Arteriosclerosis, Thrombosis, and Vascular Biology

BRIEF REVIEW

Aortic Valve Stenosis

From Basic Mechanisms to Novel Therapeutic Targets
Philip Roger Goody,* Mohammed Rabiul Hosen, Dominik Christmann, Sven Thomas Niepmann, Andreas Zietzer, Matti Adam,
Florian Bönner, Sebastian Zimmer, Georg Nickenig, Felix Jansen*

ABSTRACT: Aortic valve stenosis is the most prevalent heart valve disease worldwide. Although interventional treatment options
have rapidly improved in recent years, symptomatic aortic valve stenosis is still associated with high morbidity and mortality.
Calcific aortic valve stenosis is characterized by a progressive fibro-calcific remodeling and thickening of the aortic valve
cusps, which subsequently leads to valve obstruction. The underlying pathophysiology is complex and involves endothelial
dysfunction, immune cell infiltration, myofibroblastic and osteoblastic differentiation, and, subsequently, calcification. To date,
no pharmacotherapy has been established to prevent aortic valve calcification. However, novel promising therapeutic targets
have been recently identified. This review summarizes the current knowledge of pathomechanisms involved in aortic valve
calcification and points out novel treatment strategies.

Key Words:  aortic valve ◼ aortic valve stenosis ◼ calcification ◼ heart valve disease ◼ inflammation ◼ valvular endothelial cells ◼ valvular interstitial cells

A
ortic valve stenosis (AVS) is the most common which is comprised of valvular endothelial cells (VECs)
acquired heart valve disease in the western world.1,2 on both sides (ventricularis and fibrosa). The interstitium
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Its prevalence is >2% in patients >60 years of age.3
When symptomatic, the 2-year mortality rate of severe
AVS is around 50%.4,5
In most humans, the aortic valve consists of 3 semilunar
leaflets, also called cusps (tricuspid aortic valve). There is a
congenital variant of bicuspid aortic valves (BAV) with a prev-
alence of 0.5% to 2 %.6 These patients develop severe AVS
early in life, in contrast to humans with tricuspid valves, and
show pronounced calcification owing to higher mechanical
strain on the valve cusps.7,8 Furthermore, genetic variants in
patients with BAV were identified. These variants include
mutations in NOTCH1 and in the GATA family of zinc finger
proteins, leading to accelerated calcification and changes
in the ECM (extracellular matrix) through dysregulation of
matrix metalloproteinases, respectively.9–11 The underlying
molecular mechanisms discussed in this review are likely to
apply to both bicuspid and tricuspid aortic valves, although
there is a great need to study differences in calcification
processes of these 2 variants.
In healthy humans, the valve cusps are less than a
millimeter thick and are covered by an endothelial layer,
of the valve consists of 3 distinct layers: the laminae
fibrosa, spongiosa, and ventricularis. The main cell popu-
lation found here is composed of valvular interstitial cells
(VICs).12 To meet the different physicomechanical needs
of the valve, these layers have a complex ECM. The lam-
ina fibrosa, representing the largest part of the valve and
the main load-bearing structure, has abundant type I col-
lagen fibrils. The lamina ventricularis consists of collagen
and radially aligned elastin, which help to distribute radial
forces during valve opening. In between lies the lamina
spongiosa that is largely made up of glycosaminoglycans
(GAGs), which are believed to have a lubricating function
for the outer layers.13,14
AVS develops through an initial endothelial injury and
dysfunction, immune cell infiltration, myofibroblastic/
osteoblastic differentiation of VICs, and subsequent cal-
cification.15 This inhibits separation due to stiffening or
fusion of the cusps and consequently causes a reduction
in valve opening with increased blood flow velocity.16 Once
fibrosis and calcification of the valve have commenced,
the majority of patients develop severe AVS.17,18 The only

 
Correspondence to: Philip Roger Goody and Felix Jansen, Heart Center Bonn, Department of Medicine II, University Hospital Bonn, Venusberg-Campus 1, 53127,
Bonn, Germany, Emails philip.goody@ukbonn.de, felix.jansen@ukbonn.de
*These authors contributed equally to this article.
For Sources of Funding and Disclosures, see page 896.
© 2020 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at www.ahajournals.org/journal/atvb

Arterioscler Thromb Vasc Biol. 2020;40:885–900. DOI: 10.1161/ATVBAHA.119.313067 April 2020   885
 Goody et al
Nonstandard Abbreviations and Acronyms
Brief Review - AL
α-SMA α-smooth muscle actin
ALP alkaline phosphatase
AVS aortic valve stenosis
BAV bicuspid aortic valve
BMP bone morphogenic protein
CAVD calcific aortic valve disease
CDH11 cadherin-11
DPP-4 dipeptidyl peptidase-4
ECM extracellular matrix
EndMT endothelial to mesenchymal transition
eNOS endothelial nitric oxide synthase
ENPP1 ectonucleotide pyrophosphatase/phos-
phodiesterase 1
EVs extracellular vesicles
FGF-2 fibroblast growth factor 2
HOTAIR HOX transcript antisense RNA
ICAM-1 intercellular adhesion molecule 1
IGF-1 insulin-like growth factor 1
IL interleukin
lncRNA long noncoding RNA
LRP5 LDL receptor–related protein 5
MALAT1 metastasis-associated lung adenocarci-
noma transcript 1
MAPK mitogen-activated protein kinase
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MGP matrix-γ-carboxyglutamic acid protein
miRNA microRNA
ncRNA noncoding RNA
NF-κB nuclear factor ‘kappa-light-chain-
enhancer’ of activated B-cells
OPG osteoprotegerin
ox-LDL oxidized low-density lipoprotein
P2Y purinoceptor
RANK receptor activator of nuclear factor
kappa B
RANKL receptor activator of nuclear factor
kappa B ligand
Runx2 Runt-related transcription factor 2
SMAD small mothers against decapentaplegic
TGF transforming growth factor
TNF tumor necrosis factor
TUG1 taurine upregulated gene 1
VCAM-1 vascular cell adhesion protein 1
VEC valvular endothelial cell
VIC valvular interstitial cell
Wnt wingless and Int-1
possible treatment for severe aortic valve stenosis is
either surgical aortic valve replacement or transcatheter
aortic valve replacement. Currently, interventional implan-
tation methods such as transfemoral or transapical valve
886   April 2020 Arterioscler Thromb Vasc Biol. 2020;40:885–900. DOI: 10.1161/ATVBAHA.119.313067
Aortic Valve Stenosis
Highlights
• Calcific aortic valve stenosis is a disease with high
prevalence and mortality.
• Currently, no medical treatment is available to halt
disease progression or reverse calcification.
• Lack of mechanistic understanding hampers devel-
opment of specific anti-inflammatory and anticalcific
drugs.
• This review summarizes our current knowledge of
disease pathology and highlights potential drug-
gable pathways.
replacement are on the rise. In fact, recently, 2 large pro-
spective, multicenter studies showed that transcatheter
aortic valve replacement can outperform surgical aortic
valve replacement, even in low-risk patients.19,20 However,
although these interventions yield very good results, there
are still peri-procedural risks, including left bundle branch
block, acute kidney injury, disabling stroke, bleeding com-
plications, and death.19,20 At this time, no therapeutic or
preventative medical treatment is available for AVS. It
is, therefore, fundamentally important to understand the
mechanisms of disease initiation and progression in order
to identify possible therapeutic targets and develop new
strategies to ameliorate this frequent and severe illness.
Here, we summarize current knowledge about the
pathophysiological mechanisms that lead to calcific AVS.
Additionally, we will highlight some innovative treatment
options with the potential to limit aortic valve calcification
and reduce the burden of this disease.
Phases of Calcific Aortic Valve Disease
The initiation and progression of calcific aortic valve dis-
ease (CAVD) can be seen as 2 distinct phases, with dif-
ferent immunologic, metabolic, physicomechanical, and
cellular responses leading to end-stage calcification as
first described by Aikawa et al in 2007.21–23 In the first of
these phases (initiation), there is lipid deposition, accom-
panied by injury and inflammation. In the second phase
(propagation), osteogenic differentiation and calcifica-
tion are responsible for disease progression (Figure 1).21
Initiation Phase
Endothelial Dysfunction
Mechanical and (oscillatory) shear stress can induce
endothelial dysfunction of the VEC layer, which leads
to the deposition of lipoproteins and the infiltration of
immune cells.24,25 The integrity of the endothelial layer is
an important factor of valvular homeostasis (Figure 1).26
During embryogenesis and valve formation, cardiac cush-
ions develop, which are a local expansion of the ECM.27
These cushions are invaded by endocardial endothelial
 Goody et al Aortic Valve Stenosis

