Unveiling Selected Influences on Chronic Kidney Disease Development and Progression.

Currently, more and more people are suffering from chronic kidney disease (CKD). It is estimated that CKD affects over 10% of the population worldwide. This is a significant issue, as the kidneys largely contribute to maintaining homeostasis by, among other things, regulating blood pressure, the pH of blood, and the water-electrolyte balance and by eliminating unnecessary metabolic waste products from blood. What is more, this disease does not show any specific symptoms at the beginning. The development of CKD is predisposed by certain conditions, such as diabetes mellitus or hypertension. However, these disorders are not the only factors promoting the onset and progression of CKD. The primary purpose of this review is to examine renin-angiotensin-aldosterone system (RAAS) activity, transforming growth factor-ß1 (TGF-ß1), vascular calcification (VC), uremic toxins, and hypertension in the context of their impact on the occurrence and the course of CKD. We firmly believe that a deeper comprehension of the cellular and molecular mechanisms underlying CKD can lead to an enhanced understanding of the disease. In the future, this may result in the development of medications targeting specific mechanisms involved in the decline of kidney function. Our paper unveils the selected processes responsible for the deterioration of renal filtration abilities.

Inhibition of Slc39a14/Slc39a8 reduce vascular calcification via alleviating iron overload induced ferroptosis in vascular smooth muscle cells.

BACKGROUND: Vascular calcification (VC) is an independent risk factor for cardiovascular diseases. Recently, ferroptosis has been recognised as a novel therapeutic target for cardiovascular diseases. Although an association between ferroptosis and vascular calcification has been reported, the role and mechanism of iron overload in vascular calcification are still poorly understood. Specifically, further in-depth research is required on whether metalloproteins SLC39a14 and SLC39a8 are involved in ferroptosis induced by iron overload. METHODS: R language was employed for the differential analysis of the dataset, revealing the correlation between ferroptosis and calcification. The experimental approaches encompassed both in vitro and in vivo studies, incorporating the use of iron chelators and models of iron overload. Additionally, gain- and loss-of-function experiments were conducted to investigate iron's effects on vascular calcification comprehensively. Electron microscopy, immunofluorescence, western blotting, and real-time polymerase chain reaction were used to elucidate how Slc39a14 and Slc39a8 mediate iron overload and promote calcification. RESULTS: Ferroptosis was observed in conjunction with vascular calcification (VC); the association was consistently confirmed by in vitro and in vivo studies. Our results showed a positive correlation between iron overload in VSMCs and calcification. Iron chelators are effective in reversing VC and iron overload exacerbates this process. The expression levels of the metal transport proteins Slc39a14 and Slc39a8 were significantly upregulated during calcification; the inhibition of their expression alleviated VC. Conversely, Slc39a14 overexpression exacerbates calcification and promotes intracellular iron accumulation in VSMCs. CONCLUSIONS: Our research demonstrates that iron overload occurs during VC, and that inhibition of Slc39a14 and Slc39a8 significantly relieves VC by intercepting iron overload-induced ferroptosis in VSMCs, providing new insights into the VC treatment.

Overexpression of miR-204-5p Alleviates Osteogenic Differentiation and Calcification of Human Aortic Vascular Smooth Muscle Cells by Targeting Calcium/Calmodulin-dependent Protein Kinase 1.

ABSTRACT: Vascular calcification (VC), a major complication in chronic kidney disease (CKD), is predominantly driven by osteoblastic differentiation. Recent studies have highlighted the crucial role of microRNAs in CKD's pathogenesis. Here, our research focused on the effects of miR-204-5p and its molecular mechanisms within VC. We initially found a notable decrease in miR-204-5p levels in human aortic vascular smooth muscle cells stimulated with inorganic phosphate, using this as a VC model in vitro. Following the overexpression of miR-204-5p, a decrease in VC was observed, as indicated by alizarin red S staining and measurements of calcium content. This decrease was accompanied by lower levels of the osteogenic marker, runt-related transcription factor 2, and higher levels of α-smooth muscle actin, a marker of contractility. Further investigation showed that calcium/calmodulin-dependent protein kinase 1 (CAMK1), which is a predicted target of miR-204-5p, promotes VC. Conversely, overexpressing miR-204-5p reduced VC by suppressing CAMK1 activity. Overexpressing miR-204-5p also effectively mitigated aortic calcification in an in vivo rat model. In summary, our research indicated that targeting the miR-204-5p/CAMK1 pathway could be a viable strategy for mitigating VC in CKD patients.

