scholarly journals Endoplasmic Reticulum Stress Mediates Vascular Smooth Muscle Cell Calcification via Increased Release of Grp78-Loaded Extracellular Vesicles

2020 ◽  
Author(s):  
Malgorzata Furmanik ◽  
Rick van Gorp ◽  
Meredith Whitehead ◽  
Sadia Ahmad ◽  
Jayanta Bordoloi ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Er Stress ◽  
Vascular Calcification ◽  
Bone Mineralization ◽  
Extracellular Vesicle ◽  
Dependent Manner ◽  
The Matrix ◽  
Stress Dependent

Objective: Vascular calcification is common among aging populations and mediated by vascular smooth muscle cells (VSMCs). The endoplasmic reticulum (ER) is involved in protein folding and ER stress has been implicated in bone mineralization. The role of ER stress in VSMC-mediated calcification is less clear. Approach and Results: mRNA expression of the ER stress markers PERK (PKR (protein kinase RNA)-like ER kinase), ATF (activating transcription factor) 4, ATF6, and Grp78 was detectable in human vessels with levels of PERK decreased in calcified plaques compared to healthy vessels. Protein deposition of Grp78/Grp94 was increased in the matrix of calcified arteries. Induction of ER stress accelerated human primary VSMC-mediated calcification, elevated expression of some osteogenic markers (Runx2, Osterix, ALP, BSP, and OPG), and decreased expression of SMC markers. ER stress potentiated extracellular vesicle (EV) release via SMPD3. EVs from ER stress-treated VSMCs showed increased Grp78 levels and calcification. Electron microscopy confirmed the presence of Grp78/Grp94 in EVs. siRNA knock-down of Grp78 decreased calcification. Warfarin-induced Grp78 and ATF4 expression in rat aortas and VSMCs and increased calcification in an ER stress-dependent manner via increased EV release. Conclusions: ER stress induces vascular calcification by increasing release of Grp78-loaded EVs. Our results reveal a novel mechanism of action of warfarin, involving increased EV release via the PERK-ATF4 pathway, contributing to calcification. This study is the first to show that warfarin induces ER stress and to link ER stress to cargo loading of EVs.

scholarly journals Sclerostin as Regulatory Molecule in Vascular Media Calcification and the Bone–Vascular Axis

Toxins ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 428 ◽  
Author(s):  
Annelies De Maré ◽  
Stuart Maudsley ◽  
Abdelkrim Azmi ◽  
Jhana O. Hendrickx ◽  
Britt Opdebeeck ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Bone Formation ◽  
Vascular Smooth Muscle ◽  
Vascular Calcification ◽  
Bone Mineralization ◽  
Progressive Increase ◽  
Feedback Mechanism ◽  
Vascular Pathology ◽  
Dependent Manner ◽  
Local Production

Sclerostin is a well-known inhibitor of bone formation that acts on Wnt/β-catenin signaling. This manuscript considers the possible role of sclerostin in vascular calcification, a process that shares many similarities with physiological bone formation. Rats were exposed to a warfarin-containing diet to induce vascular calcification. Vascular smooth muscle cell transdifferentiation, vascular calcification grade, and bone histomorphometry were examined. The presence and/or production of sclerostin was investigated in serum, aorta, and bone. Calcified human aortas were investigated to substantiate clinical relevance. Warfarin-exposed rats developed vascular calcifications in a time-dependent manner which went along with a progressive increase in serum sclerostin levels. Both osteogenic and adipogenic pathways were upregulated in calcifying vascular smooth muscle cells, as well as sclerostin mRNA and protein levels. Evidence for the local vascular action of sclerostin was found both in human and rat calcified aortas. Warfarin exposure led to a mildly decreased bone and mineralized areas. Osseous sclerostin production and bone turnover did not change significantly. This study showed local production of sclerostin in calcified vessels, which may indicate a negative feedback mechanism to prevent further calcification. Furthermore, increased levels of serum sclerostin, probably originating from excessive local production in calcified vessels, may contribute to the linkage between vascular pathology and impaired bone mineralization.

scholarly journals P0906SALUBRINAL IMPROVES OSTEOCLAST DIFFERENTIATION AND VASCULAR CALCIFICATION IN UREMIC MILLENNIUM THROUGH INHIBITION OF THE ENDOPLASMIC RETICULUM (ER) STRESS CONDITION

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Cai-Mei Zheng ◽  
Hui-Wen Chiu ◽  
Kuo-Cheng Lu ◽  
Chien-Lin Lu ◽  
Mai-Szu Wu ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Bone Loss ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Er Stress ◽  
Vascular Calcification ◽  
Vascular Smooth Muscle Cells ◽  
Osteoclast Differentiation ◽  
Muscle Cells ◽  
Uremic Toxins

