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 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.

scholarly journals Stressor-Induced “Inflammaging” of Vascular Smooth Muscle Cells via Nlrp3-Mediated Pro-inflammatory Auto-Loop

2021 ◽  
Vol 8 ◽  
Author(s):  
Jaqueline Herrmann ◽  
Mengdi Xia ◽  
Manasa Reddy Gummi ◽  
Anna Greco ◽  
Annika Schacke ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Cardiovascular Mortality ◽  
Ex Vivo ◽  
Muscle Cells ◽  
Sterile Inflammation ◽  
Subsequent Reduction

Calcification of the vessel wall as one structural pathology of aged vessels is associated with high cardiovascular mortality of elderly patients. Aging is linked to chronic sterile inflammation and high burden of reactive oxygen species (ROS), leading to activation of pattern recognition receptors (PRRs) such as Nlrp3 in vascular cells. The current study investigates the role of PRR activation in the calcification of vascular smooth muscle cells (VSMCs). Therefore, in vitro cell culture of primary rat VSMCs and ex vivo aortic stimulations were used to analyze osteogenic, senescence and inflammatory markers via real-time PCR, in situ RNA hybridization, Western Blot, photometric assays and histological staining. Induction of ROS and DNA-damage by doxorubicin induces a shift of VSMC phenotype toward the expression of osteogenic, senescence and inflammatory proteins. Induction of calcification is dependent on Nlrp3 activity. Il-1β as a downstream target of Nlrp3 induces the synthetic, pro-calcifying VSMC phenotype. Inhibition of PRR with subsequent reduction of chronic inflammation might be an interesting target for reduction of calcification of VSMCs, with subsequent reduction of cardiovascular mortality of patients suffering from vessel stiffness.

Expanded Haemodialysis Therapy of Chronic Haemodialysis Patients Prevents Calcification and Apoptosis of Vascular Smooth Muscle Cells in vitro

2017 ◽  
Vol 45 (1-3) ◽  
pp. 131-138 ◽  
Author(s):  
Kevin Willy ◽  
Matthias Girndt ◽  
Jakob Voelkl ◽  
Roman Fiedler ◽  
Peter Martus ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Calcification ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Dialysis Patients ◽  
Serum Samples ◽  
Dialysis Membranes

Background: Vascular calcification is a common phenomenon in patients with chronic kidney disease and strongly associated with increased cardiovascular mortality. Vascular calcification is an active process mediated in part by inflammatory processes in vascular smooth muscle cells (VSMC). These could be modified by the insufficient removal of proinflammatory cytokines through conventional high-flux (HF) membranes. Recent trials demonstrated a reduction of inflammation in VSMC by use of dialysis membranes with a higher and steeper cut-off. These membranes caused significant albumin loss. Therefore, the effect of high retention Onset (HRO) dialysis membranes on vascular calcification and its implications in vitro was evaluated. Methods: In the PERCI II trial, 48 chronic dialysis patients were dialyzed using HF and HRO dialyzers and serum samples were collected. Calcifying VSMC were incubated with the serum samples. Calcification was determined using alizarin red staining (AZR) and determination of alkaline phosphatase (ALP) activity. Furthermore, apoptosis was evaluated, and release of matrix Gla protein (MGP), osteopontin (OPN) and growth differentiation factor 15 (GDF-15) were measured in cell supernatants. Results: Vascular calcification in vitro was significantly reduced by 24% (ALP) and 36% (AZR) after 4 weeks of HRO dialysis and by 33% (ALP) and 48% (AZR) after 12 weeks of dialysis using HRO membranes compared to HF dialysis. Apoptosis was significantly lower in the HRO group. The concentrations of MGP and OPN were significantly elevated after incubation with HF serum compared to HRO serum and healthy controls. Similarly, GDF-15 release in the supernatant was elevated after incubation with HF serum, an effect significantly ameliorated after treatment with HRO medium. Conclusions: Expanded haemodialysis therapy reduces the pro-calcific potential of serum from dialysis patients in vitro. With a markedly reduced albumin filtration compared to high cut-off dialysis, use of the HRO dialyzers may possibly provide a treatment option for chronic dialysis patients to reduce the progression of vascular calcification.

scholarly journals Dependence of proliferative vascular smooth muscle cells on CD98hc (4F2hc, SLC3A2)

2009 ◽  
Vol 206 (11) ◽  
pp. 2397-2406 ◽  
Author(s):  
Per Fogelstrand ◽  
Chloé C. Féral ◽  
Ramin Zargham ◽  
Mark H. Ginsberg
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Intimal Hyperplasia ◽  
Ex Vivo ◽  
Muscle Cells ◽  
Amino Acid Transporters ◽  
Vessel Morphology

Activation of vascular smooth muscle cells (VSMCs) to migrate and proliferate is essential for the formation of intimal hyperplasia. Hence, selectively targeting activated VSMCs is a potential strategy against vaso-occlusive disorders such as in-stent restenosis, vein-graft stenosis, and transplant vasculopathy. We show that CD98 heavy chain (CD98hc) is markedly up-regulated in neointimal and cultured VSMCs, and that activated but not quiescent VSMCs require CD98hc for survival. CD98hc mediates integrin signaling and localizes amino acid transporters to the plasma membrane. SMC-specific deletion of CD98hc did not affect normal vessel morphology, indicating that CD98hc was not required for the maintenance of resident quiescent VSMCs; however, CD98hc deletion reduced intimal hyperplasia after arterial injury. Ex vivo and in vitro, loss of CD98hc suppressed proliferation and induced apoptosis in VSMCs. Furthermore, reconstitution with CD98hc mutants showed that CD98hc interaction with integrins was necessary for the survival of VSMCs. These studies establish the importance of CD98hc in VSMC proliferation and survival. Furthermore, loss of CD98hc was selectively deleterious to activated VSMCs while sparing resident quiescent VSMCs, suggesting that activated VSMCs are physiologically dependent on CD98hc, and hence, CD98hc is a potential therapeutic target in vaso-occlusive disorders.