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Figure 1. Phases of calcific aortic valve disease (CAVD).

Pathomechanisms of initiation and progression of CAVD can be seen as distinct disease phases: endothelial dysfunction and lipid deposition,
inflammation, fibrosis, and calcification. Different stimuli, for example, mechanical/shear stress, induce endothelial dysfunction of valvular
endothelial cells (VECs), which allows lipoprotein infiltration, specifically LDL (low-density lipoprotein) and Lp(a) (lipoprotein[a]), and
consequently extravasation of immune cells into the interstitium of valve leaflets. A dysregulation of the eNOS (endothelial nitric oxide synthase)
pathway induces the production of reactive oxygen species (ROS), which stimulates the oxidation of infiltrated lipids into ox-LDL (oxidized LDL)
and oxidized phospholipids (ox-PLs). These molecules are further transformed into lysophosphatidylcholine (lysoPC), promoting apoptosis
of valvular interstitial cells (VICs) and release of apoptotic bodies leading to diffuse calcification. Following the oxidation of lipids in the valve,
immune cells infiltrate the tissue and are activated. Osteogenic differentiation of VICs is stimulated by macrophages and T cells via secretion
of TNF (tumor necrosis factor), IL (interleukin)-1β, IL-6, and RANKL (receptor activator of nuclear factor kappa B). Calcification is further
promoted by the release of so-called matrix vesicles /calcifying microvesicles from macrophages and VICs. BMP-2 (bone morphogenic protein
2) and Runx2 (runt-related transcription factor 2) induce osteogenic differentiation. Notch1 protects against osteoblastic differentiation via
inhibition of BMP-2 expression. ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) catalyze the hydrolysis of ATP to AMP and
pyrophosphate (PPi). Wnt (wingless and Int-1) induces osteoblastic differentiation. TGF indicates transforming growth factor.

cells, which can differentiate into mesenchymal valve
progenitor cells—a precursor of VICs—in a process called
endothelial to mesenchymal transition (EndMT).7,27,28
Notch signaling increases the levels of TGF (transform-
ing growth factor)-β in these cells, which, in turn, activates
the transcription factors Zinc finger protein SNAI1 and 2
(Snail/Slug) and results in a decrease of VE-cadherin
(vascular endothelial cadherin; Figure 1).29 This leads to
a weakening of the endothelial barrier function, owing to
the loss of adherens junctions.29 This process of trans-
differentiation is also believed to occur during CAVD in
adults.30 VECs differentiate into endothelial-derived VICs,
which in turn can also differentiate into osteoblastic VICs,
promoting calcification. Interestingly, VICs can inhibit this
transdifferentiation, which was shown through cocul-
ture experiments in vitro.30 It is thought that a low rate

