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\begin{document}

\title{The antagonistic effects and mechanisms of microRNA-26a action in
hypertensive vascular remodeling}



\author[1]{Wenqian Zhang}%
\author[1]{Qiaozhu Wang}%
\author[1]{Xin Xing}%
\author[1]{Lijun Yang}%
\author[1]{Min Xu}%
\author[1]{Chunhui Cao}%
\author[1]{Rong Wang}%
\author[1]{Weicheng Li}%
\author[1]{Xiaolin Niu}%
\author[2]{Dengfeng Gao}%
\affil[1]{Xi'an Jiaotong University Second Affiliated Hospital}%
\affil[2]{2nd affiliated hospital of Xi'an Jiaotong University}%


\vspace{-1em}



  
  \date{May 25, 2024}


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\selectlanguage{english}
\begin{abstract}
Background and Purpose: Hypertensive vascular remodeling (VR) is
responsible for end-organ damage and is the result of increased
extracellular matrix accumulation and excessive vascular smooth muscle
cell (VSMC) proliferation. MicroRNA-26a (miR-26a), a non-coding small
RNA, is involved in multiple cardiovascular diseases. We aimed to
validate the effect and mechanisms of miR-26a in hypertensive VR.
Experimental Approach: Spontaneously hypertensive rats (SHRs) were
injected intravenously with recombinant adeno-associated virus-miR-26a.
In vitro experiments, angiotensin II (AngII)-induced VSMCs were
transfected with miR-26a mimic or inhibitor. Key Results: We found
miR-26a downregulated in the thoracic aorta and plasma of SHRs.
Overexpression of miR-26a inhibited extracellular matrix deposition by
targeting connective tissue growth factor (CTGF) and mitigated VSMC
proliferation by regulating the enhancer of zeste homolog 2 (EZH2)/p21
pathway both in vitro and in vivo. AngII-mediated Smad3 activation
suppressed miR-26a expression, which in turn promoted Smad3 activation
via targeted regulation of Smad4, leading to further downregulation of
miR-26a. Conclusion and Implications: Our study reveals that AngII
stimulates a Smads/miR-26a positive feedback loop, which further reduces
miR-26a expression, leading to collagen production and VSMC
proliferation and consequently, VR. MiR-26a has an antagonistic effect
on hypertensive VR and can be a strategy for treating hypertensive VR.%
\end{abstract}%



\sloppy


\textbf{Abbreviations}

AngII: angiotensin II

Col: collagen

CTGF: connective tissue growth factor

CVF: collagen volume fraction

ECM: extracellular matrix

EZH2: enhancer of zeste homolog 2

GAPDH: glyceraldehyde-3-phosphate dehydrogenase

H\&E: hematoxylin-eosin

LD: lumen diameter

miR: microRNA

MT: media thickness

MuT: mutant-type

OD: optical density

PBS: phosphate buffered saline

PCNA: proliferating cell nuclear antigen

p-Smad3: phospho-Smad3

qRT-PCR: quantitative real-time PCR

rAAV: recombinant adeno-associated virus

SBP: systolic blood pressure

SHR: spontaneously hypertensive rat

siRNA: small interfering RNA

VR: vascular remodeling

VSMC: vascular smooth muscle cell

WKY: Wistar Kyoto rat

\textbf{Introduction}

Vascular remodeling (VR) persists throughout the course of hypertension
and is characterized by chronic, continuous, and complex changes in
vascular structure and abnormalities in vascular functions
(Hayashi\emph{et al.} , 2009; Lemarie \emph{et al.} , 2010; Zhang
\emph{et al.} , 2019). VR, as an important indicator of the progression
of hypertension, is closely related to the severity and prognosis of the
disease (Mulvany, 2012; Magnussen, 2017; Brown \emph{et al.} , 2018).
Angiotensin II (AngII)-induced dysfunction of vascular smooth muscle
cells (VSMCs) plays a critical role in hypertensive VR (Das \emph{et
al.} , 2018), but the potential molecular mechanisms remain poorly
understood. Therefore, study of new therapeutic targets for hypertensive
VR and exploring potential molecular mechanisms are of benefit.

MicroRNAs (miRs) are highly conserved, small non-coding RNAs that
modulate the function of VSMCs, such as their proliferation,
differentiation, and migration (Chen \emph{et al.} , 2018a; Chen
\emph{et al.} , 2018b; Wang \emph{et al.} , 2019a; Wang \emph{et al.} ,
2019b). Moreover, miR expression is highly related to vascular
remodeling and angiogenesis (Henn \emph{et al.} , 2019). Cai \emph{et
al.} found that miR-24 attenuated VR under diabetic conditions (Cai
\emph{et al.} , 2019). Downregulation of miR-224 aggravated VR in acute
coronary syndrome (Xu \emph{et al.} , 2019). MiR-1 overexpression
mitigated pulmonary VR, thereby protecting against the progression of
pulmonary hypertension (Sysol \emph{et al.} , 2018).

