Vascular calcification (VC) is an important pathological development progress in chronic kidney disease (CKD) and may increase mortality but lacks effective treatments. N6-methyladenosine (m6A) has been verified to be the most prevalent internal chemical RNA modification in mammalian mRNAs. The M6A-modified mRNA degradation process is mediated by the reader YTHDF2 in an m6A-dependent manner. Nevertheless, the exact role and molecular mechanism of YTHDF2 in VC remain unclear. This study aimed to investigate the potential role of YTHDF2 in the osteogenic differentiation of vascular smooth muscle cells (VSMCs). It was found that YTHDF2 was markedly downregulated in both in vivo and in vitro calcified models. Functionally, YTHDF2 plays a protective role in VC. The overexpression of YTHDF2 inhibited the transdifferentiation of VSMCs from a contractile to an osteogenic phenotype, thus decreasing the expression of mineralization regulatory proteins and calcium deposition. Conversely, YTHDF2 deficiency aggravated this process. At the mechanistic level, YTHDF2 suppressed osteogenic transdifferentiation of VSMCs by regulating the Runt-related transcription factor 2 (Runx2). RNA immunoprecipitation-qPCR (RIP-qPCR) confirmed the binding of YTHDF2 to Runx2, and luciferase reporter assays confirmed the presence of the m6A site in Runx2. In addition, an actinomycin D assay showed that the half-life of Runx2 mRNA was dramatically shortened in VSMCs overexpressing YTHDF2. These results suggest that YTHDF2 directly binds to the m6A modification site of Runx2 to mediate the mRNA degradation that prevents VC by inhibiting the osteogenic development of VSMCs. Therefore, YTHDF2 can be considered a potential therapeutic target for managing VC.

Arterial pathological remodeling, central to arterial diseases including atherosclerosis and aortic aneurysms, is characterized by vascular smooth muscle cell (VSMC) phenotypic switching with concomitant loss of contractile markers. Uncovering the molecular changes initiating phenotypic transition may advance the understanding of vascular pathogenesis and provide new therapeutic strategies.

To construct a cross-species integrative model of VSMC transition in arterial diseases including atherosclerosis and aortic aneurysm, identify key genes regulating phenotypic switching in the trajectory from contractile to other phenotypes, and further validate their function in arterial remodeling models.

Public single-cell RNA-seq datasets were analyzed to map VSMC transcriptional dynamics and identify regulated gene expression patterns during transition. The changes were further checked using experimental animal aneurysm samples and PDGF-BB treated VSMCs. Functional validation included in vitro siRNA-mediated knockdown using primary VSMCs and in vivo gene-manipulated (AAv-shRNA/Adv-overexpression) wire-injury models.

Dysregulation of cytoskeletal-related genes (Fblim1, Tns1, and Synpo2) may cause disarrangement of actin cytoskeleton, and were identified as checkpoint process before VSMCs transition initiation. Knockdown of target genes suppressed contractile markers, enhanced proliferation, migration, and disrupted cytoskeleton architecture in VSMCs. In animal models, gene down-regulation exacerbated pathological remodeling while over-expression partially reverted these effects.

The findings highlight the critical role of cytoskeleton-related genes in arterial diseases that function as a critical checkpoint in preventing VSMC pathological phenotypic switching.

Vascular calcification is prevalent in chronic kidney disease (CKD), but existing medical treatments fail to achieve satisfactory therapeutic effects. Vascular calcification is now recognized as an active multifactorial process involving diverse mechanisms. Alpha-ketoglutarate (AKG), an intermediate in tricarboxylic acid cycle, has been demonstrated to extend lifespan and ameliorate age-related osteoporosis. However, whether AKG inhibits vascular calcification remains unknown.

Here, mineral deposition was studied with AKG treatment in rodent and human vascular smooth muscle cells (VSMCs) under osteogenic conditions in vivo and in vitro.

AKG treatment remarkably ameliorated calcification of rat and human arterial rings ex vivo and aortic calcification in CKD rats and mice. Mechanistically, AKG treatment upregulated DNA demethylase ten-eleven translocation 2 (TET2) expression during vascular calcification. Knockdown of TET2 by siRNA and pharmacological inhibition of TET2 by Bobcat339 promoted vascular calcification in rat VSMCs. Bobcat339 also enhanced rat aortic ring calcification. Conversely, TET2 overexpression ameliorated vascular calcification in rat VSMCs, rat aortic rings and CKD rats. Furthermore, VSMC-specific TET2 deficiency promoted aortic calcification in CKD mice. Both TET2 siRNA and Bobcat339 independently counteracted the inhibitory effect of AKG on vascular calcification of rat VSMCs. Inhibitory effect of AKG administration on vascular calcification was reduced in TET2 knockout mice. TET2 overexpression reduced the levels of the NLRP3 inflammasome pathway, cleaved Caspase-1 and IL-1β protein expression in VSMCs and NLRP3 agonist Nigericin-induced cell calcification.

Our study demonstrate that AKG attenuates vascular calcification partially via upregulation of TET2 and inhibition of NLRP3 inflammasome, indicating the critical role of epigenetic modifier in vascular calcification. Modulation of TET2 may become a promising strategy for the treatment of vascular calcification.

Background/Objectives: Under physiological conditions, vascular smooth muscle cells (VSMCs) are in a quiescent contractile state, but under pathological conditions, such as atherosclerosis, they change their phenotype to synthetic, characterized by increased proliferation, migration, and production of an extracellular matrix. Furthermore, VSMCs can undergo calcification, switching to an osteoblast-like phenotype, contributing to plaque instability. Methods: In this study, we analyzed the phenotypic changes in VSMCs during the transition from a physiological to a pathological state, a key process in the progression of atherosclerosis, using confocal and transmission electron microscopy, real-time PCR, and intracellular calcium quantification. Results: Confocal and transmission electron microscopy revealed a prominent remodeling of the actin cytoskeleton, increasing autophagic vacuoles in synthetic VSMCs and the deposition of calcium microcrystals in calcified cells. Immunofluorescence analysis revealed differential expression of α-SMA (contractile marker) and galectin-3 (synthetic marker), confirming the phenotypic changes. Real-time PCR further validated these changes, showing upregulation of RUNX-2, a marker of osteogenic transition, in calcified VSMCs. Conclusions: This study highlights the dynamic plasticity of VSMCs and their role in atherosclerosis progression. Understanding the characteristics of these phenotypic transitions can help develop targeted therapies to mitigate vascular calcification and plaque instability, potentially countering cardiovascular disease.