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 Goody et al
of EndMT functions to replenish defunct VICs but that a
higher rate of EndMT contributes to the progression of
Brief Review - AL
disease.30,31 The role of EndMT in vivo is not yet entirely
clear but warrants further investigation. The endothelial
cells on the fibrosa (=aortic) and ventricularis sides of
the valve have different phenotypes, as demonstrated in
porcine aortic valve specimens.32 Furthermore, a differ-
ence in the expression of eNOS (endothelial nitric oxide
synthase) was observed in calcified versus noncalcified
human aortic valve samples.33 The expression level was
reduced overall in calcified valves, with higher expres-
sion found on the ventricularis side. Additionally, eNOS
expression levels are higher in the ventricular endothe-
lium of porcine aortic valves.33 This could be one reason
why more pronounced calcification is frequently observed
on the aortic side of the cusps. Thus, endothelial cell het-
erogeneity could play a major role in the initiation of dis-
ease, and local differences in gene and noncoding RNA
(ncRNA) expression could provide further insight into the
disease mechanisms.
Lipid Deposition
There is growing evidence that the deposition of lipopro-
teins strongly induces chronic inflammation, which is a
hallmark of the early stages of CAVD and can be visualized
long before calcification occurs by fluorodeoxyglucose
positron emission tomography/computed tomography
(FDG-PET/CT).34 Different apolipoproteins, such as apoB,
apoA1, and apo(a), as well as a dysregulation of the eNOS
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pathway and increased nicotinamide adenine dinucleo-
tide phosphate (NADPH) oxidase levels, were observed
in explanted human aortic valves (Figure 1).35–37 The for-
mation of reactive oxygen species may act as a stimulus
of calcification, as shown in a vascular smooth muscle
cell (VSMC) calcification model.38 This increased oxida-
tive stress also promotes the formation of ox-LDLs (oxi-
dized low-density lipoproteins) and oxidized phospholipids,
which triggers calcification in vascular cells.38 Oxidized
phospholipids can be transformed into lysophosphatidyl-
choline by Lp-PLA2 (lipoprotein-phospholipase A2), lead-
ing to apoptosis in VICs.39,40 Lp-PLA2 levels are higher in
stenotic aortic valve tissue.40,41 Ox-LDL induces upregula-
tion of cell adhesion molecules, including ICAM-1 (inter-
cellular adhesion molecule 1) and VCAM-1 (vascular cell
adhesion protein 1), and consequently, there is greater
adhesion and extravasation of immune cells.35,42–44
Inflammation
Oxidized lipids also trigger an inflammatory response in val-
vular tissue (Figure 1). Following the uptake of oxidized lip-
ids, local macrophages, CD4+ and CD8+ T lymphocytes,
and mast cells are activated.24,45 This is in part a result of
the activation of PRRs (pattern recognition receptors), such
as TLRs (toll-like receptors) and the NF-κB (nuclear fac-
tor κB) pathway.46 TLR2 and TLR4 are expressed in VICs
and treatment of the cells with lipopolysaccharide, a proin-
flammatory TLR ligand, induces an osteogenic response in
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Aortic Valve Stenosis
isolated VICs.47 Furthermore, activation of TLR3 in human
VICs with Poly(I:C) (polyinosinic:polycytidylic acid) can
induce an upregulation of BMP (bone morphogenic pro-
tein)-2, TGF-β1 and ALP (alkaline phosphatase), thus lead-
ing to calcium deposition.48 Monocytes and macrophages
stimulate the osteogenic differentiation of VICs as well as
calcification via secretion of TNF (tumor necrosis factor),
followed by activation of NF-κB and IL (interleukin)-1β and
IL-6, possibly due to an induction of the homeobox protein
MSX2 and DR4 (death receptor 4).49–52
It was recently shown that another pattern recognition
receptor, namely CLEC4E (C-type lectin domain family 4
member E), is highly expressed in human stenotic aortic
valve tissue.53 This receptor is found on phagocytic cells,
such as monocytes and macrophages and can recognize
cell debris and cholesterol crystals, promote their incor-
poration, intracellular degradation, and consequent vas-
cular inflammation and atherogenesis.54
Localized cell death, as a consequence of endothe-
lial dysfunction and inflammation, results in the release
of apoptotic bodies that cause microcalcification, which
is further promoted by the release of extracellular vesi-
cles (EV) from macrophages and VICs.55–58 This process
shows similarities to bone formation, where EVs filled
with calcium and inorganic phosphate are shed. These
calcium-phosphate complexes within the EV can form
hydroxyapatite crystals, which grow in size and ultimately
destroy the EV. The hydroxyapatite crystals come into
contact with the ECM, where they function as nucleation
sites for further macrocalcification.55,56 Such calcification
triggers an even more pronounced immune response,
creating a vicious circle and consequently leading to the
propagation phase of the disease.
Propagation Phase
Myofibroblastic and Osteoblastic Differentiation
After the initial inflammatory phase, where lipid deposi-
tion and chronic inflammation initiate CAVD, the face of
the disease changes, and calcification becomes the driv-
ing force. Differentiation of VICs into myofibroblastic and
osteoblastic phenotypes is regarded as a fundamental
step during the propagation phase. The initiation of this
differentiation seems to be regulated by cytokines that
are secreted by immune cells.59 During progression of
the disease into the propagation phase, calcific stimuli
become more independent of immune cells, and the cal-
cification process is regulated by a number of calcific
pathways. In an elegant study by the Aikawa lab, pro-
teomic analyses confirmed differential protein expression
amongst the 3 layers of the aortic valve cusps.60 VICs had
a greater potential for calcification on the fibrosa side of
the valve, possibly owing to the activation of pathogenic
pathways, such as collagen biosynthesis and modifica-
tion, ECM degradation, GAG metabolism, and lipid metab-
olism.52 These results demonstrate a large spatiotemporal
 Goody et al
heterogeneity within the valvular tissue contributing to
regional differences in calcification patterns and dem-
onstrates the great plasticity of this cell type. VICs can
differentiate into myofibroblast- and osteoblast-like cells.
Calcification is a result of both of these differentiation
pathways, although the relative contribution of each of
these pathways in vivo is currently unclear.12 Myofibroblas-
tic differentiation can be achieved in vitro by stimulating
VICs with TGF-β1 and can be inhibited by FGF (fibro-
blast growth factor)-2.61 This differentiation leads to an
increased expression of α-SMA (α-smooth muscle actin),
vimentin, and smooth muscle myosin.62 Downstream
effects include activation of the canonical MAPK (mito-
gen-activated protein kinase) pathway via SMAD (small
mothers against decapentaplegic) and noncanonical p38
activation.63 This leads to increased binding of LDL to
GAGs and nodule formation as well as increased ALP
activity, respectively.63,64 A further effect of TGF-β1 treat-
ment is enhanced expression of CDH11 (cadherin-11),
leading to its binding with α-SMA and consequent nodule
formation.65 CDH11 was found to be highly expressed in
diseased aortic valves of humans and in a mouse-model
of AVS.66 CDH11 participates in mesenchymal to osteo-
chondral differentiation and was found to be expressed
on 91% of myofibroblastic and 96% of osteoblastic
cells.66 Increased ALP activity and osteocalcin expression
with increased calcification can also occur upon treat-
ment with TGF-β3 and BMP-2, BMP-4, BMP-7 or an
osteogenic medium, containing ascorbate-2-phosphate,
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dexamethasone, and β-glycerol phosphate.67 The latter is
commonly used to induce osteoblastic differentiation in
vitro. These treatments lead to an increase in ALP activ-
ity and expression of osteoblast markers, such as Runx2
(runt-related transcription factor 2) and BMP-2. BMP-2
is a potent initiator of osteoblastic differentiation, whereas
Runx2 is seen as the master transcriptional regulator for
osteogenesis.68,69 This process seems to be passage-
dependent, in that cells from higher numbers of passages
show less potential for calcification.70 Cells treated with a
so-called procalcifying medium, containing NaH2PO4 and
L-ascorbic acid, showed ALP- and passage-independent
calcification.70
Calcification of valve tissue can arise as a result of
osteoblastic differentiation of VICs and lead to osteo-
genic calcification with formation of bone-like struc-
tures.71,72 This pathway only accounts for around 13% of
calcific deposits in tricuspid aortic valves, whereas BAV
have a >2-fold higher incidence of osteogenic calcifica-
tion.72 The majority of mineral deposition is acquired via
dystrophic calcification (ca. 83%), which is speculated
to be the result of diffuse calcium precipitation on cel-
lular debris after apoptosis of VICs and is suggested to
be regulated in part by TGF-β.71,73 A possible explana-
tion for increased osteogenic calcification in BAVs could
be higher biomechanical strain on the valve cusps when
compared with tricuspid aortic valves. This can lead to
Arterioscler Thromb Vasc Biol. 2020;40:885–900. DOI: 10.1161/ATVBAHA.119.313067
Aortic Valve Stenosis
downregulation of the long ncRNA (lncRNA) HOTAIR
resulting in an increase in ALP and BMP-2 levels, pro-
Brief Review - AL
moting osteoblastic differentiation of VICs.74,75 The exact
mechanisms of dystrophic calcification are still poorly
understood and warrant extensive research to unravel
the underlying pathways.
Molecular Calcific Pathways
In the initiation phase of CAVD, osteoblastic differen-
tiation seems to be coordinated by infiltrating macro-
phages, which has been demonstrated by in vivo imaging,
as mentioned above.23 During the later stages of disease,
other pathways become more important. These include
the RANK (receptor activator of nuclear factor kappa
B)/RANKL (RANK ligand)/OPG (osteoprotegerin),
Wnt (wingless and Int-1)/β-catenin, and Notch signal-
ing pathways (Figures 1 and 2).16 Notch1 gained interest
when 2 families with frame-shift mutations in NOTCH1,
resulting in a premature stop codon, were reported.76 In
these families, a high rate of BAV and severe aortic valve
calcification was observed. Further studies in mice and
VICs demonstrated that functional Notch1 represses
BMP-2 expression and Runx2 transcriptional activity,
thus protecting VICs from osteoblastic differentiation
and calcification (Figure 1).77 The Wnt/β-catenin path-
way appears to be a positive regulator of osteoblastic
differentiation, in which binding of Wnt to the LRP5 (LDL
receptor–related protein 5) leads to activation of the
canonical Wnt/β-catenin pathway.78 Calcium deposition
leads to stiffening of the cusps, which increases mechan-
ical strain and injury and further induces activation of
the Wnt/β-catenin pathway, eventually leading to even
more osteoblastic differentiation of VICs through ENPP
(ectonucleotide pyrophosphatase/phosphodiesterase
1; Figures 1 and 2). ENPP1 hydrolyzes extracellular
ATP generating inorganic phosphate and thus promot-
ing calcification.79 A single nucleotide polymorphism
in intron 9 of ENPP1 was associated with CAVD, and
higher expression levels of ENPP1 have been detected
in stenotic valves by quantitative polymerase chain reac-
tion.79 Loss of extracellular ATP leads to more apopto-
sis of VICs because ATP can act as a survival signal by
binding to purinergic P2Y2 (purinoceptor) receptors on
VICs.79 Diminished P2Y2 signaling increases levels of
IL-6, which in turn promotes the expression of BMP-2
and leads to greater calcification of VICs.80
Another pathway involved in cardiovascular calci-
fication is mediated by fetuin-A and MGP (matrix-γ-
carboxyglutamic acid protein).81 MGP is activated by
carboxylation and phosphorylation in a vitamin K–depen-
dent manner, and low levels of vitamin K lead to inefficient
carboxylation of MGP and thus more coronary calcifica-
tion.81 There is evidence that patients taking vitamin K
antagonists show more vascular calcification and higher
rates of aortic valve degeneration.82,83 Dietary intake of
menaquinone (vitamin K2) was found to reduce vascular
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Figure 2. Mechanisms of calcification in calcific aortic valve disease.