MiR-26a, a highly conserved post-transcriptional regulator, can inhibit
the proliferation and migration of VSMCs after vascular injury
(Tan\emph{et al.} , 2017) and protect VSMCs against oxidative stress
(Peng\emph{et al.} , 2018). Thus, miR-26a may be a therapeutic target
for vascular dysfunction. Moreover, the serum level of mir-26a was found
altered in patients with hypertension (Yang \emph{et al.} , 2018).
However, the role and molecular mechanisms of miR-26a in hypertensive VR
need to be clarified.

Our research aimed to elucidate the role of miR-26a in hypertensive VR
and to reveal the potential miR-dependent mechanisms. We present
evidence that miR-26a plays a protective role in hypertensive VR.
AngII-activated Smad3 inhibited miR-26a expression, which in turn
promoted Smad3 activation by targeting Smad4, thereby forming a
Smads/miR-26a positive feedback loop and further downregulating miR-26a.
Downregulation of miR-26 further led to VR by promoting connective
tissue growth factor (CTGF) expression and the enhancer of zeste homolog
2 (EZH2) /p21 pathway.

\textbf{Materials and Methods}

\textbf{2.1 Animals and experimental protocols}

Seven-week-old male spontaneously hypertensive rats (SHRs; 190\selectlanguage{ngerman}±10 g) and
Wistar Kyoto rats (WKYs; 200±10 g) were purchased from Beijing Wei Tong
Lihua Experimental Animal Center. Ten-week-old male C57BL/6 mice (25±2
g) were purchased from the Laboratory Animal Center of Xi'an Jiaotong
University. Animals were housed in a room with a controlled environment
(12/12-h light/dark cycle, 22-25°C, 50-60\% relative humidity) with free
access to normal diet and water. After study, all animals were
anesthetized by inhalation of 1-2.5\% isoflurane, and then euthanized by
cervical dislocation. All animal experimental protocols were approved by
the local and national ethical committee and conformed to NIH guidelines
and the recommendations made by the British Journal of Pharmacology.

After 1 week of adaptation, SHRs were randomly divided into 3 groups of
8 rats each: recombinant adeno-associated virus (rAAV)-miR-26a (150 \selectlanguage{greek}μ\selectlanguage{english}l,
i.v.); rAAV-GFP (150 \selectlanguage{greek}μ\selectlanguage{english}l, i.v.) (a total dose of~viral particles was
2 \selectlanguage{ngerman}×\selectlanguage{english} 10\textsuperscript{11}~v.g.; the rAAV vectors we used were rAAV of
type-9; rAAV of miR-26a and vector controls was synthesized by Han Heng
Biotechnology, China); and SHR-Ctrl (normal saline 150 \selectlanguage{greek}μ\selectlanguage{english}l, i.v.). Six
WKY rats were normal controls (normal saline 150 \selectlanguage{greek}μ\selectlanguage{english}l, i.v.). Systolic
blood pressure (SBP) of the caudal artery of rats was measured every 3
to 5 days by using a non-invasive BP analysis system (BP-2000 SERIESII,
Visitech, USA). After 3 weeks, plasma and thoracic aortas of rats were
collected.

For mice experiments, C57BL/6 mice (n = 6 per group) received AngII
(Merck, USA, 2.0 mg/kg/d) or normal saline subcutaneously by implanted
ALZET\selectlanguage{ngerman}® 2002 minipumps under isoflurane anesthesia. Two weeks later,
plasma and thoracic aortas of mice were collected.

\textbf{2.2 Histology}

Thoracic aorta sections were fixed in 4\% paraformaldehyde, then
embedded in paraffin. Morphological characteristics of thoracic aorta
were assessed by hematoxylin-eosin (H\&E) staining. The media thickness
(MT) of thoracic aorta was defined as the region between the internal
and external elastic laminae. The MT/lumen diameter (LD) represented the
level of VR. Thoracic aorta fibrosis was assessed by Masson's trichrome
staining. Collagen volume fraction (CVF) was quantified by measuring the
ratio of blue fibrotic area to total thoracic aorta area.

\textbf{2.3 Immunohistochemical staining}

Aortic tissue sections (4\selectlanguage{english} \selectlanguage{greek}µ\selectlanguage{english}m) were deparaffinized, and endogenous
peroxidase activity was quenched with
H\textsubscript{2}O\textsubscript{2}. The sections were incubated with
anti-\selectlanguage{greek}α-\selectlanguage{english}actin (dilution 1:200) and anti-proliferating cell nuclear
antigen (PCNA; dilution 1:100) antibody at 4\selectlanguage{ngerman}°C overnight, then with
secondary antibody. After staining, each section was analyzed by
confocal microscopy (DS-Fi1-Eclipse, Nikon).

