Lipoxin Alleviates Diabetic Vascular Calcification via the YAP Pathway

Abstract Background: Vascular calcification is highly prevalent in patients with diabetes and has detrimental consequences. However, no effective prevention and treatment methods are currently available. Extensive evidence has demonstrated the protective effect of lipoxin (LX) against vascular diseases. However, whether LX prevents diabetic vascular calcification remains unknown. Here, we tested the hypothesis that LX alleviated osteogenic differentiation and subsequent calcification of vascular smooth muscle cells (VSMCs).Methods: In vitro, human aortic smooth muscle cells (HASMCs) were incubated in osteogenic medium (OM) with advanced glycation end products (AGEs) and LX to further determine the underlying mechanisms. An in vivo diabetic mouse model was established using a combination of a high-fat diet and multiple formulations of low-dose streptozotocin (STZ). Cell culture, alkaline phosphatase (ALP) staining, ALP activity, Alizarin red staining, von kossa staining, determination of calcium content, western blot analysis, immunohistochemistry, and immunofluorescence staining and statistical analysis were used in our study. Results: AGEs dose-dependently induced calcification and expression of osteogenesis-related markers, including Runt-related transcription factor 2 (RUNX2), osteopontin (OPN), and type I collagen (COL1), coupled with the activation of yes-associated protein (YAP). Mechanistically, YAP activation enhanced the AGE-induced osteogenic phenotype and calcification, but inhibition of YAP signalling alleviated this trend. Consistent with the in vitro results, diabetes promoted YAP expression as well as the subcellular localisation of the protein in the nucleus in the arterial tunica media. Interestingly, treatment with LX reduced vascular osteogenesis and calcification in diabetic mice, which was correlated with the reduced YAP levels. In addition, LX significantly inhibited COL1 accumulation and modulated the extracellular matrix. Our results further demonstrated that a pharmacological agonist of YAP reversed LX-mediated protection against osteogenic phenotypic conversion and calcification in VSMCs.Conclusions: These results demonstrate that LX attenuates transdifferentiation and calcification of VSMCs in diabetes mellitus via the YAP signalling axis, suggesting that LX is a potent therapeutic strategy to prevent diabetic vascular calcification.

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Abstract 418: Co-Culture of Endothelial Cells, Smooth Muscle Cells and Monocytes in Collagen Scaffold: A Novel i n vitro Model of Atherosclerosis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.418 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Martin Liu ◽

Angelos Karagiannis ◽

Matthew Sis ◽

Srivatsan Kidambi ◽

Yiannis Chatzizisis

Keyword(s):

Endothelial Cells ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Type I Collagen ◽

Smooth Muscle Actin ◽

Muscle Cells ◽

Type I ◽

Collagen Gels ◽

Human Coronary Artery

Objectives: To develop and validate a 3D in-vitro model of atherosclerosis that enables direct interaction between various cell types and/or extracellular matrix. Methods and Results: Type I collagen (0.75 mg/mL) was mixed with human artery smooth muscle cells (SMCs; 6x10 5 cells/mL), medium, and water. Human coronary artery endothelial cells (HCAECs; 10 5 /cm 2 ) were plated on top of the collagen gels and activated with oxidized low density lipoprotein cholesterol (LDL-C). Monocytes (THP-1 cells; 10 5 /cm 2 ) were then added on top of the HCAECs. Immunofluorescence showed the expression of VE-cadherin by HCAECs (A, B) and α-smooth muscle actin by SMCs (A). Green-labelled LDL-C particles were accumulated in the subendothelial space, as well as in the cytoplasm of HCAECs and SMCs (C). Activated monocytes were attached to HCAECs and found in the subendothelial area (G-I). Both HCAECs and SMCs released IL-1β, IL-6, IL-8, PDGF-BB, TGF-ß1, and VEGF. Scanning and transmission electron microscopy showed the HCAECs monolayer forming gap junctions and the SMCs (D-F) and transmigrating monocytes within the collagen matrix (G-I). Conclusions: In this work, we presented a novel, easily reproducible and functional in-vitro experimental model of atherosclerosis that has the potential to enable in-vitro sophisticated molecular and drug development studies.

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Overexpression of Decorin by Rat Arterial Smooth Muscle Cells Enhances Contraction of Type I Collagen In Vitro

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/01.atv.0000107026.98626.3b ◽

2004 ◽

Vol 24 (1) ◽

pp. 67-72 ◽

Cited By ~ 25

Author(s):

Hannu Järveläinen ◽

Robert B. Vernon ◽

Michel D. Gooden ◽

Aleksandar Francki ◽

Stephanie Lara ◽

...