Intracellular Na+ regulation of Na+ pump sites in cultured vascular smooth muscle cells

1989 ◽  
Vol 256 (4) ◽  
pp. C786-C792 ◽  
Author(s):  
J. C. Allen ◽  
S. S. Navran ◽  
C. L. Seidel ◽  
D. K. Dennison ◽  
J. M. Amann ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Muscle Cells ◽  
Ouabain Binding ◽  
Na Pump ◽  
Site Number

Enzymatically dispersed cells from canine saphenous vein and femoral artery were grown in fetal calf serum and studied at day 0 (freshly dispersed) through confluence in primary culture. Intracellular Na levels (Nai), but not intracellular K (Ki), were increased after 24 h in culture and then decreased to a steady state by 4 days. Na+ pump site number [( 3H] ouabain binding) increased through day 3 and remained elevated. Nai was still elevated at 2 days when the Na+ pump site number began to increase. Total pump turnover (maximum ouabain-inhibited 86Rb uptake) reflected the increase in Na+ pump site number. These key events precede the observed increases in both protein production and cellular proliferation. If the same cells are maintained in defined medium, without fetal calf serum, Nai, Ki, and the number of [3H]ouabain binding sites do not change with time. These data are consistent with the suggestion that the initial mitogenic response of vascular smooth muscle cells to fetal calf serum involves an increased Na+ influx, and a Nai accumulation, caused by low Na+ pump density. The synthesis of new pump sites effects a decrease in the accumulated Nai, which may be related to cell proliferation.

scholarly journals Dysfunctional vascular smooth muscle cells mediate early and late-stage neuroinflammation and Tau hyperphosphorylation

2021 ◽  
Author(s):  
Jorge A. Aguilar-Pineda ◽  
Karin J. Vera-Lopez ◽  
Pallavi Shrivastava ◽  
Rita Nieto-Montesinos ◽  
Miguel A. Chávez-Fumagalli ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Ex Vivo ◽  
Muscle Cells ◽  
Dependent Manner ◽  
Tau Hyperphosphorylation

SUMMARYDespite the emerging evidence implying early vascular contributions to neurogenerative syndromes, the role of vascular smooth muscle cells (VSMCs) in the pathogenesis of Alzheimer’s disease is still not well understood. Herein, we show that VSMCs in brains of AD patients and the animal model of the disease, are deficient in multiple VSMC-contractile markers which correlated with Tau accumulation in brain arterioles. Ex vivo and in vitro experiments demonstrated that VSMCs undergo dramatic phenotypic transitions under AD-like conditions, adopting pro-inflammatory and synthetic phenotypes. Notably, these changes coincided with Tau hyperphosphorylation at residues Y18, T205 and S262. We also observed that loss of VSMC markers occurred in an age-dependent manner, and that expression of Sm22α and α-Sma proteins were inversely correlated with CD68 and Tau accumulation in brain arterioles of 3xTg-AD mice. Together, these findings further support the contribution of VSMCs in AD pathogenesis, and nominate VSMCs as potential novel therapeutic target in AD.Graphical Abstract

scholarly journals Development of the BioHybrid Assay: Combining Primary Human Vascular Smooth Muscle Cells and Blood to Measure Vascular Calcification Propensity

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2097
Author(s):  
Armand M. G. Jaminon ◽  
Asim C. Akbulut ◽  
Niko Rapp ◽  
Rafael Kramann ◽  
Erik A. L. Biessen ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Calcification ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Matrix Gla Protein ◽  
Plasma Samples ◽  
Cvd Risk

Background: Vascular calcification is an active process that increases cardiovascular disease (CVD) risk. There is still no consensus on an appropriate biomarker for vascular calcification. We reasoned that the biomarker for vascular calcification is the collection of all blood components that can be sensed and integrated into a calcification response by human vascular smooth muscle cells (hVSMCs). Methods: We developed a new cell-based high-content assay, the BioHybrid assay, to measure in vitro calcification. The BioHybrid assay was compared with the o-Cresolphthalein assay and the T50 assay. Serum and plasma were derived from different cohort studies including chronic kidney disease (CKD) stages III, IV, V and VD (on dialysis), pseudoxanthoma elasticum (PXE) and other cardiovascular diseases including serum from participants with mild and extensive coronary artery calcification (CAC). hVSMCs were exposed to serum and plasma samples, and in vitro calcification was measured using AlexaFluor®-546 tagged fetuin-A as calcification sensor. Results: The BioHybrid assay measured the kinetics of calcification in contrast to the endpoint o-Cresolphthalein assay. The BioHybrid assay was more sensitive to pick up differences in calcification propensity than the T50 assay as determined by measuring control as well as pre- and post-dialysis serum samples of CKD patients. The BioHybrid response increased with CKD severity. Further, the BioHybrid assay discriminated between calcification propensity of individuals with a high CAC index and individuals with a low CAC index. Patients with PXE had an increased calcification response in the BioHybrid assay as compared to both spouse and control plasma samples. Finally, vitamin K1 supplementation showed lower in vitro calcification, reflecting changes in delta Agatston scores. Lower progression within the BioHybrid and on Agatston scores was accompanied by lower dephosphorylated-uncarboxylated matrix Gla protein levels. Conclusion: The BioHybrid assay is a novel approach to determine the vascular calcification propensity of an individual and thus may add to personalised risk assessment for CVD.

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