Department of Reproductive Endocrinology, University Hospital Zürich, Switzerland
P041
Inhibition of microRNA-221 by Estradiol Contributes to its Differential Effects on
Smooth Muscle Cell Growth and Endothelial Cell Capillary Formation
Lisa Rigassi, Elisabeth Unterleutner, Federica Barchiesi, Bruno Imthurn, Raghvendra K Dubey
Results
C
50
0
E2 10nM
*
50
0
AC
A221
MC
M221
B
0h
500 μm
24h
100
*
C
PDGF MC M221
*
200
C
100
C
PDGF
MC
M221
CyclinD1
β-Actin
0
200
C
E
PDGF MC M221
*
*
C
PDGF
MC
M221
0h
100
24h
0
C
E2 10nM
*
B
A
A221
MC
B
M221
1.0
Fig. 1 Effect of E2, miR-221 antimir and miR-221 mimic on
HUVEC activity. HUVECs were transfected with miR-221
antimir and mimic (A221, M221) and the respective controls
(AC, MC) or treated with 10nM E2. A. Capillary formation:
images were taken 18h after cell seeding on 2D-Matrigel,
30min after E2 treatment or 24h post-transfection. B. Scratch
wound assay: with or without E2 or 48h after transfection.
*
0.5
0.0
*
2.0
§
1.5
1.0
0.5
0.0
0
500 μm
50
0
0
C
400
0
10 100
PDGF + E2 (nM)
*
§
300
200
100
AC
AC A221
PDGF
*
100
§
*
50
10
C
E2 (nM)
0
10
100
PDGF + E2 (nM)
Fig. 4 Relative expression of
miR-221 in HUVECs (A) and
HCASMCs (B), treated for
24h with or without 10100nM E2 and 20ng/ml
PDGF-BB, was examined
using RT-PCR after RNA
extraction. U48 and U49
were used as endogenous
controls.
10
100
0
0
200
24h
0
C
AC
0
10 100
PDGF + E2
*
150
*
AC
§
500 μm
*
§
50
24h
0h
AC A221
PDGF
100
100
AC
50
A221
0
0
10 100
PDGF + E2 (nM)
AC
AC A221
PDGF
PDGF + E2 (nM)
E
C
50
0
AC
100
0h
C
0
4. E2 Inhibits miR-221 Expression
in HUVECs and HCASMCs
miR-221
relative expression
0
50
150
*
500 μm
PDGF MC M221
miR-221
relative expression
50
200
150
100
100
§ 100
C
Wound closure
Area % of control
100
150
Wound closure
Area % of control
*
150
*
C
*
D
50
C
0
Wound closure
Area % of control
Human Umbilical Vein ECs (HUVECs) and Human
Coronary Artery SMCs (HCASMCs) were treated
with or without E2 (10-100nM) and PDGF-BB
(20ng/ml) prior to RNA extraction and RT-qPCR. To
assess the role of miR-221, both cell types were
transfected with 25 nM miR-221 mimic and antimir
or their respective controls. Cell counts and a BrdU
ELISA kit were employed to study HCASMC
proliferation. HCASMC migration was assessed
using scratch wound assay. Matrigel capillary
formation and scratch wound assay were used to
investigate HUVEC activity. CyclinD1 protein
expression was quantified by Western Blotting.
Experiments were repeated three times in
triplicates.
*p<.05 to respective control; §p<.05 as indicated.
D
200
*
250
PDGF + E2 (nM)
100
*
A
PDGF
50
B
300
0
300
BrdU incorporation
rlu % of control
100
150
Wound closure
Area % of control
Capillary length
% of control
100
*
150
Capillary length
% of control
*
150
*
Cell number
% of control
M221
BrdU incorporation
rlu % of control
MC
Fig. 2 Effect of PDGF and miR-221 mimic
on HCASMC proliferation and migration.
HCASMCs were transfected with control
and miR-221 mimics (MC, M221) or treated
with 20ng/ml PDGF-BB. A. Cells were
counted 3 days after treatment or
transfection. B. BrdU incorporation ELISA.
CyclinD1 protein expression (C) and
Wound scratch assay (D, E): 48h after
treatment or transfection.
