Abstract 581: Lipid Peroxide Derived Dicarboxylic Acids Induce Atherosclerotic Calcification

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Aladdin Riad ◽  
Chandrakala A Narasimhulu ◽  
Dmitry Litvinov ◽  
Xueting Jiang ◽  
Irene F Ruiz ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Azelaic Acid ◽  
Lipid Peroxide ◽  
Muscle Cells ◽  
The United States ◽  
Arterial Calcification ◽  
Plaque Burden ◽  
Alizarin Red ◽  
Ldl Particles

Background: Cardiovascular diseases, including atherosclerosis, are the leading cause of death in the United States. Atherosclerotic lesions are formed by deposition of lipids in the intima of arteries. Upon exposure to oxidative stresses, low-density lipoprotein (LDL) is converted to highly atherogenic oxidized LDL (ox-LDL) particles, which contribute to disease development and progression. Advanced disease stages may result in calcification of lesions. This calcification process is important, as it has been shown to be associated with stable plaques that are less prone to rupture. Calcification is present in lipid rich domains of lesions, however neither the composition of the mineralized calcium deposits nor its relationship to lipid peroxidation is known. Hypothesis: Lipid peroxide derived AZA induces calcification of smooth muscle cells thereby providing the link between calcification and overall plaque burden, and association of calcification with the lipophilic region of the lesion. Methods: In this study, the potential of lipid peroxide-derived lipophilic dicarboxylic acid (DCA, e.g. azelaic acid) to promote calcification upon exposure to vascular smooth muscle cells was tested. Using 13-Hydroperoxyoctadecadienoic acid (13-hydroperoxylinoleic acid, 13-HPODE) and thin-layer and gas chromatography[[Unable to Display Character: –]]mass spectrometry we characterized the conditions where HPODE is decomposed to aldehyde product 9-oxo-nonanoic acid and its corresponding DCA azelaic acid (AZA). Results: Both free AZA and intracellular delivery of AZA via lysophosphatidylcholine (lysoPtdCho) micelles induced calcification of aortic smooth muscle cells, as determined by Von Kossa and alizarin red staining. HPODE treatment resulted in the cellular conversion to ONA and AZA as determined by GC-MS. Conclusion: These results demonstrate that DCAs could contribute to atherosclerotic calcification thus accounting for the latter’s relationship to plaque burden and association with lipids. This study also challenges the dogma that arterial calcification represents the deposition of calcium phosphate. Our future work aims to delineate the association of calcium with lipid rich plaques and lipid oxidation with calcification in animal and human atherosclerosis.

scholarly journals Globular adiponectin inhibits osteoblastic differentiation of vascular smooth muscle cells through the PI3K/AKT and Wnt/β-catenin pathway

2021 ◽  
Author(s):  
Yun Zhou ◽  
Li-Long Wei ◽  
Rui-Ping Zhang ◽  
Cheng-Wu Han ◽  
Yongtong Cao
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Osteoblastic Differentiation ◽  
Alizarin Red S ◽  
Regulation Mechanism ◽  
Alizarin Red ◽  
Globular Adiponectin

AbstractLipid metabolism is closely related to the improvement of vascular calcification (VC) in chronic kidney disease (CKD). Globular adiponectin (gAd) has been reported to be involved in the development of VC in CKD, but the detailed regulatory role remains unclear. The present study is aimed to investigate the biological function and the underlying regulation mechanism of gAd in the process of VC during CKD. Vascular smooth muscle cells (VSMCs) calcification was determined by Alizarin Red S staining. Protein signaling related with VC was tested by western blotting. The expression and intracellular localization of runt-related transcription factor 2 (Runx2) was detected by immunofluorescence and uraemic rat with VC was established by a two-step nephrectomy. Combined with the results of Alizarin Red S staining, we discovered that β-glycerophosphate (β-Gp)-induced the osteoblastic differentiation of VSMCs was significantly reversed by gAd treatment. Along with the VSMCs calcification and the increase of Runx2 in β-Gp-exposed VSMCs, the activities of protein kinase B (AKT) and Wnt/β-catenin pathway were enhanced, but that were counteracted by the exposure of gAd in rat and human VSMCs. After administration with agonists of the Wnt (SKL2001) and AKT (SC79), there appeared more osteoblastic differentiation and higher expression of Runx2 in gAd-treated VSMCs, but showing lower impact in the presence of SC79 than that in the presence of SKL2001. In the in vivo experiments, intravenous injection of gAd also significantly inhibited VC and Runx2 level in uraemic rat in a dose-dependent manner, possibly through regulating Wnt/β-catenin pathway. This study demonstrates that gAd ameliorates osteoblastic differentiation of VSMCs possibly by blocking PI3K/AKT and Wnt/β-catenin signaling transduction. The findings provide an important foundation for gAd in treating VC in kidney diseases.