Abstract Background and Aims Chronic kidney disease (CKD) is a globally increasing health problem especially in aged era. CKD-mineral and bone disorder (CKD-MBD) becomes a principal consequence of CKD, which includes mineral abnormalities, vascular calcifications (VC) and renal osteodystrophy (ROD), and patients eventually present with fractures and cardiovascular disease (CVD) (Figure 1). Thus, bone loss in CKD patients relates closely with cardiovascular calcification. The endoplasmic reticulum (ER) stress is triggered by many clinical conditions unique to CKD, including uremic toxins, chronic inflammation, metabolic acidosis, proteinuria, malnutrition, etc. In our study, we hope to evaluate the ER stress inhibitor, salubrinal as targeted therapy for CKD related bone loss and vascular calcification. Methods: Isolation of primary osteoclasts from long bone primary cell culture: Femoral and tibia bone from 8-week-old Sprague–Dawley rats were removed and used as primary osteoclast cultures. Osteoclast precursor cells were seeded in 96-well plates (2.0 x 104 cells/well) and cultured for 6 days in alpha minimum essential medium (α-MEM) supplemented with 10% FBS, 50 ng/ml M-CSF and 50 ng/ml RANKL in the absence or presence of uremic toxins. The whole process of osteoclast development in cell cultures was divided into first M-CSF–dependent growth of osteoclast progenitor’s phase and latter phase is RANKL induced terminal differentiation phase. The experiment is performed with the approval of the Laboratory Animal Center of the Taipei Medical University in Taipei, Taiwan. Primary Human Aorta Vascular Smooth Muscle Cell Culture: Primary human aorta vascular smooth muscle cells (VSMC) were isolated by ScienCell Research Laboratories, Inc. (Carlsbad, CA) and purchased through Innoprot (Derio, Spain). We examined the role of uremic toxin on ER stress and autophagy using osteoclasts and vascular smooth muscle cells cultured with uremic toxins including PTH, p-cresylsulfate (pCS) and p-cresylglucuronide (pCG), indoxyl sulfate, asymmetric dimethylarginine (ADMA), and homocysteine. The cells are treated with salubrinal with or without toxins and see how ER stress and autophagy effects on survival and differentiation of osteoclasts and VSMC cells. Results We found that ER stress and autophagy related mRNA were increased in osteoclasts using GEO database analysis, revealed that ER stress and autophagy play an important role in osteoclast differentiation. Further, we revealed that uremic toxins increased the autophagy and ER stress status and increased the osteoclasts differentiation. Treatment of the osteoclasts under uremic millennium with salubrinal, an ER stress inhibitor, inhibited the ER stress and osteoclast differentiation. Uremic toxins also increased the ER stress in human vascular smooth muscle cells (VSMCs). After treated with salubrinal, the calcification and proliferation improved in these cells. Conclusion ER stress increases osteoclast differentiation and vascular calcification in uremic millennium, and salubrinal improves these conditions through inhibition of ER stress. Thus, salubrinal probably might be used as targeted therapy for vascular calcification and bone loss in CKD patients.

scholarly journals ER stress dependent microparticles derived from smooth muscle cells promote endothelial dysfunction during thoracic aortic aneurysm and dissection

2017 ◽  
Vol 131 (12) ◽  
pp. 1287-1299 ◽  
Author(s):  
Li-Xin Jia ◽  
Wen-Mei Zhang ◽  
Tao-Tao Li ◽  
Yan Liu ◽  
Chun-Mei Piao ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Endothelial Dysfunction ◽  
Aortic Aneurysm ◽  
Er Stress ◽  
Thoracic Aortic Aneurysm ◽  
Mechanical Stretch ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Aortic Aneurysm And Dissection ◽  
Stress Dependent

The degeneration of vascular smooth muscle cell(s) (SMC) is one of the key features of thoracic aortic aneurysm and dissection (TAAD). We and others have shown that elevated endoplasmic reticulum (ER) stress causes SMC loss and TAAD formation, however, the mechanism of how SMC dysfunction contributes to intimal damage, leading to TAAD, remains to be explored. In the present study, in vitro assay demonstrated that elevated mechanical stretch (18% elongation, 3600 cycles/h) stimulated the ER stress response and microparticle(s) (MP) production from both SMC and endothelial cell(s) (EC) in a time-dependent manner. Treatment of EC with isolated MP led to anoikis, which was determined by measuring the fluorescence of the ethidium homodimer (EthD-1) and Calcein AM cultured in hydrogel-coated plates and control plates. MP stimulation of EC also up-regulated the mRNA levels of inflammatory molecules (i.e. Vascular cellular adhesion molecular-1 (VCAM-1)), intercellular adhesion molecular-1 (ICAM-1), interleukin-1β (IL-1β), and interleukin-6 (IL-6)). Use of an ER stress inhibitor or knockout of CHOP decreased mechanical stretch-induced MP production in SMC. In vivo, administration of an ER stress inhibitor or knockout of CHOP suppressed both apoptosis of EC and the infiltration of inflammatory cells. Moreover, TAAD formation was also suppressed by the administration of an ER stress inhibitor. In conclusion, our study demonstrates that elevated mechanical stretch induces MP formation in SMC leading to endothelial dysfunction, which is ER stress dependent. The inhibition of ER stress suppressed EC apoptosis, inflammation in the aorta, and TAAD development.

