Simulating early calcific aortic valve disease within novel in vitro 3D tissue platform

An insufficient understanding of calcific aortic valve disease (CAVD) pathogenesis remains a major obstacle in developing treatment strategies for this disease. The aim of the present study was to create engineered environments that mimic the earliest known features of CAVD and apply this in vitro platform to decipher relationships relevant to early valve lesion pathobiology. Glycosaminoglycan (GAG) enrichment is a dominant hallmark of early CAVD, but culture of valvular interstitial cells (VICs) in biomaterial environments containing pathological amounts of hyaluronic acid (HA) or chondroitin sulfate (CS) did not directly increase indicators of disease progression such as VIC activation or inflammatory cytokine production. However, HA-enriched matrices increased production of vascular endothelial growth factor (VEGF), while matrices displaying pathological levels of CS were effective at retaining lipoproteins, whose deposition is also found in early CAVD. Retained oxidized low-density lipoprotein (oxLDL), in turn, stimulated myofibroblastic VIC differentiation and secretion of numerous inflammatory cytokines. OxLDL also increased VIC deposition of GAGs, thereby creating a positive feedback loop to further enrich GAG content and promote disease progression. Using this disease-inspired in vitro platform, we were able to model a complex, multistep pathological sequence, with our findings suggesting distinct roles for individual GAGs in outcomes related to valve lesion progression, as well as key differences in cell–lipoprotein interactions compared with atherosclerosis. We propose a pathogenesis cascade that may be relevant to understanding early CAVD and envision the extension of such models to investigate other tissue pathologies or test pharmacological treatments.

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Reproducible In Vitro Tissue Culture Model to Study Basic Mechanisms of Calcific Aortic Valve Disease: Comparative Analysis to Valvular Interstitials Cells

Biomedicines ◽

10.3390/biomedicines9050474 ◽

2021 ◽

Vol 9 (5) ◽

pp. 474

Author(s):

Andreas Weber ◽

Melissa Pfaff ◽

Friederike Schöttler ◽

Vera Schmidt ◽

Artur Lichtenberg ◽

...

Keyword(s):

Tissue Culture ◽

Aortic Valve ◽

Aortic Valve Disease ◽

Valve Disease ◽

Natural Conditions ◽

Calcific Aortic Valve Disease ◽

Culture Model ◽

3D Environments ◽

Tissue Culture Model

The hallmarks of calcific aortic valve disease (CAVD), an active and regulated process involving the creation of calcium nodules, lipoprotein accumulation, and chronic inflammation, are the significant changes that occur in the composition, organization, and mechanical properties of the extracellular matrix (ECM) of the aortic valve (AV). Most research regarding CAVD is based on experiments using two-dimensional (2D) cell culture or artificially created three-dimensional (3D) environments of valvular interstitial cells (VICs). Because the valvular ECM has a powerful influence in regulating pathological events, we developed an in vitro AV tissue culture model, which is more closely able to mimic natural conditions to study cellular responses underlying CAVD. AV leaflets, isolated from the hearts of 6–8-month-old sheep, were fixed with needles on silicon rubber rings to achieve passive tension and treated in vitro under pro-degenerative and pro-calcifying conditions. The degeneration of AV leaflets progressed over time, commencing with the first visible calcified domains after 14 d and winding up with the distinct formation of calcium nodules, heightened stiffness, and clear disruption of the ECM after 56 d. Both the expression of pro-degenerative genes and the myofibroblastic differentiation of VICs were altered in AV leaflets compared to that in VIC cultures. In this study, we have established an easily applicable, reproducible, and cost-effective in vitro AV tissue culture model to study pathological mechanisms underlying CAVD. The valvular ECM and realistic VIC–VEC interactions mimic natural conditions more closely than VIC cultures or 3D environments. The application of various culture conditions enables the examination of different pathological mechanisms underlying CAVD and could lead to a better understanding of the molecular mechanisms that lead to VIC degeneration and AS. Our model provides a valuable tool to study the complex pathobiology of CAVD and can be used to identify potential therapeutic targets for slowing disease progression.

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Calcific Aortic Valve Disease: Molecular Mechanisms And Therapeutic Approaches

European Cardiology Review ◽

10.15420/ecr.2015.10.2.108 ◽

2015 ◽

Vol 10 (2) ◽

pp. 108 ◽

Cited By ~ 28

Author(s):

Daniel Alejandro Lerman ◽

Sai Prasad ◽

Nasri Alotti ◽

◽

...