Genome-wide association studies have identified common genetic variants at ≈300 human genomic loci linked to coronary artery disease susceptibility. Among these genomic regions, the most impactful is the 9p21.3 coronary artery disease risk locus, which spans a 60-kb gene desert and encompasses ≈80 SNPs (single nucleotide polymorphism) in high linkage disequilibrium. Despite ≈2 decades since its discovery, the role of the 9p21.3 locus in cells of the vasculature remains incompletely resolved.

We differentiated induced pluripotent stem cells (iPSCs) from risk, nonrisk donors at 9p21.3, and isogenic knockouts into vascular smooth muscle cells (VSMCs). We performed single-cell transcriptomic profiling, including coembedding and comparison with publicly available human arterial data sets. We conducted functional characterization using migration and calcification assays and confirmed our findings on iPSC-VSMCs derived from additional donors. Finally, we used overexpression of ANRIL followed by gene expression analysis.

We demonstrated that iPSC-VSMCs harboring the 9p21.3 risk haplotype preferentially adopt an osteochondrogenic state and show remarkable similarity to fibrochondrocytes from human artery tissue. The transcriptional profile and functional assessment of migration and calcification capacity across iPSC-VSMC lines from multiple donors concordantly resemble an osteochondrogenic state. Importantly, we identified numerous transcription factors driving different VSMC state trajectories. Additionally, we prioritized LIMCH1 and CRABP1 as signature genes critical for defining the risk transcriptional program. Finally, overexpression of a short isoform of ANRIL in 9p21.3 knockout cells was sufficient to induce the osteochondrogenic transcriptional signature.

Our study provides new insights into the mechanism of the 9p21.3 risk locus and defines its previously undescribed role in driving a disease-prone transcriptional and functional state in VSMCs concordant with an osteochondrogenic-like state. Our data suggest that the 9p21.3 risk haplotype likely promotes arterial calcification, through altered expression of ANRIL, in a cell type-specific and cell-autonomous manner, providing insight into potential risk assessment and treatment for carriers.

Heterotopic ossification of tendons and ligaments causes pain and dysfunction, significantly reducing quality of life. However, its underlying mechanisms remain elusive. In addition to injury, tissue organization and stiffness have been implicated in heterotopic ossification. Collagen XII, a member of the fibril-associated collagens with interrupted triple helices (FACIT) family, plays a crucial role in maintaining the structural integrity and function of tendons and ligaments. Its deficiency alters tissue stiffness and predisposes ligaments to rupture. In this study, we investigated whether collagen XII contributes to the development of heterotopic ossification. Three-dimensional microcomputed tomography (3D-μCT) and X-ray analyses revealed heterotopic bone formation in the knee and ankle ligaments, but not in tendons, of Col12a1-deficient mice, with a 100 % incidence in mice older than 19 weeks. Histological analysis showed the presence of Alcian blue- and Toluidine blue-positive fibrochondrocyte-like cells in Col12a1-deficient ligaments, which were subsequently replaced by bone tissue, as indicated by Alizarin red staining. Real-time qPCR analysis of knee ligaments demonstrated a slight increase in chondrogenic markers and a significant upregulation of osteogenic markers in Col12a1-deficient mice compared with wild-type controls. In vitro chondrogenesis and osteogenesis assays using primary tenocytes from wild-type and Col12a1-deficient mice revealed that collagen XII deficiency enhanced osteogenic potential, whereas chondrogenic potential remained comparable. Our findings indicate that collagen XII deficiency specifically induces heterotopic bone formation in knee and ankle ligaments, occurring via fibrochondrocytes rather than through endochondral or intramembranous ossification.

Genome-wide association studies have identified common genetic variants at ~300 human genomic loci linked to coronary artery disease (CAD) susceptibility. Among these genomic regions, the most impactful is the 9p21.3 CAD risk locus, which spans a 60 kb gene desert and encompasses ~80 SNPs in high linkage disequilibrium. Despite nearly two decades since its discovery, the role of the 9p21.3 locus in cells of the vasculature remains incompletely resolved.

We differentiated induced pluripotent stem cells (iPSCs) from risk and non-risk donors at 9p21.3 into vascular smooth muscle cells. We performed single-cell transcriptomic profiling, including co-embedding and comparison with publicly available human arterial datasets. We conducted functional characterization using migration and calcification assays and confirmed our findings on iPSC-VSMCs derived from additional donors. Finally, we used overexpression of ANRIL followed by gene expression analysis.

We demonstrated that iPSC-VSMCs harboring the 9p21.3 risk haplotype preferentially adopt an osteochondrogenic state and show remarkable similarity to fibrochondrocytes from human artery tissue. The transcriptional profile and functional assessment of migration and calcification capacity across iPSC-VSMCs lines from multiple donors concordantly resemble an osteochondrogenic state. Importantly, we identified numerous transcription factors driving different VSMC state trajectories. Additionally, we prioritized LIMCH1 and CRABP1 as signature genes critical for defining the risk transcriptional program. Finally, overexpression of a short isoform of ANRIL in non-risk cells was sufficient to induce the osteochondrogenic transcriptional signature.

Our study provides new insights into the mechanism of the 9p21.3 risk locus and defines its previously undescribed role in driving a disease-prone transcriptional and functional state in VSMCs concordant with an osteochondrogenic-like state. Our data suggest that the 9p21.3 risk haplotype likely promotes arterial calcification, through altered expression of ANRIL, in a cell-type specific and cell-autonomous manner, providing insight into potential risk assessment and treatment for carriers.

