Abstract 12584: Transcription Factor EB Regulates Vascular Smooth Muscle Calcification

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Ryo Ishiwata ◽  
Yuji Morimoto
Keyword(s):  
Transcription Factor ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Protein Expression ◽  
Vascular Calcification ◽  
Inorganic Phosphate ◽  
Calcium Deposition ◽  
Nuclear Fraction ◽  
Lysosomal Activity ◽  
Transcription Factor Eb

Introduction: Vascular calcification is a strong predictor of cardiovascular-related mortality. Hyperphosphatemia causes phenotypic switches of vascular smooth muscle cells (VSMCs), leading to medial calcification. Recent studies have suggested that dysfunction of the autophagy-lysosomal pathway in VSMCs is a cause of vascular calcification, but the process through which this pathway fails is elusive. Transcription factor EB (TFEB) is a master regulator of lysosome biogenesis; its function in VSMCs is unknown. Hypothesis: We assessed the hypothesis that the dysfunction of TFEB in VSMCs is a cause of vascular calcification. Methods and Results: We induced vascular calcification in wild type mouse aorta with an ex vivo hyperphosphatemia model . Addition of inorganic phosphate at a 1.7 mmol/L for five days decreased TFEB protein expression (0.23 ± 0.10-fold vs. day0, n = 5-7). Immunohistochemistry and alizarin red staining showed that a decrease in TFEB expression in the tunica media was correlated with the formation of calcification. VSMCs were isolated from rat aorta and were cultured for seven days in vitro . Addition of inorganic phosphate dose-dependently decreased TFEB protein expression both in whole cell lysate and in nuclear fraction (0.07± 0.03-fold vs. control, n = 5 ; 0.01 ± 0.003-fold vs. control, n = 4, respectively) while it rather increased mRNA expression of Tfeb (4.48 ± 0.95-fold, n = 7 vs. control). The Decrease in TFEB protein by inorganic phosphate was correlated with the accumulation of TFEB in the SDS-insoluble fraction, suggesting the formation of protein aggregates. The Knockdown of TFEB in VSMCs by siRNA exacerbated phosphate-induced calcium deposition (2.93 ± 0.85-fold, n = 4 vs. negative siRNA). The addition of inorganic phosphate significantly decreased lysosomal activity determined by LysoTracker dye; and treatment with 20 nmol/L of bafilomycin A, a lysosome inhibitor, further accelerated phosphate-induced calcium deposition (13.6 ± 3.69-fold, n = 7 vs. vehicle). Conclusion: In conclusion, TFEB expression in VSMCs is downregulated at the protein level by hyperphosphatemia. The diminution of functional TFEB predisposes to vascular calcification, presumably through downregulating lysosomal activity.

Abstract 391: Mir-A-3p Targets Atf3 to Reduce Calcium Deposition in Vascular Smooth Muscle Cells

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Nakwon CHOE ◽  
Duk-hwa Kwon ◽  
Juhee Ryu ◽  
SERA SHIN ◽  
Hosouk Joung ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Calcification ◽  
Vascular Smooth Muscle Cells ◽  
Metabolic Diseases ◽  
Mrna Level ◽  
Muscle Cells ◽  
Calcium Deposition

Vascular calcification, the ectopic deposition of calcium in blood vessels, develops in association with various metabolic diseases and atherosclerosis and is an independent predictor for morbidity and mortality of these diseases. Here we report that reduction of microRNA-A-3p (miR-A-3p) causes an increase in ATF3, activating transcription factor 3, a novel osteogenic transcription factor, in vascular smooth muscle cells. Both miRNA and mRNA microarrays were performed with rat vascular smooth muscle cells and reciprocally regulated pairs of miRNA and mRNA were selected after bioinformatic analysis. Inorganic phosphate significantly reduced the expression of miR-A-3p in A10 cells. The transcript level was also reduced in vitamin D3-administered mouse aortas. miR-A-3p mimic reduced calcium deposition, whereas miR-A-3p inhibitor increased it. The Atf3 mRNA level was upregulated in cellular vascular calcification model, and miR-A-3p reduced the Atf3 mRNA and protein levels. Transfection with Atf3 could recover the miR-A-3p-induced reduction of calcium deposition. Our results suggest that reduction of miR-A-3p may contribute to the development of vascular calcification by de-repression of ATF3

scholarly journals Fibulin-3 Attenuates Phosphate-Induced Vascular Smooth Muscle Cell Calcification by Inhibition of Oxidative Stress