scholarly journals Exosomal miR-151-3p Induces Calcium Deposition in Vascular Smooth Muscle Cells by Inhibiting Atg5

2021 ◽  
Author(s):  
Li Chen ◽  
Rongrong Zhang ◽  
Jinyin Li ◽  
Yiping Gao ◽  
Shilong Mao
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Calcium Deposition ◽  
Alizarin Red ◽  
Potential Biomarker

Abstract Background: Calcium deposition in vascular smooth muscle cells (VSMCs) can lead to the rigidity of the vasculature and an increase of risk in cardiac events. This study aimed to explore the role of exosomal microRNA-151-3p (miR-151-3p) in the regulation of VSMC calcification. Methods: A cellular calcification model was established using the mouse primary aortic VSMCs by β-glycerophosphate treatment. The calcium deposition was evaluated by Alizarin Red staining. The expression of miR-151-3p in exosomes was evaluated by qRT-PCR. The relationship between miR-151-3p and Atg5 was determined by bioinformatics analysis and dual-luciferase gene reporter assay. The exosome derived from mouse VSMCs transfected with miR-151-3p mimics/inhibitor were isolated and used to stimulate VSMCs. The expression of Atg5, α-SMA, OPN, Runx2 and BMP2 was evaluated by western blot. An animal model was established to investigate the role of miR-151-3p in exosomes.Results: MiR-151-3p was significantly upregulated in the exosomes of VSMCs treated with β-glycerophosphate. Exosomes derived from calcific VSMCs increased the calcium deposition of general VSMCs without any treatment. Exosomes derived from miR-151-3p mimics transfected VSMCs increased the expression of Runx2 and BMP2, while reduced the expression of α-SMA and OPN in general VSMCs. and exosomes derived from miR-151-3p inhibitor transfected VSMCs reversed these effects in vitro. Meanwhile, miR-151-3p served as a ceRNA of Atg5 by directly binding to the 3'UTR of Atg5. Moreover, the expression of α-SMA, OPN, Runx2 and BMP2 in vivo was consistent with the results in VSMCs in vitro.Conclusion: Our study revealed that miR-151-3p in VSMCs-derived exosomes might induce calcium deposition through regulating Atg5 expression, suggesting that miR-151-3p might be a potential biomarker for vascular calcification.

scholarly journals Molecular and Cellular Mechanisms that Induce Arterial Calcification by Indoxyl Sulfate and P-Cresyl Sulfate

Toxins ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 58 ◽  
Author(s):  
Britt Opdebeeck ◽  
Patrick C. D’Haese ◽  
Anja Verhulst
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Left Ventricular ◽  
Uremic Toxins ◽  
Indoxyl Sulfate ◽  
Arterial Calcification ◽  
Cellular Mechanisms

The protein-bound uremic toxins, indoxyl sulfate (IS) and p-cresyl sulfate (PCS), are considered to be harmful vascular toxins. Arterial media calcification, or the deposition of calcium phosphate crystals in the arteries, contributes significantly to cardiovascular complications, including left ventricular hypertrophy, hypertension, and impaired coronary perfusion in the elderly and patients with chronic kidney disease (CKD) and diabetes. Recently, we reported that both IS and PCS trigger moderate to severe calcification in the aorta and peripheral vessels of CKD rats. This review describes the molecular and cellular mechanisms by which these uremic toxins induce arterial media calcification. A complex interplay between inflammation, coagulation, and lipid metabolism pathways, influenced by epigenetic factors, is crucial in IS/PCS-induced arterial media calcification. High levels of glucose are linked to these events, suggesting that a good balance between glucose and lipid levels might be important. On the cellular level, effects on endothelial cells, which act as the primary sensors of circulating pathological triggers, might be as important as those on vascular smooth muscle cells. Endothelial dysfunction, provoked by IS and PCS triggered oxidative stress, may be considered a key event in the onset and development of arterial media calcification. In this review a number of important outstanding questions such as the role of miRNA’s, phenotypic switching of both endothelial and vascular smooth muscle cells and new types of programmed cell death in arterial media calcification related to protein-bound uremic toxins are put forward and discussed.