Vascular calcification, a devastating vascular complication accompanying atherosclerotic cardiovascular disease and chronic kidney disease, increases the incidence of adverse cardiovascular events and compromises the efficacy of vascular interventions. However, effective therapeutic drugs and treatments to delay or prevent vascular calcification are lacking.

This study was designed to test the therapeutic effects and mechanism of Moscatilin (also known as dendrophenol) from Dendrobium huoshanense (an eminent traditional Chinese medicine) in suppressing vascular calcification in vitro, ex vivo and in vivo.

Male C57BL/6J mice (25-week-old) were subjected to nicotine and vitamin D3 (VD3) treatment to induce vascular calcification. In vitro, we established the cellular model of osteogenesis of human aortic smooth muscle cells (HASMCs) under phosphate conditions.

By utilizing an in-house drug screening strategy, we identified Moscatilin as a new naturally-occurring chemical entity to reduce HASMC calcium accumulation. The protective effects of Moscatilin against vascular calcification were verified in cultured HASMCs. Unbiased transcriptional profiling analysis and cellular thermal shift assay suggested that Moscatilin suppresses vascular calcification via binding to interleukin 13 receptor subunit A2 (IL13RA2) and augmenting its expression. Furthermore, IL13RA2 was reduced during HASMC osteogenesis, thus promoting the secretion of inflammatory factors via STAT3. We further validated the participation of Moscatilin-inhibited vascular calcification by the classical WNT/β-catenin pathway, among which WNT3 played a key role in this process. Moscatilin mitigated the crosstalk between WNT3/β-catenin and IL13RA2/STAT3 to reduce osteogenic differentiation of HASMCs.

This study supports the potential of Moscatilin as a new naturally-occurring candidate drug for treating vascular calcification via regulating the IL13RA2/STAT3 and WNT3/β-catenin signalling pathways.

Highly differentiated mature vascular smooth muscle cells (VSMCs) are the predominant type of cells constituting arterial walls, which are essential for maintaining the structural and functional integrity of blood vessels. VSMCs demonstrate a notable degree of adaptability following vascular damage, a characteristic that plays a crucial role in the progression of vascular remodeling. Advances in single-cell RNA sequencing in both healthy and pathological vascular tissues have offered profound insights into the complexity of VSMCs, revealing a more intricate diversity than previously recognized. In response to injury, VSMCs undergo dedifferentiation and exhibit pluripotent markers. This review summarizes the researches that have employed lineage tracing alongside single-cell sequencing analysis to explore the dynamics of vascular damage. The primary focus of this study was on the process of dedifferentiation in VSMCs, with particular attention to its underlying mechanisms. The discussion included the impact of microenvironmental cues, the control of transcription factors, and the various molecular pathways involved in VSMCs dedifferentiation. Herein, we provide a comprehensive analysis of cells dedifferentiated from adult VSMCs upon vascular injury.

The effective and translational strategy to regenerate knee meniscal fibrocartilage remained challenging. Herein, we first identified vascular smooth muscle cells (VSMCs) transdifferentiated into fibrochondrocytes and participated in spontaneous meniscal regeneration using smooth muscle cell lineage tracing transgenic mice meniscal defect model. Then, we identified low-intensity pulsed ultrasound (LIPUS) acoustic stimulus enhanced fibrochondrogenic transdifferentiation of VSMCs in vitro and in vivo. Mechanistically, LIPUS stimulus could up-regulate mechanosensitive ion channel Piezo1 expression and then activate the transforming growth factor β1 (TGFβ1) signal, following repression of the Notch signal, consequently enhancing fibrochondrogenic transdifferentiation of VSMCs. Finally, we demonstrated that the regular LIPUS stimulus enhanced anisotropic native-like meniscal fibrocartilage tissue regeneration in a beagle canine subtotal meniscectomy model at 6 months postoperatively. The single-cell RNA sequencing analysis confirmed the role of VSMC fibrochondrogenic transdifferentiation in meniscal regeneration.

Vascular calcification (VC) is a dynamic, tightly regulated process driven by cellular activity and resembling the mechanisms of bone formation, with specific molecules playing pivotal roles in its progression. We aimed to investigate the involvement of the bone morphogenic proteins (BMP-2, BMP-4, BMPR-1a/1b, and BMPR-2) system in this process. Our study used an advanced in vitro model that simulates the biological environment of the vascular wall, assessing the ability of a phosphate mixture to induce the osteoblastic switch in human coronary artery smooth muscle cells (HCASMCs).

HCASMCs were grown in mono- and co-culture with human coronary artery endothelial cells (HCAECs) in a double-flow bioreactor (LiveBox2 and IVTech), allowing static and dynamic conditions through a peristaltic pump. The VC was stimulated by incubation in a calcifying medium for 7 days. A BMP system Real-Time PCR was performed at the end of each experiment.

In monocultures, BMP-2 expression increased in calcified HCASMCs in static (p = 0.01) and dynamic conditions. BMP-4 and the biological receptors were expressed in all the experimental settings, increasing mainly in dynamic flow conditions. In co-cultures, we observed a marked increase in BMP-2 and BMP-4, BMPR-1a (p = 0.04 and p = 0.01, respectively), and BMPR-2 (p = 0.001) in the calcifying setting mostly in dynamic conditions.

The increase in BMP-2/4 in co-culture suggests that these genes might promote the switch towards an osteogenic-like phenotype, data also supported by the rise of both BMPR-1a and BMPR-2. Thus, our findings provide insights into the mechanisms by which dynamic co-culture modulates the BMP system activation in an environment mimicking in vivo VC's cellular and mechanical characteristics.

