Abstract
Background:
Vascular injury is an early manifestation of hypertension. microRNAs (miRNAs) play an important role in cardiovascular disease, but their implication in vascular injury remains unclear. Using small and total RNA sequencing, we identified in murine mesenteric arteries (MAs) a conserved angiotensin (Ang) II-upregulated Dlk1-Dio3 miRNA miR-431 that correlated with blood pressure (BP), and an Ang II-downregulated BP-correlated conserved putative miR-431 target, the transcriptional factor ETS homologous factor (
Ehf
). miR-431 might be involved in vascular remodeling as
Ehf
regulates expression of extracellular matrix genes including alpha-1 type I collagen (
Col1a1
) and of other Dlk1-Dio3 miRNAs. In this study, we proposed to validate the miR-431-
Ehf
-
Col1a1
interaction
in vitro
and
in vivo
, and determine whether miR-431 inhibition antagonizes angiotensin (Ang) II-induced hypertension and vascular injury.
Methods and Results:
Transfection of miR-431 mimics into human aortic smooth muscle cells decreased
Ehf
expression (0.13±0.05 fold,
P
<0.001) and increased
Col1a1
(1.7±0.5 fold,
P
<0.01), whereas miR-431 inhibitors increased
Ehf
(1.5±0.2 fold,
P
<0.001) and decreased
Col1a1
(0.89±0.11 fold,
P
<0.05).
Ehf
siRNA transfection increased 1.2±0.1 fold
Col1a1
(
P
<0.01). Co-transfection of miR-431 mimics with luciferase reporter vectors that contain the wild-type but not mutated miR-431 human
EHF
3’ UTR binding site decreased 0.51±0.01 fold (
P
<0.05) luciferase expression compared to scrambled mimics. miR-431 inhibitor IV injection in mice at day 0 and 7 of Ang II infusion decreased miR-431 (0.16±0.05 fold,
P
<0.01),
Col1a1
(0.58±0.11 fold,
P
<0.05), increased
Ehf
(2.9±0.8 fold,
P
<0.05) in MAs, delayed BP elevation (
P
<0.01), improved endothelium-dependent relaxation (33±8 vs 64±7%,
P
<0.05) and reduced vascular stiffness (strain at 140mmHg: 0.68±0.02 vs 0.58±0.02 ΔD/D,
P
<0.01) compared to scrambled mimics-injected Ang II-infused mice.
Conclusion and Perspectives:
miR-431 and its target
Ehf
may act as master regulators in the pathophysiology of vascular damage in hypertension. miR-431 inhibition has the potential to serve as a novel therapeutic approach for treatment of vascular damage and hypertension.