. 2016;119:422-433 Advertisement Article InformationMetrics © 2023 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000607PMID: 36946881 Originally publishedMarch 22, 2023 PDF download Advertisement

Mast cells participate in smooth muscle cell reprogramming and atherosclerotic plaque calcification

BackgroundCalcification, a key feature of advanced human atherosclerosis, is positively associated with vascular disease burden and adverse events. We showed that macrocalcification can be a stabilizing factor for carotid plaque molecular biology, due to inverse association with immune processes. Mast cells (MCs) are important contributors to plaque instability, but their relationship with macrocalcification is unexplored. With a hypothesis that MC activation negatively associates with carotid plaque macrocalcification, we aimed to investigate the link between MCs and carotid plaque vulnerability, and study MC role in plaque calcification via smooth muscle cells (SMCs). MethodsPre-operative computed tomography angiographies of patients (n = 40) undergoing surgery for carotid stenosis were used to characterize plaque morphology. Plaque microarrays (n = 40 and n = 126) were used for bioinformatic deconvolution of immune cell populations. Tissue microarrays (n = 103) were used to histologically validate the contribution of activated and resting MCs in plaques. ResultsActivated MCs and their typical markers were negatively correlated with macrocalcification. The ratio of activated vs. resting MCs was increased in low-calcified plaques from symptomatic patients. There was no modulating effect of medication on MC ratios. In vitro experiments showed that SMC calcification attenuated MC activation, while both active and resting MCs stimulated SMC calcification and induced dedifferentiation towards a pro-inflammatory-, osteochondrocyte-like phenotype, without modulating their migro-proliferative function. ConclusionsIntegrative analyses from human plaques showed that MC activation is inversely associated with macrocalcification and positively with parameters of plaque vulnerability. Mechanistically, MCs induce SMC osteogenic reprograming, while matrix calcification in turn attenuates MC activation, offering new therapeutic avenues for exploration.

Meet the First Authors.

Meet the First Authors.

Roles of Vascular Smooth Muscle Cells in Atherosclerotic Calcification.

The accumulation of calcium in atherosclerotic plaques is a prominent feature of advanced atherosclerosis, and it has a strong positive correlation with the total burden of atherosclerosis. Atherosclerotic calcification usually appears first at the necrotic core, indicating that cell death and inflammatory processes are involved in calcification. During atherosclerotic inflammation, various cell types, such as vascular smooth muscle cells, nascent resident pericytes, circulating stem cells, or adventitial cells, have been assumed to differentiate into osteoblastic cells, which lead to vascular calcification. Among these cell types, vascular smooth muscle cells are considered a major contributor to osteochondrogenic cells in the atherosclerotic milieu. In this review, we summarize the molecular mechanisms underlying the osteochondrogenic switch of vascular smooth muscle cells in atherosclerotic plaques.

Mechanisms of the Wnt Pathways as a Potential Target Pathway in Atherosclerosis.

The proteins of the Wnt family are involved in a variety of physiological processes by means of several canonical and noncanonical signaling pathways. Wnt signaling has been recently identified as a major player in atherogenesis. In this review, we summarize the existing knowledge on the influence of various components of the Wnt signaling pathways on the initiation and progression of atherosclerosis and associated conditions. We used the PubMed database to search for recent papers on the involvement of the Wnt pathways in atherosclerosis. We used the combination of "Wnt" and "atherosclerosis" keywords to find the initial papers, and chose papers published after 2018. In the first section of the paper, we describe the general mechanisms of the Wnt signaling pathways and their components. The next section is dedicated to existing studies assessing the implication of Wnt signaling elements in different atherogenic processes, such as cholesterol retention, endothelial dysfunction, vascular inflammation, and atherosclerotic calcification of the vessels. Lastly, various therapeutic strategies based on interference with the Wnt signaling pathways are considered. We also compare the efficacy and availability of the proposed treatment methods. Wnt signaling can be considered a potential target in the treatment and prevention of atherosclerosis. Therefore, in this review, we reviewed evidences showing that wnt signaling is an important signal for developing appropriate treatment strategies for atherosclerosis.

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.

TXNIP Suppresses the Osteochondrogenic Switch of Vascular Smooth Muscle Cells in Atherosclerosis.

The osteochondrogenic switch of vascular smooth muscle cells (VSMCs) is a pivotal cellular process in atherosclerotic calcification. However, the exact molecular mechanism of the osteochondrogenic transition of VSMCs remains to be elucidated. Here, we explore the regulatory role of TXNIP (thioredoxin-interacting protein) in the phenotypical transitioning of VSMCs toward osteochondrogenic cells responsible for atherosclerotic calcification. The atherosclerotic phenotypes of Txnip-/- mice were analyzed in combination with single-cell RNA-sequencing. The atherosclerotic phenotypes of Tagln-Cre; Txnipflox/flox mice (smooth muscle cell-specific Txnip ablation model), and the mice transplanted with the bone marrow of Txnip-/- mice were analyzed. Public single-cell RNA-sequencing dataset (GSE159677) was reanalyzed to define the gene expression of TXNIP in human calcified atherosclerotic plaques. The effect of TXNIP suppression on the osteochondrogenic phenotypic changes in primary aortic VSMCs was analyzed. Atherosclerotic lesions of Txnip-/- mice presented significantly increased calcification and deposition of collagen content. Subsequent single-cell RNA-sequencing analysis identified the modulated VSMC and osteochondrogenic clusters, which were VSMC-derived populations. The osteochondrogenic cluster was markedly expanded in Txnip-/- mice. The pathway analysis of the VSMC-derived cells revealed enrichment of bone- and cartilage-formation-related pathways and bone morphogenetic protein signaling in Txnip-/- mice. Reanalyzing public single-cell RNA-sequencing dataset revealed that TXNIP was downregulated in the modulated VSMC and osteochondrogenic clusters of human calcified atherosclerotic lesions. Tagln-Cre; Txnipflox/flox mice recapitulated the calcification and collagen-rich atherosclerotic phenotypes of Txnip-/- mice, whereas the hematopoietic deficiency of TXNIP did not affect the lesion phenotype. Suppression of TXNIP in cultured VSMCs accelerates osteodifferentiation and upregulates bone morphogenetic protein signaling. Treatment with the bone morphogenetic protein signaling inhibitor K02288 abrogated the effect of TXNIP suppression on osteodifferentiation. Our results suggest that TXNIP is a novel regulator of atherosclerotic calcification by suppressing bone morphogenetic protein signaling to inhibit the transition of VSMCs toward an osteochondrogenic phenotype.

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

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