Vascular calcification frequently occurs in patients with chronic conditions such as chronic kidney disease (CKD), diabetes, and hypertension and represents a significant cause of cardiovascular events. Thus, identifying effective therapeutic targets to inhibit the progression of vascular calcification is essential. 4-Octyl itaconate (4-OI), a derivative of itaconate, exhibits anti-inflammatory and antioxidant activity, both of which play an essential role in the progression of vascular calcification. However, the role and molecular mechanisms of 4-OI in vascular calcification have not yet been elucidated. In this study, we investigated the effects of exogenous 4-OI on vascular calcification using vascular smooth muscle cells (VSMCs), arterial rings, and mice. Alizarin red staining and western blot revealed that 4-OI inhibited calcification and osteogenic differentiation of human VSMCs. Similarly, 4-OI inhibited calcification of rat and human arterial rings and VitD3-overloaded mouse aortas. Mechanistically, RNA sequencing analysis revealed that 4-OI treatment is most likely to affect heme oxygenase 1 (HMOX-1) mRNA expression. The study demonstrated that 4-OI treatment increased HMOX-1 mRNA and protein levels, but suppressed inflammation and oxidative stress in VSMCs under osteogenic conditions. Moreover, HMOX-1 knockdown by siRNA or treatment with the HMOX-1 inhibitor ZnPP9 significantly reversed the suppression effect on calcification of VSMCs and aortas of VitD3-overloaded mice by 4-OI. Furthermore, HMOX-1 knockdown by siRNA markedly abrogated the inhibitory effect of 4-OI on inflammation in VSMCs. These findings suggest that 4-OI alleviates vascular calcification and inhibits oxidative stress and inflammation through modulation of HMOX-1, indicating its potential as a therapeutic target for vascular calcification.

Vascular calcification (VC), associated with high cardiovascular mortality in patients with chronic kidney disease (CKD), involves osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs). O-GlcNAcylation, a dynamic post-translational modification, is closely linked to cardiovascular diseases, including VC. However, the exact role and molecular mechanism of O-GlcNAc signaling in abnormal mineral metabolism-induced VC remain unclear. In the current study, we found that the levels of O-GlcNAc transferase (OGT) and global protein O-GlcNAcylation were significantly upregulated in the artery tissues of mouse calcification models and CKD patients with VC. To further delineate the in vivo role of OGT in VC, we generated Ogt smooth muscle cell-specific knockout mice and challenged them with 5/6 nephrectomy (5/6 Nx) or high-dose vitamin D3 to induce VC. Deletion of Ogt in VSMCs led to alleviated VC in response to 5/6 Nx or VD3. Moreover, elevated O-GlcNAcylation, induced by Thiamet-G, facilitated osteogenic transdifferentiation in VSMCs in response to phosphate, whereas OSMI-1, which reduces O-GlcNAcylation, exhibited an opposite phenotypic effect. Mechanistically, O-GlcNAc signaling enhanced the osteogenic conversion of VSMCs through regulation of canonical Wnt/β-catenin pathway. Indeed, β-catenin was O-GlcNAcylated by OGT and further increased its transcriptional activity in VSMCs. Furthermore, pharmacological activation of Wnt/β-catenin signaling largely reversed the diminished aortic calcification caused by Ogt ablation. Our findings demonstrate that smooth muscle O-GlcNAc signaling plays an important role in regulating hyperphosphatemia-induced VC and reveal that O-GlcNAcylation of β-catenin protein modulates its content and activity in VSMCs.

Vascular smooth muscle cell (VSMC) plasticity is a state in which VSMCs undergo phenotypic switching from a quiescent contractile phenotype into other functionally distinct phenotypes. Although emerging evidence suggests that VSMC plasticity plays critical roles in the development of vascular diseases, little is known about the key determinant for controlling VSMC plasticity and fate.

We found that smooth muscle cell-specific deletion of Lkb1 in tamoxifen-inducible Lkb1flox/flox;Myh11-Cre/ERT2 mice spontaneously and progressively induced aortic/arterial dilation, aneurysm, rupture, and premature death. Single-cell RNA sequencing and imaging-based lineage tracing showed that Lkb1-deficient VSMCs transdifferentiated gradually from early modulated VSMCs to fibroblast-like and chondrocyte-like cells, leading to ossification and blood vessel rupture. Mechanistically, Lkb1 regulates polypyrimidine tract binding protein 1 (Ptbp1) expression and controls alternative splicing of pyruvate kinase muscle (PKM) isoforms 1 and 2. Lkb1 loss in VSMC results in an increased PKM2/PKM1 ratio and alters the metabolic profile by promoting aerobic glycolysis. Treatment with PKM2 activator TEPP-46 rescues VSMC transformation and aortic dilation in Lkb1flox/flox;Myh11-Cre/ERT2 mice. Furthermore, we found that Lkb1 expression decreased in human aortic aneurysm tissue compared to control tissue, along with changes in markers of VSMC fate.

Lkb1, via its regulation of Ptbp1-dependent alterative splicing of PKM, maintains VSMC in contractile states by suppressing VSMC plasticity.

Endothelial cell (EC) calcification is an important marker of atherosclerotic calcification. ECs play a critical role not only in atherogenesis but also in intimal calcification, as they have been postulated to serve as a source of osteoprogenitor cells that initiate this process. While the role of transglutaminase 2 (TG2) in cellular differentiation, survival, apoptosis, autophagy, and cell adhesion is well established, the mechanism underlying the TG2-mediated regulation of EC calcification is yet to be fully elucidated.

The TG2 gene was overexpressed or silenced by using siRNA and recombinant adenovirus. RT-PCR and WB were used to analyze the relative expression of target genes and proteins. 5-BP method analyzed TG2 activity. mCherry-eGFP-LC3 adenovirus and transmission electron microscopy analyzed EC autophagy level. Calcium concentrations were measured by using a calcium colorimetric assay kit. Alizarin red S staining assay analyzed EC calcification level. Elisa analyzed IL-6 level. Establishing EC calcification model by using a calcification medium (CM).