Mechanical and shear stress induce endothelial dysfunction of the valvular endothelial cell (VEC) layer leading to endothelial to mesenchymal
transition (EndMT) of VECs to myofibroblastic cells and endothelial-derived valvular interstitial cells (VICs). VICs can inhibit this transdifferentiation.
TGF (transforming growth factor)-β inhibits expression of CD31 and activates the transcription factors Snail/Slug, resulting in a decrease of
VE-Cadherin (vascular endothelial cadherin) and loss of adherens junctions. TNF (tumor necrosis factor) α inhibits eNOS (endothelial NOS)
and CD31 expression and stimulates expression of α-SMA (α smooth muscle actin), necessary for myofibroblastic differentiation. VICs can also
undergo myofibroblastic or osteoblastic differentiation. Myofibroblastic differentiation is stimulated by cholesterol, CDH11 (cadherin-11), and
TGF-β1 and inhibited by FGF-2 (fibroblast growth factor 2). Myofibroblastic VICs undergo Casp (caspase) mediated apoptosis, which leads to the
release of apoptotic bodies and diffuse calcification. Osteoblastic differentiation can be induced by TGF-β 1 and 3, RANK (receptor activator of
nuclear factor kappa B), BMP (bone morphogenic proteins; BMP-2, BMP-4, BMP-7), Wnt (wingless and Int-1)/β-catenin, SMADs (small mothers
against decapentaplegic), ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1), TLRs (toll-like receptors) and others. Noncoding RNA
(NcRNAs) can either stimulate or inhibit differentiation, while Notch1 signaling seems to inhibit differentiation. Calcification is further promoted by
the release of calcifying extracellular vesicles from VICs. Myofibroblastic differentiation leads to increased ALP (alkaline phosphatase) expression/
activity and expression of α-SMA and vimentin. Osteoblastic differentiation leads to an increase of Runx2, BMPs, ALP, and osteocalcin levels.
Cardiovascular osteoclast-like cells (green) have impaired mineral resorption capacity, thus also aiding calcification and show low levels of TRAP
(tartrate-resistant acid phosphatase) and CTSK (cathepsin K).

