The role of macrophage polarization in vascular calcification.

Vascular calcification is an important factor in the high morbidity and mortality of Cardiovascular and cerebrovascular diseases. Vascular damage caused by calcification of the intima or media impairs the physiological function of the vascular wall. Inflammation is a central factor in the development of vascular calcification. Macrophages are the main inflammatory cells. Dynamic changes of macrophages with different phenotypes play an important role in the occurrence, progression and stability of calcification. This review focuses on macrophage polarization and the relationship between macrophages of different phenotypes and calcification environment, as well as the mechanism of interaction, it is considered that macrophages can promote vascular calcification by releasing inflammatory mediators and promoting the osteogenic transdifferentiation of smooth muscle cells and so on. In addition, several therapeutic strategies aimed at macrophage polarization for vascular calcification are described, which are of great significance for targeted treatment of vascular calcification.

Hypomethylation of the LncRNA H19 promoter accelerates osteogenic differentiation of vascular smooth muscle cells by activating the Erk1/2 pathways.

OBJECTIVE: Vascular calcification is a common chronic kidney disease complication. This study aimed to investigate the function of long non-coding RNA (LncRNA) H19 in vascular calcification to explore new therapeutic strategies. METHODS: We induced osteogenic differentiation and calcification of vascular smooth muscle cells (VSMCs) using ß-glycerophosphate. Then, we detected the LncRNA H19 promoter methylation status and Erk1/2 pathways using methylation-specific polymerase chain reaction and western blotting, respectively. RESULTS: Compared with the control group, high phosphorus levels induced VSMC calcification, accompanied by increases in LncRNA H19 and the osteogenic marker Runx2 and reduction of the contractile phenotype marker SM22a. LncRNA H19 knockdown inhibited osteogenic differentiation and calcification of VSMCs. However, the suppressed role of VSMC calcification caused by shRNA H19 was partially reversed by simultaneous activation of the Erk1/2 pathways. Mechanically, we found that the methylation rate of CpG islands in the LncRNA H19 promoter region was significantly lower in the high-phosphorus group, and the hypomethylation state elevated LncRNA H19 levels, which in turn regulated phosphorylated Erk1/2 expression. CONCLUSIONS: LncRNA H19 promoted osteogenic differentiation and calcification of VSMCs by regulating the Erk1/2 pathways. Additionally, hypomethylation of LncRNA H19 promoter CpG islands upregulated LncRNA H19 levels and subsequently activated Erk1/2 phosphorylation.

Sox9 Accelerates Vascular Aging by Regulating Extracellular Matrix Composition and Stiffness.

BACKGROUND: Vascular calcification and increased extracellular matrix (ECM) stiffness are hallmarks of vascular aging. Sox9 (SRY-box transcription factor 9) has been implicated in vascular smooth muscle cell (VSMC) osteo/chondrogenic conversion; however, its relationship with aging and calcification has not been studied. METHODS: Immunohistochemistry was performed on human aortic samples from young and aged patients. Young and senescent primary human VSMCs were induced to produce ECM, and Sox9 expression was manipulated using adenoviral overexpression and depletion. ECM properties were characterized using atomic force microscopy and proteomics, and VSMC phenotype on hydrogels and the ECM were examined using confocal microscopy. RESULTS: In vivo, Sox9 was not spatially associated with vascular calcification but correlated with the senescence marker p16 (cyclin-dependent kinase inhibitor 2A). In vitro Sox9 showed mechanosensitive responses with increased expression and nuclear translocation in senescent cells and on stiff matrices. Sox9 was found to regulate ECM stiffness and organization by orchestrating changes in collagen (Col) expression and reducing VSMC contractility, leading to the formation of an ECM that mirrored that of senescent cells. These ECM changes promoted phenotypic modulation of VSMCs, whereby senescent cells plated on ECM synthesized from cells depleted of Sox9 returned to a proliferative state, while proliferating cells on a matrix produced by Sox9 expressing cells showed reduced proliferation and increased DNA damage, reiterating features of senescent cells. LH3 (procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3) was identified as an Sox9 target and key regulator of ECM stiffness. LH3 is packaged into extracellular vesicles and Sox9 promotes extracellular vesicle secretion, leading to increased LH3 deposition within the ECM. CONCLUSIONS: These findings highlight the crucial role of ECM structure and composition in regulating VSMC phenotype. We identify a positive feedback cycle, whereby cellular senescence and increased ECM stiffening promote Sox9 expression, which, in turn, drives further ECM modifications to further accelerate stiffening and senescence.

