Mirtron MicroRNA-1236 Inhibits VEGFR-3 Signaling

During Inflammatory Lymphangiogenesis

Dennis Jones, Yonghao Li, Yun He, Zhe Xu, Hong Chen, Wang Min

Objective—Vascular endothelial growth factor receptor(VEGFR)-3 is a critical regulator of developmental and adult
vasculogenesis and lymphangiogenesis through its interactions with select members of the VEGF family. The goal of
this study was to investigate how VEGFR-3 expression is regulated during inflammatory lymphangiogenesis.
Methods and Results—In this study, we present for the first time evidence that VEGFR-3 can be negatively regulated by
a mirtron, hsa-miR–1236 (miR-1236), which is expressed in primary human lymphatic endothelial cells. In human
lymphatic endothelial cells, miR-1236 is upregulated in response to IL-1␤, a negative regulator of VEGFR-3. miR-1236
binds the 3⬘ untranslated region of Vegfr3, resulting in translational inhibition. Overexpression of miR-1236
significantly decreased expression of VEGFR-3, but not VEGFR-2, in human lymphatic endothelial cells. Compared to
a control miR, overexpression of miR-1236 also led to decreased VEGFR-3 signaling. However, VEGFR-2–specific
signaling was not affected. miR-1236 can attenuate human lymphatic endothelial cell migration and tube formation, as
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well as in vivo lymphangiogenesis.

Conclusion—Our data suggest that miR-1236 may function as a negative regulator of VEGFR-3 signaling during
inflammatory lymphangiogenesis. (Arterioscler Thromb Vasc Biol. 2012;32:633-642.)
Key Words: angiogenesis 䡲 vascular biology 䡲 VEGFR-3 䡲 lymphangiogenesis 䡲 miR-1236

T he vascular endothelial growth factor (VEGF) family of

ligands and its tyrosine kinase receptors are critical
regulators of angiogenesis and lymphangiogenesis. Of the
VEGF receptors, VEGF receptor (VEGFR)-3 has been shown
to be critical for developmental angiogenesis and lymphan-
giogenesis, as VEGFR-3 null mice die due to a failure in
remodeling of the primary vascular plexus.1 VEGFR-3 ex-
pression is maintained in blood endothelial cell lymphatic
precursors but in adulthood becomes restricted to lymphatic
endothelial cells with the exception of few blood capillaries
in some organs.2 As a receptor for VEGF-C, VEGFR-3 is
essential for proper development of the lymphatic vascula-
ture.3–5 The lymphatic vasculature is an integral part of
normal physiology. Lymphatic vessels act as a conduit for
immune cells for assistance in immune surveillance, return
extravasated interstitial fluid to the blood, and absorb dietary
lipids in the intestine.2,6 – 8 In the adult mouse, VEGFR-3 is
critical for signaling in response to VEGF-C/D–induced
lymphatic vessel growth,9 which occurs mostly as a result of
inflammation.2 Recent work has demonstrated that the tran-
scription factors Prox1, NF-␬B, and Tbx1 are critical for
VEGFR-3 gene expression.10,11 However, little is known
about the mechanisms by which VEGFR-3 expression is
controlled during inflammatory lymphangiogenesis12 and is
the subject of this study.
Recently, posttranscriptional regulators of gene expression
have gained attention. MicroRNAs (miRNAs) are endoge-
nous, nonprotein coding small RNAs that play important
roles in the posttranscriptional regulation of target genes by
directing target mRNAs for translational repression or desta-
bilization. The collection of miRNA-class regulatory RNAs
in invertebrates and mammals was expanded by the finding of
short hairpin introns known as mirtrons.13,14 Mirtrons bypass
Drosha cleavage by using the spliceosome to generate their
precursor ends (pri-miRNAs). Although generated differently
from conventional miRNAs, mirtrons are also cleaved by
Dicer to generate functional miRNAs that can also regulate
genes posttranscriptionally. Recent work has indicated a role
for miRNAs in angiogenesis15 and determination of lym-
phatic endothelial cell lineage fate from blood endothelial
cells by regulation of Prox-1.16,17 However, it is not known if
miRNAs can directly regulate VEGFR-3—a critical receptor
involved in lymphangiogenesis and angiogenesis.
In this study, we show that miR-1236 is expressed in
endothelial cells and binds to the 3⬘ untranslated region
(UTR) of the VEGFR-3 gene, resulting in translational

Received on: September 10, 2011; final version accepted on: December 14, 2011.
From the Interdepartmental Program in Vascular Biology and Therapeutics (D.J., Y.L., Y.H., Z.X., W.M.), Department of Immunobiology (D.J.), Yale
University School of Medicine, New Haven, CT; State Key Laboratory of Ophthalmology (Y.L., W.M.), Zhongshan Ophthalmic Center, Sun Yat-sen
University, Guangzhou, China; Cardiovascular Biology Research Program (H.C.), Oklahoma Medical Research Foundation, Oklahoma City, OK.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA/243576/-/DC1.
Correspondence to Wang Min, Interdepartmental Program in Vascular Biology and Therapeutics, Department of Pathology, Yale University School
of Medicine, 10 Amistad St., New Haven, CT 06520. E-mail wang.min@yale.edu
© 2012 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.111.243576