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 Goody et al
calcification and mortality.84 The effects of vitamin K sup-
plementation are currently being investigated in multiple
clinical trials (VitaVask [Vitamin K1 to Slow Vascular Cal-
cification in Hemodialysis Patients], iPACK-HD [Inhibiting
the Progression of Arterial Calcification With Vitamin K
in HemoDialysis Patients], BASIK2 [Bicuspid Aortic Valve
Stenosis and the Effect of Vitamin K2 on Calciummetab-
olism on 18F-NaF PET/MRI], VitaK-CAC [The Effects of
Vitamin K2 Supplementation on the Progression of Coro-
nary Artery Calcification]).
Osteoclastic Differentiation
Although there is an absence of real osteoclasts in the cardio-
vascular system, monocytes/macrophages can differentiate
into cardiovascular osteoclast-like cells, displaying mineral
resorption capacity.85,86 Indeed, during bone formation and
homeostasis, bone preosteoclasts, which further mature into
multinucleated bone-resorbing osteoclasts, also arise from
the monocyte cell lineage.87,88 In bone, osteoblasts are the
natural counterplayer to osteoclasts, promoting bone forma-
tion.89 RANKL and MCSF (macrophage colony-stimulating
factor), which are produced by osteoblastic stromal cells in
bone tissue, are necessary for osteoclast differentiation and
maturation.90 Binding of RANKL to RANK on marrow stro-
mal cells and preosteoclasts leads to osteoclast differentia-
tion and demineralization of the bone matrix. This effect is
counteracted by OPG, which acts as a soluble decoy recep-
tor.22 Conversely, in human VICs, RANKL binding can lead
to osteoblastic differentiation.91 This difference could be due
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to the fact that although there is a high density of preosteo-
clasts in bone tissue, thus favoring RANKL’s ability to induce
osteoclastic differentiation, this cell type is absent in the car-
diovascular system.92 The calcific effect is further enhanced
by high levels of RANKL and extremely low levels of OPG in
calcific aortic valves compared with healthy valves.91
In the cardiovascular system, osteoclast-like cells can
arise from proinflammatory (M1) macrophages or anti-
inflammatory (M2) macrophages, although there is growing
evidence of the existence of a greater variety of macrophage
subsets.85,86,93 The action of cardiovascular osteoclast-like
cells is counterbalanced by VSMCs and VICs, which can
both differentiate into an osteoblastic cell type.94,95 Osteo-
blastic VSMCs are able to secrete RANKL and MCSF
required for osteoclastic differentiation of macrophages,
which can also be used in vitro to induce osteoclastic differ-
entiation.94,96 These osteoclast-like cells express TRAP (tar-
trate-resistant acid phosphatase) and CTSK (cathepsin K),
which are involved in bone resorption and mineral uptake97,98
In atherosclerotic plaques, M2-derived cardiovascu-
lar osteoclast-like cells cluster around calcific deposits
and show a diminished mineral uptake capacity due to
decreased expression of TRAP and CTSK.97 Calcific
aortic valves contain osteoclast-like cells, which appear
to arise from M1 polarized macrophages.99,100 Calci-
fied aortic valves also show a pronounced infiltration of
IFN (interferon)-γ–secreting CD8+ T cells, and calcific
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Aortic Valve Stenosis
regions of the valve display high expression of CD8+
T-cell gene transcripts when compared with noncalci-
Brief Review - AL
fied valve parts.100–102 IFN-γ is a well-known inductor
of M1 polarization and is widely used in vitro to achieve
M1 polarization. Stimulation of CD14+ monocytes in vitro
with IFN-γ leads to impaired osteoclastic activity with low
expression of cathepsin K, TRAP, and RANKL.
Impaired resorption capacity of cardiovascular osteo-
clast-like cells seems to play a major role during cardio-
vascular calcification by tipping the balance towards a
procalcifying milieu and thus contributing considerably to
cardiovascular disease burden. Activating cardiovascular
osteoclast-like cells could be a promising route of halt-
ing or even reversing cardiovascular calcification through
improved mineral uptake by these cells.
Intercellular Communication via EVs During
Aortic Valve Calcification
Various cell types play roles during CAVD. These cells
communicate with each other and can influence pheno-
typic switching, as shown by Hjortnaes et al.30 They dem-
onstrated in coculture experiments that EndMT of VECs is
inhibited by the presence of VICs, and VECs significantly
decreased the activation of VICs in vitro. Interestingly, the
presence of elevated extracellular calcium and phosphate
levels induces the release of calcifying EVs by VICs, and
spheroid mineralized EVs are produced from VICs under
mechanical strain (Figures 1 and 2).103,104 Patients under-
going a transcatheter aortic valve replacement proce-
dure showed an increase of circulating platelet-derived
EVs and a decrease in endothelial-derived EVs.105 These
findings demonstrate that intercellular communication,
including through EVs, can potentially regulate cellular
functions and phenotypes during aortic valve calcification.
EVs are known to be actively involved during car-
diovascular calcification processes and can promote
or inhibit calcification through differences in their lipid
composition and cargo.106 This cargo includes surface
molecules, proteins, and nucleic acids, such as DNA,
mRNA, and ncRNAs, which can be transferred between
cells.106 By doing so, EVs are able to modify the function
or fate of the target/recipient cells.107,108 Within this con-
text, ncRNAs are considered essential effectors of EVs
because they regulate gene expression.109,110 Studies
have shown that various stress signals in cardiovascu-
lar disorders, including endothelial cell injury, trigger the
release of EVs and transport of ncRNAs.111,112
The role of EVs during CAVD is currently unclear, but
their role during atherosclerosis, cardiovascular calcifica-
tion, and endothelial regeneration has already been well
studied. Due to similarities of these diseases, EVs are
likely to be of importance also in CAVD initiation and pro-
gression. Further investigation is warranted to elucidate
the mechanisms involved.
April 2020   891
 Goody et al Aortic Valve Stenosis

Role of ncRNAs in Aortic Stenosis acts as a negative regulator of osteogenic differentiation

by repressing its direct target, Runx2, resulting in impaired
Brief Review - AL

Growing evidence suggests that ncRNAs are key regula-

tors of osteogenesis in atherosclerosis, but knowledge
about ncRNAs in CAVD remains very limited.113,114 Since
their characterization during the human genome project as
nonprotein-coding sequences, ncRNAs have gained inter-
est as regulators of many biological as well as pathological
processes, leading to the rise of a new discipline in biologi-
cal research.115,116 Although the involvement of ncRNA in
CAVD is still not completely understood, many studies have
identified microRNAs (miRNAs, miRs) and lncRNAs (long
non-coding RNAs) that have specific functions within the
cells of the aortic valve. The first functional study of miRNAs
in AVS was carried out on miR-141. Ectopic expression of
miRNA-141 in porcine VICs repressed TGF-β stimulated
myofibroblast transformation in a BMP-2-dependent man-
ner.118 Zhang et al119 revealed that miR-30b is significantly
downregulated in CAVD samples and regulates human aor-
tic valvular calcification and apoptosis via Runx2, Smad1,
and caspase-3. In an attempt to further understand the func-
tional role of miRNAs in the osteoblastic differentiation of
VICs, Wang et al120 analyzed miRNA levels in human calci-
fied aortic valves and BMP-2-stimulated human VICs. They
showed downregulation of miR-204 in both samples and
demonstrated through in vitro experiments that miR-204
Downloaded from http://ahajournals.org by on April 8, 2022
expression of ALP and osteocalcin.
In addition to miRNAs, lncRNAs have also been found
to be involved in CAVD. The lncRNA H19 was the first
lncRNA to be described in CAVD.121 Gain-of-function and
loss-of-function experiments showed that NOTCH1 gene
expression is inhibited by H19, through interference with
the binding of p53 to the NOTCH1 promoter.122 This, in
turn, induces expression of the pro-osteogenic genes
RUNX2 and BMP-2.122 Xiao et al123 showed an increase
in MALAT1 (metastasis-associated lung adenocarci-
noma transcript 1) expression in CAVD patients. MALAT1
acts as a sponge for the protective miRNA, miR-204,
thereby reversing the inhibition of SMAD4 by miR-204
and promoting osteogenesis, as shown by an increase in
osteoblast markers, such as ALP.123 Similarly to MALAT1,
taurine upregulated gene 1 (TUG1), was found to be
significantly upregulated in calcified aortic valves and to
directly interact with miR-204-5p.124 In a recent study
by Carrion et al,75 the lncRNA HOX transcript antisense
RNA (HOTAIR) was shown to be downregulated human
BAV cells after cyclic stretch and Wnt/β-catenin signal-
ing. Furthermore, silencing HOTAIR resulted in increased
expression of calcification inducing genes, including

Figure 3. Graded wire injury model of aortic valve stenosis in mice.