Our findings demonstrated that CM increased TG2 activity and expression, which activated the NF-κB signaling pathway, and induced IL-6 autocrine signaling in ECs. Furthermore, IL-6 activated the JAK2/STAT3 signaling pathway to suppress cell autophagy and promoted ECs calcification.

ECs are not only critical for atherogenesis but also believed to be a source of osteoprogenitor cells that initiate intimal calcification. Previous research has shown that TG2 plays an important role in the development of VC, but the mechanism by which it exerts this effect is not yet fully understood. Our results demonstrated that TG2 forms complexes with NF-κB components inhibition of autophagy promoted endothelial cell calcification through EndMT. Therefore, our research investigated the molecular mechanism of EC calcification, which can provide new insights into the pathogenesis of atherosclerosis.

Prolyl 4-hydroxylase subunit alpha 2 (P4HA2), a key enzyme modulating the post-transcription of proteins, was reported to be a causative gene in IA. Nevertheless, the exact function and mechanism of P4HA2 in the formation and rupture of IA is elusive. The current study first explored the expression of P4HA2 and its association with the clinicopathological demonstrations in patients with IA. In addition, an in vitro model of IA was established using H2O2 to stimulate vascular smooth muscle cells (VSMCs). The behaviors of treated VSMCs were evaluated using CCK-8, Wound healing, and Transwell assays. The expression of genes was detected by RT-qPCR and Western blot. Interaction between genes was confirmed using Luciferase Reporter assay and Co-immunoprecipitation (Co-IP) assay. Our results revealed that P4HA2 expression was upregulated in IA, especially ruptured IA; high P4HA2 expression correlates with unfavorable clinicopathological parameters. Through the in vitro experiments, it was discovered that P4HA2 knockdown rescued VSMCs from H2O2-induced viability impairment, enhancement in migration and apoptosis, switch from contractile phenotype, and augmentation of oxidative stress and inflammation. Mechanistically, P4HA2 was found to trigger the prolyl hydroxylation of YAP1 to negatively regulate the transcriptional activity of YAP1 in H2O2-challenged VSMCs. The effect of P4HA2 on H2O2-challenged VSMCs could be annulled by the mutation of YAP1 hydroxylation sites. In summary, P4HA2 served as a contributing factor during IA progression through its suppression on YAP1 activity by prolyl hydroxylation.

Coronary artery disease (CAD) poses a substantial threat to global health, leading to significant morbidity and mortality worldwide. It has a significant genetic component that has been studied through genome-wide association studies (GWAS) over the past 17 years. These studies have made progress with larger sample sizes, diverse ancestral backgrounds, and the discovery of multiple genomic regions related to CAD risk. In this review, we provide a comprehensive overview of CAD GWAS, including information about the genetic makeup of the disease and the importance of ethnic diversity in these studies. We also discuss challenges of identifying causal genes and variants within GWAS loci with a focus on non-coding regions. Additionally, we highlight tissues and cell types relevant to CAD, and discuss clinical implications of GWAS findings including polygenic risk scores, sex-specific differences in CAD genetics, ethnical aspects of personalized interventions, and GWAS guided drug development.

Vascular calcification is a common phenomenon in various vascular diseases, where its presence heralds increased occurrence of adverse disease events, which invariably lead to increased morbidity and mortality in patients. Although the impact of calcification has become apparent, adequate and early detection of the most damaging form of early microcalcification is still in its infancy, preventing reliable identification of locations that would benefit from intervention. In this review, we will provide an overview of the current state-of-the-art noninvasive calcification imaging and its persisting limitations. We discuss promising approaches that may address these limitations in the future. In this context particular attention will be paid to imaging modalities such as CT, PET, and ultrasonography and molecular and cellular mechanisms and agents involved in physiological bone formation.

Atherosclerosis (AS) is a complex pathology process involving intricate interactions among various cells and biological processes. Vascular smooth muscle cells (VSMCs) are the predominant cell type in normal arteries, and under atherosclerotic stimuli, VSMCs respond to altered blood flow and microenvironment changes by downregulating contractile markers and switching their phenotype. This review overviews the diverse phenotypes of VSMCs, including the canonical contractile VSMCs, synthetic VSMCs, and phenotypes resembling macrophages, foam cells, myofibroblasts, osteoblasts/chondrocytes, and mesenchymal stem cells. We summarize their presumed protective and pro-atherosclerotic roles in AS development. Additionally, we underscore the molecular mechanisms and regulatory pathways governing VSMC phenotypic switching, encompassing transcriptional regulation, biochemical factors, plaque microenvironment, epigenetics, miRNAs, and the cytoskeleton, emphasizing their significance in AS development. Finally, we outline probable future research directions targeting VSMCs, offering insights into potential therapeutic strategies for AS management.

Cardiovascular calcification, characterized by hydroxyapatite deposition in the arterial wall and heart valves, is associated with high cardiovascular morbidity and mortality. Cardiovascular calcification is a hallmark of aging but is frequently seen in association with chronic diseases, such as chronic kidney disease (CKD), diabetes, dyslipidemia, and hypertension in the younger population as well. Currently, there is no therapeutic approach to prevent or cure cardiovascular calcification. The pathophysiology of cardiovascular calcification is highly complex and involves osteogenic differentiation of various cell types of the cardiovascular system, such as vascular smooth muscle cells and valve interstitial cells. In vitro cellular and ex vivo tissue culture models are simple and useful tools in cardiovascular calcification research. These models contributed largely to the discoveries of the numerous calcification inducers, inhibitors, and molecular mechanisms. In this review, we provide an overview of the in vitro cell culture and the ex vivo tissue culture models applied in the research of cardiovascular calcification.