633
 634 Arterioscler Thromb Vasc Biol March 2012
inhibition of VEGFR-3. This work reveals that miR-1236
may represent a posttranscriptional mechanism by which
VEGFR-3 expression is fine-tuned in lymphatic endothelial
cells. Our work is the first example of a miRNA that targets
VEGFR-3 and may have important implications for the control
of VEGFR-3 during inflammatory lymphangiogenesis.
Methods
DNA Constructs/Reporter Gene Assays
From human lymphatic endothelial cells (HLEC), cDNAs encoding
the entire 3⬘UTR of VEGFR-3 (1.69kb) was amplified by reverse
transcription-PCR from HLEC RNA and was directionally sub-
cloned into the NotI-XhoI sites downstream of the Renilla luciferase
in the psiCHECK2 vector that also contains a constitutively ex-
pressed firefly luciferase gene. Construct was confirmed by sequenc-
ing at WM Keck Facility at Yale University. Next, the region
complementary to the miR-1236 predicted seed sequence in position
144 to 150 of the human VEGFR-3 3⬘UTR, GGAAGA, was
scrambled to TCTAGA (mut Vegfr3 3⬘UTR), using the
QuikChange™ Site-Directed Mutagenesis Kit. This construct was
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also confirmed by sequencing. Luciferase output was quantified with
a Berthold Lumat LB 9501 luminometer and renilla luciferase
activity was normalized to the corresponding firefly luciferase
activity using a Dual-Glo Luciferase Assay System (Promega) and
reported as the percentage of control cells cotransfected with the
same concentration of control mimic or miR-1236 mimic. Experi-
ments were performed 4 times using triplicates.
VEGFR-3 Lentiviral Expression Construct
Full-length human VEGFR-3 was amplified using PCR from a
VEGFR-3 mammalian expression plasmid, and BamHI and Not1
restriction sites were introduced at either end of the cDNA. The PCR
product was subcloned into the target lentiviral vector pLex (Open-
Biosystems), resulting in pLex-VEGFR-3. The VEGFR-3 cDNA
insert was confirmed by DNA sequencing and VEGFR-3 protein
expression from pLex-VEGFR-3 was confirmed by Western blotting
after transient transfection into 293T cells.
MiRNA Analysis
MiRNAs were isolated from cells using miRNeasy kit (Qiagen)
according to the manufacturer’s instructions. Quantitative reverse
transcription-PCR (qRT-PCR) assays were performed using a
TaqMan miRNA assay kit (Applied Biosystems) for the mature
miRNA using the target-specific stem-loop reverse transcription
primer. The forward primer sequences for hsa-miR–1236 and
RNU6B were purchased from Applied Biosystems and used for PCR
validation. qRT-PCR was performed by using iQ SYBR Green
Supermix on C1000 Thermal Cycler (Bio-Rad).
Statistical Analyses
All data are expressed as mean⫾SEM. Statistical analysis was
performed with a 2-tailed Student’s paired t test. Statistical signifi-
cance for probability values are as follows: *⬍0.05; **⬍0.01;
***⬍0.001. For analysis, positive staining and area was quantified
with Image Pro Plus Software (MediaCybernetics).
The detailed methods that we have published are provided in the
online-only Data Supplement. These methods include lymphatic
endothelial cell (EC) culture, transfection, immunoblotting for
VEGFR2/3 signaling, immunofluorescence, cell migration/tube for-
mation assays, and in vivo lymphangiogenic assays as we described
previously.18
Results
Inflammatory Lymphangiogenesis In Vivo
Is Transient
We have observed that lymphangiogenesis is transient in all
models we have examined, including VEGF-induced cornea
lymphangiogenesis,18 VEGF-induced lymph node (LN) lym-
phangiogenesis,18 and inflammation-mediated LN lymphan-
giogenesis by immunization with oxazolone (Figure 1A). In
the oxazolone skin painting model, oxazolone-induced in-
flammation elicits a profound LN lymphangiogenic re-
sponse.19 In this system, LN lymphangiogenesis peaked on
days 3 to 4 followed by regression by day 7 (Figure 1A with
quantifications in Figure 1B). We determined the levels of
lymphangiogenic growth factors (VEGF-A and VEGF-C) by
qRT-PCR and found that their expressions in LNs were even
higher at day 7 compared to day 3 postoxazolone challenge
(Figure IA in the online-only Data Supplement). Rececently,
it has been shown that inflammation can decrease VEGFR-3
protein and transcript during oxazolone challenge, concomi-
tant with the expression of inflammatory cytokines.20,21
Therefore, we hypothesized that inflammatory cytokines may
inhibit oxazolone-induced lymphangiogenesis. Consistent
with a recent report,20 we found that proinflammatory cyto-
kines such as tumor necrosis factor-␣ (TNF), interleukin-1␤
(IL-1␤) and interferon ␥ (IFN-␥) were increased after oxa-
zolone challenge (Figure IB in the online-only Data Supple-
ment) with IL-1␤ at a much higher level. Although TNF and
IFN-␥ have been shown to drive and inhibit lymphangiogen-
esis, respectively,22,23 the effect of IL-1␤ on lymphangiogen-
esis is not well-characterized. We determined a direct effect
of IL-1␤ on lymphangiogenesis in a VEGF-C–induced HLEC
tube formation assay. In the presence of IL-1␤, VEGF-C-
induced tube formation in a Matrigel was significantly
suppressed (Figure 1C with quantification in Figure 1D). To
determine the mechanism for decreased tube formation by
IL-1␤, we analyzed protein expression of the VEGF-C
receptors, VEGFR-2 and VEGFR-3. As we reported re-
cently,18 protein analysis of VEGFR-3 by Western blot
revealed 3 bands (Figure 1E). Although the band at 175 kDa
is considered to be the intracellular and unglycosylated
precursor, the band with molecular weight 125 kDa repre-
sents the mature form of VEGFR-3.24,25 Stimulation of HLEC
with IL-1␤ for 24 hours strongly reduced expression of
VEGFR-3 but not VEGFR-2 (Figure 1E). A similar effect
was found by stimulation with other inflammatory cytokines,
namely IL-1␣ and TNF (Figure IC in the online-only Data
Supplement). However, IL-1␤ did not significantly affect
VEGFR-3 mRNA as measured by qRT-PCR (Figure 1F).
These results suggested that IL-1␤ may regulate VEGFR-3 at
a posttranscriptional level.
Induction of MiR-1236 by IL-1␤
The results above prompted us to examine if VEGFR-3
expression can be regulated by miRNA. To this end, Target-
Scan (www.targetscan.org) and other miRNA prediction
algorithms were used to search for miRNA binding sites in
the VEGFR-3 3⬘UTR. In silico analysis revealed a highly
conserved site in the VEGFR-3 3⬘UTR as a putative target of
a 7mer-m8 site in the seed region of miR-1236 (Figure 2A).
We chose miR-1236 as a candidate because of its relatively
high context percentile score (73%, as TargetScan defines a
favorable score as being 50 –100) and the possibility of being
functionally testable in vivo, as the predicted binding site is
also conserved in mice. In contrast, miR-132, miR 323 to 5p,
 Jones et al miR-1236 Inhibits VEGFR-3 635
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Figure 1. Inflammatory lymphangiogenesis in vivo is transient. A, B, 4% oxazolone in acetone was administered on shaved skin on the
abdomen in proximity to inguinal and brachial lymph nodes (LNs) of WT mice. LNs were collected at day 4 and 7. Lymphatic vessels
were visualized by immunostaining with anti-LYVE-1 and representative images are shown in A. Scale bar, 200 ␮m. LYVE-1-positive
area were quantified in B. Data are mean⫾SEM, n⫽10 for time points, *P⬍0.05 and **P⬍0.01. C, D, Tube formation assays in a Matri-
gel. Human lymphatic endothelial cells (HLEC) were stimulated with interleukin-1␤ (IL-1␤) (5 ng) for 24 hours. Cells were then seeded to
Matrigel and cultured with 3% serum⫹VEGF-C (100 ng/mL) for 6 hours and representative images are shown in C. Branch points as
indicated by arrowheads were quantified in D. Data are mean⫾SEM from 3 independent experiments, **P⬍0.01. Scale bar, 100 ␮m. E,
F, Effects of IL-1␤ on vascular endothelial growth factor receptor (VEGFR)-3 protein and mRNA. HLEC were stimulated with IL-1␤ (5 ng)
for 24 hours. VEGFR-3 and VEGFR-2, and ICAM-1 proteins were detected by Western blot with respective antibodies. Beta-actin was
used as a loading control and relative levels of VEGFR-3 are indicated below the blot with untreated group as 1.0 (E). VEGFR-3 mRNA
was assessed by qRT-PCR with normalization to gapdh mRNA (F). Data are mean⫾SEM from 3 independent experiments.