\textbf{2.4 Cell culture}

VSMCs were prepared from thoracic aorta of 50-day-old female
Sprague-Dawley rats. Aortic VSMCs were cultured in Dulbecco's modified
Eagle's medium (Gibco, USA) with 10\% fetal bovine serum (BI, USA) and
1\% penicillin/streptomycin at 37\selectlanguage{english} degC in a 5\%
CO\textsubscript{2}humidified incubator. Immunohistochemical staining
revealed positivity for smooth muscle \selectlanguage{greek}α-\selectlanguage{english}actin (Wanleibio, China) and
negativity for CD105 (Wanleibio, China), indicating that primary VSMCs
of rats were successfully isolated. Cells in passages 3-9 were used for
all experiments.

\textbf{2.5 Transfection}

After 24-h incubation in serum-free medium, VSMCs were transfected with
the miR-26a mimic (JTS Scientific, China) or miR-26a inhibitor (JTS
Scientific, China) or a corresponding negative control (NC) or siRNA
(Wanleibio, China) or SB431542 (MCE, China) according to the
manufacturer's instructions. After 24- or 48-h transfection, cells were
treated with AngII (10\textsuperscript{-7} mol/L for 24 h).

\textbf{2.6 CCK-8 assay}

VSMCs were cultivated in 96-well plates, with
4\selectlanguage{ngerman}×10\textsuperscript{3}cells in each well, and 5 duplicate wells were
designed for each group. A 10-\selectlanguage{greek}μ\selectlanguage{english}L amount of CCK-8 solution (Wanleibio,
China) was added after 24 h and incubated for 1 h under 5\%
CO\textsubscript{2} at 37. A microplate reader (BioTek, USA) was used
to detect optical density (OD) values at 450 nm, which represents cell
proliferation.

\textbf{2.7 Cell cycle assay}

Each group of VSMCs was washed twice with ice-cold phosphate buffered
saline (PBS); cells were harvested by centrifugation and re-suspended in
PBS, then fixed and permeabilized with 70\% ethanol. Cells were washed
twice in ice-cold PBS to remove ethanol, then treated with 500 \selectlanguage{greek}μ\selectlanguage{english}l
propidium iodide (PI; Wanleibio, China) in the dark at 4\selectlanguage{ngerman}°C for 30 min.
Flow cytometry (ACEABio, NovoCyte, USA) was used to detect cell cycle.

\textbf{2.8 Immunofluorescence cell staining}

Cultured VSMCs were washed three times with PBS and fixed with 4\%
paraformaldehyde for 15 min, followed by permeabilization with 0.1\%
Triton-X-100 (Beyotime, China) for 30 min. Then cells were washed three
times with PBS and incubated with anti-phospho-Smad3 (anti-p-Smad3)
antibody (1:200 dilutions, ab52903, Abcam, England) overnight at 4°C,
followed by secondary goat anti-rabbit IgG-Cy3 antibody (1:200
dilutions, A0516, Beyotime, China) incubated for 1 h at room
temperature. Nuclear staining of the cells involved using
4'6-diamidino-2-phenylindole dihydrochloride (DAPI, Beyotime, China) for
5 min at room temperature. Images were captured by fluorescence
microscopy (OLUMPUS, Japan).

\textbf{2.9 ELISA}

Levels of collagen I and III (Col I and III) were detected by using
ELISA kits (Cloud Clone Corp., China) according to the manufacturer's
recommendations, and the absorbance at 450~nm was read. The relative Col
I and III levels were calculated according to the standard
concentration-absorbency curve.

\subsubsection*{2.10 Quantitative real-time PCR
(qRT-PCR)}

{\label{quantitative-real-time-pcr-qrt-pcr}}

Total RNA from plasma, tissues or cells was extracted with Trizol
reagent (Thermo Fisher Scientific, USA). cDNA was synthesized by using
the miRNA Reverse Transcription System kit (TaKaRa, Japan) or 2×SYBR
real-time PCR premixture (Bioteke Corp., China), following the
manufacturer's protocol. Real-time PCR was performed with the
StepOnePLus Real-Time PCR System. The primer sequences and conditions
for amplification are in Supplemental Table S2 and S2, respectively. U6
or \selectlanguage{greek}β-\selectlanguage{english}actin was the internal reference. The relative expression of mRNAs
and miR was calculated by the 2\textsuperscript{-^^Ct}method.