Induction of aortic valve stenosis in mice is achieved by inserting a coronary wire into the left ventricle (LV) via the carotid artery. For mild and
moderate injury, a straight guidewire is pushed through the aortic valve 20 or 50× and rotated 50 or 100× (C and A). To achieve severe injury,
a spring wire with a 15° angled tip is pushed through the valve 20× and then rotated 200× (D and A). A and B, Parasternal long-axis views of
using ultra high-frequency echocardiography with straight or angled wire. E, Incidence of acute aortic insufficiency (AI) after wire injury. F, Peak
velocities over the aortic valve after sham, mild, moderate, or severe injury, compared with baseline. n.d. indicates not determined; and ns, not
significant. Adapted from Niepmann et al117 with permission. Copyright ©2019, Springer-Verlag GmbH Germany, part of Springer Nature.

892   April 2020 Arterioscler Thromb Vasc Biol. 2020;40:885–900. DOI: 10.1161/ATVBAHA.119.313067
 Goody et al
ALP, thereby confirming its involvement in aortic valve
calcification.
Current Challenges—Model Systems
Although a number of interesting pathways leading to
CAVD have already been identified, the in vivo and in
vitro data obtained can sometimes be unreliable, and
conflicting results have been observed from different
laboratories. One explanation for this is the variety of dif-
ferent cellular and in vivo systems and protocols used
to examine CAVD. The following section summarizes the
current problems, challenges, and opportunities of exist-
ing model systems.
In Vitro Models of Aortic Valve Calcification
Multiple studies have been undertaken on aortic valve–
derived cells that were isolated from humans or animals.
VECs and VICs are usually obtained from large animals,
such as pigs, sheep, and cows.125–127 It is also possible to
isolate these cells from murine aortic valves.80 These ani-
mal cells have a major drawback because there might be
large differences in the myofibroblastic and osteoblastic
pathways between animals and humans.12
One major problem for using human cells lies in the
availability of aortic valve material, especially healthy valve
tissue to be used as control samples. Diseased human
valve tissue can be acquired from patients undergoing
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surgical aortic valve replacement due to CAVD. From this
material, VECs and VICs can be isolated for in vitro exper-
iments via collagenase treatment and magnetic-assisted
cell sorting.12 A good source of control samples is donor
hearts that could not be transplanted.68 This poses a prob-
lem for most researchers since there are only few trans-
plantation centers where these samples can be obtained.
A second source can be the explanted hearts of trans-
plant recipients, if the aortic valve is not affected by the
underlying disease.67 Another, more widely used control
sample are noncalcified aortic valves, which have been
explanted because of aortic valve insufficiency. However,
these patients often present with aortic root aneurysms,
which has to be taken into account when analyzing the
acquired data.128 Pediatric valves that were removed
because of an underlying congenital heart defect have
also been used as a source material.129 Indeed, the most
ideal control samples maybe those from patients present-
ing with aortic dissection,130 but an inherent problem is
that these patients present as acute emergencies and
have to be operated on quickly, making the acquisition
of informed consent difficult. It is also possible to divide
an aortic valve cusp into calcific, fibrotic, and healthy tis-
sue by using a near-infrared fluorescent calcium tracer.60
A problem here lies in underlying genetic variants that
can affect the whole valve and might distort the results
obtained. Cadavers are the least desirable source of
Arterioscler Thromb Vasc Biol. 2020;40:885–900. DOI: 10.1161/ATVBAHA.119.313067
Aortic Valve Stenosis
valve material, especially for cell isolation or RNA analysis
because of the variable quality of the source material.
Brief Review - AL
In Vitro Calcification
The protocols for inducing calcification in vitro vary
immensely between labs. Both osteogenic media and
procalcifying media have been used. The incubation of
isolated VICs with these media leads to an in vitro cal-
cification of cells, which can be visualized by alizarin
red staining and further quantified by quantitative poly-
merase chain reaction or protein analysis of osteogenic
markers, for example, ALP. Osteogenic medium shows a
passage-dependent effect on VICs, where cells from a
higher passage show diminished calcification potential.
This passage dependency could not be seen when using
procalcifying medium.70 In addition, different growth fac-
tors such as TGF-β1, TGF-β3, BMP-2, BMP-4, BMP-7,
and others have been used to induce myofibroblastic or
osteoblastic differentiation.12,67 Cell culture materials vary
between glass and plastic, or cells are grown on a coat-
ing of collagen I, II, or III, or fibronectin.131 VICs cultured
on hard surfaces, such as plastic, show the tendency to
spontaneously differentiate into a myofibroblastic phe-
notype.132 Therefore, studies have been undertaken
using 3-dimensional (3D) collagen hydrogels with differ-
ent densities.133,134 A hybrid of methacrylated gelatin and
methacrylated hyaluronic acid can also be used and has
shown promising results. Using this material, seeded VICs
remained quiescent without treatment but could still dif-
ferentiate into either myofibroblast-like cells upon TGF-
β stimulation or osteoblast-like cells upon treatment with
osteogenic medium.30,135 Van der Valk et al105 even went
one step further. They analyzed the layer-specific mechan-
ical properties of human AV tissue and created a 3D bio-
printed CAVD model using UV-crosslinked methacrylated
gelatin/methacrylated hyaluronic acid hydrogels. It is not
surprising that applying these different culture and calcifi-
cation techniques can lead to immensely different results.
In Vivo Models of AVS
Most animals do not naturally develop CAVD, but
model systems have been developed in pigs and mice
that induce AVS through a high-cholesterol diet.136,137
Another strategy has been to create genetic variants
that acquire stenosis.138 The reproducibility of these
systems is a problem because not all of the genetically
modified or high-fat diet animals develop AVS. In addition
to diet-induced and genetically induced AVS, Honda et
al139 developed a wire injury model, where a guidewire is
inserted into the left ventricle of the heart under echocar-
diographic guidance via the right common carotid artery
of C57BL/6 mice. The wire is then rotated to create an
endothelial injury. Induction of AVS can be detected by
measuring the velocity of blood flow by echocardiogra-
phy.139 This method has recently been improved upon
to yield more consistent results.117 It is now possible to
induce mild, moderate, or severe stenosis by changing
April 2020   893
 Goody et al