miR 200b, and miR200c, which have a higher percentile
score than miR-1236, but do not have predicted 3⬘UTR
target sites that are conserved in mice, making it difficult
to test the effectiveness in vivo. Using qRT-PCR with
TaqMan primers that recognize the mature miRNA spe-
cies, we detected a pan-endothelial expression of miR-
1236. HLEC had higher expression of miR-1236 than
human vein endothelial cells and human aortic EC but
lower than human SMCs (Figure 2B). Expression of
miR-1236 was specific to human cells, using mouse ECs as
a control (Figure 2B). Because lymphatic cells express
endogenous VEGFR-3, we used HLEC to determine the
effect of miR-1236 on VEGFR-3 expression. Next, we
determined if miR-1236 is regulated by inflammatory
cytokines in HLEC. Stimulation of HLEC with inflamma-
tory cytokines that downregulate VEGFR-3 caused an
upregulation of miR-1236 (Figure IIA in the online-only
Data Supplement). Stimulation of HLEC with IL-1␤ led to
the most significant (⬎3-fold) upregulation of miR-1236,
peaking at 24 hours poststimulation (Figure 2C). Expression of the
host gene of miR-1236, negative elongation factor-E, was also
confirmed in HLEC. Furthermore, we observed a trend in the
upregulation of host gene, negative elongation factor-E upon stim-
ulation with IL-1␤ (Figure IIB in the online-only Data Supplement).
The kinetics of miR-1236 induction is consistent with IL-1␤-
induced downregulation of VEGFR-3 at 24 hours (Figure 1E).
These data suggest that IL-1␤–induced miR-1236 may negatively
regulate VEGFR-3 expression in HLEC.
 636 Arterioscler Thromb Vasc Biol
vegfr3 3’UTR
A Species
5'......... GCUAGGAAGAGCAGGACU... Human
... GCUAGGAAGAGCAGGACU... Chimpanzee
... GCUGGGAAGAGCAGAACU... Rhesus
... UCCAGGAAGAGUCAAACU... Mouse
... UCCAGGAAGAGUCAAACU... Rat
|||| |||
3 '...CUCUGUUCCCCUUCUCC.... hsa-miR-1236
B 0.4
0.3
hsa-miR-1236 /U6
0.2
0.1
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0.0
mEC HUVEC HLEC HAEC HVSMC
C *
1.00
hsa-miR-1236 /U6
0.75
0.50
0.25
0.00
- 6h
IL-1β
Figure 2. Endogenous expression and induction of miR-1236 by
interleuklin-1␤ (IL-1␤). A, Cross species sequence alignment of
the putative miR-1236 binding site located in the 3⬘untranslated
region (UTR) (nucleotide 140 –157 following the stop codon
shown) of the vascular endothelial growth factor receptor
(VEGFR)-3 transcript. B, Expression of miR-1236 in mouse en-
dothelium (mEC), human venous endothelial cell (HUVEC),
human lymphatic EC (HLEC), human arterial EC (HAEC), and
human vascular smooth muscle cells (HVSMC). TaqMan primers
were used to probe miR-1236 expression, which was normal-
ized to RNU6B expression using the comparative Ct method. C,
Expression of miR-1236 in HLEC after stimulation with IL-1␤ (5
ng) for 6 hours and 24 hours compared to unstimulated cells.
Data in B and C are mean⫾SEM from duplicates in 3 indepen-
dent experiments. *P⬍0.05.
Endogenous miR-1236 in Primary Human
Lymphatic Endothelial Cells Regulates VEGFR-3
Expression by Binding to the VEGFR-3 3ⴕUTR
We were able to efficiently transfect siRNA into HLEC
(Figure III in the online-only Data Supplement). To verify if
miR-1236 targets VEGFR-3, HLEC were transfected with a
control anti-miR or anti-miR–1236. Inhibition of endogenous
miR-1236 was confirmed by qRT-PCR (Figure 3A). Total
VEGFR-3 was modestly increased in HLEC (Figure 3B),
suggesting that endogenous miR-1236 in primary human
lymphatic endothelial cells may partially contribute to regu-
lation of VEGFR-3 protein expression. To further determine
how miR-1236 regulates VEGFR-3 expression in HLEC, we
took an overexpression approach. Introduction of miR-1236
March 2012
to HLEC in culture resulted in significant downregulation of
total VEGFR-3 protein, but not mRNA, indicating that
miR-1236 targets the VEGFR-3 gene for translational inhi-
bition (Figure 3C and 3D). Importantly, neither VEGFR-2
transcript nor protein was significantly decreased after over-
expression of miR-1236 (Figure 3C and 3D).
In order to confirm VEGFR-3 as a bona fide direct target
of miR-1236, we sought to determine whether miR-1236 was
able to bind to and repress VEGFR-3 mRNA. Therefore, the
entire 1.69 kb VEGFR-3 3⬘UTR, containing the predicted
consensus miR-1236 binding site was cloned into the psi-
CHECK2 luciferase reporter construct. Next, we mutated the
putative binding site of the original construct by substituting
three base pairs (Figure 3E). We then tested these predicted
miRNA/mRNA interactions based on luciferase activity as
subsequently measured in COS cells. In contrast to cotrans-
fection of the VEGFR-3 reporter construct together with a
control miRNA, cotransfection of the VEGFR-3 reporter
construct together with miR-1236 significantly reduced lu-
ciferase activity (Figure 3F). This decrease in luciferase
activity was seen in a concentration-dependent manner (not
shown). Furthermore, cotransfection of miR-1236 with a
mutant VEGFR-3 3⬘UTR reporter construct into which only
a 3-bp mutation in the miR-1236 binding site was introduced
did not repress luciferase activity as seen by miR-1236 on the
wild-type construct (Figure 3F, mut VEGFR-3 3⬘UTR). In
addition, no significant repression was found by cotransfec-
tion of the psiCHECK2 vector backbone together with
miR-1236 (Figure 3F). Taken together, these data suggest
that miR-1236 directly binds to VEGFR-3 to negatively
24h
regulate VEGFR-3 expression.
MiR-1236 Negatively Regulates
VEGFR-3–Dependent Signaling in Primary
Human Lymphatic Endothelial Cells
We then determined if miR-1236 regulates VEGFR-3 signal-
ing in HLEC. PI3K-Akt, and MAPK (ERK1/2) signaling
pathways are reported to be the major downstream effectors
in VEGFR-3 signaling. We have recently shown in HLEC
that VEGF-A activates VEGFR-2, whereas VEGF-C acti-
vates VEGFR-3 strongly and VEGFR-2 weakly. However, a
mutant form of VEGF-C (C156S, CS) specifically binds to
and activates VEGFR-3 but not VEGFR-218 (also see Figure
4). We have also reported that VEGF-C and VEGF-CS
strongly activate the VEGFR-3-Akt axis but not the PLC-␥
axis. In contrast, VEGF-A strongly activates VEGFR-2–
PLC-␥/ERK1/2 pathways.18 Therefore, we examined effects
of miR-1236 on VEGF-C, VEGF-CS, and VEGF-A-induced
signaling in HLEC. Overexpression of miR-1236 in HLEC
significantly reduced the total level of VEGFR-3 as well as
VEGF-C or VEGF-CS–induced phosphorylation of
VEGFR-3 (Figure 4A and 4B). The reduced phosphorylation
of VEGFR-3 was due to reduced total levels of VEGFR-3, as
overexpression of VEGFR-3 lacking the 3⬘UTR rescued
VEGFR-3 signaling (Figure IV in the online-only Data
Supplement). MiR-1236 also weakly reduced VEGF-C–in-
duced phosphorylation of VEGFR-2 without effects on the
total levels of VEGFR-2 (Figure 4A), perhaps partially due to
heterodimerization between VEGFR2 and VEGFR3.26 MiR-
 Jones et al miR-1236 Inhibits VEGFR-3 637
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Figure 3. Endogenous miR-1236 in primary human lymphatic endothelial cells regulates VEGFR-3 protein expression. A-B. Effects of
anti-miR-1236 on vascular endothelial growth factor receptor (VEGFR)-3 expression. Human lymphatic endothelial cells (HLEC) were
transfected with control (Ctrl) anti-miR or anti-miR–1236 (60 nmol/L) for 48 hours. Expression of miR-1236 was determined by quantita-
tive reverse transcription-PCR (qRT-PCR) (A). Data are representative of 3 independent experiments. **P⬍0.01. VEGFR-3 and VEGFR-2
protein expression was determined by Western blot with respective antibodies (B). Similar results were obtained from additional 3
experiments. C, D, Effects of miR-1236 on VEGFR-3 expression. Primary HLEC were transfected with control miRNA mimics or miR-
1236 mimics (40 nmol/L) for 48 hours. Western blot was used to detect VEGFR-3 and VEGFR-2 protein (C). Relative levels of VEGFR-3
(normalized with VEGFR-2) are indicated below the blot with Ctrl miR as 1.0. D, qRT-PCR for VEGFR-2 and VEGFR-3 mRNA and data
are mean⫾SEM from 3 independent experiments. E, Construct design. cDNAs encoding the entire 3⬘untranslated region (UTR) of
VEGFR-3 including the region complementary to the miR-1236 predicted seed sequence in position 144 to 150 of the human VEGFR-3
3⬘UTR (wild-type VEGFR-3 3⬘UTR). The putative binding sequence, GGAAGA, was scrambled to TCTAGA (mut VEGFR-3 3⬘UTR). F,
Luciferase reporter assay COS-7 cells. Ctrl or miR-1236 mimics (60 nmol/L) were cotransfected with the wild-type or mutated 3⬘ UTR
of VEGFR-3, or with the psiCHECK2 vector backbone. Renilla luciferase signal is normalized to the internal firefly luciferase. Data repre-
sent the mean⫾SEM from 4 independent experiments. **P⬍0.01.

1236 –mediated reduction of p-VEGFR-2 was also rescued by
overexpression of VEGFR-3 (Figure IV in the online-only
Data Supplement). Moreover, VEGF-C and VEGF-CS–in-
duced phosphorylation of downstream effectors Akt and
ERK1/2 were also decreased by miR-1236 (Figure 4A and
4B). In contrast, miR-1236 had no significant effect on
VEGF-A–induced VEGFR-2–PLC-␥/ERK1/2 signaling (Fig-
ure 4C). With the exception of VEGFR-3, total levels of
VEGFR-2, Akt, PLC-␥, and ERK1/2 were not altered by
miR-1236 (Figure 4A– 4C). These data suggest that miR-
1236 specifically regulates VEGFR-3– dependent signaling in
lymphatic endothelial cells.
MiR-1236 Regulates VEGFR-3–Dependent
Functions in Primary Human Lymphatic
Endothelial Cells
VEGFR-3 has been shown to be important for growth,
survival, and migratory signals in HLEC.27 Therefore, we
 638 Arterioscler Thromb Vasc Biol March 2012

A ctrl miR miR-1236 B C

ctrl miR miR-1236 ctrl miR miR-1236
0 5 15 30 0 5 15 30 VEGF-C (min) 0 5 15 30 0 5 15 30 VEGF-CS (min) 0 5 15 30 0 5 15 30 VEGF-A (min)
p-VEGFR-3 p-VEGFR-3 p-VEGFR-3
1.0 2.5 2.3 3.3 0.2 0.3 0.4 0.6 1.0 2.4 3.0 3.8 1.0 1.5 1.6 1.7

VEGFR-3 VEGFR-3 VEGFR-3

1.0 1.2 1.2 1.1 0.3 0.4 0.3 0.3 1.0 1.1 1.0 1.1 0.4 0.5 0.4 0.3 1.0 1.1 1.1 1.0 0.3 0.3 0.2 0.2

p-VEGFR-2
p-VEGFR-2 p-VEGFR-2
1.0 5.5 3.0 2.5 1.0 6.5 2.8 2.5
1.0 3.5 4.2 2.8 0.7 2.9 2.7 2.2
VEGFR-2 VEGFR-2
VEGFR-2

p-Akt p-Akt p-PLC-γ

ns
1.0 2.4 4.5 2.5 0.7 1.2 2.5 1.9 1.0 5.2 2.2 1.5 1.0 4.0 2.0 1.4
1.0 3.2 3.3 3.5 0.9 1.9 1.7 1.8
PLC-γ
Akt Akt

p-ERK p-ERK p-ERK

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1.0 3.3 4.5 2.8 0.8 1.3 2.4 1.9 1.0 2.0 3.2 3.8 0.9 1.2 1.7 1.6 1.0 5.0 3.5 2.3 1.0 4.2 3.7 2.6