{\label{total-rna-from-plasma-tissues-or-cells-was-extracted-with-trizol-reagent-thermo-fisher-scientific-usa.-cdna-was-synthesized-by-using-the-mirna-reverse-transcription-system-kit-takara-japan-or-2sybr-real-time-pcr-premixture-bioteke-corp.-china-following-the-manufacturers-protocol.-real-time-pcr-was-performed-with-the-steponeplus-real-time-pcr-system.-the-primer-sequences-and-conditions-for-amplification-are-in-supplemental-table-s2-and-s2-respectively.-u6-or-ux3b2-actin-was-the-internal-reference.-the-relative-expression-of-mrnas-and-mir-was-calculated-by-the-2ct-method.}}

\textbf{2.11 Western blot analysis}

Protein was extracted from tissues or cells by using RIPA lysate (Hat
Biotechnology, China). After quantification with bicinchoninic acid,
equal amounts of protein were loaded and separated by SDS-PAGE, then
transferred electrophoretically to polyvinylidene difluoride membranes,
which were incubated with primary antibodies overnight at 4, followed
by a 1:5000 dilution of goat anti-rabbit IgG antibody (EK020, Zhuangzhi
Biotech, China) at room temperature. Primary antibodies were for CTGF
(1:1000, ab6992), Smad3 (1:1000, ab40854), Smad4 (1:1000, ab40759),
p-Smad3 (1:500, ab193297), cyclin D2 (1:1000, ab230883), p21 (1:500,
ab109199; all Abcam, England); EZH2 (1:500, 5246; CST, USA); Col I
(1:500, WL0088), Col III (1:500, WL0318), \selectlanguage{greek}β-\selectlanguage{english}actin (1:500, WL01372; all
Wanleibio, China), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH;
1:1000, AF7021, Affinity Biosciences, USA). Protein bands were
visualized by using a potent ECL kit (KF001, Affinity Biosciences, USA)
and the Gene Company imaging system (China). GAPDH or \selectlanguage{greek}β-\selectlanguage{english}actin was an
internal reference.

\textbf{2.12 Chromatin immunoprecipitation (ChIP) assay}

1\% formaldehyde was used to cross-link VSMCs for 10 min at room
temperature, then glycine (0.125M) was added for 5 min. The ChIP assay
was performed with Smd3 or lgG antibody (Abcam, USA) and the ChIP kit
(Wanleibio, China) according to the manufacturer's instructions. Bound
DNA fragments were subjected to PCR. For PCR, 2 \selectlanguage{greek}μ\selectlanguage{english}L of DNA was amplified
with 2\selectlanguage{ngerman}×Taq PCR Master-mix (Bioteke Corp., China), using a primer pair
specific to the binding region of the miR-26a promoter. The primer
sequences and the condition are in Supplemental Table S1 and S2,
respectively. Finally, PCR products were visualized on 2\% agarose gels.

\textbf{2.13 Luciferase reporter assay}

The mutant-type (MuT) 3\selectlanguage{english}'-UTR of EZH2, CTGF and Smad4 was constructed.
The WT or MuT 3\selectlanguage{english}'-UTR of EZH2, CTGF and Smad4 were inserted into pMIRGLO
vectors (Promega, USA). The miR-26a mimic or miR-negative control and WT
or MuT 3\selectlanguage{english}'-UTR of EZH2, CTGF and Smad4 vector were co-transfected into
HEK293T cells (Wanleibio, China). After 48 h, cells were lysed, and
luciferase activity was determined by using a luciferase assay kit
(Promega, USA) and multi-function microplate reader (TECAN, M200Pro,
Switzerland).

\textbf{2.14 Data and statistical analysis}

The data and statistical analysis comply with the recommendations of the
British Journal of Pharmacology on experimental design and analysis in
pharmacology (Curtis \emph{et al.} , 2018). All data are expressed as
mean +- SD and GraphPad Prism 6 software was performed to statistical
analysis. To determine differences between groups with one factor, data
were tested using unpaired Student's t-test for two group's comparison,
and one\selectlanguage{english}-way ANOVA with Tukey's post hoc test analysis for multiple group
comparisons. To determine differences between groups with multiple
factors, data were tested using two-way ANOVA followed by Bonferroni's
test. Post hoc tests were run only when F achieved P \textless{} 0.05.
Differences were considered statistically significant at P \textless{}
0.05. All measurements were undertaken only for n \selectlanguage{english}[?] 5.

\textbf{Results}

\textbf{3.1 Ang II regulated miR-26a expression both in vivo and in
vitro}

MiR-26a expression was reduced in AngII-treated VSMCs dose- and
time-dependently as compared with the control (Fig. 1a, b), so AngII
negatively regulated the expression of miR-26a. In vivo, the relative
level of miR-26a was lower in the thoracic aorta and plasma of
AngII-treated mice than the control (Fig. 1c), which indicated Ang II
regulated miR-26a expression both in vivo and in vitro.