Prevotella copri promotes vascular calcification via lipopolysaccharide through activation of NF-κB signaling pathway.

Emerging evidence indicates that alteration of gut microbiota plays an important role in chronic kidney disease (CKD)-related vascular calcification (VC). We aimed to investigate the specific gut microbiota and the underlying mechanism involved in CKD-VC. We identified an increased abundance of Prevotella copri (P. copri) in the feces of CKD rats (induced by using 5/6 nephrectomy followed by a high calcium and phosphate diet) with aortic calcification via amplicon sequencing of 16S rRNA genes. In patients with CKD, we further confirmed a positive correlation between abundance of P. copri and aortic calcification scores. Moreover, oral administration of live P. copri aggravated CKD-related VC and osteogenic differentiation of vascular smooth muscle cells in vivo, accompanied by intestinal destruction, enhanced expression of Toll-like receptor-4 (TLR4), and elevated lipopolysaccharide (LPS) levels. In vitro and ex vivo experiments consistently demonstrated that P. copri-derived LPS (Pc-LPS) accelerated high phosphate-induced VC and VSMC osteogenic differentiation. Mechanistically, Pc-LPS bound to TLR4, then activated the nuclear factor κB (NF-κB) and nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome signals during VC. Inhibition of NF-κB reduced NLRP3 inflammasome and attenuated Pc-LPS-induced VSMC calcification. Our study clarifies a novel role of P. copri in CKD-related VC, by the mechanisms involving increased inflammation-regulating metabolites including Pc-LPS, and activation of the NF-κB/NLRP3 signaling pathway. These findings highlight P. copri and its-derived LPS as potential therapeutic targets for VC in CKD.

Relationship between arginine methylation and vascular calcification.

In patients on maintenance hemodialysis (MHD), vascular calcification (VC) is an independent predictor of cardiovascular disease (CVD), which is the primary cause of death in chronic kidney disease (CKD). The main component of VC in CKD is the vascular smooth muscle cells (VSMCs). VC is an ordered, dynamic activity. Under the stresses of oxidative stress and calcium-­phosphorus imbalance, VSMCs undergo osteogenic phenotypic transdifferentiation, which promotes the formation of VC. In addition to traditional epigenetics like RNA and DNA control, post-translational modifications have been discovered to be involved in the regulation of VC in recent years. It has been reported that the process of osteoblast differentiation is impacted by catalytic histone or non-histone arginine methylation. Its function in the osteogenic process is comparable to that of VC. Thus, we propose that arginine methylation regulates VC via many signaling pathways, including as NF-B, WNT, AKT/PI3K, TGF-/BMP/SMAD, and IL-6/STAT3. It might also regulate the VC-related calcification regulatory factors, oxidative stress, and endoplasmic reticulum stress. Consequently, we propose that arginine methylation regulates the calcification of the arteries and outline the regulatory mechanisms involved.

12,13-diHOME attenuates high glucose-induced calcification of vascular smooth muscle cells through repressing CPT1A-mediated HMGB1 succinylation.

Diabetes is closely associated with vascular calcification (VC). Exorbitant glucose concentration activates pro-calcific effects in vascular smooth muscle cells (VSMCs). This study enrolled 159 elderly patients with type 2 diabetes and divided them into three groups, T1, T2 and T3, according to brachial-ankle pulse wave velocity(BaPWV). There were statistically significant differences in the waist circumference, waist hip ratio, systolic blood pressure, 12,13-diHOME (a lipokin) concentration among T1, T2 and T3. 12,13-diHOME levels were positively correlated to high density lipoprotein cholesterol and total cholesterol, but negatively correlated to with waist circumference, waist hip ratio, systolic blood pressure and baPWV. Studies in vitro showed that 12,13-diHOME effectively inhibits calcification in VSMCs under high glucose conditions. Notably, 12,13-diHOME suppressed the up-regulation of carnitine O-palmitoyltransferase 1 (CPT1A) and CPT1A-induced succinylation of HMGB1. The succinylation of HMGB1 at the K90 promoted the protein stability and induced the enrichment of HMGB1 in cytoplasm, which induced the calcification in VSMCs. Together, 12,13-diHOME attenuates high glucose-induced calcification in VSMCs through repressing CPT1A-mediated HMGB1 succinylation.