ERK ERK ERK

Figure 4. miR-1236 regulates vascular endothelial growth factor receptor (VEGFR)-3 dependent signaling in primary human lymphatic
endothelial cells. A, C, Human lymphatic endothelial cells (HLEC) were transfected with control (ctrl) miR or miR-1236. After 48 hours,
cells were subsequently serum-starved overnight followed by treatment with vascular endothelial growth factor (VEGF)-C (100 ng/mL)
(A), VEGF-C Cys156Ser (VEGF-CS, 250 ng/mL) (B), or VEGF-A (50 ng/mL) (C) for indicated times. Phospho- and total VEGFR-3,
VEGFR-2, Akt, ERK, and PLC␥ were detected by Western blot with respective antibodies. Relative levels of phospho- and total pro-
teins were quantified by taking untreated ctrl miR as 1.0. Similar results were obtained from additional two experiments.

determined if miR-1236 repression of VEGFR-3 signaling
would have negative functional outcomes in HLEC on 2
well-known functions for lymphatic endothelial cells-
migration and tube formation. A control miR or miR-1236
was transfected into HLEC, and cell migration was assessed
by a scratch wound assay. Overexpression of miR-1236
significantly reduced scratch closure by HLECs in response
to VEGF-C compared to the control miR (Figure 5A with
quantification in 5B). Proliferation might minimally contrib-
ute to the effect in this assay as cell cycle analysis showed
miR-1236 weakly inhibited VEGF-C (but not VEGF-A)-
induced cell proliferation (Figure V in the online-only Data
Supplement). Next, we determined the effect of miR-1236 on
tube formation by HLEC in a Matrigel assay, in which
endothelial cells align to form elongated tube-like structures.
Compared to the control miR, overexpression of miR-1236 in
HLEC led to a significant reduction in the ability of HLEC to
form tube-like structures in a Matrigel in response to
VEGF-C (Figure 5C with quantification in 5D). Taken
together, these results suggest that miR-1236 can attenuate
VEGF-C/VEGFR-3 mediated functions in HLEC.
MiR-1236 Reduces Lymphangiogenesis and
Angiogenesis In Vivo
Development of the murine retina is well described.28 Inter-
estingly, VEGFR-3 is expressed on the developing retina.29
Because there is no murine homologue to miR-1236, we
injected miR-1236 into mouse tissues to determine an in vivo
effect. Intravitreous injection of FITC-labeled siRNA was
taken up by the retina (Figure VI in the online-only Data
Supplement). After a single intravitreous injection of miR-
1236, we found that miR-1236 was detectable up to 5 days
(Figure 6A), indicating that miR-1236 was stable in the
vitreous chamber and retina. Importantly, VEGFR-3, but not
VEGFR-2 or neuropilin-2 (NRP-2, a VEGF-A coreceptor),
was decreased from mouse retinas that were injected with
miR-1236, relative to control mimics (Figure 6B). Isolectin
B4 staining revealed a tortuous and disorganized pattern of
blood vessels in the right eye (miR-1236) compared to the left
eye (control miR) (Figure 6C). Furthermore, miR-1236 –
treated retina showed dysregulated vascularization with a
reduced number of sprouts on the leading edge (tip cells) and
(Figure 6C with quantification in 6D), consistent with the
phenotypes on VEGFR-3 inhibition as described by Tammela
et al.29 To assess the effects of miR-1236 on ear skin
lymphangiogenesis, we treated 12 days-old mice undergoing
postnatal ear lymphangiogenesis with 2 injections of miR-
1236 and monitored vascular growth 8 days later. At the site
of injection, LYVE-1 positive lymphatic vessels in the ear
were decreased approximately 30% relative to the control
miR (Figure 6E with quantification in 6F). These results
suggest that overexpression of miR-1236 can decrease lym-
phangiogenesis in vivo, through inhibition of VEGFR-3
signaling.
Discussion
In the study presented here, we define a novel mechanism of
VEGFR-3 posttranscriptional regulation mediated by cyto-
kine and microRNA. It was recently found that in the setting
of acute skin inflammation in mice, VEGFR-3 mRNA and
protein is strongly downregulated in inflamed lymphatics.
This decrease correlates with the appearance of inflammatory
cytokines, including IL-1␤.20 IL-1␤, a multifunctional medi-
ator of the inflammatory response, is among the cytokines
 Jones et al miR-1236 Inhibits VEGFR-3 639

A 0h 24 h

--- ---- - ---- -- --- -------

- B
Ctrl miR

30

Wound Healing (% closure)

------- -- -
-------- ---- 20
- Ctrl miR
miR-1236

-- - ----- -- - --------- **
-- - 10
-
miR-1236

--- - --- ----- -- ------ - 0

- -- VEGF-C
D
C
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Ctrl miR miR-1236 25

Branch points/Field
Ctrl miR
20 miR-1236
15 **
10
5
0
VEGF-C
Figure 5. miR-1236 regulates vascular endothelial growth factor receptor (VEGFR)-3 dependent functions in primary human lymphatic
endothelial cells. A, B, Human lymphatic endothelial cell (HLEC) monolayer migration assay. Forty-eight hours after transfection with
control (Ctrl) miR or miR-1236, confluent monolayers of HLEC were subjected to “wound” injury assay in the presence of 3%
serum⫹vascular endothelial growth factor (VEGF)-C (100 ng/mL) for 24 hours. Representative images are shown in A. Scale bar,
100 ␮m. Cell migration distances (mm) were measured and % wound closure was quantified in B. Data are mean⫾SEM from dupli-
cates (10 different areas in each well) of 3 independent experiments. **P⬍0.01. C, D, Tube formation assays in a Matrigel. HLEC were
transfected with Ctrl miR or miR-1236 for 48 hours. Cells were then seeded to Matrigel and cultured with 3% serum⫹VEGF-C (100
ng/mL) for 6 hours and representative images are shown in C. Branch points as indicated by arrowheads were quantified in D. Data are
mean⫾SEM from 3 independent experiments, **P⬍0.01. Scale bar, 100 ␮m.

produced during oxazolone-induced inflammation.20 Despite
IL-1␤ having a lymphangiogenic response in vivo, it was
reported to be indirect, through production of VEGFs.30 –32 To
determine the direct effects of IL-1␤ on HLEC, we intro-
duced IL-1␤ to HLEC in culture. Interestingly, IL-1␤ down-
regulated VEGFR-3 protein but not the transcript in cultured
lymphatic ECs, leading to decreased HLEC function. To
investigate a potential role for miRNA in VEGFR-3 regula-
tion in HLEC, in silico analysis using TargetScan predicted a
conserved binding site in the 3⬘ UTR of VEGFR-3, comple-
mentary to hsa-miR–1236. Hsa-miR–1236 is an experimen-
tally cloned mirtron, first mentioned by Berezikov et al using
a computational screen for conserved mammalian mirtrons.33
We cloned the 3⬘ UTR of VEGFR-3 and subsequent lu-
ciferase assays identified a miR-1236 binding site between
nucleotides 144 and 150 of the human VEGFR-3 3⬘ UTR.
This region of the 3⬘UTR is conserved across species includ-
ing analyses of the human, chimpanzee, rhesus macaque,
mouse, and rat VEGFR-3 UTRs. From our in vitro studies,
we conclude that the VEGFR-3 transcript is a target of
miR-1236. miR-1236 was able to decrease VEGFR-3 protein,
but not VEGFR-3 transcript. Compared to the control miR,
miR-1236 caused reduction of VEGFR-3 protein and led to a
significant reduction in the signaling response to the lymp-
hangiogenic growth factor, VEGF-C. No significant differ-
ence was found after stimulation with a VEGFR-2 specific
ligand, VEGF-A, suggesting a VEGFR-3 specific effect.
Importantly, miR-1236 –mediated inhibition on VEGFR-3
signaling resulted in reduced lymphatic EC migration and
tube formation, as well as lymphangiogenesis in vivo.
Another important finding is that miR-1236 is inducible by
IL-1␤ signaling in lymphatic endothelial cells. These results
support a model that miR-1236 functions as a negative
regulator of VEGF-C/VEGFR-3 signaling and inflammation-
induced lymphangiogenesis (Figure 6G). Inflammatory cyto-
kines such as IL-1␤ contribute to initial lymphangiogenesis,
in part, by inducing expression of VEGFs and adhesion
molecules such as ICAM-1, which recruits inflammatory
cells. IL-1␤ also induces miR-1236, which in turn suppresses
VEGFR-3– dependent signaling. Negative feedback mecha-
nisms have been previously demonstrated for VEGF/VEGFR
signaling. Specifically, VEGF-A via VEGFR-2 induces acti-
 640 Arterioscler Thromb Vasc Biol March 2012