\textbf{3.2 miR-26a had a protective effect on hypertensive VR}

To evaluate the role of miR-26a in hypertensive VR, rAAV vectors were
constructed to overexpress miR-26a in SHRs. As expected, rAAV-miR-26a
transfection significantly increased the expression of miR-26a in aortas
and plasma of SHRs (Fig. 2a). Meanwhile, rAAV-miR-26a prevented systolic
blood pressure (SBP) from continuously increasing in SHRs (Supplemental
Fig. S1). To evaluate VR, thoracic aortas underwent H\&E staining. As
compared with normal WKYs, in SHRs, the media of the thoracic aorta was
significantly thickened. However, miR-26a overexpression could reduce
the medial thickening of the thoracic aorta caused by hypertension. The
MT/LD ratio declined profoundly with miR-26a overexpression (Fig. 2b, c;
rAAV-miR-26a vs rAAV-GFP and SHR-Ctrl was 5.430 +- 0.2943 vs 6.793 +-
0.4130 and 7.575 +- 1.584; P\textless{}0.05). Immunohistochemistry for
smooth muscle \selectlanguage{greek}α-\selectlanguage{english}actin showed well-distributed smooth muscle and intact
intima in WKY rats. However, VSMCs formed intima hyperplasia and parts
of the vascular walls protruded into the aortic lumen in the SHR-Ctrl
and rAAV-GFP groups. rAAV-miR-26a treatment significantly improved
smooth muscle alignment and integrity (Fig. 2b).

\textbf{3.3 miR-26a ameliorates extracellular matrix (ECM) production in
vivo and in vitro.}

ECM is mainly composed of collagen, so we observed and quantified CVF in
vivo by Masson's trichrome staining. Trichrome staining and CVF were
reduced in SHRs transfected with miR-26a as compared with rAAV-GFP and
SHR-Ctrl groups (Fig. 3a, b; 31.26 \selectlanguage{ngerman}± 3.902 vs 54.25 ± 5.987 and 56.17 ±
7.218, P\textless{}0.05). Then, we transfected miR-26a mimic and
inhibitor into AngII-induced VSMCs to evaluate the effect of miR-26a on
collagen production in vitro. On qRT-PCR, miR-26a was significantly
elevated by miR-26a mimic treatment and markedly inhibited by miR-26a
inhibitor treatment in AngII-treated VSMCs; neither of their negative
controls (NCs) promoted or suppressed miR-26a expression (Fig. 3c). As
expected, miR-26a mimic overexpression significantly suppressed and
miR-26a inhibition enhanced the mRNA and protein expression of Col I and
Col III according to ELISA, qRT-PCR and western blot analysis (Fig.
3d-f). In short, miR-26a inhibited collagen deposition both in vivo and
in vitro.

\textbf{3.4 miR-26a inhibits VSMC proliferation in vivo and in vitro.}

The development of hypertensive VR is associated with the excessive
proliferation of VSMCs. To determine whether miR-26a affected VSMC
proliferation, immunohistochemistry for PCNA was used to test VSMC
proliferation in thoracic aortas of SHRs and WKY rats. Cells with brown
staining were defined as PCNA-positive cells. The proportion of
PCNA-positive cells (ratio of number of PCNA-positive cells to total
number of sampled cells) was lower with rAAV-miR-26a overexpression than
in vector- and vehicle-treated controls (Fig. 4a, b; 0.08801 ± 0.01623
vs 0.4440 ± 0.04262 and 0.4496 ± 0.01413, P\textless{}0.05). We used
CCK\selectlanguage{english}-8 assay and flow cytometry to measure the proliferation of VSMCs in
vitro. Upregulation of miR\selectlanguage{english}-26a reduced the proliferation of
AngII-induced VSMCs and retained cells in the G1 phase (Fig. 4c-e).
miR-26a inhibitor treatment had the opposite effect, promoting cell
proliferation.

\textbf{3.5 miR-26a suppresses ECM deposition by directly targeting
CTGF.}

CTGF is essential for the synthesis of collagen (Liang \emph{et al.} ,
2014). We hypothesized that miR-26a attenuates ECM accumulation by
regulating CTGF. In vivo data shown in Figure 5a support this
hypothesis: CTGF protein level was significantly downregulated in SHR
thoracic aortas with rAAV-miR-26a overexpression. The effect of miR-26a
was further confirmed in vitro: miR-26a mimic suppressed CTGF expression
in AngII-induced VSMCs and miR-26a inhibitor increased CTGF protein and
mRNA levels in vitro (Fig. 5b, c). Bioinformatics analysis by using
Targetscan (www.targetscan.org) predicted CTGF as a potential target
gene of miR-26a (Fig. 5d). By using a reporter construct with the
putative 3\selectlanguage{english}'-UTR miR-26a binding site of CTGF downstream of the
luciferase gene, we found that miR-26a reduced luciferase activity,
which provides experimental validation of CTGF as a target for miR-26a.
Accordingly, after mutation of the binding site, the activity of
luciferase was not altered (Fig. 5e), which confirms that the binding of
miR-26a to the 3\selectlanguage{english}'-UTR site was necessary for silencing CTGF