A combined circulating microRNA panel predicts the risk of vascular calcification in community-dwelling older adults with age strata differences.

BACKGROUND: Older adults have a higher risk of developing vascular calcification (VC). Circulating miRNAs can be potential risk indicators. However, prior studies used single miRNA mostly, whereas miRNA panels were rarely evaluated. We aimed to examine whether a miRNA panel outperformed each miRNA alone, and analyzed whether advanced age affected VC risk predictive performance offered by the miRNA panel. METHODS: We prospectively enrolled older adults (age ≥65 years) during their annual health checkup in 2017, and examined their VC severity followed by analyzing sera for VC regulatory miRNAs (miR-125b-5p, miR-125b-3p, and miR-378a-3p). We used multiple regression analyses to determine associations between each miRNA or a 3-combind panel and VC risk, followed by area under the receiver-operating-characteristics curve (AUROC) analysis. Participants were further divided to those of 65-75 and ≥75 years for comparison. RESULTS: From 199 older adults screened, 169 (median age, 73.3 years) with available calcification assessment were analyzed, among whom 74.6 % having VC. Those with VC had significantly lower circulating miR-125b-5p, miR-125b-3p, and miR-378a-3p levels than those without. Regression analyses showed that the 3-combined miRNA panel exhibited significant associations with VC risk, with significantly higher AUROC than those of models based on individual miRNA. Importantly, in those ≥75 years, the miRNA-predicted risk of VC was more prominent than that in the 65-75 years group. CONCLUSION: A miRNA panel for VC risk prediction might outperform individual miRNA alone in older adults, and advanced age modified the association between circulating miRNAs and the risk of VC.

The transcription factor GATA6 accelerates vascular smooth muscle cell senescence-related arterial calcification by counteracting the role of anti-aging factor SIRT6 and impeding DNA damage repair.

Arterial calcification is a hallmark of vascular pathology in the elderly and in individuals with chronic kidney disease (CKD). Vascular smooth muscle cells (VSMCs), after attaining a senescent phenotype, are implicated in the calcifying process. However, the underlying mechanism remains to be elucidated. Here, we reveal an aberrant upregulation of transcriptional factor GATA6 in the calcified aortas of humans, mice with CKD and mice subjected to vitamin D3 injection. Knockdown of GATA6, via recombinant adeno-associated virus carrying GATA6 shRNA, inhibited the development of arterial calcification in mice with CKD. Further gain- and loss-of function experiments in vitro verified the contribution of GATA6 in osteogenic differentiation of VSMCs. Samples of human aorta exhibited a positive relationship between age and GATA6 expression and GATA6 was also elevated in the aortas of old as compared to young mice. Calcified aortas displayed senescent features with VSMCs undergoing premature senescence, blunted by GATA6 downregulation. Notably, abnormal induction of GATA6 in senescent and calcified aortas was rescued in Sirtuin 6 (SIRT6)-transgenic mice, a well-established longevity mouse model. Suppression of GATA6 accounted for the favorable effect of SIRT6 on VSMCs senescence prevention. Mechanistically, SIRT6 inhibited the transcription of GATA6 by deacetylation and increased degradation of transcription factor Nkx2.5. Moreover, GATA6 was induced by DNA damage stress during arterial calcification and subsequently impeded the Ataxia-telangiectasia mutated (ATM)-mediated DNA damage repair process, leading to accelerated VSMCs senescence and osteogenic differentiation. Thus, GATA6 is a novel regulator in VSMCs senescence. Our findings provide novel insight in arterial calcification and a potential new target for intervention.

lncRNA FAS-AS1 served as a diagnostic biomarker of end-stage renal disease and mediated vascular calcification via regulating oxidative stress and inflammation.

