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.

scholarly journals Prevention of Vascular Calcification by Magnesium and Selected Polyphenols

2021 ◽  
Vol 2021 ◽  
pp. 1-5
Author(s):  
Haile Mehansho ◽  
Satya Majeti ◽  
Gabe Tzeghai
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Calcification ◽  
Vascular Smooth Muscle Cells ◽  
Heart Diseases ◽  
In Vitro Study ◽  
Muscle Cells ◽  
Calcium Deposition

Arterial vascular calcification (VC) represents formation of calcium phosphate deposits on the interior of arteries, which could restrict blood flow leading to heart health problems, including morbidity and mortality. VC is a complex and tightly regulated process that involves transformation of vascular smooth muscle cells (VSMCs) to bone-like cells and subsequent deposition of calcium as hydroxyapatite. Natural bioactives, including quercetin (Q), curcumin (C), resveratrol (R), and magnesium (Mg), have been reported to inhibit VC. Thus, we conducted an in vitro study using rat vascular smooth muscle cells (rVSMCs) to evaluate the protective effect of natural bioactives found in OptiCel, that is, Mg combined with polyphenols (PPs), Q, C, and R. Calcification was induced by culturing rVSMCs in a high phosphate (HP) medium. The addition of Mg and Q + C + R separately decreased the HP-induced calcium deposition by 37.55% and 42.78%, respectively. In contrast, when Mg was combined with Q, C, and R, the inhibition of calcium deposition was decreased by 92.88%, which is greater than their calculated additive inhibition (80.33%). These results demonstrate that the combination of Mg with selected PPs (Q, C, and R) is more effective than when used separately. The findings also suggest the combination has a synergistic effect in inhibiting VC, which is a risk factor for cardiovascular disease. Thus, regular consumption of these natural bioactives could have a beneficial effect in reducing the development of heart diseases.

MO583IN VITRO AND EX VIVO EXPERIMENTS OF VASCULAR CALCIFICATION

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Jana Holmar ◽  
Heidi Noels ◽  
Joachim Jankowski ◽  
Setareh Orth-Alampour
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Calcification ◽  
Vascular Smooth Muscle Cells ◽  
Incubation Time ◽  
Fetal Calf Serum ◽  
Ex Vivo ◽  
Calf Serum

Abstract Background and Aims Vascular calcification (VC) is one major complication in patients with chronic kidney disease whereas a misbalance in calcium and phosphate metabolism plays a crucial role. The mechanisms underlying VC have not been entirely revealed to date. Therefore are the studies aiming at the identification and characterization of the mediators/uremic toxins involved in VC ongoing and highly relevant. However, currently many different protocols being used in the studies of vascular calcification processes. This complicates the comparison of study outcomes, composing systematic reviews, and meta-analyses. Moreover, the reproducibility of data is hampered, and the efficiency in calcification research through the lack of a standardized protocol is reduced. In this study, we developed a standardized operating protocol for in vitro and ex vivo approaches to aiming at the comparability of these studies. Method We analysed in vitro and ex vivo experimental conditions to study VC. Vascular smooth muscle cells (HAoSMCs) were used for in vitro experiments and aortas from Wistar rats were used for ex vivo experiments. The influence of the following conditions was studied in detail: • Phosphate and calcium concentrations in calcifying media. • Incubation time. • Fetal calf serum (FCS) concentration. The degree of calcification was estimated by quantification of calcium concentrations that were normalized to protein content (in vitro) or to the dry weight of the aortic ring (ex vivo). Additionally, the aortic rings were stained using the von Kossa method. Optimal conditions for investigating medial vascular calcification were detected and summarized in the step-by-step protocol. Results We were able to demonstrate that the degree and the location of VC in vascular smooth muscle cells and aortic rings were highly dependent on the phosphate and CaCl2 concentration in the medium as well as the incubation time. Furthermore, the VC was reduced upon increasing fetal calf serum concentration in the medium. An optimized protocol for studying vascular calcification in vitro and ex vivo was developed and validated. The final protocol (Figure 1) presented will help to standardize in vitro and ex vivo approaches to investigate the processes of vascular calcification. Conclusion In the current study, we developed and validated a standardized operating protocol for systematic in vitro and ex vivo analyses of medial calcification, which is essential for the comparability of the results of future studies.