Associations between pan-immune-inflammation value and abdominal aortic calcification: a cross-sectional study.

Background: Abdominal aortic calcification (AAC) pathogenesis is intricately linked with inflammation. The pan-immune-inflammation value (PIV) emerges as a potential biomarker, offering reflection into systemic inflammatory states and assisting in the prognosis of diverse diseases. This research aimed to explore the association between PIV and AAC. Methods: Employing data from the National Health and Nutrition Examination Survey (NHANES), this cross-sectional analysis harnessed weighted multivariable regression models to ascertain the relationship between PIV and AAC. Trend tests probed the evolving relationship among PIV quartiles and AAC. The study also incorporated subgroup analysis and interaction tests to determine associations within specific subpopulations. Additionally, the least absolute shrinkage and selection operator (LASSO) regression and multivariable logistic regression were used for characteristics selection to construct prediction model. Nomograms were used for visualization. The receiver operator characteristic (ROC) curve, calibration plot and decision curve analysis were applied for evaluate the predictive performance. Results: From the cohort of 3,047 participants, a distinct positive correlation was observed between PIV and AAC. Subsequent to full adjustments, a 100-unit increment in PIV linked to an elevation of 0.055 points in the AAC score (ß=0.055, 95% CI: 0.014-0.095). Categorizing PIV into quartiles revealed an ascending trend: as PIV quartiles increased, AAC scores surged (ß values in Quartile 2, Quartile 3, and Quartile 4: 0.122, 0.437, and 0.658 respectively; P for trend <0.001). Concurrently, a marked rise in SAAC prevalence was noted (OR values for Quartile 2, Quartile 3, and Quartile 4: 1.635, 1.842, and 2.572 respectively; P for trend <0.01). Individuals aged 60 or above and those with a history of diabetes exhibited a heightened association. After characteristic selection, models for predicting AAC and SAAC were constructed respectively. The AUC of AAC model was 0.74 (95%CI=0.71-0.77) and the AUC of SAAC model was 0.84 (95%CI=0.80-0.87). According to the results of calibration plots and DCA, two models showed high accuracy and clinical benefit. Conclusion: The research findings illuminate the potential correlation between elevated PIV and AAC presence. Our models indicate the potential utility of PIV combined with other simple predictors in the assessment and management of individuals with AAC.

l-thyroxine attenuates extracellular Hsp90α-induced vascular endothelial calcification in diabetes mellitus, as revealed by parallel metabolic profiles.

BACKGROUND AND AIMS: Diabetic atherosclerotic vascular disease is characterized by extensive vascular calcification. However, an elevated blood glucose level alone does not explain this pathogenesis. We investigated the metabolic markers underlying diabetic atherosclerosis and whether extracellular Hsp90α (eHsp90α) triggers vascular endothelial calcification in this particular metabolic environment. METHODS: A parallel human/animal model metabolomics approach was used. We analyzed 40 serum samples collected from 24 patients with atherosclerosis and from the STZ-induced ApoE-/- mouse model. A multivariate statistical analysis of the data was performed, and mouse aortic tissue was collected for the assessment of plaque formation. In vitro, the effects of eHsp90α on endothelial cell calcification were assessed by serum analysis, Western blotting and immunoelectron microscopy. RESULTS: Diabetic ApoE-/- mice showed more severe plaque lesions and calcification damage. Stearamide, oleamide, l-thyroxine, l-homocitrulline and l-citrulline are biomarkers of diabetic ASVD; l-thyroxine was downregulated in both groups, and the thyroid sensitivity index was correlated with serum Hsp90α concentration. In vitro studies showed that eHsp90α increased Runx2 expression in endothelial cells through the LRP1 receptor. l-thyroxine reduced the increase in Runx2 levels caused by eHsp90α and affected the distribution and expression of LRP1 through hydrogen bonding with glutamine at position 1054 in the extracellular segment of LRP1. CONCLUSIONS: This study provides a mechanistic link between characteristic serum metabolites and diabetic atherosclerosis and thus offers new insight into the role of extracellular Hsp90α in promoting vascular calcification.