PURPOSE: Vascular calcification is a frequently occurring complication of end-stage renal disease (ESRD). This study focused on the significance of long non-coding RNA Fas cell surface death receptor-antisense 1(lncRNA FAS-AS1) in ESRD-related vascular calcification aiming to explore a potential biomarker for the detection. METHODS: The study enrolled 65 healthy individuals, 79 ESRD patients (48 patients with vascular calcification), and 93 early-stage (I-IV) chronic kidney disease (CKD) patients. The expression of FAS-AS1 in serum was evaluated by real-time quantitative polymerase chain reaction (PCR). The diagnostic potential of FAS-AS1 was assessed in discriminating ESRD patients, vascular calcification, and the severity of vascular calcification. In vitro, the vascular smooth muscle cells (VSMCs) were treated with a hyperphosphatemia medium to evaluate the effect of FAS-AS1 on VSMCs calcification. RESULTS: Elevated serum FAS-AS1 was observed in ESRD patients, which could discriminate from healthy individuals and early-stage CKD patients. FAS-AS1 was associated with the development of ESRD and the occurrence of vascular calcification. FAS-AS1 was also upregulated in vascular calcification patients, especially the patients with severe calcification, which showed diagnostic significance in evaluating vascular calcification degrees. Calcified VSMCs showed significantly increased levels of Ca2+, reactive oxygen species (ROS), tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6), which was attenuated by silencing FAS-AS1. CONCLUSIONS: FAS-AS1 discriminated ERSD patients and was associated with the occurrence of vascular calcification. The knockdown of FAS-AS1 suppressed hyperphosphatemia-induced vascular calcification via alleviating oxidative stress and inflammation.

ENPP1 in Blood and Bone: Skeletal and Soft Tissue Diseases Induced by ENPP1 Deficiency.

The enzyme ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) codes for a type 2 transmembrane glycoprotein that hydrolyzes extracellular ATP to generate pyrophosphate (PPi) and adenosine monophosphate, thereby contributing to downstream purinergic signaling pathways. The clinical phenotypes induced by ENPP1 deficiency are seemingly contradictory and include early-onset osteoporosis in middle-aged adults and life-threatening vascular calcifications in the large arteries of infants with generalized arterial calcification of infancy. The progressive overmineralization of soft tissue and concurrent undermineralization of skeleton also occur in the general medical population, where it is referred to as paradoxical mineralization to highlight the confusing pathophysiology. This review summarizes the clinical presentation and pathophysiology of paradoxical mineralization unveiled by ENPP1 deficiency and the bench-to-bedside development of a novel ENPP1 biologics designed to treat mineralization disorders in the rare disease and general medical population.

Upregulation of NOR-1 in calcified human vascular tissues: impact on osteogenic differentiation and calcification.

Cardiovascular calcification is a significant public health issue whose pathophysiology is not fully understood. NOR-1 regulates critical processes in cardiovascular remodeling, but its contribution to ectopic calcification is unknown. NOR-1 was overexpressed in human calcific aortic valves and calcified atherosclerotic lesions colocalizing with RUNX2, a factor essential for osteochondrogenic differentiation and calcification. NOR-1 and osteogenic markers were upregulated in calcifying human valvular interstitial cells (VICs) and human vascular smooth muscle cells (VSMCs). Gain- and loss-of-function approaches demonstrated that NOR-1 negatively modulates the expression of osteogenic genes relevant for the osteogenic transdifferentiation (RUNX2, IL-6, BMP2, and ALPL) and calcification of VICs. VSMCs from transgenic mice overexpressing NOR-1 in these cells (TgNOR-1VSMC) expressed lower basal levels of osteogenic genes (IL-6, BMP2, ALPL, OPN) than cells from WT littermates, and their upregulation by a high-phosphate osteogenic medium (OM) was completely prevented by NOR-1 transgenesis. Consistently, this was associated with a dramatic reduction in the calcification of both transgenic VSMCs and aortic rings from TgNOR-1VSMC mice exposed to OM. Atherosclerosis and calcification were induce in mice by the administration of AAV-PCSK9D374Y and a high-fat/high-cholesterol diet. Challenged-TgNOR-1VSMC mice exhibited decreased vascular expression of osteogenic markers, and both less atherosclerotic burden (assessed in whole aorta and lesion size in aortic arch and brachiocephalic artery) and less vascular calcification (assessed either by near-infrared fluorescence imaging or histological analysis) than WT mice. Our data indicate that NOR-1 negatively modulates the expression of genes critically involved in the osteogenic differentiation of VICs and VSMCs, thereby restraining ectopic cardiovascular calcification.