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Type I Collagen ◽

Muscle Cells ◽

Type I ◽

Arterial Smooth Muscle ◽

Arterial Smooth Muscle Cells

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Mechanical Stress-Induced Orientation and Ultrastructural Change of Smooth Muscle Cells Cultured in Three-Dimensional Collagen Lattices

Cell Transplantation ◽

10.1177/096368979400300605 ◽

1994 ◽

Vol 3 (6) ◽

pp. 481-492 ◽

Cited By ~ 104

Author(s):

Keiichi Kanda ◽

Takehisa Matsuda

Keyword(s):

Smooth Muscle ◽

Tensile Stress ◽

Smooth Muscle Cells ◽

Type I Collagen ◽

Dynamic Stress ◽

Three Dimensional ◽

Muscle Cells ◽

The Other ◽

Type I ◽

Oriented Parallel

The effect of tensile stress on the orientation and phenotype of arterial smooth muscle cells (SMCs) cultured in three-dimensional (3D) type I collagen gels was morphologically investigated. Ring-shaped hybrid tissues were prepared by thermal gelation of a cold mixed solution of type I collagen and SMCs derived from bovine aorta. The tissues were subjected to three different modes of tensile stress. They were floated (isotonic control), stretched isometrically (static stress) and periodically stretched and recoiled by 5% above and below the resting tissue length at 60 RPM frequency (dynamic stress). After incubation for up to four wk, the tissues were investigated under a light microscope (LM) and a transmission electron microscope (TEM). Hematoxylin and eosinstained LM samples revealed that, irrespective of static or dynamic stress loading, SMCs in stress-loaded tissues exhibited elongated bipolar spindle shape and were regularly oriented parallel to the direction of the strain, whereas those in isotonic control tissues were polygonal or spherical and had no preferential orientation. In Azan-stained samples, collagen fiber bundles in isotonic control tissues were somewhat retracted around the polygonal SMCs to form a random network. On the other hand, those in statically and dynamically stressed tissues were accumulated and prominently oriented parallel to the stretch direction. Ultrastructural investigation using a TEM showed that SMCs in control and statically stressed tissues were almost totally filled with synthetic organelles such as rough endoplasmic reticulums, free ribosomes, Golgi complexes and mitochondria, indicating that the cells remained in the synthetic phenotype. On the other hand, SMCs in dynamically stressed tissues had increased fractions of contractile apparatus, such as myofilaments, dense bodies and extracellular filamentous materials equivalent to basement membranes, that progressed with incubation time. These results indicate that periodic stretch, in concert with 3-D extracellular collagen matrices, play a significant role in the phenotypic modulation of SMCs from the synthetic to the contractile state, as well as cellular and biomolecular orientation.

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

10.21203/rs.3.rs-172981/v1 ◽

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.

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IL-8 reduces VCAM-1 secretion of smooth muscle cells by increasing p-ERK expression when 3-D co-cultured with vascular endothelial cells

Clinical & Investigative Medicine ◽

10.25011/cim.v34i3.15186 ◽

2011 ◽

Vol 34 (3) ◽

pp. 138 ◽

Cited By ~ 5

Author(s):

Zhi Zhang ◽

Guang Chu ◽

Hong-Xian Wu ◽

Ni Zou ◽

Bao-Gui Sun ◽

...

Keyword(s):

Endothelial Cells ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Type I Collagen ◽

Vascular Endothelial Cells ◽

Umbilical Vein ◽

Muscle Cells ◽

Type I ◽

Vascular Endothelial ◽

Culture Model

Objective: The goal of this study was to investigate the crosstalk between vascular endothelial cells (ECs) and smooth muscle cells (SMCs) using a three-dimensional (3-D) co-culture model. In addition, the role of IL-8 in this crosstalk was investigated. Methods: A 3-D co-culture model was constructed using a Transwell chamber system and type I collagen gel. Human umbilical artery smooth muscle cells (HUASMCs) were suspended in the gel and added to the upper compartment of the Transwell. Human umbilical vein endothelial cells (HUVECs) were then grown on the surface of the gel. The growth of HUASMCs was tested with a CFDA SE cell proliferation kit. IL-8 and other bioactive substances were investigated by ELISA and real-time PCR. The alteration of p-ERK expression related to the change in IL-8 levels was also examined by Western blot analysis. Results: The proliferation rate of HUASMCs in the 3-D co-culture model was 0.679 ± 0.057. Secretion and transcription of VEGF, t-PA, NO and VCAM-1 in the 3-D co-culture model were different than in single (2-D) culture. When 3-D co-cultured, IL-8 released by HUVECs was significantly increased (2.35 ± 0.16 fold) (P﹤0.05) and the expression of VCAM-1 from HUASMCs was reduced accordingly (0.55±0.09 fold). In addition, increasing or decreasing the level of IL-8 changed the level of p-ERK and VCAM-1 expression. The reduction of VCAM-1, resulting from increased IL-8, could be blocked by the MEK inhibitor, PD98059. Conclusion: Crosstalk between HUVECs and HUASMCs occurred and was probably mediated by IL-8 in this 3-D co-culture model.

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

10.21203/rs.3.rs-152330/v1 ◽

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.

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

Frontiers in Physiology ◽

10.3389/fphys.2021.676727 ◽

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.

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

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfab090.003 ◽

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.

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