Keyword(s):

Aortic Valve ◽

Aortic Valve Disease ◽

Molecular Mechanisms ◽

Cell Model ◽

Human Monoclonal Antibody ◽

Valve Disease ◽

Vascular Lesions ◽

Calcium Deposition ◽

Calcific Aortic Valve Disease

Calcification occurs in atherosclerotic vascular lesions and in the aortic valve. Calcific aortic valve disease (CAVD) is a slow, progressive disorder that ranges from mild valve thickening without obstruction of blood flow, termed aortic sclerosis, to severe calcification with impaired leaflet motion, termed aortic stenosis. In the past, this process was thought to be ‘degenerative’ because of time-dependent wear and tear of the leaflets, with passive calcium deposition. The presence of osteoblasts in atherosclerotic vascular lesions and in CAVD implies that calcification is an active, regulated process akin to atherosclerosis, with lipoprotein deposition and chronic inflammation. If calcification is active, via pro-osteogenic pathways, one might expect that development and progression of calcification could be inhibited. The overlap in the clinical factors associated with calcific valve disease and atherosclerosis provides further support for a shared disease mechanism. In our recent research we used an in vitro porcine valve interstitial cell model to study spontaneous calcification and potential promoters and inhibitors. Using this model, we found that denosumab, a human monoclonal antibody targeting the receptor activator of nuclear factor-κB ligand may, at a working concentration of 50 μg/mL, inhibit induced calcium deposition to basal levels.

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Two Photon Excited Fluorescence Microscopy Metrics are Sensitive to Early Phenotypic Changes in Calcific Aortic Valve Disease In Vitro and Ex Vivo

Structural Heart ◽

10.1080/24748706.2020.1717199 ◽

2020 ◽

Vol 4 (sup1) ◽

pp. 94-95

Author(s):

Ishita Tandon ◽

Kartik Balachandran

Keyword(s):

Aortic Valve ◽

Fluorescence Microscopy ◽

Aortic Valve Disease ◽

Ex Vivo ◽

Valve Disease ◽

Calcific Aortic Valve Disease ◽

Phenotypic Changes ◽

Two Photon

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Standardization of Human Calcific Aortic Valve Disease in vitro Modeling Reveals Passage-Dependent Calcification

Frontiers in Cardiovascular Medicine ◽

10.3389/fcvm.2019.00049 ◽

2019 ◽

Vol 6 ◽

Cited By ~ 17

Author(s):

Shinji Goto ◽

Maximillian A. Rogers ◽

Mark C. Blaser ◽

Hideyuki Higashi ◽

Lang H. Lee ◽

...

Keyword(s):

Aortic Valve ◽

Aortic Valve Disease ◽

Valve Disease ◽

Calcific Aortic Valve Disease ◽

In Vitro Modeling

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Human Aortic Valve Interstitial Cells Display Proangiogenic Properties During Calcific Aortic Valve Disease

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.120.314287 ◽

2020 ◽

Author(s):

Nicolas Gendron ◽

Mickael Rosa ◽

Adeline Blandinieres ◽

Yoann Sottejeau ◽

Elisa Rossi ◽

...

Keyword(s):

