Mechanosensitive microRNA-181b Regulates Aortic Valve Endothelial
Matrix Degradation by Targeting TIMP3
Coulter Department of Biomedical Engineering, Emory University and Georgia Institute of Technology, Atlanta, GA, USA;
Department of Cardiology, Emory University, Atlanta, GA, USA
(Received 5 August 2016; accepted 4 February 2017; published online 24 February 2017)
Associate Editors Craig Simmons and Ajit P. Yoganathan oversaw the review of this article.
Abstract—Calciﬁc aortic valve disease (CAVD) is a major
cause of morbidity in the aging population, but the underlying
mechanisms of its progression remain poorly understood.
Aortic valve calciﬁcation preferentially occurs on the ﬁbrosa,
which is subjected to disturbed ﬂow. The side-speciﬁc
progression of the disease is characterized by inﬂammation,
calciﬁc lesions, and extracellular matrix (ECM) degradation.
Here, we explored the role of mechanosensitive microRNA-
181b and its downstream targets in human aortic valve
endothelial cells (HAVECs). Mechanistically, miR-181b is
upregulated in OS and ﬁbrosa, and it targets TIMP3, SIRT1,
and GATA6, correlated with increased gelatinase/MMP
activity. Overexpression of miR-181b led to decreased TIMP3
and exacerbated MMP activity as shown by gelatinase assay,
and miR-181b inhibition decreased gelatinase activity
through the repression of TIMP3 levels. Luciferase assay
showed speciﬁc binding of miR-181b to the TIMP3 gene.
Overexpression of miR-181b in HAVECs subjected to either
LS or OS increased MMP activity, and miR-181b inhibition
abrogated shear-sensitive MMP activity. These studies sug-
gest that targeting this shear-dependent miRNA may provide
a novel noninvasive treatment for CAVD.
Keywords—microRNA, Aortic valve, Extracellular matrix,
Matrix metalloproteinase, Shear stress, Endothelium.
Calciﬁc aortic valve disease (CAVD) is a major cause
of morbidity among the aging population and a major
risk factor for myocardial infarction and other cardio-
Once diagnosed, the only
available treatment is valve repair or replacement.
Stenosis, the endpoint of CAVD, is characterized by
inﬂammation, sclerotic and calciﬁc lesions, and extra-
cellular matrix (ECM) degradation.
many genetic factors (e.g., bicuspid aortic valve) and
environmental factors (e.g., left ventricular hypertro-
phy) lead to increased risk for CAVD. Although CAVD
poses a major medical problem for the aging popula-
tion, the mechanisms underlying its initiation and pro-
gression are not well-deﬁned, and few advancements
have been made in therapeutic options for the disease.
The sclerosis and calciﬁcation of the aortic valve is a
side-speciﬁc phenomenon, correlated with the diﬀerential
shear stresses observed during the cardiac cycle. Each side
of the valve experiences a distinct shear proﬁle sensed by
the overlying endothelium. The ventricularis layer faces
the ventricle and experiences a high-magnitude and pul-
satile, unidirectional, laminar shear stress (LS), and rarely
calciﬁes. The ﬁbrosa layer facing the aorta experiences
low magnitude and oscillatory shear stress (OS). This
endothelial layer exhibits a highly inﬂammatory pheno-
type, expressing abundant adhesion molecules and other
inﬂammatory markers, and downregulating anti-in-
ﬂammatory signals such as Klf2. The ﬁbrosa preferen-
tially exhibits sclerosis and calciﬁcation, with large
nodules and macrophage inﬁltration mimicking
atherosclerosis observed in the spongiosa and intersti-
tium near this valve face. In vitro, LS conditions applied
to aortic valve endothelial cells upregulate CAVD-pro-
tective genes such as KLF2, KLF4 and eNOS, whereas
OS conditions upregulate pro-CAVD genes and para-
crine mediators such as BMP4, Cathepsin K, and matrix
metalloproteinases MMP-2 and MMP-9.
Notably, the matrix metalloproteinases (MMPs)
have been correlated with valve disease in various
Address correspondence to Hanjoong Jo, Coulter Department of
Biomedical Engineering, Emory University and Georgia Institute of
Technology, Atlanta, GA, USA. Electronic mail: hanjoong.jo@
Jack M. Heath and Joan Fernandez Esmerats have contributed
equally to this work.
Cardiovascular Engineering and Technology, Vol. 9, No. 2, June 2018 (
2017) pp. 141–150
2017 Biomedical Engineering Society