Cell number
% of control
A221
100 μm
0
Methods
AC
400
Cell number
% of control
E2 10nM
A
BrdU incorporation
Rlu % of control
A
C
3. Downregulation of miR-221 Inhibits
HCASMC Growth Similar to E2
2. miR-221 Induces HCASMC Growth
Similar to PDGF-BB
Wound closure
Area % of control
1. miR-221 Antimir Mirrors the Actions of
E2 on HUVEC Activity, whereas miR-221
Mimic has Opposite Effects
Capillary length
% of control
MicroRNAs play a key role in vascular remodeling
associated
with
cardiovascular
disease.
MicroRNA-221 (miR-221) actively contributes to
injury-induced neointima formation by inhibiting
endothelial cell (EC) growth and promoting smooth
muscle cell (SMC) growth. Since estradiol (E2)
prevents neointimal thickening by differentially
modulating EC and SMC growth, we hypothesize
that E2 mediates its vasoprotective actions by
downregulating miR-221 expression and
abrogating its effects on SMC and EC growth.
Wound closure
Area % of control
Objective
0
10
PDGF
100
AC
CyclinD1
CyclinD1
β-Actin
β-Actin
AC
A221
Fig. 3 Effect of miR-221 antimir and E2 on HCASMC proliferation and
migration. Cells were transfected with control and miR-221 antimirs
(AC, A221) or treated with or without 100nM E2 in presence of 20ng/ml
PDGF-BB. A. Cells were counted 3 days after treatmen or transfection.
B. BrdU incorporation ELISA. CyclinD1 protein expression (E) and
Wound scratch assay (C, D): 48h after treatment or transfection.
Outcomes
miR-221
Capillary formation
Wound closure
vascular
protection
Disclosures: None
Acknowledgement: This project is supported by the SNF Grant 31003A_138067 and IZZERO_142213/1
MC
M221
100
Migration
Proliferation
- Cell number
- DNA synthesis
- CyclinD1
150
B
*
§
C
*
50
0
150
250
MC
M221
§
§
200
150
*
100
50
0
MC
M221
*
D
§
100
50
0
C
E2 10nM
300
MC
M221
200
§
*
§
*
100
0
C
0
10
100
PDGF + E2 (nM)
Fig. 5 Cells were transfected with miR221 mimic (M221) and the respective
control (MC) and treated with E2
simultaneously. E2-induced capillary
formation (A) and wound closure (B) in
HUVECs were abrogated in presence
of M221. Inhibitory effects of E2 on
PDGF-induced HCASMCs proliferation
(C, cell count after 3 days) and
migration (D, scratch wound assay,
24h) were also reversed by M221.
6. E2 Reduces miR-221 Expression via ERα
A
miR-221
relative expression
miR-221
A
Wound closure
Area % of control
HCASMC
Microvessel length
% of control
HUVEC
Cell number
% of control
E2
Wound closure
Area % of control
Our findings provide the first evidence that
E2 inhibits miR-221 production in
HCASMCs and HUVECs and these effects
contribute, in part, to the antimitogenic
actions of E2 on HCASMCs and the
capillary formation inducing effects of E2 in
HUVECs. Modulation of miR-221 by E2
represents a novel mechanism by which E2
may mediate its differential effects on SMC
and EC growth and confer vascular
protection
5. miR-221 Mimic Abrogates the Growth Effects of E2
in both HCASMCs and HUVECs
1.0
*
*
B
C
1.0
1.0
*
*
*
0.5
0.5
0.5
0.0
0.0
0.0
C
E2 PPT DPN G-1
PDGF
C
E2 PPT C
- MPP
E2 PPT
+ MPP
C
E2
- ICI
C
E2
+ ICI
Fig. 6 Relative expression of miR-221 in HCASMCs. A. Treatment for
24h with 100nM E2, and Estrogen Receptor (ER) agonists PPT (ERα),
DPN (ERβ) and G-1 (GPER) in presence of 20ng/ml PDGF-BB. ERα
specific antagonist MPP (500nM, B) and unspecific ER antagonist ICI182-782 (ICI, 1uM, C) were added 30min prior agonists and PDGF-BB.
U48 and U49 were used as endogenous controls.