Vascular calcification is quite common in patients with end-stage chronic kidney disease and is a major trigger for cardiovascular complications in these patients. These complications significantly impact the survival rate and long-term prognosis of individuals with chronic kidney disease. Numerous studies have demonstrated that the development of vascular calcification involves various pathophysiological mechanisms, with the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) being of utmost importance. High phosphate levels, bone morphogenetic protein 2 (BMP2), and runt-related transcription factor 2 (RUNX2) play crucial roles in the osteogenic transdifferentiation process of VSMCs. This article primarily reviews the molecular mechanisms by which high phosphate, BMP2, and RUNX2 regulate vascular calcification secondary to chronic kidney disease, and discusses the complex interactions among these factors and their impact on the progression of vascular calcification. The insights provided here aim to offer new perspectives for future research on the phenotypic switching and osteogenic transdifferentiation of VSMCs, as well as to aid in optimizing clinical treatment strategies for this condition, bearing significant clinical and scientific implications.

Sex hormones play a pivotal role as endocrine hormones that exert profound effects on the biological characteristics and vascular function of vascular smooth muscle cells (VSMCs). By modulating intracellular signaling pathways, activating nuclear receptors, and regulating gene expression, sex hormones intricately influence the morphology, function, and physiological state of VSMCs, thereby impacting the biological properties of vascular contraction, relaxation, and growth. Increasing evidence suggests that abnormal phenotypic changes in VSMCs contribute to the initiation of vascular diseases, including atherosclerosis. Therefore, understanding the factors governing phenotypic alterations in VSMCs and elucidating the underlying mechanisms can provide crucial insights for refining interventions targeted at vascular diseases. Additionally, the varying levels of different types of sex hormones in the human body, influenced by sex and age, may also affect the phenotypic conversion of VSMCs. This review aims to explore the influence of sex hormones on the phenotypic switching of VSMCs and the development of associated vascular diseases in the human body.

Vascular calcification is associated with increased mortality in patients with cardiovascular disease. Secondary calciprotein particles are believed to play a causal role in the pathophysiology of vascular calcification. The maturation time (T50) of calciprotein particles provides a measure of serum calcification propensity. We compared T50 between patients with ST-segment-elevated myocardial infarction and control subjects and studied the association of T50 with cardiovascular risk factors and outcome.

T50 was measured by nephelometry in 347 patients from the GIPS-III trial (Metabolic Modulation With Metformin to Reduce Heart Failure After Acute Myocardial Infarction: Glycometabolic Intervention as Adjunct to Primary Coronary Intervention in ST Elevation Myocardial Infarction: a Randomized Controlled Trial) and in 254 matched general population controls from PREVEND (Prevention of Renal and Vascular End-Stage Disease). We also assessed the association between T50 and left ventricular ejection fraction, as well as infarct size, the incidence of ischemia-driven reintervention during 5 years of follow-up, and serum nitrite as a marker of endothelial dysfunction.

Patients with ST-segment-elevated myocardial infarction had a significantly lower T50 (ie, higher serum calcification propensity) compared with controls (T50: 289±63 versus 338±56 minutes; P<0.001). In patients with ST-segment-elevated myocardial infarction, lower T50 was associated with female sex, lower systolic blood pressure, lower total cholesterol, lower LDL (low-density lipoprotein) cholesterol, lower triglycerides, and higher HDL (high-density lipoprotein) cholesterol but not with circulating nitrite or nitrate. Ischemia-driven reintervention was associated with higher LDL (P=0.03) and had a significant interaction term for T50 and sex (P=0.005), indicating a correlation between ischemia-driven reintervention and T50 above the median in men and below the median in women, between 150 days and 5 years of follow-up.

Serum calcification propensity is increased in patients with ST-segment-elevated myocardial infarction compared with the general population, and its contribution is more pronounced in women than in men. Its lack of/inverse association with nitrite and blood pressure confirms T50 to be orthogonal to traditional cardiovascular disease risk factors. Lower T50 was associated with a more favorable serum lipid profile, suggesting the involvement of divergent pathways of calcification stress and lipid stress in the pathophysiology of myocardial infarction.

Primary hyperparathyroidism (pHPT) afflicts our aging population with an incidence approaching 50 per 100 000 patient-years at a female:male ratio of ~3:1. Decisions surrounding surgical management are currently driven by age, hypercalcemia severity, presence of osteoporosis, renal insufficiency, or hypercalciuria with or without nephrolithiasis. Cardiovascular (CV) disease (CVD) is not systematically considered. This is notable since the parathyroid hormone (PTH) 1 receptor (PTH1R) is biologically active in the vasculature, and adjusted CV mortality risk is increased almost threefold in individuals with pHPT who do not meet contemporary recommendations for surgical cure. We provide an overview of epidemiology, pharmacology, and physiology that highlights the need to: (i) identify biomarkers that establish a healthy 'set point' for CV PTH1R signaling tone; (ii) better understand the pharmacokinetic-pharmacodynamic (PK-PD) relationships of PTH1R ligands in CV homeostasis; and (iii) incorporate CVD risk assessment into the management of hyperparathyroidism.

Vascular calcification is a pathological process commonly associated with atherosclerosis, chronic kidney disease, and diabetes. Paraspeckle protein NONO is a multifunctional RNA/DNA binding protein involved in many nuclear biological processes but its role in vascular calcification remains unclear. Here, we observed that NONO expression was decreased in calcified arteries of mice and patients with CKD. We generated smooth muscle-specific NONO-knockout mice and established three different mouse models of vascular calcification by means of 5/6 nephrectomy, adenine diet to induce chronic kidney failure, or vitamin D injection. The knockout mice were more susceptible to the development of vascular calcification relative to control mice, as verified by an increased calcification severity and calcium deposition. Likewise, aortic rings from knockout mice showed more significant vascular calcification than those from control mice ex vivo. In vitro, NONO deficiency aggravated high phosphate-induced vascular smooth muscle cell osteogenic differentiation and apoptosis, whereas NONO overexpression had a protective effect. Mechanistically, we demonstrated that the regulation of vascular calcification by NONO was mediated by bone morphogenetic protein 2 (BMP2). NONO directly bound to the BMP2 promoter using its C-terminal region, exerting an inhibitory effect on the transcription of BMP2. Thus, our study reveals that NONO is a novel negative regulator of vascular calcification, which inhibits osteogenic differentiation of vascular smooth muscle cell and vascular calcification via negatively regulating BMP2 transcription. Hence, NONO may provide a promising target for the prevention and treatment of vascular calcification.

