This document summarizes research investigating the correlation between heparanase (HPA) expression and endothelial-to-mesenchymal transition (EndMT) in vascular remodeling. The researchers found that overexpression of HPA in endothelial cells enhanced EndMT development. In shear stress experiments, HPA overexpression increased expression of mesenchymal markers and decreased expression of endothelial markers. Treatment with cytokines that induce EndMT also increased mesenchymal markers and decreased endothelial markers in HPA overexpressing cells compared to wild type cells. Experiments using variable shear stress mimicking arterial bifurcations demonstrated a link between shear stress and EndMT in vascular remodeling. Future work will further study the relationship between the endothelial glycocalyx, shear stress, and

1. Endothelial-to-Mesenchymal Transition and Its Role in Vascular Remodeling
Cody Heiser1, Darshil Choksi1, Peter Voyvodic1, and Aaron B. Baker, Ph.D.1
1Department of Biomedical Engineering, University of Texas, Austin, TX
Introduction
Cardiovascular disease results in one-third of annual deaths in the United States1.
Atherosclerosis of coronary arteries contributes greatly to this figure. Despite therapies such as
balloon angioplasty, coronary angiography, and stenting, wound healing often causes re-
occlusion of the vasculature. This phenomena, known as restenosis, results in post-operative
complications and often failure of the therapy altogether. Restenosis occurs when the smooth
muscle cells (SMC) in the intima layer of the vessel are allowed to proliferate and migrate in the
absence of the monolayer of endothelial cells (EC) that lines the wall of the lumen. ECs function
in part to prevent neointimal formation. However, when they are damaged due to stenting or
other pathological conditions, they can give way to vascular remodeling. A defining characteristic
of ECs is an extracellular layer of proteoglycans and glycoproteins that lines the vasculature.
This glycocalyx contains heparan-sulfate proteoglycans
(HSPG) that have heparan-sulfate chains on their
extracellular domain (Figure 1). Baker et al. showed in
2009 that the overexpression of the enzyme heparanase
(HPA) that cleaves heparan-sulfate chains of the
glycocalyx, leads to greater neointimal development2.
Another process that promotes vascular remodeling is the
endothelial to mesenchymal transition (EndMT). ECs can
express a mesenchymal phenotype under pathological
conditions3. Our objective is to investigate the correlation
between HPA expression and EndMT in vascular
remodeling.
Figure 1. Cross-sectional image of
arterial lumen and glycocalyx
pHPA Flow Experiment
We began by performing a shear stress experiment
on wild-type (WT) and pHPA HUVECs. Heparanase
overexpressing cells were transfected with a HPA
overexertion promoter from a lentiviral vector. The
multichannel flow setup developed by Voyvodic et al.
allowed us to study the activity of HPA in a
physiological environment (Figure 2)4. Cells were
seeded into multichannel flow chambers. The system
was kept in an incubator at 37 °C with a 5% CO2
atmosphere. Flow was applied for 24 hours at 12
dynes/cm2 shear stress. Control samples were
cultured in static media conditions. All samples were
analyzed for mRNA expression via real-time PCR and
normalized to the static WT group.
Figure 2. Multichannel flow setup for
shear stress experiments
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*
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pHPA Flow Results
The samples were evaluated for endothelial and mesenchymal markers, as well as HPA. The
PCR data confirms that the pHPA HUVECs express heparanase approximately 300 times more
than WT HUVECs (Figure 3). Both WT and pHPA samples in the shear stress environment
expressed more of an endothelial genotype than those in static culture. Endothelial nitric oxide
synthase (eNOS), PECAM, KLF2, and KLF4 levels were elevated in flow-exposed HUVECs.
Alpha-smooth muscle actin (α-SMA), a mesenchymal cell marker, had decreased expression in
the flow plates.
WT
Control
(A)
WT
EndMT
(B)
pHPA
Control
(C)
pHPA
EndMT
(D)
TGF-β2
- + - +
IL-1β
- + - +
Drug Treatment Experiment
Maleszewska et al. showed that the
combination of transforming growth
factor-β2 (TGF-β2) and interleukin-
1β (IL-1β) induces EndMT5. We
exploited this relationship to further
examine EndMT in pHPA HUVECs.
Our control group was fed with
HUVEC media (MCDB131 + 7.5%
FBS + 1% P/S + 1% L-glutamine +
HUVEC supplements), while our Figure 4. Experimental groups for IL-1β and TGF-β2 treatment
WT pHPATGFβ2/
IL1β
+ +- -
PECAM
αSMA
eNOS
SM22α
Snail
VCAM1
Figure 5. Immunostaining of drug treated HUVECs
Figure 3. Results of mRNA gene expression
0.0
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Static Flow
mRNARelativetoStaticWT
PECAM-1 WT
pHPA
EndMT group was fed with HUVEC media supplemented with 10 ng/mL TGF- β2 and 10 ng/mL
IL-1β (Figure 4). Both treatments were conducted on WT and pHPA HUVECs, in triplicate. The
experiment was simultaneously conducted on flow plates for immunostaining (Figure 5).