scholarly journals Loss of PARP-1 attenuates diabetic arteriosclerotic calcification via Stat1/Runx2 axis

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Peng Li ◽  
Ying Wang ◽  
Xue Liu ◽  
Bin Liu ◽  
Zhao-yang Wang ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Macrophage Polarization ◽  
Muscle Cells ◽  
Plaque Burden ◽  
Metabolic Complications ◽  
Runx2 Expression ◽  
Parp 1

AbstractAccelerated atherosclerotic calcification is responsible for plaque burden, especially in diabetes. The regulatory mechanism for atherosclerotic calcification in diabetes is poorly characterized. Here we show that deletion of PARP-1, a main enzyme in diverse metabolic complications, attenuates diabetic atherosclerotic calcification and decreases vessel stiffening in mice through Runx2 suppression. Specifically, PARP-1 deficiency reduces diabetic arteriosclerotic calcification by regulating Stat1-mediated synthetic phenotype switching of vascular smooth muscle cells and macrophage polarization. Meanwhile, both vascular smooth muscle cells and macrophages manifested osteogenic differentiation in osteogenic media, which was attenuated by PARP-1/Stat1 inhibition. Notably, Stat1 acts as a positive transcription factor by directly binding to the promoter of Runx2 and promoting atherosclerotic calcification in diabetes. Our results identify a new function of PARP-1, in which metabolism disturbance-related stimuli activate the Runx2 expression mediated by Stat1 transcription to facilitate diabetic arteriosclerotic calcification. PARP-1 inhibition may therefore represent a useful therapy for this challenging complication.

MO441CALPROTECTIN IS A NOVEL CONTRIBUTING FACTOR IN VASCULAR CALCIFICATION AND A PREDICTOR OF CARDIOVASCULAR OUTCOME IN CKD PATIENTS*

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Ana Amaya Garrido ◽  
José M Valdivielso ◽  
Stanislas Faguer ◽  
Arnaud Del Bello ◽  
Benedicte Buffin-Meyer ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Calcification ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Calcium Deposition ◽  
Alizarin Red ◽  
Serum Proteome ◽  
Ckd Stage

Abstract Background and Aims Vascular calcification, leading to aortic stiffening and heart failure, is decisive risk factor for cardiovascular (CV) mortality in patients with chronic kidney disease (CKD). Promoted by bone mineral disorder and systemic inflammation in CKD patients, vascular calcification is a complex mechanism involving osteochondrogenic differentiation of vascular smooth muscle cells (VSMCs) and abnormal deposition of minerals in the vascular wall. Despite intensive research efforts in recent years, available treatments have limited effect and none of them prevent or reverse vascular calcification. The aim of this study was to analyse the serum proteome of CKD stage 3-4 patients in order to unravel new molecular changes associated to CV morbid-mortality and to decipher the role of novel candidates on vascular calcification to provide potential new therapeutic agents. Method In this study we used serum samples from two independent cohorts: 112 CKD stage 3-4 patients with a 4 years follow-up for CV events and 222 CKD stage 5 patients exhibiting a broad range of calcification degree determined by histological quantification in the epigastric and/or iliac artery. Serum proteome analysis was performed using tandem mass-spectrometry in a subcohort of 66 CKD3-4 patients and validation of protein candidates was performed using ELISA in the two full cohorts. Human primary vascular smooth muscle cells and mouse aortic rings were used for calcification assays. Calcium content was quantified using QuantiChrom calcium assay kit and calcium deposition was visualized by Alizarin Red and Von Kossa staining. Results Among 443 proteins detected in the serum of CKD3-4 patients, 134 displayed significant modified abundance in patients with CV events (n=32) compared to patients without (n=34). One of the most prominent changes was increased level of calprotectin (up to 8.6 fold, P<.0001). Using ELISA, we validated that higher serum calprotectin levels were strongly associated with higher probability of developing CV complications and increased mortality in CKD stage 3-4 patients (Figure A). Moreover, we showed that higher serum calprotectin was associated with increased vascular calcification levels in CKD stage 5 patients (Figure B). In vitro, calprotectin promoted calcification of human VSMCs (p<0.0001) (Figures C-D) and in mouse aortic rings (p<0.0001) (Figure E-F). Interestingly, these effects were significantly attenuated by paquinimod, a calprotectin inhibitor (Figures C-F). Conclusion Circulating calprotectin is a novel predictor of CV outcome and mortality in CKD patients. Calprotectin also shows calcification-inducing properties and its blockade by paquinimod alleviates its effects. Future experiments will consist in deciphering the signalling pathways involved in the regulation of calcification by calprotectin and evaluating in vivo the therapeutic potential of paquinimod on the development of medial vascular calcification lesions associated with CKD.