A 300 n=5
C Isolectin B4 E

.....
hsa-miR-1236/U6

...
200

Ctrl miR

Ctrl miR
100

miR-1236 .

miR-1236
0 ..
Ctrl miR miR-1236
(Left eye) (Right eye)

B ctrl miR
D F G
30

LY VE-1+Vascular Area(x 10 5 pixel)

miR 1236 100 2
n=5 Inflammation
n=3
% retina vascularization

VEGFR-3 (IL-1β)
* n=5 *
Tip cell sprouts/hpf
75 20
1.0 0.4 **
VEGF-C
VEGFR-2 50 1
Downloaded from http://atvb.ahajournals.org/ by guest on July 18, 2018

10
VEGFR-3 miRs
NRP-2 25

Actin 0 0 0
Ctrl miR miR-1236 Ctrl miR miR-1236 Ctrl miR miR-1236 Lymphangiogenesis

Figure 6. miR-1236 regulates vascular endothelial growth factor receptor (VEGFR)-3 and (lymph)angiogenesis in vivo. A, B, Four day-
old pups received an intraorbital injection of control miR, left eye (0.3 ␮g) or miR-1236, right eye (0.3 ␮g). Retinas were harvested on
day 4 postinjection and miR-1236 was assessed by quantitative reverse transcription-PCR (qRT-PCR) with normalization to RNU6B (A).
VEGFR-3, VEGFR-2, neuropilin (NRP)-2, and ␤-actin were detected by Western blot with respective antibodies (B). C, D, Retinas were
harvested on day 4 postinjection and dissected, fixed, and stained with isolectin B4. Retina edges at low power images (10⫻) are indi-
cated by dashed lines (left panel). A 20 ⫻ images are shown in the middle. Tip cell sprouts at high power images (40⫻) are indicated
by dots (right panel). Scale bar, 100 ␮m. % retina vascularization areas toward edge were quantified from the low power images (10⫻)
and number of tip cell sprouts (indicated by dots) was quantified from the high power images (40⫻, right) are presented in D. Data in
A–D presented are from 5 retinas for each group. **P⬍0.01. E, F, Control miR or miR-1236 (0.3 ␮g) was injected into the left ear and
right ear, respectively, of 12 days-old mice. Ears of 20-day-old mice were collected and stained with anti-LYVE-1 with quantification of
LYVE-1⫹ areas in F (n⫽3 mice per group). *P⬍0.05. Scale bar, 50 ␮m. G, A model for miR as a negative regulator in inflammation/IL-
1␤ mediated inhibition of VEGFR-3– dependent lymphangiogenesis (see text for details).

vation/expression of Notch, which in turn suppresses vivo caused a significant reduction in tip cells and dysregu-
VEGFR-2 expression/activity, therefore negatively regulating lated angiogenesis of blood vessels. Moreover, lymphatic
angiogenesis.34–36 In addition, VEGFR-2/3 is also negatively density was decreased during postnatal vascular development
regulated by many other mechanisms, including phospha- in tissues examined. These results are consistent with the
tases, endocytosis, and ubiquitination.37–39 VEGFR-2 has importance of VEGFR-3 for lymphangiogenesis and
been recently shown to be regulated by microRNA-424 and angiogenesis.29
microRNA.16,40 Our study has demonstrated, for the first MiR-1236 is an evolutionary recent miRNAas it is only
time, that VEGFR-3 signaling is feedback regulated by found in Pongo pygmaeus, Pan troglodytes, and Homo
microRNA. Our data suggests that miR-1236 activity could sapiens. Because mirtrons are thought to be newly evolved
play a role in the regulation of VEGFR-3 expression in regulators in various animal species with a preexisting ca-
lymphatic and blood vasculature during human hemangio- nonical miRNA pathway, it is thought that the contribution of
genesis. Modulation of VEGFR-3 levels using miR-1236 may mirtrons to the miRNA-mediated regulatory network is
represent a promising approach for the treatment of human smaller than that of canonical miRNAs, owing to their
disease in which a decrease in VEGFR-3 would be beneficial. generally modest expression levels.41 Our data are consistent
We cannot rule out other targets of miR-1236, as exoge- with this finding. By Northern blot, absolute expression of
nous VEGFR-3 failed to completely rescue tube formation miR-1236 is low in HLEC. Furthermore, this may explain
and migration despite rescuing VEGFR-3 signaling (Figure why knockdown of miR-1236 had a modest effect on
IV in the online-only Data Supplement). However, analysis VEGFR-3 protein and function in HLEC (Figure VII in the
of miR-1236 targets did not yield signature genes necessary online-only Data Supplement). It is possible that there may be
for lymphatic signaling in response to VEGF-C such as more mirtrons and conventional miRNAs that target
neuropilin-2. Furthermore, we confirmed that neuropilin-2 VEGFR-3 mRNA to add to the regulation provided by more
levels were not altered by miR-1236 in HLEC and in retina highly expressed miRNAs in the traditional pathway. Indeed,
(Figure 6). Because 3⬘ UTR analysis revealed that this site is knockdown of Dicer in HLECs led to an increase of
evolutionarily conserved in vertebrates, we decided to test the VEGFR-3 in steady state conditions (not shown), supporting
effect of miR-1236 in vivo. Administration of miR-1236 in this hypothesis. Nevertheless, our study indicates that miR-