Vascular calcification has been linked to bone mineral density (BMD). This study aimed to investigate the association between BMD and abdominal aortic calcification (AAC).

Data from the 2013-2014 National Health and Nutrition Examination Survey (NHANES) were utilized. Participants lacking BMD and AAC score data were excluded. BMD at the femoral neck was measured using dual-energy X-ray absorptiometry. AAC scores were assessed using the Kauppila scoring system, with AAC defined as a score greater than zero, and severe AAC defined as a score greater than six. Weighted multivariable regression analysis and subgroup analysis were conducted to examine the independent relationship between BMD and AAC score, AAC, and severe AAC. A total of 2965 participants were included. After adjusting for multiple covariates, BMD showed a negative association with higher AAC scores (β = -0.17, 95% CI -0.29, -0.05, p = 0.0066). The odds of having AAC and severe AAC decreased by 9% and 16%, respectively, for every one-unit increase in BMD (AAC: odds ratio [OR] = 0.91, 95% CI 0.82, 1.00, p = 0.0431; severe AAC: OR = 0.84, 95% CI 0.71, 0.99, p = 0.0334).

Low BMD is associated with higher AAC scores and an increased risk of AAC and severe AAC. Considering the detrimental impact of low BMD on cardiovascular health, individuals with AAC should be evaluated for osteopenia and osteoporosis in clinical settings.

Distinguishing quiescent from rupture-prone atherosclerotic lesions has significant translational and clinical implications. Electrochemical impedance spectroscopy (EIS) characterizes biological tissues by assessing impedance and phase delay responses to alternating current at multiple frequencies. We evaluated invasive 6-point stretchable EIS sensors over a spectrum of experimental atherosclerosis and compared results with intravascular ultrasound (IVUS), molecular positron emission tomography (PET) imaging, and histology. Male New Zealand White rabbits (n = 16) were placed on a high-fat diet, with or without endothelial denudation via balloon injury of the infrarenal abdominal aorta. Rabbits underwent in vivo micro-PET imaging of the abdominal aorta with 68Ga-DOTATATE, 18F-NaF, and 18F-FDG, followed by invasive interrogation via IVUS and EIS. Background signal-corrected values of impedance and phase delay were determined. Abdominal aortic samples were collected for histology. Analyses were performed blindly. EIS impedance was associated with markers of plaque activity including macrophage infiltration (r = .813, p = .008) and macrophage/smooth muscle cell (SMC) ratio (r = .813, p = .026). Moreover, EIS phase delay correlated with anatomic markers of plaque burden, namely intima/media ratio (r = .883, p = .004) and %stenosis (r = .901, p = .002), similar to IVUS. 68Ga-DOTATATE correlated with intimal macrophage infiltration (r = .861, p = .003) and macrophage/SMC ratio (r = .831, p = .021), 18F-NaF with SMC infiltration (r = -.842, p = .018), and 18F-FDG correlated with macrophage/SMC ratio (r = .787, p = .036). EIS with phase delay integrates key atherosclerosis features that otherwise require multiple complementary invasive and non-invasive imaging approaches to capture. These findings indicate the potential of invasive EIS to comprehensively evaluate human coronary artery disease.

Tumoral calcinosis is an extremely rare genetic disease caused by mutations in three genes, GALNT3, FGF23, and KL, which disrupt phosphorus metabolism. The hallmark of this condition is the formation of tumors in the soft tissues around the joints. Other phenotypic features of tumoral calcinosis are dental involvement and brain and vascular calcifications. The clinical case reported herein presents for the first time to the scientific community the c.202A>G (p.Thr68Ala) mutation of the FGF23 gene, associated with a hyperphosphatemic variant of tumoral calcinosis and multiple severe vascular aneurysms. A female patient underwent multiple surgeries for tumor formations in her soft tissues that first appeared at the age of 12 months. On this occurrence, the patient was found to have hyperphosphatemia, low phosphate clearance, increased tubular reabsorption with normal levels of total and ionized calcium, vitamin D3, and parathyroid hormone, and no effect of treatment with sevelamer hydrochloride and a low-phosphate diet. At the age of 39, the patient underwent imaging studies due to edema and a pulsating formation in the neck area, which revealed multiple vascular aneurysms with thrombosis, for which she received operative and interventional treatment. In this connection, and because of the established phosphorus metabolism disturbance, a genetic disease was suspected. The sequence analysis and deletion/duplication testing of the 358 genes performed on this occasion revealed that the woman was homozygous for a variant of the c.202A>G (p.Thr68Ala) mutation of the FGF23 gene. The established mutation is not present in population databases. The presented clinical case is the first and only one in the world to demonstrate the role of this type of FGF23 gene mutation in the development of a hyperphosphatemic variant of tumoral calcinosis characterized by aggressive formation of multiple vascular aneurysms.

Control of vascular smooth muscle cell (SMC) gene expression is an essential process for establishing and maintaining lineage identity, contractility, and plasticity. Most mechanisms (epigenetic, transcriptional, and post-transcriptional) implicated in gene regulation occur in the nucleus. Still, intranuclear pathways are directly impacted by modifications in the extracellular environment in conditions of adaptive or maladaptive remodeling. Integration of extracellular, cellular, and genomic information into the nucleus through epigenetic and transcriptional control of genome organization plays a major role in regulating SMC functions and phenotypic transitions during vascular remodeling and diseases. This review aims to provide a comprehensive update on nuclear mechanisms, their interactions, and their integration in controlling SMC homeostasis and dysfunction. It summarizes and discusses the main nuclear mechanisms preponderant in SMCs in the context of vascular disease, such as atherosclerosis, with an emphasis on studies employing in vivo cell-specific loss-of-function and single-cell omics approaches.