scholarly journals MicroRNA-223-3p inhibits vascular calcification and the osteogenic switch of vascular smooth muscle cells

2021 ◽  
Vol 296 ◽  
pp. 100483
Author(s):  
Yingchun Han ◽  
Jichao Zhang ◽  
Shan Huang ◽  
Naixuan Cheng ◽  
Congcong Zhang ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Calcification ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells

Abstract 17763: Inhibition of Bone Morphogenetic Protein Signaling Reduces Endothelial-Mesenchymal Transition and Vascular Smooth Muscle Cell Calcification Associated with Matrix Gla Protein Deficiency

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Megan F Burke ◽  
Caitlin O’Rourke ◽  
Trejeeve Martyn ◽  
Hannah R Shakartzi ◽  
Timothy E Thayer ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Bone Morphogenetic Protein ◽  
Vascular Calcification ◽  
Bmp Signaling ◽  
Matrix Gla Protein ◽  
Wild Type ◽  
Mesenchymal Transition ◽  
Morphogenetic Protein ◽  
Endothelial Mesenchymal Transition

Background: Matrix Gla protein (MGP) is an extracellular matrix protein that inhibits bone morphogenetic protein (BMP) signaling in vitro. MGP deficiency induces vascular calcification associated with osteogenic transdifferentiation of endothelial cells (via endothelial-mesenchymal transition, EndMT) and vascular smooth muscle cells (VSMCs). We previously reported that treatment with two pharmacologic inhibitors of BMP signaling reduced aortic calcification in MGP-/- mice. We hypothesized that BMP signaling is essential for EndMT and VSMC osteogenic transdifferentiation induced by MGP deficiency. Methods and Results: Aortic levels of mRNAs encoding markers of osteogenesis (Runx2 and osteopontin) and EndMT (nanog, Sox2, and Oct3/4) were greater in MGP-/- than in wild-type mice (P<0.01 for all). Aortic expression of markers of VSMC differentiation (α-smooth muscle actin, transgelin, and calponin) was less in MGP-/- than in wild-type mice (P<0.001 for all). Treatment of MGP-/- mice with the BMP signaling inhibitor, LDN-193189, reduced expression of both osteogenic and EndMT markers (P<0.05 for all) but did not prevent VSMC de-differentiation. Depletion of MGP in cultured wild-type VSMCs with siRNA specific for MGP (siMGP) was associated with a 30-40% reduction in levels of mRNAs encoding markers of VSMC differentiation (P<0.05 for all), an effect that was not prevented by LDN-193189. Incubation in phosphate-containing media induced greater calcification in siMGP-treated VSMCs than in cells treated with control siRNA (P<0.0001). Treatment with LDN-193189 reduced calcification in siMGP-treated VSMCs (50%, P=0.0003). Conversely, infection of MGP-/- VSMCs with adenovirus specifying MGP increased expression of markers of VSMC differentiation by 60-80% (P<0.01 for all) and decreased calcification by 74% (P=0.03). Conclusions: Inhibition of BMP signaling suppresses osteogenic and EndMT gene programs in MGP-/- mice and reduces calcification of siMGP-treated VSMCs. However, MGP deficiency induces VSMC de-differentiation via a BMP-independent mechanism. These findings suggest that the processes underlying vascular calcification in MGP deficiency are mediated by both BMP signaling-dependent and -independent mechanisms.

