A Novel Ex Vivo Model of Aortic Valve Calcification. A Preliminary Report

Background: No pharmacological treatment exists to prevent or stop the calcification process of aortic valves causing aortic stenosis. The aim of this study was to develop a robust model of induced calcification in whole aortic valve leaflets which could be suitable for studies of the basic mechanisms and for testing potentially inhibitory drugs.Methods: Pig hearts were obtained from a commercial abattoir. The aortic valve leaflets were dissected free and randomized between experimental groups. Whole leaflets were cultured in individual wells. Two growth media were used for cultivation: standard growth medium and an antimyofibroblastic growth medium. The latter was employed to inhibit contraction of the leaflet into a ball-like structure. Calcification was induced in the growth medium by supplementation with an osteogenic medium. Leaflets were cultivated for four weeks and medium was changed every third day. To block calcification, the inhibitor SNF472 (a formulation of the hexasodium salt of myo-inositol hexaphosphate hexasodium salt) was used at concentrations between 1 and 100 µM. After cultivation for four weeks the leaflets were snap frozen in liquid nitrogen and kept at −80 °C until blind assessment of the calcium amount in leaflets by inductively coupled plasma optical emission spectroscopy. For statistical analysis, a Kruskal–Wallis test with Dunn’s post-test was applied.Results: Osteodifferentiation with calcium accumulation was in principle absent when standard medium was used. However, when the antimyofibroblastic medium was used, a strong calcium accumulation was induced (p = 0.006 compared to controls), and this was blocked in a dose-dependent manner by the calcification inhibitor SNF472 (p = 0.008), with an EC50 of 3.3 µM.Conclusion: A model of experimentally induced calcification in cultured whole leaflets from porcine aortic valves was developed. This model can be useful for studying the basic mechanisms of valve calcification and to test pharmacological approaches to inhibit calcification.

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The Congenital Bicuspid Aortic Valve Can Experience Fluid Shear Stresses Associated With Sclerosis

ASME 2011 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2011-53926 ◽

2011 ◽

Author(s):

Choon Hwai Yap ◽

Neelakantan Saikrishnan ◽

Swetha Rathan ◽

Gowthami Tamilselvan ◽

Nikolay V. Vasilyev ◽

...

Keyword(s):

Aortic Valve ◽

Ex Vivo ◽

The Elderly ◽

Shear Stresses ◽

Dependent Manner ◽

Aortic Valve Calcification ◽

Fluid Shear ◽

Valve Calcification ◽

Mechanical Environment ◽

High Prevalence

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Fluid Shear Stress Characteristics of the Ventricular Surface Versus the Aortic Surface of the Aortic Valve: An In Vitro Study

ASME 2011 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2011-53931 ◽

2011 ◽

Author(s):

Choon Hwai Yap ◽

Neelakantan Saikrishnan ◽

Gowthami Tamilselvan ◽

Ajit P. Yoganathan

Keyword(s):

Aortic Valve ◽

Fluid Shear Stress ◽

Ex Vivo ◽

Shear Stresses ◽

Dependent Manner ◽

Aortic Valve Calcification ◽

Fluid Shear ◽

Valve Calcification ◽

Ventricular Surface

Aortic valve calcification is a degenerative disease with high prevalence, especially amongst the elderly, and is a major cause of morbidity and mortality. Ex vivo experiments has shown that aortic valve leaflets are sensitive to their mechanical environment in a magnitude dependent manner. Fluid shear stresses, specifically, has been shown to affect inflammatory and remodeling responses relevant to aortic valve calcification [1,2]. Consequently, it has been proposed that the phenomenon of diseased calcium nodules developing exclusively on the aortic surface as opposed to the ventricular surface is due to the exposure of the aortic surface to disturbed shear stresses, whereas undisturbed shear stresses on the ventricular surface do not trigger calcification [3,4]. Additionally, it has been observed that the non-coronary leaflet of the AV is more susceptible to calcification, which is hypothesized to be due to reduced shear stresses from the lack of diastolic coronary flow [5].

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An Assessment of Ca-Naphthenate-Epon Mixtures as Standards for Calcium Quantification in Aortic Valve Leaflets

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100054558 ◽

1982 ◽

Vol 40 ◽

pp. 390-391

Author(s):

Kathryn N. Colonna ◽

Sydney S. Breese ◽

Susan C. Sellers ◽

J. David Deck

Keyword(s):

Aortic Valve ◽

Aortic Valves ◽

X Ray ◽

Aortic Valve Leaflets

Qualitative x-ray microanalytical studies used to demonstrate calcium in bioprosthetic aortic valves have shown that it occurs in a range of morphological forms. A consistent and reproducible standard for measuring calcium was necessary to investigate whether these forms represented varying concentrations of calcium. To provide such a standard, we tested a series of calcium naphthenate-epon mixtures.