Aortic Valve ◽

Aortic Valve Disease ◽

Conditioned Media ◽

Valve Disease ◽

Calcific Aortic Valve Disease ◽

Aortic Valves ◽

Valve Interstitial Cells ◽

Endothelial Colony Forming Cells

Objective: The study’s aim was to analyze the capacity of human valve interstitial cells (VICs) to participate in aortic valve angiogenesis. Approach and Results: VICs were isolated from human aortic valves obtained after surgery for calcific aortic valve disease and from normal aortic valves unsuitable for grafting (control VICs). We examined VIC in vitro and in vivo potential to differentiate in endothelial and perivascular lineages. VIC paracrine effect was also examined on human endothelial colony-forming cells. A pathological VIC (VIC p ) mesenchymal-like phenotype was confirmed by CD90 + /CD73 + /CD44 + expression and multipotent-like differentiation ability. When VIC p were cocultured with endothelial colony-forming cells, they formed microvessels by differentiating into perivascular cells both in vivo and in vitro. VIC p and control VIC conditioned media were compared using serial ELISA regarding quantification of endothelial and angiogenic factors. Higher expression of VEGF (vascular endothelial growth factor)-A was observed at the protein level in VIC p -conditioned media and confirmed at the mRNA level in VIC p compared with control VIC. Conditioned media from VIC p induced in vitro a significant increase in endothelial colony-forming cell proliferation, migration, and sprouting compared with conditioned media from control VIC. These effects were inhibited by blocking VEGF-A with blocking antibody or siRNA approach, confirming VIC p involvement in angiogenesis by a VEGF-A dependent mechanism. Conclusions: We provide here the first proof of an angiogenic potential of human VICs isolated from patients with calcific aortic valve disease. These results point to a novel function of VIC p in valve vascularization during calcific aortic valve disease, with a perivascular differentiation ability and a VEGF-A paracrine effect. Targeting perivascular differentiation and VEGF-A to slow calcific aortic valve disease progression warrants further investigation.

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In vitro 3D model and miRNA drug delivery to target calcific aortic valve disease

Clinical Science ◽

10.1042/cs20160378 ◽

2017 ◽

Vol 131 (3) ◽

pp. 181-195 ◽

Cited By ~ 12

Author(s):

Casper F.T. van der Ven ◽

Pin-Jou Wu ◽

Mark W. Tibbitt ◽

Alain van Mil ◽

Joost P.G. Sluijter ◽

...

Keyword(s):

Drug Delivery ◽

Aortic Valve ◽

Aortic Valve Disease ◽

3D Model ◽

In Vitro Models ◽

Valve Disease ◽

Calcific Aortic Valve Disease ◽

Valve Interstitial Cells ◽

Delivery Strategies

Calcific aortic valve disease (CAVD) is the most prevalent valvular heart disease. Valve interstitial cells (VICs) mediate calcification in the aortic valve (AV) leaflets, leading to aortic stenosis (AS) and eventually heart failure. Aortic valve replacement (AVR) surgery is the only available treatment. Drug-based therapies and the in vitro models to study CAVD are inadequate or lacking. Here, we present a forward-looking review of 3D CAVD models, miRNA-based therapeutics and controlled drug-delivery strategies.

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Label-Free Multiphoton Microscopy for the Detection and Monitoring of Calcific Aortic Valve Disease

Frontiers in Cardiovascular Medicine ◽

10.3389/fcvm.2021.688513 ◽

2021 ◽

Vol 8 ◽

Author(s):

Ishita Tandon ◽

Kyle P. Quinn ◽

Kartik Balachandran

Keyword(s):

Aortic Valve ◽

Aortic Valve Disease ◽

Multiphoton Microscopy ◽

Valve Disease ◽

Label Free ◽

Calcific Aortic Valve Disease ◽

Treatment Standard ◽

Non Destructive

Calcific aortic valve disease (CAVD) is the most common valvular heart disease. CAVD results in a considerable socio-economic burden, especially considering the aging population in Europe and North America. The only treatment standard is surgical valve replacement as early diagnostic, mitigation, and drug strategies remain underdeveloped. Novel diagnostic techniques and biomarkers for early detection and monitoring of CAVD progression are thus a pressing need. Additionally, non-destructive tools are required for longitudinal in vitro and in vivo assessment of CAVD initiation and progression that can be translated into clinical practice in the future. Multiphoton microscopy (MPM) facilitates label-free and non-destructive imaging to obtain quantitative, optical biomarkers that have been shown to correlate with key events during CAVD progression. MPM can also be used to obtain spatiotemporal readouts of metabolic changes that occur in the cells. While cellular metabolism has been extensively explored for various cardiovascular disorders like atherosclerosis, hypertension, and heart failure, and has shown potential in elucidating key pathophysiological processes in heart valve diseases, it has yet to gain traction in the study of CAVD. Furthermore, MPM also provides structural, functional, and metabolic readouts that have the potential to correlate with key pathophysiological events in CAVD progression. This review outlines the applicability of MPM and its derived quantitative metrics for the detection and monitoring of early CAVD progression. The review will further focus on the MPM-detectable metabolic biomarkers that correlate with key biological events during valve pathogenesis and their potential role in assessing CAVD pathophysiology.