the wire type, tip angle, and the number of rotations (Fig-
ure 3).117 Mouse aortic anatomy differs from humans in
Brief Review - AL
that they do not have a trilayer architecture.140 It is, there-
fore, fundamentally necessary to reproduce any findings
from mice in human valves or human cells.
In Vivo Imaging
In vivo imaging is highly attractive because it can monitor
disease development over time in serial exams. Recent
advances in ultrahigh-frequency ultrasound make it
possible to assess the cardiovasculature in small ani-
mals with high accuracy. AVS is evaluated by continu-
ity equation, peak velocity, and mean pressure gradients
in suprasternal and parasternal long-axis views. Left
ventricular systolic and diastolic function can be char-
acterized by 2-dimensional echocardiography, M-mode,
pulsed-wave–Doppler, and tissue-Doppler. Image acqui-
sition and interpretation is quick and noninvasive, making
this imaging modality ideal for routine follow-ups.117
Even more robust in serial measurements, magnetic
resonance imaging can provide additional information on
3D flow and 3D structures. A comprehensive magnetic
resonance imaging approach, simultaneously assess-
ing changes in valvular, left ventricular, and aortic function
and morphology was recently proposed.141 High-resolution
magnetic resonance imaging (9,4 T) can visualize valve ori-
fice area, thickness of the cusps, 3D transvalvular flow, and
aortic regurgitation. Furthermore, left ventricular parameters
can be calculated. Mice with AVS demonstrate a turbulent
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flow pattern over the stenotic valve with increased aortic
strain and increased aortic and left ventricular wall thick-
ness.141 These noninvasive techniques and protocols are
already used in clinical scanners and might, therefore, serve
as important tools for translational research approaches.142
Sex Differences
The influence of sex on the development of CAVD has
been described, with males harboring a 2-fold risk increase
compared with females of acquiring AVS and higher rates
of aortic valve calcium density progression.18,143 These
findings stimulated studies investigating the calcium load
of stenotic aortic valves in a sex-dependent manner. Mul-
tidetector computed tomography unveiled higher calcium
content in male patients with AVS while showing similar
AVS severity as females.144 Following up on this study,
Simard et al145 were able to show that women with com-
parable AVS severity and valve weight density presented
with a higher grade of valvular fibrosis and significantly
less calcium density. However, the majority of in vitro and
in vivo studies have been carried out on valvular cells or
animals without addressing potential sex differences. One
of the few studies looking into sex-related differences in
gene expression of porcine aortic valve cusps revealed
183 significantly differentially expressed genes involved
in disease-relevant pathways, such as apoptosis, ossifica-
tion, and inflammation. The authors further confirm their
findings in sex-specific in vitro experiments with porcine
894   April 2020 Arterioscler Thromb Vasc Biol. 2020;40:885–900. DOI: 10.1161/ATVBAHA.119.313067
Aortic Valve Stenosis
VICs, demonstrating sex-related differences in prolifera-
tion and apoptosis.146 Sex hormones may be responsible
for many of the observed effects since it is known that
androgens play an important role during bone formation
as well as in the regulation of osteoblast and osteoclast
activity.147 Cardiovascular calcification was also shown to
occur at a higher rate in apolipoprotein E knockout mice
treated with androgens.148 Reinforcing these findings,
treatment of murine VSMCs with testosterone can induce
calcium deposition of VSMCs.149
Sex-related differences in molecular pathways are
most likely highly relevant during initiation and progres-
sion of CAVD and should be investigated more thor-
oughly to identify differences in disease pathology in
males and females, potentially leading to a sex-depen-
dent therapeutic approach in the future.
Standardization
Many different in vivo and in vitro systems have been
established in laboratories all over the world. This het-
erogeneity makes the reproducibility and comparabil-
ity of studies difficult. A more standardized and uniform
approach would be desirable for the future and could help
with the interpretation of data. A Collaborative Research
Initiative on Aortic Disease (TRR259, Deutsche Forschun-
gsgemeinschaft), consisting of the Universities of Bonn,
Cologne, and Düsseldorf (Germany) has been established
in the last year and aims to standardize in vivo and in vitro
models and use these to elucidate disease pathways and
therapeutic targets to develop potential therapies.
Future Directions and Novel Therapeutic
Strategies
Much has been learned in the last years about aortic
valve calcification, but the lack of ideal in vivo model
systems and standardized in vitro approaches have left
many questions unanswered. New model systems, such
as the wire injury mouse model or 3D cell culture models,
could lead to a better understanding of disease initia-
tion and progression. There are ongoing clinical studies
that are testing treatments targeting the pathways identi-
fied in the last few years. Denosumab and bisphospho-
nates are currently being investigated in the SALTIRE II
trial (Scottish Aortic Stenosis and Lipid Lowering Trial,
Impact on Regression). Administration of denosumab, a
human monoclonal antibody against RANKL that inhibits
its binding to RANK, mimics the role of OPG, thus pos-
sibly inhibiting osteoblastic differentiation in aortic valve
tissue.22 Bisphosphonates reduce bone resorption and
have been shown to inhibit vascular and aortic valve cal-
cification.150 They can also directly interfere with the pro-
duction of inflammatory cytokines involved in CAVD.151
In 2013, Thanassoulis et al152 identified a single
nucleotide polymorphism in the lipoprotein(a) locus
(rs1045872), which was associated with aortic valve
 Goody et al
calcification independent of coronary artery calcification
or coronary artery disease. Levels of Lp(a) (lipoprotein[a])
and genetic variants had already previously been shown
to be associated with coronary artery disease.153 Mecha-
nistically, deposition of Lp(a), which contains apo(a), apo-
lipoprotein B100, and oxidized phospholipids in the vessel
intima, can promote atherosclerotic lesion progression and
inflammation.154 As described earlier, oxidized lipoproteins
play a crucial role during CAVD progression. In a recent
study by Zheng et al,155 it could be shown that high levels
of Lp(a) and oxidized phospholipid correlate with disease
progression in CAVD. Furthermore, the authors could also
demonstrate their osteogenic effects on VICs in vitro.
Lipid-lowering agents, such as statins and ezetimibe, were
shown not to be effective in halting progression of CAVD
and even showed increased cancer rates in the SEAS trial
(Simvastatin and Ezetimibe in Aortic Stenosis).156–158 One