2018 ◽  
Vol 46 (4) ◽  
pp. 1305-1316 ◽  
Author(s):  
Trang T. D. Luong ◽  
Nadeshda Schelski ◽  
Beate Boehme ◽  
Manousos Makridakis ◽  
Antonia Vlahou ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Mrna Expression ◽  
Vascular Calcification ◽  
Muscle Cells ◽  
Additional Treatment ◽  
Calcium Deposition

Background/Aims: Fibulin-3, an extracellular matrix glycoprotein, inhibits vascular oxidative stress and remodeling in hypertension. Oxidative stress is prevalent in chronic kidney disease (CKD) patients and is an important mediator of osteo-/chondrogenic transdifferentiation and calcification of vascular smooth muscle cells (VSMCs) during hyperphosphatemia. Therefore, the present study explored the effects of Fibulin-3 on phosphate-induced vascular calcification. Methods: Experiments were performed in primary human aortic smooth muscle cells (HAoSMCs) treated with control or with phosphate without or with additional treatment with recombinant human Fibulin-3 protein or with hydrogen peroxide as an exogenous source of oxidative stress. Results: Treatment with calcification medium significantly increased calcium deposition in HAoSMCs, an effect significantly blunted by additional treatment with Fibulin-3. Moreover, phosphate-induced alkaline phosphatase activity and mRNA expression of osteogenic and chondrogenic markers MSX2, CBFA1, SOX9 and ALPL were all significantly reduced by addition of Fibulin-3. These effects were paralleled by similar regulation of oxidative stress in HAoSMCs. Phosphate treatment significantly up-regulated mRNA expression of the oxidative stress markers NOX4 and CYBA, down-regulated total antioxidant capacity and increased the expression of downstream effectors of oxidative stress PAI-1, MMP2 and MMP9 as well as BAX/BLC2 ratio in HAoSMCs, all effects blocked by additional treatment with Fibulin-3. Furthermore, the protective effects of Fibulin-3 on phosphate-induced osteogenic and chondrogenic markers expression in HAoSMCs were reversed by additional treatment with hydrogen peroxide. Conclusions: Fibulin-3 attenuates phosphate-induced osteo-/ chondrogenic transdifferentiation and calcification of VSMCs, effects involving inhibition of oxidative stress. Up-regulation or supplementation of Fibulin-3 may be beneficial in reducing the progression of vascular calcification during hyperphosphatemic conditions such as CKD.

Uremic Patients with Increased Vascular Calcification Score Have Serum with High Calcific Potential: Role of Vascular Smooth Muscle Cell Osteoblastic Differentiation and Apoptosis

2019 ◽  
Vol 48 (2) ◽  
pp. 142-149 ◽  
Author(s):  
Paola Ciceri ◽  
Andrea Galassi ◽  
Carlo Alfieri ◽  
Piergiorgio Messa ◽  
Mario Cozzolino
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Vascular Calcification ◽  
Osteoblastic Differentiation ◽  
Calcium Deposition ◽  
Calcification Score ◽  
Uremic Serum ◽  
Uremic Patients ◽  
Apoptosis And Necrosis