Roles of vitamin K­dependent protein in biomineralization (Review).

Vitamin K (VK), a fat­soluble vitamin, is well known as an anticoagulant in the clinic. It is essential for the post­translational activation of VK­dependent proteins (VKDPs) because hydroquinone VK is a cofactor of glutamine carboxylase. At present, 17 VKDPs are known, which are mainly involved in coagulation and calcification. When Glu residues are carboxylated to Gla residues, these proteins gain a higher calcium­binding ability, which explains why VK has an important role in blood coagulation and biomineralization. However, the current view on the role of VK and several VKDPs in biomineralization remains inconsistent. For instance, conflicting results have been reported regarding the effect of osteocalcin gene knockout on the bone of mice; matrix Gla protein (MGP) promotes osteoblasts mineralization but inhibits vascular smooth muscle cell mineralization. The present review aimed to summarize the existing evidence that several VKDPs, including osteocalcin, MGP, Gla­rich protein and growth arrest specific 6 are closely related to calcification, including bone health, vascular calcification and lithiasis. The current review discussed these controversies and provided suggestions for future studies on VKDPs, i.e. taking into account dietary habits, geographical environments and genetic backgrounds.

Integrated omics analysis of coronary artery calcifications and myocardial infarction: the Framingham Heart Study.

Gene function can be described using various measures. We integrated association studies of three types of omics data to provide insights into the pathophysiology of subclinical coronary disease and myocardial infarction (MI). Using multivariable regression models, we associated: (1) single nucleotide polymorphism, (2) DNA methylation, and (3) gene expression with coronary artery calcification (CAC) scores and MI. Among 3106 participants of the Framingham Heart Study, 65 (2.1%) had prevalent MI and 60 (1.9%) had incident MI, median CAC value was 67.8 [IQR 10.8, 274.9], and 1403 (45.2%) had CAC scores > 0 (prevalent CAC). Prevalent CAC was associated with AHRR (linked to smoking) and EXOC3 (affecting platelet function and promoting hemostasis). CAC score was associated with VWA1 (extracellular matrix protein associated with cartilage structure in endomysium). For prevalent MI we identified FYTTD1 (down-regulated in familial hypercholesterolemia) and PINK1 (linked to cardiac tissue homeostasis and ischemia-reperfusion injury). Incident MI was associated with IRX3 (enhancing browning of white adipose tissue) and STXBP3 (controlling trafficking of glucose transporter type 4 to plasma). Using an integrative trans-omics approach, we identified both putatively novel and known candidate genes associated with CAC and MI. Replication of findings is warranted.

CircHIPK3 relieves vascular calcification via mediating SIRT1/PGC-1α/MFN2 pathway by interacting with FUS.