Abstract 17038: Twist1 Modulates Smooth Muscle Cell Phenotypes in Atherosclerosis

Circulation ◽  
2018 ◽  
Vol 138 (Suppl_1) ◽  
Author(s):  
Marie A Guerraty ◽  
Sylvia T Nurnberg ◽  
Vraj Shah ◽  
Daniel J Rader
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Risk Allele ◽  
Muscle Cells ◽  
Chromosome 9 ◽  
Alizarin Red ◽  
Plaque Stability ◽  
Atherosclerotic Lesions ◽  
Mesenchymal Transformation

Introduction: Genome-wide association studies have identified rs2107595, a non-coding locus on chromosome 9 between HDAC9 and Twist1 genes, as a risk allele for several vascular phenotypes, including Coronary Artery Disease (CAD). Rs2107595 has, more specifically, been associated with stable CAD over myocardial infarction phenotypes. Recent work has shown that rs2107595 risk allele increases Twist1 expression in smooth muscle cells (SMCs) by creating an RBPJ binding site. In other cell types, Twist1 is known to maintain cells in a de-differentiated state and to promote epithelial to mesenchymal transformation, driving tumor progression and metastasis. Hypothesis: Twist1 modulates SMC differentiation to promote an immature proliferative state over a differentiated (osteoblastic) state. This shift in phenotype promotes features of plaque stability in vivo . Methods: Twist1 expression plasmid (pCMV6-TWIST1) was transfected into A7r5 rat smooth muscle cells. To assess proliferation, cells were counted at 24, 48, 72, and 96 hours. To assess calcification, A7r5 cells were cultured in calcification media (2mM NaPhos) for 10 days and stained with Alizarin Red. In vivo studies were performed in Twist1 fl/fl tamoxifen-inducible MYH11-Cre C57BL/6 mice on ApoE-/- background fed a Western diet for 16 weeks to induce atherosclerotic lesions. Immunohistochemistry with SM22a identified lesion SMCs, and alizarin red was used to identify calcifications. Results: Ectopic overexpression of Twist1 in A7r5 SMCs decreased proliferation at 48h and 72h (80%, p=0.014). Twist 1 overexpression also decreased the total area of calcification (33% reduction, p=0.007). In vivo , both control and Twist 1 KO mice show similar burden of atherosclerosis. However, there is a decrease in sub-endothelial SMCs in atherosclerotic lesions by SM22a staining in the Twist1 KO. Additionally, Twist1 KO mice have more prominent and larger focal calcifications. Conclusions: Twist1 promotes SMC proliferation and decreases calcification in vitro , and may affect the presence of subendothelial SMCs and calcification in vivo . This provides a compelling link that rs2107595 may promote plaque stability in CAD by increasing Twist1 to modulate SMC phenotypes.

Abstract 15560: Induced Pluripotent Stem Cell (iPSC) Derived Smooth Muscle Cells as a Model to Study Vascular Calcification

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Samantha K Atkins ◽  
Romi Brouwhuis ◽  
Sasha A Singh ◽  
Masanori Aikawa ◽  
Elena Aikawa
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Calcification ◽  
Time Course ◽  
Muscle Cells ◽  
Alizarin Red S ◽  
Alizarin Red ◽  
Pearson’S Correlation ◽  
Marker Proteins ◽  
Pearson's Correlation