\textbf{3.6 EZH2/p21 pathway mediates miR-26a dysregulation-induced VSMC
proliferation.}

To explore the mechanisms of miR-26a-inhibited proliferation of VSMCs,
we investigated the regulation of miR-26a of proliferation-related
genes. Considering that rAAV-miR-26a overexpression decreased EZH2
protein level in the thoracic aorta of SHRs (Fig. 6a), we assumed that
miR-26a inhibiting VSMC proliferation is mediated by EZH2. Consistent
with in vivo findings, in AngII-induced VSMCs, the expression of EZH2 at
both the protein and mRNA levels was significantly decreased after
transfection with miR-26a mimic (Fig. 6b, e). Furthermore, Targetscan
revealed that the miR-26a binding site within the 3'UTR of EZH2 mRNA is
highly conserved (Fig. 6f). We further performed luciferase reporter
assay and found reduced luciferase activity with miR-26a mimic
transfection. However, miR-26a did not affect activity of the binding
site mutation, which confirms that EZH2 is a target of miR-26a (Fig.
6g).

p21 belongs to a cell-cycle regulator that contributes to cell growth
inhibition, and the expression of p21 is indirectly inhibited by EZH2
(Lu \emph{et al.} , 2011; Li \emph{et al.} , 2019). In our study, p21
expression was negatively related to the expression of EZH2 both in
vitro and in vivo (Fig.~6a, c), which suggests that miR-26a upregulated
p21 level by directly suppressing EZH2 expression. In other words,
miR-26a inhibited VSMC proliferation by regulating the EZH2/p21 pathway.

miR-26a can also regulate cell proliferation by directly targeting
cyclin D2 (Zhou \emph{et al.} , 2016). To explore whether miR-26a
regulated cyclin D2 expression in VSMCs, we transfected the miR-26a
mimic and inhibitor in AngII-induced VSMCs. The expression of cyclin D2
was markedly upregulated in AngII-induced VSMCs on transfection with
miR-26a inhibitor and was reversed by transfection with the miR-26a
mimic (Fig. 6d). Thus, miR-26a mitigates excessive VSMC proliferation by
targeting the EZH2/p21 pathway and cyclin D2.

\textbf{3.7 AngII regulates miR-26a expression via Smad3 activation.}

PROMO analysis revealed three potential Smad3 binding sites in the
miR-26a promoter (Fig.~7a). ChIP assay with VSMC lysates and anti\selectlanguage{english}-Smad3
antibody validated that Smad3 directly binds to the promoter of miR\selectlanguage{english}-26a
to suppress its expression. Additionally, Smad3 exhibited stronger
binding to the miR-26a promoter in AngII-induced VSMCs as compared with
controls (Fig.~7b, c). Thus, AngII promoted Smad3 binding to the miR-26a
promoter, thereby inhibiting miR-26a transcription. To further elaborate
whether miR-26a downregulation was mediated by Smad3 in VSMCs, we used
loss-of-function studies. We first constructed three siRNAs fragments of
Smad3 to transfect VSMCs. Both qRT-PCR and western blot analysis
confirmed that siRNA-3 silenced Smad3 the best (Supplemental Fig. S2a,
b). Next, we used siRNA or SB431542, which inhibited Smad3 expression or
activation, respectively, and observed increased miR-26a expression in
AngII-induced VSMCs (Fig. 7d-f), which indicates that Smad3 negatively
regulates miR-26a expression. These results indicate that AngII
regulates miR-26a expression via Smad3 activation.

\textbf{3.8 miR-26a inversely regulates the activation of Smad3}

We transfected AngII-induced VSMCs with an miR-26a mimic, which showed
high\selectlanguage{english}-transfection efficiency (Fig. 8a). Also, transfection of an miR-26a
mimic significantly decreased p-Smad3 protein expression (Fig. 8b).
Additionally, upregulation of miR-26a inhibited nuclear translocation of
p-Smad3 (Fig. 8c). These results implied that miR-26a can also inversely
inhibit Smad3 activation.

\textbf{3.9 miR-26a inhibits Smad3 activation by targeting Smad4}

We next sought to determine how miR-26a regulates Smad3 activation.
MiRanda (www.microRNA.org) analysis indicated that Smad4 contains a
binding site for miR-26a (Fig. 9a). We confirmed that Smad4 was a direct
target of miR-26a by luciferase reporter assay (Fig. 9b). Consistently,
Smad4 expression was significantly downregulated with miR-26a mimic
overexpression, whereas miR-26a inhibitor treatment increased the
protein level of Smad4 in AngII-induced VSMCs (Fig. 9c, d).