BACKGROUND: Circular RNAs (circRNAs) have been reported to regulate the biological processes of human diseases. CircHIPK3 has been implicated in vascular calcification, but the downstream regulatory mechanisms remain unclear. Our study aimed to understand the regulatory function of circHIPK3 in vascular calcification. METHODS: CircHIPK3 expression in atherosclerosis (AS) serum samples and vascular smooth muscle cells (VSMCs) calcification model was assessed by quantitative real-time polymerase chain reaction (qRT-PCR). The binding relationships between fused in sarcoma (FUS) and circHIPK3 or sirtuin 1 (SIRT1) were verified by RNA immunoprecipitation (RIP) assay and RNA pull-down assays. Alkaline phosphatase (ALP) activity and alizarin red staining assays were performed to evaluate the biological effect of ß-glycerophosphate (ß-GP) and circHIPK3 on calcium deposition. qRT-PCR and western blot assays were used to examine the effect of ß-GP, circHIPK3, SIRT1, mitofusin 2 (MFN2), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) on VSMCs calcification and the expression of calcification-related proteins. RESULTS: In AS serum samples and VSMCs calcification model, the expression of circHIPK3 was significantly reduced. CircHIPK3 overexpression inhibited ALP activity and calcium deposition in ß-GP-induced VSMCs. Moreover, circHIPK3 could recruit FUS to further stabilize SIRT1 mRNA. CircHIPK3 promoted MFN2 expression to alleviate VSMCs calcification via activating SIRT1/PGC-1α signaling. CONCLUSION: The positive regulation of circHIPK3/FUS/SIRT1/PGC-1α/MFN2 signaling pathway contributed to the alleviate VSMCs calcification, revealing a novel regulatory axis for vascular calcification.

Cardioprotective function of sclerostin by reducing calcium deposition, proliferation, and apoptosis in human vascular smooth muscle cells.

BACKGROUND: Sclerostin is an inhibitor of the Wnt/b-catenin pathway, which regulates bone formation, and can be expressed in vascular smooth muscle cells (VSMCs). Type 2 diabetes (T2D) is associated with an increased risk of cardiovascular disease (CVD) and increased serum and tissue expression of sclerostin. However, whether the role of sclerostin is detrimental or protective in the development of CVD is unknown. Therefore, our aims are to determine the level of sclerostin in T2D patients with/without CVD and in controls, both at serum and vascular tissue, and to analyze the role of sclerostin in VSMCs under calcified environments. METHODS: Cross-sectional study including 121 controls and 139 T2D patients with/without CVD (48/91). Sclerostin levels in serum were determined by ELISA, and sclerostin expression was analyzed by RT-qPCR and immunohistochemistry in calcified and non-calcified artery of lower limb from T2D patients (n = 7) and controls (n = 3). In vitro experiments were performed in VSMCs (mock and sclerostin overexpression) under calcifying conditions analyzing the sclerostin function by determination of calcium and phosphate concentrations, and quantification of calcium deposits by Alizarin Red. Proliferation and apoptosis were analyzed by MTT assay and flow cytometry, respectively. The regulation of the expression of genes involved in bone metabolism was determined by RT-qPCR. RESULTS: A significant increase in serum sclerostin levels in T2D patients with CVD compared to T2D patients without CVD and controls (p < 0.001) was observed. Moreover, higher circulating sclerostin levels were independently associated with CVD in T2D patients. Increased sclerostin expression was observed in calcified arteries of T2D patients compared to non-calcified arteries of controls (p = 0.003). In vitro experiments using VSMCs under calcified conditions, revealed that sclerostin overexpression reduced intracellular calcium (p = 0.001), calcium deposits (p < 0.001), cell proliferation (p < 0.001) and promoted cell survival (p = 0.015). Furthermore, sclerostin overexpression exhibited up-regulation of ALPL (p = 0.009), RUNX2 (p = 0.001) and COX2 (p = 0.003) and down-regulation of inflammatory genes, such as, IL1ß (p = 0.005), IL6 (p = 0.001) and IL8 (p = 0.003). CONCLUSIONS: Sclerostin could play a protective role in the development of atherosclerosis in T2D patients by reducing calcium deposits, decreasing proliferation and inflammation, and promoting cell survival in VSMCs under calcifying conditions. Therefore, considering the bone-vascular axis, treatment with anti-sclerostin for bone disease should be used with caution.

MicroRNA regulators of vascular pathophysiology in chronic kidney disease.