1236 is a miR that targets VEGFR-3, and should provide a
foundation for us to define more miRNAs regulating
VEGFR-3 signaling and lymphangiogenesis in the context of
inflammation. Moreover, the seemingly dual role of IL-1␤
and other inflammatory cytokines in the promotion and
resolution of lymphangiogenesis is intriguing. Although
IL-1␤ and other inflammatory cytokines mediate inflam-
mation, the kinetics and interplay between these cytokines
and lymphangiogenic factors needs further investigation.
Acknowledgments
We thank Dr Mark Saltzman and Christopher Cheng for labeled
RNAs.
Sources of Funding
This work was supported by NIH grant R01 HL065978 and a
National Nature Science Foundation of China ((81170863). Dennis
Jones is supported by a National Research Service Award (National
Cancer Institute, Ruth Kirschstein Predoctoral Fellowship F31 CA
Downloaded from http://atvb.ahajournals.org/ by guest on July 18, 2018
136316).
Disclosures
None.
References
1. Dumont DJ, Jussila L, Taipale J, Lymboussaki A, Mustonen T, Pajusola
K, Breitman M, Alitalo K. Cardiovascular failure in mouse embryos
deficient in VEGF receptor-3. Science. 1998;282:946 –949.
2. Tammela T, Alitalo K. Lymphangiogenesis: Molecular mechanisms and
future promise. Cell. 2010;140:460 – 476.
3. Kaipainen A, Korhonen J, Mustonen T, van Hinsbergh VW, Fang GH,
Dumont D, Breitman M, Alitalo K. Expression of the fms-like tyrosine
kinase 4 gene becomes restricted to lymphatic endothelium during devel-
opment. Proc Natl Acad Sci U S A. 1995;92:3566 –3570.
4. Joukov V, Pajusola K, Kaipainen A, Chilov D, Lahtinen I, Kukk E,
Saksela O, Kalkkinen N, Alitalo K. A novel vascular endothelial growth
factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR
(VEGFR-2) receptor tyrosine kinases. Embo J. 1996;15:1751.
5. Karkkainen MJ, Haiko P, Sainio K, Partanen J, Taipale J, Petrova TV,
Jeltsch M, Jackson DG, Talikka M, Rauvala H, Betsholtz C, Alitalo K.
Vascular endothelial growth factor C is required for sprouting of the first
lymphatic vessels from embryonic veins. Nat Immunol. 2004;5:74 – 80.
6. Oliver G. Lymphatic vasculature development. Nat Rev Immunol. 2004;
4:35– 45.
7. Alitalo K, Tammela T, Petrova TV. Lymphangiogenesis in development
and human disease. Nature. 2005;438:946 –953.
8. Cueni LN, Detmar M. The lymphatic system in health and disease.
Lymphat Res Biol. 2008;6:109 –122.
9. Veikkola T, Jussila L, Makinen T, Karpanen T, Jeltsch M, Petrova TV,
Kubo H, Thurston G, McDonald DM, Achen MG, Stacker SA, Alitalo K.
Signalling via vascular endothelial growth factor receptor-3 is sufficient
for lymphangiogenesis in transgenic mice. Embo J. 2001;20:1223–1231.
10. Flister MJ, Wilber A, Hall KL, Iwata C, Miyazono K, Nisato RE, Pepper
MS, Zawieja DC, Ran S. Inflammation induces lymphangiogenesis
through up-regulation of VEGFR-3 mediated by NF-kappaB and Prox1.
Blood. 2010;115:418 – 429.
11. Chen L, Fulcoli FG, Tang S, Baldini A. Tbx1 regulates proliferation and
differentiation of multipotent heart progenitors. Circ Res. 2009;105:
842– 851.
12. Jones D, Min W. An overview of lymphatic vessels and their emerging
role in cardiovascular disease. J Cardiovasc Dis Res. 2011;2:141–152.
13. Okamura K, Hagen JW, Duan H, Tyler DM, Lai EC. The mirtron
pathway generates microRNA-class regulatory RNAs in Drosophila. Cell.
2007;130:89 –100.
14. Ruby JG, Jan CH, Bartel DP. Intronic microRNA precursors that bypass
Drosha processing. Nature. 2007;448:83– 86.
15. Suarez Y, Fernandez-Hernando C, Yu J, Gerber SA, Harrison KD, Pober
JS, Iruela-Arispe ML, Merkenschlager M, Sessa WC. Dicer-dependent
Jones et al miR-1236 Inhibits VEGFR-3 641
endothelial microRNAs are necessary for postnatal angiogenesis. Proc
Natl Acad Sci U S A. 2008;105:14082–14087.
16. Pedrioli DM, Karpanen T, Dabouras V, Jurisic G, van de Hoek G, Shin
JW, Marino D, Kalin RE, Leidel S, Cinelli P, Schulte-Merker S, Brandli
AW, Detmar M. miR-31 functions as a negative regulator of lymphatic
vascular lineage-specific differentiation in vitro and vascular devel-
opment in vivo. Mol Cell Biol. 2010;30:3620 –3634.
17. Kazenwadel J, Michael MZ, Harvey NL. Prox1 expression is negatively
regulated by miR-181 in endothelial cells. Blood. 2010;116:2395–2401.
18. Jones D, Xu Z, Zhang H, He Y, Kluger MS, Chen H, Min W. Functional
analyses of the nonreceptor kinase bone marrow kinase on the x chro-
mosome in vascular endothelial growth factor-induced lymphangio-
genesis. Arterioscler Thromb Vasc Biol. 2010;30:2553–2561.
19. Liao S, Ruddle NH. Synchrony of high endothelial venules and lymphatic
vessels revealed by immunization. J Immunol. 2006;177:3369 –3379.
20. Huggenberger R, Siddiqui SS, Brander D, Ullmann S, Zimmermann K,
Antsiferova M, Werner S, Alitalo K, Detmar M. An important role of
lymphatic vessel activation in limiting acute inflammation. Blood. 2011;
117:4667– 4678.
21. Vigl B, Aebischer D, Nitschke M, Iolyeva M, Rothlin T, Antsiferova O,
Halin C. Tissue inflammation modulates gene expression of lymphatic
endothelial cells and dendritic cell migration in a stimulus-dependent
manner. Blood. 2011;118:205–215.
22. Baluk P, Yao LC, Feng J, Romano T, Jung SS, Schreiter JL, Yan L,
Shealy DJ, McDonald DM. TNF-alpha drives remodeling of blood
vessels and lymphatics in sustained airway inflammation in mice. J Clin
Invest. 2009;119:2954 –2964.
23. Kataru RP, Kim H, Jang C, Choi DK, Koh BI, Kim M, Gollamudi S, Kim
YK, Lee SH, Koh GY. T lymphocytes negatively regulate lymph node
lymphatic vessel formation. Immunity. 2011;34:96 –107.
24. Pajusola K, Aprelikova O, Pelicci G, Weich H, Claesson-Welsh L, Alitalo
K. Signalling properties of FLT4, a proteolytically processed receptor
tyrosine kinase related to two VEGF receptors. Oncogene. 1994;9:
3545–3555.
25. Bando H, Brokelmann M, Toi M, Alitalo K, Sleeman JP, Sipos B, Grone
HJ, Weich HA. Immunodetection and quantification of vascular endo-
thelial growth factor receptor-3 in human malignant tumor tissues. Int J
Cancer. 2004;111:184 –191.
26. Dixelius J, Makinen T, Wirzenius M, Karkkainen MJ, Wernstedt C,
Alitalo K, Claesson-Welsh L. Ligand-induced vascular endothelial
growth factor receptor-3 (VEGFR-3) heterodimerization with VEGFR-2
in primary lymphatic endothelial cells regulates tyrosine phosphorylation
sites. J Biol Chem. 2003;278:40973– 40979.
27. Makinen T, Veikkola T, Mustjoki S, Karpanen T, Catimel B, Nice EC,
Wise L, Mercer A, Kowalski H, Kerjaschki D, Stacker SA, Achen MG,
Alitalo K. Isolated lymphatic endothelial cells transduce growth, survival
and migratory signals via the VEGF-C/D receptor VEGFR-3. Embo J.
2001;20:4762– 4773.
28. Stahl A, Connor KM, Sapieha P, Chen J, Dennison RJ, Krah NM,
Seaward MR, Willett KL, Aderman CM, Guerin KI, Hua J, Lofqvist C,
Hellstrom A, Smith LE. The mouse retina as an angiogenesis model.
Invest Ophthalmol Vis Sci. 2010;51:2813–2826.
29. Tammela T, Zarkada G, Wallgard E, Murtomaki A, Suchting S, Wir-
zenius M, Waltari M, Hellstrom M, Schomber T, Peltonen R, Freitas C,
Duarte A, Isoniemi H, Laakkonen P, Christofori G, Yla-Herttuala S,
Shibuya M, Pytowski B, Eichmann A, Betsholtz C, Alitalo K. Blocking
VEGFR-3 suppresses angiogenic sprouting and vascular network for-
mation. Nature. 2008;454:656 – 660.
30. Nakao S, Kuwano T, Tsutsumi-Miyahara C, Ueda S, Kimura YN,
Hamano S, Sonoda KH, Saijo Y, Nukiwa T, Strieter RM, Ishibashi T,
Kuwano M, Ono M. Infiltration of COX-2-expressing macrophages is a
prerequisite for IL-1 beta-induced neovascularization and tumor growth.
J Clin Invest. 2005;115:2979 –2991.
31. Watari K, Nakao S, Fotovati A, Basaki Y, Hosoi F, Bereczky B, Higuchi
R, Miyamoto T, Kuwano M, Ono M. Role of macrophages in inflam-
matory lymphangiogenesis: enhanced production of vascular endothelial
growth factor C and D through NF-kappaB activation. Biochem Biophys
Res Commun. 2008;377:826 – 831.
32. Lin CH, Lu J, Lee H. Interleukin-1beta expression is required for lyso-
phosphatidic Acid-induced lymphangiogenesis in human umbilical vein
endothelial cells. Int J Inflam. 2010;2011:351010.
33. Berezikov E, Chung WJ, Willis J, Cuppen E, Lai EC. Mammalian mirtron
genes. Mol Cell. 2007;28:328 –336.
 642 Arterioscler Thromb Vasc Biol March 2012
34. Williams CK, Li JL, Murga M, Harris AL, Tosato G. Up-regulation of the
Notch ligand Delta-like 4 inhibits VEGF-induced endothelial cell
function. Blood. 2006;107:931–939.
35. Ridgway J, Zhang G, Wu Y, Stawicki S, Liang WC, Chanthery Y,
Kowalski J, Watts RJ, Callahan C, Kasman I, Singh M, Chien M, Tan C,
Hongo JA, de Sauvage F, Plowman G, Yan M. Inhibition of Dll4 sig-
nalling inhibits tumour growth by deregulating angiogenesis. Nature.
2006;444:1083–1087.
36. Noguera-Troise I, Daly C, Papadopoulos NJ, Coetzee S, Boland P, Gale
NW, Lin HC, Yancopoulos GD, Thurston G. Blockade of Dll4 inhibits
tumour growth by promoting non-productive angiogenesis. Nature. 2006;
444:1032–1037.
37. Nakamura Y, Patrushev N, Inomata H, Mehta D, Urao N, Kim HW,
Razvi M, Kini V, Mahadev K, Goldstein BJ, McKinney R, Fukai T,
Ushio-Fukai M. Role of protein tyrosine phosphatase 1B in vascular
endothelial growth factor signaling and cell-cell adhesions in endothelial
cells. Circ Res. 2008;102:1182–1191.
Downloaded from http://atvb.ahajournals.org/ by guest on July 18, 2018
38. Bruns AF, Herbert SP, Odell AF, Jopling HM, Hooper NM, Zachary IC,
Walker JH, Ponnambalam S. Ligand-stimulated VEGFR2 signaling is
regulated by co-ordinated trafficking and proteolysis. Traffic. 2010;11:
161–174.
39. Karpanen T, Heckman CA, Keskitalo S, Jeltsch M, Ollila H, Neufeld G,
Tamagnone L, Alitalo K. Functional interaction of VEGF-C and VEGF-D
with neuropilin receptors. Faseb J. 2006;20:1462–1472.
40. Chamorro-Jorganes A, Araldi E, Penalva LO, Sandhu D, Fernandez-
Hernando C, Suarez Y. MicroRNA-16 and microRNA-424 regulate
cell-autonomous angiogenic functions in endothelial cells via tar-
geting vascular endothelial growth factor receptor-2 and fibroblast
growth factor receptor-1. Arterioscler Thromb Vasc Biol. 2011;31:
2595–2606.
41. Martin R, Smibert P, Yalcin A, Tyler DM, Schafer U, Tuschl T, Lai EC.
A drosophila pasha mutant distinguishes the canonical microRNA and
mirtron pathways. Mol Cell Biol. 2009;29:861– 870.
 Mirtron MicroRNA-1236 Inhibits VEGFR-3 Signaling During Inflammatory
Downloaded from http://atvb.ahajournals.org/ by guest on July 18, 2018