Introduction: Vascular calcification is the top cause of mortality worldwide. Vascular smooth muscle cells (SMCs) are major contributors to vascular calcification. There is an urgent need for a large scale and inexhaustible source of functional SMCs to study mechanisms of cardiovascular calcification. Here, we describe a new platform utilizing iPSCs differentiated towards SMCs that can calcify, to model ectopic calcification and identify mechanisms of SMCs’ contribution in this pathological process. Methods: We differentiated human iPSCs towards an induced-SMC (iSMC) phenotype in a 10-day protocol. We verified successful iSMC differentiation through immunofluorescent staining for SMC marker proteins αSMA and MYH11, FACS using CD140b, and proteomics analysis on whole cell lysate at days 0, 3, 5, 7, and 10. A set of 19 canonical SMC proteins were used to generate a Pearson’s correlation over the differentiation time course. After differentiation, we cultured the iSMCs in osteogenic media (OM) for 14 days. Alizarin Red S staining performed on day 14 confirmed the presence of calcification. Three independent experiments were performed for all data analyses. Results: Typical SMC morphology with positive staining for αSMA and MYH11 was detected at day 10 by immunofluorescence (Fig 1A). FACS analysis on markers for iPSCs (TRA-1-60), ECs (CD31), and SMCs (CD140b) demonstrated that 93.9% of cells were positive for CD140b after 10 days of differentiation (Fig 1B, C). Time course proteomics showed a strong Pearson’s correlation with of iSMCs with SMC marker proteins (R = 0.89) by day 10 (Figure 1D). Calcification in osteogenic media was observed by day 14 in culture using Alizarin Red S staining (Fig 1E, F), with 4.8-fold higher calcification in OM as compared to normal media (NM). Conclusions: Investigated iSMCs have promising future applications in cardiovascular research and may serve as an inexhaustible resource for studying vascular SMC calcification.

P1233M2 MACROPHAGES PROMOTE MOUSE AORTIC SMOOTH MUSCLE CELLS CALCIFICATION BY SGK1-TGFB1 AXIS

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Beibei Wu ◽  
Xiaodong Zhang ◽  
Yuqiu Liu ◽  
Xiao liang Zhang
Keyword(s):  
Smooth Muscle ◽  
Western Blot ◽  
Smooth Muscle Cells ◽  
Vascular Calcification ◽  
Muscle Cells ◽  
Immunofluorescence Staining ◽  
Alizarin Red ◽  
Aortic Smooth Muscle Cells ◽  
Aortic Smooth Muscle ◽  
Receptor Inhibitor

Abstract Background and Aims Vascular calcification is an independent risk factor for all-cause mortality in patients with CKD. Macrophages play an important role in vascular calcification, which involve in the osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs). Recent research shows SGK1 is a highly attractive candidate for developing VSMCs calcification. Previous studies indicated that TGFβ1 induces vascular calcification by regulating osteo-/chondrogenic transdifferentiation of VSMCs. This study focus on the effects of macrophages-derived SGK1 on VSMCs transdifferentiation via regulating TGFβ1 expression. Method Before induced into M2 phenotype with IL-4, RAW 264.7 cells were treated with SGK1 inhibitor EMD638683 for 24 hours to reduce SGK1 expression. The above cells were grouped as: M0, M2, M2+EMD to explore the expression of SGK1 and TGFβ1 by using western blot, qRT-PCR and immunofluorescence staining, respectively. The culture medium (CM) of the above group macrophages was collected. Then mouse aortic smooth muscle cells (MAoSMCs ) were cultured in these supernatants with normal medium or osteogenic medium (OM) with or without TGFβ1 receptor inhibitor SB-431542 for 3 weeks to explore the transdifferentiation and calcification of MOVAS by using western blot, RT-qPCR, immunofluorescence staining, calcium quantification, Alizarin Red and Von Kossa Staining. Results As Alizarin Red and Von Kossa Staining shown, MAoSMCs contained more calcium deposits in the M2-CM group compared with either in the group of NC nor M0-CM. Consistent with the above results, We also found that M2-CM promoted MAoSMCs transdifferentiation, which was characterized by markedly increase of expression of osteo-/chondrogenic markers (Runx2, ALPL, FGF23) and decrease of the MAoSMCs marker (SM22α). Exploring the mechanism of the above phenomenon we found the expression of SGK1 and TGFβ1 were significantly increased in M2 group compared with M0 group. Interestingly, both SGK1 inhibitor EMD638683 which reduced TGFβ1 expression in M2 and TGFβ1 receptor inhibitor SB-431542 could partially blocked MAoSMCs osteo-/chondrogenic transdifferentiation and calcification. Furthermore, recombinant mouse TGFβ1 Protein increased calcium content in MAoSMCs by using calcium quantification, Alizarin Red and Von Kossa Staining and promoted MAoSMCs osteo-/chondrogenic transdifferentiation, which was characterized by markedly increase of expression of osteo-/chondrogenic markers (Runx2, ALPL, FGF23) and decrease of the MAoSMCs marker (SM22α). Conclusion Our findings shed light M2 macrophages promotes MAoSMCs osteo-/chondrogenic transdifferentiation and calcification by up-regulating TGFβ1 expression.

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