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Ozaki procedure: Ex vivo simulation

10.1510/mmcts.2021.075 ◽

2021 ◽

Keyword(s):

Aortic Valve ◽

Ex Vivo ◽

Aortic Valves ◽

Autologous Pericardium ◽

Video Tutorial ◽

Novel Technique ◽

Valve Reconstruction

Replacements for diseased aortic valves are limited. Repair of the aortic valve is performed by only a few surgeons. A novel technique of aortic valve reconstruction using autologous pericardium shows promising results. In this video tutorial, we demonstrate the Ozaki procedure using an ex vivo low fidelity simulation.

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Abstract 295: Hemodynamic Shear Stress Alterations Contribute to Calcific Aortic Valve Disease in a Frequency- and Magnitude-Dependent Manner

Circulation Research ◽

10.1161/res.111.suppl_1.a295 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Ling Sun ◽

John LeCluyse ◽

Brian Robillard ◽

Philippe Sucosky

Keyword(s):

Shear Stress ◽

Aortic Valve ◽

Aortic Valve Disease ◽

Ex Vivo ◽

Fold Increase ◽

Endothelial Activation ◽

Valve Disease ◽

Dependent Manner ◽

Molecular Signaling ◽

Calcific Aortic Valve Disease

INTRODUCTION: Calcific aortic valve disease (CAVD) is an active process presumably triggered by interplays between atherogenic risk factors, molecular signaling networks and hemodynamic cues. While our earlier work demonstrated that progressive alterations in fluid wall-shear stress (WSS) on the fibrosa could trigger leaflet inflammation, the mechanisms of CAVD pathogenesis secondary to side-specific WSS abnormalities are poorly understood. HYPOTHESIS: Supported by our previous studies, we hypothesize that valve leaflets are sensitive to both WSS magnitude and pulsatility and that abnormalities in either promote CAVD development. OBJECTIVE: This study aims at elucidating ex vivo the contribution of isolated and combined alterations in WSS magnitude and pulsatility to valvular calcification. METHODS: The fibrosa and ventricularis of porcine leaflets were subjected simultaneously to different combinations of WSS magnitude and pulsatility (i.e., physiologic, sub- and supra-physiologic levels) for 48 hours in a double-sided shear stress bioreactor. Endothelial activation (ICAM-1, VCAM-1), paracrine expression (TGF-β and BMP-4), and proteinase/collagenase expression (MMP-2, cathepsin L) were detected by immunohistochemistry, while osteogenic differentiation (α-SMA) was assessed via western blot. RESULTS: Regardless of the magnitude or frequency, non-physiologic WSS conditions did not result in endothelial activation. Tissue exposure to either supra-physiologic WSS magnitude or pulsatility significantly upregulated paracrine (74-fold increase), proteinase (4-fold increase), collagenase (5-fold increase) and α-SMA (23-fold increase) expressions relative to the levels measured under physiologic WSS. In contrast, combined alterations in WSS magnitude and pulsatility downregulated those responses. CONCLUSION: This study demonstrates the sensitivity of aortic valve leaflets to both WSS magnitude and pulsatility and the ability of supra-physiologic WSS magnitude or pulsatility to trigger events involved in early CAVD pathogenesis. The results provide new potential insights into the mechanisms of CAVD secondary to hypertension and Paget’s disease, which are associated with abnormal blood flow and leaflet WSS.

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P4662Advanced glycation end products accumulate in aortic valve tissue during calcification development in the absence of diabetes mellitus. A spectroscopic study

European Heart Journal ◽

10.1093/eurheartj/ehz745.1044 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

N Anousakis-Vlachochristou ◽

K Toutouzas ◽

M Kyriakidou ◽

E Varela ◽

A Kapelouzou ◽

...