Background/Aims: Uremic patients experience premature vascular ageing that causes cardiovascular morbidity. In this study, we investigated the relationship between uremic serum calcific potential induced by high phosphate (Pi) and vascular calcification score (VCS). Methods: Vascular smooth muscle cells (VSMCs) were cultured with 3.5 mM Na3PO4 (Pi) with 10% uremic serum and calcium deposition, markers of osteoblastic transformation, and apoptosis were evaluated. Results: Culture with uremic serum and high-Pi significantly induced calcification (0.21 ± 0.03 vs. 8.05 ± 0.6; ctr vs. Pi; OD/mg protein; p < 0.01). We next stratified patients with respect of the degree of VCS in 2 groups: absence of vascular calcification (VC) “no VC group” and presence of VC “VC group”. We found that there was a significant correlation between VCS and uremic serum calcific potential induced by high Pi in vitro (p < 0.01). Interestingly, uremic sera of the “VC group” were more effective than sera from the “no VC group”, in downregulating α-actin and SM22α, after treatment with high-Pi (41.3 ± 4.7 vs. 23.3 ± 2.9 and 25.6 ± 6.8 vs. 8.14 ± 2.3; VC vs. no VC group, α-actin and SM22α respectively; Δ intensity area; p < 0.01). Similarly, sera from “VC group” were more effective than sera from “no VC group” in adjuvanting the high-Pi effect of increasing osteoblastic markers, such as bone morphogenic protein 2 (BMP2), osteocalcin (OC), and runt-related transcription factor 2 (RUNX2; 39.1 ± 11.3 vs. 5.0 ± 2.6 BMP2; 12.2 ± 4.2 vs. 1.7 ± 0.3 OC; 2.9 ± 0.4 vs. 1.2 ± 0.2 RUNX2; VC vs. no VC group respectively; p < 0.05). We found a similar pattern with significantly higher apoptosis and necrosis induction by sera from the “VC group” compared to the “no VC group” (2.05 ± 0.33 vs. 1.29 ± 0.13 and 54.1 ± 19.5 vs. 27.4 ± 10.6; Pi; VC group vs. no VC group; enrichment factor of apoptotic or necrotic fragments, respectively; p < 0.05). Conclusions: We conclude that VCS of end-stage renal disease patients significantly correlates with serum-calcific potential induced by high Pi. In addition, uremic patients with higher VCS have sera with a higher potential to induce VSMC osteoblastic trans-differentiation, apoptosis, and necrosis.

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&lt;.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&lt;0.0001) (Figures C-D) and in mouse aortic rings (p&lt;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.

scholarly journals Attenuating Effects of Pyrogallol-Phloroglucinol-6,6-Bieckol on Vascular Smooth Muscle Cell Phenotype Changes to Osteoblastic Cells and Vascular Calcification Induced by High Fat Diet

Nutrients ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2777
Author(s):  
Myeongjoo Son ◽  
Seyeon Oh ◽  
Ji Tae Jang ◽  
Chul-Hyun Park ◽  
Kuk Hui Son ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cell ◽  
Vascular Calcification ◽  
Muscle Cell ◽  
Vascular Smooth Muscle Cell ◽  
High Fat Diet ◽  
Calcium Deposition ◽  
High Fat

Advanced glycation end products/receptor for AGEs (AGEs/RAGEs) or Toll like receptor 4 (TLR4) induce vascular smooth muscle cell (VSMC) phenotype changes in osteoblast-like cells and vascular calcification. We analyzed the effect of Ecklonia cava extract (ECE) or pyrogallol-phloroglucinol-6,6-bieckol (PPB) on VSMC phenotype changes and vascular calcification prompted by a high-fat diet (HFD). HFD unregulated RAGE, TLR4, transforming growth factor beta (TGFβ), bone morphogenetic protein 2 (BMP2), protein kinase C (PKC), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signals in the aorta of mice. ECE and PPB restored the increase of those signal pathways. AGE- or palmitate-treated VSMC indicated similar changes with the animal. HFD increased osteoblast-like VSMC, which was evaluated by measuring core-binding factor alpha-1 (CBFα-1) and osteocalcin expression and alkaline phosphatase (ALP) activity in the aorta. ECE and PPB reduced vascular calcification, which was analyzed by the calcium deposition ratio, and Alizarin red S stain was increased by HFD. PPB and ECE reduced systolic, diastolic, and mean blood pressure, which increased by HFD. PPB and ECE reduced the phenotype changes of VSMC to osteoblast-like cells and vascular calcification and therefore lowered the blood pressure.