Coronary artery disease (CAD) is a severe comorbidity in chronic kidney disease (CKD) due to heavy calcification in the medial layer and inflamed plaques. Chronic inflammation, endothelial dysfunction and vascular calcification are major contributors that lead to artherosclerosis in CKD. The lack of specific symptoms and signs of CAD and decreased accuracy of noninvasive diagnostic tools result in delayed diagnosis leading to increased mortality. MicroRNAs (miRNAs) are post-transcriptional regulators present in various biofluids throughout the body. In the circulation, miRNAs have been reported to be encapsulated in extracellular vesicles and serve as stable messengers for crosstalk among cells. miRNAs are involved in pathophysiologic mechanisms including CAD and can potentially be extended from basic research to clinical translational practice.

Bone-derived PDGF-BB drives brain vascular calcification in male mice.

Brain vascular calcification is a prevalent age-related condition often accompanying neurodegenerative and neuroinflammatory diseases. The pathogenesis of large-vessel calcifications in peripheral tissue is well studied, but microvascular calcification in the brain remains poorly understood. Here, we report that elevated platelet-derived growth factor BB (PDGF-BB) from bone preosteoclasts contributed to cerebrovascular calcification in male mice. Aged male mice had higher serum PDGF-BB levels and a higher incidence of brain calcification compared with young mice, mainly in the thalamus. Transgenic mice with preosteoclast-specific Pdgfb overexpression exhibited elevated serum PDGF-BB levels and recapitulated age-associated thalamic calcification. Conversely, mice with preosteoclast-specific Pdgfb deletion displayed diminished age-associated thalamic calcification. In an ex vivo cerebral microvascular culture system, PDGF-BB dose-dependently promoted vascular calcification. Analysis of osteogenic gene array and single-cell RNA-Seq (scRNA-Seq) revealed that PDGF-BB upregulated multiple osteogenic differentiation genes and the phosphate transporter Slc20a1 in cerebral microvessels. Mechanistically, PDGF-BB stimulated the phosphorylation of its receptor PDGFRß (p-PDGFRß) and ERK (p-ERK), leading to the activation of RUNX2. This activation, in turn, induced the transcription of osteoblast differentiation genes in PCs and upregulated Slc20a1 in astrocytes. Thus, bone-derived PDGF-BB induced brain vascular calcification by activating the p-PDGFRß/p-ERK/RUNX2 signaling cascade in cerebrovascular cells.

Exploring a new mechanism between lactate and VSMC calcification: PARP1/POLG/UCP2 signaling pathway and imbalance of mitochondrial homeostasis.

Lactate leads to the imbalance of mitochondria homeostasis, which then promotes vascular calcification. PARP1 can upregulate osteogenic genes and accelerate vascular calcification. However, the relationship among lactate, PARP1, and mitochondrial homeostasis is unclear. The present study aimed to explore the new molecular mechanism of lactate to promote VSMC calcification by evaluating PARP1 as a breakthrough molecule. A coculture model of VECs and VSMCs was established, and the model revealed that the glycolysis ability and lactate production of VECs were significantly enhanced after incubation in DOM. Osteogenic marker expression, calcium deposition, and apoptosis in VSMCs were decreased after lactate dehydrogenase A knockdown in VECs. Mechanistically, exogenous lactate increased the overall level of PARP and PARylation in VSMCs. PARP1 knockdown inhibited Drp1-mediated mitochondrial fission and partially restored PINK1/Parkin-mediated mitophagy, thereby reducing mitochondrial oxidative stress. Moreover, lactate induced the translocation of PARP1 from the nucleus to the mitochondria, which then combined with POLG and inhibited POLG-mediated mitochondrial DNA synthesis. This process led to the downregulation of mitochondria-encoded genes, disturbance of mitochondrial respiration, and inhibition of oxidative phosphorylation. The knockdown of PARP1 could partially reverse the damage of mitochondrial gene expression and function caused by lactate. Furthermore, UCP2 was upregulated by the PARP1/POLG signal, and UCP2 knockdown inhibited Drp1-mediated mitochondrial fission and partially recovered PINK1/Parkin-mediated mitophagy. Finally, UCP2 knockdown in VSMCs alleviated DOM-caused VSMC calcification in the coculture model. The study results thus suggest that upregulated PARP1 is involved in the mechanism through which lactate accelerates VSMC calcification partly via POLG/UCP2-caused unbalanced mitochondrial homeostasis.