Figure 6. Western analysis of drug treatment
Drug Treatment Results
Western blots were quantified using Metamorph
and normalized to the WT-Control group. The
EndMT groups were expected to exhibit
elevated levels of mesenchymal markers such
as α-SMA, N-Cadherin, SM22α, and Calponin,
with decreased expression of endothelial
markers such as Snail, VCAM, PECAM, and
eNOS. For both control and EndMT treatments,
pHPA groups exhibited higher mesenchymal
markers and lower endothelial markers than
WT groups (Figure 6). N-Cadherin levels
provide the only exception, where WT groups
are slightly higher in expression than pHPA
groups. This inconsistency may be due to
irregularity in cell-cell junction formation during
EndMT. Unexpected differences in expression
of control and EndMT treatment were seen in
snail and calponin levels as well, which may be
attributed to the activity of the cytokines used in
the study. Syndecan-1, a transmembrane
HSPG, was also expressed less in pHPA
groups than WT groups. Future work may use
variable concentrations of EndMT-inducing
cytokines to determine their effectiveness.
Diamond Flow Experiment
To investigate the behavior of pHPA cells under variable shear stress, we performed a flow
experiment using diamond flow plates that create 12 dynes/cm2 shear stress on straight
channels and 6 dynes/cm2 shear stress at junctions, mimicking arterial bifurcations. Flow was
applied using the multichannel dampening setup (Figure 2) for 24 hours4. Samples were then
immunostained for endothelial and mesenchymal protein markers. The variable shear stresses
contribute to a wide range of protein expression at different locations in the bifurcation (Figure 7).
References
(1) James, D.; Rafii, S. Maladapted Endothelial Cells Flip the Mesenchymal Switch. Sci. Transl.
Med. 2014, 6, 227fs12.
(2) Baker, A. B.; Groothuis, A.; Jonas, M.; Ettenson, D. S.; Shazly, T.; Zcharia, E.; Vlodavsky, I.;
Seifert, P.; Edelman, E. R. Heparanase Alters Arterial Structure, Mechanics, and Repair
Following Endovascular Stenting in Mice. Circ. Res. 2009, 104, 380–387.
(3) Medici, D.; Kalluri, R. Endothelial-Mesenchymal Transition and Its Contribution to the
Emergence of Stem Cell Phenotype. Semin. Cancer Biol. 2012, 22, 379–384.
(4) Voyvodic, P. L.; Min, D.; Baker, A. B. A Multichannel Dampened Flow System for Studies on
Shear Stress-Mediated Mechanotransduction. Lab. Chip 2012, 12, 3322–3330.
(5) Maleszewska, M.; Moonen, J.-R. A. J.; Huijkman, N.; van de Sluis, B.; Krenning, G.;
Harmsen, M. C. IL-1β and TGFβ2 Synergistically Induce Endothelial to Mesenchymal Transition
in an NFκB-Dependent Manner. Immunobiology 2013, 218, 443–454.
Figure 7. Immunostaining of pHPA and WT HUVECs after diamond plate flow experiment
Conclusions and Future Work
Vascular remodeling results from a variety of confounding factors in pathological and
atheroprone conditions. The glycocalyx of the endothelial monolayer contributes to shear stress-
responsive mechanotransduction2. Endothelial-to-mesenchymal transition of ECs also plays a
major role in vascular remodeling3. Our first shear stress experiment confirmed that our pHPA
HUVECs overexpressed heparanase (Figure 3). We also observed increased endothelial
expression in flow-exposed cells, and a mesenchymal phenotype in static culture. We then
utilized the relationship between IL-1β/TGF-β2 treatment and EndMT to study the contribution of
heparanase to the process. In EndMT-treated HUVECs, mesenchymal markers were higher in
pHPA cells than in WT cells. Endothelial markers were lower in the pHPA-EndMT group than the
WT-EndMT group as well (Figure 6). These data suggest that heparanase overexpression
enhances EndMT development in endothelial cells. In order to further explore the effects of
shear stress on EndMT, our experiment with the diamond flow plates allowed us to mimic arterial
bifurcations. The variable shear stress contributed to a wide range of phenotypes in the ECs,
suggesting a strong link to EndMT and vascular remodeling (Figure 7). Future work will further
investigate the complex relationship between the endovascular glycocalyx, shear stress, and
endothelial-to-mesenchymal transition. A shear stress study involving EndMT-inducing cytokines
would allow us to observe all three factors simultaneously.