Lymphangiogenesis
Dennis Jones, Yonghao Li, Yun He, Zhe Xu, Hong Chen and Wang Min

Arterioscler Thromb Vasc Biol. 2012;32:633-642; originally published online January 5, 2012;
doi: 10.1161/ATVBAHA.111.243576
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 SUPPLEMENTAL MATERIAL

Mirtron MicroRNA-1236 Inhibits VEGFR-3 Signaling during Inflammatory Lymphangiogenesis

Dennis Jones1, Yonghao Li1,2, Yun He1, Zhe Xu1, Hong Chen3, Wang Min1,2,4

1
Interdepartmental Program in Vascular Biology and Therapeutics, Department of Immunobiology, Yale
University School of Medicine, 10 Amistad St., New Haven, CT 06520. 2State Key Laboratory of
Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
3
Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City,
OK 73104.
4
Corresponding author: Dr. Wang Min, Interdepartmental Program in Vascular Biology and
Therapeutics, Department of Pathology, Yale University School of Medicine, 10 Amistad St., New
Haven, CT 06520. Tel: 203-785-6047; Fax: 203-737-2293; Email: wang.min@yale.edu.

  1  
SUPPLEMENTAL MATERIALS AND METHODS

Cell Culture and growth factors. HLEC (HMVEC-dLyAd) were purchased from Lonza. HLEC were
cultured in EGM-2 MV media. HUVEC were maintained in M199 media supplemented with 20% FBS,
100 U/ml penicillin, 100µg/ml streptomycin, 2 mM L-glutamine, and 50µg/ml EC growth supplement. All
flasks for ECs were coated with 0.1% gelatin. COS-7 cells were cultured in DMEM with 10% FBS. All
cells were cultured at 37°C in 5% CO2. Recombinant human VEGF-A, VEGF-C, VEGF-C (Cys156Ser),
IL-1α, IL-1β, TNFα, IFN-g and IL-6 were purchased from R&D Systems.

Transfections. COS-7 cells were co-transfected with 0.25 µg of the indicated reporter construct and 60
nM of miRIDIAN miR-1236 mimic (miR-1236) or 60 nM negative control mimic sequences (Ctrl mimic)
(Dharmacon and Ambion) using Lipofectamine 2000 (Invitrogen). HLEC were plated on 12 or 24-well
dishes or slides, cultured overnight and subsequently transfected with 40 nM (final concentration) Ctrl
mimic or miRNA-1236 mimic using Oligofectamine (Invitrogen). Cells were harvested 48 h later and
subjected to analyses. For miRNA, inhibitor experiments, 60 nm of control inhibitor or miRNA-1236
inhibitor was used. Control siRNA and Dicer siRNA were purchased from Dharmacon and were
transfected using Oligofectamine (Invitrogen) Cells were harvested 48-72 h later and subjected to
analyses.

Antibodies. Antibodies against phospho-VEGFR-2 (pY1054/1059), VEGFR-2, VEGFR-3, phospho-

PLC-γ (pY783) and PLC-γ, phospho-Akt (Ser-473), Akt, phospho-ERK1/2 (Thr202/Tyr204), ERK 1/2,
and β-actin were purchased from Cell Signaling Technology; p-VEGFR-3 was from Cell Applications,
Inc. Anti-mouse VEGFR-3 was from eBioScience.

Cell surface immunostaining. HLEC were transfected with control mimic or miR-1236 mimic, control
inhibitor or miR-1236 inhibitor as indicated, stained with directly APC-conjugated mAbs against VEGFR-
3 (R&D Systems) and analyzed on a FACS Calibur (BD Biosciences) using FloJo analysis software,
collecting 10,000 gated cells per sample.

RNA Analysis. MicroRNAs analyses were described in the text. Total RNAs were isolated from cells or
lymph nodes using RNeasy kit (Qiagen) according to the manufacturer’s instructions. Reverse
transcription was done using a standard procedure (SuperScript First-Strand Synthesis System;
Invitrogen) using 0.5-1 mg total RNA. qRT-PCR was performed by using iQ SYBR Green Supermix on
C1000 Thermal Cycler (Bio-Rad). Specific primers for Vegfr3, Vegfr2, and nelf-e were used. Gapdh
RNA was used as an internal control.

Western Blotting. All protein samples were boiled for 5 minutes, resolved in 8-10% polyacrylamide
gels and transferred to polyvinylidene difluoride membrane and blocked with 5% milk diluted in PBS
containing 0.05 % Tween 20 (PBST). Membranes were then immunoblotted with the specified
antibodies (1:1000 dilution in PBS containing 0.05 % Tween 20 (PBST) using horseradish peroxidase–
conjugated secondary antibodies (1:2,000 dilution; GE Healthcare Life Sciences/Amersham
Biosciences) and enhanced chemiluminescence detection system (GE Healthcare Life Sciences,
Amersham Biosciences). Densitometry of original film used for western blot quantifications.

Immunofluorescence microscopy (IF). Whole mount tissue sections were harvested, fixed with 2%
PFA in PBS for 15 minutes at room temperature, permeabilized with 0.1% triton-X buffer, blocked in 5%
rabbit or horse serum diluted in PBS overnight. Tissues were stained at 4 °C overnight using specified
antibodies, followed by Alexa Fluor 488- or 594-conjugated secondary antibodies (donkey anti-goat,
donkey anti-rabbit, donkey anti- mouse (Invitrogen Molecular Probes, Eugene, OR). Tissues were

  2  
mounted in mounting media with DAPI (VECTASHIELD). Slides were observed using a Zeiss Axiovert
200 fluorescence microscope (Carl Zeiss MicroImaging; Thornwood, NY), and images were captured
using Openlab3 software (Improvision, Lexington, MA). For lymph nodes, 5 -µm serial sections cut from
frozen, OCT-embedded tissues were fixed in -20 °C acetone for 10 minutes, dried for 15 minutes,
followed by the same blocking/antibody protocol for cells as listed above. Positive staining was
quantified with Image Pro Plus Software(MediaCybernetics). The degree of positive staining is
expressed as a fraction of the total area of the lymph node.

Lymphatic EC migration. For monolayer migration, HLEC were seeded onto gelatin-coated 12-well
tissue culture plates and grown to confluence. Briefly, HLEC were transfected with mimic. On the next
day, serum starved cells were subjected to “wound injury” assay with a 200 µl plastic pipette tip. Fresh
media containing 3% serum alone or serum in addition to specified growth factors and were further
cultured for 24h. The LEC migration in culture was determined by measuring wound areas in cell
monolayers. Wound images were captured by a digital camera under a Zeiss Axiovert microscope
(10X). Wound healing (% closed) was measured and analyzed by NIH Image 1.60
(http://rsbweb.nih.gov/nih-image/).

Lymphatic EC tube formation. LEC tube formation on Matrigel was performed as we described
previously 1. HLEC were seeded onto gelatin coated 6-well tissue culture plates were and grown to
70% confluence. HLEC were transfected with mimic. 48 h later, 80,000 serum-starved cells were plated
on Matrigel (0.2 ml) in a 24-well plate. 6h later, cells were visualized and photographs were taken using
a Zeiss Axiovert microscope. The number of branch points was measured and analyzed by Image Pro
Plus Software. Tube length and area per field (20x) were calculated using Image Pro Plus Software.

Lymphatic EC DNA Analysis. Briefly, 70% ethanol was added to each sample, followed by RNAse
digestion at 37 degrees for 30 minutes. 10µg/ml propidium iodide solution was added. Cells were
analyzed on a FACS calibur.