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.

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 Nano-Hydroxyapatite Accelerates Vascular Calcification by Inhibiting Lysosomal Acidification in Smooth Muscle Cells.

2021 ◽  
Author(s):  
Qi Liu ◽  
Yi Luo ◽  
Yun Zhao ◽  
Pingping Xiang ◽  
Jinyun Zhu ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Calcification ◽  
Chemical Properties ◽  
Muscle Cells ◽  
Calcium Deposition ◽  
Rapid Progression ◽  
Autophagic Flux

Abstract Background: Vascular calcification (VC) is a common characteristic of aging, diabetes, chronic renal failure, and atherosclerosis. The basic component of VC is hydroxyapatite (HAp). Nano-sized HAp (nHAp) has been identified as the initiator of pathological calcification of vasculature. However, whether nHAp can induce calcification in vivo and the mechanism of nHAp in the progression of VC remains unclear.Results: We discovered that nHAp existed both in vascular smooth muscle cells (VSMCs) and their extracellular matrix (ECM) in the calcified arteries from patients. Synthetic nHAp had similar morphological and chemical properties as natural nHAp recovered from calcified artery. nHAp induced rapid progression of VC by stimulating osteogenic differentiation and accelerating mineralization of VSMCs in vitro. Synthetic nHAp could also directly induce VC in vivo. Mechanistically, nHAp was internalized into lysosome, which impaired lysosome vacuolar H+-ATPase for its acidification, therefore blocked autophagic flux in VSMCs. The accumulated autophagosomes and autolysosomes were converted into calcium-containing exosomes which were secreted into ECM and accelerated vascular calcium deposit. Inhibition of exosome release in VSMCs decreased calcium deposition. Conclusions: Our results illustrated a novel mechanism of nHAp-induced vascular calcification. Understanding the role of nHAp in autophagy-lysosome-exosome pathway in SMCs could have great clinical significance in preventing the progression of VC.

scholarly journals RAGE Differentially Altered in vitro Responses in Vascular Smooth Muscle Cells and Adventitial Fibroblasts in Diabetes-Induced Vascular Calcification

2021 ◽  
Vol 12 ◽  
Author(s):  
Amber M. Kennon ◽  
James A. Stewart
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
P38 Mapk ◽  
Vascular Calcification ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Adventitial Fibroblasts ◽  
Rage Expression

The Advanced Glycation End-Products (AGE)/Receptor for AGEs (RAGE) signaling pathway exacerbates diabetes-mediated vascular calcification (VC) in vascular smooth muscle cells (VSMCs). Other cell types are involved in VC, such as adventitial fibroblasts (AFBs). We hope to elucidate some of the mechanisms responsible for differential signaling in diabetes-mediated VC with this work. This work utilizes RAGE knockout animals and in vitro calcification to measure calcification and protein responses. Our calcification data revealed that VSMCs calcification was AGE/RAGE dependent, yet AFBs calcification was not an AGE-mediated RAGE response. Protein expression data showed VSMCs lost their phenotype marker, α-smooth muscle actin, and had a higher RAGE expression over non-diabetics. RAGE knockout (RKO) VSMCs did not show changes in phenotype markers. P38 MAPK, a downstream RAGE-associated signaling molecule, had significantly increased activation with calcification in both diabetic and diabetic RKO VSMCs. AFBs showed a loss in myofibroblast marker, α-SMA, due to calcification treatment. RAGE expression decreased in calcified diabetic AFBs, and P38 MAPK activation significantly increased in diabetic and diabetic RKO AFBs. These findings point to potentially an alternate receptor mediating the calcification response in the absence of RAGE. Overall, VSMCs and AFBs respond differently to calcification and the application of AGEs.

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.

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.

Sign in / Sign up

Export Citation Format

Share Document