PFKFB3-driven vascular smooth muscle cell glycolysis promotes vascular calcification via the altered FoxO3 and lactate production.

A link between increased glycolysis and vascular calcification has recently been reported, but it remains unclear how increased glycolysis contributes to vascular calcification. We therefore investigated the role of PFKFB3, a critical enzyme of glycolysis, in vascular calcification. We found that PFKFB3 expression was upregulated in calcified mouse VSMCs and arteries. We showed that expression of miR-26a-5p and miR-26b-5p in calcified mouse arteries was significantly decreased, and a negative correlation between Pfkfb3 mRNA expression and miR-26a-5p or miR-26b-5p was seen in these samples. Overexpression of miR-26a/b-5p significantly inhibited PFKFB3 expression in VSMCs. Intriguingly, pharmacological inhibition of PFKFB3 using PFK15 or knockdown of PFKFB3 ameliorated vascular calcification in vD3 -overloaded mice in vivo or attenuated high phosphate (Pi)-induced VSMC calcification in vitro. Consistently, knockdown of PFKFB3 significantly reduced glycolysis and osteogenic transdifferentiation of VSMCs, whereas overexpression of PFKFB3 in VSMCs induced the opposite effects. RNA-seq analysis and subsequent experiments revealed that silencing of PFKFB3 inhibited FoxO3 expression in VSMCs. Silencing of FoxO3 phenocopied the effects of PFKFB3 depletion on Ocn and Opg expression but not Alpl in VSMCs. Pyruvate or lactate supplementation, the product of glycolysis, reversed the PFKFB3 depletion-mediated effects on ALP activity and OPG protein expression in VSMCs. Our results reveal that blockade of PFKFB3-mediated glycolysis inhibits vascular calcification in vitro and in vivo. Mechanistically, we show that FoxO3 and lactate production are involved in PFKFB3-driven osteogenic transdifferentiation of VSMCs. PFKFB3 may be a promising therapeutic target for the treatment of vascular calcification.

In silico analysis and verification of critical genes related to vascular calcification in multiple diseases.

Identifying a functional molecular therapeutic target of vascular calcification (VC) that will not affect normal osteogenic differentiation is a challenge. To address this aim, we screened the differentially expressed genes (DEGs) in different VC conditions, including endothelial-osteogenic transition (EOT) (GSE167962), chronic kidney disease (CKD), and atherosclerosis (AS) (GSE159832). KEGG pathways, protein-protein interactions, and hub genes were also analyzed. The intersecting DEGs among the EOT, CKD, and AS groups were verified by quantitative reverse transcription polymerase chain reaction and immunohistochemistry in a DOCA-salt hypertension mouse model. The phosphoinositide 3-kinase-protein kinase B signaling pathway, ECM-receptor interaction, chemokine signaling pathway, and focal adhesion were enriched in EOT and AS-induced VC. ECM-receptor interaction, PPAR signaling pathway, apelin signaling pathway, AMPK signaling pathway, adipocytokine signaling pathway, and cholesterol metabolism were enriched in CKD and AS-induced VC. C4b, Cebpa, Lyz2, and Spp1 were also upregulated in EOT, CKD, AS, and hypertension. This study identified promising molecular targets for VC therapy.

The role of miR-433-3p in vascular calcification in type 2 diabetic patients: targeting WNT/ß-Catenin and RANKL/RANK/OPG signaling pathways.