In vivo assays. All animal studies were approved by the Institutional Animal Care and Use Committee
(IACUC) of Yale University. Male or female littermates BL/6 mice were used for all experiments. 12 d
old mice received IP injections (3.5 µg) followed by an additional injection 4 days later. 20 d old mice
were killed and ears or diaphragms were dissected and stained with CD31 or LYVE-1 specific antibody,
followed by tissue fixation in 4% PFA. For retina studies, 4-day-old pups received an intraorbital
injection of microRNA (0.3 µg). Retinas were harvested 5 days later, dissected, fixed, and stained with
isolectin B4 (Sigma). Oxazolone immunization was performed as skin painting of 50 µl per LN area of
4% Oxazolone (Sigma-Aldrich) in acetone. Oxazolone was administered on shaved skin on the
abdomen in proximity to inguinal and brachial lymph nodes. Oxazolone was applied once. LNs were
harvested at different time 4 and 7 days after application.

Statistical analyses. All data are expressed as mean ± SEM. Statistical analysis was performed with a
two-tailed Student’s paired t-test. Statistical significance for p-values are as follows: *, < 0.05; **, <0.01;
***, <0.001. For analysis, positive staining and area was quantified with Image Pro Plus Software
(MediaCybernetics).

1. Jones D, Xu Z, Zhang H, He Y, Kluger MS, Chen H, Min W. Functional analyses of the bone
marrow kinase in the X chromosome in vascular endothelial growth factor-induced
lymphangiogenesis. Arterioscler Thromb Vasc Biol. 2010;30:2553-2561.

  3  
Supplemental Figure I

A 0.006 C
0.0035
- IL-1α IL-1β IFNγ TNF-α IL-6
0.005 0.0030

0.004 0.0025

vegf-c/gapdh
vegf-a/gapdh

0.0020 VEGFR-3
0.003
0.0015
0.002
0.0010 VEGFR-2
0.001
0.0005
0.000 0.0000
Day 0 Day 3 Day 7 Day 0 Day 3 Day 7

B
0.15 0.0003 0.7
0.6
0.5
0.10 0.0002
ifn-g/gapdh

il-1b/gapdh
tnf/gapdh

0.4
0.3
0.05 0.0001
0.2
0.1
0.00 0.0000 0.0
Day 0 Day 3 Day 7 Day 0 Day 3 Day 7 Day 0 Day 3 Day 7

Supplemental Fig.I. A. Real-time RT-PCR analyses were performed using RNA from whole LN (inguinal) harvested from mice at day 0
(unstimulated), day 3 and 7 after oxazolone challenge (n=2 per group). SYBR Green-based primers were used to detect (lymph)angiogenic
factor genes (A) and inflammatory cytokine genes (B). (C) HLEC were unstimulated(-) or stimulated for 24 hrs with IL-1α (20ng/ml),
IL-1β (5 ng/ml), IFN-γ (10 ng/ml), TNF-α (10 ng/ml), or IL-6 (10ng/ml). VEGFR-3 and VEGFR-2 proteins were detected by Western blot with
respective antibodies.
 Supplemental Figure II

A B
0.7 0.25

0.6
0.20
0.5

hsa-miR-1236 /U6

nelf-e/gapdh
0.15
0.4
0.3 0.10
0.2
0.05
0.1
0.0 0.00
- IL-1α IL-1β TNFα IL-1β (hrs) - 6 24

Supplemental Fig.II. Expression and regulation of endogenous miR-1236 in primary human lymphatic endothelial cells.
A. Expression of miR-1236 in HLEC.TaqMan primers were used to probe miR-1236 expression with normalization
to RNU6B using the comparative Ct method for unstimulated cells(-) or cells stimulated for 24 hrs with IL-1α
(20ng/ml), IL-1β (5 ng/ml), or TNF-α (10 ng/ml). B. Quantification, as assessed by real-time RT-PCR, of nelf-e mRNA
levels in HLEC after stimulation stimulated with IL-1β (5 ng) for 6 and 24 h. Unstimulated HLEC(-) were used as reference.
Nelf-e mRNA was normalized to gapdh mRNA Data are representative of 3 independent experiments.
Supplemental Figure III

FITC dsRNA

Unlabeled
dsRNA

Supplemental Fig.III. Transfection efficiency of labeled RNA. Unlabeled and FITC-labeld dsRNA (20 nM)
were transfected into HLEC for 48 h. Brightfield and Immunofluorescent images were taken.
 Supplemental Figure IV

A Ctrl Lentivirus VEGFR-3 Lentivirus D Ctrl lentivirus VEGFR-3 lentivirus

- VEGF-C - VEGF-C
36

36

36

36

Ctrl miR
CM

CM

CM

CM
12

12

12

12
pVEGFR-3

VEGFR-3

pVEGFR-2

miR-1236
VEGFR-2
B Ctrl Lentivirus VEGFR-3 Lentivirus
2
p-VEGFR-3/VEGFR-3

E
40 Ctrl Lentivirus
1 VEGFR-3 Lentivirus
Branch Point/Field

30
*
0
20
36

36

36

36
CM

CM

CM

CM
12

12

12

12

- VEGF-C - VEGF-C 10
C Ctrl Lentivirus VEGFR-3 Lentivirus
1.25
0
p-VEGFR-2/VEGFR-2

1.00
Ctrl miR miR-1236

0.75 Supplemental Fig.VI. VEGFR-3 rescued VEGF-C signaling. HLECs were transfected with ctrl miR or miR1236.
After 24 h, HLEC were infected with VEGFR-3 lentivirus for 24 h. Cells were then serum starved for 24 h and left
0.50
unstimulated or stimulated with VEGF-C (100ng/ml) for 20 minutes and subjected to Western blot with phospho-
0.25 specific antibodies/total antibodies (A). Quantification of p-VEGFR-3/VEGFR-3 and p-VEGFR-2/VEGFR-2 shown in
0.00
B and C, respectively. D-E. Tube formation assays in a Matrigel. Cells were then seeded to Matrigel and cultured
with 3% serum + VEGF-C (100 ng/ml) for 6 h and representative images are shown in D. Branch points as
36

36

36

36
CM

CM

CM

CM

indicated by arrowheads were quantified from three independent experiments in E. Data are mean ± SEM ,
12

12

12

12

- VEGF-C - VEGF-C *, p<0.05 comparing Ctrl lentivirus vs VEGFR-3 groups. Scale bar, 100 µm.
 Supplemental Figure V

A VEGF-C B VEGF-A
80 Ctrl Mimic
100
Ctrl Mimic 70 miR-1236

% cells gated
miR-1236 60

% cells gated
75
50
40
50
30
20
25
10
0
0 S
G0/G1 G2/M
G0/G1 S G2/M

Supplemental Fig.V. Effects of miR-1236 on HLEC proliferation. 48 h after transfection with ctrl miR
or miR1236, primary HLECs were serum starved overnight and stimulated with 100 ng VEGF-C (A) or
VEGF-A (B) for 24 hrs and subjected to FACS analysis and the relative G1, S, and G2/M compartments
calculated. Data are representative of three independent experiments.
Supplemental Figure VI

Ctrl RNA FITC dsRNA

Supplemental Fig.VI. Uptake of RNA in retina. 4 day old pups received an intraorbital injection of 0.3µg of control dsRNA
at left eye or FITC-labeled dsRNA at right eye. Retinas were harvested on day 4 post-injection and fluorescence was visualized.
Representative images from 3 pairs of mice.
 Supplemental Figure VII

A 0 48 h C

Ctrl AM

Ctrl AM
Anti- miR 1236

Anti- miR 1236

-

B D
100 * 25 *
Wound Healing (% closure)

80 20

Branch points/Field
60 15

40 10

20 5

0 0
Ctrl AM Anti-miR 1236 Ctrl miR Anti-miR 1236

Supplemental Fig.VII. Marginal effects of anti-miR-1236 on in vitro lymphangiogenesis.

A-B. HLEC were transfected with ctrl anti-miR or anti-miR 1236. 48 h after transfection with ctrl anti- miR or
anti-miR-1236, confluent monolayers of HLEC were subjected to “wound” injury assay in the presence of 3%
serum + VEGF-C (100 ng/ml) for 48 h. Representative images are shown in A. Scale bar, 100 µm. Cell migration
distances (mm) were measured and % wound closure was quantified in B. Data are mean±SEM from duplicates
(10 different areas in each well) of three independent experiments. *, P<0.05. C-D. Tube formation assays in a
Matrigel. HLEC were transfected with ctrl anti-miR or anti-miR-1236 for 24 h. Cells were then seeded to Matrigel
and cultured with 3% serum + VEGF-C (100 ng/ml) for 6 h and representative images are shown in C. Branch points
as indicated by arrowheads were quantified in D. Data are mean ± SEM from three independent experiments,
*, p<0.05. Scale bar, 100 µm.