Docosahexaenoic Acid, a Peroxisome Proliferator-Activated Receptor-α Activator, Induces Apoptosis in Vascular Smooth Muscle Cells by Activation of P38 Mitogen-Activated Protein Kinase

Hypertension ◽  
2000 ◽  
Vol 36 (suppl_1) ◽  
pp. 679-679
Author(s):  
Quy N Diep ◽  
Rhian M Touyz ◽  
Ernesto L Schiffrin
Keyword(s):  
Smooth Muscle ◽  
Cytochrome C ◽  
Docosahexaenoic Acid ◽  
Vascular Smooth Muscle ◽  
Morphological Changes ◽  
Mitogen Activated Protein Kinase ◽  
Peroxisome Proliferator ◽  
Dependent Manner ◽  
Mitogen Activated Protein ◽  
P38 Mapks

9 Omega-3 fatty acids (n-3 FAs) exert a blood pressure-lowering effect in hypertension, possibly by influencing vascular structure. We previously demonstrated that n-3 FAs might induce vascular smooth muscle cell (VSMC) apoptosis, which could exert an effect on structure of blood vessels. This study investigated signaling pathways through which n-3 FAs mediate apoptosis in VSMCs. Cultured Mesenteric VSMCs from Sprague Dawley rats were stimulated with docosahexaenoic acid (DHA), a representative n-3 FA. Morphological changes of apoptosis and DNA fragmentation were examined by phase-contrast microscopy and fluorescence microscopy with Hoechst 33342 staining. To clarify possible pathways of apoptosis, expression of phosphorylated p38 mitogen-activated protein kinases (p38 MAPKs), bax, bcl-2, cytochrome C and peroxisome proliferator-activated receptors-α (PPARs-α) was evaluated by Western blot analysis. DHA treatment induced cell shrinkage, cell membrane blebbing and apoptotic bodies in VSMCs. DHA increased apoptosis (%) in a time-dependent manner to 1.5±0.1, 3.6±0.5, 7.1±0.4, 22.5±0.6, 50.8±1.8 and 61.4±0.9 after 0, 1, 3, 6, 17, and 24 h, respectively. DHA time-dependently activated p38 MAPKs, bax, PPARs-α and cytochrome C with maximal effects obtained after 5, 30 min, 1 h and 3 h, respectively to 551±42, 245±55, 310±12 and 407±14.7 % of controls, respectively. SB-203580 (10 -5 M) and SB-202190 (10 -5 M), selective p38 inhibitors, reduced DHA-elicited apoptosis and expression of PPARs-α, but had no effect on expression of bax or cytochrome C. The present results indicate that DHA induces apoptosis in VSMCs through at least two distinct mechanisms: (i) a p38-dependent pathway that regulates PPAR-α and (ii) a p38-independent pathway via dissipation of mitochondrial transmembrane potential. The death-signaling pathway mediated by DHA may involve an integration of these multiple pathways. By triggering VSMC apoptosis, DHA could play a pathophysiological role in vascular remodeling in cardiovascular disease.

Abstract 126: Differential Effects of Respectively Blocking Two BET Bromodomains on Vascular Smooth Muscle Cell Phenotype Transformation

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Mengxue Zhang ◽  
Bowen Wang ◽  
Craig Kent ◽  
Lian-Wang Guo
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cell ◽  
Muscle Cell ◽  
Vascular Smooth Muscle Cell ◽  
Molecular Mechanisms ◽  
Cell Phenotype ◽  
Dependent Manner ◽  
Stat3 Phosphorylation ◽  
Phenotype Transformation

Introduction: Intimal hyperplasia (IH) occurs primarily due to vascular smooth muscle cell (SMC) transformation from quiescent to pathogenic phenotypes (e.g. proliferation and inflammation). Identification and effective targeting of key epigenetic factors governing SMC pathogenic transformation may lead to novel therapeutic methods for prevention of IH. We previously found that globally blocking the bromo- and extra-terminal (BET) epigenetic “reader” family abrogated SMC phenotype transformation and IH. We further investigated the functions of the two BET bromodomains (Bromo1 and Bromo2). Hypothesis: Bromo1 and Bromo2 play different roles in SMC pathogenic transformation. Methods and Results: We pre-treated rat primary aortic SMCs (for 2h) with Olinone or RVX208, inhibitors specific for Bromo1 and Bromo2 respectively, and then stimulated SMC phenotype transformation. Whereas RVX208 abrogated PDGF-BB-stimulated SMC proliferation (BrdU assay) in a dose dependent manner, Olinone enhanced SMC proliferation at high concentrations (>20 μM). RVX208 at 50 μM reduced TNFα-induced SMC inflammation (MCP-1 ELISA) by 80%,but Olinone at the same concentration slightly increased MCP-1. Furthermore, whereas RVX208 abolished PDGF-BB or TNFα-induced STAT3 phosphorylation (Western blotting), Olinone slightly increased phospho-STAT3. Conclusions: Our results reveal that blocking two BET bromodomains respectively produces distinct effects on SMC phenotype transformation, suggesting their differential epigenetic functions. Further elucidation of the underlying molecular mechanisms should contribute to precise targeting of the BET family for optimal mitigation of IH.

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.

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