To explore the effect of Smad4 on Smad3 activation, we examined
siRNA-mediated knockdown of reduced level of Smad4 in VSMCs. We selected
siRNA with the best silencing effect on Smad4 to transfect AngII-treated
VSMCs (Supplemental Fig. S2c, d). Smad4 expression was highly relevant
for the expression and nuclear translocation of p-Smad3. Inhibition of
Smad4 could reduce p-Smad3 protein expression and decreased the nuclear
translocation of p-Smad3 (Fig. 9e-h). Therefore, Smad4 promoted the
activation of Smad3. In summary, miR-26a regulated Smad3 activation by
targeting Smad4.

Combined with previous results (Fig. 7-9), a positive feedback loop
between miR-26a and Smad3/4 may be involved in hypertensive VR (Fig.
10).

\textbf{Discussion}

In this study, we evaluated the role and mechanisms of miR-26a in
controlling hypertensive VR (Fig. 10). MiR-26a was significantly
downregulated in the thoracic aorta and plasma of SHRs with VR.
Overexpression of miR-26a effectively reduced ECM deposition and VSMC
hyperproliferation both in vivo and in vitro. Also, AngII potentiated
Smad3 binding to the miR-26a promoter, inhibiting miR-26a transcription,
which in turn promoted Smad3 activation by upregulating Smad4, thereby
further downregulating miR-26a. These findings reveal a possible new
positive feedback loop between miR-26a and Smad3/4, which may be related
to hypertensive VR. Moreover, miR-26a mitigated ECM accumulation and
excessive VSMC proliferation in AngII-induced VSMCs by targeting CTGF
and the EZH2/p21 pathway, respectively. Our study suggests that miR-26a
is a protective molecule in hypertensive VR and can be considered a
promising target for treating hypertensive VR.

The crucial finding of this study is that miR-26a plays a protective
role in hypertensive VR. Previous studies have shown that miR-26a is
closely related to cardiovascular diseases. Li \emph{et al.} found low
expression of miR-26a in plasma of patients with acute myocardial
infarction as compared with healthy people (Li \emph{et al.} , 2015).
Zhang \emph{et al.} showed that miR-26a plays a role in the process of
myocardial fibrosis after acute myocardial infarction by inhibiting
phosphatase and tensin homolog expression, enhancing matrix
metalloproteinase 9 level and promoting the PI3K/AKT pathway
(Zhang\emph{et al.} , 2018). Chiang \emph{et al.} discovered that
miR-26a ameliorates cardiac dysfunction and fibrosis in myocardial
infarction (Chiang \emph{et al.} , 2020). In this study, we first
observed that miR-26a expression was downregulated in the thoracic aorta
and plasma of SHRs. We next used rAAV-miR-26a to overexpress miR-26a in
the thoracic aorta and plasma of SHRs. The overexpression of miR-26a
reduced media thickening of the thoracic aorta and diminished the
severity of VR induced by hypertension. However, due to the limitations
of this experiment, we have not been able to obtain data on the extent
of SHR aortic remodeling before miR-26a treatment. Therefore, our
conclusion was based on the hypothesis that there was no difference in
VR of SHRs in each group before intervention. Meanwhile, we found that
overexpression of miR-26a appears to have an effect on blood pressure,
it is unknown whether miR-26a directly reduces VR or if it is secondary
to reduced blood pressure, which can continue to study in future.

One of the characteristics of VR is pathological remodeling of ECM,
which is often accompanied by increased ECM release and collagen
deposition (Ricard-Blum \emph{et al.} , 2019). ECM is a structural
scaffold of the blood vessel wall, which controls cellular functions in
the pathological environment. When stimulated or injured, the deposition
of ECM protein, especially Col I, alters the collagen/elastin ratio and
hemodynamics, leading to increased vascular stiffness and affecting
blood vessel function (Lee \emph{et al.} , 2015). We found the CVF of
the thoracic-aorta media layer reduced in miR-26a-overexpressed SHRs. In
vitro studies showed that miR-26a mimic treatment inhibited but miR-26a
inhibitor treatment promoted Col I and III expression in AngII-induced
VSMCs. CTGF belongs to matricellular protein expressed by various cells
in response to stimuli and is an important molecule prominently
implicated in increased deposition of ECM (Li \emph{et al.} , 2016;
Petrosino \emph{et al.} , 2019). Our results showed that that CTGF is a
downstream target gene of miR-26a and the level of miR-26a was inversely
associated with CTGF expression both in vivo and in vitro, so miR-26a
may diminish ECM deposition by targeting CTGF. Additionally, the Col I
is a downstream target gene of miR-26a (Wei \emph{et al.} , 2013).
MiR-26a may directly inhibit collagen synthesis to reduce ECM
deposition, which needs further study.