Vascular smooth muscle cells (VSMCs) play a critical role in regulating vasotone, and their phenotypic plasticity is a key contributor to the pathogenesis of various vascular diseases. Two main VSMC phenotypes have been well described: contractile and synthetic. Contractile VSMCs are typically found in the tunica media of the vessel wall, and are responsible for regulating vascular tone and diameter. Synthetic VSMCs, on the other hand, are typically found in the tunica intima and adventitia, and are involved in vascular repair and remodeling. Switching between contractile and synthetic phenotypes occurs in response to various insults and stimuli, such as injury or inflammation, and this allows VSMCs to adapt to changing environmental cues and regulate vascular tone, growth, and repair. Furthermore, VSMCs can also switch to osteoblast-like and chondrocyte-like cell phenotypes, which may contribute to vascular calcification and other pathological processes like the formation of atherosclerotic plaques. This provides discusses the mechanisms that regulate VSMC phenotypic switching and its role in the development of vascular diseases. A better understanding of these processes is essential for the development of effective diagnostic and therapeutic strategies.

Vascular calcification is a severe complication of cardiovascular diseases. Previous studies demonstrated that endothelial lineage cells transitioned into osteoblast-like cells and contributed to vascular calcification. Here, we found that inhibition of cyclin-dependent kinase (CDK) prevented endothelial lineage cells from transitioning to osteoblast-like cells and reduced vascular calcification. We identified a robust induction of CDK1 in endothelial cells (ECs) in calcified arteries and showed that EC-specific gene deletion of CDK1 decreased the calcification. We found that limiting CDK1 induced E-twenty-six specific sequence variant 2 (ETV2), which was responsible for blocking endothelial lineage cells from undergoing osteoblast differentiation. We also found that inhibition of CDK1 reduced vascular calcification in a diabetic mouse model. Together, the results highlight the importance of CDK1 suppression and suggest CDK1 inhibition as a potential option for treating vascular calcification.

Prior studies have established a connection between folate intake and cardiovascular disease (CVD). Abdominal aortic calcification (AAC) has been introduced as a good predictor of CVD events, but no previous study has investigated the relationship between dietary folate intake and severe AAC. Therefore, the study aims to explore the association between dietary folate intake and severe AAC in the United States (US) middle-aged and elderly population.

This study employed cross-sectional data from the 2013-2014 National Health and Nutrition Examination Survey (NHANES) to examine the relationship between dietary folate intake and severe AAC. Two 24-h dietary recall interviews were conducted to assess dietary folate intake and its sources, while a DXA scan was used to determine the AAC score. To analyze the association between dietary folate intake and severe AAC, a multivariable logistic regression model was applied, and a subgroup analysis was performed.

Our analysis utilized data from 2640 participants aged 40 years and above, including 288 individuals diagnosed with severe AAC. After adjusting for confounding factors, we observed an inverted L-shaped association between folate intake and severe AAC. Upon further adjustment for specific confounding factors and covariates, the multivariable-adjusted odds ratios (ORs) and corresponding 95% confidence intervals (CIs) for the second, third, and fourth quartiles of folate intake, using the first quartile as the reference, were as follows: 1.24 (0.86-1.79), 0.86 (0.58-1.27), and 0.63 (0.41-0.97), respectively. Subgroup analysis results were consistent with the logistic regression models, indicating concordant findings. Moreover, no significant interaction was observed in the subgroup analyses.

The study findings suggest an inverted L-shaped association between dietary folate intake and severe AAC. However, additional prospective investigations are necessary to explore the impact of dietary folate intake on severe AAC in patients.

Smooth muscle cells, endothelial cells and macrophages display remarkable heterogeneity within the healthy vasculature and under pathological conditions. During development, these cells arise from numerous embryological origins, which confound with different microenvironments to generate postnatal vascular cell diversity. In the atherosclerotic plaque milieu, all these cell types exhibit astonishing plasticity, generating a variety of plaque burdening or plaque stabilizing phenotypes. And yet how developmental origin influences intraplaque cell plasticity remains largely unexplored despite evidence suggesting this may be the case. Uncovering the diversity and plasticity of vascular cells is being revolutionized by unbiased single cell whole transcriptome analysis techniques that will likely continue to pave the way for therapeutic research. Cellular plasticity is only just emerging as a target for future therapeutics, and uncovering how intraplaque plasticity differs across vascular beds may provide key insights into why different plaques behave differently and may confer different risks of subsequent cardiovascular events.

Vascular calcification is a major risk factor for cardiovascular disease mortality, with a significant prevalence in chronic kidney disease (CKD). Pharmacological inhibition of histone acetyltransferase has been proven to protect against from vascular calcification. However, the role of Histone Deacetylase 2 (HDAC2) and molecular mechanisms in vascular calcification of CKD remains unknown. An in vivo model of CKD was established using mouse fed with a high adenine and phosphate diet, and an in vitro model was produced using human aortic vascular smooth muscle cells (VSMCs) stimulated with β-glycerophosphate (β-GP). HDAC2 expression was found to be reduced in medial artery of CKD mice and β-GP-induced VSMCs. Overexpression of HDAC2 attenuated OPN and OCN upregulation, α-SMA and SM22α downregulation, and calcium deposition in aortas of CKD. The in vitro results also demonstrated that β-GP-induced osteogenic differentiation was inhibited by HDAC2. Furthermore, we found that HDAC2 overexpression caused an increase in LC3II/I, a decrease in p62, and an induction of autophagic flux. Inhibition of autophagy using its specific inhibitor 3-MA blocked HDAC2's protective effect on osteogenic differentiation in β-GP-treated VSMCs. Taken together, these results suggest that HDAC2 may protect against vascular calcification by the activation of autophagy, laying out a novel insight for the molecular mechanism in vascular calcification of CKD.