Keyword(s):

Aortic Valve ◽

Control Group ◽

Aortic Valve Calcification ◽

Aortic Valves ◽

Valve Calcification ◽

Valve Tissue ◽

Glycation End Products ◽

End Products ◽

Ft Ir

Abstract Background Advanced glycation end products (AGPs) promote human aortic smooth muscle cell calcification in vitro. Moreover, reduction of AGPs levels and inhibition of RAGE signaling decrease vascular calcification in vivo in animal studies. The role of AGPs in aortic valve calcification has not been investigated. Purpose We sought to investigate the role of AGPs in aortic valve calcification, in the absence of diabetes mellitus (DM). Methods We used human and animal cohorts. Firstly, we obtained aortic valves from patients without DM that underwent aortic valve replacement due to aortic valve stenosis. We studied the valves with Fourier-Transformed Infrared spectroscopy (FT-IR, Nicolet 6700 spectrometer, with Attenuated Total Reflection-ATR accessory, each spectrum consisted of 120 co-added spectra) in order to evaluate chemical changes. In the animal cohort, New Zealand male rabbits where randomized in calcification diet (normal chow+cholesterol 0,5%+3500 IU ergocalciferol/kg daily) and control group and sacrificed at 2, 4, 6, 8 10 and 12 weeks. The valves were longitudinally assessed with FT-IR. Results A total of 200 human aortic valves were studied (age 64–78). All patients demonstrated characteristic vibrations at the area about 1165 cm-1, where the C-O-C bonds absorb, attributed to AGPs. Thirty six rabbit valves were used, 3 per group. Glucose levels were within normal range and did not differ between groups. The FT-IR spectra of the rabbit aortic valves showed increasing intensity of the C-O-C band at 1165 cm-1 in experimental group in comparison to control group. The band at 1744 cm-1 is attributed to aldehyde formation due to oxidative stress and inflammation. Shifts and shape changes were detected at the bands of amide I and II at 1650 cm-1 and 1550 cm-1, respectively, concerning protein misfolding, fiber formation and sclerosis. The bands in the region 1299–900 cm-1 correspond to phosphate groups of phospholipidsand the formed calcium phosphate salts and non-biological hydroxyapatite Ca3(PO4)2 formation. All vibrations increased significantly longitudinally during experimental diet period. Representative FT-IR spectra of valves Conclusions Advanced glycation end products are detected in human calcified aortic valves irrespectively of DM. Moreover, AGPs correlate with presence and gradual development of aortic valve calcification in experimental rabbit model, along with acidosis, oxidation and protein secondary misfolding. Accumulation of AGPs in valve tissue is implicated in mechanisms of disease development.

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Citrullinated vimentin mediates development and progression of lung fibrosis

Science Translational Medicine ◽

10.1126/scitranslmed.aba2927 ◽

2021 ◽

Vol 13 (585) ◽

pp. eaba2927

Author(s):

Fu Jun Li ◽

Ranu Surolia ◽

Huashi Li ◽

Zheng Wang ◽

Gang Liu ◽

...

Keyword(s):

Lung Tissue ◽

Lung Fibrosis ◽

Ex Vivo ◽

Lavage Fluid ◽

Surface Expression ◽

Lung Fibroblasts ◽

Dependent Manner ◽

Ex Vivo Model ◽

Molecular Pattern ◽

Citrullinated Vimentin

The mechanisms by which environmental exposures contribute to the pathogenesis of lung fibrosis are unclear. Here, we demonstrate an increase in cadmium (Cd) and carbon black (CB), common components of cigarette smoke (CS) and environmental particulate matter (PM), in lung tissue from subjects with idiopathic pulmonary fibrosis (IPF). Cd concentrations were directly proportional to citrullinated vimentin (Cit-Vim) amounts in lung tissue of subjects with IPF. Cit-Vim amounts were higher in subjects with IPF, especially smokers, which correlated with lung function and were associated with disease manifestations. Cd/CB induced the secretion of Cit-Vim in an Akt1- and peptidylarginine deiminase 2 (PAD2)–dependent manner. Cit-Vim mediated fibroblast invasion in a 3D ex vivo model of human pulmospheres that resulted in higher expression of CD26, collagen, and α-SMA. Cit-Vim activated NF-κB in a TLR4-dependent fashion and induced the production of active TGF-β1, CTGF, and IL-8 along with higher surface expression of TLR4 in lung fibroblasts. To corroborate ex vivo findings, mice treated with Cit-Vim, but not Vim, independently developed a similar pattern of fibrotic tissue remodeling, which was TLR4 dependent. Moreover, wild-type mice, but not PAD2−/− and TLR4 mutant (MUT) mice, exposed to Cd/CB generated high amounts of Cit-Vim, in both plasma and bronchoalveolar lavage fluid, and developed lung fibrosis in a stereotypic manner. Together, these studies support a role for Cit-Vim as a damage-associated molecular pattern molecule (DAMP) that is generated by lung macrophages in response to environmental Cd/CB exposure. Furthermore, PAD2 might represent a promising target to attenuate Cd/CB-induced fibrosis.