Another pathological manifestation of hypertensive VR is excessive VSMC
proliferation, which is determined by the cell cycle (Deniset \emph{et
al.} , 2018; Lu \emph{et al.} , 2018; He \emph{et al.} , 2019). EZH2
accelerates the cell cycle, whereas p21 arrests the cell cycle
(Karimian\emph{et al.} , 2016; Dai \emph{et al.} , 2018). EZH2/p21 has
been found a critical signaling pathway to regulate cell proliferation
(Lu \emph{et al.} , 2011; Li \emph{et al.} , 2019). We found that
transfection with rAAV-miR-26a suppressed PCNA and EZH2 expression, but
p21 was upregulated in the thoracic aorta of SHRs. Similarly, in vitro
experiments showed that miR-26a inhibited the progression of the cell
cycle accompanied by downregulated protein level of EZH2, whereas p21
level was correspondingly increased in AngII-induced VSMCs; miR-26a
inhibitor treatment had the opposite result. Furthermore, EZH2 is a
target gene of miR-26a, so miR-26a participates in VSMC proliferation by
targeting the EZH2/p21 pathway. Cyclin D2 can promote the G1/S
transition (Gul \emph{et al.} , 2018; Pei \emph{et al.} , 2018), and
previous studies with luciferase reporter assay showed that cyclin D2 is
also a target gene of miR-26a (Zhou \emph{et al.} , 2016). Consistently,
we found that miR-26a suppressed cyclin D2 expression in AngII-induced
VSMCs. Thus, miR-26a may be involved in excessive VSMC proliferation by
directly regulating cyclin D2 apart from targeting the EZH2/p21 pathway.

Our study confirmed that AngII regulates miR-26a level dose- and
time-dependently. However, how AngII regulates miR-26a expression is
unclear. Some studies demonstrated that miRs are subject to
sophisticated regulation, and superposition of subtle changes in the
expression of several molecules may be responsible for miRs exerting
their effects (Krol \emph{et al.} , 2010; Liang \emph{et al.} , 2014).
Previous studies determined that miR-26a inhibits TGF\selectlanguage{greek}β-\selectlanguage{english}dependent Smad
signaling (Leeper \emph{et al.} , 2011). We focused on TGF\selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}independent
Smad signal regulatory mechanisms of miR-26a. In this study, we
validated that Smad3 could directly suppress miR-26a expression, and
AngII could promote this effect; AngII regulates miR-26a by activating
Smad3. Previous studies reported a functional crosstalk between miR-26a
and other molecules via a feedback loop (Jiang \emph{et al.} , 2018;
Liu\emph{et al.} , 2018). We found that miR-26a downregulation enhanced
Smad4 expression via targeted regulation and then induced Smad3
activation and p-Smad3 nuclear translocation. Smad3 activation caused
further downregulation of miR-26a, thereby forming a Smads/miR-26a
positive feedback loop. Once the loop is activated, it will run
repeatedly, eventually leading to increased ECM accumulation, excessive
VSMC proliferation and VR.

To sum up, miR-26a plays a protective role in hypertensive VR. The
effect of miR-26a on VR was mediated by activation of a Smads/miR-26a
positive feedback loop. In addition, miR-26a inhibited ECM deposition by
targeting CTGF and attenuated VSMC proliferation by regulating the
EZH2/p21 pathway. We clarify the functions and mechanisms of miR-26a in
hypertensive VR and suggest that miR-26a may be a novel therapeutic
target of hypertensive VR.

\textbf{Acknowledgements}

The authors thank the Research and Experiment Center of the Second
Affiliated Hospital of Xi'an Jiaotong University and the Experimental
Animal Center of the Medical Department of Xi'an Jiaotong University for
technical assistance and experimental equipments. This work was
supported by the National Natural Science Foundation of China (81570382,
81872563).

\textbf{Conflict of interest}

The authors declare that they have no competing interests.

\textbf{Author contributions}

D.G. conceived and designed this study. W.Z., Q.Z., X.X., L.Y., M.X.,
C.C., R.W. and W.C. conducted the experiments and analysed the data.
D.G. and W.Z. drafted the manuscript and coordinated manuscript
revisions. Prof. X. N. provided suggestion for the experiments. The
manuscript has been reviewed and approved by all authors.

\textbf{Declaration of transparency and scientific rigour}

This Declaration acknowledges that this paper adheres to the principles
for transparent reporting and scientific rigour of preclinical research
as stated in the BJP guidelines for Design \& Analysis, Immunoblotting
and Immunochemistry, and Animal Experimentation, and as recommended by
funding agencies, publishers and other organisations engaged with
supporting research.

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