BACKGROUND: Vascular calcification (VC) is a major predictor of cardiovascular diseases that represent the principal cause of mortality among type-2 diabetic patients. Accumulating data suggest the vital role of some microRNAs on vascular calcification as an epigenetic regulator. Thus, we assessed herein, the role of serum miR-433-3p in vascular calcification in type-2 diabetic patients. METHODS: Twenty healthy subjects (control group) and forty diabetic patients (20 without VC and 20 with VC) were involved in the study. miR-433-3p gene expression was measured. Runx2, Dickkopf-1 (DKK1), ß-catenin, Receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG) levels in serum were assessed by ELISA technique. RESULTS: Diabetes patients had significantly lower levels of miR-433-3p expression in comparison to the control group, with the lowest levels being found in diabetic patients with VC. Furthermore, Runx2, ß-catenin, and RANKL levels were significantly increased with concomitant lower DKK1 and OPG levels detected in the two diabetic groups especially those with VC. CONCLUSION: Collectively, the study documented that down-regulation of miR-433-3p may contribute to the development of VC through activating WNT/ß-Catenin and RANKL/RANK/OPG signaling pathways.

SIRT3-and FAK-mediated acetylation-phosphorylation crosstalk of NFATc1 regulates Nε-carboxymethyl-lysine-induced vascular calcification in diabetes mellitus.

BACKGROUND AND AIMS: Arterial calcification is the predictor of cardiovascular risk in diabetic patients. Nε-carboxymethyl-lysine (CML), a toxic metabolite, is associated with accelerated vascular calcification in diabetes mellitus (DM). However, the mechanism remains elusive. This study aims to explore the key regulators involved in CML-induced vascular calcification in DM. METHODS: We used Western blot and immuno-staining to test the expression and localization of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) in human samples, a diabetic apolipoprotein E-deficient (ApoE-/-) mouse model, and a vascular smooth muscle cells (VSMC) model. Further, we confirmed the regulator of NFATc1 phosphorylation and acetylation induced by CML. The role of NFATc1 in VSMCs calcification and osteogenic differentiation was explored in vivo and in vitro. RESULTS: In diabetic patients, CML and NFATc1 levels increased in the severe calcified anterior tibial arteries. CML significantly promoted NFATc1 expression and nuclear translocation in VSMCs and mouse aorta. Knockdown of NFATc1 significantly inhibited CML-induced calcification. CML promoted NFATc1 acetylation at K549 by downregulating sirtuin 3 (SIRT3), which antagonized the focal adhesion kinase (FAK) induced NFATc1 phosphorylation at the Y270 site. FAK and SIRT3 affected the nuclear translocation of NFATc1 by regulating the acetylation-phosphorylation crosstalk. NFATc1 dephosphorylation mutant Y270F and deacetylation mutant K549R had opposite effects on VSMC calcification. SIRT3 overexpression and FAK inhibitor could reverse CML-promoted VSMC calcification. CONCLUSIONS: CML enhances vascular calcification in DM through NFATc1. In this process, CML increases NFATc1 acetylation by downregulating SIRT3 to antagonize FAK-induced NFATc1 phosphorylation.

Phosphorus May Induce Phenotypic Transdifferentiation of Vascular Smooth Muscle Cells through the Reduction of microRNA-145.

Phosphorus is a vital element for life found in most foods as a natural component, but it is also one of the most used preservatives added during food processing. High serum phosphorus contributes to develop vascular calcification in chronic kidney disease; however, it is not clear its effect in a population without kidney damage. The objective of this in vivo and in vitro study was to investigate the effect of high phosphorus exposure on the aortic and serum levels of miR-145 and its effect on vascular smooth muscle cell (VSMCs) changes towards less contractile phenotypes. The study was performed in aortas and serum from rats fed standard and high-phosphorus diets, and in VSMCs exposed to different concentrations of phosphorus. In addition, miR-145 silencing and overexpression experiments were carried out. In vivo results showed that in rats with normal renal function fed a high P diet, a significant increase in serum phosphorus was observed which was associated to a significant decrease in the aortic α-actin expression which paralleled the decrease in aortic and serum miR-145 levels, with no changes in the osteogenic markers. In vitro results using VSMCs corroborated the in vivo findings. High phosphorus first reduced miR-145, and afterwards α-actin expression. The miR-145 overexpression significantly increased α-actin expression and partially prevented the increase in calcium content. These results suggest that miR-145 could be an early biomarker of vascular calcification, which could give information about the initiation of the transdifferentiation process in VSMCs.