explanation is the lack of Lp(a)-lowering capabilities.159
Lp(a)-lowering agents could provide a way of slowing or
halting disease progression of CAVD, and clinical trials on
the effects of niacin, PCSK9 (proprotein convertase sub-
tilisin/kexin type 9) inhibitors, and antisense oligonucle-
otides against apo(a) mRNA are currently underway.160
Targeting CAVD with repurposed substances also
appears to be a viable route. In this regard, Choi et al161
studied the influence of DPP-4 (dipeptidyl peptidase-4)
inhibitors, such as sitagliptin, which is widely used for the
treatment of type 2 diabetes mellitus, on calcification of
VICs. Depletion of NO in VECs, mimicking endothelial dys-
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function, resulted in activation of the NF-κB pathway in
VICs. NF-κB acts as a transcription factor for DPP-4, which
can decrease IGF-1 (insulin-like growth factor 1) levels by
enzymatic degradation, leading to more osteoblastic differ-
entiation of VICs. This effect was abrogated with DPP-4
inhibitors. In a rabbit model of CAVD, animals fed with high-
cholesterol diet and vitamin D2, and treated with sitagliptin,
showed beneficial results on transvalvular peak velocity,
pressure gradients, and valve orifice area.161 These results
could be the basis for human trials with DPP-4 inhibitors.
Given the prominent role of macrophages in all stages
of CAVD, an elegant way to inhibit inflammation and val-
vular and, in fact, also vascular calcification would be to
specifically target macrophages or T cells responsible
for macrophage polarization. As discussed earlier in this
review, an impaired mineral uptake capacity of cardio-
vascular osteoclast-like cells, which are derived from
macrophages, is suggested to play an important role in
cardiovascular calcium deposition. Activation of osteo-
clast-like cells to promote mineral uptake could not only
halt progression of calcific cardiovascular diseases but
also lead to regression in calcification. The inherent prob-
lem with general activation of osteoclasts throughout the
human body would be loss of bone mass with conse-
quent osteoporosis. To our knowledge, there is no spe-
cific activator of cardiovascular osteoclasts available at
this point in time. Further studies are needed to identify
Arterioscler Thromb Vasc Biol. 2020;40:885–900. DOI: 10.1161/ATVBAHA.119.313067
Aortic Valve Stenosis
possible mechanisms to selectively activate cardiovascu-
lar osteoclast-like cells.
Brief Review - AL
Because the macrophage continuum seems to be
comprised of more subsets than just classical proinflam-
matory and anti-inflammatory (M1 and M2) macrophages,
a better understanding of mineral uptake capacity of
osteoclast-like cells derived from specific subsets is nec-
essary. If a specific macrophage subset were to show
enhanced mineral uptake capacity, one could imagine
strategies to polarize cardiovascular macrophages into
this subset, thus enhancing mineral uptake by osteoclast-
like cells arising from this subset. This could possibly be
achieved by administering cytokines or synthetic peptides,
mimicking their action and thus promoting a specific mac-
rophage subset polarization, or using existing antibodies
against cytokines to block polarization into macrophage
subsets with diminished mineral uptake capacity.
Disease-modifying antirheumatic drugs, such as the
TNF-α antibody adalimumab, were shown to be able
to influence synovial macrophage polarization to anti-
inflammatory macrophages in vitro in an IL-10/STAT3
(Signal Transducer and Activator of Transcription 3)
dependent manner.162 Influencing macrophage polariza-
tion, therefore, seems to be a viable route and might be
able to promote more effective calcium uptake. In the
CANTOS trial (Canakinumab Antiinflammatory Throm-
bosis Outcome Study), treatment with Canakinumab, a
monoclonal antibody against IL-1,β was able to reduce
cardiovascular mortality. In addition to having proinflam-
matory effects, it was shown that IL-1β produced by
vascular macrophages is able to directly induce calcifica-
tion of vascular mesenchymal cells.163 Whether observed
effects in the CANTOS trial were purely due to modula-
tion of inflammatory pathways or also due to direct inhibi-
tion of osteoblastic differentiation remains unclear.
Key proteins during the propagation phase include
RunX2 and BMP-2, whose expression is directly influ-
enced by different miRNAs in CAVD. Targeting these could
be of special interest to break through the vicious circle
of calcification. One interesting way of lowering their con-
centration in aortic valvular cells could be via the use of
EV- or liposome-bound miRNA sponges, small molecule
inhibitors, antisense oligonucleotides, or miRNA-mimics,
although targeting the aortic valve can present as a prob-
lem for treatments such as these. Administering molecules
that also interfere with bone metabolism could lead to an
increase in osteoporosis. Transcriptome analyses and next-
generation sequencing could help identify target proteins
that are solely expressed on valvular cells so that EVs or
liposomes with homing properties could be designed to
administer drugs with minimal off-target effects.
CONCLUSIONS
CAVD is a highly complex pathology, where an interplay
between metabolic factors, immune cells, and resident
April 2020   895
 Goody et al
endothelial and interstitial cells drive progression of the
disease. A lack of suitable in vivo model systems and
Brief Review - AL
the great heterogeneity of in vitro methods used has
limited the reliability of the knowledge gained so far.
Newer methods with higher reproducibility will improve
our understanding of this disease and undoubtedly lead
to novel treatment strategies.
ARTICLE INFORMATION
Received October 16, 2019; accepted February 14, 2020.
Affiliations
From the Heart Center Bonn, Department of Medicine II, University Hospital
Bonn, Germany (P.R.G., M.R.H., D.C., S.T.N., S.Z., G.N., F.J.); Clinic for Internal Medi-
cine II, University Hospital Cologne, Germany (M.A.); and Clinic for Cardiology,
Pulmonology, and Angiology, University Hospital Düsseldorf, Germany (F.B.).
Acknowledgments
We thank Dr Deborah Goody for valuable discussions and Dr Meghan Campbell
for critical reading of the article.
Sources of Funding
P.R. Goody is funded by the Else-Kröner-Fresenius Foundation, F. Jansen is
funded by the Deutsche Forschungsgemeinschaft (JA 2351/2-1 and SFB
TRR [Sonderforschungsbereich/Transregio] 259/1) and the Corona founda-
tion. M.R. Hosen is funded by the Corona foundation. STN is funded by the
Else-Kröner-Fresenius Foundation. A. Zietzer is funded by the BONFOR (Bonn
forscht) program. F. Bönner is funded by the Deutsche Forschungsgemeinschaft
(BO 4264/1-1 and SFB TRR 259/1), G. Nickenig, S. Zimmer, and M. Adam are
supported by the Deutsche Forschungsgemeinschaft (SFB TRR 259/1).
Disclosures
None.
Downloaded from http://ahajournals.org by on April 8, 2022
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