Orphan Nuclear Receptor NR4A3 Promotes Vascular Calcification via Histone Lactylation.

BACKGROUND: Medial arterial calcification is a chronic systemic vascular disorder distinct from atherosclerosis and is commonly observed in patients with chronic kidney disease, diabetes, and aging individuals. We previously showed that NR4A3 (nuclear receptor subfamily 4 group A member 3), an orphan nuclear receptor, is a key regulator in apo (apolipoprotein) A-IV-induced atherosclerosis progression; however, its role in vascular calcification is poorly understood. METHODS: We generated NR4A3-/- mice and 2 different types of medial arterial calcification models to investigate the biological roles of NR4A3 in vascular calcification. RNA-seq was performed to determine the transcriptional profile of NR4A3-/- vascular smooth muscle cells under ß-glycerophosphate treatment. We integrated Cleavage Under Targets and Tagmentation analysis and RNA-seq data to further investigate the gene regulatory mechanisms of NR4A3 in arterial calcification and target genes regulated by histone lactylation. RESULTS: NR4A3 expression was upregulated in calcified aortic tissues from chronic kidney disease mice, 1,25(OH)2VitD3 overload-induced mice, and human calcified aorta. NR4A3 deficiency preserved the vascular smooth muscle cell contractile phenotype, inhibited osteoblast differentiation-related gene expression, and reduced calcium deposition in the vasculature. Further, NR4A3 deficiency lowered the glycolytic rate and lactate production during the calcification process and decreased histone lactylation. Mechanistic studies further showed that NR4A3 enhanced glycolysis activity by directly binding to the promoter regions of the 2 glycolysis genes ALDOA and PFKL and driving their transcriptional initiation. Furthermore, histone lactylation promoted medial calcification both in vivo and in vitro. NR4A3 deficiency inhibited the transcription activation and expression of Phospho1 (phosphatase orphan 1). Consistently, pharmacological inhibition of Phospho1 attenuated calcium deposition in NR4A3-overexpressed vascular smooth muscle cells, whereas overexpression of Phospho1 reversed the anticalcific effect of NR4A3 deficiency in vascular smooth muscle cells. CONCLUSIONS: Taken together, our findings reveal that NR4A3-mediated histone lactylation is a novel metabolome-epigenome signaling cascade mechanism that participates in the pathogenesis of medial arterial calcification.

New insight into air pollution-related cardiovascular disease: an adverse outcome pathway framework of PM2.5-associated vascular calcification.

Despite the air quality has been generally improved in recent years, ambient fine particulate matter (PM2.5), a major contributor to air pollution, remains one of the major threats to public health. Vascular calcification is a systematic pathology associated with an increased risk of cardiovascular disease. Although the epidemiological evidence has uncovered the association between PM2.5 exposure and vascular calcification, little is known about the underlying mechanisms. The adverse outcome pathway (AOP) concept offers a comprehensive interpretation of all of the findings obtained by toxicological and epidemiological studies. In this review, reactive oxygen species generation was identified as the molecular initiating event (MIE), which targeted subsequent key events (KEs) such as oxidative stress, inflammation, endoplasmic reticulum stress, and autophagy, from the cellular to the tissue/organ level. These KEs eventually led to the adverse outcome, namely increased incidence of vascular calcification and atherosclerosis morbidity. To the best of our knowledge, this is the first AOP framework devoted to PM2.5-associated vascular calcification, which benefits future investigations by identifying current limitations and latent biomarkers.

Assessment for Carotid Atherosclerotic Plaque Using Vessel Wall Magnetic Resonance Imaging: A Multireader ROC Study to Determine Optimal Sequence for Detecting Vessel Wall Calcification.