scholarly journals The Bioactive Substance Secreted by MSC Retards Mouse Aortic Vascular Smooth Muscle Cells Calcification

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Shuangshuang Wang ◽  
Maoqing Tong ◽  
Siwang Hu ◽  
Xiaomin Chen
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Intracellular Calcium ◽  
Vascular Calcification ◽  
Calcium Content ◽  
Calcium Deposition ◽  
Expression Ratio ◽  
Alizarin Red ◽  
Alp Activity ◽  
Study Results

Background. Vascular calcification, which is associated with low-level chronic inflammation, is a complication that occurs during aging, atherosclerosis, chronic kidney disease, diabetes mellitus, and hyperlipaemia. In this study, we used conditioned media from mesenchymal stem cells (MSC-CM), a source of autologous cytokines, to test the hypothesis that MSC-CM inhibits vascular smooth muscle cell (VSMC) calcification by suppressing inflammation and apoptosis. Methods. VSMCs were treated with β-glycerophosphate (β-GP) to induce calcification and MSC-CM was used as a treatment. Calcium deposition was evaluated using alizarin red and von Kossa staining after a 7-day induction period. Intracellular calcium contents were measured via the o-cresolphthalein complexone method, and alkaline phosphatase (ALP) activity was determined using the para-nitrophenyl phosphate method. The expressions of specific-osteogenic markers, inflammatory cytokines, and apoptosis-associated genes/proteins were examined by real-time polymerase chain reaction or western blotting. Results. MSC-CM inhibited β-GP-induced calcium deposition in VSMCs and decreased intracellular calcium content and ALP activity. Additionally, MSC-CM suppressed the β-GP-induced increases in BMP2, Msx2, Runx2, and osteocalcin expression. Additionally, MSC-CM decreased the expression of TNF-α, IL-1β, and IL-6 in VSMC. MSC-CM also partly blocked β-GP-induced VSMC apoptosis, which was associated with an increase in the Bcl-2/Bax expression ratio and a decrease in caspase-3 expression. Conclusion. Our study results suggest that MSC-CM can inhibit VSMC calcification. This suggests a potential novel clinical application for MSCs in the treatment of vascular calcification and associated diseases.

scholarly journals Infection of Porphyromonas gingivalis Increases Phosphate-Induced Calcification of Vascular Smooth Muscle Cells

Cells ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 2694
Author(s):  
Hyun-Joo Park ◽  
Yeon Kim ◽  
Mi-Kyoung Kim ◽  
Hae-Ryoun Park ◽  
Hyung-Joon Kim ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Porphyromonas Gingivalis ◽  
Vascular Calcification ◽  
Vascular Smooth Muscle Cells ◽  
Ex Vivo ◽  
Muscle Cells ◽  
Calcium Deposition ◽  
Periodontal Infection

Accumulating evidence suggests a link between periodontal disease and cardiovascular diseases. Vascular calcification is the pathological precipitation of phosphate and calcium in the vasculature and is closely associated with increased cardiovascular risk and mortality. In this study, we have demonstrated that the infection with Porphyromonas gingivalis (P. gingivalis), one of the major periodontal pathogens, increases inorganic phosphate-induced vascular calcification through the phenotype transition, apoptosis, and matrix vesicle release of vascular smooth muscle cells. Moreover, P. gingivalis infection accelerated the phosphate-induced calcium deposition in cultured rat aorta ex vivo. Taken together, our findings indicate that P. gingivalis contributes to the periodontal infection-related vascular diseases associated with vascular calcification.

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