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Ex vivo Model of Human Aortic Valve Bacterial Colonization

BIO-PROTOCOL ◽

10.21769/bioprotoc.2316 ◽

2017 ◽

Vol 7 (11) ◽

Author(s):

Alejandro Avilés-Reyes ◽

Irlan Freires ◽

Pedro Rosalen ◽

José Lemos ◽

Jacqueline Abranches

Keyword(s):

Aortic Valve ◽

Ex Vivo ◽

Bacterial Colonization ◽

Ex Vivo Model ◽

Human Aortic Valve

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The influence of bicuspid aortic valves on the dynamic pressure distribution in the ascending aorta – a Porcine Ex-vivo Model

The Thoracic and Cardiovascular Surgeon ◽

10.1055/s-0032-1332378 ◽

2013 ◽

Vol 61 (S 01) ◽

Cited By ~ 1

Author(s):

A Juraszek ◽

T Dziodzio ◽

M Stoiber ◽

D Fechtig ◽

V Gschlad ◽

...

Keyword(s):

Pressure Distribution ◽

Ex Vivo ◽

Dynamic Pressure ◽

Ascending Aorta ◽

Ex Vivo Model ◽

Aortic Valves ◽

Bicuspid Aortic Valves

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Abstract 6147: A Tissue Stem Cell Niche Regulates Bicuspid Aortic Valve Disease

Circulation ◽

10.1161/circ.118.suppl_18.s_1071 ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Nalini M Rajamannan

Keyword(s):

Stem Cell ◽

Aortic Valve ◽

Bicuspid Aortic Valve ◽

Cross Talk ◽

Aortic Valve Disease ◽

Stem Cell Niche ◽

Ex Vivo ◽

Valve Disease ◽

Aortic Valves ◽

Cell Niche

Bicuspid aortic valve disease is the most common congenital cardiac malformation and indication for surgery for aortic valve patients. The age of onset for the development of stenosis is earlier in bicuspid aortic valves than tricuspid aortic valves. Understanding the cellular mechanisms of this valve lesion, will help to develop novel approaches towards slowing disease progression. This study hypothesizes that bicuspid aortic valve disease develops secondary to a tissue stem cell niche to activate a cross-talk mechanism which induces Notch1 cleavage and Lrp5 mediated bone formation specific to the bicuspid aortic valve. Human ex vivo bicuspid valves versus control aortic valves were tested for Notch1 expression by RTPCR, Western Blot and Immunohistochemistry. eNOS null bicuspid mice: control (n=20), cholesterol (n=20), cholesterol + Atorvastatin (n=20), were tested for the development of aortic stenosis by Visual Sonics Echo, Immunohistochemistry for Notch1, Wnt, Lrp5, Osteocalcin, PCNA and RTPCR for Notch1, Lrp5, Cbfa1, Osteocalcin. In vitro studies were performed to characterize Wnt secretion from aortic valve endothelial cells and gene expression for Notch1, Lp5 and osteocalcin from the valve myofibroblast cells. This study characterizes the secretion of Wnt3a (>300-fold, p<0.01) from aortic valve endothelium in the presence of abnormal nitric oxide regulation and lipids as measured by eNOS enzymatic activity and tissue nitrite levels. Osteoblastogenesis in the adjacent myofibroblast cell is activated via Notch1 cleavage(p<0.001) and upregulation of the Wnt3a/Lrp5 receptor. Human ex vivo valves express Notch1 cleavage as compared to normal valves from heart transplant(p<0.01). Cholesterol treated eNOS mice develop severe stenosis with cleavage of Notch1, increase in Lrp5, Wnt3a, cyclin, Cbfa1, and Osteopontin,(3-fold increase(p<0.01) which was not present in the controls and normalized in the statin treated valves. Targeting the Notch1/Wnt3a/Lrp5 pathway in bicuspid valvular calcification presents a novel approach towards treating this disease. The importance of this cross talk mechanism is demonstrated in three models of aortic valve disease and will have important clinical implications. This research has received full or partial funding support from the American Heart Association, AHA National Center.

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