INTRODUCTION: We aimed to compare conventional vessel wall MR imaging techniques and quantitative susceptibility mapping (QSM) to determine the optimal sequence for detecting carotid artery calcification. METHODS: Twenty-two patients who underwent carotid vessel wall MR imaging and neck CT were enrolled. Four slices of 6-mm sections from the bilateral internal carotid bifurcation were subdivided into 4 segments according to clock position (0-3, 3-6, 6-9, and 9-12) and assessed for calcification. Two blinded radiologists independently reviewed a total of 704 segments and scored the likelihood of calcification using a 5-point scale on spin-echo imaging, FLASH, and QSM. The observer performance for detecting calcification was evaluated by a multireader, multiple-case receiver operating characteristic study. Weighted κ statistics were calculated to assess interobserver agreement. RESULTS: QSM had a mean area under the receiver operating characteristic curve of 0.85, which was significantly higher than that of any other sequence (p < 0.01) and showed substantial interreader agreement (κ = 0.68). A segment with a score of 3-5 was defined as positive, and a segment with a score of 1-2 was defined as negative; the sensitivity and specificity of QSM were 0.75 and 0.87, respectively. CONCLUSION: QSM was the most reliable MR sequence for the detection of plaque calcification.

Association between lower extremity arterial calcification and coronary arterial calcification in a population at increased risk of cardiovascular disease.

INTRODUCTION: There is conflicting evidence whether lower extremity arterial calcification coincides with coronary arterial calcification (CAC). The aims of this study were to investigate the associations between (1) femoral and crural calcification with CAC, and (2) femoral and crural calcification pattern with CAC. RESEARCH DESIGN AND METHODS: This cross-sectional study included 405 individuals (74% men, 62.6±10.9 years) from the ARTEMIS cohort study at high risk of cardiovascular disease (CVD) who underwent a CT scan of the femoral, crural and coronary arteries. High CVD risk was defined as history/presence of cerebrovascular disease, coronary artery disease, abdominal aortic aneurysm, renal artery stenosis, peripheral artery disease or CVD risk factors: diabetes mellitus type 2, hypertension, hyperlipidemia. Calcification score within each arterial bed was expressed in Agatston units. Dominant calcification patterns (intimal, medial, absent/indistinguishable) were determined via a CT-guided histologically validated scoring algorithm. Multivariable-adjusted multinomial logistic regression analyses were used. Replication was performed in an independent population of individuals with diabetes mellitus type 2 (Early-HFpEF cohort study). RESULTS: Every 100-point increase in femoral and crural calcification score was associated with 1.23 (95% CI=1.09 to 1.37, p<0.001) and 1.28 (95% CI=1.11 to 1.47, p=0.001) times higher odds of having CAC within tertile 3 (high) versus tertile 1 (low), respectively. The association appeared stronger for crural versus femoral arteries. Moreover, the presence of femoral intimal (OR=10.81, 95% CI=4.23 to 27.62, p<0.001), femoral medial (OR=10.37, 95% CI=3.92 to 27.38, p<0.001) and crural intimal (OR=6.70, 95% CI=2.73 to 16.43, p<0.001) calcification patterns were associated with higher odds of having CAC within tertile 3 versus tertile 1, independently from concomitant calcification score. This association appeared stronger for intimal versus medial calcification patterns. The replication analysis yielded similar results. CONCLUSIONS: Higher femoral and crural calcification scores were associated with higher CAC. Moreover, the presence of femoral intimal, femoral medial and crural intimal calcification patterns was associated with increased CAC. It appears that arterial calcification is a systemic process which occurs simultaneously in various arterial beds.

Carbonylation of Runx2 at K176 by 4-Hydroxynonenal Accelerates Vascular Calcification.