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Knockdown of CD44 expression decreases valve interstitial cell calcification in vitro

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00123.2018 ◽

2019 ◽

Vol 317 (1) ◽

pp. H26-H36 ◽

Cited By ~ 1

Author(s):

Lauren Baugh ◽

Matthew C. Watson ◽

Erica C. Kemmerling ◽

Philip W. Hinds ◽

Gordon S. Huggins ◽

...

Keyword(s):

Hyaluronic Acid ◽

Aortic Valve ◽

Aortic Valve Disease ◽

Time Frame ◽

Valve Disease ◽

Nodule Development ◽

Calcific Aortic Valve Disease ◽

Cd44 Expression ◽

Sirna Knockdown

The lack of pharmaceutical targets available to treat patients with calcific aortic valve disease (CAVD) necessitates further research into the specific mechanisms of the disease. The significant changes that occur to the aortic valves extracellular matrix (ECM) during the progression of CAVD suggests that these proteins may play an important role in calcification. Exploring the relationship between valve interstitial cells (VICs) and the ECM may lead to a better understand of CAVD mechanisms and potential pharmaceutical targets. In this study, we look at the effect of two ECM components, collagen and hyaluronic acid (HA), on the mineralization of VICs within the context of a two-dimensional, polyacrylamide (PAAM) model system. Using a novel, nondestructive imaging technique, we were able to track calcific nodule development in culture systems over a 3-wk time frame. We saw a significant increase in the size of the nodules grown on HA PAAM gels as compared with collagen PAAM gels, suggesting that HA has a direct effect on mineralization. Directly looking at the two known receptors of HA, CD44 and receptor for HA-mediated motility (RHAMM), and using siRNA knockdown revealed that a decrease in CD44 expression resulted in a reduction of calcification. A decrease in CD44, through siRNA knockdown, reduces mineralization on HA PAAM gels, suggesting a potential new target for CAVD treatment. NEW & NOTEWORTHY Our in vitro model of calcific aortic valve disease shows an interaction between the hyaluronic acid binding protein CD44 with the osteogenic factor OPN as a potential mechanism of aortic valve calcification. Using siRNA knockdown of CD44, we show an upregulation of OPN expression with a decrease in overall mineralization.

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Asporin Reduces Adult Aortic Valve Interstitial Cell Mineralization Induced by Osteogenic Media and Wnt Signaling Manipulation In Vitro

International Journal of Cell Biology ◽

10.1155/2020/2045969 ◽

2020 ◽

Vol 2020 ◽

pp. 1-19

Author(s):

Anisha Polley ◽

Riffat Khanam ◽

Arunima Sengupta ◽

Santanu Chakraborty

Keyword(s):

Aortic Valve ◽

Aortic Valve Disease ◽

Interstitial Cell ◽

Valve Disease ◽

Periodontal Ligament Cells ◽

Calcific Aortic Valve Disease ◽

Disease Manifestation ◽

Regulatory Pathways ◽

Replacement Surgery

Worldwide, calcific aortic valve disease is one of the leading causes of morbidity and mortality among patients with cardiac abnormalities. Aortic valve mineralization and calcification are the key events of adult calcific aortic valve disease manifestation and functional insufficiency. Due to heavy mineralization and calcification, adult aortic valvular cusps show disorganized and dispersed stratification concomitant with deposition of calcific nodules with severely compromised adult valve function. Interestingly, shared gene regulatory pathways are identified between bone-forming cells and heart valve cells during development. Asporin, a small leucine-rich proteoglycan (43 kDa), acts to inhibit mineralization in periodontal ligament cells and is also detected in normal murine adult aortic valve leaflets with unknown function. Therefore, to understand the Asporin function in aortic cusp mineralization and calcification, adult avian aortic valvular interstitial cell culture system is established and osteogenesis has been induced in these cells successfully. Upon induction of osteogenesis, reduced expression of Asporin mRNA and increased expression of bone and osteogenesis markers are detected compared to cells maintained without osteogenic induction. Importantly, treatment with human recombinant Asporin protein reduces the mineralization level in osteogenic media-induced aortic valvular interstitial cells with the concomitant decreased level of Wnt/β-catenin signaling. Overall, all these data are highly indicative that Asporin might be a novel biomolecular target to treat patients of calcific aortic valve disease over current cusp replacement surgery.

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