BACKGROUND: Vascular calcification, which is characterized by calcium deposition in arterial walls and the osteochondrogenic differentiation of vascular smooth muscle cells, is an actively regulated process that involves complex mechanisms. Vascular calcification is associated with increased cardiovascular adverse events. The role of 4-hydroxynonenal (4-HNE), which is the most abundant stable product of lipid peroxidation, in vascular calcification has been poorly investigated. METHODS: Serum was collected from patients with chronic kidney disease and controls, and the levels of 4-HNE and 8-iso-prostaglandin F2α were measured. Sections of coronary atherosclerotic plaques from donors were immunostained to analyze calcium deposition and 4-HNE. A total of 658 patients with coronary artery disease who received coronary computed tomography angiography were recruited to analyze the relationship between coronary calcification and the rs671 mutation in aldehyde dehydrogenase 2 (ALDH2). ALDH2 knockout (ALDH2-/-) mice, smooth muscle cell-specific ALDH2 knockout mice, ALDH2 transgenic mice, and their controls were used to establish vascular calcification models. Primary mouse aortic smooth muscle cells and human aortic smooth muscle cells were exposed to medium containing ß-glycerophosphate and CaCl2 to investigate cell calcification and the underlying molecular mechanisms. RESULTS: Elevated 4-HNE levels were observed in the serum of patients with chronic kidney disease and model mice and were detected in calcified artery sections by immunostaining. ALDH2 knockout or smooth muscle cell-specific ALDH2 knockout accelerated the development of vascular calcification in model mice, whereas overexpression or activation prevented mouse vascular calcification and the osteochondrogenic differentiation of vascular smooth muscle cells. In patients with coronary artery disease, patients with ALDH2 rs671 gene mutation developed more severe coronary calcification. 4-HNE promoted calcification of both mouse aortic smooth muscle cells and human aortic smooth muscle cells and their osteochondrogenic differentiation in vitro. 4-HNE increased the level of Runx2 (runt-related transcription factor-2), and the effect of 4-HNE on promoting vascular smooth muscle cell calcification was ablated when Runx2 was knocked down. Mutation of Runx2 at lysine 176 reduced its carbonylation and eliminated the 4-HNE-induced upregulation of Runx2. CONCLUSIONS: Our results suggest that 4-HNE increases Runx2 stabilization by directly carbonylating its K176 site and promotes vascular calcification. ALDH2 might be a potential target for the treatment of vascular calcification.

Paraspeckle protein NONO attenuates vascular calcification by inhibiting bone morphogenetic protein 2 transcription.

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.

Coronary Artery Calcification: Current Concepts and Clinical Implications.

Coronary artery calcification (CAC) accompanies the development of advanced atherosclerosis. Its role in atherosclerosis holds great interest because the presence and burden of coronary calcification provide direct evidence of the presence and extent of coronary artery disease; furthermore, CAC predicts future events independently of concomitant conventional cardiovascular risk factors and to a greater extent than any other noninvasive biomarker of this disease. Nevertheless, the relationship between CAC and the susceptibility of a plaque to provoke a thrombotic event remains incompletely understood. This review summarizes the current understanding and literature on CAC. It outlines the pathophysiology of CAC and reviews laboratory, histopathological, and genetic studies, as well as imaging findings, to characterize different types of calcification and to elucidate their implications. Some patterns of calcification such as microcalcification portend increased risk of rupture and cardiovascular events and may improve prognosis assessment noninvasively. However, contemporary computed tomography cannot assess early microcalcification. Limited spatial resolution and blooming artifacts may hinder estimation of degree of coronary artery stenosis. Technical advances such as photon counting detectors and combination with nuclear approaches (eg, NaF imaging) promise to improve the performance of cardiac computed tomography. These innovations may speed achieving the ultimate goal of providing noninvasively specific and clinically actionable information.

Aortic calcification accelerates cardiac dysfunction via inducing apoptosis of cardiomyocytes.

Vascular calcification (VC) is a known predictor of cardiovascular events in patients with atherosclerosis and chronic renal disease. However, the exact relationship between VC and cardiovascular mortality remains unclear. Herein, we investigated the underlying mechanisms between VC progression, arterial stiffness, and cardiac dysfunction. C57BL/6 mice were administered intraperitoneally vitamin D3 (VD3) at a dosage of 35×104 IU/day for 14 days. At day 42, VC extent, artery elasticity, carotid artery blood flow, aorta pulse propagation velocity, cardiac function, and pathological changes were evaluated. Heart apoptosis was detected using TUNEL and immunohistochemistry staining. In vitro, rat cardiomyocytes H9C2 were exposed to media from calcified rat vascular smooth muscle cells (VSMCs) cultured in calcification medium, and then H9C2 apoptosis and gene expression related to cardiac function were assessed. VD3-treated mice displayed a significant aortic calcification, increased pulse propagation velocity of aortae, and reduced cardiac function. Aortae showed increased calcification and elastolysis, with increased heart apoptosis. Hearts demonstrated higher levels of ANP, BNP, MMP2, and lower levels of bcl2/bax. Moreover, calcified rat VSMC media induced H9C2 apoptosis and upregulated genes expression linked to cardiac dysfunction. Our data provide evidence that VC accelerates cardiac dysfunction, partially by inducing cardiomyocytes apoptosis.

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.

Pravastatin promotes type 2 diabetes vascular calcification through activating intestinal Bacteroides fragilis to induce macrophage M1 polarization.

BACKGROUND: Pravastatin is an oral lipid-lowering drug, commonly used by patients with diabetes that is positively correlated with the occurrence of vascular calcification (VC), but the mechanism is unclear. METHODS: In this study, 16S rRNA sequencing and qRT-PCR wereused to detect the differential gut bacteria. Metabolomics and ELISA were used to analyze the differential metabolites. qRT-PCR and western blotting (WB) were used to detect genes expression. Flow cytometry was used to analyze macrophage phenotype. Immunohistochemistry was used to analyze aortic calcification. RESULTS: We found that gut Bacteroides fragilis (BF) increased significantly in patients who took pravastatin or type 2 diabetes (T2D) mice treated with pravastatin. In vitro experiments showed that pravastatin had little effect on BF but significantly promoted BF proliferation in vivo. Further analysis showed that ArsR was an important gene for pravastatin to regulate the activation of BF, and overexpression of ArsR significantly promoted the secretion of 3,4,5-trimethoxycinnamic acid (TMCA). Importantly, pravastatin significantly promoted BF secretion of TMCA and significantly increased TMCA secretion in T2D patients or T2D mice. TMCA had little effect on vascular smooth muscle cell calcification but significantly promoted macrophage M1 polarization, which we had demonstrated that M1 macrophages promoted T2D VC. In vivo studies found that pravastatin significantly upregulated TMCA levels in the feces and serum of T2D mice transplanted with BF and promoted the macrophage M1 polarization in bone marrow and the osteoblastic differentiation of aortic cells. Similar results were obtained in T2D mice after intravenous infusion of TMCA. CONCLUSIONS: Promoting intestinal BF to secrete TMCA, which leads to macrophage M1 polarization, is an important mechanism by which pravastatin promotes calcification, and the result will be used for the optimization of clinical medication strategies of pravastatin supplying a theoretical basis and experimental basis.

[Clinical significance of uremic toxin indoxyl sulfate and inflammation in the development of vascular calcification and cardiovascular complications in stage C3-C5D chronic kidney disease].

AIM: To clarify the role of the uremic toxin indoxyl sulfate (IS) and inflammation in the development of vascular calcification and cardiovascular complications in chronic kidney disease (CKD). MATERIALS AND METHODS: One hundred fifteen patients aged 25 to 68 years with CKD stage C3-C5D were examined. Serum concentrations of IS, interleukin 6 (IL-6), tumor necrosis factor (TNF-α), troponin I, parathyroid hormone were determined by enzyme immunoassay using kits from BluGene biotech (Shanghai, China), Cloud-Clone Corp. (USA), ELISA Kit (Biomedica, Austria). RESULTS: An increase in the serum concentration of IS, IL-6, TNF-α was revealed, which was significantly associated with a deterioration in renal function and changes in the morphological and functional parameters of the heart and aorta. CONCLUSION: High concentrations of IS, IL-6, TNF-α, which are closely associated with an increase in renal failure and cardiovascular complications, indicate their significant role in vascular calcification, which underlies the damage to the cardiovascular system in CKD.